A method for simultaneously preparing and separating leaf protein and glycoprotein

Abstract

The present invention discloses a method for simultaneously preparing and separating leaf protein and glycoprotein, belonging to the technical field of deep processing of plant leaf protein. After crushing plant leaves, the present invention adds an isostatic flow autoclave for reaction, uses ultracentrifugation to remove grass dregs and chlorophyll, filters through a hydrophilic composite membrane of polytetrafluoroethylene membrane, purifies the protein in plant leaves by using resin, and elutes with different ethanol aqueous solution concentrations to simultaneously prepare glycoprotein and leaf protein. The method of the present invention reduces the use and loss of organic reagents, has lower costs, has a high yield and high purity of the prepared glycoprotein, and has excellent antioxidant properties, can be widely applied to the food field, and has extremely high economic value and application value.

Description

Technical Field

[0001] The present invention relates to a method for simultaneously preparing and separating leaf protein and glycoprotein, and belongs to the technical field of deep processing of plant leaf protein. Background Art

[0002] Glycoproteins extracted from plants are a class of natural proteins rich in glycosyl groups, which contain rich bioactive components and potential medical, health care and beauty applications. The unique properties of plants as biological resources make them an important source of glycoproteins. Glycoproteins extracted from plants are natural and green, originating from natural plant resources and being closer to human physiological characteristics. By deeply exploring the glycoprotein composition of different plants and exploring their mechanisms of action in regulating the immune system, antioxidant, anti-inflammatory, anti-aging and other aspects, researchers have provided a solid scientific basis for the future development of application products of plant-extracted glycoproteins.

[0003] Plant tissues are rich in cell walls, cell membranes, cytoplasm and other parts, which contain abundant proteins, and these proteins often bind to glycosyl groups to form the structure of glycoproteins. This natural glycoprotein structure not only endows it with special biological activities, but also shows broad application prospects in the fields of functional foods, health products and beauty products. The differences in the activities and functions of glycoproteins are mainly manifested in the biological activities of their glycosyl groups, which endow glycoproteins with many unique biological functions. When preparing products, glycoproteins extracted from plant proteins are often widely used in the fields of medicine, health care and beauty products. Due to their natural source, antioxidant properties and cell-regulating functions, glycoproteins have become ideal ingredients for preparing functional foods, health products and beauty products. These products may involve multiple aspects such as anti-aging, immune regulation, skin care, etc., expanding new possibilities for the application of plant proteins, and at the same time promoting in-depth research on the interaction mechanism between glycoproteins and plant proteins.

[0004] It is difficult to separate glycoproteins from plant proteins, mainly because there are many different proteins in plant tissues, and glycoproteins are only a small part of them. The difficulty lies in the need for efficient technical means to selectively extract and purify this type of protein without affecting the integrity of other proteins. Due to the wide variety and complex structure of plant proteins, separating glycoproteins with a relatively high purity often requires the combination of multiple biochemical and separation techniques, such as column chromatography, Sephadex, etc., making it a complex and time-consuming task.

[0005] Patent CN110343156B discloses a method for extracting and purifying black bean glycoprotein, which uses water extraction and alcohol precipitation, and then uses DEAE 52 cellulose ion column chromatography for preliminary separation, and dextran gel column G 100 purification. Patent CN111171126A discloses a method for extracting coix seed glycoprotein, which also uses water extraction and alcohol precipitation, and assists Sevage reagent to remove free protein and dialysis purification. Patent CN115232191A discloses a method for extracting Codonopsis pilosula glycoprotein with antioxidant activity, which uses Tris HCl buffer extraction, Sevage reagent to remove free protein, and ammonium sulfate precipitation to remove polysaccharides to remove impurities. Patent CN103554237B discloses an active glycoprotein extracted from purslane and its preparation method, which uses NaCl extraction, ultrafiltration concentration, and ethanol precipitation. The above method consumes a large amount of reagents to extract glycoproteins, has a low yield, and is not easy to achieve industrial production under low-cost conditions.

[0006] Therefore, there is an urgent need to develop a method that can efficiently prepare and separate glycoproteins while also reducing the loss of other proteins in plants. This method can be widely used in the food field and has extremely high economic and application value. Summary of the invention

[0007] To solve the above problems, the present invention adds isostatic flow autoclave high-pressure extraction after crushing plant leaves, uses ultracentrifugation to remove grass residues and chlorophyll, filters through polytetrafluoroethylene membrane (PTFE) hydrophilic composite membrane, uses resin to purify protein in plant leaves, elutes through different ethanol aqueous solution concentrations, and simultaneously prepares glycoprotein eluate and leaf protein eluate, and then concentrates and dries to obtain glycoprotein and leaf protein respectively. The method of the present invention realizes the simultaneous preparation and separation of glycoprotein and leaf protein in plant leaves, improves the preparation and separation efficiency of protein, and is suitable for industrial production.

[0008] Leaf-eating grass, also known as leafy vegetables, has high protein content and a complete range of amino acids.

