Influence of Lipid Fermentation Wastewater on Yield and Nutritional Profile of Edible and Medicinal Mushrooms

Abstract

Utilizing agricultural waste to produce mushrooms may be a cost-effective and environmentally friendly proposition to address the nutritional and health demands of the growing global population. Mushrooms can grow on a range of substrates and their selection is based on their availability and cost. In this study, five types of local waste were mixed: olive crop residues (OC), coffee residue (CR) or rice husk (RH) with wheat straw (WS) and beech wood shavings (BW), respectively. Then, the mixtures were sprayed with 20% w/w lipid fermentation wastewater (LFW) from Rodosporidium toruloides that was used as an alternative substrate-moistening method. Afterwards, these mixtures were tested for cultivating Pleurotus spp., Ganoderma spp. and Lentinula edodes. The results showed that the substrate significantly affected the incubation period and the biological efficiency (BE), with OC mixed substrates proving to be the most favorable across the different species. Pleurotus spp. had the shortest cultivation times and the highest BE, while G. lucidum required the longest incubation periods and had the lowest BE, particularly on CR substrates. The study also found that substrates affected mushroom morphology. Nutritional analysis revealed significant differences in protein, polysaccharides, lipids, ash and energy content, depending on the species and substrate. High protein levels were found in P. eryngii (28.05–29.58% d.w.) and G. resinaceum (28.71–29.90% d.w.). The elevated total phenolic compounds (28.47–40.17 mgGAE/g) values in carposomes from CR and OC substrates for Ganoderma spp., L. edodes, P. pulmonarius and P. ostreatus, along with antioxidant activity (DPPH, ABTS, FRAP) assays, highlighted the crucial role of substrate composition in enhancing the medicinal properties of mushrooms. The mixed substrates also influenced the fatty acid (FA) and polysaccharide composition, with WS increasing unsaturated FAs and glucose (<69.8%) being the primary monosaccharide. The study suggests that using the spraying method of 20% w/w LFW as a moisture agent in these substrates is effective for mushroom production.

1. Introduction

Mushrooms are becoming an increasingly important component of global diets due to their high nutritional value and medicinal properties [1]. They are a good source of protein, vitamins and minerals [2]. Additionally, they have anti-cholesterol, anticancer and antitumor properties, and are useful for managing diabetes and lung diseases [3]. Among fungi, Pleurotus, Lentinula and Ganoderma species are widely cultivated and well known for their nutritional or/and medicinal value [1]; they are efficient colonizers and bio-converters of lignocellulosic residues into mushrooms with unique biological and pharmacological characteristics [4].

The chemical properties of the substrate are key factors that influence both the quantity and quality of mushrooms, ultimately determining their nutritional profile [5]. Therefore, understanding the substrate’s chemical composition is crucial before using it in mushroom cultivation. As the rapid growth of agro-industrial activities has led to the excessive production of agro-waste and significant global quantities of lignocellulosic residues, their re-utilization, especially for those requiring microbial involvement for biodegradation and potentially posing environmental risks (such as coffee pulp, husks, olive wastes and olive crop residues), is essential for maintaining environmental balance [6]. Utilizing these wastes as substrates, either individually or in mixtures, is particularly important. Substrate mixtures are often more beneficial, promoting faster growth and higher yields than homogeneous substrates due to their balanced chemical composition [7]. Although paddy straw and wheat straw are the traditional substrates for Pleurotus spp., different biological efficiencies have been reported by various authors, viz., 11.07, 75–100 and 97% for the latter [4,8,9].

In addition to waste management, water scarcity has emerged as a pressing issue affecting both arid regions and areas with abundant rainfall due to limited supply and poor water quality [10]. Sustainable water use focusing on conservation, environmentally friendly practices, suitable technologies, economic feasibility and social acceptance is crucial for agriculture, especially in water-scarce areas [10]. Therefore, innovations in irrigation management are essential, as agriculture remains the largest consumer of water in these regions. Crude glycerol, a renewable, abundant and low-cost byproduct of biodiesel production, can serve as a primary carbon source in industrial fermentations [11] and utilizing it helps offset the low market price of glycerol. Furthermore, glycerol-containing wastewater is generated from bioethanol, alcoholic beverage production and oleochemical processes like soap and fatty acid production [11]. Microbial lipids (SCOs) produced by yeasts, algae, fungi and bacteria are gaining attention as feedstocks for non-conventional biodiesel or as substitutes for valuable fats like cocoa-butter and fish oils [12]. However, lipid fermentation wastewater (LFW) disposal and treatment are one of the major problems in microbial fermentation because in many instances they contain non-negligible quantities of salts [6]. The chemical composition of LFW can vary, leading to different treatment requirements and outcomes. In some cases, this wastewater could be repurposed as maceration water in the solid-state fermentation of edible and medicinal mushrooms [6,13,14]. This aligns with international zero-waste and water-saving policies [15].

Cultivated mushrooms typically have less fat and digestible polysaccharides but more protein than most vegetables [16], making them a valuable addition to low-calorie diets. Interestingly, the nutrient content of mushroom fruiting bodies, particularly proteins, can vary depending on the type of agro-waste used during cultivation [17]. Mushroom production serves as an excellent example of how biological processes can recover food proteins from lignocellulosic materials on both small and large scales [18]. In addition to their protein content, mushrooms contain lipids, which make up 6–8% of their dry weight (d.w.). These include essential polyunsaturated fatty acids that are crucial for human basal metabolism and offer various health benefits [19].

Beyond their nutritional value, crude polysaccharides extracted from various mushroom species, such as G. applanatum [20], L. edodes [21], G. lucidum [22] and P. sajor-caju [23], have shown promising antibacterial, antioxidant and immunomodulatory properties. Mushrooms also contain various polyphenolic compounds, which are recognized for their excellent antioxidant activity [24]. Pleurotus species are also known for their antioxidant properties, with recent research highlighting the antioxidant activity in species such as P. ostreatus and P. eryngii [25].

The objective of this study was to evaluate the effect of replacing soaking in tap water with spraying using 20% w/w LFW from Rodosporidium toruloides on carposome production and the synthesis of metabolic compounds in Ganoderma spp., Pleurotus spp. and L. edodes (including valuable polysaccharides, antioxidants and lipids). This solid-state fermentation was performed on various strains of the above basidiomycetes across a range of agro-industrial residues [14] using completely eco-friendly and sustainable processes. The concentration of 20%, w/w LFW was selected in our previous study as a generally accepted concentration for the experiments concerning the mushroom cultivation procedure due to the lack of prior studies or established guidelines. Although alternative concentrations may produce different results, this one was selected to examine basic mushrooms’ physiological parameters for subsequent investigation and optimization of cultivation and fruiting. Although the soaking of substrates in LFW was also tested, the spraying method achieved the desired moisture level, creating optimal conditions for mushroom growth and contributing to water conservation compared to soaking in LFW or tap water, while giving the best results. Therefore, in this study, the results concerning the yield and the nutritional data of the carposomes produced in olive crop residues, coffee residue, or rice husk mixed with wheat straw and beech wood shavings sprayed with 20% w/w are presented.

2. Materials and Methods

2.1. Fungal Species, Substrates and Culture Conditions

The mushroom strains used in solid-state fermentations were P. ostreatus (AMRL 135), P. eryngii (AMRL 161), P. pulmonarius (AMRL 177), G. applanatum (AMRL 341), G. resinaceum (AMRL 325), G. lucidum (AMRL 330) and L. edodes (AMRL 121) of the Laboratory of Edible Fungi/Institute of Technology of Agricultural Products/Hellenic Agricultural Organization—Dimitra. The strains were maintained on potato dextrose agar (PDA, Merck, Darmstadt, Germany) at 4 ± 1 °C and they were grown at 26 ± 1 °C prior to spawn production. Before the experiments, each species’ grain spawn was created in 500-mL Erlenmeyer flasks with 180 g of boiled millet (Panicum miliaceum), as previously described Philippoussis et al. [8].

Substrate formulations were composed on a dry substrate weight basis. The substrates included olive crop residues (leaves and branches) (OC), coffee residue (CR), rice husk (RH), beech wood shavings (BW), wheat straw (WS), wheat bran (WB) and soybean flour (SF) (additives). All substrates were derived by various Greek farms and industries, except for CR, which was obtained from espresso coffee preparation at a local coffee shop. The different substrates were prepared by combining residues (dry substrate weight) as follows: (1) CR:80, WS:18 (coffee residue + wheat straw, named CW); (2) CR:80, CB:18 (coffee residue + beech wood shavings, CB); (3) OC:70, WS:12 (olive crop + wheat straw, OW); (4) OC:70, BW:12 (olive crop + beech wood shavings, OB); (5) RH:70, WS:12 (rice husk + wheat straw, RW) and (6) RH:70, BW:12 (rice husk + beech wood shavings, RB). Wheat bran and soybean flour were used as supplements to obtain a final C/N ratio of 20–30. Calcium carbonate was also added to the substrates (1% w/w, in terms of dry weight) to obtain a pH = 6.0–7.5. This process is detailed in the previous study by Diamantis et al. [14]. The substrates were then sprayed with 20% w/w lipid fermentation wastewater (LFW), serving as an alternative method for substrate moistening.

