Chemo-characterization and optimization of macro and micro nutrients for exopolysaccharides and mycelia growth in pleurotus tuberregium ( RUMPH , EX FR )

Exopolysaccharides (EPS) are membrane bound polysaccharides found in large family of lower organisms including fungi and released as slime/capsules into immediate environments. Some roles ascribed to EPS in fungi are substrate adhesion, defense, adsorption and storage of nutrients (Wotton, 2004). EPS are high molecular weight polymers of diverse homo or heterosugars (Zhang et al., 2004; Zhao et al., 2014). The immunomodulatory, antimicrobials, antioxidant, antitumor, hypoglycemic and anti-tyrosinase properties of EPS have been reported (Hwang, et al., 2005; Borges et al., 2013; Barakat & Sadik, 2014; Zhao et al., 2014). Therefore, EPS has potential applications in pharmaceutical, neutraceutical, and probiotical for many devastating diseases such as diabetes and cancer. Bae et al. (2005) reported potentials of EPS in cosmetics, cosmeceutical, and nutricosmetical industries for skin treatment. On addition, EPSs are use industrially, as emulsifiers, thickeners, stabilizers, and gelling agents (Mahapatra & Banerjee, 2013).


Introduction
Exopolysaccharides (EPS) are membrane bound polysaccharides found in large family of lower organisms including fungi and released as slime/capsules into immediate environments.Some roles ascribed to EPS in fungi are substrate adhesion, defense, adsorption and storage of nutrients (Wotton, 2004).EPS are high molecular weight polymers of diverse homo or heterosugars (Zhang et al., 2004;Zhao et al., 2014).The immunomodulatory, antimicrobials, antioxidant, antitumor, hypoglycemic and anti-tyrosinase properties of EPS have been reported (Hwang, et al., 2005;Borges et al., 2013;Barakat & Sadik, 2014;Zhao et al., 2014).Therefore, EPS has potential applications in pharmaceutical, neutraceutical, and probiotical for many devastating diseases such as diabetes and cancer.Bae et al. (2005) reported potentials of EPS in cosmetics, cosmeceutical, and nutricosmetical industries for skin treatment.On addition, EPSs are use industrially, as emulsifiers, thickeners, stabilizers, and gelling agents (Mahapatra & Banerjee, 2013).
Exopolysaccharides have been isolated in diverse fungi using different techniques and biomass materials and still possess similar medicinal properties (Borges et al., 2013;Zhao et al., 2014;Anike et al., 2015).Advantages of bio-exopolysaccharides over chemosynthetic EPS are: they are renewable, non-toxic and biodegradable (Freitas et al., 2011).Fungi of the Pleurotus genus are nutritious because of their reported high content of proteins, fibers, low calorie, vitamins and minerals content (Reis et al., 2012).Their extracts in various solvents and bioactive have been tested for various pharmacological properties for therapeutic and preventive purposes and these properties are associated to their polysaccharides content (Hwang, et al., 2005;Borges et al., 2013;Barakat & Sadik, 2014;Zhao et al., 2014).
Pleurotus tuberregium (Fr.), the king tuber mushroom, is a lesser known edible fungus that belongs to the Basidiomycotina and mainly distributed in tropical and subtropical regions such as Africa, Australia, and China with history of use as food and medicine (Dso, 1977).On Nigerian village settings, it is use mainly as a substitute for meat protein in stew and soups, and is cherish as a delicacy in many dishes as a flavor and important nutrients in urban.Ot can be cooked alone as mushroom soup or with other vegetables or assorted porridge dishes.Extracts from P. tuberregium have been associated with antitumor and superoxide radical scavenging activities and for treating headaches, fever and stomach upsets (Zhang et al., 2004;Dyetayo, 2011).Ots EPS has been shown to possess pharmacological activities such as antihyperglycemic, antihyperlipidemic and antioxidant properties (Huang et al., 2012;Bamigboye et al., 2016).
supplements for its EPS production in submerged fermentation.Furthermore, chemometric methods such as Duncan multiple range test, rank-sum and cluster analysis for characterization and identification of most suitable culturing conditions and nutrients in fungi are underexplored.Therefore, characterization, identification using chemometrics and optimizing environmental conditions and nutrient supplements for optimal PtEPS and PtMG in submerged fermentation is sought in the present study to maximize P. tuberregium utilization for EPS and MG production.

