酪蛋白胃肠模拟水解液活性检测及细胞吸收机制初步研究

薛海燕; 李珊; 杜枘宣; 韩波; 张颖

doi:10.13386/j.issn1002-0306.2017.18.003

酪蛋白胃肠模拟水解液活性检测及细胞吸收机制初步研究

陕西科技大学食品与生物工程学院

基金项目:

国家自然科学基金项目(31301405)；陕西省科技统筹计划项目(2013KTZB02-02-05(2))；陕西省教育厅专项项目(16JK1101)；

详细信息

作者简介:
薛海燕 (1979-) , 女, 博士, 副教授, 研究方向:食品免疫检测技术, E-mail:xuehaiyan@sust.edu.cn。;

中图分类号: TS201.21
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出版历程
- 收稿日期: 2017-02-27

The bioacitve detection and mechanism of cell-based absorptionon simulating gastrointestinal hydrolysis of casein

Institute of Food Biotechnology, Shaanxi University of Science and Technology

摘要

摘要: 目的:研究牛乳酪蛋白在胃肠模拟水解条件下水解物的生物活性,以及这些肽类在细胞水平的吸收机制,为蛋白质生理作用机制研究奠定基础。方法:以牛乳酪蛋白为原料,对其进行体外模拟胃肠消化实验,通过检测消化液的DPPH自由基清除率和抑菌圈大小,分析其抗氧化和抗菌活性;建立Caco-2单层细胞模型对水解液进行过膜吸收,RP-HPLC分析过膜前后消化液组成,研究吸收情况。结果:模拟胃和肠消化时间均在2 h时,牛乳酪蛋白消化液的DPPH自由基清除率达到最大,分别为46.40%和56.7%,并且此时对大肠杆菌和金黄色葡萄球菌抑菌性能最强;细胞吸收实验表明2 h胃模拟水解液肽氮生物利用度达到7.79%,2 h肠模拟水解液达到15.11%,之后基本持平;亲水性的多肽能被成功吸收进入单细胞层下侧,但疏水性多肽大部分不易通过,易被降解或阻止。结论:牛乳酪蛋白在模拟消化条件下能产生生物活性肽类,并能被肠细胞吸收。
- 酪蛋白 /
- 胃肠模拟消化 /
- 抗氧化活性 /
- Caco-2细胞
Abstract: Objective: To study on bioative detetion and cell-based absorption mechanisms of gastrointestinal hydrolysis derived from bovine casein. It can further elucidate physiological mechanisms of food protein to humanbeing.Methods: Bovine milk casein was hydrolyzed in vitro simulating gastrointestinal digestion.The hydrolysate's antioxidant and antimicrobial activity were detected through the DPPH free radical clearance rate analysis and the bacteriostatic ring size determination. In addation, Caco-2 monolayer cell model was optimizated and established to simulate the membrane absorption of hydrolysate. RP-HPLC was used to analyze the digestive juices before and after across the membrane.Results: It showed the DPPH free radical clearance rates reached maximum, 46.40% of 2 h-Gastricand 56.7% of 2h-Gastroin-testinal digestions from bovine casein.At the same time, the inhibition activity on Escherichia coli and Staphylococcus aureus also achieve maximum. Experiment results showed that hydrolyzed 2 h stomach simulation peptide absorption nitrogen bioavailability of 7.79%, 2 h hydrolyzed imtestines simulation of 15.11%, after the flat.The peptide can be successfully absorb water into single cell layer under the side, but most hydrophobic peptide by not easily, easy degradation or stop.Conclusion: The gastrointestinal hydrolysate of casein can produce bioactive peptides as well as absorb by intestinal cell monolayer.
- casein /
- gastrointestinal digestion /
- antioxidant activity /
- Caco-2 cells

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参考文献(25)

