梅鹿辄葡萄自然发酵酵母菌群研究

利用WL营养培养基对梅鹿辄葡萄自然发酵过程中分离到的98株酵母菌进行初步鉴定,进而对代表性的菌株进行5.8S rDNA-ITS区PCR产物的限制性内切酶酶切分析.结果表明,供试的98株酵母菌属于3属3个种,分别为酿酒酵母(Saccharomyces cerevisiae)、东方伊萨酵母(Issatchenkia orientalis)、克鲁维毕赤酵母(Pichia kluyver),3种酵母菌分别占分离自该葡萄品种总酵母菌株的80.61％、13.26％、6.13％.在自然发酵的初期和中期,酵母菌的比例因是否添加SO2而有所差异,但发酵末期均以S.cerevisiae为优势菌.     

Yeast dynamics during spontaneous wine fermentation of the Catalanesca grape

Although the use of starter cultures in winemaking has improved the reproducibility and predictability of wine quality, the main drawback to this practice is the lack of the typical traits of wines produced by spontaneous fermentation. In this study, we identified for the first time the yeast population occurring during spontaneous fermentation of the Catalanesca white grape, a variety from Campania (Italy). Yeasts were identified using molecular tools: PCR-DGGE and partial sequence analysis of the 26S rRNA gene from isolates. Eighteen different species belonging to 11 different genera were identified. Hanseniaspora spp., Issatchenkia spp. and Candida spp. were the dominant yeasts during the early stages of fermentation, while the middle and end phases were dominated by Saccharomyces cerevisiae. Four species of Issatchenkia spp., rarely isolated from wine fermentation, were found in this study accounting for the 33.5% of the total isolates. The RAPD-PCR screening of the isolates followed by partial rRNA gene sequencing proved to be a very effective approach to first differentiate the isolates and then identify yeast species involved in a wine making procedure. The results show very high yeast diversity in this natural wine fermentation and also highlight the possibility of considering interesting autochthonous strains for starter selection.     

Molecular identification and osmotolerant profile of wine yeasts that ferment a high sugar grape must

The objective of this study was to examine the Saccharomyces and non-Saccharomyces yeast populations involved in a spontaneous fermentation of a traditional high sugar must (Vino cotto) produced in central Italy. Molecular identification of a total of 78 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and sequencing of the D1/D2 domain of the 26S rRNA gene. In addition, the isolates were differentiated by RAPD-PCR. Only a restricted number of osmotolerant yeast species, i.e. Candida apicola, Candida zemplinina and Zygosaccharomyces bailii, were found throughout all the fermentation process, while Saccharomyces cerevisiae prevailed after 15 days of fermentation. A physiological characterization of isolates was performed in relation to the resistance to osmotic stress and ethanol concentration. The osmotolerant features of C. apicola, C. zemplinina and Z. bailii were confirmed, while S. cerevisiae strains showed three patterns of growth in response to different glucose concentrations (2%, 20%, 40% and 60% w/v). The ability of some C. apicola and C. zemplinina strains to grow at 14% v/v ethanol is noteworthy. The finding that some yeast biotypes with higher multiple stress tolerance can persist in the entire winemaking process suggests possible future candidates as starter for Vino cotto production.     

冰葡萄在自然发酵过程中酵母的动态变化

为了探讨云南德钦产区冰葡萄自然发酵过程中酵母的动态变化,采用WL培养基对不同阶段发酵液中的511株酵母进行形态划分。通过分析5.8S rDNA-ITS、18S rDNA以及26S rDNA的D1/D2区基因序列对酵母进行分子鉴定。结果表明:发酵过程中共存在5种酵母,葡萄破碎后立即取样Cryptococcus flavescens,Cryptococcusamylolentus,Hyphopichiapseudoburtonii,分别占50%,16.7%和33.3%。48h后,Cryptococcus amylolentus迅速减少甚至消失,Hyphopichiapseudoburtonii成为优势菌种,同时分离到Hanseniasporauvarum。待发酵启动后,发酵液中只存在Hanseniasporauvarum与Saccharomyces cerevisiae。随着发酵的进行,Hanseniasporauvarum所占的比例呈动态减少趋势。当还原糖下降至172 g/L,酒精含量上升为11.6%时,发酵液中只分离到Saccharomyces cerevisiae,直至发酵结束。     

