Phenolic and Cellulose-Rich Fractions from Subcritical Water Treated Beer Bagasse

Phenolic and Cellulose-Rich Fractions from Subcritical Water Treated Beer Bagasse | 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 Phenolic and Cellulose-Rich Fractions from Subcritical Water Treated Beer Bagasse Paula Gomez-Contreras, Catalina Obando, Pedro Freitas, Laia Martin-Perez, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4610399/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Of the three types of waste generated in beer processing, beer grain spent (BGS) or beer bagasse is the most abundant and has a high potential for valorisation. In this work, defatted BGS was subjected to an extraction process with subcritical water (SWE) at different temperatures (110, 130, 150 and 170° C) to obtain extracts rich in phenols and the cellulosic fractions. Furthermore, the obtained cellulose fractions were also purified by means of a greener methodology using hydrogen peroxide (H 2 O 2 ). The results showed that the extraction conditions affected the composition and properties of the fractions. The dry extracts obtained at 170°C were richer in phenolics (24 mg GAE. g − 1 defatted beer bagasse (DB), but with lower antioxidant capacity (71 mg DB.mg − 1 DPPH). This extract (E-170) also showed the highest antibacterial potential (lower MIC values) against L. innocua (80 mg·mL − 1 ) and E. coli (140 mg·mL − 1 ) than those obtained at lower temperatures. The purification of cellulose from the SWE residues, using hydrogen peroxide revealed that DB is not a good source of cellulose material since the bleached fractions showed low yields (20–25%) and low cellulose purity (42–67%), even after four bleaching cycles (1 h) at pH 12 and 8% H 2 O 2 . Despite this, the subcritical water extraction method highlights the potential of a simple processes as technological option to convert underutilized side streams like beer bagasse into added-value, potential ingredients for innovative food and pharmaceutical applications. phenolic compounds cellulose fibres antioxidant antimicrobial Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Global beer consumption has experienced a notable increase over the last 50 years, reaching 150 billion liters per year, surpassing wine consumption by seven (Rodriguez et al ., 2023). It is estimated that by 2025, it will be worth $ 502.9 billion and have an annual growth rate of 19.9% in response to growing demand and the wide variety of new styles and flavors (Pokrivčák et al ., 2019). During the beer brewing process, three types of waste are generated: spent beer grains (SBG or bagasse), spent hops and spent brewer's yeast. It is estimated that between 14 and 20 kg of bagasse, 0.2 and 0.4 kg of hops, and 1.5 and 3 kg of residual yeast are generated for every 100 liters of beer produced (Rodriguez et al. , 2023). Consequently, the beer bagasse constitutes approximately 85% of the total byproducts generated (Puligundla & Mok, 2021). This residue presents outstanding nutritional properties that make it a valuable source of high-value compounds and includes hulls of barley malt grains, parts of the pericarp, and layers of the barley seed coat. It contains fibers (soluble and insoluble), proteins, lipids and phenolic compounds, which can be free or bound to dietary fiber (Bonifacio-Lopes et al. , 2022). Among phenolic acids, ferulic, p-coumaric and caffeic predominate in a bound state (Ikram et al. , 2017). The chemical composition of BSG depends on the type of barley, the time of harvest, and the malting and mashing conditions, being rich in cellulose (12–25% in dry basis), hemicellulose (20–25%), lignin (10–28%), starch (2–7%), proteins (20%), lipids (6–10%) and minerals (2–5%) (Lynch et al. , 2016), together with polyphenols (0.7-2%). So, the extraction of different fractions from beer bagasse could be of great interest to obtain different valued biomolecules to be use for the pharmaceutical, packaging or food industry. Among the various new green extraction and separation technologies, ultrasound-assisted, microwave-assisted, enzyme-assisted and subcritical and supercritical fluid extraction has been recently used, surpassing conventional methods such as maceration, infusion, and Soxhlet extraction (Messinese et al ., 2023; Panzella et al. , 2020). In subcritical water extraction (SWE), the physicochemical properties of water, such as the relative dielectric constant and polarity, decrease significantly with increasing temperature so that, under subcritical conditions, water can dissolve polar and non-polar compounds (Galamba et al ., 2019). Furthermore, viscosity and surface tension decrease with increasing temperature, resulting in higher extraction efficiency (Wiboonsirikul & Adachi, 2008; Zakaria & Kamal, 2016). Additionally, water is an economical, non-toxic, non-hazardous, and safe solvent that works as a solvent and catalyst to take advantage of and transform biomass into bioactive products (Abdelmoez et al. , 2007; Sarkar et al. , 2022). In the last few years, this technology have shown an increasing interest in the scientific community (from 300 articles published in 1995–2000 to 5,000 articles in the 2021–2023 period), thus showing SWE is a promising technology for extracting target compounds such as proteins, polyphenols or phenolic acids from different sources. SWE has been applied in a variety of agro-industrial wastes such as winery, tea, shellfish and tobacco wastes, carrot leaves, cotton flowers, peels (chestnut, almond, potato, citrus, mandarin, mango, kiwifruit), seeds (papaya), pomace (apple, pomegranate, kiwifruit), among others (Majeed et al. , 2024). These studies showed that SWE is a sustainable and energy-effective way of biomass processing because it promotes selective extraction and reactions that convert waste materials and by-products into valuable products. These hold great potential for the creation of novel food products and waste reduction while enhancing the overall sustainability of food production. The extraction of different compounds from BSG have been studied by few researchers for different purposes, which were mainly focused on the recovery of proteins and polyphenols. Thus, Devnani et al (2023) reviewed the effect of extraction techniques and conditions on the composition, physicochemical, and techno-functional properties of the obtained BSG protein extracts. Guido and Moreira (2017) focused on the current extraction methods used to obtain phenolic compounds from BSG, ranging from more traditional (the conventional solid–liquid extractions employing organic solvents, alkaline, and enzymatic reactions) to advanced techniques such as pressurized fluid, supercritical and microwave-assisted and ultrasound-assisted extractions. Camacho-Núñez et al .,(2023) addressed the extraction of cellulose from BSG by means of alkaline hydrolysis and bleaching reactions, and its further conversion into cellulose acetate for packaging applications. Alonso-Riaño et al., (2021) used SWE as hydrolytic medium to recover proteins and specific polyphenols from craft BSG for pharmaceutical and cosmetic applications. They found 185 o C to be the best temperature to maximize the extraction of protein and aldehyde phenolic compounds (vanillin, syringic and protocatechuic aldehyde) while lower temperatures (160 o C ) promoted the extraction of hydroxycinnamic acids, such as ferulic acid and p-coumaric. Nevertheless, no studies using SWE for cellulose extraction at different temperatures have been found neither the characterization of the obtained phenolic rich fraction in terms of phenolic content, antioxidant and antimicrobial properties, to the best of our knowledge. The main objective of the present work is to fractionate the beer bagasse residue by subcritical water extraction (SWE) at different temperatures (ranging between 110–170 o C C), analyzing the compositional and functional properties of soluble (extracts) and insoluble (residues), thus contributing to valorize the BGS waste in the context of a circular economy. Thus, soluble extracts and cellulose-rich insoluble fractions were obtained as a function of the temperature. The insoluble fraction was also submitted to a green bleaching process to purify cellulose. 2. Materials and Methods 2.1 Materials Petroleum ether (40–60°C bp), phosphorous pentoxide (P 2 O 5 , 98.2%), sodium hydroxide (NaOH), glucose, and arabinose were purchased from Sigma-Aldrich (USA). D (+)-Xylose was supplied by Merck KGaA (Darmstadt, Germany). Ethanol (98%), hydrogen peroxide (H 2 O 2 , 30%), sulphuric acid (H 2 SO 4 , 98%), and sodium carbonate (Na 2 CO 3 , 99.5%) were obtained from Panreac Quimica S.L.U (Castellar del Vallés, Barcelona, Spain). 2.2 Residue preparation Beer bagasse was supplied by a brewery factory located in Valencia and dried at 60 o C ± 2°C for five days in a forced-air oven (J.P Selecta, s. a., Barcelona, Spain). After that, it was milled using a Thermomix (Model TM6 Vorwerk España M.S.L S.C) and sieved to obtain particles under 0.71 mm and cold stored. The defatting process was performed under reflux with petroleum ether for 8 hours at 60 o C, with stirring using a 1:4 ratio of dry sample to solvent. Then, it was allowed to settle, decanted and filtered with a 125 mm paper filter, washing with pure solvent, to separate the defatted residue that was left to solvent evaporation at room temperature in an extractor hood for 16 h, when constant weight was reached. The liquid phase was dried by adding anhydrous sodium sulfate and then left for 48 h and filtered under vacuum. The oil was recovered by evaporating petroleum ether in the vacuum rotary evaporator (Rotary Evaporators, Heidolph Instruments GmbH & Co. KG, Walpersdorfer, Germany). 2.3 Subcritical Water Extraction Subcritical extraction (SWE) of defatted bagasse was carried out with a ratio solids-water of 1:8, using a pressure reactor (Model 1-TAP-CE, 5 L capacity, Amar Equipment PVT. LTD, Mumbai, India). The temperature-pression conditions used were 110°C -1 bar, 130°C -2 bar, 150°C-4.5 bar, and 170°C-8 bar, applying 50 rpm in all cases, for 30 minutes. After each extraction step, the defatted sample dispersions were filtered through a filter with pore size less than 0.5 mm (Filterlab, Barcelona, Spain). Thus, two fractions were obtained from each SWE process: one insoluble residue (R) and the soluble extracts (E). The extracts, named E-110, E-130, E-150, and E-170, were lyophilized at -60°C and 0.8 mbar and stored in desiccators (P 2 O 5 , 0% relative humidity) at 4 ◦C. The respective mass yields of extracted solids and solid residues were determined with respect to the initial defatted bagasse. To determine the extracts yield, three aliquot samples of the liquid extracts were dried at 105ºC until a constant weight to determine water:solid ratio and the total solids extracted was calculated by multiplying by the total water mass in the reactor. To determine the yield in the extraction residues (R-110, R-130, R-150, and R-170), these were washed with distilled water, filtered, and dried at 40 ◦C for 48 hours to determine their weight yield; then these were stored at 4 ◦C until further use. 2.4 Bleaching process The insoluble fractions obtained from SWEs were bleached following the method described by Freitas et al (2023), using hydrogen peroxide as bleaching agent. 4% (wt) H 2 O 2 solution was prepared while pH was fitted to 12 (using NaOH). The lignocellulosic residues were treated with this bleaching solution using a solvent-solid ratio of 30:1, at 40°C for 1 h, in four consecutive cycles, filtering and washing the sample with distilled water after each cycle. After the four cycles, the cellulose fractions were filtered and washed with abundant deionized water to remove residues of the bleaching solution and then dried at 50ºC overnight. The bleached samples were labeled as BR-110-1C to BR-110-4C, BR-130-1C to BR-130-4C, BR-150-1C to BR-150-4C and BR-170-1C to BR-170-4C. The mass yield (%) and whiteness index (WI) were determined in all samples using equations ( 1 ) and ( 2 ) at each bleaching cycle to control the sample development. The color coordinates L* (lightness), a* (red green), and b* (yellowish-blue) of each bleached fraction were obtained with a CM-3600d spectro-colorimeter (Minolta Co., Tokyo, Japan), using a D65 illuminant and 10º observed. $$Yield= \frac{Weight of bleached cellulose}{Weight of material before bleaching}$$ 1 $$WI=100- \sqrt{{(100-\text{L}\text{*})}^{2}+{\text{a}\text{*}}^{2}+{\text{b}\text{*}}^{2}}$$ 2 2.5 Physico-chemical analysis of beer bagasse, soluble and insoluble fractions. The amount of protein in raw beer bagasse BB, extracts (E-110, E-130, E-150, E-170), and extraction residues (R-110, R-130, R-150, R-170) was measured using the Dumas combustion method (Leco, St. Joseph, MI, USA) by duplicate. A conversion factor 4.74 was applied to calculate protein content from total nitrogen (Krul, E.S, 2019) In the same way, all samples were subjected to thermogravimetric analysis (TGA). A TGA/SDTA 851e analyzer (Mettler Toledo, Schwarzenbach, Switzerland) working under nitrogen flow (20 mL/min) was used to obtain the weight loss vs. temperature curves (TGA) and the first derivatives (DTGA). Samples (3–5 mg) previously conditioned sample in P 2 O 5 were placed in 70 µL alumina crucible and heated from 25 to 900°C at 10 K/min. Three replicates per sample were obtained. 