Sunflower meal valorization through enzyme-aided fractionation and production of emerging prebiotics

Sunflower meal valorization through enzyme-aided fractionation and production of emerging prebiotics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sunflower meal valorization through enzyme-aided fractionation and production of emerging prebiotics Milica Simović, Katarina Banjanac, Milica Veljković, Valentina Semenčenko, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3975794/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Graphical Abstract Abstract Sunflower meal, a plentiful and underutilized oil industry by-product, is currently discarded as waste or used as cheap component of animal feed with poor protein content and high content of complex carbohydrates. To harness its great potential for valorization, we propose an efficient method through enzyme-aided fractionation yielding 47.8% of hemicellulosic fraction, with simultaneous generation of various other potentially valuable fractions (including polyphenol-rich fraction, protein isolate, pectin, and lignin). According to the monomeric composition the main type of extracted hemicellulose was xylan with the common feature of a backbone of β -(1→4)-linked xylose residues, with a common substitution with arabinose moieties and some glucuronic acid residues. Subsequently, the xylan fraction underwent enzymatic treatment using commercial xylanase (ROHALASE ® SEP-VISCO) to produce highly valuable compounds - emerging prebiotics xylo-oligosaccharides (XOS). Under optimized reaction conditions (70°C, pH 6 and enzyme concentration of 0.005% v/v using 5% w/v xylan solution) a yield of XOS with a polymerization degree DP<50 reached approximately 52.3% after 2 hours (majority of obtained product had DP<6 with predominance of XOS2 and XOS3 and without significant xylose generation). sunflower meal waste valorization enzyme-aided fractionation xylo-oligosaccharides emerging prebiotic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Sunflower (Helianthus annuus L.) stands as one of the world's most widespread cultivated oil crops, alongside soybean and rapeseed (1, 2). Its good nutritional properties assure their wide utilization in food sector (1); however, its greatest economic importance comes from the high oil content in the seeds (3), and its potential for production of a high-quality oil that represents one of the most preferable and widely used vegetable oil in the world (1, 4). Despite ongoing efforts to enhance the oil extraction process, a significant portion of solid residues (hulls, meals, and cakes) continues to be generated worldwide (5). These oilseed residues are usually discarded as a waste or utilized as a cheap component of animal feed for various livestock species, with a neglectable application in human diet (6). Sunflower meal (SFM), the primary by-product of sunflower oil extraction, holds great potential as a source of proteins, dietary fibers, phenolic compounds, and minerals, yet remains underutilized (7). Its profitable industrial application is currently limited due to its comparatively low protein content (8), high levels of undigestible carbohydrates (9), and finally the abundance of antinutritional factors (10)[9], especially phenolics that may form complexes with proteins thus leading to reduced functionality and digestibility of meals (5, 11). Accordingly, there has been growing interest in their profitable reutilization aligning with principles of circular economy and sustainable development. Valorization of SFM could result in the preparation of multiple products with added value for the food and nutraceutical industries or agriculture (11). The most commonly utilized methods for valorization of SFM are based on the extraction of protein fraction (8), which follows constant increase in demand for relatively cheap and adequate protein sources (12). This is not surprising having in mind the nutritional and technological advantages of SFM proteins (13, 14). Nevertheless, in view of fully achieving biorefinery goals the polyphenolic and carbohydrate fractions should also be considered for valorization. Phenolic compounds are frequently eliminated within the first steps of protein isolation process, but recent data show researchers are shifting towards their utilization upon gaining new knowledge about the utility of these fractions (10, 15). However, the systematic valorization of carbohydrate fraction, which makes up to 52% of dry matter, depending on the oil extraction process (16), is still insufficiently studied. Conventional extraction methods to valorize carbohydrate fraction in different by-products and waste materials, based on multi-step extractions using acids and alkalis, come with many limitations such as loss in fiber functionality and laborious operation with low extraction efficacy and, more importantly, environmental unacceptability, since the rigid lignocellulose structure requires severe pretreatments to facilitate efficient extraction of mentioned fractions (17). Pectic compounds are usually extracted using mineral or organic acids (18, 19), while traditional hemicellulose extraction methods are mostly based on alkaline extraction (sodium or potassium hydroxides) of xylan, with or without delignification step (20-22). Delignification is usually provided with sodium chlorite, hydrogen peroxide or peroxyacetic acid, with the aim of obtaining purer xylan fractions, since alkaline treatment also partially solubilizes present lignin (23). These methods caused unavoidable hemicellulose degradation, and therefore, alternative means of isolation were purposed, such as organic solvent treatments using different organic solvents (e.g. dimethyl sulfoxide, mixtures of dimethyl sulfoxide with other compounds and dioxane) but these methods provided significantly lower yields of wanted compounds most probably being restricted to water soluble fraction of xylan (24). Additionally, application of complementary emerging technologies as microwaves, ultrasound or enzymatic assistance is growing nowadays (25-27). In the field of sunflower carbohydrate valorization, only sunflower heads were previously utilized for pectin extraction (28-31), while SFM was not subjected to either pectin or xylan extraction to date. Xylan is commonly studied for textile, food and biomedical applications, however recently it is widely utilized for production of non-digestible oligomers, known as xylo-oligosaccharides (XOS) a promising functional ingredient with diverse applications in the food and pharmaceutical industry. These xylose-based oligosaccharides are naturally occurring in vegetables, fruits or honey (32), but at insufficient quantities. XOS are viewed as promising emerging prebiotics, that have a crucial role in maintaining gut health, and simultaneously are well-suited for incorporation in a wide variety of food and feed products. This is due to their exceptional application characteristics, including stability at high temperatures (up to 100 ºC) and across a broad pH range (2.5–8), as well as their good sweetening power with minimal caloric impact (33). XOS may be produced using commercially available xylan, however, in order to enable their economically viable production nowadays major focus has been placed on utilization of lignocellulosic biomass, which represents cheap source of xylan (34). Up to date, large number of lignocellulosic materials (wheat and rice straw, wheat and barley brans, sorghum and grape stalks, sugarcane bagasse, corncob, beechwood and birchwood) have been studied as xylan sources for XOS production with varying efficiency, mostly depending on the type of present xylan, and employed conversion methods (35). Therefore, different methods of XOS production and great diversity in potential xylan substrates lead to wide spectrum of different XOS structures with varied substituents of xylose backbone and degrees of polymerization that consequently have great impact on its prebiotic and other functional properties. The main aim of this work was to propose a promising pathway for SFM valorization primarily through enzyme-aided extraction of xylan that would be further used in production of valuable xylo-oligosaccharides (XOS) as emerging prebiotics. Thus, enzymatic hydrolysis of obtained xylans will be achieved using highly promising enzymatic preparation Rohalase ® SEP-Visco, a thermostable bacterial xylanase from Trichoderma reesei, that has not been previously applied for similar purposes. This way it is expected to provide value added prebiotic-rich fraction of SFM. Additionally, our enzyme-aided fractionation method will deliver other fractions enriched in other compounds, such as polyphenols, proteins, pectin, lignin and cellulose, that can be utilized in future as food additives or in the field of nutraceuticals and functional cosmetics ingredients. 2. Materials and methods 2.1. Materials Partially dehulled sunflower meal (SFM) utilized in this study was kind donation of Victoriaoil LTD (Šid, Serbia). Analytical grade chemicals, including ethanol, acetone, hydrochloric acid, sodium chlorite, and sodium hydroxide, ammonium acetate were obtained from Centrohem (Stara Pazova, Serbia). HPLC grade solvents (acetonitrile and water), tri-fluoroacetic acid (TFA), hydroxylamine chloride, pyridine, hexamethyldisilazane (HMDS), pullulan standard set, phenyl- β -D-glucoside and 1-phenyl-3-methyl-5-pyrazolone (PMP) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Xylo-oligosaccharides (XOS) standards (xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose) were purchased from Megazyme LTD, Wicklow, Ireland. Enzymes used within the study were: Alcalase ® 2.4L, Novozymes (Bagsværd, Denmark) and Rohalase ® SEP-Visco that was kind donation of AB Enzymes GmbH (Darmstadt, Germany). 2.2. Sunflower meal compositional analysis 2.2.1. Analysis of dry matter (DM) Dry matter (DM) was determined gravimetrically drying the samples until constant weight according to the AOAC method 950.01 (36). 2.2.2. Analysis of ash content The ash content was determined according to method number 923.03 by the slow combustion of the sample at 550 °C in a muffle furnace (L47, 1200°C, Naber Industrieofenbau, Lilienthal, Germany) (37). 2.2.3. Analysis of oil content The oil content was obtained according to the Soxhlet method number 920.39 (37), on a FatExtractor E-500 (BÜCHI Labortechnik, Flawil, Switzerland). 2.2.4. Analysis of Total Protein Content The protein content was determined by the standard micro-Kjeldahl method (official Method 920.87) as the total N multiplied by 6.25 on BÜCHI Kjeldahl System (Auto Kjeldahl Distillation Unit K-350 and Speed Digester K-439, BÜCHI Labortechnik, Flawil, Switzerland). 2.2.5. Analysis of Sugar Content The content of total sugars, reducing sugars and sucrose was determined by the Luff-Schoorl method based on the reaction between reducing sugars and alkaline solution of copper sulphate, with a subsequent reduction of cupric copper to cuprous oxide. In the method, Cu 2+ ions that had not been reduced are determined iodometrically. Furthermore, total sugars were then determined by converting non-reducing sugars into reducing sugars through acid hydrolysis (38). 2.2.6. Analysis of Dietary Fiber Content The content of hemicellulose, cellulose, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were determined by the Van Soest detergent method modified by Mertens using the Fibertec system FOSS 2010 Hot Extractor (FOSS Tecator, Hoeganaes, Sweden) (39). After filtering and drying, the NDF, ADF and ADL were calculated as a percentage of the original sample. The content of hemicellulose was obtained as the difference between NDF and ADF contents, while the cellulose content was calculated as the difference between ADF and lignin contents. 2.3. Sunflower meal fractionation 2.3.1. Method of conventional sunflower meal fractionation The schematic representation of the fractionation process applied to SFM is shown in Figure 1. Preparation of alcohol-insoluble residue of sunflower meal (AIR-SFM) was provided by triple extraction using 70% v/v aqueous ethanol solution (1:10 w/v). Slurry was stirred for 30 min on a magnetic stirrer and recovered by vacuum filtration. The solid residue (AIR-SFM) was washed using 96% ethanol and acetone, respectively. Combined ethanol extracts were concentrated using rotary vacuum evaporator and represent polyphenol-rich fraction. Next step of fractionation was performed using hot diluted hydrochloric acid (HCl, pH adjusted to 1.5) at 1:25 w/v ratio and heated at 90 °C for 1.5 h. The extracted material was recovered by vacuum filtration and solid residue was rinsed with water. The obtained pectin was recovered by cold ethanol precipitation with 4 volumes of 96% ethanol overnight. The obtained precipitates were recovered by centrifugation (10 min on 6000 rpm). Solid residue (Dpect-SFM) was dried by solvent exchange (ethanol, acetone). Next step in fractionation scheme was delignification step performed using 1.87% w/v solution of sodium chlorite with 1.87% v/v acetic acid at 1:32 w/v substrate to solvent ratio at 70 °C for 2 h. The delignified solid residue (DL-SFM) was recovered by vacuum filtration rinsed with water until neutral pH and dried using solvent exchange (ethanol, acetone). Solubilized lignin was lyophilized. Final step in xylan extraction was alkaline extraction using 4 M potassium hydroxide solution at 1:20 (w/v) ratio with continuous mixing at room temperature during 4 h. The obtained extract was separated by vacuum filtration and adjusted to pH 6 using concentrated acetic acid, and thereafter recovered by cold ethanol precipitation with 4 volumes of 96% ethanol overnight. The obtained precipitate was recovered by centrifugation (10 min on 6000 rpm) and lyophilized afterwards (xylan). The solid residue (cellulose-rich residue) was rinsed with distilled water until neutral pH is reached and afterwards dried using solvent exchange (ethanol, acetone). The obtained precipitates were recovered by centrifugation. 