酪蛋白胶束结构研究方法综述

酪蛋白是乳中蛋白质的主要成分,约占其80%。由于酪蛋白胶束对乳制品的功能特性起重要作用,因此其研究一直备受关注,但酪蛋白胶束的内部结构至今没有确切的定义,而是许多学者提出了各种理论模型。文章综述了酪蛋白胶束结构、解离与聚集的多种研究方法,为以后学者研究酪蛋白提供借鉴。      

胰蛋白酶水解酪蛋白进程研究

研究了碱性务件下胰蛋白酶对酪蛋白的水解，用甲醛固定法对蛋白质的水解度进行测定，并用水解度（DH）表征其反应历程。分析了底物浓度、酶浓度、时间、pH和温度对DH、水解速度的影响。     

酪蛋白胶束结构和理化性质的研究进展

酪蛋白是乳中一大类蛋白质的总称,约占乳蛋白质量分数的80%。牛乳中的酪蛋白不是单独存在的,而是和矿质元素相互缠绕在一起而形成的复合物。牛乳的酸凝、酶凝以及液态乳稳定性的变化,从本质上来说都是酪蛋白胶束发生的一系列变化所导致的。本文概述了组成酪蛋白胶束中四种酪蛋白单体各自的结构和理化特征。综述了胶束的组成、胶束的流体力学直径、容积度、密度、水合性、分子间距等几个重要的特征参数。最后总结了核壳模型、双结合模型、亚单元模型和Holt模型等酪蛋白胶束的理论结构模型的特征及各种的缺陷,以期对乳制品的生产和加工提供一些思路。     

酪蛋白胶束结构和理化性质的研究进展

酪蛋白是乳中一大类蛋白质的总称,约占乳蛋白质量分数的80%。牛乳中的酪蛋白不是单独存在的,而是和矿质元素相互缠绕在一起而形成的复合物。牛乳的酸凝、酶凝以及液态乳稳定性的变化,从本质上来说都是酪蛋白胶束发生的一系列变化所导致的。本文概述了组成酪蛋白胶束中四种酪蛋白单体各自的结构和理化特征。综述了胶束的组成、胶束的流体力学直径、容积度、密度、水合性、分子间距等几个重要的特征参数。最后总结了核壳模型、双结合模型、亚单元模型和Holt模型等酪蛋白胶束的理论结构模型的特征及各种的缺陷,以期对乳制品的生产和加工提供一些思路。      

牦牛乳酪蛋白胶束琥珀酰化修饰研究

以琥珀酸酐为酰化试剂,牦牛乳酪蛋白为原料,对其进行了酰化修饰。系统研究了多种因素对酰化程度的影响,采用响应面法优化了修饰工艺,研究了酪蛋白乳化性变化。结果显示,牦牛乳酪蛋白胶束琥珀酰化修饰最佳条件为琥珀酸酐与酪蛋白配比0.6∶1(g/g),温度42℃,反应时间49min,pH8.8,酰化程度为86.01%。修饰后,牦牛乳酪蛋白的乳化稳定性和乳化活性分别提高了161.64%和98.54%。酪蛋白酰胺化修饰反应条件温和,易调节,修饰程度高,修饰酪蛋白的乳化性显著改善。该研究可为牛乳酪蛋白的酰化改性提供参考依据。      

葡萄糖对脱脂乳酪蛋白胶束稳定性的影响研究

研究了葡萄糖添加量、体系pH、热处理温度、热处理时间对脱脂牛乳酪蛋白胶束稳定性的影响。研究表明,在接近中性pH条件下,少量葡萄糖分子可降低脱脂乳酪蛋白胶束的稳定性,大量葡萄糖分子可增强其稳定性。添加葡萄糖后,在高温热处理的诱导下,酪蛋白沉淀的pH升高。随着热处理时间的延长,脱脂乳中酪蛋白胶束表现出聚集行为,致使粒径增大,浊度和沉淀率总体呈现上升趋势。该研究结论可为乳品加工提供参考依据。      

