Thesis final (wilson 2009-080-085)

Production of Bioactive Peptides from Red

Bean, Mung Bean, and Soy Bean using

Bromelain Enzyme

Wilson Dwinanda Putra

2009-080-085

Thesis

Faculty of Biotechnology

Atma Jaya Catholic University of Indonesia

Jakarta

2013

i

Production of Bioactive Peptides from Red Bean,

Mung Bean, and Soy Bean using Bromelain

Enzyme

Wilson Dwinanda Putra 2009-080-085

Thesis

As partial fulfillment for the degree of

Bachelor of Science in Faculty of Biotechnology,

Atma Jaya Catholic University of Indonesia

Faculty of Biotechnology

Atma Jaya Catholic University of Indonesia Jakarta

2013

ii

INTELLECTUAL PROPERTY STATEMENT FORM

I,

Name : Wilson Dwinanda Putra

NIM : 2009-080-085

Certify that my thesis is my own original work and no portion of my thesis has

been copyrighted previously unless properly referenced.

If there is a breach of items above, I will take full responsibility to Atma Jaya

Catholic University of Indonesia for any legal action that might be caused.

Jakarta, January 31st 2013

Wilson Dwinanda Putra

iii

APPROVAL FORM

We hereby certify that:

Name : Wilson Dwinanda Putra

NIM : 2009-080-085

Thesis title : Production of Bioactive Peptides from Red Bean, Mung

Bean, and Soy Bean using Bromelain Enzyme

Date of exam : January 31st, 2013

Has passed the thesis exam and confirmed that this thesis had been thoroughly

examined, improved, and approved by advisors.

Approved by

Prof . Dr. Ir. Maggy T. Suhartono Dr. Noryawati Mulyono, S.Si.

Advisor Co-Advisor

Acknowledged

Dr. Diana Elizabeth Waturangi, M.Si.

Dean

iv

PREFACE

This thesis is a final work as partial fulfillment for the degree of Bachelor of

Science in Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia

titled “Production of Bioactive Peptides from Red Bean, Mung Bean, and Soy

Bean using Bromelain Enzyme” was conducted in Biochemistry and Enzyme

Technology Research Laboratory, Faculty of Biotechnology, Atma Jaya Catholic

University of Indonesia, from June 2012 until January 2013.

Author would firstly like to thank God for His blessings until this thesis

research is completed. Author also like to thank all people who were involved in

this research, Prof. Dr. Ir. Maggy T. Suhartono and Dr. Noryawati Mulyono, S.Si.

as advisors, Yanti, Ph.D. as examiner, technicians in Faculty of Biotechnology,

Atma Jaya Catholic University of Indonesia for their valuable comments,

suggestions, and feedbacks in technical and data analysis.

Furthermore, many people are appreciated for their assistance for data

collection process. Author would like to thank partners, Maureen Novia and

Yosep Sutanto, for their cooperation in this research, family for their love and

supports, all of my friends for times we have spent together, and all other people

that indirectly involved in the research.

Lastly, may this thesis be useful for readers and for future research about

similar topic or any other related field.

Jakarta, January 31st 2013

Wilson Dwinanda Putra

v

BIOGRAPHY

Wilson Dwinanda Putra was born in Jakarta, November 14th, 1991 as the

third son from Djap Darmanto and Tjong Lindawaty. He graduated from IPEKA

Sunter 2 Senior High School, Jakarta in 2009 and obtained his degree in Bachelor

of Science in January 2013 from Faculty of Biotechnology, Atma Jaya Catholic

University of Indonesia.

During his course in Atma Jaya Catholic University of Indonesia, Author

was involved in several commitees, including “BONSAI” charity event in 2009,

“ELBOW” entrepreneurship seminar in 2010, Science Club Fieldtrip as public

relation division in 2011, Science Club event division coordinator (2011 to 2012),

and “MICROVONE” charity concert in 2012.

Beside his organizational experiences, he have been worked several times

in Atma Jaya Faculty of Biotechnology, as assistant in Basic Chemistry

Laboratory in 2010, Biochemistry Laboratory in 2011, Biostatistics in 2011, and

Food Technology Laboratory in 2012. He completed a short internship program at

PT Sosro Tbk., Bekasi as a Chemical Analyst Staff from January until February

2012.

vi

CONTENTS

Page

List of Tables ............................................................................................ vii

List of Figures ........................................................................................... viii

List of Appendices .................................................................................... ix

PRODUCTION OF BIOACTIVE PEPTIDES FROM RED BEAN, MUNG

BEAN, AND SOY BEAN USING BROMELAIN ENZYME

Abstract ........................................................................................... 1

Introduction ..................................................................................... 1

Literature Review ............................................................................ 2

Materials & Methods ....................................................................... 3

Results ............................................................................................. 5

Discussions ...................................................................................... 8

Conclusion ....................................................................................... 11

References ....................................................................................... 11

Appendices ...................................................................................... 13

vii

LIST OF TABLES

Page

1 BSA concentration for standard curve .................................................. 4

2 Tyrosin concentration for standard curve .............................................. 4

3 Composition of SDS-PAGE gel ........................................................... 5

4 Increment of DPPH scavenging activity (0.173 U/ml) .......................... 8

5 Increment of ferric reducing activity ...................................................... `8

6 Increment of ACE inhibitory activity ................................................... 8

7 Bioactivity of many plant and animal samples in various studies .......... 10

viii

LIST OF FIGURES

Page

1 The chemical structure of the compound DPPH free radical and non-

radical .................................................................................................. 3

