The objective of this study was to develop an economical, simple, and large-scale separation method for IgY from egg yolk. Egg yolk diluted with 9 volumes of cold water was centrifuged after adjusting the pH to 5.0. The supernatant was added with 0.01% charcoal or 0.01% carrageenan and centrifuged at 2,800 x g for 30 min. The supernatant was filtered through a Whatman no. 1 filter paper and then the filtrate was concentrated to 20% original volume using ultrafiltration. The concentrated solution was further purified using either cation exchange chromatography or ammonium sulfate precipitation. For the cation exchange chromatography method, the concentrated sample was loaded onto a column equilibrated with 20 mM citrate-phosphate buffer at pH 4.8 and eluted with 200 mM citrate-phosphate buffer at pH 6.4. For the ammonium sulfate precipitation method, the concentrated sample was twice precipitated with 40% ammonium sulfate solution at pH 9.0. The yield and purity of IgY were determined by ELISA and electrophoresis. The yield of IgY from the cation exchange chromatography method was 30 to 40%, whereas that of the ammonium sulfate precipitation was 70 to 80%. The purity of IgY from the ammonium sulfate method was higher than that of the cation exchange chromatography. The cation exchange chromatography could handle only a small amount of samples, whereas the ammonium sulfate precipitation could handle a large volume of samples. This suggests that ammonium sulfate precipitation was a more efficient and useful purification method than cation exchange chromatography for the large-scale preparation of IgY from egg yolk.
Two new primer sets of a 766-and a 344-bp fragment were introduced into the conventional Bruce-ladder PCR assay. This novel multiplex PCR assay rapidly and concisely discriminates Brucella canis and Brucella microti from Brucella suis strains and also may differentiate all of the 10 Brucella species.The alphaproteobacterial genus Brucella consists of 10 species: B. abortus, B. canis, B. suis, B. ovis, B. neotomae, B. melitensis, B. ceti, B. pinnipedialis, B. microti, and B. inopinata (3, 5, 16, 17). Brucella species show a host preference, but some strains can be transmitted among a variety of animals, including humans (11,12,14,18).To accelerate effective prevention and control of brucellosis, a fast and accurate identification method is necessary. Many studies have developed PCR-based assays for the discrimination of Brucella species (2,4,7,8,10). Recently, López-Goñi et al. reported that the Bruce-ladder PCR assay could differentiate most Brucella species, including marine mammal and vaccine strains B. abortus S19, B. abortus RB51, and B. melitensis Rev.1 (6, 10, 13). However, these assays did not solve the problem of erroneous identification of B. canis isolates (such as B. suis) and basic differentiation between two marine mammal Brucella species (B. ceti and B. pinnipedialis) (9, 13, 15). Therefore, the aim of this study was to develop a fast, simple, and accurate one-step multiplex PCR assay to differentiate 10 Brucella species using 22 reference strains and 106 field isolates in South Korea.To discriminate between B. canis and B. suis, alignments of their whole-genome sequence or each biovar short-gun
The objective of this study was to develop a simple and economical protocol for separating ovotransferrin from egg white. Egg white was separated from the yolk and diluted with the same volume of distilled water. To prevent denaturation during the separation process, ovotransferrin in 2x-diluted egg white was converted to its holo-form by adding 20 mM FeCl3.6H2O solution (0.25 to 3 mL/100 mL). The pH of egg white was adjusted to pH 7.0, 8.0, or 9.0, and NaHCO3 and NaCl were added to 50 mM and 0.15 M, respectively (final concentrations) to facilitate iron binding to ovotransferrin. The iron-bound ovotransferrin was separated from the egg white using different concentrations of ethanol (30 to 50%). Ethanol at 43% (final concentration) and pH at 9.0 were the best conditions for separating iron-bound ovotransferrin from 2x-diluted egg white solution. Almost all egg white proteins including ovalbumin were precipitated at 43% ethanol, but most of the iron-bound ovotransferrin remained in the supernatant. Holo-ovotransferrin in the 43% ethanol solution started to precipitate as the concentration of ethanol increased, but the optimal condition for precipitating ovotransferrin was when the ethanol concentration reached 59% (final). The precipitated holo-ovotransferrin was dissolved with distilled water, and AG1-X(2) ion exchange resin (at 3x iron content in ovotransferrin) was used to remove iron bound to ovotransferrin after pH adjustment to 4.7 using 500 mM citric acid. The apo-ovotransferrin obtained using this protocol was >80% in purity and around 99% in yield. The protocol developed is simple, economical, and appropriate for a large-scale production of ovotransferrin from egg white. Also, the isolated ovotransferrin can be applied in human foods, because the only solvent used in this process is ethanol. Furthermore, the AG1-X2 ion exchange resin and ethanol used in this process can be regenerated and recovered.
