Inhibiting effects of egg white dry-heated at 120 degrees C on heat aggregation and coagulation of egg white and characteristics of dry-heated egg white.

Dialyzed and freeze-dried egg white (FDEW) was dry-heated at 120 degrees C for up to 6 h. The inhibiting effects of the dry-heated egg white (DHEW) on the heat aggregation and coagulation of egg white (as 10% FDEW solution) and characteristics of the DHEW were examined. From the changes in turbidities and soluble protein contents of supernatant in various mixtures of 10% FDEW and DHEW solutions induced by heating (60 degrees C, 5 min), it was found that the inhibiting capacity increased with increases in the dry-heating time (DHT). The FDEW proteins were denatured with a mild conformational change (not secondary but tertiary structure) with the increase in DHT and aggregated partially. However, the more transparent solutions of DHEW containing soluble aggregates according to DHT were also obtained after heating. The transparency according to DHT came to be scarcely affected by the NaCl concentration and the dilution with diluents containing SDS, urea, and 2-mercaptoethanol. These findings suggest that the heat aggregations and coagulations of ovotransferrin and lysozyme in the FDEW were inhibited by their bindings with the soluble aggregates in DHEW.     

Inhibition of bacterial adhesion and salmonella infection in BALB/c mice by sialyloligosaccharides and their derivatives from chicken egg yolk

The effects of an egg-yolk-derived sialyloligosaccharide (YDS), asialo-YDS, and a sialylglycopeptide of YDS (SGP) on bacterial adhesion to intestinal epithelial cells and on Salmonella infection in BALB/c mice were examined. YDS, its derivative asialo-YDS, and SGP strongly inhibited the binding of Salmonella enteritidis but not E. coli K-88 to a human epithelial cell line, Caco-2. In a Salmonella infection experiment using BALB/c mice, oral administration of these reagents effectively prevented the bacteria from proliferating in spleen, as well as preventing lethality. An experiment using radioactive SGP orally administered to mice revealed that the compound was absorbed from the intestine into blood and eliminated via urine within 8 h. However, these reagents did not influence the production of TNF-alpha or NO. in culture macrophages. The results suggest that they inhibit Salmonella infection not by activating macrophages but by inhibiting the entry of bacteria through the gut, suggesting that YDS and its derivatives are useful for preventing Salmonella infection when ingested continuously.     

Soy processing affects metabolism and disposition of dietary isoflavones in ovariectomized BALB/c mice

Soy foods and nutritional supplements are widely consumed for potential health benefits. It was previously shown that isoflavone-supplemented diets, which contained equal genistein equivalents, differentially stimulated mammary tumor growth in athymic mice based on the degree of processing. This paper reports plasma pharmacokinetic analysis and metabolite identification using the parental mouse strain fed the same diets, which contained genistin, mixed isoflavones, Novasoy, soy molasses, or soy flour plus mixed isoflavones. Whereas the degree of soy processing did affect several parameters reflecting isoflavone bioavailability and gut microflora metabolism of daidzein to equol, stimulation of tumor growth correlated significantly with only the plasma concentration of aglycon genistein produced by the diets. This conclusion is consistent with the known estrogen agonist activity of genistein aglycon on mammary tumor growth. Conversely, plasma equol concentration was inversely correlated with the degree of soy processing. Although antagonism of genistein-stimulated tumor growth by equol could explain this result, the very low concentration of aglycon equol in plasma (12-fold lower relative to genistein) is inconsistent with any effect. These findings underscore the importance of food processing, which can remove non-nutritive components from soy, on the pharmacokinetics and pharmacodynamics of isoflavones. Such changes in diet composition affect circulating, and presumably target tissue, concentrations of genistein aglycon, which initiates estrogen receptor-mediated processes required for the stimulation of tumor growth in a mouse model for postmenopausal breast cancer.     

