摘要
酪蛋白是是牛乳中最主要的蛋白质,占牛乳中蛋白质总量的80%。近年来研究表明,酪蛋白中除了富含人体必需氨基酸等营养物质外,还蕴藏着多种功能性生物活性肽。近年来随着高血压发病率日趋严重,人们健康意识的不断增强,源于酪蛋白中降压肽因其安全性高、来源广泛、无副作用的优点在高血压防治的过程中具有十分重要的作用,越来越成为国内外研究的热点。
本课题以酪蛋白为原料,采用蛋白酶水解,确定最佳的水解工艺参数,并获得了较高ACE抑制活性的水解物,对水解液进行了分离纯化。通过以上研究得出以下结论：
首先建立了体外快速测定降血压肽的血管紧张素转换酶(ACE)抑制活性的高效液相色谱分析方法。在优化后的条件下,可通过测定由ACE水解马尿酰组氨酰亮氨酸后产生的马尿酸的量得到抑制肽的活性。采用色谱条件为：柱温为25℃,流动相采用甲醇+0.02 mol/L醋酸铵=15+85(V/V),流速1.0ml/min,检测波长228nm。此时,马尿酸浓度与其峰面积呈良好的线性关系(R2为0.9999),马尿酸的平均回收率为99.17%-100.26%。样品处理后经高效液相色谱保留时间定性,峰面积定量。该方法简便快速,具有较好的重复性和精确性,为降血压产品的研制提供了简便可靠的检测手段。
论文在ACE抑制率的测定过程需要大量使用ACE,因此,本文对猪肺中ACE的提取条件进行了优化,得出最佳提取条件：pH为7.6；去杂质硫酸铵饱和度为26%；沉淀硫酸铵饱和度为45%；质液比为1：2.5,在此条件下得到酶液的总酶活力为132.2U,酶比活力为0.362U/mg蛋白,并对粗酶液进行SephadexG-100分离纯化,300g新鲜猪肺最终可制得ACE1027.68 U,比活力为1.085U/mg蛋白,酶活回收率为60.8%。
采用碱性蛋白酶、中性蛋白酶、胰蛋白酶、Alcalase (Novozymes)水解酪蛋白制备ACE抑制肽,并采取高效液相色谱法测定ACE抑制肽的抑制率,最终确定选择碱性蛋白酶作为水解用酶,制备ACE抑制肽。采用四因素三水平正交实验对碱性蛋白酶水解酪蛋白的条件进行了优化,结果显示在水解温度50℃、E/S为6.0%、pH10.0条件下水解6h的酶解产物对ACE的抑制效果最好,达到95.60%。
采用DA201-C大孔树脂吸附对水解样品进行脱盐处理,用不同浓度乙醇进行梯度洗脱。结果表明脱盐最佳工艺为样品初始浓度为20mg/ml,脱盐流速为0.5 BV/h,解吸剂为75%的乙醇溶液,脱盐处理后的酶解物灰分降至1.34%,抑制率有所提高,脱盐率为92.78%,氮回收率84.22%。
用凝胶层析技术SephadexG-25对脱盐处理后的酪蛋白的碱性蛋白酶水解物进行分离纯化,在如下分离条件下进行凝胶过滤分离取得较好的的分离效果：样品浓度：30mg/ml;上样量：3ml；流速：0.6ml/min;洗脱剂：0.05mol/LHAc-NaAc缓冲液(pH4.0)。制备得到的具有较高活性ACE抑制肽组分D,抑制率为92.3%,其组分蛋白质浓度为0.828mg/ml。再经SephadexG-15进行进一步分离纯化得到更高活性的组分F,抑制活性为96.49%,其组分蛋白质浓度为0.567mg/mL该组分的ACE抑制活性受pH值影响较大,总体上酸性条件下损失大于碱性条件,对热及消化道酶系统都具有较高的稳定性。
Casein is the main protein in milk, it is accounted for 80% of the total protein. Recently, many rsearches demonsrtated that casein was not only soucre of nutrients such as essential amino acids for individual gorwth, it also contains many functional and biological activity of peptides. with the growing incidence of hypertension in recent years, people's health consciousness is growing, blood pressure peptides which derived from casein has received the most attention for its safe,wide variety of sources and no side effects advantages in the therapy of hypertension.
