Leaf protein, extraction method and application

Abstract

The invention relates to a leaf protein, an extraction method and application. The leaf protein extraction method comprises the following steps: pulping plant leaves to obtain a first slurry, and centrifuging to obtain a first precipitate and a first supernatant; adding water into the first precipitate, adjusting the pH value to be greater than or equal to 7, and carrying out heating treatment to obtain a first precipitate dispersion liquid; adding an organic solvent into the first precipitate dispersion liquid, and centrifuging to obtain a second precipitate and a second supernatant; respectively recovering the residual organic solvent in the second precipitate and the second supernatant to obtain a third precipitate and a third supernatant; and refining the third supernate to obtain the leaf protein. The invention also provides the leaf protein extracted by the extraction method. The invention also provides an application of the leaf protein in preparation of a product for reducing blood sugar and / or a product for improving an insulin secretion function and / or a product for improving food bulkiness. The method solves the problem that the purity, the yield and the functionality cannot be considered at the same time by the existing leaf protein extraction method.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and particularly relates to a leaf protein, an extraction method and an application thereof. Background Art

[0002] Globally, there is an increasing demand for the exploration of new plant-based food resources, which has prompted people to continuously search for and develop underutilized plant species. Rumex patientia L.×R. tianschanicus cv. Rumex K-1 (also known as Rumex patientia) is a perennial polygonaceae plant cultivated by Chinese scientific researchers. It has a high protein content (24%-40% on a dry basis), a rich amino acid composition (dominated by glutamic acid and aspartic acid), and a variety of bioactive substances (such as flavonoids, vitamin C, linolenic acid, etc.). Its safety has been verified through multiple toxicology tests and it has been approved as a new food raw material. The phenolic substances (such as flavonoid compounds) in Rumex patientia have significant hypoglycemic activity. Studies have shown that these substances can delay the digestion and absorption of carbohydrates by inhibiting the activities of α-glucosidase and α-amylase, thereby reducing the postprandial blood glucose level. In addition, the phenolic substances in Rumex patientia also have antioxidant effects, which can reduce the damage of free radicals to pancreatic islet β cells and further improve insulin secretion function. The strong adaptability of Rumex patientia in extreme environments makes it a sustainable plant protein resource, especially showing great potential in the fields of plant-based foods, functional additives and pharmaceutical carriers.

[0003] As a high-protein plant resource, although the extraction technology of Rumex patientia has been developed for many years, it still faces the core problem that it is difficult to synergistically optimize purity, yield and functional properties. In traditional methods, the alkali dissolution and acid precipitation method, as the mainstream process, can achieve a purity of 60% - 80% (dry basis) at most, and the yield is usually less than 50%. However, although high temperature (temperature above 75°C) and strong alkali (pH greater than 12) conditions can increase the yield to 63% and the purity can reach 81%, they cause complete degradation of the protein and loss of functional properties, and important indicators such as the solubility and emulsification of the protein extracted under conventional conditions are insufficient; in recent years, the improved processes have still not broken through the bottleneck: although ultrasonic assistance can increase the yield to 64.3%, the purity is only 59.23%. The direct heating method is simple to operate, but it causes thermal denaturation and destroys the nutritional value; the acid heating method has a risk of chemical residue, and the salting-out method (such as ammonium sulfate method) is limited due to low purity (34%) and poor separation efficiency. Existing technologies generally have a "triple contradiction" - high purity (about 80%) depends on extreme conditions, resulting in protein damage, mild conditions are difficult to balance yield and purity, and it is difficult to have both the improvement of functional properties and the retention of active substances. In addition, the low extraction efficiency of phenolic substances leads to the underutilization of hypoglycemic activity, highlighting the structural defects of the current technical system in the co-extraction of components and the preservation of functions.

[0004] Therefore, there is an urgent need for a method that can simultaneously achieve the extraction of high-purity, high-yield, and highly functional Portulaca oleracea L. protein. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a leaf protein, an extraction method, and an application to solve the problem that the existing leaf protein extraction methods cannot simultaneously take into account purity, yield, and functionality.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: An extraction method of leaf protein, comprising the following steps: S1. Pulverize plant leaves to obtain a first slurry, and centrifuge to obtain a first precipitate and a first supernatant; S2. Add water to the first precipitate and adjust the pH value to be greater than or equal to 7, and perform heat treatment to obtain a first precipitate dispersion; S3. Add an organic solvent to the first precipitate dispersion, and centrifuge to obtain a second precipitate and a second supernatant; S4. Recover the residual organic solvent in the second precipitate and the second supernatant respectively to obtain a third precipitate and a third supernatant; S5. Refine the third supernatant to obtain leaf protein.

[0007] According to the above technical means, by adopting the "acid / alkali / organic solvent gradient purification" process, through staged regulation of acidic pretreatment, alkaline re-dissolution, and gradient organic solvents for selective precipitation, proteins are effectively separated from other components (such as phenolic substances, polysaccharides, cellulose, etc.). Under mild conditions, the protein purity reaches 68% - 95% (dry basis), the protein yield is not less than 50%, and at the same time, the ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit is retained. It effectively solves the problem that the existing leaf protein extraction methods cannot simultaneously take into account purity, yield, and functionality.

