Fibril assemblies in aqueous whey protein mixtures

Fibril formation in mixtures of whey proteins upon heating at pH 2 was investigated. Fibrils were found to coexist with other structures, such as spherulites. These spherulites consist of radially oriented fibrils. At total protein concentrations above 6 wt %, transparent gels were formed. Changing the ratio between the various whey proteins did not affect this gelation concentration as long as beta-lactoglobulin (beta-lg) was present, suggesting that beta-lg was dominant in the gelation. Pure alpha-lactalbumin and pure bovine serum albumin did not form fibrils, nor did they gel upon heating at pH 2 and 80 degrees C for up to 10 h. They did however induce a decrease in the beta-lg concentration needed for gel formation upon heating at pH 2. Our results suggest that beta-lg is the only fibril forming protein at the conditions used and that no mixed fibrils are formed.     

Micrometer-sized fibrillar protein aggregates from soy glycinin and soy protein isolate

Long, fibrillar semiflexible aggregates were formed from soy glycinin and soy protein isolate (SPI) when heated at 85 degrees C and pH 2. Transmission electron microscopy analysis showed that the contour length of the fibrils was approximately 1 microm, the persistence length 2.3 microm, and the thickness a few nanometers. Fibrils formed from SPI were more branched than the fibrils of soy glycinin. Binding of the fluorescent dye Thioflavin T to the fibrils showed that beta-sheets were present in the fibrils. The presence of the fibrils resulted in an increase in viscosity and shear thinning behavior. Flow-induced birefringence measurements showed that the behavior of the fibrils under flow can be described by scaling relations derived for rodlike macromolecules. The fibril formation could be influenced by the protein concentration and heating time. Most properties of soy glycinin fibrils are comparable to beta-lactoglobulin fibrils.     

Glucose slows down the heat-induced aggregation of β-lactoglobulin at neutral pH

The behavior of β-lactoglobulin (β-Lg) during heat treatments depends on the environmental conditions. The influence of the presence or absence of a reducing sugar, namely, glucose, on the modification of the protein during heating has been studied using fluorescence, polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), and transmission electron microscopy. Glycated products were formed during heating 24 h at 90 °C and pH 7. The fluorescence results revealed an accumulation of the advanced Maillard products and the formation of aggregates during heating. PAGE and SEC data suggested that the products in the control samples were essentially composed of covalently linked fibrillar aggregates and that their formation was faster than that for glycated samples. We showed that glucose affected the growing step of covalent aggregates but not the initial denaturation/aggregation step of native protein. Glucose-modified proteins formed a mixture of short fibrils and polydisperse aggregates. Our results revealed that β-Lg forms fibrils at neutral pH after heating and that glucose slows the formation of these fibrils.     

Growth kinetics of amyloid-like fibrils derived from individual subunits of soy β-conglycinin

The amyloid-like fibrillation of soy β-conglycinin subunits (α, α', and β) upon heating (0-20 h) at 85 °C and pH 2.0 was characterized using dynamic light scattering, circular dichroism (CD), binding to amyloid dyes (Thioflavin T and Congo red), and atomic force microscopy. The fibrillation of all three subunits was accompanied by progressive polypeptide hydrolysis. The hydrolysis behaviors, fibrillation kinetics, and morphologies of amyloid-like fibrils considerably varied among α, α', and β subunits. Faster hydrolysis rates and special fragments were observed for the α and α' subunits compared to the β subunit. However, the order of the fibrillation rate and capacity to form β-sheets was α' > β > α, as evidenced by CD and Thioflavin T data. Moreover, sequential growth of twisted screw-structure fibrils, leading to macroscopic fibrils with distinct morphological characteristics, was observed for β-conglycinin and individual subunits. The different fibrillation kinetics and morphologies of α, α', and β subunits appear to be associated with the differences in the amino acid composition and typical sequence of peptides. Besides, the disruption of ordered structure of fibrils occurred upon further heating (6-20 h) due to extensive hydrolysis. These results would suggest that all subunits are involved in the fibrillation of β-conglycinin, following multiple steps including polypeptide hydrolysis, assembly to amyloid structure, and growth into macroscopic fibrils with a fibril shaving process.     

