Growth Parameters of Agaricus brasiliensis Mycelium on Wheat Grains in Solid-state Fermentation
Agaricus brasiliensis mycelium is rich in antitumoral molecules such proteoglycans and ergosterol and this is the first study to report production of mycelium and ergosterol by A. brasiliensis in solid-state fermentation. The aim of this research was to study the biomass content and several growth parameters of A. brasiliensis mycelium on grounded wheat grain as the substrate in solid-state fermentation with ergosterol as the main growth indicator. The growth parameters were determined by the respirometric activity in the columns of a bioreactor with forced aeration and the outlet air composition was determined by gas chromatography. Ergosterol was extracted, measured by liquid chromatography and used for calculating biomass production. Then, the respirometric activity and ergosterol production data were used to estimate biomass production with the program Fersol (F). The growth parameters resulted in an achieved specific growth velocity of 0.016 h-1 at 18 h and biomass yield (YX/O) = 0.160 g of biomass g-1 of consumed O2 at 302 h of fermentation. The analysis resulted in ergosterol values of 6.71 mg g-1 of fungi biomass (Dry Weight: DW) from submerged fermentation and 1.95 mg g-1 of solid-state-fermented wheat grains. The correlation with biomass production allowed us to estimate a value of 0.29 g g-1 of fungi biomass per gram of the solid-state-fermented wheat grains. The importance of this study is allow calculation of the fungi biomass percentage in solid-state-fermented wheat grains and determination of the growth parameters of the main mycelium A. brasiliensis in this substrate, which can be milled to obtain a bio-flour to produce nutraceutical foods with beneficial effects due the presence of ergosterol which has recognized antitumor activity.
to cite this article:
H.S. Dalla-Santa, R. Rubel, F.M.D. Vitola, J.A. Rodriguez-Leon, O.R. Dalla-Santa, D. Brand, D.C. Alvarez, R.E.F. Macedo, J.C. Carvalho and C.R. Soccol, 2012. Growth Parameters of Agaricus brasiliensis Mycelium on Wheat Grains in Solid-state Fermentation. Biotechnology, 11: 144-153.
Received: February 06, 2012;
Accepted: May 24, 2012;
Published: July 03, 2012
A. brasiliensis is a medicinal and edible mushroom with strong antitumor
activity and immunostimulant properties (Stamets, 2000;
Wasser, 2011). Pharmaceutical industries are interested
in A. brasiliensis for the development of drugs and nutraceuticals because
of these pharmacological activities (Okwulehie et al.,
2007). This fungus produces several important bioactive molecules such as
proteoglycans, lectins and ergosterol which are found in the fruiting body,
mycelium and diffuse through the fermentation medium (Hamedi
et al., 2007; Fan et al., 2007). Polysaccharide
and ergosterol are the most investigated bioactive molecules produced by A.
brasiliensis because of their health benefits (Lima
et al., 2008). Recent research has shown that ergosterol from A.
brasiliensis exhibits antitumor activity such as inhibition of angiogenesis
and reduction of tumor growth (Takaku et al., 2001).
This mushroom which is also known as Himematsutake, Sun-mushroom, A. blazei,
A. subrufescens, or A. brasiliensis and refer to refer as A.
brasiliensis in the present work, based on recent studies that clarify the
problems of nomenclature of this specie (Wasser, 2011).
Ergosterol is a sterol who does not occur in plant or animal cells, however
is abundant in yeast and specially in the membrane of fungi (Weete,
1973). Ergosterol plays an important role in the human body as a precursor
of vitamin D which is formed in response to the exposure of ultraviolet radiation
(sunlight) to the sterols present in the skin. Vitamin D enhances calcium absorption
in animals for the formation of strong bones and teeth, prevention of rickets
and osteoporosis and maintenance of immune activity (Stamets,
2005). There is an increasing worldwide demand for ergosterol for the production
of pharmaceutical-grade, food-grade and beverage-grade vitamin D2,
than the levels of vitamin d are low in a lot of foods (Pal
et al., 2011). Because of this demand, Submerged Fermentation (SF)
was recently assessed for optimizing ergosterol and or polysaccharide production
by A. brasiliensis (Fan et al, 2003; Zou,
2005; Gao and Gu, 2007; Hamedi
et al., 2007). SF is reportedly the best process for obtaining fungal
mycelium and the desired biomolecules because of the shorter fermentation time,
lesser space requirement and better controlled fermentation parameters (Karmakar
and Ray, 2010; Fan et al., 2007; Gao
and Gu, 2007).
Solid-State Fermentation (SSF) requires low capital investment because of low
energy and waste output (Onyango et al., 2011;
Jamal et al., 2012) and is, therefore, advantageous
over SF. Furthermore, sometimes, the produced metabolites by SSF are more thermostable
than those produced by SF, such α-amylase (Regulapati
et al., 2007). Other advantages are the economy and reduction in
contamination problems due low-water indices used in the substrate which can
be used for nutrition and support for the microorganism growth (Soccol
and Vandenberghe, 2003; Rubel et al., 2008).
Furthermore, this method can be used to produce functional foods such as cereals
when the substrates are nutritionally valuable and free of toxic components
(Rubel et al., 2010). The use of A. brasiliensis
mycelium produced by SF are recently used for formulation of nutraceutical foods
(Ribeiro and Salvadori, 2003; Camelini
et al., 2005). On the other hand, recently studies verified the beneficial
biological effects of SSF wheat grain ingestion with the mushroom mycelium in
mice (Dalla-Santa et al., 2009, 2010).
