Inulin-type fructans (ITF) are water-soluble mixtures of oligo- or polysaccharides composed of β-D-fructosyl units linked by (2–1) glycosidic bonds [93]^[85]. Based on the degree of polymerization (DP), ITF can be further divided into long-chain inulin (DP > 10) and short-chain oligofructose, also called FOS (DP < 10) [94]^[86]. The changes in the gut microbiota caused by ITF are suggested to indirectly prevent gastrointestinal and systemic infections in both animal and human studies. The possible mechanism suggested to be responsible for the protective role of ITF is the bacterial antagonism and competition of so-called positive microbiota with pathogens as well as the trophic effects on the intestinal epithelium [92]^[84]. In vitro and animal studies show a great promise of the ITF intake on gut barrier integrity. However, there is no consensus with human studies to date.

A recent in vitro study was proposed to understand the mechanism of action of ITF on AMP-activated protein kinase (AMPK) and TJ assembly [95]^[87]. The authors investigated the effect of FOS under two conditions, inflammatory and non-inflammatory, using a human intestinal epithelial cell line derived from a colorectal adenocarcinoma patient (T84). It was reported that FOS in a dose of 0.1 mg/mL was capable of inducing AMPK phosphorylation via a calcium-sensing receptor (CaSR), independently from the gut microbiota. Moreover, FOS reversed the ability of LPS to suppress AMPK activity and TJ assembly through the same pathway [95]^[87]. A study with a porcine cell line was performed by Uerlings et al. [96]^[88]. The authors compared the effect of digested inulin (after the hydrolysis-dialysis steps) and fermented inulin on the expression of gut barrier genes. Digested inulin upregulated adherence and TJ gene expression levels (occludin, claudin-3 and ZO-1, while CDH1, mucin 1 (MUC1) and reduced the expression of claudin-3, which can suggest a beneficial impact of inulin directly on the gut epithelial barrier. Fermented inulin induced higher expression of CDH1, claudin-1, -3, epidermal growth factor receptor (EGFR) and MUC1 compared to digested inulin [96]^[88]. Such changes indicated that the fermentation step might endorse the production of beneficial metabolites, such as butyrate, which enhance the integrity of the gut barrier.

Several in vivo studies using various animal models aimed at evaluating and understanding the effect of ITF on the gut barrier were reported. Cani et al. [97]^[89]. hypothesized that prebiotic-induced modulation of gut microbiota normalizes intestinal permeability by a mechanism involving glucagon-like peptide-2 (GLP-2), consequently reducing inflammation and metabolic disorders during obesity and diabetes. To verify this assumption, they performed a series of studies with ob/ob mice. Mice treated with oligofructose exhibited lower plasma LPS and cytokines concentrations compared to the placebo group. Moreover, a decreased hepatic expression of inflammatory and oxidative stress markers was detected in the prebiotic group, which was associated with reduced intestinal permeability and improved integrity of TJs. Prebiotics also increased GLP-2 production, and the GLP-2-dependent mechanism was proposed to be responsible for improved gut barrier functions [97]^[89]. The effect of FOS on the functions of gut barrier in the methionine-choline-deficient mice with nonalcoholic steatohepatitis was reported by Matsumoto et al. [98]^[90]. In this study, only the mice supplemented with 5% FOS maintained a normal gut microbiome. Moreover, positive morphological changes were observed after FOS intake. Mice from the prebiotic group had significantly higher villus and longer small intestines compared to control mice. Additionally, the expression of TJ proteins examined via ZO-1 staining increased in mice fed with FOS, confirming the positive effect on the gut barrier [97]^[89]. FOS was also found to be a promising therapeutic and prophylactic agent in mucositis induced by 5-fluorouracyl in BALB/c mice [97]^[89]. In the study of Carvalho et al. [99]^[91], intestinal permeability, TJ expression (ZO-1 and occludin) and propionate concentration within physiologic levels remained unchanged despite the induction of mucositis. Moreover, in the groups fed with FOS, increased levels of butyrate and reduced bacterial translocation and inflammatory infiltrate were observed. Notably, treatment with FOS before and during the induction of the disease was found to be most efficient in maintaining histological and morphometric parameters as well as IgA production and protecting the gut barrier. On the other hand, the FOS did not induce mucus production, which was reduced in the animals with induced mucositis [99]^[91]. Beisner et al. [100]^[92] designed an in vivo study aimed to evaluate whether inulin can improve the gut barrier function damaged by the Western diet given to mice. The authors applied the Western diet enriched with 10% inulin in mice for 12 weeks. The supplementation with inulin induced expression of Paneth cell α-defensins and matrix metalloproteinase-7 (MMP7), which were reduced by the Western diet. Additionally, the intestinal permeability, measured using polyethylene glycol 4000 (PEG4000), was reduced in mice fed with inulin.

