Study of thermal aggregation of globulin from common buckwheat (Fagopyrum esculentum Moench) by size-exclusion chromatography and laser light scattering

The heat-induced aggregation of common buckwheat (Fagopyrum esculetum Moench) globulin (BWG) was studied using size-exclusion chromatography (SEC) combined with on-line multiangle laser light scattering (MALLS) and quasielastic light scattering (QELS). The unheated BWG was found to exist mainly as a hexamer, with an estimated weight-average molecular weight (M(w)) of 342 000, close to that deduced from the genomic cloned data of 13S buckwheat globulin. The QELS data predicted that the hexamer exists as two annular trimeric rings (diameter approximately 10.8 nm) placed on top of each other, forming an oblate cylinder (height approximately 9.1 nm). Upon heating, hexamers and trimers were dissociated and then associated to form extended small aggregates, finally forming compact, large macroaggregates. N-Ethylmaleimide would favor macroaggregate formation and increased the molar masses and hydrodynamic radii of the soluble aggregates, suggesting a different aggregation process in the presence of the sulfhydryl-blocking agent. A plot of log hydrodynamic radius versus log molar mass showed changes in the slope during heat treatment, suggesting conformational transformation in the heat-denatured and aggregated BWG molecules.     

Limited aggregation behavior of β-conglycinin and its terminating effect on glycinin aggregation during heating at pH 7.0

In this work, different thermal aggregation behaviors of soy β-conglycinin and glycinin at pH 7.0 were characterized with size exclusion chromatography and low-angle light scattering. Limited aggregation that grew via the consumption of "monomers" was detected in β-conglycinin, forming soluble aggregates. For glycinin, the association between the aggregates that led to the appearance of insoluble materials was observed. Heated with β-conglycinin, the assembly between the glycinin aggregates was terminated and its solubility was recovered. The structure of the soluble and insoluble aggregates was analyzed by small-angle X-ray scattering and dynamic light scattering. Unlike the β-conglycinin soluble aggregates that possessed limited size and less compact conformation, particles with a denser core and a less dense outer shell were found in the glycinin insoluble aggregates. Evidence is presented to reveal the transition between the soluble and insoluble aggregates and the role of β-conglycinin in the solubilization of the soy protein aggregates during heating.     

Effects of caseins on thermal stability of bovine beta-lactoglobulin

Casein fractions have been shown to act as molecular chaperones and inhibit aggregation of whey proteins in dilute solutions (< or =1% w/v). We evaluated if this approach would stabilize protein solutions at higher concentration and thermal processing temperatures desired for beverage applications. Mixtures of beta-lactoglobulin (BLG) (6% w/v) with either beta-casein (BCN) (0.01-2% w/v) or alpha s-casein (ACN) (2% w/v) were adjusted to pH 6.0 and heated (70-90 degrees C) for 20 min, cooled, and then analyzed to determine the degree of aggregation. Aggregation was determined by solution turbidity as optical density (OD) at 400 or 600 nm. The addition of 0.05% (w/v) BCN or greater caused a drop in turbidity for solutions heated at 70-90 degrees C. In contrast, inhibition was observed in BLG-ACN mixtures at 70 degrees C but not at > or =75 degrees C. Moreover, prolonged heating (90 min) of BLG with 2% (w/v) BCN (pH 6.0) at 90 degrees C produced a clear solution while BLG-ACN solutions formed translucent gels after heating for 15 min. The weight-averaged molar mass and root-mean-square (rms) radius of soluble aggregates were determined by size exclusion chromatography in conjunction with multiangle laser light scattering (SEC-MALS). SEC-MALS confirmed the turbidity results by showing that the BLG-BCN mixture (8% w/v protein) produced aggregates with lower molar mass and smaller rms radius (majority 20-40 nm). These results showed that BCN is a feasible component to stabilize higher concentrations of whey proteins in beverages.     

