Subchronic exposure to fit-for-purpose polystyrene nanoplastics impairs gut and liver health at low doses with non-monotonic effects in a diet-dependent manner.

ABSTRACT Intestinal paracellular permeability was analyzed ex vivo by incubation of tissue segments at 0°C with the fluorescent dyes FM1‐43FX (FM) or TRITC‐dextran 3 kDa lysine‐fixable (TD3L) and confocal microscopy in (i) healthy mice and (ii) mice submitted to chronic stress or lipid diets. In the small intestine of healthy mice, FM staining was restricted to the apical surface of enterocytes but fully penetrated around Goblet cells, enteroendocrine cells, tuft cells, and apoptotic cells. The same cell types were similarly labeled in the colon when located on the tissue surface but not within the crypts. TD3L exhibited a comparable labeling pattern but also showed moderate staining of the basolateral surface of enterocytes at the tips of small intestinal villi, and also substantial penetration around colonic epithelial cells at the surface or top of crypts. The study reveals patterns of permeability likely corresponding to the “leak” pathway of paracellular transport through the intestinal epithelium, because transcellular endocytosis is blocked at 0°C. This pathway is found around specific cell populations involved in the luminal detection of food, antigens, microbes, or their secretions. These trigger immune, neural, and tissue responses that maintain intestinal homeostasis. Chronic stress induced by glucocorticoid exposure increased FITC‐dextran 4 kDa permeability in vivo. Using FM, increased paracellular permeability was also detected ex vivo and selectively localized in the colon of stressed mice. A single oral administration of a lipid‐rich food also increased ex vivo permeability around jejunal enterocytes. Pathophysiological increases in paracellular permeability are therefore detectable using the FM methodology.

Irritable bowel syndrome (IBS) is a multifactorial disorder of the gut-brain axis, characterized by visceral hypersensitivity (VH). Psychobiotics, through GABA synthesis, are good candidates to alleviate gastrointestinal discomfort. Here, we analyzed the GABA-producer Lactococcus lactis CNCM I-5388 as an active-enzyme postbiotic to relieve VH mediated by psychological stress. L. lactis CNCM I-5388 was inactivated by ethanol while maintaining its glutamate decarboxylase (GAD) activity. This EtOH-treated nonviable form was given daily orally for 1, 5, or 10 days to female Wistar rats in comparison with viable L. lactis CNCM I-5388 or vehicle. Visceral sensitivity was measured by electromyography before and after partial restraint stress (PRS). GABA was quantified in the stomach collected from rats and in the gastric compartment of TIM-1 human gut model in fed state. A daily treatment for 5 and 10 days by L. lactis CNCM I-5388 both in its viable and nonviable forms counteracted VH promoted by PRS. However, only viable L. lactis CNCM I-5388 tended to reduce VH after a single administration. After 5-day treatment, only under PRS conditions, the production of GABA within the stomach was enhanced in rats treated with viable or nonviable L. lactis CNCM I-5388. This increase was confirmed by using the TIM-1 human gut model. We found that a postbiotic with an active-GAD enzyme of L. lactis CNCM I-5388, similarly to its viable psychobiotic form, exerts anti-VH properties in an IBS-like rat model. These effects are associated with GABA production in the stomach where the low pH promotes GAD activity.

To address the overuse of antimicrobials in poultry production, new functional feed ingredients, i.e. ingredients with benefits beyond meeting basic nutritional requirements, can play a crucial role thanks to their prophylactic effects. This study evaluated the effects of the supplementation of arginine, threonine and glutamine together with grape polyphenols on the gut integrity and functionality of broilers facing a stress condition. 108 straight-run newly hatched Ross PM3 chicks were kept until 35 days and were allocated to 3 treatments. Broilers in the control group were raised in standard conditions. In experimental groups, birds were administered with corticosterone in drinking water (CORT groups) to impair the global health of the animal and were fed a well-balanced diet supplemented or not with a mix of functional amino acids together with grape extracts (1 g/kg of diet—CORT + MIX group). Gut permeability was significantly increased by corticosterone in non-supplemented birds. This corticosterone-induced stress effect was alleviated in the CORT + MIX group. MIX supplementation attenuated the reduction of crypt depth induced by corticosterone. Mucin 2 and TNF-α gene expression was up-regulated in the CORT + MIX group compared to the CORT group. Caecal microbiota remained similar between the groups. These findings indicate that a balanced diet supplemented with functional AA and polyphenols can help to restore broiler intestinal barrier after a stress exposure.

