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     

Comparing the Impact of Environmental Factors During Very High Gravity Brewing Fermentations

The impact of the initial dissolved oxygen, fermentation temperature, wort concentration and yeast pitching rate on the major fermentation process responses were evaluated by full factorial design and statistical analysis by JMP 5.01 (SAS software) software. Fermentation trials were carried out in 2L‐EBC tall tubes using an industrial lager brewing yeast strain. The yeast viability, ethanol production, apparent extract and real degree of fermentation were monitored. The results obtained demonstrate that very high gravity worts at 22°P can be fermented in the same period of time as a 15°P wort, by raising the temperature to 18°C, the oxygen level to about 22 ppm, and increasing the pitching rate to 22 × 10⁶ cell/mL. When diluting to obtain an 11.5°P beer extract, the volumetric brewing capacity increased 91% for the 22°P wort fermentation and 30% using the 15°P wort. After dilution, the fermentation of the 22°P wort resulted in a beer with higher esters levels, primarily the compound ethyl acetate.     

啤酒高浓酿造中氨基酸对酵母发酵性能和啤酒色值的影响

探讨在24?°P高浓啤酒发酵过程中8?种氨基酸（Met、Phe、Trp、Arg、His、Ile、Leu、Lys）的不同添加量（分别为原麦汁中相应氨基酸含量的0.5、1?倍和2?倍）对酵母生理特性、发酵性能和啤酒色值的影响。结果表明：8?种氨基酸的补充可显著提高麦汁发酵度、乙醇产量,促进酵母生长,提高酵母活细胞率,改善啤酒色值。其中,补充1?倍氨基酸的高浓麦汁发酵性能较好,与对照组相比,发酵度、乙醇产量、最大悬浮酵母细胞数和发酵结束时的酵母活细胞率分别提高了6%、17%、11%和10%。添加氨基酸的高浓酿造啤酒经稀释后,啤酒色泽依然鲜亮,且添加1?倍氨基酸酿造而成的啤酒经稀释后色差（ΔE）最小,色泽最接近青岛纯生啤酒。     

Vitamins in brewing: presence and influence of thiamine and riboflavin on wort fermentation

Thiamine and riboflavin vitamers are present in a wide range of foods including beer. These vitamers play critical roles in a variety of enzymatic complexes and can promote and maintain metabolism. Currently, the presence and role of these vitamers in the malting and brewing industry have not been widely explored. This research investigated the effects of various fermentation conditions that may lead to the variations in the vitamin content in beer observed by previous researchers. The present research found that during fermentation, the thiamine content of wort is quickly utilized within the first 6 h of a standard fermentation and the uptake of this vitamin is not affected by increases in wort gravity. While no significant changes were observed in extracellular phosphorylated vitamers of thiamine, both free thiamine and thiamine diphosphate accumulated intracellularly during the wort fermentation. Meanwhile extracellular riboflavin vitamers were only poorly utilized during beer fermentations, however flavin mononucleotide rapidly accumulated intracellularly and more so under aerobic conditions. When yeast was exposed to an all‐malt high‐gravity wort, the thiamine or riboflavin utilization was not affected. However, thiamine utilization was reduced in adjunct‐driven high‐gravity worts. Notwithstanding the lowered thiamine uptake under high‐gravity conditions; there were some minor improvements in fermentation performance and yeast viability. The addition of thiamine to an all‐malt wort did appear to enhance yeast viability, both under normal and high‐gravity conditions. Copyright © 2016 The Institute of Brewing & Distilling     

ABSENCE OF NUTRITIONAL REQUIREMENT FOR UNSATURATED FATTY ACIDS BY BREWER'S YEAST IN MALT WORT

Addition to de-aerated malt wort of ergosterol dispersed in the non-lipid detergent Tergitol allows satisfactory growth of oxygen-requiring cells of brewing yeast. Any nutritional requirement for unsaturated fatty acid for viability and growth of such cells (as distinct from effect on metabolite balance, e.g. of esters) can therefore be supplied by the normal constituents of a typical malt wort.     

