Development of chitosan-coated agar-gelatin particles for probiotic delivery and targeted release in the gastrointestinal tract.

This study reports the development of a novel and simple formulation for probiotic delivery using chitosan-coated agar-gelatin gel particles. This methodology involves the production of agar-gelatin particles by thermally treating a mixture of agar and gelatin solutions at high temperatures (121 °C) and subsequently coating with chitosan. The particles were able to protect the probiotic strain Lactobacillus plantarum NCIMB 8826 during incubation for 2 h in simulated gastric fluid (pH 2), as no statistically significant loss (P > 0.05) in cell concentration was observed, and also resist dissolution in simulated intestinal fluid (pH 7.2). Interestingly, this protection is related to the fact that the intense thermal treatment affected the physicochemical properties of agars and resulted in the formation of a strong and tight polymer network, as indicated by the X-ray diffraction (XRD) analysis. Using an in vitro faecal batch fermentation model simulating the conditions of the distal part of the large intestine (pH 6.7-6.9), it was demonstrated by quantitative real-time PCR that the majority of L. plantarum cells were released from the agar-gelatin particles within 30 to 48 h. Overall, this work led to the development of a novel methodology for the production of probiotic-containing particles, which is simpler compared with current encapsulation technologies and has a lot of potential to be used for the controlled release of probiotics and potentially other solid bioactives in the large intestine.Key Points• Chitosan gel particles is a simple and scalable method of probiotic encapsulation.• Autoclaving agar-gelatin particles increases their stability at low pH.• Chitosan gel particles protected L. plantarum during gastrointestinal conditions.• Probiotics could be controlled release in the colon using chitosan gel particles.

Spray dried formulation of mesalamine embedded with probiotic biomass for the treatment of ulcerative colitis: in-vitro and in-vivo studies.

This study is using the targeted approach and anti-inflammatory action of the probiotic biomass to lessen the side effects of therapeutic agents of ulcerative colitis. The aim of the present study is to prepare mesalamine loaded eudragit S-100 with probiotic microparticles by spray drying method. The in-vitro release of the optimized formulation was 90.55 ± 2.42 in 24 hr, which display controlled drug release of mesalamine at a particular region. Mesalamine loaded eudragit S-100 with probiotic microparticles (F12) presented average particle size of 4.91 µm. The statistical analysis was done by one way ANOVA and then comparison test of Bonferroni was done and p values <.05 were considered as significant. The effects of spray dried microparticles over inflamed Caco-2 cell were also evaluated by determining the concentration of IL-8. From in-vivo study it was seen that pretreatment of mesalamine with probiotic prevents DNBS (Dinitrobenzenesulfonic acid) induced colitis in rats and represents protective action against ulcerative colitis because of its antioxidant and anti-inflammatory actions. The results give the foundation for a combination of targeted approach along with the anti-inflammatory potential of the probiotic which might help to decrease the problems which are seen with the traditional cure and management of ulcerative colitis.

Bacteria-Carried Iron Oxide Nanoparticles for Treatment of Anemia.

The efficiency of maghemite nanoparticles for the treatment of anemia was sensibly higher when nanoparticles were incorporated onto the probiotic bacterium Lactobacillus fermentum (MNP-bacteria) than when administrated as uncoated nanoparticles (MNP). Plasma iron and hemoglobin, intestine expression of divalent metal transporter 1 (DMT1) and duodenal Cytochrome b (DcytB), as well as hepatic expression of the hormone hepcidin were fully restored to healthy levels after administration of MNP-bacteria but not of MNP. A magnetic study on biodistribution and biodegradation showed accumulation of maghemite nanoparticles in intestine lumen when MNP-bacteria were administrated. In contrast, MNP barely reached intestine. In vivo MRI studies suggested the internalization of MNP-bacteria into enterocytes, which did not occur with MNP. Transmission electronic microscopy confirmed this internalization. The collective analysis of results point out that L. fermentum is an excellent carrier to overcome the stomach medium and drive maghemite nanoparticles to intestine, where iron absorption occurs. Due the probiotic ability to adhere to the gut wall, MNP-bacteria internalize into the enterocyte, where maghemite nanoparticles are delivered, providing an adequate iron level into enterocyte. This paper advances a new route for effective iron absorption in the treatment of anemia.

