- Email: [email protected]
Vitamins, phenolics and antioxidant activity of culinary prepared Suillus luteus (L.) Roussel mushroom
LWT - Food Science and Technology 59 (2014) 701e706
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Vitamins, phenolics and antioxidant activity of culinary prepared Suillus luteus (L.) Roussel mushroom _ a, *, Emilia Bernas a, Aleksandra Skrzypczak a, Grazyna Jaworska a, Krystyna Pogon Ireneusz Kapusta b a b
Department of Raw Material and Processing of Fruit and Vegetables, University of Agriculture in Cracow, Balicka Street 122, 30-149 Cracow, Poland w, Rejtana 16 c Street, 35-959 Rzeszo w, Poland Department of Technology and Analysis of Plant Derived Products, University of Rzeszo
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2014 Received in revised form 18 July 2014 Accepted 25 July 2014 Available online 6 August 2014
Unblanched and blanched Suillus luteus (L.) Roussel mushrooms were braised to consumption consistency with rape seed oil (100 g/kg) and NaCl (5 g/kg) and 2 types of culinary product were obtained. Evaluation was carried out on fresh mushrooms, as well as mushroom products as follows: unstored; stored at 20 C for 48 h; and stored at 4 C for 48 and 96 h. Braising led to increases in dry matter, basic nutritional components and tocopherols, and decreases in: total polyphenols (46e61%), total flavonoids (71e81%), vitamin C (71e81%), thiamine (71e81%), riboflavin (78%), vitamin B3 (0e13%), vitamin B6 (13 e70%) and antioxidant activity using ABTS, DPPH and FRAP assays (70e77%). Storage led to further losses of vitamin C and B-group vitamins. Products stored for 48 h at room temperature showed high total viable counts as well as the presence of yeasts, lactic acid bacteria and Bacillus strains; however, coldstored products showed no microbiological contamination. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Braising Edible mushrooms Nutrients Nutraceuticals
1. Introduction There has recently been increased interest in the consumption of forest mushrooms as awareness of their composition and health promoting properties has grown. Fresh mushrooms are rich in vitamins, particularly B, D and E, and in bioactive compounds such as polyphenols and carotenoids. They are also a good source of dietary fibre, mineral salts and fairly high quality protein. Moreover, forest mushrooms have a unique flavour, adding to the attraction for consumers (Jaworska & Bernas, 2009; Reis, Barros, Martins, & Ferreira, 2012; Ribeiro et al., 2008). Suillus luteus (L.) Roussel mushrooms are widely consumed in central Europe, particularly in Poland, Czech Republic and Germany. Their slimy skin requires peeling prior to thermal treatment, which usually consists of stir-frying followed by braising in their own juices. Data on composition and properties of S. luteus is lacking. Thermal treatment affects the nutritional value of food, in particular causing losses in constituents such as vitamins, unsaturated fatty acids and mineral compounds. On the positive side, it improves the digestibility of proteins, causes starch to paste and produces desirable sensory traits through the Maillard reaction (Kalogeropoulos, Mylona, Chiou, Ioannou & Anrikopoulos, 2007; * Corresponding author. Tel.: þ48 12 6624754; fax: þ48 12 6624757. ). E-mail address: [email protected] (K. Pogon http://dx.doi.org/10.1016/j.lwt.2014.07.040 0023-6438/© 2014 Elsevier Ltd. All rights reserved.
Murcia, Jiminez & Martinez-Tome, 2009). S. luteus (L.) Roussel mushrooms have not been the subject of detailed analysis in the literature. The aim of this work was to determine the effect of culinary treatment and different storage conditions on the basic chemical composition and levels of biologically active compounds such as polyphenols and vitamins in S. luteus (L.) Roussel mushrooms. 2. Material and methods 2.1. Chemicals All solevts were of HPLC analytical grade and were procured ˛ skie, Poland). Standards and reagents from Chempur (Piekary Sla such as Folin-Ciocalteu reagent, ABTS, DPPH, TPTZ, gallic acid, (þ)-catechin, Trolox, tocopherols, L-ascorbic acid and B-group vitamins were procured from Sigma Chemical Co. (St.Louis, MO, USA). For microbiological analysis special culture media supplied by Merck (Darmstadt, Germany) were used. Triple distilled and HPLC purified water was employed for the entire study. 2.2. Material The material examined comprised fresh S. luteus (L.) Roussel mushrooms (hereafter referred to as S. luteus) with caps 4e8 cm in
702
G. Jaworska et al. / LWT - Food Science and Technology 59 (2014) 701e706
diameter, together with culinary products obtained from them. The mushrooms were purchased from an approved supplier of forest mushrooms and were processed approximately 10 h after harvest. After sorting, mushroom were rinsed in cold water, had their caps peeled and were cut into 1 cm cubes. Half the batch was blanched in water at 98 C for 90 s. After preliminary treatment, mushrooms were braised under cover with a small amount (100 g/ kg) of 100% pure rape seed oil for 10 min. Two types of culinary product were thus obtained: unblanched mushrooms with oil (100 g/kg) and salt (5 g/kg) e product NB; and blanched products with oil (100 g/kg) and salt (5 g/kg) e product BL. The following amounts of products were obtained from 1000 g raw material (mushrooms and oil) after culinary processing: 750 g (product NB) and 870 g (product BL). After braising, the products were packed in containers and stored for 48 h at 20 C, and for 48 and 96 h at 4 C. Analysis was performed on: fresh mushrooms; unstored braised products; and braised products stored for 48 h at 20 C, and for 48 and 96 h at 4 C. The entire braised products were analysed. Two independent replications of products were obtained and analysis was carried out on every product in three replications (n ¼ 6). 2.3. Proximate composition The proximate composition of fresh mushrooms and culinary products was determined according to the AOAC. (2005) method. The dry matter was estimated by drying at 105 C (930.04), the crude protein (N 4.38) was estimated by the Kjeldahl method (978.04), the crude fat was determined by extracting a sample with diethyl ether in a Soxhlet apparatus (920.39) and the ash content was determined by incineration at 485 C (920.05). Total carbohydrates were calculated by difference using the formula: Total carbohydrates ¼ (g dry weight) e (g crude protein) e (g fat) e (g mineral salts). The energy value was calculated using the formula: Energy (kJ) ¼ 17 (g raw protein) þ 15 (g total carbohydrates) þ 38 (g fat). 2.4. Antioxidants Analysis of total polyphenols and total flavonoids was performed in methanolic extracts (800 g/kg) acidified with HCl (5 g/kg) by spectrophotometry using the Folin-Ciocalteu reagent and the solutions of AlCl3 and NaNO2 The content of these compounds was determined according to the standard curves prepared for the gallic acid and (þ)-catechin respectively. Absorbance was measured at l ¼ 675 nm for total polyphenols and at l ¼ 510 nm for total flavonoids. Phenolic acids were analyzed using UPLC/PDA/TDQ/MS proce ski, Kolniak-Ostek, and Wojdyło (2012). dure according to Oszmian Lyophilized mushroom samples were extracted with MeOH (700 g/ kg) at 100 C in an accelerated solvent extractor (Speed Extractor E916 BÜCHI, Flawil, Switzerland) at a working pressure of 100 bar. Structural information and general phenolic profiles were gathered using a Waters Aquity UPLC system (Waters Associates, Milford, MA) consisting of binary solvent manager, sample manager, PDA detector and triple quadrupole detector (TQD) operating in the negative electrospray mode. Ion source parameters were as follows: cone voltage 35 V, capillary voltage 3 kV, extractor 3 V, RF lens 100 mV, source temperature 120 C, desolvation temperature 350 C, desolvation gas flow 222 ml/s, cone gas flow 29 ml/s, and collision gas flow 0.005 ml/s. Collision cell parameters were as follows: entrance -2, exit 0.5, and collision energy 22 eV. Parameters of quadrupole 1 were set to achieve maximal mass resolution: both LM and HM resolutions were set to 15, and ion energy was set to 0.8. Phenolic acids and flavonoids were separated on a 100 mm