[0009] The first object of the present invention is to provide a method for preparing and separating glycoprotein and leaf protein, comprising the following steps:

[0010] S1 Raw material pretreatment: adding sodium metabisulfite and CaCl2·2H2O to crush plant leaves, and high pressure homogenization to obtain straw pulp;

[0011] S2 high pressure extraction: adding the straw pulp into an isostatic pressure autoclave for high pressure extraction to obtain the treated straw pulp;

[0012] S3: remove chlorophyll: ultracentrifuge the grass pulp at 15000-17000 r / min for 60-90 min to obtain grass juice;

[0013] S4 Microfiltration: Filter the grass juice using a hydrophilic composite membrane of polytetrafluoroethylene membrane, with a filtration pressure of 0.05 - 0.2 Mpa, to obtain the filtered grass juice;

[0014] S5 Resin Purification: Use resin to adsorb the filtered grass juice, first elute with an ethanol aqueous solution of 20 - 25% v / v, collect the glycoprotein eluate, then elute with an ethanol aqueous solution of 65 - 75% v / v and collect the eluate, collect the leaf protein eluate;

[0015] S6 Rotating Cross - Flow Membrane Separation: Ultrafilter the glycoprotein eluate and the leaf protein eluate respectively to obtain a glycoprotein concentrate and a leaf protein concentrate;

[0016] S7 Drying: Dry the glycoprotein concentrate and the leaf protein concentrate respectively to obtain leaf protein and glycoprotein.

[0017] In one embodiment, the plant leaves in S1 are Patrinia heterophylla.

[0018] In one embodiment, wet grinding is used in S1, and the fineness of the grass residue after grinding is 80 - 120 mesh.

[0019] In one embodiment, the dosage ratio of sodium metabisulfite, CaCl2·2H2O, and plant leaves in S1 is 1.0 - 2.0 g : 100 - 200 mM : 1 kg.

[0020] In one embodiment, the reaction conditions of the isostatic pressure flow autoclave in S2 are under 500 - 600 MPa and at 50 - 60 °C for high - pressure extraction for 40 - 50 min.

[0021] In one embodiment, the pore size of the PTFE hydrophilic composite membrane in S4 is 0.2 - 1 μm.

[0022] In one embodiment, the temperature during ultrafiltration in S4 is controlled not to exceed 30 °C, and the pressure is 0.03 - 0.6 Mpa.

[0023] In one embodiment, the mass ratio of the grass juice to the resin in S5 is 1:(0.5 - 10); the adsorption flow rate is 1 BV / h. After adsorption, first elute with an ethanol aqueous solution of 20 - 25% v / v for 2 - 3 BV, the desorption flow rate is 1 BV / h, collect the eluate to obtain glycoprotein; then elute with an ethanol aqueous solution of 65 - 75% v / v for 2 - 3 BV, the desorption flow rate is 1 BV / h, collect the eluate to obtain leaf protein. BV represents the column volume.

[0024] In one embodiment, the pore size of the ultrafiltration membrane in S6 is 0.01 - 0.1 μm.

[0025] In one embodiment, the pressure of ultrafiltration in S6 is 0.01 - 0.1 Mpa, the pumping - stopping ratio is (8 - 9):(1 - 2), that is, pumping for 8 - 9 minutes and pausing for 1 - 2 minutes; the rotational speed of the ultrafiltration membrane separation turntable is 10 - 20 r / min; the ultrafiltration membrane material is polyvinylidene fluoride (PVDF) or PTFE.

[0026] The second object of the present invention is to provide a glycoprotein prepared by any of the above - mentioned methods.

[0027] The present invention also provides a composition containing the above - mentioned glycoprotein.

[0028] The present invention also provides the use of the above - mentioned glycoprotein or the above - mentioned composition in the preparation of protein products.

[0029] In one embodiment, the protein product is food, health food or medicine.

[0030] In one embodiment, the protein product is a protein supplement, feed or food additive.

[0031] In one embodiment, the protein product has at least one of the following functions:

[0032] (a) Antioxidation;

[0033] (b) Immunity regulation;

[0034] (c) Anti - inflammation.

[0035] The third object of the present invention is to provide a method for improving the antioxidant property of plant - derived glycoprotein, which is characterized by comprising the following steps:

[0036] S1 Raw material pretreatment: Mix and crush sodium metabisulfite, CaCl2·2H2O and plant leaves, and perform high - pressure homogenization to obtain grass pulp;

[0037] S2 High - pressure extraction: Add the grass pulp into an isostatic - flow high - pressure autoclave for high - pressure extraction to obtain the treated grass pulp;

[0038] S3 Chlorophyll removal: Centrifuge the treated grass pulp at 15000 - 17000 r / min for 60 - 90 min to obtain grass juice;

[0039] S4 Microfiltration: Filter the grass juice using a polytetrafluoroethylene membrane hydrophilic composite membrane to obtain the filtered grass juice;

[0040] S5 Resin purification: Adsorb the filtered grass juice using resin; after adsorption, first elute with an ethanol aqueous solution with a concentration of 20 - 25% v / v to collect the glycoprotein eluate; then elute with an ethanol aqueous solution with a concentration of 65 - 75% v / v to collect the leaf protein eluate;

[0041] S6 Rotary cross-flow membrane separation: Ultrafilter the glycoprotein eluate and leaf protein eluate respectively to obtain a glycoprotein concentrate and a leaf protein concentrate;

[0042] S7 Drying: Dry the glycoprotein concentrate and the leaf protein concentrate respectively to obtain leaf protein and glycoprotein with good antioxidant properties.

