Immunopotentiator containing high-activity microbial protein peptide and preparation method of immunopotentiator

Abstract

The invention relates to the technical field of biological pharmacy, in particular to an immunopotentiator containing high-activity microbial protein peptide and a preparation method of the immunopotentiator. The immunopotentiator containing the high-activity microbial protein peptide comprises the following raw materials: beta-glucan, composite immune peptide, a freeze-drying protective agent, a metal organic framework and the balance of a stabilizer. The preparation method comprises the following steps: firstly, constructing an immune activation core skeleton by using beta-glucan, and activating a phosphorylation pathway by using Kluyveromyces marxianus source peptide in the composite immune peptide; the silkworm chrysalis protein peptide is in targeted combination with a receptor compound, so that the phagocytic rate of macrophages is increased; the recombinant lactoferrin peptide destroys a lipid bilayer of a microbial cell membrane through a cationic structural domain, activates a TLR4 pathway to promote lymphocyte differentiation, and ensures that the systematic immune defense efficacy is improved; specific antibacterial protein contained in the Fusarium brachypodium FXFB001 fermentation active protein extract can directly destroy a pathogen cell membrane structure and activate a TLR2 / 4 pathway to enhance innate immune response at the same time.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceuticals, in particular, to an immune enhancer containing high-activity microbial protein peptides and a preparation method thereof. BACKGROUND

[0002] Biopharmaceuticals refers to the use of organisms, biological tissues or their components, combined with genetic engineering, cell engineering and other modern biological technologies, to manufacture drugs for disease prevention, diagnosis and treatment. Moreover, biopharmaceuticals produced by genetic engineering, cell culture and other biological technologies have the characteristics of high pharmacological activity and small toxic and side effects, and are widely used in the treatment of diseases such as cancer, AIDS and diabetes.

[0003] The immune enhancer belongs to biopharmaceuticals, and biological macromolecules such as cytokines, antibodies and vaccines produced by biological technology methods directly act on the human immune system to enhance its function, such as the preparation method of a microbial immune enhancer in CN109091505A, using Saccharomyces cerevisiae as raw material, adding sodium chloride for autolysis induction, using isopropyl alcohol, acetone organic solvent and serratia peptidase for treatment, which can remove the remaining lipid and mannose protein, and obtain a purified immune enhancer matrix, and the obtained product will not pollute the water body; adding branched chain amino acids such as isoleucine and arginine can not only directly kill microorganisms but also regulate immune function, and after adding, in the intestinal epithelium, endogenous antibacterial peptides secreted to the surface and immunoglobulin A jointly play the immune barrier function and carry out immune regulation.

[0004] The problem is that in the above preparation method, the purification process excessively depends on organic solvent treatment and serratia peptidase degradation, which can destroy the key active substances (such as β-1, 3-glucan) in the cell wall of Saccharomyces cerevisiae with immune stimulating effect, resulting in lack of effective components for directly promoting T / B lymphocyte proliferation and differentiation or activating the surface pattern recognition receptors (such as TLR and Dectin-1) of macrophages, and the immune enhancement effect is not ideal. SUMMARY

[0005] The purpose of the present application is to ensure that the immune enhancer has effective components for promoting cell proliferation and differentiation, thereby improving the immune enhancement effect of the enhancer.

[0006] The purpose of the present application is to provide an immune enhancer containing high-activity microbial protein peptides and a preparation method thereof, by retaining the intact active structure of β-glucan and introducing high-efficiency immune peptides, avoiding the destruction of immune active substances by organic solvents, thereby ensuring the immune enhancement effect.

[0007] To achieve the above purpose, one of the purposes of the present application is to provide an immune enhancer containing high-activity microbial protein peptides, which comprises the following raw materials in mass ratio: beta-glucan 25-30%, complex immunopeptide 5-10%, freeze-drying protective agent 3-6%, metal organic framework 38-44%, and the rest is stabilizer; The complex immunopeptide comprises Kluyveromyces marxianus-derived peptide, silkworm chrysalis protein peptide, recombinant lactoferrin peptide and Fusarium oxysporum f. sp. cubense FXFB001 fermentation active extract, and the mass ratio of the Kluyveromyces marxianus-derived peptide, the silkworm chrysalis protein peptide, the recombinant lactoferrin peptide and the Fusarium oxysporum f. sp. cubense FXFB001 fermentation active protein extract is 3:2:2:3.

[0008] As a further improvement of the technical solution, the freeze-drying protective agent is mannitol.

[0009] As a further improvement of the technical solution, the metal organic framework comprises Mn-MOF-74 and ZIF-8, and the mass ratio of the Mn-MOF-74 and the ZIF-8 is 3:2.

[0010] As a further improvement of the technical solution, the stabilizer comprises citrate and ascorbyl palmitate, and the mass ratio of the citrate and the ascorbyl palmitate is 1:1.

