The Influence of Substrate and Strain on Protein Quality of Pleurotus ostreatus

Abstract

Background: The effect of substrate and strain on the nutritional and functional properties of mushroom flours and protein concentrates (PCs) has not thoroughly been investigated. Methods: The proteins of P. ostreatus flours (strains AMRL144 and 150) were isolated following alkaline extraction (pH 12) and isoelectric precipitation (pH 4) as it was determined by the solubility curves. The protein quality of the flours and PCs were evaluated by determining the protein solubility index (PSI) and their functional properties, such as water (WAC) and oil absorption capacity (OAC), foam capacity (FC), foam stability (FS) and emulsion stability (ES). The amino acid (AA) composition of the flours was determined by reversed-phase liquid chromatography after protein hydrolysis and o-phthalaldehyde derivatization. Results: The PSIs of the flours and PCs of P. ostreatus were comparable to those of soy protein flours and protein concentrates. The highest AA concentration was found for both strains when cultivated on the barley and oat straw (BOS) substrate, showing a similar trend as the protein content. A principal component analysis (PCA) indicated an impact of the strain on the functional properties. Conclusions: Both strains can produce high quality proteins especially when cultivated on wheat straw (WS). The FS was positively correlated with the P. ostreatus strain AMRL150 whereas the FC was positively correlated with AMRL144.

1. Introduction

Global mushroom cultivation is continuously increasing due to their nutritional value and culinary properties [1]. Agaricus bisporus, Lentinula edodes and Pleurotus spp. are the most cultivated mushrooms worldwide [2,3]. Edible mushrooms constitute a rich source of carbohydrates and protein [4,5]. The high molecular mass carbohydrates primarily consist of polysaccharides that have been shown to exert multiple benefits for human health [6]. Among them, β-glucans are the most important, with various biological functions [7]. Moreover, Pleurotus spp. contain all nine essential amino acids, making them a viable alternative source instead of animal protein, which is ideal for vegetarians [8]. The fat fraction of edible mushrooms, although quantitatively low, contains valuable unsaturated (oleic acid) and polyunsaturated (linolenic acid) fatty acids [4,9]. Several bioactive compounds have been also detected in mushrooms such as phenolics, tocopherols, ergosterol, vitamins B1, B2 and C, and minerals [10,11,12].

Among mushrooms, Pleurotus spp. are the most suitable higher fungi that can be used to colonize and fructify on several types of agro-industrial residues and wastes [5,13]. Apart from wheat straw, which is the most common cultivation substrate, other materials, such as barley and oat straw, beech wood shavings, coffee residue, rice bark, etc., have been examined as alternative substrates [5,13,14,15].

Compared to plant proteins, which lack some essential amino acids, mushrooms are considered to cover the dietetic requirements [16]. Moreover, their high glutamic acid, aspartic acid and sulfur amino acid (methionine and cystine) contents contribute to the meaty taste of processed foods and thus may be served as flavor enhancers [17]. Pleurotus spp. have been widely studied for their role in the development of functional foods [18,19,20,21]. You et al. (2022) [21] summarized the uses of P. ostreatus in the food industry. Among them, mushroom powder has been used as a meat replacement in chicken nuggets and beef salami; as a partial wheat flour replacement in biscuits; and as a substrate to produce novelty alcoholic drinks with unique flavors.

The manufacturing of PCs has been studied to enhance the uses of P. ostreatus. The species, strain and growth substrate of the mushrooms, together with the growing environment and maturity stage, all have a significant impact on PC quality [18,21]. Moreover, the protein extraction conditions directly affect the functionality of the produced PCs due to the molecular changes that may occur and are linked with the disruption or stabilization of chemical bonds, mainly non-covalent interactions [22]. The most commonly used approach for protein isolation is based on alkaline extraction followed by isoelectric precipitation (AE-IP) of the protein fraction. Due to the severe alkaline conditions (pH 8.0–12.0), protein denaturation and exposure of hydrophobic bonds may occur, which result in decreased solubility of the extracted proteins. Moreover, partial protein degradation may occur, resulting in increased proportions of low molecular weight proteins and free amino acids that are susceptible to degradation, thereby influencing the amino acid composition of the PCs [23,24]. It has been found [25] that by applying a nanoparticulation strategy of the protein fraction received after AE-IP, the protein dispersibility in water and the emulsion capacity can be improved. It was also found that a combination of different extraction conditions (i.e., type of solvent, salt addition and pH) could effectively separate plant proteins based on their solubility characteristics, whereas enzymatic hydrolysis is mostly preferred due to the gentle pH and temperature conditions, which enhance the solubility of the protein isolates [16,26].

