**5.4. The feasibility of** *D***(acid)-,** *D***(bile)-,** *z***(acid)- and** *z***(bile)-values for selection of probiotic strains and for determining the mechanisms of resistance to acid and bile salts stress**

Table 5, shows that accurate tabulation of the *D*(acid)- or *D*(bile)-values and their respective *z*(acid)- or *z*(bile)-values is tremendously helpful in evaluating the resistance and susceptibility of probiotics to acidic pH and high bile salt concentrations, respectively. Both the estimated *D*(acid)- and *D*(bile)-values validated that the most acid- and bile-resistant strain is *B. bifidum*  followed by *B. infantis*, *B. longum*, and final *B. adolescentis.* It is also possible to observe in Table 5, that, increasing the bile salt concentration from 0.15 to 0.60% had a greater impact on survival than decreasing the pH values from 4.5 to 3.0, with the *D*(bile)-values of *B. bifidum*  decreasing from 17.40 to 1.40 min and the *D*(acid)-values decreasing from 23.80 to 1.10 min. Similar trends are observed with all other *Bifidobacterium* strains. However, decreases of depicted *z*(acid)-value in the pH value (pH<4.5) or increases of depicted *z*(bile)-value in the bile salt concentrations (% ox-bile) are expected to cause a 1-log reduction in their respective *D*values. In practice, *z*(acid)- or *z*(bile)-value measures how the sensitivity of probiotic strains is to small changes in [H+] and/or [OH- ] or bile salts. As for probiotics to gain intestinal colonization in humans or animals for their proclaimed therapeutic health benefits, obviously, they have to tolerate inhibitory substances secreted by the host, such as gastric acids (in the stomach) and bile salts (in the small intestine).

Of all ions, H+ and OH<sup>−</sup> are the most mobile, and minor changes in their concentrations show significant effects on microorganisms. Most organisms survive better when these ions are present in approximately equal concentrations, that is, pH 7.0. Although many bacteria tolerate higher pH values, only a few are acid tolerant or acidophilic. In addition, many other bacteria are tolerant of small pH variations, especially in the pH range of 6.0 to 9.0. For instance, if the pH of the medium changes rapidly, there may be a transient change in the intracellular pH, and this is usually readjusted to the original pH within 30 min. Consequently, any damage produced by adverse pH is not actually due to the H+ and/or

OH−, but to the effect of these ions on the proportion of undissociated weak acids or bases, which penetrate more readily into the bacterial cell than the ionized forms. In contrast, bile salts are biological detergents synthesized in the liver from cholesterol, conjugated to either glycine or taurine, and are then secreted into the intestine where they facilitate fat absorption. Bile salts are well known to be toxic for many cells as they disrupt the lipid bilayer structure of the cellular membranes. Many earlier studies revealed that the autochthonous gastrointestinal microbiota must develop strategies to protect themselves against bile salts.

*Bifidobacterium* in Human GI Tract:

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 303

**Figure 9.** Linear regressions of the loss of CFU for the selected bifidobacteria strains when exposed to high bile salt (oxgall) concentrations of 0.60%, 0.45%, 0.30% and 0.15%, respectively: (a) *B*. *bifidum*, (b) *B*.

The individual *z*(acid)- or *z*(bile)-values calculated from their *D*(acid)- and *D*(bile)-values ranged from 1.11 – 1.55 pH units and 0.40 – 0.49%, respectively (Table 5). Although the combination of both the low acidic pH and bile salts is not assessed, it is assumed that at pH < 3.0, and 0.60% of oxbile, the combined effects could be more synergistic and even greater in magnitude for probiotic bacteria to survive. Additionally, the *D*(acid)- and *D*(bile)-values reveal a modern and efficient sorting order of the more-resistant probiotic strains to these two distinct hostile GI tract conditions in humans. Many authors have investigated the effect of bile on survival of LAB. For example, Kim *et al*., (1999) examined the effect of bile concentration in the range of 0 – 0.4% on survival of *Lb. lactis* and found bile to be toxic at concentrations over 0.04%. Shimakawa *et al*., (2003) reported that 0.2% oxgall in the growth medium inhibited growth of *B. breve* strain Yakult. Others detected that all bacterial cells were killed by 0.2% bile and higher (Olejnik *et al.*, 2005). However, Khalil *et al*., (2007) reported higher resistance to bile salts, with viability of

strains apparently increasing when exposed to high levels of oxgall (0.4%).

*longum*, (c) *B*. *infantis*, (d) *B*. *adolescentis*.

**Figure 8.** Linear regressions of the loss of CFU for the selected bifidobacteria strains when exposed to simulated gastric acidity of pH 3.0, pH 3.5, pH 4.0 and pH 4.5, respectively: (a) *B*. *bifidum*, (b) *B*. *longum*, (c) *B*. *infantis*, (d) *B*. *adolescentis*.

against bile salts.

(c) *B*. *infantis*, (d) *B*. *adolescentis*.

OH−, but to the effect of these ions on the proportion of undissociated weak acids or bases, which penetrate more readily into the bacterial cell than the ionized forms. In contrast, bile salts are biological detergents synthesized in the liver from cholesterol, conjugated to either glycine or taurine, and are then secreted into the intestine where they facilitate fat absorption. Bile salts are well known to be toxic for many cells as they disrupt the lipid bilayer structure of the cellular membranes. Many earlier studies revealed that the autochthonous gastrointestinal microbiota must develop strategies to protect themselves

**Figure 8.** Linear regressions of the loss of CFU for the selected bifidobacteria strains when exposed to simulated gastric acidity of pH 3.0, pH 3.5, pH 4.0 and pH 4.5, respectively: (a) *B*. *bifidum*, (b) *B*. *longum*,

**Figure 9.** Linear regressions of the loss of CFU for the selected bifidobacteria strains when exposed to high bile salt (oxgall) concentrations of 0.60%, 0.45%, 0.30% and 0.15%, respectively: (a) *B*. *bifidum*, (b) *B*. *longum*, (c) *B*. *infantis*, (d) *B*. *adolescentis*.

