Physical and chemical interactions in cold gelation of food proteins

pH-Induced cold gelation of whey proteins is a two-step process. After protein aggregates have been prepared by heat treatment, gelation is established at ambient temperature by gradually lowering the pH. To demonstrate the importance of electrostatic interactions between aggregates during this latter process, beta-lactoglobulin aggregates with a decreased iso-electric point were prepared via succinylation of primary amino groups. The kinetics of pH-induced gelation was affected significantly, with the pH gelation curves shifting to lower pH after succinylation. With increasing modification, the pH of gelation decreased to about 2.5. In contrast, unmodified aggregates gel around pH 5. Increasing the iso-electric point of beta-lactoglobulin via methylation of carboxylic acid groups resulted in gelation at more alkaline pH values. Comparable results were obtained with whey protein isolate. At low pH disulfide cross-links between modified aggregates were not formed after gelation and the gels displayed both syneresis and spontaneous gel fracture, in this way resembling the morphology of previously characterized thiol-blocked whey protein isolate gels (Alting, et al., J. Agric. Food Chem. 2000, 48, 5001-5007). Our results clearly demonstrate the importance of the net electric charge of the aggregates during pH-induced gelation. In addition, the absence of disulfide bond formation between aggregates during low-pH gelation was demonstrated with the modified aggregates.     

Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk

Traditionally, milk has been heat treated to control microorganisms and to alter its functionality, for example, to increase its heat stability. Pressure treatment has been considered as a possible alternative for microorganism control, but some of the functionality-related milk protein interactions have not been explored. The present study used two novel two-dimensional polyacrylamide gel electrophoresis (2D PAGE) methods to explore the differences in the irreversible disulfide bond changes among the milk proteins after four common heat treatments and after 30-min pressure treatments of milk at 200, 400, 600, and 800 MPa at ambient temperature (22 degrees C). The pasteurizing heat treatment (72 degrees C for 15 s) denatured and aggregated only a few minor whey proteins, but the high heat treatments (100 degrees C for 120 s, 120 degrees C for 120 s, and 140 degrees C for 5 s) formed disulfide-bonded aggregates that included a high proportion of all of the whey proteins and kappa-casein (kappa-CN) and a proportion of the alpha(s2)-CN. Pressure treatment of milk at 200 MPa caused beta-lactoglobulin (beta-LG) to form disulfide-bonded dimers and incorporated beta-LG into aggregates, probably disulfide-bonded to kappa-CN. The other whey proteins appeared to be less affected at 200 MPa for 30 min. In contrast, pressure treatment at 800 MPa incorporated beta-LG and most of the minor whey proteins, as well as kappa-CN and much of the alpha(s2)-CN, into aggregates. The accessibility of alpha(s2)-CN and formation of complexes involving alpha(s2)-CN, kappa-CN, and whey proteins in the pressure treated milk is an important novel finding. However, only some of the alpha-lactalbumin was denatured or incorporated into the large aggregates. These and other results show that the differences between the stabilities of the proteins and the accessibilities of the disulfide bonds of the proteins at high temperature or pressure affect the formation pathways that give the differences among the resultant aggregates, the sizes of the aggregates, and the product functionalities.     

Heat-induced redistribution of disulfide bonds in milk proteins. 2. Disulfide bonding patterns between bovine beta-lactoglobulin and kappa-casein

Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with kappa-casein (kappa-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys(121) of beta-lactoglobulin (beta-LG) has been implicated. The reaction at 60 degrees C between beta-LG A and an activated kappa-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [beta-LG(102-124)] and kappa-CN Cys(88) and others between kappa-CN Cys(88) or kappa-CN Cys(11) and beta-LG Cys(160). Only the latter two novel disulfide bonds were identified in heated (90 degrees C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to beta-LG(102-135):kappa-CN(11-13) and beta-LG A(61-69):kappa-CN(87-97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, beta-LG(1-40) and beta-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions.     

