Migration of chicken egg-white protein ovalbumin-related protein X and its alteration in heparin-binding affinity during embryogenesis of fertilized egg.

Ovalbumin-related protein X (OVAX) is a 50 kDa egg-white protein which has heparin-binding affinity. In this study, migration of OVAX and its heparin-binding performance during embryogenesis of fertilized egg were investigated to explore a possible involvement in chick embryo development. Western blotting of egg yolks at different stages using an anti-OVAX antibody showed that OVAX accumulates in yolk during incubation of fertilized egg. Immunohistochemical analysis of embryo resided in 10-day-incubated eggs showed that OVAX existed in almost all tissues of the embryo. These suggest that OVAX is incorporated from egg white into the embryo through yolk sac. Heparin-sepharose chromatography, isothermal titrating calorimetry using fondaparinux as a ligand, and zeta potential measurement indicated that OVAX retained the heparin-binding affinity (Kd = 0.185 ± 0.037) even after 10 D incubation of fertilized egg, although the affinity was slightly decreased during egg incubation because of acidification of molecular surface charge. In conclusion, although heparin-binding ability of egg-white OVAX slightly decreases during embryogenesis, OVAX incorporated into embryo can retain heparin-binding affinity. Our findings provide a new insight that OVAX participates in the functions of heparin during embryogenesis.

Enhanced Antibacterial Activity of Lactoperoxidase-Thiocyanate-Hydrogen Peroxide System in Reduced-Lactose Milk Whey.

The product of the lactoperoxidase system (LPOS) has been developed as a preservative agent to inhibit foodborne bacteria, but its action was, heretofore, limited to several original compounds in milk. This research was conducted to analyze the application of the lactoperoxidase system against Escherichia coli in fresh bovine milk and its derivative products to determine the strength of antibacterial activity. Lactoperoxidase was purified from bovine whey using the SP Sepharose Big Beads Column. The enzymatic reaction involving lactoperoxidase, thiocyanate, and hydrogen peroxide was used to generate the antibacterial agent from LPOS. This solution was then added to milk, skimmed milk, untreated whey, reduced-LPO whey, reduced-lactose whey, and high-lactose solution containing E. coli at an initial count of 6.0 log CFU/mL. LPOS showed the greatest reduction of bacteria (1.68 ± 0.1 log CFU/mL) in the reduced-lactose whey among the products tested. This result may lead to a method for enhancement of the antimicrobial activity of LPOS in milk and derived products.

Extending Shelf Life of Indonesian Soft Milk Cheese (Dangke) by Lactoperoxidase System and Lysozyme.

Dangke, a type of fresh soft cheese made of bovine and buffalo milk, is a traditional dairy product used in South Sulawesi, Indonesia. It is prepared from fresh milk using the conventional method, which easily destroys the quality. This study was conducted to assess whether using lactoperoxidase system and lysozyme as preservative agents could suppress the growth of bacteria in dangke. The pH value, total microbial count, and hardness of dangke were determined to measure the quality. Lactoperoxidase and lysozyme were purified from fresh bovine milk, and their purity was confirmed using SDS-PAGE. The combination of lactoperoxidase system and lysozyme was able to remarkably suppress the total microbial count in dangke from 7.78 ± 0.67 to 5.30 ± 0.42 log CFU/ml during 8 h of storage at room temperature. Preserving dangke in this enzyme combination affected its hardness, but there was no remarkable change in the pH value. Results of this study may provide knowledge to utilize a new method to preserve the quality of dangke.

Structural change of ovalbumin-related protein X by alkali treatment.

Chicken egg white protein ovalbumin (OVA) undergoes a conversion to a more thermostable form by alkali treatment, which is assumed to be involved in the physiological functions of OVA. Ovalbumin-related protein X (OVAX), a chicken egg white protein, has 77% sequence similarity to OVA and binds to heparin. In this study, structure characteristics and heparin-binding affinity of alkali-treated OVAX were investigated. Cation-exchange chromatography using SP Sepharose resin showed that alkali treatment (pH 10, 55°C) of OVAX induces the occurrence of a distinct OVAX form with a less positive-charge (acidic OVAX). Circular dichroism and tryptophan-fluorescence analyses showed that the newly-formed acidic OVAX form has an 8% lower α-helical content than its native counterpart, while there is no significant difference in steric environments around tryptophan residues between the 2 forms. The OVAX structure built by homology-modeling showed that OVAX possess a basic cluster domain with α-helix equivalent to 7% of total secondary structures, which does not contain any tryptophan residues. These results suggest that, during alkali treatment, OVAX undergoes mainly a conformational change of the α-helical basic cluster domain and thereby forms acidic OVAX. Acidic OVAX induced by alkali treatment exhibited weaker interactions with Heparin Sepharose resin than native OVAX did. Our results suggest that OVAX basic cluster domain is likely a specific binding site of heparin. Consequently, it is suggested that alkali treatment causes the collapse of the OVAX heparin binding site, which might participate in regulating the functions of heparin.

