Download Production of exopolysaccharide from mycelial culture of Grifola

FEMS Microbiology Letters 251 (2005) 347–354
www.fems-microbiology.org
Production of exopolysaccharide from mycelial culture
of Grifola frondosa and its inhibitory effect on matrix
metalloproteinase-1 expression in UV-irradiated
human dermal fibroblasts
Jun Tae Bae a, Gwan Sub Sim a, Dong Hwan Lee a, Bum Chun Lee a, Hyeong Bae Pyo a,
Tae Boo Choe b, Jong Won Yun c,*
a
R&D Center, Hanbul Cosmetics Co., Chungbuk 369-830, Republic of Korea
Department of Microbial Engineering, Konkuk University, Seoul 143-701, Republic of Korea
Department of Biotechnology, Daegu University, Kyungsan, Kyungbuk 712-714, Republic of Korea
b
c
Received 10 June 2005; received in revised form 13 August 2005; accepted 16 August 2005
First published online 30 August 2005
Edited by D. Mattanovich
Abstract
Exopolysaccharide (EPS) was prepared by submerged mycelial culture of a newly isolated mushroom Grifola frondosa HB0071 in
a 5-l stirred-tank fermenter. This fungus produced a high concentration of biomass (24.8 g l1 at day 4), thereby achieving high EPS
concentration (7.2 g l1 at day 4). EPS was proven to be a proteoglycan consisting of 85.6% carbohydrates (mostly glucose) and
7.3% proteins with a molecular weight of 1.0 · 106 Da. The photoprotective potential of EPS was tested in human dermal fibroblasts
(HDF) exposed to ultraviolet-A (UVA) light. It was revealed that EPS had an inhibitory effect on human interstitial collagenase
(matrix metalloproteinase, MMP-1) expression in UVA-irradiated HDF without any significant cytotoxicity. The treatment of
UVA-irradiated HDF with EPS resulted in a dose-dependent decrease in the expression level of MMP-1 mRNA (by maximum
61.1% at an EPS concentration 250 lg ml1). These results suggest that EPS obtained from mycelial culture of G. frondosa
HB0071 may contribute to inhibitory action in photoaging skin by reducing the MMP 1-related matrix degradation system.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Exopolysaccharide; Grifola frondosa; Human dermal fibroblasts; Matrix metalloproteinase; Skin aging; UVA-irradiation
1. Introduction
Much interest has been generated in biotechnological
methods for the production of polysaccharides for
application in the food, pharmaceutical, cosmetic and
other industries [1–3]. Most of the polysaccharides with
*
Corresponding author. Tel.: +82 53 850 6556; fax: +82 53 850
6559.
E-mail address: [email protected] (J.W. Yun).
various physiological activities frequently originate from
fungi, especially mushrooms [4–6]. Some kinds of mushroom polysaccharides such as Lentinan (from Lentinus
edodes), Schizophyllan (from Schizophyllum commune),
and Krestin (from Coriolus versicolor) are currently
available to the pharmaceutical industry [7–9].
Grifola frondosa is a Basidiomycete fungus belonging
to the order Aphyllopherales, and the family Polyporaceae. Fruit body and liquid-cultured mycelia of this
mushroom have been reported to contain useful
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.08.021
348
J.T. Bae et al. / FEMS Microbiology Letters 251 (2005) 347–354
antitumor polysaccharides in various fractions of the
culture filtrates [10–12].
A wide variety of applications of polysaccharides
have been reported, including thickening and stabilizing
agents in the chemical industry, and immunostimulating
and antitumor agents for clinical use [13–15]. Apart
from these applications, polysaccharides have been used
as a substance that enhances the skins natural ability to
heal and protect itself against infection [2]. Recently,
several polysaccharides have been used as alternative
ingredients for enhancing collagen biosynthesis and
increasing cell proliferation [16,17].
Ultraviolet irradiation plays an important role in
altering the dermis and activating a family of degradative enzymes called matrix metalloproteinase (MMPs),
thereby inducing photodamage and skin aging. These
enzymes target the components of the extracellular matrix (ECM) such as collagen, laminin, fibronectin and
proteoglycan [18,19]. The expression of MMPs in UVirradiated fibroblasts is known to be initiated by reactive
oxygen species (ROS) and by activation of a cell surface
growth factor and cytokine receptors [20,21].
