Top Banner
Studies in the Enzymatic depolymerisation of natural polysaccharides A THESIS SUBMITTED TO THE UNIVERSITY OF MUMBAI FOR THE Ph. D. (Technology) DEGREE IN CHEMICAL ENGINEERING SUBMITTED BY SATISH DASHARATH SHEWALE UNDER THE GUIDENCE OF Prof. A. B. Pandit INSTITUTE OF CHEMICAL TECHNOLOGY UNIVERSITY OF MUMBAI MATUNGA, MUMBAI-400 019. INDIA JUNE-2008
268

Studies in depolymerization of natural polysaccharides--PhD thesis

Nov 18, 2014

Download

Documents

satish_shewale
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Page 1: Studies in depolymerization of natural polysaccharides--PhD thesis

Studies in the Enzymatic depolymerisation of

natural polysaccharides

A THESIS SUBMITTED

TO THE

UNIVERSITY OF MUMBAI

FOR THE

Ph. D. (Technology) DEGREE

IN CHEMICAL ENGINEERING

SUBMITTED BY SATISH DASHARATH SHEWALE

UNDER THE GUIDENCE OF Prof. A. B. Pandit

INSTITUTE OF CHEMICAL TECHNOLOGY

UNIVERSITY OF MUMBAI

MATUNGA, MUMBAI-400 019.

INDIA

JUNE-2008

Page 2: Studies in depolymerization of natural polysaccharides--PhD thesis

STATEMENT BY THE CANDIDATE

As required by the University Ordinance 770, I wish to state that the work

embodied in this thesis titled “Studies in the Enzymatic depolymerisation of

natural polysaccharides”, forms my own contribution to the research work

carried out under the guidance of Prof. A. B. Pandit at the Institute of Chemical

Technology, University of Mumbai, Matunga, Mumbai. This work has not been

submitted for any other degree of this or any other university. Whenever

references have been made to the previous work of others, it has been clearly

indicated as such and included in the Bibliography.

Satish D. Shewale

(Research Student)

Certified by,

Prof. Aniruddha B. Pandit

(Research supervisor)

UGC Scientist ‘C’ (Professor’s Grade),

Chemical Engineering Division,

Institute of Chemical Technology,

University of Mumbai,

Matunga, Mumbai – 400 019.

Date:

Place: Matunga, Mumbai – 400 019

Page 3: Studies in depolymerization of natural polysaccharides--PhD thesis

CERTIFICATE

The research work described in this thesis has been carried out by Mr. Satish D.

Shewale under my supervision. I certify that this is his bonafide work. The work

described is original and has not been submitted for any other degree of this or any

other university. Further that he was a regular student and has worked under my

guidance as a full time student at Institute of Chemical Technology, University of

Mumbai, Matunga, Mumbai – 400 019 until the submission of the thesis to the

University of Mumbai.

Prof. Aniruddha B. Pandit

(Research Supervisor)

UGC Scientist ‘C’ (Professor’s Grade)

Chemical Engineering Division,

Institute of Chemical Technology,

University of Mumbai,,

Matunga, Mumbai – 400 019.

Date:

Place: Matunga, Mumbai-400 019.

Page 4: Studies in depolymerization of natural polysaccharides--PhD thesis

List of publications

1. Satish D. Shewale; Aniruddha B. Pandit “Hydrolysis of soluble starch using B.

licheniformis α-amylase immobilized on superporous CELBEADS”,

Carbohydrate Research, 2007, 342 (8), Pg. 997-1008.

2. Satish D. Shewale; Aniruddha B. Pandit “Enzymatic production of glucose

from different qualities of sorghum and application of ultrasound to enhance

the yield”, Carbohydrate Research, Forwarded for publication.

3. Satish D. Shewale; Aniruddha B. Pandit “Enzymatic production of maltose

from different qualities of sorghum and application of ultrasound to enhance

the yield”, Carbohydrate Research, To be Forwarded for publication.

Page 5: Studies in depolymerization of natural polysaccharides--PhD thesis

Dedicated to my family . . . .

Page 6: Studies in depolymerization of natural polysaccharides--PhD thesis

Acknowledgement qÉÉhÉxÉÉcrÉÉ LMÇüSU eÉÏuÉlÉmÉëuÉÉxÉÉiÉ ÌMüiÉÏiÉUÏ lÉuÉÏlÉ aÉÉPûÏpÉåOûÏ WûÉåiÉÉiÉ. MükÉÏMükÉÏ uÉÉOûiÉ AzÉÉ aÉÉPûÏpÉåOûÏ fÉÉsrÉÉ lÉxÉirÉÉ iÉU MüÉrÉ fÉÉsÉÇ AxÉiÉÇ ? MüÉ AzÉÏ uÉåaÉuÉåaÉVûÏ qÉÉhÉxÉÇ AÉmÉsrÉÉ AÉrÉÑwrÉÉiÉ mÉëuÉåzÉ MüUiÉÉiÉ AÉÍhÉ irÉÉÇcrÉÉ mÉëåqÉÉcÉÉ PûxÉÉ qÉlÉÉuÉU EqÉOûuÉÑlÉ eÉÉiÉÉiÉ ? LMüÉ ÌPûMüÉhÉÏ rÉÉcÉ xÉÇSpÉÉïiÉ AqÉÚiÉÉ mÉëÏiÉqÉ ÌMüiÉÏ NûÉlÉ ÍsÉWÕûlÉ aÉåsrÉÉiÉ –

EqÉUÉ Så CxÉ MüÉaÉeÉ Måü E¨Éå CzMåü iÉåUÉ AÇaÉÑPûÉ sÉÉrÉÉ, MüÉælÉ ÌWûxÉÉoÉ cÉÑMüÉLaÉÉ

(qÉÉfrÉÉ AÉrÉÑwrÉÉcrÉÉ MüÉaÉSÉuÉU iÉÑfrÉÉ mÉëåqÉÉlÉå AÉmÉsÉÉ AÇaÉPûÉ EqÉOûuÉsÉÉrÉ irÉÉcÉÉ ÌWûzÉåoÉ MüÉåhÉ SåhÉÉU) ZÉUÇ iÉU AzÉÉcÉ mÉSkÉiÉÏlÉå ÌMüiÉÏiÉUÏ qÉÉhÉxÉÉÇcÉå mÉëåqÉPûxÉå AÉmÉsrÉÉ AÉrÉÑwrÉÉuÉU EqÉOûiÉ eÉÉiÉÉiÉ. irÉÉÇcÉÉ ÌWûzÉÉåoÉ AÉmÉsrÉÉsÉÉWûÏ SåiÉÉ rÉåiÉ lÉÉWûÏ, ±ÉrÉcÉÉ mÉërɦÉWûÏ Müà lÉrÉå. mÉhÉ Wåû PûxÉå AÉmÉsrÉÉsÉÉ eÉaÉhÉ ÍzÉMüuÉiÉÉiÉ Wåû qÉÉ§É lÉÉMüUiÉÉ rÉåiÉ lÉÉWûÏ. AÉmÉsÉå AWÇûMüÉU, sÉÉåpÉ, CwÉÉï, MüÉåhÉirÉÉiÉUÏ LZÉÉ±É uÉthÉÉuÉU ÌmÉMüsrÉÉ mÉÉlÉÉmÉëqÉÉhÉå AÉmÉxÉÔMü aÉVÕûlÉ mÉQûiÉÉiÉ. xÉÇSpÉï : uÉVûhÉÉuÉUcÉÏ qÉÉhÉxÉÇ, QûÊ. UÉeÉåÇSì qÉÉlÉå UICT is the place, where I have spent about 6 years of my life and these years

are mainly responsible for my way of being today. So firstly I thank UICT and

all the people associated with UICT. I thank all the teaching staff of UICT in

M. Chem. Engg. for taking my chemical engineering knowledge to new

heights.

I express my sincere gratitude to my research supervisor Prof. Aniruddha B.

Pandit for his constant encouragement, bearing with me and supporting me

in all my downtimes, not spoon feeding me in the research work and bringing

the best in me. I always amazed by his knowledge, his way of thinking, calm

nature, and switching gears from one research area to other in the lab

meeting. His enthusiasm always motivated me and compelled me to do at my

best. I have been enormously fortunate to have a supervisor who has always

had the time and patience to answer the many (often quite stupid and tedious

I think) questions I have had. I am also thankful to him for checking several

versions of the article that needs to be forwarded for publication, reports,

chapters of thesis etc. without getting bore of mistakes in them.

I would like to thank Prof. A. M. Lali for providing CELBEADS for the

present work and allowing me to use lab facilities from his lab.

Page 7: Studies in depolymerization of natural polysaccharides--PhD thesis

I would like to thank Prof. D. N. Bhowmick for allowing me to use facilities

from his lab.

I am grateful to UGC for providing research fellowship during tenure of Ph D

work.

My special thanks are due to Mr. Potdar from workshop, who always being

there in case of any instrument problems. Without his efforts, it would have

been really very difficult to complete this work.

I thank my very special friends during the entire time, i spent in UICT,

Yogesh Doshi, my “saala” – “Awaara” Balu and Ajit (my langoti yaar in

chemical engineering) for their timely help and constant encouragement. I am

really very thankful to god to give me such wonderful friends.

I would like to thank all my ABP labmates Parag, Virendra, Gopal, Rajesh,

Shashank (KS), Mohan, Amit (ask him any problem about computer,

amazingly he has solution), Pratap, Prashant (Birra), Ambu, Parag (kanthu),

Preeti, Shailesh (Sher), Shankar, Vishwa, Haresh, Naresh, Ajaykumar,

Pramod, Hemant, Sandip, Shravan, Bijal, Aditi, Apoorva, and all others who

have newly joined the ABP group.

My special thanks to all AML labmates, especially Kishor (for giving me the

taste of Trekking), Amol, Amit, Pratap, Pooja, Archana, Ann, Ganesh, Umesh,

Amrutraj, Abhijar, Monika, Rashmi, Sandip, and all others for making the

working in lab joyful.

Special thanks to my dear friends, Atul (Ghatotkach) from Physics lab,

Mahendra (Mamu) from BNT lab, Anup, Rajesh, Santosh from GDY lab for

their wonderful company.

Special thanks to my Masters group Sunil (Barakya), Yoko, Ninad, Balu,

Randheer (Bhaiyya) and Shreenivas for their constant encouragement and

timely help.

Page 8: Studies in depolymerization of natural polysaccharides--PhD thesis

I also thank Chindarkar, Subhash, and Parab for their cooperation during my

work and their always helping nature.

I would like to thank my parents ‘aai and nana’ for providing all the support

needed; their support and faith in me always encouraged me. Sweet

memories of my aaji come to my mind. She would have been very happy to

me for completing the highest qualification. I also thank my younger brother

and my best friend ‘Chetan’ for everything he did for me. I also thank my

elder brother ‘Suresh’, Mugdhavahini, my darling neice ‘Lekha’, and my in

laws ‘mama, mami, Smita and all others’ for their love for me.

I would like to thank my dear wife ‘Supriya’ for her love, encouragement,

understanding, and awesome support throughout the Ph D work. Thank you

for bearing with my late hours, work weekends, bad moods (which being

consequences of not getting results), and going for treks with freinds. Now,

last but certainly not least my son, ‘Malhar’. He always is a source of joyful

moments for me. His presence around me always kept me in fresh mood to

do my work.

It is quite possible that I might have missed name of few people in spite of

their valuable assistance, both from a professional and personal perspective. I

thank all of them.

Satish D. Shewale

Page 9: Studies in depolymerization of natural polysaccharides--PhD thesis

CONTENTS

1. Introduction 1

2. Overview of starch and starch hydrolysis products 7

2.1. Starch 8

2.1.1. Starch composition 11

2.1.1.1. Amylose 11

2.1.1.2. Amylopectin 12

2.1.1.3. Other minor components 14

2.1.2. Starch granule structure 15

2.1.3. Gelatinization of starch 18

2.1.4. Starch production and applications 20

2.2. Starch hydrolysis products 21

2.2.1. Starch hydrolyzing enzymes 22

2.2.1.1. Bacterial α-amylase 27

2.2.1.2. Fungal α-amylase 28

2.2.1.3. Glucoamylase 29

2.2.1.4. β-amylase 29

2.2.1.5. Pullulanase 30

2.2.1.6. Isoamylase 30

2.2.1.7. Glucose isomerase 30

2.2.2. Maltodextrins 31

2.2.2.1. Production 31

2.2.2.2. Application 34

2.2.3. Glucose syrup 35

2.2.3.1. Production 35

2.2.3.2. Applications 39

2.2.4. Dextrose hydrolysate, crystalline dextrose and liquid dextrose 40

2.2.4.1. Production 40

2.2.4.2. Applications 44

2.2.5. Maltose syrup 45

2.2.5.1. Production 45

Page 10: Studies in depolymerization of natural polysaccharides--PhD thesis

2.2.5.2. Applications 46

2.2.6. Fructose syrup 47

2.2.5.1. Production 47

2.2.5.2. Applications 48

3. Sorghum: Literature Review 49

3.1. Introduction, Origin and Geographical distribution of sorghum 50

3.2. Taxonomy 52

3.3. Production, cultivation area and yield of sorghum 55

3.3.1. Trends in production, cultivation area and yield in the world 55

3.3.2. Trend in cultivation area, production and yield of sorghum 62

in India and different states of India

3.4. Plant anatomy and growth 66

3.4.1. Botanical parts of sorghum plant 66

3.4.2. Growth of sorghum plant 69

3.4.3. Optimum growth requirement of sorghum plant 71

3.5. Grain morphology 73

3.6. Utilization of sorghum 80

3.6.1. Food use 80

3.6.2. Industrial utilization 85

3.6.2.1. Animal feed 85

3.6.2.2. Alcohol industry 87

3.6.2.3. Starch industry 89

3.6.2.4. Other industries 90

3.7. Insect pests, Diseases and weeds on sorghum 91

3.8. Factors affecting industrial utilization of sorghum 91

3.8.1. Gelatinization of starch 92

3.8.2. Protein digestibility 94

3.8.3. Starch digestibility 95

3.8.4. Tannin content in sorghum 99

3.9. Production of ethanol from sorghum: Literature review 99

3.10. Production of starch from sorghum: Literature review 105

Page 11: Studies in depolymerization of natural polysaccharides--PhD thesis

4. Hydrolysis of soluble starch using B. licheniformis α-amylase 108

immobilized on superporous CELBEADS

4.1. Introduction and literature review 109

4.1.1. Immobilized enzymes 111

4.1.2. Immobilization of bacterial α-amylase 113

4.1.3. Objectives 114

4.2. Experimental 115

4.2.1. Materials 115

4.2.2. Methods 116

4.2.2.1. Measurement of protein concentration and 116

reducing sugar concentration.

4.2.2.2. HPTLC analysis. 116

4.2.2.3. Immobilization of B. licheniformis α-amylase (BLA) 118

on CELBEADS.

4.2.2.4. Amylolytic activity measurement. 120

4.2.2.4. A. Free BLA. 120

4.2.2.4. B. Immobilized BLA. 120

4.2.2.5. Measurement of kinetic constants of free 121

and immobilized BLA.

4.2.2.6. Hydrolysis of soluble starch using immobilized BLA 122

in batch mode.

4.2.2.7. Thermostability and reusability of immobilized BLA. 123

4.2.2.8. Hydrolysis of soluble starch using immobilized BLA 123

in packed bed or expanded bed mode.

4.2.2.9. Measurement of residence time in the packed bed. 125

4.3. Result and Discussion 125

4.3.1. Immobilization of bacterial α-amylase on CELBEADS. 125

4.3.2. pH and temperature dependence of activity of free and 126

immobilized BLA, and their catalytic properties

4.3.3. Effect of reaction conditions on hydrolysis of soluble 129

starch using immobilized BLA and saccharide composition

4.3.3.1. Effect of pH. 129

4.3.3.2. Effect of temperature. 132

Page 12: Studies in depolymerization of natural polysaccharides--PhD thesis

4.3.3.3. Effect of initial starch concentration, [S]0 and [IEU]/[S]0. 134

4.3.4. Comparison of saccharide composition of starch hydrolysate 138

using free and immobilized BLA

4.3.5. Thermostability and reusability of immobilized BLA 142

4.3.6. Semiempirical model for prediction of saccharide composition 144

4.3.7. Effect of mode of operation on hydrolysis of soluble starch 147

4.3.8. Hydrodynamic stability of immobilized BLA 149

4.3.9. Hydrolysis of sorghum slurry using immobilized BLA. 151

4.4. Conclusions 152

5. Enzymatic production of glucose from sorghum 154

5.1. Introduction and literature review 154

5.2. Experimental 157

5.2.1. Materials 157

5.2.2. Analytical methods 158

5.2.2.1. Measurement of protein concentration, reducing sugar 158

concentration and concentrations of malto-oligosaccharides.

5.2.2.2. Measurement of moisture content of sorghum flour 158

5.2.2.3. Measurement of particle size distribution of 158

sorghum flour

5.2.2.4. Measurement of starch content of sorghum flour 158

5.2.3. Amylolytic activity measurement 159

5.2.3.1. Free bacterial α-amylase (BLA) 159

5.2.3.2. Free amyloglucosidase (AG) 159

5.2.3.3. Free pullulanase (PL) 160

5.2.4. Thermostability study of amyloglucosidase (AG) 160

5.2.5. Optimization of AG: PL ratio for saccharification 161

5.2.6. Experimental work for production of glucose from sorghum 161

5.2.6.1. Optimization of liquefaction of sorghum flour 163

5.2.6.2. Liquefaction of sorghum of different varieties 164

5.2.6.3. Effect of prior ultrasound treatment on the 165

liquefaction of sorghum

5.2.6.4. Optimization of saccharification. 165

Page 13: Studies in depolymerization of natural polysaccharides--PhD thesis

5.3. Results and Discussion 167

5.3.1. Studies in the liquefaction process 167

5.3.1.1. Optimization of liquefaction process 167

5.3.1.1. A. Effect of pH. 167

5.3.1.1. B. Effect of BLA concentration. 167

5.3.1.1. C. Effect of CaCl2 concentration. 168

5.3.1.1. D. Effect of sorghum slurry concentration 169

5.3.1.1. E. Effect of liquefaction temperature. 170

5.3.1.1. F. Liquefaction of sorghum of different varieties 171

5.3.1.2. Effect of prior ultrasound treatment on liquefaction. 173

5.3.2. Optimization of saccharification 174

5.3.2.1. Properties of free amyloglucosidase and pullulanase 174

5.3.2.2. Thermostability of amyloglucosidase and 176

optimization of operating temperature for saccharification

5.3.2.3. Optimum ratio of amyloglucosidase units to 178

pullulanase units for saccharification

5.3.2.4. Optimization of amyloglucosidase concentration 180

5.3.3. Saccharification of sorghum liquefact 181

5.3.4. Effect of ultrasound treatment on particle size distribution 183

5.3.5. Studies on effect of different process parameters on 184

% saccharification

5.3.5.1. Effect of washing of cake obtained after hot filtration 184

5.3.5.2. Effect of ultrasound treatment on % saccharification 185

5.3.6. Economics of the process of production of glucose from 190

sorghum of different varieties

5.4. Conclusions 194

6. Enzymatic production of maltose syrup from sorghum 196

6.1. Introduction and literature review 197

6.2. Experimental 197

6.2.1. Materials 197

6.2.2. Analytical methods 198

6.2.2.1. Measurement of protein concentration, reducing sugar 198

concentration and concentrations of malto-oligosaccharides.

Page 14: Studies in depolymerization of natural polysaccharides--PhD thesis

6.2.2.2. Measurement of moisture content of sorghum flour 198

6.2.2.3. Measurement of particle size distribution of 198

sorghum flour

6.2.2.4. Measurement of starch content of sorghum flour 198

6.2.3. Amylolytic activity measurement 199

6.2.3.1. Free bacterial α-amylase (BLA) 199

6.2.3.2. Free Barley β-amylase (BBA) 199

6.2.3.3. Free pullulanase (PL) 199

6.2.4. Thermostability study of Free Barley β-amylase (BBA) 200

6.2.5. Production of glucose from sorghum: Experimental work 201

6.2.5.1. Liquefaction of sorghum flour 202

6.2.5.2. Optimization of saccharification. 202

6.3. Results and Discussion 203

6.3.1. Optimization of saccharification 203

6.3.1.1. Properties of free Barley β-amylase (BBA) 203

and pullulanase

6.3.1.2. Thermostability of Barley β-amylase and 205

optimization of operating temperature for saccharification

6.3.2. Saccharification of sorghum liquefact 208

6.3.3. Studies on effect of different process parameters on 210

% saccharification

6.3.4. Economics of the process of production of maltose syrup 211

from sorghum of different varieties

6.4. Conclusions 215

6.5. Alternative approaches for value addition to sorghum 216

References 218

Appendix A. Analytical methods 233

Appendix B. Matlab code to find kinetic constants 243

Synopsis

Page 15: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 1: Introduction

Studies in the Enzymatic depolymerisation of natural polysaccharides

1

1. Introduction

Page 16: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 1: Introduction

Studies in the Enzymatic depolymerisation of natural polysaccharides

2

Sorghum (Sorghum bicolor L. Moench) is an important drought resistant

cereal crop and fifth largest produced cereal in the world after wheat, rice, barley and

maize. Production of sorghum in 2007-2008 in the world was 64 Million Metric Tons

(www.fas.usda.gov). Leading sorghum producing countries were United States

(19.9%), Nigeria (15.5%), India (11.3%), Mexico (9.8%), Sudan (7%), and Argentina

(5.4%) (www.fas.usda.gov). Sorghum is a staple food crop for many of the world’s

poorest people, and constitutes a major source of energy and proteins for millions of

people in Africa and Asia. In India, sorghum is grown in the kharif (rainy season) and

rabi (postrainy season). Rabi crop is almost entirely used for human consumption,

whereas kharif crop is not very popular for human consumption and is largely used

for animal feed, starch, and by the alcohol industry. Maharashtra is the largest

sorghum producing state in India with production of 5.8 million Tonnes in 2001-2002.

Sorghum ranks third in the major food grain crops in India.

Sorghum is also termed as “Nature-cared crop” because it has strong

resistance to harsh environments such as dry weather and high temperature in

comparison to other crops, it is usually grown as a low-level chemical treatment crop

with limited use of pesticides and it has a potential to adapt itself to the given natural

environment. Sorghum is valued because of its ability to grow in areas with marginal

rainfall and high temperatures (i.e. semi arid tropics and sub tropical regions of the

world), where it is difficult to grow any other cereal, and also because of its relatively

short growing season requirement, thus its suitability for double cropping and crop

rotation systems (Smith and Frederiksen, 2000).

Though, production of sorghum is high in India, demand for the sorghum is

decreasing with change in the way of living due to increased urbanization, increased

per capita income of the population, and easy availability of other preferred cereals in

Page 17: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 1: Introduction

Studies in the Enzymatic depolymerisation of natural polysaccharides

3

sufficient quantities at affordable prices. Hence, in addition to being a major source

of staple food for humans, it also serves as a source of feed for cattle and other

livestock in case of scarcity of maize, but at lower prices. Also, about 10-20 % of the

production gets wasted due to damage and inadequate transport and storage facilities.

Industrial grade damaged sorghum grains (inclusive of 30-55% sound grains) are

available in large quantity at Food Corporation of India (FCI) at 10 times lower rate

than the fresh grains (Suresh et al., 1999a). Damage includes chalky appearance,

cracked, broken, mold, infection etc. These damaged grains are not suitable for

human consumption. Several mold-causing fungi are producers of potent mycotoxins

that are harmful to health and productivity of human and animal (Bandyopadhyay et

al., 2000). Hence, damage caused by insect infection and attack of fungus (blackened

sorghum or grain mold) because of wet and humid weather makes sorghum grains

even unfit for animal consumption.

Hence, an industrial application is needed to be exploited for normal and

blackened sorghums in order to make sorghum cultivation economically viable for

farmers, through value added products. There is very small amount of research done

on value addition to sorghum through; production of glucose (Devarajan and Pandit,

1996; Aggarwal et al., 2001), production of ethanol (Wu et al., 2007; Suresh et al.,

1999a,b; Zhan et al., 2003 and Zhan et al., 2006) and isolation of starch (Yang and

Sieb, 1996; Xie and Seib, 2002; Higiro et al., 2003; Perez-Sira and Amaiz, 2004; Park

et al., 2006). The reason for the lower level of industrial exploitation can be

attributed to reduced sorghum starch digestibility (Lichtenwalner et al., 1978; Rooney

and Pflugfelder, 1986; Chandrashekar and Kirleis, 1988; Zhang and Hamaker, 1998;

Elkhalifa et al., 1999; Ezeogu et al., 2005) and reduced protein digestibility (Duodu et

Page 18: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 1: Introduction

Studies in the Enzymatic depolymerisation of natural polysaccharides

4

al., 2003) after cooking i.e. heat-moisture treatment of sorghum flour. There is no

literature available on value addition products using blackened sorghum.

Research aim

Objective of the present work was a production of value addition products:

glucose and maltose from different qualities of sorghum i.e. healthy, germinated and

blackened. In the present work, sorghum flour was used directly for liquefaction and

saccharification rather than isolating starch and using it for liquefaction and

saccharification as the yields of starch isolation from sorghum were reported to be

around 50–60% i.e rest part (40–50%) gets wasted or does not fetch much price.

Such methodology of direct hydrolysis was first used by Kroyer in 1966 using corn

grits for the production of glucose.

Thesis Outline

Starch structure and chemistry, action of different types of enzymes used for

starch hydrolysis are described in chapter 2. Production, properties and application of

different starch hydrolysis products are also discussed in Chapter 2. Attempt is made

to cover almost everything related to sorghum in chapter 3. In the chapter 3, origin,

geographical distribution, taxonomy, plant anatomy, plant growth and grain

morphology of sorghum are described. Trends in production, cultivation area and

yield of sorghum in the world and India are also briefly discussed in chapter 3.

Applications of sorghum viz. food use and industrial utilization, and problem areas

(Gelatinization of sorghum starch, protein digestibility, starch digestibility and tannin

content) and factors affecting them in industrial utilization of sorghum are also briefly

reviewed in chapter 3. Then lastly literature on production of ethanol and starch from

Page 19: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 1: Introduction

Studies in the Enzymatic depolymerisation of natural polysaccharides

5

sorghum is reviewed in chapter 3.

Due to several advantages (like reusability of the enzyme, continuous

operation of the system, easy separation of product from the enzyme etc.) that

immobilized enzyme have over the free enzyme, it was first decided to develop a

process for the production of glucose from sorghum flour using immobilized

enzymes. This process constitutes following steps viz. 1. Gelatinization of 15 %

sorghum slurry in boiling water for 10 min. 2. Circulating slurry through the bed of

immobilized B. licheniformis α-amylase (BLA) and amyloglucosidase (AG). But

before studying this, it was necessary to first immobilize BLA on beads and study its

catalytic characteristics. Chapter 4 covers studies in the immobilization of BLA on

rigid superporous (pore size ∼ 3 µm) cross-linked cellulose matrix (CELBEADS) and

hydrolysis of soluble starch using immobilized BLA.

Chapter 5 covers the work to add value to three different varieties of sorghum

viz. normal healthy, germinated, and blackened through production of glucose and to

intensify glucose production (yield) by means of ultrasound treatment. Liquefaction

(using B. licheniformis α-amylase) and saccharification (using amyloglucosidase)

processes were optimized with the use of normal sorghum flour as a starting material

for the production of glucose. Effect of ultrasound treatment on the sorghum slurry

prior to liquefaction was studied on the process of liquefaction and saccharification at

optimized conditions.

Chapter 6 details the work to add value to three different varieties of sorghum

viz. normal healthy, germinated, and blackened through production of maltose and to

intensify its production (yield) by means of ultrasound treatment. Liquefaction part

remains same as that in the chapter 5. Saccharification (using β-amylase and/or

pullulanase) process was optimized with the use of normal sorghum flour as a starting

Page 20: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 1: Introduction

Studies in the Enzymatic depolymerisation of natural polysaccharides

6

material for the production of maltose. Effect of ultrasound treatment on the sorghum

slurry prior to liquefaction and use of pullulanase during saccharification was studied

on the process of saccharification at optimized conditions.

List of the references used in the present work and synopsis are provided at the

end of thesis. All analytical methods are provided in the appendix A and code in

Matlab used to find kinetics in hydrolysis of soluble starch using immobilized BLA is

also reported in the appendix B.

Page 21: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

7

2. Overview of starch and starch hydrolysis products

Page 22: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

8

2.1. Starch

Starch is an abundant carbohydrate distributed worldwide in green plants,

where it accumulates as microscopic granules. Starch is a food reserve that sustains

initial plant growth. Man harvests this reserve and with little preparation may use it

directly or after separation and purification by relatively simple processes. Starch has

been important ingredient of human diet over centuries mainly as a high calorie food

source. (Zobel, 1992) Starch pastes and gels are used to control consistency and

textures of sauces, soups and spreads. In addition to its use in human food, it has

become a very important biopolymer and is used in many industries as a feedstock

material. Sweetener and fermentation industries are two of the main consumers of the

starch. Nutritive sweeteners are mainly products of enzymatic hydrolysis of starch,

namely maltodextrins, high maltose syrup, maltose, glucose syrup, dextrose, and

fructose, which are used in food and pharmaceutical industry.

Starch biosynthesis is a complex process (Ball, 1995, 1998; Buleon et al.,

1998; Denyer et al., 2001; Emes et al., 2003; Smith et al., 1997; Tester and Karkalas,

2002 as cited in Tester et al., 2004a). Sucrose (derived from photosynthesis) is the

starting point for alpha-glucan deposition. In the cell cytosol the sucrose is converted

to uridine diphosphate glucose (UDP-glucose) and fructose by sucrose synthase, the

UDP-glucose being subsequently converted to glucose-1-phosphate (G-1-P) in the

presence of pyrophosphate (PPi) by UDP-glucose pyrophosphorylase. Then this is

itself converted to glucose-6-phosphate (G-6-P) by phosphoglucomutase. The G-6-P

is translocated across the amyloplast (the intra-cellular organelle responsible for

starch biosynthesis in storage tissues) membrane by specific translocators and is

converted to G-1-P by phosphoglucomutase. There is some evidence that, in cereals at

least, G-1-P may be (a) translocated directly into the amyloplast or (b) be converted

Page 23: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

9

to, and translocated as, adenosine diphosphate glucose (ADP-glucose) generated as a

consequence of cytosol based adenosine diphosphate (ADP)-glucose

pyrophosphorylase activity in the presence of adenosine triphosphate (ATP). Using

amyloplast located ADP-glucose pyrophosphorylase, G-1-P within the amyloplast is

(also) converted to ADP-glucose and provides glucose residues for amylose and

amylopectin biosynthesis. Starch synthases (of which there are commonly considered

to be two major classes, ‘granule bound’ and ‘soluble’, with a number of isoforms of

each) add glucose units to the nonreducing ends of amylose and amylopectin

molecules. Granule bound starch synthase can elongate maltooligosaccharides to form

amylose and is considered to be responsible for the synthesis of this polymer. Soluble

starch synthase is considered to be responsible for the synthesis of unit chains of

amylopectin. Starch branching enzyme creates branching in amylopectin by linking

linear chains (branches) to the growing amylopectin molecule. (Tester et al., 2004a)

Starch granules are synthesized in a broad array of plant tissues and within

many plant species. Characteristics of starch granule viz. size (~1–100 µm in

diameter), shape (round, lenticular, polygonal), size distribution (uni- or bi-modal),

association as individual (simple) or granule clusters (compound) and composition

(α-glucan, lipid, moisture, protein and mineral content) are mainly reflection of their

botanical origin (Table 2.1; Tester et al., 2004a). For industrial production of starch,

most important sources of starches are cereal grains, pulses and tubers, with maize

and potatoes contributing the major proportion in the world.

Page 24: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

10

Table 2.1. Characteristics of starch granules from different botanical sources (Tester et al., 2004a). Starch Type Shape Distribution Size (µm)

Barley Cereal Lenticular (A-type), Bimodal 15–25, 2–5

spherical (B-type)

Maize Cereal Spherical/polyhedral Unimodal 2–30

(waxy and normal)

Amylomaize Cereal Irregular Unimodal 2–30

Millet Cereal Polyhedral Unimodal 4–12

Oat Cereal Polyhedral Unimodal 3–10(single)

80 (compound)

Pea Legume Rentiform (single) Unimodal 5–10

Potato Tuber Lenticular Unimodal 5–100

Rice Cereal Polyhedral Unimodal 3–8 (single)

150 (compound)

Rye Cereal Lenticular (A-type) Bimodal 10–40

Spherical (B-type) 5–10

Sorghum Cereal Spherical Unimodal 5–20

Tapioca Root Spherical/Lenticular Unimodal 5–45

Triticale Cereal Spherical Unimodal 1–30

Sago Cereal Oval Unimodal 20–40

Wheat Cereal Lenticular (A-type) Bimodal 15–35

Spherical (B-type) 2–10

Page 25: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

11

2.1.1. Starch composition

The chemical composition of starch granules, which depends on its biological

origin has a considerable impact on starch processing technologies. Starch granules

are mainly composed of α-glucan, lipids, protein and moisture. Starch granules

consist of two types of α-glucan viz. amylose and amylopectin, which represent

approximately 98–99% of the dry weight. Percentage content of amylose and

amylopectin varies according to the botanical origin of the starch. Starches are

defined as waxy when amylose content is less than 15%, normal when amylose

content is in the range of 15-35% and high-amylose (amylo-) when amylose content

exceeds ~36% (Tester et al., 2004a).

2.1.1.1. Amylose

Amylose is a relatively long, linear α-glucan containing around 99% α(1→4)

and 1% α(1→6) linkages and differs in size and structure depending on botanical

origin. Amylose has a molecular weight of approximately 1 × 105 – 1 × 106, a degree

of polymerisation (DP) of 324–4920 with around 9–20 branch points equivalent to 3–

11 chains per molecule. Each chain contains approximately 200–700 glucose residues

equivalent to a molecular weight of 32,400–113,400. (Tester et al., 2004a) Structure

of amylose is shown in the Fig. 2.1. Buleon et al. (1998) concluded that though few

branch points (i.e. α(1→6) linkages) are present in the amylose, they do not

significantly alter the solution behavior of amylose chains, which remains identical to

that of strictly linear chains. One end of the linear chain has a free C1 hydroxyl group

and is the reducing end. Another specific feature of interest concerning amylose is its

capacity to bind iodine. Amylose complex with iodine and produces deep blue color,

this property is generally used as qualitative method to identify the presence of starch.

Page 26: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

12

Figure 2.1. Basic structure of amylose and amylopectin. Adapted from Tester et al.

(2004a)

2.1.1.2. Amylopectin

Amylopectin is a much larger molecule than amylose with a molecular weight

of 1 × 107 – 1 × 109 and a heavily branched structure built from about 95% α(1→4)

and 5% α(1→6) linkages with DP typically within the range 9600–15,900. In

common with amylose, the molecular size, shape, structure and polydispersity of the

molecule varies with botanical origin. Unlike amylose, however, there is great

additional variation with respect to the unit chain lengths and branching patterns.

Amylopectin unit chains are relatively short compared to amylose molecules with a

broad distribution profile. (Tester et al., 2004a) Structure of amylopectin is shown in

the Fig. 2.1.

The individual chains can be specifically classified in terms of their lengths

(chain lengths, CL) and consequently position within starch granules. The A and B1

chains are the most external (exterior) and form double helices (and crystallites)

within the native granules. Their CL is typically, 12–24 depending on genetic origin

Page 27: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

13

and starches with ‘A-type’ crystallinity, (most cereals) having shorter chain lengths on

average than ‘B-type’ starches (like potato). With the exterior chains of amylopectin

(A- and B1) comprising a range from CL 12–24 as previous mentioned, the A-type

chains are typically CL 12–16 and B1 CL 20–24. Amylopectin molecules from high

amylose starches contain relatively high proportions of very long chains. With respect

to the structure of amylopectin (Fig. 2.2), the A-chains of amylopectin are α - (1 → 6)

linked by B-chains which in turn can be linked to other B-chains or the ‘backbone’ of

the amylopectin molecule, the single C-chain (which contains a sole reducing group).

Depending on the CL and correspondingly the number of (radial) clusters traversed

within the native granule, B chains are referred to as B1–B4 (one to four clusters).

Typical CLs for A, B1–B4 chains for different starches (after debranching with

isoamylase) are 12–16, 20–24, 42–48, 69–75 and 101–119, respectively. The ratio of

A- to B-chains depends on the starch source and is typically of the order of ,1:1 to .2:1

on a molar basis or ,0.5:1 to .1:1 on a weight basis. (As reviewed by Tester et al.,

2004a)

Figure 2.2. Schematic representation of a section of amylopectin indicating the

branching pattern of unit α - (1 →4) chains (A, B1–B3) joined together by α - (1 →6)

linkages (branch points). (Hizukuri, 1986 as cited in Tester et al., 2004a)

Page 28: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

14

2.1.1.3. Other minor components

Minor components associated with starches correspond to three categories of

materials: (i) particulate material, composed mainly of cell-wall fragments; (ii)

surface components, removable by extraction procedures; and (iii) internal

components. The main constituents of surface components are proteins, enzymes,

amino acids and nucleic acids. Mineral fractions are negligible in cereal starches in

contrast to tuber starches. Cereal starches contain phosphorus that is mainly in the

form of phospholipids. (Buleon et al., 1998)

Lipids represent the most important fraction associated with the starch

granules. According to Morrison (1981) three categories of cereal lipids can be

distinguished, i.e. nonstarch, lipids on surface of starch granule and intragranular

lipids. The non-starch lipids comprise triacylglycerols, diacylgalactosylglycerols,

small amounts of free fatty acids, tocopherols, and sterols. Cereal starch granules

contain internal (or intragranular) lipids, which are exclusively monoacyl lipids i.e.

free fatty acids (FFA) and lysophospholipids (LPL), and can complex with amylose.

Internal lipids are proportional to the amylose fraction in all normal cereal starches

and the LPL may account for up to 2% of starch weight in high amylose cereal

starches. (Morrison, 1988) Since the lipid fraction within starch granules is

insufficient to saturate entire quantity of amylose, amylose exists in two forms; free

amylose and amylose-lipid complex (Tester et al., 2004a). Amylose-lipid complex is

usually composed of a typical left-handed amylose helix, in which the aliphatic part of

the lipid is included i.e. fatty acid chains occupy a hydrophobic core located within

the single amylose helix (Nebesny et al., 2002; Tester et al., 2004a).

Lipids on the surface of starch granules comprise triglycerides, glycolipids,

phospholipids and free fatty acids derived from the amyloplast membrane and non-

Page 29: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

15

starch sources. These differ from internal lipids, which are composed exclusively of

the FFA and LPL. (Tester et al., 2004a) Normal and high-amylose cereal starches

contain more internal, than granule surface lipids, whereas waxy cereal starches,

potato and bean starches contain small amounts of granule surface lipids and probably

no internal ones (Morrisson, 1981, 1988; Nebesny et al., 2002).

Monoacyl lipids will induce the formation of amylose–lipid complexes during

gelatinization. They will restrict swelling, dispersion of the starch granules and

solubilization of amylose, thus generating opaque pastes with reduced viscosity and

increased pasting temperatures. (Buleon et al., 1998)

In commercially available purified starches, moisture content ranges from 10

to 18% depending on the source, with cereal starches at the lower end and tuber

starches at the Upper end. Purified starches contain low levels of protein (<0.5%)

which largely represent the residues of biosynthetic enzymes involved in the synthesis

of starch. (Tester et al., 2004b)

2.1.2. Starch granule structure

There are few reviews (Oates 1997; Buleon et al., 1998; Tester et al., 2004a),

which have analyzed and discussed starch granule structure. Starch granules are

roughly spherical. These granules are synthesized by plants as semi-crystalline

matrices, where crystallinity is generated by registration of amylopectin double

helices into crystalline lamellae interspersed with amorphous lamellae comprising α-

(1→6) branch regions of amylopectin and amylose (Fig. 2.3). Both amylose chains

and exterior chains of amylopectin molecule (A and B1) form double helices, which

in turn associate to form crystalline domain. From Fig. 2.3 it is apparent that the

alternating crystalline-amorphous lamellae provide the basis of semi-crystalline

Page 30: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

16

(‘dark’) growth rings which comprise about 16 crystalline lamellae, which are of

approximately the same width as the interspersed amorphous (‘light’) growth rings

(~140nm). (Buleon et al., 1998; Tester et al., 2004a, 2004b)

Three types of starches, designated as type A, type B, and type C, have been

identified based on X-ray diffraction patterns. These depend partly on the chain

lengths making up the amylopectin lattice, the density of packing within the granules,

and the presence of water. Although type A and type B are real crystalline

modifications, type C is a mixed form. (Sajilata et al., 2006) The important features of

the types of starches (Sajilata et al., 2006) are as follows.

Type A. The type A structure has amylopectin of chain lengths of 23 to 29 glucose

units. The hydrogen bonding between the hydroxyl groups of the chains of

amylopectin molecules results in the formation of outer double helical structure. In

between these micelles, linear chains of amylose moieties are packed by forming

hydrogen bonds with outer linear chains of amylopectin. This pattern is very common

in cereals.

Type B. The type B structure consists of amylopectin of chain lengths of 30 to 44

glucose molecules with water inter-spread. This is the usual pattern of starches in raw

potato and banana.

Type C. The type C structure is made up of amylopectin of chain lengths of 26 to 29

glucose molecules, a combination of type A and type B, which is typical of peas and

beans.

Schematic representation of starch granule architecture is given in the Fig. 2.3.

Page 31: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

17

Starch granule B A Figure 2.3. (A) Structure of amylopectin. (B) Organization of the amorphous and crystalline regions of the structure generating the concentric layers that contribute to the “growth rings” those are visible by light microscopy. (C) Orientation of the amylopectin molecules in a cross section of an idealized entire granule. (D) Shows the likely double helix structure taken up by neighboring chains and giving rise to the extensive degree of crystallinity in granule (www.lsbu.ac.uk; van der Maarel et al., 2002)

Page 32: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

18

Literature till now hasn’t completely understood the precise location of

amylose in the starch granule. Amylose may be located in bundles between

amylopectin clusters and randomly interspersed among clusters in both crystalline and

amorphous regions. In high-amylose starches amylose probably forms double helices

and crystalline domains accordingly. The location of amylose with respect to the

crystalline and amorphous regions is dependent on the botanical source of the starch.

In wheat starch, amylose is mainly found in the amorphous region, but in potato

starch it may be partly co-crystallized with amylopectin. Large amylose molecules

that are present in the granule core are able to participate in double helices with

amylopectin, whereas smaller amylose molecules present at the granule periphery are

able to leach from the granule (Oates, 1997).

2.1.3. Gelatinization of starch

Gelatinization of starch is crucial step for many starch based industrial and

food applications. For example, gelatinization of starch is required prior to enzymatic

hydrolysis to make starch accessible for the amylolytic enzymes. Industrial

gelatinization process is usually carried out with a 30–35% dry solids starch slurry.

Higher starch concentrations may yield higher volumetric efficiencies and lower

energy consumption (Baks et al., 2008), but are difficult to handle mechanically.

The starch granules are very organized (specific to their botanical origin) with

amylose having helical coil like structure and amylopectin having tree like structure.

When the temperature of an aqueous starch-water suspension is increased,

gelatinization takes place. Starch granules absorb water, swell, lose crystallinity

(crystalline form gets transformed into amorphous form), and leach amylose during

thermal gelatinization. Gelatinization is used as a collective term for the changes that

starch granules faces when they are heated in the presence of water. These changes

Page 33: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

19

include loss of birefringence, change in x-ray diffraction pattern, absorption of water

and swelling, change in the shape and size of granule, and leaching of amylose.

Ultimately, granule structure is completely lost and a thin paste (<~4%) or gel (>~4%)

is formed. At the molecular level, the gelatinization event is initiated by water

‘plasticising’ amorphous rearrangements and ultimately hydrating double helices as

they unravel as a consequence of the elevated temperature. Gelatinization temperature

of starch is mainly dependent on its source or botanical origin. The gelatinisation

process is shown schematically in Figure 2.4. Gelatinization of starch is affected by

the starch-water ratio. When the moisture content of the starch-water mixture is low,

complete swelling and disruption of the starch granules is not possible and only part

of the crystallinity of the starch granules is lost. (Leach, 1965; Tester et al., 2004b;

Baks et al., 2008)

Figure 2.4. Idealized diagram of the swelling and gelatinisation of a starch granule in the presence of water. Adapted from Tester et al. (2004b).

Page 34: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

20

Starch can also be gelatinized by the application of high pressures and or

temperatures in the presence of water. During high-pressure gelatinization,

disintegration of the granules is less (or the granules even remain intact), amylose

solubilization is less, and swelling of the granules is limited as compared to thermal

gelatinization. (Baks et al., 2008)

2.1.4. Starch production and applications

Sources for industrial production of starch are plant seeds, roots, and tubers.

Basic process steps include cleaning, steeping, grinding, separation, and isolation of

finished products. Products include starch, and co-products based on protein, fiber,

and oil components. In 2005 world starch production was 60 million tones, out of

which ~ 70% starch had origin as maize (www.starch.dk). Maize can be processed

through Dry or Wet milling operations for the production of starch. However in

industry wet milling process is more popular because products by Dry mill process

consists of protein adhering to starch and also due to this protein it is also not suitable

for hydrolysate manufacture due to increased refining cost. (Zobel, 1992)

Applications of starch and modified starches are summarized in Table 2.2.

Physical and chemical modifications of starch resulted into wide range of

additional products. Physical modification includes pregelatinization, grinding, and

solvent treatment; these modifications impart some degree of granule dispersibility in

cold water. Since swelling occurs in cold water, these starches are used in instant food

mixes as a thickening agent. Chemical modifications of starch include reactions of

substitution or derivatization, cross-linking, oxidation, and hydrolysis. Derivatized

starches can have lowere granule gelatinization temperature and gels that show less

firming with age, as well as freeze thaw stability and better clarity. Due to good film

forming property hydroxypropyl starch give smooth paper surfaces and ink holdout.

Page 35: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

21

Cross linking is most commonly accomplished using adipic acid and phosphorous

oxychloride. It is common practice to combine both derivatization and crosslinking in

granules to achieve needed functionality. Oxidized starch, thin boiling starch and

dextrins provide examples of starches that have been depolymerized to varying

degrees. (Zobel, 1992)

Table 2.2. Applications of starch (www.starch.dk)

Food Beverage Animal Feed Plastic Pharmacy Building

Mayonnaise Soft drinks Pellets Biodegradable plastic Tablets Mineral fibre

tiles

Baby food Beer By products Dusting powder Gypsum board

Bread Alcohol Concrete Buns Coffee Gypsum plaster Confectionery Agriculture Textile Paper Various Meat sausages Jelly gums Seed coating Warp Corrugated

board Foundries

Meat rolls and loaves

High-boiled sweets Fertiliser Fabrics Water

treatment Ketchup Jellies Yarns Cardboard Coal Marchmallows Soups Marmalade Paper Detergent

Snacks Jam Fermentation Non-Wowen Printing paper Oil drilling

Pizza sauces Ice cream Vinegar Hygienic diapers Stain remover

Sauces Dairy cream Enzymes Baby diapers Packaging material Glue

Low fat foods Fruit fillings Sanitary napkins Foamed starch

2.2. Starch hydrolysis products

Starch has become a very important biopolymer and is used in many industries

as a feedstock material. Sweetener and fermentation industries are two of the main

consumers of the starch. Nutritive sweeteners are mainly starch hydrolysis products

namely maltodextrins, high maltose syrup, maltose, glucose syrup, dextrose, which

are used in food and pharmaceutical industry (Table 2.3). Two excellent textbooks

(Schenck and Hebeda, 1992; Kearsley and Dziedzic, 1995) on the basics concepts of

the production of starch hydrolysate were mainly referred to write this entire section

Page 36: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

22

2.2. Three steps in which enzymes are used in starch hydrolysis and processing are as

follows:

1. Liquefaction of starch (combination of starch gelatinization and dextrinization of

gelatinized starch)

2. Saccharification of starch liquefact (containing oligosaccharides) to glucose or

maltose

3. Isomerization of glucose

Table 2.3. Different types of nutritive sweetener

Nutritive sweetener DE Class (Degree of polymerization)

Maltodextrins <20 Malto-oligosaccharides (3-9)

High maltose syrup 20-50 Maltose, glucose, Malto-oligosaccharides

Maltose 53 Sugar (2)

Glucose syrup 20-95 Glucose, maltose, malto-oligosaccharides

High fructose syrup - Fructose, glucose

Dextrose 100 Sugar (1)

2.2.1. Starch hydrolyzing enzymes

Starch is a polymer of glucose units, in which glucose units are linked together

by α(1→4) and α(1→6) glucosidic linkages. Glucose (monomer), maltose (dimer),

fructose (isomer of glucose) and malto-oligosaccharides are useful in many

applications. Hence these become commercially and industrially important products.