Effects of Different Lipase Inactivation Treatments on Physicochemical Properties of Naked Oat Globulins

Lipase inactivation is an essential treatment for oat processing, because of the negative effects of lipase on nutrient preservation and storage extension. The effects of different lipase inactivation treatments including hot air roasting, infrared roasting, normal‐pressure steaming, and high‐pressure steaming on the physicochemical properties of oat globulins were investigated. Results showed that normal‐pressure steaming had little effect on solubility of oat globulins; hot air roasting increased foaming capacity of oat globulins but did not change their foaming stability; and all the inactivation treatments increased the surface hydrophobicity and content of total sulfhydryls of oat globulin but decreased exposed sulfhydryl groups. In addition, oat globulin granules from the hot air roasting treatment were distributed more evenly in oat globulin powder compared with the control group. All treatments except normal‐pressure steaming changed the molecular weight of oat globulin subunits, which made the bands of 66,000 and 45,000 disappear from SDS‐PAGE. These results indicated that normal‐pressure steaming was ideal to maintain good solubility of oat globulins, and hot air roasting was ideal to maintain relatively good foaming properties. The treatments changed physicochemical properties of oat globulins by influencing protein aggregation and subunit composition that resulted in different content of sulfhydryl groups and surface hydrophobicity.     

Visualization of single and aggregated hulless oat (Avena nuda L.) (1-->3),(1-->4)-beta-D-glucan molecules by atomic force microscopy and confocal scanning laser microscopy

Surfactants were used to disperse oat beta-glucan. Atomic force microscopy (AFM) images of the resulting samples revealed a distribution of extended chainlike molecules and allowed, for the first time, direct visualization of single oat beta-glucan molecules with cross-sectional heights of about 0.44 nm. The number-average contour length (L(n)) and root-mean-square end-to-end distance ((R(ee)2)(1/2)) measured from the AFM images were 938 and 912 nm, respectively. The calculated persistence length (L(p)) was 526 nm. The weight-average molecular weight (M(w)) calculated from single beta-glucan molecules was 4.43 x 10(5). Samples without surfactant showed a strong tendency to form aggregates. The sample concentration, reserving time, and calcofluor as well as freezing could affect the formation of aggregates. These aggregates were visualized by both AFM and confocal scanning laser microscopy. The shape of the aggregates changed from small dots with diameters of approximately 20-50 nm to microfibrils over 3 microm long with the increasing of the concentration of oat beta-glucan from 10 to 100 microg/mL. The particle size distribution obtained by a laser particle size analyzer was 926 nm, which confirmed the size of oat beta-glucan molecules obtained from AFM images.     

Influence of heating on oil-in-water emulsions prepared with soybean soluble polysaccharide

The effect of heating on the physicochemical properties of emulsions prepared with soybean soluble polysaccharide (SSPS) was investigated. The emulsions were stable after heating at 90 degrees C for up to 30 min. Heating at different pH values or in the presence of CaCl2 (<10 mM) did not affect the stability; however, at higher concentrations of calcium ions, the emulsion particle size increased. Two fractions, a high molecular weight (HMF) and a low molecular weight (LMF) fraction, were separated from the crude SSPS preparation by gel fitration. Emulsions prepared with SSPS/HMF (MW = 310-420 kDa) showed little change in size with heating, while the protein impurities of the SSPS/LMF fraction formed aggregates by heating at pH 7. Analysis of the heat-induced aggregation of the two fractions of SSPS suggested that the changes in SSPS functionality with heating can be attributed to the protein impurities (LMF) present in the SSPS.     

Effect of pH and ionic strength modifications on thermal denaturation of the 11S globulin of sunflower (Helianthus annuus)

Helianthinin, the main storage protein of sunflowers, has low water solubility and does not form a gel when heated; this behavior is different from other 11S globulins and limits its food applications. To understand this particular behavior, changes on helianthinin association-dissociation state induced by modifications in pH and ionic strength were analyzed. The influence of these different medium conditions on its thermal stability and tendency to form aggregates was also studied. Helianthinin behavior at different pH values and ionic strengths is similar to other 11S globulins except that it remains in a trimeric form at pH 11. Helianthinin thermal stability is higher than other 11S globulins but is lower than oat 11S globulin. Alkaline pH produces a 10 degrees C decrease of its denaturation temperature and also of the cooperativity of denaturation process, but it does not affect the denaturation activation energy. The decrease in thermal stability with the pH increase is also manifested by its tendency to form aggregates by SH/SS interchange reactions. When thermal treatments at alkaline pH are performed, all helianthinin subunits form aggregates, characterized by a higher proportion of beta-polypeptides than alpha-polypeptides, which is an indication that aggregation is accompanied by dissociation. Treatments at 80 degrees C are sufficient to induce aggregation but not to produce denaturation, and in these conditions hexameric forms remain after the treatment.     

