酶解水牛奶制备抗凝血肽的工艺研究
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摘要
目的:本文以新鲜水牛奶为原料,研究木瓜蛋白酶、中性蛋白酶及碱性蛋白酶复合酶解、混合酶解水牛奶的作用,比较不同水解方式的作用效果;并采用凝血酶滴定改进法测定水解液抗凝血活性。方法:分别进行三种蛋白酶单因素水解实验,确定各种酶的最适水解条件;在此基础上进行复合酶解与混合酶解实验,测定水解度和抗凝血活性。结果:木瓜蛋白酶的最适水解条件为:水解温度60℃、水解时间2.0 h、p H5.0、料水比1∶2(v/v)、酶用量6000 U/(m L底物);中性蛋白酶的最适条件为:水解温度40℃、水解时间3.0 h、料水比1∶2(v/v)、p H6.0、酶用量6000 U/(m L底物);碱性蛋白酶的最适条件为:水解温度50℃、水解时间4.0 h、料水比1∶2(v/v)、p H8.0、酶用量6000 U/(m L底物)。复合酶解法制备的水解液其抗凝血活性均高于混合酶解,最高抗凝血活性为52.0 ATU/m L。结论:本实验条件下,加酶组合为"中性蛋白酶-木瓜蛋白酶-碱性蛋白酶"的复合酶解制备的水解液具有最高的抗凝血活性,即52.0 ATU/m L。
Objective: Research on buffalo milk affected by the systems about mixed enzyme and composite enzyme including papain,neutral protease and alkaline protease,comparing with the result in different ways of hydrolysis. Method: First,the experiment went on with the single factor of three proteases hydrolyzing milk to explore the optimum condition of enzymatic hydrolysis.On this basis,the degree of hydrolysis was determinated through composite enzymatic hydrolysis and mixed enzyme and anticoagulant activity was determinated.Consequence: The results showed that optimum condition of papain were as follows:the ratio of material to water for 1∶ 2,p H = 5.0,temperature 60 ℃,time 2 h,enzyme dosage 6000 u/m L.Neutral protease of were as follows: the ratio of material to water for 1∶ 2,p H = 6.0,temperature 40 ℃,time of 2 h,enzyme dosage 6000 u/m L.Optimum condition of alkaline protease were as follows: the ratio of material to water for 1 ∶ 2,p H = 8.0,temperature 50 ℃,time 4 h,enzyme dosage 6000 u/m L. Composite enzymatic hydrolysis anticoagulant activity was higher than by mixed enzymatic hydrolysis.The highest anticoagulant activity was 52 ATU/m L. Conclusion: In the experimental conditions,the degree and anticoagulant activity by composite enzymatic hydrolysis were higher than by mixed enzymatic hydrolysis. The enzymatic hydrolysis solution,which was prepared by the enzyme combination of the composite enzyme of neutral protease and papain protease and alkaline protease,had the highest anticoagulant activity,was 52.0 ATU/m L.
Objective: Research on buffalo milk affected by the systems about mixed enzyme and composite enzyme including papain,neutral protease and alkaline protease,comparing with the result in different ways of hydrolysis. Method: First,the experiment went on with the single factor of three proteases hydrolyzing milk to explore the optimum condition of enzymatic hydrolysis.On this basis,the degree of hydrolysis was determinated through composite enzymatic hydrolysis and mixed enzyme and anticoagulant activity was determinated.Consequence: The results showed that optimum condition of papain were as follows:the ratio of material to water for 1∶ 2,p H = 5.0,temperature 60 ℃,time 2 h,enzyme dosage 6000 u/m L.Neutral protease of were as follows: the ratio of material to water for 1∶ 2,p H = 6.0,temperature 40 ℃,time of 2 h,enzyme dosage 6000 u/m L.Optimum condition of alkaline protease were as follows: the ratio of material to water for 1 ∶ 2,p H = 8.0,temperature 50 ℃,time 4 h,enzyme dosage 6000 u/m L. Composite enzymatic hydrolysis anticoagulant activity was higher than by mixed enzymatic hydrolysis.The highest anticoagulant activity was 52 ATU/m L. Conclusion: In the experimental conditions,the degree and anticoagulant activity by composite enzymatic hydrolysis were higher than by mixed enzymatic hydrolysis. The enzymatic hydrolysis solution,which was prepared by the enzyme combination of the composite enzyme of neutral protease and papain protease and alkaline protease,had the highest anticoagulant activity,was 52.0 ATU/m L.
