摘要： 为确定酶解法提取菲律宾蛤仔（Ruditapes philippinarum）抗肿瘤活性多肽的最佳工艺条件，选用5种蛋白酶在相同的料液比、酶解时间和酶添加量、各自最适温度和pH的条件下酶解菲律宾蛤仔。通过甲醛电位滴定法和Cell counting kit-8(CCK-8)方法分别测定了菲律宾蛤仔酶解液氨基态氮含量和酶解多肽的抗肿瘤活性，分析了氨基态氮含量与A549抑制率之间的相关性。在单因素试验的基础上，以人非小细胞肺癌A549抑制率为指标，正交分析优化酶解工艺条件。结果显示，菲律宾蛤仔的5种酶解产物中，风味蛋白酶的酶解液中氨基态氮含量最高，而胃蛋白酶获得的酶解多肽抗肿瘤活性最高。胃蛋白酶酶解菲律宾蛤仔正交实验确定酶解最佳工艺条件为：料液比（w/w）1:1，pH 2.0，酶解时间1 h，酶添加量2 000 U/g，酶解温度37 ℃。当设定最佳酶解工艺获得的酶解多肽浓度为20 mg/mL时，其对A549的抑制率为99.83%。研究表明，酶的种类对菲律宾蛤仔酶解液氨基态氮含量和酶解多肽的抗肿瘤活性有较大影响，菲律宾蛤仔酶解液中氨基态氮含量与A549抑制率没有直接的正相关性。

关键词: 菲律宾蛤仔, 酶解多肽, A549, 抗肿瘤活性

Abstract: In order to develop the optimum process of anti-cancer peptides preparation from Ruditapes philippinarum, five proteases were selected to hydrolyze the clams under the same solid-liquid ratio, reaction time, enzyme dosage, optimum temperature and pH conditions. The amino nitrogen content and antitumor activity were measured using formaldehyde potentiometric titration and Cell counting kit-8(CCK-8) methods, respectively, and the correlation between the two indexes was analyzed. Enzymolysis conditions were optimized using single factor experiment and orthogonal design. The results showed that the enzymatic peptides of flavored protease had the highest content of amino nitrogen; whereas the enzymatic peptides of pepsin had the highest antitumor activity. Optimal hydrolysis conditions of pepsin were confirmed as: solid-liquid ratio 1:1(w/w), pH value 2.0, reaction time 1 h, enzyme in solid matter 2 000 U/g, and, enzymolysis temperature 37 ℃. At the dose of 20 mg/mL, the enzymatic peptides that were obtained under the optimal conditions had an inhibition rate of 99.83% on A549. Thus the types of enzymes exert significant influence on the antitumor activity of enzymatic peptides, and there was no direct positive correlation between the content of amino nitrogen and the inhibition rate on A549 in the enzymolysis solution of Ruditapes philippinarum.

Key words: Ruditapes philippinarum, enzymolysis polypeptides, A549, anticancer activity