In order to manufacture these products α(1→4) and α(1→6) glucosidic linkages must

be cleaved. This cleavage of the glucosidic linkages can be achieved by either acid

hydrolysis or enzymatic hydrolysis.

Previously heat and acid treatments were used for generating starch hydrolysis

products. Though effective, these methods were not specific and undesirable by-

products and off-flavors were also formed due to harsh reaction conditions. Enzymes

Page 37: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

23

catalyze reaction under moderate conditions of pH and their use has led to better

controlled processes making fewer by-products. (Teague and Brumm, 1992)

Processes involving biocatalysts are potentially energy saving and are very well suited

for a wide range of industrial applications. Moreover enzymes can be produced

industrially in an ecologically safe way and are renewable, degradable and non-toxic

(Uhlig, 1998).

Enzymes (i.e. proteinaceous catalyst) are used throughout industries to convert

starch into products for use in beverages, food, pharmaceuticals, and industrial

ethanol production (Teague and Brumm, 1992). They are present in all living cells,

where they perform vital functions by controlling the metabolic processes and causing

breakdown of biomaterials into simpler compounds. Most biocatalysts have limited

stability, and they lose their activity in due course of time. (Davidson, 1999) The

essential role of enzymes in almost all physiological processes stems from two key

features of enzymatic catalysis: (1) enzymes greatly accelerate the rates of chemical

reactions; and (2) enzymes act on specific molecules, referred to as substrates, to

produce specific reaction products. Together these properties of rate acceleration and

substrate specificity afford enzymes the ability to perform the chemical conversions

of metabolism with the efficiency and fidelity required for life. (Copeland, 2000) In

some cases, enzyme action is specific to certain bonds in the compounds with which,

they react (Roberts, 1989). Enzymes are classified based on the types of reactions

catalyzed (Copeland, 2000) into following:

EC 1 Oxidoreductases: catalyze oxidation/reduction reactions

EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group)

EC 3 Hydrolases: catalyze the hydrolysis of various bonds

EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation

Page 38: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

24

EC 5 Isomerases: catalyze isomerization changes within a single molecule

EC 6 Ligases: join two molecules with covalent bonds

An exceptionally significant field, in which enzymes have proved to be of

great value over the last two decades, was the starch industry. In 1950s, fungal

amylase was used in the manufacture of syrup, containing a range of sugars, which

could not be otherwise prepared using conventional acid hydrolysis. But it was in

1960, a breakthrough occurred through the application of enzyme glucoamylase that

caused a complete break down of starch to glucose (Blanch, 1996).

Starch degrading enzymes i.e. “Amylases” can be generally defined as the

enzyme which hydrolyses the O-glycosyl linkage of starch. The α-amylase family, is

a large enzyme family that constitutes about 20 enzymes having different reaction and

product specificities, including exo/endo specificity, preference for hydrolysis or

transglycosylation, α(1→4) or α(1→6) glycosidic bond specificity and glucan

synthesizing activity. Plants, animals and microorganism use starch as source of

energy and carbon. Microorganisms mainly bacteria, fungi produce various starch

hydrolyzing enzymes in different environmental niche in order to degrade these large

macromolecule (Vorgias and Antranikian, 1997).

Enzymes of interest to starch hydrolysis industry are hydrolases like α-

amylase, glucoamylase, and β-amylase, which can be derived from bacteria, fungi, or

plants and their classification, are summarized in the Table 2.4. Action pattern of all

enzymes is diagrammatically represented in the Fig. 2.5. Nigam and Singh (1995),

Guzman-Maldonado & Paredas-Lopez (1995) and Kuriki & Umanaka (1999) have

reviewed amylolytic enzymes involved in starch processing and products derived

from starch. Van der Maarel et al. (2002) have reviewed properties and applications

of starch-converting enzymes of the α-amylase family. The starch hydrolytic enzymes

Page 39: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

25

comprise 30% of the world’s enzyme consumption (Van der Maarel et al., 2002). Use

of the enzymes in the starch processing is shown in the Fig. 2.6.

Table 2.4. Summary of starch hydrolyzing enzymes

Enzyme Source Action pattern and optimum reaction conditions Product

Bacillus amyloliquefaciens (i.e. B. Substilus)

Cleaves internal α(1→4) glucosidic bonds to give α-dextrins and mainly maltose (G2), G3, G6 and G7 oligosaccharides. Neutral pH and 70 °C

B. licheniformis

Cleaves internal α(1→4) glucosidic bonds to give α-dextrins and produces mainly G5 – G9 and small amounts of G3, G4 and G5 oligosaccharides. 6 - 7 pH and 85 °C

Bacterial α-Amylase EC 3.2.1.1. Endo-enzyme

B. Stearothermophilus

Cleaves internal α(1→4) glucosidic bonds to give α-dextrins and major products are G2, G3, G5 and G6. 5.5–6.5 pH and 80 °C

Maltodextrins

Fungal α-amylase EC 3.2.1.1. Exo-enzyme

Aspergillus niger, Aspergillus oryzae, Rhizopus oryzae

Cleaves internal α(1→4) glucosidic bonds and predominantly produces maltose from starch with significant quantities of glucose and maltotriose. For Aspergillus, 4 pH and 60 °C For Rhizopus, 5-5.2 pH and 40 °C

Maltose syrup, glucose syrup

β-Amylase EC 3.2.1.2 Exo-enzyme

Barley malt, Soybean, wheat

Cleaves second α(1→4) glucosidic bond from non reducing end and give β-limit dextrins and β-maltose. 5.2 pH, 50-60 °C

Maltose syrup, maltose

Glucoamylase EC 3.2.1.3 Exo-enzyme

Aspergillus niger, Rhizopus niveus, Rhizopus oryzae, Rhizopus sp.

Cleaves first α(1→4) or α(1→6) glucosidic bond from non reducing end to give β-glucose. For A. Niger, 4-5 pH, 60 °C

Glucose syrup, glucose

Pullulanase EC 3.2.1.41

B. acidopullulyticus Klebsiella planticola

Only α(1→6) glucosidic bond are cleaved to give straight chain maltodextrins. For K. planticola, 6.5 pH and 50 °C For B. acidopullulyticus, 4.5-5 pH and 60 °C

Linear dextrins, Maltose syrup

Isoamylase EC 3.2.1.68

Pseudomonas amyloderamosa

Cleaves α(1→6) glucosidic bond. pH 3-4 and 45-55 °C

Linear dextrins, maltose

Glucose isomerase EC 5.3.1.18

Actinoplanes missouriensis B. coagulans Microbacterium arborescen

Isomerizes glucose into fructose 7 – 8 pH and 60 °C

High fructose syrup

Page 40: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

26

Figure 2.5. Schematic representation of action pattern of starch hydrolyzing enzymes (Guzman-Maldonado & Paredas-Lopez, 1995; and Kuriki & Umanaka, 1999 as cited in Nair, 2006)

Page 41: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

27

Figure 2.6. The use of enzymes in processing starch (www.lsbu.ac.uk)

2.2.1.1. Bacterial α-amylase

Bacterial α-amylase (1, 4-α-D-glucan glucanohydrolases, EC 3.2.1.1,

glycogenase) is an endo-hydrolase which cleave any internal α(1→4) glucosidic

bonds. As the name indicates oligosaccharides produced by hydrolysis of starch using

bacterial α-amylase have reducing group in α configuration. It can bypass but cannot

cleave α(1→6) glucosidic branch points. It can be produced commercially from

following three sources viz. B. Amyloliquefaciens, B. Licheniformis, and B.

Stearothermophilus. Bacterial α-amylase is a thermostable enzyme and work up to

110 °C in presence of substrate. Since this is an organometallic enzyme, it requires

35% in cold water pH 6, 40 ppm Ca2+

Starch slurry Bacterial α-amylase, 1500 U/kg, 105 °C, 5 min

Gelatinization

Gelatinized Starch (< 1 DE) 95 °C, 2 h Liquefaction

Liquefied starch (11-15 DE) 0.3% D-glucose 2.0% maltose

97.7% oligosaccharides

Starch granules

Saccharification pH 4.5 glucoamylase, 150 U/kg pullulanase, 100 U/kg 60 °C, 72 h

Glucose syrup (99 DE) 97% D-glucose 1.5% maltose 0.5% isomaltose 1% other oligosaccharides

Maltose syrup (44 DE) 4% D-glucose 56% maltose 28% maltotriose 12% other oligosaccharides

pH 5.5 fungal α-amylase, 2000 U/kg 50 ppm Ca2+, 60 °C, 72 h

Page 42: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

28

supplementation of Ca2+ ions during liquefaction.

Bacterial α-amylase is particularly used in the liquefaction of starch and

production of maltodextrins. Liquefied starch by means of bacterial α-amylase is

further used for production of glucose and maltose syrup. During liquefaction α(1→4)

bonds are hydrolyzed in random manner and it results into reduction in the viscosity

of reaction mixture and increase in the DE which is a measure of degree of starch

hydrolysis.

The maximum DE obtainable during liquefaction starch using bacterial α-

amylase is around 40, but prolonged treatment leads to the formation of maltulose (4-

α-D-glucopyranosyl-D-fructose), which is resistant to hydrolysis by glucoamylase

and α-amylases (www.lsbu.ac.uk/biology/enztech/starch.html). DE values of 10-16

are used in most commercial processes, where liquefact is supposed to be

saccharified. Liquefaction till DE of 10-16 is required to reduce the viscosity of the

gelatinized starch to ease subsequent processing and also to avoid further possible

retrogradation of gelatinized starch. Recently, Haki and Rakshit (2003) have

discussed the source microorganisms and properties of bacterial α-amylase in the

context of calcium independence, pullulanase and β-amylase in the context of

thermostability. The industrial needs for such specific thermostable enzyme and

improvements required to maximize their application in the future are also suggested.

(Teague and Brumm, 1992; Olsen, 1995)

2.2.1.2. Fungal α-amylase

Fungal α-amylase (1, 4-α-D-glucan glucanohydrolases, EC 3.2.1.1,

glycogenase) is an exo-amylase which hydrolyzes α(1→4) linkages and

predominantly produces maltose from starch with significant quantities of glucose and

maltotriose. It has ability to bypass α(1→6) glucosidic bonds i.e. it does not attack

Page 43: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

29

α(1→6) bond, but overlook it and attack next α(1→4) bonds in the chain. It is used in

the production of high maltose syrup from liquefied starch. Prolonged incubation of

liquefied starch with fungal α-amylase results in the production of large amounts of

maltose. (Teague and Brumm, 1992; Olsen, 1995)

2.2.1.3. Glucoamylase

Glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3, sacchorogenic

amylase, amyloglucosidase) cleaves first α(1→4) glucosidic bond from non reducing

end of starch and glycogen in exo-manner and then liberates β-glucose unit. Its

speciality exist in the ability to cleave α(1→6) glucosidic bond. Hydrolysis proceeds

in the stepwise manner. Maltotriose (G3) and particularly maltose are hydrolyzed at

lower rates than higher saccharides and α(1→6) linkages are broken more slowly than

α(1→4). It has activity towards α(1→2), α(1→3), α(1→4), and α(1→6) glucosidic

bonds. At low starch concentrations, glucoamylase completely degrades starch to

glucose and this fact has been used for development of starch assay. At high

concentration of glucose, it catalyzes polymerization of glucose into higher polymers

generally referred as condensation or reversion products. Reversion products include

isomaltose, isomaltotriose, isomaltotetraose, kohibiose, nigerose, maltose, α,β-

trehalose, and panose. As practically complete conversion of starch to glucose is

possible using amyloglucosidase, it is also referred as saccharifying amylase. It is

normally used in the production of glucose syrup and dextrose. (Teague and Brumm,

1992; Olsen, 1995)

2.2.1.4. β-amylase

β-amylase (1,4-α-D-glucan maltohydrolase, EC 3.2.1.2) is a exo-acting

enzyme. It cleaves second α(1→4) glucosidic bond from non reducing end of starch

Page 44: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

30

or glycogen polymer and releases β-maltose unit. β-amylase can neither cleave

α(1→6) glucosidic bond (unlike glucoamylase) nor bypass it (unlike fungal α-

amylase). Hence hydrolysis of starch by β-amylase results into production of maltose

and β-limit dextrins. (Teague and Brumm, 1992)

2.2.1.5. Pullulanase

Pullulanase (α-dextrin endo-1,6-α-glucosidase; EC 3.2.1.41; limit dextrinase,

debranching enzyme, amylopectin 6-glucanohydrolase, pullulan 6-glucanohydrolase)

cleaves α(1→6) glucosidic bonds in amylopectin, pullulan, and glycogen. It

debranches amylopectin to produce linear dextrins and pullulan (a polysaccharide

with a repeating unit of maltotriose that is α(1→6) linked) to produce maltotriose.

Group II pullulanase (α-amylase–pullulanase or amylopullulanase) hydrolyze both

α(1→4) and α(1→6) glucosidic bonds and produces maltose and maltotriose (van der

Maarel et al., 2002).

2.2.1.6. Isoamylase

Isoamylase (Glycogen 6-glucanohydrolase, EC 3.2.1.68, debranching enzyme)

is cleaves α(1→6) linkages. It is the only enzyme that debranch glycogen. These

enzymes have no effect on pullulan (this fact distinguishes it from pullulanase), while

it cleaves all the α(1→6) linkages in amylopectin and glycogen. (Olsen, 1995; van der

Marrel et al., 2002)

2.2.1.7. Glucose isomerase

Glucose isomerase (EC 5.3.1.18) catalyzes Isomerization of D-glucose to D-

fructose. An excellent review on this enzyme and its industrial application has been

written by Bhosale et al., (1996).

Page 45: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

31

2.2.2. Maltodextrins

Maltodextrins, which are partially hydrolyzed starch products, have been on

the market since the first commercial product Frodex 15 (later called Lo-Dex 15) was

introduced by the American Maize Products Company in 1959. The United States

Food and Drug Administration define maltodextrin as (21 CFR paragraph 184.1444):

a non-sweet, nutritive saccharide polymer that consists of D-glucose units linked

primarily by α(1→4) bonds and that has a DE (dextrose equivalent) of less than 20.

Maltodextrin is a saccharide mixture that consists of maltose, malto-oligosaccharides

and linear or branched dextrins. Starch hydrolysis products are commonly

characterized by their degree of hydrolysis, expressed as the dextrose equivalent

(DE), which is the percentage of reducing sugar calculated as dextrose on dry-weight

basis. (Alexander, 1992)

Marchal et al., (1999) provides an excellent review article, which focuses on

the production of maltodextrins with more defined saccharide compositions; glucose

and maltose syrups, and cyclodextrins are not considered here. Design of the desired

saccharide composition and production possibilities for maltodextrins have been

discussed in detail by Marchal et al., (1999).

2.2.2.1. Production

Maltodextrin is usually produced by liquefaction process. Liquefaction of

starch is a combination of gelatinization of starch and dextrinization of gelatinized

starch. Liquefaction of starch can be accomplished by the use of acid or enzyme.

Single stage and double stage processes are normally used for the production of

maltodextrins from starch. In single stage process, either acid or enzyme conversion is

performed at high temperature (~105 °C) using thermostable bacterial α-amylase. The

dual stage process involves first liquefaction at high temperature (105 °C) with acid or

Page 46: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

32

enzyme to a low DE (usually < 3), followed by treatment at high temperature (~120-

130 °C in jet cooker) to ensure complete gelatinization of starch. Second stage

involves hydrolysis using bacterial α-amylase at temperature around 85-105 °C.

(Teague and Brumm, 1992; Alexander, 1992)

Random nature of acid catalyzed hydrolysis limits its utility as a means of

producing low DE products or liquefact suitable for further conversion. Low DE

conversion products of acid hydrolysis contain more glucose than their enzymatically

produced counterparts at the same DE. This leads to increased sweetness,

hygroscopicity, and susceptibility to color development upon heating, generally

undesirable for this type of product. Furthermore, they have a low stability caused by

the presence of linear chains that have not been shortened sufficiently to avoid

retrogradation. Hence, though partial hydrolysis of starch has traditionally been

carried out using acids, acid hydrolysis is being replaced by enzymatic hydrolysis for

the production of tailor-made maltodextrins. (Reeve, 1992; Teague and Brumm, 1992;

Marchal et al., 1999)

Only dextrose equivalent of maltodextrins has been shown to be inadequate to

predict product performance in various applications. Maltodextrins with the same DE

can even have different properties in various applications that reflect differences in

their molecular composition. The saccharide composition of a maltodextrin

determines both its physical and biological functionality. Factors influencing the

saccharide composition are type and source of enzyme, source of starch, starch

concentration, temperature, organic solvents, pressure, immobilization, downstream

processing and extraction of products during hydrolysis (Marchal et al., 1999) and

grouped according to potential and costs in Fig. 2.7.

Page 47: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

33

Figure 2.7. Different ways to influence the saccharide composition of a starch hydrolysate grouped according to potential and costs. (Adapted from Marchal et al., 1999)

Dextrose equivalent Starch Maltodextrin a 0 5 10 15 20

Viscosity/ Bodying agent Browning reaction

Cohesiveness

Freezing point depression Hygroscopicity

Sweetness

Prevention of coarse crystals Solubility

Osmolality

Figure 2.8. Increase or decrease in functional properties of maltodextrins as a function of DE (Alexander et al., 1992)

Potential

Costs

Enzymes

Temperature Downstream processing

Source of starch

Concentration Immobilization

Extraction of products

Pressure

Organic solvents

Page 48: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

34

2.2.2.2. Application

Maltodextrin is a versatile ingredient, which finds numerous applications in

the food processing and pharmaceutical industries. These applications are based on a

wide range of functional properties like freezing point depression, hygroscopicity,

osmolality, prevention of coarse crystals, solubility, sweetness, viscosity, and

absorption by human. The dextrose equivalent (DE) value and saccharide composition

of maltodextrin determines the variation in the effect of the above-mentioned

parameters. (Marchal et al., 1999). Increase or decrease in these properties as function

of DE is illustrated in Fig. 2.8.

Maltodextrins are used as spray drying aid/ flavor encapsulation, bulking

agent, texture provider, fat replacer, tablet expicient, film former, sport beverage,

parenteral and enteral nutrition products (Alexander, 1992; Marchal et al., 1999).

Spray drying aid/ flavor encapsulation. Preparation of dried flavors by encapsulating

flavor or flavor oil.

Carrier / Bulking agent. Used in dry mix products (includes puddings, soup, and

frozen desserts.) and dry mix beverages. Particularly advantageous in dry

soup mixes due to less hygroscopicity.

Nutritional. It is directly used as rehydration / energy beverage, sport drink and as a

carbohydrate source in the nutritional fluids like energy bar, infant formula

(i.e. non milk or non lactose based fluids), enteral products, parental nutrition

product, oral nutritional supplement due to easy digestibility, instant

dispersibility in cold water, and low osmolality.

Medical. Used in production improved excipients for tableting or directly as tablet

excipients, isotonic solution that can be directly infused into veins of patient.

Page 49: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

35

Fat replacer. It produces soft reversible gel and gives creamy fatlike mouthfeel. Gel

can be used to replace part of fat or oil in high fat products like ice cream and

spoonable salad dressings

Desserts. Used as ingredient in the preparation.

Dairy. Maltodextrin is used extensively in coffee whiteners, imitation sour creams,

imitation cheeses and whipped toppings.

Confectionery. It is perfect for candy coating and soft-centre candies, for frosting and

glazing, for nut and snack coating, in lozenges and for binding, plasticizing

and crystal inhibition. In hard candies, it reduces the hygroscopic

characteristics.

2.2.3. Glucose syrup

Glucose syrup is a purified concentrated aqueous solution of nutritive

saccharides obtained from starch. Glucose syrups are largely composed of glucose

and maltose (concentration of glucose is largest and followed by concentration

maltose) and have DE values between 20 and 80. Major source of starch for

production glucose syrup is corn. (Howling 1992)

2.2.3.1. Production (Reeve, 1992; Teague and Brumm, 1992; Howling, 1992)

Acid conversion

When a suspension of starch in water is heated with acid (normally HCl or

H2SO4) at a temperature exceeding that required for starch gelatinization (normally

water boiling conditions at atmospheric pressure), rapid hydrolysis takes place, with

breakage of both α(1→4) and α(1→6) glucosidic linkages. It is normally regarded as

a random type of hydrolysis. Rate of hydrolysis is a function of temperature and

concentrations of both acid and starch. Acid catalyzed starch hydrolysis is remarkably

Page 50: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

36

reproducible. Provided the reaction conditions (i.e. time, temperature, acid

concentration, starch concentration) are kept constant.

Random nature of acid hydrolysis puts important constraint on the glucose

syrups produced. If a DE of less than 30 is obtained, due to random action there

remains a proportion of glucose polymers of sufficient chain length to give rise to

retrogradation, resulting in the formation of insoluble starch particles. Second

limitation is that in the production of glucose syrup with DE above 55, acid promote

dehydration and condensation in addition to hydrolysis that results into undesirable

products like formic acid, gentiobiose, hydroxymethylfurfural, and levulinic acid.

These undesirable products serve as impurities and also impart undesirable color to

syrup.

Hence the range of glucose syrup produced by acid catalyzed hydrolysis is

limited to the range of 30 - 55 DE. In this range of DE, products are reproducible in

terms of saccharide composition and high quality (i.e. color stability and clarity).

Typical sugar composition of glucose syrups produced by acid catalyzed hydrolysis

(Howling, 1992) is shown the table 2.5.

Table 2.5. Typical Sugar Composition of Acid Converted Glucose Syrups

(Howling, 1992)

Sugar 30 DE 42 DE 55 DE

Dextrose % on dry basis 10 19 31

Maltose % on dry basis 9 14 18

Trisaccarides % on dry basis 10 11 13

Higher sugars % on dry basis 71 56 48

Page 51: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

37

Acid - enzyme conversion

The range of glucose syrup i.e. 30-55 DE, produced with acid conversion, is

widened by use of enzyme in a dual conversion with acid used for liquefaction. Acid

catalyzed hydrolysate of 20-40 DE was enzymatically hydrolyzed at temperature of

55-60 °C. Second stage enzymatic hydrolysis can be done using bacterial α-amylase,

amyloglucosidase, fungal α-amylase, β-amylase individually or combination of two

enzymes, depending upon the properties of required glucose syrup i.e. DE value and

saccharide composition (mainly of glucose, maltose and maltotriose). pH of the acid

catalyzed hydrolysate was adjusted to appropriate value (4.5 for amyloglucosidase,

5.5 for fungal α-amylase and 6 for bacterial α-amylase).

Low DE dextrose syrup was produced by hydrolysis of 17-20 DE acid

catalyzed substrate using bacterial α-amylase. Higher end of spectrum, 55-80 DE

were made by hydrolysis of 17-20 DE acid substrate or 40-42 DE acid substrate using

amyloglucosidase alone or in combination with fungal α-amylase. Table 2.6 shows

saccharide composition, enzyme and substrate used for a range of acid-enzyme syrups

(Howling, 1992).

Table 2.6. Composition of acid-enzyme converted glucose syrups. (Howling, 1992).

DE of Acid catalyzed

hydrolysate, 1st stage 17 20 40 40

Enzyme used in second

stage

Bacterial

α-amylase

β-amylase fungal α-amylase

+ glucoamylase

β-amylase

+ glucoamylase

DE 26 42 63 63

Dextrose % on db i.e.

dry basis 4 6 36 37

Maltose % on db 4 45 30 32

Higher sugars % on db 92 49 34 31

Page 52: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

38

Enzyme-Enzyme conversion

This method was developed after the discovery of thermostable Bacterial α-

amylase. Liquefaction of starch was performed using bacterial α-amylase at 90-100

°C and hydrolysate of 12-15 DE was produced. Then this hydrolysate was further

saccharified using enzyme combinations described in the earlier section on Acid-

enzyme conversion at 55-60 °C to produce glucose syrup of desired DE and

saccharide composition.

Progress in the processes from acid to acid-enzyme to enzyme-enzyme not

only provided better control over saccharide composition but also reduced the

formation of color precursor 5-HMF (5-hydroxymethylfurfural) and the ash content in

the glucose syrup (Howling, 1992).

Refining and evaporation (Howling, 1992).

Refining means the process by which filtered starch hydrolysate product is

purified and deodorized by removing trace impurities that remained after separation

of bulk of the protein and lipid by filtration. Impurities that remain consist of protein

or protein hydrolysate, peptides and amino acids, color precursors, and flavor and

odor contaminants. In enzyme-enzyme process based glucose syrup, calcium sulphate

or calcium phosphate haze forms due to the presence of calcium, which is usually

added as a cofactor for bacterial α-amylase. Also, syrup might contain sulphate and

phosphate picked up from filer aid and / or tap water. Hence demineralization is also

required, which gave an improved product in terms of color removal and color

stability. This demineralization step will also remove color precursors, which are

basically ionic. This refining step will be required and will be mostly same for all

other starch hydrolysate products also.

After refining, glucose syrups require concentration to about 80% solids for

Page 53: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

39

shipment. This dry wt concentration is a compromise between minimum water

content, viscosity, resistance to microbial contamination, and ability to pump and

store at temperatures that do not reduce shelf life by color formation. Evaporation is

carried out at as low temperature as possible to minimize color formation.

2.2.3.2. Applications (Howling, 1992)

Applications are based on a wide range of functional properties like freezing

point depression, hygroscopicity, osmolality, prevention of coarse crystals, solubility,

sweetness, and viscosity, which are mainly dependent on DE. The increase or

decrease in these properties with an increase in DE is same as that shown in the Fig.

2.8 for maltodextrins. Glucose syrups are used in a variety of food and food

applications. In general confectionary area is the dominant worldwide market for

glucose syrups.

Confectionary. Glucose syrups are used in various confectionary products like hard

candies, toffees/ caramel/ fudge, Gums and jellies, fondants, marshmallows, and

chewing gum. Properties of concern are sweetness, hygroscopicity, and viscosity.

Nondairy creamer. Product is made by mixing 50% glucose syrup solids (25-30 DE)

with 40% hardened vegetable oil, emulsifier, stabilizer, flavor etc at 82 °C and

spray dried.

Fruit preserves. Due to preservative nature of carbohydrate solids in excess of 67%

concentration, they are used in products like jams, preserves, conserves, and

marmalades.

Candied fruit. 63 DE syrup used due to sweetness, preservative property & low

viscosity.

Frozen desserts.

Bakery goods. Used in preparation of bakery products like bread, rolls, doughnuts,

Page 54: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

40

cookies, crackers, pies, and cake.

Breakfast cereals. 42 DE syrup used to coat cereal products due to good film forming

property and also imparts good shelf life and enhancement in flavor of the

product.

Alcoholic beverages. Used in alcoholic beverages to provide body, viscosity,

sweetness control and a source of fermentable carbohydrates. High DE syrup is

also used as priming sugars in brewing industry.

Pharmaceutical industry. Used in three area: 1. Fermentations that produce antibiotics

and fine chemicals, 2. Medicated confectionary and 3. as a carrier for liquid cough

mixtures and medicines.

2.2.4. Dextrose hydrolysate, crystalline dextrose and liquid dextrose

Dextrose hydrolysate is one of the most important starch hydrolysis products.

It contains D-glucose in excess of 90% on dry basis (db) with a typical value between

95 and 96 % db D-glucose. Dextrose hydrolysate after refining and concentration

serves as feedstock to dextrose crystallization process. Dextrose hydrolysate is

sometimes also termed as high DE glucose syrup. The products produced from this

feedstock include crystalline dextrose, liquid glucose and high fructose syrup. Liquid

dextrose is a syrup rich in D-glucose that exceeds content of D-glucose in dextrose

hydrolysate and is typically 99% db or greater. (Mulvihill, 1992)

2.2.4.1. Production (Reeve, 1992)

Acid catalyzed starch hydrolysis process is incapable of yielding more than

85-90% dextrose under practical conditions because of concurrent reversion and

dehydration reactions. The acid-enzyme process will yield 93% dextrose, since acid

hydrolysis produces highly branched saccharides resistant to action of glucoamylase.

Page 55: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

41

Hence dextrose hydrolysates are today produced almost exclusively by totally

enzymatic processes (Fig. 2.6), which give dextrose levels of 95-96%.

There are mainly two steps involved in the enzymatic production of Dextrose

hydrolysate from starch:

1. Liquefaction of starch using bacterial α-amylase

2. Saccharification of liquefied starch using amyloglucosidase with or without

pullulanase

Liquefaction

Starch liquefaction is defined as the combination of two processes:

1. Complete gelatinization (including hydration) of starch polymer, ensuring

accessibility to hydrolytic attack.

2. Dextrinization to a degree that prevents retrogradation on further processing.

In the enzymatic liquefaction of starch, bacterial α-amylase is used, which is

thermostable and hydrolyzes only internal α(1→4) linkages in the starch. Most

commonly used bacterial α-amylase is from B. licheniformis. Other sources of

bacterial α-amylase are discussed earlier in the section 4.2.1. Hydrolysis of starch

using bacterial α-amylase is random in nature except that linkages near either end of

polymer chain and those close to a branch point are resistant. Hydrolysis of starch

using bacterial α-amylase produces malto-oligosaccharides, linear and branched

dextrins. Though enzymatic starch hydrolysate contains high molecular wt glucose

polymers, it is less susceptible to retrogradation because high mol wt fraction in

hydrolysate is more highly branched due to inability of bacterial α-amylase to cleave

α(1→6) linkages.

High pH (6 – 6.5) and calcium requirement (~100-400 ppm) to ensure enzyme

stability are disadvantages of this process. Added calcium salts must be removed at

Page 56: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

42

the cost of increased refining expenses. Production of maltulose precursors is

enhanced by high pH (pH > 6), high temperature (>110 °C), long residence times in

the liquefaction, and is more pronounced at high DE values. Maltulose precursors are

produced due to chemical isomerization of the reducing end glucose units during

enzyme liquefaction and autoclaving of starch in the enzyme-enzyme process of

glucose manufacture. The precursors are hydrolyzed during subsequent

saccharification with amyloglucosidase to form maltulose as one of the products. Acid

liquefaction prior to saccharification does not result in maltulose precursor formation.

(Dias and Panchal, 1987)

In the liquefaction, liquefact with DE preferably between 7 and 15 should be

produced under conditions unfavorable to maltulose precursor development. Inability

of amyloglucosidase to hydrolyze maltulose will otherwise lead to a reduction in the

final dextrose level attainable. Liquefaction is usually performed with reaction time of

1-2 h.

Saccharification of liquefied starch

After liquefaction, pH of the reaction mixture is reduced to 4.5 and

temperature decreased to 60 °C. Saccharification of starch liquefact is performed with

or without pullulanase to produce dextrose hydrolysate. Saccharification time is

usually 24-96 h depending upon enzyme dosage. Amyloglucosidase is an exo-

amylase with ability to cleave both α(1→4) and α(1→6) linkages. Hence it seems that

100% dextrose is possible theoretically. Amyloglucosidase produces 100% dextrose

(db) with very low concentration (<1% w/v) of starch as substrate solution. As starch

concentration increases maximum dextrose attainable decreases (at 30% w/v starch

concentration it is 96%). During saccharification at higher concentrations (necessary

for an economic process), the dextrose level rises to the maximum indicated and then

Page 57: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

43

begins to fall. This occurs due to reverse reactions catalyzed by amyloglucosidase,

which form disaccharides by repolymerization of dextrose. Reversion reactions are

favored by high substrate concentration. These reversion reactions will proceed till

equilibrium is reached. Here, it should be remembered that maximum attainable

dextrose is same, irrespective of amyloglucosidase concentration in the reaction

mixture. But increase in the dosage of amyloglucosidase will also enhance the rate of

reversion and enzyme inactivation by heating to 80 °C can become necessary to avoid

a rapid reduction in dextrose content.

Hydrolysates obtained from 30-35% w/v starch slurry are economically and

technically accepted. After completion of liquefaction and saccharification of starch

slurry (30-35% w/v), a 33-39% w/v solution is obtained with DE of 97-98 and

saccharide composition of 95-96%db dextrose, 1.2-2% maltose, 1-2% isomaltose, 0.4-

0.8% maltotriose and 0.6-1% higher saccharides. Normal refining (carbon, strong

cation exchange) will remove glucoamylase activity before significant reversion

occurs.

Dextrose content in the dextrose hydrolysate can be further increased by using

debranching enzyme i.e. pullulanase along with amyloglucosidase. Pullulanase will

cleave α(1→6) linkages faster (which are cleaved, but relatively slowly by

amyloglucosidase) and make linear chains available for faster hydrolysis by

amyloglucosidase. Use of pullulanase also reduces quantity of glucoamylase resistant

oligosaccharides, which are normally present. By adding pullulanase, dosage of

amyloglucosidase can be reduced to maintain hydrolysis rate. The consequent

reduction in the reversion rate will result into an increase in the dextrose level (>1%).

Choice of pullulanase usage in saccharification in combination with amyloglucosidase

is dictated by process economics and dextrose levels required.

Page 58: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

44

Dextrose monohydrate (α-D-glucose monohydrate) crystals are produced by

crystallization of saturated D-glucose solutions at temperatures below 50 °C.

Crystallization at higher temperature gives the anhydrous α and β forms. (Mulvihill

1992)

Dissolving crystalline dextrose monohydrate in water (remelting) gives high

purity syrup of 71% ds and 99.5% db D-glucose has been the usual method of

production. Liquid chromatographic separation using ion-exchange resins produces a

stream rich in D-glucose by separating it from higher saccharides present in

hydrolysate streams. The chromatographically produced liquid dextrose is nearly

equivalent to remelt dextrose in D-glucose content and is acceptable in many

applications. (Mulvihill 1992)

2.2.4.2. Applications (Mulvihill 1992)

Dextrose hydrolysate, crystalline dextrose and liquid dextrose are mainly used

in foods, fermentation, industry, and pharmaceutical uses.

Foods. In food applications are similar as mentioned for glucose syrup.

Industrial Applications. Liquid glucose and solids are used in adhesives, building

materials, chemical (mainly sorbitol) manufacture, fermentation, tobacco, and

leather.

Pharmaceutical applications. Anhydrous dextrose recrystallized from dissolved

monohydrate crystal and treated to remove pyrogens is utilized in intravenous

solutions. Used as excipients in making direct compression tablets.

Page 59: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

45

2.2.5. Maltose syrup

Maltose syrup is a purified concentrated aqueous solution of nutritive

saccharides obtained from starch, containing maltose as principal sugar. Maltose

syrup is classified into high maltose, extra high maltose, and high conversion syrup.

Composition of maltose syrups is shown in the Table 2.7.

Table 2.7. Typical composition of maltose syrups (Reeve 1992)

Maltose syrup DE Glucose concn.

%db

Maltose concn.

%db

High maltose 40-46 <4 45-55

Extra high maltose 50-55 <4 70-80

High conversion 60-70 30-35 30-45

2.2.5.1 Production (Reeve 1992)

Production of maltose syrup from starch involves following two steps:

1. Liquefaction of starch using bacterial α-amylase

2. Saccharification of liquefied starch using fungal α-amylase or β-amylase with or

without pullulanase.

Liquefaction part essentially remains same as discussed in the section 2.2.4.1.

except that liquefaction is allowed to proceed to produce liquefact with DE of 7-10.

High maltose syrups are produced by saccharification of starch liquefact with

fungal α-amylase or β-amylase. Fungal α-amylase is endo-amylase, which cleaves

α(1→4) bonds yielding large quantities of maltose and maltotriose. β-amylase is exo-

amylase, which cleaves second α(1→4) bond from non-reducing end to produce

maltose. Since it does not have the ability to bypass α(1→6) bond, saccharification

with β-amylase yields maltose and β-limit dextrins. β-amylase is used in

Page 60: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

46

saccharification at 5.5 pH and 60 °C, whereas fungal α-amylase is utilized at 5.5 pH

and 50-55 °C.

For the production of very high maltose syrup with maltose content of 70-

85%, debranching enzyme, pullulanase or isoamylase, must be used along with β-

amylase. Debranching enzyme cleaves α(1→6) linkages and produces linear dextrins,

which are further saccharified by β-amylase to produce maltose. Major constraint on

maltose level attainable is the liquefact DE. As the liquefact DE increases, quantity of

maltose that can be produced reduces. Therefore, liquefact DE should be as low as

possible. Liquefact DE of 5-10 is normally suitable to attain 70-80% maltose level.

Reaction conditions are pH of 5.5 and temperature of 60 °C.

High conversion syrup, usually of 60-70 DE are formulated to be of maximum

sweetness and fermentability, while resisting crystallization at 4 °C and 80-83% dry

wt concentration. Dextrose content is limited to a maximum of 40% db to avoid

crystallization. Maltose is the bulk of remaining saccharides. High conversion syrups

are produced by the combined use of amyloglucosidase and fungal α-amylase or β-

amylase in the saccharification of liquefied starch. Choice between fungal α-amylase

or β-amylase has been done mainly on economic rather than technical basis. Ratio

between amyloglucosidase and maltogenic enzyme must be varied in order to meet

required dextrose and maltose content. Low DE liquefact increases the

maltose/dextrose ratio.

2.2.5.2. Applications

Maltose syrup is increasingly gaining popularity among diabetic and diet-

concern people. Maltose syrup invariably scores as a favorite alternative to sugar for

health conscious but sweet-loving people. It is often used in bread, cake and beer

brewage because of its well volatility. Meanwhile, Maltose Syrup is also widely used

Page 61: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

47

in other fields such as candy, drinks, bakery goods, frozen foodstuff and seasoning

and so on. Its applications are mostly same as that of glucose syrup.

2.2.6. Fructose syrup

2.2.6.1. Production

High fructose syrup (HFS) is normally produced by enzymatically isomerizing

refined dextrose hydrolysate (containing 94-96% db dextrose). Isomerization is done

by passing the hydrolysate stream through a column of immobilized glucose

isomerase. Dextrose hydrolysate stream with 94-96% dextrose is clarified, refined

with carbon or ion-exchange resins and concentrated to 45% w/v. Clarification is

extremely important as it removes particles in the stream that are detrimental to

column performance, while refining removes calcium ions detrimental to the activity

of immobilized glucose isomerase. Clarified and refined dextrose hydrolysate is then

adjusted to pH of 7-8.5 and supplemented with 0.5-5 mM magnesium ions. Lower

cost is obtained if inlet pH is 7-7.5, whereas maximum throughput is obtained at pH

8.3. The magnesium maximizes and stabilizes the enzyme activity while

counteracting the inhibitory effect of residual calcium ions. The system will tolerate

calcium up to 0.075 mM (3 ppm) as long as magnesium concentration is maintained

in 20 fold excess over calcium. Sulfite or bisulfite salts are added to feed and is

usually aerated. These two adjustments lengthen enzyme half life. Isomerization is

performed as a continuous process at temperature of 53-61 °C. Feed flow rate is a

function of column age, decrease in activity with time, operating conditions and

immobilization technology employed. It is maintained in order to have 42-45%

fructose in the product stream. This is HFS-42. This HFS-42 is fractionated to

produce HFS-90 containing 90% fructose. HFS-42 and HFS-90 are blended to

Page 62: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 2: Overview of starch and starch hydrolysis products

Studies in the Enzymatic depolymerisation of natural polysaccharides

48

produce syrup with desired fructose/dextrose ratio; mostly HFS-55 with 55% fructose.

Syrup is further filtered, refined and evaporated to 77-80% w/v. (Teague and Brumm

1992; White 1992)

2.2.6.2. Applications

HFS-55 is approved to replace 50% of the sucrose in carbonated beverages.

Functional attributes of HFS include sweetness, viscosity, humectancy,

fermentability, resistance to crystallization (HFS > 55%), browning/ flavor

development, and colligative properties (e.g. freezing point depression and osmotic

pressure). Extensive list of applications of HFS (White 1992) is as follows:

Alcoholic beverages and Brewing. Beer, brandy, cordials, liqueurs, wine.

Animal feed.

Baking industry and snack foods. Biscuits, breads, cakes, caramel color, cookies,

crackers, doughnuts, extracts and flavors, frosting/icing, pies, potato chips,

pretzels, rolls etc.

Nonalcoholic beverages. Carbonated, fruit drinks/ juices, powdered mixes

Canners and packers. Berries, candied fruits, fruit fillings, fruit pectin, soups, tomato

sauces, vegetables

Cereals. Breakfast cereals

Chemicals, drugs, pharmaceuticals. Acids, amino acids, antibiotics, food and drug

coatings, drugs, enzyme, fermentation processes, lecithin, mannitol, medicinal

syrups, organic acids, shampoo, pharmaceuticals.

Page 63: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

49

3. Sorghum: Literature Review

Page 64: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

50

3.1. Introduction, Origin and Geographical distribution of sorghum

Sorghum (Sorghum bicolor L. Moench) is an important drought resistant

cereal crop and fifth largest produced cereal in the world after wheat, rice, barley and

maize. Production of sorghum in 2007-2008 in the world was 64 Million Metric

Tons (www.fas.usda.gov). Leading sorghum producing countries were United States

(19.9%), Nigeria (15.5%), India (11.3%), Mexico (9.8%), Sudan (7%), and Argentina

(5.4%) (www.fas.usda.gov). Sorghum ranks third in the major food grain crops in

India. In India, Maharashtra is a largest sorghum producing state with share of around

50%. Sorghum is a staple food for about 300 millions people worldwide. The seed or

caryopsis of sorghum provides a major source of calories and protein for millions of

people in Africa and Asia. In addition to being a major source of staple food for

humans, it also serves as an important source of cattle feed and fodder. It is grown by

United States, Australia and other developed countries for animal feed. Sorghum

grows comparatively quicker and gives not only good yields of grain but also very

large quantities of fodder.

Sorghum is believed to be originated in equatorial Africa, where a large

variability in wild and cultivated species is still found today. It was probably

domesticated in Ethiopia between 5000 and 7000 years ago. From there, it was

distributed along trade and shipping routes around the African continent, and through

the Middle East to India at least 3000 years ago. It is believed that from India it was

carried to China along the silk route and through coastal shipping to South-East Asia.

Sorghum was first taken to America through the slave trade from West Africa. It was

introduced into the United States for commercial cultivation from North Africa, South

Africa and India at the end of the 19th century and subsequently spread to South

America and Australia. It is now widely cultivated in dry areas of Africa, Asia, the

Page 65: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

51

Americas, Europe and Australia between latitudes of up to 50°N in North America

and Russia and 40°S in Argentina. (Kimber et al., 2000; Balole & Legwaila, 2005)

Sorghum is distributed throughout the tropical, semi-tropical, arid and semi

arid regions of the world. Sorghum is also found in temperate regions and at altitudes

of up to 2300 meters in the tropics. It has a potential to compete effectively with

crops like maize under good environmental and management conditions. It is one of

the most widely grown dry land food grains in India. It does well even in low rainfall

areas. Sorghum is also termed as “Nature-cared crop” because it has strong resistance

to harsh environments such as dry weather and high temperature in comparison to

other crops, it is usually grown as a low-level chemical treatment crop with limited

use of pesticides and it has a potential to adapt itself to the given natural environment.

Sorghum is valued because of its ability to produce in areas with marginal rainfall

(400 – 600 mm) and high temperatures (i.e. semi arid tropics and sub tropical regions

of the world), where it is difficult to grow any other cereal, and also, because of its

relatively short growing season requirement, thus its suitability for double cropping

and crop rotation systems (Smith and Frederiksen, 2000).

In Africa, a major growing area runs across West Africa south of the Sahara,

through Sudan, Ethiopia and Somalia. It is grown in upper Egypt and Uganda,

Kenya, Tanzania, Burundi, and Zambia. It is an important crop in India, Pakistan,

Thailand, in central and northern China, Australia, in the dry areas of Argentina and

Brazil, Venezuela, USA, France and Italy. Sorghum is called by various names in

different places in the world. Sorghum is known by various names in Africa: as

guinea-corn, dawa or sorgho in West Africa, durra in the Sudan, mshelia in Ethiopia

and Eritrea, mtama in East Africa, kaffircorn in South Africa and mabele or amabele

in several countries in Southern Africa. It is called jowar in India, kaolian in China

Page 66: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

52

and milo in Spain. In the Indian subcontinent, it is known as jowar (Hindi), jwari

(Maharashtra), jonna (Andhra Pradesh), cholam (Tamil Nadu) and jola (Karnataka).

3.2. Taxonomy

Pliny (ca. 60 to 70 A. D.) was the first to give a written description of sorghum

and after that there was hardly a mention of it until the sixteenth century. Moench in

1794 established the genus Sorghum and brought the sorghums under the name

Sorghum bicolor. Harlan and de Wet (1972) developed a simplified classification that

has real practical utility for sorghum workers. Sorghum (L.) Moench comprises about

20-30 species. Sorghum Bicolor (L.) Moench is primarily cultivated specie. Other

perennial species being Sorghum almum (Columbus grass), Sorghum halepense

(Johnson grass) and Sorghum propinquum. Subspecies of Sorghum Bicolor (L.)

Moench are arundinaceum, bicolor and drummondii. (Dahlberg, 2000)

Sorghum bicolor (L.) Moench subspecies bicolor i.e. grain sorghum contains

all of the cultivated sorghum and is sub classified into different races on the basis of

grain shape, glume shape, and panicle shape. Five basic races are Bicolor, Guinea,

kafir, Caudatum, and Durra. There are 10 intermediate races, which are caused by

hybridization of 2 or more basic races. (Dahlberg, 2000)

The description of the 5 basic races (Dahlberg, 2000) in short is as follows,

1. Bicolor: The most primitive cultivated sorghum, characterized by open

inflorescences and long clasping glumes that enclose the usually small grain at

maturity. Cultivars are grown in Africa and Asia, some for their sweet stems to make

syrup or molasses, others for their bitter grains used to flavor sorghum beer, but they

are rarely important. They are frequently found in wet conditions.

2. Guinea: It is characterized by usually large, open inflorescences with branches

Page 67: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

53

often pendulous at maturity; the grain is typically flattened and twisted obliquely

between long gaping glumes at maturity. Guinea sorghum occurs primarily in West

Africa, but it is also grown along the East African rift from Malawi to Swaziland and

it has also spread to India and the coastal areas of South-East Asia. Many subgroups

can be distinguished, e.g. with cultivars especially adapted to high or low rainfall

regimes. In the past the grain was often used as ship’s provisions because it stored

well.

3. Kafir: It is characterized by relatively compact panicles that are often cylindrical in

shape, elliptical sessile spikelets and tightly clasping glumes that are usually much

shorter than the grain. Kafir sorghum is an important staple across the eastern and

southern savanna from Tanzania to South Africa. Kafir landraces tend to be

insensitive to photoperiod and most commercially important male sterile lines are

derived from kafir type sorghum.

4. Caudatum: It is characterized by turtle-backed grains that are flat on one side and

curved on the other; the panicle shape is variable and the glumes are usually much

shorter than the grain. Cultivars are widely grown in north-eastern Nigeria, Chad,

Sudan and Uganda. The types used for dyeing also belong here and are known as

‘karan dafi’ by the Hausa people in Nigeria.

5. Durra: It is characterized by compact inflorescences, characteristically flattened

sessile spikelets, and creased lower glumes; the grain is often spherical. Cultivars are

widely grown along the fringes of the southern Sahara, western Asia and parts of

India. The durra type is predominant in Ethiopia and in the Nile valley in Sudan and

Egypt. It is the most specialized and highly evolved of all races and many useful

genes are found in this type. Durra cultivars range in maturity from long to short-

season. Most of them are drought resistant.

Page 68: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

54

Different intermediate races are guinea-bicolor, caudatum-bicolor, kafir-

bicolor, durra- bicolor, guinea- caudatum, guinea-kafir, guinea-durra, kafir-caudatum,

durra-caudatum, kafir-durra. Hybrid races exhibit various combinations and

intermediate forms of the characteristics of the 5 basic races. Durra-bicolor is found

mainly in Ethiopia, Yemen and India, guinea-caudatum is a major sorghum grown in

Nigeria and Sudan, and guinea-kafir is grown in East Africa and India. Kafir-

caudatum is widely grown in the United States and almost all of the modern North

American hybrid grain cultivars are of this type. Guinea-caudatum with yellow

endosperm and large seed size is used in breeding programmes in the United States.

The species Sorghum bicolor covers a wide range of varieties, from white and yellow

to brown, red and almost black. Classification and characterization of sorghum is

given briefly in Dahlberg (2000).

Taxonomical hierarchy of Sorghum bicolor (L.) Moench (www.itis.gov) is as

follows:

Kingdom Plantae -- Plants

Subkingdom Tracheobionta -- Vascular plants

Sperdivision Spermatophyta –Seed plants

Division Magnoliophyta -- Flowering plants

Class Liliopsida -- Monocotyledons

Subclass Commelinidae

Order Cyperales

Family Poaceae -- graminées, grass family

Genus Sorghum Moench -- sorghum

Species Sorghum bicolor (L.) Moench -- black amber, broomcorn,

chicken corn, shatter cane, shattercane, sorghum, wild cane

Subspecies (ssp.)

