Shiitake Culinary-Medicinal Mushroom, Lentinus edodes (Agaricomycetes), Supplementation Alters Gut Microbiome and Corrects Dyslipidemia in Rats

Abstract

Recent interest in diet-induced modulation of the gut microbiome has led to research on the impact that dietary fibers can have on host health. Lentinus edodes mushroom–derived fibers may act as an appropriate substrate for gut microbe digestion and metabolism. The metabolites that gut microbes excrete can modulate host energy balance, gut absorption, appetite, and lipid metabolism. In the present study, we explored the dynamics of the gut microbiome of hypercholesterolemic rats supplemented with L. edodes. Wistar rats were offered a chow maintenance diet (CMD; CON group) or the same CMD ration with cholesterol (1.5% w/w) and cholic acid (0.5% w/w) added to induce hypercholesterolemia (day 1 to day 24). Hypercholesterolemic rats were subsequently offered either the same cholesterol–cholic acid diet (HC-CON group) or were supplemented with L. edodes (5% w/w; LE group) for 42 days (day 25 to day 66). At the end of the experiment, serum triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol concentrations were determined. Colon digesta were subjected to DNA extraction and subsequent 16S rRNA gene sequencing. Raw sequences were quality filtered and statistically analyzed using QIIME and LEfSe tools. Triglyceride concentrations were lower (P = 0.002) in the LE group than in the CON and HC-CON groups. Total cholesterol and LDL cholesterol concentrations were slightly decreased, whereas HDL cholesterol concentrations were increased by L. edodes supplementation compared with the HC-CON group. The gut microbiome of the LE group had higher species richness characterized by increased abundance of Clostridium and Bacteroides spp. Linear discriminant analysis identified bacterial clades that were statistically different among treatment groups. In conclusion, manipulation of gut microbiota through the administration of L. edodes could manage dyslipidemia.

Full text

International Journal of Medicinal Mushrooms, 21(1):79–88 (2019)
79
1521-9437/19/$35.00 © 2019 Begell House, Inc. www.begellhouse.com
Shiitake Culinary-Medicinal Mushroom, Lentinus
edodes (Agaricomycetes), Supplementation Alters
Gut Microbiome and Corrects Dyslipidemia in Rats
Haseeb Anwar,a,* Jan S. Suchodolski,b Muhammad I. Ullah,c Ghulam Hussain,a
Muhammad Z. Shabbir,d Imtiaz Mustafa,a & Muhammad U. Sohaile,*
aDepartment of Physiology, Government College University, Faisalabad, Pakistan; bGastrointestinal Laboratory,
Department of Small Animal Clinical Sciences, Texas A&M University, College Station, Texas, USA; cFaculty of
Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan; dQuality Control Laboratory, University of
Veterinary and Animal Sciences, Lahore, Pakistan; eBiomedical Research Center, Qatar University, Qatar
*Address all correspondence to: Muhammad Umar Sohail, Biomedical Research Center, Qatar University, Qatar; drumarsohail@gmail.com; or
Haseeb Anwar, Department of Physiology, Government College University, Faisalabad 38000, Pakistan; drhaseebanwar@gcuf.edu.pk
ABSTRACT: Recent interest in diet-induced modulation of the gut microbiome has led to research on the impact that
dietary bers can have on host health. Lentinus edodes mushroom–derived bers may act as an appropriate substrate
for gut microbe digestion and metabolism. The metabolites that gut microbes excrete can modulate host energy balance,
gut absorption, appetite, and lipid metabolism. In the present study, we explored the dynamics of the gut microbiome of
hypercholesterolemic rats supplemented with L. edodes. Wistar rats were offered a chow maintenance diet (CMD; CON
group) or the same CMD ration with cholesterol (1.5% w/w) and cholic acid (0.5% w/w) added to induce hypercho-
lesterolemia (day 1 to day 24). Hypercholesterolemic rats were subsequently offered either the same cholesterol–cholic
acid diet (HC-CON group) or were supplemented with L. edodes (5% w/w; LE group) for 42 days (day 25 to day 66).
At the end of the experiment, serum triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, and low-
density lipoprotein (LDL) cholesterol concentrations were determined. Colon digesta were subjected to DNA extraction
and subsequent 16S rRNA gene sequencing. Raw sequences were quality ltered and statistically analyzed using QIIME
and LEfSe tools. Triglyceride concentrations were lower (P = 0.002) in the LE group than in the CON and HC-CON
groups. Total cholesterol and LDL cholesterol concentrations were slightly decreased, whereas HDL cholesterol concen-
trations were increased by L. edodes supplementation compared with the HC-CON group. The gut microbiome of the
LE group had higher species richness characterized by increased abundance of Clostridium and Bacteroides spp. Linear
discriminant analysis identied bacterial clades that were statistically different among treatment groups. In conclusion,
manipulation of gut microbiota through the administration of L. edodes could manage dyslipidemia.
