羊胰酶提取工艺及水解酪蛋白动力学研究
|
摘要
目前国内生产的胰酶主要是猪胰酶,羊胰酶还是空白;羊胰酶属清真产品,为广大穆斯林消费者专用。本研究以羊胰脏为试验材料,采用有机溶剂法进行羊胰酶的提取,优选异丙醇为提取剂和沉淀剂;通过单因素试验和正交设计,对主要影响因素和工艺参数进行了研究,确定了胰酶提取的主要工艺流程和参数,优选了保护剂、激活剂和脱脂剂;参照中华人民共和国药典,测定了羊胰酶活力和理化指标;并研究了羊胰酶水解酪蛋白动力学特性。研究结果如下:
1.工艺流程:采用有机溶剂法,以冻藏的羊胰脏为原料,经过胰脏的破碎、激活、提取、沉淀、脱脂、真空干燥等工艺过程后得到羊胰酶。
2.提取工艺参数:优选提取剂浓度、激活时间、激活温度、激活pH值、提取温度和提取时间的最佳提取条件及参数。通过单因素试验,选出影响羊胰酶提取过程的主要因素及其参数范围,进一步通过L_(18)(3~7)正交试验,以胰蛋白酶活力为依据,筛选出的最佳工艺参数:提取剂异丙醇浓度10%、提取温度15℃、提取时间5h、激活pH6、激活温度15℃、激活时间5h。
3.优选保护剂和激活剂:筛选麦芽糖、淀粉和氯化钠为保护剂,Ca~(2+)-胰酶粉为激活剂,均得到高活力的羊胰酶。
4.沉淀工艺参数:优选沉淀剂浓度、沉淀温度、沉淀pH和静置时间的最佳沉淀条件及参数。通过单因素试验,选出影响胰酶沉淀的主要因素及其参数范围,进一步通过L_9(3~4)正交试验,以胰蛋白酶活力为依据,筛选出的最佳工艺参数:沉淀剂浓度60%、沉淀温度5℃、沉淀pH值6、静置时间1h。
5.优选丙酮为最佳脱脂剂,按上述最佳工艺提取的羊胰酶,通过胰酶活力的测定,胰蛋白酶的活力为2654U/g,胰淀粉酶的活力13800U/g,胰脂肪酶的活力为10400U/g,羊胰酶的收率为11.2%,脂肪残留为17.1mg/g,干燥失重为3.2%,均符合药典的规定。
6.羊胰酶水解酪蛋白的动力学特征:最适温度40℃、最适pH值8;通过热稳定性的研究,酶活力随时间延长逐渐降低,温度越高,胰酶失活越快,热稳定性越差;通过pH稳定性的研究,酶活力随时间延长逐渐降低,最适pH时,胰酶失活最快,pH稳定性越差;Ca~(2+)激活羊胰酶,并提高胰酶的稳定性,Na~+对胰酶活力无影响,EDTA可以抑制胰酶活性。
综上所述,采用异丙醇提取羊胰酶,工艺简单、提取效率高、产品酶活高、成本低、生产周期短。提高了生产效率和原料利用率,节约资源,减少污染。利用先进的生物分离工程技术进行羊胰酶的生产工艺研究,提高动物副产品综合利用中的回收率和保存率,在生化制药业中以及动物副产品综合利用中具有积极的意义。
Now production of pancreatin is mainly pig pancreatin,Ovine pancreatin in domestic blank, Ovine pancreatin is Muslim product for the Muslim consumers,The raw materials of the experiment is ovine pancreas, pancreatin extracting was studied by the methods of organic solvents, isopropyl alcohol as optimum extraction agent and precipitation agent.Through the single factor and the orthogonal test, the major effect factor and the process parameter of the pancreatin extracting method were studied, the main technical process and the parameter of pancreatin’s product has determined; protectant,activator and degreaser were studied;Through the pancreatin activities and physics chemistry Indicators were determined by the chinese pharmacopoeia,Kinetics of Ovine pancreatin hydrolyse casein were studied, the research results are as follows:
1. Technical process: During the experiment which product ovine pancreatin by organic solvents method, take the frozen ovine pancreas as the raw material, during the pancreas broken,activation,extract,precipitation,remove fat,vacuum drying to get the pancreatin.
2.Parameters of Extracting process: Optimizing activating time, activating temperature, activating pH value, extraction agent concentration, extracting temperature and extracting time of the best condition and parameters. Through the single factor test, determined the main impact factors and parameters, then using L_(18)(3~7)orthogonal test, take the activities of trypsin as the index, screened out the best process parameter were: extraction agent concentration 10%,extracting temperature 15℃, extracting time 5h, activating pH6,activating temperature15℃and activating time 5h.
3. Optimizing protectants and activators :Starch, maltose and sodium chloride as protectants and select the CaCl_2 -Pancreatin for the best activator.
4.Parameters of precipitating process : Optimizing Precipitation agent concentration, Precipitation temperature, Precipitation pH value and Static placed time of the best condition and parameters. Through the single factor test,Determined the main impact factors and parameters, then using L_9(3~4)orthogonal test, take the activities of trypsin as the index, screened out the best process parameter were: Precipitation agent concentration 60%,Precipitation temperature 5℃,Precipitation pH value 6 , Static placed time1h.
