Chemicals and Instruments
Chemicals used in this study including methanol, distilled water (Aq), n-hexane, dimethyl sulfoxide (DMSO), Folin-Ciocalteu (FC) reagent, anhydrous sodium carbonate, gallic acid (GA), aluminium trichloride hexahydrate, sodium acetate, DPPH were of AR grade. All the above reagents and potato dextrose agar (PDA), glucose (Glc) and agar were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. Standard antibiotic drugs, gentamycin and terbinafine were obtained from Pharmagen, Hefei. Laminar flow cabinet was produced by Stramline Laboratory. Petri plates and glass columns were bought from Hefei Meifeng Chemical Instrument Co. Ltd. Autoclaver was produced by Shanghai ShenAn medical equipment factory
Collection and Identification of Materials
Lycoperdon pyriforme was collected from south wollo, Sayint Adjibar woreda, Waro kebele, Ethiopia and identified by a botanist in the Department of botany, Wollo University, Dessie. The puffball samples were frozen at ‒20 °C, prior to freeze-drying procedure. Freeze dried samples were ground to a fine powder, wrapped in plastic bags and stored in a dark place at room temperature, until further use.
Determination of Extractive value
Naturally all solvents are divided into two types such as polar and non-polar based on dielectric constant. In the present study, both polar and non-polar solvents were used to obtain the output of the edible wild mushroom, Lycoperdon pyriforme. To obtain the output, 100 grams of dried powder of Lycoperdon pyriforme was extracted separately and dipped in a beaker containing all the hot and non-polar solvents such as distilled water, methanol and hexane. Leave all the beakers for 48 hours, the supernatant collected separately from each goat. The same process is repeated three to five times until the soaked powder changes color, then, the extract obtained dried at room temperature and the extract extracted under reduced pressure . The appropriate dried residues were dispersed in 5% of DMSO, prior to analysis.
The extractive value (EV) (%) was calculated by using the following formula:
Total phenol content
The total phenol (TP) content of LycMtOH content was determined , converted to a 96 source plate reader (Multiskan Ascent, Thermo Electron Corporation). Folin-Ciocalteu reagent (125 μl, 0.1 M) was added to the blended extracts (25 μl). After 10 minutes, 100 μl of 7.5% w / v sodium carbonate was added and the reaction mixture was incubated for 2 hours. Absorbance read at 690 nm. TP is defined as mg gallic acid equivalents (GAE) / g of dry weight (d.w.).
Total flavonoid content
The total flavonoid (TF) content of LycMeOH was measured spectrophotometrically, at a 96-well plate reader, using a modified method with . The appropriate sample (30 μl) was mixed with methanol (90 μl), aluminumtrichloride (6 μl, 0.75 M), sodium acetate (6 μl, 1 M) and purified aqua (170 μl). Absorbance is measured at 414 nm, after incubating for 30 minutes. The results are shown to be equivalent to mg quercetin (QE) / g of dry weight (d.w.).
DPPH scavenging activity
The free radical extract capacity of mushroom extracts is measured on the basis of stable disposal activity 1, 1- diphenyl- 2- picrylhyorazyl (DPPH) free radical according to the modified method described by . 1ml of plant extracted with different concentrations such as (10, 25, 50, 75, 100 µg / ml) taken from a separate test tube. For each test tube, 1 ml of DPPH solution 0.1 mM is added to ethanol. At the same time, a randomized sample was prepared and BHT (1- 100 µg / ml) was used as an indicator. A mixture of 1 ml ethanol and 1 ml of DPPH solution was used as a control. The reaction was triple and the absorption drop was measured at 517 nm after 30 minutes in the dark using a UV-Vis spectrophotometer. % of free radical block chain arithmetic is calculated using the following formula. Decreased absorption indicates an increase in free radical release.
Pure cultures of Staphylococcus aureus, Candida albicans, Escherichia coli and Bacillus subtilis were obtained from Bless Agri Food Laboratory Services PLC.
Assessment of Antimicrobial activity
The antimicrobial activity of Stump puffball (Lycoperdon pyriforme) extracted was tested using two types of disc distribution methods which were sinus blockers (ZOI)  and a small concentration of prevention (MICs), ZOI analyzes quality and MIC is quantitative analysis antimicrobial function .
In this study, three types of bacteria and one type of fungus were used to determine the antimicrobial activity of stump puffball (Lycoperdon pyriforme). The agar cultures of S. aureus, B. subtilis, C. albicans, E. coli prepared to evaluate the effects of Stump puffball antimicrobial. 50 ml of nutrient broth medium is poured into the Erlenmeyer flask, four flask prepared for tested samples. Flakes including agar medium sterilized at autoclave at 121 ° C for 15 minutes.
For antibacterial testing, bacterial cultures have been raised to 35 ° C for 24 hours by injection into the Nutrient Broth . Petri containers containing 20 ml of Nutrient Agar were prepared, previously vaccinated with 100 μl of standard suspension (1%, containing 106-107 cfu / ml) and the same volume of contaminated water was used as a controller. Three springs (5.0 mm wide) were cut from the agar in a hollow state. Prepared stump puffball solutions were filled in wells. Incubated plates were placed 24 hours at 35 ° C, after which the antimicrobial activity of the samples was observed. At the end of the incubation period, measurements are made basically from the edge of the site to the end of the spring.
The same method was used for all mold samples. C. albicans are grown in Potato dextrose broth (PDB) at 25 ° C for 48 hours. 100 μl of various fungal cultures (1%, containing 106-107 cfu / ml) were added to various flasks containing 25 sterile PDA at 45 ° C and poured into Petri containers. Agar is allowed to harden at 4 ° C for 1 hour. The springs (5.0 mm wide) were cut from the agar in a hollow state. Incubated plates 24 h at 25 ° C. PDA plates with added salt are used as controls. The plates are then incubated at 25 ° C for two days. ZOI restricted area is calculated.
Minimum inhibitory concentration (MIC)
A standard agar purification solution with double dilution was used. Extracts are incorporated into nutrient agar at concentrations ranging from 0.39 mg / ml to 25 mg / ml. A non-extract controller was also configured. 10 μL of tested organic matter, previously purified to 10 CFU / ml, was used for injection plates. These are incubated at 37 ° C 24 hours initially and another 24 hours before growth and recording. The minimum inhibitory concentrations (MICs) of each microorganism extract have been considered as a plate of agar with very low concentration without growth .