[0043] Beneficial effects

[0044] In the present invention, after crushing plant leaves, an isostatic flow autoclave reaction is carried out. Ultracentrifugation is used to remove grass residues and chlorophyll, and filtration is carried out through a PTFE hydrophilic composite membrane. Resin is used to purify the protein in plant leaves, and elution is carried out with ethanol aqueous solutions of different concentrations. At the same time, glycoprotein and leaf protein are prepared, and then glycoprotein and leaf protein are obtained through concentration and drying respectively.

[0045] The glycoprotein and leaf protein of the present invention have the following advantages:

[0046] (1) In the present invention, organic reagent ethanol or Sevage reagent is not used for the extraction of glycoprotein, and only low-concentration ethanol aqueous solution is used during protein concentration and separation, reducing reagent loss and environmental impact;

[0047] (2) In the present invention, the purification of glycoprotein is achieved by combining ultrafiltration and resin. Both are suitable for industrial production, and the cost is relatively low. The resin can be recycled;

[0048] (3) The glycoprotein and leaf protein prepared by the present invention have high purity and yield and light color; specifically, the yield of glycoprotein reaches more than 13.1%, the purity reaches more than 76.9%, the yield of leaf protein reaches more than 67.4%, the purity reaches more than 85.2%, and the total protein yield reaches more than 75.1%;

[0049] (4) The glycoprotein and leaf protein prepared by the present invention have high purity and yield and light color; specifically, the yield of glycoprotein reaches more than 12.5%, the purity reaches more than 76.9%, and at the same time, leaf protein is prepared and separated, the yield reaches more than 64.3%, the purity reaches more than 85.0%, and the total yield reaches more than 77.1%. Description of the drawings

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0051] Figure 1It is the elution separation curve of the glycoprotein and leaf protein of the present invention;

[0052] Figure 2 It is the silver staining map and SDS electrophoresis map of the glycoprotein of the present invention, and the SDS electrophoresis map of the leaf protein;

[0053] Figure 3 It is the liquid phase map of the glycoprotein of the present invention;

[0054] Figure 4 They are the liquid phase map and molecular weight map of the standard protein; Figure 4 (a) is the liquid phase map of the standard protein; Figure 4 (b) is the standard curve of molecular weight vs. peak elution time;

[0055] Figure 5 It is the infrared spectrum map of the glycoprotein of the present invention. Detailed implementation mode

[0056] The present invention will be further described below in conjunction with examples, but the implementation modes of the present invention are not limited thereto. Any extraction of glycoprotein is within the scope of the present invention.

[0057] 1. Qualitative test of protein

[0058] (1) Biuret reaction

[0059] Add biuret reagent (0.1 g / mL NaOH and 0.01 g / mL CuSO4 solution) to the protein solution of 2 mg / mL, and detect the color change.

[0060] (2) Detection of acidic polysaccharides

[0061] Drop cetyltrimethylammonium bromide (CTAB) solution into the protein solution of 2 mg / mL, and detect the color change.

[0062] (3) Detection of tannic acid by ferric chloride reaction

[0063] Drop 1% v / v ferric chloride solution into 3 mL of the protein solution of 2 mg / mL, mix well, and detect the color change.

[0064] (4) Iodine-potassium iodide reaction

[0065] Drop 1% v / v iodine-potassium iodide solution into the protein solution of 2 mg / mL, and detect the color change.

[0066] (5) Infrared spectrum detection

[0067] Mix 1 - 2 mg of the protein sample with 100 mg of potassium bromide, grind it in a mortar, take a little and press it into a tablet in a mold, and perform infrared spectrum scanning in the range of 400 - 4000 cm -1 interval.

[0068] (6) High performance liquid chromatography

[0069] Prepare a 2 mg / mL protein solution (containing 1% w / v SDS) with 0.1 M sodium phosphate buffer at pH 6.8, and filter it through a 0.45 μm filter membrane. Use a TSK G4000-SW chromatographic column, and the mobile phase is 0.1 M sodium phosphate buffer containing 0.1% w / v SDS at pH 6.8. The flow rate is 0.7 mL / min -1 , and record the absorbance at 220 nm.

[0070] (7) SDS-PAGE and periodic acid-silver staining method

[0071] The electrophoresis conditions are 4% stacking gel and 12.5% separating gel; the working voltage of the stacking gel is 80 V, and the working voltage of the separating gel is 120 V. Glycoprotein staining uses the periodic acid-silver nitrate staining method, with glucoamylase as the positive control and whey protein as the negative control. After SDS gel electrophoresis, the gel is fixed with 10% v / v acetic acid and 25% v / v isopropanol solution for 30 min, 10 g / L periodic acid solution is added and reacted at 4 °C for 1 h. After the reaction, it is repeatedly rinsed with distilled water (about 3 h), silver stained with 250 mL 2.5 g / L silver nitrate solution for 10 min (add 100 μL 37% formaldehyde before use), washed with distilled water 3 times, and then developed with 250 mL 25 g / L anhydrous sodium carbonate solution (add 50 μL 37% formaldehyde before use). Before the whey protein changes color, soak it in 14.6 g·L -1 EDTA disodium solution for 10 min to terminate the reaction.

