Sample running. Substrate preparation Two types of substrates namely,

Sample                                                                                                                                   

The
specimen was collected from forest bed of Tripura, North-East India. The
mushroom sample was identified by comparing the descriptions with the work of
Pegler (1977), Purkayastha and Chandra (1985). Pure mycelial culture was
obtained from junction point of stipe and pileus of fresh fruit body and
maintained on Potato Dextrose Agar (PDA) medium. Remaining mushroom samples was
dried in hot air oven within the range of 45ºC to 55ºC for 24 hour and
preserved in polyethylene bag by adding 1, 4-dichlorobenzene as disinfectant
for further analysis (Debnath et al. 2017).

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Mother spawn
preparation

Wheat grain was used for mother spawn preparation. Healthy grains were
first washed with tap water for surface cleaning. Then boil the grains with equal volume of water for
15-20 min and drain off excess water. After draining
excess water, surfaces dry the grain in shade for a few hours (4h). Then mix
properly the grain thoroughly with calcium sulfate (2%) and calcium carbonate
(0.5%) on dry weight (w/w) basis of grain. After mixing fill 300g-350g grain
chemical mixture in 500 ml containers and plug it. Followed by sterilization
and then allow the containers to cool to room temperature. Inoculate the
containers with the culture of mushroom mycelia using laminar air flow. Then the container was incubated with their caps loosely in
an incubator at 28°C 15-20 days for mycelium running.

Substrate preparation

Two types of substrates namely, sawdust (SD), paddy straw (PS) was
prepared. Firstly soak the substrates in hot water for overnight and then dry
it. After drying add 2% of calcium sulfate, 0.5% calcium carbonate on
substrates and mixed thoroughly and moisture was increased by adding water.

Preparation and
culture of spawn packet

The spawn packets were prepared separately for each type substrate
(1 Kg). Polyethylene bag 22.5 cm X 30 cm size was filled with prepared
substrate and packed tightly. Then two tea spoonfuls of prepared mother spawn
containing mycelia was placed through the hole of each packet. The inoculated
packets were again plugged and covered properly. The inoculated packets were
kept on rack in an incubation room at room temperature (23°C – 27°C) and
relative humidity 70 – 80%. Mycelium running rate of each type of substrates
was observed and data was recorded. After completion of mycelium running spawn
packets was opened in upper sight and also hole around the bag and sprayed water
twice a day.

Cropping and harvesting
determination of mushroom

The appearing of first primordia formation and harvesting time mainly
depends upon substrate which was used. Matured mushroom identified by curl
margin of the cap was harvested by twisting to uproot from the base. Mushrooms was
harvested when pileus of mushroom turning down at the edges.

Determination of
growth and yield

Growth
was determined by the growing pattern of mycelium. After the growing of
mycelium inoculated substrate was kept in a dark spawn running room at 25ºC
with 70 % relative humidity. Yield of mushroom was determined by number of
fruit body, size of fruit body, colour of fruit body before and after drying,
and yield per packet.

Productivity and biological
efficiency

Productivity
(P) was determined by the formula of P % = FWM/FWC × 100, where, FWM was mushroom
fresh weight and FWC was compost fresh weight.

Biological
efficiency (BE) was determined by the formula of BE % = FWM/ DWC × 100, where
DWC corresponds to compost dry weight.

Mushroom extraction preparation

Preparation of methanolic extracts of mushroom
was done by slightly modified method of Mau et al.11.
The dried powdered mycelium (5g) was extracted by grinding with 50 mL of
methanol with the help of pastle and morter. After filtering through Whatman
No.4 filter paper, the mycelium was then extracted twice with addition of 50 mL
of methanol in each. The methanolic extract was then evaporated at 40ºC to
dryness in rotary evaporator (Rotavap: PBV-7D). The dried extract was used
directly for determination of antioxidant activities.

Antibacterial activity:

Test microorganisms

Antimicrobial activity was tested against one gram
positive bacteria, Staphylococcus aureus (MTCC 96) and one gram negative bacteria, Escherichia
coli (MTCC 40). Test
bacterial strains were procured from IMTECH Chandigarh, India.

Test of antibacterial activity

The
antibacterial tests were carried out by the disc diffusion method (Collins and Lyne, 1987).
Using 100 µl of suspension of bacteria. The discs (4 mm) were then impregnated
with 100 µl of mushroom extract and then placed on the inoculated agar.

