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Production and partial characterization of the exopolysaccharide from Pleurotus sajor caju

Production and partial characterization of the exopolysaccharide from Pleurotus sajor caju Purpose Microbial exopolysaccharides (EPSs) are very important because they are used in biotechnological applications in different industrial areas. The aim of the study was to determine the best EPS producer Pleurotus sp., to optimize EPS production and to perform partial purification and characterization of the produced EPS. Methods After the production conditions were optimized, the EPS was isolated and partially purified. EPS was characterized by HP-TLC, H-NMR, FT-IR, and TGA. Hydroxyl, superoxide, and DPPH radical scavenging activities of the EPS were also investigated spectrophotometrically. Result The best EPS producer and its incubation period in submerged fermentation were determined as Pleurotus sajor caju and on 5 days, respectively. Culture conditions to increase EPS production were optimized as follows (in per liter): 90 g of glucose, 2+ 10 g of yeast extract, 10 g of peptone, and 100 mM of Mg . The optimal initial pH, temperature, and an agitation rate of culture −1 were determined as 5.0, 25 °C, and 150 rate min , respectively. The highest EPS production was determined as 33.32 ± −1 1.6gL . After isolation of EPS, one active fraction was obtained by gel filtration chromatography. EPS is composed mainly of glucose according to HP-TLC analysis. Conclusion To the results, EPS had a complex structure by having carbohydrate and protein contents. The produced EPS had high degradation temperature as well as high antioxidant activity. . . . . Keywords Pleurotus sajor caju Submerged fermentation EPS production EPS characterization Antioxidant activity Introduction important components that determine the hardness and mor- phological characteristics of the fungal cell wall and which Polysaccharides, a group of valuable biopolymers, have appli- can be excreted into the culture medium depending upon the cations at a wide range of industrial fields, like food and phar- culture conditions (Gern et al. 2008). Many microorganisms maceuticals. In recent years, extracellular polysaccharide synthesize EPS in the form of amorphous loam that binds to (EPS) production has been prominent with numerous fungal the cell surface or is present extracellularly. EPSs help the cell submerged cultures since it has biological and pharmacologi- in a wide variety of functions. EPSs provide protection against cal activities such as anti-tumor, antioxidant, and hypoglyce- biotic stress and protection against abiotic stresses that may mic activities (Han et al. 2006;Song etal. 2008;Li etal. 2010; include nutrient limitation, temperature, light intensity, or pH. Zhao et al. 2012; Cao et al. 2018). Polysaccharides are For example, in acidophilic or thermophilic species, EPSs help to adapt to extreme conditions (Antón et al. 1988). The genus Pleurotus is widely cultivated and commercial- * Raziye Ozturk Urek ized in the world. It involves many biologically active com- raziye.urek@deu.edu.tr ponents, such as polysaccharides, proteins, enzymes, dietary fibers, and vitamins (Wang et al. 2001). Large amounts of Seda Ilgin sedailginn@gmail.com biomass and EPS production, which can be used for biotech- nological areas, occur in the submerged cultures of the Faculty of Science, Chemistry Department, Biochemistry Division, Pleurotus species. For instance, water-soluble polysaccharide Dokuz Eylül University, 35160 Buca, Izmir, Turkey of Pleurotus ostreatus could be a potent immune stimulant for Graduate School of Natural and Applied Sciences, Biotechnology use in nutraceuticals or medicine against both pathogens and Department, Dokuz Eylül University, 35160 Buca, Izmir, Turkey 1202 Ann Microbiol (2019) 69:1201–1210 cancer (Ooi and Liu 2000). However, EPS of Pleurotus cooled. Inoculated flasks were incubated on the rotary shaker −1 pulmonarius showed in vitro antioxidant activity (Shen et al. at 150 rate min and25 °Cfor 2weeks. 2013). Likewise, it was observed that EPS produced via To increase EPS production, sucrose, glycerol, and sorbitol −1 Pleurotus eryngii has antioxidant and antitumor activities as 10 g L were used as carbon source instead of glucose. The (Jing et al. 2013). A few works on EPS production have also optimum concentration was determined after glucose was been carried out by submerged fermentation (SmF) of identified as the best carbon source. The effects of varying Pleurotus spp. (Silveira et al. 2015). To improve EPS produc- nitrogen sources and concentrations, such as peptone, yeast tion, some nutritional factors have been investigated on the extract, ammonium nitrate, and urea, on EPS production were 2+ SmF of fungi. investigated. Then, different concentrations of Mg ion (4, 7, The purpose of this study is to achieve high efficiency EPS 10, 30, 50, 100, 250 mM), various pH levels (3, 4, 5, 6, 7, 8, production in SmF using white-rot fungi Pleurotus spp. In the 9), different fermentation temperatures (20, 25, and 30 °C), −1 first step of this study, the best producer Pleurotus strain and and agitation rate (100, 150, and 180 rate min )were inves- its incubation period were determined. In the next step, effects tigated. All samples were harvested on the 5th day of incuba- 2+ of carbon and nitrogen sources, and concentrations, Mg ion tion and analyzed for EPS production. concentration, pH, temperature, and agitation rate were inves- EPS samples taken from the flasks were centrifuged at tigated to optimize EPS production conditions in SmF. After 10000×g for 20 min, and then, the upper phase was filtered production at an optimum condition in SmF, EPS was extract- using a 0.45-μm membrane filter. The supernatant was used ed and partially purified by Sepharose CL-6B column. In the for analysis below. The dry weight of cell (CDW) was mea- last step of the study, partial purified EPS was characterized by sured after washing the mycelial pellet with distilled water and chromatographic (TLC), spectroscopic (FT-IR and H-NMR), drying at 70 °C to obtain invariable value. For EPS isolation, and TGA analyses. Also, antioxidant features, like hydroxyl, the obtained culture filtrate was mixed with four volumes of superoxide, DPPH radical scavenging activities, reducing absolute cold ethanol, admixed vigorously, and put for 24 h at power, and chelating activity of the partial purified EPS, were 4 °C. The precipitated crude EPS was obtained by centrifuga- investigated. tion at 10000×g for 20 min and then dried at 60 °C overnight to obtain constant value. Protein content of cell-free supernatant of production me- dia was determined by Bradford (1976) using bovine serum Materials and methods albumin standard. The content of reducing sugar and nitrogen was determined by DNS method and phenol-hypochlorite as- Maintenance of microorganisms say, respectively (Miller 1959; Weatherburn 1967). Four different Pleurotus strains were used in the study: Partial purification and characterization of produced Pleurotus djamor (Rumph. Ex Fr.) Boedijn (MCC15), P. EPS ostreatus (Jacq.) Pleurotus Kumm. (MCC16), Pleurotus sajor caju (Fr.) Singer (MCC29), and P. eryngii (DC.) Gillet EPS precipitated with ethanol was handled with Sevag reagent (MCC58). Pleurotus djamor, P. ostreatus,and P. sajor caju (1:4 n-butanol/chloroform, v/v)(Staub 1965). After centrifu- −1 were maintained on potato dextrose agar (PDA) (39 g L , gation, water phase was ultra-filtered (10 kDa) and filtrate was pH 5.6) at 25 °C for 7 days; P. eryngii was grown on malt admixed with absolute cold ethanol; the precipitated EPS was −1 extract:peptone:agar (MPA) (30:3:15 g L ,pH 5.6) at 25 °C centrifuged and dried. The EPS (100 mg) was disintegrated in for 14 days (Akpinar and Ozturk Urek 2017). 