In the present study, optimal hydrolysis conditions of casein of casein hydrolysates were determined, and it can obtained higher activity hydrolyzates.Then,we used some separation and purification methods to achieve higer ACE inhibitory peptide activity. The following were the results:
First, High performance liquid chromatography method was established for the anlysis of angiotensin converting enzyme(ACE) inhibitory activity of antihypertensive peptides fast. With this method, the activity of the inhibitory peptides was determined with the amount of hippuric acid which was released from substrate Hippuryl-His-leu (HHL) by ACE.The chromatographic conditions were as follows:column temperature:25℃,Methanol+0.02mo 1/L acetic acid solution (15+85(V/V)) was identified as the flow of mobile phase,the detection wavelength was 228nm,velocity was 1.0 ml/min;At this time, an excellent lineari ty(R2=0.9999) was observed. The hippuric acid reovery efficieney is 99.17%-100.26%. The samples were treated with HPLC,the qualitative and the quantitative analy ses were carried out by chromatography based on the retention time and peak area respectively. This method is simple, rapid and reproducible. This reliable method can be used for analysis of ACE activityof all kinds of antihypertensive peptide.
In this article, we would required a large use of ACE in the determin-ation of ACE inhibition process, Therefore, in this study, ACE was fracti-onated from pig's lung organism, the results the optimum extraction con-ditions:pH 7.6;the ammonium sulfate saturation which remove impurities is 26%;the ammonium sulfate saturation which precitipiate is 45%;The ratio bewteen quality and solution is 1:2.5. Under these conditions,the to-tal enzyme activty was 132.2U, the specific activity was 0.362U/mg pro-tein.The enzyme solution was purified by filtration on Sephadex G-100 column,300g feash pig's lung can obtained ACE which the total ACE ac-tivity is 1027.68 U, the specific activity was 1.085, enzyme activity reco- very was 60.8%.
ACE inhibitory peptides were prepared from casein protein by four commercial proteases (alcalase, trypsin, neutrase, alcalase(Novozymes)), and their ACE inhibitory activity were determined in vitro by high-performance liquid chromatography. Finally alcalase was used to hydrolyze casein for the production of ACE inhibitory peptides. The optimum hydrolysis conditions of casein of alcalase were studied by the orthogonal design. The result showed that the ACE inhibitory activity of the alcalase's hydrolysates of casein can reach 95.60% under hydrolytic temperature 50℃, E/S is 6.0% and hydrolytic pH10.0 conditions after hydrolyzed 6 hour.
DA201-C Macroporous were used for desalting of ACE inhibitory and antioxidative peptides from the hydrolysis by gradient ethanol elution. The results indicated that the optimum condition for desalting was obtained as follows:loading sample concentration of 20 mg/ml,sampling flow rate of 0.5 BV/h and 75% alcohol aseluent. Under this condition,the ash content was decreased to 1.34% and ACE inhibitory activity was improved, desalination ratio reached to 92.78% and nitrogen recovery ratio was 84.22%.
The Alcalase hydrolyzate of casein were separated by molecular sieve chromatographic column Sephadex G-25. The best separation condition was charaters:concentration of sample,30 mg/ml;injection volume,3 ml;flow,0.6ml/min;eluting reagent,0.05 mol/L HAc-NaAc buffer(pH 4.0). The ACE peptides obtained from the Sephadex G-25 process is the fraction D which its inhibitory activity is 92.3%,component protein concentration is 0.828mg/ml.This fraction were further fractionated with ultrafiltration Sephadex G-15,in order to obtain the fraction F with higher ACE inhibitory activity. The ACE inhibitory activity was 96.49% when the component protein concentration is 0.567mg/ml. The ACE inhibitory activity of this fraction was susceptible to pH. Overall, The ACE inhibitory activity under acid environment was prone to loss than that of alkaline,and it was shown high stability against different temperature and gastrointestinal proteases systerm.
引文
[1]Hughes. ITIsolation of all endogenous compound from the brain with the pharmacological properties similar to morphine[J]. BrainRes,1975,88(3):295-298.
[2]乐国伟,施用晖,杨凤.肽在动物蛋白质营养中的作用—小肽在动物氨基酸吸收中的作用[J].四川农业大学学报.1996,14：19-27.
[3]苏秀兰.生物活性肽的研究进展[J].内蒙古医学院学报,2006,5(28)：471-475.
[4]廖海艳,刘湘虎.生物活性肽的研究现状与应用前景[J].畜牧兽医科技信息.2008,2：17-20.
[5]Kilara A, Panyam D. Peptides from milk proteins and their properties[J]. Critical Reviews in Food Science and Nutrition,2003,43:607-633
[6]XU RJ. Bioactive peptides in milk and their biological and health implications[J]. Food Rev Int,1998,14:1-9.
[7]马歌丽,何红,王俊伟.酪蛋白磷酸肽提取工艺[J].食品研究与开发,2009,9(30)：122-126.
