Báo cáo khoa học " Antioxidant and antimicrobial activities of shiitake (Lentinula edodes) extracts obtained by organic solvents and supercritical fluids " pptx

Antioxidant and antimicrobial activities of shiitake (Lentinula
edodes) extracts obtained by organic solvents and supercritical fluids
Cı
´
ntia Sorane Good Kitzberger
a
, Artur Sma
ˆ
nia Jr.
b
, Rozangela Curi Pedrosa
c
,
Sandra Regina Salvador Ferreira
a,
*
a
Chemical and Food Engineering Department – Federal Universtity of Santa Catarina – Laboratory of Thermodynamics and Supercritical Fluid
Extraction (LATESC/EQA –UFSC), CP 476, CEP 88040-900, Floriano
´
polis, SC, Brazil
b
Microbiology and Parasitology Department – UFSC, Brazil
c
Biochemistry Department – UFSC, Brazil
Received 10 March 2006; received in revised form 20 May 2006; accepted 16 June 2006
Available online 17 August 2006
Abstract
Shiitake mushroom contains several therapeutic actions such as antioxidant and antimicrobial properties, carried by the diversity of
its components. In the present work, extracts from shiitake mushroom were obtained using different extraction techniques: high-pres-
sure operations and low-pressure methods. The high-pressure technique was applied to obtain shiitake extracts using pure CO

2
and
CO
2
with co-solvent in pressures up to 30 MPa. Organic solvents such as n-hexane, ethyl acetate and dichloromethane were further-
more used to produce the shiitake extracts in low-pressure extraction process. The different extraction procedures were evaluated for
antioxidant activity by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) essays and the results compared with data from Folin–Denis
method, used to measure the total phenolic content. Antimicrobial activities of the extracts were also subjected to preliminary screen-
ing against four strains of bacteria and one fungal strain using agar dilution method. The results indicate that the fractions obtained
with CO
2
using ethanol as co-solvent, at 40 °C, 20 MPa and 15% EtOH, and for dichloromethane in low-pressure technique had sim-
ilar antioxidant activities. Furthermore, only the supercritical fluid extracts had antimicrobial activity against Micrococcus luteus and
Bacillus cereus. The shiitake extraction yields were up to 3.81% w/w and up to 1.01% w/w for supercritical fluid extraction with eth-
anol as co-solvent and with pure CO
2
, respectively, while the low-pressure extraction indicates yields up to 1.25% w/w for n-hexane as
solvent.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Supercritical fluid extraction; Shiitake (Lentinula edodes); Antioxidant; Antimicrobial; Organic solvent extraction
1. Introduction
Shiitake ( Lentinula edodes) is the second largest culti-
vated and most popular edible mushroom in world, reach-
ing a production of 7.5 million ton in 2000 (Royse, 2005).
Additionally, L. edodes present several functional proper-
ties, such as antitumor and hypocholesterolemic actions,
and antimicrobial and antioxidant potentials that have
been intensively investiga ted (Hatvani, 2001; Manzi & Piz-
zoferatto, 2000; Mau, Chao, & Wu, 2001; Shimada , Mor-
ita, & Sugiyama, 2003; Yang, Lin, & Mau, 2002).

Antioxidant compounds reduce the action of reactive
oxygen species (ROS) in tissue damage. The oxidation
proceeds in lipids with polyunsaturated fatty acids, gener-
ating ROS such as hydroxyl radicals (Halliwell & Gutter-
idge, 1989). Natural products with antioxidant activity
are used to aid the endogenous protective system, increas-
ing interest in the antioxidative role of nutraceutic prod-
ucts (Kanter, 1998 ). Furthermore, Cheung, Cheung, and
Ooi (2003) found at organic solvent extracts from mush-
room, a direct correlation between antioxidant activity
0260-8774/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2006.06.013
*
Corresponding author. Tel.: +55 48 3331 9448; fax: +55 48 3331 9687.
E-mail address: (S.R.S. Ferreira).
www.elsevier.com/locate/jfoodeng
Journal of Food Engineering 80 (2007) 631–638
and total phenolic content, although the antioxidant
action is raised by other substances such as tocopherols
and b-carotene.
Antimicrobial activity of shiitake extracts have also been
investigated because mushrooms are considered a source of
natural antibiotics (Sma
ˆ
nia et al., 1995). Several mushroom
by-products have been used against human pathogens, for
the activation of immunologic system and to improve
human health due to antioxidant and antitumor actions
(Wasser & Weis, 1999). According to Hirasawa, Shouji,
Neta, Fukushima, and Takada (1999), chloroform shiitake

