Adding-value to Ganoderma lingzhi by producing enzymes and antioxidant compounds under submerged fermentation using different culture media

Agregando valor ao Ganoderma lingzhi através da produção de enzimas e compostos antioxidantes sob fermentação submersa em diferentes meios de cultura

Abstract

Ganoderma lingzhi is widely reported for its medicinal properties, presenting several bioactive substances with potential pharmaceutical and industrial application. This study aimed to evaluate the production of mycelial biomass, extracellular enzymes and antioxidant compounds by G. lingzhi under submerged fermentation. G. lingzhi was cultured in Polysaccharide (POL) and Melin-Norkrans (MNM) media for 7 days. The cellulases, xylanases, pectinases, laccases, and proteases activities were quantified in the culture broth, while the antioxidant potential was evaluated for the mycelial biomass. G. lingzhi showed higher biomass production in MNM. However, it exhibited similar microstructural characteristics in both culture media. In the POL there was greater activity of CMCase (0.229 U/mL), xylanase (0.780 U/mL), pectinase (0.447 U/mL) and proteases (16.13 U/mL). FPase did not differ (0.01 U/mL), and laccase was detected only in MNM (0.122 U/mL). The biomass water extract from MNM showed high levels of phenolic compounds (951.97 mg AGE/100 g). DPPH^• inhibition (90.55%) and reducing power (0.456) were higher in MNM medium, while ABTS^•+ inhibition (99.95%) and chelating ability (54.86%) were higher in POL. Thus, the MNM medium was more favorable to the production of mycelial biomass and phenolic compounds, while the POL medium favored the synthesis and excretion of hydrolytic enzymes.

Keywords:
fermentation processes; cellulases; ligninases; proteases; phenolic compounds

Resumo

O Ganoderma lingzhi é amplamente conhecido por suas propriedades medicinais, apresentando diversas substâncias bioativas com potencial aplicação farmacêutica e industrial. Este estudo teve como objetivo avaliar a produção de biomassa micelial, enzimas extracelulares e compostos antioxidantes por G. lingzhi através de fermentação submersa. G. lingzhi foi cultivado em meios de Polissacarídeo (POL) e Melin-Norkrans (MNM) por 7 dias. As atividades de celulases, xilanases, pectinases, lacases e proteases foram quantificadas no caldo de cultura, enquanto o potencial antioxidante foi avaliado para a biomassa micelial. G. lingzhi mostrou maior produção de biomassa em MNM. No entanto, exibiu características microestruturais semelhantes em ambos os meios de cultura. No POL, houve maior atividade de CMCase (0,229 U/mL), xilanase (0,780 U/mL), pectinase (0,447 U/mL) e proteases (16,13 U/mL). FPase não diferiu (0,01 U/mL), e lacase foi detectada apenas em MNM (0,122 U/mL). O extrato aquoso de biomassa de MNM apresentou altos níveis de compostos fenólicos (951,97 mg AGE/100 g). A inibição do DPPH^• (90,55%) e o poder redutor (0,456) foram maiores no meio MNM, enquanto a inibição do ABTS^•+ (99,95%) e a capacidade quelante (54,86%) foram maiores no POL. Assim, o meio MNM foi mais favorável para a produção de biomassa micelial e compostos fenólicos, enquanto o meio POL favoreceu a síntese e excreção de enzimas hidrolíticas.

Palavras-chave:
processos fermentativos; celulases; ligninases; proteases; compostos fenólicos

1. Introduction

The mushroom species belonging to the Ganoderma genus are widely used in Asian culture due to their beneficial effects on human health, holding significant importance in the industrial sector and contributing to economic development (Yao et al., 2020). The specific epitope 'Ganoderma lucidum' was long erroneously used to describe the Lingzhi mushroom widely cultivated in Asia. However, it is currently known that this actually corresponds to G. sichuanense and G. lingzhi, which have been used as binomials for the species (Du et al., 2023).

G. lingzhi has been described with significant potential in the bioconversion of lignocellulosic residues, owing to the action of lignocellulolytic enzymes produced and excreted by fungal hyphae during their development (Gouvêa et al., 2023). Lignocellulolytic enzymes are carbohydrate-active enzymes (CAZy) and hold considerable importance in the organism's carbohydrate metabolism, being highly active during mycelial development, with their synthesis varying according to the substrate used as a source for fungal growth (Zhou et al., 2018).