0.2 M NaCl buffer, and fulled onto a Sepharose CL-6B col- umn (1.6 cm × 90 cm). The column was eluted with this buffer −1 Production, optimization, and isolation of EPS at a flow rate of 0.6 mL min . Five dextran molecules (25000, 80000, 270000, 670000, and 1100000 Da) were used Pleurotus spp. were grown on PDA and MPA slants and then as molecular-weight standards. The dextran standards transferred to basal medium by 1 cm of the five agar plates (2.5 mg) were prepared in 0.2 M NaCl buffer, and loaded. with a sterilized cutter. Submerged fermentation was per- The protein level in the gel fractions was measured at formed in 250-mL Erlenmeyer flasks including 100 mL of 280 nm, while the carbohydrate level was recorded at 490 nm. basal medium. The basal medium composition was as follows The total carbohydrate, protein, uronic acid, and pyruvate −1 in (g L ): glucose, 10; NH NO , 0.724; KH PO ,1.0; contents of the EPS were investigated by the phenol-sulfuric 4 3 2 4 MgSO .7H O, 1.0; KCl, 0.5; yeast extract, 0.5; acid method, Bradford method, borate-sulfuric acid-carbazole 4 2 FeSO .7H O, 0.001; ZnSO .7H2O, 0.0028; CaCl .2H O, assay, and 2,4-dinitrophenyllhydrazine assay, respectively 4 2 4 2 2 0.033; peptone, 10 (Bazalel et al. 1997). The medium pH (Dubois et al. 1956;Healyetal. 1996; Friedemann and was adjusted to 6.0 and sterilized at 121 °C for 20 min then Haugen 1943). 1203 Ann Microbiol (2019) 69:1201–1210 To determine monosaccharide composition of the partial in Fig. 1a, our results indicated that P. sajor caju was the best −1 purified EPS, standard sugars (glucose, mannose, galactose, strain for the EPS production (2.62 ± 0.16 g L , on the 5th rhamnose, and xylose) were analyzed by HP-TLC (CAMAG). day of incubation period). The maximum EPS productions by Ethyl acetate:acetic acid:water (2:2:1) was used as mobile P. eryngii, P. djamor,and P. ostreatus were achieved on the phase on TLC silica gel 60 F254 plates (MERCK, 6th, 3rd, and 7th days of incubation period as 2.1 ± 0.35, 1.4 ± −1 Germany) with solution as staining reagent (2% 0.3, and 1.2 ± 0.25 g L , respectively. Our results showed that naphtoresorcinol in 10% ethanolic phosphoric acid). EPS EPS production depends on microorganism’s strain. Likewise, (50 mg) was hydrolyzed with 2 N H SO (4 mL) at 100 °C Nehad and El-Shamy (2010) reported that the yield of EPS 2 4 in oil bath for 2 h, then it was neutralized with NaOH (1 g) and produced by different fungal strains was different. loaded on plates and analyzed. After the determination of the best EPS producer as P. sajor For spectroscopic analysis, EPS, FT-IR, and H-NMR were caju, the effects of incubation time and pH of the medium on used. The FT-IR spectrum was monitored on the Perkin Elmer EPS production were investigated. Figure 1 b shows that max- −1 1 −1 Spectrum BX, in the 4000–400 cm spectral region. For H- imum EPS production was determined as 2.62 ± 0.16 g L on NMR spectroscopy, the sample was recorded, as solution in the 5th day of incubation, while maximum CDW was obtain- −1 dimethyl sulfoxide, on a MERCURY Plus-AS 400 spectrom- ed as 4.06 ± 0.4 g L on the 7th day. pH value of SmF medi- eter at 400 MHz and 30 °C. The chemical shifts were um dependent on incubation period was not a significant expressed in ppm. change up to 7 days, and then it slightly increased to 8.17 ± A thermal gravimetric analysis of EPS was carried out with 0.07 on the 12th day. Perkin Elmer-Diamond TG/DTA. About 3–5mg of dryEPS Chemical composition analyses of SmF medium were car- sample was loaded on a platinum pan and its energy level was ried out during the incubation period (Fig. 2). Maximum pro- scanned in the ranges of 30–600 °C under a nitrogen atmo- tein and nitrogen levels were detected as 78.09 ± 3.2 ppm and −1 sphere with a temperature gradient of 10 °C min . 459.9 ± 17 ppm on the 9th and12th days of incubation, respec- tively. Level of reducing sugar on the 2nd day of incubation Evaluation of antioxidant activity of partial purified was determined as 593.4 ± 29 ppm and limited conditions EPS were reached on the 5th day. After the determination of the best EPS producer as P. sajor Hydroxyl and superoxide radical scavenging activities of EPS caju and the incubation day (5th) for EPS production were measured according to the methods of Halliwell et al. (1987) and Liu et al. (1997), respectively. DPPH and chelating activity methods were based on Kumaran (2006) and Decker and Welch (1990), respectively. The reducing power of EPS 2.5 was detected to the method of Oyaizu (1986). 1.5 Statistical analysis 1 0.5 All experiments were carried out three times (n = 3) and re- P. ostreatus P. eryngii P. djamor P. sajor caju peated three times. Each value is the average of three parallel Pleurotus strains experimental studies. Data were given as mean ± standard deviation (SD). The data were assayed by analysis of variance 4.5 10 (ANOVA) to identify the significantly different groups at P < 0.05 by one-way ANOVA test utilizing SPSS software 3.5 statistical program (SPSS for windows ver. 21.00, USA). 2.5 1.5 Results and discussion 3 0.5 Optimization of fermentation conditions 0 0 23579 12 Incubation period (day) EPS production capacities of four different Pleurotus spp. CDW (g/l) EPS (g/l) pH were investigated in order to determine the best EPS producer in the basal medium of SmF and the incubation day of max- Fig. 1 a Production of EPS by Pleurotus strains. b Production of EPS by imum EPS production. They were incubated on the shaker P. sajor caju and CDW levels during incubation period. The values are −1 mean ± SD of three separate experiments incubator at 150 rates min and 25 °C for 2 weeks. As shown Concentration (g/L) Concentration (g/L) pH 1204 Ann Microbiol (2019) 69:1201–1210 Fig. 2 Glucose, nitrogen, and 700 100 protein levels of medium during incubation period of P. sajor caju. The values are mean ± SD of three separate experiments 0 0 02468 10 12 14 Incubation period (day) Glucose Nitrogen Protein −1 −1 according to the yield of EPS, components of the SmF medi- production (3.97 ± 0.15 g L ) and CDW (6.03 ± 0.32 g L ) −1 um and fermentation conditions were optimized. To find out was 90 g L . the most appropriate carbon source in EPS production by P. The carbon source is required for energy and structural sajor caju, three different carbon sources named sorbitol, su- molecules. Therefore, the concentration and type of carbon −1 crose, and glycerol were separately provided at 10 g L in- source are important for all bioprocesses. The carbon source stead of glucose employed in the basal medium. Betwixt the and its concentration have a major impact on the production investigated carbon sources, maximum EPS production and and yield of EPS. Glucose, sucrose, maltose, lactose, fructose, −1 CDW were obtained in glucose medium as 2.62 ± 0.16 g L galactose etc. are generally used as carbon sources in the cul- −1 and 2.83 ± 0.14 g L ,respectively (Fig. 3a). ture medium (Mahapatra and Banerjee 2013). In most studies, After selecting glucose as the best carbon source for EPS glucose, sucrose, and maltose have been chosen as the most production, the optimum concentration of glucose was deter- necessary carbon source for the fungal EPS production (Kim mined for EPS production (Fig. 3b). For this purpose, various et al. 2005; Zhang and Cheung 2011). These results suggest glucose concentrations were used in medium and the results that various carbon sources may have some catabolic repres- showed that optimum glucose concentration for EPS sion effects in various EPS syntheses. It is also an indication that different fungal strains have different carbon source in- take characteristics or that these carbon sources can be easily utilized by fungi. Furthermore, the structure and concentration 3.5 of the carbon source used can regulate secondary metabolism, such as catabolic repression (Görke and Stülke 2008). 