Graduate Thesis Or Dissertation
 

Rapid assay for Bacillus proteinases in raw milk as detected by a simple casein denaturation method

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  • A casein agar diffusion method was developed to detect and quantify pertinent levels of proteinases produced in raw milk supplies by heat resistant Bacillus sporeformers. In order to optimize the required heat treatment conditions of raw milk samples, trials that involved a combination of different temperatures and times were evaluated. A heat treatment of 75°C for 20 min was the most effective for recovering the highest number of surviving spores. A sporulation broth containing five different minerals and supplemented with 0.2% nonfat dry milk was used to maximize spore production in all heat-treated samples. A β-casein based assay detected proteinase activity from raw milk samples that ranged from 0.093 to 4.034 units/mg which corresponded to zones of β-casein precipitation in the β-casein agar of 5.0 and 15.0 mm respectively, and was compared to Protease Type VIII (from B. licheniformis). This assay correlated well with the fluorescein isothiocyanate casein-labeled assay (FITC), R=0.995 (Protease Type VIII). Proteases of Bacillus origin such as Protease Type IX, X, XV and XXXI were also evaluated but were rejected in favor of a broader range of activity expressed by Protease Type VIII. For an initial set of 370 raw milk samples, no quality deterioration, such as coagulation or bitter taste was observed in heat-treated (75°C for 20 min) and incubated samples (7.2°C for 10 days). However, during the winter season, 18 of 75 incubated samples (7.2°C for 10 days) tasted slightly bitter and exhibited a slight degree of casein precipitation. One sample coagulated but exhibited no proteinase activity on the β-casein agar gel, hence it was considered a false negative. The positive results for proteinase activity from raw Grade A samples tested by the β-casein agar diffusion method did not correlate either with fresh spore counts (R=0.21) or post-heat treatment incubation counts (R=0.03) or with psychrotrophic sporeformer counts (R=0.06). The β-casein agar diffusion method is simple, rapid and sensitive to Bacillus spp. proteinases, but was unreliable in projecting results related to the psychrotrophic sporeformer count. Consequently, further research is required to establish optimum conditions (time and/or temperature) and inoculum volumes into sporulation broth for attainment of a more positive correlation between β-casein agar precipitation zones and psychrotrophic sporeformer populations of either raw or processed milk samples.
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