Improved Vinegar Production Through Acetobactor aceti for Immobilization

IMPROVED VINEGAR PRODUCTION THROUGH ACETOBACTER ACETI

FOR IMMOBILIZATIONNEELIMA GARG*, KAUSHLESH K. YADAV, EJAZ BEG, SANJAY KUMAR, ANIL

VERMA

Post Harvest Management Central Institute for Subtropical

Horticulture, Rehmankhera, P.O. Kakori, Lucknow-227107

*E-mail: [email protected]

ABSTRACTVinegar is a sour-tasting condiment and preservative prepared by

two successive microbial processes, the first being an alcoholic

fermentation affected by yeasts and the second an oxidation of

alcohol by Acetobacter aceti. Generally using traditional protocol,

it takes around 3 months for preparing vinegar from sugarcane

juice. The present study was undertaken to reduce the time for

acetification using cell immobilization technique. From sugarcane

juice, 9.6% (v/v) alcohol was obtained in 7 days using

Saccharomyces cerevisiae. Acetobacter aceti naturally isolated from

mother of vinegar was immobilized on sterile bamboo segment (15-

16cm) and wood shaving (30 g) for 4-5 days. The final Acetobacter

aceti counts on bamboo segments and wood shaving was 2.8 x106

and 1.4x106cells/ gm, respectively. The fermented sugar cane

juice containing alcohol was inoculated with immobilized

Acetobacter cells and incubated under aerated conditions at room

temperature (35+ 5OC). After 22 days, 10.5% of acetic acid with

98% fermentation efficiency was produced using bamboo segments

while 9.4 % acetic acid was produced with wood shavings. Thus,

good quality sugarcane vinegar could be prepared in total 29

days. This protocol is very simple and may be followed by farmers

and small scale processors working on vinegar production.

Key word: Acetobacter, vinegar, immobilization, bamboo, wood

shaving, acetic acid

INTRODUCTIONVinegar may be defined as a condiment made from various sugary

and starchy materials by alcoholic and subsequent acetic

fermentation (Cruess 1958). It is traditionally used as a food

preservative. Vinegar can be produced by different methods and

from various raw materials. Wine (white, red, and sherry wine),

cider, fruit must, malted barley and/ or pure alcohol are used as

substrate. Vinegar production ranges from traditional methods

employing wood casks and surface culture to submerged

fermentation in acetators (Morales et al. 2001). Acetic acid is

formed by anaerobic conversion of sugars to ethanol by yeast

fermentation, conversion of ethanol to hydrated acetaldehyde and

dehydrogenation to acetic acid by aldehyde dehydrogenase (Nichol

1979; Canning 1985).

Whether naturally produced during fermentation or intentionally

added, vinegar retards microbial growth and contributes sensory

properties to a number of foods. The wide diversity of products

containing vinegar (sauces, ketchup, mayonnaise, etc.) have

favored an increase in vinegar production (De Ory et al. 2002).

Acetic acid is the predominant flavoring and antimicrobial

component in vinegar. Earlier processes used for making vinegar

were the Orleans process (which is also known as the slow

process), the quick process (which is also called the generator

process) and the submerged culture process (Klemps et al., 1987).

The quick process and submerged culture processes are used for

commercial vinegar production today. Sugarcane juice is a

substrate of choice for natural vinegar because of its high sugar

content and availability. Therefore, in order to make this

process suitable for a low cost cottage industry, it is essential

to produce natural vinegar at low cost and in as short a time as

possible ( Allgeier and Hildebrandt 1960). Cell immobilization

provides a means to improve upon the fermentation process by

increasing biomass, option of reusability, protection of cells

from toxic effects of low pH, temperature, inhibitors etc. (as

ionic/hydrophobic interactions of the immobilization matrix

induces increased stability and a buffered zone is provided by

the immobilization materials). Cell immobilization also helps in

early clarification of the product. Further, the choice of

immobilization material in the form of inexpensive easily

available inert biological materials can help reduce the cost of

the process. Almost all the methods of immobilization, namely,

gel entrapment, adsorption on the surfaces of the various

carriers, cross linking were tried for alcohol fermentation

(Kennedy et al 1985). The present study was undertaken to develop

an efficient process for acetification using free cells of

Saccharomyces cerevisiae and immobilized cells of Acetobacter and

comparing different materials for immobilization of the Acetobacter

cells for vinegar production from sugarcane juice.

MATERIALS AND METHODS Raw material: Sugarcane juice was obtained from IISR Lucknow.

Microorganisms: Saccharomyces cerevisiae culture was obtained from

culture collection of Microbiology lab, CISH and Acetobacter aceti

was isolated from mother vinegar and identified as per the

protocol described in Bergeys Manual of Determinative

Bacteriology (Holt et al.1994)

Media: Yeast Extract Potato Dextrose Agar and Actobacter agar

were prepared as per method of Speck, (1985).

