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Vitamin D2 content and antioxidant properties of fruit body and mycelia of edible mushrooms by UV-B irradiation
Journal of Food Composition and Analysis 42 (2015) 38–45
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Original Research Article
Vitamin D2 content and antioxidant properties of fruit body and mycelia of edible mushrooms by UV-B irradiation Shih-Jeng Huang a, Chun-Ping Lin b, Shu-Yao Tsai b,c,* a
Department of Health Food, Chung Chou University of Science and Technology, Yuanlin, Changhua 51003, Taiwan, ROC Department of Health and Nutrition Biotechnology, Asia University, 500, Lioufeng Rd., Wufeng, Taichung 41354, Taiwan, ROC c Department of Nutrition, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan, ROC b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 October 2012 Received in revised form 3 February 2015 Accepted 24 February 2015 Available online 20 March 2015
Our objective was to study the effect of ultraviolet-B (UV-B) light irradiation on the vitamin D2 content of several edible fruit bodies and mycelia and their antioxidant properties. Eleven species of fresh mushroom fruiting bodies, including species from each of the six genera, Agaricus, Agrocybe, Auricularia, Hypsizigus, Lentinula and Pholiota, and five species from Pleurotus genus, were irradiated with UV-B light for 2 h. For three species of mushroom fruiting bodies with excellent vitamin D2 yield, their mycelia were obtained by liquid culture, and subjected to the same time as the UV-B irradiation. Vitamin D2 content of irradiated fruit bodies significantly increased from 0–3.93 to 15.06–208.65 mg/g, of which the amount in golden oyster mushroom increased by a maximum of 204.7 mg/g. Vitamin D2 content in irradiated mycelia of golden oyster, oyster and pink oyster mushrooms increased from 0.28–5.93 to 66.03– 81.71 mg/g, respectively. The three irradiated mycelium polysaccharide contents decreased in a range from 1.3% to 24.6%. Overall, EC50 values of non-irradiated and irradiated fruiting bodies and mycelia were 0.92–4.94, 0.20–6.90 and 0.02–0.84 mg/mL for reducing power, scavenging ability and chelating ability, respectively. Although UV-B irradiation influenced the content of ergothioneine, flavonoids and total phenols, these irradiated samples still contained a sufficient amount of these antioxidant components. ß 2015 Elsevier Inc. All rights reserved.
Keywords: UV-B irradiation Vitamin D2 Ergocalciferol Antioxidant properties Antioxidant components Pleurotus citrinopileatus Pleurotus ostreatus Pleurotus salmoneostramineus Food analysis Food composition
1. Introduction Edible and medicinal mushrooms contain many physiologically active substances, including polysaccharides, triterpenoids, ergosterol, vitamin D and ergothioneine (Kalac, 2013). Vitamin D is important in human nutrition because deficiency in infancy/ childhood can result in rickets, while in adults vitamin D deficiency is associated with osteoporosis (Holick, 2007). The vitamin D receptor has been detected in a broad range of tissues; therefore, it
Abbreviations: UV-B, ultraviolet-B; EC, effective concentration; HPLC, high-performance liquid chromatograph; DPPH, 2,2-diphenyl-1-picrylhydrazyl; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; EDTA, ethylenediaminetetraacetic acid; FB0, fresh fruiting bodies UV-B irradiated for 0 h; FB2, fresh fruiting bodies UV-B irradiated for 2 h; MY0, freshly harvested mycelia UV-B irradiated for 0 h; MY2, freshly harvested mycelia UV-B irradiated for 2 h. * Corresponding author at: Department of Health and Nutrition Biotechnology, Asia University, 500, Lioufeng Rd., Wufeng, Taichung 41354, Taiwan, ROC. Tel.: +886 4 2332 3456x20028; fax: +886 4 2332 1126. E-mail address: [email protected] (S.-Y. Tsai). http://dx.doi.org/10.1016/j.jfca.2015.02.005 0889-1575/ß 2015 Elsevier Inc. All rights reserved.
has been suggested that vitamin D may play a role in cardiovascular and autoimmune diseases and cancer prevention (Mullin and Dobs, 2007; Kikkinen et al., 2009). Natural food sources for vitamin D include certain fish species, egg, milk and mushrooms (Holden et al., 2008). Vitamin D exists as two distinct forms: vitamin D3 (cholecalciferol) is oily fish, egg and red meat, and vitamin D2 (ergocalciferol) is found in yeasts and mushrooms (Shrapnel and Truswell, 2006). Ultraviolet light can be applied to mushrooms to enhance their vitamin D2 content (Jasinghe and Perera, 2005, 2006; Teichmann et al., 2007; Ko et al., 2008; Roberts et al., 2008; Simon et al., 2011; Koyyalamudi et al., 2009, 2011; Kalaras et al., 2012). Most studies on mushroom fruiting bodies try to enhance their vitamin D content. Low vitamin D2 content occurs naturally in many mushrooms, with a high potential for large increases based on the presence of ergosterol by ultraviolet light treatment. However, information about the application of ultraviolet light on mushroom mycelium is not available. Since the mycelium is easily produced as compared to fruiting body and is readily irradiated due to its larger flat surface, it has great potential for the
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development of health foods containing a high content of vitamin D. Using ultraviolet irradiation on mushrooms to increase vitamin D content not only provides another option for people to supplement vitamin D from non-animal sources but also helps vegetarians to maintain vitamin D status. Mushrooms are a good source of beneficial components (such as ergothioneine, total phenols, flavonoids, and polysaccharides) that may be destroyed by UV irradiation, but there are no data. Given the growing use of irradiation to enhance vitamin D, the need to demonstrate an effect on these components is valuable. Eleven species of fresh mushroom fruiting bodies are commercially available in Taiwan, including species from each of the six genera of Agaricus, Agrocybe, Auricularia, Hypsizigus, Lentinula and Pholiota and five species from Pleurotus genus. In addition, limited information about the vitamin D2 content of these mushrooms is available and there have been no reports on the vitamin D2 content of mycelia. We irradiated fresh fruiting bodies and freshly harvested mycelia with ultraviolet light to study its effect on the vitamin D and polysaccharides content. We also studied the antioxidant properties of ethanolic extracts from irradiated fruiting bodies and mycelia, including reducing power, scavenging ability on radicals, and chelating ability on ferrous ions, and compared them to nonirradiated samples. The content of potential antioxidant components (ergothioneine, flavonoids and total phenols) was also determined. 2. Materials and methods 2.1. Fruiting bodies and mycelia Eleven species of fresh mushrooms included Brazilian (Agaricus blazei), popular (Agrocybe cylindracea), wood ear (Auricularia polytricha), hon-shimeji (Hypsizigus marmoreus), shiitake (Lentinula edodes), sticky pholiota (Pholiota nameko), king oyster (Pleurotus eryngii), golden oyster (Pleurotus citrinopileatus), ferulae (Pleurotus ferulae), oyster (Pleurotus ostreatus) and pink oyster (Pleurotus salmoneostramineus) mushrooms were obtained from QYo Bio-Technology Farm, Changhua County, Taiwan (1218E, 248N, 556 m). Immediately after harvest, the whole fresh fruiting bodies (10–15 kg each) were kept at 4 8C in a cooler and transported to the laboratory for irradiation treatment within 1 h. We selected the three highest levels of vitamin D2 of irradiated fruiting bodies, performing cultured mycelia. Three species of mycelia (golden oyster, oyster, and pink oyster mushrooms) cultures were obtained from the inner living tissues of the fruit bodies in potato dextrose
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agar (Difco Laboratories, Sparks, MD, USA) medium at 25 8C. For the production of mycelia, the culture was inoculated into 250-mL flask containing 90 mL of liquid medium and incubated at 25 8C and 100 rpm. The liquid medium (1 L) consisted of 20 g of glucose, 5 g of yeast extract, 0.068 g of KH2PO4, and 2.3 g of MgSO47H2O. After 7 days of incubation, the mycelia were harvested, washed five times with deionised water, and fresh mycelia was obtained by wringing water from 400-mesh cloth. Both fruiting bodies and mycelia were irradiated in fresh and moist state on the day of harvest. 2.2. Irradiation of mushrooms Fresh fruiting bodies (FB, 500 g) and moist mycelia (MY, 400 g) were randomly selected by being placed in a single layer in 30 cm 30 cm stainless steel trays. For UV-B irradiation, 15 trays from each treatment were done for each species mushroom. Untreated fresh fruiting bodies and moist mycelia were controls. Photographs of the fresh fruiting bodies are displayed in Fig. 1. A tray of mushrooms was placed 19 cm from the source of irradiation, a UV-B lamp (280–360 nm, Sankyo Denki, G15T8E, Tokyo, Japan) for 2 h at ambient temperature (25 8C). The UV-B irradiation intensity was measured by using a UVX 31 radiometer (UVP, Upland, CA, USA) to be 0.36 mW/cm2 and the irradiation doses for 2 h were 25.9 kJ/m2. During all steps of preparation, care was taken that samples were not exposed to incidental UV light. After UV-B irradiation, all samples were stored frozen at 20 8C until freeze drying, then ground in an RT-34 pulverizing machine (Rong Tsong Precision Technology Co., Taichung, Taiwan), sequentially ground and sieved until all particles were <0.4 mm. The powdered samples were dispensed into 500-mL matte plastic bottle, surrounded with aluminium foil, and stored in darkness at 4 8C before use. The moisture content was determined by moisture loss from freeze-drying until constancy was achieved. 2.3. Vitamin D2 assay Vitamin D2 was extracted and analysed according to the method of Tung et al. (2007) with some modification. Mushroom powder (5 g) was mixed with 10 mL of dimethyl sulfoxide (Merck, Darmstadt, Germany) and ultrasound-oscillated at 45 8C for 30 min. Then 10 mL of methanol and water (1:1, v/v) and 20 mL of hexane were added and the mixture was ultrasound-oscillated at 45 8C for 30 min and centrifuged at 3000 g for 10 min. The residue was extracted twice with 20 mL of hexane and centrifuged.
Fig. 1. Photograph of golden oyster (a), wood ear (b), pink oyster (c), sticky pholiota (d), king oyster (e) and ferulae (f) before UV-B irradiation. The top row of each quadrant represents sector mushroom; the bottom row of each quadrant represents stick mushroom.
