Mitteilungen Klosterneuburg 53 (2003): 113-122

Deacidification of using wine yeast and bacteria starter cultures

SUSANNE BERGER, KAROLINE PISCHINGER und SILVIA WENDELIN

HoÈ here Bundeslehranstalt und Bundesamt fuÈ r Wein- und Obstbau A-3400 Klosterneuburg, Wienerstraûe 74 e-mail: [email protected] Present investigations deal with the malic acid reducing capacity of selected wine yeast strains of the species Saccha- romyces cerevisiae and bacteria starter cultures of the species Oenococcus oeni. Deacidification capacity and in- fluence of bacteria present in seven of ten commercial dry yeast strains compared to pure culture of yeast strains were evaluated on the basis of 'Rheinriesling' must fermentations. Analysis of malic acid reduction in must and wine showed basically yeast borne activity which was shown to be amplified in musts with pH-value 3.1 and 3.4 re- spectively. Some of the investigated dry yeast preparations were found to contain low amounts of bacteria cultivable on appropriate media. Results concerning the malic acid degradation velocity in red did not correlate with the amount of bacteria cell number in dry yeast preparations. Mash from of the 'Blauer Portugieser' cultivar was fermented with two commercial dry yeast preparations. Malolactic fermentation variants were induced using three different bacteria starter cultures, simultaneously to a spontaneous fermentation variant. At 16 8C malolactic fer- mentation finished successfully several days later compared to variants at 21 8C. Both yeast and bacteria reduced monomeric anthocyanins in red wines. After four months of storage on fine lees, the influence of microorganisms on phenols and biological stability was evaluated. Bacteria developed during spontaneous malolactic fermentation showed highest viability using temperatures of 21 8C and 16 8C compared to Oenococcus oeni starter culture. Du- ring wine storage on fine lees, the amino acid tyrosine increased to the highest amount compared to arginine and ly- sine. Amounts of caffeic acid, a precursor for volatile phenols, cannot be related to yeast or bacteria strain influence. Keywords: Wine, deacidification, yeasts, malolactic fermentation, starter cultures, Saccharomyces cerevisiae, Oeno- coccus oeni

Abbau der AÈ pfelsaÈure mit Weinhefen und Bakterien-Starterkulturen. Die aÈpfelsaÈureabbauende KapazitaÈt von se- lektionierten StaÈmmen der Art Saccharomyces cerevisiae und von Bakterien der Art Oenococcus oeni sind Gegen- stand vorliegender Untersuchungen in Weinen der Sorte 'Rheinriesling' und 'Blauer Portugieser'. Die Analysen be- staÈtigten, dass der AÈ pfelsaÈureabbau teilweise auf die metabolische AktivitaÈt der untersuchten Hefen zuruÈ ckzufuÈh- ren ist. Diese AktivitaÈt trat in Mosten mit einem pH-Wert von 3.4 im Vergleich zu Mosten mit einem pH-Wert von 3.1 verstaÈrkt auf. In sieben von zehn TrockenhefepraÈparaten wurden Bakterien nachgewiesen. Die Kinetik des biologischen SaÈureabbaus im Rotwein der Sorte 'Blauer Portugieser' korrelierte nicht mit der HoÈhe der nachge- wiesenen Bakterienzellzahlen. Trauben der Sorte 'Blauer Portugieser' wurden mit zwei kommerziellen HefestaÈm- men vergoren. Der anschlieûend durchgefuÈ hrte biologische SaÈureabbau mit drei Bakterien-Starterkulturen im Ver- gleich zu spontanem biologischen SaÈureabbau verlief in allen Varianten erfolgreich, war jedoch bei 16 8C um einige Tage verzoÈgert im Vergleich zu 21 8C. Hefen und Bakterien reduzierten die monomeren Anthocyane in den Rot- weinen in unterschiedlichem Ausmaû. Nach einer Lagerung von vier Monaten auf der Feinhefe wurde der Einfluss der Mikroflora auf die biologische StabilitaÈt und auf die Phenole evaluiert. Die Bakterien-Spontanflora wies im Vergleich zu Oenococcus oeni Starterkulturen die hoÈchste Lebendzellzahl auf. Der Gehalt der AminosaÈure Tyrosin stieg am staÈrksten an. Auch die Konzentration an Arginin und Lysin nahm zu. KaffeesaÈure kann auf Grund mikro- biologischen Einflusses in fluÈ chtige Phenole umgewandelt werden. Saccharomyces cerevisiae oder Oenococcus oeni hatten keinen Einfluss auf Zu- oder Abnahme des KaffeesaÈuregehaltes in den Weinen. SchlagwoÈ rter: Wein, EntsaÈuerung, Hefen, biologischer SaÈureabbau, Starterkulturen, Saccharomyces cerevisiae, Oe- nococcus oeni

