麦芽中酵母超前絮凝（PYF）活力新评价指标的建立与应用研究
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摘要
麦芽PYF(酵母超前絮凝)因子是麦芽中含有的某种可以影响酵母絮凝性能的多糖物质,会在啤酒后熟期导致酵母发生提前絮凝(Premature Yeast Flocculation,PYF)现象,从而引发发酵不完全、成熟度差等严重质量后果。
目前国内外各大啤酒集团为了保证同一啤酒品牌多地生产的口味一致性,在麦芽采购方面均实行集中管理、统一采购,年进口采购量达百万吨。如果一旦采购到有PYF问题的麦芽将直接影响到数十家啤酒厂的酵母生理状态及发酵过程的酿造性能,并最终对啤酒成品的风味一致性产生极大的负面影响,随之造成的经济损失及声誉损失不可估量。
意识到这一问题的严重性后,啤酒制造企业也逐渐尝试把麦芽PYF指标纳进麦芽采购评价里,并根据各自酵母的絮凝敏感特性及生产质量需求来设定麦芽PYF指标采购标准。然而目前通行的麦芽PYF活力评价方法都必须依赖酵母的絮凝表现来间接评价,其反映的是麦芽PYF活力、酵母絮凝性能以及活力状态对酵母絮凝表现的一个综合指标,缺乏客观性而不被麦芽制造商所认可；同时由于各啤酒集团使用的酵母菌种絮凝性差异非常大,依赖于各自菌种特性建立的麦芽PYF活力检测方法各成体系,这也给把麦芽PYF指标纳入采购标准带来了一定的障碍。
因此本研究从对麦芽PYF多糖的提取纯化着手,首次利用竞争ELISA检测技术建立一种快速、灵敏、适应于现场的麦芽PYF活力分析方法,该检测方法及操作系统稳定性较好,可信度较高,有别于目前使用的PYF活力检测方法需要利用酵母的特点,可转化应用为一种客观、适应于第三方评估的麦芽大宗采购质量评价指标；并通过对不同批次麦芽PYF活力的实际应用检测,对麦芽PYF的诱发机理与调控机制展开初步探索,形成一条从基础到应用的纵向研究体系。
主要研究成果如下：
(1)首先根据测定应用酵母的絮凝特性分别从酵母悬浮液的初始浓度、缓冲体系的pH值、钙离子浓度几个方面对麦芽PYF微量光密度测定方法进行优化改良,建立了一种快速、灵敏的适用于判定微量样品麦芽PYF活力的方法,用以判定麦芽PYF多糖各提纯步骤的纯化效率。
(2)依据麦芽PYF多糖是以极其松散的方式结合在酵母细胞壁之间、极易被洗脱的性质特点,利用PYF强阳性麦芽进行发酵,然后以酵母泥为吸附基团,通过NaCl溶液对回收酵母的洗脱建立了一种快速、高效的麦芽PYF粗多糖提取方式。
利用切向流超滤技术对麦芽PYF多糖粗提液进行大幅度的浓缩、除盐及小分子糖类的祛除,然后利用DEAE-Sephadex A-25离子交换树脂及Sephadex G-100葡聚糖凝胶柱层析等处理进行纯化。
通过紫外吸收光谱扫描、SDS-PAGE试验分析此种多糖含有蛋白结构,分子量约为40kDa。但通过脱蛋白试验表明蛋白成分不是引发PYF现象的必要组成部分。最后利用高效液相色谱技术鉴定证明得到了一种麦芽PYF纯品,加入到酵母细胞中后能够显著的促进酵母的絮凝。
(3)分别以牛血清白蛋白(BSA)和匙孔血蓝蛋白(KLH)为载体,采用高碘酸钠法合成了麦芽PYF多糖的完全抗原,并利用紫外可见光谱扫描方法证明完全抗原合成成功。
(4)利用PYF-BSA制备包被抗原,PYF-KLH制备免疫抗原,通过免疫Balb/c小鼠和新西兰大白兔制备抗体,利用间接ELISA检测两种抗血清的效价分别可达1：25600和1：204800。并以兔多抗为基础通过ELISA最佳反应条件的建立、麦芽检测样品制备方法的优化,初步建立了一套适用于实际麦芽样品PYF多糖竞争ELISA分析方法的操作程序,其中竞争ELISA反应体系的主要工作参数为：包被抗原浓度为50μg/mL,兔抗血清稀释度为10000倍,酶标二抗稀释度为10000倍。
(5)从生产现场选取不同麦芽样品分别利用竞争ELISA分析方法和原来使用的快速发酵法进行了重复性、重现性及灵敏度试验的对比验证,证明麦芽PYF因子竞争ELISA检测方法其重复性及重现性远远大于快速发酵法,其变异系数分别小于5%和10%,说明该检测方法及操作系统稳定性较好,可信度较高。
(6)根据麦芽PYF值对本论文所选用酵母絮凝性能的影响程度,建立以竞争ELISA法为依据的麦芽采购建议标准如下：
麦芽样品OD450小于0.513的可判定为PYF强阳性麦芽,说明此批麦芽对酵母絮凝性能有较大影响,不予接受；
麦芽样品OD450处于0.513-0.579之间的可判定为PYF弱阳性麦芽,说明此批麦芽对酵母絮凝性能有一定影响,可商定折价采购；
麦芽样品OD450大于0.579的可判定为PYF阴性麦芽。
(7)综合利用竞争ELISA法和快速发酵法初步探讨了麦芽搭配、洗麦、剥皮等降低麦芽PYF活力工艺措施的可行性。其中麦芽洗涤、原料除尘及剥皮可有效降低麦芽PYF活力,并充分证明了麦芽PYF多糖极易溶于水,且在水中保留了导致酵母产生PYF潜力的研究观点。
同时发现麦芽PYF活力不具备简单的加和性,因此进行麦芽搭配时,不能单纯依靠计算理论值来判定搭配后的麦芽PYF活力,必须对配方麦芽重新进行PYF活力检测；不同PYF强度的麦芽与PYF阴性麦芽搭配时,其对配方麦芽PYF活力的降低效果差异比较明显：如果使用PYF强阳性麦芽进行搭配,即使在全麦芽比例中只占10%,也会强烈诱发PYF。因此在实际生产时如果麦芽PYF活力比较严重,就不再适宜进行搭配使用。