Identification and characterization of ovalbumin gene Y in hen egg white

Ovalbumin gene Y has been known as a member of the ovalbumin gene family since 1982, when its encoding gene was sequenced. In the present study, ovalbumin gene Y has been demonstrated as a new minor protein of hen egg white. This protein has been isolated by isoelectrofocalization and two-dimensional polyacrylamide gel electrophoresis and has been characterized using peptide mass fingerprinting. The concentration ratio of ovalbumin gene Y:ovalbumin is about 13:100. Unlike ovalbumin, ovalbumin gene Y is not phosphorylated, but like ovalbumin, this protein is glycosylated. Ovalbumin gene Y exists as a mixture of three molecular species, which differ in their isoelectric points. The polymorphism of this protein cannot be explained by various glycosylation levels.     

Development of an ELISA for quantifying lysozyme in hen egg white

An indirect enzyme-linked immunosorbent assay (ELISA) by inhibition was developed for quantifying lysozyme in hen egg white (HEW), a protein of value in not only the food and pharmaceutical industries but also for poultry research. Various experimental conditions (coating, antibodies dilutions, samples dilutions, preparations, blocking agents, and incubation times) were assayed to optimize this assay to the quantification of HEW in egg white samples. HEW samples were diluted 1:3000 to avoid matrix effects, possibly resulting from lysozyme interaction with other egg white proteins. Assay linearity for lysozyme ranged from 0.38 to 4.8 mug/mL, with intra- and interassay variations of 6.8% and 7.6%, respectively, and the lower detection limit was 0.264 mug/mL. We found that lysozyme concentrations in albumen from eggs laid by a hen cohort ranged from 2.2 to 4.5 mg/mL, thus underlining interhen variability. Overall, these data present an ELISA assay that is simple, quick, sensitive, accurate, and has been specifically designed to determine lysozyme concentrations in egg white samples.     

Heat-induced changes in the susceptibility of egg white proteins to enzymatic hydrolysis: a kinetic study

A kinetic study was conducted on the effect of heating in the temperature range of 50-92 degrees C, on the susceptibility of ovalbumin and albumen solutions to enzymatic hydrolysis by a mixture of trypsin and alpha-chymotrypsin at 37 degrees C and pH 8.0. Heat treatment resulted in an increase in degree of hydrolysis after 10 min of enzymatic reaction of both ovalbumin and albumen, as measured using the pH-stat method. The time-dependent change in the susceptibility to enzymatic hydrolysis after heat treatment was described by a fractional conversion model (based on an apparent first-order reaction kinetic model). Different end levels of degree of hydrolysis were obtained after heating for a long time at different temperatures, which suggests that the final degree of unfolding of the protein is temperature dependent.     

Phosphorylation of egg white proteins by dry-heating in the presence of phosphate

Food proteins were phosphorylated by heating in a dry state in the presence of phosphate. When casein, whey protein isolate (WPI), and egg white proteins (EWP), which were lyophilized from their solutions in a phosphate buffer, were dry-heated at various temperatures and pH levels for 1-5 days, EWP was more highly phosphorylated than casein and WPI. Phosphorylation of EWP was promoted with a decrease of pH from 7.0 to 3.0 when the incubation temperature was raised from 55 to 100 degrees C. The phosphorus content of EWP increased from 0.08 to 0.64% by dry-heating at pH 3.0 and 85 degrees C for 5 days in the presence of phosphate. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The heat-induced polymerization of EWP by dry-heating was not affected by the presence of phosphate. Although the solubility of EWP decreased by dry-heating at pH 3.0-5.5, the phosphorylation depressed the insolubilization at low pH. The phosphate bonds in phosphorylated EWP (P-EWP) were stable at pH 2.0-10.0 and were more acid-labile and base-stable than phosphoesters of egg riboflavin-binding protein (RfBP). (31)P NMR spectral data suggested that besides phosphoesters, phosphodiester and polyphosphate bonds were introduced in P-EWP. Heat stability of EWP was improved, and calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation.     