KEY WORDS: Lentinus edodes, medicinal mushrooms, dyslipidemia, metabolic disorders, microbiome, 16S rRNA
sequencing
ABBREVIATIONS: ANOSIM, analysis of similarities; CMD, chow maintenance diet; CON, control group; CYP7A1, cholesterol
7-α-hydroxylase 1; GIT, gastrointestinal tract; HC, hypercholesterolemia-only group; HDL, high-density lipoprotein; LDA, linear
discriminant analysis; LDL, low-density lipoprotein; LE, L. edodes diet; OTU, operational taxonomic unit
I. INTRODUCTION
The mammalian gastrointestinal tract (GIT) harbors a dense, dynamic, and complex microbial community
commonly known as the microbiome. The host–microbiome consortium is intimately interactive, with
mutually shared interests. Microbes prevent local infections and systemically monitor metabolism and
neuroendocrine processes.1 The scientic literature suggests a signicant role of the GIT microbiome in
metabolic diseases such as diabetes, obesity, and dyslipidemia.2 Changes in blood lipid proles and predictive
metabolic responses to diet and drugs are associated with the GIT microbiome through currently unknown
processes. Our lifestyle and dietary habits inuence the structure and activity of our gut microbial ecology. It

International Journal of Medicinal Mushrooms
Anwar et al.
80
is generally believed that dietary interventions improve bacterial diversity and composition that indirectly
correlate with improvement in metabolic markers such as glucose and lipid homeostasis. Researchers have
observed that the continuous consumption of animal protein or plant products for at least > 7 days alters
the phylogenetic structure and functional capacity of our microbiome.3 Dietary bers from the plant origin
act as a substrate for several symbiotic microbes and support a link between phylogenetic composition
and host metabolism and trigger immune modulatory activities.
Shiitake culinary-medicinal mushroom, Lentinus edodes (Berk.) Singer (=Lentinula edodes;
Marasmiaceae, Agaricomycetes), is the most cultivated mushroom in Asia and is known for its nutritional
and medicinal properties. L. edodes contains proteins, essential fats, polyphenols, vitamins, and polysac-
charides.4 This mushroom is increasing in importance as a popular functional food due to its nutritional and
medicinal properties.5 L. edodes is a good source of carbohydrates and protein. The mushroom is a source
of vitamins, especially vitamin B, which includes B1 (thiamine), B2 (riboavin), B12 (niacin), and B5 (pan-
tothenic acid), as well as provitamin D2 (ergosterol).6 Dietary bers present in L. edodes can modulate the
transcriptional prole of some genes involved in cholesterol metabolism and can help in the management of
dyslipidemia.7 Furthermore, water-soluble compounds present in L. edodes can be used to treat infections
and autoimmune and metabolic diseases. These therapeutic characteristics may be attributed to the lentinan,
phenol, and polysaccharide compounds present in L. edodes.8,9 Our previous studies reveal that dietary bers
and polyphenolic compounds present in different foods can preferentially modulate gut microbial ecology
in diabetic and obese rat models.10,11 Although previous studies have shown cholesterol-lowering effects of
L. edodes,12 no connection has been established between the microbiome composition and lipid prole in
L. edodes–supplemented rats. Therefore, the present study was designed to assess changes in the gut micro-
biome of hypercholesterolemic rats supplemented with L. edodes.
II. MATERIALS AND METHODS
A. Animals and Diets
Twenty-four adult Wistar rats of the same weight (200 ± 20 g) and mixed sex were procured from the
University of Agriculture in Faisalabad, Pakistan. All rats were housed in individual cages under standard
management conditions (24 ± 2°C, 12-hour light/12-hour dark cycle). The rats were fed either a chow
maintenance diet (CMD) (CON group; n = 8) or a hypercholesterolemic diet (n = 16) for 24 days. The
hypercholesterolemic diet contained cholesterol (1.5% w/w) and cholic acid (0.5% w/w), replaced by
starch in the CMD (Table 1). After conrmation of hypercholesteremia with the help of commercially
available kits, the hypercholesterolemic rats were divided into 2 groups: the HC-CON group (fed only
the hypercholesterolemic diet) and the LE group (fed the HC diet further supplemented with L. edodes
at the dose of 5% w/w). Dietary treatments were continued from day 25 to day 66, and the lipid prole
was assessed at the end of the study period. The rats were killed by decapitation, and trunk blood and
colon digesta were collected. All procedures involving animals were approved by the Ethics Committee
of Government College University, Faisalabad, Pakistan (reference no. GCUF/ERC/131).
