Abstract
Obesity is one of the major public health problems worldwide, mainly resulting from unhealthy lifestyles and diet. Gut microbiota dysbiosis may also lead to obese humans and animals. Modulating gut bacteria through fecal transplantation, the use of probiotics or certain dietary supplements, could normalize gut microbiota and subsequently alleviate obesity. Daily consumption of Fuzhuan brick tea (FBT) or its extracts have been observed to alleviate obesity in humans and experimental animals. In this study, high-fat diet-induced dysbiosis of gut microbiota in C57BL/6J mice was partially reversed by consumption of Eumtium cristatum, the dominant fungi during the manufacturing and storage of FBT. E. cristatum was able to modulate both gut fungi and bacteria composition, based on the analysis of microbiota composition of mice fecal samples. E. cristatum increased acetate and butyrate-producing bacteria in mice gut, and produced five times more butyrate than both obese and normal mice. Our results suggested that E. cristatum may be used as a fungi probiotic to beneficially modulate gut microbiota and to alleviate obesity in humans.
Introduction
Obesity is a disease associated with numerous health problems, mainly caused by a shift in lifestyles towards less physical expenditure and more hyper-caloric foods1. It is closely linked with chronic and low-grade inflammation, which may cause insulin resistance, type 2 diabetes, fatty liver disease or cardiovascular disease2–4. Based on two recent population analysis from 1975-2016 or 1980-2015, about 10% of the world population, including over 100 million young people, were obese5. Obesity epidemic becomes a major threat to public health in modern human history6. Obesity treatment may include bariatric surgery or changing of lifestyles to increase exercise, which reduce calorie intake7. Anti-obesity drugs, such as phentermine or orlistat, could suppress appetite or inhibit the absorption and utilization of fat8. Alternatively, regulating intestinal microbes has recently been used for the treatment of obesity, because microbiota dysbiosis plays prominent roles in the development of obesity9–12. A variety of probiotics, either individually or more often used as cocktails, were shown to alleviate obesity in a high-fat diet (HFD) fed rodents. For example, both Bifidobacterium and Lactobacillus were shown to attenuate HFD-induced obesity in animals and humans, including weight loss, reduced visceral fat and improved glucose tolerance13–15. Most of these bacteria produce short-chain fatty acids (SCFAs), such as acetate and butyrate, which reduce gut luminal pH and maintain an acidic environment16,17. Previous studies also highlighted the importance of SCFAs to improve chronic inflammatory diseases and to promote colonocyte health18. SCFAs were reported to suppress production of pro-inflammatory cytokines IL-1β, enhance IL-10 expression and activate regulatory T cells(Treg), to maintain intestinal homeostasis19.
Humans have prepared and consumed a variety of fermentation products, such as pickles, sauerkraut, sourdough bread, artisanal cheeses, yogurts and kefir, craft beers and kombucha (fermented tea), in which various bacteria and fungi species are present, dated back to the seventh millennium before Christ20,21. Fermentation is very useful to preserve food and increase their flavor and nutrition22. Fuzhuan brick tea (FBT, also named Fu brick tea) is a fermented tea in the presence of a dominant fungi species named Eurotium cristatum, which is yellow-colored and is thus also called “golden flora” in Chinese23. Human consumption of FBT dated back to the 16th century24, while recent pilot dietary interventions on humans in China have also shown that FBT consumption could improve metabolic disease conditions, which were summarized in Supplementary Table 1. FBT consumption or polysaccharides extracted from FBT were shown to reduce obesity, hyperlipidemia and arterial stiffening, as well as to improve metabolic syndrome in animals25–28. The water or ethanol extracts of FBT reduced obesity through normalizing gut microbiota in mice25,29.
The dominant fungi E. cristatum in FBT play an important role in the flavor, color and health benefits of FBT30. We hypothesized that live E. cristatum in FBT may survive in the gastrointestinal tract during tea consumption. It might have served as a fungi probiotic to modulate gut microbiota, which led to the observed anti-obesity effects of FBT. In this study, we evaluated the effects of dietary supplementation of live E. cristatum to prevent diet-induced obesity in mice. Our study supports the use of E. cristatum as a fungi probiotic to modulate gut microbiota against obesity, which has been used by humans for about 600 years.