Aggregation of a proline-rich protein induced by epigallocatechin gallate and condensed tannins: effect of protein glycosylation

Astringency is one of the most important organoleptic qualities of numerous beverages, including red wines. It is generally thought to originate from interactions between tannins and salivary proline-rich proteins (PRPs). In this work interactions between a glycosylated PRP, called II-1, and flavan-3-ols were studied in aqueous solutions and at a colloidal level, by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). The flavan-3-ols were a monomer, epigallocatechin gallate (EGCG), and polymerized flavan-3-ol fractions extracted from grape seeds. In aqueous solutions containing EGCG and protein II-1, protein aggregation took place when protein concentration and the EGCG/protein ratio exceeded a threshold. The aggregates had a small size, comparable with the dimensions of protein monomers, and formed stable dispersions (no phase separation). Most proteins remained free in solution. This behavior is in sharp contrast with the phase separation observed for nonglycoslated PRP in the same conditions. Moreover, this slight aggregation of II-I in the presence of EGCG was disrupted by the addition of 12% ethanol. Increasing the flavan-3-ol molecular weight strongly enhanced II-I/tannin aggregation: the threshold was at a lower protein concentration (0.2 mg/mL) and a lower tannin/protein ratio. Still, in most cases, and in contrast with that observed with a nonglycosylated PRP, the aggregates remained of discrete size and stable. Only at low ethanol content (2%) did the addition of tannin polymers finally lead to phase separation, which occurred when the molar ratio of tannins to proteins exceeded 12. This systematic effect of ethanol confirmed the strong effect of cosolvents on protein/tannin interactions.     

Characterization of commercial and experimental sodium caseinates by multiangle laser light scattering and size-exclusion chromatography

A range of sodium caseinate samples were characterized by a multiangle laser light scattering (MALLS) system or by the use of MALLS as an on-line detector with size-exclusion chromatography (SEC). Sodium caseinate solutions, analyzed using a MALLS system alone, gave weight-average molar mass (M(w)) values in the range 1200-4700 kDa and z-average root-mean-square radius (R(g)) values ranged from approximately 50 to 120 nm. When these solutions were ultracentrifuged at 90000g for 1 h, a cloudy top layer was formed; the subnatant was carefully removed and analyzed by SEC-MALLS. The M(w) values were found to be in the range approximately 30-575 kDa, and R(g) values ranged from approximately 22 to 49 nm. During SEC, the MALLS system detected some very large-sized material that eluted close to the void volume; this material was hardly detected by the concentration detectors, i.e., ultra-violet (UV) and differential refractive index (DRI). The intensity of the light scattering (LS) signal from this very large sized material was greatly reduced in the subnatant. SEC of sodium caseinate samples revealed two main peaks with M(w) of approximately 420-750 kDa and 39-69 kDa, respectively. The R(g) values were very large for a protein molecule, and initial calculations suggested that the shape of caseinate molecules was likely to be highly elongated.     

Thermal aggregation of patatin studied in situ.

In this work dynamic light scattering was used to study the thermal aggregation of patatin in situ, to elucidate the physical aggregation mechanism of the protein and to be able to relate the aggregation behavior to its structural properties. The dependence of the aggregation rates on the temperature and the ionic strength suggested a mechanism of slow coagulation, being both diffusion and chemically limited. The aggregation rate dependence on the protein concentration was in accordance with the mechanism proposed. The aggregation rates as obtained at temperatures ranging from 40 to 65 degrees C correlated well with unfolding of the protein at a secondary level. Small-angle neutron scattering and dynamic light scattering results were in good accordance; they revealed that native patatin has a cylindrical shape with a diameter and length of 5 and 9.8 nm, respectively.     

Stability of ingested methylcellulose in the rat determined by polymer molar mass measurements by light scattering

Methylcellulose (MC) is ingested by humans in food and pharmaceutical formulations. The functional properties of MC like those of other linear polymers depend primarily on polymer length or molar mass for largely linear polymers. Although many studies in animals and humans have shown complete excretion of MC, in vitro human fecal fermentation studies indicate that MC can be degraded and presumably lose some of its functionality. In this study, MC polymer distribution in the feces from rats fed a diet containing 8% methylcellulose were compared to the fed MC. The water-soluble polymers in the feces were separated by a size exclusion chromatography (SEC) and the polymer distributions determined by multiple angle laser light scattering (MALLS). Detection of the fluorescent MC-calcofluor complex was used to confirm the identity of the eluting MC peak. All dietary MC was recovered in the feces. There is a small shift (P < 0.06) in the weight-averaged molecular weight of polymer distribution of MC extracted from the feces to 2.71 +/- 0.15 x 10(5) g/mol from 3.15 +/- 0.02 x 10(5) g/mol in the standard. There is also an increase in the polydispersity from 1.21 in the standard to 1.8 in the fecal extract. The distribution of the substituted methoxylated glucose monomers by gas chromatography also confirms the stability of MC fed to rats. The amount of actual hydrolysis is estimated to be about 0.1 glycosidic linkage/molecule. MC is not easily determined by standard dietary fiber methods, and SEC with MALLS and/or fluorescence may be a useful alternative.     

