The industrial application of postbiotics based on non-viable probiotic cells (paraprobiotics), requires robust inactivation technologies that ensure replicative death while preserving bioactive bacterial structures and functions. Here, we evaluated cobalt-60 gamma (γ) irradiation as an industrially scalable approach to produce functional postbiotics from freeze-dried biomasses of twelve probiotic strains packaged in sealed multilayer pouches. Viable counts of untreated biomasses ranged from 10.8 to 11.3 log10 CFU g-1. γ irradiation at 10, 15, or 20 kGy resulted in complete loss of culturability for all strains (below the detection limit), indicating that 10 kGy was sufficient for full inactivation under the tested conditions. Membrane integrity, assessed by flow cytometry according to ISO 19344:2015, was largely preserved immediately after irradiation and remained stable during storage at 4 °C up to 18 months, with strain-dependent but generally moderate declines in the active population. Intracellular functionality was probed via β-galactosidase activity, which remained detectable in irradiated biomasses and did not differ significantly from matched untreated controls. Moreover, the adhesion capacity of selected adhesive strains to differentiated Caco-2 monolayers was retained after γ irradiation. Using Bifidobacterium bifidum MIMBb23sg as a model, γ irradiation outperformed industrial pasteurization by preserving membrane integrity, adhesion, and β-galactosidase activity; both inactivation methods maintained comparable antioxidant responses in a Caco-2 cellular antioxidant activity assay. Overall, γ irradiation enables the production of non-culturable yet structurally and functionally competent bacterial biomasses, supporting its potential for next-generation postbiotic ingredients.

The commercialization of a probiotic microorganism requires genetic and phenotypic consistency across production lots. However, the large-scale industrial production of probiotic microbial biomasses over years poses a risk of genetic drift, potentially affecting the probiotic's ability to confer health benefits. This study assessed the stability of Lacticaseibacillus paracasei DG (DSM 34154), a commercial probiotic. Seven isolates from different commercial lots over 10 years, one 8-year-old laboratory subculture, one isolate from human feces, and the DG strain deposited at DSMZ were analyzed. Shotgun and PacBio sequencing were combined to obtain the complete genome of the 10 isolates. Comparative analysis showed that the seven commercial isolates had identical genomes, differing from the DSMZ isolate by one synonymous transition and one non-synonymous transversion. The laboratory subculture strain had two additional mutations. Phenotypic analyses, including antibiotic resistance, carbohydrate fermentation profile, survival to simulated gastrointestinal transit, immunomodulatory capacity, and radical scavenging ability, found no significant differences among isolates. Overall, this study demonstrates the substantial equivalence of L. paracasei DG over 10 years of industrial production, indicating that the current industrial practices help prevent genome alterations that could compromise probiotic performance. Similar studies should be part of continuous monitoring and quality control measures for the probiotic products on the market.IMPORTANCEThe genetic and functional stability of probiotic strains during years of industrial production is essential but has not been clearly demonstrated for many strains. This study shows that careful industrial practices can maintain the genetic integrity and functionality of probiotics. Using advanced genome sequencing and detailed laboratory tests, we confirmed that the probiotic Lacticaseibacillus paracasei DG (DSM 34154) has remained stable over a decade of production, consistently delivering its health-promoting properties. These findings support the quality and reliability of probiotic products, fostering consumer trust and highlighting the importance of continuous monitoring in probiotic manufacturing to sustain quality assurance.

Probiotics are exploited for adjuvant treatment in IBS, but reliable guidance for selecting the appropriate probiotic to adopt for different forms of IBS is lacking. We aimed to identify markers for recognizing non-constipated (NC) IBS patients that may show significant clinical improvements upon treatment with the probiotic strain Lacticaseibacillus paracasei DG (LDG). To this purpose, we performed a post-hoc analysis of samples collected during a multicenter, double-blind, parallel-group, placebo-controlled trial in which NC-IBS patients were randomized to receive at least 24 billion CFU LDG or placebo capsules b.i.d. for 12 weeks. The primary clinical endpoint was the composite response based on improved abdominal pain and fecal type. The fecal microbiome and serum markers of intestinal (PV1 and zonulin), liver, and kidney functions were investigated. We found that responders (R) in the probiotic arm (25%) differed from non-responders (NR) based on the abundance of 18 bacterial taxa, including the families Coriobacteriaceae, Dorea spp. and Collinsella aerofaciens, which were overrepresented in R patients. These taxa also distinguished R (but not NR) patients from healthy controls. Probiotic intervention significantly reduced the abundance of these bacteria in R, but not in NR. Analogous results emerged for C. aerofaciens from the analysis of data from a previous trial on IBS with the same probiotic. Finally, C. aerofaciens was positively correlated with the plasmalemmal vesicle associated protein-1 (PV-1) and the markers of liver function. In conclusion, LDG is effective on NC-IBS patients with NC-IBS with a greater abundance of potential pathobionts. Among these, C. aerofaciens has emerged as a potential predictor of probiotic efficacy.