2.5.1 Analysis of structural components in the Insoluble fraction Cellulose, hemicellulose, acid-insoluble lignin content of defatted beer bagasse (DB), insoluble fractions (R), and bleached samples (BR) were analyzed according by the method of National Renewable Energy Laboratory (NREL/TP-510-42618—2008) (Sluiter, 2025). The test consisted of a two-stage hydrolysis with 72% sulfuric acid, of which one results in a soluble fraction in which the sugar content (glucose, xylose, and arabinose) was measured by high-resolution liquid chromatography. (HPLC, Agilent Technologies, model 1120 Compact LC, Waldbronn, Germany) and a RezexTM RCM-Monosaccharide Ca2 + column (300 × 7.8 mm). On the other hand, the insoluble fraction was used to quantify the acid insoluble lignin content by thermogravimetric method. The cellulose content was obtained from the quantified glucose and hemicellulose from the sum of quantified xylose and arabinose, as described by Sluiter (2025). Before hydrolysis, the raw material (DB) and the insoluble fractions (R) were subjected to the extractives determination the using the standard NREL method (NREL/TP-510-42619—2008) (Sluiter, 2025). This procedure was performed using a Soxhlet set-up, which consists of two stages: a first extraction with water for 6 h, followed by a second extraction with ethanol at 60°C for 6 h. The thermal stability analysis was carried out on all samples by triplicate using the TGA 1 Stare System analyzer, (Mettler Toledo, Greifensee, Switzerland), previously conditioned in phosphorous pentoxide (P2O5) for two weeks. The analysis was conducted from 25 to 900ºC at 10 K/min with a nitrogen flow of 10 ml per minute. In addition, the ash content was analyzed using the UNE-EN 14775 standard. 2.5.2. Total phenolic content, antioxidant and antibacterial properties of soluble fraction. The total phenol content was determined using the Folin Ciocalteu method. Briefly, 0.5 ml of each extract was mixed with 6 ml of distilled water, and 0.5 ml of Folin reagent (2N) was added. After one minute, 1.5 ml of 20% Na 2 CO 3 solution and distilled water were added to a volume of 10 ml. After stirring, they were kept in the dark for 2 hours. The absorbance at 725 nm was then measured in triplicate using a UV-Vis spectrophotometer (Evolution 201, Thermo Scientific). Gallic acid was used as a standard, and the results were expressed as mg L − 1 gallic acid equivalents (GAE) using a standard curve (R 2 = 0.9991) of gallic acid (2–20 mg.L − 1 ). The TPC value of milled raw material was also determined by previously extracting phenols with methanol at a solid to liquid ratio of 1:50 at room temperature and dark for 24 h under constant stirring. The antiradical capacity of the extracts was determined using the 2,2-diphenyl-1-pikryl-hydrazyl (DPPH) free radical method. For each extract, a solution of DPPH in methanol at a concentration of 6.22 × 10 − 2 mM was mixed with different extract concentrations until reaching a final volume of 4 ml. The resulting solutions were kept in the dark at room temperature for 12 h, after which the absorbance at 515 nm was measured. The initial and final concentrations of DPPH in the reaction medium were calculated from a calibration curve fitted by linear regression (R 2 = 0.9992). The antiradical activity was evaluated by the EC 50 parameter, which represents the amount of antioxidants necessary to reduce the initial concentration of DPPH by 50%, when the stability of the reaction is reached. This value was expressed as mg of dried extract per mg of DPPH and also, in mg defatted beer bagasse per mg of DPPH for comparison purposes. EC 50 values were determined from graphs showing the percentage of [DPPH] remaining as a function of the amount of solid extract relative to the amount of DPPH, using the Eq. 1 : \({\%\left[DPPH\right]}_{remaining}=\frac{{\left[DPPH\right]}_{t}}{{\left[DPPH\right]}_{t=0}}x 100\) (Eq. 1 ) Regarding the antimicrobial capacity of the extracts. the minimum inhibitory concentration (MIC) of the different extracts was determined for two bacteria: the Gram-positive bacterium Listeria innocua and the Gram-negative Escherichia coli . This analysis followed the method outlined by Freitas et al ., (2023). Standard 96-well microtiter plates with a total volume of 200 µL were employed for this analysis. For both bacterial strains, stored at -20ºC, a stock solution was prepared by transferring bacterial amounts using an inoculation loop to a volume of 10 mL of TSB and incubated at 37 o C for 24 hours. Subsequently, 10 µL of the stock solution was taken and transferred to a tube containing 10 mL of TSB to prepare the corresponding work solution with a concentration of 10 5 CFU.mL-1. This concentration was confirmed through serial dilution and counting. For each bacterium, 100 µL of the bacterial solution with an initial concentration of 10 5 CFU.mL-1 was added to each well. Thereafter, different volumes of each extract solution, with 200 mg.mL − 1 , were added in each well while the final volume was adjusted to 200 µL with TSB to obtain different extract dilutions. The plates were then incubated at 37 o C for 24 hours. Afterward, 100 µL from each well was transferred to TSA plates and incubated at 37 o C C for 24 hours for final counting. The MIC for each extract was determined as the lowest extract concentration at which no bacterial growth was observed on the TSA plate. This analysis was performed in duplicate. 2.6. Statistical analysis The Statgraphics Centurion XVII-64 program (Manugistics Corp., Rockville, Md.) was used to perform statistical analyses using an analysis of variance (ANOVA) with a confidence level of 95%. The Fisher test was applied to detect possible differences in treatment responses, using a critical value of 5% to determine significance. 3. Results and Discussion 3.1. Yields and composition of the different SWE fractions Figure 1 shows the flow chart of the brewer spent grain (BSG) or beer bagasse fractionation throughout the SWE, giving rise to soluble extracts (E) and insoluble residues (R) at each temperature. The latter were submitted to a bleaching step to purify cellulose while the extracts were freeze-dried to obtain extract powders. Images of the different obtained products, together with the obtained mass yield of each process step are also shown in Fig. 1 . Previously to the SWE, a defatting step of the BSG yielded around 8% oil from the dried bagasse, being this value in agreement with that reported in the literature (Faulds et al ., 2008; Del Rio et al. , 2013). The main beer bagasse lipid compounds has been reported, these being triglycerides (55–67%), free fatty acids (18–30%), such as palmitic, oleic and linoleic acids and free steroids (5%), such as sitosterol and campesterol. These lipid compounds have a wide range of nutraceutical, pharmaceutical and cosmetic applications (Del Rio et al ., 2013). The values of mass yields of solid extracts (E-110, E-130, E-150 and E-170) and dried residues (R-110, R-130, R-150 and R-170) of the SWE process performed at different temperatures, shown in Fig. 1 , indicate that the extraction yield increased (from 7–41%) when the extraction temperature rose from 110°C to 170°C. This is mainly explained by the changes in the water solvent properties when the temperature increased, which reduces the strength of hydrogen bonds and leads to an important reduction in dielectric constant, this becoming closer to the dielectric constant value of some organic solvents, such as methanol (ɛ = 33) or ethanol (ɛ = 25) (Carr et al. , 2011; He et al. , 2012). The sum of both yields (extract and residue) at a given temperature closed the mass balance, thus indicating a low mineralisation degree of the organic matter present at the processing conditions used. The TGA curves of the defatted bagasse (DB), extracts and residues obtained after SWE at the different temperatures is shown in Fig. 2 a, together with de derivative curves (Fig. 2 b). The DB presented three main degradation steps: the first mainly corresponding to the loss of bound water; a second step associated with the degradation of polysaccharides with different thermostability such as hemicelluloses (150–350 o C ), celluloses (275–350 o C ) and a part of lignin (160–900 o C ), and the third one, related to the degradation of residual lignin and secondary metabolites from the previously thermo-degraded compounds, as previously described by other authors for lignocellulosic biomass (Freitas et al. , 2023; Carichino et al. , 2023). The major weight losses took place between 225 and 625 o C (80%), in line with the lignocellulosic nature of this residue, in agreement with the results obtained by other authors (Ortiz et al. , 2020; Carichino et al. 2023). Very similar TGA patterns were obtained for every lignocellulosic residue. Nevertheless, it is remarkable that the highest extraction temperatures (150 and 170 o C) gave rise to the samples with the highest peak temperatures (temperature of the maximum degradation rate), which indicates that these were the most enriched in cellulose that shows peak temperature between 330–350 o C (Zhang et al ., 2012). In contrast, the TGA and DTGA curves of the extracts revealed a more complex compositional profile, exhibiting several thermodegradation steps. The extract obtained at the highest temperature showed a higher proportion of compounds that degrade at higher temperature according to a greater extraction of polymeric components, such as hemicellulose or lignin. A higher final mass residue was also observed in comparison with the untreated sample (defatted bagasse), which can be due to the extract enrichment in minerals or formation of degraded organic matter from the soluble compounds. Some compositional differences in the extracts and residues obtained at each temperature can be observed in Tables 1 and 2 . Table 1 Chemical composition of defatted beer bagasse (DB) and insoluble fractions after SWE at different temperatures. Sample Extractive (%) Protein (%) Ash (%) Lignin* (% ) Cellulose (%) Hemicellulose (%) DB 13,1 ± 0,5 ᵃ 22 ± 2 a 3,71 ± 0,01 ᶜ 9,5 ± 1,6 ᵃᵇ 17 ± 2 ᵃ 17,9 ± 0,6 d R-110 20,9 ± 1,5 ᵇ 28 ± 2 ᵇ 3,12 ± 0,11 ᵇ 9,1 ± 0,4 ᵃ 16 ± 2 ᵃ 15 ± 2 ᶜ R-130 21,79 ± 0,05 ᵇ 26,2 ± 0,2 ᵃᵇ 2,79 ± 0,04 ᵇ 11,56 ± 0,08 ᵇ 20 ± 2 ᵃ 14,9 ± 1,2 ᶜ R-150 34,5 ± 0,9 ᵈ 26 ± 2 ᵇ 2,27 ± 0,11 ᵃ 14,6 ± 0,5 ᶜ 21 ± 2 ᵃ 7,8 ± 1,2 ᵇ R-170 28 ± 3 ᶜ 35,7 ± 0,2 ᵃᵇ 3,1 ± 0,2 ᵇ 29,9 ± 0,3 ᵈ 30 ± 3 ᵇ 2,01 ± 0,08 ᵃ a,b,c ..Different superscript letters (a,b,c) in the same column indicate significant differences (p < 0.05) * acid insoluble lignin In Table 1 , the total (water and ethanol) extractive content, protein, ashes, cellulose, hemicellulose and acid insoluble lignin contents of the DB can be observed, together the values obtained for the different SWE solid residues. The obtained values for raw brewer´s spent grain were within the range previously reported (around 16–22% for cellulose, 24–28% hemicellulose and 9–27% total lignin) (Verni et al. , 2020; Alonso-Riaño et al. , 2023; Qazanfarzadeh et al . 