引文
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[17]方富永,苗艳丽,陈绍红,等.酪蛋白双酶复合酶解与混合酶解作用研究[J].中国食品学报,2013,13(5):111-116.
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[20]潘丽军,张丽,钟昔阳,等.双酶分步水解法制备谷氨酰胺活性肽的研究[J].食品工业科技,2009(4):184-187.
[2]黄元元,戴秋云.抗凝血多肽及类肽研究进展[J].中国生化药物志,2007,28(5):349-352.
[3]李旭霞,李庆伟.抗凝血蛋白药物的研究进展[J].辽宁师范大学学报:自然科学版,2003,26(2):187-191.
[4]Jo H Y,Jung W K,Kim S K.Purification and characterization of a novel anticoagulant peptide from marine echiuroid worm,Urechis unicinctus[J].Journal of Biotechnology,2008,43(2):179-184.
[5]Indumathi P,Mehta A.A novel anticoagulant peptide from the Nori hydrolysate[J].Journal of Functional Foods,2016,20:606-617.
[6]黎观红,乐国伟,施用晖,等.利用自建的活性肽数据库搜寻食物蛋白质中潜在的生物活性肽[J].食品与发酵工业,2004,30(1):85-88.
[7]Fiat A M,Migliore-Samour D,Jollès P,et al.Biologically active peptides from milk proteins with emphasis on two examples concerning antithrombotic and immunomodulating activities[J].Dairy Sci,1993,76:301-310.
[8]刘恭,高维东,纪银莉,等.牦牛乳酪蛋白抗凝血肽制备工艺研究[J].食品研究与开发,2014,17:65-68.
[9]苗艳丽,刘卓勤,孙瑞雄,等.酪蛋白抗凝血肽的制备及其体外抗凝血、溶栓作用研究[J].中草药,2016,47(11):1875-1881.
[10]胡永倩.花生肽的酶法制备及其抗凝血活性研究[D].郑州:河南工业大学,2015.
[11]周向军,吴红艳,颜彩映,等.蛋清肽的工艺优化、抗氧化作用及特性[J].食品与生物技术学报,2013,32(8):844-853.
[12]刘静波,王菲,王翠娜,等.Alcalase酶解蛋清粉制备抗凝血肽的工艺优化[J].吉林大学学报(工学版),2012,42(1):250-255.
[13]任清,张晓平.酶膜反应器制备燕麦蛋白质ACE抑制肽的研究[J].中国食品学报,2011,11(4):24-35.
[14]陈钧辉,李俊,张太平,等.生物化学实验[M].第4版.北京:科学出版社,2008:71-72.
[15]陈华友,邢自力,李媛媛,等.凝血酶滴定法测定水蛭素活性的改进[J].生物技术,2002,12(06):24-25.
[16]钟超,吴晖,赖富饶.酶解菲牛蛭制备抗凝血肽的工艺优化[J].现代食品科技,2012,28(2)164-167,155.
[17]方富永,苗艳丽,陈绍红,等.酪蛋白双酶复合酶解与混合酶解作用研究[J].中国食品学报,2013,13(5):111-116.
[18]Zhao Y,Li B,Liu Z,et al.Antihypertensive effect and purification of an ACE inhibitory peptide from sea cucumber gelatin hydrolysate[J].Process Biochemistry,2007,42(12):1586
[19]杜林,李亚娜.生物活性肽的功能与制备研究进展[J].中国食物与营养,2005(8):18-21.
[20]潘丽军,张丽,钟昔阳,等.双酶分步水解法制备谷氨酰胺活性肽的研究[J].食品工业科技,2009(4):184-187.