extract have bactericide activity against Streptococcus
mutans (cause tooth decay) and Prevotella intermedia
(agent of periodontal disease).
Natural products with biological activity are normally
present in plants, mushroom and several other sources,
therefore, the use of extraction techniques is important
to select substances or group of components of interest.
Then, the evaluation of the extraction process related
to its efficiency to reach target components from a solid
matrix is of considerable relevance. According to Spigno
and de Faveri (2006), solvent extractions are normally
used for antioxidant recovery from food material, but
supercritical fluid extraction (SFE) represents a viable
alternative for solute extraction from natural matrixes
because it offers solvent free products and prevails
thermo degradation (Va
´
gi, Sima
´
ndi, Suhajda, & He
´
thelyi,
2005).
Therefore, the aim of this work was to investigate the
antioxidant and antimicrobial activities of extracts
obtained from shii take mushroom using classical organic
solvent extraction (COSE) with different solvents and using
SFE. In the SFE, pure CO
2
was applied at different condi-

tions of temperature and pressure. Also, CO
2
was used in a
mixture with ethanol, dichloromethane and ethyl acetate as
co-solvents at 40 °C and 20 MPa and concentration of co-
solvent up to 20% w/w.
2. Material and methods
2.1. Sample preparation
The raw material used in this work consisted of dried
shiitake mushroom (L. edodes) purchased from Ind. &
Com. Guinishi (Suz ano, SP, Brazil). The shiitake, with
moisture content of 5.2% w/w, was stored at room temper-
ature, and samples of the mushroom were grounded in a
domestic blender immediately before the extractions. The
particle size of the grounded material was classified in a
sieve separator and the fraction of mesh À80 +100, was
selected to settle the bed of L. edodes inside the extractor.
The fixed bed was formed with 40.0 ± 0.5 · 10
À3
kg of trit-
urated shiitake, placed slowly inside the extractor to obtain
a uniform bed and avoid wall effects and channeling. The
particles mean diameter was evaluated by electronic micro -
scope and the results indicate a particle diameter of
0.214 mm.
2.2. Supercritical fluid extraction (SFE)
The high-pressure unit used for the SFE with CO
2
and
solvent mixtures (CO

2
plus co-solvent) was modified from
the unit detailed by Danielski, Michielin, and Ferreira (in
press). In the present work, a co-solvent pump (Constamet-
ric, 3200, EUA), was connected to the extraction line in
order to supply the modifier (organi c solvent at high-pres-
sure) at pre-established flow rate, to mixture with CO
2
flow
before the extraction vessel. The co-solvent pump works
with flow rate from 0.01 to 9.99 mL/min. Ethanol (EtOH),
ethyl acetate (EtAc) and dichloromethane (DCM) were
used as co-solvents. The EtOH was used with concentra-
tions of 5%, 10% and 15% w/w, DCM at 10%, 15% and
20% and EtAc was used at 15% w/w. The process used
CO
2
99.9% pure delivered at pressure up to 6 MPa (White
Martins, Brazil). The extracting condition was 20 MPa and
40 °C for the operations with CO
2
plus modifier at different
concentrations, while assays with pure CO
2
were carried
out at 30, 40 and 50 °C and from 15 to 30 MPa, at constant
flow rate of 3.33 (±0.02) g/min. The experimental proce-
dure for the high-pressure operation and the unit compo-
nents were described elsewhere by Michielin, Bresciani,
Danielski, Yunes, and Ferreira (2005) and a fixed mass

of 45 g of grounded shiitake mushroom was used to form
the fixed bed of particles for the high-pressure extractions.
Samples were collected at 3 h extraction time and weighed
in an analytical balance.
2.3. Classical organic solvent extraction (COSE)
Different solvents, n-hexane (Hx), dichloromethane
(DCM) and ethyl acetate (EtAc), in ascending polarity of
0, 3.1 and 4.4 (Mahjoor, 2005), were used to fractionate
the soluble compounds from the shiitake mushroom. The
COSE method used to obtain the shiitake extract consists
in a cold maceration of the mushroom to avoid thermal
degradation. The extraction was performed with dried shii-
take powder (100 g) placed in ethanol for six days. The
resulting extract was evaporated at reduced pressure up
to 10% of the initial volume to obtain the crude extract
(CE), the ethanolic fraction. Then, the CE was partitioned
with n-hexane, dichloromethane and ethyl acetate using
60 mL each (Cheung et al., 2003). The organic solvents
used were 99% pure (CAQ Ind. & Com., SP, Brazil).
2.4. Extract composition
The identification and the relative quantification of the
components present in the extracts were achieved by chro-
matographic analysis. Extract samples obtained with CO
2
at 40 °C and 15 MPa and with COSE, using DCM and
EtAc were quantified in a gas chromatograph (Agilent
model 6890) equipped with mass detector (Agilent, model
5973). The samples were dissolved in dichloromethane
and injected (1.0 lL) for analyses following the conditions:
initial temperature of 50 °C and final temperature of

