超声预处理对金枪鱼皮胶原ACE抑制肽消化稳定性的影响及消化产物的分离纯化、鉴定
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摘要
以金枪鱼皮为原料,超声预处理后酶解制得高血管紧张素转化酶(Angiotensin converting enzyme,ACE)抑制肽,研究ACE抑制肽的模拟胃肠道消化稳定性,并对消化产物进行分离纯化和鉴定。结果表明,超声处理后不同酶解时间酶解液的ACE抑制活性均有所提高;超声预处理20、40 min后,酶解5 min酶解液的消化产物的ACE抑制活性显著高于其他组(p <0.05)。采用葡聚糖凝胶G-15对E5U20和E5U40进行分离纯化,两组酶解液均可分离为3个组分,其中E5U20组组分2的IC_(50)值最小为0.393 mg/mL。采用半制备高效液相色谱对E5U20组分2进行分离纯化,得到8个组分,其中峰3的IC_(50)值最小为0.102 mg/mL。通过质谱法对峰3进行鉴定,测得峰3序列为GPSGPPGP,来源于α1肽链第842～849的位置,符合高ACE抑制活性的多肽构效特点,目前尚无该氨基酸序列的报道。
In this study,tuna skin was prepared by enzymatic hydrolysis after ultrasonic pretreatment to produce hydrolysate with ACE inhibitory activity.This hydrolysate was used as raw material,the gastrointestinal digestion in vitro of hydrolysate was tested.The results showed that all peptides from tuna skin keep high ACE inhibitory activity after digested. After 20 min and 40 min of ultrasonic pretreatment,the ACE inhibitory activity of digested products by enzymatic hydrolysis for 5 min was significantly higher than that of other groups( p < 0.05).E5U20 and E5U40 was separated into three components by Sephadex G-15,the peak F2 of E5U20 digestible products was best,of which IC_(50) value was 0.393 mg/mL.And then,the F2 was further separated by semi-high performance liquid chromatographic. There were 8 peaks after separated,peak 3 had a lowest IC_(50) of 0.102 mg/mL. Finally,the peak 3 was identified by mass spectrometry,the results showed that peak 3 peptide sequence was GPSGPPGP,this sequence was derived from 842 ～ 849 of collagen α1 chain. And these amino acids were consistent with the structure-activity characteristics of ACE inhibitory peptides,and the sequences were not reported by now.
In this study,tuna skin was prepared by enzymatic hydrolysis after ultrasonic pretreatment to produce hydrolysate with ACE inhibitory activity.This hydrolysate was used as raw material,the gastrointestinal digestion in vitro of hydrolysate was tested.The results showed that all peptides from tuna skin keep high ACE inhibitory activity after digested. After 20 min and 40 min of ultrasonic pretreatment,the ACE inhibitory activity of digested products by enzymatic hydrolysis for 5 min was significantly higher than that of other groups( p < 0.05).E5U20 and E5U40 was separated into three components by Sephadex G-15,the peak F2 of E5U20 digestible products was best,of which IC_(50) value was 0.393 mg/mL.And then,the F2 was further separated by semi-high performance liquid chromatographic. There were 8 peaks after separated,peak 3 had a lowest IC_(50) of 0.102 mg/mL. Finally,the peak 3 was identified by mass spectrometry,the results showed that peak 3 peptide sequence was GPSGPPGP,this sequence was derived from 842 ～ 849 of collagen α1 chain. And these amino acids were consistent with the structure-activity characteristics of ACE inhibitory peptides,and the sequences were not reported by now.
引文
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[22]顾敏.青养蛋白抗氧化肽的分离纯化及胃肠道消化特性研究[D].广州:华南理工大学,2014.
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[24]王娟.酶法制备羊乳酪蛋白ACE抑制肽及分离纯化[D].西安:陕西科技大学,2014.
[25]刘轶,马良,张宇昊.预处理对胶原蛋白水解和血管紧张素转化酶抑制肽段释放的影响[J].食品科学,2014,35(21):21-25.
[26]贾俊强,马海乐,王振斌,等.降血压肽的构效关系研究[J].中国粮油学报,2009,24(5):110-114.
[27]Mizuno S,Nishimura S,Matsuura K,et al.Release of short and proline-rich antihypertensive peptides from casein hydrolysate with an Aspergillus oryzae protease[J].Journal of Dairy Science,2004,87(10):3183-3188.
[2]Rahman M S,Al-Saidi G S,Guizani N.Thermal characterisation of gelatin extracted from yellowfin tuna skin and commercial mammalian gelatin[J].Food Chemistry,2008,108(2):472-481.
