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.     

Gelation of edible blue-green algae protein isolate (Spirulina platensis Strain Pacifica): thermal transitions, rheological properties, and molecular forces involved

Proteins isolated from blue-green algae Spirulina platensis strain Pacifica were characterized by visible absorption, differential scanning calorimetry (DSC), viscometry, and dynamic oscillatory rheological measurements. Unique thermal unfolding, denaturation, aggregation, and gelation of the algal protein isolate are presented. DSC analysis showed that thermal transitions occur at about 67 and 109 degrees C at neutral pH. Calcium chloride stabilized the quaternary structure against denaturation and shifted the transitions at higher temperatures. Viscometric studies of Spirulina protein isolate as a function of temperature showed that the onset of the viscosity increase is closely related to the dissociation-denaturation process. Lower viscosities were observed for the protein solutions dissolved at pH 9 due to an increased protein solubility. Solutions of Spirulina protein isolate form elastic gels during heating to 90 degrees C. Subsequent cooling at ambient temperatures caused a further pronounced increase in the elastic moduli and network elasticity. Spirulina protein isolate has good gelling properties with fairly low minimum critical gelling concentrations of about 1.5 and 2.5 wt % in 0.1 M Tris buffer, pH 7, and with 0.02 M CaCl(2) in the same buffer, respectively. It is suggested that mainly the interactions of exposed hydrophobic regions generate the molecular association, initial aggregation, and gelation of the protein isolate during the thermal treatment. Hydrogen bonds reinforce the network rigidity of the protein on cooling and further stabilize the structure of Spirulina protein gels but alone are not sufficient to form a network structure. Intermolecular sulfhydryl and disulfide bonds were found to play a minor role for the network strength of Spirulina protein gels but affect the elasticity of the structures formed. Both time and temperature at isothermal heat-induced gelation within 40-80 degrees C affect substantially the network formation and the development of elastic modulus of Spirulina protein gels. This is also attributed to the strong temperature dependence of hydrophobic interactions. The aggregation, denaturation, and gelation properties of Spirulina algal protein isolate are likely to be controlled from protein-protein complexes rather than individual protein molecules.     

Effect of Transglutaminase,Citrate Buffer,and Temperature on a Soft Wheat Flour Dough System

Transglutaminase (TGase) can improve the functional characteristics of proteins by introducing covalent bonds inter‐ or intrachains. Temperature and pH interfere with the protein structure and the catalytic activity of enzymes. Because these three factors can act synergistically, TGase, citrate buffer, and temperature were evaluated for their effects on the rheological and chemical changes in low‐protein wheat flour dough. Dough strength, measured by microextension test, significantly increased with increasing levels of TGase (8 U/g of protein), with changes in pH of the citrate buffer (pH 6.5), and by the effect of interaction between these factors. The same trend was observed in the size‐exclusion HPLC measurements, indicating that these two parameters have the effect of increasing gluten protein aggregation. Temperature had a significant effect on dough extension, measured by microextension test. The changes in secondary structure of gluten protein were investigated by FTIR second‐derivative spectra (amide I region, 1,600–1,700 cm⁻¹) and showed an increase in β‐sheet structures initiated by TGase, citrate buffer pH, and their interaction.     

Raman spectroscopy determines structural changes associated with gelation properties of fish proteins recovered at alkaline pH

Structural changes of alkali-treated rockfish protein isolate (AKPI) during frozen storage were elucidated using a Raman spectrometer and scanning electron microscope (SEM). The results were compared to conventional surimi (CS). No significant textural difference was noted between AKPI stored at pH 5.5 and 7.0. The strongest texture was found for AKPI frozen with cryoprotectants and CS, while the weakest texture was observed in AKPI frozen without cryoprotectants. SEM revealed the most discontinuity in gels of AKPI with no cryoprotectants and a more aggregated microstructure after storage at pH 5.5 than at neutral pH. Raman spectral analysis demonstrated refolding of AKPI by pH readjustment to 7.0, although the refolded structure was not identical to that before the pH shift. CS showed higher alpha-helix content (approximately 50%) than AKPI (approximately 20-30%). Frozen storage induced a decrease and an increase in the alpha-helix content of CS and AKPI samples, respectively. AKPIs were slightly less stable than CS during frozen storage.     

