Inulin protects against the harmful effects of dietary emulsifiers on mice gut microbiome

Introduction

Inflammatory bowel diseases are a chronic, heterogeneous group of diseases that cause inflammation in the gastrointestinal tract, including Crohn’s disease and ulcerative colitis (Malik, 2015). With the increase in the prevalence of inflammatory bowel diseases, they have become an important public health problem worldwide (Ng et al., 2017). Although the etiology of inflammatory bowel disease is not completely known, alteration in the gut microbiota composition, called dysbiosis, is thought to be an important factor in pathogenesis (Khan et al., 2019). Chronic intestinal inflammation usually occurs in the terminal ileum and colon, which is the region of the highest intestinal microflora concentration (Akram, Garud & Joshi, 2019).

A variety of factors induce microbiota dysbiosis and encroachment, including consumption of the Western-style diet. The Western-style diet includes high amounts of protein, saturated fat, sugar, processed foods, and food additives, while it includes low consumption of vegetables, fruits, and fiber (Schreiner et al., 2020). Emulsifiers are a class of food additives used in many processed foods to extend shelf life and improve organoleptic properties. Microbiota dysbiosis caused by emulsifiers is considered to be an important factor in the association between consumption of ultra-processed foods and the development of chronic inflammatory diseases (Vo, Lynch & Roberts, 2019).

Recent studies have reported that various food emulsifiers and thickeners such as carboxymethyl cellulose (CMC), polysorbate 80 (P80) and carrageenan increase intestinal permeability, bacterial translocation and the passage of bacterial components such as lipopolysaccharides into the systemic circulation, thus causing intestinal and systemic inflammation (Shang et al., 2017; Viennois et al., 2020). It has been stated that emulsifiers may play a role in the development of various diseases from inflammatory bowel disease to metabolic syndrome by changing the composition of fecal microbiota, and the amount and composition of short-chain fatty acids (SCFA) (Chassaing et al., 2015; Furuhashi et al., 2020).

Prebiotics are non-digestible nutritional components usually included in dietary fiber that can be fermented by the gut microbiota and selectively stimulate the growth and/or activity of health-related gut bacteria (Quigley, 2019). Inulin is a general term for all linear fructans (inulin–type fructans) with different degrees of polymerization. The length of the fructosyl chains of inulin molecules can range from 2–60 fructose units. The main sources of inulin are chicory, asparagus, onions, garlic, banana, rye, barley, and artichokes. Inulin protects the microbial population, which helps the host to defend against pathogen translocation, supports the epithelial barrier function, and increases the production of SCFA and has positive effects on the prevention of symptoms of IBD and immune modulation (Akram, Garud & Joshi, 2019; Tawfick et al., 2022).

The protective effect of inulin on the gut microbiome and inflammation caused by emulsifiers have not been investigated before. This study mainly aimed to investigate the effect of CMC and lecithin on intestinal inflammation, as well as the potential of inulin to prevent emulsifier-induced disruption of host microbiota homeostasis and how close inulin get the emulsifier groups to control.

Materials & Methods

Results

Intestinal histopathology and inflammation

Histopathological examination of the small intestine revealed a normal microscopic appearance in the control group. In the CMC and lecithin groups, shortening and bulking of the villus were observed in the ileum of the mice. In the CMC+inulin and lecithin+inulin groups, elongation was observed in the villus. Intestinal flora was significantly available in the intestinal lumen in the CMC and lecithin groups, but this microbial migration did not increase serum inflammatory parameters. Inulin administration decreased amount of bacteria in both the CMC and lecithin groups, but did not result in complete improvement (Fig. 1). The results of statistical analysis of histopathological score, villus length, crypt length, and villus thickness are shown in Table 2.

Table 2:

Histopathological examination and inflammatory markers of groups (mean ± SD).

