Microwave-Assisted Pretreatment of Corncob Using Potash Alum (AlK(SO4)2) Catalyst for Enhanced Xylooligosaccharide (XOS) Extraction with High Prebiotic and Antioxidant Properties

Microwave-Assisted Pretreatment of Corncob Using Potash Alum (AlK(SO4)2) Catalyst for Enhanced Xylooligosaccharide (XOS) Extraction with High Prebiotic and Antioxidant Properties | 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 Microwave-Assisted Pretreatment of Corncob Using Potash Alum (AlK(SO4)2) Catalyst for Enhanced Xylooligosaccharide (XOS) Extraction with High Prebiotic and Antioxidant Properties Umar Seno Aji, Hana Nur Fitriana, Haqqyana Haqqyana, Khaswar Syamsu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6158861/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jul, 2025 Read the published version in Waste and Biomass Valorization → Version 1 posted 5 You are reading this latest preprint version Abstract This study explores the potential of corncob waste valorization through microwave-assisted hydrolysis for xylooligosaccharides (XOS) extraction using potash alum (AlK(SO₄)₂) as a catalyst. The process was optimized through a One-factor-at-a-time (OFAT) approach, evaluating potash alum concentration, heating time, and reaction temperature. Optimal conditions (20 µmol/mL potash alum, 5 minutes, 160°C) yielded 12.32 g/L XOS in the hydrolysate, with ethanol precipitation achieving a 16.97% (g/g) purified XOS yield. Potash alum was chosen as an alternative catalyst due to its potential cost-effectiveness and milder environmental impact compared to strong acids, though further comparative analysis is warranted. The purified XOS exhibited strong antioxidant activity (94.16 ± 0.54% DPPH radical scavenging) and high total phenolic content (32.63 ± 0.29 µg GAE/mg of XOS). In vitro prebiotic assays demonstrated XOS’s capacity to enhance probiotic growth, particularly Lactobacillus fermentum , which showed the highest prebiotic index and prebiotic activity within 24 hours. These findings suggest that corncob-derived XOS can serve as a functional ingredient with prebiotic and antioxidant properties, supporting its application in health-promoting food and nutraceuticals. Xylooligosaccharide (XOS) corncob waste potash alum prebiotic antioxidant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Statement of Novelty Xylooligosaccharides (XOS) are valuable prebiotic compounds typically extracted using strong acids or enzymes, which can be costly and have environmental drawbacks. This study examines the use of potash alum (AlK(SO₄)₂) as a catalyst in microwave-assisted hydrolysis for XOS extraction. While potash alum has been explored in biomass processing, its application in XOS production remains underreported. This approach utilizes corncob, an abundant agricultural byproduct, as a feedstock and operates under relatively mild reaction conditions. The extracted XOS retains antioxidant activity (94.16% DPPH radical scavenging), high phenolic content, and demonstrates a high prebiotic index and prebiotic activity, particularly in stimulating Lactobacillus fermentum growth. These findings contribute to ongoing efforts to develop alternative catalysts for lignocellulosic biomass valorization and more sustainable XOS production methods. 1. Introduction The increasing global emphasis on environmental sustainability has driven research into the valorization of agricultural residues, particularly those that are abundant yet underutilized. Corncob waste, a major byproduct of the maize industry, remains largely underexploited despite its high hemicellulose content, particularly xylan. Traditionally, corncob is either discarded or used in low-value applications such as animal feed or fuel, missing the opportunity to convert it into high-value bioactive compounds. One such compound is xylooligosaccharides (XOS)—non-digestible carbohydrates with well-documented prebiotic and antioxidant properties [ 1 ]. XOS are sugar oligomers derived from the hydrolysis of xylan-rich hemicellulose. These oligomers feature a branched structure with diverse substituents such as arabinose, uronic acids, and acetyl groups, which are linked through β-(1,4) glycosidic bonds [ 2 ]. Recent studies highlight XOS’s potential in promoting gut health by selectively stimulating beneficial bacteria such as Lactobacillus and Bifidobacterium species, as well as its antioxidant properties that make it valuable for functional food and nutraceutical applications [ 3 – 5 ]. However, conventional XOS production methods—including enzymatic, alkali, and acid hydrolysis—face significant challenges, such as high operational costs, long reaction times, and environmental concerns related to chemical waste disposal. There is a critical need for more efficient, cost-effective, and environmentally friendly XOS extraction techniques. Microwave-assisted hydrolysis has emerged as a promising alternative due to its ability to rapidly break down biomass with reduced energy consumption and minimal byproduct formation [ 6 , 7 ]. Additionally, studies have shown that integrating microwave irradiation with inorganic salts can enhance biomass pretreatment efficiency [ 8 , 9 ]. One such inorganic salt, potash alum (AlK(SO₄)₂), has been reported to facilitate lignin degradation by raising the dipole moment in plant cells, leading to β-O-4 ether cleavage and hemicellulose release [ 10 – 12 ]. Unlike strong acids or expensive enzymes, potash alum is non-toxic, cost-effective, and widely used in water purification [ 13 ], making it an attractive alternative for biomass hydrolysis. Despite its potential, its application as a catalyst in microwave-assisted XOS production has not been explored, leaving a significant gap in the development of efficient and scalable XOS extraction methods. This study aims to bridge this gap by investigating the feasibility of potash alum-catalyzed microwave-assisted hydrolysis for XOS production from corncob waste. By optimizing key reaction parameters—potash alum concentration, heating time, and temperature—using a One-factor-at-a-time (OFAT) approach, we seek to enhance XOS yield while maintaining its functional properties. Furthermore, we evaluate the prebiotic and antioxidant activities of the purified XOS, demonstrating its potential as a functional food ingredient. Our findings provide a sustainable and scalable strategy for agricultural waste utilization while introducing an alternative catalyst that could improve existing biomass pretreatment processes. 2. Materials and Methods 2.1 Materials Corncob was collected from farmers in Subang, West Java, Indonesia. The materials were naturally dried for 2–3 days to reduce moisture content before processing. Corncob was then cut into 5 cm lengths and ground using a ring flaker (Pallmann Maschinenfabrik GmbH & Co. KG, Germany). To further reduce particle size, the material underwent additional grinding processes using a hammer mill and a disk mill (both from Pallmann Maschinenfabrik GmbH & Co. KG, Germany). The final product was passed through a sieve to obtain particles within the size range of 40 to 60 mesh (250–420 µm). This study's catalyst used for treatment was potash alum (Merck, Darmstadt, Germany). 2.2 Optimization of XOS extraction using a combination of potash alum and microwave pretreatment The extraction procedure for XOS consists of three distinct steps. In the first step, the concentration of potash alum was optimized, with the heating temperature and time fixed at 160°C and 5 minutes, respectively. Various concentrations of potash alum, ranging from 0 to 80 µmol/mL, were tested. In the second step, the heating temperature was optimized using the potash alum concentration determined in step 1, while the heating time was maintained at 5 minutes. Heating temperatures ranged from 140°C to 180°C. In the third step, the heating duration (3, 5, and 7 minutes) was optimized using the potash alum concentration from step 1 and the heating temperature identified in step 2. Oven-dried samples measuring (250–420 µm) will be mixed with alum solution with the concentration required for each layer while maintaining a solid-to-solution ratio of 1:10 in a closed container. After microwave treatment, it is cooled to room temperature, and the dissolved and insoluble fractions are separated using a vacuum filter. The pulp was rinsed using distilled water until it reached normal pH and dried at 60 o C for further analysis. The soluble fraction (hydrolysate), which contains a lot of carbohydrate degradation, was analyzed for the content of reducing sugars, xylose, and XOS. This hydrolyzate is then processed further to be separated and purified. 2.3 Separation and purification of XOS powder Following the pretreatment using a microwave-assisted potash alum catalyst, the hydrolyzate is concentrated using a rotary evaporator until it reaches one-third of the volume. Unhydrolized xylans were separated using ethanol precipitation, following the method of Vieira et al. [ 14 ], with modifications. A two-step precipitation process was applied in this study, where one volume of ethanol was used for the first precipitation, followed by the addition of two volumes for the second. The supernatants from the second precipitation were collected, and ethanol was removed using a rotary evaporator. Each phase obtained at the purification step is freeze-dried and stored for further testing. 2.4 Combined severity factor (CSF) In the microwave-assisted pretreatment, combined severity factor (CSF) calculation was used to integrate the effects of potash alum concentration, temperature, and reaction time in a single parameter. The CSF applied in this study is defined by Eq. 1 : CSF = log {t.exp [(TH − TR)/14.75]} – pH (1) In this equation, t represents the reaction time in minutes, TH refers to the heating temperature in degrees Celsius (°C), TR is the reference temperature set at 100°C, pH signifies the solution's acidity level, and 14.75 is a constant value related to the activation energy based on first-order kinetic modeling [ 15 ]. 2.5 Analytical methods 2.5.1 Analysis of the chemical compositions of corncob The Van Soest method was employed to simultaneously analyze the hemicellulose, lignin, and cellulose contents, providing an estimation of the lignocellulose composition of corncob [ 16 ]. 2.5.2 Determination of XOS, xylose, and reducing sugar concentration and yield in the pretreatment hydrolysate The total reducing sugars were measured using the 3,5-dinitrosalicylic acid (DNS) method using a UV-Vis spectrophotometer (UV-1800, Shimadzu Scientific Instruments Inc., Kyoto, Japan) [ 4 ]. Xylose and XOS concentrations were analyzed using a D-xylose assay kit (Megazyme, Wicklow, Ireland), with absorbance measured at 340 nm using a microplate reader (TECAN Infinite® 200 Pro). To determine the XOS content, the hydrolyzate is first hydrolyzed to xylose with 4% H₂SO₄ at 121°C for 1 hour, and xylose after hydrolysis is quantified [ 17 ]. The XOS concentration was calculated as the difference between xylose before and after acid hydrolysis of the hydrolyzate, following the procedure described in previous studies [ 18 ]. The hydrolysate's xylose to XOS ratio (xylose/XOS) was determined using a modified calculation method from Hao et al. [ 19 ]. 2.5.3 Determination of XOS fraction The purification product was dissolved and filtered using SEPARA® syringeless filters (Zola Pedosa, Italy). The solution was then analyzed using thin layer chromatography (TLC) Silica Gel 60F254 plates (20 × 20 cm, EMD/Merck, Darmstadt, Germany) and developed using an eluent consisting of n-butanol, acetic acid, and water in a 2:1:1 (v/v/v) ratio [ 20 ]. The spots were visualized using a diphenylamine-aniline-phosphoric acid reagent (Merck KGaA, Darmstadt, Germany) in acetone, followed by heating at 120°C for 15 minutes. The standards used included pure xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6), all sourced from Megazyme (Wicklow, Ireland). 2.5.4 Determination of total phenolic content (TPC) The Folin–Ciocalteu (F-C) assay has been used for decades to measure total phenolics in natural products. Based on an oxidation/reduction mechanism, this method is commonly employed to evaluate antioxidant activity. Total phenolic content was measured using a modified spectrophotometric technique. XOS powder was dissolved in dimethyl sulfoxide (DMSO) at 2 mg/mL concentration. A 0.5 mL sample aliquot was mixed with 2.5 mL of F–C reagent (Merck) and 5 mL of Na₂CO₃. The mixture is then dissolved in water until it reaches a final volume of 50 mL. The sample was then incubated in a water bath at 40°C for 30 minutes before spectrophotometric analysis. Absorbance at 750 nm was measured using a UV-Vis spectrophotometer (Model UV-1800, Shimadzu Corp., Kyoto, Japan). A blank was prepared using the same mixture, but the sample was replaced with 0.5 mL DMSO. The samples were prepared in duplicate, and the mean absorbance value was recorded. The same technique was applied for gallic acid. A calibration curve was constructed, and the total phenolic content was reported in terms of gallic acid equivalents (mg GA per gram of extract) [ 21 ]. 2.5.5 Determination of antioxidant activity The antioxidant activity of the XOS modification method, as described by Bian et al. [ 2 ], was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. A 0.1 mM DPPH solution in ethanol was mixed with an equal volume of the XOS solution, and the reaction was carried out in the dark with continuous stirring for 30 minutes. The absorbance of the mixture was measured at 517 nm using a UV-Vis spectrophotometer (UV-1800, Shimadzu Scientific Instruments Inc., Kyoto, Japan), and the antioxidant activity was determined using Eq. 2 . $$\:\text{A}\text{n}\text{t}\text{i}\text{o}\text{x}\text{i}\text{d}\text{a}\text{n}\text{t}\:\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:\:\left(\%\right)\:=\:1\:-\frac{absorbance\:sample}{absorbance\:control}\:x\:100\%$$ 2 2.5.6 Determination of prebiotic index and activity The probiotic cultures tested included Lactobacillus fermentum , Lactobacillus rhamnosus , and Bifidobacterium sp. For comparison, tests were also conducted on pathogenic bacteria such as Salmonella typhimurium, Escherichia coli , and Staphylococcus aureus to evaluate their ability to utilize XOS and other available carbon sources and the antibiotic effect of XOS. The fermentation was conducted in a basal medium containing peptone (10 g/L), MnSO₄ (100 mg/L), MgSO₄ (50 mg/L), NH₄Cl (2 g/L), KH₂PO₄ (2 g/L), and Tween 80 (1 mL/L)) using four different carbon sources: glucose, xylose, a combination of glucose and xylose (1:1 ratio), and corncob XOS. The fermentations were incubated at 37°C, agitated at 180 rpm, and monitored at 0, 3, 6, 12, 24, 48, and 72 hours by measuring optical density at 600 nm. The prebiotic index was evaluated to assess each of four different carbon sources (2% w/v) for probiotics in basal medium, which was calculated using formula (1) to determine the efficacy of each extract as a carbon source for probiotics. Log P24 represents the probiotic bacterial optical density after 24 hours of incubation, while Log P0 is the optical density before incubation [ 22 ]. $$\:Prebiotic\:index=\:\frac{\left(Log\:P24-Log\:P0\right)\:extract}{\left(Log\:P24-Log\:P0\right)glucose}$$ 1 The prebiotic activity was assessed to determine each XOS extract’s selectivity for supporting the growth of the probiotic rather than the pathogen. Probiotics and pathogens culture were inoculated into basal medium supplemented with XOS extract, using a 1% (v/v) inoculum, and filled into a 15 mL conical tube, followed by incubation at 37°C. Samples were taken before and after 24 hours of incubation, and measured the optical density at 600 nm. $$\:Prebiotic\:activity=\left(\frac{\left(Log\:P24-Log\:P0\right)extract}{\left(Log\:P24-Log\:P0\right)glucose}\right)-\left(\frac{\left(Log\:E24-Log\:E0\right)extract}{Log\:E24-LogE0)glucose}\right)$$ 2 Log P24 represents the probiotic bacterial count after 24 hours of incubation, while Log P0 indicates the initial probiotic count before incubation. Similarly, Log E24 refers to the pathogen bacterial count after 24 hours, and Log E0 is the initial count of the pathogen before incubation [ 23 ]. 