2.1 mm i.d., 1.7 mm Acquity BEH column (Waters Associates, Milford, MA) using the following linear, 8.5 min gradient from 80 to 100% of solvent B (acetonitrile solution (400 g/kg) containing 1 g/kg formic acid) in solvent A (water containing 1 g/kg formic acid) with a flow of 0.35 ml/min. Vitamin C content was established according to HPLC methods EN 14130:2003 (2003). The analysis was carried out on a Thermo Scientific DIONEX ULTIMATE 3000 UHPLC chromatograph with DAD Detector and Chromeleon software (Waltham, MA, USA). Sample was injected into a Onyx Monolithic C 18 column (100 4.6 mm) with CHO-7649 precolum (Phenomenex, Torrance, CA, USA). The isocratic elution was carried out using 0.1 mol/l phosphoric acid, the flow rate was 1 ml/min. The absorbance was monitored at 254 nm. Tocopherol content was established using a normal phase HPLC. The analysis was carried out on a Merck HITACHI (Tokyo, Japan) chromatograph with on-line degasser L-7612, Programmable Autosampler L-7250, pump L-7100, fluorescent detector FL Detector L-7480, Interface D-7000 and D-7000 HPLC e System e Manager (HSM) software. Sample was injected into a LUNA 5 mm NH2 250 4.6 column with AJO-4302 precolum (Phenomenex, Torrance, CA, USA). The isocratic elution was carried out using mixture of n-hexane and 2-propanol (95:5) at flow rate of 2.5 ml/min. The wavelengths of excitation/emission were 290/330. a-, b-, g- and dtocopherol content was established using an external standards. Vitamin E activity was calculated as a a-tocopherol equivalents (aTE) per kg of dry weight. Vitamin E activity ¼ 1 atocopherol þ 0.5 b-tocopherol þ 0.1 g-tocopherol þ 0.03 d e tocopherol. 2.5. Antioxidant activity Antioxidant activity was determined in methanol extracts (800 g/kg) prepared with heat treatment against the DPPH radical (Pekkarinen, Heinonen, & Hopia, 1999) and against ABTS radical cation (Re et al., 1999), as well as by the Ferric Reducing/Antioxidant Power method (Benzie & Strain, 1996). The value of the antioxidant activity was determined according to the standard curve prepared for Trolox Eqivalent for ABTS and DPPH and to the standard curve prepared for iron ions (II) for FRAP method. 2.6. B-group vitamins Vitamin B1 and B2 content was established according to HPLC methods EN 14122:2003 (2003) and EN 14152:2003 (2003) respectively. Thiamine and riboflavin were detected using a Merck HITACHI (Tokyo, Japan) liquid chromatograph with fluorescence detector. Analysis was carried out on an Onyx Monolithic C 18 column (100 4.6 mm) with CHO-7649 precolum (Phenomenex, Torrance, CA, USA) and was conducted at excitation/emission wavelengh 360/503 nm. Elution was carried out using linear 12 min gradient from 12% to 100% of solvent B (acetonitrile) in solvent A (water) with a flow of 1 ml/min. Vitamin B3 content was established using a normal phase HPLC using a Merck liquid chromatograph with UV/Vis detector (L-7420, Merck HITACHI, Tokyo, Japan). The analysis was carried out on a LUNA 5 mm NH2 250 4.6 column with AJO-4302 precolum (Phenomenex, Torrance, CA, USA). Niacin was determined at a wavelength of 220 nm. Isocratic elution at flow rate of 0.1 ml/min was performed. Mobile phase consisted of mixture (1:99) of methanol and Pic-A (tetrabutylammonium hydrogen sulphate) solution in water (0.005 mol/l). Vitamin B6 content was determined using the chromatographic method (EN 14164, 2008). The analysis was carried out on a Merck HITACHI (Tokyo, Japan) liquid chromatograph fitted with a
G. Jaworska et al. / LWT - Food Science and Technology 59 (2014) 701e706
703
Table 1 Proximate composition of braised Suillus luteus (g or MJ per kg of fresh weight, mean value ± sd, n ¼ 6). Type of product
Dry weight
Proteins
Fat
Total carbohydrates
Ash
Energy (MJ)
Raw mushrooms Not blanched (NB) Blanched (BL) LSD (P < 0.05)
45.4 ± 1.3 157 ± 11 162 ± 13 e
11.4 ± 0.3 14.7 ± 1.4 14.8 ± 1.6 5.54
3.2 ± 0.1 109 ± 3 110 ± 4 e
22.5 ± 0.5 25.2 ± 0.6 27.9 ± 0.9 0.18
8.3 ± 0.2 9.6 ± 0.2 8.7 ± 0.1 0.11
0.66 ± 0.04 4.55 ± 0.04 4.67 ± 0.04 0.008
fluorescence detector, on a Phenosphere-Next 150 4.60 mm monolithic column with Sewrity Guard Cartridge (4 2.0 mm) precolumn (Phenomenex, Torrance, CA, USA). Measurements were performed at excitation/emission wavelengths of 290/390 nm. Mobile phase was a mixture of sulphuric acid (0.015 mol/l) and trichloroacetic acid (TCA, 0.005 mol/l). Isocratic elution was performed at a flow rate of 0.9 ml/min. 2.7. Microbiological analysis The amount of 1.00 ± 0.01 g of mushroom sample was transferred to a bottle containing 99 ml of sterile distilled water, and vigorously shaken (30 s). Next, serial dilution of the samples from 104 to 106 were prepared in sterile distilled water, from which cultures were carried out on the appropriate media. The yeasts and molds number was determined according to PN-90/A-75052.08, the coli index was evaluated in the LPB broth (with lactose and bromocresol purple), and the number of lactic acid bacteria was assessed according to PN-90/A-75052/07. The TVC (Total Viable Count) and the amount of spore-forming bacteria (Bacillus) were determined according to PN-EN ISO 4833:2004/Ap1:2005 by culturing the sample diluted 1:1000000, respectively before and after 15 min of boiling. 2.8. Statistical analysis The results of the investigation were analyzed statistically using one e way analysis of variance based on the F e Snedecor test and t e Student test and the least significant difference (LSD) was established at P < 0.05. The Statistica 8.0 (Stat e Soft) program was used for statistical calculations. Linear correlation coefficiency between the results of antioxidant compounds and antioxidant activity values was also established. 3. Results and discussion 3.1. Proximate composition The chemical composition of culinary products made from fresh S. luteus is given in Table 1. 1 kg fresh mushrooms contained 45.4 g dry matter, of which total carbohydrates comprised 50%, crude proteins 25%, ash 18% and fat 7%. The low content of energy components in fresh mushrooms was reflected in their low calorific
value (0.66 MJ/kg). Compared with other forest mushrooms, S. luteus had low dry weight, while the proportions of individual components were similar to those in Boletus edulis, Cantharellus cibarius and Romaria botrytis (Manzi, Marconi, Aguzzi & Pizzoferrato, 2004; Pereira, Barros, Martins, & Ferreira, 2012). During culinary treatment, part of the moisture content evaporated and mushrooms were saturated in rape seed oil, resulting in a significant increase in dry weight and its components, as well as in calorific value. The greatest change was in fat content, which increased 35-fold after culinary processing. Levels of the remaining components increased by 12e30%, with calorific value rising to 45.5e46.7 MJ/kg. Product NB contained significantly more ash but less total carbohydrates than product BL, which is explained by the fact that blanching (product BL) resulted in the loss of soluble constituents.
3.2. Antioxidant compounds Total polyphenols and flavonoids are given in Table 2. 1 kg dry mass of S. luteus contained 8.16 g total polyphenols expressed as gallic acid equivalents (GAE) and 3.69 g total flavonoids (as (þ)-catechin). According to the available literature, Suillus species are rich in polyphenols compared with other mushrooms. Pereira et al. (2012) determined 58 mg polyphenols (expressed as GAE) and 33 mg flavonoids (expressed as (þ)-catechin equivalents) in 1 g of methanol extract of Suillus variegatus mushrooms. Culinary treatment resulted in decreases in total polyphenols and total flavonoids of 46e61% and 77e81% respectively. Phenolic compounds show a marked tendency to degrade during thermal processing; considerable losses (up to 70%) have been recorded in many vegetables during cooking or frying in small amounts of oil (Kalogeropoulos et al., 2007; Murcia et al., 2009). Preliminary treatment can also be a factor in such losses. Mushrooms exhibit high phenol oxidase and phenol peroxidase activity, which causes polyphenols to oxidise when mushroom tissue is broken. Among the mushroom products examined by Barros, Baptista, Correia, Sa Morais, and Ferreira (2007), cooked products had lower levels of polyphenols and flavonoids than dried or frozen products. S. luteus products obtained from blanched mushrooms showed significantly greater losses of total polyphenols and flavonoids than those obtained from raw mushrooms, which was no doubt due to exposure to high temperature and partial leaching of these compounds during blanching. Storage did not result in statistically significant
Table 2 Antioxidant compounds of braised Suillus luteus after culinary treatment and after storage (per kg of dry weight, mean value ± sd, n ¼ 6). Type of product Raw mushrooms Not blanched (NB) Blanched (BL) LSD (P < 0.05) nd e not detected.
Type of storage
Total polyphenols (g GAE)
Total flavonoids (g (þ)-catechin)
Caffeic acid (mg)
Vitamin C (mg)
0 After storage 0 After storage
8.16 ± 4.40 ± 4.35 ± 3.22 ± 3.49 ± 0.212
3.96 ± 0.32 0.90 ± 0.01 0.86 ± 0.08e0.90 ± 0.01 077 ± 0.02 0.72 ± 0.02e0.76 ± 0.05 0.136
51 ± 2 46 ± 1 nd 42 ± 2 nd 2.6
256 ± 12 6±1 nd 13 ± 3 nd 3.5
0.93 0.17 0.14e4.40 ± 0.17 0.14 0.11e3.59 ± 0.14
704
G. Jaworska et al. / LWT - Food Science and Technology 59 (2014) 701e706
Table 3 Tocopherols of braised Suillus luteus (mg per kg of dry weight, mean value ± sd, n ¼ 6). Type of product
Raw mushrooms Not blanched (NB)
Blanched (BL)
Type of storage
0 48 48 96 0 48 48 96
h/20 C h/4 C h/4 C h/20 C h/4 C h/4 C
LSD (P < 0.05)
Vitamin value (a-TE)
Tocopherol
a
g
d
12.7 ± 0.2 230.7 ± 0.9 221.3 ± 0.6 225.5 ± 1.1 237.2 ± 0.6 218.2 ± 3.0 210.0 ± 1.6 217.3 ± 1.1 212.4 ± 1.0 15.47
2.4 ± 0.1 337.1 ± 0.2 329.2 ± 2.1 317.1 ± 3.4 316.4 ± 4.0 327.1 ± 4.4 309.4 ± 7.2 318.2 ± 5.2 314.6 ± 7.6 30.59
nd 4.8 ± 5.1 ± 2.6 ± 2.2 ± 5.0 ± 4.9 ± 1.9 ± 1.9 ± 2.11
Total 0.2 0.3 0.1 0.2 0.1 0.3 0.2 0.1
15.1 ± 0.2 572.7 ± 1.1 555.6 ± 0.23 535.2 ± 3.2 566.0 ± 4.1 551.2 ± 7.6 524.3 ± 7.1 537.4 ± 5.6 548.9 ± 8.2 55.71
12.9 ± 1.1 254.5 ± 1.1 245.0 ± 0.9 247.3 ± 1.7 258.9 ± 1.2 251.0 ± 3.3 241.1 ± 15.1 249.2 ± 1.8 253.9 ± 0.4 6.21
nd e not detected.