The effect of pitching rate on fermentation,maturation and flavour compounds of beer produced on an industrial scale

The aim of the study was to determine the effect of the initial number of yeast cells in the wort on the process of fermentation, maturation and the content of the volatile components of beer, as well as the viability and vitality of the yeast biomass. The experiments were performed on an industrial scale, with fermentation and maturation in cylindro‐conical fermentation tanks with a capacity of 3800 hL. Yeast for pitching was collected after secondary fermentation (third passage) and wort pitching levels were 5 × 10⁶, 7 × 10⁶ and 9 × 10⁶ cells/mL. During fermentation and maturation, the changes in the content of the extract, yeast growth, yeast vitality and selected volatile components were investigated. Experiments showed that the yeast inoculum had a significant impact on the course of the fermentation and metabolic changes. With increasing numbers of cells introduced into the wort, the content of the esters and fusel alcohols increased, while the acetaldehyde concentration decreased. These changes affected the final quality of the beer. Copyright © 2015 The Institute of Brewing & Distilling     

高浓酿造技术研究进展

高浓酿造技术的主要优点通过有效降低糖化的用水，仅利用现有糖化、发酵和储存设备即可大幅度提高啤酒产量，缺点包括：降低糖化锅原料及煮锅酒花利用率，泡沫稳定性下降，酸洗效应，降低酵母存活率，酵母回用代数下降以及需要及时调整麦汁中二阶离子的浓度。     

FERMENTATION OF WORTS MADE FROM 100% RAW SORGHUM AND ENZYMES

Worts made from raw sorghum and enzymes were successfully fermented even though the level of FAN present (51 mg/l) is well below that essential for fermentation of wort made from malted barley. Changes in typical fermentation parameters such as specific gravity, pH uptake of free amino nitrogen (FAN) and ammonium ions mirrored the increase in yeast cell concentration. Yeast viability remained high throughout the fermentation. Under identical fermentation conditions, malted barley worts showed typical fermentation profiles. However, malted barley worts with specific gravity maintained by the addition of D-glucose, but in which the FAN was diluted to a level similar to that found in a wort made from sorghum and enzymes, fermented more slowly and failed to attenuate fully. Five consecutive fermentations, using yeast cropped from the preceding to pitch the current fermentation were conducted. The specific gravity profiles were essentially the same in all five fermentations. Final values of pH, yeast in suspension, yeast viability and FAN were also indistinguishable. The yeast crop taken from fermentations of worts made from raw sorghum and enzymes represented a 5-fold increase over the initial pitching rate. When compared to commercial beers, the beers derived from fermentation of worts made from raw sorghum and enzymes contained lower levels of ethyl acetate, and higher levels of both 2- and 3-methyl butanol. In the beers derived from sorghum, isobutanol was always less than 20% of the total higher alcohol concentration.     

The Effects of Linoleic Acid Supplementation of Cropped Yeast on its Subsequent Fermentation Performance and Acetate Ester Synthesis

For beer wort fermentation the addition of unsaturated fatty acids has sometimes been suggested as an alternative to wort oxygenation. This can however negatively affect the synthesis of acetate esters and consequently beer flavour. This work investigates the effect of supplementing a cropped yeast with an unsaturated fatty acid on the fermentation performance of the pitching yeast. Cropped yeast is in a different physiological state to yeast pitched in unfermented wort. Using a synthetic medium for the fermentations, it was found that the incubation of cropped yeast with linoleic acid resulted in two important changes in the yeasts composition: (1) the ratio of unsaturated fatty acids to total fatty acids increased from 0.53 to 0.66 and (2) the ratio of trehalose to glycogen increased from 0.17 to 0.49. The performance of this yeast in subsequent fermentations was compared to unsupplemented yeast under three conditions: medium pre‐aeration, de‐aerated medium and de‐aerated medium with newly added unsaturated fatty acid. It was found that the supplemented pitching yeast showed growth, attenuation and ethanol formation profiles similar to those obtained with unsupplemented yeast in pre‐aerated medium, which simulated the normal brewing practice. Compared to fermentations with unsaturated fatty acids added to the medium, the supplemented cropped yeast did not induce a reduction in acetate ester synthesis. Results indicated that the supplementation of cropped yeast with unsaturated fatty acids could be an interesting alternative to wort oxygenation to restore the optimal membrane fluidity of the yeast.     

