Simple Separation of Immunoglobulin from Egg Yolk by Ultrafiltration

A water-soluble fraction (WSF) was prepared by filtering 10 times diluted yolk (pH 5.0) stored at 4–6°C through Whatman No. 52 or No.1 filter paper for delipidation. However, solid-phase delipidation using an octadecyl column after filtration through a cellulose powder column was more practical for industrial application. After ultrafiltration (UF) of the WSF (pH 9.0,1.5M NaCl), results showed: Amicon 80–85% recovery, 74–99% purity; Harp 72–74% recovery, 81–84% purity; A/G 75% recovery, 89% purity; Koch 88% purity.     

Immunoglobulins from Egg Yolk: Isolation and Purification

Simple water dilution was employed for the separation of water-soluble plasma proteins from egg yolk granules. An optimum recovery of immunoglobulin Y (IgY, 93–96%) in water-soluble fraction was obtained by sixfold water dilution at pH between 5.0–5.2 with incubation time of 6 hr at 4°C. Among the factors studied, pH was found to be the most important factor affecting IgY recovery. Active IgY of high purity with good recovery was obtained by a combination of several techniques including salt precipitation, alcohol precipitation, ultrafiltration, gel filtration and ion exchange chromatography. Salt precipitation, ultrafiltration, and get filtration is the recommended sequence. Over 100 mg of electrophoretically pure IgY was routinely obtained from one egg.     

Egg Yolk Antibody (Ig Y) Stability in Aqueous Solution with High Sugar Concentrations

Effect of sugars on the stabilization of hen egg yolk immunoglobulin (IgY) under various processing conditions was investigated. By adding 30–50% (w/v) sucrose or invert sugar to an IgY solution heat denaturation of the IgY antibody at 75–80°C was markedly suppressed. A high concentration of sugar was also effective to retain the IgY activity under acidic conditions of pH 3 or high pressure of 5,000 kg/cm² at 60°C. Addition of high concentrations of sucrose may be a simple means to stabilize IgY for processing and preservation.     

Effect of wheat sprout powder incorporation on lipid oxidation and physicochemical properties of beef patties

The aim of this study was to determine the effects of incorporation of wheat sprout flours (WSF) on lipid oxidation and some quality characteristics of beef patties. Two wheat sprout flour samples (WSF1 and WSF2) were obtained by germination, drying and milling of two different wheat varieties. The beef patties were stored at +4 °C for 6 days or at ?18 °C for 180 days and analysed for their proximate composition, pH, sensory properties, colour parameters and lipid oxidation. TBA formation in WSF‐added patties was reduced approximately by as much as 20% when compared to that of the control at the 180th day of the storage, while favourable results were detectable in the storage period at +4 °C. Again, up to 4% WSF1 or WSF2 incorporation gave acceptable sensorial scores to the beef patties. As a conclusion, it was confirmed that WSF could be incorporated into beef patties to improve their quality parameters and retard lipid peroxidation.     

抗人大肠杆菌蛋黄抗体的分离纯化及理化性质研究

以人源大肠杆菌(ATCC23985)免疫产蛋母鸡所产免疫蛋为原料，通过蛋黄水提物制备、乙醇分级沉淀、离子交换色谱和凝胶排阻色谱对其中的免疫蛋黄抗体进行了分离纯化，并对其基本理化性质进行了分析。结果表明：蒸馏水-氯仿法是制备蛋黄水提物的最优方法，而35％的乙醇添加量能够很好地将水提物中的抗体沉淀出来。经DEAE-Sepharose柱和SephacrylS-200柱连续纯化的抗体在电泳检测时只有1个区带。研究获得的抗人大肠杆菌卵黄抗体的等电点为5.5，包含分子质量分别为65900u和24600u的2类亚基。该抗体的氨基酸组成与普通蛋黄抗体的氨基酸组成差异不大。     