Yacon flour (YF) (Smallanthus sonchifolius), which is a source of FOS, was incorporated into the diet of rats with colon cancer by the Brazilian group [101,102]^[93][94]. In the first study [101]^[93], YF in a dose delivering 7.5% of FOS was added for eight weeks to the diet of rats with induced colon cancer. Rats with cancer presented significantly worsened parameters of the gut barrier compared to the healthy control. The animals fed with YF also had significantly reduced urinary excretions of mannitol and lactulose compared to the control group. The ratio of lactulose to mannitol change reduced by 40% after YF incorporation, suggesting the positive effect of prebiotics on the gut barrier. The second study by the same group confirmed these findings [102]^[94]. In this study, the dose of YF was reduced to provide 5% of FOS. After the intervention, the urinary excretion of lactulose was not affected; however, the improvement in mannitol excretion was noted. Consequently, the lactulose to mannitol ratio was reduced in the animals fed with YF, contrary to the rats without prebiotic.

The in vivo studies showed a great potential of application of ITF to restore the gut barrier. However, despite these promising results, and although ITF is one of the most studied prebiotics, there is a limited number of clinical studies evaluating its effect on the gut barrier. Olguin et al. [103]^[95] conducted a randomized, double-blind, controlled clinical trial in patients with burn injuries, supplemented with 6 g/day of oligofructose or placebo for 15 days. Gut permeability was evaluated by the sugar absorption test before and after the intervention. The ratio of lactulose to mannitol was significantly elevated in the burn patients, suggesting the impaired gut barrier. The sugar absorption test results improved progressively from day 1 to 21, irrespectively on the applied supplement, suggesting no effect of the prebiotic intake. However, as the author reported [103]^[95], the sucrose excretion was highly increased in the patients, suggesting the major defects of permeability in the upper gut, mainly at the gastric level. Therefore, it cannot be surprising that the prebiotic, which works mainly in the colon, did not alleviate the intestinal permeability in the upper intestinal tract.

Another study investigated the effect of the inulin-enriched pasta on the intestinal permeability measured by SAT as well as GLP-2 and zonulin concentrations in healthy young subjects [104]^[96]. Twenty young men were enrolled to consume pasta containing 11% of inulin or control pasta without prebiotics for five weeks. After the intervention, the lactulose recovery was lower in the inulin group, while there was no difference in the mannitol excretion. Consequently, the ratio of lactulose to mannitol was reduced in the individuals consuming prebiotics. Moreover, the inulin consumption also resulted in reduced zonulin and increased GLP-2 values. All the results indicate that the incorporation of inulin into the pasta has a beneficial effect on the gut barrier in healthy men [104]^[96]. These results are quite surprising considering that the study was performed in healthy subjects which did not have any gut problems and did not have impaired gut barriers. It is rather impossible that the barrier can be “more tight” than the perfectly physiological barrier.

