酶解牦牛血发酵液制备抗氧化肽工艺优化
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摘要
为进一步提高牦牛血枯草芽胞杆菌发酵液多肽含量,对牦牛血发酵液进行酶解,以酶解液的·OH清除率、多肽含量为主要指标,筛选酶解发酵液的最适蛋白酶,通过单因素和响应面试验优化酶解工艺,并对比菌酶联合制备产物与发酵液体外抗氧化活性。结果表明,碱性蛋白酶酶解发酵液最佳条件为:酶解时间3 h、p H值9.5、酶解温度60℃和酶底比190 U·g-1,此条件下制备得到酶解液多肽含量为5.52mg·mL~(-1),较发酵液提高2.39倍。菌酶联合制备产物对·O2-及脂质过氧化的IC50分别为6.22、4.87mg·mL~(-1),发酵法制备产物对·O2-及脂质过氧化的IC50分别为8.42、11.71 mg·mL~(-1),且菌酶联合制备产物的还原力优于发酵制备产物。因此,菌酶联合制备牦牛血抗氧化肽法优于传统单一发酵法。本研究结果对提高牦牛血抗氧化肽得率,增加牦牛产品附加值具有重要的意义。
In order to increase the peptides yield of Yak blood fermented by Bacillus subtilis,broth was hydrolyzed with protease. The ·OH scavenging race and the yield of peptide were used as main indexes to select suitable protease. The enzymatic hydrolysis conditions were optimized by single factor test and response surface analysis. The results showed that the optimum conditions for alkaline protease were as follow: hydrolysis for 3 h,190 U·g- 1of enzyme / substrate ratio,p H 9. 5,60 ℃. Under such conditions,the yield of peptides was 5. 52 mg·mL~(-1),which was 2. 39 times higher than that prepared by fermentation only. The in vitro antioxidant activities of peptides prepared by different methods were compared. Results showed that the IC50 for O2-and lipid peroxidation of peptides prepared by the combination of bacteria and enzyme were 6. 22 mg·mL~(-1)and 4. 87 mg·mL~(-1),respectively. The IC50 for O2-and lipid peroxidation of peptides prepared by single fermentation were 8. 42 mg·mL~(-1)and 11. 71 mg·mL~(-1). And the reducing power of peptides prepared by the combination of bacteria and enzyme was also better than peptides prepared by single fermentation.Therefore,the combination of bacteria and enzyme for producing antioxidant peptides is an effective approach,which will be significant to improve the yield of Yak blood antioxidant peptides and increase the added value of Yak blood.
In order to increase the peptides yield of Yak blood fermented by Bacillus subtilis,broth was hydrolyzed with protease. The ·OH scavenging race and the yield of peptide were used as main indexes to select suitable protease. The enzymatic hydrolysis conditions were optimized by single factor test and response surface analysis. The results showed that the optimum conditions for alkaline protease were as follow: hydrolysis for 3 h,190 U·g- 1of enzyme / substrate ratio,p H 9. 5,60 ℃. Under such conditions,the yield of peptides was 5. 52 mg·mL~(-1),which was 2. 39 times higher than that prepared by fermentation only. The in vitro antioxidant activities of peptides prepared by different methods were compared. Results showed that the IC50 for O2-and lipid peroxidation of peptides prepared by the combination of bacteria and enzyme were 6. 22 mg·mL~(-1)and 4. 87 mg·mL~(-1),respectively. The IC50 for O2-and lipid peroxidation of peptides prepared by single fermentation were 8. 42 mg·mL~(-1)and 11. 71 mg·mL~(-1). And the reducing power of peptides prepared by the combination of bacteria and enzyme was also better than peptides prepared by single fermentation.Therefore,the combination of bacteria and enzyme for producing antioxidant peptides is an effective approach,which will be significant to improve the yield of Yak blood antioxidant peptides and increase the added value of Yak blood.
