牛乳酪蛋白体外模拟消化液的ACE抑制活性及其肠道吸收
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摘要
本文对牛乳酪蛋白进行体外模拟消化,以ACE抑制活性为指征,LC-MS/MS确定水解物肽谱,并采用大鼠肠道模型对其吸收进行初步研究。结果为人工胃液消化、人工肠液消化、胃肠联合消化三种消化方式的水解度均随时间的延长而增大,单胃和单肠消化的ACE抑制率均随水解时间的延长先快速增加,随后逐渐降低,而胃肠联合的ACE抑制率则是先降低再增加,胃肠联合消化产生水解度最高为25.51%,其ACE抑制率在2 h时达到最高值为68.03%;LC-MS/MS测定消化液的肽谱,分析得出模拟消化后可能产生的ACE抑制肽有IPP、RYLGY、LHLPLP、AYFYPEL、RPKHPIKHQ及WQVLPNAVPAK;使用FITC标记酪蛋白进行体外模拟消化,SDS-PAGE和Tricine-SDS-PAGE都表明FITC-酪蛋白经过胃肠消化后荧光标记稳定存在,且水解物的分子量在5 ku以下;大鼠肠道吸收模型发现标记后的牛乳酪蛋白经模拟消化后在肠道吸收率大小依次为十二指肠吸收>空肠吸收>回肠吸收>结肠吸收,主要在十二指肠吸收。
Milk casein was digested by in vitro simulated gastro-intestinal fluid, and ACE inhibitory activity was used as an indicator in this study. Peptide spectrum of hydrolyzate was determined by LC-MS/MS, and its absorption was studied by intestinal model in rats. The results showed that the digestibility of artificial gastric juice digestion, artificial intestinal juice digestion and the combination of gastrointestinal juice increased with time, the ACE inhibition rates of monogastric and single intestinal digestion increased rapidly with the prolongation of hydrolysis time, followed by a gradual decrease, while the combination of gastric and intestinal digestion ACE inhibitory rate decreased first and then increased. The highest degree of hydrolysis of gastrointestinal combined digestion was 25.51%, and the ACE inhibition rate reached the highest value of 68.03% at 2 h; The peptide profiles of digestive juice were determined by LC-MS/MS. The results showed that ACE inhibitory peptides(including IPP, RYLGY, LHLPLP, AYFYPEL, RPKHPIKHQ and WQVLPNAVPAK) could be produced after simulated digestion. In vitro simulated digestion was performed using FITC labeled casein, SDS-PAGE and Tricine-PAGE showed that the fluorescent labeling FITC-casein was stable after the gastrointestinal digestion, and the molecular weight of the hydrolyzate was under 5 k Da. The model of intestinal absorption in the rat showed that the intestinal absorptivity order of the labeled casein in the intestinal tract after digestion was as follows: duodenum absorption > jejunal absorption > ileal absorption > colon absorption, mainly absorbed in the duodenum.
Milk casein was digested by in vitro simulated gastro-intestinal fluid, and ACE inhibitory activity was used as an indicator in this study. Peptide spectrum of hydrolyzate was determined by LC-MS/MS, and its absorption was studied by intestinal model in rats. The results showed that the digestibility of artificial gastric juice digestion, artificial intestinal juice digestion and the combination of gastrointestinal juice increased with time, the ACE inhibition rates of monogastric and single intestinal digestion increased rapidly with the prolongation of hydrolysis time, followed by a gradual decrease, while the combination of gastric and intestinal digestion ACE inhibitory rate decreased first and then increased. The highest degree of hydrolysis of gastrointestinal combined digestion was 25.51%, and the ACE inhibition rate reached the highest value of 68.03% at 2 h; The peptide profiles of digestive juice were determined by LC-MS/MS. The results showed that ACE inhibitory peptides(including IPP, RYLGY, LHLPLP, AYFYPEL, RPKHPIKHQ and WQVLPNAVPAK) could be produced after simulated digestion. In vitro simulated digestion was performed using FITC labeled casein, SDS-PAGE and Tricine-PAGE showed that the fluorescent labeling FITC-casein was stable after the gastrointestinal digestion, and the molecular weight of the hydrolyzate was under 5 k Da. The model of intestinal absorption in the rat showed that the intestinal absorptivity order of the labeled casein in the intestinal tract after digestion was as follows: duodenum absorption > jejunal absorption > ileal absorption > colon absorption, mainly absorbed in the duodenum.
