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

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.