A method for enhancing the binding of pea protein isolate to 2,5-dimethylpyrazine using nanocellulose

Abstract

This invention belongs to the field of flavor enhancement technology and provides a method for enhancing the binding of pea protein isolate to 2,5-dimethylpyrazine using nanocellulose. The method involves mixing pea protein isolate, nanocellulose, and water; the resulting first mixture is then hydrated and gelled sequentially to obtain a gel system; this gel system is then mixed with 2,5-dimethylpyrazine to obtain a second mixture, which is then combined to enhance the aroma of 2,5-dimethylpyrazine. This invention introduces nanocellulose, achieving efficient adsorption and stable retention of the flavor compound 2,5-dimethylpyrazine in the pea protein isolate system.

Description

Technical Field

[0001] This invention relates to the field of flavor enhancement technology, and more particularly to a method for enhancing the binding of pea protein isolate to 2,5-dimethylpyrazine using nanocellulose. Background Technology

[0002] As the global food industry shifts towards sustainability and health, plant-based meat products made from plant proteins such as pea protein isolate (PPI) have developed rapidly. However, they still generally suffer from insufficient meat aroma and poor flavor stability, hindering consumer acceptance. In the flavor construction of plant-based meat products, pyrazine compounds are key flavor components that impart roasted and nutty aromas to meat and baked goods. Among them, 2,5-dimethylpyrazine is widely used to mimic the meat aroma characteristics of plant-based meat products due to its low olfactory threshold and clear flavor directionality. However, its significant hydrophobicity and high volatility make it prone to volatilization loss or degradation during heat processing and storage, making it difficult to maintain stable characteristic flavors. Simply relying on increasing the amount added not only increases costs but also easily leads to flavor imbalance. Therefore, achieving efficient adsorption and stable retention of pyrazine flavor substances in protein systems is a key scientific issue in the flavor regulation of plant-based meat products. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a method for enhancing the binding of pea protein isolate to 2,5-dimethylpyrazine using nanocellulose. The method provided by the present invention achieves efficient adsorption and stable retention of the flavor compound 2,5-dimethylpyrazine in the pea protein isolate system.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for enhancing the binding of pea protein isolate to 2,5-dimethylpyrazine using nanocellulose, comprising the following steps: Pea protein isolate, nanocellulose and water were mixed, and the resulting first mixed solution was hydrated and gelled sequentially to obtain a gel system. The gel system is mixed with 2,5-dimethylpyrazine to obtain a second mixed solution, which is then compounded to enhance the aroma of 2,5-dimethylpyrazine.

[0005] Preferably, the nanocellulose includes one or more of microcrystalline cellulose, nanocrystalline cellulose, bacterial cellulose, and cellulose nanofibers.

[0006] Preferably, the microcrystalline cellulose has a length of 22.82±2.95μm, a diameter of 121.15±52.16nm, and an aspect ratio of 272.49±163.98.

[0007] Preferably, the nanocrystalline cellulose has a length of 273.12±77.98 nm, a diameter of 44.23±13.74 nm, and an aspect ratio of 6.59±2.52.

[0008] Preferably, the bacterial cellulose has a length of 5.04±1.38μm, a diameter of 71.32±22.18nm, and an aspect ratio of 68.72±26.32.

[0009] Preferably, the cellulose nanofibers have a length of 137.93±41.35 nm, a diameter of 26.99±6.69 nm, and an aspect ratio of 5.34±1.79.

[0010] Preferably, the mass ratio of pea protein isolate to nanocellulose is 10~15:0.5~1.0.

[0011] Preferably, the hydration time is 10-14 hours; the gelation temperature is 90-100°C and the time is 20-40 minutes.

[0012] Preferably, the ratio of pea protein isolate to 2,5-dimethylpyrazine used in preparing the gel system is 0.5~2g:0.05~0.25mmol.

[0013] Preferably, the composite temperature is 37°C and the time is 14~18h.

[0014] This invention provides a method for enhancing the binding of pea protein isolate to 2,5-dimethylpyrazine using nanocellulose.

[0015] This invention introduces nanocellulose; the abundant hydroxyl and carboxyl groups on the surface of nanocellulose can crosslink with pea protein isolate through hydrogen bonding and hydrophobic interactions, assisting in the construction of a dense three-dimensional network structure and exposing more adsorption sites. Simultaneously, the re-exposed polar groups can directly capture the aroma molecule 2,5-dimethylpyrazine through hydrogen bonding and other interactions, improving the protein system's efficient absorption of aroma molecules. Furthermore, the non-covalent interaction between pea protein isolate and nanocellulose can disrupt the original weak equilibrium within the protein, further inducing a conformational shift from a relatively loose state to a tightly ordered one, thus achieving stable retention of aroma molecules within the protein system. Attached Figure Description

[0016] Figure 1 FTIR spectra of PPI, PPI-MFC, PPI-CNC, PPI-BC, and PPI-CNF; Figure 2 The proportions of protein secondary structures for PPI, PPI-MFC, PPI-CNC, PPI-BC, and PPI-CNF; Figure 3 Intermolecular β-sheets for PPI, PPI-MFC, PPI-CNC, PPI-BC, and PPI-CNF; Figure 4 The surface hydrophobicity of PPI, PPI-MFC, PPI-CNC, PPI-BC and PPI-CNF; Figure 5 The active thiol and total thiol content of PPI, PPI-MFC, PPI-CNC, PPI-BC and PPI-CNF; Figure 6 The ultraviolet spectra of PPI, PPI-MFC, PPI-CNC, PPI-BC, and PPI-CNF are shown. Figure 7 The UV second derivative spectra of PPI, PPI-MFC, PPI-CNC, PPI-BC, and PPI-CNF are shown. Figure 8 Synchronous fluorescence spectra of PPI, PPI-MFC, PPI-CNC, PPI-BC and PPI-CNF (Δλ=15nm and Δλ=60nm); Figure 9 The mesoscopic structural characterization results of PPI, PPI-MFC, PPI-CNC, PPI-BC and PPI-CNF are shown. In the figure, A is Zeta potential, B is solubility, C is polydispersity index, D is average particle size and turbidity, and E is SDS-PAGE electrophoresis image. Different letters ad indicate that the difference between the two groups is statistically significant (p<0.05). Figure 10 The macroscopic structural characterization results of PPI, PPI-MFC, PPI-CNC, PPI-BC and PPI-CNF are shown. In the figure, A is the apparent viscosity, B is the storage modulus, C is the loss modulus, D is the XRD pattern, E is the TGA curve, F is the DTG curve, G is the gel SEM image, H is the gel porosity image and I is the macroscopic morphology of the gel. Figure 11The diagrams characterize the regulatory effect of nanocellulose on the aroma binding ability of pea protein isolate. A represents the binding ability of the PPI / nanocellulose complex to 2,5-dimethylpyrazine; B compares the binding abilities of pure PPI and pure nanocellulose to 2,5-dimethylpyrazine; C shows the thermal stability curve of the PPI / nanocellulose complex binding to 2,5-dimethylpyrazine; D is a thermal visualization of the PPI / nanocellulose complex binding to 2,5-dimethylpyrazine; E is a Pearson correlation analysis diagram; ABA represents aroma binding rate; PtS represents solubility; Zeta represents Zeta potential; PDI represents polydispersity index; PS represents particle size; FL represents surface hydrophobicity; TSH represents total thiol groups; ESH represents active thiol groups; and Tur represents turbidity. F is the principal component analysis diagram, and G is the UV-Vis absorption spectrum. Different letters (ad) indicate statistically significant differences between the two groups (p < 0.05). Detailed Implementation

