Fruiting Bodies - American Chemical Society

Article pubs.acs.org/JAFC

Mycochemical Changes Induced by Selenium Enrichment in P. ostreatus Fruiting Bodies Jorge A. Carrasco-Gonzalez,† Sergio O. Serna-Saldívar,† and Janet A. Gutierrez-Uribe*,† †

Tecnológico de Monterrey, Escuela de Ingenierı ́a y Ciencias, Centro de Biotecnologı ́a-FEMSA, Av. Eugenio Garza Sada 2501 Sur, Col. Tecnológico C.P. 64849, Monterrey, Nuevo León, Mexico ABSTRACT: The effects of selenium enrichment on the biological efficiency, phenolic compounds, amino acid profile, antioxidant capacity, and cellular antioxidant activity (CAA) were evaluated in Pleurotus ostreatus fruiting bodies (FB) harvested during three sequential flushes. Sodium selenate was used to reach selenium content of 17.5 or 5.8 mg/kg in the sorghum straw substrate. Biological efficiency and total selenium content increased. One of the main differences among treatments was in ergothioneine content, an indicator of oxidative stress that was positively related with valine and isoleucine contents and negatively related to leucine and phenylalanine. Besides ergothioneine, nucleosides derived from adenine and uracyl were the major peaks observed in all treatments, and coumaric and ferulic acids were found in the bound phenolics extract. Selenium enrichment also affected the antioxidant capacity, and particularly the methanolic extract obtained from the second flush of FB cultivated in selenium-enriched substrate (17.5 mg/kg) had the best CAA. KEYWORDS: Pleurotus ostreatus, selenization, nutraceutical, antioxidant, ergothioneine

■

this study was to analyze the effect of selenization and flushing on the P. ostreatus fruiting body (FB) mycochemical profile and antioxidant capacity and cellular antioxidant activity (CAA). Sodium selenate was used to reach a selenium content of 17.5 or 5.8 mg/kg in the red sorghum straw substrate.

INTRODUCTION Pleurotus ostreatus is an edible mushroom with high nutritional and nutraceutical values that can be economically and easily cultivated on a wide variety of agro-industrial wastes.1 Its nutraceutical value is due to mycochemicals related with immunomodulatory, anticholesterolemic, anticancer, and antioxidant activities.2 Ergothioneine is one of the most relevant mycochemicals, due to its good bioavailability, stability under physiological conditions, and powerful antioxidant activity.3 Pleurotus species are a good source of selenium, due to their ability to absorb and accumulate minerals from the substrate.4 However, it is known that selenium absorption in mushrooms depends on factors such as substrate type, selenium concentration, strain, and harvest or flush.5,6 Selenium has been related to a positive effect on biological efficiency of P. ostreatus because it increases the synthesis and activity of ligninolytic enzymes such as laccase and peroxidase.7 Additionally, selenium protects fungal cells against the lipid peroxidation generated under stress conditions.6,8 Selenium has the ability to replace sulfur in methionine, cysteine, and ergothioneine to generate selenized amino acids such as selenomethionine, selenocysteine, and selenoneine, respectively, which have been related to antioxidant and immunomodulatory activities.9−11 Selenium from P. ostreatus was more bioavailable than the inorganic counterpart because organic forms are effectively incorporated for the synthesis of antioxidant enzymes (glutathione peroxidase, superoxide dismutase), which diminish the number of tumor nodules of lung cancer.12,13 Additionally, it has been described that selenium enrichment for the cultivation of Pleurotus fruiting bodies (FB) affects both the content of this mineral and the mycochemical profile.14,15 Recently, it has been proved that mushrooms do not synthesize or absorb flavonoids but instead nucleosides and amino acid derivatives, which have been identified in polar solvents extracts.16,17 Therefore, the aim of © 2017 American Chemical Society

■

MATERIALS AND METHODS

Selenization of P. ostreatus Fruiting Bodies. The P15 strain of P. ostreatus (Setas Cultivadas, Ciudad de México, Mexico) was used. The substrate used for the cultivation was red sorghum straw obtained from Montemorelos, Nuevo León, Mexico. The bale of sorghum straw was triturated into 3−5 cm fragments using a hammer mill (Pulvex M200, Ciudad de México, Mexico). The manipulation of the strain was carried out under aseptic conditions using a laminar flow hood. Red sorghum grains hydrated to 75% were used for secondary mycelial growth (spawn). Subsequently, sorghum grains were introduced into autoclavable flasks with 2% of calcium sulfate and 0.5% calcium carbonate (w/w) and sterilized for 20 min at 121 °C. Sterilized grains were inoculated aseptically with 2 cm2 of primary inoculum and then incubated at 25 °C for 14 days. For mycelial expansion on substrate, red sorghum straw was used. Five kilograms of sorghum straw (10% moisture) was pasteurized with water in a metal container at 80 °C for 90 min, and then the water was drained. The resulting wet straw contained a final moisture of 70%. The pasteurized substrate was tempered to 40 °C in a conditioning room before inoculation with 500 g of secondary inoculum. The inoculated substrate was packaged in polypropylene bags with a 0.5 μm filter (Unicorn, Plano, TX, USA) and sealed with a heat sealer. A solution of 1000 ppm of sodium selenate (S8295, Sigma-Aldrich, St. Louis, MO, USA) was injected to reach the following selenium doses: 5.8 or 17.5 mg/kg of dry substrate, which corresponded to low (LSe) or high (HSe) Se concentration, respectively. Received: Revised: Accepted: Published: 4074

