Bionic composite material model for simulating solid-phase biological catalysis as well as preparation method and application of bionic composite material model

Abstract

The invention discloses a bionic composite material model for simulating solid-phase biological catalysis as well as a preparation method and application of the bionic composite material model, and belongs to the field of biological catalysis and biological materials. The model is composed of an insoluble porous fiber substrate and solid-phase target biological macromolecules immobilized on the insoluble porous fiber substrate, and through the simplified controllable structure, the state that the target macromolecules are embedded in a solid-phase network in a real system is simulated. The method solves the problems that the traditional solid-phase catalysis research is difficult to repeat and unclear in mechanism due to complex and uncontrollable matrix, can be used as a standardized research platform, and is used for deeply revealing a degradation mechanism, optimizing a catalysis process, screening an efficient enzyme preparation and evaluating a pretreatment method.

Description

Technical Field

[0001] The present invention belongs to the field of biocatalysis and biomaterial technology, and specifically relates to a biomimetic composite material model for simulating and studying the enzymatic hydrolysis or degradation mechanism of biomacromolecules (such as polysaccharides and proteins) in a complex, insoluble solid matrix, as well as a preparation method thereof and its application in enzyme preparation screening, process optimization and mechanism research. Background Art

[0002] In many fields, including biomass resource utilization, food processing, and biomedicine, enzymes are often involved in the catalytic degradation of specific biomacromolecules in solid matrices. For example, in the production of biofuels from agricultural and forestry waste, cellulase is required to degrade cellulose; in the food industry, proteases are needed to degrade solid protein matrices; and in drug delivery, specific enzymes are needed to degrade the polysaccharide or protein backbones used as carriers.

[0003] However, the study of the mechanism of such solid-phase biocatalytic processes faces huge challenges, and its shortcomings are mainly reflected in:

[0004] 1. Existing research models are out of touch with reality: Most enzymology research is conducted in homogeneous liquid systems, which cannot simulate the complex processes of enzyme molecules in real environments, such as diffusion, adsorption, substrate binding, and product mass transfer within porous, insoluble solid matrices. These physical limitations are often the rate-limiting step in the entire catalytic reaction.

[0005] 2. Uncontrollability of real systems: Directly studying real, complex solid-phase systems (such as plant cell walls and food matrices) makes it difficult to replicate experimental results due to their diverse components, heterogeneous structures, and large batch differences. Furthermore, it is impossible to effectively decouple multiple influencing factors (such as physical structural barriers, inhibition or synergistic effects of coexisting chemical substances), resulting in stagnant mechanism research and heavy reliance on trial and error in process development.

[0006] Therefore, the field urgently needs a universal, controllable, and reproducible model system capable of simulating the core scenario of "target biomacromolecules present as a solid phase within an insoluble porous fiber matrix." Such a system would become a powerful research tool for deconstructing complex solid-phase biocatalytic processes and promote the transition from empirical optimization to quantitative mechanism-driven design in related fields. Summary of the Invention

[0007] The purpose of the present invention is to provide a universal biomimetic composite material model and its preparation method and application, aiming to overcome the common difficulties in studying solid-phase biocatalytic processes in the existing technology, and to provide a standardized model platform for in-depth understanding of the degradation mechanism of various biomacromolecules in solid-phase matrices, optimization of catalytic processes, screening of high-efficiency enzyme preparations and evaluation of substrate pretreatment methods.

[0008] The working principle of the present invention is to construct a simplified and controllable biomimetic composite material model by simulating the core physical structure of "target biomacromolecules as a solid phase, fixed or embedded in an insoluble porous fiber network" that is commonly found in various real biological systems. The model adopts a modular design concept and is prepared through a two-step method of "substrate construction + functional molecule immobilization". By comparing the model with the corresponding real complex systems and utilizing the differences in catalytic kinetics, micromorphology and chemical structure changes between the two, the key physical and chemical bottleneck factors that limit catalytic efficiency in the real system (such as dense network structure, mass transfer barriers, nonspecific adsorption, etc.) can be inferred.

[0009] The object of the present invention is achieved through the following technical solutions:

[0010] A biomimetic composite material model for simulating solid-phase biocatalysis, an insoluble porous fiber substrate; and

[0011] One or more target biomacromolecules exist in a solid phase, and the target biomacromolecules are immobilized on the surface or in the internal pores of the porous fiber substrate.