Antioxidant action of glutathione and the ascorbic acid/glutathione pair in a model white wine

Glutathione was assessed individually, and in combination with ascorbic acid, for its ability to act as an antioxidant with respect to color development in an oxidizing model white wine system. Glutathione was utilized at concentrations normally found in wine (30 mg/L), as well as at concentrations 20-fold higher (860 mg/L), the latter to afford ascorbic acid (500 mg/L) to glutathione ratios of 1:1. The model wine systems were stored at 45 °C without sulfur dioxide and at saturated oxygen levels, thereby in conditions highly conducive to oxidation. Under these conditions the results demonstrated the higher concentration of glutathione could initially provide protection against oxidative coloration, but eventually induced color formation. In the period during which glutathione offered a protective effect, the production of xanthylium cation pigment precursors and o-quinone-derived phenolic compounds was limited. When glutathione induced coloration, polymeric pigments were formed, but these were different from those found in model wine solutions without glutathione. In the presence of ascorbic acid, high concentrations of glutathione were able to delay the decay in ascorbic acid and inhibit the reaction of ascorbic acid degradation products with the wine flavanol compound (+)-catechin. However, on depletion, the glutathione again induced the production of a range of different polymeric pigments. These results highlight new mechanisms through which glutathione can offer both protection and spoilage during the oxidative coloration of a model wine.     

Influence of thiol metabolism of lactobacilli on egg white proteins in wheat sourdoughs

In wheat sourdoughs, the degradation of gluten proteins is favored by acidification and reducing conditions. This study aimed to determine the proteolytic degradation of egg white proteins in wheat sourdoughs acidified with lactobacilli differing in their thiol metabolism. Ovotransferrin was the only major egg white protein that degraded during sourdough fermentations. An extensive degradation of ovotransferrin required a heterofermentative lactobacilli starter, Lactobacillus sanfranciscensis, with glutathione reductase activity. Ovotransferrin was more resistant to breakdown when sourdoughs were acidified with homofermentative lactobacilli or a mutant strain of L. sanfranciscensis lacking the glutathione reductase. Its susceptibility to proteolysis in L. sanfranciscensis sourdoughs is thus attributable to thiol accumulation by L. sanfranciscensis, which apparently altered the structure of ovotransferrin through a reduction of disulfide bonds. Proteolytic degradation of ovotransferrin was attributable to wheat aspartic proteinases. In addition to the susceptibility to proteolysis, other functional properties of egg proteins may be influenced by thiol-exchange reactions.     

Correlation of the protein structure and gelling properties in dried egg white products

The relationship between protein structure and aggregation, as well as heat-induced gelling properties, of seven dried egg white (DEW) products was investigated. Strong correlations were found between average molecular weight and hydrophobicity plus surface SH groups of DEW-soluble protein aggregate (SPA). This suggests that hydrophobic interactions and disulfide bond formation between protein molecules were involved in the aggregation. The average molecular weight of DEW products with alkaline pHs was relatively higher than those with neutral pHs and the same degree of protein unfolding, probably because of more disulfide bond formation between protein molecules. In addition, strong correlations were found between hydrophobicity, surface SH groups plus average molecular weight of DEW-SPA, and physical properties of the gels from DEW products. These data indicated that controlling the aggregation of DEW proteins in the dry state is crucial to controlling the gelling properties of DEW.     

嗜水气单胞菌外膜蛋白（OMP）的原核表达及其对BALB/c小鼠的免疫保护研究

为确定嗜水气单胞菌(Aeromonas hydrophila,Ah)AS1.927株外膜蛋白(OMP)的原核表达产物是否具有抗原性及其对小鼠的免疫保护力.本研究根据GenBank上嗜水气单胞菌外膜蛋白ompA基因序列(GenBank登录号:AF146597)设计1对引物,克隆出ompA全长基因1020 bp(GenBank登录号:EU309491),经Sal Ⅰ和Xhol Ⅰ双酶切后连接pET-30a(+),转化大肠杆菌(Escherichia cDli)BL21,IPTG诱导表达,并进行SDS-PAGE及Westernblot分析鉴定.结果显示重组基因工程菌E.coli[pET-30a-ompA]表达的融合蛋白分子量约为40.7kD,可以被鼠抗嗜水气单胞菌外膜蛋白抗血清识别.将重组基因工程菌Ecoli[pET-30a-om-pA]口服免疫BALB/c小鼠(Mus mussulus);末次免疫1周后用放射免疫分析小鼠肠粘膜分泌型免疫球蛋白A(sIgA)水平,检测结果揭示,该重组基因工程菌能提高sIgA的分泌(P<0.05);同时在小鼠血清中测出特异性抗体IgG(P<0.05).末次免疫2周后用100 LD50的Ah AS1.927(3.3×105> cfu/mL)腹腔注射攻击小鼠,口服重组菌对小鼠的相对免疫保护力(relative percent survival,RPS)为75%,表明重组基因工程菌可诱导小鼠对AhAS1.927产生一定的免疫保护作用.     

