Degradation of free and sulfur-dioxide-bound acetaldehyde by malolactic lactic acid bacteria in white wine

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1 Journal of Applied Microbiology ISSN ORIGINAL ARTICLE Degradation of free and sulfur-dioxide-bound acetaldehyde by malolactic lactic acid bacteria in white wine J.P. Osborne, A. Dubé Morneau and R. Mira de Orduña Department of Chemistry, The University of Auckland, Auckland, New Zealand Department of Food Science, University of Guelph, Guelph, Ontario, Canada Keywords acetaldehyde, lactic acid bacteria, malolactic fermentation, sulfur dioxide, wine, yeast. Correspondence R. Mira de Orduña, Department of Food Science, University of Guelph, Guelph, Ontario, Canada NG W /0977: received 0 August 005, revised 7 November 005, accepted December 005 doi:0./j x Abstract Aims: Acetaldehyde is the major carbonyl compound formed during winemaking and has implications for sensory and colour qualities of wines as well as for the use of the wine preservative SO. The current work investigated the degradation of acetaldehyde and SO -bound acetaldehyde by two commercial Oenococcus oeni starters in white wine. Methods and Results: Wines were produced by alcoholic fermentation with commercial yeast and adjusted to ph. and.6. While acetaldehyde was degraded rapidly and concurrently with malic acid at both ph values, SO - bound acetaldehyde caused sluggish bacterial growth. Strain differences were small. Conclusions: Efficient degradation of acetaldehyde can be achieved by commercial starters of O. oeni. According to the results, the degradation of acetaldehyde could not be separated from malolactic conversion by oenococci. While this may be desirable in white winemaking, it may be necessary to delay malolactic fermentation (MLF) in order to allow for colour development in red wines. SO -bound acetaldehyde itself maybe responsible for the sluggish or stuck MLF, and thus bound SO should be considered next to free SO in order to evaluate malolactic fermentability. Significance and Impact of the Study: The current study provides new results regarding the metabolism of acetaldehyde and SO -bound acetaldehyde during the MLF in white wine. The information is of significance to the wine industry and may contribute to reducing the concentration of wine preservative SO. Introduction Acetaldehyde is a potent volatile flavour compound that can be found in many foods and beverages, including cheese, yoghurt and wines (Fugelsang 997). Quantitatively, it is the most important carbonyl compound present in wine accounting for 90% of the total aldehydes with levels typically ranging from 0 to 00 mg l ) (McCloskey and Mahaney 98; Romano et al. 994). It is mainly formed as yeast by-product during alcoholic fermentation (AF) (Margalith 98; Romano et al. 994) or through chemical oxidation reactions (Ebeler and Spaulding 999; Liu and Pilone 000). It is a volatile and flavour-active (Nykanen 986) compound with a sensory threshold of approx. 00 mg l ) in table wines and chemically very reactive. For example, acetaldehyde can participate in the formation of flavour-active acetals during the aeration of wines (Ribéreau-Gayon et al. 998) and influence ageing and colour stability of red wines. Specifically, acetaldehyde accelerates polymerization reactions between anthocyanins and phenolics, or between phenolics, leading to condensation products that have a higher colour intensity and stability (Timberlake and Bridle 977; Somers and Wescombe 987; Saucier et al. 997). Acetaldehyde also strongly binds to SO (Somers and Wescombe 98; Romano and Suzzi 99), a 474 Journal compilation ª 006 The Society for Applied Microbiology, Journal of Applied Microbiology 0 (006)

