Nutritional value, bioactive compounds and antioxidant properties of three edible mushrooms from Poland

1" " Nutritional value, bioactive compounds and antioxidant properties of three edible mushrooms from Poland Sandrina A. Helenoa,b, Raíssa Carolina Ferreirac, Amilcar L. Antonioa, Maria-João R.P. Queirozb, Lillian Barrosa,*, Isabel C.F.R. Ferreiraa* aCentro de Investigação de Montanha (CIMO), ESA, Instituto Politécnico de Bragança, Campus de Santa Apolónia, apartado 1172, 5301-854 Bragança, Portugal. bCentro de Química, Universidade do Minho, Campus de Gualtar 4710-057 Braga, Portugal. cIFSULDEMINAS, Campus Inconfidentes, Praça Tiradentes, 416, Inconfidentes-MG, Brasil * Authors to whom correspondence should be addressed (Isabel C.F.R. Ferreirae-mail: [email protected]; telephone +351-273-303219; fax +351-273-325405; Lillian Barrosemail: [email protected]; telephone +351-273-303309; fax +351-273-325405). Running title: Valorization of edible mushrooms from Poland 2" " ABSTRACT Mushrooms contain a multitude of biomolecules with nutritional and/or biological activity. Among the bioactive molecules, phenolic compounds and tocopherols are the most responsible for their antioxidant activity. In the present work, Boletus edulis, Lentinus edodes and Xerocomus badius, three edible mushroom species originated from Poland, were analyzed for their chemical composition and antioxidant activity. Carbohydrates were the most abundant macronutrients, followed by proteins and ash. Fructose, mannitol and trehalose were the prevalent sugars, but glucose was only found in B. edulis. Polyunsaturated fatty acids predominated over mono and saturated fatty acids. Palmitic, oleic and linoleic acids were abundant in the three samples. αand βTocopherols were quantified in all the samples, but γ-tocopherol was only identified in X. badius. Oxalic and fumaric acids were quantified in the three samples; quinic acid was only present in L. edodes, and malic and citric acids were only found in X. badius. p-Hydroxybenzoic, protocatechuic and cinnamic acids were quantified in all the species, while p-coumaric acid was only found in B. edulis. This species and X. badius revealed the highest antioxidant properties, being B. edulis more effective in radicals scavenging activity and reducing power, and X. badius in lipid peroxidation inhibition, which is related with the highest amounts in phenolic compounds and tocopherols, respectively. KEYWORDS Wild mushrooms; Nutritional value; Chemical composition; Antioxidant activity 3" " 1. Introduction Over the last decades, the consumption of mushrooms has significantly increased due to the scientific evidence of their ability to help the organism in the combat and prevention of several diseases (Ferreira, Barros, & Abreu, 2009; Kalac, 2012). Fruiting bodies of mushrooms are consumed as a delicacy for their texture and flavor, but also for their nutritional properties that makes them even more attractable (Lindequist, Niedermeyer, & Julich, 2005; Kalac, 2012). Mushrooms are also described as an excellent choice to include in low caloric diets since they have high amounts of dietary fiber, minerals, vitamins, water, protein, carbohydrates, and low content in lipids (Mattila et al., 2001; Heleno, Barros, Sousa, Martins, & Ferreira, 2009; Kalac, 2012). Furthermore, mushrooms contain a huge variety of bioactive compounds, and proved to be effective mainly as antioxidants, anticancer and antimicrobial agents (Barros, Ferreira, & Baptista, 2008; Ferreira, Vaz, Vasconcelos, & Martins, 2010; Alves et al., 2012). Among the bioactive molecules, phenolic acids