Implications of recovery procedures on structural and rheological properties of schizophyllan produced from date syrup

This study investigates the effects of different recovery procedures on high molar mass schizophyllan produced by Schizophyllum commune using low value agricultural residues. Recovered extracellular polysaccharides (EPSs) were compared in terms of purity, sugar composition, degree of branching, molecular weight, and rheological properties. Performing different recovery methods, such as re-dissolving in water and re-precipitation with ethanol on produced EPS, provided schizophyllan with purity similar to the commercial grade. Besides, Freeze-thawing cycles allowed the fractionation of schizophyllan based on branching degree and solubility. The EPSs with higher purity and lower degree of branching (less conformational flexibility) showed higher viscosity. This study evidences the possibility of producing EPSs with excellent rheological properties using low value agricultural side products. Furthermore, our results demonstrate the importance of recovery methods for tailoring the purity, molecular structure and macroscopic properties of the produced polysaccharides for specific applications.


Introduction
Schizophyllan is an exopolysaccharide (EPS) produced by Schizophyllum commune, a white-rot fungus and ubiquitous mushroom. It is a non-ionic, water-soluble homoglucan, which consists of a backbone of β-13-glucose residues with β-16-linked glucose side chains at every third residue on average [1]. Schizophyllan is known to be a biological response modifier and to act as a non-specific stimulator of the immune system. Because of its immunotherapeutic properties, it has been used as an immune effect enhancer in anticancer, vaccines and also as a bioactive ingredient in cosmetics [2]. Simultaneously, due to the high molecular weight and the very stiff triple helical conformation in water, schizophyllan aqueous solutions exhibit well recognized rheological properties, which suggest its potential application as thickener in enhanced oil recovery [3] and as biodegradable bioflocculant [4]. The biological activity and rheological properties of aqueous schizophyllan solutions are mainly under the influence of purity, molecular structure, molecular weight, and the macromolecular conformation in solution [5][6][7][8][9].
There is evidence that extracellular β-glucans produced from the same strain and under the same conditions but yet subjected to different post-fermentation treatments (downstream processing), may show different solution properties and macromolecular features [8,10]. In spite of the importance of post fermentation treatments and their influence on the properties and the macromolecular features of recovered schizophyllan, limited investigations have mainly focused on this subject. Rau et al. [11] investigated the effect of cross-flow filtration, an effective procedure for washing out low molecular substances (salts, mono-and oligosaccharides, peptides) and concentrating the cell free schizophyllan solution, on molecular weight and shear viscosity of schizophyllan solutions and showed that molecular weight and shear viscosity of the β-glucan solution decreased after this procedure. Zentz and Muller [8] found that impurities from the fermentation broth can unfavorably affect the solution properties of schizophyllan and that aggregation of the exopolysaccharide molecules result from the thermal treatment of the unpurified fermentation broth (higher molecular weight). These observations indicate that downstream processing greatly influences the macromolecular features of schizophyllan, which as a consequence can affect its macroscopic properties and potential applications.
We recently demonstrated a simplified process at laboratory scale level for the recovery of schizophyllan produced from date syrup and corn steep liquor by S. commune ATCC 38548 [12]. The efficient fermentation of the low-cost residues for schizophyllan production along with the importance of post-fermentation processing on schizophyllan properties encouraged us to further investigate the influence of the recovery and purification processes on the molecular features and rheological properties of schizophyllan. In this study, the effect of recovery procedures on the produced schizophyllan was investigated by comparing two parallel processes: (i) a two-step redissolution and precipitation in ethanol and (ii) three cycles of freeze-thawing of the crude extracellular polysaccharide. The purity, monosaccharide composition, degree of β-(13)(16)-glycosidic branching, molecular weight, and rheological properties were compared, in order to elucidate the structure-property relationships of the recovered and purified exopolysaccharides.

