# Screening of probiotic strains to improve visceral hypersensitivity in irritable bowel syndrome by using in vitro and in vivo approaches

E. Torres-Maravilla, F.A. Carvalho, S. Holowacz, J. Delannoy, L. Lenoir, E.

Jacouton, F. Barbut, P. Langella, L.G. Bermúdez-Humarán, A.-J.

Waligora-Dupriet

► To cite this version:

E. Torres-Maravilla, F.A. Carvalho, S. Holowacz, J. Delannoy, L. Lenoir, et al.. Screening of probiotic strains to improve visceral hypersensitivity in irritable bowel syndrome by using in vitro and in vivo approaches. Beneficial Microbes, 2024, 15 (3), pp.293-310. 10.1163/18762891-bja00006. hal-05071287

HAL Id: hal-05071287

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RESEARCH ARTICLE

# Screening of probiotic strains to improve visceral hypersensitivity in irritable bowel syndrome by using *in vitro* and *in vivo* approaches

E. Torres-Maravilla¹,² ID, F.A. Carvalho³ ID, S. Holowacz⁴ ID, J. Delannoy² ID, L. Lenoir⁴, E. Jacouton⁴ ID, F. Barbut²,⁵ ID, P. Langella¹ ID, L.G. Bermúdez-Humarán¹* ID and A.-J. Waligora-Dupriet²* ID

¹Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France; ²Université Paris Cité, INSERM, UMRS 1139, 3PHM, 75006 Paris, France; ³INSERM U1107 NeuroDol, Université Clermont Auvergne, 3iHP, Clermont-Ferrand, France; ⁴PiLeJe Laboratoire, 31-35 Rue de la Fédération, 75015 Paris, France; ⁵Service de Microbiologie de l'environnement, UHLIN (Unité d'Hygiène et de Lutte contre les Infections Nosocomiales), National Reference Laboratory for Clostridium difficile, Groupe Hospitalier de l'Est Parisien (HUEP) Site Saint-Antoine, 75012 Paris, France; *luis.bermudez@inrae.fr; anne-judith.waligora@u-paris.fr

Received 20 June 2023 | Accepted 27 March 2024 | Published online 30 April 2024 |
Published in issue 3 June 2024

## Abstract

Oral administration of probiotics has been proposed as a promising biotherapy to prevent and treat different diseases related to gastrointestinal disorders, such as irritable bowel syndrome (IBS). Due to the increasing research area on the characterisation of new probiotic bacterial strains, it is necessary to perform suitable *in vitro* experiments, using pertinent cellular models, in order to establish appropriate readout profiles based on IBS symptoms and subtypes. In this work, a collection of 30 candidate strains, belonging mainly to the *Lactobacillus* and *Bifidobacterium* genera, were screened using three different sets of *in vitro* experiments with different readouts to identify promising probiotic strains with: (1) the ability to inhibit the synthesis of IL-8 production by TNF-α stimulated HT-29 cells, (2) immunomodulatory properties quantified as increased IL-10 levels in peripheral blood mononuclear cell (PBMCs), and (3) the ability to maintain epithelial barrier integrity by increasing the trans-epithelial/endothelial electrical resistance (TEER) values in Caco-2 cells. Based on these criteria, three strains were selected: *Lactobacillus gasseri* PI41, *Lacticaseibacillus rhamnosus* PI48 and *Bifidobacterium animalis* subsp. *lactis* PI50, and tested in a murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS), which mimics some of the symptoms of IBS. Among the three strains, *L. gasseri* PI41 improved overall host well-being by preventing body weight loss in DNBS-treated mice and restored gut homeostasis by normalising the intestinal permeability and reducing pro-inflammatory markers. Therefore, the potential of this strain was confirmed in a second murine model known to reproduce IBS symptoms: the neonatal maternal separation (NMS) model. The PI41 strain was effective in preventing intestinal permeability and reducing colonic hypersensitivity. In conclusion, the set of *in vitro* experiments combined with *in vivo* assessments allowed us to identify a promising probiotic candidate strain, *L. gasseri* PI41, in the context of IBS.

## Keywords

*Lactobacillus* – probiotics – IBS – gut permeability – serotonin
# 1 Introduction

Irritable bowel syndrome (IBS) is a gastrointestinal disorder with associated chronic abdominal pain and changes in stool appearance or frequency (Saha, 2014). IBS patients have been classified into specific subtypes based on bowel habits: IBS associated to diarrhoea (IBS-D), IBS associated to constipation (IBS-C), IBS with alternation between diarrhoea and constipation (IBS-M), and IBS unsubtyped (IBS-U) (Lacy and Patel, 2017). It should be noted that patients with acute bacterial gastroenteritis develop IBS symptoms, referred as post-infection IBS (PI-IBS); as the symptoms are consistent with IBS-D, the pathology tends to be classified as IBS-D rather than another subtype of IBS (Enck et al., 2016; Neal et al., 2002). The physiopathology of IBS is only partially understood, and two mechanisms have been described, the central mechanisms and the peripheral mechanisms. Central mechanisms involve genetic mutation of *SCN5A* gene (sodium voltage-gated channel alpha subunit 5), altered serotonin metabolism, alterations in brain-gut function (stress and visceral hypersensitivity) and dietary influence (Benjak Horvat et al., 2021) while peripheral mechanisms involve changes in gastrointestinal motility, intestinal permeability (Ivashkin et al., 2021), local immune response disorders, immune activation, bile salt metabolism disorders, post-infectious changes, chronic infections and intestinal microbiota disturbances (Benjak Horvat et al., 2021). Human gastrointestinal (GI) tract harbours trillions of microbes, including beneficial commensal bacteria that play an important role in healthy individuals. In IBS cases, the reduction of microbiome diversity, gut barrier deficiency, gut-brain signalling disorders, and immune disorders are significantly related to the abnormal function of the GI tract (El-Salhy et al., 2020). In fact, gut microbiota of IBS-D and IBS-C patients frequently shows dysbiosis (i.e. microbiota unbalance), with a significant increase in *Clostridiales*, and *Verrucomicrobia* and a reduction of *Actinobacteria* (Lo Presti et al., 2019), *Prevotella* and *Faecalibacterium* species (Ivashkin et al., 2021; Rajilić-Stojanović et al., 2011) and probiotic species (Chong et al., 2019). Consequently, biotherapy trough faecal microbiota transplantation, administration of probiotics and synbiotics has been proposed to modulate the gut microbiota to prevent and/or to treat some IBS subtypes (Fong et al., 2020; Salinas et al., 2021; Swanson et al., 2020; Torres-Maravilla et al., 2021). The efficacy and safety of probiotic strains for the treatment of IBS are supported by an increasing number of clinical studies (Sisson et al., 2014). Probiotics may improve bloating symptoms by decreasing the production of intestinal gas, promoting gut motility, and relieving abdominal pain scores (Benjak Horvat et al., 2021; Konstantis et al., 2023). *Bifidobacterium* and lactic acid bacteria species appear to be safe for patients, encouraging their evaluation for management of IBS. Some probiotic strains stabilise the gut microbiota and prevent gut permeability, normalise the abnormal interleukin (IL)-10/IL-12 ratio produced by peripheral blood mononuclear cells (PBMCs) in *in vivo* models of IBS (Guyonnet et al., 2007; Kajander et al., 2008; Laval et al., 2015; Martín et al., 2016; O'Mahony et al., 2005). In addition, probiotics can have central effects via the modulation of local expression of cannabinoid receptor type 2 and tryptophan hydrolase 1 isoform, and directly alter the visceral perception of nociceptive stimulants (Perez-Burgos et al., 2015), thus reducing visceral pain.

Recent systematic reviews and network meta-analyses revealed that only few species (*Bacillus coagulans*, *Lactiplantibacillus plantarum*, *Lactobacillus acidophilus* and *Bifidobacterium longum*) relief symptoms of IBS (Li et al., 2020; Zhang et al., 2022). Francavilla et al. (2010) reported that *Lacticaseibacillus rhamnosus* LGG significantly reduced the frequency and severity of abdominal pain in children with IBS; Ait Abdellah et al. (2022) reported that after a 4-week supplementation with *Lactobacillus gasseri* LA806, a decrease of ≥30% in both abdominal pain score and global IBS symptom score was attained in 61.5% of patients. Although the efficacy and safety of probiotics have been confirmed by numerous studies, the best probiotic species for the treatment of IBS have not yet been identified (Zhang et al., 2022). As it has been seen, there is a renewed growing interest in isolating and characterising new probiotic candidates targeting IBS. To achieve this task, adequate screening strategies on the earliest stages of characterisation (*in vitro* and *in vivo* experiments) of probiotic bacteria should be considered, to ensure the success of the beneficial effects of probiotic bacteria in further evaluations as clinical trials (Alard et al., 2018; Huang et al., 2021; Mallappa et al., 2019; Torres-Maravilla et al., 2022b). In this work, we screened 30 strains (mostly lactic acid bacteria and *Bifidobacterium* species) using three different *in vitro* cellular models for IBS that allowed the identification of candidate strains for subsequent *in vivo* experiments: measurement of anti-inflammatory activity by IL-8 blockage in HT-29 cells, immunomodulation of IL-10/IL-12 in PBMCs and *in vitro* intestinal permeability in Caco-2 cells. The candidate strains were then selected and tested in two murine models that overlap with some symptoms of IBS, i.e. the DNBS model
that results in an increase of intestinal permeability and immune activation, and the neonatal maternal separation (NMS) model that is characterised by an increase in intestinal permeability and colonic hypersensitivity.

