Impact of winemaking practices on arginine and citrulline metabolism during and after malolactic fermentation

AIMS: To study arginine degradation and carcinogenic ethyl carbamate precursor citrulline formation during and after malolactic fermentation (MLF). METHODS AND RESULTS: MLF was induced in white wine with two commercial Oenococcus oeni strains under different winemaking conditions regarding the type of alcoholic fermentation (spontaneous, induced) and the lees management (racked, on lees). Arginine degradation and citrulline formation did not occur during malic acid degradation in any treatment. In five of the six treatments in which arginine degradation took place, it occurred 3 weeks after malic acid depletion and significant amounts of citrulline were formed. Presence of yeast lees in wines led to increased citrulline formation. Conclusions: This study suggests that arginine metabolism is inhibited in oenococci at low pH values (< 3.5) and that in the postalcoholic fermentation phase, citrulline formation from arginine degradation can be avoided if MLF is induced by pure cultures of O. oeni with inhibition of the bacterial biomass after malic acid depletion. Residual yeast lees in the wine have been identified as a significant risk factor for increased citrulline formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Conclusions drawn from this study allow reducing the risk of carcinogenic ethyl carbamate formation from citrulline excretion by wine lactic acid bacteria.     

Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods

Aims: To develop rapid methods allowing enumeration of lactic acid bacteria producing biogenic amines in wines and to analyse wine samples by the methods. Methods and Results: Methods based on quantitative PCR targeting bacterial genes involved in histamine, tyramine and putrescine production were developed and applied to detect and quantify the bacteria producing these biogenic amines in wine. Analysis of 102 samples revealed low populations of the targeted bacteria in grape must samples, an increased bacteria biomass in wine samples after alcoholic fermentation, reaching the highest population levels (above 10⁶ cells ml^?1) during spontaneous malolactic fermentation. A minimum of 10³ ml^?1 producing cells was required for production of more than 1 mg l^?1 of biogenic amines. Accumulation of putrescine in wine was correlated with the presence of bacteria carrying an ornithine decarboxylation pathway. Trials of winemaking showed that the use of selected bacteria for inducing malolactic fermentation was efficient to limit the proliferation of undesirable bacteria and the production of biogenic amines. Conclusion: Methods using quantitative PCR are efficient to enumerate biogenic amines‐producing cells in wine. Significance and Impact of the Study: The methods can help to better control and to improve winemaking conditions in order to avoid biogenic amine production.     

A mathematical model of the link between growth and L-malic acid consumption for five strains of Oenococcus oeni

In winemaking, after the alcoholic fermentation of red wines and some white wines, L-malic acid must be converted into L-lactic acid to reduce the acidity. This malolactic fermentation (MLF) is usually carried out by the lactic acid bacteria Oenococcus oeni. Depending on the level of process control, selected O. oeni is inoculated or the natural microbiota of the cellar is used. This study considers the link between growth and MLF for five strains of O. oeni species. The kinetics of growth and L-malic acid consumption were followed in modified MRS medium (20 °C, pH 3.5, and 10 % ethanol) in anaerobic conditions. A large variability was found among the strains for both their growth and their consumption of L-malic acid. There was no direct link between biomass productivities and consumption of L-malic acid among strains but there was a link of proportionality between the specific growth of a strain and its specific consumption of L-malic acid. Experiments with and without malic acid clearly demonstrated that malic acid consumption improved the growth of strains. This link was quantified by a mathematical model comparing the intrinsic malic acid consumption capacity of the strains.     