February April 26, April 27, April 27,

20, 2017 2017 2017 2017 DOI: 10.1021/acs.jafc.7b00715 J. Agric. Food Chem. 2017, 65, 4074−4082

Article

Journal of Agricultural and Food Chemistry The bags were incubated at 25 °C for 21 days, and the growth and appearance of contaminants were monitored every 3 days. After the incubation period, the fruiting stage was induced by exposure to light (photoperiod of 12 h), watering to maintain a relative humidity of 80% using an AquaFog Hydro SS 700 (Jaybird Manufacturing Inc., Centre Hall, PA, USA) and gas exchange generated by punching 12 holes in the bags. The fruiting bodies were harvested when the stipe was developed and they had a wavy and cracked pileus. From each culture bag, the fruiting bodies of the first three flushes were considered to obtain the biological efficiency (BE). The BE was calculated on the basis of the sum of the fresh fruiting bodies’ weight divided by the dry weight of the substrate. The fruiting bodies were dehydrated in a fruit dryer set at 50 °C (Excalibur 3900B, Sacramento, CA, USA) during 24 h, milled using a coffee grinder, and stored at −20 °C in hermetic metalized plastic bags. Quantification of Total Selenium. Samples of 0.2 g were digested with 3 mL of nitric acid in Teflon vessels and microwaved (Mars 5 Xtraction CEM, Matthews, NC, USA) as previously reported.15 The following protocol was used: 15 min of temperature increase, 15 min at 180 °C (hold time), and 20 min of cooling. An aliquot of the hydrolysate was taken and filtered through a 13 mm (membrane GHP 0.45 μm Acrodisc) filter using a syringe. Total Se concentration was analyzed by inductively coupled plasma mass spectrometry (Thermo Scientific XSeries 2 ICP-MS) using the mass of Se 78 and 82. Rhodium was used as an internal standard. Ergothioneine and Phenolic Compounds Analysis. ENTER Ergothioneine Extraction. Ergothioneine extraction was carried out using 0.5 g of P. ostreatus powder and 10 mL of 70% ethanol in water with 100 μM betaine monohydrate (B2754, Sigma-Aldrich), 10 mM dithiothreitol (D0672, Sigma-Aldrich), and 100 mM 2-mercapto-1methylimidazole (CBR00498 Sigma-Aldrich). The samples were vigorously vortexed for 2 min, and then 2 mL of 70% ethanol in water with 1% sodium dodecyl sulfate was added, vortexed again for 2 min, and centrifuged for 10 min at 1677g and 4 °C. The supernatant was recovered and concentrated in an evaporator at 45 °C (GeneVac EZ-2 series, Gardiner, NY, USA). The extract was resuspended in 2 mL of 50% methanol and stored at −20 °C until further analysis.18 Phenolic Compounds Extraction. The extraction of phenolic compounds was performed using 500 mg of mushroom powder, which was stirred vigorously for 2 min with 15 mL of 80% methanol (v/v) and then sonicated (Branson 2510 CT) for 15 min.19 Samples were placed at −20 °C for 90 min, the supernatant was recovered, and the extraction was repeated twice. The recovered methanol was concentrated under vacuum at 45 °C (GeneVac EZ-2 series). The extract was resuspended in 2 mL of 50% methanol and centrifuged at 1677g for 10 min, and the supernatant was recovered. Due to the high abundance of ergothioneine in the extracts, a fractionation using solid phase extraction cartridges (Strata, 55 μm, 6 mL, 8B-S001-HCH, Phenomenex, Torrance, CA, USA) was performed afterward. The cartridge column was conditioned with aqueous 10 mM HCl, and then the extract previously diluted (1:5) with aqueous HCl 10 mM was passed. The cartridge was washed with aqueous 10 mM HCl to remove most of the ergothioneine, and then 5 mL of 100% methanol was used to elute the substances of interest for further analysis. The solvent was dried in a GeneVac set at 45 °C, resuspended in 2 mL of methanol 50%, and stored at −20 °C until further analysis. To obtain the bound phenolics, 20 mL of sodium hydroxide (4 M) was added to the remaining pellet of methanolic extraction.19 The tubes were saturated with nitrogen just after adding sodium hydroxide to remove the oxygen of the headspace and then incubated for 90 min at 25 °C. The pH was adjusted to 2 using hydrochloric acid (6 N), and 15 mL of hexane was added, vortexed for 1 min, and centrifuged for 10 min at 1677g to remove the upper phase (corresponding to the hexane). Subsequently, samples were washed three times with ethyl acetate as follows: added 10 mL of ethyl acetate was vortexed for 1 min and centrifuged for 10 min at 1677g, and the upper phase corresponding to ethyl acetate was recovered. The ethyl acetate was concentrated in the GeneVac at 45 °C for 5 h, and the pellet was resuspended with 2 mL of methanol 50%. Subsequently, the extract

