To Örebro University

Background: This study investigates the impact of fermentation supernatants (FSs) from Pleurotus eryngii whole mushrooms (PEWS), as well as its subcomponents, digested (PEWSD) and extracted (PEWSE) forms, on intestinal barrier function and immune modulation in lipopolysaccharide (LPS) -stimulated Caco-2 cells.

Methods: Gene expression of tight junction (TJs) genes, cytokines, and key immune/metabolic receptors was assessed via qRT-PCR, while cytokine protein levels were measured using ELISA to explore post-transcriptional regulation.

Results: LPS challenge significantly downregulated TJs zonula occludens-1 (ZO-1,) occludin, and claudin-1, compromising epithelial integrity. Treatment with FS-PEWS notably restored ZO-1 and occludin expression, outperforming FS-PEWSD and FS-PEWSE, which only partially mitigated the LPS-induced damage. FS-PEWS further demonstrated potent immunomodulatory effects, upregulating anti-inflammatory IL-10 and pro-inflammatory cytokines such as IL-8 and TNF-α. The activation of key receptors like TLR-2 and mTOR suggests that FS-PEWS modulates critical immune and metabolic pathways, such as NF-kB signaling, to maintain immune homeostasis. Although mRNA expression of pro-inflammatory cytokines was altered, no corresponding protein release was detected, suggesting potential post-transcriptional regulation.

Conclusions: FS-PEWS preserves intestinal barrier integrity and modulates immune responses, particularly in low-grade inflammation, highlighting the whole food matrix's role in enhancing its bioactivity and functional food potential.

Pleurotus eryngii (PE), an edible mushroom rich in bioactive compounds, has been shown to exert immunomodulatory, anti-inflammatory, antioxidant, anti-carcinogenic, anti-microbial, antihepatotoxic and hypolipidemic activities, all important for the well-being of the ageing population. This study assessed in vitro the prebiotic-like effects of multiple forms of this mushroom. An in vitro static batch fermentation was performed for 24 h with faecal inocula from five apparently healthy older adults in the presence of the following PE forms: whole food matrix (PEWS), in vitro digested (PEWSD) and rich in beta-glucans extract (PEWSE). The changes in bacterial communities upon fermentation at family, genera and species level were detected via 16S rRNA Next Generation Sequencing and Quantitative real-time PCR. Short-chain fatty acids (SCFAs) were quantified using gas chromatography (GC), whereas other metabolites were analysed through ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). DEseq2 analysis indicated that PEWS presence exhibited the largest impact on faecal microbial families' and genera's abundance compared to negative (noncarbon source) and positive (inulin) controls. Only PEWS significantly increased Bifidobacterium spp. and F. prausnitzii populations, while all three forms robustly increased Bacteroides spp. levels and levels of butyrate, acetate and propionate acids. Statistical significance was set at p < 0.05. Overall, the findings highlight the beneficial effect of PE on intestinal health of older adults supporting its potential incorporation into innovative functional foods. However, additional in vivo studies are required to substantiate these findings before translating them into dietary guidelines or clinical applications.

Background: This study explores the potential of the Pleurotus eryngii mushroom fermentation supernatant (FS-PEWS) as an intervention for mitigating sodium deoxycholate (SDC)-induced intestinal barrier dysfunction and inflammation.

Methods: FS-PEWS was assessed for its protective effects against SDC-induced barrier dysfunction and inflammation using an in vitro Caco-2 cell model and ex vivo colonic biopsies from healthy adult donors, where barrier integrity, permeability, immunomodulation and receptor-mediated pathways were evaluated.

Results: In Caco-2 cells, SDC exposure downregulated ZO-1, occludin, and claudin-1 expression, with FS-PEWS restoring ZO-1 and claudin-1 levels while maintaining cell viability. In colonic biopsies from healthy adults, FS-PEWS maintained tissue integrity and selectively mitigated transcellular permeability without affecting paracellular permeability when combined with the stressor. Additionally, FS-PEWS exhibited potent anti-inflammatory effects, reducing pro-inflammatory cytokines, e.g., TNF-α, IL-6, and IL-1β and modulating receptor-mediated pathways, i.e., TLR-4, dectin-1.

Conclusions: These results demonstrate the potential of FS-PEWS to sustain intestinal barrier function and modulate immune responses under stress, highlighting its therapeutic potential for managing gut barrier dysfunction and inflammation associated with microbial metabolite-induced disruptions.