Immobilization of Acetobacter aceti

Acetobacter aceti used for acetic acid production was isolated from

mother of vinegar, multiplied on nutrient agar and immobilized on

sterile bamboo segment (15-16 cm length) and wood shaving (30 gm)

for 4-5 days till Acetobacter cells were completely adsorbed. The

final Acetobacter aceti counts on bamboo segments and wood

shaving were 2.82 x 106 and 1.43 x 106cells/ gm, respectively.

Protocol followed for vinegar preparation

Vinegar was prepared in two steps

Step 1. Conversion of sugarcane juice in to alcohol

The Saccharomyces yeast strain was used for ethanol production. Ten

litres of sugarcane juice (filtered with muslin cloth, TSS 21°B)

was taken in a 20 litre capacity cylindrical glass jar and

inoculated with an active culture of Saccharomyces cerevisiae at 2%

inoculum level. It was then incubated under partially anaerobic

conditions using fermentation lock at 37C for 8-10 days till the

alcoholic fermentation is complete. Ethanol production in the

fermented juice was determined by the method of Caputi et al.,

(1969).

Step 2. Conversion of alcohol into acetic acid

The fermented sugar cane juice as obtained by previous step was

allowed to settle for 2 days at 4°C and the supernatant was

siphoned off. The final ethanol content in the fermented juice

was 8.3% (v/v). It was then inoculated with immobilized Acetobacter

cells on bamboo or wood shaving and incubated under stationary

aerated conditions at room temperature. In another treatment

immobilized wood shavings were packed in sterile food grade

polypropylene column and fermented juice was recirculated using a

peristaltic pump (Fig 1).

Bio-chemical Analyses: The pH, TSS and titratable acidity (as

acetic acid) were monitored regularly as per the methods of AOAC

(1995). Ethanol concentration in the fermented juice was

determined spectrophotometrically by method of Caputi et al.

(1968). The alcohol fermentation efficiency was calculated and

expressed as alcohol actually produced compared with the

theoretical yield calculated from of the conversion reaction of

glucose to ethanol where 1 g sugar yields 0.51 g ethanol.

Steps followed for preserving vinegar: Vinegar was filtered

through a layer of muslin cloth to remove the bacterial film. The

bottles were sealed vinegar was pasteurized at 60 C and filled

it into dry sterilized bottles. It was bottled and placed in hot

water bath at 71C for at least 30 minutes, finally the

sterilized bottles were stored in cool and dry place. The acetic

fermentation efficiency was calculated and expressed as acetic

acid actually produced compared with the theoretical yield

calculated from of the conversion reaction of ethanol to acetic

acid, where 1 g ethanol yields 1.304 g acetic acid.

RESULTS AND DISCUSSION The microbial isolate was observed as acid producing Gram

negative rods. Results of carbohydrate fermentation test,

catalase test\ peroxidase test, oxidase test, motility test,

gelatine hydrolysis test, citrate utilization test, ammonia

production test and urea test confirmed that the isolated

microorganism was Acetobacter aceti (Table 1). Latter is a gram

negative bacteria which is motile by peritrichous flagella. It is

obligately aerobic, possessing only the ability for respiratory

metabolism with no fermentative ability. Acetobacter aceti does not

form endospores( Bergeys 1957 ). This bacterium is ubliquitous in

the environment, existing in soil, water, flowers, fruits and on

honey bees in essence, wherever sugar fermentation is occurring.

Acetobacter aceti produces acetic acid from ethanol in alcoholic

niches in the environment. Acetate and lactate are oxidized to

CO2 and H2O by the organism. The optimal temperature for growth is

between 25 to 30 C while optimum pH between 5.4 to 6.3 (De Ley

et al., 1984).

The fermentation of the clarified juice by S. cerevisiae resulted in

production of 8.3% (v/v) alcohol in 7 days with 77.5% ethanol

fermentation efficiency and the total soluble sugar (TSS) of the

juice was reduced from 21°B to 9.6°B and acidity increased from

zero to 1.9 %.

Acetobacter aceti was immobilized as per protocol mentioned in

earlier section (Fig 2). Wood shaving and Bamboo containing

immobilized Acetobacter were dipped in fermented sugar cane juice

as described above. After 20 days of submerged aerobic

fermentation, the TSS decreased from 21 to 10.2OB (Table 2 ) and

10.5% of acetic acid was produced with the bamboo, while 9.4 %

acetic acid was produce with wood shaving immobilized material

(Fig.3). Zhou et al. (2013) have reported immobilization of yarrowia

lipolytica lipase on bamboo charcoal to resolve (r,s)-phenylethanol

in organic medium. In current study bamboo pieces served as

suitable substrate for immobilization. Better fermentation

efficiency with bamboo immobilization might be because of the

increased air spaces compared to wood shaving. Three months

storage study indicated no loss in acidity or deterioration in

taste. Ghommidh et al. (1986 ) reported that acid yield

improvements can be achieved using high rates of aeration during

continuous production. However, no further increase in acetic

acid yield was achieved using dynamic aerobic conditions in a

continuous production system suggesting that stationary

fermentation is sufficient for vinegar production .