PYF is a phenomenon whereby flocculation competent yeast settles out of the fermentation medium abnormally early during primary fermentation, resulting in a poorly attenuated wort and a final product with undesirable flavor characteristics.
At present the beer groups implement centralized management and unified purchase in malt procurement, in order to guarantee taste consistency of the production. If once purchasing the malt with PYF problems, the quality of malt will directly affect yeast physiological performance and finally affect negative impacts on the beer flavor consistency of dozens of brewery, which will cause inestimable economic losses and reputation losses.
Aware of the seriousness of this problem, beer manufacturing enterprise gradually tried to incorporate PYF into malt procurement evaluation, and set purchasing standards for PYF index according to the yeast flocculation sensitive properties and quality demands. However, current PYF evaluation method must rely on yeast flocculation performance and is a comprehensive index which reflects the malt PYF activity, yeast flocculation property and yeast vitality on yeast flocculation performance. Malt manufacturers refuse agreeing with this provision because the assay method lacks of objectivity. At the same time each beer group uses yeast strains with different flocculation properties, so PYF assay methods are different, which also brings some obstacles to incorporate the malt PYF index into purchasing standards.
In this study, a rapid and sensitive PYF assay method using competitive ELISA detection technology was established for the first time. Compared to the current PYF assay methods, the new one has the advantages of stable, high reliable, and no need to use yeast, which can be applied to the third party assessment to evaluate malt quality. The causative and regulation mechanism of PYF was also researched according to assaying PYF activity of different batches of malt.
The main research results are as follows:
(1)A rapid and sensitive PYF activity assay method to determine purification efficiency was established at first on the basis of previous study by adjusting several indexes, such as the concentration of yeast suspension, the pH and the concentration of Ca2+of buffer solution.