Cholesterol Reduction in Liquid Egg Yolk using β-Cyclodextrin

A process to reduce cholesterol in liquid egg yolk using β-cyclodextrin (CD) was optimized by response surface methodology, based on cholesterol reduction and yield. The most important factors influencing cholesterol reduction were dilution of egg yolk to a defined water:solid ratio and CD concentration. In the final process, egg yolk was adjusted to pH 10.5, diluted to a water:solid ratio of 2.9, heated to 50°C, and CD added at a CD:cholesterol molar ratio of 4.0. The slurry was mixed for 6.5 min at 1800 rpm, cooled to 4°C and centrifuged. Cholesterol in the supernatant was reduced by 89.2%. The yield, cholesterol and CD in the final product were adjusted by varying centrifugal force and CD concentration.     

Enzymatic Production of Ribonucleotides from Autolysates of Kluyveromyces marxianus Grown on Whey

After 20h fermentation of medium containing 5% (w/v) dehydrated whey, at 30°C, pH 4.5, yeast cells were harvested, diluted in 0.1M KH₂PO₄, and autolyzed at different pHs (6.5–7.5) and temperatures (45–55°C). Phosphodiesterase (0.2–1.0% w/v, 65°C, pH 6.5, 6h) and adenyl deaminase (0.5-1.0% w/v, 60°C, pH 5.5, 4h) were added to the autolysates. After heat treatment (100°C, 15 min), samples were analyzed by RP-HPLC and LC/MS. Production of 5′-ribonucleotides was maximized at 50°C, pH 6.5. Yields of 5′-AMP (800 μg/g of biomass) and 5′-GMP (2000 μg/g) increased considerably after addition of 1.0% phosphodiesterase. 5′-IMP increased only after addition of 1.0% adenyl deaminase.     

Effects of specific egg yolk antibody (IgY) on the quality and shelf life of refrigerated Paralichthys olivaceus

BACKGROUND: The spoilage of fishery food has been attributed to limited types of microorganisms called specific spoilage organisms (SSO). Unlike traditional food‐preserving techniques which usually exploit broad‐spectrum antimicrobial agents, here, based on the specific antimicrobial activity of egg yolk antibodies (IgY) against two SSO in refrigerated fish (Shewanella putrefaciens and Pseudomonas fluorescens), a novel strategy for fish preservation was suggested and evaluated. RESULTS: During storage of Paralichthys olivaceus fillets at 4 ± 1 °C, the bacteria growth (including total microorganisms and the two SSO) in test groups was significantly inhibited in comparison to that of controls (P < 0.05). This antibacterial activity of the specific IgY was also confirmed by chemical analysis (pH, total volatile base nitrogen and 2‐thiobarbituric acid value) and sensory evaluation, and the shelf life of samples was extended approximately from 9 days to 12–15 days in the presence of the specific IgY. CONCLUSION: These results indicated a significant antimicrobial activity of the anti‐SSO IgY for refrigerated fish products, which allowed us to suggest its potential as a bio‐preservative for seafood. Copyright © 2011 Society of Chemical Industry     

绿色高效连续分离技术制备蛋黄中活性蛋白质和磷脂

为了实现蛋黄功能性成分的充分利用，采用绿色溶剂从新鲜蛋黄中高效连续分离具有生物活性的卵黄免疫球蛋白（immunoglobulin Y, IgY）、卵黄高磷蛋白（phosvitin, PV）和磷脂（phospholipids, PL）。首先采用水稀释法（稀释倍数为1:10）分离水溶性组分与脂蛋白，通过35%饱和(NH₄)₂SO₄和0.5% NaCl（w/v）对上清液进行盐析并用8% PEG 6000纯化IgY。脂质组分中的PV在pH5.0，90 ℃条件下的NaCl溶液（w/v）中进一步纯化。采用乙醇/乙酸乙酯比例为1:2（v/v）提取蛋黄中全部脂类，进一步采用乙酸乙酯在0 ℃下提纯蛋黄总脂中的PL。经脱盐、冷冻干燥后，IgY、PV和PL的纯度分别为97.38%、78.63%和85.94%。IgY和PV的得率分别为6.15、10.15 mg/mL。蛋黄总脂中PL的含量为35.18%。因其良好的多不饱和脂肪酸/饱和脂肪酸比率（0.70）和相对较低的n-6/n-3比率（5.37），该PL产品可应用于食品加工中。本方法简便、环保并且高效，可从蛋黄中依次分离出高纯度IgY、PV和PL，并为蛋黄的综合利用提供技术支持。     

