A vertical diffusion method for quantitative analysis of Nisin in canned food was discussed.
Bacillus stearothermophilus IAM 1035 was used as test organisms for this study.
Results obtained may be summarized as follows:
1. Dispense 9ml of nutrient agar (meat extract 0.5%, Polypeptone 1.0%, NaCl 0.5%, Agar-agar 1.5%, pH 7.0) in sterile 16mm×160mm cotton-stoppered tubes. Sterilize in an autoclave for 15 minutes at 121°C.
2. Spore suspensions of
B. stearothermophilus are prepared in nutrient agar slants with incubation at 55°C for five to seven days until 80 per cent or more cells are in the spore state. Spores are suspended in sterile saline to give the desired concentration (6×10
6-6×10
5/ml), and heat-shocked for 10 minutes at 80°C before putting them into the test media.
3. An one ml portion of spore suspension is added to the test media which have been melted in water bath. After thoroughly mixing, the media are dispensed in sterile 5mm×75mm cotton-stoppered tubes and soiidified at vertical position in cold water.
4. The sample adjusted to pH 6.0 was stratified on the agar media and stored for 120 minutes at 30°C.
5. After incubation for 18 hours at 55°C, the length of inhibited zone is measured through a projector (×20) and read at the level of 0.01mm. The concentration of Nisin is obtained from the standard curve.
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