Isolation and characterization ofvinegar culture (Acetobacteraceti) from indigenous sources

Tahir Zahoor, Farzana Siddique and Umar FarooqInstitute of Food Science and Technology, University of Agriculture,

Faisalabad, Pakistan

Abstract

Purpose – The Food and Agriculture Organization (FAO) of the United Nations has established thatvinegar is a liquid only obtained by fermentation. Although in Pakistan mostly synthetic vinegar ismarketed, production of vinegar through fermentation at industrial level is also carried out. However,vinegar produced by this method tends to be of inferior quality due to uncontrolled conditions. Inindustry a mixed culture of Acetobacter is used for the production of acetic acid but no attention isgiven towards its proper maintenance and culture is contaminated with other kinds of microorganismsand still no work has been done along these lines in Pakistan. There is a need to develop pure vinegarcultures for vinegar production so that the use of synthetic vinegar may be avoided as it is prohibitedin most countries. Keeping in view all of these points, the present work was conducted to isolate a pureculture of Acetobacter aceti, the maintenance of this culture and finally its utilization in fermentationfor vinegar production.

Design/methodology/approach – In this study efforts were made to isolate vinegar culture(Acetobacter aceti ) from sugar cane juice, rotten apples, flowers, wine, canal water and vinegar as aprimary source for Acetobacter by continuous sub-culturing on standard medium glucose, yeastextract and calcium carbonate (GYC). The culture was identified on the basis of colony characteristicsand morphology. It was finally confirmed by different biochemical/enzymatic tests and furtherspecified by nutritional and temperature requirements for the growth. The isolated strain was laterused for the production of vinegar through fermentation.

Findings – Among canal water, crushed apples, sugar cane juice, alcohol, vinegar and flowers, thealcohol and vinegar were found to be the most suitable sources for isolation of Acetobacter spp. Thecolonies of purified culture were found to be pale to off-white, circular, raised, convex, smooth and not.3 mm in diameter with morphology of Gram-ve, ellipsoidal, rods, squat bacilli, roundish, single, inpairs and in chains. The isolated and identified spp. gave excellent results for the production ofvinegar and this vinegar was more acceptable rather than commercially available fermented vinegar.

Practical implication – At industrial level good quality vinegar can be produced by using pureculture of Acetobacter aceti for acetic acid fermentation.

Originality/value – The research carried out is one of an original type as no work has been done inPakistan previously. Further, for the accuracy of the results, all the practices were carried out intriplicates.

Keywords Fermentation, Food products, Acids, Pakistan

Paper type Research paper

IntroductionThe profound and sweeping involvement of microbes in the natural world isinescapable. Although our daily encounters with them usually go unnoticed, humanand microbial life are clearly intertwined on many levels. It is no wonder that long agohumans realized the power of microbes and harnessed them for metabolic tasks. Thepractical applications of microorganisms in manufacturing products, or carrying out a

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British Food JournalVol. 108 No. 6, 2006

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0007-070XDOI 10.1108/00070700610668405

particular decomposition process, belong to the large and diverse area ofbiotechnology.

The microorganisms that oxidize ethanol to acetic acid, commonly called acetic acidbacteria, belong to genus Acetobacter. Acetic acid bacteria are polymorphous; cells areellipsoidal to rod shaped, straight or slightly curved, 0.5-0.8mm by 0.9-4.2mm in size,occurring singly, in pairs, or in chains. There are non-motile forms and motile formswith polar or peritrichous flagella. They are obligatorily aerobic, some producepigments and some produce cellulose (Rehm and Reed, 1983).

Acetobacter cause rot in apples, pears and pink disease in pineapples. Some canoxidize both glucose and ethanol simultaneously (Sokollek et al., 1998), these arenon-pathogenic towards humans, some can fix nitrogen in plants (Loganathan, 1999)and also cause acetification, ropiness, turbidity, off-flavors and discoloration in beer.One strain of the Acetobacter can also kill yeast. The actual history of the use of A. acetifor the production of acetic acid from ethanol is not known, however members of thegenus have been used industrially since the 1850s (Edberg, 1992). At an industrial levela mixed culture of Acetobacter is used for the production of acetic acid and moreattention is required for its proper maintenance. Normally the culture is contaminatedwith other kinds of microorganisms at industrial level. Keeping in view minimizingcontamination and microbial hazards the plan was to isolate a pure culture ofAcetobacter aceti, and to maintain the pure culture to the best level of production.

