**4. Discussion**

High variability of strains and unlimited possibilities of creating experimental conditions in scientific research significantly impair the comparison and discussion of results achieved in various studies [35]. There are several criteria, which need to be met to classify a strain as a "probiotic". The key ones among functional criteria include tolerance to gastric juice and bile, and capability of adhesion to colonic mucosa [36,37]. Probiotic bacterial strains have to survive unfavorable conditions encountered during their gastrointestinal passage to be able to colonize the colon and to exert a positive effect on consumer/host health [32]. However, as indicated in scientific research, not all strains classified as "probiotic" meet these criteria [38,39]. Both manufacturers of probiotic preparations and pediatricians exclude the administration of a probiotic preparation in the form of a gelatin capsule to young children. Giving toddlers a capsule is not advisable because they can easily choke by swallowing it. It is strictly recommended to remove the capsule and suspend probiotic with water and administrate it in this form. For this reason described, experiments showed results of survivability of strains lacking early protection against bile salts and low pH [40–43].

*Lb. rhamnosus* GG is a well-characterized probiotic strain [44]. It is a commensal, which colonizes the gastrointestinal tract in humans [45]. In 1985, *Lb.* GG was patented as a probiotic partly due to its resistance to low pH and to bile salts [46]. The exact mechanism of these bacteria effecting the organism of the host remains unknown; however, bacteria of the *Lb. rhamnosus* species are implied to exhibit antimicrobial, antiviral, and diarrheapreventing properties [47,48]. Pitino et al. [34] demonstrated that *Lb. rhamnosus* strains isolated from cheese showed high survivability in MRS broth with pH 5.0 during simulated

dynamic digestion in the stomach. In our study, the *Lb.* GG strain also showed growth in MRS broth with pH 5.0 (Figure 3, Table 1). In a clinical survey conducted by Hibberd et al. [48], in 73% of volunteers to whom *Lb. rhamnosus* was administered orally in a dose of 10<sup>10</sup> CFU for 28 days, reduced numbers of these bacteria were detected in feces, i.e., from 1.4 × 10<sup>3</sup> to 1.3 × 10<sup>8</sup> CFU [48]. Other *Lb. rhamnosus* strains isolated from wine showed growth after 24 h incubation at pH 3.5, likewise *Lb. rhamnosus* isolated from meat [49]. Goldin et al. [44] proved that *Lb. rhamnosus* GG survived incubation in the medium with pH 3.0. Results of other studies indicate high resistance of this strain to a bile salt concentration of 1.5% [49]. In our study, the *Lb. rhamnosus* GG strain showed no growth at bile salt concentrations of 1, 2 or 3% (Table 4).

Bacteria of the genus *Bifidobacterium* possess the GRAS (Generally Recognised As Safe) status and constitute part of the natural microflora of the gastrointestinal tract of humans (likewise other probiotic bacteria, e.g., those from the genus *Lactobacillus*); therefore, they are often used as components of commercial dietary supplements [3]. In our experiment, the BB-12 strain showed no growth either in MRS broth with pH 2.0 or with pH 3.0 (Figure 3b, Table 1). In MRS broth with pH 4.0, we observed the growth of BB-12 bacteria already after 36 h (Table 1). Analyses conducted in our study showed no growth of BB-12 strain under simulated gastrointestinal conditions. De Castro-Cislaghi et al. [30] observed a reduction in the cell count of the BB-12 strain in the presence of bile salts in the concentration of 1%, from the initial value of ca. 9.5 log CFU/g to ca. 9 log CFU/g after 3 h incubation. In addition, they demonstrated that resistance to various pH values and concentrations of bile salts is a variable, strain-specific feature.

Probiotic lactic bacteria of the species *Lb. casei* have been widely applied in the production of fermented foods [50]. The administration of lyophilized preparations of these bacteria is believed to reduce the blood level of cholesterol and to impair proliferation of cancer cells [51–53]. Apart from the *Lb. casei* strain, commercial preparation 3 tested in our study contained inulin, which was supposed to support its viability. Growth tests conducted in the model MRS culture medium demonstrated that the phase of cell adaptation to conditions of the culture medium was one of the longest in the case of this strain (ca. 25 h, Figure 3, Table 2). This was the only strain which showed no growth in MRS broth with pH 4.0 (Table 1). Cells of *Lb. casei* began to divide already at pH 5.0, although their adaptation phase was again one of the longest compared to the other strains (Figure 3, Table 2).