Heat-induced soy-whey proteins interactions: formation of soluble and insoluble protein complexes

The aggregation behavior during heating of a solution containing soy protein and whey protein isolate (WPI) was studied using rheology, confocal microscopy, gel filtration, and electrophoresis. Soy/WPI mixtures formed gels at 6% total protein concentration with a high elastic modulus (G') and no apparent phase separation. The ratio of soy to WPI was fundamental in determining the type of network formed. Systems containing a high soy to WPI ratio (>70% soy protein) showed a different evolution of the elastic modulus during heat treatment, with two apparent stages of network development. Whey proteins formed disulfide bridges with soy proteins during heating, and at low ratios of soy/WPI, the aggregates seemed to be predominantly formed by 7S, the basic subunits of 11S and beta-lactoglobulin. Size exclusion chromatography indicated the presence of high molecular weight soluble complexes in mixtures containing high soy/WPI ratios. Results presented are the first evidence of interactions between soy proteins and whey proteins and show the potential for the creation of a new group of functional ingredients.     

Re-formation of fibrils from hydrolysates of β-lactoglobulin fibrils during in vitro gastric digestion

In this study, in vitro digestion of β-lactoglobulin (β-Lg) fibrils and the re-formation of fibril-like structures after prolonged enzymatic hydrolysis (up to 48 h) were investigated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), thioflavin T fluorescence photometry, and transmission electron microscopy (TEM). Pure β-Lg fibrils that had been formed by heat treatment at pH 2.0 were rapidly hydrolyzed by pepsin in the simulated gastric fluid (pH 1.2), and some new peptides that were suitable for further fibril formation were produced. TEM showed that the new fibrils were long and straight but thinner than the original fibrils, and both TEM and MALDI-MS indicated that the peptides in the new fibrils were shorter/smaller than the peptides in the original fibrils. The formation of new fibrils was found to be affected more by pH than by enzyme activity or temperature.     

Whey protein nanofibrils: the environment-morphology-functionality relationship in lyophilization, rehydration, and seeding

Amyloid-like fibrils from β-lactoglobulin have potential as efficient thickening and gelling agents for food and biomedical applications, but the link between fibril morphology and bulk viscosity is poorly understood. We examined how lyophilization and rehydration affects the morphology and rheological properties of semiflexible (i.e., straight) and highly flexible (i.e., curly) fibrils, the latter made with 80 mM CaCl(2). Straight fibrils were fractured into short rods by lyophilization and rehydration, whereas curly fibrils sustained little damage. This was reflected in the viscosities of rehydrated fibril dispersions, which were much lower for straight fibrils than for curly fibrils. Lyophilized straight or curly fibrils seeded new fibril growth, but viscosity enhancement due to seeding was negligible. We believe that the increase in fibril concentration caused by seeding was counterbalanced by a decrease in fibril length, reducing the ability of fibrils to form physical entanglement networks.     

Heat-induced changes in the ultrasonic properties of whey proteins

The physical aggregation of commercial whey protein isolate (WPI) and purified beta-lactoglobulin was studied by ultrasound spectroscopy. Protein samples were dialyzed to achieve constant ionic strength backgrounds of 0.01 and 0.1 NaCl, and gelation was induced in situ at constant temperatures (from 50 to 75 degrees C) or with a temperature ramp from 20 to 85 degrees C. Changes in the ultrasonic properties were shown in the early stages of heating, at temperatures below those reported for protein denaturation. During heating, the relative ultrasound velocity (defined as the difference between sample velocity and reference velocity) decreased continuously with temperature, indicating a rearrangement of the hydration layer of the protein and an increase in compressibility of the protein shell. At temperatures <50 degrees C the ultrasonic attenuation decreased, and <65 degrees C both velocity and attenuation differentials showed increasing values. A sharp decrease in the relative velocity and an increase in the attenuation at 70 degrees C were indications of "classical" protein denaturation and the formation of a gel network. Values of attenuation were significantly different between samples prepared with 0.01 and 0.1 M NaCl, although no difference was shown in the overall ultrasonic behavior. WPI and beta-lactoglobulin showed similar ultrasonic properties during heating, but some differences were noted in the values of attenuation of WPI solutions, which may relate to a less homogeneous distribution of aggregates caused by the presence of alpha-lactalbumin and other minor proteins in WPI.     