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The combined filtrate was rotary evaporated at 40 8C to dryness, redissolved in 1 mL of methanol (LC grade, Merck), and filtered using a 0.45-mm polyvinylidene difluouride filter (Millipore, Billerica, MA, USA) prior to injection onto high-performance liquid chromatograph (HPLC). The HPLC system consisted of a Hitachi L-2130 pump, Hitachi L2400 UV detector, and LiChrospher 100 RP-18e column (4.6 mm 250 mm, 5 mm, Merck). The mobile phase was methanol/H2O (95:5, v/v) at a flow rate of 1.0 mL/min and UV detection was at 254 nm. The content of vitamin D2 was calculated on the basis of the calibration curve of authentic vitamin D2 (97% pure, Sigma-Aldrich, St. Louis, MO, USA). 2.4. Polysaccharide assay For golden oyster, oyster and pink oyster mushrooms, the water-soluble polysaccharide content of four treatments of each of three species (UV-B irradiated fruiting bodies and mycelia for 2 h [FB2 and MY2] and non-irradiated fruiting bodies and mycelia [FB0 and MY0], respectively) was determined. Freeze-dried mushroom powder (1 g) was refluxed with 50 mL of deionised water for 30 min. The mixture was cooled to room temperature and filtered through Whatman No. 4 filter paper. The residue was then refluxed with two additional 10 mL portions of deionised water for 30 min, cooled and filtered. The combined filtrate was dialyzed by using a Cellu Sep T2 tubular membrane (MWCO: 6,000–8,000, Membrane Filtration Products, Inc., Seguin, TX, USA) for 24 h, resulting in a water-soluble polysaccharide sample (Tseng et al., 2008). The polysaccharide content was then determined by a phenol–sulphuric acid assay according to Dubois et al. (1956). 2.5. Preparation of extracts for antioxidant properties and components For ethanolic extraction, a subsample (10 g) from four treatments of FB0, FB2, MY0, and MY2 was extracted by stirring with 100 mL of 95% (v/v) ethanol at 25 8C at 150 rpm for 24 h and filtering through Whatman No. 1 filter paper. The residue was then extracted with two additional 100 mL portions of ethanol and filtered. The combined ethanolic extracts were then rotary evaporated at 40 8C to dryness and weighed. The dried extract was used directly for analysis of antioxidant components or was weighed to make an ethanolic solution of a concentration of 50 mg extract/mL and stored at 4 8C for further use. 2.6. Determination of antioxidant properties Reducing power was determined according to the method of Oyaizu (1986). Each extract (0.5–20 mg/mL) in ethanol (2.5 mL) was mixed with 2.5 mL of 200 mM sodium phosphate buffer (pH 6.6, J. T. Baker Chemical Co., Phillipsburg, NJ, USA) and 2.5 mL of 1% potassium ferricyanide (Sigma-Aldrich), and the mixture was incubated at 50 8C for 20 min. After 2.5 mL of 10% trichloroacetic acid (Sigma-Aldrich) was added, and the mixture was centrifuged at 200 g for 10 min. The upper layer (5 mL) was mixed with 5 mL of deionised water and 1 mL of 0.1% ferric chloride (SigmaAldrich), and the absorbance was measured at 700 nm against a blank. The reducing power assayed is the ability of the extracts to form a coloured complex with ferricyanide, which is an electron acceptor. Scavenging ability on 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich) radicals was determined on the basis of Shimada et al. (1992). Each extract (0.5–20 mg/mL) in ethanol (4 mL) was mixed with 1 mL of methanolic solution containing DPPH (SigmaAldrich) radicals, resulting in a final concentration of 0.2 mM
DPPH. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was then measured at 517 nm against a blank. The scavenging ability assayed is the ability of the extracts to react with DPPH radicals and reduce most DPPH radical molecules. Chelating ability was determined according to the method of Dinis et al. (1994). Each extract (0.5–20 mg/mL) in ethanol (1 mL) was mixed with 3.7 mL of methanol and 0.1 mL of 2 mM ferrous chloride (Fluka Chemical Corp., Ronkonkoma, NY, USA). The reaction was initiated by the addition of 0.2 mL of 5 mM ferrozine (Sigma-Aldrich). After 10 min at room temperature, the absorbance of the mixture was determined at 562 nm against a blank. Ferrous ions play an important role as catalysts in the oxidative process, leading to the formation of hydroxyl radicals and hydroperoxide decomposition by Fenton reaction. The chelating ability assayed is the ability of the extracts to inhibit the complex formation of ferrozine with ferrous ions. Effective concentration at 50% (EC50) value (mg extract/mL) is the effective concentration at which the absorbance was 0.5 for reducing power; DPPH radicals were scavenged by 50%; and ferrous ions were chelated by 50%, respectively. EC50 value was obtained by interpolation from linear regression analysis. 2.7. Determination of antioxidant components Ergothioneine was extracted and analysed according to the method of Dubost et al. (2007) with slight modifications. Sample powder (1 g) was mixed with 20 mL of 10 mM 1,4-dithiothreitol (Sigma-Aldrich), 100 mM betaine (Sigma-Aldrich) and 100 mM 2mercapt-1-methylimidazole (Sigma-Aldrich) in 70% ethanol, and the resulting mixture was vortexed for 90 s. Sodium dodecyl sulphate (1%, 4 mL, J.T. Baker) was added and the mixture was centrifuged at 3000 g for 10 min. The combined filtrate was rotary evaporated at 40 8C to dryness, redissolved in 1 mL of deionised water, and filtered prior to HPLC injection in the same manner as in the vitamin D2 assay. The HPLC instrument used was the same as for the vitamin D2 assay. HPLC with separation was performed on Kinetex PFP columns (4.6 mm 250 mm, 5 mm, Phenomenex, Inc., Torrance, CA, USA). The mobile phase was 50 mM sodium phosphate in water with 3% acetonitrile (LC grade, Merck) and 0.1% triethylamine (Sigma-Aldrich) adjusted to a pH of 7.3, at a flow rate of 1.0 mL/min and UV detection was at 254 nm. The content of ergothioneine was calculated on the basis of the calibration curve of each authentic ergothioneine (98% pure, Sigma-Aldrich). Flavonoids were determined according to the method of Zhishen et al. (1999). An aliquot (0.5 mL) of appropriately diluted sample or standard solutions of rutin (94% pure, Sigma-Aldrich) was added to the flask, which contained and 0.1 mL of 5% aqueous sodium nitrite (Sigma-Aldrich) solution. After 6 min, 0.1 mL of 10% aqueous aluminium chloride (Fluka) solution was added and after another 6 min, 1 mL 5% aqueous sodium hydroxide solution was added to the mixture. Thereafter, the mixture was diluted with the addition of 0.8 mL of deionised water and thoroughly mixed. The absorbance of the mixture was determined at 510 nm against a blank. Total phenols were determined according to the method of Taga et al. (1984). Each methanolic extract (20 mg) was dissolved in a solution of 5 mL of 1.3% HCl in methanol/deionised water (60:40, v/ v) and the resulting mixture (100 mL) was added to 2 mL of 20 mg/ mL aqueous sodium carbonate solution. After 3 min, 100 mL of 50% Folin-Ciocalteu phenol reagent (Sigma-Aldrich) was added to the mixture. After 30 min standing, the absorbance was measured at 750 nm against a blank. The content of total phenols was calculated on the basis of the calibration curve of gallic acid (98% pure, Sigma-Aldrich).
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2.8. Statistical analysis All the measurements were determined in triplicate through all steps of extraction and analysis for each component. The experimental data were subjected to an analysis of variance (ANOVA) for a completely random design (CRD) using Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA, 2009) to determine the least significant difference among means at the level of P < 0.05.
3. Results and discussion 3.1. Vitamin D2 content Without UV-B irradiation, fresh fruiting bodies contained low amounts of vitamin D2 (0–3.93 mg/g) and three species (Brazilian, wood ear and sticky pholiota mushrooms) did not contain vitamin D2 (Table 1). After UV-B irradiation for 2 h, the vitamin D2 content of fruiting bodies increased from 0–3.93 mg/g to 15.06–208.65 mg/ g dry weight. The first, second and third highest vitamin D2 content after UV-B irradiation was golden oyster, oyster and pink oyster mushrooms, respectively. The increase of vitamin D2 content in these three irradiated Pleurotus mushrooms was 44- to 83-fold as compared to that in the non-irradiated controls. Fig. 2 shows the high-performance liquid chromatography (HPLC) chromatogram for vitamin D2 for the treated and untreated samples of each species mushrooms. Mau et al. (1998) reported 12.48, 6.58 and 7.58 mg/g dry weight of vitamin D2 in button (Agaricus bisporus), shiitake (L. edodes) and straw mushrooms (Volvariella volvacea) after 2 h of UV-B irradiation (14.7 kJ/m2), respectively. Jasinghe and Perera (2005) reported 53.9, 184, 79.5 and 56.5 mg/g dry weight of vitamin D2 in shiitake, oyster, abalone (Pleurotus cystidiosus) and button mushrooms after 2 h of UV-B irradiation (25.2 kJ/m2), respectively. After the exposure to UV-B light, at a dose of 25 kJ/m2, the concentration of vitamin D2 was increased to 36.7, 68.6 and 106.4 mg/g dry weight for pileus, middle, and gillus parts of shiitake mushroom, respectively (Ko et al., 2008). The gill side of whole shiitake mushrooms exposed to 0, 25, 50 and 75 kJ/m2 increased to 2.8, 13.8, 40.7 and 61.9 mg/g dry weight, respectively (Ko et al., 2008). However, Simon et al. (2011) found that the vitamin D2 content increased from 5 to 410 mg/100 g dry weight by UV-B irradiation at a dose of 10.6 kJ/m2. These variations in vitamin D2 conversion might be due to the cultivar differences, different environmental conditions for mushrooms and different irradiation intensities and doses used. Phillips et al. (2012) found vitamin D4 should be Table 1 Effect of UV-B irradiation on vitamin D2 content of fresh fruiting bodies. Mushroom
Contenta (mg/g dry matter) Control (0 h)
Brazilian mushroom Popular Wood ear Hon-shimeji Shiitake Sticky pholiota King oyster Ferulae Golden oyster Oyster Pink oyster a
c
LOD 0.95 0.14 LODc 1.62 0.12 0.35 0.05 LODc 1.56 0.17 1.65 0.19 3.93 0.44 0.83 0.06 2.13 0.21
b b b b b b b b
2 hb 22.13 1.98 42.36 6.31 a 60.29 3.59 15.06 0.96 a 15.10 1.55 a 61.78 2.58 28.71 0.96 a 52.30 4.79 a 208.65 6.08 a 69.00 1.96 a 93.29 11.56 a
Number of analysed samples n = 3. All values are presented as means SD (n = 3). Means with different letters within a row differ significantly (P < 0.05). b Irradiation intensity = 0.36 mW/cm2. c Limit of detection.