Heat-induced formation of intermolecular disulfide linkages between thaumatin molecules that do not contain cysteine residues

Thaumatin, a sweet protein that contains no cysteine residues and eight intramolecular disulfide bonds, aggregates upon heating at pH 7.0 above 70 degrees C, and its sweetness thereby disappears. The aggregate can be solubilized by heating in the presence of both thiol reducing reagent and SDS. This molecular aggregation depended on the protein concentration during heating and was suppressed by the addition of N-ethylmaleimide or iodoacetamide, indicating a thiol-catalyzed disulfide interchange reaction between heat-denatured molecules. An amino acid analysis of the aggregates suggested that the cysteine and lysine residues were reduced, and the formation of a cysteine residue and a lysinoalanine residue was confirmed. The reduction and formation of these residues stoichiometrically satisfied the beta-elimination of a cystine residue. The disulfide interchange reaction was catalyzed by cysteine; that is, a free sulfhydryl residue was formed via beta-elimination of a disulfide bond. Intermolecular disulfide bonds were probably formed between thaumatin molecules upon heating at pH 7.0, which led to the aggregation of thaumatin molecules.     

Effects of ionic strength and sulfate upon thermal aggregation of grape chitinases and thaumatin-like proteins in a model system

Consumers expect white wines to be clear. During the storage of wines, grape proteins can aggregate to form haze. These proteins, particularly chitinases and thaumatin-like proteins (TL-proteins), need to be removed, and this is done through adsorption by bentonite, an effective but inefficient wine-processing step. Alternative processes are sought, but, for them to be successful, an in-depth understanding of the causes of protein hazing is required. This study investigated the role played by ionic strength (I) and sulfate toward the aggregation of TL-proteins and chitinases upon heating. Purified proteins were dissolved in model wine and analyzed by dynamic light scattering (DLS). The effect of I on protein aggregation was investigated within the range from 2 to 500 mM/L. For chitinases, aggregation occurred during heating with I values of 100 and 500 mM/L, depending on the isoform. This aggregation immediately led to the formation of large particles (3 μm, visible haze after cooling). TL-protein aggregation was observed only with I of 500 mM/L; it mainly developed during cooling and led to the formation of finite aggregates (400 nm) that remained invisible. With sulfate in the medium chitinases formed visible haze immediately when heat was applied, whereas TL-proteins aggregated during cooling but not into particles large enough to be visible to the naked eye. The data show that the aggregation mechanisms of TL-proteins and chitinases are different and are influenced by the ionic strength and ionic content of the model wine. Under the conditions used in this study, chitinases were more prone to precipitate and form haze than TL-proteins.     

Aggregates and gel network structure of globin hydrolysates

A gel with excellent functional properties was prepared successfully using the hydrolysates of globin. In the present study, the structures of intermediate aggregates and gel network were observed directly with an electronic microscope. It was shown clearly that the intermediate aggregates were in a thin rod shape with a length of 130--140 nm, which was in good accordance with the results of the light scattering obtained in a previous study. The diameter of intermediate aggregates was 4--5 nm. Each unit of the intermediate aggregate was composed of beta-chain and peptide beta-1 in a ratio of 1:1. Its molecular weight was 26922 Da, and it had a diameter of 4.1 nm. The thin rod-shaped aggregates were formed with units through the hydrophobic interaction. The length of intermediate aggregate was >30--33 times the diameter. Furthermore, the cross-linked structure formed by peptide alpha-1 and the thin rod-shaped aggregates was also confirmed by the photography of the electronic microscope. These results supported the model proposed in previous papers as proper to depict exactly the formation and structure of the gel network of globin hydrolysates.     

