体外消化对金瓜籽抗氧化肽抗氧化活性的影响
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摘要
本实验以金瓜籽为原料,以还原力、羟自由基清除率、DPPH自由基清除率为考察指标,采用响应面试验探讨了制备金瓜籽抗氧化肽的工艺参数,并研究了其经体外模拟人体胃肠道消化后的游离氨基酸组成、分子质量分布及抗氧化活性变化。结果表明:制备金瓜籽抗氧化肽的最优工艺条件为底物(金瓜籽粕)质量分数为6%、添加碱性蛋白酶3%、在pH 8.5的体系中55℃酶解90 min,此条件下所得羟自由基清除率、DPPH自由基清除率、还原力分别为(58.19±0.80)%、(60.27±1.73)%和0.49±0.01;分子质量小于3 kDa、3～5 kDa及大于5 kDa的抗氧化肽经体外模拟胃肠道消化后分别含游离氨基酸41.72%、36.94%和33.93%,其主要为分子质量小于500 Da的肽段,占比分别为73.50%、60.15%和53.30%;与消化前相比,消化产物对羟自由基的清除能力分别提高了26.04%、29.06%和34.43%,还原力分别提高了50.98%、64.53%、67.35%。该研究可为金瓜籽抗氧化肽作为功能性抗氧化剂的生产和应用提供参考。
In this study, response surface methodology was used to optimize the enzymatic hydrolysis conditions for the preparation of antioxidant peptides from marrow seed meal for improved reducing power and scavenging capacity against hydroxyl and DPPH radicals. The changes in the free amino acid composition, molecular mass distribution and antioxidant activity of the antioxidant peptides after simulated gastrointestinal digestion in vitro were studied. The results showed that the optimal conditions were obtained as follows: substrate concentration 6%, alkaline protease 3%, and enzymatic hydrolysis at 55 ℃ for 90 min at pH 8.5. The scavenging percentages against hydroxyl and DPPH radicals and reducing power of the hydrolysate obtained under these conditions were(58.19 ± 0.80)%,(60.27 ± 1.73)% and 0.49 ± 0.01, respectively.The antioxidant peptides with a molecular mass of less than 3 kDa, 3-5 kDa and more than 5 kDa were separated from the hydrolysate. After simulated gastrointestinal digestion, these antioxidant peptides contained 41.72%, 36.94% and 33.93%free amino acids, respectively, and the digested peptides consisted mainly of peptide fragments with a molecular mass of less than 500 Da account for 73.50%, 60.15% and 53.30% of the total amount, respectively. The hydroxyl radical scavenging capacity increase by 26.04%, 29.06% and 34.43%, respectively,,and the reducing power increased by 50.98%, 64.53%and 67.35%, respectively when compared with those before digestion. The results of this study provide useful data for the production and application of antioxidant peptides from marrow seeds as a functional antioxidant.
In this study, response surface methodology was used to optimize the enzymatic hydrolysis conditions for the preparation of antioxidant peptides from marrow seed meal for improved reducing power and scavenging capacity against hydroxyl and DPPH radicals. The changes in the free amino acid composition, molecular mass distribution and antioxidant activity of the antioxidant peptides after simulated gastrointestinal digestion in vitro were studied. The results showed that the optimal conditions were obtained as follows: substrate concentration 6%, alkaline protease 3%, and enzymatic hydrolysis at 55 ℃ for 90 min at pH 8.5. The scavenging percentages against hydroxyl and DPPH radicals and reducing power of the hydrolysate obtained under these conditions were(58.19 ± 0.80)%,(60.27 ± 1.73)% and 0.49 ± 0.01, respectively.The antioxidant peptides with a molecular mass of less than 3 kDa, 3-5 kDa and more than 5 kDa were separated from the hydrolysate. After simulated gastrointestinal digestion, these antioxidant peptides contained 41.72%, 36.94% and 33.93%free amino acids, respectively, and the digested peptides consisted mainly of peptide fragments with a molecular mass of less than 500 Da account for 73.50%, 60.15% and 53.30% of the total amount, respectively. The hydroxyl radical scavenging capacity increase by 26.04%, 29.06% and 34.43%, respectively,,and the reducing power increased by 50.98%, 64.53%and 67.35%, respectively when compared with those before digestion. The results of this study provide useful data for the production and application of antioxidant peptides from marrow seeds as a functional antioxidant.
