End Of Malolactic Fermentation Research Articles

Exploring Metschnikowia pulcherrima as a Co-Fermenter with Saccharomyces cerevisiae: Influence on Wine Aroma during Fermentation and Ageing

Non-Saccharomyces yeasts, particularly Metschnikowia pulcherrima, are considered alternatives to SO2 in winemaking, combating specific microorganisms. The sensory profile of the wine is contingent upon the type of yeast, the fermentation conditions, and the concentration and mode of application with Saccharomyces cerevisiae strains (whether pure or used in mixed/sequential co-fermentation). This study assessed the aroma in red wines produced with S. cerevisiae (Sc) and M. pulcherrima (Mp, non-Sc), incorporating variations in the method of addition and the inclusion or exclusion of SO2. The enological parameters of the wines were slightly affected. Volatile compounds were analysed in the wines through gas chromatography–mass spectrometry (GC-MS) at three moments: at the end of malolactic fermentation (MLF) and after 6 and 9 months of bottle ageing. Sequential fermentation of Sc and Mp reduced the concentration of most identified alcohols and acids, which is favourable, as these compounds can yield undesirable aromas at high concentrations. Regardless of the yeast mixture and Mp dose, a majority of the acetate esters and ethyl esters were quantified at concentrations above their perception thresholds, thus enhancing the sensory quality of the wines. Sensory analysis of wines showed generally positive evaluations. Using non-Saccharomyces as an alternative to SO2 improves the aromatic profile of wines.

Evolution in the Bottling of Cabernet Sauvignon Wines Macerated with Their Own Toasted Vine-Shoots.

This work studies, for the first time, the effect of the use of Cabernet Sauvignon vine-shoots as an enological additive (called "Shoot Enological Granule", SEG) in wines of the same variety. SEGs were added in two doses (12 and 24 g/L) at the end of malolactic fermentation, and after that, wines were bottled for six months. The phenolic and volatile composition and sensory profiles of wines were analyzed at bottling and after six months. The results showed a decrease in the total content of phenolic compounds with bottle time; however, stilbenes─specifically trans-resveratrol─were maintained at significant levels in SEG wines. In contrast, the total content of volatile compounds, mainly esters, increased with bottle aging. Finally, in terms of sensory profile, SEG wines showed a clear differentiation between the descriptors and the control, with more-integrated aromas after bottle time with more toasted, nutty vanilla notes, as well as silkier and less bitter tannins, compared to the control.

Wine production from ripen pond apple (Annona glabra L.) fruit

Pond apple (Annona glabra L.) trees were widely distributed in swamp regions of Mekong Delta, Vietnam. Pond apple fruits turned from green to yellow when ripening. Ripen pond apple fruits contained numerous phenolic constituents with valuable phytochemical benefits. However, ripen pond apple fruits were not successfully utilized as other commercial fruits. This research examined the possibility of wine production utilized from ripen pond apple fruits. Different various technical variables of fermentation affecting to the quality of pond apple wine were thoroughly examined. Ripen pond apple fruits were naturally collected from Soc Trang province, Vietnam. Ripen pond apple fruits were peeled, blended, deseeded, crushed, enzyme-treated (pectinase 25 mg/L), added with sugar (5-13% w/w), pasteurized (sulphite 30 mg/L), inoculated with yeast Saccharomyces pastorianus ratio (0.1-0.5%), macerated temperature (14-22oC) in different time (6-14 days). Malolactic fermentation was performed in anaerobic condition at 12oC in different durations (4-20 weeks). At the end of malolactic fermentation, wine was racked and clarified with different fining agents (bentonite, polyvinylpyrrolidone, wheat gluten, gelatin, kaolin) at 0.03% (v/v). Results showed that must should be added with 9% sugar and 0.4% yeast inoculation, fermentation temperature of 16oC in 10 days. Malolactic fermentation could be terminated at 12 weeks. Gelatin revealed the best candidate among different clarifying agents to remove turbidity in pond apple wine while retaining the most total phenolic content and antioxidant capacity. Under above technical variable conditions, fermentation gave the high ethanol content (4.26±0.02 % v/v); the total phenolic content (32.79±0.00 mg GAE/100 ml), 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging (11.84±0.01 %), overall acceptance (8.34±0.01 score) meanwhile low turbidity (24.41±0.00 NTU) was also noticed. High ethanol content and phytochemical retention contributed to the high sensory score of pond apple wine. These quality parameters were acceptable for an alcoholic drink. Ripen pond apple fruit would be a promising carbohydrate source to convert into a new fruit wine with a pleasant alcoholic flavor and attractive appearance.

