Viable cell yield from active dry yeast products and effects of storage temperature and diluent on yeast cell viability

Active dry yeast (ADY) products are commonly fed in the dairy industry, but research regarding quality control for such products is limited. The objectives of this study were to determine yeast viability in field samples relative to manufacturers’ guarantees (experiment 1), measure the effects of high-temperature storage on yeast viability (experiment 1), and determine the effect of vitamin-trace mineral (VTM) premix on yeast viability (experiment 2). Commercially available ADY products were acquired in triplicate through normal distribution channels and stored at 4°C upon receipt. Initial samples were evaluated for colony-forming units and compared with product label guarantees. Only 1 of the 6 products sampled in experiment 1 met product guarantees for all 3 samples. To determine effects of storage temperature and duration on viability, ADY samples were stored in an incubator at 40°C with ambient humidity for 1, 2, and 3 mo. High-temperature storage significantly decreased viability over the 3-mo period; approximately 90% of viable cells were lost each month. Three of the 5 products sampled in experiment 2 met product guarantees. Fresh samples of 4 of these 5 ADY products were mixed in duplicate with ground corn (GC) or a VTM premix to achieve a target concentration of 2.2 × 10⁸ cfu/g. For each product, GC and VTM samples were stored at ambient temperature (22°C) and at an elevated temperature (40°C) for 2 wk. No differences in viable yeast count were observed between GC and VTM samples immediately after mixing or after storage at ambient temperature. Yeast viability in GC and VTM samples decreased during storage at an elevated temperature. There also was a significant interaction of diluent and storage temperature; VTM samples had higher cell viability than GC samples when subjected to high-temperature storage. Results suggest that (1) ADY products failed to consistently meet product guarantees; (2) viability of ADY products was greatly diminished during storage at 40°C for 2 wk; and (3) the loss in viability at elevated temperatures may be attenuated when ADY products are diluted with a premix containing VTM.     

Measurement of nuclear DNA content in fission yeast by flow cytometry

Cell division cycle (cdc) mutants of Schizosaccharomyces pombe are arrested at specific points in the cell cycle when grown at restrictive temperature. Flow cytometry of such cells reveals an anomalous increase in the DNA fluorescence signal, which represents a problem in experiments designed to determine the cell cycle arrest point. The increased fluorescence signal is due to cytoplasmic constituents and has been attributed to mitochondrial DNA synthesis (S. Sazer and S. W. Sherwood, J. Cell Sci.97: 509–516, 1990). Here we have studied the cdc10 mutant by flow cytometry using different DNA-binding fluorochromes and found no evidence that the increased fluorescence signal was caused by mitochondrial DNA synthesis. To determine more accurately the nuclear DNA content we have developed a novel method to remove most of the cytoplasmic material by exposing the cells to Triton X-100 and hypotonic conditions after cell wall digestion. The DNA fluorescence from cells treated in this way was more constant with time of incubation at restrictive temperature in spite of a considerable increase in cell size. With this method we could determine that the recently isolated temperature sensitive orp1 mutant is arrested with a 1C DNA content. Premature and abnormal mitosis (‘cut’) could be observed for the orp1 mutant after only 4 h at restrictive temperature. © 1997 John Wiley & Sons, Ltd.     

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。     

啤酒发酵过程中酸类物质的变化与酵母活力关系的研究

研究了发酵过程中部分有机酸、脂肪酸含量的变化趋势与酵母活力的关系，以及使用不同代数的酵母对发酵液有机酸和脂肪酸含量、组成的影响；并且对比了不同使用代数酵母泥离心液和发酵液相应有机酸、脂肪酸含量的差值，以及差值与酵母活力的关系。     

Improvement of the slide culture technique for the assessment of yeast viability

This work aimed to improve the slide culture technique (SCT) for the assessment of yeast viability. Thus, all the steps of the SCT were standardized: a sample of 20 μL containing 1 × 10⁵ cells/mL was placed in a ~ 20 × 20 mm YPD agar block and incubated for 16–24 h, at 25°C. It was proposed the use of calcofluor white (CFW) to facilitate the microscopic observation of yeast cells. The viability of cell populations in different physiological states (healthy, ethanol stressed and starved cells), assessed by SCT (without or with CFW), did not differ significantly (p  < 0.01). In addition, the viability of healthy and ethanol stressed cells determined by the SCT and the standard plate count technique (PCT) did not differ significantly (p  < 0.01). In conclusion, the improved SCT is a fast and reliable alternative to PCT for the evaluation of yeast viability in research and in the industry. Copyright © 2017 The Institute of Brewing & Distilling     

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.     

Flow Cytometry—A New Tool for Direct Control of Fermentation Processes

With flow cytometric cell cycle analysis, fermentation processes become transparent. This implies that we can control the process, by determining growth phases of the yeast population at specific times during the fermentation process, and that the process can be optimised and regulated directly.     