2.5 In this study, the capability of P. sajor caju to use various nitrogen sources for EPS production was analyzed. For this 1.5 CDW (g/l) purpose, P. sajor caju cells were grown in basal medium con- EPS (g/l) −1 taining glucose (90 g L ) as a carbon source, and four diverse 0.5 nitrogen sources, namely, peptone, yeast extract, ammonium nitrate, and urea, were added at various concentrations. Sorbitol Glucose Sucrose Glycerol Figure 4a shows that maximum EPS production was achieved Different Carbon Sources (g/L) −1 −1 −1 in 10 g L peptone, 10 g L yeast extract, and 0.724 g L −1 −1 NH NO as 4.01 ± 0.38 g L , 3.56 ± 0.3 g L , and 3.09 ± 4 3 −1 0.27 g L ,respectively.Figure 4b indicates that maximum −1 −1 CDW was determined in 12 g L peptone and 10 g L yeast −1 −1 extract medium as 5.93 ± 0.5 g L and 8.51 ± 0.77 g L , 4 respectively. Our findings are similar to those of other re- searchers, which show comparatively low mycelial growth and EPS production with inorganic nitrogen sources com- CDW (g/l) pared with organic nitrogen sources (Yang et al. 2003). EPS (g/l) −1 Similarly, nitrogen concentrations between 1 and 10 g L are sufficient for fungal EPS production (Yuan et al. 2012). 5 1020406090 120 180 2+ Mg concentration effects on EPS production and myce- Glucose concentration (g/L) 2+ lial growth were investigated (Fig. 5). Addition of Mg to the Fig. 3 a Effect of carbon sources on EPS production by P. sajor caju. b culture medium may increase EPS production. The maximum Effect of glucose concentration on EPS production by P. sajor caju.The −1 amount of 31.08 ± 3.0 g L EPS was obtained with the values are mean ± SD of three separate experiments Concentration (g/L) Concentration (g/L) Reducing Sugar and Nitrogen Levels (ppm) Protein Level (ppm) 1205 Ann Microbiol (2019) 69:1201–1210 stabilization of the plasma membrane, and its uptake into the cell is also dependent on ATP. 4 To study the effect of the initial culture pH on EPS produc- tion, P. sajor caju was cultivated in SmF with various starting pH (3.0–9.0); after 5 days, it was analyzed for EPS production (Fig. 6a). The results showed that the optimum pH for EPS −1 −1 production (33.32 ± 1.6 g L ) and CDW (7.49 ± 0.5 g L ) was 5 and 7, respectively. In general, fungi preferred low pH 0.5 0.724 1.5 3 6 10 12 for EPS production with a range between pH 3.0 and 6.5 Nitrogen Concentration (g/L) (Feng et al. 2010). In order to determine the optimum temper- Peptone Yeast extract NH4NO3 Urea ature, P. sajor caju was cultivated at different temperatures in the shaking incubator for 5 days; the optimal temperature for EPS production was detected at 25 °C (Fig. 6b). The maxi- mum CDW was achieved at 30 °C. Similarly, many microor- ganisms produce maximal amounts of EPS at temperatures slightly lower than optimal growth as observed for 3 Klebsiella sp. (Farres et al. 1997). Many researchers have reported different optimal temperature values for EPS produc- tion; changes in environmental factors appear to give rise to 0.5 0.724 1.5 3 6 10 12 Nitrogen Concentration (g/L) Peptone Yeast extract NH4NO3 Urea Fig. 4 a Effect of nitrogen sources and concentrations on EPS production and b CDW by P. sajor caju. The values are mean ± SD of three separate 40 experiments supplementation of 100 mM MgSO .7H O. The maximum 4 2 −1 2+ 10 CDW (19.25 ± 1.89 g L ) was achieved in 50 mM Mg . Therefore, 100 mM MgSO .7H O was selected as the best 4 2 concentration for the EPS production by P. sajor caju.This pH can be explained by the fact that sulfates may also have effects CDW EPS on EPS production (Liu and Miao 2017). Raposo et al. (2014) expressed that the highest yield of EPS was observed when 21 mM MgSO was added to the culture medium of Porphyridium cruentum; the total yield of EPS in microalgae cultivation suggests that it may be affected by varying MgSO concentrations. Many researchers have deemed this bio- element to be suitable for mycelial growth and EPS produc- tion, usually in liquid cultures of several basidiomycetes (Chardonnet et al. 1999; Hwang et al. 2003). It is known that 20 25 30 Temperature (°C) cations affect EPS synthesis both qualitatively and quantita- 2+ EPS CDW tively. Mg is required for all fungi and used as a cofactor in many enzymatic reactions. It is also involved in the 100 150 180 4 7 10 30 50 100 250 Agitation Rate (rpm) 2+ Mg Concentration (mM) CDW EPS CDW EPS Fig. 6 The effect of a initial pH, b temperature, and c agitation rate on 2+ Fig. 5 Effect of different Mg ion concentrations on EPS production by EPS production and CDW by P. sajor caju. The values are mean ± SD of P. sajor caju. The values are mean ± SD of three separate experiments three separate experiments CDW (g/L) EPS (g/L) Concentration (g/L) Concentration (g/L) Concentration (g/L) Concentration (g/L) 1206 Ann Microbiol (2019) 69:1201–1210 some changes in EPS efficiency, contingent upon the type of was reported by Shen et al. (2013), and one fraction was fungus (Ruas-Madiedo et al. 2002). To find the agitation rate isolated by purification of polysaccharide from P. effect on the EPS production, P. sajor caju was cultivated in a pulmonarius. Many research findings indicated that in EPS medium with various agitation rates (100, 150, produced by fungi, glycoprotein generally occurs (Hwang −1 180 rate min ). Figure 6c shows that maximum EPS produc- et al. 2003;Heet al. 2012). tion and CDW were obtained at an agitation rate of The molecular weight of the produced EPS was detected as −1 150 rate min . Additionally, aeration and agitation are impor- the low-molecular-weight EPS of approximately 25 ± 3 kDa. tant factors to ease the oxygen transfer and enable nutrient Similar observations were made by Oh et al. (2007)and transport to microorganisms. The presence of oxygen and Cheng et al. (2011) in other fungi fermentations. The molec- the rate at which the culture medium is admixed might have ular weight of EPS produced by P. sajor caju (Fr.) Singer 4 −1 a direct effect on the production polysaccharide, and an in- (CCB 019) was determined as 6.4 × 10 gmol according crease in EPS production often results from the increase in to Silveira et al. (2015). Differences in Mw may indicate dif- oxygen supply (Dassy et al. 1991; Bayer et al. 1990). The ferences in the methods used in isolation and production pro- EPS surrounding the cell can serve as a barrier for oxygen cedures, or for the determination of Mw. and nutrient transfer. Admixing culture at high rates can in- crease the availability of both nutrients and oxygen. Partial characterization of EPS The obtained results showed that the EPS yield was the highest when the concentrations of glucose, yeast extract, pep- HP-TLC is an effective chromatographic method for detecting 2+ −1 tone, and Mg were 90, 10, 10 g L , and 100 mM, respec- monosaccharide composition. On HP-TLC analysis, the Rf tively. Thus, the yield of EPS in the optimized culture medium values of the standards were as follows: glucose 0.456 ± was found to be about 13.33 times higher than that obtained 0.04, galactose 0.445 ± 0.05, mannose 0.503 ± 0.05, xylose when the basal culture medium was used. According to the 0.551 ± 0.06, and rhamnose 0.563 ± 0.04 (Fig. 7a). Our results −1 optimized conditions, the highest EPS was 33.32 ± 0.3 g L . showed that Fr-I mainly included glucose. Many fungal EPSs The EPS obtained in this study is 6.45 times more than that are composed of glucose monosaccharide (Lin and Chen produced by P. eryngii and 5.24 times more than P. 2007). Themonosaccharidecomposition was veryimportant pulmonarius (Jing et al. 2013;Shenetal. 