2023). In the extraction residues, the hemicellulose content was very low at temperatures greater than 150 o C, in accordance with the selective dissolution of hemicellulose under the subcritical water conditions (Cocero et al. , 2018; Ruthes et al ., 2017). Thus, the hemicellulose started to be removed from the beer bagasse matrix when using temperatures greater than 130 o C, reaching very low values at 170 o C (2%). At these temperatures, the lignin content significantly increased, which confirmed that this fraction of the biomass was not released under SWE. as it has been previously observed by other authors working with BSG (Alonso-Riaño et al , 2023). On the other hand, the increment in the cellulose significantly increased (p < 0.05) in the residues treated at the highest temperature (R-170) in comparison with the untreated DB. The insoluble-acid lignin in the solid residues accounted for the 87, 101, 99 and 160% of the total lignin in the raw material for 110, 130, 150 and 170ºC, respectively. Thus, the obtained lignin values are surely overestimated as the outcome of such gravimetric analysis is highly disturbed by the presence of non-lignin acid-insoluble material, e.g. proteins (Erven et al ., 2017). The corrected lignin (calculated by substracting the protein content) was not given because in most cases, negatives values were obtained. According to Alonso-Riaño et al . (2023), changes in the lignin structure took place during the SWE such as condensation reactions and structural alterations. In both extract and residue fractions, the greater mass loss in TGA curves was observed for the temperature range of 200–700 o C, where the lignin is mainly degraded, in line with the formation of secondary metabolites from the previously thermo-degraded compounds. This thermal degradation behaviour agreed with that found in the literature for other lignocellulosic residues (Freitas et al ., 2023). So, the application of SWE led to a selective fractionation of DB, giving rise to aqueous extracts richer in different compounds of lower molecular (sugars, phenolic compounds and minerals) and polysaccharide and lignin-rich insoluble residues. Table 2 Total phenolic content (TPC), antioxidant activity (EC50), protein content and ashes of the aqueous extracts (E) obtained from SWE process at different temperatures. (mean values ± standard deviation). E-110 E-130 E-150 E-170 % Protein (db) 15,1 ± 0,1 a 16,6 ± 0,2 a 22,4 ± 1,2 b 28,7 ± 0,6 c %Ashes (db) 1,54 ± 0,06 c 1,5 ± 0,1 c 1,20 ± 0,04 b 0,46 ± 0,01 a TPC 1 (mg GAE/g extract) 16,8 ± 0,1 a 22 ± 2 a 17,91 ± 0,07 b 59,1 ± 0,2 c TPC 2 (mg GAE/g DB) 1,27 ± 0,08 a 3,2 ± 0,3 ab 6,34 ± 0,02 ab 24,18 ± 0,08 b EC 50 1 (mg extract/mg DPPH) 15 ± 3 a 19 ± 2 a 48 ± 4 b 71,1 ± 0,4 c MIC (mg/ml) against L.innocua 264 198 168 80 MIC (mg/ml) against E. Coli 234 204 162 140 a,b,c…different superscripts in the same row indicates significant differences among extracts (p < 0.05) The ash content of the DB, extracts and residues (shown in Tables 1 and 2 ) showed that minerals were mainly present in the insoluble residues, whereas small amounts were released to the extracts. The value obtained per DB was in the range of the ash content reported by other authors for beer spent grain (2–5 g/100 g dry DB), being the most abundant constituents phosphorous, magnesium, calcium and potassium (Ortiz et al. , 2020). In Table 2 , the protein content of DB is also shown (around 22%), being this value in the range of previously reported values for beer bagasse (Rodriguez et al. , 2023; Alonso-Riaño et al. ,2021) considering a fat-free basis. The partition of the protein content during the SWE gave rise to greater content in the insoluble residues, thus suggesting a low solubility of the bagasse proteins under the used water subcritical conditions, especially at the lowest SWE temperatures. At 110 o C, the 95% of the total protein remained in the insoluble residue, this percentage decreasing to around 53% at 170 o C. These proteins are extracted and/or hydrolysed during the thermal treatment, leading to peptide chains of different sizes or free amino acids or even amino acid decomposition, especially at high temperatures, producing different carboxylic acids and other nitrogen containing compounds such as ethanolamine (Trigueros et al. , 2023; Rogalinski et al , 2005). Therefore, SWE treatment of beer bagasse can be considered as an efficient extraction method to recover the protein fraction of the BSG generated in the beer industry, the maximum recovery of solubilised protein in the SWE extracts being of 47% at 170 o C. 3.2. Functional properties of the SWE extracts: Antioxidant and Antibacterial properties. In Table 2 , the Total phenolic content (TPC) and antioxidant activity of the aqueous extracts obtained from SWE process at different temperatures is displayed. The antioxidant activity of the different extracts was determined through the total phenolic content by the Folin–Ciocalteu method and the EC 50 parameter with DPPH radical, which quantifies the amount of extract needed to reduce the initial concentration of the radical up to 50%. The TPC determined in the solid extracts (TPC 1 ) was also referred per mass unit of defatted bagasse (TPC 2 ). The TPC values increased from 16 to 59 mg GAE/g dried extract as the extraction temperature rose. Similarly, Rahman et al (2021) observed that BSG aqueous extract obtained at 160 o C showed highest TPC values than that obtained at 100 and 140 o C, the main phenolics compounds being flavan-3-oles, hydroxycinnamic acids (such as chlorogenic, gallic, protocatechuic, ferulic and p-coumaric acids) and flavonols. The found TPC values in defatted bagasse (7,57 mg GAE/g DB) are in the range of those reported by other authors (0.89-15 mg GAE/g sample), depending on the solvent and extraction method used (Santi et al ., 2018). Nevertheless, when expressed per mass unit of DB, TPC 2 values in the extracts were lower than the TPC value of DB, except for the extract obtained at the highest temperature (E-170). During the SWE extraction, an increment in the TPC content occurred as temperature increases due to the promotion of hydrolysis of lignin/phenolics-carbohydrate complexes, fostering the decomposition of these structures and releasing phenolic acids. Likewise, the neoformation of antioxidant compounds under severe SWE conditions has also been described (Plaza et al . 2010a,b). This neo-formed antioxidant compounds could be also quantified as phenols by the unspecific Folin-Ciocalteu reagent. These compounds are formed through Maillard and/or caramelization reactions, producing 5-HMF and sugar condensation compounds, and exhibit different bioactivities, including antioxidant activity (Trigueros et al ., 2023). On the other hand, the thermal degradation of the phenolic compounds at high temperatures could also occur. Specifically, flavonoids, one of the main phenolic compounds in the beer bagasse, are highly thermo-sensitive. Therefore, the extent of the different phenomenon occurred during SWE, depending on the composition of each natural matrix, will determine the final content and nature of phenolics in the extracts. Thus, the marked increment in the TPC observed at 170 o C could be attributed to the high progress of the hydrolytic phenolic release, compared to the potential degradation ratio, as well as to the neo-formation of higher amounts antioxidant species. The antioxidant capacity, measured throughout the EC 50 values are also shown in Table 2 . This value increased when the temperature rose, thus indicating a decrease in the radical scavenging capacity of the extracts. This decrease in the antioxidant capacity when the temperature rose, despite the promotion of higher phenolic content, can be attributed to the different phenolic profile in each extract with different radical scavenging capacity. The antimicrobial potential of the DB extracts was also studied against the Gram-negative E.Coli bacteria and the Gram positive L. Innocua , which are well-known pathogenic microorganisms responsible for food poisoning. The minimal inhibitory concentration (MIC values) of the extracts with both bacteria were determined and shown in Table 2 . The antibacterial effectiveness increased when the extraction temperature rose, being the gram positive bacteria (Listeria) more sensitive to the extracts. The E-170 MIC value for E.Coli was similar to that found for SWE extract of almond peel (90 mg/ml) obtained at 160 o C (Freitas et al ., 2023). Barbosa-Pereira et al. (2014) also reported the antimicrobial efficiency of the polyphenols from the brewery waste stream against S. Aureus, L. monocytogenes, Salmonella spp . and E.Coli bacteria, while ferulic and caffeic acids and flavonoids being the main responsible for the observed antimicrobial activity. The obtained results indicate that the SWE extracts from beer bagasse are excellent candidates to be used as antioxidants or antilisteria compounds in food preservation or in the pharmaceutical sector. 3.3. Bleaching of the Extraction Residues The extraction residues (R-110, R-130, R-150 and R-170) were bleached to recover the cellulose fraction, as they can be used for different applications in the material developing and pharmaceutical sectors. The bleaching treatment was carried out using a greener bleaching agent than the usual chlorine bleaches, to minimize the environmental impact of the process. Thus, the insoluble fractions were submitted to four successive 1 h cycles with 4% H 2 O 2 solution at pH 12. To evaluate the efficiency of the process, the white index (WI) and the yield of the process was determined in cycle for the different samples (Fig. 3 ). As expected, the application of four successive bleaching cycles significantly decreased yield and increased the WI values, in accordance with the progressive purification of cellulose in each cycle. Table 3 Chemical composition (%wt) of the insoluble fractions subjected to the four bleaching cycles with 4% hydrogen peroxide. Sample Ashes (%) Lignin (%) Protein (%) Cellulose (%) Hemicellulose (%) BR-110-1C 6,96 ± 0,09 a,1 14,69 ± 0,03 a,1 11,3 ± 0,5 a,3 52 ± 2 a,1 41 ± 5 a,1 BR-110-2C 5,9 ± 0,3 b,1 15,9 ± 0,8 a,2 8,2 ± 0,3 b,2 44 ± 2 a,1 33 ± 4 b,1 BR-110-3C 6,20 ± 0,08 b,1 13,88 ± 1,09 a,2 5,1 ± 0,2 c,2 57 ± 5 a,1 30 ± 3 c,1 BR-110-4C 6,2 ± 0,3 b,1 11,5 ± 0,9 b,1 1,9 ± 0,3 d,2 53 ± 4 a,1 26 ± 3 d,1 BR-130-1C 6,5 ± 0,3 b,1 16,2 ± 0,5 ab,2 21 ± 0,6 a,1 62 ± 3 a,1 28 ± 3 a,2 BR-130-2C 7,07 ± 0,07 b,2 17,3 ± 0,4 bc,1 11,3 ± 0,4 b,1 65 ± 4 a,2 31 ± 6 a,1 BR-130-3C 6,5 ± 0,3 b,1 17,97 ± 0,04 c,3 7,7 ± 0,2 c,1 67 ± 3 a,1 22 ± 3 ab,2 BR-130-4C 5,8 ± 0,2 a,1 15,6 ± 0,8 a,2 2,5 ± 0,1 d,1 62 ± 5 a,12 15 ± 2 b,2 BR-150-1C 5,07 ± 0,41 a,2 20,88 ± 0,06 a,3 16,2 ± 0,9 a,2 58 ± 6 a,1 15 ± 2 a,3 BR-150-2C 7,4 ± 0,5 a,2 15,35 ± 0,11 b,23 11,6 ± 0,4 b,1 64 ± 5 a,2 14 ± 2 a,2 BR-150-3C 5,5 ± 1,4 a,1 15 ± 2 b,2 7,3 ± 0,2 c,1 62 ± 5 a,1 - BR-150-4C 4,9 ± 0,7 a,1 14,77 ± 1,02 b,2 2,9 ± 0,7 d,1 71 ± 6 a,2 - BR-170-1C 5,5 ± 0,4 a,2 21,9 ± 0,3 a,4 6,7 ± 0,2 a,4 60 ± 3 a,1 - BR-170-2C 5,7 ± 0,3 a,1 14,4 ± 0,2 b,3 4,8 ± 0,3 b,3 42 ± 2 c,1 - BR-170-3C 5,7 ± 0,4 a,1 11,5 ± 0,2 c,1 2,9 ± 0,3 c,3 44 ± 2 c,2 - BR-170-4C 6,2 ± 0,2 a,1 10,2 ± 0,2 d,1 1,2 ± 0,2 d,3 53 ± 4 b,1 - a,b,c, different letters indicate significant differences(p < 0.05) between samples at the same extraction temperature 1,2,3..: different numbers indicate significant differences (p < 0.05) between samples at the same bleaching cycle The cellulose purification progress was monitored through the analysis of lignin and sugars by means of the NREL method (Sluiter 2005 and 2008). After removal of the water (which includes soluble sugars) and ethanol extractives in the samples (between 13–34%), the acid insoluble lining and hydrolyzed sugars were quantified in the different samples. Glucose was the major component, followed by xylose and arabinose. As established in the NREL method, hemicellulose content was considered as total xylose and arabinose and total glucose was attributed to the cellulose content. The obtained values are given in Table 3 . The hemicellulose content was selectively removed when successive bleaching cycles were applied (p < 0.05) in every sample. This hemicellulosic fraction significantly decreased when using 3–4 cycles in BR-110 and BR-130 samples, and completely disappeared in BR-150 and BR-170 samples, after two and one bleaching cycles, respectively. Nevertheless, no significant increase in the cellulose content of the samples occurred during the successive cycles, except for R-170, in which the cellulose content significantly decreased with successive cycles. This suggests that cellulose is progressively degraded through the bleaching cycles with hydrogen peroxide. In fact when referring the cellulose content per mass unit of initial DB, a progressive decrease was observed, ranging from 16 g cellulose/ 100 g DB in the non-bleached residues to 7–13 g cellulose/g DB in the fourth bleaching cycle of the different samples. Degradation of cellulose by the oxidative action of hydrogen peroxide has been reported by other authors (Vismara et al. , 2009) through free radical mechanisms forming alpha hydroxyalkyl radicals and subsequent chain scission. This process is largely affected by the substrate composition and the presence of catalysers or inhibitors of the reaction. Therefore, the use of hydrogen peroxide as bleaching agent of BD cellulosic fractions did not yield proper results, since an important part of cellulose is degraded during the delignification process. In general, the acid insoluble lignin content decreased when successive cycles were applied, especially after the fourth bleaching cycle. Nevertheless, as commented on above, it has to be taken into account that these values were affected by the protein content of the samples. As can be observed, the protein is progressively removed by successive bleaching cycles, especially in sample BR-170 where higher protein solubilization occurred in the SWE step. The TGA and DTGA curves of the insoluble and bleached residues are shown in Fig. 4 . All samples exhibited a first weight loss step 25 and 125 o C corresponding to the loss of bonded water, and the typical degradation steps of lignocellulosic residues, previously commented. The TGA curves of the bleached fractions showed the expected differences in the thermal behavior with respect the non-bleached samples, related with the compositional changes that occurred in the bleaching step. The partial removal of hemicellulose during the bleaching cycles are reflected on the TGA curves where the double peak in DTGA curves of polysaccharides became a single peak, mainly attributed to cellulose degradation, and the temperature of the maximum degradation rate increased from 280 to 300 o C in BR-110 and BR-130 samples. Nevertheless, no relevant changes in the cellulose purification degree can be deduced from the scarce increase in the weight loss step attributed to this polymer, remaining other compounds whose degradation overlapped with the cellulose degradation, as also observed in the analyzed composition. In sample BR-170, very few changes in the TGA curve were observed after the first bleaching cycle, coherently con the small composition changes reflected in Table 3 . Therefore, the successive cycles reduced the bleaching mass yield but did not significantly promote cellulose purification, but its degradation. In the other cases the bleaching cycles promoted the removal of hemicellulose, but also did not result in higher cellulose purity due to its partial degradation. The cellulose degradation products probably contributed to the increase in the final residual mass obtained for most of bleached samples. So, the oxidative process applied with hydrogen peroxide in an alkaline medium seems to partially degrade cellulose generating other compounds and reducing the process yield. 