Abstract: Maitake (Grifola frondosa) is a medicinal mushroom known for its peculiar biological
activities due to the presence of functional components, including dietary fibers and glucans, that
can improve human health through the modulation of the gut microbiota. In this paper, a Maitake
ethanol/water extract was prepared and characterized through enzymatic and chemical assays. The
prebiotic potential of the extract was evaluated by the growth of some probiotic strains and of a
selected probiotic consortium. The results revealed the prebiotic properties due to the stimulation of
the growth of the probiotic strains, also in consortium, leading to the production of SCFAs, including
lactic, succinic, and valeric acid analyzed via GC-MSD. Then, their beneficials effect were employed
in evaluating the vitality of three different healthy and tumoral colorectal cell lines (CCD841, CACO-2,
and HT-29) and the viability rescue after co-exposure to different stressor agents and the probiotic
consortium secondary metabolites. These metabolites exerted positive effects on colorectal cell lines,
in particular in protection from reactive oxygen species.
1. Introduction
The nutritional and medicinal effects of mushrooms are recognized worldwide, in
particular in Asian and northern and Central American countries. Indeed, over 270 fungal
species are identified for their biological activities, such as anti-inflammatory, antimicrobial,
and antioxidant properties [1]. Nevertheless, an important role has always been recognized
in the preservation of the healthy state of the gastrointestinal tract [2]. Accordingly,
nowadays, mushrooms are used not only in the pharmaceutical industry but also in the
nutraceutical and cosmeceutical industries [1]. This is due to their high protein and low-fat
contents, as well as to the presence of several vitamins, and minerals [1], but most of
all, it is due to the presence of important functional components such as dietary fibers,
chitin, and glucans [3]. In particular, glucans are polysaccharides composed of chemically
heterogeneous glucose molecules, classified in - or -glucans depending on the glycosidic
linkage [4]. -glucans are known for their beneficial effects in lowering blood pressure,
reducing glycemia, and acting as antitumoral and antioxidant agents [5]. Furthermore,
they could be used by the intestinal microbiota as prebiotics [2], defined as “non-digestible
dietary food ingredients that, when passing through the colon, will benefit the host by
selectively stimulating the growth and/or activity of one or a limited number of beneficial
bacteria in situ” [6]. Therefore, they are currently the most sought after functional food [2].
Among the mushrooms with health booster properties, there is Grifola frondosa (knows
as Maitake, Hen of the wood, or Signorina mushroom) [7]. It belongs to the Polyporaceae family [8], and it is characterized by caps with a smoky brown color [7]. Generally, it
grows in temperate regions, such as Japan, Europe, northeastern states of America, and
subtropical regions at high elevations [7]. Regarding the chemical composition, Maitake is
characterized by 3.8% water-soluble polysaccharides; among them, 13.2% correspond to
(1!3, 1!6)- -D-glucans [7]. Typically, the water-soluble polysaccharides can be divided
into two subpopulations based on the molecular weight, i.e., 722.7 kDa and 19.6 kDa [9].
Other components are starch, oligofructose, fructooligosaccharides (FOS), lactulose,
galactomannan, polydextrose, and dextrin [10]. Because of these characteristics, researchers
are trying to combine the possible beneficial effects of the mushroom with the helpful
action of probiotics (i.e., live microorganisms that confer a health benefit to the host when
administered in adequate amounts [11]). Generally, this type of fermentation is applied to
several food ingredients because probiotics can enhance the nutritional value of food [5]
and support positive health effects, such as immune modulation and the maintenance of a
state of eubiosis in the context of the gut microbiota composition. Furthermore, mushrooms
themselves could improve the antioxidant condition through the modulation of the gut
microbiota [10]. As a consequence, an important role in the status of intestinal mucosal
epithelial cells could be played by the released bacterial secondary metabolites generated
by mushrooms’ fermentation, in particular, short-chain fatty acids (SCFAs) [12], including
acetate, propionate, butyrate, and valerate [13]. In the presence of a medium-rich fiber
diet, SCFA concentration in the intestine is around 10 mmol/L, so the intestinal epithelial
cells are constantly exposed to these metabolites, mediating the crosstalk between the
microbiota and the host [14]. It is known that a fiber-rich diet could also prevent the
development of colorectal cancer (CRC). Interestingly, the SCFA butyrate could especially
act as an antitumoral agent through the modulation of several transduction pathways,
including the cellular proliferation pathway [12]. For example, butyrate inhibited the
proliferation of a colorectal tumoral cell line, decreasing the presence of the phosphorylated
extracellular-regulated kinase 1/2 (p-ERK 1/2), which is classified as a survival signal [14].
