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: sandra@enq.ufsc.br (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. 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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. 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