changes in total polyphenols and flavonoids regardless of storage time and temperature. UPLC/PDA/TQD/MS analysis of the phenolic acids profile of fresh and braised S. luteus revealed three peaks with retention times: 3.37 s, 4.67 s and 4.77 s. Analysis of UV and MS spectra established that the peaks represented derivatives of caffeic acid, and the fragmentation pattern led to identification of peaks 1 and 2 as glucuronides of caffeic acid. After plotting the calibration curve for caffeic acid, the areas beneath the peaks were read; 1 kg of fresh mushrooms contained 51 mg of sum of caffeic acids derivatives, culinary products 5e9 mg less. Levels of caffeic acid were significantly lower in blanched than in unblanched products. In their analysis of Suillus. collinitus and Suillus mediterraneensis mushrooms, Vaz, et al., (2011) found levels of total phenolic acids ranging from 4.4 to 20.7 mg per kg dry weight; while phenolic acids such as protocatechuic acid, p-hydroxybenzoic acid and cinnamic acid were present. For their part, Riberio et al. (2008) found p-hydroxybenzoic acid present in Suillus granulatus mushrooms. Bernas, Jaworska, and Lisiewska (2006) stated that average levels of vitamin C in mushrooms do not exceed 70 mg per kg edible parts. Vitamin C content in the culinary products examined is given in Table 2. 1 kg dry weight of mushrooms was found to contain 256 mg vitamin C. Valentao et al. (2005) were of the opinion that Suillaceae contain more ascorbic acid than any other mushrooms, finding average levels of 520 mg vitamin C per kg fresh weight of S. collinitus mushrooms. Pereira et al. (2012), on the other hand, recorded 60 mg ascorbic acid per kg dry weight of Suillus verigatus mushrooms. Braising resulted in virtually total (95e98%) loss of vitamin C; any traces remaining after braising disappeared completely after 48-h storage regardless of the storage temperature. As a mixture of tocopherols, vitamin E is the most important antioxidant in the lipid fraction of edible mushrooms (Heleno, Barros, Sousa, Martins, & Ferreira, 2010). The presence of a- and g-tocopherols was detected in fresh S. luteus mushrooms, whereas
there was no evidence of b- and d-tocopherols. Total tocopherols were 15.1 mg per kg dry weight, of which a-tocopherol comprised 84%. Vitamin E activity was determined at 12.9 mg a-TE. Compared with other mushroom species examined by Heleno et al. (2010) and Pereira et al. (2012), mushrooms of the Suillaceae family contained substantial amounts of tocopherols, although d-tocopherol was not detected in any of the species examined. S. verigatus was found to contain 1.5 mg tocopherol per kg fresh weight, 95% of which was gtocopherol (Heleno et al., 2010), whereas S. collinitus contained 8.8 mg tocopherols per kg dry weight, consisting of g-tocopherol (63%), b- tocopherol (25%) and a small amount of a-tocopherol (Pereira et al., 2012). Braising S. luteus mushrooms resulted in 36- and 20-fold increases in tocopherols and vitamin E activity respectively due to the use of rape seed oil, a rich source of tocopherols (Table 3). According to Lechner, Reiter, and Lorbeer (1999), rape seed oil contains 0.37 g tocopherols per kg, comprising of g-tocopherol (59%), a-tocopherol (36%) and d-tocopherol (5%); b- tocopherol was not found. The results for tocopherol content in culinary products are therefore in line with what would be expected given the composition of rape seed oil used in braising; for the same reason, no differences were found in tocopherol levels between blanched and unblanched braised products. It can be assumed that braising did not cause any loss of tocopherols since these compounds are very thermally stable (Rossi, Alamprese, & Ratti, 2007). 100 g fried S. luteus mushrooms contained approximately twice the amount of tocopherol as a portion of cooked spinach (Kim, Giraud, & Driskell, 2007). There were slight fluctuations in tocopherol content and vitamin E activity during storage of culinary products obtained from S. luteus. 3.3. Antioxidant activity The antioxidant profile of culinary products obtained from S. luteus is given in Table 4. Antioxidant activity per kg dry weight of S.
Table 4 Antioxidant activity of braised Suillus luteus (per kg of dry weight, mean value ± sd, n ¼ 6). Type of product Raw mushrooms Not blanched (NB)
Blanched (BL)
LSD (P < 0.05)
Type of storage
0 48 48 96 0 48 48 96
h/20 C h/4 C h/4 C h/20 C h/4 C h/4 C
Antioxidant activity against ABTS (mmol TE)
Antioxidant activity against DPPH (mmol TE)
Ferric reducing/antioxidant potential e FRAP (mmol Fe2þ)
7.78 ± 2.37 ± 1.97 ± 2.31 ± 2.40 ± 1.82 ± 1.42 ± 1.87 ± 1.81 ± 0.112
3.48 ± 0.91 ± 0.91 ± 0.93 ± 0.77 ± 0.78 ± 0.70 ± 0.75 ± 0.76 ± 0.101
9.15 ± 2.74 ± 2.74 ± 2.83 ± 2.85 ± 2.27 ± 2.33 ± 2.38 ± 2.37 ± 0.322
0.10 0.05 0.05 0.13 0.03 0.03 0.10 0.06 0.07
0.20 0.12 0.07 0.03 0.06 0.02 0.04 0.07 0.02
0.28 0.06 0.05 0.09 0.17 0.04 0.09 0.07 0.05
G. Jaworska et al. / LWT - Food Science and Technology 59 (2014) 701e706
luteus measured by the ABTS and DPPH methods was 7.78 and 3.48 mmol TE respectively, using the FRAP method it was 9.15 mmol Fe2þ. Compared with other mushroom species, Suillaceae exhibit moderate antioxidant activity as determined by DPPH assay and b-carotene bleaching (Heleno et al., 2010; Pereira et al., 2012). Culinary treatment of S. luteus mushrooms resulted in substantial reductions in antioxidant activity, ranging from 70 to 74% and 75e77% in unblanched and blanched products respectively. Reductions of 0%e95% have been recorded in cooked or fried vegetables, the degree of loss depending on the raw material and the time and method of treatment (Kalogeropoulos et al., 2007; Murcia et al., 2009). The greater reduction in antioxidant activity in blanched compared with unblanched products correlates with the greater loss of antioxidant components in the former. Slight fluctuations in antioxidant activity were observed in culinary products during storage. Statistically significant decreases (17e22%) were noted in products stored for 48 h at 20 C (ABTS method), and in product NB stored for 96 h at 4 C (DPPH method). In the culinary products examined there was a strong linear correlation between total polyphenol content and antioxidant activity measured using the three methods (correlation coefficient r ¼ 0.93e0.95), and between total flavonoids and antioxidant activity (r ¼ 0.98e0.99). No positive correlation was found between tocopherol content and antioxidant activity.
705
Table 6 Microbiology of braised Suillus luteus after storage. Type of product After 48 h at 20 C Not blanched (NB) Blanched (BL) After 96 h at 4 C Not blanched (NB) Blanched (BL)
Total viable count (TVC)
Yeast and molds
Lactic acid bacteria
E.coli
Bacillus
þþþ þþþ
þ þ
þþ þþ
Ø Ø
þ þ
Ø Ø
Ø Ø
Ø Ø
Ø Ø
Ø Ø
Ø e no growth. þ e below 104 cfu per g. þþ e between 104 e 106 cfu per g. þþþ e over 106 cfu per g.
20e56% and 13e45% respectively in B. edulis when blanched in various solutions. Storage resulted in further losses of some B-group vitamins in S. luteus products. After cold storage, reductions were observed in the levels of thiamin (7e45%), riboflavin (5e65%), niacin (33e44%) and vitamin B6 (1e15%) compared with levels immediately after braising. Losses of vitamins B1 and B2 after 48 h of storage were similar regardless of storage temperature. Levels of vitamin B3, on the other hand, did not change, while vitamin B6 content, especially that of pyridoxine, actually increased in products stored at room temperature. This was probably due to the growth of microorganisms capable ski & Py of synthesising this compound (Moszczyn c, 1998). Jaworska and Bernas (2009) reported 30e43% losses of vitamin B1 but no loss of vitamin B2 in frozen B. edulis stored for 12 months.