Sorghum bicolor ssp. Arundinaceum – Common wild sorghum

Sorghum bicolor (L.) Moench ssp. bicolor – grain sorghum

Sorghum bicolor ssp. drummondii – Sudangrass

Page 69: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

55

Synonyms for Sorghum bicolor – Black amber, broom-corn, broomcorn, chicken

corn, common wild sorghum, Drummond broomcorn, durra, Egyptian millet, feterita,

forage sorghum , great millet, guinea corn, jowar, Kaffir-corn, Kaffircorn, milo,

shallu, shatter cane, shattercane, sorghum, Sudan Grass, sweet sorghum and wild

cane.

Synonyms for Sorghum bicolor ssp. Bicolor – Holcus bicolor, Holcus sorghum,

Sorghum bicolor var. caffrorum, Sorghum caffrorum, Sorghum cernuum, Sorghum

dochna, Sorghum dochna var. technicum, Sorghum drummondii, Sorghum durra,

Sorghum saccharatum, Sorghum subglabrescens, Sorghum vulgare, Sorghum vulgare

var. caffrorum, Sorghum vulgare var. durr, Sorghum vulgare var. roxburghii,

Sorghum vulgare var. saccharatum and Sorghum vulgare var. technicum.

3.3. Production, cultivation area and yield of sorghum

Sorghum is an important cereal crop which is grown globally for food and

feed purposes. It is most widely grown in the semi-arid tropics, where water

availability is limited and is frequently subjected to drought. About 100 countries

grow sorghum.

3.3.1. Trends in production, cultivation area and yield in the world

Sorghum cultivation is distributed throughout the world (Figs. 3.1 and 3.2). In

Asia, it is grown in China, India, Korea, Pakistan, Thailand and Yemen. Australia

and USA grow the crop too. Here one thing should be remembered that these

developed countries cultivate sorghum for animal feed, whereas, developing countries

in Asia and Africa cultivate it for use as human feed. In Southern and Eastern Africa,

the sorghum-growing countries are Botswana, Eritrea, Kenya, Lesotho, Madagascar,

Malawi, Mozambique, Namibia, Somalia, South Africa, Swaziland, Tanzania, Zambia

Page 70: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

56

and Zimbabwe. In West and Central Africa, the crop is grown in Benin, Burkina

Faso, Burundi, Cameroon, Central African Republic, Chad, Egypt, Gambia, Ghana,

Guinea, Guinea-Bissau, Ivory Coast, Mali, Mauritania, Morocco, Niger, Nigeria,

Rwanda, Senegal, Sierra Leone, Sudan, Togo, Tunisia and Uganda. In Latin America,

the sorghum-growing countries are Argentina, Brazil, Colombia, El Salvador,

Guatemala, Haiti, Honduras, Mexico, Nicaragua, Peru, Uruguay and Venezuela. In

Europe, it is grown in France, Italy, Spain, Albania and Romania. (Deb et al., 2004)

Page 71: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

57

Figure 3.1. Distribution of sorghum area, 1999-2001. (Source: Deb et al., 2004)

Figure 3.2. Distribution of sorghum production, 1999-2001. (Source: Deb et al.,

2004)

19.9

15.5

11.39.87

17.9

5.45 4.2 4

United States NigeriaIndia MexicoSudan OthersArgentina EthiopiaAustralia China

Figure 3.3. % production of sorghum country-wise with world production of 64.5

Million Tones in 2007-2008. (Source of data: www.fas.usda.gov)

Page 72: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

58

Production of sorghum in 2007-2008 in the world was 64 Million Metric Tons

(www.fas.usda.gov). Foremost sorghum producing countries were United States,

Nigeria, India, Mexico, Sudan, and Argentina with production in million metric tones

of 12.8 (19.9%), 10 (15.5%), 7.3 (11.3%), 6.3 (9.8%), 4.5 (7%) and 3.5 (5.4%),

respectively in 2007-2008 (Fig. 3.3) (www.fas.usda.gov).

Table 3.1 shows top fifteen countries in terms of sorghum cultivation area,

sorghum production and yield. India has the largest area under sorghum cultivation of

10.06 million ha (Table 3.1, Fig. 3.1). The second largest sorghum cultivating

country is Nigeria, followed by Sudan, USA and Niger. More than 90% of the

world’s sorghum area lies in the developing countries, mainly in Africa and Asia.

The area under sorghum in countries across the world has recorded a mixed trend over

the last three decades (Table 3.2). Area significantly declined in many major

sorghum-growing countries like Argentina, China, India and USA (Table 3.2).

However, sorghum-growing countries like Brazil, Mali, Mexico, Niger, Sudan and

Tanzania experienced notable increases in area at the end of the 20th century

compared to the early 1970s, and this increase has been consistent over the last three

decades. Though Nigeria experienced a decline in area under sorghum in the early

1980s, it increased in the early 1990s and, at the end of the 20th century, was 42%

higher than in the early 1970s. Niger is at 5th position in sorghum cultivation area, but

is at 18th position in production due to very poor yield i.e. 217 kg/ha. Countries like

Australia, Burkina and Egypt shows neither significant increase nor significant

decrease in sorghum cultivation area.

Page 73: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

59

Table 3.1. Area, production and yield of sorghum in different countries, 1999-2001. Rank

Country

Area

('000 Ha)

Production

('000 T)

Yield

(kg/Ha)

Area

wise

Production

wise

Yield

wise

India 10055.7 8231.7 818.6 1 2 72

Nigeria 6816 7647.3 1122 2 3 54

Sudan 4306.5 2441 566.8 3 7 89

USA 3352.7 13379.8 3990.7 4 1 12

Niger 2286.2 500.9 219.1 5 16 98

Mexico 1992.4 6092 3057.6 6 4 19

Burkina Faso 1301.9 1130.6 868.4 7 10 68

Ethiopia 1189.8 1377.6 1157.9 8 9 53

China 941.5 2947.7 3130.9 9 6 16

Chad 879.4 529.6 602.2 10 15 85

Mali 718.5 649.5 903.8 11 14 67

Argentina 690.5 3159.1 4575.3 12 5 8

Tanzania 638.9 653.6 1023 13 13 57

Australia 601.8 1810 3007.8 14 8 20

Brazil 452.8 742.9 1640.4 15 12 38

Egypt 162.7 945.1 5810.3 30 11 6

Colombia 66.2 212.2 3203 41 27 15

France 59.8 364.2 6094.3 42 21 5 Italy 33.5 216.6 6457.8 53 26 3 Uruguay 26.8 95 3539.1 54 39 13

Spain 8.5 44.1 5164.1 64 50 7

Yugoslavia, 2.2 9.1 4102.7 75 67 11

Israel 1.1 13.4 12663.5 79 60 1 Croatia 0.1 0.5 4115.4 89 86 10

Peru 0.1 0.3 3268.9 90 90 14

New Caledonia 0.1 0.1 1366.7 91 95 43

Algeria 0.1 0.4 6400 92 87 4 Kazakhstan 0.1 0.2 4392.6 93 91 9

Jordan Negligible 0.3 11710.5 98 89 2 Other countries 5273.4 5406.2 975.4 - - -

World 41859.3 58556.5 1398.9 - - -

Countries belonging to top 15 positions in Area, Production and Yield are mentioned. Source of data: Deb et al. (2004)

Page 74: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

60

Table 3.2. Trend in area, production and yield of sorghum in major sorghum producing countries during 1971 – 2001. Source of data: Deb et al. (2004) and www.fas.usda.gov

Country Average Area ('000 Ha) 1971-73 1981-83 1991-93 1999-2001

Argentina 2074 2411 721 690 Australia 629 671 460 602

Brazil 50 117 159 453 Burkina Faso 1038 1073 1417 1302

China 5072 2704 1368 941 Egypt 205 166 144 163 India 16335 16469 12574 10056 Mali 373 534 875 719

Mexico 1077 1520 1305 1992 Niger 531 1075 2315 2286

Nigeria 4792 2216 4535 6816 Sudan 1974 3682 5345 4307

Tanzania 338 500 642 639 USA 6077 5101 4160 3353

Average production ('000 Tones) 1971-73 1981-83 1991-93 1999-2001 2005-07 2007-08

Argentina 4140 7935 2626 3159 2800 3500 Australia 1181 1160 915 1810 1769 2700

Brazil 85 224 274 743 1698 1575 Burkina Faso 489 626 1280 1131 1679 1800

China 8680 7343 5151 2948 2324 2600 Egypt 846 623 740 945 900 900 India 7929 11578 10588 8232 7340 7300 Mali 284 452 716 649 n. a. n.a.

Mexico 2799 5286 4582 6092 5733 6300 Niger 200 345 424 501 683 800

Nigeria 3072 3589 4832 7647 10333 10000 Sudan 1527 2300 3323 2441 4058 4500

Tanzania 172 493 619 654 853 900 USA 21951 18614 16839 13380 9518 12827

Average yield (kg/Ha) 1971-73 1981-83 1991-93 1999-2001

Argentina 1953 3306 3635 4585 Australia 1912 1738 1931 3003

Brazil 2231 1953 1739 1639 Burkina Faso 471 583 903 867

China 1711 2716 3765 3124 Egypt 4120 3747 5149 5811 India 485 703 839 819 Mali 765 848 833 902

Mexico 2601 3485 3513 3056 Niger 370 322 185 217

Nigeria 637 1620 1064 1122 Sudan 775 619 616 568

Tanzania 509 1134 965 1027 USA 3625 3596 4001 3986

Page 75: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

61

In terms of annual production during 1999-2001 (Table 3.1), USA tops the list

with 13.38 million T, followed by India (8.23 million T), Nigeria (7.65 million T),

Mexico (6.09 million T) and Argentina (3.16 million T). Here it should be noted that

India was second largest producer of sorghum in 1999-2001 with 8.23 million T. But

at present in the 2007-2008, Nigeria is the second largest producer of sorghum with

10 million T, whereas in India production of sorghum decreased to 7.3 million T

(Table 3.2). Though, USA is largest sorghum producing country, its annual

production is steadily decreasing mainly because of decrease in the sorghum

cultivation area. In 2007-2008 annual production of sorghum in USA is around 60 %

of that in 1971-72; whereas annual production sorghum in Nigeria is about three times

of that in 1971-72 (Table 3.2). In India annual sorghum production has been steadily

decreasing followed by decrease in the sorghum cultivation area.

None of these major sorghum producing countries have highest global yields

e.g. India, Nigeria, Sudan, and Niger have yields of 819, 1120, 567 and 220 kg/ha,

respectively (Table 3.1). Whereas the largest sorghum producing country (USA) has

yield of 3990 kg/ha (Table 3.1). Highest sorghum yields during 1999-2001 (Table

3.1) were recorded by Israel (12664 kg/ha), followed by Jordan (11711 kg/ha), Italy

(6458 kg/ ha) and Algeria (6400 kg/ha). Maximum yield of 12664 of Israel is around

15 times the sorghum yield of India. Thus, while Asian and African countries like

India and Nigeria had the largest area devoted to sorghum cultivation, countries in

West Asia (like Israel and Jordan) and Europe (Italy and France) reaped the highest

yields. It should be noted that Israel and Jordan are not major sorghum-growing

countries. The average area under the crop during 1999-2001 was 1006 ha and

production 13 400 t in Israel, and 30 ha and 300 t in Jordan.

Page 76: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

62

3.3.2. Trend in cultivation area, production and yield of sorghum in India and

different states of India

In India sorghum is grown in the kharif (rainy season) and rabi (postrainy

season). The share of kharif is higher both in terms of area under cultivation and

production. The kharif sorghum crop accounts for 55% of the total area under

cultivation and 68% of the total production. It can be seen from the Figure 3.4 that

cultivation area under sorghum initially increased from 1950-51 to 1960-61. India

was having maximum land under sorghum cultivation during 1960 to 1970. But,

thereafter continuous decline in the sorghum cultivation area was observed. Reason

for this could be attributed to increase in the % coverage under irrigation (Fig. 3.4).

As the irrigation facility is improved, tendency of farmers to grow cash crops like

sugarcane, cotton etc increases.

0

2

4

6

8

10

12

14

16

18

20

1 11 21 31 41 51

Year

Sorghum cultivation area (million ha)

% coverage under irrigation

1950-51 1960-61 1970-71 1980-81 1990-91 2000-01

Figure 3.4. Trend in sorghum cultivation area (million ha) and % coverage under irrigation (Source of data: www.agricoop.nic.in)

Page 77: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

63

Similarly, it can be seen from Fig. 3.5 that annual sorghum production

increased from 1950 to 1980. The decade 1980-90 has seen peak production of

sorghum in the country. But, after that annual sorghum production steadily decreased

from 11 to 7.3 million tones. This decrease is primarily due to decrease in the

sorghum cultivation area. It can be seen from the Fig. 3.6 that sorghum yield in the

country is increasing continuously.

0

2

4

6

8

10

12

14

1 11 21 31 41 51

Year

Prod

uctio

n (M

illio

n To

nes)

1950-51 1960-61 1970-71 1980-81 1990-91 2000-01

Figure 3.5. Trend in annual sorghum production (Source of data: agricoop.nic.in) Average annual area under sorghum in India declined from 16 million ha in

the early 1970s to 10 million ha in the late 1990s (Fig. 3.4). Sorghum production was

increasing until the early 1980s but declined after that Yield of sorghum has increased

over time (Figs. 3.5 and 3.6). Average sorghum yield in the late 1990s was 826 kg/ha

against 543 kg/ha in the early 1970s. Decrease in sorghum production was primarily

due to the decrease in area under sorghum.

Page 78: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

64

Table 3.3. Trend in Area, production and yield of sorghum in different states of India

during 1972 – 2002. (Source of data. Deb et al., 2004)

Average Area ('000 ha) State

1972-75 1981-84 1991-94 1998-2002

Andhra Pradesh 2709.9 2102.2 1057.2 721.6

Gujarat 970.6 956.6 444.6 206.1

Karnataka 2037.3 2205.7 2159.2 1885

Madhya Pradesh 2122.7 2138 1363.9 690.6

Maharashtra 5718 6588.7 5857 5019.8

Rajasthan 971.7 968.3 714.6 588.4

Tamilnadu 665.3 688.7 500.8 402.6

India (Total) 16139.3 16469 12703.5 10012.3

Average Production ('000 Tones)

Andhra Pradesh 1363.9 1326.4 815.6 559.2

Gujarat 321.4 544.7 267.6 190

Karnataka 1578 1726.3 1842.7 1707.7

Madhya Pradesh 1598 1747.7 1277.3 575.9

Maharashtra 2577.7 4740.7 5351.3 4388

Rajasthan 337.3 451.7 243.1 153.8

Tamilnadu 504 492 508.3 403.9

India (Total) 8826.3 11578 10773.3 8272

Average Yield (kg/ha)

Andhra Pradesh 506.7 630 770 779.3

Gujarat 333.3 570 616.7 896.7

Karnataka 763.3 783.3 856.7 906

Madhya Pradesh 750 816.7 936.7 828.7

Maharashtra 436.7 720 906.7 875.3

Rajasthan 350 463.3 330 363.6

Tamilnadu 760 710 1013.3 1001.7

India (Total) 543.3 706.7 846.7 826

Page 79: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

65

0

200

400

600

800

1000

1200

1 11 21 31 41 51

Year

Yie

ld (k

g/He

ctar

e)

1950-51 1960-61 1970-71 1980-81 1990-91 2000-01

Figure 3.6. Trend in the sorghum yield (Source of data: agricoop.nic.in)

50.28.1

7.5

5.9 3.8 3.3 2.20.30.10.2

18.5

MaharashtraKarnatakaAndhra PradeshMadhya PradeshTamilnaduUttar PradeshRajasthanGujaratHaryanaOrissaOthers

Figure 3.7. State wise distribution of sorghum production during 2001-2002 (Source of data: agricoop.nic.in)

Maharashtra was largest sorghum producing state in 2001-2002 with share of

around 50% (Fig. 3.7). The trends in the area, production and yield of sorghum in

major sorghum-growing states in India are presented in Table 3.3. The area under

sorghum in the late 1990s (1998-2002) declined by 1 to 60% in major sorghum-

growing states (Andhra Pradesh, Gujarat, Madhya Pradesh, Rajasthan and Tamilnadu)

compared to the early 1970s, early 1980s and early 1990s. In fact, the niche of

Page 80: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

66

sorghum production primarily remains in the two states of Maharashtra and

Karnataka, where area under sorghum production stands at a total of 7 million ha

(Table 3.3).

3.4. Plant anatomy and growth

Sorghum is a self-pollinating plant and its drought resistance is higher than

that of corn. The height of the plant varies from 0.5 m to 5 m. Sorghum produces one

or several tillers, which emerge initially from the base and later from the stem nodes.

The long, wide leaves grow from the stalk. The seed is small and round. A seed head

of about 25 cm to 36cm is seen on the top of the stalk of a mature sorghum plant. The

flower is a panicle, usually erect, but sometimes recurved to form a goose neck.

Grain sorghum has a large, erect stem terminating in a semi compact or compact head

or panicle.

3.4.1. Botanical parts of sorghum plant

Botanical parts of sorghum plant are shown in the Fig. 3.8. Sorghum plant

consists of following botanical parts (Plessis, 2008):

Root system

The roots of the sorghum plant can be divided into a primary and secondary

system. The primary roots are those which appear first from the germinating seed.

The primary roots provide the seedling with water and nutrients from the soil. Primary

roots have a limited growth and their functions are soon taken over by the secondary

roots. Secondary roots develop from nodes below the soil surface. The permanent root

system branches freely, both laterally and downwards into the soil. If no soil

impediments occur, roots can reach a lateral distribution of 1 m and a depth of up to 2

Page 81: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

67

m early in the life of the plant. The roots are finer and branch approximately twice as

much as roots from maize plants.

Figure 3.8. Botanical parts of sorghum plant

Leaves

Sorghum leaves are typically green, glasslike and flat, and not as broad as

maize leaves. Sorghum plants have a leaf area smaller than that of maize. The leaf

blade is long, narrow and pointed. The leaf blades of young leaves are upright but the

blades tend to bend downwards as leaves mature. Stomata occur on both surfaces of

the leaf. A unique characteristic of sorghum leaves is the rows of motor cells along

the midrib on the upper surface of the leaf. These cells can roll up leaves rapidly

during moisture stress. Leaves are covered by a thin wax layer and develop opposite

one another on either side of the stem. Environmental conditions determine the

number of leaves, which may vary from 8 to 22 leaves per plant.

Stem

The stem of the plant is solid and dry, to succulent and sweet. Stalk is the main

Page 82: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

68

stem of plant. Under favorable conditions more internodes develop, together with

leaves, producing a longer stem. The stem consists of internodes and nodes. A cross

section of the stem appears oval or round. The diameter of the stem varies between 5

and 30 mm. The internodes are covered by a thick waxy layer giving it a blue-white

color. The waxy layer reduces transpiration and increases the drought tolerance of the

plants. The root band of nodes below or just above the soil surface develops prop

roots. The growth bud develops lateral shoots. Sometimes the growth buds higher up

the stem may also develop lateral shoots.

Inflorescence (panicle)

The inflorescence of sorghum, the panicle, may be compact or open. The

shape and color of the panicle varies between cultivars. Panicles are carried on a main

stem or peduncle with primary and secondary branches on which the florets are borne.

The peduncle is usually straight and its length varies from 75 to 500 mm. Each

panicle contains from 800 to 3000 kernels, which are usually partly enclosed by

glumes. The colour of the glumes may be black, red, brown or tan. The flowers of

sorghum open during the night or early morning. Those at the top of the panicle open

first and it takes approximately 6 to 9 days for the entire panicle to flower. Because of

the structure of the flower, mainly self-pollination takes place. A small percentage of

cross-pollination (approximately 6 %) occurs naturally.

Seed

The ripe seed (grain) of sorghum is usually partially enclosed by glumes,

which are removed during threshing and/or harvesting. The shape of the seed is oval

to round and the colour may be red, white, yellow, brown or shades thereof. If only

the pericarp is coloured, the seed is usually yellow or red. Pigmentation in both the

Page 83: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

69

pericarp and testa results in a dark-brown or red-brown colour. Grain structure is

explained in the section 3.5. i.e. grain morphology.

Better drought tolerance of sorghum than most other grain crops (Plessis

2008) can be attributed to:

1. An exceptionally well-developed and finely branched root system, which is

very efficient in the absorption of water.

2. It has a small leaf area per plant, which limits transpiration.

3. The leaves fold up more efficiently during warm, dry conditions than that of

maize.

4. It has an effective transpiration ratio of 1:310, as the plant uses only 310 parts

of water to produce one part of dry matter, compared to a ratio of 1:400 for

maize.

5. The epidermis of the leaf is corky and covered with a waxy layer, which

protects the plant form desiccation.

6. The stomata close rapidly to limit water loss. During dry periods, sorghum has

the ability to remain in a virtually dormant stage and resume growth as soon as

conditions become favorable. Even though the main stem can die, side shoots

can develop and form seed when the water supply improves.

3.4.2. Growth of sorghum plant

Growth of sorghum plant is mainly distributed in following three stages

(Plessis, 2008):

1. Vegetative stage (0 to 30 days after sowing) – Identification of growth stage is

done according to leaf development

Page 84: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

70

2. Reproductive stage (30 to 60 days after sowing) – Identification of growth

stage is done according to development of grain kernels

3. Grain filling and physiological maturity (60 to 90 days after sowing).

Growth of sorghum plant is not very rapid up to the 8-inch height, while the

plant establishes a root system and starts to take up nutrients much more rapidly.

Shortly after reaching the 8-inch height, the growing point of the plant changes from

producing leaves to producing the head. For a medium-maturity sorghum, this occurs

in about 30 to 35 days after emergence. This is a critical point in the development of

the plant since the plant’s total number of leaves is determined at this stage. At this

point, when the plant has completed about 5 percent of its growth, it has taken up 10

to 15 percent of the nutrients it will use during the entire season. During the next 30 to

35 days, until flowering, the plant grows rapidly. It produces much of the leaf area,

which will be important during the grain-filling period. During this time, the head

develops and the stalk grows rapidly. First, the lower portion of the stalk grows,

pushing the head up into the flag leaf sheath into the boot stage. Later, the upper stalk

(the peduncle, which holds the head) grows rapidly, pushing the head out of the flag

leaf sheath, where flowering and pollination can occur. If something happens during

this stage of growth, the head may not fully emerge from the sheath, may not be fully

pollinated, or may cause problems at combining. This period (from when the head

first starts to form until lowering) is a time for rapid growth and rapid nutrient uptake.

At flowering, the plant will have produced about half of its total weight at maturity;

however, between 60 and 70 percent of the total nutrient uptake already will have

occurred. The final stage of growth, from flowering to physiological maturity, is the

important grain-filling period. During this time, total production of the plant is going

into the grain. Materials stored in the stalk are being moved into the grain, and the

Page 85: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

71

plant is taking up approximately the final one-third of the nutrients. If drought occurs,

both uptake and growth may be limited. The end of this period occurs when the grain

is no longer increasing in dry weight. This physiological maturity is not necessarily

the harvest maturity. At physiological maturity, the grain moisture will be 25 to 40

percent, and it must dry considerably before it can be harvested and placed in

conventional storage. For high moisture grain or early harvest and artificial drying,

sorghum can be harvested at any time after physiological maturity. (Vanderlip, 1993,

1998)

3.4.3. Environmental requirement of sorghum plant

The optimum growth requirements of sorghum plants, in order to exploit its

inherit yield potential, are a deep well-drained fertile soil, a medium to good and

fairly stable rainfall pattern during the growing season, temperate to warm weather

(20 – 35 °C) and a frost-free period of approximately 120 to 140 days.

Following environmental conditions are necessary for growth of the sorghum

plant (Kimber, 2000):

1. Day length. Sorghum is a short-day plant, which means that the plant requires short

days (long nights) before proceeding to the reproductive stage. Traditional

varieties initiate the reproductive stage, when the day lengths return to 12 hours.

The optimum photoperiod, which will induce flower formation, is between 10 and

11 hours. Photoperiods longer than 11 to 12 hours stimulate vegetative growth.

The tropical varieties are usually more sensitive to photoperiod than the quick,

short-season varieties. Sorghum plants are most sensitive to photoperiod during

flower initiation.

Page 86: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

72

2. Rain fall. Sorghum can grow in rain weather and also does well in semiarid areas. It

is more important in areas which are too dry for maize. In strongly seasonal

climates, when the rains stop, the plant often begins flowering. In natural flood

irrigation areas such as the decrue system regions of West Africa, the entire life

cycle is completed during the dry season. Sorghum is produced in areas with

rainfall conditions varying from 400 mm to 800 mm.

3. Altitude. Sorghum can grow at altitudes from sea level to 3000 m. The high figure

is probably due to humanly controlled selection.

4. Temperature. Sorghum is a warm-weather crop, which requires high temperatures

for good germination and growth. Seeds germinate well at 10 to 35 °C. Optimum

temperature for germination is 30 °C; being a crop of tropics, it is tolerant of high

temperatures. Breeding efforts have extended its range into cooler area. Base

temperatures vary with cultivars. Temperature plays an important role in growth

and development after germination. A temperature of 27 to 30 °C is required for

optimum growth and development. The temperature can, however, be as low as

21 °C, without a dramatic effect on growth and yield. Exceptionally high

temperatures cause a decrease in yield. Flower initiation and the development of

flower primordia are delayed with increased day and night temperatures. Plants

with four to six mature leaves that are exposed to a cold treatment (temperatures

less than 18 °C) will form lateral shoots. However, for plants in or beyond the

eight-leaf stage, apical dominance will prevent the formation of lateral shoots.

Temperatures below freezing are detrimental to sorghum and may kill the plant.

At an age of 1 to 3 weeks, plants may recover if exposed to a temperature of 5 °C

below freezing point, but at 7 °C below zero, plants are killed. Plants older than 3

weeks are less tolerant to low temperatures and may die off at 0 °C.

Page 87: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

73

5. Soil Requirement. Sorghum plants tolerate a wide range of soils. Sorghum is

mainly grown on low potential, shallow soils with high clay content, which

usually are not suitable for the production of maize. Sorghum usually grows

poorly on sandy soils, except where heavy textured subsoil is present. The

tolerable range of pH of soil varies from 5.0 to 8.5. Sorghum can better tolerate

short periods of water logging compared to maize. Soils with a clay percentage of

between 10 and 30 % are optimum for sorghum production.

3.5. Grain morphology

The caryopsis (seed) consists of three distinct anatomical components (Fig.

3.9): pericarp (outer layer), endosperm (storage tissue), and germ (embryo) with

percentage of total mass of 4.3-8.7, 8-11 and 81-86.5, respectively. Waniska and

Rooney (2000) have reviewed and given the grain morphology in detail.

Pericarp thickness ranges from 8 µm to 160 µm and varies within an

individual mature caryopsis. The outer layer or pericarp originates from the ovary

wall and is comprised of three segments viz. epicarp, mesocarp, and endocarp. The

outermost layer (epicarp) is generally covered with a thin layer of wax. The epicarp is

two or three cell layers thick and consists of rectangular cells that often contain

pigmented material. The thickness of the mesocarp, the middle structure, varies from

the very thin cellular layer (containing small amount of starch granules) to 3 or 4

cellular layers containing a large amount of starch granules. Sorghum is the only food

grade crop that is reported to contain starch in this anatomical section. The innermost

endocarp is composed of cross cells and tube cells. The inner tube cells conduct

water during grain germination, whereas, the outer cross cells form a layer that

impedes moisture loss.

Page 88: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

74

A stylar area is located on the tip of the caryopsis, opposite the germ. The

black layer, or hilum, is the colored placenta scar tissue that develops at the germ tip,

when the caryopsis reaches physiological maturity. Cell walls of sorghum pericarp,

aleurone, and endosperm exhibit a blue autofluorescence, which is mainly due to

esters of ferulic acid. The seed coat or testa is derived from the ovule integuments.

The thickness of the testa ranges from 8 µm to 40 µm and varies within individual

caryopses. The thickest area usually is observed below the style and the thinnest on

the side.

The endosperm tissue is triploid, resulting from the fusion of a male gamete

with two female polar cells. It is composed of the aleurone layer, peripheral, corneous

and floury areas. The aleurone is the outer cover and consists of a single layer of

rectangular cells adjacent to the testa or tube cells. The cells possess a thick cell wall,

large amounts of proteins (protein bodies, enzymes), ash (phytin bodies), and oil

(spherosomes). The peripheral area is composed of several layers of dense cells

containing more protein and smaller starch granules than the corneous area. Both the

peripheral and corneous areas appear translucent, or vitreous, and they affect

processing and nutrient digestibility. Waxy sorghums contain larger starch granules

and less protein in the peripheral endosperm than regular sorghums.

The corneous and floury endosperm cells are composed of starch granules,

protein matrix, protein bodies, and cell walls rich in cellulose, β-glucans, and

hemicellulose. Starch granules and protein bodies are embedded in the continuous,

protein matrix in the peripheral and corneous areas. The protein bodies are largely

circular and 0.4–2.0 µm in diameter. High-lysine cultivars contain fewer and smaller

protein bodies than do regular sorghums, and thus contain significantly less alcohol

soluble kafirins. The starch granules are polygonal and often contain dents from the

Page 89: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

75

protein bodies. Size of starch granules varies from 4 µm to 25 µm, the average being

15 µm. Granules present in the corneous endosperm are smaller and angular whereas

those in the floury endosperm are larger and spherical. The opaque, floury endosperm

(located near the center of the caryopsis) has a discontinuous protein phase, air voids,

and loosely packaged, round, starch granules. The presence of air voids diffracts

incoming light giving an opaque or chalky appearance.

The germ is diploid due to the sexual union of one male and one female

gamete. It consists of two major parts: the embryonic axis and scutellum (Fig. 3.9).

The protein of the germ contains high levels of lysine and tryptophan that are

excellent in quality. The embryonic axis contains the new plant and is divided into a

radicle and plumule. Upon germination and development, the radicle forms primary

roots whereas the plumule forms leaves and stems. The scutellum is the single

cotyledon and contains reserve nutrients, i.e., moderate amounts of oil, protein,

enzymes, and minerals, and serves as the bridge or connection between the endosperm

and germ. The vitreous endosperm has a continuous protein matrix, which is attached

to the starch granules, protein bodies, and cell walls. The floury endosperm has a

discontinuous protein matrix with many small voids between the starch granules. Rate

of endosperm development is faster in sorghum with hard endosperm than that with

softer endosperm.

Page 90: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

76

Figure 3.9: Diagrammatic representation of sorghum grain (Source: Chandrashekar and Mazhar, 1999)

Page 91: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

77

Sorghum grain consists of starch, proteins, fibers, lipids etc. Physical (i.e.

distribution in pericarp, germ, and endosperm) and chemical composition of sorghum

grain is given in the Table 3.4.

Table 3.4. Composition of sorghum seed in % (Source: Waniska and Rooney, 2000)

Caryopsis Endosperm Germ Pericarp Caryopsis Range

100 –

84.2 81.7 – 86.5

9.4 8.0 – 10.9

6.5 4.3 – 8.7

Protein Range Distribution

11.3 7.3 – 15.6 100

10.5 8.7 – 13.0 80.9

18.4 17.8 – 19.2 14.9

6.0 5.2 – 7.6 4.0

Fiber Range Distribution

2.7 1.2 – 6.6 100

– – –

– – –

– – –

Lipid Range Distribution

3.4 0.5 – 5.2 100

0.6 0.4 – 0.8 13.2

28.1 26.9 – 30.6 76.2

4.9 3.7 – 6.0 10.6

Ash Range Distribution

1.7 1.1 – 2.5 100

0.4 0.3 – 0.4 20.6

10.4 – 68.6

2.0 – 10.8

Starch Range Distribution

71.8 55.6 – 75.2 100

82.5 81.3 – 83 94.4

13.4 – 1.8

34.6 – 3.8

Carbohydrates in sorghum are composed of starch, soluble sugar and fiber

(pentosans, cellulose, and hemicellulose). Starch is most abundant and others have

low content.

Starch

Description of starch is given in detail in the chapter 2. Sorghum starch has

properties and uses similar to those of maize starch and the procedure for wet milling

of sorghum is similar to the one used for maize. Pigments in the sorghum pericarp

discolors the starch, yielding light pink color. Bleaching with NaClO2 or rinsing with

NaOH or methanol produces acceptable color. Normal sorghum starch contains 23 –

30 % amylose. Average molecular weights of amylose and amylopectin were 1 to 3 ×

105 and 8 to 10 × 106 kD, respectively. Sorghum that has three recessive wx genes

Page 92: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

78

produces caryopses that contain mostly amylopectin and are termed as waxy sorghum.

Waxy sorghum consists of 1% amylose. Heterowaxy sorghum (inclusive one or two

wx genes) consists of 5 to 19 % amylose. Lichtenwalner et al. (1978) reported that %

amylose in normal (WxWxWx), heterowaxy1 (WxWxwx i.e. single wx gene),

heterowaxy2 (Wxwxwx i.e. two wx genes) and waxy (wxwxwx) sorghum were 24,

23, 17.3 and 1, respectively. Starch isolated from corneous endosperm has lower

iodine binding capacity and higher gelatinization temperature than that isolated from

floury endosperm. Gelatinization temperature range of for sorghum starch is 71 – 80

°C. (Waniska and Rooney, 2000)

Protein

The second major component of sorghum and millet grains is protein. Protein

content and composition varies due to genotype, water availability, temperature, soil

fertility and environmental conditions during grain development. The protein content

of sorghum is usually 11-13% but sometimes higher values are reported (Dendy,

1995). Grain proteins are broadly classified into four fractions according to their

solubility characteristics: albumin (water soluble), globulin (soluble in dilute salt

solution), prolamin (soluble in alcohol) and glutelin (extractable in dilute alkali or

acid solutions).

The structural and functional properties of kafirins are reviewed by Belton et

al. (2006). In common with other cereals, the major storage proteins (kafirins) in the

grain of sorghum are soluble in alcohol–water mixtures and therefore defined as

prolamins. Belton et al 2006 have classified prolamins into four groups, called α, β, γ

-kafirins (based on their relationships to the zeins revealed by their amino acid

compositions and sequences, their molecular masses and their immunochemical cross-

reactions), and δ-kafirin (related to the d-zeins of maize, which has been identified

Page 93: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

79

from the sequences of cloned DNAs but has not been characterised at the protein

level).

Prolamins (kafirins) constitute the major protein fractions in sorghum,

followed by glutelins. Lack of gluten is characteristic of sorghum protein

composition, and traditionally, the bread which cannot be baked from sorghum and

millet is only cake bread. (Leder, 2004)

Fat and Lipids

The crude fat content of sorghum is 3 percent, which is higher than that of

wheat and rice but lower than that of maize. The germ and aleurone layers are the

main contributors to the lipid fraction. The germ itself provides about 80 percent of

the total fat. As the kernel fat is mostly located in the germ, in sorghum mutants with

a large embryo fraction the fat content is higher (5.8 to 6.6 percent) than normal.

Neutral lipid fraction was 86.2 percent, glycolipid 3.1 percent, and phospholipid 10.7

percent in sorghum fat. Fatty acid was significantly higher in kafir, caudatum and

wild sorghum than in the bicolor, durra and guinea groups. On the other hand,

caudatum types had the lowest linoleic acid and bicolor, durra and guinea varieties

had more than wild and kafir sorghum. Oleic and linoleic acids were negatively

correlated with each other. The fatty acid composition of sorghum fat (linoleic acid 49

percent, oleic 31 percent, palmitic 14 percent, linolenic 2.7 percent, stearic 2.1

percent) was similar to that of corn fat but was more unsaturated.

Page 94: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

80

3.6. Utilization of Sorghum

Utilization of the sorghum can be classified mainly in following two

categories viz. Human Food, and industrial use (includes Animal Feed, alcohol

industries etc.). It can be seen from Table 3.5 that in 1979-81, an estimated 39

percent of global production was used as human food and 54 percent for animal feed,

whereas in 1992–94, 42 percent of total utilization was for human food and 48 percent

for animal feed. The proportion of food utilization has gradually increased as a result

of a greater food use in Africa and the substitution of sorghum by other grains (mainly

maize) as feed elsewhere.

3.6.1. Food use

Worldwide, approximately 27 million tons of sorghum was consumed as food

each year during the 1992–94 period (Table 3.5), almost the entire amount in Africa

and Asia. It is a key staple cereal in many parts of the developing world, especially in

the drier and more marginal areas of the semi-arid tropics. Per capita annual food

consumption of sorghum in rural producing areas is more stable, and usually

considerably higher, than in urban centers. And within these rural areas, consumption

tends to be highest in the poorest, most food-insecure regions. Sorghum is eaten in a

variety of forms that vary from region to region. In general, it is consumed as whole

grain or processed into flour, from which traditional meals are prepared. There are

four main sorghum- based foods:

• Flat bread, mostly unleavened and prepared from fermented or unfermented dough

in Asia and parts of Africa;

• Thin or thick fermented or unfermented porridge, mainly consumed in Africa;

• Boiled products similar to those prepared from maize grits or rice;

• Preparations deep-fried in oil.

Page 95: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

81

Table 3.5. Sorghum utilization by type and region (Source: ICRISAT/FAO, 1996)

Human Food

Animal Feed

Other uses

Total utilization

Per capita annual Food use

million tons

million tons

million tons

Million tons kg

1979–81 average Developing countries 25 14.7 4.4 44.2 7.7 Africa 9 0.8 2.3 12.1 18.8 Asia 15.7 7.4 2 25.1 6.1 Central America an the caribbean 0.4 7 0.2 7.6 3.6 South America 0.1 3.7 0.3 4.1 0.3 Developed Countries 0.3 20.4 0.6 21.2 0.2 North America 0.1 10.5 0.2 10.8 0.5 Europe 0 2.8 0 2.8 0 USSR (Former) 0 2.5 0 2.5 0 Oceania 0 0.4 0 0.4 0 World 25.3 35.1 5 65.4 5.7 1989–91 average Developing countries 25.1 14.5 3.7 43.3 6.2 Africa 11.5 0.9 1.8 14.2 18.2 Asia 13.3 6.1 1.6 21 4.6 Central America an the caribbean 0.4 8.4 0.3 9.1 2.7 South America 0 2.7 0.2 2.9 0.1 Developed Countries 0.4 16.8 0.5 17.7 0.3 North America 0.2 10.9 0.2 11.3 0.8 Europe 0 1.2 1.2 1.4 0 USSR (Former) 0 0.3 0 0.3 0 Oceania 0 0.8 0 0.8 0 World 25.5 31.3 4.2 61.1 4.8 1992–94 average Developing countries 26.4 14.8 5.5 46.7 6.2 Africa 12.8 1.3 3.2 17.3 18.6 Asia 13.3 5.6 2 20.9 4.1 Central America an the caribbean 0.4 7.5 0.3 8.3 2.9 South America 0 3.1 0.3 3.4 0.1 Developed Countries 0.3 15.8 0.7 16.8 0.2 North America 0.1 11.1 0.3 11.5 0.5 Europe 0 1.1 0.2 1.3 0 CIS 0 0.1 0 0.1 0 Oceania 0 0.8 0 0.8 0 World 26.7 30.6 6.2 63.5 4.8

Page 96: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

82

Per capita annual consumption of sorghum and its importance as a food

security crop is highest in Africa. For example, per capita annual consumption is 90–

100 kg in Burkina Faso and Sudan; sorghum provides over one-third of the total

calorie intake in these two countries. In Asia, sorghum continues to be a crucial food

security crop in some areas (e.g., rural Maharashtra in India, where per capita annual

consumption is over 70 kg). (ICRISAT/FAO, 1996)

However, both production and food utilization have fallen during the 1980s

and early 1990s, because of shifting consumer preferences. As incomes rise,

consumers are shifting to wheat and rice which taste better and are easier and faster to

cook. This trend is accentuated by rapid urbanization and the growing availability of

a range of convenience foods based on wheat and rice. ICRISAT/FAO (1996) have

discussed sorghum economy in detail. Several previous reviews have addressed the

subject of traditional foods from sorghum in depth, for example Murty and Kumar

(1995), Rooney and Waniska (2000), and Rooney and Serna-Saldivar (2000).

The most common and simplest food prepared from sorghum and millets is

porridge. In all cultures traditionally depending on cereals, a range of treatments of

the whole seed before milling and sifting has been applied. The treatments procedures

are steeping, fermentation, malting, alkali or acid treatment, popping, roasting (dry or

wet), parboiling, and drying. One of the aims of seed treatment is to remove the

polyphenolic compounds from the seed. Others are to improve storage quality, or to

make many kinds of snacks and other popular foods. The traditional art of food

preparation is not standardized and routine procedures have been passed on to the

women through generations. The stiff porridge prepared from maize or cereal

mixture (maize, sorghum, pearl millet, finger millet, etc.) in Kenya, Uganda and

Tanzania is commonly called ugali. The most important fermented thin porridge that

Page 97: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

83

is consumed in Nigeria and parts of Ghana is ogi. In much of Northern Africa a

steamed, granulated product called couscous, made from cereal flours (mostly wheat)

is highly popular. In West Africa, sorghum, pearl millet, maize, and fonio are used to

prepare couscous, although pearl millet is preferred. Sorghum noodles are an

important food product in China. Sorghum is used for tortilla preparation either alone

or in combination with maize in Honduras, Nicaragua, Guatemala, El Salvador and

Mexico. Roti is an unfermented dry roasted pancake made in India from wheat,

sorghum, pearl millet and maize flour. Sorghum grain is used in the production of

two types of beer: clear beer and opaque beer. The latter is a traditional, low-alcohol

African beer that contains fine suspended particles. Sorghum is traditionally a major

ingredient in home-brewed beer. Small quantities are used in the beer industries in

Mexico and USA. Sorghum is a good source of starch, cellulose, and glucose syrup.

Although domestication was primarily for food (and also for beer and sweet stems in

Africa, and for brooms in China), crop residues have been valued as animal fodder,

building materials, and fuel. By applying hydrothermic technologies (flaking,

puffing, extrusion, micronizing) new sorghum and millet products of good quality and

good taste can be produced. (Leder, 2004)

Taylor et al. (2006) have reviewed role of sorghum in nutrition and health of

human, and novel food and non food uses of sorghum. In the developed countries,

nowadays there is a growing demand for gluten-free foods and beverages from the

people with coeliac disease and other intolerances to wheat, who cannot eat products

from wheat, barley, or rye. Coeliac disease is a syndrome characterised by damage to

the mucosa of the small intestine caused by ingestion of certain wheat proteins and

related proteins in rye and barley. The gliadins and glutenins of wheat gluten have

been shown to contain protein sequences that are not tolerated by coeliacs. The

Page 98: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

84

average worldwide prevalence has been estimated as high as 1: 266. Estimates place

the number of persons with coeliac disease in the USA at roughly 3 million. The

cornerstone treatment for coeliac disease is the total lifelong avoidance of gluten

ingestion. This means that wheat, rye, and barley have to be avoided, including durum

wheat, spelt wheat, kamut, einkorn, and triticale. Sorghum is often recommended as a

safe food for coeliac patients, because it is only distantly related to the Triticeae tribe

cereals wheat, rye and barley, being a member of the Panicoideae sub-family which

also includes maize and most millets. Sorghum therefore, provides a good basis for

gluten-free breads and other baked products like cakes and cookies (biscuits) and in

snacks and pasta. (Taylor et al., 2006)

Sorghum can contain substantial levels of a wide range of phenolic

compounds, which have health promoting properties, in particular their antioxidant

activity. Their use as nutraceuticals and in functional foods are reviewed in the paper

by Dykes and Rooney (2006). In addition to the potential health benefits of sorghum

phenolics, sorghum wax may also have unique health properties. Long-chain fatty

alcohols, aldehydes and acids are

interconverted in cellular metabolism, so that all three classes might lower

cholesterol. Policosanols (fatt alcohols in sorghum wax) are a promising resource for

the prevention and therapy of cardiovascular disease. Crude lipid extract from whole

kernel sorghum, which comprised a wide range of lipid substances including plant

sterols and policosanols, lowered cholesterol absorption and plasma non-HDL

cholesterol in hamsters. (Taylor et al., 2006).

Taylor et al. (2006) reviewed literature on novel and non traditional sorghum

foods like Gluten-free leavened breads (starch bread and additives, flour breads and

additives, effect of cultivar and Theoretical basis for sorghum functionality in gluten-

Page 99: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

85

free bread making), Cakes and cookies, Tortillas, snack foods, parboiled sorghum,

and noodles.

3.6.2. Industrial Utilization

The main industries using sorghum in India are the animal feed sector, alcohol

distilleries, and starch industries. Only rainy-season sorghum is used for industrial

purposes. Post rainy-sorghum is a highly valued food grain, and thus too expensive to

be used as industrial raw material. Statistics on industrial demand (‘000 t) for

sorghum in India is summarized in Table 3.6.

3.6.2.1. Animal feed

About 48 percent of world sorghum grain production was fed to livestock

(human food use constitutes about 42 percent). In contrast to food utilization, which is

relatively stable, utilization for feed sorghum changes significantly in response to two

factors: rising incomes, which stimulate the consumption of livestock products, and

the price competitiveness of sorghum vis-à-vis other cereals, especially maize. While

sorghum is generally regarded as an inferior cereal when consumed as food, the

income elasticities for livestock products (and hence the derived demand for feed) are

generally positive and high.

Demand for animal feed is concentrated in the developed countries and in middle-

income countries in Latin America and Asia, where demand for meat is high and the

livestock industry is correspondingly intensive. Over 85 percent of the use of sorghum

as animal feed occurs in Developed countries. (ICRISAT/ FAO)

Another important factor is consumer preference for meat colour. Maize

contains higher carotene levels than sorghum, so meat from maize-fed animals tends

to be more yellow than meat from sorghum fed animals. In Japan for example,

Page 100: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

86

consumers generally prefer white-coloured meat. Therefore, sorghum is a valued

ingredient in some compound feed rations (for poultry, pigs and some breeds of beef

cattle). In contrast, sorghum is discounted by producers in India because consumers

there generally prefer poultry meat and egg yolks with a deeper yellow colour.

(ICRISAT/ FAO)

Table 3.6. Summary of industrial demand (‘000 t) for sorghum in India (Kleih et al.,

2007).

Industry 19981 20102

Poultry feed

Broilers 86-129 570-1150

Layers 312-468 1100-1830

Others 20-30 156-234

Dairy feed 160-240 290-570

Alcohol 90-100 200-500

Starch 50 30-80

Total 718-1017 2346-4364

1. These figures reflect the average utilization of sorghum during the 1990s, based on past inclusion rates and current requirements of raw material, rather than on specific data for 1998. The poultry and starch industries use sorghum only when maize is expensive or not readily available

2. Figures are projected ones

The limited inclusion of sorghum in poultry feed and its relatively low status

as a raw material can be accounted to disadvantages of sorghum as given in the Table

3.7.

Page 101: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

87

Table 3.7. Industry-perceived advantages and disadvantages of using sorghum in

poultry feed (Kleih et al., 2007).

Advantages Disadvantages

• Low cost • Lower energy content than maize

• Energy alternative to maize • Risk of aflatoxins (often associated with

• Easy availability blackened grain)

• Good pelletability • Risk of tannins

• Not always available

• Problems with grinding; mash becomes

powdery reducing feed intake by birds

• Low palatability and digestibility

• Varying quality; grain often infested with

weevils, fungi, etc.

• Sorghum lacks the carotenoid pigments

present in yellow maize, which are necessary

for egg yolk colour

• Feed including sorghum is more difficult to

sell

• Absence of standard varieties in the market

3.6.2.2. Alcohol industries

Sorghum has potential for being used in the production of bio-industrial

products, including bioethanol. Sorghum is a starch-rich grain with similar

composition to maize, and, as with all cereals, its composition varies significantly due

to genetics and environment. Starch ranges of 60–77% and 64–78% have been

reported for sorghum and maize, respectively. As such, sorghum grain would be

appropriate for use in fermentation similar to the use of maize for the production of

bioethanol. Its use may be of particular benefit in countries where rainfall is limiting

and maize does not grow well. Taylor et al. (2006) concluded from the available data

Page 102: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

88

that 1.2–2.3 million metric tons sorghum was used for ethanol production, 3.7–7.5%

of the grain used for ethanol production was sorghum, and 0.13–0.25 billion gallon

(0.49–0.95 billion litres) of ethanol originated from sorghum. (Taylor et al., 2006)

While discussing the potential for using sorghum in alcohol production, one

must keep in mind that in India, molasses (a byproduct of sugar manufacture using

sugarcane) constitutes the most important raw material in this industry. It is estimated

that about 95% of the alcohol manufactured in India is from molasses and the rest

comes from grains, and roots and tubers. Although, the quantity of sorghum grain

presently used by the alcohol sector in India is comparatively low (Table 3.6), it

seems to be the most "enthusiastic" user of the crop as an industrial raw material.

