Extraction and purification process of high-purity protein mulberry SOD

Abstract

The present application relates to the technical field of extraction and purification process of bioactive protease from plant source, in particular to an extraction and purification process of high-purity SOD from Morus notabilis Hoo. The present application uses Morus notabilis Hoo as raw material, first inactivates endogenous degrading enzyme by microwave instantaneous fixation, and locks the activity of raw material by low-temperature vacuum drying; then realizes efficient dissolution of SOD by ultrasonic-complex enzyme coupling wall breaking, and protects the activity by copper and zinc ions throughout the process; then realizes directional enrichment by PEG2000 / ammonium sulfate aqueous two-phase system, removes impurities by ceramic microfiltration-ultrafiltration membrane grading, and refines by DEAE anion exchange chromatography and Sephadex G-75 gel filtration chromatography, and finally obtains the finished product by low-temperature freeze-drying. The present application constructs a unique process system of multi-process synergistic coupling, and the finished product SOD has high purity, enzyme activity and total recovery rate, good stability, green and mild process, and is suitable for industrialized scale production in the fields of food, health care products and medicine.

Description

Technical Field

[0001] This invention relates to the field of plant-derived bioactive protease extraction and purification technology, and in particular to a green industrial production process for preparing high-purity, high-activity SOD using protein-rich mulberry leaves as raw material, employing microwave inactivation of endogenous enzymes, ultrasonic-composite enzyme coupling for cell wall disruption, two-phase aqueous directional enrichment, membrane fractionation, and ion exchange-gel filtration dual-layer chromatography. Background Technology

[0002] Superoxide dismutase (SOD) is a type of functional protease containing copper-zinc / manganese metal cofactors. It can efficiently scavenge superoxide anion free radicals in the body and has antioxidant, anti-aging, anti-inflammatory, and immunomodulatory effects. It is widely used in functional foods, health foods, cosmetics, and the biopharmaceutical industry. Mulberry leaves have a high SOD enrichment, are renewable raw materials, and have low costs for large-scale cultivation, making them a high-quality plant-derived SOD raw material.

[0003] The existing SOD extraction and purification process for protein mulberry has significant technical shortcomings and industry bottlenecks:

[0004] (1) Conventional hot water extraction and high-temperature cooking processes are at high temperatures, which can easily destroy the spatial conformation of SOD, resulting in a large loss of enzyme activity and a generally low specific activity of the finished product. At the same time, the endogenous protease in mulberry leaves is not pre-inactivated, and SOD continues to degrade during the extraction process, further reducing the yield and activity.

[0005] (2) Single enzymatic hydrolysis and ordinary organic solvent extraction have low cell wall breaking efficiency and incomplete SOD dissolution; moreover, the organic solvent residue is large and the environmental protection is poor, which does not meet the requirements for food and pharmaceutical grade production.

[0006] (3) Traditional salting out and single-layer chromatography purification methods can only remove a small amount of impurities and cannot effectively separate a large number of interfering impurities such as polyphenols, flavonoids, soluble polysaccharides, and pigments in mulberry leaves. The purity of the products is generally less than 60%, which is difficult to meet the high-end application access standards.

[0007] (4) Existing processes are mostly simple superposition of unit operations, without collaborative coupling design between processes, with long process steps, large intermediate losses, and a total recovery rate of less than 35%, resulting in high production costs and the inability to achieve continuous industrial production.

[0008] (5) Conventional purification processes lack enzyme-active ion protection systems, and SOD is easily deactivated by pH fluctuations and room temperature treatment, resulting in poor storage stability of the finished product.

[0009] Existing literature and patents mostly employ ultrasonic extraction, enzymatic hydrolysis, single aqueous two-phase or ordinary membrane separation individually. None of them have formed a complete coupled process of microwave-controlled activation-ultrasonic enzymatic hydrolysis synergistic cell disruption-aqueous two-phase directional enrichment-membrane fractionation-dual-layer gradient polishing. Furthermore, no integrated technology solution with low loss, high purity and high recovery specifically for protein mulberry has been found. Summary of the Invention

[0010] In view of this, the purpose of this invention is to overcome the defects of existing technologies, such as low activity retention rate of protein mulberry SOD extraction, insufficient purification purity, low process recovery rate, lack of process synergy, poor product stability, and difficulty in large-scale production. The invention provides a high-purity protein mulberry SOD extraction and purification process with mild process conditions, green and non-toxic solvents, synergistic effect of multiple processes, and precise and controllable parameters.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0012] This invention provides a method for preparing SOD, comprising:

[0013] S1: Mix the buffer solution, complex enzyme and crude protein powder, sonicate, extract, centrifuge, and collect the supernatant to obtain crude SOD extract;

[0014] S2: Mix the aqueous two-phase system with the crude SOD extract, separate the layers, and collect the upper phase enrichment.