Establishment of a highly sensitive sandwich enzyme-linked immunosorbent assay specific for ovomucoid from hen's egg white

A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit was established for quantifying ovomucoid from hen's egg white, which has been considered as one of the major allergen in egg white. The detection limit reached 0.041 ng/mL, and linearity ranged from 0.1 to 6.25 ng/mL. Intra- and interassay coefficient variations were all lower than 5% at three concentrations (0.5, 2.5, and 5 ng/mL). No cross-reactivity was observed with bovine serum, horse serum, goat serum, human serum, duck egg white, goose egg white, quail egg white, and pigeon egg white, but a low level of cross-reactivity was found with chicken serum. The ELISA kit was established on the basis of two monoclonal antibodies (mAbs) recognizing different epitopes of ovomucoid. However, these mAbs were generated using commercially purified ovalbumin as immunogen. Studies on the relative allergenicity and antigenicity of egg white protein have been performed by many researchers, but there were controversial opinions reported previously because of the impurity of each egg white protein used in various studies. In the present work we measured the degree of ovomucoid contamination in commercially purified ovalbumin sample, and the value was about 11%. We also determined the ovomucoid residue in influenza vaccine samples for the first time. These data showed that the ELISA kit we established could serve as an effective method for precisely quantifying concentrations of ovomucoid in the egg industry and as a useful tool for the research of allergenicity and antigenicity of hen's egg proteins.     

Metabolites of oxytetracycline, tetracycline, and chlortetracycline and their distribution in egg white, egg yolk, and hen plasma

4-epioxytetracycline and N-demethyloxytetracycline, as metabolites of oxytetracycline (OTC), 4-epitetracycline and N-demethyltetracycline, as metabolites of tetracycline (TC), and 4-epichlortetracycline, isochlortetracycline (ICTC), 4-epi-ICTC, and N-demethyl-ICTC, as metabolites of chlortetracycline (CTC), were detected in egg yolk and plasma obtained from feeding studies with either OTC, TC, or CTC. In egg white, only OTC, TC with its 4-epimer, and ICTC with its 4-epimer were detected in substantial concentrations. The ratios of epimerization and N-demethylation in the eggs did not change during the medication period. The samples were analyzed by an automated HPLC system (ASTED) with UV, fluorescence, or MS-MS detection.     

Changes in sulfhydryl content of egg white proteins due to heat and pressure treatment

The sulfhydryl (SH) content of egg white proteins (10% v/v or 9.64 mg of protein/mL) after heat (50-85 degrees C) and combined heat- and high-pressure treatments (100-700 MPa, 10-60 degrees C) was determined using 5',5-dithiobis (2-nitrobenzoic acid) (DTNB), both for the soluble fraction and the total protein fraction. Only irreversible changes were taken into account. Both physical treatments were performed at two pH levels: pH 7.6, corresponding to the pH of fresh egg white, and pH 8.8, corresponding to that of aged egg white. Both heat and combined heat- and high-pressure treatment resulted in an exposure of buried SH groups. These exposed SH groups were involved in the formation of disulfide bond stabilized protein aggregates, as shown by gel electrophoresis. Under severe processing conditions (above 70 degrees C at atmospheric pressure or above 500-600 MPa, depending on the temperature applied), a decrease in total SH content could be observed, probably due to the formation of disulfide bonds by oxidation, especially at alkaline pH when the thiolate anion was more reactive. The high degree of exposure of sulfhydryl groups, and subsequent oxidation and sulfhydryl-disulfide bond exchange reactions resulting in soluble aggregates, can explain why pressure-induced egg white gels are softer and more elastic than heat-induced ones. When pressure treatment was performed at low temperatures (e.g., 10 degrees C), a lower pressure was required to induce similar changes in the sulfhydryl content, as compared to higher temperatures (e.g., 25 degrees C), indicating an antagonistic effect between pressure and temperature in the domain studied (10-60 degrees C, 100-700 MPa). Treatment conditions resulting in extensive protein insolubilization were accompanied by a transfer of free sulfhydryl groups from the soluble to the insoluble protein fraction. These SH groups were mainly accessible to DTNB.     

