Carboxyl- or Dopamine-Modified Hydrated Silica for Flavourzyme Immobilization: Towards Efficient Debittering of Corn Peptides

Carboxyl- or Dopamine-Modified Hydrated Silica for Flavourzyme Immobilization: Towards Efficient Debittering of Corn Peptides | 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 Carboxyl- or Dopamine-Modified Hydrated Silica for Flavourzyme Immobilization: Towards Efficient Debittering of Corn Peptides Xu Zhao, Yuqin Cheng, Lei Cai, Chun Cui This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7756046/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract This study investigated the surface modification of hydrated silica to enhance the immobilization efficiency of Flavourzyme, a protease complex widely used in the food industry. Hydrated silica was functionalized with carboxyl and dopamine groups to improve its enzyme loading capacity and activity recovery. The carboxyl-modified hydrated silica (CHS) exhibited a higher immobilization rate of 62.93%, while the dopamine-modified hydrated silica (DHS) demonstrated superior enzyme activity recovery at 73.68%. Both CHS- and DHS-immobilized Flavourzyme displayed excellent thermal stability and reusability, retaining over 40% of their initial activity after ten consecutive reaction cycles. Furthermore, the immobilized enzymes were applied to reduce the bitterness of corn peptides, with the CHS-immobilized Flavourzyme exhibiting the most effective debittering performance. These results highlight the potential of surface-modified hydrated silica as a promising carrier for enzyme immobilization and its application in improving the sensory properties of food protein hydrolysates. Hydrated silica surface modification immobilized Flavourzyme corn peptides debittering Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Immobilized enzyme technology has revolutionized modern biocatalysis by significant enhancing enzyme stability, reusability, and operational convenience, making it an indispensable tool in industrial bioprocessing (Tadesse and Liu 2025 ). The core principle of this technology is the physical confinement of enzymes to solid supports, which streamlines their separation from the reaction mixture and enables efficient recycling in repeated industrial cycles (Tadesse and Liu 2025 ). Over the past decade, extensive research has been devoted to exploring diverse enzyme carriers, including natural and synthetic polymers, inorganic materials, and advanced composite systems, each offering unique advantages for specific biocatalytic applications (Mohidem et al. 2023 ). Among these candidates, hydrated silica has emerged as a highly favored carrier material, attributed to its remarkable combination of high surface area, superior biocompatibility, and versatile surface chemistry that can be readily tailored for optimal enzyme immobilization (Lim and Jo 2025 ). Building on these properties, recent advancements have focused on modifying the native surface of hydrated silica to further amplify enzyme loading capacity and catalytic efficiency. For example, the affinity-induced immobilization of β-glucosidase onto amino-functionalized silica not only significantly enhanced enzyme activity and stability but also demonstrated exceptional potential in the biotransformation of ginsenoside Rb1, highlighting the synergy between surface engineering and enzyme performance (Wu et al. 2021 ). Complementary studies have also shown that surface modification strategies, such as incorporating functional groups like amino and carboxyl, play a pivotal role in modulating enzyme immobilization efficiency and activity recovery, thus expanding the applicability of hydrated silica in complex biocatalytic workflows (Xiao et al. 2017 ; Zhao et al. 2017 ). Flavourzyme, a commercial enzyme preparation containing fungal proteases and aminopeptidases, has become indispensable in the food industry (Gu et al. 2022 ). Its unique proteolytic profile enables the efficient hydrolysis of proteins into smaller peptides and free amino acids. This not only elevates the nutritional quality of food products but also significantly enhances their sensory attributes, particularly flavor. For instance, in Cantonese bacon and Chinese sausage, Flavourzyme has been shown to drive proteolysis, yielding peptides that contribute to desirable sensory attributes such as umami taste and reduced astringency (Feng et al. 2014 ; Zhang et al. 2017 ). These findings highlight Flavourzyme’s role as an essential tool in the production of protein hydrolysates, flavor enhancers, and functional foods. However, the industrial application of free Flavourzyme is limited by its relatively short operational stability and the high costs associated with repeated enzyme additions (Zhao et al. 2025b ). To address these limitations, immobilizing Flavourzyme on suitable carriers has emerged as a promising solution. This approach can enhance the enzyme’s stability, reusability, and catalytic efficiency, thereby making it more cost-effective for large - scale industrial processes. Corn peptides (CPs), produced by hydrolyzing corn protein, are highly valued in the food and nutraceutical sectors for their nutritional benefits, including antioxidant, antimicrobial, and dipeptidyl peptidase IV (DPP-IV) inhibitory activities (Yao et al. 2023 ; Yu et al. 2023 ; Zhu et al. 2019 ). However, the bitter taste, which mainly originates from hydrophobic amino acids and bitter peptides formed during hydrolysis, restricts their application in food products. Reducing bitterness while preserving nutrition is a major challenge. Traditional debittering methods have drawbacks (Mirzapour-Kouhdasht et al. 2023 ). For example, activated carbon can adsorb bitter compounds from CPs, but its effectiveness is limited and it may also remove beneficial components (Suh et al. 2000 ). The inclusion complex of neohesperidin dihydrochalcone and glucosyl-β-cyclodextrin shows promise in masking bitterness of CPs (Dong et al. 2017 ), but its high cost and complexity limit food industry applications. Immobilized enzymes offer a promising alternative. They enable precise hydrolysis, targeting specific peptide bonds that cause bitterness. This method can reduce bitterness while keeping CPs' nutritional value intact, making them more suitable for food products. The primary objective of this study was to develop an efficient and reusable immobilized Flavourzyme system for the debittering of corn peptides. To enhance the efficiency of the enzyme immobilization process and improve the recovery of enzyme activity, the surface of hydrated silica was modified through carboxylation and dopamylation. The research delved into the exploration of kinetic parameters and structural characterization of Flavourzyme immobilized using different modified carriers. Furthermore, the study compared the impact of Flavourzyme immobilized on various modified carriers on the debittering effect of corn peptides. This study highlights the critical role of carrier modification in enhancing the performance of immobilized enzymes, offering valuable insights for the optimization of industrial processes aimed at producing high-quality, low-bitterness corn peptides. 2. Materials and methods 2.1 Materials Hydrated silica was procured from Evonik Degussa (China) Investment Co., Ltd. Glutaraldehyde, 6-aminocaproic acid, and dopamine hydrochloride were obtained from Shanghai Macklin Biochemical Technology Co., Ltd. Flavourzyme ® 1000L was kindly provided by Novozymes (China) Biotechnology Co., Ltd. Corn peptides were acquired from Guangzhou Kejin Biotechnology Co., Ltd. All other reagents used were of analytical grade. 2.2 Surface modification of hydrated silica (HS) 2.2.1 Carboxylation modification of HS The carboxylation modification of HS was performed following the method of (Li et al. 2020) with slight modifications. Briefly, 1 g of HS was pre-treated with 10 mL of 5% glutaraldehyde (GA) solution at 37°C for 4 h, followed by washing with deionized water and drying. Subsequently, 1 g of the pre-treated HS was incubated with 10 mL of 4% 6-aminocaproic acid solution at 60°C for 2 h under shaking. After the reaction, the carrier was thoroughly washed with deionized water until the filtrate reached neutrality. Finally, the carrier was dried to obtain the carboxy-modified HS (CHS). The reaction conditions for carboxylation, including the concentration of aminocaproic acid solution (2, 4, 6, 8, 10%), reaction temperature (30, 40, 50, 60, 70°C), and reaction time (1, 2, 3, 4, 5 h), were optimized based on the surface carboxyl group density. 2.2.2 Dopamine modification of HS The dopamine modification of HS was conducted according to our previously reported method (Zhao et al. 2025b). Similarly, 1 g of HS was pre-treated with 10 mL of 5% GA solution at 37°C for 4 h. The pre-treated carrier was then thoroughly washed with deionized water and dried. Next, the pre-treated carrier was immersed in 10 mL of dopamine solution, which was prepared by dissolving 2 g of dopamine hydrochloride in 0.1 M Tris buffer (pH 8.5). The mixture was reacted for 12 h at 25°C under shaking. After the reaction, the carrier was sequentially washed with anhydrous ethanol and deionized water until the filtrate became colorless and transparent. Finally, the carrier was dried to obtain dopamine-modified HS (DHS). The modification conditions, including the concentration of dopamine hydrochloride solution (1, 2, 3, 4, 5 mg/mL), reaction temperature (25, 30, 35, 40, 45, 50°C), and reaction time (0, 5, 10, 15, 20, 25 h), were optimized based on the surface amino group density. 2.3 Determination of surface functional group density of modified HS 2.3.1 Determination of surface carboxyl group density The surface carboxyl group density of CHS was determined using the Boehm titration method with slight modifications (Tkachenko et al. 2024). Initially, 1 g of CHS was soaked in 0.5% HCl for 40 min to remove residual Cl⁻, followed by washing with deionized water until the filtrate reached neutrality. The sample was then dried to a constant weight at 105°C. Subsequently, 1 g of the treated sample was accurately weighted into a 250 mL iodine flask, and 100 mL of 0.1 M calcium acetate solution was added. The mixture was shaken at 25°C for 16 h. After the reaction, 10.00 mL of the supernatant was transferred to a conical flask. Then, 2-3 drops of phenol red-thymol blue indicator were added, and the solution was titrated with 0.1 M NaOH solution until it turned a stable purple-rose color that persisted for 30 s. Three parallel determinations were performed, and the volume of NaOH consumed was recorded. The carboxyl group density was calculated using Equation (1): Where C is the concentration of NaOH, (mol/L); V is the volume of NaOH consumed (μL); M is the mass of the specimen, (g). 2.3.2 Determination of surface amino group density The surface amino group density of DHS was determined following the method established by (Melnyk et al. 2023). Accurately weigh 0.5 g of DHS into a 50 mL conical flask. Add 10 mL of deionized water and 30 μL of methyl red indicator, and mix thoroughly. Titrate with 0.1 mol/L HCl standard solution to determine the amino group density. The calculation formula is shown in equation (2): Where C is the concentration of HCl (mol/L); V is the volume of HCl consumed (μL); and M is the mass of the sample (g). 2.4 Preparation of immobilized Flavourzyme The immobilization of Flavourzyme on modified HS was performed as follows: For DHS, the immobilization was conducted according to our previously reported method (Zhao et al. 2025b), which involved mixing the enzyme solution with the modified carrier under specific conditions to facilitate enzyme adsorption onto the carrier surface. For CHS, an 8 mg/mL Flavourzyme solution was prepared using PBS buffer (pH 8.0, 0.1 M). The modified carrier was added to the enzyme solution at a solid-to-liquid ratio of 1:10. The mixture was incubated at 25°C for 12 h to allow the enzyme to attach to the carrier. After the reaction, the immobilized Flavourzyme was separated from the supernatant, washed with the PBS buffer, and dried at room temperature. It was then stored at 4°C for subsequent use. The immobilized Flavourzyme obtained by the two methods were named DHS-F and CHS-F, respectively. To assess the efficiency of the immobilization process, the soluble protein content in the supernatant before and after immobilization was determined using a BCA Protein Assay Kit. The immobilization rate was calculated using the following formula: where C 0 and C t are the protein contents (mg/mL) before and after immobilization, and V 0 and V t are the volumes of the enzyme solution (mL) before and after immobilization. The enzyme activity of both free and immobilized Flavourzyme was determined by the Folin-Ciocalteu method. The enzyme activity unit (U) was defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute at 40°C and pH 7.5. To evaluate the impact of immobilization on enzyme activity, the enzyme activity recovery was calculated as follows: 2.5 Determination of enzyme kinetic parameters Free and immobilized Flavourzyme with the same enzyme activity were separately added to casein solutions at concentrations of 1, 2, 5, 10, 20, 40, and 80 mg/mL. Reactions were carried out under the respective optimal reaction conditions, and enzyme activity was measured. The Michaelis constant (K m ) and maximum reaction rate (V max ) values were calculated using the Lineweaver-Burk double reciprocal plot. A double reciprocal plot was constructed with the reciprocal of substrate concentration on the x-axis and the reciprocal of reaction rate on the y-axis to determine the K m and V max values for the immobilized Flavourzyme. 2.6 Structural characterization of immobilized Flavourzyme 2.6.1 SEM Analysis The surface morphology of the immobilized Flavourzyme was observed using a scanning electron microscope (SEM, Merlin, Zeiss, Germany). A suitable concentration of the sample solution was prepared using water as the dispersant. The sample solution was then dropped onto an aluminum foil sample preparation table. After drying, the sample was coated with a thin layer of gold to enhance conductivity. 2.6.2 FTIR Spectroscopy Fourier transform infrared (FTIR) spectroscopy was used to identify functional groups and investigate the interactions between the enzyme and the carrier. Dried and milled samples were mixed with KBr at a ratio of 1:100 and pressed into tablets. KBr was used as a blank control to subtract background signals. FTIR spectra were conducted using a Nicolet IS50 spectrometer (Thermo Fisher Scientific, USA) over a wavelength range of 400–4000 cm⁻¹ with a resolution of 4 cm⁻¹ and 32 scans. 