113 Mitteilungen Klosterneuburg 53 (2003): 113-122 Berger et al.

DeÂgradation de l'acide malique par des levures de vin et des cultures starter de bacteÂries. La capacite des souches seÂlectionneÂes du genre Saccharomyces cerevisiae et des bacteÂries du genre Oenococcus oeni de deÂgrader l'acide ma- lique fait l'objet des preÂsents examens des vins des ceÂpages 'Rheinriesling' et 'Blauer Portugieser'. Les analyses con- firment que la deÂgradation de l'acide malique est partiellement due aÁ l'activiteÂmeÂtabolique des levures examineÂes. Cette activite s'est manifesteÂe plus intenseÂment dans des mouÃts d'un pH de 3,4 que dans les mo«ts d'un pH de 3,1. Dans quelques preÂparations de levure seÁche, on a deÂtecte des bacteÂries qui ne jouent treÁs vraisemblablement aucun rzÂle aÁ la fermentation. La cineÂtique de la deÂgradation biologique de l'acide dans le vin rouge du ceÂpage 'Blauer Por- tugieser' ne preÂsente aucune correÂlation avec le nombre des bactereis deÂtecteÂs dans les souches commerciales. Les ma- ceÂrations avec les raisins du ceÂpage 'Blauer Portugieser' ont eÂte effectueÂes avec deux souches de levure commerciale. LadeÂgradation biologique de l'acide aÁ l'aide de trois cultures starter de bacteÂries s'est deÂrouleÂe de manieÁre efficace par comparaison aÁladeÂgradation biologique spontaneÂe de l'acide, mais a eÂte retardeÂe de quelques jours aÁ une tem- peÂrature de 16 8C par rapport aÁ une tempeÂrature de 21 8C. Les levures et les bacteÂries reÂduisaient d'une manieÁre dif- feÂrente les anthocyanes monomeÁres dans les vins rouges. L'influence de la microflore sur la stabilite biologique et sur les pheÂnols a eÂteÂeÂvalueÂe apreÁs un stockage de quatre mois sur lies fines. CompareÂe aÁ Oenococcus oeni des cultures commerciales, la flore spontaneÂe preÂsentait l'indice de germination le plus eÂleveÂ. La teneur en acide amine tyrosine preÂsentait la plus forte augmentation. La concentration d'arginine et de lysine a eÂgalement augmenteÂ. GracËe aÁ l' in- fluence microbiologique, l'acide cafeÂique peut nÂtre transforme en pheÂnols volatils. Saccharomyces cerevisiae ou Oe- nococcus oeni n'ont eu aucune influence sur l'augmentation ou la baisse de la teneur des vins en acide cafeÂique. Mots cleÂs: vin, deÂsacidification, levures, fermentation malolactique, cultures starter, Saccharomyces cerevisiae, Oe- nococcus oeni