Feasibility studies of concomitant administration of optimized formulation of probiotic-loaded Vancomycin hydrochloride pellets for colon delivery.

Objective of this study was to develop Vancomycin HCl pellets loaded with Saccharomyces boulardii (S.b.) for pH-dependent system and CODES™ for augmenting the efficacy of Vancomycin HCl in the treatment of colitis. Pellets were prepared by extrusion-spheronization. In the pH-dependent system, the pellets were coated with Eudragit FS 30D. These pellets exhibited spherical form and a uniform surface coating. The CODES™ system consisted of three components: core containing mannitol, drug and probiotic, an inner acid-soluble coating layer, and an outer layer of enteric coating material. Statistical factorial design was used to optimize both formulations. Scanning electron micrographs of coated pellets revealed uniform coating. In vitro drug release of these coated pellets was studied sequentially in various buffers with (2%) and without rat cecal content for a period of 12 h. From the optimized pH-dependent formulation, F6 (20% w/w coating level and 15% w/v concentration of polymer), higher amount of probiotic was released in earlier time phase (first 5 h) as compared to the CODES™ and so R5 [containing acid-soluble inner coating layer (15% w/w coating level and 12% w/v concentration of Eudragit E100), and an outer layer of enteric coating material (12% w/w coating level and 10% w/v concentration of Eudragit L100)] was considered as the best formulation after confirming in vivo X-ray studies conducted on rabbits, suggesting that Vancomycin HCl and S.b. may be co-administered as pellets [CODES™] to enhance the effectiveness of Vancomycin HCl in the treatment of colitis without its associated side effects, which can only be confirmed after clinical trials.

Divergent immunomodulating effects of probiotics on T cell responses to oral attenuated human rotavirus vaccine and virulent human rotavirus infection in a neonatal gnotobiotic piglet disease model.

Rotaviruses (RVs) are a leading cause of childhood diarrhea. Current oral vaccines are not effective in impoverished countries where the vaccine is needed most. Therefore, alternative affordable strategies are urgently needed. Probiotics can alleviate diarrhea in children and enhance specific systemic and mucosal Ab responses, but the T cell responses are undefined. In this study, we elucidated the T cell and cytokine responses to attenuated human RV (AttHRV) and virulent human RV (HRV) in gnotobiotic pigs colonized with probiotics (Lactobacillus rhamnosus strain GG [LGG] and Bifidobacterium lactis Bb12 [Bb12]), mimicking gut commensals in breastfed infants. Neonatal gnotobiotic pigs are the only animal model susceptible to HRV diarrhea. Probiotic colonized and nonvaccinated (Probiotic) pigs had lower diarrhea and reduced virus shedding postchallenge compared with noncolonized and nonvaccinated pigs (Control). Higher protection in the Probiotic group coincided with higher ileal T regulatory cells (Tregs) before and after challenge, and higher serum TGF-ß and lower serum and biliary proinflammatory cytokines postchallenge. Probiotic colonization in vaccinated pigs enhanced innate serum IFN-α, splenic and circulatory IFN-γ-producing T cells, and serum Th1 cytokines, but reduced serum Th2 cytokines compared with noncolonized vaccinated pigs (Vac). Thus, LGG+Bb12 induced systemic Th1 immunostimulatory effects on oral AttHRV vaccine that coincided with lower diarrhea severity and reduced virus shedding postchallenge in Vac+Pro compared with Vac pigs. Previously unreported intestinal CD8 Tregs were induced in vaccinated groups postchallenge. Thus, probiotics LGG+Bb12 exert divergent immunomodulating effects, with enhanced Th1 responses to oral AttHRV vaccine, whereas inducing Treg responses to virulent HRV.