3.4. B complex vitamins Levels of B-group vitamins in fresh S. luteus mushrooms and the culinary products obtained from them are given in Table 5. 1 kg (dry weight) of fresh mushrooms contained 11.2 mg, 38.8 mg, 348 and 5.82 mg of B1, B2, B3 and B6 respectively. Edible mushrooms are considered a good source of thiamin and riboflavin, with levels exceeding those found in edible plants (Bernas et al., 2006; Mattila et al., 2001). Pleurotus ostreatus ranks among the species most abundant in vitamin B1 (0.9e4.25 mg in 100 g dry weight), whereas levels in B. edulis and Agaricus bisporus are 1.25 mg and 0.56 respectively (Bernas et al., 2006). Levels of Riboflavin in 100 g (dry weight) of mushrooms vary from 0.90 mg in shii-take to 9.36 mg in B. edulis. Mattila et al. (2001) found high levels of niacin (43e53 mg per 100 g dry weight) in cultivated mushrooms: A. bisporus and P. ostreatus. Information on vitamin B6 levels in edible mushrooms is lacking. Braising S. luteus mushrooms resulted in significant losses of most B-group vitamins: 46e53% in thiamin, 78% in riboflavin and 51e69% in vitamin B6. Losses of vitamin B3, on the other hand, were 0e13%, which was not a statistically significant difference. Levels of vitamins B1 and B6 were significantly lower in blanched than in unblanched products; however, riboflavin and niacin levels did not vary according to the type of pretreatment applied. Jaworska and Bernas (2009) noted reductions in levels of vitamins B1 and B2 of
3.5. Microbiological analysis There is scant information available on the microbiological quality of mushrooms, fresh or processed. Microbiological analysis of culinary products was carried out after 48 h of storage, on the assumption that fried products would not be consumed later than this period. The results are given in Table 6. After 48 h storage at room temperature, braised S. luteus was not suitable for consumption. In all samples, total viable count exceeded that allowed by Polish Public Health Regulations (27 December 2000). Yeasts, moulds and lactic acid bacteria and Bacillus were detected. Products cold-stored for 96 h, on the other hand, showed no microbial presence in the dilutions used for analysis, making them safe for consumption. Blanching had no effect on reducing microbial contamination in products after storage. 4. Conclusion The present work describes the properties of fresh and culinary processed S. luteus. Compared with other species of mushroom, the dry weight of fresh S. luteus is low but levels of antioxidant
Table 5 Vitamins B of braised Suillus luteus (per kg of dry weight, mean value ± sd, n ¼ 6). Type of product
Raw mushrooms Not blanched (NB)
Blanched (BL)
LSD (P < 0.05) nd e not detected.
Type of storage
B1 (mg)
B2 (mg)
B3 (mg)
0 48 48 96 0 48 48 96
11.2 ± 1.6 6.0 ± 0.7 4.3 ± 0.4 4.4 ± 0.2 3.9 ± 0.3 5.3 ± 0.1 3.5 ± 0.0 5.0 ± 0.1 2.9 ± 0.7 0.12
38.8 ± 3.1 8.5 ± 0.9 6.4 ± 1.0 7.8 ± 0.3 3.0 ± 0.2 8.6 ± 0.0 7.2 ± 0.5 8.0 ± 0.7 8.2 ± 0.5 0.36
348 ± 302 ± 322 ± 203 ± 186 ± 347 ± 345 ± 223 ± 195 ± 51.6
h/20 C h/4 C h/4 C h/20 C h/4 C h/4 C
12 8 5 3 4 3 4 5 5
B6 Pyridoxine (mg)
Pyridoxiamine (mg)
Pyridoxal (mg)
Sum (mg)
nd 44 ± 48 ± 41 ± 39 ± nd 43 ± nd nd 1.3
3.5 ± 2.6 ± 4.4 ± 2.4 ± 2.3 ± 1.7 ± 5.0 ± 1.7 ± 1.7 ± 0.66
2.4 ± 1.7 ± 1.4 ± 1.1 ± 0.9 ± 1.1 ± 0.9 ± 0.9 ± 0.8 ± 0.32
5.8 ± 2.9 ± 4.6 ± 2.5 ± 2.4 ± 1.8 ± 5.1 ± 1.8 ± 1.8 ± 0.71
1 2 1 3 1
0.2 0.1 0.1 0.1 0.0 0.0 0.1 0.1 0.0
0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.1
0.2 0.1 0.1 0.1 0.2 0.1 0.2 0.1 0.1
706
G. Jaworska et al. / LWT - Food Science and Technology 59 (2014) 701e706
compounds and B-group vitamins are high. On the basis of this study, braising can be recommended as a method of preparing mushrooms for consumption since it resulted in an increase of calorific value and tocopherol levels. Furthermore, braising did not lead to greater losses of thermolabile compounds than other methods of processing mushrooms and vegetables. For reasons of microbiological safety, culinary products should be cold stored. Acknowledgement This study was financed by the Ministry of Education and Science under a research Project No. N N 312 241739. This study was financed by the University of Agriculture [Project number BM2012/KSiPOW/4732]. References AOAC. (2005). Official methods of analysis (18th ed.). Maryland: AOAC International. Barros, L., Baptista, P., Correia, D. M., Sa Morais, J., & Ferreira, I. C. F. R. (2007). Effects of conservation treatment and cooking on the chemical composition and antioxidant activity of portuguese wild edible mushrooms. Journal of Agricultural and Food Chemistry, 55, 4781e4788. Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of „antioxidant power”: the FRAP assay. Analytical Biochemistry, 239, 70e76. Bernas, E., Jaworska, G., & Lisiewska, Z. (2006). Edible mushrooms as a source of valuable nutritive constituents. Acta Scientarum Polonorum, Technologia Alimentaria, 5(1), 5e20. EN 14122. (2003). Foodstuffs-Determination of vitamin B1 by HPLC. Brussels: CEN. EN 14130. (2003). Foodstuffs e Determination of vitamin C by HPLC. Brussels: CEN. EN 14152. (2003). Foodstuffs-Determination of vitamin B2 by HPLC. Brussels: CEN. EN 14164. (2008). Foodstuffs-Determination of vitamin B6 by HPLC. Brussels: European Committee for Standardization, CEN. EN ISO 4833:2004/Ap1:2005. (2005). Microbiology of foodstuffs and feeds. Brussels: CEN. Heleno, S. A., Barros, L., Sousa, M. J., Martins, A., & Ferreira, I. C. F. R. (2010). Tocopherols composition of Portuguese wild mushrooms with antioxidant capacity. Food Chemistry, 119, 1443e1450. Jaworska, G., & Bernas, E. (2009). The effect of preliminary processing and period of storage on the quality of frozen Boletus edulis (Bull: Fr.) mushrooms. Food Chemistry, 113(4), 936e943. Lechner, M., Reiter, B., & Lorbeer, E. (1999). Determination of tocopherols and sterols in vegetable oils by solid-phase extraction and subsequent capillary gas chromatographic analysis. Journal of Chromatography A, 857(1e2), 231e238. Kalogeropoulos, N., Mylona, A., Chiou, A., Ioannou, M. S., & Andrikopoulos, N. K. (2007). Retention and distribution of natural antioxidants (a-tocopherol,
polyphenols and terpenic acids) after shallow frying of vegetables in virgin olive oil. LWT, 40(6), 1008e1017. Kim, Y. N., Giraud, D. W., & Driskell, J. A. (2007). Tocopherol and carotenoid contents of selected Korean fruits and vegetables. Journal of Food Composition and Analysis, 20(6), 458e465. Manzi, P., Marconi, S., Aguzzi, A., & Pizoferrato, L. (2004). Commercial mushrooms: nutritional quality and effect of cooking. Food Chemistry, 84, 201e206. Mattila, P., Konko, K., Eurola, M., Philava, J. M., Astola, J., Vahteristo, L., et al. (2001). Contents of vitamins, mineral elements, and some phenolis compounds in cultivated mushrooms. Journal of Agriculture and Food Chemistry, 49, 2343e2348. ski, P., & Py Moszczyn c, R. (1998). Vitamin biochemistry: B-Complex vitamins and coenzymes. Warsaw: Polish Scientific Publishing, ISBN 8301125012. Murcia, M. A., Jiminez, A. M., & Martinez-Tome, M. (2009). Vegetables antioxidant losses during industrial processing and refrigerated storage. Food Research International, 42(8), 1046e1052. ski, J., Kolniak-Ostek, J., & Wojdyło, A. (2012). Characterization and content Oszmian of flavonol derivates of Allium ursinum L. plant. Journal of Agriculture and Food Chemistry, 61, 176e184. Pekkarinen, S. S., Heinonen, I. M., & Hopia, A. I. (1999). Flavonoids, quercetin, myricetin, kaemferol and (þ) catechin and antioxidants in methyl linoleate. Journal of the Science of Food and Agriculture, 79(4), 499e506. Pereira, E., Barros, L., Martins, A., & Ferreira, I. C. F. R. (2012). Towards chemical and nutritional inventory of Portuguese wild edible mushrooms in different habitats. Food Chemistry, 130(2), 394e403. PN-90/A-75052.07-08. (2008). Processed fruits, vegetables and meats. Methods of microbiological analysis. Enumeration of yeasts and molds. Enumeration of lactic acid bacteria. Warsaw: Polish Normalization Committee (in Polish). Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 26, 1231e1237. Regulation of Polish Minister of Public Health. (27 December 2000). On the list of acceptable amounts of additives and other foreign substances added to foods or drugs, and contaminants that may be present in foodstuffs or stimulants. Act No. 9, pos. 72. Reis, F. S., Barros, L., Martins, A., & Ferreira, I. C. F. R. (2012). Chemical composition and nutritional value of the most widely appreciated cultivated mushrooms: an inter-species comparative study. Food and Chemical Toxicology, 50(2), 191e197. Ribeiro, B., Lopes, R., Andrade, P. B., Seabra, R. M., Goncalves, R. F., Baptista, P., et al. (2008). Comparative study of phytochemicals and antioxidant potential of wild edible mushroom caps and stipes. Food Chemistry, 110, 47e56. Rossi, M., Alamprese, C., & Ratti, S. (2007). Tocopherols and tocotrienols as free radical-scavengers in refined vegetable oils and their stability during deep-fat frying. Food Chemistry, 102(3), 812e817. Valentao, P., Lopes, G., Valentae, M., Barbosa, P., Andrade, P., Silva, B., et al. (2005). Quantitation of nine organic acids in wild mushrooms. Journal of Agricultural and Food Chemistry, 53, 3626e3630. Vaz, J. A., Barros, L., Martins, A., Morais, J. S., Vasconcelos, M. H., & Ferreira, I. C. F. R. (2011). Phenolic profile of seventeen Portuguese wild mushrooms. LWT, 44, 343e346.