## 2 Material and methods

### Strains and culture conditions

Thirty previously isolated strains belonging mainly to *Lactobacillus* and *Bifidobacterium* species from the PiLeJe Laboratoire collection were used in this study (Table 1). The strain of *L. rhamnosus* LGG (ATCC 53103) was used as a positive control in *in vitro* permeability assays. All bacteria were grown in Man-Rogosa-Sharpe (MRS) medium (DIFCO, France) at 37 °C. For *Bifidobacterium* strains, MRS was supplemented with 0.5 mg/ml cysteine (Sigma-Aldrich, St. Quentin Fallavier, France) at 37 °C in a Coy chamber under anaerobic conditions (5% H₂, 5% CO₂, and 90% N₂). *Saccharomyces*

*boulardii* strain PI31 was grown in Sabouraud medium (DIFCO) at 37 °C. Briefly, cells were collected 1 h after the beginning of the stationary phase, harvested by centrifugation (3,500×g), washed twice, re-suspended in phosphate-buffered saline (PBS; Gibco, Thermo Scientific, Graffenstaden, France) containing 15% glycerol, and stored at -80 °C until further assays. Colony forming units (cfu) were estimated by serial dilution on agar plates. Three independent growth experiments were performed for each strain. Preparation of bacterial inoculum for administration to mice was performed as described above and lyophilised.

### Experiments with HT-29 cells

The human colon carcinoma cell line HT-29 was grown in 24-well culture plates in Dulbecco's modified Eagle's medium (DMEM) (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated foetal bovine serum (FBS; Gibco, Thermo Scientific), 1% glutamine at 37 °C in a 10% CO₂-air atmosphere. The medium was changed

<table><caption>TABLE 1 List of strains used in this study.<fnref n="1" /></caption><thead><tr><th>Strain</th><th>Identification</th><th>Origin</th><th>Strain</th><th>Identification</th><th>Origin</th></tr></thead><tbody><tr><td>1</td><td>LA306<br><i>Bifidobacterium animalis</i><br>subsp. <i>lactis</i></td><td>Human</td><td>17</td><td>PI37<br><i>Lactobacillus gasseri</i></td><td>Human</td></tr><tr><td>2</td><td>LA702<br><i>Lacticaseibacillus</i><br><i>rhamnosus</i></td><td>Dairy<br>food</td><td>18</td><td>PI38<br><i>Lactobacillus gasseri</i></td><td>Human</td></tr><tr><td>3</td><td>LA803<br><i>Bifidobacterium bifidum</i></td><td>Human</td><td>19</td><td>PI39<br><i>Lactobacillus gasseri</i></td><td>Human</td></tr><tr><td>4</td><td>LA804<br><i>Bifidobacterium animalis</i><br>subsp. <i>lactis</i></td><td>Human</td><td>20</td><td>PI40<br><i>Lactobacillus gasseri</i></td><td>Human</td></tr><tr><td>5</td><td>LA805<br><i>Bifidobacterium breve</i></td><td>Human</td><td>21</td><td>PI41<br><i>Lactobacillus gasseri</i></td><td>Human</td></tr><tr><td>6</td><td>PI17<br><i>Lactobacillus gasseri</i></td><td>Human</td><td>22</td><td>PI42<br><i>Lactobacillus gasseri</i></td><td>Human</td></tr><tr><td>7</td><td>PI22<br><i>Bifidobacterium bifidum</i></td><td>Human</td><td>23</td><td>PI43<br><i>Lactobacillus gasseri</i></td><td>Human</td></tr><tr><td>8</td><td>PI28<br><i>Lacticaseibacillus</i><br><i>rhamnosus</i></td><td>Human</td><td>24</td><td>PI44<br><i>Lactiplantibacillus plantarum</i></td><td>Human</td></tr><tr><td>9</td><td>PI29<br><i>Bifidobacterium bifidum</i></td><td>Human</td><td>25</td><td>PI45<br><i>Lacticaseibacillus casei</i></td><td>Human</td></tr><tr><td>10</td><td>PI30<br><i>Lactiplantibacillus</i><br><i>plantarum</i></td><td>Human</td><td>26</td><td>PI46<br><i>Lacticaseibacillus casei/paracasei</i></td><td>Human</td></tr><tr><td>11</td><td>PI31<br><i>Saccharomyces boulardii</i></td><td>Vegetable</td><td>27</td><td>PI47<br><i>Lacticaseibacillus casei/paracasei</i></td><td>Human</td></tr><tr><td>12</td><td>PI32<br><i>Bifidobacterium infantis</i></td><td>Human</td><td>28</td><td>PI48<br><i>Lacticaseibacillus rhamnosus</i></td><td>Human</td></tr><tr><td>13</td><td>PI33<br><i>Bifidobacterium animalis</i><br>subsp. <i>lactis</i></td><td>Human</td><td>29</td><td>PI49<br><i>Limosilactobacillus reuteri</i></td><td>Human</td></tr><tr><td>14</td><td>PI34<br><i>Lactobacillus crispatus</i></td><td>Human</td><td>30</td><td>PI50<br><i>Bifidobacterium animalis</i> subsp.<br><i>lactis</i></td><td>Human</td></tr><tr><td>15</td><td>PI35<br><i>Limosilactobacillus</i><br><i>reuteri</i></td><td>Human</td><td>31</td><td>LGG<br><i>Lacticaseibacillus rhamnosus</i><br>LGG*</td><td></td></tr><tr><td>16</td><td>PI36<br><i>Limosilactobacillus</i><br><i>fermentum</i></td><td>Baking<br>leaven</td><td></td><td></td><td></td></tr></tbody></table>

<footnote n="1">¹ All strains belong to PiLeJe Collection, * ATCC (American Type Culture Collection) 53103.</footnote>
every two days. Experiments were started on day 7 after seeding, when cell confluence was reached. HT-29 cell were co-incubated with bacteria at a multiplicity of infection (MOI) of 1:40. Cells were simultaneously stimulated with recombinant human TNF-α (5 ng/ml; Peprotech, Cranbury, NJ, USA) for 6 h at 37 °C in 10%/CO₂ (Kechaou et al., 2013). We used 1 mM of butyrate (Sigma-Aldrich) as positive control due to its anti-inflammatory activity (Lenoir et al., 2020). After co-incubation, cell supernatants were collected and frozen at -80 °C until subsequent analysis of IL-8 concentrations by ELISA (Biolegend, San Diego, CA, USA) according to the manufacturer's instructions.

### Experiments with PBMCs

Human PBMCs were extracted from buffy coat cells of five healthy donors (all males; aged <65 years, with a body mass index of <30; negative for HIV and hepatitis A and B viruses). Buffy coat cells were obtained from the Etablissement Français du Sang (agreement C CPSL UNT_18/EFS/030) as previously described (Neau et al., 2016). PBMCs were adjusted and seeded in 48-well plates at a density of 6.75 × 10⁵ cells/well in Iscove's modified Dulbecco's medium (Sigma-Aldrich) supplemented with 10% FBS, 1% L-glutamine, 0.1% penicillin-streptomycin, and 0.1% gentamicin. Bacteria from three independent cultures were added at a MOI of 1:10 in 50 µl of PBS. Plates were incubated for 48 h at 37 °C in 5% CO₂. Samples were finally stored at -80 °C until further analysis of IL-10 and IL-12p70 concentrations by ELISA-Multiplex (Bio-Rad, Hercules, CA, USA).

### In vitro permeability assay in Caco-2 cells

Caco-2 cells were grown on Transwell semi-permeable filter support (12 mm-diameter wells, polystyrene membranes with 0.4 µm pores, Costar, Corning, MI, USA) and seeded at 2.9 × 10⁴ cells per well with Trans-Epithelial Electrical Resistance (TEER) readings of >1,500 ohms/cm² (8-10 days, HTS robot REMS autosampler; World Precision Instruments, Sarasota, FL, USA). TEER was measured before adding 1 × 10⁶ cfu of each bacterial strain onto the apical surface and co-incubated for 3 h at 37 °C/10% CO₂, prior treating the medium from basolateral part of the well with TNF-α (100 ng/ml) for an additional 21 h. Experiments were performed at least in duplicate. The *L. rhamnosus* LGG strain was used as a positive control (Laval et al., 2015). The resulting data are presented as a TEER ratio:

$$TEER \ ratio = \frac{TEER \ treatment \ T24/TEER \ treatment \ T0}{TEER \ control \ T24/TEER \ control \ T0}$$

### DNBS model and administration of strains

Specific pathogen-free male C57BL/6 mice (6-8 weeks old, Janvier, Le Genest Saint Isle, France) were maintained under normal breeding conditions at the animal care facilities of Infectiologie Expérimentale des Rongeurs et des Poissons (IERP) of the INRAE-Jouy-en-Josas. They were housed in cages of five. Our experiments were performed in accordance with the European Union legislation on animal welfare and were approved by COMETHEA, our local committee on animal experimentation (n°16744-201807061805486 v2).