[0011] The second object of the present application is also to provide a preparation method for preparing the immunopotentiator containing high-activity microbial protein peptides. Step S1: waste beer yeast is suspended in a pH 7.4 phosphate buffer at a concentration of 10%, pre-cooled at 4 DEG C, then treated by ultrasonic, and then precipitated by centrifugation and column chromatography to obtain beta-glucan; Step S2: 3% Kluyveromyces marxianus JY-1 is inoculated in a 5% bovine milk casein solution, and then hydrolyzed to obtain Kluyveromyces marxianus-derived peptide; Defatted silkworm chrysalis protein is dissolved in deionized water, the pH is adjusted to 8.0, alkaline protease is added, and hydrolysis is carried out at 50 DEG C for 2-4 h; after inactivation by boiling water, the supernatant is obtained by centrifugation to obtain silkworm chrysalis protein peptide; The bovine lactoferrin peptide gene is cloned into the Pichia pastoris expression vector pPIC9K, and then electrotransformed into the X-33 host bacteria; the fermentation broth is obtained by methanol induction during the shake flask fermentation stage; and the recombinant lactoferrin peptide is obtained by cation exchange chromatography purification of the fermentation broth; Fusarium oxysporum f. sp. cubense FXFB001 is inoculated in PDB culture medium, and then fermented at 28 DEG C with 180 rpm oscillation for 6 days; the supernatant is obtained by ultrasonic crushing and centrifugation of the fermentation broth; the 40-60% saturation precipitation component is obtained by ammonium sulfate fractionation precipitation method; and the Fusarium oxysporum f. sp. cubense FXFB001 fermentation active protein extract with a molecular weight of 15-25 kDa is obtained by DEAE ion exchange chromatography and Sephadex G-75 gel chromatography purification. Step S3: the above-mentioned Kluyveromyces marxianus-derived peptide, silkworm chrysalis protein peptide, recombinant lactoferrin peptide and the active protein extract of Fusarium oxysporum FXFB001 are mixed in a mass ratio of 3:2:2:3 to obtain a composite immune peptide, and then the beta-glucan is mixed with the composite immune peptide to form an electrostatic complex; Then, the electrostatic complex is dispersed in a 2-methyl imidazole zinc salt solution to obtain a complex solution; The complex solution is reacted at room temperature for 1h to form a ZIF-8 primary coating layer, and then is transferred into a manganese acetate / 2,5-dihydroxyterephthalic acid solution to react for 2h to form an outer layer of Mn-MOF-74, thereby constructing a metal organic framework; Step S4: a freeze-drying protective agent and a stabilizer are further added to the complex solution, and the solution is subjected to sterilization filtration treatment to obtain an immune enhancer.

[0012] As a further improvement of the technical solution, in step S1, the ultrasonic treatment is performed by using a 15-25 kHz amplitude horn ultrasonic system, and the yeast particle size is reduced to 1.47-2.07 μm by treating in an ice bath for 10-30 min. Then, NaOH is added to a final concentration of 2.0 M, and the solution is stirred at 60℃ for 2h. After centrifugation, the precipitate is neutralized to neutral in dilute hydrochloric acid, and then subjected to Sephacryl S-400 column chromatography. The 45-65 kDa fraction is collected and freeze-dried to obtain beta-glucan.

[0013] As a further improvement of the technical solution, in step S2, the hydrolysis treatment is performed by inoculating Kluyveromyces marxianus JY-1, and then hydrolyzing at pH 6.5 and 37℃ for 18-30h. The peptide fragments with a molecular weight of less than 3 kDa are removed by ultrafiltration to obtain Kluyveromyces marxianus-derived peptides.

[0014] As a further improvement of the technical solution, in step S2, the methanol induction time is 84-108h.

[0015] As a further improvement of the technical solution, in step S3, the mixing is performed at pH 5.8 and 40℃ for 1-3h to obtain an electrostatic complex.

[0016] As a further improvement of the technical solution, in step S4, the sterilization filtration treatment is performed by sterilization filtration through a 0.22 μm filter, pre-freezing at-50℃, and then vacuum freeze-drying to obtain an immune enhancer.

[0017] In the present application, the following raw materials are included: Beta-glucan, composite immune peptide, freeze-drying protective agent, metal organic framework and stabilizer, wherein: The natural helical conformation of the beta-glucan is retained as an immune activation core framework, triggering a signal pathway by specific binding to the macrophage surface receptor, inducing pro-inflammatory cytokine secretion; In the complex immune peptide, the Kluyveromyces marxianus-derived peptide activates the macrophage phosphorylation pathway; the silkworm chrysalis protein peptide targets the receptor complex to enhance the nuclear translocation efficiency and improve the phagocytosis rate of macrophages; the recombinant lactoferrin peptide destroys the microbial cell membrane lipid bilayer while activating the macrophage TLR4 pathway and promoting lymphocyte differentiation; the specific antibacterial protein contained in the fermentation active protein extract of Fusarium oxysporum f. sp. cubense FXFB001 can directly destroy the cell membrane structure of pathogens, and at the same time, enhance the innate immune response by activating the TLR2 / 4 pathway; In the metal organic framework, the inner layer of ZIF-8 maintains structural integrity at physiological pH, protecting the activity of the immune peptide through confinement effect; the outer layer of Mn-MOF-74 dissociates in response to the acidic environment of lysosomes, releasing Mn²⁺, which acts as an endogenous activator to drive type I interferon synthesis, while Zn²⁺ penetrates into the interstitial space of lymphocytes to promote proliferation and differentiation. The freeze-drying protectant is used to prevent activity loss, and the stabilizer is used to inhibit oxidative damage caused by reactive oxygen species.