have attracted special attention since they are reported as strong antioxidants and the main responsible for the antioxidant properties of mushrooms (Ferreira et al., 2009; Palacios et al., 2011). Lentinus edodes (Berk.) Pegler and Boletus edulis Bull. are two of the most consumed and popular mushrooms worldwide; being L. edodes the second most cultivated mushroom (Chang & Miles, 2004) and B. edulis considered as the tastiest one among the Boletus genus (Jaworska & Bernas, 2009). Several authors described these two mushroom species as being rich in nutrients and bioactive molecules, such as phenolic acids and tocopherols, that are related with their antioxidant activity (Cheung, Cheung, & Ooi, 2003; Cheung & Cheung, 2005; Heleno et al., 2011; Özyürek, Bener, Güçlü, & Apak, 2014). Xerocomus badius is one of the most consumed mushroom in Poland. 4" " In the present work, the chemical composition of the mentioned mushroom species (L. edodes, B. edulis and X. badius), originated from Poland, was evaluated. Furthermore, the chemical compounds found in each sample were related with their antioxidant properties, measured as free radical scavenging activity, reducing power and lipid peroxidation inhibition. 2. Materials and methods 2.1. Samples Commercial samples of Boletus edulis Bull., Lentinus edodes (Berk.) Pegler and Xerocomus badius (Fr.) E.-J.Gilbert, three edible dried mushrooms, were obtained in a local market in Poland, in November 2012. The specimens were kept at -20 ºC until further analysis. 2.2. Standards and Reagents Acetonitrile 99.9%, n-hexane 95% and ethyl acetate 99.8% were of HPLC grade from Fisher Scientific (Lisbon, Portugal). The fatty acids methyl ester (FAME) reference standard mixture 37 (standard 47885-U) was purchased from Sigma (St. Louis, MO, USA), as also other individual fatty acid isomers and standards of sugars (L-(+)- arabinose, D-(+)-mannitol, D-(+)-trehalose), tocopherols (α-, β-, and γ-isoforms), organic acids (malic, oxalic quinic, citric and fumaric acids), phenolic compounds (phydroxybenzoic, protocatechuic, p-coumaric and cinnamic acids), trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid). Racemic tocol, 50 mg/mL, was purchased from Matreya (PA, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA). 5" " 2.3. Chemical composition 2.3.1. Nutritional value The samples were analysed for chemical composition (moisture, proteins, fat, carbohydrates and ash) using the AOAC procedures (AOAC, 1995). The crude protein content (N×4.38) of the samples was estimated by the macro-Kjeldahl method; the crude fat was determined by extracting a known weight of powdered sample with petroleum ether, using a Soxhlet apparatus; the ash content was determined by incineration at 600±15 °C. Total carbohydrates were calculated by difference. Energy was calculated according to the following equation: Energy (kcal) = 4×(g protein+g carbohydrate)+9×(g fat). 2.3.2. Free sugars Free sugars were determined by a High Performance Liquid Chromatography (HPLC) system consisted of an integrated system with a pump (Knauer, Smartline system 1000, Berlin, Germany), degasser system (Smartline manager 5000) and auto-sampler (AS2057 Jasco, Easton, MD, USA), coupled to a refraction index detector (RI detector Knauer Smartline 2300) as previously described by the authors (Heleno et al., 2009). Sugars were identified by comparing the relative retention times of sample peaks with standards. Data were analysed using Clarity 2.4 Software (DataApex, Prague, Czech Republic). The chromatographic separation was achieved with a Eurospher 100-5 NH2 column (5 µm, 250 mm × 4.6 mm i.d., Knauer) operating at 35 ºC (7971 R Grace oven). The mobile phase was acetonitrile/deionized water, 70:30 (v/v) at a flow rate of 1 mL/min. Quantification was based on the RI signal response of each standard, using the internal standard (IS, raffinose) method and by using calibration curves obtained from commercial standards of each compound. The results were expressed in g per 100 g of dry weight. 