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    Application of lysozyme in formation of multilayer emulsion containing caseinate : Master of Food Technology, Massey University, Palmerston North, New Zealand
    (Massey University, 2018) Chen, Zhenghao
    The properties of oil-in-water emulsions stabilized by caseinate-lysozyme complexes were investigated. Complexes were prepared by the mixture of 0.8 wt.% sodium caseinate with various amounts of lysozyme (0-0.5% W/W) at neutral pH. The emulsions formed by the serum solutions were not stable at low lysozyme concentrations (0-0.2 % W/W), but were stable at high concentrations (0.3-0.5 % W/W). The effects of lysozyme on caseinate-stabilized O/W emulsions were studied. Multilayer emulsions (containing 0.4 wt.% caseinate and 0-0.5 wt.% lysozyme) were created by mixing a primary caseinate stabilized emulsion with lysozyme solutions. The emulsions were evaluated at pH 3.3 and 6.8. At neutral pH, the emulsions were stable in the initial presence of 0.1 wt.% lysozyme due to bridging flocculation, but unstable at high lysozyme concentration of 0.1-0.5 wt.% . In acidified emulsions, lysozyme had no effect on caseinate-stabilized emulsions. Therefore, at neutral pH, complexes formed with high caseinate-lysozyme ratio could not create stable emulsions. On the other hand, caseinate-stabilized emulsions could only stay stable when the lysozyme to caseinate weight ratio was 1:2.
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    The potential use of hen egg white lysozyme as an antimicrobial agent in foods : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology at Massey University
    (Massey University, 1995) Rushizha, Edgar
    The potential use of lysozyme as an antimicrobial agent in foods was investigated in model food systems(brainheart infusion broth) using factorial designed experiments and in mussel and cottage cheese food systems. Optical density or absorbance was used as a tool to monitor the growth response of Listeria monocytogenes and C. tyrobutyricum in brain heart infusion broth under the combined influence of pH(5.5, 6.5), lysozyme (0.2mg/ml, 3mg/ml) and different chelating agents(ethylene diaminetetraacetic acid(EDTA), glycine, gluco delta lactone(GDL), citric acid, sodium phosphate dibasic(SPDB) and sodium hexametaphosphate(SHMP)(10mM, 25mM). Using 2 3 full factorial design experiments, the yield of the organisms (expressed as the area under the curve of a plot of change in optical density at 600nm vs time) was taken as the quantitative response variable for each treatment. These yield values were then used for (a) statistical analysis to determine which of the single or interactive factors tested significantly reduced the yield, (b) formulation of a mathematical regression equation which could be used to predict microbial growth within the limits of the factors studied. Diagnostic plots were constructed to evaluate further how well the statistical model fit the observed yield values. Plots of residuals versus predicted yield values appeared to suggest that a transformation of the response would improve the fit of the models. No other serious reservations were suggested by the diagnostic plots. Goodness of fit of the models was also evaluated by the R-squared values. Significant two-way and three-way interactions between lysozyme, pH and EDTA, GDL, citric acid and glycine were exhibited. Response surface methodology(RSM) was used to (a) characterize the response of L. monocytogenes to variation in treatment combinations and (b) show non-linearity of models(or interaction of factors). Generally yield was minimal in treatment where pH was low, with high lysozyme and chelator. Based on equal molar concentrations, the antimicrobial activity of the different chelating agents was in the order EDTA > GDL > citric acid > glycine > adipic acid > SHMP > SPDB. The same ranking was true for the degree to which each chelating agent enhanced lysozyme activity. Based on broth culture studies, the chelating agents EDTA, GDL, glycine, citric acid and adipic acid were demonstrated to have potential for use as antimicrobial agents in combination with lysozyme in food systems. Results of a 2 5 factorial design indicated that the 5 factors, lysozyme, GDL, pH, inoculum level and temperature were important in the inhibition of L. monocytogenes. Results of the broth culture studies gave a good reflection of the survival of L. monocytogenes in the food system. The variable combinations interacted to decrease the growth of L. monocytogenes and extended the lag phase duration. However C. tyrobutyricum was more tolerant to the different treatment combinations other than EDTA. A study of protein interference demonstrated that the antimicrobial activity of the lysozyme-GDL preservation system was not inhibited by the presence of proteins. The food system study demonstrated that the lysozyme-GDL treatment combination has potential for use as a preservative in refrigerated low pH ready-to-eat foods. The susceptibility of L. monocytogenes to lysozyme-GDL treatment in both broth culture and food systems increased as the temperature was reduced(25C-5C) and as the pH decreased(pH6.5-pH5.5). Food system studies demonstrated that modified atmosphere packaging(96.58%N2 , 2.09%O2 and 1.34% CO2) has no influence of the growth of L. monocytogenes. The susceptibility of L. monocytogenes to lysozyme-GDL was a stable characteristic, remaining unchanged during the entire study. Attempts to select for greater lysozyme-GDL resistance by testing populations grown from lysozyme-GDL survivors isolated at the end of the food system study was unsuccessful. There was no evidence that L. monocytogenes was resistant to the lysozyme-GDL treatment.