Intestinal immune activation and permeability was induced by DNBS according to the protocol described in Martín et al. (2014) with minor modifications (Supplementary Figure S1). Briefly, mice were anesthetised by intraperitoneal injection of 0.1% ketamine (Imalgene 1000, Merial, France) and 0.06% xylazine (Rompun, Bayer, La Garenne-Colombes, France). Inflammation was triggered by intra-rectal injection of 200 mg/kg of DNBS (ICN Biomedical Inc., Irvine, CA, USA) resuspended in 30% ethanol-PBS (DNBS-PBS) (day 1). Mice in the non-inflamed group received 30% EtOH-PBS alone. Thirteen days after DNBS injection (day 14), 200 µl of PBS solution containing 1 × 10⁹ cfu of the strain tested, or 200 µl of PBS, were administered intra-gastrically daily for 10 days. Low-grade inflammation was reactivated 21 days after the first DNBS injection (recovery period) with a second injection of 100 mg/kg of DNBS solution (day 21). During this reactivation period, the mice lost weight until the end-point of the experiment (day 24). The study groups were non-inflamed control group (EtOH-PBS), inflamed control group (DNBS-PBS), *L. gasseri* PI41 (DNBS-PI41), *L. rhamnosus* PI48 (DNBS-PI48) and *B. animalis* subsp. *lactis* PI50 (DNBS-PI50). Low-grade inflammation was reactivated 21 days after the first DNBS injection (recovery period) with a second injection of 100 mg/kg of DNBS solution. After euthanasia, the colon was removed, opened longitudinally and immediately assessed for visible damage, macroscopic scores, and myeloperoxidase (MPO) activity levels and intestinal permeability integrity by using fluorescein isothiocyanate-dextran (FITC-Dextran) were characterised as previously described (Barone et al., 2018; Martín et al., 2014). We quantified inflammation by assessing the levels of the pro-inflammatory cytokines IL-4, IL-5, IL-6, and IL-22 from spleen by enzyme-linked immunosorbent assay (ELISA, Biolegend). For lipocalin-2, faeces samples were homogenised (50 mg/ml) in reaction buffer (0.5% W/V NaHCO₃, pH 8.3) and ceramic beads (diameter: 1.4 and 2.8 mm) using a Precellys tissue homogeniser in cold
conditions. Samples were centrifuged for 20 min, and the supernatant was frozen at -80 °C. Lipocalin-2 was assayed using Duoset murine Lcn-2 ELISA kit (R&D Systems, Minneapolis, MN, USA). Faecal protease activity was determined photometrically by using azocasein as a proteolytic substrate (Maeda *et al.*, 2014).

### Intestinal tight junction proteins

Total RNA was isolated from 20-30 mg of colon samples with a RNeasy Mini Kit (Qiagen, Hilden, Germany). DNAse column treatment was used to eliminate any potential DNA contamination. RNA quantity and quality were confirmed with a NanoDrop apparatus (Thermo Scientific) and by agarose gel electrophoresis. Only samples with intact RNA were used for subsequent cDNA synthesis with iScript reverse transcriptase (BioRad): 1 µg of the total RNA preparation was used for each sample. Real-time quantitative PCR (qPCR) was performed with diluted cDNA (10×) in triplicate and with an iQ5 Real-Time Detection System (Bio-Rad). The reaction mixture consisted of Sofast Evagreen Supermix (Bio-Rad), primers at 0.5 µM and 2 µl of diluted cDNA. Values are expressed as normalised relative fold differences with respect to the housekeeping gene, ribosomal protein L19 (*Rpl19*) and TATA box binding protein (*Tbp*) by the 2⁻⁵ΔΔCT method. All procedures were performed according to the manufacturers' instructions. The entire list of primers used can be found in Supplementary Table S1.

### Neonatal maternal separation (NMS) model and administration of strains

A stress during childhood period, called NMS paradigm, was applied on C57BL/6 mice as previously described (Miquel *et al.*, 2016). Briefly, specific pathogen-free C57BL/6 mice were bred in the animal facility of Université Clermont Auvergne. Stress was induced by NMS of pups for 3 h each day from day 2 after birth until day 14. Pups were placed in individual boxes, in a separate room set up with similar environmental conditions. After this period, NMS-induced visceral sensitivity was assessed by performing a colorectal distension (CRD) method described by Larauche *et al.* (2012). Only mice (male or female) that showed the highest responses to CRD after NMS were used in this study. After the NMS and CRD procedures, mice were fed with 200 µl containing 1 × 10⁹ cfu of *L. gasseri* PI41 and/or PBS for 10 days. Each group, the hyperpermeability-induced control group (NMS-PBS) and the hyperpermeability-induced *L. gasseri* PI41-supplemented group (NMS-PI41), consisted of eight male and six female C57BL/6J mice. The experimental

protocols were approved by local ethics committee (protocol number: CE110-12, CE111-12 and EU0116-3460) and followed the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain (IASP). Assessment of intestinal permeability (FITC and intestinal tight junction proteins gene expression) and sensibility (i.e. CRD), and faecal lipocalin-2 and proteolytic activities were determined as mentioned above. Serum serotonin assay was performed on samples from NMS-mice groups by ELISA according to the manufacturer's (LDN, Nordhorn, Germany).

### Statistical analyses

All results were expressed as means ± standard error of the mean (SEM). A one-way ANOVA was performed for normal samples and multiple comparisons were carried out using Dunn's test. For non-normal samples and/or with unequal variances, non-parametric tests were performed within groups (Kruskal-Wallis test) and multiple comparisons were carried out using Dunn's test using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA). An alpha level of 0.05 was used.

## 3 Results

### *In vitro* probiotic screening

Anti-inflammatory properties of potential probiotic strains on TNF-α-stimulated HT-29 cells
Sixteen of the strains were able to reduce significantly IL-8 production by TNF-α-stimulated HT-29 cells (*P* < 0.05, Figure 1). The highest anti-inflammatory effect was observed with *L. plantarum* PI30 at 69%, similar to the percentage inhibition of 1 mM butyrate (71%), used as anti-inflammatory reference in the experiment. Five *L. gasseri* strains (PI17, PI41, PI42, PI43, and PI37) displayed a significant IL-8 inhibition values of around 59%, followed by three *L. rhamnosus* strains (LA702, PI48, PI28, with 63, 38 and 35% of IL-8 inhibition, respectively), two *L. plantarum* strains (PI30 and PI44), one *L. crispatus* (PI34) and one *L. reuteri* (PI49). Only four *Bifidobacterium* strains were able to inhibit IL-8 production, *B. infantis* PI32 by almost 60%, two *B. bifidum* (PI22 and PI32), and two *Bifidobacterium animalis* subsp. *lactis* (LA804 and PI50, 49 and 32% of IL-8 inhibition, respectively).
<figure><img src="image_4.png" /><figcaption>FIGURE 1 Effect of bacterial and yeast strains on interleukin (IL)-8 production by tumour necrosis factor (TNF)-α-stimulated HT-29 cells. HT-29 cells were stimulated with 5 ng of TNF-α, incubated during 6 h. HT-29 cells were used to normalise percentage values. Butyrate 1 mM was used as reference of anti-inflammatory activity (IL-8 blockage). Results are presented as mean ± standard error of the mean (n = 6). ANOVA comparison was performed following Dunnett's test. \* *P* < 0.05, \*\* *P* < 0.01, \*\*\* *P* < 0.001 indicate significant differences between strain groups and the control (PBS) group.</figcaption></figure>

Human PBMC stimulation to classify the immuno-modulatory profile of strains by measuring the IL-10/IL-12 ratio

The reference concanavalin A (ConA), known to stimulate T cells, was able to significantly increase the IL-10/IL-12 ratio (*P* < 0.05) compared to untreated cells (RPMI medium) (Figure 2A). Among all strains, only PI44 and PI28 were able to significantly increase the IL-10/IL-12 ratio (*P* < 0.05), no significant differences were found for the *Saccharomyces* PI31 strain and the rest of the bacterial strains tested. However, strains PI41, PI48 and PI50 were able to significantly stimulate IL-10 production compared to untreated cells by almost 300 and 60 pg/ml, respectively (Figure 2B).

Ability to maintain barrier integrity in Caco-2 cells under pro-inflammatory status (TNF-α-stimulated cells) TNF-α-induced barrier disruption in Caco-2 cells decreased to TEER values of 0.75 (Figure 3). Thirteen of the strains were protective against permeability *in vitro* (TEER values >1.0). Among all, *B. breve* LA805 and *L. rhamnosus* PI48 showed the best activity by increased the TEER values by 1.31 (*P* < 0.05), similar levels of LGG strain known for its positive effects on the intestinal barrier. Six strains belonged to *Bifidobacterium* species were able to significantly protect the barrier integrity (*P* < 0.05), three *B. bifidum* strains (PI28, PI29 and LA803), two *B. animalis* subsp. *lactis* strains (PI50 and LA804) and one *B. breve* strain (LA805) and one *B. infantis* strain (PI32). On other hand, three strains of

*L. gasseri* were able to maintain the barrier integrity *in vitro* (PI37, PI41, PI42), followed by *L. rhamnosus* (PI48 and PI28) and one *S. boulardii* (PI31).

Based on the best results from each *in vitro* assay, we found that most of the anti-inflammatory strains belonging to the LAB, and *Bifidobacterium* strains are the most potent fortifiers of the epithelial barrier *in vitro*. Meanwhile, nine strains of the collection displayed both properties, five LAB strains (*L. gasseri* PI37, PI41, PI42, and *L. rhamnosus* PI28, PI48) and four *Bifidobacterium* strains (*B. animalis* subsp. *lactis* PI50, LA804, *B. animalis* subsp. *infantis* PI32, and *B. bifidum* PI22). Only three strains were able to inhibit IL-8 production (HT-29 cells), immunomodulate IL-10 (PBMCs) and increase TEER values (Caco-2 cells): *L. gasseri* PI41, *L. rhamnosus* PI48, and *B. animalis* subsp. *lactis* PI50 (Supplementary Figure S2). These three candidate strains were selected for further evaluation in preclinical murine models of IBS.

*Impact of Lactobacillus and Bifidobacterium strains on the overall health of mice in a DNBS model of low-grade intestinal inflammation*

Intrarectal injection of DNBS induced a 1% weight lost, a decrease in macroscopic scores of ~0.8, an increase in colonic myeloperoxidase (an enzyme found in the intracellular granules of neutrophils) of 3.2 units, as well as in the faecal-proteolytic (~1.6 fold change) and Lcn-2 (~180 pg/ml) activities (Figure 4), while the treatment with bacteria tended to restore all these parame-
<figure><img src="image_5.png" /><figcaption>FIGURE 2 Determination of interleukin (IL)-10 and IL-12 cytokines by ELISA after co-incubation for 24 h of bacteria with PBMCs from five donors. (A) IL-10/IL-12 ratio after incubation of strains with PBMCs. (B) IL-10 immunomodulation after incubation of strains with PBMCs. Concanavalin A (ConA) was used as a positive control. Results are presented as mean ± standard error of the mean (3 independent tests per donor). ANOVA comparison was performed following Dunnett's test. \* *P* < 0.05 and \*\*\* *P* < 0.001 indicate significant differences between the RPMI medium group and the strains.</figcaption></figure>

ters. *L. gasseri* PI41 and *L. rhamnosus* PI48 treated mice recovered weight and macroscopic scores in a significant manner (*P* < 0.05), while *B. animalis* subsp. *lactis* PI50 did not show this effect (Figure 4A-B). Moreover, MPO (a colonic marker of inflammation) was significantly reduced (*P* < 0.05) by the three selected strains to reach similar units as the untreated EtOH-PBS group (~1.8 MPO units) (Figure 4C). Only *L. rhamnosus* PI48 was able to significantly reduce faecal proteolytic activ-

ity by a factor of 0.5 (*P* < 0.05) (Figure 4D). Fecal-Lipocalin-2 levels were reduced by oral administration of all three strains, although this change was not significant (Figure 4E).