(2)A barley malt factor inducing premature yeast flocculation (PYF) was successfully concentrated using lager yeast as an affinity particle, followed by tangential flow filtration technology, anion-exchange chromatography and gel filtration chromatography. The purified PYF polysaccharide is proved as a single component by HPLC which could significantly promote yeast flocculation, and consists of protein component by UV spectrum assay and SDS-PAGE, with a relative molecular mass of40kDa. It was also found that protein structure is not the necessary trigger component of PYF phenomenon through the protein dispel test.
(3)PYF-carrier protein conjugates (PYF-BSA/PYF-KLH) were artificially synthesized by the method using NaI04, and the conjugates of PYF-KLH was determined by UV scanning technology.
(4)Polyclonal antibodies were raised through immunizing Balb/c mice and New Zealand white rabbits with PYF-KLH, and the titers of antiserum had reached1:25600and1:204800separately. The optimal working concentration was optimized by ELISA as follows:the dilution ratio of coated antigen was50μg/mL, the dilution ratio of rabbit antiserum and IgG was both10000.
(5)A series of PYF samples from brewery have been determined by the competitive ELISA, and the results showed more sensibility, reliability (C.V.<5%) and reproducibility (C.V.<10%) than the former method.
(6)According to the influence of the malt PYF activity on the yeast flocculation performance, the malt purchase standard based on this competitive ELISA method was established as follows:
OD450of the malt sample<0.513:refuse;
OD450of the malt sample>0.579:accept;
OD450of the malt sample between0.513and0.579:discount purchase.
(7)Methods to decrease the content of PYF activity including malt collocation^malt wash and peel off were studied and proved to be effective. It was also found that the malt polysaccharide was water-soluble and continued to have the PYF patent in the malt-wash water.
Because the PYF activity of malt didn't have additive property, so after mixture it must be assayed again. It was also showed that the PYF activity of mixture malts display a marked difference when the PYF(-)-malt was mixed with different PYF(+)-malts. If mixed with severe PYF(+) malt, the PYF activity will be very high even if accounts for only of10%.So it was strongly recommended that severe PYF(+)malt would not be suitable used for malt collocation.
目前国内外各大啤酒集团为了保证同一啤酒品牌多地生产的口味一致性,在麦芽采购方面均实行集中管理、统一采购,年进口采购量达百万吨。如果一旦采购到有PYF问题的麦芽将直接影响到数十家啤酒厂的酵母生理状态及发酵过程的酿造性能,并最终对啤酒成品的风味一致性产生极大的负面影响,随之造成的经济损失及声誉损失不可估量。
