Insufficient wine acidity can affect wine quality and stability. To overcome this problem, DL-malic acid can be added to the grape juice prior to fermentation. We have investigated the effect of DL-malic acid on wine fermentations and its influence on the final concentration of L-malic acid, naturally present in grape juice. To this end yeast strains that metabolise L-malic acid in different ways were tested and compared; namely, Schizosaccharomyces pombe (efficient L-malic acid degrader), Saccharomyces cerevisiae (non-degrader), hybrid strain S. cerevisiae x S. uvarum (intermediate degrader) and Saccharomyces uvarum (promoting L-malic acid synthesis). In all cases, D-malic acid passively entered the yeast cells and did not undergo malo-alcoholic fermentation. However, its presence in the juice, as a component of the mixture of D- and L- malic acid (DL-malic acid), reduced the amount of L-malic acid that can be degraded or synthesised by yeasts during malo-alcoholic fermentation.Nedovoljna kiselost vina može utjecati na njegovu kakvoću i stabilnost. Da bi se riješio taj problem, može se moštu prije vrenja dodati DL-jabučna kiselina. Ispitan je utjecaj DL-jabučne kiseline na fermentaciju vina i njezin utjecaj na konačnu koncentraciju L jabučne kiseline koja se nalazi u moštu. Stoga su ispitani i uspoređeni sojevi kvasca koji metaboliziraju L-jabučnu kiselinu, i to Schizosaccharomyces pombe (djelotvoran razgrađivač L-jabučne kiseline), S. cerevisiae (ne razgrađuje), hibridni soj S. cerevisiae x S. uvarum (djelomično razgrađuje) i S. uvarum (promotor sinteze jabučne kiseline). U svim slučajevima D-jabučna kiselina pasivno ulazi u stanice kvasca i ne provodi jabučno-alkoholno vrenje. Međutim, njezina prisutnost u grožđanom soku, kao dio smjese D- i L jabučne kiseline (DL-jabučna kiselina) snizuje količinu jabučne kiseline koja se može razgraditi ili sintetizirati s kvascima tijekom jabučno-alkoholnog vrenja

