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The taxonomic position of the L. casei group has been the subject of some debate. Bergey's Manual of Systematic Bacteriology lists four subspecies of L. casei (Kandler and Weiss, 1986) and was reclassified in three species by Collins et al. (1989) on the basis of DNA homology data. Mori et al. (1997) reported sequences from the 16S rRNA that allows differentiation of these species. So protocols for bacterial typing using polymerase chain reaction (PCR) techniques are becoming increasingly valuable.

Though increasing use of gene sequences for taxonomy, classification of Lactobacillus isolates has begun to emerge in a more stable fashion. This has been supported, where appropriate, by biochemical, morphological, and physiological traits. Review of the literature concerning Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus zeae reveals that the classification of these bacteria has been controversial and that only recently proposals have been made to handle the ongoing controversy. Given the importance of these bacteria as probiotics or as fermentation contaminants, it is important to have a universally accepted taxonomy to avoid confusion when identifying, examining, and discussing these bacteria. The essence of the debate over the classification of these Lactobacillus species is as follows.

Based on 16S rRNA gene sequences (Collins et al, 1989), many subspecies formerly known as L. casei were reassigned to other species. L. casei subsp. alactosus and L. casei subsp. pseudoplantarum were reassigned to L. paracasei subsp. paracasei, and L. casei subsp. tolerans

Chapter 1 ______________________________________________________________________________ 20 was renamed L. paracasei subsp. tolerans. Finally, L. casei subsp. rhamnosus was elevated to the species level and named L. rhamnosus. Soon after, Dellaglio et al. (1991) requested that the type designation of L. casei ATCC 393 be rejected, the neotype L. casei ATCC 334T be recognized, and the name L. paracasei be rejected. Later, Dicks et al. (1996) called for become the type strain of the L. casei ATCC 393 to be designated L. zeae. It also was suggested that L. casei ATCC 334Tbecome the type strain of the L. casei spp. That includes all L. paracasei isolates (Dicks et al., 1996). Further support for this claim came later from Felis et al. (2001) and Dellaglio et al., (2002).

Various studies have supported the need for taxonomic changes in this branch of the genus Lactobacillus (Collins et al., 1989, / 1991; Dellaglio et al., 1991; Dicks et al., 1996; Mori et al., 1997; Tynkkynen et al., 1999; Chen et al., 2000). Collectively, these studies indicated that L. zeae (basonym L. casei) ATCC 393, L. zeae (basonym L. rhamnosus) ATCC 15820, and L. zeae isolates from a common group. The remaining L. casei isolates, including ATCC 334T, and L. paracasei isolates from another group, and L. rhamnosus isolates, except ATCC 15820, belong to a third group. Although the International Committee on Systematic Bacteriology recently endorsed these findings (Biavati 2001; Klein 2001), the proposed changes are not widely known and therefore have not found common usage.

Chapter 1 ______________________________________________________________________________ 21

1.4. Gut Microflora

The human colon is the body’s most metabolically active organ. This is because of the resident microbiota, which comprises 1012 bacterial cells for every gram of gut contents. There are numerous publications purporting that probiotic are active in the gut after ingestion, others have questioned such claims and the beneficial effects that probiotics are said to confer their hosts.

In terms of the microbiology of different digestive tract areas, there is variability both in terms of composition and activity. The lumen of the human stomach is essentially sterile due to a low gastric pH. However, micro-organisms are known to reside in the mucosal layer that overlies the gastric epithelium. This includes Helicobacter pylori, which has attracted a great deal of research interest. This organism uses its flagellae to invade the gastric mucus layer and thereafter adhere to epithelial cells. In conjunction with a production of ammonia, this allows effective colonization of the stomach (Rathbone and Heatley, 1992).

In the small intestine, the transit time of gut contents tends to maintain bacterial numbers at below 106 / ml of contents. Intestinal secretions like pancreatic enzymes and physiochemical variables such as pH and Eh also contribute towards the type of microflora that develop. Facultatively anaerobic and aerotolerant bacteria such as streptococci, staphylococci and lactobacilli dominate the upper small gut with bacterial numbers showing a progressive increase.

In comparison to other regions of the gastrointestinal tract, the human large intestine is an extremely complex microbial ecosystem, with at least several hundred different bacterial species being present. The environment is favourable for bacterial growth with a slow transit time, ready availability of nutrients and favourable pH. The vast majority (>90%) of the total cells in the body are present as bacteria in the colon. It is thought that over 60% of the faecal mass exists as

Chapter 1 ______________________________________________________________________________ 22 prokaryotic cells. Generally, the various components of the large intestinal microbiota may be considered as exerting pathogenic effects or they may have potential health promoting values. Given that the microbiota has components that are positive for human health, there is currently much interest in the use of diet to specifically increase groups perceived as health promoting. As such, the gastrointestinal flora and its activities are a major focus for functional food developments.