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expected to occur in mushrooms exposed to UV light, such as commercially produced vitamin D enhanced products. Since the UV irradiation acts only on the surface of mushrooms, it is probably important to expose a larger fraction of mushrooms to UV irradiation. However, the round shape of fruiting bodies would result in poor exposure to UV-B irradiation. The use of mycelia with larger flat surface might resolve the problem of exposure area. Therefore, further study was to measure the effect of UV-B irradiation on the vitamin D2 content of three fresh mycelia of Pleurotus spp., whose fruiting bodies showed the highest vitamin D2 content after UV-B irradiation in Fig. 3. After UV-B irradiation for 2 h, the vitamin D2 content in fresh mycelia of golden oyster, oyster and pink oyster mushrooms increased from 2.72, 5.93 and 0.28 mg/g to 81.67, 81.71 and 66.03 mg/g, respectively (Fig. 3). It showed that the vitamin D2 content of both fruiting bodies and mycelia was 0.28–5.93 mg/g and their content could be increased to 66.03–208.65 mg/g after UV-B irradiation for 2 h. The treatment of UV-B irradiation for 2 h appears to have been effective for increasing the vitamin D2 content of both fruiting bodies and mycelia. Vitamin D2 is derived by photo-irradiation from its precursor ergosterol. This increase in vitamin D2 content in mushrooms is due to photosynthetic/thermal processes occurring from exposure of ergosterol to UV light (Simon et al., 2013). Sterols are known to be involved in the stabilization of membranes to UV light (Jasinghe and Perera, 2005). Both initial moisture content and temperature of irradiation could influence the conversion of ergosterol (Perera et al., 2003). In a preliminary study, after UV-B irradiation for 2 h, the vitamin D2 content in dry mycelia of golden oyster, oyster and pink oyster mushrooms was from 2.72, 5.93 and 0.28 mg/g to 34.73, 27.12 and 18.52 mg/g, respectively. Jasinghe and Perera (2005) reported that the optimal moisture content for the conversion of ergosterol vitamin D2 (22–27 mg/g dry weight) was around 70–80% on a wet weight basis of shiitake mushroom. However, in this study, fresh fruiting bodies and fresh mycelia used contained 87–90% moisture. It appears that the conversion of ergosterol to vitamin D2 could occur in both dry (dead) and fresh (live) mushroom fruiting bodies and mycelia. Furthermore, fresh fruiting bodies and mycelia with higher moisture content could more effectively enhance such vitamin D2 conversion. 3.2. Polysaccharide content Polysaccharide content in three fresh fruiting bodies and mycelia was affected by UV-B irradiation (Table 2). Polysaccharide content in three fresh mycelia and golden oyster fruiting bodies decreased after UV-B irradiation for 2 h, whereas that in fruiting bodies of oyster and pink oyster mushrooms increased after UV-B irradiation for 2 h. The UV-B irradiation might cause a decomposition of polysaccharides in mycelia but a synthesis of polysaccharides in two fruiting bodies of oyster and pink oyster, except for that of golden oyster mushrooms. In other words, with regard to polysaccharide content, fruiting bodies and mycelia responded differently to UV-B irradiation. However, for golden oyster, UV-B irradiation caused the degradation of polysaccharide in both fruiting bodies and mycelia. For each species, the polysaccharide content in mycelia was higher than that in its corresponding fruiting bodies. Unfortunately, there is no publication available regarding the effect of UV-B irradiation on mushroom polysaccharides. 3.3. Antioxidant properties The ethanolic extracts were obtained from freeze-dried samples and their yields were expressed as the g/100 g of samples on the basis of dry weight. For golden oyster, the yields for irradiated and non-irradiated fruiting bodies and mycelia were
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Fig. 2. HPLC chromatograms showing vitamin D2 profiles for each species of mushroom. Peaks: 1, vitamin D2; 2, ergosterol. Marked: a, non-irradiated sample; b, irradiated sample. The HPLC system consisted of a solvent degasser, gradient pump, temperature controlled autosampler, and UV detector (Hitachi Ltd., Tokyo, Japan), with a LiChrospher 100 RP-18 e column, 4.6 mm 250 mm, 5 mm (Merck KGaA, Darmstadt, Germany); mobile phase 950 mL, methanol: 50 mL H2O; flow rate 1.0 mL/min; detection at 254 nm.
comparable (13.4–14.1 g/100 g). Besides, the yields from irradiated and non-irradiated mycelia of oyster (15.5–17.4 g/100 g) and pink oyster (14.1–14.2 g/100 g) were higher than those from their corresponding fruiting bodies (9.62–9.80 g/100 g and 9.14–9.43 g/ 100 g, respectively). In addition, no difference in the yields of irradiated or non-irradiated fruiting bodies and mycelia was observed, except that the yield of irradiated oyster mycelia (17.4 g/ 100 g) was higher than that of the non-irradiated control (15.5 g/ 100 g).
The antioxidant properties assayed herein are summarized in Table 3, and the EC50 values (mg ethanolic extracts per mL) were calculated for comparison. With regard to reducing power, the EC50 values of irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster (0.92–4.73 mg/mL) were lower than those of the non-irradiated controls (1.02–4.94 mg/mL), except that the EC50 value of non-irradiated golden oyster mycelia (0.99 mg/mL) was higher than that of the irradiated mycelia (1.45 mg/mL). Ethanolic extracts from UV-B irradiated fruiting bodies and
S.-J. Huang et al. / Journal of Food Composition and Analysis 42 (2015) 38–45 a
200
a
FB0 FB2 MY0 MY2
100
Vitamin D2 (µg/g)
b a b b
50
c
c
c
d
c
0 Golden oyster
Oyster
d
Pink oyster
Fig. 3. Content of vitamin D2 of UV-B irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster mushrooms. Number of analysed samples n = 3. All values are presented as means SD (n = 3). Means with different letters within a group differ significantly (P < 0.05). FB0 and FB2, fresh fruiting bodies UV-B irradiated for 0 and 2 h, respectively; MY0 and MY2, freshly harvested mycelia UV-B irradiated for 0 and 2 h, respectively.
mycelia were more effective than those from the non-irradiated controls, except for non-irradiated golden oyster mycelia being more effective. Furthermore, three mycelia sample were more effective than their corresponding fruiting bodies in reducing power. For the scavenging ability on DPPH radicals, the EC50 values of irradiated fruiting bodies of oyster and pink oyster and irradiated mycelia of golden oyster and pink oyster (0.97–6.90 mg/mL) were higher than those of the non-irradiated controls (0.27–3.41 mg/ mL). No difference in the EC50 values between irradiated and nonirradiated samples was observed for golden oyster fruiting bodies and oyster mycelia. Ethanolic extracts from UV-B irradiated fruiting bodies and mycelia were less effective than those from non-irradiated fruiting bodies and mycelia, except that UV-B irradiation had no effect on the scavenging ability of golden oyster fruiting bodies and oyster mycelia. Three fruiting bodies were more effective than their corresponding mycelia in scavenging ability on DPPH radicals. For chelating ability on ferrous ions, the EC50 values of irradiated and non-irradiated golden oyster fruiting bodies and mycelia were 0.02–0.04 mg/mL, indicating that their ethanolic extracts were excellent chelators for ferrous ions. The EC50 values of irradiated pink oyster fruiting bodies and mycelia (0.11– 0.20 mg/mL) were lower than those of the non-irradiated controls (0.23–0.29 mg/mL). However, the EC50 values of irradiated oyster fruiting bodies and non-irradiated mycelia were higher than those
Table 2 Content of total water-soluble polysaccharides of UV-B irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster mushrooms. Contenta (mg/g dry matter)
Golden oyster Oyster Pink oyster
FB0b
FB2b
MY0b
MY2b
19.92 0.12 c
19.01 0.09 d
68.48 0.12 a
51.61 0.14 b
48.89 0.49 d 24.57 0.12 d
53.23 0.35 c 33.88 0.05 c
56.06 0.16 a 123.01 0.20 a
55.31 0.05 b 112.34 0.09 b
a Number of analysed samples n = 3. All values are presented as means SD (n = 3). Means with different letters within a row differ significantly (P < 0.05). b FB0 and FB2, fresh fruiting bodies UV-B irradiated for 0 and 2 h, respectively; MY0 and MY2, freshly harvested mycelia UV-B irradiated for 0 and 2 h, respectively.