[0072] (8) Determination of polysaccharide content

[0073] The phenol-sulfuric acid method is used to determine the sugar content in the sample. Dilute the sample to an appropriate concentration, add 1 mL 5% phenol and 5 mL concentrated sulfuric acid, react at room temperature for 20 minutes, and measure the absorbance at 490 nm.

[0074] (9) Determination of protein content

[0075] Determined using a Bradford protein concentration assay kit.

[0076] (10) Calculation of the yields of protein and glycoprotein and glycoprotein content

[0077] Yield of leaf protein = (content of crude protein in leaf protein / content of protein in fresh leaves) * 100%;

[0078] Yield of glycoprotein = (content of crude protein in glycoprotein / content of protein in fresh leaves) * 100%;

[0079] The content of glycoprotein = (mass of sugar + mass of protein) / (total mass of glycoprotein) * 100%;

[0080] The total yield = yield of leaf protein + yield of glycoprotein.

[0081] (11) Detection of in vitro antioxidant activity of glycoprotein

[0082] Determination of DPPH radical scavenging ability: Prepare a protein sample solution with a concentration of 0.5 mg / mL, and react it with 2 mL of DPPH solution (0.2 mM, 95% ethanol aqueous solution) in the dark for 30 min (A0): React 2 mL of 95% v / v ethanol aqueous solution for 30 min (A1); React 2 mL of DPPH solution with 2 mL of distilled water for 30 min (A2). Measure the absorbance at 517 nm to obtain A0 - A2, using VC as a control. The calculation formula is:

[0083] DPPH scavenging rate (%) = (1 - (A0 - A1) / A2) × 100

[0084] Determination of hydroxyl radical scavenging ability: Prepare protein sample solutions with concentrations of 0, 0.1, 0.5, 1, 1.5 mg / mL, and react each with 0.5 mL of 9 mmol / L FeSO4 and 8.8 mM H2O2 for 10 minutes. Then add 0.5 mL of 9 mmol / L salicylic acid ethanol and react for 30 min. Measure the absorbance value at a wavelength of 510 nm. A0 is the absorbance with distilled water instead of the sample; A i Sample absorbance; A j Absorbance with distilled water instead of FeSO4 solution. Use VC as a control. The calculation formula is:

[0085] Hydroxyl radical scavenging rate (%) = (1 - (A i -A j ) / A0) × 100

[0086] 2. Sources of equipment and materials

[0087] Isostatic press flow autoclave: Purchased from Avure Technologies AB, USA, Isostatic press Flow Autoclave System;

[0088] PTFE hydrophilic composite membrane: Purchased from Microporous BioTechnology Co., Ltd., model: FPL010B51;

[0089] D3520 series macroporous resins: Purchased from Tianjin Yunkai Resin Co., Ltd., including D3520;

[0090] Rotating cross-flow membrane separation equipment: Model MXZ400 / 24;

[0091] Whey protein: purchased from Roquette Frères (France);

[0092] Glucoamylase: purchased from Aladdin, liquefied type, 100,000 u / mL.

[0093] Example 1: Separation and preparation of glycoprotein and leaf protein

[0094] A method for simultaneously preparing and separating glycoprotein and leaf protein comprises the following steps:

[0095] S1. Raw material pretreatment: plant leaves, sodium pyrosulfite and CaCl2·2H2O are mixed, wherein the amount ratio of sodium pyrosulfite, CaCl2·2H2O and plant leaves is 1.0g:100:1kg, wet-crushing the edible grass, and homogenizing at 100Mpa for 20min to obtain grass pulp;

[0096] S2 high pressure extraction: the straw pulp obtained in S1 was subjected to an isostatic pressure autoclave at 500 MPa and 60°C for 40 min to obtain treated straw pulp;

[0097] S3: remove chlorophyll: ultracentrifuge the grass pulp treated with S2 at 17000r / min for 60min to remove chlorophyll and grass residue to obtain grass juice;

[0098] S4 microfiltration: the grass juice obtained in S3 is filtered using a 0.2 μm polytetrafluoroethylene (PTFE) hydrophilic composite membrane at a pressure of 0.1 MPa; the grass juice after filtration;

[0099] S5 resin purification: The grass juice filtered by S4 was adsorbed by D3520 series macroporous resin, with the weight ratio of grass juice to resin being 1:3 and the adsorption flow rate being 1BV / h. After adsorption, 2BV was eluted with 25% v / v ethanol aqueous solution at a decomposition flow rate of 1BV / h to collect the glycoprotein eluate; 2BV was eluted with 75% v / v ethanol aqueous solution at a decomposition flow rate of 1BV / h to collect the leaf protein eluate; the column volume was 20mL.