[0015] S3: The upper phase enrichment solution is filtered sequentially through a microfiltration membrane and an ultrafiltration membrane to separate and obtain a refined SOD concentrate;

[0016] S4: The SOD purified concentrate is subjected to ion exchange chromatography, and the elution fractions with SOD characteristic activity are collected in segments to obtain the SOD active peak solution.

[0017] S5: The SOD active peak solution was subjected to Sephadex G-75 gel filtration chromatography to obtain pure SOD enzyme;

[0018] The buffer solution contains a metal ion protectant, which includes copper ions and / or zinc ions;

[0019] The complex enzyme includes at least one of cellulase, neutral protease, and pectinase.

[0020] The protein-rich mulberry powder is sterilized by microwave;

[0021] The polymer in the aqueous two-phase system is PEG, and the hydrophilic salt is at least one of ammonium sulfate and K2HPO4.

[0022] In some specific embodiments of the present invention, the preparation method of the above-mentioned protein mulberry crude powder includes:

[0023] Fresh protein mulberry leaves are microwave-sterilized, then vacuum-dried, pulverized, and sieved to obtain the protein mulberry coarse powder.

[0024] In some specific embodiments of the present invention, the power of the microwave in the above preparation method is 260 W, 270 W, 280 W, 290 W, 300 W, 310 W, 320 W, 350 W, 400 W, 500 W, 600 W, 700 W or 800 W.

[0025] In some specific embodiments of the present invention, the blanching time in the above preparation method is 60 s, 65 s, 70 s, 75 s, 80 s, 85 s, 90 s, 95 s, 100 s, 105 s, 110 s, 115 s or 120 s.

[0026] In some specific embodiments of the present invention, the vacuum drying conditions of the above preparation method are 40°C, 41°C, 42°C, 43°C, 44°C or 45°C; and the vacuum degree is 0.08 MPa, 0.09 MPa or 0.1 MPa.

[0027] In some specific embodiments of the present invention, the above preparation method further includes a step of vacuum freeze-drying SOD purified enzyme.

[0028] In some specific embodiments of the present invention, the vacuum freeze-drying conditions of the above preparation method include: -55°C, vacuum degree below 10 Pa, for 24 h.

[0029] In some specific embodiments of the present invention, in S1 of the above preparation method:

[0030] The ratio of the crude protein mulberry powder to the buffer solution is 1:8, 1:9, 1:10, 1:11 or 1:12.

[0031] The buffer solution is a phosphate buffer solution with pH 6.8, 6.9, 7, 7.1 or 7.2;

[0032] The metal ion agent includes copper ions and zinc ions, wherein the concentration of copper ions in the buffer solution is 0.1 mmol / L, and the concentration of zinc ions in the buffer solution is 0.1 mmol / L.

[0033] The mass of the compound enzyme is 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, or 1.2% of the crude protein mulberry powder;

[0034] The ultrasonic power is 350 W, 360 W, 370 W, 380 W, 390 W, 400 W, 410 W, 420 W, 430 W, 440 W or 450 W, and the temperature is 32℃, 34℃, 36℃, 38℃, 40℃, 42℃, 44℃, 46℃, 48℃ or 50℃.

[0035] In some specific embodiments of the present invention, in S2 of the above preparation method:

[0036] The PEG in the aqueous two-phase system has a mass fraction of 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, or 18%.

[0037] The PEG includes PEG2000;

[0038] The mass fraction of the hydrophilic salt in the aqueous two-phase system is 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, or 27%.

[0039] The pH of the aqueous two-phase system is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7;

[0040] The volume ratio of the aqueous two-phase system to the crude SOD extract is 1:1.

[0041] In some specific embodiments of the present invention, in S3 of the above preparation method:

[0042] The microfiltration membrane has a pore size of 0.2 μm or a molecular weight cutoff of 100 kDa;

[0043] The ultrafiltration membrane has a molecular weight cutoff of 30 kDa;

[0044] The temperature of the filter is ≤25℃.

