**3. Results**

*3.1. Survivability and Growth of Bacteria Present in Commercial Probiotic Preparations in MRS Broth*

Bacteria were cultured in the MRS broth deemed optimal for the growth of LAB and bifidobacteria [29–31] to observe how they react in optimal conditions.

Curves of changes in optical density (OD, λ = 600 nm) during culture of commercial preparations of probiotic bacteria in MRS broth with various pH values are shown on Figure 3. The viable cell counts of the tested probiotic bacteria determined by the plate method are illustrated in Table 1.

**Figure 3.** Changes in the optical density (OD600) during culture of commercial probiotics preparations: (**a**) *Lb. rhamnosus* GG ATCC 53103; (**b**) *Bifidobacterium* BB-12; (**c**) *Lb. casei*; (**d**) *Lb. acidophilus*; (**e**) *Lb. plantarum* in MRS medium with different pH (2.0; 3.0; 4.0; 5.0; 6.2), (*p* < 0.05). The number of repeats for each treatment *n* = 3. Letters marked with the same color define a homogeneous group within one pH value among the tested variants of the experiment (a given color correlates with the pH value and a given letter correlates with a given homogeneous group within pH range).


**Table 1.** Growth of commercial probiotic strains (log CFU mL−<sup>1</sup> ± SD) in MRS medium with different pH.

"-"—no growth/less than 4 log CFU mL−1.

The OD of *Lb*. GG culture in MRS broth with pH 2.0 and pH 3.0 was decreasing throughout the experiment (from the initial value of ca. 0.5 to the final value of ca. 0.36) (Figure 3). The results obtained using the plate method (Table 1) indicate that during the first 12 h of the experiment at pH 2.0 or 3.0, the number of *Lb*. GG decreased to less than 4 log CFU ml−1. It was found that the other tested strains reacted in a similar way, except *Lb. casei* and *Lb. plantarum* in MRS with pH 3.0 (Table 1). The highest OD values were noted for this probiotic strain in MRS broth with pH 5.0 and pH 6.2 (Figure 3). The number of *Lb.* GG in the MRS with pH 5.0 after 48 h of the experiment increased by about 1.34 log order, while at the optimal pH (pH 6.2) by 2.2 log order.

The course of the growth curve plotted for the BB-12 strain at pH 4.0 indicated that the bacteria needed a lot of time to adapt to medium conditions before their cells began to divide (Figure 3). By the 36th hour of the experiment, a decrease in the number of strain BB-12 from the initial 7.13 log CFU mL−<sup>1</sup> to 4.24 log CFU mL−<sup>1</sup> was observed, while in the last twelve hours there was an increase in the number of bacteria by about one logarithmic order, which is also visible on the course of the OD curve (Figure 3). Only *Lb.* GG, *Lb. acidophilus* and *Lb. plantarum* strains showed an increase in cell number during incubation in MRS at pH 4.0 (Table 1).

Growth curves plotted for the BB-12 strain in MRS media with pH 5.0 and 6.2. had a similar course and indicated intensive cell proliferation (Figure 3). For all tested strains, the number of cells was increased during incubation in MRS at pH 5.0 and pH 6.2 (Table 1). The increase in the viable cell number at pH 5.0 was 1.25–3.46 log orders and at pH 6.0 about 3.0 log orders.

For most of the probiotic strains tested, the course of the growth curves was characteristic and included lag phase, log phase, and stationary phase. Worthy of notice is, however, that growth curves plotted for bacteria cultured under experimental conditions differed for each preparation (Figure 3).

Statistical analysis showed that there was not significant difference between the OD value of *L. rhamnosus* GG growing in MRS pH 2.0, 3.0, as well as in MRS pH 5.0 and 6.2 (Figure 3). It was also shown that in the case of the BB-12 strain growing in MRS, the same homologous group was for growth in pH 2.0, 3.0 and 4.0, and, simultaneously, the OD values for this strain cultivating in MRS pH 5.0 and 6.2 belonged to the same homologous group (Figure 3). An analogous situation was observed for the MRS culture of *Lb. casei* (Figure 3). However, in the case of cultivation with the use of an *Lb. acidophilus* strain, three homologous groups were observed—the first for growth in pH 2.0 and 3.0, second for pH 4.0 and the third for growth in pH 5.0 and 6.2 (Figure 3).

Selected lag phase and log phases, initial and maximal OD values, and coefficients of the specific growth rate of the analyzed probiotic preparations are summarized in Table 2.