Although several investigators have studied different
fractions of polysaccharides from G. frondosa and reported their anti-tumor activities, biological activities
in respect of skin aging have not been extensively demonstrated [12,22,23]. In the present study, we have produced EPS from a submerged mycelial culture of the
newly isolated G. frondosa HB0071 and investigated its
inhibitory effect on matrix metalloproteinase (MMP-1)
expression upon UVA-irradiation in cultured human
dermal fibroblasts.
2. Materials and methods
2.1. Microorganism and media
G. frondosa HB0071 was isolated from the mountainous district in Chungbuk province, Korea. The stock
culture was maintained on potato dextrose agar (PDA)
slants. Unless otherwise specified, slants were incubated
at 27 C for 5 days and then stored at 4 C. The seed culture was grown at 27 C on a rotary shaker incubator at
120 rpm for 5 days in a 250-ml flask containing 50 ml of
medium: per litre, 30 g glucose, 6 g yeast extract, 2 g
polypeptone, 0.5 g MgSO4 Æ 7H2O, 0.5 g K2HPO4, and
0.2 g MnSO4 Æ 5H2O.
2.2. Fermentations for EPS production
G. frondosa HB0071 was initially grown on PDA
medium in a petri dish, and then transferred to the seed
culture medium by punching out 5 mm of the agar plate
culture with a sterilized house-developed cutter [24]. The
seed cultures were grown in a 250-ml flask containing
50 ml of basal medium at 27 C on a rotary shaker incubator at 120 rpm for 3 days. The second flask culture
experiments were performed in a 500-ml flask containing
100 ml of the media after inoculating with 3% (v/v) of
the seed culture under the aforementioned culture conditions. The fermentation media were inoculated with 3%
(v/v) of the seed culture and then cultivated at 27 C in
a 5-l stirred-tank fermenter (Best-Korea, Daejeon,
Korea). Fermentations were conducted at 27 C, aeration rate 1.0 vvm, agitation speed 150 rpm, pH 5.5,
and working volume 3-l. The seed cultures were transferred to the fermentation medium and were cultivated
for 5 days.
2.3. Preparation of EPS
The fermentation broth was centrifuged at 8000g for
20 min, and the resulting supernatant was filtered
through Whatman filter paper No. 2 (Whatman International Ltd., Maidstone, UK) and mixed with four volumes of absolute ethanol, stirred vigorously and left
overnight at 4 C. The precipitated EPS was collected
by centrifugation at 8000g for 10 min, discarding the
supernatant. The residue was re-precipitated with four
volumes of ethanol and the precipitate of pure EPS
was freeze-dried in a lyophilizer.
2.4. Culture of human dermal fibroblasts
Human dermal fibroblasts (HDF), isolated from human neonatal foreskin, were purchased from Modern
Tissue Technologies Inc. (Seoul, Korea). HDF were cultured on Dulbeccos modified Eagles medium/Hams F12 nutrient mixture (DMEM/F-12; 3:1 v/v, Sigma) containing 10% fetal bovine serum (FBS), penicillin
(100 IU ml1), and streptomycin (100 lg ml1) at
37 C in a humidified atmosphere containing 5% CO2.
Fibroblast cultures were subcultured by trypsinization
and used between the sixth and tenth passages.
2.5. UVA irradiation
HDF (1.5 · 105 per well) were seeded into 35B plates
(CORNING, Corning Inc., NY, USA) and cultured
overnight. Prior to irradiation, when cells were 70–
80% confluent, they were washed twice with phosphate
buffered saline (PBS). UVA simulator (Jhonsam Inc.,
Seoul, Korea), filtered for the emission of UVA (320–
400 nm), was used at a tube-to-target distance of
15 cm. The dose of UVA radiation, determined with a
UV radiometer (International light Inc., Newburyport,
MA, USA) was set at 6.3 J/cm2. During irradiation,
control cells were treated identically, except for the
exposure to UV light. After irradiation, fresh serum-free
medium containing EPS at different concentrations were
added to cells at 37 C for 24 h.