	Groups (n:6)
	Sample	Control	CMC	Lecithin	CMC+Inulin	Lecithin+Inulin	P value
Histopathology scores	Ileum	0.0 ±0.0a	1.0 ±0.25b	1.0 ±0.25b	0.5 ±0.22a	0.7 ±0.21b	<0.01**
	Colon	0.2 ±0.16a	1.3 ±0.21b	1.7 ±0.21b	0.5 ±0.22ac	0.8 ±0.16c	<0.001***
Villus length (µm)	Ileum	399.8 ±9.58a	265.7 ±10.36b	250.3 ±7.64b	291.0 ±6.25c	298.8 ±4.79c	<0.001***
	Colon	103.0 ±4.65a	83.3 ±1.11b	77.8 ±3.12b	90.3 ±0.91c	87.0 ±6.16c	<0.001***
Villus thickness (µm)	Ileum	43.5 ±2.20a	56.2 ±1.07b	57.5 ±1.17b	43.3 ±0.66a	41.7 ±1.25a	<0.001***
	Colon	48.7 ±1.72a	54.3 ±0.55c	53.3 ±1.74bc	49.2 ±0.70a	50.0 ±0.70ab	<0.001***
Crypt lenght (µm)	Ileum	67.3 ±2.94a	42.8 ±3.06b	30.7 ±2.58c	52.7 ±2.80d	57.8 ±1.47e	<0.001***
	Colon	27.8 ±5.49a	15.3 ±2.50bc	11.7 ±1.86b	17.0 ±2.09c	16.8 ±1.94c	<0.001***
Goblet cell count	Ileum	55.8 ±2.24a	40.5 ±0.88b	42.3 ±1.14b	54.5 ±0.56a	51.0 ±2.38a	<0.001***
	Colon	108.8 ±2.35a	89.0 ±0.63b	92.5 ±1.47b	102.5 ±1.23c	103.5 ±1.66c	<0.001***
Lcn-2	Stool	4.7 ±3.49	9.7 ±3.29	5.7 ±3.77	4.1 ±2.98	6.2 ±3.54	0.077
IL-6	Serum	105.8 ±8.41	96.3 ±11.73	101.5 ±10.10	111.6 ±2.98	102.3 ±7.42	0.063
IL-10	Serum	261.2 ±40.52a	179.5 ±29.47b	186.1 ±25.1b	238.6 ±40.52a	220.8 ±32.58a	0.001**

DOI: 10.7717/peerj.17110/table-2

Notes:

a, b, c, dThere are statistical differences between the means having the different superscripts on the same line.

**p < 0.05.

***p < 0.001.

Masson’s trichrome staining of the ileum and colon revealed a decrease in the amount of collagen in the connective tissue in the CMC and lecithin groups compared to the control group, and it was observed that the administration of inulin slightly normalized the connective tissue (Fig. 2). In PAS staining, the number of goblet cells decreased significantly in the CMC and lecithin groups in the ileum and colon. Inulin administration increased the number and size of goblet cells in both CMC+inulin and lecithin+inulin groups (Fig. 3). There was no significant difference between the groups in terms of serum cytokines IL-6 and IL-10 and fecal levels of the inflammatory marker (P>0.05) (Table 2). However, lipocalin levels, which were higher in CMC group (9.7 ± 3.29 ng) compared to control group (4.7 ± 3.49 ng) decreased to 4.1 ± 2.98 ng when inulin was administered, and the result was significant in the pairwise comparison (P = 0.01).

Fecal microbiota analysis

A total of 1,148,814 high-quality reads were obtained from 25 samples (ranging from 28,345 to 60,283 reads per sample). No significant difference was observed for ASVs and Shannon index, however, both tended to be lower, especially in CMC group. Faith’s phylogenetic diversity was significantly lower in CMC group compared to the lecithin and CMC+inulin groups (P = 0.028 for both comparisons; Fig. 4A). This result indicates that CMC administration decreased the phylogenetic diversity, while inulin administration leads to a significant increase in Faith’s pd compared to the CMC group.