Hyaluronic acid (HA) is a key component of the respiratory mucosa. By acting as a natural moisturizer, it provides hydration to the airways. In normal conditions, high molecular weight HA molecules form viscous gels providing a protective shield against external insults. This is particularly important in the upper airways where the HA protective barrier helps to prevent environmental agents to reach the lungs. Most respiratory diseases are characterized by inflammatory processes causing degradation of HA into small fragments which reduces the HA barrier effect and increases the risk of exposure to external insults. Dry powder inhalers (DPIs) are efficient devices used to deliver therapeutic molecules in the form of dry powder to the respiratory tract. PolmonYDEFENCE/DYFESA™ is a novel formulation based on HA delivered to the airways using the PillHaler® DPI device. In this study we report the results of in vitro inhalation performances of PolmonYDEFENCE/DYFESA™ as well as its mechanism of action in human cells. We found that the product targets the upper airways and that HA molecules form a protective barrier on cell surface. Furthermore, exposure to the device is safe in animal models. The promising pre-clinical results of this study provide the bases for future clinical investigation.

Diverticular disease (DD) is a common gastrointestinal condition. Patients with DD experience a huge variety of chronic nonspecific symptoms, including abdominal pain, bloating, and altered bowel habits. They are also at risk of complications such as acute diverticulitis, abscess formation, hemorrhage, and perforation. Intestinal dysbiosis and chronic inflammation have recently been recognized as potential key factors contributing to disease progression. Probiotics, due to their ability to modify colonic microbiota balance and to their immunomodulatory effects, could present a promising treatment option for patients with DD. Lactobacillus paracasei CNCM I 1572 (LCDG) is a probiotic strain with the capacity to rebalance gut microbiota and to decrease intestinal inflammation. This review summarizes the available clinical data on the use of LCDG in subjects with colonic DD.

The immunomodulatory potential of probiotics and (poly)phenols (PP) is recognized; however, studies regarding microorganisms-PP synergisms are yet to be explored. Here, we investigated the cooperation between probiotics and berry-derived PP extracts in modulating the cytokine responses in dendritic cells. Bacteria elicited immune responses in a strain-dependent manner. PP extracts showed different modulation of cytokine triggered by bacteria. Also with LPS, used as pro-inflammatory stimulus, PP from blueberry (BB) and cranberry (CB) most efficiently reduced IL12 production. L. paracasei LPC-S01 and B. bifidum MIMBb23sg resulted the best bacterial association in abrogating IL12 and increasing IL10. The use of PP fraction from BB50f and CB1 with the LPC-S01 + MIMBb23sg association resulted the most efficient combinations in terms of anti-inflammatory activity. These results provide bases for further investigation in vivo, in the perspective to develop food supplements that might conceivably deliver the single and combined benefits of probiotics and berry (poly)phenols.

Burning mouth syndrome (BMS) is defined as “idiopathic orofacial pain with intraoral burning or dysesthesia recurring daily for more than 2 hours per day and more than 3 months, without any identifiable causative lesions, with or without somatosensory changes” in International Classification of Orofacial Pain, 2020. Worldwide prevalence of BMS was estimated to be 1.73% in population-based studies, while female and elderly are at higher risk of BMS. The aim of this narrative review is to clarify the main etiopathogenetic factors of BMS investigated so far in the scientific literature. There is growing evidence of an important role of peripheral neuropathology in BMS, supported by immunohistochemical studies which have demonstrated a significant loss of epithelial and subepithelial nerve fibers. Other possible etiopathogenetic factors emerging from literature are laryngopharyngeal reflux and hormonal and salivary changes related to aging and menopause. Finally, the role of the oral microbiota in BMS has not yet been thoroughly investigated. Further studies are necessary to investigate the probably multifactorial etiopathogenesis of primary BMS, a pathology which has a serious impact on the quality of life of our patients, a disease we find ourselves treating without the adequate therapy and the necessary knowledge.

