酵母提前絮凝的研究进展

介绍了国内外关于酵母提前絮凝的研究进展及导致酵母提前凝的因素和酵母提前絮凝的假说机理,归纳了酵母絮凝的检测方法,提出了预防酵母提前絮凝的措施及酵母提前絮凝的研究方向。     

Optimised quantification of the antiyeast activity of different barley malts towards a lager brewing yeast strain

The brewing of beer involves two major biological systems, namely malted barley (malt) and yeast. Both malt and yeast show natural variation and assessing the impact of differing malts on yeast performance is important in the optimisation of the brewing process. Currently, the brewing industry uses well-established tests to assess malt quality, but these frequently fail to predict malt-associated problem fermentations, such as incomplete fermentations, premature yeast flocculation (PYF) and gushing of the final beer product. Antimicrobial compounds, and in particular antiyeast compounds in malt, may be one of the unknown and unmeasured malt factors leading to problem fermentations. In this study, the adaptation of antimicrobial assays for the determination of antiyeast activity in malt is described. Our adapted assay was able to detect differing antiyeast activities in nine malt samples. For this sample set, malts associated with PYF during fermentation and gushing activity in beer showed high antiyeast activity. Both PYF and gushing are malt quality issues associated with fungal infection of barley in the field which may result in elevated antimicrobial activity in the barley grain. Also, two more malts that passed the normal quality control tests were also observed to have high antiyeast activity and such malts must be considered as suspect. Based on our results, this assay is a useful measure of malt quality as it quantifies the antiyeast activity in malt which may adversely impact on brewery fermentation.     

Optimization of a Small‐scale Fermentation Test to Predict the Premature Yeast Flocculation Potential of Malts

Rapid and early detection of malts likely to cause premature yeast flocculation (PYF) is of major importance both to the brewer and the maltster. To date, the most reliable and widely applied diagnostic tests for predicting the PYF status of malts involve conducting a small scale fermentation test, which usually takes several days to be completed. This study investigated the impact of selected factors on the ability of such a test to distinguish between PYF positive and PYF negative malts and the fermentation time after which the test results, and therefore the PYF status of the malts, could be confirmed. The results obtained suggest that the PYF potential of malts could be predicted 40 hours post pitching by adding 6 mg.L^?1 linoleic acid and using a highly flocculent PYF sensitive yeast strain.     

A Method to Detect Anti‐metabolic Factors in Fermentations

Premature yeast flocculation (PYF) has been described as the rapid settling of yeast cells during fermentation despite the presence of sufficient nutrients. PYF can cause negative impacts on beer quality and thus can be quite costly to brewers and maltsters. To investigate the causative agent of PYF, small‐scale fermentations were undertaken in both test tubes and cuvettes (15 and 3.5 mL respectively) using worts prepared from PYF‐positive and PYF‐negative malt samples. Fermentations were carried out using six malts, for up to seven days. Turbidity and extract values were monitored for all samples. The small scale (test tube) assay exhibited clear yeast cell flocculation differences between malts. In the cuvette assay the wort fermented, but the yeast cells settled out of suspension rapidly. While this property made the cuvette assay unsuitable for detecting PYF malt, it did allow for measurement of impaired sugar uptake by the yeast independent of yeast in suspension effects. All wort samples fermented in the cuvette assay showed a similar decline in apparent extract (p > 0.05), indicating that (at least in the samples studied) premature yeast flocculation was not caused by a decline in yeast activity. We believe the simple cuvette assay reported here could have application in the measurement of anti‐metabolic factors in fermenting media.     

麦芽PYF因子活力评价及其与酵母絮凝联系研究

通过考察标准培养基下动态培养下面絮凝啤酒酵母的生长进程、酵母悬浮液初始浓度和用量、缓冲液pH、体系水质、PYF因子提取方式等多因素对测定结果的影响,确立了麦芽PYF因子活力测定体系的最佳参数。麦芽PYF因子加和性及其与酵母絮凝性能关系的进一步分析结果表明:PYF因子具有很好的加和性,对酵母絮凝起促进作用。     

