Flow cytometric analysis of Saccharomyces cerevisiae autolytic mutants and protoplasts.

Simple methods, based on the technique of flow cytometry, have been developed for the phenotypic characterization of yeast autolytic mutants and for the analysis of the formation and regeneration of the yeast protoplasts. The expression of lytic mutations determined uptake of the fluorescent dye propidium iodide, which could be carefully monitored by flow cytometry. Mixed populations of lysed and viable cells were precisely quantified and sorted, and the technique was also applied to demonstrate protection from lysis of mutant cells with cell wall defects, in the presence of osmotic stabilizers. Protoplast formation and regeneration was monitored by analysing relative cell size; this was facilitated by the preparation of homogeneous protoplast preparations. The technique of flow cytometry proved superior to other conventional methods for these types of study.     

Rapid Assessment of Yeast Viability and Yeast Vitality During Alcoholic Fermentation

A flow cytometric method based on the measurement of the efflux percentage of carboxyfluorescein after staining of yeast cells with carboxyfluorescein diacetate is reported. This efflux percentage was followed throughout alcoholic fermentations performed with various pH and temperature conditions. The results obtained show that the efflux percentage is a good indicator of the energetic state of yeast cells and varies through the fermentation in a similar manner to the specific ethanol production rate. These results suggest that this flow cytometric method can be applied to assess viability and to predict vitality of yeast cells in alcoholic fermentations.     

Relationship among seminal quality measures and field fertility of young dairy bulls using low-dose inseminations

Optimal use of genetically superior bulls through artificial insemination (AI) is highly dependent on precise assessment of seminal quality which allows for reasonable estimations of field fertility with normal or low-dose inseminations. In the present study, seminal measures such as sperm motility and morphology, sperm viability, sperm DNA fragmentation, and the ability of the sperm to display an acrosome reaction were tested. The relationships between field fertility and the seminal measures were investigated using 3 ejaculates from each of 195 bulls (156 Holstein and 39 Jersey) participating in a progeny test program. A range of AI doses, varying from 2 × 10⁶ to 15 × 10⁶ sperm/straw, was obtained by a controlled dilution process applied to each ejaculate. The different AI doses were distributed at random among 75,610 experimental first inseminations in 4,721 herds and 208 AI technicians. Most of the seminal measures appeared to contain a predictive value for the nonreturn to estrus at 56 d post-AI (NRR56) regardless of the number of sperm per AI dose and can be regarded as noncompensable sperm traits. But, due to correlations between the individual measures, the best model for describing (and predicting) NRR56 was based on sperm concentration and viability in the neat (raw) semen, and post-thaw sperm viability. The statistical models for describing NRR56 included the following explanatory variables: strength of the estrus, number of sperm per AI dose, breed, parity, and random components representing herds and AI technicians. The present results show that the most precise estimation of a bull’s NRR56 can be achieved through flow cytometric detection of sperm concentration and viability in neat semen as well as flow cytometric detection of post-thaw sperm viability.     

流式细胞术快速检测直投式发酵剂菌体活力

利用荧光染料标记直投式发酵剂菌体细胞并结合流式细胞术快速检测和分析菌体细胞的活力以及生存状态,对科学地评价各种微生物发酵剂的品质具有现实意义。研究选用保加利亚乳杆菌（Lactobacillus bulgaricus）和嗜热链球菌（Streptococcus thermophilus）两种直投式发酵剂以及制备的新鲜菌体细胞和热处理菌体细胞作为分析检测对象,利用流式细胞术结合羧基荧光素双乙酸酯（5-（6）-carboxyfluoresceindiacetate,5（6）-cFDA）和碘化丙啶（两种荧光染料检测了这些菌体的细胞活力。研究结果表明,5（6）-cFDA和PI双染色方法适合检测L. bulgaricus直投式发酵剂中菌体的存活力,该发酵剂中菌体存活率仅为4.7%,活力较低;而利用5（6）-cFDA单染色方法检测S. thermophilus直投式发酵剂中菌体存活力的效果较好,其菌体存活力接近于90%,活力较强;同时,发酵活力验证也得到了相同的结果;另外,研究证实了PI不能很好地区分S. thermophilus的死活细胞。因此,流式细胞术可作为直投式发酵剂生产行业快速检测评价产品质量的可靠方法。     

COMPARISON OF METHODS FOR THE DETERMINATION OF CELL VIABILITY IN STORED BAKER'S YEAST

The classical plate method for discriminating between viable and nonviable yeast cells in stored baker's yeast was compared with dead cell staining techniques using methylene blue and three fluorochrome stains. The increase of dead yeast cells during storage of baker's yeast for up to 16 days at 5°C, 20°C and 35°C was determined. During prolonged storage, especially at 35°C, the death rate increased rapidly and the yeast cake began to soften because of autolysis. In these conditions the choice of method for determining the proportion of dead cells proved to be of great importance. Useful results for yeast stored for some time at 35°C could be obtained only by the fluorochrome technique using primuline, acridine orange or acriflavine as fluorochromes.     

