beta-Lactoglobulin A with N-ethylmaleimide-modified sulfhydryl residue, polymerized through intermolecular disulfide bridge on heating in the presence of dithiothreitol

Roles of sulfhydryl groups on thermal aggregation of beta-lactoglobulin A (betaLG A) at pH 7.5 were investigated. It is known that betaLG A modified at Cys(121) with N-ethylmaleimide (NEM-betaLG A) does not form an aggregate by heating and that dithiothreitol (DTT) reduces cystine residues and induces the intermolecular sulfhydryl/disulfide interchange reaction and/or oxidation. NEM-betaLG A was heated in the presence of DTT. The molecules were linked together with an intermolecular disulfide bridge, and the polymer formed increased with increase in DTT concentration. The largest portion of polymer was formed when DTT was added at around the same molar concentration as that of NEM-betaLG A. Then, polymer formation decreased with further increase in DTT concentration. The results suggest that sulfhydryl/disulfide residues other than Cys(121), generated from cysteine residues, can induce intermolecular sulfhydryl/disulfide interchange reactions to polymer and that thiol compounds, for example, added DTT, are capable of starting such reactions.     

Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase

Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.     

Protein recovery in soymilk and various soluble fractions as a function of genotype differences, changes during heating, and homogenization

Harovinton, a variety of tofu type soybean, and 11 derived null soybean genotypes lacking specific glycinin (11S) and beta-conglycinin (7S) protein subunits were investigated to determine whether changes in protein composition affected the protein recovery in soymilk and its soluble fractions after various centrifugation steps. As both heating and homogenization have a marked effect on the increase in protein solubility, the changes occurring during these processing steps were studied for each soybean genotype. Harovinton and 11S-null genotypes showed significantly higher protein yields than the other genotypes evaluated. Subunits of group I (A(1), A(2)) of glycinin had a negative impact on protein solubility in all treatments, but this effect was the greatest in unheated soymilk samples. Samples containing a high beta-conglycinin to glycinin ratio showed an effect of heating on the solubility of the protein, as beta-conglycinin subunits aggregate with heating. The presence of the alpha' subunit of beta-conglycinin aids in the recovery of protein in the supernatant prepared from lines containing group I (A(1,4) A(2)) glycinin. The results of this study will help determine which specific protein composition will confer an increased stability in soymilk and soymilk-derived products.     

Effects of microfluidization treatment and transglutaminase cross-linking on physicochemical, functional, and conformational properties of peanut protein isolate

Peanut protein isolate (PPI) was treated by high-pressure microfluidization (40, 80, 120, and 160 MPa) and/or transglutaminase (TGase) cross-linking. It was found that individual microfluidization at 120 MPa was more effective in improving the solubility, emulsifying properties, and surface hydrophobicity of PPI than at other pressures (e.g., 40, 80, or 160 MPa). Individual TGase cross-linking also effectively changed the physicochemical and functional properties of PPI. Microfluidization (120 MPa) or TGase cross-linking caused the unfolding of PPI structure, resulting in the decrease of α-helix and β-turns levels and the increase of β-sheet and random coil levels, as proved by Fourier transform infrared (FTIR) and circular dichroism (CD) spectra. Compared with individual treatments, microfluidization followed by TGase cross-linking significantly (p < 0.05) improved the emulsion stability during long-term storage (20 days). Moreover, the combined treatments led to looser structure of PPI and resulted in more obvious changes in physicochemical properties.     

