Free Amino Group Content Research Articles

Molecular docking explores heightened immunogenicity and structural dynamics of acetaldehyde human immunoglobulin G adduct.

Acetaldehyde is a metabolite of ethanol, an important constituent of tobacco pyrolysis and the aldehydic product of lipid peroxidation. Acetaldehyde induced toxicity is mainly due to its binding to cellular macromolecules resulting in the formation of stable adducts accompanied by oxidative stress. The aim of this study was to characterize structural and immunological alterations in human immunoglobulin G (IgG) modified with acetaldehyde in the presence of sodium borohydride, a reducing agent. The IgG modifications were studied by various physicochemical techniques such as fluorescence and CD spectroscopy, free amino group estimation, 2,2-azobis 2-amidinopropane (AAPH) induced red blood cell hemolysis as well as transmission electron microscopy. Molecular docking was also employed to predict the preferential binding of acetaldehyde to IgG. The immunogenicity of native and acetaldehyde-modified IgG was investigated by immunizing female New Zealand white rabbits using native and modified IgG as antigens. Binding specificity and cross reactivity of rabbit antibodies was screened by competitive inhibition ELISA and band shift assays. The modification of human IgG with acetaldehyde results in quenching of the fluorescence of tyrosine residues, decrease in free amino group content, a change in the antioxidant property as well as formation of cross-linked structures in human IgG. Molecular docking reveals strong binding of IgG to acetaldehyde. Moreover, acetaldehyde modified IgG induced high titer antibodies (>1:12800) in the experimental animals. The antibodies exhibited high specificity in competitive binding assay toward acetaldehyde modified human IgG. The results indicate that acetaldehyde induces alterations in secondary and tertiary structure of IgG molecule that leads to formation of neo-epitopes on IgG that enhances its immunogenicity.

Anti-Oxidative and Anti-Aging Activities of Porcine By-Product Collagen Hydrolysates Produced by Commercial Proteases: Effect of Hydrolysis and Ultrafiltration.

To investigate methods for improving the processing of porcine waste, porcine skin was hydrolyzed using different commercially available proteases (Alcalase, Flavorzyme, Neutrase, Bromeline, Protamex, and Papain) under several optimal conditions. Following enzymatic hydrolysis, the collagen hydrolysates (CHs) were fractionated by molecular weight (3 kDa) via membrane ultrafiltration. The CHs were analyzed for physical properties (pH, protein recovery, free amino group content, molecular weight distribution, and amino composition) as well as for functional properties (antioxidant activities and anti-aging activities). Among the CHs, CHs hydrolyzed by Alcalase (CH-Alcalase) exhibited the highest degree of hydrolysis compared to other CHs. Both “CH-Alcalase” and “CH-Alcalase < 3 kDa” fractions showed a considerably high antioxidant activity and collagenase inhibition activity. Therefore, resulting bioactives have potential for development as antioxidants and anti-aging ingredients in the food, cosmetics, and pharmaceuticals, from animal by-products.

Investigation of the Mechanism of Conformational Alteration in Ovalbumin as Induced by Glycation with Different Monoses through Conventional Spectrometry and Liquid Chromatography High-Resolution Mass Spectrometry.

Glycation between ovalbumin (OVA) and different monoses under mild dry heating at 37 °C was studied. The content of free amino groups decreased dramatically, and the conformational changes based on fluorescence and circular dichroism spectra were evident in glycated OVA. The glycated sites and the average degree of substitution per peptide molecule per site were determined using liquid chromatography high-resolution mass spectrometry. Lysine and arginine were the predominant glyaction sites, in which Lys207 was a relatively reactive site for glycation in all of the conjugates. In general, the extent of glycation of aldose was higher, and its alterations on the steric layouts of protein were more drastic than those of ketose. The configuration of hydroxyl groups at C-4 in sugar epimers might be important for the glycation reactivity and conformational modification in the glycated proteins. These insights would have important implications for the creation of sweetened food products with desirable structures and excellent quality control.