Dietary fibres are key modulators of gut health, including barrier integrity and immune function, yet these effects are not sufficiently characterised. β-Glucans and rhamnogalacturonan-I (RG-I) have been shown to shape microbial composition and immune signalling, but their specific contributions to barrier integrity require further elucidation. Paper I examines how faecal fermentation supernatants (FS) from Pleurotus eryngii (PE) cultivated on distinct substrates influence intestinal barrier integrity in an LPS-stimulated Caco-2 model, revealing substrate-driven variations in tight junction modulation. Paper II describes the comparative effects of whole PE, its digested derivatives and a β-glucan-enriched extract on gut microbiota composition and metabolism in elderly individuals, using an in vitro fermentation model, highlighting the superior modulatory potential of the whole PE. Paper III explores the immunoregulatory properties of PE’s FS in an LPS-stimulated Caco-2 model, demonstrating their role in cytokine signal-ling and barrier’s protection. Paper IV evaluates the capacity of PE FS to mitigate bile acid-induced barrier dysfunction in in vitro and ex vivo colonic models, underscoring their protective effects against hyperpermeability. Paper V investigates the prebiotic potential of carrot RG-I in a human intervention study, showing its ability to enhance bifidobacteria populations, modulate immune responses and support barrier integrity. In conclusion, this thesis advances the understanding of dietary fibre bioactivities beyond conventional microbiota analyses. By demonstrating that β-glucans and RG-I modulate epithelial and immune functions alongside microbial interactions, it establishes a mechanistic foundation for precision nutrition strategies targeting gut health through an integrated, systems-based approach.

Aim: The cell matrix of plant foods has received little attention in prebiotic fiber research. We aimed to understand the impact of the plant cell matrix in dried chicory root on its breakdown in the human gut to explain its reported beneficial effects on gut and metabolic health.

Methods: We applied in vitro digestion and fermentation models together with an ex vivo gut barrier integrity model. Plant cell matrix intactness in the upper gastrointestinal tract was investigated by scanning electron microscopy. Colonic breakdown of inulin, and chicory root cubes and powder was assessed by gut microbiota analysis using 16S rRNA gene amplicon sequencing and determining the kinetics of changes in pH, gas, and short- chain fatty acid (SCFA) production. Finally, effects on gut barrier integrity were explored by exposing colonic biopsies to fermentation supernatants in an Ussing chamber model.

Results: The plant cell matrix of dried chicory root cubes remained intact throughout upper gastrointestinal transit. Dried chicory root fermentation resulted in higher final relative abundances of pectin-degrading Monoglobus and butyrate-producing Roseburia spp. compared to inulin and a seven-fold increase in Bifidobacterium spp. in donors where these species were present. Dried chicory root cubes yielded similar total SCFAs but higher final butyrate levels than chicory root powder or isolated inulin with less gas produced. No uniform but donor-specific effects of fermentation supernatants on the maintenance of gut barrier integrity were detected.

Conclusion: The intact plant cell matrix of dried chicory root affected its colonic breakdown kinetics and microbiota, underpinning its beneficial effect in vivo.

BACKGROUND: Many patients with glioma and their caregivers seek complementary and alternative medicine (CAM) methods to comfort themselves, cope with cancer medication side effects, and feel they are taking control of their disease.

OBJECTIVE: To summarize existing evidence on safety and efficacy of CAM treatments for gliomas.

METHODS: We performed an exhaustive electronic literature search for in vitro, animal, and clinical studies (English language, all years available) on CAM modalities for gliomas.

RESULTS: A total of 378 studies (315 unique articles) were analyzed. Distribution was as follows: in vitro-274 (73%), animal-77 (20%), and clinical-26 (7%, 2491 patients). Most studies were conducted in China (n = 135, 43%), followed by the United States (n = 62, 20%) and Spain (n = 17, 5%-6%). Resveratrol was the most commonly investigated CAM therapy in the in vitro (n = 62) and in vivo (n = 17) setting. Safety/toxicity was examined in 21% of in vitro (cytotoxic at same dose in 48%), 39% of in vivo (no evidence of organ toxicity), and 50% of clinical studies (adverse events reported in 6). Cytotoxicity was the most frequent end point among in vitro (60%) and animal studies (56%), followed by synergistic action with chemotherapy and inhibition of invasiveness and migration. Finally, 7 of 26 studies found no clinical effect, whereas 5 reported possible impact on progression-free or overall survival, 3 demonstrated decrease or arrest of tumor progression, and 2 showed positive impact on symptoms and quality of life.

CONCLUSION: These findings will hopefully educate providers and patients and stimulate further research in the field of CAM therapy for gliomas.

