Analysing the Biocontrol Attribute of Indigenous Mushroom concentrates against Pathogenic bacterial spp.

 

Arun Karnwal^1*, Aradhana Dohroo², Shivani Sharma³

¹School of Bioengineering and Biosciences, Lovely Professional University, Jalandhar-Delhi, G.T. Road, National Highway 1, Phagwara, Punjab, India 144411.

²Baddi University of Emerging Sciences and Technologies, Baddi, Himachal Pradesh, India-173405.

³Bhojia Institute of Life Sciences, Baddi, Himachal Pradesh, India-173405.

 

ABSTRACT:

A. bisporus mushrooms are well known for their nutritional and medicinal values. A. bisporus is a source of protein (about 40% on dry basis), ergo sterol, a precursor of vitamin D, several minerals, carbohydrate and fat. In the present investigation studies were conducted on the effect of A. bisporus (U-03) extracts on human pathogenic bacteria in-vitro condition. Highest antimicrobial activity was observed in methanol extract (19.5%) against P. aeruginosa as compared to ethanol extract (11.25%). Similar results was obtained for S. aureus (16%) and B. cereus (13%) in methanol extract. Least microbial growth inhibition was observed in B. cereus (13%) followed by P. aeruginosa (11.25%) with respect to control in ethanol extract. Highest antimicrobial activity with ethanolic extracts was recorded against S. aureus (18.42%) as compared control and other tested bacterial isolates. No antimicrobial activity observed in P. aeruginosa in ethanol extract but least inhibition was observed in methanol extract (20mm). The present study has shown that the extracts of the A. bisporus mushrooms have shown promising antimicrobial activities against the tested organisms.

 

 

INTRODUCTION:

Mushrooms have been considered delicacy and preferred both for taste and flavor. They have a long history of medicinal usages. A new term was coined as “nutraceutical”¹ to explain their medicinal components. Availability of HMW and LMW compounds in mushroom make it as natural antibiotic source². High molecular weight (HMW) compounds are peptides and proteins³ whereas Low molecular weight (LMW) compounds are primarly categorized in secondary metabolites¹ category those includes various types of steroids, sesqui-terpenes and other terpenes, anthraquinone, quinolines, and benzoic acid derivatives⁴. However, LMW also carried few primary metabolites i.e. oxalic acid and others those can be used as antimicrobial agents^1,5,6.

 

A. bisporus consists of a huge variety of these kinds of biomolecules having dietary and/or therapeutic traits^6,7,8. As a result of all these traits, they have been known for valuable dietary  foods, as well as, a resource intended for the production of remedies and nutraceuticals^8,9. Fruiting bodies, mycelia, and spores of A. bisporus hold a complete range of bioactive biomolecules that has immuno-modulatory, cardiovascular, liverprotective, antifibrotic, anti-inflammatory, antidiabetic, antiviral, antioxidant, antitumor, and antimicrobial properties^10,11,12.

 

A. bisporus, being one of the most commonly white button mushrooms contributes about 40-45% to the world mushroom production³. A. bisporus is a low temperature species requiring 23±2 C for its vegetative growth and 16±2 C for its fruiting and widely consumed mushrooms in the world is cultivated in more than seventy countries^2,10. Antimicrobial activity of A. bisporus must have been due to the presence of essential bioactive components. Catechin which is one of phenolic components has been found to exhibit antimicrobial, antioxidant, anticancer and antiallergy properties^3,7. Caffeic acid and rutin has been shown to exhibit antimicrobial activity⁴. Gallic acid is a bioactive compound which is widely present in plants is also found in A. bisporus. It is a strong natural antioxidant; it has also been shown to have anti-inflamatory, antitumor, antibacterial and antifungal activity property^1,4,8. Methanolic extract of A. bisporus exhibit higher activity against Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Staphylococcus epidermidis and lower activity against Micrococcus luteus, Micrococcus flavus. Research scholars^2,6 reported the antimicrobial activity of different Agaricus species against various bacterial spp. They demonstrated that Agaricus bitorquis and Agaricus essettei methanolic extracts showed an inhibitory effect on various gram-positive bacteria in lab condition.  Agaricus silvicola methanolic extract also shown antimicrobial properties against Bacillus cereus, Bacillus subtilis and against Staphylococcus aureus although lower than the standard ampicillin^6,8. The mycelium of Agaricus cf. nigrecentulus and Tyromyces duracinus (ethyl acetate extracts) showed activity only against Staphylococcus saprophyticus⁷.

