Effect of extrusion temperature on the solubility and molecular weight of lentil bean flour proteins containing low cysteine residues

Lentil flour was extruded at die temperatures of 135, 160, and 175 degrees C. The soluble protein content in the extrudates decreased by 40.1% in the extracting buffer (1% sodium dodecyl sulfate in 50 mM sodium phosphate buffer, pH 6.9) as the extrusion die temperature was increased to 175 degrees C. The most insoluble proteins in the extrudates extruded at die temperatures of up to 175 degrees C could be resolubilized by using sonication. The total disulfide content and sulfhydryl content in the extrudates decreased. The SDS-PAGEs showed that the molecular weight distribution of proteins in the lentil flour changed little before and after extrusion as well as during reduction. The results from this study show that the extrusion temperature had less effect on the solubility and molecular weight of the lentil proteins, which contain a lower level of cysteine residues than wheat proteins.     

碱性蛋白酶Alcalase凝固大豆分离蛋白的分子间作用力

为了进一步揭示蛋白酶凝固豆乳的机理，该文通过添加不同化学试剂研究碱性蛋白酶Alcalase凝固大豆分离蛋白(SPI)过程中的分子间作用力。结果发现凝固过程中的分子间作用力主要是氢键和疏水作用，而离子键和二硫键对凝固过程影响不大。大豆蛋白质分子间的交联主要由次级键起作用，同时需要克服由负电荷引起的静电斥力，这就解释了为什么与无机盐和酸相比，Alcalase得到的SPI凝固物强度低。根据以上结论，该文还对豆乳凝固酶当前的筛选策略进行了评价。     

Mechanisms of heat-mediated aggregation of wheat gluten protein upon pasta processing

During pasta processing, structural changes of protein occur, due to changes in water content, mechanical energy input, and high temperature treatments. The present paper investigates the impact of successive and intense thermal treatments (high temperature drying, cooking, and overcooking) on aggregation of gluten protein in pasta. Protein aggregation was evaluated by the measurement of sensitivity of disulfide bonds toward reduction with dithioerythritol (DTE), at different reactions times. In addition to the loss in protein extractability in sodium dodecyl sulfate buffer, heat treatments induced a drastic change in disulfide bonds sensitivity toward DTE reduction and in size-exclusion high-performance liquid chromatography profiles of fully reduced protein. The protein solubility loss was assumed to derive from the increasing connectivity of protein upon heat treatments. The increasing degree of protein upon aggregation would be due to the formation of additional interchain disulfide bonds.     

Heat-induced redistribution of disulfide bonds in milk proteins. 1. Bovine beta-lactoglobulin

Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.     

Phosphorylation Does Not Improve the Solubility of Rice Protein

The present study tried to phosphorylate rice protein (RP), a known insoluble food ingredient, and determine the improvement of its solubility. RP was allowed to react with sodium trimetaphosphate (STMP) at pH 11.5 and 35°C, and the results indicated that 20.6% of the RP seryl residues were phosphorylated. Interestingly, the solubility of phosphorylated RP (2.6%) was not improved compared with that of RP (2.5%) at pH 7. The involvement of hydrophobic interactions and disulfide bonds in phosphorylated RP solubility was further evaluated. The phosphorylation of RP in the presence of urea as a chaotropic agent for weakening the hydrophobic effect resulted in 22.0% phosphoseryl residues but still did not increase RP solubility. The reduction of RP disulfide bonds prior to phosphorylation resulted in 31.3% phosphoseryl residues and increased RP solubility to 8.3% at pH 7, indicating that disulfide bonds within RP could be responsible for the failure to increase its solubility after phosphorylation.     

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.     

