磷酸化大豆多肽螯合钙的制备工艺优化及其对成骨细胞的活性影响

廖子蔚; 陈阳; 陈秀云; 黄茜茜; 龙林; 孙代华; 胡辉

doi:10.13386/j.issn1002-0306.2022050142

磷酸化大豆多肽螯合钙的制备工艺优化及其对成骨细胞的活性影响

Optimization of the Preparation Process of Phosphorylated Soybean Polypeptide Chelated Calcium and Its Effect on the Activity of Osteoblasts

摘要

摘要: 为了探究磷酸化大豆多肽螯合钙（PSPCC）的生理活性，提高磷酸化大豆多肽螯合钙的钙螯合量，本研究通过单因素实验和响应面试验相结合的方式优化磷酸化大豆多肽螯合钙的制备工艺，并通过体外实验评价了磷酸化大豆多肽螯合钙对成骨细胞的活性影响。结果表明，制备磷酸化大豆多肽螯合钙的最佳工艺条件为：三聚磷酸钠与大豆多肽的质量比1:2，磷酸化反应温度52 ℃，磷酸化反应pH7.0，磷酸化反应时间9.7 h，磷酸化大豆多肽与氯化钙的质量比2:1，螯合反应pH8.0，螯合反应温度50 ℃，螯合反应时间1.5 h，钙螯合量最大为107.25±0.10 mg/g；MTT法结果显示，磷酸化大豆多肽螯合钙组（D组）成骨细胞相对增殖率是大豆多肽组（A组）的1.6倍（第3 d）；ALP染色实验表明，D组染色阳性率是空白组的33.6倍。采用ALP试剂盒对各样品第7 d的ALP活性进行检测，结果显示D组ALP活力是空白组的6.2倍。通过茜素红染色实验发现，D组钙结节数量是空白组的94倍。本研究使用响应面法对磷酸化大豆多肽螯合钙的制备工艺优化合理，并初步证明了磷酸化大豆多肽螯合钙对成骨细胞具有显著的促增殖、分化作用（P<0.05），且作用效果高于其它样品，为后续磷酸化大豆多肽螯合钙的进一步开发利用提供了一定的理论基础。

Abstract: This study aimed to explore the physiological activity of phosphorylated soybean polypeptide chelated calcium (PSPCC) and improve its calcium chelation capacity. To be specific, the preparation process of PSPCC was optimized by single factor test and response surface methodology. And the effect of phosphorylated soybean peptide chelated calcium on osteoblast activity was evaluated by in vitro experiments. The results showed that, the optimum process of PSPCC was as follows: Mass ratio of sodium tripolyphosphate to soybean polypeptide 1:2, phosphorylation reaction temperature 52 ℃, phosphorylation reaction pH7.0, phosphorylation reaction time 9.7 h, mass ratio of phosphorylated soybean polypeptide to calcium chloride 2:1, chelation reaction pH8.0, chelation reaction temperature 50 °C, and chelation reaction time 1.5 h. The maximum calcium chelation amount of PSPCC was 107.25±0.10 mg/g under the above optimal conditions. The results of MTT assay showed that the relative proliferation rate of osteoblasts in the PSPCC group (group D) was 1.6 times that of the soybean polypeptide group (group A) (the third day). In particular, the ALP staining experiment showed that the positive rate of staining in group D was 33.6 times that of the blank group. In addition, the ALP activity of each sample was detected by the ALP kit on the 7th day, and the results showed that the ALP activity of the group D was 6.2 times that of the blank group. And it was found that the number of calcium nodules in the group D was 94 times higher than that of the blank group through the alizarin red staining experiment. It was reasonable that the preparation process of PSPCC was optimized by response surface methodology, which was preliminarily proved that PSPCC had a significant effect on promoting proliferation and differentiation of osteoblasts (P<0.05), and the effect of PSPCC on promoting proliferation and differentiation was higher than that of other samples. This result could provide a theoretical basis for the further exploitation and utilization of PSPCC.