New enological procedures contribute to accurate wine capacity of Saccharomyces cerevisiae to degrade malic making and a higher quality on the wine market. Pro- acid amounts during alcoholic fermentation. Dry yeast cessing standards are getting higher, demanding precise preparations contain bacteria contaminants at amounts 2 5 winemakers' skills and process monitoring from vi- of10 cfu/g to 10 cfu/g of dry yeast (RADLER et al., neyard to filled bottles. 1985). Bacterial contaminants in dry yeast preparations Balance oftartaric acid and malic acid is important for could contribute to malic acid reduction. further product stability. High numbers ofviable bacteria cells in wine might Acidification and deacidification of must or wine repre- cause off-flavours like bitterness and buttery taste or sent useful tools to adjust the quality. Malic acid will be deteriorate red colour. Analysis ofbacteria viability du- degraded by chemical or microbiological procedures in ring storage is necessary because viable starter cultures case ofhigh vintage acidity or forbiological stabiliza- are a potential risk for wine stability. tion ofred wines. Certain substances can cause wine aging damages and Microbiological tools such as dry yeasts ofthe species therefore it is of interest to analyse their concentrations Saccharomyces cerevisiae used for alcoholic fermenta- with respect to dry yeast and bacteria starter culture va- tion and bacteria starter cultures ofthe species Oeno- riants. The influence of the fine lees microflora on wine coccus oeni both degrade malic acid. Metabolism ofma- quality can be evaluated on the basis ofamino acids, lic acid by yeast leads to the formation of carbonic phenols and anthocyanins. acids like pyruvate, L-lactic acid and relatively high Some amino acids are released to wines during lees con- amounts ofD-lactic acid, succinic acid and acetic acid. tact. On one hand, amino acids could be used by bacte- Homofermentative and heterofermentative lactic acid ria for their nutrition. On the other hand, sulphuric bacteria metabolize malic acid (RIBEÂ REAU-GAYON et al., molecules like cysteine could damage wine flavour. 1998). Malic acid degradation can be generated sponta- The uncoloured ethylcaffeic acid is a cinnamic acid. neously in case ofnative bacterial floradevelopment, Cinnamic acid alterates the colour ofwine towards yel- but accurately selected bacteria starter cultures do em- low or brown. Saccharomyces cerevisiae, lactic acid bac- phasize dominance of Oenococcus oeni species. Product teria and Dekkera bruxellensis produce cinnamate de- diversity ofsupplied bacterial cultures increases process carboxylase and are able to transform caffeic acid to un- reliability. Addition ofhigh cell numbers of10 6 cfu (co- desired volatile phenols. lony forming units)/ml are recommended. Present ex- Monomeric anthocyanin concentrations indicate colour periments with must of'Rheinriesling' investigate the stability. Some yeast strains preserve monomeric antho-