J.T. Bae et al. / FEMS Microbiology Letters 251 (2005) 347–354
2.6. RNA isolation and RT-PCR
RNA was extracted using a RNeasy Mini Kit (Qiagen, Maryland, USA) according to the suppliers
instructions. First, a reverse-transcriptase polymerase
chain reaction (RT-PCR) was performed to synthesize
cDNA using an Omniscript RT Kit (Qiagen, Hilden,
Germany) according to the manufacturers instructions.
PCR was then performed with each cDNA of MMP-1,
b-actin fragments, primers, and Tag DNA polymerase.
The primers used were as follows: MMP-1
5 0 -AAAGGGAATAAGTACTGGGC-3 0 (sense)
5 0 -AATTCCAGGAAAGTCATGTG-3 0 (anti-sense)
b-actin
5 0 -ATGCAGAAGGAGATCACTGC-3 0 (sense)
5 0 -CTGCGCAAGTTAGGTTTTGT-3 0 (anti-sense).
The primer sets yielded PCR products of 237 and
248 bp for MMP-1 and b-actin, respectively. Reactions
were carried out in an automatic heat-block DNA thermal cycler (ASTEC PC801, ASTEC Inc, Tokyo, Japan)
for 25 cycles: denaturation for 30 s at 94 C; annealing
for 30 s at 50 C; extension for 60 s at 72 C. PCR products were electrophoresed on a 1.5% agarose gel in TAE
(40 mM Tris acetate, 1-mM EDTA) and visualized by
ethidium bromide staining. The level of each gene
mRNA expression was expressed as the ratio of the
intensity of each gene PCR product to the corresponding b-actin PCR product as a reference molecule for
measuring of mRNA stability and normalized to the
control sample.
2.7. Analytical methods
2.7.1. Estimation of mycelial growth and EPS production
The precipitate of EPS was lyophilized and the total
weight of EPS was estimated. The dry weight of mycelium was measured after repeated washing of the mycelial
pellets with distilled water and drying overnight at 70 C
to a constant weight. For a quantitative measurement of
glucose, the filtrate was analyzed by high performance
liquid chromatography (HPLC) using a Sugar-Pak column (300 · 6.5 mm, Waters Co., Milford, MA, USA)
equipped with an evaporative light scattering detector
(ELSD, Alltech Associates, Deefield, IL, USA).
2.7.2. Analysis of carbohydrates and amino acids
The total sugar content of EPS was determined by the
phenol-sulphuric acid method using glucose as the standard [25]. The sugar composition was analyzed by
HPLC system (Waters 2695 Separations Module,
Waters Co., Milford, MA, USA) with a Sugar-Pak 1
column and an ELSD detector. The total protein was
349
determined by the Lowry method with bovine serum
albumin as the standard [26]. The composition of amino
acids was analyzed by the HPLC system with AccQ Æ Tag Amino Acid Analysis column (150 · 3.9 mm,
Waters Co., Milford, MA, USA) and photo array detector (996, Waters Co., Milford, MA, USA).
2.7.3. Molecular weight determination
The molecular weight of EPS was estimated on the
basis of the calibration curve made by the HPLC system
with a Shodex OHpak KB-804 column (300 · 0.8 mm,
Showa Denko K.K., Tokyo, Japan) using distilled water
as a mobile phase (column temperature 50 C; flow rate,
0.8 ml min1; injection vol., 20 ll). The eluate was monitored by an ELSD detector. The column was standardized with dextrans of diverse molecular mass (Polymer
Standards Service Inc., Silver Spring, MD, USA).
2.7.4. Morphological measurements
The morphological properties of the mycelia were
evaluated using an image analyzer (WINA Tech Co.,
Ansan, Korea) with software coupled to a light microscope through a charged coupled device (CCD) camera
(Toshiba Co., Japan). The samples were fixed with an
equal volume of fixative (13 ml of 40% formaldehyde,
5 ml glacial acetic acid with 200 ml of 50% ethanol).
Each fixed sample (0.1 ml) was transferred to a slide,
air dried and stained with methylene blue (0.3 g methylene blue, 30 ml 95% ethanol in 100 ml water) [27].
