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Comparative Analysis of the Rats’ Gut Microbiota Composition in Animals with Different Ginsenosides Metabolizing Activity Wei-Wei Dong, Fang-Ling Xuan, Fei-Liang Zhong, Jun Jiang, Songquan Wu, Donghao Li, and Lin-Hu Quan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04848 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016
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Journal of Agricultural and Food Chemistry
Comparative Analysis of the Rats’ Gut Microbiota Composition in Animals with Different Ginsenosides Metabolizing Activity Wei-Wei Dong, Fang-Ling Xuan, Fei-Liang Zhong, Jun Jiang, Songquan Wu*, Donghao Li*, Lin-Hu Quan*
Key Laboratory of Natural Resource of the Changbai Mountain and Functional Molecular (Yanbian University), Ministry of Education, Park Road 977, Yanji City, Jilin Province 133002, China *Corresponding author Address: Yanbian University Park Road 977, Yanji City, Jilin Province 133002, China (Tel) 86-433-2436452 (Fax) 86-433-2432456 (E-mail)
[email protected] (LH
Quan),
[email protected] [email protected] (S Wu)
1
ACS Paragon Plus Environment
(D
Li),
Journal of Agricultural and Food Chemistry
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ABSTRACT: Following oral intake of Panax ginseng, major ginsenosides are metabolized
2
to deglycosylated ginsenosides by gut microbiota before absorption into the blood. As the
3
composition of gut microbiota varies between individuals, metabolic activities are
4
significantly different. We selected 6 rats with low efficiency metabolism (LEM) and 6 rats
5
with high efficiency metabolism (HEM) from 60 rats following oral administration of Panax
6
ginseng extract, and analyzed their gut microbiota composition using Illumina HiSeq
7
sequencing of the 16S rRNA gene. The components of gut microbiota between the LEM and
8
HEM groups were significantly different. Between the 2 groups, S24-7, Alcaligenaceae, and
9
Erysipelotrichaceae occupied most OTUs of the HEM group, which was notably higher than
10
the LEM group. Furthermore, we isolated Bifidobacterium animalis GM1 that could convert
11
the ginsenoside Rb1 to Rd. The result implies that these specific intestinal bacteria may
12
dominate the metabolism of Panax ginseng.
13
KEYWORDS: ginsenoside, LC-MS/MS, metabolism, Panax ginseng, rats gut microbiota
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INTRODUCTION
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In Asian countries, Traditional Chinese Medicines (TCMs) have been used to cure various
16
diseases for thousands of years,1 with oral administration being the most common intake
17
method. Therefore, contact inevitably occurs with the gastrointestinal tract, with the
18
majority of active components metabolized by gut bacteria.2 The gut microbiota, the
19
trillions of microbes residing in the human intestine, plays a crucial role in many biological
20
functions, including immunity, nutrition, and metabolism.3,4 As an important “microbial
21
organ”, they are nearly ten–fold greater than the total of our somatic and germ cells.
22
Additionally, they are also involved in diverse processes including the metabolic function
23
of TCMs.5
24
Panax ginseng is a type of TCM that is widely used as a functional food, and is found in tea,
25
powder, and capsules.6 The major pharmacological components in Panax ginseng are
26
ginsenosides,
27
immunomodulatory, anti-tumor, anti-aging, and anti-inflammatory activities.7-11 Following
28
oral intake of Panax ginseng, major ginsenosides are metabolized to deglycosylated
29
ginsenosides via intestinal microbiota before absorption into the blood.12,13 For example,
30
major ginsenosides Rb1, Rc, Rb2, Rb3, Rd, Rg1 and Re are mainly metabolized to
31
deglycosylated ginsenosides compound K, Rh1, and F1 by intestinal bacteria.14-18 The
32
deglycosylated ginsenosides possess potent pharmacological activity compared with the
33
major ginsenosides. For example, the deglycosylated ginsenoside compound K displays
34
anti-cancer, anti-diabetic, and anti-obesity activities and the ginsenosides Rh1 and F1 exhibit
35
anti-aging, anti-oxidant, and anti-allergy function to a greater degree than major
which
possess
various
biological
functions
3
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anti-diabetic,
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ginsenosides.19-23 This indicates that gut microbiota play an important role in metabolizing
37
ginsenosides and producing bioactive metabolites. The conditions of the host, such as diet,
38
stress, and even environmental exposure could lead to the differences of gut microbiota
39
composition and function which affects the efficiency of metabolism and absorption of
40
ginsenosides.24 Therefore, studies on the relationship between the community structure of gut
41
microbiota and the metabolism of ginsenosides are significant.
42
In this study, we used LC-MS/MS to analyze the colonic content samples of 60 rats after
43
oral administration of Panax ginseng extract. From these, we selected 6 rats with low
44
efficiency metabolism (LEM) and 6 rats with high efficiency metabolism (HEM), and then
45
compared the gut microbiota composition of the LEM and HEM groups using Illumina
46
HiSeq sequencing of 16S rRNA gene.