5. Optimizing degreaser : Acetone as the best degreaser . According to the confirmation test of above process parameter, the result indicated that the yield of pancreatin was 11.2%,the activities of trypsin,amylopsin,lipase respective were 2654 U/g,13800U/g,10400 U/g,Loss on drying 3.2%,Fat content 17.1mg/g, the results fit for the chinese pharmacopoeia.
6. Kinetics of Ovine pancreatin hydrolyse casein were studied: The optimum temperature was measured 40℃, the optimum pH was measured 8.Through the research of Thermal stability, pancreatin activity decreased with the time gradually increased, The higher the temperature, the faster inactivation of trypsin, The poor thermal stability;Through the research of pH stability, pancreatin activity decreased with the time gradually increased, At the optimum pH, the fastest inactivation of trypsin, The poor pH stability. According to research the affect of trypsin activity by Ca~(2+),Na~+and EDTA: Ca~(2+) can activate ovine pancreatin,and to improve the stability of the ovine pancreatin,Na~+ does not affect the activity of trypsin, but EDTA has Strong Inhibition effect to the activity of trypsin.
In summary,Using the isopropyl alcohol extraction ovine pancreatin powder,Simple process,higher yield ,higher activity of three enzymes, Lower cost and Short production cycle. Improve the production efficiency and the utilization rate of raw materials, conservation of resources, reduce pollution.Use advanced bioseparation engineering to produce pancreatin, increase the yield and enzyme activity which has positive significance in biochemical pharmaceutical industry and comprehensive utilization of animal by-products. Key words: Ovine Pancreas; Pancreatin; Isopropyl alcohol; Extract;
1.工艺流程:采用有机溶剂法,以冻藏的羊胰脏为原料,经过胰脏的破碎、激活、提取、沉淀、脱脂、真空干燥等工艺过程后得到羊胰酶。
2.提取工艺参数:优选提取剂浓度、激活时间、激活温度、激活pH值、提取温度和提取时间的最佳提取条件及参数。通过单因素试验,选出影响羊胰酶提取过程的主要因素及其参数范围,进一步通过L_(18)(3~7)正交试验,以胰蛋白酶活力为依据,筛选出的最佳工艺参数:提取剂异丙醇浓度10%、提取温度15℃、提取时间5h、激活pH6、激活温度15℃、激活时间5h。
3.优选保护剂和激活剂:筛选麦芽糖、淀粉和氯化钠为保护剂,Ca~(2+)-胰酶粉为激活剂,均得到高活力的羊胰酶。
4.沉淀工艺参数:优选沉淀剂浓度、沉淀温度、沉淀pH和静置时间的最佳沉淀条件及参数。通过单因素试验,选出影响胰酶沉淀的主要因素及其参数范围,进一步通过L_9(3~4)正交试验,以胰蛋白酶活力为依据,筛选出的最佳工艺参数:沉淀剂浓度60%、沉淀温度5℃、沉淀pH值6、静置时间1h。
5.优选丙酮为最佳脱脂剂,按上述最佳工艺提取的羊胰酶,通过胰酶活力的测定,胰蛋白酶的活力为2654U/g,胰淀粉酶的活力13800U/g,胰脂肪酶的活力为10400U/g,羊胰酶的收率为11.2%,脂肪残留为17.1mg/g,干燥失重为3.2%,均符合药典的规定。
6.羊胰酶水解酪蛋白的动力学特征:最适温度40℃、最适pH值8;通过热稳定性的研究,酶活力随时间延长逐渐降低,温度越高,胰酶失活越快,热稳定性越差;通过pH稳定性的研究,酶活力随时间延长逐渐降低,最适pH时,胰酶失活最快,pH稳定性越差;Ca~(2+)激活羊胰酶,并提高胰酶的稳定性,Na~+对胰酶活力无影响,EDTA可以抑制胰酶活性。
综上所述,采用异丙醇提取羊胰酶,工艺简单、提取效率高、产品酶活高、成本低、生产周期短。提高了生产效率和原料利用率,节约资源,减少污染。利用先进的生物分离工程技术进行羊胰酶的生产工艺研究,提高动物副产品综合利用中的回收率和保存率,在生化制药业中以及动物副产品综合利用中具有积极的意义。
Now production of pancreatin is mainly pig pancreatin,Ovine pancreatin in domestic blank, Ovine pancreatin is Muslim product for the Muslim consumers,The raw materials of the experiment is ovine pancreas, pancreatin extracting was studied by the methods of organic solvents, isopropyl alcohol as optimum extraction agent and precipitation agent.Through the single factor and the orthogonal test, the major effect factor and the process parameter of the pancreatin extracting method were studied, the main technical process and the parameter of pancreatin’s product has determined; protectant,activator and degreaser were studied;Through the pancreatin activities and physics chemistry Indicators were determined by the chinese pharmacopoeia,Kinetics of Ovine pancreatin hydrolyse casein were studied, the research results are as follows:
1. Technical process: During the experiment which product ovine pancreatin by organic solvents method, take the frozen ovine pancreas as the raw material, during the pancreas broken,activation,extract,precipitation,remove fat,vacuum drying to get the pancreatin.
2.Parameters of Extracting process: Optimizing activating time, activating temperature, activating pH value, extraction agent concentration, extracting temperature and extracting time of the best condition and parameters. Through the single factor test, determined the main impact factors and parameters, then using L_(18)(3~7)orthogonal test, take the activities of trypsin as the index, screened out the best process parameter were: extraction agent concentration 10%,extracting temperature 15℃, extracting time 5h, activating pH6,activating temperature15℃and activating time 5h.
