Valorization of Grape Pomace for Trametes versicolor Mycelial Mass and Polysaccharides Production

Abstract

Polysaccharides and protein–polysaccharide complexes produced from the fungal strain Trametes versicolor have demonstrated bioactive properties that depend on the substrate, the fermentation conditions and also the fungal strain. Likewise, the submerged and controlled fermentation of medicinal mushrooms elicits numerous advantages over traditional processes to produce mycelia and added-value products, along with the exploitation of biodiversity. This study evaluated the growth profile of an indigenous T. versicolor isolate using commercial nutrients that were subsequently replaced with renewable resources, specifically, grape pomace extract (GPE), under static and shaking conditions. The effect of elicitor addition was also assessed using GPE. The process productivity was significantly improved, yielding 21 g/L of biomass. Agitation proved beneficial for all examined cases regarding biomass productivity and substrate consumption rates. The chemical and antioxidant profile of crude intracellular and extracellular polysaccharides was determined, whereby intracellular extracts indicated >50% antioxidant activity. FTIR analysis validated the preliminary chemical characterization of the extracts, whereas the amino acid profile of IPS extracts was also included. Evidently, our study elaborates on the development of a bioconversion concept to valorize wine-making side-streams to formulate added-value products with potential bioactive attributes.

1. Introduction

Medicinal fungi are an indispensable part of traditional diets worldwide, particularly in Asian and Mediterranean countries. Bioactive components in several mushroom species have been previously investigated regarding established and potential health benefits. These include immunomodulatory properties and antitumor, anticancer and antioxidant activities, in addition to their high nutritional value and prebiotic activity [1,2,3].

Mushrooms mainly consist of vitamins, minerals, essential amino acids, polyphenols and fatty acids but also indicate a major source of carbohydrates, specifically glucans, that could exert a prebiotic effect [4]. Likewise, the research interest in polysaccharides and protein–polysaccharides complexes of medicinal fungi has escalated in recent years, considering that fungal extracts can be introduced in the development of functional foods [5]. Extracts from medicinal fungi can derive from the cultivation of fruiting bodies and basidiocarps or with controlled fermentation of mycelial mass. Traditional but also sophisticated and green methods have been evaluated to generate extracts [5,6], which have been assessed during in vitro, in vivo and clinical trials [3].

Trametes versicolor, also referred to as Coriolus versicolor and Turkey tail mushroom, belongs to Basidiomycota and has been consumed as food or tea since the ancient era [7]. Exopolysaccharide and intracellular polysaccharide production from T. versicolor have been previously investigated in the open literature, demonstrating numerous health benefits [8,9]. Polysaccharide K (PSK) and polysaccharide P (PSP) designate well-established compounds extracted from T. versicolor with evidenced bioactive properties. In view of the unexplored diversity of T. versicolor strains, the growth conditions and production of polysaccharides should be undertaken for novel and indigenous isolates [3].

Submerged or solid-state fermentations of medicinal mushrooms under controlled conditions elicit several advantages and have been previously used for the growth of T. versicolor to generate high-added value products, including enzymes (e.g., laccase, lignin peroxidase, manganese peroxidase) [1], biofuels (e.g., bioethanol) [10] and fungal biomass for edible membranes [11]. However, to the best of our knowledge, limited research has been performed on the utilization of agro-industrial waste and specifically, grape pomace as substrates in submerged fermentation of T. versicolor to produce mycelial mass, along with intracellular and extracellular polysaccharides.

In the context of the bioeconomy, the development of cost-effective bioprocesses should undertake the valorization of waste and by-product streams as onset feedstocks that generate products with diversified end applications. The processes following the principles of a circular economy should combine the exploitation of renewable resources and the formulation of value-added products, aiming to enhance economic feasibility, which is contingent on the end applications. For instance, the market value of T. versicolor mycelial powder is estimated at 25,000 USD/ton, whereas the market value for extracts could be higher, considering the bioactive properties of polysaccharides. The cost of raw materials, which also affects process economics, could be mitigated; for instance, the solid waste disposal of grape pomace (approximately 35 USD/ton) could be saved in the case of a biorefining process. Integrated biorefining processes could also moderate transport costs, estimated at 5 USD/ton [12], as these are associated with the distance, the annual operating days and the origin of solid waste. Likewise, circular bioeconomy scenarios will elicit a positive impact on society, the economy and the environment [12].

The aim of our study was to evaluate the growth pattern of a newly isolated T. versicolor strain using synthetic media and grape pomace extract as an alternative substrate obtained using a mild treatment process. Fungal proliferation and polysaccharide synthesis were investigated along with the addition of elicitors. Preliminary characterization and structural analysis of extracellular and intracellular extracts were carried out to elucidate bioactive properties and potential food applications. Our study elaborates on an innovative utilization route for the combined production of mycelial mass and polysaccharides using food industry by-products. On top of that, limited research exists on the controlled fermentation of T. versicolor using renewable substrates and the subsequent evaluation of crude mycelial extracts. Our focus is to design holistic bioprocesses that foster the pillars of the bioeconomy with the exploitation of agro-industrial resources to generate high added-value products that will be introduced in food formulations and elicit health benefits.