B. Procurement and Processing of L. edodes
In this study, L. edodes fruiting bodies were procured from the Institute of Horticulture Science at the
University of Agriculture (Faisalabad, Pakistan) and were identied by the Department of Botany at
Government College University (Faisalabad, Pakistan). L. edodes fruiting bodies were dried in an oven
at 37°C and then milled into powder.

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Effects of Lentinus edodes on Microbiome and Dyslipidemia 81
C. Serum Lipid Prole Analysis
At the end of the experiment, blood serum was harvested by blood centrifugation at 2000 × g at 4°C for 10
minutes. Total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride concentrations in
the serum were determined enzymatically using commercially available reagent kits (Randox Laboratories,
Kearneysville, WV). Low-density lipoprotein (LDL) cholesterol concentrations were calculated as fol-
lows: Total cholesterol − HDL cholesterol = LDL cholesterol.
D. Microbiome Analysis
Genomic DNA was extracted from colon digesta using the QIAmp DNA Mini Kit (Qiagen, Valencia,
CA). Genome concentrations (in ng/μL) and purity (A260/280 and A260/230) were assessed using a
NanoDrop spectrophotometer (ND 1000; Wilmington, DE). Libraries of the V4 hypervariable region of
the 16S rRNA gene were prepared using primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R
(5′-GGACTACVSGGGTATCTAAT-3′). The PCR amplication products were puried on 2% agarose
gels and subsequently calibrated using Ampure XP beads. Amplicon libraries were sequenced at the MR
DNA Laboratory (Shallowater, TX) using the Illumina MiSeq instrument (Illumina, San Diego, CA).
Raw sequence data obtained from the MiSeq instrument were trimmed, quality ltered, denoised, and
chimera depleted. Demultiplexing and clustering as operational taxonomic units (OTUs) were performed
at 97% similarity using QIIME (V1.9) default settings. The amplicon sequences were converted into
FASTQ and were submitted to the National Center for Biotechnology Information short-read archive.
E. Statistical Analysis
Serum lipid prole readings were converted to mean per group, and the standard deviation was calcu-
lated for each group. Differences in mean values were calculated using analysis of variance and Tukey’s
multiple-comparison test. Microbiome data were subjected to alpha and beta diversity using an even sample
depth of 19,793 sequences per sample. Analysis of similarity (ANOSIM; P = 0.05) was performed to
detect differences in microbiome composition among different treatment groups. Normal distribution of
TABLE 1: Composition of Diets
Dietary Contents Chow Maintenance Diet
(% w/w)
Hypercholesterolemic Diet
(% w/w)
Lentinus edodes Diet
(% w/w)
Starch 76 74 69
Protein 10 10 10
Oil (cooking) 10 10 10
Vitamin and mineral premixa4 4 4%
Cholic acid —0.5 0.5
Cholesterol —1.5 1.5
L. edodes — — 5
aVitamin-mineral premix (each kg contained): 35 g calcium, 0.2 g folic acid, 0.03 g copper sulfate, 200,000 IU vitamin A, 32 g
phosphorus, 0.89 g iron, 0.08 g selenium, 96,000 IU vitamin D, 9.44 g sodium, 0.39 g manganese, 0.39 g cobalt, 350 IU
vitamin E, 8.64 g magnesium, 0.22 g zinc, 0.87 g potassium iodide, and 0.6% vitamin B (350 IU vitamin B1, 85,000 IU
vitamin B2, 67,000 IU vitamin B6, and 350 IU vitamin B12).

International Journal of Medicinal Mushrooms
Anwar et al.
82
the data was assessed using the Kolmogorov–Smirnov test. A nonparametric Kruskal–Wallis H test was
applied to the data, and signicance was observed at P values < 0.05. The Galaxy-based LEfSe tool was
used for linear discriminant analysis (LDA) of the microbiome data, and a histogram and a cladogram
were constructed.13
III. RESULTS
A. Serum Lipid Prole
The serum lipid prole, consisting of triglycerides, total cholesterol, HDL cholesterol, and LDL cholesterol
concentrations, is presented in Table 2. Total cholesterol and LDL cholesterol levels were high (P ≤ 0.05)
in the HC-CON group compared to the CON group, whereas HDL cholesterol decreased (P ≤ 0.05) in the
HC-CON group compared to the CON and LE groups, and triglyceride concentrations were signicantly
higher (P ≤ 0.05) in the HC-CON group than in the LE group.
