葡萄酒苹果酸-乳酸菌精氨酸代谢研究概况

葡萄酒苹果酸-乳酸菌的精氨酸代谢会导致葡萄酒中氨基甲酸乙酯含量的增加,从而严重影响葡萄酒的饮用安全性。近年来研究表明,葡萄酒苹果酸-乳酸菌的精氨酸代谢途径是精氨酸脱亚氨基酶途径(Arginine deiminasepathway,简称ADI途径)。系统分析苹果酸-乳酸菌的ADI途径、精氨酸转运机制、ADI途径酶的调节等方面的研究进展,阐明葡萄酒苹果酸-乳酸菌的精氨酸代谢对酿造优质葡萄酒具有重要的理论和实际意义。     

The inhibitory activity of wine yeast starters on malolactic bacteria

Malolactic fermentation is a process that is influenced by various factors that can inhibit the growth of the malolactic bacteria. Inhibitory metabolites produced by yeast may have an important role in the correct development of malolactic fermentation. For these reasons, we have investigated the effects of such metabolites on the growth of malolactic bacteria under different environmental conditions, to aid in our understanding of the significance of these interactions in the wine-making environment. Our screening methods to detect interactions between yeast and malolactic bacteria showed a variable and wide diffusion of yeast inhibitory activity on the growth of the malolactic bacteria. However, this first approach to determine this inhibitory activity of yeast gave an overestimation when compared to the results obtained under actual wine-making conditions. The evaluation of malic acid consumption indicated that under inhibitory conditions a partial L-malic acid degradation was seen, indicating that the malolactic activity continued without bacterial growth. However, these yeast-inhibiting effects in addition to other environmental factors could cause a complete failure of malolactic fermentation.     

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

AIMS: Acetaldehyde is the major carbonyl compound formed during winemaking and has implications for sensory and colour qualities of wines as well as for the use of the wine preservative SO(2). The current work investigated the degradation of acetaldehyde and SO(2)-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, SO(2)-bound acetaldehyde caused sluggish bacterial growth. Strain differences were small. CONCLUSIONS: Efficient degradation of acetaldehyde can be achieved by commercial starters of O. oeni. According to the results, the degradation of acetaldehyde could not be separated from malolactic conversion by oenococci. While this may be desirable in white winemaking, it may be necessary to delay malolactic fermentation (MLF) in order to allow for colour development in red wines. SO(2)-bound acetaldehyde itself maybe responsible for the sluggish or stuck MLF, and thus bound SO(2) should be considered next to free SO(2) in order to evaluate malolactic fermentability. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides new results regarding the metabolism of acetaldehyde and SO(2)-bound acetaldehyde during the MLF in white wine. The information is of significance to the wine industry and may contribute to reducing the concentration of wine preservative SO(2).     

Predominance of malolactic fermentation overd-glucose utilization inLactobacillus plantarum andLactobacillus curvatus wine strains

Summary Two selected wine strains of the genusLactobacillus (L. plantarum 197 andL. curvatus 783) were tested for their ability to complete malolactic fermentation (MLF) in a synthetic medium (PBM-broth) supplemented withL-malic acid (7.5–74.6 mM) andD-glucose (5.5–55 mM). The 24 directed fermentation assays, 12 for each bacterial strain, were carried out at 20°C and pH 3.5. MLF was completed (residualL-malic acid 0.2 mM) in eight days in 19 of the 24 fermentation assays, even in the presence of 74.6 mML-malic acid or 55.5 mMD-Glucose utilization was generally simultaneous to MLF but was completed (residual concentrations 0.2 mM) only in 6 of the 24 fermentation assays. These results support the use of these strains in directed MLF assays at the very differentL-malic acid andD-glucose concentrations tested.     