Results
E. cristatum may survive in mouse intestine
In order to clarify if E. cristatum could survive during tea brewing and consumption, 104 spores of E. cristatum isolated from FBT, were heated in water containing 20% (v/v) glycerol at 50, 70, 80, 85, 90 and 100 °C for 2, 5 or 10 min, respectively (Supplemental Fig.1). E. cristatum survived even after 10 min in 90 °C and 2 min in 100 °C. E. cristatum was next mixed with drinking water and fed to Kunming mice. E. cristatum could be isolated from mouse feces, even without feeding of E. cristatum for two weeks (Supplemental Fig. 2). There were no significant differences in body weight among mice fed with or without E. cristatum, as well as mice fed with the water extracts or filtered water extracts of Fuzhuan brick tea. These results suggested that E. cristatum from Fuzhuan brick tea is non-toxic and could survive in mice intestine.
E. cristatum reduced HFD-induced obesity in C57BL/6J mice
The HFD-induced C57BL/6J obese mouse model was used to explore if live E. cristatum may have any anti-obesity effect, in comparison to mice that received normal chow diet (NCD). The high-fat diet mice that received FBT or filtered Fuzhuan brick tea (FFBT), in which live E. cristatum were removed by filtering the FBT extracts using a 0.22 μm filter, were also used as controls (Fig. 1a-e). The mice in HFD group showed significant increases in body and liver weights, and epididymal fat accumulation, compared with mice in the NCD group (Fig. 1a-e). In contrast, the high-fat diet mice that received E. cristatum (103 CFU/day) in drinking water showed significantly decreased body weight gain, liver weight and epididymal fat accumulation. The FFBT-treated mice also showed significant decrease in liver and epididymal fat, while FBT-treated mice only had statistically-insignificant lower body weight, in comparison to HFD-fed mice.
E. cristatum increased glucose tolerance in HFD-fed mice
The effects of E. cristatum, FBT or FFBT consumption on glucose homeostasis of HFD-fed mice were next determined (Fig. 2a-c). Compared to HFD-fed mice that received only drinking water, the HFD-fed mice with daily consumption of E. cristatum, FBT or FFBT had reduced fasting blood glucose levels (FBG) and increased glucose tolerance. Since endotoxemia controls the production of pro-inflammatory cytokines in target tissues and may lead to chronic inflammation in HFD-fed mice31, the serum lipopolysaccharide (LPS) levels in these mice were further examined (Fig. 2d). Both FBT and FFBT consumption reduced the LPS level in HFD-fed mice, while E. cristatum had no significant effect.
E. cristatum reduced inflammation in HFD-fed mice
HFD-induced obese mice have been observed to produce higher levels of pro-inflammatory cytokines in hepatic tissues, including interleukin-1-β (IL-1β) and interleukin-6 (IL-6), while the level of the anti-inflammatory cytokine interleukin (IL-10) was reduced in obese animals32. The IL-1β and IL-10 in hepatic tissues of the sacrificed mice were thus measured by enzyme-linked immune sorbent assay (ELISA) (Fig. 2e-f). The HFD-fed mice produced about 300 pg/mL IL-1β, while the consumption of E. cristatum, FBT or FFBT reduced the IL-1β level to about 200 pg/mL, similar to the NCD-fed mice. Similarly, the levels of anti-inflammatory cytokine IL-10 were also restored to those in NCD-mice through consumption of E. cristatum, FBT or FFBT.