A study on the effect of oligofructose-enriched inulin on the gut barrier in patients T1D was reported by Ho et al. [105]^[97]. The authors designed a randomized, placebo-controlled trial in children 8 to 17 years of age with T1D supplemented with prebiotic for 12 weeks. After three months, a significant increase in the relative abundance of Bifidobacterium in the prebiotic group was noted, which was no longer present after the 3-month washout. Moreover, a modest improvement in the intestinal permeability, measured by the sugar absorption test, was observed, accompanied by a higher content of C-peptide. After three-month prebiotic intake, a decrease in intestinal permeability was noted, while in the placebo group, an increase in their intestinal permeability was observed, although these differences did not reach statistical significance [105]^[97]. Importantly, at the baseline, only one-third of the patients had elevated values of the sugar absorption test.

Another study was conducted with subjects with at least one criterium for higher risk of type 2 diabetes (T2D). In this proof-of-concept study, 24 adults at risk of T2D were supplemented with 10 g/day of inulin or placebo for six weeks [106]^[98]. No effect of the prebiotic supplementation was observed in the intestinal permeability markers, plasma endotoxin concentration or LPS-binding protein concentration, despite a significant bifidogenic effect. It could be explained by the fact that the participants of this study were relatively healthy (i.e., did not have impaired glucose tolerance or confirmed T2D).

Celiac disease is suggested to belong to diseases characterized by the impaired gut barrier [107]^[99]. A randomized, placebo-controlled study was proposed to evaluate the effect of 12-week supplementation of a gluten-free diet (GFD) with oligofructose-enriched inulin (10 g per day) on intestinal permeability in children with celiac disease treated with a GFD. The integrity of the gut barrier was assessed by the analysis of the zonulin, intestinal fatty acid-binding protein, claudin-3, calprotectin and GLP-2, as well as SAT. The authors reported the lack of the substantial effect of prebiotic supplementation on the gut barrier. Although the intake of oligofructose-enriched inulin increased the excretion of mannitol, which may suggest an increase in the epithelial surface, the majority of children in this study seem to have normal values for intestinal permeability tests before the intervention. The inclusion criterium was that children were following a GFD for at least six months, which could be enough to restore the gut barrier [108]^[100]. Notably, for individuals with elevated values, improvement in calprotectin and SAT was observed after the prebiotic intake. Although the results seem to be promising, as the authors underlined, it is difficult to draw a conclusion based on the small number of individuals with an impaired gut barrier [108]^[100].

To summarize, the in vitro and in vivo studies showed great promise in the effects of ITF on the gut barrier, which was not fully confirmed in clinical trials. However, the majority of studies were performed with participants without the ongoing impairment in the gut barrier integrity. Therefore, there is a need to design studies with participants with elevated intestinal permeability to check whether the effects of ITF observed in animal studies can be translated into humans.

Galactooligosaccharides (GOS) consist of β-linked galactose moieties with galactose or glucose at the reducing end. Known GOS contain β-(1→2), β-(1→3), β-(1→4), or β-(1→6) linked galactose moieties and usually have a DP of 3–8. GOS are produced from lactose by β-galactosidase in a kinetically controlled reaction between enzymatic hydrolysis and transgalactosylation [109]^[101]. GOS are not digested in the upper gut and exhibit a strong bifidogenic effect. Commonly, GOS are used as a substitute for HMOs in infant formulas [110,111]^[102][103]. Many studies showed that the intake of GOS can improve gut health [112]^[104] thus it can be assumed that GOS can also affect the gut barrier function.

Akbari et al. [113]^[105] compared the effect of different formulations of GOS (syrup and purified GOS) and DP on the epithelial integrity in an in vitro model of intestinal barrier dysfunction. Vivinal^® GOS syrup was found to be the most efficient in the protection of monolayer integrity and TJ reassembly. Notably, GOS syrup was rich in oligosaccharides with DP of 2–3 and comparing the obtained effects with ITF of different DP; it could be seen that smaller DP are of greater importance for the protection of barrier integrity than oligosaccharides of longer chains. The authors concluded that the microbiota-independent effect of oligosaccharides depends on the oligosaccharide structure, DP and concentration of each fraction [113]^[105].