[0017] This invention discloses a method for enhancing the binding of pea protein isolate to 2,5-dimethylpyrazine using nanocellulose, comprising the following steps: Pea protein isolate, nanocellulose and water were mixed, and the resulting first mixed solution was hydrated and gelled sequentially to obtain a gel system. The gel system is mixed with 2,5-dimethylpyrazine to obtain a second mixed solution, which is then compounded to enhance the aroma of 2,5-dimethylpyrazine.

[0018] Unless otherwise specified, the raw materials used in this invention are preferably commercially available products.

[0019] This invention involves mixing pea protein isolate, nanocellulose, and water, and then sequentially hydrating and gelling the resulting first mixed solution to obtain a gel system. In this invention, the protein isolate preferably has a protein content of ≥80%, specifically 80%. In this invention, the nanocellulose preferably includes one or more of microcrystalline cellulose (MFC), nanocrystalline cellulose (CNC), bacterial cellulose (BC), and cellulose nanofibers (CNF). In this invention, the microcrystalline cellulose preferably has a length of 22.82±2.95 μm, specifically 22.82 μm; a diameter preferably of 121.15±52.16 nm, specifically 121.15 nm; and an aspect ratio preferably of 272.49±163.98, specifically 272.49.

[0020] In this invention, the length of the nanocrystalline cellulose is preferably 273.12±77.98 nm, more preferably 273.12 nm; the diameter is preferably 44.23±13.74 nm, more preferably 44.23 nm; and the aspect ratio is preferably 6.59±2.52, more preferably 6.59.

[0021] In this invention, the length of the bacterial cellulose is preferably 5.04±1.38μm, more preferably 5.04μm; the diameter is preferably 71.32±22.18nm, more preferably 71.32nm; and the aspect ratio is preferably 68.72±26.32, more preferably 68.72.

[0022] In this invention, the length of the cellulose nanofiber is preferably 137.93±41.35nm, more preferably 137.93nm; the diameter is preferably 26.99±6.69nm, more preferably 26.99nm; and the aspect ratio is preferably 5.34±1.79, more preferably 5.34.

[0023] In this invention, the preferred mass ratio of pea protein isolate to nanocellulose is 10-15:0.5-1.0, and more preferably 14:0.8.

[0024] In this invention, the mixing of pea protein isolate, nanocellulose, and water preferably includes the following steps: mixing pea protein isolate and water to obtain an aqueous solution of pea protein isolate; mixing the aqueous solution of pea protein isolate with nanocellulose; the preferred ratio of pea protein isolate to water is 10-15g:100mL.

[0025] In this invention, the mass concentration of pea protein isolate in the first mixed solution is preferably 10-15 w / v, specifically preferably 10 w / v%, 11 w / v%, 12 w / v%, 13 w / v%, 14 w / v%, or 15 w / v; the mass concentration of nanocellulose is preferably 0.5-1.0 w / v, specifically preferably 0.5 w / v%, 0.6 w / v%, 0.7 w / v%, 0.8 w / v%, 0.9 w / v%, or 1.0 w / v.

[0026] In this invention, the hydration temperature is preferably room temperature, i.e., no additional heating or cooling is required, the hydration time is preferably 10-14 hours, more preferably 12 hours, and the hydration is preferably carried out under stirring conditions, preferably magnetic stirring.

[0027] In this invention, the gelation temperature is preferably 90-100°C, more preferably 95°C, and the gelation time is preferably 20-40 min, more preferably 30 min. The gelation is preferably carried out under water bath conditions. After the gelation is completed, the invention preferably further includes cooling to room temperature in an ice bath.

[0028] After obtaining the gel system, the present invention mixes the gel system with 2,5-dimethylpyrazine, and then composites the resulting second mixed solution to achieve aroma enhancement of 2,5-dimethylpyrazine.

[0029] In this invention, the ratio of pea protein isolate to 2,5-dimethylpyrazine used to prepare the gel system is 0.5~2g:0.05~0.25mmol.

[0030] In this invention, the gel system is preferably diluted before mixing with 2,5-dimethylpyrazine; the diluent used for dilution preferably includes a K2HPO4-KH2PO4 buffer solution, the concentration of which is preferably 0.01 mol / L and the pH is preferably 7.2. In this invention, the 2,5-dimethylpyrazine is preferably used in the form of a 2,5-dimethylpyrazine solution.

[0031] In this invention, the mass concentration of pea protein isolate in the second mixed solution is 0.05~0.20 w / v, specifically preferably 0.05 w / v%, 0.06 w / v%, 0.07 w / v%, 0.08 w / v%, 0.09 w / v%, 0.10 w / v%, 0.11 w / v%, 0.12 w / v%, 0.13 w / v%, 0.14 w / v%, 0.15 w / v%, 0.16 w / v%, 0.17 w / v%, 0.18 w / v%, 0.19 w / v%, or 0.20 w / v; the concentration of 2,5-dimethylpyrazine is 0.05~0.25 mmol / L, specifically preferably 0.0 5mmol / L, 0.06mmol / L, 0.07mmol / L, 0.08mmol / L, 0.09mmol / L, 0.10mmol / L, 0.11mmol / L, 0.12mmol / L, 0.13mmol / L, 0.14mmol / L, 0.15mmo l / L, 0.16mmol / L, 0.17mmol / L, 0.18mmol / L, 0.19mmol / L, 0.20mmol / L, 0.21mmol / L, 0.22mmol / L, 0.23mmol / L, 0.24mmol / L or 0.25mmol / L.

