In course of present study, the air-dried aspen wood (Populus tremula) was milled to a fraction size of 2.5 mm and then dried at 100°C–150°C in an oven to a constant weight. The up mentioned species was chosen due its wide distribution all over the world.

To obtain polysaccharides 10 g of wood sawdust was treated with delignifying mixture acetic acid-hydrogen peroxide-water in a 250 mL three-necked glass batch equipped with a reflux condenser under a constant stirring (300 rpm). Reagents ratio of the delignifying mixture was selected basing on the previously conducted studies [6,25], and specifically with an acetic acid (Reagent, Samara, Russia) content of 30 wt.%, hydrogen peroxide (Lego, Dzerzhinsk, Russia) of 6 wt.% and a liquid/sawdust ratio of 15. In addition, the impact of an addition water-soluble catalyst (NH₄)₆Mo₇O₂₄ (Sigma Tech., Moscow, Russia) by varying its concentration in the range of 0.5–1.5 wt.% (relative to an absolutely dry sample) and delignification conditions, such as temperature (90°C or 100°C) and duration (3–4 h) of treatment on the isolation and properties of the hemicelluloses, were explored. The subsequent procedure for the isolation of hemicelluloses is similar to the studies [7,24].

As a result, the composition and structure of the obtained hemicelluloses samples (Table 1) were characterized by a set of modern physicochemical methods of analysis. In addition, various model compounds were used to reveal the impact of the hemicellulos’s composition and structure on their antioxidant and flocculation activities.

Uronic acids were determined using modified carbazole and tetraborate methods [26]. The calibration curve was performed using the stock solution of the analytical standard of D-galacturonic acid (Sigma Aldrich, St. Louis, MO, USA). The samples with specified concentration were diluted in distilled water and further treated with 0.025 M sodium tetraborate (SCRS, Krasnoyarsk, Russia) in sulfuric acid (Reagent, Samara, Russia) and carbazole solution.

In typical procedure, 6 mL of sodium tetraborate dissolved in sulfuric acid was slowly added to 1 mL of the investigated sample. These operations allows to decompose the hemicellulose’s samples into monomeric compounds.

To proceed the reaction with galacturonic acid the 0.5 mL of 0.015% carbazole-ethanol solution reagent was added and reaction mixture was shaken gently at first and then thoroughly vortexed for 30 s followed by incubation at 80°C for 10 min. Finally, the cooled sample solutions optical absorbance were spectrophotometrically measured at 530 nm with distilled water used as a reference solution.

The monosaccharide units composition of the hemicellulose was determined by the hydrolysis of initial samples in a 10% solution of H₂SO₄ [27] followed by derivatization according to method [28]. Analysis of derivated hydrolysates was conducted using gas chromatograph VARIAN-450 GC (Varian, Inc., Palo Alto, CA, USA), equipped with a VF-624ms capillary column (30 m × 0.32 mm). As an analytical standards xylose, mannose, arabinose, galactose, glucose, sorbitol (Panreac, Darmstadt, Germany) were used.

To evaluate the molecular weight properties of the isolated hemicelluloses, gel permeation chromatography analysis was performed using an Agilent 1260 Infinity II GPC/SEC system (Agilent Technologies, Santa Clara, CA, USA) equipped with the refractometer detector and three Agilent PL Aquagel-OH columns. The sample eluent solutions (5 mg/mL in 0.1 M aqueous NaNO₃ (AppliChem, Darmstadt, Germany) with 250 ppm of NaN₃ (AppliChem, Darmstadt, Germany)) were filtered through a membrane (Millipore, Burlington, MA, USA). The calibration was performed using polyethylene glycol (PEG) Agilent standard (Agilent Technologies, Santa Clara, CA, USA) with Mp 106–500,000 g/mol. The eluent flow rate during the analysis was 1 mL/min. The molecular weight distribution characteristics were depicted basing on the data registered and processed by Agilent GPC/SEC MDS software version 2.2.

The absorbance units in the Infra-Red spectra of hemicellulose samples were registered by Shimadzu IRTracer-100 (Shimadzu, Kyoto, Japan) and characterized in 2000–800 cm⁻¹ region, witnessing the structural changes of hemicelluloses depended on specific isolation conditions. In a typical procedure, the 3 mg of sample were pressed with 1 g of potassium bromide into tablets.