Similarly to ITF, several in vivo studies were performed to evaluate the effect of GOS on the gut barrier using various animal models. Barrat et al. [114]^[106] conducted a study with neonatal rats fed with milk formula with or without the mixture of GOS and inulin (ratio 88:12) from the 7th to 20th day of life. Supplementation with prebiotic formula resulted in an increase in bacterial translocation at day 18, which was not observed anymore at day 40. However, the colonic permeability assessed by an Ussing chamber and the expression of TJ genes were not altered by the prebiotic intake. Contrary, the expression of ZO-1 was reduced by 40%, and trophic effects in cecal mucosa were observed in a GOS/inulin group. The explanation of the increased bacterial translocation is unclear, but one of the possible reasons could be the rapid fermentation of the prebiotics leading to the higher concentration of organic acids, which can impair the gut barrier. However, it was not confirmed in this study [114]^[106]. In another study, Zhong et al. [115]^[107] evaluated the effect of GOS supplementation in enteral nutrition in rats with induced acute severe pancreatitis. Pancreatitis was accompanied by intestinal barrier dysfunction. In the groups supplemented with GOS, the number of faecal bifidobacteria, sIgA level in intestinal mucus, intestinal occludin mRNA level at days 4 and 7 and the intensity of intestinal epithelial apoptosis at day 7 was significantly higher compared to the standard enteral formula. The authors reported that prebiotic intake could decrease the bacterial translocation in mesenteric lymph nodes and livers and alleviate pancreatic inflammation [115]^[107]. A study analyzing the effect of GOS on the gut barrier in rats with alcohol withdrawal syndrome was proposed by Yang et al. [116]^[108]. The chronic consumption of alcohol for five weeks increased the serum concentrations of diamine oxidase (DAO), D-lactate and LPS, indicating the impairment of the gut barrier. The withdrawal of alcohol decreased the concentrations of the gut barrier markers; however, the values were still higher than in control, healthy groups. The alcohol abstinence did not restore the villus length and crypt depth. The supplementation of the diet with GOS for three weeks ameliorated the gut barrier functions, and despite no effect in crypt morphology, the villus to crypt ratios increased significantly, suggesting that GOS can be applied as an auxiliary therapy for improvement of gut health after long-term alcohol consumption [116]^[108]. Another situation when the increase in intestinal permeability is observed is ageing. An interesting study was reported by Arnold et al. [117]^[109], who evaluated the effect of GOS in age-associated gut permeability. The authors confirmed that older mice had increased intestinal permeability compared to younger animals. However, GOS supplementation (71.8 g per kg) managed to alleviate these effects in old animals. Moreover, in old mice fed with GOS, an increase in mucus abundance was noted. Notably, the GOS intake had no effect in young mice, which is understandable since the animals had no gut barrier impairment [117]^[109].

Pigs provide a very good, clinically relevant model for studies on the human intestinal tract [118]^[110]. Therefore, several in vivo studies were performed using pigs, aimed to mimic the effects of prebiotics which then could be expected in humans. An example of such a study is the research of Alizadeh et al. [119]^[111]. The study was performed with piglets whose intestinal maturation is similar to the human neonates and infants. Animals were fed with GOS from 3 to 26 days, which resulted in an increase in colonic fermentation, decrease in pH and bifidogenic effect. The expression of the TJ genes was upregulated in the piglets fed with GOS as well as a higher level of defensin porcine β-defensin-2 in the colon, suggesting that the GOS-supplemented formula promotes the gut development and defence mechanism in the intestine in neonates.

