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. / Graduation date: 1991
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27151 |
| Date | 09 January 1991 |
| Creators | Feijoo, Sergio C. |
| Contributors | Bodyfelt, F. W. (Floyd W.), 1937- |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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