Distribution of phenolic yeasts and production of phenolic off-flavors in wine fermentation

The activity of wine yeasts to decarboxylate ferulic and p-coumaric acids is one of their biological properties related to the production of phenolic off-flavors (POF) in wine-making. We examined POF productivity in 116 strains of wine yeast, 74 strains of wild yeast (Saccharomyces cerevisiae) and 23 strains of non-Saccharomyces yeast, and found that a majority of these yeasts were POF-producing strains. The frequency distribution of POF-producing strains was 81 to 95% in wine yeasts, 85 to 97% in wild yeasts and 78 to 83% in non-Saccharomyces yeasts based on the POF test with addition of ferulic and p-coumaric acids to grape juice medium. The Rhodotorula, Candida, Cryptococcus, Pichia, Hansenula, and Brettanomyces strains had high or moderate POF productivity among the 20 non-Saccharomyces species. The decomposition rate of ferulic acid correlated with POF production and the critical concentration of phenolic acid (free form) in grape must was estimated to be more than 10 mg/l. Segregation of POF phenotype and Southern blot analysis of phenolic wine yeasts suggest that POF production is controlled by the POF gene (PAD1). The results showed the frequent distribution of phenolic yeasts in the wine-making environment. These suggest the importance of controlling POF production by using wine yeast strains of low POF productivity. The grapes must be prepared by a suitable process to prevent the increase in phenolic acid content.     

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.     

耐高渗酵母选育及其在葡萄酒中的应用研究

高醇、高糖、高硫等条件会影响酵母菌的生物活性。为筛选出耐高酒精度、高糖度、高SO2浓度的葡萄酒酿造优良菌株,该实验采集葡萄酒糟、黄酒糟、酒醅,经过富集筛选分离纯化得到8株酵母菌,经高酒精度、高糖度、高SO2耐受试验后初步筛选出2株菌株,一株耐高糖,在含糖量60%条件下21.5 h仍可启动发酵;一株耐高酒精度,在酒精度20%vol条件下21.3 h仍可启动发酵;2株菌株均可在300 mg/L SO2条件下在6.7 h启动发酵。经生理生化试验初步鉴定两株菌分别为酿酒酵母（Saccharomyces cerevisiae）和膜醭毕赤氏酵母（Pichia membranefaciens）。在浓缩葡萄汁初始糖含量28%条件下接种酿酒酵母,高泡期补料至含糖量34%接种膜醭毕赤氏酵母,20 ℃发酵15 d,得到的葡萄酒酒精度为18.1%vol。     

杨梅果酒酿造酵母的筛选、鉴定及耐受性研究

从杨梅果园周边环境及杨梅自然发酵醪中共分离得到152株酵母菌,通过TTC显色法、杨梅汁杜氏管法及三角瓶发酵法进行三级筛选,共得到4株酵母在产气性能、色泽及风味方面比较突出,分别为110号、147号、151号及152号,经过分子生物学鉴定,确定4株菌分别为克鲁弗毕赤酵母、有孢汉逊酵母、库德里阿兹威氏毕赤酵母和酿酒酵母。将筛选到的4株酵母进行耐受性测试,其中152号耐受性最好,在酒精含量18%vol、SO_2含量250 mg/L、糖含量50%和pH1.5的条件下均有较好的产气性能,非常适合杨梅果酒的酿造;110号、147号和151号可用于生产低醇果汁饮料、酿造低酒精度果酒或与产酒力高的果酒酵母混合发酵时增香。     

Cork-borne bacteria and yeasts as potential producers of off-flavours in wine

Bacteria and yeasts were found to be present within cork lenticels, covered by mucous or fibrous substances. They survived heating, peroxide treatment and contact with the alcohol and sulfur dioxide of wine. 187 bacteria and 36 yeast strains were isolated from cork stoppers of wine bottles and, during various stages of production, from corkwood and new cork stoppers. After culturing, a number of isolates showed the ability to modify the aroma of model systems consisting of dilute or full strength wine and pulverised cork. The aromas produced by isolates of varying cork origin are tabled. A small number of isolates methylated 2,4,6-trichlorophenol, yielding 2,4,6-trichloroanisole, responsible for the typical cork taint. During the boiling of cork slabs, the internal temperature on the inside of a box made from cork slices did not exceed 87°C.     