47 48
MATERIALS AND METHODS
49 50
Chemicals and Materials. HPLC-grade deionized water, methanol (MeOH), and
51
acetonitrile (ACN) were obtained from Fisher Scientific (Pittsburgh, USA). LC-MS grade
52
formic acid was obtained from ROE Scientific Inc Co. (Dover, DE, USA). Other reagents
53
used were of analytical grade. Standard ginsenosides (Rg1, Re, Rf, Rb1, Rb2, Rd,
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20(S)-Rg2, Rc, 20(S)-Rh1, F1, F2, 20(S)-Rg3, 20(S)-PPT, compound K, and 20(S)-Rh2)
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were purchased from the Chinese National Institute (Beijing, China). Fresh 4-year old
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ginseng roots were supplied by JiAn, China.
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Panax ginseng Extract Preparation. One-hundred grams of dried and pulverized Panax 4
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ginseng root powder was extracted with 1,000 mL 80% (v/v) ethanol solution at 82 °C
59
using reflux condensation. This process was repeated twice, and the combined extract was
60
evaporated to dryness at 40 °C in a rotary evaporator. The Panax ginseng extract was then
61
ready for the subsequent experiment.
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Animal Experiments. Adult male Sprague-Dawley rats (7 weeks old, weight: 220 ± 20 g)
63
were obtained from the Changchun Yisi Experimental Animal Research Center (Changchun,
64
China). The rats were housed at a temperature of 24 ± 2 °C with a 12 h light/dark cycle.
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After a one-week acclimatization period at our facility, 60 rats were orally administered
66
100 mg/kg bodyweight of the total saponins in the Panax ginseng extract by gastric
67
intubation. After treatment, the colonic content samples were collected at 12 h. All samples
68
were stored at -80 °C prior to analysis. Animal welfare standards and experimental
69
procedures were conducted according to the guidelines of Yanbian University’s Animal
70
Ethics.
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LC–MS/MS Analysis of Metabolites. Colonic content samples (0.5 g) were mixed with
72
cold saline (5 mL) and then extracted 3 times by ultra-sonication with water-saturated
73
n-butanol for 30 min. The extracted solution was centrifuged at 5,000 × g for 10 min at
74
4 °C. The extract was concentrated by evaporation under vacuum. The resultant residue
75
was then removed with methanol solution. Samples were passed through 0.22-µm filters
76
and analyzed using LC–MS/MS.
77
HPLC separation was conducted on an Agilent 1260 series LC system (Agilent
78
Technologies, USA). A reversed-phase column was achieved with an Agilent Poroshell
79
ZORBAX SB-C18 column (4.6 mm × 150 mm, 5 µm) with a C18 guard column (4.6 mm × 5
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12.5 mm, 5 µm). The mobile phase consisted of (A) 0.1% formic acid in water, and (B)
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0.1% formic acid in acetonitrile, with a gradient elution of 0–15 min (23–30% B), 15–34
82
min (30–44% B), 34–46 min (44–68% B), 46–61 min (68–85% B), 61–66 min (85–80% B),
83
and 66–73 min (80–23% B). Two microliters of the sample were injected at a flow rate of
84
0.5 mL/min, and measured at 203 nm. In this metabolism study, all mass spectrometric
85
experiments were conducted on an Agilent 6420 triple quadrupole mass spectrometer in
86
positive ion mode, and monitored by MS/MS detection in MRM mode, to detect the
87
metabolites of Panax ginseng saponins in colonic content samples. The parameters of the
88
acquisition system were set as follows: drying gas (N2) flow rate, 8 L/min; drying gas
89
temperature, 350 °C; nebulizer, 15 psi; capillary voltage, 4000 V; and scan range, m/z
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100-1400. The dwell time was set at 100 µs. Data acquisition and qualitative analysis were
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accomplished through Mass Hunter workstation software (version B.06.06). The
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positive-ion fragments were detected to analyze ginsenosides.
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Experimental Design. Ginsenosides in colonic content samples from 60 rats were
94
quantified by LC-MS/MS detection in MRM mode. The data indicated that metabolic
95
activities in individuals were significantly different (Figure 1A). Based on the level of
96
metabolism, we selected 6 rats with LEM (sample no. A7, A39, A28, A17, A36, and A53)
97
and 6 rats with HEM (sample no. B12, B22, B23, B25, B35, and B49) from 60 rats to
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analyze their gut microbiota composition.
