ORIGINAL ARTICLE

Characterization of yeast starter cultures used in householdalcoholic beverage preparation by a few ethniccommunities of Northeast India

Alak K. Buragohain & Bhaben Tanti & Hridip K. Sarma &

Pranjan Barman & Kishore Das

Received: 17 May 2012 /Accepted: 22 August 2012 /Published online: 13 September 2012# Springer-Verlag and the University of Milan 2012

Abstract In this study, we report the characterization oftraditional household starter cultures from a few ethnicgroups of northeast India. Pure cultures obtained from thestudy have been deposited in the Microbial Type CultureCollection (MTCC), India. These isolates have been analyzedfor their growth characteristics, sensitivity to temperature andpH, alcohol tolerance, alcohol production, and alcohol dehy-drogenase (ADH) content. The pure cultures obtained fromdifferent starter cultures revealed the presence of Debaryomy-ces,Wickerhamomyces, and Candida along with the ferment-ing yeast Saccharomyces. The growth behavior at differenttemperatures, pH and alcohol tolerance revealed numerousfacts and behavior of the yeast strains associated with tradi-tional alcoholic fermentation. All the isolates were found to bethermotolerant up to 37 °C, fairly pH-resistant, good in ADHsecretion, and with appreciable alcohol production. For all thestrains studied, the Saccharomyces cerevisiae MTCC 3976strain from the Tea Tribes of Assam and the Wickerhamomy-ces anomalusMTCC 3979 from the Apatani Tribe of Aruna-chal Pradesh were found to be exceptional in terms of

thermotolerance, alcohol tolerance, alcohol production, andADH activity, and hence may be identified as potential strainsfor industrial fermentation.

Keywords Northeast India . Starter culture . Indigenousalcohol fermentation . Yeast . Alcohol dehydrogenase .

Alcohol tolerance . Thermotolerance

Introduction

The northeastern part of India is well known for the produc-tion of household liquors, which is associated with theregion’s rich indigenous knowledge system, with the knowl-edge extricably linked to its social, cultural, environmental,and institutional contexts (Sharma and Mazumdar 1980).The various ethnic tribes of Northeast India represent aconcoction of various aborigines, which include Mongoloid,Chinese and Aryan descent (Ghosh 1992). The methods forwine and beverage production among the tribes differ andfollow their own indigenous protocols employing differentstarter cultures, although most of them use similar substratesfor fermentation (Tanti et al. 2010). These locally producedalcohols and alcoholic beverages have several limitations,like bad odor, turbidity, toxic metabolites, texture, and in-consistency which not only lower the quality and yield butalso contribute to undesirable traits, rendering problems forcommercialization of the fermented products (Tsuyoshi etal. 2004).

It is now well known that alcoholic fermentationsuccessively involves different microorganisms, althoughyeasts are the most prominent species (Demuyter et al.2004). Under natural fermentations, a progressive pat-tern of yeast growth is usually observed. Many ecolog-ical studies have shown that several species of yeasts

A. K. Buragohain (*) : B. Tanti :H. K. Sarma : P. Barman :K. DasDepartment of Molecular Biology and Biotechnology,Tezpur University,Tezpur 784 028 Assam, Indiae-mail: alak.buragohain@yahoo.in

Present Address:B. TantiCytogenetics and Plant Breeding Laboratory,Department of Botany, Gauhati University,Guwahati 781 014 Assam, India

Present Address:H. K. Sarma : P. BarmanDepartment of Biotechnology, Gauhati University,Guwahati 781 014 Assam, India

Ann Microbiol (2013) 63:863–869DOI 10.1007/s13213-012-0537-1

with low fermentative capability such as Hanseniaspora,Kloeckera, Pichia, and Candida are active during earlystages of fermentation (Fleet and Heard 1993). The endproduct of fermentation (i.e. ethanol) is highly toxic to thesurvival of these yeasts and therefore they die off, leavingSaccharomyces cerevisiae strains to continue the fermentationprocess until the end (Mills et al. 2002). In addition, yeastsisolated from spontaneous and natural fermentation processesare mostly prototrophic, homothallic, heterozygous, and mostoften aneuploids (Puig et al. 2000). Genetic instability inyeasts may alter useful properties of industrially importantstrains resulting in problems during biotechnologicalscale-up and process optimization by lowering the qual-ity of products or may even producing toxic metabolitesduring fermentation in indigenously prepared alcoholand alcoholic beverages which are harmful to humanbeings (Ramirez et al. 2004). Given that persistence ofmetabolically active nonculturable populations of yeasts oreven the existence of non-Saccharomyces and indigenousbacteria in natural or spontaneous fermentations may affectperformance as well as final product texture, a better under-standing of the role of these populations in natural fermenta-tion processes in this region becomes critical.