Microencapsulation of new probiotic formulations for gastrointestinal delivery: in vitro study to assess viability and biological properties.

The paper describes the preparation of new probiotic formulations based on chitosan-coated alginate microcapsules containing three different probiotic strains, Lactobacillus plantarum PBS067, Lactobacillus rhamnosus PBS070, and Bifidobacterium animalis subsp. lactis PBS075 taken individually and as a mixture of them. The effects of microencapsulation on the viability of the strains in conditions simulating the gastrointestinal tract and under industrial processes conditions were studied. In addition, an evaluation of their probiotic properties was also investigated by in vitro tests on the human intestinal cell line HT-29 to explore the effect of microencapsulation on health beneficial effect of the considered strains. Non-encapsulated cells were completely destroyed when exposed to simulated gastric juice and other stress conditions, while encapsulated cells exhibited a significantly higher resistance to artificial intestinal juice and heat and osmotic treatment. Moreover, in this study, the effect of the various microencapsulated probiotic strain formulations was compared with analogous formulations also containing the ß-glucan Pleuran. The microencapsulation effectively protected the selected bacteria, as single strain and as a mixture of the three strains in both the formulations with and without Pleuran, from simulating gastrointestinal tract and industrial process conditions in delivering the viable cells without any significant adverse effect on their functionalities. The comparative study of the immunomodulatory properties of each single strain and the mixture of the three strains revealed a synergistic effect of the probiotic mixture, but no appreciable difference between the two kinds of formulations could be detected, as the effect of Pleuran is covered by the higher potential of the probiotic strains.

Ability of synbiotic encapsulated Saccharomyces cerevisiae boulardii to grow in berry juice and to survive under simulated gastrointestinal conditions.

The probiotic yeast, Saccharomyces cerevisiae boulardii, was microencapsulated in a mixture of alginate-inulin-xanthan gum, and its ability to grow in berry juice and survive 4 weeks of storage at 4 °C was determined. Exposure of the yeast in these forms to artificial gastrointestinal conditions was also assessed. Encapsulation significantly enhanced cell viability after fermentation and storage compared with the free yeast (7.59 log10 colony forming units/ml versus 6.98 log10 colony forming units/ml, respectively) and protected it from exposure to a simulated gastrointestinal transit after 4 weeks of storage. Conversely, a dramatic loss of viability was exhibited by free yeast after 4 weeks of storage, and viability values closer to zero (0.23 log10 cfu/ml) were found after the simulated gastrointestinal treatment. Microcapsules were capable of absorbing a certain amount of polyphenols and anthocyanins. This work, based on use of microencapsulated probiotic yeasts, might represent the starting point for the development of new functional foods or functional ingredients. Microcapsules were capable to absorb, from berry juice, a certain amount of anthocyanins which, maintaining their native form after the in vitro gastrointestinal transit, might in vivo therein be transformed into other, simpler molecules, with beneficial effect on microflora and human health too.

Polymerase chain reaction detection of Lactobacillus acidophilus in human oral cavity and fecal samples after 2-week consumption of yoghurt.

OBJECTIVE: To investigate whether short-term daily consumption of yoghurt leads to colonization by Lactobacillus acidophilus in a group of human subjects who were initially totally devoid of L. acidophilus in their oral cavities. MATERIAL AND METHODS: Twenty-three volunteers consumed yogurt containing L. acidophilus during a 14-day trial stage. Oral and fecal samples were collected at the clearance stage and at the post-yoghurt intake stage until L. acidophilus was found. Standard polymerase chain reaction methods using specific primers were adopted for the detection and identification of L. acidophilus. RESULTS: The isolation frequency decreased rapidly 72 h after stopping intake of yoghurt. After 1 week, L. acidophilus was absent in all oral samples. Non-significant differences were found between the survival rates of L. acidophilus in samples of saliva, plaque, tongue surface, and buccal mucosa. L. acidophilus was also found to remain in the gastrointestinal tract for longer than in the oral cavity. CONCLUSION: Allochthonous L. acidophilus is not likely to permanently colonize the oral cavity and intestine.

