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UBC Theses and Dissertations

Minimization of extracellular enzyme production by Pseudomonas fluorescens in a model milk system by controlled oxygen atmospheres Lee, Karoline K.


Pseudomonas fluorescens, the predominant aerobic spoilage microorganism in raw milk, proliferate and produce heat resistant extra cellular enzymes during storage. The critical upper 02 level which would minimize the synthesis of proteinases and lipases by P. fluorescens in a model milk system was determined in our study. Two litre volumes of Ultra-High Temperature sterilized milk (2 % m.f.) in 4 litre carboys were equilibrated with a continuous supply of atmospheric oxygen between 1 - 21 %. Milk was then inoculated with P. fluorescens biotype A to approximately 1 x 104CFU rriL-1 to reflect the psychrotrophic populations normally encountered in milk and stored under controlled 02 atmospheres at 4 °C up to 18 days. The atmospheric composition was analyzed with gas chromatography. Milk samples were collected every two days for determination of dissolved 02, P. fluorescens numbers, proteolysis, lipolysis, pH, proteinase and lipase activity. The dissolved 02 tension in milk decreased despite a constant supply of 02 during mid-exponential growth phase of P. fluorescens when populations reached 5.0 - 6.5Logi() CFU mL4 under all atmospheric 02 levels tested. The growth rate of P. fluorescens was enhanced during the first four days of storage under decreased atmospheric 02 concentrations at 1.2, 4.8, 5.3, and 13.4 % when compared to the aerobic control. Lower levels of proteinase and lipase activities were detected in milk stored under all decreased atmospheric 02 conditions (1.2, 4.8, 5.3, 9.6,10.2, 10.3, 13.4, and 16.9 % 02) when compared to the aerobic control (20.2, 20.7, and 20.9 % 02). As a result, lower degree of proteolysis and lipolysis were observed in milk stored under decreased atmospheric 02 conditions. Deviation from initial milk pH reflecting the predominant effect of either proteolysis or lipolysis was minimized when the initial dissolved 02 tension in milk was decreased. Greater inhibitory effect on proteinase than lipase production by P. fluorescens was observed under decreased 02 atmospheres. P. fluorescens under 10 % atmospheric 02 concentration consistently showed slow growth rates during initial storage, low proteinase and lipase activities and as a result, low degree of proteolysis and lipolysis in milk. Initial dissolved 02 tension of 4.1 ppm (unstirred sampling) and 6.1 ppm (stirred sampling) were measured in milk under 10% atmospheric 02 concentration which was observed to be the critical upper limit, of the levels tested, which minimized extra cellular enzyme production by P. fluorescens, as well as delaying its growth rate during initial storage, in UHT-sterilized milk. Compared to storage of raw milk under aerobic atmosphere, decreased atmospheric 02 conditions may provide a better environment for storage of raw milk when the predominant flora consists of aerobic P. fluorescens. Conditions for an improved f3-naphthyl caprylate assay for lipase activity were established at final concentrations of 15 mM sodium taurocholate and 8 mM 13-naphthylcaprylate in the reaction mixture and a solvent system of 60:40 ethyl acetate to ethanol. A final concentration of ethylenediamine tetraacetic acid between 2 and 5 rnM minimized degradation of lipases by proteinases and thus enhanced lipase activity during the assay procedure.

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