Microbial community structure were analysed by employing Bray-Curtis dissimilarity which is based on abundance and unweighted UniFrac distance which encounters the presence/absence of ASVs. Results showed that all groups were clustered separately, which was more pronounced in both inulin-administered groups according to Bray-Curtis dissimilarity (PERMANOVA, P < 0.05; Fig. 4B). Unweighted UniFrac distances also revealed significant differences for all comparisons except for CMC+inulin vs. control group, which may be explained by a shift from a CMC-associated microbiome to control group after inulin administration (PERMANOVA, P < 0.05; Fig. S1A, Table S1).

It was observed that Bacteroidetes phylum was dominant in all groups, followed by Firmicutes, and the rest consisted of Campylobacterota, Actinobacteria, Verrucomicrobia, Proteobacteria and Patescibacteria phylum. Actinobacteria and Verrucomicrobia phylum were found to be higher in CMC+inulin group than in the other groups (Fig. 4C). Muribaculaceae, Prevotellaceae, Lachnospiraceae, Helicobacteraceae, and Bacteroidaceae were the most abundant families among all groups. CMC exposure and inulin administration led to an increase in Prevotellaceae levels compared to the control and lecithin groups (Fig. S2).

We used the LEfSe algorithm to determine significantly differentiated taxa between groups. In general, the gut microbiome in the lecithin group was likely less affected compared to CMC group (Figs. 5A–5B, Figs. 6A–6B). Marvinbryantia, a member of Lachnospiraceae, and Fournierella, a member of Ruminococcaceae family significantly increased and Lactobacillus significantly decreased in the CMC group compared to the control group (Fig. 5A, Table S2). Lecithin group showed a significant enrichment in Eubacterium ruminantium and decrease in Bifidobacterium compared to the control group (Fig. 5B). In addition, the increased Helicobacter in the lecithin group decreased with the administration of inulin (Fig. 6B, Fig. S1B). CMC reduced Actinobacteria, Bifidobacterium, Lactobacillus, Verrucomicrobiota, and Akkermansia, whereas administration of inulin reversed this effect (Fig. 4C, Fig. 6A, Table S2). Administration of inulin resulted in an increase in Prevotellaceae NK3B31, Prevotellaceae UCG 001, Gordonibacter, Christensenellaceae, Clostridia UCG 014, Eubacterium nodatum group, and Peptostreptococcales in both CMC and lecithin groups (Figs. 6A–6B, Table S2).

Discussion

The Western-style diet is characterized by a high intake of fat, sugar, protein, processed foods, and low dietary fiber. Processed foods are industrial formulations that contain many food additives, including flavor enhancers, colorants, and emulsifiers (Monteiro et al., 2018). It has been reported that emulsifiers and thickeners may have health effects by disrupting the barrier function of intestinal mucosa, alterations the functional properties of the gut microbiota, and causing mucosal inflammation (Halmos, Mack & Gibson, 2019). Prebiotics play an important role in regulating the microbiota by inhibiting the production of SCFA, the proliferation of beneficial bacteria, and preventing colonization of pathogenic microorganisms (Sanders et al., 2019). This study examined the effects of different emulsifiers on the intestinal microbiota and intestinal inflammation and the potential of inulin as a means of preventing disruption of host microbiota homeostasis. Our primary hyphothesis was to test whether inulin administration improve emulsifier-induced microbial community structure by getting it closer to that of control mice.

High dietary fiber intake has been associated with lower body weight and prevention of weight gain. These effects have been reported to be due to suppresion of hunger, reduction in food intake, alterations in microbiota, acceleration of gastrointestinal transit, reduction in nutrient absorption, and changes in SCFA (Chen et al., 2018; Schäfer et al., 2021). It has been reported that inulin administration reduced food intake and weight gain in mice with high-fat diet and a streptozotocin-induced diabetes model (Shao et al., 2020). In our study, the inulin groups receiving both CMC and lecithin had lower weight gain than the ones in CMC- and lecithin-only groups, in parallel with the decrease in food intake. Consistent with a previous study in which mice were administered CMC, P80, lecithin, and gum arabic, no significant difference in food intake was observed between CMC, lecithin, and control groups (Sandall et al., 2020). We found that the difference in weight gain between the groups was not statistically significant as in previous studies (Daniel, Gewirtz & Chassaing, 2023).