BACKGROUND:Gelsectan® is a formulation of xyloglucan (XG), pea protein, grape seed extract (PPGS) and xylo-oligosaccharides (XOS). Our aim was to examine the effect of Gelsectan® on rectal sensitivity in an animal model, abdominal pain in irritable bowel syndrome with diarrhoea (IBS-D) subjects and intestinal permeability in both conditions. METHODS:Animals: Wistar rats received gavage with XOS, XG + PPGS or XG + PPGS + XOS, as a single dose or for 7 days before a partial restraint stress (PRS). Visceromotor response to rectal distension and total gut paracellular permeability to 51Cr-EDTA were assessed. Humans: IBS-D and control patients were involved. After initial colonoscopy with biopsy sampling Gelsectan® was administered to IBS-D patients for 12 weeks. Stool count and pain scores were documented. After treatment, colonoscopy was repeated. The permeability of biopsy samples was measured in Ussing-chambers. Adherent mucus layer, Muc-2 expression as well as TNFα, Interferon IFNγ were evaluated by histology/immunohistochemistry and ELISA assays, respectively. RESULTS:Animal studies: In control rats, PRS significantly increased visceromotor response as well as gut paracellular permeability. Single dose administration of XG + PPGS + XOS failed to reverse PRS, but 7 days of oral treatment reversed PRS-induced rectal hypersensitivity and gut hyperpermeability. Human studies: Gelsectan® treatment significantly reduced and abdominal pain. Intestinal permeability in IBS-D patients was elevated compared with controls, Gelsectan® restored permeability in the ascendent colon. Periodic acid-Schiff-stained mucus layer was significantly thinner in IBS-D patients compared with controls, In both segments, mucus thickness and the proportion of Muc-2 positive cells were not affected by Gelsectan® treatment. IFNγ tissue level in the sigmoid colon shows modest mucosal inflammation in IBS-D. CONCLUSIONS:Gelsectan® prevented rectal hypersensitivity in rats, abdominal pain in human and intestinal hyperpermeability in rat and human studies respectively. These effects involve restoration of gut permeability. Based on this translational study, Gelsectan® can be considered as an effective therapy for IBS-D symptoms.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by its main symptom, visceral hypersensitivity (VH), which is aggravated by stress. Gut-brain interactions and gut bacteria may alleviate IBS symptoms, including VH. gamma-amino butyric acid (GABA), produced notably by lactic acid bacteria (LAB), shows promising result in IBS symptoms treatment. In bacteria, GABA is generated through glutamate decarboxylase (GAD) metabolism of L-glutamic acid, maintaining intracellular pH. In mammals, GABA acts as an inhibitory neurotransmitter, modulating pain, stress, and anxiety. Therefore, utilizing GABA-producing LAB as a therapeutic approach might be beneficial. Our previous work showed that a GABA-producing Lactococcus lactis strain, NCDO2118, reduced VH induced by acute stress in rats after a 10-day oral treatment. Here, we identified the strain CNCM I-5388, with a four-fold higher GABA production rate under the same conditions as NCDO2118. Both strains shared 99.1% identical GAD amino acid sequences and in vitro analyses revealed the same optimal pH for GAD activity; however, CNCM I-5388 exhibited 17 times higher intracellular GAD activity and increased resistance to acidic pH. Additionally, in vivo experiments have demonstrated that CNCM I-5388 has faster anti-VH properties in rats compared with NCDO2118, starting from the fifth day of treatment. Finally, CNCM I-5388 anti-VH effects partially persisted after 5-day treatment interruption and after a single oral treatment. These findings highlight CNCM I-5388 as a potential therapeutic agent for managing VH in IBS patients.