Survival and proteolytic capacity of probiotics in a fermented milk enriched with agave juice and stored in refrigeration

In this study, the survival and proteolysis of lactic acid bacteria Lactobacillus casei Shirota and Lactobacillus rhamnosus GG were measured in fermented milks enriched with agave juice and inulin. Fermentations were terminated at pH 4.5 and stored for 21 days at 4 °C. Survival was determined by viable count in MRS-agar. The production of free amino groups was performed by the TNBS method and peptide separation was performed by SDS-PAGE. During fermentation, pH decreased faster in milk with agave juice than with inulin. There was no significant difference in the concentration of free amino groups (0.632 ± 0.007 and 0.627 ± 0.007 mg/L) between the two fermentation systems for any microorganism. The concentration of lactic acid bacteria in the presence of agave juice was higher than the recommended concentration for probiotic foods (7.59 log CFU/mL for L. rhamnosus GG and 8.26 log CFU/mL for L. casei Shirota). The production of free amino groups and peptides of low molecular weight continued in refrigeration and was higher in systems with agave juice. This could represent a proteolytic activation in the presence of this carbon source. The results showed that agave juice might be a functional ingredient with prebiotic character in symbiotic systems.

Fabrication of Cellulose Nanocrystal/Chitosan Hydrogel for Controlled Drug Release.

In this work, a novel nanocomposite hydrogel based on cellulose nanocrystal (CNC) and chitosan (CS) was fabricated and applied as a carrier for the controlled delivery of theophylline. CNC was firstly periodate-oxidized to obtain dialdehyde nanocellulose (DACNC). Then, chitosan was crosslinked using DACNC as both the matrix and crosslinker in different weight ratios, to fabricate CNC/CS composites. The prepared composites were characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction pattern (XRD), scanning electron microscopy (SEM), zeta potential measurement and swelling ratio tests. FT-IR results confirmed the successful reaction between the free amino groups on chitosan and the aldehyde groups on DACNC. With the increase of chitosan percentage in the hydrogel, the isoelectric point was shifted towards an alkaline pH, which was probably caused by the higher content of free amino groups. The swelling ratio of the composite also increased, which may have been due to the decrease of crosslinking density. Because the swelling ratio of the drug-loaded hydrogels differed under varied pH values, the cumulative drug release percentage of the composite hydrogel was achieved to approximately 85% and 23% in the gastric (pH 1.5) and intestinal (pH 7.4) fluids, respectively. Therefore, CNC/CS hydrogel has application potential as a theophylline carrier.

Enhancement of Hydrophobicity of Fish Skin Gelatin via Molecular Modification with Oxidized Linoleic Acid

Fish gelatin possesses hydrophilicity in nature since it contains a few hydrophobic amino acids and a large portion of hydrophilic amino acids. Low hydrophobicity of fish gelatin results in poor water vapor barrier and water resistance of gelatin films. This can limit its usage as packaging material. To overcome this drawback, the molecular attachment of hydrophobic domains can effectively improve the hydrophobicity of fish gelatin. In this study, fish skin gelatin modified with oxidized linoleic acid (OLA) prepared under various conditions using different molar ratios of OLA/free amino group content was characterized. When OLA was prepared at 60 and 80°C for various oxidation times (0–24 h), the peroxide value (PV) increased continuously up to 9 and 12 h when reaction was performed at 80 and 60°C, respectively. Consequently, the PV decreased until the end of the reaction (24 h). Thiobarbituric acid reactive substances (TBARS) of OLA were increased sharply up to 12 h, regardless of reaction temperatures. Thus, primary and secondary lipid oxidation products mainly occurred within the first 12 h. As gelatin was modified with different OLA at various OLA/free amino group molar ratios, the one modified with OLA prepared at 60°C for 24 h at a molar ratio of 10 : 1 had the highest increases in surface hydrophobicity and carbonyl content with the coincidentally lowest free amino group content, compared with control gelatin (without OLA modification). Fourier transforms infrared (FTIR) spectra also reconfirmed the presence of fatty acid covalently attached to resulting gelatin. Therefore, OLA could be used to modify gelatin and increase its hydrophobicity.