Children with autism spectrum disorder (ASD) often suffer gastrointestinal disturbances consistent with gut microbiota (GM) alterations. Treatment with pro/prebiotics may potentially alleviate gut symptoms, but the evidence for prebiotics is scarce. This study aims to evaluate the effects of edible mushrooms (Pleurotus, Basidiomycota) and prebiotic compounds on GM composition and metabolite production in vitro, using faecal samples from autistic and non-autistic children. Specific microbial populations were enumerated after 24 h of fermentation by quantitative PCR, and the metabolic production was determined by gas chromatography. Higher levels of Prevotella spp. and Bifidobacterium spp. were measured in neurotypical children compared to ASD children. A total of 24 h fermentation of Pleurotus eryngii and P. ostreatus mushroom powder increased the levels of Bifidobacterium, while known prebiotics increased the levels of total bacteria and Bacteroides in both groups. Only P. eryngii mushrooms resulted in significantly elevated levels of total bacteria Bacteroides and Feacalibacterium prausnitzii compared to the negative control (NC) in the ASD group. Both mushrooms induced elevated levels of butyrate after 24 h of fermentation, while short-chain fructooligosaccharides induced increased levels of acetate in the ASD group, compared to NC. Overall, this study highlights the positive effect of edible mushrooms on the GM and metabolic activity of children with ASD.

Edible mushrooms contain biologically active compounds with antioxidant, antimicrobial, immunomodulatory and anticancer properties. The link between their anticancer and immunomodulatory properties with their possible prebiotic activity on gut micro-organisms has been the subject of intense research over the last decade. Lyophilized Pleurotus eryngii (PE) mushrooms, selected due to their strong lactogenic effect and anti-genotoxic, immunomodulatory properties, underwent in vitro static batch fermentation for 24 h by fecal microbiota from eight elderly apparently healthy volunteers  (>65 years old). The fermentation-induced changes in fecal microbiota communities were examined using Next Generation Sequencing of the hypervariable regions of the 16S rRNA gene. Primary processing and analysis were conducted using the Ion Reporter Suite. Changes in the global metabolic profile were assessed by 1H NMR spectroscopy, and metabolites were assigned by 2D NMR spectroscopy and the MetaboMiner platform. PLS-DA analysis of both metataxonomic and metabolomic data showed a significant cluster separation of PE fermented samples relative to controls. DEseq2 analysis showed that the abundance of families such as Lactobacillaceae and Bifidobacteriaceae were increased in PE samples. Accordingly, in metabolomics, more than twenty metabolites including SCFAs, essential amino acids, and neurotransmitters discriminate PE samples from the respective controls, further validating the metataxonomic findings.

The human gut microbiota (GM) is a complex microbial ecosystem that colonises the gastrointestinal tract (GIT) and is comprised of bacteria, viruses, fungi, and protozoa. The GM has a symbiotic relationship with its host that is fundamental for body homeostasis. The GM is not limited to the scope of the GIT, but there are bidirectional interactions between the GM and other organs, highlighting the concept of the "gut-organ axis". Any deviation from the normal composition of the GM, termed "microbial dysbiosis", is implicated in the pathogenesis of various diseases. Only a few studies have demonstrated a relationship between GM modifications and disease phenotypes, and it is still unknown whether an altered GM contributes to a disease or simply reflects its status. Restoration of the GM with probiotics and prebiotics has been postulated, but evidence for the effects of prebiotics is limited. Prebiotics are substrates that are "selectively utilized by host microorganisms, conferring a health benefit". This study highlights the bidirectional relationship between the gut and vital human organs and demonstrates the relationship between GM dysbiosis and the emergence of certain representative diseases. Finally, this article focuses on the potential of prebiotics as a target therapy to manipulate the GM and presents the gaps in the literature and research.

Recent studies have revealed the crucial role of several edible mushrooms and fungal compounds, mainly polysaccharides, in human health and disease. The investigation of the immunomodulating effects of mushroom polysaccharides, especially beta-glucans, and the link between their anticancer and immunomodulatory properties with their possible prebiotic activity on gut micro-organisms has been the subject of intense research over the last decade. We investigated the immunomodulating effects of Pleurotus eryngii mushrooms, selected due to their high beta-glucan content, strong lactogenic effect, and potent geno-protective properties, following in vitro fermentation by fecal inocula from healthy elderly volunteers (>60 years old). The immunomodulating properties of the fermentation supernatants (FSs) were initially investigated in U937-derived human macrophages. Gene expression as well as pro- (TNF-alpha, IL-1 beta) and anti-inflammatory cytokines (IL-10, IL-1R alpha) were assessed and correlated with the fermentation process. The presence of P. eryngii in the fermentation process led to modifications in immune response, as indicated by the altered gene expression and levels of the cytokines examined, a finding consistent for all volunteers. The FSs immunomodulating effect on the volunteers' peripheral blood mononuclear cells (PBMCs) was verified through the use of cytometry by time of flight (CyTOF) analysis.