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of their corresponding samples. Fruiting bodies and mycelia appear to be considerably effective in the chelating ability on ferrous ions, as evidenced by their low EC50 values of <1 mg/mL and <0.05 mg/mL (for golden oyster). Lee et al. (2007) found that the EC50 values of the ethanolic extracts from golden oyster fruiting bodies and mycelia were 2.28 and 8.29 mg/g for reducing power, 1.33 and 7.92 mg/g for scavenging ability, and 6.49 and 17.32 mg/g for chelating ability, respectively. Tsai et al. (2009) reported that the EC50 values of the ethanolic extracts from oyster fruiting bodies were 6.76, 5.58 and 3.48 mg/g for reducing power, scavenging and chelating abilities, respectively. The EC50 values of the ethanolic extracts from golden oyster and oyster were lower than those previously published. However, the antioxidant properties of UV-B irradiated mushrooms have not been published. Therefore, this is the first time to report the effect of UV-B irradiation on reducing power, scavenging and chelating abilities. Overall, EC50 values in the ethanolic extracts of non-irradiated and irradiated fruiting bodies and mycelia were 0.92–4.94, 0.20– 6.90 and 0.02–0.84 mg/mL for reducing power, scavenging ability on DPPH radicals and chelating ability on ferrous ions, respectively. These fruiting bodies and mycelia were effective in the reducing power and scavenging ability on DPPH radicals and considerably effective in the chelating ability on ferrous ions. UV-B treatment could slightly enhance the reducing power and chelating ability but slightly decrease the scavenging ability of fruiting bodies and mycelia. Although antioxidants such as BHA, ascorbic acid and atocopherol are good for reducing power and scavenging ability on DPPH radicals, and EDTA is a good chelator for ferrous ions, they are additives and used or present in milligram levels in foods. However, the ethanolic extracts from UV-B irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster could be used in gram levels as food or a food ingredient. Therefore, these ethanolic extracts from both UV-B irradiated fruiting bodies and mycelia might also serve as possible protective agents in the human diet in addition to their high vitamin D2 content. 3.4. Antioxidant components Phenolic compounds are widely distributed in mushrooms. One phenolic compound, ergothioneine (2-mercaptohistidine trimethylbetaine), is a naturally occurring amino acid which is synthesized in some bacteria and fungi but not in animals (Melville et al., 1955). It has been documented that ergothioneine is an antioxidant in vivo (Hartman, 1990) and a cellular protector against oxidative damage (Aruoma et al., 1999). Non-irradiated fruiting bodies and mycelia contained 1.42–3.46 and 0.63–1.39 mg/g of ergothioneine, respectively (Table 4). The ergothioneine content of fruiting bodies was much higher than that of their corresponding mycelia. Generally, UV-B irradiation had an influence on the ergothioneine content of fruiting bodies and mycelia, except for oyster fruiting bodies. After UV-B irradiation for 2 h, the ergothioneine content of irradiated fruiting bodies and mycelia contained 0.71–3.13 and 0.63–0.66 mg/g of ergothioneine, respectively. Although UV-B irradiation lowered the ergothioneine content in fruiting bodies and mycelia, these irradiated samples were still an abundant source of ergothioneine. Flavonoids act in plants as antioxidants, antimicrobials, photoreceptors, visual attractors, feeding repellents, and light screening substances. Non-irradiated fruiting bodies and mycelia contained 5.62–11.96 and 2.26–8.81 mg/g of flavonoids, respectively (Table 4). UV-B irradiation seems to have had a slight influence on the flavonoid content of fruiting bodies and mycelia. These results are consistent with the finding of Liu et al. (2011) that post-harvest irradiation with UV-B enhanced the amounts of
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Table 3 EC50 values of ethanolic extracts from UV-B irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster mushrooms in antioxidant properties. EC50 valuea (mg extract/mL) FB0b
FB2b
MY0b
MY2b
Reducing power Golden oyster Oyster Pink oyster
2.05 0.01 a 4.94 0.02 a 2.93 0.02 a
1.97 0.06 b 4.73 0.08 b 2.51 0.03 b
1.02 0.03 c 0.99 0.01 d 1.76 0.01 c
0.92 0.01 d 1.45 0.03 c 1.04 0.02 d
Scavenging ability Golden oyster Oyster Pink oyster
0.20 0.01 b 0.83 0.01 c 0.84 0.01 d
0.25 0.01 b 1.44 0.03 b 0.97 0.01 c
0.27 0.01 b 5.10 0.11 a 3.41 0.05 b
1.35 0.51 a 5.06 0.08 a 6.90 0.02 a
Chelating ability Golden oyster Oyster Pink oyster
0.02 <0.01 c 0.84 0.04 a 0.23 0.01 b
0.03 <0.01 b 0.74 0.01 b 0.20 0.02 c
0.04 <0.01 a 0.42 0.01 d 0.29 0.01 a
0.04 <0.01 a 0.59 0.02 c 0.11 0.01 d
a EC50 value: the absorbance was 0.5 for reducing power; 2,2-diphenyl-1-picrylhydrazyl radicals were scavenged by 50%; and ferrous ions were chelated by 50%, respectively. EC50 value was obtained by interpolation from linear regression analysis. Number of analysed samples n = 3. All values are presented as means SD (n = 3). Means with different letters within a row differ significantly (P < 0.05). b FB0 and FB2, fresh fruiting bodies UV-B irradiated for 0 and 2 h, respectively; MY0 and MY2, freshly harvested mycelia UV-B irradiated for 0 and 2 h, respectively.
flavonoids, phenols and total phenols in the tomato fruiting. Irradiated fruiting bodies and mycelia contained 5.31–7.26 and 1.78–8.21 mg/g of flavonoids, respectively. However, for golden oyster fruiting bodies and mycelia, after UV-B irradiation for 2 h, no significant difference in flavonoid content between irradiated and non-irradiated samples was observed. Total phenols are the major naturally occurring antioxidant components found in fruiting bodies and mycelia (Table 4). The high amount of total phenols (6.36–18.92 mg/g) in the ethanolic extracts might explain their higher effectiveness in reducing power and scavenging ability on DPPH radicals. UV-B irradiation had no to slight influence on total phenol content of three fruiting bodies, but showed different effects on total phenol content of three mycelia. After UV-B irradiation for 2 h, total phenol content of golden oyster and oyster mycelia increased, while that of pink oyster mycelia decreased. Lee et al. (2007) found that the total phenol content of the ethanolic extracts from golden oyster fruiting bodies and mycelia was 8.62 and 5.84 mg/g, respectively, much lower than those in Table 4. Tsai et al. (2009) reported that the EC50 values of the ethanolic extracts from oyster fruiting bodies were 7.11 mg/g,
slightly lower than that in Table 4. These results are consistent with their effectiveness in antioxidant properties assayed. However, the total phenol content of UV-B irradiated mushrooms has not been published. Therefore, this is the first report on the effect of UV-B irradiation on their total phenol content. Phenols such as butylated hydroxytoluene (BHT) and gallate are known to be effective antioxidants (Madhavi et al., 1996). Due to their scavenging abilities on free radicals and chelating abilities on ferrous ions, phenols might possess good antioxidant, antimutagenic and anticancer properties (Ahmad and Mukhtar, 1999). Tsai et al. (2007) found that the content of total antioxidant components was moderately to highly correlated (r = 0.636– 0.907) with antioxidant properties. Although UV-B irradiation has a slight influence on the content of ergothioneine, flavonoids, total phenols of fruiting bodies and mycelia, these irradiated samples still contained sufficient amount of these antioxidant components. Accordingly, in addition to their high vitamin D2 content, UV-B irradiated fruiting bodies and mycelia might also be potential antioxidants for use in food products and could be developed as a new dietary supplement and functional foods.
Table 4 Content of ergothioneine, flavonoids and total phenols of ethanolic extracts from UV-B irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster mushrooms. Contentsa (mg/g) FB0b
FB2b
MY0b
MY2b
Ergothioneine Golden oyster Oyster Pink oyster
3.46 0.03 a 1.42 0.05 b 1.48 0.02 a
3.13 0.03 b 1.99 0.01 a 0.71 0.02 b
1.39 0.03 c 0.84 0.01 c 0.63 0.02 c
0.63 0.01 d 0.66 0.01 d 0.59 0.02 d
Flavonoids Golden oyster Oyster Pink oyster
5.62 0.34 b 11.96 0.50 a 7.92 0.03 a
5.31 0.20 b 7.26 0.06 b 6.53 0.49 b
8.81 0.09 a 2.26 0.11 c 6.28 0.13 b
8.21 0.37 a 1.78 0.20 c 3.23 0.22 c
Total phenols Golden oyster Oyster Pink oyster
18.92 0.27 a 7.56 0.06 c 13.32 0.21 a
18.25 0.09 b 7.15 0.21 c 13.49 0.18 a
11.37 0.06 d 9.65 0.20 b 7.32 0.02 b
13.08 0.18 c 11.58 0.22 a 6.36 0.25 c
a b
Number of analysed samples n = 3. All values are presented as means SD (n = 3). Means with different letters within a row differ significantly (P < 0.05). FB0 and FB2, fresh fruiting bodies UV-B irradiated for 0 and 2 h, respectively; MY0 and MY2, freshly harvested mycelia UV-B irradiated for 0 and 2 h, respectively.
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4. Conclusion Based on the results, 2 h of UV-B irradiation not only significantly increased the vitamin D2 content of mushroom fruiting bodies, but it could also significantly enhance its content of vitamin D2 of mycelium. UV-B irradiation could also slightly enhance the reducing power and chelating ability, but slightly decrease the scavenging ability of fruiting bodies and mycelia. Obviously, the amount of vitamin D of fruiting bodies and mycelia increased was not consistent. For developing product with rich vitamin D2, irradiated golden oyster fruiting bodies were the best choice; but thinking about the cost, the mycelia of irradiated golden oyster or oyster maybe were the better choices, because of the cultivation time that mushroom mycelia could be harvested is only a few days, but for fruiting bodies is a few months. However, mushrooms possess many bioactive compounds that must be taken into consideration when mushrooms are irradiated by UV-B. Overall, in addition to their high vitamin D2 content, UV-B irradiated fruiting bodies and mycelia might also be potential antioxidants that could be developed as a new dietary supplement. In the future, further exploring the cause of different impacts between mushroom fruiting bodies and mycelia with UV-B irradiation will help to develop more suitable products. Acknowledgments The study was supported by the Ministry of Science and Technology, Taiwan, Republic of China (NSC 98-2313-B-468-004MY3) and Asia University (102-asia-05). We thank Mr. Shih-Wen Fang, Q-Yo Bio-Technology Farm for providing mushrooms. References Ahmad, N., Mukhtar, H., 1999. Green tea polyphenols and cancer: biologic mechanisms and practical implications. Nutr. Rev. 57, 78–83. Aruoma, O.I., Spencer, J.P.E., Mahmood, N., 1999. Protection against oxidative damage and cell death by the natural antioxidant ergothioneine. Food Chem. Toxicol. 37, 1043–1053. Dinis, T.C.P., Madeira, V.M.C., Almeida, L.M., 1994. Action of phenolic derivatives (acetaminophen, salicylate, and 5-amino salicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys. 315, 161–169. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Dubost, N.J., Ou, B., Beelman, R.B., 2007. Quantification of polyphenols and ergothioneine in cultivated mushrooms and correlation to total antioxidant capacity. Food Chem. 105, 727–735. Hartman, P.E., 1990. Ergothioneine as an antioxidant. Methods Enzymol. 186, 310– 318. Holden, J.M., Lemar, L.E., Exler, J., 2008. Vitamin D in foods: development of the US Department of Agriculture database. Am. J. Clin. Nutr. 87 (Suppl.), 1092S– 1096S. Holick, M.F., 2007. Vitamin D deficiency. N. Engl. J. Med. 357, 266–281. Jasinghe, V.J., Perera, C.O., 2006. Ultraviolet irradiation: the generator of vitamin D2 in edible mushrooms. Food Chem. 95, 638–643. Jasinghe, V.J., Perera, C.O., 2005. Distribution of ergosterol in different tissues of mushrooms and its effect on the conversion of ergosterol to vitamin D2 by UV irradiation. Food Chem. 92, 541–546. Kalac, P., 2013. A review of chemical composition and nutritional value of wild growing and cultivated mushrooms. J. Sci. Food Agric. 93, 209–218.
