Antibacterial, Antioxidant and Anti-Inflammatory Effect with GCMS Profile of Edible Mushroom Fermented by LAB

INTRODUCTION

Around the world, people create and eat fermented food, with probiotic bacteria probably the most prevalent type of fermentation. Because of their distinct flavor and its universal recognition as a cuisine marvel, mushrooms are used all over the world (Bakratsas et al., 2021). In addition to their organoleptic quality, therapeutic qualities, and socioeconomic relevance, mushrooms are regarded as a delicacy with high nutritional and functional value and are also recognized as nutraceutical foods. In recognition of its anticancer properties, lactic fermentation has long been utilized in many parts of the world for maintaining the fruiting bodies of edible mushrooms (Atila et al., 2021). The Agaricus bisporus mushroom is among the most widely grown in the globe (Ramos et al., 2019). Diverse antimicrobial substances, primarily secondary metabolites along with certain primary metabolites as oxalicacid, peptides, and proteins, may be found in mushrooms. The most extensively researched species, Lentinus edodesis, appears to exhibit antibacterial properties against both bacteria and fungi (Ergonul et al., 2013). They also guard towards inflammation and the growth of tumors. The most significant polysaccharide in contemporary medicine is glucan, a highly well-known and adaptable metabolite with a broad range of biological activities (Alves et al., 2012). In addition to a variety of amino acids, dietary mushrooms are a good source of riboflavin (vitamin B2), niacin, folates and trace amounts of vitamin C, B1, B12, D, and E. (Pereira et al., 2012) Strong immune boosters have been discovered in a protein from A. polytricha and a lectin from A. bisporus. It has been demonstrated that A. bisporus extract stops breast cancer cells from proliferating (Ferreira et al., 2010; Jabłońska et al., 2019). Although lactic acid fermentation of mushrooms is common in Southeast Asia and Eastern Europe, it is not commonly used in industry. Therefore, the purpose of this work is to investigate the in vitro antioxidant and antibacterial activity of the acetone and methanol extract of the Russula cyanoxanth, Lactarius piperatus, Amanita rubescens, and Cantharellus cibarius mushrooms (Kosanić et al., 2016).

MATERIALS AND METHODS

Raw material processing

Experimental study on preservation of produced edible mushrooms has focused on improving the procedure thoroughly using curd samples. Commercially procured Agaricus bisporus and Calocybe indica were the two types of mushrooms used in these investigations. Typically, only the mushroom caps are utilized for lactic fermentation; the stems are thrown away. The edible parts of the mushrooms used in this study were chopped into short pieces, measuring between 3.5 and 4.5 cm and then blanched in boiling water for 60 sec. For fermentation, just about 25 g of stems and fruiting bodies were used. When a washing procedure is done correctly, dirt can be removed during dry cleaning, and surface microorganisms can be partially reduced (Liu et al., 2016).

Starter cultures

The majority of investigations on fermented mushrooms used starter cultures of Lactic Acid Bacteria (LAB), which are obtained from curd samples on MRS agar, to carry out lactic fermentation. The curd sample was serially diluted and plated on MRS agar and subsequently subcultured on MRS broth. The cell pellet was collected by centrifugation and the OD at 600 nm was adjusted to 1 using phosphate buffer.

Fermentation

In a sterile vial, 50 g of edible mushroom parts are combined with 5 mL of LAB at 1x10^-8 CFU sample and thoroughly mixed. It is advised to use the raw material in a 1:2 ratio to the brine that contains 2% salt and 1% sucrose in this about 25 mL sterilized brine solution. The samples, which were stored at 30ºC for 25 days (Bernas and Jaworska, 2012).

Extraction

10 mL of the sample were obtained, and it went through a centrifuge for 15 min at 10,000 rpm. After 3 hr, the clear aqueous phase was gathered and extracted using 10 mL of ethanol. Following a 10-min centrifugation at 5000 rpm, the supernatant was filtered through Whatman filter paper No. 2, dried in a Rotary Evaporator, and then reconstituted in 1 mg/mL of double-distilled water.

Total Antioxidant activity

The antioxidant capability of the isolated biological compound assessed using 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) at a concentration of 1 mg/mL. A 100 µg quantity of ascorbic acid served as the evaluation's standard. 0.2 mL of DPPH reagent was incorporated to 2 mL of ethanol solution in a test tube, and everything was thoroughly mixed. The reaction tube filled with 0.1 mL of the fermented aqueous sample. After that, the tube was covered with aluminum foil and allowed to sit at room temperature for 20 min and OD recorded at 517 nm, absorbance was measured against ethanol blank. The percentage was calculated by equation 1

Metal Chelating Activity

Metal chelating activity carried out on the extract's by mixing 1mL of ethanol with 100 µL of the recovered fermented mushroom with 50 µL of 2 mM ferrous chloride, 0.2 mL of 5 mM ferrozine was added to initiate the reaction. After 10 min in a dark environment, absorbance at 562 nm was then measured. EDTA is used as standard. For the control, distilled water was utilized. The equation that follows was used to determine the metal chelating efficiency.