The effect of microbial arabinoxylanases on premature yeast flocculation

The use of barley malt infected with microorganisms can induce premature yeast flocculation (PYF) during fermentation. To understand this further, it is necessary to determine the role of microbial enzymes influencing PYF. In this study, the induction of PYF by two arabinoxylanases from Bacillus subtilis (A and B) and one from Aspergillus brasiliensis (F) was investigated. The results showed that all three arabinoxylanases could induce premature yeast flocculation. Husk from PYF negative (PYF-) malt pre-treated with arabinoxylanases could induce severe PYF. An increase in enzyme activity resulted in an increase in of PYF. Bound ferulic acid – an important component inducing PYF – increased significantly in the 40% (v/v) ethanol precipitate of wort. However, the ratio of ferulic acid/arabinoxylan in the 40% ethanol precipitate decreased with an increase in arabinoxylanase F, but increased with arabinoxylanases A and B. The addition of the three arabinoxylanases at the beginning of the PYF- mashing process showed that the arabinoxylanases A and B could induce PYF with greater intensity than arabinoxylanase F. It can be inferred that the bacterial arabinoxylanases produced more PYF factors than from Aspergillus brasiliensis. The results of this study may provide new insights for further research and focus on the role of bacteria in PYF. © 2020 The Institute of Brewing & Distilling     

Premature yeast flocculation factors from barley malt present in both malt husk and the non‐husk part

In the present study, small‐scale fermentations of seven malts from different maltsters in China were used to monitor their premature yeast flocculation (PYF) potential. Husk exchange was applied between PYF negative malt (PYF‐) and PYF positive malt (PYF+) to investigate the PYF factors potentially present in the husk. The results showed that PYF factors were present in both malt husk and the non‐husk part. The two factors showed various ratios among different PYF+ malts, and either of them could induce PYF if it reached the threshold. Moreover, the major antimicrobial substances damaging the yeast cells were in the non‐husk part. Copyright © 2014 The Institute of Brewing & Distilling     

The role of ferulic acid and arabinoxylan in inducing premature yeast flocculation

Premature yeast flocculation (PYF) is a universal phenomenon and has caused serious problems in the brewing industry. For brewing quality control, it is of great value to investigate the PYF factors that induce this destructive phenomenon. In the present study, two barley malts (PYF+ and PYF?), made from the same barley cultivar by one maltster in China, were selected for PYF factor investigations. The results showed that considerably higher amounts of the bound ferulic acid (FA), rather than arabinoxylan (AX), existed in the PYF+ wort compared with the PYF? wort. To better understand the role of these polysaccharides in PYF+ and PYF? wort, they were fractional precipitated with graded ethanol concentrations. The AX and FA contents in each fraction, as well as the molecular weight profiles and monosaccharide composition, were determined. Furthermore, the PYF?inducing activities of each fraction were measured using a small‐scale fermentation. It was found that the 40% ethanol‐precipitated fraction of both the PYF+ and PYF? wort, and the 60% fraction from the PYF+ wort, could induce the severe PYF phenomenon when added to the PYF? wort. These fractions were proposed to contain PYF factors of suitable molecular weight and a sufficient amount of the polysaccharides. In addition, it was found that the bound FA in arabinoxylan behaved as an important PYF factor in barley malt. The results of this work may contribute to the further study of the PYF mechanism. Copyright © 2015 The Institute of Brewing & Distilling     

FLOCCULENCE OF BREWERY YEASTS AND THEIR SURFACE PROPERTIES: CHEMICAL COMPOSITION,ELECTROSTATIC CHARGE AND HYDROPHOBICITY

The flocculence of a top and a bottom fermentation brewery yeast was studied through the cell surface properties: surface composition (X-ray photoelectron spectroscopy), surface charge (microelectrophoresis) and hydrophobicity (adhesion tests). Aerobic Cultures and fermentation simulating industrial conditions were performed. The flocculence (tendency to flocculate in favourable conditions) has been determined by a test which separated the occurrence of flocculation and the measurement of its manifestation (suspension clearing); the flocculating agents were ethanol and calcium. The top fermentation strain does not flocculate with calcium alone but is very sensitive to ethanol (0.01% (v/v)). The lower flocculence observed for cells cultured on malt extract as compared with cells cultured on yeast extract is accompanied by a more negative zeta potential. The bottom fermentation strain which has a more negative surface flocculates only in the presence of calcium but ethanol enhances the process. The variation of flocculence according to the culture medium and harvesting time is correlated to the variation of the phosphate surface concentration. This is not paralleled by a variation of the zeta potential which does not decrease below a value of about-35 mV as the phosphate surface concentration increases. The increase of flocculence as a function of culture time correlates also with an increase of the cell hydrophobicity; this is more obvious for the cultures than for the fermentations.     