The Ganoderma genus arouses significant interest due to its potential for developing new antioxidants with low toxicity and possible pharmaceutical applications (Wang et al., 2019). In general, antioxidant activities of this mushrooms are direct associated to phenolic compounds (Kiss et al., 2021). When studied G. lingzhi basidiomata showed a high antioxidant capacity of G. lingzhi mycelial biomass from submerged fermentation, with large amounts of polyphenols, including flavonoids, and presence of ascorbic acid and superoxide dismutase, reflecting in a remarkable scavenging activity against hydroxyl, superoxide, DPPH^• and ABTS radical, as well as strong ferric reducing power and ferrous ion chelating activity (Meng et al., 2018).

Submerged fermentation technology indeed presents a promising alternative for obtain valuable primary and secondary fungal metabolites (Chicatto et al., 2014), standing out from solid fermentation due to its more compact space and shorter incubation time, compensating the limited availability of fungal resources, since the fruiting bodies of G. lingzhi require a long time to grow and are rare in nature (Si et al., 2019). Recently, our research group reported the potential of G. lingzhi CC22 strain in producing micelial biomass via submerged fermentation using basal GYP medium, with variations in enzyme content according to the concentration of the cultivation medium components (Pessoa et al., 2023). Hence, the objective of this study was to conduct submerged fermentation of G. lingzhi using different culture media to compare the excretion of enzymes with biotechnological interest and the production of mycelial biomass with antioxidant activity.

2. Materials and Methods

2.1. Submerged fermentation

Ganoderma lingzhi CC22 (=Ganoderma sichuanense) was reactivated and maintained in Petri dishes containing potato dextrose agar (PDA) medium and incubated at 28°C. Submerged fermentations were carried out in Polysaccharide medium (POL) and Melin-Norkrans Medium (MNM). The POL medium consisted of (NH₄)₂SO₄ (5.0 g/L); MgSO₄.7H₂O (0.2 g/L); K₂HPO₄ (1.0 g/L); yeast extract (2.0 g/L); soy peptone (1.0 g/L); CaCO₃ (1.0 g/L); sucrose (40.0 g/L), pH 5.5 (Cavazzoni and Adami, 1992). The MNM medium was composed of sucrose (20.0 g/L); (NH₄)₂HPO₄ (0.93 g/L); KH₂PO₄ (0.5 g/L); K₂HPO₄ (0.7 g/L); CaCl₂.2H₂O (0.05 g/L); NaCl (0.025 g/L); MgSO₄.7H₂O (0.150 g/L); 1.2 mL of FeCl₃ (1%); 100 µL of Thiamine-HCl; 0.1% Brazil nut extract; malt extract (3.0 g/L), pH 5.5 (Marx, 1969).

In Erlenmeyers (250 mL) containing 100 mL of each medium, 5 mycelium discs (Ø = 7 mm) were inoculated and incubated in a shaker (Thermo SCIENTIFIC/MaxQ 6000) at 120 rpm, 28 ± 2ºC, in the absence of light for 7 days. After cultivation, the fermented broth and mycelial biomass were separated by centrifugation at 3,857 × g (Thermo SCIENTIFIC, SORVALL LYNX 4000) for 20 minutes at 4ºC. The culture broth was stored at -20ºC until enzymatic analysis. The mycelial biomass was washed with distilled water and lyophilized for later extraction of bioactive compounds.

At the end submerged fermentation, the fermented broths were submitted to pH determination using a pH meter (Tecnal®, Brazil) (Lutz, 2008). Mycelial biomass production was estimated gravimetrically, in a drying oven at 70ºC.

2.2. Mycelial biomass morphology

The morphological characteristics of the mycelial biomass were recorded photographically, using a cell phone, and ultrastructurally by scanning electron microscopy (SEM) (Jeol JSM IT500HR). For the SEM analyses, at the end of the fermentation process, the mycelia were collected and washed three times with distilled water, with subsequent fixation for 24 hours at room temperature (25°C) in modified Karnovsky solution (2% formaldehyde and 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 7.4). After fixation, the samples were submitted to 4 successive washes in sodium cacodylate buffer (0.2 M, pH 7.4) and post-fixed for 2 hours in a solution of 1% osmium tetroxide and 0.8% potassium ferrocyanide (1:1, v/v) at room temperature (25°C).