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Original Research Article
Vitamin D2 content and antioxidant properties of fruit body and mycelia of edible mushrooms by UV-B irradiation Shih-Jeng Huang a, Chun-Ping Lin b, Shu-Yao Tsai b,c,* a
Department of Health Food, Chung Chou University of Science and Technology, Yuanlin, Changhua 51003, Taiwan, ROC Department of Health and Nutrition Biotechnology, Asia University, 500, Lioufeng Rd., Wufeng, Taichung 41354, Taiwan, ROC c Department of Nutrition, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan, ROC b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 October 2012 Received in revised form 3 February 2015 Accepted 24 February 2015 Available online 20 March 2015
Our objective was to study the effect of ultraviolet-B (UV-B) light irradiation on the vitamin D2 content of several edible fruit bodies and mycelia and their antioxidant properties. Eleven species of fresh mushroom fruiting bodies, including species from each of the six genera, Agaricus, Agrocybe, Auricularia, Hypsizigus, Lentinula and Pholiota, and five species from Pleurotus genus, were irradiated with UV-B light for 2 h. For three species of mushroom fruiting bodies with excellent vitamin D2 yield, their mycelia were obtained by liquid culture, and subjected to the same time as the UV-B irradiation. Vitamin D2 content of irradiated fruit bodies significantly increased from 0–3.93 to 15.06–208.65 mg/g, of which the amount in golden oyster mushroom increased by a maximum of 204.7 mg/g. Vitamin D2 content in irradiated mycelia of golden oyster, oyster and pink oyster mushrooms increased from 0.28–5.93 to 66.03– 81.71 mg/g, respectively. The three irradiated mycelium polysaccharide contents decreased in a range from 1.3% to 24.6%. Overall, EC50 values of non-irradiated and irradiated fruiting bodies and mycelia were 0.92–4.94, 0.20–6.90 and 0.02–0.84 mg/mL for reducing power, scavenging ability and chelating ability, respectively. Although UV-B irradiation influenced the content of ergothioneine, flavonoids and total phenols, these irradiated samples still contained a sufficient amount of these antioxidant components. ß 2015 Elsevier Inc. All rights reserved.
Keywords: UV-B irradiation Vitamin D2 Ergocalciferol Antioxidant properties Antioxidant components Pleurotus citrinopileatus Pleurotus ostreatus Pleurotus salmoneostramineus Food analysis Food composition
1. Introduction Edible and medicinal mushrooms contain many physiologically active substances, including polysaccharides, triterpenoids, ergosterol, vitamin D and ergothioneine (Kalac, 2013). Vitamin D is important in human nutrition because deficiency in infancy/ childhood can result in rickets, while in adults vitamin D deficiency is associated with osteoporosis (Holick, 2007). The vitamin D receptor has been detected in a broad range of tissues; therefore, it
Abbreviations: UV-B, ultraviolet-B; EC, effective concentration; HPLC, high-performance liquid chromatograph; DPPH, 2,2-diphenyl-1-picrylhydrazyl; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; EDTA, ethylenediaminetetraacetic acid; FB0, fresh fruiting bodies UV-B irradiated for 0 h; FB2, fresh fruiting bodies UV-B irradiated for 2 h; MY0, freshly harvested mycelia UV-B irradiated for 0 h; MY2, freshly harvested mycelia UV-B irradiated for 2 h. * Corresponding author at: Department of Health and Nutrition Biotechnology, Asia University, 500, Lioufeng Rd., Wufeng, Taichung 41354, Taiwan, ROC. Tel.: +886 4 2332 3456x20028; fax: +886 4 2332 1126. E-mail address: [email protected] (S.-Y. Tsai). http://dx.doi.org/10.1016/j.jfca.2015.02.005 0889-1575/ß 2015 Elsevier Inc. All rights reserved.
has been suggested that vitamin D may play a role in cardiovascular and autoimmune diseases and cancer prevention (Mullin and Dobs, 2007; Kikkinen et al., 2009). Natural food sources for vitamin D include certain fish species, egg, milk and mushrooms (Holden et al., 2008). Vitamin D exists as two distinct forms: vitamin D3 (cholecalciferol) is oily fish, egg and red meat, and vitamin D2 (ergocalciferol) is found in yeasts and mushrooms (Shrapnel and Truswell, 2006). Ultraviolet light can be applied to mushrooms to enhance their vitamin D2 content (Jasinghe and Perera, 2005, 2006; Teichmann et al., 2007; Ko et al., 2008; Roberts et al., 2008; Simon et al., 2011; Koyyalamudi et al., 2009, 2011; Kalaras et al., 2012). Most studies on mushroom fruiting bodies try to enhance their vitamin D content. Low vitamin D2 content occurs naturally in many mushrooms, with a high potential for large increases based on the presence of ergosterol by ultraviolet light treatment. However, information about the application of ultraviolet light on mushroom mycelium is not available. Since the mycelium is easily produced as compared to fruiting body and is readily irradiated due to its larger flat surface, it has great potential for the
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development of health foods containing a high content of vitamin D. Using ultraviolet irradiation on mushrooms to increase vitamin D content not only provides another option for people to supplement vitamin D from non-animal sources but also helps vegetarians to maintain vitamin D status. Mushrooms are a good source of beneficial components (such as ergothioneine, total phenols, flavonoids, and polysaccharides) that may be destroyed by UV irradiation, but there are no data. Given the growing use of irradiation to enhance vitamin D, the need to demonstrate an effect on these components is valuable. Eleven species of fresh mushroom fruiting bodies are commercially available in Taiwan, including species from each of the six genera of Agaricus, Agrocybe, Auricularia, Hypsizigus, Lentinula and Pholiota and five species from Pleurotus genus. In addition, limited information about the vitamin D2 content of these mushrooms is available and there have been no reports on the vitamin D2 content of mycelia. We irradiated fresh fruiting bodies and freshly harvested mycelia with ultraviolet light to study its effect on the vitamin D and polysaccharides content. We also studied the antioxidant properties of ethanolic extracts from irradiated fruiting bodies and mycelia, including reducing power, scavenging ability on radicals, and chelating ability on ferrous ions, and compared them to nonirradiated samples. The content of potential antioxidant components (ergothioneine, flavonoids and total phenols) was also determined. 2. Materials and methods 2.1. Fruiting bodies and mycelia Eleven species of fresh mushrooms included Brazilian (Agaricus blazei), popular (Agrocybe cylindracea), wood ear (Auricularia polytricha), hon-shimeji (Hypsizigus marmoreus), shiitake (Lentinula edodes), sticky pholiota (Pholiota nameko), king oyster (Pleurotus eryngii), golden oyster (Pleurotus citrinopileatus), ferulae (Pleurotus ferulae), oyster (Pleurotus ostreatus) and pink oyster (Pleurotus salmoneostramineus) mushrooms were obtained from QYo Bio-Technology Farm, Changhua County, Taiwan (1218E, 248N, 556 m). Immediately after harvest, the whole fresh fruiting bodies (10–15 kg each) were kept at 4 8C in a cooler and transported to the laboratory for irradiation treatment within 1 h. We selected the three highest levels of vitamin D2 of irradiated fruiting bodies, performing cultured mycelia. Three species of mycelia (golden oyster, oyster, and pink oyster mushrooms) cultures were obtained from the inner living tissues of the fruit bodies in potato dextrose
39
agar (Difco Laboratories, Sparks, MD, USA) medium at 25 8C. For the production of mycelia, the culture was inoculated into 250-mL flask containing 90 mL of liquid medium and incubated at 25 8C and 100 rpm. The liquid medium (1 L) consisted of 20 g of glucose, 5 g of yeast extract, 0.068 g of KH2PO4, and 2.3 g of MgSO47H2O. After 7 days of incubation, the mycelia were harvested, washed five times with deionised water, and fresh mycelia was obtained by wringing water from 400-mesh cloth. Both fruiting bodies and mycelia were irradiated in fresh and moist state on the day of harvest. 2.2. Irradiation of mushrooms Fresh fruiting bodies (FB, 500 g) and moist mycelia (MY, 400 g) were randomly selected by being placed in a single layer in 30 cm 30 cm stainless steel trays. For UV-B irradiation, 15 trays from each treatment were done for each species mushroom. Untreated fresh fruiting bodies and moist mycelia were controls. Photographs of the fresh fruiting bodies are displayed in Fig. 1. A tray of mushrooms was placed 19 cm from the source of irradiation, a UV-B lamp (280–360 nm, Sankyo Denki, G15T8E, Tokyo, Japan) for 2 h at ambient temperature (25 8C). The UV-B irradiation intensity was measured by using a UVX 31 radiometer (UVP, Upland, CA, USA) to be 0.36 mW/cm2 and the irradiation doses for 2 h were 25.9 kJ/m2. During all steps of preparation, care was taken that samples were not exposed to incidental UV light. After UV-B irradiation, all samples were stored frozen at 20 8C until freeze drying, then ground in an RT-34 pulverizing machine (Rong Tsong Precision Technology Co., Taichung, Taiwan), sequentially ground and sieved until all particles were <0.4 mm. The powdered samples were dispensed into 500-mL matte plastic bottle, surrounded with aluminium foil, and stored in darkness at 4 8C before use. The moisture content was determined by moisture loss from freeze-drying until constancy was achieved. 2.3. Vitamin D2 assay Vitamin D2 was extracted and analysed according to the method of Tung et al. (2007) with some modification. Mushroom powder (5 g) was mixed with 10 mL of dimethyl sulfoxide (Merck, Darmstadt, Germany) and ultrasound-oscillated at 45 8C for 30 min. Then 10 mL of methanol and water (1:1, v/v) and 20 mL of hexane were added and the mixture was ultrasound-oscillated at 45 8C for 30 min and centrifuged at 3000 g for 10 min. The residue was extracted twice with 20 mL of hexane and centrifuged.
Fig. 1. Photograph of golden oyster (a), wood ear (b), pink oyster (c), sticky pholiota (d), king oyster (e) and ferulae (f) before UV-B irradiation. The top row of each quadrant represents sector mushroom; the bottom row of each quadrant represents stick mushroom.