2 compound with antimicrobial and antioxidant properties formed by yeast to varying degrees and commonly added as a preservative in winemaking (Boulton et al. 996). Binding to SO effectively reduces the volatility of acetaldehyde, and thus perception of its aroma in wines. Because the sherry-like aroma of acetaldehyde is undesired in most table wines, and because it has been suggested that acetaldehyde-bound SO has only weak antimicrobial and antioxidant functions (Burroughs and Sparks 97; Romano and Suzzi 99), more SO is generally added to wines containing high acetaldehyde concentrations. However, growing consumer awareness of the adverse health reactions associated with SO (Yang and Purchase 985; Snelten and Schaafsma 99) has prioritized efforts to reduce SO contents of wines. Moreover, acetaldehyde has toxicological relevance itself. It readily binds to proteins (Tuma and Sorrell 985) and DNA (Hemminki and Suni 984) and may cause mutagenesis and carcinogenesis (Dellarco 988). Acetaldehyde formation can be reduced by appropriate yeast strain selection and prevention of oxidation during vinification (Romano et al. 994). However, after AF and removal of the yeast, few alternatives for the reduction of acetaldehyde remain. Wine lactic acid bacteria (LAB) are responsible for malolactic fermentation (MLF), a secondary fermentation leading to wine deacidification, and their possible contribution to acetaldehyde degradation has been indicated early (Somers and Wescombe 987). Nevertheless, little is known about the degradation of acetaldehyde by wine LAB and about its kinetics to date. Recently, it was shown that homo- and heterofermentative wine LAB of the genera Lactobacillus and Oenococcus were able to degrade free and SO -bound acetaldehyde in a wine-mimicking buffer system, whereas strains of the genus Pediococcus did not (Osborne et al. 000). The aim of the current study was to investigate the metabolism of acetaldehyde during MLF in white wine at different ph values and its relation to the degradation of malic acid as well as the degradation of SO -bound acetaldehyde. For this, two commonly applied commercial malolactic Oenococcus oeni starters were tested under practical conditions in white wine. Materials and methods Micro-organisms Saccharomyces cerevisiae var. bayanus strain Red Star Première Cuvée (PC) is available from Lesaffre Yeast Corporation (Milwaukee, WI, USA). The O. oeni strains EQ54 and VFO are commercially available from Lallemand Inc. (Montreal, Quebec, Canada) and Chr. Hansen A/S (Hoershom, Denmark), respectively. Grape must, inoculation procedures, alcoholic and malolactic fermentations Twenty litres of white grape must (Grapetise; Pacific Beverages, Bayswater, Australia) were used as the medium for vinification. The must had a natural soluble solid content of Brix and ph.5. Before induction of AF, the soluble solids were adjusted to 7 Brix with sucrose (Sigma) and the must was sterile filtered through 0.45 lm pore size nylon filters (Whatman, Brentford, UK). Both yeast inoculations in must and bacterial inoculations in the final wine were carried out according to manufacturer s recommendations, i.e. 50 mg l ) of yeast and 0 mg l ) of bacteria. AF was performed at 8 C until dryness was reached (< Brix). After cooling overnight at 4 C, the wine was racked off the yeast lees, and the concentration of l-malic acid was adjusted to 4. g l ). This wine was used without any further adjustments to investigate the degradation of free acetaldehyde. To study the degradation of SO -bound acetaldehyde, a molar excess of SO was calculated from the given acetaldehyde concentration and added to the wines as potassium metabisulfite. After a period of days to allow for complete binding, free SO was removed by nitrogen purging the wine using a -lm pore size HPLC inlet filter. After repeated sterile filtration of all wines, aliquots of l were distributed in l glass bottles for duplicate incubations. Samples were taken periodically, centrifuged (0 000 g for 5 min) and the supernatant was frozen ()8 C) until analysed. Food grade nitrogen was used as inert cover gas during samplings. Analytical methods During fermentations, growth was followed by determining the optical density at 650 nm of samples taken after mixing. Dryness of wines after AF was determined by a chemical colour reaction for reducing sugars (Clinitest; Bayer, Elkhart, IN, USA). Acetaldehyde was quantified enzymatically with a commercial test kit (R-Biopharm, Marshall, MI, USA). SO was measured iodometrically by the Ripper procedure (Amerine and Ough 974). Malic acid was analysed with formic acid as internal standard by ion exchange HPLC (Dionex Summit HPLC System; Dionex Corporation, Sunnyvale, CA, USA). Samples were filtered through 0.-lm nylon filters (Millipore, Bedford, MA, USA) and 0 ll was directly injected. Separations took place on a ID mm Supelcogel H column preceded by a ID mm Supelguard C60H (Supelco, Sigma- Aldrich, St Louis, MO, USA) precolumn with the same filling and a 0.5-lm in-line filter (Upchurch, Oak Harbour, WA, USA). Separations were carried out under isocratic conditions with a 0.% H PO 4 mobile phase (HPLC grade, Journal compilation ª 006 The Society for Applied Microbiology, Journal of Applied Microbiology 0 (006)

