Factors Affecting the Hydroxycinnamate Decarboxylase/Vinylphenol Reductase Activity of Dekkera/Brettanomyces: Application for Dekkera/Brettanomyces Control in Red Wine Making

ABSTRACT:  The growth of Dekkera/Brettanomyces yeasts during the ageing of red wines—which can seriously reduce the quality of the final product—is difficult to control. The present study examines the hydroxycinnamate decarboxylase/vinylphenol reductase activity of different strains of Dekkera bruxellensis and Dekkera anomala under a range of growth-limiting conditions with the aim of finding solutions to this problem. The yeasts were cultured in in-house growth media containing different quantities of growth inhibitors such as ethanol, SO₂, ascorbic acid, benzoic acid and nicostatin, different sugar contents, and at different pHs and temperatures. The reduction of p -coumaric acid and the formation of 4-ethylphenol were periodically monitored by HPLC-PDA. The results of this study allow the optimization of differential media for detecting/culturing these yeasts, and suggest possible ways of controlling these organisms in wineries.     

Mousy off-flavour production in grape juice and wine by Dekkera and Brettanomyces yeasts

The ability of four known species of Dekkera and Brettanomyces yeasts to produce mousy off-flavour in grape juice and wine was investigated for the first time. Using a sensory assessment technique the twelve type strains of Dekkera and Brettanomyces , representing four species, were found to grow and to be capable of producing mousy off-flavour in a grape juice medium; however, differences between strains were apparent. Four strains representing the two species of Dekkera (D. bruxellensis and D. anomala ) known to be associated with the spoilage of wine and other fermented beverages were further investigated for mousy off-flavour production in a red and white wine supplemented with nutrients. D. anomala and only one of the three D. bruxellensis strains tested grew in both wines and generally produced a moderate level of off-flavour, whereas the remaining two strains of D. bruxellensis , despite slowly losing viability (10- to 100-fold) over the 52–55 day period, produced detectable off-flavour. This work demonstrates the general ability of Dekkera and Brettanomyces yeasts to produce mousy off-flavour and confirms the importance of these yeasts for off-flavour production in grape juice and wine.     

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     

Dekkera, Brettanomyces and Eeniella: electrophoretic comparison of enzymes and DNA-DNA homology

The taxonomic status of various species of Dekkera, Brettanomyces and Eeniella was examined by electrophoretic comparison of enzymes, by deoxyribonucleic acid homology and by physiological characterization. These studies demonstrated that two teleomorphic Dekkera species, D. anomala and D. bruxellensis (synonym D. intermedia), and four anamorphic Brettanomyces species, B. anomalus (synonym B. claussenii), B. bruxellensis (synonym B. abstinens, B. custersii, B. intermedius, B. lambicus), B. custersianus and B. naardenensis, can be recognized. The anamorphic genus Eeniella remained as a separate, monotypic taxon.     

Detection and identification of Brettanomyces/Dekkera sp. yeasts with a loop-mediated isothermal amplification method

Primer sets for a loop-mediated isothermal amplification (LAMP) method were developed to specifically identify each of the four Brettanomyces/Dekkera species, Dekkera anomala, Dekkera bruxellensis, Dekkera custersiana and Brettanomyces naardenensis. Each primer set was designed with target sequences in the ITS region of the four species and could specifically amplify the target DNA of isolates from beer, wine and soft drinks. Furthermore, the primer sets differentiated strains of the target species from strains belonging to other species, even within the genus Brettanomyces/Dekkera. Moreover, the LAMP method with these primer sets could detect about 1 x 10(1) cfu/ml of Brettanomyces/Dekkera yeasts from suspensions in distilled water, wine and beer. This LAMP method with primer sets for the identification of Brettanomyces/Dekkera yeasts is advantageous in terms of specificity, sensitivity and ease of operation compared with standard PCR methods.     