2 Renin angiotensin system (RAS) ......................................................... 3

3 DPPH scavenging activity (%) of bean flour substrates hydrolyzed

with bromelain enzyme ........................................................................ 6

4 Ferric reducing activity of bean flour substrates hydrolyzed with

bromelain enzyme ................................................................................ 7

5 ACE inhibitory activity (%) of bean flour hydrolyzed with bromelain

enzyme ................................................................................................. 7

6 SDS-PAGE result of hydrolysates of bean samples with different

hydrolysis time ..................................................................................... 8

ix

LIST OF APPENDICES

Page

1 Procedure of ACE inhibitory activity ..................................................... 14

2 DPPH scavenging activity (%) of bean flour substrates (10 mg/ml)

hydrolyzed with bromelain enzyme (0.272 U/ml) ................................. 14

3 Tyrosin standard curve ......................................................................... 15

4 Bradford standard curve ....................................................................... 15

1

Produksi Peptida Bioaktif dari Kacang Merah, Kacang Hijau,

dan Kacang Kedelai menggunakan Enzim Bromelain

Production of Bioactive Peptides from Red Bean, Mung Bean, and

Soy Bean using Bromelain Enzyme

WILSON DWINANDA PUTRA

Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia

Jalan Jenderal Sudirman 51, Jakarta 12930

Abstract. Food proteins have been known to contain peptide sequences capable of modulating

health-related benefits in human. These proteins, termed bioactive peptides, can be used as safe, effective, natural antioxidant and antihypertension drug. The purposes of this study were to

produce bioactive peptides derived from the various beans using bromelain enzyme and evaluate

the effect of bioactive peptides as antioxidant and antihypertension agents. Bromelain enzyme

extracted from pineapple and bean protein extracts were mixed to produce bioactive peptides at

different hydrolysis time. 1,1-diphenyl 2-picrylhydrazyl (DPPH) scavenging activity and ferric

reducing power were used to evaluate the effect of peptides as antioxidants. Antihypertensive

capabilities were measured regarding inhibition of angiotensin-I converting enzyme (ACE) by

sample. The highest activity for DPPH scavenging assay was mung bean (94.90 %) and for ferric

reducing power assay was red bean (1.367) at 5 min hydrolysis time compared with ascorbic acid

and α-tocopherol. It also showed that all samples have ACE inhibitory activity, while the highest

activity was mung bean (96.67 %), which is higher than captopril as positive control. Enzyme activity, protein concentration and hydrolysis time variation seem to play role in bioactivity. This

research showed the bioactive peptides produced from beans could act as potent radical scavengers

and antihypertension drugs.

Keywords: bioactive peptides; antioxidant; antihypertension

Abstrak. Protein makanan telah diketahui memiliki sekuens peptida yang dapat

memberikan efek yang menguntungkan bagi manusia, Protein ini, yang disebut peptida bioaktif,

dapat digunakan sebagai antioksidan dan obat antihipertensi alami yang aman dan efektif. Tujuan

dari penelitian kali ini adalah untuk memproduksi peptida bioaktif dari berbagai jenis kacang-

kacangan menggunakan enzim bromelain dan mengetahui efek peptida bioaktif sebagai

antioksidan dan agen antihipertensi. Enzim bromelain diekstraksi dari nanas lalu dicampurkan

dengan ekstrak protein kacang-kacangan dalam waktu hidrolisis berbeda untuk menghasilkan peptida bioaktif. Uji aktivitas penangkapan radikal 1,1-diphenyl 2-pikrilhidrazil (DPPH) dan

kekuatan reduksi ferri digunakan untuk mengukur aktivitas antioksidan. Kemampuan

antihipertensif diukur berdasarkan penghambatan enzim konversi angiotensin-I (ACE) oleh

sampel. Aktivitas tertinggi untuk uji penangkapan DPPH adalah kacang hijau (94.90 %) dan untuk

uji kekuatan reduksi ferri adalah kacang merah (1.367) dengan waktu hidrolisis 5 menit,

dibandingkan dengan asam acrobat dan α-tokoferol. Hasil lain juga menunjukan semua sampel

memiliki aktivitas penghambatan enzim ACE, dimana yang tertinggi adalah kacang hijau (96.67

%), yang lebih tinggi dibandingkan kaptopril sebagai kontrol positif. Aktivitas enzim, konsentrasi

protein, dan waktu hidrolisis juga berperan dalam bioaktivitas. Penelitian ini menunjukkan peptida

bioaktif yang dihasilkan dari kacang-kacangan dapat menjadi agen penangkap radikal bebas dan

obat antihipertensi yang potensial. Kata kunci: peptida bioaktif; antioksidan; antihipertensi

INTRODUCTION

Nowadays, consumer preferences for

processed foods, concern in use of synthetic

compounds and chemical additives, and

increased knowledge of the relationship

between diet and health raise the demand for

natural and functional food ingredients. Food

proteins, besides their nutritional roles,

contain peptide sequences encrypted in their

2

primary structure that are capable of

modulating specific physiological functions

that are very useful to be used in medical field

and any other fields (Correa et al. 2011).

These protein fragments, termed bioactive

peptides, are inactive within the sequence of

the native protein, and could be released

through enzymatic hydrolysis in vivo and in

vitro (Correa et al. 2011). After release,

bioactive peptides might exert

antihypertensive, antibacterial, anticancer, and antioxidant activities, which have potential

applications in food science, technology, and

nutritional science (Shahidi & Zhong 2008).