Influence of pH on Egg Yolk Lipid Oxidation

The influence of pH on lipid oxidation of liquid egg yolk was studied. Liquid yolk samples were prepared to contain 60% yolk, 40% distilled water, 0.012% sodium azide, and pH was adjusted from the normal pH of 6.0 to 2.0, 3.0, 4.0, and 5.0 with 1N HCl. Samples were agitated at room temperature (ca 23°C) for 20 days and lipid oxidation measured using the distillation thiobarbituric acid test. No increase in thiobarbituric acid reactive substances occurred in the control (pH 6) or pH 5 yolk samples. The degree of oxidation significantly increased (P<0.01) in the more acidic samples in the following order: pH 3 > pH 2 > pH 4. Addition of 1.2% EDTA significantly lowered (P<0.01) the degree of oxidation but did not inhibit oxidation completely.     

Isolation of immunoglobulin in yolk (IgY) and rabbit serum immunoglobulin G (IgG) specific against bovine lactoferrin by immunoaffinity chromatography

Hens were intramuscularly immunized and rabbits were subcutaneously immunized once every two weeks for 6 weeks using bovine lactoferrin (LF) as antigen. Antibody titers of both yolk (IgY) and rabbit serum (IgG) were as high as 1.68×10⁸ at the 6th and 8th weeks, respectively, after the initial immunization treatment. However, antibody titer against LF in yolk was 9.4×10⁷ at 16 weeks. While antibody titer of rabbit serum declined sharply to 2.1×10⁷ at the 12th week and to 2.6×10⁶ at the 13th week after the initial immunization. The purification efficiency (specific activity of purified antibody against LF/specific activity of the corresponding antiserum or yolk against LF) of rabbit serum IgG purified by laboratory-prepared LF-Sepharose 4B immunoaffinity column (0.05 mg LF/ml wet gel) was about 2400, similar to that of IgY purified by LF-Sepharose 4B immunoaffinity column. Different amounts (0–15.0 mg) of IgY purified by LF-Sepharose 4B immunoaffinity chromatography were applied to the same column to determine the binding capacity (q_m) and dissociation constant (K_d) of LF-Sepharose 4B immunoaffinity gel for IgY specific against LF. It was found that q_m was 0.81 mg IgY/ml wet gel (1.620 mg IgY/mg LF) and K_d was 6.4×10⁻⁶ M as determined by Langmuir-type adsorption isotherms.     

Antibacterial activity of egg yolk antibody (IgY) against Listeria monocytogenes and preliminary evaluation of its potential for food preservation

BACKGROUND: Egg yolk antibody (IgY) is a unique type of immunoglobulin found in egg yolks, and many reports have described its ability to inhibit corresponding antigen bacteria. The objective of this study was to evaluate the antibacterial activity of IgY specific to Listeria monocytogenes, an important food pathogen to both humans and animals, as well as its potential use for food preservation. RESULTS: Specific IgY was generated by immunising Leghorn chickens with whole cells of L. monocytogenes, and its inhibitory effect on bacterial growth was tested in liquid medium and food samples. After 8 h of incubation with specific IgY, there was a significant decrease in the growth (absorbance at 600 nm) of L. monocytogenes in comparison with controls. IgY also inhibited the growth of L. monocytogenes inoculated onto fresh or smoked salmon samples. Compared with those of blanks, numbers of L. monocytogenes were reduced by more than 2 log units after 15 days of storage at 6 ± 1 °C in the presence of specific IgY. CONCLUSION: The results suggest the potential application of specific IgY as a natural antimicrobial agent for food preservation. Copyright © 2011 Society of Chemical Industry     