[8]吴蕾,庞广昌,张建辉.乳源性生物活性肽的研究进展[J].食品研究与开发,2009,4(30)：181-186.
[9]Ondetti M A, Rubin B, Cushman D W. Science,,1977,196:441-444.
[10]沈小琴,李朝慧,罗永康.乳清蛋白酶解ACE抑制肽分离纯化技术的研究[J].中国乳品工业,2005,11(33)：4-6.
[11]赵秀丽,陈捷,崔艳丽等.中国14省市高血压现状的流行病学研究[J].2006,16(86)：1148-1153.
[12]Kirkutis A, Norkiene S, Griciene P, et a.l Prevalence of hypertension in Lithuanian mariners. Proc West Pharmacol Soc,2004,47:71-75.
[13]李端,殷明.药理学[M].北京：人民卫生出版社,2006,214-215.
[14]陈修,陈维洲,曾贵云.心血管药理学[M].北京：人民卫生出版社,2003,346-348.
[15]贾敏.血管紧张素转换酶抑制剂的作用与临床应用[J].广西大学学报,1997,9(22)：277-281.
[16]Zimmerman Ben G, Dunham Earl W. Tissue renin-angiotensin system:a site of drug action?[J]. Ann Rev Pharmacol Toxicol,1997,37:53-69.
[17]杨严俊,李进伟.兔肺血管紧张素转换酶的分离提取及特性研究[J].食品与发酵工业.2002,8(29)：57-61.
[18]Cushman D W, CheungH S. Spectrophotometric assay and properties of the angiotensin Ⅰ-converting enzyme of rabbit lung [J]. Biochem Pharmaco,1971,20:1637-1648.
[19]Frederic E. Dorer, Joseph R. Kahn, Kenneth E. Lentz, Melvin Levine and Leonar D T.Ske Ggs. Purification and Properties of Angiotensin-Converting Enzyme from Hog Lung[J]. Cir culation research,1972,31:356-366.
[20]陈兰英,田敏.猪肺血管紧张素转换酶的提纯[J].生物化学杂志,1988,4(4)：345-352.
[21]孙凤祥,曲梅花,高佩林.猪肺血管紧张素转换酶的制备[J].中国生化药物杂志,2002,3(23)： 122-123.
[22]Ferreira S H.. A bradykinin-potentiating factor(BPF)present in the venom of Bothro psjararaca.Brit J Pharmacol,1965,24:163-169.
[23]李桂琴,刘晶,韩清波.蛋白酶水解牛乳蛋白的研究[J].中国乳品工业,2003,6(31)：11-14.
[24]辛志宏,吴守一,马海乐等.从麦胚蛋白质中制备降血压肽的研究[J].食品科学,200324(10)： 120-123.
[25]Yasunori Nakamura..Purification and Characterization of Angiotensin I-Converting Enzyme Inhibitors from Sour Milk[J]. Journal of Dairy Science,1995,78:1253-1257.
[26]李晓晖.牛乳中酪蛋白的结构特性及其应用[J].食品工业,2002,2：29-32.
[27]张建忠,郦一心.酪蛋白和酪蛋白制品的开发[J].中国乳品工业,1998,6(26)：31-32.
[28]范志红,南庆贤,乳中蛋白质和肽对人体的保护作用,中国乳品工业,2000,28(5)：30-33.
[29]周根来.酪蛋白磷酸肽的应用研究进展[J].饲料研究,2009,1(7)：10-15.
[30]Naovnki Yamainoto, Atsuko Akino, Toshiaki Takano. Antihypertensive effect of the peptides derived from casein by an extraccllnlar protcinase from lactobacillus helveticns CP790[J]. Dairv Sci,1994,77:917-922.
[31]Hidcaki Kaiaki, Kanio Doi, Shigecrn Snganoet al. Antihypertensive effect of tryptic hvdrolvsate of milk casein in spomtaneously hypertensivc rats[J].comp biochem Physiol,1990, 96(2):367-371.
[32]Susurnu Maruvarna,Hideo Suzuki.A peptide Inhibitor of angiotcnsin Ⅰ—converting enzyme in the tryptic hydrolysate of casein[J].Agrie Biolhere,1982,46(5):1393-1394.
[33]Maruyama S,Nakagomi K,Tomizuka N.Angiotensin—Ⅰ-converting enzyme inhibitor derived from an enzymatic hydrolysate of casein(Ⅱ):Isolation and bradykinin.-potentiating activity on uterus and the ileum of rats.Agric.Bio L Chem.,1985,49(5):1405-1409
[34]Yamamoto N,Maeno M,Takano T.Purification and characterization of an antihypertensive peptides from a yoghout-like product fermented by Lactobacillus helveticus CPN4[J].Journal of dairy science,1999,82:1388-1393.
