Production of schizophyllan from distiller’s dried grains with solubles by diverse strains of Schizophyllum commune

Production of schizophyllan by diverse strains of Schizophyllum commune

Properties of schizophyllan solutions from S. commune strains ATCC 20165 and CBS 266.60

Schizophyllan from S. commune strains ATCC 20165 and CBS 266.60 was structurally analyzed by Heteronuclear Single Quantum Coherence-NMR (Figure 2). The spectra were essentially identical to those of both a commercial schizophyllan standard and schizophyllan from commercial strain ATCC 38548. Spectra showed two anomeric sugar signals at 4.55 ppm and 4.20 ppm, due to the β-1,3-linked glucose and β-1,6-linked glucose, respectively. These signals correlated to overlapping ¹³C signals at 103.4 ppm, consistent with β-linked glucosyl residues. Other carbohydrate signals were apparent in the 2.7 – 4.0 ppm region for ¹H, and 55 – 85 ppm for ¹³C nuclei. Characteristic methylene –CH₂ signals were apparent at 3.40 and 3.60 ppm, coupled to a single ¹³C signal at 61 ppm. These were assigned to the C-6 position of the backbone glucose residues carrying a 1,6-glucose branch. These data are consistent with the isolated polysaccharides being schizophyllan.

The solution viscosity properties of 0.3% (w/v) aqueous solutions of schizophyllan from strains ATCC 20165 and CBS 266.60 were compared with those of commercial strain ATCC 38548 (Figure 3). These strains exhibited nearly identical viscosity and shear thinning properties, characteristic of the pseudoplastic flow behavior of schizophyllan solutions (Rau 1999).

HPSEC was used to characterize the molecular weight of schizophyllan. Schizophyllan produced by S. commune strains ATCC 20165 and CBS 266.60 exhibited heterodisperse peaks with maximum molecular weights of 4.6 × 10⁶ and 5.2 × 10⁶, respectively. The values are consistent with literature values of 6–12 × 10⁶ (Rau 1999).

The interfacial tension of 0.3% (w/v) aqueous solutions of schizophyllan from S. commune strains ATCC 20165 and CBS 266.60 were 52 ± 1.9 and 55 ± 2.8 dy/cm, respectively (standard deviations shown). By comparison, schizophyllan from commercial strain ATCC 38548 showed similar values of 58 ± 3.5 dy/cm, while pure water exhibits an interfacial tension of 72 dy/cm (Dunlap et al. 2011). Thus, schizophyllan solutions from strains ATCC 20165 and CBS 266.60 showed relatively low surface activity, similar to commercial preparations (Rau 1999).

Strains and culture conditions

Schizophyllum commune strains used in this study were obtained from the American Type Culture Collection, Manassas, VA (ATCC strains), the ARS Culture Collection at the National Center for Agricultural Utilization Research, Peoria IL (NRRL strain) and the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS strains). Malt extract (ME) basal medium contained 2% (w/v) malt extract and 0.1% (w/v) peptone. Strains were grown on ME solid medium containing 2% (w/v) glucose and 2.5% (w/v) agar at 28°C for 7–10 days. A 7 mm × 7 mm square of mycelium was used to inoculate 250 mL of ME basal medium containing 2% (w/v) glucose in a 500 ml fluted Erlenmeyer flask with three 10 mm glass beads. This preinoculum culture was incubated at 240 rpm for 4–5 days at 30°C. Experimental cultures containing 1% (w/v) of DDGS in 150 mL of ME basal medium (without glucose) in 500 mL flasks were inoculated with 1.5 mL of preinoculum (1% v/v) and incubated at 240 rpm for 8 days at 30°C. DDGS was obtained from the National Corn-to-Ethanol Research Center, Edswardville, IL. All experiments were carried out in triplicate and standard deviations are shown.

Isolation of schizophyllan

Whole culture suspensions were homogenized for 20 sec (Power Gen 700, Fisher Scientific) and centrifuged at 6,166 × g for 1 h at 4°C. The supernatants were collected and the insoluble pellets (containing mycelia and residual DDGS) were resuspended in 100 mL of deionized water, homogenized, and centrifuged as before. The pellets were dried under vacuum for 48 h at 60°C. The supernatants were combined and one volume of 95% ethanol was added. After 1 h at 4°C, precipitates were collected by centrifugation at 6,166 × g for 1 h at 4°C, air-dried overnight to reduce the amount of ethanol, and then lyophilized.

NMR analysis

Solution NMR spectra were recorded on a Bruker AMX 500 spectrometer at normal probe temperature with standard instrument settings. Deuterated dimethylsulfoxide (d6-DMSO) was used as the solvent. All chemical shifts were referenced to tetramethylsilane at 0 ppm.

Molecular weight determinations

Schizophyllan molecular weights were determined by size exclusion chromatography as previously described, using a Shodex SB-806 M high performance size exclusion chromatography (HPSEC) column (Showa Denko, Tokyo, Japan) and eluted with 0.05 M sodium nitrate at a flow rate of 0.5 ml/min (Leathers et al. 2010). The column was calibrated with a set of eight pullulan molecular weight standards ranging from 5.8 × 10³ Da to 1.66 × 10⁶ Da (Showa Denko, Tokyo, Japan). Separations were monitored using a Shodex OR-1 optical rotation detector (Showa Denko).

Solution viscosity properties

Solution viscosity was measured using a TA Instruments (New Castle, DE) ARES LS-1 controlled strain rheometer with a 25 mm titanium parallel plate. All tests were performed at 25°C using a peltier plate. Steady rate sweeps were used to determine the viscosity of samples from 0.01-100 s^-1. The Cross model was used to determine the zero shear viscosity, which was 460 Pa.s.

Surface activity

Surface activity was determined using the pendant drop method (Dunlap et al. 2011). Samples were analyzed using the FTA 4000 surface tension instrument (First Ten Angstorms Inc., USA). Measurements were made using 22 gauge blunt needles with 7 μL drops. The reported values are the average of triplicate cultures.