Effect of Specific Mechanical Energy on Protein Bodies and α-Zeins in Corn Flour Extrudates

Zeins, the storage proteins of corn, are located in spherical entities called protein bodies. The disruption of protein bodies and zein release during extrusion may influence the texture of corn-based extruded foods. In this work, chemical and microscopic studies were conducted on corn flour that had been extruded under mild to extreme conditions to determine the specific mechanical energy (SME) required to break apart protein bodies and release α-zein, and to assess changes in protein-protein interactions. Transmission electron microscopy with immunolocalization of α-zein revealed that starch granules and protein bodies remained intact under mild processing conditions (SME 35–40 kJ/kg), but under harsher conditions, protein bodies were disrupted and α-zein was released. At SME ≈100 kJ/kg, protein bodies appeared highly deformed and fused together with the α-zein released, whereas at higher SME, protein bodies were completely disrupted and α-zein was dispersed and may have formed protein fibrils. Protein in extrudates was less soluble in urea and SDS than in unprocessed corn flour, but it was readily extracted with urea, SDS, and 2-ME. This was likely due to protein aggregation upon processing due to a prevalence of hydrophobic interactions and disulfide bonds. This research directly relates SME during extrusion to chemical and structural changes in corn proteins that may affect the texture of corn-based, ready-to-eat food products.     

0℃贮藏下南美白对虾品质变化研究

为分析冰藏冷链运输条件下南美白对虾品质和微观结构的变化,本研究测定了0℃贮藏条件下南美白对虾的色差、质构、挥发性盐基氮(TVB-N)及其组分含量和蛋白质图谱的变化情况,并对其肌肉的微观结构进行观察。结果表明,随着0℃贮藏时间的延长,南美白对虾L^*值、质构以及肌原纤维蛋白和肌基质蛋白含量呈下降趋势。0℃贮藏条件下,TVB-N值与贮藏时间呈正相关;贮藏4 d后南美白对虾色泽、质构和肌肉微观结构开始出现明显变化,肌原纤维蛋白和肌基质蛋白含量明显下降,而碱溶性蛋白含量呈增加趋势;贮藏6 d后蛋白条带降解明显。综合考虑,建议0℃贮藏条件下南美白对虾的货架期为4 d。本研究结果为冷藏条件下南美白对虾提供了品质评价的方法。     

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.     

Rheological and proteolytic properties of Monterey Jack goat's milk cheese during aging

To enhance the understanding of the quality traits of goat's milk cheeses, rheological and proteolytic properties of Monterey Jack goat's milk cheese were evaluated during 26 weeks of 4 degrees C storage. As expected with aging, beta-casein levels decreased with concomitant increases in peptide levels and were correlated with changes in rheological properties of the cheese. Hydrolysis of the protein matrix resulted in more flexible (increased viscoelastic properties) and softer (decreased hardness, shear stress, and shear rigidity) cheeses. During the first 4-8 weeks of storage, cheese texture changed significantly (P < 0.05) and then stabilized. Characterization of rheological and proteolytic properties of the goat's milk semihard cheese during aging provided insight into the changes occurring in the protein matrix, the relationship to structure, and a shift in cheese quality.     

Elucidation of Fermentation Effect on Rice Noodles Using Combined Dynamic Viscoelasticity and Thermal Analyses

Natural fermentation of whole polished rice grains (indica) is a traditional processing method widely applied in China and South Asia to improve the texture of rice noodles. To elucidate the effects of fermentation on noodle texture, thermal analysis of rice flour and rheological measurement were performed on a gel made from the rice flour with the same solids concentration as practical noodles. The test method proved useful for monitoring starch granule swelling, gel forming, and retrogradation of rice gels during rice noodle production. Dynamic viscoelasticity in a temperature ramp sweep test showed that starch granules of the fermented sample swelled more and were more resistant to breakdown than those of the nonfermented sample. Differential scanning calorimetry revealed that the gelatinization temperature of fermented rice flour shifted to a lower temperature. The decreased protein content after fermentation also played a role in the modified rheological and thermal properties of fermented rice flour. Both fermented and nonfermented rice flour formed an elastic gel just after heating to the conclusion temperature of gelatinization (T_c). The fermented rice flour gel formed earlier with a well‐formed structure; however, it had slower retrogradation during aging. The result also indicated that the retrogradation of amylopectin impaired the desired texture of rice noodles.     

