【作者】 张子健；

【导师】 张柏林；

【作者基本信息】 河北农业大学 ， 农产品加工及贮藏工程， 2010， 硕士

【摘要】 ACE抑制肽具有降血压功能,采用酶解或微生物发酵是目前获取乳源性ACE抑制肽的主要手段。干酪乳杆菌属于同型发酵乳酸菌,有较好的产酸活性及蛋白水解活力,广泛用于多种乳制品生产上。本文从ACE抑制活性菌株的筛选和鉴定、发酵乳ACE抑制率的影响因素、ACE抑制肽的胃肠耐受性及乳酸菌产ACE抑制肽的机制等方面对干酪乳杆菌的ACE抑制活性进行了研究,旨在为应用干酪乳杆菌到相关食品中提供依据。主要研究结果如下:1、D400和Y5-2b在经pH=2.5的人工胃液消化4 h后,活菌率仍能达到89.09%和86.28%,而经过人工肠液和0.3%胆盐消化后活菌率则仍能达到96%~98%,均表现为良好的耐胃肠环境性能。D400无论在乳中的生长状态,发酵性能还是蛋白水解活力方面都优于Y5-2b,ACE抑制活性试验证明, D400发酵乳的ACE抑制率高于Y5-2b。因此D400被选作本次研究的试验菌株。基于生理生化和16S rRNA基因序列的试验数据,乳杆菌D400被鉴定为干酪乳杆菌(Lactobacillus casei)。2、随着L.casei D400细胞生物量和发酵乳滴定酸度的上升,菌株的蛋白水解活力以及对ACE的抑制率随之上升,表明随着菌株生长,含在乳蛋白中的ACE抑制肽逐渐被释放出来,菌株的蛋白水解活力与ACE抑制活性有着明显的相关性。3、单因素试验表明,发酵温度、发酵时间和无脂乳固形物浓度都会影响菌株的蛋白水解活力,影响其ACE抑制效率。优化后的L.casei D400在乳中的发酵条件是:无脂乳固形物浓度为10%,发酵时间为24 h,发酵温度为37°C,在这样的条件下,L.casei D400的ACE抑制率是81.15%。4、模拟胃肠环境试验结果表明,经胃蛋白酶和胰蛋白酶的作用后,L.casei D400的ACE抑制率由消化前的81.15%下降到消化后的67.83%,下降程度不是很大,说明L.casei D400产生的ACE抑制肽具有一定的耐受胃肠道内蛋白酶消化能力。5、对L.casei D400发酵乳中的氨基酸组分与含量分析表明,疏水性氨基酸含量在总氨基酸中的比重也由发酵前的22.29%上升为48.50%,脯氨酸含量在总氨基酸中的比重大幅上升,由发酵前的1.67%上升为18.75%,且相对增加量高于其它氨基酸。亮氨酸和苏氨酸的含量相对增加较多,异亮氨酸、精氨酸、赖氨酸、缬氨酸其次,且新生成了蛋氨酸和苯丙氨酸,结合已知ACE抑制肽的序列结构,符合了产ACE抑制肽的条件。因此,L.casei D400产ACE抑制肽的原因是其具有水解蛋白产生脯氨酸及其他疏水性氨基酸和芳香族氨基酸的能力。更多还原

【Abstract】 ACE inhibitory peptide has a function on lowering blood pressure. Enzymatic or microbial fermentation is the primary means of obtaining ACE inhibitory peptides from milk. Lactobacillus casei belong to the same type fermentation of lactic acid bacteria, has good acid production activity and protein hydrolysis activity, widely used in the various of dairy products. This paper studies screening and identification of the Lactobacillus casei, the determination of proteolytic activity of the strain, ACE inhibitory activity of the strain and contents of amino acids in fermentation milk whey and other aspects, which establishes screening model of Lactobacillus casei with ACE inhibitory activity. Major findings are as follows:1. After 4 hours digestion by the artificial gastric juice( pH = 2.5), the viable rate of D400 and Y5-2b can reach 89.09% and 86.28%, and the viable rate of digestion is still 96% to 98% after the artificial intestinal juice and 0.3% concentration bile salt. Therefore, D400 and Y5-2b have good resistance to gastrointestinal environmental performance. Further more, D400 in terms of the growth state in the milk, acid production or proteolytic activity areas were higher than Y5-2b, ACE inhibitory activity of D400 fermented milk was higher than Y5-2b. therefore, selected L.casei D400 as the experimental strains. Based on experiment data of physiological, biochemical and 16S rRNA gene sequence, determins D400 and Y5-2b as Lactobacillus casei.2. With the cell biomass and titratable acidity of L.casei D400 fermented milk increased, the proteolytic activity and the ACE inhibition rate rised, which indicated with the growth of cell biomass, ACE inhibitory peptide included in the milk protein gradually released, proteolytic activity and ACE inhibition has significant correlation.3. Single factor test showed that the fermentation temperature, fermentation time and the concentration of non-fat milk solids will affect the proteolytic activity and the efficiency of ACE inhibition of the strain. Optimized L.casei D400 in milk fermentation conditions are: SNF concentration of 10%, fermentation time 24 h, fermentation temperature 37°C. In such conditions, ACE inhibition rate of L. casei D400 is 81.15%.4. After the digestion of pepsin and trypsin, ACE inhibition rate of L.casei D400 decreased from 81.15% before to 67.83%, indicating ACE inhibitory peptides produced by L.casei D400 has a certain tolerance to the gastrointestinal tract protease digestion. 5. The amino acids content in the L.casei D400 fermented milk analysis show that the hydrophobic amino acids in the proportion of the total amino acids increased from the fermentation before 22.29% to 48.50%, proline proportion in the total amino acid has increased significantly from 1.67% before to 18.75%, and the relative increase contents was higher than other amino acids. Leucine and threonine content were relatively large increase, isoleucine, arginine, lysine, valine followed, and found methionine and phenylalanine. Combination structure of ACE inhibitory peptide sequence which has known, the conditions of producing ACE inhibitory peptides formed. Therefore, the capacity of L.casei D400 produce ACE inhibitory peptides is because of its hydrolyzed protein activity producing proline, other hydrophobic amino acids and aromatic amino acid.更多还原