意识到这一问题的严重性后,啤酒制造企业也逐渐尝试把麦芽PYF指标纳进麦芽采购评价里,并根据各自酵母的絮凝敏感特性及生产质量需求来设定麦芽PYF指标采购标准。然而目前通行的麦芽PYF活力评价方法都必须依赖酵母的絮凝表现来间接评价,其反映的是麦芽PYF活力、酵母絮凝性能以及活力状态对酵母絮凝表现的一个综合指标,缺乏客观性而不被麦芽制造商所认可；同时由于各啤酒集团使用的酵母菌种絮凝性差异非常大,依赖于各自菌种特性建立的麦芽PYF活力检测方法各成体系,这也给把麦芽PYF指标纳入采购标准带来了一定的障碍。
因此本研究从对麦芽PYF多糖的提取纯化着手,首次利用竞争ELISA检测技术建立一种快速、灵敏、适应于现场的麦芽PYF活力分析方法,该检测方法及操作系统稳定性较好,可信度较高,有别于目前使用的PYF活力检测方法需要利用酵母的特点,可转化应用为一种客观、适应于第三方评估的麦芽大宗采购质量评价指标；并通过对不同批次麦芽PYF活力的实际应用检测,对麦芽PYF的诱发机理与调控机制展开初步探索,形成一条从基础到应用的纵向研究体系。
主要研究成果如下：
(1)首先根据测定应用酵母的絮凝特性分别从酵母悬浮液的初始浓度、缓冲体系的pH值、钙离子浓度几个方面对麦芽PYF微量光密度测定方法进行优化改良,建立了一种快速、灵敏的适用于判定微量样品麦芽PYF活力的方法,用以判定麦芽PYF多糖各提纯步骤的纯化效率。
(2)依据麦芽PYF多糖是以极其松散的方式结合在酵母细胞壁之间、极易被洗脱的性质特点,利用PYF强阳性麦芽进行发酵,然后以酵母泥为吸附基团,通过NaCl溶液对回收酵母的洗脱建立了一种快速、高效的麦芽PYF粗多糖提取方式。
利用切向流超滤技术对麦芽PYF多糖粗提液进行大幅度的浓缩、除盐及小分子糖类的祛除,然后利用DEAE-Sephadex A-25离子交换树脂及Sephadex G-100葡聚糖凝胶柱层析等处理进行纯化。
通过紫外吸收光谱扫描、SDS-PAGE试验分析此种多糖含有蛋白结构,分子量约为40kDa。但通过脱蛋白试验表明蛋白成分不是引发PYF现象的必要组成部分。最后利用高效液相色谱技术鉴定证明得到了一种麦芽PYF纯品,加入到酵母细胞中后能够显著的促进酵母的絮凝。
(3)分别以牛血清白蛋白(BSA)和匙孔血蓝蛋白(KLH)为载体,采用高碘酸钠法合成了麦芽PYF多糖的完全抗原,并利用紫外可见光谱扫描方法证明完全抗原合成成功。
(4)利用PYF-BSA制备包被抗原,PYF-KLH制备免疫抗原,通过免疫Balb/c小鼠和新西兰大白兔制备抗体,利用间接ELISA检测两种抗血清的效价分别可达1：25600和1：204800。并以兔多抗为基础通过ELISA最佳反应条件的建立、麦芽检测样品制备方法的优化,初步建立了一套适用于实际麦芽样品PYF多糖竞争ELISA分析方法的操作程序,其中竞争ELISA反应体系的主要工作参数为：包被抗原浓度为50μg/mL,兔抗血清稀释度为10000倍,酶标二抗稀释度为10000倍。
(5)从生产现场选取不同麦芽样品分别利用竞争ELISA分析方法和原来使用的快速发酵法进行了重复性、重现性及灵敏度试验的对比验证,证明麦芽PYF因子竞争ELISA检测方法其重复性及重现性远远大于快速发酵法,其变异系数分别小于5%和10%,说明该检测方法及操作系统稳定性较好,可信度较高。
(6)根据麦芽PYF值对本论文所选用酵母絮凝性能的影响程度,建立以竞争ELISA法为依据的麦芽采购建议标准如下：
麦芽样品OD450小于0.513的可判定为PYF强阳性麦芽,说明此批麦芽对酵母絮凝性能有较大影响,不予接受；
麦芽样品OD450处于0.513-0.579之间的可判定为PYF弱阳性麦芽,说明此批麦芽对酵母絮凝性能有一定影响,可商定折价采购；
麦芽样品OD450大于0.579的可判定为PYF阴性麦芽。
(7)综合利用竞争ELISA法和快速发酵法初步探讨了麦芽搭配、洗麦、剥皮等降低麦芽PYF活力工艺措施的可行性。其中麦芽洗涤、原料除尘及剥皮可有效降低麦芽PYF活力,并充分证明了麦芽PYF多糖极易溶于水,且在水中保留了导致酵母产生PYF潜力的研究观点。
同时发现麦芽PYF活力不具备简单的加和性,因此进行麦芽搭配时,不能单纯依靠计算理论值来判定搭配后的麦芽PYF活力,必须对配方麦芽重新进行PYF活力检测；不同PYF强度的麦芽与PYF阴性麦芽搭配时,其对配方麦芽PYF活力的降低效果差异比较明显：如果使用PYF强阳性麦芽进行搭配,即使在全麦芽比例中只占10%,也会强烈诱发PYF。因此在实际生产时如果麦芽PYF活力比较严重,就不再适宜进行搭配使用。
PYF is a phenomenon whereby flocculation competent yeast settles out of the fermentation medium abnormally early during primary fermentation, resulting in a poorly attenuated wort and a final product with undesirable flavor characteristics.
At present the beer groups implement centralized management and unified purchase in malt procurement, in order to guarantee taste consistency of the production. If once purchasing the malt with PYF problems, the quality of malt will directly affect yeast physiological performance and finally affect negative impacts on the beer flavor consistency of dozens of brewery, which will cause inestimable economic losses and reputation losses.
