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Volatile thiols, such as 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), are among the most potent aroma compounds found in wine and can have a significant effect on wine quality and consumer preferences. At optimal concentrations in wine, these compounds impart flavours of passionfruit, grapefruit, gooseberry, blackcurrant, lychee, guava and box hedge. The enzymatic release of aromatic thiols from grape-derived, non-volatile cysteinylated precursors (Cys-4MMP and Cys-3MH) and the further modification thereof (conversion of 3MH into 3MHA) during fermentation, enhance the varietal characters of wines such as Sauvignon Blanc. Wine yeast strains have limited and varying capacities to produce aroma-enhancing thiols from their non-volatile counterparts in grape juice. Even under optimal fermentation conditions, the most efficient thiol-releasing Saccharomyces cerevisiae wine strain known realizes less than 5% of the thiol-related flavour potential of grape juice. The objective of this study was to develop a wine yeast able to unleash the untapped thiol aromas in grape juice during winemaking. To achieve this goal, the Escherichia coli tnaA gene, encoding a tryptophanase with strong cysteine-beta-lyase activity, was cloned and overexpressed in a commercial wine yeast strain under the control of the regulatory sequences of the yeast phosphoglycerate kinase I gene (PGK1). This modified strain expressing carbon-sulphur lyase activity released up to 25 times more 4MMP and 3MH in model ferments than the control host strain. Wines produced with the engineered strain displayed an intense passionfruit aroma. This yeast offers the potential to enhance the varietal aromas of wines to predetermined market specifications.

Protein haze formation in white wine is dependent on the presence of both wine protein and other unknown wine components, termed factor(s) X. The ability to reconstitute protein haze upon heating artificial model wine solutions (500 mg/L thaumatin, 12% ethanol, 4 g/L tartaric acid) to which candidate components were added was employed to identify factor(s) X. No protein haze was formed in the absence of additives. The individual or combined addition of caffeic acid, caftaric acid, epicatechin, epigallocatechin-O-gallate, gallic acid, or ferulic acid at typical white wine concentrations did not generate protein haze. However, PVPP fining of commercial wines resulted in a reduction in protein haze, suggesting that phenolic compounds may play a modulating role in haze formation. To elucidate the nature of the unknown factor(s) wine was fractionated and fractions were back-added to model wine and tested for their essentiality. Wine fractions were generated by ultrafiltration, reverse-phase chromatography, and mixed-mode anion-exchange and reverse-phase chromatography. The only purified fraction containing the essential component(s) was free of phenolic compounds, and analysis by mass spectrometry identified sulfate anion as the dominant component. Reconstitution with KHSO4 using either commercially available thaumatin or wine proteins confirmed the role of sulfate in wine protein haze formation. The two main wine proteins, thaumatin-like protein and chitinase, differed in their haze response in model wines containing sulfate. Other common wine anions, acetate, chloride, citrate, phosphate, and tartrate, and wine cations, Fe(2+/3+) and Cu(+/2+), when added at typical white wine concentrations were not found to be essential for protein haze formation.

Acetic acid bacteria (AAB) are ubiquitous organisms that are well adapted to sugar and ethanol rich environments. This family of Gram-positive bacteria are well known for their ability to produce acetic acid, the main constituent in vinegar. The oxidation of ethanol through acetaldehyde to acetic acid is well understood and characterised. AAB form part of the complex natural microbial flora of grapes and wine, however their presence is less desirable than the lactic acid bacteria and yeast. Even though AAB were described by Pasteur in the 1850s, wine associated AAB are still difficult to cultivate on artificial laboratory media and until more recently, their taxonomy has not been well characterised. Wine is at most risk of spoilage during production and the presence of these strictly aerobic bacteria in grape must and during wine maturation can be controlled by eliminating, or at least limiting oxygen, an essential growth factor. However, a new risk, spoilage of wine by AAB after packaging, has only recently been reported. As wine is not always sterile filtered prior to bottling, especially red wine, it often has a small resident bacterial population (

Sulfur compounds in wine can be a 'double-edged sword'. On the one hand, certain sulfur-containing volatile compounds such as hydrogen sulfide, imparting a rotten egg-like aroma, can have a negative impact on the perceived quality of the wine, and on the other hand, some sulfur compounds such as 3-mercaptohexanol, imparting fruitiness, can have a positive impact on wine flavor and aroma. Furthermore, these compounds can become less or more attractive or repulsive depending on their absolute and relative concentrations. This presents an interesting challenge to the winemaker to modulate the concentrations of these quality-determining compounds in wine in accordance with consumer preferences. The wine yeast Saccharomyces cerevisiae plays a central role in the production of volatile sulfur compounds. Through the sulfate reduction sequence pathway, the HS(-) is formed, which can lead to the formation of hydrogen sulfide and various mercaptan compounds. Therefore, limiting the formation of the HS(-) ion is an important target in metabolic engineering of wine yeast. The wine yeast is also responsible for the transformation of non-volatile sulfur precursors, present in the grape, to volatile, flavor-active thiol compounds. In particular, 4-mercapto-4-methylpentan-2-one, 3-mercaptohexanol, and 3-mercaptohexyl acetate are the most important volatile thiols adding fruitiness to wine. This paper briefly reviews the metabolic processes involved in the production of important volatile sulfur compounds and the latest strategies in the pursuit of developing wine yeast strains as tools to adjust wine aroma to market specifications.