4. Conclusions Despite being rich in polysaccharides, proteins and phenolic compounds, BSG is still underutilized in the food, materials or pharmaceutical sectors. New sustainable approaches, such as the use of subcritical water extraction could be a possible technology to fractionate this waste obtaining bioactive agents, proteins and cellulose fractions from the beer bagasse while contributing to the circular economy. The use of subcritical water treatment of defatted beer bagasse allowed to obtain bioactive aqueous extracts (7–41% mass yield of the defatted BSG) with radical scavenging capacity and antimicrobial activities. The highest extraction temperature (170 o C) gave rise to the highest extract yield while provide the extracts with greater polyphenol content and antibacterial effect, but with lower DPPH radical scavenging capacity. In contrast, the extraction at 150°C was optimal for producing extracts (35% mass yield) with the greatest radical scavenging capacity. Likewise, the extract obtained at 170 o C was the richest in protein, which could be separated by precipitation from the liquid extract. Beer bagasse can be considered as a relatively poor source of cellulose in comparison with other agro-industrial residues. Considering the yield of the different process steps to purify cellulose, (19, 17, 14 and 13% for samples treated a 110, 130, 150 and 170 o C) and the similar degree of cellulose purity obtained after the 4 bleaching cycles (50, 60, 70 and 50%, respectively for samples treated a 110, 130, 150 and 170 o C ), the best treatment to obtain cellulose would be the extraction at 150 o C, followed by two bleaching cycles with hydrogen peroxide. These conditions allows for the removal of most of the hemicellulose and led to a cellulose purity degree without significant differences with respect to that obtained in the successive cycles. 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08:42:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4610399/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4610399/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61073727,"identity":"a9128e67-26df-4626-b4d2-a13608d58c46","added_by":"auto","created_at":"2024-07-25 09:01:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":323990,"visible":true,"origin":"","legend":"\u003cp\u003eFlow chart of the beer bagasse fractionation and the process step yields (PSY: g outgoing solids. 100 g\u003csup\u003e−1\u003c/sup\u003e of incoming dried material) for the defatting step and SWE carried out at the different temperatures.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4610399/v1/0d071acd54bdfb50364bfcfe.png"},{"id":61073728,"identity":"93a80dc3-e1d2-4416-9290-d254882fa872","added_by":"auto","created_at":"2024-07-25 09:01:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98173,"visible":true,"origin":"","legend":"\u003cp\u003eTGA (a) and DTGA (b) of the defatted beer bagasse (DB), the active extracts (E) and the insoluble fractions (R) obtained from SWE at the different temperatures.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4610399/v1/cf05f16eb9d1781f2ac08cc1.png"},{"id":61073729,"identity":"f92ff975-4d6b-47df-b56d-48e4fc96cb6a","added_by":"auto","created_at":"2024-07-25 09:01:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":117588,"visible":true,"origin":"","legend":"\u003cp\u003eVisual appearance, white index (WI ) (full lines) and Yield (%) (dashed lines) of the insoluble residues submitted to different bleaching cycles.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4610399/v1/465f8f1cab762f6f979afce6.png"},{"id":61073730,"identity":"e2e29b5e-b4e1-4bf0-89e3-6b74d3d1d0b2","added_by":"auto","created_at":"2024-07-25 09:01:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1084061,"visible":true,"origin":"","legend":"\u003cp\u003eTGA (a) and DTGA (b) curves of the insoluble residues (R) and the bleached residues (BR) obtained from SWE at 110ºC (a), 130°C (b), 150ºC (c) and 170ºC (d) submitted to different bleaching cycles.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4610399/v1/045f7c3d73eec924f258e404.png"},{"id":62235917,"identity":"eea0e6a7-ae19-41f6-897f-07b9f7acc9fe","added_by":"auto","created_at":"2024-08-12 01:05:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2149334,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4610399/v1/9177b397-740b-4633-95c4-00974177d7df.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003ePhenolic and Cellulose-Rich Fractions from Subcritical Water Treated Beer Bagasse\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlobal beer consumption has experienced a notable increase over the last 50 years, reaching 150\u0026nbsp;billion liters per year, surpassing wine consumption by seven (Rodriguez \u003cem\u003eet al\u003c/em\u003e., 2023). It is estimated that by 2025, it will be worth \u003cspan\u003e$\u003c/span\u003e502.9\u0026nbsp;billion and have an annual growth rate of 19.9% in response to growing demand and the wide variety of new styles and flavors (Pokrivč\u0026aacute;k \u003cem\u003eet al\u003c/em\u003e., 2019). During the beer brewing process, three types of waste are generated: spent beer grains (SBG or bagasse), spent hops and spent brewer's yeast. It is estimated that between 14 and 20 kg of bagasse, 0.2 and 0.4 kg of hops, and 1.5 and 3 kg of residual yeast are generated for every 100 liters of beer produced (Rodriguez \u003cem\u003eet al.\u003c/em\u003e, 2023). Consequently, the beer bagasse constitutes approximately 85% of the total byproducts generated (Puligundla \u0026amp; Mok, 2021). This residue presents outstanding nutritional properties that make it a valuable source of high-value compounds and includes hulls of barley malt grains, parts of the pericarp, and layers of the barley seed coat. It contains fibers (soluble and insoluble), proteins, lipids and phenolic compounds, which can be free or bound to dietary fiber (Bonifacio-Lopes \u003cem\u003eet al.\u003c/em\u003e, 2022). Among phenolic acids, ferulic, p-coumaric and caffeic predominate in a bound state (Ikram \u003cem\u003eet al.\u003c/em\u003e, 2017). The chemical composition of BSG depends on the type of barley, the time of harvest, and the malting and mashing conditions, being rich in cellulose (12\u0026ndash;25% in dry basis), hemicellulose (20\u0026ndash;25%), lignin (10\u0026ndash;28%), starch (2\u0026ndash;7%), proteins (20%), lipids (6\u0026ndash;10%) and minerals (2\u0026ndash;5%) (Lynch \u003cem\u003eet al.\u003c/em\u003e, 2016), together with polyphenols (0.7-2%). So, the extraction of different fractions from beer bagasse could be of great interest to obtain different valued biomolecules to be use for the pharmaceutical, packaging or food industry.\u003c/p\u003e \u003cp\u003eAmong the various new green extraction and separation technologies, ultrasound-assisted, microwave-assisted, enzyme-assisted and subcritical and supercritical fluid extraction has been recently used, surpassing conventional methods such as maceration, infusion, and Soxhlet extraction (Messinese \u003cem\u003eet al\u003c/em\u003e., 2023; Panzella \u003cem\u003eet al.\u003c/em\u003e, 2020). In subcritical water extraction (SWE), the physicochemical properties of water, such as the relative dielectric constant and polarity, decrease significantly with increasing temperature so that, under subcritical conditions, water can dissolve polar and non-polar compounds (Galamba \u003cem\u003eet al\u003c/em\u003e., 2019). Furthermore, viscosity and surface tension decrease with increasing temperature, resulting in higher extraction efficiency (Wiboonsirikul \u0026amp; Adachi, 2008; Zakaria \u0026amp; Kamal, 2016). Additionally, water is an economical, non-toxic, non-hazardous, and safe solvent that works as a solvent and catalyst to take advantage of and transform biomass into bioactive products (Abdelmoez \u003cem\u003eet al.\u003c/em\u003e, 2007; Sarkar \u003cem\u003eet al.\u003c/em\u003e, 2022). In the last few years, this technology have shown an increasing interest in the scientific community (from 300 articles published in 1995\u0026ndash;2000 to 5,000 articles in the 2021\u0026ndash;2023 period), thus showing SWE is a promising technology for extracting target compounds such as proteins, polyphenols or phenolic acids from different sources. SWE has been applied in a variety of agro-industrial wastes such as winery, tea, shellfish and tobacco wastes, carrot leaves, cotton flowers, peels (chestnut, almond, potato, citrus, mandarin, mango, kiwifruit), seeds (papaya), pomace (apple, pomegranate, kiwifruit), among others (Majeed \u003cem\u003eet al.\u003c/em\u003e, 2024).\u003c/p\u003e \u003cp\u003eThese studies showed that SWE is a sustainable and energy-effective way of biomass processing because it promotes selective extraction and reactions that convert waste materials and by-products into valuable products. These hold great potential for the creation of novel food products and waste reduction while enhancing the overall sustainability of food production.\u003c/p\u003e \u003cp\u003eThe extraction of different compounds from BSG have been studied by few researchers for different purposes, which were mainly focused on the recovery of proteins and polyphenols. Thus, Devnani \u003cem\u003eet al\u003c/em\u003e (2023) reviewed the effect of extraction techniques and conditions on the composition, physicochemical, and techno-functional properties of the obtained BSG protein extracts. Guido and Moreira (2017) focused on the current extraction methods used to obtain phenolic compounds from BSG, ranging from more traditional (the conventional solid\u0026ndash;liquid extractions employing organic solvents, alkaline, and enzymatic reactions) to advanced techniques such as pressurized fluid, supercritical and microwave-assisted and ultrasound-assisted extractions. Camacho-N\u0026uacute;\u0026ntilde;ez \u003cem\u003eet al\u003c/em\u003e.,(2023) addressed the extraction of cellulose from BSG by means of alkaline hydrolysis and bleaching reactions, and its further conversion into cellulose acetate for packaging applications. Alonso-Ria\u0026ntilde;o et al., (2021) used SWE as hydrolytic medium to recover proteins and specific polyphenols from craft BSG for pharmaceutical and cosmetic applications. They found 185 \u003csup\u003eo\u003c/sup\u003eC to be the best temperature to maximize the extraction of protein and aldehyde phenolic compounds (vanillin, syringic and protocatechuic aldehyde) while lower temperatures (160 \u003csup\u003eo\u003c/sup\u003eC ) promoted the extraction of hydroxycinnamic acids, such as ferulic acid and p-coumaric. Nevertheless, no studies using SWE for cellulose extraction at different temperatures have been found neither the characterization of the obtained phenolic rich fraction in terms of phenolic content, antioxidant and antimicrobial properties, to the best of our knowledge.