The increasing demand for alternative protein sources to replace animal proteins has brought mushrooms to be the top choice for the food industry and consumers. However, there is still limited information on the extraction of mushroom proteins and thus its effects on the proteins’ functional and nutritional properties [21]. Moreover, in the framework of a circular and sustainable economy, mushrooms can be cultivated on several types of agro-industrial residues and wastes; however, this can impact their protein quality. Thus, our aim was to assess the protein quality of P. ostreatus flours and protein concentrates and investigate the effect of the cultivation substrate and strain on their nutritional value and functional properties. For this purpose, two strains of P. ostreatus (AMRL150 and AMRL144) were cultivated on two different substrates, namely, wheat straw (WS) and a mixture of barley with oat straw (BOS). The amino acid composition of flours was used to estimate their nutritional value and the protein quality index (PSI) was used to evaluate the protein quality of the flours and PCs. Mid-infrared spectroscopy was chosen in the present study because it is a rapid and efficient technique for determining the type of functional group as well as the effect of the chemical environment on the structure of the molecules. The water (OAC) and oil absorption capacity (OAC), the foam capacity (FC) and stability (FS), and the emulsion stability (ES) of FLs and PCs of P. ostreatus were determined to correlate them with the different strains and/or substrates.

2. Materials and Methods

2.1. Reagents

L-glutamic acid (Glu), L-alanine (Ala), L-serine (Ser), L-aspartic acid (Asp) and L-lysine (Lys) were purchased from Acros Organics (Geel, Belgium); L-valine (Val), L-isoleucine (Ile), L-phenylalanine (Phe), L-arginine (Arg), L-threonine (Thr), L-methionine (Met), L-histidine (His), L-leucine (Leu) and cystine (Cys-Cys) were purchased from Sigma-Aldrich (St. Louis, MO, USA); L-tyrosine (Tyr) and glycine (Gly) were purchased from Merck (Darmstadt, Germany), all with a purity level ≥ 98%; and norvaline, o-phthalaldehyde (OPA) and β-mercaptoethanol (MCE) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile and methanol were purchased from J.T. Baker (Phillipsburg, NJ, USA). A hydrochloric acid solution (37%), sodium tetraborate decahydrate (Na₂B₄O₇⋅10H₂O) and sodium phosphate dibasic (Na₂HPO₄) were purchased from Merck (Darmstadt, Germany) and bovine serum albumin (BSA) was purchased from BDH (BDH Chemicals LTD, Dubai, United Arab Emirates).

2.2. Raw Materials

Two strains of P. ostreatus (Jacq.: Fr.) Kumm. (AMRL144, 150) belonging to the Laboratory of Edible Fungi/ITAP/ELGO-Dimitra were examined in the present study. Both strains were cultivated on wheat straw (WS), as control substrate, supplemented with wheat bran at a ratio of 80:20 (m/m, in terms of dry mass) and barley and oat straw (BOS) supplemented with wheat bran at a ratio of 70:30 to obtain a final C/N ratio of 20–27/1. All residues were derived from different Greek farms and industries. Grain spawn production, substrate preparation and mushroom cultivation conditions were as previously described by Melanouri et al. (2022) [13]. Mushrooms were harvested by hand during the first flush, frozen at −20 ± 0.5 °C and then they were lyophilized (in a HetoLyoLab 3000, Heto-Holten Als, Allered, Denmark) and ground (in a Janke and Kunkel IKA-WERKE analytical mill (Staufen, Germany)) for further analysis.

2.3. Preparation of P. ostreatus Protein Concentrates

The protein concentrates of P. ostreatus FLs were prepared using alkaline extraction followed by isoelectric precipitation based on the conditions described by Gonzales et al. (2021) [18]. Briefly, mushroom flour was dispersed in distilled water in a 1:10 (m/v) ratio and the pH was adjusted to 12 using 2 Μ NaOH to achieve maximum protein solubility. The suspension was centrifuged at 4427× g for 20 min at 4 °C (Hermle, Z326K, Wehingen, Germany) and the pH of the supernatant was adjusted to 4 with 2 Μ HCl. The centrifugation step was repeated once. The precipitate was freeze dried to obtain a lyophilized powder (Unicryo MC 2L-60 °C, GmbH, Munich, Germany) for further analyses. The efficiency of the procedure was estimated by calculating protein extraction yield using the following equation:

$$Yield (\%)=\left(\frac{EP}{TP}\right)\times 100$$

(1)

where EP is the amount of extracted protein (g) and TP is the amount of total protein (g) in the P. ostreatus flour, as determined by Kjeldahl method (AOAC 920.87) [27].

2.4. Protein Solubility

The assessment of the protein solubility of the P. ostreatus flours was performed based on the procedure described by Gonzales et al. (2021) [18]. In brief, 0.25 g of the sample was dispersed in 25 mL of distilled water and the pH was adjusted from 2 to 12 by adding HCl or NaOH. After centrifugation (4427× g for 30 min at 4 °C), the amount of soluble protein in the supernatant was determined by the Lowry method [28]. The concentration of soluble protein was calculated using a BSA standard curve (0.05 to 0.30 mg/mL).

