Extraction process of plant polypeptide with blood sugar regulating function

Abstract

The invention relates to the field of polypeptide extraction, and discloses a process for extracting plant polypeptide with a blood sugar regulating function, which comprises the following steps: S1, raw material pretreatment; s2, electric field assisted microwave pretreatment; s3, performing enzymolysis; s4, electric field assisted separation and purification; s5, concentrating and drying; the permeability of cell membranes and cell walls is improved through the synergistic effect of the electric field and microwaves, the release efficiency of inclusions is greatly improved, microenvironment regulation and control of the enzymatic reaction are achieved through dynamic changes of the polymer capable of responding to pressure, and the more efficient enzymatic reaction is achieved by combining initial adjustment of the microwaves and the electric field.

Description

Technical Field

[0001] The invention relates to the field of polypeptide extraction, and more specifically, to a process for extracting plant polypeptides with the function of regulating blood sugar. Background Art

[0002] There are peptides in plants that can regulate blood sugar function. For example, bitter melon peptides are active peptides extracted from bitter melon. Studies have shown that they may have a positive effect on blood sugar regulation through various mechanisms (such as promoting insulin secretion and improving insulin sensitivity).

[0003] The current plant peptide extraction process has poor effects on raw material pretreatment and incomplete plant cell disruption, resulting in low efficiency of subsequent enzymatic extraction. These problems need to be solved. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a process for extracting plant polypeptides with blood sugar regulating function.

[0005] A process for extracting plant polypeptides having the function of regulating blood sugar comprises the following steps:

[0006] S1: Raw material pretreatment;

[0007] Fresh bitter melon is sliced ​​and freeze-dried to remove moisture and improve the stability of the raw material; the freeze-dried bitter melon is ground into fine powder to increase the surface area for subsequent processing;

[0008] S2: electric field-assisted microwave pretreatment;

[0009] The bitter melon powder was suspended in a buffer solution and the mixture was treated with a pulsed electric field (PEF), which helps to destroy the cell structure and improve the efficiency of enzymatic hydrolysis;

[0010] Subsequently, microwave radiation heating is applied, which can accelerate heat transfer and improve the uniformity of reaction temperature;

[0011] S3: enzymatic hydrolysis;

[0012] By adding immobilized enzymes, under the combined action of electric field and microwave, the enzymatic reaction is enhanced, the enzymatic hydrolysis time is shortened, and the extraction efficiency and activity of polypeptides are improved.

[0013] S4: electric field assisted separation and purification;

[0014] The polypeptide extract is initially separated by electrophoresis, and then further purified by ultrafiltration to screen out polypeptides within the target molecular weight range.

[0015] S5: Concentration and drying;

[0016] The purified peptide solution is vacuum concentrated and freeze-dried to dry the peptide into powder form to ensure its activity and stability.

[0017] Preferably: in step S2, the immobilized enzymes are pepsin and trypsin immobilized on a carrier.

[0018] Preferably, the carrier is a mesoporous material with biocompatibility.

[0019] Preferably, the pore size of the mesoporous material is 2-50 nanometers.

[0020] Preferably, the mesoporous material is bio-silica or calcium phosphate based material.

[0021] Preferably, the immobilized enzyme is immobilized in the pores of the mesoporous material by physical adsorption or chemical bonding.

[0022] Preferably: the preparation process of the immobilized enzyme is as follows:

[0023] The enzyme solution is mixed with the cleaned carrier, and the enzyme is adsorbed onto the carrier through capillary action of the pores and surface attachment;

[0024] Surface modification of mesoporous materials to introduce functional groups (amino or carboxyl) that can covalently bind to specific residues (hydroxyl, amino) of enzymes;

[0025] A cross-linking agent (glutaraldehyde) was used to form covalent bonds between the enzyme and the support to increase the stability of the immobilization.

[0026] Preferably: the periphery of the carrier is coated with a pressure-responsive polymer.

[0027] Preferably: the coating process of the pressure responsive polymer is as follows:

[0028] Modification of natural polymers followed by biodegradable synthetic polymers to obtain desired mechanical response properties by adjusting their molecular weight and copolymer unit composition;

[0029] Use a spray device to evenly spray the polymer solution on the surface of the carrier;

[0030] Preferably, the natural polymer is chitosan or gelatin.

[0031] Preferably, the biodegradable synthetic polymer is polylactic-co-glycolic acid (PLGA).

[0032] The beneficial effect of the present invention is that the plant polypeptide extraction process with blood sugar regulating function proposed by the present invention improves the permeability of cell membrane and cell wall by the synergistic effect of electric field and microwave, so that the release efficiency of the contents is greatly improved.

[0033] Microwaves can directly interact with water molecules and ions in the material through intermolecular dipole interactions and ion conductivity effects, resulting in rapid oscillation of molecules inside the medium. This oscillation generates heat, which can quickly and evenly heat the reaction system. Rapid temperature rise not only speeds up the enzymatic reaction, but also reduces the degradation of thermally unstable components.