[0012] The insoluble porous fiber substrate is composed of one or more materials selected from cellulose, hemicellulose, lignin, chitin, collagen, and polylactic acid.

[0013] The target biomacromolecules are selected from polysaccharides (such as starch, cellulose, pectin), proteins (such as casein, soy protein, collagen), nucleic acids or a combination thereof.

[0014] The mold may optionally further comprise a filler, which is calcium carbonate or kaolin.

[0015] The binder is a biocompatible polymer, preferably selected from one or a combination of chitosan, sodium carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), guar gum, and xanthan gum. The binder is preferably added in an amount of 0.5% to 5.0% of the dry weight of the porous fiber substrate. This range effectively enhances bonding while not overfilling the pores and affecting enzyme accessibility.

[0016] Furthermore, the fiber raw materials of the porous fiber substrate are selected according to the different simulation objects: when used to simulate the degradation of plant biomass, the insoluble fiber is preferably selected from one or more of tobacco fiber, wood pulp fiber, bamboo pulp fiber, cotton fiber, and sugarcane bagasse fiber; when used to simulate animal-derived food matrix or tissue, the insoluble fiber is preferably collagen fiber; when used to simulate the degradation of fungi or crustaceans, the insoluble fiber is preferably chitin or chitosan fiber.

[0017] Furthermore, the immobilized target biomacromolecule is selected according to the specific research purpose: when studying starch enzymatic hydrolysis, the target biomacromolecule is preferably corn starch, potato starch, cassava starch, or natural starch granules directly extracted from the target biomass; when studying cellulose degradation, the target biomacromolecule is preferably microcrystalline cellulose (MCC), amorphous cellulose or bacterial nanocellulose; when studying protein digestion, the target biomacromolecule is preferably casein, soy protein isolate, whey protein or collagen in the form of solid phase particles.

[0018] Furthermore, a key technical feature of the present invention is that the ratio between the immobilized target biomacromolecule and the porous fiber substrate is controllable to simulate the substrate abundance in different real systems. Preferably, the ratio of the dry weight of the target biomacromolecule to the dry weight of the porous fiber substrate is 1:1000 to 30:100 (i.e., 0.1% to 30%); a lower ratio (such as 0.1%-5%) is suitable for simulating scenarios where the target macromolecule is scarce in the matrix and enzyme accessibility is the main bottleneck; a higher ratio (such as 5%-30%) is suitable for simulating scenarios where the target macromolecule is one of the main components of the matrix. At the same time, by controlling the immobilization process, the coating thickness of the target macromolecule on the substrate surface can also be controlled within the range of 5 to 100 microns.

[0019] Substantially different from existing composite materials designed to optimize combustion or consumption characteristics (such as reconstituted tobacco), the core purpose and structure of this invention is to serve as a controllable research tool. Through a separate "base-substrate" construction, it intentionally creates a controllable physical barrier and enzyme action site. Its function is to serve as a research platform, not a final product.

[0020] In a preferred embodiment, the raw material of the insoluble porous fiber substrate is selected from one or more of plant fibers (such as cellulose, hemicellulose, and lignin), animal fibers (such as chitin and collagen), and biodegradable polymers (such as polylactic acid). When simulating the degradation of plant biomass, tobacco fibers, wood pulp fibers, or bamboo pulp fibers are preferred; when simulating animal-derived matrices, collagen fibers are preferred.

[0021] In another preferred embodiment, the target biomacromolecule is selected from polysaccharides (such as starch, cellulose, pectin), proteins (such as casein, soy protein, collagen), nucleic acids or a combination thereof.

[0022] To enhance the practicality of the model, the model may further comprise optional components. For example, a binder may be included to enhance mechanical strength, preferably chitosan, sodium carboxymethylcellulose, or polyvinyl alcohol, preferably in an amount of 0.5% to 5.0% of the base's dry weight. Furthermore, fillers (such as calcium carbonate and kaolin) may be included to adjust the base's porosity and physical properties, as specifically demonstrated in Example 1, but this component is optional.