2.5. Protein Solubility Index

The protein quality of the P. ostreatus flours and PCs was evaluated based on the procedure described by Liu (2022) [29], with some modifications. In brief, 0.80 ± 0.02 g of finely ground flour (0.30 mm mesh) was placed in a pre-weighed 50 mL Falcon tube. After the addition of 40 mL of a NaOH solution (5 mM), the suspension was agitated and centrifuged at 69× g for 1 h at room temperature and then at 2000× g for 10 min. The supernatant was gently decanted and the precipitate was freeze dried to obtain a dried lyophilized residue. The nitrogen content of the dried residue (N_R) and the sample flour (N_S) was determined by the Kjeldahl method. All samples were tested at least in triplicate. The protein solubility index (%) was calculated using Equation (2):

$$\%PSI=\frac{(N_S-N_R)}{N_S}\times 100\%$$

(2)

2.6. Amino Acid Composition

2.6.1. Acidic Hydrolysis of P. ostreatus Flour for Amino Acid Analysis

The amino acid content of the P. ostreatus flours was determined after acidic hydrolysis of the proteins based on the procedure described by Dai et al. (2014) [30]. During acidic hydrolysis, Asn and Gln are deaminated to Asp and Glu, respectively, whereas Trp is destroyed, particularly in sample that are more than 5% carbohydrates [31].

Briefly, 125 ± 3 mg of flour or an equal amount of water for the blank, 5 mL of 6 M HCl and 625 μL of norvaline (internal standard, 1 nmol/μL) were placed in screw-cap glass tube. After sparging with nitrogen, the tubes were placed in an oven at 110 °C for 24 h. Subsequently, 0.4 mL of the hydrolysate was mixed with 0.4 mL of 6 M NaOH and 0.2 mL HCl 0.5 M in a 2 mL glass vial and filtered through a 0.45 μm PVDF membrane. Each sample was hydrolyzed in triplicate.

2.6.2. Amino Acid Derivatization and HPLC Analysis

The on-line derivatization reaction was performed according to the Application Note 5991-5571EN (Agilent, Santa Clara, CA, USA) [32], which was slightly modified to adapt it to our autosampler. Briefly, 8 μL of an amino acid standard solution or sample were mixed with 20 μL of a borate buffer (0.4 M) and 4 μL of the OPA reagent (10 mg/mL in borate containing 10% MCE). After mixing for 1 min, 160 μL of injection diluent (mobile phase A containing 0.4% H₃PO₄) was added and 10 μL was injected. Chromatographic separation of the derivatized amino acids was carried out on a Perkin Elmer Flexar HPLC system equipped with an on-line degasser, a quaternary pump, an autosampler, a column oven and a photodiode array detector. Separation was performed on a Luna C18(2) column (250 mm × 4.6 i.d., 5 μm, Phenomenex) set at 40 °C. Mobile phase A had a pH 8.2 and contained 10 mM Na₂HPO₄, 10 mM Na₂B₄O₇ and 5 mM NaN₃. Mobile phase B contained acetonitrile/methanol/water (45:45:10, v:v:v). The gradient elution used was as follows; 0 min: 4% B; 0.8 min: 4% B; 33.4 min: 57% B; 33.5 min: 100% B; 39.9 min: 100% B. The initial condition was reached in 0.1 min and the column was equilibrated for 8 min. The flow rate was held constant at 1.5 mL/min. The signal was measured at 338 nm. Amino acid standards were prepared in 0.1 M HCl and calibration curves were constructed (90–900 pmol/μL) using norvaline as the internal standard. The results were expressed as mg of amino acid per g of dry matter.

2.7. Crude Protein Content

The crude protein of the P. ostreatus flours and PCs was calculated on the basis of their nitrogen content, as determined by the Kjeldahl method (AOAC 920.87) [27]. The converting factor used to convert nitrogen to protein content was 4.38 [33], which is lower than the commonly used factor of 6.25 due to the high content of non-protein nitrogen compounds, such as chitin, that are typically present in mushrooms [17].

2.8. FTIR Spectroscopy

The spectra of the P. ostreatus flours were recorded in triplicate (three different sub-samples) using a spectrophotometer (Ostec corporation group, Moscow, Russia) equipped with a Mercury–Cadmium-Telluride (MCT) detector (Tekran® Instruments Corporation, Seattle, WA, USA) and the Attenuated Total Reflectance (ATR) technique using a diamond crystal. The recording parameters were as follows: spectral region, 4000–400 cm⁻¹; resolution, 4 cm⁻¹; 64 scans; and speed of interferometer moving mirror, 0.3164 mm·s⁻¹. A background spectrum was recorded using only the diamond crystal before each spectrum was taken. The OMNIC (ver.8.2.387; Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for spectrum manipulation: the spectrum was “automatically smoothed” using the Savitzky–Golay algorithm (2nd order, 5-point window) and “automatic baseline correction” (2nd order polynomial fit) was performed. Finally, the averaged spectrum of each sample was calculated from the triplet spectra.