2013). To to determine the produced EPS function. Osińska-Jaroszuk et al. (2015), the maximum produced EPS The FT-IR spectrum is a method used to detect functional yield from Ascomycota and Basidiomycota fungi ranges from groups and characterize covalent binding information. Typical −1 0.12 to 42.24 g L , which is mostly dependent on the strain FT-IR spectrum for Fr-I is presented in Fig. 7b.Fr-Idisplayed and culture conditions used. a stretching intense distinctive peak at approximately the re- −1 Recently, it is important to investigate the chemical com- gion of 3388 cm for the carbohydrate ring. Small band at −1 position and physicochemical properties of EPS produced 2948 cm was assigned to the stretching vibration of the since the introduction of natural polymers for industrial appli- methylene group (C-H), commonly found in hexoses. A −1 cations increases the interest in EPS production. After optimi- distinguishing peak absorption band emerged at 1638 cm zation of EPS production conditions, the produced EPS was was ascribed to the stretching vibration of the carboxyl group −1 isolated and its chemical characterization was assayed. The (C=O). A meager peak about 1110 cm corresponding to the chemical composition of 1 mg isolated EPS was formed by glycosidic link bond (C-O-C) was determined in the spectrum 2.26 ± 0.41 μg protein, 219.75 ± 10 μg total carbohydrate, of the polysaccharide (Copikova et al. 2001). The stretching −1 28.59 ± 0.32 μg reducing sugar, 15.73 ± 1.1 μg nitrogen, vibration peak of 1040 cm implied the presence of C-H-O 1.01 ± 0.01 μg pyruvate, and 6.7 ± 0.5 μg uronic acid. linkage position. The weak absorption peak at 985 has coin- Results of chemical composition analyses demonstrated the cided to N-H of primary amine. The FT-IR spectrum showed complex structure of produced EPS. that proteins and polysaccharides were present in EPS com- position. Additionally, it was determined that MgSO did not Isolation and partial purification of EPS bear an impact on the structure of EPS in the FT-IR spectrum. This data was also supported by Liu and Miao (2017). The produced EPS in optimum SmF conditions by P. sajor According to the H-NMR spectrum of Fr-I, signals at 2.2– caju of 90 g glucose, 10 g peptone, 10 g yeast extract, 100 mM 2.8 ppm were ascribed to peaks of C-H (data not shown). The 2+ Mg per liter, pH 5, at 25 °C, and 150 rpm was isolated. C-C signals were established in the region 3.0–4.2 ppm, Isolated EPS was mixed with Sevag reagent then ultrafiltrated which were ascribed to protons of the C-2-C-6 glycoside ring and applied on gel filtration chromatography (Sepharose CL- of hexoses. The NH signals at 7.1–7.5 ppm demonstrated 6B). Active one fraction of EPS (Fr-I), which consisted of amino acid and peptide structure. The obtained results dem- polysaccharides and proteins, was co-eluted. Fr-I was revealed onstrated that EPS produced by P. sajor caju had functional to be glycoprotein based on preliminary data. A similar result groups, bonds, and structures which are present in 1207 Ann Microbiol (2019) 69:1201–1210 218,3 %T 3388 2368 40 1110 625 0,0 4000,0 3000 2000 1500 1000 500 200,0 cm-1 Fig. 7 a HP-TLC analysis of EPS and standards. Solvent system-ethyl (3), (4), (5), and (6) mannose, glucose, galactose, xylose, and rhamnose, acetate butanol:acetic acid:water (2:1:1), spray reagent; 2% respectively. b FT-IR spectrum of EPS. The values are mean ± SD of three naphtoresorcinol in 10% ethanolic phosphoric acid. Spot (2) Fr-I; (1), separate experiments glycoprotein-type polysaccharide. It was deduced that EPS Antioxidant activities of partial purified EPS from P. possessed a complicated structure with carbohydrate and pro- sajor caju tein contents. The presence of thermal durability of the polysaccharide Free radicals, especially hydroxyl radicals and other derived is a paramount property for its applications. TGA measures radicals, are highly potent oxidant molecules capable of the mass change of the sample by temperature change and is reacting with many biomolecules in living cells and leading a very effective system for analyzing samples with mass to significant biological detriment and lipid peroxidation. attain or loss throughout heating. Degradation of Fr-I oc- As seen from Fig. 8, the high hydroxyl free radical scav- curred by two stages as noticed in TGA. At first part, enging activity of EPS increased from 10 ± 1.08 to 61.3 ± 6.769% of weight decrement was documented from 38.16 5.4% in terms of concentration, which was higher than that to 78.74 °C owing to the loss of alcohol molecules and of 50.8 ± 4.8% for Boletus edudis, 49.4 ± 4.2% for Pholiota humidity. At the second step of degradation, 11.196% adipose, and 26.2 ± 2.4% for Antrodia camphorate at −1 weight loss was observed between 169.94 and 369.44 °C. 5mg mL (Lin et al. 2012). DPPH free radical is largely used The decomposition temperature of Fr-I was around to assess the free radical scavenging activities of the usual 276.91 °C. Degradation temperatures of the purified EPSs substance (Leong and Shui 2002). As shown in Fig. 8,the by P. pulmonarius and Lactobacillus plantarum MTCC EPS had an obvious DPPH scavenging ability increase from 9510 were 217 and 260 °C, respectively (Shen et al. 2013; 7.4 ± 0.3% to 41.83 ± 4.0% in terms of concentration. Radical Ismail and Nampoothiri 2010). The complex structure of the scavenging activity of EPS was much higher than 20 ± 0.3% EPS produced has been shown to provide high degradation of Boletus aereus EPS (Zhen et al. 2014). The scavenging temperature. activities of superoxide radical of EPS produced by P. sajor 1208 Ann Microbiol (2019) 69:1201–1210 Fig. 8 OH, DPPH, O radicals 70 1.6 scavenging (%), and chelating 1.4 activities (%) and reducing power (A )of produced EPS by P. 1.2 sajor caju. The values are mean ± SD of three separate experiments 1 0.8 0.6 0.4 0.2 0 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 OH radical DPPH Superoxide A700 caju are shown to be dose-dependent in Fig. 8. The superoxide EPS was able to scavenge superoxide anion, hydroxyl, and radical scavenging impact of EPS ranged from 1.31 ± 0.2% at DPPH radicals, and showed great potential as an antioxidant −1 −1 0.25 mg mL to 18.83 ± 1.5% at 5 mg mL , which was in vitro. This research contributes important knowledge about similar to Cordyceps militaris SU5-08 and Bacillus edudis. EPS production, which can be used as functional food, anti- It has also been indicated that the decomposition energy of oxidant, or a valuable compound for biotechnological appli- O-H bonds may be related to the superoxide anion scavenging cations, such as cosmetics, medicine, and pharmaceutics, from mechanism (Tsiapali et al. 2001). The reducing power of com- P. sajor caju. pound may serve as a substantial sign of its potential antiox- Acknowledgments This study was supported by the Scientific Research idant activity (Kumar et al. 2004). As shown in Fig. 8,our Project of Dokuz Eylül University (project number = 2014.KB.FEN.037). results indicated that EPS had greater reducing power. Compared with other studies, the reducing power of Fr-I at −1 Compliance with ethical standards 3.5 g mL was also higher than some researcher’sdata (Ma et al. 2013;Li et al. 2013; Gao et al. 2013). The iron-chelating Conflict of interest The authors declare that they have no conflict of ability of EPS was associated with sample concentration. The interest. iron-chelating ability of EPS was determined as 19.8 ± 1.7% −1 at 1.5 mg mL . The obtained results show that the EPS has Research involving human participants and/or animals Not applicable. promising antioxidant capacities. This can be ascribed to the Informed consent Not applicable. hydroxyl and other related functional molecules in EPS ob- tained from P. sajor caju. These groups can donate electrons to reduce radicals to a more stable form or react with free References radicals to terminate the radical chain reaction (Nakiboglu et al. 2007). 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Process Biochem 49: Weatherburn MW (1967) Phenol-hypochlorite reaction for determination 1047–1053 of ammonia. Anal Chem 39:971–974 Yang FC, Huang HC, Yang MJ (2003) The influence of environmental Publisher’snote Springer Nature remains neutral with regard to conditions on the mycelial growth of Antrodia cinnamomea in sub- jurisdictional claims in published maps and institutional affiliations. merged cultures. Enzym Microb Technol 33:395–402 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Production and partial characterization of the exopolysaccharide from Pleurotus sajor caju