Effects of rare sugar d-allulose on heat-induced gelation of surimi prepared from marine fish.

BACKGROUND: d-Allulose (Alu), the C3-epimer of d-fructose, is a non-caloric sweetener (0.39 kcal g-1 ) with a suppressive effect on postprandial blood glucose elevation. The aim of this study was to investigate the effects of Alu used as a sweetener and gel improver instead of sucrose on heat-induced gelation of surimi. RESULTS: The puncture test of a heat-induced surimi gel showed that with 50 g kg-1 Alu the gel had 15% and 6% higher gel strength than the corresponding gel with sucrose (Suc) and with sorbitol (Sor), respectively. In addition, Alu-gel had 26% and 25% higher water-holding capacity (WHC) than Suc- and Sor-gel. Heating of myofibrillar protein with Alu, unlike Suc and Sor, facilitated the formation of both disulfide and non-disulfide crosslinks that might be associated with the mechanical properties and WHC of Alu-gel. CONCLUSION: Alu improves the mechanical properties and WHC of the heat-induced surimi gel. Furthermore, Alu is low in calories compared with Suc (4.0 kcal g-1 ) and Sor (3.0 kcal g-1 ). Thus Alu will be an alternative of Suc or Sor for developing surimi-based products with health benefits. © 2017 Society of Chemical Industry.

Effect of D-psicose used as sucrose replacer on the characteristics of meringue.

Excessive intake of sugar-rich foods leads to metabolic syndrome. D-Psicose (Psi) not commonly found in nature, is noncalorie sweetener with a suppressive effect on the blood glucose level. Thus, Psi has the potential to be utilized as a sucrose (Suc) replacer in sugar-rich foods, including meringue-based confectionery (MBC). In this study, we investigated the effect of Psi on the physical and chemical properties of meringue. Meringue was made by whipping egg white and Suc (at a weight ratio of 1:1) and baking at 93 °C for 2 h. Thirty percent of the total weight of Suc was replaced with D-ketohexoses such as Psi, D-fructose, D-tagatose, and D-sorbose. The meringues containing D-ketohexoses had higher specific volume than the meringue not containing D-ketohexoses (Ct-meringue). Baking of meringue caused differences between Psi and the other D-ketohexose meringues. Meringue containing Psi (P30-meringue) had the highest breaking stress (7.00 × 10(5) N/m(2)) and breaking strain (4.40%), resulting in the crunchiest texture. In addition, P30-meringue also had the highest antioxidant activity (491.84 µM TE/mg-meringue determined by ABTS method) and was the brownest due to a Maillard reaction occurring during baking. The replacement of Suc with Psi improved the characteristics of baked meringue. Thus, Psi was found to be useful in modifying the physical and chemical properties of MBC.

Transepithelial transports of rare sugar D-psicose in human intestine.

D-Psicose (Psi), the C3-epimer of D-fructose (Fru), is a noncalorie sugar with a lower glycemic response. The trans-cellular pathway of Psi in human enterocytes was investigated using a Caco-2 cell monolayer. The permeation rate of Psi across the monolayer was not affected by the addition of phlorizin, an inhibitor of sugar transporter SGLT1, whereas it was accelerated by treatment with forskolin, a GLUT5-gene inducer, clearly showing that GLUT5 is involved in the transport of Psi. The permeability of Psi was suppressed in the presence of D-glucose (Glc) and Fru, suggesting that the three monosaccharides are transported via the same transporter. Since GLUT2, the predominant sugar transporter on the basolateral membrane of enterocytes, mediates the transport of Glc and Fru, Psi might be mediated by GLUT2. The present study shows that Psi is incorporated from the intestinal lumen into enterocytes via GLUT5 and is released to the lamina propria via GLUT2.

Multiple biological functions of novel basic proteins isolated from duck egg white: duck basic protein small 1 (dBPS1) and 2 (dBPS2).