**Table 2.** Selected lag and log phase lengths, initial and final OD600 in log phase, and coefficient of specific growth rate for the bacteria present in commercial probiotic preparations tested in MRS medium.

In the case of the *Lb. rhamnosus* GG ATCC 53103 strain, the highest value of the specific growth rate coefficient (μ = 0.084) was determined in MRS broth with pH 6.2. The μ values computed for the culture of these bacteria in MRS broth with pH 4.0 and 5.0 reached 0.042 and 0.063, respectively. In the case of MRS broth with pH 4.0, bacterial cells needed 19 h to adapt to medium conditions, whereas in MRS broth with higher pH values (5.0 or 6.2), cells of this probiotic began proliferation immediately after culture initiation. The growth curve plotted for *Lb.* GG in the medium with pH 4.0 had the longest phase of logarithmic growth of 29 h, whereas at pH 5.0 and pH 6.2, the length of this phase reached 19 and 16 h, respectively. In MRS broth with pH 4.0 and pH 5.0, the final OD value increased three times, whereas in the medium with pH 6.2 the final OD value of culture increased nearly four times in comparison with initial OD (Table 2)

The value of the μ coefficient for the growth of BB-12 strain cells increased along with increasing active acidity of the culture medium (μ = 0.093 at pH 5.0 and μ = 0.114 at pH 6.2). The growth of the cells of this strain in the medium with pH 4.0 revealed a long, nearly 40 h phase of adaptation of the cells to conditions of the medium (Figure 3). In MRS broth with pH 5.0 and 6.2, the adaptation phase lasted ca. 3 h. The length of the logarithmic growth phase noted for BB-12 in pH 5.0 was 16.5 h and in pH 6.2 was 12 h (Table 2). The highest (4.7-fold) increase in OD value of BB-12 strain culture was observed in MRS broth with pH 5.0 (Table 2).

The *Lb. casei* strain showed no growth in MRS broth with pH 4.0 (Figure 3), whereas in MRS with pH 5.0 and 6.2 the length of lag phase was the same (24.5 h). The value of μ coefficient calculated for the culture incubated at pH 6.2 was higher by 0.036 compared to the culture incubated at pH 5.0 (Table 2).

The culture of the *Lb. acidophilus* strain was characterized by the highest value of the growth rate coefficient in MRS broth with pH 6.2 (μ = 0.053). After cell introduction into the MRS broth with pH 4.0, the lag phase lasted ca. 25 h, whereas in the other media (with pH 5.0 and 6.2) it was definitely shorter and reached ca. 6 h. In MRS broth with pH 5.0, the final OD value increased 2.6-fold compared to the initial value (for comparison, in MRS broth with pH 6.2, the log phase lasted 28 h and OD increased over 4-fold) (Table 2).

No growth of the *Lb. plantarum* strain was observed in MRS broth with pH 2.0 and 3.0 (Table 1, Figure 3). The value of the μ coefficient determined for the *Lb. plantarum* strain cultured in MRS broth with pH 4.0, 5.0 and 6.2 reached 0.019, 0.039 and 0.058, respectively (Table 2). Cells of this strain started division immediately after culture onset, regardless of medium pH. The log phase lasted 29 h for the culture incubated at pH 4.0, as well as 17 and 12 h for cultures incubated at pH 5.0 and 6.2, respectively.

#### *3.2. Survivability and Growth of Bacteria Present in Commercial Probiotic Preparations in a Food Matrix Simulating Gastric Passage*

Food retention in the stomach usually lasts ca. 1–3 h [32] and liquid foods are retained. Once food has been ingested and its digestion has begun, pH value successively decreases [33,34].

Curves depicting changes in OD values during the incubation of the cultures of tested preparations in the medium simulating conditions likely to occur in the stomach of a small child after consumption of a chicken–vegetable soup (CVS), and after taking a probiotic preparation in a suspension of spring water, are shown on Figure 4. Changes in the cell number of the tested strains during incubation in the gastric medium are presented in Table 3.

**Figure 4.** Changes in the optical density during culture of commercial probiotic strains in the food matrix under simulated condition of the gastric passage (**a**) *Lb. rhamnosus* GG ATCC 53103; (**b**) *Bifidobacterium* BB-12; (**c**) *Lb. casei*; (**d**) *Lb. acidophilus*; (**e**) *Lb. plantarum*, (*p* < 0.05). The number of repeats for each treatment *n* = 3. Letters marked with the same color define a homogeneous group within one pH value among the tested variants of the experiment (a given color correlates with the pH value and a given letter correlates with a given homogeneous group within pH range).