[0008] Among them, since the whole process of the process avoids strong denaturing conditions (such as SDS, high temperature), it ensures that the Rubisco subunit is not degraded. The macromolecule or its aggregate structure has high surface activity, effectively differentiating the emulsifying and foaming properties of the protein.

[0009] Verification of Functional Characteristics Association ① High solubility: The synergistic effect of alkaline re-dissolution (S2) and ultrafiltration refining (S5) reduces the formation of aggregates, thereby improving the protein dispersion.

[0010] ② High emulsifying property: The exposure of the hydrophobic domain of Rubisco (heating in S2) and the removal of impurities (organic solvent in S3) synergistically enhance the oil-water interface adsorption capacity.

[0011] ③ High foaming property: The hydrophilic-hydrophobic balance on the protein surface (regulated by the S3 gradient solvent) promotes the formation and stability of foaming.

[0012] It has been determined that the protein purity (dry basis) of the leaf protein product obtained by the above extraction method is 68-95%, and the protein yield is not less than 50%; the protein gel electrophoresis under reducing conditions contains ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit bands. The protein product has high solubility, high emulsifying property and high foaming property. The protein product has obvious hypoglycemic activity.

[0013] Preferably, the plant leaves are selected from at least one of Rumex patientia, Rumex obtusifolius, Rumex K-1, Medicago sativa and Morus alba leaves.

[0014] Preferably, the plant leaves include fresh leaves or leaf powder.

[0015] Preferably, in S1, before centrifugation, it further includes: adjusting the pH value of the first slurry to acidic.

[0016] Preferably, in S1, before centrifugation, it further includes: adjusting the pH value of the first slurry to 3.0-5.0.

[0017] Preferably, in S1, before centrifugation, it further includes: adjusting the pH value of the first slurry to 3.0-4.5.

[0018] Preferably, the pH value of the first slurry is adjusted with sodium hydroxide or hydrochloric acid. Among them, the pH value of the first slurry is related to the Rumex patientia in different growth periods and storage conditions.

[0019] Preferably, the mass ratio of solids to water in the first slurry is 1:5-1:30.

[0020] Preferably, centrifugation is carried out under the condition of a centrifugal force of 2000-4000 g to obtain the first precipitate and the first supernatant.

[0021] Preferably, the mass ratio of the first precipitate to water is 1:5-1:30.

[0022] Preferably, the temperature of the heat treatment is 40-70 °C.

[0023] By precisely controlling the heating temperature threshold, the heating temperature of S2 is lower than or equal to the thermal denaturation temperature of Rubisco (usually about 60-70 °C), which not only effectively avoids the loss of function, but also can effectively regulate the oil-water interface adsorption ability of the protein under the condition of partial denaturation.

[0024] Preferably, an alkaline substance is used in S2 to adjust the pH value to be greater than or equal to 7.

[0025] Preferably, the alkaline substance is selected from at least one of alkali metal hydroxides, alkaline earth metal hydroxides, alkali metal carbonates, alkali metal bicarbonates, and alkali metal phosphates.

[0026] Preferably, the alkali metal hydroxide is selected from sodium hydroxide (NaOH) and / or potassium hydroxide (KOH).

[0027] Preferably, the alkaline earth metal hydroxide is selected from magnesium hydroxide (Mg(OH)2) and / or calcium hydroxide (Ca(OH)2).

[0028] Preferably, the alkali metal carbonate is selected from at least one of lithium carbonate (Li2CO3), sodium carbonate (Na2CO3), and potassium carbonate (K2CO3).

[0029] Preferably, the alkali metal bicarbonate is selected from at least one of sodium bicarbonate (NaHCO3) and potassium bicarbonate (KHCO3).

[0030] Preferably, the alkali metal phosphate is selected from at least one of sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), trisodium phosphate (Na3PO4), potassium dihydrogen phosphate (KH2PO4), and dipotassium hydrogen phosphate (K2HPO4).

[0031] Preferably, the pH value of the first precipitate is 7 - 12.

[0032] Preferably, the organic solvent is selected from at least one of ethanol, methanol, isopropanol, and acetone. Among them, when the extracted product is used for food, the organic solvent is selected from at least one of ethanol, isopropanol, and acetone.

[0033] Preferably, the concentration of the organic solvent in the mixture after adding the organic solvent to the first precipitate dispersion is 65 - 85% (v / v).

[0034] Preferably, the concentration of the organic solvent in the mixture after adding the organic solvent to the first precipitate dispersion is 70 - 80% (v / v).

[0035] Preferably, centrifugation is carried out under the condition of a centrifugal force of 2000 - 4000 g to obtain the second precipitate and the second supernatant.

[0036] Preferably, the residual organic solvent in the second precipitate and the second supernatant is recovered by vacuum evaporation respectively; The pressure of the vacuum evaporation is 20 - 40 kPa, and the temperature is 30 - 55 °C.

[0037] Preferably, the refining method is selected from at least three combinations of pH adjustment, centrifugation, membrane separation, water addition modulation, and evaporation concentration.

[0038] Preferably, the pH adjustment is carried out by using at least one of hydrochloric acid, phosphoric acid, citric acid, fumaric acid, adipic acid, tartaric acid, lactic acid, acetic acid and malic acid to adjust the pH value of the third supernatant to acidic or neutral.