Carlo Zambonelli

Fabio Coloretti

Lorena Castellari

Sandra Rainieri

Vincenzo Tini

English

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UDC 547.476.2:663.252.4 original scientific paperISSN 1330-9862(FTB-1159)The Effect of DL-Malic Acid on the Metabolism of L-MalicAcid during Wine Alcoholic FermentationFabio Coloretti1, Carlo Zambonelli1*, Lorena Castellari2,Vincenzo Tini1 and Sandra Rainieri1,**1Dipartimento di Protezione e Valorizzazione Agroalimentare (DIPROVAL), Università di Bologna, VillaLevi, Reggio Emilia, Italy2Centro di Assistenza Tecnicologica in Enologia e Viticoltura, Tebano, Faenza, Italy**Present address: Department of Biotechnology, Graduate School of Engineering,Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 JapanReceived: June 10, 2002Accepted: November 7, 2002SummaryInsufficient wine acidity can affect wine quality and stability. To overcome this pro-blem, DL-malic acid can be added to the grape juice prior to fermentation. We have investi-gated the effect of DL-malic acid on wine fermentations and its influence on the final con-centration of L-malic acid, naturally present in grape juice. To this end yeast strains thatmetabolise L-malic acid in different ways were tested and compared; namely, Schizosaccha-romyces pombe (efficient L-malic acid degrader), Saccharomyces cerevisiae (non-degrader),hybrid strain S. cerevisiae x S. uvarum (intermediate degrader) and Saccharomyces uvarum(promoting L-malic acid synthesis). In all cases, D-malic acid passively entered the yeastcells and did not undergo malo-alcoholic fermentation. However, its presence in the juice,as a component of the mixture of D- and L- malic acid (DL-malic acid), reduced the amountof L-malic acid that can be degraded or synthesised by yeasts during malo-alcoholic fer-mentation.Key words: D-malic acid; DL-malic acid; L-malic acid; malo-alcoholic fermentation; yeastIntroductionL-malic acid, together with tartaric acid, are themain organic acids of grapes and wine. During grapejuice fermentation yeasts belonging to different speciescan affect the concentration of L-malic acid in differentways. Schizosaccharomyces yeasts can convert L-malicacid to ethanol (malo-alcoholic fermentation) very effi-ciently, using up all the compound available in the me-dium. The key enzyme of this reaction is the malic en-zyme (EC 1.1.1.38). The high substrate affinity of thisenzyme for L-malic acid, as well as the presence of anactive transport system for L-malic acid, are responsiblefor the high metabolisation efficiency of L-malic acid inSchizosaccharomyces yeasts (1,2). Saccharomyces cerevisiaestrains generally do not affect L-malic acid concentration(low metabolisation). Only some selected strains werefound to degrade up to 50 % of the starting concentra-tion of grape juice malic acid (3). The low metabolic effi-ciency of L-malic acid in S. cerevisiae strains is attributedto the low substrate affinity of the S. cerevisiae malic en-zyme as well as to the absence of an active transportsystem for L-malic acid. L-malic acid, in fact, enters thecells of S. cerevisiae strains only by passive diffusion.317F. COLORETTI et al.: Effect of DL-Malic Acid on the Metabolism of L-Malic Acid, Food Technol. Biotechnol. 40 (4) 317–320 (2002)* Corresponding author; E-mail: carlo.zambonelli@libero.itSaccharomyces uvarum strains can synthesise L-malic aciddepending on the starting concentration of the com-pound in grape juice (4). The mechanism of L-malic acidsynthesis in S. uvarum has not yet been elucidated.L-malic acid plays a fundamental role in wine acid-ity. Wines produced in hot countries often lack acidity.Low grape juice acidity affects the body, the colour aswell as the microbial stability of the wine. According tothe legislation of the country where the wine is pro-duced, DL-malic acid can be added to grape juice to in-crease wine acidity. The choice of adding DL-malic acidis mainly driven by economic concern, since L-malicacid is an expensive compound.Since L-malic acid is naturally present in grapes, aswell as in most fruits, research concerning wine acidityhas been focused on this isomeric form and little isknown about the influence of D-malic acid on wine fer-mentations. D-malic acid, in fact, is not found in natureand it can only be chemically synthesised (5).The aim of this work was to establish whether theaddition of DL-malic acid affects the metabolism ofL-malic acid, naturally present in grape juice, duringwine fermentation.Materials and MethodsThe following yeast strains were used: Schizosaccha-romyces pombe strain 4z (Istituto di Microbiologia agra-ria, Università di Firenze, Italy); S. cerevisiae strain DI-PROVAL 6167 (Università di Bologna, Italy); S. uvarumstrain DIPROVAL 12233; S. cerevisiae X S. uvarum inter-specific hybrid strain DIPROVAL 35G2 X 12233. The ef-fect of these strains on the metabolism of L-malic acidhas been described in previous studies (4,6–8).In order to test the activity of the malic enzyme inthe metabolism of L-malic acid or D-malic acid indepen-dently from the presence or absence of a specific trans-port system, the yeast cells were permeabilised