The promising results obtained in vivo required confirmation in the randomized clinical trials. Pedersen et al. [120]^[112] applied GOS in a dose of 5.5 g per day for 12 weeks in 29 men with T2D. The prebiotic intake had no effect on the intestinal permeability estimated based on the urinary recovery of ₅₁Cr-EDTA, although a decreasing trend was observed. However, again, the number of participants with elevated permeability at the baseline was lower than expected (only 28%), which can explain the lack of significant differences. In another study, 5 g of GOS was consumed by 151 volunteers with obesity for three weeks [121]^[113]. The effect was compared to the probiotic strains Bifidobacterium adolescentis IVS-1 (autochthonous and selected via in vivo selection) and Bifidobacterium lactis BB-12 (commercial probiotic allochthonous to the human gut), used individually or as synbiotic combinations. None of the treatments had a significant effect on intestinal permeability. However, when the hyperpermeability was induced by a high dose of aspirin, GOS or probiotics intake improved the gut barrier tightness. Importantly, no synergistic effect was observed when GOS and probiotic were applied simultaneously.

Oligosaccharides are a big group of compounds differing in the nature of monomeric sugars. Apart from the above-mentioned ITF and GOS, there are many oligosaccharides that could potentially serve as prebiotics. The structure, characteristic and occurrence in nature was described elsewhere [122]^[114]. The examples of studies applying less popular oligosaccharides to improve the gut barrier function will be presented below.

Ducray et al. [123]^[115] evaluated the effect of Saccharomyces cerevisiae fermentate prebiotic in the protection of intestinal barrier integrity in rats during heat stress. Prebiotic was applied for two weeks before the exposition to heat. Heat stress resulted in inhibition of TJ proteins expression, a decrease of Paneth and goblet cells, a decrease of beneficial and increase of pathogenic bacteria. The intake of prebiotic before the heat treatment reduced the negative changes in the gut caused by heat, limiting the disruption of Paneth and goblet cells homeostasis and maintaining expression of TJ proteins [123]^[115]. Xylooligosaccharides (XOS) are sugar oligomers composed of xylose units linked by β-(1,4) bonds, with DP ranging from 2 to 10. The effect of XOS on the gut barrier, pancreatic islet and salivary gland inflammation in non-obese diabetic (NOD) mice were evaluated [124]^[116]. XOS supplementation delayed diabetes onset and decreased cellular infiltrations in their pancreatic islets and salivary glands. Importantly, XOS intake reduced gut permeability markers in the small and large intestines, which was accompanied by the upregulation of the mucus-related genes. The authors concluded that supplementation with XOS in early life could regulate the barrier function in a microbiota-independent manner [124]^[116].

HMOs play an important role in infant health and development; thus, in the last few decades, many studies were conducted to characterize the beneficial biological functions of HMOs and uncover the mechanistic pathways by which they are exerted [125]^[117]. This research was expanded to search for similar structures in other mammals. The widespread of the dairy industry has prompted studies into the therapeutic value of bovine milk oligosaccharides (BMOs) [126]^[118]. Hamilton et al. [127]^[119] verified the hypothesis that BMOs could prevent the deleterious effect of the high-fat diet on the gut microbiota and intestinal permeability and attenuate the development of the obese phenotype in mice. Mice were fed with a high-fat diet with or without BMO or inulin (6%/kg). BMO significantlyattenuated weight gain, decreased adiposity and decreased caloric intake. Notably, BMO and inulin intake abolished the increase in paracellular and transcellular permeability in the small and large intestines caused by the high-fat diet.

Another example of a prebiotic applied for the improvement of the gut barrier was isomaltodextrin (IMD) [128]^[120]. IMD was given to mice with low-grade chronic inflammation induced by LPS. Mice fed with IMD had decreased plasma concentrations of endotoxin, reduced macrophage infiltration into adipocytes, and increased expression of mucin 2, mucin 4 and the TJ protein claudin 4. However, the intestinal permeability measured by D-mannitol recovery was not affected by the oligosaccharide intake [128]^[120]. Gao et al. [129]^[121] evaluated the effect of Lycium barbarum polysaccharides (LBPs), alone or together with aerobic exercise, on the intestinal microbiota, gut barrier function and hepatic inflammation in rats with nonalcoholic fatty liver disease (NAFLD). Intake of LBP in a dose of 50 mg/kg for eight weeks together or without aerobic activity upregulated the expression of ZO-1 and occludin in the colon. Moreover, it reduced gut-derived LPS and hepatic LPS-binding proteins. The authors summarized that LBP could be a promising auxiliary therapy for NAFLD.