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Vitamins, phenolics and antioxidant activity of culinary prepared Suillus luteus (L.) Roussel mushroom _ a, *, Emilia Bernas a, Aleksandra Skrzypczak a, Grazyna Jaworska a, Krystyna Pogon Ireneusz Kapusta b a b
Department of Raw Material and Processing of Fruit and Vegetables, University of Agriculture in Cracow, Balicka Street 122, 30-149 Cracow, Poland w, Rejtana 16 c Street, 35-959 Rzeszo w, Poland Department of Technology and Analysis of Plant Derived Products, University of Rzeszo
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2014 Received in revised form 18 July 2014 Accepted 25 July 2014 Available online 6 August 2014
Unblanched and blanched Suillus luteus (L.) Roussel mushrooms were braised to consumption consistency with rape seed oil (100 g/kg) and NaCl (5 g/kg) and 2 types of culinary product were obtained. Evaluation was carried out on fresh mushrooms, as well as mushroom products as follows: unstored; stored at 20 C for 48 h; and stored at 4 C for 48 and 96 h. Braising led to increases in dry matter, basic nutritional components and tocopherols, and decreases in: total polyphenols (46e61%), total flavonoids (71e81%), vitamin C (71e81%), thiamine (71e81%), riboflavin (78%), vitamin B3 (0e13%), vitamin B6 (13 e70%) and antioxidant activity using ABTS, DPPH and FRAP assays (70e77%). Storage led to further losses of vitamin C and B-group vitamins. Products stored for 48 h at room temperature showed high total viable counts as well as the presence of yeasts, lactic acid bacteria and Bacillus strains; however, coldstored products showed no microbiological contamination. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Braising Edible mushrooms Nutrients Nutraceuticals
1. Introduction There has recently been increased interest in the consumption of forest mushrooms as awareness of their composition and health promoting properties has grown. Fresh mushrooms are rich in vitamins, particularly B, D and E, and in bioactive compounds such as polyphenols and carotenoids. They are also a good source of dietary fibre, mineral salts and fairly high quality protein. Moreover, forest mushrooms have a unique flavour, adding to the attraction for consumers (Jaworska & Bernas, 2009; Reis, Barros, Martins, & Ferreira, 2012; Ribeiro et al., 2008). Suillus luteus (L.) Roussel mushrooms are widely consumed in central Europe, particularly in Poland, Czech Republic and Germany. Their slimy skin requires peeling prior to thermal treatment, which usually consists of stir-frying followed by braising in their own juices. Data on composition and properties of S. luteus is lacking. Thermal treatment affects the nutritional value of food, in particular causing losses in constituents such as vitamins, unsaturated fatty acids and mineral compounds. On the positive side, it improves the digestibility of proteins, causes starch to paste and produces desirable sensory traits through the Maillard reaction (Kalogeropoulos, Mylona, Chiou, Ioannou & Anrikopoulos, 2007; * Corresponding author. Tel.: þ48 12 6624754; fax: þ48 12 6624757. ). E-mail address: [email protected] (K. Pogon http://dx.doi.org/10.1016/j.lwt.2014.07.040 0023-6438/© 2014 Elsevier Ltd. All rights reserved.
Murcia, Jiminez & Martinez-Tome, 2009). S. luteus (L.) Roussel mushrooms have not been the subject of detailed analysis in the literature. The aim of this work was to determine the effect of culinary treatment and different storage conditions on the basic chemical composition and levels of biologically active compounds such as polyphenols and vitamins in S. luteus (L.) Roussel mushrooms. 2. Material and methods 2.1. Chemicals All solevts were of HPLC analytical grade and were procured ˛ skie, Poland). Standards and reagents from Chempur (Piekary Sla such as Folin-Ciocalteu reagent, ABTS, DPPH, TPTZ, gallic acid, (þ)-catechin, Trolox, tocopherols, L-ascorbic acid and B-group vitamins were procured from Sigma Chemical Co. (St.Louis, MO, USA). For microbiological analysis special culture media supplied by Merck (Darmstadt, Germany) were used. Triple distilled and HPLC purified water was employed for the entire study. 2.2. Material The material examined comprised fresh S. luteus (L.) Roussel mushrooms (hereafter referred to as S. luteus) with caps 4e8 cm in
702
G. Jaworska et al. / LWT - Food Science and Technology 59 (2014) 701e706
diameter, together with culinary products obtained from them. The mushrooms were purchased from an approved supplier of forest mushrooms and were processed approximately 10 h after harvest. After sorting, mushroom were rinsed in cold water, had their caps peeled and were cut into 1 cm cubes. Half the batch was blanched in water at 98 C for 90 s. After preliminary treatment, mushrooms were braised under cover with a small amount (100 g/ kg) of 100% pure rape seed oil for 10 min. Two types of culinary product were thus obtained: unblanched mushrooms with oil (100 g/kg) and salt (5 g/kg) e product NB; and blanched products with oil (100 g/kg) and salt (5 g/kg) e product BL. The following amounts of products were obtained from 1000 g raw material (mushrooms and oil) after culinary processing: 750 g (product NB) and 870 g (product BL). After braising, the products were packed in containers and stored for 48 h at 20 C, and for 48 and 96 h at 4 C. Analysis was performed on: fresh mushrooms; unstored braised products; and braised products stored for 48 h at 20 C, and for 48 and 96 h at 4 C. The entire braised products were analysed. Two independent replications of products were obtained and analysis was carried out on every product in three replications (n ¼ 6). 2.3. Proximate composition The proximate composition of fresh mushrooms and culinary products was determined according to the AOAC. (2005) method. The dry matter was estimated by drying at 105 C (930.04), the crude protein (N 4.38) was estimated by the Kjeldahl method (978.04), the crude fat was determined by extracting a sample with diethyl ether in a Soxhlet apparatus (920.39) and the ash content was determined by incineration at 485 C (920.05). Total carbohydrates were calculated by difference using the formula: Total carbohydrates ¼ (g dry weight) e (g crude protein) e (g fat) e (g mineral salts). The energy value was calculated using the formula: Energy (kJ) ¼ 17 (g raw protein) þ 15 (g total carbohydrates) þ 38 (g fat). 2.4. Antioxidants Analysis of total polyphenols and total flavonoids was performed in methanolic extracts (800 g/kg) acidified with HCl (5 g/kg) by spectrophotometry using the Folin-Ciocalteu reagent and the solutions of AlCl3 and NaNO2 The content of these compounds was determined according to the standard curves prepared for the gallic acid and (þ)-catechin respectively. Absorbance was measured at l ¼ 675 nm for total polyphenols and at l ¼ 510 nm for total flavonoids. Phenolic acids were analyzed using UPLC/PDA/TDQ/MS proce ski, Kolniak-Ostek, and Wojdyło (2012). dure according to Oszmian Lyophilized mushroom samples were extracted with MeOH (700 g/ kg) at 100 C in an accelerated solvent extractor (Speed Extractor E916 BÜCHI, Flawil, Switzerland) at a working pressure of 100 bar. Structural information and general phenolic profiles were gathered using a Waters Aquity UPLC system (Waters Associates, Milford, MA) consisting of binary solvent manager, sample manager, PDA detector and triple quadrupole detector (TQD) operating in the negative electrospray mode. Ion source parameters were as follows: cone voltage 35 V, capillary voltage 3 kV, extractor 3 V, RF lens 100 mV, source temperature 120 C, desolvation temperature 350 C, desolvation gas flow 222 ml/s, cone gas flow 29 ml/s, and collision gas flow 0.005 ml/s. Collision cell parameters were as follows: entrance -2, exit 0.5, and collision energy 22 eV. Parameters of quadrupole 1 were set to achieve maximal mass resolution: both LM and HM resolutions were set to 15, and ion energy was set to 0.8. Phenolic acids and flavonoids were separated on a 100 mm