2. Materials and Methods

2.1. Fungal Strain and Growth Conditions

The medicinal mushroom Trametes versicolor, utilized in this study, is an isolate from the wild fruitbody of the species acquired from Kefalonia Island [11]. The strain T. versicolor CCIU 2013 is maintained at the culture collection of the Laboratory of Food Chemistry and Industrial Fermentations (under the direction of Dr. Kopsahelis) of the Department of Food Science and Technology at the Ionian University (Greece), and also at the established Fungal Culture Collection of the Mycetotheca (ATHUM 9921, under the direction of Dr. Gonou) in the National and Kapodistrian University of Athens (Greece). The strain was kept in inclined potato dextrose agar (PDA, Condalab) slants, filled with paraffin oil at 5.0 ± 1.0 °C, and at −20.0 ± 1.0 °C in 20% (w/w) glycerol. For the fermentation inoculum, fresh mycelium derived after sequential growth on PDA Petri dishes was further used in liquid precultures. A specific volume (150 mL) of synthetic glucose-based medium (pH 6.2) was dispensed in Erlenmeyer flasks (500 mL) for inoculum liquid precultures. The liquid preculture medium included various nutrients and elements, particularly, glucose, 10 g/L; yeast extract, 1.5 g/L; peptone, 1.5 g/L; KH₂PO₄, 1 g/L; MgSO₄·7H₂O, 0.5 g/L; CaCl₂·2H₂O, 0.23 g/L; FeCl₃·6H₂O, 0.08 g/L; MnSO₄·H₂O, 0.04 g/L and ZnSO₄·7H₂O, 0.02 g/L (pH 6.1) [11].

Prior to inoculation, the flasks were sterilized for 20 min at 121 ± 1 °C. Subsequently, inoculation was performed with two 10-day-old PDA agar plugs (6 mm diameter) as previously described [11]. The liquid precultures were incubated at 25.0 ± 0.5 °C (10 days) under agitation (150 rpm) in an orbital shaker (ZWYR-200D, LABWIT, Shanghai, China). Once the incubation was complete, the precultures were homogenized in aseptic conditions and utilized as inoculum (10%, v/v) in the subsequent fermentations.

2.2. Fermentation Supplements and Submerged Fermentations (SmFs)

Conventional synthetic media were initially used during the submerged fermentations (SmFs) of T. versicolor, to evaluate growth on diversified carbon sources, namely, fructose, glucose and a mix of fructose and glucose in equal amounts (i.e., mixed sugars). The sugar concentration was adjusted at ~10 g/L; in the case of mixed sugars, fructose and glucose were in equal amounts. Synthetic media consisted of (in g/L): yeast extract at 2.5; peptone at 3.5; CaCO₃ at 2.0; KH₂PO₄ at 1.0; MgSO₄·7H₂O at 0.5; CaCl₂·2H₂O at 0.23; MnSO₄·H₂O at 0.04; ZnSO₄·7H₂O at 0.02 and FeCl₃·6H₂O at 0.08.

The basis of carbon selection was to simulate the composition of nutrient feedstocks obtained from grape pomace, which is a low-cost agro-industrial substrate. Grape pomace (including skins and seeds) was collected from a local vineyard, as a side-stream of the wine-making process of the red variety Mavrodaphni (Kefalonia, Greece). A previously described method was used to formulate grape pomace extract (GPE) using an aqueous extraction process. Briefly, the extraction of soluble, free sugars from grape pomace was performed at 40 °C for 2 h at a solid-to-liquid ratio of 1:10 (w/v) with deionized water [13]. The GPE contained ~5–6 g/L of glucose and ~5–6 g/L of fructose along with trace amounts of sucrose (0.4–0.5 g/L).

For each examined case, i.e., synthetic media and GPE, 30 mL of the substrate was dispensed in Erlenmeyer flasks (100 mL), sterilized (121 °C, 20 min) and inoculated with the fungal preculture (10%, v/v). Prior to sterilization, the pH value of the media was fixed at 6.2 ± 0.1 using 5 N HCl or 5 N NaOH. The same pH value was used during the experiments with GPE so that it simulated the synthetic media and avoided the impact of lower initial pH values. After inoculation, the flasks were incubated both in static and agitated conditions (150 rpm) in an orbital shaker (ZWYR-200D, LABWIT, Shanghai, China) at 25.0 ± 0.5 °C. The total volume of duplicate samples was collected in five-day intervals, based on preliminary experiments, to monitor the fermentation profile of T. versicolor.

2.3. Analytical Methods

2.3.1. Determination of Total Dry Weight (TDW)

As earlier stated, the samples were withdrawn at designated time points (every five days) to estimate fermentation variables. The total volume of each flask was measured with a volumetric cylinder and then filtered under vacuum (Whatman^® No 1, Buckinghamshire, UK) to remove the mycelia mass from the fermentation broth. Fungal mycelia were washed twice with deionized water, transferred into pre-weighed McCartney vials (25 mL) and dried at 60 ± 0.5 °C [14]. The results are presented as total dry weight (TDW, g/L). Additionally, dried mycelial mass was used to assess the intracellular polysaccharide content. The mycelia-free broth was kept at −20 °C and used for the analysis of residual sugar and exopolysaccharide production.

The productivity of the process (Q_X, g/L/d) was determined using the equation:

$${\mathrm{Q}}_{\mathrm{X}}=\raisebox{1ex}{$({\mathrm{X}}_{\mathrm{t}}-{\mathrm{X}}_0)$}\!\left/ \!\raisebox{-1ex}{${\mathrm{T}}_{\mathrm{t}}$}\right.$$

where X_t and X₀ indicate the concentrations of total dry weight (g/L) at a specific fermentation time point and inoculation time, respectively, whereas T_t corresponds to the fermentation time point (d).