was passed through a filter of 0.45 μm and stored at −20 °C until further analysis. Chromatographic Method for Phenolic Compounds and Ergothioneine Analysis. Ergothioneine and phenolic compounds were analyzed by HPLC-PDA (Agilent Technologies, 1260 Series, Santa Clara, CA, USA) using a method previously reported for phenolic acids.20 The following standards from Sigma-Aldrich were used: L-(+)-ergothioneine (E7521), gallic acid (G7384), protocatechuic acid (1579310), p-hydroxybenzoic acid (240141), caffeic acid (C0625), ferulic acid (PHR1791), p-coumaric acid (C9008), phenylalanine (P2126), adenosine (A9251), and syringic acid (C6881). The separation was performed using water at pH 2 (adjusted with trifluoroacetic acid) (solvent A) and methanol 100% (solvent B) in a Luna C-18 250 mm × 4.6 mm, 5 μm, column (Phenomenex Inc.) at 25 °C and a flow of 0.8 mL/min. The gradient was as follows: 15% B at 0 min, 58% B at 10 min, 80% B at 14 min, and 100% B at 20 min and post-time of 5 min with the initial conditions. The detection signal used was 280 nm. The identification of the compounds was confirmed by HPLC-MSTOF (Agilent 1100). The chromatographic conditions were the same used for analysis by HPLC-PDA. The ionization was effected by electrospray in positive and negative modes, using nitrogen gas (350 °C), detecting over a range of m/z 150−1500, fragmentor of 50 V, skimmer at 40 V, OCT RF at 250 V, gas temperature of 300 °C, drying gas at 10 L/min, nebulized pressure of 40 psig, and capillary voltage of 4000 V. Analysis of Amino Acid Released after 24 h of Protease Digestion. The amino acid extraction process was performed by enzymatic digestion using a protease (type XIV) isolated from Streptomyces griseus (Sigma-Aldrich P-5147). Amounts of 0.2 g of P. ostreatus and 0.02 g of enzyme were suspended in 15 mL of distilled water (70 units of enzyme/mL). The samples were incubated at 37 °C for 24 h, and then the samples were centrifuged at 1677g for 10 min at 4 °C. The supernatant was recovered and passed through a 0.22 μm filter.21 The amino acids released after protease digestion were determined by HPLC-fluorescence using the Waters AccQ Tag Amino Acid Analysis method (Millipore Corp., Milford, MA, USA). The samples were diluted 1:10 with water (HPLC grade) and subsequently derivatized with WatersAccQ reagent and analyzed using a C-18 column (Waters AccQ HPLC Nova Pak, 4 μm, 3.9 × 150 mm). For quantification, an external amino acids standard mix (AccQ Waters) and selenomethionine (Sigma-Aldrich) were employed. Antioxidant Tests. The antioxidant capacity of methanolic extracts of mushroom powder was determined using the oxygen radical absorbance capacity (ORAC) assay.22 The results were expressed as micromoles of Trolox equivalents (TE) per gram of mushroom powder. CAA was evaluated using Caco-2 cells, which were seeded in a 96well microplate (1 × 104−1 × 105 cells/well) when reaching their exponential growth phase. After incubation during 24 h, the cell culture medium was discarded, and the cells were gently washed with 100 μL of PBS (37 °C). The methanolic extracts were tested at five 1:10 serial dilutions starting with 1 μg/mL (based on the mushroom powder). The extracts were diluted in 100 μL of cell culture medium with 60 μM dichlorodihydrofluorescein diacetate (DCFH-DA). After 20 min of incubation at 37 °C and 5% of CO2, the medium was removed and the cells were washed again with 150 μL of warm PBS. Then 100 μL of PBS with 94.5 μg/mL of 2,2′-azobis(2amidinopropane) dihydrochloride (AAPH) was added to detect fluorescence (485/538 nm) during 2 h.23 CAA units were calculated with the formula

∫ SA/∫ CA) × 100

CAA units = 100 − (

where ∫ SA (sample area) is the integrated area under the curve for the sample fluorescence versus time and ∫ CA (control area) is the integrated area from the control curve. 4075

DOI: 10.1021/acs.jafc.7b00715 J. Agric. Food Chem. 2017, 65, 4074−4082

Article

Journal of Agricultural and Food Chemistry

■

RESULTS AND DISCUSSION Biological Efficiency and Days to Harvest. There was no change in the color or morphology of FB due to selenium enrichment, but it induced a delay in the harvest time of the second and third flushes (2 and 3 days, respectively), compared to the control (Figure 1A). The delayed harvest time is

total Se, ergothioneine, and amino acids released after protease digestion. No selenomethionine was detected in the enzymatically digested samples. Ergothioneine concentration was the response that explained 99.9% of the variation among samples, and the highest contents were found in the control FB from flushes 2 and 3 as well as the flush from HSe (Figure 2A, right quadrants). The selenium content of control P. ostreatus fruiting bodies was in the range of 0.35−1.50 μg/g DW (Figure 2B), similar to and lower than those reported for cultivated on nonenriched

Figure 1. (A) Effect of selenium on days to harvest of the three flushes of P. ostreatus fruiting bodies obtained from control, nonselenized; LSe, low selenium concentration (5.8 mg Se/kg substrate dw); or HSe, high selenium concentration (17.5 mg Se/kg substrate dw). (B) Biological efficiency of each flush for the different treatments. Statistical analysis for panel A was performed by treatments for each flush and for panel B by treatment including the three flushes. Different letters denote significant differences (α = 0.05).

