**1. Introduction**

The definition of "probiotic" provided by the International Scientific Association of Probiotics and Prebiotics states that probiotics are "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" [1]. The best known and the most thoroughly investigated probiotic strains are representatives of *Lactobacillus* (*Lb.*) and *Bifidobacterium* genera [2–4]. The role of probiotics is to, i.a., alleviate symptoms of lactose intolerance, ameliorate outcomes of food allergies, and reduce cholesterol concentration in blood [5–7]. The administration of probiotic preparations is recommended, e.g., during and after antibiotic therapy to aid the reconstruction of natural enteric microflora [8–10].

It is estimated that from 11 to 30% of children treated with antibiotics (mainly β-lactam ones and vancomycin) suffer from intestinal discomfort and diarrheas [11–13]. Diarrhea is especially dangerous for small children/toddlers as it may cause malfunction of the water–electrolyte balance of their bodies within a short period of time [14]. Sometimes, however, children suffer from post-antibiotic diarrhea despite their diet supplementation with probiotic strains [15]. This is, probably, caused by reduced survivability of individual probiotic strains under varying conditions of the alimentary tract. Oral administration of at least 10<sup>7</sup> cells of a probiotic strain per milliliter or gram of food should ensure a positive

**Citation:** Stasiak-Róza ´ ˙ nska, L.; Berthold-Pluta, A.; Pluta, A.S.; Dasiewicz, K.; Garbowska, M. Effect of Simulated Gastrointestinal Tract Conditions on Survivability of Probiotic Bacteria Present in Commercial Preparations. *Int. J. Environ. Res. Public Health* **2021**, *18*, 1108. https://doi.org/10.3390/ijerph 18031108

Academic Editor: Paul B. Tchounwou and Diana María Cardona Mena

Received: 20 November 2020 Accepted: 22 January 2021 Published: 27 January 2021

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effect to the host even when some of them do not survive the unfavorable conditions of the gastrointestinal passage [16]. Most of the commercial probiotics are registered as "dietary supplements" and therefore do not have to comply with quality requirements obligatory for drugs [17]. After oral administration, the probiotic strains are exposed to low pH of the stomach and bile salts in the enteral section of the alimentary tract of the host. Many of them often fail to survive conditions of the passage [18,19]. Metabolic and biochemical activity of probiotics during gastrointestinal passage can be sustained through earlier encapsulation of their cells [20,21]. However, probiotic preparation producers recommend removal of the protective capsule before giving it to toddlers and making a suspension of probiotic powder with water to avoid choking or strangulation during swallow. Such information can be found on preparation leaflets. Another means of protecting probiotics against adverse effects of the gastrointestinal conditions is their administration together with a prebiotic [22]. The best known and the most commonly used prebiotics include inulin and oligofructose [23].

The aim of this study was to examine the survivability and possibility to growth of strains obtained from commercial probiotic preparations (without protective capsule) under conditions simulating gastrointestinal tract.

#### **2. Materials and Methods**

#### *2.1. Commercial Probiotic Preparations*

The study was conducted with 5 commercial preparations, available on the Polish market, which contained lyophilized cells of one-strain of probiotic bacteria, namely:

Preparation 1—*Lactobacillus rhamnosus* GG ATCC 53103 (3 × 10<sup>9</sup> CFU/one dose, 4.5 × 10<sup>9</sup> CFU/g);

Preparation 2—*Bifidobacterium* BB-12 (4 × 10<sup>9</sup> CFU/one dose, 1.7 × 10<sup>10</sup> CFU/g); Preparation 3—*Lactobacillus casei* (4 × 10<sup>8</sup> CFU/one dose, 2.1 × 10<sup>9</sup> CFU/g); Preparation 4—*Lactobacillus acidophilus* (2 × 10<sup>9</sup> CFU/one dose, 3.1 × 10<sup>10</sup> CFU/g); Preparation 5—*Lactobacillus plantarum* (4 × 10<sup>8</sup> CFU/one dose, 1.8 × 10<sup>9</sup> CFU/g).

In the case of preparations 3, 4 and 5, producers did not provide any information about the number of strain or its origin. This information is a trade secret. Preparation 2 contained a prebiotic in the form of fructooligosaccharide (FOS), whereas preparations 3 and 4 contained inulin, and preparations 1 and 5 did not contain prebiotic. The preparations originated from various Polish producers and were registered as dietary supplements. Their production dates were similar. Preparation 1 was in the form of a lyophilizate in a paper sachet, whereas the other preparations were encapsulated in gelatin capsules. Preparation 1 was poured out of the sachet prior to testing. All gelatin capsules (which normally protect probiotics from outside the GIT environment) from preparations 2, 3, 4, and 5 were removed before using probiotic preparations in experiments; this is typically the intake procedure for probiotic preparations for toddlers (12–18 months) to avoid choking.

#### *2.2. Growth Media and Solutions*

MRS broth (Sigma-Aldrich, St. Louis, MO, USA), agar 15 gL−<sup>1</sup> (for seeding step), pH 6.2. The broth was sterilized at a temperature of 121 ◦C for 15 min.

Gastric electrolyte solution (GES) [24,25], composed of [gL−1]: NaCl 4.8, NaHCO3 1.56, KCl 2.2, CaCl2 0.22, pepsin 1. The solution was sterilized at a temperature of 121 ◦C for 20 min; after sterilization GES was supplemented with a filter sterile pepsin solution in water (P6887; Sigma-Aldrich, 0.22 μL, Sartorius Poland Sp. z o o.) to final concentration 1 gL−1.

