Process for extracting cereal polypeptide for enhancing immunity
By using sodium alginate gel carrier to wrap the enzyme and increase the outer stable layer, the problem of enzyme inactivation during the enzymatic extraction of cereal protein polypeptides is solved, the stability of the enzyme and the efficiency of peptide extraction are improved, and the production cost is reduced.
Patent Information
- Application Number
- CN202411791313.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-04-11
AI Technical Summary
During the enzymatic extraction of cereal protein polypeptides, the enzyme is prone to inactivate under strong stirring or dynamic fluid conditions, resulting in a decrease in reaction efficiency and limited durability and reuse ability of immobilized enzyme particles.
Sodium alginate is used as the enzyme immobilization carrier, and the enzyme is wrapped in sodium alginate gel particles through the gelation process, and materials such as polylactic acid-glycolic acid copolymer (PLGA) and crosslinking agent (glutaraldehyde) are added to the outer layer to improve mechanical strength.
It improves the activity stability and reusability of enzymes, reduces the frequency of replacement of immobilized enzyme particles, improves the efficiency and yield of peptides, and reduces production costs.
Smart Images
Figure CN119799827A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of polypeptide extraction, and more specifically to a process for extracting immunity-enhancing cereal polypeptides. Background Art
[0002] Cereals are not only rich in starch and other substances, but also contain proteins that can enhance immunity. These proteins need to be enzymatically hydrolyzed to produce peptides that can be absorbed by the human body.
[0003] When enzymatically extracting such grain protein peptides, immobilized enzyme technology is widely used in various biocatalytic reactions to improve enzyme stability, reusability and production efficiency. However, in actual processes, this method faces the following challenges: enzyme inactivation and reduced reaction efficiency under strong stirring or dynamic fluid conditions. The durability and reusability of particles are limited due to mechanical damage and enzyme activity attenuation. Summary of the invention
[0004] In order to solve the above technical problems, the present invention provides a process for extracting cereal polypeptides for enhancing immunity.
[0005] A process for extracting grain polypeptides for enhancing immunity comprises the following steps:
[0006] S1: Raw material preparation and processing;
[0007] Dissolve wheat flour in water, adjust the pH to alkaline to increase the solubility of the protein, and then separate the soluble protein components by high centrifugal force, and collect the supernatant;
[0008] S2: enzymatic hydrolysis;
[0009] Immobilized enzymes were added into the supernatant one by one for enzymatic hydrolysis;
[0010] S3: peptide extraction and separation;
[0011] After the enzymatic hydrolysis is completed, the mixed system is centrifuged and membrane filtered to remove macromolecules and insoluble matter;
[0012] S4: purification;
[0013] The solution containing the polypeptide is purified to obtain a targeted polypeptide with immunity enhancement effect.
[0014] Preferably: in step S2, the immobilized enzyme is an enzyme fixed on a carrier, and the enzyme includes pepsin, trypsin and alkaline protease.
[0015] Pepsin: It has good initial deconstruction effect under weak acidic conditions (pH 3-4).
[0016] Trypsin: It can further hydrolyze proteins and increase the breakage of polypeptide chains under neutral to slightly alkaline conditions (pH 7-8).
[0017] Alkaline protease: Applicable under alkaline conditions (pH 8-10) and used to overcome protein parts that are difficult to hydrolyze.
[0018] Preferably, in step S2, the mixture is first treated with pepsin, and then deeply hydrolyzed with trypsin and alkaline protease respectively.
[0019] Preferably: the preparation process of the immobilized enzyme is as follows:
[0020] S21: preparing each monomer enzyme solution;
[0021] Prepare solutions of pepsin, trypsin and alkaline protease respectively;
[0022] S22: preparing sodium alginate solution;
[0023] Dissolve sodium alginate in deionized water to form a uniform solution;
[0024] S23: enzyme solution mixing;
[0025] Slowly add the prepared enzyme mixture solution to the sodium alginate solution to ensure even distribution;
[0026] S24: gelation;
[0027] Using the titration method, the mixed solution is added dropwise into the calcium chloride solution to cause a cross-linking reaction to form gel particles. During this process, the enzyme is encapsulated inside the gel;
[0028] S25: curing and washing;
[0029] The generated sodium alginate gel particles are solidified and washed with a buffer solution to remove excess surface residual substances and uncaptured enzyme molecules;
[0030] S26: Add outer stabilization layer;
[0031] A stabilizing layer is wrapped around the sodium alginate gel particles to improve the mechanical strength.