[0023] The method for preparing the biomimetic composite material model of the present invention comprises the following steps:

[0024] Preparation of porous fiber substrate: One or more insoluble fibers are prepared by wet molding, freeze drying or electrospinning to form a network skeleton with a controllable pore structure:

[0025] Functional molecule immobilization: One or more target biomacromolecules are immobilized on the surface or internal pores of a prefabricated porous fiber substrate in a solid phase form including particles, microspheres, fibers through coating, impregnation, co-precipitation or in situ synthesis.

[0026] The application of the present invention is to use the bionic composite material as a standardized simulation platform for simulating and studying the degradation mechanism of various biomacromolecules in a solid matrix, optimizing catalytic processes, screening high-efficiency enzyme preparations, and evaluating substrate pretreatment methods.

[0027] Furthermore, the biomimetic composite material can be used in methods for evaluating or screening biocatalysts (such as enzymes) acting on solid substrates, or in evaluating pretreatment methods for improving the accessibility of target macromolecules in solid substrates.

[0028] More specifically, the main technical features of the present invention are:

[0029] (1) Product Features: A layered biomimetic composite material for simulating solid-phase biocatalysis, comprising:

[0030] A porous fiber substrate: prepared by one or more insoluble fibers (such as cellulose, hemicellulose, chitin, collagen, etc.) through wet molding, freeze drying or electrospinning to form a network skeleton with a controllable pore structure.

[0031] One or more target biomacromolecules are present in a solid-phase form (e.g., particles, microspheres, fibers, etc.) and immobilized on the surface or within the pores of the porous fibrous substrate by coating, impregnation, co-precipitation, or in situ synthesis. The target biomacromolecules are selected from polysaccharides (e.g., starch, cellulose, pectin, chitin), proteins (e.g., casein, soy protein, collagen), nucleic acids, or combinations thereof.

[0032] Optional Components: A small amount of binder may be optionally included to enhance bonding, or other specific chemical components may be included to simulate a more complex microenvironment. Depending on the specific simulation requirements, the composite material may optionally include fillers such as calcium carbonate and kaolin to adjust the substrate's porosity, density, or pH microenvironment.

[0033] (2) Method characteristics: A modular preparation method comprising:

[0034] The first step is substrate preparation: according to research needs, specific fiber raw materials are selected, and a porous fiber substrate with target porosity and mechanical strength is prepared through a specific process.

[0035] The second step is functional molecule immobilization: select the target biomacromolecule and load it in a controllable manner onto a prefabricated porous substrate in a solid phase through specific technical means.

[0036] The concrete effects of the present invention are:

[0037] Taking an artificial tobacco leaf model (ATL), a specific embodiment of the present invention, as an example, the enzymatic hydrolysis process of tobacco starch (target macromolecule) on a tobacco fiber substrate was studied and compared with real tobacco leaves (NTL), and the following results were obtained:

[0038] Beneficial effects:

[0039] (1) The simulation and deconstruction effects were significant: the starch degradation rate of ATL (20.15%) was much higher than that of NTL (10.46%), which quantitatively revealed that the complex structure of NTL was the main bottleneck.

[0040] (2) Research platform standardization: The enzymatic hydrolysis process of ATL can well fit the first-order kinetic model (R 2 >0.90), while NTL showed a significant deviation, proving its effectiveness as an “ideal control”.

[0041] (3) In-depth mechanism revelation: Through multi-scale characterization such as SEM, FTIR, and XRD, it is clearly revealed that the cell wall and lignin in NTL are the specific structural obstacles that restrict the action of enzymes.

[0042] These results demonstrate that the general model building strategy proposed in this invention is successful and can be extended to other systems, such as:

[0043] a. Simulated cellulose degradation: Using wood fiber as the substrate and loading microcrystalline cellulose, it is used to study the mechanism of action of cellulase.

[0044] b. Protein digestion in simulated food: Soybean fiber was used as the substrate and loaded with casein particles to study the digestion kinetics of proteases in a simulated food matrix.

[0045] The innovation of the present invention compared with the prior art is:

[0046] (1) Methodological innovation: For the first time, a universal, modular biomimetic composite model construction strategy was proposed and implemented to simulate and deconstruct the biocatalytic process of any target biomacromolecule in an insoluble solid matrix. This methodology is highly flexible and widely applicable and can be extended to multiple fields such as biofuels, food science, and biomedicine.