Calcium effects on the functionality of a modified whey protein ingredient

The primary objective for this study addressed the effects of supplemental calcium on the functional properties of a modified whey protein ingredient (mWPC), prepared by acidification to pH 3.35, followed by extended heat treatment, gelation, and spray drying. In the presence of added calcium (mWPC-Ca2+), protein solutions showed increased thickening capacity, especially under refrigeration temperatures, compared to dispersions made with mWPC alone. A rheological assessment included the determination of (i) power law parameters, (ii) viscoelastic properties, and (iii) the effects of heating and cooling on these protein systems. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) banding profile suggested that various disulfide-linked molecular forms of beta-lactoglobulin, bovine serum albumin, and immunoglobulin were likely formed during manufacturing of the mWPC ingredient based on the patterns obtained when electrophoresis was performed in the absence of beta-mercaptoethanol compared to those observed with commercial WPC samples. An enhanced water-holding capacity was measured in mWPC-Ca2+ dispersions. Differential scanning calorimetry established that the addition of calcium salts caused a 2-fold increase in the amount of bound or unfreezeable water compared to mWPC controls. The physical appearance of the network structure varied significantly upon visualization with scanning electron microscopy, in which case the formation of large, rounded, spherical structures was noted in mWPC-Ca2+ samples, ascribed to an increased surface tension caused by the higher salt content. Ultimately, such attributes may afford distinct advantages for whey-based ingredients intended for application within food systems, especially under cold processing conditions.     

Molecular structure, physicochemical characterization, and in vitro degradation of barley protein films

Barley protein films were prepared by thermopressing using glycerol as a plasticizer. The combined effects of heating temperature and amount of plasticizer interacted to determine protein conformation and, subsequently, the properties of the film matrix. The film barrier and mechanical properties were systematically investigated using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), SDS-PAGE, and protein solubility tests. These experiments demonstrated that heat treatment induced barley protein unfolding and then protein aggregation and the formation of covalent disulfide bonds to enhance film strength. Increasing the amount of plasticizer reduced protein denaturation and limited protein interactions, resulting in significantly improved film flexibility at the cost of reduced film moisture barrier property and tensile strength. In vitro degradation experiments demonstrated that barley films were resistant in gastric conditions, yet can still be completely degraded by intestinal enzymes, and they possess low cytotoxicity to Caco-2 cells. The prepared barley films have potential for development as delivery systems for gastric-sensitive bioactive compounds to the intestine for release.     

自由基引起的氧化对牛乳清蛋白凝胶特性的影响

为了深入了解蛋白氧化对凝胶特性的影响,以此探讨乳清蛋白氧化对其功能性质的影响机制,该文主要研究了氧化对乳清蛋白凝胶质地、流变学特性和微观结构变化的影响。试验采用羟基自由基氧化体系,在不同H2O2浓度(1～20mmol/L)及不同FeCl3浓度(0.1～1mmol/L)对乳清蛋白分别氧化3h,通过质构仪、流变仪和扫描电镜对凝胶特性和微观结构进行研究。结果显示:同未氧化乳清蛋白相比,在所有氧化条件下,凝胶硬度降低了90%以上,贮藏模量(G')值降低了17%以上,复合模量(G*)值降低了20%以上;高浓度氧化条件下,弹性降低了20%以上。氧化明显改变了凝胶的微观结构,随着氧化剂的加入,导致了疏松多孔且不规则凝胶的形成。上述结果表明,氧化对蛋白凝胶质地和凝胶形成能力起着很大的破坏作用,并影响着其微观结构。     

Evaluation of Textural and Structural Properties of Barbari Bread During Storage

This study was performed using three Barbari flours (strong, medium, and weak) with different physical, chemical, and rheological properties. Determination of texture firmness of Barbari breads (A, B, and C, made of strong, medium, and weak flours, respectively) during storage was carried out with a texture analyzer and evaluating the bread crumb properties and changes during storage with a nondestructive ultrasonic technique. The bread microstructure was assessed with scanning electron microscopy, and the general process of starch gelatinization and retrogradation was evaluated with differential scanning calorimetry. The bread sensory properties were evaluated by 10 trained panelists. Barbari A, made from strong flour, had less firmness, lower transition of ultrasonic wave velocity, lower value of elastic modulus, reduced value of enthalpy, lower average temperature, larger pore diameter and area of images, and higher point total in sensory evaluation than Barbari B and Barbari C, particularly the latter, as storage time progressed. Barbari A's desirable quality characterization and longer shelf life were owing to the qualities of the flour, which enabled the production of dough with the appropriate properties. Eventually, the results of device‐based and sensory tests were significantly correlated. Ultrasonic nondestructive testing is recommended over other methods for assessing the texture, cell structure, and elastic properties of bread after baking and during storage, because it is fast, nondestructive, and less expensive than other methods and can be used during production.     