Less popular oligosaccharides and prebiotics were also applied in clinical trials. Westerbeck et al. [130]^[122] compared the efficacy of neutral GOS, FOS and acidic oligosaccharides (AOS) on intestinal permeability in 113 preterm infants. Prebiotics were added to the enteral formula between the 3rd and 30th days of life. At the baseline, the low birth weight was associated with increased lactulose to mannitol ratio. Irrespectively on the applied type of feeding (breastfeeding or the prebiotic-enriched enteral formulas), the lactulose to mannitol ratio decreased in the first week of life. Importantly, the lack of effect of prebiotics on intestinal permeability could be explained by high doses of antibiotics used in the intensive care unit (75% of all infants), which could inhibit the bifidogenic effect [131]^[123].

Another candidate for prebiotic are arabinoxylans (AX). The structure of AX consists of a backbone of β-(1,4)-linked xylose residues, which are substituted with arabinose residues on the C(O)-2 and/or C(O)-3 position. Salden et al. [131]^[123] evaluated if the supplementation of the diet with AX could reduce the intestinal permeability in individuals with obesity. The consumption of the AX for six weeks did not affect intestinal permeability analysed using a multi-sugar absorption test. Moreover, the expression of TJ proteins was not changed after AX intake. However, gene transcription of occludin was upregulated in the group supplemented with 7.5 g of AX, while the transcription of claudin-3 and claudin-4 were upregulated in the group consuming 15 g of AX [130]^[122]. Maybe a higher dosage and increase of the intervention period could give more promising results.

Βeta-glucans are soluble fibres derived from the cell walls of algae, bacteria, fungi, yeast and plants. Depending on the origin, they can have different types of glucan linkages. The structure and the effect of β-glucan supplementation on gut health were reviewed and summarized by Shoukat and Sorrentino [132]^[124].

The effect of yeast β-glucan on gut inflammation in dextran sulfate sodium (DSS)-induced colitis in mice was evaluated by Han et al. [133]^[125]. The intake of 5% of yeast β-glucan for seven days reduced clinical symptoms, inflammatory infiltrates and cell apoptosis in the colon epithelium. Moreover, the prebiotic administration ameliorated intestinal permeability and the structural integrity of TJs, which were highly impaired by DSS treatment. Mice fed with β-glucan increased the expression of the TJ genes up to 90% [133]^[125]. These results suggest that β-glucan could be beneficial in restoring the increased intestinal permeability in intestinal inflammatory diseases. Porcine models were used for the analysis of the effect of β-glucan on the gut barrier [134,135]^[126][127]. In the first study, barley-derived β-glucan was applied in different dosages in weaning pigs [134]^[126]. The high dose of β-glucan resulted in an increase in mannitol permeability and tissue conductance. Although it suggests that a high dose of β-glucan altered paracellular permeability, an increase in the epithelium surface area would also increase mannitol permeability. However, no differences in villus height, mucosal height, crypt depth or intestinal wet weight or length were noted. Therefore, it can be assumed that β-glucans exert effects on barrier function by altering the permeability of the transcellular pathway. In the second study, the influence of the β-glucan on nutrient composition and mucus permeability was evaluated [135]^[127]. In vitro digestion indicated that 90% of the β-glucan in the diet was released in the proximal small intestine. The intake of β-glucan for three days reduced the mucosa permeability to 100 nm latex beads and lipid.

