Examining the Impact of Substrate Composition on the Biochemical Properties and Antioxidant Activity of Pleurotus and Agaricus Mushrooms

: The composition of the substrate is one of the most critical factors inﬂuencing the quality as well as the nutritional value and bioactive content of mushrooms. Therefore, the effects of various substrates, such as barley and oat straw (BOS), beech wood shavings (BWS), coffee residue (CR), rice bark (RB) and wheat straw (WS, control substrate), on the biochemical properties (lipid, protein, polysaccharide, glucan, ash, and mineral content, fatty acids and tocopherols composition), total phenolic compounds and antioxidant activity of Pleurotus mushrooms, P. ostreatus (strains AMRL 144, 150) and P. eryngii (strains AMRL 166, 173-6), cultivated in ‘bag-logs’, was examined. Proximate analysis of A. bisporus and A. subrufescens grown on two different composts (C/N ratios of 10 and 13) was conducted, too. The whole carposomes, pilei and stipes were analyzed. Results showed that BOS, RB, BWSs and CR improved the antioxidant activity of Pleurotus species and their nutritional characteristics. Both pilei and stipes were rich in polysaccharides (27.51–67.37 and 22.46–39.08%, w / w , for Pleurotus and Agaricus spp., respectively), lipids (0.74–8.70 and 5.80–9.92%, w / w ), proteins (6.52–37.04 and 25.40–44.26, w / w, for Pleurotus and Agaricus spp., respectively) and total phenolic compounds (10.41–70.67 and 7.85–16.89 mg gallic acid equivalent/g for Pleurotus and Agaricus spp., respectively), while they contained important quantities of unsaturated FAs of nutritional and medicinal importance. Pilei were richer in proteins, total phenolic compounds and enhanced antioxidant activity and reducing power than stipes, whereas stipes were richer in IPSs and glucans compared to the corresponding pilei. Thus, mushroom cultivation could upgrade rejected agro-industrial residues and wastes to new uses as substrates for the production of mushrooms with speciﬁc nutritional and medicinal attributes.


Introduction
Mushrooms have long been used as food or food flavoring material due to their unique flavor and aroma [1,2], and they have gained extra attention due to their low calories and fat, and also because they contain a variety of polysaccharides, proteins and fibers [1,[3][4][5][6] that are essential nutrition components. Also, mushrooms of the Basidiomycetes family have long been known for their seemingly beneficial medical usage, most notably in traditional Chinese and Japanese medicine [4]. However, knowledge about the composition and nutritional value of culinary mushrooms, mainly wild-growing ones, remained limited until the last decade as compared to vegetables and medicinal mushroom species [7]. measured using a Jasco V-530 UV-Visible spectrophotometer (Tokyo, Japan), except for total protein determination which took place in a 96-cell microplate reader spectrophotometer.

Total Intra-Cellular Polysaccharide (IPS) Determination
IPS determination was conducted according to Diamantopoulou et al. [64] and Liang et al. [65]. In particular, 20 mL of 2.5 M HCl was used to hydrolyze 0.1 g of dried powder mushrooms at 100°C for 20 min. The entire mixtures were neutralized to a pH of 7 using 2.5 M NaOH. The DNS assay was then performed on samples that contained total sugars after they had been filtered (through No.2 Whatman filters, Whatman plc, Kent, UK) [66]. The IPS content (expressed as glucose equivalents) was determined, measuring the absorbance at 540 nm. All samples were analyzed in triplicate.
The composition of individual carbohydrates of the produced IPSs was performed by HPLC analysis as described by Diamantopoulou et al. [64]. Filtered aliquots of the neutralized samples with NaOH were analyzed by a Waters Association 600E apparatus at a 30.0 cm × 7.8 mm column Aminex HPX-87H (Bio-Rad, Hercules, CA, USA). The mobile phase used was H 2 SO 4 at 0.005 M with a flow rate 0.8 mL/min, while the column temperature was 65 ± 1 • C. Individual simple sugars and sugar alcohols were detected using an RI detector (differential refractometer 410-Waters).

Quantitative Evaluation of a-and β-Glucan Content
α-glucans content of the fruiting bodies of edible mushrooms was determined using a Yeast Beta-Glucan assay kit (Megazyme, Wicklow, Ireland) following its procedure. Then, β-glucans content was determined by subtracting α-glucans content from total glucans content. Each sample was analyzed in triplicate.

Total Protein Determination
The crude protein content of dried mushroom species was determined according to the Bradford assay [67]. For this purpose, 50 mg of each sample was extracted in 1.5 mL of 50 mM EDTA (ethylenediaminetetraacetic acid) using an ultrasonic bath, for 60 min at 25 ± 0.5 • C. The mixtures were vortexed thoroughly and centrifuged at 10,000 rpm for 10 min. A total of 10µL of each supernatant was diluted in 240 µL Coomassie Brilliant blue solution and incubated for 10 min at 25 ± 0.5 • C, then compared to the reagent blank; the absorbance was measured at 620 nm using a 96-cell microplate reader spectrophotometer. A standard curve of BSA (0.1-1.5 mg/mL) was made. All samples were analyzed in triplicate.