\u003c/p\u003e \u003cp\u003eThe main objective of the present work is to fractionate the beer bagasse residue by subcritical water extraction (SWE) at different temperatures (ranging between 110\u0026ndash;170 \u003csup\u003eo\u003c/sup\u003eC C), analyzing the compositional and functional properties of soluble (extracts) and insoluble (residues), thus contributing to valorize the BGS waste in the context of a circular economy. Thus, soluble extracts and cellulose-rich insoluble fractions were obtained as a function of the temperature. The insoluble fraction was also submitted to a green bleaching process to purify cellulose.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003ePetroleum ether (40\u0026ndash;60\u0026deg;C bp), phosphorous pentoxide (P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e, 98.2%), sodium hydroxide (NaOH), glucose, and arabinose were purchased from Sigma-Aldrich (USA). D (+)-Xylose was supplied by Merck KGaA (Darmstadt, Germany). Ethanol (98%), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 30%), sulphuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 98%), and sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, 99.5%) were obtained from Panreac Quimica S.L.U (Castellar del Vall\u0026eacute;s, Barcelona, Spain).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Residue preparation\u003c/h2\u003e \u003cp\u003eBeer bagasse was supplied by a brewery factory located in Valencia and dried at 60 \u003csup\u003eo\u003c/sup\u003eC \u0026plusmn; 2\u0026deg;C for five days in a forced-air oven (J.P Selecta, s. a., Barcelona, Spain). After that, it was milled using a Thermomix (Model TM6 Vorwerk Espa\u0026ntilde;a M.S.L S.C) and sieved to obtain particles under 0.71 mm and cold stored.\u003c/p\u003e \u003cp\u003eThe defatting process was performed under reflux with petroleum ether for 8 hours at 60 \u003csup\u003eo\u003c/sup\u003eC, with stirring using a 1:4 ratio of dry sample to solvent. Then, it was allowed to settle, decanted and filtered with a 125 mm paper filter, washing with pure solvent, to separate the defatted residue that was left to solvent evaporation at room temperature in an extractor hood for 16 h, when constant weight was reached. The liquid phase was dried by adding anhydrous sodium sulfate and then left for 48 h and filtered under vacuum. The oil was recovered by evaporating petroleum ether in the vacuum rotary evaporator (Rotary Evaporators, Heidolph Instruments GmbH \u0026amp; Co. KG, Walpersdorfer, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Subcritical Water Extraction\u003c/h2\u003e \u003cp\u003eSubcritical extraction (SWE) of defatted bagasse was carried out with a ratio solids-water of 1:8, using a pressure reactor (Model 1-TAP-CE, 5 L capacity, Amar Equipment PVT. LTD, Mumbai, India). The temperature-pression conditions used were 110\u0026deg;C -1 bar, 130\u0026deg;C -2 bar, 150\u0026deg;C-4.5 bar, and 170\u0026deg;C-8 bar, applying 50 rpm in all cases, for 30 minutes. After each extraction step, the defatted sample dispersions were filtered through a filter with pore size less than 0.5 mm (Filterlab, Barcelona, Spain). Thus, two fractions were obtained from each SWE process: one insoluble residue (R) and the soluble extracts (E). The extracts, named E-110, E-130, E-150, and E-170, were lyophilized at -60\u0026deg;C and 0.8 mbar and stored in desiccators (P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e, 0% relative humidity) at 4 ◦C. The respective mass yields of extracted solids and solid residues were determined with respect to the initial defatted bagasse. To determine the extracts yield, three aliquot samples of the liquid extracts were dried at 105\u0026ordm;C until a constant weight to determine water:solid ratio and the total solids extracted was calculated by multiplying by the total water mass in the reactor. To determine the yield in the extraction residues (R-110, R-130, R-150, and R-170), these were washed with distilled water, filtered, and dried at 40 ◦C for 48 hours to determine their weight yield; then these were stored at 4 ◦C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Bleaching process\u003c/h2\u003e \u003cp\u003eThe insoluble fractions obtained from SWEs were bleached following the method described by Freitas \u003cem\u003eet al\u003c/em\u003e (2023), using hydrogen peroxide as bleaching agent. 4% (wt) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution was prepared while pH was fitted to 12 (using NaOH). The lignocellulosic residues were treated with this bleaching solution using a solvent-solid ratio of 30:1, at 40\u0026deg;C for 1 h, in four consecutive cycles, filtering and washing the sample with distilled water after each cycle.\u003c/p\u003e \u003cp\u003eAfter the four cycles, the cellulose fractions were filtered and washed with abundant deionized water to remove residues of the bleaching solution and then dried at 50\u0026ordm;C overnight. The bleached samples were labeled as BR-110-1C to BR-110-4C, BR-130-1C to BR-130-4C, BR-150-1C to BR-150-4C and BR-170-1C to BR-170-4C. The mass yield (%) and whiteness index (WI) were determined in all samples using equations (\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and (\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) at each bleaching cycle to control the sample development. The color coordinates L* (lightness), a* (red green), and b* (yellowish-blue) of each bleached fraction were obtained with a CM-3600d spectro-colorimeter (Minolta Co., Tokyo, Japan), using a D65 illuminant and 10\u0026ordm; observed.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$Yield= \\frac{Weight of bleached cellulose}{Weight of material before bleaching}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$WI=100- \\sqrt{{(100-\\text{L}\\text{*})}^{2}+{\\text{a}\\text{*}}^{2}+{\\text{b}\\text{*}}^{2}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Physico-chemical analysis of beer bagasse, soluble and insoluble fractions.\u003c/h2\u003e \u003cp\u003eThe amount of protein in raw beer bagasse BB, extracts (E-110, E-130, E-150, E-170), and extraction residues (R-110, R-130, R-150, R-170) was measured using the Dumas combustion method (Leco, St. Joseph, MI, USA) by duplicate. A conversion factor 4.74 was applied to calculate protein content from total nitrogen (Krul, E.S, 2019)\u003c/p\u003e \u003cp\u003eIn the same way, all samples were subjected to thermogravimetric analysis (TGA). A TGA/SDTA 851e analyzer (Mettler Toledo, Schwarzenbach, Switzerland) working under nitrogen flow (20 mL/min) was used to obtain the weight loss vs. temperature curves (TGA) and the first derivatives (DTGA). Samples (3\u0026ndash;5 mg) previously conditioned sample in P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e were placed in 70 \u0026micro;L alumina crucible and heated from 25 to 900\u0026deg;C at 10 K/min. Three replicates per sample were obtained.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Analysis of structural components in the Insoluble fraction\u003c/h2\u003e \u003cp\u003eCellulose, hemicellulose, acid-insoluble lignin content of defatted beer bagasse (DB), insoluble fractions (R), and bleached samples (BR) were analyzed according by the method of National Renewable Energy Laboratory (NREL/TP-510-42618\u0026mdash;2008) (Sluiter, 2025). The test consisted of a two-stage hydrolysis with 72% sulfuric acid, of which one results in a soluble fraction in which the sugar content (glucose, xylose, and arabinose) was measured by high-resolution liquid chromatography. (HPLC, Agilent Technologies, model 1120 Compact LC, Waldbronn, Germany) and a RezexTM RCM-Monosaccharide Ca2\u0026thinsp;+\u0026thinsp;column (300 \u0026times; 7.8 mm). On the other hand, the insoluble fraction was used to quantify the acid insoluble lignin content by thermogravimetric method. The cellulose content was obtained from the quantified glucose and hemicellulose from the sum of quantified xylose and arabinose, as described by Sluiter (2025).\u003c/p\u003e \u003cp\u003eBefore hydrolysis, the raw material (DB) and the insoluble fractions (R) were subjected to the extractives determination the using the standard NREL method (NREL/TP-510-42619\u0026mdash;2008) (Sluiter, 2025). This procedure was performed using a Soxhlet set-up, which consists of two stages: a first extraction with water for 6 h, followed by a second extraction with ethanol at 60\u0026deg;C for 6 h.\u003c/p\u003e \u003cp\u003eThe thermal stability analysis was carried out on all samples by triplicate using the TGA 1 Stare System analyzer, (Mettler Toledo, Greifensee, Switzerland), previously conditioned in phosphorous pentoxide (P2O5) for two weeks. The analysis was conducted from 25 to 900\u0026ordm;C at 10 K/min with a nitrogen flow of 10 ml per minute. In addition, the ash content was analyzed using the UNE-EN 14775 standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Total phenolic content, antioxidant and antibacterial properties of soluble fraction.\u003c/h2\u003e \u003cp\u003eThe total phenol content was determined using the Folin Ciocalteu method. Briefly, 0.5 ml of each extract was mixed with 6 ml of distilled water, and 0.5 ml of Folin reagent (2N) was added. After one minute, 1.5 ml of 20% Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e solution and distilled water were added to a volume of 10 ml. After stirring, they were kept in the dark for 2 hours. The absorbance at 725 nm was then measured in triplicate using a UV-Vis spectrophotometer (Evolution 201, Thermo Scientific). Gallic acid was used as a standard, and the results were expressed as mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e gallic acid equivalents (GAE) using a standard curve (R \u003csub\u003e2\u003c/sub\u003e = 0.9991) of gallic acid (2\u0026ndash;20 mg.L \u0026minus;\u0026thinsp;1 ). The TPC value of milled raw material was also determined by previously extracting phenols with methanol at a solid to liquid ratio of 1:50 at room temperature and dark for 24 h under constant stirring.\u003c/p\u003e \u003cp\u003eThe antiradical capacity of the extracts was determined using the 2,2-diphenyl-1-pikryl-hydrazyl (DPPH) free radical method. For each extract, a solution of DPPH in methanol at a concentration of 6.22 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e mM was mixed with different extract concentrations until reaching a final volume of 4 ml. The resulting solutions were kept in the dark at room temperature for 12 h, after which the absorbance at 515 nm was measured. The initial and final concentrations of DPPH in the reaction medium were calculated from a calibration curve fitted by linear regression (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9992). The antiradical activity was evaluated by the EC\u003csub\u003e50\u003c/sub\u003e parameter, which represents the amount of antioxidants necessary to reduce the initial concentration of DPPH by 50%, when the stability of the reaction is reached. This value was expressed as mg of dried extract per mg of DPPH and also, in mg defatted beer bagasse per mg of DPPH for comparison purposes. EC\u003csub\u003e50\u003c/sub\u003e values were determined from graphs showing the percentage of [DPPH] remaining as a function of the amount of solid extract relative to the amount of DPPH, using the Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({\\%\\left[DPPH\\right]}_{remaining}=\\frac{{\\left[DPPH\\right]}_{t}}{{\\left[DPPH\\right]}_{t=0}}x 100\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eRegarding the antimicrobial capacity of the extracts. the minimum inhibitory concentration (MIC) of the different extracts was determined for two bacteria: the Gram-positive bacterium \u003cem\u003eListeria innocua\u003c/em\u003e and the Gram-negative \u003cem\u003eEscherichia coli\u003c/em\u003e. This analysis followed the method outlined by Freitas \u003cem\u003eet al\u003c/em\u003e., (2023). Standard 96-well microtiter plates with a total volume of 200 \u0026micro;L were employed for this analysis.\u003c/p\u003e \u003cp\u003eFor both bacterial strains, stored at -20\u0026ordm;C, a stock solution was prepared by transferring bacterial amounts using an inoculation loop to a volume of 10 mL of TSB and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 24 hours. Subsequently, 10 \u0026micro;L of the stock solution was taken and transferred to a tube containing 10 mL of TSB to prepare the corresponding work solution with a concentration of 10\u003csup\u003e5\u003c/sup\u003e CFU.mL-1. This concentration was confirmed through serial dilution and counting. For each bacterium, 100 \u0026micro;L of the bacterial solution with an initial concentration of 10\u003csup\u003e5\u003c/sup\u003e CFU.mL-1 was added to each well. Thereafter, different volumes of each extract solution, with 200 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, were added in each well while the final volume was adjusted to 200 \u0026micro;L with TSB to obtain different extract dilutions. The plates were then incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 24 hours. Afterward, 100 \u0026micro;L from each well was transferred to TSA plates and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC C for 24 hours for final counting. The MIC for each extract was determined as the lowest extract concentration at which no bacterial growth was observed on the TSA plate. This analysis was performed in duplicate.