2.3.2. Evaluation of Sugar and Free Amino Nitrogen Concentration

Glucose and fructose were quantified using high-performance liquid chromatography analysis (HPLC, Agilent, Santa Clara, CA, USA) equipped with an ROA-organic acid H+ (300 mm × 7.8 mm, Phenomenex, Torrance, CA, USA) column coupled to a differential refractometer (RID). The operating conditions were as follows: sample volume of 10 μL; mobile phase of 10 mM H₂SO₄; flow rate of 0.6 mL/min and column temperature of 65 °C. The samples were diluted and filtered (Whatman^®, Buckinghamshire, UK, Uniflo syringe filters, 0.2 μm) prior to analysis [15].

The consumption rate of sugars (Q_S, g/L/d) was determined using the equation:

$${\mathrm{Q}}_{\mathrm{S}}=\raisebox{1ex}{$({\mathrm{S}}_{\mathrm{t}}-{\mathrm{S}}_0)$}\!\left/ \!\raisebox{-1ex}{${\mathrm{T}}_{\mathrm{t}}$}\right.$$

where S_t and S₀ indicate the sugar concentration (g/L) at specific fermentation time point and inoculation time, respectively, whereas T_t corresponds to the fermentation time point (d).

The concentration of free amino nitrogen (FAN) was estimated using the ninhydrin colorimetric method, as previously described in [16], to assess nitrogen consumption.

2.3.3. Preparation of Extracellular Polysaccharide Extracts (EPSs)

The broth obtained after filtration was processed with ice-cold ethanol at a ratio of 1:4 to obtain EPSs via precipitation. The mixture was vigorously mixed and incubated overnight at 4 °C. After incubation, centrifugation (Rotina 420R, Hettich Zentrifugen, Tuttlingen, Germany) was performed (4200× g for 10 min), and the supernatant was discarded to collect the precipitate. EPSs were estimated gravimetrically after freeze-drying the precipitate and expressed in g/L.

2.3.4. Preparation of Intracellular Polysaccharide Extracts (IPSs)

The crude IPS fraction of mycelia was recovered using hot water extraction, based on a previously published method [17]. Briefly, a known amount of dried mycelial biomass was extracted at 100 °C (in boiling water), using magnetic stirrers, at a solid-to-liquid ratio (1:10, w/v) for 1 h in screw cap vials. The liquid extract was then filtered and freeze-dried to evaluate the extraction yield. After lyophilization, the crude dried extract was redissolved in deionized water at a concentration of 20 mg/mL and maintained at −20 °C to be further analyzed.

Ethanolic extraction was also assessed for crude IPSs, following a previous method with slight modifications [18]. Briefly, dried mycelia mass was extracted with 95% ethanol, at a solid-to-liquid ratio (1:10, w/v), under agitation (150 rpm) in an orbital shaker for 24 h at room temperature. The ethanolic extract was filtered, the ethanol was removed using a rotary evaporator (40 °C) and the extraction yield was determined gravimetrically. The extract was resuspended at a concentration of 20 mg/mL and stored at −20 °C until further analysis.

2.3.5. Compositional Characterization and Antioxidant Activity of Crude IPSs and EPSs

The content of polysaccharides in the crude extracts was measured using the phenol–sulfuric method with glucose as the standard [19]. The protein content was assessed using the Lowry method, and bovine serum albumin (BSA) was applied for the standard curve [20]. The antioxidant activity was determined using the DPPH• (2,2-diphenyl-1-picrylhydrazyl) scavenging radical method and an ABTS spectrophotometric assay. The percent inhibition (I%_DPPH•) of free radicals was estimated with the following equation:

$${\mathrm{I}\mathrm{\%}}_{\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}•}=\frac{{\mathrm{A}\mathrm{B}\mathrm{S}}_{\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}•}-{\mathrm{A}\mathrm{B}\mathrm{S}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{{\mathrm{A}\mathrm{B}\mathrm{S}}_{\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}•}}\times 100$$

where ABS_DPPH• corresponds to the absorbance of the blank and ABS_sample corresponds to the sample absorbance. The calibration curve was generated using Trolox, and the antioxidant activity was expressed as μg of Trolox equivalents per mg of dry fungal extract.

Τhe working solution for the ABTS spectrophotometric method is composed of 2,2′-amino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (7 mM) and potassium persulfate (2.45 mM). The reagents were mixed 12–16 h prior to analysis and placed in a dark cabinet at room temperature to form ABTS•+. The solution was diluted with methanol to achieve an absorbance of 0.7 ± 0.05 at 734 nm. Then, 2.85 mL of the solution was mixed with 0.15 mL of the sample, and the absorbance was read after 10 min at 734 nm. The changes in absorbance were monitored to quantify the inhibition percentage (I%_ABTS•+).

$${\mathrm{I}\mathrm{\%}}_{\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S}•+}=\frac{{\mathrm{A}\mathrm{B}\mathrm{S}}_{\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S}•+}-{\mathrm{A}\mathrm{B}\mathrm{S}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{{\mathrm{A}\mathrm{B}\mathrm{S}}_{\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S}•+}}\times 100$$