2.5.7 Functional group determination of untreated and treated biomass The functional groups in the biomass were examined using an attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectrometer (Perkin Elmer FTIR Spectrometer–Spectrum Two, C 106456, Perkin Elmer Inc., Waltham, MA, USA). About 0.1 mg of biomass was placed on a diamond plate and compressed using a force gauge until the spectral data appeared on the computer screen. The analysis covered the range of 4000 to 400 cm⁻¹ with a resolution of 4 cm⁻¹, 40 scans, and a force gauge setting of 90. The Perkin Elmer software was used to transform the IR spectra from transmittance to absorbance values. 2.5.8 Crystallinity determination of untreated and treated biomass using X-ray diffractogram Dried samples of treated and untreated biomass were examined using an X-ray diffractometer (XRD) (Shimadzu XRD-7000 MaximaX series, Shimadzu Corp., Kyoto, Japan) with copper as the radiation source. The scanning angle was 5–30º, operated at 40.0 kV and 30.0 mA, with a reading speed of 2.0º per minutes and a step size of 0.02º. The degree of crystallinity was calculated using the Ruland-Vonk method, which reduces the amorphous component by comparing the area above the amorphous profile, which refers to the total sample area [ 24 ]. 2.5.9 Surface morphology analysis of untreated and treated biomass The surface appearance of treated and untreated biomass was viewed using a JSM IT 200 Scanning Electron Microscope (JEOL Ltd., Tokyo, Japan). The sample is placed in a solid stainless tube using double-sided tape to ensure adhesion. After being coated with a thin layer of gold, the sample is placed in the sample port. After being coated with a thin layer of gold, the sample was positioned in the sample port of microscope. Images captured at 500 and 1000x magnifications. 2.5.10 Statistical analysis The catalysts impact on the concentrations of XOS, xylose, and reducing sugars after microwave pretreatment was assessed using a one-way analysis of variance (ANOVA). Parameters with significant F-values were further analyzed using Duncan’s Multiple Range Test (DMRT) at a 95% confidence level. 3. Result and Discussion 3.1 Optimization of potash alum concentration To optimize XOS recovery during microwave-assisted pretreatment, varying concentrations of potash alum (AlK(SO₄)₂) catalyst (0, 20, 40, 60, and 80 µmol/mL) were evaluated. The irradiation temperature and time were fixed at 160°C and 5 minutes, respectively. The potash alum concentration significantly influenced the yields of total reducing sugars, xylose, and XOS (p < 0.05). The addition of potash alum enhanced all parameters up to an optimal concentration. As shown in Fig. 1 , potash alum improved the total reducing sugar concentration. Without the catalyst, the reducing sugar content, including xylose and glucose, was low (3.67 g/L), suggesting that hydrolysis by high-temperature water alone was minimal under these conditions, consistent with previous findings [ 25 ]. When potash alum concentration increased from 20 to 40 µmol/mL, the reducing sugar concentration rose from 14.67 to 29.95 g/L, highlighting the catalyst's role in enhancing hydrolysis. Further increases in catalyst concentration continued to raise the reducing sugar yield, though with diminishing returns, likely due to saturation of available hydrolysis sites [ 25 ]. The XOS yield showed a different pattern. It increased significantly at 20 µmol/mL, reaching 12.32 g/L, but declined sharply to 3.21 g/L at 60 µmol/mL. Although a slight increase to 4.21 g/L was observed at 80 µmol/mL, this change was insignificant. The reduced XOS yield at higher catalyst levels suggests that excessive potash alum catalyzed further hydrolysis of XOS into smaller sugars, primarily xylose [ 26 ]. Xylose yield increased with potash alum concentration up to 40 µmol/mL but decreased at higher levels. The yield peaked at 16.48 g/L at 40 µmol/mL, then dropped to 16.35 g/L and 15.58 g/L at 60 and 80 µmol/mL, respectively. This trend indicates that moderate catalyst concentrations promote hemicellulose breakdown and XOS conversion to xylose, but excessive catalyst leads to xylose degradation into byproducts like furfural [ 25 , 27 ]. Despite the decline in XOS and xylose yields at higher potash alum concentrations, the total reducing sugar yield continued to increase, indicating additional sugar production from cellulose hydrolysis. At 20 µmol/mL potash alum (CSF − 1.19), the xylose/XOS ratio was 1.09, indicating efficient XOS production with minimal degradation. This agrees with Akpinar et al. [ 28 ], who noted that mild acid conditions favor XOS formation. However, at 60 µmol/mL (CSF − 1.00), the ratio surged to 5.10, consistent with Pramasari et al. [ 4 ], who reported that harsher acid environments promote extensive XOS hydrolysis into xylose. These findings underscore moderate catalyst loading optimally depolymerizes hemicellulose without excessive XOS breakdown. The synergistic effect of Brønsted and Lewis acids from aluminum salt catalysts, such as (AlK(SO₄)₂) in a water-only medium, promotes the cleavage of intermolecular linkages in lignocellulosic complexes. This mechanism facilitates the dissolution of hemicellulose into XOS and subsequently into reducing sugars like xylose and glucose. Luo et al. [ 12 ] discussed the role of water and aluminum sulfate (Al₂(SO₄)₃) in the selective catalytic transformation of corn stover hemicellulose into xylose. Water molecules form hydrogen bonds with the intermolecular linkages of hemicellulose-cellulose-lignin in biomass, weakening these linkages and enabling hemicellulose release. Aluminum salts enhance this effect by hydrolyzing in water to produce species like [Al(OH)₂(H₂O)x]⁺, which act as Lewis acids, promoting selective hemicellulose solubilization. Aluminum species may also form hydrogen-bonded complexes with -OH 1 and C 1 –O 6 of the xylan unit, stabilizing xylose and reducing its degradation while promoting its formation with the assistance of Brønsted acid species (H⁺ ions) from the aluminum salt hydrolysis. Based on this Luo et al.'s study, a proposed reaction mechanism for XOS extraction using potash alum is shown in Fig. 2 . Previous studies reported that Al₂(SO₄)₃ in a water system significantly improved hemicellulose-to-xylose conversion, with yields reaching 85.1 wt% from corn stover hydrolysis [ 12 ]. Hermiati et al. [ 26 ] combined microwave heating with Al₂(SO₄)₃ in a water medium for sugarcane trash pretreatment, observing substantial increases in reducing sugar and xylose yields with increasing catalyst loads. However, no prior studies have explored using potash aluminum from lignocellulosic materials for XOS production. 3.2 Optimization of heating temperature Microwave pre-treatment of corncob was conducted at temperatures of 140, 160, and 180°C for 5 minutes. A potash alum concentration of 20 µmol/mL was selected as it was identified as the optimal value for maximizing XOS yield. The statistical analysis revealed that temperature changes significantly impacted XOS production (p < 0.05). The results showed total reducing sugars increased with temperature, reaching a peak yield of 37.74 g/L at 180°C (Fig. 3 ). A similar trend was observed for xylose, which exhibited a positive correlation with higher temperatures, achieving a maximum of 18.95 g/L at 180°C. These findings suggest that elevated temperatures enhance reducing sugars and xylose release from biomass. Pre-treating biomass at higher temperatures under microwave irradiation disrupts the fibre structure, facilitating the breakdown of hemicellulose [ 29 ]. As a low molecular weight, short-branched polymer, hemicellulose contains linkages with lower activation energy, making its structure particularly susceptible to thermal degradation under high temperatures [ 10 , 29 ]. This leads to hemicellulose becoming readily solubilized, enabling further hydrolysis into its monomeric components, such as xylose, or forming oligosaccharides like XOS. While higher temperatures increased the overall release of sugar, XOS exhibited a different behaviour under high heating. The XOS yield increased from 3.38 g/L at 140°C to 11.18 g/L at 160°C but declined sharply to 5.59 g/L at 180°C. Notably, the xylose yield increased significantly beyond 160°C, suggesting that XOS degraded into smaller molecules, such as xylose, at higher temperatures. A similar result was also reported in previous studies [ 30 , 31 ]. Temperature strongly correlates with CSF, with higher temperatures resulting in higher CSF values because pretreatment conditions become more severe. At low temperatures (140°C, CSF − 1.78), the xylose/XOS ratio was relatively low (1.58), indicating a balanced production of XOS without significant degradation. However, at 160°C (CSF − 1.19), the ratio increased slightly to 1.72, as XOS degradation co-occurred with its production. Qin et al. [ 32 ] reported that low temperature produced large soluble polymers and xylose, while the XOS with a degree of polymerization (DP) of 2–5 remains low. As the temperature increases, the production rate of XOS surpasses that of xylose, leading to a decline in the xylose/XOS ratio. At 180°C (CSF − 0.61), the ratio spiked to 3.39, coinciding with the sharp decline in XOS yield and a significant increase in xylose yield. A further temperature rise enhances the rate of XOS degradation, causing an increase in the xylose/XOS ratio. It was previously reported that increased temperature facilitated the formation of X2-X3 oligosaccharides by promoting the hydrolysis of higher DP (X4-X6) XOS into lower DP XOS and xylose [ 33 ]. Therefore, while elevated temperatures were beneficial to hemicellulose degradation and enhanced overall sugar release, an optimal temperature of 160°C is critical for maximizing XOS yield while minimizing degradation. 3.3 Optimization of heating time Figure 4 illustrates the yields of reducing sugars, xylose, and XOS resulting from microwave pre-treatment of corncob at irradiation times ranging from 3 to 7 minutes. The experiments were conducted using a potash alum concentration of 20 µmol/ml and a constant heating temperature of 160°C. The CSF values increased with longer irradiation times, rising from − 1.42 at 3 minutes to -1.19 at 5 minutes and further to -1.05 at 7 minutes. This increase in CSF indicates a gradual rise in the severity of the treatment over longer microwave heating durations. The results show that all three products achieved their highest yields at 5 minutes of irradiation, while the shortest duration of 3 minutes produced the lowest yields. Specifically, at 5 minutes, the reducing sugar concentration peaked at 14.42 g/L, xylose reached 12.99 g/L, and XOS achieved its maximum value at 11.47 g/L. Extending the microwave heating to 7 minutes reduced all yields, likely due to degradation under prolonged exposure. Previous studies also reported similar results, noting that prolonged heating did not improve XOS production but instead resulted in increased degradation of XOS into xylose [ 4 , 30 ]. The Xylose/XOS ratio gradually decreased with time, starting at -1.24 at 3 minutes, reducing to -1.13 at 5 minutes, and reaching − 1.02 at 7 minutes. This declining trend indicates longer heating time favoured XOS retention relative to xylose formation. Additionally, longer acid hydrolysis times have been reported to increase the rate of XOS hydrolysis into lower molecular weight oligosaccharides and monosaccharides [ 34 , 35 ]. Lin et al. [ 33 ] observed that prolonging the irradiation time led to higher levels of X2 and X3, while reducing the levels of X5 and X6. These findings suggest that the DP distribution for the XOS also depends on heating time. Extending the heating duration enhances hemicellulose breakdown, but sufficient heating time is required to achieve optimal XOS yields. In this study, five minutes of irradiation time was identified as an optimal duration to produce the highest yields of XOS. 3.4 Catalytic effects of potash alum catalyst 3.4.1 Corncob chemical component changes The chemical composition of corncob biomass undergoes significant changes after pretreatment with a 20 µmol/mL potash alum catalyst at 160°C for 5 minutes, compared to the untreated material (Table 1 ). The hemicellulose content decreased sharply by 22%, from 36.9% ± 0.5 to 14.9% ± 0.1, indicating the breakdown and solubilization of hemicellulose into oligomers, simpler sugars, and other by-products during the treatment. In contrast, the cellulose content increased by 15.7%, rising from 42.6% ± 0.1 to 58.3% ± 0.2, while lignin content grew by 4.3%, from 8.4% ± 0.1 to 12.7% ± 0.3. This increase in cellulose and lignin proportions results primarily from the substantial loss of hemicellulose, reducing the overall biomass mass and leaving cellulose and lignin to comprise a greater percentage of the remaining material. Table 1 Changing in chemical components of untreated and treated corncob Components Corncob Untreated Treated Cellulose (%) 42.6 ± 0.1 58.3 ± 0.2 Hemicellulose (%) 36.9 ± 0.5 14.9 ± 0.1 Lignin (%) 8.4 ± 0.1 12.7 ± 0.3 Ash (%) 3.5 ± 0.2 5.5 ± 1.0 The observed shifts in biomass composition differ from findings by Simanullang et al. [ 10 ], who reported a 31% reduction in hemicellulose, a 5% reduction in lignin, and an 11.1% reduction in cellulose when using a higher potash alum concentration (30 µmol/mL) and a more severe pretreatment temperature (200°C) on sugarcane trash. These more intense conditions led to the production of reducing sugars rather than oligomers (XOS), as targeted in this study. The discrepancy highlights how pretreatment parameters influence the extent of biomass hydrolysis and the nature of the final products. The differing outcomes between these studies can be attributed to the distinct catalytic mechanisms of potash alum, which behaves as a Lewis acid during biomass pretreatment. Potash alum (AlK(SO₄)₂) provides aluminum ions that act as Lewis acid sites, breaking ether bonds within hemicellulose structures by coordinating with oxygen atoms in the polymer chains. In acidic environments, metal ions from the catalyst function analogously to hydronium ions in Lowry-Brønsted acids, enhancing the depolymerization of hemicellulose into smaller sugars [ 36 ]. A related Lewis acid, aluminum sulfate (Al 2 (SO 4 ) 3 ), has been shown to selectively hydrolyze hemicellulose with minimal impact on cellulose and lignin, as demonstrated by Luo (2020). In that study, the hydrolysis of Al 2 (SO 4 ) 3 generated H + ions, accelerating hemicellulose breakdown into xylose. Simultaneously, the formation of active aluminum species [Al(OH) 2 (H 2 O) x ] + promoted selective hemicellulose dissolution and protected xylose from further degradation through complex formation with hydroxyl groups in xylan units. [ 12 ]. This dual catalytic action illustrates how Lewis acids, including potash alum, drive the selective transformation of biomass components depending on treatment severity and target products. 3.4.2 Corncob functional group changes The treated corncob sample underwent microwave-assisted pretreatment using a potash alum catalyst at 160°C for 5 minutes with a catalyst concentration of 20 µmol/mL. This process induced significant structural and chemical changes, as evidenced by differences in the FTIR spectra between untreated and treated samples (Fig. 5 ), particularly at key peaks corresponding to functional groups involved in the biomass structure. At peak 1, the frequency shifted from 3340.52 cm⁻¹ to 3333.84 cm⁻¹, while intensity increased from 94.84–95.51%, demonstrating the existence of O-H stretching vibrations in cellulose [ 37 ]. The frequency reduction suggests stronger hydrogen bonding interactions, likely due to enhanced interaction between hydroxyl groups. Simultaneously, the increased intensity implies a higher concentration of accessible O-H functional groups. Peak 2 exhibited a frequency decrease from 2916.19 cm⁻¹ to 2903.95 cm⁻¹, with a slight rise in intensity from 96.75–97%. This peak represents C-H stretching vibrations in alkanes, and the changes indicate subtle modifications in the local chemical environment, potentially resulting from hemicellulose deconstruction [ 38 ]. Minimal changes at peaks 3 (1602.89 cm⁻¹) and 4 (1513.22 cm⁻¹ to 1513.26 cm⁻¹) suggest stability in C = C aromatic ring vibrations, possibly related to limited lignin removal. The small intensity reduction at peak 4 (from 96.83–96.41%) further supports this hypothesis [ 37 ]. A significant frequency shift occurred at peak 5, from 1369.72 cm⁻¹ to 1424.67 cm⁻¹, indicating significant structural alterations involving C-H deformation or C-O stretching associated with alcohols or esters. This shift suggests substantial transformations in hemicellulose or other non-cellulosic polymers. Peak 6 displayed a slight frequency decrease (from 1317.13 cm⁻¹ to 1315.9 cm⁻¹) with stable intensity, indicating minor changes in functional group environments. Peak 7 (1243.5 cm⁻¹) remained nearly unchanged in frequency, with a slight intensity increase, highlighting the stability of C-O stretching vibrations in alcohols or esters. At peak 8 (1158.53 cm⁻¹ to 1159.28 cm⁻¹), the frequency remained stable, but intensity decreased, suggesting a reduction in the quantity of C-O-C groups. This region corresponds to the β-glycosidic linkage in xylan, a key component of hemicellulose [ 39 ]. Conversely, peak 9 (1032.72 cm⁻¹ to 1030.73 cm⁻¹) showed a decreased frequency with increased intensity, indicating greater involvement of C-O-C groups, possibly due to structural rearrangements following hemicellulose breakdown. Peak 10 (897.52 cm⁻¹) associated with O–H bending showed minimal frequency change but slightly increased intensity, reflecting stability in β-glycosidic linkages within cellulose [ 40 , 41 ]. Finally, peak 11 exhibited a substantial frequency shift from 528.07 cm⁻¹ to 558.85 cm⁻¹, suggesting significant structural changes potentially involving interactions between biomass components and the alum catalyst. The emergence of two new peaks at 519.32 cm⁻¹ and 441.87 cm⁻¹, replacing the single peak at 441 cm⁻¹ in the untreated sample, indicates the formation of new structures or modifications involving inorganic compounds. Together, these spectral changes illustrate the intricate chemical transformations driven by potash alum catalysis, particularly in hemicellulose deconstruction and selective functional group modifications, enhancing cellulose accessibility while stabilizing certain structural elements. 