During the present investigations, yeasts from starter cul-tures responsible for brewing processes used by differenttribes were comprehensively characterized, as no scientificinvestigation was available that could throw light on andaddress the various problems associated with householdwine-making in Northeast India.

Materials and methods

Collection of starter cultures

Starter cultures were collected from 12 ethnic groups repre-senting four states of Northeast India, viz., Assam, Manipur,

Nagaland, and Arunachal Pradesh (Fig. 1), and baker’s yeastwas used as reference strain.

Pure culture of yeast from starter culture

One gram of starter culture was dissolved in 10 ml distilledwater, diluted 1,000-fold and inoculated in YPD-agar media[1 % Yeast extract, 2 % Bactopeptone, 2 % Dextrose and 2 %Agar (Difco) supplemented with Chloramphenicol 100 mg/L(Sigma) and 50 mg/L Chlorotetracycline (Sigma)] at 30 °C.Three successive subcultures were made after every 7 daysinoculation by repeated streaking, and pure cultures wereobtained.

Yeast isolates were identified and deposited at the Micro-bial Type Culture Collection (MTCC), Institute of MicrobialTechnology (IMTECH), India. The identified yeast isolateswere stored in 20 % (v/v) glycerol at –70 °C and for routineuse on YPD agar slants at 4 °C.

Thermotolerance, alcohol tolerance and pH sensitivityof the yeast strains

For thermal stability assay, 1 μL of pure culture in YPD at acell density of 2×106 cells/mL was spotted on YPD-agarplates and incubated at different temperatures (viz., 25, 30,37, and 42 °C) and their growths were observed.

Alcohol tolerance of the isolates were measured by inoc-ulating 4×106 cells/mL into 50 mL YPD broth containingdifferent concentrations of ethanol (4, 8, 12, and 16 %) andincubated at 30 °C in a shaking incubator at 200 rpm for72 h. Growths were observed by measuring optical density(OD) at 595 nm.

pH sensitivity of the strains were observed by measuringOD595 of inoculated cultures at variable pH (viz., 4.0, 4.5, 5.0,5.5, 6.0, 6.5, and 7.0) using UV-VIS spectrophotometer(Hitachi). pHs in the inoculated media were optimized by0.5 M Na2HPO4.

Fig. 1 Northeast India showingthe collection sites of startercultures

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Alcohol production by the yeast isolates

Amounts of 15 μL from preinocula containing 2×106 cells/mL were inoculated into 250-mL Erlenmeyer flasks contain-ing 100mLYPS broth (Yeast extract, Peptone and Starch 2%)with 4 % Sucrose, 0.5 % Bromophynol Blue at pH 7.2, andincubated at 30 °C for 72 h and 200 rpm. The resulting weightloss (in g) was obtained by measuring weights before incuba-tion and after 24, 36, and 72 h of incubation.

Protein profiling of isolated yeast strains using SDS-PAGE

The whole cell proteins from all the yeast isolates wereextracted following the method of Auty et al. (2004) andresolved using SDS-PAGE 12.5 % resolving gel and 4.5 %stacking gel (Sambrook et al. 1989), along with 10 μl of astandard 14.3–97.4 kDa protein ladder (Bangalore Genei,India). The gels were stained with Commassie Brilliant Blue(CBB R-250, Sigma B-2025) and documented.

Raising anti-ADH antibodies and Western blot

Antibodies against pure ADH (Sigma A 7011; Sigma Aldrich,USA) were raised in rabbits as reported earlier by Harlow andLane (1988). For immunization, rabbits were injected withADH antigen at multiple positions, followed by 2 boosterdoses. Two weeks after the second booster, blood was collect-ed, allowed to clot at 37 °C, and incubated overnight at 4 °C.Serum was collected by centrifuging incubated samples at8,000 g for 30 min and stored with 0.02 % Sodium azide at−20 °C. The generated anti-ADH polyclonal antibodies werediluted 1:2,000-fold in TBST buffer before use.