The individual *z*(acid)- or *z*(bile)-values calculated from their *D*(acid)- and *D*(bile)-values ranged from 1.11 – 1.55 pH units and 0.40 – 0.49%, respectively (Table 5). Although the combination of both the low acidic pH and bile salts is not assessed, it is assumed that at pH < 3.0, and 0.60% of oxbile, the combined effects could be more synergistic and even greater in magnitude for probiotic bacteria to survive. Additionally, the *D*(acid)- and *D*(bile)-values reveal a modern and efficient sorting order of the more-resistant probiotic strains to these two distinct hostile GI tract conditions in humans. Many authors have investigated the effect of bile on survival of LAB. For example, Kim *et al*., (1999) examined the effect of bile concentration in the range of 0 – 0.4% on survival of *Lb. lactis* and found bile to be toxic at concentrations over 0.04%. Shimakawa *et al*., (2003) reported that 0.2% oxgall in the growth medium inhibited growth of *B. breve* strain Yakult. Others detected that all bacterial cells were killed by 0.2% bile and higher (Olejnik *et al.*, 2005). However, Khalil *et al*., (2007) reported higher resistance to bile salts, with viability of strains apparently increasing when exposed to high levels of oxgall (0.4%).


*Bifidobacterium* in Human GI Tract:

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 305

As compared to previous studies, the practicality of *D*(acid)-, *D*(bile)-, *z*(acid)- and *z*(bile)-values as new kinetic-measurements applied in this study, are indeed, quick to identify comparably higher survival of bifidobacteria cells (> 4.1 log CFU/ml after 2.5 h) at elevated bile salt concentrations of 0.6% (w/v), thereby confirm also that the individual *Bifidobacterium* strains are resistant to harsh intestinal conditions in the following order: *B. bifidum* > *B. infantis* > *B. longum* > *B. adolescentis*. A number of researchers reported that *B. infantis* had the highest survival rates followed by *B. bifidum, B. breve and B. longum,* when exposed to bile salt at concentrations ranging from 0 to 3 g/l. In contrast, the literature contains also one preliminary report that *B. longum* exhibited the highest tolerance to bile salts followed by *B. bifidum* and *B. infantis,* which was almost the exact opposite in order of their tolerance to acidic pH. These contrasting observations may reflect the strain-specific resistance to acid or bile salts stress. It also indicates that tolerance is strain- rather than species-specific.

Likewise, the source of isolation of the probiotic strains is particularly influential too.

Apart from the isolation, enumeration, unequivocal taxonomical characterization, screening and selection of tolerant strains of bifidobacteria to gastric acid and bile salts studies, the assessment of the tolerant bifidobacteria to bile salts and low pH has been made possible by use of *D*- and *z*-value concept. After log-conversion, inactivation followed first-order kinetic law whereby validating the kinetic assumptions of the latter concept. The projected *z*(acid) and *z*(bile)-values were all fairly similar for the bifidobacteria strains and suggested the effect of increasing the bile salt concentration or decreasing the pH on the *D*(acid)- and *D*(bile)-values. This approach is useful for measuring the resistance and sensitivity of lactic acid bacteria or bifidobacteria to these two hostile gastrointestinal conditions. The approach pursued in this chapter would be extremely useful for predicting the suitability of bifidobacteria and/or other LAB as probiotics for use in real life situations. While the mechanisms of probiotic survival in the GI tract could be more complex, the practical utility of the *D*(acid)- and/or

We are grateful to Ms. Masa Vidovic and the entire staff of InTech for helpful advices and the opportunity given to us. We would also like to thank Mr. Richard Shigwedha for the

**6. Conclusion** 

**Author details** 

Nditange Shigwedha\*

**Acknowledgement** 

good cooperation.

Corresponding Author

 \*

*D*(bile)- and their *z*(acid)- and *z*(bile)-values is significant.

 and Li Jia *University of Namibia & Meat Corporation of Namibia, Namibia* 

**Table 5.** Selected *Bifidobacterium* strains and their calculated *D*(acid)-, *D*(bile)-, *z*(acid)- and *z*(bile)-values.

As compared to previous studies, the practicality of *D*(acid)-, *D*(bile)-, *z*(acid)- and *z*(bile)-values as new kinetic-measurements applied in this study, are indeed, quick to identify comparably higher survival of bifidobacteria cells (> 4.1 log CFU/ml after 2.5 h) at elevated bile salt concentrations of 0.6% (w/v), thereby confirm also that the individual *Bifidobacterium* strains are resistant to harsh intestinal conditions in the following order: *B. bifidum* > *B. infantis* > *B. longum* > *B. adolescentis*. A number of researchers reported that *B. infantis* had the highest survival rates followed by *B. bifidum, B. breve and B. longum,* when exposed to bile salt at concentrations ranging from 0 to 3 g/l. In contrast, the literature contains also one preliminary report that *B. longum* exhibited the highest tolerance to bile salts followed by *B. bifidum* and *B. infantis,* which was almost the exact opposite in order of their tolerance to acidic pH. These contrasting observations may reflect the strain-specific resistance to acid or bile salts stress. It also indicates that tolerance is strain- rather than species-specific. Likewise, the source of isolation of the probiotic strains is particularly influential too.