冷榨菜籽蛋白的碱性电解水提取工艺及其结构性能表征

为探寻一种油料蛋白提取方法,该研究以双低冷榨菜籽饼为原料,碱性电解水为溶剂,通过单因素与响应面试验确定了碱性电解水提取冷榨菜籽饼分离蛋白（Alkaline electrolyzed water extracted Rapeseed Protein,ARP）的优化条件,并与相同条件下碱溶酸沉法提取菜籽分离蛋白（Ultrapure water extracted Rapeseed Protein,URP）的结果进行对比。结果表明: 提取工艺各环节因素对菜籽蛋白提取率影响的主次顺序分别为：碱性电解水pH值、温度、料液比、时间;提取优化工艺条件为温度45 ℃、碱性电解水pH值11.5、料液比1:10 g/mL和提取时间60 min,蛋白质提取率为59.34%。与相同条件下碱溶酸沉法相比,碱性电解水提取的菜籽分离蛋白游离巯基和二硫键含量高、表面疏水性强、粒径小、ζ电位绝对值大,同时其提取率、溶解性、持油性、乳化性和起泡性均显著得到改善（P＜0.05）;此外,ARP内源荧光光谱强度更高,二级结构更有序。由此可知,该研究结果为碱性电解水提取冷榨菜籽饼蛋白提供参考,不仅提高了提取率,而且最大程度地减少了对菜籽蛋白结构和功能特性的破坏。     

Effects of designed sulfhydryl groups and disulfide bonds into soybean proglycinin on its structural stability and heat-induced gelation

The gel-forming ability of glycinin is one of soybean's most important functional properties. The proglycinin A1aB1b homotrimer was engineered to introduce sulfhydryl groups and disulfide bonds, and their effects on the structural stability and the heat-induced gelation were evaluated. On the basis of the crystal structure, five mutants were designed and prepared: R161C and F163C forming an interprotomer disulfide bond with the inherent free cysteine residue of Cys377, N116C/P248C forming a new intraprotomer disulfide bond, and N116C and P248C introducing a new sulfhydryl group. Mutants of R161C, F163C, and N116C/P248C formed a new disulfide bond as expected. N116C/P248C was significantly more stable than the wild type against chemical and thermal denaturation and more resistant to alpha-chymotrypsin digestion, whereas F163C showed significantly increased thermal stability. All mutants exhibited greater hardness of heat-induced gels than wild type, and in particular, N116C/P248C gave the hardest gel. This result indicates that it is possible to increase hardness of glycinin gel by introduction of cysteine residues using protein engineering.     

Enzyme-aided modification of chicken-breast myofibril proteins: effect of laccase and transglutaminase on gelation and thermal stability

The effect of laccase and transglutaminase (TG) on cross-linking, gelation, and thermal stability of salt-soluble chicken-breast myofibril proteins was investigated at pH 6. Both enzymes modified the protein pattern detected by SDS-PAGE. Identification of proteins by peptide mass mapping showed that myosin heavy chain (MHC) and troponin T were the most affected proteins. These proteins faded or disappeared as a function of the incubation time with both enzymes on SDS-PAGE. The molecular weight of actin was not, however, affected by either enzyme. The effects that the enzymes had on the gel formation of chicken-breast myofibrils were studied in 0.35 and 0.60 M NaCl solutions at 3% protein content and a constant temperature of 40 degrees C by using a small deformation viscoelastic measurement. TG substantially increased the storage modulus (G') of 3% protein in 0.35 M NaCl. Without the enzymes, gelation was insignificant in 0.35 M NaCl. The increased solubility of the proteins at 0.60 M NaCl intensified gelation with TG. G' increased 32 and 64% at dosages of 10 and 100 nkat of TG, respectively. Also, laccase increased G' of the gel in 0.60 M salt concentration. However, a high laccase dosage decreased the magnitude of G' below the control level. Differential scanning calorimetric (DSC) measurements indicated slightly reduced myosin heat stability after TG pretreatment and increased actin heat stability with both enzymes. Maximum transition temperatures did not alter with either enzyme.     

