Productivity and some properties of immunoglobulin specific against Streptococcus mutans serotype c in chicken egg yolk (IgY)

Hens were immunized on thighs by using whole cells of Streptococcus mutans MT8148 serotype c strain as antigen through intramuscular (im) and subcutaneous (sc) routes to investigate the difference of immunization reactions and the changes in yolk antibody activities against antigen after initial immunization. Several properties of crude IgY were examined to evaluate the stability during food processing. Results showed that the specificity of IgY of im treated hens was nearly 10 times as high as those of sc treated antibody. IgY from the hens immunized with the serotype c strain showed significant cross-reactions against serotypes e and f, while minor reactions against serotypes a, b, d, and g were observed. In thermal stability tests, IgY activity in both yolk and crude IgY decreased with the increasing temperature, from 70 to 80 degrees C, but the thermal denaturation rates between those two samples were not significantly different. The addition of high levels sucrose, maltose, glycerol, or 2% glycine displayed effective protection against thermal denaturation of IgY. Lyophilized yolk-5% gum arabic powder showed better stability against proteases.     

Chitosan-alginate microcapsules for oral delivery of egg yolk immunoglobulin (IgY)

Chitosan-alginate microcapsules were evaluated as a method of oral delivery of IgY antibodies. Physical characteristics, encapsulation efficiency (EE%), the loading capacity for IgY (IgY loading percentage, %, w/w of microcapsules), gastro-resistance, and release characteristics of these microcapsules in vitro under varying pH were investigated. Optimum physical factors were established for preparation of homogeneous, spherical, and smooth microcapsules. IgY loading% was not significantly altered by pH of the encapsulation medium. Encapsulation efficiency was highest (73.93%) at a pH of 3.5, above which EE% decreased significantly (p < 0.05). IgY was released from microcapsules upon exposure to simulated intestinal fluid (SIF, pH 6.8), and decreasing pH increased significantly IgY release (p < 0.05). The stability of IgY in simulated gastric fluid (SGF, pH 1.2) was greatly improved by encapsulation in chitosan-alginate microcapsules, and the residual activity was not affected by pH of the encapsulation medium. Moreover, microencapsulated IgY was significantly resistant to pepsin hydrolysis. This approach may enable intact IgY to reach target microorganisms within the lower digestive tract.     

Isolation and characterization of immunoglobulin in yolk (IgY) specific against hen egg white lysozyme by immunoaffinity chromatography

Six hens were intramuscularly (im) immunized once a week for 3 weeks using chicken egg white lysozyme (LS) as antigen. Antibody (immunoglobulin in yolk, IgY) ELISA values of 10(3)-fold diluted yolk were almost as high as 1.879 in the sixth week and maintained a value of 0.756 in the eighth week after the initial immunization treatment. The purification efficiency (specific activity of purified IgY against LS/specific activity of antibody in yolk against LS) of IgY specific against LS isolated by laboratory-prepared LS-bound (IgY-) Sepharose 4 Fast Flow immunoaffinity column was approximately 3380. By applying various amounts (0-22 mg) of the thusly obtained IgY specific against LS to the immunoaffinity column, the binding capacity (q(m)) and dissociation constant (K(d), M(-1)) of such immunoaffinity gel for IgY against LS were found to be 0.68 mg of IgY/mL of wet gel (0.54 mg of IgY/mg of LS) and 7.13 x 10(-6) M, respectively, as determined by Langmuir-type adsorption isotherms.     

Effects of hen egg yolk immunoglobulin in passive protection of rainbow trout against Yersinia ruckeri

Anti-Yersinia ruckeri egg yolk immunoglobulin (IgY) was transferred to egg yolk after immunization of White Leghorn hens with formalin-killed whole cells of serovar 1 (RS1154) and serovar 2 (RS1153)Y. ruckeri and its lipopolysaccharide (LPS). The IgY was specific for its homologous LPS in western immunoblot, whereas some protein bands were commonly recognized, even by IgY from eggs of unimmunized hens. Purified LPS from both Y. ruckeri serovar types 1 and 2 had a very poor immunogenicity. The IgY activity was stable when processed into pellet form by a microbial transglutaminase treatment and showed a considerable resistance against acid pepsin for at least 2 h. Feeding specific anti-serovar 1 Y. ruckeri IgY to fish either before or after immersion infection produced marginal reductions in mortalities and in intestine infection. The same IgY did passively protect rainbow trout against infection when administered by intraperitoneal injection 4 h before an immersion challenge.     