[0047] (2) Product concept innovation: A new, customizable research tool, the biomimetic composite material model, was invented. Its structure (substrate + immobilized macromolecules) and function (simulated solid-phase catalysis) are not reported in the existing technology.

[0048] (3) Expansion of application areas: This model is used as a standardized platform, and its application is no longer limited to mechanism research, but has been expanded to high-throughput screening of enzyme preparations in the industrial field, rapid evaluation of pretreatment processes, etc., and has huge potential for industrial application.

[0049] The core of the present model lies in the layered or controlled distribution of "substrate + functional molecules." The substrate is intentionally fabricated to possess a controllable porous network, its primary function being to provide pathways for enzyme diffusion and adsorption. Functional molecules, such as macromolecular substrates, are also immobilized so that they are exposed to the enzyme, rather than uniformly intermingled. This structure is key to achieving its "simulated solid-phase catalysis" function. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 ; Schematic diagram of the process of preparation and analytical application of the bionic composite material model constructed by the present invention.

[0051] Figure 2 : In Example 2, the single factor experimental results that affect the starch degradation rate are shown.

[0052] Figure 3 : In Example 3, a comparison diagram of starch degradation kinetics of the artificial model (ATL) and the natural model (NTL); wherein, sub-figure (A) shows the change of total starch content over time, and sub-figure (B) is the fitting result of the first-order kinetic model; (a1, b1) in the sub-figures represent the artificial model (ATL), and (a2, b2) represent the natural model (NTL).

[0053] Figure 4 : In Example 3, chemical and crystal structure characterization diagrams of the two models before and after enzymatic hydrolysis; wherein, sub-figure (a) is the Fourier transform infrared spectroscopy (FTIR) spectrum, and sub-figure (b) is the X-ray diffraction (XRD) spectrum.

[0054] Figure 5: In Example 3, XRD and FTIR patterns of the artificial model and natural model before and after amylase treatment.

[0055] (a) Infrared spectrum; (b) X-ray diffraction pattern with peak coverage. Specific implementation plan

[0056] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Figure 1 The general preparation and analysis process of the biomimetic composite material model proposed in this invention is demonstrated.

[0057] Example 1: Preparation of a biomimetic composite material model (artificial tobacco leaf model, ATL) for simulating enzymatic hydrolysis of starch in tobacco waste

[0058] This embodiment provides a specific method for preparing the biomimetic composite material model of the present invention. It should be noted that the calcium carbonate filler in step (1) is a preferred but optional component of the present invention, and its function is to adjust the physical properties of the substrate. In other embodiments (Examples 4 and 5), such fillers may not be included, which reflects the modular and customizable characteristics of the present invention.

[0059] (1) Substrate Preparation: 85.0% pulverized tobacco fiber (including tobacco leaf and tobacco stem fibers), 10.0% lignocellulose (DP 1000) as a reinforcing agent, 2.0% chitosan as a binder, and 3.0% calcium carbonate (particle size 2–5 μm) as a filler were weighed by weight. The fiber components were processed in a laboratory pulper and then mixed uniformly under high-speed stirring to prepare a fiber slurry with a solids concentration of 15–18%.

[0060] (2) Wet Forming: The fiber slurry was transferred to a standard sheeter and wet-formed on an 80-mesh filter. Most of the water was removed by vacuum filtration to form a wet paper sheet. The wet paper sheet was then pressed on a flat press at a pressure of 0.5 MPa for 5 minutes to further dehydrate the paper and strengthen the bonding between the fibers.

[0061] (3) Drying of substrate: The pressed substrate was placed in a blast drying oven at 80°C and dried to a constant weight, obtaining a quantitative value of 50±2 g / m 2 , a porous fiber substrate with a thickness of 0.12–0.15 mm.

[0062] (4) Starch immobilization: Tobacco starch extracted and purified from tobacco waste is prepared into a deionized water suspension of a set concentration. The starch suspension is evenly coated on the surface of the porous fiber substrate prepared in step (3) using a laboratory doctor bar.

[0063] (5) Finished Product Drying and Preparation: The starch-coated composite sheet is dried in a vacuum oven at 40°C for 8-12 hours to gently remove moisture without gelatinization and allow the starch granules to adhere to the fiber substrate. After drying, the biomimetic composite material model (ATL) of the present invention is obtained.