For western blotting, 25 μg of whole cell protein wassubjected to SDS-PAGE and transferred to 0.45-μM pore sizenitrocellulose membrane (Amersham, USA). Transferredblots were rinsed in TBS containing 10 mM Tris-Cl and150 mM Na2Cl (pH 7.4), and blocked overnight using 5 %BSA in 100 mL TBST buffer. The blots were further rinsedwith TBST and incubated for 1 h with primary anti-ADHantibody produced in the rabbit. After rinsing again withTBST for 10 min, the blots were incubated with secondaryantibody, i.e., goat ant-rabbit IgG-alkalyne phophatase conju-gate for 1 h. The secondary antibody conjugate was diluted1:1,500 times before use. Blots were later treated with fluo-rescent conjugated BCIP/NBT (Banglore Genei) and docu-mented using Gel-Doc system (Bio-Rad 2000).

Quantization of yeast ADH by slot blot analysis

Slot blot analysis for yeast ADH secreted by the isolates wasconducted as described by Loeffler et al. (2000). Hybondnitrocellulose membranes were soaked in TBST buffer andplaced in the slot blot apparatus (Hoefer, Germany) and

allowed to dry for 2 min. Then, 25 μg of whole cell proteinalong with standard concentration of commercial ADH (0.1,0.5, 1.0, 5.0, 10, 100, and 1,000 ng/mL) were loaded on to theslot blot under denaturing condition. Blots were hybridizedwith biotinylated florescent-conjugated antibodies BCIP/NBT(Banglore Genei). Hybridization and detection were con-ducted according to the manufacturer’s recommendation.

Results

Isolation and identification of yeast

Continuous sub-culturing of yeast isolated from 12 differentstarter cultures representing 12 native ethnic tribes of north-east India along with baker’s yeast as reference yielded theisolation of 13 purified strains. Despite our repeatedapproaches, however, cwe ould not isolate more than oneyeast type representing each starter cake. Isolated yeaststrains were identified and deposited at the Microbial TypeCulture Collection (MTCC), Institute of Microbial Technol-ogy, India (Table 1). Interestingly, one of the isolates, Can-dida glabrata MTCC 3982, obtained from the Nepalicommunity of Assam appeared red in coloration, whichsuggested that the strain was an adenine-deficient (ade-)mutant, in its wild form (Poulter and Rikkerink 1983). Allthe other isolates showed a smooth, protruded phenotype,except for Wickerhamomyces anomalus MTCC 3979 thatappeared as papillae during solid culture (Fig. 2a, b).

Thermotolerance, alcohol tolerance and pH sensitivity

All isolates except Candida glabrataMTCC 3985, Candidaglabrata MTCC 3986 and Wickerhamomyces anomalusMTCC 3979 showed sustainably stable growth up to 37 °C. The growth decreased significantly beyond 37 °C, andnone of the strains showed growth at 42 °C; hence, most ofthe strains were found to be thermotolerant up to 37 °C.

Alcohol tolerance activity of isolated yeast strains wasobserved to be highly variable in nature. Highest alcoholtolerance (16 % ethanol v/v in media) was expressed bySaccharomyces cerevisiae MTCC 3976 isolated from thecakes of the Tea Tribe of Assam. Similar findings could beobserved for the reference strain Saccharomyces cerevisiaeMTCC 3980 and the lone Wickerhamomyces anomalusMTCC 3979 that portrayed appreciable growth rates athigher concentrations of alcohol. Interestingly, none of theCandida isolates could thrive at alcohol concentrationsabove 12 % (Fig. 3). The most deficient in alcohol toleranceactivity among all the isolated strains was Debaryomyceshansenii var. hansenii MTCC 3977, obtained from the Mei-tei community of Manipur which did not show any growthbeyond 4 % alcohol concentration.

Ann Microbiol (2013) 63:863–869 865

Similar confronting observations could also be no-ticed for pH sensitivity of the isolated yeast strainswhereby most of the isolates (except Candida glabrataMTCC 3984 and Candida glabrata MTCC 3985) fa-vored an acidic pH for attaining maximum growth(Fig. 4). Growth in all the strains decreased with anincrease in pH. The pH of the final fermentation prod-ucts stood at around 5.5–6.0. Exceptional observationscould be noticed for Candida glabrata MTCC 3987 thatrecorded an atypical growth at more acidic pH (5.0–5.5). On the other hand, two other isolates, Candidaglabrata MTCC 3984 and Candida glabrata MTCC3985, grew well only at a less acidic condition (pH6.5). The isolates Debaryomyces hansenii var. hanseniiMTCC 3977 and Debaryomyces hansenii var. hanseniiMTCC 3978 expressed maximum growth between pH5.5 and 6.0, while Wickerhamomyces anomalusMTCC 3979favored maximum growth only at an acidic pH of 5.5.

