矢车菊素-3-葡萄糖苷与大豆蛋白相互作用的多重光谱分析及分子对接

采用多重光谱技术和分子对接技术，研究矢车菊素-3-葡萄糖苷（cyanidin-3-glucoside，C3G）与β-伴大豆球蛋白和大豆球蛋白的相互作用。结果表明，C3G以静态、动态组合模式强烈的猝灭β-伴大豆球蛋白/大豆球蛋白的内源荧光，且C3G对大豆球蛋白的结合亲和力强于β-伴大豆球蛋白。C3G与β-伴大豆球蛋白/大豆球蛋白结合的主要相互作用力不同，但n（结合位点数）表明C3G和大豆蛋白以物质的量比1∶1形成稳定的复合物。C3G能够诱导大豆蛋白二级结构部分展开，促使部分α-螺旋转变为β-折叠，使大豆蛋白多肽链解折叠；并降低β-伴大豆球蛋白色氨酸残基微环境疏水性，而对大豆球蛋白氨基酸残基微环境没有明显影响。C3G的大部分酚羟基参与成键，其与大豆蛋白的结合依靠疏水作用力和氢键为主导的多种作用力维持。大豆球蛋白对C3G具有更好的稳定、递送性能，但可能不利于C3G生物活性的发挥。     

高效液相色谱法分析中国荷斯坦奶牛乳蛋白多态性

采用高效液相色谱法检测牛乳中6种主要乳蛋白的多态性。奶样来自102头泌乳中国荷斯坦奶牛。研究发现,酪蛋白中αs2-酪蛋白存在A型和B型两种类型,β-酪蛋白存在A1,A2,B,C和F5种类型,κ-酪蛋白存在A/E型和B型两种类型,而αs1-酪蛋白仅有1种,未发现其多态性;乳清蛋白中,β-乳球蛋白存在多态性,有A,B和C3种类型,但未发现α-乳白蛋白的多态性。研究结果表明,在本试验条件下,中国荷斯坦奶牛乳中除αs1-酪蛋白和α-乳白蛋白外,αs2-酪蛋白、β-酪蛋白、κ-酪蛋白以及β-乳球蛋白均存在多态性,以β-酪蛋白类型最多。     

帕米尔牦牛乳蛋白分离及α-乳白蛋白纯化研究

帕米尔牦牛分布在新疆克孜勒苏柯尔克孜自治州和喀什地区境内的帕米尔高原,帕米尔牦牛乳蛋白含量高,营养丰富,是当地牧民重要的食物来源。该研究利用等电点沉淀法对新疆帕米尔牦牛乳中大分子蛋白进行分离,得到乳清蛋白和酪蛋白,对其含量进行测定,并采用硫酸铵分级沉淀和离子交换层析等方法对α-乳白蛋白(α-lactalbumin,α-La)进行纯化。结果表明,帕米尔牦牛乳中总蛋白含量为(2.81±0.23) g/100mL,其中乳清蛋白和酪蛋白质量比为43∶57;帕米尔牦牛乳蛋白质包含α-La、β-乳球蛋白(β-lactoglobulin,β-Lg)、酪蛋白、血清白蛋白、乳铁蛋白和免疫球蛋白G等多种活性蛋白;另外,用饱和度为80%的硫酸铵可将α-La和β-Lg有效分离;再用DEAE-Sepharose离子交换层析进一步纯化,可除去β-Lg,得到高纯度的α-La。该研究可为新疆帕米尔牦牛产业发展及牦牛乳产品开发提供理论基础。     

山羊乳蛋白质及其组学分析

为探索山羊乳蛋白质的基本组成,利用蛋白质组学技术——功能强大的分离技术,结合高分辨率的质谱分析,以获得极其重要的乳蛋白信息。采用毛细管电泳法解析山羊乳中的宏量蛋白质,采用蛋白质组学技术分析山羊乳中乳清蛋白和乳脂肪球膜蛋白的种类和功能分布,以及乳粉加工工艺对山羊乳蛋白质组成的影响,并以牛乳蛋白质作比较。结果发现,通过蛋白质组学技术鉴定出山羊乳乳清蛋白103种,乳脂肪球膜蛋白121种;在热处理和均质过程中,乳清蛋白是首先变性的蛋白质,尤其是β-乳球蛋白,而乳脂肪球膜蛋白含量降低的同时会引起其中的β-乳球蛋白、β-酪蛋白及α-酪蛋白的含量显著增加。综上所述,蛋白质组学技术为山羊乳蛋白质的进一步研究奠定了基础,为研究加工过程中乳蛋白组分的变化提供了机会。     