Nanoplastics (NPLs), the presence in the environment of which was considered only “highly plausible” until recently, have become the focus of environmental and ecotoxicological studies. However, up to know, little is known about the potential risks of NPLs to human health. In this review, we provide an overview of the evidence of a specific impact of NPLs on human digestive health reported to date. We focus on the different sources of oral exposure to NPLs, including food packaging, food and beverages. We then summarize the toxicological effects of such exposure on the digestive ecosystem in vitro and in vivo in rodents. Importantly, gut toxicity results should be assessed with the type of NPL model materials employed, due to the critical influence of their chemical and physical properties. As a result, we thoroughly describe NPLs with their source, chemical composition and physicochemical behavior to emphasize the lack of NPL characterization and/or model materials. Finally, we propose avenues for interdisciplinary studies at the interface of toxicology and chemistry, with a view to achieving appropriate scientific assessments of the risks to gut health posed by NPLs, and improvements in their management.

Background: Escherichia coli is the predominant non-pathogenic facultative microbe of the human intestine, although some strains are diarrhoeagenic in humans. E. coli-derived lipopolysaccharide (LPS) induces diarrhoea, intestinal barrier impairment, bacterial translocation and intestinal inflammation. Associations with the mucoprotectant xyloglucan exhibit antidiarrhoeal effects. This study evaluated and compared the effects of xyloglucan in combination with gelatin or gelose (agar-agar) on jejunal permeability and inflammation using an in vivo rat model of E. coli LPS-induced enteritis. Methods: Xyloglucan (12.5 mg/kg) plus gelatin (250 mg/kg) or gelose (250 or 500 mg/kg) were administered orally 2 hours before intraperitoneal injection with E. coli LPS. Following euthanasia, jejunal segments were removed for intestinal permeability measurement in Ussing chambers and inflammatory tone evaluation by myeloperoxidase activity assay. Results: LPS administration increased jejunal permeability and increased mucosal inflammation in male Wistar rats. Xyloglucan plus gelatin 250 mg/kg and xyloglucan plus gelose 500 mg/kg significantly attenuated LPS-induced jejunal hyperpermeability and myeloperoxidase activity. Conclusion: Xyloglucan, a known mucosal barrier protector, in combination with gelatin or gelose, has beneficial and comparable effects on intestinal permeability and inflammation following E. coli LPS insult in male rats.

Fermented milk plays an important role in the diet of many populations around the world, particularly those of the Eastern Mediterranean countries where its consumption contributes to the populations' food security and welfare. This review explores 12 examples of traditional fermented milk products from countries bordering the Eastern Mediterranean Sea. Each product has unique physicochemical, microbiological, and sensory characteristics, but they also share commonalities. They can be classified into four groups mainly based on the presence or not of a draining step or mixing with other raw materials (cereals). Lactobacilli are the dominant identified genera. Moreover, fermented milk products can be considered as a source of probiotics and prebiotics, offering health benefits. This work aims to preserve these special food products. Encouraging studies on microbial populations, fermentation, flavour development, sanitary and health qualities of these milks is essential. Increasing such knowledge will help better understand and appreciate culturally significant fermented milk products.

Labneh Ambaris is a traditional Lebanese dairy product typically made using goat milk in special earthenware jars. Its production is characterized by the regular additions of milk and coarse salt, all while draining the whey throughout a process that lasts for a minimum of 2 mo. In this study, 20 samples of labneh Ambaris, all produced by spontaneous fermentation, were studied. They were collected at the end of fermentation from different regions in Lebanon. Physicochemical and sensory properties were studied and microbial diversity was analyzed using culture-dependent and independent techniques. The V3-V4 region of the 16S rRNA gene and the ITS2 region were sequenced by DNA metabarcoding analyses for the identification of bacteria and yeast communities, respectively. Out of 160 bacterial and 36 fungal taxa, 117 different bacterial species and 24 fungal species were identified among all labneh Ambaris samples studied. The remaining ones were multi-affiliated and could not be identified at the species level. Lactobacillus was the dominant bacterial genus, followed by Lentilactobacillus, Lactiplantibacillus, Lacticaseibacillus, and Lactococcus genera, whereas Geotrichum and Pichia were the dominant fungal genera. The 20 samples tested had varying levels of salt, protein, and fat contents, but they were all highly acidic (mostly having a pH < 4). According to the sensory scores generated by classical descriptive analysis, all samples were described as having basic similar characteristics such as goat smell and flavor, but they could be differentiated based on various intensities within the same descriptors like salty and acidic. This work could be considered as a base toward obtaining a quality label for labneh Ambaris.