After this period, the excess post-fixation solution was washed 3-times with the same buffer used in the previous steps. Subsequently, the samples were dehydrated in a gradual series of ethanol diluted in distilled water (30%, 50%, 70%, 80%, 90% and 3x 100%), for 20 min each. Next, the mushroom was submitted to Leica EM CPD300 critical point drying and subsequently adhered to carbon tapes on aluminum stubs and subjected to gold metallization in the JEOL Smart Coater (∼20 nm) (Souza, 2007). The micrographs were obtained at different magnification, through a scanning electron microscope (Jeol JSM IT500HR), using a voltage acceleration of 5kV, in secondary electron modes.

2.3. Enzymatic activities in the culture broth

The FPase (exoglucanase), CMCase (endoglucanase), xylanase and pectinase activities were determined using the substrates Whatman nº 1 quantitative filter paper, carboxymethylcellulose (1% w/v), xylan (1% w/v) and citrus pectin (1% w/v), respectively (Ghose and Bisaria, 1987). Activities were estimated from the quantification of reducing sugars using dinitrosalicylic acid (DNS) (Miller, 1959). Results were expressed in activity units per mL of extract (U/mL), where U was defined as the amount of enzyme required to release 1.0 mmol of reducing sugars.

Laccase activity was determined according to the methodology proposed (Wolfenden and Willson, 1982). from the oxidation of 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in spectrophotometric (SYNERGY H1, BioTek, USA) readings at 420 nm, for 90 seconds at 5-second intervals. The laccase activity results were expressed in activity units per mL of extract (U/mL), where U is the amount of enzyme capable of oxidizing 1.0 µM of substrate per minute.

Proteolytic activity was determined according to the methodology (Hamada et al., 2017). Initially, the samples were incubated with 12 µL of DTT (dithiothreitol, 3mM, Sigma-Aldrich) and EDTA (ethylenediaminetetraacetic acid, 2mM, Sigma-Aldrich), and 108 µL of sodium acetate buffer (50 mM, pH 5.0) for 10 minutes at room temperature (25 ± 2ºC). Subsequently, 60 µL of azocasein (1% w:v) were added, followed by incubation for 1 hour at 37ºC. After this period, the reaction was stopped by the addition of 90 µL of TCA (trichloroacetic acid, 20%). The reaction product was centrifuged at 8,680 × g for 10 minutes. The supernatants were basified with NaOH (sodium hydroxide, 2 M) and read spectrophotometrically (SYNERGY H1, BioTek, USA) at 420 nm. The results were expressed in units of activity per mL of extract (U/mL), where U was defined as the amount of enzyme capable of increasing the absorbance by 0.01 units.

2.4. Extractions of antioxidants

The mycelial biomass was crushed and subjected to extraction in water (1:10 w/v), according to the methodology (Narasimhan et al., 2013). Subsequently, the extracts were lyophilized (LÍOTOP, K108) and resuspended at a concentration of 10 mg/mL to carry out assays of quantification of metabolites and antioxidant activity.

2.5. Total Phenolic Compounds (TPC)

The content of total phenolic compounds in the extracts from biomass was quantified (Pires et al., 2017). In this assay, 20 μL of extracts, 200 μL of water, 20 μL of Folin-Ciocalteu, and 60 μL of sodium carbonate solution (10%) were added to a 96-well microplate. The reaction mixture remained at rest for 30 min, in the dark, followed by spectrophotometric (SYNERGY H1, BioTek, USA) readings at 760 nm. Gallic acid was used as a standard and the results were expressed in milligrams of Gallic Acid Equivalence (GAE) per 100 g of sample.

2.6. Flavonoids

Flavonoid content in the extracts from biomass was determined (Chang et al., 2012). The sample (25 μL) was homogenized in water (152.5 μL) and sodium nitrite (5%, 7.5 μL). After 6 minutes, aluminum chloride (10%, 15 μL) was added, and incubated for 5 minutes at room temperature. Then, sodium hydroxide (1 M, 50 μL) was added and the reaction mixture was incubated for 15 minutes at room temperature. The absorbance was read at 510 nm in a spectrophotometer (SYNERGY H1, BioTek, USA) and the results were obtained based on a quercetin standard curve.

