甘薯ACE抑制肽的制备及其降血压活性研究
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摘要
甘薯加工产生的大量废液中富含蛋白,而甘薯蛋白是具有显著的加工和保健特性的优质植物蛋白,其经回收利用可减少资源的浪费及环境污染,因此,研究甘薯蛋白酶解产物的降血压活性具有重要的理论意义和实践价值。本研究采用胃蛋白酶水解甘薯蛋白制备ACE抑制肽,并对其进行分离、纯化和结构的测定,同时探讨了甘薯蛋白肽对原发性高血压大鼠血压的影响。
利用胃蛋白酶水解甘薯蛋白制备ACE抑制肽,通过单因素试验和四元二次回归正交旋转组合设计,考察底物浓度、酶与底物浓度比、pH值和温度对ACE抑制率的影响,确定了酶解甘薯蛋白的最适条件为:底物浓度2.3%,酶与底物浓度比3.7%,pH值2.3,温度37℃,时间8h。甘薯ACE抑制肽经超滤后分为三个分子量范围的组分即0-3kDa、3-5kDa、5-10kDa,三种组分的ACE抑制率均随着浓度的上升逐渐增大呈现剂量依赖性。其中分子量小于3kDa的组分ACE抑制效果最好IC50值为0.6661mg/ml,而分子量为5-10kDa的组分活性最差。通过MALDI-TOF-MS检测甘薯ACE抑制肽(0-3kDa组分)的分子量在212Da-2550Da之间,其中分子量为1384Da、1131Da、985Da及815Da的多肽含量较多。通过反相高效液相色谱法测定其氨基酸组成,发现其中疏水性、芳香族和支链氨基酸含量较高,分别为29.8g/100g Peptide、8.41 g/100g Peptide和12.94 g/100g Peptide。
此外,甘薯蛋白肽对原发性高血压大鼠(SHR)具有短期和长期降压功效,且可显著低降低SHR血清中ET-1的水平,而对SHR的心率、体重和血脂水平未产生不利影响。一次给药三个剂量组(600mg/kg·bw、1500mg/kg·bw和2000mg/kg·bw)的甘薯蛋白肽最大降幅均出现在灌胃4h后,分别为23.59mmHg、16.91mmHg和16.18mmHg。随后血压开始回升,12h时基本回复到原有水平。长期给药甘薯蛋白肽24天,6天之后三个剂量组SHR的收缩压与空白对照组相比均显著降低(P<0.05),分别为13.01mmHg、10.54mmHg和8.46mmHg。其中甘薯蛋白肽的低剂量与中剂量给药组(600mg/kg·bw和1500mg/kg·bw)血压下降值无显著差异(P>0.05)。
无论是一次还是多次给药甘薯蛋白肽与卡托普利的混合物(1500mg/kg·bw+5 mg/kg·bw)对SHR血压的降低均具有协同效果,优于单独使用卡托普利的功效,且可明显降低SHR血清中ET-1与Ang II的水平,而未见对SHR的生长状况、心率和血脂水平未产生不利影响。一次给药蛋白肽与卡托普利的混合物和单独给药卡托普利在3到6h内保持降压活性(P<0.05),与灌胃蒸馏水的空白对照组相比,两组的最大降幅均出现在灌胃3h后,分别为15.08mmHg和9.55mmHg。连续给药24天甘薯蛋白肽与卡托普利的混合物(1500mg/kg·bw +5mg/kg·bw),发现二者联用之后对SHR的血压有显著影响,经6天后较之空白对照组联用组SHR的收缩压为164.16mmHg显著下降了20.03mmHg,继续给药,联用组和卡托普利单独给药组的血压值与空白对照组相比均有显著性差异(P<0.05),灌胃24天后联用组的血压降幅为28.87mmHg。研究表明,甘薯蛋白肽具有较好的降血压效果,可作为一种新型的保健食品进行开发和利用。
The waste solution produced in sweet potato processing contains large amounts of sweet potato protein. Recycling sweet potato protein from waste solutions can reduce the waste of resources and the pollution to the environment. Furthermore, sweet potato protein has significant bioactive functionality and it is a high-quality plant protein with good processing characteristics. Therefore, it is of great theoretical and practical value to study the antihypertensive activity on the hydrolyzates of sweet potato protein. The objective of this work was to obtain ACE inhibitory peptide from sweet potato protein using pepsin as well as to research its isolation, purification and structural analysis, at the same time explored the effect on the SBP of SHRs from sweet potato protein peptide.
