生物活性肽—酪蛋白磷酸肽（CPP）的研制

摸索了2709碱性蛋白酶水解酪蛋白制备CPP的最佳工艺条件：底物浓度10%、用酶量1500u/g反应温度45℃、pH10.5、反应时间150min。采用选择沉淀法分离CPP,在pH4.5添加1.1?Cl2(w/v)和50%（v/v)乙醇,室温沉淀4h ,得到CPP产品的N/P、得率分别为6.39%和13.94%。     

响应曲面法优化酪蛋白磷酸肽的制备

采用Plackett-Burman设计法和响应面分析法相结合,以CPP(酪蛋白磷酸肽)得率作为指标,进行碱性蛋白酶水解酪蛋白的条件优化.结果表明,底物浓度、温度和pH是影响CPP得率的主要因素.最佳水解条件为温度55℃、底物浓度16％、pH10.8、酶底比1.5％,水解时间4.5h,在此条件下,CPP得率可达33.2mg/mL.     

酪蛋白磷酸肽酶解工艺条件的优化研究

酪蛋白磷酸肽(CPP)是以牛乳酪蛋白为原料,通过生物技术制得的具有生物活性的多肽,能有效促进人体对钙、铁、锌等二价矿物营养素的吸收和利用,可用于各种营养、保健食品。本研究对不同种类的酶水解牛乳酪蛋白,优选出CPP得率最高的LC-1酶,对其工艺条件进行优化,确定其最佳酶解条件为:pH8.0、温度50℃、底物浓度10%、酶解时间2h。通过中试验证,CPP含量为22.3%。     

Fe3O4@ZrO2磁纳米粒子在酪蛋白磷酸肽富集中的应用

本实验将制备好的Fe₃O₄@ZrO₂磁性纳米粒子作为载体,对经胰蛋白酶消化后的酪蛋白磷酸肽（Casein Phosphopeptides,CPP）进行高效地选择性富集。实验以CPP的N/P（摩尔比）以及磁纳米粒子吸附量为评价指标,进行单因素实验以便选取较优的实验因素,分析酪蛋白水解度、吸附pH、吸附时间、吸附温度及肽溶液初始浓度这五个因素对Fe₃O₄@ZrO₂磁性纳米粒子选择性吸附CPP的能力的影响,优化CPP富集的技术参数。结果表明,磁性材料最佳富集工艺系数为:酪蛋白的水解度为22%,反应pH=4.5,吸附温度为30 ℃,吸附时间为50 min,肽溶液初始浓度为50 mg/mL;在此条件下,可得到N/P（摩尔比）为4.87的CPP,磁纳米粒子的吸附量为94.37 mg/g,且用NaOH(pH13)溶液进行解析,CPP的洗脱率可达95%以上。综上,Fe₃O₄@ZrO₂磁性纳米粒子呈现出优异的选择性富集CPP的潜力,对高质量高纯度CPP 的生产具有重要的指导意义。     

酶催化浸出米糠油

对水酶催化浸出米糠油进行了研究。影响浸出最显著因素是酶催化时间、水中米糠浓度、己烷体积、酶用量。米糠和水混合物在 pH =4 .5 ,温度 5 0℃左右、催化反应 6h ,2 %纤维素酶 ,2 %果胶酶 ,水糠比率 5∶1(v/w)、己烷 /米糠 (v/w) 2 5∶1,油回收率达 10 0 %     

毛细管区带电泳测定酪蛋白磷酸肽方法的研究

用毛细管区带电泳(CZE)对酪蛋白磷酸肽(CPP)进行了分离和测定。研究出的适宜电泳操作条件为:工作电压30KV、柱温25℃、毛细管长度50cm、内径70μm、进样量5sec(气压进样)、紫外检测波长200nm、缓冲液pH值9.2测定不同N/P样品的CPP含量分别为42.2％、50.0％、51.0％。     

酪蛋白磷酸肽制备工艺研究

研究从水解液中制备酪蛋白磷酸肽的工艺。通过N/P和得率为评价指标,通过单因素试验和正交试验,确定了胰蛋白酶制备CPPs的条件:酪蛋白浓度为4%,底物与酶的比例为100∶1,水解温度40℃,水解时间60min。CPPs的沉淀分离条件为CaCl2终浓度1.0g/L,乙醇浓度50%vol,沉淀pH值为4.5,沉淀时间6.5h。     