 

A. bisporus mushrooms are well known for their nutritional and medicinal values. They are also valued for waste management as most of them grow on lignocellulosic materials of agricultural origin, forest litter and garden litter. A. bisporus is a source of protein (about 40% on dry basis), ergo sterol, a precursor of vitamin D, several minerals, carbohydrate and fat. It was demonstrated that extract of A. bisporus exhibit anti-microbial properties and anti-oxidant properties against some gram positive as well as gram negative bacteria^2,5,13. They used different assay methods to determine the antimicrobial property of A. bisporus extracts i.e. micro-dilution assay, Disc-diffusion assay, Agar-streak dilution assay based on radial-diffusion assay¹⁴.

 

The above cited description points to the nutritional and medicinal values and subsequent studies on A. bisporus (U-03) antimicrobial properties however, there is hardly such report available in the vicinity of industrial hub of Himachal Pradesh. Thus, present piece of work highlights the antimicrobial activity of mushroom under prevalent environmental conditions using regional resources and human pathogenic bacterial isolates.

 

MATERIAL AND METHODS:

Sample collection:

For the isolation of different gram positive and gram negative bacteria sewage samples were collected from different sewage treatment plants Baddi, Himachal Pradesh (India) in sterile beaker and collected samples were transferred to sterile plastic bags in aseptic conditions.

 

Isolation of different gram positive and gram negative bacteria:

For isolation of bacteria from sewage samples two media i.e. Nutrient agar/Nutrient broth and Mac-conkey Agar/Mac-conkey broth were used⁴.  Serial dilution agar plating (SDA) method was used for the isolation of microorganisms from sewage samples. 1ml of sewage sample was added to 9ml of distilled water to make the final volume 10ml. Serial dilutions were done ranging from10^-2, 10^-3,…….10^-7 by pipetting 1ml into additional dilution blanks. Finally, 1ml aliquot of various dilutions was added to sterile petriplates having 15ml of sterile, cool, molten nutrient agar (NAM)/Mac-conkey agar media. The dilutions 10^-4 to 10^-7 were selected for enumeration of bacteria. Upon solidification, the plates were incubated in inverted position for 3-4 days at 37±1°C after incubation, different types of colonies were appeared on plates. Three replication of each dilution were maintained.

 

Identification of microorganisms:

After 3-4 days of incubation the isolated colonies appeared on medium were identified on the basis of Bergy’s manual of determinative bacteriology¹⁵. Biochemical characterization of the isolated bacteria were carried out by using Gram staining, urease production, citrate utilization, indole production, oxidation reaction, casein hydrolysis, starch hydrolysis, HCN production, catalase test, H₂S production, lipolysis activity, lipolysis activity, 3-ketolactose production, and gelatin liquefaction. Isolated and identified bacteria were maintained on slants and kept at 4±1°C till further use. Slants were revived periodically to maintain the colonies in active conditions⁴.

 

Screening of anti-bacterial activity of A. bisporus (U-03):

Collection of A. bisporus (U-03):

Fresh and healthy fruiting bodies of A. bisporus (U-03) were collected from the beakers grown in laboratory at Bhojia Institute of Life Sciences (BILS), Himachal Pradesh (India) and after that fruiting bodies were kept at 47±1°C in hot air oven to get fully dried till constant dry weight.