3. Optimizing protectants and activators :Starch, maltose and sodium chloride as protectants and select the CaCl_2 -Pancreatin for the best activator.
4.Parameters of precipitating process : Optimizing Precipitation agent concentration, Precipitation temperature, Precipitation pH value and Static placed time of the best condition and parameters. Through the single factor test,Determined the main impact factors and parameters, then using L_9(3~4)orthogonal test, take the activities of trypsin as the index, screened out the best process parameter were: Precipitation agent concentration 60%,Precipitation temperature 5℃,Precipitation pH value 6 , Static placed time1h.
5. Optimizing degreaser : Acetone as the best degreaser . According to the confirmation test of above process parameter, the result indicated that the yield of pancreatin was 11.2%,the activities of trypsin,amylopsin,lipase respective were 2654 U/g,13800U/g,10400 U/g,Loss on drying 3.2%,Fat content 17.1mg/g, the results fit for the chinese pharmacopoeia.
6. Kinetics of Ovine pancreatin hydrolyse casein were studied: The optimum temperature was measured 40℃, the optimum pH was measured 8.Through the research of Thermal stability, pancreatin activity decreased with the time gradually increased, The higher the temperature, the faster inactivation of trypsin, The poor thermal stability;Through the research of pH stability, pancreatin activity decreased with the time gradually increased, At the optimum pH, the fastest inactivation of trypsin, The poor pH stability. According to research the affect of trypsin activity by Ca~(2+),Na~+and EDTA: Ca~(2+) can activate ovine pancreatin,and to improve the stability of the ovine pancreatin,Na~+ does not affect the activity of trypsin, but EDTA has Strong Inhibition effect to the activity of trypsin.
In summary,Using the isopropyl alcohol extraction ovine pancreatin powder,Simple process,higher yield ,higher activity of three enzymes, Lower cost and Short production cycle. Improve the production efficiency and the utilization rate of raw materials, conservation of resources, reduce pollution.Use advanced bioseparation engineering to produce pancreatin, increase the yield and enzyme activity which has positive significance in biochemical pharmaceutical industry and comprehensive utilization of animal by-products. Key words: Ovine Pancreas; Pancreatin; Isopropyl alcohol; Extract;
引文
[1]铁平.胰酶与胰酶制剂[J].药物园地,1999(2):2-3.
[2]中华人民共和国药典委员会.中华人民共和国药典(2部)[S].北京:化学工业出版社,2005,631-633.
[3]赵小兰.胰酶制备新工艺的研究[J].青海医药杂志,2000,4(30):56-57.
[4]JoeR.Kimmel,L. Joseph Hayden. Isolation and Characterization of a New Pancreatic Polypeptide Hormone[J].The Journal of biological chemistry, 1975, 25(24): 9369-9376.
[5]Duke GE, Kimmel JR, et a1.The influence of avian pancreatic polypeptide on gastric secrection and motility in laying hens[J]. Poultry Science, 1985, 64(6): 1231-1235.
[6] Duke GE. Recent studies on regulation of gastric motility in turkeys[J]. Poultry Science,1992, 72(1): 1-8.
[7]Duke GE,Kimmel JR, Redig PT, et al. Influence of exogenous avian pancreatic polypeptide on gastrointestinal motility in turkeys[J]. Poultry Science, 1979,58(1):239-246.
[8]朱晓彤,傅伟龙.禽胰多肽对粤黄公鸡生长的影响[J].华南农业大学学报,2002,23(2):65-69.
[9]曲瑞瑶,顾增发.侧脑室注射牛胰多肽对家兔糖耐量和血浆胰岛素浓度的影响[J].生理学报,1984, (36)4:331-335.
[10]王境岩,朱庆庚,徐长法.生物化学[M].北京:高等教育出版社,2002.
[11]郭勇.酶的生产与应用[M].北京:化学工业出版社,2003.
[12]王思新,焦中高,孙治军.固定化细胞技术在果酒生产中的应用[J].中国食品与营养,2003(1): 40-42.
[13]江西食品发酵工业科学研究所编.酶制剂生产和在食品工业中的应用.北京:轻工业出版社,1997:138-139.
[14]万峰,张莹,杨东来.猪胰脂肪酶制备工艺研究[J].畜牧兽医科技信息,2007(6):38-39.
[15]张海燕,丁玉,尹瑞卿等.脂肪酶酶活性的最新研究[J].生物学通报,2007,42(3):16-17.
[16]黄素珍.应用胰酶提高肉品利用价值的研究[J].中国畜产与食品,1999,6(4):148-149.
[17]晓萍.消化酶的临床应用[J].中国医院用药评价与分析,2004,4(6):378-379.
[18]皇甫建林,陆志强.胰激肽释放酶对糖尿病心血管白主神经功能异常的疗效观察[J].中国临床医学,2003,10(5):651-652.
[19]上海市达吉临床研究协作组.多种消化酶制剂治疗消化不良的多中心、随机、安慰剂、双盲、平行对照研究[J].胃肠病学,2003,8:79.
[20]欧阳安力,周燕,华平等.胰酶对皮肤角质细胞分离和传代的影响[J].生物工程学报,2002, 18(1):59-62.