Determination of Total Lipids and Fatty Acids
Total lipids were determined by a modified version of the method of Folch [68]. For this purpose, 0.5 g of ground mushroom was suspended in a 10 mL chloroform:methanol (2:1 v/v) mixture, mixed thoroughly and let stand for 7 days. The solution was then filtrated and the solvents were removed in a rotary evaporator (at 50 ± 0.5 • C) under vacuum (RE 300 evaporator Stuart-RE 300 DB digital water bath). What remained were the crude lipids.
The fatty acid methyl esters preparation was performed in a two-stage reaction (to avoid trans-isomerization) using sodium methoxide and methanol/hydrochloride according to the AFNOR method [69]. Fatty acid methyl esters were identified by reference to authentic standards. For this purpose, methyl esters were suspended in hexane and analyzed by GC in a Varian CP-3800 chromatograph equipped with flame ionization detector (Agilent Technologies, Santa Clara, CA, USA) in which an Agilent J&W Scientific DB23 capillary column (model n. 30.0 m × 0.32 mm, film thickness 0.25 µm) (Agilent Technologies, Santa Clara, CA, USA) was used. Helium was used as a carrier gas with a column flow rate of 2.0 mL/min. The set-up conditions were as follows: Initial oven temperature was set at T = 150 • C, held for 18 min, subsequently rammed to T = 185 • C at a rate of 5 • C/min and held for 2 min. Then, the oven temperature was moved to T = 210 • C at a flow rate of 5 • C/min and held for 2 min, then increased to T = 240 • C at 10 • C/min. The injector and flame ionization detector temperatures were set at T = 260 • C and T = 270 • C, respectively. Individual fatty acid methyl esters were identified by comparison of their retention times with external standard (Supelco 37 Component fatty acid methyl esters Mix, CRM47885) retention times. The content of each fatty acid was expressed as a percentage using the peak area.

Minerals
The concentrations of the elements Ca, Mg, Na, Fe, Cu, Zn, B and Mn of the mushroom samples were determined following standard protocols for the atomic absorption spectrophotometer (AAS) (SpectrAA 220); Na was determined using a flame photometer 410 (Corning) [70], while P was determined using an LLG-uniSPEC 2 Spectrophotometer, as described by Kalra and Maynard [71].

Determination of Tocopherols
Tocopherol content was determined according to Barros et al. [72] using BHT (butylhydroxytoluene) (~10 mg/mL) with the samples prior to the extraction procedure. A total of 500 mg of each sample was homogenized with 4 mL of methanol by vortex mixing, and then hexane was added and it was vortexed for another 1 min. After that, 2 mL of saturated NaCl aqueous solution was added, and the mixtures were then homogenized, centrifuged and the upper layer was transferred to a vial. The combined extracts from 3 extractions were dried under a nitrogen stream, redissolved in 1 mL of hexane, dehydrated with sodium sulphate, filtered and transferred into a dark vial for HPLC analysis. The determination of α-, β-, γ-, and δ-tocopherols and tocotrienols was performed according to ISO 9936 [73], using high-performance liquid chromatography with fluorescence detection. In brief, a JASCO HPLC system (JASCO International Co., Ltd., Tokyo, Japan) was used, consisting of a quaternary pump (PU-2089 Plus), an autosampler (AS-1555) and a fluorescence detector (FP-920). Separation was accomplished with a Pinnacle DB Silica column (250 mm × 4.6 mm i.d., 5 µm, Restek, Bellefonte, PA, USA) using isocratic elution with n-Hexane/1,4-Dioxane (97:3 v/v). The flow rate was set at 1.5 mL/min, and the injection volume was 20 µL. The excitation and emission wavelengths were set at 295 nm and 330 nm, respectively. The content of each tocol was calculated using the calibration factor of a standard solution of (±)-α-tocopherol (Merck, Darmstadt, Germany) and expressed in mg/kg.

Total Phenolic Compounds and Antioxidant Activity
Methanolic extracts were prepared as follows: 250 mg of fresh mushrooms were extracted with 5 mL of methanol in an ultrasonic bath (SKYMEN, JP-060S, Shenzhen, China) for 15 min at 25 • C, followed by vortex and centrifugation (3500 rpm, 15 min, 25 ± 0.5 • C; Micro 22R, Hettich, Germany). The extraction was repeated three times, and the supernatants were stored at 4.0 ± 0.5 • C for further analysis.

Determination of Total Phenolic Compounds (TPC)
The TPC in the mushroom samples were estimated using the Folin-Ciocalteu assay as described by Slinkard and Singleton [74] and were measured at 760 nm using gallic acid for the standard curve. Briefly, 0.5 mL of each sample (methanolic extract) was diluted in 10.5 mL H 2 O and mixed with 8 mL Na 2 CO 3 (75g/L) and 1 mL of Folin-Ciocalteu reagent. The mixtures were vortexed and allowed to react in the dark for 2 h. Samples were measured in three replicates. Results were expressed as gallic acid equivalent (GAE) µg/g dry weight (dw) of biomass.

Antioxidant Activity: Ferric Reducing Antioxidant Power (FRAP)
The sample's capacity to convert Fe 3+ to Fe 2+ ions is the basis for this method [75]. FRAP working solution was freshly prepared by mixing 25 mL acetate buffer (300 mM/L, pH 3.6), 5 mL TPTZ solution (10 mM/L 2, 4, 6-tripyridyl-s-triazine in 40 mM/L HCl) and 2.5 mL FeCl 3 ·6H 2 O (20 mM/L in distilled water) solution. A total of 300 µL of each extract was added to 2700 µL of FRAP solution and the mixtures were vortexed and incubated at 37 • C for 10 min. The ferric-tripyridyltriazine (Fe 3+ -TPTZ) complex was reduced to the ferrous (Fe 2+ -TPTZ) form at low pH in the presence of TPTZ (Sigma Aldrich, St. Louis, MO, USA), resulting in a vivid blue color. The absorbance was measured at 593 nm against a blank for each sample. Trolox was used to obtain a standard curve and the antioxidant activity was expressed in mmol trolox equivalents per 100 g of dry weight. Samples were measured in three replicates.
2.9.3. Antioxidant Activity: Scavenging Activity of ABTS + Radical Free radical scavenging activity was determined according to Re et al. [76] with some modifications. ABTS˙+ [2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid] radicals were produced by the reaction between 7 mM ABTS˙+ in water and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12-16 h before use. Prior to use, the solution was diluted with ethanol to get an absorbance of 0.7000 ± 0.05 at 734 nm. Free radical scavenging activity was assessed by mixing 50 µL of each methanolic sample with 2 mL of the diluted ABTS˙+ working solution. The mixtures were vortexed and the decrease in absorbance was measured against the blank (2 mL ABTS˙+ with 50 µL methanol). Trolox was used as calibration standard and the results were expressed as mg trolox equivalents per 1 L of the extract. Samples were measured in three replicates. The scavenging ability on DPPH˙free radicals was determined according to [77]. Briefly, 0.1 mL of each methanolic extract was added to 3.9 mL DPPH˙(60 µM in methanol) in test tubes and vortexed. The mixtures were left in the dark for 30 min and the reduction of the DPPH˙was determined by measuring the absorbance at 515 nm. DPPH˙methanolic solution was used as a blank and the results were expressed as mmol trolox equivalents per 100 g of dry matter. Samples were measured in three replicates.