引文
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[16]武慧超,陆洋,杜守颖,等.新旧工艺仙灵骨葆胶囊中淫羊藿大鼠肠吸收特性对比研究[J].中国中药杂志,2011,36(19):2648-2652WU Hui-chao,LU Yang,DU Shou-ying,et al.Comparative study on absorption kinetics in intestines of rats of Epimedii Folium of Xianlinggubao capsules prepared by different processes[J].China Journal of Chinese Materia Medica,2011,36(19):2648-2652
[17]吴军,翁春梅,刘晓东,等.外翻肠囊法研究灵仙新苷肠吸收动力学[J].中国新药杂志,2014,23(5):601-605WU Jun,WENG Chun-mei,LIU Xiao-dong,et al.Intestinal absorption of clematichinenoside AR in rat everted gut sacs[J].Chinese Journal of New Drugs,2014,23(5):601-605
[18]庞广昌,陈庆森,胡志和,等.蛋白质的消化吸收及其功能评述[J].食品科学,2013,34(9):375-391PANG Guang-chang,CHEN Qing-sen,HU Zhi-he,et al.Bioactive peptides:absorption,utilization and functionality[J].Food Science,2013,34(9):375-391
[19]贾俊强.超声对酶法制备小麦胚芽ACE抑制肽的影响及其作用机理[D].镇江:江苏大学,2009JIA Jun-qiang.Effect and mechanism of ultrasound on enzymatic preparation of ACE-inhibitory peptides from wheat germ protein[D].Zhenjiang:Jiangsu University,2009
[20]李东平.酶解水牛乳蛋白制备降血压肽的研究[D].南宁:广西大学,2012LI Dong-ping.Study on preparing ACE inhibitory peptides from buffalo milk protein with enzymatic hydrolysis[D].Nanning:Guangxi University,2012
[21]祝倩,郭宇星,潘道东.利用Caco-2细胞模型评价乳源ACE抑制肽小肠吸收机制的研究进展[J].食品科学,2013,34(9):330-335ZHU Qian,GUO Yu-xing,PAN Dao-dong.Progress in research on intestinal absorption mechanism of Milk-derived ACE inhibitory peptides using Caco-2 cell model[J].Food Science,2013,34(9):330-335
[22]刘文涛,于盼盼,李泽鸿.抗狂犬病毒单克隆抗体的纯化及标记[J].中国兽药杂志,2011,45(4):5-8LIU Wen-tao,YU Pan-pan,LI Ze-hong.Purification and application of the rabies viral monoclonal antibody[J].Chinese Journal of Veterinary Drug,2011,45(4):5-8
[23]曹颖,李永吉,吕邵娃.大鼠在体肠灌流模型在中药研究中的应用[J].中医药学报,2010,38(3):134-136CAO Ying,LI Yong-ji,LV Shao-wa.Application of rat intestinal perfusion model in traditional chinese medicine research[J].Acta Chinese Medicine and Pharmacology,2010,38(3):134-136
[24]Maclean C,Moenning U,Reichel A,et al.Closing the gaps:a full scan of the intestinal expression of p-glycoprotein,breast cancer resistance protein,and multidrug resistance-associated protein 2 in male and female rats[J].Drug Metabolism and Disposition,2008,36(7):1249-1254
[25]张倩.中药大分子活性成分示踪及鹿茸蛋白的示范性研究[D].长春:吉林大学,2012ZHANG Qian.The establishment of active compound tracing method and its application in the research of pilose antler protein[D].Changchun:Jilin University,2012
[26]Fagerholm U,Johansson M,Lennern?s H.Comparison between permeability coefficients in rat and human jejunum[J].Pharmaceutical Research,1996,13(9):1336-1342
[2]Silva S V,Malcata F X.Caseins as source of bioactive peptides[J].International Dairy Journal,2005,15(1):1-15