2.9. Functional Properties

2.9.1. Water and Oil Absorption Capacity

The water absorption capacity (WAC) and oil absorption capacity (WAC) were determined according to Zhang et al. (2021) [22]. Specifically, 0.1 g of flour or protein concentrate was suspended in 0.60 mL of water or soya oil and the mixture was vortexed for 5 min. The suspensions were allowed to stand for 30 min and centrifuged at 1000× g for 30 min. Subsequently, the excess water or oil was decanted and their volume was measured with a graduated pipette [34]. All tests were performed in triplicate. The WAC and OAC were calculated as follows:

$$\%WAC=\frac{\mathrm{mL} of absorbed water}{weight of sample \left(\mathrm{g}\right)}\times 100\%$$

(3)

$$\%OAC=\frac{\mathrm{mL} of absorbed oil}{weight of sample \left(\mathrm{g}\right)}\times 100\%$$

(4)

2.9.2. Foam Properties

The foaming properties of the P. ostreatus flours and PCs were assessed by suspending 1 g of sample in 100 g of water. Then, an aliquot (15 mL) of the suspension was homogenized (Ingenieubruro CAT, M. Zipperer GmbH, Ballrechten-Dottingen, Germany) for 5 min at 8000 rpm. The solution was immediately transferred to a 50 mL graduated cylinder to measure the foam volume. The foam capacity (FC) was calculated as follows:

$$\%FC=\frac{V_F }{V_L}\times 100\%$$

(5)

where V_F is the volume of foam formed and V_L is the volume of the initial solution.

The foam stability (FS) was calculated as the foam volume remaining after 30 min of storage (V₃₀) divided by the foam volume produced immediately after homogenization (V_F) [22,33]. All tests were performed in triplicate.

$$\%FS=\frac{V_{30} }{V_F}\times 100\%$$

(6)

2.9.3. Emulsion Properties

The emulsion stability (ES) of the P. ostreatus flours and PCs powders was determined according to Lam et al. (2017) [19]. In brief, 1 g of sample was suspended in in 100 g of water and 5 g of this solution was homogenized for 5 min at 8000 rpm with 5 g of soya oil. The emulsion was immediately transferred to a 10 mL graduated cylinder and after 30 min the height of the aqueous phase was recorded (V_A) and subtracted from the initial volume of the aqueous phase before homogenization (V_B).

$$\%ES=\frac{V_B-V_A}{V_B}\times 100\%$$

(7)

2.10. Statistical Analysis

Analysis of variance (ANOVA) was carried out to assess the significant (p < 95%) differences in the protein content, PSI, amino acid content and functional properties between the strains and the substrates used in the present study. To check the assumptions of ANOVA, Levene’s test was used to assess the homogeneity of the variances and Shapiro–Wilk’s test was used to assess the normality. When the assumptions were violated, robust ANOVA was performed using the Walrus module in Jamovi (version 2.3.21.0) software (https://www.jamovi.org/ accessed on 17 March 2024). A principal component analysis (PCA) was performed in the Metaboanalyst platform [35] to analyze and visualize the interactions between the samples (flours and PCs) and the variables (functional properties). The data were log-transformed, mean-centered and divided by the square root of the standard deviation of each variable (Pareto scaling).

3. Results and Discussion

3.1. Protein Solubility

The influence of pH on the protein solubility of flour from P. ostreatus cultivated on different substrates is shown in Figure 1. In all cases, a U-shaped solubility profile was developed with minimum solubility at pH 4 and maximum solubility at pH 11–12. Most plant proteins also exhibit a U-shaped solubility curve due to their acidic nature connected to the greater amount of Asp and Glu residues than of Lys, Arg and His residues [22,36]. The lowest solubility (at pH 4) was noted for AMRL150 cultivated on WS (0.65 ± 0.08 mg/mL) (Table S1). Obviously, pH 4, where the minimum solubility of proteins was observed, is near their isoelectric point, which favors their precipitation. For this reason, pH 4 was selected to accomplish the protein separation from the rest of the compounds in the current study. González et al. (2021) [18] also reported that a pH between 3.0 and 4.0 coincides with the lowest solubility of flour protein from P. ostreatus cultivated on sorghum forage. The extraction of proteins from our samples was performed at pH 12. The solubility of 150WS-F (Table S1) at pH 12 is significantly higher than at 11, whereas for the rest of the P. ostreatus flours tested, the solubility did not differ significantly between the aforementioned pHs. Among all cases, the maximum solubility (1.67 ± 0.19 mg/mL) was noted for the strain 150 cultivated on BOS at pH 11. In the limited literature on the extraction of P. ostreatus proteins [21], the conventional alkaline extraction followed by acidic precipitation around the isoelectric point was usually employed [18,34].

3.2. Protein Content and Extraction Yield

The protein content (% m/m, dry mass) of the P. ostreatus flours and PCs is presented in

Table 1. Protein content (mean ± standard deviation, n = 3) of P. ostreatus flours and protein concentrates. Different superscript letters in each column denote significantly different contents (p ≤ 0.05) between samples.

