卵黄高磷蛋白磷酸肽的制备及其对小鼠免疫功能的影响
|
摘要
为了初步探索卵黄高磷蛋白磷酸肽(PPP)是否具有免疫作用,本研究在制备了PPP的基础上,考察了PPP对小鼠体内外免疫功能的影响,并初步分析PPP影响机体免疫功能的机理。本研究包括以下三个试验。
试验一,PPP的制备。本试验首先从鸡蛋黄中提取卵黄高磷蛋白(PV),经胰蛋白酶水解,水解液经截留分子量3 kDa和1 kDa的超滤膜超滤,取分子量在1 kDa-3 kDa之间的截留液冷冻干燥得到PPP。经SDS-PAGE电泳测PV的纯度,用N/P(摩尔比)检测PPP的磷酸丝氨酸残基密度。结果表明:所制备的PV样品与纯品电泳模式相近,在1 kDa-3 kDa范围内存在PPP电泳条带,且N/P比例为5.02。说明所提取PV样品相对较纯,PPP样品含有磷酸丝氨酸残基。
试验二,PPP对小鼠体外免疫功能的影响。为了评价PPP对细胞增殖、吞噬功能、细胞因子以及免疫球蛋白生成的影响,分离小鼠的脾淋巴细胞和腹腔巨噬细胞并进行培养。结果显示:添加LPS或ConA后,显著地促进了细胞的增殖、细胞吞噬功能、细胞因子的分泌以及免疫球蛋白的生成(p＜0.05)。终浓度为0.02 mg/mL的PV组不能显著地影响细胞功能(p＞0.05)。然而,终浓度为0.02 mg/mL的PPP组和0.1 mg/mL PPP组显著地提高了刺激指数(SI)和吞噬指数(PI),以及IgA、IL-1β、IL-2和IL-6的生成。LPS或ConA诱导组比无丝裂原组刺激作用显著增强。结果表明,PPP显著提高了SI、PI、细胞因子以及免疫球蛋白生成的影响,无丝裂原组相比,LPS或ConA诱导下,以上各指标均显著提高。
试验三,PPP对小鼠体内免疫功能的影响。为了确定胃饲PPP对小鼠免疫功能的影响,将48只小鼠随机分到3个处理中,每个处理设4个重复,每个处理的小鼠在基础日粮的基础上分别隔天灌胃0.2mL生理盐水(对照组)、PPP和PV溶液,按体重喂给0.03mg/(g·d)。14天后取样确定小鼠的生长性能和免疫功能。结果显示:与对照组和PV组相比,PPP组小鼠的体增重极显著提高(p＜0.01),血清总蛋白、白蛋白、球蛋白和葡萄糖均升高,但差异不显著(p＞0.05),而血清尿素氮、总胆固醇和甘油三酯呈下降趋势,脾脏指数和胸腺指数呈显著提高(p＜0.05)。PPP组的SI和PI显著提高(p＜0.05),血清和脾淋巴细胞培养液中的IgA、IL-2和IL-6浓度,以及血清和腹腔巨噬细胞培养液中的IL-1β均显著地上升(p＜0.05)。PV组与对照组相比,这些指标均无显著变化(p＞0.05)。在LPS或ConA诱导下,培养液上清中的SI、PI、IgA、IL-1β、IL-2和IL-6均有显著的提高(p＜0.05)。结果表明,胃饲PPP不仅提高了体增重,脾脏指数和胸腺指数,还显著增强了小鼠SI和PI,血清和脾淋巴细胞培养液中的IgA、IL-2和IL-6浓度,以及血清和腹腔巨噬细胞培养液中的IL-1β浓度。与无丝裂原组相比,LPS或ConA诱导组以上各指标均显著提高。
综合以上各试验可看出,制备所得的PPP不仅提高了小鼠的生长性能,还增强了小鼠体内外免疫功能。
Effects of phosvitin phosphopeptides(PPP) from enzymic hydrolysates of phosvitin (PV) which come from chicken egg-yolk on immune functions of mice were investigated. On the basis of preparing PPP,the study focuses on the immune functions of mice in vivo or vitro.The whole study consisted of three experiments.
Experiment 1:Preparing he PPP
First,the phosvitin extracted from chicken egg-yolk was hydrolyzed by trypsin.Then, the hydrolysate was ultrafiltrated by ultrafiltration membrane.The filtrate of MW between 1kDa-3kDa was collected and lyophilized to get the PPP.PPP purity was determined by SDS-PAGE and N/P(molar ratio).The result indicated that electrophoresis strip of extracted PV was similar to the pure of PV.Electrophoresis of PPP showed that its MW was between 1-3kDa,N/P was 5.02.It was concluded that,the purity of PV and PPP prepared here was high enough for further study.
Experiment 2:Effects of PPP on Immune Functions of Mice in vitro
The Mice's spleen lymphocytes and peritoneal macrophages were isolated and cultured to evaluate the effects of PPP on cell stimulation index(SI),phagocytosis index(PI) and cytokine secretions and immunoglobulin formation.Results showed that LPS or ConA administration significantly stimulated cell proliferation,phagocytosis,cytokine secretion and immunoglobulin formation in vitro(p＜0.05).PV administration(0.02 mg/mL) did not significantly affect the cell functions.However,PPP administration(0.02 mg/mL and 0.1 mg/mL) significantly stimulated the SI and elevated the concentrations of IgA,IL-2 and IL-6(p＜0.05) in culture supematant.The stimulating effects of PPP for LPS/ConA-induced cells were stronger than uninduced cells.These results suggested that PPP administration enhanced the SI,PI,cytokine secretions and immunoglobulin formation.When the LPS or ConA existed,the indicators were more obvious effects.
