wine vinegar production using base wines made with different yeast species

J Sci Food Agric 1998, 78, 290È294

Wine Vinegar Production Using Base Wines Madewith Diþerent Yeast SpeciesMaurizio Ciani

Dipartimento di Biologia Vegetale, sez Microbiologia applicata, Universita degli Studi di Perugia, 06121Perugia, Italy

(Received 19 August 1997 ; revised version received 19 January 1998 ; accepted 10 March 1998)

Abstract : The inÑuence of base wines obtained by the fermentation of di†erentyeast species on acetic acid bacteria growth and on the analytical proÐle of vine-gars was investigated. Results show that the substrates for wine vinegar pro-duction exerted a strong inÑuence on both acetic acid bacteria growth andanalytical proÐle of vinegars. The base wine obtained from the alcoholic fermen-tation of Saccharomyces cerevisiae was not always the best substrate. The fermen-tate made with Candida stellata positively inÑuenced the acetic acid bacteriagrowth and the quality of vinegar, while the wine obtained from the fermentationof Kloeckera apiculata was a good substrate for acetic acid bacteria growth andacetic acid production and could be used for “ordinaryÏ vinegar production.

1998 Society of Chemical Industry(

J Sci Food Agric 78, 290È294 (1998)

Key words : wine vinegar ; base wine ; acetic acid bacteria growth ; quality.

INTRODUCTION

Biological vinegar could be obtained from di†erentalcoholigenous raw materials. By far the largest percent-age of vinegar is alcohol vinegar, which is producedfrom diluted puriÐed ethanol (Ebner and Follmann1983), but the interest for wine or apple vinegars is nowgrowing among the consumers. Wine vinegar, in partic-ular, is generally recognised to have a higher organolep-tic value in comparison with other vinegars (Carnaciniand Gerbi 1992). Several recent studies (Guerrero et al1994 ; Gerbi et al 1995, 1996 ; Gonzalez-Vinas et al 1996)were carried out in order to characterise and deÐne theprincipal parameters of quality of wine vinegar.

Wine vinegar is obtained by double fermentation : thealcoholic fermentation is performed by yeasts, while theacetic acid bacteria carry out the acetic acid fermenta-tion (oxidative fermentation). The characteristics andcomposition of the base wine could interfere with aceticacid fermentation and a†ect the quality of vinegar. Thepresent study is directed at evaluating the inÑuence ofdi†erent yeast species used for the base wine productionon the growth of acetic acid bacteria and on the analyti-cal proÐle of vinegars.

EXPERIMENTAL

Microorganisms

Acetobacter aceti strain no 20 and Gluconobacteroxidans strain no 92 isolated from artisanal vinegars(Ciani and Rosini 1990) were used in this study.

Media

White grape must from a Trebbiano toscano cultivar(pH 3É10) diluted with distilled water until 10% w/v ofsugar was used for alcoholic fermentation. Grape wasadded with ammonium Phosphate (0É25 g litre~1) toreconstitute the nitrogen concentration and sterilised at90¡C for 20 min. The di†erent base wines were obtainedby alcoholic fermentation carried out in 14-litre cylin-drical vessels (BiolaÐtte type) by the following yeastspecies belonging to Industrial Yeast Collection ofDipartimento di Biologia Vegetale sez MicrobiologiaApplicata Universita di Perugia (DBVPG) : Kloeckeraapiculata DBVPG 3071, Hanseniaspora uvarumDBVPG 3019, Saccharomycodes ludwigii DBVPG 3079,T orulaspora delbrueckii DBVPG 6168, Candida stellata

2901998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain(

InÑuence of base wine on wine vinegar production 291

DBVPG 4124, Saccharomyces cerevisiae DBVPG 2189.The analytical proÐles of base wines made with di†erentyeast species are showed in Table 1. Ethanol concentra-tion of fermentates was brought at 70 ml litre~1. Basewines were Ðltered sterilised through 0É22 lm pore-sizemembranes (Millipore Corp, Bedford, MA, USA).

Fermentation condition

Acetic acid fermentations were carried out in 1-litreÑasks, each containing 200 ml of di†erent base wines at27¡C. The aerobiosis condition was maintained by agi-tation of the Ñasks on a rotary shaker (150 rev min~1).Two percent inoculum of 5-day-old acetic acid bacteriastrains, pre-incubated in the same base wine in aero-biosis condition at 27¡C, were used to give an initial cellconcentration of 0É1 optical density (OD, absorbance at520 nm). Fermentations were recorded by evaluatingthe OD (absorbance at 520 nm) and the pH value.When the pH value become constant, the fermentationwas considered to be Ðnished. For chemical analysis,samples were Ðltered through 0É22 lm pore-size mem-branes (Millipore) and stored at [20¡C.