E. cristatum altered the fungi diversity in mice gut
Since consumption of E. cristatum alone had significant anti-obesity effects of the HFD-fed mice (Fig. 1-2), we hypothesized that E. cristatum may be able to modulate mice gut microbiota through its interaction with commensal fungi and bacteria33,34. Therefore the composition, abundance and function of mice gut microbiota were next analyzed by high-throughput sequencing of the internal transcribed spacer 2 (ITS2) and 16S rRNA of caecal stool samples of the above C57BL/6J mice. After removing unqualified sequences, a total of 741,205 raw reads and an average of 37,060 ± 23,543 reads per mouse sample were obtained for ITS2 rDNA regions. Rarefaction of chao1 and observed OTUs indicated that the sequencing depth covered most of the fungi diversity, including rare new phylotypes (Supplemental Fig. 4a-b).
To evaluate fungi diversity in mice gut, the fungi β diversity was measured. Constrained principal-coordinate analysis (CPCoA) based on the similarity index revealed distinct clustering of fungi composition for each mice group (Fig. 3a). E. cristatum- or FBT-treated HFD-fed mice were closer to NCD-fed mice. Further hierarchical clustering analysis did not reveal distinct separation of each mice group. Fungi taxonomic profiling in the genus level of intestinal fungi revealed that Euroteomycetes were more abundant in E. cristatum-treated mice than those of other groups, which suggested that E. cristatum had survived in mice intestine (Fig. 3c-d). Statistical analysis of metagenomic profiles (STAMP) of gut fungi also revealed several OTUs belonging to Scolecobasidium, Eurotium, Penicillium and Cosmospora, which were significantly altered in different mice groups (Fig. 3e-f).
Dietary E. cristatum beneficially altered the gut bacteria
The V4 region of 16S rRNA of mice gut bacteria were also obtained, which included a total of 765,760 raw reads and an average of 30,630±17,252 reads per mouse sample. The rarefaction curves analysis of chao1 and observed OTUs revealed that most of the commensal bacteria diversity were covered (Supplemental Fig. 4). The β diversity of the gut bacteria was also evaluated using CPCoA-based Bray–Curtis similarity index, which revealed distinct clustering of bacteria composition for each mice group (Fig. 4a). Compared with other groups, E. cristatum-treated HFD-fed mice were closer to NCD-fed mice in both primary and secondary ordination axes. The inter-sample correlation calculated by Pearson coefficient analysis revealed that the gut bacteria of NCD and E. cristatum groups were more closely related (Fig. 4b).
Bacterial taxonomic profiling in the phylum level of intestinal bacteria from different mice groups were next analyzed (Fig. 4c). The gut microbiota of HFD-fed mice were characterized by an increased Firmicutes-to-Bacteroidetes ratio (F/B), which was consistent the previous reports35,36. Notably, the treatment of FBT in HFD-fed mice reversed the F/B ratio, while FFBT and E. cristatum intervention exhibited limited effects on the relative abundances of Firmicutes and Bacteroidetes and the ratio of F/B (Fig. 4d). STAMP was used to identify the specific bacterial phylotypes of each mouse. Compared with NCD-fed mice, there were 132 bacterial OTUs were significantly altered in HFD-fed mice, in which 118 OTUs increased and 14 OTUs decreased. For HFD-fed mice treated with FBT, FFBT or E. cristatum, there were 67 (18 increased and 49 decreased), 96 (26 increased and 66 decreased), 85 (36 increased and 49 decreased) altered OTUs, respectively, compared with HFD-fed mice. Interestingly, 43, 51 and 45 bacterial OTUs in groups of E. cristatum, FBT and FFBT, were altered towards NCD-fed mice, respectively. Analysis of the gut bacteria altered by E. cristatum consumption indicated that SCFAs-producing bacteria Lactobacillus increased over 14 folds, in comparison to HFD-fed mice (Fig. 4e). Bifidobacterium, the other SCFAs-producing bacteria, increased dramatically. In contrast, Odoribacteracae37, Parabacterioide12, Sutterella38 and Clostridium, decreased in HFD-fed mice, which were negatively correlated with diet-induced obesity. These results suggested that E. cristatum, as well as FBT and FFBT, may modulate the gut bacteria in HFD-fed mice and resulted in a microbiota composition close to that of NCD-fed mice.