[0064] Example 2: Optimizing the catalytic process of α-amylase using the biomimetic composite material model (ATL) prepared in Example 1 This example demonstrates the application of the model of the present invention in optimizing biocatalytic process parameters.

[0065] (1) Experimental design: The ATL prepared in Example 1 was cut into 3 mm × 5 mm pieces. Before the Box-Behnken design was conducted, the reasonable range of each factor was determined by single-factor experiments. The typical results are shown in Figure 2. Figure 2 As shown in the figure, temperature, time and enzyme concentration all have a significant effect on the degradation rate. Based on the single-factor experimental results, Box-Behnken design (BBD) was used to optimize three key process parameters: reaction temperature (A: 25, 35, 45°C), reaction time (B: 6, 8, 24h) and enzyme concentration (C: 6, 9, 12U / g).

[0066] (2) Enzymatic hydrolysis: According to the 17 experimental points designed by BBD, 20 g of ATL fragments were accurately weighed and placed in a sealed reaction bag. According to the experimental design, the corresponding volume of crude α-amylase extract (total enzyme activity 108 ± 0.27 U / mL, diluted to reach the target enzyme concentration) was added, and an appropriate amount of buffer was supplemented to accurately control the total water content of the system to 25%. The sealed bag was placed in a constant temperature incubator at the corresponding temperature and reacted for the corresponding time. Three parallel reactions were set for each experimental point.

[0067] (3) Result analysis: After the reaction, the samples were immediately inactivated and the residual total starch content was determined by dual-wavelength colorimetry to calculate the starch degradation rate. The experimental data were subjected to regression analysis and variance analysis using Design Expert 12.0 software.

[0068] (4) Optimization results: The obtained regression model was significant (P < 0.0001), R 2 The optimal process conditions predicted by the model were: reaction temperature 35°C, reaction time 8 hours, and enzyme concentration 9 U / g. Three validation experiments were conducted under these conditions, and the average starch degradation rate measured was 20.15%, very close to the model-predicted value of 20.6%, demonstrating the reliability and effectiveness of process optimization using the proposed model. For details on the BBD experiment design and results, please see the "Experimental and Experimental Data" table at the end of this article.

[0069] Example 3: Using the biomimetic composite material model (ATL) prepared in Example 1 to illustrate the rate-limiting mechanism of starch enzymatic hydrolysis in natural tobacco leaves (NTL)

[0070] This example demonstrates the use of the model described in the present invention for mechanistic studies and as a comparative benchmark.

[0071] (1) Comparative Experimental Setup: Two groups of samples were prepared: one group was the ATL prepared in Example 1 (initial starch content 2.63%), and the other group was natural tobacco leaf (NTL) samples that had undergone the same pulverization process (initial starch content measured to be 6.53%). Both groups of samples were subjected to enzymatic hydrolysis reactions simultaneously under the optimal process conditions obtained in Example 2 (35°C, 8 h, 9 U / g enzyme concentration, 25% moisture content).

[0072] (2) Kinetic comparison analysis: Samples were taken at multiple time points, such as 0, 2, 4, 6, 8, and 10 hours after the start of the reaction. The changes in the total starch content in the two groups of samples were measured and compared. The results showed that after 10 hours, the starch degradation rate of ATL reached 20.15%, while that of NTL was only 10.46%. Figure 3 As shown in (A), the starch content in ATL decreased rapidly and greatly, while that in NTL decreased relatively slowly. The data were fitted with a first-order kinetic model, as shown in Figure 3 As shown in (B), it was found that the degradation process of ATL was highly consistent with the model (R 2 >0.90), while the degradation process of NTL showed significant deviation, indicating that its degradation process was affected by more complex limiting factors.

[0073] (3) Multi-scale structural comparative analysis:

[0074] Microscopic morphology (SEM): SEM characterization of samples before and after enzymatic hydrolysis. Figure 4 As shown in the figure, the enzyme degrades ATL in a lamellar exfoliation manner, forming a porous network; while for NTL, the enzyme's attack is limited to the particle surface, forming a large number of pitting pits, and its internal core structure is preserved.