Biological functions of duck basic protein small 1 (dBPS(1)) and 2 (dBPS(2)) were investigated by in vitro experiments. Results of agarose gel retardation assay indicated that dBPS(1) and dBPS(2) associate with RNA. Addition of NaCl or urea induced partial dissociation of dBPS(1)/dBPS(2)-RNA complex, implying that electrostatic interaction, hydrophobic interaction, and hydrogen bonds are involved in the association of dBPS(1)/dBPS(2) to RNA. dBPS(1) and dBPS(2) inhibited pancreatic lipase activity with the fifty percent inhibitory concentration (IC(50)) of 250 and 100 µg/mL, respectively. Peptic hydrolysates of dBPS(1) and those of dBPS(2) showed a potent angiotensin I-converting enzyme (ACE) inhibition with an IC(50) of 22.5 and 49.6 mg/L. The most potent ACE-inhibitory peptide was a nanopeptide (EKKGFCAGY) from dBPS(1) and an octapeptide (KYCPKVGY) from dBPS(2). These multiple biological functions of dBPS(1) and dBPS(2) may contribute to reducing the risk of lifestyle diseases.

Effects of mono- and disaccharides on the antimicrobial activity of bovine lactoperoxidase system.

The effects of mono- and disaccharides on the antimicrobial activity of the lactoperoxidase (LPO) system against Salmonella Enteritidis were investigated. The results clearly reveal that most of the sugars inhibit the antimicrobial activity of the LPO system. The inhibitory potency varies depending on the structure of sugar. L-Fructose and D-allose were strongly inhibitive to the action of the LPO system, while sucrose was the weakest inhibitor. The decreased antimicrobial activity is due to the reduction of LPO catalytic activity by sugar. An inhibitory kinetic study showed the noncompetitive inhibitor. D-Allose and L-fructose yielded strikingly low K(i) values of 0.36 and 0.42 mM, respectively, while the K(i) values of the other sugars ranged from 1.37 to 3.60 mM. Since LPO activity is inhibited by the saccharides, the sugar content in food should be considered when the LPO system is applied to the preservation of food.

Structural and physicochemical characteristics of novel basic proteins isolated from duck egg white.

Novel basic proteins, duck basic protein small 1 (dBPS(1)) and 2 (dBPS(2)), were isolated from duck egg white by cation-exchange and gel filtration chromatography. Protein sequence analyses indicated that they possessed 39 amino acid residues with three disulfide bonds. The amino acid sequence of dBPS(1) showed 45% identity with dBPS(2). The amino acid sequence of dBPS(2) was the same as cygnin, a small protein from black swan, and strongly homologous with meleagrin from turkey and chicken. Phylogenic relationships implied that dBPS(1) and dBPS(2) share a common ancestry with cygnin and meleagrin. Based on MALDI-TOF mass spectra, the molecular masses of dBPS(1) and dBPS(2) were 4,373, and the 4,486 Da. pI of dBPS(1) and dBPS(2) elucidated by isoelectric focusing were 9.35 and 9.44. FT-IR spectra classified these proteins as (beta) proteins. Both dBPS(1) and dBPS(2), possessed high heat stability, Td 101.2 and 98.3 degrees C. Indirect ELISA results showed that the dBPS(1)/dBPS(2)-related proteins were distributed in the oviduct and gallbladder.

Influence of a rare sugar, d-Psicose, on the physicochemical and functional properties of an aerated food system containing egg albumen.

d-Psicose (Psi) might be an ideal sucrose (Suc) substitute for food products due to its sweet taste, easy processing, and functional properties (noncaloric and low glycemic response). In the present study, the effects of Psi on foaming properties of egg white (EW) protein and the quality of butter cookies were analyzed to find a better use of Psi in aerated food systems. The results showed that Psi could improve the foaming properties of EW protein with increasing whipping time in comparison to Suc and d-fructose (Fru). The addition of Psi to butter cookies, as partial replacement of Suc, had no influence on the cook loss while significantly contributing to a color change of the cookie crust through a nonenzymatic browning reaction. Furthermore, Psi-containing cookies possessed the highest antioxidant capacity in all tested cookies using two assays of radical scavenging activity and ferric reducing power. It was found that there was a close correlation between the crust color and the antioxidant activity of the cookie. The results suggest that the addition of Psi enhanced the browning reaction during cookie processing and, consequently, produced a strong antioxidant activity.

Cloning and characterization of the gene encoding endo-beta-1,3-glucanase from Arthrobacter sp. NHB-10.

The gluA gene, encoding an endo-beta-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-beta-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-beta-1,3-glucanases, but GluA was partially similar to two fungal exo-beta-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two beta-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-beta-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.

Rheological characteristics of heat-induced custard pudding gels with high antioxidative activity.