2.3.6. Amino Acid Analysis in the Crude IPS Extracts

Amino acid analysis was carried out using high-performance liquid chromatography (HPLC) after derivatization of the IPS extracts with ortho-phthalaldehyde (OPA) and fluorenylmethoxy chloroformate (FMOC). OPA was prepared by diluting 10 mg in 1 mL borate buffer (0.4 M, pH = 10.2) and 23 μL 3-mercaptopropionic acid. FMOC (2.5 mg) was diluted in 1 mL acetonitrile. An Agilent G1329A autosampler was used for the derivatization reaction following the Agilent 5990-4547EN Application Note injection program. Elution was carried out using an Agilent 1200 series coupled with a diode array detector (DAD) according to the Agilent 5990-4547EN Application Note with some modifications. The derivatized amino acids were separated with a Zorbax Eclipse Plus C18 column (4.6 × 150 mm, 5 μm) and a gradient elution system consisting of 10 mM Na₂HPO₄: 10 mM Na₂B₄O₇, pH 8.2: 5 mM NaN₃ (Mobile Phase A) and acetonitrile: methanol: water (45:45:10, v:v:v) (Mobile Phase B). The gradient program was the following: 98% Mobile Phase A for 0 to 0.84 min, 43% A at 33.4 min, 0% A at 33.5–39.3 min and 98% A at 39.4–60 min. The flow rate was 1 mL/min, and the column temperature was 40 °C. The absorbance was recorded at 262 and 338 nm, and peak identification was based on standard compounds (Figures S1 and S2).

2.3.7. Fourier-Transform Infrared Spectroscopy (FTIR)

A structural analysis of crude EPS and IPS extracts was undertaken with FTIR spectroscopy using an Agilent Cary 630 FTIR Spectrometer equipped with diamond ATR (attenuated total reflectance). The spectra were acquired after 32 scans at room temperature in the frequency range 4000–500 cm⁻¹ with a resolution of 2 cm⁻¹. Data were compiled and refined using MicroLab PC 5.6.2135.0 software (Agilent Technologies, Santa Clara, CA, USA).

2.3.8. Statistical Analysis

Fermentations were performed in triplicates (n = 3). The statistical analysis was carried out with Microsoft Excel 2018. Values are presented as average ± standard deviation, while significant differences (significance level α = 0.05) were evaluated using the Student’s t-test.

3. Results

3.1. Growth Performance on Commercial Carbon Sources and Synthetic Media

The initial step of this study was to determine the growth pattern of the newly isolated T. versicolor strain on commercial carbon sources and synthetic media that provided all the essential nutrients for fungal growth. Glucose, fructose and the combination of glucose and fructose were used during static and shaking conditions, and the results are presented in the following figures. Fermentations were monitored until the carbon source was depleted. The results correspond to the fermentation time points that maximum values of biomass were achieved. More specifically, Figure 1a presents total dry weight (TDW) production, and Figure 1b,c present the process productivity (Q_X, g/L/d) and rate of sugar consumption (Q_S, g/L/d), respectively. Glucose utilization was not affected by static or shaking conditions since biomass production and productivity were in the same range and were not deemed different (p > 0.05). The same pattern was observed for fructose, as TDW production between static and shaking conditions (9.05 and 8.90 g/L, respectively) did not differ significantly (p > 0.05). Higher biomass production was observed during static fermentations in the case of mixed sugars (8.49 g/L) compared with agitated conditions (7.70 g/L); however, the statistical analysis did not indicate a significant difference. Notably, agitation had a significant effect (p < 0,05) on biomass productivity, which increased almost two-fold for fructose and mixed sugars. In all the examined cases, the conversion yield of sugars to biomass ranged from 0.82–0.95 g/g. Apparently, agitated conditions confer an impact on the oxygen transfer rate and shear stress, which has been evidenced to affect the size of pellets and metabolites [4]. On top of that, filamentous fungi are primarily aerobic; hence, oxygen transfer facilitates respiratory activities, leading to rapid growth and increased productivity.

The effect of carbon and nitrogen sources on T. versicolor growth and metabolites production has been evaluated in previous studies. For instance, Nguyen et al. evaluated the effect of numerous carbon sources on the mycelia growth of T. versicolor, demonstrating that fructose and xylose resulted in optimal growth rates [21]. Twelve culture media were examined for their influence on the growth of six Coriolus versicolor (synonym of T. versicolor) strains, along with several parameters (e.g., temperature, pH and the C/N ratio) [22]. The authors stated that dextrin and yeast extract were the optimum sources; however, fructose performed equally for some specific strains. Tavares et al. studied five commercial media for exopolysaccharide (EPS) production, along with the effect of glucose concentration and pH value, indicating that yeast malt extract medium exhibited the highest production [23].

Similarly, the influence of static and shaking conditions on laccase, manganese peroxidase and lignin peroxidase by T. versicolor FPRL 28A INI was used during the development of a treatment process to remove phenolics from olive mill wastewater [24]. The authors reported that aeration affected total phenolic removal and was correlated with laccase production, which was higher in shaking conditions, indicating the effectiveness of shaking conditions. Increased production of ligninolytic enzymes and biomass during shaking conditions was also demonstrated for a T. polyzona isolate, hence indicating higher productivity [25].

In our previous study that evaluated the growth of T. versicolor on lactose, biomass production was not statistically different between static and shaking conditions, whereas static conditions affected the sugar consumption rate [11]. Hence, similar to other mushroom strains, there is a strain specificity for optimum carbon sources and fermentation conditions for T. versicolor. However, our results provided proof to proceed with the substitution of synthetic media with the grape pomace-based substrate.