Alcohol production

All the isolated yeast strains differed significantly from oneanother in terms of alcohol production (Fig. 5). The maxi-mum amount of alcohol production could be observed forthe isolate Saccharomyces cerevisiae MTCC 3976 obtainedfrom the Tea Tribe of Assam. Intermediate expression ofalcohol productivity could be observed for the Candidaglabrata MTCC 3983, Candida glabrata MTCC 3981 andCandida glabrata MTCC 3988 respectively. The otherthree, i.e., Candida glabrata MTCC 3984, Candida gla-brata MTCC 3985 and Candida glabrata MTCC 3986showed the least amount of alcohol production. The Wick-erhamomyces anomalus MTCC 3979 portrayed a moderateexpression of alcohol productivity comparable to the Deba-ryomyces isolate. All the isolated strains depicted fermenta-tion activity after 36 h of incubation as was evident fromweight loss and alcohol production during fermentation.

AH

AP

AD

NP

KH

BY

BY

NP

TT

MA MS

KA

AN AN

BR DM

a b

Fig. 2 Colony morphology, color and phenotype characteristics ofyeasts isolated in YPD (Dextrose used as substrate) from starter culturecakes. a KH (C. glabrata MTCC 3987); BR (C. glabrata MTCC3984); NP (C. glabrata MTCC 3982); AN (C. glabrata MTCC3986); KA (C. glabrata MTCC 3988) and MS (C. glabrata MTCC

3983). bMA (D. hansenii var. hanseniiMTCC 3977); DM (D. hanseniivar. hansenii MTCC 3978); AP (W. anomalus MTCC 3979); AD (C.glabrata MTCC 3985); TT (S. cerevisiae MTCC 3976); AH (C. gla-brata MTCC 3981); BY (S. cerevisiae MTCC 3980)

Table 1 Sources of yeast strainsand MTCC code

aReference strain

Strain type MTCC code Source of collection Lab. code

Saccharomyces cerevisiaea 3980 Baker’s yeasta BY

Saccharomyces cerevisiae 3976 Tea Tribe of Assam TT

Debaryomyces hansenii var. hansenii 3977 Meitei community of Manipur MA

Debaryomyces hansenii var. hansenii 3978 Dimasa Tribe of Assam DM

Wickerhamomyces anomalus 3979 Apatani Tribe of Arunachal Pradesh AP

Candida glabrata 3981 Ahom community of Assam AH

Candida glabrata 3982 Nepali community of Assam NP

Candida glabrata 3983 Mishing Tribe of Assam MS

Candida glabrata 3984 Bodo Tribe of Assam BR

Candida glabrata 3985 Adivasi Tribe of Assam AD

Candida glabrata 3986 Angami Tribe of Nagaland AN

Candida glabrata 3987 Khasi Tribe of Meghalaya KH

Candida glabrata 3988 Karbi Tribe of Assam KA

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Protein profiling and western blot analysis

Analysis of crude whole-cell protein extracts of all the 12isolated strains along with reference based on SDS-PAGErevealed very little information. However, some bands werecomparable and certain differences could be observed.

The expression of ADH by the isolates was found to behighly variable as evident from Fig. 6. The maximum ADHactivity was observed for the strain Saccharomyces cerevi-siae MTCC 3976 obtained from the Tea Tribe of Assam.The Candida isolates on the other hand, despite their rela-tively poor alcohol tolerance, delineated a reasonably higherexpression profile of the ADH. Debaryomyces hansenii var.hansenii MTCC 3977 and D. hansenii var. hansenii MTCC

3978 depicted a moderate expression profile of ADH asevident from the band intensity along with the Wickerhamo-myces anomalus MTCC 3979.

Analysis of alcohol dehydrogenase by slot blot

Quantitative analyses of ADH secreted through slot blottingyielded appreciable amounts of information which confirmedthat almost all the strains expressed a higher level of ADH(>1,000 ng/mL).Candida glabrataMTCC 3983 obtained fromthe Mishing Tribe of Assam expressed the least amount ofADH activity corresponding to <100 ng ADH/ml in fermenta-tion media. The maximum quantity of ADH secretion werefound to be expressed by Saccharomyces cerevisiae MTCC