[0032] In this invention, the composite temperature is preferably 37°C, the composite time is preferably 14~18h, and more preferably 16h; the composite is preferably carried out under light-protected conditions.

[0033] The following detailed description, in conjunction with embodiments, illustrates the method provided by the present invention for enhancing the binding of pea protein isolate to 2,5-dimethylpyrazine using nanocellulose, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1. Materials and Reagents Pea protein isolate (80% protein content) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., China. Microcrystalline cellulose (MFC) and three different sizes of nanocellulose (CNC, BC, CNF) were purchased from Guilin Qihong Technology Co., Ltd., China. Flavor compound standard, namely 2,5-dimethylpyrazine (98% purity), was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. All other chemicals used in this experiment were of at least analytical grade or higher purity.

[0035] 2. Preparation of composite gel PPI solutions (final concentration 14%, w / v) were mixed with MFC, CNC, BC, and CNF (final concentration 0.8%, w / v), respectively, and magnetically stirred at room temperature for 12 h to ensure complete hydration of PPI, resulting in composite solution samples (PPI, PPI-MFC, PPI-CNC, PPI-BC, PPI-CNF). The solution samples were transferred to 10 mL beakers, sealed, and heated in a 95 °C water bath for 30 min, followed by cooling to room temperature in an ice bath. The prepared gel samples were then stored at 4 °C for 12 h for later use.

[0036] 3. Characterization methods 3.1 Characterization of nanocellulose The zeta potentials of the four fiber samples were determined using a Zetasizer analyzer. XRD diffraction patterns were obtained by placing the four lyophilized fiber powders onto a glass slide in an X-ray diffractometer. The microstructures of the four fiber samples (MFC, CNC, BC, and CNF) were observed under an atomic force microscope, and their dimensions were quantitatively analyzed using Nanoscope analysis 1.5 software.

[0037] 3.2 Multi-scale structural testing 3.2.1 Microstructure (1) Protein secondary structure The freeze-dried protein gel sample was mixed with spectroscopically pure KBr at a mass ratio of 1:100, ground, and pressed into thin sheets. Absorbance was collected using an infrared spectrometer (FTIR Vertex 70, Bruker, Germany) in transmission mode, with a spectral range of 4000-400 cm⁻¹. -1 The resolution is 4cm. -1 32 scans were performed. Peakfit 4.12 software was used to determine the amide I band (1700-1600 cm⁻¹). -1 The FTIR spectrum of the protein was used to calculate the secondary structure of the protein by fitting the data.

[0038] (2) Protein functional groups Take 15 mg of lyophilized protein gel sample and add it to 5 mL of test solution (Tris-Gly buffer for determining exposed thiol content, Tris-Gly-8M Urea buffer for determining total thiol content). Add 50 μL of DTNB solution, incubate at room temperature for 1 h, then centrifuge (25℃, 13600 g, 10 min), collect the supernatant, and record the absorbance value at 412 nm. The thiol content is calculated according to Formula 1: SH (μmol / g) = 75.53 × A 412 ×D / C Formula 1; In Formula 1: A 412 denoted as sample absorbance, D as dilution factor, and C as protein content.

[0039] (3) Hydrophobicity 8-Aniline-1-naphthalenesulfonic acid (ANS) was used as a fluorescent probe to determine the surface hydrophobicity of samples. Protein buffers of different concentrations (0.005, 0.010, 0.015, 0.020, 0.025, 0.030 mg / mL) were prepared using K₂HPO₄-KH₂PO₄ buffer (0.01 mol / L, pH=7.6). ANS solution was added to each buffer, and the mixtures were shaken and incubated in the dark at 25°C for 10 min. Fluorescence intensity was recorded using a fluorescence spectrophotometer (FL 6500, PerkinElmer, USA). The spectral range was 400-700 nm, the excitation wavelength was 380 nm, and the slit width was 10 nm. A linear regression was performed with protein sample concentration as the independent variable and fluorescence intensity as the dependent variable; the slope of the regression line represented the surface hydrophobicity.

[0040] (4) Amino acid microenvironment The amino acid microenvironment of protein solution samples (0.05 mg / mL) was detected using a UV-2600i spectrophotometer (Shimadzu, Japan) in the wavelength range of 220-400 nm. The second derivative of the acquired data was calculated using Origin 2024 image processing software. 2 A / d 2 B).

[0041] The microenvironmental changes of tyrosine (Δλ=15nm) and tryptophan (Δλ=60nm) in a 0.1 mg / mL protein solution were detected using a fluorescence spectrophotometer (FL 6500, PerkinElmer, USA). The excitation wavelength was set to 200-400 nm, with a wavelength interval Δλ of 15 nm or 60 nm, and a slit width of 5 nm.

[0042] 3.2.2 Mesoscopic Structure (1) Particle size and polydispersity index After centrifugation (25℃, 3500 rpm, 5 min), the supernatant of the protein solution samples was collected and diluted to 0.1 mg / mL. The particle size and polydispersity index were measured using a nanolaser particle size analyzer (ZSU 3100, Malvern Panalytical Science, UK).

[0043] (2) Zeta potential Measurements were performed using a Malvern Zeta potentiometer (ZSU 3100, Malvern Panalytical Science, UK).

[0044] (3) Turbidity The protein solution sample was diluted 500 times with deionized water and shaken thoroughly for 2 min. The absorbance of the diluted solution was measured at 600 nm using a microplate reader (VICTOR NivoHH3500500, PerkinElmer, USA).

[0045] (4) Solubility Protein solution samples were diluted 400-fold with deionized water and centrifuged (4℃, 10000g, 30min), and the supernatant was collected. The protein concentration in the supernatant was determined using a Bradford protein assay kit (Solarbio, Beijing, China).

