超声预处理辅助酶解玉米胚芽ACE抑制肽的研究
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摘要
旨在研究不同工作模式的超声预处理对玉米胚芽蛋白酶解制备血管紧张素转换酶(Angiotensin-I Converting Enzyme,ACE)抑制肽的影响。以蛋白转化率和高活性肽占比为指标,利用聚能逆流双频、发散三频和对振双频的超声设备,对玉米胚芽粕进行预处理,得到最优的超声预处理模式;采用单因素逐级优化法来确定最佳超声预处理参数;在最优超声处理条件下,优化酶解反应条件。结果表明分子量在300~1000 Da的多肽ACE抑制活性最高,IC50值为0.78 mg/m L;最优的超声模式为20/40 k Hz交替双频,最佳超声预处理参数为功率密度100 W/L、底物浓度为8%、超声时间20 min、超声温度30℃,酶解条件为加酶量2000 U/g蛋白、酶解时间2.5 h。在最优条件下,蛋白转化率为85.00%,相比于未超声组的73.01%提高了16.42%;高活性肽占比为29.63%,相比于未超声组的26.00%提高了13.96%。因此,逆流双频超声波辅助酶解法能有效提高蛋白转化率和产物ACE抑制活性,有利于ACE抑制肽的制备。
This study aimed to research the effects of ultrasonic pretreatment of different working modes on the preparation of ACE-inhibitory peptides from corn germ meal.According to protein conversion and proportion of highly active peptides,dualfrequency countercurrent energy-gathered ultrasound,triple-frequency divergence ultrasound and dual-frequency relative vibration ultrasound were used to pretreat corn germ meal and the best ultrasonic mode was selected. The ultrasonic preprocessing parameters were determined by single-factor step optimization method. Under ideal ultrasonic pretreatment conditions,enzymatic reaction conditions were optimized.Results showed that the ACE inhabitary activity of peptides of 300 ~1000 Da was the highest,IC50 of which was 0.78 mg/m L; the most effective ultrasonic mode was 20/40 k Hz alternate dual-frequency and the optimum parameters of the ultrasonic pretreatment were the power density of 100 W/L,the substrate concentration of 8%,ultrasonic time of 20 min,ultrasonic temperature of 30 ℃.Enzymolysis conditions were enzyme dosage of2000 U/g protein,enzymolysis time of 2.5 h.Under the optimal conditions,the protein conversion rate was 85.00%,which was16.42% higher than 73.01% in the control group( without ultrasound),and the proportion of highly active peptides was29.63%,which was 13.96% higher than 26.00% in the control group.The results showed that dual-frequency ultrasound could effectively improve the yield and the ACE inhabitary activity of the product,which was beneficial to the preparation of ACE inhibitory peptides.
This study aimed to research the effects of ultrasonic pretreatment of different working modes on the preparation of ACE-inhibitory peptides from corn germ meal.According to protein conversion and proportion of highly active peptides,dualfrequency countercurrent energy-gathered ultrasound,triple-frequency divergence ultrasound and dual-frequency relative vibration ultrasound were used to pretreat corn germ meal and the best ultrasonic mode was selected. The ultrasonic preprocessing parameters were determined by single-factor step optimization method. Under ideal ultrasonic pretreatment conditions,enzymatic reaction conditions were optimized.Results showed that the ACE inhabitary activity of peptides of 300 ~1000 Da was the highest,IC50 of which was 0.78 mg/m L; the most effective ultrasonic mode was 20/40 k Hz alternate dual-frequency and the optimum parameters of the ultrasonic pretreatment were the power density of 100 W/L,the substrate concentration of 8%,ultrasonic time of 20 min,ultrasonic temperature of 30 ℃.Enzymolysis conditions were enzyme dosage of2000 U/g protein,enzymolysis time of 2.5 h.Under the optimal conditions,the protein conversion rate was 85.00%,which was16.42% higher than 73.01% in the control group( without ultrasound),and the proportion of highly active peptides was29.63%,which was 13.96% higher than 26.00% in the control group.The results showed that dual-frequency ultrasound could effectively improve the yield and the ACE inhabitary activity of the product,which was beneficial to the preparation of ACE inhibitory peptides.