2.3.2. Enzyme-aided method of sunflower meal fractionation Alcalase ® 2.4 L treatment of AIR-SFM was performed in shaken Erlenmeyer flask (reaction volume of 500 mL) at orbital shaker (120 rpm) at 50 °C. AIR-SFM was suspended in 100 mM sodium-phosphate buffer (pH 7.5) in ratio 1:20 (w:v), and the enzyme concentration was 2.5% (v/w) calculated on AIR-SFM. In optimization experiments one parameter was varied at a time in following ranges: temperature 40-60°C, substrate to buffer ratio: 1:10-1:30 (w/v) and enzyme concentration: 0.625-2.5% (v/w) calculated on substrate. Concentration of proteins in supernatant was monitored during 2 h. At a predefined time, aliquots of reaction mixture were taken and incubated at 100 °C for 5 min to stop the reaction, centrifuged and then analyzed by determination of protein concentration (mg eq BSA per g of AIR-SFM) using Lowry method (40). For the enzyme-aided SFM fractionation under the selected optimal conditions, AIR-SFM was suspended in 100 mM sodium phosphate buffer (pH 7.5) at ratio1:20 w/v. The reaction was started with addition of 2.5% of Alcalase ® 2.4L, and reaction was performed with constant mixing at 50 °C for 2 h. The solid residue from the reaction mixture (DP-SFM) was recovered by vacuum filtration, and immediately rinsed with distilled water, and then dried with solvent exchange (ethanol and acetone) to rinse the enzyme and stop the reaction. The protein rich filtrate fraction was boiled at 90 °C to stop the reaction and afterwards concentrated using rotary evaporator and finally lyophilized (protein-rich fraction). 2.4. FTIR analysis FTIR analysis of both samples of extracted xylans was performed. KBr discs were prepared mixing the xylan samples with KBr (1:100) and pressed. FT-IR spectra were performed in a Bruker IFS66v equipment (Bruker, US). Data were collected in absorbance mode using a frequency range of 4000–400 cm -1 , and resolution of 4 cm -1 (mid infrared region) with 250 co-added scans (26). 2.5. Determination of monomeric composition For determination of monomeric composition of SFM and obtained xylans previously developed method was utilized (26). Samples (30 mg) were hydrolyzed using 1.5 mL of 2 M TFA at 110 °C under inert conditions for 4 h. Upon hydrolysis, 300 μL of the hydrolyzed samples were evaporated. After evaporation of the acid, 400 μL of phenyl- β -D-glucoside internal standard (0,5 mg/mL) is added, mixture is evaporated. To carry out the derivatization, 250 μL of hydroxylamine chloride (2.5%) in pyridine was first added, shaken vigorously and kept for 30 min at 70 °C to form the oximes. Subsequently, 250 μL of HMDS and 25 μL of TFA were added. It was shaken vigorously and kept in the oven at 50 °C for 30 min. Samples were centrifuged (10,000 rpm, 2 min), the supernatant was collected and kept at 4 °C until analysis by GC-FID. The analysis was carried out using an Agilent Technologies 7820A Gas Chromatograph (Agilent Technologies, Wilmington, DE, USA). A VF-5HT capillary column, phase bound (5% phenylmethylpolysiloxane, 30 m x 0.250 mm x 0.10 μm; Agilent J&W, Folson, CA, USA) was used. The injector temperature was 280 ºC and the detector temperature was 385ºC. Nitrogen was used as carrier gas with a flow rate of 1 mL/min. The temperature programme was: initial temperature 120 ºC, ramp of 3 ºC/min up to 380 ºC. Data acquisition and processing was carried out using Agilent ChemStation software (Wilmington, DE, USA). GC quantification was performed using β -phenyl-glucoside (0.5 mg/mL) as internal standard and mixtures of the sugars of interest in a range of concentrations between 2 and 0.02 mg/mL. 2.6. Determination of the molecular mass distribution (Mw) by High Performance Size-exclusion Chromatography with evaporative light scattering detector (HPSEC-ELSD). Molecular weight (Mw) distribution was estimated according to previously published procedure (41). Samples (20 mg/mL) were prepared by dissolving in distilled water for 30 min at elevated temperature (50 °C), than mixed with mobile phase (0.04 M ammonium acetate) in ratio 1:9, filtered and separated by High Performance Size Exclusion Chromatography with Evaporative Light Scattering Detector (HPSEC-ELSD) (Agilent Technologies, Boeblingen, Germany) using TSK-Gel guard column (6.0 mm × 400 mm) and two TSK-Gel columns connected in series, G5000 PWXL (7.8 mm × 300 nm, 10 μm), and G2500 PWXL (7.8 mm × 300 nm, 6 μm) (Tosoh Bioscience, Stuttgart, Germany). Pullulan standard set (Mw 0.342-788 kDa, 2-0.2 mg/mL) was utilized as standard for determination of molecular weight distribution. 2.7. Enzymatic production of xylo-oligosaccharides (XOS) The obtained xylans are finally utilized to synthesize XOS in shaken Erlenmeyer flask (reaction volume of 20 mL). The reaction mixtures were prepared by dissolving obtained xylans in 100 mM sodium phosphate buffer (pH 6) to reach the concentration of 1% and 5% (w/v). The reaction was catalyzed by means of Rohalase ® SEP-Visco in concentration range 0.005-0.05% (v/v) at 50 °C with constant orbital shaking (120 rpm). Reactions were monitored during 2 h. At a predefined time, aliquots of reaction mixture were taken and incubated at 100 °C for 5 min to stop the reaction. Part of the samples were derivatized, filtered and analyzed by high performance liquid chromatography (HPLC), and selected samples were analysed by means of HPSEC-ELSD and Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS). 2.8. Determination of total reducing sugars The total reducing sugars in the reaction mixture was determined using the method reported by Miller (1959) using the dinitrosalicylic acid reagent. The concentration of reducing sugars was calculated from the standard curve of xylose (1-10 mM) (42). 2.9. High performance liquid chromatography analysis of reaction mixture Prior to HPLC analysis samples were derivatized using the PMP in accordance with procedure published by Wang et al. (43). After derivatization samples were filtered and analyzed using Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham, USA) equipped with a reverse phase column (ZORBAX Eclipse Plus C18, 4.6 × 150 mm, 5 µm) at 30 °C, using with the mobile phase of 100 mM ammonium acetate buffer (pH 5.5):acetonitrile (80:20) with constant flow rate of 0.5 mL/min (44). Detection of products and standards was carried out by a UV detector at 245 nm. Data collection and processing were performed using Chromeleon 7.2. software. The concentration of the obtained compounds was calculated using the PMP-derivatized standard samples of XOS (XOS2-XOS6). 2.10. MALDI-TOF mass spectrometry The samples were ten times diluted in water and mixed in a 20:5 ratio (matrix:sample). The utilized matrix was 2,5-dihydroxybenzoic acid (DHB) with concentration 10 mg/mL in methanol+10% H 2 O. All mixtures were treated with strong cation exchange resin to eliminate present salts and avoid possible inhibition of the compounds of interest ionization. Analyses have been done in positive ion detection mode in the range from 50 to 5000 Da, with application of an ion deflection up to 400 to prevent mass matrix signals (which are the most intense) to saturate the detector. MALDI-TOF-MS analyses were performed on a Voyager DE-PRO mass spectrometer (Applied Biosystems, Foster City, CA) at the Interdepartmental investigation service (SIdI-UAM) of Madrid. 2.11. Statistical analysis All experiments are performed in duplicate and the results of experiments were provided as mean ± standard deviation. 3. Results and discussion 3.1. Chemical characterization of sunflower meal In the preliminary experiment, we thoroughly analyzed the compositional characteristics of partially dehulled sunflower meal (SFM), which served as the substrate for our study (Tables 1 and 2 ). The dry matter (DM) of the SFM was found to be 91.5%, while SFM was composed of 42.8% of proteins, 47.0% of carbohydrates, 2.8% of fats and 7.4% of ash (all calculated on dry matter). Table 1 Chemical composition of partially dehulled SFM Component Concentration Dry matter (%) 91.5 Simple sugars (% DM) 5.5 Hemicellulose (% DM) 12.7 Cellulose (% DM) 13.5 Lignin (% DM) 6.8 Proteins (% DM) 42.8 Fats (% DM) 2.8 Ash (% DM) 7.4 These results are in accordance with the ranges presented in previously published data on SFM, since data on chemical composition considerably vary depending on applied sunflower seed processing methods ( 45 ). Namely, dry matter (DM) values for different SFM ranged from 88.0 to 93.8%. Protein content was in the wide range from 26.4 to 40.3% per total weight, while total mineral content was found to be rather constant (5.5 to 7.8%) in all examined samples ( 45 ). On the other hand, data concerning detailed carbohydrate composition of SFM are quite scarce since most of the studies focuses on protein fraction of the meal ( 46 ). Namely, majority of data about SFM carbohydrates are expressed through crude fiber (CF) content (11.5 to 29.7% ( 45 )) or dietary fiber (DF) content (35.8–51.0% DM ( 46 )). Carbohydrate analysis of dehulled SMF in our study revealed that they are comprised of simple sugars (5.5% DM) where sucrose content was predominant, with smaller amounts of monosaccharides (glucose and fructose) and oligosaccharides (most probably raffinose and stachyose ( 46 )) and, on the other hand, high amount of polysaccharides. Among the polysaccharides, SFM contains 12.7% DM hemicellulose, 13.5% DM cellulose, and 6.8% DM lignin. The remaining portion (up to 8.5% DM) can be likely attributed to pectin bearing in mind that the obtained results on SFM monomeric composition showed that galacturonic acid is present in moderate quantities, 14.35% of total monosaccharides (Table 2 ). Similarly, regarding the monomeric composition of SFM, it can be seen that xylose (21.8%), together with arabinose (21.1%) and glucose (22.3%), represents most frequent monomeric building block for present polysaccharides, and therefore shows that SFM represents a highly potent substrate for xylan extraction. Table 2 Monomeric composition of partially dehulled SFM polysaccharides (% total identified monosaccharides). DP-xylan: deproteinized xylan Component Concentration (%) SFM Xylan DP-xylan Xylose 21.8 ± 1.9 53.7 ± 5.4 63.4 ± 1.1 Arabinose 21.1 ± 0.8 11.2 ± 0.1 9.7 ± 0.9 Rhamnose 3.6 ± 0.2 3.4 ± 0.3 3.3 ± 0.1 Fructose 2.4 ± 0.4 1.1 ± 0.6 1.7 ± 0.1 Galactose 10.6 ± 0.4 11.8 ± 7.3 5.8 ± 0.6 Mannose 3.9 ± 0.3 2.0 ± 0.2 0.5 ± 0.1 Glucose 22.3 ± 2.9 10.4 ± 1.1 8.0 ± 0.2 Uronic acids * 14.4 ± 0.4 6.5 ± 0.5 7.7 ± 0.1 * Galacturonic and glucuronic acids 3.2. Sunflower meal fractionation and xylan extraction In the preliminary experiment, extraction of xylan was performed using most frequently utilized method of alkaline treatment, based on previously published literature data on xylan extraction ( 22 ). However, this method proved to be unsuccessful since product with low purity of the isolated xylan was obtained (with 61.5% of DM being proteins), most probably due to complex composition of SFM that includes proteins and other carbohydrates. This treatment causes disruption of the complex structure by cellulose swelling and hydrolysis of uronic and acetic acid esters linkages, and dissolving hemicellulose and lignin ( 47 ). Additionally, these alkaline conditions simultaneously favor protein extraction ( 48 ). Therefore, more complex multi-step approach of SFM fractionation to obtain different carbohydrate fractions and protein isolate, was approached (Fig. 1 ). Conventional method of SFM fractionation assumed the primary removal of extractables (polyphenolic compounds, simple sugars, and colorants) using 70% ethanol solution (Fig. 1 ). The resulting extract proved to be rich in polyphenols (predominantly chlorogenic and caffeic acid) that could be applied as antioxidant agent in different formulations and potentially exhibit prebiotic activity on gut and skin microbiota ( 49 ). Since presence of pectin in SFM was assumed, the step of pectin extraction was introduced according to the work of Cebin et al. ( 35 ), in order to make better utilization of all present fractions. Also, bearing in mind that further steps (e.g. alkaline treatment) could impose structural and functional modifications on native pectin structure (47), this step was performed at the beginning of the fractionation process. In this step, pectin containing fraction (14 g) was obtained (extraction yield of 15.34%), but it should be noted here that obtained fraction might have poor purity, since great amount of proteins (10 g) were extracted alongside with pectin, that would introduce a need for further purification of obtained fraction. The remained depectinized solid residue (DPect-SFM) was thereafter subjected to delignification treatment. This step was introduced since it was determined that SMF possess around 6.8% of lignin calculated on DM that can be solubilized under alkaline conditions. For this treatment, sodium chlorite with acetic acid addition was chosen based on previously published data concerning its performance and selectivity ( 35 ). Under optimized conditions, one-step delignification of DP-SFM was performed for 2 h and resulted in approximately 18 g of lignin-containing fraction with high quantity of proteins (11.7 g proteins). With this in mind, additional experiment was performed without the delignification step, yet lack of this step highly affected the quality of finally obtained xylan. Finally, the next step - alkaline treatment was therefore performed on delignified solid residue (DL-SMF) using 4 M potassium hydroxide (1:20) for 2 h at room temperature. This step yielded 6.5 g DM of xylan (yield of 7.1% and recovery yield of 56%) and around 34 g DM of cellulose rich fraction. The obtained xylan had 52.5% of carbohydrates (mainly composed of xylose 53.7%) and 25.3% of proteins, calculated on DM. At the end, it can be concluded that high share of proteins ended up in different carbohydrate rich fractions. Therefore, to prevent this protein loss and on the other way to improve the purity, as well as a xylan yield and purity, a new method for fractionation was proposed. This method included utilization of enzymes that will help protein separation and enable easier and more efficient fractionation of carbohydrates. 