3.2. Assessment of Grape Pomace Extract (GPE) as Fermentation Substrate

The following step of our study entailed the substitution of commercial, synthetic media with fermentation substrates formulated from wine-making by-product streams, namely, grape pomace extract (GPE). Figure 2 and Figure 3 present the macroscopic observation and the profile change in sugar and biomass production during static and shaking conditions, respectively. The composition of GPE was ~6 g/L of glucose, ~6.5 g/L of fructose and trace amounts of sucrose (~0.5 g/L), whereas the free amino nitrogen (FAN) concentration was 15–20 mg/L.

It can be easily observed that fermentation in static and shaking conditions influenced fungal morphology, as depicted in Figure 2a and Figure 3a. In agitated conditions, the mushroom grew in the form of pellets, whereas during static conditions, filamentous mycelia were formed on the surface.

Glucose and FAN consumption, both in the static and shaking cultures, started within the first five days of cultivation at an equal rate (Q_GLU = 0.34 g/L/d), whereas fructose consumption was initiated at a lower rate and after glucose had been almost depleted from the media. However, during shaking conditions, the consumption rate of fructose was higher (0.30 g/L/d) compared with the static conditions (0.23 g/L/d). As a result, total fructose consumption increased from 54% in static conditions to almost 69% during shaking conditions, entailing the production of 8.3 g/L and 9.8 g/L of TDW in static and shaking conditions, respectively. Apparently, the utilization of GPE impeded the profile of fructose consumption compared with the synthetic media (e.g., fructose and mixed sugars), even though biomass production increased. Complete fructose consumption in synthetic media is postulated to connect with the fungal adaptation to the sole carbon substrate provided. In the case that mixed sugars were used, fructose was consumed after glucose, which was also observed with GPE but without complete fructose consumption. On the other hand, GPE contained phenolic compounds and low amounts of lipids from grape pomace, which possibly stimulated fungal proliferation.

The evaluation of mixed carbon sources has previously demonstrated an effect on fungal growth. For instance, Bakratsas et al. used several combinations of glucose and xylose to simulate the composition of lignocellulosic hydrolysates, noting that the combination of sugars entailed lower biomass of Pleurotus ostreatus compared with glucose, whereas xylose consumption was inhibited during the first days [26]. The addition of lignocellulosic feedstocks in submerged cultures of T. versicolor, namely, grape seeds, grape stalks and barley bran, induced the production of laccase compared with the control media [27]. Likewise, increased mycelial biomass was noted when beet wastes (6.45 g/L) and tomato pomace (4.82 g/L) were used in the fermentation of T. versicolor compared with simple carbon sources [28].

The beneficial effect that agro-industrial substrates have on T. versicolor most probably relates to the enzyme secretion and gene expression that regulate carbon metabolism. In this context, several mixtures of hexoses and xylose were evaluated for bioethanol production using T. versicolor along with enzyme production. The results showed that glucose, mannose and xylose were consumed in parallel but at different rates depending on the fermentation stage [29]. Zhang et al. investigated the transcriptomic profile of T. versicolor on poplar wood, demonstrating that significant changes were observed in the upregulation and downregulation of several genes compared with glucose, indicating the different mechanisms that govern substrate degradation [30].

In our previous studies, GPE extract was evaluated as a substrate for the cultivation of Phellinus sp., Sepedonium sp. and Ganoderma lucidum strains [13,31]. A strain specificity was shown since, depending on the strain, biomass production increased or decreased compared with the synthetic media. However, the effect of agitation on the consumption rate of sugars and process productivity was evident in the case of G. lucidum [31].

In the context of improving the process productivity and inducing fructose consumption, the addition of 0.25% Tween 80 as an elicitor was undertaken. Agitated conditions were selected, based on the increased consumption of fructose. Initially, experiments were performed at an initial total sugar concentration of ~12.5 g/L. Biomass production increased to 4.8 g/L after four days of fermentation, but most importantly, the Tween addition had a pronounced effect on biomass productivity and the sugar consumption rate, as illustrated in Figure 4.

Subsequently, considering the superior fungal growth performance using the Tween 80 addition, a similar experiment using a higher initial concentration of ~30 g/L was performed, and the results for biomass and substrate utilization are presented in Figure 5.

The obtained results showed that 94% of the initial sugars were consumed up to the 15th day of incubation, leading to the production of 21 g/L of TDW. A sequential utilization of fructose and glucose was observed. It is worth noting that at the end of fermentation, both sugars were completely metabolized. Moreover, consumption rates were significantly improved; consumption of total sugars reached 1.85 g/L/d, and biomass productivity reached 1.38 g/L/d, which was higher among all cases examined.

The effect of elicitors and the addition of other compounds has been previously investigated related to the effect on fungal biomass and the production of polysaccharides. For instance, the supplementation of media with Tween 80 in the culture of Cordyceps sinensis displayed a profound effect on EPS synthesis [32]. Increased EPS biosynthesis and improved bioactive properties were also obtained after the addition of 0.25% Tween 80 in the cultivation of G. lucidum [33]. Wang et al. undertook studies regarding the effect of tyrosol on T. versicolor, stating that biomass reached 14.5 g/L in the tyrosol-induced incubations [34]. Hence, the novelty of our study relates to the beneficial outcome that the Tween 80 addition conveyed on T. versicolor growth using substrates obtained from agro-industrial resources. In addition, these experiments indicate the potential to further elucidate the effect of elicitor addition on the production of polysaccharides including compounds that will allow for novel food applications.

