小麦面筋蛋白酶解产物中苦味肽的释放机制与调控

The research and development of bioactive peptides from wheat gluten (WG) hydrolysates contribute to the wheat industry chain to exploit high value-added products. However, the enzyme-hydrolyzed protein had strong bitterness, which became a technological problem for the producers. At present, the research on enzymatic debittering mainly focus on the screening or combination of proteases at home and abroad, but very little work has been done on the mechanisms of the change in the bitterness intensity of peptide powder. Based on the previous research, the key scientific problems in the release process of bitter peptides are systematically studied. The aim of this project is to collect bitter peptides from enzymatic hydrolysates by organic solvent extraction method, and analyze the main characteristics (the weight, amino acid composition, molecular weight distribution, surface hydrophobicity, bitterness intensity) of bitter peptides through the use of the advanced equipments (automatic kieldahl apparatus, high performance liquid chromatograph, fluorescence spectrometer, electronic tongue). The release characteristics of bitter peptides from WG hydrolysates using single-enzyme treatment are investigated at the different degrees of hydrolysis. Effect of the enzyme cleavage sites of endopeptidase on the release characteristics of bitter peptides is revealed by partial least squares regression analysis. Furthermore, bitter peptides are separated, purified and identified by mass spectrum. The amino acid sequence, chain length and average hydrophobicity of bitter peptides are determined. The regulating mechanisms of the release characteristics of bitter peptides are elucidated during the composite enzymatic hydrolysis or the step-by-step enzymatic hydrolysis. These knowledge will provide the experimental basis and theoretical guidance for the regulation of peptide powders from WG.

小麦面筋蛋白生物活性肽的研究与开发，有助于小麦产业链向高附加值产品延伸，但酶解产物苦味较重成为困扰生产者的一个技术难题。目前，国内外关于酶法脱苦的研究多集中于蛋白酶的筛选与复配，对肽粉苦味变化的内在机制缺乏深入研究。本项目在前期研究的基础上，针对苦味肽释放过程中的关键科学问题开展系统研究。采用有机试剂萃取技术，收集酶解产物中苦味肽，借助凯氏定氮仪、高效液相色谱仪、荧光光谱仪、电子舌等先进设备分析苦味肽的含量、氨基酸组成、分子量分布、表面疏水值、苦味强度等主要特征，探讨单酶酶解产物中苦味肽在不同水解度下的释放特征，运用偏最小二乘回归分析揭示内切蛋白酶专一性酶切位点对苦味肽释放特征的影响；通过进一步分离纯化、质谱鉴定，明确苦味肽的氨基酸序列、肽段长度和平均疏水性，阐明复合酶解和分步酶解调控小麦面筋蛋白酶解产物中苦味肽释放特征的具体机制，为小麦面筋蛋白肽粉的品质调控提供试验依据和理论指导。

酶解产物苦味较重是小麦面筋蛋白活性肽工业化生产需要首先解决的关键性问题，也是困扰食品企业的一个技术难题。目前，国内外关于酶法脱苦的研究多集中于蛋白酶的筛选与复配、脱苦效果以及对产品食用品质的影响，有关不同蛋白酶调控酶解产物中苦味肽释放特征的研究几乎空白，酶解产物苦味强度变化的内在机制缺乏深入研究。本项目在前期研究的基础上，针对苦味肽释放过程中的关键科学问题开展系统研究。采用乙醇分级萃取技术，收集酶解产物中苦味肽，借助凯氏定氮仪、高效液相色谱仪、荧光光谱仪、质谱仪等先进设备分析苦味肽的含量、阈值、氨基酸组成、分子量分布、表面疏水值、苦味强度等主要特征，探讨单酶酶解产物中苦味肽在不同水解度下的释放特征；通过进一步分离纯化、质谱鉴定，明确苦味肽的氨基酸序列、肽段长度和平均疏水性，确定复合酶解和分步酶解调控小麦面筋蛋白酶解产物中苦味肽释放特征的具体机制。研究结果表明：采用乙醇分级沉淀法、大孔吸附树脂色谱、SephadexG-25凝胶层析等技术可以高效萃取酶解产物中的苦味肽，新的萃取技术有利于更加准确的研究苦味肽的释放特征。与酶切位点的差异相比，水解度的不同对苦味肽的释放含量有更强的影响。伴随水解度的增大，苦味肽的释放含量会显著提高。当蛋白酶酶切位点的作用效果更有利于疏水基团的暴露，将会在一定程度上促进苦味肽释放含量的提高。在内切酶和端解酶复合酶解过程中，酶解产物中苦味肽的含量和苦味强度均明显下降。苦味肽的总疏水性氨基酸比例、总苦味氨基酸的比例、分子量在500-1000Da范围内的多肽含量及表面疏水性的显著降低导致苦味阈值增大，且苦味肽阈值的变化对酶解产物感官强度的变化有较大影响。对比聚集体酶解产物中苦味肽，分步酶解酶解产物中苦味肽的含量更低，在氨基酸组成中疏水性氨基酸的比例更低，表面疏水性更低，苦味阈值更大。通过上述研究，阐明了复合酶解和分步酶解技术的脱苦原理，为小麦面筋蛋白肽粉的品质调控提供试验依据和理论指导。