Bioactive peptides have been isolated from

other food sources of protein, including egg,

fish, oyster, cereals (rice, wheat, barley and

maize) and soy bean (Wang & Gonzales

2005).

The purposes of this study were to produce

bioactive peptides derived from the various

beans using bromelain enzyme extracted from pineapple and evaluate the effect of bioactive

peptides as antioxidant and antihypertension

agents.

LITERATURE REVIEW

A. Pineapple & Bromelain

Pineapple (Ananas comosus) is a plant

originating from Brazil (South America) and

one of Bromeliaceae family (another example;

Tillandsia usneoides & Puya raimondii).

Pineapple contains many essential minerals

like manganese as a enzymatic reaction cofactor for energy production and antioxidant

activities. One of the enzymes that use these

minerals is superoxide dismutase (SOD),

which also acts as an antioxidant (Subroto

2008).

Pineapple plant has many functions

especially for solving health problems, such as

antiinflammatory, antimicrobial, therapeutic

activity in blood clotting, also inhibition of

tumor growth (Bartholomew et al. 2003).

Besides the health related functions, the

availability and cheap price of pineapple fruit are the good points that make this fruit is quite

popular around communities (Sunarjono

2000).

So many side effects found while using

synthetic compounds, now the use of natural

materials are important safety requirements

before the products commercially applied.

Pineapple fruit is one plant that is easily

obtained in any season and already known has

many benefits, like meat tenderizer beside

papain enzyme (Sunarjono 2000). Bromelain

may also be used in a variety of other

conditions like hay fever, ulcerative colitis,

removal of dead and damaged tissue after

debridement in burns, preventing the

collection of fluid in the lung pulmonary

edema, relaxing muscles, stimulating muscle

contractions, slowing clotting of blood,

improving the absorption of antibiotics,

preventing cancer, shortening delivery time,

and helping the body to get rid of fat.

Bromelain has even been widely studied as a complementary cancer treatment, as it

contains powerful antioxidant components

(Priya et al. 2011).

Bromelain is a mixture of cysteine

proteases and non proteases components,

extracted from pineapple plant (Ananas

comosus) and divided into two types, stem

(E.C. 3.4.22.32) and fruit bromelain (E.C.

3.4.22.33) (Maurer 2001). Bromelain is stable

at pH 3 - 8 (optimum pH 4.5 - 7.5) and at

temperatures up to 60 °C (optimum temperature 35 - 45 °C). Bromelain powder

can be stored originally packed and tightly

closed up to two years at < 8 °C without

reduction of enzyme activity (Poh & Majid

2011).

B. Bioactive Peptides

Beans have long been recognized as a

complementary source of protein with whole

grains, like rice and wheat. Beans also contain

nutrients other than protein, like minerals, vitamins, complex carbohydrates and dietary

fiber (Astawan 2009). Red bean (Phaseolus

vulgaris L.) contains 22.3 g protein per 100 g

dry weight, which is similar to the mung bean

(Vigna radiata) (20-25 g per 100 g dry

weight), while soybean (Glycine max)

contains highest protein, 35 g protein per 100

g dry weight (Astawan 2009).

Bioactive peptides from enzymatic

hydrolysis (treated with protease:

alcalase,trypsin, etc) could posses

antioxidative properties against free radicals. Free radicals are atoms or groups of atom that

have one or more unpaired electrons. The

existence of unpaired electrons causes the

highly reactive compounds looking for a

partner. This radical is often associated with

cell damage, tissue damage, and the aging

process (Fang et al. 2002).

Antioxidants are chemical compounds that

can contribute electron(s) or hydrogen atom

(proton) to free radicals, so that free radicals

may be quenched and turn into more stable form. According to sources there are two

kinds of antioxidant, natural antioxidant and

3

artificial antioxidant (synthetic, like butylated

hydroxytoluene (BHT) and butylated

hydroxyanisole (BHA), which prevent

oxidative rancidity (lipid peroxidation) of

foods). Synthetic antioxidants often show side

effects such as induces cancer or tumour, so

natural antioxidants could be answers for this

problems (Race 2009).

The human body has no supply of

antioxidants within the muscle, so that if there

are excess radicals in the body, the body needs extra antioxidants or exogenous antioxidants

from outside the body (Aini 2007). There are

so many antioxidant mechanisms, include

radical scavenging (both hydrogen-donating

capability (for example, 1,1-diphenyl-2-picryl

hydrazyl) and free radical quenching activity),

inhibition of lipid peroxidation, metal ion

chelation, or a combination of these properties

(Sarmadi & Ismail 2010).

Figure 1 The chemical structure of the compound

DPPH free radical and non-radical (Molyneux 2004).

Antioxidants are naturally found in many

types of plants, such as the berries (blueberry,

acai berry, goji berry), pomegranate, peppers,

beets (Carlsen et al. 2010). There is concern

over the possibility of unknown side effects of

synthetic antioxidants has become an

alternative natural antioxidant that is needed.

Antioxidants are expected to have features such as safe in use, do not give flavor, odor,

and color of the product, effective at low

concentrations, resistant to a wide range of

processing products, and available at cheap

price (Aini 2007).