[0045] In some specific embodiments of the present invention, in S4 of the above preparation method:

[0046] The ion exchange chromatography was DEAE-Sepharose Fast Flow anion exchange chromatography;

[0047] The chromatographic equilibration solution for the ion exchange chromatography was a pH 7.2 phosphate buffer solution.

[0048] The gradient elution buffer for the ion exchange chromatography is a pH 7.2 phosphate buffer containing 0–0.5 mol / L NaCl, and the elution flow rate is 2 mL / min.

[0049] In some specific embodiments of the present invention, in S5 of the above preparation method:

[0050] The elution buffer for the Sephadex G-75 gel filtration chromatography was pH 7.0 phosphate buffer, and the elution flow rate was 1.5 mL / min.

[0051] In some specific embodiments of the present invention, the above preparation method includes the steps of microwave instantaneous blanching and low-temperature vacuum drying for synergistic activation control: firstly, the endogenous protease and polyphenol oxidase of mulberry leaves are inactivated by instantaneous high temperature to inhibit SOD degradation and oxidative inactivation from the source; then, low-temperature vacuum drying is performed to avoid high-temperature oxidation and activity decay caused by conventional hot air drying, thereby achieving maximum activation at the raw material end.

[0052] In some specific embodiments of the present invention, the ultrasonic-composite enzyme coupling cell wall disruption of the above preparation method has a synergistic effect: cellulase degrades the cell wall cellulose skeleton, and neutral protease moderately decomposes intercellular junction proteins; at the same time, the ultrasonic cavitation effect generates microjets and local micropores, which help the enzyme preparation to quickly penetrate into the cell and break the cell wall mass transfer barrier. Compared with enzymatic hydrolysis alone or ultrasonication alone, the SOD dissolution rate is significantly improved, and the whole process is low temperature and mild, without destroying the enzyme protein conformation, achieving a synergistic release effect of 1+1>2.

[0053] In some specific embodiments of the present invention, the Cu prepared by the above method 2+ / Zn 2+ In-situ preservation of SOD activity by complex ions: SOD homologous metal ions are introduced into the extraction buffer in advance to stabilize the structure of the SOD active center, buffer the pH fluctuations of the buffer system and the conformational dissociation during the subsequent separation process, and greatly improve the overall enzyme activity retention rate.

[0054] In some specific embodiments of the present invention, the above preparation method achieves PEG2000 / ammonium sulfate aqueous two-phase directional enrichment + membrane fractionation coupling for impurity removal: the aqueous two-phase system utilizes hydrophobic interactions and differences in partition coefficients to selectively enrich SOD in the upper phase, while significantly removing polysaccharides, polyphenols, and large molecular weight proteins; then, suspended impurities are removed by microfiltration and SOD is precisely retained by 30kDa ultrafiltration, and small molecule pigments, inorganic salts, and monosaccharides are removed in a fractional manner. The two-step coupling achieves efficient crude purification and impurity removal, significantly reducing the subsequent chromatography load.

[0055] In some specific embodiments of the present invention, the above preparation method achieves anion exchange + gel filtration dual-layer gradient polishing: DEAE ion exchange separates homologous impurities based on charge differences, and gradient elution precisely captures the SOD activity peak; Sephadex G-75 desaltes based on molecular size, removes protein polymers and trace amounts of isomeric proteins, and the two-stage chromatography linkage achieves molecular-level purification, stably reaching a high purity of over 98%.

[0056] In some specific embodiments of the present invention, the above preparation method achieves synergistic stability through low temperature and freeze-drying throughout the entire process: extraction, separation, and purification are all carried out in a room temperature / low temperature environment to avoid thermal denaturation; vacuum freeze-drying does not use high-temperature drying, which completely preserves the natural spatial structure of SOD, and the finished powder has slow enzyme activity decay and long shelf life when stored at room temperature.

[0057] The present invention has the following beneficial effects:

[0058] (1) Source control and low loss throughout the process: Microwave inactivation of endogenous degrading enzymes, followed by in-situ metal ion preservation and low-temperature process throughout the process, inhibits SOD inactivation from raw materials to finished products in multiple dimensions, and the overall enzyme activity retention rate is ≥85%, which is far superior to traditional high-temperature process.

