棉籽蛋白酶解物的制备及其抗菌活性

doi:10.3864/j.issn.0578-1752.2016.15.016

摘要/Abstract

摘要： 【目的】通过体外模拟动物胃肠道消化环境，对两种棉籽蛋白的酶解产物抗菌活性进行研究，为棉籽蛋白精深加工、高值化利用提供理论依据。【方法】以棉籽粕为原料，用Osborne法和传统的碱溶酸沉法分别制备棉籽蛋白（清蛋白、球蛋白、醇溶蛋白及谷蛋白）和棉籽分离蛋白（cottonseed protein isolate，CPI），用凯氏定氮法测定所得棉籽蛋白的蛋白含量，Tricine-SDS-PAGE测定蛋白亚基组成，通过扫描电镜观察蛋白的表面微观结构，选取蛋白含量较高的球蛋白（cottonseed globulin，CPG）和CPI进行研究。通过体外模拟动物胃肠道消化环境，用胃蛋白酶和胰蛋白酶依次对CPG和CPI进行酶解；以大肠杆菌和金黄色葡萄球菌为受试菌，分别以抗生素卡那霉素（Kanamycin，Kan）及未经酶解的蛋白溶液为对照，研究棉籽蛋白酶解产物的抗菌活性，同时用高效液相色谱法（high performance liquid chromatography，HPLC）检测酶解产物中小肽的分子量大小及分布。【结果】碱溶酸沉法的提取率为（70.52±2.40）%，所得CPI的蛋白含量为（89.53±0.66）%；用Osborne法分别得到清蛋白、球蛋白、醇溶蛋白和谷蛋白等4种棉籽蛋白，提取率为（67.55±1.16）%，其中CPG的蛋白含量最高为（82.57±1.02）%。Tricine-SDS-PAGE图谱表明CPG的亚基组成与CPI较接近；扫描电镜观察到的蛋白表面微观结构差别较大，CPI为颗粒大小较均匀整齐的蜂窝状结构，CPG由大小不均一的蛋白颗粒构成；相对于CPI，CPG中氨基酸保留较为完全。相同加酶量处理情况下，CPI的水解度高于CPG，且棉籽分离蛋白的小分子肽段（MW≤0.8 kDa）含量（70%—85%）显著高于棉籽球蛋白（40%—60%）。抗菌活性检测结果表明，当胃蛋白酶-胰蛋白酶加酶量为5 000—5 000 U时，两种蛋白酶解产物的抗菌能力最强，此时CPI和CPG的水解度分别为（24.72±1.07）%和（19.26±0.39）%，二者酶解产物对大肠杆菌的抑制能力均高于对金黄色葡萄球菌的抑制能力；CPI酶解产物的抗菌活性比CPG高，两者未经消化的蛋白溶液均无抗菌能力。通过分子量测定结果分析得到CPI酶解产物的抗菌活性物质的分子量在0.57—0.75 kDa，而CPG分子量分布在0.66—0.78 kDa。【结论】两种方法制备的蛋白在提取率方面没有显著差异（P＞0.05），在亚基组成方面，Osborne法分级的蛋白间差异较大，但CPG和CPI组成较为相似；二者经体外模拟消化后的酶解产物均具有一定的抗菌活性，CPI酶解产物的抗菌活性高于CPG。