The present article aims to review research papers that focus on the parietal adsorption activity of wine yeast and on its contribution to the enhancement of wine safety and quality. There is a great diversity among yeasts for their parietal adsorption activity: the outermost layer of the cell wall has a chemical composition that notably varies from yeast to yeast. Parietal mannoproteins can contain phosphate, pyruvate, or glucuronic acid, which impart negative charges, modifying the electrostatic and ionic interactions with wine components. The following could give a good reason to propose a specific selection of wine yeasts based on their parietal adsorption activity to improve wine safety and quality: (a) ochratoxin A content of wines is greatly reduced by expressly selected yeasts, sequestering the toxin during winemaking; (b) yeast influences concentration and composition of phenolic compounds in wine, above all by adsorbing them on cell wall; (c) among grape pigments, anthocyanins, in particular, may be adsorbed by yeast cell wall; and (d) yeast can also interact with wine colour producing anthocyanin-β- d -glucosidase, pyruvic acid, and acetaldehyde or releasing mannoproteins and different polysaccharides. Genomic strategies could also be used to obtain a further enhancement of the adsorption/non-adsorption activity of wine yeasts. Based on winemaking requirements and on parietal adsorption activity, a specific selection of yeasts might be performed: (a) to protect wine colour during red winemaking, (b) to remove residual wine colour during white winemaking, (c) to selectively remove ochratoxin A, and (d) to protect phenolic compounds responsible for antioxidant activity.

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 SO2. The current work investigated the degradation of acetaldehyde and SO2-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 3.3 and 3.6. While acetaldehyde was degraded rapidly and concurrently with malic acid at both pH values, SO2-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. SO2-bound acetaldehyde itself maybe responsible for the sluggish or stuck MLF, and thus bound SO2 should be considered next to free SO2 in order to evaluate malolactic fermentability. Significance and Impact of the Study: The current study provides new results regarding the metabolism of acetaldehyde and SO2-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 SO2.

Nisin is a bacteriocin used against food spoilage bacteria. Sulphur dioxide is a potent antioxidant as well as an antimicrobial agent widely used in the wine industry. In this study we describe the effect of these important antibacterial agents on the growth of a collection of 64 lactic acid bacteria (23 Oenococcus, 29 Lactobacillus, 3 Leuconostoc and 9 Pediococcus), 23 acetic acid bacteria and 20 yeast isolates, most of them recovered from wine. Minimal inhibitory concentrations (MIC) and minimal bactericide concentrations of nisin, potassium metabisulphite and ethanol were determined. Nisin MIC(50) values for the tested isolates were as follows: 0.024, 12.5, 200 and > or micro for oenococci, lactobacilli-pediococci-leuconostoc, acetic acid bacteria and yeasts, respectively. Synergistic effects on bacterial growth inhibition were observed, and potassium metabisulphite MIC(50) values decreased from one to three orders of dilution when it was combined with subinhibitory concentrations of nisin in the growth media. This effect was observed in all lactic acid bacteria species of our study. Significant differences in nisin sensitivity were observed between Gram-positive and Gram-negative bacteria, and between Oenococcus oeni and other species of lactic acid bacteria. It is concluded that appropriate combinations of nisin and metabisulphite could control the growth of spoilage bacteria in wine and therefore allow a decrease in the levels of sulphur dioxide currently used by the wine industry.

A qualitative study of volatile compounds in three commercial yeast extracts and autolysates was performed by solid-phase microextraction-gas chromatography with mass spectrometric and olfactometric detection; their addition to white wines and their effect on wine aroma composition were investigated by analytical, olfactometric and sensory evaluations. More than 160 volatile compounds were detected in the headspace of the commercial powders (some not previously reported in literature), and their olfactory characters were described. Yeast derivatives strongly modified wine aroma composition, either affecting the volatility of wine aroma compounds or by releasing exogenous volatiles. Dosage appeared to be fundamental: low amounts increased the volatility of some esters, giving more flowery and fruity notes to the wine; higher amounts increased fatty acid content in the wine headspace, producing yeasty, herbaceous and cheese-like smells. Sensory tests demonstrated that yeast derivatives would not be suitable for wines with a typical varietal aroma.

A liquid chromatography method with photodiode array and mass spectrometry detection was applied to study the changes in non-anthocyanin phenolic compounds from 47 red wines as a result of different technological processes. The procedures used include: malolactic fermentation in barrels or in stainless steel containers, the ageing of wines or not in the presence of lees, including periodic stirring of the lees, racking, clarification, cold stabilization and filtration. Wine samples were taken before and after malolactic fermentation and during the 3 months of the experiment until they had been aged for 14 months in oak barrels. Cluster analysis, analysis of variance and stepwise discriminant analysis were applied to the wine data. During malolactic fermentation, disappearance of the tartaric esters of caffeic acid (caftaric acid) and p-coumaric acid (coutaric acid) was detected, coinciding with an increase in the corresponding free acids. With the statistical treatments used, the greatest differences in the low molecular weight phenolic compounds in the wines were due to ageing time, whereas the winemaking practice did not significantly influence phenolic compounds. A significant change in the concentration of most phenolic compounds was observed in the wine at the end of the 14 months of ageing., This study was supported by the Spanish Ministerio de Educación y Ciencia (Projects PETRI 95-0759 OP and AGL2003-02436).