Isolation of ACE inhibitory peptide from sweetpotato protein was performed by utilizing pepsin. The effect of concentration of substrate, enzyme to substrate concentration ratio, pH value and temperature on the ACE inhibition ratio was studied by one- factor experiment and optimum design of quaternary quadratic rotation orthogonality experiment to get the best enzymatic hydrolysis condition. Results indicate that the optimal processing conditions for preparing ACE inhibitory peptides with pepsin are as follows: concentration of substrate 2.3 %, enzyme to substrate concentration ratio 3.7 %, pH value 2.3, temperature 37℃, time 8 h.
Sweet potato protein peptide was separated into three fractions by ultra - filtration technology, and the range of molecular weight of the peptides are 0 - 3 kDa, 3 - 5 kDa, 5 - 10kDa. With the increase in concentrations of peptides, the ACE inhibition ratio had a marked increase, indicating dose dependent. In addition, ACE inhibitory activity of the peptide below 3 kDa, of which IC50 value ratio was 0.6661mg/ml, standed out very much, while the peptide whose molecular weight distribution was between 5– 10 kDa showed the lowest activity. After further demineralization and purify, the molecular weight range of sweet peptides below 3 kD was 212 Da - 2551 Da based on the data of MALDI - TOF - MS, of which the peptides with molecular weight 1384 Da, 1131 Da, 985 Da and 815 Da occupied most. By RP - HPLC, the composition of amino acids in sweet potato peptide was measured. The result showed sweet potato peptide has high contents of hydrophobic, aromatic and branched-side chains amino acids, the content were 29.8g/100g Peptide, 8.41 g/100g Peptide and 12.94 g/100g Peptide, respectively.
The result showed that sweet potato peptide could steadily reduce SBP of SHRs both once and long - term dose. And it also turns out the heart rate, weight and blood lipids levels of SHRs were not influenced by both once and long - term dose. Furthermore, the content of ET -1 in SHR serum was significantly reduced after administration for 24 days. The once - oral administration animal experiments showed that the highest blood pressure of SHR drop was 23.59 mmHg, 16.91 mmHg, 16.18 mmHg at a dosage of 600 mg/kg·bw, 1500 mg/kg·bw and 2000 mg/kg·bw, respectively, after 1h administration. With time increased, the SBP of SHRs rose again, and nearly returned to the original level after 12 h. The long term oral administration design was conducted for 24 days. There were significant differences of SBP of SHRs for 6 days’administration when comparing the three dose groups with the control group. And the blood pressure of SHR drop was 13.01 mmHg, 10.54 mmHg, 8.46 mmHg, respectively. Moreover, There was no remarkable difference between the medium-dosage group and the low one.
Regardless once administration or long - term administration, with a mixture of sweet potato peptide and captopril ( 1500 mg/kg·bw + 5 mg/kg·bw) had a synergistic effect on the SBP of SHRs. And its effect was superior to that of using captopril alone. And there was no adverse impact on the growth condition , heart rates and lipid levels of SHRs. Meanwhile, the content of ET - 1 and Ang II in SHR serum was significantly reduced after administration for 24 days. Sweet potato peptides mixed with captopril and captopril alone filled in stomach by mouth both kept step-down activity between 3h and 6h (P < 0.05). The highest blood pressure of two groups’SHR drop both appeared after 3h administration, which were 15.08 mmHg and 9.55 mmHg. Simultaneously, the hypertension effect of sweet potato peptides mixed with captopril was better than that of captopril used alone. The results of long-term oral administration indicated that a mixture of sweet potato peptides and captopril had significant antihypertensive action. The blood pressure of the combination group was 164.16mmHg significantly dropped 20.03 mmH, and if continuing to give medicine, the blood pressure of combination group and the positive control group both had significant variation compared with blank control (P < 0.05). And the blood pressure drop of SHR was 28.87 mmHg after 24 days’administration.
