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5~40 μmol/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 high-gravity 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 5-40 μmol/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.
[1] GUAN Y, XU X, LIU C F, et al.Evaluating the physiology and fermentation performance of the lager yeast during very high gravity brewing with increased temperature[J].LWT, 2023, 173:114312.
[2] PICKERELL A T W.The influence of free alpha-amino nitrogen in Sorghum beer fermentations[J].Journal of the Institute of Brewing, 1986, 92(6):568-571.
[3] JONES M, B S, PIERCE J S, et al.Absorption of amino acids from wort by yeasts[J].Journal of the Institute of Brewing, 1964, 70(4):307-315.
[4] VERBRUGGEN N, HERMANS C.Proline accumulation in plants:A review[J].Amino Acids, 2008, 35(4):753-759.
[5] TSOLMONBAATAR A, HASHIDA K, SUGIMOTO Y, et al.Isolation of baker′s yeast mutants with proline accumulation that showed enhanced tolerance to baking-associated stresses[J].International Journal of Food Microbiology, 2016, 238:233-240..
[6] HEEMS D, LUCK G, FRAUDEAU C, et al.Fully automated precolumn derivatization, on-line dialysis and high-performance liquid chromatographic analysis of amino acids in food, beverages and feedstuff[J].Journal of Chromatography A, 1998, 798(1-2):9-17.
[7] MAYADUNNE R, NGUYEN T T, MARRIOTT P J.Amino acid analysis by using comprehensive two-dimensional gas chromatography[J].Analytical and Bioanalytical Chemistry, 2005, 382(3):836-847.
[8] CATALDI T R I, NARDIELLO D.Determination of free proline and monosaccharides in wine samples by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)[J].Journal of Agricultural and Food Chemistry, 2003, 51(13):3737-3742.
[9] COSTIN J W, BARNETT N W, LEWIS S W.Determination of proline in wine using flow injection analysis with tris(2,2′-bipyridyl)ruthenium(II) chemiluminescence detection[J].Talanta, 2004, 64(4):894-898.
[10] DAĞDEVIREN S, ALTUNAY N, SAYMAN Y, et al.A new method of UA_CPE coupled with spectrophotometry for the faster and cost-effective detection of proline in fruit juice, honey, and wine[J].Food Chemistry, 2018, 255:31-40.
[11] GORINSTEIN S, ZEMSER M, VARGAS-ALBORES F, et al.Proteins and amino acids in beers, their contents and relationships with other analytical data[J].Food Chemistry, 1999, 67(1):71-78.
[12] LONG D F, WILKINSON K L, POOLE K, et al.Rapid method for proline determination in grape juice and wine[J].Journal of Agricultural and Food Chemistry, 2012, 60(17):4259-4264.
[13] ROBERT-PEILLARD F, BOUDENNE J L, COULOMB B.Development of a simple fluorescence-based microplate method for the high-throughput analysis of proline in wine samples[J].Food Chemistry, 2014, 150:274-279.
[14] 王增妹, 王金晶, 李磊, 等.外源海藻糖对啤酒酵母在热胁迫下的保护作用[J].食品与生物技术学报, 2020, 39(8):43-50.
WANG Z M, WANG J J, LI L, et al.Protective effects of exogenous trehalose on brewer′s yeast under heat stress[J].Journal of Food Science and Biotechnology, 2020, 39(8):43-50.