花青素与小麦蛋白相互作用及对蛋白质结构的影响

利用荧光光谱法和傅里叶变换红外光谱法,研究中性条件下黑豆皮中的花青素（矢车菊素-3-O-葡糖苷（cyanidin-3-O-glucoside,C3G））与小麦蛋白麦醇溶蛋白（gliadin,Gli）及麦谷蛋白（glutenin,Glu）的相互作用。荧光结果表明：C3G对Gli、Glu均有较强的荧光猝灭作用,对Glu的荧光猝灭机制属于静态猝灭,而与Gli的猝灭方式为动态猝灭和静态猝灭的结合,C3G与Gli和Glu的结合常数（KA）分别为20.827×104、14.690×104 L/mol,结合位点（n）分别为1.263和1.159（298 K）,说明与Gli作用较强。热力学研究表明：C3G与Gli主要通过疏水作用结合;与Glu作用主要通过范德华力和氢键作用结合。同步荧光光谱分析表明：C3G与Gli的结合位点更接近色氨酸残基,而与Glu的结合位点更接近酪氨酸残基。傅里叶变换红外光谱表明：C3G能够与Gli、Glu结合并相互作用,使蛋白构象发生变化。     

槲皮素与β-乳球蛋白B的结合机制及其溶解性能

为解决槲皮素水溶性差的问题,采用β-乳球蛋白B作为载体,基于多光谱、等温滴定量热仪和分子模拟等实验手段,探究槲皮素和β-乳球蛋白B的结合机制,以及结合后β-乳球蛋白B的二级结构和槲皮素溶解度的变化。结果表明：槲皮素可与β-乳球蛋白B结合,该结合过程中氢键和范德华力起主要作用;三维荧光、同步荧光以及紫外-可见吸收光谱表明,槲皮素的结合使β-乳球蛋白B的二级结构发生了改变;圆二色谱和傅里叶变换红外光谱结果表明,槲皮素的结合诱导该蛋白中部分α-螺旋转变为β-折叠;分子模拟结果表明,槲皮素在β-乳球蛋白B上的结合位点位于一个由α-螺旋与β-转角所形成的空穴中,在该结合位点中有8 个氨基酸残基参与了与槲皮素的结合,其中第125位的苏氨酸残基提供了较强的氢键,其余残基则提供了范德华力;基于高效液相色谱检测发现在与β-乳球蛋白B结合后,槲皮素的溶解度增大至原来的1 844 倍左右。综上所述,以β-乳球蛋白B为载体结合的槲皮素水溶性显著提高。     

超高压处理对乳清分离蛋白结构及致敏蛋白含量的影响

以乳清分离蛋白为研究对象,通过测定圆二色性光谱、巯基含量以及内源性荧光光谱等研究了不同超高压水平(100、200、400和600 MPa)对其二级、三级结构的影响,并采用水解度测定、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以及2个标志性致敏蛋白(β-乳球蛋白和α-乳白蛋白)含量的检测来解析超高压对乳清分离蛋白致敏性的影响。结果表明,超高压处理能够使乳清分离蛋白的α-螺旋和β-转角部分转化为β-折叠和无规则卷曲,可以增强乳清分离蛋白的巯基含量,在400 MPa时,表面巯基含量提高了104.82%,也造成了乳清分离蛋白内源性荧光强度的显著变化以及最大吸收波长的红移,电泳图谱以及水解度未显示出明显差异。通过酶联免疫吸附实验原理检测致敏蛋白含量发现,超高压可以使α-乳白蛋白含量显著减少,但是,400 MPa的超高压处理却使β-乳球蛋白含量增加。综上表明,超高压处理能够显著改变乳清分离蛋白的二级、三级结构,暴露出结构内部的疏水基团,并对致敏蛋白产生影响。     

