*2.1.2. Isolation and screening via stress-shock*

286 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

*2.1.1. Isolation via stress-shock procedure* 

*Bifidobacterium* morphology.

probiotics.

**salts** 

may cause diarrhoea, due to the increased osmotic pressure.

**2. Screening and isolation procedures of bifidobacteria strains** 

factors. Endogenous factors mainly operate through hormones on the intestinal motility. The well-known endogenous factors include: decreased peristaltic movements during exercises and menstrual cycle. Dietary factors, like non-digestible fibres and polyalcohols (sorbitol), may retain water and thus increases stool bulk. High amounts of these factors

Molecular methods have shown that the average percentage of bifidobacteria in the GI tract of humans is approximately 3% of total microbiota, or they occur at a concentration of 109 – 1010 CFU/g of faeces (Jia *et al*., 2010). As to achieve intestinal colonization in humans or animals, bifidobacteria have to endure inhibitory substances secreted by the host, such as gastric acid in the stomach and bile salts (in the small intestine). Although, both the gastric pH (pH < 3) and bile salts are strongly bacteriocidal, some resistant bifidobacteria can handle the low pH's ranges of the stomach and also survive the effects of bile salts in the small intestine of humans. These can be isolated and screened for their leading roles as

**2.1. Isolation and cultivation of bifidobacteria resistant to acidic pH and bile** 

Selection of acid and bile resistant bifidobacteria has been based on the stressing isolation method developed by Chung *et al*., (1999). Faecal samples are collected from infants and/or adults. The tube containing the faecal sample is promptly screened for the isolation of resistant strains, as follows: Faecal samples (0.8 g each) are inoculated into 8 ml of Transga-lactooligosaccharide-propionate (TP) medium as an enrichment medium for the bifidobacteria. After an anaerobic incubation for 12 h at 37 °C, 0.8 ml of the incubated cultures is transferred into fresh TP medium with pH adjusted to 2.0 and incubated anaerobically for another 12 h at 37 °C. After the acid exposure, an aliquot (0.8 ml) of the incubation medium is transferred into fresh TP medium supplemented with 1.5% ox-gall, and the incubation continued for another 2 h at 37 °C. The resulting incubation medium is serially diluted and plated on TP agar, to select colonies of the resistant bifidobacteria strains. To isolate reference strains, serially diluted *Bifidobacterium* cells grown in the regular TP medium are plated on TP agar medium. In most of the isolation studies, *B*. *adolescentis, B. longum, B. infantis, B. bifidum* and owner identified *Bifidobacterium* strains (commonly called "own isolates" in microbiology) are used as reference strains. The reference strains are utilized for the convenience of comparison to the resistant strains. Microscopic analysis (1000 with immersion oil) is routinely performed to confirm

In addition, *Bifidobacterium* cells are examined for their biochemical and morphological characteristics according to the Bergey's Manual of Determinative Bacteriology. The cultures Briefly, faecal samples of 3 to 5 days old new-born babies are collected and taken to the laboratory for immediate analysis and isolation of bifidobacteria. About 2 g of each faeces sample is placed in a sterile test-tube (30 ml) and closed tightly with a rubber-stopper. For optimal survival of these highly sensitive anaerobic bacteria, the samples are treated within 15 min after faeces emission, or else the samples are kept in an anaerobic environment until analysis (maximum of 10 h). Screening for the isolation of resistant strains is as follows: faecal samples (2 g each) are inoculated into 10 ml test-tubes of Raffinose-Bifidobacterium (RB) broth (pH 6.8). After an anaerobic incubation for 12 h at 38.5 ºC, 1 ml of the incubated culture is transferred into 10 ml of fresh RB medium with pH adjusted to 3.0 and incubated anaerobically for 2 h at 38.5 ºC. After the acid exposure, an aliquot (1 ml) of the incubation medium is transferred into 10 ml of fresh RB medium supplemented with 1% ox-gall, and the incubation continues for another 2 h at 38.5 ºC. The resulting incubation medium is serially diluted (10-folds) in a pre-reduced Ringer solution with 5 – 10% glycerol for the inhibition of the cellulolytic activity of the fungus. An aliquot of 100 µl from each dilution is plated directly on RB and MRS agars using the surface streak method and incubated anaerobically at 38.5 °C for 3 – 4 days to determine colonies of the resistant *Bifidobacterium* strains.

Likewise, the isolate designated *B. longum* GB-03 was isolated from a pharmaceutical product called Golden Bifid (containing a combination of unspecified *Bifidobacterium* spp*.*, *Streptococcus thermophilus* and *Lactobacillus bulgaricus*) using a similar approach. The first step is crucial to reveal that a single piece (0.5 g) has to be dissolved in 0.2 ml test-tube of sterilized distilled water before being inoculated into 10 ml test tube of fresh RB-medium.