However, a correlation factor to determine the amount of biomass in the wheat grains or ergosterol and mycelium production by A. brasiliensis during SSF has not been described before. Therefore, the aim of the present research was to study the biomass content and several growth parameters of A. brasiliensis mycelium grown during SSF on grounded wheat grains with ergosterol as the main growth indicator.
MATERIALS AND METHODS
Microorganism and inoculum preparation: The strain used in this work
was A. brasiliensis LPB-03 which is preserved in the Biotechnological
Processes Laboratory of the Biotechnology and Bioprocess Engineering Division
(Federal University of Parana) (Dalla-Santa et al.,
The mushroom strain was grown in Petri dishes on potato dextrose agar for 10
days at 30°C. Following cultivation, 5 pieces of mycelium (1 cm2)
were cut and inoculated into 50 mL of fermentation medium containing (in g L-1)
glucose (20), yeast extract (3.95), MgSO4.7H2O (0.3) and
K2HPO4.3H2O (0.5). The pH was adjusted to 6.0±0.2
(Fan et al., 2003) and the medium was sterilized
at 121°C for 15 min. After inoculation of the 5 pieces of mycelium, incubation
under shaker conditions was carried out at 30°C and 120 rpm for 7 days.
The pre-inoculum was obtained by straining the mycelium produced in this first
step through a 0.5 mm nylon mesh with a spatula to break pellets and produce
small mycelium pieces. A suspension of broken mycelium was obtained by washing
the biomass with 50 mL of distilled sterilized water during filtration. This
suspension was used for the second step of the inoculum preparation, in which
50 mL of the suspension was used to inoculate 500 mL (10%, v/v) of fermentation
medium containing (in g L-1) glucose (35), yeast extract (2.5), peptone
(5), KH2PO4 (0.88) and MgSO4.7H2O
(0.5). The pH was adjusted to 5.5 (Tang and Zhong, 2002)
and incubation was performed under conditions similar to those described for
the pre-inoculum preparation. The cultivated medium was filtered with 500 mL
of distilled sterilized water and the obtained mycelium suspension was used
as the inoculum for SF and SSF.
In order to estimate the biomass production of A. brasiliensis in SSF, SF was carried out using the same inoculum. The data obtained for ergosterol production in SF were used to determine the correlation factor between biomass and ergosterol production.
Biomass production in SF: Biomass production of A. brasiliensis in SF was carried out in Erlenmeyer flasks using 5% (v/v) of the inoculum prepared as described above. After 7 days of incubation, the biomass was filtered, washed with distilled water and vacuum-dried (10 mmHg, 45°C) for 24 h. About 0.5 g of the dried biomass was collected for analysis of moisture and ergosterol content. All analyses were performed in triplicate.
Ergosterol extraction and analysis from the SF biomass: Ergosterol extraction
was performed according to the method described by De Carvalho
et al. (2006) with some modifications. Initially, 0.5 g of the biomass
produced by SF was weighed, transferred to a glass flask and 2 mL of analytical-grade
ethanol and 1 mL of NaOH (2 M) were added.
||Column setup for respirometric studies, 1: Air pump, 2: Air
distribution system, 3: Humidifiers 4: Fermentation columns which were immersed
in a water bath with controlled temperature, 5: Drying column attached to
a column exit, 6: Sampling valve, 7: Gas chromatograph and 8: Computer with
data acquisition and control software
The material was shaken and incubated for 1 h at 70°C with periodic agitation
followed by the addition of 2 mL of HCl (1 M), 1 mL of KHCO3 (1 M)
and 2 mL of n-hexane with subsequent stirring. The mixture was agitated, transferred
to glass tubes and centrifuged at 3,000 g for 10 min at 20°C to facilitate
the separation of light and heavy phases. The light phase (n-hexane) was separated
and 2 more extractions with 2 and 1 mL of n-hexane were carried out. The organic
extracts were pooled and filtered through a 0.22 μm membrane (GS ester
cellulose), evaporated in vacuo at 200 mmHg and 35°C and resuspended
in 200 μL of n-hexane.
Analysis of the organic extract was performed in a ProStar high-performance
liquid chromatograph (HPLC; Varian, Inc.) with a C18 column and a
photodiode array (PDA) detector set at 282 nm. HPLC-grade solvents were used
(Sigma). The conditions of elution and chromatographic analysis were set according
to a previous report (De Carvalho et al., 2006).
The peak areas which were used to quantify ergosterol values, were obtained
from the chromatographic analysis of the ergosterol standards and from the calibration
curve. This resulted in a linear regression and the formula:
where, ERG is ergosterol (ppm) and A is the peak area, with R2 =
Biomass production in SSF columns: Airflow and initial moisture levels for the SSF columnar system were optimized in a previous experiment (data not shown) and these conditions were used for subsequent respirometric and kinetic studies.
Wheat grains were ground in a knife mill and sieved to obtain particles with
0.8-2 mm diameter for use as the substrate for SSF. The granulometry was determined
by a previous test which indicated that particles smaller or greater than 0.8-2
mm would be difficult for mycelium penetration and further growth. The substrate
was soaked in water for 12 h and sterilized at 121°C for 50 min. The initial
pH of the substrate was adjusted to 5.9±0.1 with 0.1 N HCl and the initial
moisture was adjusted to 39% with sterile water mixed with the inoculum suspension
at an inoculation rate of 5% (v/w). The inoculated substrate was packed into
12 columns (40-mm diameter and 200-mm height) with a bed height of 120 mm (volume,
150 mL). Each column was filled with 45 g of the inoculated substrate and incubated
for 14 days at 30°C. The fermentation system consisted of glass columns
immersed in a water bath at 30°C and an airflow of 0.22 N mL g-1
substrate min per column (milliliter of air in standard conditions, per gram
of wet substrate, per minute) (Raimbault, 1998) (Fig.