The disrupted mucosal barrier promotes intestinal inflammation by increasing intestinal permeability and exposure to luminal contents, and initiating an immunological response (Michielan & D’Incà, 2015). When mice genetically predisposed to inflammatory bowel disease (IL-10 gene-deficient) were administered a 2% w/v CMC solution for 3 weeks, increased bacterial growth in the intestinal mucosa, decreased villus length, increased inter-villus space, migration of bacteria to the bottom of the crypts, and increased bacterial adhesion to the mucosa, which were similar to inflammatory bowel disease in humans were observed (Swidsinski et al., 2009). Similarly, in another study it was reported that CMC and P80 induced colitis in genetically susceptible mice, but not in wild-type. In addition, they led to chronic intestinal inflammation, shortened the colon and damage to the epithelium in both types mice. It has been reported that emulsifier exposure increased bacterial adhesion in the colon twofold, reduced the contact distance of some bacteria to the epithelium, and this invasion of the microbiota was associated with a decrease in mucus thickness (Chassaing et al., 2015).

In agreement with previous studies, (Chassaing et al., 2015; Swidsinski et al., 2009) it was found that both CMC and lecithin caused shortening and bulking of the villus in the ileum. Intestinal flora was significantly available in the intestinal lumen in the CMC and lecithin groups, but this microbial migration did not increase serum inflammatory parameters. In a similar study, CMC was reported to increase the immigration of microbiota into the epithelium, which was not associated with serum inflammatory parameters (Daniel, Gewirtz & Chassaing, 2023). We evaluated the effect of inulin on this condition for the first time, and observed that administration of inulin elongated the villus and decreased amount of bacteria in the colon lumen in both the CMC and lecithin groups, but did not result in complete improvement.

Mucus is a viscous fluid rich in mucin glycoprotein secreted by goblet cells. Mucus secretion from goblet cells is regulated by intestinal microorganisms and host-spesific metabolites. Mucin-2 produced by goblet cells organizes the mucus layer and transmits tolerogenic signals to dendritic cells, limiting the immunogenicity of intestinal antigens (Okumura & Takeda, 2018). CMC and P80 have been reported to damage to the mucus layer, increase bacterial adhesion, and have a more proinflammatory microbiota composition (Chassaing et al., 2015). In a recent study, the composition of the microbiota altered in terms of mucin-destroying bacteria, the colon mucus became thinner, and intestinal permeability increased in mice given CMC for 12 weeks. When mice were administered galactooligosaccharide to improve the damaged intestinal microbiota, mucin-2 gene expression increased, mucus layer thickness and intestinal barrier function were improved (Xu et al., 2019). In another study, the number of goblet cells decreased in mice given CMC and P80, and the mucus barrier was strengthened when A. muciniphila was given (Daniel, Gewirtz & Chassaing, 2023). Also, in this study, it was found that the number of goblet cells decreased in the CMC and lecithin groups when compared with the ones in control group. As a new solution suggest, we administered the inulin to CMC and lecithin groups and observed increases the number and size of goblet cells. Similarly, it was reported that cecal mucin levels increased in mice given 2.5g/kg and 5g/kg body weight of standard inulin or short chain inulin by increasing the relative abundance of butyrate-producing bacteria (Zhu et al., 2017).

An important consequence of inflammatory bowel disease is intestinal fibrosis. As fibrosis progresses, some segments of the intestine become narrowed, which affect severely the structure and functionality of the intestine and worsen the patients’ quality of life. An essential fibrosis-initiating factor is chronic inflammation. Inflammatory bowel disease frequently results in intestinal fibrosis, which is typically described as excessive buildup of scar tissue in the gut wall (Wang et al., 2022). In our study, no fibrosis was observed, even a slight decrease in the amount of collagen in the connective tissue of the propria mucosa was observed in the CMC and lecithin groups compared with the control group. This may indicate that intestinal permeability increases in these groups.