Sample	Protein Content (% m/m on a Dry Mass Basis)
	Mushroom Flour (F)	Protein Concentrate (PC)
150WS	17.2 ± 0.1 a	37.2 ± 0.0 a
150BOS	24.6 ± 0.4 b	43.1 ± 0.4 b
144WS	18.7 ± 0.5 c	39.8 ± 0.6 c
144BOS	22.7 ± 0.2 d	43.5 ± 0.1 b

. The protein content differed significantly between the strains and the substrates. Regarding the mushroom flours, the highest and lowest values were recorded for AMRL150 cultivated on BOS (24.6 ± 0.4%) and on WS (17.2 ± 0.1%), respectively. The BOS substrate significantly enhanced the protein content of both P. ostreatus strains. Diamantopoulou et al. (2023) [37] also reported that the maximum protein concentration for the carposomes of the same P. ostreatus strains was achieved when cultivated on BOS and on beech wood shavings (BWS). Following the alkaline extraction at pH 12 and precipitation at the isoelectric point (pH 4), the produced PCs had almost doubled their protein content. The highest protein content was achieved in the PC from the AMRL150 and 144 strains cultivated on BOS (43.1 ± 0.4% and 43.5 ± 0.1%, respectively). The mean protein extraction yield (calculated using Equation (1)) was equal to 25 ± 2%, which agrees with that reported in the literature. Specifically, Gonzales et al. (2021) [18] found that for the same extraction ratio as in the present study (mushroom-to-solvent ratio of 1:10), the extraction yield reached 25% and by increasing the ratio from 1:10 to 1:20, no significant difference in the protein solubility was found.

3.3. Protein Solubility Index (PSI)

Protein solubility is a critical feature that determines the uses and applications of protein concentrates and isolates in the food industry. It is one of the most important functional properties of proteins that is governed by their water-binding capacity as well as by the degree of protein molecule denaturation. For this reason, protein solubility is used to estimate the overall protein quality [36,38]. Moreover, protein solubility mostly affects the protein concentrate’s foaming, emulsifying and gelation capacities. Several methods have been developed to evaluate protein solubility as a measure of protein quality based on diverse criteria such as the nitrogen solubility index (NSI), protein dispersibility index (PDI), protein solubility in 0.2% KOH (PS-KOH), urease activity, trypsin inhibitor activity and protein solubility index (PSI) [26,29]. In the present study, the PSI was used to estimate the protein quality of the P. ostreatus flours and PCs. This choice was based on the small sample size that the PSI procedure requires compared to the others found in the literature, as well as on its good reproducibility. Figure 2 presents the PSIs of the P. ostreatus flours and PCs, which were calculated using Equation (2). In all cases, the PCs presented lower PSI values than their corresponding flour. The highest PSI value was noted for the mushroom flour from AMRL150 cultivated on both substrates and the lowest for AMRL144 cultivated on WS. A profound effect of substrate was observed, especially in the case of the PCs. The produced PCs of both mushroom strains when cultivated on WS were characterized by the smallest PSI values. Given that the PSI value is inversely proportional to the actual solubility, it can be inferred that the PCs should possess better functional properties than the flours. Moreover, a better protein quality is achieved when both strains were cultivated on WS. In the literature [29], a series of soy products were evaluated using the PSI procedure. It was found that for full-fat soy flour, low-fat soy flour and soy protein concentrate, the PSI values were equal to 86.0%, 55.5% and 13.0%, respectively. Thus, the quality of P. ostreatus flours (mean PSI: 71.2%) and PCs (mean PSI: 17.7%) is close to that of soy protein, supporting the possibility for many applications in new food formulations and meat alternative foods [21].

3.4. Amino Acid Composition

The AA composition of the P. ostreatus flours is shown in

Table 2. Amino acid composition of the protein of flours from P. ostreatus strains AMRL150 and 144 cultivated on different substrates (WS: wheat straw; BOS: barley and oat straw). Values (mg/g on a dry mass basis) are expressed as mean ± standard deviation (n = 3).