considered as a negative factor for the industry, but HSe and LSe had higher total BE (126 and 117%, respectively) than observed for the control (108%), mainly due to the difference observed in BE of the first flush (Figure 1B). Therefore, selenization of the P. ostreatus cultures is a low-cost strategy with high industrial potential because it increased mushroom production with a production cost of only U.S. $0.17 per kg of fresh FB. The total BE values of HSe and LSe were higher than that obtained in the P. ostreatus cultivated from coffee husks enriched with selenium (66%)6 but similar to that obtained by cultivation in a mixture of cottonseed hulls and wheat bran (125.6%).24 Biomass production decreased in all treatments according to the flush number. It has been widely reported that the biological efficiency depends on the nutrient content in the culture medium, but little is known about the selenization effect.25 The fruiting bodies obtained from the selenized treatments were distinguished by having a slight smell of garlic, which has been previously related to sulfur compounds generated by selenization.26 Total Selenium, Ergothioneine, and Amino Acids Contents. To evaluate the main differences among fruiting bodies obtained from the three flushes, a principal component analysis (PCA) was performed with the data obtained from the

Figure 2. (A) Principal component analysis of amino acids, selenium, and ergothioneine contents of P. ostreatus fruiting bodies from different flushes (1−3) of control, high, and low selenium (HSe and LSe) treatments. (B) Ergothioneine and total selenium contents. (C) Positive correlation between ergothioneine and valine or isoleucine contents. (D) Negative correlation between ergothioneine and leucine or phenylalanine contents. Statistical analysis for panel B was performed by treatment and flush. Different letters denote significant differences (α = 0.05). 4076


Article


Figure 3. (A) Chromatogram obtained at 280 nm from phenolic acids standards. Peaks: 1, gallic acid; 6, protocatechuic acid; 7, hydroxybenzoic acid; 8, caffeic acid. (B) Chromatogram obtained at 280 nm of compounds found in the methanolic extract from the second flush of P. ostreatus cultivated in a substrate enriched with low selenium (LSe) concentration (5.8 mg Se/kg dw). Peaks: 2, 5′-deoxy-5′-(methylsulfinyl)adenosine; 3, glutamic acid 5-2-(α-hydroxy-p-tolyl)hydrazide; 4, 1-(2,3-di-O-acetyl-2-C-methyl-5-O-p-methylbenzoyl-β-D-ribofuranosyl)uracil; 5, phenylalanine.

substrates such as coffee husks (0.12−0.96 μg/g DW) and wheat straw (2.7−3.4 μg/g DW), respectively.5,6,15 As expected, the highest selenium concentration (15.1−15.2 μg/g DW) was observed for HSe fruiting bodies from flushes 1 and 2 (Figure 2A, upper quadrants). A consumption of only 3.6 g of dry mushrooms from HSe could supply the recommended daily selenium intake of 55 μg.27 For HSe, the flush number significantly affected the selenium content of P. ostreatus; fruiting bodies of the third flush had 51% less than those harvested for flushes 2 and 1. Interestingly, no significant differences of selenium content were observed in the three different flushes of fruiting bodies corresponding to the LSe treatment. It has been previously reported that the selenium absorption rate is not constant or directly proportional to the salt concentration used to enrich the substrate.6

The selenium contents of spent mushroom compost from selenized and nonselenized treatments were similar (0.7−0.6 μg/g DW), indicating the wide ability of P. ostreatus to bioabsorb organic and inorganic selenium associated with the substrate (data not shown). In the case of selenized substrates, the mineral was more available because it was dissolved in water and the mycelium can absorb it effectively by passive absorption.28 Selenium content in plants is linked to fiber and protein components; therefore, mushrooms first need to hydrolyze fiber components to absorb the mineral.29,30 This study reported for the first time the effect of selenium and flush number on the ergothioneine content of P. ostreatus. Interestingly, the control fruiting bodies from flushes 2 and 3, along with flush 3 from HSe, showed the highest ergothioneine 4077


Article


Table 1. Comparison of ESI-MS and UV−Vis Data of Phenolic Acids and Peaks Detected at 280 nm in Methanolic Extract from P. ostreatus m/z peak

λmax (nm)

(+)

1 2 3

217, 270 218, 280 258

171.07 (M + H) 314.10 (M + H) 268.11 (M + H), 290.09 (M + Na), 306.07 (M + K), 557.19 (2M + Na)

4

218, 278

5

220, 270

166.09 (M + H), 331.17 (2M + H)

6 7 8

218, 280 256 218, 324

155.07 (M + H) ND 181.08 (M + H)

(−)

accurate mass

compound ID

459.13 (M − H) 164.07 (M − H), 329.14 (2M − H)

gallic acid 5′-deoxy-5′-(methylsulfinyl)adenosine glutamic acid 5-2-(α-hydroxy-p-tolyl)hydrazide

170.11 313.08 267.12

1-(2,3-di-O-acetyl-2-C-methyl-5-O-pmethylbenzoyl-β-D-ribofuranosyl)uracil phenylalanine