引文
[1]杨忠.崇明金瓜种质资源的评价与利用[D].上海:上海交通大学,2010:1-7.
[2]宋忠明,蔡希义,朱忠南,等.崇明金瓜种质资源鉴定及加工性状的综合评价[J].上海农业学报,2007,23(3):132-134.
[3]KORHONEN H,PIHLANTO A.Bioactive peptides:production and functionality[J].International Dairy Journal,2006,16(9):945-960.DOI:10.1016/j.idairyj.2005.10.012.
[4]TONG L M,SASAKI S,MCCLEMENTS D J,et al.Mechanisms of the antioxidant activity of a high molecular weight fraction of whey[J].Journal of Agricultural and Food Chemistry,2000,48(5):1473-1478.DOI:10.1021/jf991342v.
[5]李景军,陈正行,毛健.抑制羟自由基的粟水溶性蛋白胁迫培养基试验[J].江苏大学学报(自然科学版),2008,29(1):9-12.
[6]MENDIS E,RAJAPAKSE N,KIM S K.Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate[J].Journal of Agricultural and Food Chemistry,2005,53(3):581-587.DOI:10.1021/jf048877v.
[7]GUERRA A,ETIENNE-MESMIN L,LIVRELLI V,et al.Relevance and challenges in modeling human gastric and small intestinal digestion[J].Trends in Biotechnology,2012,30(11):591-600.DOI:10.1016/j.tibtech.2012.08.001.
[8]WU H C,CHEN H M,SHIAU C Y.Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel(Scomber austriasicus)[J].Food Research International,2003,36(9/10):949-957.DOI:10.1016/S0963-9969(03)00104-2.
[9]AMAROWICZ R,NACZK M,SHAHIDI F.Antioxidant activity of various fractions of non-tannin phenolics of canola hulls[J].Journal of Agricultural and Food Chemistry,2000,48(7):2755-2759.DOI:10.1021/jf9911601.
[10]AHMADI F,KADIVAR M,SHAHEDI M.Antioxidant activity of Kelussia odoratissima Mozaff.in model and food systems[J].Food Chemistry,2007,105(1):57-64.DOI:10.1016/j.foodchem.2007.03.056.
[11]SZO?TYSIK M,NIEDBALSKA J,DABROWSKA A,et al.Use of enzymatic hydrolysis of casein for the preparation of peptides with antioxidative activity[J].Przemysl Chemiczny,2012,91(5):1014-1019.
[12]RUIZ JáG,RAMOS M,RECIO I.Angiotensin converting enzymeinhibitory activity of peptides isolated from Manchego cheese.stability under simulated gastrointestinaldigestion[J].International Dairy Journal,2004,14(12):1075-1080.DOI:10.1016/j.idairyj.2004.04.007.
[13]张苏平,邱伟强,卢祺,等.全自动氨基酸分析仪法测定4种贝类肌肉中谷胱甘肽和游离氨基酸含量[J].食品科学,2017,38(4):170-176.DOI:10.7506/spkx1002-6630-201704027.
[14]高瑀珑,郑锐,王玉梅,等.菜籽分离蛋白分子质量分布及酶解条件的研究[J].中国粮油学报,2014,29(6):38-45.
[15]徐贤.高压预处理-酶解制备大豆蛋白抗氧化肽的研究[D].郑州:郑州轻工业学院,2009:11-20.
[16]桂兴芬,吕文华,康爱英.生物化学[M].3版.郑州:郑州大学出版社,2008:194-267.