6" " 2.3.3. Fatty acids Fatty acids were determined after a trans-esterification procedure as described previously by the authors (Heleno et al., 2009), using a gas chromatographer (DANI 1000, Contone, Switzerland) equipped with a split/splitless injector and a flame ionization detector (GC-FID at 260 ºC) and a Macherey–Nagel (Düren, Germany) column (50% cyanopropyl-methyl-50% phenylmethylpolysiloxane, 30 m × 0.32 mm i.d. × 0.25 µm df). The oven temperature program was as follows: the initial temperature of the column was 50 ºC, held for 2 min, then a 30 ºC/min ramp to 125 ºC, 5 ºC/min ramp to 160 ºC, 20 ºC/ min ramp to 180 ºC, 3 ºC/min ramp to 200 ºC, 20 ºC/min ramp to 220 ºC and held for 15 min. The carrier gas (hydrogen) flow-rate was 4.0 mL/min (0.61 bar), measured at 50 ºC. Split injection (1:40) was carried out at 250 ºC. Fatty acid identification was made by comparing the relative retention times of FAME peaks from samples with standards. The results were recorded and processed using CSW 1.7 software (DataApex 1.7, Prague, Czech Republic). The results were expressed in relative percentage of each fatty acid. 2.3.4. Tocopherols Tocopherols were determined following a procedure previously described by the authors (Heleno, Barros, Sousa, Martins, & Ferreira, 2010). Analysis was performed by HPLC (equipment described above), and a fluorescence detector (FP-2020; Jasco, Easton, MD, USA) programmed for excitation at 290 nm and emission at 330 nm. The chromatographic separation was achieved with a Polyamide II normal-phase column (5 µm, 250 mm × 4.6 mm i.d., YMC Waters), operating at 35 ºC. The mobile phase used was a mixture of n-hexane and ethyl acetate (70:30, v/v) at a flow rate of 1 mL/min. The compounds were identified by chromatographic comparisons with authentic standards. 7" " Quantification was based on the fluorescence signal response of each standard, using the IS (tocol) method and by using calibration curves obtained from commercial standards of each compound. The results were expressed in µg per 100 g of dry weight. 2.3.5. Organic acids Organic acids were determined following a procedure previously described by the authors (Reis et al., 2013). Analysis was performed by ultra-fast liquid chromatograph (UFLC) coupled to photodiode array detector (PDA), using a Shimadzu 20A series UFLC (Shimadzu Corporation, Kyoto, Japan). Separation was achieved on a SphereClone (Phenomenex) reverse phase C18 column (5 µm, 250 mm × 4.6 mm i.d.) thermostatted at 35 ºC. The elution was performed with sulphuric acid 3.6 mM using a flow rate of 0.8 mL/min. Detection was carried out in a PDA, using 215 nm and 245 as preferred wavelengths. The organic acids were quantified by comparison of the area of their peaks recorded at 215 nm with calibration curves obtained from commercial standards of each compound. The results were expressed in g per 100 g of dry weight. 