Methionine oxidation enhances κ-casein amyloid fibril formation

The effects of protein oxidation, for example of methionine residues, are linked to many diseases, including those of protein misfolding, such as Alzheimer's disease. Protein misfolding diseases are characterized by the accumulation of insoluble proteinaceous aggregates comprised mainly of amyloid fibrils. Amyloid-containing bodies known as corpora amylacea (CA) are also found in mammary secretory tissue, where their presence slows milk flow. The major milk protein κ-casein readily forms amyloid fibrils under physiological conditions. Milk exists in an extracellular oxidizing environment. Accordingly, the two methionine residues in κ-casein (Met(95) and Met(106)) were selectively oxidized and the effects on the fibril-forming propensity, cellular toxicity, chaperone ability, and structure of κ-casein were determined. Oxidation resulted in an increase in the rate of fibril formation and a greater level of cellular toxicity. β-Casein, which inhibits κ-casein fibril formation in vitro, was less effective at suppressing fibril formation of oxidized κ-casein. The ability of κ-casein to prevent the amorphous aggregation of target proteins was slightly enhanced upon methionine oxidation, which may arise from the protein's greater exposed surface hydrophobicity. No significant changes to κ-casein's intrinsically disordered structure occurred upon oxidation. The enhanced rate of fibril formation of oxidized κ-casein, coupled with the reduced chaperone ability of β-casein to prevent this aggregation, may affect casein-casein interaction within the casein micelle and thereby promote κ-casein aggregation and contribute to the formation of CA.     

Effect of pH, NaCl, CaCl2 and temperature on self-assembly of β-lactoglobulin into nanofibrils: a central composite design study

The ability of certain globular proteins to self-assemble into amyloid-like fibrils in vitro opens opportunities for the development of new biomaterials with unique functional properties, like highly efficient gelation and viscosity enhancement. This work explored the individual and interacting effects of pH (1 to 3), NaCl (0-100 mM), CaCl(2) (0-80 mM) and heating temperature (80 to 120 °C) on the kinetics of β-lactoglobulin self-assembly and the morphology of resulting nanofibrils. Statistically significant (p < 0.05) interactions included CaCl(2)*temperature, NaCl*pH, CaCl(2)*pH, temperature*pH and NaCl*CaCl(2). Particularly notable was the very rapid self-assembly at pH 3 and the highly nonlinear effect of pH on self-assembly kinetics. Nanofibril morphologies ranged from long and semiflexible or curled and twisted to short and irregular. There did not seem to be a link between the kinetics of fibril formation and the morphology of fibrils, except at pH 3, where self-assembly was very rapid and fibrils were short and irregular, suggesting haphazard, uncontrolled self-assembly.     

Formation of carbon particles from a gas phase: Nucleation phenomenon

Highly divided C can be formed by heat treatment of hydrocarbons in absence of O (thermal decomposition) or in its presence (flames). This variety of C, named soot or carbon black, has a considerable polluting effect. Moreover, considering the increasing cost of energy sources, energy loss under the form of unburnt species has to be reduced. To decrease or to suppress soot formation, it is often necessary to know the mechanism of formation and, in particular, the phenomena involved in the nucleation step. The mechanism of soot formation in flames is basically identical in premixed flames and in diffusion flames. However, as the different chemical stages are well separated, from a spatial point of view, in premixed flames, most investigations deal with this type of flame. In the oxidation zone of the flame (blue zone), a part of the hydrocarbon is burnt out while another part undergoes complex reactions leading to polyacetylenes and later to polycyclic hydrocarbons with lateral chains. In the same region, ions are formed by chemiionization. In the zone of formation of C particles (yellow zone), polycyclic hydrocarbons are dehydrogenated and give polyaromatic hydrocarbons; their partial pressure increases until they reach a supersaturation high enough to induce nucleation of liquid microdroplets. The microdroplets are formed by homogeneous nucleation as well, probably, as the nucleation on the positive ions formed in the oxidation zone of the flame; they are converted by growth, association and chemical transformation into solid soot particles. In thermal systems, the mechanisms involved are identical except that nucleation on ions may be disregarded. In both systems, nucleation of microdroplets is a fast and discontinuous phenomenon. Such an understanding in C particle formation might be efficiently used to reduce noticeably atmospheric pollution and energy loss by soot formation.     

玉米秸秆热解动力学分析(简报)

该文采用耐驰STA449同步热分析仪,研究了玉米秸秆在升温速率为5、10、15、20、30 K/min,反应终止温度1273K的热分解反应,结果表明玉米秸秆热解过程可分为失水预热解、热解和碳化3个阶段,随着升温速率增加,反应的特征温度和最大失重速率增加,差示扫描（DSC）曲线整体向下倾斜,升温速率过大时,出现失水滞后现象。分别用FWO法、FRL法和Kissinger法对玉米秸秆热解进行了动力学计算,其热解活化能为（161±23）kJ/mol,通过Malek法确定了玉米秸秆热解满足J-M-A方程,反应机理为随机成核随后生长,确定了反应级数n和指前因子对数lgA的范围,并利用Matlab软件对实验数据进行拟合,验证了机理的正确性,并确定了玉米秸秆热解反应的动力学参数。该文结果对工程应用有指导意义。     