The present study showed that, Sweet potato peptide has good antihypertensive effect, and is an ideal source for development and utilization.
利用胃蛋白酶水解甘薯蛋白制备ACE抑制肽,通过单因素试验和四元二次回归正交旋转组合设计,考察底物浓度、酶与底物浓度比、pH值和温度对ACE抑制率的影响,确定了酶解甘薯蛋白的最适条件为:底物浓度2.3%,酶与底物浓度比3.7%,pH值2.3,温度37℃,时间8h。甘薯ACE抑制肽经超滤后分为三个分子量范围的组分即0-3kDa、3-5kDa、5-10kDa,三种组分的ACE抑制率均随着浓度的上升逐渐增大呈现剂量依赖性。其中分子量小于3kDa的组分ACE抑制效果最好IC50值为0.6661mg/ml,而分子量为5-10kDa的组分活性最差。通过MALDI-TOF-MS检测甘薯ACE抑制肽(0-3kDa组分)的分子量在212Da-2550Da之间,其中分子量为1384Da、1131Da、985Da及815Da的多肽含量较多。通过反相高效液相色谱法测定其氨基酸组成,发现其中疏水性、芳香族和支链氨基酸含量较高,分别为29.8g/100g Peptide、8.41 g/100g Peptide和12.94 g/100g Peptide。
此外,甘薯蛋白肽对原发性高血压大鼠(SHR)具有短期和长期降压功效,且可显著低降低SHR血清中ET-1的水平,而对SHR的心率、体重和血脂水平未产生不利影响。一次给药三个剂量组(600mg/kg·bw、1500mg/kg·bw和2000mg/kg·bw)的甘薯蛋白肽最大降幅均出现在灌胃4h后,分别为23.59mmHg、16.91mmHg和16.18mmHg。随后血压开始回升,12h时基本回复到原有水平。长期给药甘薯蛋白肽24天,6天之后三个剂量组SHR的收缩压与空白对照组相比均显著降低(P<0.05),分别为13.01mmHg、10.54mmHg和8.46mmHg。其中甘薯蛋白肽的低剂量与中剂量给药组(600mg/kg·bw和1500mg/kg·bw)血压下降值无显著差异(P>0.05)。
无论是一次还是多次给药甘薯蛋白肽与卡托普利的混合物(1500mg/kg·bw+5 mg/kg·bw)对SHR血压的降低均具有协同效果,优于单独使用卡托普利的功效,且可明显降低SHR血清中ET-1与Ang II的水平,而未见对SHR的生长状况、心率和血脂水平未产生不利影响。一次给药蛋白肽与卡托普利的混合物和单独给药卡托普利在3到6h内保持降压活性(P<0.05),与灌胃蒸馏水的空白对照组相比,两组的最大降幅均出现在灌胃3h后,分别为15.08mmHg和9.55mmHg。连续给药24天甘薯蛋白肽与卡托普利的混合物(1500mg/kg·bw +5mg/kg·bw),发现二者联用之后对SHR的血压有显著影响,经6天后较之空白对照组联用组SHR的收缩压为164.16mmHg显著下降了20.03mmHg,继续给药,联用组和卡托普利单独给药组的血压值与空白对照组相比均有显著性差异(P<0.05),灌胃24天后联用组的血压降幅为28.87mmHg。研究表明,甘薯蛋白肽具有较好的降血压效果,可作为一种新型的保健食品进行开发和利用。
The waste solution produced in sweet potato processing contains large amounts of sweet potato protein. Recycling sweet potato protein from waste solutions can reduce the waste of resources and the pollution to the environment. Furthermore, sweet potato protein has significant bioactive functionality and it is a high-quality plant protein with good processing characteristics. Therefore, it is of great theoretical and practical value to study the antihypertensive activity on the hydrolyzates of sweet potato protein. The objective of this work was to obtain ACE inhibitory peptide from sweet potato protein using pepsin as well as to research its isolation, purification and structural analysis, at the same time explored the effect on the SBP of SHRs from sweet potato protein peptide.
