Studies on Changes in Stored Shell Eggs

The content of the ovomucin gel obtained from the gel parts of stored thick white decreased during storage. Changes of the content of the ovomucin gel (A) was much larger than that of the ovomucin gel (B). The content of the ovomucin sol obtained from the sol parts of stored thick white increased during storage.

The hexose and hexosamine contents of the ovomucin gel (B) decreased to about one half and the sialic acid content decreased to one eighth after 20 days storage at 30°C On the other hand, the carbonhydrate contents of the ovomucin sol (B) increased during storage and those obtained from sol parts of the stored (20 days) thick white were higher than those of the control ovomucin gel (B) obtained from the newly laid thick white. The amino acid composition of the ovomucin gel (B) and sol (B) did not show a great deal of change during storage.

It is suggested from these results that the properties of the ovomucin gel (B) changed greatly during storage; one portion of the ovomucin gel (B), the carbohydrate-rich component, solubilized to the sol parts of stored thick white and the other portion, the carbohydrate-poor component, remained insoluble.     

Solubilization and Characterization of Ovomucin without Chemical Modification

1. The egg white, thick and thin fractions, was solubilized in 1.0% SDS solution by vigorous mixing and subjected to gel filtration on a Sepharose 4B column, eluted with 1.0% SDS. The isolated thick and thin ovomucins were found by analytical disc electrophoresis to be free from contamination with lysozyme.

2. In the velocity sedimentation the two ovomucin fractions behave similarly, both comprising at least two components with sedimentation coefficients 35 S and 30 S.

3. The chemical compositions of the two ovomucin fractions showed only notable difference in that the carbohydrate content of the thick white ovomucin was somewhat higher than that of the thin white ovomucin. The amino acid profiles of the two fractions were similar.

     

The Release of Carbohydrate Rich Component from Ovomucin Gel during Storage

The fractionation pattern of OMG₀, ovomucin gel(B) in fresh egg white, by density gradient column electrophoresis showed two peaks. Each peak was shown to migrate as a single component, with a mobility of either the fast or slow moving component of ovomucin gel(B). Each peak was named as F-component and S-component.

Carbohydrate and sulfate contents of F-component were much higher than these of S-component. The carbohydrate content of F-component and S-component was found to be about 50 and 15 percents of dry matter, respectively. Serine and threonine contents in F-component were much higher than those in S-component.

The fractionation pattern of OMG₂₀, ovomucin gel(B) in egg white stored for 20 days at 30°C, by density gradient electrophoresis showed only one peak which corresponded to S-component, and that of OMS₂₀, ovomucin sol (B) in egg white stored for 20 days at 30°C, showed two peaks which corresponded to F- and S-component.

Ability of F-component to interact with lysozyme was greater than that of S-component.     

Studies on the Dissociation of the Soluble Ovomucin by Sonication

The soluble ovomucin obtained from the liquid part of thick white by gel filtration on a Sepharose 4B was an aggregated and polymerized molecule (intrinsic viscosity was 365 ml/g and molecular weight was 8.3 × 10⁶) and it was unable to dissociate the soluble ovomucin into two components without modifications.

Molecular weight and reduced viscosity of the soluble ovomucin decreased markedly with time of sonication. By the sonication for 10 min, it was successful to fractionate it into carbohydrate rich and poor component by density gradient electrophoresis, cellulose acetate electrophoresis and DEAE-cellulose column chromatography.

Concerning carbohydrate and amino acid compositions of two components obtained from the sonicated soluble ovomucin, it was found that the carbohydrate poor component corresponded to the reduced S-component or the reduced α-ovomucin, and the carbohydrate rich component to the reduced F-component or the reduced β-ovomucin.

It was considered that the sonicated soluble ovomucin was an intermediate of the aggregated, polymerized ovomucin (the soluble ovomucin) and the monomeric ovomucin (the sonicated and reduced soluble ovomucin).     