To assess intestinal barrier function, the relative fluorescence units (RFU) of FITC-dextran were measured. A significant alteration of intestinal permeability was observed in the DNBS group (4000 RFU increase, *P* < 0.05), compared to the untreated group. Administration
<figure><img src="image_6.png" /><figcaption>FIGURE 3 Protective effect of bacterial and yeast strains on the integrity of intestinal barrier as measured by Trans-Epithelial Electrical Resistance (TEER). TEER was measured on Caco-2 cells grown on trans-well plates. TEER was measured prior to bacteria/yeast treatment on the apical surface and the tumour necrosis factor (TNF)-α stimulation (100 ng/ml) on basolateral part of the well, and at the end of TNF-α stimulation 21 h at 37 °C. Caco-2 cells were incubated at 37 °C/10% CO₂. Results are presented as mean ± standard error of the mean (n = 6). Analysis was performed by ANOVA test followed by Dunnett's test between DMEM + TNF-α group and bacterial treatments; \* *P* < 0.05.</figcaption></figure>

of *L. gasseri* PI41, *L. rhamnosus* PI48 and *B. animalis* PI50 significantly decreased intestinal hyperpermeability to levels similar to the EtOH-PBS group (1000 RFU, *P* < 0.05, Figure 5A). To better understand the impact of bacterial treatment on gut permeability in mice, the expression of proteins of the cellular junction complex was measured by qPCR. DNBS downregulated mRNA levels of *cingulin* (*P* = 0.0444), *claudin-2* (*Clnd-2*) (*P* = 0.0010) and *Tjp1* (*P* = 0.0129), but the occludin genes were not affected (Figure 5B-E). Only *B. animalis* subsp. *lactis* PI50 tended to normalise *Clnd-2* gene mRNA transcripts, with a 1.5-fold change (*P* < 0.05). In contrast, there were no significant differences observed with *L. gasseri* PI41 and *L. rhamnosus* PI48. At the systemic level, DNBS induced a pro-inflammatory T-helper response measured by the increase in IL-5 and IL-6, but not IL-4, IL-22 (Figure 6) and IFN-γ (data not shown). Interestingly, *L. gasseri* PI41 and *L. rhamnosus* PI48 appeared to stimulate an anti-inflammatory response through the blocking of IL-5 and IL-6 (*P* > 0.05). Although DNBS tended to induce IL-4 and IL-22, no significant changes were found between treatments.

### Impact of *Lactobacillus gasseri* PI41 in a NMS model of IBS

Due to the results obtained with *L. gasseri* PI41 in the DNBS model, reducing intestinal permeability and pro-inflammatory markers, we decided to further anal-

yse this strain in a second murine model mimicking IBS without impacting inflammation profile: the NMS model (Miquel *et al.*, 2016). As expected, the stress induced in mice by NMS altered intestinal permeability. Interestingly, the oral administration of *L. gasseri* PI41 restored the barrier function, significantly reducing FITC-dextran tracers by 36% (*P* < 0.05) in serum (Figure 7A). Furthermore, mice treated with *L. gasseri* PI41 showed a significant reduction of faecal Lcn-2 levels by 70% (*P* = 0.0234). However, there was no significant change in faecal proteolytic activity (–0.8 fold-change, *P* = 0.0669) (Figure 7B-C). Interestingly, *L. gasseri* PI41 (NMS-PI41) ameliorated colonic hypersensitivity, as observed by a significant 36% reduction in intracolonic pressure variation to a CRD (*P* < 0.0001) at the highest distension volumes tested (60–80 mmHg) (Figure 7D). As colonic hypersensitivity is the result of neurotransmitter-mediated gut-brain signalling impairment, we measured serotonin (5-HT: 5-hydroxytryptamine) levels in blood serum samples, and found that *L. gasseri* PI41 reduced serotonin levels by 30% in male but not in female mice (Figure 7E). Finally, due to the impact of *L. gasseri* PI41 in protecting the integrity of the intestinal barrier, the expression of cell junction proteins was determined but no significant differences were observed (*P* > 0.05) (Supplementary Figure S3).
<figure><img src="image_7.png" /><figcaption>FIGURE 4 Effect of three bacterial strains in a DNBS model of low-grade intestinal inflammation. (A) Body weight variation of mice, (B) macroscopic score, (C) myeloperoxidase (MPO) units (U)/mg of tissue, (D) faecal proteolytic activity, and (E) faecal lipocalin-2. Results are presented as means ± standard error of the mean. Comparisons involved the Kruskal-Wallis non-parametric test, followed by a Dunn's Multiple Comparison test. \* *P* < 0.05, \*\* *P* < 0.01, \*\*\* *P* < 0.001 indicate significant differences compared to DNBS-PBS, ns: not significant.</figcaption></figure>

4 Discussion and conclusions

IBS is characterised by a wide variability of clinical
features. As there is no specific curative treatment,
different approaches are being investigated, such as
modulation of the gut microbiota by the use of pro-
biotic strains, especially to treat the IBS-D subtype
(and/or the PI-IBS classified as IBS-D) (Laval et al., 2015;
Moayyedi et al., 2010; Zhang et al., 2016). Here, we com-
bined in vitro approaches, focusing on immunomod-
ulatory properties and in vitro intestinal permeabil-
ity, to characterise new potential probiotic strains for
IBS management. Elevated levels of pro-inflammatory
cytokines, such as TNF-α, IL-12 and IL-8 have been
correlated with IBS-D (Choghakhori et al., 2017; Mit-
selou et al., 2020; Zhen et al., 2015) and PI-IBS (Patel
et al., 2017). For this reason, we first identified certain
bacterial strains capable of blocking IL-8 secretion by
TNF-α-stimulated HT-29 cells, belonging to L. gasseri,
L. reuteri, and B. animalis species. In previous exper-
iments, the ability to inhibit IL-8 production led to

the identification of anti-inflammatory properties of
some probiotic candidate bacteria (Benítez-Cabello et
al., 2020; Hernández-Delgado et al., 2021; Kechaou et
al., 2013; Torres-Maravilla et al., 2016). On the other
hand, IL-10 seems to have an important role and effect
in IBS (Hong et al., 2021; Mauras et al., 2018). Indeed,
decreased levels of IL-10 in plasma have been reported
in patients with IBS (Liebregts et al., 2007; Zhu et al.,
2019). Among the strains with an anti-inflammatory
effect, four strains were able to stimulate IL-10 pro-
duction by PBMCs, one *Bifidobacterium* (PI50) one *L.
rhamnosus* (PI48), one *L. gasseri* (PI41) and one *L. plan-
tarum* (PI44) strain. Immunomodulatory effects have
been reported for those species, for example: (1) *L.
gasseri* has been shown to exert immunomodulation *via*
the toll-like receptor 2 and NOD2-mediated PI3K/Akt
signalling pathway (Sun et al., 2020); (2) *L. rhamno-
sus* mediates immune response through the produc-
tion of exopolysaccharides (EPS) (Nowak et al., 2020);
(3) *Bifidobacterium* species produce short chain fatty
acids (SFCAs, i.e. acetate) that stimulate the immune
<figure><img src="image_8.png" /><figcaption>FIGURE 5 Regulation of intestinal tight junction proteins in the low-grade intestinal inflammation model. The expression levels of apical junction protein were determined by real-time qPCR in non-inflamed controls (EtOH-PBS), inflamed controls (DNBS-PBS), *Lactobacillus gasseri* PI41 (DNBS-PI41), *Lacticaseibacillus rhamnosus* PI48 (DNBS-PI48) and *Bifidobacterium animalis* subsp. *lactis* PI50 (DNBS-PI50) treated mice. Comparisons included the non-parametric Kruskal-Wallis test, followed by a Dunn's Multiple Comparison test. \* *P* < 0.05 compared to DNBS-PBS.</figcaption></figure>

system (Ruiz *et al.*, 2017) and/or (4) inhibition of the NF-κB pathway (Lenoir *et al.*, 2020), among others. Since increased intestinal permeability is observed in a subset of IBS patients (Zhou *et al.*, 2009), we tested the ability of bacteria to protect the intestinal barrier on Caco-2 cells. Nine anti-inflammatory strains (mainly from *L. gasseri*, *B. animalis* subsp. *lactis* and *L. rhamnosus* species) were able to protect the intestinal barrier and increased TEER values. These values reflect the cell layer integrity and barrier function across Caco-2 cells (Anderson *et al.*, 2010). It seems that bacteria-epithelial cell interactions enhance the integrity of the intestinal barrier through stimulation of mucus production and