Experiment 3:Effects of PPP on Immune Function of Mice in vivo
To determine the effects of orally administrated PPP on immune functions of mice.A total of 48 mice were randomly assigned to three treatments with 4 replications.Mice were administrated with 0.2 mL saline(control group),PPP and PV solutions(0.03 mg/g.BW.d), respectively.After 14 days,all mice were killed and sampled to investigate the growth performance and immune parameters.Results showed that the weight gain of PPP group was significantly improved(p＜0.05).The concentrations of serum total protein,albumin, globulin and glucose in PPP group tended to be increased than control or PV groups (p＞0.05),However,the concentrations of serum urea nitrogen,total cholesterol and triglyceride tended to be decreased.The spleen index and thymus index of PPP group were significantly improved(p＜0.05).Results form primary culture of spleen lymphocytes and peritoneal macrophages indicated that LPS and ConA significantly stimulated the SI and PI, and increased the concentrations of IgA,IL-1β,IL-2 and IL-6 in culture supematant (p＜0.05).The SI and PI were also significantly improved by PPP administration(p＜0.05). In addition,the concentrations of IgA,IL-2 and IL-6 in serum and spleen lymphocytes culture,the IL-1βin peritoneal macrophages culture were both actutely elevated(p＜0.05). However,none of these parameters altered by PV administration(p＞0.05).These results suggested that orally PPP administration not only improved the performance,but enhanced the SI,PI,cytokine secretions and immunoglobulin formation.When the LPS or ConA existed,the indicators were more obvious effects.
In summary,PPP prepared from the PV not only improved the performance,but enhanced the whole body immune functions in mice.
试验一,PPP的制备。本试验首先从鸡蛋黄中提取卵黄高磷蛋白(PV),经胰蛋白酶水解,水解液经截留分子量3 kDa和1 kDa的超滤膜超滤,取分子量在1 kDa-3 kDa之间的截留液冷冻干燥得到PPP。经SDS-PAGE电泳测PV的纯度,用N/P(摩尔比)检测PPP的磷酸丝氨酸残基密度。结果表明:所制备的PV样品与纯品电泳模式相近,在1 kDa-3 kDa范围内存在PPP电泳条带,且N/P比例为5.02。说明所提取PV样品相对较纯,PPP样品含有磷酸丝氨酸残基。
试验二,PPP对小鼠体外免疫功能的影响。为了评价PPP对细胞增殖、吞噬功能、细胞因子以及免疫球蛋白生成的影响,分离小鼠的脾淋巴细胞和腹腔巨噬细胞并进行培养。结果显示:添加LPS或ConA后,显著地促进了细胞的增殖、细胞吞噬功能、细胞因子的分泌以及免疫球蛋白的生成(p＜0.05)。终浓度为0.02 mg/mL的PV组不能显著地影响细胞功能(p＞0.05)。然而,终浓度为0.02 mg/mL的PPP组和0.1 mg/mL PPP组显著地提高了刺激指数(SI)和吞噬指数(PI),以及IgA、IL-1β、IL-2和IL-6的生成。LPS或ConA诱导组比无丝裂原组刺激作用显著增强。结果表明,PPP显著提高了SI、PI、细胞因子以及免疫球蛋白生成的影响,无丝裂原组相比,LPS或ConA诱导下,以上各指标均显著提高。
试验三,PPP对小鼠体内免疫功能的影响。为了确定胃饲PPP对小鼠免疫功能的影响,将48只小鼠随机分到3个处理中,每个处理设4个重复,每个处理的小鼠在基础日粮的基础上分别隔天灌胃0.2mL生理盐水(对照组)、PPP和PV溶液,按体重喂给0.03mg/(g·d)。14天后取样确定小鼠的生长性能和免疫功能。结果显示:与对照组和PV组相比,PPP组小鼠的体增重极显著提高(p＜0.01),血清总蛋白、白蛋白、球蛋白和葡萄糖均升高,但差异不显著(p＞0.05),而血清尿素氮、总胆固醇和甘油三酯呈下降趋势,脾脏指数和胸腺指数呈显著提高(p＜0.05)。PPP组的SI和PI显著提高(p＜0.05),血清和脾淋巴细胞培养液中的IgA、IL-2和IL-6浓度,以及血清和腹腔巨噬细胞培养液中的IL-1β均显著地上升(p＜0.05)。PV组与对照组相比,这些指标均无显著变化(p＞0.05)。在LPS或ConA诱导下,培养液上清中的SI、PI、IgA、IL-1β、IL-2和IL-6均有显著的提高(p＜0.05)。结果表明,胃饲PPP不仅提高了体增重,脾脏指数和胸腺指数,还显著增强了小鼠SI和PI,血清和脾淋巴细胞培养液中的IgA、IL-2和IL-6浓度,以及血清和腹腔巨噬细胞培养液中的IL-1β浓度。与无丝裂原组相比,LPS或ConA诱导组以上各指标均显著提高。
综合以上各试验可看出,制备所得的PPP不仅提高了小鼠的生长性能,还增强了小鼠体内外免疫功能。
Effects of phosvitin phosphopeptides(PPP) from enzymic hydrolysates of phosvitin (PV) which come from chicken egg-yolk on immune functions of mice were investigated. On the basis of preparing PPP,the study focuses on the immune functions of mice in vivo or vitro.The whole study consisted of three experiments.
Experiment 1:Preparing he PPP
First,the phosvitin extracted from chicken egg-yolk was hydrolyzed by trypsin.Then, the hydrolysate was ultrafiltrated by ultrafiltration membrane.The filtrate of MW between 1kDa-3kDa was collected and lyophilized to get the PPP.PPP purity was determined by SDS-PAGE and N/P(molar ratio).The result indicated that electrophoresis strip of extracted PV was similar to the pure of PV.Electrophoresis of PPP showed that its MW was between 1-3kDa,N/P was 5.02.It was concluded that,the purity of PV and PPP prepared here was high enough for further study.