Material and methodsSample collection and storageAll samples (sugar-cane juice, canal water, rotten apples and flower) were collected andtransported to the Microbiology and Biotechnology Laboratory, Institute of FoodScience and Technology, University of Agriculture, Fisalabad. The samples werestored at refrigeration temperature (48C).

Media preparationMedia (nutrient agar, standard medium GYC and SM medium) used for microbialgrowth were prepared according to the method as described by Harrigan and McCance(1976) and Holt et al. (1994). The pH of media was adjusted by using N/10 NaOH andN/10 HCl.

Nutrient agar medium. It is general-purpose medium and was used for identificationof Acetobacter aceti. Medium composition:

. peptone ¼ 10 g;

. sodium chloride ¼ 5 g;

. beef extract ¼ 5 g;

. yeast extract ¼ 5 g;

. agar ¼ 15 g; and

. distilled water ¼ 1; 000 mL.

After dissolving all ingredients completely in distilled water by slight heating, themedium was autoclaved at 1218C for 15 minutes at 15 lb pressure. The medium wasthen cooled to 50-558C and pH of the medium was maintained up to 7.2. Medium waspoured into sterilized petri dishes and allowed to solidify.

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Standard medium GYC. This is the medium used for the growth and maintenance ofAcetobactor spp. Medium composition:

. agar ¼ 20 g;

. glucose ¼ 20 g;

. CaCO3 ¼ 20 g;

. yeast extract ¼ 10 g; and

. distilled water ¼ 1; 000 mL.

All the ingredients were mixed in distilled water by slight heating and autoclaved asstated earlier. Medium was cooled, mixed thoroughly and poured in to sterile petri dishes.

SM medium. Composition of SM medium:. yeast extract ¼ 5 percent;. glucose ¼ 5 percent;. agar ¼ 1:5 percent;. ethyl alcohol ¼ 2 percent; and. distilled water ¼ 100 percent;

All the ingredients, except ethanol, were dissolved in distilled water. After sterilization,filtered (from 0.2 micron filter) alcohol was added under aseptic conditions into themedium after cooling up to 50-558C. Agar plates were prepared and culture wasinoculated on the surface of agar plates. Incubation was made at 308C for 48 hours andgrowth was observed.

Isolation of Acetobacter spp. from different indigenous sourcesIsolation of Acetobacter spp. was carried out as follows.

Inoculation and incubation. From each indigenous source the isolation ofAcetobacter aceti was accomplished by inoculating the sample on standard mediumGYC and the petri dishes were incubated at 308C for 48 hours. Inoculation was madethrough the pouring method as recommended by Cappuccino and Sherman (1996).

Morphological examination. Morphological examination of the culture isolates wasmade according to the method described by Cappuccino and Sherman (1996).

Colony morphology. The colony characteristics including color, size, shape andelevation were studied after incubation.

Gram’s staining. Gram’s staining was performed according to the proceduredescribed by Cappuccino and Sherman (1996), Harrigan and McCance (1976) and Awanand Rahman (2002). All slides were examined under 40 £ and 100 £ oil immersionlens and results were recorded.

Purification of culture isolates. All the colonies, which appeared after 48 hours ofincubation on the surface of standard medium GYC plates, were especially examined formorphological characteristics. The required colonies were inoculated on to the surface ofstandard medium GYC for specific culture isolates until a pure growth was obtained.

Identification of isolatesCulture isolates were identified on the basis of colony characters, growth charactersand morphological characteristics, which later on were confirmed by observing growth

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on different carbon sources, nutrient agar medium and studying the effect of ethanol,NaCl, different levels of glucose and effect of different temperatures on the growth ofculture as described by Holt et al. (1994) and Krieg and Holt (1984). Certain biochemicaltests were also performed for the identification of pure culture according to the methoddescribed by Cappuccino and Sherman (1996) and Ashecraft et al. (2001).