In this study, we analyzed a commercially available Maitake (Grifola frondosa) dried
extract, characterized for its -glucan content. Based on the specifics, we tested the prebiotic
property on several probiotic strains, comprising both Lactobacillus and Bifidobacterium
genera. Then, we studied a powerful probiotic consortium that, in the presence of Maitake
preparation, released SCFAs. The fermentation products were then tested for their effects
on the vitality of both healthy and tumoral colorectal cell lines. Finally, the rescue of the
viability of a cell line after induced stresses was evaluated.
2. Materials and Methods
2.1. Preparation of the Maitake Extract
The Maitake (Grifola frondosa Dicks. Gray) extract was obtained from Amita HC Italia
S.r.l. (Solaro, Milan, Italy). The original mushrooms came from China, and they were wild
at the time of the manual collection (from August to November). Then, the sporophorum
part was selected for the extraction of polysaccharides.
The commercial Maitake dried extract was prepared as follows. The Maitake sporophorums
were first ground and weighted. Then, to obtain an extract enriched in polysaccharides,
the obtained material was resuspended in a solution of ethanol:water (20:80 ratio),
and the separation was allowed. After overnight incubation, the precipitate was collected
and then dried at 50 C to eliminate the ethanol. Finally, the obtained extract was blended
and sieved to create a brownish fine powder characterized by a particle size lower than
180 m.
2.2. Characterization of the Maitake Extract
2.2.1. Determination of Starch Molecules Content
The starch molecules content was estimated through the Megazyme kit K-TSHK
(Megazyme Inc., Chicago, IL, USA) as described by the manufacturer’s instructions.
A starting weight of 100 mg of Maitake extract powder was used for the measurement.
Then, 0.2 mL of aqueous ethanol (80% v/v) was added and then stirred using a magnetic
stirrer. An amount of 2 mL of 2 M KOH was added, and the solution was stirred for
20 min in an ice-water bath. Then, 8 mL of 1.2 M sodium acetate buffer (pH 3.8) was
added, together with 0.1 mL of thermostable -amylase (from Megazyme kit) and 0.1 mL
of amyloglucosidase (20 U, from Megazyme kit). All the contents were stirred and then
incubated at 50 C for 30 min. The obtained solution was centrifuged at 3000 rpm for
10 min, and 0.1 mL of the supernatant was analyzed.
2.2.2. Determination of - and -Glucans Content
- and -glucans content was estimated through the Megazyme kit K-YBGL (Megazyme
Inc., Chicago, IL, USA) as described by the manufacturer’s instruction. A starting weight of
100 mg of Maitake extract powder was used for the measurement. Then, 2 mL of ice-cold
2 M KOH was added and then stirred using a magnetic stirrer at 4 C for 20 min. In total,
1.2 M sodium acetate buffer was added, and then amyloglucosidase (1630 U/mL) plus
invertase (500 U/mL) (200 L) (from Megazyme kit) was added. All the contents were
mixed and then incubated at 40 C for 30 min. The obtained solution was centrifuged at
2000 rpm for 10 min and 0.1 mL of the supernatant was analyzed for the glucose presence.