B. Microbiome Prole
The raw counts of 1,196,925 sequences were agglomerated to 21 phyla, 40 classes, 73 orders, 141 families,
and 258 genera. After ltration of low-count taxa by including only taxa with at least 0.01% sequence
reads in at least one treatment group, we obtained 7 phyla, 13 classes, 17 orders, 32 families, and 68
genera. Firmicutes, Clostridia, Clostridiales, Lachnospiraceae, and Blautia were the most abundant bacte-
rial clades belong to different levels of taxonomic hierarchy. The increased representation of Firmicutes
in the CON and LE groups was mostly explained by the increased abundance of Lachnospiraceae and
Ruminococcaceae families and the genus Blautia (OTU 32,730) (Table 3). Within phylum Actinobacteria,
the Bidobacteriaceae family and Bidobacterium genus were differently represented in all the study
groups. Bacteroidetes were the most abundant phyla in the L. edodes–supplemented group. Table 3 presents
percentage differences in bacterial sequences determined using the Kruskal–Wallis rank test.
A UniFrac-based principal coordinate analysis (PCoA) revealed differences in the cecal microbial com-
munities of rats fed a L. edodes–supplemented diet and clustered separately from those of the CON and
HC-CON groups (Fig. 1). Similarly, the alpha rarefaction analysis revealed higher interindividual variability
in the microbiomes of rats fed L. edodes compared with the CON and HC-CON groups. Species richness
was highest in the LE group and lowest in the HC-CON group. An ANOSIM revealed a signicantly dif-
ferent (P = 0.0001) microbiome clustering pattern in the PCoA plots. The pairwise ANOSIM P value was
0.0002 for CON versus HC-CON, 0.0001 for CON versus LE, and 0.0002 for HC-CON versus LE. These
TABLE 2: Overall Serum Lipid Prole in Different Groups
Parameter (mg/dL) Treatment Group P Value
CON HC-CON LE
Total cholesterol 252.8a ± 6.54 262.5b ± 4.50 258.5ab ± 6.67 0.019
HDL cholesterol 79.0b ± 28.8 43.2a ± 16.14 70.3b ± 19.84 0.013
LDL cholesterol 173.7a ± 34.4 219.2b ± 16.11 188.5ab ± 23.73 0.008
Triglycerides 203.8ab ± 2.39 208b ± 2.41 196.87a ± 3.56 0.002
Means within a row lacking a common superscript differ (P ≤ 0.05). CON, control group; HC-CON, hypercholesterolemic control
group; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LE, Lentinus edodes–fed hypercholesterolemic group.

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Effects of Lentinus edodes on Microbiome and Dyslipidemia 83
TABLE 3: Percentage Abundance of Predominant Bacterial Taxa Identied in Different Treatment Groups
Taxa Treatment Group
CON HC-CON LE CON
Phylum
Actinobacteria 9.340 23.570 8.333 0.056
Bacteroidetes 0.345 0.413 3.958 0.030
Euryarchaeota 6.756 4.153 9.469 0.408
Firmicutes 80.184 60.617 74.458 0.027
Class
Actinobacteria 9.340 23.570 8.333 0.056
Alphaproteobacteria 0.033 0.270 0.044 0.041
Bacilli 1.930 7.691 2.237 0.616
Bacteroidia 0.345 0.412 3.957 0.030
Clostridia 64.821 35.653 53.124 0.019
Erysipelotrichia 9.415 4.954 4.611 0.126
Order
Bacteroidales 0.345 0.412 3.957 0.030
Bidobacteriales 7.463 18.756 4.969 0.044
Clostridiales 64.821 35.653 53.122 0.019
Coriobacteriales 1.708 0.715 0.978 0.291
Erysipelotrichales 9.415 4.954 4.611 0.126
Lactobacillales 1.919 7.590 2.151 0.663
Methanobacteriales 6.756 4.153 9.469 0.408
Family
Bacteroidaceae 0.100 0.107 1.533 0.023
Bidobacteriaceae 7.463 18.756 4.969 0.044
Clostridiaceae 2.757 2.216 7.900 0.069
Erysipelotrichaceae 9.415 4.954 4.611 0.126
Eubacteriaceae 6.103 2.454 6.633 0.068
Lachnospiraceae 41.156 27.562 30.940 0.078
Lactobacillaceae 1.867 3.751 2.057 0.633
Methanobacteriaceae 6.756 4.153 9.469 0.408
Porphyromonadaceae 0.223 0.269 1.716 0.036
Ruminococcaceae 4.521 1.045 3.482 0.005
Genus
Bacteroides 0.070 0.070 1.469 0.024
Bidobacterium 7.463 18.756 4.969 0.044
Blautia 35.202 21.820 16.045 0.042
Clostridium 2.534 2.045 7.665 0.050

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Anwar et al.