Comparison of a new digital imaging technique for yeast cell counting and viability assessments with traditional methods

In this study, a new rapid automated yeast cell counter was assessed. The cell counter (Aber Countstar) uses bright‐field microscopy and a dye‐exclusion method. This study's aim was to determine whether this method could be effectively employed in an automated slide based counter to assess viability and to compare this method with results from traditional microscopy and from a radio‐frequency impedance‐based instrument (Aber Compact Lab Yeast Analyser). Excellent correlations were observed between methods. The instrument performed well over a range of yeast concentrations (R² = 0.9913 correlation between automated and manual live cell concentrations). Cell diameters compared well with manual recordings. The instrument was also able to track decreasing cell viability in conjunction with the haemocytometer at viabilities over 20%. The radio‐frequency impedance based instrument exhibited the smallest deviation between 10 repeats, followed by the cell counter (SD: ± 1.08 × 10⁷; ± 5.97 × 10⁷, respectively). Manual counts using a haemocytometer exhibited the largest error between repeats (SD: ± 2.63 × 10⁸) and also required substantially more time (2.28 min) compared with the cell counter (7 sec). The automated cell counter successfully reduced inter‐operator errors, a major hindrance with manual analyses. Tests carried out at a brewery in the UK demonstrated that the cell counter provides consistent counts for assorted yeast strains. External tests highlighted the instrument's ease of use and consistency among different strains of brewing yeast and various stages in the brewing process. Copyright © 2015 The Institute of Brewing & Distilling     

Evaluation of Light Scattering and Autofluorescent Properties of Brewer's Worts for Flow Cytometric Analysis of Yeast Viability

Flow cytometric methods for determining yeast viability are currently available. For effective analysis of yeast in breweries it is important that the light scattering properties of the sample medium (wort) do not interfere with that of target yeast cells. For this reason, a number of wort samples were analysed for their light scattering and autofluorescent properties, as well as their ability to bind the yeast viability dye, oxonol. Worts were found to produce light scattering that was sufficiently different from yeast, such that the two were clearly distinguishable by flow cytometry. Although oxonol bound to wort particles, computer software techniques allowed determination of yeast viability in worts.     

Flow Cytometry for Age Assessment of a Yeast Population and its Application in Beer Fermentations

An expeditious method of yeast age estimation was developed based on selective bud scar staining (Alexa Fluor 488‐labelled wheat‐germ agglutinin) and subsequent fluorescence intensity measurement by flow cytometry. The calibration curve resulting from the cytometric determination of average bud scar fluorescence intensities vs. microscopically counted average bud scar numbers of the same cell populations showed a good correlation and allowed routine cell age estimation by flow cytometry. The developed method was applied for yeast age control in traditional batch and continuous beer fermentations. At the pitching rates used in industrial beer fermentations, our results support former findings by locating a gradient of increasing yeast age from the top to the bottom zone of the fermenter cone. The results also indicate that in continuous beer fermentation, the increasing bud scar fluorescence of immobilized cells could help to schedule the replacement of aged biomass, prior to loss of viability or deterioration of process performance and product quality.     

Detection of Proteinases in Saccharomyces cerevisiae by Flow Cytometry

The physiological state of a yeast population used for inoculation determines how rapidly the cells adapt to new environmental conditions, begin proliferating and utilising extract. The decision as to whether a yeast culture is suitable for re‐pitching should not be based only on viability determinations since this can be misleading. Increased proteolytic activity in a yeast population indicates the onset of senescence. A flow cytometric method has been developed for measuring a wide variety of proteinases in Saccharomyces cerevisiae employing a commercially available casein‐dye conjugate. The detection of intracellular proteinase activity gives an early indication of apoptotic events and allows improved assessment of the physiological state of a yeast population. This knowledge will assist the industry to optimize the selection of yeast and its subsequent fermentation performance. Yeast cell autolysis with all its negative consequences for beer quality and stability will thus be minimised.     

峨眉山野生猕猴桃酿酒酵母筛选研究

从成熟野生猕猴桃自身分离出酿酒酵母菌种,然后与实验室现有优良酿酒酵母菌种一起进行野生猕猴桃酒发酵对比,筛选出MY20酵母菌株为最佳猕猴桃酒酿造菌株,综合菌株在猕猴桃发酵过程中产酸,产酒及其他一些发酵特征比较得出MY20优于其它几种菌株。通过后期驯化,MY20有望成为工业化猕猴桃酒的酿制专用优良菌株。     

Technical note: Flow cytometric identification of bovine milk neutrophils and simultaneous quantification of their viability

