Growth, physiological characteristics and plasmid profiles of Bifidobacterium species Public Deposited

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  • The fecal flora of healthy bottle or breast-fed infants was examined for the presence of Bifidobacterium. Identification was based on the presence of fructose-6-phosphate phosphoketolase, which is found only in these bacteria. No bifidobacteria were recovered from bottle-fed infants. However, bifidobacteria were readily isolated from 15 day to 3 month old breast-fed infants. Further characterization revealed B. breve and B. longum were the dominant species in feces of breast-fed infants, but atypical strains also were found. A whey-based medium (7% sweet whey, 0.05% cysteine and 0.3% yeast extract, WCY-0.3) was developed to grow Bifidobacterium species without use of anaerobic incubation conditions. Freshly pasteurized WCY-0.3 was inoculated with 0.2% (10⁶ to 10⁷ CFU/ml) of the following active cultures of bifidobacteria: B. bifidum 15696, B. breve 15700, B. longum 15707, B. breve 15698, B. longum L10, B. longum L12, and B. longum 3j. Following incubation for 12 hours, most strains reached cell densities of 10⁹ to 5 x 10⁹ CFU/ml, except B. bifidum 15696 and B. longum 3j. Addition of Oxyrase to the WCY (WC with any level of yeast extract) at 0.03 unit/ml (WCYO) reduced the lag phase of all strains, allowing maximum populations to be reached more quickly. A higher population density (2 to 7 times) could be achieved in the WCOY-0.3 medium with strains 15696, 15700, 15707, and L10 by incorporating 1.9% sodium glycerophosphate or trimagnesium phosphate with incubation for 12 hours at 37°C. Also, viability of these strains was retained throughout a 24-hour incubation period, in contrast to rapid death of cells grown without the neutralizing agents. Inoculation of WCY-0.3 or WCOY-0.3 medium with frozen concentrates (10⁷ to 10⁸ CFU/ml) of bifidobacteria allowed equal growth of all species, except B. bifidum 15696, which grew much better in WCOY-0.3 than in WCY- 0.3. Survival stability of whey-based medium-grown bifidobacteria when resuspended in pasteurized skim milk and refrigerated at 4°C was strain dependent and enhanced by the presence of 0.05% cysteine; generally ATCC strains were more stable than strains freshly isolated from baby feces. In this regard, B. breve 15700, B. longum 15707, and B. breve 15698 did not lose viability in 11% skim milk with 0.05% cysteine within 10 days of storage. Stability of whey-based medium-grown bifidobacteria in WCY with 15% glycerol during six months storage at -40°C was strain dependent. Bifidobacterium bifidum 15696, B. breve 15700, B. longum 15707, B. breve 15698, and B. longum L12 did not lose viability; however B. bifidum L6 lost about 50% viability, while B. longum L10, B. breve T10, and B. breve T2 lost about one log population density. The plasmid profiles of 35 strains of bifidobacteria from human sources were examined. Only one strain, B. breve 15698, harbored a 5.8Kb plasmid. A curing process using UV-light treatment to remove the plasmid was carried out but characterictics of the cured strain were identical to those of the parent strain, indicating the plasmid is cryptic.
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