2.6.3 SDS-PAGE Analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the molecular weight distribution and the integrity of the enzyme after immobilization. The analysis was performed with minor adjustments to the method described by (Zhao et al. 2024). Samples were prepared into a solution with a mass concentration of 10 mg/mL. A volume of 400 μL of the sample solution was mixed with 100 μL of loading buffer. The mixture was then heated at 100°C for 5 min to denature the proteins, followed by centrifugation to remove precipitates. Subsequently, 10 μL of the supernatant was loaded onto the gel. The electrophoresis was initially run at 60 V for 30 min for pre-electrophoresis, after which the voltage was increased to 120 V until the electrophoresis was complete. The gel was stained with Thomas Brilliant Blue R-250 and the staining was decolorized using a decolorizing solution until the protein bands were clearly visible. 2.6.4 TGA Analysis Thermogravimetric analysis (TGA) was used to evaluate the thermal stability of the immobilized Flavourzyme. Approximately 5-10 mg of sample was accurately weighed and analyzed using a Mettler TGA/DSC1/1100SF thermogravimetric analyzer. Nitrogen was used as the carrier gas with a flow rate of 20 mL/min. The heating rate was set at 20°C/min, and the temperature ranged from 25°C to 700°C. 2.7 Preparation of debittered corn peptides (DCP) To prepare the debittered corn peptides (DCP), a corn peptides solution was first prepared at a solid-to-liquid ratio of 1:10 (w/v). The solution was thoroughly stirred to ensure complete dissolution of the corn peptides. The pH of the solution was then adjusted to 8.0. Subsequently, 2% (E/S) of Flavourzyme and an equivalent amount of immobilized Flavourzyme with the same enzyme activity were added. The mixture was incubated at 37°C for 8 h to allow the enzymatic reaction to proceed. After the reaction, the mixture was heated in a boiling water bath for 15 min to inactivate the enzyme. The immobilized Flavourzyme was then filtered out, and the resulting DCP solution was obtained. Finally, the DCP solution was lyophilized to yield DCP powder, which was stored for further analysis or use. The efficiency of the enzyme digestion was evaluated based on protein recovery and degree of hydrolysis. The protein content was determined using the Kjeldahl method, while the amino acid nitrogen content was quantified by the formol titration method (Zhao et al. 2025c). 2.8 Sensory evaluation of debittered corn peptides (DCP) 2.8.1 Quantitative descriptive analysis (QDA) A panel of potential assessors was interviewed and screened for their sensory acuity and bitterness sensitivity. From this pool, 10 assessors (5 male and 5 female, aged 20-30) were selected based on their ability to discern and describe bitterness accurately. The assessors participated in training sessions to familiarize themselves with the bitterness attributes of corn peptide solutions and to ensure evaluation consistency. All sensory evaluations were carried out in a specialized sensory analysis laboratory equipped with climate control set at 25°C, industrial white lighting for color consistency, and individual tasting booths to eliminate environmental interference. For the QDA, 1% untreated corn peptides (CK) and DCP solutions were prepared separately. Quinine sulfate solutions at concentrations of 5, 10, and 15 mg/L, corresponding to scores of 5, 10, and 15, respectively, were used as reference standards. The assessors evaluated and recorded the bitterness intensity of both CK and DCP solutions. Each sample was randomly tasted three times, with water rinsed between tastings to ensure palate cleansing. 2.8.2 Time-Intensity method (TI) The TI assessment was performed following the methodology outlined by (Zhao et al. 2025a). Prior to the assessment, evaluators were instructed to rinse their mouths with water to eliminate any lingering tastes. Quinine sulfate solutions at concentrations of 5, 10, and 15 mg/L, corresponding to scores of 5, 10, and 15, respectively, served as reference standards for bitterness. Evaluators were directed to take 5 mL of 1% CP or DCP solution into their mouths, hold it for 5 seconds, and then spit it out. They were tasked with rating the perceived intensity of the taste at specific time intervals: 5, 10, 20, 30, 45, 60, 75, 90, 120, 150, 180, and 210 seconds, until the sensations of bitterness subsided. To mitigate carry-over effects between samples, a 20-minute break was enforced, during which participants rinsed their mouths with purified water. To prevent order effects, the presentation order of the samples was randomized. Each sample was subjected to three independent trials to ensure the reliability of the study's results. 2.9 Determination of molecular mass distribution and free amino acids (FAAs) The molecular weight distribution was determined using HPLC, following our previous method (Zhao et al. 2025b). The FAAs were measured with reference to the previous method with slight modifications (Xiang et al. 2024). Equal volumes of DCP solution were mixed with a 10% (w/v) trichloroacetic acid (TCA) solution. After standing at 4°C for 1 h, the mixture was centrifuged to remove proteins, and the supernatant was collected for free amino acid analysis using a fully automated amino acid analyzer (Hitachi L-8900). The dose-over-threshold (DoT) value for each amino acid was calculated as the ratio of its concentration to its taste threshold (Xiang et al. 2023). 2.10 Reusability of immobilized Flavourzyme To evaluate the feasibility of immobilized Flavourzyme in industrial applications, the reusability of the enzyme was investigated. Under optimized reaction conditions, immobilized Flavourzyme was used repeatedly for the hydrolysis of corn peptides solutions. The operational stability of the Flavourzyme was assessed through enzyme activity measurements after each cycle. The immobilized Flavourzyme was used for ten consecutive cycles. The initial enzyme activity was set as the baseline (100%), and the residual relative activity for each subsequent cycle was calculated based on this baseline. 2.11 Statistical Analysis All experiments were conducted at least in triplicate. Data were reported as "mean ± standard deviation". Differences between the means of multiple groups were examined by analysis of variance (ANOVA) along with Duncan's test using SPSS 20.0 software (SPSS Inc., Chicago, USA) at a significance level of 0.05 (p < 0.05). 3. Results and Discussion 3.1 Optimization of hydrated silica surface modification In this study, 6-aminocaproic acid was employed as the carboxyl donor to achieve surface carboxylation modification of hydrated silica through the condensation reaction between its amino group and the aldehyde groups on the HS carrier. As shown in Fig. 1 A, the surface carboxyl group density of the carrier increased with the concentration of 6-aminocaproic acid until it reached a plateau at 6% concentration. This increase is attributed to the enhanced reaction kinetics due to higher reactant concentrations. However, concentrations exceeding 6% did not lead to further significant improvements in modification efficiency, likely due to increased system viscosity, restricted molecular diffusion, and steric hindrance (Chen et al. 2023 ). Consequently, an 8% concentration of 6-aminocaproic acid was identified as optimal. Figure. 1B and 1C demonstrated the effects of reaction time and temperature on carboxylation efficiency. The findings indicated that reaction temperature significantly influenced modification efficiency, aligning with the Arrhenius equation (Eshaghi and Pouransari 2025 ). The surface carboxyl group density initially increased with temperature and then plateaus. The maximum carboxyl group density was achieved at 50°C, which may be attributed to the accelerated reaction kinetics and the shift in equilibrium towards product formation due to increased temperature. Similarly, the surface carboxyl group density increased with reaction time, but excessively long reaction times can lead to side reactions within the grafted carboxyl groups. Therefore, the optimal reaction time was determined to be 2 h, with the maximum carboxyl group density reaching 56.22 µmol/g. For the dopamine modification of HS, the surface amino group density initially increased with the concentration of dopamine and then stabilizes (Fig. 1 D). The peak surface amino group density was reached at approximately 3 mg/mL of dopamine, beyond which higher concentrations did not further enhance the modification efficiency. This is consistent with the findings reported by (Zheng et al. 2021 ), where high concentrations were found to enhance π-π stacking between dopamine molecules, leading to the formation of aggregates and a reduction in the effective reaction concentration. Additionally, the increased system viscosity at higher concentrations can hinder dopamine diffusion to the carrier surface (Sureshkumar and Lee 2011 ). Figure 1 E revealed that the surface amino group density decreased with increasing reaction temperature. The highest surface amino group density was achieved at 25–35°C, after which it declined with further increases in temperature. This suggests that lower temperatures are more conducive to the effective grafting of dopamine onto the carrier, while higher temperatures may lead to side reactions or degradation of dopamine, thereby reducing the modification efficiency. Figure 1 F shows that the surface amino group density increased with reaction time, initially at a higher rate and then plateauing. The optimal modification was achieved after a 15-hour reaction, at which point the surface amino group density achieved 61.25 µmol/g. According to (Mao et al. 2023 ), in the early stages of the reaction, dopamine molecules rapidly graft onto the carrier via Schiff base reactions. Over time, grafted dopamine may undergo oxidative self-polymerization to form a polydopamine layer. However, excessively long reaction times can lead to excessive polydopamine growth, which may mask some active amines. Therefore, a 12-h reaction time was determined to be optimal to balance the grafting and polymerization processes effectively. 3.2 Immobilization of Flavourzyme To assess the immobilization process's efficiency, both the immobilization rate and enzyme activity recovery were calculated. The results showed that DHS-F had an immobilization rate of 42.01% and enzyme activity recovery of 73.68%. In comparison, CHS-F demonstrated a higher immobilization rate of 62.93%, but its enzyme activity recovery was slightly lower at 68.22%. These findings suggest that CHS-F achieved better immobilization efficiency, which may be due to the carboxyl groups on the carrier providing more binding sites for the enzyme, facilitating a higher adsorption capacity. However, the increased density of enzyme molecules on the CHS surface could lead to steric hindrance and conformational changes in the enzyme, masking the active sites and reducing accessibility to the substrate, thus lowering enzyme activity recovery. The Michaelis constant (K m ), a key parameter in enzymatic reaction kinetics, reflects the enzyme's affinity for the substrate and its catalytic efficiency. A smaller K m value indicates stronger substrate affinity and higher catalytic efficiency (Wang et al. 2024b ). As shown in Fig. 2 A, both the free flavoruzyme (FF) and immobilized Flavourzyme groups exhibited typical saturation kinetics. Their catalytic efficiency increased significantly with substrate concentration initially, then gradually plateaued. This aligns with previous research on casein hydrolysis (Dehkordi et al. 2022 ), the importance of substrate concentration in enzymatic catalysis. To quantify the enzymatic reaction kinetics, Lineweaver-Burk reciprocal plotting was used to determine K m and V max . Figure 2 B revealed that immobilized Flavourzyme's K m values were higher than those of FF. Specifically, FF's K m was 24.48 mmol/L, while CHS-F and DHS-F's K m values increased to 26.86 mmol/L and 30.46 mmol/L, respectively (Table 1 ). This implies that immobilization reduced the enzyme's substrate affinity, possibly due to conformational changes in the enzyme structure, steric hindrance around the active site, altered microenvironment at the enzyme-carrier interface, or restricted substrate diffusion towards the active site. Despite this, the V max values increased post-immobilization. FF's V max was 349.66 mmol/(L·min), whereas CHS-F and DHS-F's V max values rose to 356.34 mmol/(L·min) and 372.20 mmol/(L·min), respectively. These changes may be attributed to altered substrate diffusion efficiency and reduced enzyme flexibility due to immobilization (Zhang et al. 2025 ). Table 1 Immobilization rate, enzyme activity recovery, and kinetic parameters of free Flavourzyme (FF), carboxylated-modified hydrated silica immobilized Flavourzyme (CHS-F) and dopamine-modified hydrated silica mobilized Flavourzyme (DHS-F). Immobilization rate (%) Enzyme activity recovery (%) [V max ] (mmol·(L/min) −1 ) K m (mmol/L) FF - - 349.66 24.48 CHS-F 62.93 ± 0.72 68.22 ± 1.58 356.34 26.86 DHS-F 42.01 ± 0.14 73.68 ± 1.16 372.20 30.46 3.3 SEM Analysis The SEM results, as depicted in Fig. 3 , revealed notable morphological changes post-modification. Carboxylation increased the surface roughness of HS, while dopamine modification resulted in a uniform dopamine coating on the silica surface, forming a distinct layered structure. The surfaces of CHS-F and DHS-F displayed typical flocculent protrusions, which are consistent with the microscopic features of protein aggregates. These features directly indicated effective enzyme loading. In contrast, the original carrier exhibited a smooth and homogeneous surface after lyophilization. The enzyme-immobilized carriers showed notably heterogeneous structures with dense granular depositions. Notably, dopamine-modified DHS-F demonstrated a more uniform organic-inorganic composite structure. The dopamine coating enhanced the carrier's surface roughness and optimized the distribution of immobilization sites through multiple interactions between its catechol groups and enzyme molecules. 3.4 FTIR Analysis FTIR spectroscopy was employed to verify the successful immobilization of Flavourzyme onto HS particles by detecting covalent bond formation. As shown in Fig. 4 A, the FTIR spectrum of HS particles displayed a prominent peak at 1105 cm − 1 , attributed to the asymmetric stretching vibration of Si-O-Si, which is a characteristic absorption peak of HS (Singh and White 2020 ). For both CHS-F and DHS-F, characteristic bands were observed at 2935 cm − 1 , 2913 cm − 1 , and 2855 cm − 1 , corresponding to the C-H stretching vibrations in -CH 2 - groups, which are common in protein structures (Mekonnen 2023 ). Additionally, absorption peaks at 1658 cm − 1 and 1633 cm − 1 were identified, which are associated with amide I bands (C = O stretching), respectively (Hussain et al. 2023 ). These peaks indicate the formation of amide bonds, which are crucial for enzyme immobilization. The free and immobilized Flavourzyme showed significant absorption at these wavelengths, confirming the integrity of the enzyme's secondary structure post-immobilization. The presence of these characteristic bands in both the immobilized derivatives and FF spectra demonstrates that Flavourzyme was successfully immobilized via covalent amide bonds on the modified HS carriers. 