It was reported that fecal lipocalin-2 levels in mice given CMC and P80, increased only slightly in wild-type mice, but increased sharply (approximately 10-fold) in genetically susceptible mice. Emulsifiers did not cause significant colitis in nongenetically susceptible mice, but caused epithelial damage and changes in colon morphology and low-grade inflammation (Chassaing et al., 2015). In another study, dietary CMC intake increased lipocalin levels compared to the control (9.5 ± 2.8 vs 2.7 ± 0.8 ng/g feces, respectively). In addition, it was found that inflammation decreased when galactooligosaccharides were administered to mice to improve the damaged gut microbiota (Xu et al., 2019). In this study, there was no statistically significant difference between the groups in terms of lcn-2, but the increased lcn-2 in the CMC group compared to the control (9.7 ± 3.29 vs 4.7 ±3.4 9 ng, respectively) was decreased with inulin administration (4.1 ± 2.98 ng) (P = 0.012). Similarly, it was reported that addition of A. muciniphila decreased lcn-2 levels in mice given CMC (Daniel, Gewirtz & Chassaing, 2023). In human study, it was observed that administration of 15g/day CMC does not affect levels of lipopolysaccharide and flagellin in feces and does not cause a difference in serum inflammatory parameters (Chassaing et al., 2022). In this study, no significant difference was found between groups in serum levels of IL-6 and IL-10 in mice.

Intestinal dysbiosis can cause impaired mucosal immune response and production of proinflammatory cytokines in genetically susceptible hosts, contributing to inflammation in inflammatory bowel diseases (Amoroso et al., 2020). The effects of twenty different emulsifiers on the microbiota were invastigated in an ex vivo study, and reported that CMC and P80 had a significant effect on the microbiota, whereas lecithin had a mild effect, and there was no significant difference between the groups in terms of alpha diversity (Naimi et al., 2021). In this study, it was observed that there was no significant difference between the groups in terms of alpha diversity, but the CMC changed the community structure more. Microbial community composition changed significantly after inulin administration in both the lecithin and CMC groups, but was more obvious in the CMC group. Moreover, the inulin effect was also higher in the CMC group in terms of the richness and evenness of the microbial community. Indeed, this might be due to the CMC-induced major alterations in the gut microbiome. From an ecological perspective, lower colonization resistance in CMC-associated microbiome could lead to this remarkable change in gut microbiome after inulin administration, as the major taxonomic groups such as Clostridiales and Lactobacillales decreased upon CMC exposure (Caballero-Flores, Pickard & Núñez, 2022). Qualitatively unweighted UniFrac distance measurements showed that community structure changed significantly in both CMC and lecithin groups compared to the control. A similar study reported that CMC and P80 exposure differed from the ones in control group in terms of unweighted UniFrac distance measurements (Daniel, Gewirtz & Chassaing, 2023).