[0034] Microwave energy can change the structure and reaction kinetics of biomacromolecules, which may make enzyme molecules more active or change the accessibility of substrates, thereby improving reaction efficiency.

[0035] Electric fields (especially pulsed electric fields, PEF) can change the potential difference on the membrane of microorganisms or plants, leading to membrane pores. In this process, the permeability of the cell membrane increases, allowing more enzymes to contact the substrate, improving the efficiency of the enzymatic reaction. This mechanism also makes it easier to release soluble substances in the cell, increasing the amount of substrate that the enzyme can act on.

[0036] The electrophoretic effect induced by the electric field can cause the directional migration of charged ions or polar molecules, which can enhance the contact frequency between the substrate and the enzyme. It also helps to maintain the uniformity of the hydrogen ion concentration in the reaction system, which is beneficial to the optimal pH conditions for the enzymatic reaction.

[0037] When the electric field and microwaves work together, they can not only quickly and evenly heat the reaction system and accelerate the reaction rate, but also make the interaction between enzymes and substrates more effective by adjusting the structural environment of cells and molecules. The synergistic effect of this physical method and biochemical process significantly improves the extraction efficiency and peptide activity.

[0038] The microenvironmental regulation of enzymatic reactions is achieved through the dynamic changes of polymers that can respond to pressure. Combined with the initial regulation of microwaves and electric fields, pressure-responsive polymers can effectively regulate the openness and flexibility of the environment during operation, achieving a controllable, environmentally friendly and continuous reaction mode, and realizing more efficient enzymatic reactions.

[0039] Microwaves and electric fields are used to activate the pressure-responsive polymer before entering the reactive phase. These forms of energy cause tiny vibrations and expansions in the polymer chain segments, adjusting the opening and closing properties of some of their chemical structures.

[0040] Temperature and vibration effects: The heat and vibrational nature of the electric field generated by microwave heating keep the polymer dynamically in a certain flexible state. The polymer may undergo local expansion or morphological changes, but it will not completely close the contact channel between the enzyme and the substrate.

[0041] Protect enzyme activity: Under specific microwave and electric fields, the polymer maintains a certain permeability and semi-open state, allowing limited enzyme activity regulation and protecting its stability under adverse conditions.

[0042] Pore ​​optimization: This state allows real-time adjustment of conditions to ensure that the enzyme's active site is not affected by a sealed or hostile environment.

[0043] Mechanical stirring or introduction of ultrasound: After the initial treatment with microwaves and electric fields, the polymer is further stimulated by applying slight pressure changes such as mechanical stirring or ultrasound.

[0044] Structural changes promote reactions: Moderate deformation of pressure-responsive polymers can expand the polymer pores, increase the contact area between the substrate and the enzyme, and eliminate any unnecessary restrictions. This process allows more substrates to enter the active center of the enzyme, thereby accelerating the catalytic reaction.

[0045] Reaction regulation: The reaction speed and efficiency can be optimized by adjusting the stimulus intensity and time, and dynamic regulation can be achieved, that is, the progress of the reaction can be adjusted as needed.

[0046] Closure protection at the end: Once the reaction reaches the ideal state (sufficient product generation), stop the external force stimulation, so that the polymer structure returns to the initial state. This also limits excessive contact with the substrate and excessive reaction, and prolongs the possibility of enzyme reuse. DETAILED DESCRIPTION

[0047] The subject matter described herein will now be discussed with reference to example embodiments. It should be understood that the discussion of these embodiments is only to enable those skilled in the art to better understand and implement the subject matter described herein, and the functions and arrangements of the elements discussed may be changed without departing from the scope of protection of the contents of this specification. Each example may omit, replace or add various processes or components as needed. In addition, the features described relative to some examples may also be combined in other examples.

[0048] Example 1

[0049] In this embodiment, a plant polypeptide extraction process with blood sugar regulating function is proposed, comprising the following steps:

[0050] S1: Raw material pretreatment;

[0051] Fresh bitter melon is sliced ​​and freeze-dried to remove moisture and improve the stability of the raw material; the freeze-dried bitter melon is ground into fine powder to increase the surface area for subsequent processing;

[0052] S2: electric field-assisted microwave pretreatment;

[0053] The bitter melon powder was suspended in a buffer solution and the mixture was treated with a pulsed electric field (PEF), which helps to destroy the cell structure and improve the efficiency of enzymatic hydrolysis;

[0054] Subsequently, microwave radiation heating is applied, which can accelerate heat transfer and improve the uniformity of reaction temperature;

[0055] S3: enzymatic hydrolysis;

[0056] By adding immobilized enzymes, under the combined action of electric field and microwave, the enzymatic reaction is enhanced, the enzymatic hydrolysis time is shortened, and the extraction efficiency and activity of polypeptides are improved.