84
Collinsella 1.478 0.577 0.728 0.226
Dorea 2.017 0.821 2.138 0.086
Eubacterium 6.061 2.425 6.521 0.068
Lachnoclostridium 4.562 2.347 8.352 0.205
Lactobacillus 0.318 3.685 2.029 0.383
Methanobrevibacter 6.756 4.153 9.469 0.408
Paludibacter 0.038 0.066 1.120 0.044
Ruminococcus 4.207 0.887 2.923 0.005
Medians were considered statistically signicant at P ≤ 0.05 (Kruskal–Wallis rank test). Only bacterial clades representing
> 1% of the genomic sequences in at least one treatment group are presented here. CON, control group; HC-CON, hypercho-
lesterolemic control group; LE, Lentinus edodes–fed hypercholesterolemic group.
TABLE 3: (continued)
FIG. 1: (A) Chao1 alpha diversity measured at 19,793 sequences per sample obtained from colon microbiome
samples of rats belonging to different groups. (B) Principal coordinate analysis plots of the unweighted Unifrac dis-
tance matrix. Control group, middle line; hypercholesterolemic control group, lower line; and Lentinus edodes–fed
hypercholesterolemic group, upper line.
results suggest that treatment groups distinctly clustered and reect unique microbiome signatures for
each treatment group.
Further, LDA-based LEfSe observed the relative abundances of all bacterial clades among different
treatment groups. The histogram and cladogram constructed on the Galaxy tool expressed spatial structure
differences of taxonomic hierarchy among treatment groups (Figs. 2 and 3). The analysis graphically dif-
ferentiated statistically abundant bacterial clades with respect to the class/group of interest. Based on the
LDA effect-size algorithm, which treats each sample with each treatment group to compare all bacterial
clades, the control group was predominantly occupied with phylum Firmicutes, class Clostridia, and genera
Blautia and Ruminococcus. The L. edodes–supplemented group was predominantly occupied with phylum
Bacteroidetes, class Bacteroidia, and genus Butyricimonas. The HC-CON group was mainly dominated
by phylum Actinobacteria, order Bidobacteriales, and genus Bidobacterium.14

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Effects of Lentinus edodes on Microbiome and Dyslipidemia 85
FIG. 2: Histogram demonstrating LDA (P = 0.05) of relative abundances of all bacterial clades among treatment
groups as observed using the Galaxy-based LEfSe tool. A pairwise Wilcoxon test was used to make comparison
between subclasses at a threshold of 4.0 on the logarithmic LDA score for discriminative features. HC-CON,
hypercholesterolemic control group; LDA, linear discriminant analysis; LE, Lentinus edodes–fed hypercholester-
olemic group.
IV. DISCUSSION
In this study, we evaluated the interaction between the gut microbiota and cholesterol metabolism in
hypercholesterolemic rats treated with L. edodes. We observed that L. edodes supplementation in hyper-
cholesterolemic rats corrected dyslipidemia and promoted the growth of specic gut microbes. Previously,
it was reported that supplementation of L. edodes decreases liver enzymes, which supports the hepato-
protective effect of L. edodes; furthermore, these researchers reported the immunomodulatory and anti-
oxidant activities of L. edodes supplementation in hypercholesterolemic rats as well.15 Continuous feeding
of a hypercholesterolemic diet supplemented with cholesterol (1.5%) and cholic acid (0.5%) for 24 days
induced hypercholesterolemia in rats. Thereafter, continuous supplementation of L. edodes for 42 days
(day 25 to day 66) to the hypercholesterolemic rats partially or completely lowered triglyceride concen-
trations and increased HDL cholesterol (Table 2). These ndings are in agreement with a previous report
by Yang et al.,16 who observed that supplementation with L. edodes signicantly lowered serum total
cholesterol, LDL cholesterol, and tryglycerides. Furthermore, they observed upregulation of the choles-
terol 7-α-hydroxylase 1 (CYP7A1) gene by L. edodes supplementation. The CYP7A1 gene is considered a
key regulator of bile acid synthesis and cholesterol metabolism.17 Therefore, upregulation of CYP7A1 by
L. edodes supplementation ultimately regulated cholesterol metabolism. An alternative mechanism for
control of dyslipidemia is the conversion of cholesterol into coprostanol by bacterial metabolism in the
GIT.18 Several microbes residing in the GIT can promote cholesterol metabolism and its conversion to
coprostanol, which is eventually excreted in stool.19 The dry ber content of mushrooms is reported to
induce a transcriptional response in Caco-2 cells, suggesting a possible cholesterol-lowering effect. In

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Anwar et al.