[0046] (5) SDS-PAGE analysis The five protein mixtures were then respectively mixed with Omni-Easy TM Equal volumes of fast-dissolving protein loading buffer were mixed and incubated in boiling water at 100°C for 3 minutes. SDS-PAGE electrophoresis was then performed using a constant current electrophoresis apparatus (DYY-6C, Liuyi Co., Ltd., Beijing, China) at 50 mA and 120 V. After electrophoresis, the bands were stained with Coomassie Brilliant Blue fast staining solution for 12 hours. Subsequently, ultrapure water was used for destaining until the bands were clearly visible. Electrophoresis images were acquired using a gel imaging system (Bio-Rad Universal Hood II, USA), and band optical density was evaluated using Image Lab 3.0 image processing software.

[0047] 3.2.3 Macrostructure (1) Rheological properties A hybrid rheometer (Discovery HR-1, TA Instruments, USA) equipped with a 40mm parallel steel plate clamp was used to record the rheological curves of the solution samples at a 1000μm slit. The frequency scan parameters were set as follows: strain 1%, angular frequency 0.1-100 rad / s, and temperature 25℃. Flow scans were performed after the frequency scans, with the shear rate increasing from 0.1 1 / s to 100 1 / s.

[0048] (2) Crystal structure The freeze-dried gel powder was placed on a glass slide of an X-ray diffractometer (SmartLab SE, Rigaku, Japan), and XRD patterns were obtained under Cu-Kα radiation (λ=0.1548nm). The scanning range was 5-90° (2θ), the scanning speed was 5° / min, the voltage was 40kV, and the current was 40mA.

[0049] (3) Conformational thermal stability Thermogravimetric analysis was performed using a thermal analyzer (TGA 5500, TA Instruments, USA) to heat the freeze-dried protein gel samples from 30°C to 600°C at a rate of 10°C / min.

[0050] (4) Gel network structure After freeze-drying, the gel sample was placed on a tray and vacuum-plated with gold for 60 seconds. The microstructure of the gel sample was observed using a scanning electron microscope (SEM, S-4800, Hitachi, Japan) with 150x magnification.

[0051] 3.3 Evaluation of the binding affinity between the nanocellulose-pea protein isolate complex and 2,5-dimethylpyrazine 3.3.1 Preparation of the embedding complex Five different protein solutions were dispersed in K₂HPO₄-KH₂PO₄ buffer (0.01 mol / L, pH 7.2), and 2,5-dimethylpyrazine stock solutions were prepared using methanol (chromatographic grade) as the solvent. Each protein solution was then thoroughly mixed with an appropriate amount of 2,5-dimethylpyrazine stock solution to achieve a final protein concentration of 0.1 w / v and a final aroma concentration of 0.1 mmol / L. The mixtures were incubated at 37°C in the dark for 16 hours to ensure complete binding of the protein and aroma compounds, thus preparing the encapsulated complex.

[0052] 3.3.2 Aroma Binding Rate Aroma binding rates were determined using headspace solid-phase microextraction-gas chromatography (HS-SPME-GC). The equilibrated embedded complex was transferred to a headspace vial and placed in a 37°C water bath. Simultaneously, aged Carboxen / poly-dimethylsiloxane fibers (50 / 30 µm, 2 cm, DVB / CAR / PDMS) were inserted into the headspace, exposing the fiber coating. Dynamic extraction was performed for 10 min to capture volatile aroma compounds. Qualitative and quantitative analyses of the aroma compounds were conducted using a gas chromatograph (GC-2010Plus, SHIMADZU, Japan) equipped with a Wax capillary column (30 m × 0.25 mm × 0.25 μm). Aroma compound binding rates were calculated according to Formula 2. Aroma binding rate (%) = 100% Formula 2; In Formula 2: A0 is the chromatographic peak area of ​​2,5-dimethylpyrazine in the protein-containing sample; A1 is the chromatographic peak area of ​​2,5-dimethylpyrazine in the protein-free control solution.

[0053] 3.3.3 Aroma Combination Heat Stability To investigate the regulatory effect of processing temperature on aroma binding stability, a temperature gradient experimental design was employed: Pre-equilibrated (37℃, 16h) embedded complexes were transferred to headspace vials and heat-treated for 10 min at 50℃ (water bath), 80℃ (water bath), and 120℃ (oil bath), with 37℃ water bath heat treatment serving as a control. After heat treatment, the headspace vials were immediately immersed in an ice-water bath for rapid cooling to terminate the temperature effect, and then transferred to a constant temperature of 37℃ for a second equilibration of 16h. After equilibration, the peak area of ​​free 2,5-dimethylpyrazine in the headspace was determined using HS-SPME-GC, and the aroma binding rate was calculated according to Formula 2 to evaluate the binding stability of proteins and aroma compounds at different heat processing temperatures.

[0054] 4. Results and Discussion 4.1 Physicochemical properties of nanocellulose In terms of size characteristics, MFC is a micron-sized fiber with a length, diameter, and aspect ratio of 22.82 μm, 121.15 nm, and 272.49, respectively. CNC is a rigid short rod with dimensions of 273.12 nm (length), 44.23 nm (diameter), and 6.59 (aspect ratio). BC is a flexible long fiber with dimensions of 5.04 μm (length), 71.32 nm (diameter), and 68.72 (aspect ratio). CNF is a flexible short fiber with the smallest size among the three types of nanocellulose, specifically 137.93 nm (length), 26.99 nm (diameter), and 5.34 (aspect ratio). Furthermore, the different cellulose samples also exhibited significant differences in surface electrical properties and crystal structure. The Zeta potentials of MFC, CNC, BC, and CNF were -18.40 mV, -32.46 mV, -39.68 mV, and -26.04 mV, respectively, with crystallinity of 48.13%, 66.29%, 62.66%, and 16.77%. Nanofibers with smaller aspect ratios can more effectively improve the protein gel network. This is likely due to their higher surface hydroxyl density and larger specific surface area, which provides more active sites, thereby enhancing interactions with protein molecules, promoting intermolecular cross-linking, and ultimately improving the uniformity and stability of the three-dimensional gel network.

[0055] 4.2 The regulatory effect of nanocellulose on the multi-scale structure of pea protein isolate 4.2.1 Microstructure (1) Protein secondary structure Amide I band (1600-1700cm) -1 This is mainly attributed to the C=O stretching vibration in protein molecules. Figure 1 As shown), its different sub-bands are closely related to specific secondary structures: 1600-1640cm -1 (β-fold), 1627-1638cm -1 (Intermolecular β-sheet), 1640-1650 cm -1 (Irregular curl), 1650-1660cm -1 (α-helix) and 1660-1700cm -1 (β-turn).