[21]李可.胰激肽释放酶联合葛根素治疗视网膜静脉阻塞[J].眼视光学杂志,1999,1(3):161.
[22]De Cock R, FickerLA, Dart JG, eta1.Topical heparin in the treatment of lingneousconjunctivitis[J].Ophthalmology, 1995, 102 (11):1654 -1658.
[23]El-Kad, I RawlingsAV, FeinberC,etal.Broad specificity alkaline proteases efficiently reduce the visual scaling associated with soap-induced xerosis[J]. Arch Dermatol Res, 2001, 293 (10):500-512.
[24]Kijima T,Izumi T. Fluorescence spectroscopic study of alpha-chymotrypsin relevant to the enantioselectivity for optical resolution of amino acid esters in organic solvents[J]. Biotechnology Letters,2004,26(8):655-658.
[25]白清芬,明秀丽.甲硝哇配伍糜蛋白酶侧弯窿封闭治疗慢性盆腔炎疗效观察[J].实用医技杂志,2003,10 (3):204.
[26]徐凤彩.酶工程[M].北京:中国农业出版社,2001:66-67.
[27]何兆雄,张天民.动物生化制药基础[M].北京:中国商业出版社,1985:284-285.
[28]林国平.胰腺激活提取对胰酶原粉收率和激肽释放酶活性的影响[J].中国生化药物杂志,2004,25(2):105-107.
[29]李良铸,李明哗.最新生化药物制备技术[M].北京:中国医药科技出版社,2001:180.
[30]马翠娥,郑昌学,徐亦农等.高活力胰酶制备工艺的研究[J].中国生化药物杂志,1993,65(3):11-13
[31]周秀芹.环保型棉织物退浆用胰酶的制备技术[J].现代纺织技术,2006,5:4-6.
[32]杨东来,高学军.猪胰淀粉酶的制备工艺研究[J].药物生物技术,2006,13(3):211-213.
[33]刘军.胰酶工艺研究[J].中国生化药物杂志,1993,64(2):40-41.
[34]蒋丽萍.松子仁蛋白提取工艺的研究.科技论坛,2005,23(8):28-30.
[35]刘高强.稀碱法提取马尾松毛虫幼虫蛋白质的研究.食品科技,2006, (5):57.
[36]夏永林.羽毛蛋白的提取及其提取物特性分析.国外丝绸,2007,2(3):73-76.
[37]张跃林.蚌锰蛋白质的提取、组成及分类研究.中国食物与营养,2006,8(6):46-47.
[38]Hordur G. Kristinsson, Barbara A. Rasco. Kinetics of hydrolysis of Atlantic salm On (Salmo salary ) muscle proteins by alkaline proteases and avisceral serine protease mixture. Process Biochemistry, 2000(36):131-139.
[39]刘德富,陆梅,卢军等.猪胰五种酶制品的联产.食品科学,1995,16(3):23-27.
[40]Floreto EAT, Baer R C, Brown P B. The effect of soybean-based diets, with and without amino acid supplementation,on growth and biochemical composition of juvenile American-lobster,Homarus americanus Aquaculture, 2000(189):235.
[41]Gildberg A.Enzymatic processing of marine raw materials.Process Biochemistry,1993 (28) :1-15
[42]安正仑,高学军,孙福忠.猪胰酶制备工艺研究.饲料与添加剂,2006, (11):74-75.
[43]许浩鸿,余蓉,李晓红等.制备高活力胰酶的安全低污染新工艺.华西药学杂志,2005,20 (3):245-246.
[44]朱碧英,毋瑾超.不同酶解条件对鳀鱼蛋白水解物苦味及氨基酸组成的影响.中国水产科学,2001,8(3):73-76.
[45]林秀年,纪学斌,杨胜钦.酵素水解条件对水解淘汰鸡肉品质的影响.食品科学,1995, (6) :97-103.
[46]董士远,刘尊英.水生动物酶在水产品加工应用中的研究进展.食品与发酵工业,2005, 31(10):111-114.
[47]傅渝滨,李勤耕,邓春平等.从猪胰脏中提取胰酶的优化工艺探索[J].重庆医科大学学报,1995,20(4):305.
[48]何执中,何执静.高活力胰酶的生产[J].中国生化药物杂志,1994,15(3):190.
[49]何新霞,冯高德,张旭等.胰酶生产工艺的研究[J].现代应用药学,1994,11(3):35.
[50]汪晓英.猪胰脏中的胰酶的提取及激肽释放酶的分离纯化[J].昆明医学院学报,2002, (1):107.
[51]赵玉红.超声波辅助提取松子仁蛋白的工艺研究.中国林副特产,2008,2(1):92.
[52]郭勇.酶的生产与应用[M].北京:化学工业出版社出版,2003:20-22,79-122.
[53]迟玉杰,阎丽.实用蛋白质制备技术[M].哈尔滨:哈尔滨工业大学出版社,1998:22-23.
[54]罗贵民.酶工程[M].北京:化学工业出版社,2002:23-29.
[55]徐凤彩.酶工程[M].北京:中国农业出版社,2001:84.
[56]欧阳平凯.化工产品手册(生物化工产品)[M].北京:化学工业出版社,1999:80-115.
[57]彭立凤.壳聚糖的制备及其在废水处理中的应用[J].化学界,1999,40(4):176-179.