3.2. Optimization of protein extraction from AIR-SFM using Alcalase ® 2.4L Generally, extraction of proteins from lignocellulosic plant materials presents a great challenge. Consequently, an enzyme-aided extraction method is frequently proposed as environmentally friendly technique that can be used to facilitate protein extraction from different plant sources. Enzymes provide simultaneous disruption of the cell wall and extract proteins by detaching of the structural protein complexes from large polysaccharide matrices or/and degrading those to smaller molecular mass proteins and peptides ( 50 ). To maximize the efficiency of protein extraction from lignocellulosic plant materials, the key process parameters such as pH, temperature, reaction time, substrate and enzyme concentration should be determined. In this part of the study commercial protease preparation (Alcalase® 2.4L) was employed to achieve maximum protein extraction efficiency from AIR-SFM (containing 37.8 g proteins). First, the influence of temperature (40–60°C) on the protein extractability was examined since it potentially could provide positive effects on both enzyme activity and extractability of proteins. The other parameters were kept constant (AIR-SFM to 100 mM sodium phosphate buffer (pH 7.5) ratio 1:20 with enzyme concentration of 2.5% calculated on substrate). Even though the enzyme possesses wide optimum range in alkaline medium (pH 6–10), buffer pH 7.5 was chosen since higher pH values may induce xylan and lignin extraction ( 51 ). According to presented results (Fig. 2 ), it can be seen that almost exact protein concentrations were obtained at 50°C and 60°C (approximately 300 mg/g AIR-SFM determined as eq BSA), while reaction at 40°C showed lower extraction yields. Therefore, the next experiments will be performed at 50°C. To promote diffusion of proteins from AIR-SFM into solution, a minimum amount of solvent needed should be determined. By adding large amount of solvent during extraction process, the extraction efficiency will likely be increased but it will raise process costs. Therefore, a study of the influence of AIR-SFM to buffer ratio on extraction process was performed. The obtained results indicate that when ratio was increased from 1:10 (w/v) to 1:20 (w/v) slightly enhanced protein extraction efficiency was achieved (approximately 300 mg/g of AIR-SFM), while further ratio increment did not induce any changes in obtained results (Fig. 2 ). Accordingly, an AIR-SFM to buffer ratio of 1:20 was selected for further experiments. Finally, the influence of enzyme concentration was examined. A typical proteases dosage from 0.5 to 5% per g of lignocellulosic plant material could be found in several studies where protease-aided protein extraction from soybean, rapeseed, peanut, lupin, rice bran and sunflower were examined ( 52 , 53 ). In case of AIR-SFM treatment with Alcalase® using different amount of enzyme (0.625-2.5% on substrate), the highest protein concentration of 300 mg/g AIR-SFM was obtained using 2.5% Alcalase® 2.4L (Fig. 2 ). 3.3. Protease-aided fractionation of sunflower meal Upon determination of optimal conditions for protein removal from the AIR-SMF, the modified xylan extraction process was developed (Fig. 1 ). After the removal of extractables and enzyme catalyzed deproteinization step, the 42.9 g DM of solid phase (DR-SFM) that contained 7.4 g proteins was obtained. It can be seen that deproteinization step was highly successful since 30,5 g of proteins (corresponding to 80.54% from total proteins of AIR-SFM) was removed to protein-rich fraction. The subsequent step in the fractionation process involved depectinization under identical conditions to those used in conventional fractionation. This step yielded 30.1 g DM of DpectDP-SFM that contained 3.8 g proteins (12.7%), while in conventional fractionation method corresponding fraction (Dpect-SFM) contained 47.0% proteins calculated on DM. The pectin-rich fraction had 12.8 g DM (extraction yield 14%) and only 3.6 g proteins, that showed great improvement in terms of achieved purity of the fraction. During the delignification step, 27.5 g DM of DLDP-SFM was obtained, and together with the lignin fraction a small quantity of proteins was removed. Finally, the DP-xylan 5.6 g DM was isolated. The yield of enzyme-aided fractionation process was 6.1%, while the recovery yield was 47.8%. Obtained DP-xylan contained 92.2% of carbohydrates mostly composed of xylose (63.4%) and approximately 7% of proteins (Table 2 ). These results prove the validity of modification the fractionation process, that is the introduction of the deproteinization step. 3.4. Characterization and comparison of obtained xylans In view of summarizing the advantages of newly developed method for SFM fractionation and xylan extraction, a more thorough comparison of obtained xylan fractions by displaying the most important parameters (Table 3 ). Thus, slightly higher quantities, and accordingly higher yields of obtained xylan fractions were achieved within conventional fractionation method. These results are expected and in accordance with previously published data. For example, Sporck et al. showed that enzymatic approach gave 2.4-times lower yields than alkaline method due to higher number of extraction steps and extraction selectivity ( 17 ). Table 3 Characterization of obtained xylans Xylan DP-xylan Quantity (g) 6.7 ± 0.3 5.8 ± 0.6 Dry matter (%) 96.8 ± 1.1 95.0 ± 2.2 Yield (%) 7.1 ± 0.5 6.1 ± 0.6 Recovery yield (%) 56.0 ± 2.2 47.8 ± 0.9 Carbohydrate content (%) 52.5 ± 2.3 92.2 ± 3.3 Protein content (%) 25.3 ± 1.2 7.1 ± 1.1 However, higher yields using the conventional method are obtained at the expense of the purity of the samples that can be seen when it comes to carbohydrate and protein content. Namely, conventionally obtained xylan has carbohydrate content of 52.5% DM corresponding to total amount of 3.4 g of carbohydrates while, on the other hand, DP-xylan obtained according to the newly proposed method exhibited great purity, since it had 92.2% DM of carbohydrates. This corresponds to 5.1 g of carbohydrates that is almost 1.5 times more than in previous case. In terms of achieved recovery yields, results obtained in this study were better than in the case of alkaline-sulfite pretreated sugarcane bagasse (recovered yields of 53% and 22% for conventional and enzymatic treatment, respectively) ( 17 ). Likewise, similar yields (4.5–8.5%) were obtained by Rowley et al., using delignified corn stover by means of different methods ( 24 ). The analyzed infrared spectra of the both obtained xylans show characteristic bands for xylan-rich compounds (Fig. 3 a). Namely, bands occurring at 3414 cm − 1 that can be assigned to the stretching vibrations of the O-H groups and the bands occurring at 2926 cm − 1 that are generally assigned to the -CH 2 antisymmetric stretching, while the band at 2850 cm − 1 was a result of -CH 2 symmetric stretching ( 54 ). Bands occurring at 1644 cm − 1 can be assigned to the absorbed water ( 55 ). The difference between two xylan samples can be seen around 1510 cm − 1 , where lignin can be observed in the xylan spectra due to the aromatic skeletal vibration ( 56 ). This band is expectedly more pronounced in case of conventionally extracted xylan, implying the lower purity of the sample (Fig. 3 a). The bands occurring at 1450 cm − 1 in some samples could be assigned to the presence of the methyl groups while spectral peaks that are visible at 1044 cm − 1 of C-O stretching in the C-O-C ether linkages (the first is the inter sugar units and the second results from intra sugar of alcoholic functional group). The peaks at 897 cm − 1 can be attributed to the stretching vibration modes (both symmetric and antisymmetric) of C-O in the ether linkage and can prove the β- configuration of 1→4 glycosidic bonds between xylose units of the xylan chain ( 54 ). Other bands at lower wavenumbers, such as 690 cm − 1 , are attributed to the out-of-plane C-H deformations ( 54 ). The signals around 1249 cm − 1 and 1736 − 1 indicate the vibrational band of the single bond C-O stretching band and C = O stretching related to the acetyl groups present in the xylan, respectively ( 57 ). Additionally, the estimation of molecular weight (Mw) distribution (four fragments) and their corresponding relative abundances (%) are shown in Fig. 3 b. It can be seen that obtained both xylans possess similar Mw distribution within the four fragments. There were two predominant fragments consisting of a very wide Mw ranges (245–8000 kDa and 4-245 kDa, respectively) followed distantly by two fragments with a narrow range, between 1 and 4 kDa and 0.15-1 kDa (corresponding to mono- and oligosaccharides). These results show that introduction of deproteinization step did not impose negative influence on the structure of extracted xylan. 3.5. Hydrolysis of obtained xylans towards xylo-oligosaccharides production After extraction and characterization of SFM xylans, the final step in SFM valorization was to obtain emerging prebiotics, XOS by means of enzymatic conversion. For this purpose, bacterial xylanase Rohalase® SEP-Visco was chosen as optimal commercial preparation owing to the fact that it produces negligible amounts of xylose (preliminary study), thus, ensures production of complex XOS mixture without need of extensive purification. This enzyme was applied for the first time for XOS production, and therefore, the first experiments enabled insight in parameters for optimal activity of the enzyme preparation needed for XOS production. It was concluded that optimum conditions for XOS production were slightly acidic to neutral reaction medium, while the optimum temperature was found to be around 70°C (supplementary data). After determination of these process parameters, the influence of substrate and enzyme concentrations on XOS production was examined. As it can be clearly seen from the time course graph (Fig. 4 a), enzyme preparation proved to have significantly high activity, and that even small amounts of the enzyme (0.005% v/v) can be utilized for this purpose. Consistent maximum XOS concentrations were achieved in all experiments, regardless of enzyme concentration. Hence, the lowest examined enzyme concentration was adopted as optimal for the next set of experiments featuring the higher concentrations of substrate in order to examine the initial kinetics of the XOS synthesis reaction, and accordingly to see differences in their structures throughout the whole reaction time course. As it was expected, higher concentrations of XOS were achieved with increment of offered substrate concentration, reaching concentrations of reducing sugars of approximately 20 and 31 mM for conventionally and enzyme-aided extracted xylan (DP-xylan), respectively when 5% (w/v) substrate solution was utilized. Obtained concentration did not exactly matched the increment of the substrate, however, this can be explained by the fact that higher xylan concentrations decreased XOS yields due to the enzyme inhibition caused by higher reaction mixture viscosity and density, that was previously noticed by Bian et al. ( 58 ). Nevertheless, it can be concluded that purer DP-xylan represents the substrate of choice for XOS production. The results presented in this way (overall produced reducing sugars) give us a comprehensive picture of the XOS reaction process, however, in order to better see what type of compounds were synthesized, as well as a compound polymerization degree, additional characterization of obtained reaction mixtures for DP-xylan was performed. To determine the ranges of degrees of polymerization from obtained XOS HPSEC-ELSD was conducted. For this purpose, reaction mixtures for the reaction times 0, 15, 60 and 120 min were chosen for further analysis. It can be clearly seen the release of smaller compounds during the reaction course hydrolysis of high molarity DP-xylan (Fig. 5 a), namely oligosaccharides (Mw < 6 kDa, corresponding to DP < 50). Namely, the abundancy of oligosaccharides lower than 6 kDa was around 10% in initial reaction mixture (0 min), while the oligosaccharide abundance was increasing with the reaction reaching 43.0%, 48.3% and 53.2% after 10, 60 and 120 min, respectively. In order to quantify the obtained products, HPLC analysis was performed, although this analysis may only detect oligosaccharides up do DP 6. From the Fig. 5 b, it can be seen that total XOS rather quickly (15 min) reaches equilibrium concentration 7.5 mg/mL that represents XOS yield of approximately 15% (calculated on total DP-xylan content). This concentration was higher than previously reported 5.29 mg/mL after 12 h using xylan-rich fraction isolated from sugarcane bagasse ( 57 ) and 5.05 mg/mL from oil palm empty fruit bunch fibers isolated xylan after 12 h [58], but lower than 28.6% of xylan extracted from corncobs reached by Teng et al. ( 59 ) and 19.1% of XOS from sugarcane bagasse by Valladares-Diestra et al. ( 60 ). Significant discrepancies in achieved yields are mainly consequence of the xylan structure and composition, which depends on the type of employed extraction treatment as well as utilized enzyme. Besides concentration, the composition of obtained XOS may vary greatly between different systems, as well as under different conditions within the same system. During the reaction of