Statistical Analysis
All experiments were repeated at least twice, and within experiments triplicate bags were used to generate each data point. Statgraphics was used for statistical analysis. The data were compared using analysis of variance (ANOVA) and Pearson's linear correlation at the 5% significance level. Significant differences between means were determined by honest significant difference (HSD-Tukey test) at the level of p < 0.05. Data were reported as mean values ± standard deviation of three independent replicates (p < 0.05, 95%).

Results and Discussion
Chemical composition of carposomes.

Intra-Cellular Polysaccharide (IPS) Content and Profile
The total IPS content in Pleurotus ( Figure 1) and Agaricus species (Table 1) revealed that Pleurotus spp. (27.51-67.37%, w/w) contained significantly higher quantities of polysaccharides than those of Agaricus (22.46-39.08%, w/w), and this finding was consistent with previous studies [78][79][80]. Regarding Pleurotus spp., the P. eryngii species produced higher amounts of IPSs than those of P. ostreatus, in all tested substrates except for BWS, where P. ostreatus 150 was the greatest IPS producer (45.84%, w/w). Particularly, the highest IPS values for the carposome were obtained when P. ostreatus 144 and P. eryngii 173-6 were cultivated on CR, whereas WS was the best substrate for P. ostreatus 150 and P. eryngii 166. It seems, therefore, that substrate composition affected the ability of fungi to produce IPSs and that there was a strain-specific preference for particular substrates. Nevertheless, all substrates and strains supported IPS production greater than 30%, w/w, but strains 150 and 173-6 performed better in most of the cases. It is worth mentioning that, in all Pleurotus species, IPS content was even greater in the stipes than in the pilei. In contrast, Agaricus stipes produced lower IPS amounts than the pilei and the carposomes. Moreover, the compost with the lowest C/N ratio favored IPS synthesis in Agaricus carposomes, but the IPS concentration was relatively low.
The total IPS content in Pleurotus ( Figure 1) and Agaricus species (Table 1) revealed that Pleurotus spp. (27.51-67.37%, w/w) contained significantly higher quantities of polysaccharides than those of Agaricus (22.46-39.08%, w/w), and this finding was consistent with previous studies [78][79][80]. Regarding Pleurotus spp., the P. eryngii species produced higher amounts of IPSs than those of P. ostreatus, in all tested substrates except for BWS, where P. ostreatus 150 was the greatest IPS producer (45.84%, w/w). Particularly, the highest IPS values for the carposome were obtained when P. ostreatus 144 and P. eryngii 173-6 were cultivated on CR, whereas WS was the best substrate for P. ostreatus 150 and P. eryngii 166. It seems, therefore, that substrate composition affected the ability of fungi to produce IPSs and that there was a strain-specific preference for particular substrates. Nevertheless, all substrates and strains supported IPS production greater than 30%, w/w, but strains 150 and 173-6 performed better in most of the cases. It is worth mentioning that, in all Pleurotus species, IPS content was even greater in the stipes than in the pilei. In contrast, Agaricus stipes produced lower IPS amounts than the pilei and the carposomes. Moreover, the compost with the lowest C/N ratio favored IPS synthesis in Agaricus carposomes, but the IPS concentration was relatively low.  In some previous studies by other researchers, a higher total amount of carbohydrates was detected in A. bisporus and A. brasiliensis (62.20 and 63.83%, respectively) [80] or in A. bisporus (6.46% f.w.) [79] than in this study, but in another study [78] the total carbohydrates in A. bisporus were 34.5% w/w, a value similar to ours. Comparable experimental results have been also previously presented for P. eryngii, 49-60% d.w. total IPSs, when it was cultivated on six different types of agro-industrial wastes, and~42%, w/w, for P. ostreatus and P. pulmonarius carposomes cultivated on spent mushroom substrate [43]. Total carbohydrates in P. ostreatus and P. eryngii were higher (66.40-74.02% and 70.52%, respectively) [80] than in this study. Also, an IPS concentration as high as 66.54% d.w. for Pleurotus has been reported [81]. These variations in IPS values may be attributed, apart from the substrate's synthesis, to the use of different strains and the different analytical methodologies adopted. It has been also demonstrated that there is a positive correlation between substrate C/N, cellulose and P. ostreatus carbohydrates content [82]. Indeed, the highest IPSs in P. ostreatus 150 were detected when it was cultivated on WS, the substrate with the highest ratio C/N and cellulose (analyses presented in [44]), although in the present research P. ostreatus 144 achieved its highest IPS value when it was cultivated on CR, the substrate with the lowest ratio C/N and cellulose.
The results from carbohydrates composition analysis are summarized in Table 2. Glucose was the main constituent present in all Pleurotus and Agaricus carposomes, stipes and pilei (up to 61%, w/w). Fructose was found in smaller quantities than glucose (10-25%, w/w) in all samples, whereas other compounds, such as mannitol and arabitol, were detected in lower concentrations (or not at all) in some samples. Polyols (mainly mannitol, trehalose and arabitol) contained in mushrooms contribute to a sweet taste; therefore, their high content would generate a moderately perception to fresh mushrooms and not the typical mushroom taste [83]. Mannitol production is a major feature of the growth of several mushroom carposomes and mycelia [84], while its role and requirements are likely to differ depending on the fungus. Previous researchers showed that glucose, mannitol and trehalose were the main soluble carbohydrates of A. bisporus extracts [84], and that glucose was the most abundant among mannose, galactose, fucose, ribose and galactose [85], all monosaccharides that were not detected in the present study. In other studies, mannitol and trehalose were the most abundant monosaccharides of A. bisporus and Pleurotus spp., respectively [79], and wild Agaricus spp. and P. cystidiosus contained mainly glucose and rhamnose, following by xylose and mannose, whereas galactose and fructose were detected in very low percentages [86]. Additionally, glucose was the main sugar in the stipes of P. ostreatus and P. eryngii, while small amounts of galactose and mannose were also detected [87]. In other studies, glucose content was determined up to 80% w/w for P. ostreatus [88,89] and up to 50% w/w for A. bisporus [85]. In general, differences in sugars composition had been observed between cultivated and wild samples of the same mushroom species, probably due to the different cultivation techniques used [79,86]. Table 2. Carbohydrate composition (% w/w) of total IPSs produced by P. ostreatus (AMRL 144, 150) and P. eryngii (AMRL 166, 173-6) cultivated in five substrates (WS, BOS, RB, BWS, CR) and by A. bisporus (AMRL 209) and A. subrufescens (AMRL 235) cultivated on compost of two different C/N ratios (10 and 13). Mushroom parts tested: carposomes (c), pilei (p) and stipes (s).