Physicochemical and nitrogen solubility properties of Bacillus proteinase hydrolysates of sodium caseinate incubated with transglutaminase pre- and post-hydrolysis

Sodium caseinate (NaCN), hydrolyzed with Protamex, a Bacillus proteinase preparation, to 0.5, 1.3, and 17.5% degrees of hydrolysis, was incubated with transglutaminase (TGase) for 3, 42, and 290 min at enzyme/substrate ratios of 1, 1, and 10% (w/w), respectively, pre- and post-hydrolysis. The electrophoretic, reversed-phase high-performance liquid chromatography (RP-HPLC) and nitrogen solubility profiles of the modified products were investigated. Combinations of hydrolysis and incubation with TGase generated products displaying novel physicochemical and nitrogen solubility properties. Significant changes in sodium dodecyl sulfate (SDS) and urea polyacrylamide gel electrophoresis profiles were apparent in the modified caseinate samples. Extensive TGase cross-linking resulted in polymers that were unable to enter the resolving gel during SDS polyacrylamide gradient gel electrophoresis. Extensive combined enzymatic modification resulted in peptides eluting earlier on RP-HPLC than limited combined enzymatic modification or limited hydrolysis. Combination of enzymatic treatments resulted in significantly (P < 0.005) improved solubility around pH 4.6, compared to incubation with TGase or hydrolysis of NaCN alone.     

Reactivity of fish and microbial transglutaminases on glutaminyl sites of peptides derived from threadfin bream myosin

Fish liver transglutaminase (FTG), a Ca(2+)-dependent enzyme, exhibits different characteristics from the Ca(2+)-independent microbial transglutaminase (MTG), leading to potential differences in their substrate specificity and reactivity. The ability of these enzymes to catalyze isopeptide bond formation by incorporating 5-(biotinamido)pentylamine (BPNH2) into peptides derived by tryptic digestion of threadfin bream (TB)-myosin was investigated to identify reaction sites and substrate specificity using a peptidomic strategy. BPNH2 was incorporated into TB-myosin peptides to a greater extent by MTG than FTG. Peptides derived from TB-myosin heavy chain (MHC) shared highest similarity to amberjack-MHC on the basis of a Mascot database search. Amino acid sequences and modification sites of BPNH2-tagged peptides were identified by tandem mass spectrometry based on the amberjack-MHC sequence. The BPNH2 modification sites catalyzed by both TGases were at the myosin rod. Most of the BPNH2 peptides contained charged amino acids (E, R, K) at the glutaminylamide site of reactive glutamine (Q*). The alpha-acrylamide site of Q* contained E, F, or L on peptides catalyzed by both enzymes, I, Q, or A on peptides catalyzed only by FTG, and V on a peptide catalyzed only by MTG. These results demonstrate the different structural requirements for glutaminyl substrates between these two enzymes.     

Micrometer-sized fibrillar protein aggregates from soy glycinin and soy protein isolate

Long, fibrillar semiflexible aggregates were formed from soy glycinin and soy protein isolate (SPI) when heated at 85 degrees C and pH 2. Transmission electron microscopy analysis showed that the contour length of the fibrils was approximately 1 microm, the persistence length 2.3 microm, and the thickness a few nanometers. Fibrils formed from SPI were more branched than the fibrils of soy glycinin. Binding of the fluorescent dye Thioflavin T to the fibrils showed that beta-sheets were present in the fibrils. The presence of the fibrils resulted in an increase in viscosity and shear thinning behavior. Flow-induced birefringence measurements showed that the behavior of the fibrils under flow can be described by scaling relations derived for rodlike macromolecules. The fibril formation could be influenced by the protein concentration and heating time. Most properties of soy glycinin fibrils are comparable to beta-lactoglobulin fibrils.     