Double-concentrated J broth (2 × JB) [24], was composed of [gL−1]: peptone 10, yeas<sup>t</sup> extract 30, K2HPO4 6, glucose 4. The broth was sterilized at a temperature of 121 ◦C for 20 min. Glucose solution was filtered (filter pore diameter—0.22 μm, Sartorius Poland Sp. z o o.) and added to 2 × JB after sterilization. Bile salts were subjected to mild sterilization (117 ◦C, 10 min) and added to sterilized 2 × JB. Concentration of bile salts (Sigma-Aldrich, B8631) was adjusted to 1%, 2%, and 3%.

Chicken–vegetable soup (CVS) composed of [gL−1]: chicken breast fillet 200, onion 35, carrot 100, celery root 30, and parsley root 65. The CVS was prepared in a Termomix Vorwerc cooker, at a temperature of 100 ◦C, for 60 min, with a mixing rate knob in position 1. The CVS was filtered (filter pore diameter—0.45 μm).

Spring water was recommended for small children, sterilized at temperature 121 ◦C for 20 min.

*Lactobacillus* and *Bifidobacterium* BB-12 were enumerated by the pour plate technique on MRS and MRS modified by adding 0.2% (*w*/*v*) lithium chloride and 0.3% (*w*/*v*) sodium propionate (MRS-LP), respectively [26].

#### *2.3. Study Design and Culture Conditions*

The experiment was divided into three stages: control cultures, stomach stage, and gastrointestinal stage.

To observe how tested bacteria react in optimal conditions (control), *Lactobacillus* was incubated in MRS broth, while *Bifidobacterium* on MRS-LP, deemed optimal for their growth, was adjusted to pH 6.2 either with HCl 5M or with NaOH 1M at a temperature of 37 ◦C, for 48 h. In order to limit the access of oxygen to the *Bifidobacterium*, cultures were carried out without shaking and, additionally, the access of air was cut off with a layer of water agar. Incubations in MRS adjusted to pH 2.0, 3.0, 4.0 or 5.0 in the same growth conditions were also performed to have a positive control of probiotics growth. In the tested preparations, the initial number of viable cells of the probiotic strain was initially determined by inoculating petri dishes with MRS medium (preparations 1, 3, 4 and 5, respectively) and with MRS-LP Agar medium (preparation 2). The obtained results were expressed as CFU/g of each preparation (2.1.)

To simulate conditions occurring in the stomach, 100 mL of GES was mixed with 100 mL of CVS and 100 mL of sterile spring water containing 1 dose of a given probiotic preparation (1, 2, 3, 4 or 5). Each of the five mixtures was cultured at final pH 2.0, 3.0, 4.0, and 5.0; temperature 37 ◦C, for 3 h (Figure 1). The acidity of the solutions was adjusted using the HCl 5M.

**Figure 1.** Scheme of the variants tested under gastric stage of experiment (GES—gastric electrolyte solution, CVS—chicken– vegetable soup).

The gastrointestinal stage consisted of mixing 100 mL of GES with 100 mL of CVS and 100 mL of spring water suspension containing a given probiotic, at final pH 3.0. The mixture was shaken in a reciprocating shaker 50 rpm, at 37 ◦C for 30 min (stomach stage) [27]. Afterwards, the suspension was mixed with 2 × JB (1:1, *v*/*v*). Survivability of all preparations was examined in the presence of bile salts with concentrations of 1, 2 or 3 (%), at 37 ◦C for 6 h; the final pH of mixture medium was 5.5 (regulated using NaOH 1M) (Figure 2).

**Figure 2.** Scheme of the variants tested under gastrointestinal stage of experiment, (GES—gastric electrolyte solution, CVS—chicken–vegetable soup, 2 × JB—2 × concentrated J broth).

Optical density (OD) of individual cultures of probiotic preparations was measured every 30 min in a Bioscreen C MBR apparatus with the length of wave λ = 600 nm. Each culture variant was conducted in three independent replications. Simultaneously the count of bacteria on MRS Agar pH 6.3 (*Lactobacillus*) and MRS-LP Agar pH 6.3 (BB-12) plates was determined. Incubation was provided in anaerobic jars containing AnaeroGen® (Argenta, Poland) [26]. The samples for spread on petri dishes was taken from time points: 0; 12; 24; 36 and 48 h for MRS conditions, 0; 1; 2; 3 h for gastric stage, 0; 2; 4; 6 h for gastrointestinal stage. In each variant of the experiment, three independent series of replicates were performed for each analyzed sample inoculated into petri dishes, differentiated by the type of preparation (1; 2; 3; 4; 5), variant type (MRS control, stomach, gastrointestinal (GI), pH (2.0; 3.0; 4.0; 5.0 or 6.2) or bile salt concentration (1%; 2%; 3%) and measuring point (h) 0; 12; 24; 36; 48 for MRS stage, 0; 1; 2; 3 for stomach, 0; 2; 4; 6 for GI). In the case of the optical density (OD) measurement in Bioscreen Apparatus, measurements were made every 30 min for each individual sample, and each sample variant was performed in three independent measurement series.

#### *2.4. Calculation of Coefficient of Specific Growth Rate*

The coefficient of the specific growth rate (μ) in time (t) was calculated from the formula: μ(t) = (ln ODf − ln ODi) / (tf − ti), where: ODf—final OD in the log phase, ODi—initial OD in the log phase, tf—time of log phase termination, ti—time of log phase onset [28].
