Secondary structural studies of bovine caseins: temperature dependence of β-casein structure as analyzed by circular dichroism and FTIR spectroscopy and correlation with micellization

To obtain a molecular basis for the similarities and dissimilarities in the functional, chemical, and biochemical properties between β-casein and the other caseins, three-dimensional models have been presented. Secondary structural prediction algorithms and molecular modeling techniques were used to predict β-casein structure. The secondary structure of bovine β-casein was re-examined using Fourier transform infrared and circular dichroism spectroscopies to test these predictions. Both methods predict a range of secondary structures for β-casein (28–32% turns, 32–34% extended) at 25°C. These elements were highly stable from 5 to 70°C as viewed by circular dichroism. More flexible conformational elements, tentatively identified as loops, helix and short segments of polyproline II, were influenced by temperature, increasing with elevated temperatures. Another view is that as temperature decreases, these elements are lost (cold denaturation). Several distinct transitions were observed by circular dichroism at 10, 33 and 41°C, and another transition, extrapolated to occur at 78°C. Calculations from analytical ultracentrifugation indicate that the 10, 33 and 41°C transitions occur primarily in the monomeric form of the protein. As β-casein polymers are formed, and increase in size, the transitions at higher temperature may reflect changes in the more flexible conformational elements as they adjust to changes in surface charge during polymer formation. The transition at 10°C may represent an actual general conformational change or cold denaturation. Over the range of temperatures studied, the sheet and turn areas remain relatively constant, perhaps forming a supporting hydrophobic core for the monomers within the micelle-like polymer. This interpretation is in accord with the known properties of β-casein, and those predicted from molecular modeling.     

羧甲基茯苓多糖对酪蛋白pH稳定性与生物活性的调控与机制研究

目的? 探究羧甲基茯苓多糖以不同比例与酪蛋白复配时对酪蛋白稳定性、结构与理化性质以及生物活性的影响。方法? 通过羧甲基衍生化,提升茯苓多糖的溶解性并表征其结构;以不同比例将羧甲基茯苓多糖与酪蛋白进行物理共混,测定复合物在不同pH值下的浊度、zeta电位、蛋白质溶解性、相图;通过激光共聚焦显微镜和旋转流变仪比较不同酪蛋白样品在酸性条件下的聚集特性与理化特性;通过自由基清除能力与非酶糖基化产物的抑制能力比较不同酪蛋白样品的生物活性。结果? 本研究所得羧甲基茯苓多糖的分子量为2.43×105 g/mol,通过取代度（0.84）和红外光谱中的羧甲基特征吸收峰可以确定羧甲基多糖的成功制备。当羧甲基茯苓多糖与酪蛋白共混后,能够有效提高酪蛋白的pH稳定性,显著影响了酪蛋白在酸性条件下的浊度、提高蛋白溶解性,这可能是由于多糖-蛋白质间静电互作力影响了复合物分子的表面电荷分布。其中,当二者以5: 1（w/w）复合时,羧甲基茯苓多糖表现出最好的稳定效果。随后的结构与理化性质研究表明,引入多糖,特别当二者以5: 1（w/w）复合时,复合物具有较低的聚集程度、表观粘度与模量,表明此时复合物的凝胶网络结构具有更好的结构稳定性。生物活性结果表明,复合物,特别以5: 1（w/w）复合时,表现出最强的抗氧化活性和非酶糖基化产物的抑制能力。结论 本研究所得羧甲基茯苓多糖-酪蛋白复合物在酸性环境下具有更好的结构稳定性与生物活性,为稳定的酸性乳制品的开发与加工提供理论基础和技术支持。     

Effect of water content on the mobility of solute molecules and of protein side chains in casemate preparations

The effect of water content on the mobility of small solute molecules and of casein side chains was studied by electron spin resonance in caseinate/water systems. The immobilization of nitroxide probes of different sizes and properties were followed during progressive dehydration of concentrated caseinate preparations (3 g H₂O/g dry protein). Above a characteristic minimum moisture content a linear relationship was observed between the rotational diffusivity and water content. The slope of the straight lines depended upon the size of the probe and upon the nature of the interactions with the protein. Nitroxide labels were covalently bound to caseinate and the changes in the flexibility of the side chains as hydration changed were followed. The results are discussed in relation to the solvent properties of water, and to recent suggestions concerning the significance, for stability, of the glass transition phenomenon in such materials.     

Removal of Emulsified Oil from Water by Coagulation and Foam Separation

Abstract

A new method of emulsified oil separation for oily wastewater incorporating simple operation and shortened treatment time is necessary for improved wastewater treatment in some manufacturing plants. In the present study, the removal of emulsified oil from water by coagulation and foam separation using poly aluminum chloride (PAC) and milk casein was examined. By adding casein before the foam separation process, the oil removal was dramatically improved. By using surfactant (LAS) as a frother, the dosage of casein was drastically reduced. Furthermore, for processing actual oily water, LAS was unnecessary because a sufficient amount of surfactants for foaming was included in the wastewater. For treatment of the actual oily wastewater collected from a steel manufacturing plant, the optimum condition for PAC and casein was 30 mg‐Al/L and 10 mg/L, respectively, and the oil concentration decreased from 170 mg/L to 2.2 mg/L. After examining several types of oily wastewater, 96–99% of oil removal efficiency was obtained by adjusting the dosages of PAC and casein. Coagulation and foam separation using casein has shown a high potential as an alternative method to dissolved air flotation (DAF) for processing emulsified oil water.     

酪蛋白磷酸肽(CPP)促进铁吸收的研究

体外消化试验表明CPP能显著提高FeCL_3溶液中Fe~(3+)的溶解性(P<0.01),并显著促进Fe~(3+)还原为Fe~(2+)(P<0.05),上述作用受CPP浓度和纯度影响较大,受pH值影响不显著(P>0.05)。加入CPP使可溶性铁和二价铁的透析率均显著高于对照组(P<0.01)。CPP与Fe~(3+)结合能力受磷酸基密度、浓度影响较大,受pH值影响不显著(P>0.05)。     

酪蛋白酶解制备酪蛋白磷酸肽研究 I．过程分析与优化

以pH－stat技术跟踪间歇搅拌反应釜中酪蛋白－碱性蛋白酶抑制备酪蛋白磷酝酸肽（CPPs）过程特性,利用水解度（DH）表征其反应历程。分析了温度（T）、pH值、底物深度（[S]）、酶浓度（[E]）及时间(t)等参数对DH的影响,并通过多元线性回归求得其关联式,确定出最适酶解条件为：T＝40℃,pH＝10,[E]＝0．2％,[S]＝2．5g/L,t=30min。     

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%。     

底物亲和法分离纯化蕲蛇胃蛋白酶

利用酶(胃蛋白酶)与其底物(酪蛋白)的亲和性,从蕲蛇胃中提取得到一种胃蛋白酶.让底物和酶在pH 2.5的乳酸缓冲液中充分结合,然后调pH至4.0(底物的等电点)沉淀底物和酶的结合物,随后让沉淀物再溶解于乳酸缓冲液中,添加低浓度的SDS将底物和酶分离,最后用DEAE离子交换色谱柱进行纯化,经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,分离得到的胃蛋白酶为电泳纯,其分子量为33KD.酶的最适pH值小于3.0,最适温度为50℃.该结果证明胃蛋白酶能利用其与酪蛋白的亲合性及SDS沉淀作用有效地分离,这种胃蛋白酶能在食品生产的加热、强酸处理过程中应用.