3.5 SDS-PAGE Analysis SDS-PAGE electrophoresis analysis of the supernatant from the immobilization systems of CHS-F and DHS-F (Fig. 4 B) revealed significant differences in protein band intensity and number compared to the free enzyme solution. After immobilization, the enzyme solution showed markedly lighter and fewer bands, which are attributed to the immobilization of key active enzymes in Flavourzyme, including neutral proteinase 1 (42.4 kDa), neutral proteinase 2 (19.0 kDa), and leucine aminopeptidase A (36.2 kDa) (Yust et al. 2007 ). Notably, the enzyme proteins with molecular weights ranging from 10 to 28 kDa, which primarily correspond to endopeptidases such as neutral proteinase 2 (19.0 kDa) and leucine aminopeptidase A (36.2 kDa), were almost completely immobilized. This reduction in enzyme protein content in the supernatant indicates efficient immobilization of these enzymes onto the carrier surface. 3.6 TGA Thermogravimetric analysis (TGA) was conducted to evaluate the thermal stability of the modified carrier and immobilized enzyme, with the results presented in Fig. 4 C. All samples exhibited slight mass loss below 150°C, attributed to the evaporation of physically adsorbed water on the particle surface (Yust et al. 2007 ). Notably, no significant mass loss occurred below 600°C for any of the samples, indicating excellent thermal stability of HS within this temperature range, consistent with previous literature (Huang et al. 2010 ). CHS demonstrated comparable thermal stability to that of HS. However, after immobilization, the mass loss rate of CHS-F increased significantly. At 155.7°C, the organic components on the CHS surface began to thermally decompose, leading to a higher mass loss rate compared to the HS. At 209.3°C, the immobilized Flavourzyme molecules on the carrier surface started to decompose, further increasing the mass loss rate. Specifically, the mass loss rates of the HS and CHS were 7.15% and 7.55%, respectively, while that of CHS-F increased significantly to 9.68%. This indicates that the enzyme protein molecules were successfully immobilized on the CHS surface, and their thermal decomposition caused the additional mass loss. In contrast, DHS exhibited enhanced thermal stability below 222°C compared to HS. Nevertheless, beyond 222°C, the mass loss rate of the modified carrier accelerated considerably due to the thermal decomposition of organic components in the dopamine coating. At 158.2°C, the immobilized Flavourzyme molecules on the modified carrier surface began to thermally decompose, intensifying the mass loss. By 550°C, the organic components on the particle surface, including GA and enzyme molecules, had completely decomposed. The mass loss rates of the HS and DHS were 7.68% and 8.32%, respectively, while that of DHS-F rose significantly to 20.23%. This confirms the successful immobilization of enzyme protein molecules on the DHS surface and highlights that their thermal decomposition caused the significant mass loss. When comparing CHS and DHS, it is evident that both carriers exhibit excellent thermal stability below 600°C. However, their thermal degradation behaviors differ significantly after enzyme immobilization. CHS-F shows earlier decomposition of enzyme molecules, while DHS-F experiences more significant mass loss at higher temperatures due to the decomposition of both enzyme and dopamine coating. These findings indicate that the choice of modified carrier can significantly influence the thermal stability and decomposition profile of the immobilized enzyme system. 3.7 Preparation of DCP and QDA Analysis Figure 5 A illustrates the appearance of debittered corn peptides prepared through enzymatic digestion using free and immobilized Flavourzyme. Notably, the enzymatic treatment did not induce significant precipitation in the corn peptides solution. However, remarkable color changes were observed in DF-CP. While the CK, FF-CP and CF-CP groups displayed a dark yellow color, the DF-CP group exhibited a dark brown hue attributed to dopamine's oxidative polymerization. The dark brown color is a typical characteristic of polydopamine coatings, arising from electron delocalization between aromatic rings during polydopamine formation (de Souza et al. 2025 ). As shown in Table 2 , the degree of hydrolysis (DH) varied among the different DCP samples. Both the FF-CP and CF-CP groups achieved a DH of over 7%, while the DF-CP group had the lowest DH at 6.5%, indicating differing catalytic efficiencies of the two immobilized enzymes. All groups demonstrated high protein recovery rates above 97%, suggesting that the enzyme treatment effectively converted proteins into smaller peptides and amino acids with minimal loss. The QDA scores revealed a significant reduction in the bitterness of the hydrolyzed corn peptides, aligning with the degree of hydrolysis. The CF-CP group exhibited the lowest bitterness intensity, while the DF-CP group had the highest. These findings suggest that the CF-CP group achieved the best debittering effect, efficiently diminishing the bitterness of the corn peptides. Table 2 Protein recovery, degree of hydrolysis (DH), QDA score, and parameters of the TI curve of debittered corn peptides. Parameter CP FF-CP DF-CP CF-CP Protein recovery (%) - 98.56 ± 1.49 a 97.78 ± 2.23 a 98.24 ± 1.98 a DH (%) - 7.02 ± 0.66 b 6.52 ± 0.12 a 7.21 ± 0.38 b QDA score 23.41 ± 0.69 d 4.10 ± 0.17 b 3.40 ± 0.36 a 5.81 ± 0.35 c I max 11.56 ± 0.77 a 7.25 ± 1.09 b 7.81 ± 0.66 b 7.38 ± 0.11 b T start 5.00 ± 0.00 a 5.00 ± 0 a 5.00 ± 0.00 a 5.00 ± 0.00 a T max 45.00 ± 0.00 a 28.75 ± 1.31 b 30.00 ± 0.00 b 30.00 ± 0.00 b T plateau 15.00 ± 0.00 b 16.25 ± 0.31 a 15.00 ± 0.00 b 15.00 ± 0.00 b T ext 180.00 ± 0.00 a 120.00 ± 0.00 b 120.00 ± 0.00 b 120.00 ± 0.00 b R increase (×10 − 2 ) 15.00 ± 2.17 c 20.60 ± 1.33 a 19.50 ± 0.71 a 17.50 ± 3.43 b R decrease (×10 − 3 ) -57.00 ± 2.53 b -56.00 ± 5.39 b -64.00 ± 4.88 a -58.00 ± 7.41 a Area before (×10 2 ) 10.85 ± 0.48 a 3.47 ± 0.70 b 4.09 ± 0.37 b 3.96 ± 0.61 b Area after (×10 2 ) 14.68 ± 0.60 a 4.79 ± 0.98 b 5.24 ± 0.13 b 5.14 ± 0.07 b Area (×10 2 ) 25.53 ± 1.79 a 8.26 ± 0.25 b 9.33 ± 1.28 b 9.09 ± 0.52 b 3.8 Time-intensity (TI) analysis Time-intensity (TI) curves are essential in sensory analysis as they map the dynamic sensory traits, such as bitterness, of products over time, thereby enhancing our understanding of consumer experience (François et al. 2007 ). In this study, TI experiments were conducted to measure the overall bitterness intensity of FF-CP, DF-CP, CF-CP, and a control (CK) over time, with the results presented in Fig. 5 B. All samples exhibited a rapid increase in bitterness intensity followed by a gradual decrease, a pattern consistent with previous studies on bitterness TI curves (Huang et al. 2024 ). Notably, FF-CP, DF-CP, and CF-CP demonstrated significantly lower bitterness intensity and shorter duration compared to CK, highlighting their effectiveness in reducing bitterness. The TI curves of FF-CP and CF-CP partially overlapped, indicating similar bitterness-time characteristics and slightly lower maximum bitterness than DF-CP. In contrast, CK samples consistently showed the highest bitterness intensity throughout the testing period. Table 2 shows that there are significant differences between FF-CP, DF-CP, CF-CP, and CK in specific TI parameters. Specifically, FF-CP, DF-CP, and CF-CP exhibited lower values in these parameters compared to CK. This indicates that CK had the highest bitterness intensity, the longest time to reach maximum bitterness, and the longest persistence of bitterness, along with the highest overall content of bitter compounds. These results clearly demonstrate that enzymatic treatment is effective in reducing the bitterness of corn peptides and also explain the shorter persistence of bitterness in the treated samples. When comparing DF-CP and CF-CP, it was observed that DF-CP had a significantly higher rate of increase in bitterness intensity before reaching I max but a lower rate of decrease after I max . This difference likely relates to variations in hydrolysis efficiency. Consistent with the degree of hydrolysis, CHS-F exhibits higher hydrolysis efficiency than DHS-F, giving CHS-F a slight advantage over DHS-F in reducing CP bitterness. This suggests that the choice of enzyme and carrier can significantly influence the effectiveness of bitterness reduction in CP solutions. 3.9 Molecular weight distribution Figure 5 C presents the molecular weight distribution of CP and DCP. The results indicate that the majority of the molecular weight fractions in all samples are distributed in the range of < 500 Da and 500–1000 Da. Specifically, CP has a relatively higher content of the 500–1000 Da and 1000–5000 Da fractions compared to the other samples. In contrast, FF-CP and CF-CP show an increase in the < 500 Da fraction and a decrease in the 500–1000 Da and 1000–5000 Da fractions. DF-CP exhibits a similar trend to CF-CP but with a slightly lower proportion of the < 500 Da fraction and a slightly higher proportion of the 500–1000 Da and 1000–5000 Da fractions. It has been shown shown that bitterness is positively associated with medium molecular weight fractions (500–1000 Da), and negatively correlated with fractions below 500 Da (Xiang et al. 2024 ). This suggests that the bitterness of hydrolysates is mainly influenced by medium molecular weight peptides. As the degree of hydrolysis increases, larger molecular weight peptides may be decomposed into smaller peptides and free amino acids, thereby reducing bitterness (Liu et al. 2016 ). The changes in molecular weight distribution observed in the samples align with this understanding. Samples with a higher proportion of medium molecular weight fractions (e.g., CP and DF-CP) tend to have stronger bitterness, while samples with a higher proportion of low molecular weight fractions (< 500 Da) such as FF-CP and CF-CP exhibit reduced bitterness. This suggests that the DCP generated by Flavourzyme and CHS-F are more hydrolyzed, have lower molecular weights and higher amino nitrogen content are more effective in reducing bitterness taste. The differences in molecular weight distribution among the samples can be attributed to the specificities of the proteases used for hydrolysis, which in turn affect the sensory properties of the hydrolysates. 3.10 FAAs Analysis To investigate the taste contributions of FAAs, their changes were monitored during debittering (Fig. 5 D). In CP, Ala, Cys, Val, Ile, Leu, and Tyr were predominant, with average contents exceeding 10 mg/g. The taste contribution of individual amino acid was evaluated by dose-over-threshold (DoT) value (Xiang et al. 2023 ). Notably, Tyr contributed strongly to bitterness, with its content being 26 times higher than its taste threshold. Leu and Ile also significantly contributed to bitterness, with DoT values above 5.0. For umami-tasting FAAs, their DoT values all exceeded 7.5 due to their low taste thresholds, making their umami taste detectable. After enzymatic digestion with free Flavourzyme and immobilized enzymes, the FAAs contents in DCP significantly increased, especially for Asn, Gln, Val, Met, Leu, and Phe, which rose by 5–20 times. CF-CP also saw increases in these amino acids, though slightly lower than FF-CP. In contrast, DF-CP had notably lower FAAs contents than FF-CP and CF-CP, consistent with hydrolysis degree results. The DoT values for the Tyr in DCP decreased, particularly in CF-CP, where it reduced by 16.33%. The increase in FAAs, including bitter-tasting ones like Leu, Ile, Val, Met, and Phe, suggests extensive protein hydrolysis. Despite higher FAA levels, bitterness reduced because the enzyme selectively cleaved peptide bonds, decreasing bitter peptide formation (Sun et al. 2022 ). Additionally, the release of more non - bitter amino acids may have masked bitterness. Asn and Gln increased significantly due to Flavourzyme and CHS - F hydrolysis, while DHS-F showed no significant increase. This implies FF and CHS-F preferentially act on Gln-containing peptide bond sites but less efficiently on neighboring Glu, indicating enzymatic selectivity in amino acid release (Wang et al. 2024a ). This selective process likely reduces bitterness while enhancing the product's umami through Gln accumulation. 3.11 Reusability The reusability of immobilized enzymes is crucial for enhancing productivity and reducing costs in continuous operations and automated production (Ishak et al. 2025 ). As shown in Fig. 6 A, the DHS-F retained 72.75% of its relative enzyme activity even after the fifth use, and maintained over 50% activity after ten uses. For CHS-F in hydrolyzing CP (Fig. 6 B), it preserved 63.31% of its activity at the fifth cycle and 42.78% after ten cycles. These results indicate that both CHS-F and DHS-F exhibit good stability and reusability, retaining over 40% of their activity after multiple uses, despite a gradual decline in activity with increasing use cycles. 4. Conclusion In this study, we successfully explored the surface modification of hydrated silica and the subsequent immobilization of Flavourzyme, and applied these immobilized enzymes to the debittering of corn peptides. The modification of hydrated silica with carboxyl and dopamine groups significantly improved its surface properties and enzyme immobilization efficiency. CHS demonstrated a higher immobilization rate of 62.93%, while DHS showed better enzyme activity recovery at 73.68%. The successful immobilization of the enzymes was confirmed by SEM, FTIR and SDS-PAGE. TGA analysis showed that the immobilized Flavourzyme on both CHS and DHS exhibited good thermal stability. The enzymatic treatment effectively reduced the bitterness of corn peptides, with CHS-F achieving the lowest bitterness intensity. Moreover, the immobilized Flavourzyme exhibited excellent reusability, retaining over 40% of its activity even after ten cycles of use. These findings provide valuable insights into optimizing the de-bittering process of corn peptides and highlight the potential of modified HS carriers and immobilized enzymes for industrial applications. Overall, this study offers a promising approach to improving the sensory quality and nutritional value of corn peptides through effective enzyme immobilization and carrier modification strategies. Declarations Data Availability Data are contained within the article. Funding This study was supported by the Guangdong province general colleges and universities characteristic innovation category project (2021KTSCX003) and Shandong province science and technology-based small and medium-sized enterprises innovation ability enhancement project mission statement (2022TSGC2558). Institutional Review Board Statement The Human Ethics Committee of South China University of Technology does not have a practice of approving sensory experiments on food. Therefore, this experiment was performed in accordance with the Declaration of Helsinki and informed consent was obtained from all assessors. Competing interests The authors declare no competing interests. Author Contribution X.Z.: conceptualization, methodology,validation, formal analysis, investigation, data curation, writing – original draft.Y.Q.C.: methodology, formal analysis.L.C.: validation, sensory analysis.C.C.: conceptualization, supervision, funding acquisition, writing – review & editing. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Oct, 2025 Editor assigned by journal 03 Oct, 2025 Submission checks completed at journal 02 Oct, 2025 First submitted to journal 30 Sep, 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7756046","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":528156280,"identity":"2200afe1-d745-4b60-9cbe-d4c72f388735","order_by":0,"name":"Xu Zhao","email":"","orcid":"","institution":"South China University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Zhao","suffix":""},{"id":528156281,"identity":"842d8b27-af25-4b1f-96f0-9dddc7135bd5","order_by":1,"name":"Yuqin Cheng","email":"","orcid":"","institution":"South China University of 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Technology","correspondingAuthor":true,"prefix":"","firstName":"Chun","middleName":"","lastName":"Cui","suffix":""}],"badges":[],"createdAt":"2025-10-01 03:38:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7756046/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7756046/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94484487,"identity":"817bae7f-3a6c-4608-89f2-937f6cf0cb17","added_by":"auto","created_at":"2025-10-27 16:36:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":459184,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of carboxyl (A-C) and dopamine (D-F) modification of hydrated silica.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7756046/v1/8f55c3680e5cc00725913baf.jpg"},{"id":94484233,"identity":"6eac300e-f781-4ca0-83f0-61f64e267569","added_by":"auto","created_at":"2025-10-27 16:35:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":644905,"visible":true,"origin":"","legend":"\u003cp\u003eMichaelis–Menten (A) and Lineweaver–Burk (B) plots of free Flavourzyme (FF), carboxy-modified hydrated silica immobilized Flavourzyme (CHS-F), and dopamine-modified hydrated silica immobilized Flavourzyme (DHS-F).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7756046/v1/cc269e7f784e5b542e0bbf49.jpg"},{"id":94490233,"identity":"4f2a9013-8481-405b-b0ce-2918a79354b7","added_by":"auto","created_at":"2025-10-27 17:08:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":837488,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of hydrated silica (HS), carboxy-modified hydrated silica (CHS), dopamine-modified hydrated silica (DHS), CHS-immobilized Flavourzyme (CHS-F), and DHS-immobilized Flavourzyme (DHS-F).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7756046/v1/b7c299134134b0b1f059ad03.jpg"},{"id":94484809,"identity":"1aa7a441-331e-4e0e-a9d3-a8badd413a79","added_by":"auto","created_at":"2025-10-27 16:38:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":743144,"visible":true,"origin":"","legend":"\u003cp\u003eFourier-transform infrared (FTIR) spectra (A), Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (B), and thermogravimetric (TGA) curve (C) for hydrated silica (HS), carboxy-modified hydrated silica (CHS), dopamine-modified hydrated silica (DHS), CHS-immobilized Flavourzyme (CHS-F), and DHS-immobilized Flavourzyme (DHS-F) and free Flavourzyme (FF).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7756046/v1/a7a9eb56afbc304acb181584.jpg"},{"id":94483963,"identity":"04920299-25b2-44ad-b69b-e531661b2e63","added_by":"auto","created_at":"2025-10-27 16:32:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":696758,"visible":true,"origin":"","legend":"\u003cp\u003eAppearance (A), bittering time-intensity (TI) curves (B), molecular weight distribution (C), and free amino acids (FAAs) contents (D) of untreated corn peptide (CK/CP) and debittered corn peptides prepared using free Flavourzyme (FF-CP), carboxy-modified hydrated silica immobilized Flavourzyme (CF-CP), and dopamine-modified hydrated silica immobilized Flavourzyme (DF-CP).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7756046/v1/01d1bc8699e1c63d51684755.jpg"},{"id":94484485,"identity":"1d8de844-0c2b-477a-af08-a62bb4e928b1","added_by":"auto","created_at":"2025-10-27 16:36:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":552021,"visible":true,"origin":"","legend":"\u003cp\u003eReusability of dopamine-modified hydrated silica immobilized Flavourzyme (DHS-F, A) and carboxy-modified hydrated silica immobilized Flavourzyme (CHS-F, B) and in the hydrolysis of corn peptides.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7756046/v1/e1f3f3c29430fa4579eb392f.jpg"},{"id":94491635,"identity":"8784b0b1-6749-4bc6-bba3-104cf98f08d5","added_by":"auto","created_at":"2025-10-27 17:24:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4828238,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7756046/v1/d91682d6-bbda-4dc8-b80d-25ba8141adec.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Carboxyl- or Dopamine-Modified Hydrated Silica for Flavourzyme Immobilization: Towards Efficient Debittering of Corn Peptides","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eImmobilized enzyme technology has revolutionized modern biocatalysis by significant enhancing enzyme stability, reusability, and operational convenience, making it an indispensable tool in industrial bioprocessing (Tadesse and Liu \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The core principle of this technology is the physical confinement of enzymes to solid supports, which streamlines their separation from the reaction mixture and enables efficient recycling in repeated industrial cycles (Tadesse and Liu \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Over the past decade, extensive research has been devoted to exploring diverse enzyme carriers, including natural and synthetic polymers, inorganic materials, and advanced composite systems, each offering unique advantages for specific biocatalytic applications (Mohidem et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among these candidates, hydrated silica has emerged as a highly favored carrier material, attributed to its remarkable combination of high surface area, superior biocompatibility, and versatile surface chemistry that can be readily tailored for optimal enzyme immobilization (Lim and Jo \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Building on these properties, recent advancements have focused on modifying the native surface of hydrated silica to further amplify enzyme loading capacity and catalytic efficiency. For example, the affinity-induced immobilization of β-glucosidase onto amino-functionalized silica not only significantly enhanced enzyme activity and stability but also demonstrated exceptional potential in the biotransformation of ginsenoside Rb1, highlighting the synergy between surface engineering and enzyme performance (Wu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Complementary studies have also shown that surface modification strategies, such as incorporating functional groups like amino and carboxyl, play a pivotal role in modulating enzyme immobilization efficiency and activity recovery, thus expanding the applicability of hydrated silica in complex biocatalytic workflows (Xiao et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFlavourzyme, a commercial enzyme preparation containing fungal proteases and aminopeptidases, has become indispensable in the food industry (Gu et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Its unique proteolytic profile enables the efficient hydrolysis of proteins into smaller peptides and free amino acids. This not only elevates the nutritional quality of food products but also significantly enhances their sensory attributes, particularly flavor. For instance, in Cantonese bacon and Chinese sausage, Flavourzyme has been shown to drive proteolysis, yielding peptides that contribute to desirable sensory attributes such as umami taste and reduced astringency (Feng et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These findings highlight Flavourzyme\u0026rsquo;s role as an essential tool in the production of protein hydrolysates, flavor enhancers, and functional foods. However, the industrial application of free Flavourzyme is limited by its relatively short operational stability and the high costs associated with repeated enzyme additions (Zhao et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e). To address these limitations, immobilizing Flavourzyme on suitable carriers has emerged as a promising solution. This approach can enhance the enzyme\u0026rsquo;s stability, reusability, and catalytic efficiency, thereby making it more cost-effective for large - scale industrial processes.\u003c/p\u003e\u003cp\u003eCorn peptides (CPs), produced by hydrolyzing corn protein, are highly valued in the food and nutraceutical sectors for their nutritional benefits, including antioxidant, antimicrobial, and dipeptidyl peptidase IV (DPP-IV) inhibitory activities (Yao et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the bitter taste, which mainly originates from hydrophobic amino acids and bitter peptides formed during hydrolysis, restricts their application in food products. Reducing bitterness while preserving nutrition is a major challenge. Traditional debittering methods have drawbacks (Mirzapour-Kouhdasht et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For example, activated carbon can adsorb bitter compounds from CPs, but its effectiveness is limited and it may also remove beneficial components (Suh et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The inclusion complex of neohesperidin dihydrochalcone and glucosyl-β-cyclodextrin shows promise in masking bitterness of CPs (Dong et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), but its high cost and complexity limit food industry applications. Immobilized enzymes offer a promising alternative. They enable precise hydrolysis, targeting specific peptide bonds that cause bitterness. This method can reduce bitterness while keeping CPs' nutritional value intact, making them more suitable for food products.\u003c/p\u003e\u003cp\u003eThe primary objective of this study was to develop an efficient and reusable immobilized Flavourzyme system for the debittering of corn peptides. To enhance the efficiency of the enzyme immobilization process and improve the recovery of enzyme activity, the surface of hydrated silica was modified through carboxylation and dopamylation. The research delved into the exploration of kinetic parameters and structural characterization of Flavourzyme immobilized using different modified carriers. Furthermore, the study compared the impact of Flavourzyme immobilized on various modified carriers on the debittering effect of corn peptides. This study highlights the critical role of carrier modification in enhancing the performance of immobilized enzymes, offering valuable insights for the optimization of industrial processes aimed at producing high-quality, low-bitterness corn peptides.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHydrated silica was procured from Evonik Degussa (China) Investment Co., Ltd. Glutaraldehyde, 6-aminocaproic acid, and dopamine hydrochloride were obtained from Shanghai Macklin Biochemical Technology Co., Ltd. Flavourzyme\u003csup\u003e\u0026reg;\u003c/sup\u003e 1000L was kindly provided by Novozymes (China) Biotechnology Co., Ltd. Corn peptides were acquired from Guangzhou Kejin Biotechnology Co., Ltd. All other reagents used were of analytical grade.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Surface modification of hydrated silica (HS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1 Carboxylation modification of HS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe carboxylation modification of HS was performed following the method of (Li et al. 2020) with slight modifications. Briefly, 1 g of HS was pre-treated with 10 mL of 5% glutaraldehyde (GA) solution at 37\u0026deg;C for 4 h, followed by washing with deionized water and drying. Subsequently, 1 g of the pre-treated HS was incubated with 10 mL of 4% 6-aminocaproic acid solution at 60\u0026deg;C for 2 h under shaking. After the reaction, the carrier was thoroughly washed with deionized water until the filtrate reached neutrality. Finally, the carrier was dried to obtain the carboxy-modified HS (CHS). The reaction conditions for carboxylation, including the concentration of aminocaproic acid solution (2, 4, 6, 8, 10%), reaction temperature (30, 40, 50, 60, 70\u0026deg;C), and reaction time (1, 2, 3, 4, 5 h), were optimized based on the surface carboxyl group density.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2 Dopamine modification of HS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dopamine modification of HS was conducted according to our previously reported method (Zhao et al. 2025b). Similarly, 1 g of HS was pre-treated with 10 mL of 5% GA solution at 37\u0026deg;C for 4 h. The pre-treated carrier was then thoroughly washed with deionized water and dried. Next, the pre-treated carrier was immersed in 10 mL of dopamine solution, which was prepared by dissolving 2 g of dopamine hydrochloride in 0.1 M Tris buffer (pH 8.5). The mixture was reacted for 12 h at 25\u0026deg;C under shaking. After the reaction, the carrier was sequentially washed with anhydrous ethanol and deionized water until the filtrate became colorless and transparent. Finally, the carrier was dried to obtain dopamine-modified HS (DHS). The modification conditions, including the concentration of dopamine hydrochloride solution (1, 2, 3, 4, 5 mg/mL), reaction temperature (25, 30, 35, 40, 45, 50\u0026deg;C), and reaction time (0, 5, 10, 15, 20, 25 h), were optimized based on the surface amino group density.