For the total glucan measurement, 100 mg of Maitake extract powder was used. An amount
of 2 mL of ice-cold 12 M sulfuric acid was added and then stirred. The tubes were then
placed at 4 C for 2 h in agitation. Then, 10 mL of water was added to each sample, which
was placed in a hot water bath (100 C) for 2 h. After cooling at room temperature, 6 mL of
10 M KOH was added, and the content was mixed well. Then, the volume was adjusted
to 100 mL with 200 mM sodium acetate buffer at pH 5. In total, 100 L of the sample was
incubated with 100 L of a mixture of exo-1,3- -glucanase (20 U/mL) plus -glucosidase
(4 U/mL) at 40 C for 60 min, and the glucose was determined with GOPOD reagent (all
of the reagents used were in the Megazyme kit). Absorbance was measured at 510 nm.
A 0.1 mL aliquot of 1 mg/mL glucose standard solution was incubated in triplicate with
GOPOD reagent; 0.1 mL of acetate buffer (200 mM, pH 5) was also incubated with 3.0 mL
of GOPOD reagent as a blank sample. Finally, the -glucan content was determined by
subtracting the -glucan content from the total glucan content.
The calculationsweremade throughMega-Calc sheet (Megazyme Inc., Chicago, IL,USA).
2.2.3. Determination of Polyphenols Content
The total phenolic content of the Maitake extract was measured using the Folin–
Ciocalteau phenol assay previously described [15]. A standard solution of Gallic Acid
(GA) ranging from 0 to 100 g/mL was used for the calibration. The GA solutions were
prepared in 80% methanol (Sigma, Milano, Italy), and the absorbance values were measured
at 765 nm. For the quantification, 0.5 mL of Folin-Ciocalteau phenol reagent (1:10 dilution)
and 1 mL of distilled water were added to 100 L of mushroom sample. The solutions were
mixed and incubated at room temperature for 1 min. Then, 1.5 mL of 20% sodium carbonate
(Na2CO3) solution was added to the sample and mixed. After the incubation for 120 min,
absorbance was recorded at 765 nm against the blank sample. Results were expressed as
mg of Gallic Acid Equivalent (GAE)/g of Maitake powder. All the measurements were
made in triplicate.
2.2.4. Determination of Protein Content
The protein content of Maitake extract was determined according to the Bradford
method [16]. A calibration curve using bovine serum albumin as a standard was performed
to determine the protein concentration of the extract.
2.2.5. Determination of Fructans and Reducing Sugars Content
The quantification of fructans in the Maitake extract powder was developed using the
fructan assay procedure kit Megazyme [17] in accordance with the manufacturer’s instruc tions. The fructans concentration was calculated considering the fructose, glucose, and sucrose
contents in the mushroom extract before and after the hydrolysis with fructanase. The
samples were treated with a specific sucrase/maltase enzyme to completely hydrolyze saccharides
to D-glucose and D-fructose. The reference values of the samples were determined
by a direct analysis of D-glucose plus D-fructose using the hexokinase/phosphoglucose isomerase/
glucose 6-phosphate dehydrogenase analytical procedure. The amount of NADPH
formed in this reaction is stechiometric with the amount of D-glucose plus D-fructose.
NADPH formation is measured by the increase in absorbance at 340 nm. The fructan
content of the samples was determined after hydrolyzation to D-fructose and D-glucose
by endo- and exo-inulinases, and then D-fructose and D-glucose content was measured as
described above. The fructan content was determined by subtracting absorbance values
of the reference from those of the sample. Beforehand, each enzymatic assay sample was
heated for 30 min at 50 C to ensure sample complete dissolution.2.3. Bacterial Strains and Culture Conditions
The bacterial strains used in this study are reported in Table 1. The strains provided
by a private collection of the company Roelmi HPC (Origgio, Italy) were previously
selected and characterized for the probiotic features by [18]. The probiotic strains were
routinely grown on MRS broth (Conda Lab, Madrid, Spain) supplemented with 0.03%
L-cysteine (Sigma-Aldrich, Milano, Italy) for 48 h, at 37 C, under anaerobic conditions
using an Anaerocult GasPack System (Merck, Darmstadt, Germany). A modified MRS
medium (mMRS), as described by [19], without glucose and supplemented with 0.03%
L-cysteine, was used for the growth trials. The pH of the medium was adjusted to 6.8 before
sterilization (121 C for 20 min). Maitake extract was added to mMRS at the concentration
of 2% w/v as a carbon source.