The results of the effect of β-glucan on the gut barrier in clinical trials are in general contradictory to animal studies. On the one hand, Skouroliakou et al. [136]^[128] showed that barley-derived β-glucan-enriched snacks consumed for a month by healthy volunteers had no effect on intestinal permeability. However, this is another study with subjects without any gut barrier problems, and positive change cannot be expected. The reply for that was a series of studies reported by Mall et al. [137^{[129][130][131]},138,139], who evaluated the effect of β-glucan on gut health in various groups with increased gut permeability. In the first study, the authors compared the efficacy of yeast-derived β-glucan and AX on restoring colonic hyperpermeability in elderly subjects with gastrointestinal symptoms [137]^[129]. No difference in intestinal permeability was detected between young subjects and the elderly group without GI symptoms based on the SAT. The effect of nondigestible polysaccharides was assessed ex vivo in Ussing chambers mounted with colonic biopsies. The elderly with gastrointestinal symptoms had higher intestinal permeability compared to the subjects without gastrointestinal manifestation. β-glucan significantly attenuated hyperpermeability in the elderly with GI symptoms but not in healthy controls. The possible explanation given by the authors for the increase in intestinal permeability was the potential contamination of β-glucan with LPS during production from yeast or the induction of the burst of reactive oxygen species caused by β-glucan [137]^[129]. At the same time, AX decreased paracellular and transcellular hyperpermeability across the colonic mucosa of healthy controls but did not attenuate transcellular permeability in the elderly with GI symptoms. This study showed that different prebiotics have different effects on the gut barrier, and the response is dependent on the initial state of the gut [137]^[129]. In another study in elderly subjects with permeability induced by anti-inflammatory drugs, Mall et al. [138]^[130] applied oat β-glucan and AX in a dose of 12 g per day for six weeks. At the baseline, the gastroduodenal permeability, small intestinal permeability and colonic permeability were elevated. The administration of prebiotics had no effect on gut barrier function. In the third study, Mall et al. [139]^[131] investigated whether β-glucan alleviate hyperpermeability in patients with CD. β-glucan significantly attenuated mast cell-induced paracellular hyperpermeability in both patients with CD and controls. However, transcellular hyperpermeability was only significantly attenuated in villus epithelium. Ussing chamber experiments further demonstrated that the inhibitory effect of β-glucan was more pronounced in follicle-associated epithelium compared to villus epithelium in patients with CD and control. The pronounced effect of β-glucan in follicle-associated epithelium-culture was confirmed by in vitro studies, demonstrating the beneficial effects of β-glucan on intestinal barrier function.

Polyphenols are a large group of secondary metabolites characterized by multiple phenol units. These bioactive compounds are widely present in various crops, fruit and vegetables and are strong antioxidants, suggested to reduce the risk of noncommunicable diseases [140]^[132]. According to the new definition of prebiotics, polyphenols also belong to this group since microbiota metabolize polyphenols in the gut, and their derivatives confer health benefits. The number of studies performed on polyphenols increased dramatically in recent decades; however, it is a very big group of compounds in which activity can differ significantly between the classes. More studies were conducted by in vivo animal models, but still, some clinical trials were performed, and a few more were registered on clinicaltrail.gov [141]^[133]. These studies differ in terms of the type of animals or subject population, disease state, duration and dose and type of bioactive compounds. A recent review of Plamada and Vodnar [142]^[134] presents polyphenol-gut microbiota interactions. The effect of polyphenols on the gut barrier functions was summarized in an excellent review [140]^[132]; therefore, in this review, only a brief summary will be presented.