[0039] Preferably, the pH adjustment is implemented according to the processing suitability of the obtained protein in the food system.

[0040] Among them, the pH adjustment is related to the functional requirements of the final product, such as foaming property, emulsifying property, etc. Different food systems have different requirements for the functionality of raw materials. For example, for acidic emulsion system foods, the pH range can be adjusted to 3.5 - 6; for neutral foaming system foods or neutral emulsion system foods, the pH range can be adjusted to 7 and near-neutral ranges such as 6.5 - 7.5, etc.

[0041] Preferably, the refining step includes a centrifugal force range of 2000 - 10000 g during centrifugation.

[0042] Preferably, the refining step includes a solid-liquid ratio of 1:5 - 1:15 during water addition and modulation.

[0043] Preferably, during evaporation and concentration in the refining step, it can be single-effect evaporation, multi-effect evaporation, or mechanical vapor recompression evaporation.

[0044] Among them, the conditions for single-effect evaporation: the vacuum degree is -0.06 to -0.09 MPa, and the heating temperature does not exceed 70°C. The conditions for multi-effect evaporation: such as 2 - 3 effects, the temperature difference per effect is controlled at 10 - 15°C, and the last effect is set between 50 - 60°C. The conditions for mechanical vapor recompression: the compression ratio is controlled between 1.8 - 2.2, the operating pressure is -0.02 to -0.05 MPa, and the evaporation temperature is 60 - 70°C.

[0045] Preferably, when the refining step involves membrane filtration, it can include one or a combination of continuous tangential flow filtration (CTFD), ultrafiltration (UF), and electrodialysis (ED).

[0046] Preferably, a fourth supernatant and a fourth precipitate are also produced during centrifugation in the refining step; the drying is freeze-drying or pulverization treatment is added during drum drying.

[0047] Preferably, the first supernatant is used as a beverage raw material. When the first supernatant is directly used as a beverage raw material, the formula can be directly adjusted and then sterilized and filled.

[0048] Preferably, the first supernatant is dried to obtain a first by-product, which contains acid-soluble proteins, polypeptides, amino acids, soluble sugars and organic acids, and the first by-product is used as a raw material for solid beverages.

[0049] Preferably, the drying methods for drying the first supernatant include: ① Spray drying: the inlet air temperature is 130-190°C, and the outlet air temperature is 60-80°C; ② Freeze drying: the vacuum degree is 10-100 Pa, and the temperature is -20 - -40°C; ③ Vacuum drying: the temperature is 40-60°C, and the vacuum degree is 100-1000 Pa.

[0050] Preferably, the third precipitate is dried to obtain a second by-product, and the second by-product contains fibers or secondary proteins.

[0051] Preferably, the drying methods for drying the third precipitate include: ① Hot air or tube bundle drying: the temperature is 50-80°C, ② Drum drying: the temperature is 120-150°C.

[0052] The present invention also provides a leaf protein extracted by the extraction method as described in the present invention, and the second by-product is used as a dietary fiber food raw material or feed.

[0053] Preferably, the IC of the α-amylase enzyme activity in the leaf protein 50 is not higher than 6.11 mg / mL, and the IC of the α-glucosidase activity 50 is not higher than 5.63 mg / mL.

[0054] Preferably, the leaf protein is rich in flavonoids, including quercetin (and its glycosides), baicalin, and kaempferol (and its glycosides).

[0055] Preferably, the foaming property of the leaf protein under neutral conditions is not lower than 170%.

[0056] Preferably, the emulsifying property of the leaf protein under the conditions of pH 4.5 and 50% oil phase fraction is not lower than 28 m 2 / g, and the emulsion stability (10 min) is not lower than 69%.

[0057] Preferably, the emulsifying property of the leaf protein under neutral and 50% oil phase fraction conditions is not lower than 85 m 2 / g, and the emulsion stability (10 min) is not lower than 87%.

[0058] Preferably, the solubility of the leaf protein under neutral conditions is not lower than 78%.

[0059] Preferably, the ultraviolet-visible spectrum of the aqueous solution of the leaf protein contains four peaks, and their peak values are respectively located at 270-280 nm, 403-405 nm, 497-500 nm, and 653-660 nm.

[0060] Preferably, in the leaf protein, the proportion of Rubisco small subunit (13 - 15 kDa) in the protein components of protein gel electrophoresis under reducing conditions is about 8 - 12%.

[0061] The present invention also provides an application of the leaf protein extracted by the extraction method as described in the present invention in the preparation of hypoglycemic products and / or products for improving insulin secretion function and / or products for improving food fluffiness.

