Oyster Mushroom Spores Ghost Preparation for Medicinal, Biotechnological and Forensic Applications

Different types of edible mushrooms are produced
worldwide using different cultivation systems. In most cases...

mushrooms contain small amounts of arabitol, a sugar alcohol, which may cause gastrointestinal upset in some people [22,23].
It causes hypersensitivity for individuals in direct contact with its spores particularly for long time [3,24,25].
Evacuating microbes from their cytoplasmic content is a natural phenomenon [26].   [27] and others reported that pores could be introduced to the microbial cell walls and/or membranes as a result of different mechanisms [26,[28][29][30][31], such as evacuating the gram-negative bacteria via bacteriophage infection [26]. Bacteriophage E lysis gene is used for evacuating bacterial cells and turning them into ghost cells via controlling their expression using heat sensitive promoter [26,28,[31][32][33][34].
SDS was proved to have the ability to perturb and destabilize cell wall/plasma membranes of both yeasts and fungi at very low concentrations [51]. SDS is also able to degrade the cell wall. H 2 O 2 has oxidizing activity and able to degrade the genetic material instead of nucleases [38]. NaOH is also known for its damaging effect on microbial cell walls and a degrading capability for both DNA and RNA. It can deactivate microbial cells and can be used alone as antimicrobial agent as proved from the previous studies of microbial ghost cells preparations. NaHCO 3 has a recognizable effect as salt and alkali as proved by Amara and Steinbüchel (2013). It can deactivate various microbes or slow the growth of others in particular concentrations and for that it could be used in Spirulina platansis selective media. In this way, it has substituted CaCO 3 in preparing ghost microbes other than Gram-negative bacteria. It might be useful to highlight that Amara and Steinbüchel have selected NaHCO 3 based on a natural phenomenon that the Lake becomes dominant with S. platensis during the summer due to the evaporation of its water content and the increase in its salt content, where carbonate is the major constituent. Both of pH and salinity were responsible for eradicating various algal species and keeping the lake clean with an exception that S. platensis could grow as a dominant cyanobacteria turning the colour of the lake to the cyanogreen colour [52].
In this study, for the first time, the spores of the oyster mushroom were evacuated from their content and turned into ghost particles using the concept of Sponge Like Protocol.

Isolation of the Oyster Mushroom
Mushroom fruiting bodies from Borg El-Arab region, Alexandria, Egypt, were collected during winter season and stored at 4 o C. Under sterile conditions, small tissues of the wild fruiting bodies were transferred to the PDA plates. The mycelia tissue was placed in the center of the plate, so that growth can radiate away from it. The plates were incubated at 28 o C for 7 days. After full colonization, the plates were stored at 4 o C.

Morphological Identification
The fungal isolate was identified morphologically after growing on rice straw by observing its macroscopic features including: Cab and stem morphology, spore-print and microscopic shape of spores.

Oyster Mushroom Spawn Preparation
Spawn of the mushroom was prepared in 250 ml bottles using sorghum grains. The grains were washed with water thoroughly to clean the dirt, then boiled in water until being semi soft, after cooling to room temperature, the excess water was drained off and the grains were mixed with 5% (w/w) CaSO 4. The bottles were filled to 3/4 with sorghum grains and sterilized by autoclaving at 121 o C and 1.5 bar for 15 minutes. The sorghum grains were inoculated with actively growing mycelium of oyster mushroom from PDA plates and incubated at 28°C. Mycelial growth were allowed for 12 to 15 days until the mycelium fully covered the grains.

Oyster Mushroom Cultivation
Pleurotus sp. was cultivated using the perforated polythene bag method with minor modifications [53,54]. Dried rice straw was chopped into 5 to 7cm length and soaked in water for 4 hours in the presence of 5% (w/w) gypsum, and excess water was drained off. The substrate was sterilized by autoclaving at 121 o C and 1.5 bar for 20min. About half kilogram of the substrate was placed in 40cm width X 60cm length polyethylene bags and spawned with 10% of the mushroom mycelia grown on sorghum grains. Spawning was done in 3 layers each above 5 cm layer of the rice straw substrate.
The bags were subsequently placed into spawn running room at 25°C±2 under dark conditions. After the completion of spawn running, polythene bags were placed into the fructification room at 23°C±2 and 75-85% relative humidity. The bags were cut open on the sides without disturbing the beds and sprayed twice a day with water for maintaining a high moisture level. The bags were exposed to light for 5-7 hours/day and left until the appearance of the mushroom pinheads. The fruiting bodies appear two weeks after bags perforation for 3-successive flushes. Mushroom was matured within 2-3 days after pinhead's initiation. Mushroom was harvested by twisting the fruiting body to displace it from the base.