632 C.S.G. Kitzberger et al. / Journal of Food Engineering 80 (2007) 631–638
250 °C, with heating rate of 5 °C/min; detector temperature
295 °C, injector temperature of 290 °C; hydrogen as carrier
gas at 2 mL/min flow rate. The chromatograph was
equipped with a 30 m column HP-5MS with inner diameter
0.25 mm and 0.25 lm film thickness. The extract compo-
nents were evaluated using the database for natural prod-
ucts Standard Reference Data Series of the National
Institute of Standard and Technology (NIST – Mass-Spec-
tral Library with Windows search program – Version 2),
where the mass spectrometer results were compared.
2.5. Antioxidant activity
2.5.1. DPPH assay method
The free radical scavenging activity of the shiitake
extract was evaluated as described by Mensor et al.
(2001). Briefly, the mushroom extract was mixed with a
0.3 mM 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH)
ethanol solution, to give final concentrations of 5, 10, 25,
50 and 100 lg of extract per mL of DPPH solution. After
30 min at room temperature, the absorbance values were
measured at 518 nm and converted into percentage of anti-
oxidant activity (% AA). This activity was also expressed
as the inhibition concentration at 50% (IC
50
), i.e., the con-
centration of the test solution required to give a 50%
decrease in the absorbance of the test solution compared
to that of a blank solution. Rutin was used as a standard
control.
2.5.2. Total phenolic method (Folin–Denis)

The shiitake extracts that indicates antioxidant poten-
tial, represented by IC
50
results lower than 200 lg/mL,
were submitted to the Folin–Denis test for the determina-
tion of total phenolic content. This procedure uses Folin–
Denis reagent, prepared with sodium tungstate de hydrate,
molybdatophosphoric acid and phosphoric acid in water,
according to method 9110 (AOAC, 1980). Initially a stan-
dard curve was prepared with tannic acid (0.1–1.0 mg/
100 mL), with the addition of 5 mL of Folin–Denis
reagent and 10 mL of sodium carbonate saturated solu-
tion. The various concentration solutions were filtered
and its absorbance values were measured in spectropho-
tometer at 760 nm. The total phenolic content for the
shiitake extracts was measured placing 5 mg of each
extract, dissolved in 1 mL of methanol. To the extract
solution was added the other reagents according to the
procedure for the standard curve. The blank consisted
of a solution only with the Folin–Denis reagents (without
the extract). The total phenolic content was calculated
based on equivalent to tannic acid (ETA) according to
Eq. (1).
Phenolic content ðg ETA=100 g extractÞ
¼
read ðmg=mLÞÂ10
sample weight ðgÞ

ð1Þ
were read (mg/mL) is the value of tannic acid concentra-

tion obtained in the standar d curve for the tested extract.
2.6. Antimicrobial activity
2.6.1. Microorgani sms tested
The shiitake extracts obtained with SFE (pure CO
2
and
with co-solvent) and with COSE, were submitted to eval-
uation of antimicrobial activity with the bacteria strains:
Escherichia coli ATCC 25922 (American Type Culture
Collection), Staphylococcus aureus ATCC 25923, Micro-
coccus luteus (MIP 200401 – Department of Microbiology
and Parasitology – UFSC, Brazil) and Bacillus cereus
ATCC11778; and the yeast strain Candida albicans ATCC
14053. The cultures were incubated at 36 °C for 18 h and
then diluted in culture broth to contain 10
6
CFU/mL.
Agar Mueller–Hinton and culture broth were used for
the bacterial growing. All bacterial cultures were incu-
bated in aerobic conditions (Sma
ˆ
nia et al., 1995; Sma
ˆ
nia,
Sma
ˆ
nia, Delle Monache, Pizzolatti, & Delle Monache,
2006).
2.6.2. Agar diffusion method
The agar diffusion method was performed using cotton

swabs for each bacterial suspension (10
6
CFU/mL) and
inoculated in plates where the bacteria’ were spread uni-
formly on the agar surface. The agar surface was perfo-
rated with 7 mm diameter holes, aseptically cut and filled
with the various shiitake extracts: SFE with CO
2
, SFE with
CO
2
/co-solvent and COSE with different solvent s. The
extracts were used in the concentration of 10 mg extract/
mL of DMSO (dimethylsulphoxide) because DMSO does
not offer inhibition to the microorganism growth. The
plates were incubated at 36 °C for 18 h and next, examined
to verify the inhibition. A positive result was defined as an
inhibition zone (halo size) of 9 mm or more around the
holes, therefore indicating the presence of antibacterial
substance in the extracts tested (Sma
ˆ
nia, Delle Monache,
Sma
ˆ
nia, & Cuneo, 1999).
2.6.3. Minimum inhibition concentration (MIC)
The antimicrobial activity of the extracts was evaluated
through the determination of the minimum inhibition con-
centration (MIC) by the microdilution method in culture
broth. The shiitake extracts that present inhibition zone

