优良本土戴尔有孢圆酵母的筛选及其在冰葡萄酒酿造中的应用

通过测定10株戴尔有孢圆酵母（Torulaspora delbrueckii）（编号为TD1～TD10）对葡萄糖、乙醇、酒石酸、SO2的耐受性能及其产β-葡萄糖苷酶性能,筛选发酵性能良好的戴尔有孢圆酵母,并将筛选菌株与酿酒酵母（Saccharomyces cerevisiae）ST混合发酵冰葡萄汁,最后对冰葡萄酒的基本理化指标进行测定。结果表明,除菌株TD2外,其他菌株均能够耐受葡萄糖500 g/L、乙醇体积分数4%、酒石酸16 g/L及SO2 350 mg/L,其中菌株TD6与TD9产β-葡萄糖苷酶活力较高,为优良本土戴尔有孢圆酵母。将菌株TD6、TD9分别与酿酒酵母ST混合发酵冰葡萄汁时均能在30 d内完成酒精发酵,且冰葡萄酒的基本理化指标均符合国标GB/T 25504—2010《冰葡萄酒》规定。此外,戴尔有孢圆酵母的接种对整个发酵过程的发酵速率与酿酒酵母的生长起到了一定的抑制作用,并且可以降低冰葡萄酒中乙酸和乙醇含量,提高总糖含量。     

Evaluation of the potential of commercial non-Saccharomyces yeast strains of Torulaspora delbrueckii and Lachancea thermotolerans in beer fermentation

This work evaluated the potential of three commercial non-Saccharomyces yeast strains Torulaspora delbrueckii (Biodiva and Prelude) and Lachancea thermotolerans (Concerto) for beer fermentation. The fermentation performance, volatile and non-volatile profiles were compared. Growth behaviours of all three yeast strains exhibited similar trends during the initial fermentation phase although a marked population decline was detected in strains Prelude and Concerto, which also showed a rapid utilisation of maltose, while strain Biodiva was unable to consume maltose and consumed lesser amounts of amino acids. Additionally, terpenoids inherently absent in the wort such as β-caryophyllene and geranyl acetone were produced in all beers, significantly higher in beers fermented with strain Prelude. For volatile profiles, Prelude and Concerto produced more ethanol and significantly higher amounts of acetate esters and long-chained ethyl esters. Strain Biodiva, on the other hand, produced higher amounts of isoamyl alcohol and ethyl butanoate.     

Isolation and characterization of the carbon catabolite‐derepressing protein kinase Snf1 from the stress tolerant yeast Torulaspora delbrueckii

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae snf1Δ mutant strain. DNA sequence analysis revealed that the fragment contained a complete open reading frame (ORF), which shares a high similarity with the S. cerevisiae energy sensor protein kinase Snf1. The cloned TdSNF1 gene was able to restore growth of the S. cerevisiae snf1Δ mutant strain on media containing nonfermentable carbon sources. Furthermore, cells of the Tdsnf1Δ mutant were unable to proliferate under nonfermenting conditions. Finally, protein domain analysis showed that TdSnf1p contains a typical catalytic protein kinase domain (positions 41–293), which is also present in other Snf1p homologues. Within this region we identified a protein kinase ATP‐binding region (positions 48–71) and a consensus Ser/Thr protein kinase active site (positions 160–172). The GenBank Accession No. for the sequenced DNA fragment is HM131845. Copyright © 2010 John Wiley & Sons, Ltd.     

戴尔有孢圆酵母WA19与酿酒酵母F33混合发酵在樱桃酒酿造中的应用

以自主筛选的戴尔有孢圆酵母WA19为研究对象,考察其与酿酒酵母F33混合发酵对樱桃酒品质的影响,同时以酿酒酵母F33单独发酵的樱桃酒、商业化戴尔有孢圆酵母Viniflora Prelude和酿酒酵母F33混合发酵的樱桃酒为对照。研究结果表明,戴尔有孢圆酵母WA19和酿酒酵母F33之间不存在相互抑制,2?种酵母均能快速适应酒体环境,迅速增殖,整个发酵周期为8?d。而酿酒酵母F33与Viniflora Prelude混合发酵则出现了相互抑制的现象。此外,戴尔有孢圆酵母WA19和酿酒酵母F33混合发酵显著提高了多种挥发性组分的含量,如β-苯乙醇、丁酸乙酯、异戊酸乙酯、己酸乙酯、里那醇等,能够增强樱桃酒的果香、花香和发酵香气,使樱桃酒的香气特征更加突出。鉴于戴尔有孢圆酵母WA19的优异性能,有望开发成为樱桃酒专用的产香酵母。     

