Alcalase水解酪蛋白制备磷酸肽和非磷肽的研究

研究了Alcalase水解酪蛋白制备磷酸肽和非磷肽的作用条件,同时分析了酪蛋白磷酸肽(CPPs)和酪蛋白非磷肽(CNPPs)的相对分子质量分布和氨基酸组成。结果表明,Alcalase水解酪蛋白的最佳条件是:底物浓度5%,加酶量0.03mL/g蛋白质,温度60℃,pH9.0。乙醇浓度和酪蛋白的水解度(DH)对CPPs的得率和N/P(摩尔比)均有影响:当乙醇浓度为70%,DH为18%时,CPPs得率达到最高,为23.94%,N/P(摩尔比)是9.61,CNPPs得率为76.06%。CPPs和CNPPs的相对分子质量都分布在200～5000之间,其中低于700的肽含量最高,分别为61.52%和67.26%。CNPPs中疏水性氨基酸含量远高于CPPs。     

凝乳酶水解酪蛋白生产酪蛋白糖巨肽工艺条件的研究

酪蛋白糖巨肽(casein glycomacropeptide,简称CGMP)是K-酪蛋白经酶解后生成的一类舍有糖链的多肽,具有许多功能特性.本文研究了凝乳酶水解酪蛋白生产CGMP的工艺条件,通过正交实验得到最佳工艺条件是底物浓度10mg/mL、酶的添加量为0.6%、酶解时间为2.5h.唾液酸回收率为80.7%,蛋白质回收率为3.03%,糖基化程度(唾液酸/蛋白质)为77.6μg/mg.     

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。     

酪蛋白生物活性肽的特性及应用前景

酪蛋白是一种营养蛋白,不仅具有营养功能,而且还具有重要的生理功能,是生物活性肽的重要来源.到目前为止已有数十种生物活性肽从酪蛋白中水解并得到确认.酪蛋白糖巨肽(Caseino Glycomacro peptide,CGMP)是从乳清中κ-酪蛋白经凝乳酶降解产生的含有糖链的多肽片段,具有独特的营养特性和生理功能,可广泛地应用于功能性食品和生物医药领域.     

一种连续分离纯化酪蛋白磷酸肽的新工艺

为建立一条制备酪蛋白磷酸肽(CPPs)的新工艺路线。采用酶解与超滤耦联技术初步分离制备酪蛋白生物活性肽,然后通过树脂层析将酪蛋白生物活性肽中的酪蛋白磷酸肽和非磷酸肽分离。选用碱性蛋白酶Alcalase在切向流酶膜反应器中(膜截留分子质量10kD)连续酶解3h、酶解温度50℃、pH8.5。重点研究大孔弱碱性阴离子交换树脂D303对酪蛋白生物活性肽的分离纯化工艺,并采用单因素试验优化CPPs的分离纯化工艺参数,确定D303树脂的较佳动态吸附解吸条件：上样流速1BV/h、上样量100mL、洗脱温度45℃、洗脱酸浓度0.15mol/L HCl,获得磷酸肽N/P比为7.21,P洗脱得率为93.11%。     

响应面法优化酪蛋白磷酸肽生产工艺

在单因素试验的基础上,根据Box-Behnken的中心组合实验设计原理,采用3因素3水平的响应曲面分析法,建立了胰蛋白酶水解酪蛋白制备酪蛋白磷酸肽的二次多项数学模型,并以水解度为响应值作响应面和等高线,得到酶水解酪蛋白制备酪蛋白磷酸肽的优化工艺条件为:水解时间为6 h,酶底比为2‰,温度50℃,底物质量分数为4%,pH值为8.4,旋转振荡速度120 r/min,在此条件下实际水解度为62.84%。     

酪蛋白源降胆固醇肽的酶解制备工艺优化

以酪蛋白为原料,采用中性蛋白酶、碱性蛋白酶以及胰蛋白酶对酪蛋白进行水解,确定制备降胆固醇肽的最佳蛋白酶;通过单因素实验和响应面试验,研究水解pH、水解温度、酶与底物比、底物浓度和水解时间对酪蛋白水解度和胆固醇胶束溶解度抑制率的影响,确定最佳水解条件;而后通过超滤和凝胶过滤层析确定降胆固醇肽的初步分离工艺。结果表明:制备酪蛋白源降胆固醇肽的最佳水解工具酶是中性蛋白酶,其最佳酶解条件为反应温度51.3 ℃,酶与底物浓度比6.47%,pH6.34,底物浓度5 g/100 mL,反应时间3.5 h,胆固醇抑制率为58.25%±0.59%;Sephadex G-10分离酪蛋白降胆固醇肽条件为上样浓度80 mg/mL,上样体积2.5 mL,洗脱速度3.5 mL/min;经酶解、超滤及层析后制备的酪蛋白源降胆固醇肽峰1和峰2样品在100 μg/mL的胆固醇溶解度抑制率为24.2%±0.24%和4.3%±0.16%。经酶解制备分离后,获得具有抑制降固醇胶束溶解活性的降胆固醇肽,为降胆固醇肽的开发提供理论研究基础。     

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.     

