Study of oat globulin conformation by Fourier transform infrared spectroscopy.

The conformation of oat globulin dispersions (10% in D2O) under the influence of pH, chaotropic salts, protein structure perturbants, and heating conditions was studied by Fourier transform infrared (FTIR) spectroscopy. The FTIR spectrum of oat globulin showed major bands from 1670 to 1634 cm(-1), corresponding to the four major types of secondary structures, that is, beta-turns, beta-sheets, alpha-helices, and random coils. At extreme acidic and alkaline pH conditions, there were changes in intensity in the bands attributed to beta-sheet structures (1626, 1634, and 1682 cm(-1)), and shifts of the bands to higher or lower wavenumbers, indicating changes in conformation. In the presence of some chaotropic salts, the 1626 and 1634 cm(-1) bands were shifted upward, with a marked decrease in the intensity of the 1634 cm(-1) peak. The addition of several protein structure perturbants led to a slight shift in the alpha-helix/random coil bands and a marked reduction in the beta-sheet peaks, suggesting protein unfolding. Heating under aggregating conditions led to slight shifts in all of the major bands and progressive changes in the intensity of the alpha-helix, beta-sheet, and beta-turn peaks, suggesting protein denaturation. This was accompanied by marked increases in intensity of the two intermolecular beta-sheet bands (1682 and 1624-1626 cm(-1)) associated with the formation of aggregated strands. The IR spectra of soluble and insoluble aggregates showed a redistribution of native and extensively denatured proteins in the two fractions.     

Physicochemical and structural properties of oat globulin polymers formed by a microbial transglutaminase

Oat globulin was polymerized by a microbial transglutaminase (TG), and some physicochemical and functional properties of polymers were studied. Reversed-phase HPLC revealed that the number of epsilon-(gamma-glutamyl) lysine isopeptide bonds formed after 4 h of enzyme incubation was 2.21 micromol/g of protein. SDS-PAGE showed that the oat globulin acidic polypeptides (AP) were more susceptible to polymerization than the basic polypeptides (BP), and the reactivities of both AP and BP were enhanced by the addition of other substrate proteins. Differential scanning calorimetry showed that both the denaturation temperature and denaturation enthalpy were decreased after TG treatment. Fourier transform infrared spectroscopy revealed marked increases in the intensity of two intermolecular beta-sheet bands associated with aggregate formation but little conformational changes in the polymerized protein. TG incubation led to progressive changes in flow properties of oat globulin dispersions, indicating enhanced pseudoplasticity and increased viscosity and yield stress.     

Acid-induced gelation of enzymatically modified, preheated whey proteins

Low-pH whey protein gels are formulated using a sequential protocol of heat treatment, enzyme incubation, and cold-set acidification. The heat-induced disulfide and enzyme-catalyzed epsilon-(gamma-glutamyl)lysine linkages, both at neutral pH, produce a polymerized protein solution. The molecular weights of these samples show an exponential increase with protein concentration. The additional enzyme-catalyzed cross-links cause little change in molecular weight from that of heat-treated samples at low protein concentrations, indicating predominant intramolecular cross-linking. Enzyme treatment at higher protein concentration however causes increase in molecular weight, possibly due to formation of intermolecular cross-links. Acidification of the polymerized protein solutions through glucono-delta-lactone acid leads to gel formation at pH 4. The elastic (G') and viscous (G' ') moduli of gels with and without enzyme treatment show similar frequency dependence, indicating comparable microstructures, consistent with all samples exhibiting similar fractal dimensions of approximately 2 obtained independently using rheology and confocal microscopy. A substantial increase in fracture strain and stress of the gel is achieved by enzyme treatment. However, the elastic modulus (G') is only slightly larger after enzyme treatment compared with heat-treated samples. These results indicate that factors responsible for fracture properties may not be apparent in the gel microstructure and linear viscoelastic properties.     

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.     