Amino Acid (AA)	P. ostreatus Strains and Substrates 1
	150WS	150BOS	144WS	144BOS
Asp + Asn	18.82 ± 0.85 a	25.34 ± 6.74 a	22.81 ± 1.86 a	24.85 ± 0.86 a
Glu + Gln	28.80 ± 0.69 a	39.92 ± 6.32 b	33.00 ± 0.92 a	39.60 ± 3.05 b
Ser	10.61 ± 0.71 a	13.60 ± 1.39 b	11.68 ± 0.79 a	13.56 ± 1.30 b
His	6.28 ± 0.34 a	8.66 ± 0.17 b	6.43 ± 0.20 a	8.28 ± 0.80 b
Gly	9.89 ± 3.48 a	13.59 ± 4.30 a	10.10 ± 4.14 a	12.01 ± 3.30 a
Thr	9.82 ± 0.44 a	13.04 ± 1.93 b	10.97 ± 0.32 a	12.45 ± 1.22 b
Arg	20.89 ± 0.80 a	30.58 ± 1.94 b	21.60 ± 2.27 a	27.70 ± 1.37 b
Ala	12.93 ± 0.52 a	18.28 ± 1.85 b	14.35 ± 1.43 a	17.72 ± 1.52 b
Tyr	5.74 ± 0.25 a	8.33 ± 1.85 b	6.81 ± 1.10 a	8.97 ± 0.67 b
Val	9.98 ± 0.26 a	13.48 ± 1.88 b	11.78 ± 0.22 a	13.54 ± 0.48 b
Met	3.55 ± 0.32 a	5.01 ± 0.22 b	3.97 ± 0.17 a	4.14 ± 0.56 b
Cys-Cys	8.90 ± 0.98 a	4.68 ± 1.70 a	3.61 ± 1.74 a	6.54 ± 2.53 a
Phe	8.43 ± 0.24 a	10.74 ± 1.84 b	9.64 ± 0.51 a	11.09 ± 0.45 b
Ile	8.34 ± 0.56 a	10.61 ± 1.08 a	9.64 ± 0.88 a	10.15 ± 1.30 a
Leu	14.71 ± 1.81 a	17.96 ± 0.94 a	17.11 ± 3.35 a	18.18 ± 2.80 a
Lys	24.77 ± 6.67 a	32.98 ± 2.48 a	30.82 ± 12.51 a	31.50 ± 5.55 a
Sum	202.45 ± 4.46 a	266.20 ± 11.50 b	224.32 ± 6.78 a	259.98 ± 6.79 b
Essential AA	106.77 ± 11.44 a	143.06 ± 12.48 b	121.96 ± 20.43 a	137.03 ± 14.53 b
Essential AA (%)	52.70 ± 0.11 a	53.80 ± 0.09 a	54.30 ± 2.40 a	52.70 ± 0.10 a
SAA 2 (Met + Cys-Cys)	12.45 ± 1.3 a	9.69 ± 1.92 a	7.58 ± 1.91 a	10.68 ± 3.09 a
AAA 3 (Phe + Tyr)	14.17 ± 0.49 a	19.07 ± 3.69 b	16.45 ± 1.61 a	20.06 ± 1.12 b
Sum of acidic amino acids (Asp + Glu)	47.62 ± 1.54 a	65.26 ± 13.06 b	5 5.81 ± 2.78 a	64.45 ± 3.91 b
Sum of basic amino acids (Lys + Arg + His)	51.94 ± 7.81 a	72.22 ± 5.49 b	58.85 ± 14.98 a	67.48 ± 7.72 b

¹ Different lowercase letters denote significant (p < 0.05) differences for each row. ² SAA: sulfur-containing amino acids. ³ AAA: amino acids with aromatic side chain.

. The total AA content ranged from 266.20 ± 1.90 mg/g (on a dry mass basis) for AMRL150 cultivated on BOS to 202.45 ± 1.67 mg/g for the same strain cultivated on WS. The highest concentration was found for both strains when cultivated on the BOS substrate, showing a similar trend as the protein content (Table 1). Glu, Arg, Asp and Lys were the predominant amino acids in all cases. In the literature, the sum of the acidic amino acids Glu and Asp predominates in the composition of mushroom protein flours [17] and usually dominates the composition of plant proteins [26]; in the present study, a higher content of the basic amino acids Lys, His and Arg was noted. The percentage of the total essential AAs (EAAs) constituted more than 50% of the amino acid composition of the mushroom samples under investigation. As can be observed in Table 2, the EAAs His, Thr, Arg, Val, Phe and Met together with the non-essential amino acids Glu + Gln, Ser, Ala, and Tyr were present in significantly higher amounts when both strains were cultivated on the BOS substrate. Similarly, on the same substrate, the amount of the EAAs, the sum of the acidic amino acids (aspartic and glutamic acid), the sum of the basic amino acids (histidine, arginine and lysine), as well as the amount of amino acids with an aromatic side chain (phenylalanine and tyrosine) were enhanced. On the contrary, in the literature [39], it was found that when maize or pumpkin straw was used for the growth of P. ostreatus, the nitrogen content and the amino acid content were not affected by the substrate.

To assess the protein quality of the P. ostreatus flours, the amino acid score (AAS) (Table S2) and the essential amino acid index (EAAI) were calculated based on Li et al. (2022) and Vierra et al. (2018) [40,41] using whole egg protein as a reference. The amino acid with the lowest AAS is the first-limiting AA. According to Table S2, the first-limiting AA in all samples was Met, whereas the highest AASs (>100) were observed for Thr and Lys. The latter indicates that both amino acids fit the ideal protein standard proposed by the FAO/WHO/UNU [42]. The EAAI values for AMRL150 ranged from 86.3 ± 4.5% to 93.4 ± 6.2% when grown on the BOS and WS substrates, respectively. The corresponding values of EAAI for AMRL144 were 90.0 ± 4.0% and 99.5 ± 6.8% when grown on BOS and WS, respectively. According to the literature [43], EAAI values greater than 90% indicate high quality protein, whereas 70–89% indicates moderate quality. Obviously, both strains can produce high quality protein, especially when cultivated on WS.