[0032] Preferably, in step S26, polylactic acid-glycolic acid copolymer (PLGA) is used to spray the sodium alginate gel particles to form a stabilizing layer on the sodium alginate gel particles.
[0033] Preferably, during the spraying process, a cross-linking agent (glutaraldehyde) is added to strengthen the bonding strength of the coating.
[0034] Preferably: in step S23, nanocellulose or graphene is added to the sodium alginate solution to enhance the filler and improve the physical strength of the gel.
[0035] Sodium alginate gel as an enzyme immobilization carrier can ensure the activity of the enzyme and facilitate recovery, mainly based on its chemical structure and physical properties. The following is a detailed description:
[0036] Chemical composition and structure:
[0037] Sodium alginate is a natural polysaccharide polymer, mainly composed of two units, mannuronic acid (M-block) and guluronic acid (G-block), connected by β-1,4-glycosidic bonds.
[0038] These sugar acid groups are highly hydrophilic, making them fully soluble in water and forming stable solutions.
[0039] Calcium ion cross-linking:
[0040] When the sodium alginate solution comes into contact with a solution containing calcium ions (such as calcium chloride), the calcium ions replace the sodium ions between the alginate chains, and the G-block and the calcium ions coordinate with each other to form a network structure similar to an "egg-box model".
[0041] This structure is stable and forms insoluble gel particles, providing a physical space for enzyme binding.
[0042] Principle of ensuring enzyme activity
[0043] Mild immobilization conditions:
[0044] The gelation process requires only neutral conditions and is carried out at room temperature. It does not involve violent chemical reactions or extreme pH values, and therefore has little effect on the conformation and active sites of the enzyme.
[0045] Avoid protein denaturation:
[0046] The mesh structure provides a microporous environment that keeps the enzyme molecules in a hydrated state while avoiding excessive mechanical stress, thereby reducing the risk of inactivation.
[0047] Biocompatibility:
[0048] As a material of natural origin, sodium alginate has good biocompatibility and does not produce precipitation or reaction by-products that adversely affect enzyme activity.
[0049] Principle of easy recycling
[0050] Gel granulation morphology:
[0051] The immobilized enzyme exists in the reaction medium in the form of particles, which are relatively large and can be easily separated and recovered by simple filtration or sedimentation methods.
[0052] Reversibility of AC Dissociation:
[0053] When recovery or regeneration is required, calcium alginate gel can be depolymerized into sodium alginate solution by complexing calcium ions with a chelating agent (such as sodium citrate), which greatly simplifies the enzyme recovery process.
[0054] Reusability:
[0055] Multiple operational experiments have shown that calcium alginate gel can be reused multiple times under appropriate conditions, and its structural reversibility and stable chemical properties ensure this.
[0056] In summary, sodium alginate effectively ensures the activity of the enzyme and the recovery and treatment after the reaction through its chemical structure and gel formation mechanism.
[0057] The beneficial effects of the present invention are as follows: the present invention proposes a grain polypeptide extraction process for enhancing immunity, which adopts a combined enzymatic hydrolysis treatment method with high enzymatic hydrolysis efficiency, improves the utilization rate of the enzyme by means of immobilized enzymes, and effectively improves the mechanical strength of the carrier by treating the carrier, improves the durability, and can reduce the frequency of replacement of immobilized enzyme particles.
[0058] The outer polymer coating and gel modification of sodium alginate gel particles enable the particles to resist mechanical impact and stirring wear. By improving the diffusion characteristics of the particles to the substrate, the enzyme activity is guaranteed and the reaction speed is promoted. The stable structure ensures that the particles have good effects after repeated use, reducing the frequency of replacement and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS
[0059] Figure 1 is a bar graph of the grinding rates of each group of particles in Example 2 of the present invention;
[0060] Figure 2 It is a line graph of the polypeptide yields of each group in Example 2 of the present invention. DETAILED DESCRIPTION
[0061] 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.
