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。     

Development of a quantitative PCR for detection of Lactobacillus plantarum starters during wine malolactic fermentation

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at 3.6 × 10(6) CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above 10(5)/ml for approx. 10 days, after which cell numbers decreased to levels of 10(3) CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. 1 × 10(2) CFU/ml was detected. The minimum detection level for quantitative PCR in this study was 1 × 10(2) to 1 × 10(3) CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.     

Performance and diacetyl production of commercial strains of malolactic bacteria in wine

This study compares 11 commercial cultures of Leuconostoc oenos and Lactobacillus plantarum in Cabernet Sauvignon, Pinot Noir and Chardonnay wines. Performance of the cultures was found to be greatly influenced by wine type. Better survival of the bacteria was observed in Cabernet Sauvignon and Pinot Noir wines. The time necessary to complete malolactic fermentation (MLF) was 65 ± 14 d for Chardonnay, 71 ± 3 d for Cabernet Sauvignon, and 25 ± 8 d for Pinot Noir. The maximal rate of malate utilization was 0·4 g d^-1 for Pinot Noir, and 0·2 g d^-1 for the two other wine types. Final diacetyl concentration was lower in Chardonnay wines (highest 0·58 mg l^-1) compared to the other wines (highest 5·8 mg l^-1). Malic and citric acid were co-metabolized by all strains. None of the strains metabolized glycerol. Significant differences in final diacetyl concentration of wine vinified with the different strains were found. Panelists could reliably differentiate MLF wines from non-MLF wines, irrespective of their diacetyl content, indicating that diacetyl is not the only important MLF flavour.     

Effects of yeast proteolytic activity on Oenococcus oeni and malolactic fermentation

Alcoholic fermentation of synthetic must was performed using either Saccharomyces cerevisiae or a mutant Deltapep4, which is deleted for the proteinase A gene. Fermentation with the mutant Deltapep4 resulted in 61% lower levels of free amino acids, and in 62% lower peptide concentrations at the end of alcoholic fermentation than in the control. Qualitative differences in amino acid composition were observed. Changes observed in amino acids in peptides were mainly quantitative. After alcoholic fermentation, each medium was inoculated with Oenococcus oeni. Malolactic fermentation in the medium with the Deltapep4 strain took 10 days longer than the control. This difference may have been due to a difference in the nitrogen composition of the two media. Free amino acids and amino acids in peptides were poorly consumed by O. oeni. Thus, the qualitative aspects of nitrogen composition, which depend in part on yeast metabolism, may be a determinant for the optimal growth of O. oeni in wine.     

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).     

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.     

The use of alternative technologies to develop malolactic fermentation in wine

The development of the malolactic fermentation, bioconversion of L-malic acid to L-lactic acid, is a difficult and time-consuming process that does not always proceed favorably under the natural conditions of wine. Traditional fermentations are used worldwide to produce high-quality wines, although delay or failure is not an unusual outcome. During recent years several technologies have been proposed to induce biological deacidification of wines by using malolactic bacteria, principally Oenococcus oeni and Lactobacillus sp. These alternative technologies usually involve the use of high densities of cells or enzymes, free or immobilized onto different matrices. Immobilization materials, several types of bioreactors, and the properties of many specific systems are discussed in this review.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.     

Evolution during wine aging of colour and tannin differences induced by wine starters

The ability of threeSaccharomyces cerevisiae strains, with different colour adsorption aptitude, to induce and maintain colour differences in wines obtained from the Calabrian Gaglioppo and Magliocco black grape varieties was studied during one year of aging. The evolution of wine tannin content was also considered. Total polyphenols, colour parameters and total tannin values exhibited, both for Gaglioppo and Magliocco wines, significant (P < 0.05) or highly significant (P < 0.01) differences among strains. It is interesting to note that yeasts appear to exhibit a different adsorption aptitude for anthocyanins and tannins. The strain that gave wine with high values for the colour parameters was not the same as the one that produced wine with high values of tannins. The obtained results suggest that the choice of yeast strain in winemaking affects, in a significant way, the phenolic composition of wines with direct consequences on their colour and tannin content. Moreover, the interaction between grape cultivar and yeast is close and important, because grape variety, due to its phenolic composition, modulates yeast strain adsorption activity.     

The effect of polysaccharide-degrading wine yeast transformants on the efficiency of wine processing and wine flavour