Use of Candida tropicalis ATCC 9968 to Adjust the pH of a Natural Lactic Acid Fermentation of Cornmeal

After 3 days of a natural lactic acid fermentation of whole kernel cornmeal, the pH was 3.78 with a titratable acidity of 0.91% at 32°C (1:4 w/v solids to water). These solids were diluted (1:12 w/v), autocalved at 121°C for 15 min and fermented with C. tropicalis for 7 days at 32°C, bringing the pH to 6.5 and 0.03% acidity. After both fermentations, the % relative nutritive value increased significantly (from 74.09% to 81.22%) and so did riboflavin (from 0.22 to 0.56 mg/100g). Both thiamin and niacin decreased significantly (0.42 to 0.20 mg/100g and 2.13 to 1.94 mg/100g, respectively).     

Strength of Protein Gels Prepared with Microbial Transglutaminase as Related to Reaction Conditions

Influence of gelling reaction conditions on the strength of several protein gels prepared with microbial transglutaminase (TGase) was investigated. A method was developed to gel proteins and measure gel breaking strength in a micro well plate. Enzyme concentration range for maximum gel breaking strength varied from 10 to 40 units/g protein. Maxima gel breaking strengths were achieved at 50°C for SPI, caseinate and gelatin and 65°C for egg yolk and egg white proteins. Optimum pH resulting in strong gels was pH 9 for SPI, caseinate, and egg yolk, and pH 6 for gelatin and egg white. Adjusting pH was promoted in egg white the formation of ?-(γ-glutamyl)lysine crosslinks and increased its gel breaking strength.     

Effects of mineral oil coating on internal quality of chicken eggs under refrigerated storage

The selected internal qualities (weight loss, Haugh unit, yolk index, and albumen pH) of noncoated and mineral oil‐coated chicken eggs during 15 weeks of storage at 4 °C and/or during 5 weeks of storage at 25 °C were evaluated. Results indicated that, without refrigeration, the noncoated and mineral oil‐coated eggs rapidly changed from AA to C and B grades as measured by Haugh unit, respectively, after 5 weeks of storage. However, the AA quality of the noncoated eggs could be maintained under refrigerated storage (4 °C) for at least 5 weeks. The mineral oil coating and refrigerated storage (4 °C) synergistically minimised weight loss and preserved the albumen and yolk qualities of chicken eggs during a long‐term storage. At 4 °C, the mineral oil‐coated eggs preserved the initial AA grade for at least 15 weeks with l.19% weight loss.     

Electrophoretic and Chromatographic Changes in Egg Yolk Proteins Due to Heat

Polyacrylamide gel electrophoresis and diethylaminoethyl-cellulose ion-exchange chromatography were used to observe changes in egg yolk products which were heat treated at 3°C intervals from 54 to 84°C for 3.5 min. The USDA pasteurization temperatures of 61.0, 63.3°C for plain, 10% sugared, and 10% salted egg yolk, respectively, had little effect on their electrophoretograms. Only the γ-livetins in plain yolk were slightly affected. Higher temperatures progressively altered these proteins until they disappeared from the electrophoretograms from plain yolk ca 73°, sugared yolk ca 76°, and salted yolk ca 79°C. The lipovitellin fraction and phosvitins were similarly affected by increasing temperatures. Sugar and salt at 10% levels had a protective effect against heat, allowing respectively ca 3° and 6°C higher temperatures before heat damage occurred. The chromatograms from plain and sugared yolk were little affected by pasteurization. However, major changes were noted between unheated and pasteurized (63.3°C) salted yolk. Patterns for salted yolk, pasteurized at 63.3 and 68.0°C were similar, which further indicated that this product can be pasteurized at 68.0°C without significant damage.     