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe Statgraphics Centurion XVII-64 program (Manugistics Corp., Rockville, Md.) was used to perform statistical analyses using an analysis of variance (ANOVA) with a confidence level of 95%. The Fisher test was applied to detect possible differences in treatment responses, using a critical value of 5% to determine significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Yields and composition of the different SWE fractions\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the flow chart of the brewer spent grain (BSG) or beer bagasse fractionation throughout the SWE, giving rise to soluble extracts (E) and insoluble residues (R) at each temperature. The latter were submitted to a bleaching step to purify cellulose while the extracts were freeze-dried to obtain extract powders. Images of the different obtained products, together with the obtained mass yield of each process step are also shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Previously to the SWE, a defatting step of the BSG yielded around 8% oil from the dried bagasse, being this value in agreement with that reported in the literature (Faulds \u003cem\u003eet al\u003c/em\u003e., 2008; Del Rio \u003cem\u003eet al.\u003c/em\u003e, 2013). The main beer bagasse lipid compounds has been reported, these being triglycerides (55\u0026ndash;67%), free fatty acids (18\u0026ndash;30%), such as palmitic, oleic and linoleic acids and free steroids (5%), such as sitosterol and campesterol. These lipid compounds have a wide range of nutraceutical, pharmaceutical and cosmetic applications (Del Rio \u003cem\u003eet al\u003c/em\u003e., 2013).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe values of mass yields of solid extracts (E-110, E-130, E-150 and E-170) and dried residues (R-110, R-130, R-150 and R-170) of the SWE process performed at different temperatures, shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, indicate that the extraction yield increased (from 7\u0026ndash;41%) when the extraction temperature rose from 110\u0026deg;C to 170\u0026deg;C. This is mainly explained by the changes in the water solvent properties when the temperature increased, which reduces the strength of hydrogen bonds and leads to an important reduction in dielectric constant, this becoming closer to the dielectric constant value of some organic solvents, such as methanol (ɛ = 33) or ethanol (ɛ = 25) (Carr \u003cem\u003eet al.\u003c/em\u003e, 2011; He \u003cem\u003eet al.\u003c/em\u003e, 2012). The sum of both yields (extract and residue) at a given temperature closed the mass balance, thus indicating a low mineralisation degree of the organic matter present at the processing conditions used.\u003c/p\u003e \u003cp\u003eThe TGA curves of the defatted bagasse (DB), extracts and residues obtained after SWE at the different temperatures is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, together with de derivative curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The DB presented three main degradation steps: the first mainly corresponding to the loss of bound water; a second step associated with the degradation of polysaccharides with different thermostability such as hemicelluloses (150\u0026ndash;350 \u003csup\u003eo\u003c/sup\u003eC ), celluloses (275\u0026ndash;350 \u003csup\u003eo\u003c/sup\u003eC ) and a part of lignin (160\u0026ndash;900 \u003csup\u003eo\u003c/sup\u003eC ), and the third one, related to the degradation of residual lignin and secondary metabolites from the previously thermo-degraded compounds, as previously described by other authors for lignocellulosic biomass (Freitas \u003cem\u003eet al.\u003c/em\u003e, 2023; Carichino \u003cem\u003eet al.\u003c/em\u003e, 2023). The major weight losses took place between 225 and 625 \u003csup\u003eo\u003c/sup\u003eC (80%), in line with the lignocellulosic nature of this residue, in agreement with the results obtained by other authors (Ortiz \u003cem\u003eet al.\u003c/em\u003e, 2020; Carichino \u003cem\u003eet al.\u003c/em\u003e 2023). Very similar TGA patterns were obtained for every lignocellulosic residue. Nevertheless, it is remarkable that the highest extraction temperatures (150 and 170 \u003csup\u003eo\u003c/sup\u003eC) gave rise to the samples with the highest peak temperatures (temperature of the maximum degradation rate), which indicates that these were the most enriched in cellulose that shows peak temperature between 330\u0026ndash;350 \u003csup\u003eo\u003c/sup\u003eC (Zhang \u003cem\u003eet al\u003c/em\u003e., 2012). In contrast, the TGA and DTGA curves of the extracts revealed a more complex compositional profile, exhibiting several thermodegradation steps. The extract obtained at the highest temperature showed a higher proportion of compounds that degrade at higher temperature according to a greater extraction of polymeric components, such as hemicellulose or lignin. A higher final mass residue was also observed in comparison with the untreated sample (defatted bagasse), which can be due to the extract enrichment in minerals or formation of degraded organic matter from the soluble compounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSome compositional differences in the extracts and residues obtained at each temperature can be observed in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\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 defatted beer bagasse (DB) and insoluble fractions after SWE at different temperatures.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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=\"left\" 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 \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExtractive\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLignin*\u003c/p\u003e \u003cp\u003e(% )\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCellulose\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHemicellulose\u003c/p\u003e \u003cp\u003e(%)\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\u003eDB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13,1\u0026thinsp;\u0026plusmn;\u0026thinsp;0,5 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3,71\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01 ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e9,5\u0026thinsp;\u0026plusmn;\u0026thinsp;1,6 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17,9\u0026thinsp;\u0026plusmn;\u0026thinsp;0,6 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eR-110\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20,9\u0026thinsp;\u0026plusmn;\u0026thinsp;1,5 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3,12\u0026thinsp;\u0026plusmn;\u0026thinsp;0,11 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e9,1\u0026thinsp;\u0026plusmn;\u0026thinsp;0,4 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eR-130\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21,79\u0026thinsp;\u0026plusmn;\u0026thinsp;0,05 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26,2\u0026thinsp;\u0026plusmn;\u0026thinsp;0,2 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2,79\u0026thinsp;\u0026plusmn;\u0026thinsp;0,04 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11,56\u0026thinsp;\u0026plusmn;\u0026thinsp;0,08 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14,9\u0026thinsp;\u0026plusmn;\u0026thinsp;1,2 ᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eR-150\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e34,5\u0026thinsp;\u0026plusmn;\u0026thinsp;0,9 ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2,27\u0026thinsp;\u0026plusmn;\u0026thinsp;0,11 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14,6\u0026thinsp;\u0026plusmn;\u0026thinsp;0,5 ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7,8\u0026thinsp;\u0026plusmn;\u0026thinsp;1,2 ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eR-170\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;3 ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35,7\u0026thinsp;\u0026plusmn;\u0026thinsp;0,2 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3,1\u0026thinsp;\u0026plusmn;\u0026thinsp;0,2 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e29,9\u0026thinsp;\u0026plusmn;\u0026thinsp;0,3 ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;3 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2,01\u0026thinsp;\u0026plusmn;\u0026thinsp;0,08 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea,b,c\u003c/sup\u003e..Different superscript letters (a,b,c) in the same column indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e* acid insoluble lignin\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the total (water and ethanol) extractive content, protein, ashes, cellulose, hemicellulose and acid insoluble lignin contents of the DB can be observed, together the values obtained for the different SWE solid residues. The obtained values for raw brewer\u0026acute;s spent grain were within the range previously reported (around 16\u0026ndash;22% for cellulose, 24\u0026ndash;28% hemicellulose and 9\u0026ndash;27% total lignin) (Verni \u003cem\u003eet al.\u003c/em\u003e, 2020; Alonso-Ria\u0026ntilde;o \u003cem\u003eet al.\u003c/em\u003e, 2023; Qazanfarzadeh \u003cem\u003eet al\u003c/em\u003e. 2023). In the extraction residues, the hemicellulose content was very low at temperatures greater than 150 \u003csup\u003eo\u003c/sup\u003eC, in accordance with the selective dissolution of hemicellulose under the subcritical water conditions (Cocero \u003cem\u003eet al.\u003c/em\u003e, 2018; Ruthes \u003cem\u003eet al\u003c/em\u003e., 2017). Thus, the hemicellulose started to be removed from the beer bagasse matrix when using temperatures greater than 130 \u003csup\u003eo\u003c/sup\u003eC, reaching very low values at 170 \u003csup\u003eo\u003c/sup\u003eC (2%). At these temperatures, the lignin content significantly increased, which confirmed that this fraction of the biomass was not released under SWE. as it has been previously observed by other authors working with BSG (Alonso-Ria\u0026ntilde;o \u003cem\u003eet al\u003c/em\u003e, 2023). On the other hand, the increment in the cellulose significantly increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the residues treated at the highest temperature (R-170) in comparison with the untreated DB.\u003c/p\u003e \u003cp\u003eThe insoluble-acid lignin in the solid residues accounted for the 87, 101, 99 and 160% of the total lignin in the raw material for 110, 130, 150 and 170\u0026ordm;C, respectively. Thus, the obtained lignin values are surely overestimated as the outcome of such gravimetric analysis is highly disturbed by the presence of non-lignin acid-insoluble material, e.g. proteins (Erven \u003cem\u003eet al\u003c/em\u003e., 2017). The corrected lignin (calculated by substracting the protein content) was not given because in most cases, negatives values were obtained. According to Alonso-Ria\u0026ntilde;o \u003cem\u003eet al\u003c/em\u003e. (2023), changes in the lignin structure took place during the SWE such as condensation reactions and structural alterations.\u003c/p\u003e \u003cp\u003eIn both extract and residue fractions, the greater mass loss in TGA curves was observed for the temperature range of 200\u0026ndash;700 \u003csup\u003eo\u003c/sup\u003eC, where the lignin is mainly degraded, in line with the formation of secondary metabolites from the previously thermo-degraded compounds. This thermal degradation behaviour agreed with that found in the literature for other lignocellulosic residues (Freitas \u003cem\u003eet al\u003c/em\u003e., 2023).\u003c/p\u003e \u003cp\u003eSo, the application of SWE led to a selective fractionation of DB, giving rise to aqueous extracts richer in different compounds of lower molecular (sugars, phenolic compounds and minerals) and polysaccharide and lignin-rich insoluble residues.