Malolactic fermentation (EML) is regarded as an important metabolic process for the production of quality wines, particularly in cool climate winegrowing areas, improving their sensory characteristics (reduction of acidity, increase of aromatic complexity) and microbiological stability. This bioconversion is called secondary fermentation because it occurs in wine after the alcoholic fermentation (primary fermentation) when yeast lees release nutrients (amino acids, vitamins) for bacterial growth. Different malolactic bacteria genera (Pediococcus, Oenococcus, Leuconostoc, Lactobacillus) are present in low concentrations on grapes and grape leaves and some of these remain at low cell density during alcoholic fermentation as a function of SO2 concentration and operative parameters used (wine cellar temperature, kind of container, maceration). On the contrary of how it happens for the alcoholic fermentation, malolactic starters cultures aren't widely used because of their reduced effectiveness. This can be due to the modalities of selection of the bacterial strains that often come from enological regions very different from those of employment. In order to obtain selected strains able to metabolize malic acid even at low temperatures (10degC), several natural isolates of lactic acid bacteria were obtained from different high quality Italian musts and wines. The ability of microrganisms to metabolize malic acid at lower temperatures obviates the necessity of warming cellar (a practise which increases the possibility of undesiderable microbial activity) and could have a significant impact on wine quality and energetic consumes of winemaking process. Moreover, the availability of bacterial strains which are autocthonous and, at the same time, technologically efficient, in agreement with the tendencies of wine market, could represent a valid instrument for producing typical wine. As the kinectics of malolactic fermentation are related to the number of viable bacteria, the evolution of microbial strains isolated was evaluated at two different temperatures (10degCC and 25degC) and the related kinetics constants (ko) calculated. Among the natural isolates of lactic acid bacteria, two bacterial strains (strains b and K), both belonging to Lactobacillus genus, show a good growth rate even at low temperature (criophylic strains) and will be screened for further enological characteristics (tolerance to SO2 , ethanol, and fumaric acid) to verify if they could be viable microorganisms for economic commercial scale control of malolactic fermentation process.

The volatile thiols, in particular 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) are potent aroma shown to contribute strongly to the varietal aroma of Sauvignon Blanc wines. The thiols 4MMP and 3MH exist as non-volatile, aroma-inactive cysteine bound conjugates in the grape must and during fermentation the thiol is cleaved from the precursor. However, no cysteine conjugate for 3MHA has been identified. In this work we showed that 3MHA is formed from 3MH by the wine yeast Saccharomyces cerevisiae during fermentation. Furthermore, the alcohol acetyltransferase, Atf1p, the enzyme involved in the formation of the ester ethyl acetate, was shown to be the main enzyme responsible for the formation of 3MHA. Both a laboratory yeast and a commercial wine yeast overexpressing the ATF1 gene produced significantly more 3MHA than the wild-type. Although an atf1Δ laboratory yeast strain showed reduced 3MHA formation, it was not abolished, indicating that other enzymes are also responsible for its formation. Therefore, overexpression of the ATF1 gene in a wine yeast presents the possiblity of modulating both the thiol and ester aromas in wine.

The effects of malolactic fermentation (MLF) on the concentration of volatile compounds released by yeasts during the production of red wine were investigated by inoculation with four commercial starters of Oenococcus oeni. Volatile compounds in wine at the end of MLF were extracted, analyzed by GC-MS and GC, and compared with those extracted form a noninoculated reference sample. Several esters known to play a role in the aroma profile of red wine, such as C4-C8 ethyl fatty acid esters and 3-methylbutyl acetate, were found to increase with MLF, and their final concentration was dependent on the bacterial starter employed for the induction of MLF. The overall increase of ethyl fatty acid esters was generally larger than the one observed for acetate esters. Ethyl lactate, 3-hydroxybutanoate, 2-phenylethanol, methionol, and gamma-butyrolactone were also increased by bacterial metabolism. The impact of MLF on other volatiles or red wine, including several higher alcohols, fatty acids, and nitrogen compounds, was generally negligible.

The volatile thiol 4-mercapto-4-methylpentan-2-one (4MMP) is a potent contributor to wine aroma. In grape juice, 4MMP is bound to cysteine as a non-volatile compound and requires the action of yeast during fermentation to release the aroma active thiol. A method was developed to measure 4MMP release from the precursor by headspace solid-phase microextraction and separation by gas chromatography with atomic emission detection to screen the ability of wine yeast to release 4MMP. Yeast commonly used in white wine making were grown with the precursor at two different temperatures, and the amount of 4MMP released was measured. The results demonstrate that yeast strain selection and fermentation temperature can provide an important tool to enhance or modulate the grape-derived aromas formed during wine fermentation.

The kinetics of both malolactic fermentation in Chardonnay wine by encapsulating Lactobacillus casei cells in pectate gel and lyophilized Oenococcus oeni culture has been carried out. The influence of acidity, sulfur dioxide content, and organic acid content on the malolactic activity of the bacteria has been controlled. Encapsulated bacteria degraded 30%, of malic acid in white wine, deacidifying it from pH 3.15 to 3.40, whereas the lyophilized culture degraded 48% of malic acid, deacidifying from pH 3.15 to 3.60. The degree of conversion of malic acid in wine by the encapsulated cells was twice as high as that obtained by the free Lactobacillus casei cells. The operational stability of calcium pectate gel capsules was 6 months. It has been proved that the encapsulated biocatalyst increases the rate of fermentation, and induces the fermentation to take place at high ethanol concentrations. The proposed encapsulated biocatalyst is an attractive material for industrial applications in continuous winemaking processes.

L. PRIPIS-NICOLAU, G. DE REVEL, A. BERTRAND AND A. LONVAUD-FUNEL. 2004. Aims: During malolactic fermentation (MLF), the secondary metabolisms of lactic acid bacteria (LAB) contributeto the organoleptic modification of wine. To understand the contribution of MLF, we evaluated the capacity ofvarious wine LAB to metabolize methionine.Methods and Results: Using gas chromatography (GC) coupled either with mass spectrometry (MS) or a flamephotometry detector in sulphur mode (FPD), we studied this metabolism in laboratory media and wine. Inlaboratory media, several LAB isolated from wine were able to metabolize methionine. They formed methanethiol,dimethyl disulphide, 3-(methylsulphanyl)propan-1-ol and 3-(methylsulphanyl)propionic acid. These are known tohave powerful characteristic odours and play a role in the aromatic complexity of wine. In various red wines, afterMLF only the 3-(methylsulphanyl)propionic acid concentration increased significantly, as verified with severalcommercial starter cultures. This compound, which is characterized by chocolate and roasted odours, couldcontribute to the aromatic complexity produced by MLF.Conclusions: This study shows that LAB isolated from wine, especially Œnococcus œni strains, the major species inMLF, are able to metabolize methionine to form volatile sulphur compounds.Significance and Impact of the Study: This is the first study to demonstrate the capacity of wine LAB tometabolize methionine.Keywords: aroma, lactic acid bacteria, metabolism, methionine, volatile sulphur compounds, wine.