Simultaneous yeast–bacteria inoculum. A feasible solution for the management of oenological fermentation in red must with low nitrogen content

The simultaneous inoculum of yeasts and bacteria is a feasible solution for improving fermentation in wines with a harsh chemical composition, capable of inhibiting microbial activity. Considering the risk of wine spoilage due to lactic bacteria, co-inoculum is suggested in white wines with a low pH. However, climate change has also caused problems in achieving malolactic fermentation in red wines, due to the high concentration of ethanol and the low nutrient content. In this work, 5 pairs of commercial oenological starters were tested in simultaneous fermentation, using 4 red musts with a low nitrogen content, and compared with a traditional winemaking process. The simultaneous inoculum caused a slowdown in the activity of yeasts, although no problems in the accomplishment of alcoholic fermentations were observed. More reliable malolactic fermentation was performed in the co-inoculum trials, while, in traditional winemaking, some failures in the degradation of malic acid were observed. Microbiological analyses agreed with these observations. No differences were found in yeast density during alcoholic fermentation, demonstrating the absence of negative interaction between the yeast and the bacteria. However, simultaneous fermentation is not without risks; the highest increases of acetic acid were noted in the co-inoculum trials. The addition of yeast and bacteria to must with a serious lack of nutrients would appear to be a promising alternative to traditional fermentation; however, careful control of the chemical composition of must is mandatory to obtain reliable microbiological activity in the first stages of winemaking.     

Microbiology of the malolactic fermentation: Molecular aspects

Abstract Malolactic fermentation conducted by lactic acid bacteria follows alcoholic fermentation during winemaking, and several positive effects make it indispensable for most wines. Research has focused on the growth and physiology of lactic acid bacteria in wine; resulting in the design of malolactic starter cultures. Future work on these starters will concentrate on aromatic changes as additional criteria for strain selection. Although the main features of the malolactic enzyme and its gene are known, the detailed mechanism of the malolactic reaction remains unclear. Cloning and expression of this activity in enological strains of Saccharomyces cereuisiae might be one of the next most important advances in the control of malic acid degradation in wine.     

苹果酸酶基因转化酿酒酵母的研究进展

微生物降酸是现代葡萄酒酿造工艺中重要环节之一。利用现代生物技术将粟酒裂殖酵母中的苹果酸酶基因和苹果酸通透酶基因共同转化到酿酒酵母中,构建苹果酸-酒精酵母,使之既能进行酒精发酵,又能分解苹果酸。主要对近些年粟酒裂殖酵母苹果酸酶性质、基因结构及其转化酿酒酵母的研究做了回顾与总结,并指出了有待于解决的问题。     

Growth and Arginine Metabolism of the Wine Lactic Acid Bacteria Lactobacillus buchneri and Oenococcus oeni at Different pH Values and Arginine Concentrations

During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth of Lactobacillus buchneri CUC-3 and Oenococcus oeni strains MCW and Lo111 in wine were investigated. In contrast to L. buchneri CUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth of L. buchneri CUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.     

Implications of new research and technologies for malolactic fermentation in wine

The initial conversion of grape must to wine is an alcoholic fermentation (AF) largely carried out by one or more strains of yeast, typically Saccharomyces cerevisiae. After the AF, a secondary or malolactic fermentation (MLF) which is carried out by lactic acid bacteria (LAB) is often undertaken. The MLF involves the bioconversion of malic acid to lactic acid and carbon dioxide. The ability to metabolise l-malic acid is strain specific, and both individual Oenococcus oeni strains and other LAB strains vary in their ability to efficiently carry out MLF. Aside from impacts on acidity, LAB can also metabolise other precursors present in wine during fermentation and, therefore, alter the chemical composition of the wine resulting in an increased complexity of wine aroma and flavour. Recent research has focused on three main areas: enzymatic changes during MLF, safety of the final product and mechanisms of stress resistance. This review summarises the latest research and technological advances in the rapidly evolving study of MLF and investigates the directions that future research may take.     

