冰乙醇分级离心法分离纯化鸡卵黄免疫球蛋白(IgY)的研究

采用冰乙醇分级离心法提取鸡卵黄免疫球蛋白 (Immunoglobulin of egg yolk,IgY).通过低温乙醇沉淀、DEAE 葡萄糖凝胶 A-50 离子交换层析、Tris-HCl 缓冲液洗脱及 Sephadex G200 凝胶过滤等逐步纯化免疫球蛋白.研究卵黄稀释倍数、pH 值、乙醇添加量以及盐浓度对纯化效果的影响.分离纯化后所得的提取物经检测 IgY 纯度可达98%,且很好地保持了免疫球蛋白的活性.     

鸡卵黄抗体(IgY)的制备工艺研究

对鸡卵黄抗体(简称IgY)的制备工艺进行研究。通过低温乙醇沉淀、DEAE葡萄糖凝胶A-50离子交换层析、Tris-HCl缓冲液洗脱及Sephadex G200凝胶过滤等逐步纯化免疫球蛋白。研究卵黄稀释倍数、pH值、乙醇添加量以及盐浓度对纯化效果的影响。分离纯化后所得的提取物经检测IgY纯度可达98%。用大肠杆菌(E.coli)对其活性进行测定,效果良好,此方法能很好保持免疫球蛋白的活性。     

鸡卵黄免疫球蛋白(IgY)分离纯化的研究

对鸡卵黄免疫球蛋白(IgY)的分离纯化进行了研究.工艺流程为:卵黄→8倍水稀释并搅拌10 min→用HCl调Ph至5.2后在4 ℃静置2 h去沉淀→在上清夜中加入95 %冰乙醇(-20 ℃)使终浓度为60 %(v/v)→4 ℃搅拌20 min后离心(22000 r/min,4 ℃,25 min)→收集沉淀并加入0.028 mol/L NaCl溶液在4 ℃下过滤除去脂蛋白沉淀→收集的滤液用SDS-PAGE法进行纯化得纯度为98 %的1gY.用大肠杆菌(E.coli)对其活性进行测定,效果良好,此方法能保持免疫球蛋白的活性.     

利用二次回归正交组合设计法构建卵黄高磷蛋白磷酸肽提取工艺数学模型的研究

研究卵黄高磷蛋白磷酸肽纯化工艺并构建出纯化工艺的数学模型。以N／P摩尔比作为考察的指标,利用二次回归正交组合设计法优化卵黄高磷蛋白磷酸肽提取工艺参数。在利用乙醇钙沉淀法纯化卵黄高磷蛋白磷酸肽的技术研究中,以N／P摩尔比为衡量指标,二次回归正交组合设计为手段,获得卵黄高磷蛋白磷酸肽纯化技术的数学模型为：y=0．6994z1＋0．0787z2-0．0043z1^2-0．004z2^2-8．4。经F检验,方程的显著水平为0．05,且方程不失拟。将此数学模型进行规划求解知乙醇钙纯化法的最优工艺参数Z1乙醇浓度为75％,乙醇体积与提取前PPP体积比乙为15：1（V：V）;验证试验所得到的样品N／P摩尔比为15．67,与通过回归方程计算所得到的14．69相差无几,更验证了回归方程的有效性。经乙醇钙沉淀法纯化后所得到的卵黄高磷蛋白磷酸肽磷酸肽含量为5．25％,N／P摩尔比为15.98。     