流式细胞技术快速检测果汁中的霉菌、酵母菌

研究应用流式细胞技术（flow cytometry,FCM）快速检测果汁饮料中霉菌、酵母菌的方法。通过正交实验和方差分析,对样品前处理技术条件进行优化,去除了影响FCM检测的基质颗粒,使FCM检测限达到101cfu/mL数量级,检测时间从5d缩短到100min。从绘制的标准曲线可以看出,FCM与平板法线性相关,符合性好。FCM将以更加灵敏、准确、快速、操作简便的优点成为一种可替代平板法来检测果汁中霉菌、酵母菌的自动化仪器检测新技术。     

FACTORS AFFECTING THE SURVIVAL OF LYOPHILIZED BREWERY YEAST STRAINS

Some of the factors that contribute to the loss of viability of brewery yeast strains during lyophilization (freeze-drying) have been investigated. A lyophilization technique for the maintenance of brewery yeast strains with higher viabilities than those previously reported has been developed. Three lyophilized strains of ale yeast still had a survival rate of 60% after periods of storage of up to three years, while a lager yeast strain maintained a viability of approximately 50% during storage for eighteen months.     

啤酒有机酸类物质研究进展

有机酸类物质是啤酒中一类重要的化合物,其组成和含量不仅影响啤酒的风味,而且与酵母的活力也有较密切的联系。文中参照国内外研究资料,从有机酸对啤酒风味的影响以及有机酸的代谢与酵母活力之间的联系阐述有机酸类物质对于提高啤酒质量的重要性。     

Optimisation of High Gravity and Diet Beer Production in a German Brewery by Flow Cytometry

Increasing the quantity of beer production without diminishing the quality of the product is a key concern of the beer producing industry. Modifications to the brewery's equipment and settings are the most commonly used methods to improve the brewing process, while the supreme importance of the physiological state of the beer producing organisms, the yeast cells, for the productivity of the brewing process is often poorly recognised. The work described here was designed to optimise two processes: the inoculation regime used to produce high gravity bottom-fermenting beer, and the production of high quality diet beer. To achieve these aims, flow cytometry was used to follow changes in the distribution of DNA, neutral lipid and 3β-hydroxsterol contents in Saccharomyces carlsbergensis strains during inoculation, fermentation and storage. This allowed potential time-saving alterations in the process to be identified. Double staining techniques proved that vigorous fermentative activity and long-term survival capacity during main and secondary fermentation requires intense multiplication of the yeast cells during inoculation. The production of high gravity beer was then enhanced by altering the schedule of the wort additions, and thus increasing the yeast's activities related to multiplication. To produce diet beer, oligosaccharides that remain after the standard brewing process are degraded by adding small amounts of wort, usually during secondary fermentation. However, during this period of fermentation the physiological activity of the yeast cells is hampered by low carbon and high ethanol concentrations. Adding small batches of wort at carefully defined time points and in optimised amounts, even during the main fermentation, improves the physiological state of the yeast cells and rapidly decreases the carbon concentration within the fermentation tank. Both of these factors help to promote quick fermentation to a high quality diet beer. Thus, the flow cytometric investigations provided a reliable basis for identifying effective means of improving the process regime for brewing both of these products.     

啤酒酵母浓度实时在线检测方法的研究(一)

酵母的存在会改变发酵液的电特性,发酵液在无线电频率范围内的电特性可用电容率和电导率充分地描述。实验证实发酵液在无线电频率范围内的电容率增量是测量频率和生物量浓度的函数。基于对发酵液电容率分布的研究,本文提出了测量酵母浓度的新方法。用本方法不用取样就能对发酵液中的酵母浓度进行实时在线测量,而且测得的是活的酵母浓度。本文制作的电极直接插入 发酵器中并满足高温蒸汽 灭菌条件。本方法在生化制药、食品发酵、啤酒酿造、污水检测等工业领域里有很好的推广应用前景