2.7. Antioxidant activities

The antioxidant activity of the extracts from biomass was determined by the methods of DPPH^•, ABTS^•+, chelating ability and reducing power, according to methodologies adapted for 96-well microplates (Khatua et al., 2017). For Inhibition of DPPH^•, 180 μL of DPPH^• (2,2-diphenyl-1-picrylhydrazyl, 4 mg/mL) were incubated with 20 μL of extracts for 30 minutes in the dark. The absorbances were obtained by spectrophotometric (SYNERGY H1, BioTek, USA) readings at 595 nm. Results were expressed in percent inhibition.

In the ABTS^•+ (2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical inhibition method, a stock solution was prepared by the reaction of ABTS (7 mM) and potassium persulfate (2.45 mM) for 16 hours, in the dark at room temperature. Then, the solution was diluted in ethanol until the absorbance between 0.8 and 1.0 at 754 nm. The assay consisted of incubating 180 μL of ABTS^•+ with 20 μL of extracts for 5 minutes, in the dark, followed by absorbance reading at 754 nm (SYNERGY H1, BioTek, USA). Results were expressed in percent inhibition.

The chelating ability on the Fe²⁺ ion was performed by incubating 100 μL of the samples with 5 μL of iron II chloride (3 mM), 10 μL of ferrozine (0.12 mM) and 85 μL of distilled water, for 15 minutes, followed by spectrophotometric readings at 562 nm. Results were expressed in percent inhibition.

To determine the reducing power, 10 μL of the samples were incubated with 25 μL of phosphate buffer (0.2 M, pH 6.6) and 25 μL of potassium ferrocyanide (1%) for 20 minutes at room temperature (25 ± 2ºC). Subsequently, 25 μL of trichloroacetic acid (10% v/v), 85 μL of distilled water and 8.5 μL of iron III chloride were added, followed by incubation for 15 minutes and spectrophotometric reading at 750 nm. The results were expressed in absorbance units.

2.8. Statistical analysis

Enzymatic and antioxidant activity assays were performed in triplicate. The results were submitted to analysis of variance (ANOVA) and means were compared by Tukey’s test at 5% of probability, using the statistical program Statistica v. 7.0 (Statsoft, USA).

3. Results

3.1. pH and mycelial biomass production

The POL culture medium presented a greater pH stability throughout the fermentation, with a low reduction at the end of the cultivation (7^th day), while the MNM exhibited a considerable acidification (Figure 1). The production of mycelial biomass was significantly different between the culture media, where in MNM (1.35 ± 0.02 g/100 mL) there was a biomass production about 3 times higher than in POL (0.42 ± 0.01 g/100 mL) (Figure 1). These results highlight the potential of the MNM medium in the production of Ganoderma lingzhi biomass.

Figure 1
Final (7^th day) pH and mycelial biomass production in the submerged fermentation of Ganoderma lingzhi in POL and MNM media. Capital letters compare biomass values and lowercase letters represent pH.

3.2. Morphological characterization

After fermentation of G. lingzhi in POL and MNM culture media, a variation in the morphology of agglomerates of mycelia in suspension, called pellets, was observed (Figure 2 and 3). It is also possible to highlight rheological and color variation of the biomass and the culture medium after fermentation (Figure 2 and 3).

Figure 2
Scanning electron micrograph of the mycelial biomass of Ganoderma lingzhi cultivated in POL medium. (A) crystals (red arrow); (B) connecting clamp (circle); (C) chlamydospore-like structures (orange arrow).

Figure 3
Scanning electron micrograph of the mycelial biomass of Ganoderma lingzhi cultivated in MNM medium. (A) crystals (red arrow); (B) connecting clamp (circle); (C) chlamydospore-like structure (orange arrow).

From the scanning electron micrograph of the mycelial biomass, similar microstructures were also observed between the culture media, such as tubular, long and smooth hyphae, structures similar to calcium oxalate crystals (Figures 2A and 3A) and clamp connection (Figure 2B and 3-B). The main morphological difference observed, depending on the nutrient media, is the presence of spherical structures, where in the POL medium they have a smooth and irregular surface (Figure 2C). In the MNM medium, small ornate structures were found in anamastosis (Figure 3C).