Isolation of ACE inhibitory peptide from sweetpotato protein was performed by utilizing pepsin. The effect of concentration of substrate, enzyme to substrate concentration ratio, pH value and temperature on the ACE inhibition ratio was studied by one- factor experiment and optimum design of quaternary quadratic rotation orthogonality experiment to get the best enzymatic hydrolysis condition. Results indicate that the optimal processing conditions for preparing ACE inhibitory peptides with pepsin are as follows: concentration of substrate 2.3 %, enzyme to substrate concentration ratio 3.7 %, pH value 2.3, temperature 37℃, time 8 h.
Sweet potato protein peptide was separated into three fractions by ultra - filtration technology, and the range of molecular weight of the peptides are 0 - 3 kDa, 3 - 5 kDa, 5 - 10kDa. With the increase in concentrations of peptides, the ACE inhibition ratio had a marked increase, indicating dose dependent. In addition, ACE inhibitory activity of the peptide below 3 kDa, of which IC50 value ratio was 0.6661mg/ml, standed out very much, while the peptide whose molecular weight distribution was between 5– 10 kDa showed the lowest activity. After further demineralization and purify, the molecular weight range of sweet peptides below 3 kD was 212 Da - 2551 Da based on the data of MALDI - TOF - MS, of which the peptides with molecular weight 1384 Da, 1131 Da, 985 Da and 815 Da occupied most. By RP - HPLC, the composition of amino acids in sweet potato peptide was measured. The result showed sweet potato peptide has high contents of hydrophobic, aromatic and branched-side chains amino acids, the content were 29.8g/100g Peptide, 8.41 g/100g Peptide and 12.94 g/100g Peptide, respectively.
The result showed that sweet potato peptide could steadily reduce SBP of SHRs both once and long - term dose. And it also turns out the heart rate, weight and blood lipids levels of SHRs were not influenced by both once and long - term dose. Furthermore, the content of ET -1 in SHR serum was significantly reduced after administration for 24 days. The once - oral administration animal experiments showed that the highest blood pressure of SHR drop was 23.59 mmHg, 16.91 mmHg, 16.18 mmHg at a dosage of 600 mg/kg·bw, 1500 mg/kg·bw and 2000 mg/kg·bw, respectively, after 1h administration. With time increased, the SBP of SHRs rose again, and nearly returned to the original level after 12 h. The long term oral administration design was conducted for 24 days. There were significant differences of SBP of SHRs for 6 days’administration when comparing the three dose groups with the control group. And the blood pressure of SHR drop was 13.01 mmHg, 10.54 mmHg, 8.46 mmHg, respectively. Moreover, There was no remarkable difference between the medium-dosage group and the low one.
Regardless once administration or long - term administration, with a mixture of sweet potato peptide and captopril ( 1500 mg/kg·bw + 5 mg/kg·bw) had a synergistic effect on the SBP of SHRs. And its effect was superior to that of using captopril alone. And there was no adverse impact on the growth condition , heart rates and lipid levels of SHRs. Meanwhile, the content of ET - 1 and Ang II in SHR serum was significantly reduced after administration for 24 days. Sweet potato peptides mixed with captopril and captopril alone filled in stomach by mouth both kept step-down activity between 3h and 6h (P < 0.05). The highest blood pressure of two groups’SHR drop both appeared after 3h administration, which were 15.08 mmHg and 9.55 mmHg. Simultaneously, the hypertension effect of sweet potato peptides mixed with captopril was better than that of captopril used alone. The results of long-term oral administration indicated that a mixture of sweet potato peptides and captopril had significant antihypertensive action. The blood pressure of the combination group was 164.16mmHg significantly dropped 20.03 mmH, and if continuing to give medicine, the blood pressure of combination group and the positive control group both had significant variation compared with blank control (P < 0.05). And the blood pressure drop of SHR was 28.87 mmHg after 24 days’administration.