[0057] S4: electric field assisted separation and purification;

[0058] The polypeptide extract is initially separated by electrophoresis, and then further purified by ultrafiltration to screen out polypeptides within the target molecular weight range.

[0059] S5: Concentration and drying;

[0060] The purified peptide solution is vacuum concentrated and freeze-dried to dry the peptide into powder form to ensure its activity and stability.

[0061] in:

[0062] In step S2, the immobilized enzymes are pepsin and trypsin immobilized on a carrier.

[0063] The carrier is a mesoporous material with biocompatibility.

[0064] The pore size of the mesoporous material can be selected to be 2, 10, 20, 30, 40 or 50 nanometers, and is preferably 30 nanometers in the present embodiment.

[0065] The mesoporous material may be bio-silicon or calcium phosphate-based material; bio-silicon is preferred in this embodiment.

[0066] Immobilized enzymes are enzymes that are fixed in the pores of mesoporous materials by physical adsorption or chemical bonding.

[0067] The preparation process of immobilized enzyme is as follows:

[0068] The enzyme solution is mixed with the cleaned carrier, and the enzyme is adsorbed onto the carrier through capillary action of the pores and surface attachment;

[0069] Surface modification of mesoporous materials to introduce functional groups (amino or carboxyl) that can covalently bind to specific residues (hydroxyl, amino) of enzymes;

[0070] A cross-linking agent (glutaraldehyde) was used to form covalent bonds between the enzyme and the support to increase the stability of the immobilization.

[0071] The periphery of the carrier is coated with a pressure-responsive polymer, and the coating process of the pressure-responsive polymer is as follows:

[0072] Modification of natural polymers followed by biodegradable synthetic polymers to obtain desired mechanical response properties by adjusting their molecular weight and copolymer unit composition;

[0073] Use a spray device to evenly spray the polymer solution on the surface of the carrier;

[0074] The natural polymer is chitosan or gelatin; chitosan is preferred in this embodiment.

[0075] The biodegradable synthetic polymer was polylactic-co-glycolic acid (PLGA).

[0076] Example 2

[0077] This example is used to study the effect of the synergistic effect of microwaves and electric fields in the immobilized enzyme system. By using biosilicon (SiO2) as a carrier to immobilize the enzyme and chitosan as a pressure-responsive polymer coating, the positive effects of microwave and electric field pretreatment on enzyme activity and reaction efficiency are explored. The activation characteristics of microwaves and electric fields acting on polymers can promote the optimization of polymer structure, enhance the substrate accessibility of enzymes, and thus improve the overall enzymatic reaction efficiency.

[0078] 1. Experimental steps

[0079] 1. Materials and reagents

[0080] Enzyme (trypsin);

[0081] Bio-silicon (SiO2 nanoparticles, pore size about 10 nanometers);

[0082] Chitosan (molecular weight about 50,000);

[0083] Microwave instruments and electric field generators;

[0084] Phosphate buffered saline (PBS);

[0085] Glutaraldehyde.

[0086] 2. Preparation of immobilized enzyme

[0087] 2.1. Bio-silicon pretreatment:

[0088] The SiO2 nanoparticles were dispersed in distilled water by ultrasonic.

[0089] Drying by evaporation method gives dry cluster particles.

[0090] 2.2. Trypsin immobilization:

[0091] Prepare enzyme solution (2 mg / mL) in PBS.

[0092] Add the biosilica particles to the enzyme solution and mix by shaking at room temperature for 12 hours. Remove the unfixed enzyme by centrifugation and wash with PBS until the eluate has no enzyme activity.

[0093] 2.3, Chemical bonding:

[0094] Treat with 0.1% glutaraldehyde solution to form a covalent connection between the enzyme and the carrier, preventing the enzyme from falling off and ensuring its stability.

[0095] 3. Preparation of chitosan coating

[0096] 3.1. Chitosan solution:

[0097] Dissolve 1% chitosan in 1% acetic acid solution and stir well.

[0098] The bio-silicon particles with immobilized enzymes were added thereto and mixed for 1 hour.

[0099] 3.2 Coating formation:

[0100] The mixture was centrifuged to remove excess chitosan.

[0101] The mixture was dried at 65°C to form a solid coating.

[0102] 4. Microwave and electric field treatment

[0103] 4.1. Sample pretreatment:

[0104] The bio-silica-enzyme-chitosan composite material was placed in a microwave instrument.

[0105] The samples were treated at a microwave frequency of 2.45 GHz and a power of 50 W for 2 minutes.

[0106] 4.2. Electric field effect:

[0107] A voltage of 1 kV / cm was applied using an electric field generator for 1 minute.

[0108] Comparison group:

[0109] Untreated bio-silica-enzyme-chitosan composite material was used as a control.