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

TT MA DM AP BY AH NP MS BR AD AN KH KA

Opt

ical

den

sity

at

595

nm

Yeast strains

4%

8%

12%

16%

Fig. 3 Alcohol tolerance depicted by the yeast isolates to increasingconcentrations of alcohol (ethanol) supplied exogenously to YPDmedia under in-vitro controlled conditions: TT (S. cerevisiae MTCC3976); MA (D. hansenii var. hansenii MTCC 3977); DM (D. hanseniivar. hansenii MTCC 3978); AP (W. anomalus MTCC 3979); BY (S.

cerevisiae MTCC 3980); AH (C. glabrata MTCC 3981); NP (C.glabrata MTCC 3982); MS (C. glabrata MTCC 3983); BR (C. gla-brata MTCC 3984); AD (C. glabrata MTCC 3985); AN (C. glabrataMTCC 3986); KH (C. glabrata MTCC 3987); KA (C. glabrata MTCC3988)

0

20

40

60

80

100

120

TT MA DM AP BY AH NP MS BR AD AN KH KA

Pro

tein

con

cent

rati

on in

µg/

ml

Yeast strains

4

4.5

5

5.5

6

6.5

7

7.5

Fig. 4 Sensitivity of the yeastisolates to variable levels of pHin liquid YPD media incubatedat 30 °C under in vitrocontrolled conditions: TT (S.cerevisiae MTCC 3976); MA(D. hansenii var. hanseniiMTCC 3977); DM (D. hanseniivar. hansenii MTCC 3978); AP(W. anomalusMTCC 3979); BY(S. cerevisiaeMTCC 3980); AH(C. glabrata MTCC 3981); NP(C. glabrata MTCC 3982); MS(C. glabrata MTCC 3983); BR(C. glabrata MTCC 3984); AD(C. glabrata MTCC 3985); AN(C. glabrata MTCC 3986); KH(C. glabrata MTCC 3987); KA(C. glabrata MTCC 3988)

Ann Microbiol (2013) 63:863–869 867

3976, Debaryomyces hansenii var. hansenii MTCC 3977,Candida glabrata MTCC 3981, Candida glabrata MTCC3981, Candida glabrataMTCC 3987, and Candida glabrataMTCC 3988 (Fig. 7a). On the other hand, significant variabil-ity could be observed for thermostability of the expressedADH isozymes among the yeast isolates as was evidencedby slot blotting under denaturing conditions (Fig. 7b). ADHsecreted by Wickerhamomyces anomalus MTCC 3979,

Candida glabrata MTCC 3981, Candida glabrata MTCC3982, Candida glabrataMTCC 3985, and Candida glabrataMTCC 3988were fairly thermostable compared to their coun-terparts secreted by Saccharomyces cerevisiae MTCC 3976.ADH thermostability was totally absent for Candida glabrataMTCC 3983 obtained from the Mishing Tribe of Assam.

Discussion

This is the first report to characterize the yeast from startercultures used for the production of household liquor by

0

1

2

3

4

5

6

7

TT MA DM AP BY AH NP MS BR AD AN KH KAW

eigh

t los

s in

gra

ms

Yeast strains

24 hours

36 hours

72 hours

Fig. 5 Substrate utilization andalcohol production by the yeastisolates. Sucrose (20 %) wasadded to YPD liquid media assole carbon source: TT (S.cerevisiae MTCC 3976); MA(D. hansenii var. hanseniiMTCC 3977); DM (D. hanseniivar. hansenii MTCC 3978); AP(W. anomalusMTCC 3979); BY(S. cerevisiaeMTCC 3980); AH(C. glabrata MTCC 3981); NP(C. glabrata MTCC 3982); MS(C. glabrata MTCC 3983); BR(C. glabrata MTCC 3984); AD(C. glabrata MTCC 3985); AN(C. glabrata MTCC 3986); KH(C. glabrata MTCC 3987); KA(C. glabrata MTCC 3988)

97.4 kDa

14.3 kDa

20.1 kDa

29.0 kDa

43.0 kDa

66.0 kDa

M 1 2 3 4 5 6 7 8 9 10 11 12 13

Fig. 6 Western blot analysis of ADH in the isolated yeasts. Thenitrocellulose electro-blots treated with BCIP/NBT were photographedand documented using a Gel Documentation System (Bio-Rad 2000,USA). Lane M Protein molecular weight (PMWM, Genei, India); 1MA (D. hansenii var. hansenii MTCC 3977); 2 DM (D. hansenii var.hansenii MTCC 3978); 3 AD (C. glabrata MTCC 3985); 4 BR (C.glabrata MTCC 3984); 5 BY (S. cerevisiae MTCC 3980); 6 TT (S.cerevisiae MTCC 3976); 7 MS (C. glabrata MTCC 3983); 8 NP (C.glabrata MTCC 3982); 9 AH (C. glabrata MTCC 3981); 10 AP (W.anomalus MTCC 3979); 11 AN (C. glabrata MTCC 3986); 12 KA (C.glabrata MTCC 3988); 13 KH (C. glabrata MTCC 3987)