Experiment 2:Effects of PPP on Immune Functions of Mice in vitro
The Mice's spleen lymphocytes and peritoneal macrophages were isolated and cultured to evaluate the effects of PPP on cell stimulation index(SI),phagocytosis index(PI) and cytokine secretions and immunoglobulin formation.Results showed that LPS or ConA administration significantly stimulated cell proliferation,phagocytosis,cytokine secretion and immunoglobulin formation in vitro(p＜0.05).PV administration(0.02 mg/mL) did not significantly affect the cell functions.However,PPP administration(0.02 mg/mL and 0.1 mg/mL) significantly stimulated the SI and elevated the concentrations of IgA,IL-2 and IL-6(p＜0.05) in culture supematant.The stimulating effects of PPP for LPS/ConA-induced cells were stronger than uninduced cells.These results suggested that PPP administration enhanced the SI,PI,cytokine secretions and immunoglobulin formation.When the LPS or ConA existed,the indicators were more obvious effects.
Experiment 3:Effects of PPP on Immune Function of Mice in vivo
To determine the effects of orally administrated PPP on immune functions of mice.A total of 48 mice were randomly assigned to three treatments with 4 replications.Mice were administrated with 0.2 mL saline(control group),PPP and PV solutions(0.03 mg/g.BW.d), respectively.After 14 days,all mice were killed and sampled to investigate the growth performance and immune parameters.Results showed that the weight gain of PPP group was significantly improved(p＜0.05).The concentrations of serum total protein,albumin, globulin and glucose in PPP group tended to be increased than control or PV groups (p＞0.05),However,the concentrations of serum urea nitrogen,total cholesterol and triglyceride tended to be decreased.The spleen index and thymus index of PPP group were significantly improved(p＜0.05).Results form primary culture of spleen lymphocytes and peritoneal macrophages indicated that LPS and ConA significantly stimulated the SI and PI, and increased the concentrations of IgA,IL-1β,IL-2 and IL-6 in culture supematant (p＜0.05).The SI and PI were also significantly improved by PPP administration(p＜0.05). In addition,the concentrations of IgA,IL-2 and IL-6 in serum and spleen lymphocytes culture,the IL-1βin peritoneal macrophages culture were both actutely elevated(p＜0.05). However,none of these parameters altered by PV administration(p＞0.05).These results suggested that orally PPP administration not only improved the performance,but enhanced the SI,PI,cytokine secretions and immunoglobulin formation.When the LPS or ConA existed,the indicators were more obvious effects.
In summary,PPP prepared from the PV not only improved the performance,but enhanced the whole body immune functions in mice.
引文
[1]Jiang Bo,Mine yoshinori.Preparation of Novel Function Oligophosphopetides from Hen Egg Yolk Phosvitin[J].Agric Food Chem,2000,48(4):990-994
[2]刘小丽.卵黄高磷蛋白及其磷酸肽的制备和研究[D].西北大学,2003
[3]Kitamura H,H.Otani,T.Oshida,et al.Milk immunoglobulin levels in sows given a diet containing acommercially available casein phosphopeptide preparationg,CPP-Ⅰ,during pregnancy.[J].Milchwissengschaft,2002,57(9-10):486-489
[4]Bo JIANG,Yoshinori MINE.Phosphopeptedes Derived from Hen Egg Yolk Phosvitin:Effect of Molecular Size on the Calcium-binding Properties[J].Biosci.Biotechnol.Biochem,2001,65(5):1187-1190
[5]陈劼.卵黄高磷蛋白磷酸肽的制备与钙结合性质的研究[D].无锡轻工业大学,2000
[6]Inwook Choi,Changhwa Jung,Heedon Choi et.al.E□ectiveness of phosvitin peptides on enhancing bioavailability of calcium and its accumulation in bones[J].Food Chemistry 2005(93):577-583
[7]Lee Y.S.et al.Phosphopeptides and solube calcium in the small intestine of rats given a casein diet[J].British Journal of Nutrition,1980,43(3):457-467
[8]Mykkanen HM,and RH Wasserman.Enchanced absorption of calcium by casein phosphopeptedes in rachitic and normal chicks[J].Nutr,1980,110,2141-2148
[9]Hata I,J Ueda,H Otani.lmmunostimulatory action of a commercially available casein phosphopetide preparation,CPP-Ⅲ,in cell cultures.Milchwissengschaft,1999,54(1):3-7
[10]Kishimoto T,D Katayama,H.Otani.Effect of β-casein(1-28)on proliferation responses and secretory functions of human immunocompetent cell lines[J].Biosci Biotechnol Biochem,2004,68(10):2091-2095
[11]刘志皋主编.食品营养学[M].北京,中国轻工业出版社,1991,77-81
[12]Friedman M Nutitionan value of proteins from different food sources[J]A review.J.Agric Food Chem,1996,44:6-29
[13]BYRNE B M,VAN HET SCHIP A D,VAN DE KLUNDERT J A M,et al.Amino acid sequence of phosvitin derived from the nucleotide sequence of part of the chicken vitellogenin gene[J].Biochem,1984,23:4275-4279
[14]高真.蛋制品工艺学[M].北京:中国商业出版社,1992
