1. Introduction
Agavins are branched neo-fructans found in
Agave plants, which contain a mixture of β(2-1) and β(2-6) linkages [
1,
2]. The degree of polymerization (DP) and the chemical structure of agavins become more complex as the plant ages. Plants from two to four years old have a high content of agavins with low DP and simpler chemical structures, while plants from five to seven years old contain a large proportion of high-DP agavins and highly-complex chemical structures [
3].
Agavins act as prebiotics inducing benefits to host health by providing specific changes in the composition and/or activity of the gut microbiota [
4]. Due to their structural complexity, endogenous gastrointestinal enzymes cannot degrade agavins during their passage through the stomach and the small intestine; so they reach both the cecum and colon, where they are fermented by saccharolytic microbiota present in these sites, producing short chain fatty acids (SCFA), mostly acetate, propionate, and butyrate. SCFA are very important because they reduce body weight gain, through G-protein-coupled receptors (GPRs), influencing the secretion of hormones involved in appetite control [
5,
6,
7]. In addition, SCFA increment through agavins fermentation in both cecum and gut induces a pH drop; which might change the intestinal microbiota structure [
8,
9].
On the other hand, earlier investigations showed that mice fed with standard or high-fat diets with agavins of low DP led to body weight loss [
10,
11,
12]. However, the microbial mechanisms remain unclear [
12,
13]. New molecular techniques that enable analysis of non-cultivable bacteria are starting to be applied in studies investigating the impact of prebiotics on the cecal microbiota. For example, investigations examined the effects of oligofructose (linear fructans) on cecal microbiota using the 16S rRNA gene sequencing technique, showed that the intake of oligofructose in mice not only stimulated the growth of
bifidobacteria and
lactobacilli, but also increased other bacteria such as
Streptococcus,
Clostridium,
Enterococcus,
Olsenella,
Akkermansia, and
Allobaculum [
14,
15]. The abundance of specific taxa, such as
Bifidobacterium spp. and
Akkermansia muciniphila has been negatively associated with inflammation in adipose tissue, circulating glucose, leptin, triglycerides, and insulin [
16], whereas the enrichment of
Allobaculum has been associated with body weight loss in obese mice [
17]. On the other hand, Firmicutes and Bacteroidetes are usually the most abundant members of the cecal microbiota; however, the ratio of these bacterial groups can change over time or by different factors, such as age, environment, or diet, and especially those with a high fat content [
18,
19,
20].
In the present work we used agavins from four-year-old
Agave tequilana plants containing a high proportion of short DP fructans, and studied the response on the microbiota of mice, continuing our previous study on prebiotic supplementation in overweight mice [
21]. Here we present changes of cecal microbiota after a diet shift and agavins supplementation, and the possibility of their association with body weight loss in overweight mice. Our hypothesis was that agavins supplementation might improve the host health, through the enrichment of probiotic bacteria, in relation to the diet shift alone.
To our knowledge, this is the first report on the global effects of a diet shift and agavins supplementation on the cecal microbiota composition through a 16S rRNA analysis in overweight mice. Finally, agavins (branched fructans) effects were compared to oligofructose (linear fructans), which was used as a positive control to evaluate the cecal microbiota changes.
2. Materials and Methods
2.1. Animals and Diets
Forty-two male C57BL/6 mice (12 weeks old at the beginning of the experiment were obtained from the Universidad Autonoma Metropolitana, Mexico City, Mexico) and housed in a temperature and humidity controlled room with a 12 h light-dark cycles. Mice were maintained in individual cages since water intake containing the fructans was measured every day. The animals were subject to a two-phase trial, the first to gain weight and the second to lose weight (
Supplementary Figure S1). In the first phase mice were fed with standard (
n = 12; 5053 Lab Diet, St. Louis, MO, USA) or high-fat diets (
n = 30; 58Y1 Test Diet, St. Louis, MO, USA) for five weeks. The standard diet (5053 Lab Diet) contained 62.4% calories from carbohydrates (28.6% starch, 3.24% sucrose, 1.34% lactose, 0.24% fructose, and 0.19% glucose), 24.5% from proteins, and 13.1% from fat. The high-fat diet (58Y1 Test Diet) had 20.3% calories from carbohydrates (16.15% maltodextrin, 8.85% sucrose, and 6.46% powdered cellulose), 18.1% from proteins, and 61.6% from fat (31.7% lard and 3.2% soybean oil). In the second phase, healthy control mice were kept with the standard diet (ST-ST10;
n = 8), and the overweight mice were shifted to the standard diet alone (HF-ST10;
n = 8) or supplemented with agavins (HF-ST + A10;
n = 8) or oligofructose (HF-ST + O10;
n = 8) for five more weeks. Food and water were provided ad libitum throughout the experiment.