[0018] Compared with the prior art, the present application has the following beneficial effects: In the immune enhancer containing high-activity microbial protein peptides and the preparation method thereof, first, a beta-glucan is used to construct an immune activation core framework, which retains a helical conformation that specifically anchors the macrophage surface Dectin-1 receptor, triggers a Syk-CARD9-NF-κB signal cascade, and initiates pro-inflammatory cytokine secretion; then, in the complex immune peptide, the Kluyveromyces marxianus-derived peptide (containing HPHPHLSF / PFPEVFG sequence) activates the MAPK-p38 phosphorylation pathway; the silkworm chrysalis protein peptide (SEQ ID NO. 2 / 3 / 8) targets the TLR2 / TLR4-MD-2 receptor complex, enhances the NF-κB nuclear translocation efficiency, and improves the phagocytosis rate of macrophages; the recombinant lactoferrin peptide destroys the microbial cell membrane lipid bilayer through the cationic domain, while activating the TLR4 pathway to promote lymphocyte differentiation; the specific antibacterial protein contained in the fermentation active protein extract of Fusarium oxysporum f. sp. cubense FXFB001 can directly destroy the cell membrane structure of pathogens, and at the same time, enhance the innate immune response by activating the TLR2 / 4 pathway; Further, the inner layer of ZIF-8 protects the active structure of the immune peptide at physiological pH through confinement effect; the outer layer of Mn-MOF-74 dissociates in response to the acidic environment of lysosomes, releasing Mn²⁺ that activates the cGAS-STING pathway to drive type I interferon synthesis, while Zn²⁺ penetrates into the interstitial space of lymphocytes to enhance metabolic proliferation; finally, the stabilizer maintains functional integrity, ensuring improved systemic immune defense efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Flow chart of the present application. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0021] One of the purposes of the embodiments of the present application is to provide an immune enhancer containing high-activity microbial protein peptides, which comprises the following raw materials in terms of mass ratio: β-glucan 25-30%, complex immune peptide 5-10%, freeze-drying protective agent 3-6%, metal organic framework 38-44%, and the balance is a stabilizer; The complex immune peptide comprises a Kluyveromyces marxianus-derived peptide (the Kluyveromyces marxianus-derived peptide is a specific immune-regulating peptide produced by hydrolyzing casein in cow milk with Kluyveromyces marxianus JY-1 strain, and the core active sequence (including HPHPHLSF, PFPEVFG, etc.) is proved by experiments to be able to significantly promote the proliferation of macrophages and enhance the secretion of cytokines such as IL-6 and TNF-α, thereby activating systemic immune response), silkworm chrysalis protein peptide (the silkworm chrysalis protein peptide is a specific immune-regulating peptide obtained by directional enzymatic hydrolysis of defatted silkworm chrysalis protein with alkaline protease, and the core sequence (such as SEQ ID NO. 2 / 3 / 8) activates the NF-κB signaling pathway by targeting the combination of macrophage TLR2 / TLR4 receptors, thereby enhancing the phagocytic function of immune cells and the secretion capacity of inflammatory factors), recombinant lactoferrin peptide (the recombinant lactoferrin peptide is a bovine lactoferrin-derived peptide expressed and optimized by Pichia pastoris engineering strain, and is subjected to 0030-α signal peptide enhanced secretion and cation exchange chromatography purification, and has broad-spectrum antibacterial activity (targeted destruction of microbial cell membranes) and immune-regulating function (activation of macrophage TLR4 pathway and promotion of lymphocyte differentiation)), and short Fusarium oxysporum FXFB001 fermentation active protein extract, and the mass ratio of the Kluyveromyces marxianus-derived peptide, the silkworm chrysalis protein peptide, the recombinant lactoferrin peptide and the short Fusarium oxysporum FXFB001 fermentation active protein extract is 3:2:2:3.

[0022] Further, the freeze-drying protective agent is mannitol, which is used to maintain the stability of active ingredients during freeze-drying.

[0023] Further, the metal organic framework includes Mn-MOF-74 (Mn-MOF-74 (MOF-74(Mn) is a metal organic framework (MOF) material composed of manganese ions (Mn) and organic ligands (such as terephthalic acid), with a chemical formula of Mn-DOBDC or CPO-27(Mn). It has a highly ordered pore structure and large surface area, and is widely used in gas storage, separation and catalysis) and ZIF-8 (zeolitic imidazolate framework-8, a metal organic framework material formed by self-assembly of zinc ions and 2-methyl imidazole ligands, with high specific surface area, adjustable pore size and excellent chemical stability), and the mass ratio of Mn-MOF-74 to ZIF-8 is 3:2.

[0024] Further, the stabilizer includes citrate and ascorbic acid palmitate, and the mass ratio of citrate to ascorbic acid palmitate is 1:1, citrate is used to maintain the self-assembly pH 5.8 environment; ascorbic acid palmitate prevents active oxygen damage.

[0025] Please refer to Figure 1 The second purpose of the embodiment of the present application is to provide a preparation method for preparing the immune enhancer containing high-activity microbial protein peptides, which comprises the following steps: Step S1: the discarded beer yeast (water content ≤8%) is suspended in a pH 7.4 phosphate buffer at a concentration of 10%, pre-cooled at 4°C, then treated by ultrasonic treatment, and then precipitated by centrifugation and column chromatography to obtain β-glucan.

[0026] Specifically, the ultrasonic treatment is performed by using a 15-25 kHz amplitude horn ultrasonic system (power density 300 W / cm²) for 10-30 min in an ice bath, so that the particle size of the yeast is reduced to 1.47-2.07 μm, and the internal structure is exposed; Then NaOH is added to a final concentration of 2.0M (2.0M solution means that there are 2.0 moles of solute per liter of solution), stirred at 60°C for 2h, and after centrifugation, the precipitate is neutralized to neutral with dilute hydrochloric acid, and then subjected to Sephacryl S-400 column chromatography, and the 45-65kDa fraction is collected, which has the best immune activation ability, and the high-purity β-glucan (purity ≥98.8%) is obtained by freeze-drying.