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Determination of surface functional group density of modified HS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1 Determination of surface carboxyl group density\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe surface carboxyl group density of CHS was determined using the Boehm titration method with slight modifications (Tkachenko et al. 2024). Initially, 1 g of CHS was soaked in 0.5% HCl for 40 min to remove residual Cl⁻, followed by washing with deionized water until the filtrate reached neutrality. The sample was then dried to a constant weight at 105\u0026deg;C. Subsequently, 1 g of the treated sample was accurately weighted into a 250 mL iodine flask, and 100 mL of 0.1 M calcium acetate solution was added. The mixture was shaken at 25\u0026deg;C for 16 h. After the reaction, 10.00 mL of the supernatant was transferred to a conical flask. Then, 2-3 drops of phenol red-thymol blue indicator were added, and the solution was titrated with 0.1 M NaOH solution until it turned a stable purple-rose color that persisted for 30 s. Three parallel determinations were performed, and the volume of NaOH consumed was recorded. The carboxyl group density was calculated using Equation (1):\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cimg 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\" style=\"width: 525px; height: 47.5781px;\" width=\"525\" height=\"47.5781\"\u003e\u003c/strong\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eWhere C is the concentration of NaOH, (mol/L); V is the volume of NaOH consumed (\u0026mu;L); M is the mass of the specimen, (g).\u003c/p\u003e\n\u003cp\u003e2.3.2 Determination of surface amino group density\u003c/p\u003e\n\u003cp\u003eThe surface amino group density of DHS was determined following the method established by (Melnyk et al. 2023). Accurately weigh 0.5 g of DHS into a 50 mL conical flask. Add 10 mL of deionized water and 30 \u0026mu;L of methyl red indicator, and mix thoroughly. Titrate with 0.1 mol/L HCl standard solution to determine the amino group density. The calculation formula is shown in equation (2):\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 531px; height: 49.0414px;\" width=\"531\" height=\"49.0414\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere C is the concentration of HCl (mol/L); V is the volume of HCl consumed (\u0026mu;L); and M is the mass of the sample (g).\u003c/p\u003e\n\u003cp\u003e2.4 Preparation of immobilized Flavourzyme\u003c/p\u003e\n\u003cp\u003eThe immobilization of Flavourzyme on modified HS was performed as follows: For DHS, the immobilization was conducted according to our previously reported method (Zhao et al. 2025b), which involved mixing the enzyme solution with the modified carrier under specific conditions to facilitate enzyme adsorption onto the carrier surface. For CHS, an 8 mg/mL Flavourzyme solution was prepared using PBS buffer (pH 8.0, 0.1 M). The modified carrier was added to the enzyme solution at a solid-to-liquid ratio of 1:10. The mixture was incubated at 25\u0026deg;C for 12 h to allow the enzyme to attach to the carrier. After the reaction, the immobilized Flavourzyme was separated from the supernatant, washed with the PBS buffer, and dried at room temperature. It was then stored at 4\u0026deg;C for subsequent use. The immobilized Flavourzyme obtained by the two methods were named DHS-F and CHS-F, respectively. To assess the efficiency of the immobilization process, the soluble protein content in the supernatant before and after immobilization was determined using a BCA Protein Assay Kit. The immobilization rate was calculated using the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 463px; height: 53.5949px;\" width=\"463\" height=\"53.5949\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere C\u003csub\u003e0\u003c/sub\u003e and C\u003csub\u003et\u003c/sub\u003e are the protein contents (mg/mL) before and after immobilization, and V\u003csub\u003e0\u003c/sub\u003e and V\u003csub\u003et\u003c/sub\u003e are the volumes of the enzyme solution (mL) before and after immobilization.\u003c/p\u003e\n\u003cp\u003eThe enzyme activity of both free and immobilized Flavourzyme was determined by the Folin-Ciocalteu method. The enzyme activity unit (U) was defined as the amount of enzyme required to hydrolyze casein to produce 1 \u0026mu;g of tyrosine per minute at 40\u0026deg;C and pH 7.5. To evaluate the impact of immobilization on enzyme activity, the enzyme activity recovery was calculated as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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Mu8vCyK5cGv7eMZaAPjJw1owtw4a+vM+a6uNfPeR8MGdfa973at477wr3NDKrQmglZveupInAbTGx1ocz3Hv2fijH6CWpZTVV5l6LLVrpav1Y69DF/1+Mn57gHeCLO1r+O/Xk8eq7rcuAdp4j9t6K907Wxqrvbd2xSpPwkPqrLavSe2tHWvXoXaoroMWD4c5Uu5qfVmzsSblT5v2mFSRS0CsiJfHRq3zNglEd3Lz8k6itVP5bv7gZbAht2LgVnngAx9YBE+3+dPcOxk7hVJybf0jFk7FoxZTWy/v10YC4shY01iwxHJRsijboP1EiNgmCtI7Jb+fxJ6Jm6Jo2jKWLnF8lJKfZaHQ2/KV3AM0rHSsfhQS15RkDQIzrGJi1iga8WGsH6xlrD5+Q1AsGyBHCWgHMNro8QJgAcs+AIj1zJqhPcVhXzFOCkKMFXcv0El+aFPQrCisdGiTGUsP2pFYogBQclGHkkabQuLWBDTkS1x0wAAwZLziEbk1zYfN06aLLlAAQACg3OTypJe+9KWddZQywzN3qs2YwUGASAAAEABJREFU3Lgf1XHI8/5x97IeyWsT6xyZWQvm0CHCPItNY1F78pOfXFhnX/3qVxd8tW31wUJtbBSV8XHfkhXwoswe7DBBrsbkAA8gA1XWi3hD/CtzFb8JEHHr29u5Mrnoxd2ZS7F+PtABWvDiww/gmzytX4cWwFffgBPr8rZt2wqXKTCCD/MhHxjQvzI0AHJ0JDI0L+hzk8qTuEtZB/fZZ59yl7vcpcijbyhsaxFPbSIX680a6ytD/dpzyQovrGqshubVmkOHInU1XkCS/Lx38iQWbvNjnzV/DhIsotYMoCu0wu88Wjt77rln99GR8XunyZpsvDtoSSx93gP61Ni8i/IjWf/GGnsImXKJm3c8UP6eeRysXwcnVljyRtMeZP6AUfTNpzEZf/SxkivQi29rRpyy/WGIjrk4YMGAYc6M/alPfWpx+ANmo75768d4rElA2aHYmow65qfdO3kqoqy9ejesOfPc5q/VvXEbk/6Ftfh4iJzbZJ2JqV2rPofokLe1Yr15j4bqmBsys/aUO8gwOFiH1ou85aSJQNECE7jtdOPlMaFtwpATjwW9nI5397q+UusDRZNucm2Oy5UPF4sYF5aUtq3NkGJq8/J+fSRAOQkep/S9MxRI9ERhO31T8GLFbGgUdpS72jQpDy80YCOvTVxTAtJtnABBW7aSe4AGn8AghUchSzZ/ygUIA2xYF8VjUTxPeMITuo8RxBgCNsootGgHQAUvaFO03LLybGz2CxYchyKuRoraRwcsRz4koKxZqfRNGYvNQltMFlroRCKPZzzjGd0PU6sDmAMd3iMB/MCW/Eja6RNgMV5xYr5AtumyYCqXKAHgFY+eJXXJyd7nHVMGZIkVBHLUAbTEZ5IN/uX1k3g4QElsIh6972hZKz6q8IGIjzwCHER7YwXMgDhjcDA0PvOjfyDL2hH/5itfoBuYA3YpNO5NoOEc5zhHMV5z5+Mfc4eOOFB94F+ZGDk8WqvGhQ/AmxIkT4BSXeMUy+iwoB+8aAuskbE5B/JY1/VvfZCtGEF0JK5fll7XmG/55gHg1pexyNMHUGRt4KlNxo62tcIC2pYBf0C1tQ3cmkvrjfWcXAFIB6NoQ34+8CKTyCN3MjBPZKM82pg/BxbWQYccNIEec8ta6KBj3sUTBj3jkYcn90CmMUe5d8JXzN5TeQ46ZAh4+Z1Ih0Zxq/YC6woYICvvpDZ42n///YsPgdC33rxbLKPorTSZc/sTTMDSOwQ87WWwgz4cnK0bsrK2WMPkS4CjPdGB0Jx6HwEZbZRL9k57pXrG4yClX2WRgGjvo8OFsUf+Wl7No/fMQRpdhw37QZvsP95/5euRWIi9O95/a3qoD4d6et8cRHmttdgXtRNywYAUZdNcJwLFWmv3taLFMUTQYhGAbEMYKs+8Y0rAZNtUuCqOWbp2Oaw0FP7aUUxK4yRgs6IQKAobaVuXoqSYWRrEANZaC0tPW4d1gFWMwqMU2rK4pxAAAZtm5K30CvBQ3EOJhcQ7H2U2JdayeAYI4j6uNmn7QfDDesITEc9te+5hYCjaskDGPfmwFgIPkaf/oBNXYKDlg0VPfUCg1tp9YepZ4obTDvAgP30DQMokbZVL6gCG7iOpr54xUhjyPcsP2g4I3IIUiPJRyV4JwKEFDKhH6aMXqeVHOZ6izNxo6xlIM16HC88OmfpH38GeNRcIVgZoW5fuJbIjV/cSHoALis6z+MwYGx6UkYsyAEiedWpdy1NGSevTs/bm2dWz/vXpvk3am0v01Yky9CVgzVjkAxv4U19Zm6wbdbwjtS7G6UU5eUaZdUVpkpG+yc+99lFfuXmKZ1fvA/7xQt7atXwAgfLVsbbUM7/4Rz+e0ZLQx1MkbVmtWOG4bVnIWLf1qz5Z4wstdeWba+31SX4he++aNviIObY2WLXl95P+7D39fM8s1a6R8CG+0rqKQ2CUxRXwBvgYkbjnyRzQpvMcBtVjTWd9JXcA2fqzfnyIBOzYD9VjaTRP9k7jledg4hpJXS5wQAh/kb8eVzIdRVeZA8So8tXm0+fk4MAwREvoBgstg0K/3Fq1X9g/HFD75eOeJwLFcY2VtacDzxYb9M/qwR2BceZwJlCbqDpM5sy1lGc/US5Mq22+GBXtpMjn/mHBEbsAEFnMTM5cXxLTtcUvj4vNQhpC0dwHhx9+ePe7WdrhXz+jkjGgzfTOnO5l4AqQH23cA4NiFfCrLtcPflxZNLhOuISV+79FaKvMWIzJM56VRzJe+SyEkadvJzouQBZKsvVMvqwdNh7uBvXNiWuk7du3I9f91hse5eOry+z9Yy7xq5zLzdXYvfiqsiibEzTkG4v8SHjCt/lVh8zxGuWuTkBOitw11ox5B76UqatdJG5M+ehyB8g3H/iQjy/tyRA98pEvUejWo83MuIwFPbJHJ1KsBXE2+EFfOzSGEnoADoXpGnX0rc94Hrp6T8TFsSYNlUeeDZf1GN94ivxZudqsY90DwpTjrPC2Xnx4B60z46X8AIb16ivpzrcEKHN7JOuq/Rq42xUjtv8IhbC/0VP6tPeywrICeu4nlkmgpZ/vmUUWiASmPUt+4J9XgkXYs/HRTcCt50j2T7rA+zTt3imUZLngJ/pbqyu9TzfivaVJR8Ee9LN9gp40duEkbb1J98IeWE1Zh4fqwhw8VZOAKu+BNdYH20M0I29VQNEkMhcHMeCAuZ05nB/f5ElcUEy2hKOukxKLmoVAcBKk7PTkNEVpM90zkzPrUvjaSczsXBlOGQAJVxj3CBTNjcW1xeUR5lUWHoLjJmG2Dh7QIlhlhC+ug0lX/4CB8qGkPVM+0y7gIJ7CMxCkvsUCmHKVMKtzMSvnFgYwAVNK3unP4jF2Lyewo414JfygxZXEWstloD+05QPnYojE87gHcMiJJYpsLUz8OM0DTMr1A4BzVXFncNkZC3p44ppy+uMyktcm4AsIIj/zKA7DSylmzOI173i3qYgTIkPyp0DR8eKgDyBbnNqbQ2sHsFCHgjWX1gEeuGScijwDiXjgcvCS4MP8a2esDibi4chIPfVtVE6l5ohLB79AKMBmTsy10yd+ubOcYsVvWLvGxcVHLvowDuMzdjKUN5S48FibbIZtuRO15NAiH39kYuP0bMPAO7dFa5FTNpRYc1iYyFQ5AG8+rcVxiRzUX89kbNa/9SZ2eT37mhXa3muHEe9z+/t5s8Jf8rFxJAB4cWuzVNtHWdmWzf0KGrA6sv7bY+kFYNEey4BAFy+XpD2c9bLdzxxygUtGAvToDe9M/2BFBowiAJV9U2r3Tns+DwQa9J+9UzhL25eyXZnIy1iEHoXe0D+wDRPhD96hYwBymIh+UmeahD79JbGq99voB16wbljU++XtM2+EfRo/ZNmWjbpfFlA0YCAjkvgWYC2IWwQWHOsehco0DdAAMYRIKUddIID7TOyCuA4vBMUp4NWL4hRjoTG1AzDRTkAxumI0LDBJcLqBAxHACGVPaTNxAwPAIOCC1zjNoCfWQL9ABF7UEb8jfoUiV6efBPk7feHfi+wHT/VjUasroBg45dZDD/gRM+PFAd64CIBBY917772L8QN4npnXLXx0JDyJ1fCCAUJcDfK5ctQFtrnRmOuNg/VQOXeROBp0uT0oL/3oD1jQFzcAl4X6Tn3oANNkIa9N+nWyBTpZ2byQ5sn4uTiAdAANYBfDI94EQMM/OoADIGNejEdb80rO5KWOsaADUFsvQBP3CnqAM3kCHuad28OpSDtjtR6AaX3YVBwmxAOaI3EyAKkXDDg1r8bqkODl014fAJax69+pXtwQuerDiwl0W8tkKm8oWePoAXFtOfmxEODJIYjF0kHBxgy42lzMB5kAvTYRBwRgq6UT9zZSNG3o8jx7+bkjxiUuK/XXM5kbINx6s/bWs69Zoc0V6jAoPsweOCt8JR8bTwJ0nr2CZ8E+u6tGUGst9ANrnn0UoHHwp2/7B99peALs7KNDbmB6hHHEXkxn0W9DNNGgV+zb9k6eH3rUO8aopMzeac+LvdOeP27vHOpnpXmMJ4GFXOEWB8aWHrzBaGZPpD/oGnhAW+Nv6467B+hgKnqD/u7XJR84CAYaJc9oo706dCJ9HvnjrssCipQnFBpJkKlJiw4E04q7YFWhXC06iwx4dAIIEGRj5ZKyiJwcIGsgVBAuM7RBsPCw7BA8K1n0YfHK9xJR3mJAKEqLBZoWrM01R1l7BobkUcrAFEWMFpM3QAHcUGj6BEIobi5oJmT1+kk9fQIuygAlix2i98zthl8AlTy4IAEQ1leARp2hRD5Ab1sGdADJJpbF02JRzkJIvmRNhsCR9srGJeBC/ByQxapH9uqzcDJZCyS3EOW1yZiBSfPKnWAc5ArgGzsztjkxD8YLiKnr4wKAzMsMIAMz5oBczDeQaAwWuCB0ANp6QcOYAXzzAfQ7faIPABs/8Bk8eunI2zyi6WTl2TzZrPBLRk7HXjZ9WLfKxMYAaayUxmYNiOOwUQZ9AJDl1IZF3pG/nCsrt0MMQGudA8n6O/TQQ7v/VR7rOVnaUKxbBwNrFrCc1A863icb/bhkzlparLjGk+lYJWWQMtid1kC7D8S9fR7QsD8yGviwBACrded4z6i/mmut09GstRa4A3j1kRRPj2TPY4ygw+yd9IS9E7YAbulNwHI1PE5qC3MEFuIpY8hodThdRK/SjQw75Eu/TrL4DfULXMMu9CI93tZhmANCGWiUt2Wj7sUC85xGmNuoepG/R9xMc621Fso1EiBoMZWBv1qnWwgmU2wW6x6F3ZJiCaI8mcMJCnizOHyB2dYbuq+1dr+bFmW11rjtrqx7FpkXQr+RKFMAdBTatxgpedYcCt8EdQSP+ocL2YJoJ7PW2smtrOAPQAOaWduMH1h0EmDhs7EtlyQA7UTDnYp/7QEvwd5cmp6Xk1hyhQaQS8gQ4APEWLC4RS1GJ+Vaj56DWuuSTABroAUQLM1frbWgKYbT6VIRayqrHhBKFl4SZfHyOWUCpyyE2kaqtRZgCr/ojEqsiE6rgqy5SLj7rTngutaj+R/Vflw+17UDg0MKMM1lKW4FbfLSj/E46NhcbDp+0gIP4+gCk+Y0xjrqCni2dAByMsl02W5tpBxSDrvLGmj3gbhn7OD1Y+1jvOF9ckjmeYk6017tLaPq0lsMBLXWJR1QBv7oKod/Rbxi9k54gZGE/uIhtHcCX/ZO1jF7J5BoH4crJu2daK801Vo7/uEh46Xv6A73pZTu55HotpXo1TLlH1zEYMOiyRDHsCa5pwvpGtbGPrlal6fLlgUU+515ZilxXUkCILhnLRo/VcCC1tKh/JmRWaQMVtyZhTCN9aylM+7e6cRHAW0SV2mhDbVjWRIbyBrJzeanPdp6FD3QE7F3bdlK7gEIwJhsuMXJzEnFl0u1Lm+yo3+boUXNXcbCxiLnhGHBR53lXPHmNNXKMO59Zem0B1iP2nCAN5uUl7/tt9ba/XCxjaXWxbGaeydI64FrFv/47pWtqYoAABAASURBVM+XA0zwEFfj9EK1ffTvveSAucOCF9CBAmAkn37doWcbkwPIUFmbZ31wwbPwsngrYyX11VycClmrvfA2QOWR0G9liTchETHOUVcxs0HDVV+j6mb+YSVlkDKY1zXg/W+T/QTY4h1itHHw5G1hFLEHtnWnuecVsafzVkV9z3RBACf7j/3cQT/quNKh9nS4wHM/2TvF/Nk7eaqUOyyjF3sn3Ty0d6q7XolxiF4NcItPYwPY1qtPnjbGGLH8DBGReMSAfGFc8Mpq+181UMSAyeeuZf3zPE2ygMRhUPQGw3oy1A6AhJpZ1LgrmVeH6i03z6KqtRaWqZZvi5mFDRgboskE7pTgoxBm+liYUddLwAIGyQMNkU9G3ObxvJwrly3rrVgHNAAIrtTl0GjrWshiOQAQFlHAFtiodRGMtXUn3bOe2mSAede2PmBCttz0TjlOqm25oF+nQnPPmiieoy1Hz4vG2smVrQxodGIE5IBBX3cLV3CqVO5l5dpmhUZfXiTzZm7jedSVu4ULGm9kDggHmBvVRr4QC5sCeXoel7zY3BE2u3H1bJjAa1uHxdQ7YTNu8/N+5RLIlimB3VkCDt72a7+UAaDVWosDuTAcOnK5shFnTYcCK9HWXs7QIXxIHhe3vVtIkOdIeLGPs85FXntlMOFRYzRq8/v3Q3tnv856PbPI0mnwgY8nV9sPHUDnAdE8aUEPffH99G+bWFrNI7wS3wpEG1cgFn+S50lpTYCiAFNxZgYzqcMoF9/oIwUfLQBCJlUZOhS6e4mC9tEMYbgSjPzVJsAI2GKl9IEFNyCaYgoInNnbcz+J7TNO4EEZc7ir5EVwojBBrEXc0PIBMqDGRHtuE8DsRACgtvntPeAjUBbwAVz0ATC1dSbd64PrOurZBLhwxfORvZc2ypZzJUNuVC+v2BBzZ0wsruYYCPRCi020CYmLQN/VRsASaC4kAFusovJILLgAuZjUyCNfVlZuCC+htlFmfQBQrI1crYCkMpuRUzJ+PI9LZGuj4y73RZ3NbJoXyhwBcWQwjj7LphdeXKS1FHXFUlpDZCOPFdZY+2tR6IE1ZTNQL1NKICWQEliNBAAvX+PSt7UuGgzs2fZoFsYh2owf9rGhMnsbq549GrBx6KcPHaLDCymsyh4mrl8+Omj6QXEAVZy5vDaxeuqTK7rdO9WfZu9saa3XPaMIQxFvGnnShYxh0R9AHvdx5U2CPUIOkR9XBhm0AG1GgsiHQ+g7OrhNZM8b6XcnGVqiflzxR1ey6EbeuOtEoEjhUt5MnMzwrCxtAgbEAjABAyJ+soMiwwgztjztKD8fZFCSPrsXH2dytaPE0RQQKi7CogqmDdZiNeBYYKUsljI3+9CBcPXBRE5Jo0+R4sUXykAgUywBawM8oAv1m0w/q8NKBxiwWgI8XpLFXnb+15e74jh82OAFYuEDltCwINBjKfVymECgjgnfOI0BNRY9E8R87qtoX0wDcuLiWODIwlhaOYhZY4FCi8ULHUkspTEaPwsmGQIryoAichCvB8jpV75Uay2+LPfBB9BlIcofSlzDDgPi6biHyRJdddH08wmAp/FbzKx/vu7y8UattQBsQJQx+nFVQcDm1FfTwHOttQDW8vEU4QC+KBdvB+Dpq036NF82JHMZZcZhblgdgUzgy1yKm/Q1us0F/144Llx9WaPRPq6Cpc0NoObr68gfd/WiWkfWM7A8VNe8miMbHYDe1gGogVJy9HM8TtU+tmrrOAl6r7z8LJhtWd6nBFICKYGVSMBe1B7Gg0attdjP4zmuPIiMDMAZXQ7sRZmrPdfHkXQt4wpd7CfP/DIAHaFOrbUAij7A8+sU8IGft7NH8yDWughY1ZVi7+RlxK+8SDwzPmSJvZMeoBuifC2udDNAx2iFnoM8QwV9LdHBYsrt2T5GZFwQvrR58+YiNIveFtY0JGfuYaFgcBPa/WS89AsMIRSqX76cZ3qRzhSGBWhO03YiUGT1s1D8bIjYNhPeJpNnssUTci16Zj3zbOLFCUgEB5AQpsVFkbMktrR8QQRQUdIt8xYdczVBt/mQOYXJDczaRgBOLRaJCYKY0cQXMADAEY56QCOgamGZJDwDBhao+L+2n/YeuLGoLUqTbmFrD8SymOHVQvG/4mKp9BJYACxUXgC0gA8uXx+A+DjDycmLp60XCd94boGiiQWigSA0IsXiBWaMAZCwgJWzxBkTyyFXc/SvTGKpIgNgyvOoRHYAtp9tAHD87l+AUW1Y9GwCZIEHINFPBpkb5V4MoNgLJnQAP+JAjVu55HRpLfhNQ+EIDijAMdM5PtVpk81Gf9ZUm+8eWOfaBbbw46TssAPMOo1aIwC/+FLg1Xi06yd843HTpk39osFnwM66s8atsaFKAKR5wWO/3Pwbk/eNzIFY8xr1rAfz4GBgbUV+XmdXAuZKSMU4j0Fwz5NhfuN5Pa72XlZyBzMKvN+HQzXFr9yBuF++Xs9+Murggw8u04SGrBcPSXd6CYiJrrUW+ytARI+2rWuthY63x9oLfbFrz6JDWnBC1/GSwQL2fPuifQ8OaOnZ+1gbGR3oirbMPZr0sD3b3im2UriWsrVKdCtArA/jptsZpegtCXCmS+hIRh39MnRw3fu5MLgAeKbzlbWJ3md4Ylxq89t7Os+YGCLa/KF7/YpXZJDql8NEsBRjTb9s1PNEoAicTErAC3Bnsvt1oek2D8gC+tq89h6AoiiDYYAOimcajzi0KPNVdNsWALE42zzAo88XiyF3LjpiClmutDFZLFzyRyXlJlTfrIVADBAGJAYQs2hZxyh9QM1PorB+BU2AAljw8y/AJmBrceEhkhOSU0S0cQVaAnx5lvRvAUY71kPgTxlw6ad5vKD9F085EKZ/NDyPStzSXozow5Xc2vpcyECSMptDv1xMqL6UWwMOHW17915Ac+OFZ6pnPSVLZUPJ2NAdKrOGWBH1h5bTmHrmz8sqX/ICA/3K2mRTcgoUq9jmj7s3X37CwUHA3NtY+vUdQnxZH2ulLa+1FrK2DsWV9DcUMT9CNQB7oLdtO4v3LMrex6HEhTSLPK+WJzFZwCFrNVr2RYrN2vA8Lpl76zbq8G7E/VpcrWng0JUC8V601gmHbWC11lqsTwdc4SBr0TcaaA+tBYpPHLPxUsTqZpptCdg722TP7nNMz9G9jCsO6/Y9ef16vFL2fd4kQJLbtF+n1lp4o+hca3Oo3IF+1N7Zr7+SZ+8M40477qF7701Lnz6mg+k9B/8h/nmT6E/eybZte28vIR9Aj0GuLevfwxdwTV+WPIGAuDkByvvtRj1PBIqjGq5nPnDIpCu+gXWJa5awp9ls15OveaDN9c8d4CTEneyr8hWOa+6acd07KbMyctVbc0z9yxnoXnvtVVhLbYhefu765bQfVdfmABwCE6w9TvGj6s5Kvk2V5dZ7SyZCPSSHDiEMs8LnWvLBIgjssICg6wDl5zyGlIPyNjkAUHbyHI5Z2dyvVXLQ8M47gDiY2l9bCwZronLKnZKxV7A8rFX/rCFAKlBgHQhb8p7olweKIlyrvpJOSmAeJeAdYtzwfq5kfA6KXPJD4Vzj6M0kUBRn6ARi4xTbxsQbG+i4wWTZZAlYIE5lrJBOFj7cmNxq96hBwQsh4DYgGyfcWneOk5lGEhQe6whrMrcjK9M07UbVcVhCh4WOch1lRR3V/v8rnyudRRhIYlGX3PMyOKAAU0IYtm/fXoAFIMvHbOJu8ezrR3mRWL+EZMSzL8yFXnBtqcuKB+QDRH7bExDiClKPlU97J2q0JbG3Qcu9vH7iqtVOPe6ztlyf8vGkb30Ir3CwVdfBw8du4mEdftWV8KjM2D27kgW3K6uecu+nuHB9oKuee3SMAU9k1fLjHh+sgOqjJ35bvpAL8lAm9ILMeVXi/ceD+Chjwb/5IFtrV+iQttzn8sgbfbTQjoQ/+WRuPJEfV+vBGrAmXB16KC2Hq6jTXvGEprGKcRf3TTb6wD8Zki/e4tnch7yMJdaScaAVVn51jNOYyNSzcZGtuSNb8tdXJP3gg36S571EH5/KyNVck6P1RG7mQBu8a+c55sRYtTcG9PBgvQ3JTt1dk7KX9ZQAS6CQDvPOq2LOrY1p+6y1dnGdvJi+zxDKNE1b+wLXfngvHd6naRd1ZhIoAoU+VmCuZdkBFoPhvK5OAsAG07gPZ1gWVkdtvloDiE5r3L7cZFwiqxmheBrhBdbzauhwV4u3ZFFcDZ1ZaOtDN9ZsmyOlKmwFWBDjKq5XOAGFi1e/sQpwS2JRxRjzNPiRfOEfYpEAIB8piYm18YqX1g7Q56a3j4jJk7il9IE2ICQ8AG0uIQdT+W1i/SJ38bXijIRCACrq+HhNaAJgiC88U/bABdDggylARGy2tvhkVRUeY4OnKAAgLiAAxq8vGKNYZVZo5YCF2Cey4bbz7gI6gBg3FTniJRLg5ItLa87PQxm/EBn1gRY8ATVixwXfRztXwFo8OqDlUEKZ6UOcE/4dLoExNMiQ3MwbRae9sdmrfdhoboA0+eOSPoVpMAj065ln8jWvwofEeBu795KcjJPy8zEhGfvAy9yIj3aYIjNuOmO11pSLeTY3+sKndWQNAnURhiPOjMyM3TyxvIpbtw7MnTVGPnj24QJFDTDGszUgtMq60Lc1Jr5fPD3aEfYCDJov8cwOlPi1zsgRWMVjpvmTAG+ePcKaEE5nPTqsLHekXMv0lYPFNG29Aw6G1mY/hG+a9jMJFMXWeUG9QGL1oOBpBpN1JkuAXCknJuxal28tm9zDxq0hVlOMLEU3FNM5iyPbCDwBJdyNYlSBNQAI3yxMYvIoTMqfBVeMDmuOckCBArcHiGEDWFjagEsWKTE2XPA8DubMvXxWS+AN4AcEzKu9RHwcIEa5AwE2TvS9D4CkTVy/kQATFiBgU8C9fYnVGRgA2vALQNh4xdlxs4tBEpPsMIY+ntBzBRoEmfta3diFNhiLEIX2MCHembvaOwrAiv/iCdBG8L+YR7Gw2qIdCRgFrBxQyM64gFXgjmvX2rauAS+W82jnCogC2PhwQPdTZGKygGWx0QAahWYuzSG5kT8QxYLmS34yBp7w4WMCdIcSuVkLgJz5GKqjbwYCYzC3Qi8oWDIW/8wayW3NGgkoyjdOihNYdHgAbgFmB4p+rLF9MPq1Bq0r4FyoAMDmwGjd4NFcm0syATaBVh/w4YF1n8LHB4BJ9sAvEEvGgKHDkWe0JIcKfbMeW4dc/fgVksGqCdgrzzR/EgDUvDuRrEP7ynJHyhMgZtOvlkzT1t5hza6kL/RnEihiLFNKICUwHxLwIREAwRJFcfpoyci46MXI+ZrPpscdAjSGaw64Ya0Rk8k65tmGB4gBcE7jXIoUPYDoQy8fKwFYACflLlHcDptcnk7Wkt9qY3nDC0sRMAHw4CsSsAJAugJurG3AEsUPJAAH6ANLwF20i6tAcqAmno2XFR/YQgcoUA7YshBEvf7V2PyyAysbqxmL41BzVRg/AAAH2ElEQVR9AA3IxhtZAj0+qpNnzH26457xSrGgxXIBZAMwLGWeyQ3IkQcksm6SM/Do3hhH0QfirQUWyVF1rBmyMm94MB6gFZA1v2IcAX5WWXySoTkEeM29dmRkzrQb1U/kA53mGbi0HoE34BMY94sJ1guACxiacxZHeWSOF/XMNwWOVwcGdQF7axbAdKBw4NFevyyZDk2s6NaSQ4S+jEN5pt1WAjM38ASKMzclyVBKYL4kQGmzqAEfQIYPhLhAgSUjrbWWWqvbwiUneaDgueJ8Ie9nreRJQKWTOVcnoFhrLfpQJtW6SMv9qESZcx8DpVx9Yu/QbOvr35fBLG1clyx5yoEBIMW9RPFT+u7HJTwaCysfy5dYOGEF49pEGQAEIHFhageQRVl7JVMp8mqtBQiqdbJMok17JSdJnisZcUGTm9hPMjS3LJ7yJP2PsyiSl7XArc3ChnY/Ae2+KGXNFY8ljlCdWmvBg9AZANhcsNAqk/Qd64cVr9bpx11rLeiVo/7IDZhnyeZuZmFk1VTMQq4foJg72odb8iORlftaF/sPurUuPivT3pry0RC5AZ3GycKsPFNKYFYksMesMJJ8pARSAvMvAVYn7hLWvEngiruTm461hsvExxOUKSkBD9yKrGvcqfKWk1ibuGnj62sWMAq7pcF6xx3NTQQ0cEUr37x5cwH2uEM979ixowASwAGrJWUvdi/qqxMJqOFyZJljKYv8/hUo0Z7rHKhWzsLFMssypR95bRJTy4IlrhEP2vuQQvwci1tbdyX3voAGcFlzgRygCXBlFSY7X2qjC/QAV+7bpA3LJhdvmx/3gF0AcD9tReZcstzv7qMea6M1RBaspiyBylgSxS6SGeDvq3H1WPaUk5kPR8hFfCTrtT6VWYv4An49S8YhdpQFm3WT5dTvsyqThESwdrNIs7DKI3dj1NYz4KoPlk3PysmNLMyjutYwfvXNSm59qJspJTArEkigOCszkXykBOZMAj4qoOjF9YkB8/GAxE0LCAFiW7duLX6Sxc82COin6FkKKWgxWyyGYua04371EQoxsUyK06OIWWXksW6x/AEp3MOsVYAmWkAndydeAClAhCvbFe1t27YVsY7oROLyZQniGuRqBMTwSKEDCGLVtOVOB8a4GPFlLD5cYG3yc0ue3aMLyLEYGTMroTwWTbQAGJY47khAFvgCKAEN9bgzgWLWRH3JaxMrnQ9zADZflXPxAlU+gpLnmVzwGyA32vvwwu+fcvX76Q39+uBD7CUXK0BDzuLuAGfjFocJ7HLTitXkSpbPCsvqF7TjCrgBQsAWYBT5rsoAUGDMl6FiVrm28epZHe0AVcDP/52DZZUslUkAHdeuH/U3fpZPa4BclONNrKkYxE2bNhUgGsBXT0ynD3CsxwCr4ifxQDbGLQaRlRktyQ8WOxw4ONS6aCn0ERAQKk4SXW1dyRYA9GwNkhe+rD/zjF+WaxZMtDOlBGZJArsjUJwl+ScvKYG5lQD3nP9jA3AA/AEeEuAE0Pihc3F3rHmsZYAGQMNK5eMDXwMDjtpIhxxySBE/FgJTnys3rGXAg/a+3hX7B9QBRkCAjwx8IYsXX0azPvlggVURbV8hUtxB2xUI8oEEyyVwBEwCnPgGdoEobcWucYcDldyp+gEMAFk/SaEd6xOaEiAGoLCSegaa0QCI0AMExQSySgIP4tfU4+LVLxnUughM5EcCWgBDwI68gREyMi48A6HAFTmKxYt2rvgzTpZS9QAYYNeHO/vuu2/nkgVO3QPcDgEAF4CsvQ89gF/8+/iGlVF+m9QxJv2j1ZYBm3jTNysgIA5ksyaSqa+2fSwDrGvHymydiEn1LJkvcsQfUOmQAoyTi3JrzpjMjdhNoNiaQUMsqkOFD2NYHtUHVvEbawSI9UGQMsm8Aapt+IA24iYBQnR9rAD8mQsHDyCaZVy/1ouQCgcc/BofgA0Io58pJTArEkigOCszkXykBOZMAhQeQDaUgCRgIcoo0bY+JRxlcWWNo1yJiZsQyPQBgGepbS8eMNpxC4qLi2d91VpLv3802gRgAGfa+WAG4OLm1A4f8iX01dVWmTr6M0blEjejcgkQQde9pK2xqYcWN6vk2ZhqXQSFPnJgfeS+124o1VoL2tqiSQ7q4UtepJYf5Z6jzFW/rlLISz2gHI/yyYQc5EvyJOW1LvIsP1LIA1+17lxOJtFWH57JEa24J3+yQs94PNcadEoX56oP7dDCd9QvC3/o6BvYlK+OdaY/+fFc6yJN9eW1ybwC0SzbwhIAXP0skO/+I8foH11l2pOVPvXtmXw1IL/IM1bjkp8pJTBLEthjlphJXlICKYGUwDgJcEWzGHEvs1pRruPqz0MZdzoLIZc56xUwNA/j2qhjECfLKswC2bfMbtQxJd8pgXESSKA4TjpZtm4SSMIpgZVIgKWNNYabkVt1JTQ2WhuWLVYnwLgfR7nRxjIP/HI/C5UQO8tSOA9jyjGkBMZJIIHiOOlkWUogJTBTEhD35YeYfbgwU4ytIzPiDn3skCBxHYW8DNLiHsUSRuzoMprOe9Uc35xKIIHinE5sDislkBJICaQEUgIpgZTAaiWQQHG1Esz2KYGNKoHkOyWQEkgJpARSAhMkkEBxgoCyOCWQEkgJpARSAimBlMBGkMB68JhAcT2kmjRTAimBlEBKICWQEkgJzIEE/g8AAP//ivvX4wAAAAZJREFUAwDYupwild8FLAAAAABJRU5ErkJggg==\" style=\"width: 625px; height: 50.9615px;\" width=\"625\" height=\"50.9615\"\u003e\u003c/p\u003e\n\u003cp\u003e2.5 Determination of enzyme kinetic parameters\u003c/p\u003e\n\u003cp\u003eFree and immobilized Flavourzyme with the same enzyme activity were separately added to casein solutions at concentrations of 1, 2, 5, 10, 20, 40, and 80 mg/mL. Reactions were carried out under the respective optimal reaction conditions, and enzyme activity was measured. The Michaelis constant (K\u003csub\u003em\u003c/sub\u003e) and maximum reaction rate (V\u003csub\u003emax\u003c/sub\u003e) values were calculated using the Lineweaver-Burk double reciprocal plot. A double reciprocal plot was constructed with the reciprocal of substrate concentration on the x-axis and the reciprocal of reaction rate on the y-axis to determine the K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e values for the immobilized Flavourzyme.\u003c/p\u003e\n\u003cp\u003e2.6 Structural characterization of immobilized Flavourzyme\u003c/p\u003e\n\u003cp\u003e2.6.1 SEM Analysis\u003c/p\u003e\n\u003cp\u003eThe surface morphology of the immobilized Flavourzyme was observed using a scanning electron microscope (SEM, Merlin, Zeiss, Germany). A suitable concentration of the sample solution was prepared using water as the dispersant. The sample solution was then dropped onto an aluminum foil sample preparation table. After drying, the sample was coated with a thin layer of gold to enhance conductivity.\u003c/p\u003e\n\u003cp\u003e2.6.2 FTIR Spectroscopy\u003c/p\u003e\n\u003cp\u003eFourier transform infrared (FTIR) spectroscopy was used to identify functional groups and investigate the interactions between the enzyme and the carrier. Dried and milled samples were mixed with KBr at a ratio of 1:100 and pressed into tablets. KBr was used as a blank control to subtract background signals. FTIR spectra were conducted using a Nicolet IS50 spectrometer (Thermo Fisher Scientific, USA) over a wavelength range of 400\u0026ndash;4000 cm⁻\u0026sup1; with a resolution of 4 cm⁻\u0026sup1; and 32 scans.\u003c/p\u003e\n\u003cp\u003e2.6.3 SDS-PAGE Analysis\u003c/p\u003e\n\u003cp\u003eSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the molecular weight distribution and the integrity of the enzyme after immobilization. The analysis was performed with minor adjustments to the method described by (Zhao et al. 2024). Samples were prepared into a solution with a mass concentration of 10 mg/mL. A volume of 400 \u0026mu;L of the sample solution was mixed with 100 \u0026mu;L of loading buffer. The mixture was then heated at 100\u0026deg;C for 5 min to denature the proteins, followed by centrifugation to remove precipitates. Subsequently, 10 \u0026mu;L of the supernatant was loaded onto the gel. The electrophoresis was initially run at 60 V for 30 min for pre-electrophoresis, after which the voltage was increased to 120 V until the electrophoresis was complete. The gel was stained with Thomas Brilliant Blue R-250 and the staining was decolorized using a decolorizing solution until the protein bands were clearly visible.\u003c/p\u003e\n\u003cp\u003e2.6.4 TGA Analysis\u003c/p\u003e\n\u003cp\u003eThermogravimetric analysis (TGA) was used to evaluate the thermal stability of the immobilized Flavourzyme. Approximately 5-10 mg of sample was accurately weighed and analyzed using a Mettler TGA/DSC1/1100SF thermogravimetric analyzer. Nitrogen was used as the carrier gas with a flow rate of 20 mL/min. The heating rate was set at 20\u0026deg;C/min, and the temperature ranged from 25\u0026deg;C to 700\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e2.7 Preparation of debittered corn peptides (DCP)\u003c/p\u003e\n\u003cp\u003eTo prepare the debittered corn peptides (DCP), a corn peptides solution was first prepared at a solid-to-liquid ratio of 1:10 (w/v). The solution was thoroughly stirred to ensure complete dissolution of the corn peptides. The pH of the solution was then adjusted to 8.0. Subsequently, 2% (E/S) of Flavourzyme and an equivalent amount of immobilized Flavourzyme with the same enzyme activity were added. The mixture was incubated at 37\u0026deg;C for 8 h to allow the enzymatic reaction to proceed. After the reaction, the mixture was heated in a boiling water bath for 15 min to inactivate the enzyme. The immobilized Flavourzyme was then filtered out, and the resulting DCP solution was obtained. Finally, the DCP solution was lyophilized to yield DCP powder, which was stored for further analysis or use. The efficiency of the enzyme digestion was evaluated based on protein recovery and degree of hydrolysis.\u0026nbsp;The protein content was determined using the Kjeldahl method, while the amino acid nitrogen content was quantified by the formol titration method (Zhao et al. 2025c).\u003c/p\u003e\n\u003cp\u003e2.8 Sensory evaluation of debittered corn peptides (DCP)\u003c/p\u003e\n\u003cp\u003e2.8.1 Quantitative descriptive analysis (QDA)\u003c/p\u003e\n\u003cp\u003eA panel of potential assessors was interviewed and screened for their sensory acuity and bitterness sensitivity. From this pool, 10 assessors (5 male and 5 female, aged 20-30) were selected based on their ability to discern and describe bitterness accurately. The assessors participated in training sessions to familiarize themselves with the bitterness attributes of corn peptide solutions and to ensure evaluation consistency. All sensory evaluations were carried out in a specialized sensory analysis laboratory equipped with climate control set at 25\u0026deg;C, industrial white lighting for color consistency, and individual tasting booths to eliminate environmental interference. For the QDA, 1% untreated corn peptides (CK) and DCP solutions were prepared separately. Quinine sulfate solutions at concentrations of 5, 10, and 15 mg/L, corresponding to scores of 5, 10, and 15, respectively, were used as reference standards. The assessors evaluated and recorded the bitterness intensity of both CK and DCP solutions. Each sample was randomly tasted three times, with water rinsed between tastings to ensure palate cleansing.\u003c/p\u003e\n\u003cp\u003e2.8.2 Time-Intensity method (TI)\u003c/p\u003e\n\u003cp\u003eThe TI assessment was performed following the methodology outlined by (Zhao et al. 2025a). Prior to the assessment, evaluators were instructed to rinse their mouths with water to eliminate any lingering tastes. Quinine sulfate solutions at concentrations of 5, 10, and 15 mg/L, corresponding to scores of 5, 10, and 15, respectively, served as reference standards for bitterness. Evaluators were directed to take 5 mL of 1% CP or DCP solution into their mouths, hold it for 5 seconds, and then spit it out. They were tasked with rating the perceived intensity of the taste at specific time intervals: 5, 10, 20, 30, 45, 60, 75, 90, 120, 150, 180, and 210 seconds, until the sensations of bitterness subsided. To mitigate carry-over effects between samples, a 20-minute break was enforced, during which participants rinsed their mouths with purified water. To prevent order effects, the presentation order of the samples was randomized. Each sample was subjected to three independent trials to ensure the reliability of the study\u0026apos;s results.\u003c/p\u003e\n\u003cp\u003e2.9 Determination of molecular mass distribution and free amino acids (FAAs)\u003c/p\u003e\n\u003cp\u003eThe molecular weight distribution was determined using HPLC, following our previous method (Zhao et al. 2025b). The FAAs were measured with reference to the previous method with slight modifications\u0026nbsp;(Xiang et al. 2024). Equal volumes of DCP solution were mixed with a 10% (w/v) trichloroacetic acid (TCA) solution. After standing at 4\u0026deg;C for 1 h, the mixture was centrifuged to remove proteins, and the supernatant was collected for free amino acid analysis using a fully automated amino acid analyzer (Hitachi L-8900). The dose-over-threshold (DoT) value for each amino acid was calculated as the ratio of its concentration to its taste threshold\u0026nbsp;(Xiang et al. 2023).\u003c/p\u003e\n\u003cp\u003e2.10 Reusability of immobilized Flavourzyme\u003c/p\u003e\n\u003cp\u003eTo evaluate the feasibility of immobilized Flavourzyme in industrial applications, the reusability of the enzyme was investigated. Under optimized reaction conditions, immobilized Flavourzyme was used repeatedly for the hydrolysis of corn peptides solutions. The operational stability of the Flavourzyme was assessed through enzyme activity measurements after each cycle. The immobilized Flavourzyme was used for ten consecutive cycles. The initial enzyme activity was set as the baseline (100%), and the residual relative activity for each subsequent cycle was calculated based on this baseline.\u003c/p\u003e\n\u003cp\u003e2.11 Statistical Analysis\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted at least in triplicate. Data were reported as \u0026quot;mean \u0026plusmn; standard deviation\u0026quot;. Differences between the means of multiple groups were examined by analysis of variance (ANOVA) along with Duncan\u0026apos;s test using SPSS 20.0 software (SPSS Inc., Chicago, USA) at a significance level of 0.05 (p \u0026lt; 0.05).\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Optimization of hydrated silica surface modification\u003c/h2\u003e\u003cp\u003eIn this study, 6-aminocaproic acid was employed as the carboxyl donor to achieve surface carboxylation modification of hydrated silica through the condensation reaction between its amino group and the aldehyde groups on the HS carrier. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the surface carboxyl group density of the carrier increased with the concentration of 6-aminocaproic acid until it reached a plateau at 6% concentration. This increase is attributed to the enhanced reaction kinetics due to higher reactant concentrations. However, concentrations exceeding 6% did not lead to further significant improvements in modification efficiency, likely due to increased system viscosity, restricted molecular diffusion, and steric hindrance (Chen et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Consequently, an 8% concentration of 6-aminocaproic acid was identified as optimal. Figure. 1B and 1C demonstrated the effects of reaction time and temperature on carboxylation efficiency. The findings indicated that reaction temperature significantly influenced modification efficiency, aligning with the Arrhenius equation (Eshaghi and Pouransari \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The surface carboxyl group density initially increased with temperature and then plateaus. The maximum carboxyl group density was achieved at 50\u0026deg;C, which may be attributed to the accelerated reaction kinetics and the shift in equilibrium towards product formation due to increased temperature. Similarly, the surface carboxyl group density increased with reaction time, but excessively long reaction times can lead to side reactions within the grafted carboxyl groups. Therefore, the optimal reaction time was determined to be 2 h, with the maximum carboxyl group density reaching 56.22 \u0026micro;mol/g.