Gamma-irradiation influence on the structure and properties of calcium caseinate-whey protein isolate based films. Part 2. Influence of polysaccharide addition and radiation treatment on the structure and functional properties of the films

The influence of gamma-irradiation (32 kGy) followed by the addition of polysaccharides (potato starch, soluble potato starch, and sodium alginate) and heating on the properties of the films based on calcium caseinate (CC)-whey proteins isolate (WPI) and the gels formed with CaCl(2) was evaluated. Radiation induced an improvement of the mechanical and barrier properties of all films. The polysaccharides' effect on the irradiated and non-irradiated CC-WPI gels could be predicted as the sum of their separate effects on CC and on WPI, apart from the alginate interaction with the irradiated CC-WPI. The better properties of the films achieved after admixing polysaccharides to the formerly irradiated protein solution correspond to the smaller strength of gels. Properties of the films and gels prepared using the irradiated proteins and alginate differed depending on whether alginate was admixed before or after irradiation. Results were related to the protein structure, interaction with polysaccharides, and the film's microstructure.     

Light-scattering study of the structure of aggregates and gels formed by heat-denatured whey protein isolate and beta-lactoglobulin at neutral pH

The structure of aggregates and gels formed by heat-denatured whey protein isolate (WPI) has been studied at pH 7 and different ionic strengths using light scattering and turbidimetry. The results were compared with those obtained for pure beta-lactoglobulin (beta-Lg). WPI aggregates were found to have the same self-similar structure as pure beta-Lg aggregates. WPI formed gels above a critical concentration that varied from close to 100 g/L in the absence of added salt to about 10 g/L at 0.2 M NaCl. At low ionic strength (<0.05 M NaCl) homogeneous transparent gels were formed, while at higher ionic strength the gels became turbid but had the same self-similar structure as reported earlier for pure beta-Lg. The length scale characterizing the heterogeneity of the gels increased exponentially with increasing NaCl concentration for both WPI and pure beta-Lg, but the increase was steeper for the former.     

Collagen fibril alignment and deformation during tensile strain of leather: a small-angle X-ray scattering study

The distribution and effect of applied strain on the collagen fibrils that make up leather may have an important bearing on the ultimate strength and other physical properties of the material. While sections of ovine and bovine leather were being subjected to tensile strain up to rupture, synchrotron-based small-angle X-ray scattering (SAXS) spectra were recorded edge-on to the leather at points from the corium to the grain. Measurements of both fibril orientation and collagen d spacing showed that, initially, the fibers reorient under strain, becoming more aligned. As the strain increases (5-10% strain), further fibril reorientation diminishes until, at 37% strain, the d spacing increases by up to 0.56%, indicating that significant tensile forces are being transmitted to individual fibrils. These changes, however, are not uniform through the cross-section of leather and differ between leathers of different strengths. The stresses are taken up more evenly through the leather cross-section in stronger leathers in comparison to weaker leathers, where stresses tended to be concentrated during strain. These observations contribute to our understanding of the internal strains and structural changes that take place in leather under stress.     

Improvement of the material property of shark type I collagen by composing with pig type I collagen

Fibril reconstruction process, that is, the nucleation and growth of mixed type I collagen fibril of shark and pig, progressed faster than that of the individual collagen species of shark or pig. The reconstructed mixed collagen fibril had a greater resistance to return to the solution or to melt into gelatin in comparison with the counterpart consisting solely of shark collagen. The denaturation temperature of the mixed collagen gel was about 10 degrees C higher than that of shark, and about 5 degrees C lower than that of pig. By scanning electron microscopy, the diameter of mixed collagen fibril showed an intermediate range between shark and pig collagen fibril. The breaking strength of the mixed collagen gel was tougher than that of pig, but weaker than that of shark. Other physicochemical properties of the mixed type I collagen gel were observed to be at intermediate positions between those of shark and pig type I collagen gels.     

