Functional Properties of Peptides Produced by Bacterial Enzymes from Traditional Fermented Foods

几千年来，人们一直用传统的和自然的发酵方法来制作食物。自然存在的微生物，如细菌、酵母菌和霉菌，产生用于制作食物的蛋白水解酶。传统的发酵谷物和豆类食品中含有多种芽孢杆菌。本研究旨在从发酵食品中收集微生物，研究细菌酶的蛋白水解潜力和功能特性。与泰国、越南、中国和柬埔寨签署了基于《生物多样性公约》的合作备忘录，因此本研究的数据可以公开分发。分离得到的细菌经16S rDNA鉴定，并进行系统发育树分析。纯化酶的分子量为33 kDa， n端氨基酸序列为AQSVPYGISQIKAPA。然而，这是第一次从传统发酵小麦粉食品中分离出该菌。纯化后的蛋白酶将酸性酪蛋白分解成c端有亲疏水氨基酸的片段，特别是Arg、Glu、Val和Ile。枯草芽孢杆菌DB和SR的浓缩粗酶可通过水解免疫印迹法检测到的致敏麦胶蛋白片段，降低麦胶蛋白（小麦过敏原）的致敏性。此外，枯草芽孢杆菌DB的浓缩粗酶也能在1 h内将牛奶过敏原β-乳球蛋白（β-lactoglobulin）消化成小片段，培养3 h后这些片段消失。然后对片段进行n端氨基酸序列分析；发现该酶能水解Gln^<35>-Ser^<36>位置的β-乳球蛋白的主要致敏表位。因此，这些酶可用于生产低过敏性小麦粉或牛奶食品。

For many thousands of years, people have prepared foods by traditional and natural fermentation. Naturally occurring microorganisms such as bacteria, yeast and moulds produce the proteolytic enzymes for making foods. A variety of Bacillus sp. have been involved in traditional fermented cereal and legume foods. This study aimed to collect microorganisms from fermented foods and characterize proteolytic potentials and functional properties of bacterial enzymes. Memorandum of Understanding (MOU) of collaboratory based on the Convention on Biological Diversity was decided with Thailand, Vietnam, China and Cambodia, the data from this research could therefore be publicly distributed. The isolated bacteria were identified by 16S rDNA and phylogenetic-tree analysis was also performed. The molecular mass and N-terminal amino acid sequence of purified enzyme were 33 kDa and AQSVPYGISQIKAPA, respectively. However, this was the first report in which it was isolated from traditional fermented wheat flour food. The purified protease digested acid casein into fragments with hydrophilic and hydrophobic amino acids at C-terminal, in particular Arg, Glu, Val and Ile.The concentrated crude enzyme of B. subtilis DB and SR could reduce allergenicity of gliadin (wheat allergen) by hydrolyzing the allergenic gliadin fragments detected by immunoblotting. Furthermore, the concentrated crude enzyme of B. subtilis DB could also digest β-lactoglobulin (milk allergen) into small fragments within 1 h and these fragments were disappeared after 3-h incubation. The fragments were then analyzed for N-terminal amino acid sequence ; it was found that this enzyme could hydrolyze the major allergenic epitope of β-lactoglobulin at Gln^<35>-Ser^<36> position. Therefore these enzymes can be applied for the production of hypoallergenic wheat flour or milk food products.