胶原多肽螯合钙的制备及其壮骨、骨质疏松防治作用研究
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摘要
猪皮中含有丰富的胶原蛋白，是制备胶原多肽的优质原料。本实验以猪皮为原料，采用蛋白酶水解法制备胶原多肽并确定最佳工艺，通过与最适钙源螯合并筛选最佳工艺制备出胶原多肽螯合钙，以小鼠为实验对象，并以碳酸钙和葡萄糖酸钙两种常规补钙剂作为阳性对照，对制得的胶原多肽螯合钙进行骨质疏松的防治及壮骨作用研究。
选用猪皮为原料，采用中性蛋白酶、碱性蛋白酶、木瓜蛋白酶水解提取胶原多肽，对影响酶作用的pH值、温度、酶浓度、底物浓度、时间等因素进行单因素和正交试验，得出水解的最佳工艺条件。在此基础上任选两种酶并改变添加顺序对猪皮进行酶解，以水解度为依据得出最佳组合。结果表明：(1)中性蛋白酶水解猪皮制备胶原多肽的最适pH为6.5，最适温度为45℃，最适酶量为0.5％，最适底物浓度为15％。碱性蛋白酶水解猪皮制备胶原多肽的最适pH值为11，最适温度为55℃，最适酶量为0.5％，最适底物浓度为35％。木瓜蛋白酶水解猪皮制备胶原多肽的最适pH为5，最适温度为60℃，最适酶量为0.9％，最适底物浓度为30％。水解能力大小为：碱性蛋白酶＞木瓜蛋白酶＞中性蛋白酶。(2)通过分步酶水解试验发现，复合酶水解能力均高于各自单一酶水解能力，且添加顺序对水解度的值有显著影响。以木瓜蛋白酶+碱性蛋白酶的水解效果最好。(3)对水解产物的分子量分析结果表明，其分子量要选远地小于胶原蛋白，证明其已被分解，且其分子量呈连续分布，大多分布在几千到4万千道尔顿之间。
选用以最佳水解条件制备出的胶原多肽为原料，找出与其螯合的最佳钙源并研究制备胶原多肽螯合钙的最佳工艺条件。结果表明，最佳钙源为CaCl_2，最佳的螯合条件是pH值为8.0，肽钙质量比为4∶1。采用有机溶剂萃取法使胶原多肽螯合钙在有机溶剂中沉淀出来，并讨论了萃取时有机溶剂的用量对产品得率的影响，结果得出在无水乙醇与蒸馏水体积比为9∶1时产品得率最高。最后用紫外扫描和红外光谱对产品进行鉴定，结果表明该螯合产物不同于胶原多肽和CaCl_2，是一种新的物质。
动物实验分为三个小试验，即治疗试验组(A组)，预防试验组(B组)，为壮骨试验组(C组)。以葡萄糖酸钙、碳酸钙为阳性对照研究胶原多肽螯合钙在防治骨质疏松和壮骨方面的效果。结果表明，胶原多肽螯合钙无论是在对骨质疏松的防治中还是在促进骨骼生长、壮骨作用中，其效果均优于葡萄糖酸钙和碳酸钙，是一种优良的功能性补钙制剂。
Collagen is one of the important and polyfunctional protein from pig skin. The optium hydrolysis conditions of pig skin with single enzyme and compound enzyme, and the suitable calcium chelating condition with collagen hyhrolysate were studied in this paper. Furthermore,animial experiments were used for validating the collagen polypeptide calcium was a first-rate Ca additive ,which was better than Calcium carbonate and calcium gluconate.
Firsrtly, neutral proteinase, alkaline proteinase, papain were used to hydrolyze fresh pig skin respectively. The effects of pH value ,temperature, enzyme content and substrate content on hydrolysis were studied by single factor test and orthogonal, design with degree of hydrolysis (DH) as only guide was adopted, the optimum condition of hydrolysis parameters was determined. On the base of the optimum condition single proteinase, any two proteinases were chosen from the neutral proteinase, alkaline proteinase and papain, through the analysis of experiment date ,the optimum compounding were determined. The results showed as follows: (1) The suitable hydrolysis parameters of neutral proteinase was pH value 6.5,temperature 45℃,enzyme content 0.5%,suhstrate content 15%. The suitable hydrolysis parameters of alkaline proteinase was pH value 11,temperature 55℃,enzyme content 0.5%,suhstrate content 35%. The suitable hydrolysis parameters of papain was pH value 5,temperature 60℃,enzyme content 0.9%,suhstrate content30%.The ability of hydrolysis was alkaline proteinase >papain>neutral proteinase. (2) The compounding have the bigger DH than their single enzyme, and the sequence of addition inflents DH .The compounding of papain+alkaline has the biggest DH. (3) The result of the studied on the molecular weight of collagen hydrolysis and collagen indicate that the molecular weight of collagen is about 300000 daltons and narrow distribution, the molecular weight of collagen hydrolysis is smaller than collagen ,it's molecular weight is several thousands to forty thousands, and has a wide distribution.
Secondly, the products of optimum compounding and the suitable calcium were choosed as the material of collagen polypeptide calcium ,according to chelated rate as only guide ,the synthetic conditions of calcium chelate was studied with an empHasis on the chelating reaction.The result showed that the optimal calcium was CaCl_2,the optimal reaction condition was pH value 8,the molar ratio is 4: 1.Consequently, the chelate products were purified and analyzed by common chemical method .Extration solvent was also evaluated .The effect of volume ratio of organic solvent to filtrate was studied.The highest recovery rate was obtained at 9:1 of ethyl alcohl to distilled water.The product was testified by ultraviolet (UV) and infra red (IR) .
