Use of Group-Specific and RAPD-PCR Analyses for Rapid Differentiation of <Emphasis Type="Italic">Lactobacillus</Emphasis> Strains from Probiotic Yogurts

The increasing interest in probiotic lactobacilli implicates the requirement of techniques that allow a rapid and reliable identification of these organisms. In this study, group-specific PCR and RAPD-PCR analyses were used to identify strains of the Lactobacillus casei and Lactobacillus acidophilus groups most commonly used in probiotic yogurts. Group-specific PCR with primers for the L. casei and L. acidophilus groups, as well as L. gasseri/johnsonii, could differentiate between 20 Lactobacillus strains isolated from probiotic yogurts and assign these into the corresponding groups. For identification of these strains to species or strain level, RAPD profiles of the 20 Lactobacillus strains were compared with 11 reference strains of the L. acidophilus and L. casei group. All except one strain could be attributed unambigously to the species L. acidophilus, L. johnsonii, L. crispatus, L. casei, and L. paracasei. DNA reassociation analysis confirmed the classification resulting from the RAPD-PCR.     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Prevention of Pathogenic Escherichia coli Infection in Mice and Stimulation of Macrophage Activation in Rats by an Oral Administration of Probiotic Lactobacillus casei I-5

Lactobacillus casei I-5 isolated from an alcohol fermentation broth enhanced immunity and prevented pathogenic infection as a probiotic. Mice fed with I-5 cells for 11 days prior to an intraperitoneal challenge with pathogenic Escherichia coli Juhl exhibited a high survival rate compared with the control group. Rats fed with I-5 cells for 10 days significantly increased the phagocytosis of peritoneal macrophages. In a cell culture system employing peritoneal macrophages from rats, the I-5 administration activated NF-κB stimulated by LPS. It also enhanced LPS-stimulated IL-12 and TNF-α production, but not IL-6 production. These results show that L. casei I-5 effectively prevented infection by pathogenic E. coli possibly through the activation of peritoneal macrophages. The strain would be useful to prevent pathogenic microbial infections in humans and farm animals.     

The Acid-Dependent and Independent Effects of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R,and Lacticaseibacillus rhamnosus CLR2 on Clostridioides difficile R20291

Clostridioides difficile infections (CDI) result from antibiotic use and cause severe diarrhea which is life threatening and costly. A specific probiotic containing Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacticaseibacillus rhamnosus CLR2 has demonstrated a strong inhibitory effect on the growth of several nosocomial C. difficile strains by production of antimicrobial metabolites during fermentation. Though there are several lactobacilli shown to inhibit C. difficile growth by processes relying on acidification, this probiotic has demonstrated potency for CDI prevention among hospitalized patients. Here, we describe the acid-dependent and independent mechanisms by which these strains impair the cytotoxicity of a hypervirulent strain, C. difficile R20291 (CD). These bacteria were co-cultured in a series of experiments under anaerobic conditions in glucose-rich and no-sugar medium to inhibit or stimulate CD toxin production, respectively. In glucose-rich medium, there was low CD toxin production, but sufficient amounts to cause cytotoxic damage to human fibroblast cells. In co-culture, there was acidification by the lactobacilli resulting in growth inhibition as well as ≥ 99% reduced toxin A and B production and no observable cytotoxicity. In the absence of glucose, CD produced much more toxin. In co-culture, the lactobacilli did not acidify the medium and CD growth was unaffected; yet, the amount of detected toxin A and B was decreased by 20% and 41%, respectively. Despite the high concentration of toxin, cells exposed to the supernatant from the co-culture were able to survive. These results suggest that in addition to known acid-dependent effects, the combination of L. acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2 can interfere with CD pathogenesis without acidification: (1) reduced toxin A and B production and (2) toxin neutralization. This might explain the strain specificity of this probiotic in potently preventing C. difficile-associated diarrhea in antibiotic-treated patients compared with other probiotic formulae.