[58]杨通在,刘亦龙,杨君.阳离子型改性高分子絮凝剂对轻工废水的处理[J].工业水处理,1998,18(5):27-29.
[59]张廷红,万海清.壳聚糖提取动物胰脏中胰酶的工艺研究.西南科技大学学报, 2005,20 (1):72-74.
[60]吴晓英,张聚宝,林影等.胰酶制备工艺研究[J].广东药学院学报,2005,21 (1):64-67.
[61]Fleming I. Cytochrome p450 and vascular homeostasis[J].Circ Res, 2001, 89: 753.
[62]Zhang F, Qian JQ, WangDW. Arachidonate CYP hydroxyl-lases of kidney contribute to formation of hypertension and maintenance of blood pressure [J]. Acta Pharmacol Sin,2002, 23: 497.
[63]Wang H, L in L, J iang J , et al. Up-regulation of endothelial Nitric-oxide synthase by endothelium-derived hyperpolarizing factor involves mitogen-activated protein kinase and p- rotein kinase C signaling pathways [J]. J Pharmacol Exp Ther,2003, 307: 753.
[64]林立,王红,陆再英等.内皮源性超极化因子对内皮一氧化氮合酶基因表达的调节[J].中国药理学通报,2003,19(9):1003.
[65]Abboudi, Mouhsine El, Beaulieu, et al, Mothed for producing pancreatin which contains low amounts of residual organic solvent and product thereof. [P]. US: 5861291.
[66]Schultze, Hans, Isolation of pancreatin [P]. US: 4623624.
[67]中华人民共和国卫生部,中国国家标准化管理委员会.中华人民共和国国家标准食品卫生检验方法,理化部分[S].中国标准出版社,2004.
[68]许学书,吴南,翟汉迁等.胰酶水解干酪素的动力学行为.华东理工大学报,2000,26(4): 346-349.
[69]Cohen T, Gertler A and Birk Y. Pancreatic proteolytic enzymes from carp(Cyprinus carpio)-I. Purification and physical properties of trypsin, chymotrypsin,elastase and carboxypeptidase B[J]. Comparative Biochemistry and Physiology,1981,69B:639-646.
[70]Hjelmeland K, Raa J.Characteristics of two trypsin type isozymes isolated from the Arctic fish capelin(Mallotus villosus)[J]. Comparative Biochemistry and Physiology,1982,71B:557-562.
[71]Yoshinaka R M,Suzuki S T and Ikeda S. Enzymatic characterization of anionic trypsin of catfish(Parasilurus asotus)[J]. Comp Biochem.Physiol.1984.77B:1-6.
[72]Simpson B K and Haard N F. Purification and characterization of trypsin from the Greenland cod(Gadus ogac). Kinetic and thermodynamic characteristics[J]. Canadian Journal of Biochemistry and Cell Biology,1984,62:894-900.
[73]刘欣华,张敏,胡世兴.胰蛋白酶预防后发性白内障的实验研究[J].眼外伤职业眼病杂志,2004,24(4):369-370.
[74]宋东奎,安书虹.胰蛋白酶加乙二胺四乙酸钠溶解草酸钙结石实脸研究[J].郑州大学学报(医学版),2002,37(4) :483-484.
[75]黄素珍.应用胰酶提高肉品利用价值的研究[J].中国畜产与食品,1999,6(4):148-149.
[76]王友同,吴文俊.胰脏综合利用的求索.生化药物杂志,1984, (4):5-14.
[77]Lconhard H. Hans K, Friedrich K.Simulteneous separation of insulin and pancreatin from swine pancreas, Ger.Offen 2400334.1974
[78]王益民,陈玮,任涛等.胰岛素-胰酶联产工艺研究初报.生化药物杂志,1986,(4):56- 57.
[79]唐爱星,何泽超,郭丽.絮凝法生产胰酶.中国生化药物杂志,2005,26(3):166-168.
[80]孙春亮.胰酶活化机制的研究进展.国外医学外科学分册,1996,23(5):265-267.
[81]许学书,吴南,翟汉迁等.胰酶水解干酪素的动力学行为[J].华东理工大学学报,2000,26 (4):346-348.
[82]胡红嫣.异丙醇的应用及国内外市场[J].当代石油化工,2003,11 (5):17-20.
[83]李良铸,李明晔.最新生化药物制备技术[M].北京:中国医学科技出版社,2002,179-183.
[84]林元藻,王凤山,王转花等.生化制药学.北京:人民卫生出版社,1998,191.
[85]范秀娟,张达磊,李桂生.温度及不同浓度乙醇对胰酶活力的影响.中国生化药物杂志, 2001,22(2):95-96.
[86]A.V.Berdutina, A.D.Neklyudov, A.I.Ivankin, et al. Comparison of proteolytic activities of the enzyme complex from mammalian pancreas and pancreatin[J]. Applied Biochemistry and Microbiology, 2000, 36(4): 363-367.
[87]罗红,周昕.工业胰酶生产研究.中国生化药物杂志,1994,15(4):283-284.
[88]刘湘新,唐小武,刘进辉等.胰酶的制备与生物活性测定.中兽医医药杂志,2006,(6):35- 37.
[89]游文素.胰酶酶活力测定方法分析.药品检测,2000,9(5):23-24.
[90]王增禄,卢军,茹炳根等.猪胰脏多酶联产实验研究.第四军医大大学学报,1995,16 (4):294-299.