86
FIG. 3: The cladogram represents the key bacterial clades in a taxonomic tree specied by the hierarchical feature in
respective treatment group.
the palliative setting, it reduces the hepatic triglyceride, likely because Dgat1 was downregulated. Thus,
they reduce the triglyceride accumulation in the liver.20 Therefore, researchers concluded that beta glucans
play an immense role in the regulation of fat metabolism, which helps to reduce the blood cholesterol
level and ultimately helps to reduce body weight.21
The gut microbiome plays a crucial role in host immunity and metabolism. Individuals with micro-
bial dysbiosis (characterized by low bacterial richness) have marked overall adiposity, dyslipidemia,
and insulin resistance compared with individuals with high bacterial richness.22 In the present study, we
observed high bacterial richness in L. edodes–supplemented rats compared with the CON and HC-CON
groups. Polysaccharide compounds present in L. edodes alter the spatial structure of the gut microbiome,
particularly increasing the number of Bacteroidetes, Butyricimonas, Alistipes, and Helicobacter.23 These
bacterial clades are more abundantly clustered in lean subjects in comparison with the phylum Firmicutes,
which is more often associated with obesity-associated metabolic syndromes.24 In the present study, few
bacterial clades were distinguished between rats with high and low bacterial richness, even between the
HC-CON and LE groups. Particularly, rats supplemented with L. edodes had a higher abundance of phylum
Bacteroidetes and genera Clostridium and Bacteroides. It has been reported that signicant sources of
probiotics in mushrooms are nondigestible polysaccharides, which not only inhibit the growth of patho-
genic bacteria in the gut but additionally promote the growth of probiotic bacteria.25 It has been observed

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Effects of Lentinus edodes on Microbiome and Dyslipidemia 87
that the change from a low-fat/high-ber diet to a high-fat/sugar diet reduces the Bacteriodes spp. in the
gut, with a counter increase in the Bacilli and Erysipelotrichia from the Firmicutes phylum in animal
models.14 Members of the Bacteroides genus are well-reported organisms involved in carbohydrate and
lipid metabolism; they express enzymes like glycoside hydrolases, polysaccharide lyases, and glycosyl
transferases. Meanwhile, they also suppress the inhibition of lipoprotein lipase activity in adipocytes.26
The role of mushrooms in enhancing these Bacteroides spp. and the partial role of these bacteria in lipid
metabolism in the gut of the animal models was recently reviewed and summarized, and Bacteroides
spp. isolated from human feces are known to reduce cholesterol into coprostanol.27 Similarly, Clostridium
spp. are also known for cholesterol metabolism and for lowering plasma cholesterol levels.28 In addition,
supplementation of phenolic compounds or bers enhances Butyricimonas availability in the GIT and
lowers cholesterol diet-induced inammation or insulin resistance in obese subjects.29,30 Furthermore,
butyrate-producing bacteria, including Butyricimonas, have also been related to metformin treatment–asso-
ciated anti-inammatory, blood lipid–lowering, and weight-lowering benets.31 Bidobacterium is another
important bacterial genus that alleviates dyslipidemia and insulin resistance.32 However, in the present
study, its OTU count was higher in the HC-CON group than in the CON and LE groups. These ndings
suggest that interactions between the gut microbiota and cholesterol metabolism are partially unspecied
under some conditions and that some patterns of dysbiosis associated with cholesterol metabolism have
not been fully characterized.