Nowadays government policies on licensing alcohol production and trade are

changing and also government is promoting production of grain based alcohol.

Hence, present scenario is providing an opportunity for sorghum to gain greater

acceptability as a raw material in the alcohol industry. Some distillers indicated a

preference for varieties with a higher starch content and less protein. Distilleries had

no objection to using severely blackened grain as long as the starch content was

acceptable. In general, like most other industrial users, distilleries purchase rainy-

season sorghum through traders or brokers in main producing centers. Problem about

this system could be the misuse of the position by brokers to "control" the market. In

this context, contract farming may be an option providing better linkages between

producers and industrial users. (Kleih et al., 2007)

Maharashtra Government is in favor of promoting grain-based alcohol

production to create a demand for rainy-season sorghum. It must be remembered in

this context that rain-damaged or blackened sorghum could be a favorable raw

material for alcohol production because of its lower market price. Maharashtra, the

Page 103: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

89

main producer of rainy season sorghum, regularly faces the problem of finding

suitable users of blackened sorghum which constitutes 40-60% of its produce,

depending on the rainfall pattern during grain maturity. Advantages and

disadvantages of using sorghum in alcohol production are given in the Table 3.8.

Literature on use of sorghum for alcohol production is reviewed briefly in section 3.9.

Table 3.8. Advantages and disadvantages of using sorghum in alcohol production

(Kleih et al., 2007)

Advantages Disadvantages

• No major technical constraints with • Sorghum is a food grain, and may not be

modern technology available for alcohol production in times of

• Causes least pollution food shortages

• Good quality alcohol free from sulphates • Some producers in Maharashtra face

and aldehydes present in molasses based difficulties in selling grain-based alcohol,

alcohol largely due to the State-imposed export pass

• Can create demand for damaged grain fee. This difficulty is localized.

• Possible regular sourcing of grain from • Cost of molasses based alcohol is lower than

rainy-season crop grain based alcohol

• Byproduct of grain alcohol production

can be used as animal feed

.

3.6.2.3. Starch industry

In general the wet milling of sorghum is similar to that of maize. A thorough

review of early research on wet milling of sorghum can be found in Munck (1995).

However, a problem particular to sorghum is the presence of polyphenolic pigments

the pericarp and/or glumes, which, stains the isolated starch. Due to this reason

sorghum is not much popular in starch producing industries. Sorghum is used for

starch production only when maize is not available. Literature on use of sorghum for

starch production is reviewed briefly in section 3.10.

Page 104: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

90

In 2004, starch production in the world was around 60 million t. 70 % of the

starch was produced from corn. Other raw materials being wheat, sweat potato,

cassava and potato. (www.starch.dk/isi/stat/rawmaterial.html) In India, 0.7 million t

of starch was produced in 1998, out of which 0.6 million t was produced from maize

and 0.1 million t was from cassava. (Kleih et al., 2007)

3.6.2.4. Other industries

Malting and brewing

Malting and brewing with sorghum to produce lager and stout, often referred

to as clear beer as opposed to traditional African opaque beer, has been conducted on

a large, commercial scale since the late 1980s, notably in Nigeria. Nigeria brews in

excess of 900 million litres of beer annually. Brewing with sorghum is now also

taking place in east Africa, southern Africa, and the USA. (Taylor et al., 2006)

In India, easily available barley malt is preferred as the principal raw material

for brewing. Sorghum is not currently used for beer production in India either as malt

or as an adjunct. (Kleih et al., 2007)

There has been extensive research and development work and several

excellent reviews published covering enzymes in sorghum malting, sorghum malting,

and brewing technology (Agu and Palmer, 1998; Hallgren, 1995; Owuama, 1997,

1999; Taylor and Dewar, 2000, 2001). Major outstanding problem areas (like use of

tannin sorghum in malting and brewing, starch gelatinization, the role of the

endosperm cell walls and beta-amylase activity in malt) in sorghum brewing that are

specific to characteristics of sorghum grain and sorghum malt are reviewed by Taylor

et al. (2006).

Page 105: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

91

3.7. Insect pests, Diseases and weeds on sorghum

Chandrashekar and Satyanarayana, 2006 have reviewed available information

on the mechanisms of resistance to insect pests and fungal pathogens in sorghum and

millets. Teetes and Pendleton (2000), Frederiksen (2000) and Stahlman and Wicks

(2000) have discussed Insect pests of sorghum, Diseases and disease management in

sorghum, and Weeds and their control in grain sorghum, respectively.

3.8. Problem areas and factors affecting them in industrial utilization of

sorghum

Sorghum has the distinct advantage (compared to other major cereals) of being

drought resistant and many subsistence farmers in these regions cultivate sorghum as

a staple food crop for consumption at home. Therefore sorghum acts as a principal

source of energy, protein, vitamins and minerals for millions of the poorest people

living in these regions. In this way, sorghum plays a crucial role in the world food

economy as it contributes to rural household food security. (Duodu et al., 2003)

However there are few problem areas in the utilization of sorghum food as well as

industrial utilization and are discussed here.

3.8.1. Gelatinization of starch

Gelatinization of the starch is most important initial step, due to which starch

becomes more susceptible to enzyme action and completely digestible by starch

hydrolyzing enzymes. Phenomenon of starch gelatinization is discussed briefly in the

section 2.1.3.

Sorghum starch gelatinization temperature ranges were reported to be 67–73

°C and 71–81 °C for sorghums grown in southern Africa and in India, respectively

Page 106: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

92

(Taylor et al., 2006). Gelatinization temperature of sorghum starch is reported to be

in the range of 75–80 °C (Palmer, 1992) and 60–80 °C (Wu et al., 2007).

Gelatinization temperature of sorghum starch is far higher than the range

quoted for barley starch of 51–60 °C. This factor becomes important in the malting

and brewing of sorghum. Due to this difference in the gelatinization temperature the

simultaneous gelatinisation and hydrolysis of starch that occurs when mashing barley

malt, is problematical with sorghum malt. Sorghum grain or sorghum malt is first

cooked to gelatinize the starch and then the starch is hydrolyzed using barley malt,

commercial enzymes or a combination of the two.

Taylor et al. (2006) have reviewed the literature on starch gelatinization with

context of its use in malting and brewing. In a study of 30 sorghum varieties, Dufour

et al. (1992) found a few with low gelatinisation temperatures, approaching that of

barley. More recently, Beta et al. (2000a–c) found that Barnard Red, a traditional

South African sorghum variety which was selected for its good malting and opaque

beer characteristics, had a low onset starch gelatinisation temperature of 59.4 °C and

gave high paste viscosity, even though the starch had a normal amylose-amylopectin

ratio. It is suggested that waxy sorghums gelatinize more rapidly, have a relatively

weak endosperm protein matrix and are more susceptible to hydrolysis by amylases

and proteases than normal endosperm sorghums and hence should be better for

brewing (Del Pozo-Insfran et al., 2004). Figueroa et al. (1995) investigated mashing

of 20 sorghum adjuncts of varying endosperm structure with barley malt. They found

that the waxy and heterowaxy types gave much shorter conversion times (time to

starch disappearance as indicated by iodine yellow colour) than normal types. They

attributed this to the lower starch gelatinisation temperatures, 69.6 °C for waxy type,

71.1 °C for the heterowaxy type and 71.1–73.3 °C for the normal types. Interestingly,

Page 107: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

93

Ortega Villican and Serna-Saldivar (2004) found that when brewing with waxy

sorghum adjunct, highest beer ethanol content and lowest residual sugar content were

obtained if the adjunct was first heated at 80 °C, then pressure cooked.

Chandrashekar and Kirleis (1988) showed that the degree of starch

gelatinisation (using the β-amylase and pullulanase method) was lower in hard

endosperm sorghum (with high kafirin protein content) than in soft endosperm types

(low kafirin content). The addition of the reducing agent 2-mercaptoethanol during

cooking markedly increased the degree of gelatinisation. However, increase in the

degree of starch gelatinization was more in harder high kafirin sorghum than that in

the softer low kafirin sorghum. They concluded that the endosperm protein matrix

which envelops the starch granules limits starch gelatinisation. They also reported

that after treatment of pepsin with uncooked sorghum flour (soft sorghum), flour

particles lose their structural integrity and only free starch granules with some

adhering protein bodies remain indicating that the integrity of sorghum particles is

maintained by protein. Their SEM work showed that flour particles from the hard

grains were most often covered with cell wall and when exposed the starch granules

seemed to be surrounded by numerous protein bodies. In contrast, in the soft grains,

cell walls appeared sloughed off the particle and far fewer protein bodies surrounding

the starch granule. Starch granules, protein bodies and cell wall appear to be linked

together by strands of protein. This gets supported by the fact that organized structure

in the both hard and soft grains was lost due to treatment with pepsin. They suggested

that the linking proteins that hold the particle together are strands of glutelin. Thus,

the protein matrix in the hard grain contains both protein bodies and matrix strands,

whereas soft grains contains large amount of strand protein.

Page 108: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

94

3.8.2. Protein digestibility

Sorghum plays a crucial role in the world food economy as it contributes to

rural household food security (ICRISAT/ FAO). A nutritional constraint to the use of

sorghum as food is the poor digestibility of sorghum proteins on cooking.

Digestibility may be used as an indicator of protein availability. It is essentially a

measure of the susceptibility of a protein to proteolysis. A protein with high

digestibility is potentially of better nutritional value than one of low digestibility

because it would provide more amino acids for absorption on proteolysis. In vivo

studies using pepsin and in vitro studies show that the proteins of wet cooked

sorghum are significantly less digestible than the proteins of other similarly cooked

cereals like wheat and maize. (Duodu et al., 2003) In an excellent review on factors

affecting sorghum protein digestibility, Duodu et al. (2003) divided these factors into

two broad categories:

Exogenous factors: These refer to factors that arise out of the interaction of sorghum

proteins with non-protein components like polyphenols, non-starch polysaccharides,

starch, phylates and lipids.

Endogenous factors: These refer to factors that arise out of changes within the

sorghum proteins themselves and do not involve interaction of the proteins with non-

protein components.

Duodu et al. (2003) have discussed all these factors in detail in the review.

Since protein digestibility is not topic of interest in the present work, this will not be

discussed in detail here.

Page 109: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

95

3.8.3. Starch digestibility

Starch digestibility is an important parameter from the context of feeding

value of sorghum as well as production of ethanol from sorghum. Sorghum flour with

high starch digestibility will have good food value as well as will be a good candidate

for production of ethanol.

Lichtenwalner et al. (1978) reported that amylose content decreased and in

vitro starch digestibility using amyloglucosidase increased with incremental increases

of the waxy gene in sorghum. Due to pronase treatment of the sorghum flour, starch

digestibility increases in all four types of sorghum and slightly lesser than that of

isolated sorghum starch. The pronase treatment significantly increased the rate of

starch hydrolysis because it hydrolyzed the protein matrix (which surrounds starch

granules) and increased the surface area of the starch in contact with

amyloglucosidase. Their data is shown in the Table 3.9.

Table 3.9. Effect of the waxy gene of kafir and pronase treatment on in vitro starch

hydrolysis (mg glucose/g starch) using amyloglucosidase. Lichtenwalner et al. (1978)

Genotype Amylose Ground grain Pronase pretreated Purified

content % ground grain starch

Normal (WxWxWx) 24 434 540 550

Heterowaxy (WxWxwx) 23.1 467 565 598

Heterowaxy (Wxwxwx) 17.3 545 703 745

Waxy (wxwxwx) 1 741 966 1035

Rooney and Pflugfelder (1986) have reviewed factors affecting starch

digestibility with special emphasis on sorghum and corn. The digestibility of starch is

affected by the composition and physical form of the starch, protein ~ starch

interactions, the cellular integrity of the starch-containing units, antinutritional factors

Page 110: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

96

and the physical form of the feed or food material. Starch exists inside the endosperm

of cereals enmeshed in a protein matrix, which is particularly strong in sorghum (Fig.

3.10). Starch digestibility is affected by the plant species, the extent of starch-protein

interaction, physical form of the granule, inhibitors such as tannins, and the type of

starch. Among the cereals, sorghum generally has the lowest raw starch digestibility

due to restrictions in accessibility to starch caused by endosperm proteins.

High-amylose corn (amylomaize) has poor digestibility in both raw and

cooked forms, while waxy cereal starches are among the most digestible of all

starches. Digestibility of a starch is generally inversely proportional to amylose

content i.e. directly proportional to waxyness of the sorghum. They have also

explained briefly the reasons behind lower digestibility of sorghum as compared to

corn. (Rooney and Pflugfelder, 1986)

Page 111: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

97

Figure 3.10. SEM images of intermediate texture sorghum kernels. A) Endosperm

cross section (P = pericarp, AL = aleurone cell layer, PE = peripheral endosperm, CE

= corneous endosperm; approx 200X). B) Corneous endosperm area (SV = starch

void, SG = starch granule; approx 1,000X). C) Protein and starch of corneous

endosperm (PM = protein matrix, PB = protein bodies, SG = starch granule; approx

2,000X). D) Starch of floury endosperm (approx 4,000X). (Source: Rooney and

Pflugfelder, 1986)

Page 112: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

98

Zhang and Hamaker (1998) found that digestibility (using porcine pancreatic

α-amylase) of cooked isolated sorghum starches was markedly higher than starch

from cooked sorghum flours. They observed that pepsin pretreatment before cooking

increased the starch digestibility of sorghum flour by 7–14%, but no significant

increase in starch digestibility was seen when pepsin treatment was performed after

cooking. These authors also reported that after cooking with reducing agent, 100 mM

sodium metabisulfite, starch digestibility of sorghum flours increased significantly.

Elkhalifa et al. (1999) also observed that in vitro starch digestibility (IVSD) of the

treated gruel initially increased in the presence of cysteine, sodium metabisulphite or

ascorbic acid; however, at high levels of cysteine or sodium metabisulphite the IVSD

was low. Ezeogu et al. (2005) reported that starch digestibility (using pancreatic

porcine α-amylase) was significantly higher in floury sorghum endosperm than

vitreous endosperm and cooking with reducing agent, 2-mercaptoethanol, increased

starch digestibility in sorghum, and more with vitreous endosperm flours.

The fact that reducing agents improved sorghum starch digestibility suggests

that disulphide bond cross-linking within the kafirin-containing endosperm protein

matrix is responsible for the reduced gelatinisation in sorghum. This is the same

mechanism that has been implicated in the reduced protein digestibility of cooked

sorghum (Duodu et al., 2003). This interpretation is supported by the work of Ezeogu

et al. (2005) who found evidence of disulphide bond cross-linked prolamin proteins in

high proportion and extensive polymerization through disulphide bonding of

prolamins on cooking of sorghum through SDS-PAGE, with the formation of high

molecular weight polymers (M. Wt > 100k). Also formation of web-like or sheet-like

protein structures due to a disulphide-mediated protein polymerization process during

mashing or heat moisture treatment is reported by Hamaker and Bugusu (2003) and

Page 113: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

99

Wu et al. (2007). Ezeogu et al. (2005) also observed that pressure cooking the flours

improved starch digestibility of vitreous (hard) and floury (soft) endosperm maize and

sorghum flours and markedly so for sorghum vitreous endosperm flour. They

suggested that pressure cooking could have physically disrupted the protein matrix.

3.8.4. Tannin content in sorghum

Tannin is located in the testa portion of sorghum grain. Tannins confer

valuable agronomic properties on sorghum, including protection against insects, birds

and weather damage. However, tannins inactivate extracted malt amylases (Beta et

al., 2000a–c; Daiber, 1975), significantly reducing starch breakdown and sugar

production during brewing (Daiber, 1975). Tannins are well known for their adverse

effect on starch digestibility because of their ability to interact with proteins

(including hydrolytic enzymes), metal ions, and polysaccharides (Wu et al., 2007).

Wu et al. (2007) found that the liquefaction of starch in tannin sorghums was more

difficult and slower than in normal and waxy sorghums. Wu et al. (2007) also

confirmed that tannin contents had a strong adverse effect on conversion efficiency of

sorghum to ethanol. Taylor et al. (2006) have reviewed the use of tannin sorghum in

malting and brewing.

3.9. Production of ethanol from sorghum: Literature review

Demand of ethanol is consistently increasing as an alternative energy source.

Ethanol is normally manufactured from sugarcane molasses. This conventional

substrate is no longer cheap and good quality raw material due to its decontrol by

Indian government. Also in order to compete in international market, there is need to

improve the alcohol quality. Hence, since 1990s industries are diverting from use of

Page 114: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

100

molasses to starchy substrates for production of ethanol. Also, due to several

environmental issues associated with production of ethanol from molasses,

government is promoting grain based alcohol. In this section, literature review is only

limited to use of sorghum for ethanol production and not the use other starchy

substrates like corn, wheat, tapioca, rice etc.

Recently, Taylor et al. (2006) has reviewed literature on production of ethanol

from sorghum.

Suresh et al. (1999a) developed a simultaneous saccharification and

fermentation (SSF) system for producing ethanol from damaged sorghum (50% sound

and 50% damage grains). They have reported ethanol yields of 91.5% and 78.6% of

the theoretical ethanol yield with use of VSJ1 strain and standard strain MTCC 170

for damaged sorghum. These authors later utilized a similar SSF method to compare

ethanol production from damaged (50% sound and 50% damaged grains) and high

quality sorghum (Suresh et al., 1999b). It was must be noted that the latter method

involved no cooking step. Raw flour starch was saccharified by Bacillus subtilis

amylase and fermented by Saccharomyces cerevisiae. The damaged portion included

kernels that were broken, cracked, attacked by insects, dirty or discolored. The high-

quality sorghum flour was obtained locally. They found that using a level of 25 %

(w/v) substrate yielded 3.5% (v/v) ethanol from the damaged grain sample. For

comparison, the high-quality sorghum flour yielded 5.0% (v/v) ethanol. The values of

optimum pH and temperature were reported to be 5.8 and 35 °C respectively for SSF

process for damaged sorghum. The damaged grain sample was reported to be ten

times cheaper than high-quality grain and thus may be an economical way to produce

ethanol even though yields were lower. The authors further emphasized that

utilization of raw starch (i.e. without cooking) would save energy.

Page 115: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

101

Zhan et al. (2003) investigated the impact of genotype and growth

environment on the fermentation quality of sorghum. Eight sorghum hybrids grown in

two different locations were used to produce ethanol. The process included following

steps: heating with thermostable α-amylase at 95 °C and then 80 °C (liquefaction),

incubation with amyloglucosidase at 60 °C (saccharification), inoculation with S.

cerevisiae and fermentation for 72 h at 30 °C. It was found that ethanol

concentrations varied relatively narrowly (about 5%) across the 16 samples. Genotype

and production environment had a significant effect on chemical composition and

physical properties of the sorghum used in this study, which in turn significantly

affected ethanol yields. The correlation between ethanol concentration and starch

content was positive, as expected, but low (r = 0.35, P > 0.05), while a much more

distinct negative correlation between ethanol concentration and protein content was

found (r = -0.84, P < 0.001). Since protein and starch content are inversely

proportional, it is not surprising that opposite correlations for these two measures to

ethanol production would be found. But, protein content does not have a significant

effect on the percentage of the theoretical ethanol yield. However, it is interesting

that protein had a much stronger relationship to ethanol yield than did starch. More

research is needed to determine exactly what components in the grain, and their

interactions, are responsible for ethanol yields in sorghum. It is possible that during

the initial heating steps, a disulphide-mediated protein polymerization process

occurred, resulting in web-like or sheet-like protein structures, as described by

Hamaker and Bugusu (2003). Under these conditions, some of the starch might be

trapped in these protein webs, and its full gelatinisation and degradation by amylases

might be hampered. Evidence for this is provided by the work of Zhan et al. (2006)

who investigated cooking sorghum using supercritical-fluid-extrusion (SCFX) to

Page 116: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

102

gelatinize the starch. In SCFX supercritical carbon dioxide is used in place of water as

the blowing agent. Using SCFX increased ethanol yields by around 5% compared to

non-extrusion cooked sorghum. An improvement in the bioconversion of sorghum

starch was accounted to the release of starch from the protein matrix due to SCFX and

enhancing the availability of starch for conversion to fermentable sugar. Literature

review on ethanol production is summarized in Table 3.9 with reaction conditions and

remarks as parameters.

In addition to breeding sorghum specifically for fermentation quality, pre-

processing the grain can be used to improve ethanol yields and process efficiency.

Corredor et al. (2006) investigated decorticating sorghum prior to starch hydrolysis

and ethanol fermentation. In general, decortication decreased the protein content of

the samples up to 12% and increased starch content by 5–16%. Fiber content was

decreased by 49–89%. These changes allowed for a higher starch loading for ethanol

fermentation and resulted in increased ethanol production. Ethanol yields increased 3–

11% for 10% decorticated sorghum and 8–18% for 20% decorticated sorghum. Using

decorticated grain also increased the protein content of the distillers dried grains with

solubles (DDGS) by 11–39% and lowered their fiber content accordingly. Using

decorticated sorghum may be beneficial for ethanol plants as ethanol yield increases

and animal feed quality of the DDGS is improved. The bran removed before

fermentation could be used as a source of phytochemicals (Awika et al., 2005) or as a

source of kafirin and wax.

Page 117: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

103

Table 3.9. Summary of literature review on ethanol production from sorghum

Reference Reaction conditions Remarks

Suresh et al., 1999a

200 mL slurry autoclaved at 121 °C for 30 min and a 3% inoculum of A. niger (NCIM 1248) and 7% inoculum of yeast was added to it. 2-12% starch concn, 150 rpm, 30 °C, 5 days

Damaged sorghum (50% sound and 50% damaged grains used in this work)

Suresh et al., 1999b

100 ml slurry and 0.3% peptone 0.1% KH2PO4 and 0.1% (NH4)2SO4; pH 5.8. The crude amylase broth (10 ml) of B. subtilis VB2 and 6% S. cerevisiae VSJ4 suspension was added to slurry and incubated at 35 °C and 200 rpm for 4 days.

Optimized conditions being pH 5.8, 35 °C and 25% w/v slurry concn. High quality and damaged sorghum produced 5% v/v and 3.5% v/v ethanol production with no cooking step.

Zhan et al., 2003

16 different varieties of sorghum. Positive correlation between ethanol concn and starch content and negative correlation between ethanol concn and protein content.

Zhan et al., 2006

100 mL slurry, pH 5.8. Liquefaction: 95 °C for 45 min, 80 °C for 30 min (0.01 mL amylase/g of starch in both steps of liquefaction). Saccharification: 60 °C (150 U/g of starch) for 30 min. 50 rpm for all steps. Fermentation: 20 g ground sorghum, 0.3 g peptone, 0.1 g KH2PO4, and 0.1 g (NH4)2SO4 at pH 3.8. Medium was inoculated with 6% yeast suspension (1×106 cells/mL) and incubated 200 rpm for 72 h at 30 °C.

Extrusion could break disulphide protein bonds and disrupt the protein matrix, gelatinize starch, and make more starch available for enzyme hydrolysis, and consequently, increase ethanol yield and fermentation efficiency.

Wu et al., 2007

Ethanol yields varied by 22% and conversion efficiencies by 9% among 70 sorghum samples. Positive effect of starch content on fermentation efficiency and negative effect of protein, tannin, crude fiber, and ash content on fermentation efficiency was observed.

Zhao et al., 2008

30 g flour was mixed with 100 mL distilled water. 10 µL liquozyme was added and slurry was digested for 45 min at 95 °C. Slurry cooled to 80 °C and second dosage of 10 µL liquozyme was added and liquefaction was continued for additional 30 min at 120 rpm. Saccharification: 100 µL Spirizyme, 120 rpm, 60 °C, 30 min. pH adjusted to 4.2 and inoculated with 5 mL of 48 h yeast pre-culture.

During mashing cross-linked microstructure, which could hold starch granules or polysaccharides inside or retard or prevent the access of enzymes to starch get formed. Severe cross-linking in mashed sample was most likely because of a combination of heat-induced cross linking and cross-linking because of protein-tannin interactions. Tight and open microstructures were observed with low conversion sorghum and high conversion sorghum, respectively.

Page 118: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

104

Wu et al. (2007) have performed ethanol production from 70 genotypes and

elite hybrids of sorghum using dry grind process and identified factors impacting

ethanol production. They have observed variation in the ethanol yield by 22% and

conversion efficiencies by 9% among 70 sorghum samples, indicating significant

effect of sorghum genotype on fermentation efficiency to ethanol. They reported

positive effect of starch content on fermentation efficiency and negative effect of

protein, tannin, crude fiber, and ash content on fermentation efficiency. Protein

digestibility of waxy, normal sorghum (60-68 %) were higher that that for high tannin

sorghum (28%); Higher fermentation efficiencies were observed for waxy, normal

sorghum (89-90%) than those of high tannin sorghum (85%). After mashing sorghum

protein were appeared to produce highly extended, strong web like micro structures

(in accordance with results of Hamaker et al., 2003) into which small starch granules

were tightly trapped. These changes related protein structure during mashing could

contribute to incomplete gelatinization and hence hydrolysis of starch and conversion

efficiency to ethanol. DSC thermograms of waxy sorghum starch consists of single

endothermic peak (60-80 °C), whereas that of normal sorghum starch showed

presence of two endothermic peaks; one, in 60-80 °C corresponds to amylopectin, and

second, in 85-105 °C corresponds to amylose-lipid complex. Waxy sorghum gives

higher conversion efficiencies to ethanol than normal sorghum; this mainly happens

due to presence of amylose-lipid complex. Wu et al. (2007) concluded that major

factors adversely affecting conversion efficiency to ethanol being condensed tannin,

high viscosity, low protein digestibility, protein-starch interactions, and amount of

amylose-lipid complex.

Zhao et al. (2008) have characterized the changes in sorghum protein in

digestibility, solubility, and microstructure during mashing and to relate those changes

Page 119: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

105

to ethanol fermentation quality of sorghum by using 9 sorghum cultivars (2 contained

tannin and rest were tannin free. They have observed that protein solubility and in

vitro protein digestibility decreased significantly after mashing. Tendency of

sorghum proteins to form highly extended, strong web like microstructures during

mashing was confirmed using CFLSM (confocal laser-scanning microscopy) images.

Formation of web like structure was earlier reported by Hamaker and Bugusu (2003)

and Wu et al. (2007). They reported that the cultivar with the lowest conversion

efficiency formed a tightly cross-linked microstructure, which could hold starch

granules or polysaccharides inside or retard or prevent the access of enzymes to

starch, and severe cross-linking in this sample was most likely because of a

combination of heat-induced cross linking and cross-linking because of protein-tannin

interactions. More open web-like microstructures were observed in cultivars with

higher conversion efficiencies upon mashing. Protein digestibility of the unmashed

sorghum, Solubility and the SE-HPLC area of proteins extracted from mashed

samples were highly correlated with ethanol fermentation. Since protein cross linking

plays a significant role in the fermentation, it was expected that γ-kafirin (%) would

relate significantly with conversion efficiency. But, it neither correlated to ethanol

yield nor conversion efficiency significantly. They concluded that protein cross-

linking does play a role in the production of ethanol from sorghum, albeit through

indirect measures of protein cross-linking (i.e. reduction in protein digestibility after

mashing, which is due to protein cross linking).

3.10. Production of starch from sorghum: Literature review

In general the wet milling of sorghum is similar to that of maize. A thorough

review of early research on wet milling of sorghum can be found in Munck (1995).

Page 120: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

106

Recently, Taylor et al. (2006) have shortly reviewed literature on starch production

from sorghum. However, a problem particular to sorghum is that where polyphenolic

pigments are present in the pericarp and/or glumes, they stain the starch (Beta et al.,

2000a–c).

In recent years several new developments in sorghum wet milling have been

reported. Perez-Sora and Lares-Amaiz (2004) investigated alkaline reagents for

bleaching the starch and found a mixture of sodium hypochlorite and potassium

hydroxide to be the most effective. To improve the economics of sorghum wet-

milling Yang and Seib (1995) developed an abbreviated wet-milling process for

sorghum that required only 1.2 parts fresh water per part of grain and that produced

no waste water. The products of this abbreviated process were isolated starch and a

high moisture fraction that was diverted to animal feed.

Buffo et al. (1997) investigated the impact of sulphur dioxide and lactic acid

steeping on the wet-milling properties of sorghum and reported that the amount of

lactic acid used during steeping had the most impacted wet-milling quality

characteristics such as starch yield and recovery. These authors also investigated the

relationships between sorghum grain quality characteristics and wet-milling

performance in 24 commercial sorghum hybrids (Buffo et al., 1998). Perhaps not

surprisingly, they found that grain factors related to the endosperm protein matrix and

its breakdown and subsequent release of starch granules as important factors in wet-

milling of sorghum. Related to this, Mezo-Villanueva and Serna-Saldivar (2004)

found that treatment of steeped sorghum and maize with protease increased starch

yield, with the effect being greater on sorghum than maize.

Wang et al. (2000) optimized the steeping process for wet-milling sorghum

and reported the optimum steeping process to utilize 0.2% sulphur dioxide, 0.5%

Page 121: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 3: Sorghum: Literature Review

Studies in the Enzymatic depolymerisation of natural polysaccharides

107

lactic acid at a temperature of 50 °C for 36 h. Using this steeping process, wet-milling

of sorghum produced starch with an lightness value (Black and white samples will

have lightness value of 0 and 100 respectively. Lightness value is measured by

chroma meter and normally denoted as L) of 92.7, starch yield of 60.2% (db), and

protein in starch of only 0.49% (db). Beta et al. (2000a–c) found that both polyphenol

content and sorghum grain properties influence sorghum starch properties.

Using sorghum grits as the starting material for wet-milling rather than whole

sorghum produced lower yields, but the isolated starch was higher in quality.

Sorghum starch matching the quality of a commercial corn starch was successfully

produced by wet-milling sorghum grits (Higiro et al., 2003).

Xie and Seib (2002) developed a limited wet-milling procedure for sorghum

that involved grinding sorghum with in the presence of 0.3% sodium bisulphite

solution. This procedure produced starch with an L value of 93.7 and a starch

recovery of 78%. Large grain sorghum hybrids wet-milled by this ‘‘no steep’’

procedure were reported to produce high-quality starches with L values from 93.1 to

93.7 (compared to 95.2 for a commercial corn starch). Some of the large grain hybrids

tested showed promise for easy recovery of the germ by flotation in a similar fashion

as is done for maize (Xie et al., 2006).

Park et al. (2006) reported the use of ultrasound to rapidly purify starch from

sorghum. This procedure resulted in very high-purity starch with only 0.06% residual

protein in the starch. New developments in wet-milling procedures for sorghum as

well as breeding sorghum hybrids with improved wet milling characteristics should be

of benefit for the industrial use of sorghum starch, either directly for the production of

bioethanol or other industrial uses such as the production of activated carbon (Diao et

al., 2002) or isolation of phytosterols from wet-milled fractions (Singh et al., 2003).

Page 122: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

108

4. Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Page 123: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

109

4.1. Introduction and Literature review

Most green plants produce starch as a means of energy storage. Starch is a

polymer composed of glucose units linked by α(1→4) glucosidic bonds and α(1→6)

linkages. It consists of two types of polymers: amylose and amylopectin. Amylose is

roughly linear molecule containing ~ 99% α(1→4) and ~ 1% α(1→6) bonds with

molecular weight of 65 101101 ×−× . Amylopectin is a much larger molecule

(molecular weight of 97 101101 ×−× ) and is a branched polymer with ~ 95% α(1→4)

and ~ 5% α(1→6) bonds. Linear chains of 12-120 glucose units (linked by α(1→4)

glucosidic bonds) are connected by α(1→6) glucosidic linkages.

Starch has become a very important biopolymer and is used in many industries

as a feedstock material. In several industrial processes, enzymes are used to

transform starch to useful and value added biochemicals. Sweetener and fermentation

industries are two of the main consumers of the starch. Nutritive sweeteners are

mainly starch hydrolysis products namely maltodextrins, high maltose syrup, maltose,

glucose syrup, dextrose, which are used in food and pharmaceutical industry.

Complete starch hydrolysis results into glucose as a final product. The first

commercial maltodextrin was Frodex 15 (later called Lo-Dex 15), introduced by

American Maize Products Company in 1959 (Alexander, 1992).

Starch hydrolysis products are commonly characterized by their degree of

hydrolysis, expressed as the dextrose equivalent (DE), is the percentage of reducing

sugar calculated as the dextrose on dry weight basis. Theoretical DE is defined as the

percentage of reducing sugar (glucose equivalent) to total reducing sugar (glucose

equivalent) produced after complete hydrolysis. Pure starch has DE of zero and

dextrose has DE of 100.

The term maltodextrins is used for the saccharide mixtures of dextrose

Page 124: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

110

equivalent (DE) less than 20, which consist of maltose, malto-oligosaccharides and

linear or branched dextrins (Alexander, 1992). Since maltodextrins are products of

partial hydrolysis of natural starch, their saccharide composition varies with nature

and concentration of enzyme/s, extent of hydrolysis and botanical origin of starch

used for hydrolysis. Maltodextrins with the same average DE value can have different

saccharide composition (Kennedy and Cabral, 1987). DE value of a maltodextrin has

been shown to be inadequate to predict product performance in various applications

(Chronakis, 1988). The saccharide composition of maltodextrins determines it’s both

physical and biological functionality, and there are different parameters (like type and

source of enzyme, source of starch, starch concn, temperature, organic solvents,

immobilization of enzymes, downstream processing etc.) influencing the saccharide

composition of maltodextrins (Marchal et al., 1999). Design of the desired saccharide

composition and production possibilities for maltodextrins were briefly discussed

(Marchal et al., 1999). Aspects related to the saccharide composition include

hygroscopicity, gelation, sweetness, stability, fermentability in food products,

osmolality, and absorption of maltodextrins by humans (Marchal et al., 1999).

Maltodextrins find applications in various industries like confectionary industry,

Beverage industries, papermaking industry etc. Maltodextrins are also used as carrier

or bulk agents, texture provider, spray-drying aid, flavor encapsulating aid, fat

replacer, tablet expicient, film former, freeze-control agent, sport beverage, to prevent

crystallization and to supply nutritional value i.e. parenteral and enteral nutrition

products. (Alexander, 1992; Marchal et al., 1999) All these aspects of Maltodextrin

are discussed in detail in the chapter 2.

Though partial hydrolysis of starch has traditionally been carried out using

acids, acid hydrolysis is being replaced by enzymatic hydrolysis for the production of

Page 125: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

111

tailor-made maltodextrins (Schenck and Hebeda, 1992; Marchal et al., 1999). The

most widely used enzymes for production of maltodextrins using partial hydrolysis of

starch are α-amylase from B. licheniformis, B. stearothermophilus and B.

amyloliquefaciens. Production of maltodextrin from starch is reviewed in detail in the

chapter 2.

Soluble enzymes are widely used for various industrial applications. However,

these enzymes can be used only once since they can’t be separated from the reaction

mixture. Further, most often the presence of enzyme in the final product is

undesirable. In such cases soluble enzymes must be deactivated or killed, and many

times is to be separated from the product at the end of the process. This is generally

carried out by pH adjustment, using either an acid or a base, which eventually leads to

effluent problems. These problems can be overcome by using ‘insoluble’ enzymes,

which can be separated easily from the reaction mixture after the reaction is over.

4.1.1. Immobilized enzymes

The process of making enzymes insoluble through their localization on some

or the other kind of solid surface is called immobilization. Immobilized enzymes are

defined as “enzymes which are physically confined or localized in a certain defined

region of space with retention of their catalytic activities, and which can be used

repeatedly and continuously” (Chibata, 1978). This can be achieved by using a solid

support on to which the soluble enzyme is ‘confined’ and thus is separated from the

bulk phase containing substrate and eventually the product.

Advantages of an immobilized enzyme over the soluble enzyme are:

1. Reusability of the enzyme

2. Continuous operation of the system

3. Easy separation of product from the enzyme

Page 126: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

112

4. Less effluent problems

5. Increased stability and activity of the enzyme in some cases

Activity of an enzyme stems from its tertiary structure and surface topology.

Enzyme as a protein has specific structure and any changes in it can affect the

bioactivity of the enzyme. Thus its interaction with any surface or molecule can play

an important part in determining the enzyme activity. By the same rule, surface

properties of the solid matrix on to which the enzyme is immobilized plays an

important role. Ideal solid support should have the following properties:

1. Large surface area to achieve higher immobilization yields

2. Hydrophilic character to avoid denaturation of enzyme

3. High rigidity to withstand high pressure drop in packed bed columns

4. Chemical, mechanical and thermal stability

5. Resistance to microbial attack

6. Permeability to allow easy diffusion of substrates and products in and out of

the matrix pores

Morphologically, matrices can be classified as a) Porous and b) Non-porous.

a) Non-porous matrices have low surface area therefore immobilization yields are

low. In order to increase the enzyme loading fine particles could be used. However,

it is difficult to separate these particles from the reaction mixture. Small particles

also lead to high pressure drops when used in packed bed modes.

b) Porous supports on the other hand have large surface area for enzyme coupling.

However, the porous support must allow easy accessibility in the pores to substrate

and product molecules and should minimize internal diffusional resistances.

Various chemistries can be used for the immobilization of enzymes on solid

supports. These techniques can be divided into three major classes (Kennedy and

Page 127: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

113

Cabral, 1987):

1. Support binding method

1.1. Physical adsorption

1.2. Ionic binding

1.3. Metal linking method using inorganic supports

1.4. Covalent binding

2. Entrapment method

2.1. Gel entrapment

2.2. Microencapsulation

3. Cross-linking method

4.1.2. Immobilization of bacterial α-amylase

Ivanova and Dobreva (1994) studied the hydrolysis of starch, maltopentaose

and maltohexaose using soluble and immobilized alpha amylase. They found that the

hydrolysis product profiles, obtained using soluble and immobilized enzyme differed

significantly. When soluble enzyme was used for starch hydrolysis, considerable

amounts of low molecular weight saccharides, identified as glucose, maltose and

malto-oligosaccharides up to maltopentaose were produced. Immobilized alpha

amylase on the other hand produced higher quantity of malto-oligosaccharides than

the soluble enzyme. Soluble enzyme could partially hydrolyze maltopentaose into

maltotriose, maltose and glucose, whereas immobilized alpha amylase could not

hydrolyze maltopentaose. Soluble enzyme hydrolyzed maltohexaose completely,

while immobilized alpha amylase exhibited an increase in the production of glucose

from maltohexaose and maltopentaose was obtained as the major hydrolysis product.

Reasons for a different saccharide composition with immobilized enzyme compared

to free enzyme can be attributed to diffusion limitation, which increases with the

degree of polymerization (DP) of oligosaccharide, enhancing the heterogeneous

hydrolysis (Tarhan, 1989). The other reason is that, immobilization alters three

Page 128: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

114

dimensional structure of the enzyme; which causes changes in its affinity towards the

substrates, thus increasing its product specificity (Ivanova and Dobreva, 1994).

There are several attempts (Kvesitadze and Dvali, 1982; Tarhan, 1989; Roig et

al., 1993; Ivanova and Dobreva, 1994; Tumturk et al., 2000; Lali et al., 2002;

Karandikar, 2004) to immobilize bacterial α-amylase and application of it to

hydrolyze starch. Small size pores (< 0.1 µm) in typical matrix supports offer high

diffusional resistances to the large starch molecules and limits their accessibility to

active immobilized enzyme sites inside the pores leading to low reaction rates.

However these diffusional resistances can be overcame or significantly reduced by

use of matrix support with large pores diameters (70-80 nm for Kvesitadze and Dvali,

1982; 7-550 nm for Siso et al., 1990; ∼5000 nm for Lali et al., 2002 and Karandikar,

2004) for immobilization of enzymes. Hence in the present work, Bacillus

licheniformis α-amylase (BLA) was immobilized on superporous (pore diameter ∼ 3

µm) CELBEADS, in order to minimize diffusional resistances.

It is reported (Marchal et al., 1999) that there is no significant influence of pH

(in the range of 5.1 and 7.6) and significant influence of temperature on saccharide

profile of starch hydrolysate produced using free BLA (Maxamyl). But there is no

literature available on the effect of pH, temperature and initial starch concn on the

saccharide composition of starch hydrolysate produced using immobilized bacterial

amylase.

4.1.3. Objectives

Hence, in the present work, the effect of different parameters like pH,

temperature, initial starch concn and ratio of concn of enzyme units to initial starch

concn on the saccharide profile of starch hydrolysate produced using immobilized

BLA has been also studied in a batch mode. Thermostability and reusability of

Page 129: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

115

immobilized BLA was also studied. Also, a semiempirical model has been used for a

priori prediction of saccharide composition of starch hydrolysate with respect to time.

Also, in this work the possibility of production of glucose using gelatinized sorghum

slurry has been explored.

4.2. Experimental

4.2.1. Materials

3,5-Dinitrosalicylic acid (DNSA), soluble starch, maltose, dextrose, MeCN for

chromatography LiChrosolv and other chemicals were purchased from Merck Ltd

(India). Bacillus licheniformis α-amylase (BLA) (EC number 3.2.1.1) was gifted by

Advance Enzyme Technologies Pvt Ltd (India).

CELBEADS, a rigid superporous cross-linked cellulose matrix, were prepared

indigenously according to patent (Lali and Manudhane, 2003) and made available for

the present work. Properties of the CELBEADS are given in the Table 4.1.

Table 4.1. Properties of CELBEADS (Lali and Manudhane, 2003)

Properties Description

Mean Bead size 200 µm (100-350 µm)

Spherecity 0.7-0.9

Nature Rigid aerogel

Average pore size ~ 3 µm

Total volume porosity ~ 57%

Bulk density (water) 1438 kg/m3

Page 130: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

116

4.2.2. Methods

4.2.2.1. Measurement of protein and reducing sugar concentration.

The protein concn of the free enzyme was determined using modified Folin

Lowry method (Lowry et al., 1951) using BSA (0-0.6 mg/mL) as a standard. The

reducing sugar concn was measured using DNSA method (Miller, 1959) using

dextrose (0-1 mg/mL) as a standard. Details of modified Folin Lowry method and

DNSA method are provided in the Appendix A.

Theoretical dextrose equivalent (DE) of the starch hydrolysate is defined as

following,

10018)n(162 ehydrolysatstarch of wt mol averagednumber

180.6 i.e. glucose anhyd of wt molDE ×+×

= (4.1)

where n is the average degree of polymerization (DP) of starch hydrolysate, which

can be calculated by following formula;

ehydrolysatstarch in equiv) (glucosesugar reducing ofconcn sidaseamylogluco using hydrolysis completeafter equiv) (glucosesugar reducing ofconcn n =

By using above formula, DE of dextrose, maltose and starch can be calculated to be

100, 53 and 0 respectively. The values of DE reported later in the text are those

calculated using Eq. 4.1.

4.2.2.2. HPTLC analysis.

Oligosaccharide separation of starch hydrolysate samples was performed using

20 cm × 10 cm TLC sheets (silica gel 60, Merck Ltd, India). The samples were

applied to the TLC sheet (prewashed with MeOH) using applicator AS 30 (DESAGA,

Heidelberg, Germany), equipped with a 10 µL microsyringe (Hamilton, Switzerland).

Best resolution was obtained by triple development. Mobile phase MeCN: 0.02 M

Na2HPO4 of composition 70:30 (v/v) was used for 1st and 2nd development; whereas

Page 131: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

117

mobile phase MeCN: 0.01 M Na2HPO4 of composition 80:20 (v/v) was used for 3rd

development. TLC sheet, after triple development, was stained by dipping for 4

seconds in the diphenylamine-aniline-phosphoric acid reagent (40 mL acetone, 0.8 g

diphenylamine, 0.8 mL aniline and 6 mL 85% H3PO4) and then keeping at 120 °C for

10 min. Densitometry was performed using HPTLC densitometer CD 60 (DESAGA,

Heidelberg, Germany) and computer. Sample densitogram is shown in Fig. 4.1 (A)

and TLC image is shown in the Fig. 4.1. (B) Samples of starch hydrolysate were

quantified by use of external standards of Glucose (G1) and Maltose (G2) from Merck

India Ltd and Maltotriose (G3), Maltotetraose (G4), Maltopentaose (G5),

Maltohexaose (G6) and Maltoheptaose (G7) from Sigma (St Louis, MO, USA).

Concentrations of malto-octaose (G8), maltononaose (G9) and maltodecaose (G10) in

starch hydrolysate reported later in the text are those, which were determined by using

standard curve of G7. Details of HPTLC method are given in the appendix A.

15 40 50 70 80 mm

G1

G2

G3

G4

G5

G6

G7G8G9G10

Figure 4.1. A. Densitogram of starch hydrolysate of DE 10, produced using immobilized BLA at 55 °C, pH 5.2 and [S]0 = 90 mg/mL. G1 is Glucose and G2-G10 are maltooligosaccharides with degree of polymerization 2-10 respectively. B. TLC image showing chromatographic separation of bands corresponding to glucose and malto-oligosaccharides

AB

Page 132: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

118

4.2.2.3. Immobilization of B. licheniformis α-amylase (BLA) on CELBEADS.

Surface hydroxyl groups of CELBEADS were activated using epichlorohydrin

(ECH), while ethylenediamine (EDA) was used as a spacer arm to prevent the

possible steric hindrances between the immobilized enzyme and the substrate.

Activation and coupling procedures (Lali et al., 2002; Hermanson et al., 1992) were

followed for immobilization. Chemistry of the immobilization is shown in Fig. 4.2.

CELBEADS (10 mL) were washed with 200 mL of distilled water and suction dried

to moist cake on a sintered glass funnel. The wet matrix was then added to a conical

flask containing 2 M NaOH (34.5 mL), NaBH4 (0.1275 g) and ECH (3.75 mL). To

this flask another 2 M NaOH (34.5 mL) and ECH (17 mL) were added in small

portions over a period of 2 h under mild stirring. The flask mixture was shaken on an

orbital shaker overnight at room temperature. The matrix was then filtered on a

sintered glass funnel and washed extensively with 200 mL each of 0.1 M HOAc, 0.2

M NaHCO3 and distilled water sequentially. The washed and suction dried epoxy-

activated matrix (ECH-CELBEADS) was then added to a flask containing a mixture

of 0.2 M NaHCO3 (22.5 mL) and EDA (15 mL). The mixture was shaken at 50 °C on

an orbital shaker for 24 h. The resulting matrix (ECH-EDA-CELBEADS) was filtered

and washed successively with 200 mL each of 0.1 M HOAc, 0.2 M NaHCO3 and the

distilled water.

Page 133: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

119

CH2 CH CH2

OO H2N CH2 CH2 NH2

CHOH

CH CH NH2O CH2N CH2

CHOH

CH CH CH2 NH2O CH2HOC CHO(CH2)3 NaBH4

CHOH

NH CH CH2O CH2 NH (CH2)3 CHO

N

CH2

H2N Enzyme NaBH4CH

OH

NH CH2 CH2O CH2 NH (CH2)3 CHOCH2

CH

OH

NH CH2 CH2O CH2 NH (CH2)3 CH2CH2 NH Enzyme

CH2 CH CH2

OCl CH2 CH CH2

OOOH

+

EP-CELBEADS EP-EDA-CELBEADSEDA

+ +

EP-EDA-CELBEADS GA Sodium borohydride

EP-EDA-GA-CELBEADS

EP-EDA-GA-CELBEADS

+

Enzyme

+

Sodium borohydride

Immobilized enzyme

CELBEADS EP EP-CELBEADS

Figure 4.2. Chemistry of immobilization of enzyme on CELBEADS

The matrix (ECH-EDA-CELBEADS) was then further activated overnight for

enzyme conjugation using 30 mL of 12.5% w/v aqueous glutaraldehyde (GA). The

activated matrix (ECH-EDA-GA-CELBEADS) was washed well with distilled water

to remove traces of glutaraldehyde. Activated matrix was mixed with 20 mL of 100

fold diluted solution (15 mg protein) of BLA in 0.1 M phosphate buffer (pH 7.5) and

kept under shaking condition overnight at 5 °C. After immobilization, NaBH4 (0.08

Page 134: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

120

g) was added and kept under shaking condition for further 30 min. The enzyme

immobilized matrix was filtered on a sintered glass funnel and then washed with 200

mL each of phosphate buffer, 1 M NaCl solution and distilled water sequentially. The

protein concn and free enzyme units in the supernatants and washes were determined.

Amount of protein and free enzyme units immobilized on the matrix were calculated

by material balance.

4.2.2.4. Amylolytic activity measurement.

4.2.2.4. A. Free BLA.

Soluble starch was added to 0.1 M acetate buffer (pH 5.6) to have 9 mg/mL

concn and then gelatinized in a stoppered conical flask by heating in boiling water for

6 min. Mixture of 0.5 mL of the gelatinized starch solution (9 mg/mL), 1.4 mL of

acetate buffer (0.1 M, pH 5.6) and 0.1 mL of 10000 fold diluted free BLA was

incubated at 55 °C (optimum enzyme activity temperature; found separately) in a

water bath for 20 min. The reaction was stopped by adding 1 mL of DNSA reagent.