Collection of Oyster Mushroom Spores
The stems of the fresh oyster mushroom fruiting bodies were cut at the base of the fan-shaped cap. Then, the oyster mushroom cap was placed on a clean white paper with the underside down on the paper. A glass cup was placed over the mushroom cap and kept in a cool and dry place for 24 to 48 hrs. The glass cup and the mushroom cap were removed to find a spore print of the mushroom cap where the spores collected on the paper. Finally, the spores were collected in an Eppendorf tube by scrapping off the paper using sterile scalpel.

Determination of the MIC and MGC Concentrations for NaOH, SDS, NaHCO 3 and H 2 O 2 and Spore Viability
Standard broth microdilution susceptibility assay was used for determining the MIC values for each of NaOH, SDS, NaHCO 3 and H 2 O 2 [35,36]. Ten Eppendorf tubes, each contain 3 mg of the spores in one ml of water were used. Different stocks of the used chemical compounds represent: 10% of NaOH (non-autoclaved), SDS (autoclaved), NaHCO 3 (autoclaved), and 30% of H 2 O 2 (commercial grade purchased from a pharmacy). After overnight incubation, direct plat count were performed by spreading 100 µl of each spores-treated solution on PDA medium and the plates were

Determination of DNA /Protein Concentrations
The concentration of DNA in the supernatant, after each step for each randomization experiment, was determined spectrophotometrically by measuring the absorbance at 260/280nm using spectrophotometer (Biochrom LTD, Cambridge CB4 0FG, England).
An extinction coefficient 260 = 1 corresponds to 50µg dsDNA mL -1 [54]. The DNA concentration was calculated automatically by the aid of the software of the spectrophotometer [55].

Spore Cells Evaluation Using Light Microscope
Spore cells generated from different treatments were investigated using light microscopy. The spores were stained on glass slides using crystal violet stain for 1 min, and then examined under light-microscope to check the evacuation process during various steps of the treatments.

Sample Preparation for Electron Microscope Examination
For further investigation of the quality of the ghost-spores, electron microscope was used to scan the spore particles. A spore smear of each preparation was prepared, and the smear surface was then coated with approximately 15nm gold layer (SPI-Module Sputter Coater).

Scanning of the Prepared Ghost-Spores (SGs) Surface
The gold-coated samples were scanned by analytical scanning electron microscope (Jeal JSM-6360, LA) with secondary element at 10kv acceleration voltage at room temperature. The digital images were then adjusted and analyzed.

Results and Discussion
Different groups of fungi are well known for their ability to produce spores as a part of their reproduction process and for protecting their origin. Such spores are usually more resistant to harsh environmental conditions and are able to spread everywhere.
They are capable to reach our bodies particularly to our lungs. Mushrooms spores are invited candidates in forensic investigation, establishing a protocol for evacuating the mushroom spores using the concept of MIC will enable better realise to their DNA and protein contents. In some cases, the amount of the spores is  from the electron microscope as images from a to e in Figure 1. The protocol also succeeded to turn virus to ghost empty and inactive virus [38]. However, in order to prove the killing effect of different used compounds, direct cultivation on plates contain nutrients was done. This step gives better determination for the MIC and the MGC. Moreover, the usage of one ml of water and half ml of the chemical compound has led to narrowing the dilution differences, which give better MIC and MGC. In a previous study, we used 4.5ml of the medium and 0.5ml of the chemical compound, which give high dilution within the tubes. Both DNA and Protein percentages were calculated directly after the serial dilution experiment as in Table 1. The centrifugation, which happened only during collecting the spores from the treated samples to analyse them by either the electron or light microscopes, also help in evacuating the spores from their contents as shown in Figure 1 and Figure 2. This study considered as is an additional step for evacuating different microbes as well as their components such as the spore of mushrooms. Using the main concept of the cell evacuation protocol as described by  [27] by using different chemical compounds after determining their minimum inhibition concentration.