多功能植物小分子肽的研究
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摘要
本研究以植物蛋白(大豆蛋白和花生蛋白)为原料,采用微射流均质技术结合复合酶酶解技术制备功能性小分子肽,研究了微射流和复合蛋白酶酶解的最佳工艺条件,并对小分子肽的结构和功能性质进行表征和分析,初探了多功能性小分子肽的构效关系。
1、在单因素实验的基础上,采用Plackett-Burman设计,以酸溶蛋白肽的得率为响应值,对影响植物蛋白水解的因子进行筛选。本实验选取了10个相关因子进行筛选,试验结果表明底物蛋白的浓度、第一次酶解时的pH和温度这三个为主要影响因子。
2、在Plackett-Burman设计的基础上,采用响应曲面法(Response Surface Methodology, RSM)分析,以酸溶蛋白肽的得率为响应值,对底物蛋白的浓度、第一次酶解时的pH和温度3个关键因素的最佳水平进行了优化。经求解二次多项回归方程得知,底物蛋白的浓度为8.90%、第一次酶解时溶液的pH为8.04、温度为57.80℃时,其酸溶蛋白肽的得率最高为88.11%,相关系数为0.9516,所建模型极其显著(p<0.0008)。
3、采用Sephadex G-25凝胶色谱对酶解后的溶液进行分离纯化,得到小分子肽。采用HPLC法对其相对分子量进行测定,结果显示,小分子肽主要集中在相对分子量为400Da左右的范围,该组分的小分子肽具有清除DPPH功能和降胆固醇功能,其DPPH的清除率为44.78%,胆固醇胶束的抑制率为73.2%。
4、采用HPLC-MS法对M3、M4的结构进行了初步研究,结果表明,M3含有SRQ或SRK、DHI或DHL；而M4中基本上全部是氨基酸,从质谱图中找到了两种可能的氨基酸,它们是丙氨酸和异亮氨酸或亮氨酸。
5、对于功能性植物小分子肽的产品开发,现今只开发了功能性植物小分子肽口服液。以植物蛋白水解液为原料制备小分子肽口服液,各项指标均符合国家标准,且口感好,易于被人体吸收,将是新一代的保健产品。
Vegetable protein (soybean protein and peanut protein) was used as the raw material in this study. Microfluidization homogenizing technology combining with complex enzymolysis was used to prepare functional small peptides. The optimal processing condition was studied. The structure and functional properties of the small peptides were represented and analyzed. Besides, the structure-function relationship of multi-functional small peptides was initially explored.
1. Plackett-Burman design was used based on single-factor experiments. Using acid-soluble protein yield as the response value, the factors affecting vegetable protein hydrolysis were screened.10 related factors were chosen in this study to screen, and the results showed that the substrate protein concentration, pH of the first enzymolysis and temperature were the main affecting factors.
2. Response Surface Methodology analysis was used based on Plackett-Burman design. Using acid-soluble protein yield as the response value, the optimal levels of the three key factors-substrate protein concentration, pH of the first enzymolysis and temperature were optimized. The quadratic regression equation solution indicated that when the substrate protein concentration was 8.90%, pH of the first enzymolysis was 8.04, and temperature was 57.80℃, the highest acid-soluble protein yield was 88.11%, correlation coefficient was 0.9516, and the model was extremely significant(p<0.0008).
3. Sephadex G-25 gel chromatography was used to separate and purify the hydrolysate, and small peptides were obtained. Its relative molecular weight was measured by HPLC. The results showed that the small peptides main concentrated in the range of 400Da or so, of which the small peptides had the function of scavenging DPPH and lowering cholesterol. The scavenging rate of DPPH was 44.78% and inhibition of micellar cholesterol was 73.2%.
4. HPLC-MS was used to primarily study the structure of M3 and M4. The results revealed that M3 contained SQR or SRK, DHI or DHL, while M4 was almost amino acids. Two possible amino acids were found in the mass-spectrogram, they were alanine, and isoleucine or leucine.