The findings in the literature support the higher nutritional value of mushroom protein compared to that of plant proteins [16]. Ayaz et al. (2011) [44] analyzed the amino acid composition of eleven wild species in Turkey and reported a high abundance of Glu, Asp and Leu [44]. Li et al. (2022) [45] found that the powder of the edible fungus Pleurotus citrinopileatus had the highest hydrolyzed amino acid content, reaching 14.656 g/100 g, compared to that of Agrocybe chaxinggu, Flammulina velutipes, Lentinus edodes and Hericium erinaceus. It has been also found that the nutritional value of Agaricus brasilensis protein is limited by its low isoleucine and valine content; Flammulina velutipes, Pleurotus eryngii and Pleurotus ostreatus (black oyster) are deficient in leucine while Agaricus bisporus (champignon and portobello), Letinus edodes, Pleurotus djamor and Pleurotus ostreatus (white oyster) meet the essential amino acid requirements according to the WHO/FAO/UNU reference standard for protein [16]. Obviously, the literature supports our findings for the high nutritional value of P. ostreatus protein. The free amino acid (or non-protein amino acid) content of mushrooms has also been investigated due to their important role in the taste and deliciousness of the product [45,46]. In Pleurotus citrinopileatus, the free amino acids Gln, Leu, Glu and Ala predominated [47] and the substrate was found to exhibit a notable effect on the amino acid composition. Tagkouli et al. (2020) [8] also found that the free amino acid Leu predominated in the composition of P. ostreatus, P. eryngii and P. nebrodensis, followed by Val, Iso, Thr, Phe and Lys.

3.5. Spectroscopic Analysis

Figure 3 presents the spectral region (1840–840 cm⁻¹) of the ATR-FTIR spectra of the mushroom flours 144BOS, 150BOS, 144WS and 155WS. The peaks and their assignments are shown in Tables S3 and S4. Shifts were observed in the 1560–1542 (150BOS,150WS), 1163–1139 (144WS) and 925–905 cm⁻¹ (144WS) spectral regions, which are probably due to intermolecular interactions. The highest peaks were at 1739–1738, 1367–1366 and 1218–1217 cm⁻¹. The first peak has been attributed to >C=O of alkyl esters [48,49]. The second peak has been attributed to C-H deformation, and >C=O and C-O stretching of COO^-. These absorption peaks have been linked to the presence of polysaccharides such as β-glucans and salts of uronic acids, particularly glucuronic acid [48,50,51]. The third peak has been assigned to P=O stretching of phospholipids [50,52]. Nine other smaller peaks were also observed. The vibrations at 1666–1621, 1560–1542 and 1450–1440 cm⁻¹ have been attributed to the presence of proteins [48,52,53] and the C=C and C=O stretching of amino acids, carboxylates, phenols and chitin/chitosan [49,54]. The absorption at 1163–1152, 1086–1082, 1043–1039 and 991–989 cm⁻¹ have been attributes to carbohydrates [51,52,55], while the absorption at 926–905 and 893–891 cm⁻¹ have been attributed to β- and α- glycosidic bonds, mainly in α- and β-glucans [49]. The observed shifts at 1560–1542 (150 BOS, 150 WS), 1163–1139 and 926–905 cm⁻¹ (144WS) are most likely due to intermolecular interactions.

Based on the spectroscopic analysis, the P. ostreatus components found were polysaccharides (especially α- and β-glucans), proteins, phospholipids, chitin/chitosan and amino acids. Several researchers have studied the components of P. ostreatus. Polysaccharides and, in particular, α- and β-glucans [48,49,50,54,56], proteins [18,49], phospholipids [56], chitin/chitosan [54] and amino acids [57] have been identified. Thus, the spectroscopic findings are in agree with the literature.

3.6. Functional Properties

Figure 4 and Table S5 present the functional properties of the P. ostreatus flours and PCs. The water absorption capacity (WAC), also referred to as the water-binding capacity, is related to the hydrogen bonding and the hydration of ionic group abilities of protein molecules [36]. Overall, the WAC of the flours was significantly higher than that of the corresponding PCs. Among the flours, the highest and lowest WAC values were observed for the strain AMRL144 when it was cultivated on BOS (503.1 ± 1.5%) and WS (437.1 ± 2.9%), respectively. The WAC of the PCs ranged from 244.0 ± 3.6% for AMRL150 to 330.6 ± 9.2% for AMRL144 when they were cultivated on WS. The observed differences between the mushroom flours and PCs could be attributed to the higher content of soluble carbohydrates in the flours [26]. It was found that the total carbohydrate content decreased by 60% in protein concentrates due to the harsh conditions applied during the protein extraction process [18]. The WAC for flours from three strains of P. ostreatus and their corresponding protein concentrates was also studied by Cruz-Solorio et al. (2018) [34] who reported similar results to ours. Plant protein concentrates are also characterized by lower WAC values than their corresponding flours and this was attributed to the extraction procedure as well as to the protein conformation and the availability of polar amino acids for protein–water interactions [26,34]. The oil absorption capacity (OAC) of the P. ostreatus PCs was significantly higher than that of the corresponding flour. The highest OAC was observed for the PCs of AMRL150 when cultivated on either WS or BOS (540.9 ± 11.3% and 539.3 ± 4.2%, respectively). Among the flours, the flour from AMRL144 cultivated on BOS presented the lowest OAC (414.7 ± 5.0%) value. Similar findings were obtained when the WAC and OAC of flours and PCs from three strains of P. ostreatus [34] and P. tuberregium sclerotia [58] were examined. Obviously, the ability of PCs to bind with oil is directly attributed to their high concentration of non-polar amino acids.