A homozygous diploid subset of commercial wine yeast strains

Genetic analysis was performed on 45 commercial yeasts which are used in winemaking because of their superior fermentation properties. Genome sizes were estimated by propidium iodide fluorescence and flow cytometry. Forty strains had genome sizes consistent with their being diploid, while five had a range of aneuploid genome sizes that ranged from 1.2 to 1.8 times larger. The diploid strains are all Saccharomyces cerevisiae, based on genetic analysis of microsatellite and minisatellite markers and on DNA sequence analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA of four strains. Four of the five aneuploid strains appeared to be interspecific hybrids between Saccharomyces kudriavzevii and Saccharomyces cerevisiae, with the fifth a hybrid between two S. cerevisiae strains. An identification fingerprint was constructed for the commercial yeast strains using 17 molecular markers. These included six published trinucleotide microsatellites, seven new dinucleotide microsatellites, and four published minisatellite markers. The markers provided unambiguous identification of the majority of strains; however, several had identical or similar patterns, and likely represent the same strain or mutants derived from it. The combined use of all 17 polymorphic loci allowed us to identify a set of eleven commercial wine yeast strains that appear to be genetically homozygous. These strains are presumed to have undergone inbreeding to maintain their homozygosity, a process referred to previously as ‘genome renewal’.     

Ethyl carbamate precursor citrulline formation from arginine degradation by malolactic wine lactic acid bacteria

Major commercially available strains for induction of malolactic fermentation in wine were examined for arginine metabolism in a resting cell system at wine pH with the aim of evaluating their ability to excrete and utilize citrulline, a precursor of carcinogenic ethyl carbamate (urethane). All strains tested excreted citrulline from arginine degradation. Citrulline was stored intracellularly during growth in arginine rich medium and was released upon lysis of the cells. All strains were found to degrade citrulline as a sole amino acid and some of them were able to reutilize previously excreted citrulline.     

Cytofluorometric detection of wine lactic acid bacteria: application of malolactic fermentation to the monitoring

In this study we report for the first time a rapid, efficient and cost-effective method for the enumeration of lactic acid bacteria (LAB) in wine. Indeed, up to now, detection of LAB in wine, especially red wine, was not possible. Wines contain debris that cannot be separated from bacteria using flow cytometry (FCM). Furthermore, the dyes tested in previous reports did not allow an efficient staining of bacteria. Using FCM and a combination of BOX/PI dyes, we were able to count bacteria in wines. The study was performed in wine inoculated with Oenococcus oeni (10⁶ CFU ml^?1) stained with either FDA or BOX/PI and analyzed by FCM during the malolactic fermentation (MLF). The analysis show a strong correlation between the numbers of BOX/PI-stained cells determined by FCM and the cell numbers determined by plate counts (red wine: R ² ≥ 0.97, white wine R ² ≥ 0.965). On the other hand, we found that the enumeration of O. oeni labeled with FDA was only possible in white wine (R ² ≥ 0.97). Viable yeast and LAB populations can be rapidly discriminated and quantified in simultaneous malolactic-alcoholic wine fermentations using BOX/PI and scatter parameters in a one single measurement. This rapid procedure is therefore a suitable method for monitoring O. oeni populations during winemaking, offers a detection limit of <10⁴ CFU ml^?1 and can be considered a useful method for investigating the dynamics of microbial growth in wine and applied for microbiological quality control in wineries.     

Performance assessment of malolactic fermenting bacteria Oenococcus oeni and Lactobacillus brevis in continuous culture

The growth performance of malolactic fermenting bacteria Oenococcus oeni NCIMB 11648 and Lactobacillus brevis X₂ was assessed in continuous culture. O. oeni grew at a dilution rate range of 0.007 to 0.052 h⁻¹ in a mixture of 5:6 (g l⁻¹) of glucose/fructose at an optimal pH of 4.5, and L. brevis X₂ grew at 0.010 to 0.089 h⁻¹ in 10 g l⁻¹ glucose at an optimal pH of 5.5 in a simple and safe medium. The cell dry weight, substrate uptake and product formation were monitored, as well as growth kinetics, yield parameters and fermentation balances were also evaluated under pH control conditions. A comparison of growth characteristics of two strains was made, and this showed significantly different performance. O. oeni has lower maximum specific growth rate (μ_max=0.073 h⁻¹), lower maximum cell productivity (Q _x ^max=17.6 mg cell l⁻¹ h⁻¹), lower maximum biomass yield (Y _x/s ^max=7.93 g cell mol⁻¹ sugar) and higher maintenance coefficient (m _s=0.45 mmol⁻¹ sugar g⁻¹ cell h⁻¹) as compared with L. brevis X₂ (μ_max=0.110 h⁻¹; Q _x ^max=93.2 g⁻¹ cell mol⁻¹ glucose; Y _x/s ^max=22.3 g cell mol⁻¹ glucose; m _s=0.21 mmol⁻¹ glucose g⁻¹ cell h⁻¹). These data suggest a possible more productive strategy for their combined use in maturation of cider and wine.     