1). The columns were closed at both ends with cotton plugs, connected to
humidifiers and the aeration rate was adjusted twice a day using a rotameter
This procedure was performed simultaneously for kinetic studies of ergosterol and the corresponding biomass production (columns numbered from 1-10). The outlet gases were analyzed for online respirometry (columns 11 and 12). The data obtained in this analysis were used to determine the growth parameters of A. brasiliensis mycelium in SSF.
Ergosterol extraction and analysis from the SSF biomass: For the kinetic
studies on biomass production, the entire columnar content from 1-10 was withdrawn
at 0, 4, 18, 24, 36, 48, 72, 100, 196 and 302 h of fermentation and analyzed
for pH, moisture and water activity. Samples (1.0 g) were vacuum-dried (200
mmHg, 40°C) for 12 h, pulverized in an electric mill and analyzed for ergosterol
content using the same procedures described for ergosterol obtained by SF. All
analyses were performed in duplicate and data of biomass and fermented wheat
grains were presented in Dry Weight (DW).
Estimation of the correlation factor between ergosterol and biomass production: The data about ergosterol content from the biomass obtained by SF was used to correlate the biomass production from SSF from the ergosterol content of the samples.
Respirometric analysis: The respirometric analysis of A. brasiliensis
growth on wheat grains was carried out in 2 columns (numbered 11 and 12 with
initial moisture of 39% and airflow of 0.22 N mL/g/min). The outlet gases were
passed through desiccant silica gel columns and analyzed by Gas Chromatography
(GC) (Shimadzu GC-8A; Shimadzu Co., Japan). This system was linked to a program
for chromatograph control and integration (Chroma Biosystèmes, France)
followed by analysis with the Fersol mathematical model (Rodriguez-Leon
et al., 1988). The GC system had a Porapak 80/100 column at 60°C
with 2 m length, helium as the carrier gas, a thermal conductivity detector
and a sample injection volume of 300 μL for GC. The gases used for system
calibration were air, CO2 (0.0)/O2 (21.0)/N2
(79.0); mixture 1, CO2 (5.0)/O2 (5.0)/N2 (90.0)
and mixture 2, CO2 (10.0)/O2 (15.0)/N2 (75.0).
In these conditions, the retention time of each component was 0.95 min for CO2,
5.72 min for O2 and 8.02 min for N2. Exhaust gas analysis
was performed every hour with 2 replicates and Oxygen Update Rate (OUR) and
CO2 production results were collected to establish a relationship
between the online respirometric parameters in the columnar bioreactor with
forced air and the mycelium growth. The results of the respirometric analysis
were averaged for the two columns used in the assay.
Kinetic parameters: The kinetic parameters were calculated as described
by Pandey et al. (2001) and biomass production
by ergosterol analysis was used to obtain its yield based on O2 consumption
(YX/O) (g of biomass g-1 of consumed O2).
The same biomass data was correlated to dependent variables using Fersol software
(Rodriguez-Leon et al., 1988) and all data received
the suffix F. The CO2 production results, OUR determination and ergosterol
content from the kinetic study during the fermentation period were used to correlate
the biomass produced in SSF at a certain time (Xn)F which
was calculated by assuming values for its yield based on O2 consumption
(YX/O)F (g of biomass g-1 of consumed O2)
and the biomass maintenance coefficient (mX)F using Fersol.
Analytical procedures: The pH of the solid substrate was determined
with a digital pH meter by mixing 5 g of the sample with 50 mL of distilled
water. Substrate moisture was determined following standard procedures (AOAC,
2002). Water activity (aw) was measured using AquaLab CX-2.
Correlation between ergosterol and the biomass obtained by SF: The ergosterol
content of mushrooms can be correlated with the fungal biomass (Klamer
and Baath, 2004). In the present study, the following correlation was obtained:
This value was used to calculate the biomass production in SSF using the ergosterol content produced by this fermentation method.
Correlation between ergosterol and the biomass obtained by SSF: The
biomass in SSF was estimated using an indirect method because it is difficult
to separate the biomass from the substrate. Indirect methods are based on measurement
of cell components or respirometric balance while accounting for biomass composition
and stoichiometry of the process (Soccol and Vandenberghe,
Ergosterol analysis of wheat grains without fermentation resulted in a very low value, i.e., 1.74 μg g-1 which was subtracted from the ergosterol content found in the fermented samples. The results obtained from samples from the 14th day of fermentation were expressed as:
The calculation of the fungi biomass per gram of fermented dried wheat grains was obtained from the correlation between Eq. 1 and 2 and resulted in a maximum value of 0.29 biomass g-1 of fermented wheat grains (DW) at the 14th day of fermentation.
||Respirometric data of Agaricus brasiliensis mycelium
on wheat grains in the column, Column has 45 g of wheat grains and 39% initial
moisture with forced aeration, RQ: Respiratory quotient and μ: specific
||Growth parameters of Agaricus brasiliensis mycelium
on wheat grains in the column, Column has 45 g of wheat grains and 39% initial
moisture with forced aeration
Kinetic studies of A. brasiliensis growth and the SSF biomass from respirometric analysis: The respirometric data and biomass production of A. brasiliensis mycelium in SSF are shown in Fig. 2 and 3, respectively. The results were obtained from a whole column containing 45 g of wheat grains with 39% initial moisture. The biomass yield coefficient was calculated as the ratio of biomass production per amount of O2 consumed and is presented in Fig. 3.