3.4.3 Corncob crystallinity changes Lignocellulosic biomass primarily consists of hemicellulose, lignin, and cellulose. The structural arrangement of these components influences the degree of crystallinity in biomass. Cellulose contains both crystalline and amorphous regions, while hemicellulose and lignin are entirely amorphous [ 42 ]. Changes in the composition and structure of these components can significantly affect the crystallinity index of the biomass [ 43 ]. The crystallinity index of untreated corncob was measured at 45.92%, whereas microwave pretreatment combined with AlK(SO₄)₂ as a catalyst increased the crystallinity to 49.3% (Fig. 6 ). This enhancement suggests that the pretreatment process effectively dissolved some amorphous components, including hemicellulose, lignin, and non-crystalline cellulose regions. The chemical composition analysis (Table 1 ) supports this observation, showing a nearly 60% reduction in hemicellulose content after pretreatment. Additionally, the cellulose content increased by 37%, contributing to the higher crystallinity observed in the treated corncob. The X-ray diffraction (XRD) spectra reveal more intense and sharper diffraction peaks at 2θ angles of 10°–17°, 20°–25°, and 32°–37° for the pretreated corncob (Fig. 6 ), indicating enhanced crystallinity. These more defined peaks confirm the structural modifications induced by the pretreatment. The reduction in amorphous content improves cellulose accessibility by minimizing structural hindrances to hydrothermal hydrolysis, making the treated biomass more suitable for enzymatic processing and conversion into value-added products. Pretreatments involving acids or inorganic catalysts enhance the crystallinity index by selectively removing amorphous components. This structural modification facilitates enzymatic saccharification, as the enzymes can more effectively penetrate the biomass matrix and access the crystalline cellulose through the void spaces created by the solubilization of amorphous regions [ 44 ]. 3.4.4 Corncob morphological structure changes Scanning Electron Microscope (SEM) analysis was performed to assess the effect of this treatment on the surface structure and crystallinity of untreated and treated corncob biomass (Fig. 7 ). In the SEM images of untreated samples, the corncob surface appeared rough and irregular, with a dense, non-porous structure, reflecting the undegraded lignocellulosic nature of the biomass. The intact lignin fibers formed a tight network, limiting access to enzymes or chemical reagents [ 45 ]. In contrast, the SEM images of treated samples revealed significant lignin degradation and structural changes in hemicellulose and cellulose. The treated samples displayed a more porous surface, indicating that the bonds between lignin and cellulose had broken down, allowing for increased cellulose crystallinity. This observation aligns with the XRD data, which shows enhanced crystallinity in the treated samples, confirming the breakdown of amorphous components. The increased porosity in the treated samples facilitates enzymatic hydrolysis, as enzymes can more readily access the now-open cellulose structure, potentially improving biomass conversion efficiency [ 46 ]. However, the reduction of lignin and hemicellulose may impact on the thermal and chemical stability of the biomass, increasing its susceptibility to more destruction or chemical reactions during subsequent processing. 3.5 Fractionation of XOS powder The XOS hydrolysate obtained under optimal pretreatment conditions was purified using ethanol precipitation and freeze-dried to produce XOS powder. The fractionation of XOS in the powder was analyzed by thin-layer chromatography (TLC), as shown in Fig. 8 . Fractionation analysis of precipitates 1 and 2 revealed minimal differences, with both samples displaying only two prominent bands corresponding to X1 and X2. In contrast, the XOS powder from the supernatant exhibited more distinct fractionation, with clear bands ranging from X1 to X6 and prominent bands observed at X1 and X2. Stepwise ethanol precipitation during purification enhances the accumulation of low-degree polymerization (DP) XOS in the supernatant, making it suitable for use as a prebiotic [ 47 ]. However, the TLC pattern of the XOS powder from precipitates 1 and 2 and the supernatant deviates from the findings of Valls et al. [ 48 ], who reported that precipitate 1 primarily contained unhydrolyzed xylan, while precipitate 2 was enriched with long-chain xylooligomers. Despite using the same ethanol precipitation method for purification, the observed differences are likely due to substrate and production method variations. Valls et al. utilized commercial xylan as the substrate and enzymatic hydrolysis for XOS production, a method associated with high costs. In contrast, the present study extracted XOS directly from corncob biomass using microwave pretreatment with a potash alum catalyst. This difference in feedstock and processing conditions may have influenced the composition and fractionation patterns of the XOS products. 3.6 Antioxidant activity of corncob XOS powder Table 2 summarizes the scavenging capability of XOS powder derived from the first precipitant, second precipitant, and supernatant. The supernatant, which yields the highest amount of XOS (16.97%), demonstrates the strongest antioxidant activity (94.1 ± 0.54), while the first and second precipitants exhibit lower antioxidant activities. According to Valls et al. [ 48 ], the enhanced antioxidant activity in the supernatant can be attributed to its higher concentration of longer xylooligomers, which possess superior antioxidant properties. This finding is corroborated by the TLC analysis of XOS powder fractions in Fig. 7 . Moreover, the elevated antioxidant activity of the supernatant aligns with its significantly higher total phenolic content (32.63 ± 0.29 µg GAE/mg of XOS) compared to the first and second precipitants. Previous investigations have demonstrated the significant antioxidant action of hemicellulose hydrolysates, especially those enriched with polyphenols [ 49 , 50 ]. Studies have demonstrated that phenolic chemicals such as coumaric acid, syringic acid, ferulic acid, and caffeic acid neutralize free radicals by generating stable radicals [ 51 ]. These results imply that the XOS produced in this investigation has increased antioxidant features, mostly due to its enhanced phenolic content, which might have potential uses in many domains of interest. Table 2 Antioxidant activity, total phenolic content, and yield of the product after the purification process of corncob pretreatment at 160°C for 5 minutes using a 20 µmol/mL potash alum catalyst. Ethanol purification Antioxidant activities (%) Total phenolic content (µg GAE/mg of XOS) Yield XOS product (%) Precipitant 1 80.9 ± 0.62 b 4.50 ± 0.03 a 0.01 ± 0.002 a Precipitant 2 72.7 ± 0.23 a 4.29 ± 0.08 a 0.27 ± 0.05 b Supernatant 94.1 ± 0.54 c 32.63 ± 0.29 b 16.97 ± 0.35 c 3.7 Prebiotic activity of corncob XOS powder 3.7.1 In vitro growth and fermentation of XOS and other carbon sources by probiotics The ability of three probiotic strains— Lactobacillus fermentum , L. rhamnosus , and Bifidobacterium sp.—to metabolize different carbon sources was evaluated over 72 hours (Fig. 9 ). Each strain exhibited unique growth patterns depending on the substrate provided. Lactobacillus fermentum demonstrated adaptive growth on complex carbohydrates such as XOS, although overall growth was lower than the other two strains across all substrates. Interestingly, L. fermentum exhibited limited growth on glucose and a glucose-xylose combination, showing a notable aversion to these simpler sugars. However, its growth on xylose was nearly double that observed on glucose or the glucose-xylose mixture, indicating a preference for xylose. Contrary to these results, previous studies report that L. fermentum favors glucose over xylose. This preference is facilitated by carbon catabolite repression (CCR), a regulatory system determined by the catabolite control protein A (CcpA) that binds to catabolite-responsive elements (CREs) in the promoter areas of sugar metabolism operons, such as the xyl operon for xylose. When glucose is abundant, CcpA activates CCR, repressing the xyl operon and inhibiting xylose utilization [ 52 ]. However, Zhang et al. [ 53 ], observed that when CREs are deficient, CCR is incomplete, allowing L. fermentum to co-metabolize glucose and xylose, demonstrating its metabolic flexibility. In addition to glucose and xylose, L. fermentum efficiently metabolizes more complex substrates like XOS. Growth on XOS exhibited a distinct lag phase before steady growth began. Notably, the specific growth rate on XOS (0.0012) indicates its efficient utilization. The ability to metabolize these complex carbohydrates is supported by increased expression of sugar metabolism genes in the presence of XOS, highlighting L. fermentum ’s metabolic adaptability. Bifidobacterium sp. displayed significant variability in growth depending on the carbon source. Glucose supported the most rapid and highest growth, with a marked increase after 12 hours. Xylose and XOS also promoted substantial growth, with xylose showing a consistent growth pattern and XOS demonstrating a steady increase after an initial lag phase of approximately 3 hours. Moderate growth was observed on the glucose-xylose combination, with noticeable increases after 24 hours. The results reported here correspond with previous research demonstrating that glucose is the most preferred carbon source for various Bifidobacterium species, while xylose and XOS also contribute positively to growth [ 54 ]. The observed differences in substrate-dependent growth patterns highlight the importance of strain-specific metabolic capabilities and carbohydrate availability in influencing the efficiency of Bifidobacterium growth. Lactobacillus rhamnosus demonstrated distinct growth trends across various carbon sources. Glucose supported the most rapid and significant growth, with a substantial increase observed after 12 hours and continuing through 48 hours. This result is consistent with the findings of Petrut et al. [ 55 ], who identified glucose as the most favorable substrate for L. rhamnosus . The glucose-xylose combination also promoted robust growth, with consistent increases starting at 12 hours. In contrast, growth on xylose was the lowest but remained steady throughout the 72-hour incubation period, indicating a lower metabolic efficiency for xylose compared to glucose-based substrates. Moderate growth was observed on XOS derived from corncob, with the most pronounced increases between 24 and 48 hours, reflecting a delayed but sustained fermentation response. These findings highlight the metabolic versatility of L. rhamnosus , which can adapt to a range of carbon sources, with glucose supporting the most efficient growth and XOS showing potential prebiotic benefits due to its moderate but sustained fermentation. This adaptability makes L. rhamnosus a promising candidate for probiotic applications targeting carbohydrate-rich environments. All three probiotic strains exhibited positive growth on XOS, characterized by high specific growth rates. This finding is in agreement with Yan et al. [ 56 ], which demonstrated that XOS derived from various agricultural by-products effectively stimulates the growth of intestinal probiotics, including lactic acid bacteria and Bifidobacteria . These bacteria ferment XOS to generate short-chain fatty acids (SCFAs), such as butyrate, acetate, and propionate which confer numerous beneficial health effects on the host, notably increased immunological function, a reduced risk of gastrointestinal disorders, and improved gut health. 3.7.2 In vitro growth and fermentation of XOS and another carbon source by pathogen bacteria In addition to evaluating the prebiotic effects of XOS on beneficial bacteria, its influence on the growth of pathogenic bacteria, including E. coli , S. typhimurium , and S. aureus was also investigated (Fig. 10 ). This assessment aimed to determine whether different carbon sources selectively promote beneficial bacteria while inhibiting pathogen proliferation, a desirable attribute for prebiotic compounds. Growth studies on S. aureus revealed significant variability depending on the carbon source. Monosaccharides such as glucose, xylose, and a glucose-xylose mixture supported substantial growth, consistent with findings by Bacha et al. [ 57 ]. In contrast, corncob-derived XOS demonstrated minimal support for S. aureus growth, which was markedly lower than that observed with other carbon sources. Similarly, S. typhimurium exhibited distinct growth patterns when exposed to various carbon sources. Among these, xylose supported the most robust and sustained growth, showing the highest specific growth rate. Glucose and a glucose-xylose mixture provided moderate growth, with specific growth rates comparable to that of xylose. These results align with the findings of Eberl et al. [ 58 ], who noted that S. typhimurium , as a facultative anaerobe, is capable of metabolizing a broad range of simple sugars and sugar alcohols, including glucose, D-xylose, maltose, galactitol, L-arabinose, D-mannitol, L-rhamnose, D-mannose, trehalose, and D-sorbitol. The lowest specific growth rate for S. typhimurium was observed when XOS was used as the carbon source. The growth of E. coli showed moderate and comparable cell densities on glucose, xylose, and a glucose-xylose mixture. Naturally, E. coli can metabolize both glucose and xylose. However, due to carbon catabolite repression (CCR) [ 59 ], glucose is preferentially consumed when both sugars are available. Therefore, the growth of E. coli on these simple sugars was superior to its growth on XOS, a more complex carbohydrate with prebiotic properties that can inhibit pathogenic bacteria. In this study, the specific growth rate of E. coli on XOS was less than one-third of that observed with the other carbon sources. These observations underscore the metabolic preferences of the pathogenic strains tested. Simple sugars like glucose and xylose efficiently support pathogen growth, while corncob-derived XOS consistently limits it. Therefore, XOS emerges as a promising prebiotic candidate for promoting gut health and maintaining microbial balance by reducing the risk of pathogen overgrowth. Sun et al. [ 60 ] found that adding 0.25% XOS to the growth medium did not affect S. aureus alone. Still, it significantly reduced S. aureus counts when combined with probiotics like Bifidobacterium lactis . In that study, a higher XOS concentration of 4% (w/v) was needed to inhibit S. aureus growth directly. However, the current study demonstrates improved efficacy, as a 2% (w/v) XOS powder inhibited the growth of all three pathogenic bacteria tested, indicating a more potent inhibitory effect at a lower concentration. 