In our study, the *Lb. casei* strain showed no growth in conditions simulating the gastric and/or gastrointestinal passage (Tables 3 and 4). Dimitrellou et al. [32] demonstrated successive viability loss for the *Lb. casei* strain incubated in gastric media. After 3 h of incubation, they observed a decrease in the cell count of this strain by ca. 4.0 log CFU g<sup>−</sup><sup>1</sup> at the initial pH 2.0 and by 1.5 log CFU g<sup>−</sup><sup>1</sup> at pH 3.0. In addition, they showed the presence of bile salts in the concentration of 1 gL−<sup>1</sup> to evoke *Lb. casei* cell count reduction by nearly 6 log CFU g<sup>−</sup><sup>1</sup> after 6 h of incubation. In turn, Mishra and Prasad [29] proved that all seven analyzed strains of *Lb. casei* survived incubation at pH 3.0, and two of them were viable once pH was decreased to the value of 2.0. All seven analyzed strains were viable after 12 h incubation in solutions with bile salt concentrations of 1 and 2%.

Bacteria of the *Lb. acidophilus* species naturally occur in the gastric tract of humans and animals [54]. In our study, the *Lb. acidophilus* NCFM strain (preparation 4) showed no growth under experimental gastro-intestinal conditions (Table 4).

Representatives of the *Lb. plantarum* species are also implied to exhibit probiotic traits. The *Lb. plantarum* NRRL-B4496 strain is one of the main probiotics used in fermented food products [55,56]. Multiple scientific works indicate this strain to be capable of inhibiting the growth of certain pathogens that induce diseases of the alimentary tract, e.g., *Helicobacter pylori* or *Listeria monocytogenes* [56–60].

Commercial probiotic preparations are often supplemented with prebiotics, the task of which is to increase the chances of probiotic strains for the survival of adverse conditions during gastrointestinal passage and to sustain their metabolic activity [3,61,62]. The most

frequently used prebiotics include inulin [63,64], β-glucan, and fructooligosaccharides (FOS) [62,65,66].

Obtained results clearly show that, among all analyzed commercial probiotic strains, only the *Lb. plantarum* was the most resistant to the applied conditions of the culture medium. It showed a noticeable growth under both in vitro gastric conditions at pH 4.0 and 5.0, as well as in vitro intestinal conditions at all tested concentrations of bile salts. Interestingly, its preparations did not contain a prebiotic.

No OD changes of the tested commercial probiotics under the assumed experimental conditions does not have to indicate their incapability for surviving the in vivo gastrointestinal passage. However, obtained results from the plate count method show that the *Lb. plantarum* strain had the best capability for growth, which suggests it could proliferate in intestines also under in vivo conditions. Considering the fact that the tested probiotics were deprived of a gelatin capsule at the beginning of the experiment, it can be concluded that *Lb. plantarum* exhibited distinctive properties that allowed this strain to survive the simulated conditions of the passage. This is valuable knowledge, considering that exogenous probiotics share a limited capability for adhesion to cells of the intestinal epithelium and that their major part is excreted with feces.

The scheme of experiments, shown in this study, largely covers the simplified conditions during the gastrointestinal transit in the human body and does not include many factors. For some of the probiotic strains present in the tested preparations, there are literature data from several years ago showing their beneficial effect on the course and shortening of the duration of diarrhea of various origins [67]. The medical practice of using probiotic food supplements to children consists of administering them, for example, after or still during antibiotic therapy for the treatment of diarrheal disorders [67]. Recent data concerning the effect of probiotics on inflammation of gastroenteritis, which is often manifested by diarrhea, are different from those previously described. Freedman et al. [68] showed that administration of a probiotic product containing *Lactobacillus rhamnosus* R0011 and *L. helveticus* R0052, at a dose of 4.0 × 10<sup>9</sup> CFU/unit twice daily, did not reduce the incidence of diarrhea in children with gastroenteritis. Similarly, among preschool children with acute gastroenteritis, those who had taken *L. rhamnosus* GG did not show better outcomes than those children who had received placebo [69].