[0075] Chemical structure (FTIR): Compare the FTIR spectra of samples before and after enzymatic hydrolysis. Figure 5 As shown in (a), in the NTL spectrum, in addition to the starch-related absorption peak, other absorption peaks related to cell wall polysaccharides also changed significantly, while the changes in ATL were mainly concentrated on the characteristic peaks of starch.

[0076] Crystal structure (XRD): Compare the XRD patterns of samples before and after enzymatic hydrolysis. Figure 5As shown in (b), the relative crystallinity of ATL decreased significantly from 42.5% to 37.30%, indicating that the amorphous and partially crystalline regions were effectively degraded. However, the crystalline region of NTL showed stronger resistance and the change in crystallinity was not obvious.

[0077] (4) Mechanism conclusion: Through the above comparative analysis, it is concluded that although the initial starch concentration of NTL is higher, its enzymatic hydrolysis efficiency and degree are far lower than those of the simplified ATL model. This difference is not due to the properties of starch itself, but rather to the severe "enzyme accessibility" barrier caused by the complex physicochemical structure of NTL (such as dense cell walls, physical shielding and chemical adsorption of lignin, etc.). Therefore, the ATL model provided by the present invention can be used as an effective benchmark for quantifying and identifying the structural anti-degradation bottlenecks of real biomass.

[0078] Example 4: A biomimetic composite material model for simulating cellulose enzymatic hydrolysis and its preparation (using freeze-drying and impregnation methods)

[0079] This embodiment provides a model for simulating the cellulose degradation process in the field of biomass energy.

[0080] (1) Substrate preparation (freeze drying method):

[0081] Weigh 10g of bamboo pulp fiber, place it in 1L of deionized water, use a high-speed homogenizer to disperse it at a speed of 10,000rpm for 15 minutes to form a uniform fiber suspension (1% w / v). Pour this suspension into multiple culture dishes with a diameter of 5cm, and the liquid level is about 1cm. Subsequently, the culture dishes are placed in a -80℃ refrigerator and quickly frozen for 12 hours to fix the fiber structure in the ice crystal template. Finally, the frozen sample is transferred to a vacuum freeze dryer and dried for 48 hours at -50℃ and a pressure of less than 10Pa. After drying, a white sponge-like bamboo fiber substrate with a high porosity (>90%) and a three-dimensional interconnected network structure is obtained.

[0082] (2) Immobilization of target biomacromolecules (impregnation method):

[0083] 2 g of microcrystalline cellulose (Avicel PH-101, a model substrate with high crystallinity) was dispersed in 100 mL of deionized water and ultrasonically treated for 30 minutes to prepare a uniform suspension (2% w / v). The porous bamboo fiber substrate prepared in step (1) was completely immersed in the microcrystalline cellulose suspension and placed in a vacuum drying oven and repeatedly evacuated and broken the vacuum 3 times to ensure that the suspension could fully penetrate into the internal pores of the substrate. After immersion for 2 hours, the substrate was taken out, and the excess liquid on the surface was gently absorbed with absorbent paper, and then placed in a 60°C forced air drying oven to dry to constant weight. After drying, the microcrystalline cellulose particles were physically adsorbed and immobilized on the inner and outer surfaces of the bamboo fiber substrate, obtaining a bionic composite material model for simulating cellulose enzymatic hydrolysis.

[0084] The expected effects of this example are as follows: This model can be used to test the synergistic effects of cellulases from different sources (e.g., endoglucanases, exoglucanases, and β-glucosidases), or to evaluate the effects of different physical / chemical pretreatment methods (e.g., dilute acid treatment and steam explosion) on improving cellulose accessibility. Its degradation products (e.g., glucose and cellobiose) can be quantitatively detected by high-performance liquid chromatography (HPLC).

[0085] Example 5: A biomimetic composite material model for simulating food protein digestion and its preparation (using electrospinning and coprecipitation methods)

[0086] This example provides a model for simulating the protein degradation process within a solid food matrix in the human digestive tract.

[0087] (1) Substrate preparation (electrospinning method):

[0088] One gram of polylactic acid (PLA), a biodegradable polymer that mimics hydrophobic food matrices, was dissolved in 9 mL of a 7:3 volume ratio of dichloromethane (DCM) and N,N-dimethylformamide (DMF) solvent mixture to prepare a 10% (w / v) spinning solution. This solution was loaded into a syringe with a metal needle and electrospinning was performed using an electrospinning device. The process parameters were: voltage 20 kV, receiving distance 15 cm, and spinning solution feed rate 1.0 mL / h. The spun nanofibers were collected on a roller receiver covered with aluminum foil for 4 hours. The collected fiber membrane was dried in a vacuum oven at 40°C for 24 hours to remove residual solvent, resulting in a PLA nanofiber membrane substrate with a high specific surface area and nanoscale pores.