Comparison of water addition and early‐harvest strategies to decrease alcohol concentration inVitis viniferacv. Shiraz wine: impact on wine phenolics, tannin composition and colour properties

Background and Aims Regulations in some wine‐producing countries allow water addition to grape must. The research presented compared the impact of water addition to must on wine phenolics and colour relative to that of wines at the same alcohol concentration, prepared from fruit harvested earlier. Methods and Results Shiraz grapes were harvested at three stages; earlier harvest at 13.4 and 14.4°Be (H‐1, H‐2) and a later harvest at 15.6°Be (H‐3). Water was then added to H‐3 must to match the sugar concentration of H‐1 and H‐2, respectively. Two modes of water addition were followed: (i) water was directly added to the must; or (ii) juice was substituted for water. Both water addition treatments significantly decreased the concentration of phenolics throughout the winemaking process. At the end of malolactic fermentation, the concentration of phenolics, tannin and non‐bleachable pigments, tannin molecular mass and wine colour density were lower in the water addition treatments compared with that of H‐3, but in all other compositional aspects were higher than that of H‐1 and H‐2. Interestingly, where the extent of alcohol reduction by water addition was equivalent, no difference in any of the measured wine attributes was found between the two modes of water addition. Conclusions Water addition to the H‐3 treatment led to higher wine tannin, colour and non‐bleachable pigment than were found in wines made from grapes harvested at a lower sugar concentration. Significance of the Study The study presents a meaningful comparison of potential outcomes in Shiraz wine phenolics and colour for producers who aim to lower alcohol using the approaches of either water addition or early harvest.

ATR-MIR spectroscopy and multivariate analysis in alcoholic fermentation monitoring and lactic acid bacteria spoilage detection

Wine production processes still rely on post-production evaluation and off-site laboratory analyses to ensure the quality of the final product. Here we propose an at-line methodology that combines a portable ATR-MIR spectrometer and multivariate analysis to control the alcoholic fermentation process and to detect wine fermentation problems. In total, 36 microvinifications were conducted, 14 in normal fermentation conditions (NFC) and 22 intentionally contaminated fermentations (ICF) with different lactic acid bacteria (LAB) concentrations. ATR-MIR measurements were collected during alcoholic and malolactic fermentations and relative density, pH, and l-malic acid were analyzed by traditional methods. Partial Least Squares Regression could suitably predict density and pH in fermenting samples (root mean squared errors of prediction of 0.0014 g mL−1 and 0.06 respectively). With regard to ICF, LAB contamination was detected by multivariate discriminant analysis when the difference in l-malic acid concentration between NFC and ICF was in the order of 0.7–0.8 g L−1, before the end of malolactic fermentation. This methodology shows great potential as a fast and simple at-line analysis tool for detecting fermentation problems at an early stage.

Magnetic immunochromatographic test for histamine detection in wine

Histamine, a biogenic amine, is abundant in fermented foods and beverages, notably wine. A high intake of this monoamine may produce adverse reactions in humans, which may be severe in individuals with a reduced capacity to catabolise extrinsic histamine. Thus, control of histamine concentration during wine production and before distribution is advisable. Simple, rapid, point-of-use bioanalytical platforms are needed because traditional methods for the detection and quantification of histamine are expensive and time-consuming. This work applies the lateral flow immunoassay technique to histamine detection. Superparamagnetic particle labels, and an inductive sensor designed to read the test line in the immunoassay, enable magnetic quantification of the molecule. The system is calibrated with histamine standards in the interval of interest for wine production. A commercial optical strip reader is used for comparison measurements. The lateral flow system has a limit of detection of 1.2 and 1.5mg/L for the inductive and optical readers, respectively. The capability of the inductive system for histamine quantification is demonstrated for wine samples at different processing points (at the end of alcoholic fermentation, at the end of malolactic fermentation, in freshly bottled wine, and in reserve wine). The results are validated by ultra-high-performance liquid chromatography. Graphical abstract.