Celiac-related properties of chemically and enzymatically modified gluten proteins

The effects of chemical (acid-heating treatment) and enzymatic (microbial transglutaminase, TGase) modification (deamidation) of gluten proteins on their physicochemical and celiac disease-related properties were studied. Ammonia release, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sample solubility analyses were employed to check the extent of gluten modification. Among different treatments achieved, the acid-heating treatment performed at 90 degrees C for 3 h induced gluten deamidation, paralleling an increase of gluten solubility without relevant proteolysis. Changes in the immunoreactivity of celiac IgA anti-gliadin antibodies (AGAs) to modified gluten proteins were detected by using a competitive indirect enzyme-linked immunosorbent assay method. Chemical deamidation by acid-heating treatment of gluten lowered IgA-AGA immunoreactivity. IgA-AGA immunoreactivity to gliadins was increased when they were submitted to TGase-catalyzed deamidation. The acid-heating treatment of gluten reduced its cytotoxic activity on human colon adenocarcinoma LoVo cell line. These results showed that chemical deamidation of gluten may be envisaged as a way to lower the potential risk for celiac people due to widespread use of gluten as a food additive.     

Apigenin-induced suicidal erythrocyte death

Apigenin, a flavone in fruits and vegetables, stimulates apoptosis and thus counteracts cancerogenesis. Erythrocytes may similarly undergo suicidal cell death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)), ceramide formation and ATP depletion. The present study explored the effect of apigenin on eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, ceramide utilizing antibodies, and cytosolic ATP with luciferin-luciferase. A 48 h exposure to apigenin significantly increased [Ca(2+)](i) (≥ 1 μM), increased ceramide formation (15 μM), decreased ATP concentration (15 μM), decreased forward scatter (≥ 1 μM), and increased annexin V binding (≥ 5 μM) but did not significantly modify hemolysis. The effect of 15 μM apigenin on annexin V binding was blunted by Ca(2+) removal. The present observations reveal novel effects of apigenin, i.e. stimulation of Ca(2+) entry, ceramide formation and ATP depletion in erythrocytes with subsequent triggering of suicidal erythrocyte death, paralleled by cell shrinkage and phosphatidylserine exposure.     

Sorption and catalytic hydrolysis of diethatyl-ethyl on homoionic clays

Sorption and catalytic hydrolysis of the herbicide diethatyl-ethyl [N-chloroacetyl-N-(2,6-diethylphenyl)glycine ethyl ester] on homoionic Na(+)-, K(+)-, Ca(2+)-, and Mg(2+)-montmorillonite clays were studied in aqueous media. The Freundlich sorption coefficient, K(f), measured from isotherms on clay followed the order of Na(+) approximately K(+) > Mg(2+) approximately Ca (2+). Analysis of FT-IR spectra of diethatyl-ethyl sorbed on clay suggests probable bonding at the carboxyl and amide carbonyl groups of the herbicide. The rate of herbicide hydrolysis in homoionic clay suspensions followed the same order as that for sorption, indicating that sorption may have preceded and thus caused hydrolysis. Preliminary product identification showed that hydrolysis occurred via nucleophilic substitution at the carboxyl carbon, causing cleavage of the ester bond and formation of diethatyl and its dechlorinated derivative, and at the amide carbon, yielding an ethyl ester derivative and its acid. These pathways also suggest that hydrolysis of diethatyl-ethyl was catalyzed by sorption on the clay surface.     

Surfactant protein of the Streptomyces subtilisin inhibitor family inhibits transglutaminase activation in Streptomyces hygroscopicus

Transglutaminase (TGase) is widely used in the food industry for improving protein properties by catalyzing the cross-linking of proteins. In Streptomyces, TGase is secreted as a zymogen, and an activation process has been observed in liquid culture. However, the activation mechanism remains unclear. In the present study, the TGase activation process in Streptomyces hygroscopicus was investigated by biochemical approaches. In a liquid culture, Pro-TGase was secreted and gradually was converted into active TGase during the growth period; however, in a cell-free system in which cells were removed from the liquid culture, TGase activation stalled unexpectedly. Subsequently, the TGase activation process was found to be inhibited by a TGase-activating protease inhibitor (TAPI). N-Terminal amino acid sequencing and a homology search of the purified TAPI revealed that it is a member of the Streptomyces subtilisin inhibitor (SSI) family. Furthermore, it was found that TAPI (0.1 mg/mL) decreased the surface tension of water from 72 to 60 mJ/m2 within 5 min, suggesting that it possesses surface activity. This is the first report that an SSI member functions as a surfactant protein. On the basis of these findings, a model for TAPI-regulated TGase activation process was proposed. This study provides novel insights into the TGase activation process in Streptomyces.     