Custard pudding gels were prepared from fresh whole egg, milk and sugar. The effects of D-psicose (Psi), a non-calorie rare hexose, on the antioxidative activity and rheological properties of the custard pudding gels were investigated at different temperatures for comparison with those of control sugars (sucrose, Suc; D-fructose, Fru). The rheological behavior of the heat-induced pudding gels was evaluated by using breaking and creep tests. During the heat-induced gel formation, custard pudding containing Psi (15%, wt/wt) demonstrated a stronger breaking strength and higher viscoelasticity than those containing Fru and Suc. The thermodynamic parameters obtained from DSC indicated that the egg white (EW) proteins were made less thermally stable when heated in the presence of Psi than in the presence of Fru and Suc. These findings are consistent with enhanced aggregation of the EW solution in the presence of Psi. Furthermore, the Psi pudding gels possessed higher antioxidative activity than the control sugar pudding gels by using an analysis of the scavenging activity on DPPH radicals and the ferric-reducing antioxidative power. These results suggest that Psi favored cross-linking of Psi-protein molecules through the Maillard reaction which increased the formation of intermediate products to improve functionality. Custard pudding containing Psi could therefore be an effective functional sweet desert with high antioxidative activity and the outstanding gelling characteristics.

Antioxidant effects of Maillard reaction products obtained from ovalbumin and different D-aldohexoses.

Nonenzymatic glycation between ovalbumin (OVA) and seven D-aldohexoses was carried out to study the chemical and antioxidant characteristics of sugar-protein complexes formed in the dry state at 55 degrees C and 65% relative humidity for 2 d through the Maillard reaction (MR). The effects of Maillard reaction products (MRPs) modified with different aldohexoses on radical scavenging, lipid oxidation, and tetrazolium salt (XTT) reducibility were investigated. The results showed that the degree of browning and aggregation and the tryptophan-related fluorescent intensity of glycated proteins displayed a noticeable difference that depended on the sugars used for modification. All the glycated proteins exhibited higher antioxidant activity as compared to a heated control and native OVA, and the antioxidant activity was well correlated with browning development. Furthermore, the order of antioxidant activities for the seven complexes was as follows: altrose/allose-OVAs > talose/galactose-OVAs > glucose-OVA > mannose/glucose-OVAs. This implies that sugar-protein complexes with two sugars known as epimers about C-2 showed a similar antioxidant capacity. From these results, the configuration of a hydroxyl (OH) group about position C-2 did not influence the advanced cross-linking reaction, but the configuration of OH groups about C-3 and C-4 might be very important for formation of MRPs and their antioxidant behaviors.

Evaluation of the site specific protein glycation and antioxidant capacity of rare sugar-protein/peptide conjugates.

Protein-sugar conjugates generated in nonenzymatic glycation of alpha-lactalbumin (LA) with rare sugars [D-allose (All) and D-psicose (Psi)] and alimentary sugars as controls [D-glucose (Glc) and D-fructose (Fru)] were qualitatively determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Mass spectra revealed that the extent of glycation at lysine residues on LA with D-aldose molecules was very much higher than that of glycation with d-ketose molecules. To identify the specific site of glycation, the peptide mapping was established from protease V8 digestion, using a combination of computational cutting of proteins and MALDI-TOF-MS. As compared to peptide mapping, three and seven glycation sites were located in the primary structure of LA-ketose and LA-aldose conjugates, respectively. On the other hand, the antioxidant activities of protein-sugar conjugates and their peptic hydrolysates were investigated by 1,1-diphenyl-2-picrylhydrazyl radical scavenging method. The antioxidant activities of proteins/peptides glycated with rare sugars were significantly higher than those modified with the control sugars. The results indicated that the glycation degree and position were not markedly different between rare sugar and corresponding control sugar, but the antioxidant properties of protein and its hydrolysate were significantly enhanced by modifying with rare sugar.

Effects of a lactoperoxidase-thiocyanate-hydrogen peroxide system on Salmonella enteritidis in animal or vegetable foods.

Lactoperoxidase (LPO) from skim milk was purified by ion-exchange chromatography. The purified protein was used to catalyze the oxidation of thiocyanate by H2O2 in an antibacterial system (LPO system). The LPO system was used to inactivate or inhibit Salmonella enteritidis in tomato juice, carrot juice, milk, liquid whole egg, and chicken skin extract under various conditions. The system was found to be more effective against the organism in vegetable juices than in animal products, at low pH than at neutral pH, and at higher temperatures than at lower temperatures. Acid-adapted S. enteritidis cells were more susceptible than nonadapted cells. The system reduced numbers of S. enteritidis in vegetable products by up to 5.4 log units and inhibited growth of the organism in animal-derived foods during 4 h incubation at 30 degrees C. Sodium chloride (>100 mM) and polyphosphate (0.01-0.5%) enhanced the antibacterial effects of the system in tomato juice and chicken skin extract, respectively. The findings indicate that the LPO system could probably be used to prevent the growth and survival of salmonellae in minimally processed fruit and vegetable products, but combination of the system with other preservatives or treatments would be needed to effectively inhibit growth and survival of salmonellae in animal products.