[0062] Advantages of the present invention: The extraction method of the leaf protein of the present invention adopts the "acid / alkali / organic solvent gradient purification" process. By regulating acidic pretreatment, alkaline re - dissolution and gradient organic solvents (such as ethanol) for selective precipitation in stages, the protein is effectively separated from other components (such as phenolic substances, polysaccharides, cellulose, etc.). High - efficiency separation of impurities is achieved under mild conditions, avoiding the destruction of protein structure by high temperature or strong alkali, thus taking into account both purity and yield and reducing protein loss. After testing, the protein purity of the leaf protein extracted by the extraction method of the present invention reaches 68% - 95% (dry basis), and the protein yield is not less than 50%. At the same time, ribulose - 1,5 - bisphosphate carboxylase (Rubisco) subunits are retained, thus retaining the important nutritional and functional components in Atriplex hortensis L., providing key indicators for product quality control. The high yield reduces production costs, making Atriplex hortensis L. protein more competitive in the plant - based food market. At the same time, the utilization rate of Atriplex hortensis L. resources is improved, which is beneficial to the development of sustainable agriculture.

[0063] The leaf protein extracted by the extraction method of the present invention not only has excellent emulsifying properties, especially still has good emulsifying ability (not less than 25 m 2 / g) at pH 4.5, enabling it to be widely used in the production of foods such as dairy product substitutes, salad dressings, and margarines; but also has high foaming ability (not less than 170%) and good foam stability (stability not less than 30% after 30 minutes). Therefore, when used in baked foods and desserts, it can significantly improve the fluffiness and taste of the products.

[0064] The leaf protein extracted by the extraction method of the present invention retains the phenolic substances naturally present in Atriplex hortensis L. These substances have significant hypoglycemic activity and can delay the digestion and absorption of carbohydrates by inhibiting the activities of α - glucosidase and α - amylase, thereby reducing the post - meal blood glucose level. This characteristic gives the protein product prepared by the present invention potential health benefits and can be applied to functional foods. It has promotional application value in the field of food processing technology. Description of the Drawings

[0065] Figure 1 is the process flow chart of the extraction method of the leaf protein of the present invention; Figure 2 The physical picture of the leaf protein powder obtained by the extraction method of the present invention; Figure 3 The typical electrophoresis spectrum (bottom) and optical density map (top) of the Portulaca oleracea leaf protein powder obtained by the extraction method of the present invention; Figure 4 The ultraviolet spectrum of the Portulaca oleracea leaf protein powder obtained by the extraction method of the present invention; Figure 5 The non-targeted metabolome compound classification pie chart of the Portulaca oleracea leaf protein powder obtained by the extraction method of the present invention; Figure 6 The α-amylase inhibitory activity data of the Portulaca oleracea leaf protein powder obtained by the extraction method of the present invention; Figure 7 The α-glucosidase inhibitory activity data of the Portulaca oleracea leaf protein powder obtained by the extraction method of the present invention. Detailed implementation manners

[0066] The following will illustrate the implementation manners of the present invention with reference to the preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, rather than for limiting the protection scope of the present invention.

[0067] Example 1 As Figure 1 shown, a method for extracting leaf protein includes the following steps: S1. Mix fresh Portulaca oleracea leaves and water at a mass ratio of 1:10 to make a pulp, adjust the pH value of the first pulp to 4.5 with sodium hydroxide, and then centrifuge at a centrifugal force of 2500 g to obtain a first precipitate and a first supernatant; the first supernatant is spray-dried to obtain a first by-product, and the first by-product is used as a raw material for solid beverages. Among them, the inlet air temperature of the spray drying is 130-190°C, and the outlet air temperature is 60-80°C; S2. Add water to the first precipitate obtained in S1, the mass ratio of the first precipitate to water is 1:10, and adjust the pH value to 7 with sodium carbonate, heat to 70°C and stir for 50 min to obtain a first precipitate dispersion; S3. Add ethanol to the first precipitate dispersion obtained in S2 so that the concentration of ethanol in the mixture after adding ethanol is 75%, and centrifuge at a centrifugal force of 2500 g to obtain a second precipitate and a second supernatant; S4. Recover the residual ethanol in the second precipitate and the second supernatant respectively by vacuum evaporation to obtain a third precipitate and a third supernatant. Among them, the second precipitate is desolventized using a stripping machine, the direct steam pressure is controlled at about 0.02 MPa, and the steaming and drying temperature is maintained between 105 and 110 °C. The second supernatant is distilled and rectified using a packed rectifying column or an explosion-proof ethanol recovery machine, the vacuum pressure ≤ -0.075 MPa, the temperature ≤ 65 °C. The third precipitate is dried by hot air at a temperature of 50 to 80 °C to obtain a second by-product, and the second by-product is used as a raw material for dietary fiber food or feed. S5. Adjust the pH value of the third supernatant obtained in S4 to 6.5 - 7.5 using hydrochloric acid, and then successively perform tangential flow filtration (CTFD, the conditions of tangential flow filtration are: the initial transmembrane pressure is 0.1 - 0.3 MPa, the flow rate is controlled between 0.5 and 1.5 L / min / m², concentrated to 1 / 5 - 1 / 10 of the original volume) through a hollow fiber membrane made of regenerated cellulose with a molecular weight cut-off of 5 kDa and electrodialysis (ED, homogeneous ion exchange membrane, ASTOM ACS, the conditions of electrodialysis are: the voltage is 10 - 30 V, the current density is controlled between 50 and 150 A / m², until the conductivity < 1 mS / cm) to obtain an enriched protein material. The enriched protein material is successively passed through triple-effect evaporation (the conditions of triple-effect evaporation are: the vacuum degree is between -0.02 and -0.08 MPa, the material temperature is adjusted to 60 - 70 °C, 50 - 60 °C and 40 - 50 °C respectively through a heat medium) and spray drying (the conditions of spray drying: the inlet air temperature is 140 - 160 °C, the outlet air temperature is 50 - 65 °C) to obtain the protein powder of Portulaca oleracea L. leaves.