2
Materials and methods

Preparation of downstream-processed schizophyllan samples
For the production of crude schizophyllan, Schizophyllum commune ATCC 38548 was grown on PDA plates at 28 °C for 7 days. For the first subculture, an approximately 5 x 5 mm square of S. commune covered agar was used to inoculate 50 ml of sterile seed culture medium in 250 ml shake flask with 2 baffles, which was then incubated at 28 °C, 180 rpm for 2 days on a rotary shaker. The second seed culture with 100 ml medium in 250 ml shake flasks was inoculated with 10 ml of homogenized culture suspension prepared using a Potter-type homogenizer and cultivated for 2 days as described. The seed culture medium consisted of glucose (30 g l -1 ), yeast extract (3 g l -1 ), KH 2 PO 4 (1 g l -1 ), and MgSO 4 ·7H 2 O (0.5 g l -1 ) [1]. The second seed culture was added at a 7.7% (v/v) inoculum size to the optimized medium for schizophyllan production containing 7% (w/v) date syrup and 0.1% (w/v) corn steep liquor.
The culture was incubated for 180 h at agitation rate of 181 rpm at 28 °C [12]. The sugar composition of date syrup was 29.5 (%w/w) glucose, 33.2 (%w/w) fructose and 0.1 (%w/w) sucrose.
Downstream processing started with a 3-fold dilution of culture broth with distilled water and homogenization (Power Gen 700, Fisher Scientific) for 40 s, and then centrifuging at 15,000 x g for 20 min at 4 °C. In order to isolate the extracellular polysaccharides (mainly schizophyllan), an equivalent volume of 95% (v/v) ethanol was added to precipitate the schizophyllan from the supernatant. After 1 h at 4 °C, the precipitate was collected by centrifugation at 15,000 x g for 20 min at 4 °C [12].
The crude schizophyllan was further treated with two different methods (Fig. 1). In the first method, the crude schizophyllan was re-dissolved in distilled water and then purified by re-precipitation in 95% ethanol (two times).
The polysaccharide precipitate was freeze-dried, milled (by means of two 10 s-pulses in a domestic coffee grinder), and stored as EPS-r (Fig. 1). In the second method, after freeze drying of the crude schizophyllan, the method involved 3 consecutive freeze-thawing cycles of the crude schizophyllan (−80 °C for 30 min, 65 °C for 3 h), and separation of the precipitate fraction (EPS-pre) from the supernatant (EPS-sup) fraction by centrifugation (6000 × g, 15min, 4 ºC) (Fig. 1). Finally, each fraction was freeze-dried and milled as above (Fig. 1). Commercial scleroglucans (grade CS11and CS6) were kindly provided by Cargill (France) and used without further modification or purification for comparison with the recovered schizophyllan samples. The samples were analyzed for protein content by the Bradford method [13] using BSA as standard, and total carbohydrates were determined by the phenol-sulfuric acid method [14] with glucose as standard.

