Studies on Changes in Stored Shell Eggs

The intrinsic viscosity of ovomucin(B) from the thick white was higher than that of the thin white. The relative area of the fast moving component in the electrophoretic pattern of ovomucin(B) from the thick white was about twice that of the thin white. The hexose, hexosamine and sialic acid contents of ovomucin(B) from the thick white were higher than those of the thin white. The intrinsic viscosity and carbohydrate content of ovomucin from the egg white previously freed from lysozyme were low in comparison with those of ovomucin(B). The carbohydrate contents of crude lysozyme from the thick white were higher than those of the thin white, and crude lysozyme obtained from the thick white contained larger amounts of ovomucin-iike material than the thin white. From these results, discussion were made about the relation between the properties of ovomucin(B) and ovomucin-lysozyme interaction.     

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.     

Co-extraction of egg white proteins using ion-exchange chromatography from ovomucin-removed egg whites

Efficient isolation of egg white components is desired due to its potential uses. Existing methods mainly targeted on one specific protein; an attempt has been made in the study to co-extract all the valuable egg white components in a continuous process. Ovomucin was first isolated by our newly developed two-step method; the resultant supernatant obtained after ovomucin isolation was used as the starting material for ion-exchange chromatography. Anion-exchange chromatography of 100 mM supernatant yielded a flow-through fraction and three other fractions representing ovotransferrin, ovalbumin and flavoproteins. The flow-through fraction was further separated into ovoinhibitor, lysozyme, ovotransferrin and an unidentified fraction which represents 4% of total egg white proteins. Chromatographic separation of 500 mM supernatant resulted in fractions representing lysozyme, ovotransferrin and ovalbumin. This co-extraction protocol represents a global recovery of 71.0% proteins.     

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 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.     

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.     

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.     

Proteomic analysis of the chicken egg vitelline membrane

The avian vitelline membrane (VM) is a multilayered proteinaceous structure separating egg white from yolk. The innermost layer of the VM, deposited onto the oocyte plasma membrane in the ovary, corresponds to the mammalian zona pellucida (ZP). The outer layer is produced in the infundibulum, the first section of the oviduct. Using high-throughput, high-end LC-MS(n) 137 proteins were identified, only 13 of which were known previously to be components of the VM. Depending on the washing protocol, two largely overlapping, but not identical, sets of identified proteins were produced from water-washed and salt-washed VMs. Most of the components of the VM were known previously from other egg compartments, such as, for instance, the egg white proteins lysozyme C, ovalbumin, ovotransferrin, and ovomucin. Specific components of the VM not identified previously in other egg compartments included eight ZP proteins, oviductin protease, and two ATPases. The vitelline outer membrane protein (VMO) VMO II was identified as beta-defensin-11. The list of VM proteins presented in this report is by far the most comprehensive dataset available at present and complements proteomic analyses of chicken egg compartments published previously.     

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.     

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.     

Precipitation of egg white proteins below their isoelectric points by sodium dodecyl sulphate and temperature.

The precipitating of effect of sodium dodecyl sulphate (SDS) on the egg white proteins ovalbumin, conalbumin and lysozyme was studied at 25 degrees C and at different pH values. The proteins precipitated below their respective isolectric points, provided the detergent to protein ratio was appropriate. The pH profile of precipitation was different for the three proteins reflecting net charge differences. The binding of SDS to the proteins was studied with [35S]-labelled SDS and for ovalbumin a ratio of 21--28 SDS molecules/protein molecule, dependent on the concentration of SDS initially used, seem to be required for precipitation at pH 4.5. This number, however, is dependent on pH and increases with an increased positive net charge of the protein. The precipitating effect of SDS was identical for ovalbumin, conalbumin and lysozyme when compared on a gram to gram basis (0.1--0.15 g SDS/g precipitated protein). The precipitated protein was denatured as measured by differential scanning calorimetry, but was also completely redissolved if pH was increased to above the isoelectric point. The precipitating effecto f SDS was also examined at elevated temperatures. The two-phase systems of the proteins induced by SDS at 25 degrees C were heated from 25 degrees C to 90 degrees C at a rate of 1.25 degrees C/min. The precipitation behaviour was similar for the three proteins upon heating. When the SDS concentration was increased the precipitation curves were transferred towards lower temperatures and the courses of precipitation became less sharp. The synergistic effect of SDS and heat on protein precipitation was differentiated by denaturation measurements and radioactive labelling. The ratio SDS to precipitated protein gradually diminished towards higher temperatures but no purely thermal precipitation was found.     

Effects of dietary maize solubles and minerals on albumen quality of fresh and stored brown-shelled eggs

Two hundred and ten Warren SSL laying hens (producing brown eggs) were used to test the effects on Haugh Units (HU) and on egg-white foaming capacity of seven dietary treatments: control (maize, wheat, soya bean meal); 25 and 100 g kg^?1 of maize solubles (DDGS); chromium (10 mg kg^?1); chromium + zinc (10 + 1000 mg kg^?1); magnesium (4 g kg^?1); and current trace mineral supplementation. Several chemical parameters of egg-white (proteins, lysozyme, sialic acid as an indicator of ovomucin, Ca, Mg, Na and K levels) were also recorded. These analyses were performed on frozen albumen from fresh (1-day) and stored (21-day) eggs.Egg production, feed consumption and egg weight were reduced only by Mg supplementation. The HU value of fresh eggs also remained constant except in the case of Cr + Zn supplementation, which caused a small decrease.The foaming capacity of egg white was hardly affected by the dietary treatments; a slight fall in foam stability occurred only after adding trace minerals. Egg storage caused a lightening of foam and increased its stability.Lysozyme activity was decreased and the sodium content of the egg white increased by the lowest level of DDGS, Cr and Mg treatments. The Ca and Mg contents of egg-white dry matter decreased during egg storage.These results are discussed in relation to the dietary use of DDGS or Mg supplementation, the effects of egg storage and the interrelationships between the composition and the functional properties of egg-white.     