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Rats Gut Microbiota Analysis. Colonic content samples were snap-frozen in liquid
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nitrogen and stored at -80 °C. Genomic DNA was extracted from the colonic content
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samples (0.25 g) using a PowerSoil DNA Isolation Kit (MO BIO Laboratories, USA). Total 6
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genome DNA from samples was extracted via SDS method. The concentration and purity
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of the extracted DNA were measured using a Nanodrop spectrophotometer (ND-1000,
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NanoDrop Technologies, USA). The V3-V4 region of the bacteria 16S rRNA gene was
105
amplified by PCR (95 °C for 5 min, followed by 25 cycles of 95 °C for 30 s, 50 °C for 30 s,
106
72
107
(5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT
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-3′), where the barcode is an eight-base sequence unique to each sample. The PCR products
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were mixed with the same volume of 2 × loading buffer and we operated electrophoresis on
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1.8% agarose gel for detection. Samples with a bright main strip about 450 bp were chosen
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and mixed in equidensity ratios. Then, a mixture of PCR products was purified using a
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GeneJET Gel Extraction Kit (Thermo Scientific).
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The 16S rRNA gene amplicons were used to determine the diversity and structural
114
comparisons of the bacterial species in rat intestinal microbiota using Illumina HiSeq
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sequencing at Biomarker Bioinformatics Technology Co., Ltd, Beijing, China. The raw
116
reads have been submitted to the NCBI Sequence Read Archive (SRA) database
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(Accession number SRP091529). Sequencing libraries were measured using an Agilent
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2100 bioanalyzer following the manufacturer’s recommended protocol. The qualified
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libraries were amplified on cBot to generate the cluster on the flow-cell.
°C
for
40
s,
and
72
°C
for
7
min)
using
the
primers
338F
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Paired-end reads were allocated to samples based on their unique barcode. The
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overlapping regions between the paired-end reads were merged using FLASH v1.2.7 and
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raw reads were quality filtered under specific filtering conditions to obtain the high-quality
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clean tags on the basis of the QIIME (V1.7.0), quality control process. The chimera 7
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sequences were detected by comparing tags with the reference database (Gold database)
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using the UCHIME algorithm and then removed. Bioinformatics analysis of sequences was
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conducted using the QIIME software package. Sequences with ≥ 97% similarity were
127
grouped into the same operational taxonomic units (OTUs). Alpha (within sample), beta
128
(between sample) diversity, and Principal coordinate analysis (PCoA) based on unweighted
129
Unifrac distances were conducted by QIIME.
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Screening of Metabolizing Ginsenosides by Rat Intestinal Bacteria. We collected 1 g of
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rat colonic content samples and suspended in 9 mL of cold physiological saline. The
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suspension was centrifuged at 500 × g for 5 min, and then, the supernatant was centrifuged at
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10,000 × g for 20 min. The resulting precipitates were suspended in 15 mL of GAM broth and
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cultivated under anaerobic conditions (85% N2, 10% H2, 5% CO2) and incubated at 37 °C for
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48 h, and diluted to a concentration that ranged from 10-1 to 10-6. Two hundred microliters of
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the diluted suspension were inoculated on GAM agar plates, and incubated anaerobically at
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37 °C for 24 h.
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We used Esculin-GAM agar to screen rat intestinal bacteria which generated
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β-glucosidase.25 The growth medium (per 1 L) consists of 3 g of esculin and 0.2 g of ferric
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citrate in GAM agar. Intestinal bacteria that produce β-glucosidase could hydrolyze esculin,
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resulting in a reddish brown turning to dark-brown zone appeared around colonies on the
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esculin-GAM agar plates. Ultimately, single colonies from those plates were anaerobically
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cultured in the GAM broth at 37 °C until the absorbance at 600 nm reached 1.0. Ginsenoside
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Rb1 (1.0 mg/mL) reacted with suspensions at 37 °C for 72 h under anaerobic conditions in
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three replications. All of the samples were analyzed by LC–MS/MS. 8
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The
bacterial
16S
rRNA
was
amplified
with
the
universal
primers
27F
147
(5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGA
148
CTT-3′). After the PCR process, the production was sequenced by Shanghai Invitrogen
149
Biotechnology Co. Ltd., China and then we compared the sequence to the GenBank
150
databases using the BLAST algorithm. A phylogenetic tree was charted by the
151
neighbor-joining method via the MEGA 6.0 program with bootstrap values based on 1,000
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replicates.
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Statistics. Data obtained from 6 rats are shown as mean ± standard deviation. The
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differences between treatment groups were analyzed by Student’s t-test. Significant
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differences are indicated in the tables and figures by * p < 0.05, ** p < 0.01, *** p < 0.001.
156 157
RESULTS AND DISCUSSION
158 159
Characterization of Ginsenosides in the Panax ginseng Extract. A total of 15
160
reference standards of ginsenosides were analyzed in this work (Supplementary Figure 1A).