2.1 mm i.d., 1.7 mm Acquity BEH column (Waters Associates, Milford, MA) using the following linear, 8.5 min gradient from 80 to 100% of solvent B (acetonitrile solution (400 g/kg) containing 1 g/kg formic acid) in solvent A (water containing 1 g/kg formic acid) with a flow of 0.35 ml/min. Vitamin C content was established according to HPLC methods EN 14130:2003 (2003). The analysis was carried out on a Thermo Scientific DIONEX ULTIMATE 3000 UHPLC chromatograph with DAD Detector and Chromeleon software (Waltham, MA, USA). Sample was injected into a Onyx Monolithic C 18 column (100 4.6 mm) with CHO-7649 precolum (Phenomenex, Torrance, CA, USA). The isocratic elution was carried out using 0.1 mol/l phosphoric acid, the flow rate was 1 ml/min. The absorbance was monitored at 254 nm. Tocopherol content was established using a normal phase HPLC. The analysis was carried out on a Merck HITACHI (Tokyo, Japan) chromatograph with on-line degasser L-7612, Programmable Autosampler L-7250, pump L-7100, fluorescent detector FL Detector L-7480, Interface D-7000 and D-7000 HPLC e System e Manager (HSM) software. Sample was injected into a LUNA 5 mm NH2 250 4.6 column with AJO-4302 precolum (Phenomenex, Torrance, CA, USA). The isocratic elution was carried out using mixture of n-hexane and 2-propanol (95:5) at flow rate of 2.5 ml/min. The wavelengths of excitation/emission were 290/330. a-, b-, g- and dtocopherol content was established using an external standards. Vitamin E activity was calculated as a a-tocopherol equivalents (aTE) per kg of dry weight. Vitamin E activity ¼ 1 atocopherol þ 0.5 b-tocopherol þ 0.1 g-tocopherol þ 0.03 d e tocopherol. 2.5. Antioxidant activity Antioxidant activity was determined in methanol extracts (800 g/kg) prepared with heat treatment against the DPPH radical (Pekkarinen, Heinonen, & Hopia, 1999) and against ABTS radical cation (Re et al., 1999), as well as by the Ferric Reducing/Antioxidant Power method (Benzie & Strain, 1996). The value of the antioxidant activity was determined according to the standard curve prepared for Trolox Eqivalent for ABTS and DPPH and to the standard curve prepared for iron ions (II) for FRAP method. 2.6. B-group vitamins Vitamin B1 and B2 content was established according to HPLC methods EN 14122:2003 (2003) and EN 14152:2003 (2003) respectively. Thiamine and riboflavin were detected using a Merck HITACHI (Tokyo, Japan) liquid chromatograph with fluorescence detector. Analysis was carried out on an Onyx Monolithic C 18 column (100 4.6 mm) with CHO-7649 precolum (Phenomenex, Torrance, CA, USA) and was conducted at excitation/emission wavelengh 360/503 nm. Elution was carried out using linear 12 min gradient from 12% to 100% of solvent B (acetonitrile) in solvent A (water) with a flow of 1 ml/min. Vitamin B3 content was established using a normal phase HPLC using a Merck liquid chromatograph with UV/Vis detector (L-7420, Merck HITACHI, Tokyo, Japan). The analysis was carried out on a LUNA 5 mm NH2 250 4.6 column with AJO-4302 precolum (Phenomenex, Torrance, CA, USA). Niacin was determined at a wavelength of 220 nm. Isocratic elution at flow rate of 0.1 ml/min was performed. Mobile phase consisted of mixture (1:99) of methanol and Pic-A (tetrabutylammonium hydrogen sulphate) solution in water (0.005 mol/l). Vitamin B6 content was determined using the chromatographic method (EN 14164, 2008). The analysis was carried out on a Merck HITACHI (Tokyo, Japan) liquid chromatograph fitted with a
G. Jaworska et al. / LWT - Food Science and Technology 59 (2014) 701e706
703
Table 1 Proximate composition of braised Suillus luteus (g or MJ per kg of fresh weight, mean value ± sd, n ¼ 6). Type of product
Dry weight
Proteins
Fat
Total carbohydrates
Ash
Energy (MJ)
Raw mushrooms Not blanched (NB) Blanched (BL) LSD (P < 0.05)
45.4 ± 1.3 157 ± 11 162 ± 13 e
11.4 ± 0.3 14.7 ± 1.4 14.8 ± 1.6 5.54
3.2 ± 0.1 109 ± 3 110 ± 4 e
22.5 ± 0.5 25.2 ± 0.6 27.9 ± 0.9 0.18
8.3 ± 0.2 9.6 ± 0.2 8.7 ± 0.1 0.11
0.66 ± 0.04 4.55 ± 0.04 4.67 ± 0.04 0.008
fluorescence detector, on a Phenosphere-Next 150 4.60 mm monolithic column with Sewrity Guard Cartridge (4 2.0 mm) precolumn (Phenomenex, Torrance, CA, USA). Measurements were performed at excitation/emission wavelengths of 290/390 nm. Mobile phase was a mixture of sulphuric acid (0.015 mol/l) and trichloroacetic acid (TCA, 0.005 mol/l). Isocratic elution was performed at a flow rate of 0.9 ml/min. 2.7. Microbiological analysis The amount of 1.00 ± 0.01 g of mushroom sample was transferred to a bottle containing 99 ml of sterile distilled water, and vigorously shaken (30 s). Next, serial dilution of the samples from 104 to 106 were prepared in sterile distilled water, from which cultures were carried out on the appropriate media. The yeasts and molds number was determined according to PN-90/A-75052.08, the coli index was evaluated in the LPB broth (with lactose and bromocresol purple), and the number of lactic acid bacteria was assessed according to PN-90/A-75052/07. The TVC (Total Viable Count) and the amount of spore-forming bacteria (Bacillus) were determined according to PN-EN ISO 4833:2004/Ap1:2005 by culturing the sample diluted 1:1000000, respectively before and after 15 min of boiling. 2.8. Statistical analysis The results of the investigation were analyzed statistically using one e way analysis of variance based on the F e Snedecor test and t e Student test and the least significant difference (LSD) was established at P < 0.05. The Statistica 8.0 (Stat e Soft) program was used for statistical calculations. Linear correlation coefficiency between the results of antioxidant compounds and antioxidant activity values was also established. 3. Results and discussion 3.1. Proximate composition The chemical composition of culinary products made from fresh S. luteus is given in Table 1. 1 kg fresh mushrooms contained 45.4 g dry matter, of which total carbohydrates comprised 50%, crude proteins 25%, ash 18% and fat 7%. The low content of energy components in fresh mushrooms was reflected in their low calorific
value (0.66 MJ/kg). Compared with other forest mushrooms, S. luteus had low dry weight, while the proportions of individual components were similar to those in Boletus edulis, Cantharellus cibarius and Romaria botrytis (Manzi, Marconi, Aguzzi & Pizzoferrato, 2004; Pereira, Barros, Martins, & Ferreira, 2012). During culinary treatment, part of the moisture content evaporated and mushrooms were saturated in rape seed oil, resulting in a significant increase in dry weight and its components, as well as in calorific value. The greatest change was in fat content, which increased 35-fold after culinary processing. Levels of the remaining components increased by 12e30%, with calorific value rising to 45.5e46.7 MJ/kg. Product NB contained significantly more ash but less total carbohydrates than product BL, which is explained by the fact that blanching (product BL) resulted in the loss of soluble constituents.
3.2. Antioxidant compounds Total polyphenols and flavonoids are given in Table 2. 1 kg dry mass of S. luteus contained 8.16 g total polyphenols expressed as gallic acid equivalents (GAE) and 3.69 g total flavonoids (as (þ)-catechin). According to the available literature, Suillus species are rich in polyphenols compared with other mushrooms. Pereira et al. (2012) determined 58 mg polyphenols (expressed as GAE) and 33 mg flavonoids (expressed as (þ)-catechin equivalents) in 1 g of methanol extract of Suillus variegatus mushrooms. Culinary treatment resulted in decreases in total polyphenols and total flavonoids of 46e61% and 77e81% respectively. Phenolic compounds show a marked tendency to degrade during thermal processing; considerable losses (up to 70%) have been recorded in many vegetables during cooking or frying in small amounts of oil (Kalogeropoulos et al., 2007; Murcia et al., 2009). Preliminary treatment can also be a factor in such losses. Mushrooms exhibit high phenol oxidase and phenol peroxidase activity, which causes polyphenols to oxidise when mushroom tissue is broken. Among the mushroom products examined by Barros, Baptista, Correia, Sa Morais, and Ferreira (2007), cooked products had lower levels of polyphenols and flavonoids than dried or frozen products. S. luteus products obtained from blanched mushrooms showed significantly greater losses of total polyphenols and flavonoids than those obtained from raw mushrooms, which was no doubt due to exposure to high temperature and partial leaching of these compounds during blanching. Storage did not result in statistically significant
Table 2 Antioxidant compounds of braised Suillus luteus after culinary treatment and after storage (per kg of dry weight, mean value ± sd, n ¼ 6). Type of product Raw mushrooms Not blanched (NB) Blanched (BL) LSD (P < 0.05) nd e not detected.
Type of storage
Total polyphenols (g GAE)
Total flavonoids (g (þ)-catechin)
Caffeic acid (mg)
Vitamin C (mg)
0 After storage 0 After storage
8.16 ± 4.40 ± 4.35 ± 3.22 ± 3.49 ± 0.212
3.96 ± 0.32 0.90 ± 0.01 0.86 ± 0.08e0.90 ± 0.01 077 ± 0.02 0.72 ± 0.02e0.76 ± 0.05 0.136
51 ± 2 46 ± 1 nd 42 ± 2 nd 2.6
256 ± 12 6±1 nd 13 ± 3 nd 3.5
0.93 0.17 0.14e4.40 ± 0.17 0.14 0.11e3.59 ± 0.14
704
G. Jaworska et al. / LWT - Food Science and Technology 59 (2014) 701e706
Table 3 Tocopherols of braised Suillus luteus (mg per kg of dry weight, mean value ± sd, n ¼ 6). Type of product
Raw mushrooms Not blanched (NB)
Blanched (BL)
Type of storage
0 48 48 96 0 48 48 96
h/20 C h/4 C h/4 C h/20 C h/4 C h/4 C
LSD (P < 0.05)
Vitamin value (a-TE)
Tocopherol
a
g
d
12.7 ± 0.2 230.7 ± 0.9 221.3 ± 0.6 225.5 ± 1.1 237.2 ± 0.6 218.2 ± 3.0 210.0 ± 1.6 217.3 ± 1.1 212.4 ± 1.0 15.47
2.4 ± 0.1 337.1 ± 0.2 329.2 ± 2.1 317.1 ± 3.4 316.4 ± 4.0 327.1 ± 4.4 309.4 ± 7.2 318.2 ± 5.2 314.6 ± 7.6 30.59
nd 4.8 ± 5.1 ± 2.6 ± 2.2 ± 5.0 ± 4.9 ± 1.9 ± 1.9 ± 2.11
Total 0.2 0.3 0.1 0.2 0.1 0.3 0.2 0.1
15.1 ± 0.2 572.7 ± 1.1 555.6 ± 0.23 535.2 ± 3.2 566.0 ± 4.1 551.2 ± 7.6 524.3 ± 7.1 537.4 ± 5.6 548.9 ± 8.2 55.71
12.9 ± 1.1 254.5 ± 1.1 245.0 ± 0.9 247.3 ± 1.7 258.9 ± 1.2 251.0 ± 3.3 241.1 ± 15.1 249.2 ± 1.8 253.9 ± 0.4 6.21
nd e not detected.