Identification and characterization of the precursor of chicken matrix metalloprotease 2 (pro-MMP-2) in hen egg

Using zymography and mass spectrometry, we identified for the first time the precursor of chicken matrix metalloprotease 2 (pro-MMP-2) as a complex with TIMP-2 (tissue inhibitor of metalloproteinases) in egg white and yolk. Real-time polymerase chain reaction confirmed that MMP-2 and its inhibitors TIMP-2 and TIMP-3 were expressed all along the oviduct and in the liver of laying hens. We also demonstrated that the processing of pro-MMP-2 into mature MMP-2 by serine proteases does not occur in vivo, although purified pro-MMP-2 undergoes proteolytic maturation by these proteases in vitro. Moreover, the relative pro-MMP-2 activity assessed by gelatin zymography was shown to decrease in egg white during the storage of unfertilized or fertilized eggs. However, the mature form of 62 kDa MMP-2 could not be detected. The fact that MMP-2 is found as a proform in fresh eggs suggests that the activity of this metalloprotease is regulated under specific conditions during embryonic development.     

Development of an indirect competitive ELISA for flumequine residues in raw milk using chicken egg yolk antibodies

To detect flumequine in raw milk, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed. By carbodiimide conjugation, flumequine was conjugated to cationized bovine serum albumin (cBSA-flumequine) and to cationized ovalbumin (cOVA-flumequine). For the immunization of chickens, cBSA-flumequine was used, which allowed the isolation of specific chicken egg yolk immunoglobulins (IgY) for flumequine. As the coating antigen in the immunoassay, cOVA-flumequine was used. In the indirect competitive assay, standard flumequine was incubated together with the anti-flumequine antibodies. The antibody by which the lowest concentration of free flumequine that gives 50% inhibition of binding (IC50) was found in aqueous dilution was further tested for the applicability to detect flumequine in raw milk. An IC50 level in milk was reached that was about 5 times lower than in aqueous solution. So flumequine can be detected directly in raw milk at maximum residue level (50 microg/kg). No cross-reactivity was noticed with various related quinolones.     

Identification of antiadhesive fraction(s) in nonimmunized egg yolk powder: in vitro study

The presence of antiadhesive component(s) in the hen egg yolk against foodborne pathogens was anticipated from results of a previous animal study conducted by the authors. The previous work showed egg yolk powder without specific antibodies is effective in controlling Salmonella enteritidis,Salmonella typhimurium, and Escherichia coli O157:H7 colonization in laying hens. Therefore, this study was necessary to locate the activity and identify the effective component(s). In vitro experiments were conducted using confluent Caco-2 cell monolayers. S. enteritidis, S. typhimurium, and E. coli O157:H7 were investigated against the various extracted granule and plasma fractions in three different assays: adhesion elimination, adhesion prevention, and antimicrobial. This study revealed original findings and identified the protective yolk fraction against the foodborne pathogens as the granule component, high-density lipoproteins (HDL). The protective activity conveyed by HDL was confirmed to remain intact despite peptic and tryptic enzymatic digestion and to have antiadhesive but not antimicrobial effect.     

Cholecalciferol and 25-hydroxycholecalciferol content of chicken egg yolk as affected by the cholecalciferol content of feed.