114 Mitteilungen Klosterneuburg 53 (2003): 113-122 Berger et al.

cyanins during alcoholic fermentation (BERGER et al., growth was initiated in appropriate medium. 0.5 g of 2000) and are therefore useful for red wine production each dry yeast were cultivated in MRS (Man, Rogosa, (FUSTER et. al., 2002). Sharpe)-liquid medium (10 g peptone, 10 g meat ex- tract, 5 g yeast extract, 20 g glucose, 1 g Tween 80, 2 g Materials and Methods K2HPO4,5gCH3COONa x 3 H2O, 2 g diammonium citrate, 0.2 g MgS04 x7H2O, 0.05 g MnSO4, VWR, USA; adjusted to pH-value 5.4, aqua dest. ad 1000 ml, Malic acid degradation during alcoholic fer- 80 mg/ml cycloheximide, VWR, USA, added to sterili- mentation zed medium) supporting multiplication ofthe species Oenococcus, Lactobacillus and Pediococcus. Cells were st 1 Experiment: Fresh 'Riesling' must (pH-value 3.1): incubated at 28 8C for 72 hours. Numbers of viable Fresh 'Rheinriesling' must (5 l per variant), pH-value cells were determined on Orange-Serum-agar plates 3.1, was inoculated with 10 g/hl offollowingeleven (VWR, USA) supplied with 80 mg/l ofcycloheximide rehydrated commercial dry yeast strains: L1033 , L71 (VWR, USA) to suppress yeast multiplication. B, EC 1118, LRC 272, L2056, RHST (all: Lallemand, Canada), Oenoferm Klosterneuburg, Oenoferm Bou- Malolactic fermentation quet, Oenoferm Tipico, Oenoferm Interdry (all: Erb- sloÈ h-Geisenheim, ), and Fermicru VR5 (Gist Mash from the cultivar 'Blauer Portugieser' was Brocades, Netherlands). fermented with two different yeast strains, L2056 (Lal- Additionally one variant was inoculated with a pure na- lemand, Canada) and Oenoferm Klosterneuburg (Erb- tive selected Saccharomyces cerevisiae culture named sloÈ h Geisenheim, Germany). After three days the spon- ¹Rheinriesling Eisweinª, starting from a single colony taneous mash fermentation variant was inoculated with on Wallerstein-agar plates (VWR, USA), cultivated 10 g/hl of each dry yeast strain. Three different bacteria over night at 28 8C in 250 ml liquid YEP medium (20 starter cultures, Viniflora oenos (Loen. A Chr. Hansen, g peptone, DIFCO, USA; 20 g glucose; 10 g yeast ex- DK,), Bitec pro Vino (Loen. B, ErbsloÈ h Geisenheim, tract, VWR, USA; aqua dest. ad 1000 ml; autoclaved at Germany), and Uvaferm MBR alpha (Loen. C Lalle- 121 8C for 15 min.). After centrifugation at 2500 rpm, mand, Canada) were added to dry young wines. Viable the medium was discarded. The pellet was resuspended cell numbers were determined in wines by plate coun- in pasteurized must and total cell number was adjusted ting on Orange-Serum-agar plates (VWR, USA) as fol- in fresh must to 107 cfu/ml. lows: Viniflora oenos viable cell number 3.2 x 108 cfu/l in wine; Bitec pro Vino 1.3 x 106 cfu/ l in wine; Uva- 2nd Experiment: Pasteurized 'Riesling' must (pH-va- ferm MBR alpha 7.4 x 105 cfu/l in wine. Volumes used lue 3.4): for experiments were 5 l for each variant. Malolactic Each 10 g/hl ofyeast strain L71 B, EC 1118, (all: Lalle- fermentation was initiated at 21 8C and 16 8C and mo- mand, Canada), Oenoferm Bouquet, Oenoferm Tipico nitored by analysis ofmalic acid down to residual and Oenoferm Klosterneuburg (all: ErbsloÈ h-Geisen- amounts ofless than 0.1 g/l. Four weeks afteraccom- heim, Germany) were added to 600 g pasteurized plished alcoholic fermentation and first racking, wines 'Rheinriesling' must (pH-value 3.4). Fermentation la- were kept on fine lees for further four months at cellar sted ten days in both experiments. Malic acid was ana- temperature (138Cto178C). Numbers ofviable bacte- lysed by ionchromatography (Dionex, USA) in untrea- ria were then determined in wines ofall variants using ted must and in wines immediately after alcoholic fer- the plate counting method on Orange-Serum-agar pla- mentation. tes (VWR, USA). Amino acids (BERGER et al., 2000), Usually, number ofviable bacteria cells accompanying monomeric anthocyanins (EDER et al., 1990) and pheno- 5 dry yeast preparations come to numbers less than 10 lic compounds (VRHOVSÏEK et al., 1997) were determined cfu/g of dry yeast compared to Saccharomyces cell by HPLC. Colour intensity was measured using photo- 10 numbers of10 cfu/g of dry yeast (RADLER et al., metrical sample analysis (HP Hewlett Packard 8452A 1985). By direct plate counting, starting from rehydra- diode array spectrophotometer) at wavelengths of420, ted dry yeast or fermenting musts, aerobic or faculta- 520 and 620 nm. tive anaerobic bacteria were not detectable. To promote multiplication ofpotential contaminants bacterial

115 Mitteilungen Klosterneuburg 53 (2003): 113-122 Berger et al.

Results and discussion activity at pH-value 3.1, but 0.6 g/l were reduced at pH-value of3.4. Influence of yeast strain on malic acid degra- With fresh must (pH-value of 3.1), results indicate three dation during alcoholic fermentation groups of yeasts with different degrading levels of malic acid: Influence of pH-value in musts on deacidification 1. strains with high malic acid degrading activity, corre- sponding to 43 % degradation (L71B, LRC 212, Oe- 1st Experiment noferm Klosterneuburg, Oenoferm Bouquet), Variants offreshmust (pH-value 3.1) and pasteurized 2. strains reducing malic acid from 15 % to 35 % (EC must (pH-value 3.4) were fermented with 10 g/hl of 1118, L2056, Oenoferm Interdry, Fermicru VR 5 ) in- eleven rehydrated commercial dry yeast strains as pre- cluding the native selected pure strain Rheinriesling Eis- viously described and a single colony ofyeast strain na- wein, med Rheinriesling Eiswein (RR Eiswein). All strains 3. strains showing very low malic acid degrading acti- but Oenoferm Tipico displayed malic acid degrading vity of0 % to 10 % ( RHST, Oenoferm Tipico). features (Fig. 1). The major part of the initial malic These characteristics will be useful mostly for white wi- acid amount of2.3 g/l in fresh'Rheinriesling' must nes. was reduced and converted to lactic acid by L71B (1 2nd Experiment g/l), by L RC212 and Oenoferm Klosterneuburg (0.6 g/l). Strains RHST and Oenoferm Tipico showed the lo- In pasteurized 'Rheinriesling' must containing an initial west malic acid degrading activity (0.2 g/l and 0 g/l, malic acid amount of1.8 g/l, all strains increased their resp.). Oenoferm Tipico had no malic acid metabolizing deacidifying capacity from 0.6 g/l to 1 g/l of wine. In