CONCLUSIONImmobilization is a rapid and efficient technique for vinegar

production. Acetobacter aceti immobilized on bamboo segments produced

10.5% acetic acid with 98% fermentation efficiency, in 21 days.

The method may be of great use to farmers and small entrepreneurs

as they can make their vinegar with in short period with more

efficiency.

ACKNOWLEDGEMENTS Authors are thankful to the Director of Central Institute for

Subtropical Horticulture, Rehmankera, Kakori, Lucknow, for

providing necessary facilities.

REFERENCES

Allgeier R.J. and Hildebrandt F. M.; 1960. Newer developments in

vinegar manufacture. Advance Applied Microbiology, 11:163-181.

A.O.A.C.; 1984. Official Methods of Analysis. Association of Official

Analytical Chemists, Washington D.C.

Bergey D.H.; 1957. Bergey’s manual of determinative bacteriology, American

Society for Microbiology. 1860-1937.

Canning A.; 1985. Vinegar Brewing. Journal of Food Science. Sept/ Oct:

20-21.

Cruess W.V.; 1958. Commercial fruit and vegetable products: Chapter 21

– Vinegar Manufacture. 1st ed. New York: McGraw-Hill Book Company,

Inc. pp. 681-707.

Caputi J.A. and Wright D.; 1969. Collaborative study of the

determination of ethanol in wine by chemical oxidation. Journal

Association of Analytical Chemistry, 52: 85–88.

De Ory Ignacio., Romero, L.E. and Cantero, D.; 2004. Operation in

semi-continuous with a closed pilot plant scale acetifier for vinegar

production. Journal of Food Engineering, 63: 39-45.

Ghommidh C., Cutayar, J.M. and Navvarro, J.M.; 1986. Continuous

production of vinegar research strategy. Journal of Biotechnology,

8:13-18.

Holt J.G.,  Noel R., Sneath K.P., Staley J.  and Williams. Bergey's

Manual of Determinative Bacteriology - 9th edition by Lippincott

Williams and wilkins a wolters Kluwer Company, London.

Kennedy J.F. and Cabral J.M.S. 1985.

In: Immobilized Cells and Enzymes .ed. Woodward, J, IRL Press, pp.19–

37.

Klemps R., Schoberth S.M. and Sahm H.; 1987. Production of acetic acid

by Acetogenium kivui. Applied Microbiology Biotechnology, 27:229-234.

Morales M.L., Gonzalez A.G. and Troncoso A.M.; 1999. Ion-exclusion

chromatographic determination of organic acids in vinegars. Journal of

Chromatography. 831:331-331.

Nickol G. B., Peppler H.J., Perlman D.; 1979. Vinegar. In:

Microbiology Technology. 2nd ed. New York: Academic Press. pp 155-

172.84.

Speck M.; 1985. Compendium of methods for the microbiological

examination of foods. 2nd edition, American public health association,

Inc. pp. 644-649.

Shaffer P.A. and Somogyi, M.; 1995. Glucose in Sugar and Syrups (micro

method), AOAC Official Method, Washington, DC, pp. 911-913.

Zhou X., Gao Q., Feng W. andPan K.; 2013.

Immobilizationof Yarrowia lipolytica Lipaseon Bamboo Charcoal toResolve (R,S)-Phenylethanol in OrganicMedium. ChemicalEngineering & Technology. 36:1249–1254.

Table1: Summarized

table for biochemical

identification

of microbial isolate

The results of all test

indicated in table,+++ High

S. NO Name of Biochemical

test

Result

1 Carbohydrate

fermentation testa Glucose +++b Sucrose ++c Mannitol +2 Catalase test\

Peroxidase test

+++

3 Oxidase test ---4 Motility test +++3 Gelatine hydrolysis

test

---

5 Citrate utilization

test

---

6 Ammonia production

test

++

7 Urea test ++

efficiency, ++ Moderate efficiency, + Low efficiency and ---

Negative results

Table 2: change in TSS (OB) as affected by immobilization on

bamboo or wood shaving during vinegar preparation

Fermentation Days Bamboo Wood Shavings0 21.0 21.01 12.0 12.03 11.5 11.77 11.0 11.59 11.0 11.012 10.4 10.419 10.2 10.222 10.2 10.225 10.2 10.2

Fig 1: Immobilized wood shavings packed in sterile food grade

poly propylene columns and peristaltic pump

Fig 2: Immobilized Acetobacter on wood shaving and Bamboo

Fig.3: Development of acidity (% acetic acid) as affected by

immobilization on bamboo or wood shaving during vinegar

preparation. Ti- Bamboo; T2 – Wood Shavings