1 2 3 4 5 6 7 8 9 10 11

12 1 3 0.1 0.5 1.0 5.0 10 50 100 1000

12 1 3 0.1 0.5 1.0 5.0 10 50 100 1000

ADH (ng/mL)

ADH (ng/mL)

a

b

Fig. 7 a. Slot blot analysis of ADH in the isolated yeasts under nativeconditions. b Slot blot of yeast ADH under denaturing conditions Lane1 TT (S. cerevisiae MTCC 3976); 2 MA (D. hansenii var. hanseniiMTCC 3977); 3 DM (D. hansenii var. hansenii MTCC 3978); 4 AP(W. anomalus MTCC 3979); 5 BY (S. cerevisiae MTCC 3980); 6 AH(C. glabrataMTCC 3981); 7 NP (C. glabrataMTCC 3982); 8MS (C.glabrata MTCC 3983); 9 BR (C. glabrata MTCC 3984); 10 AD (C.glabrata MTCC 3985); 11 AN (C. glabrata MTCC 3986); 12 KH (C.glabrata MTCC 3987); 13 KA (C. glabrata MTCC 3988)

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different ethnic communities of Northeast India. In the presentinvestigations, it was noticed that the majority of the yeaststrains involved in alcoholic fermentation represented differ-ent genera within the same family (Saccharomycetaceae) i.e.,Debaryomyces, Wickerhamomyces and Candida, apart fromthe presence of fermenting yeast Saccharomyces.

The study has revealed some facts about the behavior ofyeast strains associated with traditional alcoholic fermenta-tion. An interesting finding was the thermotolerance of thestrains to survive and withstand higher temperatures. Alco-holic beverages produced by natural fermentation processespracticed by the ethnic communities are intoxicating as wellas highly invigorating. Under such circumstances, the lowalcohol tolerance of most Candida and Debaryomycesstrains are enigmatical, and therefore we cannot reject thepresence of other cultivable or non-cultivable Saccharomy-ces species in fermentation cakes that might thrive in con-sortia but could not be detected by isolation in the presentstudy. Much more noteworthy is the chronological lifespanof the isolated yeasts which has no significance to alcoholtolerance of a strain (Minois et al. 2005).

For pH sensitivity, it was observed that most of the yeastisolates were sensitive to changes in pH which may play animportant role in the phenotypic divergence of yeasts likeCandida (Kaur et al. 1988). The observations were crucialand it throws light on the acclimatization of the strains toindigenous fermentation protocols and the subsequent addi-tion of ingredients.

Extremely incongruous results could be observed for theCandida isolates wheremost of the strains depicted low alcoholtolerance despite higher alcohol productivity. The Debaryomy-ces strains MA and DM appeared to have fairly appreciablealcohol productivity but poor alcohol tolerability. One convinc-ing reason for such inconsistent findings might be attributed tothe presence of other cultivable or uncultivable yeasts in theconsortia, which might contribute to increased alcohol produc-tion, and which could not be detected in the present investiga-tion (Curtain 1986; Garry-Arroyo et al. 2003).

The relative expression profiles of enzyme alcohol dehy-drogenase (ADH) appeared quite amazing whereby most ofthe Candida isolates expressed appreciable amounts ofADH. It therefore becomes pertinent that the Candida iso-lates have a greater potential for conversion of acetaldehydeinto ethanol in culture, but the presence of inhibitory sub-stances secreted by other contaminating microorganismsmight lead to decreased alcohol tolerance of the strains.

The present findings have provided some good evidenceto conclude that the strain Saccharomyces cerevisiaeMTCC3976 from the Tea Tribe of Assam and WickerhamomycesanomalusMTCC 3979 from the Apatani Tribe of ArunachalPradesh were found to be exceptional in terms of all theparameters used in this study and hence may be identified aspotential strains for industrial fermentation.

Acknowledgments Authors are grateful to Dr. Bhaskarjyoti Sarmahfor his technical guidance and continuous support. The Institute ofMicrobial Technology (IMTECH), Chandigarh, India, is duly acknowl-edged for identifying the yeast strains. This work was supported by aminor grant from the University Grants Commission (UGC), India.

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