Controlled Stepwise Reduction of Disulfide Bonds and Heat-Induced Modification of Wheat Dough Proteins

A reducing solution of 2-mercaptoethanol and its oxidized form 2-hydroxyethyl disulfide, whose variable concentrations set variable disulfide reduction potentials, was applied to progressively reduce the disulfide bonds of proteins extracted from doughs made from Meneba and Robin Hood flour. Several dough proteins had disulfide bonds stronger than those of other dough proteins. A SDS-sedimentation method was applied to monitor the baking of dough into bread. Dough proteins susceptible to heat (baking) were studied by SDS-fractionation, extraction with reducing alcoholic solution, SDS-PAGE, and N-terminal protein sequencing. High or low molecular weight glutenins, α, β, and γ-gliadins, α-amylase inhibitor, and α-amylase trypsin inhibitor were identified among the dough proteins modified by heat (as shown by reduced solubility in aqueous-SDS solution). The heat-induced modification of the gliadins and glutenins might contribute to the coagulation of dough proteins, while the heat-induced modification of the amylase or trypsin inhibitors might contribute to the regulation of endogenous or exogenous amylolytic or proteolytic activities in dough or bread.     

Effect of Single‐Screw Extruder Die Temperature,Amount of Distillers' Dried Grains With Solubles (DDGS), and Initial Moisture Content on Extrudates

Corn distillers' dried grains with solubles (DDGS) was extruded with corn meal in a pilot plant single‐screw extruder at different extruder die temperatures (100, 120, and 150°C), levels of DDGS (0, 10, 20, and 30%) and initial moisture contents (11, 15, and 20% wb). In general, there was a decrease in water absorption index (WAI), water solubility index (WSI), radial expansion, and L* value with an increase in DDGS level, whereas a* value and bulk density increased. Increase in extruder die temperature resulted in an increase in WSI and WAI but a decrease in L* and bulk density. Peak load was highest at 30% DDGS as compared with 0, 10, and 20% DDGS extrudates. Die temperature of 120°C and initial moisture content of 20% resulted in least peak load. The a* value remained unaffected by changes in extruder die temperature. Radial expansion was highest at extruder die temperature of 120°C. Maximum WAI, WSI, radial expansion, and L* value were obtained at 15% initial moisture content. An increase in initial moisture content, in general, decreased L* value and bulk density but increased a* value of extrudates.     

Immunochemical and Electrophoretic Analysis of the Modification of Wheat Proteins in Extruded Flour Products

Antibodies specific for wheat proteins were used to identify protein fractions modified during extrusion of Hard Red Spring wheat flour (14% protein) under four different combinations of extrusion conditions (18 and 24% feed moisture and 145 and 175°C die temperature). Antibody binding was assessed on immunoblots of proteins extracted from flour and extrudates separated by SDS‐PAGE. Antibodies to high molecular weight glutenin subunits (HMW‐GS) and to B‐group low molecular weight glutenin subunits (LMW‐GS) recognized intact subunits from both flour and extrudates. Antibodies to C‐group LMW‐GS had diminished binding to extruded proteins. Glutenin‐specific antibodies also recognized protein in the extrudates migrating as a smear at molecular weights higher than intact subunits, indicating cross‐linked proteins. Antibodies recognized albumins or globulins in flour but not in extrudates, evidence that these fractions undergo significant modification during extrusion. Acid‐PAGE and antibody reaction of gliadins extracted in 1M urea and in 70% ethanol revealed total loss of cysteine‐containing α, β, γ‐gliadins but no obvious effects on sulfur‐poor ω‐gliadins, suggesting gliadin modification involves replacing intramolecular disulfides with intermolecular disulfide cross‐links. Identifying protein fractions modified during different extrusion conditions may provide new options for tailoring extrusion to achieve specific textural characteristics.     

Twin-Screw Extrusion Processing of Feed Blends Containing Distillers Dried Grains with Solubles (DDGS)

Extrusion trials were conducted with varying levels of distillers dried grains with solubles (DDGS) along with soy flour, corn flour, fish meal, vitamin mix, mineral mix, and net protein content adjusted to 28% using a Wenger TX-52 twin-screw extruder. The properties of extrudates were studied in experiments conducted using a full-factorial design with three levels of DDGS content, two levels of moisture content, and two levels of screw speed. Increasing the DDGS content from 20 to 60% resulted in a 36.7% decrease in the radial expansion, leading to a 159 and 61.4% increase in the unit density and bulk density of the extrudates, respectively. Increasing the DDGS content resulted in a significant increase in the water absorption index (WAI) but a significant decrease in the water solubility index (WSI) of the extrudates. Changing the screw speed and moisture content had no significant effect on the radial expansion ratio but resulted in a significant difference in the bulk density of the extrudates, which may be due to the occurrence of longitudinal expansion. Even though changing the moisture content and screw speed had no significant effect on the WSI of the extrudates, significant differences in the WAI of the extrudates were observed. The ingredient components in the blend and moisture content had an influence on the color changes of the extrudates, as did the biochemical changes occurring inside the barrel during processing. Overall, it was determined that DDGS could be included at a rate of up to 60% using twin-screw extrusion, and that viable pelleted floating feeds can be produced.     