The Aw values ranged from 0.96-0.97 even with alterations in moisture content which increased gradually during the fermentation period (Fig. 3) and reached a value of 66% at the 14th fermentation day.
The initial pH value was ~6.0 and showed slight variation during the fermentation period (302 h, Fig. 3). The pH decreased slowly to 5.16 at 72 h of fermentation followed by an increase to values proximate to the initial pH (6.1) at the end of the fermentation period.
The data for biomass, produced CO2 and consumed O2 obtained during SSF were applied to the Fersol program and are presented in Table 1. The predicted biomass at a certain time (Xn)F involved the assumption of values for its yield based on the O2 consumption (YX/O)F and the coefficient of biomass maintenance (mx)F. The software allowed determination of the equation coefficients by successive approximation.
||Online respirometric parameters and biomass production by
A. brasiliensis mycelium by solid-state culture in a column coupled
with forced aeration
|*The values of time, biomass, produced CO2 and
consumed O2 were used to estimate biomassF
production in the column (Wheat grains: 45 g, Initial moisture: 39%, Air
flow: 0.22 N mL g-1 substrate/min per column, Diameter: 40 mm
and Bed height: 120 mm) with forced aeration through the Fersol program
Based on the values of fermentation time and biomass obtained from ergosterol correlation and consumed O2, the following data were obtained using Fersol: predicted biomass yield (YX/O)F = 1.949 g of biomass g-1 of consumed O2; coefficient of biomass maintenance (mx)F = 0.0027 g of consumed O2/(g of produced biomass. h); specific growth rate (μF) = 0.086/h and correlation coefficient (R2F) = 0.955.
The development of the fungal mycelia was described according to the RQ values,
O2 consumption and CO2 and biomass production (Fig.
2, 3). The ergosterol detection was very low at the 4th
h with 0.006 mg g-1 of wheat grains and the production of CO2
started at the sixth hour of fermentation. From 6 h to 12 h, the RQ value
increased and reached a maximum of 3.94 at 11 h of fermentation (Fig.
2) which resulted from higher CO2 production and little O2
uptake in the system. After 12 h, the RQ value decreased to ~1, indicating aerobic
respiratory activity according to the theoretical value expected for this parameter
(Fig. 2). CO2 production was associated with O2
consumption. During the growth phase, the mycelium spread over the substrate
surface, showing a white cover resembling cotton. Ergosterol production began
to increase at the 18th h of fermentation, with the values varying from 0.03-0.94
mg g-1 of fermented wheat grains (dry weight) at the 72nd h (Fig.
3). The O2 uptake rate and CO2 production were very
high between 57 and 76 h, indicating intense metabolism of the fungus (Fig.
2). The maximum CO2 production occurred at 69 h of fermentation,
reaching the maximum rate of 7 mmol h-1. The development of A.
brasiliensis during this phase resulted in a maximum specific growth velocity
of 0.016 h-1 at 18 h. CO2 production decreased after 78
h, whereas O2 consumption increased gradually until 98 h. During
the fermentation periods from 105-126 h and from 156-213 h, an increase in CO2
production and O2 consumption was observed (Fig. 2).
Ergosterol analysis resulted in a continuous increase with the maximum value
achieved at the 14th fermentation day, at 302 h, with 1.95 mg g-1
of fermented wheat grains (dry weight) (Fig. 3).
Biotechnological applications of SSF are widespread and are one of the oldest
processes known for production of foods such as tempeh, koji processing, cheese
production and many secondary metabolites such enzymes and more recently fermentable
sugars aiming the production of bioethanol (Anusha et
al., 2012; Hong et al., 2011; Murad
and Azzaz, 2010; Sherief et al., 2010; Pandey
et al., 2001). However, several technical problems related to SSF
have not been solved during the scale-up process, such as difficulty controlling
temperature, pH, moisture, substrate concentration, or pO2 during
cultivation under limited water availability (Holker et
al., 2004; Singhania et al., 2009). The
observed increase in the moisture levels from initial values of 39% to 66% at
the 14th day of SSF in the kinetic study (Fig. 3) was possibly
due to the humidity of the air flushed through the columnar system. In contrast,
SSF performed in trays by Dalla-Santa et al. (2010)
resulted in maintenance or reduction of the moisture levels when fermentation
was conducted in humidified or normal air conditions, respectively.
It is also difficult to maintain pH values during SSF, as observed by the varying
pH values during fermentation (Fig. 3) which seems characteristic
of this microorganism. Similar changes were detected in another study of SF
with A. brasiliensis, with pH values varying from 5.6-4.4 on the second
day and reaching 7.84 on the sixth day (Gern, 2005) which
the author suggested was correlated with acid production and concomitant glucose
consumption. The pH variance was more intense in the above-mentioned study than
in our study which is presumably due to the differences in the mycelium grown,
nutrient absorption related to the fermentation type (SF versus SFF) and to
the chemical difference of the substrate (liquid complex medium versus wheat
grains). The wheat grains used in the present study are rich in protein and
it is well known that protein-rich substrates can act as buffers and inhibit
excessive pH changes (Chisti, 1999).
