麦芽中酵母超前絮凝多糖及其对啤酒发酵的影响研究
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摘要
酵母超前絮凝(Premature Yeast Flocculation,PYF)是指在麦汁营养成分消耗完之前,酵母出现过早絮凝的一种现象。它严重影响着酵母性能的发挥,影响啤酒发酵的正常进程。研究表明麦芽中存在一种生物大分子,能够促进酵母超前絮凝,并且对高温等外界环境不敏感,能够一直存在于发酵液中。本文利用生产中发现的一批PYF麦芽研究这种生物大分子。主要研究结果如下:
1.在前人研究的基础上,从麦芽中PYF因子的提取方法、酵母悬浮液的浓度和用量、缓冲体系的pH值、缓冲液的用量几个角度对测定方法进行优化,建立了一种快速准确的测定麦芽PYF活力的方法。该方法与传统方法本斯法、光密度法相比,平行性和稳定性最好,RSD＜3%。并且能够很好的区分不同PYF因子的絮凝性。
2.从PYF麦芽中利用EBC糖化、乙醇沉淀、木瓜蛋白酶和Sevag法、超滤等法得到PYF粗多糖;利用DEAE-Sephadex A-25离子交换树脂层析分离粗多糖,得到三个组分PYFⅠ、PYFⅡ、PYFⅢ;选取PYF活力最大的PYFⅠ(F值为53.54%)用Sephadex G-100葡聚糖凝胶柱层析处理后得到了一种多糖,能够显著的促进酵母絮凝(F值为68.90%),命名为PYFⅠ-Ⅰ。认为PYFⅠ-Ⅰ就是麦芽中一种引发酵母超前絮凝的多糖物质。
3.对提取得到的超前絮凝因子PYFⅠ-Ⅰ的理化性质及结构进行测定,结果显示其糖含量为58.3%、蛋白含量为3.8%;利用Sephadex G-100凝胶柱层析和高效液相色谱测其纯度,发现洗脱图谱均为单一窄峰,说明PYFⅠ-Ⅰ为均一组分;利用高效凝胶过滤色谱法(HPSEC)法测得PYFⅠ-Ⅰ的分子量为13598Dr;薄层层析和糖醇乙酸酯衍生物气相色谱法(GC)测得PYFⅠ-Ⅰ的单糖组成为阿拉伯糖、木糖、葡萄糖;利用红外光谱法分析其结构,发现PYFⅠ-Ⅰ存在O—H的伸缩振动、CH_3中C-H键的伸缩振动和变角振动、吡喃环的醚键C-O-C中C-O的伸缩振动等多糖特征吸收峰,在1241cm~(-1)出现了硫酸基(-O-SO_3-键)中S=O的伸缩振动引起的吸收峰,说明被测物质是一种硫酸多糖,在1639cm~(-1)出现了是NH_2的面内弯曲振动吸收峰。紫外光谱分析表明PYFⅠ-Ⅰ不含有核酸组分,有蛋白组分存在。
4.将不同量的PYFⅠ-Ⅰ添加到发酵液中,考查其对啤酒发酵度、酒精含量、酵母数、风味物质和有机酸等相关指标的影响,以研究麦芽PYF多糖对啤酒质量的影响。添加了PYFⅠ-Ⅰ的试验组发酵度、酒精含量和发酵液中悬浮的酵母数量都要低于空白组,这与文献中的报道是一致的,势必造成发酵不完全和原料的浪费;添加PYFⅠ-Ⅰ可以导致发酵液中双乙酰含量升高,高级醇、酯类、DMS和有机酸含量降低,发酵液中乙醛含量升高,这些会破坏啤酒发酵中风味物质的固有比例与含量范围,使得啤酒的口味变得不纯正,影响啤酒的质量。
5.从清洗麦芽、麦芽加和和制麦工艺三方面研究开发降低PYF麦芽中超前絮凝多糖含量的方法:麦芽经过水洗→浸泡→烘干后,PYF活力有所降低,从60%左右降低到45%左右,处理后的麦芽水分含量、色度、库值、a-氨基氮、糖化力等指标仍然处于正常范围之内;将PYF活力高的麦芽(F值为60%左右)和PYF活力低的麦芽(F值为50%左右)按一定比例混合,测定混合麦芽的F值,发现麦芽的PYF活力有很好的加和性,可以通过高低搭配来调节PYF活力;通过调整制麦工艺,缩短浸麦时间、降低浸麦度、加强浸麦通风、提高发芽温度、延长发芽时间、降低排潮排风温度、延长焙焦时间也可以使得麦芽的PYF活力降低,从72.67%降低到67.77%。
Premature Yeast Flocculation(PYF) is a phenomenon that yeast flocculated before out of nutrition in the fermentation liquid.The yeast can not demonstrate its fermentation performance fully and the fermentation process can not go smoothly in this case.It is reported that a biomacromolecule in malt which is insensitive to external environment can induce PYF.The same batch of PYF malt were used as the starting material,the research results are as follows:
1.A rapid and sensitive method to measure Premature Yeast Flocculation Activity in Malt was established on the basis of previous study by adjusting several index, such as the method of extracting the PYF polysaccharide of malt,the concentration and amount of yeast suspension,the pH and amount of buffer solution.The parallelism and stability of the new method is better than Bens and Optical Density Assay,RSD＜3%.The new method can distinguish the PYF activity of malt apparently.