     

Assessment of the Safety of Lactobacillus casei IMV B-7280 Probiotic Strain on a Mouse Model

Probiotics, in particular Lactobacillus (lactic acid bacteria, LAB) strains, are widely used in clinical practice. Despite that these probiotics have GRAS (generally regarded as safe) and qualified presumption of safety (QPS) statuses, the safety of particular strains still needs to be thoroughly studied. The aim of the study was to evaluate the safety of Lact. casei IMV B-7280 strain by investigating toxicity and the effects on gut microbiota in experimental animal model. Male BALB/c mice (7–8 weeks, weight 20–24 g) were treated with amounts of Lact. casei IMV B-7280 strain: 5 × 10⁶, 5 × 10⁸, or 5 × 10⁹ CFU/animal once per day during 7 days, or in the amount of 1 × 10¹⁰ CFU/animal once per day during 3 days (most of the proposed probiotic doses for humans—from 10⁸ to 10⁹ CFU) and monitored during 14 days. Blood tests and serum biochemistry were conducted; the cecal content from mice of the experimental and control groups were freshly collected and analyzed. At the end of the experiments (15th day), the presence of LAB in the heart, liver, kidney, and mesenteric lymph nodes and peripheral blood was determined; histology of the brain, liver, heart, fragments of the small and large intestine, and mesenteric lymph nodes was conducted. Survival rate of BALB/c mice treated with Lact. casei IMV B-7280 strain in different concentrations in toxicity experiments during 14 days was 100%. We observed no signs of toxicity as changes in gait, lethargy, sleep, somatomotor activity as well as changes in fur, eyes, skin and mucous membranes, tremors, behavior pattern, convulsions, salivation, diarrhea, and local injuries in mice from all experimental groups. After administration of probiotic strain, the number of opportunistic bacteria in cecal contents, such as Staphylococcus spp., Candida spp., Pseudomonas spp., and total aerobic and optionally anaerobic bacteria decreased compared to controls; the population of beneficial bacteria such as lactobacilli increased in cecal contents of these mice. LAB were not detected in the peripheral blood, heart, liver, kidneys, and mesenteric lymph nodes after administration of this strain to intact mice. Lact. casei IMV B-7280 strain is safe at dose up to 10¹⁰ CFU/animal during 3- and 7-day oral administration to mice and has a positive effect on the gut microbiota composition; it could be potentially considered as safe probiotic for humans.

     

Probiotic Lactobacillus fermentum UCO-979C biofilm formation on AGS and Caco-2 cells and Helicobacter pylori inhibition

The ability of the human isolate Lactobacillus fermentum UCO-979C to form biofilm and synthesize exopolysaccharide on abiotic and biotic models is described. These properties were compared with the well-known Lactobacillus casei Shirota to better understand their anti-Helicobacter pylori probiotic activities. The two strains of lactobacilli synthesized exopolysaccharide as detected by the Dubois method and formed biofilm on abiotic and biotic surfaces visualized by crystal violet staining and scanning electron microscopy. Concomitantly, these strains inhibited H. pylori urease activity by up to 80.4% (strain UCO-979C) and 66.8% (strain Shirota) in gastric adenocarcinoma (AGS) cells, but the two species showed equal levels of inhibition (~84%) in colorectal adenocarcinoma (Caco-2) cells. The results suggest that L. fermentum UCO-979C has probiotic potential against H. pylori infections. However, further analyses are needed to explain the increased activity observed against the pathogen in AGS cells as compared to L. casei Shirota.     

Efficient production and secretion of bovine β-lactoglobulin by <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

Background  

Lactic acid bacteria (LAB) are attractive tools to deliver therapeutic molecules at the mucosal level. The model LAB Lactococcus lactis has been intensively used to produce and deliver such heterologous proteins. However, compared to recombinant lactococci, lactobacilli offer some advantages such as better survival in the digestive tract and immunomodulatory properties. Here, we compared different strategies to optimize the production of bovine β-lactoglobulin (BLG), a major cow's milk allergen, in the probiotic strain Lactobacillus casei BL23.     

Draft Genome Sequence of Lactobacillus casei W56

We announce the draft genome sequence of Lactobacillus casei W56 in one contig. This strain shows immunomodulatory and probiotic properties. The strain is also an ingredient of commercially available probiotic products.     