460.15

protocatechuic acid hydroxybenzoic acid caffeic acid

154.12 138.12 180.15

165.08

Table 2. Content and Abundance Based on Area under the Curve (AUC) at 280 nm of Compounds Found in the Methanolic Extract from Three Different Flushes of P. ostreatus Fruiting Bodies Expressed in Dry Weighta

a

treatment

flush

5′-deoxy-5′-(methylsulfinyl)adenosineb (μg/g)

peak 3 (AUC)

peak 4 (AUC)

phenylalanine (μg/g)

control

1 2 3

14.2 ± 0.8cd 20.5 ± 1.7b 23.8 ± 0.1a

11.3 ± 0.1de 17.8 ± 1.5bc 34.3 ± 0.4a

43.2 ± 2.7ab 86.9 ± 7.9a 35.5 ± 3.3b

82.1 ± 2.9a 65.2 ± 6.0bc 30.4 ± 2.0e

LSe

1 2 3

20.7 ± 1.8b 15.2 ± 1.5c 11.8 ± 0.5d

14.0 ± 0.5cd 19.8 ± 2.1b 9.8 ± 0.3e

60.3 ± 11.9ab 59.2 ± 16.3ab 39.8 ± 16.5b

63.5 ± 2.6bc 57.8 ± 7.9bc 53.2 ± 1.5cd

HSe

1 2 3

11.9 ± 0.8d 14.1 ± 0.1c 12.9 ± 0.1cd

9.6 ± 0.8e 9.3 ± 0.5e 12.1 ± 0.1de

42.0 ± 2.2b 72.7 ± 12.8ab 75.1 ± 11.6ab

33.2 ± 0.7e 46.6 ± 2.6d 67.1 ± 10.0b

Statistical analysis was performed by treatment and flush; different letter(s) denote significant differences (α = 0.05). bAdenosine equivalents.

flush 1 of all treatments (Figure 2D). The lower contents in flushes 2 and 3 were related to the increase in ergothioneine content as a response to oxidative stress produced by nutrient depletion in the substrate. Phenolic Compounds Profile. The standards of gallic (peak 1), protocatechuic (peak 6), hydroxybenzoic (peak 7), and caffeic (peak 8) acids appeared in the same retention time window as the most abundant compounds detected at 280 nm in the methanolic extract, but these were not identified as phenolic acids (Figure 3). Gallic, protocatechuic, hydroxybenzoic, and caffeic acids had been reported in different Pleurotus species, even in fruiting bodies obtained from selenized substrates; however, they were analyzed only on the basis of comparisons of the retention time and UV spectra.14,37 The UV spectra of the analyzed compounds in the methanolic extract were similar to those of standards of phenolic acid compounds, but the mass spectra did not match. Peak 2 was identified as 5′-deoxy-5′-(methylsulfinyl)adenosine on the basis of MS, UV spectra, and retention time of an adenosine standard (Table 1). It was previously reported in Ganoderma lucidum mushroom. 38 Peak 3 corresponded to glutamic acid 5-2-(α-hydroxy-p-tolyl)hydrazide also known as agaritine that was previously reported in Agaricus bisporus.39 On the basis of the accurate mass, peak 4 was tentatively identified as 1-(2,3-di-O-acetyl-2-C-methyl-5-Op-methylbenzoyl-β-D-ribofuranosyl)uracil; this compound has not been previously reported in P. ostreatus, but uracyl nucleotides have been related to its umami taste.40 Phenylalanine (peak 5) was confirmed on the basis of retention time

contents (846, 875, and 815 μg/g DW, respectively) (Figure 2B). Ergothioneine contents were similar to those previously reported in P. ostreatus FB cultivated in Japan (944.1 μg/g DW) and higher than those grown in Korea and Taiwan (165.3 and 216.4 μg/g DW, respectively).18,31 It has been observed that ergothioneine concentration increases as a response to oxidative damage generated by nutrient deficiency and by supplementation of its precursor (histidine) in the substrate.32−34 Interestingly, no significant differences among the ergothioneine contents of the different flushes of LSe fruiting bodies were observed. In fact, they were similar to those obtained from the first flushes of both the control and HSe, indicating that oxidative stress was prevented. Significant correlations were found between ergothioneine and valine, isoleucine, leucine, and phenylalanine released after protease digestion. Valine and isoleucine were positively related (Spearman ρ 0.71 and 0.79, respectively) with ergothioneine content, whereas leucine and phenylalanine were negatively related (Spearman ρ −0.87 and −0.84, respectively). Ergothioneine synthesis is mediated by an intracellular redox sensor (WhiB3) that also modulates leucine and valine as a response to oxidative stress generated by lack of glucose and nitrogen.33,35,36 Oxidative stress down-regulated leucine biosynthesis to favor the accumulation of isoleucine and valine (Figure 2C). Moreover, leucine and phenylalanine concentrations have been classified as good markers for oxidation.32 Higher concentrations of leucine and phenylalanine were found in 4078


Article


Figure 4. Bound coumaric (A) and ferulic acid (B) contents of P. ostreatus fruiting bodies from three different flushes cultivated on selenized and nonselenized substrates. Control, nonselenized; LSe, low selenium concentration (5.8 mg Se/kg substrate dw); HSe, high selenium concentration (17.5 mg Se/kg substrate dw). Statistical analyses were performed by treatment and flush. Different letters denote significant differences (α = 0.05).

of standard compound and on mass spectra in positive and negative modes as previously reported.32 In control fruiting bodies, the content of 5′-deoxy-5′(methylsulfinyl)adenosine increased from 14.1 μg/g DW in flush 1 to 20.49 and 23.851 μg/g DW in the second and third flushes, respectively (Table 2). Interestingly, LSe showed the highest abundance of 5′-deoxy-5′-(methylsulfinyl)adenosine in the first flush because it decreased from 20.74 to 15.25 and 11.84 μg/g DW in the second and third flushes, respectively. The content of peak 3 showed a similar trend as 5′-deoxy-5′(methylsulfinyl)adenosine; HSe fruiting bodies had the lowest abundance, and there were no changes in the three flushes. For peak 4, the only contrasting changes were observed between the second and third flushes of the control fruiting bodies. Among the samples with the highest concentrations of phenylalanine (peak 5), there were the first flushes of control and LSe (82.15 and 63.46 μg/g DW, respectively) that also showed the highest content of this amino acid released after protease digestion (Figure 2D). In contrast, for HSe, the highest phenylalanine content was found in the third flush (67.25 μg/g DW), indicating that the lower Se content allowed the biosynthesis of this amino acid.