Growth on different carbon sourcesSodium acetate, ethanol and glucose were used as carbon sources for the growth ofAcetobactor spp. while using standard medium GYC. Agar plates were prepared andinoculated by streaking followed by incubation at 308C for 48 hours. The suitability ofthese carbon sources for the growth of culture isolates was observed.

Effect of ethanol on growth of culture isolatesDifferent levels of ethanol (1, 2, 5 and 10 percent) by following Krieg and Holt (1984)were used in SM medium to observe the effect of ethanol concentration on the growthof Acetobactor spp.

Effect of sodium chloride (NaCl) on growth of culture isolatesDifferent concentrations of NaCl (0.5, 1 and 2 percent) were used in SM medium to seethe effect of different levels of NaCl on the growth of Acetobactor spp. Inoculation wasmade by streak plate method and incubation in an inverted position at 308C for 48hours.

Effect of different levels of glucose on growth of culture isolatesDifferent levels (20, 25 and 30 percent) of D-glucose were used in SM medium in orderto observe the effect of different concentrations of glucose on the growth of cultureisolates. Inoculation was made on agar plates that were incubated in an incubator at308C for 48 hours. The effect of different percentages of glucose was observed bygrowth of Acetobactor spp. on the media containing different concentrations of glucose.

Growth on nutrient agar mediumCulture isolates were confirmed by their growth on nutrient agar by following therecommendations of Krieg and Holt (1984). Nutrient agar plates were inoculated withculture isolates and incubation was made at 308C for 48-72 hours.

Growth at different temperaturesInoculation was made on standard medium (GYC) plates by streak plate method andthese plates were incubated at different temperatures (288C, 348C, 378C) in an incubatorfor 48 hours. After 48 hours growth of culture isolates was observed and results wererecorded.

Biochemical testsThe following biochemical tests were performed to further confirm the culture isolates.

Catalase test. This test was used to detect the production of enzyme catalase. Onedrop of 3 percent hydrogen peroxide was added to a colony on agar plate. Results wererecorded by the production of bubbling on colonies.

Oxidase test. The oxidase test was performed to detect the production of oxidaseenzyme. Oxidase testing liquid was placed over the bacteria on a swab. Results were

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recorded by the development of blue color on the bacterial colony swab by followingthe method described by Ashecraft et al. (2001).

Results and discussionThe present research was conducted for isolation, identification and purification ofAcetobacter aceti as a vinegar culture. For isolation of indigenous culture, vinegar,wine, sugar cane juice, apple, flowers and canal water were used as raw sourcematerials.

Isolation of Acetobacter sppA number of colonies were observed when different samples (sugar cane juice, rottenapples, canal water, flowers, wine and vinegar) were inoculated on standard mediumGYC agar plates. The colonies showed different cultural and morphologicalcharacteristics as given in Table I. These colonies were further isolated whileresolving on the basis of morphological and microscopic examination.

In the case of sugar cane juice, canal water and rotten apple samples, most of thecolonies were small, white, spherical, pinpoint, raised and off-white. Isolates fromsugar cane juice samples were only yeast with budding characteristics.Microorganisms isolated from canal water were Gram positive, cocci, single, pair aswell as in chains while those isolated from rotten apple were Gram positive, cocci,single and yeast with bud formation. Observations indicated that there was nomicrobial growth when the flowers were incubated for 48 hours on standard GYCmedium.

A variety of microbial growth was obtained from alcohol and vinegar and most ofthe colonies were smooth, small, medium, big, white, off white, pale, spherical, raised,convex, spheroid, star shaped, rough, crinkled and flat with Gram negative, rods,single, pair, chain, bud forming (yeast), Gram variable, cocci, cluster, squat bacilli,coccobacilli, and ellipsoidal characters morphologically.