3.7.3 Prebotic index and prebiotic activity against Salmonella typhimurium Salmonella typhimurium is a Gram-negative bacterium and a significant pathogen responsible for foodborne illnesses. It belongs to the Enterobacteriaceae family and is a leading cause of gastroenteritis in humans and animals. This bacterium can survive in diverse environments and is frequently associated with contaminated food products, particularly poultry, eggs, and dairy [ 61 ]. Prebiotics prevent the growth of pathogenic bacteria by selectively encouraging the multiplication of beneficial bacteria, including Lactobacillus and Bifidobacterium . Supplementing with XOS can suppress the growth of pathogens like S. typhimurium by enhancing the growth of probiotics that produce organic acids, which lower pH and create unfavorable conditions for pathogen survival [ 60 , 62 ]. A compound is classified as a prebiotic if it has a prebiotic index greater than 1 and a positive prebiotic activity score, indicating selective support for beneficial bacteria over harmful ones [ 22 , 63 ] is shown in Table 3 . According to the data, Lactobacillus fermentum demonstrated the most positive response to corncob-derived XOS, reflected in its prebiotic index and activity scores. In contrast, Bifidobacterium sp. and Lactobacillus rhamnosus exhibited significantly lower responses. The prebiotic index values for these two strains were less than 1, and their prebiotic activity scores were negative, except for Bifidobacterium sp. at 72 hours. The prebiotic index values for all bacterial strains peaked within the first 24 hours, indicating that XOS-derived prebiotics effectively promoted bacterial growth during this period. At 48 and 72 hours, a relative decline was observed, likely due to rapid bacterial growth in the glucose-containing positive control medium, which surpassed the growth of both Lactobacillus fermentum and Lactobacillus rhamnosus in XOS. However, Bifidobacterium sp. continued to grow at 72 hours, likely because utilizing XOS as its carbon source helped sustain its logarithmic phase. In contrast, in the glucose-containing positive control medium, Bifidobacterium sp. had already reached the stationary phase. Table 3 Prebiotic index and prebiotic activity of corncob XOS L. fermentum Bifidobacterium sp. L. rhamnosus Corncob XOS Prebiotic index 24 h 1.10 0.44 0.48 48 h 0.40 0.41 0.39 72 h 0.42 0.59 0.32 Prebiotic activity 24 h 0.67 -0.19 -0.15 48 h 0.12 -0.01 -0.03 72 h 0.13 0.15 -0.12 These findings suggest that L. fermentum has a superior capability to utilize XOS from corncob compared to the other tested probiotic strains. Additionally, the prebiotic activity results indicate that the growth of beneficial bacteria is more pronounced relative to the growth of the pathogenic S. typhimurium . This highlights the potential of corncob-derived XOS as an effective prebiotic for promoting gut health and mitigating pathogen proliferation. 4. Conclusion This study highlights the effectiveness of using potash alum as a catalyst in a microwave-assisted hydrolysis process to extract XOS from corncob waste. The optimized pretreatment conditions significantly enhanced XOS yield while demonstrating a sustainable approach to agricultural byproduct valorization. The purified XOS exhibited remarkable functional properties, including strong antioxidant activity and the ability to promote beneficial probiotic growth, with L. fermentum showing the highest prebiotic response. The results suggest that the balanced catalytic action of potash alum can facilitate selective hemicellulose hydrolysis while limiting XOS degradation, contributing to a cost-effective and scalable production process. This work supports the potential of corncob-derived XOS as a multifunctional ingredient in health-related applications, promoting both gut health and oxidative stability in food and nutraceutical formulations. Future research could explore scaling up the process and investigating the synergistic effects of XOS in combination with other bioactive compounds. Declarations Author contribution Conceptualization: Hana Nur Fitriana, Umar Seno Aji, Khaswar Syamsu; methodology: Umar Seno Aji, Haqqyana, Isti Qomariah; formal analysis and investigation: Haqqyana, Dewi Sondari, Isti Qomariah; writing—original draft preparation: Umar Seno Aji, Hana Nur Fitriana, Haqqyana; writing—review and editing: Hana Nur Fitriana, Riksfardini A. Ermawar, Khaswar Syamsu; funding acquisition: Hana Nur Fitriana; resources: Riksfardini A. Ermawar, Dewi Sondari; supervision: Hana Nur Fitriana Funding This work was supported by the RIIM LPDP Grant and BRIN (Grant Numbers: B-803/II.7.5/FR/6/2022 and B-1373/III.5/PR.03.08/6/2022), received by Author Hana Nur Fitriana. Acknowledgment We sincerely thank the RIIM LPDP Grant and BRIN for their financial support. Our gratitude also goes to the Integrated Laboratory of Bioproducts, National Research and Innovation Agency (BRIN)—formerly the Indonesian Institute of Sciences (LIPI)—for providing essential facilities and invaluable scientific and technical assistance throughout this study. Data availability Data will be made available on reasonable request Declaration This article contains no studies with human participants or animals performed by any of the authors. Conflict of interest The authors declare no competing interests. References Samanta AK, Senani S, Kolte AP, et al (2012) Production and in vitro evaluation of xylooligosaccharides generated from corn cobs. Food Bioprod Process 90:466–474. https://doi.org/10.1016/j.fbp.2011.11.001 Bian J, Peng F, Peng XP, et al (2013) Structural features and antioxidant activity of xylooligosaccharides enzymatically produced from sugarcane bagasse. 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Ber Biol 22:51–59. https://doi.org/10.55981/beritabiologi.2023.806 Supplementary Files HNFGraphicalAbstract.jpeg Cite Share Download PDF Status: Published Journal Publication published 10 Jul, 2025 Read the published version in Waste and Biomass Valorization → Version 1 posted Reviewers agreed at journal 01 Apr, 2025 Reviewers invited by journal 28 Mar, 2025 Editor invited by journal 22 Mar, 2025 Editor assigned by journal 08 Mar, 2025 First submitted to journal 04 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6158861","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":435517225,"identity":"7bfccb1d-2308-4229-868f-02622b8ff48a","order_by":0,"name":"Umar Seno Aji","email":"","orcid":"","institution":"LIPI: Lembaga Ilmu Pengetahuan Indonesia","correspondingAuthor":false,"prefix":"","firstName":"Umar","middleName":"Seno","lastName":"Aji","suffix":""},{"id":435517226,"identity":"5f63f4ca-c28c-408e-8134-43b0527d166f","order_by":1,"name":"Hana Nur Fitriana","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACgwNAIsHgn5wBA2MDWISNIYEILR8KDhiTpoVxxocDiRsQYoS0HD978DOPwZ307dKH2x4w/LJh4GMnoMX+TF6yNI/Bs9ydfYntBox9aQxsPA8IOSzHAKiFOXfDGcY2CcaewwxsEoQcdv6N8W+glnQD4rXcyDGTnGFwOAGsheEHUVremFl8MEgz3NkD1JLYkMZD2C/nc4xvJPyxkTfnYX8m8eGPjZx8OwFbUEFiGwMPKepB4A+pGkbBKBgFo2AkAAAdw0X5mNX6ugAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6435-0275","institution":"LIPI: Lembaga Ilmu Pengetahuan Indonesia","correspondingAuthor":true,"prefix":"","firstName":"Hana","middleName":"Nur","lastName":"Fitriana","suffix":""},{"id":435517227,"identity":"a66a421c-8432-46dc-a66d-6f3b57cc3c8f","order_by":2,"name":"Haqqyana Haqqyana","email":"","orcid":"","institution":"LIPI: Lembaga Ilmu Pengetahuan Indonesia","correspondingAuthor":false,"prefix":"","firstName":"Haqqyana","middleName":"","lastName":"Haqqyana","suffix":""},{"id":435517228,"identity":"1c3d71e3-c3c9-47a8-8c36-860daf4a4a51","order_by":3,"name":"Khaswar Syamsu","email":"","orcid":"https://orcid.org/0000-0002-5113-8830","institution":"IPB University: Institut Pertanian Bogor","correspondingAuthor":false,"prefix":"","firstName":"Khaswar","middleName":"","lastName":"Syamsu","suffix":""},{"id":435517229,"identity":"8d949ed5-5eab-4c70-9277-268eddbe1725","order_by":4,"name":"Riksfardini Annisa Ermawar","email":"","orcid":"https://orcid.org/0000-0001-6356-9999","institution":"LIPI: Lembaga Ilmu Pengetahuan Indonesia","correspondingAuthor":false,"prefix":"","firstName":"Riksfardini","middleName":"Annisa","lastName":"Ermawar","suffix":""},{"id":435517230,"identity":"e3cd71ec-f913-407a-b622-100e422bfd59","order_by":5,"name":"Dewi Sondari","email":"","orcid":"https://orcid.org/0000-0002-4923-2027","institution":"LIPI: Lembaga Ilmu Pengetahuan Indonesia","correspondingAuthor":false,"prefix":"","firstName":"Dewi","middleName":"","lastName":"Sondari","suffix":""},{"id":435517231,"identity":"e4d9ef91-7117-4542-8f82-8be4563ad4fd","order_by":6,"name":"Isti Qomariah","email":"","orcid":"","institution":"Universitas Indonesia Fakultas Matematika dan Ilmu Pengetahuan Alam","correspondingAuthor":false,"prefix":"","firstName":"Isti","middleName":"","lastName":"Qomariah","suffix":""}],"badges":[],"createdAt":"2025-03-05 04:57:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6158861/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6158861/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12649-025-03205-3","type":"published","date":"2025-07-10T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80835146,"identity":"0c687a8f-b526-4fb9-80ac-26e5389cb400","added_by":"auto","created_at":"2025-04-17 14:41:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44370,"visible":true,"origin":"","legend":"\u003cp\u003eReducing sugar, xylose, and xylooligosaccharide (XOS) concentrations, along with the xylose/XOS ratio, measured in the soluble fraction after pretreatment of corncob using varying potash alum concentrations, with heating time and temperature fixed at 5 minutes and 160°C, respectively.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/37903e95bd8f7c1b3e62859b.jpg"},{"id":80835768,"identity":"9e7d3261-1e8b-4f0f-92d4-bc404c027ce8","added_by":"auto","created_at":"2025-04-17 14:49:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31331,"visible":true,"origin":"","legend":"\u003cp\u003ePossible Reaction of the potash alum and hemicellulose during pretreatment\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/535cda54d5918002cd4c7e8f.jpg"},{"id":80835767,"identity":"ad4f5cfb-d3c9-4b82-8dd9-88f72240cd5a","added_by":"auto","created_at":"2025-04-17 14:49:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":45861,"visible":true,"origin":"","legend":"\u003cp\u003eReducing sugar, xylose, and xylooligosaccharide (XOS) concentrations, along with the xylose/XOS ratio, measured in the soluble fraction after varying heating temperature for the pretreatment of corncob, with heating time and potash alum concentration fixed at 5 minutes and 20 µmol/mL, respectively.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/736be91ea047679e9a827e0a.jpg"},{"id":80835148,"identity":"1fbff94e-1e6d-4f18-ba09-7200e18f5c3d","added_by":"auto","created_at":"2025-04-17 14:41:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41930,"visible":true,"origin":"","legend":"\u003cp\u003eReducing sugar, xylose, and xylooligosaccharide (XOS) concentrations, along with the xylose/XOS ratio, measured in the soluble fraction after varying heating time for the pretreatment of corncob, with potash alum concentration and temperature fixed at 20 µmol/mL and 160°C, respectively.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/d9c72d64a38eec393be7a487.jpg"},{"id":80836448,"identity":"37e53d6e-c30d-4d47-ae85-beaef975f281","added_by":"auto","created_at":"2025-04-17 14:57:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37715,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of untreated and pretreated corncob biomass with 20 µmol/ml potash alum at 160 °C for 5 min\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/06657d6f7ea3d18499216ea5.jpg"},{"id":80835157,"identity":"c6711020-eda1-4ac5-8987-a2ec9c593034","added_by":"auto","created_at":"2025-04-17 14:41:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":26360,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffractogram of untreated and pretreated corncob biomass with 20 µmol/ml potash alum at 160 °C for 5 min\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/e77f6b2c6ad34c10cf8c6d1b.jpg"},{"id":80836809,"identity":"7e99e2cb-9acf-4ac9-9558-83e3f744b244","added_by":"auto","created_at":"2025-04-17 15:05:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":66536,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs of untreated corncob biomass (a) and corncob biomass pretreated with 20 µmol/mL potash alum at 160°C for 5 minutes, at magnifications of 500x and 1000x\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/cf614c4740adc8dc2f0854c2.jpg"},{"id":80835771,"identity":"c938d64b-921e-404b-92e4-3bcefc5f2428","added_by":"auto","created_at":"2025-04-17 14:49:29","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":19288,"visible":true,"origin":"","legend":"\u003cp\u003eThin-layer chromatography (TLC) results showing the XOS powder fraction after purification. STD: XOS standards (X1: xylose; X2: xylobiose; X3: xylotriose; X4: xylotetraose; X5: xylopentaose; X6: xylohexose). L1: First precipitate; L2: Second precipitate; L3: Supernatant from ethanol precipitation steps.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/d04105465538a612b6d95104.jpg"},{"id":80835773,"identity":"da964387-25f0-4861-9031-7f26dab70158","added_by":"auto","created_at":"2025-04-17 14:49:30","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":62703,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of probiotic cultures on five different carbon sources—glucose, xylose, a combination of glucose and xylose, inulin, and corncob-derived XOS—represented as OD at 600 nm. \u003cem\u003eLactobacillus fermentum\u003c/em\u003e(a), \u003cem\u003eBifidobacterium sp.\u003c/em\u003e (b), and \u003cem\u003eL. rhamnosus\u003c/em\u003e (c). Bars represent the mean ± SE from duplicate experiments.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/28504b48f675181b74f78389.jpg"},{"id":80835774,"identity":"38a02690-ad47-4089-803f-985f7f4bd5f1","added_by":"auto","created_at":"2025-04-17 14:49:30","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":59931,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of pathogen cultures on five different carbon sources—glucose, xylose, a combination of glucose and xylose, inulin, and corncob-derived XOS—represented as OD at 600 nm. \u003cem\u003eS. aureus \u003c/em\u003e(a)\u003cem\u003e, S. typhimurium \u003c/em\u003e(b)\u003cem\u003e,\u003c/em\u003e and\u003cem\u003e E. coli \u003c/em\u003e(c). Bars represent the mean ± SE from duplicate experiments.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/c840d9fd953c7414031d6127.jpg"},{"id":86699561,"identity":"81162572-5d9d-4837-b246-87b7a8810a40","added_by":"auto","created_at":"2025-07-14 16:10:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2024713,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/25756f19-3aac-4baf-8298-620849459d4d.pdf"},{"id":80836808,"identity":"9f0845c2-412e-4636-9991-3dea4efd87f0","added_by":"auto","created_at":"2025-04-17 15:05:29","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":150211,"visible":true,"origin":"","legend":"","description":"","filename":"HNFGraphicalAbstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6158861/v1/4524238c487bc195f5b0d1eb.jpeg"}],"financialInterests":"","formattedTitle":"Microwave-Assisted Pretreatment of Corncob Using Potash Alum (AlK(SO4)2) Catalyst for Enhanced Xylooligosaccharide (XOS) Extraction with High Prebiotic and Antioxidant Properties","fulltext":[{"header":"Statement of Novelty","content":"\u003cp\u003eXylooligosaccharides (XOS) are valuable prebiotic compounds typically extracted using strong acids or enzymes, which can be costly and have environmental drawbacks. This study examines the use of potash alum (AlK(SO₄)₂) as a catalyst in microwave-assisted hydrolysis for XOS extraction. While potash alum has been explored in biomass processing, its application in XOS production remains underreported. This approach utilizes corncob, an abundant agricultural byproduct, as a feedstock and operates under relatively mild reaction conditions. The extracted XOS retains antioxidant activity (94.16% DPPH radical scavenging), high phenolic content, and demonstrates a high prebiotic index and prebiotic activity, particularly in stimulating \u003cem\u003eLactobacillus fermentum\u003c/em\u003e growth. These findings contribute to ongoing efforts to develop alternative catalysts for lignocellulosic biomass valorization and more sustainable XOS production methods.