Total Glucans, α-Glucans and β-Glucans
The total αand βglucan contents in dry mushroom carposomes, pilei and stipes are presented in Table 3. All examined Pleurotus species, cultivated on different substrates, were found to contain high amounts of total glucans, ranging from 21.47 to 64.21% (w/w of total IPSs), with the percentage of β-glucans predominating. WS and CR substrates favored IPS concentration and consequently the glucans production of all Pleurotus strains, as well as BOS in the case of P. eryngii. It is interesting that stipes of all species contained significantly greater amounts of total glucans and β-glucans than carposomes or pilei, and this was more pronounced for P. ostreatus cultivated on WS and CR, as well as for P. eryngii on WS and BOS. Also, there was a positive correlation between IPS content and total glucans, as the stipes with the highest IPS content had the highest total glucans content, too. Comparing P. ostreatus with P. eryngii strains, those of P. eryngii seemed to produce slightly greater amounts of total glucans and have higher β-glucan content than the P. ostreatus ones, apart from BWS. Among the P. ostreatus fungi, strain 150 (commercial) performed better than 144, while glucan production proved to be substrate-dependent for both P. eryngii strains, a tendency that had already been detected in previous studies [90,91]. Regarding Agaricus spp., our data showed that they contained much lower total glucans than Pleurotus strains, ranging from 10.12 to 24.39% (w/w of total IPSs), and no significant difference was detected between species or between species and C/N ratio imposed. Manzi and Pizzoferrato [92] reported significantly lower β-glucan content in P. ostreatus and P. eryngii, which ranged from 0.22 to 0.38%. The results of the present study were close to those reported by Bekiaris et al. [93], who recorded 38.84-58.90% and 32.84-61.40% d.w. total glucan content for P. ostreatus and P. eryngii, respectively, after having been cultivated on WS, whereas β-glucans content ranged from 26.44% to 51.36% d.w. for both Pleurotus species.  The presence of β-glucans in mushrooms is important as they have a plethora of functional and bioactive properties, such as anti-cancer and antioxidant effects and activities against infectious diseases [54][55][56][57]94]. The high amounts of β-glucans in the mushrooms of the present study would render them a source of highly nutritious food. According to the above results, all mushroom stipes had high values of total glucans and consequently were rich in β-glucans. Considering that a piece of mushroom stipes is usually removed/discarded before packaging, due to dirt from the substrate and the difficulty for them to be swallowed and absorbed by some consumers, stipes could be used as a β-glucan source in a biorefinery scheme aiming to valorize these residues, for example, in the development of medicinal compounds and nutraceuticals [95,96], or by adding them as supplements to new mushroom crops. Similar studies conducted on Lentinula edodes (pileus and stipe) showed that the fiber content of the pileus was lower than that of the stipe, and therefore stipes contained higher amounts of β-glucans than the rest of the mushroom [97], too. As for fungi cultivation substrates other than those used in our study, fiber production in G. lucidum mushrooms seemed to be influenced variously when cultivated on soybean hulls and soybean and corn residues and the soybean residue increased to a large extent the β-glucan production compared to the soybean hulls and corn residue [90]. It was also shown that the stalks of P. eryngii species contained higher glucans content than the caps, and that the use of olive mill waste as substrate increased α-glucan levels [98]. It is therefore possible to increase the β-glucans content simply by selecting the substrate formulation.