过氧乙酸在UHT奶中的应用

研究了过氧乙酸在UHT奶的实际应用中的作用，验证了过氧乙酸对于管道、罐体、灌装设备、车间空气及人员手的消毒作用．通过试验得出过氧乙酸在UHT奶生产中的应用浓度及使用最佳条件。     

酵母菌醋酸菌混菌发酵高产醋酸工艺研究

以葡萄糖为底物,利用酵母菌和醋酸菌混合发酵生产醋酸,通过单因素试验筛选了最佳醋酸生产工艺条件。试验结果表明:pH值为5.5,醋酸菌种龄48h,接种间隔0h,醋酸菌接种量6%,酵母菌接种量0.15%,装液量50mL/250mL,发酵温度30℃,初始还原糖含量18%为最优工艺条件,在此工艺条件下醋酸的产量可达到10.09g/100mL,糖转化率达到90%。     

The influence of oxygen and organic hydrogen acceotors on glycolytic dioxide production in Brettanomyces anomalus

The yeast Brettanomyces anomalus showed the Custers effect in that under strictly anaerobic conditions, in the presence of glucose, CO₂ production was negligble. CO₂ production was stimulated by mixing anaerobic cell suspensions with an aerated glucose solution in astopped-flow cell. Glycolytic CO₂ production continued even after oxygen exhaustion. Studies using an open reaction vessel showed that the rate of glycolytic CO₂ production could be increased to a maximum level by exposing the anaerobic cell suspension to brief pulses of O₂. A cell suspension CO₂ at a maximal rate demonstrated the Pasteur effect on switching the mobile gas to a mixture conatining oxygen (5.05 KPa). In contrast to glycolytic CO₂ production in vivo nicotinamide pool responded rapidly to changes in oxygen concentration. The addition of acetaldehyde, acetone, or 3-hydroxy-butan-2-one led to a temprorary production of CO₂ at an initial rate depending on the concentration of substance added according to the Michaelis–Menten equation. The maximal rates were equal with all three substances, whereas tha apparent K_m values were different. The total amount of CO₂ produced was 22-fold greater than the amount of acetaldehyde added. Added organic hydrogen acceptors modulated the intracellular reedox balance of B. anomalus under conditions. These results are discussed in relation to the current hypothesis of the Custers effect.     

Diversity in Spoilage Yeast Dekkera/Brettanomyces bruxellensis Isolated from French Red Wine. Assessment During Fermentation of Synthetic Wine Medium

Yeast Brettanomyces bruxellensis is a contaminant found worldwide and is responsible for red wine spoilage due to the development of animal and phenolic off‐odours. During this study, 24 Brettanomyces bruxellenis isolates were obtained from red wine samples from two French wineries and these were discriminated as 23 strains. Nine strains coming from 2 wineries and 4 vintages were cultivated in synthetic wine medium for 1500 hours and they gave nine different behaviours. Four main growth patterns (with different growth steps and durations) and three main different sugar consumption profiles were obtained. Glucose and fructose were not limiting substrates for all strains. The production level of 4‐ethylphenol was found to vary from strain to strain (from 0.350 to 2.773 mg L^?1) and was independent of the biomass concentration. Some strains presented a coupled‐to‐growth production of volatile phenols, others did not. This study showed that different strains of Brettanomyces bruxellensis behaved differently, one from another, under the given conditions taking into consideration several aspects. The results thus demonstrate a large intraspecific diversity.     

食醋生产中醋酸菌保存方法改进技术研究

为更好地保藏醋酸菌,通过k（3 4）正交试验考察酒精浓度、醋酸浓度和复配营养素添加量等多个因素在液体保藏醋酸菌中作用,通过数据分析,认为底酸浓度为40g／L、初始酒度为80g／L和复配营养素添加量为5g／L是醋酸菌的最佳保藏条件。同时对固体斜面培养基和液体培养基保藏的菌种性能进行对比分析,通过发酵产酸试验、菌种生长状况对比和在斜面培养基的生长形态,认为液体培养基更适用于高浓度酒生产醋的需要。     