PICRUSt analysis of the potential function of gut microbiota in different groups
In order to study the potential function of gut microbiota in different groups, linear discriminant analysis effect size (LEfSe) was used to infer the relative abundance of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which were predicted by phylogenetic reconstruction of unobserved states (PICRUSt)39. The HFD-fed mice had a reduced capacity for energy metabolism and increased capacity of carbohydrate metabolism, consistent with a recent metagenomics-based study35. Notably, both galactose metabolism and starch metabolism were significantly lower in E. cristatum-treated mice than in those HFD-fed mice. In addition, the energy metabolic pathways, including fatty acid and nucleotide metabolisms were significantly lower in HFD-fed mice and they could be reversed by E. cristatum consumption. These metabolic changes could be also identified in the FBT and FFBT groups (Supplemental Fig. 5a-c).
E. cristatum increased SCFAs production in mice gut
The SCFAs analysis of mice fecal samples revealed dramatic differences among these mice groups (Fig. 5). Comparing to NCD-fed mice, the HFD-fed mice showed decreased levels of acetate and propionate in their feces samples, while they had a comparable level of butyrate production. In contrast, consumption of E. cristatum in HFD-fed mice significantly increased the amount of acetate and butyrate. Notably, the amount of butyrate in the E. cristatum group increased over five times over both NCD-fed and HFD-fed mice. The amount of butyrate in the mice fecal samples in FBT mice group also increased significantly. The increasing amount of acetate and butyrate in E. cristatum-treated mice was consistent with the increased acetate- and butyrate-producing bacteria in mice gut, such as Bifidobacterium and Lactobacillus (Fig. 3-4).
Determination of the optimal dosage of E. cristatum to reduce HFD-induced obesity in C57BL/6J mice
Three different dosages of E. cristatum, 104 CFU/day, 103 CFU/day and 102 CFU/day, were employed to HFD-induced C57BL/6J mice for 8 weeks. Alternatively, E. cristatum (103 CFU/day) were given to HFD-induced mice only for 3 and 5 weeks, respectively. Consumption of different dosages or various periods of E. cristatum, all decreased the body weight gain, liver weight and fat accumulation in HFD-fed mice (Fig. 6). The HFD-fed mice receiving E. cristatum (103 CFU/day) for 8 weeks gained slightly less weights than other groups. The E. cristatum (103 CFU/day, 8 weeks) group also had better blood glucose tolerance than other mice groups, while higher or lower dosage of E. cristatum (102 or 104 CFU/day, 8 weeks) were less effective to improve blood glucose homeostasis of HFD-fed mice, indicated by the OGTT test (Fig. 7). Consumption of E. cristatum for 3 or 5 weeks also increased glucose tolerance in HFD-fed mice, which suggested that short-term intervention of the microbiota dysbiosis of HFD-fed mice using E. cristatum might also be effective (Fig. 7c-d).
Discussion
One of the hallmarks of obesity is the dysbiosis of gut microbiota, and normalization of the gut microbiota through fecal transplantation, consumption of dietary fibers and consumption of certain probiotics may be instrumental to manage the obesity epidemic in the world. In the current study, we hypothesized that E. cristatum, derived from Fuzhuan brick tea, may be adopted as a promising fungi probiotic against obesity. In the HFD-fed mice model, E. cristatum was shown to alleviate HFD-induced mice obesity, by reducing inflammation and improving their glucose homeostasis. The normalization of gut microbiota and the increased production of butyrate in E. cristatum-treated obese mice contributed to the observed anti-obesity effect (Fig. 8). Fuzhuan brick tea extracts and polysaccharides have previously been shown to alleviate HFD-induced mice obesity, probably also through modulation of gut microbiota25,26,29. Our study is consistent with these previous studies, in which the FFBT-treated, HFD-fed mice showed reduced liver weight and epididymal fat, and both FBT- and FFBT-treated mice groups showed improved glucose homeostasis and attenuated inflammation, compared to HFD-fed mice.