Raman spectroscopic study of oat globulin conformation

Analysis of Raman spectra of oat globulin showed that extreme pH values caused an increase in the amide and C-H stretching band intensity, indicating changes in the secondary structures of the protein due to denaturation. Similar changes were observed when oat globulin was treated with chaotropic salts and several protein perturbants. Sodium dodecyl sulfate, beta-mercaptoethanol, and ethylene glycol also caused a shift in the amide III' band, suggesting a transition from beta-sheet to a random coil conformation. Heating at temperatures near the denaturation temperature of oat globulin led to increases in the amide and C-H band intensity, indicating unfolding of the protein. The data indicate that FT-Raman spectroscopy is suitable for studying the secondary structure of plant proteins such as oat globulin.     

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.     

Heat-induced gelation of pea legumin: comparison with soybean glycinin

Gel network formation of pea legumin (8.4% on a protein basis, pH 7.6) was monitored via dynamic rheological measurements. Gelation was performed in the absence and presence of the thiol-blocking reagent N-ethylmaleimide, at different rates of heating and cooling. Overall, it was shown that pea legumin gel formation was not effected by changes in the heating rate, and the two differently heated samples were unaffected by the addition of 20 mM NEM, which indicated that disulfide bonds were not essential within the network strands of these legumin gels. However, slowly cooling the legumin samples caused disulfide bonds to become involved within the network; this was observed by a large increase in gel strength that was then substantially reduced when repeating the sample in the presence of NEM. These experiments were repeated with soybean glycinin in order to determine whether a common model for gel formation of legumin-like proteins could be built, based upon molecular reasoning. The two proteins were affected in the same way by changes in the conditions used, but when applying a procedure of reheating and recooling the gel networks responded differently. Pea legumin gel networks were susceptible to rearrangements that caused the gels to become stronger after reheating/recooling, yet glycinin gel networks were not. It was concluded that the same physical and chemical forces drove the processes of denaturation, aggregation, and network formation. Each process can therefore be readily targeted for modification based upon molecular reasoning. Pea legumin and soybean glycinin gel networks had structurally different building blocks, however. A model of gelation aimed at texture control therefore requires additional information.     

Polymerization and gelation of whey protein isolates at low pH using transglutaminase enzyme

Dynamic and steady shear rheology is used to examine the synthesis of low-pH (approximately 4) whey protein gels obtained through a two-step process. The first step involves cross-linking of whey proteins at pH 8 and 50 degrees C using transglutaminase enzyme, while the second step entails cold-set acidification of the resulting solution using glucono-delta-lactone (GDL) acid. During the first step, the sample undergoes enzyme-catalyzed epsilon-(gamma-glutamyl)lysine bond formation with a substantial increase in viscosity. Acidification in the second step using GDL acid leads to a rapid decrease in pH with a concomitant increase in the elastic (G') and viscous (G' ') moduli and formation of a gelled network. We examine the large strain behavior of the gel samples using a relatively new approach that entails plotting the product of elastic modulus and strain (G'gamma) as a function of increasing dynamic strain and looking for a maximum, which corresponds to the yield or fracture point. We find the enzyme-catalyzed gels to have significantly higher yield/fracture stress and strain compared to cold-set gels prepared without enzyme or conventional heat-set gels. In addition, the elastic modulus of the enzyme-catalyzed gel is also higher than its non-enzyme-treated counterpart. These results are discussed in terms of the gel microstructure and the role played by the enzyme-induced cross-links.     