Materials and methods
All chemical reagents were of ADAC standard and distilled water was prepared in the Department of Chemical Sciences Laboratory, Tai Solarin University of Education, Ojagun, Ojebu-Dde, Nigeria, where all experiments were carried out in batch culturing mode in three replicates.

Microorganism sample
Sclerotia of Pleurotus tuberregium used in this study were purchased from Bodija market, Obadan, Dyo State, Nigeria.The sclerotia were washed thoroughly with tap water to remove adhering debris, dried at 30 °C to constant weight and stored at room temperature (25 ± 2 °C).A hundred gram sclerotia was soaked in dil.H 2 D for 30 min, incubated for 3 d and finally, plated on 4% malt extract agar medium at 25 °C.

Assessment of nutritional supplements
For selection of most suitable nutrients for optimal yield of PtEPS and PtMG, 50 g glucose in the positive control medium was separately substituted with 50 g of fructose, xylose, sucrose, lactose, dextrose, and maltose.Three grams, each of yeast and malt extract in control medium were replaced with each of 10 g/L yeast, peptone, ammonium oxalate ((NH 4 ) 2 C 2 D 4 ), ammonium sulphate ((NH 4 ) 2 SD 4 ), ammonium nitrate (NH 4 ND 3 ) and ammonium chloride (NH 4 Cl).For minerals, KH 2 PD 4 .H 2 D was swop with tricalcium phosphate (Ca 3 (PD 4 ) 2 ) and sodium dihydrogen phosphate (NaH 2 PD 4 ); and sulphate ion as MgSD 4 was replaced with hydrates of iron (ii) sulphate (FeSD 4. 7H 2 D), zinc sulphate (ZnSD 4. 7H 2 D) and copper (ii) sulphate (CuSD 4 .7H 2 D); and 1g each of Na, Ca, K and Mg chloride salt was added-value.Thirty millimeters of each experimental medium was sterilized at 121 0 C for 15 min at 96 psi and 0.3 ml of 10% lactic acid was added to suppress bacterial growth after sterilization.Each 30 ml medium was inoculated with a 12 mm agar plus disc of vigorously growing P. tuberregium mycelium.

Determination of PtMG and total PtEPS in dry weight
The dry weight yield of PtMG was determined by filtration of the mycelium cultured medium through No. 1. Whatman filter paper.The residue was air dried to a constant weight using a sensitive weighing balance (Baran scientific and instrument company, England).The crude PtEPS from the various cultures was isolated using precipitation method.Ten milliliters of each cultured sample were transferred into a clean test tube and centrifuged at 4500 rpm for 15 min using Cole Centrifuge model 0414-1.Five milliliters of the supernatant was carefully transferred into a clean test tube with an addition of 10 ml 96% ethanol and left overnight at 4 °C.The precipitate was recovered by centrifugation at 4500 rpm for 15 min and washed successfully with 96% ethanol/H 2 D (1:4 v/v), lyophilized (Labconco Vacutec) and weighed.

Analysis of data
Precision measures, ANDVA, and correlation analyses were carried out using SAS v. 9.2 (Statistical Analysis System, 2002).Characterization of environmental conditions and nutrients for PtMG and PtEPS yield was by chemometrics: Duncan multiple range tests, rank-sum analysis according to Moyib et al. (2015b), and Chemo-phene trees by dissimilarity analysis representative for windows (DARwin, Perrier & Jacquemoud-Collet, 2006).