[35]Maeno M,Yamamoto N,Takano T.Identification of an antihypertensive peptide from casein hydrolysate produced by a proteinase from lactobaccillus helucticus CP790[J].Journal of dairy science,1996,79:1316-1321.
[36]姜瞻梅,孔保华,田波等.食物蛋白源降血压肽的抗高血压效果研究[J].食品工业科技, 2009,8(30):329-332.
[37]赵一明,王璋,许时婴等.酪蛋白非磷肽的理化性质及ACE抑制活性[J].食品科技,2008, 5:100-104.
[38]翟光超.乳清蛋白研究进展[J].现代农业科技,2008,21:283-286.
[39]S.F. Gauthier, Y. Pouliot.Functional and Biological Properties of Peptides Obtained by Enzymatic Hydrolysis of Whey Proteins[J].Journal of Dairy Science,2003,1(86):78-87.
[40]OTTEA J,SHALABY S M,ZAKORA M,et al.Angiotensin-convert-ingenzyme inhibitor actiVity of milk protein hydrolysates:Effectof substrate,enzyme and time of hydrolysis[J].International Dairy Journal,2007,17:488-503.
[41]OTTE J,SHALABY S M A,ZAKORA M,et al.Fractionation and identification of ACE-inhibitory peptides from α-lactalbumin and β-Casein produced by thermolysin-catalysed hydrolysis[J].International DairyJournal,2007,17:1460-1472.
[42]田俊英,潘道东.水解乳清蛋白制备ACE抑制肽的工艺研究[J].食品科学,2009,24(30)152-158.
[43]SuetsunaK.Isolati on and charaeterizarion of angiotensin I-converting enzyme inhibitor dipeprides derived from Allium sativumL(garlic)[J].Nutr.Biochem,1998,9:415-419.
[44]豆康宁,董彬,王银满.大豆蛋白活性肽的生物功能与应用前景[J].粮食加工,2007, 32(2)：52-56.
[45]吴建平,丁霄霖.大豆降压肽的研制(Ⅰ)——生产高活性ACEI肽酶系的筛选[J].中国油脂,1998,2(23)：49-52
[46]Marsui T, LiCH, TanakaT, et al. Dcprcssor effect of wheat hydrolysate and its novel angiotensin converting enzyme inhibitory peptide, Ile-Val-Tyr, and the metabolism in rat and human plasma[J]..Bio Pharm Bull,2000,23(4):427-431.
[47]辛志宏,吴守一,马海乐等.从麦胚蛋白质中制备降血压肽的研究[J].食品科学,2003,20(24)：120-124.
[48]陈黎斌,杨严峻.从醋蛋水解物中分离纯化ACE抑制活性物质[J].食品与发酵工业,2007,12(33)：29-33.
[49]刘焕.大米ACE抑制肽的研究[D].无锡：江南大学,2005.
[50]姜瞻梅,霍贵成.不同食物来源的ACE抑制肽的研究现状[J].食品研究与开发,2003,1(24)：27-30.
[51]Jianping Wu, Xiaolin Ding.Hypotensive and Physiological.Effect of Angiotensin Converting Enzyme Inhibitory Peptides Derived from Soy Protein on Spontaneously Hypertensive Rats[J]. J Agric Food Chem,2001, (49):501-506.
[52]Cushman D W, Cheung H S. Spectrophotometric Assay and Properties of the Angiotensin Converting Enzyme of Rabbit Lung[J]. Biochem Pharmol,1971,20:1637-1648.
[53]姜瞻梅,田波,吴刚等.高效液相色谱法快速测定降血压肽的血管紧张素转换酶抑制活性[J].食品与发酵工业,2007,8(33)：122-127.
[54]Zhang R, Xu X, Chen T,et al. An assay for angiotensin-converting enzyme using capillary zone electrophoresis[J]. Anal Biochem,2000,280(2):286-290.
[55]辛志宏,马海乐,吴守一等.高效毛细管电泳测定血管紧张素转化酶抑制剂captopril的活性[J].药学学报,2003,38(11)：843-845.
[56]金化民,张茨,陈葳等.酶偶联法测定血清血管紧张素转化酶[J].中华医学检验杂志,1998,21：362-365.
[57]Shin Zl, Yu R, Park SA, et al His-His-Leu, an angiotensm I converting enzyme inhibitory peptide denved from Korean soybean paste, exerts antihypertenswe activity in vivo [J]. Agmc Food Chem,2001,49:304-309.