g/l 1,2

1

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8 2 T 6 g 1B 1 1 S 5 r t y o 5 in 7 1 2 0 u e r c R e 1 C H 2 b u rd i V L C R R L eu q e ip sw n u t T i E L r o In . E te .B . en R s n en R lo e O O O .K en O

freshmustpH=3,1 pasteur.mustpH=3,4 Fig. 1: Malic acid degradation using ten different commercial dry yeast preparations and a pure culture of a new strain named RR Eiswein (Rheinriesling Eiswein) during alcoholic fermentation of musts with pH-values of3.1 and 3.4

116 Mitteilungen Klosterneuburg 53 (2003): 113-122 Berger et al.

all variants, fermented with L71B, EC 1118, Oenoferm Table 1: Klosterneuburg, and Oenoferm Tipico, 10 % to 20 % Viable bacteria in ten dry wine yeast preparations and more malic acid were metabolized. In wines fermented in liquid culture ofa single colony fromthe strain na- with Oenoferm Tipico, malic acid reduction increased med RR Eiswein after three days incubation in MRS from 0 % to 26 % in case of pH-value 3.4 compared (pH-value 5.4) to pH-value 3.1. These results definitely indicate an in- fluence of the pH-value but also pasteurization on yeast Yeast strain bacteria Cfu per g metabolism concerning malic acid degradation. L1033 5,40E+07 At ph-value of3.4, deacidificationcapacity of Saccharo- myces was shown to increase. In white wines, deacidifi- L71B 2,20E+07 cation varies according to pH-value ofthe must. Com- EC1118 5,90E+07 plete degredation ofmalic acid has not been achieved. LRC212 8,00E+07 Nevertheless deacidifying characteristics have to be ta- RHST 1,20E+08 ken into account with respect to treatments for must L2056 1,80E+08 acidification. Oen. Klosterneuburg 3,90E+03 Hygienic quality of wine yeast Oen. Bouquet 1,00E+00 In fermentation variants using musts with a pH-value Oen. Interdry 1,00E+00 of3.1, spoilage bacteria are not able to multiply to rele- Oen. Tipico 1,00E+00 vant cell numbers under alcoholic fermentation conditi- ons. Therefore viable cells were not analysed. RR Eiswein 0,00E+00 After incubation of an aliquot of rehydrated dry yeast and - for selected Saccharomyces cerevisiae - an over- night culture from single colony, viable bacteria (cfu/g Pure culture Rheinriesling Eiswein (RR Eiswein) was yeast) were chosen to be determined in MRS after 72 free from bacteria. These results - accompanied by the hours incubation (Table 1). MRS was adjusted to pH- fact, that malic acid degradation occured in pasteurized, value 5.4 to support bacteria multiplication. bacteria free must independently of bacterial contami- In experiments with strains L 71B, EC 1118, LRC212, nation grade - indicate, that malic acid degradation to RHST, L2065 and L1033 , bacteria developed cell num- a large extent is due to Saccharomyces cerevisiae capa- bers per gram dry yeast to amounts ofapproximately city. 2x1010 cfu/g to 2 x 1011 cfu/g. Viable cell amounts of 3.9 x 106 cfu/ml after inoculation with Oenoferm Klo- Deacidification using bacteria starter culture sterneuburg and 103 cfu/mg dry yeast were analysed in variants inoculated with Oenoferm Bouquet, Oenoferm Viable bacteria Interdry, Oenoferm Tipico. Malolactic fermentation (MLF) was induced by using Bacteria contamination levels did not correlate with three different starter cultures described above and fur- scales ofmalic acid degradation. Saccharomyces cerevi- ther designated as Loen. A, Loen. B, and Loen. C. Pre- siae L71B caused the highest degradation of1 g/l, but cise application needs attention to level oftotal or free 10 number ofviable bacteria came to 2.5 x 10 cfu/ml, SO2, and the pH-value in fermented must samples as which is low compared to EC 1118, L RC212, RHST, well as bacteria temperature tolerance and kinetics of and L2056. Strain L2056 caused bacteria cell number MLF (FUSTER et al., 2000 und 2002). Number ofviable 1011 cfu/ml, but only 0.4 g malic acid per liter were re- cells developed to 106 cfu/ml in Loen. A variants, to 8 duced in must with a pH-value of3.1. Bacteria cell x105 cfu/ml in Loen. B, and 3 x 105 cfu/ml in Loen. C number in variants inoculated with strains Oenoferm variants. Klosterneuburg, Oenoferm Bouquet, Oenoferm Inter- The initial malic acid amount of2.9 g/l was totally re- dry, and Oenoferm Tipico reached 103 cfu/ml, but malic duced (Table 2) in all variants incubated at 21 8Cor acid was only reduced from 0.4 mg/l to 0.6 mg/l under 16 8C within 19 days and 28 days, respectively. A coo- given conditions. Strain Oenoferm Tipico reduced malic ler temperature of16 8C caused a 5 to 10 days retarda- acid only in must with a pH-value of3.4. tion. MLF kinetics developed differently depending on bacteria culture. Loen. C displayed best MLF velocity,