[91]周秀芹.环保型棉织物退浆用胰酶的制备.纺织导报,2006,6:82-83.
[92]龚丽芬,郑志福,陈碧娥.胰蛋白酶酶解文蛤的工艺条件[J].氨基酸和生物资源,2003, (2):45-47.
[93]Zambonino-Infante J L,Cahu C L, Peres A et al. Sea bass(Dicentrarchus labrax)larvae fed different Artemia rations;growth, pancreas enzymatic response and development of digestive function[J]. Aquaculture,1996,139(1-2):129-138.
[94]Kohla U,Saint-Paul U, Friebe J et al.Growth, digestive enzyme activities and hepatic glycogen levels in juvenile Colosoma macropomum Cuvier from South America during feeding, starvation and refeeding[J]. Aquaculture Fisheries Management,1992,23(1):189-208.
[95]胡银英,袁铿,戴志芳等.胰蛋白酶在原代细胞制备中的灵活应用.江西医学院学报,2002,42,(1):43-44.
[96]范丽.关于工业用胰酶标准的建议[J].皮革化工,1997,15 (2) :38.
[97]冯定远.酶制剂在饲料工业中的应用[M].北京中国农业科学技术出版社,2005,299-307.
[2]中华人民共和国药典委员会.中华人民共和国药典(2部)[S].北京:化学工业出版社,2005,631-633.
[3]赵小兰.胰酶制备新工艺的研究[J].青海医药杂志,2000,4(30):56-57.
[4]JoeR.Kimmel,L. Joseph Hayden. Isolation and Characterization of a New Pancreatic Polypeptide Hormone[J].The Journal of biological chemistry, 1975, 25(24): 9369-9376.
[5]Duke GE, Kimmel JR, et a1.The influence of avian pancreatic polypeptide on gastric secrection and motility in laying hens[J]. Poultry Science, 1985, 64(6): 1231-1235.
[6] Duke GE. Recent studies on regulation of gastric motility in turkeys[J]. Poultry Science,1992, 72(1): 1-8.
[7]Duke GE,Kimmel JR, Redig PT, et al. Influence of exogenous avian pancreatic polypeptide on gastrointestinal motility in turkeys[J]. Poultry Science, 1979,58(1):239-246.
[8]朱晓彤,傅伟龙.禽胰多肽对粤黄公鸡生长的影响[J].华南农业大学学报,2002,23(2):65-69.
[9]曲瑞瑶,顾增发.侧脑室注射牛胰多肽对家兔糖耐量和血浆胰岛素浓度的影响[J].生理学报,1984, (36)4:331-335.
[10]王境岩,朱庆庚,徐长法.生物化学[M].北京:高等教育出版社,2002.
[11]郭勇.酶的生产与应用[M].北京:化学工业出版社,2003.
[12]王思新,焦中高,孙治军.固定化细胞技术在果酒生产中的应用[J].中国食品与营养,2003(1): 40-42.
[13]江西食品发酵工业科学研究所编.酶制剂生产和在食品工业中的应用.北京:轻工业出版社,1997:138-139.
[14]万峰,张莹,杨东来.猪胰脂肪酶制备工艺研究[J].畜牧兽医科技信息,2007(6):38-39.
[15]张海燕,丁玉,尹瑞卿等.脂肪酶酶活性的最新研究[J].生物学通报,2007,42(3):16-17.
[16]黄素珍.应用胰酶提高肉品利用价值的研究[J].中国畜产与食品,1999,6(4):148-149.
[17]晓萍.消化酶的临床应用[J].中国医院用药评价与分析,2004,4(6):378-379.
[18]皇甫建林,陆志强.胰激肽释放酶对糖尿病心血管白主神经功能异常的疗效观察[J].中国临床医学,2003,10(5):651-652.
[19]上海市达吉临床研究协作组.多种消化酶制剂治疗消化不良的多中心、随机、安慰剂、双盲、平行对照研究[J].胃肠病学,2003,8:79.
[20]欧阳安力,周燕,华平等.胰酶对皮肤角质细胞分离和传代的影响[J].生物工程学报,2002, 18(1):59-62.
[21]李可.胰激肽释放酶联合葛根素治疗视网膜静脉阻塞[J].眼视光学杂志,1999,1(3):161.
[22]De Cock R, FickerLA, Dart JG, eta1.Topical heparin in the treatment of lingneousconjunctivitis[J].Ophthalmology, 1995, 102 (11):1654 -1658.
[23]El-Kad, I RawlingsAV, FeinberC,etal.Broad specificity alkaline proteases efficiently reduce the visual scaling associated with soap-induced xerosis[J]. Arch Dermatol Res, 2001, 293 (10):500-512.
[24]Kijima T,Izumi T. Fluorescence spectroscopic study of alpha-chymotrypsin relevant to the enantioselectivity for optical resolution of amino acid esters in organic solvents[J]. Biotechnology Letters,2004,26(8):655-658.
[25]白清芬,明秀丽.甲硝哇配伍糜蛋白酶侧弯窿封闭治疗慢性盆腔炎疗效观察[J].实用医技杂志,2003,10 (3):204.
[26]徐凤彩.酶工程[M].北京:中国农业出版社,2001:66-67.
[27]何兆雄,张天民.动物生化制药基础[M].北京:中国商业出版社,1985:284-285.