The most studied bioactive peptides are

the antihypertensive peptides that modulate

angiotensin-I converting enzyme (ACE, EC

3.4.15.1). Some of these peptides have also

been used as functional foods for

antihypertension problem (ACE inhibitor) such as „Ameals‟, „peptACE‟, and „Biozate‟,

which contain highly purified peptides (small

proteins) from milk, fish, and whey proteins,

respectively. ACE is one of the key enzymes

in the blood pressure regulation pathway

(renin-angiotensin system). ACE acts by

removing a dipeptide (two amino acids linked

together) from the C-terminal of angiotensin I

to produce angiotensin II, the latter being a

very powerful vasoconstrictor (Aluko 2008).

Figure 2 Renin-Angiotensin System (RAS) (Belovic

et al. 2012).

In disease conditions or as a result of

genetic or environmental factors, the level of

ACE is up-regulated and the resultant high levels of angiotensin II produces undesirable

rates of blood vessel contraction that leads to

the development of high blood pressure

(hypertension). Though less active than

commercial drugs, naturally occurring

hypotensive peptides are believed to be safer

than synthetic drugs as agents for the

treatment of hypertension since synthetic

drugs have some side effects. It is also

important to note that food protein-derived

ACE-inhibitory peptides can be consumed at

higher levels since there is no risk associated with an overdose when compared to

commercial drugs that need to be used at

levels below the upper tolerable limit (Aluko

2008).

MATERIALS AND METHODS

Materials. The materials used for this

research were Palembang pineapple, red bean

flour, mung bean flour, and soybean flour

(both fluor samples from high standard

commercial flours), ascorbic acid (a. acid) 320

mg, α-tocopherol (α-toco) 67 mg, captopril (12.5 mg), absolute ethanol, sodium

carbonate, L-Tyrosine, Coomasie Brilliant

Blue G-250 (CBB G-250), phosphoric acid,

bovine serum albumin (BSA), NaOH, HCl,

casein, sodium phosphate, acrylamide, NN-

methylene-bis acrylamide, Tris, sodium

dodecyl sulfate (SDS), bromphenol blue,

potassium phosphate (KH2PO4 and K2HPO4),

1,1-diphenyl-2-picrylhydrazyl (DPPH),

potassium ferricyanide, trichloroacetate

(TCA), ferric chloride, Angiotensin I-

converting enzyme (ACE), ACE substrate (Hip-L-His-L-Leu), sodium borate, sodium

chloride, and ethylacetate. In antioxidant

assays, ascorbic acid and α-tocopherol were

used as positive controls. Ascorbic acid was

diluted in distilled water and α-tocopherol was

diluted in absolute ethanol. In ACE inhibitory

4

assay, captopril was diluted in distilled water.

Other chemicals and reagents used were of the

highest analytical grade commercially

available.

Protein Extraction. Flour mixed with

water in the ratio of sample : distilled water =

1: 9. Next step, adjust the pH of solution to

8.6 by addition of 1 M NaOH solution and

stirred for 30 min at a temperature of 50 – 55

°C. Then centrifuge at 3000×g for 30 min, which will form pellet and supernatant. pH of

the supernatant was lowered by the addition of

2 M HCl solution to 4.5 for protein

precipitation. Centrifugation perfomed again

at 1500×g for 20 min. Pellet obtained was

dried using incubator at 50 °C for 72 h. Then

concentration of protein in the pellet was

measured .

Protein Concentration Assay (Bradford

1976). Bradford reagent was made by diluting 100 mg comasie brillant blue G-250 into 50

ml of absolute ethanol; 100 ml of H3PO4 85%

(w/v), and then added by aquadest until 1000

ml. The reagent was incubated overnight and

filtered to remove the insoluble matter. The

obtained filtrate was kept as stock solution

and diluted (10 times) when used.

Bovine Serum Albumin (BSA) was used as

standard analysis. A series of solution was

made as standard to make the Lambert-Beer

curve so the protein concentration could be calculated. The composition of standard

solution was shown in Table 1.

Table 1 BSA concentration for standard curve

A 0.2 ml BSA for each concentration were added by 4 ml Bradford working solution and

then incubated for 5 minutes before measured

at 595 nm. Briefly, the sample analysis was

done by adding 0.2 ml sample with 4 ml

Bradford working solution. Blank used for

this assay was 0.2 ml aquadest. Standard

curve can be seen in Appendix 4.

Extraction of Bromelain. Pineapple was

peeled and separated from the flesh and crown

of the fruit. Then pineapple core was blended

and 0.01 M potassium phosphate buffer pH 7

was added half of the total pineapple stem

weight. The mixture was filtered using

cheesecloth. Centrifugation was performed at

3000×g for 25 min. The filtrate is stored in

dark bottles in the refrigerator (4 °C) for

further use (Kosasih 2012).

Enzyme Precipitation with Ethanol. A

50 ml cold absolute ethanol was slowly added

to filtrate until ethanol level reach 60%. The mixture was incubated for 24 h 4 °C to

precipitate bromelain, then centrifuge at

4000×g for 25 min to obtain pellet and filtrate.

Pellets were weighed and dissolved using 0.02

M potassium phosphate buffer pH 7.0 as

much as the pellet weight. A 0.5 ml of the

pellet was dissolved in 0.02 M potassium

phosphate buffer pH 7.0 (10 times dilution)

(Kosasih 2012).

Protease Activity Assay (Enyard 2008). Protease activity of bromelain enzyme was

measured using casein 1% as enzyme

substrate. A 800 µL casein was mixed with

200 µL enzyme then incubated at 37°C for 10

min. A 500 µL TCA was added to the mixture

to stop the enzyme reaction. For blank

solution, TCA was added before the addition

of enzyme. Mixture was incubated 30 min in

room temperature to let TCA works before

centrifuged at 11000×g for 10 min.