分子量对酪蛋白多肽抗氧化活性的影响

在对酪蛋白酶解产物制备工艺进行优化的基础上,对不同分子量抗氧化肽(>3ku,1～3ku,<1ku)的抗氧化活性进行了评价。首先对酶的种类、酶底物比及水解时间进行了单因素实验,最终确定采用碱性蛋白酶,在pH8.0,55℃,底物浓度5%,酶底物比0.192AU/g的条件下,酶解4h所得水解物抗氧化活性最高。经过超滤和凝胶过滤层析分离获得不同分子量的抗氧化肽,并采用2,2′-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)二铵盐(ABTS+.)、羟自由基和超氧自由基清除活性评价其抗氧化性。结果表明,ABTS+.清除活性与分子量呈负相关(r=-0.898,p<0.01),分子量低于1ku组分活性最强(2mg/mL,Trolox当量为2.08±0.05mmol/L);分子量低于3ku的抗氧化肽羟自由基清除活性较高(IC50:1～3ku,4.43±0.03mg/mL;<1ku,4.35±0.06mg/mL);分子量高于3ku组分主要分布在3～5ku,超氧自由基清除活最强(10mg/mL,66.1%±1.0%)。     

高效液相色谱法测定婴幼儿配方奶粉中酪蛋白磷酸肽含量

目的建立高效液相色谱法(highperformanceliquidchromatography,HPLC)测定婴幼儿配方奶粉中酪蛋白磷酸肽(casein phosphopeptide, CPP)的含量的方法。方法对样品静置温度、静置时间、沉淀分离方式进行优化,用高效液相色谱法对婴幼儿配方奶粉中CPP的含量进行测定。结果最优的前处理条件为样品溶解后调整pH至4.6,于4℃条件下静置1 h,过滤后上清液经0.22μm滤膜过滤,以Ultimate AQ-C_(18)色谱柱(250mm×4.6mm,5μm)分离。经钼蓝比色法鉴定,保留时间为8.387min的组分为CPP。该方法在10～1000mg/L范围内线性关系良好,相关系数为0.9993,定量限为4.40 mg/L,婴幼儿奶粉中添加量为500、1000、5000 mg/kg时, CPP的回收率为96.63%%～101.42%。结论该方法样品前处理简单,稳定性和可靠性良好,检测结果符合定性定量要求,可用于婴幼儿配方奶粉中CPP含量的测定。     

酪蛋白磷酸肽(CPP)理化特性的研究

研究了CPP的溶解性、起泡性及乳化性。CPP在pH2.0～10.0范围内,溶解性除在pH4.0约为90％外,其它均高于90％,且随pH增加而增大。与酪蛋白相比,CPP具有更好的起泡性和泡沫稳定性。CPP对热及Ca~(2 )具有较好的稳定性。CPP乳化力较酪蛋白下降约20％,乳化性下降2.89％,乳化稳定性下降1.45倍。     

A biological perspective on the structure and function of caseins and casein micelles

Caseins belong to a larger group of secreted calcium phosphate-binding phosphoproteins that have a natively unfolded conformation. Nearly all members of the group are involved in aspects of calcium phosphate biology and nearly all have recognition sites for phosphorylation by the Golgi protein kinase. In the caseins these are often close together in the primary structure, forming the so-called phosphate centres. Certain highly phosphorylated phosphopeptides derived from the calcium-sensitive caseins will combine with amorphous calcium phosphate to form defined chemical complexes called calcium phosphate nanoclusters. Both the substructure of casein micelles and the partition of salts in milk can be explained quantitatively by the ability of the calcium-sensitive caseins to sequester calcium phosphate and form nanocluster-like structures. A simple stability rule for milk can be derived by applying equilibrium thermodynamics to the process of calcium phosphate sequestration. In principle, the stability rule can be extended to problems of instability encountered in milk-processing operations and to the formulation of other types of high calcium foods.     

Preparation of casein phosphorylated peptides and casein non-phosphorylated peptides using alcalase

Casein was digested with a cheaper enzyme, alcalase, to produce casein phosphorylated peptides and casein non-phosphorylated peptides concurrently. The casein hydrolyzates were separated to the two kinds of peptides by using combined treatment of CaCl₂ and ethanol. Casein phosphorylated peptides and non-phosphorylated peptides constitute some peptides with molecular weight lower than 2509 Da and 2254 Da respectively as determined using size exclusion HPLC, particularly when a degree of hydrolysis of 20% for the casein hydrolyzates was achieved. At the end, the recovery of casein phosphorylated peptides reached 24%. Phosphorus component of casein phosphorylated peptides was found to be 3.08%. The nitrogen recovery of casein non-phosphorylated peptides was about 76%.     

