1. Introduction
The gut microbiome is increasingly recognized as a primary organ with involvement in nutrient metabolism and absorption [
1]. The bacteria colonizing the digestive tract of animals can be beneficial or harmful [
2]. The beneficial bacteria, including
Bifidobacterium,
Bacteroidales and
Akkermansia muciniphila, play a vital role in maintaining gut homeostasis [
3,
4]. It is also reported that these beneficial bacteria can prioritize the use of fermenting non-digestible carbohydrates to produce bioactive metabolites such as short-chain fatty acids (SCFAs), which can be used by the host as precursors for the biosynthesis of lipids and proteins or as an energy source [
5,
6,
7,
8]. In addition to SCFAs, other gut microbial metabolites related to amino acid metabolism and carbohydrate fermentation, such as indole-like metabolites, are attracting more and more attention [
9,
10]. For instance,
Lactobacillus and
Clostridium butyricum can convert dietary tryptophan into indole-acetic acid (IAA) and indole-3-lactic acid (ILA), which have been implicated in the regulation of lymphocyte function [
11,
12].
Chinese yam, scientifically known as
Dioscorea opposita Thunb., has a long history of cultivation spanning over three thousand years in East Asia [
13]. It serves both as a staple food crop and a widely utilized ingredient in traditional Chinese medicine [
14,
15]. Studies have revealed the nutritional and medicinal properties of CY; for example, it effectively activated macrophages in vitro and promoted the production of NO, IL-6 and TNF-a [
16]. Its components can trigger tumor necrosis, apoptosis and activation of antitumor immunity in C57BL/6 mice [
17]. Remarkably, CY water decoction has exhibited efficacy in enhancing gastrointestinal peristalsis and restoring the metabolic phenotype, thus ameliorating intestinal disorders [
18]. However, there are relatively few studies on the effects of CY on gut microbiota.
CY boasts an abundance of valuable nutrients, including resistant starches, steroidal sapogenins like diosgenin, mucilage polysaccharides and flavonoids such as taxifolin [
19]. Chinese yam mucilage polysaccharides (CYPs) have been found to impede gastric emptying and small intestinal propulsion while facilitating intestinal absorption in mice with spleen deficiency [
20]. CYPs were found to modulate the gut microbiota by reducing the levels of
Desulfovibrio and
Sutterella while concurrently elevating the abundance of
Prevotella [
21]. Diosgenin, which is recognized as a significant active substance in CY, exhibits gastric-ulcer-improving properties and boosts immunity by regulating intestinal flora
in vivo, thereby safeguarding gastric mucosa cells and fortifying the gastrointestinal mucosa against harmful substances [
22]. Taxifolin, as a bioflavonoid found abundantly in Tiegun yam, possesses potent anti-inflammatory and neuroprotective effects [
23]. It also improved gut microbiota diversity and prevented
Proteobacteria from blooming [
24]. These results demonstrate that CY ingredients have a considerable influence on shaping the gut microbiota. Nevertheless, whether these components play a role in contributing to the impact of CY on gut microbiota remains unknown.
In vitro fermentative models are considered tools that allow the screening of substances to assess how they alter and are altered by gastrointestinal environments and microbial populations. They are widely applied to predict the influence of foods or nutrients on gut microbiota because these models are relatively inexpensive and without ethical concerns, which enables us to bind food, intake route and gut microbiota together [
25]. In vitro batch fermentation models have recently been utilized for studying contribution of food to gut microbiota composition and functionality [
26]. This methodology provides the investigator with the ability to access numerous substrate samples, either as actual food in its cooked or raw form or specific food components like dietary fibers or phenolic compounds [
27]. It could help us to formulate initial hypotheses and facilitate the foundation of future trials.
In this study, we used an in vitro simulated digestion and fermentation model to investigate the effects of CY on the gut microbial function and metabolism. We further aimed to compare the differences between the effects of Chinese yam and its active components (CYPs, diosgenin, taxifolin) on intestinal flora function and indole-like compounds’ metabolism. Our results provide a scientific basis for future dietary interventions using CY to achieve intestinal homeostasis.
2. Materials and Methods
2.1. Materials
Diosgenin and taxifolin were purchased from Solarbio Science & Technology (Beijing, China). Trypsin (>99%) from porcine pancreas, pepsin (>99%), mucin from porcine stomach, L-cysteine (97%) and methanol (>99%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Bile salt No. 3 (>99%), peptone and yeast extract were purchased from Aobox Biotechnology (Beijing, China). Twee-80 was purchased from Yuanye Bio-Technology (Shanghai, China). Chemicals studied in the article are listed in
Appendix A.