[0100] S6 Rotary Cross-flow Membrane Separation: The glycoprotein eluate and leaf protein eluate obtained from S5 were subjected to ultrafiltration respectively. The ultrafiltration membrane material was PTFE hydrophilic composite membrane. The size of the ultrafiltration membrane was 0.05 μm. The operating pressure was 0.06 MPa. The filtration was repeated for 8 minutes and then paused for 2 minutes. The ultrafiltration membrane separation turntable rotated at a speed of 15 r / min.

[0101] S7 drying: vacuum drying the concentrated solution obtained in S6 to obtain leaf protein and glycoprotein respectively;

[0102] The elution results of glycoprotein and leaf protein in S5 are as follows Figure 1 As shown, fromFigure 1 It can be seen that: for the first 40 mL, an ethanol aqueous solution with 25% v / v was used for elution. The curves of protein and sugar were consistent, indicating that the glycoprotein was eluted at this time. After eluting 40 mL, the sugar content was 0, and when eluting to 40 - 60 mL, leaf protein was eluted. By using different eluent concentrations, the separation of glycoprotein and leaf protein was achieved.

[0103] Example 2: Separation and preparation of glycoprotein and leaf protein

[0104] On the basis of Example 1, the addition amount of sodium metabisulfite in S1 was changed to 2.0 g / kg of raw material, and the addition amount of CaCl2·2H2O was 200 mM; the centrifugation conditions in S3 were centrifugation at 15000 r / min for 90 min, the pore size of the hydrophilic composite polytetrafluoroethylene membrane in S4 was 0.45 μm, and the pressure was 0.4 Mpa. The pore size of the ultrafiltration membrane in S6 was 0.01 μm, and leaf protein and glycoprotein were prepared.

[0105] Example 3: Separation and preparation of glycoprotein and leaf protein

[0106] On the basis of Example 1, the pore size of the hydrophilic composite polytetrafluoroethylene membrane in S4 was changed to 1 μm, and the filtration pressure was 0.6 MPa; in S5, an ethanol aqueous solution with 20% v / v was first used for elution, and then an ethanol aqueous solution with 65% v / v was used for elution; the size of the ultrafiltration membrane in S6 was 0.1 μm, and leaf protein and glycoprotein were prepared.

[0107] Comparative Example 1: Influence of ultracentrifugation speed

[0108] On the basis of Example 1, the centrifugation conditions in S3 were changed to centrifugation at 10000 r / min for 120 min, and leaf protein and glycoprotein were prepared. The purity and yield of leaf protein and glycoprotein were detected.

[0109] Comparative Example 2: Influence of different microfiltration membranes

[0110] On the basis of Example 1, the microfiltration membrane in S4 was changed to polyester membrane, polypropylene membrane, and ceramic membrane with the same pore size, and leaf protein and glycoprotein were prepared. The purity and yield of leaf protein and glycoprotein were detected.

[0111] Comparative Example 3: Other methods for preparing glycoprotein

[0112] 1. Water extraction method (reference can be made to - Research on the extraction process of glycoprotein from sweet potato leaves [J]. Cereals & Oils, 2023, 36(01): 135 - 138.)

[0113] S1 Pretreatment: Wash the naturally air - dried leaves of Atriplex hortensis, dry them in a drying oven at 70°C, crush them, and pass through a 0.15 mm sieve.

[0114] S2 Water extraction: Add distilled water to the pretreated sample according to a certain liquid-to-solid ratio, perform water bath extraction at a certain temperature for a period of time, filter while it is hot, record the volume of the filtrate, and reserve the filtrate for later use;

[0115] S3 Ethanol precipitation: Centrifuge the filtrate after water extraction at 10000 r / min for 5 min, take the supernatant, then add 95% (v / v) ethanol aqueous solution for precipitation, let it stand overnight at 4°C, centrifuge again at 10000 r / min for 20 min, and dissolve the precipitate in 60 mL of distilled water;

[0116] S4 Sevage reagent treatment: Take 3 mL of the dissolved solution, according to the volume ratio of dissolved solution:Sevage reagent = 3:1 (the volume ratio of chloroform and n-butanol in Sevage reagent is 3:1 and mix well), centrifuge at 10000 r / min for 5 min, repeat 3 times, and take the supernatant for later use;

[0117] Step 5, Dialysis: Take the solution in Step 4 and dialyze it with distilled water for 24 h, centrifuge the liquid inside the dialysis bag, and vacuum dry the supernatant to obtain the crude glycoprotein.

[0118] 2. Salt extraction method (for reference: Comparative study on extraction methods of glycoprotein from Dendrobium officinale Kimura et Migo [J]. Anhui Agricultural Science Bulletin, 2022, 28(02): 24 - 26)

[0119] S1 Pretreatment: Cut the leaves of Atriplex hortensis into pieces, add 16 times the volume of NaCl solution, soak overnight in a 4°C refrigerator, and filter to obtain the clear liquid;

[0120] S2 Ammonium sulfate fractional precipitation: Weigh 2 times the leaf weight of ammonium sulfate and slowly add it (while stirring) to the supernatant until it is completely dissolved, place it in a 4°C refrigerator overnight, centrifuge (4000 r / min, 40 min). After centrifugation, collect the precipitate. Slowly add 3 times the leaf weight of ammonium sulfate to the supernatant for secondary precipitation (while stirring) until it is completely dissolved, place it in a 4°C refrigerator overnight, centrifuge (4000 r / min, 40 min), and collect the secondary precipitate. Then add 3 times the leaf weight of ammonium sulfate to the supernatant again (while stirring) for tertiary precipitation, after placing it in a 4°C refrigerator overnight, centrifuge (4000 r / min, 40 min), and collect the tertiary precipitate;

[0121] S3 Dialysis: Redissolve the precipitate in water for dialysis, concentrate the glycoprotein extraction solution in the dialysis bag with polyethylene glycol 10000, and then mix and dry to obtain the glycoprotein.