3.3. Evaluation of Crude Intracellular (IPS) and Extracellular (EPS) Polysaccharide Synthesis

Mushroom polysaccharides extracted from the mycelia, the fruiting bodies and the culture supernatant have emerged in the spotlight of scientific interest mainly because of their bioactive properties [35,36]. However, in the vast majority of studies investigating the beneficial properties of polysaccharides, intracellular (IPS) or extracellular (EPS), the compounds of interest were obtained from basidiocarps and fruiting bodies and to a lesser extent from mycelia. On top of that, the largest share of studies used conventional synthetic media for the cultivation stage. Evidently, to sustain the resilience of food systems, the development of cascade bioprocessing should encompass the combined exploitation of renewable resources and the production of high-value components to formulate functional foods.

In view of the above, crude IPSs were extracted from fungal biomass using aqueous extraction, whereas EPSs were precipitated from the culture supernatant. Preliminary experiments were also undertaken to evaluate ethanolic extractions for the IPS fractions, but the extraction yield was lower compared with aqueous extractions, contrary to other studies. Hence, hot water extraction was selected to also allow for future inclusion in food product development. The results regarding the extraction and characterization of crude IPSs, derived from fermentations using synthetic mixed sugar media and GPE, are presented in

Table 1. Production and preliminary characterization of crude IPSs produced during SmF of T. versicolor on mixed sugars and GPE under static and shaking conditions and Tween 80 addition.

SmF Conditions	IPS Production (g/L)	IPS Content (mg/g DW *)	IPS Productivity (g/L/day)	Protein (%)	Polysaccharides (%)
Mixed sugars
static	1.13 ± 0.05	132.97 ± 0.14	0.11 ± 0.01	10.48 ± 0.01	14.07 ± 0.01
shaking	0.83 ± 0.04	107.50 ± 0.32	0.17 ± 0.01	8.96 ± 0.03	6.61 ± 0.02
GPE
static	2.59 ± 0.11	268.21 ± 0.41	0.13 ± 0.01	13.61 ± 0.54	18.52 ± 0.71
shaking	2.63 ± 0.08	257.81 ± 0.38	0.17 ± 0.01	14.43 ± 0.52	15.93 ± 0.48
shaking–Tween 80	2.96 ± 0.13	139.40 ± 0.51	0.19 ± 0.01	7.54 ± 0.34	29.10 ± 0.37

* DW: dry weight of mycelial mass.

.

When synthetic media were applied, IPS production and IPS content were higher in static compared with agitated conditions. However, the IPS productivity increased during shaking conditions. On the other hand, the application of agitation did not affect the production of crude IPSs when GPE was used. It is worth noting that when Tween 80 was added to the media, the IPS content reduced almost two-fold, yielding 139 mg/g dry weight compared with 268 and 257 mg/g dry weight, in static and shaking conditions, respectively (Table 1). Notably, when GPE was used, the IPS content was increased, compared with the mixed sugar synthetic media case.

The protein and total carbohydrate contents exhibited a lower content in synthetic media compared with GPE. Likewise, the protein content using Tween 80 and GPE was lower, but the polysaccharide content was increased. In the case that GPE was the sole substrate, the protein content was 13.6–14.4%, and total polysaccharides ranged from 15.9 to 18.5%. In the case that bioprocess targets the intracellular products, the obtained content indicates an important parameter to be optimized.

Previous studies have indicated that apart from agitation, the type of substrate and carbon source can entail modifications on the protein content. For instance, Pilafidis et al. used wine distillery effluents and brewer’s spent grain as substrates for submerged cultivation of several macro-fungi, including T. versicolor [37]. The authors reported that brewer’s spent grain influenced the protein content, whereas when wine distillery effluents were evaluated, the protein content was not affected [37].

It should be noted that during fermentations with GPE, the production of crude IPSs increased during fermentation, demonstrating maximum values along with maximum biomass production. The increased production of IPSs and also EPSs was also demonstrated by Cui et al. during the growth of C. versicolor in bioreactors using milk permeate [38].

After extraction, the samples were freeze-dried and characterized regarding their potential antioxidant activity using two different methods, and the results are displayed in

Table 2. Antioxidant activity of crude IPSs produced during SmF of T. versicolor on synthetic media (mixed sugars) and GPE under static and shaking conditions and Tween 80 addition using two different (DPPH and ABTS) in vitro assays.

SmF Conditions	IDPPH (%)	μg Trolox/mg Extract	IABTS (%)	μg Trolox/mg Extract
Synthetic media
mixed–static	18.76 ± 0.74	4.81 ± 0.74	81.35 ± 1.92	3.96 ± 0.18
mixed–shaking	8.44 ± 0.30	2.82 ± 0.30	79.73 ± 4.27	3.35 ± 0.41
GPE
static	57.56 ± 0.42	11.24 ± 0.34	91.67 ± 0.12	4.68 ± 0.18
shaking	42.75 ± 0.21	8.71 ± 0.25	84.14 ± 0.19	4.17 ± 0.21
shaking–Tween 80	35.27 ± 0.36	5.44 ± 0.29	90.05 ± 0.09	4.06 ± 0.10

.