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    Stabilization of enzymes by chemical modifications : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, New Zealand
    (Massey University, 2011) Pattamawadee, Tananchai; Tananchai, Pattamawadee
    This study focused on thermostabilization of enzymes in solution by intramolecular crosslinking of the specific functional groups within an enzyme molecule. Three model enzymes were used: α-amylase of Aspergillus oryzae (EC 3.2.1.1), β-galactosidase of Aspergillus oryzae (EC 3.2.1.23) and extracellular invertase (EC 3.2.1.26) of Saccharomyces cerevisiae. Crosslinking was examined using the following homobifunctional reagents: diisocyanates (O=C=N(CH2)nN=C=O, n = 4, 6, 8), diimidoesters (CH3O(=NH)C(CH2)nC(=NH)OCH3, n = 4, 5, 6) and diamines (NH2(CH2)nNH2, n = 0, 2, 4, 6, 8, 10, 12). The concentration of the enzymes was kept low at 0.9 μM in attempts to promote intramolecular crosslinking as opposed to intermolecular crosslinking. Only invertase could be stabilized relative to controls by crosslinking with diisocyanates. Invertase (0.9 μM) crosslinked with 1,4-diisocyanatobutane (n = 4; or butamethylene diisocyanate, BMDC) and 1,6-diisocyanatohexane (n = 6) showed enhanced thermostability. Stability was improved dramatically by crosslinking invertase with 20-30 μM of the reagent. Molecular engineering of invertase by crosslinking reduced its first-order thermal denaturation constant at 60 °C from 1.232 min-1 for the native enzyme to 0.831 min-1 for the stabilized enzyme. Similarly, the best crosslinking treatment increased the activation energy for thermal denaturation from 372 kJ mol-1 for the native invertase to 517 kJ mol-1 for the stabilized enzyme. Values of the Michaelis- Menten parameters (Km and νmax) showed a reduced efficiency of invertase after the crosslinking treatment. The nature of the crosslinking was examined using size exclusion chromatography (SEC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS) and multiple angle laser light scattering (MALLS). Depending on the conditions used, both intermolecular and intramolecular crosslinking occurred. The estimated molecular weight of the intermolecularly crosslinked invertase appeared to be much higher compared to the intramolecularly crosslinked invertase and the native invertase. In attempts to simplify certain analyses, attempts were made to remove the carbohydrate moiety from crosslinked invertase (a glycoprotein) molecule. Deglycosylation with PNGase F achieved a significant reduction of carbohydrate for the native invertase but not for the intra- and intermolecularly crosslinked invertase. Circular dichroism (CD) measurements showed that crosslinking with BMDC affected slightly the secondary structure of invertase. The nature of the crosslinking that might be occurring in invertase molecule was further studied using small model oligopeptides, small nonglycosylated enzymes (hen egg white lysozyme and pepsin) and glycoprotein models (ovalbumin). Crosslinking of the model pentapeptide (0.9 μM) suggested that crosslinking with BMDC involved reaction between BMDC and the amino group of lysine or the carboxylate at C-terminal of the pentapeptide. Using a heptapeptide (1 mM) in crosslinking with BMDC showed a changed hydrophobicity of the crosslinked peptide. The crosslinking treatment of lysozyme (3.5 mM) with BMDC clearly produced an intermolecularly crosslinked lysozyme as evidenced by SEC and SDS-PAGE. A changed net charge of lysozyme after the crosslinking treatment was demonstrated using native PAGE. Mass spectrometry was used to then prove the intramolecular crosslinking of lysozyme with BMDC. CD spectra of the intramolecularly crosslinked lysozyme showed it be more resistant to thermal unfolding relative to native lysozyme.
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    Characterisation of lysozyme-steroid glucuronide conjugates : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University
    (Massey University, 1997) Smales, Christopher Mark
    The steroid glucuronides estrone glucuronide and pregnanediol glucuronide were synthesised using the O-glycosylation reaction of a glycosyl donor with the appropriate steroid under standard Koenigs-Knorr conditions. X-ray crystallographic studies showed that the synthetic estrone glucuronide molecule had the correct stereochemistry. Estrone glucuronide and pregnanediol glucuronide conjugates of hen egg white lysozyme were prepared by both the mixed anhydride and active ester coupling procedures. Both methods gave good yields of conjugate but the active ester procedure gave a more diverse range of products. Unreacted lysozyme, which was present in all cases, was removed by a combination of cation-exchange and hydrophobic-interaction chromatography to give purified conjugate material whose lytic activity was inhibited by over 90% in the presence of excess anti-steroid glucuronide antibody. Steroid glucuronide-lysozyme conjugates purified in this way could be used in a homogeneous enzyme immunoassay system to measure the levels of urinary estrone and pregnanediol glucuronide encountered in a normal menstrual cycle. Chromatography of the conjugation reaction mixtures on an S-Sepharose (fast flow) column in the presence of 7 M urea allowed the isolation of the different conjugate products. Conjugation of lysozyme with estrone glucuronide by the mixed anhydride method gave one major derivative exclusively acylated at lysine residue 33 while acylation with pregnanediol glucuronide gave two major derivatives exclusively acylated at lysine residues 33 and 97 respectively. On the other hand, conjugation of lysozyme with the two steroid glucuronides by the active ester method gave six derivatives which were acylated at combinations of one or more of three lysine residues, 33, 97, and 116. The correlation of the protein environments of the lysine amino groups in the crystal structure of lysozyme with the acylation positions in the conjugate families suggested that these positions were determined not only by the surface accessibility and nucleophilicity of the lysine residues but also by the steroid glucuronide and the acylating reagent. Computer derived three dimensional structures of the estrone glucuronide- lysozyme and pregnanediol glucuronide-lysozyme conjugates suggested that the enzyme conjugate may be inactivated by the antibody in the immune complex by either providing a physical barrier to approach by the large bacterial substrate or by disrupting the binding of the bacterial cell wall polymer into the active site cleft. The lytic activity of the estrone glucuronide-lysozyme E3 conjugate was not inhibited in the presence of excess antibody when the small chitohexaose substrate was used, implying that the substrate could access the active site even when the conjugate was bound by antibody. The detailed characterisation of the mixed anhydride estrone glucuronide-lysozyme conjugate coupled with the current knowledge of the antigenic determinants of hen egg white lysozyme has made it possible to design, in principle, a novel sandwich solid phase immunoassay format for the measurement of estrone glucuronide levels.