[15]Chung S L,Ferrier L K.Conditions affecting emulsifyin properties of egg yolk phosvitin[J].Food Sci,1991,56(5):1259-1262
[16]Itoh T,Yutaka a,Adachi S.Comparative studies onαandβ-phosvitin from hen's egg yolk[J].Food Sci,1983,48,1775
[17]Khan M A,Nakamura S,Ogawa M,Akita E,Azakami H,Kato A.Bactericial action of egg yolk phosvitin against Escherichia coli under Thermal stress[J].Agric Food Chem,2000,48:1503-1506
[18]LOSSOJ N.NAKAIS A.As imple procedure for isolation of phosvitin from chicken Egg in Egg Uses and Processing Technologies New Developments[J].Oxon:Cab International.1994,150-157
[19]Joubert J,Cook W H.Preparation and characterization of phosvition from hen egg yolk[J].Can J Biochem Physiol,1985,36:399-408
[20]Sudararajan A,Sampath Kumar K S V,Sarma P S.A simplified procedure for the preparation of phosvitin and vitellin[J].Biochim Biophys Acta.1960,38:360-362
[21]Wallace R A,Jared D W,Eisen AZ.A general method for the isolation and purification of phosvitin from vertebrate eggs[J].Can J Biochem.1996,44:1647-1655
[22]若松利男,押甲一夫,山浦淑子.卵黄高磷蛋白的提取方法.日本专利:JP Kokai Tokkyo Koho JP:3-56500
[23]王喜波,迟玉杰,石冬冬.卵黄高磷蛋白的研究进展[J].食品科技,2004(4),43
[24]Takatoshi Iton,Yutake Abe,Susumu Aadchi.Comparative studies on the α and β phosvitin from hen's egg yolk[J].Journal of Food Science,1983,48:1755-1757
[25]张小燕,刘小丽,范晓东.卵黄高磷蛋白的提取工艺研究[J].食品科学,2002(2):85-87
[26]刘大川,钟方旭.酶水解法制备大豆肽的研究[J].中国油脂,1997,22(6):14-17
[27]王梅,沈辉.食物蛋白酶解物中的生物活性肽氨基酸与生物资源[J].1997,19(1):40-43
[28]庞广昌,陈庆森.生物活性肽一酪蛋白磷酸肽(CPPs)的研究、应用及展望[J].食品科学,1999,26(6):25-28
[29]江波,Y.Mine.卵黄高磷蛋白磷酸肽的制备及钙结合性质研究[D].无锡轻工业大学学报,2000,19(4):325-330
[30]江波,陈劼.卵黄高磷蛋白的酶解及磷酸肽的持钙性质[J].食品工业,2001,5,32-33
[31]薛正莲.生物活性肽--酪蛋白磷酸肽(CPP)的研究[J].山西食品工业,2000,4,2-4
[32]顾王英,裴元英.酪蛋白磷酸肽促钙吸收的作用及研究状况[J].中国临床医学杂志,2000,9(1):58-60
[33]Holt C,Wahlgren,NW,et al.Ability of a beta-casein phosphopeptides tmodulate the precipitation of calcium phosphate by forming amorphoudicalcium phosphate nanoclusters[J].Biocemical Journal,1996,314(3):1035.
[34]郭勇,许梓荣.酪蛋白磷酸肽的生理功能及在饲料业上的应用前景[J].上海畜牧通讯,2003,5,4-5
[35]严霞等.新型功能性饲料添加剂--酪蛋白磷酸肽[J].中国动物保健,2003,10,55-56
[36]Himyama M.Toyoto K.Yamaguchi G.et al.HPLC analysis of casein phosphopeptides(CPP)[J].Biosci Biotech Biochem.1992,56(7):1126-1127
[37]张亚非,周羽并,吴凤兰,等.酪蛋白磷酸肽对大鼠钙吸收利用的影响[J].营养学报,1994,16(1):73
[38]牟广庆,鹿保鑫,王新.酪蛋白磷酸肽(CPPs)对铁吸收影响的研究[J].黑龙江八一农垦大学学报,2001,(3):70-73
[39]M Hansen,B Sandstrom and B Lonnerdal.The effect of casein phosphopeptides on zinc and calcium absorption from high phytate infant diets assessed in rat pups and Caco-2 cells[J].1996(4)
[40]郑华.牛乳酪蛋白水解物的分析及其对小鼠生长影响的研究[D].广东,华南农业大学,1999
[41]张莉莉 大豆蛋白与酪蛋白酶解物对小鼠免疫功能影响的比较研究[D],南京农业大学,2004
[42]Otani H.,T.Kawahara,Y.Tashiro.Effects of bovine[3-casein(1-28) and its chemically synthesized partial fragments on proliferation response and immunoglobulin production in mouse spleen cultures[J].Biosci.Biotechnol.Biochem.,2001,65(11):2489-2495
[43]潘翠玲.添喂蛋白酶解物对早期断奶仔猪消化生理及免疫功能的影响[D1.南京.南京农业大学,2002
[44]Sutat as Y,E Soppi,H Korhonen,et al.Suppression of lymphocyte proliferation m vitro by bovine caseins hydrolyzed with Lactobacillus casei GG-derived enzymes[J].Allergy Clin Immunol,1996,98,(1):216-224
[45]Mao XY,QX.Nan,YH.Li,et al,Effects of different casein-derived peptides and their separated fractions on mouse cell immunity[J].Milchwissenschaft,2005a,60(1):10-14
[46]Coste M,V Rochet,J.Leonil,et al Identification of C-terminal peptides of bovine b-casein that enhance proliferation of rat lymphocytes[J].Immunol.Lett,1992,33.41-46
[47]Hansen M,Sandstroem B,Jensen M etal.Effect of casein phosphoeptides on zinc and calcium absorption from bread meals[J].Trace Elem Med Biol,1997.11(3):143-149
[48]毛宁英,南庆贤,李莹辉,等.酪蛋白酶解产物对小鼠脾T淋巴细胞体外增殖作用的影响[J].营养学报,2004a,26(2):116-118
[49]Otani H,M Monnai Inhibition of proliferation responses of mouse spleen lymphocytes by bovine milk κ-casein digests[J].Food Agric Immunol 1993,5:219-229
[50]郑华.牛乳酪蛋白水解物的分析及其对小鼠生长影响的研究[D].广东,华南农业大学,1999
[51]Otani H,S Higashryama,and I Hata.Indentification of a phosphopeptide in bovine alpha sl-casein digest as a factor influencing proliferation and immunoglobulin production in lymphocyte cultures [J].Dairy Res,1998,65(4)59-578
[52]杜念兴主编.兽医免疫学[M].北京中国农业出版社,2000
[53]Mighore-Samour D,F Floc'h,and P Jolles.Biologically active casein peptides implicated in immunomodulation[J].Dairy Res,1989,56:357-362
[54]王恬,傅永明,吕俊龙,等.小肽营养素对断奶仔猪生产性能及小肠发育的影响[J].畜牧与兽医,2003,35(6)4-8
[55]Otani H,K Nakano,and T Kawahara.Casein phosphopeptede enhances mucosal IgA response to LPS Drug Weekly[J]July 11,2003:356-357
[56]郭尧君编著.蛋白质电泳实验技术[M].科学出版社,北京,1999,123-145
[57]SHANTZ R C.D AWSON L E.Electrphoretic examination of native and phosvitin fraction of avian egg yolk[J].Poultry Sci.1974,53:969-974
[58]冯凤琴,许时婴,王璋.酪蛋白磷酸肽的分子量及持钙功能的分析[J].食品与发酵工业.1997,23(2):18.