After that, 400 µL supernatant was pipetted to a new microcentrifuge tube and 1

ml Na2CO3 and 200 µL of Folin and

Ciocalteu‟s phenol were added to the solution

then incubated at 37oC for 30 min. Mixture

was centrifuged 11000×g for 10 min.

Supernatant was measured by

spectrophotometer in 660 nm wavelength. A

series of solution were made as standard to

make the Lambert-Beer curve so that the

activity of enzyme could be calculated. The

composition of standard solution was shown

in Table 2.

Table 2 Tyrosin concentration for standard curve

All reagents were vortexed and 400 µL

were pipetted into a new micro centrifuge tube.

As much as 1 ml Na2CO3 and 200µL of Folin

and Ciocalteu‟s phenol were added to the

Tube [BSA]

(mg/ml)

BSA

volume

1 mg/ml

(ml)

Aquadest

volume (ml)

1

2

3

4

5

6

1.0

0.8

0.6

0.4

0.2

0.1

2.50

2.00

1.50

1.00

0.50

0.25

0.00

0.50

1.00

1.50

2.00

2.25

5

solution, then incubated at 37 oC for 30 min.

Mixture was centrifuged 11000×g for 10 min.

The obtained supernatant was measured at

660 nm wavelength. One unit of enzyme

activity was defined as enzyme which

produced 1µmol of tyrosin per minute.

Standard curve can be seen in Appendix 3.

Production of Bioactive Peptides. Protein extract was added with bromelain

(protein sample : enzyme = 10 : 1), and was incubated at desired hydrolysis time (0, 5, 10,

20, 30, 60 min). The reaction was stopped by

incubation at 90 °C for 10 min using water

bath.

DPPH Radical Scavenging Activity

(Bersuder et al. 1998). This assay used to

measure capability of samples as proton

(hydrogen) donors. As much as 500 μl of bean

sample solution was mixed with 500 μl of

ethanol 99.5% and 125 μl ethanol 99.5% containing 0.02% (w/v) 2,2-diphenyl-1-

picrylhydrazyl (DPPH). The mixture was kept

in the dark at room temperature for 60 min

before the absorbance was measured

spectrophotometrically at 517 nm.

Radical scavenging activity (%) =

%100

Control

SampleBlankControl

Where the control was the absorbance

value of 500 μl of distilled water & 125 μl of

ethanol 99.5% containing 0.02% DPPH & 500

μl ethanol 99.5%, and the sample containing 500 μl sample solution & 125 μl of ethanol

99.5% including 0.02% DPPH & 500 μl

ethanol 99.5%, and the blank was 500 μlof

sample solution & 125 μl ethanol 99.5% &

500 μl ethanol 99.5%.

Ferric Reducing Power Activity (Oyaiza

1986). This assay used to measure capability

of samples as electron donors. Sample

solution (0.5 ml) was mixed with 2.5 ml of 0.2

M phosphate buffer (pH 6.6) and 2.5 ml of 1% w/v potassium ferricyanide. The mixture was

incubated at 50 °C for 20 min. A total of 2.5

ml trichloroacetic acid (TCA) was added to

the mixture, followed by centrifugation 700×g

for 10 min. The upper layer (2.5 ml) of the

solution was mixed with 2.5 ml of distilled

water and 2.5 ml of 0.1% ferric chloride and

the absorbance was read at 700 nm. Increasing

absorbance of the reaction mixture indicates

increasing reducing power.

ACE Inhibitory Activity. ACE inhibitory

activity of the peptide sample was measured

with a UV spectrophotometer based on the

rate of formation of hippuryl hippurat acid-L-

histidyl-L-leusine (HHL) (Chusman &

Cheung 1971). For each measurement, 10 μl

peptide samples and 10 μl HHL 50 mM were

incubated with 10 μl ACE 0.8 mU at 37 °C for

45 min. The reaction was stopped by adding

100 μl of 1 N HCl, then 600 μl ethyl acetate

was added and homogenized using vortex for 30 seconds, and then centrifuged at 300×g for

10 min. A total of 100 μL supernatant (upper

layer) was taken and dried at a temperature of

140 °C for 10 min. The residue obtained was

dissolved with 600 μl 1 M NaCl. Absorbance

was measured at wavelength of 228 nm. The

complete procedure of measurement of ACE

inhibitory activity can be seen in Appendix 1.

ACE inhibitory activity (%) =

%100)(

AbAc

AsAc

Description: Ac = absorbance of control As = absorbance of the sample Ab = absorbance of blank

SDS-PAGE Analysis. To analyze and

visualize protein pattern, SDS-PAGE (sodium

dodecyl sulphate polyacrylamide gel

electrophoresis) was done. As much as 10 μl

sample were mixed with 40 μl sample buffer

and were incubated 100 °C for 5 min before

loading. Proteins were separated on a 6% (stacking gel) and 15% (separating gel) (Table

3) with 70 V for 3-4 h. After the run, the gels

were stained with CBB-R 250 staining

solution (15 min) and destained for 24 h

(Laemmli 1970).

