Degree Of Deamidation Research Articles

Improving solubility of rice protein powder by modifying its physicochemical properties by ultrasound-assisted protein-glutaminase

The effect of ultrasound-assisted protein-glutaminase (PG) deamidation on the physicochemical properties of rice protein (RP) was investigated. After ultrasound pretreatment, the degree of deamidation of RP reached the highest of 60.4 % at deamidating for 16 h. With the deamidating time increasing, the particle size of RP became smaller and the absolute value of ζ-potential gradually increased. For functional properties of RP, ultrasound-assisted PG deamidation improved the foaming capacity, emulsifying capacity and oil-holding capacity of RP. Based on the ameliorative physicochemical properties of RP, the properties of rice protein powder (RPP) were further determined. The solubility of RPP was significantly improved, increased by 102.6 % at 90 °C compared with the non-treated. Meanwhile, the antioxidant activity and flavor of RPP under PG deamidation were remarkably improved. In vitro digestibility of RPP also increased significantly. These results illustrated that PG deamidation could be an efficient method for improving the properties of proteins.

The effect of protein-glutaminase from Chryseobacterium proteolyticum on physicochemical and functional properties of high-temperature soybean meal protein

Protein-glutaminase (EC 3.5.1.44, PG) can significantly improve the functional properties of food proteins. However, the low yield of PG has limited industrial applications. Results showed that 0.02 % tea saponin could increase the PG yield by 18.93 %. The transcription level of the PG gene was significantly enhanced, which promoted the extracellular secretion of PG through an increase in membrane permeability. On this basis, PG was used to modify high-temperature soybean meal protein (HSMP) due to its poor properties. In this study, the deamidation degree (DD) of PG-modified HSMP was optimized to 58.61 % by the response surface method. HSMP with different DD was prepared and its physicochemical and functional properties were studied. After PG treatment, the intermolecular repulsive force of HSMP increased, the particle size distribution became uniform, and the solution system was more stable. In addition, the surface morphology of HSMP gradually became loose and porous. The solubility of HSMP significantly improved, reaching 11.34 times that of untreated HSMP at pH 5.00. Meanwhile, the emulsifying and foaming capacity of HSMP significantly improved, but the foaming stability was reduced.

Effects of deamidated gluten protein on wheat starch properties and rheological properties of starch-gluten dough

To further investigate the interaction between starch and gluten protein, deamidated gluten protein with varying degrees of organic acid (citric acid) was mixed with wheat starch in various proportions, and the solubility, pasting, and rheological properties of the blend flour were investigated. The solubility of the sample powder increased significantly as the deamidated protein content increased. The gelatinization results also demonstrated that the deamidated protein content increased the peak viscosity, trough viscosity gradually decreased, and peak viscosity time shortened. The results of the rheological measurement revealed that the elastic modulus (G′), viscous modulus (G″), and creep recovery of the mixed flour showed regular changes with the continuous change of the mixed powder proportion. Furthermore, consistent alterations were observed in the effects of proteins with varying degrees of deamidation in the same proportion on farinaceous characteristics. FTIR analysis of the gluten protein revealed that the primary secondary structures were the β-sheet and β-turn; different levels of damage were observed.

Effects of protein-glutaminase on the properties of glutinous rice flour, paste, and gel food: Based on the interactions between the deamidated protein and starch

In the present study, protein-glutaminase (PG) from Chryseobacterium proteolyticum was applied to improve the processing properties of glutinous rice flour (GRF). After PG modification, the degree of deamidation of glutinous rice protein (GRP) reached 13.6% at 2.0 h, with smaller particle size and decreased zeta potential. The interaction of GRP with starch in PG-modified GRF (PM-GRF) was changed, exhibiting in protein aggregates decreasing and exposure of starch on the surface of GRF. Compared with unmodified GRF (UM-GRF), the solubility and turbidity of PM-GRF were both increased. The rheological properties reflected that the viscosity of PM-GRF paste was increased, and the freeze-thaw stability was also enhanced. Moreover, the textural characteristics showed that the hardness of PM-GRF balls remarkably reduced and the springiness increased. These results indicate that deamidation by PG could be an efficient method for improving characteristics of GRP and GRF.