Occurrence of the killer character in yeasts associated with Spanish wine production

The occurrence of neutral, sensitive or killer phenotypes in 270 oenological yeast strains belonging to 18 species was studied. Strains were isolated from a total of 54 samples representing spontaneous fermentations at 11 wineries of the three Madrid viticultural areas (Arganda, Navalcarnero and San Martı́n de Valdeiglesias). The percentage of strains with the killer phenotype was 42·6% but this percentage varied during the fermentation: 18·8% in must, 50·0% in the middle and 58·8% by late fermentation. The highest frequency of the killer phenotype occurred in the species Saccharomyces cerevisiae (86·9%). Killer activity for this species was shown to result from toxins in the K₂ and K₃ groups.     

草莓果酒酵母菌的筛选、鉴定及耐受性研究

从自然发酵的草莓果酒中分离得到野生酵母菌,根据其菌落形态特征、生理生化实验及酒精、二氧化硫、葡萄糖、pH的耐受性实验,筛选出性能最优2株菌株（RL4和RL6）进行分子生物学鉴定。结果表明,初步鉴定菌株RL4和RL6分别为Hanseniaspora thailandica和葡萄汁有孢汉逊酵母（Hanseniaspora uvarum）。该研究为草莓果酒研制提供了优良酵母菌。     

The multifunctional regulatory proteins ABF1 and CPF1 are involved in the formation of a nuclease-hypersensitive region in the promoter of the QCR8 gene

The abundant DNA-binding proteins ABF1 and CPF1 are members of a family of global regulators with diverse chromosomal functions in the yeast Saccharomyces cerevisiae. Recent evidence suggests that these protein factors may be involved in establishing and maintaining well-defined chromatin structures in promoter regions and other genetic elements. We have investigated the involvement of ABF1 and CPF1 in chromatin organization at the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase. The promoter region of the QCR8 gene contains overlapping binding sites for ABF1 and CPF1. Nucleosome positioning studies indicate that the QCR8 gene is associated with a phased array of nucleosomes under both catabolite-repressed and derepressed growth conditions. Analysis of binding site mutants reveals that both ABF1 and CPF1 are involved in maintaining a nuclease-hypersensitive region in the QCR8 promoter. The chromatin structure at QCR8 during steady-state growth is, however, mainly dependent on binding of ABF1 to the promoter region. Implications of these findings for the role played by ABF1 and CPF1 in the regulation of mitochondrial biogenesis and other processes important for cell growth and division will be discussed.     

烟台干红葡萄酒发酵过程中酵母种类鉴定

利用WL培养基对分离自烟台葡萄果皮和干红葡萄酒发酵过程中的117株酵母菌进行归类,采用26S rDNA D1/D2区序列分析进行分子鉴定,共得到6属8种。并对发酵过程不同时期的酵母种类进行分析,结果表明发酵过程中酵母种类持续减少。     

The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter

We cloned and characterized a gene encoding isocitrate lyase from the methylotrophic yeast Pichia pastoris. This gene was isolated from a P. pastoris genomic library using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed from conserved regions in yeast isocitrate lyases. The cloned gene was sequenced and consists of an open reading frame of 1563 bp encoding a protein of 551 amino acids. The molecular mass of the protein is calculated to be 60.6 kDa with high sequence similarity to isocitrate lyase from other organisms. There is a 64% identity between amino acid sequences of P. pastoris Icl and Saccharomyces cerevisiae Icl. Northern blot analyses showed that, as in S. cerevisiae, the steady-state ICL1 mRNA levels depend on the carbon source used for cell growth. Expression in P. pastoris of the dextranase gene (dexA) from Penicillium minioluteum under control of the ICL1 promoter proved that P(ICL1) is a good alternative for the expression of heterologous proteins in this methylotrophic yeast. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ272040.     

Influence of different yeasts on the growth of lactic acid bacteria in wine

The influence of various yeasts on the growth of lactic acid bacteria in wine was tested by inoculating Lactobacillus hilgardii, L. brevis and two strains of Leuconostoc mesenteroides into experimental wines made with twelve different yeasts of the genus Saccharomyces. Wines made from juice which had been infected with several spoilage yeasts and then fermented with a wine yeast were also tested in this way. It was found that the yeasts differed considerably in their effects on bacterial growth. In some of the experimental wines bacterial growth was delayed or failed altogether. Generally, the unfavourable influence of any yeast on bacterial growth was much reduced if the wines were left in contact with the yeast cells for some weeks after the fermentation. The significance of these results in relation to the occurrence of malo-lactic fermentation in commercial wineries is discussed.     