Intense physical activity is often associated with undesirable physiological changes, including increased inflammation, transient immunodepression, increased susceptibility to infections, altered intestinal barrier integrity, and increased oxidative stress. Several trials suggested that probiotics supplementation may have beneficial effects on sport-associated gastro-intestinal and immune disorders. Recently, in a placebo-controlled human trial, the AminoAlta™ probiotic formulation (AApf) was demonstrated to increase the absorption of amino acids from pea protein, suggesting that the administration of AApf could overcome the compositional limitations of plant proteins. In this study, human cell line models were used to assess in vitro the potential capacity of AApf to protect from the physiological damages that an intense physical activity may cause. The obtained results revealed that the bacteria in the AApf have the ability to adhere to differentiated Caco-2 epithelial cell layer. In addition, the AApf was shown to reduce the activation of NF-κB in Caco-2 cells under inflammatory stimulation. Notably, this anti-inflammatory activity was enhanced in the presence of partially hydrolyzed plant proteins. The AApf also triggered the expression of cytokines by the THP-1 macrophage model in a dose-dependent manner. In particular, the expression of cytokines IL-1β, IL-6, and TNF-α was higher than that of the regulatory cytokine IL-10, resembling a cytokine profile characteristic of M1 phenotype, which typically intervene in counteracting bacterial and viral infections. Finally, AApf was shown to reduce transepithelial permeability and increase superoxide dismutase activity in the Caco-2 cell model. In conclusion, this study suggests that the AApf may potentially provide a spectrum of benefits useful to dampen the gastro-intestinal and immune detrimental consequences of an intense physical activity.

BACKGROUND AND AIM:An association between reflux and burning mouth syndrome (BMS) has been proposed. Aims of this study were: 1) to investigate the frequency of BMS in a sample of GERD patients; 2) to measure G17, in a sample of BMS patients; 3) to assess the efficacy of different therapeutical schedules for GERD in BMS patients.METHODS:We divided the study in 3 main steps. In step one, we analyzed 500 consecutive GERD patients' type and frequency of extraesophageal manifestations including BMS. In step two, we collected 124 consecutive BMS patients' symptoms and  G17.  In step three, we evaluate the efficacy of 3 different drugs on BMS.RESULTS:In step one, 204 patients complained heartburn; 31 globus pharyngeus; 52 chronic cough; 54 pharyngitis; 31 postnasal drip; 56 burning mouth symptoms; 34 noncardiac chest pain; 17 asthma and 21 sleep apnea. In step two, 29 patients had G17 ≤ 1 pg/L; 64 patients between 1 and 3; and 31 patients ≥ 3. In step three, 49 patients reported slight benefit with PPI, 75 no benefit. 61 patients reported slight benefit with sodium alginate and sodium bicarbonate, 63 no benefit. 23 reported an almost complete remission with HYCHSA, 26 slight benefit, 33 no benefit.CONCLUSIONS:Prevalence of BMS in GERD patients was similar to that reported for chronic chough and pharyngitis. Low levels of G17 were found in the majority of BMS patients. Finally, we observed a greater benefit from barrier drugs therapy than from PPI therapy in BMS patients. (www.actabiomedica.it).

The exposure of lung epithelium to environmental hazards is linked to several chronic respiratory diseases. We assessed the ability of an inhaled dry powder (DPI) medical device product (PolmonYDEFENCE/DYFESATM, SOFAR SpA, Trezzano Rosa, Italy), using a formulation of sodium hyaluronate (Na-Hya) as the key ingredient as a defensive barrier to protect the upper respiratory tract. Specifically, it was evaluated if the presence of the barrier formed by sodium hyaluronate present on the cells, reducing direct contact of the urban dust (UD) with the surface of cells can protect them in an indirect manner by the inflammatory and oxidative process started in the presence of the UD. Cytotoxicity and the protection capability against the oxidative stress of the product were tested in vitro using Calu-3 cells exposure to UD as a trigger for oxidative stress. Inflammation and wound healing were assessed using an air-liquid interface (ALI) culture model of the Calu-3 cells. Deposition studies of the formulation were conducted using a modified Anderson cascade impactor (ACI) and the monodose PillHaler® dry powder inhaler (DPI) device, Na-Hya was detected and quantified using high-performance-liquid-chromatography (HPLC). Solubilised PolmonYDEFENCE/DYFESATM gives protection against oxidative stress in Calu-3 cells in the short term (2 h) without any cytotoxic effects. ALI culture experiments, testing the barrier-forming (non-solubilised) capabilities of PolmonYDEFENCE/DYFESATM, showed that the barrier layer reduced inflammation triggered by UD and the time for wound closure compared to Na-Hya alone. Deposition experiments using the ACI and the PillHaler® DPI device showed that the majority of the product was deposited in the upper part of the respiratory tract. Finally, the protective effect of the product was efficacious for up to 24 h without affecting mucus production. We demonstrated the potential of PolmonYDEFENCE/DYFESATM as a preventative barrier against UD, which may aid in protecting the upper respiratory tract against environmental hazards and help with chronic respiratory diseases.