Endoxylanases in durum wheat semolina processing: solubilization of arabinoxylans, action of endogenous inhibitors, and effects on rheological properties

Endoxylanases seriously affect the rheological properties of durum wheat (Triticum durum Desf.) semolina spaghetti doughs prepared with, and as evaluated, by the farinograph. Under the experimental conditions, control doughs (34.9% moisture content) made from two semolinas (semA and semB) yielded a maximal consistency of 525 and 517 farinograph units (FU), with, respectively, 19.4 and 16.4% of the total level of arabinoxylans (TOT-AX) being water-extractable (WE-AX). When 75.4 Somogyi units/50 g of semolina of the endoxylanases from Trichoderma viride (XTV), rumen microorganisms (XRM), Bacillus subtilis (XBS), and Aspergillus niger (XAN) were used, the maximal consistencies at 34.9% moisture decreased for semA to 467, 436, 448, and 417 FU, respectively. This was accompanied by increased WE-AX contents of 60.8, 71.2, 70.7, and 73.0%, respectively. Similar results were observed for semB. By reducing the total water content of doughs, it was possible to recover the maximal consistency of the original doughs. Both the decrease in maximal consistency and the amount of water to be omitted were significantly related to the decrease in molecular weight (MW) of the WE-AX and the percentage of WE-AX solubilized as a result of the enzymic action. At the same time, it was clear that endogenous endoxylanase inhibitors were present in the durum wheat semolinas and that they inhibited the endoxylanases used to different degrees. Part of the differences in effects between the different endoxylanases (decrease in maximal consistency, amount of AX solubilized, MWs of the WE-AX, and amount of water that could be omitted) could be ascribed to the differences in inhibition of the endoxylanases by endogenous inhibitors.     

Effect of soil aggregate size on infiltration and erosion characteristics

Detachment of soil particles by the processes of splash from rainfall and shallow flow from surface runoff is influenced by soil cohesion, soil aggregate properties, and characteristics of this flow. We have evaluated relationships between rates of detachment, aggregate size, and tensile strength of the soil. Soil and water losses were determined in the laboratory from sieved air‐dry samples on three aggregate size ranges of two clay loam soils differing in particle‐size distribution and organic matter. Tensile strength was measured for each aggregate size range. The results showed that as clod size increased, detachment rates increased and interaggregate tensile strength decreased. Wash erosion increased as initial clod size increased despite a decrease in runoff. Final rates of loss by splash were greater from the largest clods than from the smaller clods. Finally, splashed material was larger in size than material washed off. The fact that the size of the splashed material was larger than washed‐off material shows that material in the wash suffered more impact by raindrops and thus was more likely to be fragmented.     

Physicochemical properties of soy protein: effects of subunit composition

Soy protein elastomer (SPE) exhibits elastic, extensible, and sticky properties in its native state and displays great potential as an alternative to wheat gluten. The objective of this study was to better understand the roles of soy protein subunits (polypeptides) contributing to the functional properties of SPE. Six soy protein samples with different subunit compositions were prepared by extracting the proteins at various pH values on the basis of the different solubilities of conglycinin (7S) and glycinin (11S) globulins. Soy protein containing a large amount of high molecular weight aggregates formed from α' and α subunits exhibited stronger viscoelastic solid behavior than other soy protein samples in terms of dynamic elastic and viscous modules. Electrophoresis results revealed that these aggregates are mainly stabilized through disulfide bonds, which also contributed to higher denaturation enthalpy as characterized by DSC and larger size protein aggregates observed by TEM. Besides, the most viscoelastic soy protein sample exhibited flat and smooth surfaces of the protein particles as observed by SEM, whereas other samples had rough and porous particle surfaces. It was proposed that the ability of α' and α to form aggregates and the resultant proper protein-protein interaction in soy proteins are the critical contributions to the continuous network of SPE.     