DP-xylan hydrolysis, main reaction products and their abundance change while XOS total concentration remains constant (Fig. 5 b). At the starting stages of the reaction XOS3 seems to be the most present XOS derivative, while XOS2 concentration increases during the whole examined reaction period, finally reaching same concentration after 2h. On the other hand, concentration of XOS4 decreases over the time together with less abundant XOS5 and XOS6. Once again, it should be emphasized that concentration of xylose is quite low, owing to the fact that enzymatic preparation with pronounced endo-xylanase activity was utilized. Even though, the oligosaccharides are often regarded as group of diverse compounds with degree of polymerization between 2–50 monomeric units or Mw values up to 5–7 kDa, according to different criteria ( 61 ), information about their composition is quite critical for further establishment of structure-function relationships. According to the MC (Table 2 ) the main type of extracted hemicellulose was xylan with the common feature of a backbone of β-(1→4)-linked xylose residues, with a common modification of xylans with numerous arabinose and some glucuronic acid residues on O-2 ( 62 ). Therefore, the obtained structures were additionally confirmed by means of MALDI-TOF-MS analysis since both the chain length and the expected types of glycosidic linkage contained in the XOS can be inferred. Namely, mass spectra confirmed existence of pentose-based oligosaccharides DP3-DP22 as sodium adducts ( m/z 437, 569, 701, 833, 965, 1097, 1229, 1230.0, 1362.1,1494.2, 1626.3, 1758.4, 1890.6, 2022.7, 2154.8, 2286.9, 2419.0, 2551.1) and as potassium adducts ( m/z 453.4, 585.6) with a lower abundance. These most likely correspond to xylose, according to the compositional data of DP-xylan (Table 2 ), although some of the xylose units may be substituted with arabinose which cannot be substantiated by MALDI-TOF due to equal masses for xylose and arabinose. None of these pentoses seems to be substituted with acetyl groups which is good agreement with the lack of characteristic band for acetyl groups previously observed in FTIR spectra. It should be noted that oligomers below m/z 400 are not included in the spectrum, due to hindrance of matrix peaks, and therefore this analysis cannot confirm the presence of xylobiose and small quantity of xylose that were previously detected by HPLC analysis. Thereafter, it can be seen from Fig. 6 that the most important are acidic oligosaccharides, that are present in three series, i) the most simple pentoses with 1 molecule of glucuronic acid with DP 5–17; ii) the most abundant series are oligosaccharides (methylglucuronic acid (MeGlcA) substituted XOS) are most likely present DP4-DP21 ( m/z values of 628, 759, 891, 1023, 1155, 1287, 1419, 1551, 1683, 1815, 1947, 2079, 2212.8 2344.9, 2477.0, 2609.1, 2741.2, 2873.3), and also are present the potassium adducts with DP between 5–17 (775.7, 907.8, 1039.9, 1172.0, 1304.1, 1436.2, 1568.3, 1700.5, 1832.6, 1964.7, 2096.8, 2228.9, 2361.0); iii) XOS with 2 groups of MeGlcA with DP 8–21 (1213.9, 1346.0, 1478.1, 1610.2, 1742.4, 1874.5, 2006.6, 2138.7, 2270.8, 2402.9, 2535.0, 2667.1, 2799.2, 2931.3). Finally, in small amount, a series of hexose oligomers (from DP 3 to DP17) can be recognized within the spectrum ( m/z values of 527, 689, 851, 1031, 1175, 1333, 1499, 1661, 1823, 1824.6, 1986.7, 2148.8, 2311.0, 2473.1, 2635.3, 2797.4), that might be a consequence of small share of adjuvant polysaccharide fractions within the obtained DP-xylan. 4. Conclusion Sunflower meal, an abundant and unexploited by-product of oil industry, proved to be a good source of valuable compounds that could be valorized throughout the newly proposed efficient method based on enzyme-aided fractionation. The application of enzymes not only enabled increment the yield of a purer xylan, but also to generate several other fractions (polyphenol-rich fraction, protein isolate, pectin and lignin) ready to be used to add value to different food and cosmetic preparations. Finally, high purity obtained xylan extract was successfully transformed into emerging group of prebiotic xylo-oligosaccharides. Declarations Acknowledgments The authors acknowledge and thank to AB Enzymes GmbH (Darmstadt, Germany) for their kind donation of enzyme preparation Rohalase ® SEP-Visco. This work was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia [Contract No. 451-03-47/2023-01/200287 and 451-03-47/2023-01/200135] and has received funding from Science Fund of the Republic of Serbia, programme IDEAS, project number 7750109 (PrIntPrEnzy) and the Horizon Europe 2021-2027 research and innovation programme under grant agreement ID 101060130 (TwinPrebioEnz). The graphical abstract was created with BioRender.com. Author contribution M. Simović: Investigation, Conceptualization, Methodology, Writing-original draft. K. Banjanac Investigation, Data curation. M. Veljković: Investigation, Formal analysis. V. Semenčenko: Methodology, Formal analysis. P. Lopez-Revenga: Formal analysis, Data curation A. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3975794","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274655741,"identity":"5a791aaa-9e1d-4b2f-bab7-bcfe0ca4adf3","order_by":0,"name":"Milica Simović","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYPACCQZ+IPmBsYEULZJtDIwzSNHCwGBwjFgtuu2Hn3342GYhb3y/O7GBcUctA3/7AbaPP/BoMTuTZjxzZpuE4bZjvBsbGM8cZ5A4k8A8mweflhsMxsw8ZyQSzI7xbn/A2HaMgeEGAzMzPoeZ3WD/zPwHqMW4DWQLUIs8UAsjXofd4DFmZqiQSDBgA2upYTAAamHA67AzOcWMPRUShjOO5W5sSDxzgMfwTGIzM14tx49vZvhhUCfP33x2Y8PHHXVycscPH8brMFSQwHAYaD5pEcpQR5LqUTAKRsEoGBkAAK6tSszeyBMEAAAAAElFTkSuQmCC","orcid":"","institution":"Faculty of Technology and Metallurgy, University of Belgrade, Serbia","correspondingAuthor":true,"prefix":"","firstName":"Milica","middleName":"","lastName":"Simović","suffix":""},{"id":274655742,"identity":"623c55b0-1cb0-41c8-9403-2c2a4ef43791","order_by":1,"name":"Katarina Banjanac","email":"","orcid":"","institution":"Innovation Centre of Faculty of Technology and Metallurgy, University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Katarina","middleName":"","lastName":"Banjanac","suffix":""},{"id":274655743,"identity":"bb067641-df17-45b2-a983-1d293db13ea2","order_by":2,"name":"Milica Veljković","email":"","orcid":"","institution":"Innovation Centre of Faculty of Technology and Metallurgy, University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Milica","middleName":"","lastName":"Veljković","suffix":""},{"id":274655744,"identity":"05c2f103-c752-4839-9ccd-542dc2c0de4d","order_by":3,"name":"Valentina Semenčenko","email":"","orcid":"","institution":"Maize Research Institute Zemun Polje","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Semenčenko","suffix":""},{"id":274655745,"identity":"c0d43e9f-6715-4376-89ba-490a0a2d302e","order_by":4,"name":"Paula Lopez-Revenga","email":"","orcid":"","institution":"Food Science Research Institute CIAL (CSIC-UAM)","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"","lastName":"Lopez-Revenga","suffix":""},{"id":274655746,"identity":"7bf034ca-2628-4e24-b5de-5fd545e24dc7","order_by":5,"name":"Antonia Montilla","email":"","orcid":"","institution":"Food Science Research Institute CIAL (CSIC-UAM)","correspondingAuthor":false,"prefix":"","firstName":"Antonia","middleName":"","lastName":"Montilla","suffix":""},{"id":274655747,"identity":"ba2e4670-a8ac-4841-8bd4-10444aaaef3a","order_by":6,"name":"F. Javier Moreno","email":"","orcid":"","institution":"Food Science Research Institute CIAL (CSIC-UAM)","correspondingAuthor":false,"prefix":"","firstName":"F.","middleName":"Javier","lastName":"Moreno","suffix":""},{"id":274655748,"identity":"b27cb718-1bac-4304-810d-37b2d57c40d1","order_by":7,"name":"Dejan Bezbradica","email":"","orcid":"","institution":"Faculty of Technology and Metallurgy, University of Belgrade, Serbia","correspondingAuthor":false,"prefix":"","firstName":"Dejan","middleName":"","lastName":"Bezbradica","suffix":""}],"badges":[],"createdAt":"2024-02-21 14:00:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3975794/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3975794/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51748050,"identity":"cc29abbb-f6f8-45d0-8e11-0602077b4466","added_by":"auto","created_at":"2024-02-28 11:29:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":113315,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of applied SFM fractionation methods\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3975794/v1/b4e64b3e9fac38a260a0e24e.png"},{"id":51748319,"identity":"f199c251-a618-467c-95dc-91ba48c2b4c7","added_by":"auto","created_at":"2024-02-28 11:37:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87499,"visible":true,"origin":"","legend":"\u003cp\u003eThe influence of temperature (a), AIR-SFM to buffer ratio (b) and enzyme concentration (c) on Alcalase®-aided protein extraction process\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3975794/v1/3e41172255d9b036f22fa97a.png"},{"id":51748051,"identity":"c3581cb3-8d13-4404-8a5e-26a54d7e9654","added_by":"auto","created_at":"2024-02-28 11:29:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":244733,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra (a) and distribution of molecular weights, Mw (b) of obtained xylans\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3975794/v1/4eb8777634ef7fff93c2d259.png"},{"id":51748053,"identity":"b7ba0263-7050-4eaf-9d52-f94bb6e4592b","added_by":"auto","created_at":"2024-02-28 11:29:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":178239,"visible":true,"origin":"","legend":"\u003cp\u003eTime course of XOS production using Rohalase\u003csup\u003e®\u003c/sup\u003e SEP-Visco. Influence of enzyme concentration on hydrolysis of xylan (a) and substrate concentration of xylan and DP-xylan (b).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3975794/v1/e45ff207512e06283fab400b.png"},{"id":51748054,"identity":"0d21aab4-0f8a-470d-93c3-8d29b7a26549","added_by":"auto","created_at":"2024-02-28 11:29:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":79186,"visible":true,"origin":"","legend":"\u003cp\u003eHPSEC-ELSD chromatograms of XOS reaction mixtures for reaction times 0, 10, 60 and 120 min (a). Vertical line represents 6 kDa. Time course of XOS synthesis with achieved concentrations for compounds up to XOS6 analyzed using HPLC-UV (b).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3975794/v1/946777556f52b81d28419ee0.png"},{"id":51748055,"identity":"a8dd4a7d-9ed7-47c3-8272-f74f35594d5d","added_by":"auto","created_at":"2024-02-28 11:29:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":58754,"visible":true,"origin":"","legend":"\u003cp\u003eMALDI- TOF MS analysis of DP-xylan reaction mixture in various reaction times.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3975794/v1/adeba2346ea3c88c7f1a7530.png"},{"id":51748056,"identity":"f30fa2d3-464c-4245-a00f-a28a8ed84d63","added_by":"auto","created_at":"2024-02-28 11:29:09","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"graphical-abstract","size":121407,"visible":true,"origin":"","legend":"Sunflower meal, a plentiful and underutilized oil industry by-product, is currently discarded as waste or used as cheap component of animal feed with poor protein content and high content of complex carbohydrates. To harness its great potential for valorization, we propose an efficient method through enzyme-aided fractionation yielding 47.8% of hemicellulosic fraction, with simultaneous generation of various other potentially valuable fractions (including polyphenol-rich fraction, protein isolate, pectin, and lignin). According to the monomeric composition the main type of extracted hemicellulose was xylan with the common feature of a backbone of -(1\u0026rarr;4)-linked xylose residues, with a common substitution with arabinose moieties and some glucuronic acid residues. Subsequently, the xylan fraction underwent enzymatic treatment using commercial xylanase (ROHALASE\u0026reg; SEP-VISCO) to produce highly valuable compounds - emerging prebiotics xylo-oligosaccharides (XOS). Under optimized reaction conditions (70\u0026deg;C, pH 6 and enzyme concentration of 0.005% v/v using 5% w/v xylan solution) a yield of XOS with a polymerization degree DP\u0026thinsp;\u0026lt;\u0026thinsp;50 reached approximately 52.3% after 2 hours (majority of obtained product had DP\u0026thinsp;\u0026lt;\u0026thinsp;6 with predominance of XOS2 and XOS3 and without significant xylose generation).","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3975794/v1/26cf8ee56e142ee8062d6033.png"},{"id":51975174,"identity":"b1f8e089-6326-49d5-8331-398de6f8f7ee","added_by":"auto","created_at":"2024-03-04 19:15:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1188481,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3975794/v1/f8a8b637-8e7c-4169-beaf-3d6bd4ee3976.