 

Preparation for extract:

Dried fruiting bodies of A. bisporus (U-03) were weighed to know the moisture content and then grinded in fine powder in mortar pestle. Powder of one gram dried fruit bodies were suspended in 9ml. ethanol and methanol. Extraction was accomplished by stirring solution at 150rpm for 48h at 20°C. The broth was then centrifuged at 10,000rpm for 10 minutes and the supernatant was filtered by using whatman No. 1 filter paper and extracts were collected in pre sterilized micro-centrifuge vials. Extracts were then stored at 4°C for study of antibacterial activity.

Screening of anti-bacterial activity of A. bisporus (U-03):

In-vitro anti-bacterial susceptibility tests are performed by using identified pathogenic microorganisms following agar well diffusion method^3,7. Nutrient agar medium was utilized throughout the study for the cultivation of isolated bacterial spp. Medium was autoclaved at 121°C for 30 minutes. The plates were left overnight at 37±1°C for pre incubation to check for any contamination to appear. Then after freshly grown bacteria were seeded on the medium to prepare bacterial lawns and two agar wells of 8mm diameter were made opposite to each other. One well serve as control and other serve as test. 10µl. of extract were pipetted in one well and its respective solvent without extract were pipetted in other well. After pipetting plates were kept at 20±1°C for 1 hr so that extract get absorb in the medium. Inoculated plates were then incubated at 37 C for 24 hrs to see the effect of extract on bacterial lawn and result thus obtained get compared to control treatment.

 

RESULTS AND DISCUSSION:

A large number of pharmaceutic chemicals with strong and exceptional health boosting benefits have been separated from mushrooms and marketed globally^3,5,7. Mushroom oriented remedies isolated from the mycelia or fruiting bodies are utilized in the form of tablets, capsules or concentrates.

 

In present study, the concentrated methanolic and ethanolic extracts of A. bisporus (U-03) were tested to screen the potential antibacterial properties against three human pathogens. The level of antimicrobial action was evaluated through the MIC value towards the bacterial strains⁷. The well diffusion approach primarly applied to reveal the antibacterial ability associated with mushroom extracts against chosen three human pathogenic bacteria i.e. Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa.

 

Isolation and biochemical characterization of Pseudomonas strains:

Bacterial species were isolated from sewage samples collected from Sewage treatment plant, Baddi, Himachal Pradesh (India). The SDA plate method was applied for the isolation of bacterial spp. on NAM. Macroscopic, Microscopic and biochemical methods were applied for the identification of isolated bacteria as detailed under Bergey’s Manual of Determinative Bacteriology.

 

Table 1: Macroscopic, Microscopic and Biochemical characteristics of bacterial isolates

 

Table 2: Percent inhibition of growth of bacterial isolates at various % concentration of A. bisporus (U-03) methanolic extract

 

 

Table 3: Percent inhibition of growth of bacterial isolates at various % concentration of A. bisporus (U-03) ethanolic extract

S. No.	% Concentration ethanolic extract	% Inhibition of bacterial growth
		B. cereus	S. aureus	P. aeruginosa
1	Control	0	0	0
2	25	0±0.00	1.4±0.04	3±0.14
3	50	7.50±0.21	4.64±0.18	9±0.12
4	75	9.50±0.12	`12.8±0.22	15.5±0.44
5	100	13±0.14	18.42±0.18	11.25±0.16

 

All the 3 isolates i.e. Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa were biochemically identified/characterized for Gram staining, urease production, citrate utilization, indole production, oxidation reaction, casein hydrolysis, starch hydrolysis, HCN production, catalase test, H₂S production, lipolysis activity, lipolysis activity, 3-ketolactose production, and gelatin liquefaction (Table 1).

 

Mushroom mycelial properties:

Agaricus bisporus (U-03) fruiting bodies used for extraction of extract were cultivated in laboratory under control conditions. Color of mushroom fruiting bodies was white. Mushroom spores were ovoid to circular in shape with brown color spore print. Mycelial growth was radial longitudinally, elevated in the beginning, bright white, turning into compactly twisted and cottony in consistency.