Five replicates of polypropylene-autoclavable bags per substrate and strain were filled with 1 kg of substrate, autoclaved at 121 ± 1 °C for 2 h (1.1 atm) and inoculated with 3–5% w/w (on fresh weight basis) mushroom spawn under aseptic conditions. Substrate colonization took place in an incubator at 25 ± 1 °C and 80% RH, in the dark. After complete colonization, the bags were transferred to the fruiting room (used for all stages) with specific environmental conditions (a temperature between 15 and 18 °C, relative humidity of 90% and light intensity of 700 lux) for mushroom induction. The total time taken for the colonization and carposomes formation period (first crop; earliness) was recorded, along with the number and weight of fruit bodies. The Biological Efficiency (BE %) was defined as the percentage ratio of the fresh weight of harvested mushroom over the dry weight of substrate [8,26]. Mature fruit bodies were harvested daily, counted and weighed, while the pileus diameter [average pileus diameter = (longer + shorter pileus diameter)/2] and stipes’ diameter and length (length from pileus base to end of stalk) were measured. The harvested mushrooms were dried by a Heto LyoLab 3000 freeze-dryer (Heto-Holten Als, Allerød, Denmark) and used for nutritional and bioactive compound analysis. Various parameters were measured during the experiments.

2.2. Analytical Methods

2.2.1. Protein

The proteins were determined by Bradford’s method [27]. A total of 50 mg of lyophilized mushroom samples were extracted with 50 mM EDTA using an ultrasonic bath for 60 min. The homogenate was centrifuged at 6000 rpm for 10 min. After extraction, 10 µL of different mushroom samples were mixed with 190 µL of Coosmassic Brillaint Blue solution and then incubated for 10 min at a temperature of 25 °C, and absorbance at 620 nm was recorded against the reagent blank. A standard curve of protein was made of BSA 0.1–1.5 mg/mL by measuring the absorption at 620 nm using a 96-cell microplate reader spectrophotometer. All samples were analysed in triplicate.

2.2.2. Lipid

The total cellular lipid was extracted from the lyophilized mushroom by chloroform/methanol: 2/1 (v/v) mixture and determined gravimetrically [28]. The derivatization of fatty acids to methyl-esters was performed in a two-stage reaction (to avoid trans-isomerization) using sodium methoxide and methanol/hydrochloride according to Dedousi et al. [29]. Fatty acid methyl esters were identified by reference to authentic standards. For this purpose, methyl-esters were suspended in hexane and analysed by GC in a Varian CP-3800 chromatograph equipped with a flame ionization detector (Agilent Technologies, Santa Clara, CA, USA), in which an Agilent J&W Scientific DB23 capillary column (model n.123–2332, 30.0 m × 0.32 mm, film thickness 0.25 μm) (Agilent Technologies, Santa Clara, CA, USA) was used. Helium gas 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 150 ± 1 °C, held for 18 min, subsequently rammed to 185 ± 1 °C at a rate of 5 °C/min and held for 2 min. Then, the oven temperature was moved to 210 ± 1 °C at a flow rate of 5 °C/min and held for 2 min, then increased to 240 ± 1 °C at 10 °C/min. The injector and flame ionization detector temperatures were set at 260 ± 1 °C and 270 ± 1 °C, respectively. Individual fatty acid methyl-esters were identified by comparison with their retention times with external standard (Supelco 37 Component FAME Mix, CRM47885, Darmstadt, Germany) retention times. The amounts of individual fatty acid methyl-esters identified were expressed in % of the total fatty acid areas chromatograms identified.

2.2.3. Intracellular Polysaccharides (IPS) and Ash Content

Intracellular polysaccharide (IPS) determination was conducted after acid hydrolysis, as described by Diamantopoulou et al. [30]. To perform this procedure, IPS were extracted from lyophilized mushroom (100 mg) using 5 mL of 2.5 M HCl, heated to 100 ± 1 °C for 20 min. The mixture was then neutralized to pH 7 using 2.5 M NaOH, and the final volume was adjusted to 20 mL. After filtering the mixtures, 0.5 mL of each filtrate was combined with 0.5 mL of DNS reagent, vortexed and incubated at 100 ± 1 °C for 5 min. After cooling the tubes to room temperature in a water bath, the IPS content was determined by measuring the absorbance at 540 nm. All samples were analysed in triplicate. The composition of monosaccharides of the produced IPS was performed by HPLC analysis. Thus, filtered aliquots of the neutralized samples with NaOH were analysed by a Waters Association 600E apparatus (Waters Corporation, Milford, MA, USA) at a 30.0 cm × 7.8 mm column Aminex HPX-87H (Bio-Rad, Hercules, CA, USA). The mobile phase used was H₂SO₄ at 0.005 M with a flow rate 0.8 mL min⁻¹, and the column temperature was 65.0 ± 0.5 °C. Individual simple sugars and sugar-alcohols were detected by an RI detector (differential refractometer 410-Waters). The ash contents of the mushroom samples were determined using a previously established method [31].

2.2.4. Energy Values

Energy values of 100 g of fresh mushrooms were calculated according to the following equations [32].

Energy (kcal/100 g) = 4 × (g protein + g carbohydrate) + 9 × (g lipid)

2.3. Total Phenolic Compounds and Antioxidant Activity

Methanolic extracts (sample) for the total phenolic compounds and antioxidant activity were prepared as follows: 250 mg of lyophilized mushroom were extracted with 5 mL of methanol using an ultrasonic bath (SKYMEN, JP-060S, Shenzhen, China) (15 min, room temperature) followed by vortex and centrifugation (3500 rpm, 15 min, ambient temperature; Micro 22R, Hettich, Kirchlengern, Germany). The same extraction was repeated three times and the supernatants were used for further analysis, stored at 4.0 ± 0.5 °C.

2.3.1. Analysis of Phenolic Compounds

The determination of phenolic compounds in mushroom samples was estimated using the Folin–Ciocalteau (FC) assay [33]. Briefly, 0.5 mL of each sample was diluted in 10.5 mL H₂O and mixed with 8 mL Na₂CO₃ (Alpha Aesar, Kandel, Germany) (75 g/L) and 1 mL of FC (Merck, Darmstadt, Germany) reagent. The mixtures were vortexed and allowed to react in the dark for 2 h. Absorbance was then read at 750 nm using gallic acid as standard. Each sample was analysed in triplicate.

2.3.2. Determination of Antioxidant Activities (DPPH, ABTS and FRAP Methods)

The free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was employed to evaluate free radical scavenging, based on the method detailed by [34,35], with minor modification. An aliquot of 3.9 mL of 60 mM DPPH radical (Sigma-Aldrich, Darmstadt, Germany) in methanol was added to a test tube with 0.1 mL of sample; the mixture was then incubated at room temperature for 30 min in the dark. Methanol was used instead of the mushroom sample as a control. The reaction mixture was vortex mixed at room temperature and the absorbance (Abs) was determined by measuring at 520 nm with a spectrophotometer, using trolox as a standard. Samples were prepared and measured in triplicates.

Free Radical Scavenging Activity Assay: This assay was carried out using the modified method of Re et al. [36]. The ABTS stock solution was prepared by reacting ABTS (Sigma-Aldrich, Darmstadt, Germany) aqueous solution (7 mM) with 2.45 mM aqueous solution of potassium persulfate (Merck, Darmstadt, Germany) in equal quantities; the mixture was allowed to stand in the dark at room temperature for 12–16 h before use. The working solution of ABTS was obtained by diluting the stock solution in methanol to give an absorbance of 0.70 ± 0.02 at 734 nm. Then, 2.0 mL of ABTS solution was mixed with 50 μL of the sample. The mixture was then incubated at room temperature for exactly 10 min in the dark. The control was prepared by mixing 2.0 mL of ABTS solution with 1 mL of double distilled water. The absorbance was measured against a blank at 734 nm using trolox as a standard. Samples were prepared and measured in triplicates.

Determination of Ferric Reducing Antioxidant Power (FRAP): This method is based on the ability of the sample to reduce Fe³⁺ to Fe²⁺ ions. At low pH, in the presence of TPTZ, ferric-tripyridyltriazine (Fe³⁺-TPTZ) complex is reduced to the ferrous (Fe²⁺-TPTZ) form with the formation of an intense blue colour having an absorption maximum at 593 nm. The method is described by Arnous et al. [34]. The working solution (stock solution) was prepared prior to use as follows: 25 mL of acetic acid buffer (300 mM/L, pH 3.6) was mixed with 5 mL of TPTZ (Sigma-Aldrich, Darmstadt, Germany) solution (10 mM/L 2,4,6-tripyridyltriazine in 40 mM/L HCl) and 2.5 mL of FeCl₃·6H₂O (20 mM/L in deionized water). Then, 300 μL of sample was added to 2700 μL of FRAP solution. The mixtures were stirred and incubated at 37 ± 0.5 °C for 10 min. Absorbance was measured at 593 nm, using trolox as a standard.

2.4. Data Analysis

All experiments were repeated at least twice and, within experiments, triplicate bags were used per strain and substrate to generate each data point. Variance analysis was performed by JMP 7.0.1. software (SAS, Cary Institute, Cary, NC, USA), using the Least Significant Difference (LSD) test at 5% level of probability to compare mean values of parameters tested.