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The combined filtrate was rotary evaporated at 40 8C to dryness, redissolved in 1 mL of methanol (LC grade, Merck), and filtered using a 0.45-mm polyvinylidene difluouride filter (Millipore, Billerica, MA, USA) prior to injection onto high-performance liquid chromatograph (HPLC). The HPLC system consisted of a Hitachi L-2130 pump, Hitachi L2400 UV detector, and LiChrospher 100 RP-18e column (4.6 mm 250 mm, 5 mm, Merck). The mobile phase was methanol/H2O (95:5, v/v) at a flow rate of 1.0 mL/min and UV detection was at 254 nm. The content of vitamin D2 was calculated on the basis of the calibration curve of authentic vitamin D2 (97% pure, Sigma-Aldrich, St. Louis, MO, USA). 2.4. Polysaccharide assay For golden oyster, oyster and pink oyster mushrooms, the water-soluble polysaccharide content of four treatments of each of three species (UV-B irradiated fruiting bodies and mycelia for 2 h [FB2 and MY2] and non-irradiated fruiting bodies and mycelia [FB0 and MY0], respectively) was determined. Freeze-dried mushroom powder (1 g) was refluxed with 50 mL of deionised water for 30 min. The mixture was cooled to room temperature and filtered through Whatman No. 4 filter paper. The residue was then refluxed with two additional 10 mL portions of deionised water for 30 min, cooled and filtered. The combined filtrate was dialyzed by using a Cellu Sep T2 tubular membrane (MWCO: 6,000–8,000, Membrane Filtration Products, Inc., Seguin, TX, USA) for 24 h, resulting in a water-soluble polysaccharide sample (Tseng et al., 2008). The polysaccharide content was then determined by a phenol–sulphuric acid assay according to Dubois et al. (1956). 2.5. Preparation of extracts for antioxidant properties and components For ethanolic extraction, a subsample (10 g) from four treatments of FB0, FB2, MY0, and MY2 was extracted by stirring with 100 mL of 95% (v/v) ethanol at 25 8C at 150 rpm for 24 h and filtering through Whatman No. 1 filter paper. The residue was then extracted with two additional 100 mL portions of ethanol and filtered. The combined ethanolic extracts were then rotary evaporated at 40 8C to dryness and weighed. The dried extract was used directly for analysis of antioxidant components or was weighed to make an ethanolic solution of a concentration of 50 mg extract/mL and stored at 4 8C for further use. 2.6. Determination of antioxidant properties Reducing power was determined according to the method of Oyaizu (1986). Each extract (0.5–20 mg/mL) in ethanol (2.5 mL) was mixed with 2.5 mL of 200 mM sodium phosphate buffer (pH 6.6, J. T. Baker Chemical Co., Phillipsburg, NJ, USA) and 2.5 mL of 1% potassium ferricyanide (Sigma-Aldrich), and the mixture was incubated at 50 8C for 20 min. After 2.5 mL of 10% trichloroacetic acid (Sigma-Aldrich) was added, and the mixture was centrifuged at 200 g for 10 min. The upper layer (5 mL) was mixed with 5 mL of deionised water and 1 mL of 0.1% ferric chloride (SigmaAldrich), and the absorbance was measured at 700 nm against a blank. The reducing power assayed is the ability of the extracts to form a coloured complex with ferricyanide, which is an electron acceptor. Scavenging ability on 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich) radicals was determined on the basis of Shimada et al. (1992). Each extract (0.5–20 mg/mL) in ethanol (4 mL) was mixed with 1 mL of methanolic solution containing DPPH (SigmaAldrich) radicals, resulting in a final concentration of 0.2 mM
DPPH. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was then measured at 517 nm against a blank. The scavenging ability assayed is the ability of the extracts to react with DPPH radicals and reduce most DPPH radical molecules. Chelating ability was determined according to the method of Dinis et al. (1994). Each extract (0.5–20 mg/mL) in ethanol (1 mL) was mixed with 3.7 mL of methanol and 0.1 mL of 2 mM ferrous chloride (Fluka Chemical Corp., Ronkonkoma, NY, USA). The reaction was initiated by the addition of 0.2 mL of 5 mM ferrozine (Sigma-Aldrich). After 10 min at room temperature, the absorbance of the mixture was determined at 562 nm against a blank. Ferrous ions play an important role as catalysts in the oxidative process, leading to the formation of hydroxyl radicals and hydroperoxide decomposition by Fenton reaction. The chelating ability assayed is the ability of the extracts to inhibit the complex formation of ferrozine with ferrous ions. Effective concentration at 50% (EC50) value (mg extract/mL) is the effective concentration at which the absorbance was 0.5 for reducing power; DPPH radicals were scavenged by 50%; and ferrous ions were chelated by 50%, respectively. EC50 value was obtained by interpolation from linear regression analysis. 2.7. Determination of antioxidant components Ergothioneine was extracted and analysed according to the method of Dubost et al. (2007) with slight modifications. Sample powder (1 g) was mixed with 20 mL of 10 mM 1,4-dithiothreitol (Sigma-Aldrich), 100 mM betaine (Sigma-Aldrich) and 100 mM 2mercapt-1-methylimidazole (Sigma-Aldrich) in 70% ethanol, and the resulting mixture was vortexed for 90 s. Sodium dodecyl sulphate (1%, 4 mL, J.T. Baker) was added and the mixture was centrifuged at 3000 g for 10 min. The combined filtrate was rotary evaporated at 40 8C to dryness, redissolved in 1 mL of deionised water, and filtered prior to HPLC injection in the same manner as in the vitamin D2 assay. The HPLC instrument used was the same as for the vitamin D2 assay. HPLC with separation was performed on Kinetex PFP columns (4.6 mm 250 mm, 5 mm, Phenomenex, Inc., Torrance, CA, USA). The mobile phase was 50 mM sodium phosphate in water with 3% acetonitrile (LC grade, Merck) and 0.1% triethylamine (Sigma-Aldrich) adjusted to a pH of 7.3, at a flow rate of 1.0 mL/min and UV detection was at 254 nm. The content of ergothioneine was calculated on the basis of the calibration curve of each authentic ergothioneine (98% pure, Sigma-Aldrich). Flavonoids were determined according to the method of Zhishen et al. (1999). An aliquot (0.5 mL) of appropriately diluted sample or standard solutions of rutin (94% pure, Sigma-Aldrich) was added to the flask, which contained and 0.1 mL of 5% aqueous sodium nitrite (Sigma-Aldrich) solution. After 6 min, 0.1 mL of 10% aqueous aluminium chloride (Fluka) solution was added and after another 6 min, 1 mL 5% aqueous sodium hydroxide solution was added to the mixture. Thereafter, the mixture was diluted with the addition of 0.8 mL of deionised water and thoroughly mixed. The absorbance of the mixture was determined at 510 nm against a blank. Total phenols were determined according to the method of Taga et al. (1984). Each methanolic extract (20 mg) was dissolved in a solution of 5 mL of 1.3% HCl in methanol/deionised water (60:40, v/ v) and the resulting mixture (100 mL) was added to 2 mL of 20 mg/ mL aqueous sodium carbonate solution. After 3 min, 100 mL of 50% Folin-Ciocalteu phenol reagent (Sigma-Aldrich) was added to the mixture. After 30 min standing, the absorbance was measured at 750 nm against a blank. The content of total phenols was calculated on the basis of the calibration curve of gallic acid (98% pure, Sigma-Aldrich).
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2.8. Statistical analysis All the measurements were determined in triplicate through all steps of extraction and analysis for each component. The experimental data were subjected to an analysis of variance (ANOVA) for a completely random design (CRD) using Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA, 2009) to determine the least significant difference among means at the level of P < 0.05.
3. Results and discussion 3.1. Vitamin D2 content Without UV-B irradiation, fresh fruiting bodies contained low amounts of vitamin D2 (0–3.93 mg/g) and three species (Brazilian, wood ear and sticky pholiota mushrooms) did not contain vitamin D2 (Table 1). After UV-B irradiation for 2 h, the vitamin D2 content of fruiting bodies increased from 0–3.93 mg/g to 15.06–208.65 mg/ g dry weight. The first, second and third highest vitamin D2 content after UV-B irradiation was golden oyster, oyster and pink oyster mushrooms, respectively. The increase of vitamin D2 content in these three irradiated Pleurotus mushrooms was 44- to 83-fold as compared to that in the non-irradiated controls. Fig. 2 shows the high-performance liquid chromatography (HPLC) chromatogram for vitamin D2 for the treated and untreated samples of each species mushrooms. Mau et al. (1998) reported 12.48, 6.58 and 7.58 mg/g dry weight of vitamin D2 in button (Agaricus bisporus), shiitake (L. edodes) and straw mushrooms (Volvariella volvacea) after 2 h of UV-B irradiation (14.7 kJ/m2), respectively. Jasinghe and Perera (2005) reported 53.9, 184, 79.5 and 56.5 mg/g dry weight of vitamin D2 in shiitake, oyster, abalone (Pleurotus cystidiosus) and button mushrooms after 2 h of UV-B irradiation (25.2 kJ/m2), respectively. After the exposure to UV-B light, at a dose of 25 kJ/m2, the concentration of vitamin D2 was increased to 36.7, 68.6 and 106.4 mg/g dry weight for pileus, middle, and gillus parts of shiitake mushroom, respectively (Ko et al., 2008). The gill side of whole shiitake mushrooms exposed to 0, 25, 50 and 75 kJ/m2 increased to 2.8, 13.8, 40.7 and 61.9 mg/g dry weight, respectively (Ko et al., 2008). However, Simon et al. (2011) found that the vitamin D2 content increased from 5 to 410 mg/100 g dry weight by UV-B irradiation at a dose of 10.6 kJ/m2. These variations in vitamin D2 conversion might be due to the cultivar differences, different environmental conditions for mushrooms and different irradiation intensities and doses used. Phillips et al. (2012) found vitamin D4 should be Table 1 Effect of UV-B irradiation on vitamin D2 content of fresh fruiting bodies. Mushroom
Contenta (mg/g dry matter) Control (0 h)
Brazilian mushroom Popular Wood ear Hon-shimeji Shiitake Sticky pholiota King oyster Ferulae Golden oyster Oyster Pink oyster a
c
LOD 0.95 0.14 LODc 1.62 0.12 0.35 0.05 LODc 1.56 0.17 1.65 0.19 3.93 0.44 0.83 0.06 2.13 0.21
b b b b b b b b
2 hb 22.13 1.98 42.36 6.31 a 60.29 3.59 15.06 0.96 a 15.10 1.55 a 61.78 2.58 28.71 0.96 a 52.30 4.79 a 208.65 6.08 a 69.00 1.96 a 93.29 11.56 a
Number of analysed samples n = 3. All values are presented as means SD (n = 3). Means with different letters within a row differ significantly (P < 0.05). b Irradiation intensity = 0.36 mW/cm2. c Limit of detection.