Pre-fermentative cold maceration in the presence of non-Saccharomyces strains: effect on fermentation behaviour and volatile composition of a red wine

Background and Aims This study evaluated the impact of pre-fermentative cold maceration (PCM), in the presence of two non-Saccharomyces yeasts, Metschnikowia pulcherrima MP 346 and Metschnikowia fructicola MF 98-3, and of a commercial pectic enzyme, on fermentation kinetics and on the volatile composition of a Sangiovese red wine. Methods and Results Sangiovese grape must was inoculated with MP 346 or MF 98-3 or treated with a pectic enzyme preparation during PCM, at 5°C for 24 or 72 h. A Control wine was produced by a pure culture of Saccharomyces cerevisiae. Both non-Saccharomyces strains affected the initial yeast population dynamics and the persistence of S. cerevisiae at the end of malolactic fermentation. Irrespective of the duration of PCM, the inoculum of Metschnikowia strains did not influence the rate of sugar consumption or the kinetics of malolactic fermentation. The volatile composition of the final wines was evaluated with solid-phase extraction, followed by GC/MS. The concentration of some terpenes and C13-norisoprenoids, nerol, geraniol, 8-hydroxy-linalool (cis) and 3-oxo-α-ionol, and of some esters, isoamyl lactate and ethyl isoamyl succinate, was higher in wines inoculated with Metschnikowia strains than in the Control and wine treated with pectic enzyme. Conclusions Metschnikowia yeast strains MP 346 and MF 98-3 affect wine volatile composition. Significance of the Study This study shows for the first time that an inoculum of Metschnikowia strains MP 346 and MF 98-3 during PCM is effective in modulating the volatile composition of a Sangiovese red wine.

Influence of glycosides on behavior of Oenococcus oeni in wine conditions: growth, substrates and aroma compounds.

Autochthonous Oenococcus oeni strains (MS9, MS20 and MS46) with good malolactic performance and yielding adequate diacetyl levels, were selected to investigate the effect of synthetic and grape glycosides on bacterial growth, substrate utilization and β-glucosidase (βGlu), α-arabinofuranosidase (αAra) and α-rhamnopyranosidase (αRha) activities in a wine-like medium containing 6% ethanol, pH 4.0 (WBM). Then, changes in the volatile compounds profile were evaluated at the end of malolactic fermentation (MLF) carried out by the MS46 strain in WBM containing 1mgL-1 of natural glycoside. All strains grew and efficiently degraded L-malic acid in WBM where βGlu and αAra activities were found but not αRha. In presence of a synthetic glycoside (eriodictyol 7-O-β-rutinoside) βGlu activity was significantly enhanced for two of the cultures tested (MS20 and MS460) while a low αRha activity was induced, presenting MS46 the better performance. Glycosides extracted from fermented grape musts under different conditions allowed maximum growths, L-malic acid utilization rates and glycosidase activities in the MS46 strain. Thus, βGlu, αAra and αRha activities increased between 30-50 and 3-11% respectively. This indirectly correlated to significant changes in total esters and higher alcohols at the end of MLF, which increased by up to 140 and 30% respectively. Moreover, ethyl and acetate esters formed up to 100-fold than alcohols or esters degraded highlighted the main role of this microorganism in the esters synthesis. Results obtained encourage the potential use of selected indigenous O. oeni strains as a tool to enhance wine complexity through MLF, mainly on highly fruity aroma.

The impacts of temperature, alcoholic degree and amino acids content on biogenic amines and their precursor amino acids content in red wine

The aim was to study how factors such as temperature, alcoholic degree, and amino acids supplementation are able to influence the content of tyramine, histamine, 2-phenylethylamine, tryptamine and their precursor amino acids in winemaking process. Biogenic amines and amino acids were quantified at the beginning, middle and end of alcoholic fermentation, and at the end of malolactic fermentation. In general, samples produced with amino acid supplementation did not show the highest concentrations of biogenic amines, except for histamine, which content increased with the addition of the four amino acids. The synthesis of tyramine was mainly affected by the temperature and alcoholic degree, the formation of phenylethylamine was largely influenced by alcoholic degree, and tryptamine synthesis principally depended on temperature. Interestingly, there was interaction between these three factors for the biogenic amines studied. In conclusion, winemaking conditions should be established depending on the biogenic amine which synthesis is required to be controlled.