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.     

Heat-induced gelation of chicken Pectoralis major myosin and beta-lactoglobulin

The denaturation, aggregation, and rheological properties of chicken breast muscle myosin, beta-lactoglobulin (beta-LG), and mixed myosin/beta-LG solutions were studied in 0.6 M NaCl, 0.05 mM sodium phosphate buffer, pH 7.0, during heating. The endotherm of a mixture of myosin and beta-LG was identical to that expected if the endotherm of each protein was overlaid on the same axis. The maximum aggregation rate (AR(max)) increased, and the temperature at the AR(max) (T(max)) and initial aggregation temperature (T(o)) decreased as the concentration of both proteins was increased. The aggregation profile of <0.5% myosin was altered by the presence of 0.25% beta-LG. Addition of 0.5-3.0% beta-LG decreased storage moduli of 1% myosin between 55 and 75 degrees C, but increased storage moduli (G') when heated to 90 degrees C and after cooling. beta-LG had no effect on the gel point of > or =1.0% myosin, but enhanced gel strength when heated to 90 degrees C and after cooling. After cooling, the G' of 1% myosin/2%beta-LG gels was about 1.7 times greater than that of gels prepared from 2% myosin/1% beta-LG.     

Effects of high-pressure processing at low temperature on the molecular structure and surface properties of beta-lactoglobulin

High-pressure processing (HPP) was utilized to induce unfolding of beta-lactoglobulin (beta-LG). beta-Lactoglobulin solutions at concentrations of 0.5 mg/mL, in pH 7.5 phosphate buffer, were pressure treated at 510 MPa for 10 min at either 8 or 24 degrees C. The secondary structure, as determined by circular dichroism (CD), of beta-LG processed at 8 degrees C appeared to be unchanged, whereas beta-LG processed at 24 degrees C lost alpha-helix structure. Tertiary structures for beta-LG, as determined by near-UV CD, intrinsic protein fluorescence spectroscopy, hydrophobic fluorescent probe binding, and thiol group reactivity, were changed following processing at either temperature. The largest changes to tertiary structure were observed for the samples processed at 24 degrees C. Model solutions containing the pressure-treated beta-LG showed significant decreases in surface tension at liquid-air interfaces with values of 54.00 and 51.69 mN/m for the samples treated at 24 and 8 degrees C, respectively. In comparison, the surface tension for model solutions containing the untreated control was 60.60 mN/m. Changes in protein structure during frozen and freeze-dried storage were also monitored, and some renaturation was observed for both storage conditions. Significantly, the sample pressure-treated at 8 degrees C continued to display the lowest surface tension.     

Protein-protein interactions during high-moisture extrusion for fibrous meat analogues and comparison of protein solubility methods using different solvent systems

Soy protein, mixed with gluten and starch, was extruded into fibrous meat analogues under high-moisture and high-temperature conditions. The protein solubility of samples collected at different extruder zones and extrudates made with different moistures was determined by 11 extraction solutions consisting of 6 selective reagents and their combinations: phosphate salts, urea, DTT, thiourea, Triton X-100, and CHAPS. Protein solubility by most extractants showed decreasing patterns as the material passed through the extruder, but the solution containing all 6 reagents, known as isoelectric focus (IEF) buffer, solubilized the highest levels and equal amounts of proteins in all samples, indicating that there are no other covalent bonds involved besides disulfide bonds. With regard to relative importance between disulfide bonds and non-covalent interactions, different conclusions could be made from protein solubility patterns, depending on the type of extracting systems and a baseline used for comparison. The observation points out pitfalls and limitation of current protein solubility methodology and explains why controversy exists in the literature. Using the IEF buffer system with omission of one or more selective reagents is considered to be the right methodology to conduct protein solubility study and thus recommended. Results obtained with this system indicate that disulfide bonding plays a more important role than non-covalent bonds in not only holding the rigid structure of extrudates but also forming fibrous texture. The sharpest decrease in protein solubility occurred when the mix passed through the intermediate section of the extruder barrel, indicating formation of new disulfide bonds during the stage of dramatic increase in both temperature and moisture. After this stage, although the physical form of the product might undergo change and fiber formation might occur as it passed through the cooling die, the chemical nature of the product did not change significantly.     