Optimizing environmental conditions
The pattern of trends shown in Figure 1 indicate favorable acidic medium, long incubating periods and increasing cultivating temperature till a peak at 30 °C for high yield of PtMG and PtEPS.Medium acidity of pH 6, culturing temperature of 30 °C and incubating period of 15 d were most suitable environmental conditions for production of both PtMG and PtEPS in P. tuberregium.Analysis of varance statistics shows various initial medium pHs, incubating periods and cultivating temperatures were highly significant for both the PtMG and PtEPS (p <0.0001, r 2 = 0.99) except for non-significant difference among cultivating periods of 20, 25 and 35 d for PtEPS and Pearson correlation analysis shows a strong inverse relationship among level of pH with PtMG (p<0.0001,r 2 = -0.82)and PtEPS (p<0.0001,r 2 = -0.81)yields.Therefore, as the culturing medium acidity increases, levels of PtMG and PtEPS were decreasing, supporting a favorable acidic medium for P. tuberregium growth and metabolic activities as noted earlier.Oncubating period had positive relationships with PtMG (p<0.0001,r 2 = 0.84) and PtEPS (p<0.0001,r 2 = 0.81), supporting lengthy incubation period.Cultivating temperature showed inverse relationships with both PtMG (p<0.05,r 2 = -0.59)and PtEPS (P<0.001,r 2 = -0.65)and apparently, indicates higher levels of PtMG and PtEPS at low temperatures, but highest level was obtained with a median temperature (30 o C) and hence, the low r 2 (0.59).The present results are similar to previous reports of acidic medium, a cultivating temperature above room temperature and above 10 d incubating period in fungi (Nehad & El-Shamy, 2010; Adejoye, et al. 2012;Joshi et al., 2013;Lai et al., 2014).

Optimal carbon sources for energy
Figure 2a shows glucose and lactose with highest and lowest yields, respectively, for both PtMG and PtEPS.Adejoye et al. (2012) suspicious of galactose' positive role in lactose for L. squarrosulus is somehow supported with its fourth position to glucose.The very low levels of PtMG and PtEPS observed in the negative control indicate P. tuberregium could survive in no-sugar medium but with low metabolic activities.Yields of PtMG and PtEPS were significantly distinct among the various carbon sources (p<0.0001,r 2 = 0.99).The present choice of glucose as optimal carbon source for both PtMG and PtEPS is similar to reports in fungi (Nehad & El-Shamy, 2010;Barakat & Sadik, 2014;Lai, et al., 2014) and contrasts to starch, sucrose, maltose and xylose in some other studied fungi (Joshi et al., 2013;Mahapatra & Banerjee, 2013;Anike et al., 2015).Though, many diverse explanations such as affinity of a particular fungus for a specific sugar and preference between mono-and disaccharides were given but many lack biochemical elucidation.The present results are expected for glucose, one, as the only starting and direct sugar for glycolytic pathway that generates ATP in cells respiration (Berg et al., 2007).Two, the transportation barrier varies from facilitated simple diffusion for monosaccharides to active transport for maltose (Garrett & Grisham, 2005;Berg et al., 2007).Three, enzymes  affinity for substrate suggests greater affinity of hexokinase to hexoses than glucosidase to disaccharides for breaking glycosidic bond (Garrett & Grisham, 2005;Berg et al., 2007).Therefore, the present result infers rate of metabolism of sugars, perhaps, as a potential limiting factor in MG and EPS production, which requires biochemical investigation.

Optimal nitrogen source for protein
Yeast showed highest induction and closely followed by peptone for PtEPS and PtMG yields (Figure 2b).Chemo-nitrogen sources were at the bottom level indicating their lower productivity to bio-nitrogen sources in P. tuberregium.The very low levels of PtMG and PtEPS observed in negative control indicate non-absolute reliance of P. tuberregium on nitrogen.High significant variation was observed among the assessed nitrogen sources for both PtMG and PtEPS (p<0.0001,r 2 = 0.99).The present result supports and establishes the importance of yeast as the most utilized nitrogen source and complex organic nitrogen are preferable over chemo-nitrogen by fungi (Nehad & El-Shamy, 2010;Joshi et al., 2013;Mahapatra & Banerjee, 2013;Anike et al., 2015).