Factors influencing the formation of histaminol, hydroxytyrosol, tyrosol, and tryptophol in wine: Temperature, alcoholic degree, and amino acids concentration

The validation of a HPLC–PDA–MS/MS chromatographic method for the quali/quantitative characterization of histaminol, hydroxytyrosol, tyrosol, and tryptophol in wine has been described and discussed. Four standards showed a good linearity with high correlation coefficient values (over 0.9989) and LOD and LOQ were 0.001–0.015mg/L and 0.004–0.045mg/L, respectively. Furthermore, this study reported how factors such as temperature, alcoholic degree, and amino acids concentration are able to influence the formation of these four alcohols in Monastrell wines. The quantification values of these alcohols has been detected both at the half and end of alcoholic fermentation, and at the end of malolactic fermentation. In relation to interactions between factors, several significant variations emerged (p⩽0.001). The impact of amino acids supplementation in Monastrell must it has been demonstrated, mainly in regards to histaminol and tryptophol.

Growth and volatile phenol production by Brettanomyces bruxellensis in different grapevine varieties during fermentation and in finished wine

Three red grapevine varieties, Touriga Nacional, Syrah and Cabernet Sauvignon, were evaluated for their susceptibility to spoilage by two strains of Brettanomyces bruxellensis during grape juice fermentation and in finished wine. B. bruxellensis ISA 2211 survived well during both alcoholic and malolactic fermentations. The 4-ethylphenol concentration at the end of malolactic fermentation was 176 µg L−1 for T. Nacional, 190 µg L−1 for C. Sauvignon and 149 µg L−1 for Syrah. These levels were slightly increased during 3 months of incubation, even though no culturable cells of B. bruxellensis could be detected after malolactic fermentation. However, a viable but non-culturable (VBNC) population of 3–4 log cells mL−1 was observed in all grape varieties when analyzed by flow cytometry. On the other hand, commercial varietal wines, inoculated with of B. bruxellensis ISA 1791, could attain levels of 6–8 log CFU mL−1 and 4-ethylphenol reached 1750, 1512 and 2707 µg L−1 in T. Nacional, C. Sauvignon and Syrah, respectively. Overall, the production of 4-ethylphenol was mostly dependent on the existence of growing B. bruxellensis populations, regardless of the grape variety. In finished wines, 4-ethylphenol was always produced in concentrations far above its preference threshold, indicating that T. Nacional, Syrah and C. Sauvignon are equally susceptible to the “horse sweat” taint.

PCR-based DGGE fingerprinting and identification of the microbial population in South African red grape must and wine

<p style="text-align: justify;"><strong>Aim</strong>: The aim of this study was to evaluate the microbial population present in red grape must and wine using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE).</p><p style="text-align: justify;"><strong>Methods and results</strong>: Red wine from the cultivars Pinotage and Merlot were produced and samples taken throughout alcoholic and malolactic fermentation (MLF). PCR fragments were resolved by DGGE and unique fingerprints were obtained for the bacteria and yeasts present in the wines. <em>Lactobacillus plantarum</em>, <em>Enterobacter</em> <em>sakazakii</em> (<em>Cronobacter</em> sp.) and <em>Pantoea agglomerans</em> were present in the Pinotage during both alcoholic and MLF, and in both inoculated and spontaneous fermentations. <em>E. sakazakii</em> (<em>Cronobacter</em> sp.) and <em>P. agglomerans</em> were also observed in the Merlot wines during alcoholic fermentation as well as MLF. <em>Saccharomyces cerevisiae</em> was the most dominant yeast observed in Pinotage, and was the only yeast observed in Merlot. This yeast was observed until the end of MLF.</p><p style="text-align: justify;"><strong>Conclusions</strong>: Results showed that the microbial flora that participates in the winemaking process is more diverse than commonly thought.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: This method may serve as an alternative to conventional microbiological methods for the identification of the microbial species in red grape must and wine.</p>

Effect of the Prefermentative Addition of Five Enological Tannins on Anthocyanins and Color in Red Wines