[0056] Peak fitting of the infrared spectrum yields... Figure 2 The protein secondary structure ratio shown ( Figure 2 As shown in the figure), the results indicate that the addition of nanocellulose has little effect on the content of random curls. p>0.05), but to varying degrees, it increased the β-sheet ratio (including intermolecular β-sheet) and decreased the α-helix and β-turn content. Among them, the PPI-CNF group showed the most significant changes: α-helix decreased by 4.24% compared to PPI, β-sheet increased by 19.42%, and intermolecular β-sheet increased by 14.91% ( Figure 3 (As shown).

[0057] α-helices are detrimental to protein gelation, while β-sheets facilitate the formation of three-dimensional gel networks. The high-density hydroxyl groups on the surface of low aspect ratio CNFs readily form high-energy intermolecular hydrogen bonds with C=O / NH in proteins, promoting peptide chain unwinding and remodeling into a more stable β-sheet conformation, thus forming a highly rigid and stable composite gel. Based on this, it can be inferred that low aspect ratio cellulose nanofibers may primarily improve the gelation properties of PPIs by promoting the formation of intermolecular β-sheets.

[0058] (2) Protein functional groups The addition of nanocellulose reduced the surface hydrophobicity of PPI by 12.7% (PPI-MFC), 17.2% (PPI-CNC), 20.7% (PPI-BC), and 56.4% (PPI-CNF), respectively. Figure 4 As shown; p <0.05). This is mainly due to the introduction of hydrophilic hydroxyl groups in nanocellulose, which enhances the polarity of the system and achieves the masking of hydrophobic regions and the exposure of hydrophilic sites through molecular entanglement and conformational rearrangement. In addition, the steric hindrance effect of nanocellulose also has a shielding effect on the hydrophobic microregions of proteins. It is noteworthy that the decrease in surface hydrophobicity is negatively correlated with the aspect ratio of nanocellulose. Among them, CNF has a higher surface hydroxyl density, stronger molecular entanglement ability, and a denser network structure, so its hydrophobicity decrease is the most significant. These structural characteristics enable CNF to effectively regulate the conformational changes of proteins: inducing the embedding of hydrophobic groups while promoting the exposure of hydrophilic fragments, thereby significantly reducing the surface hydrophobicity of PPI.

[0059] After introducing nanocellulose with different aspect ratios, the content of active thiol groups and total thiol groups in PPI decreased simultaneously; among them, the PPI-CNF group showed the largest decrease, decreasing by 37.7% and 40.4%, respectively (e.g., ...). Figure 5 As shown; p<0.05). It is generally believed that a decrease in total thiol groups reflects an increase in disulfide bonds in the gel system. The correlation observed in this invention may be due to the addition of nanocellulose promoting the cross-linking of protein molecules, allowing more active thiol groups to be converted into disulfide bonds through oxidation. CNFs, with their smaller aspect ratio, exhibit particularly significant regulatory effects on thiol group conversion due to their high specific surface area and strong adsorption capacity. Thiol groups, as key groups maintaining the tertiary and quaternary structures of proteins, play a crucial role in the formation and stability of gel networks by providing molecular cross-linking sites. Based on the above results, it is speculated that nanocellulose may induce PPI molecules to unfold, exposing internal thiol groups, and thereby enhancing the structural stability of the composite gel network through disulfide bond cross-linking.

[0060] (3) Amino acid microenvironment The addition of nanocellulose significantly reduced the UV absorption intensity of PPI in the 220-320nm range (e.g., Figure 6 As shown in the figure, this indicates that it can induce conformational rearrangement of PPI molecules and alter the microenvironment of aromatic amino acids. The spectral peak at 287 nm in the UV second derivative spectrum is attributed to the combined effect of tyrosine (Tyr) and tryptophan (Trp), while the peak at 296 nm originates solely from tryptophan (as shown in the figure). Figure 7 (As shown). After the addition of nanocellulose, the peak at 287 nm of the PPI-based composite solution showed a slight red shift, while the peak at 297 nm showed a blue shift, and the intensity of both peaks decreased. These spectral changes suggest that nanocellulose may induce tryptophan residues to be exposed to a hydrophilic environment, while tyrosine residues are embedded in hydrophobic regions, implying protein unfolding and reorganization of hydrophobic regions.

[0061] The dielectric properties of the tyrosine residue microenvironment can be reflected by the ratio of the positive and negative peak differences of the second derivative in UV light (r = a / b): the r value of tyrosine residues decreases with decreasing solvent polarity, while the r value of tryptophan residues is almost unaffected by solvent polarity. In this invention, the r value decreased from 1.30 (PPI) to 1.27 (PPI-MFC), 1.25 (PPI-CNC), 1.11 (PPI-BC), and 1.08 (PPI-CNF). This decreasing trend indicates that with the addition of nanocellulose, especially CNF with a small aspect ratio, the hydrophobicity of the microenvironment containing tyrosine residues gradually increases. The mechanism is speculated to be that the fibrous network of nanocellulose restricts the penetration of water molecules into the tyrosine binding domain, while simultaneously driving protein conformational rearrangement through surface charge effects, further embedding tyrosine residues in the hydrophobic core, thereby enhancing protein stability.

[0062] Synchronous fluorescence spectroscopy showed that the fluorescence intensity of tyrosine and tryptophan in the PPI-based composite solution decreased in a gradient manner as the aspect ratio of nanocellulose decreased (e.g., Figure 8As shown in the figure, nanocellulose may bind to PPI and quench its intrinsic fluorescence. The quenching effect of Δλ=60nm (tryptophan) is greater than that of Δλ=15nm (tyrosine), indicating that more tryptophan residues are embedded, further confirming the results of the UV second derivative spectroscopy. This phenomenon may originate from the tertiary conformational change of PPI induced by nanocellulose, which embeds amino acid residues in hydrophobic microregions. However, the maximum emission wavelength of the composite solution did not shift significantly, indicating that the local dielectric environment around the amino acid residues remained relatively stable. Notably, CNF exhibited the strongest fluorescence quenching effect on the two aromatic amino acids, possibly related to its smaller aspect ratio, which facilitates the formation of a denser three-dimensional entangled network. This structure can significantly reduce fluorescence intensity by enhancing steric hindrance, promoting PPI molecule aggregation and tryptophan interactions.

