响应面优化南极磷虾蛋白酶解工艺及蛋白肽组分分析
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摘要
以南极磷虾蛋白酶解液中的氨基氮含量为指标,采用甲醛电位滴定法测定胰蛋白酶、木瓜蛋白酶、碱性蛋白酶、胃蛋白酶、风味蛋白酶、枯草杆菌蛋白酶6种生物酶在其最适酶解条件下对南极磷虾蛋白酶解效果,筛选出酶解南极磷虾蛋白最佳生物酶。在单因素试验基础上,以酶解液中氨基氮含量为响应值,选择酶解温度、时间、pH为自变量,通过三因素三水平Box-Bohnken响应面分析法对筛选出来的生物酶解工艺进行优化。在优化酶解工艺的基础上,利用SDS-PAGE凝胶电泳方法分析南极磷虾酶解蛋白肽及南极磷虾蛋白的分子量分布范围。结果表明:胰蛋白酶为酶解南极磷虾蛋白的理想蛋白酶;响应面分析法建立二次多项回归方程:Y=51.07-0.83A+1.72B-2.06C+0.25AC+1.15BC-3.06A2-2.77B2-3.69C2,模型p<0.0001,而失拟项p>0.05,表明模型极显著且比较稳定,通过模型分析得最佳条件:酶解温度44.48℃、酶解时间8.52 h、pH 7.88,考虑实际应用可行性,我们在酶解温度45.0℃、酶解时间8.5 h,pH 7.9条件下实验得到的氨基氮含量为50.96±1.07mg/g,与理论预测值51.57mg/g基本一致。通过电泳分析得到南极磷虾蛋白分子量范围在15 ku以上,主要集中分子量范围为15 ku～45 ku;胰蛋白酶解多肽分子量在25 ku以下,主要分子量分布在5 ku以下。
The amino nitrogen content in the antarctic krill hydrolysate was used as an indicator.The hydrolysis results of trypsin,papain,alkaline protease,pepsin,flavor protease and subtilisin were evaluated by formaldehyde potentiometric titration.The enzymatic hydrolysis of Antarctic krill protein was optimized for the enzymatic hydrolysis of Antarctic krill.On the basis of single factor experiment,the amino acid content in the enzymatic hydrolysate was used as the response value,and the enzymatic hydrolysis temperature,time and pH were selected as independent variables.The three-factor and three-level Box-Bohnken response surface analysis method was used to screen the bio-enzymatic solution.The process is optimized.Based on the optimized enzymatic hydrolysis process,the molecular weight distribution range of Antarctic krill enzymatic protein peptide and Antarctic krill protein was analyzed by SDS-PAGE gel electrophoresis.The results showed that trypsin was the ideal protease for enzymatic hydrolysis of Antarctic krill protein;the response surface analysis method was used to establish a quadratic multiple regression equation:Y=51.07-0.83 A+1.72 B-2.06 C+0.25 AC+1.15 BC-3.06 A2-2.77 B2-3.69 C2,the model p<0.0001,and the missing term p>0.05,indicating that the model is extremely significant and stable,the best condition obtained by model analysis is:Enzymatic hydrolysis temperature was 44.48 ℃,the enzymatic hydrolysis time was 8.52 h,and the pH was 7.88.Considering the practical application feasibility,the amino nitrogen content of the experiment was 50.96±1.07 mg/g under the conditions of enzymatic hydrolysis temperature of 45.0 ℃,hydrolysis time of 8.5 h and pH 7.9,which is basically consistent with the theoretical prediction value of 51.57 mg/g.The molecular weight of Antarctic krill protein was over 15 ku by electrophoresis analysis,and the main molecular weight range was 15 ku～45 ku.The molecular weight of trypsin peptide was below 25 ku,and the main molecular weight distribution was below 5 ku.
The amino nitrogen content in the antarctic krill hydrolysate was used as an indicator.The hydrolysis results of trypsin,papain,alkaline protease,pepsin,flavor protease and subtilisin were evaluated by formaldehyde potentiometric titration.The enzymatic hydrolysis of Antarctic krill protein was optimized for the enzymatic hydrolysis of Antarctic krill.On the basis of single factor experiment,the amino acid content in the enzymatic hydrolysate was used as the response value,and the enzymatic hydrolysis temperature,time and pH were selected as independent variables.The three-factor and three-level Box-Bohnken response surface analysis method was used to screen the bio-enzymatic solution.The process is optimized.Based on the optimized enzymatic hydrolysis process,the molecular weight distribution range of Antarctic krill enzymatic protein peptide and Antarctic krill protein was analyzed by SDS-PAGE gel electrophoresis.The results showed that trypsin was the ideal protease for enzymatic hydrolysis of Antarctic krill protein;the response surface analysis method was used to establish a quadratic multiple regression equation:Y=51.07-0.83 A+1.72 B-2.06 C+0.25 AC+1.15 BC-3.06 A2-2.77 B2-3.69 C2,the model p<0.0001,and the missing term p>0.05,indicating that the model is extremely significant and stable,the best condition obtained by model analysis is:Enzymatic hydrolysis temperature was 44.48 ℃,the enzymatic hydrolysis time was 8.52 h,and the pH was 7.88.Considering the practical application feasibility,the amino nitrogen content of the experiment was 50.96±1.07 mg/g under the conditions of enzymatic hydrolysis temperature of 45.0 ℃,hydrolysis time of 8.5 h and pH 7.9,which is basically consistent with the theoretical prediction value of 51.57 mg/g.The molecular weight of Antarctic krill protein was over 15 ku by electrophoresis analysis,and the main molecular weight range was 15 ku～45 ku.The molecular weight of trypsin peptide was below 25 ku,and the main molecular weight distribution was below 5 ku.
