**5. Experimental procedures for the enumeration of bifidobacteria and determining microbial inactivation by low acidic pH or bile salts**

LAB or bifidobacteria strains can be selected or isolated from commercial or alleged "own isolates" strains, from freeze-dried cultures which are resuscitated to stationary phase in MRS broth at a ratio of 2% of the volume of the fresh broth. Decimal dilutions are put onto Raffinose–Bifidobacterium (RB) agar plates whose pH had to be adjusted to 6.8 – 7.0 with 2 N NaOH. The agar plates are then incubated anaerobically at 38.5 ºC for 3 – 4 d and number of colony forming units (CFU)/mL are determined. Two hundred microliters of each strain containing about 108 CFU/mL is aseptically transferred into test tubes containing 9 mL of diluted MRS medium with pH adjustments of 3.0, 3.5, 4.0, or 4.5, using 2 N HCl. These suspensions are incubated anaerobically at 38.5 ºC and numbers of survivors are determined after various times as shown in Figure 7 (A). Cells were harvested by centrifugation at 5 ºC, were washed with phosphate-buffered saline (PBS) and were re-suspended in diluted MRS medium without pH adjustment. After thorough mixing on a vortex mixer, the concentration of surviving cells is determined by anaerobic pour plate counts, using 2 plates of RB agar per dilution, and incubated at 38.5 ºC for 3 – 4 days.

*Bifidobacterium* in Human GI Tract:

Incubate anaerobically. Hold for 350, 700, 1050, 1400, 1750, 2100, 2450 min, respectively.

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 299

Incubate anaerobically. Hold for 250, 500, 750, 1000, 1250, 1500, 1750 min, respectively.

**Figure 7.** Schematic diagram of treatment groups for the selected bifidobacteria. A): Influence of gastric

Centrifuge @ 3000 rmp for 15 min at 5°C. Wash with Phosphate-Buffered Saline. Re-suspend in SGJ @ pH 6.8. Vortex well. Pour and/or spread plate count on RB agar @ 38.5°C / 72 hours.

*Bifidobacterium* spp. Suspended in SGJ @ ambient (38.5°C).

**B) Bile Salt Treatments: 2nd Simulated Stressing Method** 

Centrifuge @ 3000 rmp for 15 min at 5°C. Wash with Phosphate-Buffered Saline. Re-suspend in SGJ @ pH 6.8. Vortex well. Pour and/or spread plate count on RB agar @ 38.5°C / 72 hours.

*Bifidobacterium* spp*.*  Suspended in Simulated Gastric Juice (SGJ) @ ambient temp. (38.5°C).

**Treatment - pH 3.0 Treatment - pH 3.5 Treatment - pH 4.0 Treatment - pH 4.5** 

**A) Stomach pH Treatments: 1st Simulated Stressing Method** 

10

10

Log 

*<sup>z</sup> <sup>D</sup>*

Log 

*<sup>z</sup> <sup>D</sup>*

2 1

Incubate anaerobically. Hold for 150, 300, 450, 600, 750, 900, 1050 min, respectively.

**Treatment - 0.45% oxgall**

(1)

Incubate anaerobically. Hold for 50, 100, 150, 200, 250, 300, 350 min, respectively.

**Treatment - 0.60% oxgall** 

(2)

(acid pH acid pH )

*D*

2 1

*D*

BS BS

(acid 1)

(acid 2)

(bile 1)

(bile 2)

acidity and its residence time. B): Influence of bile salt(s) and its residence time.

**Treatment - 0.30% oxgall**

Incubate anaerobically. Hold for 150, 300, 450, 600, 750, 900, 1050 min, respectively.

Incubate anaerobically. Hold for 250, 500, 750, 1000, 1250, 1500, 1750 min, respectively.

**Treatment - 0.15% oxgall**

Incubate anaerobically. Hold for 50, 100, 150, 200, 250, 300, 350 min, respectively.

Incubate anaerobically. Hold for 350, 700, 1050, 1400, 1750, 2100, 2450 min, respectively.

(acid)

(bile)

Similarly, treatments for the bile salts are carried out at the final concentrations of 0.15%, 0.30%, 0.45%, and 0.60% ox-gall in diluted MRS medium (pH 6.8), exposed to appropriate times as to low pH and incubated anaerobically at 38.5 ºC (see Figure 7 (B). The cells are harvested by centrifugation, washed with PBS, re-suspended in diluted MRS medium without pH adjustment, and mixed using a vortex mixer as described for acidic pH conditions before. Numbers of CFU of bifidobacteria surviving the lytic effect of bile salts are also determined by anaerobic pour plate counts on RB agar after anaerobic incubation for 3 – 4 d at 38.5 ºC.

### **5.1. Characterizations for** *D***(acid)-,** *D***(bile)-,** *z***(acid), and** *z***(bile)-values**

*D*(acid)-value is defined as the time (in min) required at a specified acidic pH to reduce the number of cells by 90%, while *D*(bile)-value is defined as the time (in min) required at a specified concentration of bile salts to reduce the number of cells by 90%. In fact, the *D*(bile) value of any LAB or bifidobacterial strain is directly proportional to the bile salt concentrations, while the *D*(acid)-value is inversely proportional to the acidic pH.