Table 3 Composition of SDS-PAGE gel

Composition 15% (µl) 6% (µl)

Solution A 2500 1000

Solution B 1250 -

Solution C - 1250

MiliQ 1250 2750

APS 10% 100 50

TEMED 10 5

RESULTS

In this research, DPPH radical scavenging

activities of beans were analyzed with

different hydrolysis time and protein

concentration (Figure 3; Table 4). All samples

showed antioxidant capacity in scavenging

6

free radicals. Red bean, mung bean, and soy

bean hydrolysates exhibited the highest

activity at 10 mg/ml protein concentration,

both results (91.24 %, 94.90 %, 67.83 %,

respectively) were carried out at 5 min

hydrolysis time with 0.173 U/ml bromelain,

and decreased thereafter. Results also showed

that the increase of protein concentration gave

better scavenging activity in each sample

(from 0.5 mg/ml; 1 mg/ml; 2 mg/ml).

However, in 10 mg/ml with the same enzyme activity, the scavenging activity was reduced.

Higher enzyme activity (0.272 U/ml) which

used at 10 mg/ml concentration showed

higher scavenging activity (Appendix 2). It

also peaked after 5 min hydrolysis, decreased

thereafter. Due to this property, beans

hydrolysates could scavenge by donating

protons to free radicals and solution colour

were changed to yellowish colour.

Results of ferric reducing activity of

hydrolysates obtained after various hydrolysis time treatments. It showed that all samples

had antioxidant capacity in reducing ferric

into ferrous form (Figure 4; Table 5). All

samples showed highest activity at 2 mg/ml

protein concentration with 5 min hydrolysis

time (0.272 U/ml). Similarly, the results of

DPPH scavenging activity, samples were

peaked after 5 min hydrolysis and decreased

thereafter. Both antioxidant assays (DPPH &

Fe reducing) were compared to ascorbic acid

and α-tocopherol as positive controls.

For ACE inhibitory assay, all samples

could act as antihypertension agents. Mung

bean showed highest activity in different

enzyme activity treatment in 5 min hydrolysis,

90.13 % and 96.67 % (0.173 U/ml and 0.272

U/ml, respectively) with 10 mg/ml protein

concentration (Figure 5; Table 6).

Antihypertensive activity of beans increased with hydrolysis treatment (in most samples)

than without hydrolysis. Highest

antihypertensive activity in all samples and

different enzyme activity seen at 5 min

hydrolysis, decreased thereafter.

The SDS-PAGE results (Figure 6) showed

that several different molecular weights of

proteins were observed, which expected as

bioactive peptides in bean samples. The

breakdown of samples can be seen as protein

bands disappear over increasing hydrolysis time (showed by arrows). Peptide

concentrations are different depending on

their size: in the first stages of storage

peptides larger than 45 kDa were

predominant, but as time increased, the

concentration of lower MW peptides

increased.

Figure 3 DPPH scavenging activity (%) of bean flour substrates (RB: red bean; MB: mung bean; SB: soy bean) hydrolyzed

with bromelain enzyme (0.173 U/ml).

0

20

40

60

80

100

RB MB SB A.acid

(10mg/ml)

α-toco

(67mg/ml)

Scaven

gin

g acti

vit

y(%

)

(1 mg/ml)

0 min

5 min

10 min

0

20

40

60

80

100

RB MB SB A.acid

(10mg/ml)

α-toco

(67mg/ml)

Ssca

ven

gin

g a

cti

vit

y(%

)

(0.5 mg/ml)

0 min

5 min

10 min

0

20

40

60

80

100

120

RB MB SB A.acid

(10mg/ml)

α-toco

(67mg/ml)

Scaven

gin

g a

cti

cit

y(%

)

(2 mg/ml)

0 min

5 min

10 min

0

20

40

60

80

100

RB MB SB A.acid

(10mg/ml)

α-toco

(67mg/ml)

Scaven

gin

g a

cti

vit

y (

%)

(10 mg/ml)

0 min

5 min

10 min

20 min

30 min

60 min

7

Figure 4 Ferric reducing activity of bean flour substrates (RB: red bean; MB: mung bean; SB: soy bean) hydrolyzed with

bromelain enzyme.

Figure 5 ACE inhibitory activity (%) of bean flour substrates (RB: red bean; MB: mung bean; SB: soy bean hydrolyzed

with different bromelain enzyme activity.

0

20

40

60

80

100

RB MB SB C0.5 C1

AC

E in

hib

itory

acti

vit

y(%

)

(10 mg/ml; 0.173 U/ml)

0 min

5 min

10 min

20 min

30 min

60 min

0

20

40

60

80

100

RB MB SB C0.5 C1

AC

E i

nh

ibit

ory a

cti

vit

y (

%)

(10 mg/ml; 0.272 U/ml)

0 min

5 min

10

min20

min

0,0

0,5

1,0

1,5

2,0

2,5

RB MB SB A.acid

(10mg/ml)

α-toco

(67mg/ml)

Ferric

red

ucin

g a

cti

vit

y

(10 mg/ml; 0.173 U/ml)

0 min

5 min

10 min

20 min

30 min

60 min

0,0

0,5

1,0

1,5

2,0

2,5

RB MB SB A.acid

(10mg/ml)

α-toco

(67mg/ml)

Ferric

red

ucin

g a

cti

vit

y

(1 mg/ml); 0.272 U/ml)

0 min

5 min

10 min

20 min

30 min

60 min

0,0

0,5

1,0

1,5

2,0

2,5

RB MB SB A.acid

(10mg/ml)

α-toco

(67mg/ml)

Ferric

red

ucin

g a

cti

vit

y

(2 mg/ml; 0.272 U/ml)

0 min

5 min

10 min

20 min

30 min

60 min

(Captopril; mg/ml)

(Captopril; mg/ml)

8

Figure 6 SDS-PAGE result of hydrolysates (10 mg/ml; 0.272 U/ml) of bean samples with different hydrolysis time. Lane

M: LMW Marker; Lane 1-3: Red bean (0, 5, 60 min, respectively); Lane 4-6: Mung bean (0, 5, 60 min,

respectively); Lane 7-9: soy bean (0, 5, 60 min, respectively); Lane 10: Bromelain enzyme.