[0110] 2. Experimental Results

[0111] Group Processing conditions Enzyme activity (U / mg) Control group No microwaves and electric fields 80 Microwave treatment Microwave for 2 minutes without electric field 95 Electric field treatment Electric field for 1 minute, no microwave 90 Microwave + electric field Microwave 2 minutes, electric field 1 minute 110

[0112] 3. Analysis and Summary

[0113] Microwave treatment alone can significantly enhance enzyme activity, which may be due to the uniform microwave heating and non-thermal activation effects.

[0114] The electric field treatment also enhanced the enzyme activity, possibly by temporarily modifying the chitosan coating structure. In addition, the electric field may have improved the diffusion efficiency of the substrate.

[0115] The synergistic effect of microwave and electric field was the most significant, and the enzyme activity increased to the highest level. This means that the simultaneous application of the two produced a superimposed effect, optimizing the structure of the chitosan polymer to allow for more complete contact with the substrate.

[0116] This experiment verified the positive effect of microwave and electric field synergy on enzymatic reactions, especially the promotion effect through the structural adjustment of pressure-responsive chitosan. In production applications, by optimizing the combination of microwave and electric field conditions, the catalytic ability of enzymes can be effectively protected and enhanced.

[0117] The above describes an embodiment of the present embodiment, but the present embodiment is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present embodiment, ordinary technicians in this field can also make many forms, all of which are within the protection of the present embodiment.

Claims

1. A process for extracting plant polypeptides having the function of regulating blood sugar, characterized in that: The steps include: S1: Raw material pretreatment; Fresh bitter melon is sliced ​​and freeze-dried to remove moisture and improve the stability of the raw material; the freeze-dried bitter melon is ground into fine powder to increase the surface area for subsequent processing; S2: electric field-assisted microwave pretreatment; The bitter melon powder is suspended in a buffer solution and the mixture is treated with a pulsed electric field, which helps to destroy the cell structure and improve the efficiency of enzymatic hydrolysis; Subsequently, microwave radiation heating is applied, which can accelerate heat transfer and improve the uniformity of reaction temperature; S3: enzymatic hydrolysis; By adding immobilized enzymes, under the combined action of electric field and microwave, the enzymatic reaction is strengthened, the enzymatic hydrolysis time is shortened, and the extraction efficiency and activity of polypeptides are improved; S4: electric field assisted separation and purification; The peptide extract is initially separated by electrophoresis, and then further purified by ultrafiltration to screen out peptides within the target molecular weight range; S5: Concentration and drying; The purified peptide solution is vacuum concentrated and freeze-dried to dry the peptide into powder form to ensure its activity and stability.

2. The process for extracting plant polypeptides having the function of regulating blood sugar according to claim 1, characterized in that: In step S2, the immobilized enzymes are pepsin and trypsin immobilized on a carrier.

3. The process for extracting plant polypeptides having the function of regulating blood sugar according to claim 2, characterized in that: The carrier is a mesoporous material with biocompatibility.

4. The process for extracting plant polypeptides having the function of regulating blood sugar according to claim 3, characterized in that: The pore size of mesoporous materials is 2-50 nanometers.

5. The process for extracting plant polypeptides having the function of regulating blood sugar according to claim 4, characterized in that: Mesoporous materials are biogenic silica or calcium phosphate based materials.

6. The process for extracting plant polypeptides having the function of regulating blood sugar according to claim 5, characterized in that: Immobilized enzymes are enzymes that are fixed in the pores of mesoporous materials by physical adsorption or chemical bonding.

7. The process for extracting plant polypeptides having the function of regulating blood sugar according to claim 6, characterized in that: The preparation process of immobilized enzyme is as follows: The enzyme solution is mixed with the cleaned carrier, and the enzyme is adsorbed onto the carrier through capillary action of the pores and surface attachment; Surface modification of mesoporous materials to introduce functional groups capable of covalently binding to specific residues of enzymes; Cross-linking agents are used to form covalent bonds between the enzyme and the support to increase the stability of immobilization.

8. The process for extracting plant polypeptides having the function of regulating blood sugar according to claim 7, characterized in that: The periphery of the carrier is coated with a pressure-responsive polymer.

9. The process for extracting plant polypeptides having the function of regulating blood sugar according to claim 8, characterized in that: The coating process of the pressure-responsive polymer is as follows: Modification of natural polymers followed by biodegradable synthetic polymers to obtain desired mechanical response properties by adjusting their molecular weight and copolymer unit composition; Use a spray device to evenly spray the polymer solution on the surface of the carrier; Preferably, the natural polymer is chitosan or gelatin.

10. The process for extracting plant polypeptides having the function of regulating blood sugar according to claim 9, characterized in that: The biodegradable synthetic polymer is polylactic-co-glycolic acid.