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     

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。     

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.     

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。     

啤酒酵母FLO8基因敲除对酵母发酵性能的改良

利用PCR介导基因中断技术,以pUG6为模板,设计含有与flo8基因两侧序列同源的长引物,构建带有卡那抗性基因（KanMX）中断盒,转化啤酒酵母G-03,获得一株转化菌G-03/f8。对转化菌G-03/f8和原菌G-03进行生理生化性能和摇瓶发酵性能比较。该菌株在实验室常规发酵中,生理性能、发酵性能基本上与出发菌株保持一致。当用酵母提前絮凝（PYF,premature yeast flocculation）值高的麦芽糖化后的麦汁接种发酵时,G-03/f8的絮凝性能与常规发酵下的絮凝性能基本一致,此时明显优于出发菌株G-03。G-03/f8的酒精度、发酵度等低温发酵指标与常规发酵相比有小幅度的下降,但明显高于此时G-03的各项发酵指标。相对于出发菌而言,G-03/f8对高PYF值的麦汁不敏感,能够保持较好的发酵性能,因此在高PYF值麦芽的利用上有良好的应用前景。     

Isolation and partial purification of mannose-specific agglutinin from brewer's yeast involved in flocculation

Yeast cell-agglutinating activity, designated agglutinin (possible lectin), was isolated from cell walls of both non-flocculent and flocculent brewer's yeast cells. Agglutinin-mediated aggregation of yeast cells in a manner similar to flocculation with respect to specific mannose-sensitivity, pH-dependence and calcium-dependence. Agglutinating activity was found to be heat-stable and protease-insensitive. Furthermore, addition of agglutinin to flocculent cells strongly stimulated the flocculation ability of the cells, whereas addition to non-flocculent cells rendered these cells weakly flocculent. Agglutinin was found to be released from flocculent cells during the course of a flocculation assay, but not from non-flocculent cells. Presence of mannose during the assay inhibited release of agglutinin. Our results suggest that (i) mannose-specific agglutinin is continuously synthesized during growth of brewer's yeast cells, (ii) agglutinin is present in cell walls of non-flocculent cells but is unable to bind its ligand on other cells, and (iii) the ability of yeast cells to flocculate in a flocculation assay depends, among other factors, on release of agglutinin from the cells. A 10-kDa polypeptide might represent one form of agglutinin.     

INDUCTION OF PREMATURE YEAST FLOCCULATION BY A POLYSACCHARIDE FRACTION ISOLATED FROM MALT HUSK

A husk polysaccharide component from a malt causing premature yeast flocculation was isolated and characterized, and its effect on the fermentation pattern of Saccharomyces cerevisiae 2036 established. 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 molecular weight polysaccharide were identified. These components were separated by gel chromatography and their effect on the fermentation pattern established. Only the polysaccharide fraction induced premature flocculation. Sugar analysis of this fraction indicated the presence of arabinose, galactose, glucose, mannose, xylose, rhamnose and an acidic sugar component. The polysaccharide fraction showed haemoagglutination activity, which was not inhibited by any of 11 simple sugars tested.     

阿拉伯木聚糖的氧化交联与啤酒大麦麦芽过滤性能的相关性研究

本文以18个大麦麦芽样品为研究对象,以pearson相关分析法研究了大麦麦芽中过氧化物酶(Peroxidase,POD)、麦汁中键合态阿魏酸以及多聚阿拉伯木聚糖(PAX)与麦芽过滤性能的相关性。研究结果表明:麦芽过滤速度与麦芽POD酶活、麦汁粘度、麦汁中键合态阿魏酸及PAX含量呈显著负相关,相关系数分别为0.632(p<0.01)、0.521(p<0.05)、0.555(p<0.05)和0.664(p<0.01);PAX含量与键合态阿魏酸含量、麦汁粘度呈显著正相关,相关系数分别为0.590(p<0.05)和0.595(p<0.01)。这一结果表明,阿魏酸介导的AX的氧化交联与大麦麦芽过滤性能紧密相关,很可能是AX导致麦芽过滤性能差的重要途径之一。本文为改善大麦麦芽过滤性能提供了一定的理论依据。      

采用黑曲霉发酵制备阿拉伯木聚糖酶和阿魏酸酯酶的研究

采用黑曲霉发酵麦麸、蔗渣和玉米麸皮都能获得阿魏酸酯酶和阿拉伯木聚糖酶,并在第3d达到产酶高峰。但不同发酵基质产酶活性差别很大,麦麸最好,蔗渣最差。同时,酶制剂因发酵基质不同会产生底物专一性,因此,在生产酶制剂时应注意根据所降解的对象选择发酵基质。     

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     

酶解麦麸制备阿魏酸和低聚糖的研究

本研究以黑曲霉(Aspergillus niger)作菌种,采用液体深层发酵法制备出含有阿魏酸酯酶(FAE)和阿拉伯木聚糖酶(AX)的混合酶制剂,探讨了它们协同作用麦麸制备阿魏酸和低聚糖的工艺条件。结果表明,FAE和AX协同作用的最适反应pH为4.4,最适反应温度为50℃。采用混合酶制剂作用于去淀粉麦麸(DSWB)发现,通过3次降解后,麦麸降解率达55.46%,大大高于文献报道的、采用单一酶作用的结果。扫描电子显微镜(SEM)观察发现,DSWB在酶处理前后的微观结构发生了显著变化。     