Potential bioaccessibility of phenolic acids in whole wheat products during in vitro gastrointestinal digestion and probiotic fermentation.

Health benefits of whole wheat products are partially attributed by their unique phenolic compounds. This study investigated effect of simulated gastrointestinal digestion and probiotic fermentation on releasing of phenolic acids from whole wheat foods (bread, cookie, and pasta). Kinetics results showed that more phenolic acids were released within the first hour of gastric and intestinal digestions compared to the prolonged digestion. Lactobacillus rhamnosus GG, a common probiotic strain, released additional phenolic acids from the digestive residues during fermentation. Simulated digestion released more soluble trans-ferulic acid than chemical extraction in breads (17.69 to 102.71 µg/g), cookie (15.81 to 54.43 µg/g), and pasta (4.88 to 28.39 µg/g). Phenolic acid composition of whole wheat products appeared to be better estimated by digestion methods than the chemical extraction method. The unique insoluble-bound nature and fermentability of wheat phenolic acids may lead to a mechanistic understanding of whole grain consumption for potential colorectal cancer prevention.

Kinetics of Intestinal Presence of Spores Following Oral Administration of Bacillus clausii Formulations: Three Single-Centre, Crossover, Randomised, Open-Label Studies.

BACKGROUND AND OBJECTIVE: Probiotics are live microorganisms that may provide benefits including the prevention of gastrointestinal disorders and other diseases. Enterogermina is a probiotic mix of spores from four strains of Bacillus clausii (O/C, T, N/R and SIN), available in several oral formulations. The objective of this analysis was to evaluate and compare the kinetic profiles of different formulations of Enterogermina-vial [E4 once daily (OD) and E2 twice daily (BID)], capsule [EC2 three times daily (TID)], oral powder for suspension (ES6 OD) and oral powder not requiring suspension (E6 OD) from two studies from 2012 (EUDRACT 2010-024497-19 and 2010-023187-41) and one study from 2016 (EUDRACT 2015-003330-27). METHODS: B. clausii spores were counted in homogenised faecal samples (results expressed as counts per gram) or after culture at 37 °C for 24-36 h (results expressed as colony-forming units). Kinetics were assessed by area under the concentration-time curve (AUC), maximum concentration (Cmax), time to maximum concentration (Tmax) and spore presence/persistence. RESULTS: In total, 22 subjects in each of the 2012 studies and 30 subjects in the 2016 study were randomised (mean age 25.0-33.8 years across studies). The mean (±SD) absolute faecal spore counts (in millions) expressed as AUC per hour were 270.7 ± 147.7 (E2 BID) and 213.8 ± 60.2 (E4 OD) in 2012 EGKINETIC4, 312.7 ± 218.0 (EC2 TID) and 319.0 ± 221.1 (ES6 OD) in 2012 EGKINETIC6, and 212.6 ± 118.0 (E6 OD) and 293.2 ± 247.2 (ES6 OD) in 2016 EGKINETIC6OP. The kinetic profiles of the different formulations of Enterogermina were similar, with superimposable AUC and daily curve profiles in each study up to the 8th day post dose. B. clausii spore presence/persistence in the intestine of healthy volunteers did not differ between the two formulations within each of the three studies. Enterogermina was well tolerated across all formulations and studies. CONCLUSION: These results show different formulations of Enterogermina had similar kinetic profiles within each study; however, they also showed that probiotics could be associated with high variability. The European Medicines Agency guidelines are the current bioequivalence reference, although only the Tmax parameter is used for high variability drugs. Due to the specific kinetics of probiotics, new parameters of bioequivalence could be necessary, considering, for example, variability via a parameter such as AUC. TRIAL REGISTRATION: EUDRACT 2010-024497-19, 2010-023187-41 and 2015-003330-27.