Engineering nutritious proteins: improvement of stability in the designer protein MB-1 via introduction of disulfide bridges.

Protein design is currently used for the creation of new proteins with desirable traits. In this laboratory the focus has been on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations faced in this endeavor is achieving stable proteins despite a highly biased amino acid content. Reported here are the synthesis and characterization of two disulfide-bridged mutants derived from the MB-1 designer protein. Both mutants outperformed their parent protein MB-1 with their bridge formed, as shown by circular dichroism, size exclusion chromatography, thermal denaturation, and proteolytic degradation experiments. When the disulfide bridges were cleaved, the mutants' behavior changed: the mutants significantly unfolded, suggesting that the introduction of Cys residues was deleterious to MB-1-folding. In an attempt to compensate for the mutations used, a Tyr62-Trp mutation was performed, leading to an increase in bulk and hydrophobicity in the core. The Trp-containing disulfide-bridged mutants did not behave as well as the original MB-1Trp, suggesting that position 62 might not be adequate for a compensatory mutation.     

Cold-set globular protein gels: interactions,structure and rheology as a function of protein concentration

We identified the contribution of covalent and noncovalent interactions to the scaling behavior of the structural and rheological properties in a cold gelling protein system. The system we studied consisted of two types of whey protein aggregates, equal in size but different in the amount of accessible thiol groups at the surface of the aggregates. Analysis of the structural characteristics of acid-induced gels of both thiol-blocked and unmodified whey protein aggregates yielded a fractal dimension (2.3 +/- 0.1), which is in line with other comparable protein networks. However, application of known fractal scaling equations to our rheological data yielded ambiguous results. It is suggested that acid-induced cold-gelation probably starts off as a fractal process, but is rapidly taken over by another mechanism at larger length scales (>100 nm). In addition, indications were found for disulfide cross-link-dependent structural rearrangements at smaller length scales (<100 nm).     

Influence of pH and ionic strength on heat-induced formation and rheological properties of soy protein gels in relation to denaturation and their protein compositions

The influence of pH and ionic strength on gel formation and gel properties of soy protein isolate (SPI) in relation to denaturation and protein aggregation/precipitation was studied. Denaturation proved to be a prerequisite for gel formation under all conditions of pH and ionic strength studied. Gels exhibited a low stiffness at pH >6 and a high stiffness at pH <6. This might be caused by variations in the association/dissociation behavior of the soy proteins on heating as a function of pH, as indicated by the different protein compositions of the dissolved protein after heating. At pH 3-5 all protein seems to participate in the network, whereas at pH >5 less protein and especially fewer acidic polypeptides take part in the network, coinciding with less stiff gels. At pH 7.6, extensive rearrangements in the network structure took place during prolonged heating, whereas at pH 3.8 rearrangements did not occur.     

Magnetic resonance imaging, rheological properties, and physicochemical characteristics of meat systems with fibrinogen and thrombin

Magnetic resonance imaging (MRI) and textural and physicochemical analyses were carried out to evaluate the effect of fibrinogen and thrombin (Fibrimex) addition to meat systems formulated with and without NaCl. For this purpose, different model systems were elaborated: fibrinogen and thrombin (FT), meat emulsion (ME), and meat emulsion with fibrinogen and thrombin (MEFT), with 0, 1, and 2% of NaCl. The addition of fibrinogen-thrombin to meat emulsions results in a gel network with modified physicochemical and textural characteristics, increasing the hardness and springiness. The addition of NaCl at 2% to FT and MEFT systems reduced the gel hardness. MRI parameters (T2, T1, and apparent diffusion coefficient) indicated that systems with fibrinogen and thrombin (FT and MEFT) presented a structure with many and large pores, bulk water, and higher translational motion of water. Significant correlations were found between MRI, texture, and physicochemical parameters.     