Nitrogen requirements of commercial wine yeast strains during fermentation of a synthetic grape must

Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. Currently, the most common method for dealing with nitrogen-deficient fermentations is adding supplementary nitrogen (usually ammonium phosphate). However, it is important to know the specific nitrogen requirement of each strain, to avoid excessive addition that can lead to microbial instability and ethyl carbamate accumulation. In this study, we aimed to determine the effect of increasing nitrogen concentrations of three different nitrogen sources on growth and fermentation performance in four industrial wine yeast strains. This task was carried out using statistical modeling techniques. The strains PDM and RVA showed higher growth-rate and maximum population size and consumed nitrogen much more quickly than strains ARM and TTA. Likewise, the strains PDM and RVA were also the greatest nitrogen demanders. Thus, we can conclude that these differences in nitrogen demand positively correlated with higher growth rate and higher nitrogen uptake rate. The most direct effect of employing an adequate nitrogen concentration is the increase in biomass, which involves a higher fermentation rate. However, the impact of nitrogen on fermentation rate is not exclusively due to the increase in biomass because the strain TTA, which showed the worst growth behavior, had the best fermentation activity. Some strains may adapt a strategy whereby fewer cells with higher metabolic activity are produced. Regarding the nitrogen source used, all the strains showed the better and worse fermentation performance with arginine and ammonium, respectively.     

贺兰山东麓产区葡萄自然发酵液中酵母的多样性研究

为了分析宁夏贺兰山东麓葡萄自然发酵液中酵母多样性,从贺兰山东麓不同产区（广夏、立兰、银川和红寺堡）葡萄自然发酵的不同阶段分离酵母菌,并采用WL培养基与26S rDNA D1/D2序列分析法进行鉴定。结果表明,共分离到316株酵母,经鉴定被归为6个属9个种,其中在各产区均分离到有孢汉逊酵母（Hanseniaspora uvarum）、美极梅奇酵母（Metschnikowia pulcherrima）、Candida zemplinina和酿酒酵母（Saccharomyces cerevisiae）;在立兰、银川和红寺堡均分离到克鲁维毕赤酵母（Pichia kluyveri）;在红寺堡还分离到加利福尼亚假丝酵母（Candida californica）和伽状毕赤酵母（Pichia galeiformis）;库德里阿兹威氏毕赤酵母（Pichia kudriavzevii）仅存在于立兰和银川;戴尔有孢圆酵母（Torulaspora delbrueckii）仅存在于银川。宁夏贺兰山东麓葡萄自然发酵汁中酵母的多样性分析不仅为该产区酵母的开发利用奠定基础,也将为其他产区酵母筛选提供参考依据。     

Tannase activity by lactic acid bacteria isolated from grape must and wine

We examined a range of oenological lactic acid bacteria species and reference strains for their potential to degrade tannins. Bacterial tannase activity was checked by a spectrophotometric and a visual reading method. None of the strains belonging to the oenological species of the genus Lactobacillus, Leuconostoc, Oenococcus or Pediococcus were tannase producers, with the exception of Lactobacillus plantarum. All the L. plantarum strains analyzed were positive for tannase activity and their identities were reconfirmed by L. plantarum PCR-specific assay or by sequencing the 16S rDNA. Tannase activity could be considered an important criterion for the selection of malolactic starter cultures since it might confer advantages in the winemaking process by reducing astringency and haze in wine.     

Study of the oenological characteristicsand enzymatic activities of wine yeasts

Seventy-four wine strains of theSaccharomycesgenus were tested to study their oenological characteristics and enzymatic activities. These strains were isolated from spontaneous microbiota taken from cellars in the Valdepeñas region (Spain) and identified by contour homogeneous electric field (CHEF) technique. When Cluster Analysis was applied to oenological properties, only SH₂production, tolerance to 12⁰of ethanol and synthesis of killer toxin contributed to the distribution of the strains in the different statistical groups. All the strains possessed esterase activity and some also showed protease and pectinase activities. None of them hadβ-D-glucosidase activity.     