糖接枝处理改善大豆蛋白纤维聚集体泡沫稳定性

为了探究糖接枝对大豆蛋白纤维聚集行为和泡沫性质的影响,明确蛋白质结构与功能的关系,该研究以大豆蛋白(soy protein isolation,SPI)和乳糖(lactose)为原料,通过干热法制备糖接枝大豆蛋白(SPI-lactose conjugate,SPI-Lac),以及在酸性条件下加热诱导其形成纤维聚集体(p H值2.0),制备了一种糖接枝大豆蛋白纤维聚集体(SPI-lactose conjugate fibillar aggregates),并考察了糖接枝对大豆蛋白的纤维聚集行为及泡沫性质的影响。研究结果表明:大豆蛋白在酸性条件下(p H值2.0)经加热后会发生水解,同时水解产物不断聚集形成大分子的纤维聚集体。糖接枝导致大豆蛋白的水解速度下降,但荧光光强和粒径的结果表明糖接枝能增强纤维聚集能力。SPI-Lac在中性条件下的溶解度(p H值5.0—7.0)显著高于SPI(P0.05),且不同时间处理的SPI-Lac纤维聚集体均能改善SPI在酸性条件下的溶解度(p H值2.0—5.0)。此外,不同时间处理的SPI-Lac纤维聚集体在酸性条件下的起泡能力均高于SPI纤维聚集体。SPI和SPI-Lac纤维聚集体的形成会导致SPI起泡能力的下降,但是短时间酸热处理形成的纤维聚集体泡沫稳定性得到显著改善。因此,糖接枝结合短时间酸热处理制备的糖接枝大豆蛋白纤维聚集体在中性条件下的泡沫稳定性显著提高(P0.05),是合理有效的蛋白质改性方法。     

适宜含水率保持油茶籽贮藏品质

为了确定油茶籽贮藏适宜的含水率,研究了在4℃,不同含水率(7%、10%、13%、16%、20%)油茶籽贮藏期间的品质变化。结果表明,较低的含水率能较好保持油茶籽的贮藏特性及营养品质。其中,含水率为7%的油茶籽贮藏效果较好,但与10%处理效果差异不明显(P>0.05)。在整个贮藏期,含水率为7%时油茶籽可溶性蛋白下降了13.05 mg/g,油酸含量下降了2.38%,酸值、过氧化值等品质指标上升速率较慢,同时能较好保持β-谷甾醇和角鲨烯等生物活性成分;其次是10%的含水率处理。而含水率为20%的油茶籽贮藏期间可溶性蛋白下降较快,贮藏结束时为25.47 mg/g,油茶籽劣变严重,所提取的油样品质变差,营养物质含量较少,因此含水率20%的油茶籽不适宜长期贮藏。综合考虑油茶籽品质因素和处理成本,认为控制含水率在10%以下能较好保持油茶籽的贮藏品质。该研究可为科学合理地贮藏油茶籽提供参考。     

Crystallization behavior of anhydrous milk fat-sunflower oil wax blends

This research evaluates the effect of sunflower oil wax (SFOw) addition on the crystallization behavior and functional properties of anhydrous milk fat (AMF). Induction times of nucleation, melting behavior, microstructure of crystals, and hardness were evaluated for samples of pure AMF and AMF with 0.1 and 0.25% SFOw. Results from this research show that the addition of waxes induced the onset of crystallization of AMF by inducing its nucleation, as evidenced by decreased induction times of nucleation and the formation of smaller crystals. Crystal growth after tempering was also promoted by waxes, and significantly harder lipid networks were obtained. Results presented in this paper suggest that SFOw can be used as an additive to alter the physiochemical properties of low trans-fatty acid lipids.     

土壤颗粒悬液搅拌对土壤质地分析的影响

[目的]探究土壤颗粒悬液搅拌对土壤质地分析的影响,为实验室准确测定土壤机械组成提供可行的参考与指导。[方法]采用比重法测定土壤机械组成,在搅拌不同行程、搅拌不同次数、悬液静置不同时间后搅拌、不同测定时点搅拌的多种条件下所测结果进行比较分析。[结果]搅拌行程、搅拌次数、悬液静置后搅拌对黏粒含量测定无影响,对粉砂粒和砂粒含量测定均有影响。搅棒从液面下行至沉降筒底搅拌,有利于颗粒充分运动及均匀分布,黏粒、粉砂粒、砂粒含量测定稳定,精密度良好;以氢氧化钠为分散剂的悬液搅拌30次,黏粒、粉砂粒、砂粒含量测定准确,精密度良好;悬液静置的适宜时间大于1h,搅拌30次所测粉砂粒和砂粒含量的精密度呈下降趋势,搅拌45次所测粉砂粒、砂粒、黏粒含量稳定,精密度良好,与洗后即搅拌30次的测定结果一致。在第2测定时点增加搅拌1min对测定结果无影响。[结论]本试验方法测定结果准确可靠,避免了重复操作,有利于减少误差,适用于多个样品的同时检测。