The antioxidant activity was assessed using two established in vitro methods, the DPPH radical scavenging activity and the ABTS spectrophotometric method. It was observed that the extracts indicated antioxidant capacity; however, different responses were obtained between the DPPH and ABTS assays. In fact, the extracts demonstrated a high ability to reduce the ABTS radical (>80%) in all examined cases, whereas DPPH inhibition varied from 35 to 57% for the GPE-derived extracts and was even lower for synthetic media. The differences between the two assays could be associated with the presence of various compounds, reacting differently depending on the radical used. For instance, ABTS presents very low reactivity toward thiols and proteins because their mechanism is based on hydrogen atom transfer [39].

Previous studies have also noticed varying antioxidant capacity of crude extracts depending on the assay, and this could be associated with phenolic compounds [40]. Crude hot water polysaccharides obtained from T. versicolor fruiting bodies showed 74.7–77.5% inhibition of DPPH free radical [41]. In another study, Angelova et al. used four assays to estimate the antioxidant activity of crude exopolysaccharides of T. versicolor, concluding that the antioxidant activity varied depending on the method the antioxidant activity varied, still ABTS activity was higher compared to DPPH [7]. The higher antioxidant activity of T. versicolor basidiocarps compared with mycelium extracts to neutralize ABTS radicals was also shown by Knežević et al. The polysaccharides from C. versicolor have generally demonstrated 60–90% scavenging activity, which often relates to the concentration of the extract used in the assays and also the applied in vitro method [42].

Amino acid analysis of the IPS extracts produced from different carbon sources (synthetic or renewable substrates) was also carried out in order to further elucidate their composition. Variations among the different culture conditions and substrates are evident (

Table 3. Amino acid composition (mg/g) of crude IPSs produced during SmF of T. versicolor on mixed sugar synthetic media and GPE under static and shaking conditions and Tween 80 addition.

Amino Acid Content (mg/g)	Mixed Sugars Static	Mixed Sugars Shaking	GPE Static	GPE Shaking	GPE-Tween 80 Shaking
L-aspartic acid	1.73	2.76	0.65	0.83	0.78
L-glutamic acid	5.24	4.11	3.12	2.06	1.49
L-serine	3.83	1.90	0.47	0.44	n.d
Glutamine	2.58	2.58	4.19	0.89	n.d
L-histidine	1.14	2.03	0.59	0.22	0.76
Glycine	1.78	1.32	0.45	0.29	0.35
L-threonine	2.63	2.65	0.55	0.34	0.61
L-arginine	7.11	6.22	2.67	4.69	1.48
L-alanine	2.53	1.99	1.12	0.44	n.d
L-tyrosine	1.27	1.81	0.42	0.50	n.d
L-cystine	1.82	n.d	0.32	n.d.	n.d
L-valine	n.d	n.d	0.42	0.64	n.d
L-methionine	0.47	n.d	n.d	n.d	n.d
L-tryptophan	n.d	n.d	0.11	0.13	n.d
L-phenylalanine	n.d	1.83	0.19	0.35	n.d
L-isoleucine	n.d	1.07	0.20	0.39	0.89
L-leucine	2.87	2.01	n.d.	0.51	0.82
L-lysine	4.32	2.13	0.52	0.46	n.d
L-proline	4.79	1.36	n.d.	0.58	n.d

). The amino acids L-aspartic, L-glutamic, L-histidine, glycine, L-threonine, and L-arginine were present in all tested IPS samples (Table 3). Additionally, glutamic acid and arginine were detected in significant amounts in all IPS samples, ranging from 1.49 to 5.24 mg/g and 1.48 to 7.11 mg/g, respectively.

When GPE was used as a substrate, arginine (4.69 mg/g) and glutamic acid (2.06 mg/g) were the predominant amino acids found in IPS extracts produced under continuous agitation of T. versicolor cultures. The IPSs produced under static conditions were rich in glutamine (4.19 mg/g), followed by glutamic acid (3.12 mg/g), arginine (2.67 mg/g) and alanine (1.12 mg/g). The essential amino acids histidine, threonine, valine, tryptophan, phenylalanine, isoleucine and lysine were identified in both extracts, while leucine was found only in the IPSs from the shaking cultures. The addition of Tween 80 led to the purest sample in terms of the free amino acid content, which was also in accordance with the low protein content (Table 1).

The free amino acid content could affect the potential food applications of the IPS extracts, as they are linked to taste attributes. Glutamic and aspartic acids positively contribute to taste, since they both are similar to monosodium glutamate [43]. It is worth noting that the concentration of these amino acids was above the taste detection threshold [44]. Arginine and glutamine provide bitter and sweet tastes, respectively [44]. The higher antioxidant activity of IPS extracts from static conditions —in terms of inhibition toward the DPPH radical (57.6%)— could also be partially attributed to the presence of histidine, tyrosine and cystine in higher amounts (1.33 mg/g) in comparison with the one under agitation (0.72 mg/g) [45]. Evidently, these attributes could suggest diversified end applications, specifically in the development of functional food. Studies regarding the toxicity of the extracts should be undertaken; however, based on the literature, previous research on the toxicity of hot water mycelium extracts from T. versicolor indicated the lack of toxicity [46].

Table 4. Production and characterization of crude EPSs produced during SmF of T. versicolor on GPE under static and shaking conditions and Tween 80 addition.