The gut microbiota of obese humans and animals have decreased ratio of Bacteroides-to-Firmicutes, and Firmicutes are crucial in obesity-related metabolic disorder41. Therefore the treatment of obese mice with Myrciaria dubia42, melatonin43 and other probiotics reversed this ratio and alleviated obesity. The consumption of FBT in HFD-fed mice could restore this lopsided microbiota, while supplementation of FFBT and E. cristatum were unable to reverse this unbalance. Probiotic bacteria, such as SCFAs-producing Lactobacillus, Bifidobacterium and Leuconostoc were shown previously to prevent or treat gastrointestinal disorders44–46, and to have anti-obesogenic or anti-diabetic potential47–49. Our study demonstrated that E. cristatum enhanced Lactobacillus, Bifidobacterium and leuconostoc in mice gut. Akkermansia muciniphila were shown to tighten gut barrier and reversed high-fat diet-induced obesity and improve glucose tolerance under high-fat diet conditions in conventional mice15,50,51. A. muciniphila in the E. cristatum-treated HFD-fed mice group also increased slightly. These results suggested that E. cristatum could modulate gut microbiota by increasing beneficial bacteria in mice gut. Although there is a strong correlation between gut fungi and host health, few studies were reported to manage metabolic disorder induced by high-fat diet by regulating gut fungi33,34. Our study also demonstrated that E. cristatum were able to modulate both fungi and bacteria composition (Fig. 3-4).
SCFAs, generated by the fermentation of intestinal microbiota52,53,54, are important to control chronic inflammatory diseases55,56 and maintain gut homeostasis57. The generation of a large amount of butyrate in the gut of E. cristatum-treated HFD-fed mice was unprecedented, which might contribute to the observed anti-obesity effects. Locally produced butyrate played essential role for the development of intestinal and systemic immune system. For example, butyrate may directly or indirectly induce Treg differentiation, including IL-10 producing Treg cells. It was consistent with the increased level of IL-10, and the decreased level of IL-1β in E. cristatum-treated HFD-fed mice (Fig. 2). Similar effects were also observed in FBT-treated HFD-fed mice.
In conclusion, our study strongly suggested that E. cristatum had significant anti-obesity effects in HFD-fed mice by modulating gut microbiota. Therefore, it may be used as a fungi probiotic to alleviate obesity, or other metabolic diseases, such as type II diabetes involving microbiota dysbiosis. Although Fuzhuan brick tea has already been consumed by humans for hundreds of years, the potential toxicity of the direct consumption of E. cristatum in humans needs to be established (Fig. S1). Since low dosage and short period of E. cristatum consumption may also have certain anti-obesity effects in HFD-fed mice (Fig. 7), E. cristatum may be used as a promising probiotic to alleviate obesity epidemic in the near future.
Methods
Preparation of E. cristatum, FBT and FFBT
E. cristatum CB10001 was isolated from Fuzhuan brick tea (FBT) (Jiuyang Processing Factory Co., Ltd., Yiyang, Hunan, China), which was confirmed to be E. cristatum based on its 18S rRNA sequence. E. cristatum CB10001 was grown in M40Y medium and its spores were harvested and stored in 20% glycerol and kept in –80 °C. The extracts for Fuzhuan brick tea (FBT) were prepared by brewing the tea with 85 °C deionized H2O, which was previously boiled. Briefly, Fuzhuan brick tea (8 g) was brewed in 1 L deionized H2O (85 °C) in a household teapot for 15 min. The filtered Fuzhuan brick tea (FFBT) was prepared by filtering the prepared FBT through a defecator (0.22 μm, thermo, USA).
Survival of E. cristatum in elevated temperature
E. cristatum (104 CFU) were suspended in 1 mL 20% glycerol and heated for 2 min, 5 min, 10 min under 50, 70, 80, 85, 90 and 100 °C, respectively. Then E. cristatum in each sample was serially diluted and spread onto M40Y agar plate. The plates were incubated at 30 °C for 48 h, and the resulting colonies were counted.