The variation in the concn of reducing sugar was measured by DNSA method using

dextrose as a standard. One free enzyme unit (FEU) was defined as that required to

liberate one micromole of reducing sugar (glucose equiv) per min under conditions of

assay. Activity of commercial preparation of BLA (FEU/mL) was calculated using the

following formula,

min 20 mL) 0.1 (i.e.aliquot enzyme of volume (180.6) glucose of wt.mol.10000) (i.e.aliquot enzyme offactor dilution

g/mL)( equiv.) (Glucose sugars Reducing ofion Concentrat

FEU/mL××

×=

µ

4.2.2.4. B. Immobilized BLA.

End point assay method has been employed to calculate enzyme activity of

Page 135: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

121

immobilized bacterial α-amylase. Gelatinized starch solution (25 mL) of concn 90

mg/mL (0.1 M acetate buffer, pH 5.2) was incubated with 1 mL immobilized BLA at

55 °C for one h and samples were withdrawn initially and finally. Reducing sugar

concn (glucose equiv) of the samples was measured and the immobilized enzyme

units per mL of CELBEADS were determined. One immobilized enzyme unit (IEU)

was defined as that required to liberate one micromole of reducing sugar (glucose

equiv) per min under the conditions of assay. Enzyme units per ml of CELBEADS

can be calculated from the following formula,

min 60 (180.6) glucose of wt.mol.ml 25

g/ml)(hr 1in produced )equivalent (glucosesugar Reducing

CELBEADS of EU/ml×

×=

µ

Retained enzyme activity is defined as the ratio of sp activity of immobilized

enzyme to sp activity of free enzyme (calculated by assay procedure of immobilized

BLA).

4.2.2.5. Measurement of kinetic rate constants of free and immobilized BLA.

Gelatinized starch solutions (25 mL each) prepared in 0.1 M acetate buffer

(pH 5.2 for immobilized BLA and pH 5.6 for free BLA) of different initial starch

concentrations [S]0 varying in the range of 9-45 mg/mL with suitable enzyme concn

(for immobilized BLA, [IEU] = 0.5 and for free BLA, [FEU] = 0.66 i.e. [IEU equiv] =

0.738) were incubated separately at 55 °C for 2 h. The reaction was carried out in a

shaker at 180 rpm. Samples of starch hydrolysate were withdrawn at regular time

intervals of 0.5 h and analyzed for reducing sugar concn. The initial reaction rate (V)

was calculated from the slope of linear part of reducing sugar concn vs. time plot at

all initial starch concentrations for both immobilized and free BLA. Kinetic constants

Page 136: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

122

(Km and Vmax) were determined for free and immobilized BLA from Eadie-Hofstee

plot of V vs. V/[S]0 with -Km as slope and Vmax as the y-intercept. Since Vmax is not a

fundamental property of the enzyme and is dependent on the enzyme concn, it was

converted to turnover number, kcat (i.e. Vmax/[IEU] for immobilized BLA and

Vmax/[IEU equiv] for free BLA).

4.2.2.6. Hydrolysis of soluble starch using immobilized BLA in batch mode.

Suspension of soluble starch at desired concn (mg/mL) was prepared with 0.1

M acetate buffer (desired pH) and then gelatinized in a stoppered conical flask by

heating in boiling water for 6 min. Immobilized BLA was added to the freshly

prepared gelatinized starch solution to have a desired [IEU]/[S]0 and kept at desired

temperature for 8 h in the shaker at 180 rpm. Rotational speed of 180 rpm was

selected by using the following criterion: 1. All beads should be always in the

suspended form throughout the batch. 2. Selected speed should be in such a range; in

which there is no dependence of hydrolysis curve on the rotational speed (which is

observed to be beyond the speed of 150 rpm). At regular time intervals (0.5 h up to

reaction time of 3 h and then every 1 h till the end of batch hydrolysis), samples were

withdrawn and diluted to concn of 9 mg/mL to avoid retrogradation. Samples were

then analyzed for reducing sugar concn and immediately frozen. Samples were

thawed and saccharide composition of samples was determined by HPTLC. Effect of

reaction conditions on hydrolysis and oligosaccharide composition was studied by

varying pH, temperature, [S]0 and [IEU] in the range of 4.4 to 7, 37 to 70 °C, 18 to

180 mg/mL and 0.2964 to 1.86 respectively.

Page 137: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

123

4.2.2.7. Thermostability and reusability of immobilized BLA.

Immobilized BLA (5mL) was resuspended in 50 mL of 0.1 M acetate buffer

solution (pH 5.2) without the presence of soluble starch and kept under shaking

conditions (180 rpm) at 55 °C for 24 h. Similarly free BLA was diluted 10000 fold

with 0.1 M acetate buffer solution (pH 5.6) and kept under shaking conditions (180

rpm) at 55 °C without the presence of starch for 24 h. Samples of immobilized BLA

and free BLA were taken at various time intervals for the measurement of its activity.

Procedure to carry out reaction for reusability study was same as that

described in section 4.2.2.6. At end of 8 h, reaction mixture was separated from

immobilized BLA. Then immobilized BLA was first washed thoroughly with

distilled water and acetate buffer solution sequentially, and then kept under shaking

condition with acetate buffer (amount same as the reaction mixture, pH 5.2) at 55 °C

for 30 min to remove any substrate or product molecules, which could have been

trapped inside the pores. Then acetate buffer was again separated from immobilized

BLA. Fresh acetate buffer solution was added to the immobilized BLA and kept at 6

°C, and the same was used for next batch hydrolysis under same conditions on the

next day. For each batch, samples were collected at regular time intervals and

analyzed for reducing sugar concentration to see the progress of the reaction.

4.2.2.8. Hydrolysis of soluble starch using immobilized BLA in packed bed or

expanded bed mode.

Hydrolysis of gelatinized starch solution was carried out with immobilized

BLA (2 mL) packed in a 10 mm diameter and 20 cm long jacketed glass column

equipped at the two ends with adjustable 14 cm long flow adapters. The adapters were

inserted to touch the matrix bed from both the sides. The lower adapter was connected

Page 138: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

124

to a peristaltic pump that pumped the substrate solution from the mixing tank through

the column at a desired flow rate and re-circulated it back to the mixing tank. 40 mL

starch suspension was prepared in 0.1 M acetate buffer (pH 5.2). Temperature of the

starch solution was maintained at 55°C by circulating hot water through jacket around

it (Fig. 4.3). The starch solution was continuously stirred and kept in the form of

uniform solution using a magnetic stirrer. Temperature of the column was also

maintained at 55°C by circulating hot water through the jacket. Samples were drawn

at regular time intervals from the mixing tank to monitor the progress of starch

hydrolysis by analyzing the same for reducing sugar.

Figure 4.3. Experimental set-up for hydrolysis of starch using immobilized α-amylase in packed bed mode. A Water bath cum circulator B Peristaltic pump C Magnetic stirrer D Jacketed mixing tank E Packed bed of immobilized α-amylase on CELBEADS F Jacketed column

Expanded bed experiments were carried out in much the same way except that

the upper adapter was placed well above the settled matrix instead of touching the

matrix. This provided free board for the settled immobilized CELBEADS to expand

AB

C

DE

F

Page 139: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

125

when starch solution was passed up through the column at a flow rate of 1 mL/min. It

was seen that this flow rate expanded the bed about 1.4 times from the settled bed

height. Rest of the experiment was same as that for packed bed experiment. Samples

were drawn at regular time intervals and analyzed for reducing sugar concentration.

Reusability of immobilized BLA was also studied in the batch mode with and

without intermittent washing step, and in the packed and expanded mode without

intermittent washing steps by employing procedure described in the section 4.2.2.7.

4.2.2.9. Measurement of residence time in the packed bed.

5mL of de-aerated underivatized CELBEADS were packed in a 10 mm

diameter and 20 cm long glass column. A pulse of 2 mL of 10% (w/v) gelatinized

starch solution was injected in the distilled water flowing through the column at

desired flow rate (1, 2 or 3 mL/min). Eluting fractions were collected at every 15 sec

in test tubes and analyzed for starch concentration using the starch-iodine method

(described in Appendix A). The Concentration of starch in the eluting fractions

collected (C) was plotted against time (t). The value of mean residence time tm was

calculated by the formula (Fogler, 2005): )(/)( tCttCtm ∆Σ∆Σ= .

4.3. Results and Discussion

4.3.1. Immobilization of bacterial α-amylase on CELBEADS.

BLA was immobilized onto CELBEADS by covalent binding as described

earlier. It was observed that by material balance, 90% of the loaded FEUs (i.e. 300

FEUs per mL of CELBEADS) and 56% of the proteins loaded (i.e. 0.83 mg per mL of

CELBEADS) got immobilized.

The number of enzyme units immobilized per ml of CELBEADS can be

determined from material balance as mentioned earlier. But when an enzyme is

Page 140: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

126

immobilized on matrix, activity of enzyme may decrease, or change due to several

reasons;

1. Some immobilized enzyme molecules may be immobilized in such a way that

the active site could be oriented towards the support surface i.e. decreasing the

accessibility of substrate molecule to the enzyme.

2. A reactive site in the enzyme molecule may be involved in the binding to the

matrix.

3. The enzyme molecules on binding may be held in an inactive configuration.

4. The reaction conditions used for immobilization may cause denaturation or

inactivation of the enzyme.

Hence it becomes necessary to determine activity of immobilized enzyme

separately (reported in Table 4.2) in addition to FEUs immobilized per mL of

CELBEADS from material balance.

4.3.2. pH and temperature dependence of activity of free and immobilized BLA

and their catalytic properties.

After immobilization, pH-enzyme activity profile shifted towards acidic side

and optimum pH slightly decreased from 5.6 to 5.2 (Fig. 4.4). Shift in the optimum

pH towards acidic side (Kvesitadze and Dvali, 1982; Tumturk et al., 2000; Ivanova et

al., 1998) might be because of the difference in the hydronium ion concn in the bulk

solution and the microenvironment in the vicinity of immobilized enzyme molecule

(Tumturk et al., 2000). Fig. 4.5 shows that % relative activity above 55 °C was

marginally better and approximately the same over the temperature range of 55 to 70

°C after the immobilization, indicating that optimum temperature changes from 55 °C

to 55-70 °C (Fig. 4.5) upon immobilization. Fig. 4.5 also shows that % relative

Page 141: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

127

activity at temperature less than 55 °C was reduced after immobilization. This could

be because, at low temperature, starch solution is more viscous and hence the

diffusional resistance for the migration of the starch molecules through the

macropores is likely to be more at low temperature (<55 °C) as compared to higher

temperature. The increase (Tumturk et al., 2000) or no change (Marchal et al., 1999;

Ivanova et al., 1998) in the optimum temperature may be because of the improvement

in the enzyme rigidity upon immobilization by covalent binding.

Properties of free and immobilized BLA are summarized in the Table 4.2.

Activation energy (Ea) of immobilized BLA (3.18) was higher than that of free BLA

(1.63). Similar increase in the Ea after immobilization is reported and attributed to the

change in enzyme structure upon immobilization (Ivanova et al., 1998). appmK of

immobilized BLA (15 mg/mL) was 4.5 times of the freemK (3.3 mg/mL). Higher value

of Km for immobilized BLA indicates less affinity between immobilized BLA and

substrate molecules, which could be because of either similar nature of the charges

carried by the support and the substrate or structural changes in the enzyme occurring

upon immobilization or lower accessibility of substrate to the active enzyme site of

the immobilized BLA due to steric hindrances and still persisting diffusional

limitation. The apparent value of Km is reported to increase up to 2.6 times and 9

times for α-amylase (from porcine pancreas) immobilized on HEMA and styrene-

HEMA microspheres (Tumturk et al., 2000) respectively; up to 10 times for α-

amylase (B. licheniformis) immobilized on different types of matrices (Ivanova et al.,

1998). appcatk of immobilized BLA (0.93) was about half of the free

catk (1.76). Lower

value of kcat of immobilized BLA is due to lower accessibility of substrate to the

active enzyme site of the immobilized BLA, which subsequently results into lower

reaction rate. Apparent Vmax is reported to decrease marginally (Tumturk et al., 2000)

Page 142: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

128

for nonporous support, as well as significantly up to 20 times (Ivanova et al., 1998)

for porous support. This is mainly attributed to diffusional limitation that exists in the

pores of porous support.

Table 4.2. Properties of free and immobilized BLA

Parameter Free BLA Immobilized BLA

Optimum pH 5.6 5.2

Optimum Temperature (°C) 55 55-70

Protein content (mg/mL) 75 0.83a

Activity of biocatalyst

(FEU/mL)

(IEU/mL)

16500

18450b

300a

18.5

Sp activity

(FEU/mg of protein)

(IEU/mg of protein)

220

246.1b

361a

22.3

Retained enzyme activity

after immobilization n.a 9.1%

Ea (kcal/mol) 1.63c 3.18d

Km (mg/ml) 3.3 15

Vmax (µmol/(min.mL)) 1.3 0.46

kcat (min-1) 1.76 0.93 a calculated using material balance; b calculated using assay procedure adapted for immobilized BLA (i.e. IEU

equiv); c calculated using arhenius plot over temperature 32-55 °C; d calculated using arhenius plot over temperature 37-55 °C.

Page 143: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

129

5

25

45

65

85

105

4 4.4 4.8 5.2 5.6 6 6.4 6.8 7.2pH

% re

lativ

e ac

tivity

Immobilised BLA, 55 °CFree BLA, 55 °C

Figure 4.4. pH-% relative activity profile of free and immobilized BLA

5

25

45

65

85

105

30 35 40 45 50 55 60 65 70 75Temperature (°C)

% re

lativ

e ac

tivity

Immobilised BLA, pH 5.2Free BLA, pH 5.6

Figure 4.5. Temperature-% relative activity profile of free and immobilized BLA

4.3.3. Effect of reaction conditions on hydrolysis of soluble starch using

immobilized BLA and saccharide composition

4.3.3.1. Effect of pH.

Hydrolysis performed at pH 5.2 showed maximum initial hydrolysis rate as

well as maximum hydrolysis rates in the later stages of hydrolysis (Fig. 4.6),

indicating that stability of the immobilized BLA is relatively high at pH 5.2. Hence

Page 144: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

130

pH 5.2 was taken as an optimum operating pH and experiments to account the effect

of temperature, [S]0 and [IEU]/[S]0 were performed at pH 5.2. Initial hydrolysis rates

with unbuffered solution (i.e. soluble starch gelatinized in distilled water) are

comparable to that with pH 5.6; but in the later stages of hydrolysis (i.e. beyond the

reducing sugar concn of 20 mg/mL), hydrolysis rate decreases significantly as

compared to that with pH 5.6 (Fig. 4.6). This indicates that though the initial

hydrolysis rate with unbuffered solution is high, the stability of the immobilized BLA

in unbuffered solution is relatively low due to continued exposure to varying pH

conditions.

It was observed that at low DE (8.5 and 12.5), there are no significant changes

in the composition of maltodextrins (Fig 4.7, A & B). However Fig. 4.8 (A) or Fig

4.8 (D) shows that at DE of 20.5, wt % of G5 and G3 significantly increases from 7.7

to 13 and from 4.7 to 6.7 respectively with an increase in the pH from 4.4 to 7; but

there are marginal increases in the wt % of G1, G2, G4, G6 and G7. It can be also

seen from Fig. 4.8 (A) that wt % of oligosaccharides with DP higher than 7 (which

mainly constitute branched dextrins) decreases from 76.8 to 70.5. It also indicates

that increase in the pH of the reaction mixture up to 7 favors binding of higher

dextrins to immobilized BLA and the subsequent hydrolysis of the same.

Page 145: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

131

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8Time (h)

Con

cn o

f red

ucin

g su

gars

(mg/

mL)

4.4 55.2 5.45.6 67 unbuffered

pH

Figure 4.6. Concentration of reducing sugars vs. time with pH as parameter

at [S]0 = 90 mg/mL, 55 °C and [IEU]/[S]0 = 8.27e-3.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

4.3 4.8 5.3 5.8 6.3 6.870

75

80

85

90

95A

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

4.3 4.8 5.3 5.8 6.3 6.870

75

80

85

90

95B

0

1

2

3

4

5

6

7

8

9

4.3 4.8 5.3 5.8 6.3 6.860

65

70

75

80

85C

0

2

4

6

8

10

12

14

4.3 4.8 5.3 5.8 6.3 6.850

55

60

65

70

75

80D

wt p

erce

ntag

e of

G1-

G7

)

wt p

erce

ntag

e of

olig

osac

char

ides

of D

P >

7

)

pH Figure 4.7. Effect of pH on the saccharide composition at reaction conditions same as

Fig. 4.6. A, DE 8-9; B, DE 12-13; C, DE16-17; and D, DE 20-21.

□ G1, ■ G2, G3, ◊ G4, G5, ♦ G6, + G7, - oligosaccharides with DP >7.

Page 146: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

132

0

2

4

6

8

10

12

14

16

4.3 4.8 5.3 5.8 6.3 6.8pH

50

55

60

65

70

75

80A

0

2

4

6

8

10

12

14

16

35 40 45 50 55 60 65 70Temperature (°C)

50

55

60

65

70

75B

0

2

4

6

8

10

12

14

16

0 45 90 135 180[S]0

45

50

55

60

65

70

75

80C

0

2

4

6

8

10

12

0.0025 0.0085 0.0145 0.0205[IEU]/[S]0

40

45

50

55

60

65

70

75

80Dw

t % o

f G1-

G7

wt %

of o

ligos

acch

arid

es o

f DP

>7

)

Fig. 4.8. Effect of pHa, temperatureb, [S]0

c and [IEU]/[S]0d on the saccharide

composition at DE 20-21.

□ G1, ■ G2, G3, ◊ G4, G5, ♦ G6, + G7, - oligosaccharides with DP >7. a [S]0 = 90 mg/mL, 55 °C and [IEU]/[S]0 = 8.27e-3. b [S]0 = 90 mg/mL, pH 5.2 and [IEU]/[S]0 = 6.22e-3. c 55 °C, pH 5.2 and [IEU]/[S]0 = 3.3e-3. d pH 5.2, 55 °C and [S]0 = 90 mg/mL.

4.3.3.2. Effect of temperature.

Initial rate of reaction (V) increases significantly with an increase in the

temperature from 37 °C to 55 °C; but further increase in temperature results in lesser

increase in V and initial rates at temperature above 60 °C are approximately the same

(Fig. 4.5 and Fig. 4.9).

At DE of 20, wt % of G5 and G3 remains constant with an increase in the

temperature from 36 to 50 °C at 12-13 and 6-7 respectively, but further increase in

Page 147: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

133

temperature up to 70 °C significantly decreases wt % of G5 and G3 from 12.5 to 8

and from 7 to 4.8 respectively (Fig. 4.8 (B) or 4.10 (D)). At DE 20, wt % of G1, G2,

G4, G6, G7 and oligosaccharides higher than G7 increases from 0.9 to 1.35, from 2.4

to 3.1, from 2.3 to 2.6, from 3.9 to 5.7, from 0.7 to 1 and from 69 to 73.5 respectively

with an increase in the temperature from 36 °C to 70 °C (Fig. 4.8 (B)). This indicates

that increase in the temperature decreases product specificity of immobilized BLA

towards G3 and G5, whereas specificity towards G1, G2, G4, G6 and G7 increases.

This results in more homogeneous molecular weight distribution with an increase in

the operating temperature.

Similar decrease in wt % of G5 and G3 and increase in wt % of G2 and G4

with an increase in the temperature from 50 to 90 °C, for free α-amylase (B.

licheniformis, Maxamyl), are reported and is attributed to the combination of the

following aspects: (1) A decrease in product specificity of α-amylase with increasing

temperature. (2) An increase in amount of transglycosylation products with increasing

temperature. (3) A change in the ratio of rate of hydrolysis of different linear

oligosaccharides (of different DP) with increasing temperature. (Marchal et al., 1999)

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8Time (h)

Con

cn o

f red

ucin

g su

gars

(mg/

mL)

37 45 50

55 60 6570

Temperature (°C)

Figure 4.9. Concentration of reducing sugars vs. time with temperature as parameter at [S]0 = 90 mg/mL, pH 5.2 and [IEU]/[S]0 = 6.22e-3.

.

Page 148: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

134

0

0.5

1

1.5

2

2.5

35 40 45 50 55 60 65 7060

65

70

75

80

85

90

95A

0

1

2

3

4

5

6

35 40 45 50 55 60 65 7060

65

70

75

80

85

90B

0

1

2

3

4

5

6

7

8

9

10

35 40 45 50 55 60 65 7060

65

70

75

80

85C

0

2

4

6

8

10

12

14

35 40 45 50 55 60 65 7050

55

60

65

70

75D

Temperature (°C)

wt p

erce

ntag

e of

G1-

G7

wt p

erce

ntag

e of

olig

osac

char

ides

of D

P >

7)

Figure 4.10. Effect of temperature on the saccharide composition at reaction

conditions same as Fig. 4.9. A, DE 8-9; B, DE 12-13; C, DE16-17; and D, DE 20-21.

□ G1, ■ G2, G3, ◊ G4, G5, ♦ G6 and + G7, - oligosaccharides with DP >7.

4.3.3.3. Effect of initial starch concn, [S]0 and [IEU]/[S]0.

In order to compare hydrolysis curves (Fig. 4.11 (A)) at different values of

[S]0 (varying from 18 to 180 mg/mL), DE vs. time is plotted in Fig. 4.11 (B). Since

the ratio [IEU]/[S]0 was same, it was expected that all curves in Fig. 4.11 (B) would

lie on the same line. But at low value of [S]0 (18 mg/mL), [IEU] was also kept low

i.e. 0.0593 in order to maintain [IEU]/[S]0 constant i.e. 3.3e-3. Hence though the

viscosity of the starch solution was less, due to lesser concentrations of IEU and

starch, probability of contact of enzyme active site on beads with starch molecule

becomes less which result into less increase in DE (5.7) in 3 h of reaction (Fig. 4.11

(B)). But as [S]0 increases, probability of contact of enzyme active site on beads with

Page 149: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

135

starch molecule increases; but due to increase in the viscosity, diffusion resistance for

starch molecule to reach active enzyme site inside pore increases. This could be the

reason for initial increase and then the maxima in the increase in DE in 3 h of reaction

(for [S]0 of 45 and 90 mg/mL, increase in DE was 9.2 and 10 respectively; Fig. 4.11

(B)) with an increase in [S]0. Further increase in the [S]0 results into increase in the

viscosity and the diffusion resistance for starch molecule to reach active site inside

pore. This could be the reason for observed lower increase in DE (8.9), in 3 h of

reaction at high starch concn i.e. 180 mg/mL (Fig. 4.11 (B)). Decrease in the rate of

hydrolysis at high starch concentration is reported for starch hydrolysis using free α-

amylase (B. licheniformis, Termamyl) and attributed to the imposed restriction on the

free movements of starch and enzyme molecules due to viscosity effects and/or

reduced water activity (Komolprasert and Ofoli, 1991), supporting the above

conclusion.

It can be seen from Fig. 4.8 (C) and Fig. 4.12 that wt % of G1, G2 and G4

remains approximately the same with an increase in the [S]0 at any value of DE;

whereas wt % of G3 and G5 decreases (from 6.8 to 5.3 and from 13.5 to 11.1

respectively at DE of 20) with an increase in [S]0 from 18 to 90 mg/mL and further

increase in [S]0 from 90 to 180 mg/mL results in an increase in wt % of G3 and G5

(from 5.3 to 6.7 and from 11.1 to 14.2 respectively at DE of 20). It can be also seen

from Fig. 4(C) that wt % of G6 and G7 marginally increases (from 4.2 to 4.9 and from

0.3 to 0.7 respectively) with an increase in [S]0 from 18 to 180 mg/mL; whereas wt %

of higher oligosaccharides (> G7) increases (from 68.6 to 72.7 at DE of 20) with an

increase in [S]0 from 18 to 90 mg/mL and then decreases (from 72.7 to 66.7 at DE of

20) with a further increase in [S]0 from 90 to 180 mg/mL.

With an increase in [IEU]/[S]0 obviously hydrolysis takes place fast (Fig.

Page 150: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

136

4.13). It can be seen from Fig. 4.8 (D) and Fig. 4.14 that there are marginal changes

in saccharide composition with an increase in the ratio of [IEU]/[S]0.

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8Time (h)

Con

c. o

f red

ucin

g su

gars

(mg/

mL) 18 45

90 180

[S]0 (mg/mL)

0

5

10

15

20

25

0 1 2 3 4 5 6 7 8Time (h)

Dex

trose

equ

ival

ent (

DE

)

18 45

90 180

[S]0 (mg/mL)

Figure 4.11(A). Figure 4.11(B). Figure 4.11. A. Concentration of reducing sugars vs. time with [S]0 as parameter B. DE vs. time with [S]0 as parameter; at 55 °C, pH 5.2 and [IEU]/[S]0 = 3.3e-3.

0

0.5

1

1.5

2

2.5

3

3.5

0 45 90 135 18070

75

80

85

90

95

100A

0

1

2

3

4

5

6

7

8

0 45 90 135 18070

74

78

82

86

90B

0

2

4

6

8

10

12

0 45 90 135 18045

50

55

60

65

70

75

80

85C

0

2

4

6

8

10

12

14

16

0 45 90 135 18045

50

55

60

65

70

75D

wt p

erce

ntag

e of

G1-

G7

wt p

erce

ntag

e of

olig

osac

char

ides

of D

P>7

)

Initial starch concentration (mg/mL)

Figure 4.12. Effect of [S]0 on saccharide composition at reaction conditions same as

Fig. 4.11. A, DE 8-9; B, DE 12-13; C, DE16-17; and D, DE 20-21.

□ G1, ■ G2, G3, ◊ G4, G5, ♦ G6 and + G7, - oligosaccharides with DP >7.

Page 151: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

137

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8Time (h)

Con

cn o

f red

ucin

g su

gars

(mg/

mL)

3.3e-3 8.23e-3

13.8e-3 20.7e-3

[IEU]/[S]0

Figure 4.13. Concentration of reducing sugars vs. time with [IEU]/[S]0 as parameter at pH 5.2, 55 °C and [S]0 = 90 mg/mL.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0.0025 0.0085 0.0145 0.020570

75

80

85

90

95

A

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0.0025 0.0085 0.0145 0.020570

74

78

82

86

90B

0

1

2

3

4

5

6

7

8

0.0025 0.0085 0.0145 0.020560

65

70

75

80

85C

0

2

4

6

8

10

12

0.0025 0.0085 0.0145 0.020540

45

50

55

60

65

70

75

80D

[IEU]/[S]0

wt p

erce

ntag

e of

G1-

G7

wt p

erce

ntag

e of

olig

osac

char

ides

of D

P >

7

)

Figure 4.14. Effect of [IEU]/[S]0 on saccharide profile at reaction conditions same

as Fig. 4.13. A, DE 8-9; B, DE 12-13; C, DE16-17; and D, DE 20-21.

□ G1, ■ G2, G3, ◊ G4, G5, ♦ G6 and + G7, - oligosaccharides with DP >7.

Page 152: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

138

4.3.4. Comparison of saccharide composition of starch hydrolysate using free

and immobilized BLA

DE of gelatinized starch solution was ∼ 5-6. For free BLA, hydrolysis ceased

at DE of around 42-43 because BLA could not hydrolyze more α(1→4) linkages due

to the presence of branched dextrins; whereas for immobilized BLA, DE of starch

hydrolysate at hydrolysis equilibrium was marginally low (around 36-37). Reason

could be attributed to steric hindrances for branched dextrins in the vicinity of active

enzyme site. DE of starch hydrolysate can be correlated with reaction time (t) by the

following exponential equation,

Bt))exp(A(1DEDE 0 −−+= (4.2)

in which DE0 is initial DE of the gelatinized starch solution (∼ 5-6), A + DE0 is

maximum attainable DE by hydrolysis (which is around 36-37 for immobilized BLA)

and B is pseudo first order hydrolysis constant (h-1). Hydrolysis constant (B) was

correlated with operating parameters for immobilized BLA by following empirical

equation.

008314T))-33.65/(0.45.18)exp(0.521[S](-0.002[S])U]/[S]5.67e4([IEB 020

0.8920 ++=

(4.3)

In case of the free BLA, it can be seen from Fig. 4.15 that concn of G1, G2,

G3, G4 and G5 increases w. r. t. reaction time. Concn of G6 and G7 increases with an

increase in DE and attains maxima at DE of about 37 and then shows a minor

decrease with further increase in DE. G8, G9 and G10 also show similar behavior.

For immobilized BLA, it can be seen from Fig. 4.16 that concn of G1, G2, G3,

G4 and G5 increases with an increase in the hydrolysis time; whereas concn of the G6

and G7 increases up to DE of 25 and 20 respectively and then decreases (due to their

further hydrolysis) with further increase in the DE. Data of G8, G9 and G10 are not

Page 153: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

139

shown in Fig. 4.16 because, at any DE their wt % was always lower than 0.3. For

immobilized BLA, at any DE, G5 and G3 are the principal products (Fig. 4.16 and

4.17). Similar high production of G5 and G3 from soluble starch was reported

(Ivanova and Dobreva, 1994) with immobilized BLA, but variation in concn of

oligosaccharides w. r. t. DE or time has not been reported. This information will be

useful in deciding the appropriate reaction quenching time to get the final product of

desired saccharide composition.

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6 7 8time (h)

conc

n of

G1-

G5

(mg/

mL)

0

2

4

6

8

10

12

conc

n of

G6-

G10

(mg/

mL)

G1 G2

G3 G4

G5 G6

G7 G8

G9 G10

DE 4.9 13 20.2 31.3 35.3 38.2 40.1 40.9 Figure 4.15. Change in concn of oligosaccharides w. r. t. time and DE, with

free BLA. [S]0 = 90 mg/mL, [IEU equiv]/[S]0 = 8.3e-3, pH 5.6, 55 °C.

Page 154: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

140

0

5

10

15

20

25

30

35

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5time (h)

conc

n of

G1-

G5

(mg/

mL)

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

conc

n of

G6

and

G7

(mg/

mL)

G1 G2 G3 G4 G5 G6 G7

DE 5.8 13.4 19.5 24.1 25.9 28.3 30.3 33.6 36.1 36.2

Figure 4.16. Change in concn of oligosaccharides w. r. t. time and DE, with

immobilized BLA. [S]0 = 90 mg/mL, [IEU]/[S]0 = 20.7e-3, pH 5.2, 55 °C.

0

5

10

15

20

25

30

1 2 3 4 5 6 7 8 9 10Degree of polymerisation of oligosaccharide

wt %

olig

osac

char

ide

DE 13 Free BLADE 13 Immobilized BLADE 20 Free BLADE 20 Immobilized BLADE 36 Free BLADE 36 Immobilized BLA

Figure 4.17. Comparison of saccharide profile produced by free and immobilized

BLA.

Fig. 4.17 shows that at same DE, wt % of G1, G2, G3 and G5 produced by

immobilized BLA were much higher than that produced by free BLA and there was

no significant change in wt % of G4, whereas wt % of G6, G7, G8, G9 and G10

produced by immobilized BLA were significantly lower than that produced by free

Page 155: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

141

BLA. Composition of starch hydrolysate produced by free BLA (reported in this

work) is different than the earlier reports (Ivanova and Dobreva, 1994; Marchal et al.,

1999) (produced by α-amylase, B. licheniformis), which could be due to different type

of strain and different botanical source of starch (i.e. corn, tapioca, potato, wheat etc),

which mainly differ in amylose: amylopectin ratio, average molecular weight etc.

At hydrolysis equilibrium with immobilized BLA, there were only traces of

G6 and G7 (Fig. 4.16). Since BLA can hydrolyze linear G6 and G7, and not the

branched one, we can say that immobilized BLA produces linear G6 and G7 from

higher linear or branched dextrins in early stages of hydrolysis (DE<25), which gets

further hydrolyzed leaving only traces of G6 and G7 at hydrolysis equilibrium.

However presence of significant quantities of G6-G10 at hydrolysis equilibrium with

free BLA (Fig. 4.15) indicates that free BLA produces significant amount of branched

G6-G10 than linear one. Being speculative, this could be possibly because in the case

of immobilized BLA, linear part of higher dextrins must be forming productive

complex with active enzyme site rather than branched part. Reason for this could be

attributed to steric hindrances, which is a property of both porous nature of the

support and extent of branching of starch (Marchal et al., 1999). However there are no

such steric hindrances in the case of free BLA.

Few experiments were also performed on hydrolysis of G4, G5, G6 and G7

separately using immobilized BLA at 55 °C. It was observed that immobilized BLA

could not hydrolyze G4 and G5. But it completely hydrolyses G6 and principally

produces G5 and G1 (this is in agreement with Ivanova and Dobreva, 1994). It also

completely hydrolyses G7 and produces G5, G2 mainly and also small quantity of G1.

This must be because immobilized BLA hydrolyses major fraction of G7 to G5 and

G2, and rest to G6 and G1; thus produced G6 further hydrolyses to G5 and G1.

Page 156: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

142

Action pattern and subsite mapping of free BLA with modified malto-

oligosaccharide substrates have been reported by Kandra et al., 2002. Binding region

or active site of BLA is composed of five glycone, three aglycone-binding subsites

and a barrier subsite; which have different binding energies. Free BLA cleaves G5 as

a main product from non reducing end of G6, G7 and G8 with yield or bond cleavage

frequencies of 68%, 84% and 88% respectively. However as the DP of the substrate

increases, attack shifts towards reducing end and BLA cleaves G8, G9 and G10 into a

main product G3 with yield of 88%, 83% and 83% respectively. Our results on

hydrolysis of G6 and G7 using immobilized BLA are much similar to these results,

which are reported for the hydrolysis of G6 and G7 using free BLA. However, yield

of G5 were 80% and 88% for hydrolysis of G6 and G7 respectively, which are higher

than that reported with free BLA usage. (Kandra et al., 2002)

Major production of G5 by hydrolysis of G6 and G7, and higher production of

G5 and G3 from soluble starch suggests that action pattern of immobilized BLA is

more like an exoamylase with dual product specificity mainly towards G5 and G3.

Free BLA is also reported (Kandra et al., 2002) to have dual product specificity

mainly to G5 and G3 (due to the existence of the barrier subsite) using results of

hydrolysis of linear G6-G10, however effect of branching characteristic of starch was

not considered. However our results of hydrolysis of soluble starch using free BLA

do not show high specificity towards G5 and G3 (discussed earlier) and this must be

because of the absence of steric hindrance for the formation of productive complex

between free BLA and branched dextrins.

4.3.5. Thermostability and reusability of immobilized BLA

Fig. 4.18 shows that the thermostability BLA was improved after

Page 157: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

143

immobilization. Relative activity (%) of free BLA decreases from 100 to 75, whereas

it nearly remains same for immobilized BLA over the incubation period.

Immobilization of enzyme, by covalent binding, often improves thermostability

(Paolucci-Jeanjean et al., 2000). Reason could be attributed to improvement in

enzyme rigidity after immobilization.

Unlike free enzyme, immobilized enzyme can be easily separated from the

reaction mixture and reused. Hence reusability or operational stability is an important

criterion for industrial use of immobilized enzyme. Reusability study of immobilized

BLA shows that 100% activity of immobilized BLA was retained even after 8 batch

hydrolysis, which indicates good reusability. For comparison, hydrolysis curves of 1st

and 8th batch are shown in Fig. 4.19.

0

20

40

60

80

100

120

0 3 6 9 12 15 18 21 24Time (h)

% re

lativ

e ac

tivity

Free BLAImmobilized BLA

Figure 4.18. Thermostability of free and immobilized BLA.

Page 158: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

144

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8Time (h)

Con

cn o

f red

ucin

g su

gar (

mg/

mL)

1st batch hydrolysis8th batch hydrolysis

Figure 4.19. Reusability of immobilized BLA at pH 5.2, 55 °C,

[IEU]/[S]0 = 6.13 e-3, [S]0 = 90 mg/mL.

4.3.6. Semiempirical model for prediction of saccharide composition

Molecular weight distribution of soluble starch and starch hydrolysate were not

calculated in this work. Also, kinetic mechanism of starch hydrolysis is quite

complex due to the presence of multi substrates. Therefore, semiempirical equations

for the concentrations of oligosaccharides (G1-G7) vs. time were used, which are

analogous to reported (Paolucci-Jeanjean et al., 2000) semiempirical equations. It

was observed that plotting rate of formation of oligosaccharides (G1-G5) vs. concn of

oligosaccharides higher than G5 yields a straight line. This indicates that rate of

formation of oligosaccharides has an order of reaction one w. r. t. the concn of

oligosaccharides higher than G5. Since, as stated earlier oligosaccharides with DP

lower than 6 can not be hydrolyzed by immobilized BLA, there is no need for the

depletion term in the differential equations expressing the time dependent variation of

G1-G5; which are as follows,

Page 159: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

145

for i = 1-5,

)]G[[IEU](TDWdt

]Gd[ 5

1nn

i ∑=

′−=′

ik (4.4)

in which ]G[ i′ is concn (mg/mL) of oligosaccharide with DP of i, TDW is dry weight

concn of starch hydrolysate (mg/mL) and ki is kinetic constant (h-1[IEU]-1) of the

formation of oligosaccharide with DP of i. Concentration terms in the Eq. 4.4 were

made dimensionless by dividing Eq. 4.4 with TDW on both sides. Eq. 4.4 thus takes

the following form,

For i = 1-5,

)][G[IEU](1dt

]d[G 5

1nn

i ∑=

−= ik (4.5)

in which [Gi] is dimensionless concn or wt fraction of oligosaccharide with DP of i.

As stated earlier, immobilized BLA hydrolyses G6 and G7, so it becomes essential to

add depletion term ( ][IEU][Giik′ ) while constructing a differential equation

expressing the time dependent variation for G6 and G7. Differential equation for G6

and G7 are as follows,

For i = 6-7,

][IEU][G)][G[IEU](1dt

]d[Gii

i

1nn

i kki ′−−= ∑=

(4.6)

where ik′ is kinetic rate constant (h-1[IEU]-1) for hydrolysis of oligosaccharide with

DP of i. Differential equations of G8-G10 are not considered because wt % of G8-

G10 were always lower than 0.3. Values of kinetic constants k1-k7, 6k′ and 7k′ were

determined by minimizing the sum of square of the error between predicted (obtained

by simultaneously solving Eq. 4.5 and Eq. 4.6 from t = 0 h to t = time required to

attain equilibrium DE of 36.5, which was calculated using Eq. 4.2) and experimental

wt % of oligosaccharides (G1-G7). This was done by developing a code (Code is

Page 160: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

146

provided in Appendix B) in MATLAB. Kinetic constants for reaction conditions;

[IEU] = 1.86, 55 °C, pH =5.2 and [S]0 = 90 mg/mL; are k1 = 0.0069, k2 = 0.0119, k3 =

0.0191, k4 = 0.0072, k5 = 0.0508, k6 = 0.0489, k7 = 0.0148, 6k′ = 0.9725 and 7k′ =

3.2536 (unit of all kinetic constants is h-1[IEU]-1). Comparison of the experimental

and predicted wt % of oligosaccharides (G1-G7) at above-mentioned reaction

conditions is shown in the Fig. 4.20. It can be seen from Fig. 4.20 that predicted wt %

fits well with the experimental values of wt % for G1-G5; whereas for G6-G7

predicted wt % lies slightly below the experimental values of wt % up to DE of 20,

but beyond 20 DE model overpredicts wt % of G6-G7. These kinetic rate constants

were empirically correlated with [IEU], [S]0 and temperature using the following type

of correlation,

D0

CB [S](T/273)A[IEU]=k (4.7)

where A, B, C, and D are correlation constants and T is temperature in K.

Values of A, B, C and D for kinetic constants k1-k7, 6k′ and 7k′ calculated by nonlinear

regression (using POLYMATH) are given in the Table 4.3.

Table 4.3. Values of A, B, C and D for kinetic constants

Kinetic constant A B C D Correlation

coefficient k1 0.0083 -0.0807 14.58 -0.6237 0.97 k2 0.0086 -0.3975 13.28 -0.4374 0.99 k3 0.0415 -0.4727 7.30 -0.4145 0.99 k4 0.0060 -0.3673 12.42 -0.4160 0.99 k5 0.0899 -0.3777 7.26 -0.4033 0.98 k6 0.3580 0.1086 12.01 -0.9275 0.99 k7 0.0022 -0.0633 14.62 -0.0905 0.80 k'6 102 0.5063 8.84 -1.439 0.93 k'7 7.8800 0.1496 9.36 -0.6200 0.89

Page 161: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

147

0

5

10

15

20

25

30

35

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6time (h)

wt %

of G

1-G

5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

wt %

of G

6 an

d G

7

DE 5.8 13.5 19.5 24.1 25.9 28.3 30.3 33.6 36.1 36.2 Figure 4.20. Comparison of experimental and predicted saccharide composition;

[IEU] = 1.86, 55 °C, pH 5.2, [S]0 = 90 mg/mL.

Symbols represent experimental data; □ G1, ■ G2, G3, ◊ G4, G5, ♦ G6 and +

G7; continuous lines represent predicted data.

4.3.7. Effect of mode of operation on hydrolysis of soluble starch

Hydrolysis of gelatinized starch solution was performed in three different

modes of operations viz. batch mode (performed in shaker), packed bed, and

expanded bed and their hydrolysis curves are compared in the Fig. 4.21. It can be seen

from Fig. 4.21 that the mode of packed bed mode of starch hydrolysis is faster than

the batch mode. This must be happening because in contrast to reaction in a

conventional batch mode, packed bed imposes high enzyme to substrate ratio and

promotes greatly increased reaction rates.

Effect of superficial liquid velocity (i.e. flow rate / column cross sectional

area) on the hydrolysis performance was studied by performing experiments in the

packed bed mode at three different superficial velocities viz. 1.27, 2.55, and 3.82

cm/min (i.e. 1, 2 and 3 mL/min flow rates, respectively). It can be observed from Fig.

Page 162: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

148

4.21 that as the superficial velocity increases from 1.27 to 2.55 cm/min, hydrolysis

performance improved. But when it was increased from 2.55 to 3.82 cm/min, initial

reaction rates decreased and hydrolysis performance was adversely affected.

Mean residence times were measured to be 13.4, 6.3, and 3.4 min at

superficial liquid velocities of 1.27, 2.55, and 3.82 cm/min, respectively (i.e. 1, 2, 3

mL/min, respectively), for the packed bed of 5 mL of CELBEADS. This indicates that

as the liquid velocity increases, residence time decreases and flushing effect starts to

play a role. At low liquid velocities, substrate solution almost reaches all the pores in

the beads and residence time is large and obviously time required for the product

molecules to come out of pores will also be high. It could be speculated that at low

flow rates, residence time scales may be higher than reaction time scales. This could

be the reason for observing lower hydrolysis performance at low flow rate. As the

liquid velocity increases, residence time decreases and may be become comparable to

the reaction time scales. This will result into faster carriage of substrate molecules to

immobilized enzyme active sites, reaction and fast removal of product molecules from

pores to solution. This could be the reason for improvement in the hydrolysis

performance with increase in the flow rate or liquid velocity. If liquid velocity is

further increased, local residence time scales will be much lower than reaction time

scales. This will result into faster carriage of substrate molecules to immobilized

enzyme active sites, but without getting enough time for reaction they may be

removed from the active enzyme site. This is termed as “flushing effect” in the

reactor. This will obviously result into poor hydrolysis performance. This could be the

reason for poor hydrolysis performance with an increase in the superficial liquid

velocity 2.55 to 3.82 cm/min. Same reason could be attributed to poor hydrolysis

performance in the expanded bed mode hydrolysis. In the expanded bed, voidage inter

Page 163: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

149

between the beads is more. Hence, space inter between beads offers least resistance to

the flow of solution. Hence, significant part of the reactant molecules may be passing

through this voidage and obviously resulting lower reaction rates.

0

5

10

15

20

25

30

35

40

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0Time (h)

Con

cn o

f red

ucin

g su

gars

(mg/

mL)

Batch mode

Packed bed, 1.27 cm/min

Packed bed, 2.55 cm/min

Packed bed, 3.82 cm/min

Expanded bed, 1.27 cm/min

Figure 4.21. Effect of mode of operation on hydrolysis of soluble starch

at pH 5.2, 55 °C, starch concentration = 10% w/v and [IEU]/[So] = 8.9 e-3

4.3.8. Hydrodynamic stability of immobilized BLA

Reusability of immobilized enzyme is very important aspect in the industrial

utilization of it. Effect of mode of operation in performing hydrolysis of gelatinized

starch solution on the reusability of immobilized BLA is shown in the Fig. 4.22.

When mode of operation is varied, hydrodynamic conditions faced by immobilized

enzyme changes. Hence this reusability is named as Hydrodynamic stability.

Page 164: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

150

Hydrodynamic stability

0

20

40

60

80

100

0 1 2 3 4 5 6Reuse number

% in

itial

rate

Batch with intemittent washing

batch without intermittent washing

Packed bed 1 mL/min without intermittent washing

Packed bed 3 mL/min without intermittent washing

Expanded bed 1 mL/min without intermittent washing

Figure 4.22. Effect of mode of operation and intermittent mixing on reusability of immobilized BLA. pH 5.2, 55 °C, starch conc = 10% w/v, and [IEU]/[So] = 8.9 e-3 Reusability study of immobilized BLA in the batch mode with intermittent

washing shows that 100% activity of immobilized BLA was retained even after 8

batches of hydrolysis (Fig. 4.22), which indicates good reusability. But when the same

study was performed without intermittent washing (i.e. washing of beads), reduction

in the % initial reaction rate was observed from 100 to 68 (Fig. 4.22) after 6 batches

of hydrolysis. After 6 batches of hydrolysis in the packed bed mode of operation, %

initial reaction rate was observed to decrease from 100 to 28 for 1.27 cm/min

superficial liquid velocity and from 100 to 14 for 3.82 cm/min liquid velocity (Fig.

4.22). Agglomeration of the beads was visually observed after completion of the

batch, this effect was more pronounced in the packed and expanded bed than that in

the batch mode. This could be because in the batch mode, liquid velocities in the

vicinity of beads were very high as compared to those in the case of packed bed or

Page 165: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

151

expanded bed. This results into not only agglomeration of beads, but blockage of

pores due to starch molecules also. This result into decrease in the activity of

immobilized enzyme after completion of batch. When intermittent washing of beads

was done, agglomerated beads separated from each other due to high velocities and

pore blockage also get cleared. Due to this no loss in the activity was observed in the

batch mode with intermittent washing. This suggests that the packed bed mode of

operation should be carried out in a semi continuous manner with intermittent bed

washing after periodic time intervals.

4.3.9. Hydrolysis of sorghum slurry using immobilized BLA.

Due to several advantages (like reusability of the enzyme, continuous

operation of the system, easy separation of product from the enzyme etc.) that

immobilized enzyme have over the free enzyme, it was first decided to develop a

process for the production of glucose from sorghum flour using immobilized

enzymes. This process constitutes following steps viz. 1. Gelatinization of 15 %

sorghum slurry in boiling water for 10 min. 2. Circulating slurry through the bed of

immobilized BLA and amyloglucosidase.

In the present work, attempt of hydrolysis of gelatinized sorghum slurry was

made using immobilized BLA and amyloglucosidase in the batch mode (using

shaker). It was observed that use of immobilized BLA is not suitable for production of

glucose from sorghum due to following,

Change in the action pattern of BLA due to immobilization

Drastic reduction in the enzyme activity in reusability without intermittent

washing.

After mixing the gelatinised sorghum slurry with beads, it was very difficult

Page 166: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

152

to separate beads from slurry after the completion of reaction.

Technical difficulty in circulation of sorghum slurry through immobilized

enzyme bed in both packed and expanded mode due to viscosities and

pericarp particles in the slurry.

BLA is now cheaply available enzyme at cost of 250 Rs./kg

Thus, it may be more economically viable to use BLA and other enzymes in

the free form for the production of glucose from sorghum flour. The work of

enzymatic production of glucose from sorghum using enzymes in free forms is

reported in chapter 5.

4.4. Conclusions

Bacillus licheniformis α-amylase (BLA) was immobilized on superporous

CELBEADS. After immobilization, optimum pH for the enzyme action marginally

decreases and optimum temperature remains same though thermostability increases.

pH, temperature and initial starch concn has significant effect on saccharide

composition at same value of DE. Saccharide composition of starch hydrolysate

produced by immobilized BLA is different than that produced by free BLA at any

value of DE. At any DE, free BLA principally produces maltotriose, maltopentaose

and maltohexaose; whereas immobilized BLA principally produces maltotriose and

maltopentaose. Immobilized BLA has better thermostability than free BLA and found

to retain 100% activity even after 8 batches of hydrolysis. Immobilized BLA can be

used as an additional tool for production of maltodextrins solely or in combination

with variation in pH, temperature and starch concn. Used semiempirical model

predicts wt % of oligosaccharides (G1-G7) that satisfactorily fits the experimental

data of G1-G5, but over predicts wt % of G6 and G7. Such model can be used for

Page 167: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on superporous CELBEADS

Studies in the Enzymatic depolymerisation of natural polysaccharides

153

selecting operational parameters such as temperature, starch concn as design

parameter to obtain desired saccharide profile in maltodextrins. However care should

be taken because values of kinetic constant are likely to vary with change in starch

source.