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eThe increasing global emphasis on environmental sustainability has driven research into the valorization of agricultural residues, particularly those that are abundant yet underutilized. Corncob waste, a major byproduct of the maize industry, remains largely underexploited despite its high hemicellulose content, particularly xylan. Traditionally, corncob is either discarded or used in low-value applications such as animal feed or fuel, missing the opportunity to convert it into high-value bioactive compounds. One such compound is xylooligosaccharides (XOS)\u0026mdash;non-digestible carbohydrates with well-documented prebiotic and antioxidant properties [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eXOS are sugar oligomers derived from the hydrolysis of xylan-rich hemicellulose. These oligomers feature a branched structure with diverse substituents such as arabinose, uronic acids, and acetyl groups, which are linked through β-(1,4) glycosidic bonds [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Recent studies highlight XOS\u0026rsquo;s potential in promoting gut health by selectively stimulating beneficial bacteria such as \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e species, as well as its antioxidant properties that make it valuable for functional food and nutraceutical applications [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, conventional XOS production methods\u0026mdash;including enzymatic, alkali, and acid hydrolysis\u0026mdash;face significant challenges, such as high operational costs, long reaction times, and environmental concerns related to chemical waste disposal. There is a critical need for more efficient, cost-effective, and environmentally friendly XOS extraction techniques. Microwave-assisted hydrolysis has emerged as a promising alternative due to its ability to rapidly break down biomass with reduced energy consumption and minimal byproduct formation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, studies have shown that integrating microwave irradiation with inorganic salts can enhance biomass pretreatment efficiency [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne such inorganic salt, potash alum (AlK(SO₄)₂), has been reported to facilitate lignin degradation by raising the dipole moment in plant cells, leading to β-O-4 ether cleavage and hemicellulose release [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Unlike strong acids or expensive enzymes, potash alum is non-toxic, cost-effective, and widely used in water purification [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], making it an attractive alternative for biomass hydrolysis. Despite its potential, its application as a catalyst in microwave-assisted XOS production has not been explored, leaving a significant gap in the development of efficient and scalable XOS extraction methods.\u003c/p\u003e \u003cp\u003eThis study aims to bridge this gap by investigating the feasibility of potash alum-catalyzed microwave-assisted hydrolysis for XOS production from corncob waste. By optimizing key reaction parameters\u0026mdash;potash alum concentration, heating time, and temperature\u0026mdash;using a One-factor-at-a-time (OFAT) approach, we seek to enhance XOS yield while maintaining its functional properties. Furthermore, we evaluate the prebiotic and antioxidant activities of the purified XOS, demonstrating its potential as a functional food ingredient. Our findings provide a sustainable and scalable strategy for agricultural waste utilization while introducing an alternative catalyst that could improve existing biomass pretreatment processes.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eCorncob was collected from farmers in Subang, West Java, Indonesia. The materials were naturally dried for 2\u0026ndash;3 days to reduce moisture content before processing. Corncob was then cut into 5 cm lengths and ground using a ring flaker (Pallmann Maschinenfabrik GmbH \u0026amp; Co. KG, Germany). To further reduce particle size, the material underwent additional grinding processes using a hammer mill and a disk mill (both from Pallmann Maschinenfabrik GmbH \u0026amp; Co. KG, Germany). The final product was passed through a sieve to obtain particles within the size range of 40 to 60 mesh (250\u0026ndash;420 \u0026micro;m). This study's catalyst used for treatment was potash alum (Merck, Darmstadt, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Optimization of XOS extraction using a combination of potash alum and microwave pretreatment\u003c/h2\u003e \u003cp\u003eThe extraction procedure for XOS consists of three distinct steps. In the first step, the concentration of potash alum was optimized, with the heating temperature and time fixed at 160\u0026deg;C and 5 minutes, respectively. Various concentrations of potash alum, ranging from 0 to 80 \u0026micro;mol/mL, were tested. In the second step, the heating temperature was optimized using the potash alum concentration determined in step 1, while the heating time was maintained at 5 minutes. Heating temperatures ranged from 140\u0026deg;C to 180\u0026deg;C. In the third step, the heating duration (3, 5, and 7 minutes) was optimized using the potash alum concentration from step 1 and the heating temperature identified in step 2.\u003c/p\u003e \u003cp\u003eOven-dried samples measuring (250\u0026ndash;420 \u0026micro;m) will be mixed with alum solution with the concentration required for each layer while maintaining a solid-to-solution ratio of 1:10 in a closed container. After microwave treatment, it is cooled to room temperature, and the dissolved and insoluble fractions are separated using a vacuum filter. The pulp was rinsed using distilled water until it reached normal pH and dried at 60 \u003csup\u003eo\u003c/sup\u003eC for further analysis. The soluble fraction (hydrolysate), which contains a lot of carbohydrate degradation, was analyzed for the content of reducing sugars, xylose, and XOS. This hydrolyzate is then processed further to be separated and purified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Separation and purification of XOS powder\u003c/h2\u003e \u003cp\u003eFollowing the pretreatment using a microwave-assisted potash alum catalyst, the hydrolyzate is concentrated using a rotary evaporator until it reaches one-third of the volume. Unhydrolized xylans were separated using ethanol precipitation, following the method of Vieira et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], with modifications. A two-step precipitation process was applied in this study, where one volume of ethanol was used for the first precipitation, followed by the addition of two volumes for the second. The supernatants from the second precipitation were collected, and ethanol was removed using a rotary evaporator. Each phase obtained at the purification step is freeze-dried and stored for further testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Combined severity factor (CSF)\u003c/h2\u003e \u003cp\u003eIn the microwave-assisted pretreatment, combined severity factor (CSF) calculation was used to integrate the effects of potash alum concentration, temperature, and reaction time in a single parameter. The CSF applied in this study is defined by Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e1\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eCSF\u0026thinsp;=\u0026thinsp;log {t.exp [(TH\u0026thinsp;\u0026minus;\u0026thinsp;TR)/14.75]} \u0026ndash; pH (1)\u003c/p\u003e \u003cp\u003eIn this equation, \u003cem\u003et\u003c/em\u003e represents the reaction time in minutes, \u003cem\u003eTH\u003c/em\u003e refers to the heating temperature in degrees Celsius (\u0026deg;C), \u003cem\u003eTR\u003c/em\u003e is the reference temperature set at 100\u0026deg;C, \u003cem\u003epH\u003c/em\u003e signifies the solution's acidity level, and 14.75 is a constant value related to the activation energy based on first-order kinetic modeling [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Analytical methods\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Analysis of the chemical compositions of corncob\u003c/h2\u003e \u003cp\u003eThe Van Soest method was employed to simultaneously analyze the hemicellulose, lignin, and cellulose contents, providing an estimation of the lignocellulose composition of corncob [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Determination of XOS, xylose, and reducing sugar concentration and yield in the pretreatment hydrolysate\u003c/h2\u003e \u003cp\u003eThe total reducing sugars were measured using the 3,5-dinitrosalicylic acid (DNS) method using a UV-Vis spectrophotometer (UV-1800, Shimadzu Scientific Instruments Inc., Kyoto, Japan) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Xylose and XOS concentrations were analyzed using a D-xylose assay kit (Megazyme, Wicklow, Ireland), with absorbance measured at 340 nm using a microplate reader (TECAN Infinite\u0026reg; 200 Pro). To determine the XOS content, the hydrolyzate is first hydrolyzed to xylose with 4% H₂SO₄ at 121\u0026deg;C for 1 hour, and xylose after hydrolysis is quantified [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The XOS concentration was calculated as the difference between xylose before and after acid hydrolysis of the hydrolyzate, following the procedure described in previous studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The hydrolysate's xylose to XOS ratio (xylose/XOS) was determined using a modified calculation method from Hao et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Determination of XOS fraction\u003c/h2\u003e \u003cp\u003eThe purification product was dissolved and filtered using SEPARA\u0026reg; syringeless filters (Zola Pedosa, Italy). The solution was then analyzed using thin layer chromatography (TLC) Silica Gel 60F254 plates (20 \u0026times; 20 cm, EMD/Merck, Darmstadt, Germany) and developed using an eluent consisting of n-butanol, acetic acid, and water in a 2:1:1 (v/v/v) ratio [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The spots were visualized using a diphenylamine-aniline-phosphoric acid reagent (Merck KGaA, Darmstadt, Germany) in acetone, followed by heating at 120\u0026deg;C for 15 minutes. The standards used included pure xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6), all sourced from Megazyme (Wicklow, Ireland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4 Determination of total phenolic content (TPC)\u003c/h2\u003e \u003cp\u003eThe Folin\u0026ndash;Ciocalteu (F-C) assay has been used for decades to measure total phenolics in natural products. Based on an oxidation/reduction mechanism, this method is commonly employed to evaluate antioxidant activity. Total phenolic content was measured using a modified spectrophotometric technique. XOS powder was dissolved in dimethyl sulfoxide (DMSO) at 2 mg/mL concentration. A 0.5 mL sample aliquot was mixed with 2.5 mL of F\u0026ndash;C reagent (Merck) and 5 mL of Na₂CO₃. The mixture is then dissolved in water until it reaches a final volume of 50 mL. The sample was then incubated in a water bath at 40\u0026deg;C for 30 minutes before spectrophotometric analysis. Absorbance at 750 nm was measured using a UV-Vis spectrophotometer (Model UV-1800, Shimadzu Corp., Kyoto, Japan). A blank was prepared using the same mixture, but the sample was replaced with 0.5 mL DMSO. The samples were prepared in duplicate, and the mean absorbance value was recorded. The same technique was applied for gallic acid. A calibration curve was constructed, and the total phenolic content was reported in terms of gallic acid equivalents (mg GA per gram of extract) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.5 Determination of antioxidant activity\u003c/h2\u003e \u003cp\u003eThe antioxidant activity of the XOS modification method, as described by Bian et al. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. A 0.1 mM DPPH solution in ethanol was mixed with an equal volume of the XOS solution, and the reaction was carried out in the dark with continuous stirring for 30 minutes. The absorbance of the mixture was measured at 517 nm using a UV-Vis spectrophotometer (UV-1800, Shimadzu Scientific Instruments Inc., Kyoto, Japan), and the antioxidant activity was determined using Eq.\u0026nbsp;\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{A}\\text{n}\\text{t}\\text{i}\\text{o}\\text{x}\\text{i}\\text{d}\\text{a}\\text{n}\\text{t}\\:\\text{a}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}\\:\\:\\left(\\%\\right)\\:=\\:1\\:-\\frac{absorbance\\:sample}{absorbance\\:control}\\:x\\:100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.6 Determination of prebiotic index and activity\u003c/h2\u003e \u003cp\u003eThe probiotic cultures tested included \u003cem\u003eLactobacillus fermentum\u003c/em\u003e, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, and \u003cem\u003eBifidobacterium\u003c/em\u003e sp. For comparison, tests were also conducted on pathogenic bacteria such as \u003cem\u003eSalmonella typhimurium, Escherichia coli\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e to evaluate their ability to utilize XOS and other available carbon sources and the antibiotic effect of XOS. The fermentation was conducted in a basal medium containing peptone (10 g/L), MnSO₄ (100 mg/L), MgSO₄ (50 mg/L), NH₄Cl (2 g/L), KH₂PO₄ (2 g/L), and Tween 80 (1 mL/L)) using four different carbon sources: glucose, xylose, a combination of glucose and xylose (1:1 ratio), and corncob XOS. The fermentations were incubated at 37\u0026deg;C, agitated at 180 rpm, and monitored at 0, 3, 6, 12, 24, 48, and 72 hours by measuring optical density at 600 nm.\u003c/p\u003e \u003cp\u003eThe prebiotic index was evaluated to assess each of four different carbon sources (2% w/v) for probiotics in basal medium, which was calculated using formula (1) to determine the efficacy of each extract as a carbon source for probiotics. Log P24 represents the probiotic bacterial optical density after 24 hours of incubation, while Log P0 is the optical density before incubation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:Prebiotic\\:index=\\:\\frac{\\left(Log\\:P24-Log\\:P0\\right)\\:extract}{\\left(Log\\:P24-Log\\:P0\\right)glucose}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe prebiotic activity was assessed to determine each XOS extract\u0026rsquo;s selectivity for supporting the growth of the probiotic rather than the pathogen. Probiotics and pathogens culture were inoculated into basal medium supplemented with XOS extract, using a 1% (v/v) inoculum, and filled into a 15 mL conical tube, followed by incubation at 37\u0026deg;C. Samples were taken before and after 24 hours of incubation, and measured the optical density at 600 nm.\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:Prebiotic\\:activity=\\left(\\frac{\\left(Log\\:P24-Log\\:P0\\right)extract}{\\left(Log\\:P24-Log\\:P0\\right)glucose}\\right)-\\left(\\frac{\\left(Log\\:E24-Log\\:E0\\right)extract}{Log\\:E24-LogE0)glucose}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eLog P24 represents the probiotic bacterial count after 24 hours of incubation, while Log P0 indicates the initial probiotic count before incubation. Similarly, Log E24 refers to the pathogen bacterial count after 24 hours, and Log E0 is the initial count of the pathogen before incubation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.7 Functional group determination of untreated and treated biomass\u003c/h2\u003e \u003cp\u003eThe functional groups in the biomass were examined using an attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectrometer (Perkin Elmer FTIR Spectrometer\u0026ndash;Spectrum Two, C 106456, Perkin Elmer Inc., Waltham, MA, USA). About 0.1 mg of biomass was placed on a diamond plate and compressed using a force gauge until the spectral data appeared on the computer screen. The analysis covered the range of 4000 to 400 cm⁻\u0026sup1; with a resolution of 4 cm⁻\u0026sup1;, 40 scans, and a force gauge setting of 90. The Perkin Elmer software was used to transform the IR spectra from transmittance to absorbance values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.5.8 Crystallinity determination of untreated and treated biomass using X-ray diffractogram\u003c/h2\u003e \u003cp\u003eDried samples of treated and untreated biomass were examined using an X-ray diffractometer (XRD) (Shimadzu XRD-7000 MaximaX series, Shimadzu Corp., Kyoto, Japan) with copper as the radiation source. The scanning angle was 5\u0026ndash;30\u0026ordm;, operated at 40.0 kV and 30.0 mA, with a reading speed of 2.0\u0026ordm; per minutes and a step size of 0.02\u0026ordm;. The degree of crystallinity was calculated using the Ruland-Vonk method, which reduces the amorphous component by comparing the area above the amorphous profile, which refers to the total sample area [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.5.9 Surface morphology analysis of untreated and treated biomass\u003c/h2\u003e \u003cp\u003eThe surface appearance of treated and untreated biomass was viewed using a JSM IT 200 Scanning Electron Microscope (JEOL Ltd., Tokyo, Japan). The sample is placed in a solid stainless tube using double-sided tape to ensure adhesion. After being coated with a thin layer of gold, the sample is placed in the sample port. After being coated with a thin layer of gold, the sample was positioned in the sample port of microscope. Images captured at 500 and 1000x magnifications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.5.