3. Results and Discussion

3.1. Fungal Cultivation and Mushroom Yield Evaluation in Polypropylene Bags

The combination of the suitable species and substrate is essential for a successful mushroom cultivation because the length of the incubation and the sporophore induction phase are linked to contaminations of the substrate and consequent bags loss [1,5]. The time required for complete colonization and for carposome formation until the first harvest varied among the mushroom species and substrates examined (Figure 1). Both OW and OB substrates emerged as the most favorable, promoting rapid mycelial growth and demonstrating shorter earliness values across all examined species. In certain cases, these results were significantly superior to those observed with other substrates. For instance, P. pulmonarius and P. eryngii showed nearly half the earliness values on these substrates compared to CR for P. pulmonarius and RH and CR for P. eryngii. Overall, Pleurotus species had shorter cultivation periods compared to Ganoderma spp. and L. edodes, with G. lucidum showing the longest earliness values on CW (129 days) and CB (132 days). Although the additives WS and BW did not show significant differences when added to the same main substrate, WS generally produced better results, leading to shorter colonization and/or earliness periods. The current results are in accordance with from Philippoussis et al. [8], Melanouri et al. [26,37] and Dedousi et al. [38]. Specifically, Melanouri et al. [37] reported rapid mycelial growth for P. eryngii strains when cultivated in olive pulp substrate, and earliness values ranging from 35 to 63 days for Pleurotus spp. when grown in 60% coffee residue mixtures with wheat straw.

As the BE % of the substrates determines the ability of a particular strain to grow on that substrate, this parameter was further evaluated. It was noted that the BE % of P. ostreatus, P. eryngii and P. pulmonarius was found within a narrow range (

Table 1. Mushroom production and biological efficiency (BE %) of selected Pleurotus, Ganoderma and Lentinula species carposomes produced in substrates sprayed with 20% w/w LFW. Data are presented as mean values from duplicated measurements of five replicates (mean ± SD).

Mushroom Species	Substrates	ΒE (%)	Flushes	Average Fresh Weight (g)	Pileus Diameter (mm)	Stipe Length (mm)
P. ostreatus	CW *	65.45 ± 3.25 b,c **	3	168.86 ± 11.21 a,b	40.00 ± 1.21 b	31.00 ± 1.25 a
	CB	64.58 ± 3.14 b,c	3	151.76 ± 10.24 b	40.00 ± 2.14 b	30.00 ± 1.77 b
	OW	72.21 ± 2.78 a	3	176.19 ± 9.74 a	50.00 ± 2.14 a	40.00 ± 1.98 b
	OB	71.48 ± 2.74 a,b	3	174.41 ± 8.98 a,b	45.00 ± 1.44 a,b	32.00 ± 1.74 b
	RW	62.41 ± 3.33 c	2	73.64 ± 3.45 c	45.00 ± 2.12 a,b	33.00 ± 2.22 b
	RB	63.23 ± 1.65 c	2	79.04 ± 2.84 c	40.00 ± 3.54 b	30.00 ± 2.14 b
P. eryngii	CW	58.56 ± 1.47 b	2	151.08 ± 10.23 a,b	30.00 ± 2.28 b	45.00 ± 3.96 b
	CB	54.59 ± 1.63 b	2	128.29 ± 9.85 b	30.00 ± 1.99 b	45.00 ± 4.75 b
	OW	67.36 ± 2.02 a	3	164.36 ± 20.36 a	45.00 ± 3.33 a	70.00 ± 5.24 a
	OB	65.21 ± 3.55 a	3	159.11 ± 15.98 a,b	48.00 ± 2.09 a	70.00 ± 5.11 a
	RW	58.32 ± 1.78 b	2	68.82 ± 8.56 c	35.00 ± 4.01 b	55.00 ± 2.87 b
	RB	56.69 ± 2.85 b	2	70.86 ± 6.58 c	30.00 ± 3.69 b	56.00 ± 3.02 b
P. pulmonarius	CW	68.31 ± 2.18 a,b	3	155.16 ± 10.25 a,b	45.00 ± 2.96 a	25.00 ± 1.03 a
	CB	67.45 ± 3.91 a,b	3	137.40 ± 9.77 b	40.00 ± 2.58 a	20.00 ± 1.07 b
	OW	71.32 ± 2.45 a	3	166.68 ± 8.69 a	45.00 ± 2.36 a	20.00 ± 1.11 b
	OB	72.34 ± 3.78 a	3	164.58 ± 8.76 a	40.00 ± 2.44 a	25.00 ± 0.98 a
	RW	60.14 ± 2.37 b	2	90.43 ± 5.69 c	40.00 ± 2.88 a	20.00 ± 1.22 b
	RB	58.47 ± 3.85 b	2	84.16 ± 6.31 c	45.00 ± 3.69 a	25.00 ± 0.99 a
G. resinaceum	CW	2.25 ± 0.85 b	1	5.81 ± 0.88 b	45.00 ± 2.99 a	na ***
	CB	2.68 ± 0.36 a,b	1	6.30 ± 0.98 b	45.00 ± 2.56 a	na
	OW	3.98 ± 0.68 a	1	9.71 ± 1.03 a	50.00 ± 2.98 a	na
	OB	3.87 ± 0.56 a	1	9.44 ± 1.02 a	50.00 ± 3.55 a	na
	RW	0.51 ± 0.04 c	1	0.60 ± 0.06 c	46.00 ± 3.21 a	na
	RB	0.54 ± 0.03 c	1	0.68 ± 0.08 c	42.00 ± 3.55 a	na
G. lucidum	CW	2.35 ± 0.08 c	1	6.06 ± 0.58 a	50.00 ± 3.74 a	na
	CB	2.58 ± 0.08 b	1	6.06 ± 0.78 a	50.00 ± 3.69 a	na
	OW	2.98 ± 0.09 a	1	7.27 ± 0.99 a	55.00 ± 2.96 a	na
	OB	2.87 ± 0.06 a	1	7.00 ± 0.98 a	50.00 ± 2.98 a	na
	RW	0.62 ± 0.08 d	1	0.73 ± 0.05 b	45.00 ± 3.68 a	na
	RB	0.65 ± 0.04 d	1	0.81 ± 0.06 b	45.00 ± 3.58 a	na
G. applanatum	CW	0.56 ± 0.02 b,c	1	1.44 ± 0.66 a,b	45.00 ± 2.55 a,b	na
	CB	0.54 ± 0.01 c	1	1.27 ± 0.32 a,b	40.00 ± 2.66 b	na
	OW	0.61 ± 0.03 b	1	1.49 ± 0.41 a	45.00 ± 2.31 a,b	na
	OB	0.68 ± 0.02 a	1	1.66 ± 0.09 a	50.00 ± 1.88 a	na
	RW	0.42 ± 0.01 d	1	0.50 ± 0.05 b	40.00 ± 2.85 b	na
	RB	0.43 ± 0.01 d	1	0.54 ± 0.01 b	40.00 ± 2.96 b	na
L. edodes	CW	6.87 ± 0.85 b	1	16.76 ± 0.99 b	40.00 ± 2.35 a	25.00 ± 1.47 a
	CB	5.32 ± 0.32 b	1	12.98 ± 1.21 b	35.00 ± 2.47 a,b	30.00 ± 1.66 a
	OW	10.21 ± 0.66 a	1	26.58 ± 1.87 a	35.00 ± 2.85 a,b	29.00 ± 1.74 a
	OB	10.34 ± 0.79 a	1	27.41 ± 2.95 a	35.00 ± 2.36 a,b	30.00 ± 1.96 a
	RW	5.32 ± 0.25 b	1	13.73 ± 1.24 b	40.00 ± 2.32 a	29.00 ± 3.65 a
	RB	5.41 ± 0.45 b	1	12.71 ± 1.24 b	30.00 ± 2.87 b	28.00 ± 1.44 a

* CW: (coffee residue + wheat straw), CB (coffee residue + beech wood shavings), OW (olive crop + wheat straw), OB (olive crop + beech wood shavings), RW (rice husk + wheat straw) and RB (rice husk + beech wood shavings). ** Columns within the same strain not sharing the same letters are significantly different at p = 0.05. *** na: not applicable for Ganoderma spp.

) of values (62.41 to 72.21%, 54.59 to 67.36% and 58.47 to 72.34%, respectively). In contrast, L. edodes exhibited a BE % ranging from 5.32 to 10% and Ganoderma spp. had a BE of less than 5%. Several studies examining the suitability of various lignocellulosic materials and agro-industrial residues as substrates for mushroom cultivation have revealed significant variations in yield and biological efficiency [7]. Akyüz and Yildiz [39] reported a high biological efficiency (BE) of 50–73% for P. eryngii cultivated on various substrates. In contrast, Chai et al. [40] found a lower BE of 18.61% for P. pulmonarius, while Economou et al. [41] recorded a significantly higher BE than observed in this study. For P. eryngii, Sardar et al. [42] reported a BE ranging from 45 to 72% across different substrates. Mansour-Benamar et al. [43] noted a lower BE of 11–34% for P. ostreatus grown on olive mill wastes. In studies on Ganoderma, Erkel [44] reported an average BE of 15.09% on poplar, beech and oak sawdust, while Azizi et al. [45] observed a BE of 12.89% on hornbeam sawdust. Veena and Pandey [46] found a BE between 25.7 and 29.9% for G. lucidum and Ueitele et al. [47] recorded a BE of 5.32% for Ganoderma grown on corn cobs. Philippoussis et al. [48] documented a BE exceeding 40% for L. edodes on various substrates (i.e., wheat straw and oak-wood sawdust), which is notably higher than the findings of this study. These variations in BE can be attributed to differences in mushroom genotype and the composition of the substrates used.