[3]赵肖蓉.牛乳酪蛋白降血压肽的制备及活性研究[D].大连:大连工业大学,2011ZHAO Xiao-rong.Study on the preparation and activity on antihypertensive peptide from casein protein[D].Dalian:Dalian Polytechnic University,2011
[4]张艳,胡志和,闫星,等.胃蛋白酶水解酪蛋白制备ACE抑制肽的条件[J].食品科学,2010,31(14):42-46ZHANG Yan,HU Zhi-he,YAN Xing,et al.Pepsin hydrolysis of casein for the preparation of ACE inhibito peptides[J].Food Science,2010,31(14):42-46
[5]夏镇波.酪蛋白源血管紧张素转换酶抑制肽制备及分离纯化研究[D].南京:南京农业大学,2008XIA Zhen-bo.Researce on production and purification of angiotensin converting enzyme inhibitory peptides from casein[D].Nanjing:Nanjing Agricultural University,2008
[6]Maruyama S,Mitachi H,Tanaka H,et al.Studies on the active site and antihypertensive activity of angiotensin Iconverting enzyme inhibitors derived from casein[J].Agricultural and Biological Chemistry,1987,51(6):1581-1586
[7]Annamaria Perna,Amalia Simonetti,Emilio Gambacorta.Short communication:Effect of casein haplotype on angiotensin-converting enzyme inhibitory and antioxidant capacities of milk casein from Italian Holstein cows before and following in vitro digestion with gastrointestinal enzymes[J].Journal of Dairy Science,2016,99(9):6922-6926
[8]国家药典委员会.中华人民共和国药典(第二版)[M].北京:中国医药科技出版社,2005National Pharmacopoeia Commission.Chinese Pharmacopoeia(Second Edition)[M].Beijing:China Medical Science and Technology Press,2005
[9]顾浩峰,张富新,张怡.羊奶婴儿配方奶粉中蛋白质体外模拟消化研究[J].食品科学,2013,34(19):302-305GU Hao-feng,ZHANG Fu-xin,ZHANG Yi.In vitro digestion of proteins in goat milk formula[J].Food Science,2013,34(19):302-305
[10]杨文博,张英华.蛋白质水解度的测定方法研究[J].中国调味品,2014,39(3):88-90YANG Wen-bo,ZHANG Ying-hua.Study on the determination methods for hydrolysis degree of protein[J].China Condiment,2014,39(3):88-90
[11]谢瑛.生物大分子药物的标记及其药代动力学研究[D].上海:第二军医大学,2010XIE Ying.Label of biomacromolecular drugs and their pharmacokinetic studies[D].Shanghai:The Second Military Medical University,2010
[12]张歌.牛乳和羊乳蛋白质差异比较及检测方法的研究[D].西安:陕西科技大学,2013ZHANG Ge.Detection methods and comparison on protein difference of cow and goat milk[D].Xi’an:Shaanxi University of Science and Technology,2013
[13]张锐昌,王绮,张应龙,等.Tricine-SDS-PAGE测定小麦蛋白酶解物分子量分布[J].食品研究与开发,2012,33(12):168-171ZHANG Rui-chang,WANG Qi,ZHANG Ying-long,et al.Study on molecular weight distribution of wheat protein enzymolysate by Tricine-SDS-PAGE[J].Food Research and Development,2012,33(12):168-171
[14]孙波,迟玉杰,徐宁,等.Tricine-SDS-PAGE电泳检测蛋清肽分子量的研究[J].食品科学,2008,29(5):385-388SUN Bo,CHI Yu-jie,XU Ning,et al.Study on tricine-SDS-PAGE method detecting albumen polypeptides molecular weight[J].Food Science,2008,29(5):385-388