酪蛋白胶束结构及其对牛乳稳定性的影响

论述了酪蛋白的组成及两类具有代表性的酪蛋白胶束模型—亚胶束模型和内部结构模型,这两种模型可以较好地解释酪蛋白胶束的稳定性。pH值、酶、添加成分、工艺过程等能够影响酪蛋白胶束的稳定性,并直接影响到牛乳的稳定。这一方面有利于乳制品的生产如制作酸奶、干酪,一方面影响乳制品的品质。     

酪蛋白结构特性及应用研究进展

酪蛋白(casein,CN)又称乳酪素,是一种具有营养功能的蛋白质,主要存在于母牛、羊、骆驼及人等哺乳动物乳中。酪蛋白的4种单体形式分别为α_s1-CN、α_s2-CN、β-CN和k-CN,由α-螺旋、β-折叠和β-转角等形式构成空间结构,以胶束形式悬浮于水相中。酪蛋白营养价值高,具有稳定的鳌合体系,易被蛋白酶水解为小分子活性肽,在食品、医疗等领域应用广泛。该文综述酪蛋白的结构特性、来源含量及应用领域,为酪蛋白在各行业中的研究和广泛应用提供参考。     

酪蛋白的转谷氨酰胺酶氨基葡萄糖修饰与功能性变化

在37℃、pH值为7.5和氨基葡萄糖存在下,利用转谷氨酰胺酶(EC 2.3.2.13)对酪蛋白进行交联修饰制备修饰酪蛋白;用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳和高效液相色谱分析证实酪蛋白同时发生交联与糖基结合,且反应4 h时每摩酪蛋白可结合1.2摩葡萄糖.与酪蛋白相比,交联酪蛋白的溶解性质和起泡性质受损,而修饰酪蛋白产品的溶解性质得到改善,起泡性质尤其是泡沫稳定性质显著提高.在蛋白质质量浓度为1 g/L时,修饰酪蛋白的起泡能力和泡沫稳定性分别比酪蛋白提高8.6%和21%;质量浓度为100 g/L的修饰酪蛋白分散液表现出非牛顿流体特性,表观黏度显著高于交联酪蛋白或酪蛋白.     

Supramolecular structure of the casein micelle

The supramolecular structure of colloidal casein micelles in milk was investigated by using a sample preparation protocol based on adsorption of proteins onto a poly-l-lysine and parlodion-coated copper grid, staining of proteins and calcium phosphate by uranyl oxalate, instantaneous freezing, and drying under a high vacuum. High-resolution transmission electron microscopy stereo-images were obtained showing the interior structure of casein micelles. On the basis of our interpretation of these images, an interlocked lattice model was developed in which both casein-calcium phosphate aggregates and casein polymer chains act together to maintain casein micelle integrity. The caseins form linear and branched chains (2 to 5 proteins long) interlocked by the casein-stabilized calcium phosphate nanoclusters. This model suggests that stabilization of calcium phosphate nanoclusters by phosphoserine domains of α_s1-, α_s2-, or β-casein, or their combination, would orient their hydrophobic domains outward, allowing interaction and binding to other casein molecules. Other interactions between the caseins, such as calcium bridging, could also occur and further stabilize the supramolecule. The combination of having an interlocked lattice structure and multiple interactions results in an open, sponge-like colloidal supramolecule that is resistant to spatial changes and disintegration. Hydrophobic interactions between caseins surrounding a calcium phosphate nanocluster would prevent complete dissociation of casein micelles when the calcium phosphate nanoclusters are solubilized. Likewise, calcium bridging and other electrostatic interactions between caseins would prevent dissociation of the casein micelles into casein-calcium phosphate nanocluster aggregates when milk is cooled or urea is added to milk, and hydrophobic interactions are reduced. The appearance of both polymer chains and small aggregate particles during milk synthesis would also be expected based on this interlocked lattice model of casein micelles, and its supramolecule structure thus exhibits the principles of self-aggregation, interdependence, and diversity observed in nature.     