Conclusively two types of the bacteria were specially observed, one type of thebacteria belong to Gram negative, short rods in shape, single, pair and chain inarrangement and the other type was Gram negative, squat bacilli and cocci in shape,single, pair and chain in arrangement. Similar results were also obtained by Yamadaet al. (1999), Lisdiyanti et al. (2001) and Ashecraft et al. (2001) who also isolatedAcetobacter spp. and found that morphologically these are gram negative rods. Cultureisolates from alcohol, vinegar and sugar cane juice are shown in Figures 1 and 2respectively.

Purification of isolatesThe above isolated microorganisms showing Gram negative single, pair and chainforming rods and cocci from alcohol (A1 and A2) and vinegar (V1 and V2) samples asshown in Figures 3 and 4 were transferred on the surface of the standard medium(GYC) plates in order to get more number of specific microorganisms.

Exuberant growth as shown in Figure 5 was observed on standard medium (GYC).It was concluded from the study that the standard medium (GYC) was more suitablefor growth of Acetobacter spp. than any other medium. These findings were similar tothat of Holt et al. (1994) and Krieg and Holt (1984) who also recommended that most

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Acetobacter species and subspecies grow moderately to abundantly on standardmedium (GYC).

The colonies from selected samples (A1, A2, V1, V2) observed on standard medium(GYC) were Gram negative, ellipsoidal to some are rods, squat bacilli with a roundishappearance, single, pair and chains in arrangement. These were selected for furtherpurification and sub-cultured repeatedly on the surface of the standard medium (GYC)

Sr. no. Sample Colony characters Morphological characters

1 S1 Small, white, spherical Budding (yeast)2 S2 Pinpoint, white, circular Budding (yeast3 S3 Small, circular, raised, white Budding (yeast)4 S4 Small, white, spherical Budding (yeast)5 C1 Small, off-white, raised, spherical Gram þ ve, cocci, single, pair, chain,6 C2 Small, off-white, convex, circular Gram þ ve, cocci, pair, chain, cluster7 C3 Pinpoint, white, spherical, raised Gram þ ve, cocci, pair, chain, cluster8 R1 Small, white, spherical Budding (yeast), cocci, single,

Gram þ ve,9 R2 Pinpoint, white, circular Budding (yeast,

10 R3 Small, white, off-white, spherical Budding (yeast), cocci, single,Gram þ ve,

11 R4 Small, white, spherical Budding (yeast),12 F1 No growth –13 F2 No growth –14 A1 Smooth, big, white, off white raised,

sphericalGram-ve, rods, single pair, chain,ellipsoidal,

15 A2 Big, smooth, off-white, convex, circular Gram-ve, rods, single pair, chain,ellipsoidal,

16 A3 Small, white, raised, spherical Budding (yeast)17 A4 Small, big, off-white, raised Budding (yeast), Gram variable, cocci,

single pair, chain, cluster18 A5 Big, small, medium, white, off-white,

raisedBudding (yeast), Gram variable, cocci,single pair, chain, cluster

19 A6 Small, white, raised, spherical Budding (yeast)20 V1 Smooth, spheroid, convex, off-white, big Gram-ve, squat bacilli, cocci, single, pair,

chain, ellipsoidal21 V2 Smooth, circular, raised, off-white, pale,

bigGram-ve, squat bacilli, single, pair,chain, ellipsoidal,

22 V3 Star shaped, smooth, rough, crinkled,flat, convex, spherical, pale, white,off-white

Budding (yeast), Gram variable, rods,cocci, coccobacilli, single, pair, chain,cluster

23 V4 Small, big, medium, smooth, rough,off-white, white, convex, flat

Budding (yeast), Gram þ ve, Gram-ve,short rods, cocci, single, pair, chain,cluster

24 V5 Small, big, smooth, rough, pale, white,pale, flat crinkled

Budding (yeast), Gram þ ve, Gram-ve,short rods, cocci, single, pair, chain,cluster

25 V6 Pinpoint, smooth, rough, white, convex,flat

Budding (yeast), Gram þ ve, short rods,cocci, single, pair, chain, cluster

Notes: A ¼ alcohol; C ¼ canal water; F ¼ flowers; R ¼ rotten apple; S ¼ sugar cane juice;V ¼ vinegar

Table I.Colony andmorphological charactersof microorganismsisolated from differentsamples collected(indigenous sources)

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until single type (pure growth) of colonies was appeared after 48 hours of incubation at308C. The morphological and colony characteristics of pure culture isolates onstandard medium (GYC) are shown in the Table II. The colonies on the surface of thestandard medium (GYC) plates were pale to off-white, circular, raised or convex,smooth and not more than 3 mm in diameter as also described by Holt et al. (1994) andKrieg and Holt (1984).