In the gut microbiota, Firmicutes and Bacteroidetes are the most abundant phyla, whereas the phyla Actinobacteria, Proteobacteria, and Verrucomicrobia consisted of the rest (Illian, Brambilla & Parolini, 2020). Similar results were also found in this study. In a study examining the effects of various emulsifiers on the microbiota composition taxonomically, CMC and lecithin were reported to cause a decrease in the presence of Verrucomicrobia and Akkermensia and an increase in the Lachnospiraceae family (Naimi et al., 2021). In a human study, consumption of 15g CMC in healthy adults increased postprandial abdominal discomfort and altered the composition of the gut microbiota, decreased Faecalibacterium prausnitzi and Ruminococcus spp., and increased the presence of Lachnospiraceae. The study also showed that the response of the microbiota to this food additive can be highly individualized (Chassaing et al., 2022). Oligofructose-enriched inulin (15 g/day) decreased disease severity, and increased fecal butyrate levels, fecal abundance of Bifidobacteriaceae, Ruminococcaceae, and Faecalibacterium in patients with mild and moderately active ulcerative colitis (Valcheva et al., 2019). It was reported that in mice fed a high-fat diet, Lachnospiraceae increased, Bifidobacterium and Lactobacillus decreased, and the administration of both short-chain and long-chain inulin reversed this effect. In addition, the presence of Helicobacter was found to increase with consumption of high-fat diet and to be positively correlated with endotoxin, and Helicobacter decreased with administration of inulin (Li et al., 2020). Actinobacteria, including Bifidobacteria, is known to have a beneficial effect on the host health. A decrease in Bifidobacteria has been associated with inflammatory bowel disease (Binda et al., 2018). In our study, CMC reduced Actinobacteria, Bifidobacterium, Lactobacillus, Verrucomicrobiota, and Akkermansia, whereas administration of inulin reversed this effect. Marvinbryantia, which is known to be a cellulolytic member of Lachnospiraceae (Wolin et al., 2003) and Fournierella belongs to Ruminococcaceae family which were also known for their ability to ferment cellulose (Flint et al., 2012) were significantly increased in the CMC group compared to control. In addition, it was observed that the increase in Helicobacter in the lecithin group decreased with the administration of inulin. A recent study reported that in mice given CMC and P80, the level of A. muciniphila decreased compared to the control, resulting in inflammation in the colon, especially infiltration of inflammatory cells in the mucosa and submucosa. Administration of A. muciniphila reduced low-grade intestinal inflammation caused by emulsifiers and altered the microbiota composition (Daniel, Gewirtz & Chassaing, 2023). A. muciniphila is abundant in the intestinal tract and uses mucin as a source of carbon, nitrogen, and energy. A. muciniphila, the only genus belonging to the Verrucomicrobia phylum, increases the thickness of the intestinal mucosal layer and improves intestinal barrier function (Zhai et al., 2019).

It has been reported that SCFAs are significantly positively correlated with Prevotellaceae UCG 001, Parabacteroides, and Bacteroides (Zhu et al., 2022). Prevotella genus is more abundant in individuals who eat a diet rich in plant sources such as a Mediterranean and a vegetarian diet (Ley, 2016). In our study, Prevotellaceae NK3B31, Prevotellaceae UCG 001, Clostridia UCG 014, and Peptostreptococcales were found to change significantly after inulin administration in both lecithin and CMC groups. In addition, Prevotellaceae was higher in the CMC+inulin and CMC groups compared to lecithin groups. Another study reported that prophylactic administration of turmeric polysaccharide ameliorated the dextran sulfate sodium-induced gut microbiota imbalance in favor of Clostridia UCG 014, which produces the tryptophan metabolite indoleacrylic acid (Yang et al., 2021). Peptostreptococcus species also produce indoleacrylic acid, which promotes barrier function of intestinal epithelium and attenuates inflammatory responses. In IBD patients, the ability of microbes to degrade mucins and metabolize tryptophan was decreased (Wlodarska et al., 2017).

We acknowledge the following limitation. We evaluated the effects of only two emulsifiers in this study. Generalization of the microbiome-specific effect can be concluded by evaluating the effect of other emulsifiers together with dose-dependent experiments.

Conclusion

In conclusion, CMC and lecithin, which are commonly used as emulsifiers in processed foods, disrupt the intestinal barrier function and cause intestinal inflammation by alterations in the composition of the fecal microbiota. Carboxymethyl cellulose was more effective than lecithin on microbiota composition. The composition of the microbiota, particularly deteriorated by CMC exposure, was improved by administration of inulin. Although the effects of different emulsifiers have been shown in the current literature, studies on lecithin are quite limited. Additionally, nutritional components are needed to regulate the disrupted microbiota and reverse these effects. This is the first time, the benefical effect of inulin on microbiota dysbiosis caused by emulsifiers was investigated. Further clinical studies are needed to confirm these findings to the prevention of the development and progression of inflammatory bowel diseases in human.

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