[15]董宇,张英丰,杨庆,等.黄连提取物在大鼠肠外翻实验中的吸收研究[J].中国中药杂志,2008,33(9):1056-1060DONG Yu,ZHANG Ying-feng,YANG Qing,et al.Absorption of extractive Rhizoma Coptidis in rat everted gut scas[J].China Journal of Chinese Materia Medica,2008,33(9):1056-1060
[16]武慧超,陆洋,杜守颖,等.新旧工艺仙灵骨葆胶囊中淫羊藿大鼠肠吸收特性对比研究[J].中国中药杂志,2011,36(19):2648-2652WU Hui-chao,LU Yang,DU Shou-ying,et al.Comparative study on absorption kinetics in intestines of rats of Epimedii Folium of Xianlinggubao capsules prepared by different processes[J].China Journal of Chinese Materia Medica,2011,36(19):2648-2652
[17]吴军,翁春梅,刘晓东,等.外翻肠囊法研究灵仙新苷肠吸收动力学[J].中国新药杂志,2014,23(5):601-605WU Jun,WENG Chun-mei,LIU Xiao-dong,et al.Intestinal absorption of clematichinenoside AR in rat everted gut sacs[J].Chinese Journal of New Drugs,2014,23(5):601-605
[18]庞广昌,陈庆森,胡志和,等.蛋白质的消化吸收及其功能评述[J].食品科学,2013,34(9):375-391PANG Guang-chang,CHEN Qing-sen,HU Zhi-he,et al.Bioactive peptides:absorption,utilization and functionality[J].Food Science,2013,34(9):375-391
[19]贾俊强.超声对酶法制备小麦胚芽ACE抑制肽的影响及其作用机理[D].镇江:江苏大学,2009JIA Jun-qiang.Effect and mechanism of ultrasound on enzymatic preparation of ACE-inhibitory peptides from wheat germ protein[D].Zhenjiang:Jiangsu University,2009
[20]李东平.酶解水牛乳蛋白制备降血压肽的研究[D].南宁:广西大学,2012LI Dong-ping.Study on preparing ACE inhibitory peptides from buffalo milk protein with enzymatic hydrolysis[D].Nanning:Guangxi University,2012
[21]祝倩,郭宇星,潘道东.利用Caco-2细胞模型评价乳源ACE抑制肽小肠吸收机制的研究进展[J].食品科学,2013,34(9):330-335ZHU Qian,GUO Yu-xing,PAN Dao-dong.Progress in research on intestinal absorption mechanism of Milk-derived ACE inhibitory peptides using Caco-2 cell model[J].Food Science,2013,34(9):330-335
[22]刘文涛,于盼盼,李泽鸿.抗狂犬病毒单克隆抗体的纯化及标记[J].中国兽药杂志,2011,45(4):5-8LIU Wen-tao,YU Pan-pan,LI Ze-hong.Purification and application of the rabies viral monoclonal antibody[J].Chinese Journal of Veterinary Drug,2011,45(4):5-8
[23]曹颖,李永吉,吕邵娃.大鼠在体肠灌流模型在中药研究中的应用[J].中医药学报,2010,38(3):134-136CAO Ying,LI Yong-ji,LV Shao-wa.Application of rat intestinal perfusion model in traditional chinese medicine research[J].Acta Chinese Medicine and Pharmacology,2010,38(3):134-136
[24]Maclean C,Moenning U,Reichel A,et al.Closing the gaps:a full scan of the intestinal expression of p-glycoprotein,breast cancer resistance protein,and multidrug resistance-associated protein 2 in male and female rats[J].Drug Metabolism and Disposition,2008,36(7):1249-1254
[25]张倩.中药大分子活性成分示踪及鹿茸蛋白的示范性研究[D].长春:吉林大学,2012ZHANG Qian.The establishment of active compound tracing method and its application in the research of pilose antler protein[D].Changchun:Jilin University,2012
[26]Fagerholm U,Johansson M,Lennern?s H.Comparison between permeability coefficients in rat and human jejunum[J].Pharmaceutical Research,1996,13(9):1336-1342