Total Protein
The genus Pleurotus is considered to be a good protein source, but it seems that Agaricus species have satisfactory amounts of protein, too, comparing them to those reported in the literature. The total protein contents present in the carposome and also in the pileus and stipe of the Pleurotus and Agaricus mushrooms tested in this study are shown in Figure 2 and Table 1. In general, P. ostreatus strains had enhanced total protein values compared to P. eryngii ones. High protein amounts were found in P. ostreatus species cultivated on BOS and BWS (18.08-37.04%, w/w) substrates and in P. eryngii (9.00-21.50%, w/w) mushrooms cultivated on BWS, WS and BOS. Also, in the stipes, smaller amounts of protein were detected than in the pilei, in all species and substrates examined. Variations in the protein values were observed not only among Pleurotus genera, but also between strains cultivated in BOS and BWS, as well as in WS and CR substrates for P. eryngii. For example, the maximum protein value was recorded in the pileus of strain 150 (37.04%, w/w) and the minimum in the stipe of strain 173-6 (9.00%, w/w). Mushroom protein content varies greatly depending on intrinsic (e.g., strain) and agro-climatic conditions, as well as on culture configurations (the composition of the substrate, the harvest-time size of the pileus). Hoa et al. [99] reported that the protein contents of P. ostreatus cultivated in different substrate formulas, including sawdust, corncobs and sugarcane bagasse, as well as mixtures of sawdust and corncobs or sawdust and bagasse, were within the range of 19.52-29.70%, while for P. cystidiosus the protein was about 15.68-24.54% using the same substrates. Tolera and Abera [100] found 25.91% of crude protein in dried P. ostreatus mushrooms grown on cottonseed waste. Regarding P. eryngii species, low values of crude protein were detected during their cultivation in WS substrate, whereas the enrichment of the substrate with rice bran and cotton stalk increased the protein content [101]. Reports of much increased protein concentrations in these mushroom species are exceptional and come as a result of growth on nitrogen-rich substrates [102,103], such as spent beer grains supplemented with large amounts of bran, or wheat straw supplemented with sugar beet, for P. ostreatus [104]. In general, P. ostreatus strains had enhanced total protein values compared to P. eryngii ones. High protein amounts were found in P. ostreatus species cultivated on BOS and BWS (18.08-37.04%, w/w) substrates and in P. eryngii (9.00-21.50%, w/w) mushrooms cultivated on BWS, WS and BOS. Also, in the stipes, smaller amounts of protein were detected than in the pilei, in all species and substrates examined. Variations in the protein values were observed not only among Pleurotus genera, but also between strains cultivated in BOS and BWS, as well as in WS and CR substrates for P. eryngii. For example, the maximum protein value was recorded in the pileus of strain 150 (37.04%, w/w) and the minimum in the stipe of strain 173-6 (9.00%, w/w). Mushroom protein content varies greatly depending on intrinsic (e.g., strain) and agro-climatic conditions, as well as on culture configurations (the composition of the substrate, the harvest-time size of the pileus). Hoa et al. [99] reported that the protein contents of P. ostreatus cultivated in different substrate formulas, including sawdust, corncobs and sugarcane bagasse, as well as mixtures of sawdust and corncobs or sawdust and bagasse, were within the range of 19.52-29.70%, while for P. cystidiosus the protein was about 15.68-24.54% using the same substrates. Tolera and Abera [100] found 25.91% of crude protein in dried P. ostreatus mushrooms grown on cottonseed waste. Regarding P. eryngii species, low values of crude protein were detected during their cultivation in WS substrate, whereas the enrichment of the substrate with rice bran and cotton stalk increased the protein content [101]. Reports of much increased protein concentrations in these mushroom species are exceptional and come as a result of growth on nitrogen-rich substrates [102,103], such as spent beer grains supplemented with large amounts of bran, or wheat straw supplemented with sugar beet, for P. ostreatus [104]. In previous studies, sugarcane bagasse was used as substrate and enhanced the protein production of P. ostreatus [105], while the highest crude protein value for P. eryngii in one study was obtained from using cotton waste, as compared to carposomes cultivated on wheat straw, rice straw, corn cobs, sugarcane bagasse and sawdust [106]. On the other hand, in more recent research, a weak correlation (r 2 > 0.433) was found between substrate nitrogen and mushroom protein levels [82]. According to our earlier work [44], a similar tendency was also detected, particularly in the case of P. ostreatus, where the maximum protein concentration was recorded in BWS, which had a greater nitrogen content (1.49%, w/w) than the other substrates (WS, BOS and RB). Despite the fact that the CR substrate had a high nitrogen content and a low C/N ratio, as compared to the C/N ratio of BWS [44], the mushrooms produced were not protein-rich. A comparable finding was obtained in a study where one specific substrate (almond and walnut shells 1:1 w/w) with low nitrogen concentration yielded mushrooms with the greatest crude protein content, diminishing the correlation value [82]. Concerning Agaricus species, the total protein content was increased in all mushroom parts at a lower C/N ratio that contained higher nitrogen concentration, while the maximum value was detected in the A. subrufescens pileus (44.26%, d.w.) at the C/N = 10 ratio. The stipes was found also to have the lowest protein content which, however, was as high as 40.12% w/w. In contrast to our results, studies conducted using A. bisporus strains and two composts based on wheat straw and waste tea leaves (the percentages of the N content of the composts were arranged to be 0.5 and 2.3%, respectively) showed no significant difference in the protein content [40,104]. These results indicated that the protein content of carposomes is affected by the amount and the kind of nitrogen source present in the substrate [107].