It is important to follow the kinetics of growth in relation to the metabolic
activity in order to measure the biomass in SSF. In SSF, the microorganisms
are closely bound to the solid-state matrix, resulting in difficult biomass
separation and measurement. Therefore, the biomass is usually measured indirectly
with minor or major error by determining cell components such as glucosamine,
ergosterol, proteins, or nucleic acids and the development of adequate monitoring
and control processes are required (Pandey et al.,
2001; Chen et al., 2005). An advantage of
determining ergosterol content is that it can be recovered and separated by
HPLC and easily quantified by spectrophotometry, providing a sensitive biomass
index at low levels of growth (Raimbault, 1998). On
the other hand, estimation of O2 intake and CO2 evolution
rates are recently considered the most accurate determination of microorganism
growth (Pandey et al., 2007). Therefore, it was
used the ergosterol determination with a final value of 0.29 g of biomass per
gram of fermented wheat grains (dry weight) (Fig. 3) and the
respirometric analysis (Fig. 2, Table 1)
and both methods efficiently measured and estimated fungi biomass from A.
brasiliensis in the grounded wheat grains in SSF.
Owing to the lack of published research on A. brasiliensis in SSF, the
results obtained in the present work were compared with those obtained with
Monascus, since both these fungi have similar habitats; however, the
fact that each fungus has a specific metabolism was also considered. A pre-fermentation
procedure that includes increasing the inoculum concentration or inoculation
ratio has been suggested to reduce the long lag phase of 60 h observed in Monascus
(De Carvalho et al., 2006). In the present study,
use of the broken mycelium suspension resulted in an adaptive phase of less
than 6 h of fermentation, with CO2 production at 6 h and an increased
RQ value. This short lag phase is important for 2 reasons: it reduces contamination
which is one of the most recurrent problems in spawn production and the fermentation
time is reduced which is very important at the industrial scale. In normal spawn
production, pieces of solid medium covered with developed mycelium are inoculated
on grains, followed by incubation for 20-30 days, whereas the broken suspension
method usually enables the efficient development of mushrooms in SSF within
14-18 days. In this study, this method resulted in extremely rapid development
of A. brasiliensis. After 4 days, the entire substrate was recovered
by the fungal mycelium. Active fermentation of the fungus was detected from
the high and rapid increase in the RQ value (3.94) for A. brasiliensis
at 11 h, whereas the maximum RQ value for Monascus was <2 in another
study (De Carvalho et al., 2006).
Because there is a dearth of studies on Agaricus for comparison of the
growth rate based on O2 consumption and CO2 production,
the results were compared with others published for other Ascomycetes or Basidiomycetes
in other fermentations systems. Monascus sp., developed in the same type
of columnar system (De Carvalho et al., 2006)
reached 0.086/h of maximum specific growthF at 140 h, whereas A.
brasiliensis showed intense growth and specific growth velocity of 0.016/h
at 18 h, as also observed by the total and rapid coverage of mycelium on the
wheat particles. Gibberella fujikuroi and gibberellic acid (GA3)
production using coffee husk and cassava bagasse in a column bioreactor resulted
in a maximum specific growth rateF of 0.052 h-1 between
24 and 48 h of fermentation (Machado et al., 2004).
The yield and biological efficiency for all microorganisms depend on the substrate
formulations and fermentation parameters. Many authors specify the importance
of the absolute biomass amount for the calculation of growth rates and yields
in fermentation (Raimbault, 1998). Ergosterol was used
to calculate the biomass and the possible yield which resulted in a biomass
yield (YX/O) of 0.160 g of biomass g-1 of consumed O2
at 302 h of fermentation. Soccol and Vandenberghe (2003)
obtained good correlations between the values obtained experimentally for the
OUR and CO2 involved with the analyzed biomass through protein content
analysis using the Fersol program. In the present work it was chosen to use
ergosterol determination rather than protein analyses as the high protein content
in the wheat grains could induce error because of the possible incorporation
of wheat protein into the microorganism. On the other hand, yield can be calculated
based on product yield using ergosterol production per amount of consumed O2
(Fig. 3) and the maximum Y(P/O2) obtained value
was 1.07 mg of ergosterol g-1 of dried fermented wheat grains mmol-1
O2 at the 14th fermentation day.
The mushroom fruiting body contains remarkably high amounts of phytosterol,
the most important of which is ergosterol, whose distribution varies in species
and in different parts of the mushroom tissue (Jasinghe
and Perera, 2005; Krzyczkowski et al., 2009).
For example, variances in the overall ergosterol content of the fruiting body
of different types of mushrooms have been reported as follows (mg of ergosterol
g-1 of dry weight [DW]): 7.80 (0.35) in button mushrooms or Agaricus,
6.05 (0.07) in shiitake, 4.40 (0.08) in oyster mushrooms and 0.68 (0.14) in
enoki mushrooms (Jasinghe and Perera, 2005). Mattila
et al. (2002) observed a value of 6.79 mg g-1 DW of ergosterol
in shiitake mushrooms and Teichmann et al. (2007)
reported that ergosterol contents of dark cultivated mushrooms (A. bisporus/white,
A. bisporus/brown, A. bisporus/Portabella, Lentinula edodes
and Pleurotus ostreatus) varied from 3.7-5.1 mg g-1 DW to
40-108 mg/100 (Fresh Weight). Krzyczkowski et al.