The morphological characteristics (i.e., pileus diameter, stipe length and diameter) of the examined mushrooms grown on the various substrates (Table 1) were influenced by the structure and physical properties of the substrates, which are determined by the type of agricultural residues used. The pileus diameter ranged from 40 to 45 mm for P. ostreatus and P. pulmonarius and from 30 to 48 mm for P. eryngii. For Ganoderma spp., the values varied between 40 and 55 mm, with G. lucidum on the OW substrate registering the maximum value. L. edodes exhibited pileus diameters ranging from 30 to 45 mm. Regarding stipe length, P. ostreatus showed values between 30 and 40 mm, P. eryngii between 45 and 50 mm and P. pulmonarius between 20 and 25 mm, while L. edodes had stipe lengths ranging from 25 to 30 mm. Generally, the CR substrate promoted shorter stipe lengths, especially for P. eryngii (45 mm), which is known for its long and thick stem. On the other hand, this resulted in P. ostreatus and P. pulmonarius strains forming larger pilei and shorter stipes, aligning well with market demands. The results regarding the morphological characteristics in this study are consistent with the findings of several researchers. Sardar et al. [49] reported similar values for pileus diameter in a P. ostreatus strain, ranging between 52 and 69 mm, with even higher stipe lengths recorded from 45 to 68 mm, whereas Melanouri et al. [26] reported lower values of stipe diameter for P. ostreatus and P. eryngii strains with values ranging from 9.00 to 14.00 mm and 18.29 to 38.33 mm, respectively. Park et al. [50] also recorded comparable values for L. edodes, with a pileus diameter of 35.0–57.9 mm and a stipe length ranging from 23.6 to 52.6 mm.

Overall, the application of LFW in the substrates proved suitable for most of the examined species. It not only promoted high BE values, particularly for Pleurotus spp., but also resulted in carposomes with desirable morphological characteristics, even for L. edodes—a species for which substrate salinity is often considered a growth-limiting factor [51]. Therefore, the salt concentration in LFW was not inhibitory for this L. edodes strain. Regarding BE values, Melanouri et al. [6] indicated that LFW derived from Mortierella ramanniana had no significant effect on the BE of the examined mushrooms.

3.2. Nutritional Composition of Mushrooms

The macronutrient content (ash, carbohydrates, protein and lipids) and energy values for the studied edible/pharmaceutical mushrooms, with the addition of 20% w/w sprayed LFW, are presented in

Table 2. Chemical composition (%, w/w of d.w.) and energy values (per 100 g of f.w.) of by selected of Pleurotus, Ganoderma and Lentinula mushroom species during solid-state fermentation produced in substrates sprayed with 20% w/w LFW. Data are presented as mean values from duplicated measurements of five replicates (mean ± SD).

Mushroom Species	Substrate	%, w/w d.w.				(%, f.w.)
		Protein	IPS	Lipid	Ash	kcal
P. ostreatus	CW *	25.57 ± 1.23 a **	38.15 ± 1.25 a,b	6.76 ± 0.98 a	8.01 ± 0.98 a	89.42 ± 10.36 a,b
	CB	26.34 ± 0.93 a	41.08 ± 1.33 a	2.44 ± 0.83 b	7.85 ± 0.56 a	52.15 ± 5.58 c
	OW	27.49 ± 0.85 a	37.30 ± 1.85 a,b	7.18 ± 0.96 a	7.95 ± 0.87 a	94.01 ± 6.36 a
	OB	27.69 ± 1.79 a	35.90 ± 1.45 b	5.60 ± 0.74 a	7.92 ± 0.85 a	79.36 ± 4.95 a,b
	RW	27.93 ± 0.88 a	31.80 ± 1.36 c	5.58 ± 0.65 a	7.99 ± 0.64 a	77.74 ± 5.45 a,b
	RB	25.09 ± 1.87 a	29.03 ± 1.22 c	5.32 ± 0.88 a	7.69 ± 0.79 a	72.77 ± 4.85 b
P. eryngii	CW	28.17 ± 1.69 a	37.50 ± 2.31 a	6.96 ± 0.96 a	7.41 ± 0.65 a	92.41 ± 5.98 a
	CB	28.42 ± 1.45 a	35.48 ± 1.85 a,b	2.96 ± 0.85 b	7.06 ± 0.49 a	55.83 ± 5.78 c
	OW	28.85 ± 0.98 a	34.89 ± 1.96 a,b	4.92 ± 0.69 a,b	7.45 ± 0.86 a	73.50 ± 6.95 b
	OB	28.05 ± 1.36 a	35.00 ± 2.01 a,b	4.52 ± 0.75 b	7.65 ± 0.36 a	69.49 ± 4.77 b,c
	RW	29.37 ± 1.44 a	31.04 ± 1.09 b	4.04 ± 0.85 b	7.54 ± 0.78 a	64.43 ± 4.65 b,c
	RB	29.58 ± 1.56 a	30.74 ± 1.33 b	4.32 ± 0.73 b	7.32 ± 0.61 a	67.03 ± 2.36 b,c
P. pulmonarius	CW	27.25 ± 1.56 a	26.16 ± 1.87 a	3.80 ± 0.22 a,b	7.28 ± 0.61 a	59.22 ± 5.66 a,b
	CB	28.42 ± 2.31 a	25.93 ± 1.96 a	2.42 ± 0.36 b,c	7.68 ± 0.25 a	47.35 ± 4.89 b,c
	OW	27.82 ± 1.98 a	25.92 ± 1.56 a	4.16 ± 0.48 a	7.84 ± 0.41 a	62.67 ± 4.78 a
	OB	28.39 ± 1.09 a	24.94 ± 1.48 a	3.52 ± 0.65 a,b	7.64 ± 0.62 a	57.00 ± 4.63 a,b
	RW	28.43 ± 1.22 a	18.26 ± 1.55 b	1.48 ± 0.74 c	7.84 ± 0.66 a	36.10 ± 4.23 c
	RB	28.43 ± 1.36 a	18.83 ± 1.08 b	1.52 ± 0.98 c	7.33 ± 0.74 a	36.64 ± 5.67 c
G. resinaceum	CW	29.23 ± 1.55 a	38.63 ± 1.87 a,b	1.32 ± 0.98 a	5.14 ± 1.21 a	42.69 ± 3.69 a,b
	CB	29.90 ± 1.09 a	42.22 ± 2.33 a	1.01 ± 0.63 a	4.85 ± 1.36 a	41.24 ± 3.84 b
	OW	28.81 ± 2.06 a	34.56 ± 1.99 b	2.54 ± 0.48 a	5.85 ± 1.41 a	51.90 ± 4.56 a
	OB	28.71 ± 2.00 a	35.00 ± 2.07 b	2.21 ± 0.85 a	5.78 ± 1.36 a	49.07 ± 5.21 a,b
	RW	nm ***	nm	nm	nm	nm
	RB	nm	nm	nm	nm	nm
G. lucidum	CW	28.54 ± 1.22 a	33.54 ± 2.01 a	2.92 ± 0.58 a	3.25 ± 0.87 a	54.81 ± 6.98 a
	CB	29.03 ± 1.98 a	33.97 ± 2.33 a	3.14 ± 0.66 a	4.23 ± 0.69 a	57.22 ± 5.87 a
	OW	29.00 ± 1.54 a	35.74 ± 2.47 a	2.74 ± 0.85 a	3.33 ± 0.84 a	54.25 ± 4.87 a
	OB	29.22 ± 2.03 a	36.17 ± 1.98 a	2.41 ± 0.98 a	4.56 ± 0.69 a	51.59 ± 5.88 a
	RW	nm	nm	nm	nm	nm
	RB	nm	nm	nm	nm	nm
G. applanatum	CW	27.25 ± 2.01 a	33.45 ± 3.58 a	2.55 ± 0.75 a	4.84 ± 0.64 a	50.76 ± 8.45 a
	CB	28.42 ± 1.98 a	33.88 ± 2.54 a	2.12 ± 0.58 a	5.33 ± 0.74 a	47.74 ± 7.15 a
	OW	27.82 ± 1.87 a	35.09 ± 2.87 a	2.41 ± 0.96 a	5.19 ± 0.68 a	50.45 ± 7.41 a
	OB	28.39 ± 2.04 a	34.81 ± 2.45 a	2.33 ± 0.87 a	4.71 ± 0.74 a	49.96 ± 2.36 a
	RW	nm	nm	nm	nm	nm
	RB	nm	nm	nm	nm	nm
L. edodes	CW	26.02 ± 1.98 a	40.22 ± 2.03 a	2.76 ± 0.56 a	5.23 ± 0.84 a	54.46 ± 4.75 a
	CB	26.49 ± 2.03 a	40.40 ± 2.74 a	2.44 ± 0.45 a	5.65 ± 0.73 a	51.93 ± 4.67 a
	OW	28.26 ± 1.74 a	39.78 ± 2.14 a	2.78 ± 0.61 a	6.01 ± 0.45 a	55.76 ± 4.85 a
	OB	27.70 ± 1.96 a	40.83 ± 2.30 a	2.44 ± 0.25 a	5.98 ± 0.62 a	52.75 ± 5.61 a
	RW	27.56 ± 0.97 a	38.85 ± 2.15 a	2.85 ± 0.74 a	5.71 ± 0.43 a	55.61 ± 3.33 a
	RB	27.35 ± 1.09 a	39.10 ± 1.97 a	2.56 ± 0.35 a	5.62 ± 0.66 a	52.98 ± 4.51 a

* CW: (coffee residue + wheat straw), CB (coffee residue+ beech wood shavings), OW (olive crop + wheat straw), OB (olive crop + beech wood shavings), RW (rice husk + wheat straw) and RB (rice husk + beech wood shavings). ** Columns within the same strain not sharing the same letters are significantly different at p = 0.05. *** nm: not measured due to low BE%.

