**5.2. Survival of bifidobacteria in simulated acidic pH of human stomach**

Figure 8 shows the survival of selected *Bifidobacterium* strains exposed to various acidic pH levels. The bifidobacterial counts (range: 5.5 – 6.7 log CFU/ml) of all four strains at pH 3.5 after 5 h are an indication of resistance that may perhaps simulate gastric conditions. In fact, with *B. bifidum*, *B. infantis*, and *B. longum* the counts were > 2.0 log CFU/ml after exposure for 5 h, which indicates that these strains are relatively resistant at pH 3.0. However, as observed from all the experimental results in Figure 8, the *B. adolescentis* strain is more sensitive than the other three strains to all the acid treatments. For instance, numbers of *B. adolescentis* are 3.4 log CFU/ml after 10 h at pH 3.5, but below the level of exposure thereafter, while a similar count (~3.4 log CFU/ml) is observed with the other three strains after 12.5 h. This 2.5 h difference in survival at pH 3.5 is approximately the time chosen by Olejnik *et al*., (2005) to control acid resistance, as these times simulate residence time in the stomach. On this basis, this specific strain of *B*. *adolescentis* is considered a less-resistant strain with respect to gastric acidity.

Many other researchers have found pH 2.0 and pH 3.0 to be lethal and sublethal pH values respectively for lactic acid bacteria (LAB), including bifidobacteria (Khalil *et al*., 2007). It is vital to stress that probiotics are able to confer health benefits despite the brief exposure to exact acidic conditions following ingestion. Although a log-scale reduction of viability may occur, it may still mean that a sufficient number of bifidobacteria survive the gut, depending on the dose. Moreover, the exposure to acid does not mean that the potential health benefits are lost. Some cells may die, and some may be injured. However, these cells may recover later, and they may also have beneficial effects on health. The mechanism may be mediated, for example, through the components of the cell wall of the probiotics which will then be available in both dead and living cells. It should be also borne in mind that, the food matrix in which the probiotics are consumed is likely to have a strong effect on the survival of the bacteria in the gut.

In Figure 8, for example, it is possible to say that the high survival counts of *B. bifidum*, *B. infantis*, and *B. longum* exposed to pH 4.5 after 41 h is a representation of culture stability curves of the acid resistant strains (log CFU/ml) in a food matrix. While the pH of 4.5 does not represent gastric acid conditions, it is a typical representation of fermented products, and in such products, the survival counts are expected to last for much longer periods during shelf life, especially at the refrigeration temperature of 4 °C.

## **5.3. Survival of bifidobacteria in simulated bile salts nature**

Resistance to bile salts is considered an intrinsic property for probiotic strains to survive the conditions in the small intestine. The physiological bile salt concentration in the GI tract of humans is estimated to be 0.3 – 0.4% w/v (Jia *et al*., 2010). As shown in Figure 9, the linear regressions of the loss of CFU did express satisfactorily that *B. bifidum*, *B. infantis*, and *B. longum* are more-resistant strains to the bile salts. These 3 strains survived well in 0.45% (w/v) bile salts, with more than 4.5 log CFU/ml present after 10 h. Their capacity to survive high bile salt concentrations suggests the existence of defence mechanisms and confirms that strains showing antagonistic effect against enteric pathogens should be able to compete successfully with the pathogens in the GI tract. It is clear that *B. adolescentis* is again the lessresistant strain encountered with only 2.8 log CFU/ml surviving after 10 h in 0.45% bile salts (see Figure 9). Therefore, *B*. *adolescentis* is considered the less-resistant strain, while *B*. *bifidum*, *B*. *infantis*, and *B*. *longum*, in that order, are considered the more-resistant. It is well known that, the bile salt hydrolytic (BSH) activity may be the contributing factor towards the resistance of the LABs and to the toxicity of conjugated bile salts in the duodenum, and therefore, is an essential colonization factor.

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

at either pH2 or BS2.

bacteria in the gut.

where *z* is the acid pH value (in Equation 1) or bile salts value (in Equation 2) required for a ten-fold reduction in *D*-values; pH1 is the acidic value of pH 1; pH2 is the acidic value of pH 2; BS1 is the concentration of bile salts 1 (%); BS2 is the concentration of bile salts 2 (%); *D*(acid1) or *D*(bile1) is a *D*-value obtained at either pH1 or BS1; and *D*(acid2) or *D*(bile2) is a *D*-value obtained

Figure 8 shows the survival of selected *Bifidobacterium* strains exposed to various acidic pH levels. The bifidobacterial counts (range: 5.5 – 6.7 log CFU/ml) of all four strains at pH 3.5 after 5 h are an indication of resistance that may perhaps simulate gastric conditions. In fact, with *B. bifidum*, *B. infantis*, and *B. longum* the counts were > 2.0 log CFU/ml after exposure for 5 h, which indicates that these strains are relatively resistant at pH 3.0. However, as observed from all the experimental results in Figure 8, the *B. adolescentis* strain is more sensitive than the other three strains to all the acid treatments. For instance, numbers of *B. adolescentis* are 3.4 log CFU/ml after 10 h at pH 3.5, but below the level of exposure thereafter, while a similar count (~3.4 log CFU/ml) is observed with the other three strains after 12.5 h. This 2.5 h difference in survival at pH 3.5 is approximately the time chosen by Olejnik *et al*., (2005) to control acid resistance, as these times simulate residence time in the stomach. On this basis, this specific strain of *B*. *adolescentis* is considered a less-resistant strain with respect to gastric acidity.

Many other researchers have found pH 2.0 and pH 3.0 to be lethal and sublethal pH values respectively for lactic acid bacteria (LAB), including bifidobacteria (Khalil *et al*., 2007). It is vital to stress that probiotics are able to confer health benefits despite the brief exposure to exact acidic conditions following ingestion. Although a log-scale reduction of viability may occur, it may still mean that a sufficient number of bifidobacteria survive the gut, depending on the dose. Moreover, the exposure to acid does not mean that the potential health benefits are lost. Some cells may die, and some may be injured. However, these cells may recover later, and they may also have beneficial effects on health. The mechanism may be mediated, for example, through the components of the cell wall of the probiotics which will then be available in both dead and living cells. It should be also borne in mind that, the food matrix in which the probiotics are consumed is likely to have a strong effect on the survival of the

In Figure 8, for example, it is possible to say that the high survival counts of *B. bifidum*, *B. infantis*, and *B. longum* exposed to pH 4.5 after 41 h is a representation of culture stability curves of the acid resistant strains (log CFU/ml) in a food matrix. While the pH of 4.5 does not represent gastric acid conditions, it is a typical representation of fermented products, and in such products, the survival counts are expected to last for much longer periods

Resistance to bile salts is considered an intrinsic property for probiotic strains to survive the conditions in the small intestine. The physiological bile salt concentration in the GI tract of

during shelf life, especially at the refrigeration temperature of 4 °C.

**5.3. Survival of bifidobacteria in simulated bile salts nature** 

**5.2. Survival of bifidobacteria in simulated acidic pH of human stomach** 