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The precursor-product ion pairs used in the MRM mode were as follows: m/z 823.4➝643.6
162
for Rg1, 969.5➝789.2 for Re, 823.3➝481.0 for Rf, 1131.8➝789.8 for Rb1,
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1101.7➝790.7 for Rc, 1101.5➝789.5 for Rb2, 969.5➝789.5 for Rd, 807.5➝481.0 for Rg2,
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603.0➝423.3 for Rh1, 661.4➝481.2 for F1, 807.3➝627.2 for F2, 807.5➝465.0 for Rg3,
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441.0➝423.3 for PPT, 645.6➝465.2 for compound K, and 587.0➝407.0 for Rh2. We
166
analyzed the Panax ginseng extract samples using LC-MS/MS. Supplementary Figure 1B
167
represents the typical total ion chromatogram (TIC) of the extract in the positive-ion mode. 9
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Retention times, molecular mass, and characteristic MS/MS fragment ions were compared
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to those of standard ginsenosides, and the 7 major ginsenosides were identified as Rg1, Re,
170
Rf, Rb1, Rc, Rb2, and Rd, respectively.
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Characterization of Ginsenosides and their Metabolites in Colonic Content Samples.
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Following the oral intake of Panax ginseng, ginsenosides are inevitably in contact with the
173
gastrointestinal tract, and most of the active components are metabolized by gut bacteria. In
174
this study, samples of colonic content from 60 rats were collected at 12 h after the oral
175
intake of Panax ginseng extract and analyzed by LC-MS/MS. The TICs of a blank colonic
176
content sample and that of a typical colonic content sample collected at 12 h are compared
177
in Supplementary Figure 1C and D. There were 14 ginsenosides that were identified as the
178
main components in colonic content, including 7 major ginsenosides (Rg1, Re, Rf, Rb1, Rc,
179
Rb2, and Rd) and 7 deglycosylated metabolites (Rg2, Rh1, F1, F2, Rg3, PPT, and
180
compound K). The average abundance of major ginsenosides in HEM was significantly
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lower than in LEM, and deglycosylated metabolites in HEM were significantly higher than
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in LEM (Figure 1B). In addition, we analyzed the amount of individual ginsenosides in
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colonic content samples. In LEM, the amount of ginsenosides Re, Rg1, Rf, Rb1, Rc, Rb2,
184
and Rd was significantly higher than in HEM, whereas metabolites F2, PPT, and compound
185
K were significantly lower than in HEM (Figure 1C). Of the 14 ginsenosides in the colonic
186
content, the concentrations of PPT and compound K were significantly higher than the rest,
187
which indicates that ginsenosides PPT and compound K were the principal metabolites of
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Panax ginseng extract after oral administration. Recently, research into the metabolism of
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ginsenosides via gut microbiota has been reported. For example, after oral administration 10
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of single ginsenoside, the ginsenosides Rb1, Rb2, Rd, and Rc were primarily metabolized
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to compound K, and ginsenosides Re and Rg1 were metabolized to Rh1 and PPT by gut
192
microbiota.16-17,26-30 In addition, compound K has been reported to be a primary metabolite
193
in the feces after oral administration of American ginseng extract.31 Research into the in
194
vitro bioconversion of American ginseng extract via human gut microbiota reveals that
195
ginsenosides Rg3 and compound K are the major metabolites.32
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According to the metabolism of ginsenosides detected in this study and other reports of
197
metabolic pathways in previous studies, we speculated the pathway of ginsenosides by
198
intestinal microbiota (Figure 2). In the protopanaxadiol-type group, 2 metabolic pathways
199
can be charted. One way is that ginsenosides Rb1, Rc, Rb2, and Rd via selective
200
elimination of the C-20 and C-3 outer sugar moieties to moieties to produce F2 and then
201
compound K.33,34 Another way is to selectively eliminate the C-20 sugar chain to
202
ginsenoside Rg3 (Figure 2A).35 The metabolic pathways of the protopanaxatriol-type
203
ginsenosides via intestinal microbiota proposed in Figure 2B, illustrates that C-20 and C-6
204
sugar moieties of protopanaxatriol-type ginsenosides were hydrolyzed to transform PPT via
205
Rh1 or F1.36,37
206
Characterization of the Composition of Gut Microbiota in the LEM and HEM
207
Groups. To understand the correlation between the intestinal microbiota composition and
208
the gut microbiota metabolism of ginsenosides in individuals, we analyzed the gut
209
microbiota composition in colonic content of the LEM and HEM groups by performing
210
Illumina HiSeq sequencing-based analysis of bacterial 16S rRNA (V3–V4 region). A total
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number of 1456961 of 16S rRNA valid sequence reads were obtained from intestinal flora 11
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of 12 rats. An average of 121,413 sequence reads for each sample in the 2 groups was used
213
in this analysis. A total of 1,312,516 clean sequences were obtained after quality control,
214
containing 605,161,752 bp and the average length of the sequence reads was 461 bp from
215
12 rats. They were classified into different taxonomic categories by MGRAST. Alpha
216
diversity refers to the diversity or richness of the mean species diversity in a specific area.
