1. Introduction
Calcium (Ca) is an important macroelement of the human body and in majority it is deposited in bones providing the structural integrity of the skeleton. Ca homeostasis is precisely controlled with coordinated action of processes such as absorption in the intestine, reabsorption from the kidney and exchange from bones. The intestinal Ca absorption is essential to ensure the appropriate concentrations of intra- and extracellular calcium fluids without bone depletion [
1]. On the other side, bones have a metabolic function since Ca is continuously exchanged between bone and blood, and can be released from bone to maintain extracellular calcium concentrations, regardless of intake.
Calcium requirements vary throughout an individual’s life, with greater needs during the periods of rapid growth in childhood and adolescence [
2]. Bone density increases until the end of puberty, when it reaches its peak value. If a normal peak bone mass is not achieved, the individual is at a higher risk for developing osteoporosis; thus, the amount of bone accrued during the paediatric years is an important predictor of an individual’s future resistance to fractures [
3]. Nevertheless, even an adequate dietary Ca intake may not ensure a proper calcium balance. Besides the amount, its absorption is a critical factor determining Ca bioavailability for bone development and maintenance. In some cases, an adverse calcium balance is observed that may result from a poor intestine absorption caused by infection, inflammation or pathology in the intestine morphology. Among the group of the calcium deficiency risk there are individuals suffering from chronic intestinal diseases, including coeliac disease (CD).
In CD, defined as a permanent gluten (mixture of proteins found in wheat, rye, and barley) intolerance with genetic etiology [
4], a chronic intestinal inflammation and malabsorption of calcium and vitamin D are observed that together with general malnutrition affects negatively bone health [
5]. Recently, a persistent villus atrophy has been associated with serious sequelae, including osteoporotic fractures [
6]. Several studies have demonstrated a low bone mineral density (BMD) both in children and adults with CD [
7,
8]. Additionally, a risk for less-than-optimal peak bone mass acquisition and a retarded growth in CD children is observed. These adverse alterations are mainly induced by the underlying disease, but, to a certain degree, also by a gluten-free diet (GFD) [
9,
10], which is the only accepted therapy for CD [
11]. Above 80% of CD children adhering to a GFD consume lower than recommended amounts of Ca [
12,
13] and vitamin D, which could be due to the reduced nutritional quality of a GFD [
14,
15].
Nutrition plays an important role in proper bones mineralization [
16]. To reduce a risk of osteoporosis, an increased calcium intake is proposed as one of the most effective strategy. However, when Ca intake remains inadequate, an improvement of Ca absorption becomes an important method of Ca balance restoration and bone health improvement. Among the dietary compounds, prebiotics are widely studied functional ingredients which can enhance the mineral absorption and bone properties [
17,
18]. Inulin, a polydisperse carbohydrate material consisting mainly of beta (2-1) fructosyl-fructose links, is an example of prebiotic naturally occurring in tubers, bulbs and tuberous roots of several edible fruits and vegetables [
19]. Inulin and other inulin-type fructans (ITFs) are resistant to digestion in the small intestine and undergo fermentation in the large intestine, resulting in short-chain fatty acid (SCFA) production. On the other hand, they stimulate growth and/or activity of selected commensal bacteria, including the health-promoting bifidobacteria and lactobacilli [
20]. Several animal studies have shown that the acidification of the gut environment resulting from bacteria fermentation of ITFs enhanced calcium and magnesium absorption and bone mineralization [
21,
22,
23] nevertheless, the human studies have shown contentious results [
24,
25,
26]. In the present study, we hypothesised that the dietary administration of inulin would influence positively the intestinal microbiota, and by that will stimulate the absorption of Ca from colon and caecum of rats, especially in the conditions of Ca malnutrition. To verify this hypothesis, a 6-weeks nutritional experiment was performed aimed to assess the impact of dietary inulin on gastrointestinal tract parameters, characteristics and activity of gut microbiota (SCFAs, PSCFAs, microbial enzymes), and mineral utilization in growing rats fed GFD with reduced Ca content, established as an experimental model that in some extend may correspond to a dietary conditions observed in many paediatric CD patients treated with GFD.
4. Discussion
In the present study, all experimental GFDs had similar energy value, thus no effect on rats body weight gain was observed. Inulin in GFDs increased the weight of the small intestine and its acidification that suggested that inulin could be metabolised to a certain extent by aerotolerant small intestinal bacteria. Recently, van den Bogert et al. [
36,
37] demonstrated that small-intestinal streptococci
S. mitis,
S. bovis and
S. salivarius differed considerably in their carbohydrate metabolism, whereas Veillonella species utilised lactic acid produced by carbohydrate-fermenting streptococci, which contributed to the synthesis of acetic and propionic acids, and led to environment acidification.