2.3.6. Phenolic compounds Phenolic acids determination was performed using a Shimadzu 20A series ultra-fast liquid chromatograph (UFLC, Shimadzu, equipment described above) as previously described by Reis et al. (2013). Separation was achieved on an Aqua (3 µm, 150mm x 4.6 mm i.d., Phenomenex, Torrance, CA, USA) reverse phase C18 column (5 µm, 250 mm × 4.6 mm i.d.) thermostatted at 35 ºC. The mobile phase was: (A) 0.1% formic acid in water, (B) 100% HPLC-grade acetonitrile. The elution gradient established was 10% A to 15% B over 5 min, 15–25% A in B over 5 min, 25–35% A in B over 10 min, isocratic 50% B for 10 min, and re-equilibration of the column, using a flow rate of 8" " 0.5 ml/min.""Detection was carried out in a photodiode array detector (PDA), using 280 nm as the preferred wavelength. The phenolic compounds were quantified by comparison of the area of their peaks recorded at 280 nm with calibration curves obtained from commercial standards of each compound. The results were expressed in mg per 100 g of dry weight." 2.4. Antioxidant activity 2.4.1. Extracts preparation The lyophilized powder (1.5 g) was extracted by stirring with 40 mL of methanol (25ºC at 150 rpm) for 2 h and subsequently filtered through Whatman No. 4 paper. The residue was then extracted with 20 mL of methanol (25ºC at 150 rpm) for 2 h. The combined methanolic extracts were evaporated at 40ºC (rotary evaporator Bṻchi R-210) to dryness redissolved in methanol at a concentration of 50 mg/mL, and stored at 4 oC until analysis (Reis et al., 2011). 2.4.2. General! Successive dilutions of the stock solution of the phenolic extracts were made and submitted to in vitro assays already described by the authors (Heleno et al., 2009) to evaluate the antioxidant activity of the samples. The sample concentrations providing 50% of antioxidant activity (EC50) or 0.5 of absorbance (EC0.5) were calculated from the graphs of antioxidant activity percentages (DPPH, β-carotene/linoleate and TBARS assays) or absorbance at 690 nm (reducing power assay) against sample concentrations. In the Folin-Ciocalteu assay (for determination of total phenolics), the results were expressed as mg of gallic acid equivalents (GAE) per g of extract. The commercial standard trolox was used as positive control.! 9" " 2.4.3. Total phenolics by Folin-Ciocalteu assay ! The extract solution (1 mL) was mixed with Folin-Ciocalteu reagent (5 mL, previously diluted with water 1:10, v/v) and sodium carbonate (75 g/L, 4 mL). The tubes were vortex mixed for 15 s and allowed to stand for 30 min at 40 ºC for colour development. Absorbance was then measured at 765 nm.! 2.4.4. Reducing power by Ferricyanide/Prussian blue assay! The assay was performed using a Microplate Reader ELX800 Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The different concentrations of the extracts (0.5 mL) were mixed with sodium phosphate buffer (200 mmol/L, pH 6.6, 0.5 mL) and potassium ferricyanide (1% w/v, 0.5 mL). For each concentration, the mixture was incubated at 50 ºC for 20 min, and trichloroacetic acid (10% w/v, 0.5 mL) was added. The mixture (0.8 mL) was poured in the 48-wells, as also deionized water (0.8 mL) and ferric chloride (0.1% w/v, 0.16 mL), and the absorbance was measured at 690 nm. 2.4.5. Scavenging activity by DPPH assay This methodology was performed using the Microplate Reader mentioned above. The reaction mixture on 96 wells plate consisted of a solution by well of the extract solutions with different concentrations (30 µL) and methanolic solution (270 µL) containing DPPH radicals (6×10-5 mol/L). The mixture was left to stand for 30 min in the dark, and the absorption was measured at 515 nm. The radical scavenging activity (RSA) was calculated as a percentage of DPPH discolouration using the equation: % RSA = [(ADPPH-AS)/ADPPH] × 100, where AS is the absorbance of the solution containing the sample and ADPPH is the absorbance of the DPPH solution. 