UBC Theses and Dissertations
Separation of antimicrobial protein fractions from animal resources for potential use in infant feeding Al-Mashikhi, Shalan Alwan Edan
In the first part of this study, a non-ferric method for selective elimination of β-lactoglobulin from cheese whey was investigated. A new method was developed based on hexametaphosphate treatment of cheese whey. When Cheddar cheese whey was treated under the optimized conditions, i.e., 1.33 mg/mL sodium hexametaphosphate at 22°C and pH 4.07 for 1 hr, more than 80% of β-lactoglobulin was removed by precipitation. Almost all of the immunoglobulins and the major portion of α-lactalbumin were retained in the supernatant as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunochemical assays. By dialysis against distilled water 72.2% of the phosphorus was removed from the supernatant. In the second and the third part of the thesis, chromatographic methods were used for isolation of immunoglobulins and lactoferrin from whey proteins. By using gel filtration on Sephacryl S-300, 99, 83.3 and 92.1% biologically active immunoglobulin G were obtained for colostral whey, acid and Cheddar cheese whey, respectively. Lactoferrin, selectively adsorbed to the heparin-attached Sepharose, was eluted with 5 mM Veronal-HC1 containing 0.5M NaC1, at pH 7.2. 1,4-Butanediol diglycidyl ether-iminodiacetic acid on Sepharose 6B, or so-called metal chelate-interaction chromatography (MCIC), was loaded with copper ion and used for the same purpose. Of the two peaks obtained, the first yellowish peak was rich in lactoferrin, while the second peak was rich in immunoglobulins. Some of the physical and chemical properties of the proteins in these peaks, including immunochemical properties, isoelectric points, binding to bacterial lipopolysaccharides, and the mechanism of protein-metal interaction via histidine modification, and the capacity of the method were studied. The possibility of isolating immunoglobulins and lactoferrin from electrodialyzed whey was also investigated. In the fourth, fifth and sixth parts of the thesis, the method developed for isolation of immunoglobulins and lactoferrin from whey protein was applied to isolate these biologically important proteins directly from skimmilk, blood and egg white. The casein in skimmilk was found to compete with immunoglobulins for binding to copper ion in MCIC column when skimmilk was loaded in presence of 0.05 M Tris-acetate buffer containing 0.5 M NaC1, pH 8.2; however, this problem was solved by changing the equilibrating buffer to 0.02 M phosphate buffer containing 0.5 M NaC1, pH 7.0. When blood was directly applied to MCIC column, the yield of biologically active IgG was more than 95%. Ovotransferrin, strongly adsorbed to the MCIC column, was eluted with two-step elution protocols which suggests it exists in two forms. The histidine residues in immunoglobulins, caseins, transferrin and ovotransferrin were found to be involved in the mechanism of the interaction with the MCIC column.