Cultivated Mushrooms: A Comparative Study of Antioxidant Activity and Phenolic Content ^†

Abstract

This study compares the total phenolic content (TPC) and antioxidant activity (DPPH, ABTS, and FRAP assays) of methanolic extracts from five cultivated mushroom species: Lentinula edodes (LE), Pleurotus ostreatus (PO), Agaricus bisporus (AgW and AgB), and Hericium erinaceus (HE). Phenolic profiles were analyzed by HPLC. AgW demonstrated the highest TPC (46.2 mg GAE/100 g FW) and superior antioxidant activity in DPPH and FRAP assays, highlighting its phenolic compounds as the primary contributors. Catechin and hydroxybenzoic acid were identified as species-specific phenolics in PO and AgW, respectively, contributing to their antioxidant potential. These findings underscore the diversity of antioxidant mechanisms in mushrooms and their potential as natural sources of bioactive compounds.

1. Introduction

Mushrooms, the reproductive fruiting bodies of macro-fungi, belong to the phyla Ascomycota and Basidiomycota. For thousands of years, they have been utilized in traditional medicine due to their production of potent nutraceutical compounds that provide both therapeutic and dietary benefits [1,2].

The global mushroom industry encompasses edible, medicinal, and wild mushrooms. While Lentinula edodes (shiitake) is the most widely cultivated species, Pleurotus ostreatus (oyster mushrooms) and Agaricus bisporus (button mushrooms) also hold significant positions in the edible mushroom market due to their culinary versatility and nutritional profile [3]. Among edible species, Hericium erinaceus (Lion’s Mane), a medicinal and edible mushroom with a long history of use in traditional Chinese medicine for alleviating epigastric pain and gastrointestinal disorders, stands out. This species contains over 70 bioactive compounds, including β-glucans, erinacins, and hericenones, which collectively exhibit antibiotic, anticancer, antioxidant, and neuroprotective properties [4,5].

Mushrooms are renowned for their rich composition of bioactive metabolites, which provide a wide range of pharmacological benefits, particularly their strong antioxidant activity. Among these compounds, phenolic compounds—such as hydroxybenzoic acids—are notable for their therapeutic effects, largely attributed to their ability to neutralize free radicals and reduce oxidative damage [6]. Additionally, terpenoids, including sterols like ergosterol, further enhance the biochemical profile of mushrooms and contribute significantly to their antioxidant potential [7].

Given their diverse biochemical composition and health-promoting properties, mushrooms have garnered significant attention in scientific research. Their antioxidant potential is particularly valued for its role in reducing oxidative stress, a factor associated with aging and chronic diseases such as cardiovascular disorders, diabetes, and neurodegenerative conditions [8,9]. While numerous studies have evaluated individual species or focused on medicinal varieties, comparative analyses of cultivated edible mushrooms remain relatively scarce, particularly those employing advanced methodologies to correlate phenolic profiles with antioxidant capacities. This manuscript contributes to addressing these gaps by presenting a comprehensive comparative analysis of five commonly cultivated mushroom species—Lentinula edodes, Pleurotus ostreatus, Agaricus bisporus (white and brown varieties), and Hericium erinaceus—with a focus on their total phenolic content (TPC) and antioxidant activity as determined by multiple assays (DPPH, ABTS, and FRAP). In particular, this study leverages high-performance liquid chromatography (HPLC) to provide a detailed phenolic profile, identifying species-specific phenolics such as catechin and hydroxybenzoic acid and correlating these profiles with antioxidant performance.

2. Material and Methods

The five mushroom species selected for this study—Lentinula edodes (LE), Pleurotus ostreatus (PO), Hericium erinaceus (HE), and Agaricus bisporus (white and brown varieties, AgW and AgB, respectively)—are widely cultivated and highly consumed globally. They were chosen for their broad availability, rich nutritional profiles, and unique biochemical properties, as frequently highlighted in the literature. The mushrooms were obtained from “Serra da Lua–Cogumelos” and prepared for analysis to ensure consistency across samples. Upon arrival at the laboratory, the mushrooms were gently cleaned with paper towels to remove residual dirt, and three independent samples were constituted.

