克氏原螯虾虾头模拟胃肠道消化产物中ACE抑制肽的分离纯化与鉴定
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摘要
为了从克氏原螯虾虾头模拟胃肠道消化产物中制备血管紧张素转化酶(ACE)抑制肽,以克氏原螯虾虾头为原料,采用体外模拟肠胃道消化的方法对其进行酶解,以体外ACE抑制率为指标,通过超滤、凝胶色谱、离子色谱、反向高效液相色谱相结合的方法对克氏原螯虾虾头模拟胃肠道消化产物进行分离纯化与筛选,以期获得纯度较高的ACE抑制肽,并利用电喷雾静电场离子阱串联质谱对其氨基酸序列进行测定,结果表明,克氏原螯虾虾头模拟胃肠道消化产物中ACE抑制肽主要分布在分子质量低于3 000 u的超滤组分中;经过分离纯化鉴定得到1个新型ACE抑制肽,其分子质量为225 u,肽序列为Pro-Val,半数抑制浓度IC50值为0. 11 mg/m L,与模拟消化产物相比,ACE抑制肽的IC50值纯化了16. 72倍。
The present study aimed to isolate angiotensin converting enzyme( ACE) inhibitory peptides from simulated gastrointestinal digestion products of crayfish( Procambarus clarkia) head,and its amino acid sequence was identified. Procambarus clarkia head was hydrolyzed using simulated gastrointestinal digestion system,and the ACE inhibitory activities of the hydrolysates in vitro were measured. To obtain highly purified ACE inhibitory peptides,simulated gastrointestinal digestion products were separated by ultrafiltration,gel chromatography,ion-exchange chromatography,and reversed phase high-performance liquid chromatography( RP-HPLC). The results showed that ACE inhibitory peptides in simulated gastrointestinal digestion products of crayfish head were mainly distributed in ultrafiltration components with molecular weight below 3000 u. One novel ACE inhibitory peptide was isolated with a molecular weight of 225 u,and its peptide sequence was Pro-Val. The inhibitory activity of ACE inhibitory peptide,compared with simulated digestion products,almost increased by 16. 72 times( IC50= 0. 11 mg/m L).
The present study aimed to isolate angiotensin converting enzyme( ACE) inhibitory peptides from simulated gastrointestinal digestion products of crayfish( Procambarus clarkia) head,and its amino acid sequence was identified. Procambarus clarkia head was hydrolyzed using simulated gastrointestinal digestion system,and the ACE inhibitory activities of the hydrolysates in vitro were measured. To obtain highly purified ACE inhibitory peptides,simulated gastrointestinal digestion products were separated by ultrafiltration,gel chromatography,ion-exchange chromatography,and reversed phase high-performance liquid chromatography( RP-HPLC). The results showed that ACE inhibitory peptides in simulated gastrointestinal digestion products of crayfish head were mainly distributed in ultrafiltration components with molecular weight below 3000 u. One novel ACE inhibitory peptide was isolated with a molecular weight of 225 u,and its peptide sequence was Pro-Val. The inhibitory activity of ACE inhibitory peptide,compared with simulated digestion products,almost increased by 16. 72 times( IC50= 0. 11 mg/m L).
引文
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[27] KLEEKAYAI T,HARNEDY P A,O'KEEFFE M B,et al. Extraction of antioxidant and ACE inhibitory peptides from Thai traditional fermented shrimp pastes[J]. Food Chemistry,2015,176:441-447.
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[29] SUETSUNA K. Inhibitory activity against angiotensin Iconverting enzyme of peptides originating from fish and shellfish muscle[J]. Nippon Suisan Gakkaishi,1988,54(10):1 853-1 853.
[30] KIM S R,BYUN H G. The novel angiotensin I converting enzyme inhibitory peptide from rainbow trout muscle hydrolysate[J]. Fisheries and Aquatic Sciences,2012,15(3):183-190.
[31]杨湘华,李江.食源性降血压肽构效关系与制备研究进展[J].食品研究与开发,2016,37(3):217-220.
[2]管骁,彭剑秋,金周筠,等.食源性血管紧张素转化酶抑制肽的研究进展[J].食品与发酵工业,2011,37(7):135-141.
[3] ONDETTI M,RUBIN B,CUSHMAN D. Design of specific inhibitors of angiotensin converting enzyme:New class of orally active antihypertensive agents[J]. Science,1977,196(4288):441-444.
[4] BOUGATEF A,NEDJAR-ARROUME N,RAVALLEC-PL R,et al. Angiotensin I-converting enzyme(ACE)inhibitory activities of sardinelle(Sardinella aurita)by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases[J]. Food Chemistry,2008,111(2):350-356.
[5] ERDMANN K,CHEUNG B W,SCHRDER H. The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease[J]. The Journal of nutritional biochemistry,2008,19(10):643-654.