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expected to occur in mushrooms exposed to UV light, such as commercially produced vitamin D enhanced products. Since the UV irradiation acts only on the surface of mushrooms, it is probably important to expose a larger fraction of mushrooms to UV irradiation. However, the round shape of fruiting bodies would result in poor exposure to UV-B irradiation. The use of mycelia with larger flat surface might resolve the problem of exposure area. Therefore, further study was to measure the effect of UV-B irradiation on the vitamin D2 content of three fresh mycelia of Pleurotus spp., whose fruiting bodies showed the highest vitamin D2 content after UV-B irradiation in Fig. 3. After UV-B irradiation for 2 h, the vitamin D2 content in fresh mycelia of golden oyster, oyster and pink oyster mushrooms increased from 2.72, 5.93 and 0.28 mg/g to 81.67, 81.71 and 66.03 mg/g, respectively (Fig. 3). It showed that the vitamin D2 content of both fruiting bodies and mycelia was 0.28–5.93 mg/g and their content could be increased to 66.03–208.65 mg/g after UV-B irradiation for 2 h. The treatment of UV-B irradiation for 2 h appears to have been effective for increasing the vitamin D2 content of both fruiting bodies and mycelia. Vitamin D2 is derived by photo-irradiation from its precursor ergosterol. This increase in vitamin D2 content in mushrooms is due to photosynthetic/thermal processes occurring from exposure of ergosterol to UV light (Simon et al., 2013). Sterols are known to be involved in the stabilization of membranes to UV light (Jasinghe and Perera, 2005). Both initial moisture content and temperature of irradiation could influence the conversion of ergosterol (Perera et al., 2003). In a preliminary study, after UV-B irradiation for 2 h, the vitamin D2 content in dry mycelia of golden oyster, oyster and pink oyster mushrooms was from 2.72, 5.93 and 0.28 mg/g to 34.73, 27.12 and 18.52 mg/g, respectively. Jasinghe and Perera (2005) reported that the optimal moisture content for the conversion of ergosterol vitamin D2 (22–27 mg/g dry weight) was around 70–80% on a wet weight basis of shiitake mushroom. However, in this study, fresh fruiting bodies and fresh mycelia used contained 87–90% moisture. It appears that the conversion of ergosterol to vitamin D2 could occur in both dry (dead) and fresh (live) mushroom fruiting bodies and mycelia. Furthermore, fresh fruiting bodies and mycelia with higher moisture content could more effectively enhance such vitamin D2 conversion. 3.2. Polysaccharide content Polysaccharide content in three fresh fruiting bodies and mycelia was affected by UV-B irradiation (Table 2). Polysaccharide content in three fresh mycelia and golden oyster fruiting bodies decreased after UV-B irradiation for 2 h, whereas that in fruiting bodies of oyster and pink oyster mushrooms increased after UV-B irradiation for 2 h. The UV-B irradiation might cause a decomposition of polysaccharides in mycelia but a synthesis of polysaccharides in two fruiting bodies of oyster and pink oyster, except for that of golden oyster mushrooms. In other words, with regard to polysaccharide content, fruiting bodies and mycelia responded differently to UV-B irradiation. However, for golden oyster, UV-B irradiation caused the degradation of polysaccharide in both fruiting bodies and mycelia. For each species, the polysaccharide content in mycelia was higher than that in its corresponding fruiting bodies. Unfortunately, there is no publication available regarding the effect of UV-B irradiation on mushroom polysaccharides. 3.3. Antioxidant properties The ethanolic extracts were obtained from freeze-dried samples and their yields were expressed as the g/100 g of samples on the basis of dry weight. For golden oyster, the yields for irradiated and non-irradiated fruiting bodies and mycelia were
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S.-J. Huang et al. / Journal of Food Composition and Analysis 42 (2015) 38–45
Fig. 2. HPLC chromatograms showing vitamin D2 profiles for each species of mushroom. Peaks: 1, vitamin D2; 2, ergosterol. Marked: a, non-irradiated sample; b, irradiated sample. The HPLC system consisted of a solvent degasser, gradient pump, temperature controlled autosampler, and UV detector (Hitachi Ltd., Tokyo, Japan), with a LiChrospher 100 RP-18 e column, 4.6 mm 250 mm, 5 mm (Merck KGaA, Darmstadt, Germany); mobile phase 950 mL, methanol: 50 mL H2O; flow rate 1.0 mL/min; detection at 254 nm.
comparable (13.4–14.1 g/100 g). Besides, the yields from irradiated and non-irradiated mycelia of oyster (15.5–17.4 g/100 g) and pink oyster (14.1–14.2 g/100 g) were higher than those from their corresponding fruiting bodies (9.62–9.80 g/100 g and 9.14–9.43 g/ 100 g, respectively). In addition, no difference in the yields of irradiated or non-irradiated fruiting bodies and mycelia was observed, except that the yield of irradiated oyster mycelia (17.4 g/ 100 g) was higher than that of the non-irradiated control (15.5 g/ 100 g).
The antioxidant properties assayed herein are summarized in Table 3, and the EC50 values (mg ethanolic extracts per mL) were calculated for comparison. With regard to reducing power, the EC50 values of irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster (0.92–4.73 mg/mL) were lower than those of the non-irradiated controls (1.02–4.94 mg/mL), except that the EC50 value of non-irradiated golden oyster mycelia (0.99 mg/mL) was higher than that of the irradiated mycelia (1.45 mg/mL). Ethanolic extracts from UV-B irradiated fruiting bodies and
S.-J. Huang et al. / Journal of Food Composition and Analysis 42 (2015) 38–45 a
200
a
FB0 FB2 MY0 MY2
100
Vitamin D2 (µg/g)
b a b b
50
c
c
c
d
c
0 Golden oyster
Oyster
d
Pink oyster
Fig. 3. Content of vitamin D2 of UV-B irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster mushrooms. Number of analysed samples n = 3. All values are presented as means SD (n = 3). Means with different letters within a group differ significantly (P < 0.05). FB0 and FB2, fresh fruiting bodies UV-B irradiated for 0 and 2 h, respectively; MY0 and MY2, freshly harvested mycelia UV-B irradiated for 0 and 2 h, respectively.
mycelia were more effective than those from the non-irradiated controls, except for non-irradiated golden oyster mycelia being more effective. Furthermore, three mycelia sample were more effective than their corresponding fruiting bodies in reducing power. For the scavenging ability on DPPH radicals, the EC50 values of irradiated fruiting bodies of oyster and pink oyster and irradiated mycelia of golden oyster and pink oyster (0.97–6.90 mg/mL) were higher than those of the non-irradiated controls (0.27–3.41 mg/ mL). No difference in the EC50 values between irradiated and nonirradiated samples was observed for golden oyster fruiting bodies and oyster mycelia. Ethanolic extracts from UV-B irradiated fruiting bodies and mycelia were less effective than those from non-irradiated fruiting bodies and mycelia, except that UV-B irradiation had no effect on the scavenging ability of golden oyster fruiting bodies and oyster mycelia. Three fruiting bodies were more effective than their corresponding mycelia in scavenging ability on DPPH radicals. For chelating ability on ferrous ions, the EC50 values of irradiated and non-irradiated golden oyster fruiting bodies and mycelia were 0.02–0.04 mg/mL, indicating that their ethanolic extracts were excellent chelators for ferrous ions. The EC50 values of irradiated pink oyster fruiting bodies and mycelia (0.11– 0.20 mg/mL) were lower than those of the non-irradiated controls (0.23–0.29 mg/mL). However, the EC50 values of irradiated oyster fruiting bodies and non-irradiated mycelia were higher than those
Table 2 Content of total water-soluble polysaccharides of UV-B irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster mushrooms. Contenta (mg/g dry matter)
Golden oyster Oyster Pink oyster
FB0b
FB2b
MY0b
MY2b
19.92 0.12 c
19.01 0.09 d
68.48 0.12 a
51.61 0.14 b
48.89 0.49 d 24.57 0.12 d
53.23 0.35 c 33.88 0.05 c
56.06 0.16 a 123.01 0.20 a
55.31 0.05 b 112.34 0.09 b
a Number of analysed samples n = 3. All values are presented as means SD (n = 3). Means with different letters within a row differ significantly (P < 0.05). b FB0 and FB2, fresh fruiting bodies UV-B irradiated for 0 and 2 h, respectively; MY0 and MY2, freshly harvested mycelia UV-B irradiated for 0 and 2 h, respectively.