[59]李建武,萧能赓,余瑞元等.生物化学实验原理和方法[M].北京大学出版社,北京,1994,168-171
[60]MECHAMD KOLCOTT H S.Phosvitin.the principal phosphoprotein of egg yolk[J].Am.Chem.Soc 1994,23:4257-4279
[61]倪莉.鸡蛋中卵黄高磷蛋白的提取[D].无锡轻工大学学报,1999,18(2):55-59
[62]腾利荣.生物学基础实验教程[M].吉林科学技术出版社,579
[63]Otani H,and I Hata Inhibition of proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells by bovine milk caseins and their digests[J].Dairy Res,1995,62:339-348
[64]毛俊洁,等.白术多糖对小鼠淋巴细胞功能的调节作用[J].免疫学杂志,1996,12(4):233
[65]苗本春,耿美玉,李静,等.海洋硫酸多糖911免疫增强作用的探讨[J].中国海洋药物杂志,2002,5:1-4
[66]程宝鸾主编.动物细胞培养技术[M].广州:华南理工大学出版社,2000
[67]Gabriela Perdigon,Maria Elena Nader de Macias et.al.Effect of Perorally Administered Lactobacilli on Macrophage Activation in Mice[J].INFECTION AND IMMUNITY,Aug1986,404-410
[68]Mosmann T.Rapid colormeric assay for cellular growth and survival application to proliferation and cytotoxicity assays[J].Immunol Methods,1983,65:55-63
[67]黄林青,周世文,张诗平,等.蚕蛹多糖对小鼠免疫功能的影响[J].解放军药学学报,2002,18(1):11-13
[69]金伯泉主编.细胞和分子免疫学[M].北京,科学出版社,2001.
[70]毕爱华主编.医学免疫学[M].北京,人民军医出版社,2002
[71]杨汉春主编动物免疫学[M].北京,中国农业大学出版社,2003
[72]Hata I.,S.Higashiyama,H.Otani.Identification of a phosphopeptide in bovineαtsl-casein digest as a factor influencing proliferationg and immunoglobulin production in lymphocyte cultures[J].Dairy Research,1998,65:569-578
[73]Otani H.,H.Kitamura,M.Park.Enhancement of intestinal IgA levels in piglets by oral administration of a commercially avaible casein phosphopeptide preparation[J].Milchwissengschaft,2000b,55(8):429-432.