On the use of ultrafiltration for the concentration and desalting of phosvitin from egg yolk protein concentrate

An ultrafiltration‐based approach was integrated in the preparation of phosvitin (PVs) from delipidated egg yolk proteins. An attempt was made to concentrate PVs as well as to desalt by means of the diafiltration technique. Primary experiments were devoted to optimise the ultrafiltration performance as function of parameters such as the effects of pH, feed concentration and transmembrane pressure on permeate flux with the 10‐kDa molecular weight cut‐off (MWCO) polyethersulfone membrane at laboratory scale. Higher permeate flux values were observed at low concentration and at alkaline pH, whatever transmembrane pressure studied. Then, desalting of PVs was carried out at 50 °C with 10‐ and 30‐kDa MWCO membranes. The results showed that desalting of PVs was obtained with both the 10‐ and 30‐kDa MWCO membranes and with a few loss of protein in the permeate side.     

INACTIVATION OF MALT PEPTIDASES DURING MASHING*

Malt contains at least eight different peptidases: three carboxypeptidases (pH optima 4·8, 5·2, 5·7), three aminopeptidases active on aminoacyl-β-naphthylamides (pH optima in hydrolysis of peptides at pH 5·8-6 5), and two (amino)peptidases acting on Leu-Tyr and Ala-Gly at higher pH (pH optima 8·6, 7·8). We have studied the progressive inactivation of these peptidases during mashing with a temperature programme from 45° to 70° C (pH 5·8 → 5·7). All peptidases were stable at 45° C. The five aminopeptidases were inactivated at different rates during a 30-min incubation at 55° C. The carboxypeptidases retained 70–100% of their activities at this temperature, and one of them had 40% residual activity after 60 min at 70° C. Liberation of amino acids continued at a considerable rate during the incubation at 70° C, probably catalysed by the heat-stable carboxypeptidase. Malt carboxypeptidases are therefore more heat-stable in mashing conditions than the aminopeptidases. This property, in combination with their high activities and suitable pH optima, makes them the most important enzymes in the production of free amino acids in mashing.     

Edible coating affects physico‐functional properties and shelf life of chicken eggs during refrigerated and room temperature storage

Effects of chitosan, whey protein concentrate (WPC), mineral oil (MO) and/or soybean oil (SO) coating on egg quality were compared at 25 and 4 °C, respectively, during 5 and 20 weeks of storage. Storage time and temperature, and type of coating significantly affected Haugh unit, yolk index, weight loss, albumen pH and emulsifying capacity. Shelf life was extended 4 weeks by MO and SO and 2 weeks by chitosan and WPC longer than that observed for noncoated eggs at 25 °C. MO‐ and SO‐coated eggs maintained AA grade for 20 weeks at 4 °C. Weight loss of SO‐coated eggs was <1% after 5 weeks at 25 °C and after 20 weeks at 4 °C. Yolk index and emulsifying capacity were more correlated at 25 °C than at 4 °C. MO and SO were more effective coating materials, with SO providing a more cost‐effective coating for extending egg shelf life.     

Microencapsulation Protects Immunoglobulin in Yolk (IgY) Specific against Helicobacter pylori Urease

ABSTRACT: Hens were intramuscularly (im) immunized on thighs by using urease (E.C. 3.5.1.5) from Helicobactor pylori as antigen. The specificity of IgY against urease of H. pylori increased gradually after initial immunization. The collected yolk was microencapsulated with 10% or 20%β-cyclodextrin (β-CD) and gum arabic by a spray-drier. Microencapsulation was effective in protecting the IgY activity against pepsin. Liposome prepared at the lecithin/ cholesterol ratio of 1/0.25 (mole/mole) displayed satisfactory encapsulation efficiency (69%) of IgY. Increase in cholesterol content in the liposomal structure exhibited a stronger protection effect of IgY against pepsin and acid.