3.3. Enzymatic activities in the culture broth

G. lingzhi, in general, showed higher enzymatic activities in submerged fermentation in POL medium, with statistically different activities for CMCase, xylanase and pectinase enzymes. However, in this experimental condition, no laccase activity was observed. Among the evaluated enzymes, G. lingzhi showed the greatest potential for protease production, with the activity of this enzyme being about 3 times higher in POL medium, compared to MNM medium (Table 1).

Table 1
Enzymatic activities in cultivation broth of G. lingzhi cultivated in submerged fermentation with POL and MNM culture media.

Enzymes (U/mL)	Culture Media
	POL	MNM
CMCase	0.23 ± 0.10b	0.28 ± 0.12a
FPase	0.01 ± 0.00a	0.01 ± 0.00a
Xylanase	0.78 ± 0.03a	0.34 ± 0.04b
Pectinase	0.45 ± 0.01a	0.17 ± 0.02b
Laccase	N.D.	0.12 ± 0.09a
Proteases	16.13 ± 4.64a	4.80 ± 1.40b

• Results expressed as mean ± standard deviation (n = 3). Different letters show the statistical difference according to Tukey’s test (p ≤ 0.05) between the culture media evaluated. N.D.: Not detected.

Low FPase activity was observed in this study for both tested media and G. lingzhi showed higher xylanase activity in POL medium, about approximately 56% higher than in MNM medium. Regarding laccase activity, the POL medium completely inhibited the production of this enzyme by G. lingzhi (Table 1).

3.4. Antioxidant activity of mycelial biomass

The content of total phenolic compounds (TPC) in the biomass of G. lingzhi after 7 days of submerged fermentation showed differences according to the medium used, with MNM standing out, with was 32% higher than the POL medium. In contrast, flavonoids were not observed in the mycelial biomass from the submerged fermentation of G. lingzhi in the two evaluated media (Figure 4).

Figure 4
Total phenolic compounds content and antioxidant activity of Ganoderma lingzhi biomass produced in submerged fermentation in POL and MNM media. Each point (sample unit) represents a method for detecting antioxidant activity. * Indicates the culture medium with the highest activity values in most assays.

As for the reducing power, the mycelial biomass extracts from the MNM medium showed an absorbance value about 60% higher than that obtained in the POL medium (Figure 4). The chelating effects of the mycelial biomass extracts of G. lingzhi in POL and MNM medium were similar, with values in the order of 50% (Figure 4).

The free radical-scavenging activity was evaluated by two methods, DPPH^• and ABTS^•+, and the results indicate that the extracts obtained from the mycelial biomass of the POL and MNN media present a potent action in radical neutralization. The scavenging capacity of water extracts of G. lingzhi was high, both in POL and MNM medium, with ABTS^•+ activities being about 16% and 5% higher than those observed in DPPH^•, respectively (Figure 4).

4. Discussion

The pH of the medium is a vital intrinsic factor, as it affects the ionic state of the fermentative microenvironment, as well as the structure, morphology and physiological functions of fungal cells, and can modulate nutrient uptake and products biosynthesis (Dulay et al., 2021). One hypothesis for the pH reduction in the MNM medium could be due to the metabolic by-products released during the exponential growth phase of the mushroom mycelium. A previous study described that the decrease in pH can be attributed to the H⁺ ions released into the substrate or culture medium as a by-product of ammonia metabolism (Deacon, 2013). This feature was also observed when sodium nitrate and potassium sulfate were used as nitrogen sources for the growth of mycelial biomass of Pleurotus albidus (Kirsch et al., 2016). The reduction in pH at the end of the submerged fermentation of G. lucidum may be associated with the relatively high consumption of glucose, resulting in the production of certain organic acids (Fang and Zhong, 2002).

Previous studies where G. lucidum was produced in submerged fermentation in Potato Dextrose broth (PD) and obtained a maximum mycelial production of 5.22 g/L on the 6^th day (Asadi et al., 2021). The maximum production of G. lucidum mycelial biomass was 8 g/L on the 14^th day of submerged fermentation using seed medium (SM) (Wu et al., 2021). As observed for G. lingzhi grown in MNM medium in the present work there was a greater production of mycelial biomass and considerable pH reduction, possibly due to the production of organic acids (Amache et al., 2019).