Similarly to other prebiotics, inulin affects mainly caecal parameters by influencing resident microbiota, in particular anaerobic species [
38]. In the present study, the pH of a large intestinal digesta decreased whereas the weight of caecal and colonic tissues as well as their digesta increased considerably in animals fed GFDs with inulin. The observed inulin-mediated increase in the weight of tissue of the large intestinal segments could be attributed to fructans’ ability to stimulate bacterial growth and proliferation, however in the present study, the DM content of the digesta did not increase. This indicates that apart from bacterial counts, bacterial activity considerably influenced intestinal parameters. In view of the above, the increase in the weight of caecal and colonic tissues could be partially attributed to inulin fermentation products, mainly SCFAs. Butyric acid and, to some extent, also propionic acid, are the main energy substrates for colonocytes [
39]. Butyrate stimulates the physiological proliferation of enterocytes, induce histological changes in the gut epithelium and modifications of the mucosal architecture [
40]. Whereas, the noted increase in the weight of large intestinal contents could have simply resulted from the inulin-mediated bulking effect and a higher content of water in the large intestinal digesta [
41]. Nevertheless, a fructan-mediated increase in the weight of caecal digesta could have adverse consequences and could cause discomfort to the host [
42]. On the other hand, a considerable effect on intestinal parameters could also be exerted by the level of dietary Ca intake. Recent research has demonstrated that Ca plays a number of important roles in eukaryotes as well as in prokaryotic cells [
43]. Ca
2+ ions affect the growth, division and differentiation of prokaryotic cells, and in the absence of Ca
2+,
E. coli cells ceased to divide and proliferate, and they were eventually lysed and died [
44].
The caecum is a site of intensive carbohydrate fermentation as well as proteolytic activity which is largely mediated by microbiota, where ammonia is the main metabolite. In our study, an inulin-mediated decrease in a caecal ammonia concentration was noticed that could result from luminal acidification and, consequently, inhibited protein degradation in an acidic environment. Proteases are more active at neutral or slightly alkaline pH than in acidic pH [
45]. In general, a reduction in caecal ammonia concentration is a favourable change since ammonia is the most toxic form of nitrogen and a metabolic disruptor [
46]. The results of this study suggest that inulin and/or its fermentation products could facilitate to the utilisation and/or elimination of ammonia.
Changes in the concentrations of SCFAs, bacterial enzyme activities and DGGE profiles of eubacteria and
Bacteroides affirmed an intensive fermentation of inulin by caecal microbiota. The degree of polymerisation (DP) and the solubility of fructooligosaccharides are vital criteria for SCFAs formation, which is why oligosaccharides with a low DP produced high levels of butyric acid, whereas oligosaccharides with a high DP, such as inulin, produced high levels of propionic acid [
47]. In this study, the high propionate to total SCFAs ratio appears to be typical for the fermentation of long-chain inulin. On the other hand, a high concentration of propionate in rats fed inulin could be linked with an abundance of
Bacteroidetes and several
Firmicutes species which utilise succinate as a substrate for propionate synthesis through decarboxylation of methylmalonyl-CoA to propionyl-CoA [
48]. The addition of inulin to the experimental GFD increased butyrate concentration, but only in the group fed a GFD with the recommended Ca content, which suggests that such diets could create a favourable environment for
Firmicutes.
Firmicutes species, including
Faecalibacterium,
Eubacterium and
Roseburia, are the main butyrate-producers in the colon which are able to convert butyryl-CoA to butyrate in a single-step enzymatic reaction, the butyryl-CoA: acetate CoA-transferase pathway [
49]. Surprisingly, the low concentration of butyrate in group RI could be linked with changes in the utilisation of β-hydroxybutyryl-CoA. Prokaryotic cells maintain tight control of their cytosolic Ca
2+ by means of non-proteinaceous polyhydroxybutyrate-polyphosphate (PHB-PP) complexes or Ca
2+ channels. In the latter case, Ca ions are extruded by Ca
2+-translocating ATPases or electrochemical potential-driven Ca
2+ transporters [
50]. β-hydroxybutyryl-CoA is one of key elements of butyrate synthesis in both butyryl-CoA:acetate CoA-transferase and butyrate kinase pathways [
51]. However, it is also a precursor for PHB synthesis that relies on poly-3-hydroxybutyrate (PHB) synthase [
52]. It could be hypothesized that prebiotic fermentation increases acetyl-CoA synthesis and, consequently, acetoacetyl-CoA and β-hydroxybutyryl-CoA. This leads to a metabolic switch from butyrate production to the synthesis of PHB which acts as a universal regulator of internal ion concentrations by selectively transporting ions across membranes [
53]. It is likely that a calcium-deficient GFD without inulin is not a sufficient source of carbohydrates for the production of acetyl-CoA in amounts that could be metabolically economic for the butyrate→PHB switch. Therefore, bacteria cope with low Ca levels by activating a proteinaceous system of Ca transport [
50]. On the other hand, it cannot be ruled out that nearly all available butyrate was used up by the enlarged intestinal tissue and that butyrate levels in the intestinal lumen were depleted. This observation is supported by the lowest values of Ca excretion with faeces and highest Ca absorption. Further research is needed to verify the above hypotheses.