引文
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[14]李红艳,薛长湖,王灵昭,等.南极磷虾酶解液氯化钙法脱氟工艺的研究[J].食品工业科技,2011,3:330-333LI Hong-yan,XUE Chang-hu,WANG Ling-zhao,et al.Study on defluoridation technology of Antarctic krill hydrolyzate by adding calcium chloride[J].Science and Technology of Food Industry,2011,3:330-333
[15]倪玲,吴文惠,张启,等.蛋白水解酶联合自溶作用的南极磷虾多肽氨基酸分析[J].食品工业科技,2013,34(8):102-107NI Ling,WU Wen-hui,ZHANG Qi,et al.Amino acid analysis of Antarctic krill peptide obtained by protease related autolysis peptide[J].Science and Technology of Food Industry,2013,34(8):102-107
[16]张恒,邬应龙.鸡骨的酶解及酶解液的电泳分析[J].食品与发酵工业,2010,36(9):102-105ZHANG Heng,WU Ying-long.Study on the hydrolysis of chicken bone and electrophoretic analysis of the hydrolysate[J].Food and Fermentation Industries,2010,36(9):102-105
[17]GB5009.235-2016,食品中氨基酸态氮的测定[S]GB5009.235-2016,Determination of Amino Acid Nitrogen in Food[S]
[18]高颖,王彦超,常耀光,等.三种低氟南极磷虾肽的ACE抑制作用及抗氧化活性研究[J].食品工业科技,2016,37(2):82-87,92GAO Ying,WANG Yan-chao,CHANG Yao-guang,et al.ACE inhibition and antioxidant activities of three types of Antarctic krill(Euphausia superba)peptides with low fluorine content[J].Science and Technology of Food Industry,2016,37(2):82-87,92
[19]陆彩玲,唐深,刘楠楠,等.海马组织蛋白不同染色方法的比较[J].环境与职业医学,2013,30(1):26-28LU Cai-ling,TANG Shen,LIU Nan-nan,et al.Comparison of different staining methods for hippocampal proteomic analysis[J].Journal of Environmental&Occupational Medicine,2013,30(1):26-28
[20]黄薇,宋永康,余华,等.酶法制备玉米抗氧化肽[J].中国食品学报,2014,14(8):69-76HUANG Wei,SONG Yong-kang,YU Hua,et al.Enzymatic preparation of corn anti-oxidation peptide[J].Journal of Chinese Institute of Food Science and Technology,2014,14(8):69-76
[21]林燕,陈计峦,胡小松,等.酶解核桃蛋白制备抗氧化肽的研究[J].食品工业科技,2011,32(4):204-207,212LING Yan,CHEN Ji-luan,HU Xiao-song,et al.Preparation of antioxidant peptide from walnut protein by enzymatic hydrolysis[J].Science and Technology of Food Industry,2011,32(4):204-207,212
[22]王璋,许时英.食品化学[M].北京:中国轻工业出版社,2002WANG Zhang,XU Shi-ying.Food Chemistry[M].Beijing:China Light Industry Press,2002
[23]Montgomery D C.Design and Analysis of Experiments[M].London:John Wiley and Sons Inc,1991
[2]Gigliotti J C,Jaczynski J,Tou J C.Determination of the nutritional value,protein quality and safety of krill protein concentrate isolated using an isoelectric solubilization/precipitation technique[J].Food Chemistry,2008,111,209-214
[3]Parka J D,Poowakanjanab S,Parka J W.Biochemical properties of pelagic fish proteins as affected by isolation methods and gel properties by heating methods[J].Journal of Aquatic Food Product Technology,2012,21,307-320
[4]Wang L Z,Xue C H,Wang Y M,et al.Extraction of proteins with low fluoride level from Antarctic krill(Euphausia superba)and their composition analysis[J].Journal of Agricultural and Food Chemistry,2011,59,6108-6112
[5]廖鄂.南极磷虾蛋白提取技术研究[D].中国海洋大学,2014LIAO E.Study on extraction technology of Antarctic krill Abstract[D].Ocean University of China,2014
[6]郭静,李学才,于春芳,等.南极磷虾蛋白的提取及其复合纤维的性能[J].大连工业大学学报,2014,33(4):270-273GUO Jing,LI Xue-cai,YU Chun-fang,et al.Extraction of Antarctic krill protein and properties of its composite fibers[J].Journal of Dalian Polytechnic University,2014,33(4):270-273
[7]任艳.南极磷虾蛋白加工利用的初步研究[D].中国海洋大学,2009REN Yan.Preliminary study on processing the protein of Antarctic krill abstract[D].Ocean University of China,2009