The formation of pellets can be influenced by the composition of the culture medium and they are categorized as coagulative or non-coagulative. In the first case, the formation of aggregates occurs, while in the second, the development occurs independently, and the formation of pellets can occur even by a single spore (Veiter et al., 2018; Yang et al., 2009). The formation of pellets can be influenced by the composition of the culture medium and they are categorized as coagulative or non-coagulative. In the first case, the formation of aggregates occurs, while in the second, the development occurs independently, and the formation of pellets can occur even by a single spore (Veiter et al., 2018; Yang et al., 2009).

Thus, it is suggested that in the POL medium the formation of biomass occurred in a non-coagulative way, with a smaller average size of the pellets, while in the MNM medium the formation of biomass occurred in a coagulative form, showing pellets with a larger average size (Supramani et al., 2019) when working with the species G. pfeifferi in submerged fermentation, also verified variation in the morphology of the pellets according to the cultivation conditions, including variation in the composition of the culture medium and pH.

The structure of calcium oxalate crystals and clamp connections are produced by basidiomycetes to form the dikaryotic mycelial state, already reported in other species of the genus Ganoderma (Antinori et al., 2021; Rees et al., 2012). The morphological difference observed in the POL medium resembles the cuticular cells which are report as structures originate from the clamp connections of G. lucidum (Adaskaveg and Gilbertson, 1986). As for the MNM medium, small ornate structures were found in anastomosis, similar to vacuoles found in the submerged cultivation of G. resinaceum under conditions of osmotic stress induced by sodium chloride, raising the hypothesis that these structures might be those, given the variety of salts in the MNM medium (Mahmoud et al., 2007).

Low FPase activity was observed in this study (0.01 U/mL) (Table 1) for both tested media this fact may be associated with the production of other cellulolytic enzymes by G. lingzhi. Furthermore, it is important to emphasize that this enzyme substrate (filter-paper) is insoluble, which may justify the lower activities when compared to other enzymes. The extracellular extracts from different fungi showed a low capacity of FPase to convert the filter-paper substrate into reducing sugars (Santos et al., 2017). Thus, FPase activity is associate as total cellulase being lower than CMCase (endocellulase activity) one.

A previous study verified a CMCase activity of 3.8 U/mL in the submerged fermentation of Ganoderma lucidum (strain 477), using 4% olive tree sawdust as a substrate (Elisashvili et al., 2017). Thus, it is suggested that the use of lignocellulosic residues as supplementation of carbon sources, with high contents of cellulose and hemicellulose, may favor the excretion of CMCase in submerged fermentation (Elisashvili et al., 2017).

Xylanase is an enzyme of great industrial interest, being applied in the food industry, biotechnological research and the manufacture of animal feed and bioethanol (Ravindran et al., 2018). G. lucidum (strain 477) cultivated in submerged fermentation with the addition of 4% olive tree sawdust to the culture medium presented a xylanase activity of 4.7 U/mL (Elisashvili et al., 2017), reinforcing the positive influence of adding sawdust as a supplement in submerged fermentation, especially when directed towards the production of lignocellulosic fungal enzymes.

Pectinase is another enzyme of great economic interest, with application in several bioprocess in the pharmaceutical and food industry, whose production has already been reported for basidiomycetes (Araújo et al., 2021; Haile and Ayele, 2022). In a study with G. lucidum, a maximum peak of pectinase activity of 83.99 U/mL was obtained on the 4^th day of submerged fermentation (Zhang et al., 2017). However, it is important to note that the authors used inducing substrates (sugarcane bagasse and soy by-product) leading to infer their necessity in the production of these enzymes under submerged fermentation conditions (Zhang et al., 2017).

When evaluating the optimization of laccase production by G. lucidum in two different culture media, report a maximum activity of this enzyme on the 4^th day of submerged fermentation (23.36 U/L or 0.023 U/mL) in PD medium (Potato Dextrose), while in EPM medium (Enzyme-Producing) the maximum activity was on the 8th day, with a value of 240.92 U/L (0.240 U/mL), twice the value found in the present study (Qin et al., 2019). Therefore, the composition of the medium and the ability of the fungi to use available essential nutrients can positively or negatively influence enzyme production (Fang et al., 2015).