SCFAs deliver health benefits [
54]. The possible connection between selected inflammatory processes and a reduction in the populations of butyrate- and propionate-producing bacteria has turned the researchers’ attention to the metabolism of propionate and butyrate. Machiels et al. [
55] reported a decrease of in the counts of butyrate-producing species in ulcerative colitis. Based on above, it could be expected that the acidification of the large intestine resulting from SCFAs production could promote the growth and proliferation of beneficial microbiota and reduce the number of potentially pathogenic species. Our speculations were also supported by the results of a quantitative analysis of branched SCFAs which revealed that the addition of inulin to a GFDs suppressed putrefaction processes when the intake of dietary Ca was low.
In animals fed a GFD with the required Ca content, inulin exerted a similar influence on microbiota (stimulation of
B. animalis) to that reported in other studies [
56]. An analysis of eubacterial DGGE patterns indicates that members of the phylum Actinobacteria (
Olsenella and
Bifidobacterium) seem to be less susceptible to low Ca levels than
Firmicutes, in particular the family
Lachnospiraceae. Moreover, the MDS analysis based on DGGE profiles revealed that limited intake of dietary Ca was associated with two microbiological phenotypes, which points to individual variations in response to low Ca levels. Moreover, the combined effect of dietary factors (restricted dietary Ca intake and inulin intake) led to the most scattered distribution of samples. Therefore, it could be assumed that inulin together with low Ca levels trigger substantial changes in microbial structure due to individual differences in microbial composition. The observed in the present study changes in the structure and metabolic activity of caecal microbiota seem to indicate that the combination of low Ca intake and inulin has an undesirable effect on intestinal bacteria. The presence of
Bacteroides eggerthii, which was recently proposed as a colitis-promoting species [
57], could be of particular interest in further studies on maintaining a properly balanced commensal microbiota.
The inulin-mediated modification of gut microbiota was also manifested by changes in the activity of bacterial enzymes in the caecum. The experimental GFDs with inulin increased the concentration of α- and β-glucosidase and β-galactosidase, whereas the activity of β-glucuronidase was suppressed in groups fed GFDs with inulin. Similar results were reported in other studies of fructooligosaccharides [
42,
58] which demonstrated that moderate FOS content significantly reduced the caecal activity of β-glucuronidase and increased the activity of β-galactosidase. Bile flow modulates the activity of β-glucuronidase in the large intestines of rats [
59], therefore, the inulin-induced decrease in β-glucuronidase activity could be attributed to intensified intestinal peristalsis. In general, a reduction in β-glucuronidase activity is beneficial because this enzyme exerts toxic, carcinogenic, and mutagenic effects in the gastrointestinal tract [
60]. Thus, the addition of inulin to the experimental GF diets seems to selectively modulate the composition of microbiota, and it potentially eliminates harmful bacteria that enhance the activity of β-glucuronidase in the caecum.
Fermentable carbohydrates, including inulin, were found to increase Ca and Mg absorption in the large intestine [
21,
22], but this effect was dependent on the dose, structure of fermentable carbohydrates as well as the duration of the experiment. In the present study, we hypothesised that the addition of inulin to a GFD would increase Ca absorption in the large intestine, in particular in animals with low dietary Ca intake. Inulin intake stimulated Ca and Mg absorption in the group fed a calcium-deficient GFD however, the increase in Ca and Mg absorption was not accompanied by changes in their retention. Coudry et al. [
23] found that dietary inulin had a more beneficial effect on Ca absorption in shorter-term (17 days) than in long-term experiments (up to 40 days), and that inulin-mediated changes were more profound when dietary Ca was in low supply. In contrast, in another study, a 6-week prebiotic intervention (oligofructose + acacia gum) had no effect on Ca absorption or Ca retention in aged ovariectomized rats [
61]. In the present study performed on growing rats, which are characterised by a higher demand for Ca and higher Ca absorption capacity, the increase in Ca and Mg absorption could be attributed to prolonged inulin fermentation in the caecum, which was manifested by changes in large intestinal parameters, mainly an increase in SCFA synthesis and acidification of caecal and colonic contents. This indicates that acidic caecal pH promotes the solubilisation of Ca and Mg. The observed increase in Ca absorption in animals fed GFD with inulin is a promising result that could generate favourable surplus Ca for bone, however further in vivo studies are required to assess the impact of a dietary application of inulin on calcium metabolism and bone mineralisation.