**Table 3.** Growth of commercial probiotic strains (log CFU mL−<sup>1</sup> ± SD) in the food matrix under simulated condition of the gastric passage.

"-"—no growth/less than 4 log.

Gastric fluids differed in pH values, which were higher at the beginning and lower at the end of digestion. Optical density of the culture of the *Lb.* GG strain decreased insignificantly at pH 2.0 and 3.0. Already active acidity of 4.0 and 5.0 enabled the growth of these bacteria; however, in both variants of culture the OD value increased by 0.02 on average (Figure 4). A reduction in the number of *Lb.* GG cells was observed during incubation regardless of the pH of the medium. In gastric medium with pH 2.0, after just 2 h of incubation, the *Lb.* GG number decreased to less than 4 log CFU mL−1. BB-12, *Lb. casei* and *Lb. acidophilus* strains showed no growth during incubation, regardless of active acidity values, which was indicated by the course of curves depicting OD value changes in time of incubation (Figure 4). Among the commercial probiotic strains selected for this study, only *Lb. plantarum* showed significant growth in the GES and CSV medium at pH 4.0 and 5.0. In both cases, a 1.2-fold of increased OD (from the beginning till 3 h) was noticed. When analyzing changes in the number of cells of probiotic strains based on the results of the plate method (Table 3), it can be concluded that the *Lb. plantarum* strain was characterized by the highest resistance to low pH. In the gastric medium with pH 4.0 and pH 5.0, a slight increase in the cell number of this strain was noted (by 0.18 and 0.14 log

order). In gastric medium with pH 2.0 at the last 3rd hour of the experiment, the number of *Lb. plantarum* was 4.97 log CFU mL−1. Similar resistance was demonstrated only for strain BB-12.

#### *3.3. Survivability of Commercial Probiotic Strains in a Food Matrix Simulating Gastrointestinal Passage*

The passage of intestinal digesta through the section of the small intestine usually spans for 1–6 h [32]. No changes were observed in the optical density in any of the media simulating conditions occurring during digestion in the small intestine with BB-12, *Lb. rhamnosus*, *Lb. casei* and *Lb. acidophilus* strains (data not shown). The *Lb. plantarum* strain was the only one capable of proliferation under small intestine conditions, regardless of bile salts concentration (1%, 2% or 3%) (Figure 5). The log phases for *Lb. plantarum* took from 1.5 to 2 h for all tested concentrations of bile salts.

**Figure 5.** Changes in the optical density during culture of *Lb. plantarum* from commercial probiotic strains in the food matrix under simulated condition of the gastric and gastrointestinal passage; in this case it was only one homologous group signed *a* (*p* < 0.05). Each homogeneous group's corresponding pH was marked the same color as pH line axce.

The analysis of the initial and final OD values of the culture of the probiotic strain *Lb. plantarum* increased about 2-fold for concentration 1% and about 3-fold for the rest of the concentrations. The adaptation phase of the *Lb. plantarum* strain reached only 1.5 h regardless of bile concentrations.

Changes in the cell number of the tested strains during incubation in the food matrix under simulated condition of the gastrointestinal passage are presented in Table 4.

An increase in the number of cells was observed during incubation in the gastrointestinal medium irrespective of the amount of bile salt addition only in the case of *Lb. plantarum* strain. The number of *Lb. plantarum* in the gastrointestinal medium with 1% of bile after 6 h of the experiment increased by about 0.83 log order, while at 3% of bile by 0.29 order of magnitude in 1 mL. Some resistance to bile salts was found in the BB-12 strain. The final cell numbers of this strain after 6 h of incubation in gastrointestinal medium containing 1% and 2% of bile salts reached 6 log CFU mL−1. Only in the medium with the highest tested content of bile salts (3%) was a slight reduction in the number of cells from the initial 6.17 to 5.86 log CFU mL−<sup>1</sup> determined. The most sensitive to the presence of bile salts at the level of 3% were *Lb. casei* and *Lb.* GG strains (<4 log CFU mL−<sup>1</sup> from the 2nd and 4th hour of incubation, respectively).


**Table 4.** Survival of commercial probiotic strains (log CFU mL−<sup>1</sup> ± SD) in the food matrix under simulated condition of the gastrointestinal passage.

"-"—no growth/less than 4 log.