Purified protein glutaminase from Chryseobacterium proteolyticum enhances the properties of wheat gluten

Protein glutaminase (PG), originating from Chryseobacterium proteolyticum, can catalyze the deamidation of glutamine residues in plant proteins into glutamic acid, thus enhancing its functional properties. However, the low yield of PG limits its industrial production. In this study, the yield of PG in C. proteolyticum TM1040 increased by 121 %, up to 7.30 U/mL in a 15 L fermenter after medium optimization. Subsequently, purified PG was obtained by cation exchange chromatography (CEX) coupled with hydrophobic interaction chromatography (HIC). The degree of deamidation (DD) of wheat gluten after purified PG deamidation was 87.11 %, which is superior to chemical deamidation in safety and DD. The emulsifying and foaming properties of deamidated wheat gluten were 2.67 and 18.86 times higher, and the water- and oil-holding properties were 4.23 and 18.77 times higher, respectively. The deamidated wheat gluten with enhanced functional properties was used to improve the flavor and texture in baking cakes.

Beef tenderization without exacerbating the cooking loss: The way of enzymatic deamidation

Tenderization of beef using proteolytic enzymes often leads to high water loss due to harsh structural disruption. This study first verified the enzymatic deamidation (protein-glutaminase, PG) in softening grass-fed steak texture without exacerbating the cooking loss. PG deamidation at the deamidation degree (DD) of 5% (d5) or 7% (d7) significantly increased the tenderness and softened the texture whereas they negligibly affected the cooking loss. The muscle bundles were loosed while fibers were thickened probably as the result of charge induced myofibril dissociation (increased myofibril fraction index) and intermolecular repulsion. Compared to papain, the effect of PG deamidation on the water mobility was minor, though it was still seen a slight conversion of bound water into immobilized water in high DD steaks (d5 and d7). PG deamidation hardly hydrolyze individual proteins but partially altered the tertiary structure of muscle proteins, resulting in a slight decrease in denaturation enthalpies of muscle proteins including myosin, collagen and sarcoplasmic protein. These results demonstrated that PG deamidation appeared to be a novel and promising strategy in meat tenderization.

Heterologous expression of protein-glutaminase in Escherichia coli Nissle 1917 for improved flavor properties of soy protein and its implications on high-moisture extrudates

Beany flavor affects sensory experience of soy protein-based food. Deamidation is an effective approach to improve the flavor of soy protein, but excessive hydrolysis has a negative effect on its molecular structure and processing property. In this study, protein-glutaminase (PG; EC 3.5.1.44) was heterologous expressed in E. coli Nissle 1917 (EcN) and used to specifically modify soy protein isolate (SPI) for improved flavor properties of textured protein. A fluorescence-based enzyme activity screening method was developed and optimized for fermentation conditions. After screening for optimal fermentation conditions based on cell fluorescence intensity, 3.01 U/mL of PG activity was achieved. SPI was treated with PG (0–24 h), and the degree of deamidation (DD) rapidly increased to 47.4% within 0–2 h. There was no significant decrease in the molecular weight of deamidated SPI (DSPI) after specific deamidation of PG within 0–10 h. The contents of main beany flavor substances (e.g., hexanal, benzaldehyde, and 2-pentylfuran) in DSPI decreased significantly with increasing DD. Compared with soy protein, moderate deamidation did not affect the processing properties and fibrous structure of DSPI in preparing high-moisture extrudates, while the structure of the extrudate decreased after a deamidation time of more than 6 h. This finding developed a safe expression system for PG and provided a promising method to address beany flavor in soy-based meat analogues produced by high moisture extrusion.

Enhancement of functional properties, digestive properties, and in vitro digestion product physiological activity of extruded corn gluten meal by enzymatic modification.