引文
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[18]高强,杨杨,孙增春,姜花,贾雪,袁菊莲,何成奇.鹿茸血酒对去势大鼠的升雌激素与抗氧化作用的初步研究[J].四川大学学报:医学版,2014,45(1):134-137
[19]布冠好,姬莉莉,王琳珍,张艺帆,李开南,李梵,赵晓丹.鹅血红蛋白抗氧化活性肽的制备及工艺研究[J].食品工业科技,2013,34(3):201-204
[20]高子涵,张卫兵,梁琪.牦牛血粉制备氯化血红素工艺优化研究[J].食品工业与科技,2013,34(19):248-251
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[22]庞晨,张珍,张盛贵,牛莉黎,肖侠,周林,张阳,王晶,魏子昊.FeC l3盐析法提取牦牛血中G型免疫球蛋白的工艺[J].甘肃农业大学学报,2014,2(1):26-30
[23]邱翔,张磊,文勇立,王建文,刘鲁蜀,马力,吴贤智,金洁茹.四川牦牛、黄牛主要品种肉的营养成分分析[J].食品科学,2010,31(15):112-116
[24]杜昕,肖岚,李诚,温馨,田甜,周恒量,宋丽坤,刘静,范尹译.枯草芽胞杆菌发酵牦牛血制备抗氧化肽工艺优化[J].食品机械,2016,32(3):165-168
[25]张凤英.猪血红蛋白的酶解及其产物抗氧化活性研究[D].雅安:四川农业大学,2009
[26]陈昌琳,李诚,付刚,赖利,陈茂林,肖岚,杨勇,何利,胡滨.超声波辅助酶解制备猪肩胛骨降血压肽的工艺及酶解效果比较[J].食品工业科技,2015,36(2):217-221,226
[27]张宇昊,王强.Alcalase酶水解花生蛋白制备花生短肽的研究[J].农业工程学报,2007,4(23):258-263
[28]O'Loughlin I B,Murray B A,Kelly P M,FitzG erald R J,Brodkorb A.Enzymatic hydrolysis of heat-induced aggregates of whey protein isolate[J].Journal of Agriculture Food Chemistry,2012,60(19):4895-4904
[29]黄明,王璐莎.动物蛋白源抗氧化肽的研究进展[J].中国农业科学,2013,46(22):4763-4773
[30]裴小平,唐道邦,肖更生,蒋爱民,杨万根.酶法制备鸡肉蛋白抗氧化肽工艺研究[J].食品与发酵工业,2009,11(135):104-109
[31]张志清,宋燕,姜太玲,吴红洋,刘翔.Alcalase蛋白酶水解花椒籽蛋白制备抗氧化肽的条件优化[J].食品工业科技,2014,35(11):179-187
[32]刘红梅,师广波,李向东,孙中涛.复合酶法水解花生粕制备抗氧化肽的工艺优化[J].食品科学技术学报,2014,32(3):59-64
[33]于丽娜,孙杰,刘少芬,朱凤,毕洁,张初署,杨庆利.花生抗氧化水解物制备及其抗氧化活性研究[J].核农学报,2013,27(2):188-196
[34]姚晓红,吴逸飞,王新,孙宏,钱彬,张博,汤江武.菌酶协同制备菜籽肽工艺的优化及其营养成分分析[J].饲料工业,2011,32(18):47-51
[35]刘洪对,于丽娜,高俊安,孙杰,朱凤,毕洁,张初署,刘少芬.五种花生抗氧化肽体外抗氧化活性比较[J].核农学报,2013,27(8):1162-1167
[36]Zhang H C,Yu L N,Yang Q L,Sun J,Bi J,Liu S F,Zhang C S,Tang L.Optimization of a microwave-coupled enzymatic digestion precess to prepare peanut peptides[J].Molecules,2012,17(5):5661-5674
[37]宋茹,冯婷立,谢超.海产小杂鱼抗氧化肽制备工艺[J].食品科学,2011,32(12):29-33
[2]Chalamaiah M,Dinesh K B,Hemalatha R,Jyothirmayi T.Fish protein hydrolysates:proximate composition,amino acid composition,antioxidant activities and applications:a review[J].Food Chemistry,2012,135(4):3020-3038
[3]Fan J,He J,Zhuang Y,Sun L.Purification and identification of antioxidant peptides from enzymatic hydrolysates of Tilapia(Oreochromis niloticus)frame protein[J].Molecules,2012,17(11):12836-12850
[4]Vo T S,Ngo D H,Kim J A,Kim S J.An antihypertensive peptide from tilapia gelatin diminishes free radical formation in murine microglial cells[J].Journal of Agriculture Food Chemistry,2011,59(22):12193-12197
[5]李振华,龚吉军,赵延华,唐静.抗氧化肽的研究进展[J].食品研究与开发,2011,32(6):157-161
[6]方俊,卢向阳,蒋红梅,莫瑾,李宗军,马美湖.制备低分子质量猪血肽发酵条件的优化研究[J].中国食品学报,2006,6(6):4-8
[7]万佳蓉,马美湖,周传云.多菌种混合发酵猪血的研究[J].食品与机械,2007,23(1):50-53
[8]马小元.猪血发酵菌株的筛选及其产蛋白酶特性的研究[J].食品研究与开发,2012,33(6):182-184
[9]Li S,Zhao Y,Zhang L,Zhang X,Huang L,Li D,Niu C H,Yang Z N,Wang Q,Yang Z N.Antioxidant activity of Lactobacillus plantarum strains isolated from traditional Chinese fermented foods[J].Food Chemistry,2012,135(3):1914-1919