The diketone, diacetyl, is a major flavour metabolite produced by lactic acid bacteria (LAB). Of the LAB associated with wine, Oenococcus oeni is encouraged during the malolactic (ML) fermentation, a biodeacidification of wine during which the metabolism of diacetyl occurs. Diacetyl, which imparts a buttery aroma and flavour to many fermented foods and beverages, is a key flavour compound of most fermented dairy products. In wine, diacetyl has important stylistic implications. The biosynthesis of diacetyl is dependent upon citric acid metabolism and diacetyl is an intermediate metabolite which can be further reduced to acetoin and the alcohol, 2,3-butanediol. This review will focus on the sensory perception, metabolism, genetics and analysis of diacetyl during wine production. The extensive knowledge of diacetyl metabolism in dairy LAB is used to enhance the understanding of diacetyl metabolism of wine LAB. Factors which can effect the formation and concentration of diacetyl in wine are discussed. These include malolactic bacterial strain, wine chemical and physical parameters (pH, temperature, citric acid, sulfur dioxide, aeration) and the presence of yeast lees. Finally, the affects of other wine components, such as phenolics, are discussed.

Research has shown that non-Saccharomyces yeast strains can be detected throughout wine fermentation. Non-Saccharomyces yeasts can therefore influence the course of fermentation and also the character of the resultant wine. Previously it was shown that four non-Saccharomyces species, i.e. Kloeckera apiculata, Candida stellata, Candida pulcherrima and Candida colliculosa, predominated in grape must at the start of fermentation. In this study these four yeasts were used singularly and in combination with an industrial wine yeast (Saccharomyces cerevisiae strain VIN 13) to ferment must on a laboratory scale. The resultant wine was analysed for ethanol, volatile acidity, total S02 and glycerol. Results show that, in comparison with the industrial wine yeast, the non-Saccharomyces yeast strains could not ferment all the sugar. Furthermore, while the individual non-Saccharomyces-fermented wines had different chemical analyses, the wines fermented by the combinations were similar to the wine produced by the industrial yeast only. In subsequent, small-scale winemaking trials some of the wines produced by combined fermentations were judged to be of better quality than those produced by the S. cerevisiae only. However, this quality increase could not be linked to increased ester levels.

Wine fermentations are conducted by naturally occurring or selected industrial wine yeast strains of Saccharomyces cerevisiae. However, non-Saccharomyces yeasts also occur naturally in fermenting grape musts, especially in the initial stages of the fermentation. It has been speculated that these yeasts can contribute to the overall characteristics of the wine. Generally, it is accepted that Kloeckera apiculata is the predominant non-Saccharomyces yeast species in grape must. However, it was shown previously that Candida pulcherrima was the predominant non-Saccharomyces yeast species in a grape must after sedimentation and prior to inoculation with commercial wine yeast. Subsequently, this yeast was investigated in laboratory and small-scale wine fermentations of Chenin blanc wine. As it could not ferment grape juice to dryness on its own, it was used in combination with a S. cerevisiae wine yeast strain. The effect of SO2, di-ammonium phosphate (DAP), pH and temperature on the growth of C. pulcherrima was also investigated. In combined fermentations, no change in overall fermentation rate or standard wine chemical analyses could be observed in comparison to a control S. cerevisiae fermentation. However, wine production in three consecutive years showed that the wine produced by the combined fermentation was of higher quality than that produced by the S. cerevisiae only.

The ability of four commercial preparations of Oenococcus oeni lactic acid bacteria (EQ 54, Lalvin OSU, Uvaferm Alpha, and Lalvin 31) to hydrolyze wine aroma precursors was evaluated by measuring the concentration of free and bound aroma compounds at the end of malolactic fermentation carried out in model wines containing a mixture of glycosides extracted from Muscat wine. At pH 3.4 there was a decrease in glycosylated compounds matched by a concomitant increase in free forms in all starter cultures tested. When malolactic fermentation was carried out at pH 3.2, a significant decrease in the ability to hydrolyze aroma precursors was observed for two of the cultures tested (Uvaferm Alpha and Lalvin 31). Large differences in the extent of hydrolysis and in the specificity of this activity toward specific aroma precursors were observed and appeared to be related to the chemical structure of the aglycon as well as to individual characteristics of each starter culture. The amounts of glycosylated aroma compounds released during malolactic fermentation suggest that O. oeni can alter the sensory characteristics of wine through the hydrolysis of aroma precursors.

To produce a high quality wine, it is important to obtain a fine balance between the various chemical constituents, especially between the sugar and acid content. The latter is more difficult to achieve in wines that have a high acidity due to excess malic acid, since wine yeast in general cannot effectively degrade malic acid during alcoholic fermentation. An indigenous Saccharomyces paradoxus strain RO88 was able to degrade 38% of the malic acid in Chardonnay must and produced a wine of good quality. In comparison, Schizosaccharomyces pombe strain F effectively removed 90% of the malic acid, but did not produce a good quality wine. Although commercially promoted as a malic acid degrading wine yeast strain, only 18% of the malic acid was degraded by Saccharomyces cerevisiae Lalvin strain 71B. Preliminary studies on the transcriptional regulation of the malic enzyme gene from three Saccharomyces strains, i.e. S. paradoxus RO88, S. cerevisiae 71B and Saccharomyces bayanus EC1118, were undertaken to elucidate the differences in their ability to degrade malic acid. Expression of the malic enzyme gene from S. paradoxus RO88 and S. cerevisiae 71B increased towards the end of fermentation once glucose was depleted, whereas no increase in transcription was observed for Saccharomyces bayanus EC1118 which was also unable to effectively degrade malic acid.