. The results revealed that among the nutritional properties, protein content was 25.57–29.90% d.w. in all species and substrates. Variations in the protein values were observed among Pleurotus species. Specifically, for Pleurotus species, the protein concentration in P. eryngii (28.05–29.58% d.w.) and P. pulmonarius (27.25–28.43% d.w.) was higher than in P. ostreatus (25.57–27.69% d.w.). The presence of OC increased the protein content for P. ostreatus, something that was also observed in RH substrates (RW, RB) for P. eryngii. Concerning Ganoderma species and L. edodes, the protein content varied from 27.25 to 29.90% d.w. 26.02 to 28.06% d.w., respectively. Generally, mushrooms are a good source of protein, with levels typically ranging from 19 to 35% of d.w. [52]. Previous studies have reported lower protein contents in commercially available substrates, such as 23.8% d.w. for P. ostreatus, 22.1% d.w. for P. eryngii and 20.4% d.w. for L. edodes [53], or in second cycles of supplemented SMS (22% d.w.) for P. pulmonarius and P. ostreatus [41]. Other studies have shown values of 22.15% d.w. for P. eryngii, 7.6% d.w. for G. lucidum and 16.32% d.w. for P. ostreatus [54]. Hsieh and Yang [55] reported 20.61% d.w. protein content through solid state fermentation in the solid form of soy residue for G. lucidum, while Philippoussis al. [48] reported ~20% protein content in L. edodes grown on wheat straw, corncobs and oak wood. The inclusion of LFW, along with specific substrate compositions, seemed to have positively influenced the protein content of the mushrooms produced. This effect can be attributed not only to the genotype of the strains but also to the type and amount of nitrogen sources available in the substrate, particularly from the maceration medium, which included apart from salts, peptone and yeast extract. The nitrogen-rich substrates have been positively correlated with the protein content of mushrooms [56,57]. According to Dedousi et al. [29], even the addition of 2% yeast extract to the examined substrates led to the highest protein levels in the carposomes of P. ostreatus and P. eryngii across both flushes.

Edible mushrooms are highly valued as a good source of carbohydrate [58,59]. In this research, IPS (Table 2) were the most abundant macronutrients and their highest levels were found in G. resinaceum (42.22%, w/w). Regarding Pleurotus spp., in RH substrates (RW, RB) IPS obtained the lowest values and the addition of WS or BW had no discernible impact. In contrast, no significant differences were observed in IPS content for Ganoderma spp. and L. edodes cultivated in the different species, except for G. resinaceum with the highest being observed in CB substrate. Nevertheless, all substrates and strains supported IPS production of more than 30%, w/w, except P. pulmonarius. It is worth mentioning that P. ostreatus and P. eryngii produced a higher amount of IPS than those of P. pulmonarius in all tested substrates. Among the species with the highest protein content, certain mushrooms exhibited the lowest levels of IPS content, like P. ostreatus and P. pulmonarius cultivated on RH substrates (RW, RB) and G. resinaceum grown on OC substrates (OW, OB). A similar observation has been made by various researchers [41,54,60,61,62]. According to Yang et al. [63] and Kozarski et al. [64], IPS constitute about one half or more of mushroom dry matter, with contents usually ranging from 40.6 to 53.3% of d.w. [58,59].

Basidiomycota species generally exhibit significant differences in lipid content [29,30]. According to Papanikolaou and Aggelis [65], fungal lipids in non-oleaginous microorganisms could theoretically reach up to 20% w/w d.w. However, Pleurotus mushrooms are known for their low lipid content [66]. In this study, the lipid content for the Pleurotus species varied, ranging from 2.44 to 7.18% in P. ostreatus, from 2.96 to 6.96% in P. eryngii, and from 1.52 to 4.16% in P. pulmonarius (Table 2). Although no specific pattern was observed regarding the differences in lipid content among the main substrates examined, the additives appeared to have a more pronounced effect. For instance, the addition of BW seemed to reduce the lipid content of the produced mushrooms across Pleurotus species, when cultivated in CB. Notably, all examined Pleurotus species exhibited low lipid values in this substrate, with P. ostreatus and P. eryngii showing the lowest lipid contents (2.44 and 2.96% w/w, respectively)—nearly half of the values recorded in other substrates. Except for G. lucidum, which exhibited the highest lipid value at 3.14% w/w when cultivated in CB, lower lipid levels (<3% w/w) were detected for all Ganoderma spp. and L. edodes in the examined substrates. In general, the total fat content of fungi ranges from 0.6 to 18.4% (w/w) on a d.w. basis [67,68]. Ganoderma spp. have exhibited more elevated lipid content compared to our results, as documented by Hsien and Yang [55] and Mau et al. [54], reporting percentages of 4.6 and 5.13% d.w., respectively. L. edodes, with a lipid content exceeding 5% d.w. as observed by Yang et al. [63], was found to have a considerably lower lipid content of 1.32% d.w. in the study by Tseng et al. [62], even when compared to our study. Lipids, or fats, are crucial biomolecules that play key roles as structural and functional components of cell membranes, hormone regulators, thermal insulators and as part of the myelin sheath. They aid digestion and act as a source of metabolic energy [69]. Consequently, lipids are essential for human health and must be consumed in small amounts. However, excessive lipid intake can increase the risk of chronic diseases such as atherosclerosis, cardiovascular disorders, hypertension, obesity and diabetes, which is not the case for mushrooms [70].

The ash remaining after the complete incineration of mushrooms reflects their mineral composition. In this study, ash content varied among the examined species and strains, ranging from 3.25 to 8.01% of d.w. with Pleurotus spp., showing higher values. A higher ash content typically suggests a greater concentration of minerals, especially in white oyster mushrooms [71]. Earlier studies by Yamauchi et al. [72] and Diamantopoulou et al. [66] demonstrated that oyster mushrooms are mineral-rich, with potassium being the most abundant. The ash content found in this study is consistent with values reported in previous research [72,73,74,75]. The energy content of the mushrooms in this study varied: P. ostreatus ranged from 52.15 to 94.01 kcal, P. eryngii from 55.83 to 92.41 kcal, P. pulmonarius from 36.10 to 62.67 kcal, Ganoderma spp. from 41.24 to 57.22 kcal and L. edodes from 51.93 to 55.76 kcal. The lowest energy values were recorded for the mushrooms grown on CB substrate, except for P. pulmonarius and G. lucidum, while the highest were found in P. ostreatus and P. eryngii on CW. These results align with the values reported for P. pulmonarius by Economou et al. [41], who found 52.44 kcal and 42.02 kcal for P. ostreatus. In contrast, Ulziijargal and Jeng-Leun Mau [53] reported significantly higher energy values for P. eryngii and P. ostreatus, with 194.93 and 191.96 kcal, respectively. Ulziijargal and Mau [53] also found 138.33 kcal for G. lucidum, which is higher than our findings. L. edodes in our study had a calorific value of around 50 kcal, about half of what Tseng et al. [62] and Yang et al. [63] reported. These findings suggest that incorporating LFW in mushroom cultivation, especially for L. edodes, could be beneficial due to its high nutritional, medicinal and low calorific value. Mushroom quality is influenced by various factors, including the stage of development and pre-harvest conditions. These factors contribute to the variability in data reported by different researchers, even for the same mushroom species [76].

3.3. Fungal Phenolic Compounds—Antioxidant Components

Total phenolics are the major naturally occurring antioxidant components found in the methanolic extracts of several mushroom species [77,78,79]. Phenolic compounds have attracted considerable interest due to their biological properties, including antioxidants, anti-mutagenic and anti-carcinogenic effects. In this study, TPC in the extracts was quantified and expressed as milligrams of gallic acid equivalents (mg GAE) per gram of extract (

Table 3. Analytical characteristics for determination of phenolics and amounts (mg per g) of selected Pleurotus, Ganoderma and Lentinula species produced in substrates sprayed with 20% w/w LFW. Data are presented as mean values from duplicated measurements of five replicates (mean ± SD).