Effects of the Storage in an Atomosphere of Carbon Dioxide on Ovomucin

The yolk index of newly laid egg was 0.50, while that of the egg stored at 30°C for 15 days in an atmosphere of carbon dioxide at a pressure of 2 kg/cm² and in air was 0.43 and 0.25, respectively.

The carbohydrate content of ovomucin gel (B) obtained from the eggs stored in an atmosphere of carbon dioxide was higher than that of ovomucin gel (B) obtained from the eggs stored in air.

The free boundary electrophoretic pattern of ovomucin gel (B) obtained from the eggs stored in an atmosphere of carbon dioxide consisted of two peaks, and the relative mobility of each peak showed the same value as that of the corresponding each peak of ovomucin gel (B) obtained from newly laid egg white.

The fractionation pattern obtained by density gradient column electrophoresis of ovomucin gel (B) obtained from the eggs stored in an atmosphere of carbon dioxide showed two peaks, peak-F and peak-S, while that of the eggs stored in air showed a considerable diminution in peak-F.

From these results, discussion was made about the occurrence and mechanism of egg white thinning when eggs were stored in an atmosphere of carbon dioxide.     

Requirements for Nutrients and Minerals by Candida brumptii IFO 0731 Producing Succinic Acid from n-Paraffin

The effect of several materials on viscometric behaviour of the soluble ovomucin was determined with a cone plate viscometer.

The soluble ovomucin showed a rapid increase in viscosity above 1.5 mg/ml, and a high concentration of the soluble ovomucin led to gel whose viscosity was comparable to that of the insoluble ovomucin. The apparent viscosity of the soluble ovomucin decreased with an increase in NaCl concentration and upon addition of lysozyme as well as of CaCl₂. The soluble ovomucin in the presence of the sonicated β-ovomucin showed an increase in viscosity on addition of a small amount of CaCl₂.

It is assumed that a great increase in viscosity of egg white may result from removal of sodium ion and lysozyme.     

Studies on Changes in Stored Shell Eggs

Being solubilized by treatment with 0.01 m mercaptoethanol, the ovomucin gel(B) was found in free boundary electrophoresis to contain subunits which were consisted of two components. Changes in the physicochemical properties of all the insoluble ovomucin gel(B) and sol(B) obtained from stored egg white were studied after this treatment.

The fast moving component of the ovomucin gel(B) in free boundary electrophoresis decreased during storage and disappeared completely after 30 days. On the other hand, the fast moving component of the ovomucin sol(B) increased during storage.

The acid mucoprotein concentration of the ovomucin gel(B) in acrylamide gel electrophoresis decreased and that of the ovomucin sol(B) increased during storage, although the protein pattern did not show significant changes.

The interaction of the ovomucin gel(B) with lysozyme decreased whereas that of the ovomucin sol(B) increased during storage.

By summarizing these results, a model of ovomucin gel structure and a mechanism of egg white thinning were proposed.     

The Inhibitory Properties of Chicken Egg White Ovomucin against Aggregation of κ-Casein by Rennin

It has been shown that ovomucin, the structural glycoprotein of chicken egg white, has the inhibitory activity against aggregation of κ-casein by rennin.

The inhibitory activity of F-ovomucin, carbohydrate rich component, was much higher than that of S-ovomucin, carbohydrate poor component. The inhibitory activity was remarkably decreased by removal of sialic acid residues from ovomucin.     

Isolation,Characterization and Synthesis of Pimprinine,Pimprinethine and Pimprinaphine,Metabolites of Streptoverticillium olivoreticuli

The sulfated glycopeptides in ovomucin, chalazae and yolk membrane were isolated from the proteolytic digests by gel filtration on a Bio-Gel P-100 column and DEAE-Sephadex A-25 column chromatography. These sulfated glycopeptides contained N-acetylhexosamine (23.3-26.8%), hexose (23.6-24.4%), sialic acid (11.2-18.0%), sulfate (5-12.1%) and peptide (17.5-18.1%). The sulfate contents of glycopeptides in chalazae and yolk membrane were much higher than those in ovomucin, about two times in a molar ratio to hexosamine. The sedimentation patterns of each sulfated glycopeptide were single and the sedimentation constants were around 3 S, suggesting that these sulfated glycopeptides were macromolecular components. Thus, the presence of highly sulfated glycoproteins was confirmed in chalazae and yolk membrane, which were different from those in ovomucin.     