Certain aspects of the interrelationship between associations of yeasts and lactic acid bacteria during the fermentation of grape must were studied. Action bySaccharomyces pastorianus gave rise to increased formation of keto acids and residual metabolites. Addition ofLactobacillus plantarum to the fermentation process may inhibit alcoholic fermentation and considerably increased the production of acetaldehyde andl(+) andd(−)lactic acids.

Publisher Summary This chapter discusses the role of lactic acid bacteria in winemaking. The classification of enological lactic acid bacteria was established by using the conventional methods, which focus on phenotypes. According to morphological, biochemical and physiological properties, wine and grape lactic acid bacteria can be classified as lactobacilli and cocci , being either obligate and facultative heterofermentative or homofermentative. Since lactic acid bacteria are necessary to obtain malolactic fermentation, winemakers must find a solution for minimizing amine levels, even if indigenous bacteria possess decarboxylating activities. Today, a solution is the use of selected malolactic starters. O. oeni strains, free of undesirable activities, are produced and lyophilized in concentrated populations. They are massively added to wine to complete malolactic fermentation. They normally overcome the indigenous bacteria and seem to eliminate them by competition. Then after malolactic fermentation, wine is readily stabilized by sulfur dioxide, which eliminates the malolactic starter and the residual wild-type strains. It needs to know more about the ecology of micro-organisms living on grapes, how they survive on cellar equipment and how they grow in wines. Such studies were almost impossible in the past. Now the new molecular methods are opening new horizons for studying wine micro-organisms, particularly the lactic acid bacteria.

As important as the blend of base wines before bottling, one of the most important steps in the champagne-making process is the long ageing on lees. Two yeast strains of Saccharomyces cerevisiae MC001 and MC002, used in champagne wine production, were allowed to autolyse. After 8 days of autolysis, active dry yeasts adapted to wine released 1.7- to 1.8-fold more nitrogen compounds than nonadapted active dry yeast. The nitrogen content (total, proteins, peptides and amino) present in autolysates was measured for yeasts adapted to wine. The composition of free amino acids and amino acids constituting peptides showed no difference between the two strains of yeast used. Studies of intracellular proteolytic activity and release of peptides showed no correlation between these two phenomena. These results indicate that yeasts adapted to wine give results similar to those that occur in wine during ageing.

The influence of different types of winemaking technology on the contents of catechins, proanthocyanidins, and anthocyanins in Tinta Miuda red wines was studied. The Tinta Miuda red wines were made by fermentation with carbonic maceration, fermentation with stem contact, and fermentation without stem contact, respectively. The analysis of individual catechins, procyanidins, and anthocyanins in these wines was performed by HPLC, and quantification of total catechins, total oligomeric proanthocyanidins, total polymeric proanthocyanidins, and total anthocyanins was carried out by spectrophotometric methods. The wine made by carbonic maceration contained the highest amounts of both catechins and oligomeric and polymeric proanthocyanidins, followed by the wine made by fermentation with stem contact, whereas the wine made by fermentation without stem contact contained the lowest of these compounds. On the other hand, the concentrations of total anthocyanins and nearly all individual anthocyanins in the carbonic maceration wine were lower than those in the wines made by fermentation with stem contact and fermentation without stem contact. These results indicated that, although the carbonic maceration technique could retain higher amounts of catechins and proanthocyanidins in wine, it did not favor retaining or stabilizing anthocyanins in wine.

A biocatalyst was prepared by immobilization of Saccharomyces cerevisiae strain AXAZ-1 on apple pieces. It was examined by electron microscope and studied during the fermentation of grape must for batch wine-making. The immobilized yeast showed an important operational stability without any decrease of its activity even at low temperatures (1-12 degrees C). Specifically, at 6 degrees C the biocatalyst favored wine production within 8 days, which is less time than is required for the natural fermentation of grape must. At 1 degrees C wine production was effected in 1 month. This speeding up of the fermentation could be accepted and adopted by the industry for scaling up the wine-making process. Total and volatile acidities were similar to those found in dry wines. The concentrations of higher alcohols (propanol-1 and isobutyl alcohol) were low. The presence of amyl alcohols proved to be temperature dependent and decreased with the temperature decrease. The values of ethyl acetate concentrations were relatively high, up to 154 mg/L. This probably contributes to the fruity aroma and excellent taste of the produced wines. There was no indication of vinegar odor in the product given that the volatile acidity was

Glycerol is one of the most abundant components of wine. In dry wines its concentration is only exceeded by water and ethanol. Several studies have been carried out on the effects of glycerol on mouthfeel, but little attention has been given to glycero–flavour interactions. Consequently, here we have turned our attention to the aroma perception. Purge and Trap analysis and sensory measurements were used to investigate the modifications in volatility of aroma compounds in model solutions and white wine according to their glycerol concentration. In water, the amount of aroma compounds released was not affected by the presence of glycerol concentrations ranging from 5 to 50 g/L. The addition of glycerol did not modify the relative volatility of the four flavours tested. Sensory analysis showed that the perceived overall flavour of model wine and white wine was not changed by the addition of glycerol. These observations confirmed the Purge and Trap results. In conclusion, glycerol is not involved in the changes of aroma in white wine.

This study shows that the growth of Oenococcus oeni CECT 4100 in a synthetic medium is affected by phenolic compounds in different ways, depending on their type and concentration. Generally they have no effects at low concentrations, but hydroxycinnamic acids are inhibitory at high concentrations. Malolactic fermentation was stimulated in the presence of catechin and quercetin, but increasingly delayed with increasing amounts of p-coumaric acid. Gallic acid appeared to delay or inhibit the formation of acetic acid from citric acid. This could lead to a better control of malolactic fermentation and suppress the increase in volatile acidity, which is undesirable in the wine-making process.