[0059] (2) Process synergy: This invention organically couples microwave-controlled activation, ultrasonic-enzymatic hydrolysis for cell disruption, two-phase aqueous phase directional distribution, membrane fractionation for impurity removal, and dual-layer gradient purification to form a unique technical system, producing unexpected synergistic effects that cannot be achieved by existing single processes, and simultaneously and significantly improving dissolution rate, purity, and recovery rate.

[0060] (3) High purity and wide range of applications: After multi-stage coupling purification, the finished product has an SOD purity of ≥98% and a specific activity of ≥5000U / mg, which meets the standards for pharmaceutical grade and high-end health food access, breaking through the industry bottleneck of low purity in traditional processes.

[0061] (4) High recovery rate and low production cost: The process loss is controllable and the targeted enrichment effect is good. The total process recovery rate is ≥65%, which is nearly double that of the traditional process, making it suitable for large-scale mass production and cost reduction.

[0062] (5) Green and environmentally friendly, suitable for industrialization: No toxic organic solvents are used throughout the process, and there are no strong alkali and high temperature conditions. The wastewater components are simple and easy to treat. The process is continuous and the equipment is universal, which can be directly adapted to the continuous operation of plant extract production lines.

[0063] (6) Excellent product stability: freeze-drying preserves the natural protein conformation, and the enzyme activity retention rate is ≥90% after 12 months of sealed storage at room temperature. It has a long shelf life and is conducive to warehousing and market circulation. Detailed Implementation

[0064] This invention discloses a high-purity extraction and purification process for protein SOD. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0065] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0066] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0067] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0068] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0069] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0070] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0071] This invention uses protein-rich mulberry leaves as raw material. First, endogenous degrading enzymes are inactivated by microwave instantaneous blanching, followed by low-temperature vacuum drying to lock in the raw material's activity. Then, ultrasonic-composite enzyme coupling is used to achieve efficient SOD dissolution, supplemented with Cu. 2+ / Zn 2+The ions are kept active throughout the process; then, they undergo directional enrichment via PEG2000 / ammonium sulfate aqueous two-phase chromatography, followed by fractional impurity removal via ceramic microfiltration-ultrafiltration membrane, and two-stage purification using DEAE anion exchange chromatography and Sephadex G-75 gel filtration chromatography. Finally, the product is obtained by low-temperature freeze-drying. This invention constructs a unique multi-step synergistic coupling process system, overcoming the shortcomings of existing technologies such as large activity loss, low purity, and low recovery rate. The finished product has an SOD purity of ≥98%, enzyme activity of ≥5000 U / mg, and a total recovery rate of ≥65%. The process is green and mild, and the product has good stability, making it suitable for industrial-scale production in the food, health product, and pharmaceutical fields.

[0072] In practice, the extraction and purification process of the high-purity protein SOD includes the following steps:

[0073] (1) Raw material pretreatment: Select fresh protein mulberry leaves during the vigorous growth period, remove diseased and withered leaves, rinse with clean water and drain; use microwave instantaneous blanching to inactivate endogenous protease and polyphenol oxidase in mulberry leaves; then dry at low temperature under vacuum, pulverize and pass through a 40-60 mesh sieve to obtain protein mulberry coarse powder, and store at low temperature in a sealed and light-proof place for later use.

[0074] (2) Ultrasonic-enzymatic coupling cell wall breaking extraction: Add Cu-containing crude protein mulberry powder to the solution at the set material-liquid ratio. 2+ / Zn 2+ Phosphate buffer containing metal ion protectant was stirred evenly; a compound enzyme preparation was added, and ultrasonic-assisted coupling treatment was carried out simultaneously to gently break the cell wall and promote the full dissolution of intracellular SOD; after the coupling treatment was completed, the mixture was allowed to stand at low temperature for extraction, centrifuged to separate and discard the filter residue, and the supernatant was collected to obtain crude SOD extract.

[0075] (3) Directional enrichment by aqueous two-phase extraction: Prepare a PEG2000 / ammonium sulfate aqueous two-phase system in the crude SOD extract, adjust the pH of the system, stir and mix at a constant temperature, and let it stand to separate into layers naturally; use the difference in partition coefficient to make SOD directionally enriched in the upper PEG phase, discard the lower phase waste liquid rich in polysaccharides, polyphenols and miscellaneous proteins, and collect the upper phase SOD enriched liquid.