2.2. Preparation of CY and CYPs
Fresh yams were supplied by Xincheng Huai Pharmaceutical Co., Ltd. (Jiaozuo, China). CY was peeled and steam-cooked and then cut into slices and freeze-dried for 48 h. The dried CY was pulverized with a blender (KA-2610, Jworld Tech, Ansan, Korea) for 30 s, screened through a 100-mesh sieve and stored at −20 °C until use. The proximate analysis of the CY powder was performed by the Agricultural Products Testing Center (Beijing, China).
CY slices mixed with distilled water (1:2, w/v) were homogenized at a speed of 10,000 r/min for 60 s. Subsequently, the mixture was left at room temperature for 3 h and centrifuged it at 4000 rpm for 5 min. The combined supernatant was concentrated and then mixed with absolute ethanol (1:4, v/v) at 4 °C for 12 h. The collected precipitate (4000 rpm, 5 min) was dissolved in distilled water and subjected to freeze drying, obtaining crude CYPs.
2.3. In Vitro Gastrointestinal Digestion of Chinese Yam and Its Components
Our procedure was based on previous methods with a few modifications [
28]. The simulation of a system of gastric and small intestinal digestion with additions of Chinese yam and its components was achieved with an SPH-100D thermostat shaker (Shiping Instrument Corp., Shanghai, China). Firstly, 1 g of NaCl, 1.1 g of KCl, 0.15 g of CaCl
2 and 0.6 g of NaHCO
3 were dissolved in 1 L of distilled water to create the gastric medium. Then, 35.4 mg of gastric pepsin and 1.5 mL of CH
3COONa (1.0 mol/L, PH = 5) were added into 100 mL of the medium, and the pH was adjusted to 2.0 by 0.1 M HCl solution to afford the simulated gastric fluid. The preparation of small intestinal fluid involved firstly dissolving 5.4 g of NaCl, 0.65 g of KCl and 0.33 g of CaCl
2·2H
2O in 1 L of distilled water. A 4% bile solution was prepared with Bile salt No. 3. Then, 14 g of pancreatic material was added into 200 mL distilled water to prepare pancreatic fluid. A combination of 50 mL of small intestinal fluid, 100 mL of bile solution, and 50 mL of pancreatic fluid was used to prepare 200 mL of small intestinal medium.
All digestions were performed under protection from light (by covering them with foil) and oxygen (using airtight containers). The pH of the gastric medium solution was maintained at 2.0. The pH of the small intestinal medium was maintained at 7.0.
2.4. Preparation of Fecal Flora Suspension
Fresh feces were collected from healthy, 8-week-old male C57BL/6J mice. The bacterial suspension from the supernatant was obtained after the feces and sterile PBS buffer (pH 7.0) were combined and diluted at a ratio of 1:9 (w/v), homogenized for 10 min and then centrifuged (3000 rpm, 5 min) to remove contaminants.
2.5. In Vitro Colonic Fermentation Model
The digestive fluids of yam extract, bacterial suspension and mixed medium were mixed at a volume ratio of 1:3:6. All samples were placed in the anaerobic-gas-generating bags from the Mitsubishi MGC AnaeroPackTM series, sealed and left in an anaerobic environment (20% CO2, 80% N2) and then incubated at a constant temperature of 37 °C. Fermentation was conducted for 24 h, and samples were collected at three time points (0, 12 and 24 h) for analysis. The fermentation broth was then centrifuged with a high-speed refrigerator centrifuge (12,000 rpm, 10 min), and the supernatant and sediment were collected and stored at −80 °C for later use.
2.6. Analysis of pH and SCFAs
The liquid was collected after 0, 12 and 24 h of fermentation for pH detection. The fermentation product was centrifuged at 10,000× g for 10 min, and then the supernatant was transferred to a 10 mL screw-cap tube, and the pH value was measured with a pH S-3B instrument (Lai Chi Instrument Co., Ltd., Shanghai, China).