Screening for new brewing yeasts in the non‐Saccharomyces sector with Torulaspora delbrueckii as model

This study describes a screening system for future brewing yeasts focusing on non‐Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off‐flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by‐products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre‐fermentation as a bio‐flavouring agent for beers that have been post‐fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour‐forming properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Flow cytometry and cell sorting for yeast viability assessment and cell selection

Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat-killed mixtures. However, for re-hydrated HADY, Ox stained a significantly (P⩽0·05) higher proportion of cells than did PI. © 1998 John Wiley & Sons, Ltd.     

Maintaining yeast viability in continuous primary beer fermentation

Continuous fermentation is a long known and vastly studied process. The use of immobilized cell technology (ICT) is exploited in a significant number of studies owing to the associated high volumetric productivity, time savings and low capital demand. This work was aimed at solving one of the most relevant obstacles to implementing ICT on a large scale in beer fermentations, namely the control of biomass and the maintenance of cell viability in a gas‐lift bioreactor. For this purpose, foam fractionation by skimming was proposed as a tool for control of continuous biomass concentration. The consequences of foaming on lignocellulosic yeast carrier losses were assessed and discussed. A steady consumption of sugars from wort, as well as consistent ethanol production, were achieved. The viability of the suspended cells in the reactor was compared with that of the cell population in the foam using flow cytometry. Results suggest that foam might be used as a promising tool to skim non‐viable biomass out of the gas‐lift reactor, thus ensuring the maintenance of a cell culture with optimum viability. Copyright © 2014 The Institute of Brewing & Distilling     

低浓度淡爽型啤酒的酿造

以改善低浓度淡爽型啤酒品质为目的 ,提出了一种新颖的低浓度淡爽型啤酒的酿造方法 .采用二次煮出二段式糖化法 ,用 70 %麦芽和 3 0 %大米的原料配比 ,提高麦芽汁中糖与非糖的比值 ,并在糖化过程中添加啤酒酵母提取物作啤酒发酵的补充氮源 .所酿造的啤酒口味纯正 ,泡沫洁白细腻 ,持久挂杯 .     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

PITCHING RATE IN HIGH GRAVITY BREWING

The optimal pitching rate in high gravity worts (12–16°P) was about 0.3 g/l wet weight (2.3 × 10⁶ counted cells/ml) and per one percent of original wort gravity. In very high gravity worts (20–23°P) the corresponding figure was 0.4 g/l (2.9 × 10⁶ cells/ml). Higher amounts of yeast did not improve the fermentation rate. With increased original wort gravity, flocculation of the yeast weakened and the amount of cropped yeast decreased. The viability of the crop yeast was good. In the conditions used, excessive production of acetate esters occurred only with pitching rates lower than the recommended rate. As the original wort gravity increased, more fermentable extract was metabolized to ethanol rather than utilized for yeast growth. The highest ethanol yield obtained was 10.9% (v/v).     

ROLE OF WORT AERATION IN THE BREWING PROCESS PART 2: THE OPTIMAL AERATION CONDITIONS FOR THE BREWING PROCESS

Fermenting and growing activities of early yeast (yeast at the early stage of fermentation) were elevated by wort aeration, reaching their maximal level after 5 h of aeration. The lipid content of early yeast was also elevated by wort aeration, and reached a maximal level after 9 h of aeration. While certain amounts of lipid are necessary for the budding of yeast, the excess amounts of lipid synthesised between 5–9 h of aeration were not sufficient for one further budding, and so accumulated in the final yeast. It is better to set the optimal aeration conditions on the lipid content of early yeast, rather than the amount of wort aeration.     

