响应面法优化鹿鞭肽酶解工艺及体外补肾健骨活性分析

赵峻露; 李春楠; 尹馨雪; 兰梦; 张辉; 李晶峰

doi:10.13386/j.issn1002-0306.2022030363

响应面法优化鹿鞭肽酶解工艺及体外补肾健骨活性分析

Optimization of the Enzymatic Hydrolysis Process of Penis et Testis Cervi Peptides by Response Surface Methodology and Analysis on the Activity of Tonifying Kidney and Strengthing Bone in Vitro

摘要

摘要: 目的：确定碱性蛋白酶酶解鹿鞭肽最佳工艺条件，并探究其体外补肾健骨活性。方法：以酶解温度、pH、酶解时间和酶用量为影响因素，水解度为响应指标，在单因素实验的基础上，结合Box-Behnken方法进行响应面试验设计，获得酶解鹿鞭肽的最佳工艺；探究不同浓度的鹿鞭肽对小鼠睾丸间质细胞（TM3）的存活率以及睾酮（Testosterone，T）的分泌影响和对成骨细胞（MC3T3-E1）的存活率、碱性磷酸酶（Alkaline phosphatase，ALP）活力以及骨钙素（Osteocalcin，OCN）的分泌影响。结果：最佳酶解工艺条件为温度51 ℃，pH8.7，时间4.1 h，酶用量1300 U/g，此时水解度为40.36%±0.22%；体外活性实验结果表明，TM3细胞不同浓度（25、50、100、200 μg/mL）给药组与模型组相比较，均可显著提高细胞存活率和睾酮的分泌（P<0.05），并在50 μg/mL质量浓度下，鹿鞭酶解肽对TM3细胞的T分泌促进作用最强，其分泌量为42.47 ng/mL；MC3T3-E1细胞不同浓度（25、50、100、200 μg/mL）给药组与模型组相比较，细胞存活率和ALP活力、OCN分泌都有显著提高（P<0.05），并在50 μg/mL质量浓度下，ALP活力及OCN分泌作用最显著，ALP活力为6.80金氏单位/g prot，OCN分泌量为15.63 ng/mL。结论：本实验通过响应面法确定了鹿鞭肽的最佳酶解工艺，验证了鹿鞭酶解肽具有较好的体外补肾健骨活性，为以后鹿鞭的综合开发利用提供一定的理论基础。

Abstract: Objective: To determine the optimum process condition for enzymatic hydrolysis of Penis et Testis Cervi peptides by alcalase, and to explore its activity of tonifying kidney and strengthing bone in vitro. Methods: With the temperature, pH, time and dosage of enzyme as the influencing factors, and the degree of hydrolysis as the response index, the response surface test was designed based on the single factor experiments and Box-Behnken method to obtain the optimal enzymatic hydrolysis condition of Penis et Testis Cervi peptides. To investigate the effects of different concentrations of Penis et Testis Cervi peptide on the survival rate of TM3 cells and the secretion of testosterone (T), and on the survival rate of MC3T3-E1 cells, the activity of alkaline phosphatase (ALP), and the secretion of osteocalcin (OCN). Results: The optimal hydrolysis condition was temperature 51 ℃, pH8.7, time 4.1 h, enzyme dosage 1300 U/g, and the degree of hydrolysis was 40.36%±0.22%. The results of in vitro activity experiments showed that different concentrations (25, 50, 100, and 200 μg/mL) of TM3 cells compared with the model group could significantly increase cell viability and T secretion (P<0.05). At the mass concentration of 50 μg/mL, the T secretion of TM3 cells was most promoted by the enzymolysis peptide from Penis et Testis Cervi, with the secretion amount of 42.47 ng/mL. Compared with the model group, the cell viability, ALP activity and OCN secretion were significantly increased in the MC3T3-E1 cell medicated groups at different concentrations (25, 50, 100, and 200 g/mL) (P<0.05). At the mass concentration of 50 μg/mL, the activities of ALP and OCN secretion were the most significant, with ALP activity of 6.80 King/g prot and OCN secretion of 15.63 ng/mL. Conclusion: The response surface methodology was used to determine the optimal enzymatic hydrolysis process of Penis et Testis Cervi peptide, which verified that the enzymolysis peptide had good activity of tonifying kidney and strengthing bone in vitro, and would provide a theoretical basis for the comprehensive development and utilization of Penis et Testis Cervi in the future.