The *z*(acid) and/or *z*(bile)-values, on the other hand, is defined as a decrease in pH value (pH < 4.5) or an increase in bile salt concentration (% ox-bile) required to reduce the *D*-values by 1 log cycle, however, respectively. The *D*(acid)- and *D*(bile)-values can be directly calculated from the absolute values of the reciprocal of the slopes of the linear-regression equations, using a Microsoft Office–Excel software. It is essential to emphasize that, the regression lines must be applied to all the treatments, for which restriction of the *R*-squared (*R*2) value is pragmatic above 0.8920. Moreover, the *D*(acid)- and/or *D*(bile)-values can also be calculated algebraically from the regression equation derived using the method of least-squares to be able to produce the *z*(acid)- and/or *z*(bile)-values for the probiotic strains.

In order to determine the *z*(acid)- and/or *z*(bile)-values, the formula is exactly the same as that for heat resistance, replacing T (temperature) with pH values or bile salts (BS) concentrations as described by Equations (1) and (2), respectively. In both of these cases, the effect of acidic conditions and bile salts is determined from the reduction in concentration of colony-forming units. One has to pay attention that the dynamic *z*(acid)- and/or *z*(bile)-values are calculated for a period of exponential destruction of microbial cells (following the logarithmic order of death), using both Equations (1) and (2).

#### *Bifidobacterium* in Human GI Tract:

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 299

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

of RB agar per dilution, and incubated at 38.5 ºC for 3 – 4 days.

N NaOH. The agar plates are then incubated anaerobically at 38.5 ºC for 3 – 4 d and number of colony forming units (CFU)/mL are determined. Two hundred microliters of each strain containing about 108 CFU/mL is aseptically transferred into test tubes containing 9 mL of diluted MRS medium with pH adjustments of 3.0, 3.5, 4.0, or 4.5, using 2 N HCl. These suspensions are incubated anaerobically at 38.5 ºC and numbers of survivors are determined after various times as shown in Figure 7 (A). Cells were harvested by centrifugation at 5 ºC, were washed with phosphate-buffered saline (PBS) and were re-suspended in diluted MRS medium without pH adjustment. After thorough mixing on a vortex mixer, the concentration of surviving cells is determined by anaerobic pour plate counts, using 2 plates

Similarly, treatments for the bile salts are carried out at the final concentrations of 0.15%, 0.30%, 0.45%, and 0.60% ox-gall in diluted MRS medium (pH 6.8), exposed to appropriate times as to low pH and incubated anaerobically at 38.5 ºC (see Figure 7 (B). The cells are harvested by centrifugation, washed with PBS, re-suspended in diluted MRS medium without pH adjustment, and mixed using a vortex mixer as described for acidic pH conditions before. Numbers of CFU of bifidobacteria surviving the lytic effect of bile salts are also determined by

*D*(acid)-value is defined as the time (in min) required at a specified acidic pH to reduce the number of cells by 90%, while *D*(bile)-value is defined as the time (in min) required at a specified concentration of bile salts to reduce the number of cells by 90%. In fact, the *D*(bile) value of any LAB or bifidobacterial strain is directly proportional to the bile salt

The *z*(acid) and/or *z*(bile)-values, on the other hand, is defined as a decrease in pH value (pH < 4.5) or an increase in bile salt concentration (% ox-bile) required to reduce the *D*-values by 1 log cycle, however, respectively. The *D*(acid)- and *D*(bile)-values can be directly calculated from the absolute values of the reciprocal of the slopes of the linear-regression equations, using a Microsoft Office–Excel software. It is essential to emphasize that, the regression lines must be applied to all the treatments, for which restriction of the *R*-squared (*R*2) value is pragmatic above 0.8920. Moreover, the *D*(acid)- and/or *D*(bile)-values can also be calculated algebraically from the regression equation derived using the method of least-squares to be

In order to determine the *z*(acid)- and/or *z*(bile)-values, the formula is exactly the same as that for heat resistance, replacing T (temperature) with pH values or bile salts (BS) concentrations as described by Equations (1) and (2), respectively. In both of these cases, the effect of acidic conditions and bile salts is determined from the reduction in concentration of colony-forming units. One has to pay attention that the dynamic *z*(acid)- and/or *z*(bile)-values are calculated for a period of exponential destruction of microbial cells (following the

anaerobic pour plate counts on RB agar after anaerobic incubation for 3 – 4 d at 38.5 ºC.

concentrations, while the *D*(acid)-value is inversely proportional to the acidic pH.

**5.1. Characterizations for** *D***(acid)-,** *D***(bile)-,** *z***(acid), and** *z***(bile)-values** 

able to produce the *z*(acid)- and/or *z*(bile)-values for the probiotic strains.

logarithmic order of death), using both Equations (1) and (2).

**Figure 7.** Schematic diagram of treatment groups for the selected bifidobacteria. A): Influence of gastric acidity and its residence time. B): Influence of bile salt(s) and its residence time.

$$z\_{\text{(acid)}} = \frac{-(\text{acid pH}\_2 - \text{acid pH}\_1)}{\text{Log}\_{10} \left(\frac{D\_{\text{(acid 1)}}}{D\_{\text{(acid 2)}}}\right)}\tag{1}$$

$$z\_{\text{(bile)}} = \frac{\text{BS}\_2-\text{BS}\_1}{\text{Log}\_{10}\left(\frac{D\_{\text{(bile)}}}{D\_{\text{(bile2)}}}\right)}\tag{2}$$

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 at either pH2 or BS2.

*Bifidobacterium* in Human GI Tract:

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 301

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

**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** 

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

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

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

] or bile salts. As for probiotics to gain intestinal

therefore, is an essential colonization factor.

small changes in [H+] and/or [OH-

acids (in the stomach) and bile salts (in the small intestine).

**bile salts stress** 