[0068] The physical picture of the protein powder of leaves extracted in this example is as Figure 2 shown. It can be seen from Figure 2 that the protein powder of leaves is in the form of a yellowish-green powder.

[0069] Example 2 As Figure 1 shown, a method for extracting leaf protein includes the following steps: S1. Mix fresh Portulaca oleracea L. leaves and water at a mass ratio of 1:5 to make a first slurry, adjust the pH value of the first slurry to 3.0 using hydrochloric acid, and then centrifuge under a centrifugal force of 2000 g to obtain a first precipitate and a first supernatant. The first supernatant is spray-dried to obtain a first by-product, and the first by-product is used as a raw material for solid beverages. Among them, the inlet air temperature of spray drying is 130 - 190 °C, and the outlet air temperature is 60 - 80 °C. S2. Add water to the first precipitate obtained in S1. The mass ratio of the first precipitate to water is 1:5. Adjust the pH value to 11 using sodium hydroxide, heat up to 60 °C and stir for 40 min to obtain a first precipitate dispersion; S3. Add ethanol to the first precipitate dispersion obtained in S2 so that the concentration of ethanol in the mixture after adding ethanol is 85%. Centrifuge under the condition of a centrifugal force of 2000 g to obtain a second precipitate and a second supernatant; S4. Recover the residual ethanol in the second precipitate and the second supernatant respectively by vacuum evaporation to obtain a third precipitate and a third supernatant; among them, the second precipitate is desolvated using a desolventizer, the direct steam pressure is controlled at about 0.02 MPa, and the steaming and drying temperature is maintained between 105 and 110 °C. The second supernatant is distilled and rectified using a packed rectification column or an explosion-proof ethanol recovery machine, the vacuum pressure ≤ -0.075 MPa, the temperature ≤ 65 °C. The third precipitate is dried by hot air at a temperature of 50 - 80 °C to obtain a second by-product, and the second by-product is used as a dietary fiber food raw material or feed; S5. Adjust the pH value of the third supernatant obtained in S4 to 6.5 - 7.5 using hydrochloric acid, and then centrifuge under the condition of a centrifugal force of 2000 g to obtain a fourth supernatant and a fourth precipitate. The fourth precipitate is used to mix with the third precipitate to prepare the second by-product. The fourth supernatant is successively subjected to tangential flow filtration (CTFD, the conditions of tangential flow filtration are the same as those in Example 1) through a hollow fiber membrane with a molecular weight cut-off of 5 kDa made of regenerated cellulose and electrodialysis (ED, homogeneous ion exchange membrane, ASTOM ACS, the conditions of electrodialysis are the same as those in Example 1) to obtain an enriched protein material. The enriched protein material is spray-dried to obtain a protein powder from the leaves of Atriplex hortensis L.

[0070] Example 3 As Figure 1 shown, a method for extracting leaf protein includes the following steps: S1. Mix fresh Atriplex hortensis L. leaves and water in a mass ratio of 1:30 to make a pulp to obtain a first pulp. Adjust the pH value of the first pulp to 5.0 using sodium hydroxide, and then centrifuge under the condition of a centrifugal force of 4000 g to obtain a first precipitate and a first supernatant; the first supernatant is spray-dried to obtain a first by-product, and the first by-product is used as a raw material for solid beverages. Among them, the inlet air temperature of spray drying is 130 - 190 °C, and the outlet air temperature is 60 - 80 °C; S2. Add water to the first precipitate obtained in S1. The mass ratio of the first precipitate to water is 1:30. Adjust the pH value to 12 using calcium hydroxide, heat up to 40 °C and stir for 60 min to obtain a first precipitate dispersion; S3. Add ethanol to the first precipitate dispersion obtained in S2 so that the concentration of ethanol in the mixture after adding ethanol is 70%, and centrifuge under the condition of a centrifugal force of 4000 g to obtain a second precipitate and a second supernatant; S4. Recover the residual ethanol in the second precipitate and the second supernatant by vacuum evaporation respectively to obtain a third precipitate and a third supernatant; among them, the second precipitate is desolvated by a stripping machine, the direct steam pressure is controlled at about 0.02 MPa, and the steaming and drying temperature is maintained between 105 and 110 °C. The second supernatant is distilled and rectified by a packed rectification column or an explosion-proof ethanol recovery machine, the vacuum pressure ≤ -0.075 MPa, and the temperature ≤ 65 °C. The third precipitate is dried by hot air at a temperature of 50 to 80 °C to obtain a second by-product, and the second by-product is used as a raw material for dietary fiber food or feed; S5. Adjust the pH of the third supernatant obtained in S4 to 4 with citric acid and hydrochloric acid (molar ratio 1:1), then centrifuge under the condition of a centrifugal force of 2000 g to obtain a fourth supernatant and a fourth precipitate. Add water to the fourth precipitate and mix evenly. The mass ratio of the fourth precipitate to water is 1:20. Then, it is successively subjected to tangential flow filtration (CTFD, the conditions of tangential flow filtration are the same as those in Example 1) through a hollow fiber membrane with a molecular weight cut-off of 5 kDa made of regenerated cellulose and electrodialysis (ED, homogeneous ion exchange membrane, ASTOM ACS, the conditions of electrodialysis are the same as those in Example 1) to obtain an enriched protein material. The enriched protein material is successively passed through mechanical vapor recompression evaporation (conditions: pressure is 19.9 to 39 kPa, the material temperature is adjusted ≤ 65 °C by heating steam, compression ratio: 1.25 to 1.57) and spray drying (conditions: inlet air temperature is 140 to 160 °C, outlet air temperature is 50 to 65 °C) to obtain the protein powder of Portulaca oleracea leaves.