(2009) detected ergosterol peroxide values of 29.32, 17.27 and 12.60 mg/100
g for Boletus edulis (king bolete), Suillus bovinus (Jersey cow
mushroom) and B. badius (bay bolete) fruiting bodies, respectively.
These authors additionally described the ergosterol values for mycelia for several
mushrooms: H. erinaceum, Laetiporus sulphureus (chicken mushroom)
and Morchella esculenta (common morel) with 15.98, 10.07 and 13.37 mg/100
g, respectively. Few articles have described the biomass and ergosterol content
of the mycelium from the mushroom A. brasiliensis. Li
and Shen (2003) used a mixture of malt, yeast extract, glucose, peptone,
oatmeal and calcium carbonate as the substrate for mycelium production of A.
brasiliensis in SSF (%: malt extract, 2; yeast extract, 2; peptone, 0.5;
glucose, 2; water, 45.7, oatmeal, 45.7; calcium carbonate, 2) and obtained ergosterol
values ranging from 0.371-0.416 mg g-1 of dry substrate. In the present
study, an increase in the ergosterol content of the fermented wheat grains was
observed during the fermentation period which reached the maximum value of 1.95
mg of ergosterol per gram of dry fermented material after 302 h (day 14) of
fermentation. This result was presumably due to the efficient aeration system
which provided high O2 diffusion in the substrate and adequate nutrient
content in the wheat grains.
In our study, submerged fermentation yielded a value of 6.71 mg of ergosterol
per gram of dried biomass with 20 g of glucose in the Erlenmeyer flask. This
value is similar to a study by Zou (2006), reporting
a biomass-ergosterol production of 8.8 mg g-1 of DW in an Erlenmeyer
flask and 25 mg g-1 DW at 144 h in fed batch fermentation by SF of
A. brasiliensis using 20 g of sugar in the medium. Gao
and Gu (2007) optimized the ergosterol production of A. brasiliensis
in SF and achieved 0.076 g L-1, an increase of 43% compared to non-optimized
The importance of the present results is related to the recognized beneficial
effects of ergosterol on health improvement and tumor prevention (Takaku
et al., 2001), the capacity of phytosterols to lower plasma cholesterol
and LDL cholesterol (Shin et al., 2003) and the
proven beneficial effects of ingestion of wheat grains fermented with A.
brasiliensis mycelium in mice by our research group (Dalla-Santa
et al., 2009; Dalla-Santa et al., 2010)
which suggest that this fermented material is an excellent bioactive ingredient.
Moreover, SSF is a cheap and simple method to obtain this functional product.
The results presented herein allow calculation of the fungi biomass percentage
in solid-state-fermented wheat grains and determination of the growth parameters
of the main mycelium A. brasiliensis in this substrate which can be
milled to obtain bio-flour, nutraceutical foods.
In summary, regarding the results of biomass from ergosterol production in SSF, both ergosterol content and respirometric activity determination methods efficiently estimated the biomass production of A. brasiliensis in SSF. The growth parameters achieved a specific growth velocity of 0.016/h at 18 h and a biomass yield (YX/O) of 0.160 g of biomass g-1 of consumed O2 at 302 h of fermentation. Ergosterol analysis revealed that 6.71 mg of ergosterol g-1 of dried fungi biomass was obtained from SF and 1.95 mg of ergosterol/g of solid-state-fermented wheat grains (dry weight) from SSF. The correlation with biomass production allowed an estimate of 0.29 g of fungi biomass per gram of the solid-state-fermented wheat grains (dry weight) which represent an excellent bioactive material for nutraceutical products due the presence of the ergosterol with recognized beneficial effects.
H.S. Dalla-Santa and C.R. Soccol thank the Midwest State University (UNICENTRO) and the National Council of Technological and Scientific Development (Project no. 475798/2003-1), respectively, for financial support and for a scholarship under the Scientific Productivity Scheme.
Anusha, N.C., M.S. Umikalsom, T.C. Ling and A.B. Ariff, 2012.
Relationship between fungal growth morphologies and ability to secrete lipase in solid state fermentation. Asian J. Biotechnol., 4: 15-29.CrossRef |
International Official Methods of Analysis of AOAC International. Association of Analytical Communities, Gaithersburg, USA
Camelini, C.M., M. Maraschin, M.M. de Mendonca, C. Zucco, A.G. Ferreira and L.A. Tavares, 2005.
Structural characterization of β
-glucans of Agaricus brasiliensis
in different stages of fruiting body maturity and their use in nutraceutical products. Biotechnol. Lett., 27: 1295-1299.CrossRef | Direct Link |
De Carvalho, J.C., A. Pandey, B.O. Oishi, D. Brand, J.A. Rodriguez-Leon and C.R. Soccol, 2006.
Relation between growth, respirometric analysis and biopigments production from Monascus
by solid-state fermentation. Biochem. Eng. J., 29: 262-269.CrossRef | Direct Link |
Dalla-Santa, H.S., R. Rubel, F.M.D. Vitola, F. Leifa and A.L. Tararthuch et al
Kidney function indices in mice after long intake of Agaricus brasiliensis
mycelia (=Agaricus blazei
, Agaricus subrufescens
) produced by solid state cultivation. Online J. Biol. Sci., 9: 21-28.Direct Link |
Dalla-Santa, H.S., R. Rubel, L.C. Fernandes, S.J.R. Bonatto and S.R. Bello et al
., 2010. Agaricus brasiliensis
-enriched functional product promotes in mice increase in HDL levels and immunomodulate to Th1 CD4+T subsets. Curr. Trends Biotechnol. Pharm., 4: 957-970.Direct Link |
Fan, L., A.T. Soccol, A. Pandey, S. Germano, R. Rau, A. Pedroso and C.R. Soccol, 2003.