The influence of three levels of oxygen supply on the phenolic composition of Sangiovese wine was evaluated during 6 months of storage. Oxygenation reduced the total phenolics content and increased the concentration of red polymeric compounds. Saturation with oxygen every 30 days significantly improved the wine colour density as compared to the control. The individual behaviour of phenolic compounds such as (+) catechin, (−) epicatechin, quercetin, caffeic acid, and anthocyanins showed a significant depleting effect of oxygen on wine phenolic composition when wines were oxygenated every month. The supply of oxygen every 60 days limited the oxidation of low molecular weight phenolic compounds and slightly influenced the wine colour. This confirmed that oxygenation could improve the evolution of red wines during ageing, but its control is necessary to reduce detrimental effects upon single bioactive phenolic compounds.

In this work, we study two aspects of malolactic fermentation: the use of industrial lactic acid bacteria starter-cultures in a white Sauvignon wine and the effects of the bacterial activity on the composition of wines in terms of volatile substances from oakwood during partial malolactic fermentation in barrels. A sensory evaluation was made by the use of discriminative and descriptive methods. The consequences of bacterial development are thus limited, but it is nevertheless possible to make characteristic observations. The carbonyl substances were formed in connection with a more or less fast bacterial growth and a degradation of the citric acid. However, the influence of the bacterial starter-culture is difficult to establish. The concentration of the compounds resulting from wood was higher in the wines after malolactic fermentation compared to a wine not having undergone bacterial development. Greater complexity was perceived during the sensory analysis, with buttered, spiced, roasted, vanilla, and smoked notes. On the other hand, the intensity of the descriptors characteristic of grape variety decreased.

Fermentação maloláctica é o processo metabólico de degradação do ácido L-málico em ácido L-láctico e CO2. É responsável pela redução da acidez total, além de contribuir para a estabilidade biológica e modificação de flavor nos vinhos. Em operações normais de vinificação a fermentação maloláctica normalmente ocorre em vinhos tintos, mas recentemente seu uso vem aumentando em alguns vinhos brancos, tal como Chardonnay. O objetivo principal deste trabalho foi avaliar o comportamento de duas culturas comerciais de Leuconostoc oenos na indução da fermentação maloláctica. Uvas da cv. Gewürztraminer foram vinificadas e inoculadas com duas culturas lácticas, Viniflora Oenos e Vino, em diferentes níveis de açúcar residual: 55; 21,9; 1,1 e 0,9g/l. Os níveis de açúcares, em duas repetições, foram comparados com a ocorrência espontânea da fermentação maloláctica (controle). A degradação do ácido málico foi acompanhada através de cromatografia em papel. As determinações dos ácidos orgânicos foram realizadas através de cromatografia líquida de alta eficiência. Foram avaliados os açúcares redutores, °Brix, pH, acidez total e álcool. Foi observada uma baixa incidência (22,7 %) de fermentação maloláctica nos vinhos. Naqueles em que ocorreu, foi necessário longo tempo para o término, entre 56 e 92 dias. Nos estágios com 1,1 e 0,9g/l de açúcares redutores, os vinhos inoculados e os controles não realizaram a fermentação maloláctica. Os isolados de bactérias nativas foram identificados como pertencentes ao gênero Leuconostoc e devido algumas características fisiológicas encontradas nos isolados do vinho inoculado suspeitou-se da perda de viabilidade das culturas puras. O comportamento dos ácidos málico, acético, láctico, pirúvico e tartárico foi demonstrado pelos resultados. Malolactic fermentation is the metabolic process of L-malic acid degradation in L-lactic acid and CO2. It is responsible by the reduction in total acidity and also contributes to the biological stability and a flavor modification of the wines. In normal winery operation it occurs commonly in red wines. However, recently malolactic fermentation had been also used in some white wines, such as Chardonnay. The main aim of this work was to evaluate the behavior of two commercial strains of Leuconostoc oenos in the induction of malolactic fermentation. Gewürztraminer grapes were fermented and inoculated with two lactic cultures, Viniflora Oenos and Vino, in several residual sugar levels: 55.0 - 21.9 - 1.1 e 0.9g/l. The sugar levels were compared with spontaneous malolactic fermentation (control), with two repetitions. Degradation of malic acid was followed by paper chromatography. Organic acids determinations were performed using eficiency high liquid chromatography. Reducing sugars, °Brix, pH, total acidity and alcohol values were evaluated. A low incidence (22.7%) of the malolactic fermentation in wines was observed. However, in those wines in which malolactic fermentation occurred it took a long time to reach the end, between 56 and 92 days. Using 1.1 and 0.9g/l of residual sugar the inoculated wines and the control did not undergo the malolactic fermentation. The wild lactic acid bacterias were identified as being as Leuconostoc genus and, due to some physiologic characteristics observed in the inoculated wine isolated, there was a raised suspicius that the cultures had lost the viability. The behaviour of the malic, acetic, lactic, piruvic and tartaric acids were demonstrates by the results.

Biogenic amines (BA) are organic nitrogen compounds formed by decarboxylation of amino acids or by amination and transamination of aldehydes and ketones. The most important BA in wine are histamine, putrescine, cadaverine, tyramine, spermidine, spermine, tryptamine and β-phenylethylamine, and their content could vary due to different factors during wine production. They are mainly produced during malolactic fermentation by lactic acid bacteria, but could be also produced by yeasts during alcoholic fermentation. Concentration of BA in wine is important, because they act as moderators in many biological reactions in the human body. Concentrations of histamine, putrescine, cadaverine and tyramine were determined using high pressure liquid chromatography (HPLC) in eight different wines (four white and four red). The highest concentrations of cadaverine (8.228 mg/L) and tyramine (6.452 mg/L) were detected in white wine Vinko, while the highest level of putrescine (8.740 mg/L) was determined in white wine Chardonnay. In red wines putrescin concentration was significantly higher than levels of tyramine and cadaverine. Tyramine concentrations were lower in red than in white wines. Amounts of histamine were below detection limits in all samples, while concentrations of putrescine, cadaverine and tyramine were in agreement with Croatian wine legislation.