Optimal chloride for macromineral
Figure 2c shows NaCl as the optimal chloride for both PtMG and PtEPS and the comparative levels obtained in the controls suggest low importance of chlorides for production of PtMG and PtEPS.The lowest level of CaCl 2 suggests an inhibitory effect, which could be explained by its larger molecular size, lattice energy and the amount used in term of either micro or macro source.The present result supports NaCl as a choice of chloride salt for MG while few mentioned CaCl 2 but when used as micro mineral (Lai et al., 2014;Boumaaza et al., 2015).The present result indicates low demand for chlorides as obtainable in NaCl while CaCl 2 and FeCl 3 weren't favorable and their dosage, perhaps, caused an osmotic pressure in the culturing media and might led to cytotoxicity (Turkkan, 2013).

Optimal sulphate and phosphate for micronutrient
According to Figure 2d, Ca 3 (PD 4 ) 2 , induced the highest level of both PtMG and PtEPS.Figure 2e shows MgSD 4 as the optimal source for PtMG and ZnSD 4 was followed by FeSD 4 .7H 2 D and MgSD 4 for PtEPS production.The high levels of assessed sulphates signify importance of SD 4 2-for PtMG and PtEPS production and are in good utilizable forms (Srivastava, 1968).The assessed phosphate and sulphate salts were significantly demarcated for PtMG and PtEPS yields (p<0.0001,r 2 = 0.99).Ca 3 (PD 4 ) 2 as the most suitable source could replace KH 2 PD 4 in culturing medium for P. tuberregium.Since, MgSD 4 was optimal among sulphates for PtMG and followed ZnSD 4 for PtEPS, MgSD 4 was concluded as a choice of sulphate for both PtMG and PtEPS for convenience of further experiments.Similar studies merged phosphate and sulphate sources for assessment of optimal minerals and results reported so far differ greatly for comparison with the present observations due to non-similar mineral salts assessed (Srivastava, 1968;Turkkan, 2013;Lai et al., 2014).

Characterization of various environmental condition and nutrients for optimal production of PtMG and PtEPS
The 52 assessed environmental conditions and nutrients were ranked for their stimulatory effects on PtMG and PtEPS yields according to Moyib et al. (2015b).Six stimulant classes were generated, excellent stimulant (ES), good stimulant (GS), stimulant (S), fair stimulant (FS), poor stimulant (PS) and non-stimulant (NS) (Table 1).For examples, yeast, NaCl and Ca 3 (PD 4 ) 2 were classified as ES for both PtMG and PtEPS; glucose was ES for PtEPS but GS for PtMG (detailed classification is provided in Table 1).A chemo-phene tree was generated to reveal relationships and visual groupings among the 52 environment and nutrient conditions (Figure 3). Figure 3 reveals six clusters for PtEPS and each cluster was distinct for each stimulant class in Table 1 except for few odds.Examples, for PtEPS, cluster 2 consists ES with an odd GS class (-vechloride), and cluster 4 has S class with an odd ES (pH6).
Noteworthy, the odd nutrients/conditions identified by the chemo-phene tree were observed at barrier between two sequential classes in Table 1 and preferably, they should be right class in their present clusters (for example, pH 6 should be S class for PtEPS).The chemo-tree for PtMG follows similar demarcations for PtEPS but with little dissimilarity (Figure not shown).Therefore, glucose and fructose should be right placed as ES and GS, respectively.Therefore, clustering is an added value that identified misclassification of odd conditions and nutrients in rank-sum analysis and also, rank-sum analysis was able to reduce the large groupings in Duncan grouping to a manageable group size and they complement one another.
Classification of nutrients and environmental conditions for their stimulatory effects on MG and EPS is scanty in fungi.dose of MgSD 4 till 2 g/L and fell sharply at 3 g/L (Figure 4i, j).PtEPS showed a different pattern, it increases with increasing dose till 0.5 g and dropped sharply at 1.0 g and then gradually till 3 g/L, indicating higher requirement of sulphate for PtMG than PtEPS.The present results signifies Ca 3 (PD 4 ) 2 and MgSD 4 are important nutrients require in minute quantity as expected of microminerals and are within the reported range in fungi (Mahapatra & Banerjee, 2013;Lai et al., 2014).Generally, the patterns of the curves generated for PtMG and PtEPS production using regression analytical tool in SPSS (v.17) were best fitted with cubic model.