Analysis of the stress resistance of commercial wine yeast strains

Alcoholic fermentation is an essential step in wine production that is usually conducted by yeasts belonging to the species Saccharomyces cerevisiae. The ability to carry out vinification is largely influenced by the response of yeast cells to the stress conditions that affect them during this process. In this work, we present a systematic analysis of the resistance of 14 commercial S. cerevisiae wine yeast strains to heat shock, ethanol, oxidative, osmotic and glucose starvation stresses. Significant differences were found between these yeast strains under certain severe conditions, Vitilevure Pris Mouse and Lalvin T73 being the most resistant strains, while Fermiblanc arom SM102 and UCLM S235 were the most sensitive ones. Induction of the expression of the HSP12 and HSP104 genes was analyzed. These genes are reported to be involved in the tolerance to several stress conditions in laboratory yeast strains. Our results indicate that each commercial strain shows a unique pattern of gene expression, and no clear correlation between the induction levels of either gene and stress resistance under the conditions tested was found. However, the increase in mRNA levels in both genes under heat shock indicates that the molecular mechanisms involved in the regulation of their expression by stress function in all of the strains.     

Molecular and biochemical diversity of Oenococcus oeni strains isolated during spontaneous malolactic fermentation of Malvasia Nera wine

The diversity of indigenous Oenococcus oeni strains was investigated by molecular and biochemical characterization of isolates from Malvasia Nera wine, an economically important red wine of the Salento Region (Apulia, Italy), during spontaneous malolactic fermentation (MLF). A total of 82 isolates of this species, identified by species-specific PCR and 16S rDNA sequence analysis, was molecularly characterized by the amplified fragment length polymorphism (AFLP) technique. Three main groups resulted from cluster analysis and showed intraspecific homology higher than 50%, and a total of seven subgroups, with similarity values ranged from 80% to 98%, were obtained within these groups. Enzymatic activities, such as esterase, β-glucosidase, protease, and the consumption rate of l-malic acid, citric acid, acetaldehyde and arginine were assessed in the representative strains, according to AFLP analysis. The results showed different enzymatic activities and consumption rates of the tested metabolites among the strains. No correlation between molecular and biochemical data was observed. The evidence of biochemical variability observed among Malvasia Nera strains demonstrated that the wine aroma can be modulated depending on the strains involved in MLF. Hence, the heterogeneity existing within natural O. oeni populations represents an interesting ecological source that can be useful for technological purposes.     

Leuconostoc oenos and malolactic fermentation in wine: a review

This review article summarizes the state of the art on Leuconostoc oenos, the bacteria responsible for malolactic fermentation in wine. Both basic and practical aspects related to the metabolism of this microorganism and malolactic fermentation in general are critically reviewed. The former examines the role of genetics for the identification and classification of L. oenos and energetic mechanisms on solute transport (malic and lactic acid). The latter includes practical information on biomass production, optimal growth conditions and stress factors, which are important in growth optimization of malolactic starter cultures. Extensive data and references on the effect of malolactic fermentation on wine composition and sensory analysis are also included. Received 06 May 1999/ Accepted in revised form 13 July 1999     

Evolution of malolactic bacteria and biogenic amines during spontaneous malolactic fermentations in a Greek winery