[28]林国平.胰腺激活提取对胰酶原粉收率和激肽释放酶活性的影响[J].中国生化药物杂志,2004,25(2):105-107.
[29]李良铸,李明哗.最新生化药物制备技术[M].北京:中国医药科技出版社,2001:180.
[30]马翠娥,郑昌学,徐亦农等.高活力胰酶制备工艺的研究[J].中国生化药物杂志,1993,65(3):11-13
[31]周秀芹.环保型棉织物退浆用胰酶的制备技术[J].现代纺织技术,2006,5:4-6.
[32]杨东来,高学军.猪胰淀粉酶的制备工艺研究[J].药物生物技术,2006,13(3):211-213.
[33]刘军.胰酶工艺研究[J].中国生化药物杂志,1993,64(2):40-41.
[34]蒋丽萍.松子仁蛋白提取工艺的研究.科技论坛,2005,23(8):28-30.
[35]刘高强.稀碱法提取马尾松毛虫幼虫蛋白质的研究.食品科技,2006, (5):57.
[36]夏永林.羽毛蛋白的提取及其提取物特性分析.国外丝绸,2007,2(3):73-76.
[37]张跃林.蚌锰蛋白质的提取、组成及分类研究.中国食物与营养,2006,8(6):46-47.
[38]Hordur G. Kristinsson, Barbara A. Rasco. Kinetics of hydrolysis of Atlantic salm On (Salmo salary ) muscle proteins by alkaline proteases and avisceral serine protease mixture. Process Biochemistry, 2000(36):131-139.
[39]刘德富,陆梅,卢军等.猪胰五种酶制品的联产.食品科学,1995,16(3):23-27.
[40]Floreto EAT, Baer R C, Brown P B. The effect of soybean-based diets, with and without amino acid supplementation,on growth and biochemical composition of juvenile American-lobster,Homarus americanus Aquaculture, 2000(189):235.
[41]Gildberg A.Enzymatic processing of marine raw materials.Process Biochemistry,1993 (28) :1-15
[42]安正仑,高学军,孙福忠.猪胰酶制备工艺研究.饲料与添加剂,2006, (11):74-75.
[43]许浩鸿,余蓉,李晓红等.制备高活力胰酶的安全低污染新工艺.华西药学杂志,2005,20 (3):245-246.
[44]朱碧英,毋瑾超.不同酶解条件对鳀鱼蛋白水解物苦味及氨基酸组成的影响.中国水产科学,2001,8(3):73-76.
[45]林秀年,纪学斌,杨胜钦.酵素水解条件对水解淘汰鸡肉品质的影响.食品科学,1995, (6) :97-103.
[46]董士远,刘尊英.水生动物酶在水产品加工应用中的研究进展.食品与发酵工业,2005, 31(10):111-114.
[47]傅渝滨,李勤耕,邓春平等.从猪胰脏中提取胰酶的优化工艺探索[J].重庆医科大学学报,1995,20(4):305.
[48]何执中,何执静.高活力胰酶的生产[J].中国生化药物杂志,1994,15(3):190.
[49]何新霞,冯高德,张旭等.胰酶生产工艺的研究[J].现代应用药学,1994,11(3):35.
[50]汪晓英.猪胰脏中的胰酶的提取及激肽释放酶的分离纯化[J].昆明医学院学报,2002, (1):107.
[51]赵玉红.超声波辅助提取松子仁蛋白的工艺研究.中国林副特产,2008,2(1):92.
[52]郭勇.酶的生产与应用[M].北京:化学工业出版社出版,2003:20-22,79-122.
[53]迟玉杰,阎丽.实用蛋白质制备技术[M].哈尔滨:哈尔滨工业大学出版社,1998:22-23.
[54]罗贵民.酶工程[M].北京:化学工业出版社,2002:23-29.
[55]徐凤彩.酶工程[M].北京:中国农业出版社,2001:84.
[56]欧阳平凯.化工产品手册(生物化工产品)[M].北京:化学工业出版社,1999:80-115.
[57]彭立凤.壳聚糖的制备及其在废水处理中的应用[J].化学界,1999,40(4):176-179.
[58]杨通在,刘亦龙,杨君.阳离子型改性高分子絮凝剂对轻工废水的处理[J].工业水处理,1998,18(5):27-29.
[59]张廷红,万海清.壳聚糖提取动物胰脏中胰酶的工艺研究.西南科技大学学报, 2005,20 (1):72-74.
[60]吴晓英,张聚宝,林影等.胰酶制备工艺研究[J].广东药学院学报,2005,21 (1):64-67.
[61]Fleming I. Cytochrome p450 and vascular homeostasis[J].Circ Res, 2001, 89: 753.
[62]Zhang F, Qian JQ, WangDW. Arachidonate CYP hydroxyl-lases of kidney contribute to formation of hypertension and maintenance of blood pressure [J]. Acta Pharmacol Sin,2002, 23: 497.
[63]Wang H, L in L, J iang J , et al. Up-regulation of endothelial Nitric-oxide synthase by endothelium-derived hyperpolarizing factor involves mitogen-activated protein kinase and p- rotein kinase C signaling pathways [J]. J Pharmacol Exp Ther,2003, 307: 753.
[64]林立,王红,陆再英等.内皮源性超极化因子对内皮一氧化氮合酶基因表达的调节[J].中国药理学通报,2003,19(9):1003.