in the agar diffusion method were dissolved in 200 lLof
DMSO and the solution added to 1800 lL of Muller–Hin-
ton broth for the bacteria growth and nutritive broth for
fungi. Later, a series of dilutions with concentration vary-
ing from 2.0 to 0.0156 mg/mL in 100 lL was distributed
in the microdilution plates with 96 wells. The culture med-
ium plus DMSO was the growth control and the test dilu-
tion was used as sterilized control. In each test and growth
control well was added 5 lL of the bacterial or fungi inoc-
ula. All experiments were performed in duplicate and the
plates incubated for 24 h at 36 °C. Bacterial growth was
first detected by optical density (ELISA reader, CLX800
C.S.G. Kitzberger et al. / Journal of Food Engineering 80 (2007) 631–638 633
– Biotek Instruments) and afterwards by addition of 20 lL
of an alcoholic solution (0.5 mg/mL) of 2-(4-iodophenyl)-
3-(4-nitrophenyl)-5-phenyltetrazoliumcloride (INT)
(SIGMA). The plates were again incubated at 36 °C for
3 h, and in those wells where bacterial growth occurred,
INT changed from yellow to purple. Any remaining yellow
color indicated absence of growth. The MIC was consid-
ered the lower concentration of the substance that inhibited
the bacterial or the fungic growth, after incubation. The
results were expressed in mg/mL (Sma
ˆ
nia et al., 2006; Zac-
chino, 2001).
3. Results and discussion
3.1. Extraction yield
The process efficiency is quantitatively related to extrac-
tion yield. The results of shiitake extraction yield, compar-

ing different techniques, are presented in Fig. 1 for COSE
(with Hx, DCM and EtAc), and for SFE at 40 °C and
20 MPa (with pure CO
2
and with CO
2
plus different co-sol-
vents at different concentrations).
COSE data presented in Fig. 1 show a decrease in the
yield with solvent polarity for Hx, DCM and EtAc, an indi-
cation of the presence of non-polar components in the shii-
take mushroom. SFE with pure CO
2
result in yield lower
than Hx extraction, but above the values obtained by
DCM and EtAc. This result corroborates with the non-
polar characteristic of the CO
2
. The use of DCM and EtAc
as co-solvent in SFE at 15% solvent mixtures, enhances the
yield in 49% for DCM and in 59% for EtAc if compared
with pure CO
2
, indicating extraction of polar and non-
polar components.
In order to improve the process efficiency in yield
results, SFE was performed with EtOH as co-solvent
because important substances that show antioxidant activ-
ity are polar components. EtOH was also used for shiitake
maceration and present polarity of 5.1 (Mah joor, 2005).

The results obtaine d using different concentrations of
EtOH (5%, 10% and 15%) show the yield increasing near
one order of magnitude from pure CO
2
(yield of 0.57%
w/w) to CO
2
with 15% EtOH (yield of 3.81% w/w). This
behavior is due to the increase in the number of soluble
components in the mixture, reducing the selectivity and
enhancing the yield. Also, the enrichment in co-solvent
concentration improves the yield due to proportional
changes in the solvent mixture characteristics. Otherwise,
the use of DCM as co-solvent increases yield with raising
DCM concentration up to 15% (0.85% w/w) a nd than
decreases to 0.61% w/w with 20% DCM in the extracting
mixture. The increasing amount of co-solvent (20%),
enhance the interactions solute/co-solvent, reducing the
interactions with CO
2
, and therefore reducing the yield, a
behavior also discussed by Lo
´
pez, Arce, Garrido, Rios,
and Valca
´
rcel (2004) during astaxanthin extraction from
crustaceans.
Besides the evaluation of the quantitative efficiency of
extracting process, the yield values are not directly related

to their qualitative efficiency. Consequently it is important
to asses s the chemical profile and the biological activity of
the extracts.
3.2. Composition profile
Table 1 compares the relative composition of shiitake
extracts obtained by SFE with CO
2
(40 °C and 20 MPa)
and by COSE with DCM and EtAc. The identified compo-
nents and the respective molecular weights are listed in
Table 1.
Few components were identified in all samples, probably
due to the range of the GC analysis used in this work, ade-
quate for low polarity substances, and because the extracts
are complex mixtures of polar and non-polar compounds,
in agreement to the solvents used for the extractions .
The higher quantity of identified compounds in the SFE
sample is due to the non-polar characteristic of the CO
2
,
attribute adequate for the analysis performed. Also, to
observe the quality of the extracts from shiitake mush-
room, among the substances identified, were: niacinamide,
15%
CO
2
10%
5%
Hx
15%

10%
DCM
20%
15%
EtAc
0
0.5
1
1.5
2
2.5
3
3.5
4
Yield (%)
COSE SFE EtOH SFE DCM SFE SFE EtAC
Fig. 1. Yield results for shiitake extraction using different techniques: (a)
COSE with n-hexane (Hx), DCM and EtAc; (b) SFE (40 °C/20 MPa) with
EtOH as co-solvent at concentrations of 5%, 10% and 15%; (c) SFE
(40 °C/20 MPa) with DCM as co-solvent at concentrations of 10% 15%
and 20%; (d) SFE with pure CO
2
at 40 °C and 20 MPa; (e) SFE (40 °C/
20 MPa) with EtAc as co-solvent at 15% concentration.
Table 1
Relative composition profile, in % peak area, of shiitake extracts obtained
using SFE, with pure CO
2
at 40 °C and 20 MPa, and using COSE with
DCM and EtAc