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of their corresponding samples. Fruiting bodies and mycelia appear to be considerably effective in the chelating ability on ferrous ions, as evidenced by their low EC50 values of <1 mg/mL and <0.05 mg/mL (for golden oyster). Lee et al. (2007) found that the EC50 values of the ethanolic extracts from golden oyster fruiting bodies and mycelia were 2.28 and 8.29 mg/g for reducing power, 1.33 and 7.92 mg/g for scavenging ability, and 6.49 and 17.32 mg/g for chelating ability, respectively. Tsai et al. (2009) reported that the EC50 values of the ethanolic extracts from oyster fruiting bodies were 6.76, 5.58 and 3.48 mg/g for reducing power, scavenging and chelating abilities, respectively. The EC50 values of the ethanolic extracts from golden oyster and oyster were lower than those previously published. However, the antioxidant properties of UV-B irradiated mushrooms have not been published. Therefore, this is the first time to report the effect of UV-B irradiation on reducing power, scavenging and chelating abilities. Overall, EC50 values in the ethanolic extracts of non-irradiated and irradiated fruiting bodies and mycelia were 0.92–4.94, 0.20– 6.90 and 0.02–0.84 mg/mL for reducing power, scavenging ability on DPPH radicals and chelating ability on ferrous ions, respectively. These fruiting bodies and mycelia were effective in the reducing power and scavenging ability on DPPH radicals and considerably effective in the chelating ability on ferrous ions. UV-B treatment could slightly enhance the reducing power and chelating ability but slightly decrease the scavenging ability of fruiting bodies and mycelia. Although antioxidants such as BHA, ascorbic acid and atocopherol are good for reducing power and scavenging ability on DPPH radicals, and EDTA is a good chelator for ferrous ions, they are additives and used or present in milligram levels in foods. However, the ethanolic extracts from UV-B irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster could be used in gram levels as food or a food ingredient. Therefore, these ethanolic extracts from both UV-B irradiated fruiting bodies and mycelia might also serve as possible protective agents in the human diet in addition to their high vitamin D2 content. 3.4. Antioxidant components Phenolic compounds are widely distributed in mushrooms. One phenolic compound, ergothioneine (2-mercaptohistidine trimethylbetaine), is a naturally occurring amino acid which is synthesized in some bacteria and fungi but not in animals (Melville et al., 1955). It has been documented that ergothioneine is an antioxidant in vivo (Hartman, 1990) and a cellular protector against oxidative damage (Aruoma et al., 1999). Non-irradiated fruiting bodies and mycelia contained 1.42–3.46 and 0.63–1.39 mg/g of ergothioneine, respectively (Table 4). The ergothioneine content of fruiting bodies was much higher than that of their corresponding mycelia. Generally, UV-B irradiation had an influence on the ergothioneine content of fruiting bodies and mycelia, except for oyster fruiting bodies. After UV-B irradiation for 2 h, the ergothioneine content of irradiated fruiting bodies and mycelia contained 0.71–3.13 and 0.63–0.66 mg/g of ergothioneine, respectively. Although UV-B irradiation lowered the ergothioneine content in fruiting bodies and mycelia, these irradiated samples were still an abundant source of ergothioneine. Flavonoids act in plants as antioxidants, antimicrobials, photoreceptors, visual attractors, feeding repellents, and light screening substances. Non-irradiated fruiting bodies and mycelia contained 5.62–11.96 and 2.26–8.81 mg/g of flavonoids, respectively (Table 4). UV-B irradiation seems to have had a slight influence on the flavonoid content of fruiting bodies and mycelia. These results are consistent with the finding of Liu et al. (2011) that post-harvest irradiation with UV-B enhanced the amounts of
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Table 3 EC50 values of ethanolic extracts from UV-B irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster mushrooms in antioxidant properties. EC50 valuea (mg extract/mL) FB0b
FB2b
MY0b
MY2b
Reducing power Golden oyster Oyster Pink oyster
2.05 0.01 a 4.94 0.02 a 2.93 0.02 a
1.97 0.06 b 4.73 0.08 b 2.51 0.03 b
1.02 0.03 c 0.99 0.01 d 1.76 0.01 c
0.92 0.01 d 1.45 0.03 c 1.04 0.02 d
Scavenging ability Golden oyster Oyster Pink oyster
0.20 0.01 b 0.83 0.01 c 0.84 0.01 d
0.25 0.01 b 1.44 0.03 b 0.97 0.01 c
0.27 0.01 b 5.10 0.11 a 3.41 0.05 b
1.35 0.51 a 5.06 0.08 a 6.90 0.02 a
Chelating ability Golden oyster Oyster Pink oyster
0.02 <0.01 c 0.84 0.04 a 0.23 0.01 b
0.03 <0.01 b 0.74 0.01 b 0.20 0.02 c
0.04 <0.01 a 0.42 0.01 d 0.29 0.01 a
0.04 <0.01 a 0.59 0.02 c 0.11 0.01 d
a EC50 value: the absorbance was 0.5 for reducing power; 2,2-diphenyl-1-picrylhydrazyl radicals were scavenged by 50%; and ferrous ions were chelated by 50%, respectively. EC50 value was obtained by interpolation from linear regression analysis. Number of analysed samples n = 3. All values are presented as means SD (n = 3). Means with different letters within a row differ significantly (P < 0.05). b FB0 and FB2, fresh fruiting bodies UV-B irradiated for 0 and 2 h, respectively; MY0 and MY2, freshly harvested mycelia UV-B irradiated for 0 and 2 h, respectively.
flavonoids, phenols and total phenols in the tomato fruiting. Irradiated fruiting bodies and mycelia contained 5.31–7.26 and 1.78–8.21 mg/g of flavonoids, respectively. However, for golden oyster fruiting bodies and mycelia, after UV-B irradiation for 2 h, no significant difference in flavonoid content between irradiated and non-irradiated samples was observed. Total phenols are the major naturally occurring antioxidant components found in fruiting bodies and mycelia (Table 4). The high amount of total phenols (6.36–18.92 mg/g) in the ethanolic extracts might explain their higher effectiveness in reducing power and scavenging ability on DPPH radicals. UV-B irradiation had no to slight influence on total phenol content of three fruiting bodies, but showed different effects on total phenol content of three mycelia. After UV-B irradiation for 2 h, total phenol content of golden oyster and oyster mycelia increased, while that of pink oyster mycelia decreased. Lee et al. (2007) found that the total phenol content of the ethanolic extracts from golden oyster fruiting bodies and mycelia was 8.62 and 5.84 mg/g, respectively, much lower than those in Table 4. Tsai et al. (2009) reported that the EC50 values of the ethanolic extracts from oyster fruiting bodies were 7.11 mg/g,
slightly lower than that in Table 4. These results are consistent with their effectiveness in antioxidant properties assayed. However, the total phenol content of UV-B irradiated mushrooms has not been published. Therefore, this is the first report on the effect of UV-B irradiation on their total phenol content. Phenols such as butylated hydroxytoluene (BHT) and gallate are known to be effective antioxidants (Madhavi et al., 1996). Due to their scavenging abilities on free radicals and chelating abilities on ferrous ions, phenols might possess good antioxidant, antimutagenic and anticancer properties (Ahmad and Mukhtar, 1999). Tsai et al. (2007) found that the content of total antioxidant components was moderately to highly correlated (r = 0.636– 0.907) with antioxidant properties. Although UV-B irradiation has a slight influence on the content of ergothioneine, flavonoids, total phenols of fruiting bodies and mycelia, these irradiated samples still contained sufficient amount of these antioxidant components. Accordingly, in addition to their high vitamin D2 content, UV-B irradiated fruiting bodies and mycelia might also be potential antioxidants for use in food products and could be developed as a new dietary supplement and functional foods.
Table 4 Content of ergothioneine, flavonoids and total phenols of ethanolic extracts from UV-B irradiated fruiting bodies and mycelia of golden oyster, oyster and pink oyster mushrooms. Contentsa (mg/g) FB0b
FB2b
MY0b
MY2b
Ergothioneine Golden oyster Oyster Pink oyster
3.46 0.03 a 1.42 0.05 b 1.48 0.02 a
3.13 0.03 b 1.99 0.01 a 0.71 0.02 b
1.39 0.03 c 0.84 0.01 c 0.63 0.02 c
0.63 0.01 d 0.66 0.01 d 0.59 0.02 d
Flavonoids Golden oyster Oyster Pink oyster
5.62 0.34 b 11.96 0.50 a 7.92 0.03 a
5.31 0.20 b 7.26 0.06 b 6.53 0.49 b
8.81 0.09 a 2.26 0.11 c 6.28 0.13 b
8.21 0.37 a 1.78 0.20 c 3.23 0.22 c
Total phenols Golden oyster Oyster Pink oyster
18.92 0.27 a 7.56 0.06 c 13.32 0.21 a
18.25 0.09 b 7.15 0.21 c 13.49 0.18 a
11.37 0.06 d 9.65 0.20 b 7.32 0.02 b
13.08 0.18 c 11.58 0.22 a 6.36 0.25 c
a b
Number of analysed samples n = 3. All values are presented as means SD (n = 3). Means with different letters within a row differ significantly (P < 0.05). FB0 and FB2, fresh fruiting bodies UV-B irradiated for 0 and 2 h, respectively; MY0 and MY2, freshly harvested mycelia UV-B irradiated for 0 and 2 h, respectively.
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4. Conclusion Based on the results, 2 h of UV-B irradiation not only significantly increased the vitamin D2 content of mushroom fruiting bodies, but it could also significantly enhance its content of vitamin D2 of mycelium. UV-B irradiation could also slightly enhance the reducing power and chelating ability, but slightly decrease the scavenging ability of fruiting bodies and mycelia. Obviously, the amount of vitamin D of fruiting bodies and mycelia increased was not consistent. For developing product with rich vitamin D2, irradiated golden oyster fruiting bodies were the best choice; but thinking about the cost, the mycelia of irradiated golden oyster or oyster maybe were the better choices, because of the cultivation time that mushroom mycelia could be harvested is only a few days, but for fruiting bodies is a few months. However, mushrooms possess many bioactive compounds that must be taken into consideration when mushrooms are irradiated by UV-B. Overall, in addition to their high vitamin D2 content, UV-B irradiated fruiting bodies and mycelia might also be potential antioxidants that could be developed as a new dietary supplement. In the future, further exploring the cause of different impacts between mushroom fruiting bodies and mycelia with UV-B irradiation will help to develop more suitable products. Acknowledgments The study was supported by the Ministry of Science and Technology, Taiwan, Republic of China (NSC 98-2313-B-468-004MY3) and Asia University (102-asia-05). We thank Mr. Shih-Wen Fang, Q-Yo Bio-Technology Farm for providing mushrooms. References Ahmad, N., Mukhtar, H., 1999. Green tea polyphenols and cancer: biologic mechanisms and practical implications. Nutr. Rev. 57, 78–83. Aruoma, O.I., Spencer, J.P.E., Mahmood, N., 1999. Protection against oxidative damage and cell death by the natural antioxidant ergothioneine. Food Chem. Toxicol. 37, 1043–1053. Dinis, T.C.P., Madeira, V.M.C., Almeida, L.M., 1994. Action of phenolic derivatives (acetaminophen, salicylate, and 5-amino salicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys. 315, 161–169. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Dubost, N.J., Ou, B., Beelman, R.B., 2007. Quantification of polyphenols and ergothioneine in cultivated mushrooms and correlation to total antioxidant capacity. Food Chem. 105, 727–735. Hartman, P.E., 1990. Ergothioneine as an antioxidant. Methods Enzymol. 186, 310– 318. Holden, J.M., Lemar, L.E., Exler, J., 2008. Vitamin D in foods: development of the US Department of Agriculture database. Am. J. Clin. Nutr. 87 (Suppl.), 1092S– 1096S. Holick, M.F., 2007. Vitamin D deficiency. N. Engl. J. Med. 357, 266–281. Jasinghe, V.J., Perera, C.O., 2006. Ultraviolet irradiation: the generator of vitamin D2 in edible mushrooms. Food Chem. 95, 638–643. Jasinghe, V.J., Perera, C.O., 2005. Distribution of ergosterol in different tissues of mushrooms and its effect on the conversion of ergosterol to vitamin D2 by UV irradiation. Food Chem. 92, 541–546. Kalac, P., 2013. A review of chemical composition and nutritional value of wild growing and cultivated mushrooms. J. Sci. Food Agric. 93, 209–218.
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