[3]Cho S M,Gu Y S,Kim S B.Extracting optimization and physical properties of yellowfin tuna(Thunnus albacares)skin gelatin compared to mammalian gelatins[J].Food Hydrocolloids,2005,19(2):221-229.
[4]王国强,何力,贾鲁君,等.长鳍金枪鱼鱼皮胶原蛋白肽制备工艺的研究[J].食品研究与开发,2016,37(7):105-110.
[5]Woo J,Yu S,Cho S,et al.Extraction optimization and properties of collagen from yellowfin tuna(Thunnus albacares)dorsal skin[J].Food Hydrocolloids,2008,22(5):879-887.
[6]Gómez-Estaca J,Bravo L,Gómez-Guillén M C,et al.Antioxidant properties of tuna-skin and bovine-hide gelatin films induced by the addition of oregano and rosemary extracts[J].Food Chemistry,2009,112(1):18-25.
[7]Je J Y,Qian Z J,Byun H G,et al.Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis[J].Process Biochemistry,2007,42(5):840-846.
[8]Woo J W,Yu S J,Cho S M,et al.Extraction optimization and properties of collagen from yellowfin tuna(Thunnus albacares)dorsal skin[J].Food Hydrocolloids,2008,22(5):879-887.
[9]王金玲.基于水产品下脚料ACEIP生物法制备及其分离纯化、性质的研究[D].杭州:浙江大学,2008.
[10]刘轶,马良,张宇昊.胶原ACE抑制肽研究进展[J].食品科学,2013,34(13):350-355.
[11]于志鹏.蛋清源ACE抑制肽的结构鉴定及稳定性研究[D].长春:吉林大学,2011.
[12]刘培勇,李丁宁,刘亮,等.酸处理对酶法提取鲟鱼皮胶原多肽的影响[J].食品工业科技,2012,33(9):237-240.
[13]江勇,汪少芸,饶平凡.鲨鱼皮明胶水解肽的制备、分离纯化与抗氧化活性研究[J].中国食品学报,2012,12(03):28-33.
[14]任晓锋.扫频超声预处理对玉米醇溶蛋白特性及其酶法制备ACE抑制肽的影响[D].镇江:江苏大学,2014.
[15]Uluko H,Liu L,Li H,et al.Effect of power ultrasound pretreatment on peptidic profiles and angiotensin converting enzyme inhibition of milk protein concentrate hydrolysates[J].Journal of the Science of Food&Agriculture,2014,94(12):2420-2428.
[16]刘轶.预处理对酶解过程中胶原ACE抑制肽释放影响研究[D].重庆:西南大学,2015.
[17]黄丹丹,马良,韩霜,等.超声预处理影响金枪鱼皮胶原酶解工艺及机理初探[J].食品与发酵工业,2017,43(4):141-146.
[18]陈倩倩,佟立涛,钟葵,等.大米蛋白酶解物的ACE抑制活性研究[J].现代食品科技,2015(3):91-96.
[19]Sentandreu M A,ToldráF.A fluorescence-based protocol for quantifying angiotensin-converting enzyme activity.[J].Nature Protocols,2006,1(5):2423.
[20]黄丹丹,马良,蔡路昀,等.预处理对酶解过程中胶原ACE抑制肽释放的影响[J].现代食品科技,2016(7):85-90.
[21]薛海燕,薛丽欢,贺宝元,等.牛乳酪蛋白体外模拟消化液的ACE抑制活性及其肠道吸收[J].现代食品科技,2018(6).
[22]顾敏.青养蛋白抗氧化肽的分离纯化及胃肠道消化特性研究[D].广州:华南理工大学,2014.
[23]Li Guan-hong,Le Guo-wei,Shi Yong-hui,et al.Angiotensin I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects[J].Nutrition Research,2004,24(7):469-486
[24]王娟.酶法制备羊乳酪蛋白ACE抑制肽及分离纯化[D].西安:陕西科技大学,2014.
[25]刘轶,马良,张宇昊.预处理对胶原蛋白水解和血管紧张素转化酶抑制肽段释放的影响[J].食品科学,2014,35(21):21-25.
[26]贾俊强,马海乐,王振斌,等.降血压肽的构效关系研究[J].中国粮油学报,2009,24(5):110-114.
[27]Mizuno S,Nishimura S,Matsuura K,et al.Release of short and proline-rich antihypertensive peptides from casein hydrolysate with an Aspergillus oryzae protease[J].Journal of Dairy Science,2004,87(10):3183-3188.