[1]	Hogan S, Lei Z, Li J, et al.Development of antioxidant rich peptides from milk protein by microbial proteases and analysis of their effects on lipid peroxidation in cooked beef[J].Food Chemistry, 2009, 117 (3) :438-443.
[2]	Meisel H.Bioactive peptides from milk protein:a perspective for consumers and producers[J].Austr J Dairy Technol, 2001 (56) :83-91.
[3]	宫霞, 凌庆芝.乳酪蛋白源抗高血压活性肽的制备及其生理活性[J].上海交通大学学报, 2009, 27 (1) :53-56.
[4]	Plank J, Andres P, Krause I, et al.Gram scale separation of casein proteins from whole casein on a Source 30Q anionexchange resin column utilizing fast protein liquid chromatography (FPLC) [J].Protein Expression&Purification, 2008, 60 (2) :176-181.
[5]	Silva S V, Malcata F X.Caseins as source of bioactive peptides[J].International Dairy Journal, 2005, 15 (1) :1-15.
[6]	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) :375-391.
[7]	Maruyama S, Mitachi H, Tanaka H, et al.Studies on the active site and antihypertensive activity of angiotensin I-converting enzyme inhibitors derived from casein[J].Agricultural and Biological Chemistry, 1987, 51 (6) :1581-1586.
[8]	徐鑫, 何佳易, 刘国艳, 等.响应曲面法优化胰蛋白酶酶解条件制备乳源免疫活性肽[J].食品科学, 2011, 32 (19) :174-179.
[9]	刘珊珊, 敖静, 谢宁宁, 等.酪蛋白抗氧化肽的胃肠消化稳定性研究[J].中国食品学报, 2014, 14 (2) :47-53.
[10]	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) :601-605.
[11]	关溯, 陈孝, 黄民.Caco-2细胞模型-药物吸收研究的有效“工具”[J].中国药理学通报, 2004, 20 (6) :609-614.
[12]	丁龙.蛋清源ACE抑制肽的结构与完整吸收关系研究[D].长春:吉林大学, 2015.
[13]	国家药典委员会.中华人民共和国药典[M].第二版.北京, 中国医药科技出版社, 2005:158.
[14]	Guo H, Kouzuma Y, Yonekura M.Structures and properties of antioxidative peptides derived from royal jelly protein.[J].Food Chemistry, 2009, 113 (1) :238-245.
[15]	谢强, 林玉桓, 苗淑萍, 等.香芹酚对大肠杆菌和金黄色葡萄球菌细胞膜的影响[J].食品工业科技, 2014, 35 (23) :54-58.
[16]	Gianluca Picariello, Giuseppe Iacomino, Gianfranco Mamone, et al.Transport across Caco-2 monolayers of peptides arising from in vitro digestion of bovine milk proteins[J].Food Chemistry, 2013 (139) :203-212.
[17]	Hosoya K-I, Kim K-J, Lee V H.Age-dependent expression of P-glycoprotein gp17O in Caco-2 cell monolayers[J].Pharmaceutical Research, 1996, 13 (6) :885-890.
[18]	尤青, 马增春, 谭洪玲, 等.人参水提物在Caco-2模型的吸收特征[J].中国药理学通报, 2013, 29 (12) :1711-1716.
[19]	Ranaldi G, Conaalvo R, Sambuy Y, et al.Permeability characterististics of parental and clonal human intestinal Caco-2cell lines differentiated in serum supplemented and serum-free media[J].Toxicol In Vitro, 2003, 17 (5-6) :76-767.
[20]	鲁健章, 孙丽华, 周彦钢.凯氏定氮法测定鱼蛋白质含量的干扰因素分析[J].食品科学, 2010, 31 (19) :453-456.
[21]	Samaranayaka A G, Kitts D D, Li-Chan E C.Antioxidative and angiotensin-I-converting enzyme inhibitory potential of a Pacific Hake (Merluccius productus) fish protein hydrolysate subjected to simulated gastrointestinal digestion and Caco-2 cell permeation[J].Journal of Agricultural&Food Chemistry, 2010, 58 (3) :1535-1542.
[22]	Ningning Xie, Bo Wang, Liangping Jiang, et al.Hydrophobicity exerts different effects on bioavailability and stability of antioxidant peptide fractions from casein during simulated gastrointestinal digestion and Caco-2 cell absorption[J].Food Research International, 2015 (76) :518-526.
[23]	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, 2012.
[24]	莫李立, 王素军, 杨本坤.阿魏酸在Caco-2细胞模型的通透性及其在大鼠体内吸收特性研究[J].中草药, 2012, 43 (5) :947-951.
[25]	Parrot S, Degraeve P, Curia C, et al.In vitro study on digestion of peptides in Emmental cheese:analytical evaluation and influence on angiotensin I converting enzyme inhibitory peptides[J].Die Nahrung, 2003, 47 (2) :87.