117 Mitteilungen Klosterneuburg 53 (2003): 113-122 Berger et al.

Table 2: Residual amount of malic acid at various days ofter malolactic fermentation at 16 8C compared to 21 8C

g/l malic acid Microflora day 0 day 2 day 4 day 6 day 17 day 19 Lalvin 2056/L. oen. C 218C 2,9 2,2 1,4 0,2 0,1 Oen. Rouge/L.oen. C 218C 2,9 2,4 1,4 1,1 0,3 0,1 Lalvin 2056/L.oen. B 218C 2,9 2,5 2 1 0,2 0,1 Lalvin 2056/L.oen. A 218C 2,9 2,6 1,9 0,7 0,1 0,1 Lalvin 2056/spont. MLF 218C 2,9 2,9 2,9 2,8 2,5 2,6

02461719 Lalvin 2056L. oen. C 168C 2,9 2,3 2,1 1,6 0,8 0,4 Oen. Rouge/L. oen. C 168C 2,9 2,6 2,4 2,1 1,5 1,3 Oen. Rouge/L. oen. B 168C 2,9 2,7 2,2 1,7 1,1 0,6 Oen. Rouge/L. oen. A 168C 2,9 2,3 2 1,3 0,4 0,2 Oen. Rouge/spont. MLF168C 2,9 2,8 2,6 2,6 2,3 2,3

Influence on wine quality taking six days until accomplishing MLF at 21 8C, and more than three weeks to accomplish at 16 8C. Sponta- neous MLF did not start within the investigated period. The influence of bacteria starter cultures and MLF on Variants fermented with L2056 dry yeast displayed monomeric anthocyanins and colour was evaluated. higher MLF velocity compared to Oenoferm Kloster- Also amino acids and phenols were analysed after four neuburg variants. months ofstorage in contact with finelees and residual After first racking, basically a cell number reducing microflora. Amino acids were released into the wine procedure, wines were stored at cellar temperature of during storage in contact with fine lees (BERGER et al., 13 8Cto158C without sulphiting. To be able to eva- 1999). Amino acids amounts in wines (Fig. 3) were ana- luate storage resistance ofbacteria starter cultures, lysed after MLF. Amounts of released amino acids cor- numbers ofviable cells were determined fourmonths related with yeast strain (BERGER et al., 2000). Changes later (Fig. 2). Regarding initial bacteria inoculation, the ofselected aminoacids lysine, asparagine, tyrosine and viable cell number decreased in all variants during stor- ornithine were detected after four months of storage age. Malolactic fermentations realized at 21 8C, residual and compared to amounts in the original wine. Orni- cell numbers were higher compared to variants fermen- thine and tyrosine were ten times higher after storage. ted at 16 8C. Viable cell number also differed depending In variants fermented with the wine yeast Lalvin 2056, on starter culture. This result was evident in variants in- distinctively higher amounts oftyrosine were found oculated with Loen. A and particularly evident for compared to wines fermented with Oenoferm Rouge. Loen. C. Resistance to vinification conditions is related In case of Saccharomyces cerevisiae late inoculation (af- to bacteria strain characteristics (FUSTER et al., 2001). ter three days ofspontaneous fermentation),the release Spontaneous flora displayed higher resistance after stor- oftyrosine aftera storage period offourmonths was age. Numbers ofviable cells were higher than 5000 cfu/ about 20 % lower compared to fermentation initiated g dry yeast after MLF at 21 8C and MLF at 16 8C. Star- with dry yeast immediately in untreated mashes and ter cultures showed higher sensitivity compared to musts (data not shown). spontaneous flora and could present better preconditi- Choice ofbacteria starter culture had no systematic in- ons for final biological stabilization. fluence on the amino acid amounts in wines. One ofthe major phenolics in grapes and wines is cafta- ric acid (caftaric), a combined molecule of caffeic acid