[0076] (4) Membrane fractionation and purification: The upper phase enrichment solution is first filtered through a ceramic microfiltration membrane to remove suspended particles, fiber residues and macromolecular colloids; then it is fractionated and ultrafiltered through a 30 kDa molecular weight cutoff ultrafiltration membrane to retain SOD active protein and remove small molecule pigments, inorganic salts and monosaccharide impurities to obtain SOD purified concentrate.

[0077] (5) DEAE anion exchange chromatography purification: SOD purified concentrate was loaded onto a DEAE-Sepharose Fast Flow anion exchange chromatography column and the column bed was equilibrated with phosphate buffer; linear gradient elution with sodium chloride was used, protein and enzyme activity peaks were detected online, characteristic SOD activity elution peaks were collected in segments, and differentially charged proteins were removed.

[0078] (6) Sephadex G-75 gel filtration chromatography polishing: The SOD active peak collected by ion exchange is loaded onto the gel filtration chromatography column, and isocratic elution with phosphate buffer is performed to remove salt, protein polymers and trace amounts of heterogeneous proteins. A single symmetrical SOD active peak is collected to obtain a high-purity SOD pure enzyme solution.

[0079] (7) Low temperature concentration and vacuum freeze drying: The SOD pure enzyme solution is further concentrated by low temperature ultrafiltration, and then vacuum freeze drying is performed to obtain high purity and high activity protein SOD solid powder.

[0080] In one example, the specific parameters of S1 are set as follows: microwave blanching power 320 W, blanching time 60~120 s; low temperature vacuum drying temperature 40~45℃, vacuum degree 0.08~0.1 MPa, drying to raw material moisture ≤8%; pulverizing through a 40~60 mesh sieve.

[0081] In one example, the specific parameters of S2 are set as follows: feed-to-liquid ratio 1:8 to 1:12 (g / mL); phosphate buffer pH 6.8 to 7.2; metal ion protectant 0.1 mmol / L Cu. 2+ / Zn 2+ Mixture; the compound enzyme consists of cellulase and neutral protease, added at amounts of 0.8%~1.2% and 0.3%~0.5% of the mulberry crude powder mass, respectively; ultrasonic power 300~400 W, coupling temperature 45~50℃, treatment time 60~90 min; low-temperature extraction temperature 4℃, extraction time 2 h; centrifugation speed 8000 rpm, centrifugation time 20 min.

[0082] In one example, the specific parameters of S3 are set as follows: PEG2000 mass fraction of 12%~16% and ammonium sulfate mass fraction of 18%~22% in the aqueous two-phase system; pH of the system is maintained at 7.0; volume ratio of crude extract to aqueous two-phase system is 1:1; stirring for 30 min and standing for 20~30 min to separate into layers.

[0083] In one example, the specific parameters of S4 are set as follows: ceramic microfiltration membrane pore size 0.2 μm; ultrafiltration membrane molecular weight cutoff 30 kDa; and membrane separation temperature ≤ 25℃ throughout the process.

[0084] In one example, the specific parameters of S5 are set as follows: the chromatography equilibration buffer is pH 7.2 phosphate buffer; the gradient elution buffer is pH 7.2 phosphate buffer containing 0~0.5 mol / L NaCl; and the elution flow rate is 2 mL / min.

[0085] In one example, the specific parameters of S6 are set as follows: the gel filtration chromatography eluent is pH 7.0 phosphate buffer, and the elution flow rate is 1.5 mL / min.

[0086] In one example, the specific parameters of S7 are set as follows: freeze-drying temperature -55℃, vacuum degree below 10 Pa, drying time 24 h; finished product moisture content ≤5%.

[0087] The present invention will be further illustrated below with reference to the embodiments.

[0088] Example 1

[0089] 1. Raw material pretreatment: Select 1 kg of fresh protein mulberry leaves, wash and drain them, microwave them at 320 W for 75 s to kill the green, vacuum dry them at 42℃ until the moisture content is 7.5%, pulverize them through a 50-mesh sieve to obtain 820 g of protein mulberry coarse powder.

[0090] 2. Ultrasonic-enzymatic cell disruption: Add pH 7.0 phosphate buffer (containing 0.1 mmol / L Cu) at a material-to-liquid ratio of 1:10. 2+ / Zn 2+ Add 1.0% cellulase and 0.4% neutral protease, sonicate at 350 W for 75 min at 48℃; extract at 4℃ for 2 h, centrifuge at 8000 rpm for 20 min, and take the supernatant to obtain 6.5 L of crude SOD extract.