戴尔有孢圆酵母与酿酒酵母混合发酵对猕猴桃酒香气的影响

用戴尔有孢圆酵母（Torulaspora delbrueckii SY-2-2）和酿酒酵母（Saccharomyces cerevisiae RV002）作为猕猴桃酒发酵菌株,以单接种酿酒酵母、单接种戴尔有孢圆酵母为对照组,同时接种戴尔有孢圆酵母和酿酒酵母、顺序接种戴尔有孢圆酵母和酿酒酵母为实验组,采用顶空固相微萃取法结合气相色谱-质谱技术测定猕猴桃汁发酵酒样产生的挥发性香气成分。结果表明,混菌发酵组获得的挥发性香气成分种类高于单菌发酵组,且顺序接种发酵组的香气成分种类（23 种）和质量浓度（336.95 mg/L）均最高。在顺序接种发酵组中,戴尔有孢圆酵母的参与提高了猕猴桃酒中挥发性物质的种类和含量,特别是苯乙酸乙酯、苯丙酸乙酯、苯乙醇、β-大马士酮、α-松油醇、桉树油醇等物质,赋予猕猴桃酒浓郁的花果香味,丰富了猕猴桃酒的香气成分。香气感官评价表明顺序接种发酵组的花香、草本香、甜香味更加浓郁,酒香和果香次之,整体评分最高。     

Review: Pure non‐Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications

Recently there has been increased interest in using non‐Saccharomyces yeasts to ferment beer. The worldwide growth of craft beer and microbreweries has revitalised the use of different yeast strains with a pronounced impact on aroma and flavour. Using non‐conventional yeast gives brewers a unique selling point to differentiate themselves. Belgian brewers have been very successful in using wild yeasts and mixed fermentations that often contain non‐Saccharomyces yeasts. Historically, ancient beers and beers produced before the domestication of commonly used Saccharomyces strains most likely included non‐Saccharomyces species. Given the renewed interest in using non‐Saccharomyces yeasts to brew traditional beers and their potential application to produce low‐alcohol or alcohol‐free beer, the fermentation and flavour characteristics of different species of non‐Saccharomyces pure culture yeast were screened for brewing potential (Brettanomyces anomalus and bruxellensis, Candida tropicalis and shehatae, Saccharomycodes ludwigii, Torulaspora delbrueckii, Pichia kluyveri, Zygosaccharomyces rouxii). Alcohol‐free beer is already industrially produced using S. ludwigii, a maltose‐negative species, which is a good example of the introduction of non‐Saccharomyces yeast to breweries. Overall, non‐Saccharomyces yeasts represent a large resource of biodiversity for the production of new beers and have the potential for wider application to other beverage and industrial applications. Almost all of the trials reviewed were conducted with varying fermentation parameters, which plays an important role in the outcome of the studies. To understand these impacts all trials were described with their major fermentation parameters. Copyright © 2016 The Institute of Brewing & Distilling     

Effects of mediums on fermentation behaviour and aroma composition in pure and mixed culture of Saccharomyces cerevisiae with Torulaspora delbrueckii

The nutrient status and composition in mediums have a significant effect on yeast metabolism and phenotypic characteristics in wine fermentation. In this study, the effects of three frequently used mediums, including synthetic grape juice (SGJ), grape juice without grape pericarp and seeds (GJ) and grape must with grape pericarp and seeds (GMPS), on yeast fermentation behaviour and aroma compounds produced by pure and mixed culture of Saccharomyces cerevisiae T73 with Torulaspora delbrueckii TD20 were investigated after alcoholic fermentation. The results showed that high fermentation activities and cell population were always found in GJ medium irrespective of inoculated approach. More esters and higher alcohols were produced in GMPS medium fermented by pure S. cerevisiae, while SGJ medium had increased levels of fatty acids. Consistent with previous literatures, the mixed fermentation of T. delbrueckii and S. cerevisiae produced more acetate esters and fatty acids than the pure culture of S. cerevisiae, while this enological trait was only found in SGJ and GJ, not in GMPS. Our results highlighted that more attention should be paid to the fermentation medium when evaluating the enological and aromatic properties of selected yeasts used in industrial winemaking. In this regard, the combined use of GJ and GMPS medium might be a suitable choice.     