Total Lipid Determination and Fatty Acid (FA) Composition
As shown in Figure 3, the Pleurotus species presented the highest amounts of lipid content; P. ostreatus produced more lipids than P. eryngii species, and both presented the highest lipid content on CR and the lowest on the WS and BOS substrates. In P. ostreatus carposomes, the lipid content varied from 2.17 (referring to 144 on BWS) to 8.70% (referring to 150 on CR) of dry weight; it ranged from 1.52 (referring to 166 on WS) to 4.28% (referring to 173-6 on CR) for P. eryngii. This considerable variance in the lipid content of Pleurotus could be attributable to the agro-waste utilized in the production process. In the case of Agaricus species (Table 1), the lipid content ranged from 2.08 (A. bisporus, C/N = 13) to 3.54% of dry weight (A. subrufescens, C/N = 10). Stipes showed lower total lipid concentration than pilei in all species and substrates. This was consistent with the results of the comparative study conducted earlier on A. bisporus [108] that examined the differences in the nutritional characteristics between pilei and stipes and revealed higher values of lipids in pilei (2.48% w/w, in d.w.) than in the corresponding stipes (2.00% w/w, in d.w.). According to Crisan and Sands [109], most carposomes contain 1.1-8.3%, w/w, of lipids, with a mean of~4.0%, w/w. In a generalized lipid content survey of Pleurotus species, the values ranged from 1.18 to 4.40% of dry weight for P. ostreatus grown on a variety of substrates (wheat straw, corncobs, maze straw, etc.) and 5.97%, w/w, for P. eryngii cultured in wheat stalk, while Alam et al. [110] recorded 4.6%, w/w, lipid content on dried mushrooms, and 0.68% on fresh mushrooms. In the case of A. bisporus [83], 3.34-3.75% lipid content in d.w. was reported when it was grown in compost and/or casings enriched with safflower oil, values higher than our data, whereas Teklit [111] detected 2.12% lipid content in d.w. Lower lipid content has been registered for A. blazei compared to our results, as values of 2.62 and 1.85%, w/w in d.w., were recorded, respectively [112,113]. The lipid content of the examined mushrooms in the present study appeared to be within the reported range.  The distribution of FAs differed among genera, as well as between species within the same genus (Table 4). Lipid unsaturation was greater in the samples of P. ostreatus, followed by P. eryngii, A. bisporus and A. subrufescens. The concentration of saturated FAs varied from 0.08 to 13.1% (w/w), and it was significantly lower than that of the PUFA ones that ranged from 52.9 to 72.4%. This was in agreement with observations that PUFAs predominate over saturated ones in mushroom mycelia and carposomes [19,114,115]. Linoleic acid (C18:2) was found to be the most abundant FA among the species examined, followed by oleic acid (C18:1). Palmitic acid (C16:0) was the third and the second main FA for Pleurotus and Agaricus species, respectively (10.7-13.9%, w/w). Table 4. Fatty acid composition (% w/w) of total lipids produced by P. ostreatus carposomes (strains AMRL 144 and 150) and P. eryngii (strains AMRL 166 and 173-6) cultivated on five substrates (WS, BOS, RB, BWS, CR) and by A. bisporus (AMRL 209) and A. subrufescens (AMRL 235) cultivated on two composts (C/N ratios of 10 and 13). Each point is the mean value of three independent measurements (mean ± SD).  The distribution of FAs differed among genera, as well as between species within the same genus (Table 4). Lipid unsaturation was greater in the samples of P. ostreatus, followed by P. eryngii, A. bisporus and A. subrufescens. The concentration of saturated FAs varied from 0.08 to 13.1% (w/w), and it was significantly lower than that of the PUFA ones that ranged from 52.9 to 72.4%. This was in agreement with observations that PUFAs predominate over saturated ones in mushroom mycelia and carposomes [19,114,115]. Linoleic acid (C18:2) was found to be the most abundant FA among the species examined, followed by oleic acid (C18:1). Palmitic acid (C16:0) was the third and the second main FA for Pleurotus and Agaricus species, respectively (10.7-13.9%, w/w).
Aside from the three primary FAs already mentioned, seven others were identified and quantified (myristic, pentadecylic, ginkgolic, stearic, arachidic, behenic and lignoceric acids). The unsaturation index (U.I.) of FAs was high and increased in the RB substrate for P. ostreatus AMRL 144 (U.I. = 154), as well as for A. subrufescens in the compost of C/N = 13 (U.I. = 133). In P. eryngii species, PUFAs were found in lower concentrations (53.2-59.9%, w/w) than in P. ostreatus and Agaricus species (64.3-72.6%, w/w). The major FA found in all samples was linoleic acid (C18:2), whereas oleic acid (C18:1) was identified at much lower concentrations in Agaricus species (~1%, w/w). Therefore, all species examined, even if they were from different species, had similar FA profiles concerning the content of the main FAs. It has been also reported by other researchers that linoleic acid was the preponderant fatty acid in P. ostreatus, A. bisporus and many wild mushrooms [5,116]. High amounts of linoleic acid have been found not only in P. ostreatus (65.29%), but also in the fruit bodies of Lactarius salmonicolor (59.44%) and Flammulina velutipes (40.87%), as compared to other FAs [19]. Nevertheless, linoleic acid is a precursor of octen-3-ol, responsible for the attractive smell of (dried) mushrooms [83]. On the other hand, oleic acid was the predominant FA of oyster mushrooms grown on gmelina wood waste [117,118]. Although the nutritional contribution of mushroom lipids is limited for human diets, due to their low total lipid content and the low concentration of the desirable n-3 fatty acids, the unsaturation of lipids contained in mushrooms is a positive fact. Increasing the ratio of unsaturated fatty acids in the diet is important because it leads to an increase in HDL levels, known as good cholesterol, and a decrease in LDL levels, known as bad cholesterol [119], and mushrooms produced even in non-conventional substrates, as examined in the present study, can help achieve that. Table 4. Fatty acid composition (% w/w) of total lipids produced by P. ostreatus carposomes (strains AMRL 144 and 150) and P. eryngii (strains AMRL 166 and 173-6) cultivated on five substrates (WS, BOS, RB, BWS, CR) and by A. bisporus (AMRL 209) and A. subrufescens (AMRL 235) cultivated on two composts (C/N ratios of 10 and 13). Each point is the mean value of three independent measurements (mean ± SD).

Tocopherols
The tocopherols content in these mushroom species is presented in Table 5. P. ostreatus mushrooms were found to have α-tocopherol as the main tocopherol, except for P. ostreatus 150 and P. ostreatus 144, cultivated on WS and BOS substrates, respectively, where δtocopherol predominated. P. eryngii fungi, cultivated on BWS, RB and CR substrates, produced mainly α-tocopherol, whereas α-tocotrienol and β-tocotrienol were also produced in lower concentrations. On the other hand, P. eryngii 166 mushrooms, cultivated on WS and BOS substrates, were rich in δ-tocopherol. Concerning Agaricus species, α-tocopherol was the main tocopherol found in all mushroom samples and substrates, while both atocotrienol and β-tocotrienol were present. In general, the content of total tocopherols in mushrooms is 0.5-3 mg/kg [7]. Barros et al. [72] detected only αand β-tocopherol in five Agaricus species, and in all the samples β-tocopherol was the major compound. Specifically, a lower amount of α-tocopherol (749 ng/g d.w.) was detected than that in the present study. In P. ostreatus var. florida, α-, βand γtocopherols were detected (0.0002-0.0003, 0.2-0.26 and 0.05 mg/100g d.w., respectively) [120]. However, in another study, a significantly higher quantity of α-tocopherol was recorded in A. bisporus (9.2 mg/g) and P. ostreatus (0.9 mg/g) [121]. Such variations in the type and the quantity of tocopherols may be attributable to the different analytical methodologies and the different species; for example, cultivated species seem to be lower in tocopherols than wild mushrooms [79]. In any case, the high levels of α-and β-tocopherols indicate higher oxidative activity, which is associated with cardiovascular protection [86]. Table 5. Tocopherol composition (mg/kg) of P. ostreatus (AMRL 144 and 150) and P. eryngii (AMRL 166 and 173-6) carposomes (c), pilei (p) and stipes (s) cultivated on five substrates (WS, BOS, RB, BWS, CR) and of A. bisporus (AMRL 209) and A. subrufescens (AMRL 235) cultivated on compost of two different C/N ratios (10 and 13).