Modelling of Yeast in Suspension During Malt Fermentation Assays

Premature yeast flocculation (PYF) is a poorly understood condition leading to attenuation of fermentation and poor alcohol yields. The yeast in suspension in fermenting worts prepared from either normal or premature flocculating malts was continually measured during the small 15 mL fermentation test recently developed in the Dalhousie laboratories. A simple inexpensive monitoring system was constructed from a laser level, photometric cell and data logger. This system allowed non‐destructive data collection of absorbance data (over 700 data points per fermentation) at ?650 nm. A non‐linear modeling technique was applied to the data, and yielded two best‐fitting logistic models. The model parameters were quantitatively compared to determine if statistical differences between PYF and normal wort were apparent. A student's t test of the logistic parameters indicated a significant difference (p > 0.025) between control and PYF worts for the increase in absorbance at the beginning of the fermentation. A significant difference (p > 0.0001) in the inflection time of absorbance down‐curve between the control and PYF wort was also noted.     

Flocculation in industrial strains of Saccharomyces cerevisiae: role of cell wall polysaccharides and lectin‐like receptors

Yeast flocculation is the reversible aggregation of yeast cells promoted by the interaction between lectin‐like protein receptors with mannose side chains on adjacent cell walls. Flocculation is governed by several physiological factors, including the type of nutrient sugar available to yeast. We grew four industrial strains of Saccharomyces cerevisiae , representing applications in the brewing, winemaking and bioethanol sectors, to late stationary phase and quantified the cellular content of mannans, glucans and lectin‐like proteins on yeast cell surfaces. Results indicated that brewing and champagne strains showed moderate to high flocculation ability when grown with glucose, fructose, maltose or galactose, whereas winemaking and fuel alcohol strains only showed moderate flocculation when grown on maltose and galactose. All yeast strains studied were weakly flocculent when grown on mannose. With regard to lectin‐like receptors, their number played a more important role in governing yeast flocculation than the mannan and glucan contents in yeast cell walls. We conclude that all the industrial strains of S. cerevisiae belonged to New‐Flo type on the basis of their flocculation behaviour observed when cultured on different sugars. Quantification of yeast cell wall polysaccharides and receptor sites indicates that mannan and glucan levels remain almost constant, irrespective of the strain under investigation. The main difference in flocculation characteristics in industrial yeast strains appears to be due to variations in concentrations of lectin‐like cell surface receptors. Our findings may benefit brewers, winemakers and other yeast‐based technologies in design of media to prevent premature flocculation during fermentation. Copyright © 2017 The Institute of Brewing & Distilling     

Flocculation,cell surface hydrophobicity and 3‐OH oxylipins in the SMA strain of Saccharomyces pastorianus

Three‐hydroxy‐oxylipins (3‐OH oxylipins) have been previously detected in brewing yeast production strains at flocculation onset. In this work, the SMA strain of Saccharomyces pastorianus was characterized during growth in a miniature fermentation assay by measuring flocculation and cell surface hydrophobicity (CSH). Proportions of 3‐OH oxylipin were also measured concurrently during growth in the miniature fermentation assay and a defined 3‐OH oxylipin extraction protocol using ethyl acetate is presented along with a novel derivatization and gas chromatography–mass spectrometry (GC‐MS) detection approach. When the SMA strain was grown in the assay, near maximal CSH and flocculation levels were achieved by a 36 h fermentation time. Under the same culture conditions, the oxylipin 3‐OH decanoic acid (3‐OH 10:0) was identified. This oxylipin could not be detected early in the fermentation, but elevated relative levels of 3‐OH 10:0 were reached by 36 h, coinciding with increased CSH levels. It was previously presumed that the formation of 3‐OH oxylipins at flocculation onset might increase the CSH. However, results from this study suggest that 3‐OH 10:0 may not contribute to cell wall hydrophobicity. The flocculation behaviour of the SMA strain was also monitored in the presence of 3‐OH 10:0, but exposure to this oxylipin did not impact the sedimentation of this yeast, suggesting that 3‐OH oxylipins may not act as mediators of quorum sensing in this strain. Copyright © 2015 The Institute of Brewing & Distilling     