AIMS: To study the population dynamics of indigenous malolactic bacteria in a Greek winery and to examine their potential to produce detrimental levels of biogenic amines (BA) under winemaking conditions. METHODS AND RESULTS: Although the wines studied were of different vintage, grape variety and enological characteristics, molecular typing of malolactic bacteria revealed only a low number of strains within the single-species populations of Oenococcus oeni that developed during spontaneous fermentations. Strain MF1, originating primarily from the vineyards surrounding the winery invariably predominated in almost all samples. HPLC analysis showed a slight increase in the BA, putrescine, tyramine and phenylethylamine after malolactic conversion, while histamine, methylamine and ethylamine remained unaffected. No correlation could be established between the BA profiles and the bacterial compositions or the amino acid concentrations in wine samples studied. CONCLUSIONS: A certain regional bacterial flora is established in the winery that prevails in spontaneous malolactic fermentations (MLF) irrespective of the wine characteristics. In all cases, the BA content of the wines after malolactic conversion was within enologically acceptable levels. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the malolactic bacteria occurring naturally in spontaneous MLF in Greek red wines and a preliminary assessment of their impact on wine safety in relation to BA.     

Inhibition of bacterial growth and malolactic fermentation in wine by bacteriophage

Bacteriophage present in wine can attack bacterial starter cultures and inhibit the malolactic fermentation. The possibility of starter culture failure due to phage attack was studied in a commercial dry red wine of pH 3·23, inoculated with a multiple strain starter culture. During two stages of malolactic fermentation, bacterial growth and malate degradation in the wine were inhibited. A phage capable of lysing isolates of Leuconostoc oenos was isolated from the wine. The isolated phage had an icosahedral head of 42–45 nm diameter and a flexible, regularly cross-striated tail 197–207 nm long with a small baseplate. The results confirm that phage can attack bacterial starter cultures in wine at low pH.     

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.     

Role of malolactic fermentation in lactic acid bacteria

Although decarboxylation of malate to lactate by malolactic enzyme does not liberate biologically available energy (e.g., ATP, NADH), the growth rate of many malolactic bacteria is greatly enhanced by malolactic fermentation. The deacidification of the medium due to malate dissipation cannot fully account for this situation. The chemiosmotic theory postulates that another form of energy could generated by translocation of protons through the membrane coupled to end-product efflux. Konings et al. showed that this theory is indeed applicable to lactate efflux in Streptococcus cremoris at pH 7.0. A similar mechanism could account for the observed increased activity in malolactic bacteria. The study in wild type and mutant strains of Streptococcus lactis unable to carry out malolactic fermentation led us to the following conclusions: (1) under glucose non-limiting conditions, malolactic fermentation helps to maintain pH of the medium at a certain level; (2) during glucose limited growth, malolactic fermentation could be coupled with an energetic process independent from that mentioned above.     

Technological properties of indigenous wine yeast strains isolated from wine production regions of Turkey

The purpose of this study was to evaluate the important technological and fermentative properties of wine yeast strains previously isolated from different wine producing regions of Turkey. The determination of the following important properties was made: growth at high temperatures; fermentative capability in the presence of high sugar concentration; fermentation rate; hydrogen sulfide production; killer activity; resistance to high ethanol and sulfur dioxide; foam production; and enzymatic profiles. Ten local wine yeast strains belonging to Saccharomyces, and one commercial active dry yeast as a reference strain were evaluated. Fermentation characteristics were evaluated in terms of kinetic parameters, including ethanol yield (Y_P/S), biomass yield (Y_X/S), theoretical ethanol yield (%), specific ethanol production rate (q_p; g/gh), specific glucose uptake rate (q_s; g/gh), and the substrate conversion (%). All tested strains were able to grow at 37 °C and to start fermentation at 30° Brix, and were resistant to high concentrations of sulfur dioxide. 60 % of the strains were weak H₂S producers, while the others produced high levels. Foam production was high, and no strains had killer activity. Six of the tested strains had the ability to grow and ferment at concentrations of 14 % ethanol. Except for one strain, all fermented most of the media sugars at a high rate, producing 11.0–12.4 % (v/v) ethanol. Although all but one strain had suitable characteristics for wine production, they possessed poor activities of glycosidase, esterase and proteinase enzymes of oenological interest. Nine of the ten local yeast strains were selected for their good oenological properties and their suitability as a wine starter culture.