Hydroxyl radical-induced early stage oxidation improves the foaming and emulsifying properties of ovalbumin.

As the most abundant protein in chicken eggs, ovalbumin plays an important role in the processing of high value-added poultry products. The present study investigated the effects of hydroxyl radical-induced early stage oxidation on the physicochemical and interfacial properties of chicken egg white ovalbumin. Protein carbonyl content of ovalbumin increased (from 0.78 to 1.13nmol/mg) with the oxidation time (0-5h), while free sulfhydryl content (from 0.43 to 0.09nmol/mg) and free amino group content (from 0.49 to 0.43nmol/mg) decreased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the exposure of ovalbumin to hydroxyl radicals caused self-cross-linking and resulted in the formation of dimers and trimers. Accompanied by these changes, the surface hydrophobicity of ovalbumin was enhanced about 1.5-fold with the deepening of oxidation, and the value of zeta potential became more negative (from -7.15 to -20.51 mv). About 2 h of moderate oxidation improved the foaming and emulsifying properties of ovalbumin (1.2-fold to 1.8-fold), while excessive oxidation (3h) decreased these interface properties. Hydroxyl radical-induced oxidation changed the surface chemical groups and structures of ovalbumin, thereby affecting the surface properties. The foaming and emulsifying properties of ovalbumin could be improved by oxidation, increasing the application possibilities of ovalbumin in the food interface system.

Protein⁻Phenolic Interactions as a Factor Affecting the Physicochemical Properties of White Bean Proteins.

This study was conducted with an aim to determine the interactions of pure phenolic compounds (gallic acid, ferulic acid, chlorogenic acid, quercetin, apigenin, and catechin) and phenolics from plant extracts (green tea and green coffee) with protein fractions of white bean (albumins and globulins). The physicochemical properties of complexes were established through an analysis of the UV-Vis spectrum; relative content of free amino groups, thiol groups, and tryptophan residues; chromatographic (SE-HPLC) and electrophoretic (SD-PAGE, Native-PAGE) properties; and conformational changes reflected by Fourier transform infrared spectra. Further, the effect of pH and ionic strength on the solubility and stability of complexes as well as the binding capacity of phenolics to proteins were determined. Results show that, in most cases, phenolics significantly affected the measured parameters; however, the effects were strongly differentiated by the type of phenolic compounds and protein fraction that were applied. Moreover, it may be that changes in the properties of complexes are reflected in the biological nature of proteins and phenolic compounds such as their bioavailability and physiological activity. However, due to the structural complexity of proteins, and the multitudinous factors that affect their interactions, such studies are a great and long-term challenge for the domain of food science.

Modification by glyceraldehyde-3-phosphate prevents amyloid transformation of alpha-synuclein

Numerous investigations point to the relation between diabetes and neurodegenerative disorders. Alpha-synuclein is a protein involved in the development of synucleinopathies including Parkinson's disease. In the present work, alpha-synuclein was for the first time modified by the intermediate product of glycolysis, glyceraldehyde-3-phosphate (GA-3-P). The resulting product was compared with the alpha-synuclein modified by methylglyoxal (MGO). The efficiency of the modification by the aldehydes was evaluated by decrease in free amino group content. The modification products were detected using fluorescence spectroscopy. The effect of modification by two glycating agents on the amyloid transformation of alpha-synuclein was investigated. Transmission electron microscopy analysis of the aggregates produced by the native alpha-synuclein under fibrillation conditions revealed the presence of 355–441-nm fibrils. In the aggregates produced by the modified alpha-synuclein, short fibrils of 65–230 nm or 85–260 nm were detected in the case of the protein treated with MGO and GA-3-P, respectively. Investigation of the aggregates by the fluorescence assay with Thioflavin T and CD spectroscopy showed that, in contrast to native alpha-synuclein, alpha-synuclein treated with GA-3-P does not produce real amyloid structures. Consequently, modification of alpha-synuclein by GA-3-P, the metabolite whose concentration is determined by the activity of glyceraldehyde-3-phosphate dehydrogenase, prevents its amyloid transformation.