The Interaction between Ovomucin and Egg White Proteins

The interaction between ovomucin and egg white proteins has been investigated. Ovomucin interacted not only with lysozyme but also with such proteins in egg white as ovalbumin and conalbumin. The interaction increased correspondingly as the lysine content of proteins become higher. The maximum turbidity of ovomucin-lysozyme aggregates was proportionally dependent on the rate of acetylation of lysozyme, but that of ovomucin-ovalbumin aggregates was slightly dependent on the rate of acetylation of ovalbumin. The ovomucin-protein interaction decreased remarkably by the removal of sialic acid in ovomucin, but increased by the removal of polyhydroxyl group in sialic acid. From these results, the binding site of interaction was considered in detail.     

Role of magnesium chloride on the purity and activity of ovomucin during the isolation process

Purification of ovomucin is still an empirical technique and sometimes insufficient quantities of ovomucin are purified to allow characterization. Here we aimed to investigate the effect of MgCl(2) on the purity and bioactivity of ovomucin during isoelectric precipitation process and to develop an effective protocol to prepare pure ovomucin with high bioactivity. It was found that addition of MgCl(2) is an alternative approach to remove lysozyme from ovomucin, and that the hemagglutination inhibition (HI) activity of ovomucin with MgCl(2) against New Disease Virus (NDV) was about two times higher than the protein without salts. Thus, an improved procedure comprises a precipitation with 0.05 mol/L CaCl(2) followed by precipitation with 0.05 mol/L MgCl(2) was developed for the isolation of ovomucin. Better adhesion property of ovomucin was observed when low concentration of MgCl(2) was added in the designed ELASA test, whereas the adhesion property of the pure ovomucin without salts to NDV was lower. Thus, magnesium (II) plays an important role in the activity of ovomucin, and the alternative method developed in this study may significantly facilitate the further research on the mechanism of ovomucin activity.     

Nature of the Carbohydrate Side Chains and Their Linkage to the Protein in Chicken Egg White Ovomucin

Some proteolytic digests of chicken egg white ovomucin were fractionated and characterized. It was shown that there are at least three types of carbohydrate side chains in ovomucin; a chain composed of galactose, galactosamine, sialic acid and sulfate in a molar ratio of about 1: 1: 1: 1, a chain composed of galactose and glucosamine in a molar ratio of about 1:1, and a chain composed of mannose and glucosamine in a molar ratio of about 1:1. It was also shown that the carbohydrate side chain composed of galactose, galactosamine, sialic acid and sulfate is linked O-glycosidically to serine or threonine in the protein core of ovomucin.     

Interactions involved in ovomucin gel-forming properties: a rheological-biochemical approach

Different kinds of interactions involved in the properties of ovomucin gel formation from hen egg white were studied by combining physical and biochemical methods. A decrease in viscosity of the ovomucin gel was observed when it was subjected to chymotrypsin or sonication treatment. The viscosity decrease correlated with a change from non-Newtonian to Newtonian properties of the ovomucin gel. By treatment of the gel with either 5 M guanidinium HCl, 6 M urea, or 5% sodium dodecyl sulfate a change from non-Newtonian to Newtonian properties was also obtained. Although high ionic strength or sialic acid liberation from the ovomucin gel by neuraminidase treatment provoked a decrease in viscosity, it was not followed by a change in non-Newtonian properties. The results obtained suggest that different noncovalent interactions might be involved in gel formation. Electrostatic interactions (partially destroyed by sialic acid removal or 2 M NaCl) and hydrophobic interactions might be responsible for protein-mucin and mucin-mucin interactions. Other bonds susceptible to chymotrypsin treatment and sonication would be involved in the interaction between mucin subunits.     

The supramolecular organization of ovomucin. Biophysical and morphological studies.