A DNA region of Torulaspora delbrueckii containing the HIS3 gene: sequence, gene order and evolution

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae his3 mutant strain. DNA sequence analysis revealed that the fragment contained two complete ORFs, which share a high similarity with S. cerevisiae His3p and Mrp51p, respectively. The cloned TdHIS3 gene fully complemented the his3 mutation of S. cerevisiae, confirming that it encodes for the imidazoleglycerol-phosphate dehydrate of T. delbrueckii. Two additional ORFs, with a high homology to S. cerevisiae PET56 and DED1 genes, were mapped upstream and downstream from TdHIS3 and TdMRP51, respectively. This genetic organization is analogous to that previously found in Saccharomyces kluyveri and Zygosaccharomyces rouxii. The evolutionary significance of gene order in this chromosomal region is analysed and discussed.     

青岛啤酒酵母与高浓酵母筛选高浓酿造酵母融合亲株

以青岛啤酒酵母和高浓精酵母为供试菌株,筛选出生长良好的酵母,为选育具有青岛啤酒风味的高浓酵母做准备.比较了7株酵母不同糖类发酵、离子抗性、二氧化碳减重、发酵液风味品评等指标.结果表明:T1、T2和T3是传统的青岛啤酒发酵菌株,其发酵液口味符合青岛啤酒口味要求,且对Cu2+均不耐受;而G4和G6发酵减重试验和风味物质分析中的乙醛含量指标的评价均优于G5和G7菌株,且它们的发酵液的风味也接近青啤口味.因此,选择T1、T2、T3和G4、G6作100L酿造试验,进一步确定融合亲株.     

Investigation of beer‐spoilage ability of Dekkera/Brettanomyces yeasts and development of multiplex PCR method for beer‐spoilage yeasts

There have been many beer‐spoilage incidents caused by wild yeasts. Saccharomyces cerevisiae, Dekkera anomala and D. bruxellensis have been recognized as beer‐spoilage yeasts in the brewing industry. In contrast, the beer spoilage ability of Brettanomyces custersianus has not been well characterized, although this species was isolated from beer. In this study, the beer‐spoilage ability of currently described Dekkera/Brettanomyces yeast species was investigated. As a consequence, D. anomala, D. bruxellensis and B. custersianus were shown to grow in commercial beers. On the other hand, the remaining two Brettanomyces species, B. naardenensis and B. nanus, did not grow in beer. These results indicate that B. custersianus should be recognized as a beer‐spoilage species, in addition to S. cerevisiae, D. anomala, and D. bruxellensis. Therefore we developed multiplex polymerase chain reaction (PCR) for the simultaneous detection and identification of B. custersianus and the other beer‐spoilage yeast species. For this purpose, PCR primers were designed in the internal transcribed spacer region or 26S rDNA, and each PCR product was made in different sizes to easily discriminate the species from electrophoretic results. Specificity, reactivity and sensitivity of the designed primers were evaluated. As a result, the developed multiplex PCR method was shown to have high specificity and reactivity, and therefore was considered as an effective tool to identify beer‐spoilage yeast species. This tool can contribute to microbiological quality assurance in breweries. Copyright © 2015 The Institute of Brewing & Distilling     

Fed‐batch fermentation with glucose syrup as an adjunct for high‐ethanol beer brewing