Probiotic microorganisms may benefit the host by influencing diverse physiological processes, whose nature and underlying mechanisms are still largely unexplored. Animal models are a unique tool to understand the complexity of the interactions between probiotic microorganisms, the intestinal microbiota, and the host. In this regard, in this pilot study, we compared the effects of 5-day administration of three different probiotic bacterial strains (Bifidobacterium bifidum MIMBb23sg, Lactobacillus helveticus MIMLh5, and Lacticaseibacillus paracasei DG) on three distinct murine intestinal sites (ileum, cecum, and colon). All probiotics preferentially colonized the cecum and colon. In addition, probiotics reduced in the ileum and increased in the cecum and colon the relative abundance of numerous bacterial taxonomic units. MIMBb23sg and DG increased the inducible nitric oxide synthase (iNOS) in the ileum, which is involved in epithelial homeostasis. In addition, MIMBb23sg upregulated cytokine IL-10 in the ileum and downregulated the cyclooxygenase COX-2 in the colon, suggesting an anti-inflammatory/regulatory activity. MIMBb23sg significantly affected the expression of the main gene involved in serotonin synthesis (TPH1) and the gene coding for the serotonin reuptake protein (SERT) in the ileum and colon, suggesting a potential propulsive effect toward the distal part of the gut, whereas the impact of MIMLh5 and DG on serotonergic genes suggested an effect toward motility control. The three probiotics decreased the expression of the permeability marker zonulin in gut distal sites. This preliminary in vivo study demonstrated the safety of the tested probiotic strains and their common ability to modulate the intestinal microbiota. The probiotics affected host gene expression in a strain-specific manner. Notably, the observed effects in the gut were site dependent. This study provides a rationale for investigating the effects of probiotics on the serotonergic system, which is a topic still widely unexplored.

PURPOSE:Subclinical vitamin D (vitD) deficiency enhances the predisposition to a myriad of acute and chronic pathologies in many people worldwide. Due to the scarcity of vitD-rich foods, the consumption of supplements or fortified foods can be required to maintain healthy serum levels of 25-hydroxyvitamin D [25(OH)D], and the major circulating form of vitD that is commonly measured in serum to determine the vitD status. Since the vitD absorption seems to resemble that of lipids, improved emulsification in the gut could favor vitD permeation through the enterocyte membrane. Contextually, we hypothesized that a microorganism with cholecalciferol (vitD3)-solubilization properties may potentially result in enhanced serum vitD levels.METHODS AND RESULTS:Six probiotic strains were screened for their ability to create a stable suspension of vitD3 in water: Lacticaseibacillus paracasei DG, L. paracasei LPC-S01, L. paracasei Shirota, L. rhamnosus GG, Limosilactobacillus reuteri DSM 17938, and Lactobacillus acidophilus LA5. The DG strain displayed the strongest vitD3 solubilization ability and, consequently, were used in an in vivo trial where a commercial preparation of vitD3 in refined olive oil was administered by gavage to CD-1 mice with or without the concurrent administration of L. paracasei DG. ELISA measurements showed that the DG strain significantly increased the serum levels of 25(OH) D when administered once a day for 1 week in association with the vitD3 supplement.CONCLUSION:This preliminary pre-clinical study suggests that the combined administration of L. paracasei DG with an oil-based cholecalciferol supplement could contribute to the maintenance of the adequate 25(OH) D serum levels in people at risk of vitD deficiency.