Enzymatic modification is an effective means of improving the functional properties, digestive properties, and in vitro digestion product physiological activity of proteins, thus significantly expanding protein uses in various food applications. In this study, the addition of chymotrypsin (CT) at pH 9.0 and 11.0 was found to significantly improve the functional properties (solubility, foaming properties, water holding capacity, oil holding capacity, etc.) and digestive properties of extruded corn gluten meal (ECGM). Similar changes were observed when treating ECGM with glutaminase, protein glutaminase, and papain. These changes were likely due to the increase in number of carboxyl groups and the multiple effects of change in protein net charge and conformation caused by enzymatic deamidation. Of note, ECGM deamidated by CT showed the highest degree of deamidation, solubility, and gastrointestinal digestibility at pH 11.0, up to 44.92%, 43.75%, and 82.22%, respectively. In addition, CT-ECGM digestion product exhibited strong antioxidant activity and potential to promote alcohol metabolism in both a static digestion model and dynamic digestion model, even comparable to commercial corn peptides (CCP), while being inexpensive and of low bitterness compared to CCP. Meanwhile, the physiological activity enhanced as the molecular weight of digestion product decreased with the digested component having strongest activity. This study may promote the application of ECGM as a food component in the food industry or even as a substitute for CCP. © 2023 Society of Chemical Industry.

Wheat gluten deamidation: structure, allergenicity and its application in hypoallergenic noodles.

Wheat gluten (WG) containing gliadin and glutenin are considered the main allergens in wheat allergy as a result of their glutamine-rich peptides. Deamidation is a viable and efficient approach for protein modifications converting glutamine into glutamic acid, which may have the potential for allergenicity reduction of WG. Deamidation by citric acid was performed to investigate the effects on structure, allergenicity and noodle textural properties of wheat gluten (WG). WG was heated at 100 °C in 1 m citric acid to yield deamidated WG with degrees of deamidation (DD) ranging from DWG-25 (25% DD) to DWG-70 (70% DD). Fourier-transform infrared and intrinsic fluorescence spectroscopy results suggested the unfolding of WG structure during deamidation, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed molecular weight shifts at the 35-63 kDa region, suggesting that the deamidation mainly occurred on low molecular weight glutenin subunits and γ- gliadin of the WG. An enzyme-linked immunosorbent assay of deamidated WG revealed a decrease in absorbance and immunoblotting indicated that the intensities of protein bands at 35-63 kDa decreased, which suggested that deamidation of WG might have caused a greater loss of epitopes than the generation of new epitopes caused by unfolding of WG, and thereby reduction of the immunodominant immunoglobulin E binding capacity, ultimately leading to the decrease in allergenicity. DWG-25 was used in the preparation of recombinant hypoallergenic noodles, and the hardness, elasticity, chewiness and gumminess were improved significantly by the addition of azodicarbonamide. The present shows the potential for deamidation of the WG products used in novel hypoallergenic food development. © 2023 Society of Chemical Industry.

Mechanisms underlying the formation of non-thermal deamidated zein via synergistic treatment of cold plasma with acid: Improved foam performance

Zein with a modified fibrosis conformation has been demonstrated to be an effective surface component for stabilizing Pickering foam. In this study, a non-thermal deamidation method was developed using cold plasma (CP) in synergy with hydrochloric acid (HCl), tartaric acid (TA), and gluconolactone (GDL). The structure and properties of the material were investigated to better understand the mechanism of non-thermal deamidated zein synthesis. The results demonstrated that within 12 min, the degrees of deamidation (16.21, 14.14, and 8.35%) were regulated by the varying proton catalytic rates of HCl, TA, and GDL in the CP field. Following treatment, notable enhancements were observed in the solubility (0.12–0.28 mg/mL), foamability (50.00–166.67%), and foaming stability (106.67–128.00%) (P < 0.05). The increase in β-sheet, random coil, and loose structure of deamidated zein was revealed by employing FTIR and UV spectroscopy. The unfolding of structure resulted in the exposure of hydrophobic groups, thereby enhancing the protein's foaming properties. Deamidated zein has increased interfacial adsorbability, which was demonstrated by atomic force microscope and scanning electron microscopy. Better interface performance was shown by CP synergistic TA treatment for 12 min (CPT12-zein), offering the possibility of commercial manufacturing of foamed foods in the future.

Investigation and Characterization of Pickering Emulsion Stabilized by Alkali-Treated Zein (AZ)/Sodium Alginate (SA) Composite Particles.