upregulation of tight junction genes, such as *occludin* and *ZO-1* (Anderson *et al.*, 2010; Ewaschuk *et al.*, 2008; Yeung *et al.*, 2013). Probiotic effects appear to be strain-dependent, as reported previously (Kechaou *et al.*, 2013; Neau *et al.*, 2016). Here, the most anti-inflammatory strains belonged to *L. gasseri*. But only one strain of *L. gasseri*, the strain PI41, was able to enhance IL-10 on PBMCs. Many aspects of modulating the beneficial immune response are influenced by cell wall parame ters, and a single gene can have a significant impact on the exopolysaccharide-producing phenotype (Bubnov *et al.*, 2018). To go further *in vivo*, we crossed the results obtained in the *in vitro* models and selected three strains
<figure><img src="image_9.png" /><figcaption>FIGURE 6 Impact of bacterial strains on systemic T-helper balance. Cytokines secreted by splenocytes from each group, stimulated for 48 h with CD3/CD28, were measured by ELISA (n = 16 mice/group). Comparison was performed with the non-parametric Mann-Whitney test. \* *P* < 0.05, \*\* \*P* < 0.001 compared to DNBS-PBS.</figcaption></figure>

that showed the best profile: *L. gasseri* PI41, *L. rhamnosus* PI48, and *Bifidobacterium animalis* subsp. *lactis* PI50 (Supplementary Figure S2). Similar strategies have been implemented to select probiotic bacterial strains with promising outcomes. Alard *et al.* (2018) combining *in vitro* experiments (i.e. immunomodulation in PBMCs and gut permeability in Caco-2 cells) identified four strains *B. bifidum* PI22, *B. lactis* LA804, *L. salivaricus* LA307, *L. helveticus* PI5 with different characteristics and a high potential for the management of IBD. Kechaou *et al.* (2013) identified *L. plantarum* CNRZ1997 active against influenza virus infection in mice by *in vitro* screening in PBMCs and activated-TNF-α HT-29 cells.

Due to the multifactorial origin of IBS, we tested the strains in a model of IBS induced by DNBS, a model

in which intestinal permeability and pro-inflammatory markers can be assessed, as increased permeability and low-grade inflammation are observed in IBS-D subtype (including PI-IBS) (Ford *et al.*, 2020; Gwee, 2010; Liebregts *et al.*, 2007) although not in all patients (Torres-Maravilla *et al.*, 2022a). *L. gasseri* PI41 and *L. rhamnosus* PI48 decreased weight loss, maintained the T-helper balance as observed for spleen cytokine production, and reduced pro-inflammatory markers (MPO, faecal proteolytic and Lcn-2 activities). Similar results were found for *L. gasseri* KBL697 and *L. gasseri* SF1183, helping to restore weight loss, lowering macroscopic scores, reducing MPO activity as well as restoring the Th1/Th2 balance in the dextran sodium sulphate (DSS)-induced colitis mouse model (Di Luccia *et al.*, 2018; Han *et al.*, 2022) and for *L. rhamnosus* CNCM I-3690
<figure><img src="image_10.png" /><figcaption>FIGURE 7 Impact of oral administration of *Lactobacillus gasseri* PI41 in the NMS murine model. (A) Intestinal permeability measured by FITC-dextran, (B) faecal Lipocalin-2, (C) faecal proteolytic activity, (D) Colonic hypersensitivity, measured as intracolonic pressure variation (IPV) scores as a function of distention pressure at 20, 40, 60, 80 mmHG for the different groups of mice, (E) blood serum serotonin (5-HT: 5-hydroxytryptamine) levels in male and female mice. Results are presented as mean ± standard error of the mean (n = 14). Comparisons were done using Kruskal-Wallis test. \* *P* < 0.05, or \*\* *P* < 0.01, \*\*\* *P* < 0.001 indicate significant differences compared to control group.</figcaption></figure>

in a DNBS model (Martín *et al.*, 2019). *L. gasseri* and *L. rhamnosus* are generally considered as autochthonous microorganisms in the gastrointestinal tract of humans (Baltova and Dimitrov, 2014; Douillard *et al.*, 2013). Due to the fact that studies reporting the determination of probiotic properties of *L. gasseri* strains in IBS are still limited compared to *L. rhamnosus* species (i.e. *L. rhamnosus* GG (Wegh *et al.*, 2018), *L. rhamnosus* CNCM I-3690 (Martín *et al.*, 2019), *L. rhamnosus* HN001 (Bonfrate *et al.*, 2020)), we decided to further analyse the probiotic properties of *L. gasseri* PI41 in a complementary *in vivo* model that overlaps with the symptoms of IBS, the non-inflammatory NMS model. In this model, intestinal permeability and colonic hypersensitivity, which are associated with visceral pain in IBS patients, are associated with the stress generated during the neonatal maternal separation period (Miquel *et al.*, 2016). Stress plays an important role in the development

of IBS, psychological stress in early life or in adulthood could affect the sensitivity, motility, secretion and permeability of the intestinal tract, leading to IBS (Madison and Kiecolt-Glaser, 2019).

In both models, *L. gasseri* PI41 restored the impaired intestinal barrier without a significant increase in tight junction proteins, which could be explained by the fact that during intestinal inflammation, permeability increases and tight junction protein translocation occurs. For example, disruption of the epithelial barrier by pathogenic bacteria provokes changes in the location of zonula occludens-1 (ZO-1) and tight junction proteins (Yeung *et al.*, 2013). On the other hand, *L. gasseri* PI41 was able to reduce susceptibility to stress in the colorectal distension (CRD) test. Francavilla *et al.* (2010) reported that *L. rhamnosus* LGG significantly reduces the frequency and severity of abdominal pain in children with IBS; this effect is long-lasting and may
be secondary to the improvement of the intestinal barrier (Francavilla et al., 2010). We found that *L. gasseri* PI41 appears to restore serotonin (5-HT) unbalance in the NMS model; abnormal 5-HT functioning is associated with altered gut motility and enhanced nociceptive pain sensitivity, both characteristic symptoms of IBS (Grover and Camilleri, 2013). It was shown that strains from the *L. gasseri* species have beneficial effects in other *in vivo* models by regulating 5-HT-2A levels (Lee et al., 2021). In IBS, increased colonic serotonin levels and decreased inhibition of serotonin reuptake transporter (SERT) expression may contribute to visceral hypersensitivity (Gao et al., 2022). Recently, Gao et al. (2022) reported that intracolonic infusion of faecal supernatants from patients with IBS-D resulted in an increase in mucosal 5-HT compared with faecal supernatants from healthy control. Only male mice showed remarkable changes in 5-HT levels. In previous studies, it was demonstrated that female rats showed a significant increase in 5-HT efflux after methiothepin administration, unlike male rats. Females appear to rely more than males on the regulation of 5-HT efflux by terminal auto receptors (Jones and Lucki, 2005). The role of microbiota composition in 5-HT synthesis has not yet been fully elucidated, but a diminished expression of the 5-HT transporter has been related with dysbiosis in rats (El Aidy et al., 2017). Probiotic supplementation could regulate 5-HT levels by stimulating the production of microbial metabolites, such as butyrate, cholate, p-aminobenzoate, tyramine and ligands for aryl hydrocarbon receptor (AhR) (Ma et al., 2020; Reigstad et al., 2015; Yano et al., 2015). Central neuromodulators, low dose of tricyclic antidepressants, intestinal secretagogues and drugs acting on transient receptor potential vanilloid 1 (TRPV1) (Pang et al., 2022), opioid or 5-HT receptors are used to treat visceral pain in IBS (Ford et al., 2020; Yano et al., 2015), therefore, a bacterium able to regulate 5-HT could be useful as co-therapy in IBS.

In conclusion, this work provides one strategy to select probiotic candidate strains with specific targeted characteristics in the context of IBS by combining *in vitro* experiments. The broad range of immunomodulatory properties and intestinal barrier integrity tested *in vitro* allowed us to identify three novel probiotic candidate strains, including the *L. gasseri* PI41, which were validated for their efficacy in alleviating and protecting the intestinal barrier in two IBS models. Taking together these data suggest that *L. gasseri* PI41 in particular, may be useful in preventing intestinal hyper-permeability and improving colonic hypersensitivity in the context

of IBS. The impact of this strain on the role of serotonin (5-HT) needs to be further investigated.

## Supplementary material

Supplementary material is available online at:
https://doi.org/10.6084/m9.figshare.25550127

**Table S1.** Information on selected intestinal tight junction (Tj) genes used in this study.

**Figure S1.** DNBS-induced low-grade inflammation experimental protocol.

**Figure S2.** Venn diagram of selected probiotic properties based on three *in vitro* tests.

**Figure S3.** Tight junction's intestinal proteins regulation in NMS model.

## Acknowledgements

We would like to thank Claude Blondeau for her help for the writing assistance, technical editing, language editing, and proofreading of the manuscript. We wish to thank the staff of the animal facilities of Université Clermont Auvergne and INRAE Infectiology of Fishes and Rodents Facility (IERP-UE907, Jouy-en-Josas Research Center, France) in which animal experiments have been performed. IERP Facility belongs to the National Distributed Research Infrastructure for the Control of Animal and Zoonotic Emerging Infectious Diseases through *in vivo* Investigation.

## Authors' contribution

E.T.M., F.A.C., P.L., S.H., F.B., A.J.W.D., and L.G.B.H., designed all the experiments; E.T.M., J.D., L.L., and F.A.C., performed research; E.T.M., F.A.C., J.D., F.B., and E.J., contributed analytic tools; E.T.M., F.A.C., E.J., A.J.W.D and L.G.B.H., analysed data; and E.T.M., F.A.C., P.L., S.H., L.G.B.H., and A.J.W.D., wrote the paper. All read and approved the final manuscript.

## Conflict of interest

S.H., E.J., and L.L. are employees of PiLeJe Laboratoire; the other authors have no relevant financial or non-financial interests to disclose.
## Funding

The reported study was supported by the National Research Institute for Agriculture, Food and Environment (INRAE), Paris University and a contract with PiLeJe Laboratoire. This work was also supported by the Ministère de la Recherche et de la Technologie (NeuroDol laboratory (INSERM 1107)), by the Region Auvergne-Rhône-Alpes and FEDER (Thématiques émergentes, Pack Ambition Recherche; the French Government IDEX-ISITE Initiative, No. 16-IDEX-0001-CAP 20-25), and the Clermont Auvergne Métropole ('Recherche-Action' project).