红枣醋酸发酵饮料的生产工艺

介绍了以红枣为主原料经酒精发酵和醋酸发酵制成红枣醋后，再与红枣汁、蜂蜜、白糖等调配而成的红枣醋酸发酵饮料的生产工艺。     

响应面优化醋酸菌发酵条件研究

以醋酸菌作为发酵菌株,考察不同乙醇浓度、接种量、发酵温度及转速条件下发酵液的总酸含量,运用响应面优化醋酸菌发酵条件,并显微观察菌体的生长形态。结果表明：最佳发酵条件为温度31℃、转速142r/min、乙醇浓度6.1%vol、接种量4.50%,椭圆和杆状的菌体形态与发酵条件形成了对应。     

醋酸菌菌种的冷冻干燥保藏研究

该文介绍了醋酸菌菌种的真空冷冻干燥保藏方法，并从预冻温度和保护剂浓度对冻干菌的影响进行了研究，初步确定了醋酸菌菌种冻干保藏的预冻温度及保护剂浓度。比较了不同保藏方式对产酸的影响，实验表明用冷冻干燥保藏法保藏醋酸菌菌种效果良好。     

固定化醋酸菌在醋酸发酵中的应用研究

固定化醋酸菌发酵具有操作简单,周期短,稳定高效,经济节能等优点,大大提高了产酸速率和产酸量,成为了液体制醋和生产醋酸的发展方向.文章系统概述了固定化醋酸菌在醋酸发酵过程中载体材料的选择、常用的固定化方法、与游离醋酸菌发酵的比较,并对其仍存在的问题以及未来的发展前景进行了分析与展望.     

醋酸菌耐酸机制的研究进展

文章主要从醋酸菌的ABC转运子、质子动力势外排泵、乙酸过氧化反应和基因与酶调控等方面阐述了醋酸菌耐乙酸的机理,这对进一步认识醋酸菌,寻找适合生产高酸度食醋的发酵菌种及高效生产食醋有很好的指导意义。     

醋酸菌的分子分类学研究和在食醋生产中的应用

醋酸菌是革兰氏阴性,专性好氧,能将乙醇或糖类氧化为醋酸的细菌的总称。醋酸菌广泛分布于自然界中,它们能通过代谢酒精产生醋酸的特点在食品生产中已得到普遍应用。醋酸菌分类学的研究在很久以前就已经开始,但是直到近几十年随着各种生物分子技术的发展才对其有更加深入地认识。该文综述了醋酸菌的分子分类学研究发展和在食醋工业中的应用,主要介绍了各种分子分类学方法和食醋生产中使用的醋酸菌种类。     

优良果醋菌种的分离鉴定

采用稀释平板分离法,从自然发酵苹果醋醪液中分离筛选出优良菌株G-7,其产酸量为35.55g/L,传代5次性能稳定,对醋酸分解速度小;其产酯量为0.826g/L,最适发酵温度为32℃,耐酒精、耐酸性能好。株菌G-7初步鉴定为恶臭醋杆菌属。     

醋酸菌的分类进展

醋酸菌是好氧型革兰氏阴性菌,能以氧气为终端电子受体,氧化糖类、糖醇类和醇类生成相应的糖醇、酮和有机酸。从1898年Beijerinck定义第一个醋酸菌属-醋酸杆菌属(Acetobacter)以来,截止2014年初,已报道的醋酸菌共16个属,共84个种。该文总结了醋酸菌的分离、分类、鉴定与保藏方法,并对醋酸菌命名原则、种属名称演变以及醋酸菌的种属特征进行了归纳。     

Influence of acetic acid preservation of chilean mackerel (Trachurus murphy) on fish meal production

The influence of acetic acid preservation of Chilean mackerel (Trachurus murphy) on its suitability of processing a fish meal quality was studied on a pilot-plant scale and on the industrial level. In particular, the influence of acetic acid preservation of raw material on water-oil separation during cooking-pressing and resulting quality changes in fish meal produced was investigated. The results have shown that preservation of raw fish by acetic acid may improve water-oil separation after cooking, whereas the quality of oil and fish meal measured as acid value and available lysine, respectively, remain constant. A concentration of 1000–3000 μg kg^?1 of acetic acid in the raw material is recommended.