Mushroom Species	Substrate	Total Phenolic Content (mgGAE/g)	DPPH Radical Scavenging Properties (mg trx/g)	ABTS Radical Scavenging Properties (mg trx/g)	Ferric Reducing Antioxidant Power Assay (FRAP) (mg trx/g)
P. ostreatus	CW *	33.91 ± 3.31 a **	2.37 ± 0.21 a	1.78 ± 0.98 c,d	9.17 ± 0.87 d
	CB	32.31 ± 2.85 a	3.01 ± 0.09 a	7.42 ± 0.55 a	37.19 ± 2.01 a
	OW	29.05 ± 3.05 a	3.11 ± 0.54 a	2.58 ± 0.34 b,c	21.50 ± 1.02 b
	OB	28.47 ± 2.74 a	3.51 ± 0.41 a	3.51 ± 0.33 b	21.51 ± 0.25 b
	RW	27.59 ± 2.94 a	2.54 ± 0.33 a	1.96 ± 0.41 c,d	14.62 ± 0.74 c
	RB	27.00 ± 2.97 a	2.68 ± 0.93 a	0.87 ± 0.12 d	13.07 ± 0.33 c
P. eryngii	CW	34.39 ± 1.74 a	2.92 ± 0.24 a,b	2.13 ± 0.23 a,b	11.81 ± 0.87 b,c
	CB	34.72 ± 1.11 a	3.46 ± 0.15 a	2.24 ± 0.24 a	8.90 ± 0.37 e
	OW	22.10 ± 2.03 c	1.88 ± 0.34 b	1.15 ± 0.15 b,c	16.83 ± 0.59 a
	OB	26.55 ± 1.74 b,c	1.91 ± 0.66 b	1.42 ± 0.61 a,b,c	10.44 ± 0.21 c,d
	RW	28.68 ± 1.89 b	2.10 ± 0.45 b	2.19 ± 0.48 a	13.11 ± 0.31 b
	RB	26.86 ± 1.64 b	2.80 ± 0.63 a,b	1.03 ± 0.24 c	10.01 ± 0.36 d,e
P. pulmonarius	CW	38.94 ± 2.31 a,b	2.59 ± 0.64 a,b	0.10 ± 0.02 c	10.16 ± 0.23 c
	CB	37.09 ± 1.98 a,b,c	1.45 ± 0.54 b,c	0.05 ± 0.00 d	9.72 ± 0.97 c
	OW	37.78 ± 2.03 a,b,c	2.81 ± 0.78 a	1.44 ± 0.02 a	18.01 ± 0.23 a
	OB	40.17 ± 2.41 a	1.01 ± 0.06 c	0.23 ± 0.01 b	5.37 ± 0.66 d
	RW	32.52 ± 2.52 b,c	2.67 ± 0.07 a,b	0.05 ± 0.00 d	16.40 ± 1.21 a
	RB	31.60 ± 3.05 c	2.38 ± 0.12 a,b	0.02 ± 0.00 d	13.71 ± 1.01 b
G. resinaceum	CW	37.39 ± 2.98 a	2.23 ± 0.56 a	6.75 ± 0.98 a	7.55 ± 0.21 a,b
	CB	35.04 ± 3.03 a	2.43 ± 0.23 a	6.94 ± 0.61 a	6.94 ± 0.36 b
	OW	37.51 ± 2.85 a	0.99 ± 0.45 a,b	6.63 ± 0.76 a	8.29 ± 0.41 a
	OB	35.19 ± 2.69 a	1.58 ± 0.62 b	6.91 ± 0.83 a	8.05 ± 0.52 a
	RW	nm ***	nm	nm	nm
	RB	nm	nm	nm	nm
G. lucidum	CW	34.39 ± 3.03 a	2.21 ± 0.36 a	6.05 ± 0.81 a	8.60 ± 0.32 a
	CB	35.79 ± 2.98 a	2.18 ± 0.25 a	5.97 ± 0.49 a	7.71 ± 0.12 a,b
	OW	35.20 ± 2.45 a	1.18 ± 0.34 b	6.63 ± 0.37 a	7.43 ± 0.45 b
	OB	34.78 ± 2.31 a	1.04 ± 0.42 b	6.49 ± 0.71 a	7.70 ± 0.64 a,b
	RW	nm	nm	nm	nm
	RB	nm	nm	nm	nm
G. applanatum	CW	39.91 ± 2.88 a	1.72 ± 0.31 a	5.91 ± 0.64 a	6.39 ± 0.74 a
	CB	37.09 ± 2.94 a	1.92 ± 0.16 a	5.82 ± 0.85 a	6.66 ± 0.74 a
	OW	38.75 ± 2.36 a	0.82 ± 0.11 b	6.21 ± 0.45 a	6.28 ± 0.61 a
	OB	37.76 ± 3.06 a	0.82 ± 0.31 b	6.36 ± 0.78 a	6.77 ± 0.54 a
	RW	nm	nm	nm	nm
	RB	nm	nm	nm	nm
L. edodes	CW	27.66 ± 2.87 a	2.28 ± 0.12 a	0.72 ± 0.02 b	11.09 ± 0.74 a
	CB	28.18 ± 2.01 a	1.48 ± 0.10 b	0.62 ± 0.05 b,c	11.15 ± 0.56 a
	OW	24.87 ± 1.97 a	0.91 ± 0.04 c	0.91 ± 0.06 a	10.11 ± 0.61 a
	OB	25.64 ± 2.03 a	0.87 ± 0.08 c	0.94 ± 0.04 a	10.54 ± 0.23 a
	RW	24.36 ± 2.58 a	0.65 ± 0.11 c	0.51 ± 0.03 c,d	10.21 ± 0.45 a
	RB	23.33 ± 2.45 a	0.74 ± 0.21 c	0.44 ± 0.02 d	9.85 ± 0.26 a

* CW: (coffee residue + wheat straw), CB (coffee residue+ beech wood shavings), OW (olive crop + wheat straw), OB (olive crop + beech wood shavings) and RW (rice husk + wheat straw), RB (rice husk + beech wood shavings). ** Columns within the same strain not sharing the same letters are significantly different at p = 0.05. *** nm: not measured due to low BE%.

). The highest TPC values were observed in carposomes harvested from CR and OC substrates for Ganoderma spp., L. edodes, P. pulmonarius and P. ostreatus. This increase in TPC may be attributed not only to the nutrient composition of CR and OC, which have been reported to contain strong antioxidant phenolics, but also to the use of LFW. Overall, significantly high TPC values were noted, with the maximum for Pleurotus spp. being 33.9 mg GAE/g for P. ostreatus (CW), 40.17 mg GAE/g for P. pulmonarius (OB) and 34.39 mg GAE/g for P. eryngii (CW). Regarding Ganoderma spp., the highest TPC values recorded were 37.51 mg GAE/g for G. resinaceum, 35.79 mg GAE/g for G. lucidum and 39.91 mg GAE/g for G. applanatum. The wide range of TPC values observed in this study can be attributed to differences in species, substrate origin, cultivation conditions and mushroom maturity stages. For comparison, Reis et al. [80] reported a TPC maximum of 5.19 mg GAE/g for P. ostreatus and 9.11 mg GAE/g for P. eryngii, while Khatun et al. [81] measured 8.3 mg GAE/g—both lower than the values reported in this study. Zengin et al. [82] reported TPC values of 29.66 mg GAE/g for G. applanatum and 37.32 mg GAE/g for G. resinaceum, which are similar to the results obtained in this research.

In a further analysis, the antioxidant activity of the mushroom extracts was evaluated using three different assays (Table 3), acknowledging that antioxidant compounds function through various mechanisms and no single method can fully enclose all their effects. The results suggest that the choice of substrate significantly influenced the antioxidant properties of mushrooms, with CB and CW substrates showing the most consistent enhancement across different species and assays. This finding highlights the potential for optimizing substrate composition to maximize the nutritional and antioxidant properties of cultivated mushrooms, especially for their use in food supplements and nutraceuticals. Specifically, P. ostreatus did not exhibit significant differences regarding DPPH radical scavenging activities, with values ranging from 2.37–3.51 mg trx/g. However, it showed the highest ABTS scavenging activity in CB substrate (7.42 mg trx/g), which was significantly different compared to the other substrates. Furthermore, the FRAP value was the highest on the CB substrate (37.19 mg trx/g), indicating strong reducing power. For P. eryngii, CB was the most effective substrate for enhancing its antioxidant activity in both DPPH and ABTS assays, while OW provided the best reducing power. Similarly, the OW substrate resulted in the highest DPPH activity (2.81 mg trx/g) and FRAP value (18.01 mg trx/g) for P. pulmonarius. Although ABTS scavenging was the lowest across all substrates for P. pulmonarius, in OW and OB substrates it showed higher values. In the case of G. resinaceum, ABTS scavenging was relatively consistent with slightly higher activity observed in mushrooms cultivated in CB and OB (6.94 mg trx/g) substrates. Although DPPH activity was relatively low in all substrates, the highest value was observed in CB and CW (2.43 mg trx/g). Therefore, CB and OB were the most effective substrates for improving the antioxidant properties of G. resinaceum. For G. lucidum, in the CW substrate the highest FRAP value (8.60 mg trx/g) and a relatively strong ABTS scavenging activity (6.05 mg trx/g) was presented, although the DPPH activity was generally low and consistent in all substrates tested. CW was the most effective substrate for enhancing the antioxidant properties of G. lucidum. In the case of G. applanatum, DPPH scavenging activity was relatively low across all substrates. However, better results were obtained with mixtures of substrates containing CR as the main component compared to OC. The use of the BW additive, as opposed to WS, led to a slight increase in FRAP activity in the produced mushrooms, suggesting a modest improvement in their antioxidant capacity. Regarding L. edodes, CR (CW, CB) substrates were the most effective for enhancing DPPH, although ABTS and FRAP were not significantly impacted by substrate type and remained low in all substrates. These findings align with previous studies. Piljac-Žegarac et al. [83] reported lower FRAP values for L. edodes and P. ostreatus compared to this study, while Zengin et al. [82] observed high antioxidant activities in G. applanatum and G. resinaceum. Additionally, Diamantopoulou et al. [61] and Sulistiany et al. [84] reported lower antioxidant activities in G. resinaceum and Pleurotus spp., respectively. Therefore, these studies underscore the critical role of substrate optimization in maximizing the antioxidant potential of mushrooms.