Table 4 Increment of scavenging activity (0.173 U/ml) (RB: red bean; MB: mung bean; SB: soy bean)

Table 5 Increment of ferric reducing activity (RB: red bean; MB: mung bean; SB: soy bean)

Table 6 Increment of ACE inhibitory activity (RB: red bean; MB: mung bean; SB: soy bean)

DISCUSSION

Free radicals with major species of ROS

are unstable and react readily with other

groups or substances in the body, resuting in

cell or DNA damage and, thus human disease. Therefore, removal of free radicals and ROS

is probably one of the most effective defenses

of a living body against various diseases. The

beneficial effects of antioxidants are well

known in scavenging free radicals and ROS or

9

in preventing oxidative damage by

interrupting the radical chain reaction of lipid

peroxidation (Kim et al. 2006).

DPPH is a stable free radical that show

maximum absorbance at 517 nm in ethanol.

When DPPH encounters a proton-donating

substance, such as antioxidant, the radical is

scavenged. The color is changed from purple

to yellow and the absorbance is reduced. The

disappearance rate of DPPH free radicals was

used to express the free radical scavenging activity of beans protein hydrolyzed with

bromelain under optimized conditions

(Shimada et al. 1992).

Ferric reducing power activity also used to

measure the antioxidant activity based on

electron-donating activity. Compounds that

have antioxidant activity reacts with

potassium ferricyanide (Fe3+) to form

potassium ferrocyanide (Fe2+

) by reduction,

which then forms a complex react with FeCl3

to form complex with FeCl2. The resultant solution will be measured by the

spectrophotometric method at a wavelength of

700 nm (Hemalatha et al. 2010). The reducing

ability of beans indicates that they could act as

electron donors, reducing the oxidized

intermediates of lipid peroxidation processes,

and suggesting that the reducing power likely

contributes to the antioxidant activity (Zhu et

al. 2006).

Results showed there were variation of

scavenging activity and ferric reducing power between each sample and each treatment

(hydrolysis time). It is also observed that

protein concentration has effect on antioxidant

activity, and most likely the higher sample

concentration, the higher ability of samples to

act as antioxidant. But this properties must be

optimized with enzyme activity. Peptides and

protein hydrolysates obtained from the

proteolysis of various food proteins, are

reported to posses antioxidant activities. This

increasing antioxidant activity through

hydrolysis suggests that this process contributed to antioxidant activity by releasing

previously inactive peptides encrypted in the

sequence of bean samples and then act as

proton or electron donor (Correa et al. 2011).

However, the longer hydrolysis with

increasing antioxidant activity can not always

be observed. In this study, mostly at 60 min

hydrolysis time, samples showed reduction in

activity compared to unhydrolyzed (0 min)

samples. This possibly caused by shorter

peptide chain length and also functionality of hydrolysis depended on enzyme characteristic

(Klompong et al. 2007).

The distinctive behavior of protein

hydrolysates in the antioxidant activity also

could be explained by the different

stereoselectivity of the radicals and different

peptides present in samples capable of

reacting and quenching different radicals.

Also, the antioxidant effect of beans and bean-

derived peptides is not fully based on

capability to donate hydrogen or electron, and

differences is scavenging efficacy can be

attributed to solubility and diffusivity of radicals. His, Phe, Tyr, Trp, among other

aromatic and hydrophobic amino acids, seems

to be involved in the antioxidant activity

(Correa et al. 2011).

The other method for measuring

antioxidant activity is 2,2'-azinobis-3-

ethylbenzothiazoline-6-sulfonic acid) (ABTS)

radical scavenging assay. This assay is

generated by oxidation of ABTS radical

chromophore with potassium persulfate and is

reduced in the presence of such hydrogen-donating antioxidant. The solution are

measured at 734 nm wavelength to find

percentage of inhibition (Correa et al. 2011).

Direct reaction of a substance is not the

only mechanism by which the antioxidants

may display their activity. Secondary

antioxidants act through numerous possible

mechanisms. One of the most important

mechanisms of action of secondary

antioxidants is chelation of prooxidant metals.

Iron and other transition metals (copper, chromium, cobalt, vanadium, cadmium,

arsenic, nickel) promote oxidation by acting

as catalysts of free radical reactions (Koncic et

al. 2011). For example, the chelating

properties of hydrolysates and peptide

fractions can be evaluated by monitoring the

formation of the complex between ferrozine

and Fe2+ (ferrous) ion spectrophotometrically

at 500 nm. When a compound has chelating

properties, the formation of complex

(Ferrozine/Fe2+) is disrupted resulting in a

decrease of the red colour and this makes possible the estimation of the metal chelating

activity of constituents (Yamaguchi et al.

2000).

Many antihypertensive drugs have their

primary action on systemic vascular

resistance. Some of these drugs (captopril,

lisinopril) produce vasodilation by interfering

with vascular tone or by blocking the

formation of angiotensin II or its vascular

receptors. The other drugs have different

medication mechanism, such as calcium channel blocker, beta blocker, and diuretic

effect (Dilpardo 2010). Although some drugs

10

less commonly used because of a high

incidence of side effects, like itching; rash;

dry cough, therefore natural sources, are

promising to be used as treatment for patient,

including bioactive peptides.