2.1. Preparation of Methanolic Extracts and Total Phenolic Content

Methanolic extracts were prepared by mixing 2.5 g of mushrooms with 10 mL of methanol for each sample. The total phenolic content (TPC) was determined using a modified Folin–Ciocalteu method. Briefly, 150 μL of the extract was diluted with 2400 μL of nanopure water in test tubes, followed by the addition of 150 μL of 0.25 N Folin–Ciocalteu reagent. After a 3-min incubation, 300 μL of 1 N Na₂CO₃ was added. The final mixture was incubated for 2 h at room temperature in the dark, and absorbance was measured at 725 nm using a JASCO V-530 UV/VIS spectrophotometer (Jasco International, Tokyo, Japan). TPC values were calculated based on a gallic acid calibration curve (R² = 0.992, y = 3.34x + 0.099) and expressed as milligrams of gallic acid equivalents per 100 g of fresh weight (mg GAE/100 g fw).

2.2. Antioxidant Capacity Analysis

Antioxidant activity was assessed using DPPH, ABTS, and FRAP assays, following modified protocols from Yu et al. [10].

DPPH Assay: A stock solution was prepared by dissolving 24 mg of DPPH in 100 mL of methanol and diluted to achieve an absorbance of 1.1 ± 0.02 at 515 nm. In each cuvette, 2850 μL of the DPPH solution was mixed with 150 μL of Trolox standard, mushroom extract, or methanol (blank). After 2 h, absorbance was measured at 515 nm. Results were expressed as micromoles of Trolox equivalents per 100 g of fresh weight (μmol TE/100 g fw).

ABTS Assay: ABTS solution was prepared by mixing equal volumes of 4.8 mM potassium persulfate and 14 mM ABTS, incubated in the dark for 16 h at room temperature, and adjusted to an absorbance of 1.1 ± 0.02 at 734 nm. A volume of 2850 μL of the ABTS solution was mixed with 150 μL of the sample or standard. After 2 h in the dark, absorbance was measured at 734 nm, with results expressed as μmol TE/100 g fw.

FRAP Assay: For the Ferric-Reducing Antioxidant Power assay, a FRAP solution was prepared by mixing acetate buffer (0.3 M, pH 3.6), TPTZ (10 mM in 40 mM HCl), and ferric chloride (20 mM) in a 10:1:1 ratio. Then, 0.2 mL of sample, FeSO₄·7H₂O standard, or distilled water (blank) was added to the cuvette, followed by 1.8 mL of the FRAP solution. The mixtures were incubated in the dark for 30 min, and absorbance was measured at 593 nm. Results were expressed as mmol of FeSO₄·7H₂O per 100 g fw.

2.3. HPLC Analysis

For the quantification and identification of the phenolic compounds, an HPLC system (Waters, Milford, MA, USA) was used. This system includes quaternary pumps and an automatic injector with vials maintained at a temperature of 5 °C (Waters Alliance 2695) and a column oven set to 30 °C. Detection was carried out with a Waters 2996 photodiode array (PDA) detector, covering the wavelength range of 200 to 600 nm. The separation was performed using a Synergy Hydro column (Phenomenex, Torrance, CA, USA) according to the process detailed by Pereira et al. [11].

Quantitative analysis of phenolic compounds was performed by constructing calibration curves using standard solutions of known concentrations, measured at the wavelengths corresponding to the maximum absorption of each compound. For qualitative identification, the retention times of analyte peaks in the samples were compared to those of the respective standards.