Conclusions
The present chemometrics, Duncan multiple range test, ranksum analysis and hierarchical clustering in the given successive sequential order, complement one another, and were able to classify assessed conditional and nutrient sources into useful stimulatory classes for optimal yields of PtEPS and PtMG and allowed robust selection of most suitable nutrients for PtEPS and PtMG.The present results are useful for production of PtMG and PtEPS for pharmaceutical, food, cosmetics and any applicable industrial purposes.
However, such classification has been reported for proper utilization in food crops (Moyib et al., 2015a(Moyib et al., , 2015b) ) and at present extended to P. tuberregium.Nonetheless, selection of optimal conditions and nutrients based on ANDVA, response surface methodology, orthogonal matrix method, and Plackett-Burman design, for optimization of EPS production have been successful used in fungi (Borges et al., 2013;Joshi et al., 2013;Mahapatra & Banerjee, 2013) and the present chemometrics, Duncan multiple range test, rank-sum procedure and clustering analysis are also suffice, robust, simple, informative and self-explanatory in nature.

Optimization of dosages of glucose, yeast, NaCl, MgSO 4 and Ca 3 (PO 4 ) 2
Figure 4a and b show increasing glucose level from 10 to 100 g/L in a culture medium containing 10 g yeast, 1.0 g nicotinic acid, 20 g NaCl, 1 g MgSD 4 and 1 g Ca 3 (PD 4 ) 2 per liter caused an increasing in the production of PtMG till 40 g/L glucose and PtEPS production increases till 60 g/L glucose, after which both fell gradually till 100 g/L glucose.Glucose has been reported in varying amounts that ranged between 40 and 100 g/L for optimal production of EPS, of which the present selected concentrations of 40 (PtMG) and 60 g/L (PtEPS) are within the range reported for many fungi.Such high demand of glucose for EPS has been previously observed, even at higher NaCl levels (Nehad & El-Shamy, 2010;Lai et al., 2014).The higher glucose required for PtEPS over PtMG should be expected, since glucose is one of the basic units of EPS as glucans and mannogalactans (Zhang et al., 2004;Huang et al., 2012).
For yeast, 5 to 40 g/L dosages were evaluated, PtEPS and PtMG responded positively to increasing the dosage of yeast till 15 g/L and 20 g/L, respectively, after which, both fell gradually with increasing yeast dose till 40 g/L (Figure 4c, d).Therefore, in the presence of 50 g glucose and 20 g NaCl, a moderate dose of 15 g/L and 20 g/L yeast favored PtEPS and PtMG production, respectively and are comparable to 25 g/L chosen for MG and EPS, in L. squarrosulus (Anike et al., 2015) while lower concentrations of yeast have been reported in some other fungi (Nehad & El-Shamy, 2010;Joshi et al., 2013).
Concentrations of NaCl for optimization was tested from 20 to 150 g/L in a medium containing 50 g glucose, 20 g yeast, 1g each of Ca 3 (PD 4 ), MgSD 4 and nicotinic acid.Figure 4e shows increasing NaCl dose beyond 20 g/L reduced PtMG production, and growth was halted at 100 g/L, indicating salt saturation with a low yield at highest dose of 150 g/L.PtEPS production was favorable till 40 g/L NaCl and depreciated with further increasing dose (Figure 4f).The present result suggests NaCl is a value-added nutrient but at moderate concentration that shouldn't exceed 40 g/L, beyond which the growth of P. tuberregium could be stunted and PtEPS production truncated (Turkkan, 2013;Boumaaza et al., 2015).Surprisingly, level of PtEPS increases gradually with increasing dose of PD 4 3-from 0.05g to 1.00 g/L and reduces sharply at increasing dose beyond 1.0 g/L while PtMG increases sharply till a dose of 2 g/L and fell at 3 g/L (Figure 4g, h).The result shows that PD 4 3-supported production of both PtEPS and PtMG but at different doses.Production of PtMG increases with increasing

Figure 1 .
Figure 1.Trend of PtMG and PtEPS among environmental conditions in P. tuberregium.(a) Pattern of PtMG and EPS among various assessed media pH (b) pattern of PtMG and PtEPS among incubating periods (c) pattern of among cultivating temperatures.