The ability of proteins to form and stabilize foams is critical for food acceptability. A higher protein concentration enables higher foam stiffness and stability. The foam capacity of the P. ostreatus PCs was significantly higher than that of the corresponding flour. Among the PCs, the highest FC value (57.3 ± 1.5%) was observed for AMRL144 cultivated on WS (Table S5) and the lowest for AMRL150, independent of the cultivation substrate (BOS: 51.2 ± 1.3%; WS: 51.3 ± 1.8%). Among the flours, AMRL150 cultivated on BOS presented the lowest FC value (39.2 ± 0.7%). Jarpa-Parra et al. (2014) [59] found that the foam capacity of lentil protein concentrates was influenced by the environmental pH (pH of the solution: 3, 5 and 7) regardless of the pH used for the protein extraction (pH 8, 9 and 10). The dependence of FC on pH was studied by Cruz-Solorio et al. (2018) [34]. They found that the PCs of three P. ostreatus strains presented the highest FC values (220–276%) at pH 8, which decreased (by 116–200%) with the increase of the pH to 10. The lower FC values reported in our study could be attributed to the fact that we did not adjust the pH of the suspension. The observed difference between the flours and PCs may also be attributed to differences in flexibility and charge density of the protein molecules under the testing conditions. The foam stability (FS) of the PCs was significantly higher than that of the flours except for AMRL144 cultivated on BOS, which did not present a significant difference. Foam stability is dictated by a low charge density of the protein molecule, which allows for less intermolecular repulsion and thus occurs around the pI. Unlike FC, the FS of lentil protein concentrates was not influenced by the pH of the solution nor the extraction pH (pH range: 8–10) and the authors concluded that the interfacial film properties were not pH-dependent [59]. Regarding the FS of P. ostreatus flours and protein concentrates, it has been reported [34] that both exhibited better stability at alkaline pHs (8–10), whereas FS was quite reduced at pH 2.

Proteins can be excellent emulsifiers because they contain both hydrophobic and hydrophilic groups that decrease the interfacial tension between oil and aqueous phases, thus stabilizing emulsions. The emulsion stability (ES) of the P. ostreatus PCs was significantly better than that of the flours except for AMRL150 cultivated on BOS (Table S5). The research findings for three strains of P. ostreatus revealed that the ES is pH-dependent, being higher at alkaline pHs (between 8 and 10) [18]. The emulsifying properties of Phlebopus portentosus protein isolate (PPI) extracted from P. portentosus fruiting bodies using alkaline extraction and isoelectric precipitation were studied for their ability to act as emulsifiers for preparing double emulsions [25]. It was found that the low solubility of the PPI can be improved by applying a protein nanoparticulation strategy (PPIP) and the resulting PPIP can act as a single emulsifier to fabricate a double Pickering emulsion via one-step homogenization.

A principal component analysis was performed using the functional properties as well as the protein content as variables to visualize the differentiation of the mushroom flours and protein concentrates. The first two principal components accounted for 89.2% of the total variance (Figure 5a). It is evident that two clusters were formed along the x-axis (first principal component). The F-cluster (red) is positioned on the left side of the score plot whereas the PC-cluster (green) is positioned on the right side. According to the biplot (Figure 5b), the WAC content was associated with the F-cluster, whereas the protein content was associated with the PC-cluster. Within each of these clusters (F and PC), two sub-clusters can be distinguished spreading in the direction of y-axis; these correspond to samples grown with the different strains (AMRL150 and AMRL144). This separation (along the vertical axis) was attributed mostly to the variables FS and FC. The former was positively correlated with the strain AMRL150 whereas the latter one was positively correlated with the strain AMRL144.

4. Conclusions

Our findings support the high nutritional value of P. ostreatus protein from the two strains tested in the present study. All the essential amino acids studied were found in the proteins, with the basic amino acids prevailing over the acidic amino acids. In all the samples, the limiting amino acid was methionine, whereas the samples were a rich source of lysine and threonine. The EAAI values indicate that both strains can produce high quality protein especially when they are cultivated on a wheat straw substrate. The produced protein concentrates were characterized by a low protein solubility index (PSI), indicating that they possess better nutritional properties compared to the mushroom flours. The wheat straw substrate enhanced the protein quality of the produced PCs according to the PSI results. The lowest PSI was noted for AMRL144 cultivated on wheat straw, and its protein concentrate had the highest foam capacity. As already stated in the literature and based on the results of the present study, the correlation of PSI with the functional properties of PCs is worth investigating. In addition, the spectroscopic analysis of the mushroom flours revealed the presence of polysaccharides and salts of uronic acids, particularly glucuronic acid and phospholipids. The flours and PCs differed based on their functional properties. The WAC was significantly higher in the flours and the OAC, FC, FS and ES were higher in the PCs. The functional properties were positively correlated with the strain, indicating differences in protein surface hydrophobicity. The foam stability was positively correlated with the P. ostreatus strain AMRL150, whereas the foam capacity was positively correlated with AMRL144.

Since the solubility of proteins is essential for their foaming and emulsifying properties, partial hydrolysis, the addition of salt and adjustment of the pH of the protein concentrates of P. ostreatus strains may improve their function in new food formulations.