[0027] Step S2: 3% Kluyveromyces marxianus JY-1 is inoculated in a 5% milk casein solution, and hydrolysis treatment is performed, that is, after inoculation of Kluyveromyces marxianus JY-1, hydrolysis is performed at pH 6.5 and 37°C for 18-30h, and the peptide segment with a molecular weight <3 kDa is obtained by ultrafiltration, and the extracellular protease secreted by the strain can efficiently release the immunologically active peptide; Defatted silkworm chrysalis protein is dissolved in deionized water, the pH is adjusted to 8.0, alkaline protease (dosage 8000 U / g protein) is added, and hydrolysis is carried out at 50℃ for 2-4h. After boiling water inactivation, centrifugation is performed to obtain the supernatant, and silkworm chrysalis protein peptide is obtained.

[0028] The bovine lactoferricin peptide gene is cloned into the Pichia pastoris expression vector pPIC9K, and is electroporated into the X-33 host bacteria. The fermentation broth is obtained by methanol induction during the shake flask fermentation stage, and the methanol induction time is 84-108h. The fermentation broth is purified by cation exchange chromatography to obtain the recombinant lactoferricin peptide.

[0029] The Fusarium oxysporum FXFB001 is inoculated into PDB medium, and is fermented at 28℃ with 180rpm shaking for 6 days. After ultrasonic disruption and centrifugation, the supernatant is obtained. The 40-60% saturation precipitation component is obtained by ammonium sulfate fractionation precipitation. The Fusarium oxysporum FXFB001 fermentation active protein extract with a molecular weight of 15-25 kDa is obtained by DEAE ion exchange chromatography and Sephadex G-75 gel chromatography purification.

[0030] Step S3: The above-mentioned Kluyveromyces marxianus-derived peptide, silkworm chrysalis protein peptide, recombinant lactoferricin peptide and Fusarium oxysporum FXFB001 fermentation active protein extract are mixed in a mass ratio of 3:2:2:3 to obtain a composite immune peptide. Then, the β-glucan is mixed with the composite immune peptide. When mixing, stirring is carried out at pH 5.8 and 40℃ for 1-3h to obtain an electrostatic complex.

[0031] Then, the electrostatic complex is dispersed in a 2-methyl imidazole zinc salt solution to obtain a complex solution.

[0032] The complex solution is reacted at room temperature for 1h to form a ZIF-8 primary coating layer. Then, the complex solution is transferred into a manganese acetate / 2,5-dihydroxyterephthalic acid solution to react for 2h to form an outer layer of Mn-MOF-74, thereby constructing a metal organic framework.

[0033] Step S4: A freeze-drying protective agent and a stabilizer are further added to the complex solution, and sterilization filtration treatment is performed. The sterilization filtration treatment is sterilization filtration through a 0.22μm filter, pre-freezing at-50℃, and vacuum freeze-drying, thereby obtaining an immune enhancer (water content≤5%).

[0034] The immune enhancer containing high-activity microbial protein peptides and the preparation method thereof provided by the present application are further described through the following specific examples. Example 1

[0035] Step S1: The waste beer yeast (water content≤8%) is suspended in a pH 7.4 phosphate buffer at a concentration of 10%, pre-cooled at 4℃, and then subjected to ultrasonic treatment. Then, β-glucan is obtained by centrifugal precipitation and column chromatography.

[0036] Specifically, the ultrasonic treatment is to use a 15 kHz amplitude rod ultrasonic system (power density 300 W / cm2) to treat for 30 min in an ice bath, so that the particle size of the yeast is reduced to 1.47 μm; Then, NaOH is added to a final concentration of 2.0 M, and stirring is performed at 60°C for 2 h. After centrifugation, the precipitate is taken, neutralized to neutral in dilute hydrochloric acid, and then subjected to Sephacryl S-400 column chromatography. The 65 kDa fraction is collected, and high-purity β-glucan (purity ≥98.8%) is obtained by freeze-drying.

[0037] Step S2: 3% Kluyveromyces marxianus JY-1 is inoculated in a 5% milk casein solution, and hydrolysis treatment is performed. After inoculation of Kluyveromyces marxianus JY-1, hydrolysis is performed at pH 6.5 and 37°C for 18 h. Peptides with a molecular weight of <3 kDa are obtained by ultrafiltration. Kluyveromyces marxianus-derived peptides are obtained. Defatted silkworm chrysalis protein is dissolved in deionized water, the pH is adjusted to 8.0, and alkaline protease (8000 U / g of protein) is added. Hydrolysis is performed at 50°C for 4 h. After inactivation in boiling water, the supernatant is obtained by centrifugation. Silkworm chrysalis protein peptides are obtained.

[0038] The bovine lactoferricin gene is cloned into the Pichia pastoris expression vector pPIC9K, and is electroporated into the X-33 host strain. The fermentation broth is obtained by methanol induction during the shake flask fermentation stage, and the methanol induction time is 84 h. The recombinant lactoferricin is obtained by cation exchange chromatography purification of the fermentation broth.

[0039] Fusarium oxysporum FXFB001 is inoculated in PDB medium, and is fermented at 28°C and 180 rpm for 6 days. The supernatant is obtained by ultrasonic disruption and centrifugation of the fermentation broth. The 40% saturation precipitate fraction is obtained by ammonium sulfate fractionation precipitation. The Fusarium oxysporum FXFB001 fermentation active protein extract with a molecular weight of 25 kDa is obtained by DEAE ion exchange chromatography and Sephadex G-75 gel chromatography purification.