changes in total polyphenols and flavonoids regardless of storage time and temperature. UPLC/PDA/TQD/MS analysis of the phenolic acids profile of fresh and braised S. luteus revealed three peaks with retention times: 3.37 s, 4.67 s and 4.77 s. Analysis of UV and MS spectra established that the peaks represented derivatives of caffeic acid, and the fragmentation pattern led to identification of peaks 1 and 2 as glucuronides of caffeic acid. After plotting the calibration curve for caffeic acid, the areas beneath the peaks were read; 1 kg of fresh mushrooms contained 51 mg of sum of caffeic acids derivatives, culinary products 5e9 mg less. Levels of caffeic acid were significantly lower in blanched than in unblanched products. In their analysis of Suillus. collinitus and Suillus mediterraneensis mushrooms, Vaz, et al., (2011) found levels of total phenolic acids ranging from 4.4 to 20.7 mg per kg dry weight; while phenolic acids such as protocatechuic acid, p-hydroxybenzoic acid and cinnamic acid were present. For their part, Riberio et al. (2008) found p-hydroxybenzoic acid present in Suillus granulatus mushrooms. Bernas, Jaworska, and Lisiewska (2006) stated that average levels of vitamin C in mushrooms do not exceed 70 mg per kg edible parts. Vitamin C content in the culinary products examined is given in Table 2. 1 kg dry weight of mushrooms was found to contain 256 mg vitamin C. Valentao et al. (2005) were of the opinion that Suillaceae contain more ascorbic acid than any other mushrooms, finding average levels of 520 mg vitamin C per kg fresh weight of S. collinitus mushrooms. Pereira et al. (2012), on the other hand, recorded 60 mg ascorbic acid per kg dry weight of Suillus verigatus mushrooms. Braising resulted in virtually total (95e98%) loss of vitamin C; any traces remaining after braising disappeared completely after 48-h storage regardless of the storage temperature. As a mixture of tocopherols, vitamin E is the most important antioxidant in the lipid fraction of edible mushrooms (Heleno, Barros, Sousa, Martins, & Ferreira, 2010). The presence of a- and g-tocopherols was detected in fresh S. luteus mushrooms, whereas
there was no evidence of b- and d-tocopherols. Total tocopherols were 15.1 mg per kg dry weight, of which a-tocopherol comprised 84%. Vitamin E activity was determined at 12.9 mg a-TE. Compared with other mushroom species examined by Heleno et al. (2010) and Pereira et al. (2012), mushrooms of the Suillaceae family contained substantial amounts of tocopherols, although d-tocopherol was not detected in any of the species examined. S. verigatus was found to contain 1.5 mg tocopherol per kg fresh weight, 95% of which was gtocopherol (Heleno et al., 2010), whereas S. collinitus contained 8.8 mg tocopherols per kg dry weight, consisting of g-tocopherol (63%), b- tocopherol (25%) and a small amount of a-tocopherol (Pereira et al., 2012). Braising S. luteus mushrooms resulted in 36- and 20-fold increases in tocopherols and vitamin E activity respectively due to the use of rape seed oil, a rich source of tocopherols (Table 3). According to Lechner, Reiter, and Lorbeer (1999), rape seed oil contains 0.37 g tocopherols per kg, comprising of g-tocopherol (59%), a-tocopherol (36%) and d-tocopherol (5%); b- tocopherol was not found. The results for tocopherol content in culinary products are therefore in line with what would be expected given the composition of rape seed oil used in braising; for the same reason, no differences were found in tocopherol levels between blanched and unblanched braised products. It can be assumed that braising did not cause any loss of tocopherols since these compounds are very thermally stable (Rossi, Alamprese, & Ratti, 2007). 100 g fried S. luteus mushrooms contained approximately twice the amount of tocopherol as a portion of cooked spinach (Kim, Giraud, & Driskell, 2007). There were slight fluctuations in tocopherol content and vitamin E activity during storage of culinary products obtained from S. luteus. 3.3. Antioxidant activity The antioxidant profile of culinary products obtained from S. luteus is given in Table 4. Antioxidant activity per kg dry weight of S.
Table 4 Antioxidant activity of braised Suillus luteus (per kg of dry weight, mean value ± sd, n ¼ 6). Type of product Raw mushrooms Not blanched (NB)
Blanched (BL)
LSD (P < 0.05)
Type of storage
0 48 48 96 0 48 48 96
h/20 C h/4 C h/4 C h/20 C h/4 C h/4 C
Antioxidant activity against ABTS (mmol TE)
Antioxidant activity against DPPH (mmol TE)
Ferric reducing/antioxidant potential e FRAP (mmol Fe2þ)
7.78 ± 2.37 ± 1.97 ± 2.31 ± 2.40 ± 1.82 ± 1.42 ± 1.87 ± 1.81 ± 0.112
3.48 ± 0.91 ± 0.91 ± 0.93 ± 0.77 ± 0.78 ± 0.70 ± 0.75 ± 0.76 ± 0.101
9.15 ± 2.74 ± 2.74 ± 2.83 ± 2.85 ± 2.27 ± 2.33 ± 2.38 ± 2.37 ± 0.322
0.10 0.05 0.05 0.13 0.03 0.03 0.10 0.06 0.07
0.20 0.12 0.07 0.03 0.06 0.02 0.04 0.07 0.02
0.28 0.06 0.05 0.09 0.17 0.04 0.09 0.07 0.05
G. Jaworska et al. / LWT - Food Science and Technology 59 (2014) 701e706
luteus measured by the ABTS and DPPH methods was 7.78 and 3.48 mmol TE respectively, using the FRAP method it was 9.15 mmol Fe2þ. Compared with other mushroom species, Suillaceae exhibit moderate antioxidant activity as determined by DPPH assay and b-carotene bleaching (Heleno et al., 2010; Pereira et al., 2012). Culinary treatment of S. luteus mushrooms resulted in substantial reductions in antioxidant activity, ranging from 70 to 74% and 75e77% in unblanched and blanched products respectively. Reductions of 0%e95% have been recorded in cooked or fried vegetables, the degree of loss depending on the raw material and the time and method of treatment (Kalogeropoulos et al., 2007; Murcia et al., 2009). The greater reduction in antioxidant activity in blanched compared with unblanched products correlates with the greater loss of antioxidant components in the former. Slight fluctuations in antioxidant activity were observed in culinary products during storage. Statistically significant decreases (17e22%) were noted in products stored for 48 h at 20 C (ABTS method), and in product NB stored for 96 h at 4 C (DPPH method). In the culinary products examined there was a strong linear correlation between total polyphenol content and antioxidant activity measured using the three methods (correlation coefficient r ¼ 0.93e0.95), and between total flavonoids and antioxidant activity (r ¼ 0.98e0.99). No positive correlation was found between tocopherol content and antioxidant activity.
705
Table 6 Microbiology of braised Suillus luteus after storage. Type of product After 48 h at 20 C Not blanched (NB) Blanched (BL) After 96 h at 4 C Not blanched (NB) Blanched (BL)
Total viable count (TVC)
Yeast and molds
Lactic acid bacteria
E.coli
Bacillus
þþþ þþþ
þ þ
þþ þþ
Ø Ø
þ þ
Ø Ø
Ø Ø
Ø Ø
Ø Ø
Ø Ø
Ø e no growth. þ e below 104 cfu per g. þþ e between 104 e 106 cfu per g. þþþ e over 106 cfu per g.
20e56% and 13e45% respectively in B. edulis when blanched in various solutions. Storage resulted in further losses of some B-group vitamins in S. luteus products. After cold storage, reductions were observed in the levels of thiamin (7e45%), riboflavin (5e65%), niacin (33e44%) and vitamin B6 (1e15%) compared with levels immediately after braising. Losses of vitamins B1 and B2 after 48 h of storage were similar regardless of storage temperature. Levels of vitamin B3, on the other hand, did not change, while vitamin B6 content, especially that of pyridoxine, actually increased in products stored at room temperature. This was probably due to the growth of microorganisms capable ski & Py of synthesising this compound (Moszczyn c, 1998). Jaworska and Bernas (2009) reported 30e43% losses of vitamin B1 but no loss of vitamin B2 in frozen B. edulis stored for 12 months.