Pickering emulsions stabilized by food-grade colloidal particles have attracted increasing attention in recent years due to their "surfactant-free" nature. In this study, the alkali-treated zein (AZ) was prepared via restricted alkali deamidation and then combined with sodium alginate (SA) in different ratios to obtain AZ/SA composite particles (ZS), which were used to stabilize Pickering emulsion. The degree of deamidation (DD) and degree of hydrolysis (DH) of AZ were 12.74% and 6.58% respectively, indicating the deamidation occurred mainly in glutamine on the side chain of the protein. After the treatment with alkali, AZ particle size decreased significantly. Moreover, the particle size of ZS with different ratios was all less than 80 nm. when the AZ/SA ratio was 2:1(Z2S1) and 3:1(Z3S1), the three-phase contact angle (θo/w) were close to 90°, which was favorable for stabilizing the Pickering emulsion. Furthermore, at a high oil phase fraction (75%), Z3S1-stabilized Pickering emulsions showed the best long-term storage stability within 60 days. Confocal laser scanning microscope (CLSM) observations showed that the water-oil interface was wrapped by a dense layer of Z3S1 particles with non-agglomeration between independent oil droplets. At constant particle concentration, the apparent viscosity of the Pickering emulsions stabilized by Z3S1 gradually decreased with increasing oil phase fraction, and the oil-droplet size and the Turbiscan stability index (TSI) also gradually decreased, exhibiting solid-like behavior. This study provides new ideas for the fabrication of food-grade Pickering emulsions and will extend the future applications of zein-based Pickering emulsions as bioactive ingredient delivery systems.

Characterization of high internal phase emulsions stabilized by protein glutaminase-deamidated wheat gluten

In the present study, effect of deamidation by protein glutaminase (PG; EC 3.5.1.44) on structure of wheat gluten (WG) and its possible to construct high internal phase emulsions (HIPEs) system were investigated. The deamidation reaction was performed for different durations (0–48 h). The deamidation degree (DD, 0–70.5%) of WG was increased with an increase of reaction time (0–48 h). After the specific deamidation by PG, the solubility of WG was significantly improved at neutral pH while maintaining its molecular weight. Unlike native WG, deamidated WG (DWG) had the ability to form stable HIPEs. Moreover, HIPEs prepared using DWGs with a moderate DD (28.7–40.5%) exhibited remarkable characteristics in terms of their network structure, rheological behavior and friction coefficient. This study demonstrated the application of PG-modified WG for HIPE preparation and characterized their structural and functional relationship, providing a promising approach for the production of stabilized emulsions in food systems.

Effect of Protein-Glutaminase on Calcium Sulphate-Induced Gels of SPI with Different Thermal Treatments.

The effects of protein-glutaminase (PG) on calcium sulphate (CaSO4)-induced gels of soy protein isolate (SPI) with different heat treatment levels were investigated. The time-dependent degree of deamidation showed that the mild denaturation of the protein favored the deamidation. The particle size distribution showed that the heat treatment increased the SPI particle size, and the particle size distribution of the SPI shifted to the right or increased the proportion of the large particle size component as the degree of deamidation increased for each sample. Rheological analysis showed that the deamidation substantially pushed up the gel temperature and decreased the value of G'. The gel strength and water-holding capacity showed that the higher the amount of enzyme added, the more significant the decrease in gel strength, while the gel water-holding capacity increased. In summary, the deamidation of PG and heat treatment can affect the gel properties of SPI synergistically.

Improving rehydration of egg white powder through modifying its physicochemistry properties by ultrasound-assisted glutaminase deamidation