[0040] Step S3: The Kluyveromyces marxianus-derived peptides, silkworm chrysalis protein peptides, recombinant lactoferricin, and Fusarium oxysporum FXFB001 fermentation active protein extract are mixed in a mass ratio of 3:2:2:3 to obtain a composite immunopeptide. The β-glucan is mixed with the composite immunopeptide. The mixture is stirred at pH 5.8 and 40°C for 3 h to obtain an electrostatic complex.

[0041] Then, the electrostatic complex is dispersed in a 2-methyl imidazole zinc salt solution to obtain a complex solution.

[0042] The complex solution is reacted at room temperature for 1 h to form a primary ZIF-8 coating layer; then, the solution is transferred into a manganese acetate / 2,5-dihydroxyterephthalic acid solution to react for 2 h to form an outer layer of Mn-MOF-74, thereby constructing a metal-organic framework, in which the mass ratio of Mn-MOF-74 to ZIF-8 is 3:2.

[0043] Step S4: A freeze-drying protective agent (mannitol) and a stabilizer (the stabilizer includes citrate and ascorbic palmitate, and the mass ratio of citrate to ascorbic palmitate is 1:1) are further added to the complex solution, which is subjected to sterilization filtration treatment (0.22 μm sterilization filtration), pre-frozen at -50°C, and vacuum freeze-dried to obtain an immune enhancer (water content ≤5%).

[0044] It is worth noting that the mass ratio of each component in the immune enhancer is as follows: β-glucan 25%, complex immune peptide 10%, freeze-drying protective agent 3%, metal-organic framework 44%, and the balance is a stabilizer. Example 2

[0045] Step S1: The waste beer yeast (water content ≤8%) is suspended in a pH 7.4 phosphate buffer at a concentration of 10%, pre-cooled at 4°C, and then subjected to ultrasonic treatment, followed by centrifugal precipitation and column chromatography to obtain β-glucan.

[0046] Specifically, the ultrasonic treatment is performed using a 20 kHz amplitude rod ultrasonic system (power density 300 W / cm²) for 20 min in an ice bath, so that the particle size of the yeast is reduced to 1.77 μm; Then, NaOH is added to a final concentration of 2.0 M, and the mixture is stirred at 60°C for 2 h. After centrifugation, the precipitate is neutralized with dilute hydrochloric acid, and then subjected to Sephacryl S-400 column chromatography. The 55 kDa fraction is collected and freeze-dried to obtain high-purity β-glucan (purity ≥98.8%).

[0047] Step S2: 3% Kluyveromyces marxianus JY-1 is inoculated in a 5% milk casein solution, and subjected to hydrolysis treatment. After inoculation of K. marxianus JY-1, the solution is hydrolyzed at pH 6.5 and 37°C for 24 h. The solution is subjected to ultrafiltration to remove peptide fragments with a molecular weight <3 kDa, thereby obtaining K. marxianus-derived peptides. The defatted silkworm chrysalis protein is dissolved in deionized water, and the pH is adjusted to 8.0. Alkaline protease (8000 U / g protein) is added, and the mixture is hydrolyzed at 50°C for 3 h. After inactivation in boiling water, the supernatant is obtained by centrifugation, thereby obtaining silkworm chrysalis protein peptides.

[0048] The bovine lactoferricin gene is cloned into the Pichia pastoris expression vector pPIC9K, and is electrotransformed into the X-33 host strain. The fermentation liquid is obtained by methanol induction during the shake flask fermentation stage, and the methanol induction time is 96 h. The fermentation liquid is purified by cation exchange chromatography to obtain the recombinant lactoferricin.

[0049] The Fusarium oxysporum FXFB001 is inoculated into PDB medium, and is fermented at 28°C with 180 rpm shaking for 6 days. After ultrasonic disruption and centrifugation, the supernatant is obtained. The 50% saturation precipitation component is obtained by ammonium sulfate fractionation precipitation. Then, the DEAE ion exchange chromatography and Sephadex G-75 gel chromatography purification are performed to obtain the Fusarium oxysporum FXFB001 fermentation active protein extract with a molecular weight of 20 kDa.

[0050] Step S3: The above-mentioned Kluyveromyces marxianus-derived peptide, silkworm chrysalis protein peptide, recombinant lactoferricin, and Fusarium oxysporum FXFB001 fermentation active protein extract are mixed in a mass ratio of 3:2:2:3 to obtain a composite immune peptide. Then, the β-glucan is mixed with the composite immune peptide. During the mixing, the mixture is stirred at 40°C and pH 5.8 for 1 h to obtain an electrostatic complex.

[0051] Then, the electrostatic complex is dispersed in a 2-methyl imidazole zinc salt solution to obtain a complex solution.

[0052] The complex solution is reacted at room temperature for 1 h to form a primary ZIF-8 coating layer. Then, the complex solution is transferred into a manganese acetate / 2,5-dihydroxyterephthalic acid solution to react for 2 h to form an outer layer of Mn-MOF-74, thereby constructing a metal organic framework. The mass ratio of Mn-MOF-74 to ZIF-8 in the metal organic framework is 3:2.