Effects of Processing Parameters on Physical Properties of Corn Starch Extrudates Expanded Using Supercritical CO2 Injection

Corn starch was extruded with a corotating twin-screw extruder (24:1 L/D ratio, 31-mm screw diameter) and supercritical CO₂ was injected as a blowing agent. The effects of barrel temperature (80–90°C), screw speed (150–250 rpm), and water injection (30–54 g/min) on specific mechanical energy (SME) input for the process and the physical properties of extrudates, such as expansion ratio, water absorption (WA), water solubility (WS), breaking stress, and elastic modulus, were examined using a response surface methodology. Barrel temperature had the greatest effect on physical properties of extrudates but not on SME input, whereas screw speed and water injection had significant effects on SME input. Extrudates had a smooth surface, and air cells were uniform and closed, providing low WA and WS. Using superimposed contour plots, optimum barrel temperature, screw speed, and water injection rate, based on maximum expansion ratio and minimum SME input, were 94–96°C, 155–175 rpm, and 36–39 g/min, respectively.     

Extrusion of Wheat Gluten Plasticized with Glycerol: Influence of Process Conditions on Flow Behavior,Rheological Properties,and Molecular Size Distribution

Gluten-glycerol dough was extruded under a variety of processing conditions using a corotating self-wiping twin-screw extruder. Influence of feed rate, screw speed, and barrel temperature on processing parameters (die pressure, product temperature, residence time, specific energy) were examined. Use of flow modeling was successful for describing the evolution of the main flow parameters during processing. Rheological properties of extruded samples exhibited network-like behavior and were characterized and modeled by Cole-Cole distributions. Changes in molecular sizes of proteins during extrusion were measured by chromatography and appeared to be correlated to molecular size between network strands, as derived from the rheological properties of the materials obtained. Depending on operating conditions, extrudates presented very different surface aspects, ranging from very smooth-surfaced extrudates with high swell to completely broken extrudates. The results indicated that extrudate breakup was caused by increasing network density, and some gliadins may have acted as cross-linking agents. Increasing network density resulted in decreasing mobility of polymeric chains, and “protein melt” may no longer have been able to support the strain experienced during extrusion through the die. Increasing network density was reflected in increased plateau modulus and molecular size of protein aggregates. Increasing network structure appeared to be induced by the severity of the thermomechanical treatment, as indicated by specific mechanical energy input and maximum temperature reached.     

Characterization of polymeric proteins from vitreous and floury sorghum endosperm

Differences in protein content and composition between vitreous and floury endosperm were investigated using a number of different techniques. Differences in protein cross-linking between vitreous and floury endosperm were investigated using differential solubility, size exclusion chromatography (SEC), and analysis of sulfhydryl content and composition. Vitreous endosperm was found to have higher levels of total protein and kafirins, but floury endosperm had a higher proportion of gamma-kafirins than the vitreous. Floury endosperm was found to have higher levels of SDS-soluble proteins than SDS-insoluble proteins extracted using sonication than vitreous endosperm. Conversely, vitreous endosperm had a greater proportion of the insoluble proteins. SEC analysis of the polymeric proteins revealed that the insoluble proteins had more polymeric proteins than did the soluble proteins, indicating greater cross-linking and a larger Mw distribution. Vitreous endosperm was also found to have a greater percentage (i.e., a higher ratio of disulfide to total sulfhydryls) of disulfide bonds than floury endosperm. These results show that the proteins in vitreous endosperm have a higher degree of cross-linking and a greater Mw distribution than those found in floury endosperm.     