To produce a beer with a high ethanol content, preliminary research on fed‐batch fermentation profiles with glucose syrup as an adjunct during the primary fermentation period was conducted. The ethanol concentration of the beer was elevated by feeding a glucose syrup into the fermentors at a later stage of primary fermentation. Fermentation trials were carried out using a typical lager strain, SC‐9, with a pitching rate at 7.0 × 10⁶ cells/mL. An all‐malt wort (12.5°P) was employed and the primary fermentation temperature was 14 °C. Glucose syrup was supplemented when the concentration of residual reducing sugars was decreased to ~10 g/L. Results showed that the supplemented glucose was consumed rapidly and that the ethanol concentration in the final beer was raised to 67.9 g/L. Additional growth of yeast was observed after feeding accompanied by a low yield of ethanol (~0.46 g/g). Formation of diacetyl was enhanced by yeast growth and two additional peaks were obtained after feeding. The peak value of the diacetyl concentration was 1.90 mg/L. The fed‐batch fermentation resulted in a beer with an overproduction of higher alcohols and esters, indicating that brewing under these experimental conditions led to an unbalanced flavour profile. Results of optimization demonstrated that the optimal conditions were found to be 15°P for initial wort extract, 10 °C for fermentation temperature and 20 × 10⁶ cells/mL for yeast pitching rate, leading to total higher alcohols of 173.8 mg/L, total esters of 22.8 mg/L and an acetaldehyde concentration of 40.5 mg/L. A 12 day maturation and fermentation temperature of 8 °C was needed to reduce the acetaldehyde to 14.3 mg/L. Copyright © 2014 The Institute of Brewing & Distilling     

果酒酿造中优良酵母菌株的筛选

在果酒酿造中,酵母菌在发酵过程中起着重要的作用,其发酵力的好坏,副产物的多少,直接影响产品的风味,因此具有重大的研究价值。以实验室保藏的酵母J1,J2,M1,M2,F1,F2为出发菌株,通过测各菌株对苹果汁的发酵能力、发酵特性及发酵所得苹果酒品质的系统实验,选出F2为最佳苹果酒酿酒酵母,其对苹果汁的发酵能力强、发酵特性好,发酵所得苹果酒品质优,且酒体澄清透明、色泽橙红,具有苹果酒的典型风味,适用于果酒生产。     

West African sorghum beer fermented with Lactobacillus delbrueckii and Saccharomyces cerevisiae: fermentation by‐products

Physicochemical quality parameters and volatile fermentation by‐products were determined in West African sour sorghum beer (pito) fermented with pure cultures of Lactobacillus delbrueckii and Saccharomyces cerevisiae compared with pito prepared by traditional spontaneous fermentation. Levels of by‐products were also compared with those found in similar beer types. Similar levels of apparent extract, alcohol, pH, lactic acid and bitterness were obtained for pure culture and traditional fermentations, although differences were observed in colour and turbidity. Significant statistical differences were obtained for all of the volatile aroma compounds analysed. The pure culture approach resulted in a higher level of total volatile compounds (353 mg/L) of which higher alcohols accounted for 88%, predominately n‐propanol. The traditional approach had total volatiles of 229 mg/L with 86% higher alcohols but with iso‐amyl alcohol predominating. Ester levels were low in the pure culture beer but with a relatively high level of acetaldehyde. Fermenting pito with pure cultures yielded a product with similar physicochemical quality as traditional pito but with a suggestion of a more pronounced aroma whose impact on the overall product quality will require consumer acceptance and sensory evaluation. © 2019 The Institute of Brewing & Distilling     

West African sorghum beer fermented with Lactobacillus delbrueckii and Saccharomyces cerevisiae: shelf‐life and consumer acceptance

The fermentation profiles, shelf‐life and consumer acceptance of traditional West African sour sorghum beer (pito) fermented with pure commercial starter cultures of lactic acid bacteria (L. delbrueckii) and yeast (Saccharomyces cerevisiae) were evaluated. The beers from this ‘pure culture’ approach were compared with the spontaneous fermentation of pito wort. Lactic acid formation, pH change and extract utilisation were monitored during fermentation. Lactic acid content was used as a measure of sourness to establish the spoilage level of over‐sourness. Further, regression models relating sourness to the time the drink was kept were used to predict the shelf‐life. Consumer acceptance of the product was evaluated using a novel nine‐point hedonic scale. The pure culture and traditionally fermented beers followed similar lactic acid and fermentation profiles but strain‐specific differences were observed. Similar levels of pH, lactic acid level and extract utilisation were achieved. An improvement in shelf‐life of two days was found over traditionally fermented pito. There was no statistical difference between the two pito products for overall liking and taste. However, there was a preference for the aroma of the pure culture pito. It is suggested that the use of pure cultures will facilitate the scale‐up of pito production. © 2019 The Institute of Brewing & Distilling