[74]Warner,R.L.,Brooks,K.H.,and Pestka,J.J.In vitro effects of vomitoxin(deoxynivalenol) on T-cell interleukin production and IgA secretion[J].Food Chem Toxicol.1994,32,617-625
[75]Yan,D,Zhou,H.R.,Brooks,K.H.,and Pestka,J.J.Potential role for IL-5 and IL-6 in enhanced IgA secretion by Peyer's patch cells isolated from mice acutely exposed to vomitoxin[J].Toxicology 1997,122,145-158
[76]Mighore-Samour D,F Floc'h,and P Jolles.Biologically active casein peptides implicated in immunomodulation[J].Dairy Res,1998,56:357-362
[77]Hope J.C.,Campbell F & Hopkins S.J.Deficiency of IL-2 or IL-6 reduces lymphocyte proliferation,but only IL-6 deficiency decreases the contact hypersensitivity response[J].Eur J Immunol 2000 30(1):197-203
[78]齐顺章主编.动物生物化学[M].北京,农业出版社,1983
[79]ST-Gelais D,Roy D,Haches,et al.Growth of nonproteolytic lactococcus lactis in culture medium s upple mented with different case in hydrolyzates[J].Dairy Sci,1993,76(3):3327-3337
[80]Fumio Y,Kunio S.Immunological effects of dietary peptides derived from soybean protein[J].Nutr.Biochem.,1993,4(8):450-456
[81]张爱忠,姜宁,李士泽,等.不同水平小肽制剂对黑凤鸡生产性能及其血清生化指标的影响[J].中国畜牧杂志,2002,38(3):10-11
[82]王刚.小肽营养在家禽养殖中的应用[J].饲料博览,2006,01:10-12
[83]王恬,傅永明,吕俊龙,等.小肽营养素对断奶仔猪生产性能及小肠发育的影响[J].畜牧与兽医,2003,35(6):4-8
[84]罗有文,干恬,林贵娟.酪蛋白酶解物对小鼠生长和免疫功能的影响[J].饲料工业,2005,26(6):51-53
[85]Otani H.,Y.Kihara,M Park.Immunoenhancing property of a dietary casein phosphopeptide prepatation in mice[J].Food and Agric Immunol,2000a,12:165-173
[86]高小明.免疫学教程.[M].高等教育出版社,2006
[87]龚非力.医学免疫学[M].科学出版社,2000
[88]田刚,陈代文.酶解鸡蛋清小(寡)肽混合物对小鼠免疫功能的影响及其机理研究[D].四川农业大学博士学位论文,2006
[89]杨小军.灌胃大豆蛋白、面筋蛋白的胃蛋白酶酶解物对人鼠免疫功能的影响[D].南京,南京农业大学,2003
[90]Toyoshima,S,H Iwata,T Osawa.Kinetics of lymphocyte stimulation by concanavalin A[J].Nature,1976,264:447-449
[91]Mykkanen H M,R H Wassermann.Enhanced absorption of calcium by casein phosphopeptides in rachitic and normal chicks[J]Nutr,1980,110:2141-2148
[92]Street N E,T R Mosmann.Functional diversity of T lymphocytes due to secretion of different cytokine paterns[J].FASEB Journal,1991,5:171-177
[2]刘小丽.卵黄高磷蛋白及其磷酸肽的制备和研究[D].西北大学,2003
[3]Kitamura H,H.Otani,T.Oshida,et al.Milk immunoglobulin levels in sows given a diet containing acommercially available casein phosphopeptide preparationg,CPP-Ⅰ,during pregnancy.[J].Milchwissengschaft,2002,57(9-10):486-489
[4]Bo JIANG,Yoshinori MINE.Phosphopeptedes Derived from Hen Egg Yolk Phosvitin:Effect of Molecular Size on the Calcium-binding Properties[J].Biosci.Biotechnol.Biochem,2001,65(5):1187-1190
[5]陈劼.卵黄高磷蛋白磷酸肽的制备与钙结合性质的研究[D].无锡轻工业大学,2000
[6]Inwook Choi,Changhwa Jung,Heedon Choi et.al.E□ectiveness of phosvitin peptides on enhancing bioavailability of calcium and its accumulation in bones[J].Food Chemistry 2005(93):577-583
[7]Lee Y.S.et al.Phosphopeptides and solube calcium in the small intestine of rats given a casein diet[J].British Journal of Nutrition,1980,43(3):457-467
[8]Mykkanen HM,and RH Wasserman.Enchanced absorption of calcium by casein phosphopeptedes in rachitic and normal chicks[J].Nutr,1980,110,2141-2148
[9]Hata I,J Ueda,H Otani.lmmunostimulatory action of a commercially available casein phosphopetide preparation,CPP-Ⅲ,in cell cultures.Milchwissengschaft,1999,54(1):3-7
[10]Kishimoto T,D Katayama,H.Otani.Effect of β-casein(1-28)on proliferation responses and secretory functions of human immunocompetent cell lines[J].Biosci Biotechnol Biochem,2004,68(10):2091-2095
[11]刘志皋主编.食品营养学[M].北京,中国轻工业出版社,1991,77-81
[12]Friedman M Nutitionan value of proteins from different food sources[J]A review.J.Agric Food Chem,1996,44:6-29
[13]BYRNE B M,VAN HET SCHIP A D,VAN DE KLUNDERT J A M,et al.Amino acid sequence of phosvitin derived from the nucleotide sequence of part of the chicken vitellogenin gene[J].Biochem,1984,23:4275-4279
[14]高真.蛋制品工艺学[M].北京:中国商业出版社,1992
[15]Chung S L,Ferrier L K.Conditions affecting emulsifyin properties of egg yolk phosvitin[J].Food Sci,1991,56(5):1259-1262
[16]Itoh T,Yutaka a,Adachi S.Comparative studies onαandβ-phosvitin from hen's egg yolk[J].Food Sci,1983,48,1775
[17]Khan M A,Nakamura S,Ogawa M,Akita E,Azakami H,Kato A.Bactericial action of egg yolk phosvitin against Escherichia coli under Thermal stress[J].Agric Food Chem,2000,48:1503-1506
[18]LOSSOJ N.NAKAIS A.As imple procedure for isolation of phosvitin from chicken Egg in Egg Uses and Processing Technologies New Developments[J].Oxon:Cab International.1994,150-157
[19]Joubert J,Cook W H.Preparation and characterization of phosvition from hen egg yolk[J].Can J Biochem Physiol,1985,36:399-408
[20]Sudararajan A,Sampath Kumar K S V,Sarma P S.A simplified procedure for the preparation of phosvitin and vitellin[J].Biochim Biophys Acta.1960,38:360-362
[21]Wallace R A,Jared D W,Eisen AZ.A general method for the isolation and purification of phosvitin from vertebrate eggs[J].Can J Biochem.1996,44:1647-1655
[22]若松利男,押甲一夫,山浦淑子.卵黄高磷蛋白的提取方法.日本专利:JP Kokai Tokkyo Koho JP:3-56500
[23]王喜波,迟玉杰,石冬冬.卵黄高磷蛋白的研究进展[J].食品科技,2004(4),43
[24]Takatoshi Iton,Yutake Abe,Susumu Aadchi.Comparative studies on the α and β phosvitin from hen's egg yolk[J].Journal of Food Science,1983,48:1755-1757
[25]张小燕,刘小丽,范晓东.卵黄高磷蛋白的提取工艺研究[J].食品科学,2002(2):85-87
[26]刘大川,钟方旭.酶水解法制备大豆肽的研究[J].中国油脂,1997,22(6):14-17
[27]王梅,沈辉.食物蛋白酶解物中的生物活性肽氨基酸与生物资源[J].1997,19(1):40-43
[28]庞广昌,陈庆森.生物活性肽一酪蛋白磷酸肽(CPPs)的研究、应用及展望[J].食品科学,1999,26(6):25-28
[29]江波,Y.Mine.卵黄高磷蛋白磷酸肽的制备及钙结合性质研究[D].无锡轻工业大学学报,2000,19(4):325-330
[30]江波,陈劼.卵黄高磷蛋白的酶解及磷酸肽的持钙性质[J].食品工业,2001,5,32-33
[31]薛正莲.生物活性肽--酪蛋白磷酸肽(CPP)的研究[J].山西食品工业,2000,4,2-4
[32]顾王英,裴元英.酪蛋白磷酸肽促钙吸收的作用及研究状况[J].中国临床医学杂志,2000,9(1):58-60
[33]Holt C,Wahlgren,NW,et al.Ability of a beta-casein phosphopeptides tmodulate the precipitation of calcium phosphate by forming amorphoudicalcium phosphate nanoclusters[J].Biocemical Journal,1996,314(3):1035.