基于荧光及紫外光谱法对红松种鳞多酚与乳清蛋白相互作用的研究

利用紫外-可见吸收光谱法和荧光光谱法,研究了红松种鳞多酚与乳清蛋白之间的相互作用及其作用方式。紫外-可见吸收光谱表明:红松种鳞多酚与乳清蛋白发生了相互作用并生成新的复合物,改变了蛋白质芳香族残基在空间结构中所处的微环境,诱导乳清蛋白的构象发生改变。荧光光谱表明:红松种鳞多酚对乳清蛋白有较强的荧光猝灭作用且猝灭类型为生成复合物的静态猝灭作用。通过计算得出在不同温度下其二者相互作用的表观结合常数(KA)分别为:1.111×104 L/mol(25℃),2.201×104 L/mol(30℃),6.206×104L/mol(35℃),对应的结合位点数(n)分别为:0.885、0.937、1.043。由热力学参数分析确定红松种鳞多酚与乳清蛋白间的相互作用反应是吸热过程,反应的主要作用力是疏水作用力。     

β-乳球蛋白-IgG混合物的反胶束萃取平衡及其机理探讨

依据堆砌几何模型，选择琥珀酸二(2-乙基己基)酯磺酸钠(AOT)-异辛烷反胶束萃取体系，研究了β-乳球蛋白单一蛋白体系中以及β-乳球蛋白与免疫球蛋白G(1：1)混合物体系中蛋白质的分布特性，并探讨了AOT-异辛烷反胶束体系分离分级乳蛋白的机理。β-乳球蛋白在单一蛋白或混合蛋白(Wβ-lg：WlgG为1：1)体系中的反胶束萃取率均随着水相pH值增加(pH值为5．0-9．0)而降低，随水相[Na^ ]浓度升高(0．06-0．35mol／L)而逐渐增加，随水相起始蛋白质量浓度增加(0．75-3．0g／L)而降低，但免疫球蛋白G可明显促进β-乳球蛋白在反胶束有机相中的增溶。在混合蛋白质体系中，β-乳球蛋白与免疫球蛋白G可分别趋于富集在有机相和水相，显示出AOT-异辛烷反胶束体系在牛初乳乳清蛋白分离方面具有应用潜力。     

牛乳清蛋白中β-乳球蛋白的分离纯化

结合硫酸铵分段盐析和凝胶层析两种方法,从牛乳乳清蛋白中分离、提纯出β-乳球蛋白。通过SDS-聚丙烯酰胺凝胶电泳检测蛋白成分,结果显示:①在20%～30%、70%～80%盐析段只有两条带,分别是β-乳球蛋白和α-乳清白蛋白带,得到较纯的蛋白;②再用SephadexG-50凝胶过滤层析,电泳结果显示只有一条带,并与标准β-乳球蛋白带一致,得到较纯的β-乳球蛋白。     

Study of the acid and thermal stability of β-lactoglobulin–ligand complexes using fluorescence quenching

The major protein in bovine milk whey, β-lactoglobulin (β-LG), has several binding sites for ligands. Its interactions with folic acid (a hydrophilic compound), resveratrol (amphiphilic) and α-tocopherol (hydrophobic) at neutral and acidic pH and after heating to 85 °C were studied using fluorescence quenching. Binding of folic acid occurs in a hydrophobic pocket in the groove between the α-helix and the β-barrel and is disturbed by decreasing the pH from 7.0 to 2.0. Resveratrol binds to the outer surface of β-LG near Trp19–Arg124 to form complexes that are stable at acidic pH. Acidification caused the release of α-tocopherol bound to the internal cavity but had no influence on that bound to a site at the surface of β-LG. The β-LG/folic acid complex was thermally stable. Thermal denaturing improved the affinity of the protein for resveratrol but decreased somewhat its affinity for α-tocopherol. These results should help guide the development of formulations based on β-LG as a carrier of a wide range of bioactive nutrients.     