Mice experiments were conducted according to the Mexican Norm NOM-062-ZOO-1999 and approved by the Institutional Care and Use of Laboratory Animals Committee from Cinvestav-Mexico (CICUAL; protocol number 0091-14).
2.2. Agavins and Oligofructose Fructans
Agavins from four-year-old
Agave tequilana Weber blue variety plants were extracted and purified in our laboratory and presented an average DP of 8 [
21]. Oligofructose was bought from Megafarma
® (Mexico City, Mexico) and possess an average DP of 5. Agavins and oligofructose were added in the water at a concentration of 0.38 g/mouse/day [
15,
22].
2.3. gDNA Extraction
Cecal contents were collected before and after the fructans supplementation (at five and 10 weeks, respectively). At the end of first and second experimental phase, mice were anaesthetized with a 60 mg/kg intraperitoneal dose of sodium pentobarbital and the gastrointestinal tract was exposed for cecum removal. Cecal content was snap frozen in liquid nitrogen and stored at −70 °C until their use. Genomic DNA was extracted using the ZR Fungal/Bacterial DNA MiniPrep (Irvine, CA, USA), following the manufacturer’s instructions. The concentration and purity of DNA were evaluated using a Nanodrop spectrophotometer. Extracted DNA was stored at −20 °C until its use.
2.4. PCR Amplification of the V4 Region of the Bacterial 16S rRNA Gene
To assess microbial composition, the V4 region of the bacterial 16S
rRNA gene was amplified with barcoded fusion primers (F515/R806) [
23]. PCR reactions were carried out in triplicate, 25 μL reactions with 5 μM forward and reverse primers, 2 μL template DNA, and 1X of HotMasterMix (5 PRIME, Gaithersburg, MD, USA). Thermal cycling of PCR reactions consisted of an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 1 min, and extension at 72 °C for 90 s, with a final extension of 10 min at 72 °C.
2.5. Amplicon Quantitation, Pooling, and Sequencing
DNA concentration for each amplicon was measured using the Quant-iT PicoGreen dsDNA reagent and kit (Thermo Scientific, Waltham, MA, USA). Assays were carried out using 2 μL of cleaned PCR product in a total reaction volume of 200 μL in black, 96-well microtiter plates. Fluorescence was measured on a BioTek Synergy HT plate reader using the 480/520-nm excitation/emission filter pair. Following quantitation, cleaned amplicons were combined in equimolar ratios into a single tube. The final concentration of the pooled DNA was determined using the Qubit high-sensitivity dsDNA assay (Invitrogen, Carlsbad, CA, USA). Sequencing was carried out on the Illumina MiSeq platform at New York University.
2.6. Sequence Analysis
Sequences were processed and analyzed in the QIIME software package (Quantitative Insights Into Microbial Ecology, v1.8.0, La Jolla, CA, USA) following the pipeline described by Caporaso et al. [
24]. Sequences were removed from the analysis if they were <200 or >350 nt in length, had a mean quality score < 20, contained ambiguous characters, contained an uncorrectable barcode, or did not contain the primer sequence. Remaining sequences were assigned to samples by examining the 12-nt barcode. Similar sequences were clustered into operational taxonomic units (OTUs) using the open reference method. Taxonomic assignments for each OTU were made using the Greengenes database (May 2013) with a minimum identity of 97%. Finally, an OTU table was used to generate relative abundance plots and to calculate alpha and beta diversity (alpha diversity refers to the diversity within each sample, and beta diversity refers to patterns of similarities and differences among samples). All communities were rarefied up to 6525 reads per sample to calculate the bacterial diversity.
The raw sequences supporting the results of this article are available in the NCBI Sequence Read Archive repository under accession no. SRX1532779.
2.7. LEfSe Analysis
Linear discriminant analysis effect size (LEfSe) was used to detect significant changes in relative abundance of microbial taxa between overweight mice fed with the standard diet and fructans supplements. Briefly, LEfSe is an algorithm for applying 16S
rRNA gene datasets to detect bacterial organisms that are differentially abundant between two or more microbial environments [
25]. LEfSe first identifies features that are significantly different among biological classes using the non-parametric factorial Kruskal-Wallis ran-sum test, and then LEfSe utilizes linear discriminant analysis (LDA) to estimate the effect of each differentially-abundant feature.