Production of polysaccharide by culinary-medicinal mushroom Agaricus brasiliensis
S. Wasser et al
., LPB 03 (Agaromycetidae) in submerged fermentation and its antitumor effect. Int. J. Med. Mushr., 5: 17-23.Direct Link |
Gao, H. and W.Y. Gu, 2007.
Optimization of polysaccharide and ergosterol production from Agaricus brasiliensis
by fermentation process. Biochem. Eng. J., 33: 202-210.CrossRef | Direct Link |
Gern, J.C., 2005.
Production and characterization of extra-cellular polysaccharide from submerged fermentation by Agaricus brasiliensis
. Master Thesis, Federal University of Parana, Parana, Brazil.
Hamedi, A., H. Vahid and F. Ghanati, 2007.
Optimization of the medium composition for production of mycelial biomass and exo-polysaccharide by Agaricus blazei
Murill DPPh 131 using response-surface methodology. Biotechnology, 6: 456-464.CrossRef | Direct Link |
Holker, U., M. Hofer and J. Lenz, 2004.
Biotechnological advantages of laboratory-scale solid state fermentation with fungi. Applied Microbiol. Biotechnol., 64: 175-186.Direct Link |
Hong, L.S., D. Ibrahim and I.C. Omar, 2011.
Lignocellulolytic materials-as a raw material for the production of fermentable sugars via solid state fermentation. Asian J. Sci. Res., 4: 53-61.CrossRef | Direct Link |
Chen, F., Y. Jiang and F. Ouyang, 2005.
Development of bioprocess engineering in China. Biotechnology, 4: 1-6.CrossRef | Direct Link |
Chisti, Y., 1999.
Solid Substrate Fermentations, Enzyme Production, Food Enrichment. In: Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation, Flickinger, M.C. and S.W. Drew (Eds.). Vol. 5, John Wiley and Sons, New York, USA., ISBN-13: 9780471138228, pp: 2446-2462
Jamal, P., O.K. Saheed and Z. Alam, 2012.
Bio-valorization potential of banana peels (Musa sapientum
): An overview. Asian J. Biotechnol., 4 : 1-14.CrossRef | Direct Link |
Jasinghe, V.J. and C.O. Perera, 2005.
Distribution of ergosterol in different tissues of mushrooms and its effect on the conversion of ergosterol to vitamin D2
by UV irradiation. Food Chem., 92: 541-546.CrossRef | Direct Link |
Karmakar, M. and R.R. Ray, 2010.
Extra cellular endoglucanase production by rhizopus oryzae in solid and liquid state fermentation of agro wastes. Asian J. Biotechnol., 2: 27-36.CrossRef |
Klamer, M. and E. Baath, 2004.
Estimation of conversion factors for fungal biomass determination in compost using ergosterol and PLFA 18:2ω6,9. Soil Biol. Biochem., 36: 57-65.CrossRef | Direct Link |
Krzyczkowski, W., E. Malinowska, P. Suchocki, J. Kleps, M. Olejnik and F. Herold, 2009.
Isolation and quantitative determination of ergosterol peroxide in various edible mushroom species. Food Chem., 113: 351-355.CrossRef | Direct Link |
Fan, L., A.T. Soccol, A. Pandey and C.R. Soccol, 2007.
Effect of nutritional and environmental conditions on the production of EPS of Agaricus blazei
by submerged fermentation and its antitumor activity. Food Sci. Technol., 40: 30-35.CrossRef |
Li, P.J. and C.G. Shen, 2003.
Method for propagating fungi using solid state fermentation. US Patent 6558943. http://www.freepatentsonline.com/6558943.html.
Lima, L.F.O., S. Habu, J.C Gern, B.M. Nascimento and J.L. Parada et al
Production and characterization of the exopolysaccharides produced by Agaricus brasiliensis
in submerged fermentation. Applied Biochem. Biotechnol., 151: 283-294.CrossRef | Direct Link |
Machado, C.M.M., B.O. Oishi, A. Pandey and C.R. Soccol, 2004.
Kinetics of Gibberella fujikuroi
growth and gibberellic acid production by solid-state fermentation in a packed-bed column bioreactor. Biotechnol. Prog., 20: 1449-1453.CrossRef | Direct Link |
Mattila, P.H., A.M. Lampi, R. Ronkainen, J. Toivo and V. Piironen, 2002.
Sterol and vitamin D2
contents in some wild and cultivated mushrooms. Food Chem., 76: 293-298.CrossRef | Direct Link |
Murad, H.A. and H.H. Azzaz, 2010.
Cellulase and dairy animal feeding. Biotechnology, 9: 238-256.CrossRef | Direct Link |
Okwulehie, I.C., N.C. Princewill and O.C. Johnpaul, 2007.
Pharmaceutical and nutritional prospects of two wild macro-fungi found in Nigeria. Biotechnology, 6: 567-572.CrossRef | Direct Link |
Onyango, B.O., V.A. Palapala, P.F. Arama, S.O. Wagai and B.M. Gichimu, 2011.