 

Antibacterial activity of Agaricus bisporus (U-03):

Nowadays, cases of multi-drug tolerance in microorganisms are elevating and limiting the management of a large percentage of contagious infections^1,6. Therefore, right now there is an immediate requirement for the production of fresh and strong medicines to protect against current antibiotic-tolerant infections. Fungal varieties have been shown excellent potential as a resource of bioactive molecules of high remedial importance^1,5,6. Also, they are the richest resources of secondary metabolites. The antimicrobial activities of the methanolic and 70% ethanolic extracts of A. bisporus (U-03) were quantitatively analysed towards three bacterial isolates i.e. B. cereus, S. aureus and P. aeruginosa.

 

Extracts (methanolic and ethanolic) of A. bisporus (U-03) shown substantial inhibition in development of three tested bacteria at various concentrations (25%, 50%, 75%, 100%). In our results, it was recorded that methanolic extract of A. bisporus (U-03) shown maximum level of growth inhibition upto 13%, 16%, and 19.5% at 100% extract strength against B. cereus, S. aureus and P. aeruginosa, respectively (Table 2). Whereas, the 100% concentrated ethanolic extract was recorded with maximum growth inhibition upto 13%, 18.42%, and 11.25% against B. cereus, S. aureus and P. aeruginosa respectively (Table 3).

 

Results of present study demonstrated that extracts (methanolic and ethanolic) of A. bisporus (U-03) shown maximum % growth inhibition towards S. aureus whereas A. bisporus (U-03) extract prepared in methanol as solvent shown maximum bacterial inhibition activity upto 19.5% against P. aeruginosa in comparison to ethanolic extract of Agaricus bisporus (U-03) for P. aeruginosa (11.25%).  Earlier findings revealed that mushroom extracts demonstrated diversified anti-bacterial properties to protect against Gram-positive and Gram-negative infections^1,2. Present research findings in agree with the previous work done by many researchers  on anti-bacterial actions of mushrooms. In present study, P. aeruginosa, a Gram-negative bacteria, is identified as remarkably sensitive and its growth was more inhibited due to methanolic extracts compared to other tested/targeted organisms. Similar observation observed with ethanolic extract of Agaricus bisporus (U-03) against S. aureus. Present study results are supported with the earlier research^1,4 in which methanolic and ethanolic extracts of outdoor mushrooms possess effective antibacterial activities against S. aureus.

 

Earlier work carried out by scholars with mushroom extract confirmed the strong antibacterial activity of G. lucidum against Gram negative bacteria¹⁶. It was reported that the antimicrobial effect of methanolic extract of Lactarius delicious^2,17 and Sparassis crispa ¹⁰ against human pathogenic bacteria. Similar antimicrobial tendency of Morchella esculenta  and Ganoderma lucidum^2,4 were reported against S. aureus and E. coli. It was observed that extract of C. vermicularis and M. oreades presented even more growth inhibition to gram-negative bacteria (E. coli and P. aeruginosa) in comparison with gram-positive bacteria (B. subtilis and S. aureus)¹⁰. Scholars^2,9 also claimed the antibacterial capability of ethanolic extract of P. florida and P. ostreatus against various bacterial spp.

 

CONCLUSION:

From the present investigation it was concluded that A. bisporus (U-03) having a good antibacterial activity against different pathogens viz B. cereus, S. aureus and P. aeruginosa and it was found that the extract of this mushroom was more or less inhibitory to all three bacterial isolates. Hence there is need to exploitation of U-03 strain on large scale for therapeutic use which is still in infancy stage.

ACKNOWLEDGEMENT:

I am very thankful to Bhojia Institute of Life Sciences, Budh, Baddi, H.P., India for technical support to complete this study and unlimited help in all steps.

 

COMPETING INTEREST’S STATEMENT:

The author(s) declare(s) that there is no conflict of interest.

 

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Received on 07.06.2019           Modified on 04.07.2019

Accepted on 01.08.2019         © RJPT All right reserved