Physicochemical variables affecting the rheology and microstructure of rennet casein gels

The rheology and microstructure of a rennet casein system were studied in the pH range from 5.8 to 12.0 during cooling from 80 to 5 degrees C at four cooling rates: 0.5, 0.1, 0.05, and 0.025 degrees C/min. A dramatic increase in storage modulus with pH was observed during cooling at a fixed cooling rate. Continuous networks were formed for gels at pH 7.2 and above, while a discontinuous network was observed for gels below pH 6.5. The monotonic increase in storage modulus with pH could be correlated to the number of net (negative) charges and the strength of the hydrophobic interactions. At a higher pH, the protein micelles were larger due to weaker hydrophobic interactions and stronger repulsive electrostatic interactions resulting from more charges. When these protein micelles aggregated into flocs during cooling, the flocs had similar sizes at different pH values but a smaller fractal dimension at a higher pH. Consequently, for systems of the same protein and salt concentrations, more flocs were present in the gels at a higher pH, which subsequently generated more cross-links and a higher storage modulus. The pH also determined how the cooling rate affected the gel properties. At pH 5.8 and 6.5, the gels were firmest at the fastest cooling schedule, and the cooling rate did not show a trend in affecting the gel strength at the other three rates. On the other hand, a slower cooling rate generated a firmer gel at pH 7.2 and 12.0. The analysis of casein interactions suggests that the cooling rate affected the casein floc size only when repulsive interactions enabled a slow flocculation (at higher pH values) comparable with temperature change rates during cooling. For rennet casein gels of pH within the range of processed cheese products (pH 5.8 and 6.5), particle or cluster rearrangements created more uniform networks for gels cooled at slower schedules and weakened the structure.     

alpha-Casein improves the gel properties of dried egg white

The effects of addition of alpha-casein (alpha-CN) to dried egg white (DEW) were investigated by measuring transparency, hardness, and water-holding capacity (WHC) of the heat-induced gels. A DEW concentration of 8% (w/w) was required for formation of a self-supporting gel following heating at 80 degrees C for 20 min at pH 7. Solutions of alpha-CN, even up to a protein concentration of 12% (w/w), did not gel under the same conditions. The addition of alpha-CN (0.5-4%) to 8% DEW caused the increase in gel hardness gels, as compared with DEW gels alone at a total amount of protein concentrations, and the mixed gels became transparent with the increase of added alpha-CN concentrations. The 10% mixed protein solutions of alpha-CN (3-6%) and DEW (4-7%) formed transparent gels, although each protein did not gel individually at their protein concentrations. Mixture with 2:8 mixing ratio of alpha-CN to DEW at a total protein concentration of 10% showed synergistic effects in improving DEW gel properties above pH 7 and below 25 mM NaCl. The improvements (hardness, transparency, and WHC) of DEW gel by alpha-CN seem to be caused mainly by the inhibition of alpha-CN against heat coagulation of DEW protein.     

不同热处理大豆分离蛋白凝胶冻藏特性

为探究冻藏过程中不同加热温度处理大豆分离蛋白(soybean isolate protein,SPI)凝胶特性变化及评估不同热处理对SPI凝胶冻藏特性的影响。该文以65、90和135℃3个不同温度处理所得SPI为研究对象(分别记为65SPI、90SPI和USPI),采用离心法、质构分析法、可溶蛋白含量测定和电泳等方法对其冻藏过程中的凝胶持水性、凝胶硬度、凝胶弹性、可溶蛋白含量及亚基组成和凝胶作用力进行了分析研究。结果表明:随冻藏时间延长,不同温度处理SPI凝胶持水性、凝胶弹性和凝胶可溶蛋白含量呈下降趋势,而凝胶硬度呈增大趋势。凝胶持水性、弹性的下降和凝胶硬度的升高标志着凝胶品质的劣变。不同温度处理对SPI凝胶的冻前凝胶特性和冻藏特性有较大影响,65和90℃的温度处理降低了冻前SPI凝胶的持水性,增强了冻前SPI凝胶硬度,有更多的β和B亚基参与了凝胶形成,冻藏前后的亚基组成没有变化;超高温瞬时加热(ultra high temperature,UHT)处理则降低了冻前SPI凝胶硬度,冻藏过程中可溶蛋白含量大幅下降且可溶蛋白中β和B亚基含量下降。3种温度处理SPI的凝胶劣变程度均高于未处理SPI。加热处理会造成SPI发生部分或完全变性,变性后疏水基团的暴露会加快蛋白凝胶形成过程中聚集速率,进而增大粗糙凝胶结构形成的几率,而粗糙凝胶网络在冻藏过程中其劣变程度更甚于未加热SPI。由此可知,加热处理尽管在一定程度上增大了凝胶硬度,但会加速其凝胶品质冻藏劣变。     