关键词: 棉籽分离蛋白, 棉籽球蛋白, 体外模拟消化, 抗菌活性肽, 分子量分布

Abstract: 【Objective】 In order to provide a theoretical basis and technical support for efficient utilization of cottonseed protein, two methods were used to prepare cottonseed protein, then the isolated proteins were compared and digested in vitro by pepsin and trypsin, lastly the hydrolysates were detected and selected in antibacterial activity. 【Method】 In this paper, cottonseed protein isolate was prepared by the traditional method which is alkali extraction and acid precipitation and Osborne fractionation method. The protein content and molecular weight of the obtained proteins were determined by the Kjeldahl method and Tricine-SDS-PAGE respectively, microstructure of the proteins were examined by scanning electron microscopy. Amino acid composition of cottonseed proteins were analyzed by automatic amino acid analyzer (L-8900). And then proteins were digested by pepsin and trypsin in vitro in the process of digestination, the degree of hydrolysis (DH) of cottonseed protein isolate (CPI) and cottonseed globulin (CPG) hydrolysates were determined by ortho-phthalaldehyde (OPA), and then kanamycin (Kan) and protein solution without hydrolysis were used as control to determine the antibacterial activity of protein hydrolysates with Staphylococcus aureus and Escherichia coli bacteria. Meanwhile, the molecular weight distribution of the hydrolysis products were detected by high performance liquid chromatography (HPLC). 【Result】The extraction rate of CPI by the method of alkali extraction and acid precipitation was (70.52±2.40)% and its protein content reached (89.53±0.66)%. While the albumin, globulin, prolamin and glutelin were classified by Osborne fractionation method, and compared with the other three proteins, cottonseed globulin got the highest protein content that was (82.57±1.02)%. And there was no significant difference (P＞0.05) in protein extraction rate between the two preparation methods for the proteins extraction rate of Osborne fractionation method was (67.55±1.16)%. The Tricine-SDS-PAGE pattern showed that the subunits composition of CPG was more closely to CPI, compared to the other obtained proteins by Osborne fractionation method. The microstructures of the obtained proteins were different from each other. The structure of CPI showed a neat honeycomb structure, which was similar to glutenins’ microstructure, while the CPG showed a uneven granulated one. Amino acid composition analysis indicated that the amino acids of CPG were well maintained by Osborne method, compared to CPI. Cottonseed proteins were hydrolyzed by pepsin and trypsin simulated the digestive system in vitro. The hydrolysis degree of cottonseed protein hydrolysates was determined by microplate reader, and the result showed that the degree of hydrolysis of CPI hydrolysates was higher than CPG at the same amount of added enzyme, which indicated that CPI was easier to be hydrolysed than CPG. The molecular weight of cottonseed protein hydrolysates was measured by HPLC, and the result showed that the content of peptides (MW≤0.8 kDa) of CPI hydrolysates was significantly higher than CPG with the content of 70%-85% and 40%-60%, respectively. Results of antibacterial activity analysis showed that the antibacterial ability of these protein hydrolysates were strongest when the amount of pepsin-trypsin in digestion simulation system was 5 000-5 000 U (2-2), and the degree of hydrolysis of these protein hydrolysates were (24.72±1.07)% and (19.26±0.39)%, respectively, under such digested conditions, and the ability of both digestions to against E. coli were higher than S. aureus. CPI was higher than CPG in antibacterial activity, while both of the original protein without digestion treatment showed no antibacterial ability. The molecular weight determination results showed that the molecular weight of antibacterial activity peptides of cottonseed protein isolate should be in 0.57-0.75 kDa while cottonseed globulins’ were in 0.66-0.78 kDa.【Conclusion】 There was no significant difference between the two methods in the extracted yield of protein (P＞0.05), proteins classified by Osborne fractionation method showed an obvious difference in subunit composition, but the CPG and CPI showed a similarity. CPI is easier to be digested compared to the CPG and both of these two proteins’ hydrolysates showed an antibacterial activity, the hydrolysates of CPI is higher than CPGs’ in antibacterial activity.

Key words: cottonseed protein isolate, cottonseed globulin, in vitro digestion, antibacterial peptide, molecular weight distribution

韩晓燕，包郁明，辛凤姣，孔志强，Christophe Blecker，戴小枫. 棉籽蛋白酶解物的制备及其抗菌活性[J]. 中国农业科学, 2016, 49(15): 3019-3029.

HAN Xiao-yan, BAO Yu-ming, XIN Feng-jiao, KONG Zhi-qiang, Christophe Blecker, DAI Xiao-feng. Preparation and Antimicrobial Activity of Cottonseed Protein Hydrolysate[J]. Scientia Agricultura Sinica, 2016, 49(15): 3019-3029.

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