Poor rehydration is one of the limiting factors affecting the processing and functional properties of egg white powder (EWP). In order to improve rehydration, this study explored the ultrasound-assisted deamidation method (ultrasound power: 100 W, 200 W, 300 W, 400 W and 500 W) and its effects on the physicochemistry and processing properties of EWP. The degree of deamidation increased at first and then decreased with the increase of ultrasound power. After deamidation, rehydration significantly increased. SDS-PAGE results showed that glutaminase had no obvious effect on protein composition. However, circular dichroism spectroscopy, Fourier transform infrared spectroscopy and surface hydrophobicity analyses showed that deamidation contributed to the decrease in α-helix and increase in random coil structure, which generated more flexible and stretchable conformational proteins. The average particle size and ζ-potential of EWP changed significantly, indicating that ultrasound-assisted deamidation treatment caused the de-aggregation and re-aggregation of EWP. These helped to promote the wettability, solubility and dispersion and reduce the turbidity of the rehydrated solution by altering the surface hydrophobicity and increasing the flexibility of EWP molecules. Best rehydration was observed in the sample of 300 W-EWP. These results suggested that ultrasound-assisted deamidation treatment is a practical method to enhance the processability and applicability of EWP. • Deamidation treatment enhanced thermal stability of egg white powder. • Deamidation treatment made egg white particles more porous and looser. • The structure of deamidated egg white proteins became more flexible and stretchable. • Ultrasound-assisted deamidation treatment is a practical method to improve rehydration of EWP.

Impact of deamidation on the physicochemical properties and air-water interfacial behaviour of gliadin nanoparticles

Gliadin due to its low water solubility is excellently suited to make biopolymeric nanoparticles through liquid antisolvent precipitation. These gliadin nanoparticles (GNPs) can be utilized to populate and stabilize interfaces. Gliadin, by nature a protein that carries a low charge, can be altered by deamidation. Deamidation effectively alters the physicochemical properties of gliadin through decreasing the protein’s isoelectric point (IEP). The objective of this study is to explore the effect of different degrees of deamidation on gliadin nanoparticle (interfacial) characteristics. Besides altering the physical stability of GNP suspensions, deamidation did alter the functionality of GNPs as interfacial agents. GNP behaviour at the air-water interface is substantially impacted by pH. Particles made with untreated and deamidated gliadin exhibited a greater tendency to adsorb and form strong viscoelastic films at the air-water interface and better foam structures closer to their IEP. The combination of SEM, confocal Raman microscopy and drop tensiometry provides unique insights in the dominant gliadin structures and interactions at the interfacial film. This study provides crucial insights into the potential of deamidation to tailor gliadin and GNP properties for specific food applications.

The characteristics of protein-glutaminase from an isolated Chryseobacterium cucumeris strain and its deamidation application.

Protein-glutaminase (PG), a deamidation enzyme commercially derived from Chryseobacterium proteolyticum, is used to improve the solubility and other functional properties of food proteins. In this study, a new PG-producing strain, Chryseobacterium cucumeris ZYF120413-7, was isolated from soil, and it had a high PG yield and a short culture time. It gave the maximum PG activity with 0.557 U/ml on Cbz-Gln-Gly after 12 h of culture, indicating that it was more suitable for PG production. The enzyme activity recovery and purification fold were 32.95% and 161.95-fold, respectively, with a specific activity of 27.37 U/mg. The PG was a pre-pro-protein with a 16 amino acids putative signal peptide, a pro-PG of 118 amino acids, and a mature PG of 185 amino acids. The amino acid sequence identity of PG from strain ZYF120413-7 was 74 and 45%, respectively, to that of PG from C. proteolyticum 9670T and BH-PG. The optimum reaction pH and temperature of PG was 6 and 60°C, respectively. Enzyme activity was inhibited by Cu2+. The optimum PG substrate was Cbz-Gln-Gly, and the Km and Vmax values were 1.68 mM and 1.41 μM mg protein−1 min−1, respectively. Degree of deamidation (DD) of soy protein isolate (SPI) treated by purified PG was 40.75% within the first 2 h and 52.35% after 18 h. These results demonstrated that the PG from C. cucumeris ZYF120413-7 was a promising protein-deamidating enzyme for improving the functionality of food proteins.

Tailoring protein intrinsic charge by enzymatic deamidation for solubilizing chicken breast myofibrillar protein in water

Inspired by the salt-in effect, the potential use of protein-glutaminase (PG) to increase the intrinsic charges of chicken breast myofibrillar proteins (CMPs) for enhanced water solubility was tested. The degree of deamidation (DD) and solubility of CMPs increased with PG reaction time. Over 60% of CMPs were soluble in water under a DD of 6.5% due to specific conversion of glutamine to glutamic acid. PG deamidation could remarkably increase the net charge of CMPs with a merit in maintaining most of the amino acid and protein subunit compositions. Such a high electrostatic repulsion exerted a transformation of β-sheet into α-helix, unfolded the structure to expose hydrophobic residues, and allowed the dissociation of myofibril and release of subunits (myosin, actin or their oligomers), leading to a stable colloidal state. This work may foster the engineering advances of protein micro-modification in the tailor manufacture of muscle protein-based beverages.