Ovomucin participates in the ovomucin-gel-forming properties because of its shape and its ability to interact in a specific spatial organization. Purified from chicken egg-white by exclusion chromatography with Sephacryl S-300 and Sepharose CL-2B and analysed by light-scattering, it exhibited an Mr of about 40 x 10(6). This large Mr can be explained by the aggregation of polymers that can be degraded into 3 x 10(6)-Mr fragments by reduction with dithiothreitol. The values for hydrodynamic parameters such as Mr, radius of gyration, hydrodynamic radius, mass per unit length and combinations of them suggested that ovomucin is a linear and highly flexible molecule conferring upon it a random-coil-like structure in 0.2 M-NaCl solution. Analysis of the ovomucin molecules by electron microscopy revealed its linear character but also indicated a lower Mr than that obtained in the light-scattering experiments. By temperature-induced non-specific aggregation of an ovomucin solution containing other globular egg proteins, an attempt was made to find out what conditions are required for gel formation and to examine the quality of aggregation that is obtained under these conditions. Results show that the viscosity of the solution did not increase after heat treatment. Apparently, in the ovomucin gel, specific spatial organization of the ovomucin molecules is required for hydrogel formation.     

饲料糖水平对大黄鱼生长和糖代谢的影响

以初始体重为（137.5±0.4）g的大黄鱼Larimichthys crocea为实验对象,在海水浮式网箱中进行为期8周的摄食生长实验,研究饲料中糖水平对其生长、饲料利用、血液生化指标和糖代谢酶活力等的影响,以确定大黄鱼的饲料糖需求量。实验饲料按等氮（粗蛋白质45%）等能（18 kJ/g）设计,糖含量分别为1.75%、6.67%、13.64%、21.15%、26.69%和32.25%。结果表明随着饲料糖水平的升高,大黄鱼特定生长率（SGR）先升高后降低,当糖含量为26.69%时,SGR达最大值,显著高于糖含量为1.75%、6.67%、13.64%和32.25%处理组（P < 0.05）。饲料效率（FER）和蛋白质效率（PER）均在糖含量为13.64%-21.15%时显著高于其他处理组（P < 0.05）。随饲料中糖水平的升高,全鱼粗脂肪含量显著降低,在糖含量为32.25%时降至最低（10.56%）,显著低于其他处理组（P < 0.05）。肝体比和肝糖原含量均随饲料糖水平的升高而显著升高（P < 0.05）,在糖含量为32.25%时达到最大值,显著高于糖含量为1.75%和6.67%处理组（P < 0.05）。随饲料糖水平的升高,血浆甘油三酯和胆固醇水平均显著降低（P < 0.05）,而血糖水平不受饲料糖含量的影响（P>0.05）。大黄鱼血清溶菌酶、脂蛋白脂酶和肝脂酶活性均随饲料糖水平的升高显著降低（P < 0.05）,而肠淀粉酶活性表现为先升高后降低,在糖含量为26.69%时,酶活力达到最大值。随饲料糖水平的升高,大黄鱼肝脏己糖激酶活性先上升后下降,在糖含量为21.15%时达到最大值,显著高于糖含量为32.25%处理组（P < 0.05）,而丙酮酸激酶活力在糖水平为32.25%时达到最大值,显著高于糖含量为1.75%和6.67%处理组（P < 0.05）。用二次多项回归模型拟合特定生长率和饲料糖水平的关系,得到大黄鱼饲料中最适糖含量为22.7%。     