Lastly, The effect of collagen polypeptide calcium on the prevention and cure of osteoporosis and the promotion of bone's growing were testified by animal experiments.The result were showed that the collagen polypeptide calcium was one of the new supplement calcium which not only calcium but collagen, it's effect is better than Calcium carbonate and calcium gluconate on the prevention and cure of osteoporosis and bone's growing.
选用猪皮为原料，采用中性蛋白酶、碱性蛋白酶、木瓜蛋白酶水解提取胶原多肽，对影响酶作用的pH值、温度、酶浓度、底物浓度、时间等因素进行单因素和正交试验，得出水解的最佳工艺条件。在此基础上任选两种酶并改变添加顺序对猪皮进行酶解，以水解度为依据得出最佳组合。结果表明：(1)中性蛋白酶水解猪皮制备胶原多肽的最适pH为6.5，最适温度为45℃，最适酶量为0.5％，最适底物浓度为15％。碱性蛋白酶水解猪皮制备胶原多肽的最适pH值为11，最适温度为55℃，最适酶量为0.5％，最适底物浓度为35％。木瓜蛋白酶水解猪皮制备胶原多肽的最适pH为5，最适温度为60℃，最适酶量为0.9％，最适底物浓度为30％。水解能力大小为：碱性蛋白酶＞木瓜蛋白酶＞中性蛋白酶。(2)通过分步酶水解试验发现，复合酶水解能力均高于各自单一酶水解能力，且添加顺序对水解度的值有显著影响。以木瓜蛋白酶+碱性蛋白酶的水解效果最好。(3)对水解产物的分子量分析结果表明，其分子量要选远地小于胶原蛋白，证明其已被分解，且其分子量呈连续分布，大多分布在几千到4万千道尔顿之间。
选用以最佳水解条件制备出的胶原多肽为原料，找出与其螯合的最佳钙源并研究制备胶原多肽螯合钙的最佳工艺条件。结果表明，最佳钙源为CaCl_2，最佳的螯合条件是pH值为8.0，肽钙质量比为4∶1。采用有机溶剂萃取法使胶原多肽螯合钙在有机溶剂中沉淀出来，并讨论了萃取时有机溶剂的用量对产品得率的影响，结果得出在无水乙醇与蒸馏水体积比为9∶1时产品得率最高。最后用紫外扫描和红外光谱对产品进行鉴定，结果表明该螯合产物不同于胶原多肽和CaCl_2，是一种新的物质。
动物实验分为三个小试验，即治疗试验组(A组)，预防试验组(B组)，为壮骨试验组(C组)。以葡萄糖酸钙、碳酸钙为阳性对照研究胶原多肽螯合钙在防治骨质疏松和壮骨方面的效果。结果表明，胶原多肽螯合钙无论是在对骨质疏松的防治中还是在促进骨骼生长、壮骨作用中，其效果均优于葡萄糖酸钙和碳酸钙，是一种优良的功能性补钙制剂。
Collagen is one of the important and polyfunctional protein from pig skin. The optium hydrolysis conditions of pig skin with single enzyme and compound enzyme, and the suitable calcium chelating condition with collagen hyhrolysate were studied in this paper. Furthermore,animial experiments were used for validating the collagen polypeptide calcium was a first-rate Ca additive ,which was better than Calcium carbonate and calcium gluconate.
Firsrtly, neutral proteinase, alkaline proteinase, papain were used to hydrolyze fresh pig skin respectively. The effects of pH value ,temperature, enzyme content and substrate content on hydrolysis were studied by single factor test and orthogonal, design with degree of hydrolysis (DH) as only guide was adopted, the optimum condition of hydrolysis parameters was determined. On the base of the optimum condition single proteinase, any two proteinases were chosen from the neutral proteinase, alkaline proteinase and papain, through the analysis of experiment date ,the optimum compounding were determined. The results showed as follows: (1) The suitable hydrolysis parameters of neutral proteinase was pH value 6.5,temperature 45℃,enzyme content 0.5%,suhstrate content 15%. The suitable hydrolysis parameters of alkaline proteinase was pH value 11,temperature 55℃,enzyme content 0.5%,suhstrate content 35%. The suitable hydrolysis parameters of papain was pH value 5,temperature 60℃,enzyme content 0.9%,suhstrate content30%.The ability of hydrolysis was alkaline proteinase >papain>neutral proteinase. (2) The compounding have the bigger DH than their single enzyme, and the sequence of addition inflents DH .The compounding of papain+alkaline has the biggest DH. (3) The result of the studied on the molecular weight of collagen hydrolysis and collagen indicate that the molecular weight of collagen is about 300000 daltons and narrow distribution, the molecular weight of collagen hydrolysis is smaller than collagen ,it's molecular weight is several thousands to forty thousands, and has a wide distribution.
Secondly, the products of optimum compounding and the suitable calcium were choosed as the material of collagen polypeptide calcium ,according to chelated rate as only guide ,the synthetic conditions of calcium chelate was studied with an empHasis on the chelating reaction.The result showed that the optimal calcium was CaCl_2,the optimal reaction condition was pH value 8,the molar ratio is 4: 1.Consequently, the chelate products were purified and analyzed by common chemical method .Extration solvent was also evaluated .The effect of volume ratio of organic solvent to filtrate was studied.The highest recovery rate was obtained at 9:1 of ethyl alcohl to distilled water.The product was testified by ultraviolet (UV) and infra red (IR) .
Lastly, The effect of collagen polypeptide calcium on the prevention and cure of osteoporosis and the promotion of bone's growing were testified by animal experiments.The result were showed that the collagen polypeptide calcium was one of the new supplement calcium which not only calcium but collagen, it's effect is better than Calcium carbonate and calcium gluconate on the prevention and cure of osteoporosis and bone's growing.
引文
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