2.7.5. Fermentation kinetics
The specific growth rate, l (h1) was calculated from
the equation: l = (1/X)(dX/dt). Where, X is the cell concentration (g 11) at time t (h). The specific consumption
rate of substrate, QS/X (g g1 day1) was estimated by
the equation: QS/X = (dS/dt)(1/X). Where, S is the concentration of glucose (g1) at time t (day). The specific
production rate of EPS, PP/X (g g1 day1) was estimated by the equation: PP/X = (dP/dt)(1/X). Where, P
is the concentration of EPS (g 11) at time t (day).
The yield of EPS on substrate, YP/S (g g1) was estimated by the equation: YP/S = (dP/dt)(dS/dt).
2.7.6. Cytotoxicity
The cell viability was determined by the modified
method of Mosmann [28] using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay
by mitochondrial dehydrogenase in viable cells to produce a dark blue formazan product. HDF were cultured
on DMEM/F-12 containing 10% FBS and then 2 · 104
cells per well were added on a 96-well microtiter plate.
After addition of EPS at different concentrations into
each well, the 96-well plate was maintained at a CO2
incubator at 37 C for 24 h. After cultivation was completed and DMEM/F-12 was removed, 12 ll of 0.5%
MTT and 100 ll of fresh DMEM were added on the
J.T. Bae et al. / FEMS Microbiology Letters 251 (2005) 347–354
2.7.7. Enzyme-linked immunosorbent assay
The expression level of MMP-1 was assayed by an enzyme-linked immunosorbent assay (ELISA). HDF
(2 · 104 cells per well) were seeded into 48-well plates
and cultured overnight. The culture media were replaced
with DMEM/F-12 containing EPS at different concentrations. After 24-h incubation, the supernatants were
transferred into a 96-well plate and coating buffer
(Na2CO3 1.59%, NaHCO32.93%, NaN3 0.20%, MgCl2
1.02%, pH 9.6) was added with the same volume and
incubated for 24 h. The supernatants were removed
and the coated well was washed three times with PBS
containing 0.05% Tween 20 (PBST) followed by blocking with 3% bovine serum albumin in PBS for 1 h at
37 C. After washing three times with PBST, 50 ll of
1/1000 diluted primary antibody (Ab), Ab-5 in PBST
was added into each well and incubated for 60 min.
After washing the wells with PBST three times, 50 ll
of 1/1000 diluted secondary Ab, anti-mouse IgG conjugated with alkaline phosphatase in PBST, were added
and incubated for 60 min. After washing five times with
PBST, 100 ll of 1 mg ml1pNPP (p-nitrophenyl phosphate) in a diethanolamine buffer was added. The optical density was measured at 405 nm after 30 min.
Finally, cytotoxicity of the supplemented chemicals
was measured by the MTT assay.
2.7.8. Statistical analysis
All experiments were performed in triplicate. Data
were presented as mean ± standard error (SE). Experimental results were statistically analyzed by using the
Students t-test (SigmaPlot 2000). P values less than
0.05 were considered statistically significant.
3. Results and discussion
3.1. Fermentation for EPS production
Fig. 1 shows the typical time courses of mycelial
growth and EPS production in a 5-l stirred-tank fermenter. The maximum biomass (24.8 g l1) and EPS
(7.2 g l1) were achieved at 84 h, both of which were significantly higher concentrations compared with those of
previous fermentation results from G. frondosa [29,30].
20
30
25
15
20
10
15
10
5
5
0
Exopolysaccharide (g/l)
96-well plate. Again, the plate was maintained at the
CO2 incubator for 4 h to allow formazan formation.
The quantity of formazan produced can be regarded
as an indicator of cell density or viability. After dissolving formazan in 100 ll acid-isopropanol (0.04 N HCl in
isopropanol), the absorbance at 570 nm was measured
with a microplate reader (Model ELX 800, BIO-TEK
Inc., Winooski, VT, USA). The results obtained were
calculated from three sets of experiments and presented
as a percentage of control values.
Mycelial biomass and
Residual sugar (g/l)
350
0
0
2
48
72
96
120
Time (h)
Fig. 1. Time profiles of biomass and exopolysaccharide production in
submerged culture of Grifola frondosa HB0071 in a 5-l stirred-tank
fermenter. (d) mycelial biomass, (j) exopolysaccharide, (m) residual
glucose.