[0122] 3. Alcohol extraction method (for reference: Patent CN103554237B: An active glycoprotein extracted from Portulaca oleracea L. and its preparation method)

[0123] S1 Pretreatment: Take 2 g of Portulaca oleracea L., add 4 mL of 4.5% w / v NaCl solution, grind it into a paste, then add another 16 mL of NaCl solution, and stir evenly to obtain a grinding liquid;

[0124] S2 Centrifugation: Stir the above grinding liquid at 200 rpm for 4 h, then centrifuge it at 3000 rpm for 20 min to obtain supernatant and precipitate; Repeat step S1 three times to obtain supernatant, combine the supernatants to get an extract;

[0125] S3 Ultrafiltration: Use an ultrafilter to concentrate and desalt the above extract, where the cut-off molecular weight of the ultrafilter is 5 kDa;

[0126] S4 Ethanol precipitation: Add the pre-cooled absolute ethanol at 10 °C to the concentrated solution, precipitate for 4 h, then centrifuge it at 2000 rpm for 20 min to obtain a precipitate, and lyophilize the precipitate to obtain glycoprotein.

[0127] 4. Ethanol extraction and chromatography method (for reference, see Patent CN110343156B: Extraction and purification method of black bean glycoprotein)

[0128] S1: Dry and crush Portulaca oleracea L. to a powder particle size of 0.1 - 1 mm;

[0129] S2: Take the powder and add absolute ethanol according to a solid-liquid ratio of 1:5 g / mL for defatting treatment, repeat 2 - 3 times;

[0130] S3: Extract the defatted filter residue with 8 times the volume of distilled water at 80 °C for 2 h, filter to obtain the filtrate, then concentrate it 10 times with a rotary evaporator, add 4 times the volume of absolute ethanol at 4 °C, completely precipitate for 24 h, filter the precipitate, collect the filter residue, dissolve it with distilled water, freeze it at -40 °C for 1 day, and then vacuum dry it at -50 °C;

[0131] S4: Take the above freeze-dried powder, completely dissolve it with distilled water, dialyze it and concentrate it with a rotary evaporator until no glycoprotein precipitate precipitates, centrifuge it and take the supernatant;

[0132] S5: C1-type DEAE-52 cellulose column chromatography, elute with 0.2 mol / L NaCl solution; Collect the symmetric overlapping peaks and lyophilize them;

[0133] S6: Dissolve the dry powder obtained in S5 with distilled water, treat it with a Sephadex G-100 column, elute with 0.5 mol / L NaCl, collect the symmetric overlapping peaks, and lyophilize to obtain glycoprotein.

[0134] Comparative Example 4: Do not use an isostatic flow autoclave for processing

[0135] On the basis of Example 1, S2 was omitted to prepare glycoprotein. The purity and yield of the glycoprotein were detected, and the results are shown in Table 1.

[0136] Comparative Example 5: Influence of dry grinding

[0137] On the basis of Example 1, the treatment method of Portulaca oleracea was changed to dry grinding to a powder particle size of 0.1 - 1 mm to prepare glycoprotein. The purity and yield of the glycoprotein were detected, and the results are shown in Table 1.

[0138] The leaf protein and glycoprotein obtained by this method were inferior to those in the examples, probably because during the drying process, the binding mode or force between pigment substances and proteins changed, making it difficult to separate the pigments better.

[0139] Comparative Example 6: Influence of different eluents

[0140] On the basis of Example 1, in S5, it was first eluted with a 25% v / v methanol aqueous solution and then with a 75% v / v methanol aqueous solution to prepare glycoprotein. The purity and yield of the glycoprotein were detected, and the results are shown in Table 1.

[0141] The purity and yield of the leaf protein and glycoprotein prepared in Examples 1 - 3 and Comparative Examples 1, 2, 4 - 7 were detected, and the results are shown in Table 1.