Involvement of Ca2+ in the inhibition by crocetin of platelet activity and thrombosis formation

Crocetin, a unique carotenoid with potent antioxidative and anti-inflammatory activities, is a major ingredient of saffron used as an important spice and food colorant in various parts of the world. In the present study, the effects of crocetin on platelet activity and thrombosis formation were systematically investigated. Crocetin showed a dose-dependent inhibition of platelet aggregation induced by ADP, collagen, but not by arachidonic acid (AA). Crocetin significantly attenuated dense granule release, while neither platelets adhesion to collagen nor cyclic AMP level was altered by crocetin. Pretreatment with crocetin was confirmed to partially inhibit Ca (2+) mobilization via reducing both intracellular Ca (2+) release and extracellular Ca (2+) influx. Besides that, crocetin prolonged the occlusive time in electrical stimulation-induced carotid arterial thrombosis. These findings suggest that the favorable impacts of crocetin on platelet activity and thrombosis formation may be related to the inhibition of Ca (2+) elevation in stimulated platelets.     

Surface functional properties of native, acid-treated, and reduced soy glycinin. 1. Foaming properties

Foaming properties of native and chemically modified glycinin were evaluated. Effects of ionic strength and glycinin composition and concentration on foam formation and stabilization were studied. Glycinin was modified by means of combined treatments: cold or hot acidic treatments, with or without later disulfide bridges reduction. Modified proteins obtained from glycinin present different degrees of dissociation, deamidation, and as consequence, varied surface hydrophobicity and molecular size. Parameters of forming and stabilizing of foam were correlated with both deamidation and dissociation degrees of modified and native glycinin samples. A positive relationship was observed between surface behavior and foaming properties of different protein species. Results show that dissociation, deamidation, and reduction have produced structural changes on glycinin (increased surface hydrophobicity, increased net charge, decreased molecular size) which enhance the adsorption and anchorage of proteins at the air-water interface and, consequently, improve the foam forming and stabilizing capacities.     

Comparison of protein chemical and physicochemical properties of rapeseed cruciferin with those of soybean glycinin

Rapeseeds contain cruciferin (11S globulin), napin (2S albumin), and oleosin (oil body protein) as major seed proteins. The effects of oil expression and drying conditions on the extraction of these proteins from rapeseed meal were examined. The conditions strongly affected the extraction of oleosin and only weakly affected the extraction of cruciferin and napin. The protein chemical and physicochemical properties of cruciferin, the major protein present, were compared with those of glycinin (soybean 11S globulin) under various conditions. In general, cruciferin exhibited higher surface hydrophobicity, lower thermal stability, and lower and higher solubility at mu= 0.5 and mu = 0.08, respectively, than did glycinin. At the pHs (6.0, 7.6, and 9.0) and ionic strengths (mu= 0.08 and 0.5) examined, the emulsifying ability of cruciferin was worse than that of glycinin, except at mu= 0.08 and pH 7.6. The emulsifying abilities of cruciferin and glycinin did not correlate with thermal stability and surface hydrophobicity. Higher protein concentration, higher heating temperature, higher pH, and lower ionic strength were observed to produce harder gels from cruciferin. Gel hardness partly correlated with the structural stability of cruciferin.     