[0071] Example 4 As Figure 1 shown, a method for extracting leaf protein includes the following steps: S1. Mix fresh Portulaca oleracea leaves and water in a mass ratio of 1:20 to make a pulp to obtain a first pulp. Adjust the pH value of the first pulp to 4.0 with hydrochloric acid, and then centrifuge under the condition of a centrifugal force of 4000 g to obtain a first precipitate and a first supernatant; the first supernatant is spray-dried to obtain a first by-product, and the first by-product is used as a raw material for solid beverages. Among them, the inlet air temperature for spray drying is 130 to 190 °C, and the outlet air temperature is 60 to 80 °C; S2. Add water to the first precipitate obtained in S1. The mass ratio of the first precipitate to water is 1:25, and adjust the pH value to 11.8 with potassium hydroxide, and raise the temperature to 70 °C and stir for 60 min to obtain a first precipitate dispersion; S3. Add ethanol to the first precipitate dispersion obtained in S2 to make the concentration of ethanol in the mixture after adding ethanol 85%, and centrifuge under the condition of a centrifugal force of 3500 g to obtain a second precipitate and a second supernatant; S4. Recover the residual ethanol in the second precipitate and the second supernatant by vacuum evaporation respectively to obtain a third precipitate and a third supernatant; among them, the second precipitate is desolvated by a stripping machine, the direct steam pressure is controlled at about 0.02 MPa, and the steaming and drying temperature is maintained between 105 and 110 °C. The second supernatant is distilled and rectified by a packed rectifying column or an explosion-proof ethanol recovery machine, the vacuum pressure ≤ -0.075 MPa, the temperature ≤ 65 °C, and the third precipitate is dried by hot air at a temperature of 50 - 80 °C to obtain a second by-product, and the second by-product is used as a dietary fiber food raw material or feed; S5. Adjust the pH value of the third supernatant obtained in S4 to 6.5 - 7.5 with hydrochloric acid, then centrifuge under the condition of a centrifugal force of 10000 g to obtain a fourth supernatant and a fourth precipitate. Add water to the fourth precipitate and mix evenly. The mass ratio of the fourth precipitate to water is 1:20. Then, it is successively subjected to tangential flow filtration (CTFD, the conditions of tangential flow filtration are the same as those in Example 1) through a hollow fiber membrane with a molecular weight cut-off of 5 kDa made of regenerated cellulose and electrodialysis (ED, homogeneous ion exchange membrane, ASTOM ACS, the conditions of electrodialysis are the same as those in Example 1) to obtain an enriched protein material. The enriched protein material is successively passed through mechanical vapor recompression evaporation (conditions the same as in Example 3) and freeze-drying (conditions: vacuum degree of 30 Pa, temperature of -40 °C), and then pulverized to 60 meshes with a hammer mill to obtain the protein powder of Portulaca oleracea leaves.

[0072] Example 5 As Figure 1 shown, a method for extracting leaf protein includes the following steps: S1. Mix fresh Portulaca oleracea leaves and water in a mass ratio of 1:20 to make a first slurry, adjust the pH value of the first slurry to 3.5 with hydrochloric acid, and then centrifuge under the condition of a centrifugal force of 3000 g to obtain a first precipitate and a first supernatant; the first supernatant is spray-dried to obtain a first by-product, and the first by-product is used as a raw material for solid beverages. Among them, the inlet air temperature of the spray drying is 130 - 190 °C, and the outlet air temperature is 60 - 80 °C; S2. Add water to the first precipitate obtained in S1, the mass ratio of the first precipitate to water is 1:30, and adjust the pH value to 7 with sodium hydroxide, and raise the temperature to 40 °C and stir for 30 min to obtain a first precipitate dispersion; S3. Add ethanol to the first precipitate dispersion obtained in S2 to make the concentration of ethanol in the mixture after adding ethanol 65%, and centrifuge under the condition of a centrifugal force of 3000 g to obtain a second precipitate and a second supernatant; S4. Recover the residual ethanol in the second precipitate and the second supernatant by vacuum evaporation respectively to obtain a third precipitate and a third supernatant; among them, the second precipitate is desolvated by a desolventizer, the direct steam pressure is controlled at about 0.02 MPa, and the steaming and drying temperature is maintained between 105 and 110 °C. The second supernatant is distilled and rectified by a packed rectification column or an explosion-proof ethanol recovery machine, the vacuum pressure ≤ -0.075 MPa, the temperature ≤ 65 °C. The third precipitate is dried by hot air at a temperature of 50 - 80 °C to obtain a second by-product, and the second by-product is used as a dietary fiber food raw material or feed; S5. Adjust the pH of the third supernatant obtained in S4 to 4 with citric acid and hydrochloric acid (molar ratio 1:1), then centrifuge under the condition of a centrifugal force of 8000 g to obtain a fourth supernatant and a fourth precipitate. Add water to the fourth precipitate and mix evenly. The mass ratio of the fourth precipitate to water is 1:10. Then, it is successively subjected to tangential flow filtration (CTFD, the conditions of tangential flow filtration are the same as those in Example 1) through a hollow fiber membrane with a molecular weight cut-off of 5 kDa made of regenerated cellulose and electrodialysis (ED, homogeneous ion exchange membrane, ASTOM ACS, the conditions of electrodialysis are the same as those in Example 1) to obtain an enriched protein material. The enriched protein material is successively passed through triple-effect evaporation (the conditions of triple-effect evaporation are the same as those in Example 1) and vacuum drum drying (conditions: vacuum degree is 5 - 20 kPa, drum temperature is 60 - 70 °C, material temperature ≤ 65 °C, rotation speed is 1 - 5 rpm), and then pulverized to 60 meshes with a hammer mill to obtain Rumex patientia leaf protein powder.