A quantitative model for metabolic intervention using gut microbes.

As medicine shifts toward precision-based and personalized therapeutics, utilizing more complex biomolecules to treat increasingly difficult and rare conditions, microorganisms provide an avenue for realizing the production and processing necessary for novel drug pipelines. More so, probiotic microbes can be co-opted to deliver therapeutics by oral administration as living drugs, able to survive and safely transit the digestive tract. As living therapeutics are in their nascency, traditional pharmacokinetic-pharmacodynamic (PK-PD) models for evaluating drug candidates are not appropriate for this novel platform. Using a living therapeutic in late-stage clinical development for phenylketonuria (PKU) as a case study, we adapt traditional oral drug delivery models to properly evaluate and inform the engineering of living therapeutics. We develop the adapted for living therapeutics compartmental absorption and transit (ALT-CAT) model to provide metrics for drug efficacy across nine age groups of PKU patients and evaluate model parameters that are influenced by patient physiology, microbe selection and therapeutic production, and dosing formulations. In particular, the ALT-CAT model describes the mathematical framework to model the behavior of orally delivered engineered bacteria that act as living therapeutics by adapting similar methods that have been developed and widely-used for small molecular drug delivery and absorption.

The efficacy and toxicity of antineoplastic antimetabolites: Role of gut microbiota.

The incidence and mortality of cancer are rapidly growing all over the world. Nowadays, antineoplastic antimetabolites still play a key role in the chemotherapy of cancer. However, the interindividual variations in the efficacy and toxicity of antineoplastic antimetabolites are nonnegligible challenges to their clinical applications. Although many studies have focused on genetic variation, the reasons for these interindividual variations have still not been fully understood. Gut microbiota is reported to be associated with the efficacy and toxicity of antineoplastic antimetabolites. In this review, we summarize the interaction of antineoplastic antimetabolites on gut microbiota and the influences of shifted gut microbiota profiles on the efficacy and toxicity of antineoplastic antimetabolites. The factors affecting the efficacy and toxicity of antineoplastic antimetabolites via gut microbiota are also discussed. In addition, we present our viewpoints that regulating the gut microbiota may increase the efficacy and decrease the toxicity of antineoplastic antimetabolites. This will help us better understand the new mechanism via gut microbiota and promote individualized use of antineoplastic antimetabolites.

Lactobacillus rhamnosus induces CYP3A and changes the pharmacokinetics of verapamil in rats.

Verapamil, a calcium channel blocker, has been approved as the first-line drug for treatment of angina pectoris, hypertension and supraventricular tachycardia. Lactobacillus rhamnosus, one of the normal strains in human intestinal tract, is very popular in the probiotic market for conferring a health benefit on the host. This report investigated the potential of gut microbiota-drug interactions between lactobacillus rhamnosus and verapamil via using wild type (WT) and Cyp3a1/2 knockout (KO) rats. In WT rats, administration of Lactobacillus rhamnosus for 14 days decreased systemic exposure of verapamil and increased its metabolite norverapamil in vivo, and resulted in gut microbiota-drug interactions. In Cyp3a1/2 KO rats, however, this interaction disappeared. Further studies found that Lactobacillus rhamnosus induced CYP3A activity and expression, and changed the composition of gut microbiota, thus changing the pharmacokinetics of verapamil. These results demonstrated the interaction between lactobacillus rhamnosus and verapamil, and indicated that the effect of gut microbiota on metabolic enzymes cannot be ignored.

Exogenous enzymes and probiotics alter digestion kinetics, volatile fatty acid content and microbial interactions in the gut of Nile tilapia.