Water-soluble (NH₄)₆Mo₇O₂₄ catalyst has been previously demonstrated efficiency in intensifying of biomass delignification, as indicated by low values of lignin content in cellulosic residue (~1 wt.%) [6], as well as promising results with high spruce wood galactoglucomannan yield up to 48.2 wt.% [3]. However, this study reveals the effect of molybdenum catalyst concentration with varying process temperature and duration, which were investigated for the first time not only for hemicelluloses yields, but also on their composition, structure and physicochemical properties.

The target products yields were estimated by gravimetric method in relation to the total aspen wood hemicelluloses content, the results of which are presented in the Fig. 1. The total hemicelluloses in aspen wood contains up to ~20 wt.% [25].

Thus, based on the obtained data in terms of the total content of hemicelluloses in wood (Fig. 1), peculiar tendency is observed: in general, catalyst presence results in a significant increase of polysaccharides yield due to effect of the ammonium paramolybdate active catalytic complex with peracetic acid on lignocarbohydrate matrix and, consequently, hemicelluloses release. The samples of hemicelluloses isolated at 90°C and 3 h of processing, being the exception and distinguish by the lowest yield values not only in non-catalytic process conditions, but also in entire range of catalyst concentrations. Apparently, the given conditions lignocarbohydrate bonds oxidation occurs at a low intensity due to their chemical strength and insufficient duration of oxidizer exposure resulting in minor yields of purified hydrolysate hemicelluloses.

However, increasing the duration of the process to 4 h at 90°C leads to oxidation process activation, accompanied by a significant growth in target product yield to 45.6 wt.%, which is comparable with the data obtained at a 100°C process temperature (Fig. 1). Notably, in an earlier work [30] under similar synthesis conditions (90°C and 3 h), yields of pine wood hemicelluloses contains up to 58.1 wt.%, which undoubtedly indicates the importance of choosing the conditions for carrying out the delignification process from the raw materials used.

From the point of view of product yield the most preferred conditions for aspen wood delignification process are: 100°C process temperature, 3 and 4 h duration with 1.5 and 1 wt.% ammonium paramolybdate presence, accordingly. The above mentioned conditions allows obtain maximum yields of hemicelluloses reaching up to 62.55 wt.% in relation to total content in wood. However, isolation of polysaccharides not only with a high yield, but also with their native polymer structure preservation is still being crucial.

It is important to mention that xylan is the most common polysaccharide presented in deciduous biomass [31], depending on raw material nature and the extraction method xylose links contain additional side chains, for example, in the form of glucuronic acid residues [32]. In addition, according to various plant sources, the xylan hydroxyls could be easily substituted by acetyl groups [33].

The monosaccharide units composition of isolated hemicelluloses represented mainly by xylose, galactose, mannose and glucose (Fig. 2), varies depending on the oxidative delignification conditions used.

The predominant monosaccharide in the hydrolysates of all samples are xylose with some galactose and glucose, indicating that obtained polysaccharides represented mainly by galactoxylan and glucuronoxylan. However, mannose trace amounts and increased amount of galactose indicates the presence of easily hydrolysable polysaccharides residues in side chains of the xylan backbone, subjective to destruction regardless of oxidative delignification conditions. At the same time, initial presence of mannose and galactose in xylan structure provides complicated branched conformation and, as a consequence, greater solubility in aqueous medium.

Activation of peracetic acid by ammonium paramolybdate catalyst also intensifies the hydrolysis process, which accompanied with side chains cleavage, expressed by monosaccharide ratio rearrangement (Fig. 2). As a result, the purified xylan off accompanying polysaccharides was obtained at the output, however, with reduced solubility.

The structure of xylan samples depending on isolation conditions were studied by FTIR spectroscopy. As a result, most notable absorption units (a.u.) were identified, which are characteristic for polysaccharides (Fig. 3a,b). In general, the IR spectra of the samples are partially similar, but there are some differences.