[0053] Step S4: The freeze-drying protective agent (mannitol) and the stabilizer (the stabilizer includes citrate and ascorbyl palmitate, and the mass ratio of citrate to ascorbyl palmitate is 1:1) are added to the complex solution. The solution is subjected to sterilization filtration treatment, i.e., 0.22 μm sterilization filtration, pre-freezing at -50°C, and vacuum freeze-drying to obtain an immune enhancer (the water content is ≤5%).

[0054] It is worth noting that the mass ratio of each component in the immune enhancer is as follows: β-glucan 28%, composite immune peptide 7%, freeze-drying protective agent 5%, metal organic framework 40%, and the balance is the stabilizer. Example 3

[0055] Step S1: The waste beer yeast (water content ≤8%) is suspended in a pH 7.4 phosphate buffer at a concentration of 10%. The solution is pre-cooled at 4°C, and then is subjected to ultrasonic treatment. Then, the β-glucan is obtained by centrifugal precipitation and column chromatography.

[0056] Specifically, the ultrasonic treatment is to use a 25 kHz amplitude rod ultrasonic system (power density 300 W / cm2) to treat in an ice bath for 10 min, so that the particle size of the yeast is reduced to 2.07 μm. Then, NaOH is added to a final concentration of 2.0 M, and stirring is performed at 60°C for 2 h. After centrifugation, the precipitate is taken, neutralized to neutral in dilute hydrochloric acid, and then subjected to Sephacryl S-400 column chromatography. The 45 kDa fraction is collected, and high-purity β-glucan (purity ≥98.8%) is obtained by freeze-drying.

[0057] Step S2: 3% Kluyveromyces marxianus JY-1 is inoculated in a 5% milk casein solution, and hydrolysis treatment is performed. After inoculation of Kluyveromyces marxianus JY-1, hydrolysis is performed at pH 6.5 and 37°C for 30 h. Peptides with a molecular weight of <3 kDa are obtained by ultrafiltration, and Kluyveromyces marxianus-derived peptides are obtained. Defatted silkworm pupa protein is dissolved in deionized water, the pH is adjusted to 8.0, alkaline protease is added (dosage 8000 U / g protein), and hydrolysis is performed at 50°C for 2 h. After inactivation in boiling water, the supernatant is obtained by centrifugation, and silkworm pupa protein peptides are obtained.

[0058] The bovine lactoferricin gene is cloned into the Pichia pastoris expression vector pPIC9K, and is electroporated into the X-33 host strain. The fermentation broth is obtained by methanol induction during the shake flask fermentation stage, and the methanol induction time is 108 h. The recombinant lactoferricin is obtained by cation exchange chromatography purification of the fermentation broth.

[0059] Fusarium oxysporum FXFB001 is inoculated in PDB medium, and is fermented at 28°C and 180 rpm for 6 days. The supernatant is obtained by ultrasonic disruption and centrifugation of the fermentation broth. The 60% saturation precipitate fraction is obtained by ammonium sulfate fractionation precipitation. The Fusarium oxysporum FXFB001 fermentation active protein extract with a molecular weight of 15 kDa is obtained by DEAE ion exchange chromatography and Sephadex G-75 gel chromatography purification.

[0060] Step S3: The Kluyveromyces marxianus-derived peptides, silkworm pupa protein peptides, recombinant lactoferricin, and Fusarium oxysporum FXFB001 fermentation active protein extract are mixed in a mass ratio of 3:2:2:3 to obtain a composite immunopeptide. Then, the β-glucan is mixed with the composite immunopeptide. The mixing is performed at pH 5.8 and 40°C for 1 h with stirring to obtain an electrostatic complex.

[0061] Then, the electrostatic complex is dispersed in a 2-methyl imidazole zinc salt solution to obtain a complex solution.

[0062] The complex solution is reacted at room temperature for 1 h to form a primary ZIF-8 coating layer; then, the solution is transferred into a manganese acetate / 2,5-dihydroxyterephthalic acid solution to react for 2 h to form an outer layer of Mn-MOF-74, thereby constructing a metal-organic framework, in which the mass ratio of Mn-MOF-74 to ZIF-8 is 3:2.

[0063] Step S4: A freeze-drying protective agent (mannitol) and a stabilizer (the stabilizer includes citrate and ascorbic acid palmitate, and the mass ratio of citrate to ascorbic acid palmitate is 1:1) are further added to the complex solution, which is subjected to sterilization filtration treatment (0.22 μm sterilization filtration), pre-frozen at -50°C, and vacuum freeze-dried to obtain the immune enhancer (water content ≤5%).

[0064] It is worth noting that the mass ratio of each component in the immune enhancer is as follows: β-glucan 30%, complex immune peptide 5%, freeze-drying protective agent 6%, metal-organic framework 38%, and the balance is the stabilizer.