The anti-inflammatory protein denaturation

The extract's anti-inflammatory properties were investigated using the protein denaturation prevention technique at a concentration of 1 mg/mL. 0.1 mL of egg albumin, 2.8 mL of phosphate buffered saline (pH: 6.4), and 0.1 mL of fermented extracts at different concentration was prepared. PBS used as a negative control and dichlofenac used as standard drug. After 15 min of incubation at 37±2ºC, the mixtures were heated to 70 ºC for 5 min. After cooling, the vehicle was used as a blank to measure their absorbance at 660 nm. The following formula was used to determine the percentage inhibition of protein denaturation.

Antibacterial activity

100 µg of both fermented and crude mushroom extracts were placed onto a sterile disk after being dissolved in the vehicle for 30 min at room temperature. The disc had been placed on over the MH agar plates that had been inoculated with the test pathogen for 24 hr. The bacterial lawn agar Petri plates were coated with 30 μg of ofloxacin. The regions of inhibition surrounding each disc were measured in millimeters to assess sensitivity to either antibiotics or mushroom extracts.

GC-MS analysis

The GC Clarus 500 Perkin-Elmer apparatus with an autosampler (AOC-20i) and Gas Chromatograph interfaced to a Mass Spectrometer (GC-MS) were the instruments used to perform the GC-MS analysis of these extracts. The temperature was set at 250ºC for the injector and 280ºC for the transfer line. The oven temperature was programmed to rise by 10ºC every min from 60ºC to 280ºC, with a 9 min isothermal at 280ºC. Software for handling mass spectra and chromatograms is called Turbo Mass Ver. 5.0. The HP 5973 mass detector's settings included scan mode, ion ass/charge ratio, and 30-550 m/z. Every mass spectrum was compared to the NIST library.

RESULTS

Antioxidant DPPH activity of fermented mushroom

The LAB is used for fermentation of two different edible mushroom kept for 21 days changed into dark brown colour during fermentation and the pH is sustained 6 to 7. Acidification reached and pH is gradually decreased to 6.6, 6.4 and 6.2 during 7^th, 14^th and 21^st day of fermentation. During fermentation the LAB grown well and produced large filamentous like vegetative cells (Figure 1). Both the metal chelating assay and the DPPH radical inhibition assay were used to measure antioxidant effects. Comparison of the rate of probiotic fermented mushroom and raw mushroom on DPPH was given in Table 1. The DPPH antioxidant activity of raw mushroom and fermented mushroom was given in Figure 2. The percentage of free radical was 51.46±0.503% on Agaricus bisporus and 55.6±0.529% in Calocybe indica. Compare to LAB fermented Agaricus bisporus have 69.866±0.503% and significant p value was 0.000177. Likewise, Calocybe indica LAB showed 62.266±0.305% significant with p value 0.001394. The percentage of DPPH antioxidant activity of ascorbic acid was 78.46±0.416. The independent DPPH of sample and standard was significant p<0.05 except fermented sample (p>0.05).

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Metal chelating activity

The Metal chelating antioxidant activity of non-fermented mushroom is and fermented mushroom activity given in Figure 3 as percentage of inhibition. The data represent that both the fermented mushroom have greater activity than raw mushroom. The maximum activity of 72.33±0.305 was recorded in fermented Agaricus bisporus whereas non fermentred extract have 57.86±0.41%. The activity of fermented Calocybe indica was 68.4±0.2% and non-fermented sample have 54.3±0.1%. The dependent t-test among Agaricus bisporus (p=0.0002) was significant at p<0.05 and Calocybe indica was not significant (p=2.5). The independent t test among sample and standard shows except fermented Agaricus bisporus (p=0.027) all other activities were not significant p>0.05(Table 2).

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Anti-inflammatory activity

Figure 4 represent the results indeed verified the in vitro anti-inflammatory activities of LAB⁺ Agaricus bisporus stem reveals greatest anti-inflammatory (74%) Similarly in the albumin denaturation inhibition assay (plate 4), LAB⁺Calocybe indica stem extract had shown the good inhibition capacity with 70% inhibition. LAB⁺Agaricus bisporus FB have less significant activity compare to stem and recorded as 15-20% Similarly in the albumin denaturation inhibition assay, of non-fermented Calocybe indica extract had shown the less significant inhibition capacity with 30% inhibition and 56% by non-fermented Agaricus bisporus at 21^st day (Table 3).

