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UBC Theses and Dissertations

Comparison of proteinase assay methods and identification of pseudomonads in milk Kwan, Kent Kee Ho


This thesis is divided into two chapters summarized separately below. (1) Four methods (absorbance at 280 nm; the Lowry method; the fluorescamine method and the trinitrobenzenesulfonic acid method) for determining hydrolysis of milk proteins were compared. Each method was applied to the trichloracetic acid soluble fraction of milk protein, which had been digested with trypsin for various periods of time. Detectability was measured as the ratio between standard error of estimate and slope calculated from the linear regression analysis of Deming for cases when both variables were subject to error. Although it was nondimensional, the detectability thus calculated was simple and reliable for comparing assay methods which were based on different analytical principles. Detectability as well as the detection limit measured according to Schwerdtfeger showed that, of the methods compared, the fluorescamine method was most reliable and sensitive. (2) Proteolytic pseudomonads isolated from raw milk were classified by numerical taxonomy. Unweighted pair-group average-linkage cluster analysis was used to cluster 49 bacterial strains, of which 26 were Pseudomonas species as described in the Bergey's Manual of Determinative Bacteriology, based on 52 characters. The milk isolates resided in two clusters: one containing P. fluorescens and the other P. fragi. The isolates identified with P. fluorescens could hydrolyze milk proteins, butterfat; could produce phospholipase. The P. fragi-like isolates could hydrolyze milk proteins, butterfat; but could not produce phospholipase and fluorescent pigment. Studies, on hydrolytic characters of milk isolates, showed that the nature of the substrate and conditions under which the test was being conducted were critical. No relationship was found between proteolytic psychrotrophs populations and proteolysis in milk stored at 4 C. Electropherograms showed that β-casein was more susceptible to degradation by milk pseudomonads.

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