高F值南极磷虾小分子肽的制备优化及其对HepG2细胞影响研究  

Optimization and preparation of high Fischer ratio peptides from Antarctic krill and its effects on human hepatocellular carcinoma cell line HepG2

作　　者：鄢珊瑶 迟海[2] 冯广朋[2] 房文红[2] 张淑平[1] 黄艳青[2] YAN Shanyao;CHI Hai;FENG Guangpeng;FANG Wenhong;ZHANG Shuping;HUANG Yanqing(School of Materials and Chemistry,University of Shanghai for Science and Technology,Shanghai 200093,China;Key Laboratory of East China Sea and Oceanic Fishery Resource Exploitation,Ministry of Agriculture and Rural Affairs,East China Sea Fisheries Research Institute,China Fisheries Research Institute,Shanghai 200090,China)

机构地区：[1]上海理工大学材料与化学学院,上海200093 [2]中国水产科学研究院东海水产研究所农业农村部东海与远洋渔业资源开发利用重点实验室,上海200090

出　　处：《食品与发酵工业》2022年第17期175-182,共8页Food and Fermentation Industries

基　　金：山东省重大科技创新工程项目(2019JZZY010816);国家重点研发计划项目(2018YFC1406803)。

摘　　要：该实验以脱脂南极磷虾粉为原料,首先通过单因素(料液比、加酶量、温度、时间)和正交试验优化双酶酶解工艺条件,并采用超滤、活性炭脱芳手段制备南极磷虾高F值小分子肽(Antarctic krill peptides with high-Fischer ratio,HF-AKP),然后采用CCK-8法观察HF-AKP对人肝癌细胞株HepG2生长情况的影响,最后,通过流式细胞计数和ELISA方法分别检测HepG2细胞周期分布和细胞凋亡相关蛋白表达情况。结果显示,碱性蛋白酶的最佳酶解条件为料液比1∶17.5(g∶mL),加酶量5.0%(质量分数),温度55℃,酶解6 h;复合风味蛋白酶的最佳酶解条件为加酶量4.0%,酶解3 h。经双酶酶解的脱脂南极磷虾酶解液的水解度为(17.4±0.2)%,得到F值为2.4±0.2的小分子肽(Antarctic krill peptides,AKP,<5 kDa),再经活性炭脱芳处理得到F值为87.2±0.7的HF-AKP。HF-AKP对体外培养的人肝癌细胞株HepG2的细胞生长具有抑制作用,且呈剂量、时间正向依赖性;此外,HF-AKP可使HepG2细胞生长阻滞于G0/G1期;其凋亡机制可能与Bax/Bcl-2蛋白表达比值增加,以及人核因子κB(nuclear factorκB,NF-κB)蛋白表达减少有关,推测HF-AKP可能有一定抗肿瘤功效。Firstly,the enzymatic hydrolysis conditions were optimized by using single-factor(material-to-liquid ratio,amount of enzyme,temperature,time)and orthogonal test from defatted Antarctic krill powder in this work.Antarctic krill peptides with high-Fischer ratio(HF-AKP)were prepared by ultrafiltration and followed by activated carbon adsorption.Then the CCK-8 method was used to observe the effect of HF-AKP on the growth of human liver cancer cell line HepG2.Finally,the cell cycle distribution and apoptosis-related protein expression of HepG2 were detected by flow cytometry and ELISA methods,which made efforts to explore the effects of HF-AKP on HepG2 cells.The results revealed that enzymatic hydrolysis conditions of alkaline protease were as follows:the material-to-liquid ratio was 1:17.5(g:mL)and the addition amount of enzyme was 5.0%for enzymatic hydrolysis at 55℃for 6 h.The flavor protease optimal conditions were as follows:the addition amount of enzyme was 4.0%and enzymatic hydrolyzed for 3 h.After the second round hydrolysis,the degree of hydrolysis increased up to(17.4±0.2)%.Especially,after activated carbon adsorption,the Fischer ratio of defatted Antarctic krill peptides increased from(2.4±0.2)to(87.2±0.7)compared to AKP.HF-AKP had an inhibitory effect on the cell proliferation of the HepG2,which was positively dependent on dose-and time-manner.It also could block HepG2 cells in the G0/G1 phase.In addition,the mechanism of apoptosis may be related to the increase in Bax/Bcl-2 protein expression ratio and the decrease in NF-κB protein expression,so HF-AKP may have certain anti-tumor effects.

关 键 词：脱脂南极磷虾粉 双酶水解 高F值小分子肽 HEPG2细胞 

分 类 号：TS254.1[轻工技术与工程—水产品加工及贮藏工程]