蓬莱葡萄与葡萄酒产区自然条件

葡萄与葡萄酒作为蓬莱的4大支柱产业之一,近年来得到了蓬勃发展。蓬莱之所以成就今日葡萄产业的辉煌,与蓬莱悠久的酿酒葡萄栽培历史和传统有关,与当地政府对行业的高度重视有关,而蓬莱独特的地理环境和优越的自然条件则是成就这一切的基础。1蓬莱自然条件分析1.1地理位置蓬莱市     

Identification of the new polymorphisms in the promoter region of the CAST gene in cattle

Calpastatin (CAST) is a specific inhibitor of the ubiquitous calcium-dependent proteases – μ-calpain and m-calpain. This proteolytic system plays a key role in the tenderization process that occurs during postmortem storage of meat under refrigerated conditions. In the present study using comparative sequencing seven novel polymorphisms located within P3 promoter region for exon 1u of the bovine CAST gene: −357 (C/G), −556 (G/T), −557 (A/G), −580 (G/C), −750 (T/C), and two InDel at position −890 (A/−) and (GTT/−) at position −353/−351 were found. This region directs the expression of type III calpastatin mRNA, encoding the prototypical calpastatin. The genotype frequencies and haplotypes distribution were studied in 191 bulls belonging to six cattle breeds. All genotypes were distributed according to the HWE test and two major combined haplotypes were identified. The frequency of the haplotype1 varied from 0.45 in Aberdeen Angus to 0.82 in Simmental.     

不同果胶酶和酵母菌对葡萄酒中白藜芦醇及其二聚体的影响

采用高效液相色谱直接进样分析,测定了3种果胶酶处理、4种酵母发酵的葡萄酒中res、ε-viniferin、δ-viniferin等葡萄主要芪化物的含量.结果表明:7种葡萄酒中都含有较多的res、ε-viniferin和8-viniferin,不同果胶酶、酵母的选用均能影响到葡萄酒中的res、ε-viniferin和ε-viniferin的浸出效果.Uhrazyme Premium果胶酶酒样中ε-viniferin含量为2.187 mg/L,而Ex-v酒样中为1.926mg/L,796酵母酒样中res含量为1.193 mg/L,而CMS酒样仅为0.682 mg/L,δ-viniferin在D254酵母酒样中的含量为2.297 mg/L,而在Oneferm Color酒样中仅为1.850 me/L.Uhrazyme Premium果胶酶和D254、796酵母对果皮中上述芪化物的浸提效果较好.     

Contribution of wild yeasts to the formation of volatile compounds in inoculated wine fermentations

The aim of this work was to study the contribution of wild yeasts to the volatile composition of wine in inoculated fermentations. To do so, Parellada must, sterilized and inoculated with Saccharomyces cerevisiae strain Na33 (pure inoculated fermentation), inoculated Parellada must (mixed inoculated fermentation) and Parellada must that fermented with its wild yeasts (control fermentation) were used. From the results obtained in the pure inoculated fermentation it can be seen that S. cerevisiae produced appreciable quantities of isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate. However, the wild yeasts also contributed to the synthesis of esters since the total concentration of these substances was higher in the mixed inoculated fermentation than in the pure inoculated fermentation. 2-Phenylethyl acetate was only synthesized by wild yeasts when they did not compete with S. cerevisiae. The concentration of total alcohols was similar in the three samples; the important production of isobutanol and 2-phenylethanol in the control fermentation is noteworthy. As regards the acids, the greatest concentration corresponded to the mixed inoculated fermentation. The wild yeasts contributed to the synthesis of these compounds to a significant extent and S. cerevisiae synthesized appreciable amounts of short-chain fatty acids.     

中国葡萄酒产区划分浅议

中国葡萄酒产业正处在重大转折时期,对产业的地域分布进行合理划分十分必要。本文提出了产区划分的7个条件,并将中国葡萄酒产业的分布划分为13个产区,其中新疆产区又分为4个亚区,云南产区分为2个亚区。此外,一些新兴的酿酒葡萄及特色资源葡萄种植区正在形成。