The responses of various microbial populations to modifications in the physicochemical properties of a food matrix, as well as interactions between these populations already present, are the main factors that shape microbial dynamics in that matrix. This work focused on the study of microbial dynamics during labneh Ambaris production, a traditional Lebanese concentrated fermented goat milk made in jars during 3 months. This was assessed in two earthenware jars at a production facility. DNA metabarcoding of the ITS2 region as well as the V3-V4 region of the 16S rRNA gene was used to characterize the fungal and bacterial communities, respectively. Viable bacterial isolates were also identified by Sanger sequencing of the V1-V4 region of the 16S rRNA gene. Our results showed that the dominant microorganisms identified within labneh Ambaris (Lactobacillus kefiranofaciens, Lentilactobacillus kefiri, Lactococcus lactis, Geotrichum candidum, Pichia kudriavzevii and Starmerella sp.) settle early in the product and remain until the end of maturation with varying abundances throughout fermentation. Microbial counts increased during early fermentation stage, and remained stable during mid-fermentation, then declined during maturation. While microbial compositions were globally comparable between the two jars during mid-fermentation and maturation stages, differences between the two jars were mainly detected during early fermentation stage (D0 until D10). No significant sensorial differences were observed between the final products made in the two jars. Neither coliforms nor Enterobacteriaceae were detected in their viable state, starting D7 in both jars, suggesting the antimicrobial properties of the product.

Labneh Ambaris is a traditional Lebanese dairy product traditionally made using raw goat’s milk in earthenware jars, but recently the use of artisanally pasteurized milk was introduced for safety reasons. In this study, 12 samples of labneh Ambaris were studied, six made using raw goat’s milk and six others using artisanally pasteurized goat’s milk. These samples were collected during fermentation and their microbial compositions were analyzed. The 16S V3–V4 and the ITS2 regions of the rDNA were sequenced by DNA metabarcoding analyses for the identification and comparison of bacterial and fungal communities, respectively. The samples had high microbial diversity but differences in samples microbiota were unrelated to whether or not milk was pasteurized. The samples were consequently clustered on the basis of their dominant bacterial or fungal species, regardless of the milk used. Concerning bacterial communities, samples were clustered into 3 groups, one with a higher abundance of Lactobacillus helveticus, another with Lactobacillus kefiranofaciens as the dominant bacterial species, and the third with Lentilactobacillus sp. as the most abundant species. Species belonging to the Enterobacteriaceae family were detected in higher abundance in all raw milk samples than in artisanally pasteurized milk samples. As for fungal communities, the samples were clustered into two groups, one dominated by Geotrichum candidum and the other by Pichia kudriavzevii.