Effects of protein and peptide addition on lipid oxidation in powder model system

The effect of protein and peptide addition on the oxidation of eicosapentaenoic acid ethyl ester (EPE) encapsulated by maltodextrin (MD) was investigated. The encapsulated lipid (powder lipid) was prepared in two steps, i.e., mixing of EPE with MD solutions (+/- protein and peptides) to produce emulsions and freeze-drying of the resultant emulsions. EPE oxidation in MD powder progressed more rapidly in the humid state [relative humidity (RH) = 70%] than in the dry state (RH = 10%). The addition of soy protein, soy peptide, and gelatin peptides improved the oxidation stability of EPE encapsulated by MD, and the inhibition of lipid oxidation by the protein and the peptides was more dramatic in the humid state. Especially, the oxidation of EPE was almost perfectly suppressed when the lipid was encapsulated with MD + soy peptide during storage in the humid state for 7 days. Several physical properties such as the lipid particle size of the emulsions, the fraction of nonencapsulated lipids, scanning electron microscopy images of powder lipids, and the mobility of the MD matrix were investigated to find the modification of encapsulation behavior by the addition of the protein and peptides, but no significant change was observed. On the other hand, the protein and peptides exhibited a strong radical scavenging activity in the powder systems as well as in the solution systems. These results suggest that a chemical mechanism such as radical scavenging ability plays an important role in the suppression of EPE oxidation in MD powder by soy proteins, soy peptides, and gelatin peptides.     

Quantitation of 3-aminopropionamide in potatoes-a minor but potent precursor in acrylamide formation

3-Aminopropionamide (3-APA) has recently been suggested as a transient intermediate in acrylamide (AA) formation during thermal degradation of asparagine initiated by reducing carbohydrates or aldehydes, respectively. 3-APA may also be formed in foods by an enzymatic decarboxylation of asparagine. Using a newly developed method to quantify 3-APA based on liquid chromatography/tandem mass spectrometry, it could be shown that the biogenic amine was present in several potato cultivars in different amounts. Further experiments indicated that 3-APA is formed during storage of intact potatoes (20 or 35 degrees C) or after crushing of the cells. The heating of 3-APA under aqueous or low water conditions at temperatures between 100 and 180 degrees C in model systems always generated more AA than in the same reaction of asparagine, thereby pointing to 3-APA as a very effective precursor of AA. While the highest yields measured were about 28 mol % in the presence of carbohydrates (170 degrees C; aqueous buffer), in the absence of carbohydrates, 3-APA was even converted by about 63 mol % into AA upon heating at 170 degrees C under aqueous conditions. Propanoic acid amides bearing an amino or hydroxy group in the alpha-position, such as 2-hydroxypropionamide and l-alaninamide, were ineffective in AA generation indicating that elimination occurs only from the beta-position.     

In vitro protein degradation of 38 sainfoin accessions and its relationship to tannin content by different assays

This study compared 38 sainfoin and 2 Lotus accessions to their respective tannin contents, N buffer solubility, and in vitro protein degradation. Tannin contents were measured by a protein precipitation method using either bovine serum albumin or Rubisco and by the colorimetric HCl/butanol method. Precipitation of bovine serum albumin and Rubisco was highly correlated (R(2) = 0.939). Correlations between the protein precipitation variants and the HCl/butanol method were relatively low (R(2) < 0.6). Protein degradation was measured at 4 h of incubation in an inhibited in vitro system and could not be explained by any of the tannin assays (R(2) < 0.03) and only partially by N buffer solubility (R(2) ≤ 0.433). Decisive factors other than the quantity of tannins or their ability to precipitate proteins must be considered. Resistance of soluble protein toward degradation can possibly be caused by tannin protein binding.