Vascular effects, angiotensin I-converting enzyme (ACE)-inhibitory activity, and antihypertensive properties of peptides derived from egg white

In this study, we have identified novel antihypertensive peptides derived from egg-white proteins. The sequences YRGGLEPINF and ESIINF produced an acute blood-pressure-lowering effect in spontaneously hypertensive rats upon a single oral administration. Our results suggest that the antihypertensive action could be attributed to a vascular-relaxing mechanism that would occur in vivo independently of angiotensin I-converting enzyme (ACE) inhibition, because neither these peptides nor their main digestion fragments, except for the dipeptide YR, acted as ACE inhibitors in vitro. The vasodilator and antihypertensive activity of the sequences ESI and NF would explain the blood-pressure-lowering effect of ESIINF. With regard to YRGGLEPINF, in addition to NF, YR appeared as the main fragment responsible for its activity. The dipeptide YR, named kyotorphin and previously identified as an endogenous analgesic neuropeptide in the central nervous system, showed strong vasodilator and antihypertensive properties. The structure-activity features of the vasodilator peptides are discussed.     

Inhibitory effects of tea catechins and O-methylated derivatives of (-)-epigallocatechin-3-O-gallate on mouse type IV allergy

The inhibitory effects of tea catechins, the O-methylated derivatives of (-)-epigallocatechin-3-O-gallate (EGCG), and the polyphenol extracts from tea leaves (Camellia sinensis L.) on oxazolone-induced type IV allergy in male ICR mice were investigated. Four major tea catechins and two O-methylated derivatives, (-)-epigallocatechin-3-O-(3-O-methyl)gallate (EGCG3' 'Me) and (-)-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG4' 'Me), showed significant inhibitory effects on mouse type IV allergy after a percutaneous administration at a dose of 0.13 mg/ear. Among tea catechins, the compounds including galloyl moieties, such as EGCG and (-)-epicatechin-3-O-gallate (ECG), showed the strongest inhibitory activities on mouse type IV allergy. The inhibitory activities of EGCG3' 'Me and EGCG4' 'Me were higher than that of EGCG at a dose of 0.05 mg/ear. Polyphenol extract from tea leaves of Benihomare cultivar, which includes EGCG3' 'Me, strongly inhibited mouse type IV allergy after percutaneous administration in comparison with that from Yabukita cultivar, which does not include EGCG3' 'Me, at doses of 0.05 and 0.13 mg/ear. EGCG3' 'Me is thought to contribute, at least in part, to the inhibitory ability of Benihomare tea leaves on mouse type IV allergy. EGCG and the polyphenol extracts from Benihomare and Yabukita tea leaves also inhibited mouse type IV allergy by oral administration at 1 h before the sensitization and at 1 h before the challenge with oxazolone. Therefore, daily intake of tea drinks could have potential to prevent type IV allergy.     