The predominant source of vitamin D is the synthesis of cholecalciferol in the skin by the action of sunlight; however, due to the relative lack of sunlight, the intake of vitamin D from food is emphasized during winter, especially in the northern countries. Only a few foods (fish, eggs, wild mushrooms, meat, and milk) are natural sources of vitamin D. In addition, the content of vitamin D in foods is generally low, and some groups of people obtain amounts of vitamin D that are too small from their diet. The present study was designed to determine whether it is possible to increase the vitamin D content of egg yolk by giving hens feed containing elevated levels of cholecalciferol. Three cholecalciferol levels were tested: 26.6 (1064), 62.4 (2496), and 216 microgram (8640 IU)/kg feed. Egg yolk samples were taken after 0, 4, 5, and 6 weeks and were assayed for the presence of cholecalciferol and 25-hydroxycholecalciferol using an HPLC method. According to the present study, there was strong positive correlation between cholecalciferol content in poultry feed and cholecalciferol (r = 0. 995) and 25-hydroxycholecalciferol (r = 0.941) content in egg yolk.     

Antioxidative stress activity of oligophosphopeptides derived from hen egg yolk phosvitin in Caco-2 cells

The protective effects of hen egg yolk phosvitin phosphopeptides (PPPs) against hydrogen peroxide (H2O2)-induced oxidative stress were evaluated in an in vitro assay using human intestinal epithelial cells. Caco-2 cells were stimulated with 1 mM H2O2 for 6 h, and the secretion of IL-8, a proinflammatory mediator, was determined by ELISA as a biomarker of oxidative stress. The inhibition of H2O2-induced IL-8 secretion from Caco-2 cells was observed by pretreatment for 2 h with PPPs, but not with phosvitin. PPPs also suppressed the formation of malondialdehyde in H2O2-treated Caco-2 cells. Furthermore, intracellular glutathione levels and glutathione reductase activity were elevated by the addition of PPPs. The protective effects of PPPs against H2O2-induced oxidative stress were almost the same as that of glutathione, and PPPs with a high content of phosphorus exhibited higher protective activity than PPPs without phosphorus; however, phosphoserine itself did not show any significant antioxidative stress activity. These findings suggest that oligophosphopeptides from hen egg yolk phosvitin possess novel antioxidative activity against oxidative stress in intestinal epithelial cells and that phosphorus and peptide structure seem to have a key role in the activity.     

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.     

Detection of hexabromocyclododecane and its metabolite pentabromocyclododecene in chicken egg and fish from the official food control

During routine gas chromatography with electron capture detection (GC/ECD) analysis of chicken eggs, we observed that the most prominent peak in some samples did not match the retention time of any of the food contaminants screened. Subsequent GC coupled with mass spectrometry (GC/MS) studies clarified that the mass spectrum of the peak was very similar to hexabromocyclododecane (HBCD), which was also identified by GC/MS in the egg. The unknown compound was positively identified as pentabromocyclododecene (PBCDE), a metabolite of HBCD detected for the first time in foodstuffs. Studies of the analytical method used for the analysis of pesticides and contaminants showed that this cleanup method was suitable for the determination of HBCD and PBCDE, but storage of sample extracts resulted in the loss of HBCD when the sample extracts were not sufficiently purified. The concentrations of HBCD and PBCDE in the high polluted sample were 2.0 and 3.6 mg/kg egg fat. HBCD and PBCDE were also detected in two additional eggs at lower levels (<0.15 mg/kg), whereas 75 eggs did not contain these compounds (<0.02 mg/kg). We also detected HBCD and PBCDE in two samples of whitefish (Coregonus sp.), while an eel sample (Anguilla anguilla) positively tested for HBCD did not contain PBCDE. Surprisingly, the potential metabolite of HBCD, PBCDE, has not been detected before in any food or environmental sample. The present results indicate that more attention should be paid to the detection of HBCD and its metabolite PBCDE in chicken eggs.     

Development of a rapid and inexpensive assay for the nonspecific detection of antimicrobial residues in chicken egg yolks and neonatal yolk sacs