The overall kinetic data of G. frondosa HB0071 is illustrated in Table 1. The specific growth rate (l) of the fungus and the specific production rate of EPS (PP/X) were
0.491 h1 and 0.134 g g1 day1, respectively. Many
investigators have reported that most mushrooms required a long period of over 10 days for maximum formation of biomass and EPS in their submerged cultures
[5,23]. The markedly short fermentation time (4 days)
for maximum production of biomass and EPS in G.
frondosa HB0071 is a promising advantage because culture time often directly affects the productivity of EPS in
submerged culture processes of higher fungi.
During fermentation, the cells mainly form pellets
with high hairiness. It was observed that pellet size increased rapidly from the beginning of the fermentation
and reached a maximum size at day 4, achieving maximum biomass and EPS production (data not shown).
After this period, a denser and larger core region was
observed due to a lack of nutrient uptake and oxygen
supply. In the later stage of fermentation (after 5 days),
the larger pellets were finally divided into several smaller
pellets without significant hyphal fragmentation (no significant increase in the concentration of free mycelia,
data not shown).
Table 1
Fermentation results of Grifola frondosa HB0071 in a 5-l stirred tank
fermenter
Kinetic parameters
Values
Maximum biomass concentration, X (g l1)
Maximum exopolysaccharide concentration, P (g l1)
Specific growth rate, l (h1)
Specific consumption rate of substrate, QS/X (g g1 day1)
Specific production rate of exopolysaccharide,
PP/X (g g1 day1)
Yield of exopolysaccharide on substrate, YP/S (g g1)
24.02
6.501
0.491
0.534
0.134
0.251
J.T. Bae et al. / FEMS Microbiology Letters 251 (2005) 347–354
351
Fig. 2. Typical elution chromatogram in a gel permeation chromatography by HPLC (A) and determination of molecular weight of EPS (B).
Dextrans (abbreviated as D) of diverse molecular mass were used as the standard.
3.2. Characterization of EPS
The molecular mass of EPS was determined by HPLC
with Shodex OHpak KB-804 column, where a single symmetrical peak was observed (Fig. 2A). The EPS had an
extremely high molecular weight (1 · 106 Da) (Fig. 2B),
which is considerably higher than those obtained from
mycelial extracts of G. frondosa reported by Mizuno
et al. [11]. A compositional analysis revealed that EPS
was a proteoglycan consisting of 85.6% polysaccharide
and 7.3% protein. The detailed compositions of carbohydrate and amino acid in EPS are illustrated in Table 2.
The EPS consisted of 16 amino acids, mainly threonine
(24.1%), alanine (9.4%), valine (9.1%) and glutamine
(9.0%) in protein moiety, and two monosaccharides
(82.5% glucose and 9.8% galactose).
3.3. Cytotoxicity of EPS
MTT is a tetrazolium salt that is actively transported
into the cell and reduced to a formazan byproduct via
mitochondrial dehydrogenases [28]. This assay is a method to examine the level of cytotoxicity by surveying mitochondrial dehydrogenases activity in cells. The effect of
Table 2
Composition of amino acid and carbohydrate in exopolysaccharide
produced from submerged mycelial culture of Grifola frondosa HB0071
Composition (%)
Amino acid
Aspartic acid
Threonine
Serine
Glutamic acid
Glycine
Histidine
Arginine
Alanine
Proline
Tyrosine
Valine
Methionine
Lysine
Isoleucine
Leucine
Phenylalanine
6.36
24.16
3.92
9.02
3.85
2.69
1.3
9.44
6.43
3.1
9.16
6.08
4.48
3.23
3.56
3.13
Carbohydrate
Glucose
Galactose
Others
82.5
9.8
7.7
352
J.T. Bae et al. / FEMS Microbiology Letters 251 (2005) 347–354
Cell viability (%)
120
*
*
*
*
*
100
EPS did not exhibit any cytotoxicity within the dosage
range tested.