Total Phenolic Compounds (TPC), Antioxidant Studies and Total Reducing Power
The greatest TPC production was detected in pilei of P. ostreatus and P. eryngii strains and rarely in the whole carposome, especially on substrates of WS and BOS (Table 6). P. eryngii 166 was among the strains with the highest TPC, 19.20-56.47 mg GAE/g, while P. eryngii 173-6 had the lowest. Also, both P. ostreatus strains produced satisfactory amount of TPC (10.41-70.67 mg GAE/g d.w.). Regarding the impact of different substrates, their nature and their chemical composition significantly influenced the TPC in mushrooms [106,122]. CR and RB were the most favorable substrates, whereas all the strains produced the lowest TPC on BOS and WS. Moreover, Agaricus species seemed to produce significantly lower TPC than Pleurotus spp., with values ranging from 7.85 to 16.89 mg GAE/g d.w. ( Table 1). The highest TPC were detected again in pilei of Agaricus mushrooms, whereas A. subrufescens was a better phenolic compounds producer than A. bisporus. Also, A. bisporus presented a higher TPC concentration when cultivated on compost with the ratio C/N = 10, while the compost with a higher ratio (C/N = 13) favored A. subrufescens TPC production. The TPC of Pleurotus spp. in the present study were significantly higher than those detected by other researchers [86,122]. Da Paz et al. [122] cultivated Pleurotus sajor-caju on three different substrates and the TPC ranged from 56.26 to 205.23 mg/100 g d.w. Sharma et al. [86] reported TPC at 39.12-55.13 mg/100 g in Agaricus spp. and 53.20 mg/100 g in Pleurotus cystidiosus. Also, according to the literature, lower TPC were detected in P. eryngii and P. florida mushrooms (3.57 and 3.72 mg/g d.w., respectively) [123], as well as in the cap and stipe of A. bisporus (4 and 9.9 mg/100g d.w., respectively) [30]. The presence of high concentrations of phenolic compounds is responsible for the significant antioxidant properties of mushrooms. The functional medicinal compounds contained in them depend to a great extent on the substrate in which the fungi grow. If, for example, the substrate is high in functional molecules such as anthocyanidins, beta-glucans, selenium, ganoderic acid, triterpenes or cordycepin, then it is possible that the antioxidant substances in the produced mushrooms will be increased [86]. Table 6. Moisture, ash content (% w/w of dry biomass), total phenolic compounds (TPC), FRAP and scavenging ability on DPPH · and ABTS ·+ free radicals of methanol extracts of P. ostreatus (AMRL 144 and 150) and P. eryngii (AMRL 166 and 173-6) carposomes (c), pilei (p) and stipes (s), cultivated on five substrates (WS, BOS, RB, BWS, CR). Measurements of antioxidant studies are expressed as mg of gallic acid or trolox equivalence/g of mushroom dry weight (mean ± SD).  In the present investigation, the antioxidant activity of the mushroom extracts was examined using three assays, as antioxidant compounds have multiple mechanisms of action and no single approach can capture all of them. The capacity of mushroom extracts to scavenge free radical DPPH˙+ ranged from 0.35 to 8.72 mg trolox/g d.w. in P. ostreatus strains, and from 1.27 to 13.22 mg trolox/g d.w. in P. eryngii ones, while the scavenging capacity of the other free radical ABTS˙+ ranged from 1.04 to 11.27 mg trolox/g d.w. and 2.06 to 15.45 mg trolox/g d.w. in P. ostreatus and P. eyngii strains, respectively (Table 6). Moreover, the values for reducing power ranged from 0.25 to 11.06 mg trolox/g d.w. in P. ostreatus strains and from 1.27 to 13.41 mg trolox/g d.w. in P. eyngii strains. It is worth mentioning that the results of all assays revealed that pilei had higher antioxidant values than the corresponding stipes, in all strains and substrates examined. CR, RB and BWS were the substrates where the antioxidant activity of all Pleurotus strains was greater ( Table 6). As shown in Table 1, the antioxidant capacity of A. subrufescens was higher than that of A. bisporus in all assays. In general, Pleurotus spp. had better free radical scavenging ability than the Agaricus spp. This phenomenon had also been previously mentioned [124] when Pleurotus columbinus and P. sajor-caju antioxidant capacities were compared to that of A. bisporus. However, their ABTS˙+ scavenging activity was lower than that detected in this study. On the other hand, high amounts of TPC probably are linked to strong fungal extract antioxidant activity, as has already been detected by other researchers [86,[125][126][127].

Substrate/Fungi
It is remarkable that, according to our results, all tested fungi cultivated on WS developed noticeably low TPC, total reducing power and scavenging activity against ABTS˙+ and DPPH˙+ free radicals, indicating that substrate composition affects mushroom bioactive phenolics production. Previous studies have also noted that the chemical composition of the substrates used significantly affected the nutritional and bioactive compounds of mushrooms produced. For example, P. ostreatus and P. pulmonarius species cultivated on different woody substrates, such as beech sawdust, oak, linden, walnuts and poplar, showed significantly higher TPC concentrations on beech and linden [128].
The Pearson's correlation coefficient between the TPC results and those of DPPH and ABTS was characterized as moderate (r = 0.3696 and r = 0.3636, respectively, p < 0.05), and weak for those of FRAP (r = 0.2416, p < 0.05). This fact was expected, as the F-C method suffers from several interfering compounds that react with the F-C reagent to give elevated apparent phenolic content. On the other hand, correlation analysis between the results obtained with the three assays used to measure the antioxidant activity correlated significantly positively, with the greater relationship strength observed between the FRAP and ABTS results (r = 0.9469, p < 0.05).