Physical and biochemical properties of maize hardness and extrudates of selected hybrids

Protein and starch determinants of maize kernel hardness and extruded products were characterized to better define the role of endosperm texture during extrusion. Maize physical properties were correlated with total proteins and zein subclasses (p < 0.01). The extrusion process significantly altered protein solubility and increased protein fragmentation as measured by RP-HPLC and size exclusion chromatography. Harder grits and extrudates demonstrated higher amylose content, lower degree of starch damage, and fragmentation at different screw speeds than softer grits and extrudates. Differences in extrudate expansion ratio, water absorption index, water solubility index, oil absorption capacity, and breaking stress between harder and softer hybrids were related to protein aggregation and fragmentation as well as starch damage and fragmentation.     

Effects of non-covalent interactions with 5-O-caffeoylquinic acid (chlorogenic acid) on the heat denaturation and solubility of globular proteins

The non-covalent interactions between the monomeric phenolic compound chlorogenic acid (5-CQA) and bovine serum albumin (BSA), lysozyme, and alpha-lactalbumin were characterized, and their effect on protein properties was examined. 5-CQA had a low affinity for all three proteins, and these interactions seemed to show a negative cooperativity. 5-CQA-BSA binding decreased with increasing temperature, whereas pH (pH 3.0 compared to pH 7.0) and ionic strength had no pronounced effect. At high 5-CQA/protein molar ratios, both the denaturation enthalpy and temperature of BSA increased; however, covalent bonds were created at high temperatures. The presence of 5-CQA had no effect on the solubility of BSA and alpha-lactalbumin as a function of pH, whereas it decreased lysozyme solubility at alkaline pH due to covalent interactions. These results indicate that the non-covalent interactions with 5-CQA do not have pronounced effects on the functional properties of globular proteins in food systems.     

Effect of Bonding Forces on Corn Starch Isolation

Chemical treatments with a number of low‐toxicity or nontoxic reagents were applied to corn slurry to investigate the disruption or weakening of common bonding forces between corn starch and proteins, such as hydrogen bonds, disulfide bonds, electrostatic interactions, or combinations thereof, to improve the corn starch isolation process. Starch and proteins could be easily separated by disrupting disulfide bonds with 1% l ‐cysteine (w/v). The most effective reagents for hydrogen bonds and electrostatic interactions were 3M urea and pH 7.5 separately. The sequence treatment of hydrogen bonds, disulfide bonds, and electrostatic interactions (namely, sequence treatment of 1M urea, 1% l ‐cysteine, and pH 7.5) led to the highest amount of starch in corn slurry and facilitated the corn starch isolation.     

Changes in Disulfide and Sulfhydryl Contents and Electrophoretic Patterns of Extruded Wheat Flour Proteins

Twin‐screw extrusion of wheat flour and the effects on the flour proteins were studied using flour samples containing 9, 20, and 30% protein. Vital gluten containing 70% protein was used to achieve the flour protein levels. The three flour samples were extruded with a twin‐screw extruder at a combination of processing parameters (exit die temperatures of 120, 140, and 160°C, and screw speeds of 240, 320, and 400 rpm). Increasing extruder exit die temperatures resulted in increased sulfhydryl content of the 9 and 20% protein content flour samples, but appeared to have little or no effect on the 30% protein content flour sample. Similarly, disulfide content decreased, albeit disproportionately, following the same trend. Both sulfhydryl and disulfide contents of extruded samples were lower than those of the nonextruded samples and could imply denaturation of protein, aggregation through intermolecular disulfide bonds, or oxidation during extrusion processing. Total cysteine content of extruded samples decreased by ≈16% relative to nonextruded samples, but otherwise remained almost unchanged among all extruded samples. The loss of total cysteine in extruded samples could represent the generation of hydrogen sulfide, volatile organic compounds, or flavor compounds during extrusion. SDS‐PAGE analysis of total proteins showed a shift from the higher to lower molecular weight regions for certain protein bands. Both depolymerization and protein aggregation occurred at higher shear forces during extrusion.     