Competitive exclusion of intestinal pathogens by administration of beneficial and defined cultures of normal intestinal microflora is a safe and effective means of reducing the incidence and severity of chick infections with Salmonella and other intestinal pathogens. It is important that competitive exclusion cultures not carry genetic material (e.g., plasmids), which could transfer antibiotic resistance to other microflora, including pathogens. As such, safe and effective competitive exclusion cultures must be sensitive to commonly used antimicrobial agents. By necessity, intentional or accidental exposure of these beneficial microflora to antibiotics will reduce or eliminate the protection provided by competitive exclusion culture establishment. As antibiotic residues can be present from embryonic, hatchling, or maternal administration, a rapid and sensitive assay for the nonspecific detection of residues, which could interfere with competitive exclusion culture establishment, is needed. This study was conducted to develop a rapid and inexpensive bioassay to detect multiple antimicrobial residues in egg yolk and neonatal yolk sacs. Aerobic bacterial strains with known sensitivity to several antibiotics used by the poultry industry were selected and individually compared for sensitivity to enrofloxacin, gentamicin, tetracycline, ceftiofur, and tylosin concentrations in egg yolks. This assay was found to be relatively sensitive for the detection of these antimicrobials, and detection of residues was associated with reduced competitive exclusion culture (PREEMPT) establishment in one experiment. Importantly, this assay can be implemented with minimal training or equipment under commercial hatchery practices and could be used to determine embryo groups, in advance of hatch, that are not suitable candidates for competitive exclusion treatment in the hatchery.     

鸡SIgA的纯化及兔抗鸡SIgA抗体的制备

采用改良硫酸铵盐析法从鸡胆汁中分离鸡分泌型免疫球蛋白A(S IgA),M acro-prep H igh Q阴离子交换柱层析提纯,经二次过柱后获得的鸡S IgA浓度达22 m g/mL。SDS聚-丙烯酰胺凝胶电泳检测表明,重链分子质量为63~64 ku,轻链为26~28 ku,J链约16 ku。用纯化的鸡S IgA免疫兔,所得的抗血清经双向琼脂扩散,证明兔抗鸡S IgA血清效价达1¨128。     

Oligophosphopeptides derived from egg yolk phosvitin up-regulate gamma-glutamylcysteine synthetase and antioxidant enzymes against oxidative stress in Caco-2 cells

Previously, we have found phosphopeptides (PPPs) from hen egg yolk phosvitin possess a potent antioxidative activity against oxidative stress in human intestinal epithelial cells, Caco-2. However, their biological activity at the cellular level has not yet fully understood. The objective of this study is to evaluate the regulation of glutathione (GSH) biosynthesis-associated and antioxidant enzymes against oxidative stress in Caco-2 cells using an in vitro model. Treatment of 1 mM H2O2-induced Caco-2 cells with PPPs increased cellular GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit mRNA. Furthermore, intracellular glutathione reductase, glutathione S-transferase, and catalase activities were elevated by PPPs. In addition, PPPs with high content of phosphorus showed higher induction of these enzyme activities than PPPs without phosphorus. These data indicate that oligophosphopeptides from hen egg yolk phosvitin can up-regulate cellular GSH biosynthesis-associated enzymes activity and antioxidative activities, which play key roles against tissue oxidative stress in the human intestinal epithelial cells.     

Liquid chromatographic determination of fluoroquinolones in egg albumen and egg yolk of laying hens using fluorometric detection

A liquid chromatographic method was developed for the determination of ciprofloxacin, enrofloxacin, and sarafloxacin at 10-200 ppb in both egg yolk and egg albumen of laying hens. Egg yolk or albumen was acidified with 1 M phosphoric acid followed by deproteination with acetonitrile and centrifugation. The supernate was pipetted out, and the remaining protein pellet was extracted three times with acetonitrile. The supernates were combined and concentrated at 50 degrees C to <0.7 mL. The final volume was adjusted to 2 mL with 0.02 M potassium phosphate buffer, pH 2.5. Separation of the analytes was achieved using reversed-phase HPLC with fluorometric detection. The recoveries were >80% and coefficients of variation <20%. After validation, the method was applied for use in a national survey for fluoroquinolones in table eggs. Of the 276 eggs assayed, none was found positive for fluoroquinolones. The findings suggest that illegal use of fluoroquinolones in laying hens is not widespread.     