[0073] Example 6 A method for extracting leaf protein. In this example, except that the selected plant leaf is a fresh Rumex patientia leaf, the other conditions are the same as those in Example 1, and Rumex patientia leaf protein powder is obtained.

[0074] Example 7 A method for extracting leaf protein. In this example, except that the selected plant leaf is a fresh Rumex K-1 leaf, the other conditions are the same as those in Example 1, and Rumex K-1 leaf protein powder is obtained.

[0075] Comparative Example 1 A common method for extracting leaf protein, comprising the following steps: S1. Wash the fresh leaves of Portulaca oleracea with clean water and cut them into sections. Then, mix them with ethanol at -20°C and a concentration of 95% at a mass ratio of 1:4 to make a slurry. After cooling to 4°C, pass it through a 100-mesh sieve, filter the slurry below the sieve, discard the filtrate, and repeatedly wash the filter residue with ethanol until it is colorless. Obtain the filter residue, dry the filter residue at 40°C and then pass it through a 100-mesh sieve to obtain crude Portulaca oleracea protein powder; S2. Disperse the crude Portulaca oleracea protein powder obtained in S1 in distilled water to obtain a dispersion. The crude Portulaca oleracea protein powder and distilled water are in a mass ratio of 1:34. Then, add sodium hydroxide to the dispersion so that the concentration of sodium hydroxide in the dispersion is 10.6 g / L and the pH value is 12. Perform Portulaca oleracea protein extraction at 81°C for 60 min. After extraction, centrifuge at a speed of 7000 r / min, take the supernatant and adjust the pH to 3.5 with 1 mol / L hydrochloric acid, and centrifuge again at a speed of 7000 r / min. Mix the precipitates obtained twice, wash them twice with water, and freeze-dry to obtain Portulaca oleracea protein.

[0076] Comparative Example 2 A common method for extracting leaf protein includes the following steps: Pass the fresh Portulaca oleracea powder through a 60-mesh sieve, degrease it with petroleum ether, remove the solvent, and make it into crude Portulaca oleracea protein powder. Disperse it in distilled water. The crude Portulaca oleracea protein powder and distilled water are in a mass ratio of 1:25, and add sodium hydroxide to adjust the pH value to 10. Perform Portulaca oleracea protein extraction at 40°C for 25 min. After extraction, centrifuge at a speed of 3500 r / min, take the supernatant and adjust the pH to 3.5 with 1 mol / L hydrochloric acid, and centrifuge again at a speed of 3500 r / min. Mix the precipitates obtained twice, wash them twice with water, and freeze-dry to obtain Portulaca oleracea protein.

[0077] Comparative Example 3 An ultrasonic-assisted alkali solution acid precipitation method for extracting leaf protein includes the following steps: Dry the fresh leaves of Portulaca oleracea at 45°C until constant weight (moisture content is 3%), then grind the dry leaves into powder and pass it through a 120-mesh sieve to obtain crude Portulaca oleracea protein powder; Disperse the crude protein powder of Rumex patientia in distilled water at a mass ratio of the crude protein powder of Rumex patientia to distilled water of 1:80, add sodium hydroxide to adjust the pH value to 10, extract the protein of Rumex patientia at 50 °C for 0.5 h, after extraction, centrifuge at a rotation speed of 8500 r / min (8500 r / min), take the supernatant and adjust the pH to 3 with 1 mol / L hydrochloric acid, centrifuge again at a rotation speed of 8500 r / min, mix the precipitates obtained twice, adjust the pH value to 7.0, and freeze-dry to obtain the protein of Rumex patientia.

[0078] Comparative Example 4 A soy protein isolate, a commercially available soy protein isolate obtained by the alkali solubilization and acid precipitation process was used as Comparative Example 4.

[0079] Detection and analysis 1) Protein purity (dry basis) and protein yield test Protein purity (dry basis) refers to the percentage of protein in the product solids in the product.