## References

Ait Abdellah, S., Scanzi, J., Gal, C., Martin, M., Beck, M. and Ojetti, V., 2022. *Lactobacillus gasseri* LA806 supplementation in patients with irritable bowel syndrome: a multicenter study. Journal of Clinical Medicine 11: 7446. https://doi.org/10.3390/jcm11247446

Alard, J., Peucelle, V., Boutillier, D., Breton, J., Kuylle, S., Pot, B., Holowacz, S. and Grangette, C., 2018. New probiotic strains for inflammatory bowel disease management identified by combining *in vitro* and *in vivo* approaches. Beneficial Microbes 9: 317-331. https://doi.org/10.3920/bm2017.0097

Anderson, R.C., Cookson, A.L., McNabb, W.C., Park, Z., McCann, M.J., Kelly, W.J. and Roy, N.C., 2010. *Lactobacillus plantarum* MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation. BMC Microbiology 10: 316. https://doi.org/10.1186/1471-2180-10-316

Baltova, K. and Dimitrov, Z., 2014. Probiotic and cultural characteristic of strain *Lactobacillus gasseri* 4/13 of human origin. Biotechnology & Biotechnological Equipment 28: 1084-1088. https://doi.org/10.1080/13102818.2014.974303

Barone, M., Chain, F., Sokol, H., Brigidi, P., Bermúdez-Humarán, L.G., Langella, P. and Martín, R., 2018. A versatile new model of chemically induced chronic colitis using an outbred murine strain. Frontiers in Microbiology 9: 565. https://doi.org/10.3389/fmicb.2018.00565

Benítez-Cabello, A., Torres-Maravilla, E., Bermúdez-Humarán, L., Langella, P., Martín, R., Jiménez-Díaz, R. and Arroyo-López, F.N., 2020. Probiotic properties of *Lactobacillus* strains isolated from table olive biofilms. Probiotics and Antimicrobial Proteins 12: 1071-1082. https://doi.org/10.1007/s12602-019-09604-y

Benjak Horvat, I., Gobin, I., Kresović, A. and Hauser, G., 2021. How can probiotic improve irritable bowel syndrome

symptoms? World Journal of Gastrointestinal Surgery 13: 923-940. https://doi.org/10.4240/wjgs.v13.i9.923

Bonfrate, L., Di Palo, D.M., Celano, G., Albert, A., Vitellio, P., De Angelis, M., Gobbetti, M. and Portincasa, P., 2020. Effects of *Bifidobacterium longum* BB536 and *Lactobacillus rhamnosus* HN001 in IBS patients. European Journal of Clinical Investigation 50: e13201. https://doi.org/10.1111/eci.13201

Bubnov, R.V., Babenko, L.P., Lazarenko, L.M., Mokrozub, V.V. and Spivak, M.Y., 2018. Specific properties of probiotic strains: relevance and benefits for the host. EPMA Journal 9: 205-223. https://doi.org/10.1007/s13167-018-0132-z

Choghakhori, R., Abbasnezhad, A., Hasanvand, A. and Amani, R., 2017. Inflammatory cytokines and oxidative stress biomarkers in irritable bowel syndrome: association with digestive symptoms and quality of life. Cytokine 93: 34-43. https://doi.org/10.1016/j.cyto.2017.05.005

Chong, P.P., Chin, V.K., Looi, C.Y., Wong, W.F., Madhavan, P. and Yong, V.C., 2019. The microbiome and irritable bowel syndrome – a review on the pathophysiology, current research and future therapy. Frontiers in Microbiology 10: 1136. https://doi.org/10.3389/fmicb.2019.01136

Di Luccia, B., Mazzoli, A., Cancelliere, R., Crescenzo, R., Ferrandino, I., Monaco, A., Bucci, A., Naclerio, G., Iossa, S., Ricca, E. and Baccigalupi, L., 2018. *Lactobacillus gasseri* SF1183 protects the intestinal epithelium and prevents colitis symptoms *in vivo*. Journal of Functional Foods 42: 195-202. https://doi.org/10.1016/j.jff.2017.12.049

Douillard, F.P., Ribbera, A., Kant, R., Pietilä, T.E., Järvinen, H.M., Messing, M., Randazzo, C.L., Paulin, L., Laine, P., Ritari, J., Caggia, C., Lähteinen, T., Brouns, S.J.J., Satokari, R., von Ossowski, I., Reunanen, J., Palva, A. and de Vos, W.M., 2013. Comparative genomic and functional analysis of 100 *Lactobacillus rhamnosus* strains and their comparison with strain GG. PLOS Genetics 9: e1003683. https://doi.org/10.1371/journal.pgen.1003683

El Aidy, S., Ramsteijn, A.S., Dini-Andreote, F., van Eijk, R., Houwing, D.J., Salles, J.F. and Olivier, J.D.A., 2017. Serotonin transporter genotype modulates the gut microbiota composition in young rats, an effect augmented by early life stress. Frontiers in Cellular Neuroscience 11: 222. https://doi.org/10.3389/fncel.2017.00222

El-Salhy, M., Hatlebakk, J.G., Gilja, O.H., Bråthen Kristoffersen, A. and Hausken, T., 2020. Efficacy of faecal microbiota transplantation for patients with irritable bowel syndrome in a randomised, double-blind, placebo-controlled study. Gut 69: 859. https://doi.org/10.1136/gutjnl-2019-319630

Enck, P., Aziz, Q., Barbara, G., Farmer, A.D., Fukudo, S., Mayer, E.A., Niesler, B., Quigley, E.M.M., Rajilić-Stojanović, M., Schemann, M., Schwille-Kiuntke, J., Simren, M., Zipfel, S. and Spiller, R.C., 2016. Irritable bowel syndrome. Nature
Reviews Disease Primers 2: 16014. https://doi.org/10.1038/nrdp.2016.14

Ewaschuk, J.B., Diaz, H., Meddings, L., Diederichs, B., Dmytrash, A., Backer, J., Looijer van Langen, M. and Madsen, K.L., 2008. Secreted bioactive factors from *Bifidobacterium infantis* enhance epithelial cell barrier function. American Journal of Physiology – Gastrointestinal and Liver Physiology 295: G1025-G1034. https://doi.org/10.1152/ajpgi.90227.2008

Fong, W., Li, Q. and Yu, J., 2020. Gut microbiota modulation: a novel strategy for prevention and treatment of colorectal cancer. Oncogene 39: 4925-4943. https://doi.org/10.1038/s41388-020-1341-1

Ford, A.C., Sperber, A.D., Corsetti, M. and Camilleri, M., 2020. Irritable bowel syndrome. The Lancet 396: 1675-1688. https://doi.org/10.1016/s0140-6736(20)31548-8

Francavilla, R., Miniello, V., Magistà, A.M., De Canio, A., Bucci, N., Gagliardi, F., Lionetti, E., Castellaneta, S., Polimeno, L., Peccarisi, L., Indrio, F. and Cavallo, L., 2010. A randomized controlled trial of *Lactobacillus* GG in children with functional abdominal pain. Pediatrics 126: e1445-e1452. https://doi.org/10.1542/peds.2010-0467

Gao, J., Xiong, T., Grabauskas, G. and Owyang, C., 2022. Mucosal serotonin reuptake transporter expression in irritable bowel syndrome is modulated by gut microbiota via mast cell-prostaglandin E2. Gastroenterology. 162: 1962-1974. E6. https://doi.org/10.1053/j.gastro.2022.02.016

Grover, M. and Camilleri, M., 2013. Effects on gastrointestinal functions and symptoms of serotonergic psychoactive agents used in functional gastrointestinal diseases. Journal of Gastroenterology 48: 177-181. https://doi.org/10.1007/s00535-012-0726-5

Guyonnet, D., Chassany, O., Ducrotte, P., Picard, C., Mouret, M., Mercier, C.H. and Matuchansky, C., 2007. Effect of a fermented milk containing Bifidobacterium animalis DN-173 010 on the health-related quality of life and symptoms in irritable bowel syndrome in adults in primary care: a multicentre, randomized, double-blind, controlled trial. Alimentary Pharmacology & Therapeutics 26: 475-486. https://doi.org/10.1111/j.1365-2036.2007.03362.x

Gwee, K.A., 2010. Post-infectious irritable bowel syndrome, an inflammation-immunological model with relevance for other IBS and functional dyspepsia. Journal of Neurogastroenterology and Motility 16: 30-34. https://doi.org/10.5056/jnm.2010.16.1.30

Han, D.H., Kim, W.-K., Lee, C., Park, S., Lee, K., Jang, S.J. and Ko, G., 2022. Co-administration of *Lactobacillus gasseri* KBL697 and tumor necrosis factor-alpha inhibitor infliximab improves colitis in mice. Scientific Reports 12: 9640. https://doi.org/10.1038/s41598-022-13753-6

Hernández-Delgado, N.C., Torres-Maravilla, E., Mayorga-Reyes, L., Martín, R., Langella, P., Pérez-Pastén-Borja, R., Sánchez-Pardo, M.E. and Bermúdez-Humarán, L.G., 2021. Antioxidant and anti-inflammatory properties of probiotic candidate strains isolated during fermentation of agave (*Agave angustifolia* Haw). Microorganisms 9: 1063. https://doi.org/10.3390/microorganisms9051063

Hong, N., Ku, S., Yuk, K., Johnston, T.V., Ji, G.E. and Park, M.S., 2021. Production of biologically active human interleukin-10 by *Bifidobacterium bifidum* BGN4. Microbial Cell Factories 20: 16. https://doi.org/10.1186/s12934-020-01505-y

