**1.4. The small intestine**

When the partially digested food enters the small intestine, it is mixed with intestinal secretions, such as bile, pancreatic enzymes and bicarbonates. The bile in particular has a strong bactericidal effect. Together with a strong-fluid secretion by the intestinal mucosa, this also prevents extensive colonization of the small intestine. Colonization usually takes place in crypts and blind loops. In this lower part of the small intestine, the movement is slightly reduced, the bile is diluted, the pH becomes more neutral, and the oxygen tension drops rapidly. This favours the growth and/or transit of different bacteria, initially mainly aerotolerant species, and in the ileum also strict anaerobes as revealed in Figure 3 (Hartemink, 1999). There is not much carbohydrate fermentation in the small intestine in healthy humans, due to the flow rate and the little bacterial mass.

In studies undertaken in pigs, it has been reported that the conditions in the small intestine differed widely. The pH is much higher, and the bile secretion is less abundant, which results in an extensive bacterial growth in the small intestine. This also results in substantial fermentation of ingested carbohydrates. The human body is projected to produce between 20 to 30 g of bile salts per day to replace the loss occurring in the excreta (250 to 500 mg), and these are typically stored in the gall bladder (Glickman, 1980).

**Figure 3.** Appearance of bacterial species, oxygen tension and habitat in the small intestine.

### **1.5. The large intestine**

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

ingested carbohydrates in the stomach hardly occurs.

healthy humans, due to the flow rate and the little bacterial mass.

and these are typically stored in the gall bladder (Glickman, 1980).

**BILE 103 /g**

 *Streptococcus*  **DUODENUM** *Lactobacillus*

 *Enterococcus* 

 **JEJUNUM** *Bifidobacterium* *Clostridium Bacteriodes* 

**STOMACH high O2 / low pH** 

**Figure 3.** Appearance of bacterial species, oxygen tension and habitat in the small intestine.

**LARGE INTESTINE low O2 / high pH** 

**1.4. The small intestine** 

digestive enzymes can alter protein down to individual amino acids. Fermentation of

When the partially digested food enters the small intestine, it is mixed with intestinal secretions, such as bile, pancreatic enzymes and bicarbonates. The bile in particular has a strong bactericidal effect. Together with a strong-fluid secretion by the intestinal mucosa, this also prevents extensive colonization of the small intestine. Colonization usually takes place in crypts and blind loops. In this lower part of the small intestine, the movement is slightly reduced, the bile is diluted, the pH becomes more neutral, and the oxygen tension drops rapidly. This favours the growth and/or transit of different bacteria, initially mainly aerotolerant species, and in the ileum also strict anaerobes as revealed in Figure 3 (Hartemink, 1999). There is not much carbohydrate fermentation in the small intestine in

In studies undertaken in pigs, it has been reported that the conditions in the small intestine differed widely. The pH is much higher, and the bile secretion is less abundant, which results in an extensive bacterial growth in the small intestine. This also results in substantial fermentation of ingested carbohydrates. The human body is projected to produce between 20 to 30 g of bile salts per day to replace the loss occurring in the excreta (250 to 500 mg),

*Enterobacteria (E. coli)* 

 **ILEUM** Strict anaerobes

In the large intestine, the flow rate of the digesta decreases considerably. In addition, the bile is even more diluted, and the pH is close to neutral. Total logarithmic counts may reach up to 1011 bacteria/gram contents. Higher numbers have been reported, but it is physically impossible to achieve a number over 1012 bacteria/gram faecal dry weight, taking into account the average balance of faeces and the dimensions of an average bacterium. It is estimated that over 400 different bacterial species reside in the human large intestine. Of these, about 200 have been validly described, but often non-identifiable strains are reported.

In addition to the resident bacteria, transient bacteria are often isolated. The dominant floras in the large intestine are relatively stable, and they include *Bifidobacterium*, *Bacteriodes* and anaerobic cocci. Large variations also exist in the less dominant species, especially among the facultative or aerotolerant species like *E. coli* and lactobacilli. The numbers of the dominant species are also comparable in different population. Differences in counts are more often due to the methodology used, rather than actual differences. As in individuals, the counts of less dominant species differ widely between different populations. Among the dominant bacterial groups are members of the genera *Bacteriodes*, *Bifidobacterium*, *Coprococcus*, *Peptostreptococcus*, *Eubacterium* and *Ruminococcus*. Members of the following genera are often isolated and are available in lower numbers: *Fusobacterium*, *Streptococcus*, *Lactobacillus*, *Enterococcus*, *Veillonella*, *Megasphaera*, *Propionibacterium* and *Enterobacteriaceae*.

It is indispensable to emphasize here that the principal function of the GI tract includes breakdown and absorption of food components and water. In general, degradation takes place in the upper part of the GI tract, whereas the major sites of absorption are in the lower part of small intestines and the large intestines. Degradation and absorption are enhanced by the excretion of the large number of digestive enzymes, such as glycosidases, lipases, peptidases and proteinases. The colon receives digesta from the intestinal ileum approximately 5 h after food ingestion. Thereafter, rate of motion slows progressively from the caecum towards the distal colon. Concurrent with this is an increase in water absorption; thus gut contents in the proximal colon are more or less liquid in nature but have a faecal like appearance distally (Hartemink, 1999).

For most of the world's population, the standard gut transit time is 60 h, with a variety of 23 – 168 h. The colon itself has a capacity of approximately 500 ml with about 220 g contents. In general, stools weight correlates inversely with transit time. Studies with healthy volunteers have indicated that speeding up colonic transit times from 67 to 25 h resulted in an increase in stool weight from 148 to 285 g/day. Conversely, when transit time increased, stool weight decreases from 182 to 119 g/day (Hartemink, 1999). The differences are mainly due to changes in the water content of the faecal mass.

The structure of faeces is highly variable. Bacteria may constitute up to 55% of the total solids, whilst fibre and other non-digested, non-fermented compounds represent less than 17% of the weight of which about 24% is soluble material. Faecal water content may be as high as 70% of the total weight. Stool size is influenced by both dietary and endogenous 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 may cause diarrhoea, due to the increased osmotic pressure.

*Bifidobacterium* in Human GI Tract:

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 287

are grown in Man, Rogosa, and Sharpe (MRS) medium under anaerobic conditions, in a microprocessor-controlled anaerobic chamber. Cultures are incubated for 18 h at 37 °C and stored at 3 – 5 °C between transfers. For the fermentation test, 0.5 ml of 10% substrate solutions (which were membrane filtered through 0.45 µM filter), are added to 9.5 ml of Peptone Yeast-extract Fildes (PYF) basal medium (Mitsuoka, 1990). After 2.5 days of strictly anaerobic incubation, the pH of the growth medium is measured. Tubes showing pH values below 5.5 are considered to be positive for fermentation. The presence of acetate and lactate in the fermented PYF containing glucose medium is assayed by using gas chromatography

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

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.

In the morphological analysis of bifidobacteria population, *in situ,* in human faeces and/or other foods products, microscopes have been used to determine the degree of heterogeneity of these probiotic's populations. The morphology of bifidobacteria determined microscopically has been used as an aid to phenotypic differentiation within the group, while the effect of medium type, low pH and high bile salt concentrations on the bifidobacterial cell morphology has also been studied by this method. Individual

**3. Morphological identification of bifidobacteria by phase contrast** 

(GC) or high performance liquid chromatography (HPLC).

for 3 – 4 days to determine colonies of the resistant *Bifidobacterium* strains.

*2.1.2. Isolation and screening via stress-shock* 

**microscopy (PCM)** 