Components Mol (g/mol) SFE DCM EtAc
Palmitic acid 256.42 6.87 – –
Linoleic acid 280.50 87.59 41.71 12.98
Ergosterol 396.65 1.57 – –
5-Methyl-octadecane 268.52 3.37 – –
p-Menthane-1,8-diol
(hydrated terpin)
190.28 – 20.91 30.50
1,8-Terpin 172.26 – 2.94 –
Niacinamide 122.13 – – 49.78
Non-identified compounds – 0.60 34.54 6.74
634 C.S.G. Kitzberger et al. / Journal of Food Engineering 80 (2007) 631–638
a vitamin from B complex; ergosterol, a biological precur-
sor of vitamin D2 and fatty acids such as linoleic acid and
palmitic acids. Further and complementary studies are nec-
essary to evaluate all fractions (polar and non-polar com-
pounds) of the components present in the extracts.
3.3. Antioxidant activity
3.3.1. DPPH essay method
DPPH is a free radical, stable at room temperature,
which produces a violet solution in ethanol. In presence
of antioxidant compounds the DPPH is reduced producing
a non-color ethanolic solution. Fig. 2 shows the results of
antioxidant activity (AA) of shiitake extracts in different
concentrations, obtaine d using the DPPH method for sam-
ples from COSE (DCM and EtAc) and SFE with CO
2
plus
EtOH at 5%, 10% and 15%. Table 2 shows the IC
50

values
in DPPH essays where the results from the various shiitake
extract are compared with a pure flavonoid (rutin) with rec-
ognized antioxidant activity.
The shii take fractions obtained with dichloromethane
(DCM) and ethyl acetate (EtAc), solvents with intermedi-
ate polarity in classical organic solvent extraction, show
antioxidant activity of 64.83% and 92.93% AA, respec-
tively, for 250 lg/mL extract concentration. This behavior
is probably due to the presence of polar substances in the
extracts responsible for the cited acti vity, and indicates
the importance of the shiitake mushroom as a source of
valuable components. Supercritical extracts with pure
CO
2
from 30 to 50 °C and from 15 to 30 MPa were also
tested in DPPH essays and the results show a limit ed anti-
oxidant activity in 250 lg/mL extract concentration, near
11% AA. This result is possibly caused by the non-polar
characteristic of the solvent, resulting in the extraction of
mainly non-polar components, with low antioxidant
activity.
Otherwise, the use of ethanol as co-solvent in SFE at
40 °C and 20 MPa, show antioxidant activity for all co-sol-
vent concentrations tested (5%, 10% and 15% w/w of EtOH
in CO
2
), as presented in Fig. 2. The polar nature of ethanol
indicates this solvent as a viabl e co-solvent for SFE to
obtain antioxidant components. The antioxidant activity

increases with higher ethanol concentration in the SFE,
up to 72.97% AA, for 15% EtOH (250 lg/mL concentra-
tion), while the SFE with 10% EtOH was 63.96% AA, a
value approximate to the DCM fraction.
Fig. 2 also shows the effect of extract concentration in
the behavior of AA. For extract concentrations up to
125 lg/mL, the SFE with 10% and 15% EtOH show higher
values of AA. The dependence of the concentration for
EtAc extracts is practically linear, with R
2
of 0.998.
The results of the IC
50
values presented in Table 2 show
that the extracts obtained using 15% ethanol as co-solvent
in SFE is equivalent to use EtAc in classical solvent extrac-
tion, and mostly, is comparable to the results obtained by
rutin (78.43 lg/mL), a typical flavonoid with good antiox-
idant activity.
3.3.2. Total phenolic content (TPC) – Folin–Denis method
The antioxidant activity of vegetable extracts has been
correlated to their content of phenolic components (Velio-
glu, Mazza, Gao, & Ooma h, 1998) due to their property of
scavenging free radicals. Therefore, it is important to con-
sider the effect of the total phenolic quantity in the antiox-
idant activity of the shiitake extracts.
The TPC was expressed in equivalent of tannic acid
(ETA) (g/100 g of extract) and the results for the shiitake
extracts are presented in Table 3. The results indicate that
the higher the antioxidant activity, obtained for the EtAc

fraction (Fig. 2), the higher is the ETA value (Table 3).
This behavior is probably due to the EtAc capacity to sol-
ubilize flavonoid components from the shiitake, substances
detected by the Folin–Denis method (Falkenberg, Santos,
& Simo
˜
es, 2003).
Fig. 3 compares the behavior of the antioxidant activity,
through IC
50
values, and the phenolic content, using ETA
results. The results presented in the figure indicate the effi-
ciency of EtAc for the extraction of total phenolic com-
pounds and also that the use of EtOH as co-solvent in
CO
2
extraction of phenolic compounds from shiitake is
effective in concentrations above 5% w/w. Fig. 3 also shows
that high content of phenolic compounds (ETA result) with
the lowest IC
50
value (DPPH result) represent better anti-
oxidant activity. These results recommend EtAc as co-sol-
vent in SFE, in order to improve the antioxidant
0
10
20
30
40
50

60
70
80
90
100
0 50 100 150 200 250 300
AA (%)
DCM
EtAc
SFE-EtOH 5%
SFE-EtOH 10%
SFE-EtOH 15%
Concentration of extracts (
μ
g/mL)
Fig. 2. Antioxidant activity for the shiitake extracts obtained with
SFE + co-solvent and with organic solvent at low-pressure process.
Table 2
IC
50
values obtained for the shiitake extracts in DPPH assay
Extract IC
50
(lg/mL)
Dichloromethane (DCM) 183.2 ± 0.2
Ethyl acetate (EtAc) 132.1 ± 0.3
Ethanolic fraction >250
40 °C/20 MPa/5% EtOH 190.3 ± 0.1
40 °C/20 MPa/10% EtOH 158.3 ± 0.2
40 °C/20 MPa/15% EtOH 133.6 ± 0.4

Rutin 78.4 ± 0.1
C.S.G. Kitzberger et al. / Journal of Food Engineering 80 (2007) 631–638 635
performance of the supercritical extracts, although this
activity is already representative for shiitake extracts
obtained with pure CO
2
.
3.4. Antimicrobial activity
3.4.1. Agar diffusion method (ADM)
Although the agar diffusion method is sensitive to detect
microbial growth, it has a qualitative character and should
not be recommended to quantify the antimicrobial activity
of a substance based on the size of the inhibition zone
formed during the analyses (Rios, Recio, & Viller, 1988).
Anyhow, several shiitake extracts were tested in ADM in
order to provide indication for further detection of mini-
mum inhibition concentration.
Samples of supercritical extracts obtained at different
conditions of temperature and pressure and SFE with
15% ethyl acetate as co-solvent at 40 °C and 15 MPa were
tested against the bacteria S. aureus, B. cereus, M. luteus
(all Gram positive) and E. coli (Gram negative), and also
for the yeast C. albicans in the agar diffusion method.
Extracts obtained with COSE using EtAc, DCM and etha-
nol were also tested against the above microorganis ms but
no antimicrobial activity were detected in ADM assays
because the inhibition zone was non-existent or smaller
then 9 mm.
Table 4 shows the results of agar diffusion essays in
terms of size of inhibition zone (mm) for the extracts tested

against the studied microorganisms. The S. aureus was the
most resistant microorganisms for all extracts, presenting
inhibition zone (IH) only for the supercritical extracts:
40 °C/30 MPa and 50 °C/15 MPa, where some bacterial
growth was detected inside the halo, indicating a weak inhi-
bition power. For E. coli all extracts shown inhibition zone
with growth inside (IH), but the 40 °C/30 MPa extract
shown a 9 mm halo, still considered unsatisfactory to jus-
tify a MIC analysis. The extract obtained using ethyl ace-
tate in supercritical CO
2
(40 °C/20 MPa/15% EtAc)
indicate inhibition against B. cereus with a 12 mm halo, a
strong antimicrobial result caused probably by the interac-
tion between solvent mixture and shitake compounds at
high-pressure conditions. The M. luteus and the B. cereus
were the less resistant of the tested microorganisms, pre-
senting only two extracts with no inhibition: 30 °C/
15 MPa for both microorganisms and 30 °C/40 MPa for
B. cereus and CO
2
/co-solvent for M. luteus. The most sig-
nificant inhibition zones were obtained for M. luteus:
19 mm for 40 °C/30 MPa and 16 mm for 50 °C/150 MPa.
The yeast grown was partially limited only for supercritical
extracts at 30 °C/15 MPa and 40 °C/15 MPa (12 mm halo
for both extracts), while other extracts were not effective
against C. albicans. For the SFE, a low pressure
(15 MPa) contributed better for the yeast inhibition at 30
and 40 °C and had no effectiveness at 50 °C, probably

due to the solvent density influence, which decreases with
temperature increase, decreasing also the solvent extraction
capacity.
The antimicr obial analysis indicates higher efficiency of
the supercritical extracts compared with the low-pressure
extracts (COSE) for the experienced microorganisms. Also,
the extracts were more effective against Gram positive bac-
teria, such as M. luteus and B. cereus. Finally, the results
point toward the use of SFE to obtain shiitake extracts
with antimicrobial activity against different microorgan-
isms according to the extracting conditions used.
3.4.2. Minimum inhibition concentration (MIC)
Shiitake extracts that shown suitable results in agar dif-
fusion method (near 10 mm halo) were submitted to the
Table 3
Total phenolic content expressed in equivalent tannic acid (ETA) in g
ETA/100 g extract
Extract g (ETA)/100 g of extract
DCM 1.05
EtAc 2.15
ESC-EtOH 5% 0.45
ESC-EtOH 10% 1.01
ESC-EtOH 15% 1.02
0
40
80
120
160
200
DCM EtAc EtOH 5% EtOH 10% EtOH 15%

Shiitake extracts
IC50 (DPPH)
0
0.5
1
1.5
2
2.5
ETA (g/100 g extract)
IC50 (DPPH)
ETA
Fig. 3. Comparison between ETA and IC
50
values for the shiitake
fractions tested.
Table 4
Antimicrobial activity for the shiitake extracts, evaluated by agar diffusion
method
SFE (°C/MPa) Microorganisms
S. aureus E. coli M. luteus B. cereus C. albicans
30/15 0 IH 0 0 12
30/20 0 IH 14 10 0
30/30 0 9 12 10 0
40/15 0 IH 14 0 12
40/20 0 IH NT NT NT
40/30 IH 0 19 12 0
50/15 IH IH 16 14 0
50/20 0 IH 12 10 0
40/20 + EtAc 15% NT NT 0 12 0
COSE

a
00000
NT: non-tested. IH: inhibition zone (halo size) with bacterial growth
inside.
a
Classical organic solvent extraction: dichloromethane, ethyl acetate
and ethanol.
636 C.S.G. Kitzberger et al. / Journal of Food Engineering 80 (2007) 631–638
Minimum inhibition concentration (MIC) tests. The results
for the amount of extract which characterizes the minimum
inhibitory concentration are presented in Table 5.We
observed that, although the 30 °C/30 MPa extract had
shown a 10 mm halo in Table 4 (the smal lest halo selected
for MIC test), it was the most effective shiitake extract,
with the lowest MIC value (0.25 mg/mL) for B. cereus inhi-
bition, while the largest halo (19 mm in Table 4) obtained
from the 40 °C/30 MPa extract for M. luteus, resulted in
a MIC value of 1.0 mg/mL. This behavior is justified by
the fact that the sample potency to affect the microorgan-
ism growth is not directly proportional to the inhibition
zone (halo size), as discussed by Rios et al. (1988). The inhi-
bition for C. albicans occurred at the highest extract con-
centration (2.0 mg/mL), for extracts at 30 °C/15 MPa and
40 °C/15 MPa, indicating the enhancement resistance of
this microorganism to shiitake extracts, compared with
the other tested organisms.
4. Conclusions
The present study show that supercritical fluid extrac-
tion is effective to obtain shiitake extracts with good recov-
ery of antioxidant and antimicrobial activities. Also, the

results from classical solvent extra ction were useful to indi-
cate a suitable co-solvent for the SFE, in order to improve
the activity of the extracts. In SFE, the ethanol showed
strong influence as co-solvent in concentrations above 5%
w/w, with optimum value at 15% w/w, to provide antioxi-
dant activity for the shiitake extracts. Related to the anti-
microbial activity, the shiitake extracts obtained with
supercritical fluids were effective against the growth of M.
luteus and B. cereus (gram positive bacteria) and not effi-
cient against S. aureus and E. coli. For the yeast C. albi -
cans, the shii take extracts that showed antifungi activity
were obtained from supercritical CO
2
at 15 MPa and
30 °C and 40 °C.
The results of the biological activity of shiitake extracts
obtained using high-pressure and low-pressure techniques
indicate that: both extraction methods were adequate in
terms of antioxidant activity, while the high-pressure pro-
cess (with pure CO
2
and with co-solvent) was more effective
to obtain extracts effective against M. luteus and B. cereus,
while the low-pressure extracts did not show antimicrobial
activity. These results specify the supercritical technique as
the more efficient to obtain valuable extracts from shiitake
mushroom. The SFE technique is suitable to obtain func-
tional compounds from a food source, contributing to
increase in the aggregate value.
Acknowledgement

The authors thank CAPES for the financial support.
References
AOAC – Association Official Analytical Chemistry (1980). Official
methods of analysis of AOAC (14 ed.). Washington.
Cheung, L. M., Cheung, P. C. K., & Ooi, V. E. C. (2003). Antioxidant
activity and total phenolics of edible mushroom extracts. Food
Chemistry, 81, 249–255.
Danielski, L., Michielin, E. M. Z., & Ferreira, S. R. S. (in press). Horsetail
(Equisetum giganteum L.) oleoresin and supercritical CO
2
: experimen-
tal solubility and empirical data correlation. Journal of Food Engi-
neering. doi:10.1016/j.jfoodeng.2005.12.016.
Falkenberg, M. B., Santos, R. I., & Simo
˜
es, C. M. O. (2003). In
Farmacognosia: da Planta ao Medicamento. Introduc¸a
˜
oa
`
Ana
´
lise
Fitoquı
´mica
. Porto Alegre/Floriano
´
polis (pp. 229–245).
Halliwell, B., & Gutteridge, J. M. C. (1989). Free radicals in biology and
medicine (543 pp.) (2nd ed.). Oxford University Press.