鸡蛋黄中4种生物活性成分联合提取工艺

为研究从蛋黄中依次提取卵黄免疫球蛋白、卵黄高磷蛋白、蛋黄油、卵磷脂4 种活性物质的最佳工艺条件,对4 种活性物质的提取量测定。结果表明：5 g 蛋黄液用8 倍蒸馏水稀释,调pH 值为5.0,4 500×g 离心35 min,上清液分别用70%、30%硫酸铵两次盐析,为提取卵黄免疫球蛋白的最佳工艺条件,可得到33.3 mg 的卵黄免疫球蛋白,回收率91.44%,活性70.44%;沉淀物加入6 倍体积的95%乙醇和正己烷（体积比1∶1）混合有机溶剂3 000×g 离心20 min,下层胶状物加1.75 mol/L 的NaCl 溶液,加热、过滤、透析、冻干,为提取卵黄高磷蛋白的最佳工艺条件,可得49.95 mg 卵黄高磷蛋白,回收率为83%;上层油状物旋转蒸发,收集产物加入丙酮,3 000×g 离心20 min,其上层液体再旋转蒸发去除丙酮,可得蛋黄油0.97 g;下层沉淀用丙酮纯化后,真空干燥,得卵磷脂0.53 g。     

抗轮状病毒（anti—HRV）免疫蛋白的研究

本文报道了抗轮状病毒卵黄免疫球蛋白ＩｇＹ的制备、提取以及纯化的基本方法，并对其理化性质做了初步研究，急性毒性实验表明其安全无毒，是一种理想的免疫型功能食品添加剂。     

乙醇冷冻法纯化分离大豆磷脂酰胆碱研究

以大豆粉末磷脂为原料,结合乙醇萃取和乙醇冷冻纯化两个步骤提取纯化磷脂酰胆碱;重点探讨乙醇冷冻纯化步骤工艺条件,考察冷冻时间、冷冻温度、料液比、乙醇浓度等因素对乙醇冷冻纯化效果影响,并在单因素试验基础上进行正交试验,确定最佳冷冻纯化工艺为:冷冻时间6h,冷冻温度-12℃,料液比1:8,乙醇浓度97.5%,在此条件下纯化分离,磷酯酰胆碱含量由45.8%提高至70.5%、得率为81.2%。     

鸡卵黄免疫球蛋白的分离制备研究

实验研究了从高免鸡卵黄中分离纯化免疫球蛋白(IgY)的方法。确定最优条件为:卵黄十倍稀释,pH5.3,-18℃冻结6h,4℃解冻;添加3%硅藻土进行微滤;采用超滤浓缩五倍;再用45%饱和硫酸铵(pH5.3)盐析;沉淀重溶后经DEAE-Sepharose F.F.柱层析分离纯化。结果表明,用此方法可以从高免鸡蛋中得到电泳纯IgY,总活性回收率达到64.16%。     

特异性抗肠出血性大肠杆菌O157:H7内毒素鸡卵黄抗体的制备

目的:比较研究LPS和O-SP的免疫原性,制备出特异性抗内毒素(LPS)鸡卵黄免疫球蛋白(eggyolkimmunoglobulin,IgY),用于对E.coliO157:H7的免疫预防及检测。方法:分别用灭活E.coliO157:H7菌体、不同浓度内毒素(LPS)及O-特异性多糖链(O-SP)与不完全弗氏佐剂充分乳化后作抗原免疫临产蛋母鸡,以水稀释法从卵黄中提取抗体,阴离子交换色谱法实现对抗体的纯化,间接ELISA及双向免疫扩散检测抗体产生效价与活性。结果与结论:所制内毒素(LPS)和O-SP均有较好的免疫原性,刺激鸡体后可产生高效价抗体,灭活菌体、600μg/mlLPS、1200μg/mlLPS、2000μg/mlLPS、O-SP抗体产生效价最高可分别达1:32000、1:28000、1:32000、1:12000、1:40000。结合离子交换色谱,用0.185mol/L的离子强度可实现一步洗脱纯化抗体,制备出高纯度、高活性、特异性强的鸡卵黄免疫球蛋白IgY,为大肠杆菌O157:H7的感染防治提供了可靠的保证,在此实验基础之上可进一步探讨应用特异性IgY对大肠杆菌O157:H7的检测。     