杏酒酵母的筛选及发酵性能的研究

对16株野生酵母进行杏酒发酵试验,通过理化指标的测定和感官评价,筛选适合酿造杏酒的酵母.结果表明,酵母16、酵母06、酵母02是具有良好酿酒特性的菌种.其中酵母16发酵的杏酒感官质量最佳,其发酵的杏酒金黄清亮,具有优雅的果香与醇香,酒体饱满,酸甜适口;并且在杏汁中具有良好的适应能力,分别能在16％vol乙醇、600mg/L SO2、6％柠檬酸、70％含糖量的杏汁中发酵,在10℃时仍可发酵,在50℃时可以生长,为发酵杏酒的最佳菌株.     

红提葡萄与酿酒葡萄复合果酒发酵工艺优化

该研究以新疆红提葡萄为主要原料,加入酿酒葡萄（宝石解百纳、赤霞珠）后制备红提葡萄与酿酒葡萄复合果酒,探讨最佳酿酒葡萄种类,并通过单因素及响应面试验探寻红提葡萄与酿酒葡萄复合果酒的最佳发酵工艺参数。结果表明,最佳酿酒葡萄为赤霞珠,红提葡萄与赤霞珠的质量比为4∶1;最佳发酵工艺条件为发酵温度20 ℃,初始pH值4.4,初始糖浓度28 °Bx。在此优化条件下,发酵所得红提葡萄与酿酒葡萄复合果酒的感官评分为95分,酒精度为8%vol,残糖量为13 °Bx,其色泽透亮,口感酸甜,果香浓郁,典型性明显,其理化指标及微生物指标均符合国家相关标准。     

几种红葡萄酿酒过程中花色苷组成与CIELab参数的相关分析

采用高效液相色谱-质谱联用技术测定了河北昌黎产区几个酿酒葡萄品种及其品系在葡萄酒酿造过程中的花色苷组成和含量,利用CIELab法分析了葡萄酒颜色参数及变化,采用相关性分析、主成分分析挖掘二者的相互关系。结果表明,发酵过程中同一品种不同品系葡萄酿造的葡萄酒之间花色苷种类相同,二甲花翠素类花色苷的含量远高于其他四类花色苷;不同类型花色苷影响不同的CIELab颜色参数,花色苷的B环修饰与红绿颜色参数a*显著正相关,花色苷的酰基化修饰与黄蓝颜色参数b*显著负相关,聚合花色苷与亮度L*显著负相关。红葡萄酒中花色苷的含量随酿造过程逐渐下降,且在主成分图上分布存在差异。     

Effect of freezing grape berries and heating must samples on extraction of components and composition parameters of red wine grape varieties

Juices from grape samples subjected to two storage methods (fresh and frozen) and two post-homogenisation treatments (21^oC and 71^oC) were examined for extraction of grape berry components and compared to a microvinified wine made from the same lot of fresh grapes. Cabernet Franc ( Vitis vinifera ) and Cynthiana ( Vitis aestivalis ) grapes were evaluated in 2003. Cabernet Franc, Cabernet Sauvignon ( Vitis vinifera ), Chambourcin (interspecific hybrid) and Cynthiana grapes were evaluated in 2004. Fresh grapes from each variety were sampled, analysed and prepared for microvinification. Frozen grapes of all varieties were thawed and analysed at the same time. For all varieties in both years, heating the must of fresh or frozen grapes increased the titratable acidity (11–58%), tartaric acid levels (12–110%), potassium content (15–62%), darkness (31–97%), red colour (54–1517%) and total red pigment values (36–1171%) of juice as compared to the juice from unheated must. For all varieties in 2004, heating the must from fresh or frozen grapes increased total phenolics (43–619%) of juice as compared to the juice from unheated must. Compared to fresh grape processing, freezing the grapes and then processing at 21^oC or 71^oC had varying results (increases and decreases) regarding the extraction of components in all varieties and both years but usually provided juice with composition most similar to that of the red wine produced by microvinification. Although the degree of extraction of individual components in the juice is influenced by selection of grape storage and must processing procedures, consistency of handling and procedure provided consistent results with low variability.     