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.     

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.

     

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.     

Structural properties of recombinant ovalbumin and its transformation into a thermostabilized form by alkaline treatment.

The recombinant ovalbumin produced in Escherichia coli was purified from the cytoplasmic fraction and analyzed for its chemical and conformational properties. The recombinant ovalbumin displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as egg white ovalbumin. As in the egg white protein, four cysteine sulfhydryls and one cystine disulfide were contained in the recombinant protein, according to the results of amino acid analyses; the disulfide bond was found by a peptide mapping analysis to correspond to the native cystine, Cys73-Cys120. According to a gel electrophoresis analysis, the presence of the disulfide bond was accounted for by specific oxidation of the corresponding cysteine residues during purification of the cytoplasmic protein. Unlike the identity in the conformational and peptide structures, none of the post-translational modifications (N-terminal acetylation, phosphorylation, and glycosylation) that are known with egg white ovalbumin were detected in the recombinant protein. The recombinant ovalbumin was transformed into a thermostabilized form in a similar manner to the transformation of egg white protein into S-ovalbumin; alkaline treatment increased the temperature for thermostability by 8.7 degrees C. These data strongly suggest that the post-translational modifications of ovalbumin are not related to the formation mechanism for S-ovalbumin.     

Slow aggregation of lysozyme in alkaline pH monitored in real time employing the fluorescence anisotropy of covalently labelled dansyl probe

The onset of hen egg white lysozyme aggregation on exposure to alkaline pH of 12.2 and subsequent slow growth of soluble lysozyme aggregates (at 298 K) was directly monitored by steady-state and time-resolved fluorescence anisotropy of covalently attached dansyl probe over a period of 24 h. The rotational correlation time accounting for tumbling of lysozyme in solution (40 microM) increased from approximately 3.6 ns (in pH 7) to approximately 40ns on exposure to pH 12.2 over a period of 6 h and remained stable thereafter. The growth of aggregates was strongly concentration dependent, irreversible after 60 min and inhibited by the presence of 0.9 M l-arginine in the medium. The day old aggregates were resistant to denaturation by 6 M guanidine.HCl. Our results reveal slow segmental motion of the dansyl probe in day old aggregates in the absence of L-arginine (0.9 M), but a much faster motion in its presence, when growth of aggregates is halted.     

Riboflavin-binding protein exhibits selective sweet suppression toward protein sweeteners

Riboflavin-binding protein (RBP) is well known as a riboflavin carrier protein in chicken egg and serum. A novel function of RBP was found as a sweet-suppressing protein. RBP, purified from hen egg white, suppressed the sweetness of protein sweeteners such as thaumatin, monellin, and lysozyme, whereas it did not suppress the sweetness of low molecular weight sweeteners such as sucrose, glycine, D-phenylalanine, saccharin, cyclamate, aspartame, and stevioside. Therefore, the sweet-suppressing activity of RBP was apparently selective to protein sweeteners. The sweet suppression by RBP was independent of binding of riboflavin with its molecule. Yolk RBP, with minor structural differences compared with egg white RBP, also elicited a weaker sweet suppression. However, other commercially available proteins including ovalbumin, ovomucoid, beta-lactogloblin, myoglobin, and albumin did not substantially alter the sweetness of protein sweeteners. Because a prerinse with RBP reduced the subsequent sweetness of protein sweeteners, whereas the enzymatic activity of lysozyme and the elution profile of lysozyme on gel permeation chromatography were not affected by RBP, it is suggested that the sweet suppression is caused by an interaction of RBP with a sweet taste receptor rather than with the protein sweeteners themselves. The selectivity in the sweet suppression by RBP is consistent with the existence of multiple interaction sites within a single sweet taste receptor.     

高速逆流双水相色谱法纯化卵白蛋白

生物大分子的液_固色谱纯化过程中固相载体会产生产物吸附、变性等不良影响。高速逆流色谱无需固相载体 ,且具有高分便率和高回收率的优点 ,其中有机相 水相体系在分离天然产物中应用广泛 ,而应用双水相体系分离生物大分子尚处于研究阶段。双水相高速逆流色谱体系的建立与仪器设备及操作工艺条件密切相关 ,因此利用多分离柱高速逆流色谱仪 ,研究了PEG1000-无机盐双水相体系对标准蛋白质混合物以及卵白蛋白的分离。pH值和PEG浓度对不同种类蛋白质的分配系数影响不同 ,实验发现在pH9.2的150% (W/W)PEG1000 170% (W/W)磷酸钾盐体系中 ,细胞色素C、溶菌酶和肌红蛋白的分配系数差异较大 ,且分布合理 ,因而采用该体系在 0 8mL min流速 ,85 0r min转速的条件下 ,成功分离了细胞色素C、溶菌酶和肌红蛋白的混合物。实验也发现在pH9 2的 16 0 % (W/W)PEG10 0 0 17 0 % (W/W)磷酸钾盐体系中 ,鸡蛋清样品中的主要蛋白质成分:卵转铁蛋白、卵白蛋白和溶菌酶的分配系数差异最大 ,因而采用该体系在 1 8mL min流速、85 0r mi转速的条件下,200min内从鸡蛋清样品中成功分离卵白蛋白,其电泳纯度为100%,收率为95%.     

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.