Sustainable aquafeed production requires fishmeal replacement, leading to an increasing use of plant-derived ingredients. As a consequence, higher levels of antinutritional substances, such as non-starch polysaccharides and phytate, are present in aquafeeds, with negative effects on fish performance, nutrient digestibility and overall gut health. To alleviate these negative effects, providing exogenous digestive enzymes and/or probiotics can be an effective solution. In this study, we tested the effect of dietary supplementation of enzymes (phytase and xylanase) and probiotics (three strains of Bacillus amyloliquefaciens) on nutrient digestion kinetics and volatile fatty acid content along the gut, and the distal gut microbiome diversity in Nile tilapia. Chyme volatile fatty content was increased with probiotic supplementation in the proximal gut, while lactate content, measured for the first time in vivo in fish, decreased with enzymes along the gut. Enzyme supplementation enhanced crude protein, Ca and P digestibility in proximal and middle gut. Enzymes and probiotics supplementation enhanced microbial interactions as shown by network analysis, while increased the abundance of lactic acid bacteria and Bacillus species. Such results suggest that supplementation with exogenous enzymes and probiotics increases nutrient availability, while at the same time benefits gut health and contributes to a more stable microbiome environment.

Characterization of the properties of Pediococcus parvulus for probiotic or protective culture use.

Pediococcus parvulus 2.6 (previously Pediococcus damnosus 2.6, here confirmed as P. parvulus by 16S DNA sequencing) displayed antibacterial activity toward several bacterial species, including isolates found as contaminants in oats, herein genetically identified as Bacillus cereus. No inhibition of Listeria monocytogenes was found under the conditions used. Antibacterial activity was retrieved after ammonium sulfate or acetone precipitation showed it to be peptide mediated. P. parvulus 2.6 has previously shown good technological properties in oat-based products. This, together with the currently found inhibition of food spoilage microorganisms like B. cereus, makes it suitable as a food protective culture. Survival trials of P. parvulus 2.6 at conditions mimicking the gastrointestinal tract were prompted by previously found cholesterol-lowering effects in humans after consumption of oat products cofermented by using P. parvulus 2.6 and Bifidobacterium spp. Viability was measured with in vitro, gutlike simulations at 37 degrees C. High survival was shown under two of three conditions (gastric juice, bile, and small intestine juice), defined as main obstacles of the gastrointestinal tract. The critical step was bile exposure. At a concentration of 20%, viability was low, but 0.3% bile (mean concentration in the intestine) did not have a major influence on growth. Viability of P. parvulus 2.6 was significantly decreased in gastric juice at pH 1.5 (with pepsin), but it was not significantly affected at pH 2.5, and was also improved at a lower pH in 20% oat milk. Viability was judged sufficient for colonization at gutlike conditions, qualifying the strain for further probiotic studies.

A novel route for double-layered encapsulation of probiotics with improved viability under adverse conditions.

To improve the survivability of probiotics under the harsh conditions, a novel double-layered vehicle, which was developed by a one-step coaxial electrospinning procedure, was here used to encapsulate the probiotics. The morphology characterization analysis revealed that the electrospun fiber had a beaded morphology and core-shell structure. Probiotic cells were successfully encapsulated in the fibers (107 CFU/mg) and exhibited an oriented distribution along the fiber. Additionally, the encapsulation of core-shell fiber mat enhanced the tolerance of probiotic cells to simulated gastrointestinal conditions and no significant loss of viability was found (p > 0.05). Besides that, the encapsulated cells exhibited better thermal stability under heat moisture treatment, lower loss of viability (0.32 log CFU/mL) was occurred when compared with the free cells or encapsulated cells in uniaxial fiber mat. In conclusion, this double-layered vehicle presents a great potential in probiotic encapsulation and improving their resistant ability to the harsh conditions.

Enhanced viability of layer-by-layer encapsulated Lactobacillus pentosus using chitosan and sodium phytate.