Nomenclature

A, B, C, D see Eq. 4.7

[G´i] concn of oligosaccharide with DP of i (mg/mL)

[Gi] dimensionless concn or wt fraction of oligosaccharide with DP of i

[FEU] number of free enzyme units per mL of starch solution

[IEU] number of immobilized enzyme units per mL of starch solution

[IEU equiv] number of Eq. immobilized enzyme units per mL of starch solution

[S]0 initial starch concn (mg/mL)

DE dextrose equivalent of starch hydrolysate

k1-k7 kinetic constant for formation of G1-G7 (h-1[IEU]-1)

6k′ kinetic constant for depletion of G6 (h-1[IEU]-1)

7k′ kinetic constant for depletion of G7 (h-1[IEU]-1)

freecatk turnover number of free enzyme (min-1), free

maxV /[IEU]

appcatk turnover number of immobilized enzyme (min-1), app

maxV /[IEU equiv]

Km intrinsic michaelis constant (mg/mL) free

mK Km of free enzyme (mg/mL)

appmK apparent Km of immobilized enzyme (mg/mL)

n average degree of polymerization (DP) of starch hydrolysate

T temperature (K)

t reaction time (h)

TDW dry weight concn of starch hydrolysate (mg/mL)

V initial reaction rate

Vmax intrinsic maximum reaction rate free

maxV Vmax of free enzyme

appmaxV apparent Vmax of immobilized enzyme

Page 168: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

154

5. Enzymatic production of glucose from sorghum

Page 169: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

155

5.1. Introduction and literature review

Sorghum (Sorghum bicolor L. Moench) is an important drought resistant

cereal crop and fifth largest produced cereal in the world after wheat, rice, barley and

maize. Production of sorghum in 2007-2008 in the world was 64 Million Metric Tons

(www.fas.usda.gov). Leading sorghum producing countries were United States

(19.9%), Nigeria (15.5%), India (11.3%), Mexico (9.8%), Sudan (7%), and Argentina

(5.4%) (www.fas.usda.gov). Sorghum ranks third in the major food grain crops in

India. Sorghum is valued because of its ability to grow in areas with marginal rainfall

and high temperatures (i.e. semi arid tropics and sub tropical regions of the world),

where it is difficult to grow any other cereal, and also because of its relatively short

growing season requirement, thus its suitability for double cropping and crop rotation

systems (Smith and Frederiksen, 2000). Average percentage contents of starch,

proteins, moisture, fibers, lipids, and ash in the sorghum were 70.1, 11.2, 11.6, 1.82,

3.54, and 1.8 respectively (Wu et al., 2007).

Though, production of sorghum is high in India, demand for the sorghum is

decreasing with change in the way of living due to increased urbanization, increased

per capita income of the population, and easy availability of other preferred cereals in

sufficient quantities at affordable prices. Hence, in addition to being a major source

of staple food for humans, it also serves as a source of feed for cattle and other

livestock in scarcity of maize, but at lower prices. Also, about 10-20 % of the

production gets wasted due to damage and inadequate transport and storage facilities.

Industrial grade damaged sorghum grains (inclusive of 30-55% sound grains) are

available in large quantity at Food Corporation of India (FCI) at 10 times lower rate

than the fresh grains (Suresh et al., 1999a). Damage includes chalky appearance,

cracked, broken, mold, infection etc. These damaged grains are not suitable for

Page 170: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

156

human consumption. Several mold-causing fungi are producers of potent mycotoxins

that are harmful to health and productivity of human and animal (Bandyopadhyay et

al., 2000). Hence, damage caused by insect infection and attack of fungus (blackened

sorghum or grain mold) because of wet and humid weather makes sorghum grains

even unfit for animal consumption.

Hence, an industrial application is needed to be exploited for normal and

blackened sorghums in order to make sorghum cultivation economically viable for

farmers, through value added products. There is very small amount of research done

on value addition to sorghum through; production of glucose (Devarajan and Pandit,

1996; Aggarwal et al., 2001), production of ethanol (Wu et al., 2007; Suresh et al.,

1999a, 1999b; Zhan et al., 2003; Zhan et al., 2006; Zhao et al., 2008) and isolation of

starch (Yang and Sieb, 1996; Xie and Seib, 2002; Higiro et al., 2003; Perez-Sira and

Amaiz, 2004; Park et al., 2006). The reason for the lower level of industrial

exploitation can be attributed to reduced sorghum starch digestibility (Lichtenwalner

et al., 1978; Rooney and Pflugfelder, 1986; Chandrashekar and Kirleis, 1988; Zhang

and Hamaker, 1998; Elkhalifa et al., 1999; Ezeogu et al., 2005) and reduced protein

digestibility (Duodu et al., 2003) after cooking i.e. heat-moisture treatment of

sorghum flour. Literature related to production of production of ethanol and isolation

of starch from sorghum, and digestibility of sorghum starch and sorghum proteins is

reviewed in the chapter 3. There is no literature available on value addition products

to blackened sorghum.

Application of ultrasound is reported in wet milling process for isolation of

starch from corn (Zhang et al., 2005), sorghum(Park et al., 2006) and rice (Wang and

Wang 2004), and in dry corn milling ethanol production (Kinley et al., 2006; Khanal

et al., 2007).

Page 171: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

157

In the present work, sorghum flour was used directly for liquefaction and

saccharification rather than isolating starch and using it for liquefaction and

saccharification as the yields of starch isolation from sorghum were reported to be

around 50–60% i.e rest part (40–50%) gets wasted or does not fetch much price.

Such methodology of direct hydrolysis was first used by kroyer in 1966 using corn

grits for the production of glucose. Direct hydrolysis of flour of maize (Bos and Norr,

1974; Twisk et al., 1976), broken rice (Tegge and Ritcher, 1982) and sorghum (Tegge

and Ritcher, 1982; Devarajan and Pandit, 1996; Aggarwal et al., 2003) was reported

for the production of glucose.

Objectives of the present work were to optimize enzymatic liquefaction and

saccharification processes to produce glucose from three varieties of sorghum i.e.

healthy, blackened and germinated, and to study the effect of ultrasound treatment

prior to liquefaction on the performance of liquefaction and saccharification

processes.

5.2. Experimental

5.2.1. Materials

3,5-Dinitrosalicylic acid (DNSA), soluble starch, maltose, dextrose, MeCN for

chromatography LiChrosolv and other chemicals were purchased from E. Merck Ltd

(India). Commercial preparations in liquid formulation of Bacillus licheniformis α-

amylase (BLA) (EC number 3.2.1.1), Amyloglucosidase (AG) (EC number 3.2.1.3),

and Pullulanase (PL) (EC number 3.2.1.41) were gifted by Advance Enzyme

Technologies Pvt Ltd (India). Healthy sorghum and blackened sorghum were

purchased from the local market.

Page 172: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

158

5.2.2. Analytical Methods

5.2.2.1. Measurement of protein concentration, reducing sugar concentration

and concentrations of malto-oligosaccharides.

The protein concentration of the free enzyme was determined using the

modified Folin–Lowry method (Lowry et al., 1951) using BSA (0–0.6 mg/mL) as a

standard. The reducing sugar concentration was measured using the DNSA method

(Miller 1959) with dextrose (0–1 mg/mL) as a standard. Concentrations of glucose

and malto-oligosaccharides up to maltoheptaose were measured using the HPTLC

method. Details of modified folin lowry method, DNSA method and HPTLC method

are given in the Appendix A.

5.2.2.2. Measurement of moisture content

Sorghum flour was kept at 80 °C, till constant weight was obtained and

moisture content in sorghum flour was measured using mass balance. Detailed

method to measure moisture content is given in the appendix A.

5.2.2.3. Measurement of particle size distribution of sorghum flour.

Particle size distribution of the ground sorghum flour was determined by using

the Coulter Counter Particle Size Analyzer (LS 230) based on laser light diffraction.

5.2.2.4. Measurement of starch content of sorghum flour.

Sorghum grains were finely ground to the flour. Sorghum slurry (1% w/v, pH

4.5, 50 mM acetate buffer) was gelatinized for 10 min in boiling water. Then 200

units of BLA and 180 units of AG were added to gelatinized solution and reaction

mixture was kept under shaking conditions (180 rpm) at 55 °C for 24 h. Starch

content in the sorghum flour was calculated by multiplying the total reducing sugar

(glucose equiv) produced upon complete hydrolysis (i.e. at end of 24 h) by a factor of

0.9.

Page 173: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

159

5.2.3. Amylolytic activity measurement methods

5.2.3.1. Free bacterial α-amylase (BLA)

Procedure for measurement of activity of free Bacillus licheniformis α-

amylase (BLA) is detailed in the chapter 4.

5.2.3.2. Free amyloglucosidase (AG)

All enzyme assays were designed by end point assay method. It was ensured

that at the end of incubation time concentration of reducing sugar lies in the linear

part of the concentration of reducing sugars vs. time curve.

Gelatinized soluble starch solution (0.9 mL, 1% w/v, pH 4.5, 50 mM citrate

buffer) was incubated with 0.1 mL of 10000 fold diluted commercial

amyloglucosidase (AG) solution at 65 °C for 10 min. Then 1 mL of DNSA reagent

was added to reaction mixture to stop the reaction. The resulting solution was heated

in a boiling water bath for 10 min. Then 10 mL distilled water was added to the assay

mixture. Absorbance of the solution was measured against substrate blank. The

variation in the concentration of reducing sugar was measured by DNSA method

(Appendix A) using dextrose as a standard. One unit of amyloglucosidase (AGU)

was defined as that required to liberate one micromole of reducing sugar (glucose

equiv) per min under the assay conditions. Activity of commercial AG formulation

was calculated using following equation.

min 10 mL) (0.1aliquot enzyme of volume 180) (i.e. glucose of wt molfactordilution enzyme produced equiv) (glucosesugar reducing of g EU/mL

×××

min 10 mL) (0.1aliquot enzyme of volume 180) (i.e. glucose of wt mol(10000)factor dilution enzyme

mL) (1 mixtureassay of volumemin) 0at C -min 10at (C

AGU/mL

RSRS

×××

×

=

where CRS = Concn of reducing sugars (glucose equiv) in assay mixture, µg/mL

Page 174: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

160

5.2.3.3. Free pullulanase (PL).

Gelatinized soluble starch solution (0.9 mL, 1% w/v, pH 5.5, 50 mM citrate

buffer) was incubated with 0.1 mL of 500 fold diluted commercial pullulanase (PL)

solution at 60 °C for 10 min. Then 1 mL of DNSA reagent was added to the reaction

mixture to stop the reaction. The resulting solution was heated in a boiling water bath

for 10 min. Then 10 mL distilled water was added to the assay mixture. Absorbance

of the solution was measured against substrate blank. The variation in the

concentration of reducing sugar was measured by DNSA method (Appendix A) using

glucose as a standard. One unit of pullulanase (PLU) was defined as that required to

liberate one micromole of reducing sugar (glucose equiv) per min under the assay

conditions. Activity of commercial pullulanase formulation was calculated using

following equation,

min 10 mL) (0.1aliquot enzyme of volume 180) (i.e. glucose of wt molfactordilution enzyme produced equiv) (glucosesugar reducing of g EU/mL

×××

min 10 mL) (0.1aliquot enzyme of volume 180) (i.e. glucose of wt mol(500)factor dilution enzyme

mL) (1 mixtureassay of volumemin) 0at C -min 10at (C

PLU/mL

RSRS

×××

×

=

where CRS = Concn of reducing sugars (glucose equiv) in assay mixture, µg/mL

5.2.4. Thermostability study of free Amyloglucosidase.

Amyloglucosidase solution in acetate buffer (i.e. 3.63 AGU/mL, 50 mM, pH

4.5) was incubated at desired temperature under shaking conditions (180 rpm) in the

absence of substrate for 24 h. Samples of AG solution were taken at various time

intervals and were for its amylolytic activity.

Page 175: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

161

5.2.5. Optimization of AG: PL ratio for saccharification.

Gelatinized soluble starch solution (5 mL, 2% (w/v), pH 4.5, 0.05 M acetate

buffer) was incubated with 18 U of amyloglucosidase with varying units of

pullulanase (0–4.5), separately for 1 h at 55 °C. Then samples were withdrawn and

diluted ten times using 0.1 N HCl. These samples were analyzed for reducing sugar

(glucose equiv) concentration and optimum AG: PL ratio was obtained.

5.2.6. Experimental work for production of glucose from sorghum

Production of glucose from sorghum flour consists of two reaction steps:

1. Liquefaction using B. licheniformis α-amylase (BLA) in which gelatinisation of

free starch granules and dextrinization (depolymerisation) of gelatinized starch take

place simultaneously. This produces mixture of malto-oligosaccharides, linear

and branched dextrins.

2. Saccharification using amyloglucosidase (AG) in which AG cleaves first α(1→4)

linkage from non-reducing end glucose polymer and produces glucose.

Process of production of glucose syrup from sorghum flour, which is used in

the present experimental work, is shown in the Fig. 5.1. Chemistry of the process can

be also depicted from the Fig. 5.1. Chemistry of liquefaction of starch and

saccharification of liquefied starch is discussed in detail in the chapter 2.

Page 176: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

162

Figure 5.1. Process flow sheet for production of glucose syrup from sorghum

Gelatinization of free starch granules Dextrinization of gelatinized starch molecules.

Removal proteins and fibers to prevent colour formation due to solubilization of proteins

G–G–G–G–G–G–G–G–G–G–G–G–G–G Starch | G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G | G–G–G–G–G–G–G–G–G G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G

Glucose produced after saccharification of liquefied starch G

Saccharification using Amyloglucosidase (AG)

55 °C, 4.5 pH

Hot filtration

Filtration and Purification

Glucose syrup

Milling

Preparation of slurry

Grain sorghum

Removal of fine particles, lipids, proteins and unreacted starch gel

Glucose, malto-oligosaccharides, linear and branched dextrins produced after liquefaction

G–G G G–G–G–G–G–G G–G–G–G–G G | G–G–G–G–G G–G–G–G–G–G–G–G–G–G | G–G–G–G–G–G–G–G–G G–G–G–G–G–G–G–G–G–G–G–G G–G–G–G–G–G–G–G

Liquefaction using bacterial α-amylase (BLA)

85 °C, 6 pH

Page 177: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

163

Sorghum grains were milled using old fashioned flour mill (two stones of 15″

diameter × 2″ height dimensions) and this flour was used for liquefaction followed by

its subsequent saccharification.

5.2.6.1. Optimization of liquefaction of sorghum flour.

Liquefaction of sorghum flour was performed in a 250 mL stoppered conical

flask containing 100 mL magnetically stirred sorghum slurry. Experimental set-up for

liquefaction of sorghum flour is shown in the Fig. 5.2.

Figure 5.2. Experimental set up for liquefaction of sorghum flour

A Magnetic stirrer

B Conical flask containing 100 mL reaction mixture

C Silicone oil bath

D Heater

E Stirrer to for mixing in oil bath

F Temperature sensor

A

BC

D

E

Page 178: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

164

Liquefaction of sorghum slurry (10– 35% w/v in 0.05 M acetate buffer) was

performed using BLA at desired temperature (maintained by immersing the conical

flask in oil bath). Required quantity of BLA was added to sorghum slurry. Then slurry

was poured into conical flask. Oil bath temperature was maintained at 92 °C to get 85

°C temperature for sorghum slurry. Initially viscosity of the reaction mixture was very

high and as the liquefaction progresses viscosity of reaction mixture decreases; this is

an indication of increase in DE of reaction mixture.

Samples were withdrawn at time interval of 15 min and diluted to

approximately 1% (w/v) using 0.1 N HCl. Progress of liquefaction was monitored

using starch-iodine colorimetric reaction. Few drops of KI-I2 reagent (0.05% w/v I2

and 0.5% w/v KI solution) were added to few drops of diluted sample and color of

mixture was observed. As the liquefaction progresses, following different colors of

above-said mixture: deep blue, bluish violet, violet with tinge of dark red, and dark red

with tinge of violet were observed in sequence. When color becomes dark red with a

tinge of dark violet (DE of liquefact was approximately 15 at this stage), liquefaction

was considered to be completed. Samples (~ 1%w/v) were then centrifuged at 270 g

for 10 min and supernatant was analyzed for the concentration of reducing sugar.

Liquefaction process was optimized for the liquefaction time of 1.5 h using buffered

slurries of different pH values (5.2–6.7), varying concentrations of BLA (0.04–0.16%

v/w of sorghum flour) and CaCl2 (0–500 ppm), and temperature in the range of 75–95

°C.

5.2.6.2. Liquefaction of sorghum of different varieties

Healthy sorghum grains were steeped in the ordinary tap water for 12 h and then

germinated for 12 h, 24 h, 36 h, and 48 h separately. Germinated sorghum grains were

Page 179: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

165

first dried at 50 °C. Germinated and blackened sorghum were milled separately using

old fashioned flour mill. Liquefaction of geminated and blackened sorghum was

performed at optimized liquefaction conditions and compared with liquefaction of

normal healthy sorghum.

5.2.6.3. Effect of prior ultrasound treatment on the liquefaction of sorghum.

Ultrasound horn was dipped into sorghum slurry (30% w/v in 0.05 M acetate

buffer of pH 6, and CaCl2 concentration of 200 ppm) to a depth of 1 cm and slurry was

sonicated for different spans of time and at different ultrasound intensities. Ultrasound

horn (Vibra-cell, Sonics and Materials Inc., USA) having maximum power output of

750 W and operating at a frequency of 20 kHz was used in the study. Diameter of the

probe was 1.3 cm. A short ultrasound treatment of sorghum slurry was followed by the

addition of optimized amount of BLA and then subsequent liquefaction for 1.5 h was

performed at optimized conditions. After liquefaction, reaction mixture (30% w/v) was

centrifuged at 5000 g (high centrifugal force was required because of high slurry

concentration and hence viscosity) for 20 min and then supernatant was collected. This

supernatant was then analyzed for dry wt concentration of reducing sugars and reducing

sugar (glucose equiv) concentration. Dextrose equivalent (DE) is defined as following;

100mg/mL e,hydrolysatstarch ofconcn dry wt

mg/mL e,hydrolysatstarch in the C(DE)quivalent Dextrosee RS ×= (5.1)

where CRS is concentration of reducing sugar (glucose equiv)

5.2.6.4. Optimization of saccharification.

Saccharification process was first optimized for the processing temperature by

performing thermostability study of AG and also performing saccharification of

maltodextrins at different temperatures.

Page 180: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

166

At the end of liquefaction (i.e. 1.5 h), pH of the reaction mixture was reduced to

4.5 using acetic acid. Then sorghum liquefact was filtered hot using muslin cloth and

filtrate (F1) was collected. Cake obtained after hot filtration was mixed well with 10

mL distilled water and filtered. This second filtrate (F2) was mixed with first filtrate

(F1). Optimized quantity of amyloglucosidase was added to the filtrate (F1) or the

mixture of F1 and F2. Then this reaction mixture was kept under shaking conditions

(150 rpm) for 24 h under optimized conditions of temperature and enzyme

concentration. At regular time intervals, samples were withdrawn and diluted to

approximately 1% using 0.1 N HCl solution. Samples (1% w/v) were then centrifuged

at 270 g for 10 min and supernatant was analyzed for the concentration of reducing

sugar (glucose equiv). Percentage saccharification on the basis of original starch

content in the sorghum flour is defined as follows;

100Q mgin flour sorghum ofamount

mLin mixturereaction of volume mg/mLin Ccationsaccharifi % f ××

×= (5.2)

where Cf is concentration of reducing sugar (glucose equiv) at steady state (i.e. 24 h)

Q is mg of reducing sugar (glucose equiv) produced per mg of sorghum flour

using method described in 2.2.3. i.e. (% starch content /100) × 1.11

Since the calculation of % saccharification is based on the original starch content in the

sorghum flour, the term % saccharification can be also regarded as % yield of glucose.

Production of glucose from sorghum flour consists of following five steps in

sequence; 1. Ultrasound treatment on sorghum slurry, 2. Liquefaction of sorghum

slurry using B. licheniformis α-amylase, 3. Hot filtration of liquefied sorghum slurry, 4.

Washing of cake (obtained after hot filtration) followed by second filtration and mixing

of both the filtrates, and 5. Saccharification of filtrate F1 or mixture of F1 and F2 using

amyloglucosidase. Experiments were performed for production glucose using healthy,

Page 181: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

167

blackened, and germinated sorghum without or with steps 1 or 4.

5.3. Results and Discussion

5.3.1. Studies in the liquefaction process

Flour of healthy sorghum grains was used for optimization of liquefaction

process. The starch content and moisture content of the healthy sorghum flour were

estimated to be 69–70% and 10–11% respectively. Average particle size of the

sorghum flour used was 302 µm.

5.3.1.1. Optimization of liquefaction process

5.3.1.1. A. Effect of pH.

Values of the concentration of the reducing sugar (glucose equiv) obtained at 1

h after liquefaction (85 °C, 10% w/v sorghum slurry) at different values of pH of

buffered slurry; 5.2, 5.6, 6, 6.3 and 6.7 were 11.2, 12.4, 14.2, 13.4, and 11.4

respectively. This indicates that optimum pH for liquefaction of sorghum flour is 6.

Hence further study on liquefaction of sorghum flour was performed at pH of 6 (0.05 M

acetate buffer).

5.3.1.1. B. Effect of BLA concentration.

It can be seen from Fig. 5.3 that liquefaction of sorghum slurry (25% w/v) got

completed in 60 min with BLA concentration of 0.08% v/w of flour in the absence of

CaCl2 supplementation. It can be also seen from the Fig. 5.3 that when concentration

of reducing sugar was around 35 mg/mL (i.e. DE in the range of 15-17), liquefaction of

the slurry was completed according to starch-iodine reaction. The region above dotted

line in the Fig. 5.3 is liquefied region i.e. at all points in this region, liquefaction was

observed to be completed. As the BLA concentration increased, the time at which the

liquefaction was completed decreased.

Page 182: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

168

0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105Time (min)

Con

cn o

f red

ucin

g su

gar (

mg/

mL)

0.04 0.060.08 0.10.12 0.16

liquiefied

not liquified

Concn of BLA(% v/w of sorghum flour)

Figure 5.3. Effect of BLA concentration on concentration of reducing sugar (glucose

equiv) versus time curve. Reaction conditions: 25% w/v sorghum slurry, 85 °C, pH 6

and CaCl2 concn = 0 ppm.

Data points without shadow – blue color on starch-iodine reaction

Data points with shadow – disappearance of blue color (i.e. dark red with tinge of

violet) on starch-iodine reaction

5.3.1.1. C. Effect of CaCl2 concentration.

It is well known that, since BLA is an organometallic enzyme, supplementation

of Ca2+ ions improves performance of BLA. Hence, liquefaction was performed at

BLA concentration of 0.06% v/w of flour at different concentrations of CaCl2 to find

optimum CaCl2 concentration. It can be seen from Fig. 5.4 that liquefaction of

sorghum slurry (25% w/v) was completed in 60 min and all the hydrolysis curves

corresponding to CaCl2 concentration greater than 200 ppm overlapped each other (Fig.

5.4). This indicates that CaCl2 concentration of 200 ppm was optimum for liquefaction.

It is worth noting that liquefaction performance at BLA concentration of 0.08% v/w

Page 183: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

169

without supplement of CaCl2 is similar to that obtained at BLA concentration of 0.06%

v/w with CaCl2 concentration of 200 ppm (Fig. 5.3 and 5.4).

0

5

10

15

20

25

30

35

40

45

0 15 30 45 60 75 90Time (min)

Con

cn o

f red

ucin

g su

gar (

mg/

mL)

0 100 200

300 400 500

CaCl2 concn (ppm)

Figure 5.4. Effect of CaCl2 concentration on concentration of reducing sugar (glucose

equiv) versus time curve. Reaction conditions: 25% w/v sorghum slurry, 85 °C, pH 6

and BLA concn = 0.06% v/w of sorghum flour.

Data points without shadow – blue color on starch-iodine reaction

Data points with shadow – disappearance of blue color (i.e. dark red with tinge of

violet) on starch-iodine reaction

5.3.1.1. D. Effect of sorghum slurry concentration

Liquefaction was performed at various concentrations of sorghum flour varying

from 10 to 35 % w/v in the slurry. Maximum concentration of sorghum slurry that can

be used for liquefaction was observed to be 30% w/v, at which mixing of the reaction

mixture was experimentally possible and liquefaction also was successfully completed

within 1.5 h. Mixing and homogenization of the reaction mixture was visually much

less efficient at 35% w/v due to very high viscosity and it took around 2.5–3 h to

complete liquefaction. Hence, it was decided to use 30% w/v as optimum

Page 184: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

170

concentration of sorghum flour in the slurry for all further experiments.

5.3.1.1. E. Effect of liquefaction temperature.

Gelatinization temperature of sorghum starch is reported to be in the range of

75–80 °C (Palmer et al., 1992) and 60–80 °C (Wu et al., 2007). Hence, temperature

was varied in the range of 75–95 °C at 30% w/v sorghum slurry concentration for

liquefaction. Liquefaction is a combination of two processes: one, gelatinization of free

starch granules and two, dextrinization of gelatinized starch molecules (Reeve 1992).

As the temperature of liquefaction was increased from 75 to 95 °C, two effects occur

simultaneously: one, rate of gelatinization of starch increases, and two, rate of

dextrinization of starch molecules decreases due to enzyme deactivation at elevated

temperatures. Hence, there exists an optimum temperature for the liquefaction process.

Temperature of 85 °C was observed to be optimum (Fig. 5.5) for the liquefaction of

sorghum flour, at which liquefaction was completed within 1.5 h (according to starch-

iodine reaction). Though concentration of reducing sugars produced at temperatures of

75 and 80 °C were higher than that produced at a temperature of 85 °C, liquefaction

was not completed in 1.5 h (according to starch-iodine reaction). This could be because

at 75 °C and 80 °C, starch granules are not completely gelatinized (hence giving blue

color with iodine reagent). However, higher concentration of reducing sugars could be

attributed to continued depolymerisation of gelatinized starch molecules. At

temperatures of 90 °C and 95 °C, liquefaction performance was significantly poorer

than its counterparts at 85 °C (Fig. 5.5). Optimum conditions for liquefaction of

sorghum flour are summarized in the Table 5.2.

Page 185: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

171

0

10

20

30

40

50

60

0 15 30 45 60 75 90Time (min)

Con

cn o

f red

ucin

g su

gars

(mg/

mL)

75 80 85

90 95

Temperature (°C)

Figure 5.5. Effect of Temperature on concentration of reducing sugar (glucose equiv)

versus time curve. Reaction conditions: 30% w/v sorghum slurry, pH 6, CaCl2 concn =

200 ppm.

Data points without shadow – blue color on starch-iodine reaction

Data points with shadow – disappearance of blue color (i.e. dark red with tinge of

violet) on starch-iodine reaction

5.3.1.1. F. Liquefaction of sorghum of different varieties

Healthy, germinated, and blackened sorghum were found to contain 69–70, 69–

70 and 70–71% starch respectively. High starch content in the blackened sorghum

indicates that fungus has infected only pericarp of sorghum grain and not the

endosperm.

It can be seen from Fig. 5.6 that liquefaction of healthy sorghum was completed

in 1.5 h under optimized conditions. However, liquefaction progress of blackened

sorghum is slightly (7%) slower as compared to healthy sorghum. This could be

attributed to the enzyme inhibition due to mycotoxins present in the blackened pericarp.

Page 186: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

172

0

10

20

30

40

50

60

70

80

90

0 15 30 45 60 75 90Time (min)

Con

c of

redu

cing

sug

ars

(mg/

mL)

H B G1 G2 G3 G4

Figure 5.6. Effect of variety of sorghum on concentration of reducing sugar (glucose

equiv) versus time curve. Reaction conditions: 30% w/v sorghum slurry, 85 °C, pH 6

and CaCl2 concn = 200 ppm

Data points without shadow – blue color on starch-iodine reaction

Data points with shadow – disappearance of blue color (i.e. dark red with tinge of

violet) on starch-iodine reaction. H - Healthy sorghum; B - Blackened sorghum; G1,

G2, G3, G4 - healthy sorghum germinated for 12 h, 24 h, 36 h, and 48 h, respectively,

after steeping for 12 h in plain water.

It can be seen from Fig. 5.6 that as the germination time increases, liquefaction

performance also improves. However, beyond germination time of 24 h, there is no

significant improvement in the liquefaction performance. However, liquefaction of

germinated sorghum (germination time of 24 h) was observed to be completed in 1 h

only. This could be attributed to development of protease (i.e. protein matrix degrading

enzyme) and subsequent loosening of the cage of protein surrounding starch granules

during the process of germination.

Page 187: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

173

5.3.1.2. Effect of prior ultrasound treatment on liquefaction.

14

14.5

15

15.5

16

16.5

17

0 2 4 6 8 10 12sonication time (min)

DE

of li

quef

act

40% amplitude50 % amplitude100% amplitude

Figure 5.7. Effect of sonication time and ultrasound intensity on DE of liquefact.

14

14.5

15

15.5

16

16.5

17

0 5000 10000 15000 20000Power consumption (J)

DE

of li

quef

act

40% amplitude50 % amplitude100% amplitude

Figure 5.8. Variation in DE of liquefact with power consumption in sonication.

It can be seen from Fig. 5.7 that as the sonication time increases at constant

ultrasound intensity, DE of liquefact also increases. Fig. 5.8 shows that increase in the

DE is approximately same at constant power consumption, irrespective of the

Page 188: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

174

ultrasound intensity used. Since ultrasound treatment consumes large energy, it was

decided to keep sonication time low i.e. 1 min. If 1 min is considered as the optimum

time for ultrasound treatment, 100 % amplitude gives maximum increase in the DE of

the liquefact. The reason for increase in DE of liquefact due to prior ultrasound

treatment will be discussed in detail, later in the text.

5.3.2. Optimization of saccharification

5.3.2.1. Properties of free amyloglucosidase and pullulanase

% relative activity vs. pH profile and % relative activity vs. temperature profile

of Amyloglucosidase are given in the Fig. 5.9 and 5.10 respectively. Optimum pH and

optimum temperature of amyloglucosidase using assay procedure to measure enzyme

activity were 4.5 and 65 °C, respectively. Enzyme activity at optimum conditions and

protein content of commercial formulation of amyloglucosidase were 36300 AGU/mL

and 370 mg/mL.

65 °C

0102030405060708090

100110

3 3.5 4 4.5 5 5.5 6 6.5pH

Rela

tive

activ

ity (%

)

i

Figure 5.9. % relative activity vs. pH profile for amyloglucosidase

Page 189: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

175

0

10

20

30

40

50

60

70

80

90

100

30 40 50 60 70 80 90 100Temperature, °C

Rel

ativ

e ac

tivity

(%)

pH 4.5

Figure 5.10. % relative activity vs. temperature profile for amyloglucosidase

% relative activity vs. pH profile and % relative activity vs. temperature profile

of pullulanase are given in the Fig. 5.11 and 5.12 respectively. Optimum pH and

optimum temperature of pullulanase using assay procedure to measure enzyme activity

were 4 and 60 °C, respectively. Enzyme activity at optimum conditions and protein

content of commercial formulation of pullulanase were 2950 PLU/mL and 50 mg/mL.

0

10

20

30

40

50

60

70

80

90

100

110

3 3.5 4 4.5 5 5.5 6 6.5 7pH

% re

lativ

e ac

tivity

Temperature = 60 °C

Figure 5.11. % relative activity vs. pH profile for pullulanase

Page 190: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

176

0

10

20

30

40

50

60

70

80

90

100

110

30 35 40 45 50 55 60 65 70 75Temperature, °C

% re

lativ

e ac

tivity

pH 4

Figure 5.12. % relative activity vs. temperature profile for pullulanase

Properties of free amyloglucosidase (AG) and pullulanase (PL) are summarized

in the Table 5.1.

Table 5.1. Properties of amyloglucosidase and pullulanase

Parameter Free Amyloglucosidase Free pullulanase

Optimum pH 4.5 3.8–4.3

Optimum temperature (°C) 65 60

Activity 36300 AGU/mL 2950 PLU/mL

Protein content (mg/mL) 370 50

Specific activity 98 (AGU/mg of protein) 59 (PLU/mg of protein)

5.3.2.2. Thermostability of amyloglucosidase and optimization of operating

temperature for saccharification

Optimum temperature of AG using the specified assay procedure was found to

be 65 °C (Table 5.1). However, optimum operating temperature for saccharification

using AG may not be the same as that obtained using the assay procedure. Hence,

Page 191: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

177

study of thermostability of AG was essential at an optimum pH of 4.5. Thermostability

of AG was first checked at 65 °C. It was observed that % relative activity of AG

decreased to 5% (Fig. 5.13) at 65 °C within first 3 hrs only. Hence, it was decided to

check thermostability of AG at lower temperatures also to find optimum operating

temperature for saccharification of maltodextrins using AG. In addition to

thermostability check at different temperatures, experimental runs utilizing AG and

maltodextrins (DE 15, Sigma Aldrich) at different temperatures were also performed.

It can be seen from Figs. 5.13 and 5.14 that the optimum operating temperature for

saccharification of maltodextrins to glucose is around 55–60 °C.

0102030405060708090

100110

0 3 6 9 12 15 18 21 24Time (h)

Rela

tive

activ

ity (%

)

i

50 55

60 65

Temperature (°C)

Figure 5.13. Thermostability of AG at pH 4.5 at different temperatures.

Page 192: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

178

20

30

40

50

60

70

0 3 6 9 12 15 18 21 24Time (h)

Conc

n re

duci

ng S

ugar

(mg/

mL)

50 55

60 65

Temparature, °C

Figure 5.14. Change in concn of reducing sugar with time. Reaction conditions: 25 mL

of 10% maltodextrins (DE 15) solution in 50 mM acetate buffer, pH 4.5, 1.1 AGU/mL.

5.3.2.3. Optimum ratio of amyloglucosidase units to pullulanase units for

saccharification

Optimum ratio of AGU: PLU was found to be 18: 2 and about 25% increase in

the concentration of reducing sugar was observed due to the addition of optimum

quantity of pullulanase (Fig. 5.15) using starch as substrate. During saccharification of

sorghum liquefact using amyloglucosidase with and without optimum quantity of

pullulanase, increase in the concentration of reducing sugar was observed in the initial

stages of reaction. However, the concentration of reducing sugars at the end of 24 h

were the same (Fig. 5.16). Hence, it was decided to use only amyloglucosidase for

saccharification of sorghum liquefact, eliminating the use of pullulanase for

saccharification time of 24 h. If saccharification time needs to be reduced to 8 h,

pullulanase can be used along with amyloglucosidase, but obviously at the added cost

of the new enzyme.

Page 193: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

179

50

55

60

65

70

75

80

85

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5Pullulanase units (PLU)

% D

egre

e of

hyd

roly

sis

Figure 5.15. Optimization of ratio of AG units to PL units

for hydrolysis of soluble starch.

0

20

40

60

80

100

120

140

160

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28Time (h)

Con

cn o

f red

ucin

g su

gar (

mg/

mL)

0.1 AG

0.1 AG + 0.13 PL

% v of enzyme / w of saccharides

Figure 5.16. Saccharification of sorghum liquefact using amyloglucosidase

with and without pullulanase (14% w/v, pH 4.5, 55 °C)

Page 194: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

180

5.3.2.4. Optimization of amyloglucosidase concentration

Saccharification of sorghum liquefact (pH 4.5) was performed at different

concentrations of amyloglucosidase. It can be seen that at AG concentration of 0.052%

v/w of starch saccharification was completed at 24 h and attains concentration of 235

mg/mL which is same as that attained at higher concentrations of AG (Fig. 5.17).

HPTLC of starch hydrolysate at 24 h showed presence of glucose as a principle sugar

with more than 95% selectivity.

0

40

80

120

160

200

240

0 4 8 12 16 20 24Time (h)

conc

n of

redu

cing

suga

rs (m

g/m

L)

0.0260.0520.0780.104

% v of AG / w of starch

Figure 5.17. Variation in the concentration of reducing sugars versus time

with amyloglucosidase concentration as a parameter.

Optimized conditions for saccharification of sorghum liquefact are summarized

in the Table 5.2.

Page 195: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

181

Table 5.2. Optimized parameters for Liquefaction and Saccharification

Parameter Liquefaction Saccharification

Temperature (°C) 85 55

pH 6 4.5

Slurry concentration 30% w of sorghum flour / v of slurry n.a.

BLA concentration 0.06% v/w of sorghum flour i.e.

0.086% v/w of sorghum starch

n.a.

AG concentration n.a. 6 AGU/mL of liquefact i.e.

0.058 % v/ w of starch

CaCl2 concentration 200 ppm n.a.

5.3.3. Saccharification of sorghum liquefact

After liquefaction, liquefied sorghum slurry was filtered hot using muslin cloth.

Hot filtration was necessary because filtration of liquefied slurry at room temperature

yielded less filtrate volume (65 mL), whereas hot filtration yielded higher volume (72

mL). Also due to lower viscosity of liquefied slurry at higher temperature, filtration

was easy. While preparing sorghum slurry, 30 g of sorghum flour was mixed with 80

mL of acetate buffer to produce 100 mL of slurry. However, after hot filtration,

followed by liquefaction, only 72 mL filtrate was recovered. This means approximately

8 mL of liquefact was still trapped inside the cake and not available for further

saccharification. Hence, washing of the cake after hot filtration was essential. The

values of % saccharification using AG, attained after 24 h, for all the three varieties of

sorghum were dependent upon the following factors; 1. Ultrasound treatment on

sorghum slurry before liquefaction, and 2. Washing of cake (obtained after hot

filtration) followed by second filtration and mixing of both the filtrates, which are

summarized in the Table 5.3.

Page 196: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

182

Table 5.3. Summary of effect of ultrasound treatment and washing on liquefaction and

saccharification performance

Run

No

BLA concn,

% v/w

CRS, mg/mL

(DE)

F1,

mL

F2,

mL

F1 + F2,

mL

Cf

mg/mL

%

saccharification

H1 0.086 28.9 (13-14) 72 0 72 240 74 (S N; W N)

H2 0.086 27.7 (13-14) 71 15 86 229 84 (S N; W Y)

H3 0.086 31.2 (14-15) 75 0 75 251 81 (S Y; W N)

H4 0.086 28.5 (14-15) 74 15 89 235 90 (S Y; W Y)

G1* 0.086 29.6 (13-14) 73 0 73 240 75 (S N; W N)

G2* 0.086 26.7 (13-14) 73 15 88 225 85 (S N; W Y)

G3* 0.086 39.4 (16-17) 79 0 79 242 82 (S Y; W N)

G4* 0.086 36.7 (16-17) 79 11 90 232 90 (S Y; W Y)

B1 0.093 23.4 (9-10) 67 0 67 245 69 (S N; W N)

B2 0.093 24.6 (9-10) 65 17 82 231 79 (S N; W Y)

B3 0.093 22.7 (9-10) 70 0 70 240 70 (S Y; W N)

B4 0.093 24.4 (9-10) 69 16 85 226 80 (S Y; W Y)

B5 0.13 33.4 (16-17) 73 0 73 245 74 (S N; W N)

B6 0.13 32.1 (16-17) 73 15 88 233 86 (S N; W Y)

B7 0.13 32.1 (16-17) 75 0 75 239 75 (S Y; W N)

B8 0.13 30.4 (16-17) 75 14 89 234 87 (S Y; W Y)

S N: No ultrasound treatment of sorghum slurry before liquefaction

S Y: Ultrasound treatment of sorghum slurry before liquefaction for 1 min at 100

%amplitude

W N: No washing of cake after first filtration

W Y: Washing of cake after first filtration with 10 mL distilled water, followed by

second filtration

F1: Volume of filtrate after first filtration

F2: Volume of filtrate after second filtration

* Liquefaction time 1 h, Sorghum germinated for 24 h

Page 197: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

183

5.3.4. Effect of ultrasound treatment on particle size distribution

Particle size distribution of sorghum slurry was compared for three conditions;

1. Without ultrasound treatment, 2. Slurry sonicated for 1 min at 40% amplitude, and 3.

Slurry sonicated for 1 min at 100 % amplitude and is shown in the Fig. 5.18. Average

particle size was found to decrease from 302 µm to 163 µm (Slurry sonicated for 1 min

at 40% amplitude) and 115 µm (Slurry sonicated for 1 min at 100% amplitude) due to

cavitationally induced particle fragmentation. It can be also seen from Fig. 5.18 that

there are three major inflection points in the particle size distribution; which

corresponds to ~800 µm, 10-20 µm and ~1 µm. Due to ultrasound treatment of

sorghum slurry prior to liquefaction, peak area corresponding to ~800 µm decreases;

whereas, peak areas corresponding to 10-20 µm and ~1 µm increases. Inflection point

of ~800 µm must be a function of the milling process and mainly corresponds to large

pericarp particles. However, inflection point of ~1 µm may correspond to cell debris

produced due to disintegration of the cells of endosperm of sorghum. Such decrease in

the particle size distribution due to ultrasound is in agreement with the results reported

for corn slurry (Khanal et al., 2007) and for uranium ore slurry (Balasubrahmanyam et

al., 2006). Particle fragmentation increasing solid-liquid interfacial area and

enhancement in the convective diffusivity of leach solvent through micropores of ore

structure was attributed to microscopic convective motion created by acoustic

cavitation (i.e. shock wave propagation and microjet formation) at solid-liquid interface

and this results into enhancement in the rate of leaching of uranium

(Balasubrahmanyam et al., 2006).

Page 198: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

184

0

0.5

1

1.5

2

2.5

3

3.5

0.1 1 10 100 1000 10000particle size (µm)

% p

opul

atio

n

Without sonication

1 min at 40 %amplitude1 min at 100 %amplitude

Figure 5.18. Effect of prior ultrasound treatment on the particle size distribution in

30% w/v sorghum flour slurry.

5.3.5. Studies on effect of different process parameters on % saccharification

5.3.5.1. Effect of washing of cake obtained after hot filtration

Though 10 mL of distilled water was added to the cake (obtained after hot

filtration) for washing, second filtration yielded 15 mL of filtrate (H1 and H2; Table

5.3). This means washing and second filtration, followed by hot filtration has extracted

5 mL (out of 8 mL trapped inside the cake) of 1st filtrate (F1). Also it can be seen from

Table 5.3 (H1 and H2) that % saccharification increased from 74 to 84 (13% increase).

Increase in the % saccharification (which is based on the starch content in the sorghum

flour) is mainly through the recovery of oligosaccharides, which were otherwise

trapped inside the cake after hot filtration and were unavailable for further

saccharification. Similar behavior was observed in all the cases (Table 5.3).

Page 199: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

185

5.3.5.2. Effect of ultrasound treatment on % saccharification

It was observed (H1 and H3; Table 5.3) that ultrasound treatment of slurry

before liquefaction increased the volume of F1 from 72 to 75 mL (4% increase) and

increased the % saccharification from 74 to 81 (10% increase). This indicates that

ultrasound treatment prior to liquefaction improved filterability of the liquefied slurry

because of either increase in the DE of liquefact (Table 5.3) or particle disintegration

due to acoustic cavitation (Fig. 5.18). However, % increases in the volume of F1 and

% saccharification are not the same. This indicates that increase in the %

saccharification due to prior ultrasound treatment is not solely due to increase in the

volume of F1, but, there are few more factors responsible for this as discussed below.

If the step of washing of cake after 1st filtration was also provided, in addition to

ultrasound treatment prior to liquefaction, an increase in the % saccharification was

observed (H1 and H4; Table 5.3) from 74 to 90 (21% increase). Here it should be

clarified that this increase in the % saccharification (74 to 90) is a combination of two

effects; one, from 74 to 81 (9.5% increase) is due to the step of ultrasound treatment

(through the availability of additional starch granules for liquefaction and

saccharification, discussed in detail later in the text), and two, from 81 to 90 (11%

increase) is due to step of washing (through the recovery of additional oligosaccharides

from the cake after hot filtration). Similar effect of ultrasound treatment was observed

for germinated sorghum also (Table 5.3; G1, G2, G3, G4).

It is first necessary to understand the sorghum grain structure in order to

understand the reason behind the observed increase in the % saccharification due to

ultrasound treatment. Cells of inner floury endosperm are round with round starch

granules; whereas, the cells of the outer corneous endosperm are elongated with

polygonal starch granules and are filled with protein bodies (Chandrashekhar and

Page 200: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

186

Mazhar, 1999). Also, multicellular pericarp of sorghum grain unlike other cereals

consists of small starch granules (Palmer 1992). Starch granules of the floury

endosperm of sorghum are loosely associated with paper like sheets of protein material,

while in the corneous endosperm they are tightly packed within rigid protein matrix

(Chandrashekhar and Mazhar, 1999).

Treatment of sorghum flour (before cooking) with proteolytic enzymes like

pronase (Zhang and Hamaker, 1998), pepsin (Rooney and Pflugfelder, 1986) have

shown an increase in the starch digestibility by pancreatic α-amylase (Zhang and

Hamaker, 1998) and also the rate of starch hydrolysis by amyloglucosidase (Rooney

and Pflugfelder, 1986) due to hydrolysis of protein matrix surrounding starch granules.

Cooking of sorghum flour with reducing agents like sodium metabisulfite (Zhang and

Hamaker, 1998) or 2-mercaptoethanol (Chandrashekar and Kirleis, 1988) also

increased starch digestibility using pancreatic α-amylase (Zhang and Hamaker, 1998)

or degree of starch gelatinization (Chandrashekar and Kirleis, 1988) due to cleavage of

disulphide bonds linking protein surrounding starch granules. Sorghum proteins are

also reported to produce high molecular weight polymers by polymerization through

disulphide bonding of prolamins (Ezeogu et al. 2005) and large extended web like

microstructure (Hamaker and Bugusu, 2003; Wu et al., 2007) during the cooking of

sorghum flour, into which small starch granules (~ 5 µm) remain tightly trapped (Wu et

al., 2007). These changes related to protein structure during cooking of sorghum flour

with amylase, contribute to subsequent incomplete gelatinization, hydrolysis of starch,

and negative impact of protein content on conversion efficiency of sorghum to ethanol

(Wu et al., 2007).

Since the lipid fraction within starch granules is insufficient to saturate entire

quantity of amylose, amylose exists in two forms; free amylose and amylose-lipid

Page 201: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

187

complex (Tester et al., 2004). In the case of normal sorghum, this amylose-lipid

complex is reported to have endotherm or a temperature range of gelatinization (onset,

peak and ending of gelatinization) between 90 and 105 °C, whereas the remaining

starch i.e. free amylose and amylopectin has major endotherm between 60 and 80 °C

(Wu et al., 2007). Use of lysophospholipase along with amyloglucosidase in

saccharification resulted into higher degree of degradation of amylose-lipid complex

(due to hydrolysis of lysophospholipids), and an improvement in the rate and yield of

filtration of hydrolysate produced after completion of saccharification (due to partial

hydrolysis of micelles, which can clog pores of filter media) (Nebesny et al., 2002).

These findings and the presence of amylose-lipid complex in the hydrolysate (after

saccharification for 72 h using amyloglucosidase) proved by differential scanning

calorimetry (DSC) study (Nebesny et al., 2002) indicate that amylose-lipid complex

affects the final degree of saccharification that can be practically achieved.

Hence, it seems that amylose-lipid complex and the starch granules encased in

the protein matrix do not get fully gelatinized, and the ungelatinized fraction of starch

remains inaccessible for action of BLA. However, the cavitation phenomena caused by

ultrasound treatment prior to liquefaction may be releasing starch granules by

disrupting both; the protein matrix encasing starch granules and amylose-lipid complex.

This can also be observed with an increase in the peak area corresponding to the

particle diameter of 10-20 µm (Fig. 5.18); which is the diameter of sorghum starch

granule (Tester et al., 2004). These additional free starch granules get gelatinized and

are available for liquefaction, and further saccharification. This must be the reason for

an increase in the liquefaction performance and increase in the % saccharification due

to ultrasound treatment prior to liquefaction. Similar enhancement in the glucose

release using 14% w/v corn slurry (raw and cooked) due to prior sonication for 20 or 40

Page 202: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

188

s at amplitudes ranging from 180 to 299 µm has been reported (Khanal et al., 2007).