3.4. IPS and Fatty Acid (FA) Composition

The monosaccharide composition of the IPS extracts from the samples, as shown in

Table 4. Carbohydrate composition from hydrolysed total IPS synthesized by selected of Pleurotus, Ganoderma and Lentinula species produced in substrates sprayed with 20% w/w LFW. Each point is the mean value of two independent measurements.

		Carbohydrates (%, w/w of Total IPS)
Mushroom Species	Substrate	Glucose	Fructose	Mannitol
P. ostreatus	CW *	70.5 ± 2.33 a **	18.3 ± 1.11 a,b	11.2 ± 1.01 a
	CB	72.8 ± 3.05 a	22.1 ± 1.26 a	5.1 ± 0.91 b
	OW	74.5 ± 2.88 a	21.3 ± 2.34 a	4.2 ± 0.87 b
	OB	74.6 ± 2.91 a	20.2 ± 1.87 a	5.2 ± 0.88 b
	RW	70.7 ± 2.46 a	20.4 ± 2.03 a	8.9 ± 0.91 a
	RB	75.4 ± 2.34 a	14.0 ± 1.74 b	10.6 ± 0.86 a
P. eryngii	CW	75.5 ± 3.01 a	14.2 ± 1.22 a	10.3 ± 0.23 c
	CB	76.2 ± 2.99 a	15.2 ± 1.41 a	8.6 ± 0.11 d
	OW	73.1 ± 2.64 a	14.7 ± 1.23 a	12.2 ± 0.10 a
	OB	80.3 ± 2.85 a	15.2 ± 1.24 a	4.5 ± 0.03 e
	RW	72.6 ± 3.41 a	16.1 ± 1.31 a	11.3 ± 0.47 b
	RB	75.8 ± 3.03 a	14.2 ± 1.17 a	10.0 ± 0.24 c
P. pulmonarius	CW	81.7 ± 3.02 a	10.2 ± 0.23 c	8.1 ± 0.21 d
	CB	77.5 ± 3.45 a,b	11.2 ± 0.74 b,c	11.3 ± 0.12 b
	OW	77.0 ± 2.87 a,b	13.2 ± 0.64 b	9.8 ± 0.09 c
	OB	80.2 ± 2.74 a	17.5 ± 0.47 a	2.3 ± 0.03 e
	RW	70.0 ± 2.55 b	17.2 ± 1.01 a	12.8 ± 0.85 a
	RB	80.3 ± 2.31 a	17.5 ± 1.07 a	2.2 ± 0.02 e
G. resinaceum	CW	74.1 ± 3.02 a	15.3 ± 0.87 a	10.6 ± 0.87 b
	CB	73.5 ± 3.45 a	14.7 ± 0.88 a,b	11.8 ± 0.56 b
	OW	73.5 ± 3.87 a	15.2 ± 0.64 a,b	11.3 ± 0.74 b
	OB	73.1 ± 3.85 a	13.5 ± 0.73 b	13.4 ± 0.64 a
	RW	nm ***	nm	nm
	RB	nm	nm	nm
G. lucidum	CW	71.9 ± 3.69 b	13.9 ± 0.67 a	14.2 ± 0.98 a
	CB	71.5 ± 3.45 b	14.2 ± 0.47 a	14.3 ± 0.78 a
	OW	81.2 ± 3.84 a	6.3 ± 0.03 b	12.5 ± 0.45 b
	OB	84.4 ± 2.98 a	8.6 ± 0.12 b	7.0 ± 0.23 c
	RW	nm	nm	nm
	RB	nm	nm	nm
G. applanatum	CW	72.3 ± 2.47 b	14.2 ± 1.45 a	13.5 ± 1.44 a
	CB	71.6 ± 2.64 b	13.8 ± 2.10 a	14.6 ± 1.74 a
	OW	82.1 ± 3.02 a	3.7 ± 0.87 b	14.2 ± 1.23 a
	OB	81.0 ± 3.33 a	5.1 ± 0.66 b	13.9 ± 1.07 a
	RW	nm	nm	nm
	RB	nm	nm	nm
L. edodes	CW	70.7 ± 3.47 b	14.6 ± 1.01 a	14.7 ± 1.87 a,b
	CB	81.4 ± 2.35 a	10.3 ± 1.03 c	8.3 ± 1.47 c
	OW	72.3 ± 1.98 b	16.4 ± 0.97 a	11.3 ± 1.36 b,c
	OB	71.8 ± 2.87 b	11.2 ± 1.26 b,c	17.0 ± 1.26 a
	RW	69.8 ± 2.34 b	15.0 ± 1.24 a	15.2 ± 1.22 a,b
	RB	70.2 ± 2.21 b	13.6 ± 1.45 a,b	16.2 ± 1.42 a

* CW: (coffee residue + wheat straw), CB (coffee residue+ beech wood shavings), OW (olive crop + wheat straw), OB (olive crop + beech wood shavings), RW (rice husk + wheat straw) and RB (rice husk + beech wood shavings). ** Columns within the same strain not sharing the same letters are significantly different at p = 0.05. *** nm: not measured due to low BE%.

, reveals that glucose was the dominant sugar, comprising over 69.8% w/w, with fructose and mannitol being presented in much smaller amounts. Fructose was typically the second most abundant monosaccharide after glucose, except in Ganoderma spp. and L. edodes, where mannitol often took that position. Overall, the sugar profile varied significantly, depending on both the mushroom species and the specific substrate used, emphasizing the crucial role of substrate selection in optimizing IPS production for specific carbohydrate compositions. Similarly, glucose was found to be the most prevalent carbohydrate in the mycelium of P. pulmonarius [30], G. applanatum [30,85], P. ostreatus [29,63,86], G. lucidum [87,88], as well as in P. ostreatus, P. eryngii and L. edodes [89]. However, according to Diamantis et al. [90], fructose was the primary monosaccharide in P. citrinopileatus when cultivated on different ratios of spent mushroom substrate and leafy vegetable roots from hydroponic cultivation.

The main FAs found in the cultivated mushrooms examined are presented in

Table 5. FAs composition of total lipids synthesized by selected Pleurotus, Ganoderma and Lentinula species produced in substrates sprayed with 20% w/w LFW. Each point is the mean value of two independent measurements.