酪蛋白糖巨肽分离纯化方法的研究进展

酪蛋白糖巨肽(CGMP)主要是κ-酪蛋白经凝乳酶降解产生的一类含有糖链的多肽,具有许多的生理活性功能和独特的营养特性,可广泛地应用于保健食品和医药品。本文对酪蛋白糖巨肽的来源及结构作了简要概括,对其分离纯化方法及进展作出了详细论述。     

碱性蛋白酶水解劣质干酪素制备酪蛋白磷酸肽的研究

本实验采用一种微生物酶碱性蛋白酶2709水解劣质干酪素制备酪蛋白磷酸肽(CPPs),采用正交实验优化其水解条件,得出了其最佳水解条件为底物浓度15%,酶浓度800u/g,pH9.0,温度55,时间2.5h.。其N/P为4.371,产率为12.88%。     

酪蛋白磷酸肽持钙能力的研究

通过体外模拟实验考察酪蛋白磷酸肽( CPP)阻止磷酸钙沉淀的能力,分析了水解度、CPP添加量、CPP的不同等级以及食品加工过程中的其他因素对CPP持钙能力的影响,为CPP功能特性的评价以及在食品中的应用提供了依据.     

基于酪蛋白免疫快速检测的羊奶鉴伪方法建立

为了能够快速、灵敏、低成本和方便地对市场上的羊奶进行鉴伪,本文基于竞争法原理开发了牛源酪蛋白胶体金免疫层析试纸条并对其进行了评价.通过在纯羊奶中添加不同比例的牛奶,获得含有不同浓度牛源蛋白的羊奶溶液并进行免疫层析试纸条测定,评价了该方法的检测范围和灵敏度,分析了奶制品经蒸煮和酶解处理对试纸条检测结果的影响,评价了该方法...     

酪蛋白降解对牦牛乳硬质干酪苦味的影响

针对牦牛乳硬质干酪的苦味缺陷,分别以小牛皱胃酶、微生物凝乳酶和木瓜蛋白酶制作的牦牛乳硬质干酪为研究对象,利用尿素聚丙烯酰胺凝胶电泳,研究牦牛乳硬质干酪pH 4.6水不溶性酪蛋白的降解程度,且对成熟过程中的牦牛乳硬质干酪苦味进行感官评价,探究牦牛乳硬质干酪pH 4.6水不溶性酪蛋白降解对其苦味的影响。结果表明：牦牛乳硬质干酪在成熟期间酪蛋白发生了明显的降解,且αs-酪蛋白均比β-酪蛋白降解速率快。经尿素聚丙烯酰胺凝胶电泳分离后,发现木瓜蛋白酶制作的牦牛乳硬质干酪pH 4.6水不溶性酪蛋白在Pre-αs-酪蛋白区域有较强的蛋白带。木瓜蛋白酶制作的牦牛乳硬质干酪pH 4.6水不溶性酪蛋白中αs-酪蛋白和β-酪蛋白降解程度均显著或极显著高于微生物凝乳酶和小牛皱胃酶制作的牦牛乳硬质干酪（P＜0.05或P＜0.01）,木瓜蛋白酶制作的牦牛乳硬质干酪的苦味值极显著高于微生物凝乳酶和小牛皱胃酶制作的牦牛乳硬质干酪的苦味值（P＜0.01）,通过主成分分析得出3 种凝乳酶制作牦牛乳硬质干酪的苦味值和未降解β-酪蛋白和αs-酪蛋白含量成极显著负相关。这为控制牦牛乳硬质干酪品质提供了理论参考。     

Relative Proteolytic Action of Milk-Clotting Enzyme Preparations on Bovine α-, β- and β-casein

Concentrations of seven milk-clotting enzyme preparations were standardized to equal clot times. Portions of bovine α_s-, β- and κ-casein were treated with enzymes. Proteolytic activity of the coagulants on each casein fraction was determined using the TNBS (2,4,6-trinitrobenzene-sulfonic acid) procedure. Recombinant chymosin showed the lowest degree of proteolysis on α_s- and β-caseins. Excessive proteolysis of calf rennet appeared to be due to the pepsin fraction. M. miehei and M. pusillus var Lindt proteases showed similar degradation of caseins, but M. pusillus var Lindt was more proteolytic when β-casein was the substrate. C. parasitica protease showed the highest degree of proteolysis on α_s- and β-caseins but was the least proteolytic on κ-casein.     