The effects of prefermentation addition of 5 exogenous tannins with different-origin anthocyanins and color characteristics were investigated in "Cabernet Sauvignon wines" at the end of alcoholic fermentation and the end of malolactic fermentation, and after 6 mo and 9 mo of bottle aging, respectively. The results showed that the application of GSKT2 could significantly retard the degradation of most anthocyanins in the process of alcoholic fermentation and the decrease of some pyranoanthocyanins during the subsequent 3 stages, thus causing more yellowness of wine in comparison with the control. Three other condensed tannins, GSKT1, QUET, and GSET, had a positive impact only on several anthocyanin components. Four condensed tannins all contributed to more redness, suggesting that the action mechanism might be to protect wine against oxidation or contribute to form copigmented anthocyanidins, or polymeric pigments. The application of FOLT (hydrolysable tannin) did not produce any influence on wine redness even after 9 mo of bottle aging. This work provides some reasons for the reasonable application of tannin additives. The prefermentative application of condensed tannins overall could protect some pigment components from degradation and enhance wine redness. Tannin additives with different origins have different effectiveness. The tannin additive obtained from grape skins, like GSKT2, could produce significant promotion on both redness and yellowness in wine. The prefermentation addition of hydroxylase tannin like FOLT seems not to have a significant effect on wine color.

Multivariate analysis of the polyphenol composition of Tempranillo and Graciano red wines

Vitis vinifera L. cv Graciano is often used as a blending partner of Tempranillo based wines because it is considered to contribute significantly to the quality. The aim of this study is to discriminate between Tempranillo and Graciano monovarietal wines, and those made by the incorporation of 20% of Graciano variety in two different stages (at the end of malolactic fermentation and mixing the two grape varieties in the pre-fermentative maceration stage) of the winemaking process of the Tempranillo variety. To achieve this, supervised and unsupervised pattern recognition tools were applied to the data obtained in the study of the detailed polyphenolic composition, colour and other oenological parameters (143 variables). Patterns related to stages in the winemaking and ageing process, different wines and vintages can be observed using principal component analyses. Furthermore, linear discriminant analysis has been applied in order to characterise the wine samples. From the 143 variables, flavan-3-ols have exerted a profound influence on wine differentiation.

Ethyl carbamate content in wines with malolactic fermentation induced at different points in the vinification process

Ethyl carbamate (EC) is a carcinogenic compound found in fermented food and beverages such as wine. Although its carcinogenic potential in animals is known, information regarding its effects in humans remains insufficient, thus there is increasing interest in its research. EC content is higher in products with high alcohol content and in aged products. The main precursor involved in EC production in wine is urea, which is produced by metabolism of arginine by yeast, but there is also evidence that EC levels can increase after malolactic fermentation (MLF). Some lactic acid bacteria (LAB) can degrade the arginine present in must and wine via the arginine deiminase pathway, producing citrulline and carbamyl phosphate. Both compounds can react with ethanol in acidic conditions and produce EC. Our research group is studying the influence of MLF induced at different points of wine-making on the quality of the resulting wine. Among other parameters, the content of toxic compounds such as EC was evaluated. Results so far indicate that EC levels at the end of MLF were quite low (less than 3 μg/l) in all cases, i.e. below the existing legal limit (e.g. 30 μg/l in Canada). In almost all wines, EC concentrations increased after 8 months of storage as has been described by other authors. In some of the wines in which MLF was carried out by selected LAB, the increase in EC concentration was lower.

Biogenic amine production by Oenococcus oeni during malolactic fermentation of wines obtained using different strains of Saccharomyces cerevisiae

Abstract Twenty-six wild Oenococcus oeni strains were investigated for their ability to form biogenic amines during malolactic fermentation in synthetic medium and in wine. Eight strains produced histamine and tyramine in screening broth at concentrations of 2.6–5.6 mg/L and 1.2–5.3 mg/L, respectively. Based on their ability to form biogenic amines, five strains were selected to inoculate three wines obtained by the fermentation of three different Saccharomyces cerevisiae strains (A, B, and C). All bacterial strains could perform malolactic fermentation for short periods in wine C, whereas only one strain performed complete malolactic fermentation in wines A and B. Two O. oeni strains (261 and 351) produced histamine and tyramine in wine C. Time-course analysis of these compounds showed that for both strains, histamine and tyramine production began at day 10 and finished on day 25, after the end of malolactic fermentation. These results indicate that the ability of O. oeni to produce histamine and tyramine is dependent on the bacterial strain and on the wine composition, which in turn depends on the yeast strain used for fermentation, and on the length of bacteria–yeast contact time after the completion of malolactic fermentation.