固定化碱性蛋白酶生产CPP的研究

以ＧＭ２０１大孔树脂和壳聚糖为载体,以戊二醛为交联剂,对碱性蛋白酶２７０９的固定化条件,固定化酶的性质以及利用固定化酶制备ＣＰＰ进行研究。考察了交联剂的浓度,载体与酶的比例,温度以及ＰＨ等对碱性蛋白酶固空化的影响。     

A possible structure of the casein micelle based on high-resolution field-emission scanning electron microscopy

New electron micrographs, produced using the technique of Field Emission Scanning Electron Microscopy, showing the details of the micellar surface, are presented. The images show the micellar surfaces without any coating, and suggest that the surface of the micelle may have a much more complex structure than has previously been demonstrated. Although there appear to be no spherical subunits (submicelles), there is evidence for the organization of the caseins into tubular structures within the micelle. The surface is not smooth, and contains gaps between the substructures. The observations are discussed in terms of published models of micellar structure, where is it suggested how the depiction of the micellar surface can be used to explain certain factors of its reactivity and behaviour.     

生物活性肽的研究进展理论基础与展望

随着具各种生物活性的短肽的不断发现,其研究和开发日益受到各国科学家的关注。大量科学研究表明,通过选择适当的蛋白酶,水解的蛋白质可以得到大量的具有各种生物功能的生物活性肽,这些活性肽不仅具有极其广泛的活性和多样性,而且其来源丰富、成本低、安全性好。操作简单、便于工业化生产,因此已成为科学家们研究的新热点。例如：酪蛋白是哺乳动物乳中含量最丰富的蛋白质,长期以来,被人们视为一种营养蛋白,但近年来的研究结果表明,它是生物活性肽的重要来源,到目前为止,已经有数十种生物活性肽被水解和辨认出来。所以,生物活性肽非常好的研究与开发前景。     

Re-assembled casein micelles and casein nanoparticles as nano-vehicles for ω-3 polyunsaturated fatty acids

Food enrichment with nutraceuticals is an important goal, but its effectiveness in preventing diseases depends on preserving the functionality and bioavailability of the bioactive nutraceuticals. Omega-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are important nutraceutical lipids, providing protection against cardiovascular and other diseases. Caseins are the major milk proteins whose biological function is to transport calcium, protein and phosphate from mother to the neonate. Our goal was to harness the natural self-assembly properties of caseins for protecting and delivering this important, but sensitive nutraceutical, DHA. Using spectrofluorescence we have shown, apparently for the first time, that casein can bind DHA with a relatively high affinity (K_b = (8.38 ± 3.12) × 10⁶ M⁻¹), and the binding ratio was 3–4 DHA molecules per protein molecule on average. Moreover, DLS particle characterization experiments have shown the formation of nanoparticles upon addition of DHA (predissolved in ethanol) to a casein solution. When calcium and phosphate were added (at 4 °C), DHA-loaded re-formed casein micelles (r-CM) with a size of 50–60 nm were obtained and there was no significant effect of the thermal treatment (74 °C, 20 s) on particle size. When casein nanoparticles (CNP) were prepared (at room temperature and without adding calcium and phosphate), DHA-loaded CNP with a diameter of 288.9 ± 9.6 nm were formed. Both the DHA-loaded r-CM and the DHA-loaded CNP systems showed a remarkable protective effect against DHA oxidation, demonstrating good colloidal stability and bioactive conservation throughout shelf life at 4 °C. These nanotechnologies may enable the enrichment of foods and beverages for promoting health of wide populations.     

高效液相色谱法测定奶粉中酪蛋白磷酸肽(CPP)含量

建立了一种用高效液相色谱法测定奶粉中酪蛋白磷酸肽(CPP)含量的方法。结果表明:该法前处理简便,专一性好,灵敏度高,检出限为10μg/mL。在10～200μg/mL范围内线性回归方程为y=0.1113x+0.2131,R2=0.9994,回收率为95%￣105%,可用于测定奶粉中CPP的含量。     

果味牛奶中柠檬酸盐对酪蛋白稳定性的影响

从非发酵型酸乳饮料的酸化工艺出发，对以柠檬酸为酸味剂的果味牛奶中，通过不同剂量和方式添加柠檬酸盐，以提高牛乳中酪蛋白在酸性条件下的稳定性。     

酪蛋白酶解产物及其分离组分对小鼠脾淋巴细胞功能的影响

为探讨酪蛋白的胃蛋白酶、胰蛋白酶、胃蛋白酶-胰蛋白酶双酶酶解产物分离组分对小鼠脾淋巴细胞功能的调节作用,采用凝胶过滤层析法对酶解产物进行初步分离,测定了分离组分对小鼠脾淋巴细胞增殖活性及IL-2生成量的影响。结果显示,胃蛋白酶-胰蛋白酶酶解产物作用效果优于单酶酶解产物,且其分离组分间具有一定协同作用。因此,将胃蛋白酶-胰蛋白酶酶解混合物用于保健食品的生产效果更优。