The abundance of these four mycochemicals was not affected by the treatment (p > 0.05) or flush number (p > 0.05), and further studies are required to contrast the particular changes observed. Phenolic acids were released after alkaline hydrolysis. Particularly, coumaric and ferulic acids were identified on the basis of m/z, UV spectra, and retention times of standards in the bound phenolic extract of P. ostreatus fruiting bodies. It has been reported that the genus Pleurotus can synthesize the enzyme feruloyl esterase, which can release phenolic acids from the polysaccharides that form part of cell walls.41,42 The highest content of bound coumaric acid was observed in the second flush of LSe (5.56 μg/g DW) followed by HSe (3.28 μg/g DW), but the control contained 1 ng/mL (Figure 5B). The methanolic extract from flush 2 of LSe was not significantly different from the same flush obtained from HSe, but it had a negative effect on the CAA units when the concentration increased in the bioassay. Therefore, compounds that promote oxidative stress were entering into the cells more effectively than the antioxidants found in the methanolic extracts. Similarly, in flush 3 of control a higher antioxidant capacity measured through ORAC was observed but lower CAA units were reached in comparison with flushes 1 and 2.

In general, the antioxidant capacity of the extracts was slightly lower than previously reported for P. ostreatus (16.4 μmol TE/g).44 Additionally, the fruiting bodies of the third flush of control and HSe treatments were among the most stressed tissues because they had higher ergothioneine contents and this metabolite has been directly correlated with the antioxidant capacity (DPPH) of P. ostreatus.45 Ergothioneine was removed from the methanolic extracts used to measure ORAC and CAA and therefore the nucleosides, phenylalanine, glutamic acid, 5-2-(α-hydroxy-p-tolyl)hydrazide, and their interactions with other compounds must be the responsible for entering the cell and exerting effects on reactive oxygen species. This is the first study that reports the cellular antioxidant activity of P. ostreatus, which helps to clarify the effect of selenization and flushing for the development of healthier fruiting bodies. Particularly, although the BE of the second flush of LSe was low compared to the first flush, the CAA results justify its cultivation for nutraceutical purposes. 4080


Article


■

(13) da Silva, M. C. S.; Naozuka, J.; Oliveira, P. V.; Vanetti, M. C. D.; Bazzolli, D. M. S.; Costa, N. M. B.; Kasuya, M. C. M. In vivo bioavailability of selenium in enriched Pleurotus ostreatus mushrooms. Metallomics 2010, 2, 162−166. (14) Gąsecka, M.; Mleczek, M.; Siwulski, M.; Niedzielski, P. Phenolic composition and antioxidant properties of Pleurotus ostreatus and Pleurotus eryngii enriched with selenium and zinc. Eur. Food Res. Technol. 2016, 242, 723−732. (15) Gąsecka, M.; Mleczek, M.; Siwulski, M.; Niedzielski, P.; Kozak, L. The effect of selenium on phenolics and flavonoids in selected edible white rot fungi. LWT−Food Sci. Technol. 2015, 63, 726−731. (16) Gil-Ramírez, A.; Pavo-Caballero, C.; Baeza, E.; Baenas, N.; Garcia-Viguera, C.; Marín, F. R.; Soler-Rivas, C. Mushrooms do not contain flavonoids. J. Funct. Foods 2016, 25, 1−13. (17) Afrin, S.; Rakib, A.; Kim, B. H.; Kim, J. H.; Ha, Y. L. Eritadenine from edible mushrooms inhibits activity of angiotensin converting enzyme in vitro. J. Agric. Food Chem. 2016, 64, 2263−2268. (18) Chen, S.-Y.; Ho, K.-J.; Hsieh, Y.-J.; Wang, L.-T.; Mau, J.-L. Contents of lovastatin, γ-aminobutyric acid and ergothioneine in mushroom fruiting bodies and mycelia. LWT−Food Sci. Technol. 2012, 47, 274−278. (19) Li, S.; Shah, N. P. Effects of various heat treatments on phenolic profiles and antioxidant activities of Pleurotus eryngii extracts. J. Food Sci. 2013, 78, C1122−C1129. (20) Cuéllar-Villarreal, M. del R.; Ortega-Hernández, E.; BecerraMoreno, A.; Welti-Chanes, J.; Cisneros-Zevallos, L.; Jacobo-Velázquez, D. A. Effects of ultrasound treatment and storage time on the extractability and biosynthesis of nutraceuticals in carrot (Daucus carota). Postharvest Biol. Technol. 2016, 119, 18−26. (21) Cuderman, P.; Stibilj, V. How critical is the use of commercially available enzymes for selenium speciation? Anal. Bioanal. Chem. 2009, 393, 1007−1013. (22) Ou, B.; Hampsch-Woodill, M.; Prior, R. L. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food Chem. 2001, 49, 4619−4626. (23) Wan, H.; Liu, D.; Yu, X.; Sun, H.; Li, H. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants. Food Chem. 2015, 175, 601−608. (24) Yang, W.; Guo, F.; Wan, Z. Yield and size of oyster mushroom grown on rice/wheat straw basal substrate supplemented with cotton seed hull. Saudi J. Biol. Sci. 2013, 20, 333−338. (25) Alananbeh, K. M.; Bouqellah, N. A.; Al Kaff, N. S. Cultivation of oyster mushroom Pleurotus ostreatus on date-palm leaves mixed with other agro-wastes in Saudi Arabia. Saudi J. Biol. Sci. 2014, 21, 616− 625. (26) Da Silva, M. C. S.; Dias, N. M.; Rodriguez, L. J. M.; Kasuya, M. C. M. Mycelial growth of Pleurotus spp. in Se-enriched culture media. Adv. Microbiol. 2013, 3, 11−18. (27) NIH, National Institutes of Health. Selenium fact sheet for consumers (2016); https://ods.od.nihgov/pdf/factsheets/SeleniumConsumer.pdf (accessed Jan 11, 2017). (28) Damodaran, D.; Balakrishnan, R. M.; Shetty, V. K. The uptake mechanism of Cd(II), Cr(VI), Cu(II), Pb(II), and Zn(II) by mycelia and fruiting bodies of Galerina vittiformis. BioMed Res. Int. 2013, 2013, 11. (29) Feng, R.; Wei, C.; Tu, S. The roles of selenium in protecting plants against abiotic stresses. Environ. Exp. Bot. 2013, 87, 58−68. (30) He, N.; Shi, X.; Zhao, Y.; Tian, L.; Wang, D.; Yang, X. Inhibitory effects and molecular mechanisms of selenium-containing tea polysaccharides on human breast cancer MCF-7 cells. J. Agric. Food Chem. 2013, 61, 579−588. (31) Dubost, N. J.; Ou, B.; Beelman, R. B. Quantification of polyphenols and ergothioneine in cultivated mushrooms and correlation to total antioxidant capacity. Food Chem. 2007, 105, 727−735. (32) Wrona, M.; Pezo, D.; Canellas, E.; Nerín, C. Ultra high performance liquid chromatography coupled to quadruple time-offlight with MSE technology used for qualitative analysis of non-volatile