217
The Chao and Ace were an estimator of phylotype richness, and Simpson and Shannon of
218
diversity reveal both the richness and community evenness (Table 1). These results
219
suggested that the abundance and diversity of the LEM and HEM groups have no
220
significant differences. Distinct clustering of microbiota composition between the LEM and
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HEM groups was observed following UniFrac-based principal coordinates analysis (PCoA)
222
and is presented in Figure 3A. The multivariate analysis of the PCoA matrix scores show a
223
statistically notable separation between the microbiota of the 2 groups (Figure 3B). These
224
results suggested that the samples in the HEM group were well separated from the LEM
225
group.
226
Then, taxonomy-based analysis revealed the populations of the dominant intestinal
227
microbiota in the 2 groups. At the phylum level, 12 phylas were discovered in the LEM and
228
HEM groups. The major phylas were Firmicutes, Actinobacteria, Fusobacteria,
229
Spirochaetae, Bacteroidetes, and Proteobacteria, and the sequences of the 6 phylas
230
occupied more than 97% of the total amount (Table 2). Of these, the Actinobacteria
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population in HEM was significantly higher than in LEM, while the level of Bacteroidetes
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in HEM was significantly lower than in LEM, and there was no obvious difference in the
233
level of Firmicutes, Fusobacteria, Spirochaetae, and Proteobacteria between the LEM and 12
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HEM groups.
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At the family level, 42 families were detected in all of the samples, and the populations
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of Alcaligenaceae, Coriobacteriaceae, Bifidobacteriaceae, S24-7, Erysipelotrichaceae,
237
Peptostreptococcaceae and Campylobacteraceae were significantly higher in HEM than in
238
LEM, while the level of Lachnospiraceae, Prevotellaceae, Porphyromonadaceae,
239
Defluviitaleaceae, Lactobacillaceae and Veillonellaceae in HEM were significantly lower
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than in LEM (Figure 3C). Kim et al. analyzed the different metabolic activity of FPG (with
241
metabolic activity) and FNG (non-metabolic activity) that metabolite ginsenoside Rb1 to
242
compound K from a pool of 100 subjects in vitro, and compared the fecal microbiota via
243
16s rRNA sequencing analysis.38 The result indicated that the population levels of
244
Clostridiales_uc,
245
Bifidobacteriaceae et al. in FPG were higher than in FNG, but those of Lachnospiraceae,
246
Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae and Leuconostocaceae
247
were lower in FPG than in FNG.
Ruminococcaceae,
Bacteroidaceae,
Rikenellaceae
and
248
Between the 2 groups in this study, there are 114 significantly different operational
249
taxonomic units (OTUs), of which 75 OTUs are significantly higher and 39 OTUs in the
250
HEM group are significantly lower than the LEM group (Table 3 and Figure 3D). Of 75
251
OTUs, 51 OTUs, 10 OTUs and 8 OTUs belong to S24-7 (phylum Bacteroidetes),
252
Erysipelotrichaceae (phylum Firmicutes), and Alcaligenaceae (phylum Proteobacteria),
253
respectively. Of 39 OTUs, 9 OTUs and 9 OTUs belongs to Prevotella (phylum Bacteroidetes)
254
and Ruminococcaceae (phylum Firmicutes), respectively. Xiao et al. has reported that
255
Bacteroidetes plays a crucial role in the metabolism of Panax notoginseng saponins.39 In our 13
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study, S24-7 (phylum Bacteroidetes) occupied most in OTUs of the HEM group, which was
257
significantly higher than the LEM group. In previous studies,the characterized metabolism of
258
ginsenosides intestinal bacteria primarily belonged to Bacteroidaceae, Bifidobacteriaceae,
259
Fusobacteriaceae, Propionibacteriaceae and Streptococcaceae families (Supplementary Table
260
1).21,28,40-43
261
Screening and Identification of Rat Intestinal Bacteria Metabolizing Ginsenoside
262
Rb1. A total of 200 microbes were isolated from the rat colonic content samples. Among
263
these, 112 β-glucosidase-producing isolates were screened using Esculin-GAM agar. Strain
264
GM1 showed the excellent activity of converting major ginsenoside Rb1. Phylogenetic
265
analysis based on 16S rRNA gene sequences indicated that GM1 belongs to the
266
Bifidobacterium animalis sp. Lactis AD011 (100% similarity). The relationships between
267
strain GM1 and other members of the genus Bifidobacterium were also evident in the
268
phylogenetic tree (Figure 4A).