Antioxidant activities and emulsifying properties of porcine plasma protein hydrolysates modified by oxidized tannic acid and oxidized chlorogenic acid

Abstract Physicochemical characteristics, antioxidant activities and emulsifying properties of porcine plasma protein hydrolysates (PPPH) modified with oxidized tannic acid (OTA) or oxidized chlorogenic acid (OCA) were investigated. The formation of complexes was showed by increased turbidity, and both OTA and OCA were covalently attached to PPPH as shown by the significantly decrease in free amino group contents ( P P

Stabilization and cyclic utilization of chrome leather shavings.

For the sake of the safe and efficient cyclic utilization of chrome leather shavings (CLS), the temporary stabilization of CLS during the storage and transport processes was optimized. Afterwards, the large-scale stabilized CLS degradation and collagen degradation product (CDP) extraction were completed. In this research, hydroxyapatite (HAP) and calcium hydroxide (Ca(OH)2) were for the first time used as stabilizers in the stabilization of CLS. The results showed that both Ca(OH)2 and HAP displayed resistance to chrome leaching, but HAP provided better stabilization effect than Ca(OH)2. The optimized stabilization conditions giving lower chromium leaching concentration were selected: 2% HAP, temperature 300°C, reaction time 60min, and initially added water contents of 50%. The reason for HAP and Ca(OH)2 stabilization was proved by energy dispersive X-ray spectroscopy (EDS). Subsequently, the exploitation value of the stabilized CLS was described by large-scale experiment. The hydrolysis of stabilized CLS could obtain the collagen degradation product (CDP), which had 3.52% of free amino content and a molecular weight of 3.4~30kDa. The filter residue was treated by 5% sulfuric acid solution extraction and pressure filtration again. The extraction solution and final filter residue were dried separately to obtain chromium-containing organic compounds (COP) and industrial gypsum. The COP, used as a filling for tannery, contains 9.37% Cr2O3 content and 90.6% organic content. The industrial gypsum contains 68.37% CaSO4, 0.13% Cr2O3, and 31.5% residual organic or inorganic compounds, which can be used in building materials. No waste water or solid waste was discharged in this experiment process.

Comparative study in physico-chemical properties of gelatin derivatives and their microspheres as carriers for controlled release of green tea's extract

Two types of gelatin derivatives were prepared from type A and type B gelatin, respectively. Cationized gelatin was produced by conjugation of ethylenediamine into type A gelatin using carbodiimide reaction. Anionized gelatin was produced by conjugation of succinic anhydride to type B gelatin. Characteristics of four gelatin types and their microspheres fabricated by water-in-oil emulsion and glutaraldehyde crosslinking were evaluated. Cationized gelatin showed the highest positive charge and free amino group concentration due to the ethylenediamine conjugation, resulting in the highest crosslinking percentage and slowest degradation rate of cationized gelatin microspheres. Type A, B and anionized gelatin which had lower concentration of free amino groups produced the low crosslinking and fast-degrading microspheres. These gelatin microspheres were introduced as carrier for controlled release of epigallocatechin 3-gallate (EGCG), a high antioxidant green tea's extract. The cationized gelatin microspheres showed prolonged release of EGCG due to the slow degradation and ionic attraction with the negative-charged EGCG. The release of EGCG from type A, B and anionized gelatin microspheres was accelerated as a result of diffusion and microsphere's swelling mechanisms. These EGCG-loaded gelatin microspheres can be further studied as injectable drug for localized treatment of inflammation-related diseases with minimal invasion.