After fermentation, the sample was mixed thoroughly with 800 μL of deionized H2O and 200 μL of 50% H2SO4. The mixture was added to 1 mL of diethyl ether and shaken by vortex for 10 s. The supernatant was collected after centrifugation (12,000× g, 10 min) and dehydrated by anhydrous CaCl2. The resulting supernatant was analyzed on a gas chromatograph using a hydrogen flame detector (flame ionization detector, FID) equipped with an SH-Stabil wax-DA (30 m × 0.32 mm × 0.50 μm). A flame ionization detector was used with an injector temperature of 260 °C, followed by temperature programming by holding the temperature at 80 °C for 1.5 min, increasing it to 240 °C at a rate of 10 °C/min and holding at 260 °C for 20 min. Nitrogen was used throughout the measurement process as a carrier gas. The concentrations of acetate, propionate, butyrate, isobutyrate, valerate and isovalerate were calculated based on the peak area of standard samples (Sigma Aldrich, St. Louis, MO, USA). The injection volume was 1 μL, and the injection was repeated 3 times.
2.7. Determination of Indole-like Compounds
The enzyme-linked immunosorbent assay (Meimian Industrial Co., Ltd., Yancheng, China) was used to determine lactic acid (LA), tryptophan (Trp), indole-3-lactic acid (ILA) and indoleacetic acid (IAA) in the gastrointestinal digestion and fermentation in vitro system at different times (0, 12 and 24 h). We measured the absorbance of each sample using a multimode microplate reader (TECAN, Männedorf, Switzerland) at a wavelength of 450 nm. The calculation methods for determining the content of indole compounds are listed in
Supplementary Materials.
2.8. Processing and Analysis of 16S rRNA Gene Sequencing
Extracted total DNA from the fermentation fluid of CY and its active ingredients were analyzed using the TIANamp Stool DNA Kit (Tiangen, Beijing, China). Genomic DNA was utilized as a template for PCR amplification of the V3/V4 region of the bacterial 16S rRNA gene. PCR amplification products were analyzed with an RDP Classifier (
http://rdp.cme.msu.edu/, accessed on 3 April 2023) against the Silva (SSU115) 16S rRNA database using a confidence threshold of 70%. Raw fastq files were demultiplexed and quality-filtered using QIIME (version 1.17) and further classified into operational taxonomic units (OTUs) within 0.03 (equivalent to 97% similarity) difference. Community richness was evaluated using Chao1, Sobs and Ace indexes. Principal coordinates analysis (PCoA) was applied to quantify the compositional differences between microbial communities. To identify bacterial taxa that were differentially represented between groups at the genus or higher taxonomic levels, linear discriminant analysis effect size (LEfSe) was applied to the present features followed by linear discriminant analysis (LDA) to measure the effect size of each abundant taxon and two filters (
p < 0.05 and LDA score of >3.5). The heatmap was generated by the R package heatmap (R 4.2.1).
2.9. Statistical Analysis
Each experiment was repeated three times. All data are expressed as the mean ± SEM. Comparison of multiple samples was conducted by one-way analysis of variance (ANOVA) using SPSS software (version 25.0). Means with different letter superscripts indicate significant differences (p < 0.05). * p < 0.05 vs. 0 h, # p < 0.05 vs. 12 h in the tables. Plotting was performed with GraphPad Prism 7. Spearman’s rho nonparametric correlations among the gut microbes and indole compounds were then calculated, and the results were visualized with R (R 4.2.1).
4. Discussion
Research on the regulation of gut microbiota by medicinal and edible homologous plants is being supported by increasing evidence. Chinese yam, cultivated for medicinal and dietary applications, contains many bioactive components, including CYPs, diosgenin and dihydroquercetin [
14]. It has been confirmed that CY possesses the capacity to induce targeted modifications in microbial communities and metabolic profiles [
35]. In vitro digestion and colonic fermentation is regarded as an economic, convenient and reproducible method that can be used to study the effect of foods on intestinal flora and its metabolites compared with in vivo intervention trials. Researchers commonly use in vitro methods to obtain initial experimental insights and subsequently employ in vivo experiments to validate the findings [
25]. At present, the specific active ingredients in CY that play a role in regulating the flora and the microbiota-derived metabolites remain unclear. The goal of our study was to examine the effects of CY and its active ingredients (CYPs, diosgenin, taxifolin) on the structure and functional metabolism of gut microbiota through anaerobic fermentation in vitro.