[0065] The immune enhancer is prepared according to the preparation method provided in Examples 1-3, and then subjected to in vitro macrophage function testing. According to the revised Section 5.3 of the Macrophage Function Evaluation Clause of GB / T 27818-2011 Chemicals In Vitro Immunotoxicity Test Method, the standardized test of in vitro macrophage function is performed: the sample is sterilized through a 0.22 μm microporous filter membrane, and then re-dissolved in sterile PBS to a final concentration of 50±0.75 μg / mL (precision ±1.5%); The RAW264.7 mouse macrophage cell line is expanded in DMEM complete medium (10% FBS), and subcultured to the 3rd-5th generation in a constant temperature gas environment of 37±0.3°C / 5% CO2; the logarithmic growth phase cells are digested with trypsin-EDTA, and the concentration is calibrated to 5.0×10 4 cells / mL (allowable error ±5%) by a hemocytometer, and inoculated into a 96-well plate (100 μL / well). A horizontal micro-vibration table (amplitude 2 mm, 60 rpm) is used for pre-culture for 24 hours to eliminate edge effects; Experimental groups 1-3: fresh culture medium containing the immune enhancers prepared in Examples 1-3 is replaced, respectively; Positive control: 1 μg / mL LPS solution; Blank control: equal volume of PBS; After 24 hours of intervention, FITC-labeled fluorescent microspheres (Φ=1.0 μm, 10 6phagocytosis rate (formula: phagocytosis rate = (FITC+ of experimental group - FITC+ of blank group) / FITC+ of blank group x 100%) was calculated by the difference of FITC+ cell ratio, and the criterion threshold was greater than or equal to 40%. The test data was recorded in Table 1.

[0066] According to Table 1, the phagocytosis rates of the macrophages in experimental groups 1-3 containing the immune enhancer prepared in Examples 1-3 were all higher than 57.02%, indicating that the immune enhancer containing the high-activity microbial protein peptide and the preparation method thereof provided by the application can prepare the immune enhancer for promoting the improvement of immune effect.

[0067] In the application, first, the immune activation core skeleton is constructed by β-glucan, which retains the specific anchoring of the spiral conformation of the macrophage surface Dectin-1 receptor, triggers the Syk-CARD9-NF-κB signal cascade, and starts the secretion of pro-inflammatory cytokines. Then, in the complex immune peptide, the peptide from the Kluyveromyces marxianus (containing the HPHPHLSF / PFPEVFG sequence) activates the MAPK-p38 phosphorylation pathway; the silkworm chrysalis protein peptide (SEQ ID NO. 2 / 3 / 8) targets the binding of the TLR2 / TLR4-MD-2 receptor complex, enhances the NF-κB nuclear translocation efficiency, and improves the phagocytosis rate of macrophages; the recombinant lactoferrin peptide destroys the microbial cell membrane lipid bilayer through the cationic domain, and at the same time activates the TLR4 pathway to promote the differentiation of lymphocytes; the specific antibacterial protein contained in the fermentation active protein extract of the short-shank fusarium FXFB001 can directly destroy the cell membrane structure of the pathogen, and at the same time, through the activation of the TLR2 / 4 pathway, enhance the innate immune response; Further, the ZIF-8 inner layer protects the active structure of the immune peptide through the confinement effect at physiological pH; the Mn-MOF-74 outer layer responds to the lysosomal acidic environment (pH 5.0) to dissociate, and the released Mn²⁺ activates the cGAS-STING pathway to drive the synthesis of type I interferon, at the same time, Zn²⁺ penetrates into the interstitial space of lymphocytes to enhance metabolism and proliferation; finally, the stabilizer maintains the functional integrity to ensure the improvement of the systemic immune defense efficiency. Example 4

[0068] In the present application, too low mass ratio of the composite immunopeptide will lead to insufficient activation of the TLR / NF-κB pathway, and too much composite immunopeptide will lead to an increase in the particle size of the electrostatic complex formed, thereby reducing the MOF encapsulation rate, exposing the hydrophobic groups on the surface of the polypeptide, accelerating the molecular aggregation during the freeze-drying process, and causing a more serious loss of activity after storage.

[0069] To verify that the mass ratio of 5-10% of the composite immunopeptide in the immune enhancer is one of the important factors for the immune enhancer provided by the present application to promote the improvement of immune effect, this embodiment changes only the mass ratio of the composite immunopeptide based on the above-mentioned embodiment 2, sets the mass ratio of the composite immunopeptide to 2%, 4%, 5%, 7%, 10%, 12% or 15%, then prepares the immune enhancer, and tests according to the test method provided in the above-mentioned embodiment, and the results are shown in Table 2.

[0070] According to Table 2, when the mass ratio of the composite immunopeptide is 2%, 4%, 12% or 15%, which is neither 5-10%, the phagocytosis rate of macrophages after using the prepared immune enhancer is lower than that when the mass ratio of the composite immunopeptide is 5%, 7% or 10%.

[0071] Therefore, it can be explained that the mass ratio of 5-10% of the composite immunopeptide in the immune enhancer is one of the important factors for the immune enhancer provided by the present application to promote the improvement of immune effect.