Analyses

The acetic acid bacteria growth was monitored by theOD at 520 nm using LKB-Ultraspec 4050 Spectropho-tometer (Pharmacia Biotech, Uppsala, Sweden). Totaland volatile acidity were analysed according to OfficialAnalytical Methods referred by Giannesi and Matta(1987). Glucose and fructose were determined by usingspeciÐc enzymatic kit (kit no. 139106 ; BoehringerMannheim, Mannheim, Germany). Ethanol was mea-sured by gas chromatographic analysis (AOAC 1990).Ethanol acetate and acetoin were detected with GLC bydirect injection. Samples were neutralised (pH 9) with

saturated solution and 0É5 ll was injected intoNa2CO3a glass column 2 m by 2 mm (inner diameter) packedwith Carbopack B/5% carbowax 20 mesh (Supelco Inc) ;internal standard, 3-methyl-2-butanol. Nitrogen (20 mlmin~1) was used as the carrier gas. Carlo Erba Vegagas chromatograph, equipped with a Ñame ionization

detector and linked to a DP 700 Carlo Erba integratorwas used. The oven temperature ranged from 45 to160¡C (5¡C min~1). The temperature of the injector anddetector was 220¡C.

RESULTS

Results show that the substrates for wine vinegar pro-duction exerted a strong inÑuence on the growth ofacetic acid bacteria. In Table 2, Ðnal cell densities (ODat 520 nm) of the two strains of acetic acid bacteriaduring vinegar production are showed. The base winesobtained by the activity of Kl apiculata and S ludwigiiwere the best substrates for the growth of acetic acidbacteria. Acetic acid bacteria strains showed a lowergrowth in base wines made with S cerevisiae and C stel-lata yeast strains. In base wines, obtained by the fer-mentation of T delbrueckii and H uvarum, the growth oftwo strains of acetic acid bacteria was very poor.

It is interesting to notice that the growth of the twodi†erent strains of acetic acid bacteria in the same sub-strates did not vary signiÐcantly. The evaluation of theprincipal analytical and technological characters ofvinegars (Table 3) conÐrms this behaviour. Gluconobac-ter oxidans and Acetobacter aceti strains did not signiÐ-cantly a†ect the main analytical and technologicalcharacters of vinegar. On the contrary, the base winesmade with the di†erent yeast species showed a largevariability of the analytical proÐle of vinegars.

Very interesting analytical proÐles of vinegar, particu-larly regarding the volatile acidity and acetic acid yield

were obtained from base wines produced by the(Y ac)fermentation of K apiculata and C stellata, whereas theanalytical proÐle of vinegars obtained by fermentates ofT delbrueckii and S. ludwigii were very similar to thatproduced in S cerevisiae base wine.

In base wines made with H uvarum, the acetic acidbacteria showed a low amount of volatile acidity andhigh residue of ethanol (higher than 20É0 g litre~1). Thisbehaviour is probably related to the poor growth of twoacetic acid bacteria strains recorded in this substrate.

In order to assess some parameters a†ecting thequality of vinegars, the author would evaluate the Ðnal

TABLE 1Analytical proÐle of base wines used for the vinegar production

Fermentation Sugar Ethanol Acetic Acetoin Ethyl pHby residue acid acetate

(g litre~1) (ml litre~1) (g litre~1) (mg litre~1) (mg litre~1)

Kl apiculata 6É9 48É0 0É25 39É7 117É6 3É10H uvarum 4É2 46É0 0É22 44É4 381É8 3É10S ludwigii ND 65É0 0É07 39É7 45É2 3É00T delbrueckii 5É3 63É0 0É03 14É5 13É7 3É19C stellata 0É4 55É0 0É09 47É6 16É5 3É07S cerevisiae ND 64É0 0É10 31É7 49É9 3É00

292 M Ciani

TABLE 2InÑuence of di†erent base wines on the growth of two acetic acid bacteria strains during the vinegar pro-

duction

Acetic acid Base wine bybacteria

Kl apiculata H uvarum S ludwigii T delbrueckii C stellata S cerevisiae

Gluconobacter 16É2a 2É7 14É0 4É8 6É3 7É7oxidansstrain 92

Acetobacter 15É3 3É4 14É0 5É1 6É7 8É4acetistrain 20

a Final optical density (OD) ; absorbance at 520 nm.

TABLE 3Principal analytical and technological characters of vinegars obtained from di†erent fermentates

Analytical Base wine byproÐle ofvinegars Kl apiculata H uvarum S ludwigii T delbrueckii C stellata S cerevisiae

Acetobacter aceti 20Final ethanol 0É63 23É3 2É1 10É1 12É3 12É5(g litre~1)

Ethanol consumed 47É9 31É8 44É0 40É4 45É6 38É6(g litre~1)

Total acidity 55É2 37É8 49É8 47É3 56É4 47É4(g litre~1)

Volatile acidity 50É2 33É5 44É3 42É8 50É4 40É2(g litre~1)

Yaca 80É3 80É8 77É2 81É2 84É7 78É8

Gluconobacter oxidans 92Final ethanol 2É8 25É1 3É7 11É7 10É7 14É3(g litre~1)