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sunflower meal valorization through enzyme-aided fractionation and production of emerging prebiotics","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSunflower (Helianthus annuus L.) stands as one of the world's most widespread cultivated oil crops, alongside soybean and rapeseed\u0026nbsp;(1, 2). Its good nutritional properties assure their wide utilization in food sector\u0026nbsp;(1); however, its greatest economic importance comes from the high oil content in the seeds\u0026nbsp;(3), and its potential for production of a high-quality oil that represents one of the most preferable and widely used vegetable oil in the world\u0026nbsp;(1, 4). Despite ongoing efforts to enhance the oil extraction process, a significant portion of solid residues (hulls, meals, and cakes) continues to be generated worldwide\u0026nbsp;(5). These oilseed residues are usually discarded as a waste or utilized as a cheap component of animal feed for various livestock species, with a neglectable application in human diet\u0026nbsp;(6). Sunflower meal (SFM), the primary by-product of sunflower oil extraction, holds great potential as a source of proteins, dietary fibers, phenolic compounds, and minerals, yet remains underutilized\u0026nbsp;(7). Its profitable industrial application is currently limited due to its comparatively low protein content\u0026nbsp;(8), high levels of undigestible carbohydrates\u0026nbsp;(9), and finally the abundance of antinutritional factors\u0026nbsp;(10)[9], especially phenolics that may form complexes with proteins thus leading to reduced functionality and digestibility of meals\u0026nbsp;(5, 11). Accordingly, there has been growing interest in their profitable reutilization aligning with principles of circular economy and sustainable development. Valorization of SFM could result in the preparation of multiple products with added value for the food and nutraceutical industries or agriculture\u0026nbsp;(11). The most commonly utilized methods for valorization of SFM are based on the extraction of protein fraction\u0026nbsp;(8), which follows constant increase in demand for relatively cheap and adequate protein sources\u0026nbsp;(12). This is not surprising having in mind the nutritional and technological advantages of SFM proteins\u0026nbsp;(13, 14). Nevertheless, in view of fully achieving biorefinery goals the polyphenolic and carbohydrate fractions should also be considered for valorization. Phenolic compounds are frequently eliminated within the first steps of protein isolation process, but recent data show researchers are shifting towards their utilization upon gaining new knowledge about the utility of these fractions\u0026nbsp;(10, 15). However, the systematic valorization of carbohydrate fraction, which makes up to 52% of dry matter, depending on the oil extraction process\u0026nbsp;(16), is still insufficiently studied. Conventional extraction methods to valorize carbohydrate fraction in different by-products and waste materials, based on multi-step extractions using acids and alkalis, come with many limitations such as loss in fiber functionality and laborious operation with low extraction efficacy and, more importantly, environmental unacceptability, since the rigid lignocellulose structure requires severe pretreatments to facilitate efficient extraction of mentioned fractions\u0026nbsp;(17). Pectic compounds are usually extracted using mineral or organic acids\u0026nbsp;(18, 19), while traditional hemicellulose extraction methods are mostly based on alkaline extraction (sodium or potassium hydroxides) of xylan, with or without delignification step\u0026nbsp;(20-22). Delignification is usually provided with sodium chlorite, hydrogen peroxide or peroxyacetic acid, with the aim of obtaining purer xylan fractions, since alkaline treatment also partially solubilizes present lignin\u0026nbsp;(23). These methods caused unavoidable hemicellulose degradation, and therefore, alternative means of isolation were purposed, such as organic solvent treatments using different organic solvents (e.g. dimethyl sulfoxide, mixtures of dimethyl sulfoxide with other compounds and dioxane) but these methods provided significantly lower yields of wanted compounds most probably being restricted to water soluble fraction of xylan\u0026nbsp;(24). Additionally, application of complementary emerging technologies as microwaves, ultrasound or enzymatic assistance is growing nowadays\u0026nbsp;(25-27). In the field of sunflower carbohydrate valorization, only sunflower heads were previously utilized for pectin extraction\u0026nbsp;(28-31), while SFM was not subjected to either pectin or xylan extraction to date.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXylan is commonly studied for textile, food and biomedical applications, however recently it is widely utilized for production of non-digestible oligomers, known as xylo-oligosaccharides (XOS) a promising functional ingredient with diverse applications in the food and pharmaceutical industry. These xylose-based oligosaccharides are naturally occurring in vegetables, fruits or honey\u0026nbsp;(32), but at insufficient quantities. XOS are viewed as promising emerging prebiotics, that have a crucial role in maintaining gut health, and simultaneously are well-suited for incorporation in a wide variety of food and feed products. This is due to their exceptional application characteristics, including stability at high temperatures (up to 100 ºC) and across a broad pH range (2.5–8), as well as their good sweetening power with minimal caloric impact\u0026nbsp;(33). XOS may be produced using commercially available xylan, however, in order to enable their economically viable production nowadays major focus has been placed on utilization of lignocellulosic biomass, which represents cheap source of xylan\u0026nbsp;(34). Up to date, large number of lignocellulosic materials (wheat and rice straw, wheat and barley brans, sorghum and grape stalks, sugarcane bagasse, corncob, beechwood and birchwood) have been studied as xylan sources for XOS production with varying efficiency, mostly depending on the type of present xylan, and employed conversion methods\u0026nbsp;(35). Therefore, different methods of XOS production and great diversity in potential xylan substrates lead to wide spectrum of different XOS structures with varied substituents of xylose backbone and degrees of polymerization that consequently have great impact on its prebiotic and other functional properties.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe main aim of this work was to propose a promising pathway for SFM valorization primarily through enzyme-aided extraction of xylan that would be further used in production of valuable xylo-oligosaccharides (XOS) as emerging prebiotics. Thus, enzymatic hydrolysis of obtained xylans will be achieved using highly promising enzymatic preparation Rohalase\u003csup\u003e®\u003c/sup\u003e SEP-Visco, a thermostable bacterial xylanase from \u003cem\u003eTrichoderma reesei,\u0026nbsp;\u003c/em\u003ethat has not been previously applied for similar purposes. This way it is expected to provide value added prebiotic-rich fraction of SFM. Additionally, our enzyme-aided fractionation method will deliver other fractions enriched in other compounds, such as polyphenols, proteins, pectin, lignin and cellulose, that can be utilized in future as food additives or in the field of nutraceuticals and functional cosmetics ingredients.\u0026nbsp;\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePartially dehulled sunflower meal (SFM) utilized in this study was kind donation of Victoriaoil LTD (\u0026Scaron;id, Serbia). Analytical grade chemicals, including ethanol, acetone, hydrochloric acid, sodium chlorite, and sodium hydroxide, ammonium acetate were obtained from Centrohem (Stara Pazova, Serbia). HPLC grade solvents (acetonitrile and water), tri-fluoroacetic acid (TFA), hydroxylamine chloride, pyridine, hexamethyldisilazane (HMDS), pullulan standard set, phenyl-\u003cem\u003e\u0026beta;\u003c/em\u003e-D-glucoside and 1-phenyl-3-methyl-5-pyrazolone (PMP) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Xylo-oligosaccharides (XOS) standards (xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose) were purchased from Megazyme LTD, Wicklow, Ireland. Enzymes used within the study were: Alcalase\u003csup\u003e\u0026reg;\u003c/sup\u003e 2.4L, Novozymes (Bagsv\u0026aelig;rd, Denmark) and Rohalase\u003csup\u003e\u0026reg;\u003c/sup\u003e SEP-Visco that was kind donation of AB Enzymes GmbH (Darmstadt, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Sunflower meal compositional analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1. Analysis of dry matter (DM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDry matter (DM) was determined gravimetrically drying the samples until constant weight according to the AOAC method 950.01 (36).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2. Analysis of ash content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ash content was determined according to method number 923.03 by the slow combustion of the sample at 550 \u0026deg;C in a muffle furnace (L47, 1200\u0026deg;C, Naber Industrieofenbau, Lilienthal, Germany) (37).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3. Analysis of oil content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe oil content was obtained according to the Soxhlet method number 920.39 (37), on a FatExtractor E-500 (B\u0026Uuml;CHI Labortechnik, Flawil, Switzerland).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.4. Analysis of Total Protein Content\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein content was determined by the standard micro-Kjeldahl method (official Method 920.87) as the total N multiplied by 6.25 on B\u0026Uuml;CHI Kjeldahl System (Auto Kjeldahl Distillation Unit K-350 and Speed Digester K-439, B\u0026Uuml;CHI Labortechnik, Flawil, Switzerland).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.5. Analysis of Sugar Content\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe content of total sugars, reducing sugars and sucrose was determined by the Luff-Schoorl method based on the reaction between reducing sugars and alkaline solution of copper sulphate, with a subsequent reduction of cupric copper to cuprous oxide. In the method, Cu\u003csup\u003e2+\u003c/sup\u003e ions that had not been reduced are determined iodometrically. Furthermore, total sugars were then determined by converting non-reducing sugars into reducing sugars through acid hydrolysis (38).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.6. Analysis of Dietary Fiber Content\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe content of hemicellulose, cellulose, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were determined by the Van Soest detergent method modified by Mertens using the Fibertec system FOSS 2010 Hot Extractor (FOSS Tecator, Hoeganaes, Sweden) (39). After filtering and drying, the NDF, ADF and ADL were calculated as a percentage of the original sample. The content of hemicellulose was obtained as the difference between NDF and ADF contents, while the cellulose content was calculated as the difference between ADF and lignin contents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Sunflower meal fractionation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1. Method of conventional sunflower meal fractionation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe schematic representation of the fractionation process applied to SFM is shown in Figure 1. Preparation of alcohol-insoluble residue of sunflower meal (AIR-SFM) was provided by triple extraction using 70% v/v aqueous ethanol solution (1:10 w/v). Slurry was stirred for 30 min on a magnetic stirrer and recovered by vacuum filtration. The solid residue (AIR-SFM) was washed using 96% ethanol and acetone, respectively. Combined ethanol extracts were concentrated using rotary vacuum evaporator and represent polyphenol-rich fraction. Next step of fractionation was performed using hot diluted hydrochloric acid (HCl, pH adjusted to 1.5) at 1:25 w/v ratio and heated at 90 \u0026deg;C for 1.5 h. The extracted material was recovered by vacuum filtration and solid residue was rinsed with water. The obtained pectin was recovered by cold ethanol precipitation with 4 volumes of 96% ethanol overnight. The obtained precipitates were recovered by centrifugation (10 min on 6000 rpm). Solid residue (Dpect-SFM) was dried by solvent exchange (ethanol, acetone). Next step in fractionation scheme was delignification step performed using 1.87% w/v solution of sodium chlorite with 1.87% v/v acetic acid at 1:32 w/v substrate to solvent ratio at 70 \u0026deg;C for 2 h. The delignified solid residue (DL-SFM) was recovered by vacuum filtration rinsed with water until neutral pH and dried using solvent exchange (ethanol, acetone). Solubilized lignin was lyophilized. Final step in xylan extraction was alkaline extraction using 4 M potassium hydroxide solution at 1:20 (w/v) ratio with continuous mixing at room temperature during 4 h. The obtained extract was separated by vacuum filtration and adjusted to pH 6 using concentrated acetic acid, and thereafter recovered by cold ethanol precipitation with 4 volumes of 96% ethanol overnight. The obtained precipitate was recovered by centrifugation (10 min on 6000 rpm) and lyophilized afterwards (xylan). The solid residue (cellulose-rich residue) was rinsed with distilled water until neutral pH is reached and afterwards dried using solvent exchange (ethanol, acetone). The obtained precipitates were recovered by centrifugation.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2. Enzyme-aided method of sunflower meal fractionation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlcalase\u003csup\u003e\u0026reg;\u003c/sup\u003e 2.4 L treatment of AIR-SFM was performed in shaken Erlenmeyer flask (reaction volume of 500 mL) at orbital shaker (120 rpm) at 50 \u0026deg;C. AIR-SFM was suspended in 100 mM sodium-phosphate buffer (pH 7.5) in ratio 1:20 (w:v), and the enzyme concentration was 2.5% (v/w) calculated on AIR-SFM. In optimization experiments one parameter was varied at a time in following ranges: temperature 40-60\u0026deg;C, substrate to buffer ratio: 1:10-1:30 (w/v) and enzyme concentration: 0.625-2.5% (v/w) calculated on substrate. Concentration of proteins in supernatant was monitored during 2 h. At a predefined time, aliquots of reaction mixture were taken and incubated at 100 \u0026deg;C for 5 min to stop the reaction, centrifuged and then analyzed by determination of protein concentration (mg eq BSA per g of AIR-SFM) using Lowry method (40).