Anti-oxidant and nutritive properties of wild edible mushrooms from distinct regions of West Bengal, India

Introduction

Commonly, the term "mushrooms" (or "higher fungi") is employed to describe fungi that create visible fruiting bodies on a larger scale. Mushrooms are categorized within the Basidiomycetes class, with some also falling under Ascomycetes. They serve as a widely available food source worldwide. Over time, individuals have collected Wild Edible Mushrooms (WEMs) from forests or grown them, finding pleasure in their nutritional importance, healing uses, and unique flavor (Kalac, 2016) [13] . Approximately 1069 species of mushrooms have been documented as edible (Boa, 2004) [5] . Undoubtedly, the tradition of gathering and consuming wild mushrooms as a food source is deeply rooted in numerous human cultures. Mushrooms possess abundant reserves of proteins, polysaccharides, and dietary fibers, which bestow upon them the classification of low-calorie health supplements (Ao and Deb 2019; Mridu and Atri 2017; Deb et al., 2018) [2] . Mushrooms have maintained a longstanding connection with humanity, offering significant biological and economic advantages. The consumption of mushrooms is experiencing substantial worldwide growth, driven by their highly promising nutritional characteristics. These include their substantial content of carbohydrates, proteins, minerals, and vitamins, while being low in calories and fat (Rathore et al., 2017) [22] . Mushrooms have maintained a longstanding connection with humanity, offering significant biological and economic advantages. The consumption of mushrooms is experiencing substantial worldwide growth, driven by their highly promising nutritional characteristics. These include their substantial content of carbohydrates, proteins, minerals, and vitamins, while being low in calories and fat (Rathore et al., 2017) [22] . Mushroom fruiting bodies contain biochemical compounds such as phenols, flavonoids, ascorbic acid, tocopherols, and carotenoids, which offer positive effects on human health. These compounds establish mushrooms as a valuable reservoir of natural antioxidants (Sanchez, 2017; Sengkhamparn and Phonkerd, 2014) [24,25] . Inside their grown parts, edible mushrooms collect different useful compounds.

These include phenolic and flavonoid compounds, as well as things like polysaccharides and steroids. Phenolic and flavonoid compounds act as strong sources of antioxidants. They can do good things like fighting tumors, stopping viruses, fighting off harmful microbes, cardiovascular and reducing inflammation and allergies. Being surrounded by the subalpine highlands of the Eastern Himalayan region on one side and the coastal region of the Bay of Bengal on the other, West Bengal offers a broad range of forest biodiversity and ecological types (Saha et al., 2021) [23] . In ecological research, edible wild mushrooms are crucial. They work in harmony with trees to support the development of both native plantations and commercial plantations of tropical forests. The saprophytic wild edible mushrooms are essential for recycling nutrients and benefiting rural and tribal populations economically. But, till date no extensive research work on wild edible mushrooms has been taken up in West Bengal, despite, the fact that not many studies have been done on wild grown edible mushrooms. Considering the biodiversity of wild mushroom strains in West Bengal and their intriguing antioxidant attributes, an investigation was conducted on thirty (30) varieties of wild edible mushrooms. This study delved into properties such as phenolic and flavonoid content, ascorbic acid presence, and nutritional aspects like protein, carbohydrate, and moisture constituents.

Materials and methods

Thirty wild edible mushroom species were collected from different forest regions of West Bengal named as BCKV 3362 to BCKV 3391. The collected mushrooms were identified by morphological and cultural characters. 30 samples of edible wild isolates/strains and few have been identified and others are in process of identification by consultation with Directorate of Mushroom Research, Solan, and HP.

Mushroom sample preparation

Each type of basidiocarp was individually dried separately in a hot air oven at 40-60°C for 24 hours (Yuwa-amornpita et al., 2020) [32] . Then the mushrooms were turned into powder and placed in sealed plastic bags. This mushroom powder was kept for future use in experiments.

Antioxidants attributes Estimation of Total Phenolic Content (TPC)

Folin-Ciocalteu reagent method was followed to estimate the total phenol content of the wild mushroom sample extracts (Singleton and Rossi, 1965) [27] . 0.1 g of dried powder sample was added to 10 ml of 80% ethanol. The mixed solution was then filtered using Whatman's filter no. 1. The liquid left after filtering was heated in a water bath at 100°C until it dried up. The remaining substance was dissolved in 10 ml of double-distilled water. Aliquot (0.8 ml) was taken and added enough water to make it a total of 3 ml. Then, 0.5 ml of Folin-Ciocalteu reagent was added to it. After waiting for 3 minutes, 2 ml of a 20% Na2CO3 solution was added and mixed it well. The mixture was then placed in a water bath for 1 minute, and estimated at 650nm using a UV-VIS Spectrophotometer 371. Gallic acid was taken at different concentrations as the standard, and the samples were analyzed in 3 distinct replications.