燕麦麸分离蛋白的酶解对其功能性质的影响

为了改善燕麦蛋白的功能性质以扩大其在食品工业中的应用,该文以燕麦麸为原料制备了燕麦麸分离蛋白(OBPI),并利用胰蛋白酶对其进行水解,得到了3种不同水解度(4.1%、6.4%、8.3%)的酶解产物。SDS-PAGE分析结果表明OBPI中的主要蛋白成分是球蛋白,其经过胰蛋白酶处理后,球蛋白酸性亚基被部分水解而碱性亚基相对保持完整。胰蛋白酶水解显著改变了OBPI的功能性质。在所考察的水解度范围内,随着水解度的升高,酶解产物的溶解性、持水性、乳化活性及起泡能力等方面均逐渐增加;但持油性、乳化及泡沫稳定性有不同程度的降低。     

Gamma-irradiation influence on the structure and properties of calcium caseinate-whey protein isolate based films. Part 1. Radiation effect on the structure of proteins gels and films

Brookfield viscosimetry, Fourier transform infrared spectroscopy, transmission electron microscopy (TEM), and measurements of the texture strength of gels formed with CaCl2 and the mechanical and barrier properties of the film were applied in studies of gel formation and structural and mechanical properties of gels and films prepared using calcium caseinate (CC)-whey protein isolate (WPI)-glycerol (1:1:1), control, and irradiated with 60Co gamma rays using a 32 kGy dose. The irradiated gels have appeared to be more "fine-stranded" as compared to the more "particulate" control gels and lead to the formation of more rigid films with improved mechanical strength and barrier properties. This results from cross-linking and the modification of protein conformations were induced by irradiation, in particular the increase in the beta-sheet and beta-strand contents. Structural modifications taking place in CC-WPI composition are related to modifications taking place separately in CC and WPI. Improvement of the properties of the films after irradiation corresponds to the increased density of the cross-linked material because no change in the porosity of the films was observed by TEM.     

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.     

Gelling properties of heat-denatured beta-lactoglobulin aggregates in a high-salt buffer

Thermal denaturation, rheological, and microstructural properties of gels prepared from native beta-lactoglobulin (beta-LG) and preheated or heat-denatured beta-LG (HDLG) aggregates were compared. The HDLG was prepared by heating solutions of 4% beta-LG in deionized water, pH 7.0, at 80 degrees C for 30 min and then diluted to the desired concentration in 0.6 M NaCl and 0.05 M phosphate buffer at pH 6.0, 6.5, and 7.0. When reheated to 71 degrees C, HDLG formed a gel at a concentration of 2% protein. At pH 7.0, 3% HDLG gelled at 52.5 degrees C and had a storage modulus (G') of 2200 Pa after cooling. beta-LG (3%) in 0.6 M NaCl and 0.05 M phosphate buffer, pH 7.0, did not gel when heated to 71 degrees C. The gel point of 3% HDLG decreased by 10.5 degrees C and the G' did not change when the pH was decreased to 6.0. The HDLG gel microstructure was composed of strands and clumps of small globular aggregates in contrast to beta-LG gels, which contained a particulate network of compacted globules. The HDLG formed a gel at a lower concentration and lower temperature than beta-LG in the high-salt buffer, suggesting an application in meat systems or other food products prepared with salt and processed at temperatures of < or =71 degrees C.     