AUTHOR INFORMATION

Corresponding Author

*(J.A.G.-U.) Phone: 52 81 8358 1400. Fax: 52 81 8328 4262. E-mail: [email protected]. ORCID

Janet A. Gutierrez-Uribe: 0000-0003-1056-7126 Funding

This research was supported by the NutriOmics Research Chair Funds from Tecnológico de Monterrey and Consejo Nacional de Ciencia y Tecnologiá (CONACYT). The scholarship to J.A.C.-G. was provided by CONACYT and Tecnológico de Monterrey. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Liliana Santos, Marı ́a Eugenia Navarrete, Alec Núñez, and Hugo Villarreal for their invaluable support of this research. We especially thank Dharma Mushrooms for technical assistance and for providing the material for mushroom cultivation.

■

REFERENCES

(1) Yang, D.; Liang, J.; Wang, Y.; Sun, F.; Tao, H.; Xu, Q.; Zhang, L.; Zhang, Z.; Wan, X. Tea waste: an effective and economic substrate for oyster mushroom cultivation. J. Sci. Food Agric. 2016, 96, 680−684. (2) Carrasco-González, J. A.; Serna-Saldivar, S. O.; Gutiérrez-Uribe, J. A. Nutritional composition and nutraceutical properties of the Pleurotus fruiting bodies: potential use as food ingredient. J. Food Compos. Anal. 2017, 58, 69−81. (3) Cheah, I. K.; Halliwell, B. Ergothioneine; antioxidant potential, physiological function and role in disease. Biochim. Biophys. Acta, Mol. Basis Dis. 2012, 1822, 784−793. (4) Falandysz, J. Selenium in edible mushrooms. J. Environ. Sci. Health C: Environ. Carcinog. Ecotoxicol. Rev. 2008, 26, 256−299. (5) Bhatia, P.; Prakash, R.; Prakash, N. T. Selenium uptake by edible oyster mushrooms (Pleurotus sp.) from selenium-hyperaccumulated wheat straw. J. Nutr. Sci. Vitaminol. 2013, 59, 69−72. (6) da Silva, M. C. S.; Naozuka, J.; da Luz, J. M. R.; de Assunçaõ , L. S.; Oliveira, P. V.; Vanetti, M. C. D.; Kasuya, M. C. M. Enrichment of Pleurotus ostreatus mushrooms with selenium in coffee husks. Food Chem. 2012, 131, 558−563. (7) Kim, Y. H.; Lee, H. S.; Kwon, H. J.; Patnaik, B. B.; Nam, K. W.; Han, Y. S.; Han, M. D. Effects of different selenium levels on growth and regulation of laccase and versatile peroxidase in white-rot fungus Pleurotus eryngii. World J. Microbiol. Biotechnol. 2014, 30, 2101−2109. (8) Serafín-Muñoz, A. H.; Wrobel, K.; Gutierrez-Corona, J. F.; Wrobel, K. The protective effect of selenium inorganic forms against cadmium and silver toxicity in mycelia of Pleurotus ostreatus. Mycol. Res. 2007, 111, 626−632. (9) Zimmerman, M. T.; Bayse, C. A.; Ramoutar, R. R.; Brumaghim, J. L. Sulfur and selenium antioxidants: challenging radical scavenging mechanisms and developing structure-activity relationships based on metal binding. J. Inorg. Biochem. 2015, 145, 30−40. (10) Snider, G. W.; Ruggles, E.; Khan, N.; Hondal, R. J. Selenocysteine confers resistance to inactivation by oxidation in thioredoxin reductase: comparison of selenium and sulfur enzymes. Biochemistry 2013, 52, 5472−5481. (11) Yamashita, Y.; Yabu, T.; Yamashita, M. Discovery of the strong antioxidant selenoneine in tuna and selenium redox metabolism. World J. Biol. Chem. 2010, 1, 144−150. (12) Yan, H.; Chang, H. Antioxidant and antitumor activities of selenium-and zinc-enriched oyster mushroom in mice. Biol. Trace Elem. Res. 2012, 150, 236−241. 4081