Annals of Microbiology , Volume 69 (11) – Aug 1, 2019

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Publisher
Springer Journals
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Copyright © 2019 by Università degli studi di Milano
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
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1869-2044
DOI
10.1007/s13213-019-01502-6
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Abstract

Purpose Microbial exopolysaccharides (EPSs) are very important because they are used in biotechnological applications in different industrial areas. The aim of the study was to determine the best EPS producer Pleurotus sp., to optimize EPS production and to perform partial purification and characterization of the produced EPS. Methods After the production conditions were optimized, the EPS was isolated and partially purified. EPS was characterized by HP-TLC, H-NMR, FT-IR, and TGA. Hydroxyl, superoxide, and DPPH radical scavenging activities of the EPS were also investigated spectrophotometrically. Result The best EPS producer and its incubation period in submerged fermentation were determined as Pleurotus sajor caju and on 5 days, respectively. Culture conditions to increase EPS production were optimized as follows (in per liter): 90 g of glucose, 2+ 10 g of yeast extract, 10 g of peptone, and 100 mM of Mg . The optimal initial pH, temperature, and an agitation rate of culture −1 were determined as 5.0, 25 °C, and 150 rate min , respectively. The highest EPS production was determined as 33.32 ± −1 1.6gL . After isolation of EPS, one active fraction was obtained by gel filtration chromatography. EPS is composed mainly of glucose according to HP-TLC analysis. Conclusion To the results, EPS had a complex structure by having carbohydrate and protein contents. The produced EPS had high degradation temperature as well as high antioxidant activity. . . . . Keywords Pleurotus sajor caju Submerged fermentation EPS production EPS characterization Antioxidant activity Introduction important components that determine the hardness and mor- phological characteristics of the fungal cell wall and which Polysaccharides, a group of valuable biopolymers, have appli- can be excreted into the culture medium depending upon the cations at a wide range of industrial fields, like food and phar- culture conditions (Gern et al. 2008). Many microorganisms maceuticals. In recent years, extracellular polysaccharide synthesize EPS in the form of amorphous loam that binds to (EPS) production has been prominent with numerous fungal the cell surface or is present extracellularly. EPSs help the cell submerged cultures since it has biological and pharmacologi- in a wide variety of functions. EPSs provide protection against cal activities such as anti-tumor, antioxidant, and hypoglyce- biotic stress and protection against abiotic stresses that may mic activities (Han et al. 2006;Song etal. 2008;Li etal. 2010; include nutrient limitation, temperature, light intensity, or pH. Zhao et al. 2012; Cao et al. 2018). Polysaccharides are For example, in acidophilic or thermophilic species, EPSs help to adapt to extreme conditions (Antón et al. 1988). The genus Pleurotus is widely cultivated and commercial- * Raziye Ozturk Urek ized in the world. It involves many biologically active com- raziye.urek@deu.edu.tr ponents, such as polysaccharides, proteins, enzymes, dietary fibers, and vitamins (Wang et al. 2001). Large amounts of Seda Ilgin sedailginn@gmail.com biomass and EPS production, which can be used for biotech- nological areas, occur in the submerged cultures of the Faculty of Science, Chemistry Department, Biochemistry Division, Pleurotus species. For instance, water-soluble polysaccharide Dokuz Eylül University, 35160 Buca, Izmir, Turkey of Pleurotus ostreatus could be a potent immune stimulant for Graduate School of Natural and Applied Sciences, Biotechnology use in nutraceuticals or medicine against both pathogens and Department, Dokuz Eylül University, 35160 Buca, Izmir, Turkey 1202 Ann Microbiol (2019) 69:1201–1210 cancer (Ooi and Liu 2000). However, EPS of Pleurotus cooled. Inoculated flasks were incubated on the rotary shaker −1 pulmonarius showed in vitro antioxidant activity (Shen et al. at 150 rate min and25 °Cfor 2weeks. 2013). Likewise, it was observed that EPS produced via To increase EPS production, sucrose, glycerol, and sorbitol −1 Pleurotus eryngii has antioxidant and antitumor activities as 10 g L were used as carbon source instead of glucose. The (Jing et al. 2013). A few works on EPS production have also optimum concentration was determined after glucose was been carried out by submerged fermentation (SmF) of identified as the best carbon source. The effects of varying Pleurotus spp. (Silveira et al. 2015). To improve EPS produc- nitrogen sources and concentrations, such as peptone, yeast tion, some nutritional factors have been investigated on the extract, ammonium nitrate, and urea, on EPS production were 2+ SmF of fungi. investigated. Then, different concentrations of Mg ion (4, 7, The purpose of this study is to achieve high efficiency EPS 10, 30, 50, 100, 250 mM), various pH levels (3, 4, 5, 6, 7, 8, production in SmF using white-rot fungi Pleurotus spp. In the 9), different fermentation temperatures (20, 25, and 30 °C), −1 first step of this study, the best producer Pleurotus strain and and agitation rate (100, 150, and 180 rate min )were inves- its incubation period were determined. In the next step, effects tigated. All samples were harvested on the 5th day of incuba- 2+ of carbon and nitrogen sources, and concentrations, Mg ion tion and analyzed for EPS production. concentration, pH, temperature, and agitation rate were inves- EPS samples taken from the flasks were centrifuged at tigated to optimize EPS production conditions in SmF. After 10000×g for 20 min, and then, the upper phase was filtered production at an optimum condition in SmF, EPS was extract- using a 0.45-μm membrane filter. The supernatant was used ed and partially purified by Sepharose CL-6B column. In the for analysis below. The dry weight of cell (CDW) was mea- last step of the study, partial purified EPS was characterized by sured after washing the mycelial pellet with distilled water and chromatographic (TLC), spectroscopic (FT-IR and H-NMR), drying at 70 °C to obtain invariable value. For EPS isolation, and TGA analyses. Also, antioxidant features, like hydroxyl, the obtained culture filtrate was mixed with four volumes of superoxide, DPPH radical scavenging activities, reducing absolute cold ethanol, admixed vigorously, and put for 24 h at power, and chelating activity of the partial purified EPS, were 4 °C. The precipitated crude EPS was obtained by centrifuga- investigated. tion at 10000×g for 20 min and then dried at 60 °C overnight to obtain constant value. Protein content of cell-free supernatant of production me- dia was determined by Bradford (1976) using bovine serum Materials and methods albumin standard. The content of reducing sugar and nitrogen was determined by DNS method and phenol-hypochlorite as- Maintenance of microorganisms say, respectively (Miller 1959; Weatherburn 1967). Four different Pleurotus strains were used in the study: Partial purification and characterization of produced Pleurotus djamor (Rumph. Ex Fr.) Boedijn (MCC15), P. EPS ostreatus (Jacq.) Pleurotus Kumm. (MCC16), Pleurotus sajor caju (Fr.) Singer (MCC29), and P. eryngii (DC.) Gillet EPS precipitated with ethanol was handled with Sevag reagent (MCC58). Pleurotus djamor, P. ostreatus,and P. sajor caju (1:4 n-butanol/chloroform, v/v)(Staub 1965). After centrifu- −1 were maintained on potato dextrose agar (PDA) (39 g L , gation, water phase was ultra-filtered (10 kDa) and filtrate was pH 5.6) at 25 °C for 7 days; P. eryngii was grown on malt admixed with absolute cold ethanol; the precipitated EPS was −1 extract:peptone:agar (MPA) (30:3:15 g L ,pH 5.6) at 25 °C centrifuged and dried. The EPS (100 mg) was disintegrated in for 14 days (Akpinar and Ozturk Urek 2017). 