Network Formation in Gluten‐Free Bread with Application of Transglutaminase

One of the main problems associated with gluten‐free bread is obtaining a good structure. Transglutaminase (TGase), an enzyme that catalyzes acyl‐transfer reactions through which proteins can be cross‐linked could be a way to improve the structure of gluten‐free breads. The objective of this study was to evaluate the impact of TGase at different levels (0, 0.1, 1, and 10 U of TGase/g of protein) on the quality of gluten‐free bread. The recipe consisted of white rice flour (relative amount: 35), potato starch (30), corn flour (22.5), xanthan gum (1), and various protein sources (skim milk powder [SMP] [12.5], soya flour, and egg powder). The influence of the various proteins in combination with the different addition levels of TGase on bread quality (% bake loss, specific volume, color, texture, image characteristics, and total moisture) was determined. Confocal laser‐scanning microscopy (CLSM) was used to evaluate the influence of TGase on the microstructure of the bread. Baking tests showed that TGase had an effect on the specific volume of the bread. For instance, the SMP bread with 10 U of enzyme contained the most compact structure, which was reflected in the crumb texture profile analysis results (highest values) (P < 0.05), digital image analysis (highest level of cells/cm²) (P < 0.05), and CLSM micrographs (network formation). Finally, it can be concluded that it is possible to form a protein network in gluten‐free bread with the addition of TGase. However the efficiency of the enzyme is dependent on both the protein source and the level of enzyme concentration.     

Purification and substrate specificity of transglutaminases from blood and Streptoverticillium mobaraense.

A procedure for a fast and simple purification of bovine plasma transglutaminase was developed, which resulted in a homogeneous enzyme preparation. Two different procedures were developed for the purification of pig erythrocyte transglutaminase, both of which resulted in partial purification. Both enzymes were used in cross-linking reactions of alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, casein, hemoglobin, glycinin, and myosin. The substrate specificity was compared to that of bacterial transglutaminase isolated from Streptoverticillium mobaraense. The bacterial transglutaminase caused cross-linking of a wider range of proteins and, thus, exhibited a lower substrate specificity than the blood transglutaminases. In addition, differences exist in the necessity of the addition of reducing agents. These differences allow specific applications of blood and bacterial transglutaminases at protein cross-linking in single or complex protein systems.     

Effects of different sources of organic matter on soil aggregate formation and stability: A laboratory study on a Lithic Rhodoxeralf from Turkey

In this study the influences of three different organic sources on aggregate formation and stability in soil were investigated.Our approach involves establishing plot experiment in the greenhouse with a Lithic Rhodoxeralf and a variety of carbon sources having specific chemical properties applied to the soil of individual plots. Treatments include K-Humate (KH, 25, 50 and 100 kg/ha), Concentrated Plant Extract (CPE, 50, 100, 200 kg/ha) and Molasses (M, 50, 100, 200 kg/ha). After a seven-month incubation period, aggregate formation and their stability were determined and their correlation to different C sources was developed.The effect of KH on aggregate formation was significant for 2–1 mm aggregates (P < 0.05) and 0.5–0.25 mm aggregates (P < 0.01). The effect of CPE on aggregate formation was significant for 0.5–0.25 aggregates (P < 0.05), while the M had no significant effect on aggregate formation in any aggregate size. The effect of KH on aggregate stability was significant for 8–4 mm and 1–0.5 mm aggregates (P < 0.01). The effect of CPE on aggregate stability was significant for 0.5–0.25 mm aggregates (P < 0.05), while the M was significant for 8–4 mm aggregates (P < 0.05).     

Effects of Transglutaminase on Rheology,Microstructure, and Baking Properties of Frozen Dough

