MICROBIOLOGY OPTIMIZATION AND CHARACTERIZATION OF ENDOPOLYSACCHARIDES FROM PLEUROTUS Eryngii AND ITS POSSIBLE APPLICATION

an economically viable bio-technology process for conversion of various lignocellulosic wastes. The mycelium growth of mushroom species is mainly dependents upon the substrate, nutrients and the growing conditions (Girmay et al., 2016) . Several mushrooms belonging to Pleurotus species are confirmed to be producer of bioactive polysaccharide (Hao et al., 2017 ).


Introduction
Mushroom, a protein-rich wonder food helps in addressing the issues of quality food, health, and environmental sustainability (Raut, 2019).
Edible mushrooms have become attractive as "health foods" and as source materials for immunomodulators (Chen et al., 2018).They have beneficial effects such interventions ameliorate oxidative stress, hepatic lipid profiles, and reduce inflammation (Fontes et al., 2019).Mushroom cultivation is reported as The present study aimed to assess the optimization and characterization of endopolysaccharides from Pleurotus eryngii.The results of optimization of culture conditions showed that the highest growth and maximum production of endopolysaccharides of Pleurotus eryngii were achieved with using potato glucose medium, at 12 days incubation period, at temperature 30°C and with using inoculum size three discs (1cm in diameter for each) of Pleurotus eryngii.High performance liquid chromatography analysis detected that endopolysaccharides extracted from Pleurotus eryngii was composed of β-glucans, Chitin, Galactans and Mannans at concentration 36.20, 22.60, 21.60 and 19.60% respectively.Endopolysaccharides extracted from Pleurotus eryngii showed antioxidant activity.
The main polysaccharides present in mushrooms are β-glucans with potential boilogical activities.β-glucans are soluble fibers with physiological functions, such as, interference with absorption of sugars and reduction of serum lipid levels (Rahar et al., 2011).Mushrooms act as antibacterial and immune system enhancer; additionally, they are important sources of bioactive compounds.As a result of these properties, some mushroom extracts are used to promote human health (Valverde et al., 2018).
Pleurotus genus is one of most extensively studied white-rot fungi due to its exceptional ligninolytic properties.It is an edible mushroom and it also has several biological effects, as it contains important bioactive molecules (Bellettini et al., 2016).The genus Pleurotus contain many biological compunds such as polysaccharides, enzymes, protein, dietary fibre and vitamins (Shen et al., 2013).
It has been recently reported that many potential natural anti-oxidants are derived from lots of sources such as oilseeds, cereal crops, vegetables, fruits, leaves, roots, spices, herbs and mushrooms (Soares et al., 2009;Lee and Yoon, 2009).
The purpose of the present research was to evaluate the growth factors that might increase the productivity of endopolysaccharides.Also, characterization of endopolysaccharides by using HPLC.Finally, antioxidant activity were analysed in vitro.EL-Dein et al ., 2004).

Extraction of endopolysaccharides
Preparation of endopolysaccharide extract was carried out according to the method described by Lung and Tasi (2009).Endopolysaccharide extract of Pleurotus eryngii was isolated from the fresh mycelia of Pleurotus eryngii.The fresh mycelia was washed with ethanol (95%) followed by distilled water, grinding with distilled water and was put in conical flask containing distilled water.The cultured mycelia was extracted with boiling water for 1 h and was autoclaved at 121°C at 1.5 atm for 15 min and then filtered through filter paper (Whatman No. 1).Filtrates were precipitated with two volumes of 95% (v/v) ethanol and left overnight at 4°C.The resultant precipitate was recovered by centrifugation (Hettich EBA 12R, Germany) at 3000 rpm for 20 minutes (Wu et al., 2008).

Measurement of carbohydrate content
The content of endopolysaccharides was determined by phenol-sulfuric colorimetric method (Dubois et al., 1956) using glucose as standard.

Purification of endopolysaccharides
Crude endopolysaccharides were partially purified by dialysis membrane (Berg et al., 2007).showed that mycelial biomass was 8.98± 0.006 mg/ml and PEPE dry weight was 0.348± 0.03 mg/ml after 12 day and then decreasing was recorded at 14 day.The optimum incubation time was 12 day and this was consistent with Abd El-Zaher et al. ( 2015) who showed that the GYP medium was used and incubated for 12 day to produce Pleurotus ostreatus and Ganoderma resinaceum polysaccharides.