0.2 M NaCl buffer, and fulled onto a Sepharose CL-6B col- umn (1.6 cm × 90 cm). The column was eluted with this buffer −1 Production, optimization, and isolation of EPS at a flow rate of 0.6 mL min . Five dextran molecules (25000, 80000, 270000, 670000, and 1100000 Da) were used Pleurotus spp. were grown on PDA and MPA slants and then as molecular-weight standards. The dextran standards transferred to basal medium by 1 cm of the five agar plates (2.5 mg) were prepared in 0.2 M NaCl buffer, and loaded. with a sterilized cutter. Submerged fermentation was per- The protein level in the gel fractions was measured at formed in 250-mL Erlenmeyer flasks including 100 mL of 280 nm, while the carbohydrate level was recorded at 490 nm. basal medium. The basal medium composition was as follows The total carbohydrate, protein, uronic acid, and pyruvate −1 in (g L ): glucose, 10; NH NO , 0.724; KH PO ,1.0; contents of the EPS were investigated by the phenol-sulfuric 4 3 2 4 MgSO .7H O, 1.0; KCl, 0.5; yeast extract, 0.5; acid method, Bradford method, borate-sulfuric acid-carbazole 4 2 FeSO .7H O, 0.001; ZnSO .7H2O, 0.0028; CaCl .2H O, assay, and 2,4-dinitrophenyllhydrazine assay, respectively 4 2 4 2 2 0.033; peptone, 10 (Bazalel et al. 1997). The medium pH (Dubois et al. 1956;Healyetal. 1996; Friedemann and was adjusted to 6.0 and sterilized at 121 °C for 20 min then Haugen 1943). 1203 Ann Microbiol (2019) 69:1201–1210 To determine monosaccharide composition of the partial in Fig. 1a, our results indicated that P. sajor caju was the best −1 purified EPS, standard sugars (glucose, mannose, galactose, strain for the EPS production (2.62 ± 0.16 g L , on the 5th rhamnose, and xylose) were analyzed by HP-TLC (CAMAG). day of incubation period). The maximum EPS productions by Ethyl acetate:acetic acid:water (2:2:1) was used as mobile P. eryngii, P. djamor,and P. ostreatus were achieved on the phase on TLC silica gel 60 F254 plates (MERCK, 6th, 3rd, and 7th days of incubation period as 2.1 ± 0.35, 1.4 ± −1 Germany) with solution as staining reagent (2% 0.3, and 1.2 ± 0.25 g L , respectively. Our results showed that naphtoresorcinol in 10% ethanolic phosphoric acid). EPS EPS production depends on microorganism’s strain. Likewise, (50 mg) was hydrolyzed with 2 N H SO (4 mL) at 100 °C Nehad and El-Shamy (2010) reported that the yield of EPS 2 4 in oil bath for 2 h, then it was neutralized with NaOH (1 g) and produced by different fungal strains was different. loaded on plates and analyzed. After the determination of the best EPS producer as P. sajor For spectroscopic analysis, EPS, FT-IR, and H-NMR were caju, the effects of incubation time and pH of the medium on used. The FT-IR spectrum was monitored on the Perkin Elmer EPS production were investigated. Figure 1 b shows that max- −1 1 −1 Spectrum BX, in the 4000–400 cm spectral region. For H- imum EPS production was determined as 2.62 ± 0.16 g L on NMR spectroscopy, the sample was recorded, as solution in the 5th day of incubation, while maximum CDW was obtain- −1 dimethyl sulfoxide, on a MERCURY Plus-AS 400 spectrom- ed as 4.06 ± 0.4 g L on the 7th day. pH value of SmF medi- eter at 400 MHz and 30 °C. The chemical shifts were um dependent on incubation period was not a significant expressed in ppm. change up to 7 days, and then it slightly increased to 8.17 ± A thermal gravimetric analysis of EPS was carried out with 0.07 on the 12th day. Perkin Elmer-Diamond TG/DTA. About 3–5mg of dryEPS Chemical composition analyses of SmF medium were car- sample was loaded on a platinum pan and its energy level was ried out during the incubation period (Fig. 2). Maximum pro- scanned in the ranges of 30–600 °C under a nitrogen atmo- tein and nitrogen levels were detected as 78.09 ± 3.2 ppm and −1 sphere with a temperature gradient of 10 °C min . 459.9 ± 17 ppm on the 9th and12th days of incubation, respec- tively. Level of reducing sugar on the 2nd day of incubation Evaluation of antioxidant activity of partial purified was determined as 593.4 ± 29 ppm and limited conditions EPS were reached on the 5th day. After the determination of the best EPS producer as P. sajor Hydroxyl and superoxide radical scavenging activities of EPS caju and the incubation day (5th) for EPS production were measured according to the methods of Halliwell et al. (1987) and Liu et al. (1997), respectively. DPPH and chelating activity methods were based on Kumaran (2006) and Decker and Welch (1990), respectively. The reducing power of EPS 2.5 was detected to the method of Oyaizu (1986). 1.5 Statistical analysis 1 0.5 All experiments were carried out three times (n = 3) and re- P. ostreatus P. eryngii P. djamor P. sajor caju peated three times. Each value is the average of three parallel Pleurotus strains experimental studies. Data were given as mean ± standard deviation (SD). The data were assayed by analysis of variance 4.5 10 (ANOVA) to identify the significantly different groups at P < 0.05 by one-way ANOVA test utilizing SPSS software 3.5 statistical program (SPSS for windows ver. 21.00, USA). 2.5 1.5 Results and discussion 3 0.5 Optimization of fermentation conditions 0 0 23579 12 Incubation period (day) EPS production capacities of four different Pleurotus spp. CDW (g/l) EPS (g/l) pH were investigated in order to determine the best EPS producer in the basal medium of SmF and the incubation day of max- Fig. 1 a Production of EPS by Pleurotus strains. b Production of EPS by imum EPS production. They were incubated on the shaker P. sajor caju and CDW levels during incubation period. The values are −1 mean ± SD of three separate experiments incubator at 150 rates min and 25 °C for 2 weeks. As shown Concentration (g/L) Concentration (g/L) pH 1204 Ann Microbiol (2019) 69:1201–1210 Fig. 2 Glucose, nitrogen, and 700 100 protein levels of medium during incubation period of P. sajor caju. The values are mean ± SD of three separate experiments 0 0 02468 10 12 14 Incubation period (day) Glucose Nitrogen Protein −1 −1 according to the yield of EPS, components of the SmF medi- production (3.97 ± 0.15 g L ) and CDW (6.03 ± 0.32 g L ) −1 um and fermentation conditions were optimized. To find out was 90 g L . the most appropriate carbon source in EPS production by P. The carbon source is required for energy and structural sajor caju, three different carbon sources named sorbitol, su- molecules. Therefore, the concentration and type of carbon −1 crose, and glycerol were separately provided at 10 g L in- source are important for all bioprocesses. The carbon source stead of glucose employed in the basal medium. Betwixt the and its concentration have a major impact on the production investigated carbon sources, maximum EPS production and and yield of EPS. Glucose, sucrose, maltose, lactose, fructose, −1 CDW were obtained in glucose medium as 2.62 ± 0.16 g L galactose etc. are generally used as carbon sources in the cul- −1 and 2.83 ± 0.14 g L ,respectively (Fig. 3a). ture medium (Mahapatra and Banerjee 2013). In most studies, After selecting glucose as the best carbon source for EPS glucose, sucrose, and maltose have been chosen as the most production, the optimum concentration of glucose was deter- necessary carbon source for the fungal EPS production (Kim mined for EPS production (Fig. 3b). For this purpose, various et al. 2005; Zhang and Cheung 2011). These results suggest glucose concentrations were used in medium and the results that various carbon sources may have some catabolic repres- showed that optimum glucose concentration for EPS sion effects in various EPS syntheses. It is also an indication that different fungal strains have different carbon source in- take characteristics or that these carbon sources can be easily utilized by fungi. Furthermore, the structure and concentration 3.5 of the carbon source used can regulate secondary metabolism, such as catabolic repression (Görke and Stülke 2008). 