3.4. B complex vitamins Levels of B-group vitamins in fresh S. luteus mushrooms and the culinary products obtained from them are given in Table 5. 1 kg (dry weight) of fresh mushrooms contained 11.2 mg, 38.8 mg, 348 and 5.82 mg of B1, B2, B3 and B6 respectively. Edible mushrooms are considered a good source of thiamin and riboflavin, with levels exceeding those found in edible plants (Bernas et al., 2006; Mattila et al., 2001). Pleurotus ostreatus ranks among the species most abundant in vitamin B1 (0.9e4.25 mg in 100 g dry weight), whereas levels in B. edulis and Agaricus bisporus are 1.25 mg and 0.56 respectively (Bernas et al., 2006). Levels of Riboflavin in 100 g (dry weight) of mushrooms vary from 0.90 mg in shii-take to 9.36 mg in B. edulis. Mattila et al. (2001) found high levels of niacin (43e53 mg per 100 g dry weight) in cultivated mushrooms: A. bisporus and P. ostreatus. Information on vitamin B6 levels in edible mushrooms is lacking. Braising S. luteus mushrooms resulted in significant losses of most B-group vitamins: 46e53% in thiamin, 78% in riboflavin and 51e69% in vitamin B6. Losses of vitamin B3, on the other hand, were 0e13%, which was not a statistically significant difference. Levels of vitamins B1 and B6 were significantly lower in blanched than in unblanched products; however, riboflavin and niacin levels did not vary according to the type of pretreatment applied. Jaworska and Bernas (2009) noted reductions in levels of vitamins B1 and B2 of
3.5. Microbiological analysis There is scant information available on the microbiological quality of mushrooms, fresh or processed. Microbiological analysis of culinary products was carried out after 48 h of storage, on the assumption that fried products would not be consumed later than this period. The results are given in Table 6. After 48 h storage at room temperature, braised S. luteus was not suitable for consumption. In all samples, total viable count exceeded that allowed by Polish Public Health Regulations (27 December 2000). Yeasts, moulds and lactic acid bacteria and Bacillus were detected. Products cold-stored for 96 h, on the other hand, showed no microbial presence in the dilutions used for analysis, making them safe for consumption. Blanching had no effect on reducing microbial contamination in products after storage. 4. Conclusion The present work describes the properties of fresh and culinary processed S. luteus. Compared with other species of mushroom, the dry weight of fresh S. luteus is low but levels of antioxidant
Table 5 Vitamins B of braised Suillus luteus (per kg of dry weight, mean value ± sd, n ¼ 6). Type of product
Raw mushrooms Not blanched (NB)
Blanched (BL)
LSD (P < 0.05) nd e not detected.
Type of storage
B1 (mg)
B2 (mg)
B3 (mg)
0 48 48 96 0 48 48 96
11.2 ± 1.6 6.0 ± 0.7 4.3 ± 0.4 4.4 ± 0.2 3.9 ± 0.3 5.3 ± 0.1 3.5 ± 0.0 5.0 ± 0.1 2.9 ± 0.7 0.12
38.8 ± 3.1 8.5 ± 0.9 6.4 ± 1.0 7.8 ± 0.3 3.0 ± 0.2 8.6 ± 0.0 7.2 ± 0.5 8.0 ± 0.7 8.2 ± 0.5 0.36
348 ± 302 ± 322 ± 203 ± 186 ± 347 ± 345 ± 223 ± 195 ± 51.6
h/20 C h/4 C h/4 C h/20 C h/4 C h/4 C
12 8 5 3 4 3 4 5 5
B6 Pyridoxine (mg)
Pyridoxiamine (mg)
Pyridoxal (mg)
Sum (mg)
nd 44 ± 48 ± 41 ± 39 ± nd 43 ± nd nd 1.3
3.5 ± 2.6 ± 4.4 ± 2.4 ± 2.3 ± 1.7 ± 5.0 ± 1.7 ± 1.7 ± 0.66
2.4 ± 1.7 ± 1.4 ± 1.1 ± 0.9 ± 1.1 ± 0.9 ± 0.9 ± 0.8 ± 0.32
5.8 ± 2.9 ± 4.6 ± 2.5 ± 2.4 ± 1.8 ± 5.1 ± 1.8 ± 1.8 ± 0.71
1 2 1 3 1
0.2 0.1 0.1 0.1 0.0 0.0 0.1 0.1 0.0
0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.1
0.2 0.1 0.1 0.1 0.2 0.1 0.2 0.1 0.1
706
G. Jaworska et al. / LWT - Food Science and Technology 59 (2014) 701e706
compounds and B-group vitamins are high. On the basis of this study, braising can be recommended as a method of preparing mushrooms for consumption since it resulted in an increase of calorific value and tocopherol levels. Furthermore, braising did not lead to greater losses of thermolabile compounds than other methods of processing mushrooms and vegetables. For reasons of microbiological safety, culinary products should be cold stored. Acknowledgement This study was financed by the Ministry of Education and Science under a research Project No. N N 312 241739. This study was financed by the University of Agriculture [Project number BM2012/KSiPOW/4732]. References AOAC. (2005). Official methods of analysis (18th ed.). Maryland: AOAC International. Barros, L., Baptista, P., Correia, D. M., Sa Morais, J., & Ferreira, I. C. F. R. (2007). Effects of conservation treatment and cooking on the chemical composition and antioxidant activity of portuguese wild edible mushrooms. Journal of Agricultural and Food Chemistry, 55, 4781e4788. Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of „antioxidant power”: the FRAP assay. Analytical Biochemistry, 239, 70e76. Bernas, E., Jaworska, G., & Lisiewska, Z. (2006). Edible mushrooms as a source of valuable nutritive constituents. Acta Scientarum Polonorum, Technologia Alimentaria, 5(1), 5e20. EN 14122. (2003). Foodstuffs-Determination of vitamin B1 by HPLC. Brussels: CEN. EN 14130. (2003). Foodstuffs e Determination of vitamin C by HPLC. Brussels: CEN. EN 14152. (2003). Foodstuffs-Determination of vitamin B2 by HPLC. Brussels: CEN. EN 14164. (2008). Foodstuffs-Determination of vitamin B6 by HPLC. Brussels: European Committee for Standardization, CEN. EN ISO 4833:2004/Ap1:2005. (2005). Microbiology of foodstuffs and feeds. Brussels: CEN. Heleno, S. A., Barros, L., Sousa, M. J., Martins, A., & Ferreira, I. C. F. R. (2010). Tocopherols composition of Portuguese wild mushrooms with antioxidant capacity. Food Chemistry, 119, 1443e1450. Jaworska, G., & Bernas, E. (2009). The effect of preliminary processing and period of storage on the quality of frozen Boletus edulis (Bull: Fr.) mushrooms. Food Chemistry, 113(4), 936e943. Lechner, M., Reiter, B., & Lorbeer, E. (1999). Determination of tocopherols and sterols in vegetable oils by solid-phase extraction and subsequent capillary gas chromatographic analysis. Journal of Chromatography A, 857(1e2), 231e238. Kalogeropoulos, N., Mylona, A., Chiou, A., Ioannou, M. S., & Andrikopoulos, N. K. (2007). Retention and distribution of natural antioxidants (a-tocopherol,
polyphenols and terpenic acids) after shallow frying of vegetables in virgin olive oil. LWT, 40(6), 1008e1017. Kim, Y. N., Giraud, D. W., & Driskell, J. A. (2007). Tocopherol and carotenoid contents of selected Korean fruits and vegetables. Journal of Food Composition and Analysis, 20(6), 458e465. Manzi, P., Marconi, S., Aguzzi, A., & Pizoferrato, L. (2004). Commercial mushrooms: nutritional quality and effect of cooking. Food Chemistry, 84, 201e206. Mattila, P., Konko, K., Eurola, M., Philava, J. M., Astola, J., Vahteristo, L., et al. (2001). Contents of vitamins, mineral elements, and some phenolis compounds in cultivated mushrooms. Journal of Agriculture and Food Chemistry, 49, 2343e2348. ski, P., & Py Moszczyn c, R. (1998). Vitamin biochemistry: B-Complex vitamins and coenzymes. Warsaw: Polish Scientific Publishing, ISBN 8301125012. Murcia, M. A., Jiminez, A. M., & Martinez-Tome, M. (2009). Vegetables antioxidant losses during industrial processing and refrigerated storage. Food Research International, 42(8), 1046e1052. ski, J., Kolniak-Ostek, J., & Wojdyło, A. (2012). Characterization and content Oszmian of flavonol derivates of Allium ursinum L. plant. Journal of Agriculture and Food Chemistry, 61, 176e184. Pekkarinen, S. S., Heinonen, I. M., & Hopia, A. I. (1999). Flavonoids, quercetin, myricetin, kaemferol and (þ) catechin and antioxidants in methyl linoleate. Journal of the Science of Food and Agriculture, 79(4), 499e506. Pereira, E., Barros, L., Martins, A., & Ferreira, I. C. F. R. (2012). Towards chemical and nutritional inventory of Portuguese wild edible mushrooms in different habitats. Food Chemistry, 130(2), 394e403. PN-90/A-75052.07-08. (2008). Processed fruits, vegetables and meats. Methods of microbiological analysis. Enumeration of yeasts and molds. Enumeration of lactic acid bacteria. Warsaw: Polish Normalization Committee (in Polish). Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 26, 1231e1237. Regulation of Polish Minister of Public Health. (27 December 2000). On the list of acceptable amounts of additives and other foreign substances added to foods or drugs, and contaminants that may be present in foodstuffs or stimulants. Act No. 9, pos. 72. Reis, F. S., Barros, L., Martins, A., & Ferreira, I. C. F. R. (2012). Chemical composition and nutritional value of the most widely appreciated cultivated mushrooms: an inter-species comparative study. Food and Chemical Toxicology, 50(2), 191e197. Ribeiro, B., Lopes, R., Andrade, P. B., Seabra, R. M., Goncalves, R. F., Baptista, P., et al. (2008). Comparative study of phytochemicals and antioxidant potential of wild edible mushroom caps and stipes. Food Chemistry, 110, 47e56. Rossi, M., Alamprese, C., & Ratti, S. (2007). Tocopherols and tocotrienols as free radical-scavengers in refined vegetable oils and their stability during deep-fat frying. Food Chemistry, 102(3), 812e817. Valentao, P., Lopes, G., Valentae, M., Barbosa, P., Andrade, P., Silva, B., et al. (2005). Quantitation of nine organic acids in wild mushrooms. Journal of Agricultural and Food Chemistry, 53, 3626e3630. Vaz, J. A., Barros, L., Martins, A., Morais, J. S., Vasconcelos, M. H., & Ferreira, I. C. F. R. (2011). Phenolic profile of seventeen Portuguese wild mushrooms. LWT, 44, 343e346.