蛋黄主要蛋白质研究进展

卵黄免疫球蛋白、蛋黄低密度脂蛋白、蛋黄高密度脂蛋白和卵黄高磷蛋白是蛋黄中的主要蛋白质,它们一方面为鸡胚胎发育提供重要的营养物质,另一方面,还具有抗菌活性、抗氧化活性等,为鸡胚发育提供保护。此外,蛋黄蛋白质的热凝胶特点、乳化特性等,为蛋黄及其相关产品提供了良好的质构特性,在食品加工中具有广泛应用。就蛋黄主要蛋白质的来源、在蛋黄中的组织状态、分子结构、功能特性以及分离纯化等进行综述,为蛋黄蛋白质的深入研究、应用和开发等提供参考。     

Simple purification methods for an alphagalactose-specific antibody from chicken eggs

For many applications avian antibody from egg yolk (IgY) offers advantages over the well-known mammalian antibodies. Different experimental techniques for the purification of IgY from chickens immunized with an alphagalactose-containing antigen (alphaGal-trisaccharide) were compared. These included ammonium sulfate precipitation, filtration with diatomaceous earth, treatment with deoxycholate, and thiophilic and affinity chromatography. Samples were tested for overall purity, protein and lipid content, and specific activity. Evaluated on the basis of these results and the simplicity of the process, the favored purification method is ammonium sulfate precipitation of diluted egg yolk directly followed by affinity chromatography. The high lipid content of IgY preparations is greatly reduced by either thiophilic or affinity chromatography. Affinity purification of ammonium sulfate precipitated material resulted in anti-alphaGal-trisaccharide IgY preparations with approximately 1% of the original protein content but approximately 100-fold higher specific activity for the alphaGal-trisaccharide epitope.     

绿色高效连续分离技术制备蛋黄中活性蛋白质和磷脂

为了实现蛋黄功能性成分的充分利用，采用绿色溶剂从新鲜蛋黄中高效连续分离具有生物活性的卵黄免疫球蛋白（immunoglobulin Y, IgY）、卵黄高磷蛋白（phosvitin, PV）和磷脂（phospholipids, PL）。首先采用水稀释法（稀释倍数为1:10）分离水溶性组分与脂蛋白，通过35%饱和(NH₄)₂SO₄和0.5% NaCl（w/v）对上清液进行盐析并用8% PEG 6000纯化IgY。脂质组分中的PV在pH5.0，90 ℃条件下的NaCl溶液（w/v）中进一步纯化。采用乙醇/乙酸乙酯比例为1:2（v/v）提取蛋黄中全部脂类，进一步采用乙酸乙酯在0 ℃下提纯蛋黄总脂中的PL。经脱盐、冷冻干燥后，IgY、PV和PL的纯度分别为97.38%、78.63%和85.94%。IgY和PV的得率分别为6.15、10.15 mg/mL。蛋黄总脂中PL的含量为35.18%。因其良好的多不饱和脂肪酸/饱和脂肪酸比率（0.70）和相对较低的n-6/n-3比率（5.37），该PL产品可应用于食品加工中。本方法简便、环保并且高效，可从蛋黄中依次分离出高纯度IgY、PV和PL，并为蛋黄的综合利用提供技术支持。     

酪氨酸酶特异性卵黄抗体Fab’片段的制备及其对酶活力的抑制性能

针对导致黑色素沉淀的酪氨酸酶,制备了特异性卵黄抗体Fab’片段,研究其对酶活力的抑制效果并从动力学角度进行分析。结果表明,胃蛋白酶法制备Fab’片段的最适条件为：pH?4.0、反应比（卵黄抗体-胃蛋白酶）1∶150（mg/U）、反应时间8?h。经免疫亲和柱纯化后可得理化性质稳定、纯度大于95%的Fab’片段;该片段通过非竞争性结合抑制酪氨酸酶活力,IC50为11.16?μmol/L,结合常数为5.79×105,抑制效果相比完整的卵黄抗体分子大大提高。本研究可为卵黄抗体Fab’片段作为潜在的酶活力抑制剂在食品、化妆品和医药等行业中应用提供理论参考。     