Analytical and pharmacological consequences of the in vivo deamidation of trastuzumab and pertuzumab.

A liquid chromatography-tandem mass spectrometry method is presented for the quantitative determination of the in vivo deamidation of the biopharmaceutical proteins trastuzumab and pertuzumab at an asparagine in their complementarity determining regions (CDRs). For each analyte, two surrogate peptides are quantified after tryptic digestion of the entire plasma protein content: one from a stable part of the molecule, representing the total concentration, and one containing the deamidation-sensitive asparagine, corresponding to the remaining non-deamidated concentration. Using a plasma volume of 10 µL and a 2-h digestion at pH 7, concentrations between 2 and 1000µg/mL can be determined for the various protein forms with values for bias and CV below 15% and without unacceptable in vitro deamidation taking place. A considerable difference between the total and non-deamidated concentrations, and thus a substantial degree of deamidation, was observed in plasma for both trastuzumab and pertuzumab. After a 56-day forced deamidation test 40% of trastuzumab and 68% of pertuzumab was deamidated, while trastuzumab and pertuzumab showed up to 47% and 35% of deamidation, respectively, in samples collected from breast cancer patients during treatment with a combination of both drugs. A good correlation between the non-deamidated concentration results and those of a receptor binding assay indicate a loss of receptor binding for both trastuzumab and pertuzumab along with the deamidation in their CDRs. Deamidated trastuzumab also lost its capability to inhibit the growth of breast cancer cells in a cell-based viability assay, suggesting a relation between the degree of deamidation and pharmacological activity.

Synergistic strongly coupled super-deamidation of wheat gluten by glucose-organic acid natural deep eutectic solvent and the efficaciousness of structure and functionality

The synergistic coupled improvement of functional properties by natural deep eutectic solvent (NADES) super-deamidated wheat gluten (WG) was investigated. Screening by eight NADESs, glucose-citric acid NADES (G-C, molar ratio at 2:1) super-deamidated WG (SDWG-N) exhibited the exceeding emulsification properties (22.37 m2/g) and foaming stability (85.61%), which were significantly higher than those of citric acid deamidated WG (CDWG). Degree of deamidation and protein recovery of SDWG-N (26 mg/mL) reached 95.47% and 96.77% respectively at 121 °C for 14 min at the condition that G-C was diluted to 18% (v/v). Supra-molecular structural properties of G-C NADES reflected that the charge inhomogeneity of G-C promoted the affinity of water to the amide groups via ionizing more H+ and formed hydrogen bonds with amino acid residues, thus achieving the strong coupling super-deamidation of WG. SDWG-N showed higher stability as function of pH 2–12 than CDWG. Fluorescence and FTIR spectra explored more unfolded and flexible with greater exposed hydrophobic groups and lower degree of aggregation of SDWG-N, which exhibited the synergistic coupled effect of super-deamidation by G-C on WG. It suggested that SDWG-N would be used as a strong stabilizer at water/oil and gas/water interface with further potential to cooperate with other macromolecules for food use.

Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins.

Personal care products containing hydrolyzed gluten have been linked to spontaneous sensitization through the skin, however the impact of the hydrolysate characteristics on the sensitizing capacity is generally unknown. The physicochemical properties of five different wheat-derived gluten products (one unmodified, one enzyme hydrolyzed, and three acid hydrolyzed) are investigated, and the skin sensitizing capacity is determined in allergy-prone Brown Norway rats. Acid hydrolyzed gluten products exhibited the strongest intrinsic sensitizing capacity via the skin. All hydrolyzed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolyzed products is associated with product-specific sensitization in wheat tolerant rats. Sensitization to acid hydrolyzed gluten products is associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitization induced by unmodified gluten or enzyme hydrolyzed gluten. Acid hydrolysis enhances the skin sensitizing capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolyzed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.