[0089] (2) Co-precipitation and immobilization of target biomacromolecules and substrate fibers:

[0090] In this step, the target biomacromolecule (casein) and another fiber (chitosan, used to form a secondary network) are bound to the PLA substrate by coprecipitation. First, 0.5g of casein is weighed and dissolved in 100ml of pH 8.0 buffer; 0.2g of chitosan is weighed and dissolved in 100ml of 1% acetic acid solution. The PLA nanofiber membrane substrate prepared in step (1) is immersed in the chitosan solution to fully wet it. Then, the chitosan solution containing the PLA membrane is slowly added dropwise to the vigorously stirred casein solution. Due to the change in pH value and the interaction between the two macromolecules, casein and chitosan will form coprecipitates, which will be deposited and captured in the porous PLA nanofiber network. After continuous stirring for 1 hour, the composite membrane is removed and repeatedly washed with deionized water to remove unbound protein and chitosan. Finally, it is dried in a vacuum oven at 40°C to constant weight to obtain a biomimetic composite material model loaded with casein-chitosan complex.

[0091] The expected effect of this example is as follows: The model can be placed in simulated gastric fluid (containing pepsin, pH 1.5-2.5) or simulated intestinal fluid (containing trypsin, pH 7.5-8.5), and by measuring the content of peptides and amino acids released into the solution, the degradation rate and extent of protein at different digestion stages can be dynamically studied, thereby being used to evaluate the effects of different processing methods (such as heat treatment and high-pressure treatment) on protein digestibility.

[0092] Test and experimental data

[0093] BBD Three Factor Three Level Response Surface Experimental Results

[0094] (BBD three-factor three-level response surface experiment results)

[0095]

[0096]

Claims

1. A biomimetic composite material model for simulating solid-phase biocatalysis, characterized in that: include: an insoluble porous fiber substrate; as well as One or more target biomacromolecules exist in a solid phase, and the target biomacromolecules are immobilized on the surface or in the internal pores of the porous fiber substrate.

2. The bionic composite material model according to claim 1, characterized in that: The insoluble porous fiber substrate is composed of one or more materials selected from cellulose, hemicellulose, lignin, chitin, collagen, and polylactic acid.

3. The bionic composite material model according to claim 1, characterized in that: The target biomacromolecules are selected from polysaccharides including starch, cellulose, pectin, chitin, proteins including casein, soy protein, collagen, nucleic acids or a combination thereof.

4. The bionic composite material model according to claim 1, characterized in that: The porous fiber substrate further comprises one or more adhesives selected from chitosan, sodium carboxymethyl cellulose and polyvinyl alcohol, and the added amount thereof is 0.5% to 5% of the dry weight of the porous fiber substrate.

5. The bionic composite material model according to claim 1, characterized in that: The ratio of the dry weight of the target biomacromolecule to the dry weight of the porous fiber substrate is 1:1000 to 30:

100.

6. The bionic composite material model according to claim 1, characterized in that: The mold may optionally further comprise a filler, which is calcium carbonate or kaolin.

7. A method for preparing a biomimetic composite material model according to any one of claims 1 to 6, characterized in that: The following steps are involved: a) Substrate preparation: preparing the insoluble porous fiber substrate by wet forming, freeze drying or electrospinning; b) Immobilization of functional molecules: Immobilizing the target biomacromolecules on the substrate by coating, impregnation, co-precipitation or in-situ synthesis.

8. Use of the biomimetic composite material model according to any one of claims 1 to 6, characterized in that: It is used as a standardized research platform to simulate and study biocatalytic processes of biomacromolecules in solid-phase matrices.

9. The use according to claim 8, characterized in that: The application is to evaluate or screen biocatalysts acting on solid phase substrates.

10. The use according to claim 8, characterized in that: The application described is the evaluation of pretreatment methods to improve the accessibility of target macromolecules in solid substrates.

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