Probiotic Lactobacillus casei Expressing Human Lactoferrin Elevates Antibacterial Activity in the Gastrointestinal Tract

In this study, Lactobacillus casei was used to deliver and express human lactoferrin (hLF) to protect the host against bacterial infection. Full-length hLF cDNA was cloned into a Lactobacillus-specific plasmid to produce the L. casei transformants (rhLF/L. casei). Antimicrobial activity of recombinant hLF was examined in inhibition of bacteria growth in vitro. A mouse model was established to test in vivo antibacterial activity and protective effect of orally-administered probiotic L. casei transformant in the gastrointestinal tract. Trials were conducted in which animals were challenged with E. coli ATCC25922. E. coli colony numbers in duodenal fluid from the group fed with rhLF/L. casei were significantly lower than those of the group fed with wild-type L. casei or placebo (P < 0.01). Histopathological analyses of the small intestine, showed both decreased intestinal injury and increased villi length were observed in the mice fed with rhLF/L. casei as compared with the control groups (P < 0.01). Our results demonstrate that L. casei expressing hLF exhibited antibacterial activity both in in vitro and in vivo. It also provides a potentially large-scale production of hLF as applications for treatment of infections caused by clinically relevant pathogens.     

A practical random mutagenesis system for probiotic Lactobacillus casei using Tn5 transposition complexes

Aims: Despite the fact that the entire genome sequence of probiotic Lactobacillus casei has recently been available, their mechanisms of beneficial effects are poorly clarified, probably because of the lack of an efficient mutagenesis system. The aim of this study was to establish a practical random mutagenesis system of L. casei using the Tn5 transposome complexes. Methods and Results: We optimized the conditions for transformation using a plasmid pUCYIT356‐1‐Not2 and then transposition reaction using Tn5 transposome system for L. casei ATCC 27139. Tn5 insertion library of this strain being consisted of 9408 mutants was constructed by repeating the mutagenesis procedure. To examine the utility of this mutagenesis system, we screened a panel of insertion mutants for nutrient requirements. Six auxotrophic mutants were isolated and their Tn5 insertion sites were determined by inverse PCR, which demonstrated that insertions occur randomly throughout the whole bacterial genome. Conclusions: Tn5 transposome system functioned efficiently to generate transposon insertion mutants of L. casei and enabled to construct useful L. casei Tn5 insertion library at optimized conditions for transformation and transposition. Significance and Impact of the Study: The availability of this system facilitates the study of the mechanisms of beneficial effects of L. casei for human health.     

Importance of Molecular Methods to Determine Whether a Probiotic is the Source of <Emphasis Type="Italic">Lactobacillus</Emphasis> Bacteremia

There has been an increasing interest in the use of probiotic products for the prevention of Clostridium difficile infection (CDI). Bio-K+^® is a commercial probiotic product comprising three strains of lactobacilli—Lactobacillus acidophilus CL1285^®, Lact. casei LBC80R^® and Lact. rhamnosus CLR2^®—that have been applied to prevent CDI. Generally considered as safe, lactobacilli have potential to cause bacteremia, endocarditis and other infections. The source of Lactobacillus bacteremia can be normal human flora or lactobacilli-containing probiotic. The aim of this study was to assess whether probiotic lactobacilli caused bacteremia and to show the value of molecular identification and typing techniques to determine probiotic and patient strain relatedness. We report an episode of Lactobacillus bacteremia in a 69-year-old man admitted to a hospital with severe congestive heart failure. During his hospitalization, he required long-term antibiotic therapy. Additionally, the patient received Bio-K+^® probiotic as part of a quality improvement project to prevent CDI. Subsequently, Lactobacillus bacteremia occurred. Two independent blinded laboratory evaluations, using pulse field gel electrophoresis, 16S rRNA gene sequencing and DNA fingerprint analysis (rep-PCR), were performed to determine whether the recovered Lact. acidophilus originated from the probiotic product. Ultimately, the patient strain was identified as Lact. casei and both laboratories found no genetic relation between the patient’s strain and any of the probiotic lactobacilli. This clinical case of lactobacillus bacteremia in the setting of probiotic exposure demonstrates the value of using discriminatory molecular methods to clearly determine whether there were a link between the patient’s isolate and the probiotic strains.     