The objective of this study was to develop a new protocol that could be used for large-scale separation of phosvitin from egg yolk using ethanol and salts. Yolk granules, which contain phosvitin, were precipitated after diluting egg yolk with 9 volumes of distilled water. The pH of the yolk solution was adjusted to pH 4.0 to 8.0 using 6 N HCl or NaOH, and then yolk granules containing phosvitin was separated by centrifugation at 3,220 × g for 30 min. Lipids and phospholipids were removed from the insoluble yolk granules using 85% ethanol. The optimal volumes and concentration of ethanol in removing lipids from the precipitants were determined. After centrifugation, the lipid-free precipitants were homogenized with 9 volumes of ammonium sulfate [(NH(4))(2)SO(4)] or NaCl to extract phosvitin. The optimal pH and concentration of (NH(4))(2)SO(4) or NaCl for the highest recovery rate and purity for phosvitin in final solution were determined. At pH 6.0, all the phosvitin in diluted egg yolk solution was precipitated. Among the (NH(4))(2)SO(4) and NaCl conditions tested, 10% (NH(4))(2)SO(4) or 10% NaCl at pH 4.0 yielded the greatest phosvitin extraction from the lipid-free precipitants. The recovery rates of phosvitin using (NH(4))(2)SO(4) and NaCl were 72 and 97%, respectively, and their purity was approximately 85%. Salt was removed from the extract using ultrafiltration. The salt-free phosvitin solution was concentrated using ultrafiltration, the impurities were removed by centrifugation, and the resulting solution was freeze-dried. The partially purified phosvitin was suitable for human use because ethanol was the only solvent used to remove lipids, (NH(4))(2)SO(4) or NaCl was used to extract phosvitin, and ultrafiltration was used to remove salt and concentrate the extract. The developed method was simple and suitable for a large-scale preparation of partially purified phosvitin.
The aim of this study was to evaluate the effect of EDTA, lysozyme, or the combination of EDTA and lysozyme on the antibacterial activity of ovotransferrin against Escherichia coli O157:H7. Ovotransferrin solutions (20 mg/mL) containing 100 mM NaHCO3 (OS) with added EDTA (2.0 or 2.5 mg/mL), lysozyme (1.0, 1.5, or 2.0 mg/mL), or both were prepared. The antibacterial activities of OS, OSE (OS+EDTA), or OSL (OS+lysozyme) against E. coli O157:H7 in model systems were investigated by turbidity and viability tests. In addition, OSE, OSL, or OSEL (OS+EDTA+lysozyme) was applied to irradiated pork chops and commercial hams to determine whether the solutions had antibacterial activity on meat products. The effect of the initial cell population on the antibacterial activity of OSE, OSL, and OSEL was determined. Ethylenediaminetetraacetate at 2 mg/mL plus OS induced a reduction of approximately 3 to 4 log in viable E. coli O157:H7 cells in brain heart infusion broth media, and 1 mg/mL of lysozyme plus OS resulted in a reduction of approximately 0.5 to 1.0 log during a 36-h incubation at 35 degrees C. However, neither OSE nor OSEL showed a significant antibacterial effect on pork chops and hams during storage at 10 degrees C. The initial cell number in media did not affect the antibacterial activity of OSE or OSEL against E. coli O157:H7. This study demonstrates that combinations of ovotransferrin, NaHCO3, and EDTA have the potential to control E. coli O157:H7.
and Implications Irradiation of ground beef under aerobic conditions oxidized myoglobin and drastically reduced color a*-values. Under vacuum or non-oxygen conditions, however, irradiation did not influence the redness of ground beef. Also, the red color of ground beef was maintained even after the irradiated beef was exposed to aerobic conditions. Vacuum-packaged irradiated ground beef had lower metmyoglobin content and lower oxidation-reduction potential than the aerobically packaged ones. Irradiating ground beef under vacuum-packaging conditions was also advantageous in preventing lipid oxidation and aldehydes production. Vacuum-packaged irradiated beef, however, produced high levels of sulfur volatiles during irradiation and maintained their levels during storage, which resulted in the production of characteristic irradiation off-odor. Double-packaging (V3/A3: vacuum-packaging during irradiation and the first 3 days of storage and then aerobic-packaging for the remaining 3 days) was an effective alternative in maintaining original beef color (red), and minimizing lipid oxidation and irradiation off-odor. The levels of off-odor volatiles in double-packaged irradiated ground beef were comparable to that of aerobically packaged ones, and the degree of lipid oxidation and color changes were close to those of vacuum-packaged ones. Ascorbic acid at 200 ppm level was not effective in preventing color changes and lipid oxidation in irradiated ground beef under aerobic conditions, but was helpful in minimizing quality changes in doublepackaged irradiated ground beef. This suggested that preventing oxygen contact from meat during irradiation and early storage period (V3/A3 double-packaging) and double-packaging+ascorbic acid combination are excellent strategies to prevent off-odor production and color changes in irradiated ground beef. Developing methods that can prevent quality changes of irradiated beef is important for the implication of irradiation, which will improve the safety of beef.
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