| 摘要: |
| 为改善鲟鱼骨胶原的营养价值和生理功能，提高骨胶原多肽得率，提升水产品的附加值，采用碱性蛋白酶和中性蛋白酶复合酶解技术，以肽得率为指标，通过单因素试验、正交设计试验优化骨胶原多肽的酶解条件。采用CCK-8法测定鲟鱼骨胶原多肽对人源结直肠癌细胞Caco-2、乳腺癌细胞MCF-7、肝癌细胞HeGp2、肺癌细胞A549及宫颈癌细胞HeLa 5株癌细胞增殖的抑制能力，并选取抑制效果明显的肺癌细胞A549为模型，进一步检测鲟鱼骨胶原多肽作用后对其caspase-1、caspase-3以及细胞膜电位的影响。结果表明:1)鲟鱼软骨胶原肽的酶解工艺条件为:加酶量1.5×105 IU/g，骨粉与水质量比1∶25，酶解时间5 h，酶解温度50 ℃，碱性蛋白酶与中性蛋白酶酶活力比1∶2。该条件下骨胶原蛋白肽得率为(72.36±2.33)%；2)抑癌活性测定结果显示，鲟鱼骨胶原多肽通过激活凋亡蛋白酶caspase-1，使细胞线粒体膜电位发生改变，膜电位降低，诱导caspase-3酶活力增加，酶活力可达149.9 IU/μg，进而促进细胞凋亡的发生，抑制细胞的增殖。鲟鱼骨胶原多肽具有抑制癌细胞增殖的能力，有潜在的开发应用前景。 |
| 关键词: 鲟鱼软骨 蛋白酶酶解 胶原多肽 细胞凋亡 |
| DOI：10.11841/j.issn.1007-4333.2020.01.15 |
| 投稿时间：2019-05-26 |
| 基金项目:北京市鲟鱼、鲑鳟鱼创新团队(BAIC08-2019) |
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| Optimization of enzymatic hydrolysis conditions of sturgeon cartilage and study on anti-tumor activity of its enzymatic hydrolysate in vitro |
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WU Ruiyun,DU Yili,HUANG Yuxia,LI Pengfei,ZHENG Haitao,LI Pinglan
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| (College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China) |
| Abstract: |
| In order to improve the nutritional and physiological function of sturgeon bone collagen, prepare bone collagen polypeptide with high peptide yield and increase the added value of aquatic products, alkaline protease and neutral protease combined enzyme hydrolysis technique was used and the yield of peptide as the study index. The hydrolysis conditions of bone collagen polypeptide were optimized by single factor test and orthogonal design test. At the same time, the growth inhibition effects of squid collagen peptide on the human colorectal cancer cell Caco-2, breast cancer cell MCF-7, liver cancer cell HeGp2, lung cancer cell A549 and cervical cancer cell line HeLa 5 were determined by CCK-8 method. The lung cancer cell A549 with obvious inhibitory effect was then selected as the model to further detect the effect of bone collagen polypeptide on caspase-1, caspase-3 and cell membrane potential of sturgeon, and explore the mechanism of action. The results showed that the optimum enzymatic hydrolysis conditions were as follows: 150 000 U/g of enzyme, the mass ratio of bone meal to water is 1∶25, 5 h of enzymatic hydrolysis time, 50 ℃ of enzymatic hydrolysis temperature, and the ratio of alkaline protease to neutral protease activity is 1∶2. Under this condition, the yield of peptide was(72. 36±2. 33)%. The results of anti-cancer activity assay showed that sturgeon bone collagen polypeptide changed the mitochondrial membrane potential by activating apoptotic protease caspase-1, decreased the membrane potential and induced the increase of caspase-3 activity. The activity of caspase-3 reached 149. 9 IU/μg, which promoted the occurrence of apoptosis and inhibiting cell proliferation. The bone collagen polypeptide of sturgeon showed the ability to inhibit the proliferation of cancer cells and had potential development and application prospects. |
| Key words: sturgeon cartilage protease hydrolysis collagen polypeptid apoptosis |