[17]HAZIME R,NGUYEN Q H,FERRONATO C,et al.Optimization of imazalil removal in the system UV/TiO2/K2S2O8 using a response surface methodology(RSM)[J].Applied Catalysis B Environmental,2013,132/133:519-526.DOI:10.1016/j.apcatb.2012.12.021.
[18]XU Q,SHEN Y,WANG H,et al.Application of response surface methodology to optimise extraction of flavonoids from fructus sophorae[J].Food Chemistry,2013,138(4):2122-2129.DOI:10.1016/j.foodchem.2012.11.099.
[19]邬建敏.蛋白质与酶在固相表面的吸附行为对其生物活性及稳定性影响的研究[D].杭州:浙江大学,2006:41-48.
[20]谢韵.蛋白质界面吸附取向和构象的分子模拟研究[D].广州:华南理工大学,2010:41-59.
[21]ADIBI S A,MERCER D W.Protein digestion in human intestine as reflected in luminal,mucosal,and plasma amino acid concentrations after meals[J].Journal of Clinical Investigation,1973,52(7):1586-1594.DOI:10.1172/JCI107335.
[22]HERNáNDEZ-LEDESMA B,DáVALOS A,BARTOLOMéB,et al.Preparation of antioxidant enzymatic hydrolysates from alphalactalbumin and beta-lactoglobulin.identification of active peptides by HPLC-MS/MS[J].Journal of Agricultural and Food Chemistry,2005,53(3):588-593.DOI:10.1021/jf048626m.
[23]RUFIN-HENARES J A,MORALES F J.Effect of in vitro enzymatic digestion on antioxidant activity of coffee melanoidins and fractions[J].Journal of Agricultural and Food Chemistry,2007,55(24):10016-10021.DOI:10.1021/jf0718291.
[24]ZHU Lijuan,CHEN Jie,TANG Xueyan,et al.Reducing,radical scavenging,and chelation properties of in vitro digests of alcalasetreated zein hydrolysates[J].Journal of Agricultural and Food Chemistry,2008,56(8):2714-2721.DOI:10.1021/jf703697e.
[25]张淼.玉米蛋白及其水解物模拟体外消化产物抗氧化活性研究[D].呼和浩特:内蒙古农业大学,2016:34-41.
[26]PANAYIOTIDIS M I,RANCOURT R C,ALLEN C B,et al.Hyperoxia-induced DNA damage causes decreased DNA methylation in human lung epithelial-like A549 cells[J].Antioxidants&Redox Signaling,2004,6(1):129-136.DOI:10.1089/152308604771978435.
[27]QIAN Z J,JUNG W K,BYUN H G,et al.Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster,Crassostrea gigas against free radical induced DNA damage[J].Bioresource Technology,2008,99(9):3365-3371.DOI:10.1016/j.biortech.2007.08.018.
[28]YOU Lijun,ZHAO Mouming,REGENSTEIN J M,et al.Changes in the antioxidant activity of loach(Misgurnus anguillicaudatus)protein hydrolysates during a simulated gastrointestinal digestion[J].Food Chemistry,2010,120(3):810-816.DOI:10.1016/j.foodchem.2009.11.018.
[29]CHEN H M,MURAMOTO K,YAMAUCHI F.Structural analysis of antioxidative peptides from soybean beta-conglycinin[J].Journal of Agricultural and Food Chemistry,1995,43(3):574-578.DOI:10.1021/jf00051a004.
[30]MENDIS E,RAJAPAKSE N,BYUN H G,et al.Investigation of jumbo squid(Dosidicus gigas)skin gelatin peptides for their in vitro antioxidant effects[J].Life Sciences,2005,77(17):2166-2178.DOI:10.1016/j.lfs.2005.03.016.
[31]DáVALOS A,MIGUEL M,BARTOLOMéB,et al.Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis[J].Journal of Food Protection,2004,67(9):1939-1944.DOI:10.4315/0362-028X-67.9.1939.