[0063] In summary, UV second derivative spectroscopy and synchronous fluorescence jointly confirmed that nanocellulose can induce conformational reorganization of PPI molecules, causing tyrosine and tryptophan residues to be embedded in hydrophobic microdomains, reducing the polarity of their microenvironment, and this effect is enhanced as the aspect ratio of nanocellulose decreases.

[0064] 4.2.2 Mesoscopic Structure (1) Zeta potential The zeta potential reflects the effective surface charge of dispersed particles at the sliding surface, and its absolute value is a key parameter determining the electrostatic stability of protein solutions. It is generally believed that when the absolute value of the zeta potential is higher than 25 mV, the system is in a stable state. At this point, the particles, carrying sufficient net charge, generate strong electrostatic repulsion, forming significant steric hindrance and making aggregation difficult. In this invention, the absolute value of the zeta potential of the PPI-based composite solution is not only higher than that of the pure PPI solution, but also exceeds 30 mV (e.g., ...). Figure 9 (A) indicates that nanocellulose transforms the PPI solution from a critical stable state to a highly stable state. Notably, the aspect ratio of nanocellulose did not show a significant correlation with the particle surface charge characteristics (…). p >0.05).

[0065] (2) Solubility Changes in solubility are essentially a macroscopic manifestation of the thermodynamic equilibrium reached between protein-protein and protein-solvent interactions, a equilibrium primarily regulated by the average hydrophobicity and net surface charge of the protein. This invention reveals that the addition of nanocellulose significantly improves the solubility of PPI, and the solubilizing effect is negatively correlated with its aspect ratio (e.g., ...). Figure 9 (B) In this context, nanocellulose, rich in hydrophilic hydroxyl groups, can form a hydrogen bond network with polar amino acid residues in PPI molecules, while simultaneously acting on hydrophobic regions of proteins, thereby effectively reducing the average hydrophobicity of PPI (e.g., ...). Figure 4As shown), it reduces the formation of insoluble aggregates. On the other hand, nanocellulose increases the net surface charge of the system (as shown). Figure 9 (A) By enhancing the electrostatic repulsion and steric hindrance between protein molecules, protein aggregation is further inhibited. The synergistic effect of these two mechanisms promotes the solubilization process of proteins, which is macroscopically manifested as a significant increase in solubility.

[0066] (3) Particle size, turbidity and dispersion stability The particle size of PPI-based composite solutions showed a significant decreasing trend compared to PPI, with reductions of 4.76% (PPI-MFC), 6.26% (PPI-CNC), 11.12% (PPI-BC), and 16.44% (PPI-CNF), respectively. Figure 9 D in the middle; p <0.05). Similarly, the turbidity change of the PPI-based composite solution was significantly positively correlated with the aspect ratio of the nanocellulose, with the PPI-CNF group showing the largest turbidity decrease, reaching 28.42% (e.g., Figure 9 D in the middle; p <0.05). This phenomenon stems from the formation of small-sized protein aggregates: according to light scattering theory, smaller particle sizes in the dispersed phase reduce light scattering intensity, thus directly leading to a decrease in turbidity. Meanwhile, the polydispersity index of the PPI-CNF group is lower (e.g., ...). Figure 9 The presence of C in the figure further demonstrates its high dispersion stability and the formation of relatively uniform aggregates. Based on the above results, it is speculated that nanocellulose with a small aspect ratio may inhibit the excessive aggregation of PPI molecules through its surface properties and steric hindrance, thus promoting the formation of smaller and more uniformly distributed protein aggregates.

[0067] (4) Distribution of protein subunits SDS-PAGE results showed that all five protein gels exhibited typical pea globulin subunit bands (e.g., Figure 9 The E in PPI is mainly composed of 7S α′ (-72kDa), 7S α (-68 kDa), and 7S β (-53kDa) subunits, as well as 11S acidic (29-33kDa) and basic (18-22kDa) subunits. Compared with pure PPI, the electrophoretic band positions of each PPI-based composite gel did not shift significantly, indicating that the introduction of nanocellulose did not change the molecular weight of the main protein subunits in PPI and did not trigger a new polymerization reaction. However, the band intensity of the PPI-based composite gel was significantly weakened in the low molecular weight region (<25kDa), with optical density decreasing by 19.68% (PPI-MFC), 21.03% (PPI-CNC), 31.21% (PPI-BC), and 43.81% (PPI-CNF) compared to PPI. p<0.05). This may be because cellulose nanoparticles bind to proteins through electrostatic repulsion and steric hindrance, increasing the solvation degree of proteins and thus inhibiting the molecular aggregation of subunits.

[0068] The above results reveal the regulatory role of the aspect ratio of nanocellulose on the aggregation behavior of PPI: PPI molecules carry a negative charge on their surface, while CNF with a smaller aspect ratio can enhance the electrostatic repulsion between protein molecules and construct a denser fiber network in the system, producing a more significant steric hindrance effect, thereby inhibiting the disordered aggregation of PPI and improving the dispersibility, stability, and homogeneity of the composite solution. This is also consistent with the results observed in this invention, which showed that the PPI-CNF composite system has the smallest particle size and the most uniform distribution. Furthermore, according to Stokes' law, reducing the particle size can effectively reduce the sedimentation rate; at the same time, the thickening effect of CNF may increase the viscosity of the continuous phase. The synergistic effect of both significantly delays the phase separation process, further enhancing the macroscopic stability of the system. Therefore, nanocellulose with a smaller aspect ratio may play a role in stabilizing the system by enhancing steric hindrance and regulating rheological properties, thus synergistically inhibiting PPI aggregation.

[0069] 4.2.3 Macrostructure (1) Rheological behavior The steady-state shear test results of all samples showed obvious shear-thinning behavior, consistent with typical pseudoplastic fluid characteristics (e.g., Figure 10 (A) The apparent viscosity of the PPI-based composite solution was consistently higher than that of the control group, demonstrating that nanocellulose has a thickening effect. Specifically, PPI-CNF exhibited higher viscosity in the low shear rate region, indicating the formation of a denser network framework. This is attributed to the fact that CNF's low aspect ratio and high specific surface area facilitate its uniform dispersion in the protein matrix and enhance the system's fluid resistance through multi-point physical entanglement and interaction with proteins. In contrast, MFC, due to its high aspect ratio and entangled fiber morphology, is difficult to orient and easily aggregates, resulting in a loose gel network framework and reduced shear viscosity.