Effects of sugars on whey protein isolate gelation

Whey protein isolate (WPI) gels were prepared from solutions containing ribose or lactose at pH values ranging from 6 to 9. The gels with added lactose had no color development, whereas the gels with added ribose were orange/brown. Lactose stabilized the WPI to denaturation, which increased the time and temperature required for gelation, thus decreasing the fracture modulus of the gel compared to the gels with added ribose and the gels with no sugar added. Ribose, however, favored the Maillard reaction and covalent cross-linking of proteins, which increased gel fracture modulus. The decreased pH caused by the Maillard reaction in the gels containing ribose occurred after protein denaturation and gelation, thus having little if any effect on the gelation process.     

pH值和温度对燕麦蛋白溶解与聚集特性的影响

为研究燕麦蛋白的理化性质,本研究采用碱提酸沉法提取燕麦蛋白,并分析不同pH值与温度对燕麦蛋白溶解性、ζ-电位和粒径分布的影响。结果表明,在等电点附近(pH值5.0),燕麦蛋白的表面电荷数最低,蛋白粒度较大,溶解性和乳化性较低。当pH值远离等电点时,燕麦蛋白的表面电荷增加,蛋白粒度逐渐减小,溶解度升高,乳化性也随之升高;随着温度升高(55~95℃),燕麦蛋白聚集体解离,粒度变小,溶解度增加,稳定性变好;温度进一步升高(121、130℃),燕麦蛋白重新聚集形成粒径较大的聚集体,溶解度降低,稳定性变差。本研究结果为燕麦蛋白资源的利用提供了重要的理论指导。     

Apoplastic pH and monolignol addition rate effects on lignin formation and cell wall degradability in maize

Monolignol polymerization rate and apoplastic pH and may influence the formation of lignin and its interactions in cell walls. Primary maize walls were artificially lignified by gradual "end-wise" or rapid "bulk" polymerization of coniferyl alcohol at pH 4 or 5.5. Lignification efficiency was greatest for end-wise polymers at pH 5.5 (90-98%), intermediate for bulk polymers formed at either pH (54-82%), and lowest for end-wise polymers at pH 4 (41-53%). End-wise polymers had about 2.2-fold more ether inter-unit linkages and 70% fewer end-groups than bulk polymers. Low pH enhanced the formation of ether linkages in end-wise but not in bulk polymers. Differences in lignin structure did not influence the enzymatic degradability of cell walls, but lowering apoplastic pH from 5.5 to 4.0 during lignification reduced cell wall degradability by 25%. Further studies indicated this pH-dependent depression in degradability was related to cell wall cross-links formed via lignin quinone methide intermediates.     

Conformational study of globulin from common buckwheat (Fagopyrum esculentum Moench) by Fourier transform infrared spectroscopy and differential scanning calorimetry

Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) were used to study changes in the conformation of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) under various environmental conditions. The IR spectrum of the native BWG showed several major bands from 1691 to 1636 cm(-1) in the amide I' region, and the secondary structure composition was estimated as 34.5% beta-sheets, 20.0% beta-turns, 16.0% alpha-helices, and 14.4% random coils. Highly acidic and alkaline pH conditions induced decreases in beta-sheet and alpha-helical contents, as well as in denaturation temperature (Td) and enthalpy of denaturation (DeltaH), as shown in the DSC thermograms. Addition of chaotropic salts (1.0 M) caused progressive decreases in ordered structures and thermal stability following the lyotropic series of anions. The presence of several protein structure perturbants also led to changes in IR band intensities and DSC thermal stabilities, suggesting protein unfolding. Intermolecular antiparallel beta-sheet (1620 and 1681 cm(-1)) band intensities started to increase when BWG was heated to 90 degrees C, suggesting the initiation of protein aggregation. Increasing the time of the preheat treatment (at 100 degrees C) caused progressive increases in Td and pronounced decreases in DeltaH, suggesting partial denaturation and reassociation of protein molecules.     

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.