[0062] Example 1
[0063] In this embodiment, a process for extracting cereal polypeptides for enhancing immunity is proposed, comprising the following steps:
[0064] S1: Raw material preparation and processing;
[0065] Dissolve wheat flour in water, adjust the pH to alkaline to increase the solubility of the protein, and then separate the soluble protein components by high centrifugal force, and collect the supernatant;
[0066] S2: enzymatic hydrolysis;
[0067] Immobilized enzymes were added into the supernatant one by one for enzymatic hydrolysis;
[0068] S3: peptide extraction and separation;
[0069] After the enzymatic hydrolysis is completed, the mixed system is centrifuged and membrane filtered to remove macromolecules and insoluble matter;
[0070] S4: purification;
[0071] The solution containing the polypeptide is purified to obtain a targeted polypeptide with immunity enhancement effect.
[0072] Preferably: in step S2, the immobilized enzyme is an enzyme fixed on a carrier, and the enzyme includes pepsin, trypsin and alkaline protease.
[0073] Pepsin: It has good initial deconstruction effect under weak acidic conditions (pH 3-4).
[0074] Trypsin: It can further hydrolyze proteins and increase the breakage of polypeptide chains under neutral to slightly alkaline conditions (pH 7-8).
[0075] Alkaline protease: Applicable under alkaline conditions (pH 8-10) and used to overcome protein parts that are difficult to hydrolyze.
[0076] In step S2, the protein is first treated with pepsin, and then subjected to deep hydrolysis with trypsin and alkaline protease, respectively.
[0077] The preparation process of immobilized enzyme is as follows:
[0078] S21: preparing each monomer enzyme solution;
[0079] Prepare solutions of pepsin, trypsin and alkaline protease respectively;
[0080] S22: preparing sodium alginate solution;
[0081] Dissolve sodium alginate in deionized water to form a uniform solution;
[0082] S23: enzyme solution mixing;
[0083] Slowly add the prepared enzyme mixture solution to the sodium alginate solution to ensure even distribution;
[0084] S24: gelation;
[0085] Using the titration method, the mixed solution is added dropwise into the calcium chloride solution to cause a cross-linking reaction to form gel particles. During this process, the enzyme is encapsulated inside the gel;
[0086] S25: curing and washing;
[0087] The generated sodium alginate gel particles are solidified and washed with a buffer solution to remove excess surface residual substances and uncaptured enzyme molecules;
[0088] S26: Add outer stabilization layer;
[0089] A stabilizing layer is wrapped around the sodium alginate gel particles to improve the mechanical strength.
[0090] In step S26, polylactic acid-glycolic acid copolymer (PLGA) is used to spray the sodium alginate gel particles to form a stabilizing layer on the sodium alginate gel particles.
[0091] During the spraying process, a crosslinking agent (glutaraldehyde) is added to strengthen the bonding strength of the coating.
[0092] In step S23, nanocellulose or graphene is added to the sodium alginate solution to enhance the filler and improve the physical strength of the gel.
[0093] Example 2
[0094] Sodium alginate gel as an enzyme immobilization carrier has the problem of insufficient mechanical strength. By applying an additional stabilizing layer and adding nanomaterials (nanocellulose or graphene) to the outer layer of the gel particles for modification, the mechanical strength and stability of the gel can be significantly improved. This example verifies the effectiveness of these modification methods through comparative experiments.
[0095] 1. Experimental Grouping
[0096] Group A: untreated sodium alginate gel, serving as the control group.
[0097] Group B: An additional polymer stabilizing layer was added to the outer layer of sodium alginate gel.
[0098] Group C: Nanocellulose or graphene was added to the sodium alginate gel, and a stabilizing layer was added to the outer layer.
[0099] 2. Experimental steps
[0100] 1. Preparation of sodium alginate gel particles (Group A)
[0101] 1.1. Dissolve sodium alginate in deionized water to prepare a 2% w / v solution.
[0102] 1.2. Drop the solution into 0.1M calcium chloride solution to form gel particles.
[0103] 1.3. Solidify and wash the particles.
[0104] 2. Add a stabilization layer (Group B)
[0105] 2.1. Prepare gel particles according to step 1.
[0106] 2.2. Dissolve the polymer (PLGA) in ethyl acetate to form a coating solution.
[0107] 2.3. The gel particles are immersed in the coating solution, and then the outer layer is solidified by evaporation of ethyl acetate.
[0108] 3. Modification and adding stabilization layer (Group C)
[0109] 3.1. Add 1% w / v nanocellulose / graphene to the sodium alginate solution and mix well.