2.4. Growth Experiment with Single Probiotic Strains on Maitake Preparation
The probiotic bacteria described in Table 1 were pre-inoculated in MRS broth for 48 h,
at 37 C in anaerobic conditions before the setup of the prebiotic experiment. Maitake
extract powder was added to mMRS and then sterilized together before inoculation, to a
final concentration of 2% w/v.
In total, 1 mL of sterile mMRS or sterile mMRS + Maitake preparation was added to
every well of a sterile multi-well (24 wells, SPL Lifesciences, Pocheon-si, Korea). Then, each
well was inoculated with a proper volume of each probiotic pre-inoculum (around 20 L),
to achieve a final optical density (OD) at 600 nm of 0.1. Subsequently, the plates were
capped and incubated in an anaerobic jar at 37 C for 48 h. At the end of the experiment,
the OD at 600 nm was measured.
2.5. Growth Experiment with Mixed Probiotic Strains as Consortium on Maitake Preparation
The probiotic bacteria described in Table 1 were pre-inoculated in MRS broth for 48 h
at 37 C in anaerobic conditions before the setup of the experiment. Maitake extract powder
was added to mMRS and then sterilized together before inoculation, to a final concentration
of 2% w/v.The consortium was prepared in a sterile tube, mixing the selected probiotic in order
to achieve an OD at 600 nm of 0.1. After the homogenization, the proper volume of the
consortium (around 200 L) was inoculated in sterile tubes (BD, Milano, Italia) with a final
volume of 10 mL. Each tube contained only mMRS as a control or mMRS plus Maitake
extract. After the inoculum, the tubes were placed in an anaerobic jar and then incubated
for 48 h at 37 C. The growth was evaluated as optical density at 600 nm.
2.6. Extraction and Characterization of Probiotics Secondary Metabolites
2.6.1. Extraction of the Metabolites
The possible produced short-chain fatty acids (SCFAs) after Maitake fermentation
were extracted from inoculated and uninoculated tubes at the end of the fermentation using
ethyl-acetate (anhydrous, 99.8%, Sigma-Aldrich, Milano, Italy). The tubes were centrifuged
at 7000 rpm for 10 min at room temperature to separate the pellet from the supernatant.
In a glass tube (Colaver, Vimodrone, MI, Italy), 5 mL of the supernatant was acidified up
to pH 2 with HCl 6 M. Then, 5 mL of ethyl-acetate was added, followed by 20 min of
strong manual agitation. The obtained suspension was centrifuged at 4000 rpm for 20 min,
and the organic phase was withdrawn and conserved in a new glass tube. Another 5 mL
of ethyl-acetate were added to the remaining broth, followed by 5 min of strong manual
agitation. The suspension was again centrifuged, and the organic phase collected and
pooled with the one obtained in the first extraction.
2.6.2. Analysis of the Extracted Metabolites
The extracted organic phase was submitted to derivatization with BSTFA (Sigma-
Aldrich, Milano, Italy) before the gas chromatography (GC) injection. The samples and
BSTFA were mixed in a ratio of 3:1 and the reaction took place at 60 C for 20 min. After
the temperature cooled down, SCFAs were analyzed with a GC-MSD instrument, using a
Technologies 6890 N Network GC System, interfaced with a 5973 Network Mass Selective
Detector (MSD) (Agilent Technologies, Santa Clara, CA, USA). A ZB-5MS capillary column
was used (5% diphenyl-95% dimethylpolysiloxane 60 m 0.25 mm, 0.25 m; Alltech,
Lexington, KY, USA). Analyses were developed in the splitless injection mode, using
helium at 99.99% as carrier gas (Sapio, Bergamo, Italy). The program for the oven was set
at 65 C for 2 min, then 5 C min1 to 110 C, then 12 C min1 to 260 C, holding this
temperature for 10 min. Electron impact ionization spectra were obtained at 70 eV, with
recording of specific mass spectra at 73, 75, 117, 129, 132, 145, 159, 171, 173, 187, 201, 215,
229, 243, and 257 m/z. All the analyses were carried out in triplicate.