Effect of oxidation induced by hydroxyl radical‐mediated model on molecular structural and physical character of egg white powder

SummaryThe objective of this study was to determine structure and functional properties changes of oxidised egg white protein derived from hydrogen peroxide (H2O2)/ferric chloride (FeCl3) /ascorbic acid hydroxyl radical‐generating systems at room temperature, including carbonyls, sulfhydryl and total sulfhydryl groups, dityrosine, free amino, surface hydrophobicity, particle size distribution, intermolecular forces, and foamability and emulsibility. Protein carbonyl and dityrosine content of egg white protein were increased (P &lt; 0.05) with increasing the concentrations of H2O2. Oxidation decreased free sulfhydryl, total sulfhydryl groups and surface of hydrophobicity comparing with nonoxidised egg white protein. Oxidation also reduced its free amino content (P &lt; 0.05). The low concentration of H2O2 contributing the average particle size of egg white protein was smaller than the control group. However, the high concentrations of H2O2 caused that egg white protein aggregated and the average particle size became larger. To sum up, oxidation made EWP denaturation and aggregation.

Preparation, Surface Activities, and Biodegradability of a Bola‐Type Collagen Hydrolysate‐Based Siloxane Surfactant

Abstract2 bola‐type collagen hydrolysate‐based siloxane surfactants (CBES) were prepared via grafting of an epoxy‐terminated polydimethylsiloxane oligomer onto 2 collagen hydrolysates with different molecular weights. Their structure was characterized by Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR) spectroscopy, and the degree of grafting was determined by calculating the free amino content, which confirmed the successful synthesis of CBES. Subsequently, their physicochemical properties, such as particle size and isoelectric point, were evaluated. Besides, surface activities, application functionalities, and biodegradability of CBES were investigated. The results showed that CBES‐1, prepared with the lower‐molecular‐weight collagen hydrolysate, possessed superior surface activity, excellent foaming ability, and good emulsifying capacity comparable to those of a typical emulsifier such as the fatty alcohol polyoxyethylene ether (AEO‐9). In addition, their BOD5/CODCr value and CO2 evolution demonstrated that the CBES are readily biodegradable. There is, therefore, great potential for the bola‐type CBES to be applied in fields such as leather treatment and textile industries.

Investigation of conformation change of glycated ovalbumin obtained by Co-60 gamma-ray irradiation under drying treatment

Abstract Irradiation can significantly improve the incorporation of glucose into protein. In this study, the efficacy on conformation change and glycation extent of ovalbumin (OVA) powder was investigated during Co-60 irradiation treatment. Spectrophotometer was used to monitor the conformation and structure change of glycation OVA under various irradiation doses condition. A dose dependent increase in UV absorbance and development of fluorescence was observed. Free amino groups content showed that up to 5 kGy irradiation could accelerate the glycation reaction between OVA and glucose, while there were inapparent differences among the other various doses. Intrinsic, synchronous and 3D fluorescence spectra showed that the glycation reaction caused the dynamic quenching of OVA and changed the polarity of the hydrophobic microenvironment (Trp and Tyr residues) of OVA with unfolding conformation change.

Short-term frozen storage enhances cross-linking that was induced by transglutaminase in surimi gels from silver carp (Hypophthalmichthys molitrix)

To enhance the application of transglutaminase (TGase) in processing and preserving of frozen surimi, the mechanism that enhances surimi gelation induced by TGase after short-term freezing was investigated. Gel properties and cross-linking extent of surimi gels increased when surimi was frozen at −18 °C for 5–7 days. However when storage time exceeded 10 days, textural properties and water-holding capacity decreased significantly. Moreover, the difference of breaking force and hardness between surimi gels with and without TGase reached the maximum after 3–5 days of frozen storage. Free amino content of myosin increased during the first 7 days, and TGase activity increased significantly during the first 3 days. Short-term frozen storage unfolded myosin structure, decreased α-helix content, and exposed hydrophobic patches, which promoted cross-linking reactions and intermolecular hydrophobic interactions. This study provides some new ideas for the processing, storage and transport of frozen surimi and manufacture of frozen surimi-based products.