麦麸中生理活性物质研究与应用进展

阿拉伯木聚糖、植酸、阿魏酸是麦麸中重要的生理活性物质,具有抗肿瘤、抗氧化等功能,加上其很强的饱水性、稳定性,使它在食品添加剂和功能食品开发上具有广泛的应用前景。不同的分离提取方法对这些生理活性物质的得率、结构影响明显,并影响其功用,本文综述了麦麸阿拉伯木聚糖、植酸、阿魏酸的提取方法及其应用情况。     

Effect of Flour Water-Extractable Pentosans on Molecular Associations in Gluten During Mixing

Wheat gluten and mixtures of gluten and water-extractable pentosans (WEP) were mixed in a counter-rotating batch mixer at constant hydration level, in order to study the effects of feruloylated arabinoxylans (AX) on gluten mixing properties. The presence of WEP delayed the development time of gluten, suggesting a competition for water during the first stage of dough formation. Changes in macromolecular associations were followed using size-exclusion high-pressure liquid chromatography (SE-HPLC), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and solubility measurements. WEP did not affect the breakdown rate of proteins during mixing, when compared on specific energy mechanical basis. Mixing did not affect AX solubility and viscosity. Nevertheless, ferulic acid (FA) content decreased during mixing. This decrease involved mostly FA esters of compounds soluble in acetone/methanol/water (7/7/6, v/v/v) which are present in gluten. These soluble FA esters are likely to react with proteins during mixing. They would be involved, rather than feruloylated AX, in gluten breakdown during overmixing, since no covalent complex between AX and protein was detected in overmixed doughs.     

利用麦芽根提取液发酵生产酵母菌体蛋白

利用麦芽根提取液在500ml摇瓶和5L罐规模上探讨发酵生产酵母菌体蛋白的工艺可行性。实验取得了良好的效果,500ml摇瓶生产酵母的得率达到36%;5L罐规模发酵生产酵母,发酵结束后酵母干重达到2g/L,对糖的转化率达到40%。采用两种发酵规模,发酵结束后提取液中α-氨基氮的含量都下降了20%。     

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.     

Nitrogen starvation induces expression of Lg‐FLO1 and flocculation in bottom‐fermenting yeast

When exponentially growing cells of bottom‐fermenting yeast were starved for nitrogen or were grown on proline (a non‐preferred nitrogen source), flocculation was induced. This flocculation was not induced by starvation for either carbon or amino acids. Expression of Lg‐FLO1, which is required for flocculation of bottom‐fermenting yeast, was also found to be induced by starvation for nitrogen. This suggests that the flocculation of bottom‐fermenting yeast is under the control of a nitrogen catabolite repression (NCR)‐like mechanism. Copyright © 2012 John Wiley & Sons, Ltd.     

酶解麦麸制备低聚糖和阿魏酸的研究

本研究以黑曲霉(Aspergillus niger)作菌种,采用液体深层发酵法制备出含有阿魏酸酯酶(FAE)和阿拉伯木聚糖酶(AX)的混合酶制剂,探讨了它们协同作用麦麸制备阿魏酸和低聚糖的工艺条件.结果表明,FAE和AX协同作用的最适反应pH为4.4,最适反应温度为50℃.采用混合酶制剂作用于去淀粉麦麸(DSWB)发现,通过3次降解后,麦麸降解率达55.46%,大大高于文献报道的、采用单一酶作用的结果.扫描电子显微镜(SEM)观察发现,DSWB在酶处理前后的微观结构发生了显著变化.     

Effects of Xylanase and Peroxidase on Soluble and Insoluble Arabinoxylans in Wheat Bread Dough

ABSTRACT: Bread doughs supplemented with xylanase and xylanase plus peroxidase were fractionated into 4 insoluble and 3 soluble fractions. Chemical analysis and high-performance size-exclusion chromatography analysis of apparent molecular weight distribution indicated that xylanase acts on both cold-water-extractable arabinoxylans and on those that can be solubilized from cell wall fragments by hot water extraction. Peroxidase action increased the amount of insoluble small cell wall fragments, notably the amount of protein and arabinoxylan. Arabinoxylans were retained in the small cell wall fragments because cross-linking of arabinoxylans through ferulic acid residues to other arabinoxylans rendered them insoluble. Peroxidase did not affect the composition of gluten, nor was evidence obtained for peroxidase-catalyzed cross-linking of arabinoxylans to protein in the gluten and other fractions.     

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。     

絮凝法处理苯乳酸发酵液的研究

对絮凝法处理苯乳酸发酵液进行了研究。以絮凝率和苯乳酸回收率为指标,研究了7种絮凝剂对苯乳酸发酵液絮凝效果的影响,并通过单因素实验和正交实验对絮凝条件进行优化。Al2(SO4)3作为适宜的絮凝剂,其最佳絮凝条件为:pH为3.0,温度为30℃,絮凝剂用量为8g/L,以100r/min振荡35min。在此条件下发酵液脱色率达到60.88%,苯乳酸回收率达到88.13%。