Identification and quantification of aminophospholipid-linked Maillard compounds in model systems and egg yolk products

While the Maillard reaction of free amino acids and proteins is a well-established process, no defined structures from the nonenzymatic browning of aminophospholipids in foodstuffs have been described so far. Phosphatidylethanolamine (PE)-linked glucosylamines (Schiff-PE), Amadori products (Amadori-PE), 5-hydroxymethylpyrrole-2-carbaldehydes (Pyrrole-PE), and carboxymethyl (CM-PE) as well as carboxyethyl (CE-PE) derivatives were detected and quantified by liquid chromatography- electrospray mass spectrometry (LC-(ESI)MS). Model incubations of soy-PE and D-glucose were employed to firmly establish the LC-(ESI)MS procedure. Analyses of spray-dried egg yolk powders and lecithin products derived therefrom show one-fourth of the native D-glucose content of egg yolk to be transformed to Amadori-PE, corresponding to a PE derivatization quota of 11-15.5 mol %. Schiff-PE and Pyrrole-PE were present only in low amounts, no CM-PE and CE-PE could be identified in any of the investigated samples. The high glycation rate of egg yolk PE will influence the emulsifying properties and perhaps even the oxidation resistance of the respective products.     

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.     

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.     

Human immunoglobulin E (IgE) binding to heated and glycated ovalbumin and ovomucoid before and after in vitro digestion

This study focuses on the effect of heating and Maillard reaction (MR) on the in vitro digestibility and rabbit IgG- and human IgE-binding properties of ovalbumin (OVA) and ovomucoid (OM) to estimate the impact of processing on their allergenicity. With the human sera studied, heat treatment significantly reduced IgE binding to both OVA and OM, whereas MR reduced the IgE binding to OVA but increased IgE binding to OM. In contrast, heat treatment significantly favored OVA digestibility but glycation impaired it, and these treatments did not affect the digestibility of OM. The changes observed in the digestibility affected the immunogenicity of the digests accordingly, so that the higher the digestibility, the lower the antibody binding. Heat treatment and glycation by MR showed an influence on the potential allergenicity of the main egg white proteins that could be related to their resistance to denaturation and digestive enzymes.     

Antimicrobial residue detection in chicken yolk samples following administration to egg-producing chickens and effects of residue detection on competitive exclusion culture (PREEMPT) establishment

Competitive exclusion (CE) cultures may offer alternatives to antimicrobial agents for disease prophylaxis in poultry. To avoid potential transfer of antibiotic resistance, safe and effective CE cultures must, by necessity, be highly sensitive to antimicrobial residues. The following studies evaluated the effect of maternal administration of selected antibiotics on the establishment of a licensed CE culture, PREEMPT. Selected antibiotics were administered to actively laying hens for a period of 7 days (experiment 1) or 9 days (experiment 2) in drinking water [sulfadimethoxine (0.05%), enrofloxacin (0.005%), and tylosin tartrate (0.05%)] or feed (sulfadimethoxine with ormetoprim, 250 ppm). In experiment 1, fertile eggs were collected daily and subjected to bioassay for detectable antimicrobial residues in yolk. Antimicrobial residues were not detected during the 7 days of treatment or the subsequent 3 days following cessation of treatment in the control, sulfadimethoxine, sulfadimethoxine with ormetoprim, or tylosin treatment groups. However, detectable residues were observed in eggs derived from enrofloxacin-treated hens on days 6 and 7 during antibiotic administration and also on days 2 and 3 post-antibiotic administration. In experiment 2, antimicrobial residues were also only detected in yolks from hens treated with enrofloxacin. Residue detection occurred on days 2-6 of antibiotic administration, on day 9 of antibiotic administration, on days 1-3 post-antibiotic administration, and also on day 7 post-antibiotic administration. A subset of eggs from each experimental group, corresponding to days 2-6 of antibiotic administration, days 4-6 post-antibiotic administration, and days 14-16 post-antibiotic administration, were pooled for incubation, and chicks hatched from these pools of fertile eggs were treated with PREEMPT at hatch. When 48-h cecal propionate concentrations were used as an index of culture establishment, reduced (P < 0.05) efficacy was observed only in chicks derived from enrofloxacin-treated hens at either collection period. Although several antibiotics do not appear to produce detectable egg residues or interfere with CE culture establishment, these data suggest that chicks derived from enrofloxacin-treated hens may not be candidates for safe and effective CE culture treatment.