Free Radical Scavenging Activity
(DPPH) 

Free
radical scavenging activity (FRS) activity was
measured by slightly modified method of Shimada
et al.12. 4 ml dried mushroom methanolic extract (0.25- 16 mg/ml)
was mixed with 1 ml of (0.0002 M) methanolic solution containing
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (Sigma). The mixture after shaking
vigorously was allowed to stand for 30 min and the absorbance was measured at
517 nm against a blank in a spectrophotometer (Eppendorf AG 22331Hamburg). EC50
(mg/mL) is the valuable concentration at which DPPH radical were
scavenged by 50% (w/v) and was obtained by interpolation from linear regression
analysis. BHT was used as a control. Inhibition of free radical by DPPH in
percent was calculated as follows: Percentage of inhibition: (A Blank
– A Sample) / A Blank ×100, Where, A Blank
and A Sample denotes the absorbance of control and test compound
respectively.

Reducing Power 

Reducing
power was determined by the slightly modified method of Oyaizu13.Each
mycelial extract (0.5-64.0 mg/mL) in methanol (2.5 mL) was mixed with 2.5 mL of
0.2 M sodium phosphate buffer and 2.5 mL of 1% potassium ferricyanide and the
mixture was incubated at 50°C for 20 min. Then 2.5 mL of 10 % (w/v)
trichloroacetiic acid (TCA) was added. After centrifugation at 200 g for 10
min, 5 mL of upper layer was mixed with 5 mL of deionised water and 1 mL of
ferric chloride (0.1 %). The absorbance was measured against a blank in 700 nm
in spectrophotometer (Eppendorf AG 22331Hamburg). EC50 (mg/mL) is
the effective concentration at which the absorbance was 0.5 for reducing power.
BHT was used as control.

Chelating Effect on Ferrous Ion 

It
was determined by the method of Decker & Welch14. 2 ml of each
methanolic extract at various concentration (0.05-1.5 mg/ml) various
concentrations of extract in the methanol was added to a solution of 0.002 M
FeCl2 (0.05 ml). The reaction was initiated by the addition of 0.005
M ferrozine (0.2 ml). The total volume was made to 5 ml with methanol. Then,
the mixture after shaking vigorously was allowed to stand for 10 min in room
temperature and the absorbance was measured at 562 nm in spectrophotometer
(Eppendorf AG 22331 Hamburg). A mixture without extract was used as the
control. Ethylenediaminetetraacetic acid (EDTA) was used as a standard. The
inhibition percentage of Ferrozine Fe2+ complex formation was then
calculated: Metal Chelating effect (%) = {A0-A1/A0}
×100 %, Where, A0 and A1is the absorbance of
the control and the sample respectively. EC50 (mg/ml) value was
calculated from the graph of ferrous ion inhibition percentage against extract
concentration.

Fungal Phenol
Estimation 

Total phenols was determined according to the method reported by Swain
and Hillis, 195915 (Folin ciocalteau reagent method) with the
Folin-Ciocalteu reagent, using tannic acid in ethanol (80%, w/v) as standard.

Determination of Flavonoids
content

Flavonoids
was expressed as quercetin equivalents. Quercetin (0.004- 0.256 µg/ml) was used
to make the calibration curve. The standard solutions or extracts (0.5 ml) was
mixed with 1.5 ml of 95% ethanol (v/v), 0.1 ml of 10% aluminium chloride (w/v),
0.1 ml of 1 M potassium acetate and 2.8 ml of water. The volume of 10%
aluminium chloride was substituted by the same volume of distilled water in the
blank. After incubation at room temperature for 30 min, the absorbance of the
reaction mixture was measured at 415 nm (Eppendorf AG 22331 Hamburg). The mean
of three readings was used and the total flavonoid content was expressed as
milligrams of quercetin equivalents/g of extract (Kosalec et al., 2004).

Statistical
Analysis 

For each one of the
mushroom species, the assays were carried out in triplicate form. The
average data recorded for each replica were subjected to the one way ANOVA
technique using Origin 7 followed by Tukey’s Least Significant Differences
(LSD). The results were expressed as mean values ± standard deviations (SD) and
the significance was tested.

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