Aware of the seriousness of this problem, beer manufacturing enterprise gradually tried to incorporate PYF into malt procurement evaluation, and set purchasing standards for PYF index according to the yeast flocculation sensitive properties and quality demands. However, current PYF evaluation method must rely on yeast flocculation performance and is a comprehensive index which reflects the malt PYF activity, yeast flocculation property and yeast vitality on yeast flocculation performance. Malt manufacturers refuse agreeing with this provision because the assay method lacks of objectivity. At the same time each beer group uses yeast strains with different flocculation properties, so PYF assay methods are different, which also brings some obstacles to incorporate the malt PYF index into purchasing standards.
In this study, a rapid and sensitive PYF assay method using competitive ELISA detection technology was established for the first time. Compared to the current PYF assay methods, the new one has the advantages of stable, high reliable, and no need to use yeast, which can be applied to the third party assessment to evaluate malt quality. The causative and regulation mechanism of PYF was also researched according to assaying PYF activity of different batches of malt.
The main research results are as follows:
(1)A rapid and sensitive PYF activity assay method to determine purification efficiency was established at first on the basis of previous study by adjusting several indexes, such as the concentration of yeast suspension, the pH and the concentration of Ca2+of buffer solution.
(2)A barley malt factor inducing premature yeast flocculation (PYF) was successfully concentrated using lager yeast as an affinity particle, followed by tangential flow filtration technology, anion-exchange chromatography and gel filtration chromatography. The purified PYF polysaccharide is proved as a single component by HPLC which could significantly promote yeast flocculation, and consists of protein component by UV spectrum assay and SDS-PAGE, with a relative molecular mass of40kDa. It was also found that protein structure is not the necessary trigger component of PYF phenomenon through the protein dispel test.
(3)PYF-carrier protein conjugates (PYF-BSA/PYF-KLH) were artificially synthesized by the method using NaI04, and the conjugates of PYF-KLH was determined by UV scanning technology.
(4)Polyclonal antibodies were raised through immunizing Balb/c mice and New Zealand white rabbits with PYF-KLH, and the titers of antiserum had reached1:25600and1:204800separately. The optimal working concentration was optimized by ELISA as follows:the dilution ratio of coated antigen was50μg/mL, the dilution ratio of rabbit antiserum and IgG was both10000.
(5)A series of PYF samples from brewery have been determined by the competitive ELISA, and the results showed more sensibility, reliability (C.V.<5%) and reproducibility (C.V.<10%) than the former method.
(6)According to the influence of the malt PYF activity on the yeast flocculation performance, the malt purchase standard based on this competitive ELISA method was established as follows:
OD450of the malt sample<0.513:refuse;
OD450of the malt sample>0.579:accept;
OD450of the malt sample between0.513and0.579:discount purchase.
(7)Methods to decrease the content of PYF activity including malt collocation^malt wash and peel off were studied and proved to be effective. It was also found that the malt polysaccharide was water-soluble and continued to have the PYF patent in the malt-wash water.
Because the PYF activity of malt didn't have additive property, so after mixture it must be assayed again. It was also showed that the PYF activity of mixture malts display a marked difference when the PYF(-)-malt was mixed with different PYF(+)-malts. If mixed with severe PYF(+) malt, the PYF activity will be very high even if accounts for only of10%.So it was strongly recommended that severe PYF(+)malt would not be suitable used for malt collocation.
引文
[1]管敦仪.啤酒工业手册上册.北京：轻工业出版社,1985：248-251
[2]杜绿君.盘点中国啤酒工业.《中国食品与市场》2004,4：15-16
[3]中国酿酒工业协会啤酒分会.《涅槃——2009年中国啤酒产业发展分析及发展趋势》.《啤酒科技》2010,6：1-9
[4]Wolfgang Kunze.啤酒工艺实用技术.中国轻工业出版社,1998：118-123
[5]Wolfgang Kunze.啤酒工艺实用技术.中国轻工业出版社,1998：65
[6]Murray C R, Stewart G G. Experience with high gravity brewing [J]. Birrae Malto, 1991(44):52-64
[7]Wolfgang Kunze.啤酒工艺实用技术.中国轻工业出版社,1998：75
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