Sugar composition analysis of schizophyllan
The sugar composition of the recovered schizophyllan and the commercial scleroglucans (CS 11 and CS6) was  Figure S2). Different molecular parameters and distributions including the SEC weight distribution, w(log V h ), the size dependence of the weight-average molecular weight -M w (V h ), and the radius of gyration R g were thus obtained. The macromolecular size distributions are presented in terms of the end of solution preparation, volume was corrected by addition of distilled water, and the blend was left aside to reach room temperature (25 °C). In all cases, NaN 3 (0.02%, w/v) was added as preservative [17].
The rheological properties of EPSs in aqueous solutions at various concentrations were investigated with a DCR311 rheometer (Anton Paar Ltd, Austria), using a plate and plate geometry (plate diameter 50 mm, gap 1.0 mm) for both the steady shear and oscillatory tests. The steady shear measurements were made at 25 °C in the range of 5-200 s −1 .
The power law model was used to fit the experimental flow curves of EPSs at various concentrations, with the formula (eq. 1): Here, is the apparent viscosity (Pa s), Κ is the consistency index, and n is the viscosity index.
Oscillatory (dynamic) tests of EPSs at different concentrations were performed with small-amplitude oscillatory rheometry at frequencies from 10 to 0.01 Hz at 25 °C. The viscoelastic properties, storage modulus (G'), and loss modulus (G'') were determined with the data analysis software. The freshly prepared sample was mixed thoroughly and loaded immediately onto the peltier of the rheometer. A layer of light silicone oil was applied to the exposed edge of the sample to prevent water evaporation. The data presented are the means of three replicate measurements.
3 Results and discussion 3.1 Purity, composition and molecular features of the EPSs after recovery procedures While, It may not be necessary to have highly purified products in bulk biomaterials applications [18], achieving a high purity is one of the main goals during recovery methods where a high purity grade is essential for the understanding of conformation and conformational transitions associated to a certain biological activity. Indeed, the use of impure β-glucan preparations has become problematic at the time of identifying the component or structural motif inducing a biological effect [7]. Besides, tailoring the desired physicochemical properties of the recovered is also critical, which may be influenced by recovery procedures. Therefore, in order to study the effect of recovery procedures on physicochemical properties of recovered EPSs in terms of purity, monosaccharide composition, intramolecular branching, macromolecular architecture (molecular weight and size) and conformation in solution, the crude schizophyllan was subjected to different recovery methods. EPS-r was recovered by two cycles of dissolution in distilled water and precipitation in 95% ethanol, whereas EPS-pre and EPS-sup were recovered from the precipitate and the supernatant after three cycles of freeze-thawing, respectively (see Section 2.1).
Total sugars and protein content for recovered EPSs and two grades of commercial scleroglucan (CS6 and CS11) are shown in Table 1. The total carbohydrate content for ESP-sup calculated by the phenol-sulfuric method was higher than 100%; this overestimation could be due to the presence of simple sugars dragged from the culture broth [19].
The total carbohydrate content in EPS-r was approximately 93% (w/w), which was similar to commercial scleroglucan CS11 and higher than the CS6. This result suggests that the refined grade of schizophyllan could be considered a good candidate for biotechnological applications requiring high purity grade [19]. Also, the low protein content for recovered EPS-r indicated that the re-dissolving in water and re-precipitation with ethanol is an efficient method for removing protein impurity from crude EPS.
Sugar composition analysis showed that all the samples were composed mainly of glucose, which is evidently attributed to the extracellular β-D-glucans (Table 1). Relatively small quantities of Man, Gal, and GalA were detected in the EPS produced from S. commune, which can be attributed to mannan and pectin contaminants that could arise from the fungal cell walls. In agreement with total sugar and protein content results, commercial scleroclucan (CS11) was the purest sample in terms of Glc content. Again, similar glucose purity was obtained for the EPS-r, which proves that applying dissolution/precipitation procedure lead to an increase in the purity of the EPS-r compared to crude EPS. On the other hand, a rather high amount of Man, Gal and GalA-containing polysaccharides were observed in EPS-sup after freeze-thawing, which may arise from the higher solubility of such polysaccharides.
In order to ascertain if the recovery procedures also affects the intramolecular features of the recovered schizophyllan, glycosidic linkage analysis of EPSs were performed by GC/MS after partial permethylation, hydrolysis and acetylation (Table 2). Three main peaks corresponding to partially methylated alditol acetates The differences in the structure of the EPSs can be mainly derived from the degree of branching and the proportion of t-Glc, 3-linked, and 36-linked glucose in their glucan chains. As shown in Table 2, the branching features of CS11 and EPS-r are similar in terms of branching points and terminal units. This again points out that precipitation is a good method in terms of purity and molecular structure. Besides, the 36-linked/3-linked and 36-linked/t-Glc mole fractions of EPS-sup were 0.55 and 1.2, respectively, which are higher than the calculated ratios for EPSpre. This indicates that the freeze-thawing leads to the fractionation of the crude EPS into low-branched with low solubility (EPS-pre) and highly branched polysaccharides that are more soluble (EPS-sup) [20]. These results demonstrate that recovery procedures decisively influence the structural features, such as 3-to 36 linkage ratio of the β-glucan produced by S. commune which subsequently can also influence solubility, aggregate formation and polymer conformation [21].
The biological and macroscopic properties of β-(13)-D-glucans are influenced by the degree of branching and the size of the branches. It is shown that the antitumor effect of β-glucans is related to their degree of branching [2].
Also the solubility in aqueous media depends on the degree of β-(13)-(16) branching, which allows the biopolymer to become clinically applicable since this form can be safely administered via the systemic route with no toxicity and other complications [20]. Additionally, the degree of branching affects the axial ratio and the relaxation times, which directly influence the rheological properties of EPSs [22].
SEC-MALLS was used to evaluate the effect of recovery procedures on molecular weight of samples (Fig. 2). The macromolecular structural features of the EPSs were studied by size exclusion chromatography coupled to differential refractometry (DRI) and multi-angle laser light scattering (MALLS). All EPS samples show monomodal size distributions w(log V h ), with the schizophyllan samples shifted towards larger hydrodynamic sizes compared to commercial scleroglucan (CS11). The same trend can be observed for the absolute weight-average molecular weight  Table 3). The calculated -M w of schizophyllan samples and commercial scleroglucan was in the order of 1-210 6 g mol -1 and in agreement with results of previous data reported for them in DMSO so far [23,24] and as it was expected schizophyllan has higher molecular mass than scleroglucan [25]. All EPSs showed a low polydispersity, denoting a basically uniform molar mass ( Table 4).
The conformation in solution (DMSO/LiBr) of the EPSs were studied by means of the conformational plots, which display the relationship between the radius of gyration (R g ) and the weight-average molecular weight ( -M w ) [26].
According to the De Gennes scaling theory, the molecular weight and hydrodynamic size show a power-type relationship (eq. 2), where the slope of the logarithmic plots (υ g ) indicates the conformation in solution, with a theoretical value of 0.33 for a compact sphere, 0.5-0.6 for a flexible random coil and 1 for a stiff rod.
Scleroglucan exhibited a semiflexible single coil conformation in DMSO at lower molecular weights (υ g = 0.67) in accordance to previous studies [24]; however, at larger molecular weights (above Mw > 10 6 ) a conformational transition towards a stiffer rod can be observed. On the other hand all schizophyllan fractions show stiff rod conformations (with υ g slopes above 1), again reinforcing the evidence of a conformational transition in DMSO/LiBr above molecular weights of 10 6 Da for β-(13)-(16)-D-glucans with similar branching structure.
Generally, samples with higher molecular weight display higher macroscopic viscosity [22,27]; however, a clear relationship between molecular weight and viscosity of polymer solutions does not always exist, since the latter could be affected by solubility and the presence of supramolecular aggregates creating differences in microviscosity in the macromolecular dispersions [8,20]. Therefore, the independent measurement of the rheological properties is required to ascertain the macroscopic behavior of EPS gels. Regarding the effect of macromolecular spatial conformation on rheological properties, a very stiff helical structure as expected in water leads to high viscosity of schizophyllan solution [28], and the dissociation of the triple helix into single chains decrease sharply the viscosity [29].