Huang, X., Ai, F., Ji, C., Tu, P., Gao, Y., Wu, Y., Yan, F. and Yu, T., 2021. A rapid screening method of candidate probiotics for inflammatory bowel diseases and the anti-inflammatory effect of the selected strain *Bacillus smithii* XY1. Frontiers in Microbiology 12: 760385. https://doi.org/10.3389/fmicb.2021.760385

Ivashkin, V., Poluektov, Y., Kogan, E., Shifrin, O., Sheptulin, A., Kovaleva, A., Kurbatova, A., Krasnov, G. and Poluektova, E., 2021. Disruption of the pro-inflammatory, anti-inflammatory cytokines and tight junction proteins expression, associated with changes of the composition of the gut microbiota in patients with irritable bowel syndrome. PLoS ONE 16: e0252930. https://doi.org/10.1371/journal.pone.0252930

Jones, M.D. and Lucki, I., 2005. Sex differences in the regulation of serotonergic transmission and behavior in 5-HT receptor knockout mice. Neuropsychopharmacology 30: 1039-1047. https://doi.org/10.1038/sj.npp.1300664

Kajander, K., Myllyluoma, E., Rajilić-Stojanović, M., Kyrönpalo, S., Rasmussen, M., Järvenpää, S., Zoetendal, E.G., de Vos, W.M., Vapaatalo, H. and Korpela, R., 2008. Clinical trial: multispecies probiotic supplementation alleviates the symptoms of irritable bowel syndrome and stabilizes intestinal microbiota. Alimentary Pharmacology & Therapeutics 27: 48-57. https://doi.org/10.1111/j.1365-2036.2007.03542.x

Kechaou, N., Chain, F., Gratadoux, J.J., Blugeon, S., Bertho, N., Chevalier, C., Le Goffic, R., Courau, S., Molimard, P., Chatel, J.M., Langella, P. and Bermúdez-Humarán, L.G., 2013. Identification of one novel candidate probiotic Lactobacillus plantarum strain active against influenza virus infection in mice by a large-scale screening. Applied and Environmental Microbiology 79: 1491-1499. https://doi.org/10.1128/aem.03075-12

Konstantis, G., Efstathiou, S., Pourzitaki, C., Kitsikidou, E., Germanidis, G. and Chourdakis, M., 2023. Efficacy and safety of probiotics in the treatment of irritable bowel syndrome: a systematic review and meta-analysis of randomised clinical trials using ROME IV criteria. Clinical
Nutrition 42: 800-809. https://doi.org/10.1016/j.clnu.2023.03.019

Lacy, B.E. and Patel, N.K., 2017. Rome criteria and a diagnostic approach to irritable bowel syndrome. Journal of Clinical Medicine 6: 99. https://doi.org/10.3390/jcm6110099

Larauche, M., Mulak, A. and Taché, Y., 2012. Stress and visceral pain: from animal models to clinical therapies. Experimental Neurology 233: 49-67. https://doi.org/10.1016/j.expneurol.2011.04.020

Laval, L., Martin, R., Natividad, J.N., Chain, F., Miquel, S., Desclée de Maredsous, C., Capronnier, S., Sokol, H., Verdu, E.F., van Hylckama Vlieg, J.E., Bermúdez-Humarán, L.G., Smokvina, T. and Langella, P., 2015. *Lactobacillus rhamnosus* CNCM I-3690 and the commensal bacterium *Faecalibacterium prausnitzii* A2-165 exhibit similar protective effects to induced barrier hyper-permeability in mice. Gut Microbes 6: 1-9. https://doi.org/10.4161/19490976.2014.990784

Lee, S., Jung, D.H., Park, M., Yeon, S.-W., Jung, S.-H., Yun, S.-I., Park, H.-O. and Yoo, W., 2021. The effect of *Lactobacillus gasseri* BNR17 on postmenopausal symptoms in ovarianized rats. Journal of Microbiology and Biotechnology 31: 1281-1287. https://doi.org/10.4014/jmb.2105.05032

Lenoir, M., Martín, R., Torres-Maravilla, E., Chadi, S., González-Dávila, P., Sokol, H., Langella, P., Chain, F. and Bermúdez-Humarán, L.G., 2020. Butyrate mediates anti-inflammatory effects of *Faecalibacterium prausnitzii* in intestinal epithelial cells through Dact3. Gut Microbes 12: 1-16. https://doi.org/10.1080/19490976.2020.1826748

Li, B., Liang, L., Deng, H., Guo, J., Shu, H. and Zhang, L., 2020. Efficacy and safety of probiotics in irritable bowel syndrome: a systematic review and meta-analysis. Frontiers in Pharmacology 11: 332. https://doi.org/10.3389/fphar.2020.00332

Liebregts, T., Adam, B., Bredack, C., Röth, A., Heinzel, S., Lester, S., Downie-Doyle, S., Smith, E., Drew, P., Talley, N.J. and Holtmann, G., 2007. Immune activation in patients with irritable bowel syndrome. Gastroenterology 132: 913-920. https://doi.org/10.1053/j.gastro.2007.01.046

Lo Presti, A., Zorzi, F., Del Chierico, F., Altomare, A., Cocca, S., Avola, A., De Biasio, F., Russo, A., Cella, E., Reddel, S., Calabrese, E., Biancone, L., Monteleone, G., Cicala, M., Angeletti, S., Ciccozzi, M., Putignani, L. and Guarino, M.P.L., 2019. Fecal and mucosal microbiota profiling in irritable bowel syndrome and inflammatory bowel disease. Frontiers in Microbiology 10: 1655. https://doi.org/10.3389/fmicb.2019.01655

Ma, N., He, T., Johnston, L.J. and Ma, X., 2020. Host-microbiome interactions: the aryl hydrocarbon receptor as a critical node in tryptophan metabolites to brain signaling. Gut

Microbes 11: 1203-1219. https://doi.org/10.1080/19490976.2020.1758008

Madison, A. and Kiecolt-Glaser, J.K., 2019. Stress, depression, diet, and the gut microbiota: human-bacteria interactions at the core of psychoneuroimmunology and nutrition. Current Opinion in Behavioral Sciences 28: 105-110. https://doi.org/10.1016/j.cobeha.2019.01.011

Maeda, S., Ohno, K., Uchida, K., Igarashi, H., Goto-Koshino, Y., Fujino, Y. and Tsujimoto, H., 2014. Intestinal protease-activated receptor-2 and fecal serine protease activity are increased in canine inflammatory bowel disease and may contribute to intestinal cytokine expression. Journal of Veterinary Medical Science 76: 1119-1127. https://doi.org/10.1292/jvms.14-0060

Mallappa, R.H., Singh, D.K., Rokana, N., Pradhan, D., Batish, V.K. and Grover, S., 2019. Screening and selection of probiotic *Lactobacillus* strains of Indian gut origin based on assessment of desired probiotic attributes combined with principal component and heatmap analysis. LWT 105: 272-281. https://doi.org/10.1016/j.lwt.2019.02.002

Martín, R., Chain, F., Miquel, S., Lu, J., Gratadoux, J.J., Sokol, H., Verdu, E.F., Bercik, P., Bermúdez-Humarán, L.G. and Langella, P., 2014. The commensal bacterium *Faecalibacterium prausnitzii* is protective in DNBS-induced chronic moderate and severe colitis models. Inflammatory Bowel Disease 20: 417-430. https://doi.org/10.1097/01.Mib.0000440815.76627.64

Martín, R., Chamignon, C., Mhedbi-Hajri, N., Chain, F., Derrien, M., Escribano-Vázquez, U., Garault, P., Cotillard, A., Pham, H.P., Chervaux, C., Bermúdez-Humarán, L.G., Smokvina, T. and Langella, P., 2019. The potential probiotic *Lactobacillus rhamnosus* CNCM I-3690 strain protects the intestinal barrier by stimulating both mucus production and cytoprotective response. Science Reports 9: 5398. https://doi.org/10.1038/s41598-019-41738-5

Martín, R., Laval, L., Chain, F., Miquel, S., Natividad, J., Cherbuy, C., Sokol, H., Verdu, E.F., van Hylckama Vlieg, J., Bermudez-Humaran, L.G., Smokvina, T. and Langella, P., 2016. *Bifidobacterium animalis* ssp. *lactis* CNCM I-2494 restores gut barrier permeability in chronically low-grade inflamed mice. Frontiers in Microbiology 7: 608. https://doi.org/10.3389/fmicb.2016.00608

Mauras, A., Chain, F., Faucheux, A., Ruffié, P., Gontier, S., Ryffel, B., Butel, M.J., Langella, P., Bermúdez-Humarán, L.G. and Waligora-Dupriet, A.J., 2018. A new bifidobacteria expression system (BEST) to produce and deliver interleukin-10 in *Bifidobacterium bifidum*. Frontiers in Microbiology 9: 3075. https://doi.org/10.3389/fmicb.2018.03075

Miquel, S., Martín, R., Lashermes, A., Gillet, M., Meleine, M., Gelot, A., Eschalier, A., Ardid, D., Bermúdez-Humarán,
L.G., Sokol, H., Thomas, M., Theodorou, V., Langella, P. and Carvalho, F.A., 2016. Anti-nociceptive effect of *Faecalibacterium prausnitzii* in non-inflammatory IBS-like models. Science Reports 6: 19399. https://doi.org/10.1038/srep19399

Mitselou, A., Grammeniatis, V., Varouktsi, A., Papadatos, S.S., Katsanos, K. and Galani, V., 2020. Proinflammatory cytokines in irritable bowel syndrome: a comparison with inflammatory bowel disease. Intestinal Research 18: 115-120. https://doi.org/10.5217/ir.2019.00125

Moayyedi, P., Ford, A.C., Talley, N.J., Cremonini, F., Foxx-Orenstein, A.E., Brandt, L.J. and Quigley, E.M.M., 2010. The efficacy of probiotics in the treatment of irritable bowel syndrome: a systematic review. Gut 59: 325. https://doi.org/10.1136/gut.2008.167270