[34]郭勇,许梓荣.酪蛋白磷酸肽的生理功能及在饲料业上的应用前景[J].上海畜牧通讯,2003,5,4-5
[35]严霞等.新型功能性饲料添加剂--酪蛋白磷酸肽[J].中国动物保健,2003,10,55-56
[36]Himyama M.Toyoto K.Yamaguchi G.et al.HPLC analysis of casein phosphopeptides(CPP)[J].Biosci Biotech Biochem.1992,56(7):1126-1127
[37]张亚非,周羽并,吴凤兰,等.酪蛋白磷酸肽对大鼠钙吸收利用的影响[J].营养学报,1994,16(1):73
[38]牟广庆,鹿保鑫,王新.酪蛋白磷酸肽(CPPs)对铁吸收影响的研究[J].黑龙江八一农垦大学学报,2001,(3):70-73
[39]M Hansen,B Sandstrom and B Lonnerdal.The effect of casein phosphopeptides on zinc and calcium absorption from high phytate infant diets assessed in rat pups and Caco-2 cells[J].1996(4)
[40]郑华.牛乳酪蛋白水解物的分析及其对小鼠生长影响的研究[D].广东,华南农业大学,1999
[41]张莉莉 大豆蛋白与酪蛋白酶解物对小鼠免疫功能影响的比较研究[D],南京农业大学,2004
[42]Otani H.,T.Kawahara,Y.Tashiro.Effects of bovine[3-casein(1-28) and its chemically synthesized partial fragments on proliferation response and immunoglobulin production in mouse spleen cultures[J].Biosci.Biotechnol.Biochem.,2001,65(11):2489-2495
[43]潘翠玲.添喂蛋白酶解物对早期断奶仔猪消化生理及免疫功能的影响[D1.南京.南京农业大学,2002
[44]Sutat as Y,E Soppi,H Korhonen,et al.Suppression of lymphocyte proliferation m vitro by bovine caseins hydrolyzed with Lactobacillus casei GG-derived enzymes[J].Allergy Clin Immunol,1996,98,(1):216-224
[45]Mao XY,QX.Nan,YH.Li,et al,Effects of different casein-derived peptides and their separated fractions on mouse cell immunity[J].Milchwissenschaft,2005a,60(1):10-14
[46]Coste M,V Rochet,J.Leonil,et al Identification of C-terminal peptides of bovine b-casein that enhance proliferation of rat lymphocytes[J].Immunol.Lett,1992,33.41-46
[47]Hansen M,Sandstroem B,Jensen M etal.Effect of casein phosphoeptides on zinc and calcium absorption from bread meals[J].Trace Elem Med Biol,1997.11(3):143-149
[48]毛宁英,南庆贤,李莹辉,等.酪蛋白酶解产物对小鼠脾T淋巴细胞体外增殖作用的影响[J].营养学报,2004a,26(2):116-118
[49]Otani H,M Monnai Inhibition of proliferation responses of mouse spleen lymphocytes by bovine milk κ-casein digests[J].Food Agric Immunol 1993,5:219-229
[50]郑华.牛乳酪蛋白水解物的分析及其对小鼠生长影响的研究[D].广东,华南农业大学,1999
[51]Otani H,S Higashryama,and I Hata.Indentification of a phosphopeptide in bovine alpha sl-casein digest as a factor influencing proliferation and immunoglobulin production in lymphocyte cultures [J].Dairy Res,1998,65(4)59-578
[52]杜念兴主编.兽医免疫学[M].北京中国农业出版社,2000
[53]Mighore-Samour D,F Floc'h,and P Jolles.Biologically active casein peptides implicated in immunomodulation[J].Dairy Res,1989,56:357-362
[54]王恬,傅永明,吕俊龙,等.小肽营养素对断奶仔猪生产性能及小肠发育的影响[J].畜牧与兽医,2003,35(6)4-8
[55]Otani H,K Nakano,and T Kawahara.Casein phosphopeptede enhances mucosal IgA response to LPS Drug Weekly[J]July 11,2003:356-357
[56]郭尧君编著.蛋白质电泳实验技术[M].科学出版社,北京,1999,123-145
[57]SHANTZ R C.D AWSON L E.Electrphoretic examination of native and phosvitin fraction of avian egg yolk[J].Poultry Sci.1974,53:969-974
[58]冯凤琴,许时婴,王璋.酪蛋白磷酸肽的分子量及持钙功能的分析[J].食品与发酵工业.1997,23(2):18.