Solution rheology
The rheological properties of schizophyllan widely depend on their purity grade, the degree of branching, molecular weight, and the macromolecular conformation in solution, which can be clearly affected by the recovery strategies as it has been here reported [6,8,11,30]. In this direction, we have evaluated the effect of recovery procedures on steady shear flow behavior and oscillatory shear behavior of the recovered schizophyllan and compared with commercial scleroglucan.  Table 4). In this study, an increase in the EPSs concentration greatly increased the viscosity and consistency indices, which is consistent with other reports [6,9].

Steady shear flow behavior
Zhong et al. [9] also reported the pseudoplastic behavior and viscoelastic properties of solutions when the majority of high molecular weight fragments of schizophyllan were 10 6 -10 7 Da [27].
In terms of viscosity, the order of samples was CS11 < EPS-r < EPS-pre < EPS-sup. In agreement with commercial scleroglucan, EPS-r exhibited the highest consistency coefficient (K) value and lowest flow behavior index (n) which implies highest pseudoplasticity amongst the recovered schizophyllan samples ( Fig. 3 and Table 4) [17].
Conversely, the lowest K values experienced by the least purified polymer (EPS-sup, Table 4) were coherent with the low viscosity produced by the crude polymer [8]. The purity grade and degree of branching clearly affect the solution viscosity of the EPSs, as it has been reported for other β-glucans [7,19]. The solution properties of schizophyllan can be influenced by the purity grade which is very differently depending on recovery methods [8,11]. On the other hand, polysaccharides with higher branching degree and conformational flexibility encourage lateral glucose disorder and prevent intermolecular association beyond the triple helical stage, thus avoiding lateral packing and precipitation [25]. The consequences of these properties are lower flow resistance, leading to a decrease in viscosity [31,32]. Hence, the EPS-sup containing the highest degree of branching (Table 2) exhibited the lower viscosity over the shear rate range tested in this study ( Fig. 3 and Table 4) compared to EPS-pre with the lowest degree of branching. Also, lyophilization processes may be a reason for a reduced viscosity and/or diminished solubility [11,33,34] in the EPS samples. Water removal steps can significantly affect the microstructure of polysaccharide assemblies in the solid state and thus the formation of intermolecular hydrogen-bonds during rehydration may exert a significant influence on the rheological properties [35].
Schizophyllan aqueous solutions exhibit interesting rheological properties, mainly due to the high molecular weight and the stiffness of the polymer chains in aqueous solutions [2,6,8,9,18]. A remarkable pseudoplastic or shear thinning flow behavior of β-glucan solutions has been observed for all the samples at different concentrations, which provides advantages for industrial operations such as mixing and pumping [36]. Indeed, many schizophyllan applications depend on the viscosifying capacity [17,19], as for example, the efficiency of schizophyllan for oil field applications including enhanced oil recovery (EOR) closely depends on its ability to give aqueous solutions with high viscosity [34].