苹果酸降解相关基因在酿酒酵母中的表达

微生物降酸是现代葡萄酒酿造重要工艺。将裂殖酵母苹果酸通透酶基因(mae1)和苹果酸酶基因(mae2)克隆到酿酒酵母中,构建了苹果酸酒精酵母;将mae1基因和乳酸乳球菌的苹果酸乳酸酶基因(mleS)克隆到酿酒酵母中,构建了苹果酸乳酸酵母。构建的酵母重组子能够有效地分解发酵基质中的苹果酸。     

Malolactic fermentation in wine with high densities of non-proliferating Oenococcus oeni

Five strains of Oenococcus oeni (syn. Leuconostoc oenos) under non-proliferating conditions were assessed for the performance of the malolactic fermentation in wine at various initial pH values, malic acid concentration and densities of cells. We succeeded in inducing the malolactic fermentation after inoculation of high densities of O. oeni G6 even in recalcitrant wines where the traditional malolactic fermentation was inhibited by adverse environmental conditions (low pH and high concentration of malic acid). Optimal degrading conditions in wine, under different physico-chemical environments, were determined in order to achieve rapid depletion of malic acid in red wine. Off-odour compounds were not formed under these conditions, suggesting an attractive alternative for wine production. This revised version was published online in November 2006 with corrections to the Cover Date.     

Evaluation of Malolactic Bacteria Isolated from Oregon Wines

Oregon is a cool wine-producing region where grapes characteristically contain high concentrations of organic acids. To reduce the natural acidity and increase the microbiological stability and flavor complexity of the wine, malolactic fermentation is encouraged. In this study, strains of Leuconostoc oenos indigenous to Oregon wines were evaluated for their suitability to conduct malolactic fermentation in Oregon wines. Tests determined the malolactic activity of the Oregon isolates in comparison with commercial strains ML-34, PSU-1, MLT-kli, and ens 44-40 under various temperature and pH conditions. Sensitivities to sulfur dioxide, ethanol, and fumaric acid also were determined. Two Oregon strains, Er-1a and Ey-2d, were selected for commercial winemaking tests because they had greater malolactic activity under conditions of low pH (3.0) and low temperature (15 and 8°C), respectively.     

贺兰山东麓优良本土酿酒酵母筛选及发酵特性分析

【背景】商业酵母的使用造成葡萄酒同质化问题严重,发掘优良本土酿酒酵母具有十分重要的意义。【目的】从168株宁夏本土酿酒酵母菌株中筛选出性能优良、具有出色葡萄酒发酵能力的菌株。【方法】基于杜氏管发酵试验和乙醇、高糖等耐受性试验分析产H2S能力及生长曲线测定的方法,筛选出发酵力好、耐受性强、低产H2S的本土酿酒酵母进行赤霞珠葡萄酒发酵试验,测定葡萄酒样基础理化指标、酚类物质和挥发性成分,探究筛选出的酿酒酵母发酵特性。【结果】初步筛选出发酵快速,能适应13%乙醇、350 g/L葡萄糖、250 mg/L SO2、pH 1.0的生存环境且低产H2S的4株本土酿酒酵母YC-E8、QTX-D17、QTX-D7、YQY-E18。菌株YC-E8产甘油能力强,所发酵酒样香气与商业酵母XR、F33最为接近,适用于赤霞珠葡萄酒的发酵。菌株QTX-D17发酵酒样中酒精、单宁、总酚和花色苷含量最高,表现出本土酿酒酵母优良的发酵特性。菌株QTX-D7所发酵酒样香气中乙酸乙酯、辛酸乙酯、1-壬醇等物质含量较高,赋予了葡萄酒香蕉味、苹果味、菠萝味、椰子味等愉悦花果香。【结论】最终筛选出3株优良本土酿酒酵母QTX-D17...     

Integrated continuous winemaking process involving sequential alcoholic and malolactic fermentations with immobilized cells

An integrated winemaking process – including sequential alcoholic and malolactic fermentations operated continuously – was developed. For the continuous alcoholic fermentation, yeast cells (Saccharomyces cerevisiae) were immobilized either on grape stems or on grape skins, while bacterial cells (Oenococcus oeni) used for conducting continuous malolactic fermentation were immobilized on grape skins only. The produced wines were subjected to chemical analysis by HPLC (ethanol, glycerol, sugars and organic acids) and by gas chromatography (major and minor volatile compounds). The final proposed integrated continuous process permitted the production of 960 mL/d of a dry white wine, with an alcoholic strength of about 13 vol%, by using two 1.5 L tower bed reactors packed with 260 g of grape skins. The produced wines revealed a good physicochemical quality. Moreover, 67% of the malic acid concentration could be reduced in the second reactor. Both fermentative processes proved to be much more efficient than those conducted traditionally with free cells or even with immobilized cells, but in the batch mode of operation.     