Estimation of Total Flavonoid Content (TFC)

Estimation of total flavonoid content in the different samples of mushroom was done using the aluminum chloride (AlCl3) method with some modifications (Ramos et al., 2017) [21] . 0.1g dry powder sample was mixed with 10ml of methanol. After placed in a shaker at 200 rpm for 24 hours, the extract was filtered through Whatman's filter paper no.1. Then 0.5 ml of the mushroom sample extract was mixed with 1.5 ml of methanol, later the addition of 0.1 ml of AlCl3 and 0.1 ml of 1 M potassium acetate was done. Then 2.8 ml of dH2O was added to the solution and recorded the reading at 415 nm using UV-VIS Spectrophotometer 317. Quercetin was utilized at different concentrations as the standard, and the samples were examined in three distinct replications.

Estimation of Total Ascorbic acid Content

The Ascorbic acid Content in the mushroom sample extracts was determined by the colorimetric method with some modifications (de Quirós et al., 2009) [9] . 0.1 g of dried mushroom powder was dissolved in 4 ml trichloro acetic acid (TCA) (6%) solution, centrifuged at 4500 rpm for 15 minutes. The extract was then filtered and 0.1 ml of the filtrate was mixed with 0.9 ml of distilled water and added 2 ml of 2% DNPH (2,4-Dinitrophenyl hydrazine) later 0.1 ml of 10% thiourea was added. The mixture was heated for 15 minutes in a water bath and then cooled at room temperature. After, gently mix 2 ml of chilled H2SO4 and the absorbance was immediately measured at 530 nm using UV-VIS Spectrophotometer 317. Ascorbic acid was utilized at different concentrations as the standard, and the samples were analyzed in three distinct replicates.

DPPH radical scavenging activity

Analysis of radical scavenging activity of the mushroom sample extracts using the method given by Braca et al., 2001 [7] , with some modifications. Extract of each mushroom sample (0.1 ml) was mixed with 3 ml of DPPH methanol solution (0.004 mg of 2, 2-diphenyl-1picrylhydrazyl dissolved in 100 ml of methanol). The combination was left in the dark for 30 minutes, and then the absorbance at 517 nm was determined. The extraction solvent was utilized as a reference, and for the control, 100 µl of DPPH was added to 100 µl of methanol. Three separate repetitions for the samples in each treatment were done. The readings were transformed into the percentage of radical scavenging capacity using by the below equation:

Where Ao = Absorbance without extract, Ae= Absorbance with extract.

Nutritional attributes Estimation of Total Protein Content

The total protein content of the mushroom was determined as per Lowry's method (Lowry et al., 1951) [18] . 0.5 g of dried powder samples were taken in 10 ml of buffer solution (pH-7.0%). Then the samples were centrifuged at 10,000 rpm for 5 minutes at 27 and the preparation was then used for the estimation of protein as per Lowry's method. Take 0.1ml stock solution and makeup 1ml with dH2O. Then 5ml of reagent C is added to each tube and kept aside for 10 minutes (Reagent A -20g Na2Co3 + 4g NaOH in 1lit distilled water, Reagent B-1g CuSo4 in 100ml dH2o; Reagent C-Reagent A+ Reagent B). To the abovementioned solution, 0.5ml of FCR was added. It was placed in darkness for a duration of 20 minutes. Finally, readings were taken at 660 nm with a UV-VIS Spectrophotometer 371. Bovine Serum Albumin (BSA) was utilized at different concentrations as the standard, and the samples were analyzed in three distinct replicates.

Estimation of Total Carbohydrate Content

The total carbohydrate content of was assessed using Anthrone reagent method (Hedge and Hofreiter, 1962) [12] . 100 mg mushroom dry powder was mixed with 5 ml HCl (2.5N) and place in a water bath for 3 hours. Then, a pinch of Na2Co3 was added to make up to 100ml to prepare the anthrone reagent: 200 mg anthrone was mixed in 100 ml of chilled 95% H2SO4. It was prepared freshly just before use. 0.8 ml of dH2O was mixed to 0.2 ml extract of stock solution and 4ml of anthrone reagent was added. Then kept in a water bath for 10 minutes. Finally, readings were taken at 630 nm with UV-VIS Spectrophotometer 317. Glucose was utilized at different concentrations as the standard, and the samples were analyzed in three distinct replicates.

Estimation of Moisture Percentage

The moisture percentage of the freshly collected mushrooms was taken. Later the dry weight of the mushroom samples was taken after air drying. Results were expressed as grams of dried mushroom samples (Ouzouni et al., 2007) [19] .