赤霞珠葡萄自然发酵酵母菌的动态变化研究

葡萄酒发酵是一个复杂的生化过程,包含了不同酵母属种此消彼长的变化。利用WLN琼脂培养基,对陕西杨凌赤霞珠自然发酵过程中,不同阶段分离的403株酵母菌进行了归类鉴定。结果表明,其分属于9属10种:酿酒酵母(Saccharomyces cerevisiae)、葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)、东方伊萨酵母(Issatchenkia orientalis)、毕赤克鲁维酵母(Pichia kluyver)、膜璞毕赤酵母(Pichia membranefaciens)、美极梅奇酵母(Metschnikowia pulcherrima)、戴尔有孢圆酵母(Torulaspora delbrueckii)、红冬孢酵母(Rho-dosporidium mucilaginos)、假丝酵母(Candida species)和佛罗伦萨接合酵母(Zygosaccharomyces florentinus),分别占到了分离总菌株的53.00%、35.00%、1.00%、1.75%、0.75%、6.75%、0.25%、0.25%、1.00%和1.00%。     

Screening of biogenic amine production by lactic acid bacteria isolated from grape must and wine

The potential to produce the biogenic amines tyramine, histamine and putrescine, was investigated for lactic acid bacteria (LAB) of various origin, including commercial malolactic starter cultures, type strains and 78 strains isolated from Spanish grape must and wine. The presence of biogenic amines in a decarboxylase synthetic broth was determined by reverse-phase high performance liquid chromatography (RP-HPLC). Tyramine was the main amine formed by the LAB strains investigated. Leuconostoc strains were the most intensive tyramine formers. No potential to form biogenic amines was observed in Oenococcus oeni strains. Two strains of Latobacillus buchneri were associated with putrescine formation. None of the lactic acid bacteria produced histamine. According to these in vitro results, the commercial starter bacteria analyzed did not produce histamine, tyramine and putrescine.     

Changes in sour rotten grape berry microbiota during ripening and wine fermentation

This study investigated the microbiota of sour rotten wine grapes and its impact on wine fermentations. Yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were enumerated and identified on sound and sour rot grapes during the ripening stage. The alteration of the ecological balance induced by sour rot was particularly evidenced by the unequivocal increase of yeast and AAB counts on rotten grapes, since the beginning of ripening. Yeast and AAB species diversity in rotten grape samples were much higher than those found in sound grapes. LAB populations were low detected from both healthy and sour rotten grapes. The yeast species Issatchenkia occidentalis, Zygoascus hellenicus and Zygosaccharomyces bailii and the AAB species Gluconacetobacter hansenii, Gluconacetobacter intermedius and Acetobacter malorum, were recovered from damaged grapes and resulting grape juices in the winery. Acetobacter orleaniensis and Acetobacter syzygii were only recovered from sour rotten grapes. Dekkera bruxellensis and Oenococcus oeni were only recovered after wine fermentation induced by starter inoculation, irrespective of grape health, probably originating from cellar environment. After malolactic fermentation, racking and sulphur dioxide addition the only remaining species were the yeast Trigonopsis cantarellii and Saccharomyces cerevisiae, independently of the grape health status.     

不同酵母对干白葡萄酒发酵过程中香气变化的影响

选用两种酿酒酵母进行霞多丽干白葡萄酒发酵试验,酒精发酵过程中对发酵液的还原糖、可溶性固形物、总酸含量及香气物质进行分析。结果表明,两种酿酒酵母发酵过程中发酵液的还原糖和可溶性固形物均呈下降趋势,总酸含量均大于葡萄汁总酸含量;葡萄汁和发酵液中共检测出68种香气物质。相比于葡萄汁,酵母LA-DE和LA-PE发酵时发酵液中醇类、酯类、酸类、萜烯类和酚类物质含量随发酵时间延长逐渐增加,而醛类物质逐渐减少;其中酯类物质含量增加显著,发酵结束时分别为22.79 mg/L和14.57 mg/L。主成分分析结果表明,发酵过程中香气物质在前3 d变化较为明显,两种酵母发酵液在第1天、第3天、第9天香气物质差异明显。     