淀粉对竹荚鱼鱼糜流变性质和凝胶特性的影响

为探讨改善竹荚鱼鱼糜凝胶品质的最佳淀粉种类和添加量,利用流变仪、质构仪、扫描电镜等方法研究了不同来源的原淀粉及其添加量对竹荚鱼鱼糜流变性质和凝胶特性的影响。动态流变性质的结果表明,在加热过程中,竹荚鱼鱼糜的弹性模量G' 经历了2个阶段的变化,首先在20～57℃时G' 逐渐降低;然后在57～80℃由于凝胶网络的形成,使G' 增加。添加淀粉显著影响竹荚鱼鱼糜的G'。添加淀粉能够提高竹荚鱼鱼糜的凝胶强度。添加木薯淀粉、小麦淀粉对改善竹荚鱼鱼糜凝胶品质的效果最好,其添加量为4%,竹荚鱼鱼糜凝胶的凝胶强度分别提高了158%和155%。添加淀粉能提高竹荚鱼鱼糜凝胶的持水性。在相同的添加量时,木薯淀粉和小麦淀粉对提高竹荚鱼鱼糜凝胶的持水性的效果最好。添加淀粉能显著提高竹荚鱼鱼糜凝胶的白度,但淀粉的种类和添加量对鱼糜凝胶色泽的影响不显著。     

Plasma-enhanced modification of xanthan gum and its effect on rheological properties

The structure and rheological properties of xanthan gum (XG) modified in a cold plasma environment were investigated. XG was functionalized in a capacitively coupled 13.56-MHz radio frequency dichlorosilane (DS)-plasma conditions and, consecutively, in situ aminated by ethylenediamine. The surface structure of modified XG was evaluated on the basis of survey and high-resolution ESCA, FTIR, and fluorescence labeling techniques. The types of species generated in DS-plasma were reported using residual gas analysis (RGA). The aqueous solutions of modified XG were cross-linked and cured at room temperature to form stable gels. The dynamic rheological characteristics of virgin XG and functionalized and cross-linked XG were compared. It was found that parameters such as plasma treatment time and concentration of solutions can be optimized to form stable gels of XG. Thus, cold plasma technology is a novel, efficient, and nonenzymatic route to modify XG.     

Influence of sucrose on the thermal denaturation, gelation, and emulsion stabilization of whey proteins

The influence of sucrose (0-40 wt %) on the thermal denaturation and functionality of whey protein isolate (WPI) solutions has been studied. The effect of sucrose on the heat denaturation of 0.2 wt % WPI solutions (pH 7.0) was measured using differential scanning calorimetry. Sucrose increased the temperature at which protein denaturation occurred, for example, by 6-8 degrees C for 40 wt % sucrose. The dynamic shear rheology of 10 wt % WPI solutions (pH 7.0, 100 mM NaCl) was monitored as they were heated from 30 to 90 degrees C and then cooled to 30 degrees C. Sucrose increased the gelation temperature and the final rigidity of the cooled gels. The degree of flocculation in 10 wt % oil-in-water emulsions stabilized by 1 wt % WPI (pH 7.0, 100 mM NaCl) was measured using a light scattering technique after they were heated at fixed temperatures from 30 to 90 degrees C for 15 min and then cooled to 30 degrees C. Sucrose increased the temperature at which maximum flocculation was observed and increased the extent of droplet flocculation. These results are interpreted in terms of the influence of sucrose on the thermal unfolding and aggregation of protein molecules.     