5. As to the products development of functional vegetable protein, oral liquid was developed in this study. The plant protein hydrolysate was used to prepare the small peptide oral liquid, which indicators were in line with national standard. In addition, it was with good taste and could be easily assimilated, and it would be health products of a new generation.
1、在单因素实验的基础上,采用Plackett-Burman设计,以酸溶蛋白肽的得率为响应值,对影响植物蛋白水解的因子进行筛选。本实验选取了10个相关因子进行筛选,试验结果表明底物蛋白的浓度、第一次酶解时的pH和温度这三个为主要影响因子。
2、在Plackett-Burman设计的基础上,采用响应曲面法(Response Surface Methodology, RSM)分析,以酸溶蛋白肽的得率为响应值,对底物蛋白的浓度、第一次酶解时的pH和温度3个关键因素的最佳水平进行了优化。经求解二次多项回归方程得知,底物蛋白的浓度为8.90%、第一次酶解时溶液的pH为8.04、温度为57.80℃时,其酸溶蛋白肽的得率最高为88.11%,相关系数为0.9516,所建模型极其显著(p<0.0008)。
3、采用Sephadex G-25凝胶色谱对酶解后的溶液进行分离纯化,得到小分子肽。采用HPLC法对其相对分子量进行测定,结果显示,小分子肽主要集中在相对分子量为400Da左右的范围,该组分的小分子肽具有清除DPPH功能和降胆固醇功能,其DPPH的清除率为44.78%,胆固醇胶束的抑制率为73.2%。
4、采用HPLC-MS法对M3、M4的结构进行了初步研究,结果表明,M3含有SRQ或SRK、DHI或DHL；而M4中基本上全部是氨基酸,从质谱图中找到了两种可能的氨基酸,它们是丙氨酸和异亮氨酸或亮氨酸。
5、对于功能性植物小分子肽的产品开发,现今只开发了功能性植物小分子肽口服液。以植物蛋白水解液为原料制备小分子肽口服液,各项指标均符合国家标准,且口感好,易于被人体吸收,将是新一代的保健产品。
Vegetable protein (soybean protein and peanut protein) was used as the raw material in this study. Microfluidization homogenizing technology combining with complex enzymolysis was used to prepare functional small peptides. The optimal processing condition was studied. The structure and functional properties of the small peptides were represented and analyzed. Besides, the structure-function relationship of multi-functional small peptides was initially explored.
1. Plackett-Burman design was used based on single-factor experiments. Using acid-soluble protein yield as the response value, the factors affecting vegetable protein hydrolysis were screened.10 related factors were chosen in this study to screen, and the results showed that the substrate protein concentration, pH of the first enzymolysis and temperature were the main affecting factors.
2. Response Surface Methodology analysis was used based on Plackett-Burman design. Using acid-soluble protein yield as the response value, the optimal levels of the three key factors-substrate protein concentration, pH of the first enzymolysis and temperature were optimized. The quadratic regression equation solution indicated that when the substrate protein concentration was 8.90%, pH of the first enzymolysis was 8.04, and temperature was 57.80℃, the highest acid-soluble protein yield was 88.11%, correlation coefficient was 0.9516, and the model was extremely significant(p<0.0008).
3. Sephadex G-25 gel chromatography was used to separate and purify the hydrolysate, and small peptides were obtained. Its relative molecular weight was measured by HPLC. The results showed that the small peptides main concentrated in the range of 400Da or so, of which the small peptides had the function of scavenging DPPH and lowering cholesterol. The scavenging rate of DPPH was 44.78% and inhibition of micellar cholesterol was 73.2%.
4. HPLC-MS was used to primarily study the structure of M3 and M4. The results revealed that M3 contained SQR or SRK, DHI or DHL, while M4 was almost amino acids. Two possible amino acids were found in the mass-spectrogram, they were alanine, and isoleucine or leucine.
5. As to the products development of functional vegetable protein, oral liquid was developed in this study. The plant protein hydrolysate was used to prepare the small peptide oral liquid, which indicators were in line with national standard. In addition, it was with good taste and could be easily assimilated, and it would be health products of a new generation.
引文
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