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。     

Aggregation of humic substances in aqueous media as determined by light-scattering methods

Samples of peat humic acid (PHA) and surface water humic (WBHA) and fulvic acids (WBFA) extracted from Whitray Beck in North Yorkshire, UK and previously studied by analytical (UV scanning) ultracentrifugation at low concentrations (10–20 mg dm^?3) (Reid et al., 1990) have been investigated by dynamic and static light-scattering methods at concentrations up to approximately 1700 mg dm^?3. Dynamic light-scattering gave very low diffusion coefficients for the humic acids (PHA and WBHA) suggesting the presence of aggregates. Static light-scattering from the fulvic acid (WBFA) showed that the second virial coefficients of this material in various media were negative, which is characteristic of reversible aggregation. The aggregates, which consisted of approximately 20 smaller molecules, could not be dissociated completely by the detergents sodium n-dodecylsulphate or Triton X-100, although limited dissociation by the latter may occur. Sedimentation velocity measurements in the same concentration range as the light scattering gave sedimentation coefficients which increased with concentration also consistent with reversible aggregation. The studies illustrate the problems inherent in the characterization of humic substances from natural sources and demonstrate that, at least for lower molecular weight fulvic acids, static light-scattering from solutions of relatively high concentration in combination with sedimentation equilibrium measurements at low concentrations distinguishes a system having a broad molecular weight distribution from one in which aggregation occurs.     

Moisture-induced aggregation of whey proteins in a protein/buffer model system

Moisture-induced protein aggregation in a dry or intermediate-moisture food matrix can contribute to the loss of product acceptability. The present study evaluated the molecular mechanisms and controlling factors for moisture-induced whey protein aggregation in a premixed protein/buffer model system. Insoluble aggregates rapidly formed during the first 3 days of storage at 35 degrees C with a slower rate afterward. Evaluation of the insoluble aggregates by solubility tests in solutions containing SDS/urea/guanidine HCl/dithiothreitol and gel electrophoresis showed that the formation of intermolecular disulfide bonds was the main mechanism for protein aggregation, and all major whey proteins were involved in the formation of insoluble aggregates. Effects of various factors on aggregation were also investigated, including moisture content, medium pH, and the addition of NaCl. The dependence of aggregation on moisture content was bell-shaped, and the maximal extent of aggregation was achieved at a moisture content of around 70-80% on a dry weight basis.     

Polyphenol-beta-casein complexes at the air/water interface and in solution: effects of polyphenol structure

The interactions between proteins and plant polyphenols are responsible for astringency and haze formation in beverages and may participate in foam stabilization. The effect of phenolic compounds with different structures, namely, catechin (C), epicatechin (Ec), epigallocatechin (Egc), epicatechin gallate (EcG), and epigallocatechin gallate (EgcG), on the surface properties at the air/liquid interface of beta-casein, chosen as model protein, were monitored by tensiometry and ellipsometry. The formation of complexes in the bulk phase was measured by electrospray ionization mass spectrometry (ESI-MS). Adsorption of polyphenols from pure solution was not observed. Surface pressure, surface concentration, and dilational modulus of the protein adsorption layer were greatly modified in the presence of galloylated flavanol monomers (EcG and EgcG) but not of lower molecular weight polyphenols (<306 g/mol). The formation of polyphenol-protein aggregates in the bulk, as evidenced by ESI-MS and light scattering experiments, was related to the slowdown of protein adsorption.     

Heat-induced aggregation of beta-lactoglobulin as a function of pH.

The effect of pH in the range 6.0-8.0 on the denaturation and aggregation of beta-lactoglobulin (beta-lg) was investigated. Results were interpreted in terms of the reaction scheme for the denaturation and aggregation of beta-lg proposed by Roefs and De Kruif (Eur. J. Biochem. 1994, 226, 883-889). The rate of conversion of native beta-lg increased strongly at higher pH values, whereas the molecular mass of the aggregates decreased strongly. In the pH range 6.4-8.0 aggregates were formed mainly by intermolecular disulfide bonds, but even at pH 6.0, thiol/disulfide exchange reactions were involved, although to a lesser extent. The time course of the exposure of the thiol group in native beta-lg upon heating and the subsequent disappearance of this group through the formation of disulfide-linked aggregates was investigated by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and varied strongly with pH. These observations could be used, in combination with the reaction steps of the reaction scheme, to describe qualitatively the strongly pH-dependent isothermal calorimetry curves, measured at 65 degrees C.