Hyperlipidemia is connected to many other metabolic complications like diabetes, and polysaccha-
rides of mushrooms have been extensively reported to reduce hyperlipidemia and related oxidative stress,
which are key factors in the development of these metabolic disorders.33 In conclusion, the results of the
present study suggest that supplementation with L. edodes partially inhibits the development of dyslip-
idemia induced by a hypercholesterolemic diet. This partial correction in lipid prole may be attributed
to spatial distribution of gut microbiota in the supplemented rats, particularly an overexpression of genus
Butyricimonas from phylum Bacteroidetes. The diet-induced changes in bacterial diversity and species
richness indices observed in the present study can serve as a springboard for future studies to delineate
the pathophysiological role of the gut microbiome in metabolic diseases.
ACKNOWLEDGMENT
This study was partially sponsored by a Higher Education Commission seed grant for young researchers.
The authors have no conicts of interest to disclose.
REFERENCES
1. El Aidy S, Dinan TG, Cryan JF. Gut microbiota: the conductor in the orchestra of immune-neuroendocrine communication.
Clin Ther. 2015;37:954–67.
2. Hartstra AV, Bouter KE, Bäckhed F, Nieuwdorp M. Insights into the role of the microbiome in obesity and type 2 diabetes.
Diabetes Care. 2015;38:159–65.
3. David LA, Maurice CF, Carmody RN, Gootenberg DB, Button JE, Wolfe BE, Ling AV, Devlin AS, Varma Y, Fischbach MA,
Biddinger SB. Diet rapidly and reproducibly alters the human gut microbiome. Nature. 2014;505:559–63.
4. Chang ST, Wasser SP. The role of culinary-medicinal mushrooms on human welfare with pyramid model for human health.
Int J Med Mushrooms. 2012;14(2):95–134.
5. Chang ST. The world mushroom industry: trends and technological development. Int J Med Mushrooms. 2006;8:297–314.
6. Hobbs CR. Medicinal value of Lentinus edodes (Berk.) Sing. (Agaricomycetidae). A literature review. Int J Med Mushrooms.
2000;2:287–302.
7. Gil-Ramírez A, Caz V, Smiderle FR, Martin-Hernandez R, Largo C, Tabernero M, Marín FR, Iacomini M, Reglero G, Soler-
Rivas C. Water-soluble compounds from Lentinula edodes inuencing the HMG-CoA reductase activity and the expression
of genes involved in the cholesterol metabolism. J Agric Food Chem. 2016;64:1910–20.

International Journal of Medicinal Mushrooms
Anwar et al.
88
8. Chen H, Ju Y, Li J, Yu M. Antioxidant activities of polysaccharides from Lentinus edodes and their signicance for disease
prevention. Int J Biol Macromol. 2012;50:214–8.
9. Finimundy T, Gambato G, Fontana R, Camassola M, Salvador M, Moura S, Hess J, Henriques JA, Dillon AJ, Roesch-Ely
M. Aqueous extracts of Lentinula edodes and Pleurotus sajor-caju exhibit high antioxidant capability and promising in vitro
antitumor activity. Nutr Res. 2013;33:76–84.
10. Noratto GD, Garcia-Mazcorro JF, Markel M, Martino HS, Minamoto Y, Steiner JM, Byrne D, Suchodolski JS, Mertens-
Talcott SU. Carbohydrate-free peach (Prunus persica) and plum (Prunus domestica) juice affects fecal microbial ecology
in an obese animal model. PLoS One. 2014;9:e106128.
11. Sohail MU, Shabbir MZ, Steiner JM, Ahmad S, Kamran Z, Anwar H, Hussain G, Shaukat A, Suchodolski JS. Molecular
analysis of the gut microbiome of diabetic rats supplemented with prebiotic, probiotic, and synbiotic foods. Int J Diabetes
Dev Ctries. 2017;37(4):419–25.
12. Fukushima M, Ohashi T, Fujiwara Y, Sonoyama K, Nakano M. Cholesterol-lowering effects of maitake (Grifola frondosa)
ber, shiitake (Lentinus edodes) ber, and enokitake (Flammulina velutipes) ber in rats. Exp Biol Med. 2001;226:758–65.
13. Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, Huttenhower C. Metagenomic biomarker discovery
and explanation. Genome Biol. 2011;12:R60.
14. Clarke SF, Murphy EF, Nilaweera K, Ross PR, Shanahan F, O’Toole PW, Cotter PD. The gut microbiota and its relationship
to diet and obesity: new insights. Gut Microbe. 2012;3:186–202.
15. Nisar J, Mustafa I, Anwar H, Sohail MU, Hussain G, Ullah MI, Faisal MN, Bukhari SA, Basit A. Shiitake culinary-medicinal
mushroom, Lentinus edodes (Agaricomycetes): a species with antioxidant, immunomodulatory, and hepatoprotective activi-
ties in hypercholesterolemic rats. Int J Med Mushrooms. 2017;19(11):981–90.