2.4. Statistical Analysis

All analyses were conducted in triplicate. Data were analyzed using one-way analysis of variance (ANOVA) with Tukey’s HSD test (p < 0.05) to identify significant differences between mushroom properties at all test intervals. Results are presented as means ± standard deviation (SD). Statistical evaluations were performed using Statistica™ V8.0 software [12]. For evaluation of the possible correlation between phenolic content and the free radical activities of the extracts, Pearson’s correlation analysis was carried out.

3. Results

In the present study, the Total Phenolic Content (TPC) of the mushroom samples ranged from 22.3 to 46.2 mg GAE/100 g fw (Figure 1). Among the evaluated species, Agaricus varieties exhibited the highest TPC values, while LE showed a significantly lower phenolic content (p < 0.05). The results for PO and HE showed no significant differences between them. These findings align with the study by Reis et al. [13], which also reported that Agaricus white and brown varieties exhibited the highest TPC, while LE presented the lowest value, supporting the consistency of our results.

Antioxidant activity can vary based on the method and the radical or oxidant sources used. To capture this diversity, the antioxidant activity of the samples was evaluated using three methods: FRAP, ABTS, and DPPH (Figure 2).

The AgW extract demonstrated the highest antioxidant potential, with values of 3093.3 ± 138.2 µmol TE/100 g fw (DPPH) and 2.3 ± 0.3 mmol FeSO₄·7H₂O/100 g fw (FRAP), which is consistent with its high phenolic content. A strong correlation was observed between TPC and antioxidant activity measured by DPPH (r = 0.85; p < 0.05), reinforcing the role of phenolic compounds in scavenging DPPH radicals across all samples.

Interestingly, the ABTS assay revealed a different trend, with HE (7708.7 μmol TE/100 g fw) and PO (5980.9 μmol TE/100 g fw) outperforming the other species. This suggests the presence of additional non-phenolic compounds in these species that contribute significantly to their antioxidant activity.

For HE, the phenolic content was 35.8 ± 3.5 mg GAE/100 g fw, while its antioxidant activity values were 2248 ± 71.7 µmol TE/100 g fw, 378.6 ± 20.6 µmol TE/100 g fw, and 0.83 ± 0.2 mmol FeSO₄·7H₂O/100 g fw for the DPPH, ABTS, and FRAP methods, respectively. These results highlight its moderate phenolic content and the potential influence of non-phenolic compounds on its antioxidant activity. The correlations between the total phenolic content (TPC) method and the three antioxidant activity measurement methods vary depending on the type of mushroom. For instance, a positive correlation was observed between TPC and DPPH for AgW (r = 0.74) and LE (r = 0.83) mushrooms. The species PO presented a positive correlation between TPC and DPPH (r = 0.93), ABTS (r = 0.80), and FRAP (r = 0.91). Conversely, no correlations were observed for AgB and HE mushrooms.

Phenolic compounds, including gallic acid, protocatechuic acid, catechin, caffeine, caffeic acid, hydroxybenzoic acid, vanillic acid, chlorogenic acid, and coumaric acid, were identified in certain mushroom species (Figure 3). The presence of these compounds has also been described by Ferreira et al. [14]. Among these, gallic acid was detected in all samples, with the most abundance in the AgW sample (6.1 mg/100 g fw) and less abundant in LE (1.0 mg/100 g fw). Consistent with previous findings by Reis et al. [13], gallic acid was confirmed as the major phenolic component in AgW. Catenin was only found in PO samples (7.5 mg/100 g fw). Similarly, hydroxybenzoic acid (3.4 mg/100 g fw) and vanillic acid (3.6 mg/100 g fw) were only found in AgW.

4. Discussion

The bioactive composition of mushrooms is highly variable, influenced not only by species differences but also by factors such as cultivation environment and substrate composition [15]. As a result, variations are often observed between studies.

This study highlights a nuanced relationship between total phenolic content (TPC) and antioxidant activity across mushroom species, with significant variability depending on the assay method and mushroom type. These findings underscore the complexity of antioxidant mechanisms in biological systems and the diverse roles of bioactive compounds.