[0072] The above shows and describes the basic principles, main features and advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only preferred examples of the present application and are not intended to limit the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an immune enhancer containing highly active microbial protein peptides, characterized in that, Includes the following steps: Step S1: The waste brewer's yeast was suspended in a 10% phosphate buffer solution at pH 7.4, pre-cooled at 4°C, and then sonicated. β-glucan was obtained by centrifugation and column chromatography. Step S2: Inoculate 3% Kluyveromyces martensii JY-1 into a 5% bovine casein solution, and then hydrolyze it to obtain Kluyveromyces martensii peptides; Defatted silkworm pupa protein was dissolved in deionized water, the pH was adjusted to 8.0, alkaline protease was added, and hydrolysis was carried out at 50°C for 2-4 hours. After inactivation by boiling water, the supernatant was collected by centrifugation to obtain silkworm pupa protein peptides. The bovine lactoferrin peptide gene was cloned into the Pichia pastoris expression vector pPIC9K, electroporated into the X-33 host strain, and fermented with methanol during the shake-flask fermentation stage to obtain the fermentation broth. The fermentation broth was purified by cation exchange chromatography to obtain recombinant lactoferrin peptide. Fusarium septum FXFB001 was inoculated into PDB medium and fermented at 28℃ and 180 rpm for 6 days. The fermentation broth was ultrasonically disrupted and centrifuged, and the supernatant was collected. The 40-60% saturated precipitate fraction was obtained by ammonium sulfate fractionation precipitation method. The precipitate was then purified by DEAE ion exchange chromatography and Sephadex G-75 gel chromatography to obtain the fermentation active protein extract of Fusarium septum FXFB001 with a molecular weight of 15-25 kDa. Step S3: Mix the above-mentioned Max Kluyveromyces yeast-derived peptides, silkworm pupa protein peptides, recombinant lactoferrin peptides and Fusarium brevis FXFB001 fermentation active protein extract in a mass ratio of 3:2:2:3 to form an electrostatic complex. The electrostatic complex was then dispersed in a 2-methylimidazolium zinc salt solution to obtain a complex solution; The complex solution was reacted at room temperature for 1 hour to form the ZIF-8 primary coating layer; then it was transferred into manganese acetate / 2,5-dihydroxyterephthalic acid solution and reacted for 2 hours to form the outer Mn-MOF-74 layer, thus constructing the metal-organic framework. Step S4: Add lyophilization protectant and stabilizer to the complex solution, and then filter it under sterile conditions to obtain the immune enhancer; The mass percentages of each component in the immune enhancer are as follows: β-glucan 25-30%, complex immune peptides 5-10%, lyophilization protectant 3-6%, metal-organic framework 38-44%, balance is stabilizer.

2. The method for preparing the immune enhancer containing highly active microbial protein peptides according to claim 1, characterized in that: In step S1, the ultrasonic treatment is performed using a 15-25kHz amplitude-modulated ultrasonic system in an ice bath for 10-30 minutes to reduce the yeast particle size to 1.47-2.07μm. Then add NaOH to a final concentration of 2.0M, stir at 60℃ for 2 hours, centrifuge, collect the precipitate, neutralize to neutral with dilute hydrochloric acid, and then perform Sephacryl S-400 column chromatography to collect the 45-65 kDa fraction, freeze-dry to obtain β-glucan.

3. The method for preparing the immune enhancer containing highly active microbial protein peptides according to claim 1, characterized in that: In step S2, the hydrolysis treatment involves inoculating *Kluyveromyces martensii* JY-1, hydrolyzing at pH 6.5 and 37°C for 18-30 hours, and then ultrafiltration to remove peptides with a molecular weight <3 kDa to obtain *Kluyveromyces martensii* peptides.

4. The method for preparing the immune enhancer containing highly active microbial protein peptides according to claim 1, characterized in that: In step S2, the methanol induction time is 84-108h.

5. The method for preparing the immune enhancer containing highly active microbial protein peptides according to claim 1, characterized in that: In step S3, the mixture is stirred for 1-3 hours at pH 5.8 and 40°C to obtain an electrostatic complex.

6. The method for preparing the immune enhancer containing highly active microbial protein peptides according to claim 1, characterized in that: In step S3, the mass ratio of Mn-MOF-74 to ZIF-8 in the metal-organic framework is 3:

2.

7. The method for preparing the immune enhancer containing highly active microbial protein peptides according to claim 1, characterized in that: In step S4, the sterilization filtration process involves 0.22μm sterilization filtration, pre-freezing at -50℃, and then vacuum freeze-drying to obtain the immune enhancer.

8. The method for preparing the immune enhancer containing highly active microbial protein peptides according to claim 1, characterized in that: In step S4, the freeze-drying protectant is mannitol.

9. The method for preparing the immune enhancer containing highly active microbial protein peptides according to claim 1, characterized in that: In step S4, the stabilizer includes citrate and ascorbate palmitate, and the mass ratio of citrate to ascorbate palmitate is 1:

1.

10. The immunostimulant containing highly active microbial protein peptides prepared by the preparation method according to any one of claims 1-9, characterized in that, Including the following raw materials: β-glucan, complex immune peptides, lyophilization protectants, metal-organic frameworks and stabilizers, including: The β-glucan retains its natural helical conformation as a core framework for immune activation, triggering signaling pathways by specifically binding to macrophage surface receptors and inducing the secretion of pro-inflammatory cytokines. In the aforementioned complex immune peptides, the Kluyveromyces max-derived peptide activates the macrophage phosphorylation pathway; the silkworm pupa protein peptide targets and binds to the receptor complex, enhancing nuclear translocation efficiency and increasing macrophage phagocytosis rate; the recombinant lactoferrin peptide disrupts the lipid bilayer of microbial cell membranes, while simultaneously activating the macrophage TLR4 pathway and promoting lymphocyte differentiation; and the specific antimicrobial proteins contained in the Fusarium styraceae FXFB001 fermentation active protein extract can directly disrupt the pathogen cell membrane structure and enhance the innate immune response by activating the TLR2 / 4 pathway. In the metal-organic framework, the inner ZIF-8 layer maintains structural integrity at physiological pH and protects the activity of immune peptides through confinement effect; the outer Mn-MOF-74 layer dissociates and releases Mn²⁺ in response to the acidic environment of lysosomes, which acts as an endogenous activator to drive the synthesis of type I interferon, while Zn²⁺ penetrates into the intercellular space of lymphocytes to promote proliferation and differentiation. The freeze-drying protectant is used to prevent loss of activity, and the stabilizer is used to inhibit oxidative damage caused by reactive oxygen species.

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