【摘要】：蛋白水解物(Proteinhydrolysates)是蛋白質(zhì)經(jīng)酶、酸、堿等方法水解得到的混合物,其主要成分為肽類,還包含少量氨基酸、糖類、脂類、礦物質(zhì)和維生素。水解乳蛋白(Lactoalbuminhydrolysate)是一種重要的蛋白水解物,是乳蛋白經(jīng)蛋白酶或肽酶水解的產(chǎn)物,含有豐富的氨基酸和多肽,可為細胞生長提供多種營養(yǎng)成分、貼壁因子及生長因子類似物等,廣泛用于生物制藥、疫苗、食品等行業(yè)。首先,本研究以酪蛋白(Casein)為原料,采用超高壓生物反應(yīng)器結(jié)合酶水解的方法,以游離氨基酸含量、水解度、分子量為指標,從胰蛋白酶、胃蛋白酶、木瓜蛋白酶、中性蛋白酶、堿性蛋白酶和胰酶六種不同的酶中篩選出水解酪蛋白最佳的酶。再通過單因素和正交試驗對最佳用酶水解酪蛋白的最佳工藝進行了研究,結(jié)果表明胰酶水解效果較好,其氨基酸含量達到47.187%。水解度為41.06%,分子量分布在10kD以下。胰酶水解酪蛋白的最佳工藝為底物濃度15%,水解壓力為150 MPa,溫度為50℃,pH為7.5條件下水解4 h。在該條件下制備的酪蛋白水解產(chǎn)物(Casein hydrolysates,CH)的游離氨基酸含量為52.178%。其次,本研究通過SDS-PAGE、電鏡、紅外吸收光譜、X射線衍射、氨基酸分析儀、分子篩等方法對CH的理化性質(zhì)進行了分析,同時以Hyclone水解乳蛋白(Hyclone lactoalbumin hydrolysate,HLH)為對照。結(jié)果表明CH的理化性質(zhì)與HLH的結(jié)構(gòu)極其相似。即形態(tài)結(jié)構(gòu)均屬于矩陣型;分子量小于10 KD,二級結(jié)構(gòu)主要以α-螺旋和β-轉(zhuǎn)角結(jié)構(gòu)存在;主要含有蛋白質(zhì)(amid I)、羧基、纖維素、核酸、脂類和多糖;有19種相同的氨基酸,其總含量差異顯著(P0.05),分別為 24.04±0.768%和 46.202±2.263%;Cellulose-60 分子篩結(jié)果表明,CH 與 HLH均有F1、F2、F3、F4和F5 5個相同的組分,但各組分的含量有所不同。最后,本研究分別在敘利亞倉鼠腎細胞(Baby Hamster Syrian Kidney,BHK-21)貼壁/懸浮細胞的培養(yǎng)基中添加了適量的CH,研究了 CH對BHK-21細胞增殖和代謝的影響。同時以HLH、Lactoalbumin hydrolysate-35(LH35)和Lactoalbumin hydrolysate-80(LH-80)三種水解乳蛋白為對照組。結(jié)果表明CH,LH35,LH80和HLH四種水解乳蛋白對BHK-21貼壁/懸浮細胞均有明顯的促生長效果。CH對BHK-21貼壁細胞促生長效果優(yōu)于HLH,對BHK-21懸浮細胞的促細胞生長效果與HLH沒有明顯的差異,但低于LH35和LH80。水解乳蛋白對BHK-21懸浮細胞的生化、酶和氨基酸的代謝均有不同程度的影響�？傊�,本研究建立了水解乳蛋白制備工藝,按該工藝制備的水解乳蛋白可作為無血清培養(yǎng)基(Serum free medium,SFM)的添加劑,這為酪蛋白的進一步開發(fā)利用和蛋白質(zhì)水解產(chǎn)物在細胞培養(yǎng)中的應(yīng)用奠定基礎(chǔ)。
[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，

本文編號：2179356