16. Yang H, Hwang I, Kim S, Hong EJ, Jeung EB. Lentinus edodes promotes fat removal in hypercholesterolemic mice. Exp
Ther Med. 2013;6(6):1409–13.
17. Lambrinoudaki I, Kaparos G, Papadimitriou D, Sergentanis TN, Creatsa M, Alexandrou A, Logothetis E, Christodoulakos G,
Kouskouni E. Methylenetetrahydrofolate reductase C677T polymorphism is associated with central adiposity and increased
androgenicity in healthy postmenopausal women. Eur J Endocrinol. 2008;159(3):233–41.
18. Gérard P. Metabolism of cholesterol and bile acids by the gut microbiota. Pathogens. 2013;3(1):14–24.
19. Gérard P, Lepercq P, Leclerc M, Gavini F, Raibaud P, Juste C. Bacteroides sp. strain D8, the rst cholesterol-reducing
bacterium isolated from human feces. Appl Environ Microbiol. 2007;73(18):5742–9.
20. Caz V, Gil-Ramírez A, Largo C, Tabernero M, Santamaría Mn, Martin-Hernandez R, Marín FR, Reglero G, Soler-Rivas C.
Modulation of cholesterol-related gene expression by dietary ber fractions from edible mushrooms. J Agric Food Chem.
2015;63(33):7371–80.
21. Rop O, Mlcek J, Jurikova T. Beta‐glucans in higher fungi and their health effects. Nutr Rev. 2009;67(11):624–31.
22. Le Chatelier E, Nielsen T, Qin J, Prifti E, Hildebrand F, Falony G, Almeida M, Arumugam M, Batto JM, Kennedy S, Leonard
P. Richness of human gut microbiome correlates with metabolic markers. Nature. 2013;500(7464):541–6.
23. Xu X, Zhang X. Lentinula edodes-derived polysaccharide alters the spatial structure of gut microbiota in mice. PLoS One.
2015;10(1):e0115037.
24. Sohail MU, Althani A, Anwar H, Rizzi R, Marei HE. Role of gastrointestinal tract microbiome in the pathophysiology of
diabetes mellitus. J Diabetes Res. 2017;2017:9631435.
25. Bhakta M, Kumar P. Mushroom polysaccharides as a potential prebiotics. Int J Health Sci Res. 2013;3(8):77–84.
26. Jandhyala SM, Talukdar R, Subramanyam C, Vuyyuru H, Sasikala M, Reddy DN. Role of the normal gut microbiota. World
J Gastroenterol. 2015;21(29):8787–803.
27. Jayachandran M, Xiao J, Xu B. A critical review on health promoting benets of edible mushrooms through gut microbiota.
Int J Mol Sci. 2017;18(9):E1934.
28. Kobashi K, Takeda Y, Itoh H, Hase J. Cholesterol-lowering effect of Clostridium butyricum in cholesterol-fed rats. Digestion.
1983;26(4):173–8.
29. Lee SM, Han HW, Yim SY. Benecial effects of soy milk and ber on high cholesterol diet-induced alteration of gut micro-
biota and inammatory gene expression in rats. Food Funct. 2015;6(2):492–500.
30. Moreno-Indias I, Sanchez-Alcoholado L, Fernandez-Garcia JC, Castellano-Castillo D, Cardona-Diaz F, Queipo-Ortuno MI.
Extra virgin olive oil and red wine polyphenols modulate fecal microbiota and reduce metabolic risk factors in high insulin
resistant obese patients (Abstract). Endocrine Abstracts. 2016;41:EP814.
31. Lee H, Lee Y, Kim J, An J, Lee S, Kong H, Song Y, Lee CK, Kim K. Modulation of the gut microbiota by metformin
improves metabolic proles in aged obese mice. Gut Microbe. 2018;9:155–65.
32. Martínez I, Wallace G, Zhang C, Legge R, Benson AK, Carr TP, Moriyama EN, Walter J. Diet–induced metabolic improve-
ments in a hamster model of hypercholesterolemia are strongly linked to alterations of the gut microbiota. Appl Environ
Microbiol. 2009;75(12):4175–84.
33. Lo HC, Wasser SP. Medicinal mushrooms for glycemic control in diabetes mellitus: history, current status, future perspec-
tives, and unsolved problems. Int J Med Mushrooms. 2011;13(5):411–26.