Dosage
Typically an adult (60 kg) may consume 10 g of Fuzhuan brick tea per day. Therefore the dosage of a mouse to consume the Fuzhuan brick tea was set as 1.6 mg/g (1.6 mg/g = 10 g/60 kg × 10) Fuzhuan brick tea per gram of individual mouse weight, which was about 10 times of Fuzhuan brick tea a human might have consumed. Since Fuzhuan brick tea contains E. cristatum (2 × 105 CFU/g), the dosage of E. cristatum per mouse was set at 103 CFU/day first. To optimize the dosage of E. cristatum, 102 or 104 CFU/day/mouse of E. cristatum were also used.
Animals
All animals in this study were purchased from Hunan Silaikejingda Experimental Animal Company Limited (Hunan, China). They were bred in the specific pathogen-free animal (SPF) facility of the Department of Laboratory Animals at Central South University (Changsha, Hunan, China) and kept under controlled light conditions (12 h light–dark cycle), with free access to water and diet.
Kunming mice (4-week, male) were randomly distributed into five groups, and each group had five mice. They had free access to chow diet containing 13.5% fat. The mice in normal chow diet group (NCD) had free access to drinking water. The mice in E. cristatum groups (104 CFU/day) or (103 CFU/day) only had access to drinking water containing different amount of E. cristatum. The mice in the Fuzhuan brick tea group (FBT) only had access to FBT as drinking water, while the mice in the filtered Fuzhuan brick tea group (FFBT) only had access to FFBT as drinking water. All the treatment groups, including E. cristatum (104 CFU/day), E. cristatum (103 CFU/day), FBT and FFBT were given at every other 4-weeks, until the end of 16th week. The body weight of individual mouse was measured every other week and the feces of the mice were also collected every other week.
C57BL/6J mice (6-week, male) were randomly distributed into five groups, and each group contained five mice. They had free access to chow diet (13.5% fat) or high-fat diet (60% fat) (Trophic Animal Feed High-Tech Co. LTD, Jiangshu, China). E. cristatum (103 CFU/day), FBT and FFBT were given to HFD-fed mice in drinking water for 7 weeks.
Optimal dosage of E. cristatum
To identify the optimal dosage of E. cristatum, C57BL/6J mice (6-week, male) were randomly divided into 7 groups and each group had 10 mice. They had free access to chow diet (13.5% fat) or high-fat diet (60% fat). The 7 groups included: normal chow diet group (NCD), high-fat diet group (HFD), high dosage of E. cristatum group (104 CFU/day, 8 weeks), middle dosage of E. cristatum group (103 CFU/day, 8 weeks) and low dosage of E. cristatum group (102 CFU/day, 8 weeks), as well as E. cristatum group (103 CFU/day, 3 weeks) and E. cristatum group (103 CFU/day, 5 weeks), in which the HFD-fed mice were given E. cristatum for only 3 or 5 weeks, respectively.
Isolation and identification of E. cristatum from Kunming mice
The feces of Kunming mice (200 mg) were suspended in 1 mL sterile water. Then 100 μL supernatant was spread onto M40Y agar plates and cultured at 30°C for 48h. The 18S rDNA were PCR amplified from the isolated strain. The phylogenetic analysis of the 18S rRNA was performed using Mega 6 using the Neighbor-Joining method.
Oral glucose tolerance test
Overnight-fasted mice were administered a 2 g kg-1 OGTT (20% glucose solution) by oral gavage. Their blood glucose was measured by tail bleeding at time 0, 15, 30, 60, 90 and 120 min after oral gavage, using a glucose meter (Sannuo Biosensor Co., Ltd, Hunan, China).
Cytokine measurements
IL-1β, IL-10 protein levels were measured using commercial ELISA kits (eBioscience, USA).