Pain-related functional gastrointestinal disorders (FGIDs) are characterized by visceral hypersensitivity (VHS) associated with alterations in the microbiota-gut-brain axis. Since human milk oligosaccharides (HMOs) modulate microbiota, gut and brain, we investigated whether HMOs impact VHS, and explored the role of gut microbiota. To induce VHS, C57BL/6JRj mice received hourly water avoidance stress (WAS) sessions for 10 d, or antibiotics (ATB) for 12 d. Challenged and unchallenged (Sham) animals were fed AIN93M diet (Cont) or AIN93M containing 1% of a 6-HMO mix (HMO6). VHS was assessed by monitoring the visceromotor response to colorectal distension. Fecal microbiome was analyzed by shotgun metagenomics. The effect of HMO6 sub-blends on VHS and nociceptive pathways was further tested using the WAS model. In mice fed Cont, WAS and ATB increased the visceromotor response to distension. HMO6 decreased WAS-mediated electromyographic rise at most distension volumes and overall Area Under Curve (AUC=6.12±0.50 in WAS/HMO6 vs. 9.46±0.50 in WAS/Cont; P<.0001). In contrast, VHS in ATB animals was not improved by HMO6. In WAS, HMO6 promoted most microbiota taxa and several functional pathways associated with low VHS and decreased those associated with high VHS. Among the sub-blends, 2’FL+DFL and LNT+6’SL reduced visceromotor response close to Sham/Cont values and modulated serotoninergic and CGRPα-related pathways. This research further substantiates the capacity of HMOs to modulate the microbiota-gut-brain communication and identifies mitigation of abdominal pain as a new HMO benefit. Ultimately, our findings suggest the value of specific HMO blends to alleviate pain associated FGIDs such as infantile colic or Irritable Bowel Syndrome.

OBJECTIVE:To evaluate the effect of diosmectite on visceral hypersensitivity and intestinal transit in a rat chronic stress model and on the faecal microbiota.MATERIALS AND METHODS:Wistar rats (175-225 g; n=10) were randomized into four groups: diosmectite/control, diosmectite/stress, vehicle/control, and vehicle/stress. Diosmectite (500 mg/kg, PO) was administered for five days for assessment of visceral hypersensitivity and intestinal transit and for three weeks for assessment of faecal microbiota. The stress procedure was a daily chronic passive water avoidance session. Intestinal transit was evaluated by faecal output in the hour following the last stress session. Visceral sensitivity in response to colorectal distension (CRD) was assessed at baseline and 30 min after the last stress session. In another group of rats, faecal material was collected before and after treatment with diosmectite or vehicle; genomic DNA was extracted and sequenced to characterize the faecal microbiota.RESULTS:Under nonstressed conditions, diosmectite treatment did not modify intestinal transit compared to the vehicle group (p=0.33). After the stress procedure, a trend towards reduction in stress-induced stool production was observed with diosmectite compared to vehicle (6.3±1.1 vs. 4.9±1.2 respectively; p=0.38). In the control condition, the number of CRD-evoked abdominal contractions was 20±4 after diosmectite and 24±2 after vehicle (p=0.75). In the stressed condition, the number of contractions increased to 34.4±2.4 after vehicle (p<0.05 compared to control). Stress-related hypersensitivity was attenuated after diosmectite treatment (26.9±2.2; p<0.05 compared to vehicle). No relevant changes were observed in the faecal microbiotic profile after treatment with diosmectite or vehicle.CONCLUSIONS:Diosmectite treatment attenuates stress-induced visceral hypersensitivity; this effect may contribute to the therapeutic effect of diosmectite in IBS in humans.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder for which dietary interventions can be a useful treatment. In recent years, the low-FODMAP approach is gaining traction in this regard. The fermentation of these non-absorbed carbohydrates by the gut microbiota can generate toxic glycating metabolites, such as methylglyoxal. These metabolites can have harmful effects by their role in the generation of advanced glycation end products (AGEs), which activates Receptor for AGEs (AGER). Mast cells can be stimulated by AGEs and play a role in IBS. We have treated mice with lactose or fructo-oligosaccharides (FOS), with or without co-administration of pyridoxamine and investigated the colonic mucus barrier. We have found that an increased intake of lactose and fructo-oligosaccharides induces a dysregulation of the colonic mucus barrier, increasing mucus discharge in empty colon, while increasing variability and decreasing average thickness mucus layer covering the fecal pellet. Changes were correlated with increased mast cell counts, pointing to a role for the crosstalk between these and goblet cells. Additionally, AGE levels in colonic epithelium were increased by treatment with the selected fermentable carbohydrates. Observed effects were prevented by co-treatment with anti-glycation agent pyridoxamine, implicating glycation processes in the negative impact of fermentable carbohydrate ingestion. This study shows that excessive intake of fermentable carbohydrates can cause colonic mucus barrier dysregulation in mice, by a process that involves glycating agents and increased mucosal mast cell counts.