HPLC-MS/MS-Detection of Caseins and Whey Proteins in Meat Products

Screening methods for the mass spectrometric detection of caseins and whey proteins in meat products have been developed. After tryptic digestion, two α-S1-casein and two β-lactoglobulin marker peptides were measured by HPLC-MS/MS. For matrix calibrations, emulsion-type sausages with different concentrations of milk and whey protein (ppm level) were produced. The limits of detection (LODs) were below 1 ppm for milk protein and about 3 ppm for whey protein. The determination coefficients for the correlation between peak area of the marker peptides and the concentrations of milk and whey proteins were R²≥0.9899.     

The use of “lab-on-a-chip” microfluidic SDS electrophoresis technology for the separation and quantification of milk proteins

The use of a microfluidic “lab-on-a-chip” technique for the separation and quantification of milk proteins is described and compared with traditional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The microfluidic chip technique could separate all major milk proteins when standard protein solutions were used. In a milk system, α-lactalbumin, β-lactoglobulin, α_s-casein, β-casein and κ-casein were readily separated; the resolution was comparable with SDS-PAGE. However, the immunoglobulins, lactoferrin and bovine serum albumin could not be resolved from the background in the microfluidic chip technique, but were easily resolved by SDS-PAGE. Standard curves for the major whey proteins were linear with both techniques and the calculated concentrations of the major proteins in a milk sample were comparable using both microfluidic chip and SDS-PAGE techniques. These results indicate that the microfluidic chip technology may be a rapid alternative for the separation and quantification of proteins in milk protein based systems.     

Changes in proteins,physical stability and structure in directly heated UHT milk during storage at different temperatures

Changes occurring in directly heated UHT milk were studied during storage at 5, 22, 30 and 40 °C. Industrially produced UHT milk samples were analysed for changes in enzymatic activity, protein modification, destabilisation of casein micelles and relocation of milk proteins in relation to sedimentation and gel formation. Sedimentation occurred at all temperatures, and the protein composition of the sediments reflected the composition of its liquid phase; however, there was no α-lactalbumin, β-lactoglobulin or κ-casein present in sediments. Tendrils composed of β-lactoglobulin and κ-casein were seen on casein micelles after UHT treatment and grew in length prior to gelation. High degrees of lactosylation of proteins and peptides were clearly correlated with the absence of gelation and long tendrils. Gelled samples showed complete hydrolysis of intact β-casein, and limited lactosylation of β-lactoglobulin and κ-casein.     

Radiolabelling Study of the Heat-induced Interactions Between α-Lactalbumin, β-Lactoglubulin and K-Casein in Milk and in Buffer Solutions

Radiolabeled α-lactalbumin, β-lactoglobulin and κ-casein were added to milk prior to heating it at 95°C for up to 20 min. The distribution of these proteins between the micellar and serum phases and between various sized aggregates were determined chromatographically. The results from these and other supplementary experiments were consistent with a model of whey proteins denaturing in milk during heat treatment and using κ-casein as a nucleation site for the formation of heat-induced complexes involving disulfide bonding. These heat-induced complexes, like κ-casein itself, remained associated with the external surface of the casein micelle and continued to enlarge with additional heat treatment.     

Aggregation and Dissociation of Milk Protein Complexes in Heated Reconstituted Concentrated Skim Milks

Heating milk at 120°C at pH 6.55 or pH 6.85 caused the denaturation of whey proteins and increased their association with the casein micelles. The dissociation of K -, β-, and α_s-caseins (in that order by extent) from the casein micelles increased with severity of heat treatment. The effect was greater at higher pH. Gel filtration chromatography followed by gel electrophoresis of fractions showed the dissociated protein was composed of disulfide-linked k -casein/β-lactoglobulin complexes of varying composition, casein aggregates of varying sizes and some monomeric protein. When reconstituted concentrate was prepared from NFDM made from heated milk the non-sedimentable (88,000 ± g for 90 min) caseins or whey proteins/heating time profiles were altered and the rate of aggregation, as measured by turbidity of heated milks, was significantly reduced.     