2.8. SCFA and pH Determinations
A weight of 0.05 g of homogenized cecal content was placed in a conic tube. The pH was measured directly in the cecal sample through insertion of a microelectrode (PHR-146, Lazar Research Laboratories Inc., Los Angeles, CA, USA) in the tube. The pH value was read when stability was achieved; after each reading, the microelectrode was removed and rinsed with distilled water. SCFA analysis was carried out in the same sample using gas chromatography and flame ionization detection (GC-FID) [
26]. Briefly, 0.3 mL of Milli-Q water was added to the tube with cecal content. The solution was acidified with 0.05 mL of H
2SO
4 and SCFA were extracted by shaking with 0.6 mL of diethylether and subsequent centrifugation at 10,000×
g for 30 s. One microliter of the ether phase was injected directly onto a Nukol
™ capillary column (30 m × 0.32 mm; Supelco, Bellefonte, PA, USA) using an injector temperature of 180 °C and nitrogen as the carrier gas. The column temperature was initially 80 °C, then increased to 120 °C at 15 °C/min and kept at this temperature for 10 min, following an increment to 200 °C at 10 °C/min and remaining at this temperature for 10 min. The detector temperature was 230 °C. The identification and quantification of the SCFA were carried out using the retention times and calibration curves for each acid, respectively.
2.9. Statistical Analysis
Results are presented as mean ± SEM. Differences between ST5 and HF5 groups were assessed by Student’s t test. Differences between the diets were determined using a one-way ANOVA followed by Bonferroni’s multiple comparison tests. Differences were considered significant when p < 0.05. Statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA).
4. Discussion
We previously reported that agavins supplementation to a standard diet reverted the metabolic syndrome (including body weight loss) induced by high-fat diet consumption [
21]. However, we do not know that the changes originated with agavins consumption on the gut microbiota, which could be associated with this effect. Therefore, the present study describes the changes in the cecal microbiota, SCFA production, and pH values associated with body weight loss in overweight mice.
High-fat diet consumption for five weeks significantly increased the body weight gain of mice, and also led to a substantial decrease of bacterial diversity in the cecal microbiota (
Figure 2A and
Figure 3A); which is consistent with the effects of fat in reducing diversity, as previously reported [
28,
29,
30].
Firmicutes and Bacteroidetes are the most abundant members of the cecal microbiota, however, the ratio of these bacterial groups can change over time or by different factors, such environment and diet (especially those with a high fat content) [
18,
19,
20]. An increase of the ratio of Firmicutes/Bacteroidetes was seen in overweight mice, which has been associated with obesity [
18,
20].
We found that a high-fat diet enriched
Bilophila, a genus that include some opportunistic pathogens (for example
B. wadsworthia [
31]). Microbial changes under a high fat diet reduced cecal SCFA concentrations and increased pH, which is consistent with altered microbial metabolic activity, as previously reported [
32].
Supplementation with agavins or oligofructose showed an accelerated body weight loss with partially restored the cecal microbiota diversity (
Figure 2A and
Figure 3A), as well as an increase in the SCFA concentrations and acidic pH (
Table 2). This might be mediated by selected supplement addition for specific bacterial taxa that tolerate a more acidic pH [
33], since the direct effect of probiotic supplements on the microbiota have not been demonstrated [
9,
14,
34]. Moreover, acetic acid suppresses appetite [
35] and propionate and butyrate acids modulate hormones, such as GLP-1 and PYY, involved in satiety [
5,
6,
7], and this mechanism might also contribute to body weight loss.
Weight loss was associated with a decrease in the Firmicutes/Bacteroidetes ratio, as in previous reports [
18,
36], and the effect is due, in part, to a reduction of caloric intake in fructans-supplemented mice [
21]. Taxa associated with greater body weight loss included
Klebsiella and
Citrobacter (Enterobacteriaceae;
Figure 4B). Enterobacteriaceae have also been reported to increase during weight loss in obese mice [
37] and humans [
38,
39]. Other bacteria enriched by supplementation with oligofructose included
Prevotella,
Allobaculum, and
Faecalibacterium genera (
Figure 4C). Similarly, a previous study has reported an association between
Allobaculum and a reduction of body weight in obese mice [
17].
Interestingly, agavins and oligofructose supplementation led to the highest cecum SCFA, despite of structural differences between these fructans. However, fructan structure and the degree of polymerization were associated with differences in the bacteria genera enriched by agavins (branched) or oligofructose (linear). In relation to supplementation with oligofructose, agavins supplementation enriched
Citrobacter,
Klebsiella,
Pseudomonas, and
Acinetobacter, and decreased
Prevotella,
Bacteroides,
Dehalobacterium, and
Oscillospira (
Figure 4D). Nevertheless, both supplements shared the physiological response of accelerating body weight loss perhaps due to functional redundancy of the gut microbiota [
40].
5. Conclusions
In conclusion, diet supplementation of agavins restored microbiota diversity depleted by a high-fat diet, reduced the Firmicutes/Bacteroidetes ratio, enriched members of the Enterobacteriaceae, and increased the SCFA concentration in cecum, which could induce an accelerated weight loss in mice. These results could provide novel insight to develop a new supplementary strategy using agavins to modulate gut microbiota in overweight or obese individuals, which might have positive consequences on body weight loss. Furthermore, the enrichment of members of Enterobacteriaceae has not been reported previously under a prebiotic supplement, which opens opportunities to explore new probiotics.