269
The transformation of ginsenoside Rb1 via strain GM1 was analyzed by HPLC. (Figure
270
4B and C). The strain GM1 hydrolyzed the outer glucose moieties at the C-20 position of
271
ginsenoside Rb1 to transformed into ginsenoside Rd. The concentration of 1.0 mg/mL
272
ginsenoside Rb1 was transformed into 0.73 mg/mL ginsenoside Rd in 72 h, with a
273
corresponding molar conversion productivity of 86%. Ginsenosides are metabolized by
274
bifidobacteria, and it has been reported that Bifidobacterium K-50 and Bifidobacterium
275
K-103 converted ginsenoside Rc to compound K, Bifidobacterium. sp converted
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ginsenoside Rb1 or Rb2 to compound K, Bifidobacterium longum H-1 converted
277
ginsenoside Rb1 to compound K via Rd, Bifidobacterium longum RD47 converted 14
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ginsenoside Rb2 or Rc to Rd, Bifidobacterium breve K-110 converted ginsenoside Ra1 to
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Rb2, and Bifidobacterium. sp converted ginsenoside Rg3 to Rh2 and PPD.21,28,40-43
280
In conclusion, we quantitatively analyzed ginsenosides in rat colonic content samples,
281
and compared the levels of metabolized ginsenosides in individuals. The results showed
282
that ginsenoside metabolic activity was significantly different in individuals. We compared
283
the intestinal microbiota composition in colonic content samples of the LEM and HEM
284
groups using Illumina HiSeq sequencing of 16S rRNA gene, which noted significant
285
differences. Between the 2 groups, S24-7 (phylum Bacteroidetes), Alcaligenaceae (phylum
286
Proteobacteria), and Erysipelotrichaceae (phylum Firmicutes) occupied most OTUs of the
287
HEM group which was significantly higher than the LEM group. In addition, we isolated
288
Bifidobacterium animalis GM1 that could convert the ginsenoside Rb1 to Rd. The result
289
indicates that these specific intestinal bacteria may dominate the metabolism of Panax
290
ginseng.
291 292
Supporting Information
293
Ginsenosides detected by LC-MS/MS in the positive-ion mode (Supplementary Figure 1)
294
and biotransformation of ginsenosides by intestinal microbiota (Supplementary Table 1).
295
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(42) Hyun, Y. J.; Kim, B.; Kim, D. H. Cloning and characterization of ginsenoside
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and 20(R)-ginsenoside Rg3 by human intestinal bacteria and its relation to in vitro
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Funding
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This study was supported by a grant from the National Natural Science Foundation of
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China (No. 81660643; 81603365).
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FIGURE CAPTIONS
439
Figure 1. (A) Comparison of deglycosylated metabolites in 60 rats were collected in
440
colonic contents sample at 12 h following the oral administration of Panax ginseng extract.
441
(B) Average abundances of major ginsenosides and their deglycosylated metabolites in the
442
LEM and HEM groups. (C) The amount of individual ginsenosides in colonic content
443
samples in the LEM and HEM groups. The results shown represent the mean ± SD (n = 6
444
for each group; * p < 0.05, ** p < 0.01, *** p < 0.001).
445 446
Figure 2. The deglycosylated metabolic pathway of ginsenosides by gut microbiota were
447
speculated following oral administration of Panax ginseng extract. (A) The metabolic
448
pathways of the PPD-type ginsenosides and (B) the metabolic pathways of the PPT-type
449
ginsenosides.
450 451
Figure 3. Microbiota composition in the colonic content sample of the LEM and HEM
452
groups were analysed using Illumina HiSeq sequencing of the 16S rRNA gene. (A) Plots
453
shown were generated using the weighted version of the UniFrac-based PCoA. (B)
454
Multivariate analysis of variance from PCoA matrix scores between the LEM and HEM
455
groups. (C) Significant differences in family. (D) Heatmap showing the abundance of 114
456
OTUs significantly different in the LEM and HEM groups. The results shown represent the
457
mean ± SD (n = 6 for each group; * p < 0.05, ** p < 0.01, *** p < 0.001).
458 459
Figure 4. (A) The relationship between our isolates and the validated Bifidobacterium was 23
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supported by a high bootstrap value in the constructed phylogenetic tree based on the 16S
461
rRNA gene. (B) HPLC analysis of the time course of the biotransformation of ginsenoside
462
Rb1 by Bifidobacterium animalis GM1. (C) The conversion of ginsenosides Rb1 via
463
Bifidobacterium animalis GM1was quantified via HPLC analysis. Data represent the means
464
of 3 experiments, and error bars represent the standard deviation.