Antithrombotic Activity of Milk Protein Hydrolysates by Lactic Acid Bacteria Isolated from Commercial Fermented Milks

ABSTRACT In the last decade, thrombosis has been one of the pathologies with high incidence creating great concern in Health Institutes all around the world. Considering this, the aim of this research was to determine the antithrombotic activity of peptides released during lactic fermentation. Reconstituted skim milk powder was fermented by Lactobacillus casei Shirota and Lactobacillus johnsonii LA1 isolated from commercial fermented milks. The hydrolysis degree and proteolytic profile were analyzed by trinitrobenzenesulfonic acid spectrophotometry method and by peptide polyacrylamide electrophoresis gel separation. The milk fermented by Lactobacillus casei Shirota exhibited a higher concentration of free amino groups than that fermented by Lactobacillus johnsonii LA1. Additionally, in both fermentation systems peptides with molecular weights lower than 1.4 kDa were observed. The highest inhibition of thrombin (31.67±2.35%) was observed in milk fermented by Lactobacillus johnsonii LA1 at 10 hours of fermentation. Finally, no relationship was found between the antithrombotic capacity and the proteolytic profile.

Effects of high temperatures and drought during anthesis and grain filling period on wheat processing quality and underlying gluten structural changes.

Climate changes do not only affect wheat yield, but also its quality. Information on this topic gathered so far is somewhat contradictory and insufficient. Climate changes also affect wheat indirectly through their influence on the ecosystem, including insects and fungi that affect wheat technological quality. The aim of this study was to examine trends in structural and technological changes of wheat quality under conditions typical of climate changes. With this in mind, three groups of wheat varieties with the same Glu-score were examined in three production years, characterized by different production conditions. A production season characterized by climate change conditions results in lower activity of amylolytic enzymes. What is more, it results in lower content of gluten, higher gluten index value, its decrease after 1 h to 37 °C, lower number of free SH groups and higher content of free amino groups, which result in lower alveograph W, lower farinograph WA and higher extensograph dough resistance. Variability in wheat quality produced under different climatic conditions is mainly influenced by the production conditions, including their influence on ecosystem factors. The influence of wheat cultivar genetic predisposition is much less expressed. This indicates that differences among cultivars with different Glu-score might be diminished under the influence of altered production conditions, as a consequence of climate change. © 2017 Society of Chemical Industry.

Characterization of soy protein hydrolysates produced by varying subcritical water processing temperature

Abstract The objective of this study was to investigate the effect of subcritical water processing (SWP) temperature on the hydrolysis pattern and quality characteristics of soybean protein hydrolysates. Two common cultivars of soybean were subjected to SWP at temperature ranging from 150 to 250 °C and pressure of 22 MPa. The highest free amino group content and yield were obtained at a processing temperature of 190 °C for both the cultivars ( p

Development and characterization of a defatted soy flour-based bio-adhesive crosslinked by 1,2,3,4-butanetetracarboxylic acid

A new, environmentally friendly defatted soy flour-based bio-adhesive was developed by using the cross-linker 1,2,3,4-butanetetracarboxylic acid (BTCA). The reaction between BTCA and defatted soy flour was confirmed by assaying the free amino groups, by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction, and 13C nuclear magnetic resonance (13C NMR). Three-ply plywood was fabricated to measure wet shear strength, and the cross-section and thermal behavior of resultant adhesives were characterized in detail. A decrease in the free amino group content, a new ester peak in the FTIR spectra, a weaker resonance at 65.4–76.2ppm in 13C NMR, and a decrease in crystallinity confirmed that BTCA was successfully crosslinked with defatted soy flour. The resulting crosslinked bio-adhesive showed improved wet shear strength (1.36MPa) and a reduced sol fraction (24.8%). The crosslinked bio-adhesive displayed enhanced thermal stability, and had a more uniform surface based on thermogravimetric analysis and scanning electron microscopy. The results suggest BTCA can be used to prepare high-performance environmentally friendly defatted soy flour-based bio-adhesives.