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe catalysts impact on the concentrations of XOS, xylose, and reducing sugars after microwave pretreatment was assessed using a one-way analysis of variance (ANOVA). Parameters with significant F-values were further analyzed using Duncan\u0026rsquo;s Multiple Range Test (DMRT) at a 95% confidence level.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Result and Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Optimization of potash alum concentration\u003c/h2\u003e \u003cp\u003eTo optimize XOS recovery during microwave-assisted pretreatment, varying concentrations of potash alum (AlK(SO₄)₂) catalyst (0, 20, 40, 60, and 80 \u0026micro;mol/mL) were evaluated. The irradiation temperature and time were fixed at 160\u0026deg;C and 5 minutes, respectively. The potash alum concentration significantly influenced the yields of total reducing sugars, xylose, and XOS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The addition of potash alum enhanced all parameters up to an optimal concentration. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e, potash alum improved the total reducing sugar concentration. Without the catalyst, the reducing sugar content, including xylose and glucose, was low (3.67 g/L), suggesting that hydrolysis by high-temperature water alone was minimal under these conditions, consistent with previous findings [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. When potash alum concentration increased from 20 to 40 \u0026micro;mol/mL, the reducing sugar concentration rose from 14.67 to 29.95 g/L, highlighting the catalyst's role in enhancing hydrolysis. Further increases in catalyst concentration continued to raise the reducing sugar yield, though with diminishing returns, likely due to saturation of available hydrolysis sites [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe XOS yield showed a different pattern. It increased significantly at 20 \u0026micro;mol/mL, reaching 12.32 g/L, but declined sharply to 3.21 g/L at 60 \u0026micro;mol/mL. Although a slight increase to 4.21 g/L was observed at 80 \u0026micro;mol/mL, this change was insignificant. The reduced XOS yield at higher catalyst levels suggests that excessive potash alum catalyzed further hydrolysis of XOS into smaller sugars, primarily xylose [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eXylose yield increased with potash alum concentration up to 40 \u0026micro;mol/mL but decreased at higher levels. The yield peaked at 16.48 g/L at 40 \u0026micro;mol/mL, then dropped to 16.35 g/L and 15.58 g/L at 60 and 80 \u0026micro;mol/mL, respectively. This trend indicates that moderate catalyst concentrations promote hemicellulose breakdown and XOS conversion to xylose, but excessive catalyst leads to xylose degradation into byproducts like furfural [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Despite the decline in XOS and xylose yields at higher potash alum concentrations, the total reducing sugar yield continued to increase, indicating additional sugar production from cellulose hydrolysis.\u003c/p\u003e \u003cp\u003eAt 20 \u0026micro;mol/mL potash alum (CSF \u0026minus;\u0026thinsp;1.19), the xylose/XOS ratio was 1.09, indicating efficient XOS production with minimal degradation. This agrees with Akpinar et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], who noted that mild acid conditions favor XOS formation. However, at 60 \u0026micro;mol/mL (CSF \u0026minus;\u0026thinsp;1.00), the ratio surged to 5.10, consistent with Pramasari et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], who reported that harsher acid environments promote extensive XOS hydrolysis into xylose. These findings underscore moderate catalyst loading optimally depolymerizes hemicellulose without excessive XOS breakdown.\u003c/p\u003e \u003cp\u003eThe synergistic effect of Br\u0026oslash;nsted and Lewis acids from aluminum salt catalysts, such as (AlK(SO₄)₂) in a water-only medium, promotes the cleavage of intermolecular linkages in lignocellulosic complexes. This mechanism facilitates the dissolution of hemicellulose into XOS and subsequently into reducing sugars like xylose and glucose. Luo et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] discussed the role of water and aluminum sulfate (Al₂(SO₄)₃) in the selective catalytic transformation of corn stover hemicellulose into xylose. Water molecules form hydrogen bonds with the intermolecular linkages of hemicellulose-cellulose-lignin in biomass, weakening these linkages and enabling hemicellulose release. Aluminum salts enhance this effect by hydrolyzing in water to produce species like [Al(OH)₂(H₂O)x]⁺, which act as Lewis acids, promoting selective hemicellulose solubilization. Aluminum species may also form hydrogen-bonded complexes with -OH\u003csub\u003e1\u003c/sub\u003e and C\u003csub\u003e1\u003c/sub\u003e\u0026ndash;O\u003csub\u003e6\u003c/sub\u003e of the xylan unit, stabilizing xylose and reducing its degradation while promoting its formation with the assistance of Br\u0026oslash;nsted acid species (H⁺ ions) from the aluminum salt hydrolysis. Based on this Luo et al.'s study, a proposed reaction mechanism for XOS extraction using potash alum is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies reported that Al₂(SO₄)₃ in a water system significantly improved hemicellulose-to-xylose conversion, with yields reaching 85.1 wt% from corn stover hydrolysis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Hermiati et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] combined microwave heating with Al₂(SO₄)₃ in a water medium for sugarcane trash pretreatment, observing substantial increases in reducing sugar and xylose yields with increasing catalyst loads. However, no prior studies have explored using potash aluminum from lignocellulosic materials for XOS production.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Optimization of heating temperature\u003c/h2\u003e \u003cp\u003eMicrowave pre-treatment of corncob was conducted at temperatures of 140, 160, and 180\u0026deg;C for 5 minutes. A potash alum concentration of 20 \u0026micro;mol/mL was selected as it was identified as the optimal value for maximizing XOS yield. The statistical analysis revealed that temperature changes significantly impacted XOS production (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results showed total reducing sugars increased with temperature, reaching a peak yield of 37.74 g/L at 180\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A similar trend was observed for xylose, which exhibited a positive correlation with higher temperatures, achieving a maximum of 18.95 g/L at 180\u0026deg;C. These findings suggest that elevated temperatures enhance reducing sugars and xylose release from biomass.\u003c/p\u003e \u003cp\u003ePre-treating biomass at higher temperatures under microwave irradiation disrupts the fibre structure, facilitating the breakdown of hemicellulose [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. As a low molecular weight, short-branched polymer, hemicellulose contains linkages with lower activation energy, making its structure particularly susceptible to thermal degradation under high temperatures [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This leads to hemicellulose becoming readily solubilized, enabling further hydrolysis into its monomeric components, such as xylose, or forming oligosaccharides like XOS.\u003c/p\u003e \u003cp\u003eWhile higher temperatures increased the overall release of sugar, XOS exhibited a different behaviour under high heating. The XOS yield increased from 3.38 g/L at 140\u0026deg;C to 11.18 g/L at 160\u0026deg;C but declined sharply to 5.59 g/L at 180\u0026deg;C. Notably, the xylose yield increased significantly beyond 160\u0026deg;C, suggesting that XOS degraded into smaller molecules, such as xylose, at higher temperatures. A similar result was also reported in previous studies [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTemperature strongly correlates with CSF, with higher temperatures resulting in higher CSF values because pretreatment conditions become more severe. At low temperatures (140\u0026deg;C, CSF \u0026minus;\u0026thinsp;1.78), the xylose/XOS ratio was relatively low (1.58), indicating a balanced production of XOS without significant degradation. However, at 160\u0026deg;C (CSF \u0026minus;\u0026thinsp;1.19), the ratio increased slightly to 1.72, as XOS degradation co-occurred with its production. Qin et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] reported that low temperature produced large soluble polymers and xylose, while the XOS with a degree of polymerization (DP) of 2\u0026ndash;5 remains low. As the temperature increases, the production rate of XOS surpasses that of xylose, leading to a decline in the xylose/XOS ratio. At 180\u0026deg;C (CSF \u0026minus;\u0026thinsp;0.61), the ratio spiked to 3.39, coinciding with the sharp decline in XOS yield and a significant increase in xylose yield. A further temperature rise enhances the rate of XOS degradation, causing an increase in the xylose/XOS ratio. It was previously reported that increased temperature facilitated the formation of X2-X3 oligosaccharides by promoting the hydrolysis of higher DP (X4-X6) XOS into lower DP XOS and xylose [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, while elevated temperatures were beneficial to hemicellulose degradation and enhanced overall sugar release, an optimal temperature of 160\u0026deg;C is critical for maximizing XOS yield while minimizing degradation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Optimization of heating time\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the yields of reducing sugars, xylose, and XOS resulting from microwave pre-treatment of corncob at irradiation times ranging from 3 to 7 minutes. The experiments were conducted using a potash alum concentration of 20 \u0026micro;mol/ml and a constant heating temperature of 160\u0026deg;C. The CSF values increased with longer irradiation times, rising from \u0026minus;\u0026thinsp;1.42 at 3 minutes to -1.19 at 5 minutes and further to -1.05 at 7 minutes. This increase in CSF indicates a gradual rise in the severity of the treatment over longer microwave heating durations.\u003c/p\u003e \u003cp\u003eThe results show that all three products achieved their highest yields at 5 minutes of irradiation, while the shortest duration of 3 minutes produced the lowest yields. Specifically, at 5 minutes, the reducing sugar concentration peaked at 14.42 g/L, xylose reached 12.99 g/L, and XOS achieved its maximum value at 11.47 g/L. Extending the microwave heating to 7 minutes reduced all yields, likely due to degradation under prolonged exposure. Previous studies also reported similar results, noting that prolonged heating did not improve XOS production but instead resulted in increased degradation of XOS into xylose [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Xylose/XOS ratio gradually decreased with time, starting at -1.24 at 3 minutes, reducing to -1.13 at 5 minutes, and reaching \u0026minus;\u0026thinsp;1.02 at 7 minutes. This declining trend indicates longer heating time favoured XOS retention relative to xylose formation. Additionally, longer acid hydrolysis times have been reported to increase the rate of XOS hydrolysis into lower molecular weight oligosaccharides and monosaccharides [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Lin et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] observed that prolonging the irradiation time led to higher levels of X2 and X3, while reducing the levels of X5 and X6. These findings suggest that the DP distribution for the XOS also depends on heating time. Extending the heating duration enhances hemicellulose breakdown, but sufficient heating time is required to achieve optimal XOS yields. In this study, five minutes of irradiation time was identified as an optimal duration to produce the highest yields of XOS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Catalytic effects of potash alum catalyst\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Corncob chemical component changes\u003c/h2\u003e \u003cp\u003eThe chemical composition of corncob biomass undergoes significant changes after pretreatment with a 20 \u0026micro;mol/mL potash alum catalyst at 160\u0026deg;C for 5 minutes, compared to the untreated material (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The hemicellulose content decreased sharply by 22%, from 36.9% \u0026plusmn; 0.5 to 14.9% \u0026plusmn; 0.1, indicating the breakdown and solubilization of hemicellulose into oligomers, simpler sugars, and other by-products during the treatment. In contrast, the cellulose content increased by 15.7%, rising from 42.6% \u0026plusmn; 0.1 to 58.3% \u0026plusmn; 0.2, while lignin content grew by 4.3%, from 8.4% \u0026plusmn; 0.1 to 12.7% \u0026plusmn; 0.3. This increase in cellulose and lignin proportions results primarily from the substantial loss of hemicellulose, reducing the overall biomass mass and leaving cellulose and lignin to comprise a greater percentage of the remaining material.\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\u003eChanging in chemical components of untreated and treated corncob\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eComponents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCorncob\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUntreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreated\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\u003eCellulose (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHemicellulose (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLignin (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAsh (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\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\u003eThe observed shifts in biomass composition differ from findings by Simanullang et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], who reported a 31% reduction in hemicellulose, a 5% reduction in lignin, and an 11.1% reduction in cellulose when using a higher potash alum concentration (30 \u0026micro;mol/mL) and a more severe pretreatment temperature (200\u0026deg;C) on sugarcane trash. These more intense conditions led to the production of reducing sugars rather than oligomers (XOS), as targeted in this study. The discrepancy highlights how pretreatment parameters influence the extent of biomass hydrolysis and the nature of the final products.\u003c/p\u003e \u003cp\u003eThe differing outcomes between these studies can be attributed to the distinct catalytic mechanisms of potash alum, which behaves as a Lewis acid during biomass pretreatment. Potash alum (AlK(SO₄)₂) provides aluminum ions that act as Lewis acid sites, breaking ether bonds within hemicellulose structures by coordinating with oxygen atoms in the polymer chains. In acidic environments, metal ions from the catalyst function analogously to hydronium ions in Lowry-Br\u0026oslash;nsted acids, enhancing the depolymerization of hemicellulose into smaller sugars [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. A related Lewis acid, aluminum sulfate (Al\u003csub\u003e2\u003c/sub\u003e(SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e), has been shown to selectively hydrolyze hemicellulose with minimal impact on cellulose and lignin, as demonstrated by Luo (2020). In that study, the hydrolysis of Al\u003csub\u003e2\u003c/sub\u003e(SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e generated H\u003csup\u003e+\u003c/sup\u003e ions, accelerating hemicellulose breakdown into xylose. Simultaneously, the formation of active aluminum species [Al(OH)\u003csub\u003e2\u003c/sub\u003e(H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003ex\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e promoted selective hemicellulose dissolution and protected xylose from further degradation through complex formation with hydroxyl groups in xylan units. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This dual catalytic action illustrates how Lewis acids, including potash alum, drive the selective transformation of biomass components depending on treatment severity and target products.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Corncob functional group changes\u003c/h2\u003e \u003cp\u003eThe treated corncob sample underwent microwave-assisted pretreatment using a potash alum catalyst at 160\u0026deg;C for 5 minutes with a catalyst concentration of 20 \u0026micro;mol/mL. This process induced significant structural and chemical changes, as evidenced by differences in the FTIR spectra between untreated and treated samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e), particularly at key peaks corresponding to functional groups involved in the biomass structure.\u003c/p\u003e \u003cp\u003eAt peak 1, the frequency shifted from 3340.52 cm⁻\u0026sup1; to 3333.84 cm⁻\u0026sup1;, while intensity increased from 94.84\u0026ndash;95.51%, demonstrating the existence of O-H stretching vibrations in cellulose [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The frequency reduction suggests stronger hydrogen bonding interactions, likely due to enhanced interaction between hydroxyl groups. Simultaneously, the increased intensity implies a higher concentration of accessible O-H functional groups.\u003c/p\u003e \u003cp\u003ePeak 2 exhibited a frequency decrease from 2916.19 cm⁻\u0026sup1; to 2903.95 cm⁻\u0026sup1;, with a slight rise in intensity from 96.75\u0026ndash;97%. This peak represents C-H stretching vibrations in alkanes, and the changes indicate subtle modifications in the local chemical environment, potentially resulting from hemicellulose deconstruction [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMinimal changes at peaks 3 (1602.89 cm⁻\u0026sup1;) and 4 (1513.22 cm⁻\u0026sup1; to 1513.26 cm⁻\u0026sup1;) suggest stability in C\u0026thinsp;=\u0026thinsp;C aromatic ring vibrations, possibly related to limited lignin removal. The small intensity reduction at peak 4 (from 96.83\u0026ndash;96.41%) further supports this hypothesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. A significant frequency shift occurred at peak 5, from 1369.72 cm⁻\u0026sup1; to 1424.67 cm⁻\u0026sup1;, indicating significant structural alterations involving C-H deformation or C-O stretching associated with alcohols or esters. This shift suggests substantial transformations in hemicellulose or other non-cellulosic polymers. Peak 6 displayed a slight frequency decrease (from 1317.13 cm⁻\u0026sup1; to 1315.9 cm⁻\u0026sup1;) with stable intensity, indicating minor changes in functional group environments. Peak 7 (1243.5 cm⁻\u0026sup1;) remained nearly unchanged in frequency, with a slight intensity increase, highlighting the stability of C-O stretching vibrations in alcohols or esters. At peak 8 (1158.53 cm⁻\u0026sup1; to 1159.28 cm⁻\u0026sup1;), the frequency remained stable, but intensity decreased, suggesting a reduction in the quantity of C-O-C groups. This region corresponds to the β-glycosidic linkage in xylan, a key component of hemicellulose [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConversely, peak 9 (1032.72 cm⁻\u0026sup1; to 1030.73 cm⁻\u0026sup1;) showed a decreased frequency with increased intensity, indicating greater involvement of C-O-C groups, possibly due to structural rearrangements following hemicellulose breakdown. Peak 10 (897.52 cm⁻\u0026sup1;) associated with O\u0026ndash;H bending showed minimal frequency change but slightly increased intensity, reflecting stability in β-glycosidic linkages within cellulose [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Finally, peak 11 exhibited a substantial frequency shift from 528.07 cm⁻\u0026sup1; to 558.85 cm⁻\u0026sup1;, suggesting significant structural changes potentially involving interactions between biomass components and the alum catalyst. The emergence of two new peaks at 519.32 cm⁻\u0026sup1; and 441.87 cm⁻\u0026sup1;, replacing the single peak at 441 cm⁻\u0026sup1; in the untreated sample, indicates the formation of new structures or modifications involving inorganic compounds. Together, these spectral changes illustrate the intricate chemical transformations driven by potash alum catalysis, particularly in hemicellulose deconstruction and selective functional group modifications, enhancing cellulose accessibility while stabilizing certain structural elements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.4.3 Corncob crystallinity changes\u003c/h2\u003e \u003cp\u003eLignocellulosic biomass primarily consists of hemicellulose, lignin, and cellulose. The structural arrangement of these components influences the degree of crystallinity in biomass. Cellulose contains both crystalline and amorphous regions, while hemicellulose and lignin are entirely amorphous [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Changes in the composition and structure of these components can significantly affect the crystallinity index of the biomass [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe crystallinity index of untreated corncob was measured at 45.92%, whereas microwave pretreatment combined with AlK(SO₄)₂ as a catalyst increased the crystallinity to 49.3% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This enhancement suggests that the pretreatment process effectively dissolved some amorphous components, including hemicellulose, lignin, and non-crystalline cellulose regions. The chemical composition analysis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) supports this observation, showing a nearly 60% reduction in hemicellulose content after pretreatment. Additionally, the cellulose content increased by 37%, contributing to the higher crystallinity observed in the treated corncob.\u003c/p\u003e \u003cp\u003eThe X-ray diffraction (XRD) spectra reveal more intense and sharper diffraction peaks at 2θ angles of 10\u0026deg;\u0026ndash;17\u0026deg;, 20\u0026deg;\u0026ndash;25\u0026deg;, and 32\u0026deg;\u0026ndash;37\u0026deg; for the pretreated corncob (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating enhanced crystallinity. These more defined peaks confirm the structural modifications induced by the pretreatment. The reduction in amorphous content improves cellulose accessibility by minimizing structural hindrances to hydrothermal hydrolysis, making the treated biomass more suitable for enzymatic processing and conversion into value-added products.\u003c/p\u003e \u003cp\u003ePretreatments involving acids or inorganic catalysts enhance the crystallinity index by selectively removing amorphous components. This structural modification facilitates enzymatic saccharification, as the enzymes can more effectively penetrate the biomass matrix and access the crystalline cellulose through the void spaces created by the solubilization of amorphous regions [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.4.4 Corncob morphological structure changes\u003c/h2\u003e \u003cp\u003eScanning Electron Microscope (SEM) analysis was performed to assess the effect of this treatment on the surface structure and crystallinity of untreated and treated corncob biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In the SEM images of untreated samples, the corncob surface appeared rough and irregular, with a dense, non-porous structure, reflecting the undegraded lignocellulosic nature of the biomass. The intact lignin fibers formed a tight network, limiting access to enzymes or chemical reagents [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, the SEM images of treated samples revealed significant lignin degradation and structural changes in hemicellulose and cellulose. The treated samples displayed a more porous surface, indicating that the bonds between lignin and cellulose had broken down, allowing for increased cellulose crystallinity. This observation aligns with the XRD data, which shows enhanced crystallinity in the treated samples, confirming the breakdown of amorphous components. The increased porosity in the treated samples facilitates enzymatic hydrolysis, as enzymes can more readily access the now-open cellulose structure, potentially improving biomass conversion efficiency [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, the reduction of lignin and hemicellulose may impact on the thermal and chemical stability of the biomass, increasing its susceptibility to more destruction or chemical reactions during subsequent processing.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Fractionation of XOS powder\u003c/h2\u003e \u003cp\u003eThe XOS hydrolysate obtained under optimal pretreatment conditions was purified using ethanol precipitation and freeze-dried to produce XOS powder. The fractionation of XOS in the powder was analyzed by thin-layer chromatography (TLC), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Fractionation analysis of precipitates 1 and 2 revealed minimal differences, with both samples displaying only two prominent bands corresponding to X1 and X2. In contrast, the XOS powder from the supernatant exhibited more distinct fractionation, with clear bands ranging from X1 to X6 and prominent bands observed at X1 and X2. Stepwise ethanol precipitation during purification enhances the accumulation of low-degree polymerization (DP) XOS in the supernatant, making it suitable for use as a prebiotic [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, the TLC pattern of the XOS powder from precipitates 1 and 2 and the supernatant deviates from the findings of Valls et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], who reported that precipitate 1 primarily contained unhydrolyzed xylan, while precipitate 2 was enriched with long-chain xylooligomers. Despite using the same ethanol precipitation method for purification, the observed differences are likely due to substrate and production method variations. Valls et al. utilized commercial xylan as the substrate and enzymatic hydrolysis for XOS production, a method associated with high costs. In contrast, the present study extracted XOS directly from corncob biomass using microwave pretreatment with a potash alum catalyst. This difference in feedstock and processing conditions may have influenced the composition and fractionation patterns of the XOS products.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Antioxidant activity of corncob XOS powder\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the scavenging capability of XOS powder derived from the first precipitant, second precipitant, and supernatant. The supernatant, which yields the highest amount of XOS (16.97%), demonstrates the strongest antioxidant activity (94.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54), while the first and second precipitants exhibit lower antioxidant activities. According to Valls et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], the enhanced antioxidant activity in the supernatant can be attributed to its higher concentration of longer xylooligomers, which possess superior antioxidant properties. This finding is corroborated by the TLC analysis of XOS powder fractions in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Moreover, the elevated antioxidant activity of the supernatant aligns with its significantly higher total phenolic content (32.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 \u0026micro;g GAE/mg of XOS) compared to the first and second precipitants. Previous investigations have demonstrated the significant antioxidant action of hemicellulose hydrolysates, especially those enriched with polyphenols [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Studies have demonstrated that phenolic chemicals such as coumaric acid, syringic acid, ferulic acid, and caffeic acid neutralize free radicals by generating stable radicals [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. These results imply that the XOS produced in this investigation has increased antioxidant features, mostly due to its enhanced phenolic content, which might have potential uses in many domains of interest.\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\u003eAntioxidant activity, total phenolic content, and yield of the product after the purification process of corncob pretreatment at 160\u0026deg;C for 5 minutes using a 20 \u0026micro;mol/mL potash alum catalyst.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthanol purification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntioxidant activities\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal phenolic content\u003c/p\u003e \u003cp\u003e(\u0026micro;g GAE/mg of XOS)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYield XOS product\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\u003ePrecipitant 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrecipitant 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSupernatant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e94.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ec\u003c/sup\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 \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Prebiotic activity of corncob XOS powder\u003c/h2\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.7.1 In vitro growth and fermentation of XOS and other carbon sources by probiotics\u003c/h2\u003e \u003cp\u003eThe ability of three probiotic strains\u0026mdash;\u003cem\u003eLactobacillus fermentum\u003c/em\u003e, \u003cem\u003eL. rhamnosus\u003c/em\u003e, and \u003cem\u003eBifidobacterium\u003c/em\u003e sp.\u0026mdash;to metabolize different carbon sources was evaluated over 72 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Each strain exhibited unique growth patterns depending on the substrate provided. \u003cem\u003eLactobacillus fermentum\u003c/em\u003e demonstrated adaptive growth on complex carbohydrates such as XOS, although overall growth was lower than the other two strains across all substrates. Interestingly, \u003cem\u003eL. fermentum\u003c/em\u003e exhibited limited growth on glucose and a glucose-xylose combination, showing a notable aversion to these simpler sugars. However, its growth on xylose was nearly double that observed on glucose or the glucose-xylose mixture, indicating a preference for xylose.\u003c/p\u003e \u003cp\u003eContrary to these results, previous studies report that \u003cem\u003eL. fermentum\u003c/em\u003e favors glucose over xylose. This preference is facilitated by carbon catabolite repression (CCR), a regulatory system determined by the catabolite control protein A (CcpA) that binds to catabolite-responsive elements (CREs) in the promoter areas of sugar metabolism operons, such as the xyl operon for xylose. When glucose is abundant, CcpA activates CCR, repressing the xyl operon and inhibiting xylose utilization [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. However, Zhang et al. [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], observed that when CREs are deficient, CCR is incomplete, allowing \u003cem\u003eL. fermentum\u003c/em\u003e to co-metabolize glucose and xylose, demonstrating its metabolic flexibility. In addition to glucose and xylose, \u003cem\u003eL. fermentum\u003c/em\u003e efficiently metabolizes more complex substrates like XOS. Growth on XOS exhibited a distinct lag phase before steady growth began. Notably, the specific growth rate on XOS (0.0012) indicates its efficient utilization. The ability to metabolize these complex carbohydrates is supported by increased expression of sugar metabolism genes in the presence of XOS, highlighting \u003cem\u003eL. fermentum\u003c/em\u003e\u0026rsquo;s metabolic adaptability.