Conformational changes to deamidated wheat gliadins and β-casein upon adsorption to oil-water emulsion interfaces

The conformation of deamidated gliadins and β-casein in solution and adsorbed at the interface of oil-in-water emulsions was studied using synchrotron radiation circular dichroism (SRCD) and front-face-fluorescence spectroscopy. Deamidation led to partial unfolding of gliadins in solution. The α-helix content of the protein decreased from 35% (in the native form) to 16.3% while the percentage of β-sheet and unordered structure increased upon deamidation. The secondary structure of deamidated gliadins was largely unchanged upon adsorption to both tricaprin/water and hexadecane/water interfaces. In contrast, β-casein adopted a more ordered structure upon adsorption to these two oil/water interfaces, the α-helix content increased from 5.5% (in solution) to 20% and 22.5% respectively after adsorption to tricaprin/water and hexadecane/water interfaces. Both deamidated gliadins and β-casein have distinctive N-terminal hydrophilic and C-terminal hydrophobic domains. Unlike β-casein which contains no cysteine residue, gliadins have a large number of intramolecular disulphide bonds located in the C-terminal hydrophobic domain which constrains the conformational freedom of this protein upon adsorption to oil/water interfaces. The hydrophobicity of the oil phase also has an impact on the conformation of each protein upon adsorption to the oil/water interfaces - systematic trends were observed between oil phase polarity from: i) tryptophan fluorescence emission maxima, and ii) the α-helix content in the adsorbed state. Our results demonstrate that conformational re-arrangement of proteins upon adsorption to emulsion interfaces is dependent not only on the hydrophobicity of the oil phase, but more importantly on the conformational flexibility of the protein.     

Structural changes of milk proteins during heating of concentrated skim milk determined using FTIR

This work aimed to establish the impact of total solids (TS) concentration (9, 17 and 25%) of raw skim milk on the conformational properties of proteins during heating up to 121 °C. Conformational changes were determined using Fourier transform infrared spectroscopy and correlated to physicochemical properties of the system. The results showed high total solids dependence in rearrangement of β-sheet of β-lactoglobulin. Heat denaturation of whey proteins was concentration and temperature dependant. The micelle appeared to undergo temperature dependant dissociation that induced redistribution of helical and loop structures. The conformational changes were further evaluated with principal component analysis and normalising for a concentration effect for all samples. The most affected structures were located in the region 1620–1655 cm⁻¹ including intra- and inter-molecular β-sheets, α-helix/loop structures and randomly distributed structures. These findings may assist in predicting the heat stability of concentrates.     

乳清蛋白及与其它乳成分的热聚合作用机制研究进展

乳蛋白决定奶类品质,而热加工会影响乳清蛋白尤其是β-乳球蛋白的稳定性。热处理时,乳清蛋白不仅自身发生不同程度的聚合,而且通过巯基-二硫键分别与酪蛋白胶束和乳脂球膜蛋白发生结合。乳清蛋白亦可与其它乳成分如乳糖、钙盐、乳脂发生热聚合作用。本文根据乳品受热温度的不同,针对乳清蛋白间及其与其它乳成分的相互作用途径进行分析,阐明热聚合作用过程及机理,对改善乳制品的热稳定性、凝胶性等功能性质具有重要的理论意义,拓宽乳清蛋白作为配料在相关食品体系中的应用。     

Proteomic profiling of microbial transglutaminase-induced polymerization of milk proteins

Microbial transglutaminase (MTGase)-induced polymerization of individual milk proteins during incubation was investigated using a proteomics-based approach. The addition of MTGase (0.25-2.0 units/mL) caused the milk proteins to polymerize after a 3-h incubation period. Sodium dodecyl sulfate-PAGE analysis showed that the total intensities of the protein bands that corresponded to α(S)-casein, β-casein, and κ-casein decreased from 8,245.6, 6,677.2, and 586.6 arbitrary units to 1,911.7, 0.0, and 66.2 arbitrary units, respectively. Components with higher molecular weights were observed, and the intensity of these proteins increased after 3h of incubation. These results support that inter- or intramolecular crosslinking occurred in the casein proteins of MTGase-treated milk. Two-dimensional electrophoresis analysis indicated that isomers of β-casein, κ-casein, a fraction of serum albumin, α(S1)-casein, α(S2)-casein, β-lactoglobulin, and α-lactalbumin in the milk were polymerized following incubation with MTGase. In addition, MTGase-induced polymerization occurred earlier for β-casein and κ-casein isomers than for other milk proteins.