The pronounced absorption units in regions of ~1735, 1380 and 1240 cm⁻¹, characterizing vibrations of carbonyl, –C–CH₃ and –C–O– groups accordingly, witnesses that all of the obtained hemicellulose samples are acetylated [34]. Intensive signal of a.u. at 1620 cm⁻¹ commonly associated with water adsorbed by sample. Notably, a.u. at 1509 cm⁻¹ corresponding to skeletal vibrations of the lignin’s phenolic fragments is not observed for only spectrum of hemicelluloses isolated at 100°C in presence of the peracetic acid activator. This phenomenon is caused by oxidative complex interaction on lignocarbohydrate matrix at elevated temperatures, undoubtedly significant for polysaccharides release and purification. The outstanding a.u. at 1043 cm⁻¹ region refers to the C–O–C bonds stretching vibrations in xylan pyranose ring [35]. Finally, a.u. 891 cm⁻¹ characterizes C–O–C of glycosides β-configuration between first and fourth carbon atoms [36].

Such characteristics as molecular weight and conformation are being high desirable to adjust structural properties of polysaccharides, tuning of which impacts on their further application, as well as on their biological activity [37,38].

The parameters of Mark-Houwink-Sakurada equation (MHS) were obtained using the multidetector gel permeation method (Eq. (4)). (4)[η]= KMα,where [η]—intrinsic viscosity, М—molecular weight (g/mol), K and α—constants characterizing molecules branching and conformation in a solvent, data of which is represented in Table 2.

Molecular mass distribution curves and graphs based on calculated MHS equation parameters are depicted on Fig. 4a,b.

The obtained data analysis revealed that in the presence of ammonium paramolybdate, polysaccharides were mainly isolated of the conformation of a «random coil» (α ranging from 0.68 to 0.81). As it could be seen, temperature, duration and (NH₄)₆Mo₇O₂₄ catalyst concentration do not take part in transformation of polysaccharide molecules, while in a greater extent affecting on their branching.

Nevertheless, a more branched molecular structure inherent for aspen hemicelluloses obtained at 90°C as indicated by molecular weight data (ranging from 17,945 to 26,976 g/mol), polydispersity (ranging from 3.68 to 4.28), as well as lower values of constant K (ranging from 1.35 to 1.90) (Table 2) and the curved MHS graph shape (Fig. 4a).

Generally, this allows us to conclude that obtained hemicellulose samples are represented by a mixture of high-molecular xylan fraction with certain amount of oligomers, which is consistent with the monosaccharide composition data due to an increased percentage of xylose, as well as the product yields values with a maximum of ~46 wt.%. At the same time, a simultaneous prolongation of oxidative delignification to 4 h and activator concentration to 1.5 wt.% at 90°C are accompanied by high-molecular fraction degradation. It could be detected both based on a main peak shift to the low-molecular region with a noticeable decrease in the polysaccharide molecular weight to 12,162 g/mol so as the sample yield, in particular. To sum up, due to polysaccharide molecules redistribution, its conformation undergoes “random coil—semi-flexible coil” transformation (α = 0.97).

On the other hand, performance of oxidative delignification at 100°C leads to the medium molecular weight polysaccharides formation (~14 × 10³ g/mol) (Table 2). At the same time, a prolonged processing and higher ammonium paramolybdate concentration in the reaction medium do not entail significant changes in polysaccharides structure, as indicated by identical molecular weight distribution profiles and MHS curves with their slight shifts (Fig. 4b), witnessing side chains cleavage with possible partial oxidation of polysaccharide terminal groups. While the maximum of parameters varying reached, the low-molecular fraction is completely released out from obtained mixture, but although it does not affect on main polymer chain structural changes, the yield of the target product decreases from 59 to 42 wt.%.

The physicochemical properties of hemicelluloses depend on the amount of charged groups caused by carboxylic acids, which are commonly represented by carbohydrates with a carboxylic acid groups (uronic acids) [39]. They could present as both base unit or as side chains of hemicellulose, affecting its biological and antioxidant activity. Therefore, its evaluation is desirable and could indirectly point at obtained hemicelluloses application potential for drug production.

The calibration curve was depicted after spectrophotometric determination of D-galacturonic acid analytical standard (Fig. 5), and the uronic content in hemicellulose’s samples were determined basing on plotted calibration curve and trend line Eq. (5): (5)Y=0.0009 x+0.1023(R=0.995),where Y = is the measured optical adsorbance and x is the concentration of D-galacturonic acid (μg/mL), formed by hemicellulose’s hydrolysis.

Nevertheless, uronic acid content in hydrolysate sample of hemicellulose obtained with presence of Mo⁶⁺ is slightly lower than in sample of hemicellulose obtained without the catalyst (66.0 μg/mL compared to 76.6 μg/mL). The ratio of uronic acids, based on the mass of the sample used for analysis are 7.76 wt.% and 9.01 wt.%, respectively.