The present study showed that, Sweet potato peptide has good antihypertensive effect, and is an ideal source for development and utilization.
引文
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48. Aera J, Cheorun J, Kyung S K, Mooha L.Antimicrobial and human cancer cellcytotoxic effect of synthetic angiotensin-converting enzyme (ACE) inhibitory peptides. Food Chemistry, 2008, 107: 327-336.
49. Brantl V, Teschemacher H, Henschen A, Lottspeich F. Novel opioid peptides derived from casein (β- casomorphins). I. Isolation from bovine casein peptone .Hoppe Seylers Z Physiol Chemistry, 1979, 360 (9): 1211-1216.
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53. Chen Q H, Xuan G D, Fu M L, He G Q, Wang W, Zhang H B , Ruan H. Effect of angiotensin ? - converting enzyme inhibitory peptide from rice dregs protein on antihypertensive activity in spontaneously hypertensive rats . Asia Pacific Journal of Clinical Nutrition, 2007;16 (Suppl 1):281-285.
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56. Gill I, Rosina L-F. Forba X. Biologically active peptides and enzymatic approaches to their production. Enzyme and Microbial Technology, 1996, 18: 162-183.
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58. Hyun C K, Shin H K. Utilization of bovine blood plasma proteins for the production of angiotensin I converting enzyme inhibitory peptides. Process Biochemistry , 2000,36: 65-71.
59. Hata.Y., Yamamoto, N., Ohni, M. Amer. A placebo-controlled study of the effect of sour milk on blood pressure in hypertensive subjects. American Journal of Clinical Nutrition. 1996, 64:767-771.
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61. Jiang J L, Chen S W, Ren F Z, Luo Z, Steve S Z.Yak Milk Casein as a Functional Ingredient: Preparation and Identification of Angiotensin - I - Converting Enzyme Inhibitory Peptides. Journal of Dairy Research . 2007, 74:18-25.
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54. Fitzgerald R. J., Murray B A,Walsh D. J. The emerging role of dairy proteins and bioactive peptides in nutrition and health. Joural of Nutriton. 2004, 134, 4:980s-988s.
55. Groff J L, Harp J B, DiGirolamo M.Simplified enzymatic assay of angiotensin - converting enzyme in serum.Clinical Chemistry , 1993, 39: 400-404.
56. Gill I, Rosina L-F. Forba X. Biologically active peptides and enzymatic approaches to their production. Enzyme and Microbial Technology, 1996, 18: 162-183.
57. Holmquist B, Bunning P, Riordan J F. A Continuous Spectrophotometric Assay for the Angiotensin Converting Enzyme. Analytical and Bioanalytical Chemistry, 1979, 95: 540-548.
58. Hyun C K, Shin H K. Utilization of bovine blood plasma proteins for the production of angiotensin I converting enzyme inhibitory peptides. Process Biochemistry , 2000,36: 65-71.
59. Hata.Y., Yamamoto, N., Ohni, M. Amer. A placebo-controlled study of the effect of sour milk on blood pressure in hypertensive subjects. American Journal of Clinical Nutrition. 1996, 64:767-771.
60. Joen Y J, Byun H G, Kim S K. Improvement of functional properties of cod frame protein hydrolysates using ultra filtration membrane. Process Biochemistry, 1999, 4(35): 471-478.
61. Jiang J L, Chen S W, Ren F Z, Luo Z, Steve S Z.Yak Milk Casein as a Functional Ingredient: Preparation and Identification of Angiotensin - I - Converting Enzyme Inhibitory Peptides. Journal of Dairy Research . 2007, 74:18-25.
62. Jenn S T, Jia L C, Bonnie S P. ACE-inhibitory peptides identified from the muscle protein hydrolysate of hard clam (Meretrix lusoria ). Process Biochemistry , 2008(43):743-747.
63. Jung W K, Mendis E, Je J Y, Park P J, Son B W, Kim H C, Choi Y K, Kim S K. AngiotensinI-converting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats. Food Chemistry, 2006 , 94 : 26 - 32.