Effect of operating conditions on the extraction of ovomucin

Ovomucin, mainly responsible for the gelatinous property of egg white, has potential applications as a functional food and nutraceutical ingredient. A 2-step method for ovomucin preparation was recently developed. The purpose of this study was to determine the effects of various operating conditions, such as pH, NaCl concentrations and extraction volume at the second extraction, temperature, and centrifugation force, on the purity and yield of ovomucin. Our results showed that pH has a significant effect on the purity and yield of ovomucin extracts. Increasing the extraction pH from 4.0 to 5.0 could significantly (p < 0.05) increase the purity and yield of ovomucin; at pHs higher than 5.0, the purity was not affected but the yield was significantly decreased. The highest yield of ovomucin extract (308 mg/100 g of egg white) was achieved at pH 5.0 while the highest purity was achieved at pH 7.0. There is a trend that the purity of ovomucin increased (p < 0.05) but the yield of ovomucin decreased (p > 0.05) at increasing salt concentrations. Reducing extraction volume did not affect the yield of ovomucin whereas its purity was significantly decreased. The yield of ovomucin however was significantly increased at increasing settling time or centrifugation force, but the purity was less affected. Extraction of ovomucin at room temperature could significantly reduce the extraction yield compared to that at lower temperature (4 °C) but the purity was not affected.     

Fractionation and Characterization of the Sulfated Oligosaccharide Chains of Ovomucin

The O-glycosidically linked carbohydrate units of ovomucin were released from serine and threonine in peptide as oligosaccharide chains by alkali treatment with and without borohydride. Two sulfated oligosaccharides were fractionated by using gel filtration and ion-exchange chromatography. The yield of sulfated oligosaccharides released by alkali treatment was higher in the presence of borohydride than in the absence of borohydride. The sulfated oligosaccharides released by alkali treatment with borohydride were as follows: an oligosaccharide composed of N-acetylgalactosaminitol, galactose, N-acetylneuraminic acid and sulfate in a molar ratio of about 1: 1: 1: 1 and another oligosaccharide in a molar ratio of about 1:1: 0.6: 0.5.     

Avian egg's white ovomucoid as food-allergen for human

Hen eggs are considered as the most common reason of a food allergy in humans. The most important allergens of egg white proteins are as follows: ovomucoid, lysozyme, ovalbumin and ovomucin. Ovomucoid is a Kazal-type protease inhibitor which accounts for about 10% of avian egg white protein. It is a glycoprotein containing 20 through 25% carbohydrates. The molecule of ovomucoid is composed of three homologous domains. All avian ovomucoid domains contain six cysteines in similar location that form three intradomain disulfide bonds. Ovomucoid (Gal d1) is one of the major allergen in hen's egg. It is a glycoprotein comprising 186 amino acids, and it has a molecular weight of 28000 Da and an isoelectric point of 4.1. Ovomucoid has antibacterial activity resulting from its ability to inhibit bacterial proteolytic enzymes crucial for microbial growth. Many studies reveal that ovomucoid is a thermo stable molecule.     

The Binding Groups in Ovomucin-lysozyme Interaction

The ovomucin-lysozyme aggregation was remarkably affected by pH or ionic strength. The extent of interaction of F-ovomucin with lysozyme was much larger than that of S-ovomucin. The ovomucin-lysozyme interaction decreased correspondingly, at a rate depending on the time at which ovomucin was modified by neuraminidase. On the other hand, the ovomucin-lysozyme interaction disappeared completely by acetylation, succinilation, or carbamylation of lysyl ε-amino groups in lysozyme, but it was not greatly affected by guanidination of lysyl ε-amino groups in lysozyme. From these results, it was confirmed that the electrostatic interaction between the negative charges of the terminal sialic acid in ovomucin and the positive charges of lysyl ε-amino groups in lysozyme is essential for the ovomucin-lysozyme interaction.     

The Isolation and Fractionation of Chicken Egg White Ovomucin

Three fractions of chicken egg white ovomucin were obtained from ovomucin precipitated by the classical method of dilution of egg white with water. The first fraction was obtained by dilute salt extraction of the precipitated ovomucin and consisted of smaller components of ovomucin. The second fraction was obtained by extraction with 1 M KCl of the precipitate remaining after dilute salt extraction and consisted of a heterogeneous mixture of components of larger mass. The third fraction, the ovomucin remaining after concentrated salt extraction, was insoluble.

The two soluble fractions were found to be unstable with time (pH 8, μ 0.2). The rate of breakdown was slower in concentrated salt solution. The final breakdown product(s) for both fractions appeared to be a 6 S component (s) of molecular mass 1.5 × 10⁵ daltons.