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Table 1. Number of Sequence Analyzed, Operational Taxonomic Unit (OTUs), Estimated OTU Richness (ACE and Chao1) and Diversity (Simpson, and Shannon) Phylotype Group Total reads OTU Number ACE Chao1 Simpson Shannon LEM1 A17 119766 852 883.79 884.01 0.02 4.92 A28 114629 799 843.69 862.86 0.06 4.46 A36 139885 792 837.89 840.61 0.07 4.17 A39 117838 799 847.27 853.44 0.03 4.71 A53 127430 905 929.25 946.59 0.02 5.00 A7 104506 792 843.05 860.71 0.05 4.34 Mean±SD 120675±12360 823±42 864.00±32.82 874.7±34.65 0.04±0.02 4.62±0.31 HEM2 B12 148407 783 823.51 828.64 0.03 4.58 B22 136281 850 893.34 901.74 0.03 4.48 B23 123435 817 896.43 910.67 0.04 4.53 B25 99307 854 900.71 910.28 0.02 4.74 B35 119538 816 886.98 911.39 0.07 4.09 B49 105939 831 860.51 858.24 0.02 4.97 Mean±SD 122151±16547 825±26 876.91±29.79 886.82±35.06 0.04±0.02 4.57±0.29
1
LEM, 6 rats with low efficiency metabolism; 2HEM, 6 rats with high efficiency
metabolism
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Table 2. The Differences Between HEM and LEM in the Composition (percent of total sequences) of Gut Bacterial Phyla Phylum Bacteroidetes Firmicutes Fusobacteria Proteobacteria Spirochaetae Actinobacteria
Population (% of total sequences) LEM
HEM
64.61±4.75 19.21±1.82 0.06±0.05 13.82±4.21 1.03±0.41 0.23±0.04
50.11±5.07 27.62±4.36 1.15±0.69 18.32±3.17 0.68±0.49 1.32±0.30
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p value 0.04 0.08 0.14 0.41 0.56 0.002
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Table 3. Represented Bacterial Taxa Information (Genus, Family, Orders, Class, and Phylum) of Significantly Different OTUs Between the HEM and LEM Groups. Taxon OTU ID
Phylum
Class
Orders
Family
Genus
OTU15005
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU90456
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU94519
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU55556
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU146757
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU48187
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU96852
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU63427
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU166478
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU54087
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU164801
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU112204
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU86539
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU106824
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU29983
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU166641
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU79384
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU110231
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU43316
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU77640
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU45435
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU127275
Bacteroidetes
Bacteroidia
_Bacteroidales
S24-7
uncultured_bacterium
OTU92552
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU109618
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU30945
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU44759
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU8281
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU13240
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU7369
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU86203
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU86161
Proteobacteria
Betaproteobacteria
Burkholderiales
Alcaligenaceae
Parasutterella Allobaculum
OTU3398
Firmicutes
Erysipelotrichia
Erysipelotrichales
Erysipelotrichaceae
OTU91713
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU736
Actinobacteria
Actinobacteria
Bifidobacteriales
Bifidobacteriaceae
Bifidobacterium
OTU11533
Firmicutes
Erysipelotrichia
Erysipelotrichales
Erysipelotrichaceae
Allobaculum
OTU187
Firmicutes
Erysipelotrichia
Erysipelotrichales
Erysipelotrichaceae
Allobaculum Allobaculum
OTU2684
Firmicutes
Erysipelotrichia
Erysipelotrichales
Erysipelotrichaceae
OTU130125
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU120446
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
Unknown_Order
OTU21139
Cyanobacteria
Chloroplast
Unknown_Family
uncultured_bacterium
OTU57959
Proteobacteria
Gammaproteobacteria Enterobacteriales
Enterobacteriaceae
Buchnera
OTU39722
Proteobacteria
Betaproteobacteria
Burkholderiales
Alcaligenaceae
Parasutterella
OTU115943
Proteobacteria
Betaproteobacteria
Burkholderiales
Alcaligenaceae
Parasutterella
OTU85263
Proteobacteria
Betaproteobacteria
Burkholderiales
Alcaligenaceae
Parasutterella
OTU121775
Firmicutes
Erysipelotrichia
Erysipelotrichales
Erysipelotrichaceae
Allobaculum Allobaculum
OTU16810
Firmicutes
Erysipelotrichia
Erysipelotrichales
Erysipelotrichaceae
OTU46037
Proteobacteria
Betaproteobacteria
Burkholderiales
Alcaligenaceae
Parasutterella
OTU18548
Firmicutes
Erysipelotrichia
Erysipelotrichales
Erysipelotrichaceae
Allobaculum Allobaculum
OTU52565
Firmicutes
Erysipelotrichia
Erysipelotrichales