Time-dependent aggregation of casein micelle concentrates

This research focused on understanding physical and chemical changes occurring to concentrated milk protein suspensions as a function of time. Skim milk (untreated and heat treated at 90°C for 10 min) was concentrated at 6 times the original volume using osmotic stressing, a noninvasive concentration method, maintaining the serum composition as close as possible to that of native milk. A protease inhibitor cocktail, with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases, was added in selected samples. Within 9 d of storage at 4°C, the apparent viscosity increased markedly for both unheated and heated concentrated milk, but not for those in the presence of protease inhibitors. However, only unheated milk showed a significant increase in the apparent diameter of the casein micelles. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry measurements indicated a significantly lower extent of proteolysis in heated than in unheated samples. The microstructure of the aggregates was observed using field emission scanning electron microscopy, and unheated samples clearly showed aggregation of casein micelles with storage time. In heated samples, aggregation was instead triggered by heat-induced protein-protein interactions.     

The effect of ultrasound on casein micelle integrity

Samples of fresh skim milk, reconstituted micellar casein, and casein powder were sonicated at 20 kHz to investigate the effect of ultrasonication. For fresh skim milk, the average size of the remaining fat globules was reduced by approximately 10 nm after 60 min of sonication; however, the size of the casein micelles was determined to be unchanged. A small increase in soluble whey protein and a corresponding decrease in viscosity also occurred within the first few minutes of sonication, which could be attributed to the breakup of casein-whey protein aggregates. No measurable changes in free casein content could be detected in ultracentrifuged skim milk samples sonicated for up to 60 min. A small, temporary decrease in pH resulted from sonication; however, no measurable change in soluble calcium concentration was observed. Therefore, casein micelles in fresh skim milk were stable during the exposure to ultrasonication. Similar results were obtained for reconstituted micellar casein, whereas larger viscosity changes were observed as whey protein content was increased. Controlled application of ultrasound can be usefully applied to reverse process-induced protein aggregation without affecting the native state of casein micelles.     

Experimental evidence for previously unclassified calcium phosphate structures in the casein micelle

¹H-³¹P Cross-polarization magic angle spinning (CP-MAS) measurements of 40-d-old Mozzarella cheese and 20 mM EDTA-treated casein micelles revealed that each sample had immobile phosphorus with the same spectral pattern, which did not match that of native casein micelles. To identify the immobile phosphorus bodies, ¹H-³¹P CP-MAS spectra and cross-polarization kinetics measurements were undertaken on native casein micelles, EDTA-chelated casein micelles, and reference samples of β-casein and hydroxyapatite. The results showed that the immobile phosphorus bodies in the mature Mozzarella cheese had the following characteristics: they are immobile phosphoserine residues (not colloidal calcium phosphate); they are not the product of phosphoserine to colloidal calcium phosphate bridging; the phosphate is complexed to calcium; their rigidity is localized to a phosphorus site; their rigidity and bond coupling is unaffected by protein hydration; and the immobile bodies share a narrow range of bond orientations. Combining these observations, the best explanation of the immobile phosphorus bodies is that bonding structures of phosphorus-containing groups and calcium exist within the casein micelle that are not yet clearly classified in the literature. The best candidate is a calcium-bridged phosphoserine-to-phosphoserine linkage, either intra- or inter-protein.     

Activity and nature of plasminogen activators associated with the casein micelle

In fresh milk, plasminogen, the zymogen form of plasmin (PL), is the predominant form. Therefore, plasminogen activators (PA) can contribute significantly to PL activity in milk. Both tissue-type PA (tPA) and urokinase-type PA (uPA) exist in milk; however, contradictory findings have been reported for which type of PA is most closely associated with the casein micelles. Little is known about the factors that might lead to variations in the individual activities of the PA. The objective of this work was therefore to investigate possible factors that might affect the association of tPA and uPA with the casein micelle and their activities thereafter. Plasminogen activators were isolated from milk samples with different somatic cell counts following 2 different isolation protocols. Determination of uPA, tPA, and PL activities was carried out quantitatively following chromogenic assays using 2 different substrates, and qualitatively using specialized sodium dodecyl sulfate-PAGE. Different isolation methods and conditions led to differences in uPA, tPA, and PL activities. Urokinase-type PA activity was significantly higher in PA fractions isolated from milk with high somatic cell counts than from milk with low somatic cell counts. Activity results indicated that in pasteurized milk uPA could dissociate from the somatic cells and bind to casein. Moreover, a high level of PL in isolated PA fractions contributed to significantly enhanced PA activities. Overall, results confirmed the association of both uPA and tPA with the casein micelle; however, their amounts, activities, and molecular weights varied based on the nature of the milk and methods of separation, with uPA being the PA with greater potential to affect plasminogen activation in milk.     