The registered mass spectra were compared with those of the library of National
Institute of Standards and Technology (NIST) of the instrument.
2.7. Maintenance and Growth of Cell Lines for In Vitro Tests
The healthy mucosa cell line CCD841 (ATCC® CRL-1790™) and the colon cancer
cell line CACO-2 (ATCC® HTB-37™) were grown in EMEM medium supplemented with
heat-inactivated 10% FBS, 2 mM L-glutamine, 1% nonessential amino acids, 100 U/mL of
penicillin, and 100 g/mL of streptomycin and maintained at 37 C in a humidified 5%
CO2 incubator; the colon cancer cell line HT-29 (ATCC® HTB-38™) was grown in DMEM
medium supplemented with heat-inactivated 10% FBS, 2 mM L-glutamine, 100 U/mL of
penicillin, and 100 g/mL of streptomycin and maintained at 37 C in a humidified 5%
CO2 incubator.
ATCC® cell lines were validated by short tandem repeat profiles that were generated
by the simultaneous amplification of multiple short tandem repeat loci and amelogenin
(for gender identification).
All the reagents for cell cultures were supplied by EuroClone (EuroClone S.p.A, Pero,
MI, Italy).
2.8. Cell Viability Assay and Test with Stress Agents
Cells were seeded in 96-well microtiter plates at a density of 1 104 cells/well and
incubated for 24 h.
To evaluate the effect of the extracts on CCD841, CACO-2 and HT-29 viability, the
cells were treated for 24 h with 0.25, 0.5, and 1 mg/mL for each extract.
HT-29 cell line was treated with stressor compounds, H2O2 (0–8 mM), or SDS (0–0.1%),
for 24 h in order to determine the concentration responsible for an about 50% reduction in
cell viability. To evaluate a potential role of the extract in cell viability rescue, cells were
pre-treated for 1 h with 0.25, 0.5, and 1 mg/mL for each extract, and then 1 mM H2O2
or 0.0075% SDS was added to the cells with a 24 h endpoint. Following the treatment,
cell viability was assessed using an in vitro MTT-based toxicology assay kit (Sigma, St.
Louis, MO, USA): the medium was replaced with a complete medium without phenol red,
and 10 L of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide)
solution was added to each well; after 4 h of incubation, formed formazan crystals were
solubilized with 10% Triton-X-100 in acidic isopropanol (0.1 N HCl) and absorbance was
measured at 570 nm using a micro plate reader. The results were expressed as mean
values ES of at least three independent experiments.
2.9. Statistical Analysis
Regarding the growth of probiotics, all the experiments were performed in triplicate
and results were presented as mean values standard deviation. The statistical relevance of
the results was assessed by a t-Student’s test. The significance was defined as p-value < 0.01
or p-value < 0.05.
Regarding the experiments with the intestinal cell lines, all the experiments were
performed in triplicate. The results were shown as the mean value of vitality % standard
error. Statistical differences were calculated using Dunnett’s multiple comparisons test:
* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001.
3. Results
3.1. Characterization of the Principal Components of Maitake Extract
The determination of the main components of the Maitake extract was evaluated with
enzymatic assays using Megazyme methods, as described in the Materials and Methods
Section. The major components of the mushroom preparation are starch (around 50% w/w),
and glucans (around 25% w/w, comprising - and -glucans). Interestingly, the presence
of -glucans is very low with respect to the -ones, which are dominant in the extract
(around 6.2% vs. 18.8%, respectively). Furthermore, Maitake extract was also analyzed
for its content of polyphenols and protein by chemical reactions. Each extract preparation
contained at least 1.9% w/w of polyphenols and 0.02% w/w of proteins. Other components
are sugars; indeed, the extract is characterized by 1.2% w/w of fructans and 3.6% w/w
of free reducing sugars. The contents of glucans, fructans, free reducing sugars, starch,
polyphenols and protein are listed in Table 2.
