红曲糟抗热肽的制备及其对酿酒酵母热激氧化耐受性的影响

Preparation of Heat-Resistant Peptides from Hongqu Rice Distiller’s Grains and Its Effect on the Thermally Induced Oxidative Tolerance in Saccharomyces cerevi

制备红曲糟抗热肽,研究其对酿酒酵母抵御热激氧化行为的影响。以酿酒酵母热激存活率为指标,筛选蛋白酶,通过抗热肽得率优化酶解条件,对抗热肽进行质谱和抗氧化能力分析,通过磷酸戊糖通路基因表达、胞内辅酶、谷胱甘肽(glutathione,GSH)以及活性氧自由基(reactive oxygen species,ROS)含量分析,从抗氧化角度探索抗热肽对酿酒酵母的热保护作用。结果表明,红曲糟抗热肽最佳酶解工艺为:红曲糟蛋白和水固液比1∶10(m/m)、复合蛋白酶酶解、温度50℃、时间3 h、加酶量3000 U/g、pH 8.5,该条件下抗热肽得率62.44%,酿酒酵母热激存活率73.97%,较纯水组提高了22.76%。序列鉴定表明,丰度前20的肽段中16条肽段的疏水氨基酸占比50%以上。抗热肽1,1-二苯基-2-三硝基苯肼自由基半数清除量为4.53 mg/mL,2,2′-联氮双(3-乙基苯并噻唑啉-6-磺酸)阳离子自由基半数清除量为1.82 mg/mL,具有抗氧化活性。同时,抗热肽通过上调磷酸戊糖途径的基因表达量,提升NADH激酶活性,促进还原型辅酶II的转化,使GSH含量提高4.74倍,胞内ROS水平与热激前酿酒酵母基本保持一致,从而有效提高了酿酒酵母抵御热激氧化胁迫的能力。

Thermally resistant peptides were obtained from enzymatical hydrolysis of Hongqu rice distiller’s grains and evaluated for its effect on resistance to thermally induced oxidative stress in Saccharomyces cerevisiae.Various proteases were screened for the survival rate of S.cerevisiae under heat stress.The enzymatic hydrolysis conditions were optimized based on the yield of thermally resistant peptides,and the peptides were identified by mass spectroscopy and its antioxidant activity was investigated.The expression of genes in the pentose phosphate pathway and the contents of intracellular coenzyme,glutathione(GSH)and reactive oxygen species(ROS)were analyzed to explore the protective effects of the peptides on S.cerevisiae from heat-induced oxidative stress.The results indicated that the optimal enzymatic hydrolysis conditions were as follows:Hongqu rice distiller’s grains/water ratio 1:10,hydrolysis temperature 50℃,hydrolysis time 3 h,protamex dosage 3000 U/g,and pH 8.5.Under these conditions,the yield of thermally resistant peptides was 62.44%,and the survival rate of S.cerevisiae in the presence of the prepared peptides under heat stress was 73.97%,which was 22.76%higher than that of the control group(pure water).Sequence identification showed that 16 out of the top 20 most abundant peptides had hydrophobic amino acid contents of over 50%.The peptides exhibited 1,1-diphenyl-2-picrylhydrazyl(DPPH)and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)cation radical scavenging activity with a halfmaximal effective concentration(EC_(50))of 4.53 and 1.82 mg/mL,respectively,indicating good antioxidant activity.Besides,the thermally resistant peptides upregulated gene expression in the pentose phosphate pathway,elevated NADH kinase activity,increased GSH content by 5.74 times,and restored intracellular ROS to almost the same level as before heat stress treatment,thus enhancing resistance to heat-induced oxidative stress in S.cerevisiae.