118 Mitteilungen Klosterneuburg 53 (2003): 113-122 Berger et al.

cfu/ml 5000

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0 Loen.C Loen.C Loen.B Loen.A spontan Loen.C Loen.C Loen.B Leon.A spontan 21 8C 21 8C 21 8C 21 8C 21 8C 16 8C 16 8C 16 8C 16 8C 16 8C

L2056 Oen.R. L2056 L2056 L2056 L2056 Oen.R. Oen.R. Oen.R. Oen.R.

Fig. 2: Viable bacteria (cfu/ml) in 'Blauer Portugieser' after four months' storage

mg/l 80

Asparagine 70 Glutamine 60 Tyrosine Ornithine 50 Lysine

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0 Original L.oen.C Loen.B Loen.A spontan Loen.C Loen.C Loen.C Loen.B Loen.A spontan Wine 21 8C 21 8C 21 8C 21 8C 16 8C 16 8C 21 8C 16 8C 16 8C 16 8C

L2056 L2056 L2056 L2056 L2056 0en.R 0en.R Oen.R Oen.R Oen.R

Fig. 3: Content of selected amino acids in wines of 'Blauer Portugieser' after four months' storage

119 Mitteilungen Klosterneuburg 53 (2003): 113-122 Berger et al.

% 100

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0 Loen.C Loen.C Loen.C Loen.C Loen.B Loen.B Loen.A Loen.A spontan spontan 16 8C 21 8C 21 8C 16 8C 21 8C 16 8C 21 8C 16 8C 21 8C 16 8C

L2056 L2056 Oen.R Oen.R L2056 Oen.R L2056 Oen.R L2056 Oen.R Caftaric Caffeicacid Ethylcaff

Fig. 4: Percentage of caftaric acid and its derivatives in wines of 'Blauer Portugieser' after four months' storage

mg/l 300

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0 L.oenC L.oenC spontan L.oenC L.oenB L.oenB L.oenA L.oenA spontan 16 8C 21 8C 21 8C 16 8C 21 8C 16 8C 21 8C 16 8C 16 8C

L2056 L2056 L2056 Oen.R. L2056 Oen.R. L2056 Oen.R Oen.R

Fig. 5: Monomeric anthocyanins in wines of 'Blauer Portugieser' after four months' storage

120 Mitteilungen Klosterneuburg 53 (2003): 113-122 Berger et al.

mg/l 900

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0 Loen.C Loen.B Loen.A spontan Loen.C Loen.C Loen.C Loen.B Loen.A spontan 21 8C 21 8C 21 8C 21 8C 16 8C 16 8C 21 8C 16 8C 16 8C 16 8C