[0070] Within the angular frequency range of 0.1–100 rad / s, the storage modulus (G′) of all samples was higher than the loss modulus (G″), and both showed weak frequency dependence, exhibiting typical solid-like elastic characteristics of gel materials (e.g., Figure 10(BC in the text). Notably, the G′ and G″ values ​​of PPI-CNF remained the highest across the entire frequency range, indicating the formation of a more robust gel network. This difference is primarily attributed to the regulatory effect of cellulose morphology on the network structure: the low aspect ratio CNF, with its high specific surface area, provides more spatial support points in the system and forms a more stable network structure with PPI by enhancing hydrogen bonding and electrostatic interactions. This uniform and highly cross-linked network structure restricts the movement of molecular chain segments, thereby enhancing resistance to deformation.

[0071] (2) Crystal structure All samples exhibited two broadened diffraction peaks at 2θ = 9.18° (d = 9.63 Å) and 2θ = 20.24° (d = 4.38 Å), indicating that the overall gel structure is predominantly amorphous (e.g., ...). Figure 10 (D in the text). Notably, no new diffraction peaks appeared in any of the composite samples, indicating that the nanocellulose was highly dispersed in the system and did not self-aggregate to form an independent crystalline phase. This also confirms that the nanocellulose was successfully embedded in the protein network, and that there is good interfacial compatibility between the two. In terms of characteristic peak intensity, the diffraction peak at 20.24° was slightly enhanced after the introduction of nanocellulose, reflecting an improvement in the local order of the system. In particular, the enhancement was most significant in the PPI-CNF group, suggesting that CNF has a greater advantage in inducing the ordered arrangement of protein molecules. This effect may stem from the high specific surface area and low aspect ratio of CNF, which enhances the hydrogen bonding and steric hindrance effects with PPI molecules, thereby promoting the conformational transformation of protein segments to ordered stacking modes such as β-sheets. The above structural change trend and the aforementioned results of enhanced rheological properties corroborate each other, further confirming the key role of nanocellulose, especially CNF, in regulating the assembly of PPI molecules and constructing high-performance gel networks.

[0072] (3) Thermal stability Thermogravimetric curves of PPI-based composite gels all exhibited a three-stage thermal decomposition characteristic. The first stage (30–220℃) of mass change mainly stemmed from the evaporation of free and bound water; the weight loss rate of each sample in this stage showed no significant difference, indicating that nanocellulose had a limited impact on the water content of the system. The second stage (220–400℃) corresponded to the thermal degradation of the protein backbone and cellulose chains, while the third stage (above 400℃) was mainly a further oxidative decomposition process of residual carbon. Within the main decomposition range of 220–400℃, the sample mass loss rate decreased sequentially from 71.39% (PPI) to 67.75% (PPI-MFC), 65.19% (PPI-CNC), 62.12% (PPI-BC), and 59.75% (PPI-CNF) (e.g., ...). Figure 10The peak decomposition temperatures (PPI) gradually increase from 297.79℃ (PPI) to 302.69℃ (PPI-MFC), 302.95℃ (PPI-CNC), 307.97℃ (PPI-BC), and 313.98℃ (PPI-CNF) (e.g., PPI-CNF). Figure 10 The main decomposition peak gradually shifts towards the high-temperature region, indicating that low aspect ratio nanocellulose (CNF) contributes to improved thermal stability. This is mainly attributed to the small aspect ratio and high interfacial activity of CNF, which can effectively enhance the resistance of the composite system to thermal decomposition by strengthening hydrogen bonding and hydrophobic interactions with PPI molecules. Simultaneously, the dense three-dimensional network constructed by CNF can inhibit the thermal motion of polymer segments and slow down the thermal decomposition process, further confirming the stabilizing role of low aspect ratio nanocellulose in gel structure construction.

[0073] (4) Gel network structure SEM analysis revealed the significant influence of nanocellulose on the microstructure of PPI gel (e.g. Figure 10 In the G group, pure PPI gels exhibit a loose, amorphous aggregate or sheet-like morphology with a rough, porous, and wrinkled surface, lacking clearly defined regular microstructural units. After introducing nanocellulose, the composite gels form a fibrous, interwoven network structure. This morphological transformation may stem from the steric hindrance effect of nanocellulose and the hydrogen bond network formed between its high-density hydroxyl groups and proteins, which together slow down the protein aggregation rate, leading to a more compact microstructure. The gel porosity decreased from 52.68% (PPI) to 44.13% (PPI-MFC), 39.17% (PPI-CNC), 37.67% (PPI-BC), and 35.66% (PPI-CNF), respectively (e.g., ...). Figure 10 The PPI-CNF group exhibited a finer fibrous network structure, primarily due to the ultrafine fibrous morphology and high specific surface area of ​​CNF, enabling it to be highly dispersed within the PPI matrix and form sufficient interfacial contact with protein molecules. Through intermolecular interactions such as hydrogen bonds and van der Waals forces, CNF can induce PPI molecules to undergo cooperative self-assembly, forming a highly ordered fibrous network. Simultaneously, CNF, as a matrix filler, can construct a self-supporting network within the system, further enhancing the density and stability of the gel structure. The introduction of nanocellulose reduced the tilt angle of the PPI-based composite gel, and the reduction was positively correlated with the aspect ratio of nanocellulose. Specifically, the gel tilt angles of PPI-MFC, PPI-CNC, and PPI-BC were reduced by 17.39%, 30.43%, and 39.13% respectively compared to PPI, while the PPI-CNF group exhibited a 0° gel tilt angle, displaying typical gel solid-state characteristics. This indicates that the addition of nanocellulose can significantly enhance the gel-forming ability of PPI (e.g., ...). Figure 10(I in the text). In summary, nanocellulose with a smaller aspect ratio has a greater advantage in constructing stable gel structures.