Mushroom Species	Substrate/FA (%, w/w)	Lauric Acid (C12:0)	Myristic Acid (C14:0)	Pentadecanoic Acid (C15:0)	Palmitic Acid (C16:0)	Stearic Acid (C18:0)	Oleic Acid (C18:1)	Linoleic Acid (C18:2)
P. ostreatus	CW *	0.3 ± 0.0 a **	0.6 ± 0.0 c	1.8 ± 0.0 d	13.5 ± 0.8 a	1.8 ± 0.0 b,c	9.1 ± 0.2 b	70.0 ± 1.5 a
	CB	0.4 ± 0.0 a	0.6 ± 0.0 c	2.3 ± 0.0 b	13.6 ± 0.7 a	1.6 ± 0.0 d	9.6 ± 0.1 b	65.5 ± 1.8 a
	OW	0.3 ± 0.0 a	0.6 ± 0.0 c	2.1 ± 0.1 b,c	13.9 ± 0.9 a	1.8 ± 0.0 b,c	9.8 ± 0.3 a,b	68.8 ± 3.1 a
	OB	0.2 ± 0.0 a	0.4 ± 0.0 c	1.9 ± 0.1 c,d	13.8 ± 0.5 a	1.7 ± 0.0 c,d	11.0 ± 0.1 a	68.2 ± 2.2 a
	RW	0.3 ± 0.0 a	1.8 ± 0.0 a	4.0 ± 0.2 a	13.1 ± 0.8 a	2.2 ± 0.1 a	7.5 ± 0.8 c	54.0 ± 2.3b
	RB	0.4 ± 0.0 a	0.8 ± 0.0 b	1.7 ± 0.1 d	13.9 ± 0.7 a	1.9 ± 0.0 b	9.0 ± 0.8 b	69.1 ± 1.4 a
P. eryngii	CW	0.9 ± 0.0 a	1.3 ± 0.0 a	2.2 ± 0.0 a	13.1 ± 0.0 a,b	3.6 ± 0.0 a	12.3 ± 0.1 c	63.4 ± 2.5 a
	CB	0.5 ± 0.0 d	1.1 ± 0.0 b	2.0 ± 0.0 b	11.0 ± 0.7 c	3.1 ± 0.1 b	12.3 ± 0.2 c	63.1 ± 1.9 a
	OW	0.3 ± 0.0 f	0.7 ± 0.0 e	1.8 ± 0.0 c	13.4 ± 0.3 a	2.1 ± 0.1 d,e	15.9 ± 0.4 a	62.7 ± 1.7 a
	OB	0.6 ± 0.0 c	0.9 ± 0.0 c	1.9 ± 0.1 b,c	12.1 ± 0.4 b	2.0 ± 0.0 e	14.3 ± 0.2 b	61.2 ± 3.7 a
	RW	0.7 ± 0.0 b	0.4 ± 0.0 f	2.0 ± 0.0 b	12.2 ± 0.4 b	2.2 ± 0.0 d	11.3 ± 0.0 d	58.7 ± 1.2 a
	RB	0.4 ± 0.0 e	0.8 ± 0.0 d	2.2 ± 0.1 a	12.1 ± 0.0 b	2.4 ± 0.1 c	11.5 ± 0.3 d	59.3 ± 3.1 a
P. pulmonarius	CW	0.3 ± 0.0 d	1.0 ± 0.0 a	1.7 ± 0.0 b,c	13.3 ± 0.7 a	2.5 ± 0.0 a	10.8 ± 0.2 a	67.2 ± 2.8 a
	CB	0.2 ± 0.0 e	0.7 ± 0.0 d	1.9 ± 0.0 a	13.2 ± 0.7 a	2.4 ± 0.0 a	10.7 ± 0.4 a	68.3 ± 2.8 a
	OW	0.4 ± 0.0 c	0.8 ± 0.0 c	1.8 ± 0.1 a,b	13.4 ± 0.5 a	1.9 ± 0.0 c	11.2 ± 0.1 a	66.5 ± 2.5 a
	OB	0.3 ± 0.0 d	0.7 ± 0.0 d	1.8 ± 0.0 a,b	13.5 ± 0.5 a	2.5 ± 0.1 a	7.9 ± 0.4 b	69.8 ± 3.5 a
	RW	0.5 ± 0.0 b	0.9 ± 0.0 b	1.7 ± 0.0 b,c	12.7 ± 0.4 a	2.0 ± 0.0 b,c	7.0 ± 0.2 c	65.3 ± 2.0 a
	RB	0.7 ± 0.0 a	0.8 ± 0.0 c	1.6 ± 0.1 c	12.6 ± 0.0 a	2.1 ± 0.1 b	7.6 ± 0.3 b,c	67.4 ± 3.2 a
G. resinaseum	CW	0.9 ± 0.0 a	1.4 ± 0.0 b	2.5 ± 0.0 a	15.7 ± 0.9 a	5.2 ± 0.1 a	9.2 ± 0.8 a	49.6 ± 2.2 a
	CB	0.8 ± 0.0 a	1.2 ± 0.0 d	2.2 ± 0.0 b	15.3 ± 0.8 a	4.8 ± 0.2 b	9.8 ± 0.4 a	47.8 ± 2.1 a
	OW	0.8 ± 0.0 a	1.7 ± 0.1 a	2.0 ± 0.0 c	15.2 ± 0.4 a	5.3 ± 0.1 a	9.7 ± 0.8 a	50.0 ± 3.2 a
	OB	0.9 ± 0.0 a	1.3 ± 0.0 b,c	2.0 ± 0.1 c	15.6 ± 0.5 a	5.1 ± 0.1 a	9.0 ± 0.7 a	46.9 ± 3.3 a
	RW	nm ***	nm	nm	nm	nm	nm	nm
	RB	nm	nm	nm	nm	nm	nm	nm
G. lucidum	CW	0.9 ± 0.0 a	1.3 ± 0.0 a	2.2 ± 0.0 c	15.7 ± 0.4 a,b	5.7 ± 0.5 a	9.2 ± 0.7 a	64.3 ± 2.3 a
	CB	0.8 ± 0.0 a	1.2 ± 0.0 a	2.5 ± 0.0 b	15.0 ± 0.5 b	5.8 ± 0.4 a	9.8 ± 0.4 a	64.9 ± 4.1 a
	OW	0.7 ± 0.0 a	1.4 ± 0.0 a	2.5 ± 0.1 b	15.7 ± 0.3 a,b	5.3 ± 0.1 a	9.7 ± 0.4 a	63.4 ± 4.2 a
	OB	0.9 ± 0.0 a	1.5 ± 0.0 a	3.0 ± 0.0 a	15.9 ± 0.2 a	5.7 ± 0.2 a	9.0 ± 0.3 a	62.3 ± 3.3 a
	RW	nm	nm	nm	nm	nm	nm	nm
	RB	nm	nm	nm	nm	nm	nm	nm
G. applanatum	CW	0.6 ± 0.0 a	1.4 ± 0.1 a,b	3.0 ± 0.1 a	15.4 ± 1.2 a	5.7 ± 0.2 a	9.2 ± 0.1 a,b	58.2 ± 2.0 a
	CB	0.9 ± 0.0 a	1.3 ± 0.0 b	2.7 ± 0.0 b	14.8 ± 1.1 a	5.6 ± 0.3 a,b	9.8 ± 0.4 a	57.4 ± 1.7 a,b
	OW	0.9 ± 0.0 a	1.5 ± 0.0 a	2.4 ± 0.0 c	15.2 ± 1.3 a	5.2 ± 0.2 a	9.7 ± 0.3 a	52.9 ± 1.3 c
	OB	0.7 ± 0.0 a	1.6 ± 0.1 a	2.9 ± 0.1 a	15.3 ± 0.9 a	5.5 ± 0.1 a,b	9.0 ± 0.2 b	54.4 ± 1.2 b,c
	RW	nm	nm	nm	nm	nm	nm	nm
	RB	nm	nm	nm	nm	nm	nm	nm
L. edodes	CW	0.3 ± 0.0 c	0.4 ± 0.0 c	0.7 ± 0.0 d	13.2 ± 0.4 a,b	1.0 ± 0.0 d	3.0 ± 0.1 b	81.3 ± 1.4 a
	CB	0.2 ± 0.0 d	0.3 ± 0.0 d	1.1 ± 0.0 a	12.9 ± 0.3 a,b	1.7 ± 0.0 c	3.2 ± 0.1 a	79.8 ± 1.3 a
	OW	0.4 ± 0.0 b	0.7 ± 0.0 a	0.8 ± 0.0 c	13.8 ± 0.4 a	1.9 ± 0.1 b,c	2.2 ± 0.0 c	80.1 ± 1.2 a
	OB	0.5 ± 0.0 a	0.4 ± 0.0 c	0.9 ± 0.0 b	14.0 ± 0.4 a	2.1 ± 0.1 b	2.0 ± 0.0 d	78.3 ± 1.0 a
	RW	0.2 ± 0.0 d	0.5 ± 0.0 b	0.3 ± 0.0 e	12.9 ± 0.3 a,b	1.7 ± 0.0 c	2.0 ± 0.0 d	82.1 ± 3.1 a
	RB	0.2 ± 0.0 d	0.5 ± 0.0 b	1.1 ± 0.0 a	12.5 ± 0.5 b	2.4 ± 0.2 a	1.5 ± 0.0 e	79.4 ± 2.1 a

* CW: (coffee residue + wheat straw), CB (coffee residue+ beech wood shavings), OW (olive crop + wheat straw), OB (olive crop + beech wood shavings), RW (rice husk + wheat straw) and RB (rice husk + beech wood shavings). ** Columns within the same strain not sharing the same letters are significantly different at p = 0.05. *** nm: not measured due to low BE%.

. The data indicate that substrates significantly affected the lipid profile of the studied mushroom species. Saturated FAs ranged from 15.6 to 27.0%, while unsaturated FA ranged between 55.9 and 84.3% w/w. Among these, palmitic acid (C16:0) emerged as the second most prevalent saturated FA, followed by oleic acid (C18:1), a monounsaturated one. The addition of WS generally increased the linoleic acid content in most of the species examined. L. edodes was notable for its exceptionally high linoleic acid content, making it particularly valuable as a source of polyunsaturated FAs in human nutrition. Overall, the FA composition of the studied mushroom species showed that they are rich in polyunsaturated FA, especially linoleic acid, which is beneficial for health. The choice of substrate significantly influenced the FA profile. These results are consistent with previous studies on P. pulmonarius [40], P. ostreatus [91], G. lucidum [92], G. australe [93] and other basidiomycetes [94,95]. Solomko et al. [96] reported oleic acid as the dominant fatty acid in the lipids of P. ostreatus fruit bodies and mycelium, comprising 56% of total lipids, while Dedousi et al. [9] identified it as the second most abundant FA after linoleic acid. Chung et al. [97] also found that linoleic acid constituted 77–81% of the total FAs in L. edodes.

4. Conclusions

The present study underscores the importance of selecting the appropriate species and substrates for successful mushroom cultivation, as these factors significantly affect the duration of incubation and carposomes earliness, which are closely linked to substrate contamination and potential crop loss. The OW and OB substrates were particularly favorable, promoting rapid mycelial growth and shorter earliness periods across the various species, particularly for P. pulmonarius and P. eryngii. The BE varied widely among species and substrates, with Pleurotus species generally exhibiting higher BE compared to Ganoderma spp. and Lentinula edodes. The morphological characteristics of the mushrooms, including pileus diameter and stipe length, were also influenced by substrate selection, aligning with market preferences for certain species. Furthermore, the application of LFW as a maceration medium significantly improved cultivation efficiency and produced high-quality mushrooms with elevated nutritional value and antioxidant properties, and high levels of polysaccharides and polyunsaturated FA. The findings also suggest that optimizing substrate composition can significantly improve both the yield and quality of mushrooms, making them more suitable for food supplements and nutraceutical applications. Further studies on scaling up the cultivation process and examining the economic feasibility of large-scale production with optimized substrates would also be valuable for commercial applications. Finally, studying the potential of using agricultural waste products as sustainable substrates could contribute to more eco-friendly and cost-effective mushroom farming practices, aligning with broader sustainability goals.