\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\u003eTotal phenolic content (TPC), antioxidant activity (EC50), protein content and ashes of the aqueous extracts (E) obtained from SWE process at different temperatures. (mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\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\u003eE-110\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE-130\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eE-150\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eE-170\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e% Protein (db)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15,1\u0026thinsp;\u0026plusmn;\u0026thinsp;0,1 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16,6\u0026thinsp;\u0026plusmn;\u0026thinsp;0,2 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22,4\u0026thinsp;\u0026plusmn;\u0026thinsp;1,2 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28,7\u0026thinsp;\u0026plusmn;\u0026thinsp;0,6 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e%Ashes (db)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,54\u0026thinsp;\u0026plusmn;\u0026thinsp;0,06 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,5\u0026thinsp;\u0026plusmn;\u0026thinsp;0,1 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,20\u0026thinsp;\u0026plusmn;\u0026thinsp;0,04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,46\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPC\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg GAE/g extract)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16,8\u0026thinsp;\u0026plusmn;\u0026thinsp;0,1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17,91\u0026thinsp;\u0026plusmn;\u0026thinsp;0,07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e59,1\u0026thinsp;\u0026plusmn;\u0026thinsp;0,2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPC\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg GAE/g DB)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,27\u0026thinsp;\u0026plusmn;\u0026thinsp;0,08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3,2\u0026thinsp;\u0026plusmn;\u0026thinsp;0,3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6,34\u0026thinsp;\u0026plusmn;\u0026thinsp;0,02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24,18\u0026thinsp;\u0026plusmn;\u0026thinsp;0,08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg extract/mg DPPH)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e71,1\u0026thinsp;\u0026plusmn;\u0026thinsp;0,4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMIC (mg/ml)\u003c/p\u003e \u003cp\u003eagainst \u003cem\u003eL.innocua\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e264\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMIC (mg/ml)\u003c/p\u003e \u003cp\u003eagainst \u003cem\u003eE. Coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e140\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\u003ea,b,c\u0026hellip;different superscripts in the same row indicates significant differences among extracts (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e \u003cp\u003eThe ash content of the DB, extracts and residues (shown in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) showed that minerals were mainly present in the insoluble residues, whereas small amounts were released to the extracts. The value obtained per DB was in the range of the ash content reported by other authors for beer spent grain (2\u0026ndash;5 g/100 g dry DB), being the most abundant constituents phosphorous, magnesium, calcium and potassium (Ortiz \u003cem\u003eet al.\u003c/em\u003e, 2020).\u003c/p\u003e \u003cp\u003eIn Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the protein content of DB is also shown (around 22%), being this value in the range of previously reported values for beer bagasse (Rodriguez \u003cem\u003eet al.\u003c/em\u003e, 2023; Alonso-Ria\u0026ntilde;o \u003cem\u003eet al.\u003c/em\u003e,2021) considering a fat-free basis. The partition of the protein content during the SWE gave rise to greater content in the insoluble residues, thus suggesting a low solubility of the bagasse proteins under the used water subcritical conditions, especially at the lowest SWE temperatures. At 110 \u003csup\u003eo\u003c/sup\u003eC, the 95% of the total protein remained in the insoluble residue, this percentage decreasing to around 53% at 170 \u003csup\u003eo\u003c/sup\u003eC. These proteins are extracted and/or hydrolysed during the thermal treatment, leading to peptide chains of different sizes or free amino acids or even amino acid decomposition, especially at high temperatures, producing different carboxylic acids and other nitrogen containing compounds such as ethanolamine (Trigueros \u003cem\u003eet al.\u003c/em\u003e, 2023; Rogalinski \u003cem\u003eet al\u003c/em\u003e, 2005). Therefore, SWE treatment of beer bagasse can be considered as an efficient extraction method to recover the protein fraction of the BSG generated in the beer industry, the maximum recovery of solubilised protein in the SWE extracts being of 47% at 170 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Functional properties of the SWE extracts: Antioxidant and Antibacterial properties.\u003c/h2\u003e \u003cp\u003eIn Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the Total phenolic content (TPC) and antioxidant activity of the aqueous extracts obtained from SWE process at different temperatures is displayed. The antioxidant activity of the different extracts was determined through the total phenolic content by the Folin\u0026ndash;Ciocalteu method and the EC\u003csub\u003e50\u003c/sub\u003e parameter with DPPH radical, which quantifies the amount of extract needed to reduce the initial concentration of the radical up to 50%. The TPC determined in the solid extracts (TPC\u003csub\u003e1\u003c/sub\u003e) was also referred per mass unit of defatted bagasse (TPC\u003csub\u003e2\u003c/sub\u003e). The TPC values increased from 16 to 59 mg GAE/g dried extract as the extraction temperature rose. Similarly, Rahman \u003cem\u003eet al\u003c/em\u003e (2021) observed that BSG aqueous extract obtained at 160 \u003csup\u003eo\u003c/sup\u003eC showed highest TPC values than that obtained at 100 and 140 \u003csup\u003eo\u003c/sup\u003eC, the main phenolics compounds being flavan-3-oles, hydroxycinnamic acids (such as chlorogenic, gallic, protocatechuic, ferulic and p-coumaric acids) and flavonols. The found TPC values in defatted bagasse (7,57 mg GAE/g DB) are in the range of those reported by other authors (0.89-15 mg GAE/g sample), depending on the solvent and extraction method used (Santi \u003cem\u003eet al\u003c/em\u003e., 2018). Nevertheless, when expressed per mass unit of DB, TPC\u003csub\u003e2\u003c/sub\u003e values in the extracts were lower than the TPC value of DB, except for the extract obtained at the highest temperature (E-170). During the SWE extraction, an increment in the TPC content occurred as temperature increases due to the promotion of hydrolysis of lignin/phenolics-carbohydrate complexes, fostering the decomposition of these structures and releasing phenolic acids. Likewise, the neoformation of antioxidant compounds under severe SWE conditions has also been described (Plaza \u003cem\u003eet al\u003c/em\u003e. 2010a,b). This neo-formed antioxidant compounds could be also quantified as phenols by the unspecific Folin-Ciocalteu reagent. These compounds are formed through Maillard and/or caramelization reactions, producing 5-HMF and sugar condensation compounds, and exhibit different bioactivities, including antioxidant activity (Trigueros \u003cem\u003eet al\u003c/em\u003e., 2023). On the other hand, the thermal degradation of the phenolic compounds at high temperatures could also occur. Specifically, flavonoids, one of the main phenolic compounds in the beer bagasse, are highly thermo-sensitive. Therefore, the extent of the different phenomenon occurred during SWE, depending on the composition of each natural matrix, will determine the final content and nature of phenolics in the extracts. Thus, the marked increment in the TPC observed at 170 \u003csup\u003eo\u003c/sup\u003eC could be attributed to the high progress of the hydrolytic phenolic release, compared to the potential degradation ratio, as well as to the neo-formation of higher amounts antioxidant species.\u003c/p\u003e \u003cp\u003eThe antioxidant capacity, measured throughout the EC\u003csub\u003e50\u003c/sub\u003e values are also shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This value increased when the temperature rose, thus indicating a decrease in the radical scavenging capacity of the extracts. This decrease in the antioxidant capacity when the temperature rose, despite the promotion of higher phenolic content, can be attributed to the different phenolic profile in each extract with different radical scavenging capacity.\u003c/p\u003e \u003cp\u003eThe antimicrobial potential of the DB extracts was also studied against the Gram-negative \u003cem\u003eE.Coli\u003c/em\u003e bacteria and the Gram positive \u003cem\u003eL. Innocua\u003c/em\u003e, which are well-known pathogenic microorganisms responsible for food poisoning. The minimal inhibitory concentration (MIC values) of the extracts with both bacteria were determined and shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The antibacterial effectiveness increased when the extraction temperature rose, being the gram positive bacteria (Listeria) more sensitive to the extracts. The E-170 MIC value for \u003cem\u003eE.Coli\u003c/em\u003e was similar to that found for SWE extract of almond peel (90 mg/ml) obtained at 160 \u003csup\u003eo\u003c/sup\u003eC (Freitas \u003cem\u003eet al\u003c/em\u003e., 2023). Barbosa-Pereira \u003cem\u003eet al.\u003c/em\u003e (2014) also reported the antimicrobial efficiency of the polyphenols from the brewery waste stream against \u003cem\u003eS. Aureus, L. monocytogenes, Salmonella spp\u003c/em\u003e. and \u003cem\u003eE.Coli\u003c/em\u003e bacteria, while ferulic and caffeic acids and flavonoids being the main responsible for the observed antimicrobial activity. The obtained results indicate that the SWE extracts from beer bagasse are excellent candidates to be used as antioxidants or antilisteria compounds in food preservation or in the pharmaceutical sector.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Bleaching of the Extraction Residues\u003c/h2\u003e \u003cp\u003eThe extraction residues (R-110, R-130, R-150 and R-170) were bleached to recover the cellulose fraction, as they can be used for different applications in the material developing and pharmaceutical sectors. The bleaching treatment was carried out using a greener bleaching agent than the usual chlorine bleaches, to minimize the environmental impact of the process. Thus, the insoluble fractions were submitted to four successive 1 h cycles with 4% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution at pH 12. To evaluate the efficiency of the process, the white index (WI) and the yield of the process was determined in cycle for the different samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As expected, the application of four successive bleaching cycles significantly decreased yield and increased the WI values, in accordance with the progressive purification of cellulose in each cycle.\u003c/p\u003e \u003cp\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\u003eChemical composition (%wt) of the insoluble fractions subjected to the four bleaching cycles with 4% hydrogen peroxide.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAshes\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLignin\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCellulose\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHemicellulose\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBR-110-1C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6,96\u0026thinsp;\u0026plusmn;\u0026thinsp;0,09\u003csup\u003ea,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14,69\u0026thinsp;\u0026plusmn;\u0026thinsp;0,03\u003csup\u003ea,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11,3\u0026thinsp;\u0026plusmn;\u0026thinsp;0,5\u003csup\u003ea,3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ea,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e41\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003csup\u003ea,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBR-110-2C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,9\u0026thinsp;\u0026plusmn;\u0026thinsp;0,3\u003csup\u003eb,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15,9\u0026thinsp;\u0026plusmn;\u0026thinsp;0,8\u003csup\u003ea,2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8,2\u0026thinsp;\u0026plusmn;\u0026thinsp;0,3\u003csup\u003eb,2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ea,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e 