64. Karaki H. Doi K, Suam S.Antihypertensive effect of tryptic hydrolysate of milk casein in spontaneously hypertensive .Comparative Biochemistry and Physiology, 1990, 96 C (2): 367 -371.
65. Kamarudlin.M.S, Jones.D.A, Vay.L.L. Ontogenetic change in digestive enzyme activity during larval development of macrobrachium rosenberii. Aquaculture. 1994, 123: 320 -324.
66. Korhonen H, Pihlanto A.Food-derived bioactive peptides-opportunities for designing future foods. Current Pharmaceutical Design, 2003, 9: 1297 -1308.
67. Kuba M, Tanz C, Tawata S..Production of angiotensin I- converting enzyme inhibitory peptides from soybean protein with Monascus purpureus acid proteinase. Process Biochemistry, 2005, 40: 2191 -2196.
68. Kuba M, Tana S, Tawata M, Yasuda. Production of angiotensin I-converting enzyme inhibitory peptides from soybean protein with Monascus purpureus acid proteinase. Process Biochemistry, 2005, (40): 2191-2196.
69. Kim J M, Whang J H, Kim K M, Koh J H, Suh H J. Preparation of corn gluten hydrolysate with angiotensin I converting enzyme inhibitory activity and its solubility and moisture sorption.Process Biochemistry, 2004, 39:989-999.
70. Lv G S, Huo G C, Fu X Y. Expression of milk-derived antihypertensive peptide in Escherichia Coli. Jonrnal of Dairy Science, 2003, 86(6): 1927-1931.
71. Lee J R, Kwon D Y, Shin H K, Yang C B.Purification and identification of angiotensin I converting enzyme inhibitory peptide from kidney been protein hydrolysate. Food science Biotechnol, 1999, 8:172-178.
72. Laurent V, Ragnar J, Maryse D, Patrick B, Pascal J. Concentration and purification of blue whiting peptide hydrolysates by membrane processes. Journal of Food Engineering, 2007, 83(4): 581-589.
73. Martha P, Aisling Aherne, Richard J. FitzGerald and Nora M. O'Brien A 90-day subchronic toxicity study and reproductive toxicity studies on ACE-inhibiting lactotri peptide. Food and Chemical Toxicology, 2007, 45: 1468-1477.
74. Mullally M M, Meisel H, FitzGerald R J. Angiotensin - I- converting enzyme inhibitory activities of gastric and pancreatic proteins. International Dairy Journal, 1997, 7:299-303.
75. Mullally M M, Meisel H, Fitzgerald R J. Identification of novel angioteusin - I - converting enzyme inhibitory peptide corresponding to a tryptic fragment of b-lactoglobulin. FEBS Letters. 1997, 402: 99-101.
76. Marczak E D, Hachiro U, Hiroyuki F , Yang Y J, Megumi Y, Andrzej W. L, Masaaki Y. New antihypertensive peptides isolated from rapeseed. Peptides , 2003,(24): 791-798.
77. Miguel, M., Muguerza, B., Sa′nchez E., Delgado M. A, Recio I, Ramos M, Aleixandre M. A..Changes in arterial blood pressure of milk fermented by Enterococcus faecalis CECT 5728 in spontaneously hypertensive rats. British Journal of Nutrition. 2005 , 94: 34 - 36.
78. Muguerza B. , Ramos M.., Sánchez E., Manso M.A., Miguel M., Aleixandre A., Delgado M.A., Recio I . Antihyperteusive activity of milk fermented by Enterococcus faecalis stains isolated fiom raw milk . International Dairy Joural , 2006 , 16: 61-69.
79. Maeshima M, Sasaki T, Asahi T. Characterization of major proteins in sweet potato tuberous roots.Phytochemistry, 1985, 24: 1899-1902.
80. Nielsen P M, Petersen D, Dambmann C. Improved methods for determining food protein degree of hydrolysis . Journal of Food Science, 2001 ,66 (5): 642 -646.
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