引文
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[20]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.
[21]王向红,毛汉领,黄振峰.超声清洗槽内空化强度的测量[J].应用声学,2005,24(3):188-191.
[22]耿静静.超声辅助酶法制备燕麦蛋白ACE抑制肽的研究[D].镇江:江苏大学,2010.
[2]赵会燕.玉米胚芽抗氧化肽的制备及其安全性评价研究[D].长春:吉林大学,2015.
[3]张明站.玉米胚芽脱脂粕酶解条件及其产物抗氧化活性的研究[D].齐齐哈尔:齐齐哈尔大学,2013.
[4]吴迪,任健,宋春丽.酶解玉米胚芽粕制备ACE抑制肽的条件优化[J].粮油食品科技,2014,22(1):51-53.
[5]Qu W,Ma H,Jia J,et al.Enzymolysis kinetics and activities of ACE inhibitory peptides from wheat germ protein prepared with SFP ultrasound-assisted processing[J].Ultrasonics Sonochemistry,2012,19(5):1021-1026.
[6]丁青芝,马海乐,骆琳,等.脉冲超声预处理对玉米黄粉酶解制备ACEI肽的影响[J].江苏大学学报(自然科学版),2009,30(6):554-558.
[7]毛舒云,马海乐,任晓峰,等.扫频超声处理对玉米醇溶蛋白酶解特性的影响[J].食品工业科技,2013,34(14):142-147.
[8]马海乐,耿静静,骆琳,等.超声波预处理对燕麦蛋白制备ACE抑制肽的影响[J].农业机械学报,2010,41(9):133-137.
[9]Jin J,Ma H,Qu W,et al.Effects of multi-frequency power ultrasound on the enzymolysis of corn gluten meal:Kinetics and thermodynamics study[J].Ultrasonics Sonochemistry,2015,27:46-53.
[10]Wang B,Meng T,Ma H,et al.Mechanism study of dualfrequency ultrasound assisted enzymolysis on rapeseed protein by immobilized Alcalase[J].Ultrasonics Sonochemistry,2016,32:307-313.
[11]张艳艳.基于超声预处理的谷朊蛋白ACE抑制肽制备及其过程原位监测技术研究[D].镇江:江苏大学,2016.
[12]毛丽,马海乐,潘忠礼,等.超声预处理对脱脂小麦胚芽酶解制备ACE抑制肽的影响[J].食品工业科技,2016,37(12):200-205.
[13]Yang X,Li Y,Li S,et al.Effects of multi-frequency ultrasound pretreatment under low power density on the enzymolysis and the structure characterization of defatted wheat germ protein[J].Ultrasonics Sonochemistry,2017,38:410-420.
[14]Yang X,Li Y,Li S,et al.Effects of ultrasound pretreatment with different frequencies and working modes on the enzymolysis and the structure characterization of rice protein[J].Ultrasonics Sonochemistry,2017,38:19-28.
[15]吴琼英,马海乐,崔恒林,等.猪肺血管紧张素转化酶的提取纯化及其性质研究[J].食品科学,2004,25(9):71-74.
[16]陈钧辉,李俊.生物化学实验.第5版[M].科学出版社,2014.
[17]龚洋.梯度稀释补料酶膜耦合反应制备牛奶蛋白ACE抑制肽的研究[D].镇江:江苏大学,2016.
[18]Gülseren I,Güzey D,Bruce B D,et al.Structural and functional changes in ultrasonicated bovine serum albumin solutions[J].Ultrasonics Sonochemistry,2007,14(2):173-183.
[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,2009.
[20]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.
[21]王向红,毛汉领,黄振峰.超声清洗槽内空化强度的测量[J].应用声学,2005,24(3):188-191.
[22]耿静静.超声辅助酶法制备燕麦蛋白ACE抑制肽的研究[D].镇江:江苏大学,2010.