The potential application of various polysaccharides and their derivatives as antioxidant agents has been previously reported [4,40,41]. Due to the fact that unique diverse composition and structure of polysaccharides depends on the source, method of extraction and purification degree, their biological activity could vary to some extent. Possibility of regulation polysaccharides isolation parameters contributes to expand their application areas, such as food, medical and pharmaceutical industries [42,43]. The traditional methods for evaluating of polysaccharides scavenging activity nowadays are based on the hemicellulose absorption ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals so as hydroxyl radicals. Thus, the performed DPPH scavenging tests to observe a decrease in light absorption at 517 nm with an increase in the concentration of hemicelluloses in the solution, which indicates a dose-dependent nature of reagents interacted (Fig. 6а).

However, differences in the conditions of polysaccharide synthesis entail significant differences in their absorption of free radicals. The sample of hemicellulose obtained at 90°C after 4 h oxidative delignification in presence of 1.5 wt.% ammonium paramolybdate has the maximum DPPH scavenging activity (Fig. 6а). Obviously, this phenomenon is directly related to the structural composition and conformation of the molecule of this polysaccharide. Possessing the lower molecular weight and, probably, a significant amount of sterically available reactive hydroxyl end groups, due to conformational features of the molecule, these hemicelluloses actively bind with DPPH free radicals. Nevertheless, phenolic fragments in residual structure of polysaccharides obtained at 90°C also significantly contributes to its activity [44]. Such limitation does not allow to estimate objectively the antioxidant activity of unpurified polysaccharides performed by DPPH method [44,45]. At the same time, the lowest values (~12.7%) of hydroxyl radicals scavenging activity (Fig. 6b), combined with physicochemical research of samples confirms the hypothesis of accompanying structural fragments main contribution to isolated polysaccharides biological activity.

At the same time, polysaccharides without lignin phenolic fragments exhibit a lower ability to bind DPPH free radicals, which is also consistent with similar studies [37] and this study. However, these same samples, obtained precisely at a process temperature of 100°C, most actively capture hydroxyl radicals, reaching a maximum of ~26.4%. Such results of polysaccharide scavenging activity, although inferior to vitamin C control sample in both cases, anyway represent a possibility to tune properties of obtained hemicelluloses by varying the oxidative delignification parameters which modification could improve their potential as biologically active agents.

It is widely accepted that polysaccharides are capable of binding with metal ion by exchange and/or complexation since the structure of polysaccharide is relatively open and has low steric effect for that kind of interaction [46]. Most researchers are interested in the cleansing activity of polysaccharides and the mechanisms of their interaction with inorganic salts [29,47,48]. The coagulation and flocculation processes are the inexpensive, non-toxic methods for acceleration of the suspended pollutant particles in water separation. This is why such processes are commonly used for the surface water treatment. In particular, the bivalent and trivalent salts of an aluminium and iron are predominantly applied as coagulating agents in water treatment technologies among the world [49].

Therefore, the flocculating activity of obtained hemicelluloses with the same amounts of the Na⁺, Ca²⁺ and Fe³⁺ (Fig. 7) salts was studied. To simulate wastewater conditions, the 10 g/L bentonite clay water dispersion with known composition [50] was used.

As it indicated above the presence of hemicellulose in bentonite dispersion supplied by rising quantities of metal salts promotes the flocculating activity. The maxima at 62.8% has been achieved using the salt containing bivalent Ca²⁺ salt, which is slightly even without the hemicellulose sample. Still, the absence of hemicellulose in reaction media affects aggregation of pollutant’s particles insignificantly, which results in absence of the flocculation. This fact was accepted as sedimentation processes of the bentonite particles and further used for comparison of flocculation capabilities [50].

On the contrary to Ca²⁺, the impact of Na⁺ or Fe³⁺ ions on hemicellulose flocculation activity is lower—their binding activity could not reach higher than 30% at their best. Such results could be connected with nature of polysaccharide-metal ion complexation—calcium ions added to the hemicellulose solution, more likely provokes the dimerization of polymer followed by an aggregation step of chain [46,51], than monovalent Na⁺, able to form salts without connection, or trivalent Fe³⁺ sterically hindering the complexation.