Producers of probiotic products are legally required to indicate on the label only the minimum numbers of viable microorganisms at the end of shelf life expressed as colony-forming units (CFUs). Label specifications, however, describe only a fraction of the actual microbiological content of a probiotic formulation. This paper describes the microbiological components of a probiotic product that are not mentioned on the label, such as the actual number of CFUs, the presence of viable cells that cannot generate colonies on agar plates, and the abundance of dead cells. These “hidden” microbial fractions in probiotic products, the abundance of which may change during the shelf life, can promote biological responses in the host. Therefore, they should not be ignored because they may influence the efficacy and can be relevant for immunocompromised or fragile consumers. In conclusion, we propose the minimum requirements for microbiological characterization of probiotic products to be adopted for label specifications and clinical studies.

ABSTRACT Oral consumption of probiotics is practical and can be an effective solution to preserve vaginal eubiosis. Here, we studied the ability of orally administered Lactobacillus paracasei LPC-S01 (DSM 26760) to affect the composition of the vaginal microbiota and colonize the vaginal mucosa in nondiseased adult women. A total of 40 volunteers took oral probiotic (24 billion CFU) or placebo capsules daily for 4 weeks, and after a 4-week washout, they switched to placebo or probiotic capsules according to the crossover design. A total of 23 volunteers completed the study according to the protocol. Before and after capsule ingestion, vaginal swabs were collected for qPCR quantification to detect L. paracasei LPC-S01 and for 16S rRNA gene sequencing. Vaginal swabs were grouped according to their bacterial taxonomic structure into nine community state types (CSTs), four of which were dominated by lactobacilli. Lactobacillus paracasei LPC-S01 was detected in the vagina of two participants. Statistical modeling (including linear mixed-effects model analysis) demonstrated that daily intake of probiotic capsules reduced the relative abundance of Gardnerella spp. Quantitative PCR with Gardnerella vaginalis primers confirmed this result. Considering the pathogenic nature of G. vaginalis, these results suggest a potential positive effect of this probiotic capsule on the vaginal microbial ecosystem.

The fate of dietary protein in the gut is determined by microbial and host digestion and utilization. Fermentation of proteins generates bioactive molecules that have wide-ranging health effects on the host. The type of protein can affect amino acid absorption, with animal proteins generally being more efficiently absorbed compared with plant proteins. In contrast to animal proteins, most plant proteins, such as pea protein, are incomplete proteins. Pea protein is low in methionine and contains lower amounts of branched-chain amino acids (BCAAs), which play a crucial role in muscle health. We hypothesized that probiotic supplementation results in favorable changes in the gut microbiota, aiding the absorption of amino acids from plant proteins by the host. Fifteen physically active men (24.2 ± 5.0 years; 85.3 ± 12.9 kg; 178.0 ± 7.6 cm; 16.7 ± 5.8% body fat) co-ingested 20 g of pea protein with either AminoAlta™, a multi-strain probiotic (5 billion CFU L. paracasei LP-DG® (CNCM I-1572) plus 5 billion CFU L. paracasei LPC-S01 (DSM 26760), SOFAR S.p.A., Italy) or a placebo for 2 weeks in a randomized, double-blind, crossover design, separated by a 4-week washout period. Blood samples were taken at baseline and at 30-, 60-, 120-, and 180-min post-ingestion and analyzed for amino acid content. Probiotic administration significantly increased methionine, histidine, valine, leucine, isoleucine, tyrosine, total BCAA, and total EAA maximum concentrations (Cmax) and AUC without significantly changing the time to reach maximum concentrations. Probiotic supplementation can be an important nutritional strategy to improve post-prandial changes in blood amino acids and to overcome compositional shortcomings of plant proteins. ClinicalTrials.gov Identifier: ISRCTN38903788

Ability to survive the digestive process is a major factor in determining the effectiveness of a probiotic. In this study, the ability of the probiotic L. casei DG® (Lactobacillus paracasei CNCMI1572) to survive gastrointestinal transit in healthy children was investigated for the first time.

Irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, is classified according to bowel habits as IBS with constipation (IBS-C), with diarrhea (IBS-D), with alternating constipation and diarrhea (IBS-M), and unsubtyped (IBS-U). The mechanisms leading to the different IBS forms are mostly unknown. This study aims to evaluate whether specific fecal bacterial taxa and/or short-chain fatty acids (SCFAs) can be used to distinguish IBS subtypes and are relevant for explaining the clinical differences between IBS subcategories. We characterized five fecal samples collected at 4-weeks intervals from 40 IBS patients by 16S rRNA gene profiling and SCFA quantification. Finally, we investigated the potential correlations in IBS subtypes between the fecal microbial signatures and host physiological and clinical parameters. We found significant differences in the distribution of Clostridiales OTUs among IBS subtypes and reduced levels of SCFAs in IBS-C compared to IBS-U and IBS-D patients. Correlation analyses showed that the diverse representation of Clostridiales OTUs between IBS subtypes was associated with altered levels of SCFAs; furthermore, the same OTUs and SCFAs were associated with the fecal cytokine levels and stool consistency. Our results suggest that intestinal Clostridiales and SCFAs might serve as potential mechanistic biomarkers of IBS subtypes and represent therapeutic targets.

Probiotics are live microorganisms, and viability after transit through the gastrointestinal tract (GIT) is considered an inherent property of the health benefits of probiotics. The aim of the present study was to quantify the viable and total loads of Lactobacillus paracasei DG cells after passage through the GIT following the consumption of the probiotic product Enterolactis (L. casei DG®; L. paracasei CNCM I-1572; L. paracasei DG) from drinkable vials by healthy adults. We developed a novel method for discriminating and enumerating culturable L. paracasei DG cells based on the unique sticky, filamentous phenotype of this strain on MRS agar containing vancomycin and kanamycin. The identity of DG was also confirmed with strain-specific primers by colony PCR. This method was used for a recovery study of the DG strain to quantify viable cells in the fecal samples of 20 volunteers during a 1-week probiotic consumption period and a 1-week follow-up. We isolated L. paracasei DG from at least one fecal sample from all the volunteers. The highest concentration of viable DG cells [ranging from 3.6 to 6.7 log10 colony-forming unit (CFU) per gram of feces] in the feces was observed between 4 and 8 days from the beginning of Enterolactis intake and for up to 5 days after cessation of intake. As expected, the total DG count determined by real-time quantitative PCR (qPCR) was mostly higher than the viable DG cells recovered. Viable count experiments, carried out by combining ad hoc culture-based discriminative conditions and strain-specific molecular biological protocols, unambiguously demonstrated that L. paracasei DG can survive gastrointestinal transit in healthy adults when ingested as Enterolactis in drinkable vials containing no less than one billion CFU at the end of shelf life.

at all.The objective of this study is to clarify the relevance among behavioral change, the gut microbiota and gut function in an animal model.Methods: seven-week-old male Wistar rats were used.On day 1, rats were placed in a shuttle box consisted of two compartments and given inescapable footshocks.On day 14, they were placed in the same box to perform the behavioral test.Movement of rats through the gate while adaptation was recorded as 'crossings during adaptation'.Then, rats were given an avoidance/escape test to record behavior such as 'avoidance', 'error' or 'crossings during the inter-trial interval'.We divided behavior patterns into three patterns; 'PTSD-like', 'LH' (learned helplessness: similar to the depression model) and 'indeterminate'.To evaluate visceral perception, colorectal distension (CRD) was performed on days 0 (before the inescapable footshocks), 28 and 42.Responses to CRD were assessed by measuring the threshold intensity that evokes a visually identifiable contraction of the abdominal wall.The results on day 0 were used as the baseline and the differences were compared across the groups.To evaluate changes in the gut microbiota, cecal contents were analyzed at the phylum level by using PCR of 16SrRNA gene.Expression of occludin mRNA levels in the intestines was determined by using qRT-PCR.Results: 14% of rats showed LH and 50% showed PTSD-like behavior pattern.Indeterminate group was 36%.Among these groups visceral hypersensitivity was only observed in the LH group.Interestingly, each group defined by characteristic psychological behavior showed different pattern in the analyses of the microbiota.The PTSD-like group increased the ratio of Bacteroidetes and the LH group increased the ratio of Proteobacteria compared with controls.In all groups, occludin expression in the intestines was significantly decreased compared with controls.Conclusion: We demonstrated that the same stress induced different types of psychological disorders to individual rats accompanied with their characteristic patterns of the gut microbiota and gut function in the animal model.These results might strongly suggest the close connection of microbiota-gut-brain axis stimulated by psychological stress.