[58]洪宇波,史劲松,张卫明等.谷朊蛋白制备A C E抑制肽的酶解工艺优化[J].食品研 究与开发,2008,6(29)：15-21.
[59]Lv US, Huo GC, Fu XY Expression of milk-Derived Anthyipertensive Peptide m Eschverichia colt. J Dairy Sci,2003,86:1927-1931.
[60]赵骏,宫霞,郭本恒.乳酪蛋白源ACE抑制肽的分离纯化[J].中国乳品工业,2006,6(34)： 8-12.
[60]段涛,叶磊.降血压肽的研究及应用背景[J].江苏食品与发酵,2008,4：14-18.
[61]河村幸雄.大豆タンパク质变换酵素阻害血压效果作用[J].食品工业(日),1997,30：73-77.
[62]邬显章.酶的生产技术[M].吉林科技出版社.1988.
[63]徐榕榕,陶冠军,李进伟等.高效液相色谱测定血管紧张素转换酶抑制肽的活性[J],河南工业大学学报(自然科学版),2005,4(26)：18-22.
[64]赵剑虹,孙成均,毛丽莎.高效液相色谱法测定猪血清中的依维菌素[J].四川大学学报(医学版),2005；36(1)：130-131.
[65]黄家音,朱禹洁.降血压肽研究进展[J].食品与发酵工业,2006,323(6)：81-86..
[66]Bin-WhaChang,RichieLCChen,I-JenHuang,etal.Assays for Angiotensin Converting Enzyme Inhibitory Activity [J]. AnalyticalBiochemistry,2001,291,84-88.
[67]王立晖.胃蛋白酶酶解酪蛋白制备酪啡肽的研究[D].成都：西华大学,2006.
[68]于婷婷.酶法制备大豆蛋白ACE抑制肽的研究[D].吉林农业大学,2008.
[69]耿秀芳、李耀辉、李桂芝等。猪肺血管紧张素转换酶的提纯[J].,医学研究生学报2001,14(5)：428-431.
[70]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,2004,9(25)：71-75.
[71]Meisel, H. and Schlimme, E..Milk protein precursors of bioactive peptides[J].Trends Food Sc Technol.1990,1:41-50.
[72]李建武,余瑞元,袁明秀等.生物化学实验原理与方法[M].北京：北京大学出版社.160-165.
[73]郭兴凤.蛋白质水解度的测定[J].中国油脂,2000.25(6)：176-177..
[74]吕桂善,周文红.酪蛋白水解进程的研究[J].食品工业科技,2003,8：24-26.
[75]包怡红.乳清蛋白的酶解及其性质研究[[J].食品科学,2003(9)：63-65.
[76]姜瞻梅,田波,吴刚,霍贵成.酶解牛乳酪蛋白制备ACE抑制肽的研究[J].中国食品学报,2007,7(6)：39-42.
[77]孙浩,蔡兴旺.碱性蛋白酶对酪蛋白水解的最适条件[J].大连轻工业学院学报,2003,22(1)：25-27.
[78]安桂香,庄桂东,徐振凯等.食物中血管紧张素转化酶抑制肽的研究进展[J].食品研究与开发,2006,27(6)：173-174.
[79]赵新淮,冯志彪.大豆蛋白水解物水解度测定的研究[J].东北农业大学学报,1995,26(2)：178-181.
[80]张宇昊.花生短肽制备及功能活性研究[D].北京：中国农业科学院农产品加工研究所,1983.
[81]赵利,王璋,许时婴.大孔吸附树脂对酪蛋白非磷肽的脱盐和色谱分离[J].无锡轻工大学学报,2003,22(7)：69-71.
[82]潘道东,林璐DA201-C大孔吸附树脂静态吸附ACE抑制肽的研究[J].食品科学,2009,5(30)：20-24.
[83]何炳林,黄文强.离子交换与吸附树脂[M].上海：上海科技教育出版社,1995.
[84]刘大川,周俊梅.富硒菜籽蛋白肽的精制研究.[J].食品科学,2008,29(1)：129-133.
[85]李建武,余瑞元,袁明秀等.生物化学实验原理和方法[M].北京：北京大学出版社,2000,209-215.
[86]张龙翔等.生物化学实验方法和技术[M].北京：高等教育出版社,1996,117-124.
[87]汪家政,范明.蛋白质技术手册[M].北京：科学出版社,2001.
[88]杨锋,马千里,黄永春.醋蛋中ACE抑制肽的分离及稳定性研究[J].食品科学,2009,24(30)：77-80.
[89]石艳军,金义鑫,袁琳等.食品蛋白质中血管紧张素转化酶抑制肽的研究[J].生物技术通报,2004,6：6-10.