[65]Abboudi, Mouhsine El, Beaulieu, et al, Mothed for producing pancreatin which contains low amounts of residual organic solvent and product thereof. [P]. US: 5861291.
[66]Schultze, Hans, Isolation of pancreatin [P]. US: 4623624.
[67]中华人民共和国卫生部,中国国家标准化管理委员会.中华人民共和国国家标准食品卫生检验方法,理化部分[S].中国标准出版社,2004.
[68]许学书,吴南,翟汉迁等.胰酶水解干酪素的动力学行为.华东理工大学报,2000,26(4): 346-349.
[69]Cohen T, Gertler A and Birk Y. Pancreatic proteolytic enzymes from carp(Cyprinus carpio)-I. Purification and physical properties of trypsin, chymotrypsin,elastase and carboxypeptidase B[J]. Comparative Biochemistry and Physiology,1981,69B:639-646.
[70]Hjelmeland K, Raa J.Characteristics of two trypsin type isozymes isolated from the Arctic fish capelin(Mallotus villosus)[J]. Comparative Biochemistry and Physiology,1982,71B:557-562.
[71]Yoshinaka R M,Suzuki S T and Ikeda S. Enzymatic characterization of anionic trypsin of catfish(Parasilurus asotus)[J]. Comp Biochem.Physiol.1984.77B:1-6.
[72]Simpson B K and Haard N F. Purification and characterization of trypsin from the Greenland cod(Gadus ogac). Kinetic and thermodynamic characteristics[J]. Canadian Journal of Biochemistry and Cell Biology,1984,62:894-900.
[73]刘欣华,张敏,胡世兴.胰蛋白酶预防后发性白内障的实验研究[J].眼外伤职业眼病杂志,2004,24(4):369-370.
[74]宋东奎,安书虹.胰蛋白酶加乙二胺四乙酸钠溶解草酸钙结石实脸研究[J].郑州大学学报(医学版),2002,37(4) :483-484.
[75]黄素珍.应用胰酶提高肉品利用价值的研究[J].中国畜产与食品,1999,6(4):148-149.
[76]王友同,吴文俊.胰脏综合利用的求索.生化药物杂志,1984, (4):5-14.
[77]Lconhard H. Hans K, Friedrich K.Simulteneous separation of insulin and pancreatin from swine pancreas, Ger.Offen 2400334.1974
[78]王益民,陈玮,任涛等.胰岛素-胰酶联产工艺研究初报.生化药物杂志,1986,(4):56- 57.
[79]唐爱星,何泽超,郭丽.絮凝法生产胰酶.中国生化药物杂志,2005,26(3):166-168.
[80]孙春亮.胰酶活化机制的研究进展.国外医学外科学分册,1996,23(5):265-267.
[81]许学书,吴南,翟汉迁等.胰酶水解干酪素的动力学行为[J].华东理工大学学报,2000,26 (4):346-348.
[82]胡红嫣.异丙醇的应用及国内外市场[J].当代石油化工,2003,11 (5):17-20.
[83]李良铸,李明晔.最新生化药物制备技术[M].北京:中国医学科技出版社,2002,179-183.
[84]林元藻,王凤山,王转花等.生化制药学.北京:人民卫生出版社,1998,191.
[85]范秀娟,张达磊,李桂生.温度及不同浓度乙醇对胰酶活力的影响.中国生化药物杂志, 2001,22(2):95-96.
[86]A.V.Berdutina, A.D.Neklyudov, A.I.Ivankin, et al. Comparison of proteolytic activities of the enzyme complex from mammalian pancreas and pancreatin[J]. Applied Biochemistry and Microbiology, 2000, 36(4): 363-367.
[87]罗红,周昕.工业胰酶生产研究.中国生化药物杂志,1994,15(4):283-284.
[88]刘湘新,唐小武,刘进辉等.胰酶的制备与生物活性测定.中兽医医药杂志,2006,(6):35- 37.
[89]游文素.胰酶酶活力测定方法分析.药品检测,2000,9(5):23-24.
[90]王增禄,卢军,茹炳根等.猪胰脏多酶联产实验研究.第四军医大大学学报,1995,16 (4):294-299.
[91]周秀芹.环保型棉织物退浆用胰酶的制备.纺织导报,2006,6:82-83.
[92]龚丽芬,郑志福,陈碧娥.胰蛋白酶酶解文蛤的工艺条件[J].氨基酸和生物资源,2003, (2):45-47.
[93]Zambonino-Infante J L,Cahu C L, Peres A et al. Sea bass(Dicentrarchus labrax)larvae fed different Artemia rations;growth, pancreas enzymatic response and development of digestive function[J]. Aquaculture,1996,139(1-2):129-138.
[94]Kohla U,Saint-Paul U, Friebe J et al.Growth, digestive enzyme activities and hepatic glycogen levels in juvenile Colosoma macropomum Cuvier from South America during feeding, starvation and refeeding[J]. Aquaculture Fisheries Management,1992,23(1):189-208.
[95]胡银英,袁铿,戴志芳等.胰蛋白酶在原代细胞制备中的灵活应用.江西医学院学报,2002,42,(1):43-44.
[96]范丽.关于工业用胰酶标准的建议[J].皮革化工,1997,15 (2) :38.
[97]冯定远.酶制剂在饲料工业中的应用[M].北京中国农业科学技术出版社,2005,299-307.