Ethanol consumed 45É0 30É5 41É9 38É2 48É3 34É7(g litre~1)

Total acidity 52É2 34É2 45É6 47É4 60É4 42É0(g litre~1)

Volatile acidity 47É7 30É3 39É3 41É1 54É1 35É7(g litre~1)

Yac a 81É3 76É2 71É9 82É5 85É8 78É9

Acetic acid yield ; acetic acid g litre~1[ ethanol g litre~1] 46/60 ] 100.a Yac ,

TABLE 4InÑuence of di†erent base wines on acetoin production (mg litre~1) by two strains of acetic acid bacteria



Gluconobacter 123É1 153É3 135É9 119É6 366É2 190É8oxidansstrain 92


InÑuence of base wine on wine vinegar production 293

TABLE 5InÑuence of di†erent base wines on ethyl acetate production (mg litre~1) by two strains of acetic acid bacteria



Gluconobacter 105É2 163É7 80É3 338É5 659É6 518É8oxidansstrain 92


concentration of two important constituents of aro-matic proÐle of vinegars : ethyl acetate and acetoin.These compounds are considered to be typical indica-tors of quality (Amati et al 1990 ; Gerbi et al 1994), sincea high concentration in wine vinegars of quality isfound (Mecca and Di Vecchio 1977 ; Amati et al 1990).

With a regard to the acetoin production (Table 4), nosigniÐcant di†erences were found among vinegars pro-duced on base wine which is obtained by the fermenta-tion of S cerevisiae and other substrates. The onlyexception was represented by vinegars obtained on basewine made with C stellata whose levels were higher thanothers. Levels of acetoin obtained with C stellata basewine were much higher than amounts considered indis-pensable in vinegars of “qualityÏ (180 mg litre~1) (Amatiet al 1990).

The production of ethyl acetate (Table 5) seems to bemore linked to the type of fermentates than to aceticacid bacteria strains. Acetic acid fermentation, carriedout on base wine obtained by the fermentation of Cstellata, produced the largest amounts of this compoundwhich a†ects the quality of vinegar. Levels of ethylacetate (Table 5) were much higher in vinegar producedon fermentate which are made with S cerevisiae thanwith the others.

DISCUSSION

The above results showed that the base wine made withS cerevisiae was not always the best substrate for boththe acetic acid bacteria growth and the quality of vine-gars. Giudici et al (1992) suggested the use of selectedstarter cultures of Zygosaccharomyces bailii to producebase wine for balsamic vinegar, a special wine vinegar athigh osmophilic environment. Moreover, the use ofimmobilised cells of S ludwigii for continuous pro-duction of vinegar has been suggested (Saeki 1990).

The results of the present work showed that basewines obtained by the fermentation of C stellata and Klapiculata seemed to be interesting substrates for winevinegar production. Fermentate of C stellata positivelyinÑuenced the analytical and aromatic proÐle ofvinegar. The levels of acetic acid and especially of ethyl

acetate and acetoin highlighted the possibility of usingC stellata for base wine production to obtain highquality wine vinegars using traditional methods. Theimportance of acetoin and ethyl acetate on aromaticproÐle and on the complexity of vinegars are describedby several authors and related to sensory evaluation(Amati et al 1990 ; Gerbi et al 1994, 1995). The basewine made with Kl apiculata, seems positively to char-acterise the growth and the acetic acid production ofacetic acid bacteria. This yeast could be used to producebase wine for “ordinaryÏ vinegar (submerged methods) toimprove the fermentation process and the acetic acidproduction.

In order to conÐrm these results, it is necessary toverify this behaviour by evaluating the di†erent technol-ogies of vinegar production (traditional and submerged)and scale up fermentation processes.

ACKNOWLEDGEMENTS

The authors thanks Fausto Maccarelli for his technicalassistance and Ministero dellÏUniversita e della RicercaScientiÐca e Tecnologica (MURST 40%), Italy, forÐnancial support.

REFERENCES

Amati A, Carnacini A, Antonelli A, Natali N 1990 ConvegnoInternazionale SullÏaceto di V ino. Torino, Italy.

AOAC 1990 Official Methods of Analysis (Vol 2, 15th edn).Association of Official Analytical Chemists, VA, USA p 700.

Carnacini A, Gerbi V 1992 LÏaceto di vino, un prodotto datutelare e da valorizzare. Indust Bevande 21 465È477.

Ciani M, Rosini G 1990 Isolamento, identiÐcazione e primacaratterizzazione tecnologica di batteri acetici. Ann Fac AgrUniv Perugia 44 9È17.

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Gonzalez-Vinas M A, Salvador M D, Cabezudo M D 1996Taste group thresholds and sensory evaluation of Spanishwine vinegars. J Sens Stud 11 129È140.

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Mecca F, Di Vecchio L 1977 La determinazione di alcuni cos-tituenti volatili degli aceti di fermentazione mediante gas-cromatograÐa. Riv Soc Ital Sci Alim 6 177È186.

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wine vinegar production using base wines made with different yeast species

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