Effect of variety and environmental factors on gluten proteins: an analytical, spectroscopic, and rheological study

Four cultivars of winter wheat with contrasting qualities for breadmaking were selected to study the effects of environmental factors on grain protein composition and properties. They were grown in the field and under two controlled regimens designed to mimic typical "hot/dry" and "cold/wet" conditions experienced during grain development in the United Kingdom. The composition of the gluten proteins determined by SDS-PAGE and their size distribution determined by SE-HPLC were consistent with the presence of higher proportions of high M r polymers in the two varieties with good breadmaking performance (Spark and Soissons) with limited environmental effects on these parameters. Gluten protein fractions from three of the cultivars were analyzed by Fourier transform infrared (FTIR) spectroscopy and this, combined with creep measurements using a texture analyzer, showed that a conversion from beta-turns to beta-sheets occurred during extension, irrespective of the growth conditions. However, the breadmaking varieties Soissons and Spark showed greater differences related to environmental conditions than the variety Rialto, which has poorer processing quality.     

Enzyme-induced gelation of extensively hydrolyzed whey proteins by Alcalase: peptide identification and determination of enzyme specificity

Extensive hydrolysis of whey protein isolate by Alcalase was shown to induce gelation mainly via hydrophobic interactions. The aim of this work was to characterize the peptides released in order to better understand this phenomenon. The apparent molecular mass distribution indicated that aggregates were formed by small molecular mass peptides (<2000 Da). One hundred and thirty peptides with various lengths were identified by reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Alcalase was observed to have a high specificity for aromatic (Phe, Trp, and Tyr), acidic (Glu), sulfur-containing (Met), aliphatic (Leu and Ala), hydroxyl (Ser), and basic (Lys) residues. Most peptides had an average hydrophobicity of 1-1.5 kcal/residue and a net charge of 0 at the pH at which gelation occurred (6.0). Therefore, an intermolecular attractive force such as hydrophobic interaction suggests the formation of aggregates that further leads to the formation of a gel.     

Effect of galactomannans on the thermal and rheological properties of sago starch

The thermal and rheological properties of sago starch have been studied in the presence of various concentrations of locust bean gum and guar gum of various molecular masses. At the concentrations studied (<1%) the galactomannans gave rise to only a very slight increase in the gelatinization temperature (up to 0.6 degrees C), and the gelatinization enthalpy remained constant within experimental error. For the low molecular mass galactomannans, depending on the concentration, the storage modulus, G', of the mixtures remained constant or actually decreased, and tan delta remained very low (0.01-0.03 at 0.1 Hz), indicating strong elastic gels. For the higher molecular mass samples G' increased significantly; however, the loss modulus, G' ', increased proportionally to a greater extent, and at 1% galactomannan tan delta was approximately 0.20 at 0.1 Hz, indicating a reduction in elastic character. The systems were shown to undergo phase separation, and the variations in rheological properties have been discussed in the context of their phase behavior and the relative rates of the phase separation and gelation processes. The presence of galactomannans significantly improved the freeze-thaw stability.     

抛荒对冷浸稻田土壤团聚体及有机碳稳定性的影响

冷浸稻田是长江流域重要的低产稻田类型之一,近年来抛荒严重,而抛荒对冷浸稻田土壤团聚体的影响并不清楚。本研究以连年种植的冷浸稻田(CWC)、抛荒3年的冷浸稻田(CWA3)和抛荒6年的冷浸稻田(CWA6)为对象,分析抛荒后冷浸稻田土壤团聚体特征以及有机碳稳定性,以期为准确评估抛荒对长期淹水土壤的结构和有机碳的影响提供数据支持。结果表明,不论是0~25 cm土层还是25~50 cm土层,冷浸稻田土壤53μm粒级团聚体占总团聚体比例均超过40%;0~25 cm土层土壤250μm团聚体比例超过35%;53~250μm粒级团聚体比例低于20%。抛荒使0~25 cm土层53μm粒级团聚体占总团聚体比例显著增加,53~250μm粒级比例显著降低。在0~25 cm土层,抛荒使有机碳活性指数Ⅰ(LIc-Ⅰ)在53μm粒级和250μm粒级上升高,有机碳活性指数Ⅱ(LIc-Ⅱ)在53~250μm和250μm粒级上降低;而有机碳难降解指数(RIc)在53μm和53~250μm粒级上降低。土壤总有机碳随抛荒时间延长而增加。