An interesting study was conducted by Song et al. [143]^[135], who evaluated the effects of grape-seed procyanidins in controlling post-weaning diarrhoea using a rat model. After supplementation of diet with 250 mg/kg of grape-seed procyanidins, improved growth performance and reduced diarrhoea occurrence were observed. As a possible explanation for these changes, the improvement of the intestinal barrier function was proposed. The intake of grapeseed procyanidins significantly reduced urinary lactulose to mannitol ratios of weaning rats compared with the control as well as upregulated the expression of the intestinal mucosal TJ proteins. Therefore, it was concluded that the improvement of the gut barrier was responsible for the reduction of diarrhoea in weaning pigs [143]^[135]. The comparison of the restoring effect of different polyphenols was reported by Shigeshiro et al. [144]^[136]. The mouse model with DSS-induced colon damage was applied to compare the effect of curcumin, quercetin, naringenin or hesperetin on the gut barrier TJs. It was found that all polyphenols partially restored the gut damage, but the level of changes was dependent on the polyphenol tested. The naringenin was found to be most efficient in intestinal barrier amelioration compared to other analysed polyphenols. In another study, chlorogenic acid was found to be very effective in restoring the gut barrier in rats with gut injury caused by endotoxin infusion [145]^[137]. Chlorogenic acid intake decreased the lactulose to mannitol ratio, reduced ileum pathological grade and oxidative stress in the gut. A recent study showed that kiwifruit polyphenol extract applied in a dose of 50 or 100 mg/kg is capable of inhibiting the impairment of the gut barrier caused by the high-fat diet [146]^[138]. Moreover, polyphenol-rich extract upregulated the expression of TJ proteins, claudin-1, occluding and ZO-1.

Considering the clinical trials, their number is not so big yet. The MaPLE project (microbiome manipulation through polyphenols for managing gut leakiness in the elderly) is an example of a complex approach aimed to verify if the modification of a diet of elderly people, increasing the intake of polyphenols can alter the microbial ecosystem to reduce the intestinal permeability [147]^[139]. The multidisciplinary approach designed in this project helps uspeople to understand the possible mechanisms in the polyphenolsmicrobiota–gut barrier interactions. The project also includes the involvement of a metabolomic approach to understand the molecular pathways involved [148]^[140]. The initial results of this project showed that 700 mg of total polyphenols daily for 8 weeks could reduce the level of zonulin in the elderly population [149]^[141]. However, further studies are required to confirm the clinical utility of polyphenol supplementation in gut barrier protection.

Another new member of the prebiotic family is polyunsaturated fatty acids (PUFA). Although they were mentioned in the ISAPP definition as candidate prebiotics [16]^[8], there is still a lack of complete scientific evidence confirming that PUFA can be considered prebiotics [150]^[142]. Notably, with recent knowledge, not all PUFA can be considered prebiotics. Omega-3 fatty acids (eicosapentaenoic acid (EPA; C20:5), docosahexaenoic acid (DHA; C22:6) and omega-6 linoleic acid (LA; C18:2) were proposed as potential prebiotic candidates [150]^[142]. Since the effect of omega-3 fatty acids on the gut barrier was summarized in a recent review by Durkin et al. [151]^[143], in this review,ere only a few examples of clinical trials will be presented.

Van der Mewre et al. [152]^[144] evaluated the effect of long-chain PUFA supplementation of the gut integrity, cognitive development and growth in rural African infants. One hundred seventy-two children were supplemented with 200 mg DHA and 300 mg EPA or 2 mL olive oil for six months. Although the applied supplementation increased the level of circulating PUFA, no improvement in the gut integrity, growth and cognitive development was observed. In another study, the effect of PUFA on the zonulin level and LPS activity was measured in 200 pregnant women with overweight [153]^[145]. Moreover, a 2 × 2 factorial design was applied to evaluate the effect of PUFA and probiotics on intestinal permeability in pregnancy. The analysis showed that intestinal permeability increased with the progression of pregnancy in women with overweight and obesity and was reflected in LPS activity. The supplementation with PUFA, with or without prebiotic, did not affect the zonulin concentration and LPS activity.

The results of the clinical trials showed that despite the big enthusiasm of the anti-inflammatory and epithelium protective effect of PUFA obtained by in vitro and in vivo studies [154]^[146], the clinically relevant effect in humans is not easy to detect. Maybe more studies need to be performed to optimize a sufficient dose and type of PUFA, which can then ameliorate gut barrier impairment.