[0110] 3.2. Repeat step 1 to prepare nano-modified gel particles.
[0111] 3.3 Repeat step 2 to add a polymer outer layer to the particles.
[0112] 4. Mechanical strength test
[0113] Each group of gel particles was placed in a stirred reactor to simulate stirring under industrial conditions.
[0114] Particle integrity was recorded after 24 hours and particle attrition rate was analyzed.
[0115] 5. Reaction performance test
[0116] The same amount of enzyme and substrate (wheat protein extract) were added to each group, and enzymatic hydrolysis was carried out under constant temperature conditions.
[0117] The peptide yield was quantitatively determined by high performance liquid chromatography (HPLC).
[0118] 3. Experimental Results
[0119] The mechanical strength test results of each group are shown in the attached Figure 1 The experimental results of peptide yield in each group are shown in the attached Figure 2 shown.
[0120] 4. Analysis and Summary
[0121] Mechanical strength:
[0122] The particle wear rate of groups B and C was significantly reduced by surface stabilization layer and nanomaterial modification, indicating that the modification measures effectively improved the mechanical stability.
[0123] Reaction efficiency:
[0124] The peptide yield of group C was significantly higher than that of the other two groups, indicating that the introduction of nanocellulose / graphene not only improved the mechanical strength but also may have optimized the substrate diffusion capacity.
[0125] in conclusion:
[0126] Experiments have shown that adding an additional stabilizing layer and performing chemical or physical modifications on sodium alginate gel particles can significantly improve their mechanical strength, reduce wear, and increase the final reaction efficiency and peptide yield.
[0127] 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 cereal polypeptides for enhancing immunity, characterized in that: The steps include: S1: Raw material preparation and processing; Dissolve wheat flour in water, adjust the pH to alkaline to increase the solubility of the protein, and then separate the soluble protein components by high centrifugal force, and collect the supernatant; S2: enzymatic hydrolysis; Immobilized enzymes were added into the supernatant one by one for enzymatic hydrolysis; S3: peptide extraction and separation; After the enzymatic hydrolysis is completed, the mixed system is centrifuged and membrane filtered to remove macromolecules and insoluble matter; S4: purification; The solution containing the polypeptide is purified to obtain a targeted polypeptide with immunity enhancement effect.
2. The process for extracting a cereal polypeptide for enhancing immunity according to claim 1, characterized in that: In step S2, the immobilized enzyme is to immobilize the enzyme on a carrier, and the enzyme includes pepsin, trypsin and alkaline protease.
3. The process for extracting a cereal polypeptide for enhancing immunity according to claim 2, characterized in that: In step S2, the protein is first treated with pepsin, and then subjected to deep hydrolysis with trypsin and alkaline protease, respectively.
4. The process for extracting a cereal polypeptide for enhancing immunity according to claim 3, characterized in that: The preparation process of immobilized enzyme is as follows: S21: preparing each monomer enzyme solution; Prepare solutions of pepsin, trypsin and alkaline protease respectively; S22: preparing sodium alginate solution; Dissolve sodium alginate in deionized water to form a uniform solution; S23: enzyme solution mixing; Slowly add the prepared enzyme mixture solution to the sodium alginate solution to ensure even distribution; S24: gelation; Using the titration method, the mixed solution is added dropwise into the calcium chloride solution to cause a cross-linking reaction to form gel particles. During this process, the enzyme is encapsulated inside the gel; S25: curing and washing; The generated sodium alginate gel particles are solidified and washed with a buffer solution to remove excess surface residual substances and uncaptured enzyme molecules; S26: Add outer stabilization layer; A stabilizing layer is wrapped around the sodium alginate gel particles to improve the mechanical strength.
5. The process for extracting a cereal polypeptide for enhancing immunity according to claim 4, characterized in that: In step S26, the sodium alginate gel particles are sprayed with polylactic acid-glycolic acid copolymer to form a stabilizing layer on the sodium alginate gel particles.
6. The process for extracting cereal polypeptides for enhancing immunity according to claim 5, characterized in that: During the spraying process, a cross-linking agent is added to strengthen the bonding strength of the coating.
7. The process for extracting cereal polypeptides for enhancing immunity according to claim 4, characterized in that: In step S23, nanocellulose or graphene is added to the sodium alginate solution to enhance the filler and improve the physical strength of the gel.