啤酒酵母提前絮凝的研究进展

酵母提前絮凝是啤酒企业时有发生的现象,因其复杂的影响因素至今对酵母提前絮凝的发生机理及提前絮凝因子的本质没有明确的证实,也因此给整个啤酒酿造产业链造成了极大的困扰。近年来,随着环境污染及各种恶劣天气的出现,导致啤酒酵母提前絮凝发生相对增多,对麦芽企业及啤酒企业造成一定经济和名誉损失。作者综述了啤酒酵母提前絮凝的国内外最新研究进展,包括机理假说、提前絮凝因子的来源、形成机制及控制研究,以期对啤酒产业链提供有效解决酵母提前絮凝的思路和方法。     

Effects of Fermentation Parameters and Cell Wall Properties on Yeast Flocculation1

Industrial wort was fermented with a NewFlo phenotype ale yeast in lab‐scale cylindrical fermenters. The effects of various fermentation parameters and yeast cell wall properties on yeast flocculation were studied during 120 h fermentation. The evaluation of the cell volume during the fermentation revealed a non‐normal distribution (p < 0.05) at most fermentation times. Overall yeast cell size initially decreased during the first 24 h of fermentation then increased during the 24–60 h fermentation period. Cell size subsequently declined until the end of fermentation presumably due to floc settling. While yeast flocculation began after 24 h fermentation, most flocs remained in suspension until 60 h when the average turbulent shear rate caused by CO₂ evolution declined to below 8 s^?1. Both the Helm's flocculence and cell surface hydrophobicity values rapidly increased to high numbers from 24 h onward. Changes in the orthokinetic capture coefficient (α₀) value with fermentation time, measured in fermenting worts, indicated a significant increase (p < 0.001) after 24 h of fermentation. Presumably, this change was due to increases in ethanol and the decline in sugar concentration with time. Although a significant positive correlation (p < 0.05) was observed between zymolectin densities and cell surface areas, the total zymolectin level on yeast cell walls did not change significantly with fermentation time (p > 0.05). Interestingly, no significant difference existed in Helm's flocculation values of suspended and settled yeast cells (p > 0.05). The flocculation rate of LCC125 was readily inhibited by addition of glucose or maltose. Results suggest that fermentable sugar levels and shear force exert major influences on yeast flocculation during beer fermentations.     

Quality assessment of lager brewery yeast samples and strains using barley malt extracts with anti-yeast activity

Membrane active anti-yeast compounds, such as antimicrobial peptides and proteins, cause yeast membrane damage which is likely to affect yeast vitality and fermentation performance, parameters which are notoriously difficult to analyse. In this work the sensitivity of lager brewery yeast strains towards barley malt extracts with anti-yeast activity was assessed with an optimised assay. It was found that yeast, obtained directly from a brewery, was much more sensitive towards the malt extracts than the same yeast strain propagated in the laboratory. Sensitivity to the malt extracts increased during the course of a laboratory scale fermentation when inoculated with brewery yeast. As the assay was able to differentiate yeast samples with different histories, it shows promise as a yeast quality assay measuring the yeast's ability to withstand stress which can be equated to vitality. The assay was also able to differentiate between different lager yeast strains of Saccharomyces cerevisiae propagated in the laboratory when challenged with a number of malt extracts of varying anti-yeast activity. The assessment of yeast strains in the presence of malt extracts will lead to the identification of yeast strains with improved quality/vitality that can withstand malt-associated anti-yeast activity during brewery fermentations.     

DIACETYL—A REVIEW: PART I—ANALYTICAL AND BIOCHEMICAL CONSIDERATIONS: PART II—BREWING EXPERIENCE

Diacetyl and 2,3-pentanedione are normal products of yeast metabolism and are formed in every brewery fermentation. The desired level in the final beer depends on the particular flavour aimed for but, in all types of beer, flavour defects are caused by excessive concentrations of diacetyl and many brewers might be happy to have no diacetyl in the beer. Recent improvements in analytical techniques show that many of the problems associated with diacetyl are due to the occurrence of compounds which can give rise to diacetyl in the finished beer. These compounds include the so-called “precursor,” acetolactic acid, but possibly other compounds such as the bisulphite addition compound of diacetyl are also involved. Study of the factors affecting diacetyl formation and removal by yeast shows how the concentration of diacetyl in beer can be controlled, and the processes at present used to regulate the diacetyl concentration in beer are described. The yeast strain used, the condition of the pitching yeast, the wort composition, the detailed management of the fermentation and the treatment of the beer during packaging and storage can all affect the diacetyl content of the beer.     