Banana/orange composite wine was brewed in the lab by liquid fermentation using angel yeast and lactic acid bacteria as the fermentation strains, and characterized by chemical analyses. It was found that the best ratio of banana juice to navel orange juice was 1:2, and the optimum alcohol fermentation parameters were as follows: 28~30°C, 22% initial sugar content, 6% yeast, and 6 days of fermentation to reach an alcohol concentration of 11.63% v/v. The free amino acids in the composite wines were tested by automatic amino acid analyzer, and the flavor components of the composite wine were determined and analyzed by gas chromatography-mass spectrometry (GC-MS). Seven types of trace elements in the composite wine were measured by atomic absorption spectrometry. Results showed that there were 17 free amino acids, and their total concentration was up to 897.6 mg/L. A total of 16 key compounds were identified in the composite wine, 11 of which were ester, 4 of which were alcohols and 1 of which was acid. Magnesium, iron, copper and manganese elements were relatively rich in the wine, while lead was extremely low.

The flavor of alcoholic beverages is produced by a very large number of compounds (50). Among these, acetoin is important because of its involvement in the bouquet of wine, and it is the key compound in the biosynthesis of 2,3-butanediol and diacetyl. These two compounds are closely related to acetoin, representing three levels of oxidation in one four-carbon skeleton (8). Their contribution to the aroma and flavor of wine is not easily assessed. Neither 2,3-butanediol nor acetoin is strongly odorous; in fact, their threshold values in wine are very high, both being about 150 mg/liter (19, 72); however, diacetyl, which creates an off flavor in alcoholic beverages, has a characteristic odor and is detectable in wine at very low levels (threshold value, 8 mg/liter) (19). In this picture, the flavor significance of acetoin is more likely to be attributable to its potential aroma than to the odor itself. Acetoin is formed during fermentation by the microbial activity of lactic acid bacteria and yeasts. Therefore, the study of acetoin in wine is an integral part of investigation of the influence of microorganisms on the composition and quality of wine.

Different levels of insoluble solids (ISS) from apple juice were added to the apple juice must along with wine yeast strains of Saccharomyces cerevisiae and pectinolytic enzymes to study their effect on sensory quality of wine. The flavour profile of the apple wine of different treatments by quantitative descriptive analysis (QDA) showed that the wine without ISS was rated better in most of the flavour characteristics studied than those with ISS. Addition of insoluble solids imparted flavour quality characteristics of the wine which are not considered desirable. Out of various wine yeast strains, W and USD 595 performed better than UCD 505 and UCD 522. The wines without ISS were perceived having highest apple aroma, least cooked vegetables aroma, lower sweetness, lesser bitterness and lower higher alcoholic flavour. Both the addition of wine yeast strains and enzymes improved the flavour profile of the wine and from this point of view, wine made from presettled juice possessed improved flavour characteristics of the wine. Application of PCA weekly separated the wines fermented with or without insoluble solids. The flavour profile did show some interactions between insoluble solids, yeast strains and the enzymes which modified the flavour attributes of the resultant wine.

Preparation of wine from surplus apple fruits can reduces postharvest losses besides source of income. Apple wine using different sources of sugars i.e. honey, sugar, molasses and jaggary to raise the TSS of must to 24°B was prepared as per routine procedure. To impart medicinal value, spices and herb extract of hops, menth, anola, ginger and garlic @5% each was added to the must. The apparent effect of addition of extract was to delay the fermentation, not to stop it. Physico-chemical characteristics of apple wine before and after 6 months of maturation showed that the addition of extract did not affect the quality of wine adversely. From the sensory quality point of view, extract treated honey based, concentrate based or concentrate + apple juice based wines were superior to the control apple wine in most of the sensory qualities. The highest score was awarded to honey + herbs and spices extract (5%) based wine. Addition of extract increased the aldehyde, esters and total phenols which are expected to contribute the antimicrobial and antioxidant activities of the wine. The changes during maturation were desirable and in general, were the same as found in any wine. During maturation, reducing sugars, total esters, titrable acidity, ethanol and volatile acidity increased significantly while total phenols deseased but there was no effect on TSS and no significant decrease in total sugars and higher alcohols took place.

The production of wine from grape juice relies on the combined actions of both yeast and bacteria which shape the aroma and flavour of wine through the production of secondary metabolites and the biochemical transformation of many grape-derived constituents. Whereas the principal wine yeast, Saccharomyces cerevisiae, is primarily involved in the alcoholic fermentation in which glucose and fructose are converted into alcohol, the wine bacterium, Oenococcus oeni, is primarily involved in a secondary fermentation reaction where malic acid is decarboxlyated into lactic acid. This conversion, known as malolactic fermentation (MLF), results in an increase in wine pH and reduction in the sourness of the wine, while also providing microbial stability through the reduction of potential carbon sources for wine spoilage bacteria such as Lactobacilli and Pediococci1. In addition to its primary role in performing MLF, the metabolic by-products produced during the growth of O. oeni in wine have been shown to positively contribute to the flavour and mouth feel of wines which have undergone MLF.