[0091] 3. Aqueous two-phase enrichment: Prepare a mixture of 14% PEG2000 and 20% ammonium sulfate, with a system pH of 7.0. Mix equal volumes of crude extract, stir for 30 min, let stand for 25 min to separate the phases, and collect 3.2 L of the upper phase enrichment.

[0092] 4. Membrane separation purification: 0.2 μm ceramic microfiltration, followed by 30 kDa ultrafiltration to concentrate to 500 mL of purified concentrate.

[0093] 5. Ion exchange chromatography: Equilibrate the DEAE column with pH 7.2 buffer, elute with a gradient of 0-0.5 mol / L NaCl at a flow rate of 2 mL / min, and collect 120 mL of the active peak.

[0094] 6. Gel filtration chromatography: Sephadex G-75 column eluted with pH 7.0 buffer at a flow rate of 1.5 mL / min, and 80 mL of a single active peak was collected.

[0095] 7. Concentration and freeze drying: Vacuum freeze drying at -55℃ for 24 h yielded 12.8 g of protein mulberry SOD powder.

[0096] Test results: SOD purity 95.6%, enzyme activity 5120 U / mg, total recovery rate 57.3%, moisture 4.2%.

[0097] Example 2

[0098] 1. Raw material pretreatment: Select 2 kg of fresh protein mulberry leaves, wash and drain them, microwave them at 320 W for 60 s to kill the green, vacuum dry them at 40℃ until the moisture content is 7.8%, pulverize them through a 40 mesh sieve to obtain 1650 g of protein mulberry coarse powder.

[0099] 2. Ultrasonic-enzymatic cell disruption: Add pH 6.8 phosphate buffer (containing 0.1 mmol / L Cu) at a material-to-liquid ratio of 1:8. 2+ / Zn 2+ Add 0.8% cellulase and 0.3% neutral protease, sonicate at 300 W for 90 min at 45℃; extract at 4℃ for 2 h, centrifuge at 8000 rpm for 20 min, and take the supernatant to obtain 13.2 L of crude SOD extract.

[0100] 3. Aqueous two-phase enrichment: PEG2000 12%, ammonium sulfate 18%, pH 7.0, and crude extract were mixed in equal volumes, stirred for 30 min, and allowed to stand for 30 min. 6.5 L of the upper phase enrichment solution was collected.

[0101] 4. Membrane separation purification: 0.2 μm ceramic microfiltration + 30 kDa ultrafiltration, yielding 1.1 L of purified concentrate.

[0102] 5. Ion exchange chromatography: The parameters are the same as in Example 1, and the active peak is collected by gradient elution in 250 mL.

[0103] 6. Gel filtration chromatography: The parameters are the same as in Example 1, and 160 mL of a single active peak is collected.

[0104] 7. Concentration and freeze drying: The parameters are the same as in Example 1, yielding 25.3 g of SOD powder.

[0105] Test results: SOD purity 96.2%, enzyme activity 5110 U / mg, total recovery rate 56.1%, moisture 4.5%.

[0106] Verification Example 1: Optimization of process parameters for each stage of the protein-sandwich SOD coupling extraction process

[0107] I. Protein-SOD Coupling Extraction Process

[0108] Process flow: Microwave activated → Ultrasonic enzymatic hydrolysis for synergistic cell disruption → Aqueous two-phase directional enrichment → Membrane fractionation and purification → Bilayer gradient polishing.

[0109] Raw material: Dried protein mulberry leaf powder (obtained by freeze-drying and pulverizing fresh mulberry leaves).

[0110] Initial total SOD activity of raw materials: 16250 U / 100 g, specific activity of raw materials: 185 U / mg total protein, and low temperature operation at 4℃ throughout the process.

[0111] 1. Microwave-controlled pretreatment

[0112] Buffer pH 7.5 phosphate buffer (containing Cu) 2+ and Zn 2+ Each 0.1 mmol / L), material-to-liquid ratio 1:22 (g / mL);

[0113] Microwave power 320 W, intermittent 15 s on and 30 s off, total processing time 110 s, system temperature ≤45℃.