The aim of the present study was to develop a flow cytometric procedure for the quantification of the proportion of viable, apoptotic, and necrotic polymorphonuclear neutrophilic leukocytes (PMNL) isolated from both low- and high-somatic-cell-count quarter milk samples. Milk PMNL were differentiated from other cells by indirect fluorescent labeling using a primary anti-bovine granulocyte monoclonal antibody (CH138A) and an Alexa 647-labeled secondary antibody. Polymorphonuclear neutrophilic leukocytes were identified flow cytometrically based on their cytoplasmic granularity and CH138A-positivity. Additional labeling with annexin-V-fluorescein isothiocyanate and propidium iodide was used to determine milk PMNL viability. Thirty milk samples were run in parallel to assess the repeatability of the immunoassay and 6 repeated measurements per sample were performed to assess the instrument stability. Fluorescence microscopic verification of the CH138A staining pattern showed both a high sensitivity (90.9%) and specificity (92.3%). The combination of the side-scatter properties of granulated PMNL and CH138A-Alexa 647 positivity allows the distinction of labeled PMNL from other milk cells and particles that may bind nonspecifically, and from autofluorescent particles present in milk. Quantification of the proportion of PMNL and viable, apoptotic, and necrotic subpopulations in parallel samples gave repeatable results with concordance correlation coefficients varying between 0.93 and 0.99. The average coefficient of variation for repeated measurements in identical samples ranged between 4.2 and 9.7%. In conclusion, this is the first flow cytometric method suited for the simultaneous quantification of viable, apoptotic, and necrotic bovine milk PMNL in a straightforward manner.     

Genetic and physiological characterization of yeast isolated from ripe fruit and analysis of fermentation and brewing potential

‘Wild’ and spontaneously fermented beers are growing in popularity in the craft beer industry. Most of these beers are fermented by the use of either pure cultures of unconventional yeast and bacteria or spontaneous fermentation using mixed local microflora. This study examined the potential of using pure strains of new isolates of wild yeast in the fermentation of a unique beer. The microbial communities from the fruit of pindo palm, loquat, hackberry and blackberry were collected in liquid culture, then plated for isolation. Ten isolates were selected for further analysis. Strains were identified by restriction fragment length polymorphism (RFLP) analysis and analysed for growth in a simple liquid media, fermentation in a complex media, alcohol tolerance and acid tolerance. Despite identification of some strains as the same species, they displayed a wide range of physiological properties. All strains were tolerant of pH values as low as 2.4, but none were tolerant of pH 1.9. Alcohol tolerance of different strains varied from 6 to 12%. Several strains had properties that suggest potential as primary fermenters, including the alcohol fermentation of a beer wort. Organoleptic properties of beers fermented with several of the strains demonstrated potential for commercial brewing. Copyright © 2014 The Institute of Brewing & Distilling     

Flow cytometric assessment of myeloperoxidase in bovine blood neutrophils and monocytes

Myeloperoxidase (MPO) is a lysosomal peroxidase enzyme mainly stored in the azurophilic granules of neutrophils playing an important role in innate immunity for first-line protection against microorganisms in many species including cattle. As such, determination of MPO has become of great interest for the diagnosis of infectious and inflammatory diseases in multiple species such as humans. In cattle, MPO determination is rarely done because methods to assess MPO in this species are limited: functional assays have been described earlier, but so far, the quantification of MPO at the single cell level has not been done yet. In the present paper, an innovative flow cytometric method to assess MPO in blood leukocytes of dairy cattle is described. A commercial anti-bovine MPO was used following density gradient separation to isolate polymorphonuclear (PMN) and mononuclear (MN) leukocytes from blood. Identification of PMN and MN, subdivided in monocytes and lymphocytes, was based on the expression of the surface markers CH138A and CD172A. The optimized protocol was subsequently evaluated on blood samples of 17 Holstein Friesian heifers. Myeloperoxidase expression was measured flow cytometrically and visualized by fluorescence microscopic imaging of sorted PMN and MN populations. We suggest this innovative method to be useful in the field for early detection of cows at higher risk for inflammatory diseases such as mastitis and metritis during the transition period.     

Rehydration of active dried yeast: impact on strength and stiffness of yeast cells measured using microelectromechanical systems

This study investigated the rehydration of active dried yeast and the impact of temperature and wort density on the strength and stiffness of individual cells using a microelectromechanical system. Dried yeast was rehydrated using a variety of methods, including direct pitching into wort (13.6°P) at 12, 22 and 30°C, as well as propagation using YEPD media (4.2°P). Cell viability was found to broadly correlate with measurement of cell strength and stiffness. Both wort density and temperature affected viability and physical characteristics of the cells after 1 h of rehydration. Yeast cells rehydrated at low temperature and high wort density burst at a lower force (0.26 ± 0.02 μN) than cells rehydrated using high temperature and low density media (0.50 ± 0.10 μN). Cells rehydrated at higher temperatures or using low density media showed no significant difference in strength and stiffness when compared with high viability, actively fermenting yeast. Changes in yeast physiology, owing to stress responses, may contribute to the observed differences in mechanical properties. These findings have application in brewery design, as pumping, centrifugation, storage and associated shear impart mechanical stress upon yeast cells. © 2018 The Institute of Brewing & Distilling     

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。