[10]Qian B,Xing M,Cui L,Zhang S.Antioxidant,antihypertensive,and immunomodulatory activities of peptide fractions from fermented skim milk with Lactobacillus delbrueckii ssp.bulgaricus LB340[J].Journal of Dairy Research,2011,78(1):72-79
[11]Mohd A N,Mohd Y H,Long K,Yeap S K,Ho W Y,Beh S P,Abdullah M P,Alitheen N B.Antioxidant and hepatoprotective effect of aqueous extract of germinated and fermented mung bean on ethanol-mediated liver damage[J].Biomed Research International,2013,20(13):693613
[12]Mao X,Liu P,He S,Lin H.Antioxidant properties of bio-active substances from shrimp head fermented by Bacillus licheniformis OPL-007[J].Applied Biochemistry Biotechnology,2013,171(5):1240-1252
[13]杨洁芳,刘会平,张宇,袁伟.菌酶协同制备大豆肽的工艺优化及其抗氧化特性的研究[J].食品工业科技,2013,34(13):245-249
[14]孙宏.菌酶协同处理棉籽粕的营养特性、棉籽肽的制备及其抗氧化活性研究[D].杭州:浙江大学,2013
[15]方乐,葛向阳,汤江武,姚晓红,吴逸飞,孙宏.菌酶协同处理豆粕制备饲用小肽的研究[J].中国饲料,2011,1(5):17-20
[16]胡滨,陈一资,胡惠民,李诚,杨勇,何利,苏赵.猪血蛋白酶解物的抗氧化功能研究[J].核农学报,2014,28(9):1632-1640
[17]王加豪,贾睿,刘英娟,杜金梁,曹丽萍,殷国俊.猪血多肽对建鲤抗氧化能力和免疫功能的影响[J].动物营养学报,2014(12):3689-3697
[18]高强,杨杨,孙增春,姜花,贾雪,袁菊莲,何成奇.鹿茸血酒对去势大鼠的升雌激素与抗氧化作用的初步研究[J].四川大学学报:医学版,2014,45(1):134-137
[19]布冠好,姬莉莉,王琳珍,张艺帆,李开南,李梵,赵晓丹.鹅血红蛋白抗氧化活性肽的制备及工艺研究[J].食品工业科技,2013,34(3):201-204
[20]高子涵,张卫兵,梁琪.牦牛血粉制备氯化血红素工艺优化研究[J].食品工业与科技,2013,34(19):248-251
[21]刘振斌.牦牛血血红素提取工艺研究[D].兰州:甘肃农业大学,2012
[22]庞晨,张珍,张盛贵,牛莉黎,肖侠,周林,张阳,王晶,魏子昊.FeC l3盐析法提取牦牛血中G型免疫球蛋白的工艺[J].甘肃农业大学学报,2014,2(1):26-30
[23]邱翔,张磊,文勇立,王建文,刘鲁蜀,马力,吴贤智,金洁茹.四川牦牛、黄牛主要品种肉的营养成分分析[J].食品科学,2010,31(15):112-116
[24]杜昕,肖岚,李诚,温馨,田甜,周恒量,宋丽坤,刘静,范尹译.枯草芽胞杆菌发酵牦牛血制备抗氧化肽工艺优化[J].食品机械,2016,32(3):165-168
[25]张凤英.猪血红蛋白的酶解及其产物抗氧化活性研究[D].雅安:四川农业大学,2009
[26]陈昌琳,李诚,付刚,赖利,陈茂林,肖岚,杨勇,何利,胡滨.超声波辅助酶解制备猪肩胛骨降血压肽的工艺及酶解效果比较[J].食品工业科技,2015,36(2):217-221,226
[27]张宇昊,王强.Alcalase酶水解花生蛋白制备花生短肽的研究[J].农业工程学报,2007,4(23):258-263
[28]O'Loughlin I B,Murray B A,Kelly P M,FitzG erald R J,Brodkorb A.Enzymatic hydrolysis of heat-induced aggregates of whey protein isolate[J].Journal of Agriculture Food Chemistry,2012,60(19):4895-4904
[29]黄明,王璐莎.动物蛋白源抗氧化肽的研究进展[J].中国农业科学,2013,46(22):4763-4773
[30]裴小平,唐道邦,肖更生,蒋爱民,杨万根.酶法制备鸡肉蛋白抗氧化肽工艺研究[J].食品与发酵工业,2009,11(135):104-109
[31]张志清,宋燕,姜太玲,吴红洋,刘翔.Alcalase蛋白酶水解花椒籽蛋白制备抗氧化肽的条件优化[J].食品工业科技,2014,35(11):179-187
[32]刘红梅,师广波,李向东,孙中涛.复合酶法水解花生粕制备抗氧化肽的工艺优化[J].食品科学技术学报,2014,32(3):59-64
[33]于丽娜,孙杰,刘少芬,朱凤,毕洁,张初署,杨庆利.花生抗氧化水解物制备及其抗氧化活性研究[J].核农学报,2013,27(2):188-196
[34]姚晓红,吴逸飞,王新,孙宏,钱彬,张博,汤江武.菌酶协同制备菜籽肽工艺的优化及其营养成分分析[J].饲料工业,2011,32(18):47-51
[35]刘洪对,于丽娜,高俊安,孙杰,朱凤,毕洁,张初署,刘少芬.五种花生抗氧化肽体外抗氧化活性比较[J].核农学报,2013,27(8):1162-1167
[36]Zhang H C,Yu L N,Yang Q L,Sun J,Bi J,Liu S F,Zhang C S,Tang L.Optimization of a microwave-coupled enzymatic digestion precess to prepare peanut peptides[J].Molecules,2012,17(5):5661-5674
[37]宋茹,冯婷立,谢超.海产小杂鱼抗氧化肽制备工艺[J].食品科学,2011,32(12):29-33