Oscillatory shear experiments
Oscillatory shear tests were performed in the linear viscoelastic range to determine the frequency dependence of the storage modulus (G') and the loss modulus (G''). G' represents the elastic behavior of a sample, since it quantifies the deformation energy stored during the process of shearing and G'' represents its viscous behavior, which defines the energy dissipated as heat during shear [37]. Oscillatory rheology is used to differentiate between the viscous and elastic characteristics of materials [37]. Fig. 4 shows the oscillatory behavior of EPSs at different concentrations Only EPS-sup at lowest concentration showed that the viscous response was greater than the elastic parameter at all frequencies tested and other EPSs at low concentrations (1-2 (%w/v)) displayed typical viscoelastic behavior, G'' > G' at the lower frequencies and G' > G'' at the higher frequencies. The "crossover frequency" (where G' = G'') moved to a lower frequency when the polymer concentration was increased from 1 (%w/v) to 2 (%w/v). However, when the concentration increased, G' exceeded G'' over the entire accessible frequency range and the system showed gel-like behavior. [9]. Similar to flow behavior results, EPS-sup with the highest branching degree showed the lowest viscosity and elasticity, while EPS-r among schizophyllan samples, had the highest viscosity and elasticity that corresponds to maximum microstructure and high entanglement density [37].
The moduli behavior and their crossover in a typical frequency sweep plot can reveal the structural character of materials, either as gel, paste or solution [37]. An increasing importance of the elastic effect with frequency is very known characteristic of viscoelastic materials which means that the viscoelastic properties are dominated by the established network structure [39]. In all of samples solutions G'/G'' considerably increased when the frequency is incremented. However, in the opposite trend where G'/G'' is constant, this is an indication of rigidity and presence of gel-like structure that even at the highest concentration of the samples investigated in the study, was not seen [40].

Conclusion
In this study, a high molar mass schizophyllan was produced from low value agricultural residues. Performing different recovery methods, such as re-dissolving in water and re-precipitation with ethanol on produced schizophyllan, provided schizophyllan with purity similar to the commercial CS11. Besides, freeze-thawing cycles allowed fractionation of schizophyllan based on branching degree and solubility. Our results demonstrate the distinctive influence of different recovery methods on molecular structure and rheological behavior of the recovered exopolysaccharides. Furthermore, the relationship of purity and key structural information with the rheological properties of the recovered exopolysaccharides was elucidated. Our results suggest that the evaluation and standardization of recovery/purification steps within the downstream processing methods within the biotechnological processes can be considered as a promising approach for the production of tailor made exopolysaccharides with specific applications.         a For details on nomenclature, see Section 2 ( EPS: recovered extracellular polysaccharide, CS11: commercial grade of scleroglucan, EPS-r: re-dissolution in distilled water and then re-precipitation in 95% ethanol (two times) of crude EPS, EPS-sup: the supernatant fraction after freeze-thawing of crude EPS, EPS-pre: the precipitate fraction after freeze-thawing of crude EPS).
-M n , number average molecular weight; -M w , weight average molecular weight; D, dispersity index; R g , average radius of gyration a For details on nomenclature, see Section 2 ( EPS: recovered extracellular polysaccharide, CS11: commercial grade of scleroglucan, EPS-r: re-dissolution in distilled water and then re-precipitation in 95% ethanol (two times) of crude EPS, EPS-sup: the supernatant fraction after freeze-thawing of crude EPS, EPS-pre: the precipitate fraction after freeze-thawing of crude EPS).