usingthe method described by Fuck et al. (9). The test wascarried out in triplicate. Yeast cultures were grown at 25°C in YPD containing DL-malic acid (yeast extract 2 %w/v, peptone 2 % w/v, glucose 10 % w/v, DL-malicacid 1.5 % w/v). After three days they were harvestedand washed in a phosphate-potassium buffer 0.2 M,pH=7.4. The cells were then diluted in the phosphate--potassium buffer to a concentration of 5 mg/mL (dryweight). Two mL of this suspension were then trans-ferred to test tubes and 50 mL of a solution of toluol-ac-etone 1:9 were added (10). The test tubes were mechani-cally shaken to favour permeabilisation. One mL of a 15g/L L-malic or D-malic acid solution was then added togive a final concentration of 5 g/L. This would initiatethe reaction catalysed by the malic enzyme. L- andD-malic acid concentrations were determined immedi-ately after adding L- or D-malic (T0) and then after 30min.Fermentation tests were carried out in triplicate ongrape juice from Vitis vinifera cv. Trebbiano containing0.4 g/L of L-malic acid. 1.5 g/L of L-malic acid or D-ma-lic acid or 3 g/L of DL-malic acid were subsequentlyadded to three separate samples of grape juice. Grapejuice samples were then poured into 200 mL flasks andpasteurised at 60 °C for 30 min. Overnight cultures ofeach strain, grown aerobically in natural grape juice,were used as inoculum at 1 x 106 cell/mL. Flasks wereincubated at 25 °C and the fermentation progress wasmonitored by measuring the weight loss caused by CO2release. The wines obtained were cooled at 4 °C and cla-rified by filtration using porous filters (pore size 0.45µm). L-and D-malic acid and DL-malic acid concentra-tions were measured enzymatically using specific kitsfollowing the instructions of the manufacturer (Boehrin-ger, Mannheim, Germany).Data relating to the malic enzyme activity and tothe malic acid concentration at the end of fermentationwere subjected to statistical analysis using SPSS 6.1 forWinows 3.11 Base System (SPSS Inc., USA).Results and DiscussionThe assessment of the malic enzyme activity inpermeabilised yeast cells was carried out using threeyeasts with different L-malic acid degrading abilities:Schiz. pombe strain 4z (efficient degrader), S. cerevisiaestrain DIPROVAL 6167 (non-degrader) and the interspe-cific hybrid strain DIPROVAL 35G2 X 12233 (intermedi-ate degrader) (Table 1). All the yeast cultures testedgave equal values of degradation for L-malic acid. Thisdemonstrates that the ability of these yeasts to performefficient malo-alcoholic fermentation could be attributedmainly to the presence or absence of a transport system.Schizosaccharomyces pombe is the only yeast, among thetested ones, provided with an active transport systemfor malic acid and also the most efficient malic acid de-grading strain. This yeast, however, is not suitable forvinification because it produces undesirable off-flavours.Recently, recombinant S. cerevisiae wine strains express-ing the Schiz. pombe gene encoding for malate permeasehave been constructed (11,12). These strains can degradeL-malic acid more efficiently than ordinary wine strains,confirming the essential role of an active transporter onmalic acid metabolism.318 F. COLORETTI et al.: Effect of DL-Malic Acid on the Metabolism of L-Malic Acid, Food Technol. Biotechnol. 40 (4) 317–320 (2002)Table 1. Activity of malic acid enzymeYeast cells from three different strains were permeabilised andsuspended in a phosphate-potassium buffer added to the tolue-ne-acetone solution (buffer mix). Five g/L of D-malic or L-malicacid were then added to start the reaction carried out by themalic enzyme. Concentration and percentage variations ofD-malic and L-malic acids were determined in the centrifugedsupernatant of the buffer mix at the moment malic acid wasadded (t0), as well as 30 minutes later. The values represent themean of three independent replications. Within the rows, me-ans followed by the same letter are not significantly different(p<0.01).Yeast straing(L-malic acid)/(g/L) g(D-malic acid)/(g/L)t0 30 min var./% t0 30 min var./%Schiz. pombe 5.00 a 4.06 b –21 4.97 a 4.91 a 0Hybrid 5.00 a 3.98 b –23 4.99 a 4.89 a 0S. cerevisiae 4.98 a 4.14 b –20 4.98 a 4.89 a 0Negative control* 5.00 a 4.98 a 0 4.98 a 4.98 a 0* Buffer mix without addition of yeast culture.var. = variationConsistently with what had already been pointedout by Baranowski and Radler (13), the permeabilisedcells proved to be entirely inactive on D-malic acid (Ta-ble 1).Fermentation tests were carried out on Schiz. pombe,S. cerevisiae and hybrid strains as well as on Sacch.uvarum strain DIPROVAL 12233, able to synthesiseL-malic acid. The addition of L-malic, D-malic orDL-malic acid to grape juice before the beginning of fer-mentation did not affect the fermentation progress forany of the yeast strains employed (results not shown).Strains of S. cerevisiae, S. uvarum and the interspecifichybrid completed the fermentation in seven days in con-trol grape juice as well as in grape juice with addedD-malic, L-malic or DL-malic acid. Schiz. pombe strain didnot complete the fermentation process, leaving a con-centration