The increase in the glucose released of the sonicated samples was attributed to particle

size reduction, better mixing due to micro streaming effects, and the release of

additional lipid bound starch (Khanal et al., 2007). Higher conversion efficiency of

waxy sorghum to ethanol than that of normal sorghum (Wu et al., 2007) also supports

the hypothesis that higher quantity of amylose and hence, amylose-lipid complex

affects liquefaction and saccharification, and hence conversion efficiency of sorghum

to glucose and hence to ethanol. 1 to 10% increase in the DE of liquefact and ethanol

yield has been reported25 due to cavitation resulting out of sonication for 1-7.5 min

before cooking. It was claimed, but, not experimentally proved (Kinley et al., 2006)

that cavitational forces produced by sonication breaks complex proteins (i.e. proteins

not susceptible to hydrolysis to amino acids by proteolytic enzymes) to less complex

proteins, which are more bio-available to digestive systems of animals.

For normal healthy sorghum 90% saccharification has been reported (Aggarwal

et al., 2001). However, it should be clarified that experiments performed (Rooney and

Pflugfelder, 1986) for the production of glucose from sorghum (25% w/v slurry)

without any filtration step after liquefaction, as the process was optimized for

bioethanol production. Whereas, in the present work, filtration was done after

liquefaction, which result into trapping of oligosaccharides in the wet cake even after

washing procedure and hence giving lower % saccharification. When experiments

were performed in the present work for the production of glucose from healthy

sorghum without filtration step, values of % saccharification were observed to be 87–

89 and 93–95 without and with ultrasound treatment, respectively.

In the case of blackened sorghum (grain mold), marginal increase in the %

saccharification was observed due to prior ultrasound treatment. It can be seen from

Page 203: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

189

Table 5.3 that at optimized concentration of BLA, DE of liquefact obtained with

blackened sorghum was 9-10, which was much lesser than expected DE i.e. 15. Hence

BLA concentration was increased by about 40 % to get liquefact DE 16-17 (Table 5.3).

Reason could be attributed to the leaching of mycotoxins and phenolic compound in the

blackened pericarp upon sonication and inhibiting the action of BLA in liquefaction,

which require additional BLA content for liquefaction and AG content in

saccharification. Thus, lower value of blackened sorghum is partially offset by higher

BLA dosage requirement.

During germination and seedling growth of sorghum, different enzymes

including protease, endoprotease, limit dextrinase, α-amylase and endo-β-gluconase get

produced (Aisen et al., 1983). In case of germinated sorghum (germination time 24 h),

there is significant increase (~30%) in liquefact DE due to prior ultrasound treatment.

This could be attributed to weakening of cell wall and protein matrix around starch

granules due to attack of endoprotease (Aisen et al., 1983) during germination and

hence effective release of starch granules from protein matrix by ultrasound treatment.

The percentage saccharification, however, didn’t go above 90% even after germination.

Page 204: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

190

5.3.6. Economics of the process of production of glucose from sorghum of

different varieties

In the chemical project economics, cost of production plays a key role. It

represents operating expenses, which are of recurring in nature. They have significant

impact on the selling price and ultimately profitability. Operating expenses are incurred

after the plant is commissioned and the production begins. There are mainly two

parameters which being major and have direct impact on the cost of production viz. 1.

Raw materials; 2. Utilities. (Mahajani and Mokashi, 2005) In this section, processing

cost for production of glucose from sorghum was determined by considering cost of

raw materials and utilities. Cost of raw materials and utilities required for production of

glucose from healthy, blackened and germinated sorghum are compared in Table 5.4

and 5.5. Whereas comparison of processing cost is given in the Table 5.6.

Page 205: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

191

Table 5.4. Cost of raw material to process 1 kg of starch

Sorghum contains approximately 70% starch. Hence starting quantity of sorghum will be 1.43 kg

Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum Parameter Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs

Starch or sorghum

1 16 16 1.43 5 7.1 1.43 3 4.3 1.43 5 7.1

Water

2.67 0.04 0.11 3.81 0.04 0.15 3.81 0.04 0.15 3.81 0.04 0.15

BLA required for liquefaction

0.0008 250 0.2 0.0009 250 0.225 0.0013 250 0.325 0.0008 250 0.2

AG required for saccharification

0.0006 330 0.198 0.0006 330 0.198 0.0007 330 0.231 0.0006 330 0.198

Total - - 16.51 - - 7.72 - - 5 - - 7.65

Where, Q Quantity of material, kg R Rate of material, Rs/kg C Cost incurred, Rs BLA B. licheniformis α-amylase AG Amyloglucosidase

Page 206: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

192

Table 5.5. Cost of utilities to process 1 kg of starch

Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum Parameter Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs

Steam for liquefaction1,2 at 85 °C for 1.5 h

0.53 1 0.53 0.76 1 0.76 0.76 1 0.76 0.76 1 0.76

Steam for saccharification1,2, at 55 °C for 24 h

1.18 1 1.18 1.69 1 1.69 1.69 1 1.69 1.69 1 1.69

Filtration & bleaching and color removal4

- - 0.2 - - 0.429 - - 0.429 - - 0.429

Cost of steam for evaporation3 2.67 1 2.67 3.81 1 3.81 3.81 1 3.81 3.81 1 3.81

Total - - 4.58 - - 6.69 - - 6.69 - - 6.69

Note: Steam requirement for the batch process is calculated based on following assumptions

1. 20% of the enthalpy is lost to surroundings per h from reaction mixture 2. steam input is saturated vapor and steam output is saturated liquid

i.e. steam requirement = enthalpy / latent heat of water 540 kcal/kg 3. For evaporating 90% of water produced glucose syrup and Evaporation efficiency = 90% 4. Total cost of both filtrations (i.e. after liquefaction and saccharification) = 0.2 Rs/kg of starch substrate Where, Q Quantity of material, kg

R Rate of material, Rs/kg C Cost incurred, Rs

Page 207: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

193

Table 5.6. Comparison of processing cost or cost production of glucose from sorghum

Parameter Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum

Cost of Raw material 16.51 7.72 5 7.65

Cost of Utilities 4.58 6.69 6.69 6.69

Total cost 21.09 14.41 11.69 14.34

Glucose produced, kg 1.11 1 0.95 1

Processing cost, Rs/kg of glucose produced 19 14.41 12.3 14.34

Note: 100 % saccharification for pure starch (due to hydrolytic gain 1 kg starch produces 1.11 kg glucose) 90 % saccharification for health and germinated sorghum 85 % saccharification for blackened sorghum

Market cost of glucose syrup (84% b w) = 24 Rs/kg; Market cost of dry glucose = 30 Rs/kg

Page 208: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

194

From Table 5.6, it can be seen that processing cost or cost of production per kg

of glucose produced from healthy, blackened sorghum is about Rs. 4.5 less than that

from isolated starch. Also this cost is 60% and 51% of market cost of glucose syrup for

healthy sorghum and blackened sorghum, respectively. This means that the production

of glucose from sorghum is economically feasible. According to Suresh et al., 1999a,

industrial grade damaged sorghum grains (inclusive of 30-55% sound grains) are

available in large quantity at Food Corporation of India (FCI) at 10 times lower rate

than the fresh grains and it contains around 50% starch. If this industrial grade sorghum

is used as starting material then economy of the process may further improve.

5.4. Conclusions

Production of glucose from sorghum flour involves two steps viz. 1.

Liquefaction of flour using Bacillus licheniformis α-amylase and 2. Saccharification

using amyloglucosidase.

1. In the present work, use of ultrasound in the production of glucose from sorghum

flour has been explored.

2. The value of % saccharification to glucose using healthy sorghum flour (at

optimized reaction conditions for liquefaction and saccharification) was in the range

of 70- 90% depending upon following factors; 1. Ultrasound treatment of sorghum

slurry before liquefaction, and 2. Washing of cake (obtained after hot filtration)

followed by 2nd filtration and mixing of both the filtrates.

3. Ultrasound treatment to sorghum slurry prior to liquefaction appears to disrupt

hydrophobic protein matrix surrounding starch granules and amylose-lipid complex

due to physical effects of acoustic cavitation, like shock wave propagation and

microjet formation in the vicinity of liquid-solid interface. This frees starch

Page 209: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter5: Enzymatic production of glucose from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

195

granules and these additional starch granules are made available for further action

of α-amylase and amyloglucosidase. A significant increase (~8–10%) in the %

saccharification was observed.

4. In this work, blackened and germinated sorghum were also used for the production

of glucose and % saccharification was 85% and 90%, respectively, with ultrasound

treatment before liquefaction.

5. This means that integration of short ultrasound treatment (about 1 min) in the the

production of glucose from dry milled sorghum and its possible subsequent use in

the bioethanol production will result into increase in the production of glucose and

subsequently ethanol, and hence may improve the economic feasibility of the

process.

Page 210: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

196

6. Enzymatic production of maltose syrup from sorghum

Page 211: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

197

6.1. Introduction

In the section 5.1, the necessity to exploit industrial application for normal and

blackened sorghums, in order to make sorghum cultivation economically viable for

farmers through value added products, has been discussed. A little literature is

available on value addition to sorghum through; production of glucose, production of

ethanol and isolation of starch (discussed in detail in the chapter 3). However there is

no literature available on value addition to sorghum through production of maltose

syrup.

In the present work, sorghum flour was used directly for liquefaction and

saccharification in the similar fashion that used in the production of glucose from

sorghum. Liquefaction part in the process was the same as that was optimized earlier

and discussed in the chapter 5. Hence, objectives of the present work were to optimize

saccharification process to produce maltose from three varieties of sorghum i.e.

healthy, blackened and germinated, and to study the effect of ultrasound treatment

prior to liquefaction on the performance of liquefaction and saccharification

processes.

6.2. Experimental

6.2.1. Materials

3,5-Dinitrosalicylic acid (DNSA), soluble starch, maltose, dextrose, MeCN for

chromatography LiChrosolv and other chemicals were purchased from E. Merck Ltd

(India). Commercial preparations in liquid formulation of Bacillus licheniformis α-

amylase (BLA) (EC number 3.2.1.1), Barley β-amylase (BBA) (EC number 3.2.1.2),

and Pullulanase (PL) (EC number 3.2.1.41) were gifted by Advance Enzyme

Technologies Pvt Ltd (India). Healthy sorghum and blackened sorghum were

Page 212: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

198

purchased from the local market.

6.2.2. Analytical Methods

6.2.2.1. Measurement of protein concentration and reducing sugar concentration

and concentrations of malto-oligosaccharides.

The protein concentration of the free enzyme was determined using the

modified Folin–Lowry method (Lowry et al. 1951) using BSA (0–0.6 mg/mL) as a

standard. The reducing sugar concentration was measured using the DNSA method

(Miller 1959) with maltose (0–1 mg/mL) as a standard. Concentrations of glucose

and malto-oligosaccharides up to maltoheptaose were measured using the HPTLC

method. Details of modified folin lowry method, DNSA method and HPTLC method

are given in the Appendix A.

6.2.2.2. Measurement of moisture content

Sorghum flour was kept at 80 °C, till constant weight was obtained and the

moisture content in sorghum flour was measured using mass balance. Detailed

method to measure the moisture content is given in the Appendix A.

6.2.2.3. Measurement of particle size distribution of sorghum flour.

Particle size distribution of the ground sorghum flour was determined by using

the Coulter Counter Particle Size Analyzer (LS 230) based on laser light diffraction.

6.2.2.4. Measurement of starch content of sorghum flour.

Enzymatic method of Measurement of starch content of sorghum flour is

described in the 5.2.2.4.

Page 213: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

199

6.2.3. Amylolytic activity measurement methods

6.2.3.1. Free bacterial α-amylase (BLA)

Procedure for measurement of activity of free Bacillus licheniformis α-

amylase (BLA) is detailed in the chapter 4.

6.2.3.2. Barley β-amylase (BBA)

0.9 mL of 1 % (w/v) gelatinized starch solution (pH 5.5, 50 mM citrate buffer)

was incubated with 0.1 mL of 5000 fold diluted barley β-amylase (BBA) solution at

50 °C for 10 min. Then, 1 mL of DNSA reagent was added to the reaction mixture to

stop the reaction. The resulting solution was heated in a boiling water bath for 10

min. The variation in the concentration of reducing sugar was measured by DNSA

method using maltose as a standard. One enzyme unit was defined as that required to

liberate one micromole of reducing sugar (maltose equiv) per min under conditions of

assay.

min 10 mL) (0.1aliquot enzyme of volume 180) (i.e. maltose of wt molfactordilution enzyme produced equiv) (maltosesugar reducing of g BBAU/mL

×××

min 10 mL) (0.1aliquot enzyme of volume 362) (i.e. maltose of wt mol(10000)factor dilution enzyme

mL) (1 mixtureassay of volumemin) 0at C -min 10at (C

BBAU/mL

RSRS

×××

×

=

where CRS = Concn of reducing sugars (maltose equiv) in assay mixture, µg/mL

6.2.3.3. Free pullulanase (PL).

Procedure for the measurement of activity of free pullulanase (PL) is detailed

in the section 5.2.3.3.

Page 214: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

200

6.2.4. Thermostability study of free Barley β-amylase (BBA).

Solution of barley β-amylase in acetate buffer (500 fold diluted commercial

formulation i.e. 2.5 BBAU/mL, 50 mM, pH 5.5) was incubated at desired temperature

under shaking conditions (180 rpm) in the absence of substrate for 24 h. Samples of

BBA solution were taken at regular time intervals and were for its amylolytic activity.

6.2.5. Production of maltose syrup from sorghum: Experimental work

Production of maltose syrup from sorghum flour consists of two reaction

steps:

1. Liquefaction using B. licheniformis α-amylase (BLA) in which gelatinisation of

free starch granules and dextrinization (depolymerisation) of gelatinized starch take

place simultaneously. This produces a mixture of malto-oligosaccharides, linear

and branched dextrins.

2. Saccharification using barley β-amylase (BBA) with or without pullulanase, in

which BBA cleaves second α(1→4) linkage from non-reducing end of glucose

polymer and produces maltose. Use of only BBA in saccharification will result into

production of maltose and β limit dextrins, due to inability of BBA to bypass

α(1→6) linkages. If pullulanase is used along with BBA, pullulanase will cleave

α(1→6) linkage and BBA can then attack rest of the chain in the β limit dextrins.

Hence saccharification using BBA and pullulanase will result into a mostly maltose

with small quantities of glucose and maltotriose.

Process of production of maltose syrup from sorghum flour, which is used in

the present experimental work, is shown in the Fig. 6.1. Chemistry of the process has

also been depicted from the Fig. 6.1. Chemistry of liquefaction of starch and

saccharification of liquefied starch is discussed in detail in chapter 2.

Page 215: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

201

Figure 6.1. Process flow sheet for production of maltose syrup from sorghum

Gelatinization of free starch granules Dextrinization of gelatinized starch molecules.

Removal proteins and fibers to prevent colour formation due to solubilization of proteins

G–G–G–G–G–G–G–G–G–G–G–G–G–G Starch | G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G | G–G–G–G–G–G–G–G–G G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G

Saccharification using Barley β-amylase (BBA) with

or without pullulanase (PL) 50 °C, 5.5 pH

Hot filtration

Filtration and Purification

Maltose syrup

Milling

Preparation of slurry

Grain sorghum

Removal of fine particles, lipids, proteins and unreacted starch gel

Glucose, malto-oligosaccharides, linear and branched dextrins produced after liquefaction

G–G G G–G–G–G–G–G G–G–G–G–G G | G–G–G–G–G G–G–G–G–G–G–G–G–G–G | G–G–G–G–G–G–G–G–G G–G–G–G–G–G–G–G–G–G–G–G G–G–G–G–G–G–G–G

Using BBA only Using BBA and PL G–G G G–G–G G–G and small | quantity of glucose

G–G–G–G–G Maltose, β-limit dextrins

Liquefaction using bacterial α-amylase (BLA)

85 °C, 6 pH

Page 216: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

202

Sorghum grains were milled using old fashioned flour mill (two stones of 15″

diameter × 2″ height dimensions) and this flour was used for liquefaction followed by

its subsequent saccharification.

6.2.5.1. Liquefaction of sorghum flour.

Experimental set-up for liquefaction of sorghum flour is shown in the chapter

5 (Fig. 5.2). Process of liquefaction of sorghum slurry is already optimized to

produce sorghum liquefact with DE of around 15 (refer Chapter 5). Liquefaction of

sorghum flour was performed in a 250 mL stoppered conical flask containing 100 mL

sorghum slurry (30% w/v), which was magnetically stirred at the optimized

conditions of pH, temperature, CaCl2 concentration and BLA concentration for 1.5 h

i.e. 6, 85 °C, 200 ppm, and 0.086% v/w of sorghum starch, respectively.

6.2.5.2. Optimization of saccharification.

Saccharification process was first optimized for the processing temperature by

performing thermostability study of BBA and also performing saccharification of

sorghum liquefact at different temperatures.

At the end of liquefaction (i.e. 1.5 h), pH of the reaction mixture was reduced

to 5.5 using acetic acid. Then sorghum liquefact was filtered hot using muslin cloth

and filtrate (F1) was collected. Cake obtained after hot filtration was mixed well with

10 mL distilled water for washing of the cake and filtered. This second filtrate (F2)

was mixed with first filtrate (F1). Then this reaction mixture was kept under shaking

conditions (150 rpm) for 24 h under optimized conditions of temperature and enzyme

(BBA and / or Pullulanase) concentration. At regular time intervals, samples were

withdrawn and diluted to approximately 1% w/v using 0.1 N HCl solution. Samples

(1% w/v) were then centrifuged at 270 g for 10 min and supernatant was analyzed for

the concentration of reducing sugar (maltose equiv). Percentage saccharification on

Page 217: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

203

the basis of original starch content in the sorghum flour is defined as follows;

100Q mgin flour sorghum ofamount

mLin mixturereaction of volume mg/mLin Ccationsaccharifi % f ××

×= (6.1)

where Cf is concentration of reducing sugar (maltose equiv) at steady state (i.e. 24 h)

Q is mg of reducing sugar (maltose equiv) produced per mg of sorghum flour

using method described in 2.2.3. (Saccharification was performed using

BBA and PL at 5.5 pH and 50 °C) i.e. (% starch content /100) × 1.05

Since the calculation of % saccharification is based on the original starch content in

the sorghum flour, the term % saccharification can be also regarded as % yield of

maltose.

Production of maltose syrup from sorghum flour consists of following five

steps in sequence; 1. Ultrasound treatment of sorghum slurry, 2. Liquefaction of

sorghum slurry using B. licheniformis α-amylase, 3. Hot filtration of liquefied

sorghum slurry, 4. Washing of cake (obtained after hot filtration) followed by second

filtration and mixing of both the filtrates, and 5. Saccharification of filtrate F1 or

mixture of F1 and F2 using barley β-amylase with or without pullulanase.

Experiments were performed for the production maltose syrup using healthy,

blackened, and germinated sorghum without or with steps 1 or 4, and without or with

the use of pullulanase in saccharification.

6.3. Results and Discussion

6.3.1. Optimization of saccharification

6.3.1.1. Properties of free barley β-amylase and pullulanase

Percentage relative activity vs. pH profile and % relative activity vs.

temperature profile of Barley β-amylase are given in the Fig. 6.2 and 6.3,

Page 218: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

204

respectively. Optimum pH and optimum temperature of barley β-amylase using assay

procedure to measure enzyme activity were 5.5 and 50 °C, respectively. Enzyme

activity at optimum conditions (according to assay procedure) and protein content of

commercial formulation of BBA were 12500 AGU/mL and 74 mg/mL, respectively.

0102030405060708090

100110

3 3.5 4 4.5 5 5.5 6 6.5 7pH

% re

lativ

e ac

tivity

50 °C

Figure 6.2. Enzyme activity-pH temperature profile at 50 °C.

0102030405060708090

100110

30 35 40 45 50 55 60 65 70 75Temperature °C

% re

lativ

e ac

tivity

Fig 6.3. Enzyme activity-temperature profile at pH of 5.5

Page 219: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

205

Percentage relative activity vs. pH profile and % relative activity vs.

temperature profile of pullulanase are provided in the chapter 5. Optimum pH and

optimum temperature of pullulanase using assay procedure to measure the enzyme

activity were 4 and 60 °C, respectively. Enzyme activity at optimum conditions

(according to assay procedure) and protein content of commercial formulation of

pullulanase were 2950 PLU/mL and 50 mg/mL, respectively.

Properties of free barley β-amylase (BBA) and pullulanase (PL) are

summarized in the Table 6.1.

Table 6.1. Properties of barley β-amylase and pullulanase

Parameter Free barley β-amylase (BBA) Free pullulanase (PL)

Optimum pH 5.5 3.8–4.3

Optimum temperature (°C) 50 60

Activity 12500 BBAU/mL 2950 PLU/mL

Protein content (mg/mL) 74 50

Specific activity 169 (BBAU/mg of protein) 59 (PLU/mg of protein)

6.3.1.2. Thermostability of barley β-amylase and optimization of operating

temperature for saccharification

Optimum temperature for BBA using the specified assay procedure was found

to be 50 °C (Table 6.1). However, optimum operating temperature for

saccharification using BBA may not be the same as that obtained using the assay

procedure. Hence, study of thermostability of BBA was essential at an optimum pH

of 5.5. In addition to the thermostability check at different temperatures, experimental

runs on saccharification of sorghum liquefact utilizing BBA or BBA + PL at different

temperatures were also performed. Thermostability of BBA was first checked at 50

Page 220: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

206

°C. It was observed that % relative activity of BBA decreased to 3% of the original at

50 °C and to 20% of the original at 40 °C within first 3 hrs of incubation in the

absence of substrate. But, Figs. 6.5. and 6.6 shows that BBA is active till 24 h also.

This means that during the saccharification presence of substrate stabilizes BBA and

it remains active till 24 h, though in the thermostability study in the absence of

substrate BBA activity decreases drastically. It can be seen from Figs. 6.5 and 6.6 that

the optimum operating temperature for saccharification of sorghum liquefact to

produce maltose is around 50 °C.

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8Time (h)

% re

lativ

e ac

tivity

50

40

Temperature, °C

Figure 6.4. Thermostability of BBA

Fig. 6.5. and 6.6 also shows that use of pullulanase along with BBA in the

saccharification increases concentration of reducing sugars (maltose equiv.) from 174

to 204. This happens because pullulanase cleaves α(1→6) linkage and makes linear

part of the dextrin available for action of BBA.

Page 221: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

207

0

20

40

60

80

100

120

140

160

180

0 4 8 12 16 20 24 28Time (h)

Con

cn o

f red

ucin

g su

gar (

mg/

mL)

40 4550

Temperature, °C

Figure 6.5. Comparison of hydrolysis curves at different temperatures. Reaction conditions: 20 mL liquefact, 1.1 BBAU/mL; 5.5 pH and 50 °C

020406080

100120140160180200220

0 4 8 12 16 20 24 28Time (h)

Con

cn o

f red

ucin

g su

gar (

mg/

mL)

40 45

50

Temperature, °C

Figure 6.6. Comparison of hydrolysis curves at different temperatures.

Reaction conditions: 20 mL liquefact, 1.1 BBAU/mL; 0.37 PLU/mL;

5.5 pH and 50 °C

Page 222: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

208

Optimized conditions for liquefaction of sorghum flour and saccharification to

maltose syrup are shown in the Table 6.2.

Table 6.2. Optimized parameters for Liquefaction and Saccharification

Parameter Liquefaction Saccharification

Temperature (°C) 85 50

pH 6 5.5

Slurry concentration 30% w of sorghum flour / v of slurry n.a.

BLA concentration 0.06% v/w of sorghum flour i.e.

0.086% v/w of sorghum starch

n.a.

BBA concentration n.a. 0.04 %v/ w of starch

CaCl2 concentration 200 ppm n.a.

6.3.2. Saccharification of sorghum liquefact

After liquefaction, liquefied sorghum slurry was filtered hot using muslin

cloth. Necessity of hot filtration and washing of the cake after hot filtration is

discussed in the section 5.3.3. The values of % saccharification using BBA, attained

after 24 h, for all the three varieties of sorghum were dependent upon the following

factors; 1. Ultrasound treatment on sorghum slurry before liquefaction, 2. Washing of

cake (obtained after hot filtration) followed by second filtration and mixing of both

the filtrates, and 3. Use of pullulanase along with BBA during saccharification, which

are summarized in the Table 6.3.

Page 223: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

209

Table 6.3. Summary of effect of ultrasound treatment and washing on liquefaction

and saccharification performance.

Run

No

BLA concn,

% v/w

CRS,

mg/mL

F1,

mL

F2,

mL

F1 + F2,

mL

Cf

mg/mL % saccharification

H1 0.086 31 72 0 73 185 61 (S N; W N; PU N) H2 0.086 28 72 15 88 173 69 (S N; W Y; PU N) H3 0.086 28 72 15 88 188 75 (S N; W Y; PU Y) H4 0.086 34 75 0 75 195 66 (S Y; W N; PU N) H5 0.086 32 75 14 89 186 75 (S Y; W Y; PU N) H6 0.086 32 75 14 89 212 86 (S Y; W Y; PU Y) G1* 0.086 33 75 0 75 180 61 (S N; W N; PU N) G2* 0.086 32 75 14 89 172 69 (S N; W Y; PU N) G3* 0.086 32 75 14 89 195 79 (S N; W Y; PU Y) G4* 0.086 48 78 0 78 187 66 (S Y; W N; PU N) G5* 0.086 44 78 12.5 90.5 183.5 75 (S Y; W Y; PU N) G6* 0.086 44 78 12.5 90.5 209 86 (S Y; W Y; PU Y) B1 0.125 40 72 0 72 188 61 (S N; W N; PU N) B2 0.125 37 72 14 86 183 71 (S N; W Y; PU N) B3 0.125 37 72 14 86 212 83 (S N; W Y; PU Y) B4 0.125 39 75 0 75 195 66 (S Y; W N; PU N) B5 0.125 36 75 12.5 87.5 189 75 (S Y; W Y; PU N) B6 0.125 36 75 12.5 87.5 217 86 (S Y; W Y; PU Y)

S N: No ultrasound treatment of sorghum slurry before liquefaction S Y: Ultrasound treatment of sorghum slurry before liquefaction for 1 min at 100

%amplitude W N: No washing of cake after first filtration W Y: Washing of cake after first filtration with 10 mL distilled water, followed by

second filtration F1: Volume of filtrate after first filtration F2: Volume of filtrate after second filtration * Liquefaction time 1 h, Sorghum germinated for 24 h PL N : Pullulanase not used in saccharification PL Y : Pullulanase not used in saccharification pH = 5.5, temperature = 50 °C, BBA concn = 0.04 %v/ w of starch, PL concn = 0.057% v/w of starch, Liquefaction time = 1.5 h, saccharification time = 24 h.

Page 224: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

210

6.3.3. Studies on effect of different process parameters on % saccharification

Effect of washing of cake obtained after hot filtration and ultrasound treatment

before liquefaction on % saccharification remains exactly the same as discussed in

chapter 5. Hence will not be detailed here and only brief is provided.

Due to the incorporation of washing step, increase in the % saccharification

was observed. This increase in the % saccharification (which is based on the starch

content in the sorghum flour) is mainly through the recovery of oligosaccharides,

which were otherwise trapped inside the cake after hot filtration and were unavailable

for further saccharification. Similar effect of washing of cake after hot filtration was

observed for all healthy, germinated, and blackened sorghum (Table 6.3).

Increase in the % saccharification due to the step of ultrasound treatment is

through the availability of additional starch granules because of the disruption of

hydrophobic protein matrix surrounding starch granules and amylose-lipid complex

for liquefaction and saccharification, discussed in detail earlier in the chapter 5.

Similar effect of ultrasound treatment was observed for all healthy, germinated, and

blackened sorghum (Table 6.3).

Experiments to study effect of use of pullulanase were performed with

washing of cake after hot filtration and without or with ultrasound treatment before

liquefaction. It was observed that in the absence of ultrasound treatment, %

saccharification increases from 69 to 75 (Table 6.3; H2 and H3) due to the use of

pullulanase in addition to BBA in saccharification. Whereas, in the presence of

ultrasound treatment, % saccharification increases from 75 to 86 (Table 6.3; H5 and

H6) due to the use of pullulanase in addition to BBA in saccharification. This also

shows that effect of pullulanase use is more pronounced when ultrasound treatment is

provided. Reason could be attributed to the availability of additional starch granules

Page 225: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

211

due to sonication.

6.3.4. Economics of the process of production of maltose syrup from sorghum of

different varieties

In the chemical project economics, cost of production plays a key role. It

represents operating expenses, which are of recurring in nature. They have significant

impact on the selling price and ultimately profitability. Operating expenses are

incurred after the plant is commissioned and the production begins. There are mainly

two parameters which are major and have direct impact on the cost of production viz.

1. Raw materials cost, and 2. Cost of utilities. (Mahajani and Mokashi, 2005) In this

section, processing cost for the production of maltose syrup from sorghum has been

determined by considering the cost of raw materials and utilities. Cost of raw

materials and utilities required for production of maltose from healthy, blackened and

germinated sorghum are compared in Table 6.4 and 6.5. Whereas comparison of

processing cost is given in the Table 6.6.

Here it should be remembered that processing cost has been determined for

production of dried maltose syrup. In order to obtain pure maltose, selective

crystallization must be done and its cost has not considered here.

Page 226: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

212

Table 6.4. Cost of raw material to process 1 kg of starch

Sorghum contains approximately 70% starch. Hence starting quantity of sorghum will be 1.43 kg

Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum Parameter Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs

Starch or sorghum

1 16 16 1.43 5 7.1 1.43 3 4.3 1.43 5 7.1

Water

2.67 0.04 0.11 3.81 0.04 0.15 3.81 0.04 0.15 3.81 0.04 0.15

BLA required for liquefaction

0.0008 250 0.2 0.0009 250 0.225 0.0013 250 0.325 0.0008 250 0.2

BBA required for saccharification

0.0004 1500 0.6 0.0004 1500 0.6 0.0005 1500 0.75 0.0004 1500 0.6

Total - - 16.91 - - 8.12 - - 5.51 - - 8.10

Where, Q Quantity of material, kg R Rate of material, Rs/kg C Cost incurred, Rs BLA B. licheniformis α-amylase BBA Barley β-amylase

Page 227: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

213

Table 6.5. Cost of utilities to process 1 kg of starch

Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum Parameter Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs

Steam for liquefaction1,2 at 85 °C for 1.5 h

0.53 1 0.53 0.76 1 0.76 0.76 1 0.76 0.76 1 0.76

Steam for saccharification1,2, at 55 °C for 24 h

0.98 1 0.98 1.41 1 1.41 1.41 1 1.41 1.41 1 1.41

Filtration & bleaching and color removal4

- - 0.2 - - 0.429 - - 0.429 - - 0.429

Cost of steam for evaporation3 2.67 1 2.67 3.81 1 3.81 3.81 1 3.81 3.81 1 3.81

Total - - 4.38 - - 6.41 - - 6.41 - - 6.41

Note: Steam requirement for the batch process is calculated based on following assumptions

1. 20% of the enthalpy is lost to surroundings per h from reaction mixture 2. steam input is saturated vapor and steam output is saturated liquid

i.e. steam requirement = enthalpy / latent heat of water 540 kcal/kg 3. For evaporating 90% of water from produced maltose syrup and Evaporation efficiency = 90% 4. Total cost of both filtrations (i.e. after liquefaction and saccharification) = 0.2 Rs/kg of starch substrate Where, Q Quantity of material, kg

R Rate of material, Rs/kg C Cost incurred, Rs

Page 228: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

214

Table 6.6. Comparison of processing cost or cost production of maltose syrup from sorghum

Parameter Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum

Cost of Raw material 16.91 8.12 5.51 8.10

Cost of Utilities 4.38 6.41 6.41 6.41

Total cost 21.29 14.53 11.92 14.53

Maltose produced, kg Reducing sugars produced, kg

0.73 1.05

0.62 0.89

0.62 0.89

0.62 0.89

Processing cost, Rs/kg of maltose produced Rs/kg of reducing sugars

produced

29.2 20.3

23.4 16.33

19.2 13.4

23.4 16.3

Note: 100 % saccharification for pure starch (due to hydrolytic gain 1 kg starch produces 1.05 kg maltose) 85 % saccharification for healthy, blackened and germinated sorghum Reducing sugars (i.e. dry solids in maltose syrup) produced by using barley beta-amylase contains 70% maltose-assumption.

Market cost of dry maltose (imported from Japan) = 250 Rs/kg

Note: While comparing cost of dry maltose, it should be remembered that in the calculation of processing cost per kg of maltose produced, crystallization cost is not considered.

Page 229: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

215

6.4. Conclusions

Production of maltose syrup from sorghum flour involves two steps viz. 1.

Liquefaction of flour using Bacillus licheniformis α-amylase and 2. Saccharification

using Barley β-amylase without or with pullulanase.

1. In the present work, use of ultrasound in the production of maltose syrup from

sorghum flour has been explored.

2. The value of % saccharification to maltose using healthy sorghum flour (at

optimized reaction conditions for liquefaction and saccharification) was in the

range of 70- 90% depending upon following factors; 1. Ultrasound treatment of

sorghum slurry before liquefaction, 2. Washing of cake (obtained after hot

filtration) followed by 2nd filtration and mixing of both the filtrates, and 3. Use of

pullulanase in the saccharification.

3. Ultrasound treatment to sorghum slurry prior to liquefaction appears to disrupt

hydrophobic protein matrix surrounding starch granules and amylose-lipid

complex due to physical effects of acoustic cavitation, like shock wave

propagation and microjet formation in the vicinity of liquid-solid interface. This

frees starch granules and these additional starch granules are made available for

further action of α-amylase and barley β-amylase. A significant increase (~8–

10%) in the % saccharification was observed.

4. Use of pullulanase in the saccharification along with barley β-amylase increased

% saccharification through cleavage of α(1→6) linkages that results into

availability of linear chains for action of barley β- amylase.

5. This means that integration of short ultrasound treatment (about 1 min) in the

production of maltose syrup from dry milled sorghum may improve the economic

feasibility of the process.

Page 230: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

216

6.5. Alternative approaches for value addition to sorghum

In the present work, possibility of value addition to healthy, blackened and

germinated sorghum has been explored through the production of glucose and maltose

syrup. However, there are other products also, that can be produced by using sorghum

as a starting material. Flow sheet for the production of different products is provided

in the Fig. 6.7. In fact industry can switch from one product to another depending

upon the market needs.

First step in the production of any product is liquefaction, i.e. simultaneous

gelatinization of free starch granules and dextrinization of gelatinized starch using

bacterial α-amylase. Liquefaction can be continued to achieve desired DE (15, 20 or

30). Then the sorghum liquefact needs to be filtered, purified and dried to get

maltodextrins.

Flow sheet to produce glucose and maltose syrup has been already discussed

in detail in chapter 5 and 6 respectively.

Glucose syrup (DE > 96) produced can further be processed using glucose

isomerase to produce fructose syrup. (Refer Chapter 2)

Sorghum liquefact without any hot filtration can be saccharified using

amyloglucosidase to glucose syrup with DE greater than 90. Then, this syrup is

fermented to produce ethanol. After completion of fermentation, ethanol is distilled

out of this mixture. This product is termed as grain based alcohol or bioethanol.

Remanent stillage can be dried to produce DDGS, which can be used as animal feed

or can be anaerobically digested to produce bio-gas.

Page 231: Studies in depolymerization of natural polysaccharides--PhD thesis

Chapter 6: Enzymatic production of maltose syrup from sorghum

Studies in the Enzymatic depolymerisation of natural polysaccharides

217

Milling

Liquefaction using bacterial α-amylase

Hot filtration

Saccharification using barley β-amylase (BBA)

w/o or with pullulanase (PU)

Filtration and Purification

Maltose syrup

Preparation of slurry

Grain sorghum

Saccharification using amyloglucosidase w/o or with pullulanase

Filtration and Purification

Glucose syrup

Saccharification of liquefact to Glucose using amyloglucosidase

Further filtration and Purification to produce Maltodextrins of different DE 15, 20 or 30

Ethanol

DE ~ 15 DE ~ 15

Fermentation followed by distillation

Fructose syrup Glucose isomerase

Figure 6.7. Production schemes of different products from sorghum

Page 232: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

218

References

Page 233: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

219

Aggarwal N. K.; Nigam P.; Singh D.; Yadav B. S., Process optimization for the

production of sugar for the bioethanol industry from sorghum, a non-conventional

source of starch. World Journal of Microbiology & Biotechnology, 2001, 17, 411-415.

Agu, R.C., Palmer, G.H., 1998. A reassessment of sorghum for lager-beer brewing.

Bioresource Technology, 1998, 66, 253–261.

Aisen, A. O.; Palmer, G. H.; Stark, J. R. ; The Development of Enzymes During

Germination and Seedling Growth in Nigerian Sorghum. Starch, 1983, 35, 316-320.

Alexander, R. J. ‘Maltodextrins: Production, Properties and Applications.’ in Starch

Hydrolysis Products: Worldwide Technology Production and Applications. Schenck,

F. W., Hebeda, R. E. Eds.; VCH Publishers, New York, 1992, pp 233-250.

Awika, J.M., McDonough, C.M., Rooney, L.W. Decorticating sorghum to concentrate

healthy phytochemicals. J. of Agricultural and Food Chemistry, 2005, 53, 6230–6234.

Baks, T.; Bruins, M. E.; Janssen, A. E. M.; Boom, R. M. Effect of Pressure and

Temperature on the Gelatinization of Starch at Various Starch Concentrations.

Biomacromolecules, 2008, 9, 296–304.

Balasubrahmanyam, A.; Roy, S. B.; Chowdhury, S.; Hareendran, K. N. and Pandit, A.

B. Enhancement of the Leaching Rate of Uranium in the Presence of Ultrasound. Ind.

Eng. Chem. Res., 2006, 45, 7639-7648.

Ball, S.G. Recent reviews on the biosynthesis of the plant starch granule. Trends in

Glycoscience and Glycotechnology, 1995, 7, 405–415.

Ball, S.G.; van de Wal, M.H.B.J.; Visser, G.F. Progress in understanding the

biosynthesis of amylose. Trends in Plant Science, 1998, 3, 462–467.

Balole, T.V. & Legwaila, G.M. Sorghum bicolor (L.) Moench, Internet Record from

Protabase. Jansen, P.C.M. & Cardon, D. (Editors). PROTA (Plant Resources of

Tropical Africa), Wageningen, Netherlands, 2005. < http://database.prota.org>

Bandyopadhyay, R.; Butler, D. R.; Chandrashekar. A.; Reddy, R. K. and Navi, S. S.

Technical and institutional options for sorghum grain mold management: proceedings

of an international consultation, 18-19 May 2000, ICRISAT, Patancheru, India.

Page 234: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

220

(Chandrashekar, A., Bandyopadhyay, R., and Hall, A.J., eds.).

Belton, P.S.; Delgadillo, I.; Halford, N.G.; Shewry, P.R. Kafirin structure and

functionality. Journal of Cereal Science, 2006, 44, 272–286.

Beta, T., Corke, H., Taylor, J.R.N. Starch properties of Barnard Red, a South African

red sorghum of significance in traditional African Brewing. Starch, 2000a, 52, 467–

470.

Beta, T., Corke, H., Rooney, L.W., Taylor, J.R.N. Starch properties as affected by

sorghum grain chemistry. Journal of the Science of Food and Agriculture, 2000b, 81,

245–251.

Beta, T., Rooney, L.W., Marovatsanga, L.T., Taylor, J.R.N. Effect of chemical

treatments on polyphenols and malt quality in sorghum. J. of Cereal Science, 2000c,

31, 295–302.

Bhosale, S.H.; Rao, M.B.; Deshpande, V.V. Molecular and industrial aspects of

glucose isomerase. Microbiol. Rev., 1996, 60, 280–300.

Bird R. and Hopkins R. H.. The action of some alpha-amylases on amylose. Biochem.

J.., 1954, 56, 86-99.

Blanch, H. W. ‘Bioproducts and economics’ in Biochemical Engineering, Marcel-

Dekker Inc. USA, 1996, pp 627.

Bos, C. and Norr, N. J. Experiences with the DDS-Kroyer direct hydrolysis process.

Starch, 1974, 26, 181-185.

Buffo, R.A., Weller, C.L., Parkhurst, A.M. Optimization of sulfur dioxide and lactic

acid steeping concentrations for wet-milling grain sorghum. Transactions of the

American Society of Agricultural Engineers, 1997, 40, 1643–1648.

Buffo, R.A., Weller, C.L., Parkhurst, A.M. Wet-milling factors of sorghum and

relationship to grain quality. Journal of Cereal Science, 1998, 27, 327–334.

Page 235: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

221

Buleon, A.; Colonna, P.; Planchot, V.; Ball, S. Starch granules: structure and

biosynthesis. A mini Review. International Journal of Biological Macromolecules,

1998, 23, 85–112.

Chandrashekar, A. and Kirleis, A. W. Influence of protein on starch gelatinization in

sorghum. Cereal Chem., 1988, 65, 457-462.

Chandrashekar, A.; Satyanarayana, K.V. Disease and pest resistance in grains of

sorghum and millets. Journal of Cereal Science, 2006, 44, 287–304

Chandrashekhar, A.; Mazhar, H. The Biochemical Basis and Implications of Grain

Strength in Sorghum and Maize. Journal of Cereal Science, 1999, 30, 193-207.

Chibata, I. Immobilized enzymes: Research and Development. John Wiley and sons:

New York, 1978; pp 132-134.

Chronakis, I. S. On the molecular characteristics, compositional properties, and

structural –functional mechanisms of maltodextrins: A review. Crit. Rev. Food Sci.,

1988, 38, 599-637.

Copeland; R. A. Enzymes: A Practical Introduction to Structure, Mechanism, and

Data Analysis. Wiley-VCH, Inc., 1999, pp 184-186.

Corredor, D.Y.; Bean, S.R.; Schober, T.; Wang, D. Effect of decorticating sorghum

on ethanol production and composition of DDGS. Cereal Chemistry, 2006, 83, 17–21.

Dahlberg, J. A. Classification and characterization of sorghum in Sorghum: Origin,

History, Technology, and Production. Smith, C, W.; Frederiksen, R. A. Eds. Wiley,

New york, 2000.

Daiber, K.H. Enzyme inhibition by polyphenols of sorghum grain and malt. Journal

of the Science of Food and Agriculture, 1975, 26, 1399–1411.

Davidson, V. L. & Sittman, D. B. ‘Enzymes.’ in Biochemistry. Lippincott Williams &

Wilkins, USA, 1999, 59

Deb, U. K.; Bantilan, M. C. S.; Ro, A. D.; Rao P. R. Global Sorghum Production

Scenario in Sorghum genetic enhancement: research process, dissemination and

Page 236: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

222

impacts. Bantilan, M. C. S.; Deb, U. K.; Gowda, C. L. L.; Reddy, B. V. S.; Obilana,

A. B. and Evenson, R. E. Eds. 2004. International Crops Research Institute for the

Semi-Arid Tropics (ICRSAT).

Del Pozo-Insfran, D., Urias-Lugo, D., Hernandez-Brenes, C., Serna Saldivar, S.O.

Effect of amyloglucosidase on wort composition and fermentable carbohydrate

depletion in lager beers. Journal of the Institute of Brewing, 2004, 110, 124–132.

Dendy, D. A. V. Sorghum and Millets Chemistry and Technology. USA, St. Paul,

Minnesota, American Association of Cereal Chemists, Inc., 1995, pp. 406.

Denyer, K., Johnson, P., Zeeman, S., Smith, A.M. The control of amylose synthesis.

Journal of Plant Physiology, 2001, 158, 479–487.

Devarajan B.; Pandit A. B., Sorghum flour as Raw Material for Glucose Production.

J. Maharashtra Agric. Univ., 1996, 21 (1), pg. 86-90.

Diao, Y., Walawender, W., Fan, L. Activated carbons prepared from phosphoric acid

activation of grain sorghum. Biosource Technology, 2002, 81, 45–52.

Dias, F. F.; Panchal, D. C. Maltulose Formation During Saccharification of Starch.

Starch, 1987, 39, 64-66.

Dufour, J.P.; Melotte; L., Srebrnik, S. Sorghum malts for the production of a lager

beer. Journal of the American Society of Brewing Chemists, 1992, 111, 110–119.

Duodu, K.G.; Taylor, J.R.N.; Belton, P.S.; Hamaker, B.R. Factors affecting sorghum

protein digestibility. Journal of Cereal Science, 2003, 38, 117–131.

Dykes, L. and Rooney, L. W. Sorghum and millet phenols and antioxidants. Journal

of Cereal Science, 2006, 44, 236–251.

Elkhalifa, A. O.; Chandrashekar, A.; Mohamedc, B.E.; Tinay, A.H. Effect of reducing

agents on the in vitro protein and starch digestibilities of cooked sorghum. Food

Chemistry, 1999, 66, 323-326.

Page 237: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

223

Emes, M.J.; Bowsher, C.G.; Hedley, C.; Burrell, M.M.; Scrase-Field, E.S.F.; Tetlow,

I.J. Starch synthesis and carbon partitioning in developing endosperm. Journal of

Experimental Botany, 2003, 54, 569–575.

Ezeogu, L.I.; Duodua, K.G.; Taylora, J.R.N. Effects of endosperm texture and

cooking conditions on the in vitro starch digestibility of sorghum and maize flours.

Journal of Cereal Science, 2005, 42, 33–44.

Figueroa, J.D.C.; Martinez, B.F.; Rios, E. Effect of sorghum endosperm type on the

quality of adjuncts for the brewing industry. Journal of the American Society of

Brewing Chemists, 1995, 53, 5–9.

Frederiksen, R. A. Diseases and Disease management in sorghum in Sorghum:

Origin, History, Technology, and Production. Smith, C, W.; Frederiksen, R. A. Eds.

Wiley, New york, 2000.

Guzman-Maldonado & Paredas-Lopez O. Amylolytic enzymes and products derived

from starch: A review. Critical reviews in Food science and nutrition, 1995, 35, 373-

403.

Haki, G.D.; Rakshit, S.K. Developments in industrially important thermostable

enzymes: a review. Bioresource Technology, 2003, 89, 17–34.

Hallgren, L. Lager beers from sorghum. in Sorghum and Millets: Chemistry and

Technology. Dendy, D.A.V. Ed. American Association of Cereal Chemists, St. Paul,

MN, USA, 1995, pp. 283–297.

Hamaker, B. R., and Bugusu, B. A. Overview: Sorghum proteins and food quality. In:

Proc. AFRIPRO Workshop on the Proteins of Sorghum and Millets: Enhancing

Nutritional and Functional Properties for Africa. 2003, P. S. Belton and J. R. N.

Taylor, eds. Available at http://www.afripro.org.uk/papers/Paper08Hamaker.pdf.

Pretoria, South Africa.

Harlan, J. R. and de Wet, J.M.J. A simplified classification of cultivated sorghum.

Crop Science, 1972, 12, 172-176.

Page 238: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

224

Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized Affinity Ligand

Techniques. Academic Press Inc: New York, 1992. pp 78-79, 182-184.

Higiro, J., Flores, R.A., Seib, P.A. Starch production from sorghum grits. Journal of

Cereal Science, 2003, 37, 101–109.

Hizukuri, S. Polymodal distribution of the chain lengths of amylopectin and its

significance. Carbohydrate Research, 1986, 147, 342–347.

Howling, D. ‘Glucose Syrup: Production, properties and applications’ in Starch

Hydrolysis Products: Worldwide Technology Production and Applications. Schenck,

F. W., Hebeda, R. E. Eds.; VCH Publishers, New York, 1992, pp. 277-291.

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)/Food

and Agriculture Organization (FAO), 1996. The World Sorghum and Millet

Economies. Facts, Trends and Outlook. ICRISAT, Patancheru, India/ FAO, Rome.

Ivanova, V.; Dobreva, E. Catalytic properties of immobilized purified thermostable

alpha amylase from Bacillus licheniformis 44MB82-A. Process Biochem., 1994, 29,

607-612.

Ivanova, V.; Dobreva, E.; Legoy, M. D. Acta Biotechnol. 1998, 18, 339-351.

Kandra, L.; Gyemant, G.; Remenyik, J.; Hovanszki G., Liptak, A. FEBS Lett., 2002,

518, 79-82.

Kearsley, M. W. and Dziedzic, S. Z. `Handbook of Starch Hydrolysis Products and

Their Derivatives' Blackie Academic & Professional, London, 1995.

Kennedy, J. F.; Cabral, J. M. S. ‘Enzyme immobilization.’ in Biotechnology; Rehm,

H. J., Reed, G., Eds.; VCH: Germany, 1987; Vol. 7a; pp 349-404.

Khanal, S.; Montalbo, M.; Leeuwen, J.; Srinivasan, G.; Grewell, D. Ultrasound

Enhanced Glucose Release From Corn in Ethanol Plants. Biotechnology and

Bioengineering, 2007, 98, 978-985.

Page 239: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

225

Kimber, C. T. Origins of domesticated sorghum and its early diffusion to India and

China in Sorghum: Origin, History, Technology, and Production. Smith, C, W.;

Frederiksen, R. A. Eds. Wiley, New york, 2000.

Kinley, M. T.; Snodgrass, J. D. and Krohn, B. Alcohol production using sorghum. US

patent 7101691, 2006.