2.5 In this study, the capability of P. sajor caju to use various nitrogen sources for EPS production was analyzed. For this 1.5 CDW (g/l) purpose, P. sajor caju cells were grown in basal medium con- EPS (g/l) −1 taining glucose (90 g L ) as a carbon source, and four diverse 0.5 nitrogen sources, namely, peptone, yeast extract, ammonium nitrate, and urea, were added at various concentrations. Sorbitol Glucose Sucrose Glycerol Figure 4a shows that maximum EPS production was achieved Different Carbon Sources (g/L) −1 −1 −1 in 10 g L peptone, 10 g L yeast extract, and 0.724 g L −1 −1 NH NO as 4.01 ± 0.38 g L , 3.56 ± 0.3 g L , and 3.09 ± 4 3 −1 0.27 g L ,respectively.Figure 4b indicates that maximum −1 −1 CDW was determined in 12 g L peptone and 10 g L yeast −1 −1 extract medium as 5.93 ± 0.5 g L and 8.51 ± 0.77 g L , 4 respectively. Our findings are similar to those of other re- searchers, which show comparatively low mycelial growth and EPS production with inorganic nitrogen sources com- CDW (g/l) pared with organic nitrogen sources (Yang et al. 2003). EPS (g/l) −1 Similarly, nitrogen concentrations between 1 and 10 g L are sufficient for fungal EPS production (Yuan et al. 2012). 5 1020406090 120 180 2+ Mg concentration effects on EPS production and myce- Glucose concentration (g/L) 2+ lial growth were investigated (Fig. 5). Addition of Mg to the Fig. 3 a Effect of carbon sources on EPS production by P. sajor caju. b culture medium may increase EPS production. The maximum Effect of glucose concentration on EPS production by P. sajor caju.The −1 amount of 31.08 ± 3.0 g L EPS was obtained with the values are mean ± SD of three separate experiments Concentration (g/L) Concentration (g/L) Reducing Sugar and Nitrogen Levels (ppm) Protein Level (ppm) 1205 Ann Microbiol (2019) 69:1201–1210 stabilization of the plasma membrane, and its uptake into the cell is also dependent on ATP. 4 To study the effect of the initial culture pH on EPS produc- tion, P. sajor caju was cultivated in SmF with various starting pH (3.0–9.0); after 5 days, it was analyzed for EPS production (Fig. 6a). The results showed that the optimum pH for EPS −1 −1 production (33.32 ± 1.6 g L ) and CDW (7.49 ± 0.5 g L ) was 5 and 7, respectively. In general, fungi preferred low pH 0.5 0.724 1.5 3 6 10 12 for EPS production with a range between pH 3.0 and 6.5 Nitrogen Concentration (g/L) (Feng et al. 2010). In order to determine the optimum temper- Peptone Yeast extract NH4NO3 Urea ature, P. sajor caju was cultivated at different temperatures in the shaking incubator for 5 days; the optimal temperature for EPS production was detected at 25 °C (Fig. 6b). The maxi- mum CDW was achieved at 30 °C. Similarly, many microor- ganisms produce maximal amounts of EPS at temperatures slightly lower than optimal growth as observed for 3 Klebsiella sp. (Farres et al. 1997). Many researchers have reported different optimal temperature values for EPS produc- tion; changes in environmental factors appear to give rise to 0.5 0.724 1.5 3 6 10 12 Nitrogen Concentration (g/L) Peptone Yeast extract NH4NO3 Urea Fig. 4 a Effect of nitrogen sources and concentrations on EPS production and b CDW by P. sajor caju. The values are mean ± SD of three separate 40 experiments supplementation of 100 mM MgSO .7H O. The maximum 4 2 −1 2+ 10 CDW (19.25 ± 1.89 g L ) was achieved in 50 mM Mg . Therefore, 100 mM MgSO .7H O was selected as the best 4 2 concentration for the EPS production by P. sajor caju.This pH can be explained by the fact that sulfates may also have effects CDW EPS on EPS production (Liu and Miao 2017). Raposo et al. (2014) expressed that the highest yield of EPS was observed when 21 mM MgSO was added to the culture medium of Porphyridium cruentum; the total yield of EPS in microalgae cultivation suggests that it may be affected by varying MgSO concentrations. Many researchers have deemed this bio- element to be suitable for mycelial growth and EPS produc- tion, usually in liquid cultures of several basidiomycetes (Chardonnet et al. 1999; Hwang et al. 2003). It is known that 20 25 30 Temperature (°C) cations affect EPS synthesis both qualitatively and quantita- 2+ EPS CDW tively. Mg is required for all fungi and used as a cofactor in many enzymatic reactions. It is also involved in the 100 150 180 4 7 10 30 50 100 250 Agitation Rate (rpm) 2+ Mg Concentration (mM) CDW EPS CDW EPS Fig. 6 The effect of a initial pH, b temperature, and c agitation rate on 2+ Fig. 5 Effect of different Mg ion concentrations on EPS production by EPS production and CDW by P. sajor caju. The values are mean ± SD of P. sajor caju. The values are mean ± SD of three separate experiments three separate experiments CDW (g/L) EPS (g/L) Concentration (g/L) Concentration (g/L) Concentration (g/L) Concentration (g/L) 1206 Ann Microbiol (2019) 69:1201–1210 some changes in EPS efficiency, contingent upon the type of was reported by Shen et al. (2013), and one fraction was fungus (Ruas-Madiedo et al. 2002). To find the agitation rate isolated by purification of polysaccharide from P. effect on the EPS production, P. sajor caju was cultivated in a pulmonarius. Many research findings indicated that in EPS medium with various agitation rates (100, 150, produced by fungi, glycoprotein generally occurs (Hwang −1 180 rate min ). Figure 6c shows that maximum EPS produc- et al. 2003;Heet al. 2012). tion and CDW were obtained at an agitation rate of The molecular weight of the produced EPS was detected as −1 150 rate min . Additionally, aeration and agitation are impor- the low-molecular-weight EPS of approximately 25 ± 3 kDa. tant factors to ease the oxygen transfer and enable nutrient Similar observations were made by Oh et al. (2007)and transport to microorganisms. The presence of oxygen and Cheng et al. (2011) in other fungi fermentations. The molec- the rate at which the culture medium is admixed might have ular weight of EPS produced by P. sajor caju (Fr.) Singer 4 −1 a direct effect on the production polysaccharide, and an in- (CCB 019) was determined as 6.4 × 10 gmol according crease in EPS production often results from the increase in to Silveira et al. (2015). Differences in Mw may indicate dif- oxygen supply (Dassy et al. 1991; Bayer et al. 1990). The ferences in the methods used in isolation and production pro- EPS surrounding the cell can serve as a barrier for oxygen cedures, or for the determination of Mw. and nutrient transfer. Admixing culture at high rates can in- crease the availability of both nutrients and oxygen. Partial characterization of EPS The obtained results showed that the EPS yield was the highest when the concentrations of glucose, yeast extract, pep- HP-TLC is an effective chromatographic method for detecting 2+ −1 tone, and Mg were 90, 10, 10 g L , and 100 mM, respec- monosaccharide composition. On HP-TLC analysis, the Rf tively. Thus, the yield of EPS in the optimized culture medium values of the standards were as follows: glucose 0.456 ± was found to be about 13.33 times higher than that obtained 0.04, galactose 0.445 ± 0.05, mannose 0.503 ± 0.05, xylose when the basal culture medium was used. According to the 0.551 ± 0.06, and rhamnose 0.563 ± 0.04 (Fig. 7a). Our results −1 optimized conditions, the highest EPS was 33.32 ± 0.3 g L . showed that Fr-I mainly included glucose. Many fungal EPSs The EPS obtained in this study is 6.45 times more than that are composed of glucose monosaccharide (Lin and Chen produced by P. eryngii and 5.24 times more than P. 2007). Themonosaccharidecomposition was veryimportant pulmonarius (Jing et al. 2013;Shenetal. 