of unfermented sugars of 10–20 g/L (resultsnot shown).When L-malic acid was added to the grape juice,Schiz. pombe degraded the compound almost to exhaus-tion, S. cerevisiae and the interspecific hybrid degradedthe compound causing a reduction of the starting con-centration of 16 and 42 %, respectively, whereas S.uvarum synthesised the compound increasing its concen-tration (Table 2).When D-malic or DL-malic acid were added to thegrape juice, Schiz. pombe, S. cerevisiae and the hybridstrains showed a drastically reduced ability to degradeL-malic acid. When S. uvarum strain was employed, thesynthesis of L-malic acid was also drastically reduced(Table 2).The results obtained indicate that D-malic acid isable to enter the yeast cell only by passive diffusion andits presence affects the transport system for L-malic acidin Schiz. pombe, resulting in the reduction of the abilityto degrade L-malic acid. In S. cerevisiae and S. uvarumD-malic and L-malic acid enter the cell by passive diffu-sion and seem to compete with one another at metaboliclevel. D-malic acid (not susceptible to malo-alcoholic fer-mentation) presumably accumulates in the cellsreducing the activity of the yeast on L-malic acid. Wesuppose that the same happens when DL-malic acid isadded to the grape juice. According to our findings, thefinal acidity of a wine obtained from grape juice supple-mented with DL-malic acid will not correspond to theaddition of the starting concentration of the naturalL-malic acid of the grape juice and the amount supple-mented as DL-malic acid. The final acidity will be higherthan expected when S. cerevisiae or the hybrid culture S.cerevisiae x S. uvarum are used as starter cultures and lo-wer than expected when S. uvarum is used as starter cul-ture.References1. C. Osothsilp, R. E. Subden, J. Bacteriol. 168 (1986) 1439–1443.2. M. J. Souza, M. Mota, C. Leao, Yeast, 8 (1992) 1025–1031.3. C. Tortia, V. Gerbi, A. Gandini, Vignevini, 20 (1993) 15–20.4. L. Castellari, M. Ferruzzi, A. Magrini, P. Giudici, P.Passarelli, C. Zambonelli, Vitis, 33 (1994) 49–52.5. The Merk Index, 10th edition (1983) Martha Windholz Ed.Merk & Co. Inc., Kahway, NJ, USA.6. W. Balloni, G. Florenzano, Vini d’Italia, 108 (1979) 167–177.7. G. Florenzano, W. Balloni, R. Materassi, Vitis, 16 (1977)38–44.8. S. Rainieri, C. Zambonelli, V. Tini, L. Castellari, P. Giudici,S. Afr. J. Enol. Vitic, 20 (1999) 47–52.9. E. Fuck, G. Stark, F. Radler, Arch. Mikrobiol. 89 (1973) 223–231.10. J. M. Salmon, Biotechnol. Lett. 6 (1984) 43–48.11. C. Camarasa, F. Bidard, M. Bony, P. Barre, S. Dequin,Appl. Environ. Microbiol. 67 (2001) 4144–4151.12. H. Volschenk, M. Viljoen-Bloom, R. E. Subden, H. J. VanVuuren, Yeast, 18 (2001) 963–970.13. K. Baranowski, F. Radler, Antonie van Leeuwenhoek, 50(1984) 329–340.319F. COLORETTI et al.: Effect of DL-Malic Acid on the Metabolism of L-Malic Acid, Food Technol. Biotechnol. 40 (4) 317–320 (2002)Table 2. Concentration and percentage variation of L-malic acid and D-malic acid content of grape juice supplemented with 1.5 g/Lof L-malic acid, 3 g/L of DL-malic acid or 1.5 g/L of D-malic acid and fermented with four different yeast strains. The values rep-resent the mean of three independent replications. Within the columns, means followed by the same letter are not significantly dif-ferent (p<0.01).Grape juice + L-malic acid Grape juice + DL-malic acid Grape juice + D-malic acidg(L-malic acid)/(g/L) g(L-malic acid)/(g/L) g(D-malic acid)/(g/L) g(L-malic acid)/(g/L) g(D-malic acid)/(g/L)var/% var/% var/% var/% var/%Grape juice 1.76 0 1.85 0 1.44 0 0.40 0 1.50 0Schiz. pombe 0.07 a –96 0.63 a –66 1.42 a –1 0.16 a –59 1.47 a –2S. cerevisiae 1.48 b –16 1.66 b –10 1.36 a –6 0.40 b 0 1.20 a –20S. uvarum 3.45 c +96 2.03 c +10 1.33 a –8 0.40 b 0 1.33 a –11Hybrid 0.99 d –42 1.44 b –22 1.22 a –15 0.34 a –13 1.20 –20Utjecaj DL-jabu~ne kiseline na metabolizamL-jabu~ne kiseline tijekom alkoholnog vrenjaSa`etakNedovoljna kiselost vina mo`e utjecati na njegovu kakvo}u i stabilnost. Da bi se ri-je{io taj problem, mo`e se mo{tu prije vrenja dodati DL-jabu~na kiselina. Ispitan je utjecajDL-jabu~ne kiseline na fermentaciju vina i njezin utjecaj na kona~nu koncentraciju L-jabu~nekiseline koja se nalazi u mo{tu. Stoga su ispitani i uspore|eni sojevi kvasca koji metabo-liziraju L-jabu~nu kiselinu, i to Schizosaccharomyces pombe (djelotvoran razgra|iva~L-jabu~ne kiseline), S. cerevisiae (ne razgra|uje), hibridni soj S. cerevisiae x S. uvarum (djelo-mi~no razgra|uje) i S. uvarum (promotor sinteze jabu~ne kiseline). U svim slu~ajevimaD-jabu~na kiselina pasivno ulazi u stanice kvasca i ne provodi jabu~no-alkoholno vrenje.Me|utim, njezina prisutnost u gro`|anom soku, kao dio smjese D- i L-jabu~ne kiseline(DL-jabu~na kiselina) snizuje koli~inu jabu~ne kiseline koja se mo`e razgraditi ili sintetizira-ti s kvascima tijekom jabu~no-alkoholnog vrenja.320 F. COLORETTI et al.: Effect of DL-Malic Acid on the Metabolism of L-Malic Acid, Food Technol. Biotechnol. 40 (4) 317–320 (2002)

Utjecaj DL-jabučne kiseline na metabolizam L-jabučne kiseline tijekom alkoholnog vrenja

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