Evolution of the population of Saccharomyces cerevisiae from grape to wine in a spontaneous fermentation

To determine the grape or winery origin of the Saccharomyces cerevisiae involved in spontaneous fermentation, musts were collected at different stages of wine-making process and fermented. First, grapes were collected in two different vineyards and crushed at the laboratory. Second, musts were collected after crushing and clarification in the cellar. Third, musts collected in the cellar were sterilized and inoculated with tartar deposit collected in the vats. The fourth fermentation was in the cellar. For the two vineyards, two hundred of S. cerevisiae clones were isolated for each of the four fermentations, driving to a library of 1600 clones. All the library was analysed by inter-delta PCR with a basic set of primers and about 20% of the library was further analysed by inter-delta PCR with an improved set of primers. Six, and more than 30 different PCR patterns were obtained from basic- and improved-PCR analysis, respectively. The amounts of each family were analysed at the different stages of wine making. Our study demonstrates that the two vineyards present different S. cerevisiae populations. Moreover the S. cerevisiae strains involved in spontaneous fermentation in the cellar originate partly from the vineyard and partly from the winery, in amounts varying with the must.     

不同菌种对青稞酒发酵的影响

以青稞酒传统生产工艺为基础,研究了发酵过程中使用不同菌种对青稞酒感官品质和出酒率的影响。结果表明,以传统的青稞整粒固态发酵工艺,以单一菌种发酵生产的青稞酒产品其出酒率和产品感官品质几乎没有提高,而使用酿酒酵母和甜酒曲复合菌种,可以在保证产品观感质量的前提下相对于当地青稞酒生产工艺提高出酒率1倍以上。      

Clarifying agents effect on the nitrogen composition in must and wine during fermentation

The effect of static sedimentation with and without clarifying agents (silica sol and gelatine) upon the nitrogenous fraction of musts and wines was studied. Four vinifications were carried out using a Vitis vinifera cv. Cayetana white grape must. Static sedimentation reduced less than other techniques the assimilable nitrogen (FAN), however the employment of fining agents promoted a net decrease. The changes in the amino acids during fermentation were similar in all the experiments carried out. In general, during the first days there was a fast decrease followed by a slight increment and then stabilisation. This decrease fitted, in most of the cases, with first order kinetics. For most of the amino acids, the percent consumption was higher in the must settled with clarifying agents. The clarifying agent’s addition did not have the same effect on the amino acid concentration in the final wines.     

Formation of α-ketoglutaric acid by wine yeasts and its oenological significance

α-Ketoglutaric acid was measured enzymically in wines made in the laboratory from three grape varieties by pure cultures of 12 wine yeasts of the genus Saccharomyces. The results were confirmed with the same juices and 4 yeasts on pilot-plant scale in replicated 30 gallon lots. Mean values for the 12 yeasts ranged from 9 to 117 ppm (overall mean 53). In any one juice the yeasts differed by at least 10-fold in the amounts produced, and certain yeasts produced consistently high or low yields in all juices. The amounts of α-ketoglutaric acid produced depended somewhat on the grape juices used, even though these had comparable pH values, and a significant yeast-juice interaction occurred. The amount of α-ketoglutaric acid formed during fermentation at 15° was 60 per cent of that formed at 25°, and over twice as much was formed at pH 4.2 as at pH 3.0, using four yeast strains. Formation of α-ketoglutaric and pyruvic acids were not significantly correlated. The α-ketoglutaric acid content of 18 white table wines made under comparable conditions on pilot-plant scale from different grape varieties using the same yeast strain ranged from 38 to 152 ppm (mean 90). The significance of the results is discussed, particularly in relation to the binding of sulphur dioxide in wine, and recommendations are given on how to make wines which are low in α-ketoglutaric acid. Formation of α-ketoglutaric acid by three yeasts in a chemically defined medium was lower with increased amounts of nitrogen as ammonium sulphate and higher in the presence of L-glutamic acid, both being used separately as sole nitrogen sources. These findings are discussed in relation to the rǒle of α-ketoglutaric acid in nitrogen metabolism of yeasts.