Moisture percentage (%) = [(Fresh weight -dry weight) / fresh weight × 100].

Results and discussion

Antioxidants attributes Total Phenol Content

Total phenol content was recorded for 30 isolates of wild edible mushroom isolates/strains. The maximum total phenol content was recorded in BCKV 3366 (Gymnopilus sp) -29.191 mg GAE/g of dry weight, followed by BCKV 3367 (Volvariella volvaceaea) ( [6,8,24] .

Total Flavonoids Content

Total flavonoid content was recorded for 30 isolates of wild edible mushroom isolates/strains. The highest flavonoid content was observed in BCKV-3366 (Gymnopilus sp) -10.29 mg QE/g of dry weight, followed by BCKV-3367 (Volvariella volvaceae) (8.401mg QE/g of dry weight), and BCKV-3388 (Termitomyces heimii) (6.036 mg QE/g of dry weight). The least flavonoid content was recorded in BCKV-3382 (Lentinus sp) (0.862 mg QE/g of dry weight) (Table no [8,20] .

Total Ascorbicacid Content

The ascorbic acid content was significantly higher in BCKV-3387 (Termitomyces microporus) (7.84 mg/100g of dry weight) followed by BCKV-3369 (6.96 mg/100g of dry weight) and BCKV 3378 (6.88 mg/100g of dry weight). While BCKV-3371 (Termitomyces spp) showed significantly lower ascorbic acid content 2.98 mg/100g of dry weight, respectively (Table no. 1). The range of ascorbic acid content observed in our studies are in corroboration to the findings of Tsai et al., 2009 [29] .

DPPH Radical Scavenging Activity

The highest free radical scavenging activity was recorded in BCKV 3382 (Psathyrella sp) with (3.56±0.10 mg/ml of dry weight), followed by BCKV 3388 (3.44±0.10 mg/ml of dry weight), BCKV 3387 (3.132±0.06mg/ml of dry weight), and BCKV 3380 (3.223±0.03mg/ml of dry weight). While the lowest free radical scavenging activity was recorded in BCKV 3366 (Gymnopilus sp) showed (0.29±0.01mg/ml of dry weight) as shown in below (Table no [15,28] .

Nutritional attributes Total Protein Content

The highest protein content was found in BCKV-3376 (Termitomyces heimi) (43.536 g/100g of dry weight), followed by BCKV-3388 (43.243 g/100g of dry weight), BCKV-3387 (40.113 g/100g of dry weight), BCKV-3371 (38.216 g/100g of dry weight). Whereas the lowest protein content in BCKV-3382 (Psathyrella sp) (16.726 g/100g of dry weight) as shown in (Table no. 1). A similar range of total protein content found as observed by Sifat et al., 2020; Ao, and Deb, 2019 [2,26] .

Total Carbohydrate Content

The highest total carbohydrate content was observed in BCKV-3371 (Termitomyces spp) (47.31 g/100g of dry weight), followed by BCKV-3388 (Termitomyces heimi) (46.95 g/100g of dry weight). While the lowest carbohydrate content was in BCKV-3385 (Lentinus sp) ( [16,17] . [11,30] . 2, all biochemical parameters were situated on the right side of the biplot. among all the parameters the carbohydrate and moisture% on the upper quarter of the right side of the biplot are with the negative loading whereas the total phenol content, and total flavonoids content and total protein on the lower quarter of the right side of the biplot are with the positive loading besides these DPPH RSA (%) and ascorbic content was detected on the left upper side of the biplot with high positive loading. This indicates a positive correlation among DPPH, phenols, flavonoids, and ascorbic acid. The results found were similar to Keles, Koca, and Genccelep, (2011) [14] .

Multivariate analysis

Conclusion

Even though wild edible mushrooms are important for the economy and food security in both rural and urban areas, there is not enough information about the different types of wild edible mushrooms in West Bengal and their nutritional and functional characteristics. While numerous mushroom species in Bengal are safe for consumption and offer noteworthy nutritional value, there remains a traditional belief that consuming wild mushrooms can pose risks, potentially leading to danger and even fatality. Within the pharmaceutical industry, mushroom species can serve as a conveniently accessible reservoir of natural antioxidants, while also holding promise as a potential dietary supplement. The primary factors responsible for the antioxidant activity in the extracts of all mushroom species appear to be phenolic, flavonoid, and ascorbic acid compounds. It will assist in preserving human cells. An accessible supply of natural nutritional elements (protein, carbohydrates, and moisture) is the species of wild mushrooms. According to our research, methanol and ethanol extracts of various mushroom species show higher levels of antioxidants and dietary components under in-vitro conditions.

Acknowledgement

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