Identification of pOENI-1 and Related Plasmids in Oenococcus oeni Strains Performing the Malolactic Fermentation in Wine

Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly contribute to the technological performance of strains in wine.     

Isolation and Enological Characterization of Malolactic Bacteria from the Vineyards of Northwestern Spain

Thirty-five strains of malolactic bacteria were isolated from grapes and alcoholic and malolactic fermentations in two vineyards from northwestern Spain. These belonged to six species of the genera Lactobacillus and Leuconostoc. The results of their partial enological characterization showed that 47.5% utilized more than 80% of the initial malic acid.     

Malolactic fermentation in Chardonnay: growth and sensory effects of commercial strains of Leuconostoc oenos

Samples of fermenting Chardonnay juice were inoculated with five commercial cultures of Leuconostoc oenos to promote malolactic fermentation. Controls were not inoculated with malolactic starter cultures; one was held under the same conditions as the juice inoculated with malolactic starter cultures and the other was held under conditions in which malolactic fermentation was inhibited. Bacterial growth and chemical composition of the wines were monitored for eight weeks after the wines were inoculated with the yeast starter culture. The five strains of L. oenos differed in growth kinetics and rates of malic acid degradation. Significant differences were detected among the finished wines subjected to sensory evaluation.     

Lactic acid bacteria in the quality improvement and depreciation of wine

The winemaking process includes two main steps: lactic acid bacteria are responsible for the malolactic fermentation which follows the alcoholic fermentation by yeasts. Both types of microorganisms are present on grapes and on cellar equipment. Yeasts are better adapted to growth in grape must than lactic acid bacteria, so the alcoholic fermentation starts quickly. In must, up to ten lactic acid bacteria species can be identified. They belong to the Lactobacillus, Pediococcus, Leuconostoc and Oenococcus genera. Throughout alcoholic fermentation, a natural selection occurs and finally the dominant species is O. oeni, due to interactions between yeasts and bacteria and between bacteria themselves. After bacterial growth, when the population is over 10⁶CFU/ml, malolactic transformation is the obvious change in wine composition. However, many other substrates can be metabolized. Some like remaining sugars and citric acid are always assimilated by lactic acid bacteri a, thus providing them with energy and carbon. Other substrates such as some amino acids may be used following pathways restricted to strains carrying the adequate enzymes. Some strains can also produce exopolysaccharides. All these transformations greatly influence the sensory and hygienic quality of wine. Malic acid transformation is encouraged because it induces deacidification. Diacetyl produced from citric acid is also helpful to some extent. Sensory analyses show that many other reactions change the aromas and make malolactic fermentation beneficial, but they are as yet unknown. On the contrary, an excess of acetic acid, the synthesis of glucane, biogenic amines and precursors of ethylcarbamate are undesirable. Fortunately, lactic acid bacteria normally multiply in dry wines; moreover some of these activities are not widespread. Moreover, the most striking trait of wine lactic acid bacteria is their capacity to adapt to a hostile environment. The mechanisms for this are not yet c ompletely elucidated . Molecular biology has provided some explanations for the behaviour and the metabolism of bacteria in wine. New tools are now available to detect the presence of desirable and undesirable strains. Even if much remains unknown, winemakers and oenologists can nowadays better control the process. By acting upon the diverse microflora and grape musts, they are more able to produce healthy and pleasant wines.     

Quantitative study of interactions between <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Oenococcus </Emphasis><Emphasis Type="Italic">oeni</Emphasis> strains

This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained.