Antioxidant and antimicrobial activities of shiitake Lentinula edodes extracts obtained by organic solvents and supercritical fluids Cı´ntia Sorane Good Kitzberger a, Artur Smaˆnia Jr.. K
Trang 1Antioxidant and antimicrobial activities of shiitake (Lentinula edodes) extracts obtained by organic solvents and supercritical fluids
Cı´ntia Sorane Good Kitzberger a, Artur Smaˆnia Jr. b, Rozangela Curi Pedrosa c,
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-preshigh-pres-sure methods The high-preshigh-pres-sure technique was applied to obtain shiitake extracts using pure CO2and
CO2with 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 usscreen-ing agar dilution method The results indicate that the fractions obtained with CO2using ethanol as co-solvent, at 40C, 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 CO2, respectively, while the low-pressure extraction indicates yields up to 1.25% w/w for n-hexane as solvent
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 investigated (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 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: sandra@enq.ufsc.br (S.R.S Ferreira).
www.elsevier.com/locate/jfoodeng
Trang 2and 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 (Smaˆnia 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 shiitake mushroom using classical organic
solvent extraction (COSE) with different solvents and using
SFE In the SFE, pure CO2was applied at different
condi-tions of temperature and pressure Also, CO2was used in a
mixture with ethanol, dichloromethane and ethyl acetate as
co-solvents at 40C 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 (Suzano, 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 80 +100, was
selected to settle the bed of L edodes inside the extractor
The fixed bed was formed with 40.0 ± 0.5· 103kg 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 CO2and solvent mixtures (CO2plus co-solvent) was modified from the unit detailed byDanielski, 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 (organic solvent at high-pres-sure) at pre-established flow rate, to mixture with CO2flow 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
CO299.9% pure delivered at pressure up to 6 MPa (White Martins, Brazil) The extracting condition was 20 MPa and
40C for the operations with CO2plus modifier at different concentrations, while assays with pure CO2 were carried out at 30, 40 and 50C 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 CO2
at 40C 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 50C and final temperature of
Trang 3250C, with heating rate of 5 C/min; detector temperature
295C, 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% (IC50), 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 IC50 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 dehydrate,
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 contentðg ETA=100 g extractÞ
¼ read ðmg=mLÞ 10
sample weight ðgÞ
ð1Þ
were read (mg/mL) is the value of tannic acid concentra-tion obtained in the standard curve for the tested extract 2.6 Antimicrobial activity
2.6.1 Microorganisms tested The shiitake extracts obtained with SFE (pure CO2and 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 36C for 18 h and then diluted in culture broth to contain 106CFU/mL Agar Mueller–Hinton and culture broth were used for the bacterial growing All bacterial cultures were incu-bated in aerobic conditions (Smaˆnia et al., 1995; Smaˆnia, Smaˆnia, 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 (106CFU/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 CO2, SFE with
CO2/co-solvent and COSE with different solvents 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 36C 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 (Smaˆnia, Delle Monache, Smaˆnia, & 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 lL of 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 36C Bacterial growth was first detected by optical density (ELISA reader, CLX800
Trang 4– 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 36C 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 (Smaˆnia 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 extracextrac-tion yield,
compar-ing different techniques, are presented inFig 1 for COSE
(with Hx, DCM and EtAc), and for SFE at 40C and
20 MPa (with pure CO2and with CO2plus 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 CO2result 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 CO2 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 CO2, 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 (Mahjoor, 2005) The results obtained using different concentrations of EtOH (5%, 10% and 15%) show the yield increasing near one order of magnitude from pure CO2 (yield of 0.57% w/w) to CO2with 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) and 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 CO2, 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 assess 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 CO2 (40C 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 CO2, 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%
0
0.5
1
1.5
2
2.5
3
3.5
4
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
p-Menthane-1,8-diol (hydrated terpin)
Trang 5a 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 2shows the results of
antioxidant activity (AA) of shiitake extracts in different
concentrations, obtained using the DPPH method for
sam-ples from COSE (DCM and EtAc) and SFE with CO2plus
EtOH at 5%, 10% and 15%.Table 2shows the IC50values
in DPPH essays where the results from the various shiitake
extract are compared with a pure flavonoid (rutin) with
rec-ognized antioxidant activity
The shiitake 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 activity, and indicates
the importance of the shiitake mushroom as a source of
valuable components Supercritical extracts with pure
CO2from 30 to 50C and from 15 to 30 MPa were also tested in DPPH essays and the results show a limited 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
40C and 20 MPa, show antioxidant activity for all co-sol-vent concentrations tested (5%, 10% and 15% w/w of EtOH
in CO2), as presented inFig 2 The polar nature of ethanol indicates this solvent as a viable 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 R2of 0.998 The results of the IC50values presented inTable 2show 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, & Oomah, 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 3compares the behavior of the antioxidant activity, through IC50values, 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
CO2 extraction of phenolic compounds from shiitake is effective in concentrations above 5% w/w.Fig 3also shows that high content of phenolic compounds (ETA result) with the lowest IC50 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
DCM EtAc SFE-EtOH 5%
SFE-EtOH 10%
SFE-EtOH 15%
Concentration of extracts (μg/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
Trang 6performance of the supercritical extracts, although this
activity is already representative for shiitake extracts
obtained with pure CO2
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 40C 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 microorganisms 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:
40C/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 inhiinhi-bition zone with growth inside (IH), but the 40C/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 CO2 (40C/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: 30C/
15 MPa for both microorganisms and 30C/40 MPa for
B cereus and CO2/co-solvent for M luteus The most sig-nificant inhibition zones were obtained for M luteus:
19 mm for 40C/30 MPa and 16 mm for 50 C/150 MPa The yeast grown was partially limited only for supercritical extracts at 30C/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 40C 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 antimicrobial 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
0
40
80
120
160
200
Shiitake extracts
0 0.5 1 1.5 2 2.5
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
NT: non-tested IH: inhibition zone (halo size) with bacterial growth inside.
a
Classical organic solvent extraction: dichloromethane, ethyl acetate and ethanol.
Trang 7Minimum 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 30C/30 MPa extract had
shown a 10 mm halo inTable 4(the smallest 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 inTable 4) obtained
from the 40C/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 byRios et al (1988) The
inhi-bition for C albicans occurred at the highest extract
con-centration (2.0 mg/mL), for extracts at 30C/15 MPa and
40C/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 extraction 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 shiitake extracts that showed antifungi activity
were obtained from supercritical CO2 at 15 MPa and
30C 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 CO2and 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
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Table 5
MIC values of shiitake extracts determined by the microdilution method
SFE (C/MPa) MICa
Microorganisms
M luteus B cereus C albicans
NT: non-tested.
a Expressed in mg/mL of extract.
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