Moisture, Ash Content and Mineral Analysis
As shown in Table 1, the results for the moisture contents of carposomes varied from 86 to 90%, consistent with previously reported values [2,3,7,28,30,38]. For Agaricus spp., it was higher in the carposomes than in the pilei, whereas in Pleurotus spp. (Table 6) they were similar. The stipes contained the least water of all, with the minimum values being 80-82%. Ash content in Pleurotus mushrooms varied from 3.41 to 8.50% for P. ostreatus and 3.57 to 6.58% for P. eryngii strains, while Agaricus species had higher ash amounts ranging from 8.06 to 9.21% (Tables 1 and 6). It seems that the ash contents of the carposomes examined in this study were low (below 10%), but within the indicated range [82,129], whereas Ulziijargal et al. [80] reported even lower ash values for A. bisporus and A. brasiliensis, at 6.72 and 5.90%, respectively.
The contents of major and trace mineral elements are listed in Table 7. Potassium (K) was the most prevalent mineral element, with values ranging from 120 to 350 g/kg d.w. for Pleurotus species and 360 to 410 g/kg d.w. for Agaricus spp., followed by phosphorus (P) and magnesium (Mg). It is worth noting that K distribution throughout the carposomes was unequal, with the pileus having a higher concentration than the stipe, as has also been demonstrated in previous research [7]. Also, sodium (Na) was detected in low amounts (less than 12 g/kg d.w.). A decrease in Na of 18-30% content in P. ostreatus and P. eryngii was detected in mushrooms produced on RB, BWS and CR substrates. As calcium (Ca) levels are not very high in mushrooms, the Ca values ranged from 6 to 28 g/kg d.w. The mineral composition of P. ostreatus species significantly varied depending on the substrate. Specifically, the contents of K, Na, Mg and Ca were higher in mushrooms from BOS than in those grown in other substrates, while the highest value of Cu was detected on RB and BWS for P. ostreatus and P. eryngii, respectively. Trace metals presence is related to the mushroom species, as well as to the age of the fruiting bodies and mycelium [130]. The findings of this research were comparable with those of prior studies [109,129,131,132]. The low Na content along with the high K content of mushrooms indicate that they may be included in an anti-hypertensive diet; in fact, K from fruits and vegetables can lower blood pressure [129]. Generally, mushrooms' K content ranges from 182 to 395 mg/100 g, whereas the recommended daily requirement is 3100 mg/day. However, the mineral level depends on the species, the mushroom's age, the diameter of the pilei and on the substratum [133]. Table 7. Macro-and microelement contents (calculated on dry biomass) (mean ± SD) of the carposomes (c), pilei (p) and stipes (s) of P. ostreatus (AMRL 144, 150) and P. eryngii (AMRL 166, 173-6), cultivated in five different substrates (WS, BOS, RB, BWS, CR), and A. bisporus (AMRL 209) and A. subrufescens (strain AMRL 235) on two different composts (C/N ratio of 10 and 13).

Conclusions
This study indicated that the nutritional value of mushrooms could be improved by a general increase in carbohydrates, proteins and antioxidant compounds and a decrease in sodium content. The carposomes, pilei and stipes of Pleurotus species, cultivated on new substrates like BOS, BWS, RB and CR, were rich in antioxidants, IPSs, fibers, proteins, lipids, polyunsaturated fatty acids, minerals and tocopherols. P. eryngii species were found to have higher amounts of IPSs and β-glucans than P. ostreatus and Agaricus ones. In WS, BOS and CR, high amounts of IPSs were produced in all species examined, whereas BOS and BWS seemed to positively affect protein production in the P. ostreatus species. BWS, RB and CR also enhanced the production of lipids for Pleurotus mushrooms (especially for P. ostreatus cultivated on CR, which presented the highest lipid concentration); because they contain high amounts of polyunsaturated fatty acids, this may lead to an increase in HDL levels, the good cholesterol. These results confirmed that the substrate synthesis affects the final mushroom composition, so the selection of the most suitable substrates may result in the enhancement of mushrooms' nutritional value. Therefore, these alternative substrates could be employed to produce mushrooms with higher contents of proteins, polysaccharides, unsaturated lipids or antioxidants, adapted to the demands of customers for balanced diets or medical purposes. Agaricus species contained lower amounts of IPSs than Pleurotus ones, a fact that could be useful for people with prediabetes problems. Regarding A. bisporus and A. subrufescens, those mushrooms cultivated in compost with C/N = 10 had higher protein levels compared to the ones with C/N = 13, confirming that the nitrogen concentration in the substrate affects mushroom protein content. Moreover, C/N = 10 favored the antioxidant activity of A. bisporus, while C/N = 13 enhanced the antioxidant activity of A. subrufescens. These mushrooms with high phenolic content and antioxidant capacities, along with a high concentration of β-glucans, could serve as a good source not only of functional foods, but also of supplements or drugs. Additionally, due to their low Na content, they could be ideal for an anti-hypertensive diet. As far as the individual parts of mushrooms were concerned, all pilei were richer in proteins, lipids and TPC, and they showed higher antioxidant activity and reducing power than stipes. On the other hand, all stipes, a by-product of mushroom cultivation, were richer in IPSs and glucans compared to the corresponding pilei, and these results were very interesting because most studies refer only to the carposomes. Thus, it is possible that both the pilei and also the stipes of Pleurotus and Agaricus can be used, not only as food supplements, but also re-utilized in new, enriched substrates for a second-cycle cultivation of mushrooms, in the framework of a circular economy.