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\u003cp\u003e5,7\u0026thinsp;\u0026plusmn;\u0026thinsp;0,3\u003csup\u003ea,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14,4\u0026thinsp;\u0026plusmn;\u0026thinsp;0,2\u003csup\u003eb,3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4,8\u0026thinsp;\u0026plusmn;\u0026thinsp;0,3\u003csup\u003eb,3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ec,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBR-170-3C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,7\u0026thinsp;\u0026plusmn;\u0026thinsp;0,4\u003csup\u003ea,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11,5\u0026thinsp;\u0026plusmn;\u0026thinsp;0,2\u003csup\u003ec,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2,9\u0026thinsp;\u0026plusmn;\u0026thinsp;0,3\u003csup\u003ec,3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ec,2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBR-170-4C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6,2\u0026thinsp;\u0026plusmn;\u0026thinsp;0,2\u003csup\u003ea,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10,2\u0026thinsp;\u0026plusmn;\u0026thinsp;0,2\u003csup\u003ed,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,2\u0026thinsp;\u0026plusmn;\u0026thinsp;0,2\u003csup\u003ed,3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003csup\u003eb,1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\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\u003ea,b,c, different letters indicate significant differences(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between samples at the same extraction temperature\u003c/p\u003e \u003c/div\u003e\n\u003cp\u003e1,2,3..: different numbers indicate significant differences (p \u003c 0.05) between samples at the same bleaching cycle\u003c/p\u003e\n \u003cp\u003eThe cellulose purification progress was monitored through the analysis of lignin and sugars by means of the NREL method (Sluiter 2005 and 2008). After removal of the water (which includes soluble sugars) and ethanol extractives in the samples (between 13\u0026ndash;34%), the acid insoluble lining and hydrolyzed sugars were quantified in the different samples. Glucose was the major component, followed by xylose and arabinose. As established in the NREL method, hemicellulose content was considered as total xylose and arabinose and total glucose was attributed to the cellulose content. The obtained values are given in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe hemicellulose content was selectively removed when successive bleaching cycles were applied (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in every sample. This hemicellulosic fraction significantly decreased when using 3\u0026ndash;4 cycles in BR-110 and BR-130 samples, and completely disappeared in BR-150 and BR-170 samples, after two and one bleaching cycles, respectively. Nevertheless, no significant increase in the cellulose content of the samples occurred during the successive cycles, except for R-170, in which the cellulose content significantly decreased with successive cycles. This suggests that cellulose is progressively degraded through the bleaching cycles with hydrogen peroxide. In fact when referring the cellulose content per mass unit of initial DB, a progressive decrease was observed, ranging from 16 g cellulose/ 100 g DB in the non-bleached residues to 7\u0026ndash;13 g cellulose/g DB in the fourth bleaching cycle of the different samples. Degradation of cellulose by the oxidative action of hydrogen peroxide has been reported by other authors (Vismara \u003cem\u003eet al.\u003c/em\u003e, 2009) through free radical mechanisms forming alpha hydroxyalkyl radicals and subsequent chain scission. This process is largely affected by the substrate composition and the presence of catalysers or inhibitors of the reaction. Therefore, the use of hydrogen peroxide as bleaching agent of BD cellulosic fractions did not yield proper results, since an important part of cellulose is degraded during the delignification process.\u003c/p\u003e \u003cp\u003eIn general, the acid insoluble lignin content decreased when successive cycles were applied, especially after the fourth bleaching cycle. Nevertheless, as commented on above, it has to be taken into account that these values were affected by the protein content of the samples. As can be observed, the protein is progressively removed by successive bleaching cycles, especially in sample BR-170 where higher protein solubilization occurred in the SWE step.\u003c/p\u003e \u003cp\u003eThe TGA and DTGA curves of the insoluble and bleached residues are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. All samples exhibited a first weight loss step 25 and 125 \u003csup\u003eo\u003c/sup\u003eC corresponding to the loss of bonded water, and the typical degradation steps of lignocellulosic residues, previously commented. The TGA curves of the bleached fractions showed the expected differences in the thermal behavior with respect the non-bleached samples, related with the compositional changes that occurred in the bleaching step. The partial removal of hemicellulose during the bleaching cycles are reflected on the TGA curves where the double peak in DTGA curves of polysaccharides became a single peak, mainly attributed to cellulose degradation, and the temperature of the maximum degradation rate increased from 280 to 300 \u003csup\u003eo\u003c/sup\u003eC in BR-110 and BR-130 samples. Nevertheless, no relevant changes in the cellulose purification degree can be deduced from the scarce increase in the weight loss step attributed to this polymer, remaining other compounds whose degradation overlapped with the cellulose degradation, as also observed in the analyzed composition. In sample BR-170, very few changes in the TGA curve were observed after the first bleaching cycle, coherently con the small composition changes reflected in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Therefore, the successive cycles reduced the bleaching mass yield but did not significantly promote cellulose purification, but its degradation. In the other cases the bleaching cycles promoted the removal of hemicellulose, but also did not result in higher cellulose purity due to its partial degradation. The cellulose degradation products probably contributed to the increase in the final residual mass obtained for most of bleached samples. So, the oxidative process applied with hydrogen peroxide in an alkaline medium seems to partially degrade cellulose generating other compounds and reducing the process yield.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eDespite being rich in polysaccharides, proteins and phenolic compounds, BSG is still underutilized in the food, materials or pharmaceutical sectors. New sustainable approaches, such as the use of subcritical water extraction could be a possible technology to fractionate this waste obtaining bioactive agents, proteins and cellulose fractions from the beer bagasse while contributing to the circular economy.\u003c/p\u003e \u003cp\u003eThe use of subcritical water treatment of defatted beer bagasse allowed to obtain bioactive aqueous extracts (7\u0026ndash;41% mass yield of the defatted BSG) with radical scavenging capacity and antimicrobial activities. The highest extraction temperature (170 \u003csup\u003eo\u003c/sup\u003eC) gave rise to the highest extract yield while provide the extracts with greater polyphenol content and antibacterial effect, but with lower DPPH radical scavenging capacity. In contrast, the extraction at 150\u0026deg;C was optimal for producing extracts (35% mass yield) with the greatest radical scavenging capacity. Likewise, the extract obtained at 170 \u003csup\u003eo\u003c/sup\u003eC was the richest in protein, which could be separated by precipitation from the liquid extract.\u003c/p\u003e \u003cp\u003eBeer bagasse can be considered as a relatively poor source of cellulose in comparison with other agro-industrial residues. Considering the yield of the different process steps to purify cellulose, (19, 17, 14 and 13% for samples treated a 110, 130, 150 and 170 \u003csup\u003eo\u003c/sup\u003eC) and the similar degree of cellulose purity obtained after the 4 bleaching cycles (50, 60, 70 and 50%, respectively for samples treated a 110, 130, 150 and 170 \u003csup\u003eo\u003c/sup\u003eC ), the best treatment to obtain cellulose would be the extraction at 150 \u003csup\u003eo\u003c/sup\u003eC, followed by two bleaching cycles with hydrogen peroxide. These conditions allows for the removal of most of the hemicellulose and led to a cellulose purity degree without significant differences with respect to that obtained in the successive cycles.\u003c/p\u003e \u003cp\u003eThus, the subcritical water extraction method highlights the potential of a simple processes as technological option to convert underutilized side streams like beer bagasse into added-value, potential ingredients for innovative food and pharmaceutical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e5. Funding\u003c/h2\u003e \u003cp\u003eThis research was supported by MCIN/AEI/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13039/501100011033\u003c/span\u003e\u003cspan address=\"10.13039/501100011033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and by the European Union NextGenerationEU/PRTR (project TED2021-132295B-I00). The authors also thank the Generalitat Valenciana (GVA) for the grant received through the GRISOLIA program (CIGRIS/2021/033).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.G-C. and C.O made substantial contributions to the the acquisition, analysis and interpretation of data. P.V supported the methodology part and L.M-P the microbiological analysis. C.G-M. and A.C drafted the work, revised it critically and approved the version to be published\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdelmoez, W., Nakahasi, T., \u0026amp; Yoshida, H. (2007). Amino acid transformation and decomposition in saturated subcritical water conditions. Industrial \u0026amp; Engineering Chemistry Research, 46(16), 5286-5294. https://doi.org/10.1021/ie070151b\u003c/li\u003e\n\u003cli\u003eAlonso-Ria\u0026ntilde;o , P. Sanz M.T. , Benito-Roman O., Beltran , S. Trigueros, E.; (2021). 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Subcritical water extraction of bioactive compounds from plants and algae: Applications in pharmaceutical and food ingredients. Food Engineering Reviews, 8, 23-34. https://doi.org/10.1007/s12393-015-9119-x\u003c/li\u003e\n\u003cli\u003eZhang, R., Liu, H., Hou, J., Yao, Y. Ma, Y., Wang , X. (2021). Cellulose fibers extracted from sesame hull using subcritical water as a pretreatment. Arabian Journal of Chemistry,14, 6,103178.\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":"phenolic compounds, cellulose fibres, antioxidant, antimicrobial","lastPublishedDoi":"10.21203/rs.3.rs-4610399/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4610399/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOf the three types of waste generated in beer processing, beer grain spent (BGS) or beer bagasse is the most abundant and has a high potential for valorisation. In this work, defatted BGS was subjected to an extraction process with subcritical water (SWE) at different temperatures (110, 130, 150 and 170\u0026deg; C) to obtain extracts rich in phenols and the cellulosic fractions. Furthermore, the obtained cellulose fractions were also purified by means of a greener methodology using hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). The results showed that the extraction conditions affected the composition and properties of the fractions. The dry extracts obtained at 170\u0026deg;C were richer in phenolics (24 mg GAE. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e defatted beer bagasse (DB), but with lower antioxidant capacity (71 mg DB.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DPPH). This extract (E-170) also showed the highest antibacterial potential (lower MIC values) against \u003cem\u003eL. innocua\u003c/em\u003e (80 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and \u003cem\u003eE. coli\u003c/em\u003e (140 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than those obtained at lower temperatures. The purification of cellulose from the SWE residues, using hydrogen peroxide revealed that DB is not a good source of cellulose material since the bleached fractions showed low yields (20\u0026ndash;25%) and low cellulose purity (42\u0026ndash;67%), even after four bleaching cycles (1 h) at pH 12 and 8% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Despite this, the subcritical water extraction method highlights the potential of a simple processes as technological option to convert underutilized side streams like beer bagasse into added-value, potential ingredients for innovative food and pharmaceutical applications.\u003c/p\u003e","manuscriptTitle":"Phenolic and Cellulose-Rich Fractions from Subcritical Water Treated Beer Bagasse","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-25 09:01:06","doi":"10.21203/rs.3.rs-4610399/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":"9e3aee58-5bde-4446-840f-24680af44255","owner":[],"postedDate":"July 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T00:56:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-25 09:01:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4610399","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4610399","identity":"rs-4610399","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}