Gut immune stimulation by non pathogenic Gram(+) and Gram(-) bacteria. Comparison with a probiotic strain

We analyzed the gut immune stimulation induced by Gram-positive bacteria: non probiotic Lactobacillus acidophilus CRL 1462 and Lactobacillus acidophilus A9; two potentially probiotic strains: L. acidophilus CRL 924 and Lactobacillus delbrueckii subsp. bulgaricus CRL 423; comparatively with a probiotic strain: Lactobacillus casei CRL 431. We also studied Gram-negative bacteria: Escherichia coli 129 and E. coli 13-7 in BALB/c mice. All the strains increased the number of IgA+ cells. We analyzed the cytokines IFNγ, TNFα, IL-17, IL-12, IL-6 and MIP-1α. The Gram(+) strains increased the number of IL-10+ cells. Gram(−) strains did not increase IL-10+ cells, but they increased the number of IL-12+ cells. The probiotic strain increased mainly IFNγ and TNFα. In the study of the receptors TLR-2, TLR-4 and CD-206, we demonstrated that only the probiotic strain increased the number of CD-206+ cells. All the Gram(+) strains increased the number of TLR-2+ cells and the Gram(−) strains of the TLR-4+ cells. The probiotic strain induced the release of IL-6 by a preparation enriched in intestinal epithelial cells (IEC). Gram(+) and Gram(−) bacteria activated different immune receptors and induced a different cytokine profile. The probiotic strain showed a great activity on the immune cells and the enriched population in IEC, activating mainly cells of the innate immune system.     

Lactobacillus casei DN-114 001 Inhibits the Ability of Adherent-Invasive Escherichia coli Isolated from Crohn's Disease Patients To Adhere to and To Invade Intestinal Epithelial Cells

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.     

Characterization of a fibronectin‐binding protein from Lactobacillus casei BL23

Aims: To characterize the functionality of the Lactobacillus casei BL23 fbpA gene encoding a putative fibronectin‐binding protein. Methods and Results: Adhesion tests showed that L. casei BL23 binds immobilized and soluble fibronectin in a protease‐sensitive manner. A mutant with inactivated fbpA showed a decrease in binding to immobilized fibronectin and a strong reduction in the surface hydrophobicity as reflected by microbial adhesion to solvents test. However, minor effects were seen on adhesion to the human Caco‐2 or HT‐29 cell lines. Purified 6X(His)FbpA bound to immobilized fibronectin in a dose‐dependent manner. Western blot experiments with FbpA‐specific antibodies showed that FbpA could be extracted from the cell surface by LiCl treatment and that protease digestion of the cells reduced the amount of extracted FbpA. Furthermore, surface exposition of FbpA was detected in other L. casei strains by LiCl extraction and whole‐cell ELISA. Conclusions: FbpA can be found at the L. casei BL23 surface and participates in cell attachment to immobilized fibronectin. We showed that FbpA is an important, but not the only, factor contributing to fibronectin binding in BL23 strain. Significance and Impact of the Study: This is the first report showing the involvement of FbpA in fibronectin binding in L. casei BL23 and represents a new contribution to the study of attachment factors in probiotic bacteria.     

Validation of reference genes for real-time quantitative PCR studies in gene expression levels of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang

Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.     

Isolation of Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512 as novel probiotics with immunomodulatory properties

Probiotics, defined as living bacteria that are beneficial for human health, mainly function through their immunomodulatory abilities. Hence, these microorganisms have proven successful for treating diseases resulting from immune deregulation. The aim of this study was to find novel candidates to improve on and complement current probiotic treatment strategies. Of 60 lactic acid bacterial strains that were isolated from fecal samples of healthy, full‐term, breast‐fed infants, three were chosen because of their ability to activate human immune cells. These candidates were then tested with regard to immunomodulatory properties, antimicrobial effects on pathogens, required pharmacological properties and their safety profiles. To identify the immunomodulatory structures of the selected isolates, activation of specific innate immune receptors was studied. The three candidates for probiotic treatment were assigned Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512. Compared with the established allergy‐protective strain Lactococcus lactis G121, these isolates induced release of similar amounts of IL‐12, a potent inducer of T helper 1 cells. In addition, all three neonatal isolates had antimicrobial activity against pathogens. Analysis of pharmacological suitability showed high tolerance of low pH, bile salts and pancreatic enzymes. In terms of safe application in humans, the isolates were sensitive to three antibiotics (chloramphenicol, tetracycline and erythromycin). In addition, the Enterococcus isolates were free from the four major virulence genes (cylA, agg, efaAfs and ccf). Moreover, the isolates strongly activated Toll‐like receptor 2, which suggests lipopeptides as their active immunomodulatory structure. Thus, three novel bacterial strains with great potential as probiotic candidates and promising immunomodulatory properties have here been identified and characterized.     