[0074] 4.3 The regulatory effect of nanocellulose on the aroma binding ability of pea protein isolate 4.3.1 Aroma Binding Rate This invention employs HS-SPME-GC technology to evaluate the regulatory effect of nanocellulose on the binding ability of PPI to 2,5-dimethylpyrazine. Figure 11 As shown in A, the binding rate of PPI to 2,5-dimethylpyrazine is 11.10%, while the introduction of nanocellulose significantly improves the aroma binding performance of each composite system. p <0.05%. Among them, the PPI-CNF group showed the highest aroma binding rate (32.96%), followed by PPI-BC (28.08%), PPI-CNC (23.54%), and PPI-MFC (18.91%). Compared with PPI, the aroma binding capacity of each composite system increased by 70.36% (PPI-MFC), 112.07% (PPI-CNC), 152.97% (PPI-BC), and 196.94% (PPI-CNF), respectively. In contrast, the binding capacity of pure nanocellulose itself for aroma substances is extremely weak. Figure 11 B in the middle; p <0.05). This result indicates that the addition of nanocellulose can significantly enhance the binding ability of PPI to 2,5-dimethylpyrazine, and the enhancing effect is negatively correlated with the aspect ratio of nanocellulose.

[0075] Enhanced protein flavor binding capacity typically involves multi-scale, multi-mechanism synergistic regulation. At the molecular level, nanocellulose can induce protein conformational changes through surface hydrogen bonds and hydrophobic interactions, exposing more embedded binding sites. Similar phenomena have been observed in other protein-polysaccharide systems; for example, *Mesona chinensis* polysaccharide can induce a conformational change in myosin from α-helix to β-sheet, and enhance its binding capacity for aldehydes such as hexaformaldehyde by strengthening hydrophobic interactions. At the supramolecular level, nanocellulose constructs a dense three-dimensional network structure through its own entanglement and interactions with proteins, enhancing its spatial retention capacity for flavor molecules. Carboxymethyl cellulose can form a three-dimensional network with myofibrillar proteins through intermolecular interactions, exposing hydrophobic binding sites and significantly improving its binding capacity for anethole. At the macroscopic level, the introduction of nanocellulose can increase system viscosity and reduce molecular diffusion rates, thereby enhancing flavor retention. The addition of k-carrageenan can improve the viscoelasticity of the soybean protein system and enhance its adsorption capacity for 2,5-dimethylpyrazine. However, in this invention, the dominant mechanism by which nanocellulose regulates the binding of PPI to 2,5-dimethylpyrazine is not yet clear and requires further clarification through subsequent experiments.

[0076] 4.3.2 Aroma binding thermal stability Figure 11 The CD analysis revealed the thermal response behavior of the nanocellulose-PPI composite system in binding with 2,5-dimethylpyrazine. Within the temperature range of 37℃ to 120℃, all composite systems incorporating nanocellulose showed a steady increase in aroma binding rate with increasing temperature. Specifically, the aroma binding rates of the PPI and PPI-CNF groups increased significantly from 11.10% and 32.96% at 37℃ to 46.96% and 67.31% at 120℃, respectively. This phenomenon is mainly attributed to the thermal treatment inducing spatial structure unfolding and denaturation of the protein, leading to the exposure of previously embedded hydrophobic groups, thereby enhancing the hydrophobic interaction between the protein and 2,5-dimethylpyrazine. Furthermore, the increased temperature intensified protein molecule collisions and aggregation, and together with nanocellulose, constructed a denser thermally induced gel network, effectively trapping aroma molecules through steric hindrance, achieving a synergistic effect of physical encapsulation and chemisorption.

[0077] It is worth noting that the PPI-CNF group consistently exhibited the best aroma binding ability across all temperature gradients, with binding rates of 39.24%, 47.86%, and 67.31% at 50℃, 80℃, and 120℃, respectively. Figure 11 (C in the image), this trend is further confirmed in the heatmap ( Figure 11 (D in the text). This phenomenon stems from the ultra-high specific surface area and low aspect ratio of CNF. Its abundant hydroxyl and carboxyl groups, among other polar groups, can crosslink with PPI through hydrogen bonds and hydrophobic interactions, assisting in the construction of a dense three-dimensional network structure and exposing more adsorption sites. On the other hand, the non-covalent interaction between CNF and PPI can disrupt the original weak equilibrium within the protein, further inducing a conformational shift of PPI from a relatively loose state to a tightly ordered one. Simultaneously, the re-exposed polar groups can directly capture aroma molecules through hydrogen bonds and other interactions, effectively regulating protein conformation and binding site distribution. Correlation analysis showed that aroma binding rate was significantly positively correlated with protein solubility, β-sheet, and intermolecular β-sheet (p≤0.001), while it was significantly negatively correlated with surface hydrophobicity, total thiol groups, active thiol groups, and β-turn angle (p≤0.001). Figure 11 (EG in the middle).

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for enhancing the binding of pea protein isolate to 2,5-dimethylpyrazine using nanocellulose, comprising the following steps: Pea protein isolate, nanocellulose and water were mixed, and the resulting first mixed solution was hydrated and gelled sequentially to obtain a gel system. The gel system is mixed with 2,5-dimethylpyrazine to obtain a second mixed solution, which is then compounded to enhance the aroma of 2,5-dimethylpyrazine.

2. The method of claim 1, wherein, The nanocellulose includes one or more of microcrystalline cellulose, nanocrystalline cellulose, bacterial cellulose, and cellulose nanofibers.

3. The method of claim 2, wherein, The microcrystalline cellulose has a length of 22.82±2.95μm, a diameter of 121.15±52.16nm, and an aspect ratio of 272.49±163.

98.

4. The method of claim 2, wherein, The nanocrystalline cellulose has a length of 273.12±77.98 nm, a diameter of 44.23±13.74 nm, and an aspect ratio of 6.59±2.

52.

5. The method of claim 2, wherein, The bacterial cellulose has a length of 5.04±1.38μm, a diameter of 71.32±22.18nm, and an aspect ratio of 68.72±26.

32.

6. The method of claim 2, wherein, The cellulose nanofibers have a length of 137.93±41.35 nm, a diameter of 26.99±6.69 nm, and an aspect ratio of 5.34±1.

79.

7. The method of claim 1, wherein, The mass ratio of pea protein isolate to nanocellulose is 10~15:0.5~1.

0.

8. The method according to claim 1 or 7, characterized in that, The hydration time is 10-14 hours; the gelation temperature is 90-100°C and the time is 20-40 minutes.

9. The method of claim 1, wherein, The ratio of pea protein isolate to 2,5-dimethylpyrazine used in preparing the gel system was 0.5~2g:0.05~0.25mmol.

10. The method according to claim 1 or 9, characterized in that, The composite process is carried out at a temperature of 37°C for 14-18 hours.

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