Figure 2.Culture isolates on

standard medium GYCfrom sugar cane juice

Figure 1.Culture isolates on

standard medium GYCfrom alcohol and vinegar

Figure 3.Microscopic view ofculture isolates after

Gram’s staining at 100 £

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Identification of Acetobacter acetiFor the identification of Acetobacter aceti, the purified vinegar culture was furtheranalyzed on the basis of biochemical and enzymatic tests, observing growth atdifferent temperature and by using different carbon sources (sodium acetate, ethanoland glucose) as recommended by Holt et al. (1994) and Krieg and Holt (1984).

Figure 4.Microscopic view ofculture isolates afterGram’s staining at 40 £

Figure 5.Pure culture on standardmedium GYC isolatesfrom indigenous sources

Sr. no. Sample Colony characters Morphological characters

1 A1 Pale to off-white, circular, raised,convex, smooth, not .3 mm in diameter

Gram-ve, ellipsoidal, rods, squat bacilli,roundish, single, in pairs and chains

2 A2 Pale to off-white, circular, raised,convex, smooth, not .3 mm in diameter

Gram-ve, rods, roundish, single, in pairsand chains

3 V1 Pale to off-white, circular, raised,convex, smooth, not .3 mm in diameter

Gram-ve, ellipsoidal, rods, roundish,single, in pairs and chains

4 V2 Pale to off-white, circular, raised,convex, smooth, not .3 mm in diameter

Gram-ve, ellipsoidal, rods, squat bacilli,roundish, single, in pairs and chains

Notes: A1 and A2 ¼ samples from alcohol; V1 and V2 ¼ samples from vinegar

Table II.Colony andmorphological charactersof pure culture isolatesfrom alcohol and vinegar

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Catalase test. The pure culture of vinegar (A1, A2, V1 and V2) produced bubbling inthe presence of hydrogen peroxide (3 percent) that is the indication of a positive test asshown in Table III. The results obtained were similar to that of the finding of Ashecraftet al. (2001) who observed that Acetobacter aceti has the ability of catalase productionthat was also obtained in this study.

Oxidase test. The cultures A1 and A2 (alcohol) and V1 and V2 (vinegar) weresubjected to analysis of the production of oxidase enzyme. The production of blue coloron the bacterial colony swab indicates negative results for oxidase production. Theseresults coincide with the findings of Ashecraft et al. (2001) who also found thatAcetobacter aceti has negative results for oxidase production. Results are indicated inTable III.

Growth of Acetobacter aceti on different carbon sourcesGrowth of pure culture of Acetobacter aceti was observed on the medium in which ethylalcohol and sodium acetate were used as carbon sources. However on these carbonsources growth was delayed as compared to growth on standard GYC medium. Theresults are in line with the recommendations of Holt et al. (1994) who suggested alcoholand sodium acetate as carbon sources for Acetobacter aceti for its identification fromother Acetobacter spp.