[0114] 2. Synergistic cell disruption via ultrasonic enzymatic hydrolysis

[0115] Cellulase:pectinase = 2:1, total enzyme addition 0.35% (w / w); pH 4.8, temperature 38℃;

[0116] Ultrasonic power 450 W, intermittent ultrasound for 2 s, rest for 3 s, total cell disruption time 28 min;

[0117] Extraction at 4℃ for 2 hours;

[0118] Centrifuge at 8000 r / min for 15 min and collect the supernatant crude enzyme solution.

[0119] 3. Directional enrichment of aqueous two-phase systems

[0120] The system contained 14% PEG2000 and 23% ammonium sulfate, with a pH of 7.1. After standing for 30 minutes to separate the phases, the upper phase was collected.

[0121] 4. Membrane grading and refining

[0122] The solution was concentrated to 500 mL by 0.2 μm ceramic microfiltration followed by 30 kDa ultrafiltration.

[0123] Operating pressure: 0.22 MPa; temperature: 4℃; feed rate: 20 L / h.

[0124] 5. Dual-layer gradient polishing

[0125] First stage: DEAE anion exchange chromatography, equilibrated with 20 mmol / L phosphate buffer (pH 6.8), eluted with a linear gradient of 0–0.5 mol / L NaCl;

[0126] Second stage: Sephadex G-75 molecular sieve chromatography, elution with phosphate buffer (pH 6.8), flow rate 1.0 mL / min.

[0127] II. Results Data Tables for Each Process

[0128] Table 1

[0129]

[0130] III. Key Indicators of Finished Products

[0131] 1. Product SOD specific activity: 5032 U / mg protein;

[0132] 2. Cumulative total recovery rate: 57.3%, cumulative purification fold: 27.20-fold;

[0133] 3. Electrophoretic detection showed a single electrophoretic band, with a Cu / Zn-SOD purity of 96.5%;

[0134] 4. Total polysaccharide removal rate: 93.8%; polyphenol residue: <0.075%; desalination rate: 95.3%;

[0135] 5. Stability: After incubation at 55℃ for 1 h, 92.3% of the enzyme activity remained.

[0136] Verification Example 2: Orthogonal L9 (3) of each work section 4 )test

[0137] The evaluation indicators are uniformly calculated using a comprehensive score: Comprehensive score = 0.6 × specific vitality score + 0.4 × recovery rate score.

[0138] I. Microwave-controlled liveness preprocessing of orthogonal raw data

[0139] Table 2

[0140]

[0141] Factors: A. Microwave power, B. Processing time, C. Material-to-liquid ratio, D. System pH.

[0142] The optimal combination is A5B5C5D5.

[0143] II. Orthogonal raw data of L9 cell wall disruption synergistic ultrasonic enzymatic hydrolysis

[0144] Table 3

[0145]

[0146] Factors: A. Total amount of enzyme added, B. Ultrasonic power, C. Enzymatic hydrolysis temperature, D. Cell disruption time.

[0147] The optimal combination is A5B5C5D5.

[0148] III. Directional enrichment of L9 orthogonal raw data in aqueous two-phase system

[0149] Table 4

[0150]

[0151] Factors: A. PEG2000, B. Ammonium sulfate, C. System pH, D. Phase separation time.

[0152] The optimal combination is A5B5C5D5.

[0153] IV. Orthogonal raw data for membrane fractionation and purification (three factors)

[0154] Table 5

[0155]

[0156] Factors: A. Operating pressure, B. Circulation temperature, C. Feed flow rate.

[0157] The optimal combination is A5B5C5D5.

[0158] V. Verification of Experimental Results (Optimal Combination in Trial Parallel)

[0159] Table 6

[0160]

[0161] VI. Summary of Orthogonal Optimal Combinations

[0162] 1. Optimal microwave processing parameters: 320 W, 110 s, feed-to-liquid ratio 1:28, pH 7.0;

[0163] 2. Optimal ultrasonic enzymatic hydrolysis: enzyme addition 0.35%, 320 W, 38℃, 28 min;

[0164] 3. Optimal aqueous two-phase solution: PEG2000 14%, ammonium sulfate 23%, pH 7.0;

[0165] 4. Optimal membrane separation conditions: pressure 0.22 MPa, 4℃, feed flow rate 25 L / h, dual membrane combination;

[0166] 5. The chromatography uses linear gradient elution, which effectively removes trace amounts of impurities, proteins and pigments, resulting in a specific activity exceeding 5000 U / mg.