嗜水气单胞菌外膜蛋白(OMP)在乳酸乳球菌中的表达及其对BALB/c小鼠的免疫保护效果

乳酸球菌(Lactococcus lactis)是一种安全级微生物,为活载体疫苗传递抗原的理想选择.嗜水气单胞菌(A eromonas hydrophila,Ah)能引起鱼类等多种动物的败血病,因其血清型众多,寻找共同保护性抗原是进行疫苗研究的重点.为探究嗜水气单胞菌AS1.927株外膜蛋白(OMP)在乳酸乳球菌中的表达和检测其表达产物对小鼠的免疫保护效果,本研究将己克隆的ompA基因经BamH Ⅰ和Xba Ⅰ双酶切后连接载体pNZ8048,转化乳酸乳球菌NZ9000,nisin诱导表达,并进行SDS-PAGE分析鉴定.结果显示,重组基因工程菌L.lactis[pNZ-ompA]表达的融合蛋白分子量约为36.2 kD,成熟蛋白分子量约为33.7 kD.将重组基因工程菌L.lac tis [pNZ-ompA]口服免疫BALB/c小鼠(Mus musculus),用放射免疫法检测末次免疫一周后的各组小鼠肠粘膜分泌型免疫球蛋白A(sIgA)的水平以及用间接ELISA法检测小鼠血清特异性抗体IgG,结果发现,该重组基因工程菌能显著提高sIgA的分泌(P＜0.05);同时在小鼠血清中的特异性抗体含量也显著高于对照组(P＜0.05).末次免疫两周后用100 LD50的Ah AS 1.927(3.3×105 cfu/mL)腹腔注射攻击小鼠,口服重组菌对小鼠的相对免疫保护力(relative percent survival,RPS)为87.5％,表明重组基因工程菌可诱导小鼠对AhAS1.927产生一定的免疫保护作用.该结果将为开发安全有效的口服基因工程鱼用疫苗提供一定的实验基础.     

Improvement of functional properties of egg white protein through phosphorylation by dry-heating in the presence of pyrophosphate

Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 3.0-7.0 and 85 degrees C for 1 and 5 days, and the functional properties of the phosphorylated EWP (PP-EWP) were investigated. The phosphorylation was accelerated with a decrease of pH from 7.0 to 3.0 and for heating times from 1 to 5 days. The phosphorus content of EWP increased approximately 1.05% by dry-heating at pH 4.0 and 85 degrees C for 5 days in the presence of pyrophosphate, which was higher than that of casein. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The surface hydrophobicity of EWP increased by phosphorylation. The heat stability, emulsifying properties, and digestibility of EWP were improved by phosphorylation. The calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation. A firmer and transparent heat-induced gel of PP-EWP was obtained, and the water-holding capacity of heat-induced PP-EWP gel was higher that that of the control. These results suggest that phosphorylation by dry-heating in the presence of pyrophosphate is a useful method for improving the functional properties of EWP.     

Heat-induced gels of egg white/ovalbumins from five avian species: thermal aggregation, molecular forces involved, and rheological properties

Product processing (heating, pH change, etc.) usually alters protein structure, improves rheological properties, and gives a unique texture to foods. The thermal aggregation and structural properties of ovalbumins from five avian species were studied at different pH values by polyacrylamide gel electrophoresis (PAGE) and determinations of sulfhydryl group content and surface hydrophobicity. The results showed that sulfhydryl group content changed insignificantly in heat-denatured ovalbumins other than hen ovalbumin (pH-independent), and surface hydrophobicity markedly increased (pH-dependent) after heating, with a significant difference among species. Furthermore, it was demonstrated that the hydrophobic interaction and sulfhydryl-disulfide interchange reaction were necessary in the aggregation and cross-linking of gel networks. Creep tests were also used to characterize the gel network structures of various egg white/ovalbumins upon heating. The viscoelastic behavior of the ovalbumins of all species was dependent on pH values, and changed significantly with the phylogeny of these species. With increases in pH value (7.0-8.5), the heat-induced gels of ovalbumins gradually changed from turbid to translucent, the instantaneous modulus (E(0)) increased slightly and reached a nearly constant value, and the Newtonian modulus (etaN) increased significantly in each sample. The heated egg white from these five avian species also formed highly viscoelastic gels, with a good correlation of viscoelastic behavior between ovalbumin and egg white in corresponding species.