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBifidobacterium\u003c/em\u003e sp. displayed significant variability in growth depending on the carbon source. Glucose supported the most rapid and highest growth, with a marked increase after 12 hours. Xylose and XOS also promoted substantial growth, with xylose showing a consistent growth pattern and XOS demonstrating a steady increase after an initial lag phase of approximately 3 hours. Moderate growth was observed on the glucose-xylose combination, with noticeable increases after 24 hours. The results reported here correspond with previous research demonstrating that glucose is the most preferred carbon source for various \u003cem\u003eBifidobacterium\u003c/em\u003e species, while xylose and XOS also contribute positively to growth [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The observed differences in substrate-dependent growth patterns highlight the importance of strain-specific metabolic capabilities and carbohydrate availability in influencing the efficiency of \u003cem\u003eBifidobacterium\u003c/em\u003e growth.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e demonstrated distinct growth trends across various carbon sources. Glucose supported the most rapid and significant growth, with a substantial increase observed after 12 hours and continuing through 48 hours. This result is consistent with the findings of Petrut et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], who identified glucose as the most favorable substrate for \u003cem\u003eL. rhamnosus\u003c/em\u003e. The glucose-xylose combination also promoted robust growth, with consistent increases starting at 12 hours. In contrast, growth on xylose was the lowest but remained steady throughout the 72-hour incubation period, indicating a lower metabolic efficiency for xylose compared to glucose-based substrates. Moderate growth was observed on XOS derived from corncob, with the most pronounced increases between 24 and 48 hours, reflecting a delayed but sustained fermentation response. These findings highlight the metabolic versatility of \u003cem\u003eL. rhamnosus\u003c/em\u003e, which can adapt to a range of carbon sources, with glucose supporting the most efficient growth and XOS showing potential prebiotic benefits due to its moderate but sustained fermentation. This adaptability makes \u003cem\u003eL. rhamnosus\u003c/em\u003e a promising candidate for probiotic applications targeting carbohydrate-rich environments.\u003c/p\u003e \u003cp\u003eAll three probiotic strains exhibited positive growth on XOS, characterized by high specific growth rates. This finding is in agreement with Yan et al. [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], which demonstrated that XOS derived from various agricultural by-products effectively stimulates the growth of intestinal probiotics, including lactic acid bacteria and \u003cem\u003eBifidobacteria\u003c/em\u003e. These bacteria ferment XOS to generate short-chain fatty acids (SCFAs), such as butyrate, acetate, and propionate which confer numerous beneficial health effects on the host, notably increased immunological function, a reduced risk of gastrointestinal disorders, and improved gut health.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e \u003ch2\u003e3.7.2 In vitro growth and fermentation of XOS and another carbon source by pathogen bacteria\u003c/h2\u003e \u003cp\u003eIn addition to evaluating the prebiotic effects of XOS on beneficial bacteria, its influence on the growth of pathogenic bacteria, including \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. typhimurium\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e was also investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e10\u003c/span\u003e). This assessment aimed to determine whether different carbon sources selectively promote beneficial bacteria while inhibiting pathogen proliferation, a desirable attribute for prebiotic compounds.\u003c/p\u003e \u003cp\u003eGrowth studies on \u003cem\u003eS. aureus\u003c/em\u003e revealed significant variability depending on the carbon source. Monosaccharides such as glucose, xylose, and a glucose-xylose mixture supported substantial growth, consistent with findings by Bacha et al. [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In contrast, corncob-derived XOS demonstrated minimal support for \u003cem\u003eS. aureus\u003c/em\u003e growth, which was markedly lower than that observed with other carbon sources.\u003c/p\u003e \u003cp\u003eSimilarly, \u003cem\u003eS. typhimurium\u003c/em\u003e exhibited distinct growth patterns when exposed to various carbon sources. Among these, xylose supported the most robust and sustained growth, showing the highest specific growth rate. Glucose and a glucose-xylose mixture provided moderate growth, with specific growth rates comparable to that of xylose. These results align with the findings of Eberl et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], who noted that \u003cem\u003eS. typhimurium\u003c/em\u003e, as a facultative anaerobe, is capable of metabolizing a broad range of simple sugars and sugar alcohols, including glucose, D-xylose, maltose, galactitol, L-arabinose, D-mannitol, L-rhamnose, D-mannose, trehalose, and D-sorbitol. The lowest specific growth rate for \u003cem\u003eS. typhimurium\u003c/em\u003e was observed when XOS was used as the carbon source.\u003c/p\u003e \u003cp\u003eThe growth of \u003cem\u003eE. coli\u003c/em\u003e showed moderate and comparable cell densities on glucose, xylose, and a glucose-xylose mixture. Naturally, \u003cem\u003eE. coli\u003c/em\u003e can metabolize both glucose and xylose. However, due to carbon catabolite repression (CCR) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], glucose is preferentially consumed when both sugars are available. Therefore, the growth of \u003cem\u003eE. coli\u003c/em\u003e on these simple sugars was superior to its growth on XOS, a more complex carbohydrate with prebiotic properties that can inhibit pathogenic bacteria. In this study, the specific growth rate of \u003cem\u003eE. coli\u003c/em\u003e on XOS was less than one-third of that observed with the other carbon sources.\u003c/p\u003e \u003cp\u003eThese observations underscore the metabolic preferences of the pathogenic strains tested. Simple sugars like glucose and xylose efficiently support pathogen growth, while corncob-derived XOS consistently limits it. Therefore, XOS emerges as a promising prebiotic candidate for promoting gut health and maintaining microbial balance by reducing the risk of pathogen overgrowth. Sun et al. [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] found that adding 0.25% XOS to the growth medium did not affect \u003cem\u003eS. aureus\u003c/em\u003e alone. Still, it significantly reduced \u003cem\u003eS. aureus\u003c/em\u003e counts when combined with probiotics like \u003cem\u003eBifidobacterium lactis\u003c/em\u003e. In that study, a higher XOS concentration of 4% (w/v) was needed to inhibit \u003cem\u003eS. aureus\u003c/em\u003e growth directly. However, the current study demonstrates improved efficacy, as a 2% (w/v) XOS powder inhibited the growth of all three pathogenic bacteria tested, indicating a more potent inhibitory effect at a lower concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e3.7.3 Prebotic index and prebiotic activity against \u003cem\u003eSalmonella typhimurium\u003c/em\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSalmonella typhimurium\u003c/em\u003e is a Gram-negative bacterium and a significant pathogen responsible for foodborne illnesses. It belongs to the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e family and is a leading cause of gastroenteritis in humans and animals. This bacterium can survive in diverse environments and is frequently associated with contaminated food products, particularly poultry, eggs, and dairy [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Prebiotics prevent the growth of pathogenic bacteria by selectively encouraging the multiplication of beneficial bacteria, including \u003cem\u003eLactobacillus and Bifidobacterium\u003c/em\u003e. Supplementing with XOS can suppress the growth of pathogens like \u003cem\u003eS. typhimurium\u003c/em\u003e by enhancing the growth of probiotics that produce organic acids, which lower pH and create unfavorable conditions for pathogen survival [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA compound is classified as a prebiotic if it has a prebiotic index greater than 1 and a positive prebiotic activity score, indicating selective support for beneficial bacteria over harmful ones [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] is shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. According to the data, \u003cem\u003eLactobacillus fermentum\u003c/em\u003e demonstrated the most positive response to corncob-derived XOS, reflected in its prebiotic index and activity scores. In contrast, \u003cem\u003eBifidobacterium\u003c/em\u003e sp. and \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e exhibited significantly lower responses. The prebiotic index values for these two strains were less than 1, and their prebiotic activity scores were negative, except for \u003cem\u003eBifidobacterium\u003c/em\u003e sp. at 72 hours. The prebiotic index values for all bacterial strains peaked within the first 24 hours, indicating that XOS-derived prebiotics effectively promoted bacterial growth during this period. At 48 and 72 hours, a relative decline was observed, likely due to rapid bacterial growth in the glucose-containing positive control medium, which surpassed the growth of both \u003cem\u003eLactobacillus fermentum\u003c/em\u003e and \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e in XOS. However, \u003cem\u003eBifidobacterium\u003c/em\u003e sp. continued to grow at 72 hours, likely because utilizing XOS as its carbon source helped sustain its logarithmic phase. In contrast, in the glucose-containing positive control medium, \u003cem\u003eBifidobacterium\u003c/em\u003e sp. had already reached the stationary phase.\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\u003ePrebiotic index and prebiotic activity of corncob XOS\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\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eL. fermentum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eBifidobacterium sp.\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eL. rhamnosus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e\u003cb\u003eCorncob XOS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003ePrebiotic index\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003ePrebiotic activity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThese findings suggest that \u003cem\u003eL. fermentum\u003c/em\u003e has a superior capability to utilize XOS from corncob compared to the other tested probiotic strains. Additionally, the prebiotic activity results indicate that the growth of beneficial bacteria is more pronounced relative to the growth of the pathogenic \u003cem\u003eS. typhimurium\u003c/em\u003e. This highlights the potential of corncob-derived XOS as an effective prebiotic for promoting gut health and mitigating pathogen proliferation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study highlights the effectiveness of using potash alum as a catalyst in a microwave-assisted hydrolysis process to extract XOS from corncob waste. The optimized pretreatment conditions significantly enhanced XOS yield while demonstrating a sustainable approach to agricultural byproduct valorization. The purified XOS exhibited remarkable functional properties, including strong antioxidant activity and the ability to promote beneficial probiotic growth, with \u003cem\u003eL. fermentum\u003c/em\u003e showing the highest prebiotic response. The results suggest that the balanced catalytic action of potash alum can facilitate selective hemicellulose hydrolysis while limiting XOS degradation, contributing to a cost-effective and scalable production process. This work supports the potential of corncob-derived XOS as a multifunctional ingredient in health-related applications, promoting both gut health and oxidative stability in food and nutraceutical formulations. Future research could explore scaling up the process and investigating the synergistic effects of XOS in combination with other bioactive compounds.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Hana Nur Fitriana, Umar Seno Aji, Khaswar Syamsu; methodology: Umar Seno Aji, Haqqyana, Isti Qomariah; formal analysis and investigation: Haqqyana, Dewi Sondari, Isti Qomariah; writing\u0026mdash;original draft preparation: Umar Seno Aji, Hana Nur Fitriana, Haqqyana; writing\u0026mdash;review and editing: Hana Nur Fitriana, Riksfardini A. Ermawar, Khaswar Syamsu; funding acquisition: Hana Nur Fitriana; resources: Riksfardini A. Ermawar, Dewi Sondari; supervision: Hana Nur Fitriana\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the RIIM LPDP Grant and BRIN (Grant Numbers: B-803/II.7.5/FR/6/2022 and B-1373/III.5/PR.03.08/6/2022), received by Author Hana Nur Fitriana.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank the RIIM LPDP Grant and BRIN for their financial support. Our gratitude also goes to the Integrated Laboratory of Bioproducts, National Research and Innovation Agency (BRIN)\u0026mdash;formerly the Indonesian Institute of Sciences (LIPI)\u0026mdash;for providing essential facilities and invaluable scientific and technical assistance throughout this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article contains no studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSamanta AK, Senani S, Kolte AP, et al (2012) Production and in vitro evaluation of xylooligosaccharides generated from corn cobs. 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Ber Biol 22:51\u0026ndash;59. https://doi.org/10.55981/beritabiologi.2023.806\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Xylooligosaccharide (XOS), corncob waste, potash alum, prebiotic, antioxidant","lastPublishedDoi":"10.21203/rs.3.rs-6158861/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6158861/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study explores the potential of corncob waste valorization through microwave-assisted hydrolysis for xylooligosaccharides (XOS) extraction using potash alum (AlK(SO₄)₂) as a catalyst. The process was optimized through a One-factor-at-a-time (OFAT) approach, evaluating potash alum concentration, heating time, and reaction temperature. Optimal conditions (20 \u0026micro;mol/mL potash alum, 5 minutes, 160\u0026deg;C) yielded 12.32 g/L XOS in the hydrolysate, with ethanol precipitation achieving a 16.97% (g/g) purified XOS yield. Potash alum was chosen as an alternative catalyst due to its potential cost-effectiveness and milder environmental impact compared to strong acids, though further comparative analysis is warranted. The purified XOS exhibited strong antioxidant activity (94.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54% DPPH radical scavenging) and high total phenolic content (32.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 \u0026micro;g GAE/mg of XOS). In vitro prebiotic assays demonstrated XOS\u0026rsquo;s capacity to enhance probiotic growth, particularly \u003cem\u003eLactobacillus fermentum\u003c/em\u003e, which showed the highest prebiotic index and prebiotic activity within 24 hours. These findings suggest that corncob-derived XOS can serve as a functional ingredient with prebiotic and antioxidant properties, supporting its application in health-promoting food and nutraceuticals.\u003c/p\u003e","manuscriptTitle":"Microwave-Assisted Pretreatment of Corncob Using Potash Alum (AlK(SO4)2) Catalyst for Enhanced Xylooligosaccharide (XOS) Extraction with High Prebiotic and Antioxidant Properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 14:41:25","doi":"10.21203/rs.3.rs-6158861/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-01T04:39:34+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-28T19:39:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2025-03-22T12:41:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-08T10:44:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2025-03-04T23:56:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"895b4679-9989-46d1-88cb-f8a63a3ae129","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T16:06:49+00:00","versionOfRecord":{"articleIdentity":"rs-6158861","link":"https://doi.org/10.1007/s12649-025-03205-3","journal":{"identity":"waste-and-biomass-valorization","isVorOnly":false,"title":"Waste and Biomass Valorization"},"publishedOn":"2025-07-10 15:57:06","publishedOnDateReadable":"July 10th, 2025"},"versionCreatedAt":"2025-04-17 14:41:25","video":"","vorDoi":"10.1007/s12649-025-03205-3","vorDoiUrl":"https://doi.org/10.1007/s12649-025-03205-3","workflowStages":[]},"version":"v1","identity":"rs-6158861","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6158861","identity":"rs-6158861","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}