[0142] Table 1 Protein purity and yield

[0143] Yield of glycoprotein Purity of glycoprotein Yield of leaf protein Purity of leaf protein Total yield Example 1 13.1％ 80.5％ 65.4％ 86.5％ 78.50％ Example 2 12.5％ 77.3％ 65.7％ 85.2％ 78.20％ Example 3 12.8％ 76.9％ 64.3％ 85.0％ 77.10％ Comparative Example 1 - 10000 r / min 11.6％ 70.5％ 58.9％ 76.2％ 70.50％ Comparative Example 2 - Polyester membrane 8.0％ 60.7％ 56.2％ 72.5％ 64.20％ Comparative Example 2 - Polypropylene membrane 8.2％ 60.4％ 57.2％ 71.7％ 65.40％ Comparative Example 2 - Ceramic membrane 7.6％ 58.5％ 53.05％ 68.4％ 60.65％ Comparative Example 3 - Water extraction method 6.5％ 55.7％ - - - Comparative Example 3 - Salt extraction method 5.0％ 53.7％ - - - Comparative Example 3 - Alcohol extraction method 6.0％ 56.3％ - - - Comparative Example 3 - Alcohol extraction and chromatography 7.5％ 68.7％ - - - Comparative Example 4 7.2％ 63.1％ 48.4％ 62.1％ 55.6％ Comparative Example 5 9.5％ 64.2％ 55.9％ 60.7％ 65.4％ Comparative Example 6 6.4％ 65.6％ 44.8％ 67.3％ 51.2％

[0144] Note *: "-" in the table indicates no relevant data.

[0145] As can be seen from Table 1, the yield of the glycoprotein prepared in Examples 1 - 3 reached over 12.5%, the purity reached over 76.9%, while the leaf protein was prepared and separated, the yield reached over 64.3%, the purity reached over 85.0%, and the total yield reached over 77.1%, significantly higher than the extraction of glycoprotein in Comparative Examples 1 - 7. The yield and purity of the glycoprotein and leaf protein prepared using polyester film, polypropylene film, and ceramic membrane were much lower than those of the PTFE hydrophilic composite membrane; reducing the centrifugation speed would lead to a decrease in the yield and purity of the glycoprotein and leaf protein, and the color of the prepared glycoprotein and leaf protein was poor.

[0146] Example 4: Detection of glycoprotein properties

[0147] The composition and structure of the glycoprotein prepared in Examples 1 - 3 had small differences and were essentially the same. Taking the protein prepared in Example 1 as an example, its different properties were detected as follows.

[0148] 1. Glycoprotein detection

[0149] (1) Glycoprotein Verification

[0150] The glycoprotein prepared in Example 1 was detected by SDS-PAGE and periodic acid-silver staining.

[0151] The results are as follows Figure 2 As shown in (a), the bands from left to right are glycoprotein silver staining, whey protein (negative control), and saccharifying enzyme (50kDa, positive control). The diol group of the carbohydrate is oxidized to an aldehyde group by periodic acid, which can be combined with silver amine ions to develop color. The band of glycoprotein is displayed at around 15kDa. Figure (b) is the electrophoresis of leaf protein, and it can be seen that the subunits of leaf protein are 50kDa and 15kDa.

[0152] (2) Further detection of the molecular weight of glycoprotein

[0153] Liquid chromatography was used to further determine the molecular weight of the glycoprotein.

[0154] The results are as follows Figure 3 , Figure 4 , as shown by Figure 3 Molecular weight distribution of glycoprotein in liquid phase Figure 4 The molecular weight distribution of the standard protein shows that the liquid phase detection of the glycoprotein presents a single peak distribution. By comparing the linear curve of the standard protein, the molecular weight of the protein at this position can be obtained to be 14.4 kDa. This indicates that the molecular weight of the protein bound by the glycoprotein we extracted is about 14 kDa. This is consistent with the molecular weight of the protein obtained by our periodic acid-silver staining method.

[0155] (3) Analysis of chemical bonds in glycoproteins

[0156] Infrared spectroscopy is used to examine the chemical bond composition in glycoproteins.

[0157] The Fourier transform infrared spectrum of glycoprotein is shown in Figure 5 As shown, 3500-3200cm -1 The peak width is the stretching vibration of OH and NH, indicating the presence of intramolecular hydrogen bonds and extramolecular hydrogen bonds. 3000-2800cm -1 The peak is the stretching vibration of CH of sugars, 1650cm -1 Nearby is the C=O stretching vibration of the acetylamino group, 1400 cm -1 The nearby peaks are the angular vibration absorption of CH, 1250-950cm -1 The peaks are the absorption peaks of the ether bond and hydroxyl group of the pyranose ring, indicating that the sugar chain has a pyranose configuration, 891.72cm -1 This illustrates a β-type CH glycosidic bond.

[0158] 2. Glycoprotein property detection

[0159] For the glycoprotein prepared in Example 1, its basic protein properties were detected, and the results are shown in Table 2.

[0160] Table 2 Qualitative Detection of Glycoprotein

[0161]

[0162] The results showed that the glycoprotein prepared by the present invention contains acidic polysaccharides, does not contain starch, and does not contain tannic acid.

[0163] 3. Function Detection of Glycoprotein

[0164] The antioxidant activities of the glycoproteins prepared in Examples 1 to 3 and Comparative Example 3 were detected, and the results are shown in Table 3.

[0165] Table 3 In Vitro Antioxidant Activity of Glycoprotein

[0166]

[0167]

[0168] The results showed that the DPPH free radical scavenging rate of the glycoproteins prepared in Examples 1 to 3 reached more than 83.6%, and the hydroxyl free radical scavenging rate reached more than 52.7%, which was significantly higher than that of the glycoproteins prepared by methods such as water extraction method and salt extraction method.