Lactobacillus pentosus (LP) are widely used as probiotics in food products, dietary supplements, and nutraceuticals due to their health-promoting effects. To confer a functional effect, the probiotics need to survive during shelf-life and transit through the high acidic conditions of the stomach and bile salts in the small intestine. Herein, LP was firstly encapsulated in a layer-by-layer approach using chitosan (CS) and sodium phytate (SP). After digestion in simulated gastrointestinal fluid (SGF) for 120 min and 4% bile salts for 3 h, plain-LP exhibited a 7.40 and 6.09 colony forming units/ml (cfu/ml) reduction. Interestingly, two layer coated LP ((CS/SP)2-LP) exhibited less death, which reduced 4.34 and 2.33 log cfu/ml, respectively. Specially, (CS/SP)2-LP also showed a higher survival rate compared to plain-LP in heat treatment experiments, especially 65 °C. In conclusion, layer-by-layer encapsulation of LP has great potential for the protection and delivery of probiotics in food and nutraceutical products.

Oral Delivery of Probiotics Using pH-Sensitive Phthalyl Inulin Tablets.

Probiotics show low cell viability after oral administration because they have difficulty surviving in the stomach due to low pH and enzymes. For the oral delivery of probiotics, developing a formula that protects the probiotic bacteria from gastric acidity while providing living cells is mandatory. In this study, we developed tablets using a new pH-sensitive phthalyl inulin (PI) to protect probiotics from gastric conditions and investigated the effects of different compression forces on cell survival. We made three different tablets under different compression forces and measured survivability, disintegration time, and kinetics in simulated gastric-intestinal fluid. During tableting, there were no significant differences in probiotic viability among the different compression forces although disintegration time was affected by the compression force. A higher compression force resulted in higher viability in simulated gastric fluid. The swelling degree of the PI tablets in simulated intestinal fluid was higher than that of the tablets in simulated gastric fluid due to the pH sensitivity of the PI. The probiotic viability formulated in the tablets was also higher in acidic gastric conditions than that for probiotics in solution. Rapid release of the probiotics from the tablet occurred in the simulated intestinal fluid due to the pH sensitivity. After 6 months of refrigeration, the viability of the PI probiotics was kept. Overall, this is the first study to show the pH-sensitive properties of PI and one that may be useful for oral delivery of the probiotics.

The Epithelial Barrier Model Shows That the Properties of VSL#3 Depend from Where it is Manufactured.

BACKGROUND: VSL#3 has been extensively investigated and is currently recommended for the prevention and treatment of chronic pouchitis and ulcerative colitis. Nonetheless, in vitro and in vivo studies have recently shown variability in the VSL#3 efficacy often attributed to the manufacturing process. OBJECTIVE: The aim was to comparatively study the in vitro effects of two VSL#3 preparations produced in different sites (named US- and Italy-made VSL#3) on CaCo-2 epithelial barrier model in terms of trans-epithelial electrical resistance (TEER), dextran flux and expression of Tight Junctions (TJ) proteins i.e. zonulin-1 (ZO-1) and occludin, in the absence or presence of a heat stress-related damage of monolayer. METHODS: TEER was evaluated on CaCo-2 differentiated monolayers. Epithelial permeability of polarized monolayers was assessed by measuring the FITC-labeled dextran flux from the apical to basolateral chambers. ZO-1/occludin levels were analyzed by western blot analysis. A set of experiments was performed to compare the effects of both VSL#3 on TEER values, dextran flux and ZO-1/occludin expression in CaCo-2 monolayers after heat stress exposure. RESULTS: US- and Italy-made VSL#3 have opposing effects on TEER values, dextran flux, and ZO- 1/occludin expression, being all these parameters negatively influenced just by Italy-made product. US-made probiotic did not affect baseline TEER, dextran flux and ZO-1 expression and strongly increased occludin levels. Of note, pre-treatment of monolayer with US-made VSL#3, but not Italy-made product, totally prevented the heat-induced epithelial barrier integrity loss. CONCLUSION: Our data trigger the need for reassessing efficacy or safety of the Italy-made VSL#3 considering intestinal epithelial barrier plays an important role in maintaining host health.