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor the dopamine modification of HS, the surface amino group density initially increased with the concentration of dopamine and then stabilizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The peak surface amino group density was reached at approximately 3 mg/mL of dopamine, beyond which higher concentrations did not further enhance the modification efficiency. This is consistent with the findings reported by (Zheng et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), where high concentrations were found to enhance π-π stacking between dopamine molecules, leading to the formation of aggregates and a reduction in the effective reaction concentration. Additionally, the increased system viscosity at higher concentrations can hinder dopamine diffusion to the carrier surface (Sureshkumar and Lee \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE revealed that the surface amino group density decreased with increasing reaction temperature. The highest surface amino group density was achieved at 25\u0026ndash;35\u0026deg;C, after which it declined with further increases in temperature. This suggests that lower temperatures are more conducive to the effective grafting of dopamine onto the carrier, while higher temperatures may lead to side reactions or degradation of dopamine, thereby reducing the modification efficiency. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF shows that the surface amino group density increased with reaction time, initially at a higher rate and then plateauing. The optimal modification was achieved after a 15-hour reaction, at which point the surface amino group density achieved 61.25 \u0026micro;mol/g. According to (Mao et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), in the early stages of the reaction, dopamine molecules rapidly graft onto the carrier via Schiff base reactions. Over time, grafted dopamine may undergo oxidative self-polymerization to form a polydopamine layer. However, excessively long reaction times can lead to excessive polydopamine growth, which may mask some active amines. Therefore, a 12-h reaction time was determined to be optimal to balance the grafting and polymerization processes effectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Immobilization of Flavourzyme\u003c/h2\u003e\u003cp\u003eTo assess the immobilization process's efficiency, both the immobilization rate and enzyme activity recovery were calculated. The results showed that DHS-F had an immobilization rate of 42.01% and enzyme activity recovery of 73.68%. In comparison, CHS-F demonstrated a higher immobilization rate of 62.93%, but its enzyme activity recovery was slightly lower at 68.22%. These findings suggest that CHS-F achieved better immobilization efficiency, which may be due to the carboxyl groups on the carrier providing more binding sites for the enzyme, facilitating a higher adsorption capacity. However, the increased density of enzyme molecules on the CHS surface could lead to steric hindrance and conformational changes in the enzyme, masking the active sites and reducing accessibility to the substrate, thus lowering enzyme activity recovery.\u003c/p\u003e\u003cp\u003eThe Michaelis constant (K\u003csub\u003em\u003c/sub\u003e), a key parameter in enzymatic reaction kinetics, reflects the enzyme's affinity for the substrate and its catalytic efficiency. A smaller K\u003csub\u003em\u003c/sub\u003e value indicates stronger substrate affinity and higher catalytic efficiency (Wang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, both the free flavoruzyme (FF) and immobilized Flavourzyme groups exhibited typical saturation kinetics. Their catalytic efficiency increased significantly with substrate concentration initially, then gradually plateaued. This aligns with previous research on casein hydrolysis (Dehkordi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the importance of substrate concentration in enzymatic catalysis. To quantify the enzymatic reaction kinetics, Lineweaver-Burk reciprocal plotting was used to determine K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB revealed that immobilized Flavourzyme's K\u003csub\u003em\u003c/sub\u003e values were higher than those of FF. Specifically, FF's K\u003csub\u003em\u003c/sub\u003e was 24.48 mmol/L, while CHS-F and DHS-F's K\u003csub\u003em\u003c/sub\u003e values increased to 26.86 mmol/L and 30.46 mmol/L, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This implies that immobilization reduced the enzyme's substrate affinity, possibly due to conformational changes in the enzyme structure, steric hindrance around the active site, altered microenvironment at the enzyme-carrier interface, or restricted substrate diffusion towards the active site. Despite this, the V\u003csub\u003emax\u003c/sub\u003e values increased post-immobilization. FF's V\u003csub\u003emax\u003c/sub\u003e was 349.66 mmol/(L\u0026middot;min), whereas CHS-F and DHS-F's V\u003csub\u003emax\u003c/sub\u003e values rose to 356.34 mmol/(L\u0026middot;min) and 372.20 mmol/(L\u0026middot;min), respectively. These changes may be attributed to altered substrate diffusion efficiency and reduced enzyme flexibility due to immobilization (Zhang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eImmobilization rate, enzyme activity recovery, and kinetic parameters of free Flavourzyme (FF), carboxylated-modified hydrated silica immobilized Flavourzyme (CHS-F) and dopamine-modified hydrated silica mobilized Flavourzyme (DHS-F).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eImmobilization rate (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEnzyme activity recovery (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[V\u003csub\u003emax\u003c/sub\u003e] (mmol\u0026middot;(L/min)\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eK\u003csub\u003em\u003c/sub\u003e (mmol/L)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e349.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e24.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCHS-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e68.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e356.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e26.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDHS-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e73.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e372.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e30.46\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=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.3 SEM Analysis\u003c/h2\u003e\u003cp\u003eThe SEM results, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, revealed notable morphological changes post-modification. Carboxylation increased the surface roughness of HS, while dopamine modification resulted in a uniform dopamine coating on the silica surface, forming a distinct layered structure. The surfaces of CHS-F and DHS-F displayed typical flocculent protrusions, which are consistent with the microscopic features of protein aggregates. These features directly indicated effective enzyme loading. In contrast, the original carrier exhibited a smooth and homogeneous surface after lyophilization. The enzyme-immobilized carriers showed notably heterogeneous structures with dense granular depositions. Notably, dopamine-modified DHS-F demonstrated a more uniform organic-inorganic composite structure. The dopamine coating enhanced the carrier's surface roughness and optimized the distribution of immobilization sites through multiple interactions between its catechol groups and enzyme molecules.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.4 FTIR Analysis\u003c/h2\u003e\u003cp\u003eFTIR spectroscopy was employed to verify the successful immobilization of Flavourzyme onto HS particles by detecting covalent bond formation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the FTIR spectrum of HS particles displayed a prominent peak at 1105 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, attributed to the asymmetric stretching vibration of Si-O-Si, which is a characteristic absorption peak of HS (Singh and White \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For both CHS-F and DHS-F, characteristic bands were observed at 2935 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2913 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 2855 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to the C-H stretching vibrations in -CH\u003csub\u003e2\u003c/sub\u003e- groups, which are common in protein structures (Mekonnen \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, absorption peaks at 1658 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1633 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were identified, which are associated with amide I bands (C\u0026thinsp;=\u0026thinsp;O stretching), respectively (Hussain et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These peaks indicate the formation of amide bonds, which are crucial for enzyme immobilization. The free and immobilized Flavourzyme showed significant absorption at these wavelengths, confirming the integrity of the enzyme's secondary structure post-immobilization. The presence of these characteristic bands in both the immobilized derivatives and FF spectra demonstrates that Flavourzyme was successfully immobilized via covalent amide bonds on the modified HS carriers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.5 SDS-PAGE Analysis\u003c/h2\u003e\u003cp\u003eSDS-PAGE electrophoresis analysis of the supernatant from the immobilization systems of CHS-F and DHS-F (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) revealed significant differences in protein band intensity and number compared to the free enzyme solution. After immobilization, the enzyme solution showed markedly lighter and fewer bands, which are attributed to the immobilization of key active enzymes in Flavourzyme, including neutral proteinase 1 (42.4 kDa), neutral proteinase 2 (19.0 kDa), and leucine aminopeptidase A (36.2 kDa) (Yust et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Notably, the enzyme proteins with molecular weights ranging from 10 to 28 kDa, which primarily correspond to endopeptidases such as neutral proteinase 2 (19.0 kDa) and leucine aminopeptidase A (36.2 kDa), were almost completely immobilized. This reduction in enzyme protein content in the supernatant indicates efficient immobilization of these enzymes onto the carrier surface.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.6 TGA\u003c/h2\u003e\u003cp\u003eThermogravimetric analysis (TGA) was conducted to evaluate the thermal stability of the modified carrier and immobilized enzyme, with the results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. All samples exhibited slight mass loss below 150\u0026deg;C, attributed to the evaporation of physically adsorbed water on the particle surface (Yust et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Notably, no significant mass loss occurred below 600\u0026deg;C for any of the samples, indicating excellent thermal stability of HS within this temperature range, consistent with previous literature (Huang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). CHS demonstrated comparable thermal stability to that of HS. However, after immobilization, the mass loss rate of CHS-F increased significantly. At 155.7\u0026deg;C, the organic components on the CHS surface began to thermally decompose, leading to a higher mass loss rate compared to the HS. At 209.3\u0026deg;C, the immobilized Flavourzyme molecules on the carrier surface started to decompose, further increasing the mass loss rate. Specifically, the mass loss rates of the HS and CHS were 7.15% and 7.55%, respectively, while that of CHS-F increased significantly to 9.68%. This indicates that the enzyme protein molecules were successfully immobilized on the CHS surface, and their thermal decomposition caused the additional mass loss. In contrast, DHS exhibited enhanced thermal stability below 222\u0026deg;C compared to HS. Nevertheless, beyond 222\u0026deg;C, the mass loss rate of the modified carrier accelerated considerably due to the thermal decomposition of organic components in the dopamine coating. At 158.2\u0026deg;C, the immobilized Flavourzyme molecules on the modified carrier surface began to thermally decompose, intensifying the mass loss. By 550\u0026deg;C, the organic components on the particle surface, including GA and enzyme molecules, had completely decomposed. The mass loss rates of the HS and DHS were 7.68% and 8.32%, respectively, while that of DHS-F rose significantly to 20.23%. This confirms the successful immobilization of enzyme protein molecules on the DHS surface and highlights that their thermal decomposition caused the significant mass loss. When comparing CHS and DHS, it is evident that both carriers exhibit excellent thermal stability below 600\u0026deg;C. However, their thermal degradation behaviors differ significantly after enzyme immobilization. CHS-F shows earlier decomposition of enzyme molecules, while DHS-F experiences more significant mass loss at higher temperatures due to the decomposition of both enzyme and dopamine coating. These findings indicate that the choice of modified carrier can significantly influence the thermal stability and decomposition profile of the immobilized enzyme system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Preparation of DCP and QDA Analysis\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA illustrates the appearance of debittered corn peptides prepared through enzymatic digestion using free and immobilized Flavourzyme. Notably, the enzymatic treatment did not induce significant precipitation in the corn peptides solution. However, remarkable color changes were observed in DF-CP. While the CK, FF-CP and CF-CP groups displayed a dark yellow color, the DF-CP group exhibited a dark brown hue attributed to dopamine's oxidative polymerization. The dark brown color is a typical characteristic of polydopamine coatings, arising from electron delocalization between aromatic rings during polydopamine formation (de Souza et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the degree of hydrolysis (DH) varied among the different DCP samples. Both the FF-CP and CF-CP groups achieved a DH of over 7%, while the DF-CP group had the lowest DH at 6.5%, indicating differing catalytic efficiencies of the two immobilized enzymes. All groups demonstrated high protein recovery rates above 97%, suggesting that the enzyme treatment effectively converted proteins into smaller peptides and amino acids with minimal loss. The QDA scores revealed a significant reduction in the bitterness of the hydrolyzed corn peptides, aligning with the degree of hydrolysis. The CF-CP group exhibited the lowest bitterness intensity, while the DF-CP group had the highest. These findings suggest that the CF-CP group achieved the best debittering effect, efficiently diminishing the bitterness of the corn peptides.\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\u003eProtein recovery, degree of hydrolysis (DH), QDA score, and parameters of the TI curve of debittered corn peptides.