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Antibacterial activity of fermented mushroom

The antibacterial activity of ethanol extract of LAB fermented fruiting body and stem of basidiomycete Coriolus versicolor and Agaricus bisporus were examined by well diffusion method against test pathogens. The zone of inhibition against tested pathogens were given in Table 4 data reveals fermented Button mushroom-stem showed significantly greater antibacterial effect against all tested pathogens and the zone of inhibition 15.66±0.57,15.66±0.57, 15±0, 17.33±0.57 mm against Escherichia coli, P. aeruginosa, K. pneumonia, E. faecalis were recorded on Agaricus bisporus fermented by LAB⁺. The non-fermented crude extract showed <10 mm to be less significant. The antibacterial activity of fermented Calocybe indica were found to be moderately effective and less significant zone of inhibition on E. coli (12.33±0.57), P. aeruginosa (14±0) and E. faecalis (12.33±0.57) whereas not active on K. pneumoniae (4.6±0.57). Similarly, the raw extract of Calocybe indica failed to exhibit antibacterial activity. The p value of dependent t test of Agaricus sp. was 0.000467007 and Calocybe indica was 0.010124591 found to be significant. In case of the independent t test of sample of fermented Agaricus sp. and standard 0.02 (p<0.05) and non-fermented p value 0.20 (p>0.05). The independent p value of fermented Calocybe indica and standard was 0.00025 and crude was 0.001049 indicates both are significant (p<0.05).

Mass spectrum anaylsis of LAB femented Agaricus bisporus

GC MS analysis of fermented Button mushroom reveals the presence of 35 peaks (Figure 5) and 32 diffeent compounds. All the detected compounds are enlisted on Table 5. The maximum detected compounds are n-Hexadecanoic acid (14.33%), 4.87% of 2-Piperidinone (RT 11.288). Dibutyl phthalate (9.7%), 3.93% of 1,2-Benzenediol (RT 11.432),4.24% of 2-Methoxy-4-Vinylphenol and 6.32% Hydrocinnamic acid. In addition to fatty acid methylesters, spectrum shows presence of Phenylpropanoic acid or hydrocinnamic acid is a carboxylic acid and 1-Nonadecene alkenes were identified from the fermented sample. Piperidin-2-one is a delta-lactam that is piperidine with an oxo group at position 2, according to a Pub chem investigation. It has a role as L-glutamate gamma-semialdehyde dehydrogenase inhibitor. Presence of 11-octadecenoic acid, phenolic acids. A. bisporus extracts suggest that there may be several bioactive compounds that inhibit the growth of both Gram-positive and Gram negative pathogens.

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DISCUSSION

The sensory results of fermented edible mushroom such as odour, consistency was acceptable, except for the texture and colour found to be dark and juicy. All the parameters were triplicated and the bioassay pH was maintained at 6.5. Bello and Akinyele proposed an identical pattern in the use of fermenting mushroom medicinal value using fruiting bodies (Bello and Akinyele, 2007). Free radical scavenging of crude edible mushroom was lower than fermented mushroom. Both metal chelation and DPPH activity was concentration dependent. Significant protein denaturation inhibition was recorded and found to be close to standard. Bai et al., (2013) have reported that presence of phenolics aid in scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl (OH) radicals. The application of the LAB starter as fermenter also act as preservative in the food industry and enhance the use of the industrial fermentation and production of more health-oriented foods (Beganovic et al., 2014). Ascorbic acid equitant antioxidant activities of extracts of five edible mushrooms was previously reported by (Boonsong et al., 2016). Metal chelation potential of mushroom Pleurotus ostreatus was previously reported by (Oluwamodupe et al., 2013). Mushroom components like phenolics and flavonoids and ascorbic acid promotes the antioxidant potential (Nakamura et al., 2019). The data is correlates the study of (Nataraj et al., 2022) who reported metal chelating capacity of mushrooms lower than EDTA. The β-glucan-rich preparation from mushrooms has cytotoxic in nature antioxidant effects that are controlled by glucan. Its anti-inflammatory effects are achieved by inhibiting Cyclooxygenase (COX) and Nitric Oxide Synthase (NOS) inhibition. According literature survey it was reported that C. indica significantly showed anti-inflammatory, antioxidant and antimicrobial activity (Meghna et al., 2022). A number of studies showed that mushrooms extracts express a higher antimicrobial activity against pathogenic bacteria. Recently (Jankov et al., 2024) have reported the antibacterial and Synergistic activities of Agaricus bisporus against S. aureus, E. coli, B. subtilis. According to (Bartkiene et al., 2023) during fermentation A. bisporus showed decreased 1-Octen-3-ol content while benzyl alcohol, acetoin, and 2,3-butanediol increased in most fermented sample. Another study shows the FA profile of A. bisporus comprises stearic acid, palmitic, linoleic, caprylic, oleic, erucic, and eicosanoic acid (Saiqa et al., 2008). It was also reporte that Linoleic acid is converted to 13-hydroxy-9-octadecenoic acid or the antifungal 10-hydroxy-12-octadecenoic acid (Ogawa et al., 2001).

CONCLUSION

This study yielded useful data that can be used to produce fermented mushrooms on a large-scale. Additionally, a broader range of LAB for A. bisporus fermentation can be investigated in the future to suggest the most suitable LAB strains for the mushroom fermentation sector as well as the healthiest and most palatable outcome for the end user.