Kleih, U.; Ravi, S. B.; Rao, B. D. and Yoganand, B. Industrial Utilization of Sorghum

in India. Ejournal.icrisat.org, 3, 2007. This study is based upon fieldwork undertaken

in mid-1998 in the context of the project 'Sorghum in India: Technical, policy,

economic, and social factors affecting improved utilization', which was funded by the

Department for International Development (DFID) and jointly undertaken by

ICRISAT, NRCS, and NRI.

Komolprasert, V.; Ofoli, R. Y. J. Chem. Tech. Biotechnol. 1991, 51, 209-223.

Kroyer, K. Staerke, 1966, 10, 312.

Kuriki, T & Umanaka T. The concept of the α-amylase family: Structural similarity

and common catalytic mechanism. Journal of Bioscience and Bioengineering, 1999,

87, 557-565.

Kvesitadze, G. I.; Dvali, M. Immobilization of mold and bacterial amylases on silica

carriers. Biotechnol. Bioeng., 1982, 14, 1765-1772.

Lali, A. M.; Manudhane, K. S. Indian Patent Application No., 356/Mum/2003.

Lali, A. M.; Manudhane, K. S.; Motlekar, N.; Karandikar, P. A. Depolymerization of

starch and pectin using superporous matrix supported enzymes. Indian J. Biochem.

Biophys., 2002, 9, 253-258.

Leach, H. W. (1965). Gelatinization of starch. In R. L. Whistler & E. F. Paschall

(Eds.), Starch: chemistry and technology, 1. (pp. 289–306).New York: Academic

Press.

Léder, I. SORGHUM AND MILLETS, in Cultivated Plants, Primarily as Food

Sources, Füleky, G., in Encyclopedia of Life Support Systems (EOLSS), Developed

under the Auspices of the UNESCO, Eolss Publishers, Oxford ,UK.

Page 240: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

226

Lichtenwalner, R. E.; Ellis, E. B. and Rooney L. W. Effect of Incremental Dosages of

the Waxy Gene of Sorghum on Digestibility. J Anim Sci., 1978, 46, 1113-1119.

Lowry, O. D.; Roseborough, N. J.; Farr, A. L., Rondall, R. J. J Biol. Chem., 1951,

193, 265-275.

Mahajani, V. V.; Mokashi, S. M. Chemical Projects Economics. Macmillan India Ltd.

2005. pp. 150-160.

Marchal, L. M.; Beeftink, H. H.; Tramper, J. Towards a rational design of commercial

maltodextrins. Trends Food Sci. Technol., 1999, 10, 345-355.

Marchal, L. M.; van de Laar, A. M. J.; Goetheer, E.; Schimmelpennink, E. B.;

Bergsma, J.; Beeftink, H. H.; Tramper, J. Biotechnol. Bioeng., 1999, 63, 344-355.

Mezo-Villanueva, M. and Serna-Saldivar, S. O. Effect of protease addition on starch.

recovery from steeped sorghum and maize. Starch, 2004, 56, 371-378.

Miller, G. L. Anal. Chem., 1959, 31, 426–428.

Morrison, W. R. Lipids in cereal starches. A review. J. Cereal Sci., 1988, 8, 1-15.

Morrison, W. R. Starch lipids: a reappraisal. Starch/Stärke, 1981, 33, 408-410.

Mulvihill, P. J. ‘Crystalline and liquid dextrose products: Production, properties and

applications’ in Starch Hydrolysis Products: Worldwide Technology Production and

Applications. Schenck, F. W., Hebeda, R. E. Eds.; VCH Publishers, New York, 1992,

pp 121-167.

Munck, L. New milling technologies and products: Whole plant utilization by milling

and separation of the botanical and chemical components in Sorghum and Millets:

Chemistry and Technology, Dendy, D.A.V. (Ed.). American Association of Cereal

Chemists, St. Paul, MN, USA, 1995, pp. 223–281.

Murty, D.S., Kumar, K.A. Traditional uses of sorghum and millets in Sorghum and

Millets: Chemistry and Technology, Dendy, D.A.V. (Ed.). American Association of

Cereal Chemists, St. Paul, MN, USA, 1995, pp. 185–221.

Page 241: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

227

Nair, S. U. Studies in microbial pullulanases. Thesis, Ph. D. (Sci), Institute of

Chemical Technology, University of Mumbai, 2006.

Nebesny, E.; Rosicka, J. and Tkaczyk, M. Effect of Enzymatic Hydrolysis of Wheat

Starch on Amylose-Lipid Complexes Stability. Starch/Stärke, 2002, 54, 603–608.

Nigam, P.; Singh, D. Enzyme and microbial systems involved in starch processing.

Enzyme Microb. Technol., 1995, 17, 770-778.

Oates, C. G. Towards an understanding of starch granule structure and hydrolysis.

Trends in food science and technology, 1997, 8, 375-382.

Olsen, H. S. ‘Enzymatic production of glucose syrups’ in Handbook of Starch

Hydrolysis Products and Their Derivatives Kearsley, M.W. and Dziedzic, S.Z. eds.

Blackie Academic & Professional, London., 1995

Ortega Villicana, M.T.; Serna-Saldivar, S.O. Production of lager from sorghum malt

and waxy grits. Journal of the American Society of Brewing Chemists, 2004, 62, 131–

139.

Owuama, C.I. Brewing beer with sorghum. J. of Institute of Brewing, 1999, 105, 23–

34.

Owuama, C.I. Sorghum: a cereal with lager beer brewing potential. World Journal of

Microbiology and Biotechnology, 1997, 13, 253–260.

Palmer, G. H. Review: Sorghum – Food, Beverage and Brewing potential. Process

Biochemistry, 1992, 27, 145-153.

Paolucci-Jeanjean, D.; Belleville M.; Zakhia, N.; Rios, G. M. Biotechnol. Bioeng.,

2000, 68, 71-77.

Park, S. H.; Bean, S. R.; Wilson, J. D. and Schober, T. J. Rapid Isolation of Sorghum

and Other Cereal Starches Using Sonication. Cereal Chemistry, 2006, 83, 611-616.

Perez Siraa, E. E.; Amaiz, M. L. A laboratory scale method for isolation of starch

from pigmented sorghum. Journal of Food Engineering, 2004, 64, 515–519

Plessis, J. D. Sorghum production, 2008. Available at www.nda.agric.za/publications

Page 242: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

228

Radley, J. A. Starch and Its Derivatives. Chapman and Hall, Ltd., London. 1968

Reeve, A. ‘Starch hydrolysis: Processes and Equipment’ in Starch Hydrolysis

Products: Worldwide Technology Production and Applications. Schenck, F. W.,

Hebeda, R. E. Eds.; VCH Publishers: New York, 1992.

Roberts, S M. ‘An historical introduction to biocatalysis using enzymes and

microorganism.’ in Introduction to Biocatalysis Using Enzymes and Micro-

Organisms. Cambridge University press. UK, 1989, pp. 1-31

Roig, M. G.; Slade, A.; Kennedy J.F. α-amylase immobilized on plastic supports:

stabilities, pH and temperature profiles and kinetic parameters. Biomater. Artif. Cells

Immob. Biotechnol., 1993, 21, 487-525.

Rooney, L. W. and Pflugfelder, R. L. Factors affecting starch digestibility with special

emphasis on sorghum and corn. J. Anim. Sci., 1986, 63, 1607-1623.

Rooney, L.W., Serna-Saldivar, S.O. Sorghum in Handbook of Cereal Science and

Technology, Kulp, K.; Ponte, Jr., J.G. (Eds.), second ed. Marcel Dekker, New York,

2000, pp. 149–175.

Rooney, L.W., Waniska, R.D. Sorghum food and industrial utilization. in Sorghum:

Origin, History, Technology, and Production, Smith, C.W., Frederiksen, R.A. (Eds.)

Wiley, New York, 2000, pp. 689–729.

Sajilata, M.G.; Singhal, R. S.; Kulkarni, P. R. Resistant Starch-A Review.

Comprehensive reviews in food science and food safety, 2006, 5, 1-17.

Schenck, F. W. and Hebeda, R. E. Starch Hydrolysis Products: Worldwide

Technology, Production, and Applications, VCH Publishers 1992, New York.

Schenck, F. W.; Hebeda, R. E. Starch Hydrolysis Products: Worldwide Technology

Production and Applications; VCH Publishers: New York, 1992; pp 1-20.

Singh, V., Moreau, R.A., Hicks, K.B. Yield and phytosterol composition of oil

extracted from grain sorghum and its wet-milled fractions. Cereal Chemistry, 2003,

80, 126–129.

Page 243: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

229

Siso, M. I. G.; Grabber, M.; Condoret, J. S.; Combes, G. Effect of diffusional

resistances on the action pattern of immobilized α-amylase. J. Chem. Technol.

Biotechnol., 1990, 48, 185-200.

Smith, A.M.; Denyer, K.; Martin, C. The synthesis of the starch granule. Annual

Review of Plant Physiology and Plant Molecular Biology, 1997, 48, 65–87.

Smith, C, W.; Frederiksen, R. A. Preface in Sorghum: Origin, History, Technology,

and Production. Smith, C, W.; Frederiksen, R. A. Eds. Wiley, New york, 2000, pp.

viii.

Stahlman, P. W.; Wicks, G. A. Weeds and their control in grain sorghum in Sorghum:

Origin, History, Technology, and Production. Smith, C, W.; Frederiksen, R. A. Eds.

Wiley, New york, 2000.

Suresh, K.; Kiran sree, N. and Venkateswer Rao, L. Utilization of damaged sorghum

and rice grains for ethanol production by simultaneous saccharification and

fermentation. Bioresource Technology, 1999a, 68, 301-304.

Suresh, K.; Kiran sree, N. and Venkateswer Rao, L. Production of ethanol b raw

starch hydrolysis and fermentation of damaged grains of wheat and sorghum.

Bioprocess engineering, 1999b, 21, 165-168.

Tarhan, L. The effect of substrate diffusion factor on immobilized alpha amylase.

Starch, 1989, 41, 315-318.

Taylor, J. R. N.; Schober, T. J.; Bean, S. R. Novel food and non-food uses for

sorghum and millets. Journal of Cereal Science, 2006, 44, 252–271.

Taylor, J.R.N., Dewar, J. Fermented products: Beverages and porridges in Sorghum:

Origin, History, Technology, and Production, Smith, C.W., Frederiksen, R.A. (Eds.).

Wiley, New York, 2000, pp. 751–795.

Taylor, J.R.N.; Dewar, J. Developments in sorghum food technologies in Advances in

Food and Nutrition Research, vol. 43, Taylor, S. (Ed.). Academic Press, San Diego,

CA, USA, 2001, pp. 217–264.

Teague, W. M.; Brumm, P. J. ‘Commercial enzymes for starch hydrolysis products’ in

Page 244: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

230

Starch Hydrolysis Products: Worldwide Technology Production and Applications.

Schenck, F. W., Hebeda, R. E. Eds.; VCH Publishers, New York, 1992, pp. 277-291.

Teetes, G. L.; Pendleton, B. B. Insect pests of sorghum in Sorghum: Origin, History,

Technology, and Production. Smith, C, W.; Frederiksen, R. A. Eds. Wiley, New york,

2000.

Tegge, V. G. and Richter G. Sorghum and broken rice as basic materials for glucose

production. Starch, 1982, 34, 386-390.

Tester, R. F.; Karkalas, J.; Qi, X. Starch—composition, fine structure and

architecture. A Review Journal of Cereal Science, 2004a, 39, 151–165.

Tester, R. F.; Karkalas, J.; Qi, X. Starch structure and digestibility Enzme-Substrate

relationship. World’s Poultry Science Journal, 2004b, 60, 186-195.

Tester, R.F.; Karkalas, J. Starch. In: Steinbuchel, A. (Series Ed.) Vandamme, E.J., De

Baets, S., Steinbu¨chel, A. (vol. Eds.), Biopolymers, vol. 6. Polysaccharides. II.

Polysaccharides from Eukaryotes, Wiley–VCH, Weinheim, 2002, pp. 381–438.

Tumturk, H.; Aksoy, S.; Hasirci, N. Food Chem., 2000, 68, 259-266.

Twisk, P. V.; Meltze, B. W. And Cormack R. H. Production of glucose from maize

grits on commercial scale. Starch, 1976, 28, 23-25.

Uhlig, H. ‘Application of modern enzyme technology.’ in Industrial Enzymes and

Their Applications, Wiley-IEEE. USA., 1998, pp. 10.

van der Maarel M.J.E.C; van der Veen, B.; Uitdehaag, J. C. M.; Leemhuis, H.;

Dijkhuizen, L. Properties and applications of starch-converting enzymes of the α-

amylase family. Journal of Biotechnology, 2002, 94, 137-155.

Vanderlip, R. L. Growth and Development of the Sorghum Plant in Grain Sorghum

Production Handbook, Kansas State University, 1998 available at

http://www.oznet.ksu.edu.

Vanderlip, R. L. How a Sorghum Plant Develops, Kansas State University, 1993

Available at http://www.oznet.ksu.edu.

Page 245: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

231

Vorgias C E & Antranikian G. ‘Glycosyl Hydrolases from environment.’ in

Glycomicrobiology,Ronald J Doyle. Ed. Springer Publication, New York, 1997, pp.

361.

Wang, F.C., Chung, D.S., Seib, P.A., Kim, Y.S. Optimum steeping process for wet

milling of sorghum. Cereal Chemistry, 2000, 77, 478–483.

Wang, L.; Wang, Y. Application of High-Intensity Ultrasound and Surfactants in Rice

Starch Isolation. Cereal Chemistry, 2004, 81, 140-144.

Waniska, R. D. and Rooney, L. W. Structure and Chemistry of sorghum Caryopsis in

Sorghum: Origin, History, Technology, and Production. Smith, C, W.; Frederiksen,

R. A. Eds. Wiley, New york, 2000.

White, J. S. ‘Fructose syrup: Production, properties and applications’ in Starch

Hydrolysis Products: Worldwide Technology Production and Applications. Schenck,

F. W., Hebeda, R. E. Eds.; VCH Publishers, New York, 1992, pp. 277-291.

Wu, X.; Zhao, R.; Bean, S. R.; Seib, P. A.; Mclaren, J. S.; Madl, R. L.; Tuinstra, M.;

Lenz, M. C. and Wang, D. Factors Impacting Ethanol Production from Grain

Sorghum in the Dry-Grind Process. Cereal Chemistry, 2007, 84, 130-136.

Xie, X.J. and Seib, P.A. Laboratory wet-milling of grain sorghum with abbreviated

steeping to give two products. Starch/Staerke, 2002, 54, 169–178.

Xie, X.J., Liang, Y.T.S., Seib, P.A., Tuinstra, M.R. Wet-milling of grain sorghum of

varying seed size without steeping. Starch/Staerke, 2006, 58, 353–359.

Yang, P. & Sieb, P. Low-input wet-milling of grain sorghum for readily accessible

starch and animal feed. Cereal Chem., 1996, 73, 751–755.

Yang, R., Seib, P.A. Low-input wet-milling of grain sorghum for readily accessible

starch and animal feed. Cereal Chemistry, 1995, 72, 498–503.

Zhan, X.; Wang, D.; Tuinstra, M.R.; Bean, S.; Seib, P.A. and Sun, X.S. Ethanol

and lactic acid production as affected by sorghum genotype and location. Industrial

Crops and Products, 2003, 18, 245-255.

Page 246: Studies in depolymerization of natural polysaccharides--PhD thesis

References

Studies in the Enzymatic depolymerisation of natural polysaccharides

232

Zhan, X.; Wanga, D. ; Beanb, S.R.; Moc, X.,; Sunc, X.S. and Boyled, D. Ethanol

production from supercritical-fluid-extrusion cooked sorghum. Industrial Crops and

Products, 2006, 23, 304–310.

Zhang, G. and Hamaker, B. R. Low a-Amylase Starch Digestibility of Cooked

Sorghum Flours and the Effect of Protein. Cereal Chem., 1998, 75, 710-713.

Zhang, Z.; Niu, Y.; Exkhoff, S. R. and Feng, H. Sonication enhanced corn starch

separation. Starch, 2005, 57, 240-245.

Zhao, R.; Bean, S.R.; Ioerger, B. P.; Wang, D.; Boyle, D. L. Impact of Mashing on

Sorghum Proteins and Its Relationship to Ethanol Fermentation. J. Agric. Food

Chem., 2008, 56, 946–953.

Zobel H. F. ‘Starch: Sources, Production and properties’ in Starch Hydrolysis

Products: Worldwide Technology Production and Applications. Schenck, F. W.,

Hebeda, R. E. Eds.; VCH Publishers, New York, 1992, pp. 277-291.

Page 247: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix A: Analytical methods

Studies in the Enzymatic depolymerisation of natural polysaccharides

233

Appendix A : Analytical methods

A.1. Measurement of protein concentration (Lowry et al., 1951)

A.1.1. Principle and mechanism

The method is dependent on the color obtained on reaction of the Folin-

Ciocalteau phenol reagent with the tyrosine residues of the proteins, although other

chromogenic amino acids such as tryptophan, histidine and cysteine and peptide

linkages are also involved. The method is generally applicable in all cases except for

the proteins that do not contain tyrosine. In the case of the simple Lowry method, the

detergents, which may be used for extraction of proteins from the membranes, or

those, secreted during fermentations, interfere with the determinations and hence

modified method have been proposed.

The Biuret reaction with alkaline Cu (II) and the reaction of a complex salt of

phosphomolybdotungstate, called the Folin-Ciocalteau phenol reagent, which gives an

intense blue green color with the Biuret complexes of tyrosine and tryptophan. The

Folin-Lowry method is 10 times more sensitive than UV absorption at 280 nm and

100 times more sensitive than Biuret method.

A.1.2. Apparatus and Equipments

Test tubes

Pipettes

UV VIS spectrophotometer

A.1.3. Chemicals required

Folin-Ciocalteau reagent, Bovine Serum Albumin (BSA), Sodium hydroxide,

Sodium carbonate, Copper sulfate and Sodium potassium tartarate

Page 248: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix A: Analytical methods

Studies in the Enzymatic depolymerisation of natural polysaccharides

234

A.1.4 Reagents

Folin-Ciocalteau reagent: Ready reagent has been procured for the analysis.

Standard protein solution: Dissolve 100 mg of Bovine Serum Albumin (BSA) in

100 mL of distilled water

Sodium hydroxide solution: Dissolve 2 g of Sodium hydroxide in 500 mL of

distilled water

Sodium carbonate solution: Dissolve 10 g of Sodium carbonate in 500 mL of

distilled water

Copper sulfate solution: Dissolve 1.56 g of copper sulfate in 100 mL of distilled

water

Sodium potassium tartarate: Dissolve 2.37 g of sodium potassium tartarate in

100 mL of distilled water

Alkaline copper reagent: The copper reagent is prepared fresh just before use by

mixing 10 mL of sodium hydroxide solution, 10 mL of sodium carbonate

solution, 0.2 mL of copper sulfate solution and 0.2 mL of sodium potassium

tartarate solution

A.1.5. Procedure (Modified method)

1 mL of alkaline copper reagent was added to 0.1 mL of sample and vortex

immediately. The solution was incubated at room temperature (30 °C) for exactly 10

min. To this, 0.1 mL of Folin-Ciocalteau reagent (1:1 diluted with distilled water)

was added and vortexed immediately. This was allowed to stand at 30 °C for exactly

30 min. The blue color thus produced was measured with the help of a UV-VIS

spectrophotometer at 660 nm against blank sample. The standard calibration curve

was prepared in the protein concentration range of 0–1 mg/mL of BSA as shown in

Page 249: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix A: Analytical methods

Studies in the Enzymatic depolymerisation of natural polysaccharides

235

Figure A.1.

Note

Reagent with turbidity should be discarded immediately

With every new reagent, new standard curve has to be plotted

y = 0.1628x - 0.0051R2 = 0.9994

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 0.1 0.2 0.3 0.4 0.5O. D

Con

cn o

f BSA

(mg/

mL)

1

  Figure A.1. Standard curve for BSA for modified Folin Lowry method

A.2. Measurement of moisture content in the sorghum flour

A.2.1. Apparatus and Equipments

Petri dishes

Oven

A.2.2. Procedure

Petri dish is weighed. 10 g of sorghum flour was spread on the petri dish.

Then it was kept in the oven at 80 °C for drying till constant weight is obtained after

drying. Moisture content is determined by using following formula;

100W

WWcontent moisture %f

21 ×−

=  

where, Wf = weight of flour, g 

Page 250: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix A: Analytical methods

Studies in the Enzymatic depolymerisation of natural polysaccharides

236

W1 = combined weight of petri dish and flour, g

W2 = combined weight of petri dish and flour after drying is completed, g

A.3. Measurement of reducing sugar concentration (Miller et al 1951)

A.3.1. Principle and mechanism

A reducing sugar is a saccharide which has an anomeric carbon (carbonyl

carbon atom in the sugar) in the hemi-acetal or ketal form i.e. not involved in the

glycosidic linkage. This allows the sugar to act as a reducing agent. Whereas,

nonreducing sugar is a saccharide in which all anomeric carbon atoms are in the acetal

form i.e. involved in the glycosidic linkage. The aldehyde (or keto-) form or

hemiacetal (or ketal) form is available for reducing are responsible for the reducing

power of the sugars. When a sugar is oxidized, its carbonyl group (i.e. aldehyde or

ketone group) is converted to a carboxyl group.

This reducing property of sugar was used as a basis for the analysis of

reducing sugars. 3, 5-Dinitrosalicylic acid (DNSA) is an aromatic compound that

reacts with reducing sugars and other reducing molecules to form 3-amino-5-

nitrosalicylic acid, which is red brown in color and absorbs light strongly at 540 nm.

A.3.2. Apparatus and Equipments

Test tubes

Page 251: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix A: Analytical methods

Studies in the Enzymatic depolymerisation of natural polysaccharides

237

Pipettes

UV VIS spectrophotometer

A.3.3. Chemicals required

Sodium hydroxide (NaOH), 3, 5-Dinitrosalicylic acid (DNSA), Potassium

sodium tartarate (Rochelle salt)

A.3.4. Preparation of DNSA reagent

30 g Potassium sodium tartarate (Rochelle salt) and 1.6 g sodium hydroxide

were dissolved in 70 mL distilled water. 3, 5-dinitrosalicylic acid was added to the

above solution pinch by pinch in dark for dissolution, until the complete quantity (i.e.

1 gm) was added. Then the volume was made up to 100 mL by addition of distilled

water to give Dinitrosalicylic acid reagent (DNSA reagent). The reagent was filtered

and then stored in an amber colored bottle.

A.3.5. Procedure

1 mL of appropriately diluted sample was added to test tube. 1 mL of DNSA

reagent was added to test tube and the test tubes were kept in a boiling water bath for

exactly 10 min. At end of 10 min all test tubes were immediately kept in the cold

water for cooling. Then 10 mL distilled water was added to each test tube.

Absorbance of the solution was then measured using spectrophotometer (Chemito) at

540 nm against blank sample. Blank sample means 1 mL distilled water instead of

sample of solution of reducing sugar.

A standard solution of 1 mg glucose/mL was prepared. Different quantities (0

to 1 mL) of the standard solution of glucose were taken in the different test tubes and

the volume was made up to 1 mL using distilled water. 1 mL of DNSA reagent was

added to each test tube and the test tubes were kept in a boiling water bath for exactly

10 min. At end of 10 min all test tubes were immediately kept in the cold water for

Page 252: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix A: Analytical methods

Studies in the Enzymatic depolymerisation of natural polysaccharides

238

cooling. Then 10 mL distilled water was added to each test tube. Absorbance of the

solution was then measured using spectrophotometer (Chemito) at 540 nm against

blank sample. A graph of glucose concentration (mg/mL) vs. absorbance (optical

density) was plotted (Fig. A.2) and used as a standard glucose graph in the form of

calibration equation to find out concentration of reducing sugars (glucose equiv) in

the samples.

Note: DNSA reagent with turbidity should be discarded

y = 1.6585x + 0.0281R2 = 0.9999

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.2 0.4 0.6 0.8

O. D

Con

cn o

f glu

cose

(mg/

mL)

 Figure A.2. Standard curve for glucose for DNSA method

A.4. Measurement of concentrations of malto-oligosaccharides using High

Performance Thin Layer Chromatograph (HPTLC)

A.4.1. Materials

Reference standards of maltotriose, maltotetraose, maltopentaose,

maltohexaose and maltoheptaose were purchased from Sigma – Aldrich; whereas

reference standards of glucose and maltose were purchased from Merck India Ltd.

Acetonitrile for chromatography Licrosolv was used for HPTLC.

Page 253: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix A: Analytical methods

Studies in the Enzymatic depolymerisation of natural polysaccharides

239

20 cm × 10 cm TLC sheets (silica gel 60, Merck Ltd, India).

A.4.2. Procedure

Step1: Prewashing the TLC plate with methanol in the TLC chamber. Drying the plate

for ½ hr at 45°C.

Step 2: Application of samples and standards (i.e. synthetic solution containing known

weights of glucose, maltose, maltotriose, maltotetraose, maltopentaose,

maltohexaose and maltoheptaose) on the TLC plate using automatic

application device (DESAGA) and then plate is subjected to atmospheric

drying for 15 minutes.

Step 3: TLC plate is then developed in a pre-saturated (saturation time ½ hr) chamber.

Triple development resulted into good quality of chromatogram. Mobile phase

Acetonitrile: 0.02 M Na2HPO4 of composition 70:30 (v/v) was used for 1st

and 2nd development; whereas Mobile phase Acetonitrile: 0.01 M Na2HPO4

of composition 80:20 (v/v) was used for 3rd development. Intermediate drying

between two development is essential for proper separation of peaks; which

was achieved by atmospheric drying for 15 min and drying at 45°C for 15

minutes.

Step 4: Plate after triple development was dipped in the diphenylamine-aniline-

phosphoric acid reagent (which consists of 40 ml acetone, 0.8gm

diphenylamine, 0.8 ml aniline and 6 ml phosphoric acid) for 4 seconds and

then kept at 120 °C for 10 min. It resulted into colored spots corresponding to

glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltoheptaose.

Step 5: Then the TLC plate is scanned at 546 nm using densitometer to get the

chromatogram and values of peak areas corresponding to glucose, maltose,

Page 254: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix A: Analytical methods

Studies in the Enzymatic depolymerisation of natural polysaccharides

240

maltotriose, maltotetraose, maltopentaose, maltoheptaose in standards solution

and samples.

Step 6: Peak areas were plotted against the known amounts of sugar (standard)

applied to the TLC plate for glucose (G1), maltose (G2), maltotriose (G3),

maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), and

maltoheptaose (G7). From these calibration curves (Fig. A.3), concentration of

glucose and malto-oligosaccharides were determined using peak area obtained

for the reaction samples.

y = 1.02E-06x2 + 5.66E-04x

y = 9.88E-07x2 + 1.85E-04x

y = 9.26E-07x2 + 2.67E-04x

y = 7.66E-07x2 + 4.19E-04x

y = 9.56E-07x2 + 1.36E-04x

y = 1.15E-06x2 + 1.08E-04x

y = 6.40E-07x2 + 3.96E-04x

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 100 200 300 400 500 600 700 800Peak area

Con

cn o

f sug

ar (g

luco

se e

quiv

.)

G1

G2

G3

G4

G5

G6

G7

 

FigureA.3. Standard curve for glucose and malto-oligosaccharides

Page 255: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix A: Analytical methods

Studies in the Enzymatic depolymerisation of natural polysaccharides

241

A. 5. Qualitative method to see presence of starch and Quantitative method for

determination of starch concentration

A.5.1. Principle

Starch contains two polysaccharide components viz. amylose and

amylopectin. Amylose has strong affinity towards iodine and forms amylose-iodine

complex with it. Color of the amylose-iodine complex depends upon DP (degree of

polymerization) of amylose i.e. no of glucose units present in the chain and how it

varies with DP of amylose is shown in the following table.

DP (degree of polymerization) Color of amylose-iodine complex

5-10 (actually called as malto-oligosaccharides) Red

10-25 Violet

Above 25 Deep blue

Amylopectin have little affinity for iodine and gives red coloration with it.

(Radley 1968). Concentration of soluble starch was determined from a standard plot

generated using the starch-iodine method (Bird and Hopkins, 1954).

A.5.2. Chemicals required:

Sublimed I2, Potassium iodide

A.5.3. Preparation of KI-I2 reagent:

KI-I2 reagent was prepared as 0.05% w/v iodine and 0.5% w/v potassium

iodide in distilled water. The KI-I2 reagent was stored in an amber colored bottle.

Page 256: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix A: Analytical methods

Studies in the Enzymatic depolymerisation of natural polysaccharides

242

A.5.4. Method:

Standard starch solution (1% w/v) was prepared by gelatinizing the starch

slurry in boiling water for 5 minutes. Different quantities of starch solution (0 to 1ml)

were taken in different test tubes and the volume was made up to 1 ml using distilled

water. 5 ml of KI-I2 reagent was added to each test tube and the mixture was

incubated for 15 minutes in dark. The color developed was measured at 660nm on the

Chemito-2300 UV-VIS spectrophotometer. A graph starch concentration (mg/ml) vs.

absorbance (optical density) was plotted (Fig. A.4) and was used as a standard.

y = 0.7401xR2 = 0.9987

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.2 0.4 0.6 0.8absorbence

Con

cn o

f sta

rch

(mg/

mL)

 

Figure A.4. Standard curve for starch for starch-iodine method

Page 257: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix B: Code in Matlab to determine kinetic constants

Studies in the Enzymatic depolymerisation of natural polysaccharides

243

Appendix B : Code in Matlab to determine kinetic constants

function StarchhydrolysisusingimmobilizedBLA clc; A=5.78669519; C=5.78669519*5.487459631; %C= maximum DE D=0.528008427; %D=rate constant E=1.86; So=90; % Run 24 K=[0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01]; %Initial guess P=[E So A C D]; fid = fopen('Run 24.txt'); % File containing experimental data texp=fscanf(fid,'%g',[10 1]); % experimental time GCexp=fscanf(fid,'%g',[10 8]); % experimental change in composition of G1-G7 texp=texp'; GCexp=GCexp'; fclose(fid); GCexp=GCexp./100; g=GCexp(:,1); lb=[0 0 0 0 0 0 0 0 0 ]; ub=[]; %options = optimset('LargeScale', 'off'); %kvalues=fmincon(@kinetics,K,[],[],[],[],lb,ub,[],options,P,g,texp,GCexp); %K=kvalues tend=max(texp); sol = ode45(@f,[0 tend],g,[],P,K); G = deval(sol,texp); GC=G; G=G'; GC'*100 K=[0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01]; ub=[10 10 10 10 10 10 10 10 10 ]; options = optimset('LargeScale', 'off'); kvalues=fmincon(@kinetics,K,[],[],[],[],lb,ub,[],options,P,g,texp,GCexp); K=kvalues tend=max(texp); sol = ode45(@f,[0 tend],g,[],P,K); G = deval(sol,texp); GC=G; G=G'; GC'*100

Page 258: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix B: Code in Matlab to determine kinetic constants

Studies in the Enzymatic depolymerisation of natural polysaccharides

244

K=[0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01]; ub=[20 20 20 20 20 20 20 20 20 ]; options = optimset('LargeScale', 'off'); kvalues=fmincon(@kinetics,K,[],[],[],[],lb,ub,[],options,P,g,texp,GCexp); K=kvalues tend=max(texp); sol = ode45(@f,[0 tend],g,[],P,K); G = deval(sol,texp); GC=G; G=G'; GC'*100 [t,G] = ode45(@f,[0 tend],g,[],P,K); plot(t,G(:,1),'.',t,G(:,2),'-',t,G(:,3),'+',t,G(:,4),'*',t,G(:,5),'+',t,G(:,6),'^',t,G(:,7),'--'); t; G; for i=1:length(t) TDW(i)=TDW(t(i),P); end; TDW; function error = kinetics(K,P,g,texp,GCexp) sol = ode45(@f,[0 10],g,[],P,K); G = deval(sol,texp); %evaluation of G values at values of %experimental time. E=P(1);So=P(2);A=P(3);C=P(4);D=P(5); G=G'; GC=G; GCexp=GCexp'; error=[0 0 0 0 0 0 0]; M=length(texp); for i=1:7 for x = 1:M error(i)=error(i)+(GC(x,i)-GCexp(x,i))^2; end; end; error=sum(error); K; error=error*100;

Page 259: Studies in depolymerization of natural polysaccharides--PhD thesis

Appendix B: Code in Matlab to determine kinetic constants

Studies in the Enzymatic depolymerisation of natural polysaccharides

245

function dGdt=f(t,G,P,K) k1=K(1); k2=K(2); k3=K(3); k4=K(4);k5=K(5);k6=K(6);k7=K(7); kcat6=K(8); kcat7=K(9); %h1=K(10); h1=0; %value of h1 E=P(1);So=P(2);A=P(3);C=P(4);D=P(5); dGdt = [ E*(k1*(1-G(1)-G(2)-G(3)-G(4)-G(5)-h1*So/TDW(t,P)));

E*(k2*(1-G(1)-G(2)-G(3)-G(4)-G(5)-h1*So/TDW(t,P))); E*(k3*(1-G(1)-G(2)-G(3)-G(4)-G(5)-h1*So/TDW(t,P))); E*(k4*(1-G(1)-G(2)-G(3)-G(4)-G(5)-h1*So/TDW(t,P))); E*(k5*(1-G(1)-G(2)-G(3)-G(4)-G(5)-h1*So/TDW(t,P))); E*(k6*(1-G(1)-G(2)-G(3)-G(4)-G(5)-G(6)-h1*So/TDW(t,P))-kcat6*G(6));

E*(k7*(1-G(1)-G(2)-G(3)-G(4)-G(5)-G(6)-G(7)-h1*So/TDW(t,P))-kcat7*G(7)); ]; function TDW=TDW(t,P) %TDW total dry weight E=P(1);So=P(2);A=P(3);C=P(4);D=P(5); DE=A+C*(1-exp(-D*t)); acl=(180*100/DE-18)/162; TDW=162*1.11*100*So/(DE*(180*100/DE-18));

Page 260: Studies in depolymerization of natural polysaccharides--PhD thesis

INSTITUTE OF CHEMICAL TECHNOLOGY UNIVERSITY OF MUMBAI

SYNOPSIS

OF THE THESIS TO BE SUBMITTED TO UNIVERSITY OF MUMBAI

FOR THE DEGREE OF DOCTOR OF PHILOSPHY (TECHNOLOGY)

IN THE SUBJECT OF CHEMICAL ENGINEERING

Name of the Candidate

Mr. Satish D. Shewale

Title of the thesis

Studies in the Enzymatic depolymerisation of natural polysaccharides

Name and Designation of Guiding Teacher

Prof. (Dr.) Aniruddha B. Pandit UGC Scientist ‘C’ (Professor’s Grade) Chemical Engineering Division, Institute of Chemical Technology, Matunga, Mumbai 400 019.

Place of Research Work

Chemical Engineering Division, Institute of Chemical Technology, Matunga, Mumbai 400 019.

Registration Number and Date 188 (A); Dated: 29th March, 2004

Date of submission of Synopsis 18th March, 2008

Signature of the Candidate

(Satish D. Shewale)

Signature of the Guiding Teacher

Prof. (Dr.) Aniruddha B. Pandit

Page 261: Studies in depolymerization of natural polysaccharides--PhD thesis

Introduction

Sorghum (Sorghum bicolor L. Moench) is fifth largest produced and an important

cereal in world after wheat, rice, barley and maize. Production of sorghum in 2004-2005 in

world was 58 Million Tonnes. India was the second largest producer of sorghum after

U.S.A. with production of 10.8 million Tonnes in 2004-2005. Other sorghum producing

countries are Mexico, Nigeria, and China. Maharashtra is the largest sorghum producing

state in India with production of 5.8 million Tonnes. Sorghum ranks third in the major food

grain crops in India. The plant originated in equatorial Africa and is distributed throughout

the tropical, semi-tropical, arid and semi arid regions of the world. It has a potential to

compete effectively with crops like maize under good environmental and management

conditions. The greatest merit of sorghum is that it has marginal lands under moisture

stress or excessive moisture conditions. It is one of the most widely grown dry land food

grains in India. It does well even in low rainfall areas.

A few names of sorghum are milo, jowar, kafir corn, guinea corn, and cholam.

Sorghum is also termed as “Nature-cared crop” because it has strong resistance to harsh

environments such as dry weather and high temperature in comparison to other crops, it is

usually grown as a low-level chemical treatment crop with limited use of pesticides and it

has a potential to adapt itself to the given natural environment.

Though sorghum is one of the few cereals that can be grown in the semi-arid

regions of the country, demand for the sorghum is decreasing with enhanced

socioeconomic status of the population in general and easy availability of other preferred

cereals in sufficient quantities at affordable prices. Hence, in addition to being a major

source of staple food for humans, it also serves as an important source of cattle feed and

Page 262: Studies in depolymerization of natural polysaccharides--PhD thesis

fodder, but at lower prices. Also about 10-20 % of the production gets wasted due to

blackening of crop and lack of good facilities for storage etc. Hence an industrial

application is needed so that sorghum cultivation becomes economically viable for farmers

through value addition products. There is very small amount of work (Devarajan and

Pandit 1996; Aggarwal et al. 2001) done on value addition to sorghum. Hence, the

objective of the present work was a production of value addition products like glucose and

maltose from different qualities of sorghum i.e. healthy, germinated and blackened. In the

present work, we have used sorghum flour for hydrolysis instead of first isolating starch

and then its subsequent use for liquefaction and saccharification because the yields of

starch isolation from sorghum are around 50-60% i.e rest part (40 - 50%) is getting wasted

or does not fetch much price.

Constituents of sorghum are starch, proteins, moisture, fats and oil, fibers and ash

with percentage contents in the range of 65-75, 9-11, 9-13, 1-1.5, 1.5-2 and 1-2

respectively (Owuama 1997). Production of glucose from sorghum flour consists of two

steps viz. 1. Liquefaction using B. licheniformis α-amylase (BLA) 2. Saccharification using

amyloglucosidase (AG). However, enzymes can be utilized in the two forms viz. free and

immobilized. Limitations of free enzymes lies in its only once usability and effluent

problem. Immobilized enzymes are the enzymes which are physically confined or localized

in a certain defined region of space with retention of their catalytic activities and which can

be used repeatedly and continuously. Immobilized enzyme has several advantages over the

free enzyme as follows; reusability of the enzyme, continuous operation of the system,

easy separation of product from the enzyme, less effluent problems, increased stability of

the enzyme and few side products and more favorable refining conditions. However, it has

Page 263: Studies in depolymerization of natural polysaccharides--PhD thesis

disadvantages of lower reaction rates due to diffusion limitation of substrate molecules to

enzyme active site and diffusion out of product molecules from active site to bulk

solution.

Hence, firstly it was decided to develop a process for the production of glucose

from sorghum flour using immobilized enzymes. This process constitutes following steps

viz. 1. Gelatinization of 15 % sorghum slurry in boiling water for 10 min. 2. Circulating

slurry through the bed of immobilized BLA and AG. But before studying this, it was

necessary to first immobilize BLA on beads and study its catalytic characteristics.

Studies in hydrolysis of soluble starch using immobilized B. licheniformis α-amylase

In this work B. licheniformis α-amylase (BLA) is immobilized on rigid superporous

(pore size ∼ 3 µm) cross-linked cellulose matrix (CELBEADS; Lali et al. 2003) by

covalent binding method. After immobilization, it was observed that optimum operational

pH decreased slightly from 5.6 to 5.2 (because of the difference in the hydronium ion

concentration in the bulk solution and the microenvironment in the vicinity of immobilized

enzyme molecule) and optimum temperature changed from 55°C to 55 – 70°C (because of

the improvement in the enzyme rigidity upon immobilization by covalent binding).

Activity of free BLA was observed to be 16500 EU/mL at 55°C and 6 pH. Activity of

immobilized BLA was observed to be 18.5 EU/mL at 55°C and 5.2 pH.

A High performance thin layer chromatography (HPTLC) analytical method was

developed to analyze saccharide composition (i.e. concentration of malto-oligosaccharides)

of starch hydrolysate. Free BLA is reported to be endo-amylase with random attack action

pattern. However, after immobilization it behaves like exo-amylase with dual specificity

Page 264: Studies in depolymerization of natural polysaccharides--PhD thesis

towards maltopentaose and maltotriose. Immobilized BLA was observed to produce

different saccharide profile than free BLA at any value of dextrose equivalent. It was

observed that pH, temperature and initial starch concentration has a significant effect on

the saccharide profile of starch hydrolysate produced using immobilized BLA in batch

mode, whereas ratio of concentration of enzyme units to initial starch concentration has no

influence on the same. For free BLA, hydrolysis ceased at DE of around 42-43 because

BLA could not hydrolyze more α-(1-4) linkages due to the presence of branched dextrins;

whereas for immobilized BLA, DE of starch hydrolysate at hydrolysis equilibrium was

marginally low (around 36-37). While checking operation stability of immobilized BLA

without intermittent washing between two subsequent 8 hr batches, it was observed that in

the batch mode operation, the initial rate decreases to 70%, whereas in the packed bed it

decreases to about 20 %. A semi-empirical kinetic model has been used for the prediction

of saccharide composition of starch hydrolysate with respect to time.

It was observed that the use of immobilized BLA is not suitable for the production

of glucose from sorghum flour due to following,

Change in action pattern of enzyme giving different product composition profile

Drastic reduction in enzyme activity in reusability without intermittent washing

After mixing gelatinized sorghum slurry with beads, it was very difficult to

separate beads from the slurry even after the completion of reaction

BLA is now cheaply available enzyme at a cost of 250 Rs./kg

Thus, it may be more economically viable to use BLA and other enzymes in the free form

for the production of glucose from sorghum flour.

Page 265: Studies in depolymerization of natural polysaccharides--PhD thesis

Production of Glucose from sorghum flour using B. licheniformis α-amylase (BLA)

and Amyloglucosidase (AG)

Production of glucose from sorghum flour comprises two steps viz. 1. Liquefaction

using B. licheniformis α-amylase (BLA) 2. Saccharification using amyloglucosidase (AG).

In the liquefaction, gelatinization of free starch granules and dextrinization of gelatinized

starch granules occurs simultaneously. In the saccharification, AG cleaves first α-(1-4)

glycosidic bond from non reducing end and releases glucose molecules. Specialty of AG is

that it can cleave both α-(1-4) and α-(1-6) bonds and enables complete hydrolysis of starch.

Hence, Liquefaction process was first optimized. Flour was made from sorghum grains

using old fashioned flour mill. Liquefaction of sorghum flour was performed in a 250 mL

stoppered conical flask containing 100 mL magnetically stirred sorghum slurry.

Liquefaction of sorghum slurry (30% w/v in 0.05 M acetate buffer of pH 6) was performed

using BLA at 85 °C (maintained by immersing conical flask in oil bath). Progress of

liquefaction was monitored using starch-iodine colorimetric reaction. When color becomes

reddish with tinge of violet (at this stage DE of liquefact was around 15), liquefaction is

considered to be completed. Liquefaction process is optimized for the reaction time of 1.5

h using buffered slurries of different pH values (5.2-6.7), varying concentrations of BLA

(0.04-0.16% v/w of sorghum flour), and CaCl2 concentration (0-500 ppm) and temperature

in the range of 75 - 95°C. Optimized values of temperature, pH, slurry concentration, BLA

concentration and CaCl2 concentration are 85-90°C, 6, 30 % w of sorghum slurry/v of

slurry, 0.06 % v/w of sorghum flour i.e. 0.086 % v/w of sorghum starch and 200 ppm

respectively. Sorghum of different qualities i.e. healthy, blackened and germinated were

used for liquefaction. Liquefaction of healthy and blackened sorghum gets completed in

Page 266: Studies in depolymerization of natural polysaccharides--PhD thesis

1.5 h under optimized conditions, but liquefaction rate for blackened sorghum was slightly

lesser than that for healthy sorghum. However liquefaction of germinated sorghum gets

completed in 1 h only. We have also studied the effect of prior sonication on the

liquefaction performance. It was observed that sonication of sorghum slurry before

liquefaction improves liquefact DE by 10 – 25 % depending upon the sonication time and

the intensity. This must be happening because in the sorghum grain there are three

different types of endosperm viz. floury endosperm (starch granules are loosely associated

with protein material), corneous endosperm (starch granules packed inside protein bodies)

and peripheral endosperm (large amount of protein with less amount of starch); sonication

must be making starch granules free, which otherwise are packed inside protein bodies and

are inaccessible for enzyme action. It was also observed that sonication prior to

liquefaction increases filterability of the slurry after liquefaction. After liquefaction, pH of

the slurry was reduced to 4.5 and was hot filtered using muslin cloth to remove large

pericarp particles, fibers and proteins. Then filtrate was saccharified using AG.

Optimum pH and the temperature for amyloglucosidase by using assay procedure

were 4.5 and 65 °C respectively. Activity of AG was observed to be 36300 EU per mL of

commercial AG solution at pH 4.5 and 65 °C. Optimum temperature by assay procedure

may not be same as the optimum operating temperature. Hence thermo stability of AG was

studied at different temperatures and saccharification was also performed at different

temperatures. It was observed that operating optimum temperature for saccharification

using AG was 55-60°C. Other Optimized reaction conditions for saccharification to

glucose using AG were pH 4.5, 0.05 %v/ w of starch and saccharification time of 24 h. The

value of % saccharification to glucose using AG was in the range of 70- 90% depending

Page 267: Studies in depolymerization of natural polysaccharides--PhD thesis

upon following factors; 1. Sonication of sorghum slurry before liquefaction and 2.

Washing of cake (obtained after hot filtration) followed by 2nd filtration and mixing of both

the filtrates. These experiments were performed for healthy sorghum, germinated sorghum

and blackened sorghum. It was observed that sonication of slurry, prior to liquefaction

improves % saccharification by around 10 % and a similar improvement in %

saccharification was observed due to washing of cake after 1st filtration.

Production of Maltose from sorghum flour using B. licheniformis α-amylase (BLA),

Barley β-amylase (BBA) and / or pullulanase

Optimum pH and temperature for barley β-amylase (BBA) were observed to be

5.3-5.7 and 50 °C respectively. Activity of BBA was 12500 EU per mL of commercial

BBA solution at pH 5.5 and 50 °C. Optimum pH and temperature for pullulanase (PU)

were 3.8 – 4.4 and 60 °C respectively. Activity of PU was 2950 EU per mL of commercial

PU solution at pH 4 and 60 °C. BBA is an exo-amylase which cleaves 2nd α-(1-4)

glycosidic bond from non reducing end and releases maltose molecules. Limitation of

BBA is that it can neither cleave nor bypass α-(1-6) glycosidic bond. Hence use of BBA

results into the production of maltose and β-limit dextrins. Pullulanase (PU) is an endo-

amylase, which hydrolyses only α-(1-6) glycosidic bond. Hence, the combined use PU

with BBA will produce maltose in major quantity; whereas maltotriose and glucose will

get produced in lesser quantities.

For maltose production, liquefaction part remains the same. Filtrate after

liquefaction was also saccharified to maltose using barley β-amylase with or without

pullulanase. Optimized reaction conditions for the saccharification to maltose were pH 5.5,

Page 268: Studies in depolymerization of natural polysaccharides--PhD thesis

50 °C, 0.04 % v/ w of starch and reaction time of 24 h. Experiments were performed on

healthy sorghum, germinated sorghum and blackened sorghum for the production of

maltose. The value of % saccharification to maltose using BBA was in the range of 60 -

86% depending upon the following factors; 1. Sonication of sorghum slurry before

liquefaction ; 2. Washing of cake after hot filtration followed by 2nd filtration and mixing

of both filtrates and 3. Use of pullulanase along with BBA. It was observed that sonication

of slurry, prior to liquefaction improves % saccharification by around 10 % and a similar

improvement in % saccharification is observed due to washing of cake after 1st filtration.

Use of pullulanase along with BBA increases % saccharification by around 20 – 30%

depending upon whether prior sonication was done or not.

A cost comparison analysis of these methods have been carried out and it has been

shown that a significant value addition of the sorghum can be achieved by the processes

described in this work.

References

1. Devarajan B.; Pandit A. B., Sorghum flour as Raw Material for Glucose Production.

J. Maharashtra Agric. Univ., 1996, 21 (1), pg. 86-90.

2. Aggarwal N. K.; Nigam P.; Singh D.; Yadav B. S., Process optimization for the

production of sugar for the bioethanol industry from sorghum, a non-conventional

source of starch. World Journal of Microbiology & Biotechnology, 2001, 17, 411-415.

3. Owuama, C.I., Sorghum: a cereal with lager beer brewing potential. World Journal of

Microbiology & Biotechnology, 1997, 13, 253–260.

4. Lali, A. M.; Manudhane, K. S. Indian Patent Application No., 356/Mum/2003.