Initially, we observed that CY can boost the α-diversity of gut microbiota, indicating the significant effects of CY on microbiota diversity. Furthermore, the dominant bacterial populations in the broth samples at 12 h of fermentation were Proteobacteria, Firmicutes and Bacteroidota at the phylum level. After 24 h of fermentation, CY promoted the production of Firmicutes and Bacteroidota while decreasing the level of Proteobacteria. Moreover, CY could increase the abundance of
Lactobacillus and
Akkermansia while decreasing that of
Escherichia and
Proteus at the genus level. These findings suggest that CY can regulate the structure of gut microbiota. The potential prebiotic effect of CY on gut microbiota was mainly reflected in the promotion of the growth of
Lactobacillus and
Akkermansia. Lactic acid bacteria encompass a diverse group of microorganisms, and certain strains among them have been harnessed as probiotics due to their ability to metabolize tryptophan [
36]. Similarly, an increased level of lactic acid bacteria was observed with the addition of CY peel in a freshwater aquaculture carp model [
37]. Researchers have reported that
Lactobacillus can regulate the balance of the host intestinal environment and improve the host immune protection ability and nutrition metabolism by promoting the transformation and absorption of nutrients [
38].
SCFAs contribute to the acidification of the batch fermentation environment, which is consistent with the decrease in pH value. As expected, the pH values of the CY group showed a decreasing trend during the whole fermentation process, and the same trend could be observed in CYPs and taxifolin, indicating that CYPs and taxifolin could also function independently of yam. At the same time, the concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid and total SCFAs were all higher in the supernatants from the fermentation broths with the digested CY, which is related to the basal nutrient medium utilization by the increased number of SCFA-producing bacteria caused by CY. In addition, the acidic colon environment can contribute to host health by promoting the proliferation of beneficial bacteria and inhibiting the growth of pathogens.
In recent years, there has been an increasing number of studies into the small-molecule metabolites produced by gut microbes, as they play a vital role in regulating the host’s pathophysiological state [
39]. Previous research has documented alterations in the tryptophan metabolic profile when investigating the impact of yam intervention on antibiotic-induced rats [
18]. Therefore, we also explored the changes in indole-like metabolites, such as IAA and ILA, over time during in vitro fermentation. IAA is one of the metabolites of tryptophan produced from
Clostridium [
31]. Tryptophan can also be metabolized by gut bacteria like
Lactobacillus reuteri (
L. reuteri) and
Lactobacillus murinus (
L. murinus) into ILA [
40]. These indole-like compounds belong to tryptophan metabolites as well as the organic acid LA, and these indexes can be used to evaluate the potential prebiotic effect of CY. In this study, we observed that CY could promote an increase in IAA, ILA and LA concentrations in colonic fermentation, while reducing the concentration of tryptophan, which indicates that some tryptophan is converted into indole metabolites. The mediated influence of CY on promoting the production of tryptophan metabolites was very obvious, and its components were a partial effect. Nevertheless, the concentration of indole metabolites was positively correlated with the number of beneficial intestinal bacteria. These results are preliminary, and further in vivo studies are needed to determine whether these potential prebiotic effects have health benefits for humans.
In summary, this study revealed that CY could increase the output of SCFAs in the intestine, create an acidic intestinal environment and increase the richness of the flora. CY and its active components, like diosgenin and taxifolin, promoted the growth of beneficial bacteria such as Lactobacillus, Clostridium and Akkermansia. Both CY and diosgenin could promote the production of indole metabolites, which were positively correlated with the production of beneficial bacteria like Lactobacillus and Clostridium during in vitro fermentation. However, further research is needed to understand the underlying mechanisms by which the gut microbiota mediate yam metabolite production. Collectively, these findings highlight the potential importance of understanding the interaction between active ingredients of CY and gut microbiota in the context of host health.
5. Conclusions
In conclusion, during simulated gastrointestinal digestion and colon fermentation, CY and its active components were utilized by the gut microbiota, obviously improving microflora diversity at the phylum and OTU levels. CY, CYPs, diosgenin and taxifolin exerted a regulatory influence on the gut microbial composition compared with the control treatment. At the genus level, the relative abundance of beneficial bacteria like Lactobacillus, Clostridium and Bacteroides was enhanced. Concurrently, the amount of Escherichia and Proteus diminished. The addition of yam caused a significant increase in total SCFA levels, including acetic and butyric acids. Furthermore, CY and diosgenin demonstrated the capacity to stimulate the production of indole derivatives (ILA, IAA). The correlation analysis supported significant associations between specific metabolites of CY and related intestinal bacteria.