酶改性对食物蛋白过敏原性的影响

酶法改性蛋白技术主要包括酶水解和酶交联方法,它可以改变蛋白质的结构和功能特性,已广泛用于食品加工。酶水解,包括胃蛋白酶、胰蛋白酶、木瓜蛋白酶、碱性蛋白酶、糜蛋白酶,可以水解食物蛋白表面的过敏原表位,从而降低其过敏原性;酶交联,包括转谷酰胺酶、过氧化物酶、多酚氧化酶等,可以通过交联反应促使过敏原蛋白聚合而使其过敏原表位包裹在内部,因而也可以降低食物的过敏原性。总之,酶改性是降低食物蛋白过敏原性的一种有效方法,值得深入研究。     

Importance of casein micelle size and milk composition for milk gelation

The economic output of the dairy industry is to a great extent dependent on the processing of milk into other milk-based products such as cheese. The yield and quality of cheese are dependent on both the composition and technological properties of milk. The objective of this study was to evaluate the importance and effects of casein (CN) micelle size and milk composition on milk gelation characteristics in order to evaluate the possibilities for enhancing gelation properties through breeding. Milk was collected on 4 sampling occasions at the farm level in winter and summer from dairy cows with high genetic merit, classified as elite dairy cows, of the Swedish Red and Swedish Holstein breeds. Comparisons were made with milk from a Swedish Red herd, a Swedish Holstein herd, and a Swedish dairy processor. Properties of CN micelles, such as their native and rennet-induced CN micelle size and their ζ-potential, were analyzed by photon correlation spectroscopy, and rennet-induced gelation characteristics, including gel strength, gelation time, and frequency sweeps, were determined. Milk parameters of the protein, lipid, and carbohydrate profiles as well as minerals were used to obtain correlations with native CN micelle size and gelation characteristics. Milk pH and protein, CN, and lactose contents were found to affect milk gelation. Smaller native CN micelles were shown to form stronger gels when poorly coagulating milk was excluded from the correlation analysis. In addition, milk pH correlated positively, whereas Mg and K correlated negatively with native CN micellar size. The milk from the elite dairy cows was shown to have good gelation characteristics. Furthermore, genetic progress in relation to CN micelle size was found for these cows as a correlated response to selection for the Swedish breeding objective if optimizing for milk gelation characteristics. The results indicate that selection for smaller native CN micelles and lower milk pH through breeding would enhance gelation properties and may thus improve the initial step in the processing of cheese.     

The association of lipophilic phospholipids with native bovine casein micelles in skim milk: Effect of lactation stage and casein micelle size

A known biological role of casein micelles is to transport calcium from mother to young and provide amino acids for growth and development. Previous reports demonstrated that modified casein micelles can be used to transport and deliver hydrophobic probes. In this study, the distribution of lipid-soluble phospholipids, including sphingomyelins (SM) and phosphatidylcholines (PC), was quantified in whole raw milk, skim raw milk, and casein micelles of various sizes during early, mid, and late lactation stages. Low-pressure size exclusion chromatography was used to separate casein micelles by size, followed by hydrophobic extraction and liquid chromatography–mass spectrometry for the quantification of PC and SM. Results showed that the SM d18:1/23:0, d18:1/22:0, d18:1/16:0, d16:1/22:0, d16:1/23:0, and d18:1/24:0 and the PC 16:0/18:1, 18:0/18:2, and 16:0/16:0 were dominating candidates appearing in maximum concentration in whole raw milk obtained from late lactation, with 21 to 50% of total SM and 16 to 35% of total PC appearing in skim milk. Of the total SM and PC found in skim milk, 35 to 46% of SM and 22 to 29% of PC were associated with the casein micelle fraction. The highest concentrations of SM d18:1/22:0 (341 ± 17 µg/g of casein protein) and PC 16:0/18:1 (180 ± 20 µg/g of casein protein) were found to be associated with the largest casein micelles (diameter = 149 nm) isolated in milk from late lactation, followed by a decrease in concentration as the casein micelle size decreased.