3.2. Prebiotic Potential of Maitake Preparation on Lactobacillus and Bifidobacterium Strains
The possible prebiotic capability of Maitake extract at a concentration of 2% w/v
was evaluated through in vitro growth assays employing Lactobacillus and Bifidobacterium
strains, originally isolated from the human colon [18]. The initial OD600nm of each culture
was 0.1; then, it was recorded at the end of the experiment, after 48 h of anaerobic
fermentation (Figure 1). The medium containing all components except the extract was
used as a control. As shown in Figure 1, the mushroom preparation could be considered as
a prebiotic substrate for the probiotic bacteria, because all the tested strains were able to
ferment it and the difference between the growth in the control condition and the growth
on the prebiotic was statistically significant (p-value < 0.01). Among the Lactobacillus
strains, L. fermentum, L. rhamnosus, and L. reuteri showed the most positive responses
(p-value < 0.05), while, regarding the Bifidobacteria members, all the strains showed an
important growth increase in Maitake extract, in particular B. longum/infantis and B. lactis
(p-value < 0.01). The results highlight the prebiotic characteristic of the mushroom formulation.
3.3. Growth of the Probiotic Consortium on Maitake Preparation
To enhance the possible beneficial effects for the human host attributable both to
probiotics and prebiotics, a possible combination of the bacteria was studied. All the
probiotic strains previously used were inoculated at OD600nm equal to 0.1, making sure to
have a homogeneous suspension of all the considered bacteria. The growth capacity was
evaluated on the same conditions used for the experiment with single strains, i.e., control
medium and Maitake extract 2% w/v. After 48 h of anaerobic growth at 37 C, the optical
density was measured (Figure 2). Interestingly, the final OD on the mushroom preparation
had a mean value of 3.7 0.21. The prebiotic potential of Maitake preparation is also
confirmed in this kind of experiment, because of the very significant difference between
the growth on the sole medium and the one on the medium added with Maitake extract
(p-value < 0.01).Prebiotic potential of Maitake extract on probiotic consortium. The figure represents the
growth of consortium of the selected probiotic strains in presence of CTR medium and Maitake extract
at a concentration of 2% w/v. Values are represented as mean value of OD at 600 nm standard
deviation. Statistical differences were calculated using t-Student’s test: ** p-value < 0.01.
3.4. Extraction and Characterization of Probiotic Consortium Secondary Metabolites
The production of potentially beneficial secondary metabolites derived from the fermentation
of complex carbohydrates by probiotics is well documented. They are recognized
as short-chain fatty acids (SCFAs) and as branched-chain fatty acids (BCFAs) [20].
To investigate which compounds are produced by the probiotic combination after
the Maitake extract fermentation, a liquid–liquid extraction with ethyl-acetate of the broth
culture after 48 h of anaerobic fermentation was initially conducted. Then, a gas chromatography
analysis was performed, and the chromatograms were interpreted using mass
spectrometry. Each peak was compared to the example present in the NIST library.
In comparison with the control condition, samples deriving from fermented Maitake
extract presented several additional peaks (Figure 3). The highest peak, at a retention time
of 8.2 min, refers to lactic acid. The second most important peak is at 12.2 min of retention
time, and it is assigned to valeric acid. The third highest peak refers to succinic acid
(retention time of 13.6 min). Other detected molecules were the SCFA butyrate (retention
time of 10.8 min) and hydrocinnamic acid (retention time of 16.8 min), which is not a
bacterial secondary metabolite, but a Maitake component [21] probably released due to
probiotic digestion, and cinnamic acid (retention time of 19.9 min).