Evolution of red wine anthocyanins during malolactic fermentation, postfermentative treatments and ageing with lees

A comparative study was conducted on nine batches of wine, from the same initial wine, subjected to malolactic fermentation and ageing in barrels, under different technological conditions: Malolactic fermentation in barrel or in tank, with or without wine clarification, ageing with or without lees and stirring or no stirring of the lees. Samples were taken of the initial wine, of the wine at the end of malolactic fermentation, of the wines after clarifying treatments, and after 3, 6, 9, 12 and 14 months of ageing in the barrel, making a total of 48 wines. As a result of the anthocyanin analysis of all the wines studied, a total of 21 different anthocyanin compounds were detected, which can be classified into four groups: simple glucosides, acetyl glucosides, cinnamoyl glucosides and pyroanthocyanins. During MLF, it was shown that the effect of the container used seems to be more important than the metabolic activity of the bacteria responsible for the process. From application of the LSD test, significant differences were found in the concentrations of all the anthocyanin compounds identified due to ageing time and significant differences were also revealed for most anthocyanin compounds in relation to the manufacturing method, especially the presence or absence of lees.

Quantificação de 5-metiltetrahidrofolato no processamento e conservação de vinhos de uvas viníferas e híbridas

Folates are a class of vitamins that have been rediscovered in recent decades because of their association with various metabolic processes in the human organism. The main sources of folates described the scientific literature are vegetables, cereals and yeasts. The purpose of this study was to quantify 5-methyltetrahydrofolate (5-methyl-THF) in wines during wine production and aging. Red wines made from Cabernet Sauvignon, Merlot, and Pinot Noir wine grape varieties, and from Isabel (red) and Niagara (white) non-wine grape varieties were analyzed. The samples were analyzed at the beginning of fermentation, at the end of alcoholic and malolactic fermentation, and periodically at 4, 7, 12 and 24 months of age. High Performance Liquid Cromatography was used to separate, identify and quantify the folates. The results showed that the levels of 5-methyl-THF increased considerably during fermentation and continued to increase until the end of malolactic fermentation. The levels remained stable over a period of 3 to 6 months in the white wines and for 1 to 2 years in red wines, depending on the variety. The red wines proved to be a good source of 5-methyl-THF.

Determination of phenolic compounds in wines: Influence of bottle storage of young red wines on their evolution

Various analytical methods based on the HPLC-DAD technique were used to determine 38 phenolic compounds in red wines. While anthocyanins and hydroxycinnamic acids were determined by direct injection of wine samples, hydroxybenzoic acids, catechins, procyanidins and flavonols required an analytical pretreatment involving liquid–liquid partitioning with ethyl ether followed by solid-phase extraction on C 18 mini-columns. The proposed analytical methods were used to establish the phenol composition of Mencía and Brancellao, two varietal young red wines, and its influence on colour stability during storage in bottles for one year. At the end of malolactic fermentation, Mencía wine was found to contain much greater amounts of anthocyan pigments than was Brancellao wine. This resulted in a higher colour density but a weaker hue in Mencía wine than in Brancellao wine. Phenolic compounds evolved similarly in both wines during storage; changes in such compounds involved a decrease in the levels of monomeric anthocyanins, phenolic acids, epicatechin and flavonols, and an increase in those of procyanidins. The absence of a relationship between the changes in colour density and monomeric anthocyanins in both wines suggests that copigmentation and polymerization with other phenolic compounds (viz. phenolic acids, catechins and/or flavonols) prevail over degradation of the pigments.

Influence of malolactic fermentation, postfermentative treatments and ageing with lees on nitrogen compounds of red wines

A comparative study was conducted on nine batches of wine taken from the same initial wine, subjected to malolactic fermentation and ageing in barrels, under different technological conditions: malolactic fermentation in barrel or in tank, with or without wine clarification, ageing with or without lees, and stirring or no stirring of the lees. Samples were taken of the initial wine, of the wine at the end of malolactic fermentation, of the wines after clarifying treatments, and after 3, 6, 9, 12 and 14 months of ageing in the barrel, making a total of 51 wines. Only a very small decrease in amino acids was observed during malolactic fermentation, probably due to the wine releasing amino acids produced by the exocellular proteasic activity of the lactic acid bacteria. Ageing of the wine with lees modifies its nitrogen composition because amino acids are released by yeast and bacteria autolysis. The amount of amino acids released was greatest in the wines stirred weekly. All of the wines studied contained low concentrations of biogenic amines.