Nerea Jiménez-Moreno, Carmen Ancín-AzpilicuetaDepartment of Applied Chemistry, Universidad Pública de Navarra, Pamplona, SpainAbstract: Yeast walls can retain different wine compounds and so its use is interesting in order to eliminate harmful substances from the must which affect alcoholic fermentation (medium chain fatty acids) or which affect wine quality in a negative way (ethyl phenols, ochratoxin A). The aim of this study was to examine the capacity of commercial yeast cell walls in eliminating volatile phenols (4-ethylphenol and 4-ethylguaiacol) from a synthetic wine that contained 1 mg/L of each one of these compounds. The binding of these compounds to the wall was quite fast which would seem to indicate that the yeast wall-volatile compound union is produced in the outer surface layers of this enological additive. The cell walls used reduced the concentration of 4-ethylphenol and 4-ethylguaiacol, although it would seem that on modifying the matrix of the wine the number of free binding sites on the walls is also modified.Keywords: volatile phenols, yeast cell walls, wine, sorption

During fermentation in a liquid synthetic medium there was a 30% decrease in resveratrol (a phenolic compound of wine) due to the action of wine yeasts.

(1981). Effects of Fermentation Conditions and Aging Temperature on Volatile Ester Contents in Wine. Agricultural and Biological Chemistry: Vol. 45, No. 11, pp. 2645-2651.

Two new components of botrytised wine were identified: 4,5-dimethyl-3-hydroxy-2(5H)-furanone (Sotolon) and ethyl 9-hydroxynonanoate. Sotolon, the key substance of cane sugar aroma, was identified as the sugary flavor substance of botrytised wine by means of gas chromatography-mass spectrometry after column chromatographic separation on DEAE-Sephadex and silica gel. Ethyl 9-hydroxynonanoate was identified by chemical ionization and electron impact mass spectrometry. To evaluate the role of 17 volatile and 5 nonvolatile compounds characteristic of botrytised wine, these compounds were added to a normal wine. This produced a sweet, honey-like flavor similar to that of botrytised wine. The importance of Sotolon and the role of each group of flavor substances in producting this flavor was clarified by omission tests.

A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307. a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid.Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cell-free extract were optimal at pH 6.0. Divalent cations such as Mn2+, Mg2+, Co2+ and Zn2+ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg2...

Commercially produced red wines were adjusted to pH 3.0, 3.2, 3.5, 3.7, or 4.0 and examined during and after malolactic fermentation for growth of lactic acid bacteria and changes in the concentrations of carbohydrates, organic acids, amino acids, and acetaldehyde. With one exception, Leuconostoc oenos conducted the malolactic fermentation in all wines and was the only species to occur in wines at pH below 3.5. Malolactic fermentation by L. oenos was accompanied by degradation of malic, citric, and fumaric acids and production of lactic and acetic acids. The concentrations of arginine, histidine, and acetaldehyde also decreased at this stage, but the behavior of hexose and pentose sugars was complicated by other factors. Pediococcus parvulus conducted the malolactic fermentation in one wine containing 72 mg of total sulfur dioxide per liter. Fumaric and citric acids were not degraded during this malolactic fermentation, but hexose sugars were metabolized. P. parvulus and species of Lactobacillus grew after malolactic fermentation in wines with pH adjusted above 3.5. This growth was accompanied by the utilization of wine sugars and production of lactic and acetic acids.

The separation and determination of phenolic acids and aldehydes in “Fino” sherry wine, using high-perfomance liquid chromatography (HPLC), has enabled a study to be carried out of their evolution during the initial process in “fino” sherry wine production. A continuous extraction method with ethyl ether at pH 2 was chosen, by which the greatest reprodubility in the extraction of these compounds is achieved from must as well as from the wine. For stabilization of the must samples, a mixture of ascorbic acid (0.2%) and DMF (15%) was used, preventing the evolution of colour and the beginning of fermentation. These methods have permitted the study of changes in the content of the phenolic acids and aldehydes during the different stages of “fino” wine making.

The bitter and astringent taste properties of hydroxycinnamoyl tartaric acids have been responsible for incorrect speculation that these compounds influence the taste of wine. The sensory contribution of the major hydroxycinnamates found in wine was evaluated by duo—trio difference tests in model wine solutions and in white wine. The major hydroxycinnamate compound, trans-caffeoyl tartaric acid, was not detectable when added at the highest level at which it has been found in wine, 150 mg litre−1. Similarly, no significant difference in taste was produced when caffeic acid and p-coumaric acid were added at 120 mg litre−1 and 30 mg litre−1, respectively. Trans-2-S-glutathionyl caffeoyl tartaric acid, a product of oxidase activity in must, was not detectable at 50 mg litre−1. It was concluded that the hydroxycinnamates do not play any direct role in the taste of wines.

Historically, the fermentation of grape juice to wine has been carried out by indigenous yeasts found on the berry. However, in newer wine regions, e.g. the USA, inoculation with selected wine yeast strains is employed. Grape juice is high in nutritional factors and difficulties in fermentation usually arise from the inhibitory effects of the high concentration of sugar initially present and the ethanol produced. A secondary fermentation, brought about by indigenous or added lactic acid bacteria, converts malic acid to lactic acid and carbon dioxide and often occurs. This ‘malolactic’ fermentation is usually slow. For both yeast and bacterial fermentations strain selection is based more on fermentation performance than on sensory characteristics of the wine, with increased tolerance of the yeast to ethanol and of the bacteria to low pH being emphasized. Attempts to increase the malolactic fermentation rate have been made by cloning and transferring the malolactic gene from Lactobacillus to wine yeast. In early attempts to produce wines with enhanced or novel sensory characteristics a leucine-less mutant of a homothallic wine yeast has been obtained which does not produce isoamyl alcohol.

After the appearance of “Etudes sur le vin” by Pasteur, in enology lactic acid bacteria have been considered as deteriorating agents for more than 50 years. About 1920, Ferre in Burgundy and Ribereau-Gayon in Bordeaux demonstrated the enological importance of the transformation of malic to lactic acid. This notion is now generally accepted in most vinicultural areas. Malolactic fermentation is encouraged, especially for red wines, for two reasons: a) it eliminates the taste of malic acid and lowers the acidity of the wine, b) it assures the biological stability of wines conserved with a minimum of sulphurous anhydride.