Dielectric study of heat-denatured ovalbumin in aqueous solution by time domain reflectometry method.

The dielectric behavior of native and heat-denatured ovalbumins (OVAs) from three avian species in aqueous solution was examined over a frequency range of 100 kHz to 20 GHz, using the time domain reflectometry (TDR) method. For the native OVA solutions, three kinds of relaxation processes were observed at around 10 MHz, 100 MHz, and 20 GHz, respectively; these could be assigned to the overall rotation of protein molecules, the reorientations of the bound water, and the free water molecules, respectively. For the heat-denatured samples, three relaxation processes were also observed. However, the relaxation process at approximately 100 MHz originated via a different mechanism other than the reorientation of bound water, namely, the micro-Brownian motion of peptide chains of heat-denatured protein. From the observed relaxation process at approximately 100 MHz, the relaxation strength of heat-denatured OVA solution for duck was higher than that of OVA solutions for hen and guinea fowl and showed the pH dependency from pH 7.0 to 8.0 for OVAs obtained from all three species. Furthermore, the results demonstrated that the relaxation strength was closely related to surface hydrophobicity of protein molecules and gel rheological properties. It was suggested that the difference in the surface hydrophobicity of protein influenced the dielectric behavior of water around denatured protein, whereas the dielectric behavior of denatured protein could be an indication of the gel rheological properties. Such studies can aid in the understanding of the different network structures of OVA gels from three avian species.

Modification of ovalbumin with a rare ketohexose through the Maillard reaction: effect on protein structure and gel properties.

The effect of nonenzymatic glycation on the structural changes and gelling properties of hen ovalbumin (OVA) through the Maillard reaction was studied. OVA was incubated at the dry state with a rare ketohexose (D-psicose, Psi) and two alimentary sugars (D-fructose, Fru; D-glucose, Glc) at 55 degrees C and 65% relative humidity. To evaluate the modification of OVA by different reducing sugars during the glycation process, the extent of the Maillard reaction, aggregation processes, structural changes, and gelling behaviors were investigated. Reactivity of Psi with the protein amino groups was much lower than that of both Fru and Glc, whereas Psi induced production of browning and fluorescent substances more strongly than the two alimentary sugars did. Furthermore, OVA showed an increased tendency toward multimeric aggregation upon modifying with Psi through covalent bond. The modified OVAs with reducing sugar were similar to nonglycated control sample in Fourier transform infrared (FT-IR) characteristics, but significantly decreased in intensity of tryptophan-related fluorescence. The results indicate that although glycation brought about similar changes in the secondary structure without great disruption of native structure, its influence on the side chains of protein in tertiary structure could be different. Breaking strength of heat-induced glycated OVA gels with Psi was markedly enhanced by the Maillard reaction. These results suggest that Psi had a strong cross-linking activity with OVA; consequently, the glycated OVA with Psi could improve gelling properties under certain controlled conditions.

Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3.

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.

Preparation of antimicrobial reduced lysozyme compatible in food applications.

The structural and antimicrobial functions of lysozyme reduced with food-compatible reducing agents-cysteine (Cys) and glutathione (GSH)-were investigated. The disulfide bonds were partially reduced by thiol-disulfide exchange reactions under heat-induced denaturing conditions from 55 to 90 degrees C. The results showed that treatment of lysozyme with Cys and GSH resulted in the introduction of new half-cystine residues (2-3 residues/mol of protein). The released SH groups, in turn, rendered the lysozyme molecule more flexible, being accompanied by a dramatic increase in the surface hydrophobicity and exposure of tryptophan residues. As a consequence, the resulting reduced lysozymes were more capable of binding to lipopolysaccharides (LPS) and permeabilizing the bacterial outer membrane, as evidenced by the liposome leakage experiment, than were native or heated lysozyme. Both reduced lysozymes displayed significantly higher antimicrobial activity than native or heated lysozyme against Salmonella enteritidis (SE) in sodium phosphate buffer (10 mM, pH 7.2) at 30 degrees C for 1 h. Their minimal inhibitory concentrations (MICs) against the tested bacteria were about 150- and 25-fold lower than their respective MICs of native or heated lysozyme. The results suggest that partially reduced lysozyme could be used as a potential antimicrobial agent for prevention of SE attack.