[59]李建武,萧能赓,余瑞元等.生物化学实验原理和方法[M].北京大学出版社,北京,1994,168-171
[60]MECHAMD KOLCOTT H S.Phosvitin.the principal phosphoprotein of egg yolk[J].Am.Chem.Soc 1994,23:4257-4279
[61]倪莉.鸡蛋中卵黄高磷蛋白的提取[D].无锡轻工大学学报,1999,18(2):55-59
[62]腾利荣.生物学基础实验教程[M].吉林科学技术出版社,579
[63]Otani H,and I Hata Inhibition of proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells by bovine milk caseins and their digests[J].Dairy Res,1995,62:339-348
[64]毛俊洁,等.白术多糖对小鼠淋巴细胞功能的调节作用[J].免疫学杂志,1996,12(4):233
[65]苗本春,耿美玉,李静,等.海洋硫酸多糖911免疫增强作用的探讨[J].中国海洋药物杂志,2002,5:1-4
[66]程宝鸾主编.动物细胞培养技术[M].广州:华南理工大学出版社,2000
[67]Gabriela Perdigon,Maria Elena Nader de Macias et.al.Effect of Perorally Administered Lactobacilli on Macrophage Activation in Mice[J].INFECTION AND IMMUNITY,Aug1986,404-410
[68]Mosmann T.Rapid colormeric assay for cellular growth and survival application to proliferation and cytotoxicity assays[J].Immunol Methods,1983,65:55-63
[67]黄林青,周世文,张诗平,等.蚕蛹多糖对小鼠免疫功能的影响[J].解放军药学学报,2002,18(1):11-13
[69]金伯泉主编.细胞和分子免疫学[M].北京,科学出版社,2001.
[70]毕爱华主编.医学免疫学[M].北京,人民军医出版社,2002
[71]杨汉春主编动物免疫学[M].北京,中国农业大学出版社,2003
[72]Hata I.,S.Higashiyama,H.Otani.Identification of a phosphopeptide in bovineαtsl-casein digest as a factor influencing proliferationg and immunoglobulin production in lymphocyte cultures[J].Dairy Research,1998,65:569-578
[73]Otani H.,H.Kitamura,M.Park.Enhancement of intestinal IgA levels in piglets by oral administration of a commercially avaible casein phosphopeptide preparation[J].Milchwissengschaft,2000b,55(8):429-432.
[74]Warner,R.L.,Brooks,K.H.,and Pestka,J.J.In vitro effects of vomitoxin(deoxynivalenol) on T-cell interleukin production and IgA secretion[J].Food Chem Toxicol.1994,32,617-625
[75]Yan,D,Zhou,H.R.,Brooks,K.H.,and Pestka,J.J.Potential role for IL-5 and IL-6 in enhanced IgA secretion by Peyer's patch cells isolated from mice acutely exposed to vomitoxin[J].Toxicology 1997,122,145-158
[76]Mighore-Samour D,F Floc'h,and P Jolles.Biologically active casein peptides implicated in immunomodulation[J].Dairy Res,1998,56:357-362
[77]Hope J.C.,Campbell F & Hopkins S.J.Deficiency of IL-2 or IL-6 reduces lymphocyte proliferation,but only IL-6 deficiency decreases the contact hypersensitivity response[J].Eur J Immunol 2000 30(1):197-203
[78]齐顺章主编.动物生物化学[M].北京,农业出版社,1983
[79]ST-Gelais D,Roy D,Haches,et al.Growth of nonproteolytic lactococcus lactis in culture medium s upple mented with different case in hydrolyzates[J].Dairy Sci,1993,76(3):3327-3337
[80]Fumio Y,Kunio S.Immunological effects of dietary peptides derived from soybean protein[J].Nutr.Biochem.,1993,4(8):450-456
[81]张爱忠,姜宁,李士泽,等.不同水平小肽制剂对黑凤鸡生产性能及其血清生化指标的影响[J].中国畜牧杂志,2002,38(3):10-11
[82]王刚.小肽营养在家禽养殖中的应用[J].饲料博览,2006,01:10-12
[83]王恬,傅永明,吕俊龙,等.小肽营养素对断奶仔猪生产性能及小肠发育的影响[J].畜牧与兽医,2003,35(6):4-8
[84]罗有文,干恬,林贵娟.酪蛋白酶解物对小鼠生长和免疫功能的影响[J].饲料工业,2005,26(6):51-53
[85]Otani H.,Y.Kihara,M Park.Immunoenhancing property of a dietary casein phosphopeptide prepatation in mice[J].Food and Agric Immunol,2000a,12:165-173
[86]高小明.免疫学教程.[M].高等教育出版社,2006
[87]龚非力.医学免疫学[M].科学出版社,2000
[88]田刚,陈代文.酶解鸡蛋清小(寡)肽混合物对小鼠免疫功能的影响及其机理研究[D].四川农业大学博士学位论文,2006
[89]杨小军.灌胃大豆蛋白、面筋蛋白的胃蛋白酶酶解物对人鼠免疫功能的影响[D].南京,南京农业大学,2003
[90]Toyoshima,S,H Iwata,T Osawa.Kinetics of lymphocyte stimulation by concanavalin A[J].Nature,1976,264:447-449
[91]Mykkanen H M,R H Wassermann.Enhanced absorption of calcium by casein phosphopeptides in rachitic and normal chicks[J]Nutr,1980,110:2141-2148
[92]Street N E,T R Mosmann.Functional diversity of T lymphocytes due to secretion of different cytokine paterns[J].FASEB Journal,1991,5:171-177