Impact of the Long‐Term Maintenance Method of Brewer's Yeast on Fermentation Course,Yeast Vitality and Beer Characteristics

The effect of the long‐term maintenance method used with a brewer's yeast on its technological properties was determined in laboratory fermentation trials with a 12°P all‐malt wort. The trials were performed at a constant temperature and under conditions of constant substrate concentration. Two cultures of a bottom fermenting yeast, Saccharomyces pastorianus RIBM 95, were tested — one culture was maintained by subculturing on wort agar slopes at 4°C and the other culture underwent a three year storage in liquid nitrogen at minus 196°C. Parameters under investigation included yeast vitality measured as acidification power (AP), fermentation time needed to reach an alcohol level of 4%, the yeast cell count, sedimentation of the yeast during the fermentation, and the production of beer flavour compounds in green beer. The yeast culture stored for three years in liquid nitrogen displayed a higher count of suspended cells, required a shorter time to attenuate the wort to produce 4% alcohol and produced a 1.5 to 2.5‐fold higher concentration of a number of flavour compounds. The long‐term storage method did not affect the sedimentation ability and vitality of the yeast strain tested.     

不同麦汁浓度及酵母代数的电导率在线监测

采用电导率测量的方法对啤酒发酵过程进行了在线监测，并且对不同麦汁浓度、不同酵母代数进行了研究。结果表明：对于不同浓度的麦汁，其发酵过程中电导率的变化呈现明显的规律性，酵母代数对电导率的影响较大，1代酵母要比0代酵母提前约50min出现第1个拐点，即结束迟滞期；提前3h出现第2个拐点。这为研究啤酒发酵提供了一种有效的方法。     

Genome shuffling to improve fermentation properties of top-fermenting yeast by the improvement of stress tolerance

Fermentation properties of top-fermenting yeast are under the control of multiple genes difficult to manipulate directly by classical breeding, metabolic engineering, or other genetic methods with specific genes or pathways as target. Here, genome shuffling is introduced to improve fermentation performance (such as the viability of the yeast, flavor of beer, and the fermentation time) by improving wort and ethanol tolerance of top-fermenting yeast. The strategy was performed not based on polyethylene glycol (PEG)-mediated protoplast fusion but using yeast sexual and asexual reproduction by itself. The best performing strain W3-8 was selected on the selective plates after 3 rounds of genome shuffling. The fermentation time of W3-8 was not only markedly shortened, but also, most flavor compounds were distinctly improved. In particular, ethanol yield was increased by up to 67% after the 3^rd pitching compared with the control. Furthermore, W3-8 promoted desired amounts of esters and higher alcohols, in accordance with specific consumer preferences. Significant improvement in the fermentation traits of the top-fermenting yeast was achieved using genome shuffling.     

STUDIES ON THE BINDING BETWEEN YEAST AND A MALT POLYSACCHARIDE THAT INDUCES HEAVY YEAST FLOCCULATION

A factor which causes heavy yeast flocculation of Saccharomyces cerevisiae 2036 was found to be associated with the malt husk by Axcell et al.¹ In the present work it was established that the factor is a polysaccharide and immunological techniques were used to show that the factor binds to the yeast cell surface during fermentation. It was shown that the factor is present at significantly higher concentrations in wort causing premature yeast flocculation than in normal wort. A particular malt husk extract, obtained using a mild aqueous extraction procedure, induced premature flocculation when added to fermentations in normal wort. In sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the malt husk extract, 4 protein bands (42,600; 17,500, 15,100 and 13,100 daltons) and a high MW (molecular weight) polysaccharide were identified. Antibodies were raised against electroeluted proteins as well as against the homogenized polyacrylamide gel containing the polysaccharide band. Enzyme-linked immunosorbent assays (ELISA) showed that the protein components were present in similar concentrations in premature flocculent and normal wort. In contrast, the high MW polysaccharide occurred at a significantly higher concentration in wort inducing premature flocculation than in normal wort. Immunogold electron microscopy showed that the high MW polysaccharide bound extensively to the surface of flocculent cells grown in premature flocculent wort. There was markedly less labelling on yeast cells grown in normal wort. Negligible labelling occurred when the yeast cells were incubated with antibodies against the different protein components of malt husk extract.