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFF-CP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDF-CP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCF-CP\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProtein recovery (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e98.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e97.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e98.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDH (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQDA score\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003csub\u003estart\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003csub\u003eplateau\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003csub\u003eext\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e180.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e120.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e120.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e120.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR\u003csub\u003eincrease\u003c/sub\u003e (\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR\u003csub\u003edecrease\u003c/sub\u003e (\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-57.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-56.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.39\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-64.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.88\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-58.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArea\u003csub\u003ebefore\u003c/sub\u003e (\u0026times;10\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArea\u003csub\u003eafter\u003c/sub\u003e (\u0026times;10\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArea (\u0026times;10\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003eb\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=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Time-intensity (TI) analysis\u003c/h2\u003e\u003cp\u003eTime-intensity (TI) curves are essential in sensory analysis as they map the dynamic sensory traits, such as bitterness, of products over time, thereby enhancing our understanding of consumer experience (Fran\u0026ccedil;ois et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In this study, TI experiments were conducted to measure the overall bitterness intensity of FF-CP, DF-CP, CF-CP, and a control (CK) over time, with the results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. All samples exhibited a rapid increase in bitterness intensity followed by a gradual decrease, a pattern consistent with previous studies on bitterness TI curves (Huang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Notably, FF-CP, DF-CP, and CF-CP demonstrated significantly lower bitterness intensity and shorter duration compared to CK, highlighting their effectiveness in reducing bitterness. The TI curves of FF-CP and CF-CP partially overlapped, indicating similar bitterness-time characteristics and slightly lower maximum bitterness than DF-CP. In contrast, CK samples consistently showed the highest bitterness intensity throughout the testing period.\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that there are significant differences between FF-CP, DF-CP, CF-CP, and CK in specific TI parameters. Specifically, FF-CP, DF-CP, and CF-CP exhibited lower values in these parameters compared to CK. This indicates that CK had the highest bitterness intensity, the longest time to reach maximum bitterness, and the longest persistence of bitterness, along with the highest overall content of bitter compounds. These results clearly demonstrate that enzymatic treatment is effective in reducing the bitterness of corn peptides and also explain the shorter persistence of bitterness in the treated samples. When comparing DF-CP and CF-CP, it was observed that DF-CP had a significantly higher rate of increase in bitterness intensity before reaching I\u003csub\u003emax\u003c/sub\u003e but a lower rate of decrease after I\u003csub\u003emax\u003c/sub\u003e. This difference likely relates to variations in hydrolysis efficiency. Consistent with the degree of hydrolysis, CHS-F exhibits higher hydrolysis efficiency than DHS-F, giving CHS-F a slight advantage over DHS-F in reducing CP bitterness. This suggests that the choice of enzyme and carrier can significantly influence the effectiveness of bitterness reduction in CP solutions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003e3.9 Molecular weight distribution\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC presents the molecular weight distribution of CP and DCP. The results indicate that the majority of the molecular weight fractions in all samples are distributed in the range of \u0026lt;\u0026thinsp;500 Da and 500\u0026ndash;1000 Da. Specifically, CP has a relatively higher content of the 500\u0026ndash;1000 Da and 1000\u0026ndash;5000 Da fractions compared to the other samples. In contrast, FF-CP and CF-CP show an increase in the \u0026lt;\u0026thinsp;500 Da fraction and a decrease in the 500\u0026ndash;1000 Da and 1000\u0026ndash;5000 Da fractions. DF-CP exhibits a similar trend to CF-CP but with a slightly lower proportion of the \u0026lt;\u0026thinsp;500 Da fraction and a slightly higher proportion of the 500\u0026ndash;1000 Da and 1000\u0026ndash;5000 Da fractions. It has been shown shown that bitterness is positively associated with medium molecular weight fractions (500\u0026ndash;1000 Da), and negatively correlated with fractions below 500 Da (Xiang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This suggests that the bitterness of hydrolysates is mainly influenced by medium molecular weight peptides. As the degree of hydrolysis increases, larger molecular weight peptides may be decomposed into smaller peptides and free amino acids, thereby reducing bitterness (Liu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The changes in molecular weight distribution observed in the samples align with this understanding. Samples with a higher proportion of medium molecular weight fractions (e.g., CP and DF-CP) tend to have stronger bitterness, while samples with a higher proportion of low molecular weight fractions (\u0026lt;\u0026thinsp;500 Da) such as FF-CP and CF-CP exhibit reduced bitterness. This suggests that the DCP generated by Flavourzyme and CHS-F are more hydrolyzed, have lower molecular weights and higher amino nitrogen content are more effective in reducing bitterness taste. The differences in molecular weight distribution among the samples can be attributed to the specificities of the proteases used for hydrolysis, which in turn affect the sensory properties of the hydrolysates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e3.10 FAAs Analysis\u003c/h2\u003e\u003cp\u003eTo investigate the taste contributions of FAAs, their changes were monitored during debittering (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In CP, Ala, Cys, Val, Ile, Leu, and Tyr were predominant, with average contents exceeding 10 mg/g. The taste contribution of individual amino acid was evaluated by dose-over-threshold (DoT) value (Xiang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, Tyr contributed strongly to bitterness, with its content being 26 times higher than its taste threshold. Leu and Ile also significantly contributed to bitterness, with DoT values above 5.0. For umami-tasting FAAs, their DoT values all exceeded 7.5 due to their low taste thresholds, making their umami taste detectable.\u003c/p\u003e\u003cp\u003eAfter enzymatic digestion with free Flavourzyme and immobilized enzymes, the FAAs contents in DCP significantly increased, especially for Asn, Gln, Val, Met, Leu, and Phe, which rose by 5\u0026ndash;20 times. CF-CP also saw increases in these amino acids, though slightly lower than FF-CP. In contrast, DF-CP had notably lower FAAs contents than FF-CP and CF-CP, consistent with hydrolysis degree results. The DoT values for the Tyr in DCP decreased, particularly in CF-CP, where it reduced by 16.33%. The increase in FAAs, including bitter-tasting ones like Leu, Ile, Val, Met, and Phe, suggests extensive protein hydrolysis. Despite higher FAA levels, bitterness reduced because the enzyme selectively cleaved peptide bonds, decreasing bitter peptide formation (Sun et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, the release of more non - bitter amino acids may have masked bitterness. Asn and Gln increased significantly due to Flavourzyme and CHS - F hydrolysis, while DHS-F showed no significant increase. This implies FF and CHS-F preferentially act on Gln-containing peptide bond sites but less efficiently on neighboring Glu, indicating enzymatic selectivity in amino acid release (Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). This selective process likely reduces bitterness while enhancing the product's umami through Gln accumulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec35\" class=\"Section2\"\u003e\u003ch2\u003e3.11 Reusability\u003c/h2\u003e\u003cp\u003eThe reusability of immobilized enzymes is crucial for enhancing productivity and reducing costs in continuous operations and automated production (Ishak et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the DHS-F retained 72.75% of its relative enzyme activity even after the fifth use, and maintained over 50% activity after ten uses. For CHS-F in hydrolyzing CP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), it preserved 63.31% of its activity at the fifth cycle and 42.78% after ten cycles. These results indicate that both CHS-F and DHS-F exhibit good stability and reusability, retaining over 40% of their activity after multiple uses, despite a gradual decline in activity with increasing use cycles.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, we successfully explored the surface modification of hydrated silica and the subsequent immobilization of Flavourzyme, and applied these immobilized enzymes to the debittering of corn peptides. The modification of hydrated silica with carboxyl and dopamine groups significantly improved its surface properties and enzyme immobilization efficiency. CHS demonstrated a higher immobilization rate of 62.93%, while DHS showed better enzyme activity recovery at 73.68%. The successful immobilization of the enzymes was confirmed by SEM, FTIR and SDS-PAGE. TGA analysis showed that the immobilized Flavourzyme on both CHS and DHS exhibited good thermal stability. The enzymatic treatment effectively reduced the bitterness of corn peptides, with CHS-F achieving the lowest bitterness intensity. Moreover, the immobilized Flavourzyme exhibited excellent reusability, retaining over 40% of its activity even after ten cycles of use. These findings provide valuable insights into optimizing the de-bittering process of corn peptides and highlight the potential of modified HS carriers and immobilized enzymes for industrial applications. Overall, this study offers a promising approach to improving the sensory quality and nutritional value of corn peptides through effective enzyme immobilization and carrier modification strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are contained within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Guangdong province general colleges and universities characteristic innovation category project (2021KTSCX003) and Shandong province science and technology-based small and medium-sized enterprises innovation ability enhancement project mission statement (2022TSGC2558).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Human Ethics Committee of South China University of Technology does not have a practice of approving sensory experiments on food. Therefore, this experiment was performed in accordance with the Declaration of Helsinki and informed consent\u0026nbsp;was obtained from all assessors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.Z.: conceptualization, methodology,validation, formal analysis, investigation, data curation, writing \u0026ndash; original draft.Y.Q.C.: methodology, formal analysis.L.C.: validation, sensory analysis.C.C.: conceptualization, supervision, funding acquisition, writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChen XJ, Lei ZY, Liu P, Lei MJ, Xu H, Yu LJ \u0026amp; Ao MZ (2023) An aminocaproic acid-grafted chitosan derivative with superior antibacterial and hemostatic properties for the prevention of secondary bleeding. 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Comprehensive Reviews in Food Science and Food Safety. 18(1), 329-345.\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Hydrated silica, surface modification, immobilized Flavourzyme, corn peptides, debittering","lastPublishedDoi":"10.21203/rs.3.rs-7756046/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7756046/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the surface modification of hydrated silica to enhance the immobilization efficiency of Flavourzyme, a protease complex widely used in the food industry. Hydrated silica was functionalized with carboxyl and dopamine groups to improve its enzyme loading capacity and activity recovery. The carboxyl-modified hydrated silica (CHS) exhibited a higher immobilization rate of 62.93%, while the dopamine-modified hydrated silica (DHS) demonstrated superior enzyme activity recovery at 73.68%. Both CHS- and DHS-immobilized Flavourzyme displayed excellent thermal stability and reusability, retaining over 40% of their initial activity after ten consecutive reaction cycles. Furthermore, the immobilized enzymes were applied to reduce the bitterness of corn peptides, with the CHS-immobilized Flavourzyme exhibiting the most effective debittering performance. These results highlight the potential of surface-modified hydrated silica as a promising carrier for enzyme immobilization and its application in improving the sensory properties of food protein hydrolysates.\u003c/p\u003e","manuscriptTitle":"Carboxyl- or Dopamine-Modified Hydrated Silica for Flavourzyme Immobilization: Towards Efficient Debittering of Corn Peptides","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 16:05:45","doi":"10.21203/rs.3.rs-7756046/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-11T17:49:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-03T12:24:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-03T01:42:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2025-10-01T03:32:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"06979d35-91fe-4cff-824b-e4bc7252d6a0","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-03T07:41:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-27 16:05:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7756046","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7756046","identity":"rs-7756046","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}