水解乳蛋白的制备及其在BHK-21细胞培养中的应用研究
发布时间：2018-08-12 14:36
【摘要】：蛋白水解物(Proteinhydrolysates)是蛋白质经酶、酸、碱等方法水解得到的混合物,其主要成分为肽类,还包含少量氨基酸、糖类、脂类、矿物质和维生素。水解乳蛋白(Lactoalbuminhydrolysate)是一种重要的蛋白水解物,是乳蛋白经蛋白酶或肽酶水解的产物,含有丰富的氨基酸和多肽,可为细胞生长提供多种营养成分、贴壁因子及生长因子类似物等,广泛用于生物制药、疫苗、食品等行业。首先,本研究以酪蛋白(Casein)为原料,采用超高压生物反应器结合酶水解的方法,以游离氨基酸含量、水解度、分子量为指标,从胰蛋白酶、胃蛋白酶、木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和胰酶六种不同的酶中筛选出水解酪蛋白最佳的酶。再通过单因素和正交试验对最佳用酶水解酪蛋白的最佳工艺进行了研究,结果表明胰酶水解效果较好,其氨基酸含量达到47.187%。水解度为41.06%,分子量分布在10kD以下。胰酶水解酪蛋白的最佳工艺为底物浓度15%,水解压力为150 MPa,温度为50℃,pH为7.5条件下水解4 h。在该条件下制备的酪蛋白水解产物(Casein hydrolysates,CH)的游离氨基酸含量为52.178%。其次,本研究通过SDS-PAGE、电镜、红外吸收光谱、X射线衍射、氨基酸分析仪、分子筛等方法对CH的理化性质进行了分析,同时以Hyclone水解乳蛋白(Hyclone lactoalbumin hydrolysate,HLH)为对照。结果表明CH的理化性质与HLH的结构极其相似。即形态结构均属于矩阵型;分子量小于10 KD,二级结构主要以α-螺旋和β-转角结构存在;主要含有蛋白质(amid I)、羧基、纤维素、核酸、脂类和多糖;有19种相同的氨基酸,其总含量差异显著(P0.05),分别为 24.04±0.768%和 46.202±2.263%;Cellulose-60 分子筛结果表明,CH 与 HLH均有F1、F2、F3、F4和F5 5个相同的组分,但各组分的含量有所不同。最后,本研究分别在叙利亚仓鼠肾细胞(Baby Hamster Syrian Kidney,BHK-21)贴壁/悬浮细胞的培养基中添加了适量的CH,研究了 CH对BHK-21细胞增殖和代谢的影响。同时以HLH、Lactoalbumin hydrolysate-35(LH35)和Lactoalbumin hydrolysate-80(LH-80)三种水解乳蛋白为对照组。结果表明CH,LH35,LH80和HLH四种水解乳蛋白对BHK-21贴壁/悬浮细胞均有明显的促生长效果。CH对BHK-21贴壁细胞促生长效果优于HLH,对BHK-21悬浮细胞的促细胞生长效果与HLH没有明显的差异,但低于LH35和LH80。水解乳蛋白对BHK-21悬浮细胞的生化、酶和氨基酸的代谢均有不同程度的影响。总之,本研究建立了水解乳蛋白制备工艺,按该工艺制备的水解乳蛋白可作为无血清培养基(Serum free medium,SFM)的添加剂,这为酪蛋白的进一步开发利用和蛋白质水解产物在细胞培养中的应用奠定基础。
[Abstract]:Protein hydrolysate (Proteinhydrolysates) is a mixture of proteins hydrolyzed by enzyme, acid and alkali. Its main component is peptide, and it also contains a small amount of amino acids, carbohydrates, lipids, minerals and vitamins. Hydrolyzed milk protein (Lactoalbuminhydrolysate) is an important protein hydrolysate, which is hydrolyzed by protease or peptidase. It contains rich amino acids and peptides, and can provide many nutrients, adherent factors and growth factor analogues for cell growth. Widely used in biopharmaceutical, vaccine, food and other industries. First of all, using casein (Casein) as raw material, using ultrahigh pressure bioreactor with enzymatic hydrolysis method, taking the content of free amino acid, degree of hydrolysis, molecular weight as index, trypsin, pepsin, papain, neutral protease, The best casein hydrolysate was obtained from six different enzymes of alkaline protease and trypsin. The optimum hydrolysis process of casein with enzyme was studied by single factor and orthogonal test. The results showed that the hydrolytic effect of trypsin was better, and the amino acid content of trypsin was 47.187. The degree of hydrolysis is 41.06, and the molecular weight is distributed below 10kD. The optimum hydrolysis conditions for casein by trypsin were as follows: substrate concentration 15, hydrolysis pressure 150 MPA, temperature 50 鈩，
本文编号：2179356
[Abstract]:Protein hydrolysate (Proteinhydrolysates) is a mixture of proteins hydrolyzed by enzyme, acid and alkali. Its main component is peptide, and it also contains a small amount of amino acids, carbohydrates, lipids, minerals and vitamins. Hydrolyzed milk protein (Lactoalbuminhydrolysate) is an important protein hydrolysate, which is hydrolyzed by protease or peptidase. It contains rich amino acids and peptides, and can provide many nutrients, adherent factors and growth factor analogues for cell growth. Widely used in biopharmaceutical, vaccine, food and other industries. First of all, using casein (Casein) as raw material, using ultrahigh pressure bioreactor with enzymatic hydrolysis method, taking the content of free amino acid, degree of hydrolysis, molecular weight as index, trypsin, pepsin, papain, neutral protease, The best casein hydrolysate was obtained from six different enzymes of alkaline protease and trypsin. The optimum hydrolysis process of casein with enzyme was studied by single factor and orthogonal test. The results showed that the hydrolytic effect of trypsin was better, and the amino acid content of trypsin was 47.187. The degree of hydrolysis is 41.06, and the molecular weight is distributed below 10kD. The optimum hydrolysis conditions for casein by trypsin were as follows: substrate concentration 15, hydrolysis pressure 150 MPA, temperature 50 鈩，
本文编号：2179356
本文链接：https://www.wllwen.com/shoufeilunwen/benkebiyelunwen/2179356.html