Hatvani, N. (2001). Antibacterial effect of the culture fluid of Lentinus
edodes mycelium grown in submerged liquid culture. International
Journal of Antimicrobial Agents, 17, 71–74.
Hirasawa, M., Shouji, N., Neta, T., Fukushima, K., & Takada, K. (1999).
Three kinds of bacterial substances from Lentinus edodes (Berk.) Sing.
(shiitake, an edible mushroom). International Journal of Antimicrobial
Agents, 11(2), 151–157.
Kanter, M. (1998). Free radicals, exercise and antioxidant supplementa-
tion. Proceedings of the Nutrition Society, 57, 9–13.
Lo
´
pez, M., Arce, L., Garrido, J., Rios, A., & Valca
´
rcel, M. (2004).
Selective extraction of astaxanthin from crustaceans by use of
supercritical carbon dioxide. Talanta, 64, 726–731.
Mahjoor, F. (2005). Reference books and software. <-
nomenex.com> Accessed in September 2005.
Manzi, P., & Pizzoferatto, L. (2000). Beta-glucans in edible mushrooms.
Food Chemistry, 68, 315–318.
Mau, J. L., Chao, G-R., & Wu, K-T. (2001). Antioxidant properties of
methanolic extracts from several ear mushrooms. Journal of Agricul-
ture and Food Chemistry, 49, 5461–5467.
Mensor, L. L., Menezes, F. S., Leita
˜
o, G. G., Reis, A. S., dos Santos, T.
C., Coube, C. S., et al. (2001). Screening of Brazilian plant extracts for
antioxidant activity by the use of DPPH free radical method.
Phytotherapy Research, 15, 127–130.
Michielin, E. M. Z., Bresciani, L. F. V., Danielski, L., Yunes, R. A., &

Ferreira, S. R. S. (2005). Composition profile of horsetail (Equisetum
giganteum L.) oleoresin: comparing SFE and organic solvents extrac-
tion. Journal of Supercritical Fluids, 33, 131–138.
Rios, J. L., Recio, M. C., & Viller, A. (1988). Screening as methods for
natural products with antimicrobial activity: Review of the literature.
Journal of Ethnopharmacology, 23, 127–149.
Royse, D. J. (2005). Cultivation of shiitake on natural and synthetic logs.
< Accessed in July
2005.
Shimada, Y., Morita, T., & Sugiyama, K. (2003). Dietary eritadenine and
ethanolamine depress fatty acid desaturase activities by increasing liver
Table 5
MIC values of shiitake extracts determined by the microdilution method
SFE (°C/MPa) MIC
a
Microorganisms
M. luteus B. cereus C. albicans
30/15 NT NT 2.0
30/20 0.5 NT NT
30/30 1.0 0.25 NT
40/15 1.0 NT 2.0
40/30 1.0 0.5 NT
50/15 0.5 0.5 NT
50/20 1.0 NT NT
40/20 + EtAc15% >2.0 0.5 NT
NT: non-tested.
a
Expressed in mg/mL of extract.
C.S.G. Kitzberger et al. / Journal of Food Engineering 80 (2007) 631–638 637
microsomal phosphatidylethanolamine in rats. Journal of Nutrition, 3,

758–765.
Sma
ˆ
nia, A., Jr., Delle Monache, F., Sma
ˆ
nia, E. F. A., & Cuneo, R.
S. (1999). Antibacterial activity of steroidal compounds isolated
from Ganoderma applanatum (Pers.) Pat. (Aphyllophoromycetideae)
fruit body. International journal of Medicinal Mushrooms, 1,
325–330.
Sma
ˆ
nia, A., Jr., Monache, F. D., Sma
ˆ
nia, E. F., Gil, M. L., Benchetrit, L.
C., & Cruz, F. S. (1995). Antibacterial activity of a substance produced
by the fungus Pycnoporus sanguineus (Fr.) Murr. Journal of Ethno-
pharmacology, Inglaterra, 45, 177–181.
Sma
ˆ
nia, A., Jr., Sma
ˆ
nia, E. F. A., Delle Monache, F., Pizzolatti, M., &
Delle Monache, G. (2006). Derivatization does not influence antimi-
crobial and antifungal activities of applanoxidic acids and sterols from
Ganoderma spp. Zeitschrift fur Naturforschung, 61C, 31–34.
Spigno, G., & de Faveri, D. M. (2006). Antioxidants from grape stalks and
marc: influence of extraction procedure on yield, purity and antiox-
idant power of the extracts. Journal of Food Engineering, 25, 40–58.
doi:10.1016/j.jfoodeng.2005.11.020.

Va
´
gi, E., Sima
´
ndi, B., Suhajda, a
´
., & He
´
thelyi, E
´
. (2005). Essential oil
composition and antimicrobial activity of Origanum majorana L.
extracts obtained with ethyl alcohol and supercritical carbon dioxide.
Food Research International, 38, 51–57.
Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant
activity and total phenolics in selected fruits, vegetables, and grain
products. Journal of Agricultural and Food Chemistry, 46, 4113–4117.
Wasser, S. P., & Weis, A. L. (1999). Medicinal properties of substances
occurring in higher basidiomycetes mushrooms: current perspectives
(review). International Journal of Medicinal Mushrooms, 1, 31–62.
Yang, J-H., Lin, H-C., & Mau, J-L. (2002). Antioxidant properties of
several commercial mushrooms. Food Chemistry, 77, 229–235.
Zacchino, S. (2001). Estrate
´
gias para a descoberta de novos agentes
antifu
´
ngicos. In Plantas Medicinais: sob a o
´
tica da Quı

´
mica Medicinal
Moderna (pp. 436–479). Argos.
638 C.S.G. Kitzberger et al. / Journal of Food Engineering 80 (2007) 631–638