| The first objective of this work was to acquire a collection of bifidobacterial strains representing species used in cultured dairy products, to identify them at the genus-, species- and subspecies-levels, and to characterize them using both phenotypic and nucleic acid-based methods. Thirty-one strains of Bifidobacterium were obtained from six commercial starter culture companies. Based on traditional detection of fructose-6-phosphate phosphoketolase (F6PPK) activity and by PCR with genus-specific primers, twenty-two commercial strains were identified as Bifidobacterium. While only one starter culture manufacturer misidentified strains at the genus level, species misidentification was common. Based on PCR with species-specific primers, only six of twenty-two strains of Bifidobacterium were correctly identified at the species level.;Twenty of the twenty-two commercial strains of Bifidobacterium were identified as B. animalis ssp. lactis , the most common bifidobacterial (sub)species isolated from probiotic dairy products. These twenty commercial strains were characterized using a variety of phenotypic and nucleic acid-based techniques. PFGE, RAPD-PCR, and comparison of gene sequences, which are some of the more discriminatory typing methods, were unable to differentiate among this collection of highly related strains. The commercial strains exhibited a high degree of relatedness to the B. animalis ssp. lactis type strain, DSMZ 10140, and only one phenotypic difference, related to glucose utilization, was observed.;The second phase of this project was to develop an understanding of glucose utilization in B. animalis ssp. lactis. This knowledge is important when selecting a medium for detection, isolation, or enumeration of bifidobacteria from a product. Glucose non-fermenting strains of bifidobacteria may be undetected or underreported when glucose is used as the sole carbohydrate source in media, as is often the case in reports of enumeration of probiotic products. Although mentioned in passing, little detailed information is available in the literature about glucose non-fermenting strains of bifidobacteria and no study has been conducted on glucose transport by B. animalis ssp. lactis. The dominance of B. animalis ssp. lactis strains used in the manufacture of probiotic dairy products, the prevalence (50%) of glucose non-fermenting strains of B. animalis ssp. lactis identified in this work, and the common use of glucose as the sole carbon source in media for isolating or enumerating bifidobacteria emphasizes the need to characterize glucose utilization in these strains.;A low-affinity facilitated diffusion glucose transporter was identified in DSMZ 10140. Glucose uptake assays revealed: (1) saturable kinetics with a Kt of 14.8 mM, (2) stereospecificity, with greater competition by methyl-beta-glucoside than methyl-alpha-glucoside, and (3) decreased glucose uptake of more than 30% by phloretin, an inhibitor of facilitated diffusion of glucose.;While there was a correlation between growth in glucose and glucose uptake for glucose "slow" and glucose "fast" strains, not all "adapted" strains exhibited increased glucose uptake (i.e., RB 4825 and RB 5251). This suggests multiple mechanisms are responsible for a strain's inability to grow on glucose in Liver Glucose (LG) and are involved in a strain acquiring the ability to grow on glucose.;During the characterization of glucose transport in DSMZ 10140 and five commercial strains of B. animalis ssp. lactis, each strain exhibited a unique "biochemical fingerprint" of its glucose transporter. Among these six strains, glucose uptake varied with respect to levels of activity and effects of competitors, cations, and sodium chloride concentration. However, all strains were inhibited by phloretin, indicating facilitated diffusion was the primary mechanism of glucose transport.;In bifidobacteria, few glucose transporters have been described. The glucose transporters characterized in this work are the first identified in B. animalis ssp. lactis and the first identified facilitated diffusion transporters of glucose in actinobacteria, contributing to our understanding of glucose utilization and bifidobacteria physiology. (Abstract shortened by UMI.)... |