3 Sigma) at a 0. ml min ) flow rate. UV detection of organic acids was carried out at 0 nm. Results Figure shows the course of acetaldehyde concentrations during AF by S. cerevisiae Première Cuvée. Acetaldehyde increased rapidly during the exponential growth phase reaching a maximum of 08 mg l ). After several days of acetaldehyde re-utilization during late exponential and early stationary phase, a second increase could be observed. After cold stabilization and racking off the dry wine, it contained 90 mg l ) of acetaldehyde and was used to carry out MLFs. Figure presents results for the degradation of acetaldehyde by O. oeni strain EQ54 at ph. and.6. The degradation of the entire malic acid content and over 90% of the acetaldehyde occurred during the bacterial growth phase. The remaining small acetaldehyde residues observed at ph. after growth cessation were depleted during stationary phase. Degradation of both malic acid and acetaldehyde was faster at the higher ph (.6), and in both cases the metabolism of acetaldehyde was slightly delayed compared with malic acid degradation. In the case of strain VFO (Fig. ), malic acid and acetaldehyde metabolism also occurred during the bacterial growth, and the degradation kinetics were almost congruent. As for strain EQ54, degradation of both malic acid and acetaldehyde by VFO was slightly faster at ph.6 compared with incubations at ph.. Figures 4 and 5 present results of the metabolism of SO -bound acetaldehyde at ph. and.6 by strains EQ54 and VFO, respectively. Binding of the acetaldehyde present to SO as described in the Materials and Methods section led to significant inhibition of the growth of both O. oeni strains at ph. and.6. At ph.6, the expo Figure Growth and degradation of acetaldehyde and malic acid during malolactic fermentation by Oenococcus oeni VFO in white wine at ph Æ and Æ6; acetaldehyde ((), malic acid (n) and optical 4 OD 650 nm x 0 Malic aicd (g l ) Figure Growth and time course of acetaldehyde concentrations during AF by Saccharomyces cerevisiae Première Cuvée; acetaldehyde ((), optical density (s) OD 650 nm Figure 4 Growth and time course of acetaldehyde concentrations during malolactic fermentation by Oenococcus oeni EQ54 in white wine with added SO at ph Æ and Æ6; acetaldehyde ((), optical OD 650 nm x OD 650 nm x 0 Malic aicd (g l ) OD 650 nm x 0 Figure Growth and degradation of acetaldehyde and malic acid during malolactic fermentation by Oenococcus oeni EQ54 in white wine at ph Æ and Æ6; acetaldehyde ((), malic acid (n) and optical Figure 5 Growth and time course of acetaldehyde concentrations during malolactic fermentation by Oenococcus oeni VFO in white wine with added SO at ph Æ and Æ6; acetaldehyde ((), optical 476 Journal compilation ª 006 The Society for Applied Microbiology, Journal of Applied Microbiology 0 (006)

4 nential growth phase of both strains only commenced 8 days after inoculation, while at ph., significant growth was not observed until days after inoculation (both strains). At ph., acetaldehyde concentrations increased over the duration of the incubation (5 days) reaching maximum levels of 50 and 80 mg l ). However, at ph.6 the initial increase of acetaldehyde to similarly high levels was not only halted but also countered at the onset of bacterial growth. Free SO was not detected in any of the incubations with SO -bound acetaldehyde over the duration of the experiment. The acetaldehyde concentration during MLF was also followed at both ph values in an uninoculated control of the same wine. Results are shown in Fig. 6 and demonstrate that the time course of acetaldehyde concentrations at ph. and.6 did not differ considerably. Discussion Figure 6 Course of acetaldehyde concentrations in uninoculated white wine over time at ph Æ (() and Æ6 (s). This work investigated the kinetics of acetaldehyde degradation during MLF, and the effect of SO -bound acetaldehyde on acetaldehyde degradation and bacterial growth. Two common commercial starters of O. oeni were used. In practise, O. oeni is