Suitability of selected supplemented substrates for cultivation of Kenyan native wood ear mushrooms (Auricularia auricula
). Am. J. Food Technol., 6: 395-403.CrossRef | Direct Link |
Pal, U.K., P.K. Mandal, V.K. Rao and C.D. Das, 2011.
Quality and utility of goat milk with special reference to India: An overview. Asian J. Anim. Sci., 5: 56-63.CrossRef | Direct Link |
Pandey, A., C.R. Soccol and C. Larroche, 2007.
Current Developments in Solid-State Fermentation. Springer Science/Asiatech Publishers, New York, USA. and New Delhi, India
Pandey, A., C.R. Soccol, J.A. Rodriguez-Leon and P. Nigam, 2001.
Solid-State Fermentation in Biotechnology. Asiatech Publishers Inc., New Delhi, ISBN: 81-87680-06-7
Raimbault, M., 1998.
General and microbiological aspects of solid substrate fermentation. Electron. J. Biotechnol., 1: 174-188.Direct Link |
Regulapati, R., N.P. Malav and N.S. Gummadi, 2007.
Production of Thermostable α-amylases by solid state fermentation: A review. Am. J. Food Technol., 2: 1-11.CrossRef | Direct Link |
Ribeiro, L.R. and D.M.F. Salvadori, 2003.
Dietary components may prevent mutation-related diseases in humans. Mutat. Res., 544: 195-201.Direct Link |
Rodriguez-Leon, J.A., L. Sastre, J. Echevarria, G. Delgado and W.A. Bechstedt, 1988.
A mathematical approach for the estimation of biomass production rate in solid state fermentation. Acta Biotechnol., 8: 307-310.CrossRef | Direct Link |
Rubel, R., H.S. Dalla-Santa, L.C. Fernandes, J.H.C. Lima-Filho and B.C. Figueiredo et al
High immunomodulatory and preventive effects against sarcoma 180 in mice fed with Ling Zhi or Reishi mushroom Ganoderma lucidum
(W. Curt.: Fr.) P. Karst. (Aphyllophoromycetideae) mycelium. Int. J. Med. Mushrooms, 10: 37-48.CrossRef |
Rubel, R., H.S. Dalla-Santa, L.C. Fernandes, S.J.R. Bonatto and S. Bello et al
Hypolipidemic and antioxidant properties of Ganoderma lucidum
(Leyss:Fr) Karst used as a dietary supplement. World J. Microbiol. Biotechnol., 27: 1083-1089.CrossRef | Direct Link |
Sherief, A.A., N.E.A. El-Naggar and S.S. Hamza, 2010.
Bioprocessing of lignocellulosic biomass for production of bioethanol using thermotolerant Aspergillus fumigates
under solid state fermentation conditions. Biotechnology, 9: 513-522.CrossRef | Direct Link |
Shin, M.J., S.J. Rim, Y. Jang, D. Choi and S.M. Kang et al
The cholesterol-lowering effect of plant sterol-containing beverage in hypercholesterolemic subjects with low cholesterol intake. Nutr. Res., 23: 489-496.CrossRef | Direct Link |
Singhania, R.R., A.K. Patel, C.R. Soccol and A. Pandey, 2009.
Recent advances in solid-state fermentation. Biochem. Eng. J., 44: 13-18.CrossRef | Direct Link |
Soccol, C.R. and L.P.S. Vandenberghe, 2003.
Overview of applied solid-state fermentation in Brazil. Biochem. Eng. J., 13: 205-218.CrossRef | Direct Link |
Stamets, P., 2000.
Techniques for the cultivation of the medicinal mushroom royal sun Agaricus blazei
Murrill (agaricomycetidae). Int. J. Med. Mushr., 2: 151-160.Direct Link |
Stamets, P., 2005.
Notes on nutritional properties of culinary-medicinal mushrooms. Int. J. Med. Mushrooms, 7: 103-110.CrossRef | Direct Link |
Takaku, T., Y. Kimura and H. Okuda, 2001.
Isolation of an antitumor compound from Agaricus blazei
Murril and its mechanism of action. J. Nutr., 131: 1409-1413.Direct Link |
Tang, Y.J. and J.J. Zhong, 2002.
Fed-batch fermentation of Ganoderma lucidum
for hyperproduction of polysaccharide and ganoderic acid. Enzym. Microb. Technol., 31: 20-28.CrossRef | Direct Link |
Teichmann, A., P.C. Dutta, A. Staffas and J. Margaretha, 2007.
Sterol and vitamin D2
concentrations in cultivated and wild grown mushrooms: Effects of UV irradiation. LWT-Food Sci. Technol., 40: 815-822.CrossRef | Direct Link |
Wasser, S.P., 2011.
Current findings, future trends and unsolved problems in studies of medicinal mushrooms. Applied Microbiol. Biotechnol., 89: 1323-1332.CrossRef | Direct Link |
Weete, J.D., 1973.
Sterols of the fungi: Distribution and biosynthesis. Phytochemistry, 12: 1843-1864.CrossRef | Direct Link |
Zou, X., 2005.
Effects of Zn supplementation on the growth, amino acid composition, polysaccharide yields and anti-tumour activity of Agaricus brasiliensis
. World J. Microbiol. Biotechnol., 21: 261-264.CrossRef | Direct Link |
Zou, X., 2006.
Fed-batch fermentation for hyperproduction of polysaccharide and ergosterol by medicinal mushroom Agaricus brasiliensis
. Process. Biochem., 41: 970-974.CrossRef | Direct Link |