Commercial polysaccharase preparations are applied to winemaking to improve wine processing and quality. Expression of polysaccharase-encoding genes in Saccharomyces cerevisiae allows for the recombinant strains to degrade polysaccharides that traditional commercial yeast strains cannot. In this study, we constructed recombinant wine yeast strains that were able to degrade the problem-causing grape polysaccharides, glucan and xylan, by separately integrating the Trichoderma reesei XYN2 xylanase gene construct and the Butyrivibrio fibrisolvens END1 glucanase gene cassette into the genome of the commercial wine yeast strain S. cerevisiae VIN13. These genes were also combined in S. cerevisiae VIN13 under the control of different promoters. The strains that were constructed were compared under winemaking conditions with each other and with a recombinant wine yeast strain expressing the endo-beta-1,4-glucanase gene cassette (END1) from B. fibrisolvens and the endo-beta-1,4-xylanase gene cassette (XYN4) from Aspergillus niger, a recombinant strain expressing the pectate lyase gene cassette (PEL5) from Erwinia chrysanthemi and the polygalacturonase-encoding gene cassette (PEH1) from Erwinia carotovora. Wine was made with the recombinant strains using different grape cultivars. Fermentations with the recombinant VIN13 strains resulted in significant increases in free-flow wine when Ruby Cabernet must was fermented. After 6 months of bottle ageing significant differences in colour intensity and colour stability could be detected in Pinot Noir and Ruby Cabernet wines fermented with different recombinant strains. After this period the volatile composition of Muscat d'Alexandria, Ruby Cabernet and Pinot Noir wines fermented with different recombinant strains also showed significant differences. The Pinot Noir wines were also sensorial evaluated and the tasting panel preferred the wines fermented with the recombinant strains.     

The occurrence of malolactic fermentation in brandy base wine and its influence on brandy quality

AIMS: In this study we determined the extent to which lactic acid bacteria (LAB) occurred in brandy base wines, their ability to catalyse the malolactic fermentation (MLF) and the effect of MLF on the quality of the base wine and the brandy distillate. METHODS AND RESULTS: Lactic acid bacteria were isolated and enumerated from grape juice, experimental and commercially produced brandy base wines. Spontaneous MLF occurred in approximately 50% of the commercial base wines. The occurrence of MLF had an influence on the quality of the base wines and the resulting distillates. In samples where MLF occurred there was a loss of fruitiness and in the intensity of aroma. Volatile compounds like iso-amyl acetate, ethyl acetate, ethyl caproate, 2-phenethyl acetate and hexyl acetate decreased in samples having undergone MLF, while ethyl lactate, acetic acid and diethyl succinate increased in the same samples. CONCLUSIONS: Spontaneous malolactic fermentation does occur in commercial brandy base wines and it has an influence on base wine and brandy quality. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that MLF influences the quality of the base wine and the resulting distillate and with this in mind commercial base wine producers should be able to produce brandy of higher quality.     

三种酒曲酿造的崇明老白酒的挥发性风味物质分析及比较

分别采用八二酒曲、纯种米根霉曲及酿酒酵母曲、纯种米根霉曲和粘红酵母曲按崇明老白酒工艺酿造老白酒,应用顶空固相微萃取-气相色谱质谱联用法对样品进行挥发性物质的检测,并对其进行醇酯比、香气活力值(OAVs)的计算及主成分分析,筛选出较佳的酒曲,得出崇明老白酒的主体香气.辛酸乙酯、1-辛烯-3-醇、3-甲基丁酸乙酯、己酸乙酯和乙酸异戊酯的OAVs大于10,是崇明老白酒的主要呈香物质.由八二酒曲酿造的酒液挥发性物质总量最高,醇酯比为5.89∶1,呈香物质以具蘑菇香的1-辛烯-3-醇为主.米根霉和酿酒酵母曲酿造的酒液酯类、芳香族化合物种类最多,醇酯比3.24∶1,香气以丁酸乙酯的甜香、苯乙醛、苯乙醇和乙酸苯乙酯的玫瑰花香为主要特征.米根霉和粘红酵母曲酿造的酒液挥发性化合物总量最低,醇酯比为2.86,主要香气特征为辛酸乙酯的甜香和己酸乙酯的水果香.纯种曲酿造的酒液的香气类型、风味种类比例、呈香物质的丰富程度均优于八二酒曲酿造的酒液.     

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.     

Nutrient depletion modifies cell wall adsorption activity of wine yeast

Yeast cell wall is a structure that helps yeasts to manage and respond to many environmental stresses. The mannosylphosphorylation is a modification in response to stress that provides the cell wall with negative charges able to bind compounds present in the environment. Phenotypes related to the cell wall modification such as the filamentous growth in Saccharomyces cerevisiae are affected by nutrient depletion. The present work aimed at describing the effect of carbon and/or nitrogen limitation on the aptitude of S. cerevisiae strains to bind coloured polyphenols. Carbon- and nitrogen-rich or deficient media supplemented with grape polyphenols were used to simulate different grape juice conditions—early, mid, ‘adjusted’ for nitrogen, and late fermentations. In early fermentation condition, the R+G+B values range from 106 (high adsorption, strain Sc1128) to 192 (low adsorption, strain Σ1278b), in mid-fermentation the values range from 111 (high adsorption, strain Sc1321) to 258 (low adsorption, strain Sc2306), in ‘adjusted’ for nitrogen conditions the values range from 105 (high adsorption, strain Sc1321) to 194 (low adsorption, strain Sc2306) while in late fermentation conditions the values range from 101 (high adsorption, strain Sc384) to 293 (low adsorption, strain Sc2306). The effect of nutrient availability is not univocal for all the strains and the different media tested modified the strains behaviour. In all the media the strains show significant differences. Results demonstrate that wine yeasts decrease/increase their parietal adsorption activity according to the nutrient availability. The wide range of strain variability observed could be useful in selecting wine starters.     

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.