Chemical fractionation of caseins by differential precipitation: influence of pH,calcium addition,protein concentration and temperature on the depletion in α- and β-caseins

The impact of pH, calcium and casein concentrations, and temperature on the efficiency of the differential precipitation of caseins by calcium affinity was investigated, using native phosphocaseinate as starting material. We adapted one of the most recent methods for the separation of caseins that is based on the addition of calcium at alkaline pH. Increasing the pH to 11 disturbed the micellar structure by enhancing electrostatic repulsion of caseins, leading to a marked viscosity increase and a significant particle size decrease, indicating casein micelle disruption. This pH-driven increase in negative charges enhanced the affinity of individual caseins for calcium, proportionally to the number of phosphate groups carried by each casein. The addition of calcium first led to a progressive increase in the proportion of precipitated caseins, before reaching a plateau. Hence, an optimal calcium/casein molar ratio of about 40 was evidenced to optimise casein precipitation (and fractionation), leading to significant depletion of α-casein (around 80%) and, in a lesser extent, β-casein (around 65%) and κ-casein (around 55%). This method led to relative proportions of caseins significantly differing from the starting material: 31% α-casein, 45% β-casein and 24% κ-casein.     

Effects of Environmental Factors and Milk Protein Polymorphism on Composition of Casein Fraction in Bovine Milk

Test-day samples were collected from individual Holstein cows in 62 herds enrolled in the Quebec Dairy Herd Analysis Service. Samples were analyzed for protein, fat, casein, and serum protein content, somatic cell count, and relative percentages of α-, β-, and κ-casein and a-lactalbumin. Cows included in the study were phenotyped for the genetic variants of α_s1-, β-, and κ-casein. Unadjusted means for relative percentages of α_s-, β-, and κ-casein were 59.85, 31.23, and 8.93%, respectively. Least-squares analyses showed that month of test, stage of lactation, age of the cow, somatic cell count, and phenotype of the cow for β-casein contributed to variations in the relative percentages of α_s- and β-casein. Month of test, somatic cell count, and phenotype of the cow for κ-casein also had a significant effect on the relative percentage of κ-casein. When test-day milk yield; percentages of fat, protein, casein, and serum protein; casein to protein ratio; and relative percentage of α-lactalbumin were included in the model as covariates, only casein percentage did not have a significant effect on the relative percentages of α_s- and β- casein. For κ-casein, only fat percent was significant.     

Effect of minor milk proteins in chymosin separated whey and casein fractions on cheese yield as determined by proteomics and multivariate data analysis

The objective of this work was to find regressions between minor milk proteins or protein fragments in the casein or sweet whey fraction and cheese yield because the effect of major milk proteins was evaluated in a previous study. Proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry in combination with multivariate data analysis were used to study the effect of variations in milk protein composition in chymosin separated whey and casein fractions on cheese yield. By mass spectrometry, a range of proteins significant for the cheese yield was identified. Among others, a C-terminal fragment of β-casein had a positive effect on the cheese yield expressed as grams of cheese per 100 g of milk, whereas several other minor fragments of β-, α_s1-, and α_s2-casein had positive effects on the transfer of protein from milk to cheese. However, the individual effect of each identified protein was relatively low. Therefore, further studies of the relations between different proteins/peptides in the rennet casein or sweet whey fractions and cheese yield are needed for advanced understanding and prediction of cheese yield.     

干酪乳杆菌胞壁蛋白酶的分离及水解酪蛋白产物特性

通过DEAE-Sephadex A-25和Sephadex G-100对干酪乳杆菌胞壁蛋白酶粗提物进行分离纯化,分离到酶比活力为12.50U/mg的CEP-1与16.67U/mg的CEP-2两种胞壁蛋白酶。CEP-1提纯倍数达到50,回收率为33.61%;CEP-2提纯倍数为66.68,回收率55.17%。对不同时间和底物浓度下CEP-1与CEP-2的α-酪蛋白和β-酪蛋白水解特性进行研究。结果显示,酪蛋白水解产物有显著的抗ACE与抗氧化活性,产生最高ACE抑制活性的水解条件为：CEP-2水解α-酪蛋白,时间6h,酶与底物质量比1:10,此时ACE抑制活性为84.66%;在酶与底物质量比1:40,水解时间4h条件下,CEP-2水解β-酪蛋白产生最佳的O2－ ·清除能力,其IC50为0.2138mg/mL。     

Relationships Between Milk Protein Polymorphisms and Major Milk Constituents in Holstein-Friesian Cows

Milk samples from 1,908 Holstein Friesian cows were phenotyped for genetic variants of α_s1 -casein, β-casein, κ-casein, and β-lactoglobulin. The relationships between milk protein polymorphism and test day milk yield and composition were investigated. After adjustments were made for environmental effects of herd, parity number, month of test, stage of lactation, age of sample at testing, and somatic cell count, milk protein phenotypes were found to be associated with milk yield; concentrations in fat, protein, casein, and whey protein; and proportion of casein in protein. Higher test-day milk production was associated with α_s1-casein BB, β-casein A1A3, κ-casein AA, and β-lactoglobulin AA phenotypes. Fat and protein concentrations were highest in milk from cows of α_s1-casein BC, β-casein A1B, and κ-casein BB phenotypes. β-Lactoglobulin BB milk was associated with higher percentages of fat and casein and with lower percentages of total protein and whey protein. Based on desirable fat and casein contents of milk for cheese production, it would be advantageous to select for cows bearing κ-casein BB and β-lactoglobulin BB phenotypes.