猪皮胶原肽-亚铁的制备和螯合工艺

Preparation and chelation of pig skin collagen peptide-ferrous iron

目的以猪皮为原料,氯化亚铁为铁源,探讨经蛋白酶酶解后的猪皮胶原肽与亚铁离子螯合的最佳工艺条件。方法对新鲜猪皮进行脱脂处理,采用菠萝蛋白酶和碱性蛋白酶对脱脂猪皮进行酶解,制备猪皮胶原肽,并利用超滤法将其按照分子量进行分段;以螯合率和螯合物得率为指标,对猪皮胶原肽-亚铁螯合物(PCPF)的制备条件(抗氧化剂用量、肽-亚铁摩尔比、螯合pH值、螯合温度、螯合时间、肽浓度和醇沉时间)进行单因素考察,然后采用正交法进一步优化螯合条件,并采用硫化钠法对制备得到的PCPF进行定性鉴定。结果通过单因素实验和正交实验确定了猪皮胶原蛋白肽与亚铁离子螯合的最佳条件:取猪皮胶原肽溶液(0.15 g/mL),按质量比mVc∶mFeCl_(2)·4H_(2)O=0.3∶1加入抗氧化剂,用10%盐酸调pH值至5.00,按摩尔比肽∶Fe^(2+)=3∶1加入铁盐,在40℃下螯合40 min,然后用6倍体积无水乙醇醇沉,慢加快搅,冷藏静置24 h后收集沉淀,并用无水乙醇洗涤,50℃烘干后即得PCPF。验证实验表明,该螯合工艺稳定可靠。硫化钠定性检测实验表明,螯合物中不存在游离的猪皮胶原肽,进而确定猪皮胶原肽与亚铁离子己充分螯合。不同分子量猪皮胶原肽螯合活性实验表明,分子量为1~3 kDa的猪皮胶原肽的螯合活较高。结论PCPF的制备工艺稳定可靠。

Objective To determine the optimum conditions for chelating pig skin collagen peptide with ferrous ion by protease hydrolysis using pig skin as raw material and ferrous chloride as iron source.Methods The Fresh pork skin was degreased,and the pig skin collagen peptide was prepared by enzymatic hydrolysis with bromelain and alkaline protease.We prepared the pigskin collagen peptide and used the ultrafiltration method to segment them according to molecular weight.The preparation conditions of pig skin collagen peptide-ferrous chelate(PCPF)(antioxidant dosage,peptide-ferrous molar ratio,chelating pH,chelating temperature,chelating time,peptide concentration and alcohol precipitation time)were investigated by single factor with chelate rate and chelate as indexes.After that,the chelation conditions were further optimized by orthogonal method and the PCPF was qualitatively identified by sodium sulfide method.Results Through unifactor experiment and orthogonal experiment,we determined the best conditions for the chelation of pig skin collagen peptide and ferrous ion:took the pig skin collagen peptide solution(0.15 g/mL),added antioxidants with mVc∶mFeCl_(2)·4H_(2)O=0.3∶1,adjusted pH with 10%hydrochloric acid to 5.00,added iron salt with the molar ratio of peptide∶Fe ^(2+)=3∶1 to chelate for 40 min at 40℃,settled with 6 volume of anhydrous ethanol and stir slowly,collected precipitation after refrigerating for 24 h and washed with absolute ethanol,so PCPF was obtained after drying at 50℃.The validation experiment showed that the chelation process was stable and reliable;the qualitative test of sodium sulfide showed that there was no free pig skin collagen peptide in the chelate,and then confirmed that pig skin collagen peptide was chelated with ferrous ion adequately;and the chelation activity of different molecular weights showed that the chelation activity of the molecular weight of 1-3 kDa was high.Conclusion The preparation process of PCPF is stable and reliable.