2013). To to determine the produced EPS function. Osińska-Jaroszuk et al. (2015), the maximum produced EPS The FT-IR spectrum is a method used to detect functional yield from Ascomycota and Basidiomycota fungi ranges from groups and characterize covalent binding information. Typical −1 0.12 to 42.24 g L , which is mostly dependent on the strain FT-IR spectrum for Fr-I is presented in Fig. 7b.Fr-Idisplayed and culture conditions used. a stretching intense distinctive peak at approximately the re- −1 Recently, it is important to investigate the chemical com- gion of 3388 cm for the carbohydrate ring. Small band at −1 position and physicochemical properties of EPS produced 2948 cm was assigned to the stretching vibration of the since the introduction of natural polymers for industrial appli- methylene group (C-H), commonly found in hexoses. A −1 cations increases the interest in EPS production. After optimi- distinguishing peak absorption band emerged at 1638 cm zation of EPS production conditions, the produced EPS was was ascribed to the stretching vibration of the carboxyl group −1 isolated and its chemical characterization was assayed. The (C=O). A meager peak about 1110 cm corresponding to the chemical composition of 1 mg isolated EPS was formed by glycosidic link bond (C-O-C) was determined in the spectrum 2.26 ± 0.41 μg protein, 219.75 ± 10 μg total carbohydrate, of the polysaccharide (Copikova et al. 2001). The stretching −1 28.59 ± 0.32 μg reducing sugar, 15.73 ± 1.1 μg nitrogen, vibration peak of 1040 cm implied the presence of C-H-O 1.01 ± 0.01 μg pyruvate, and 6.7 ± 0.5 μg uronic acid. linkage position. The weak absorption peak at 985 has coin- Results of chemical composition analyses demonstrated the cided to N-H of primary amine. The FT-IR spectrum showed complex structure of produced EPS. that proteins and polysaccharides were present in EPS com- position. Additionally, it was determined that MgSO did not Isolation and partial purification of EPS bear an impact on the structure of EPS in the FT-IR spectrum. This data was also supported by Liu and Miao (2017). The produced EPS in optimum SmF conditions by P. sajor According to the H-NMR spectrum of Fr-I, signals at 2.2– caju of 90 g glucose, 10 g peptone, 10 g yeast extract, 100 mM 2.8 ppm were ascribed to peaks of C-H (data not shown). The 2+ Mg per liter, pH 5, at 25 °C, and 150 rpm was isolated. C-C signals were established in the region 3.0–4.2 ppm, Isolated EPS was mixed with Sevag reagent then ultrafiltrated which were ascribed to protons of the C-2-C-6 glycoside ring and applied on gel filtration chromatography (Sepharose CL- of hexoses. The NH signals at 7.1–7.5 ppm demonstrated 6B). Active one fraction of EPS (Fr-I), which consisted of amino acid and peptide structure. The obtained results dem- polysaccharides and proteins, was co-eluted. Fr-I was revealed onstrated that EPS produced by P. sajor caju had functional to be glycoprotein based on preliminary data. A similar result groups, bonds, and structures which are present in 1207 Ann Microbiol (2019) 69:1201–1210 218,3 %T 3388 2368 40 1110 625 0,0 4000,0 3000 2000 1500 1000 500 200,0 cm-1 Fig. 7 a HP-TLC analysis of EPS and standards. Solvent system-ethyl (3), (4), (5), and (6) mannose, glucose, galactose, xylose, and rhamnose, acetate butanol:acetic acid:water (2:1:1), spray reagent; 2% respectively. b FT-IR spectrum of EPS. The values are mean ± SD of three naphtoresorcinol in 10% ethanolic phosphoric acid. Spot (2) Fr-I; (1), separate experiments glycoprotein-type polysaccharide. It was deduced that EPS Antioxidant activities of partial purified EPS from P. possessed a complicated structure with carbohydrate and pro- sajor caju tein contents. The presence of thermal durability of the polysaccharide Free radicals, especially hydroxyl radicals and other derived is a paramount property for its applications. TGA measures radicals, are highly potent oxidant molecules capable of the mass change of the sample by temperature change and is reacting with many biomolecules in living cells and leading a very effective system for analyzing samples with mass to significant biological detriment and lipid peroxidation. attain or loss throughout heating. Degradation of Fr-I oc- As seen from Fig. 8, the high hydroxyl free radical scav- curred by two stages as noticed in TGA. At first part, enging activity of EPS increased from 10 ± 1.08 to 61.3 ± 6.769% of weight decrement was documented from 38.16 5.4% in terms of concentration, which was higher than that to 78.74 °C owing to the loss of alcohol molecules and of 50.8 ± 4.8% for Boletus edudis, 49.4 ± 4.2% for Pholiota humidity. At the second step of degradation, 11.196% adipose, and 26.2 ± 2.4% for Antrodia camphorate at −1 weight loss was observed between 169.94 and 369.44 °C. 5mg mL (Lin et al. 2012). DPPH free radical is largely used The decomposition temperature of Fr-I was around to assess the free radical scavenging activities of the usual 276.91 °C. Degradation temperatures of the purified EPSs substance (Leong and Shui 2002). As shown in Fig. 8,the by P. pulmonarius and Lactobacillus plantarum MTCC EPS had an obvious DPPH scavenging ability increase from 9510 were 217 and 260 °C, respectively (Shen et al. 2013; 7.4 ± 0.3% to 41.83 ± 4.0% in terms of concentration. Radical Ismail and Nampoothiri 2010). The complex structure of the scavenging activity of EPS was much higher than 20 ± 0.3% EPS produced has been shown to provide high degradation of Boletus aereus EPS (Zhen et al. 2014). The scavenging temperature. activities of superoxide radical of EPS produced by P. sajor 1208 Ann Microbiol (2019) 69:1201–1210 Fig. 8 OH, DPPH, O radicals 70 1.6 scavenging (%), and chelating 1.4 activities (%) and reducing power (A )of produced EPS by P. 1.2 sajor caju. The values are mean ± SD of three separate experiments 1 0.8 0.6 0.4 0.2 0 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 OH radical DPPH Superoxide A700 caju are shown to be dose-dependent in Fig. 8. The superoxide EPS was able to scavenge superoxide anion, hydroxyl, and radical scavenging impact of EPS ranged from 1.31 ± 0.2% at DPPH radicals, and showed great potential as an antioxidant −1 −1 0.25 mg mL to 18.83 ± 1.5% at 5 mg mL , which was in vitro. This research contributes important knowledge about similar to Cordyceps militaris SU5-08 and Bacillus edudis. EPS production, which can be used as functional food, anti- It has also been indicated that the decomposition energy of oxidant, or a valuable compound for biotechnological appli- O-H bonds may be related to the superoxide anion scavenging cations, such as cosmetics, medicine, and pharmaceutics, from mechanism (Tsiapali et al. 2001). The reducing power of com- P. sajor caju. pound may serve as a substantial sign of its potential antiox- Acknowledgments This study was supported by the Scientific Research idant activity (Kumar et al. 2004). As shown in Fig. 8,our Project of Dokuz Eylül University (project number = 2014.KB.FEN.037). results indicated that EPS had greater reducing power. Compared with other studies, the reducing power of Fr-I at −1 Compliance with ethical standards 3.5 g mL was also higher than some researcher’sdata (Ma et al. 2013;Li et al. 2013; Gao et al. 2013). The iron-chelating Conflict of interest The authors declare that they have no conflict of ability of EPS was associated with sample concentration. The interest. iron-chelating ability of EPS was determined as 19.8 ± 1.7% −1 at 1.5 mg mL . The obtained results show that the EPS has Research involving human participants and/or animals Not applicable. promising antioxidant capacities. This can be ascribed to the Informed consent Not applicable. hydroxyl and other related functional molecules in EPS ob- tained from P. sajor caju. These groups can donate electrons to reduce radicals to a more stable form or react with free References radicals to terminate the radical chain reaction (Nakiboglu et al. 2007). 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Published: Aug 1, 2019

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