\u003c/p\u003e\n\u003cp\u003eFor\u0026nbsp;the enzyme-aided SFM fractionation under the selected optimal conditions, AIR-SFM was suspended in 100 mM sodium phosphate buffer (pH 7.5) at ratio1:20 w/v. The reaction was started with addition of 2.5% of Alcalase\u003csup\u003e\u0026reg;\u003c/sup\u003e 2.4L, and reaction was performed with constant mixing at 50 \u0026deg;C for 2 h. The solid residue from the reaction mixture (DP-SFM) was recovered by vacuum filtration, and immediately rinsed with distilled water, and then dried with solvent exchange (ethanol and acetone) to rinse the enzyme and stop the reaction. The protein rich filtrate fraction was boiled at 90 \u0026deg;C to stop the reaction and afterwards concentrated using rotary evaporator and finally lyophilized (protein-rich fraction).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. FTIR analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFTIR analysis of both samples of extracted xylans was performed. KBr discs were prepared mixing the xylan samples with KBr (1:100) and pressed. FT-IR spectra were performed in a Bruker IFS66v equipment (Bruker, US). Data were collected in absorbance mode using a frequency range of 4000\u0026ndash;400 cm\u003csup\u003e-1\u003c/sup\u003e, and resolution of 4 cm\u003csup\u003e-1\u003c/sup\u003e (mid infrared region) with 250 co-added scans (26).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Determination of monomeric composition\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor determination of monomeric composition of SFM and obtained xylans previously developed method was utilized\u0026nbsp;(26). Samples (30 mg) were hydrolyzed using 1.5 mL of 2 M TFA at 110 \u0026deg;C under inert conditions for 4 h. Upon hydrolysis, 300 \u0026mu;L of the hydrolyzed samples were evaporated. After evaporation of the acid, 400 \u0026mu;L of phenyl-\u003cem\u003e\u0026beta;\u003c/em\u003e-D-glucoside internal standard (0,5 mg/mL) is added, mixture is evaporated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo carry out the derivatization, 250 \u0026mu;L of hydroxylamine chloride (2.5%) in pyridine was first added, shaken vigorously and kept for 30 min at 70 \u0026deg;C to form the oximes. Subsequently, 250 \u0026mu;L of HMDS and 25 \u0026mu;L of TFA were added. It was shaken vigorously and kept in the oven at 50 \u0026deg;C for 30 min. Samples were centrifuged (10,000 rpm, 2 min), the supernatant was collected and kept at 4 \u0026deg;C until analysis by GC-FID. The analysis was carried out using an Agilent Technologies 7820A Gas Chromatograph (Agilent Technologies, Wilmington, DE, USA). A VF-5HT capillary column, phase bound (5% phenylmethylpolysiloxane, 30 m x 0.250 mm x 0.10 \u0026mu;m; Agilent J\u0026amp;W, Folson, CA, USA) was used. The injector temperature was 280 \u0026ordm;C and the detector temperature was 385\u0026ordm;C. Nitrogen was used as carrier gas with a flow rate of 1 mL/min. The temperature programme was: initial temperature 120 \u0026ordm;C, ramp of 3 \u0026ordm;C/min up to 380 \u0026ordm;C. Data acquisition and processing was carried out using Agilent ChemStation software (Wilmington, DE, USA). GC quantification was performed using\u003cem\u003e\u0026nbsp;\u0026beta;\u003c/em\u003e-phenyl-glucoside (0.5 mg/mL) as internal standard and mixtures of the sugars of interest in a range of concentrations between 2 and 0.02 mg/mL.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.\u0026nbsp;Determination of the molecular mass distribution (Mw) by High Performance Size-exclusion Chromatography with evaporative light scattering detector (HPSEC-ELSD).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular weight (Mw) distribution was estimated according to previously published procedure\u0026nbsp;(41). Samples (20 mg/mL) were prepared by dissolving in distilled water for 30 min at elevated temperature (50 \u0026deg;C), than mixed with mobile phase (0.04 M ammonium acetate) in ratio 1:9, filtered and separated by High Performance Size Exclusion Chromatography with Evaporative Light Scattering Detector (HPSEC-ELSD) (Agilent Technologies, Boeblingen, Germany) using TSK-Gel guard column (6.0 mm \u0026times; 400 mm) and two TSK-Gel columns connected in series, G5000 PWXL (7.8 mm \u0026times; 300 nm, 10 \u0026mu;m), and G2500 PWXL (7.8 mm \u0026times; 300 nm, 6 \u0026mu;m) (Tosoh Bioscience, Stuttgart, Germany). Pullulan standard set (Mw 0.342-788 kDa, 2-0.2 mg/mL) was utilized as standard for determination of molecular weight distribution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7.\u0026nbsp;Enzymatic production of xylo-oligosaccharides (XOS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe obtained xylans are finally utilized to synthesize XOS in shaken Erlenmeyer flask (reaction volume of 20 mL). The reaction mixtures were prepared by dissolving obtained xylans in 100 mM sodium phosphate buffer (pH 6) to reach the concentration of 1% and 5% (w/v). The reaction was catalyzed by means of Rohalase\u003csup\u003e\u0026reg;\u003c/sup\u003e SEP-Visco in concentration range 0.005-0.05% (v/v) at 50 \u0026deg;C with constant orbital shaking (120 rpm). Reactions were monitored during 2 h. At a predefined time, aliquots of reaction mixture were taken and incubated at 100 \u0026deg;C for 5 min to stop the reaction. Part of the samples were derivatized, filtered and analyzed by high performance liquid chromatography (HPLC), and selected samples were analysed by means of HPSEC-ELSD and Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8.\u0026nbsp;Determination of total reducing sugars\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total reducing sugars in the reaction mixture was determined using the method reported by Miller (1959) using the dinitrosalicylic acid reagent. The concentration of reducing sugars was calculated from the standard curve of xylose (1-10 mM)\u0026nbsp;(42).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9.\u0026nbsp;High performance liquid chromatography analysis of reaction mixture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to HPLC analysis samples were derivatized using the PMP in accordance with procedure published by Wang et al. (43). After derivatization samples were filtered and analyzed using Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham, USA) equipped with a reverse phase column (ZORBAX Eclipse Plus C18, 4.6 \u0026times; 150 mm, 5 \u0026micro;m) at 30 \u0026deg;C, using with the mobile phase of 100 mM ammonium acetate buffer (pH 5.5):acetonitrile (80:20) with constant flow rate of 0.5 mL/min (44). Detection of products and standards was carried out by a UV detector at 245 nm. Data collection and processing were performed using Chromeleon 7.2. software. The concentration of the obtained compounds was calculated using the PMP-derivatized standard samples of XOS (XOS2-XOS6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10. MALDI-TOF mass spectrometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples were ten times diluted in water and mixed in a 20:5 ratio (matrix:sample). The utilized matrix was 2,5-dihydroxybenzoic acid (DHB) with concentration 10 mg/mL in methanol+10% H\u003csub\u003e2\u003c/sub\u003eO. All mixtures were treated with strong cation exchange resin to eliminate present salts and avoid possible inhibition of the compounds of interest ionization. Analyses have been done in positive ion detection mode in the range from 50 to 5000 Da, with application of an ion deflection up to 400 to prevent mass matrix signals (which are the most intense) to saturate the detector. MALDI-TOF-MS analyses were performed on a Voyager DE-PRO mass spectrometer (Applied Biosystems, Foster City, CA) at the Interdepartmental investigation service (SIdI-UAM) of Madrid.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11. \u0026nbsp;Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments are performed in duplicate and the results of experiments were provided as mean \u0026plusmn; standard deviation.\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Chemical characterization of sunflower meal\u003c/h2\u003e \u003cp\u003eIn the preliminary experiment, we thoroughly analyzed the compositional characteristics of partially dehulled sunflower meal (SFM), which served as the substrate for our study (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The dry matter (DM) of the SFM was found to be 91.5%, while SFM was composed of 42.8% of proteins, 47.0% of carbohydrates, 2.8% of fats and 7.4% of ash (all calculated on dry matter).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical composition of partially dehulled SFM\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComponent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry matter (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e91.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSimple sugars (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemicellulose (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellulose (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLignin (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProteins (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFats (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThese results are in accordance with the ranges presented in previously published data on SFM, since data on chemical composition considerably vary depending on applied sunflower seed processing methods (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Namely, dry matter (DM) values for different SFM ranged from 88.0 to 93.8%. Protein content was in the wide range from 26.4 to 40.3% per total weight, while total mineral content was found to be rather constant (5.5 to 7.8%) in all examined samples (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). On the other hand, data concerning detailed carbohydrate composition of SFM are quite scarce since most of the studies focuses on protein fraction of the meal (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Namely, majority of data about SFM carbohydrates are expressed through crude fiber (CF) content (11.5 to 29.7% (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e)) or dietary fiber (DF) content (35.8\u0026ndash;51.0% DM (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e)). Carbohydrate analysis of dehulled SMF in our study revealed that they are comprised of simple sugars (5.5% DM) where sucrose content was predominant, with smaller amounts of monosaccharides (glucose and fructose) and oligosaccharides (most probably raffinose and stachyose (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e)) and, on the other hand, high amount of polysaccharides. Among the polysaccharides, SFM contains 12.7% DM hemicellulose, 13.5% DM cellulose, and 6.8% DM lignin. The remaining portion (up to 8.5% DM) can be likely attributed to pectin bearing in mind that the obtained results on SFM monomeric composition showed that galacturonic acid is present in moderate quantities, 14.35% of total monosaccharides (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, regarding the monomeric composition of SFM, it can be seen that xylose (21.8%), together with arabinose (21.1%) and glucose (22.3%), represents most frequent monomeric building block for present polysaccharides, and therefore shows that SFM represents a highly potent substrate for xylan extraction.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMonomeric composition of partially dehulled SFM polysaccharides (% total identified monosaccharides). DP-xylan: deproteinized xylan\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComponent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eConcentration (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSFM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eXylan\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDP-xylan\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXylose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e53.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e63.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArabinose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRhamnose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFructose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGalactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMannose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUronic acids\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e*\u003c/sup\u003e Galacturonic and glucuronic acids\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Sunflower meal fractionation and xylan extraction\u003c/h2\u003e \u003cp\u003eIn the preliminary experiment, extraction of xylan was performed using most frequently utilized method of alkaline treatment, based on previously published literature data on xylan extraction (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). However, this method proved to be unsuccessful since product with low purity of the isolated xylan was obtained (with 61.5% of DM being proteins), most probably due to complex composition of SFM that includes proteins and other carbohydrates. This treatment causes disruption of the complex structure by cellulose swelling and hydrolysis of uronic and acetic acid esters linkages, and dissolving hemicellulose and lignin (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Additionally, these alkaline conditions simultaneously favor protein extraction (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Therefore, more complex multi-step approach of SFM fractionation to obtain different carbohydrate fractions and protein isolate, was approached (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConventional method of SFM fractionation assumed the primary removal of extractables (polyphenolic compounds, simple sugars, and colorants) using 70% ethanol solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The resulting extract proved to be rich in polyphenols (predominantly chlorogenic and caffeic acid) that could be applied as antioxidant agent in different formulations and potentially exhibit prebiotic activity on gut and skin microbiota (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince presence of pectin in SFM was assumed, the step of pectin extraction was introduced according to the work of Cebin et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), in order to make better utilization of all present fractions. Also, bearing in mind that further steps (e.g. alkaline treatment) could impose structural and functional modifications on native pectin structure (47), this step was performed at the beginning of the fractionation process. In this step, pectin containing fraction (14 g) was obtained (extraction yield of 15.34%), but it should be noted here that obtained fraction might have poor purity, since great amount of proteins (10 g) were extracted alongside with pectin, that would introduce a need for further purification of obtained fraction.