3.4. Effect of EPS on UVA-mediated MMP-1 expression
80
60
40
20
0
5
10
50
100
200
EPS concentrations (µg /ml)
Fig. 3. Effect of EPS on the viability of human dermal fibroblasts. The
cells were cultured in the presence of various concentrations of EPS (5–
100 lg ml1) for 24 h. The viability of the cell was measured by MTT
assay. The results were expressed as the average of triple determinations with S.D. * p < 0.05, significantly different from control.
EPS on the viability of human dermal fibroblasts was
investigated by the MTT test, in a dose-dependent manner. Cells were incubated with various EPS concentrations of 5, 10, 50, 100, and 200 lg ml1 for 24 h. In all
groups, the EPS had no significant effect either on the cell
viability or morphological change (Fig. 3). This result
indicated that human dermal fibroblasts treated with
Ultraviolet irradiation damages human skin and
causes premature skin aging (photoaging) through the
activation of matrix metalloproteinases (MMPs) which
are responsible for the degradation of collagen, gelatin
and other components of the extracellular matrix [31].
Several investigations have been undertaken to elucidate
the influence of UVA irradiation on the stimulation of
interstitial collagenase and gelatinase mRNA and their
corresponding proteins in cultured HDF [20,21]. Recently, Offord et al. [21] reported that vitamin C, vitamin
E, and carnosic acid showed photoprotective potential.
Lycopene and b-carotene did not protect on their own,
but did so in the presence of vitamin E. Their stability
in culture was improved and the rise in MMP-1 mRNA
expression was suppressed, suggesting a requirement for
antioxidant protection of the carotenoids against formation of oxidative derivatives that can influence the cellular and molecular responses.
In the present study, to estimate the effect of EPS
on MMP-1 expression in UVA-irradiated HDF
Fig. 4. Inhibitory effect of EPS on the expression of MMP-1 in the UVA-irradiated human dermal fibroblasts. The cells were cultured in the presence
of various concentrations of EPS (5–100 lg ml1) for 24 h. The results were expressed as the average of triple determinations with SD * p < 0.05,
significantly different from control. UVA dosage was 6.3 J/cm2; tRA refers to 3.5 lM of trans-retinoic acid. Total RNA extracted from HDF was
analyzed by RT-PCR and each lane in (A) corresponds to each bar in (B). The MMP-1 data were normalized to the b-actin transcript control.
J.T. Bae et al. / FEMS Microbiology Letters 251 (2005) 347–354
(6.3 J/cm2), the ELISA method was used to quantify
MMP-1 in the culture medium of HDF. The treatment
of UVA-irradiated HDF with EPS decreased the
expression of MMP-1 by 19%, 27.2%, 47.1%, and
61.1% at EPS concentrations of 5, 25, 50, and 100
(lg ml1), respectively (Fig. 4B). Surprisingly, the
inhibitory effect at a EPS concentration of 100 lg ml1
was significantly higher than that of trans-retinoic acid
(tRA), which is widely known as an inhibitor of UVAinduced MMPs. Fisher et al. [32] reported that tRA
applied to human skin inhibits subsequent activation
of activator protein (AP)1, which is essential for transcription of MMPs, and the induction of MMPs by
UVA irradiation.
It has been reported that UVA irradiation resulted in
an increase in the MMP-1 mRNA level, but did not
stimulate MMP-2 or TIMP-2 transcription [20]. Thus,
in the present study, the effect of EPS upon UVA irradiation was examined by measuring the steady-state
MMP-1 mRNA level in relation to b-actin mRNA levels. As shown in Fig. 4A, the expression of MMP-1
mRNA in UVA-irradiated HDF was significantly
reduced by EPS in a dose-dependent manner, while
b-actin mRNA remained constant.
Several investigators have reported that microbial
glucan stimulated macrophage release of wound growth
factors which modulated fibroblast collagen biosynthesis [33,34]. Recently, Kougias et al. [17] reported the
presence of at least two glucan binding sites on normal
human fibroblasts.
In conclusion, our results suggest that EPS obtained
from a mycelial culture broth of the new isolate of G.
frondosa is a potential candidate to reduce MMP activity in the skin after solar stimulation. In the future, combinations of other natural compounds may be envisaged
for more efficient photoprotection and a further study
for elucidating overall biological functions of the EPS
in vivo should be performed.
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