L2056 L2056 L2056 L2056 L2056 Oen.R Oen.R Oen.R Oen.R Oen.R

Total Phenols mg/l Color

Fig. 6: Total phenols and colour intensity in wines of 'Blauer Portugieser' after four months' storage and tartaric acid. During the process, caf- on must acidity. A pH-value of3.4 supports Saccharo- taric is splitted into caffeic acid and tartaric acid. Ethyl- myces malic acid reduction compared to pH-value 3.1. caffeic acid develops during storage. Present experi- Concerning bacterial contamination found in dry yeast, ments demonstrate a high variability in caffeic acid present investigations disclosed three quality groups of (Fig. 4) and phenols (Fig. 6) released in all variants, but dry yeast: no tendency for an influence of yeast strain, bacteria 1. high contamination level, culture or temperature. Development ofthese molecu- 2. low contamination level, and les might rather be attributed to processing (VRHOVSÏEK 3. dry yeast preparation free or nearly free of bacterial et al., 1997). contaminants. Monomeric anthocyanin amounts analysed after MLF Nevertheless, malic acid degradation intensity was not correlate with microflora (Fig. 5). In MLF variants, the found to correlate with high numbers of viable bacteria. bacterial strain Loen. C and Loen. A had lowest antho- This and the fact, that pure culture of native Saccharo- cyanin reducing properties. Regarding period ofcon- myces isolates also reduces malic acid, demonstrates tact, the influence of the used dry yeast strain seems to Saccharomyces cerevisiae to be responsible for partial be higher than the influence of selected or spontaneous malic acid metabolism. Danger ofpossible uncontrolled bacterial microflora. In variants fermented with L effects due to bacterial contaminants in dry yeast prepa- 2056, amounts ofanthocyanins were slightly higher rations seems attenuated during alcoholic fermentation, than in variants fermented with Oenoferm Rouge. but might be more important in case ofsparkling wine After MLF at a temperature of 21 8C, colour stability production after sugar addition and second fermenta- was higher compared to conditions of16 8C (Fig. 6). tion at temperatures higher than 20 8C. Such physiolo- gical conditions promote contaminants multiplication. Conclusions Bacteria starter cultures showed differences in their biotechnological quality. Malolactic fermentation was Investigated strains ofthe species Saccharomyces cerevi- accomplished at low temperatures (168C) within usual siae display different deacidifying capacities depending time delay. Oenococcus starter cultures showed slight

121 Mitteilungen Klosterneuburg 53 (2003): 113-122 Berger et al.

differences of anthocyanin reducing characteristics, CAI GAO, Y., Z HANG, G., KRENTZ, S., DARIUS, S., POWER, J. and LAGARDE, G. 2002: Inhibition ofspoilage LAB by lyso- whereas Saccharomyces strains displayed distinctive co- zyme during wine alcoholic fermentation. Austral. J. lour-preserving characteristics. Grape Wine Res. (8): 76-83 Furthermore, the use of bacteria starter cultures offers EDER, R., WENDELIN, S. und BARNA, J. 1990: Auftrennung der monomeren Rotweinanthocyane mittels HPLC - Metho- advantages regarding biological stabilization. Inocula- denvergleich und Vorstellung einer neuen Methode. Mitt. ted commercial bacteria strains showed reduced vitality Klosterneuburg 40: 69-75 compared to spontaneous flora. Relative resistance of FUSTER, A., KRIEGER, S. et REJALOT, J. 2001: AmeÂliorer la qualite et la typicite des vins aÁ l'aide de nouveaux outils biologi- spontaneous flora could be responsible for sulphurous ques. Revue FrancËaise d'ênologie (193): 28-31 acid consuming molecule formation. Inhibition of its FUSTER, A. and KRIEGER, S. 2002: ControÃle de la fermentation development can be achieved with early sulphurization malolactique - une approche pratique. Revue FrancËaise d'ênologie (194): 14-17 or by using lysozyme (CAI GAO et al., 2002) in must to RADLER, F., D IETRICH, K. und SCHOÈ NING, I. 1985: Mikrobiologi- reduce undesired bacteria flora at an early stage or in sche PruÈ fung von TrockenhefepraÈparaten fuÈ r die Weinbe- young wines. reitung. Dt. Lebensmittel-Rundsch. 81(3): 73-77 RIBE REAU-GAYON, P., D UBOURDIEU, D., DONEÁ CHE, B. et LON- VAUD, A. (1998): Traite d'ênologie. Tome 1: Microbiolo- References gie du vin : Vinifications. - Paris: Dunod, 1998 VRHOVSÏEK, U., WENDELIN, S. und EDER, R. 1997: Quantitative BERGER, S., SCHOBER, V., K ORNTHEUER, K. und FARDOSSI, A. Bestimmung von HydroxyzimtsaÈuren und Hydroxy- 1999: Einfluss des hefeverwertbaren Stickstoffes auf die zimtsaÈurederivaten (Hydroxycinnamaten) in Weiûwei- GaÈrung in Mosten der Sorten GruÈ ner Veltliner, Rhein- nen mittels HPLC. Mitt. Klosterneuburg 47: 164-172 riesling, Welschriesling, und Neuburger. Mitt. Kloster- neuburg 49: 117-123 BERGER, S., SCHOBER, V., K ORNTHEUER, K., und EDER, R. 2000: Einfluss des Hefestammes und des Weinausbaues am Ge- Received September 4, 2002 laÈger aufden spontanen biologischen SaÈureabbau. Mitt. Klosterneuburg 50: 192-201

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