\u003c/p\u003e \u003cp\u003eThe remained depectinized solid residue (DPect-SFM) was thereafter subjected to delignification treatment. This step was introduced since it was determined that SMF possess around 6.8% of lignin calculated on DM that can be solubilized under alkaline conditions. For this treatment, sodium chlorite with acetic acid addition was chosen based on previously published data concerning its performance and selectivity (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Under optimized conditions, one-step delignification of DP-SFM was performed for 2 h and resulted in approximately 18 g of lignin-containing fraction with high quantity of proteins (11.7 g proteins). With this in mind, additional experiment was performed without the delignification step, yet lack of this step highly affected the quality of finally obtained xylan. Finally, the next step - alkaline treatment was therefore performed on delignified solid residue (DL-SMF) using 4 M potassium hydroxide (1:20) for 2 h at room temperature. This step yielded 6.5 g DM of xylan (yield of 7.1% and recovery yield of 56%) and around 34 g DM of cellulose rich fraction. The obtained xylan had 52.5% of carbohydrates (mainly composed of xylose 53.7%) and 25.3% of proteins, calculated on DM. At the end, it can be concluded that high share of proteins ended up in different carbohydrate rich fractions. Therefore, to prevent this protein loss and on the other way to improve the purity, as well as a xylan yield and purity, a new method for fractionation was proposed. This method included utilization of enzymes that will help protein separation and enable easier and more efficient fractionation of carbohydrates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Optimization of protein extraction from AIR-SFM using Alcalase\u003csup\u003e\u0026reg;\u003c/sup\u003e 2.4L\u003c/h2\u003e \u003cp\u003eGenerally, extraction of proteins from lignocellulosic plant materials presents a great challenge. Consequently, an enzyme-aided extraction method is frequently proposed as environmentally friendly technique that can be used to facilitate protein extraction from different plant sources. Enzymes provide simultaneous disruption of the cell wall and extract proteins by detaching of the structural protein complexes from large polysaccharide matrices or/and degrading those to smaller molecular mass proteins and peptides (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). To maximize the efficiency of protein extraction from lignocellulosic plant materials, the key process parameters such as pH, temperature, reaction time, substrate and enzyme concentration should be determined.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this part of the study commercial protease preparation (Alcalase\u0026reg; 2.4L) was employed to achieve maximum protein extraction efficiency from AIR-SFM (containing 37.8 g proteins). First, the influence of temperature (40\u0026ndash;60\u0026deg;C) on the protein extractability was examined since it potentially could provide positive effects on both enzyme activity and extractability of proteins. The other parameters were kept constant (AIR-SFM to 100 mM sodium phosphate buffer (pH 7.5) ratio 1:20 with enzyme concentration of 2.5% calculated on substrate). Even though the enzyme possesses wide optimum range in alkaline medium (pH 6\u0026ndash;10), buffer pH 7.5 was chosen since higher pH values may induce xylan and lignin extraction (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). According to presented results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), it can be seen that almost exact protein concentrations were obtained at 50\u0026deg;C and 60\u0026deg;C (approximately 300 mg/g AIR-SFM determined as eq BSA), while reaction at 40\u0026deg;C showed lower extraction yields. Therefore, the next experiments will be performed at 50\u0026deg;C. To promote diffusion of proteins from AIR-SFM into solution, a minimum amount of solvent needed should be determined. By adding large amount of solvent during extraction process, the extraction efficiency will likely be increased but it will raise process costs. Therefore, a study of the influence of AIR-SFM to buffer ratio on extraction process was performed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe obtained results indicate that when ratio was increased from 1:10 (w/v) to 1:20 (w/v) slightly enhanced protein extraction efficiency was achieved (approximately 300 mg/g of AIR-SFM), while further ratio increment did not induce any changes in obtained results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Accordingly, an AIR-SFM to buffer ratio of 1:20 was selected for further experiments. Finally, the influence of enzyme concentration was examined. A typical proteases dosage from 0.5 to 5% per g of lignocellulosic plant material could be found in several studies where protease-aided protein extraction from soybean, rapeseed, peanut, lupin, rice bran and sunflower were examined (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). In case of AIR-SFM treatment with Alcalase\u0026reg; using different amount of enzyme (0.625-2.5% on substrate), the highest protein concentration of 300 mg/g AIR-SFM was obtained using 2.5% Alcalase\u0026reg; 2.4L (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Protease-aided fractionation of sunflower meal\u003c/h2\u003e \u003cp\u003eUpon determination of optimal conditions for protein removal from the AIR-SMF, the modified xylan extraction process was developed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After the removal of extractables and enzyme catalyzed deproteinization step, the 42.9 g DM of solid phase (DR-SFM) that contained 7.4 g proteins was obtained. It can be seen that deproteinization step was highly successful since 30,5 g of proteins (corresponding to 80.54% from total proteins of AIR-SFM) was removed to protein-rich fraction. The subsequent step in the fractionation process involved depectinization under identical conditions to those used in conventional fractionation. This step yielded 30.1 g DM of DpectDP-SFM that contained 3.8 g proteins (12.7%), while in conventional fractionation method corresponding fraction (Dpect-SFM) contained 47.0% proteins calculated on DM. The pectin-rich fraction had 12.8 g DM (extraction yield 14%) and only 3.6 g proteins, that showed great improvement in terms of achieved purity of the fraction. During the delignification step, 27.5 g DM of DLDP-SFM was obtained, and together with the lignin fraction a small quantity of proteins was removed. Finally, the DP-xylan 5.6 g DM was isolated. The yield of enzyme-aided fractionation process was 6.1%, while the recovery yield was 47.8%. Obtained DP-xylan contained 92.2% of carbohydrates mostly composed of xylose (63.4%) and approximately 7% of proteins (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results prove the validity of modification the fractionation process, that is the introduction of the deproteinization step.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Characterization and comparison of obtained xylans\u003c/h2\u003e \u003cp\u003eIn view of summarizing the advantages of newly developed method for SFM fractionation and xylan extraction, a more thorough comparison of obtained xylan fractions by displaying the most important parameters (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Thus, slightly higher quantities, and accordingly higher yields of obtained xylan fractions were achieved within conventional fractionation method. These results are expected and in accordance with previously published data. For example, Sporck et al. showed that enzymatic approach gave 2.4-times lower yields than alkaline method due to higher number of extraction steps and extraction selectivity (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterization of obtained xylans\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXylan\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDP-xylan\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eQuantity (g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDry matter (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e96.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e95.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eYield (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRecovery yield (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e56.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e47.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCarbohydrate content (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e52.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e92.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eProtein content (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHowever, higher yields using the conventional method are obtained at the expense of the purity of the samples that can be seen when it comes to carbohydrate and protein content. Namely, conventionally obtained xylan has carbohydrate content of 52.5% DM corresponding to total amount of 3.4 g of carbohydrates while, on the other hand, DP-xylan obtained according to the newly proposed method exhibited great purity, since it had 92.2% DM of carbohydrates. This corresponds to 5.1 g of carbohydrates that is almost 1.5 times more than in previous case. In terms of achieved recovery yields, results obtained in this study were better than in the case of alkaline-sulfite pretreated sugarcane bagasse (recovered yields of 53% and 22% for conventional and enzymatic treatment, respectively) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Likewise, similar yields (4.5\u0026ndash;8.5%) were obtained by Rowley et al., using delignified corn stover by means of different methods (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe analyzed infrared spectra of the both obtained xylans show characteristic bands for xylan-rich compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Namely, bands occurring at 3414 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e that can be assigned to the stretching vibrations of the O-H groups and the bands occurring at 2926 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e that are generally assigned to the -CH\u003csub\u003e2\u003c/sub\u003e antisymmetric stretching, while the band at 2850 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was a result of -CH\u003csub\u003e2\u003c/sub\u003e symmetric stretching (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Bands occurring at 1644 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be assigned to the absorbed water (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). The difference between two xylan samples can be seen around 1510 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, where lignin can be observed in the xylan spectra due to the aromatic skeletal vibration (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). This band is expectedly more pronounced in case of conventionally extracted xylan, implying the lower purity of the sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The bands occurring at 1450 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in some samples could be assigned to the presence of the methyl groups while spectral peaks that are visible at 1044 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of C-O stretching in the C-O-C ether linkages (the first is the inter sugar units and the second results from intra sugar of alcoholic functional group). The peaks at 897 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be attributed to the stretching vibration modes (both symmetric and antisymmetric) of C-O in the ether linkage and can prove the \u003cem\u003eβ-\u003c/em\u003econfiguration of 1\u0026rarr;4 glycosidic bonds between xylose units of the xylan chain (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Other bands at lower wavenumbers, such as 690 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, are attributed to the out-of-plane C-H deformations (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). The signals around 1249 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1736\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicate the vibrational band of the single bond C-O stretching band and C\u0026thinsp;=\u0026thinsp;O stretching related to the acetyl groups present in the xylan, respectively (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, the estimation of molecular weight (Mw) distribution (four fragments) and their corresponding relative abundances (%) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb. It can be seen that obtained both xylans possess similar Mw distribution within the four fragments. There were two predominant fragments consisting of a very wide Mw ranges (245\u0026ndash;8000 kDa and 4-245 kDa, respectively) followed distantly by two fragments with a narrow range, between 1 and 4 kDa and 0.15-1 kDa (corresponding to mono- and oligosaccharides). These results show that introduction of deproteinization step did not impose negative influence on the structure of extracted xylan.