Article

Journal of Agricultural and Food Chemistry oxidation markers in sliced packed mushrooms (Agaricus bisporus). J. Chromatogr. A 2016, 1432, 73−83. (33) Sajiki, K.; Pluskal, T.; Shimanuki, M.; Yanagida, M. Metabolomic analysis of fission yeast at the onset of nitrogen starvation. Metabolites 2013, 3, 1118−1129. (34) Dubost, N. J.; Beelman, R. B.; Royce, D. J. Influence of selected cultural factors and postharvest storage on ergothioneine content of common button mushrooms Agaricus bisporus (J. Lge) Imbach (Agaricomycetidae). Int. J. Med. Mushrooms 2007, 9, 163−176. (35) Pluskal, T.; Hayashi, T.; Saitoh, S.; Fujisawa, A.; Yanagida, M. Specific biomarkers for stochastic division patterns and starvationinduced quiescence under limited glucose levels in fission yeast. FEBS J. 2011, 278, 1299−1315. (36) Saini, V.; Cumming, B. M.; Guidry, L.; Lamprecht, D. A.; Adamson, J. H.; Reddy, V. P.; Chinta, K. C.; Mazorodze, J. H.; Glasgow, J. N.; Richard-Greenblatt, M.; Gomez-Velasco, A.; Bach, H.; Av-Gay, Y.; Eoh, H.; Rhee, K.; Steyn, A. J. C. Ergothioneine maintains redox and bioenergetics homeostasis essential for drug susceptibility and virulence of Mycobacterium tuberculosis. Cell Rep. 2015, 14, 572− 585. (37) Gomes-Correa, R. C.; de Souza, A. H. P.; Calhelha, R. C.; Barros, L.; Glamoclija, J.; Sokovic, M.; Peralta, R. M.; Bracht, A.; Ferreira, I. C. F. R. Bioactive formulations prepared from fruiting bodies and submerged culture mycelia of the Brazilian edible mushroom Pleurotus ostreatoroseus Singer. Food Funct. 2015, 6, 2155−2164. (38) Kawagishi, H.; Fukuhara, F.; Sazuka, M.; Kawashima, A.; Mitsubori, T.; Tomita, T. 5′-Deoxy-5′-methylsulphinyladenosine, a platelet aggregation inhibitor from Ganoderma lucidum. Phytochemistry 1993, 32, 239−241. (39) Weijn, A.; van den Berg-Somhorst, D. B.; Slootweg, J. C.; Vincken, J. P.; Gruppen, H.; Wichers, H. J.; Mes, J. J. Main phenolic compounds of the melanin biosynthesis pathway in bruising-tolerant and bruising-sensitive button mushroom (Agaricus bisporus) strains. J. Agric. Food Chem. 2013, 61, 8224−8231. (40) Phat, C.; Moon, B.; Lee, C. Evaluation of umami taste in mushroom extracts by chemical analysis, sensory evaluation, and an electronic tongue system. Food Chem. 2016, 192, 1068−1077. (41) Haase-Aschoff, P.; Linke, D.; Berger, R. G. Detection of feruloyland cinnamoyl esterases from basidiomycetes in the presence of interfering laccase. Bioresour. Technol. 2013, 130, 231−238. (42) Linke, D.; Matthes, R.; Nimtz, M.; Zorn, H.; Bunzel, M.; Berger, R. G. An esterase from the basidiomycete Pleurotus sapidus hydrolyzes feruloylated saccharides. Appl. Microbiol. Biotechnol. 2013, 97, 7241− 7251. (43) Eberhardt, M. V.; Kobira, K.; Keck, A. S.; Juvik, J. A.; Jeffery, E. H. Correlation analyses of phytochemical composition, chemical, and cellular measures of antioxidant activity of broccoli (Brassica oleracea L. var. italica). J. Agric. Food Chem. 2005, 53, 7421−7431. (44) Lam, S.; Okello, E. Determination of lovastatin, β-glucan, total polyphenols, and antioxidant activity in raw and processed oyster culinary-medicinal mushroom, Pleurotus ostreatus (higher Basidiomycetes). Int. J. Med. Mushrooms 2015, 17, 117−128. (45) Bhattacharya, M.; Srivastav, P.; Mishra, H. Optimization of process variables for supercritical fluid extraction of ergothioneine and polyphenols from Pleurotus ostreatus and correlation to free-radical scavenging activity. J. Supercrit. Fluids 2014, 95, 51−59.

4082


Fruiting Bodies - American Chemical Society

Recommend Documents