The phenolic composition varied notably among species. Gallic acid emerged as the predominant phenolic across all samples. The AgW sample, which exhibited the highest gallic acid concentration, demonstrated superior DPPH and FRAP activity, further reinforcing the strong correlation between phenolic content and antioxidant efficacy.

Interestingly, catechin was detected exclusively in PO, potentially accounting for its robust antioxidant activity across all assays. Similarly, hydroxybenzoic acid and vanillic acid were unique to AgW, contributing to its potent radical scavenging properties. These findings emphasize the diversity in phenolic profiles among mushroom species and their distinct contributions to antioxidant potential [16].

Interestingly, ABTS assay results revealed that HE outperformed other species, despite having moderate phenolic content. This suggests the significant contribution of non-phenolic bioactive compounds, such as β-glucans and terpenoids, which are known for their antioxidant properties [17]. These observations align with prior research highlighting the multifaceted nature of antioxidant mechanisms in mushrooms.

Overall, among the mushrooms analyzed, AgW displayed the highest overall antioxidant potential, highlighting its promise as a valuable source of bioactive compounds. Incorporating mushrooms like AgW into the diet may offer significant health benefits due to their strong antioxidant properties.

The novelty of this study lies in its multi-dimensional approach to elucidating the phenolic–antioxidant relationship across cultivated mushrooms, addressing inconsistencies observed in earlier research. Previous studies have often evaluated antioxidant activity using isolated methodologies, neglecting the complex interplay of phenolic and non-phenolic bioactive compounds. By integrating diverse antioxidant assays and correlating them with detailed phenolic profiling, this work not only highlights species-specific antioxidant mechanisms but also offers insights into their potential dietary and nutraceutical applications.

5. Conclusions

This study reveals significant variations in phenolic content and antioxidant activity among the different cultivated mushroom species. Among the species analyzed, AgW exhibited the highest total phenolic content (46.2 mg GAE/100 g fw) and antioxidant potential, with remarkable values of 3093.3 ± 138.2 µmol TE/100 g fw in the DPPH assay and 2.3 ± 0.3 mmol FeSO₄·7H₂O/100 g fw in the FRAP assay. The positive correlation between TPC and DPPH activity (r = 0.85) further underscores the role of phenolic compounds, particularly gallic acid, in driving antioxidant activity in AgW. Interestingly, while TPC and antioxidant activity showed a strong correlation for AgW, PO, and HE mushrooms revealed differing trends, indicating the contribution of non-phenolic compounds to antioxidant activity. HE, despite having moderate phenolic content (35.8 mg GAE/100 g fw), exhibited relatively high ABTS activity (7708.7 μmol TE/100 g fw), pointing to other bioactive compounds playing a significant role.

Gallic acid was the dominant phenolic compound detected across all mushroom species, with the highest concentration found in AgW. However, other bioactive compounds, such as catechin in PO, hydroxybenzoic acid, and vanillic acid in AgW, also contributed significantly to antioxidant potential. These findings demonstrate the complexity of antioxidant mechanisms in mushrooms, influenced not only by phenolic compounds but also by the presence of other bioactive substances.

In conclusion, AgW displayed the highest overall antioxidant activity and phenolic content, making it a promising source of bioactive compounds. Mushrooms like AgW and PO, rich in phenolic compounds such as gallic acid and protocatechuic acid, could offer substantial health benefits. The distinct antioxidant profiles of HE and other species also emphasize the importance of exploring both phenolic and non-phenolic compounds in mushrooms, as these could enhance their potential as functional foods and nutraceuticals. Further research into the synergistic effects of these compounds is necessary to optimize the health benefits of mushrooms in the diet.

These findings underscore the diversity of antioxidant pathways in mushrooms and establish a framework for optimizing their use as functional foods. This study’s novel contributions to understanding the biochemical diversity of cultivated mushrooms reaffirm their significance in the growing field of food science and health-promoting natural products.