[6] CUSHMAN D W,SON CHEUNG M H,SABO E F,et al.Development and design of specific inhibitors of angiotensin-converting enzyme[J]. American Journal of Cardiology,1982,49(6):1 390-1 394.
[7] GHASSEM M,ARIHARA K,BABJI A S,et al. Purification and identification of ACE inhibitory peptides from Haruan(Channa striatus)myofibrillar protein hydrolysate using HPLC-ESI-TOF MS/MS[J]. Food Chemistry,2011,129(4):1 770-1 777.
[8]王晓杰,丛万锁,刘晓兰,等.玉米ACE抑制肽的制备工艺及中试生产[J].食品与发酵工业,2016,42(7):158-164.
[9]毛跟年,周亚丽,贺磊,等.魔芋ACE抑制肽的分离纯化及活性检测[J].食品与发酵工业,2017,43(6):163-168.
[10]王金玲,何国庆.酶解法制备带鱼脊骨降血压肽及其降血压效果[J].食品与发酵工业,2010,36(4):54-58.
[11]刘建华,郑燕,田颖刚,等.泰和乌骨鸡肉模拟胃肠道消化液中ACE抑制肽的分离纯化及结构鉴定[J].南昌大学学报(理科版),2011,35(2):163-168.
[12]王晶晶,吉薇,吉宏武,等.远东拟沙丁鱼酶解产物的降血压效果评价及其ACE抑制肽的分离纯化与鉴定[J].食品与发酵工业,2016,42(4):63-68.
[13]苗欣宇,王祖浩,王鹏,等.红松仁清蛋白ACE抑制肽的分离纯化与结构鉴定[J].食品科学,2017,38(5):129-133.
[14]孔一颖,廖静.我国成为世界最大的小龙虾生产国[J].海洋与渔业,2017(9):30-31.
[15]王蕊.克氏原螯虾的营养保健功能及相关食品的研究与开发[J].水产科技情报,2008,35(1):24-27.
[16]陆剑锋,赖年悦,成永旭.淡水小龙虾资源的综合利用及其开发价值[J].农产品加工(学刊),2006(10):47-52.
[17]赵利,李婷,汪清,等.克氏原螯虾蛋白肽的制备及其抗氧化性的研究[J].中国调味品,2017,42(6):22-28.
[18]汪清,李婷,陈丽丽,等.克氏原螯虾蛋白肽及其美拉德反应产物抗氧化活性的研究[J].江西水产科技,2017(2):3-8.
[19]时丽霞.酶法制备克氏原螯虾活性肽及其活性初步研究[D].南京:南京农业大学,2014:12-17.
[20]胡川.酶法水解小龙虾虾壳制备小分子生物活性肽研究[D].武汉:湖北工业大学,2016:5-15.
[21] JOHNS P W,CHENG L,DOWLATI L. Analytical method for the determination of infant formula protein digestibility in vitro:US7393659[P]. 2008.
[22]朱国萍,章超桦,曹文红,等.虾头自溶产物中ACE抑制肽的分离鉴定[J].水产学报,2013,37(4):631-640.
[23] NILSANG S,LERTSIRI S,SUPHANTHARIKA M,et al. Optimization of enzymatic hydrolysis of fish soluble concentrate by commercial proteases[J]. Journal of Food Engineering,2005,70(4):571-578.
[24]陈小艺,柯丽娟,方婷,等.鲍鱼内脏胶原ACE抑制肽纯化及结构初探[J].食品科技,2017,42(4):212-217.
[25] SHARMA S,SINGH R,RANA S. Bioactive peptides:A review[J]. International Journal Bioautomation,2011,15(4):223-250.
[26] NATESH R,SCHWAGER S L,STURROCK E D,et al.Crystal structure of the human angiotensin-converting enzyme-lisinopril complex[J]. Nature,2003,421(6922):551-554.
[27] KLEEKAYAI T,HARNEDY P A,O'KEEFFE M B,et al. Extraction of antioxidant and ACE inhibitory peptides from Thai traditional fermented shrimp pastes[J]. Food Chemistry,2015,176:441-447.
[28] YAMADA A,SAKURAI T,OCHI D,et al. Novel angiotensin I-converting enzyme inhibitory peptide derived from bovine casein[J]. Food chemistry,2013,141(4):3 781-3 789.
[29] SUETSUNA K. Inhibitory activity against angiotensin Iconverting enzyme of peptides originating from fish and shellfish muscle[J]. Nippon Suisan Gakkaishi,1988,54(10):1 853-1 853.
[30] KIM S R,BYUN H G. The novel angiotensin I converting enzyme inhibitory peptide from rainbow trout muscle hydrolysate[J]. Fisheries and Aquatic Sciences,2012,15(3):183-190.
[31]杨湘华,李江.食源性降血压肽构效关系与制备研究进展[J].食品研究与开发,2016,37(3):217-220.