植物蛋白源活性肽调控酿酒酵母增殖和代谢的机理研究

Improving fermentation efficiency and beer quality during high gravity brewing is one of the important issues facing the brewer today. Recently studies increasingly indicate that nutrition supplements, particularly nitrogen, is one of efficient and simple measures to improve the fermentation performances of brewer’s yeast. Therefore, regulation of the growth and metabolism of brewer’s yeast is one of key means for improving fermentation efficiency and beer quality during high gravity brewing. The project aims at some scientific problems need to be solved urgently, such as efficient preparation of bioactive peptides, the interrelationships between chemical structure and bioactivity of peptides, and the strategy of directed regulation for brewer’s yeast and the quality of beer. Firstly, bioactive peptides with significant growth- and fermentation-promoting activity for brewer’s yeast are prepared from plant proteins by limited enzymatic hydrolysis and ultrafiltration. Secondly, series of bioactive peptides are isolated and purified from the resulting protein hydrolysates by gel filtration chromatography, and their chemical structures are identified by MALDI-TOF-MS and NMR. Moreover, the interrelationships between the chemical structure and bioactivity of target peptides will also be clarified. Lastly, the mechanism of regulating the growth and metabolism of brewer’s yeast and the feasibility of improving the quality of beer by bioactive peptides supplement during high gravity brewing will be investigated by CLSM and IRMS through fluorescent staining and isotope labeling of target peptides. Results from this project could be used to instruct beer production of brewery with improved fermentation efficiency and beer quality.

在啤酒高浓酿造中，如何提高发酵效率和啤酒质量是酿造界亟待解决的重要课题。近来的研究日益表明，营养补充，尤其是氮源补充，是一种有效且简便的提高酵母发酵性能的方法，因此，利用氮源调控酵母的增殖和代谢则是提高发酵效率和啤酒质量的关键。本项目将针对促酵母增殖活性肽的高效富集、活性肽的结构鉴定与构效关系及其对酵母代谢和啤酒质量的定向调控策略等亟需解决的科学问题，运用限制性可控酶解技术结合分级膜分离高效富集目标活性肽，然后应用凝胶过滤色谱结合基质辅助激光解析电离飞行时间质谱和核磁共振仪对目标活性肽进行分离纯化和结构鉴定，阐明其促酵母增殖活性与结构的关系，最后通过活性肽的荧光染色和同位素标记，应用激光共聚焦显微镜和稳定同位素比例质谱仪阐明活性肽在酵母胞内外的转运机制和代谢途径，明晰其定向调控酵母增殖和代谢的机理和消除高浓酿造啤酒质量缺陷的有效性，为高浓酿造中提高麦汁发酵效率和啤酒质量提供理论和方法指导。

在啤酒高浓酿造中，如何提高发酵效率和啤酒质量是酿造界亟待解决的重要课题，而氮源补充则被认为是一种有效且简便的提高酵母发酵性能的方法。本项目较为系统地研究了促酵母增殖活性肽的高效富集、活性肽的结构鉴定与构效关系及其对酵母代谢和啤酒质量的影响和定向调控策略，在获得具有显著促进酵母增殖与代谢的生物活性肽段的基础上，阐明了活性肽在酵母胞内外的转运机制和代谢途径。具体研究结果如下：.蛋白水解物的促酵母增殖活性与其水解度并无正相关关系，而是依赖于蛋白和蛋白酶种类、水解度、分子量分布和氨基酸组成。用胰酶酶解小麦面筋蛋白24h所得的酶解物和用复合蛋白酶酶解大豆分离蛋白9h所得酶解产物，在其添加量分别为1.0%和0.1%时具有最好的促进酵母增殖活性。.XAD-16树脂对于小麦面筋蛋白水解物（WGH）具有最突出的吸附和解吸能力,WGH经不同乙醇浓度洗脱所得组分的氨基酸组成和Mw分布具有明显的差异，这也是导致其在提高酵母细胞活力、出芽率及对乙醇和渗透压胁迫耐受性方面表现出不同能力的主要原因。.从植物蛋白水解物中分离鉴定得到了20余种小分子活性肽，其中LL、LLL和LML具有促进酵母增殖和提高发酵性能的活性，相关机理研究发现LL、LLL和LML补充提高了酵母的ADH酶活性和细胞膜完整性并减少胞内ROS水平；其中ROS水平的降低主要是由于抗氧化酶系统中的CAT、SOD和GSH-Px的酶活力提高以及非酶抗氧化系统中的GSH的含量升高所致。.利用GC-MSD代谢组学技术分析了二肽LL的添加对VHG发酵中酵母胞内代谢物表达的影响。PCA和PLS-DA筛选出12个主要差异代谢物，这些主要差异代谢物涉及的代谢通路主要有：倍半萜与三萜代谢途径、鞘脂类代谢、丙酮酸代谢、嘌呤代谢以及甘油脂代谢等。.在不影响啤酒非生物稳定性的前提下，补充植物蛋白水解物可使表观发酵度和乙醇产量分别提高10.4%和7.2%，发酵周期缩短4d，改善了成品啤酒的泡沫稳定性，提高了高浓酿造啤酒的风味品质。