The improving effects of transglutaminase (TGase) were investigated on the frozen dough system and its breadmaking quality. Rheological properties and microstructure of fresh and frozen doughs were measured using a Rapid Visco‐Analyser (RVA), dynamic rheometer, and scanning electron microscopy (SEM). The frozen doughs with three storage periods (1, 3, and 5 weeks at –18°C) were studied at three levels (0.5, 1.0, and 1.5%) of TGase. As the amount of TGase increased, hot pasting peak viscosity and final viscosity from the RVA decreased, but breakdown value increased. The TGase content showed a positive correlation with both storage modulus G′ (elastic modulus) and the loss modulus G″ (viscous modulus): G′ was higher than G″ at any given frequency. The SEM micrographs showed that TGase strengthened the gluten network of fresh, unfrozen dough. After five weeks of frozen storage at –18°C, the gluten structure in the control dough appeared less continuous, more disrupted, and separated from the starch granules, while the dough containing 0.5% TGase showed less fractured gluten network. Addition of TGase increased specific volume of bread significantly (P < 0.05) with softer bread texture. Even after the five weeks of frozen storage, bread volume from dough with 1.5% TGase was similar to that of the fresh control bread (P < 0.05). The improving effects of TGase on frozen dough were likely the result of the ability of TGase to polymerize proteins to stabilize the gluten structure embedded by starch granules in frozen doughs.     

Calcium- and magnesium-dependent aggregation of legume seed storage proteins.

The solubility characteristics and sedimentation behavior of total or individual globulins from legume seeds [Lupinus albus L., Pisum sativum L., and Glycine max (L.) Merr.] were investigated. The typical insolubility of globulins detected during their extraction seems to be due to the presence of a low molecular weight factor(s) in the seed extract. The solubility of the purified globulins decreases with increasing concentrations of calcium and/or magnesium, but not of other cations, showing minimum values at concentrations that vary with the particular globulin considered. Ultracentrifugation analyses revealed that the Ca(2+)- and/or Mg(2+)-induced insolubilization of the globulins involves the formation of high-order aggregates of molecules of the same or of different globulins. These macromolecular structures are dissociated under conditions of high ionic strength, suggesting the involvement of electrostatic interactions in the aggregation process. The degree of association relies heavily on the amount of Ca(2+) and/or Mg(2+) available, on the presence of chelating agents for these divalent cations, and on the ionic strength of the surrounding medium. The possible physiological significance of the findings is discussed.     

Sorption of 2,4,6-trichlorophenol in model humic acid-clay systems

Humic acids and clays are important soil components that influence the sorption and desorption of organic contaminants; however, it is unclear how humic acids influence the sorption of organic contaminants onto clays and their subsequent desorption. Sorption and desorption of 2,4,6-trichlorophenol (2,4,6-TCP) by and from humic acid-modified K(+)- and Ca(2+)-montmorillonite and -illite were compared with unmodified clays using batch equilibration methods. Commercial humic acid and the humic acid extracted from forest soil were employed in this experiment. The adsorbed amount of 2,4,6-TCP by commercial humic acid was almost twice as large as that adsorbed by the extracted soil humic acid. More 2,4,6-TCP was sorbed onto K(+)- and Ca(2+)-illite than onto K(+)- and Ca(2+)-montmorillonite. K(+) clays were more effective in adsorbing 2,4,6-TCP than Ca(2+) clays. Sorption of 2,4,6-TCP on humic acid-modified Ca(2+)- and K(+)-montmorillonite and -illite increased as compared with unmodified clays. The sorption nonlinearity of 2,4,6-TCP on humic acid-modified Ca(2+)- and K(+)-illite increased remarkably as compared with the unmodified clays. The sorption nonlinearity of 2,4,6-TCP on humic acid-modified Ca(2+)- and K(+)-montmorillonite increased slightly in contrast to unmodified montmorillonites. By comparing sorption and desorption results, we observed hysteresis for all sorbents including humic acids, clays, and humic acid-modified clays. Sorption nonlinearity and hysteresis were dependent on the structure of humic acids. Higher aromaticity of humic acids resulted in greater sorption nonlinearity and desorption hysteresis. In addition, sorption capacity (K(f)') was positively correlated with the humic acid content of the sorbents. These results show that modification of humic acids on clays can not only increase the adsorption ability of clays but also affect the sorption nonlinearity of 2,4,6-TCP, and the desorption hysteresis was probably due to the structural characteristics of humic acids.