[0169] Comparative Example 7: Influence of Different Eluent Concentrations

[0170] On the basis of Example 1, the concentrations (v / v) of the ethanol aqueous solution elution in S5 were changed to 10%, 50%; 50%, 90% respectively to prepare glycoprotein and leaf protein, and the adsorption rate, desorption rate of the resin to the protein, and the purity and yield of the prepared leaf protein and glycoprotein were detected. The results are shown in Table 4.

[0171] Table 4 Influence of Different Elution Concentrations

[0172]

[0173] The results showed that too low or too high concentrations of ethanol aqueous solution would affect the desorption and elution rates and purity of glycoprotein and leaf protein, and thus affect the yield and purity of glycoprotein and leaf protein.

[0174] The series of detailed descriptions listed above are only specific descriptions of the feasible embodiments of the present invention, and they are not intended to limit the protection scope of the present invention. Any equivalent embodiments or changes made without departing from the technical spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing and separating glycoprotein and leaf protein, characterized in that, It includes the following steps: S1 Raw material pretreatment: Mix and pulverize sodium metabisulfite, CaCl2·2H2O, and plant leaves, and perform high-pressure homogenization to obtain straw pulp; S2 High-pressure extraction: Add the straw pulp to an isostatic flow high-pressure autoclave for high-pressure extraction to obtain the treated straw pulp; S3 Chlorophyll removal: Ultracentrifuge the treated straw pulp at 15000 - 17000 r / min for 60 - 90 min to obtain straw juice; S4 Microfiltration: Filter the straw juice using a polytetrafluoroethylene membrane hydrophilic composite membrane to obtain the filtered straw juice; S5 Resin purification: Adsorb the filtered straw juice using resin; after adsorption, first elute with an ethanol aqueous solution with a concentration of 20% - 25% v / v to collect the glycoprotein eluate; then elute with an ethanol aqueous solution with a concentration of 65% - 75% v / v to collect the leaf protein eluate; S6 Rotating cross-flow membrane separation: Ultrafilter the glycoprotein eluate and the leaf protein eluate respectively to obtain the glycoprotein concentrate and the leaf protein concentrate; S7 Drying: Dry the glycoprotein concentrate and the leaf protein concentrate respectively to obtain leaf protein and glycoprotein; Among them, the plant leaf is Rumex patientia L.

2. The method according to claim 1, characterized in that, In S1, the dosage ratio of sodium metabisulfite, CaCl2·2H2O, and plant leaves is 1.0 - 2.0 g: 100 - 200 mM: 1 kg.

3. The method according to claim 1, wherein In S2, the high-pressure extraction is carried out at 500 - 600 MPa and 50 - 60 °C for 40 - 50 min.

4. The method according to claim 1, wherein In S4, the pore size of the polytetrafluoroethylene membrane hydrophilic composite membrane is 0.2 - 1 μm.

5. The method according to claim 1, wherein In S4, the pressure for filtration is 0.03 - 0.6 Mpa.

6. The method according to claim 1, wherein In S6, the material of the ultrafiltration membrane in ultrafiltration is polyvinylidene fluoride or polytetrafluoroethylene membrane, the pore size of the ultrafiltration membrane is 0.01 - 0.1 μm, and the pressure for filtration is 0.01 - 0.1 Mpa.

7. The glycoprotein or leaf protein prepared by the method according to any one of claims 1 - 6.

8. A protein composition, characterized in that, The composition contains the glycoprotein or leaf protein according to claim 7.

9. The application of the glycoprotein or leaf protein according to claim 7 and the protein composition according to claim 8 in the preparation of antioxidant products.

10. A method for improving the antioxidant property of plant-derived glycoprotein, characterized in that, It includes the following steps: S1 Raw material pretreatment: Mix and pulverize sodium metabisulfite, CaCl2·2H2O, and plant leaves, and perform high-pressure homogenization to obtain straw pulp; S2 High-pressure extraction: Add the straw pulp to an isostatic flow high-pressure autoclave for high-pressure extraction to obtain the treated straw pulp; S3 Chlorophyll removal: Ultracentrifuge the treated straw pulp at 15000 - 17000 r / min for 60 - 90 min to obtain straw juice; S4 Microfiltration: Filter the straw juice using a polytetrafluoroethylene membrane hydrophilic composite membrane to obtain the filtered straw juice; S5 Resin purification: Adsorb the filtered straw juice using resin; after adsorption, first elute with an ethanol aqueous solution with a concentration of 20% - 25% v / v to collect the glycoprotein eluate; then elute with an ethanol aqueous solution with a concentration of 65% - 75% v / v to collect the leaf protein eluate; S6 Rotating cross-flow membrane separation: Ultrafilter the glycoprotein eluate and the leaf protein eluate respectively to obtain the glycoprotein concentrate and the leaf protein concentrate; S7 Drying: The glycoprotein concentrate and the leaf protein concentrate are dried separately to obtain leaf protein and glycoprotein with good antioxidant properties; Among them, the plant leaves are Rumex patientia L.

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