preferred to induce MLF because of its relative resistance to acidity compared with other wine LAB (Henick-Kling 99), and a recent survey showed that all oenococci tested in a wine-mimicking buffer degraded acetaldehyde (Osborne et al. 000). The wine used in this study was obtained through AF of a white grape must. The acetaldehyde production kinetics, both regarding the production during exponential growth phase (Ciani 997) and the re-utilization (Farris et al. 98), as well as the final concentration of acetaldehyde after AF agreed well with the data from the literature (Fleet and Heard 99; Romano et al. 994). In this wine, acetaldehyde was degraded rapidly by the two commercial O. oeni starter strains EQ54 and VFO. Degradation of acetaldehyde was slightly slower at ph. compared with ph.6, and the differences between the strains were equally small. In all incubations, the degradation of acetaldehyde was essentially concurrent with malolactic conversion. This, together with the results from a recent survey showing that all oenococci tested were able to degrade acetaldehyde (Osborne et al. 000), has important consequences for winemaking bearing in mind that the presence of acetaldehyde may be considered advantageous or detrimental depending on wine style and the stage of vinification and ageing. For example, in most white wines, the rapid degradation of acetaldehyde is desirable in order to lower the need for SO, while the rapid reduction of acetaldehyde during MLF in red winemaking may have a negative effect on colour development. Accordingly, in red winemaking it may be beneficial to delay MLF in order to allow acetaldehyde formation and reaction with wine pigments and phenols. MLF may also counter the effects of micro-oxygenation, an area requiring further studies. Malolactic fermentations with SO -bound acetaldehyde were lengthy and led to considerable formation of acetaldehyde. Results from the noninoculated control suggest that the appreciable increase in acetaldehyde concentrations observed were attributable to the chemical oxidation of ethanol to acetaldehyde in the wine (Wildenradt and Singleton 974) caused by oxygenation during samplings, in spite of the usage of nitrogen as cover gas. Growth of both wine LAB was sluggish in all cultivations with SO - bound acetaldehyde, and the ph had an important impact on the ability of wine LAB to degrade acetaldehyde. Within the incubation period, no net reduction of acetaldehyde could be observed at ph. consistent with a study by Fornachon (96) indicating higher sensitivity towards bound SO at low ph values. Several authors have suggested that the inhibitory effect of SO -bound acetaldehyde was due to the bacterial metabolism of the acetaldehyde moiety leading to the release of free SO and subsequent inhibition by the latter (Fornachon 96; Hood 98). However, Mayer et al. (976) have maintained that already SO -bound acetaldehyde itself, and not the SO released from its degradation, would be responsible for bacterial inhibition. In this work, opportunely, the formation of acetaldehyde during incubations efficiently precluded the formation of free SO and, indeed, none could be measured over the duration of the incubations. Thus, it is suggested that in this study, too, SO -bound acetaldehyde itself was responsible for the greatly delayed onset of the growth observed. Nevertheless, SO released from the degradation of SO -bound acetaldehyde may have had an effect on incubations at ph.6 (Figs 4 and 5). It could be noted that the cultures passed from the exponential growth phase directly into the death phase, while in cultivations without added SO Journal compilation ª 006 The Society for Applied Microbiology, Journal of Applied Microbiology 0 (006)