AMINO ACID BALANCE IN YEAST FERMENTATION WITH A SYNTHETIC AMINO ACID MIXTURE AS NITROGEN SOURCE

Two brewery yeasts, one bottom- and one top-fermenting strain, were allowed to ferment an 8% glucose solution containing as nitrogen source an amino acid mixture simulating that obtained when yeast was autolysed. The amounts given were approximately twice as high as the expected requirements. After completion of fermentation the total amounts of each amino acid in the whole system, i. e., in medium and yeast, were determined. The results show that the yeast had not taken up amino acids according to its own composition. The amino acids previously found to be rapidly absorbed from brewery wort were present in the whole system in considerably smaller amounts than in the original medium, indicating that these acids had been utilized as a nitrogen souce or for other purposes. The acids which are taken up slowly from brewery wort were present in larger amounts than in the original medium, indicating that they had been synthesized despite the excess in the medium. The two strains showed relatively similar behaviour.     

Flocculation of the yeast Candida famata (Debaryomyces hansenii): An essential role for peptone

Aggregation of Candida famata (Debaryomyces hansenii) is consistent with being a form of lectin-mediated yeast flocculation. Flocculation of C. famata is unusual in that it requires the presence of peptone, either in the growth medium or added later to harvested cells in buffer. Flocculation after peptone addition was rapid, being largely complete within 10 min. Heat-killed cells also flocculated, arguing for direct participation of peptone in the flocculation binding mechanism. Flocculent C. famata cells progressively lost the ability to flocculate when washed with EDTA. Flocculation was fully restored by peptone addition; calcium addition was without effect. C. famata cells were able to agglutinate erythrocytes in the presence or absence of peptone. Pronase E-treated yeast lost both the ability to haemagglutinate and self-flocculate. Haemagglutination was not diminished by progressive EDTA washing, suggesting that surface lectins remained present and active on the yeast cell walls. Non-flocculating C. famata cells mutually flocculated with non-flocculent Schizosaccharomyces pombe cells, shown to have surface-exposed galactose residues. Mutual flocculation was lost following treatment of C. famata with Pronase E. It was concluded that the cell wall of C. famata contains lectins enabling haemagglutination and mutual flocculation but lacks carbohydrate receptors for these lectins. This yeast self-flocculates only via bridging multi-valent carbohydrates; these being present in peptone.     

利用大麦预测酵母提前絮凝现象的研究

介绍了一种用比色法来判断酵母提前絮凝现象的方法,该方法仅通过测定酵母细胞悬浮液中细胞的沉降速率来判断酵母絮凝性,无需制麦、发酵,只需简单的提取操作即可进行,克服了发酵力试验的一些缺陷。通过正交实验优化了大麦中絮凝因子的提取工艺,研究了不同菌种对大麦絮凝因子的敏感程度。通过与啤酒大生产所反映的实际情况做对照,该方法预测的结果与实际大生产有很强的对应性。     

Optimised Acidification Power Test of Yeast Vitality and its Use in Brewing Practice

The optimised acidification power test (APT) of brewer's yeast quality includes storing the yeast slurry at 2°C under beer (AP remains constant for up to 6 days), a 15 min sample equilibration to room temperature, decantation, and washing by triple centrifugation in deionised water. The final yeast pellet keeps its AP for up to 6 h at room temperature under water and thus the APT does not need to be performed immediately after yeast collection. The correct AP value (maximum acidification produced by given yeast) is determined at 25 ± 0.1°C in a 15 mL sample containing ≥5% glucose and ≥1.5 g yeast wet weight. The cell concentration is conveniently measured as absorbance (A₆₆₀). Cell flocculation and/or sedimentation that can distort APT results can be prevented by stirring the sample at ≥200 rpm. The AP of yeast of different generations used to pitch brewery fermentations in cylindroconical tanks had a very low correlation with the wort half‐attenuation time (T_1/2) due to large scatter, while each yeast generation separately showed a clear T_1/2‐AP relationship. The lowest AP of yeast cropped from cylindroconical tanks was displayed by the first cropped fraction. Post‐cropping cooling had no effect on AP. Variations in pitching yeast vitality and their effect on the outcome of a brewery fermentation can be masked by variations in pitching rate, wort composition, ambient conditions in the cylindroconical tanks and other factors.