荧光微孔板法检测啤酒酵母胞内脯氨酸含量  

Development of a novel detection method of intracellular proline in brewer’s yeast based on fluorescent microtiter plate

作　　者：于莹莹 许睿琦 钮成拓[1,2] 王金晶 郑飞云[1,2] 刘春凤 李崎[1,2] YU Yingying;XU Ruiqi;NIU Chengtuo;WANG Jinjing;ZHENG Feiyun;LIU Chunfeng;LI Qi(Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology Jiangnan University,Wuxi 214122,China;Laboratory of Brewing Science and Technology,School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,酿酒科学与工程研究室,江苏无锡214122

出　　处：《食品与发酵工业》2024年第20期321-325,共5页Food and Fermentation Industries

基　　金：江南大学教育部工业生物技术重点实验室项目(KLIB-KF202208)。

摘　　要：高浓酿造是啤酒生产中一种常用的高效经济技术,而在高浓酿造过程中啤酒酵母在各个时期存在不同的环境压力。脯氨酸被认为是啤酒酵母中的一种应激保护剂,赋予了酵母细胞更高的胁迫耐受性。酵母胞内脯氨酸含量可以作为筛选环境耐受能力强菌株的参考,因此有必要建立一种能够快速检测啤酒酵母胞内脯氨酸含量的方法。该研究基于次氯酸钠氧化和邻苯二甲醛/N-乙酰半胱胺酸荧光检测脯氨酸的通用方法,结合细胞破碎的优化,成功建立了一种可以准确测定啤酒酵母胞内脯氨酸含量的荧光微孔板法,其线性范围和回收率分别为5~40μmol/L(0.58~4.6 mg/L)和73.33%~105.5%。通过比较该检测方法与氨基酸专用高效液相色谱法,发现两者测定胞内脯氨酸含量绝对值相比无显著性差异,但是荧光微孔板法操作简便快捷。因此,该研究建立的荧光微孔板法为测定酵母胞内脯氨酸提供了一种新思路,同时为适应高浓酿造胁迫的啤酒酵母筛选提供了一种新策略。High-gravity brewing is a commonly used technique in beer production duo to its high economic efficiency.During highgravity brewing,brewer’s yeast is subjected to different environmental stresses in different brewing stages.Proline is considered as an important stress-protective agent in brewer’s yeast,which endows brewer’s yeast with better tolerance ability against stresses in high-gravity brewing.Since the intracellular concentration of proline can be used as a reference for screening of brewing yeast with good environmental tolerance ability,the establishment of a method capable of rapidly detecting the concentration of intracellular proline in brewing yeast is required.This study developed a novel fluorescent microplate method(FMM)for the detection of intracellular proline concentration in brewing yeasts based on sodium hypochlorite oxidation and o-phthalaldehyde(OPA)/n-acetyl-l-cysteine(NAC)fluorescence methods combined with the optimization of cells disruption technique.The linear ranges and the recovery rate of the novel method were 5-40μmol/L(0.58-4.6 mg/L)and 73.33%-105.5%,respectively.Compared to the high-performance liquid chromatography method,the FMM assay had no significantly different absolute quantification of intracellular proline in brewing yeasts,but the procedures in the FMM assay were much faster and more convenient.Therefore,the novel FMM method proposed in this study not only proposes a new solution for the detection of intracellular proline in brewing yeasts,but also provides a new strategy for screening the proline-accumulating brewer’s yeasts with better adaptation to stresses in high-gravity brewing.

关 键 词：脯氨酸 荧光探针 啤酒酵母 高浓酿造 

分 类 号：TS2[轻工技术与工程—食品科学与工程]