[8]Yoshikawa M,Fujita H,Matoba N,et al.Bioactive peptides derived from food proteins preventing lifestyle-related diseases[J].Bio Factors,2001,12(1-4)
[9]任娇艳,赖婷,江燕清,等.乳清蛋白肽的酶法制备,分离纯化及活性评价[J].华南理工大学学报(自然科学版),2015,43(2):1-7REN Jiao-yan,LAI Ting,JIANG Yan-qing,et al.Enzymatic preparation,purification and activity evaluation of whey protein peptides[J].Journal of South China University of Technology(Natural Science Edition),2015,43(2):1-7
[10]李荣乔.菠萝蛋白酶酶解海湾扇贝裙边制备抗氧化肽的研究[D].河北农业大学,2014LI Rong-qiao.Preparation of Antioxidant Peptides from Scallop skirt of Argopecten irradians by bromelain hydrolysis[D].Hebei Agricultural University,2014
[11]Se-kwon Kim,Yong-tae Kim,Hee-guk Byun,et al.Isolation and characterization of antioxidative peptides from gelatin hydrolysate of alaska pollack skin[J].Journal of Agricultural&Food Chemistry,2001,49(4):1984-1989
[12]ZHANG Nong,YAMASHITA Yamashiita,NOZAKIYukinori.Effect of protein hydrolysate from Antarctic krill meat on the state of water and denaturation by dehydration of lizard fish myofibrils[J].Fisheries Science[Serial Online],2002,68(3):672-679
[13]任艳.南极磷虾蛋白加工利用的初步研究[D].中国海洋大学,2009REN Yan.Preliminary study on processing the protein of Antarctic krill[D].Ocean University of China,2009
[14]李红艳,薛长湖,王灵昭,等.南极磷虾酶解液氯化钙法脱氟工艺的研究[J].食品工业科技,2011,3:330-333LI Hong-yan,XUE Chang-hu,WANG Ling-zhao,et al.Study on defluoridation technology of Antarctic krill hydrolyzate by adding calcium chloride[J].Science and Technology of Food Industry,2011,3:330-333
[15]倪玲,吴文惠,张启,等.蛋白水解酶联合自溶作用的南极磷虾多肽氨基酸分析[J].食品工业科技,2013,34(8):102-107NI Ling,WU Wen-hui,ZHANG Qi,et al.Amino acid analysis of Antarctic krill peptide obtained by protease related autolysis peptide[J].Science and Technology of Food Industry,2013,34(8):102-107
[16]张恒,邬应龙.鸡骨的酶解及酶解液的电泳分析[J].食品与发酵工业,2010,36(9):102-105ZHANG Heng,WU Ying-long.Study on the hydrolysis of chicken bone and electrophoretic analysis of the hydrolysate[J].Food and Fermentation Industries,2010,36(9):102-105
[17]GB5009.235-2016,食品中氨基酸态氮的测定[S]GB5009.235-2016,Determination of Amino Acid Nitrogen in Food[S]
[18]高颖,王彦超,常耀光,等.三种低氟南极磷虾肽的ACE抑制作用及抗氧化活性研究[J].食品工业科技,2016,37(2):82-87,92GAO Ying,WANG Yan-chao,CHANG Yao-guang,et al.ACE inhibition and antioxidant activities of three types of Antarctic krill(Euphausia superba)peptides with low fluorine content[J].Science and Technology of Food Industry,2016,37(2):82-87,92
[19]陆彩玲,唐深,刘楠楠,等.海马组织蛋白不同染色方法的比较[J].环境与职业医学,2013,30(1):26-28LU Cai-ling,TANG Shen,LIU Nan-nan,et al.Comparison of different staining methods for hippocampal proteomic analysis[J].Journal of Environmental&Occupational Medicine,2013,30(1):26-28
[20]黄薇,宋永康,余华,等.酶法制备玉米抗氧化肽[J].中国食品学报,2014,14(8):69-76HUANG Wei,SONG Yong-kang,YU Hua,et al.Enzymatic preparation of corn anti-oxidation peptide[J].Journal of Chinese Institute of Food Science and Technology,2014,14(8):69-76
[21]林燕,陈计峦,胡小松,等.酶解核桃蛋白制备抗氧化肽的研究[J].食品工业科技,2011,32(4):204-207,212LING Yan,CHEN Ji-luan,HU Xiao-song,et al.Preparation of antioxidant peptide from walnut protein by enzymatic hydrolysis[J].Science and Technology of Food Industry,2011,32(4):204-207,212
[22]王璋,许时英.食品化学[M].北京:中国轻工业出版社,2002WANG Zhang,XU Shi-ying.Food Chemistry[M].Beijing:China Light Industry Press,2002
[23]Montgomery D C.Design and Analysis of Experiments[M].London:John Wiley and Sons Inc,1991