Partially carboxymethylated feather keratins. 1. Properties in aqueous systems

Feather keratins were extracted from chicken feathers with an aqueous solution of urea and 2-mercaptoethanol. The keratin solution obtained was dialyzed to remove the reagents. Upon dialysis, extensive protein aggregation occurred. To obtain stable solutions or dispersions in water, cysteine residues were modified prior to dialysis with iodoacetamide, iodoacetic acid, or bromosuccinic acid, thereby blocking free thiol groups and introducing hydrophilic groups. For the development of biodegradable materials with good mechanical properties from these biopolymers, disulfide bonds between the keratin molecules are needed. Therefore, cysteine residues were only partially modified by using different reagent/cysteine molar ratios. The reaction rate constants of iodoacetate with glutathione and 2-mercaptoethanol were successfully used to predict the degree of modification of keratin cysteine. It was shown that, for carboxymethylated keratin, fewer aggregates were formed for higher degrees of cysteine modification, while more protein was present as oligomers. Aggregates and oligomers were stabilized through intermolecular disulfide bonds.     

Waxy Soft White Wheat: Extrusion Characteristics and Thermal and Rheological Properties

Waxy wheat flour was analyzed for its thermal and rheological properties and was extruded to evaluate its potential for extruded products. Normal soft white wheat flour was analyzed with the same methods and same extrusion conditions to directly compare differences between the two types of flour. Through DSC analysis, waxy wheat flour was found to have a higher gelatinization peak temperature of 66.4°C than normal wheat at 64.0°C, although the transition required 2.00 J/g less energy. Rapid visco‐analysis indicated that the waxy wheat flour pasted much more quickly and at lower temperatures than the normal wheat flour. Preliminary extrusion experiments were conducted to determine the optimal screw profile for waxy wheat with respect to maximum radial expansion. The optimum screw profile was used for extrusion trials with varying flour moisture (15–25% wb) and extruder screw speed (200–400 rpm) while monitoring process conditions including back pressure and specific mechanical energy. Physical properties of the extrudates were then studied. The radial expansion ratios of the waxy wheat extrudates exceeded those of the normal wheat extrudates by nearly twice as much, and it was observed that the waxy wheat flour took less energy in the form of fewer shear screw elements to expand. The waxy wheat extrudates also exhibited significantly higher water solubility and less water absorption than the normal wheat extrudates owing to solubilizing of the extrudates. The results of our study indicate that waxy wheat flour may be a viable ingredient for creating direct expanded products with less energy.     

Effects of Steam,Moisture, and Screw Speed on Physical Properties of DDGS‐Based Extrudates

A fractional factorial design with a replicated central composite point was used to investigate the effects of extrusion processing on physical properties of distillers dried grains with solubles (DDGS) based aquafeeds using a twin‐screw extruder. Extrusion cooking trials were performed with a nutritionally balanced ingredient blend for Nile tilapia, with two levels of screw speed (350 and 450 rpm), two levels of extruder water (0.236 and 0.302 kg/min), and two levels of conditioner steam (0.1 and 0.15 kg/min). The central point was 400 rpm screw speed, 0.271 kg/min extruder water, and 0.12 kg/min conditioner steam. Effects of these processing conditions on extrudate characteristics were extensively analyzed and included moisture content, water activity, thermal properties, expansion ratio, unit density, bulk density, color, water stability, sinking velocity, water absorption and solubility indices, and pellet durability index. Increasing the extruder water and conditioner steam resulted in a 5.3% decrease and nearly 8.6% rise in mass flow rate, respectively. As screw speed increased from 350 to 400 rpm, water stability and water activity increased by 13 and 58%, respectively. Increasing extruder water from 0.236 to 0.302 kg/min led to a significant increase in water stability by 12.5% and decreases in water absorption index, water activity, and expansion ratio by 13, 21, and 5.5%, respectively. As conditioner steam increased from 0.1 to 0.15 kg/min, sinking velocity and water absorption index decreased by 25 and 15%, respectively. Increasing conditioner steam from 0.1 to 0.12 kg/min resulted in 20, 5.5, 10, and 3% decreases in moisture content of the extrudates, brightness (L*), water stability, and expansion ratio, respectively. It also increased bulk density by 5.8% and unit density by 4.2%. Overall, all trials produced viable extrudates with properties appropriate for Nile tilapia feeding.