Physicochemical properties of leftover egg yolk after livetins removal

Isolation of immunoglobulin Y (IgY) from egg yolk using water-dilution method generates large quantities of leftover pellet as the co-product. Although egg yolk is well known for its great emulsion property, there is a lack of understanding on how livetins removal would affect the emulsion and rheological properties of the pellet. Therefore, the objective of this study was to determine the effect of soluble protein removal on emulsifying and rheological characteristics of the leftover egg yolk. Egg yolk pellet exhibited distinct structural and physicochemical properties after soluble protein removal. Emulsions prepared from pellet were more vulnerable to coalescence instability than that of egg yolk, although both had a similar oil droplet size. Egg yolk displayed a Newtonian behaviour, compared to a shear-thinning behaviour of pellet. Pellet showed a higher apparent viscosity as well as higher viscoelastic moduli than those of egg yolk, probably due to increased hydrogen bond and hydrophobic interactions in the pellet. Therefore, the changes on the emulsion stability and the rheological properties of egg yolk after soluble proteins removal should be considered in food formulation and processing. Further study is needed to improve the emulsion property of pellet for uses in the food industry.     

卵黄免疫球蛋白的分离提取与鉴定

对氯仿法、辛酸法、水稀释法、水-辛酸法分离卵黄免疫球蛋白(IgY)进行了比较,结果表明:水-辛酸法在9倍稀释度、pH5.3、辛酸加量1%条件下,可获得较好效果,进一步用硫酸铵沉淀脱盐后,提取率达93.4%,产率达4.67mg/mL卵黄,纯度达85%。     

Immunoglobulins from Egg Yolk: Isolation and Purification

Simple water dilution was employed for the separation of water-soluble plasma proteins from egg yolk granules. An optimum recovery of immunoglobulin Y (IgY, 93–96%) in water-soluble fraction was obtained by sixfold water dilution at pH between 5.0–5.2 with incubation time of 6 hr at 4°C. Among the factors studied, pH was found to be the most important factor affecting IgY recovery. Active IgY of high purity with good recovery was obtained by a combination of several techniques including salt precipitation, alcohol precipitation, ultrafiltration, gel filtration and ion exchange chromatography. Salt precipitation, ultrafiltration, and get filtration is the recommended sequence. Over 100 mg of electrophoretically pure IgY was routinely obtained from one egg.     

鸡卵黄免疫球蛋白的糖基化及其构效关系研究进展

鸡卵黄免疫球蛋白（egg yolk immunoglobulin,IgY）是鸡卵黄中的一种免疫球蛋白。由于其具有原料易得、制备简单、不与类风湿因子以及哺乳动物的补体发生交叉反应等优点,IgY是一种颇具潜力的抗生素替代品,但由于IgY性质不稳定,易受温度、pH值、酶的影响,从而限制了其实际应用。糖链在维持IgY理化性质、抗原特性等功能中扮演着重要角色。因此,对IgY进行糖基化修饰,从而改善其理化性质和抗原特性,不仅能使鸡蛋资源得到充分利用,也为开发蛋黄保健食品、添加剂以及相应的医药产品提供科学依据。本文归纳几种常见的IgY的糖基化方法,并论述IgY糖基化与其结构、理化性质之间的关系。     

IgG Antibody from Hen Egg Yolks: Purification by Ethanol Fractionation

A procedure was developed for large-scale preparation of IgG antibodies from egg yolks. The supernatant from egg yolks was obtained after an initial 9–fold dilution with water. The lipids in the supernatant were then almost completely eliminated from the water-soluble protein fraction containing the antibody, by precipitation with 60% ethanol and filtration. Yolk antibody was purified from the lipid-free water-soluble protein fraction by ethanol fractionation at final concentration 30% (pH unadjusted), and again at 25% (pH 7.4). The purified fraction was composed of >99% pure IgG. Recovery of antibody was calculated as 40%.