Sr. no. Test A1 A2 V1 V2

1 Catalase test þ þ þ þ2 Oxidase test 2 2 2 23 Growth on different C-sources3.1 Ethanol þ þ þ þ3.2 Na-acetate þ þ þ þ3.3 Growth in 10 percent ethanol 2 2 2 24 Effect of ethanol on growth4.1 SM medium þ 1 percent ethanol þ þ þ þ4.2 SM medium þ 2 percent ethanol þ þ þ þ4.3 SM medium þ 5 percent ethanol þ þ þ þ4.3 SM medium þ 10 percent ethanol 2 2 2 25 Effect of sodium chloride on growth5.1 SM medium þ 0.5 percent NaCl þ þ þ þ5.2 SM medium þ 1 percent NaCl þ þ þ þ5.3 SM medium þ 2 percent NaCl 2 2 2 26 Effect of D-glucose on growth6.1 0.5 percent yeast extract þ 20 percent D-glucose þ þ þ þ6.2 0.5 percent yeast extract þ 25 percent D-glucose þ þ þ þ6.3 0.5 percent yeast extract þ 30 percent D-glucose 2 2 2 27 Effect of temperature on growth7.1 Growth at 288C þ þ þ þ7.2 Growth at 348C þ þ þ þ7.3 Growth at 378C 2 2 2 28 Growth on nutrient agar 2 2 2 2

Notes: þ ¼ positive results; 2 ¼ negative results; A ¼ culture isolates from alcohol; V ¼ cultureisolates from vinegar

Table III.Results of different

confirmatory tests foridentification of

Acetobacter aceti

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Effect of ethanol on the growth of culture isolatesAt different levels of alcohol (1, 2 and 5 percent) growth of culture isolates wasobserved on agar plates while no growth was observed when 10 percent alcohol wasadded to the medium as also indicated in Table III, which relate to findings of Holt et al.(1994) who recommended the levels of alcohol tolerance (maximum 5 percent) foridentification of Acetobacter aceti.

Effect of sodium chloride on the growth of culture isolatesBy applying different levels of sodium chloride (0.5, 1 and 2 percent) in growth mediumfor identification of Acetobacter aceti, growth was obtained at 0.5 and 1 percent sodiumchloride and no growth was obtained when level of sodium chloride was increased to 2percent (Table III). It was again confirmation of Acetobacter aceti as suggested by Holtet al. (1994) and Krieg and Holt (1984).

Effect of D-glucose on the growth of culture isolatesWhen different levels of D-glucose were used in growth medium then it was found thatat 30 percent D-glucose concentration no growth was obtained while at 20 and 25percent concentrations of D-glucose the growth was observed after an incubation of 48hours at 308C as shown in the prescribed Table III. These results also lead to theidentification of Acetobacter aceti (Holt et al., 1994) and Krieg and Holt (1984).

Growth at different temperaturesAt 288C the growth was obtained by incubation of pure culture isolates (A1, A2, V1, V2)and very few colonies were observed at 348C while no growth was obtained afterincubation at 378C. The results obtained are demonstrated in Table III which confirmsthe growth of Acetobacter aceti as suggested by Holt et al. (1994) and Krieg and Holt(1984).

Growth on nutrient agar mediumPure culture of vinegar did not show the growth on nutrient agar plates. The resultsobtained are similar to the findings of Holt et al. (1994) and Ashecraft et al. (2001) whostated that in media without a carbon source for growth, e.g. yeast extract broth,peptone broth or nutrient agar no growth of Acetobacter aceti occurs.

ConclusionHence on the basis of these results it was confirmed that the culture isolated was a pureculture of Acetobacter aceti and the strain was named as TFU-1 (Tahir, Farzana andUmar). It was also suggested that the alcohol and vinegar are the best sources forisolation of acetic acid culture. In this study 30, 40, 50 and 60 percent pure isolatedculture of Acetobater aceti was also used to prepare vinegar from alcohol at laboratoryscale. The vinegar thus prepared was analyzed and compared with that ofcommercially available fermented vinegar in Pakistan for its flavor, taste, color, pH,acidity, total soluble solids, volatile acids and nonvolatile acids. On the basis of theseresults it was concluded that culture percentage significantly affected thephysico-chemical and sensory quality of vinegar. Furthermore the vinegar made atlaboratory scale from pure culture under controlled conditions was much better thanthat of commercial vinegar when analyzed organoleptically after its utilization in

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chicken soup (Siddique et al., 2004). On the basis of these results it is recommended thatproper attention must be given for fermented vinegar production at industrial scale. Bydoing this we can improve not only the quality of the product, but also its safety withutilization of pure acetic acid cultures.

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Corresponding authorUmar Farooq can be contacted at: ufd302003@yahoo.com

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