Erysipelotrichaceae
OTU24474
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU101237
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU71507
Proteobacteria
Betaproteobacteria
Burkholderiales
Alcaligenaceae
Parasutterella
OTU118120
Actinobacteria
Coriobacteriia
_Coriobacteriales
Coriobacteriaceae
uncultured_bacterium
OTU150781
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU26524
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU102138
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
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Table 3. Continued. OTU23470
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
OTU127270
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium uncultured_bacterium
OTU53163
Firmicutes
Erysipelotrichia
Erysipelotrichales
Erysipelotrichaceae
Allobaculum Collinsella
OTU158304
Actinobacteria
Coriobacteriia
Coriobacteriales
Coriobacteriaceae
OTU7977
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU17495
Proteobacteria
Betaproteobacteria
Burkholderiales
Alcaligenaceae
Parasutterella
OTU42598
Firmicutes
Erysipelotrichia
Erysipelotrichales
Erysipelotrichaceae
Allobaculum
OTU140630
Proteobacteria
Betaproteobacteria
Burkholderiales
Alcaligenaceae
Parasutterella
OTU17146
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU59779
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU68999
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU48097
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU57669
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU70947
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU135288
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU113806
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU28206
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU377
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU107292
Proteobacteria
Deltaproteobacteria
Desulfovibrionales
Desulfovibrionaceae
Desulfovibrio
OTU61594
Bacteroidetes
Bacteroidia
Bacteroidales
Prevotellaceae
uncultured_bacterium Bacteroides
OTU11348
Bacteroidetes
Bacteroidia
Bacteroidales
Bacteroidaceae
OTU23163
Bacteroidetes
Bacteroidia
Bacteroidales
Prevotellaceae
uncultured_bacterium
OTU2736
Firmicutes
Clostridia
Clostridiales
Defluviitaleaceae
uncultured_bacterium uncultured_bacterium
OTU115059
Firmicutes
Clostridia
Clostridiales
Lachnospiraceae
OTU43911
Bacteroidetes
Bacteroidia
Bacteroidales
Rikenellaceae
uncultured_bacterium
OTU40651
Bacteroidetes
Bacteroidia
Bacteroidales
Rikenellaceae
RC9_gut_group RC9_gut_group
OTU116446
Bacteroidetes
Bacteroidia
_Bacteroidales
Rikenellaceae
OTU38144
Bacteroidetes
Bacteroidia
Bacteroidales
Porphyromonadaceae
Parabacteroides
OTU91992
Bacteroidetes
Bacteroidia
Bacteroidales
Rikenellaceae
RC9_gut_group
OTU153783
Firmicutes
Clostridia
Clostridiales
Ruminococcaceae
uncultured_bacterium
OTU127622
Firmicutes
Clostridia
_Clostridiales
Ruminococcaceae
Incertae_Sedis
OTU89622
Proteobacteria
Gammaproteobacteria Enterobacteriales
Enterobacteriaceae
Buchnera
OTU134838
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU81500
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium uncultured_bacterium
OTU90048
Bacteroidetes
Bacteroidia
Bacteroidales
Prevotellaceae
OTU51872
Firmicutes
Clostridia
Clostridiales
Christensenellaceae
uncultured_bacterium
OTU38796
Bacteroidetes
Bacteroidia
Bacteroidales
Bacteroidaceae
uncultured_bacterium
OTU162980
Bacteroidetes
Bacteroidia
Bacteroidales
Porphyromonadaceae
Parabacteroides
OTU76633
Firmicutes
Clostridia
Clostridiales
Ruminococcaceae
Incertae_Sedis
OTU107932
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
uncultured_bacterium
OTU70273
Bacteroidetes
Bacteroidia
Bacteroidales
S24-7
RC9_gut_group
OTU105055
Firmicutes
Clostridia
Clostridiales
Ruminococcaceae
Incertae_Sedis
OTU135090
Proteobacteria
Deltaproteobacteria
Desulfovibrionales
Desulfovibrionaceae
uncultured_bacterium
OTU44327
Bacteroidetes
Bacteroidia
Bacteroidales
Prevotellaceae
Prevotella
OTU53682
Proteobacteria
Alphaproteobacteria
Rhodospirillales
Rhodospirillaceae
Thalassospira
OTU123062
Bacteroidetes
Bacteroidia
Bacteroidales
Prevotellaceae
uncultured_bacterium
OTU110455
Bacteroidetes
Bacteroidia
Bacteroidales
Prevotellaceae
Alloprevotella
OTU142438
Bacteroidetes
Bacteroidia
Bacteroidales
Prevotellaceae
Prevotella
OTU67198
Candidate_division_TM7 Unknown_Class
Unknown_Order
Unknown_Family
Candidatus_Saccharimonas
OTU74712
Firmicutes
Clostridia
Clostridiales
Ruminococcaceae
Incertae_Sedis
OTU109280
Firmicutes
Clostridia
Clostridiales
Ruminococcaceae
uncultured_bacterium
OTU693
Bacteroidetes
Bacteroidia
Bacteroidales
Prevotellaceae
uncultured_bacterium
OTU78381
Bacteroidetes
Bacteroidia
Bacteroidales
Bacteroidaceae
Bacteroides
OTU71345
Bacteroidetes
Bacteroidia
Bacteroidales
Prevotellaceae
Alloprevotella
OTU113741
Firmicutes
Clostridia
Clostridiales
Ruminococcaceae
uncultured_bacterium
OTU70205
Firmicutes
Clostridia
Clostridiales
Ruminococcaceae
uncultured_bacterium
OTU88733
Firmicutes
Clostridia
Clostridiales
Ruminococcaceae
uncultured_bacterium
OTU156566
Spirochaetae
Spirochaetes
Spirochaetales
Spirochaetaceae
Treponema
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