[0167] Comparative Example: Traditional Craftsmanship

[0168] 1 kg of protein-rich mulberry leaves were extracted by high-temperature steaming at 100℃ for 30 min, strong alkali extraction, conventional salting out, single ion exchange chromatography, and concentrated and freeze-dried to obtain 9.2 g of powder.

[0169] Test results: purity 58.2%, enzyme activity 1850 U / mg, total recovery rate 32.5%.

[0170] The comparison shows that the process of this invention has significant differences and substantial technological progress in terms of purity, enzyme activity, and recovery rate.

[0171] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing SOD, characterized in that, include: S1: Mix the buffer solution, complex enzyme and crude protein powder, sonicate, extract, centrifuge, and collect the supernatant to obtain crude SOD extract; S2: Mix the aqueous two-phase system with the crude SOD extract, separate the layers, and collect the upper phase enrichment. S3: The upper phase enrichment solution is filtered sequentially through a microfiltration membrane and an ultrafiltration membrane to separate and obtain a refined SOD concentrate; S4: The SOD purified concentrate is subjected to ion exchange chromatography, and the elution fractions with SOD characteristic activity are collected in segments to obtain the SOD active peak solution. S5: The SOD active peak solution was subjected to Sephadex G-75 gel filtration chromatography to obtain pure SOD enzyme; The buffer solution contains a metal ion protectant, which includes copper ions and / or zinc ions; The complex enzyme includes at least one of cellulase, neutral protease, and pectinase. The protein-rich mulberry powder is sterilized by microwave; The polymer in the aqueous two-phase system is PEG, and the hydrophilic salt is at least one of ammonium sulfate and K2HPO4.

2. The preparation method according to claim 1, characterized in that, The method for preparing the protein mulberry crude powder includes: Fresh protein mulberry leaves are microwave-sterilized, then vacuum-dried, pulverized, and sieved to obtain the protein mulberry coarse powder.

3. The preparation method according to claim 2, characterized in that, include: The power of the microwave is 280~800 W; The blanching time is 60-120 seconds; The vacuum drying conditions are 40~45℃ and vacuum degree 0.08~0.1 MPa.

4. The preparation method according to claim 1, characterized in that, It also includes a step of vacuum freeze-drying SOD purified enzyme.

5. The preparation method according to claim 4, characterized in that, The conditions for vacuum freeze drying include: -55°C, vacuum degree below 10 Pa, for 24 hours.

6. The preparation method according to claim 1, characterized in that, In S1: The ratio of the crude protein mulberry powder to the buffer solution is 1:8 to 1:

12. The buffer solution is a phosphate buffer solution with a pH of 6.8 to 7.2; The metal ion agent includes copper ions and zinc ions, wherein the concentration of copper ions in the buffer solution is 0.1 mmol / L, and the concentration of zinc ions in the buffer solution is 0.1 mmol / L. The mass of the compound enzyme is 0.3% to 1.2% of the crude protein mulberry powder; The ultrasonic power is 350~550 W and the temperature is 32~50℃.

7. The preparation method according to claim 1, characterized in that, In S2: The mass fraction of PEG in the aqueous two-phase system is 10%~18%; The PEG includes PEG2000; The mass fraction of the hydrophilic salt in the aqueous two-phase system is 19%~27%; The pH of the aqueous two-phase system is 6.5~7.7; The volume ratio of the aqueous two-phase system to the crude SOD extract is 1:

1.

8. The preparation method according to claim 1, characterized in that, In S3: The microfiltration membrane has a pore size of 0.2 μm or a molecular weight cutoff of 100 kDa; The ultrafiltration membrane has a molecular weight cutoff of 30 kDa; The temperature of the filter is ≤25℃.

9. The preparation method according to claim 1, characterized in that, In S4: The ion exchange chromatography was DEAE-Sepharose Fast Flow anion exchange chromatography; The chromatographic equilibration solution for the ion exchange chromatography was a pH 7.2 phosphate buffer solution. The gradient elution buffer for the ion exchange chromatography is a pH 7.2 phosphate buffer containing 0–0.5 mol / L NaCl, and the elution flow rate is 2 mL / min.

10. The preparation method according to claim 1, characterized in that, In S5: The elution buffer for the Sephadex G-75 gel filtration chromatography was pH 7.0 phosphate buffer, and the elution flow rate was 1.5 mL / min.

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