[32]张冠亚,黄晓君,聂少平,等.体外模拟3种消化液对铁皮石斛多糖的消化作用[J].食品科学,2014,35(23):279-283.DOI:10.7506/spkx1002-6630-201423054.
[2]宋忠明,蔡希义,朱忠南,等.崇明金瓜种质资源鉴定及加工性状的综合评价[J].上海农业学报,2007,23(3):132-134.
[3]KORHONEN H,PIHLANTO A.Bioactive peptides:production and functionality[J].International Dairy Journal,2006,16(9):945-960.DOI:10.1016/j.idairyj.2005.10.012.
[4]TONG L M,SASAKI S,MCCLEMENTS D J,et al.Mechanisms of the antioxidant activity of a high molecular weight fraction of whey[J].Journal of Agricultural and Food Chemistry,2000,48(5):1473-1478.DOI:10.1021/jf991342v.
[5]李景军,陈正行,毛健.抑制羟自由基的粟水溶性蛋白胁迫培养基试验[J].江苏大学学报(自然科学版),2008,29(1):9-12.
[6]MENDIS E,RAJAPAKSE N,KIM S K.Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate[J].Journal of Agricultural and Food Chemistry,2005,53(3):581-587.DOI:10.1021/jf048877v.
[7]GUERRA A,ETIENNE-MESMIN L,LIVRELLI V,et al.Relevance and challenges in modeling human gastric and small intestinal digestion[J].Trends in Biotechnology,2012,30(11):591-600.DOI:10.1016/j.tibtech.2012.08.001.
[8]WU H C,CHEN H M,SHIAU C Y.Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel(Scomber austriasicus)[J].Food Research International,2003,36(9/10):949-957.DOI:10.1016/S0963-9969(03)00104-2.
[9]AMAROWICZ R,NACZK M,SHAHIDI F.Antioxidant activity of various fractions of non-tannin phenolics of canola hulls[J].Journal of Agricultural and Food Chemistry,2000,48(7):2755-2759.DOI:10.1021/jf9911601.
[10]AHMADI F,KADIVAR M,SHAHEDI M.Antioxidant activity of Kelussia odoratissima Mozaff.in model and food systems[J].Food Chemistry,2007,105(1):57-64.DOI:10.1016/j.foodchem.2007.03.056.
[11]SZO?TYSIK M,NIEDBALSKA J,DABROWSKA A,et al.Use of enzymatic hydrolysis of casein for the preparation of peptides with antioxidative activity[J].Przemysl Chemiczny,2012,91(5):1014-1019.
[12]RUIZ JáG,RAMOS M,RECIO I.Angiotensin converting enzymeinhibitory activity of peptides isolated from Manchego cheese.stability under simulated gastrointestinaldigestion[J].International Dairy Journal,2004,14(12):1075-1080.DOI:10.1016/j.idairyj.2004.04.007.
[13]张苏平,邱伟强,卢祺,等.全自动氨基酸分析仪法测定4种贝类肌肉中谷胱甘肽和游离氨基酸含量[J].食品科学,2017,38(4):170-176.DOI:10.7506/spkx1002-6630-201704027.
[14]高瑀珑,郑锐,王玉梅,等.菜籽分离蛋白分子质量分布及酶解条件的研究[J].中国粮油学报,2014,29(6):38-45.
[15]徐贤.高压预处理-酶解制备大豆蛋白抗氧化肽的研究[D].郑州:郑州轻工业学院,2009:11-20.
[16]桂兴芬,吕文华,康爱英.生物化学[M].3版.郑州:郑州大学出版社,2008:194-267.
[17]HAZIME R,NGUYEN Q H,FERRONATO C,et al.Optimization of imazalil removal in the system UV/TiO2/K2S2O8 using a response surface methodology(RSM)[J].Applied Catalysis B Environmental,2013,132/133:519-526.DOI:10.1016/j.apcatb.2012.12.021.