Gut microbiota analysis
Mice feces were stored at –80 °C, and the total DNAs were extracted using a stool DNA extraction kit (Omega, USA). For each caecal stool sample, the 16S rRNA gene comprising V4 regions were amplified, using a forward primer 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and a reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’). Both primers also contained a unique 12-base barcode to tag each PCR product. The ITS2 region of fungi in the caecal stool samples were amplied using a forward primer ITS7F (5’-GTGARTCATCGARTCTTTG-3’) and a reverse primer ITS4R (5’-TCCTCCGCTTATTGATATGC-3’), which also contained a unique 12-base barcode to tag each PCR product. The high throughput sequencing was carried out using the Illumina MiSeq platform to generate 2 × 250 bp paired-end reads. The raw reads were quality filtered. The primers and barcode in each read were trimmed using QIIME58 software package. The resulting sequences were then assigned to operational taxonomic units (OTUs) with 97% identity, followed by the selection of representative sequences. The alpha and beta diversity were determined using a UniFrac analysis and a constrained principal coordinate analysis (CPCoA). The taxonomic abundance and characterize differences between groups were estimated by Linear discriminant analysis of the effect size (LEfSe). Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt)39 was performed to identify functional genes in the sampled microbial community on the basis of the data in the KEGG pathway database.
SCFA analysis
SCFAs were similarly analyzed as described before59. Briefly, 50 mg fecal sample was blended with 500 μL acetonitrile and acidified with 5% (v/v) H2SO4. Then the mixture was ultrasonically extracted for 10 min and centrifuged at 15000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter. SCFAs were measured using a gas chromatograph-mass spectrometer (GCMS-QP2010 Ultra system, Shimadzu Corporation, Japan), which was equipped with an Rtx-Wax column (30 m × 0.25 μm × 0.25 μm). The carrier gas was helium (flow 2 mL min-1, split ratio 10: 1, the volume of sampling 1 μL). The injection and ionization temperature was set at 200 °C. The standard curves of acetate, propionate and butyrate were established and the concentrations of these SCFAs (mmol/kg mouse feces) were calculated according to the standard curve.
Statistics
Statistical analysis was performed using GraphPad Prism 6.01 (GraphPad Software, Inc., San Diego, CA) unless otherwise specified. Data obtained from experiments are shown as means ± s.e.m. Differences in body weight and OGTT were analyzed by the unpaired two-tailed Student’s t-tests, and those with more than two groups were assessed with one–way ANOVA followed by Newman–Keuls post hoc tests. In the figures, the data with different superscript letters are significantly different based on post hoc ANOVA statistical analysis. Graph bars marked with different letters on top represented statistically significant results (P<0.05) based on Newman–Keuls post hoc one–way ANOVA analysis, whereas bars labeled with the same letter correspond to results that show no statistically significant differences. In the case, whereas two letters are present on top of the bar, each letter should be compared separately with the letters of other bars to determine whether the results show statistically significant differences.
Accession codes
Both sequencing data for the 16S rRNA and ITS sequences have been deposited in the NCBI’s Sequence Read Archive database under BioProject ID No PRJNA506283. The 18S rRNA of E. cristaum CB10001 has been deposited in GenBank (MK371789).
Author contributions
Y.H. and Y.D. conceived the project; Y.H., Y.D. and D.K. designed the experiments; D.K. and M.S. performed the experiments; Y.H., and D.K. analyzed the results and wrote the manuscript with inputs from all co-authors.
Competing interests
The authors declare no competing financial interests.
Materials & Correspondence
All the correspondence and material requests should be addressed to jonghuang{at}csu.edu.cn (Y.H.) or ywduan66{at}sina.com (Y.D.).
Animal protocols
All animal protocols were approved by the Animal Care and Use Committee of Central South University. All experimental procedures complied with the Guide for the Care and Use of Laboratory Animals (1996).
Additional information
Supplementary information is available online.
Competing interests
There are no conflicts to declare.
Acknowledgements
The authors thank the Center for Advanced Research for the GCMS and Minerals processing and bioengineering for Illumina MiSeq platform in Central South University. This work was supported in parts by NSFC grant 81473124 (to Y. H.), the Chinese Ministry of Education 111 Project B0803420 (to Y. D.).