Related to that, results showed ACE

inhibitory activity in all hydrolysates were

high, and some of them had higher activity

than captopril (Figure 5). This suggests that

bean samples could be potent antihypertension

drugs with very minimum side effects. It also

showed that different enzyme activity and

hydrolysis time contribute to various result of

ACE inhibitory activity. Red bean, mung

bean, and soy bean have been known for its

antioxidative characteristic, as mentioned in

previous studies. This research also showed

that proteins in each bean sample contributed

to both antioxidant and antihypertensive

capabilities (Table 7).

Table 7 Bioactivity of many plant and animal samples in various studies

The effect of bromelain on protein

breakdown in beans at the different hydrolysis

time was analyzed by SDS-PAGE (Figure 6).

Bromelain catalyzes hydrolysis of protein and

peptide amides with broad specificity for

peptide bonds and is a preference for large

uncharged residue. Molecular weight distribution of hydrolyzed protein is one of the

most important properties in producing

protein hydrolysates to be used as functional

materials, which has a direct impact on their

functional properties (Takano 2002).

SDS-PAGE results showed that so many

proteins detected with various molecular

weight. Red bean sample showed quite many

bands, mainly about 23-35 kDa (globulin

types around 22 kDa-37 kDa), 40-50 kDa

(amylase inhibitor types around 45-50 kDa) in unhydrolyzed condition (Meng & Ma 2002;

Marambe & Wanasundara 2012). After

hydrolysis, red bean sample showed reduction

in bands, except protein below 30 kDa, which

seem not being hydrolyzed. Protein with 40-

50 kDa (amylase inhibitor), showed reduction

of bands after 5 min and 60 min hydrolysis,

suggest that it turned into small proteins or

peptides that exert antioxidative capabilities.

Mung bean showed most bands than any

samples, which probably are legumin (16.5-56

kDa), vicillin (24-63.5 kDa), and globulin (22-

37 kDa) in unhydrolyzed condition (Marambe & Wanasundara 2012; Mendoza et al. 2001).

Almost all bands in mung bean showed

reduction in its thickness, started at 5 min

hydrolysis, and vicillin was probably the main

protein that exert bioactivity after being

hydrolyzed.

For soy bean, mostly below 45 kDa, which

probably were subunits of glycinine or cystein

protease types (20 kDa–40 kDa), and β-

conglycinin (63 kDa) (Wang & Gonzales

2005). All samples showed protein bands reduction afrer hydrolyzed with bromelain

enzyme into small proteins or peptides.

Cystein protease and glycinine seemed to play

main roles in antioxidant and antihypertension

capabilities. Different hydrolysis patterns

related to different specificity of bromelain

2

enzyme on each sample. This suggests that

peptide production was probably a staged

process in which low molecular weight

peptides (below 30 kDa) were formed by the

hydrolysis of midrange peptides (40 – 60 kDa)

which were once originated by high-

molecular-weight proteins or peptides.

CONCLUSION

Herein we produce bioactive peptides by

enzymatic hydrolysis using bromelain

enzyme. The bioactive peptides produced

were potent radical scavengers and

antihypertensive activity by efficiently

inhibited free radical chain reaction by

scavenging DPPH free radical and reduce ferric into ferrous form. Results showed that

each sample, especially in 5 min hydrolysis

treatment, has maximum capability as well as

positive controls. The inhibitory effect of

peptides prepared from bean on ACE activity

make these products, especially mung bean, a

potential candidates to be developed and

applied within the concept of functional foods.

Further research is needed to know the

biological effects in vivo and kinetic study.

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12

2

APPENDICES

3

Appendix 1 Procedure of ACE inhibitory activity

Volume (μl)

Blank Control Sample

Distilled water 10 10 10

NaCl 4.0 M 10 10 10

Borate buffer 0.1 M pH 8.3 50 50 50

HHL 50 mM 10 10 10

HCl 1.0 N 100 - -

Borate buffer 0.1 M pH 8.3 10 10 -

Sample solution - - 10

ACE (0.8 mU) 10 10 10

Incubated at 370C for 45 min HCl 1.0 N - 100 100

Ethylacetate 600 600 600

Homogenized using vortex for 30 seconds

Centrifuged at 300×g for 10 min

Filtrate 100 100 100

Dried in oven at 140 0C for 10 min

NaCl 1.0 M 600 600 600

Absorbance was measured at 228 nm

Appendix 2 DPPH scavenging activity (%) of bean flour substrates hydrolyzed with bromelain

enzyme (0.272 U/ml)

0

10

20

30

40

50

60

70

80

90

100

RB MB SB A.acid

(10mg/ml)

α-toco

(67mg/ml)

Sca

ven

gin

g a

cti

vit

y (

%)

Samples

0 min

5 min

10 min

20 min

30 min

60 min

14

4

Appendix 3 Tyrosin standard curve

Appendix 4 Bradford standard curve

y = 4,922x - 0,012

R² = 0,999

0

0,2

0,4

0,6

0,8

1

1,2

0 0,05 0,1 0,15 0,2 0,25

Ab

sorb

an

ce

[Tyrosin]

y = 0,094x + 0,005

R² = 0,991

0

0,02

0,04

0,06

0,08

0,1

0,12

0 0,2 0,4 0,6 0,8 1 1,2

Ab

sorb

an

ce

[Protein]

15