2.A polysaccharide named PYFⅠ-Ⅰis purified after sedimented by ethanol, remove of protein,ultrafiltration,Chromatography by DEAE-Sephadex A-25 and Sephadex G-100.The polysaccharide accelerate the flocculation of yeast apparently (F=68.90%).PYFⅠ-Ⅰis identified as the polysaccharide which can induce PYF.
3.The physicochemical property and structure of PYFⅠ-Ⅰis analysed:The content of polysaccharides and proteins was 58.3%and 3.8%;the results of Sephadex G-100 chromatography and HPLC shows that PYFⅠ-Ⅰis a single component;the molecular weight of PYFⅠ-Ⅰis 13598Dr by the method of HPSEC;the monosaccharide composition of PYFⅠ-Ⅰis analysed by TLC and GC,results show that there are Arabinose,Glucose,Xylose in PYFⅠ-Ⅰ;the results of Infrared Spectroscopy show that there are the stretching vibration of O-H,the C-H stretching vibration and angle vibration of CH_3,the C-O stretching vibration of Pyran ring,the stretching vibration of S=O and the bending vibration of NH_2.The polysaccharide is identified as a protein containing polysaccharide without nucleic acid by UV.
4.The effects of PYF polysaccharide in malt on beer fermentation were studied. The fermentation degree,alcohol content,the content of flavor compounds and organic acid in beer fermentation liquid were determined.Results showed that the quantity of suspended yeast cells,the fermentation degree and alcohol content decreased signifiCantly,which cause incomplete fermentation and raw material consumption;the content of diacetyl and acetaldehyde increased,the content of higher alcohols,esters,DMS and organic acids decreased,which make the change of content and ratio of flavor compounds,cause abnormal flavor of beer.
5.Three methods to decrease the content of PYF potysaccharide were studied.
Wash the malt:The PYF activity of malt decreased from 60%to 45%after wash the malt by water and then dry it.But the water content,colority,a-amino nitrogen, saccharification power show that the washed malt can be used to beer fermentation.
Put the malt with high PYF activity and the malt With low PYF activity together, then the F value of mixed malt were determined,the result shows that the mixed malt has a proper PYF activity for fermentation.
Adjust Malting Technology:shorten the time of malt steeping,decrease the steeping degree,enhance steeping ventilation,increase the germination temperature and time,lower the temperature of ventilation when excluding wet,extend the kilning time.The F of the malt produced by new technology is 72.67%(The F value of normal malt is 67.77%).The results show that three methods can decrease the PYF activity of malt apparently.
1.在前人研究的基础上,从麦芽中PYF因子的提取方法、酵母悬浮液的浓度和用量、缓冲体系的pH值、缓冲液的用量几个角度对测定方法进行优化,建立了一种快速准确的测定麦芽PYF活力的方法。该方法与传统方法本斯法、光密度法相比,平行性和稳定性最好,RSD＜3%。并且能够很好的区分不同PYF因子的絮凝性。
2.从PYF麦芽中利用EBC糖化、乙醇沉淀、木瓜蛋白酶和Sevag法、超滤等法得到PYF粗多糖;利用DEAE-Sephadex A-25离子交换树脂层析分离粗多糖,得到三个组分PYFⅠ、PYFⅡ、PYFⅢ;选取PYF活力最大的PYFⅠ(F值为53.54%)用Sephadex G-100葡聚糖凝胶柱层析处理后得到了一种多糖,能够显著的促进酵母絮凝(F值为68.90%),命名为PYFⅠ-Ⅰ。认为PYFⅠ-Ⅰ就是麦芽中一种引发酵母超前絮凝的多糖物质。
3.对提取得到的超前絮凝因子PYFⅠ-Ⅰ的理化性质及结构进行测定,结果显示其糖含量为58.3%、蛋白含量为3.8%;利用Sephadex G-100凝胶柱层析和高效液相色谱测其纯度,发现洗脱图谱均为单一窄峰,说明PYFⅠ-Ⅰ为均一组分;利用高效凝胶过滤色谱法(HPSEC)法测得PYFⅠ-Ⅰ的分子量为13598Dr;薄层层析和糖醇乙酸酯衍生物气相色谱法(GC)测得PYFⅠ-Ⅰ的单糖组成为阿拉伯糖、木糖、葡萄糖;利用红外光谱法分析其结构,发现PYFⅠ-Ⅰ存在O—H的伸缩振动、CH_3中C-H键的伸缩振动和变角振动、吡喃环的醚键C-O-C中C-O的伸缩振动等多糖特征吸收峰,在1241cm~(-1)出现了硫酸基(-O-SO_3-键)中S=O的伸缩振动引起的吸收峰,说明被测物质是一种硫酸多糖,在1639cm~(-1)出现了是NH_2的面内弯曲振动吸收峰。紫外光谱分析表明PYFⅠ-Ⅰ不含有核酸组分,有蛋白组分存在。
4.将不同量的PYFⅠ-Ⅰ添加到发酵液中,考查其对啤酒发酵度、酒精含量、酵母数、风味物质和有机酸等相关指标的影响,以研究麦芽PYF多糖对啤酒质量的影响。添加了PYFⅠ-Ⅰ的试验组发酵度、酒精含量和发酵液中悬浮的酵母数量都要低于空白组,这与文献中的报道是一致的,势必造成发酵不完全和原料的浪费;添加PYFⅠ-Ⅰ可以导致发酵液中双乙酰含量升高,高级醇、酯类、DMS和有机酸含量降低,发酵液中乙醛含量升高,这些会破坏啤酒发酵中风味物质的固有比例与含量范围,使得啤酒的口味变得不纯正,影响啤酒的质量。