GPE	EPS (g/L)	IDPPH (%)	μg Trolox/mg Extract	Protein (%)	Polysaccharides (%)
Static	5.35 ± 0.28	15.12 ± 0.32	2.89 ± 0.10	8.14 ± 0.41	7.78 ± 0.23
Shaking	5.32 ± 0.32	10.91 ± 0.13	2.51 ± 0.09	7.05 ± 0.28	12.12 ± 0.16
Shaking–Tween 80	12.54 ± 0.09	7.64 ± 0.11	1.84 ± 0.03	8.12 ± 0.03	21.05 ± 0.14

presents the production and preliminary characterization of EPS obtained from static and shaking conditions using GPE and the addition of Tween. Maximum production was observed along with maximum biomass in the case of GPE, reaching 5.35 and 5.32 in static and shaking conditions. Rau et al. presented the production of 4.1 g/L of EPS using a synthetic minimal medium for the cultivation of T. versicolor with a protein content of 2–3.6% w/w [8]. Similar to IPSs, agitated conditions did not influence EPS synthesis. Several basidiomycetes were studied with respect to EPS production in submerged and solid cultures, including two Trametes sp. [47]. The authors noted that the effect of agitation is species-dependent; for instance, EPS production was not affected for T. trogii, but T. versicolor demonstrated an inverse correlation between biomass and EPS [47]. The production of EPS was profoundly enhanced in the case that GPE was supplemented with Tween 80 as an elicitor, reaching 12.5 g/L. Previous studies, performed with the strain G. lucidum and Tween addition also showed beneficial results on EPS production [33,48]. The authors noted that in the group with Tween 80 treatment, specific genes were upregulated, particularly those encoding β-1,3-glucan synthase, and three genes involved in the synthesis of chitin, but the exact mechanism of EPSs remains unclear.

Subsequently, FTIR spectra characterization was conducted for all EPS and IPS extracts derived from T. versicolor fermentation on GPE, whereas two indicative spectra for EPSs and IPSs are presented in Figure 6. EPSs deriving from static and shaking cultures demonstrated similar spectra. In particular, a broad-stretched peak from 3500 cm⁻¹ to 3000 cm⁻¹ that relates to the stretching vibration of the O-H group of the polysaccharide was observed. Actually, this specific peak was consistent in both IPS and EPS extracts, regardless of the fact that the peak was sharper in the IPS spectrum. Probably, the Amide A peak (~3300 cm⁻¹) of the protein that was extracted together with the polysaccharide could contribute to the spectra [49]. The presence of protein fraction in IPS extracts, but also EPSs, was confirmed by the protein content measured with photometric methods. In addition, all extracts presented bands in the region of 2500–3000 cm⁻¹ that can correspond to the symmetric and asymmetric stretching vibrations of skeletal CH and CH2 [50]. In the area between 1500 and 1800 cm⁻¹, particularly, the bands at 1561 cm⁻¹ and 1595 cm⁻¹ for static and shaking EPSs and also at 1587 cm⁻¹ and 1591 cm⁻¹ for static and shaking IPSs, respectively, are attributed to the asymmetric stretching of carbonyl groups [51]. Similarly, this peak appears displaced to lower wavenumbers, owing to the presence of Amide I and Amide II of the protein. The bands in the region of 1000–1300 denote the presence of polysaccharides because of the stretching of carbonyl and C-H groups [51]. The strong peak at 1054 cm⁻¹ and 1066 cm⁻¹ for EPSs, and 1051 cm⁻¹ for and 1025 cm⁻¹ for IPSs, is postulated to derive from the presence of C-O-C and-OH in pyran structures. Previous studies have noted that the bands around 1041, 1153 and 891 cm⁻¹ are typical for β-glucans [52]. The peak at ~786–790 cm⁻¹ could indicate α-linked glycosyl residues.

As noted earlier, specific peaks in the IR spectra were shifted; for instance, the Amide I and Amide II bands are often assigned to 1626 and 1529 cm⁻¹, respectively. This outcome is correlated with interactions between the protein and polysaccharide complex, leading to the displacement of some peaks. However, it is unequivocal that the IR spectra confirmed the preliminary analysis of extracellular and intracellular protein–polysaccharide complexes obtained from T. versicolor fermentation.

4. Conclusions

This study introduced the utilization of grape pomace extract as a fermentation feedstock for the development of a bioconversion process using an indigenous T. versicolor isolate. Commercial glucose, fructose and the mix of glucose and fructose were initially used to assess the fermentation profiles. Fungal biomass was successfully produced, whereas agitation had a significant effect on process productivity and sugar consumption rates. Synthetic media were then replaced with low-cost substrates from renewable resources, specifically, grape pomace. Biomass production reached 8.3 and 9.8 g/L during static and shaking conditions. The addition of Tween 80 as an elicitor enhanced biomass production, which reached 21 g/L using ~30 g/L initial sugar concentration during shaking conditions. A preliminary chemical characterization of IPSs and EPSs was implemented. The antioxidant activity, when GPE was used as fermentation media, was higher for crude intracellular polysaccharides (I_DPPH up to 57%) compared with extracellular polysaccharides (I_DPPH up to 15%). FTIR structural characterization confirmed the presence of protein and carbohydrates. Additionally, HPLC analysis of IPS extracts indicated the effect of agitation on the amino acid profile. Evidently, our results demonstrate the potential to utilize agro-industrial by-products to design cascade bioprocesses that will exploit indigenous biodiversity and generate added-value compounds that could target tailor-made end applications.