5 (Figs and ), a stable stationary phase was maintained for over a week. Because the acetaldehyde concentrations decreased below the initial values in the incubations containing SO -bound acetaldehyde at ph.6, it is possible that SO was released and triggered the death phase. However, the appearance of free SO could not be substantiated analytically, which could be attributable to the limit of detection of the method applied, or because SO released was bound by other bacterial metabolites, such as diacetyl, as reported by Nielsen and Richelieu (999). In practise, the application of highly concentrated freeze-dried preparations of bacteria (Lonvaud-Funel 999) combined with a better nutrient management has led to faster and more predictable fermentations and wine quality in the previous years. Yet, MLF remains difficult to accomplish in some wines or can be slow. Besides nutritional deficiencies and inhibitors including free SO (Wibowo et al. 988; Vaillant et al. 995), the results presented in this study suggest a possible role of bound SO in the sluggish and stuck MLFs. Thus, bound SO should be considered next to free SO, if malolactic fermentability is to be assessed, especially if wine ph is low. In the dairy industry, LAB have been used to remove acetaldehyde for flavour improvement (Keenan et al. 966). Because of the simultaneous degradation of acetaldehyde and malic acid observed in this study, in white wines, this specific application of LAB would only be desirable where MLF was beneficial, i.e. in low ph wines. However, it may be possible to isolate or adopt starters in the future, which have different degradation kinetics, and could allow specific biodegradation of acetaldehyde without reduction in the malic acid levels. Acknowledgements The authors wish to thank Lallemand Inc. and Chr. Hansen for providing the malolactic strains. References Amerine, M.A. and Ough, C.S. (974) Methods for Analysis of Musts and Wine. New York: Wiley-Interscience Publication. Boulton, R.B., Singleton, V.L., Bisson, L.F. and Kunkee, R.E. (996) Principles and Practices of Winemaking. New York: Chapman & Hall. Burroughs, L.F. and Sparks, A.H. (97) Sulphite-binding power of wines and ciders (Parts I III). J Sci Food Agric 4, Ciani, M. (997) Role, enological properties and potential use non-saccharomyces wine yeasts. Recent Res Dev Microbiol, 7. Dellarco, V.L. (988) A mutagenicity assessment of acetaldehyde. Mutat Res 95, 0. Ebeler, S.E. and Spaulding, R.S. (999) Characterization and measurement of aldehydes in wine. In Chemistry of Wine Flavor ed. Waterhouse, A.L. and Ebeler, S.E. pp Washington, DC: American Chemical Society/Oxford University Press. Farris, G.A., Fatichenti, F., Deiana, P. and Madau, G. (98) Functional selection of low sulphur dioxide acceptor producers among 0 S. cerevisiae strains. J Ferment Technol 6, Fleet, G.H. and Heard, G. (99) Malolactic fermentation. In Wine Microbiology and Biotechnology ed. Fleet, G.H. pp Chur, CH: Harwood Academic Publishers. Fornachon, J.C.M. (96) Inhibition of certain lactic acid bacteria by free and bound sulphur dioxide. J Sci Food Agric 4, Fugelsang, K.C. (997) Wine Microbiology. New York: Chapman & Hall. Hemminki, K. and Suni, R. (984) Sites of reaction of glutaraldehyde and acetaldehyde with nucleosides. Arch Toxicol 55, Henick-Kling, T. (99) Malolactic fermentation. In Wine Microbiology and Biotechnology ed. Fleet, G.H. pp Chur, CH: Harwood Academic Publishers. Hood, A. (98) Inhibition of growth of wine lactic-acid bacteria by acetaldehyde-bound sulphur dioxide. Aust Grapegrow Winemak, 4 4. Keenan, T.W., Lindsay, R.C. and Day, E.A. (966) Acetaldehyde utilisation by Leuconostoc species. Appl Microbiol 4, Liu, S.-Q. and Pilone, G.J. (000) An overwiew of formation and roles of acetaldehyde in winemaking with emphasis on microbiological implications. Int J Food Sci Technol 5, Lonvaud-Funel, A. (999) Lactic acid bacteria in the quality improvement and depreciation of wine. Antonie Van Leeuwenhoek 76, 7. Margalith, P.Z. (98) Flavour Microbiology. Springfield, IL: Charles C. Thomas Publishers. Mayer, K., Pause, G. and Vetsch, U. (976) Gehalte an SO - bindenden Stoffen in Wein: Einfluss von Gärung und biologischem Säureabbau. Schweiz Z Obst Weinb, 09. McCloskey, L.P. and Mahaney, P. (98) An enzymatic assay for acetaldehyde in grape juice and wine. Am J Enol Vitic, Nielsen, J.-C. and Richelieu, M. (999) Control of flavour development in wine during and after malolactic fermentation by Oenococcus oeni. Appl Environ Microbiol 65, Nykanen, L. (986) Formation and occurrence of flavor compounds in wine and distilled alcoholic beverages. Am J Enol Vitic 7, Osborne, J.P., Mira de Orduña, R., Liu, S.-Q. and Pilone, G.J. (000) Acetaldehyde metabolism by wine lactic acid bacteria. FEMS Microbiol Lett 9, Journal compilation ª 006 The Society for Applied Microbiology, Journal of Applied Microbiology 0 (006)

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