The production of proteases by fungi has advantages in comparison to other organisms and are described as directly linked to the availability of carbon and nitrogen in the culture medium (Magalhães et al., 2019; Naureen et al., 2022). G. lucidum cultured in GYP (Glucose-Yeast-Peptone) liquid medium exhibited proteolytic activity of 23.50 U/mL, indicating that organic nitrogen sources may be more efficient for the synthesis of these enzymes, corroborating with the proteolytic activity observed for G. lucidum in the POL medium in the present study (Martim et al., 2017).

The presence of flavonoids in mushrooms presents conflicts in the literature, as a previous study asserts that mushrooms are not capable of synthesizing this chemical class due to the absence of enzymes involved in the metabolic pathway of flavonoid synthesis (Gil-Ramírez et al., 2016). However, some studies quantified flavonoids in the basidiocarp of G. lucidum from cultivation in solid-state fermentation and collected in nature (Essien et al., 2015; Rahman et al., 2020), indicating that submerged fermentation may not be favorable for flavonoid synthesis.

A previous study demonstrated that G. lucidum in submerged fermentation in a medium containing malt extract for 6 days produced TPC of 47.1 ± 1.31 mg EAG/100 g (Xie et al., 2020). These authors associated this low value with the oxidation of phenols by peroxidases and laccases secreted in the culture medium (Xie et al., 2020), corroborating with another study where they reported the correlation between the reduction of total phenolic compounds and laccase activity (Ntougias et al., 2015). Additionally, phenolic compounds can be taken up by the fungus during its development and used as a source of carbon and energy (Elisashvili et al., 2017). In these cases, obtaining phenolic compounds directly from biomass may be preferable to obtaining components from the culture medium (Elisashvili et al., 2017).

The activity of extracts in capturing DPPH^• and ABTS^•+ radicals is dependent on biochemical factors, such as the presence of phenolic compounds, which donate electrons for radical stabilization (Mohsin et al., 2011). Thus, it is suggested that the phenolic compounds present in G. lingzhi extracts are involved in the donation of hydrogens and electrons.

Extracts from Ganoderma lingzhi are described as important matrices in terms of reducing power (Lin et al., 2015), and this activity is associated with the ability of the compounds present in these extracts to donate hydrogen (Mau et al., 2002). Thus, extracts that have a higher concentration of hydrogen donor compounds, such as phenolic compounds, contribute to breaking the chain reaction of radicals.

Ferrous ions such as Fe²⁺ are known as pro-oxidants and are present in several biological systems and in foods, highlighting the importance of chelating agents in the industries (Yamauchi et al., 1988). Thus, the study of different culture media in mushroom fermentation to obtain biomolecules is necessary, since different media can directly influence the metabolic pathways that synthesize substances of interest, such as phenolic compounds (Saltarelli et al., 2015; Sharma et al., 2019).

5. Conclusions

The POL and MNM culture media induced differently the production of bioactive compounds by Ganoderma lingzhi, with the POL medium being more suitable for the synthesis of hydrolytic enzymes, while MNM stimulated greater production of biomass and phenolic compounds. However, both media provided high antioxidant activity.

Acknowledgements

The authors are grateful to the Laboratório de Cultivo de Fungos Comestíveis do Instituto Nacional de Pesquisas da Amazônia (LCFC-INPA) and to the Centro Multiusuário para Análise de Fenômenos Biomédicos da Universidade do Estado do Amazonas (CMABio-UEA) for their structure and technical support. To the Programa de Pós-Graduação em Biotecnologia (PPGBIOTEC) and the Programa de Pós-Graduação em Biodiversidade e Biotecnologia da Rede Bionorte (PPGBIONORTE) for the opportunity to carry out the doctorate. To the Conselho Nacional de Desenvolvimento Científico e Técnico (CNPq) (Process number 141036/2022-2 and DTI-A 382922/2024-7), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Process number 88887.941186/2024-00) and Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) (Process number Resolution No. 005/2022-CD/FAPEAM and No. 002/2023-CD/FAPEAM) for awarding research scholarships and doctoral scholarships. To the FAPEAM Project (PROSGRAD/2022-2023/PPGBIOTEC) and to the FAPEAM – Universal Amazonas Project (Process No. 062.00143/2020), for funding the publication and the research.

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Publication Dates

• Publication in this collection
  07 Oct 2024
• Date of issue
  2024

History

• Received
  28 Feb 2024
• Accepted
  20 Aug 2024