Human probiotic bacteria attenuate Pseudomonas aeruginosa biofilm and virulence by quorum-sensing inhibition

Abstract

This work investigated chloroform extracts from culture supernatants of two human probiotic bacteria, Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730 for the production of virulence factors and quorum sensing (QS) interference against three Pseudomonas aeruginosa strains. Both extracts inhibited biofilm biomass (up to 50%), biofilm metabolic activity (up to 39%), the production of the enzyme elastase (up to 63%) and pyocyanin (up to 77%), and decreased QS, without presenting any antibacterial acgivity. In addition, the chloroform extracts of both strains disrupted preformed biofilms of the three strains of P. aeruginosa analyzed (up to 40%). GC-MS analysis revealed that the major compounds detected in the bioactive extracts were four diketopiperazines. This study suggests that the metabolites of L. casei and L. acidophilus could be a promising alternative to combat the pathogenicity of P. aeruginosa.     

Immunogenicity of Orally Administrated Recombinant <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang Expressing <Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> Surface Adhesion Protein P23 in Mice

Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal diseases in both humans and animals. What is more, there is no promising strategy for controlling cryptosporidiosis. In this study, the P23 immunodominant surface protein of C. parvum sporozoites was stably expressed in the Lactobacillus casei Zhang strain and its immunogenicity was evaluated in a mouse model. The molecular weight (23 kDa) and immunogenicity of p23 gene expressed by L. casei Zhang were similar to that of the native P23 protein. Oral immunization with control L. casei Zhang and recombinant L. casei Zhang-p23 activated the mucosal immune system to elicit serum immunoglobulin G (IgG) and mucosal IgA in mice. Furthermore, the expression of cytokines such as IL-4, IL-6, and IFN-γ in splenocytes of mice was detected by real-time PCR after oral immunization. P23-specific immunocyte activation was also verified. These findings indicate that the live L. casei Zhang vector may be a new tool for the production of mucosal vaccines against cryptosporidiosis in animals.     

In vitro assessment of safety and probiotic potential characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from water buffalo mozzarella cheese

The aim of this study was to evaluate the safety and probiotic potential characteristics of ten Lactobacillus spp. strains (Lactobacillus fermentum SJRP30, Lactobacillus casei SJRP37, SJRP66, SJRP141, SJRP145, SJRP146, and SJRP169, and Lactobacillus delbrueckii subsp. bulgaricus SJRP50, SJRP76, and SJRP149) that had previously been isolated from water buffalo mozzarella cheese. The safety of the strains was analyzed based on mucin degradation, hemolytic activity, resistance to antibiotics and the presence of genes encoding virulence factors. The in vitro tests concerning probiotic potential included survival under simulated gastrointestinal (GI) tract conditions, intestinal epithelial cell adhesion, the presence of genes encoding adhesion, aggregation and colonization factors, antimicrobial activity, and the production of the β-galactosidase enzyme. Although all strains presented resistance to several antibiotics, the resistance was limited to antibiotics to which the strains had intrinsic resistance. Furthermore, the strains presented a limited spread of genes encoding virulence factors and resistance to antibiotics, and none of the strains presented hemolytic or mucin degradation activity. The L. delbrueckii subsp. bulgaricus strains showed the lowest survival rate after exposure to simulated GI tract conditions, whereas all of the L. casei and L. fermentum strains showed good survivability. None of the tested lactobacilli strains presented bile salt hydrolase (BSH) activity, and only L. casei SJRP145 did not produce the β-galactosidase enzyme. The strains showed varied levels of adhesion to Caco-2 cells. None of the cell-free supernatants inhibited the growth of pathogenic target microorganisms. Overall, L. fermentum SJRP30 and L. casei SJRP145 and SJRP146 were revealed to be safe and to possess similar or superior probiotic characteristics compared to the reference strain L. rhamnosus GG (ATCC 53103).