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Hydrolysis of obtained xylans towards xylo-oligosaccharides production\u003c/h2\u003e \u003cp\u003eAfter extraction and characterization of SFM xylans, the final step in SFM valorization was to obtain emerging prebiotics, XOS by means of enzymatic conversion. For this purpose, bacterial xylanase Rohalase\u0026reg; SEP-Visco was chosen as optimal commercial preparation owing to the fact that it produces negligible amounts of xylose (preliminary study), thus, ensures production of complex XOS mixture without need of extensive purification. This enzyme was applied for the first time for XOS production, and therefore, the first experiments enabled insight in parameters for optimal activity of the enzyme preparation needed for XOS production. It was concluded that optimum conditions for XOS production were slightly acidic to neutral reaction medium, while the optimum temperature was found to be around 70\u0026deg;C (supplementary data). After determination of these process parameters, the influence of substrate and enzyme concentrations on XOS production was examined. As it can be clearly seen from the time course graph (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), enzyme preparation proved to have significantly high activity, and that even small amounts of the enzyme (0.005% v/v) can be utilized for this purpose. Consistent maximum XOS concentrations were achieved in all experiments, regardless of enzyme concentration. Hence, the lowest examined enzyme concentration was adopted as optimal for the next set of experiments featuring the higher concentrations of substrate in order to examine the initial kinetics of the XOS synthesis reaction, and accordingly to see differences in their structures throughout the whole reaction time course.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs it was expected, higher concentrations of XOS were achieved with increment of offered substrate concentration, reaching concentrations of reducing sugars of approximately 20 and 31 mM for conventionally and enzyme-aided extracted xylan (DP-xylan), respectively when 5% (w/v) substrate solution was utilized. Obtained concentration did not exactly matched the increment of the substrate, however, this can be explained by the fact that higher xylan concentrations decreased XOS yields due to the enzyme inhibition caused by higher reaction mixture viscosity and density, that was previously noticed by Bian et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Nevertheless, it can be concluded that purer DP-xylan represents the substrate of choice for XOS production. The results presented in this way (overall produced reducing sugars) give us a comprehensive picture of the XOS reaction process, however, in order to better see what type of compounds were synthesized, as well as a compound polymerization degree, additional characterization of obtained reaction mixtures for DP-xylan was performed. To determine the ranges of degrees of polymerization from obtained XOS HPSEC-ELSD was conducted. For this purpose, reaction mixtures for the reaction times 0, 15, 60 and 120 min were chosen for further analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt can be clearly seen the release of smaller compounds during the reaction course hydrolysis of high molarity DP-xylan (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), namely oligosaccharides (Mw\u0026thinsp;\u0026lt;\u0026thinsp;6 kDa, corresponding to DP\u0026thinsp;\u0026lt;\u0026thinsp;50). Namely, the abundancy of oligosaccharides lower than 6 kDa was around 10% in initial reaction mixture (0 min), while the oligosaccharide abundance was increasing with the reaction reaching 43.0%, 48.3% and 53.2% after 10, 60 and 120 min, respectively.\u003c/p\u003e \u003cp\u003eIn order to quantify the obtained products, HPLC analysis was performed, although this analysis may only detect oligosaccharides up do DP 6. From the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, it can be seen that total XOS rather quickly (15 min) reaches equilibrium concentration 7.5 mg/mL that represents XOS yield of approximately 15% (calculated on total DP-xylan content). This concentration was higher than previously reported 5.29 mg/mL after 12 h using xylan-rich fraction isolated from sugarcane bagasse (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e) and 5.05 mg/mL from oil palm empty fruit bunch fibers isolated xylan after 12 h [58], but lower than 28.6% of xylan extracted from corncobs reached by Teng et al. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e) and 19.1% of XOS from sugarcane bagasse by Valladares-Diestra et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Significant discrepancies in achieved yields are mainly consequence of the xylan structure and composition, which depends on the type of employed extraction treatment as well as utilized enzyme. Besides concentration, the composition of obtained XOS may vary greatly between different systems, as well as under different conditions within the same system. During the reaction of DP-xylan hydrolysis, main reaction products and their abundance change while XOS total concentration remains constant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). At the starting stages of the reaction XOS3 seems to be the most present XOS derivative, while XOS2 concentration increases during the whole examined reaction period, finally reaching same concentration after 2h. On the other hand, concentration of XOS4 decreases over the time together with less abundant XOS5 and XOS6. Once again, it should be emphasized that concentration of xylose is quite low, owing to the fact that enzymatic preparation with pronounced endo-xylanase activity was utilized.\u003c/p\u003e \u003cp\u003eEven though, the oligosaccharides are often regarded as group of diverse compounds with degree of polymerization between 2\u0026ndash;50 monomeric units or Mw values up to 5\u0026ndash;7 kDa, according to different criteria (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e), information about their composition is quite critical for further establishment of structure-function relationships. According to the MC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) the main type of extracted hemicellulose was xylan with the common feature of a backbone of β-(1\u0026rarr;4)-linked xylose residues, with a common modification of xylans with numerous arabinose and some glucuronic acid residues on O-2 (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Therefore, the obtained structures were additionally confirmed by means of MALDI-TOF-MS analysis since both the chain length and the expected types of glycosidic linkage contained in the XOS can be inferred. Namely, mass spectra confirmed existence of pentose-based oligosaccharides DP3-DP22 as sodium adducts (\u003cem\u003em/z\u003c/em\u003e 437, 569, 701, 833, 965, 1097, 1229, 1230.0, 1362.1,1494.2, 1626.3, 1758.4, 1890.6, 2022.7, 2154.8, 2286.9, 2419.0, 2551.1) and as potassium adducts (\u003cem\u003em/z\u003c/em\u003e 453.4, 585.6) with a lower abundance. These most likely correspond to xylose, according to the compositional data of DP-xylan (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), although some of the xylose units may be substituted with arabinose which cannot be substantiated by MALDI-TOF due to equal masses for xylose and arabinose. None of these pentoses seems to be substituted with acetyl groups which is good agreement with the lack of characteristic band for acetyl groups previously observed in FTIR spectra. It should be noted that oligomers below \u003cem\u003em/z\u003c/em\u003e 400 are not included in the spectrum, due to hindrance of matrix peaks, and therefore this analysis cannot confirm the presence of xylobiose and small quantity of xylose that were previously detected by HPLC analysis. Thereafter, it can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e that the most important are acidic oligosaccharides, that are present in three series, i) the most simple pentoses with 1 molecule of glucuronic acid with DP 5\u0026ndash;17; ii) the most abundant series are oligosaccharides (methylglucuronic acid (MeGlcA) substituted XOS) are most likely present DP4-DP21 (\u003cem\u003em/z\u003c/em\u003e values of 628, 759, 891, 1023, 1155, 1287, 1419, 1551, 1683, 1815, 1947, 2079, 2212.8 2344.9, 2477.0, 2609.1, 2741.2, 2873.3), and also are present the potassium adducts with DP between 5\u0026ndash;17 (775.7, 907.8, 1039.9, 1172.0, 1304.1, 1436.2, 1568.3, 1700.5, 1832.6, 1964.7, 2096.8, 2228.9, 2361.0); iii) XOS with 2 groups of MeGlcA with DP 8\u0026ndash;21 (1213.9, 1346.0, 1478.1, 1610.2, 1742.4, 1874.5, 2006.6, 2138.7, 2270.8, 2402.9, 2535.0, 2667.1, 2799.2, 2931.3). Finally, in small amount, a series of hexose oligomers (from DP 3 to DP17) can be recognized within the spectrum (\u003cem\u003em/z\u003c/em\u003e values of 527, 689, 851, 1031, 1175, 1333, 1499, 1661, 1823, 1824.6, 1986.7, 2148.8, 2311.0, 2473.1, 2635.3, 2797.4), that might be a consequence of small share of adjuvant polysaccharide fractions within the obtained DP-xylan.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eSunflower meal, an abundant and unexploited by-product of oil industry, proved to be a good source of valuable compounds that could be valorized throughout the newly proposed efficient method based on enzyme-aided fractionation. The application of enzymes not only enabled increment the yield of a purer xylan, but also to generate several other fractions (polyphenol-rich fraction, protein isolate, pectin and lignin) ready to be used to add value to different food and cosmetic preparations. Finally, high purity obtained xylan extract was successfully transformed into emerging group of prebiotic xylo-oligosaccharides.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge and thank to AB Enzymes GmbH (Darmstadt, Germany) for their kind donation of enzyme preparation Rohalase\u003csup\u003e\u0026reg;\u003c/sup\u003e SEP-Visco. This work was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia [Contract No. 451-03-47/2023-01/200287 and 451-03-47/2023-01/200135] and has received funding from Science Fund of the Republic of Serbia, programme IDEAS, project number 7750109 (PrIntPrEnzy) and the Horizon Europe 2021-2027 research and innovation programme under grant agreement ID 101060130 (TwinPrebioEnz). \u0026nbsp;The graphical abstract was created with BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. Simović: Investigation, Conceptualization, Methodology, Writing-original draft. K. Banjanac Investigation, Data curation. M. Veljković: Investigation, Formal analysis. V. Semenčenko: Methodology, Formal analysis. P. Lopez-Revenga: Formal analysis, Data curation A. Montilla: Formal analysis, Writing - Review \u0026amp; Editing, Supervision F. J. Moreno: Writing - Review \u0026amp; Editing, Supervision D. Bezbradica: Funding acquisition, Project administration, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study will be made openly available on a data repository immediately after publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdeleke BS, Babalola OO. 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Annual Review of Plant Biology. 2010;61(1):263-89.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"sunflower meal, waste valorization, enzyme-aided fractionation, xylo-oligosaccharides, emerging prebiotic","lastPublishedDoi":"10.21203/rs.3.rs-3975794/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3975794/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSunflower meal, a plentiful and underutilized oil industry by-product, is currently discarded as waste or used as cheap component of animal feed with poor protein content and high content of complex carbohydrates. To harness its great potential for valorization, we propose an efficient method through enzyme-aided fractionation yielding 47.8% of hemicellulosic fraction, with simultaneous generation of various other potentially valuable fractions (including polyphenol-rich fraction, protein isolate, pectin, and lignin). According to the monomeric composition the main type of extracted hemicellulose was xylan with the common feature of a backbone of \u003cem\u003eβ\u003c/em\u003e-(1→4)-linked xylose residues, with a common substitution with arabinose moieties and some glucuronic acid residues. Subsequently, the xylan fraction underwent enzymatic treatment using commercial xylanase (ROHALASE\u003csup\u003e® \u003c/sup\u003eSEP-VISCO) to produce highly valuable compounds - emerging prebiotics xylo-oligosaccharides (XOS). Under optimized reaction conditions (70°C, pH 6 and enzyme concentration of 0.005% v/v using 5% w/v xylan solution) a yield of XOS with a polymerization degree DP\u0026lt;50 reached approximately 52.3% after 2 hours (majority of obtained product had DP\u0026lt;6 with predominance of XOS2 and XOS3 and without significant xylose generation).\u003c/p\u003e","manuscriptTitle":"Sunflower meal valorization through enzyme-aided fractionation and production of emerging prebiotics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 11:29:04","doi":"10.21203/rs.3.rs-3975794/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f06e901-db99-479d-9e31-f6fa73ddebf5","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-04T19:15:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-28 11:29:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3975794","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3975794","identity":"rs-3975794","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}