5.从清洗麦芽、麦芽加和和制麦工艺三方面研究开发降低PYF麦芽中超前絮凝多糖含量的方法:麦芽经过水洗→浸泡→烘干后,PYF活力有所降低,从60%左右降低到45%左右,处理后的麦芽水分含量、色度、库值、a-氨基氮、糖化力等指标仍然处于正常范围之内;将PYF活力高的麦芽(F值为60%左右)和PYF活力低的麦芽(F值为50%左右)按一定比例混合,测定混合麦芽的F值,发现麦芽的PYF活力有很好的加和性,可以通过高低搭配来调节PYF活力;通过调整制麦工艺,缩短浸麦时间、降低浸麦度、加强浸麦通风、提高发芽温度、延长发芽时间、降低排潮排风温度、延长焙焦时间也可以使得麦芽的PYF活力降低,从72.67%降低到67.77%。
Premature Yeast Flocculation(PYF) is a phenomenon that yeast flocculated before out of nutrition in the fermentation liquid.The yeast can not demonstrate its fermentation performance fully and the fermentation process can not go smoothly in this case.It is reported that a biomacromolecule in malt which is insensitive to external environment can induce PYF.The same batch of PYF malt were used as the starting material,the research results are as follows:
1.A rapid and sensitive method to measure Premature Yeast Flocculation Activity in Malt was established on the basis of previous study by adjusting several index, such as the method of extracting the PYF polysaccharide of malt,the concentration and amount of yeast suspension,the pH and amount of buffer solution.The parallelism and stability of the new method is better than Bens and Optical Density Assay,RSD＜3%.The new method can distinguish the PYF activity of malt apparently.
2.A polysaccharide named PYFⅠ-Ⅰis purified after sedimented by ethanol, remove of protein,ultrafiltration,Chromatography by DEAE-Sephadex A-25 and Sephadex G-100.The polysaccharide accelerate the flocculation of yeast apparently (F=68.90%).PYFⅠ-Ⅰis identified as the polysaccharide which can induce PYF.
3.The physicochemical property and structure of PYFⅠ-Ⅰis analysed:The content of polysaccharides and proteins was 58.3%and 3.8%;the results of Sephadex G-100 chromatography and HPLC shows that PYFⅠ-Ⅰis a single component;the molecular weight of PYFⅠ-Ⅰis 13598Dr by the method of HPSEC;the monosaccharide composition of PYFⅠ-Ⅰis analysed by TLC and GC,results show that there are Arabinose,Glucose,Xylose in PYFⅠ-Ⅰ;the results of Infrared Spectroscopy show that there are the stretching vibration of O-H,the C-H stretching vibration and angle vibration of CH_3,the C-O stretching vibration of Pyran ring,the stretching vibration of S=O and the bending vibration of NH_2.The polysaccharide is identified as a protein containing polysaccharide without nucleic acid by UV.
4.The effects of PYF polysaccharide in malt on beer fermentation were studied. The fermentation degree,alcohol content,the content of flavor compounds and organic acid in beer fermentation liquid were determined.Results showed that the quantity of suspended yeast cells,the fermentation degree and alcohol content decreased signifiCantly,which cause incomplete fermentation and raw material consumption;the content of diacetyl and acetaldehyde increased,the content of higher alcohols,esters,DMS and organic acids decreased,which make the change of content and ratio of flavor compounds,cause abnormal flavor of beer.
5.Three methods to decrease the content of PYF potysaccharide were studied.
Wash the malt:The PYF activity of malt decreased from 60%to 45%after wash the malt by water and then dry it.But the water content,colority,a-amino nitrogen, saccharification power show that the washed malt can be used to beer fermentation.
Put the malt with high PYF activity and the malt With low PYF activity together, then the F value of mixed malt were determined,the result shows that the mixed malt has a proper PYF activity for fermentation.
Adjust Malting Technology:shorten the time of malt steeping,decrease the steeping degree,enhance steeping ventilation,increase the germination temperature and time,lower the temperature of ventilation when excluding wet,extend the kilning time.The F of the malt produced by new technology is 72.67%(The F value of normal malt is 67.77%).The results show that three methods can decrease the PYF activity of malt apparently.
引文
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