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Bacteria were cultured in liquid brain heart infusion (BHI) bouillon medium for 4 days.
After single colony purification in 5% sheep blood agar, the positive colonies were grown in bouillon medium for 48 h at 37°C, and the number of colony formation units (CFU) in the culture was calculated post the inoculation.
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Skim milk powder (1.5%, w/v) was added to the bouillon culture medium to make skim milk bouillon culture medium.
Then, the diluted bacterial liquid was coated onto skim milk bouillon culture medium and incubated at 37 °C for 24 h.
The strains above were selected by the ratio of H/C and inoculated into the bouillon culture medium.
The lawn was scraped from the edge of NTG inhibition zone, and inoculated into bouillon culture medium (5 ml) and incubated at 37 °C, 220 rpm, for 4 h.
Bouillon culture medium was comprised of 1% (w/v) peptone, 0.3% (w/v) beef extract and 0.5% (w/v) NaCl, pH 7.0 ± 0.2.
Thirteen strains with the values of H/C greater than 3.4 were picked from the skim milk bouillon culture medium after NTG treatment.
The bacterial liquid with the death rate of 85% after UV treatment was selected, and coated onto the skim milk bouillon culture medium and incubated at 37 °C for 24 h.
Then pour in 11 1/8 2cups beef bouillon and lower the heat to medium.
Strain FERM P-19486 was routinely grown in the nutrient medium, which consisted of 3% (w/v) bouillon (Nissui Co., Tokyo, Japan), at 30°C for 3 d with vigorous shaking.
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