Effect of different temperatures
It was evident that temperature at 30°C was the most suitable for the highest mycelial growth and maximum production of PEPE (Figure3).This was consistent with finding of

Effect of different incubation periods
Incubation period has an essential role for high mycelia growth and high production of PEPE, So it was made to choose the optimum incubation period that gave high production of mycelial and PEPE dry weight.Figure 2

Conclusion and Recommendation
The current study indicates that endopolysaccharides extracted from Pleurotus eryngii has efficient scavenging activity.
Finally, it can be recommended that supplementation of natural products as Pleurotus eryngii mushroom is an important powerful antioxidant and can be used for medical purposes.Future studies are needed to evaluate the feasibility of Pleurotus eryngii extract for developing potent antioxidant drugs and detect their biological activities.

References:
Abd Effect of different physiological parameters on mycelial growth and endopolysaccharides production of Pleurotus eryngii Different experiments were made to select the most favorable conditions for high production of endopolysaccharides dry weight.Effect of different media on mycelial growth and endopolysaccharides production of Pleurotus eryngii Three different liquid media; potato glucose, glucose yeast peptone and mushroom complete media were used.Inoculate each medium with one fungal disc (1cm in diameter) of Pleurotus eryngii.After incubation for 7 days at 25°C, the mycelial dry weight and endopolysaccharides were determined (Nour EL-Dein et al., 2004).Effect of different incubation periods In order to select the optimum incubation period for high production of endopolysaccharides from Pleurotus eryngii, potato glucose liquid medium flasks for different incubation periods (5, 10, 12 and 14 days) were carried out (Elshamy and Nehad, 2010).At the end of incubation periods the endopolysaccharides and growth biomass of Pleurotus eryngii were determined.Effect of different temperature degrees Different temperature degrees (20, 25, 30, 35, and 37°C) were tested for 12 day incubation (Elshamy and Nehad, 2010).Effect of different inoculum size Autoclaved potato glucose liquid medium flasks were inoculated separately with different inoculum number, one, two and three discs 1cm diameter size.The inoculated flasks were incubated at 30°C for 12 day. of Chen et al. (2005).Analysis of the carbohydrate in the filtrate was performed by using High Performance Liquid Chromatography (HPLC), Shimadzu Class-VPV 5.03 (Kyoto, Japan) equipped with refractive index RID-10A Shimadzu detector.

Figure 1
Figure 1 showed that Potato Glucose liquid medium (PG) was the best medium for high mycelial biomass and endopolysaccharide extract of Pleurotus eryngii (PEPE) production which gave 29.72±0.43 and 0.164±0.01mg/ml respectively.Mycelial biomass and PEPE concentration significantly (P< 0.05) increased on PG medium compared to Glucose Yeast Peptone Media (GYP) and Mushroom Complete Media (MCM).These results were agreed with Hoa et al. (2015) who found that potato dextrose medium was more suitable for the mycelium growth of Pleurotus ostreatus, this may be due to availability of required nutrients for mushroom Pleurotus ostreatus in potato medium.

Fig. ( 4
Fig. (4):Effect of different inoculum numbers on mycelial and endopolysaccharides production dry weight of Pleurotus eryngii.Characterization of endopolysaccharides produced by Pleurotus eryngii High Performance Liquid Chromatography Analysis Endopolysaccharides were subjected to High Performance Liquid Chromatography analysis (HPLC).The HPLC chromatograph (Figure 5) showed that they consist of four peaks that referred to presence of β-glucans, Chitin, Galactans and Mannans For 1mg of endopolysaccharides structure analysis, the concentrations of β-glucans, Chitin, Galactans and Mannans were 36.20%,22.60%, 21.60% and 19.60%.According to Jahan and Singh (2019) mushrooms are a good source of insoluble fiber in the form of chitin (polymer of N-acetyl-glucosamine) and non-starch polysaccharides like β-glucans.Also, cultivated mushrooms Agaricus bisporus, Pleurotus ostreatus and Lentinula edodes fruit bodies contain chitin (Vetter, 2007).

Fig. ( 5
Fig. (5): HPLC of monomer of endopolysaccharides in culture filterate produced by pleurotus eryngii.Biological activities DPPH scavenging activity of endopolysaccharides DPPH method is usually to evaluate antioxidant activity of various natural compounds by reducing stable DPPH radicals to yellow-colored diphenylpicryl-hydrazine.DPPH radical scavenging ability is responsible for hydrogen donating efficiency of antioxidants.As shown in Figure (6) DPPH radical scavenging activity of endopolysaccharides increased gradually with increasing concentration.At 0.8 mg/ml scavenging effects of PEPE were 72.4%.It has been reported by Alam et al. (2011) that the higher scavenging activity of the acetone extract of P. citrinopileatus might be due to the hydrogen-donating components contained within it.The present results also agree with the results of Li et al. (2012) who suggested that all the polysaccharides from Hypsizygus marmoreus, Lentinus edodes, Russula vinosa Lindblad, Hohenbuehelia serotina, Auricularia auricula and Hericium erinaceus had