Neal, K.R., Barker, L. and Spiller, R.C., 2002. Prognosis in post-infective irritable bowel syndrome: a six year follow up study. Gut 51: 410-413. https://doi.org/10.1136/gut.51.3.410

Neau, E., Delannoy, J., Marion, C., Cottart, C.H., Labellie, C., Holowacz, S., Butel, M.J., Kapel, N. and Waligora-Dupriet, A.J., 2016. Three novel candidate probiotic strains with prophylactic properties in a murine model of cow's milk allergy. Applied and Environmental Microbiology 82: 1722-1733. https://doi.org/10.1128/aem.03440-15

Nowak, B., Śróttek, M., Ciszek-Lenda, M., Skałkowska, A., Gamian, A., Górska, S. and Marcinkiewicz, J., 2020. Exopolysaccharide from *Lactobacillus rhamnosus* KL37 inhibits T cell-dependent immune response in mice. Archivum Immunologiae et Therapiae Experimentalis 68: 17. https://doi.org/10.1007/s00005-020-00581-7

O'Mahony, L., McCarthy, J., Kelly, P., Hurley, G., Luo, F., Chen, K., O'Sullivan, G.C., Kiely, B., Collins, J.K., Shanahan, F. and Quigley, E.M., 2005. *Lactobacillus* and *Bifidobacterium* in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterology 128: 541-551. https://doi.org/10.1053/j.gastro.2004.11.050

Pang, Y., Ermann Lundberg, L., Mata Forsberg, M., Ahl, D., Bysell, H., Pallin, A., Sverremark-Ekström, E., Karlsson, R., Jonsson, H. and Roos, S., 2022. Extracellular membrane vesicles from *Limosilactobacillus reuteri* strengthen the intestinal epithelial integrity, modulate cytokine responses and antagonize activation of TRPV1. Frontiers in Microbiology 13: 1032202. https://doi.org/10.3389/fmicb.2022.1032202

Patel, S.R., Singh, A., Misra, V., Misra, S.P., Dwivedi, M. and Trivedi, P., 2017. Levels of interleukins 2, 6, 8, and 10 in patients with irritable bowel syndrome. Indian Journal of Pathology and Microbiology 60: 385-389. https://doi.org/10.4103/ijpm.Ijpm_544_16

Perez-Burgos, A., Wang, L., McVey Neufeld, K.A., Mao, Y.K., Ahmadzai, M., Janssen, L.J., Stanisz, A.M., Bienenstock, J.

and Kunze, W.A., 2015. The TRPV1 channel in rodents is a major target for antinociceptive effect of the probiotic *Lactobacillus reuteri* DSM 17938. Journal of Physiology 593: 3943-3957. https://doi.org/10.1113/jp270229

Rajilić-Stojanović, M., Biagi, E., Heilig, H.G., Kajander, K., Kekkonen, R.A., Tims, S. and de Vos, W.M., 2011. Global and deep molecular analysis of microbiota signatures in fecal samples from patients with irritable bowel syndrome. Gastroenterology 141: 1792-1801. https://doi.org/10.1053/j.gastro.2011.07.043

Reigstad, C.S., Salmonson, C.E., III, J.F.R., Szurszewski, J.H., Linden, D.R., Sonnenburg, J.L., Farrugia, G. and Kashyap, P.C., 2015. Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells. FASEB Journal 29: 1395-1403. https://doi.org/10.1096/fj.14-259598

Ruiz, L., Delgado, S., Ruas-Madiedo, P., Sánchez, B. and Mar-golles, A., 2017. Bifidobacteria and their molecular commu-nication with the immune system. Frontiers in Microbiol-ogy 8: 2345. https://doi.org/10.3389/fmicb.2017.02345

Saha, L., 2014. Irritable bowel syndrome: pathogenesis, diagnosis, treatment, and evidence-based medicine. World Journal of Gastroenterology 20: 6759-6773. https://doi.org/10.3748/wjg.v20.i22.6759

Salinas, E., Reyes-Pavón, D., Cortes-Perez, N.G., Torres-Maravilla, E., Bitzer-Quintero, O.K., Langella, P. and Bermúdez-Humarán, L.G., 2021. Bioactive compounds in food as a current therapeutic approach to maintain a healthy intestinal epithelium. Microorganisms 9: 1634. https://doi.org/10.3390/microorganisms9081634

Sisson, G., Ayis, S., Sherwood, R.A. and Bjarnason, I., 2014. Randomised clinical trial: a liquid multi-strain probiotic vs. placebo in the irritable bowel syndrome – a 12 week double-blind study. Alimentary Pharmacology & Thera- peutics 40: 51-62. https://doi.org/10.1111/apt.12787

Sun, L., Tian, W., Guo, X., Zhang, Y., Liu, X., Li, X., Tian, Y., Man, C. and Jiang, Y., 2020. *Lactobacillus gasseri* JMI with potential probiotic characteristics alleviates inflammatory response by activating the PI3K/Akt signaling pathway *in vitro*. Journal of Dairy Science 103: 7851-7864. https://doi.org/10.3168/jds.2020-18187

Swanson, K.S., Gibson, G.R., Hutkins, R., Reimer, R.A., Reid, G., Verbeke, K., Scott, K.P., Holscher, H.D., Azad, M.B., Delzenne, N.M. and Sanders, M.E., 2020. The international scientific association for probiotics and prebiotics (ISAPP) consensus statement on the definition and scope of syn-biotics. Nature Reviews Gastroenterology & Hepatology 17: 687-701. https://doi.org/10.1038/s41575-020-0344-2

Torres-Maravilla, E., Boucard, A.-S., Mohseni, A.H., Taghi-nezhad-S, S., Cortes-Perez, N.G. and Bermúdez-Humarán, L.G., 2021. Role of gut microbiota and probiotics in col-
orectal cancer: onset and progression. Microorganisms 9: 1021. https://doi.org/10.3390/microorganisms9051021

Torres-Maravilla, E., Holowacz, S., Delannoy, J., Lenoir, L., Jacouton, E., Gervason, S., Meynier, M., Boucard, A.-S., Carvalho, F.A., Barbut, F., Bermúdez-Humarán, L.G., Langella, P. and Waligora-Dupriet, A.-J., 2022a. Serpin-positive *Bifidobacterium breve* CNCM I-5644 improves intestinal permeability in two models of irritable bowel syndrome. Scientific Reports 12: 19776. https://doi.org/10.1038/s41598-022-21746-8

Torres-Maravilla, E., Lenoir, M., Mayorga-Reyes, L., Allain, T., Sokol, H., Langella, P., Sánchez-Pardo, M.E. and Bermúdez-Humarán, L.G., 2016. Identification of novel anti-inflammatory probiotic strains isolated from pulque. Applied Microbiology and Biotechnology 100: 385-396. https://doi.org/10.1007/s00253-015-7049-4

Torres-Maravilla, E., Reyes-Pavón, D., Benítez-Cabello, A., González-Vázquez, R., Ramírez-Chamorro, L.M., Langella, P. and Bermúdez-Humarán, L.G., 2022b. Strategies for the identification and assessment of bacterial strains with specific probiotic traits. Microorganisms 10: 1389.

Wegh, C.A.M., Benninga, M.A. and Tabbers, M.M., 2018. Effectiveness of probiotics in children with functional abdominal pain disorders and functional constipation: a systematic review. Journal of Clinical Gastroenterology 52 Suppl 1: S10-S26. https://doi.org/10.1097/mcg.0000000000001054

Yano, J.M., Yu, K., Donaldson, G.P., Shastri, G.G., Ann, P., Ma, L., Nagler, C.R., Ismagilov, R.F., Mazmanian, S.K. and Hsiao, E.Y., 2015. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell 161: 264-276. https://doi.org/10.1016/j.cell.2015.02.047

Yeung, C.-Y., Chiang Chiau, J.-S., Chan, W.-T., Jiang, C.-B., Cheng, M.-L., Liu, H.-L. and Lee, H.-C., 2013. *In vitro* pre-

vention of *Salmonella* lipopolysaccharide-induced damages in epithelial barrier function by various *Lactobacillus* strains. Gastroenterology Research and Practice 2013: 973209. https://doi.org/10.1155/2013/973209

Zhang, T., Zhang, C., Zhang, J., Sun, F. and Duan, L., 2022. Efficacy of probiotics for irritable bowel syndrome: a systematic review and network meta-analysis. Frontiers in Cellular and Infection Microbiology 12: 859967. https://doi.org/10.3389/fcimb.2022.859967

Zhang, Y., Li, L., Guo, C., Mu, D., Feng, B., Zuo, X. and Li, Y., 2016. Effects of probiotic type, dose and treatment duration on irritable bowel syndrome diagnosed by Rome III criteria: a meta-analysis. BMC Gastroenterology 16: 62. https://doi.org/10.1186/s12876-016-0470-z

Zhen, Y., Chu, C., Zhou, S., Qi, M. and Shu, R., 2015. Imbalance of tumor necrosis factor-α, interleukin-8 and interleukin-10 production evokes barrier dysfunction, severe abdominal symptoms and psychological disorders in patients with irritable bowel syndrome-associated diarrhea. Molecular Medicine Reports 12: 5239-5245. https://doi.org/10.3892/mmr.2015.4079

Zhou, Q., Zhang, B. and Verne, G.N., 2009. Intestinal membrane permeability and hypersensitivity in the irritable bowel syndrome. Pain 146: 41-46. https://doi.org/10.1016/j.pain.2009.06.017

Zhu, S.W., Liu, Z.J., Sun, Q.H. and Duan, L.P., 2019. Effect of the interleukin 10 polymorphisms on interleukin 10 production and visceral hypersensitivity in Chinese patients with diarrhea-predominant irritable bowel syndrome. Chinese Medical Journal 132: 1524-1532. https://doi.org/10.1097/cm9.0000000000000306