[18]XU Q,SHEN Y,WANG H,et al.Application of response surface methodology to optimise extraction of flavonoids from fructus sophorae[J].Food Chemistry,2013,138(4):2122-2129.DOI:10.1016/j.foodchem.2012.11.099.
[19]邬建敏.蛋白质与酶在固相表面的吸附行为对其生物活性及稳定性影响的研究[D].杭州:浙江大学,2006:41-48.
[20]谢韵.蛋白质界面吸附取向和构象的分子模拟研究[D].广州:华南理工大学,2010:41-59.
[21]ADIBI S A,MERCER D W.Protein digestion in human intestine as reflected in luminal,mucosal,and plasma amino acid concentrations after meals[J].Journal of Clinical Investigation,1973,52(7):1586-1594.DOI:10.1172/JCI107335.
[22]HERNáNDEZ-LEDESMA B,DáVALOS A,BARTOLOMéB,et al.Preparation of antioxidant enzymatic hydrolysates from alphalactalbumin and beta-lactoglobulin.identification of active peptides by HPLC-MS/MS[J].Journal of Agricultural and Food Chemistry,2005,53(3):588-593.DOI:10.1021/jf048626m.
[23]RUFIN-HENARES J A,MORALES F J.Effect of in vitro enzymatic digestion on antioxidant activity of coffee melanoidins and fractions[J].Journal of Agricultural and Food Chemistry,2007,55(24):10016-10021.DOI:10.1021/jf0718291.
[24]ZHU Lijuan,CHEN Jie,TANG Xueyan,et al.Reducing,radical scavenging,and chelation properties of in vitro digests of alcalasetreated zein hydrolysates[J].Journal of Agricultural and Food Chemistry,2008,56(8):2714-2721.DOI:10.1021/jf703697e.
[25]张淼.玉米蛋白及其水解物模拟体外消化产物抗氧化活性研究[D].呼和浩特:内蒙古农业大学,2016:34-41.
[26]PANAYIOTIDIS M I,RANCOURT R C,ALLEN C B,et al.Hyperoxia-induced DNA damage causes decreased DNA methylation in human lung epithelial-like A549 cells[J].Antioxidants&Redox Signaling,2004,6(1):129-136.DOI:10.1089/152308604771978435.
[27]QIAN Z J,JUNG W K,BYUN H G,et al.Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster,Crassostrea gigas against free radical induced DNA damage[J].Bioresource Technology,2008,99(9):3365-3371.DOI:10.1016/j.biortech.2007.08.018.
[28]YOU Lijun,ZHAO Mouming,REGENSTEIN J M,et al.Changes in the antioxidant activity of loach(Misgurnus anguillicaudatus)protein hydrolysates during a simulated gastrointestinal digestion[J].Food Chemistry,2010,120(3):810-816.DOI:10.1016/j.foodchem.2009.11.018.
[29]CHEN H M,MURAMOTO K,YAMAUCHI F.Structural analysis of antioxidative peptides from soybean beta-conglycinin[J].Journal of Agricultural and Food Chemistry,1995,43(3):574-578.DOI:10.1021/jf00051a004.
[30]MENDIS E,RAJAPAKSE N,BYUN H G,et al.Investigation of jumbo squid(Dosidicus gigas)skin gelatin peptides for their in vitro antioxidant effects[J].Life Sciences,2005,77(17):2166-2178.DOI:10.1016/j.lfs.2005.03.016.
[31]DáVALOS A,MIGUEL M,BARTOLOMéB,et al.Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis[J].Journal of Food Protection,2004,67(9):1939-1944.DOI:10.4315/0362-028X-67.9.1939.
[32]张冠亚,黄晓君,聂少平,等.体外模拟3种消化液对铁皮石斛多糖的消化作用[J].食品科学,2014,35(23):279-283.DOI:10.7506/spkx1002-6630-201423054.