16" " CONCLUSION B. edulis, L. edodes and X. badius proved to be rich sources of carbohydrates, free sugars and proteins, containing also different bioactive compounds such as organic acids, phenolic compounds and tocopherols. Polyunsaturated fatty acids predominated over mono and unsaturated fatty acids. 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In each row, different letters mean significant differences between samples (p < 0.05). Parameter Boletus edulis Lentinus edodes Xerocomus badius Ash (g/100 g dw) 5.26 ± 0.44c 6.24 ± 0.05b 7.32 ± 0.02a Carbohydrates 81.86 ± 0.41a 76.62 ± 0.07c 80.38 ±0.15b Proteins (g/100 g dw) 10.65 ± 0.47b 16.0 ± 0.05a 8.08 ± 0.14c Fat (g/100 g dw) 2.23 ± 0.02b 1.14 ± 0.01c 4.22 ± 0.03a Energy (kcal/100 g dw) 390.09 ± 1.32a 380.74 ± 0.17b 391.83 ± 0.04a Fructose Glucose 0.71 ± 0.33c 1.24 ± 0.46 2.04 ± 0.14a nd 0.95 ± 0.20b nd Mannitol 3.14 ± 1.18 c 5.16 ± 0.21b 6.20 ± 0.25a Trehalose 9.29 ± 0.51a 8.41 ± 0.24b 4.62 ± 0.02c Total Sugars (g/100 g dw) 14.38 ± 2.47b 15.61 ± 0.59a 11.77 ± 0.03c C16:0 (palmitic) 7.56 ± 0.05c 9.40 ± 0.11b 15.47 ± 0.12a C18:0 (stearic) 1.93 ± 0.08a 1.51 ± 0.05b 1.96 ± 0.15a C18:1n9c (oleic) 30.41 ± 0.01b 1.87 ± 0.41c 38.15 ± 0.01a C18:2n6c (linoleic) 57.16 ± 0.01b 82.55 ± 0.58a 42.22 ± 0.11c C18:3n3 alfa linolenic) 0.11 ± 0.00c 0.13 ± 0.02a 0.12 ± 0.01b SFA (percent) 10.59 ± 0.04c 14.12 ± 0.06b 18.52 ± 0.10a MUFA (percent) 31.90 ± 0.04b 2.62 ± 0.06b 39.03± 0.03a PUFA (percent) 57.51 ± 0.00b 83.26 ± 0.52a 42.46 ± 0.01c 22# # Table 2. Tocopherols, organic acids and phenolic compounds identified in Boletus edulis, Lentinus edodes and Xerocomus badius fruiting bodies from Poland. dwdry weight; ndnot detected. In each row, different letters mean significant differences between samples (p < 0.05). Compound Boletus edulis Lentinus edodes Xerocomus badius α-tocopherol 9.70 ± 0.99b 9.08 ± 0.97b 73.14 ± 2.54a β-tocopherol γ-tocopherol 29.20 ± 1.41b nd 18.55 ± 0.89c nd 77.62 ± 1.56a 65.47 ± 2.40 Total tocopherols (µg/100 g dw) 38.90 ± 2.40b 27.63 ± 0.08c 216.22 ± 6.49a Oxalic acid 0.75 ± 0.01a 0.22 ± 0.05c 0.29 ± 0.01b Quinic acid nd 0.50 ± 0.03 nd Malic acid nd nd 2.05 ± 0.01 Citric acid nd nd 2.11 ± 0.08 Fumaric acid 0.07 ± 0.00b 0.22 ± 0.00a 0.21 ± 0.00a Total organic acids (g/100 g dw) 0.82 ± 0.01c 0.94 ± 0.02b 4.66 ± 0.05a Protocatechuic acid 0.80 ± 0.06b 0.14 ± 0.01c 1.14 ± 0.35a p-Hydroxybenzoic acid p-Coumaric acid 0.76 ± 0.06a 0.33 ± 0.02 0.19 ± 0.01c nd 0.30 ± 0.03b nd Total phenolic compounds (mg/100 g dw) 1.89 ± 0.02a 0.33 ± 0.00c 1.44 ± 0.32b Cinnamic acid 0.31 ± 0.05a 0.07 ± 0.00c 0.29 ± 0.04b 23# # Figure 1. Antioxidant activity of Boletus edulis ( ), Xerocomus badius ( ) and Lentinus edodes ( ): A) DPPH radical-scavenging activity (EC50 values: B. edulis: 1.80 ± 0.01 mg/mL; X. badius: 2.65 ± 0.06 mg/mL; L. edodes: 7.30 ± 0.13 mg/mL); B) Reducing power (EC0.5 values: B. edulis: 0.63 ± 0.02 mg/mL; X. badius: 0.96 ± 0.01 mg/mL; L. edodes: 3.19 ± 0.03 mg/mL); C) β-carotene bleaching inhibition (EC50 values: B. edulis: 1.61 ± 0.05 mg/mL; X. badius: 1.10 ± 0.05 mg/mL; L. edodes: 3.54 ± 0.55 mg/mL); D) Lipid peroxidation inhibition (EC50 values: B. edulis: 1.71 ± 0.27 mg/mL; X. badius: 0.37 ± 0.02 mg/mL; L. edodes: 33.16 ± 0.17 mg/mL). Each value is expressed as mean ± SE (n = 3). A) C) D) C) A) B) 24# # Figure 2. Total phenolic content by the Folin-Ciocalteu assay. Each value is expressed as mean ± SE (n = 3). 0 5 10 15 20 25 30 35 40 45 50 Boletus edulis Xerocomus badius Lentinus edodes Total fenolics (mg GAE/g extract) 43.34 ± 1.08 29.12 ± 0.49 7.87 ± 0.15