[0080] Protein yield refers to the percentage of the protein mass in the protein product to the protein mass in the raw materials used for production.

[0081] Protein content test: GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Foods", with a nitrogen conversion coefficient of 6.25. Moisture test: GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Foods".

[0082] The test results of the protein purity (dry basis) and protein yield of the protein powder of Rumex patientia leaves prepared in Examples 1 to 5, the protein powder of Rumex patientia leaves prepared in Example 6, the protein powder of Rumex K-1 leaves prepared in Example 7, and the protein powder of Rumex patientia prepared in Comparative Examples 1 to 3 are shown in Table 1.

[0083] Table 1 shows the test results of protein purity (dry basis) and protein yield It can be analyzed from Table 1 that the protein purity (dry basis) of the protein powder of Rumex patientia leaves prepared in Examples 1 to 5, the protein powder of Rumex patientia leaves prepared in Example 6, and the protein powder of Rumex K-1 leaves prepared in Example 7 are all above 60%, and the protein yields of the protein powder of Rumex patientia leaves prepared in Examples 1 to 5, the protein powder of Rumex patientia leaves prepared in Example 6, and the protein powder of Rumex K-1 leaves prepared in Example 7 are all above 50%.

[0084] 2) Protein polyacrylamide gel electrophoresis test Among them, the electrophoresis samples were prepared under reducing conditions (containing 5% β-mercaptoethanol), and stacking gels with a polyacrylamide content of 4% and separating gels with a polyacrylamide content of 12% were used. Approximately 5 μg of protein was loaded onto each lane. The protein concentration of the extract was determined using the Kjeldahl method (N conversion factor 6.25). The gels after staining and decolorization were analyzed using Quantity One software. The electrophoresis results of the Portulaca oleracea leaf protein powder prepared in Examples 1 to 5 are as follows. Among them, the electrophoresis spectrogram (bottom) and optical density diagram (top) of the Portulaca oleracea leaf protein powder prepared in Example 1 are as Figure 3 shown.

[0085] As can be seen from Figure 3 , the content of the Rubisco small subunit in the electrophoresis of the Portulaca oleracea leaf protein powder prepared in Example 1 was 10%. The content of the Rubisco small subunit in the electrophoresis of the Portulaca oleracea leaf protein powder prepared in Example 2 was 10.9%. The content of the Rubisco small subunit in the electrophoresis of the Portulaca oleracea leaf protein powder prepared in Example 3 was 10%. The content of the Rubisco small subunit in the electrophoresis of the Portulaca oleracea leaf protein powder prepared in Example 4 was 11.9%. The content of the Rubisco small subunit in the electrophoresis of the Portulaca oleracea leaf protein powder prepared in Example 5 was 8%. The content of the Rubisco small subunit in the electrophoresis of the Rumex patientia leaf protein powder prepared in Example 6 was 9.7%. The content of the Rubisco small subunit in the electrophoresis of the Rumex K-1 leaf protein powder prepared in Example 7 was 10.5%. The content of the Rubisco small subunit in the electrophoresis of the Portulaca oleracea leaf protein powder prepared in Comparative Example 1 was 0%. The content of the Rubisco small subunit in the electrophoresis of the Portulaca oleracea leaf protein powder prepared in Comparative Example 2 was 3.53%. The content of the Rubisco small subunit in the electrophoresis of the Portulaca oleracea leaf protein powder prepared in Comparative Example 3 was 4.3%.

[0086] 3) UV spectrum test The specific parameter conditions for the UV spectrum test were as follows: A protein aqueous solution with a certain concentration was prepared, and a spectrophotometer (Cary 300, Agilent Technologies Inc., USA) was used to record the UV spectrum of the sample. The wavelength range was 200 to 800 nm, the scanning rate was 600.000 nm / min, the data interval was 1.000 nm, the average time was 0.100 s, and the spectral bandwidth was 2.0 nm.

[0087] The UV-visible test results of the Portulaca oleracea leaf protein powder prepared in Examples 1 to 5 are as Figure 4 shown.

[0088] As can be seen from Figure 4Analysis shows that the four peaks in the ultraviolet-visible spectrum of the Portulaca oleracea leaf protein powder prepared in Example 1 are located at 275 nm, 404 nm, 499 nm, and 655 nm; the four peaks in the ultraviolet-visible spectrum of the Portulaca oleracea leaf protein powder prepared in Example 2 are located at 280 nm, 405 nm, 497 nm, and 657 nm; the four peaks in the ultraviolet-visible spectrum of the Portulaca oleracea leaf protein powder prepared in Example 3 are located at 270 nm, 403 nm, 498 nm, and 658 nm; the four peaks in the ultraviolet-visible spectrum of the Portulaca oleracea leaf protein powder prepared in Example 4 are located at 276 nm, 404 nm, 500 nm, and 658 nm; the four peaks in the ultraviolet-visible spectrum of the Portulaca oleracea leaf protein powder prepared in Example 5 are located at 278 nm, 403 nm, 498 nm, and 654 nm. The peak position differences in the regions of 270-280 nm, 400-405 nm, 497-500 nm, and 654-658 nm among different examples respectively reflect the protein conformation (270-280 nm), the presence and state of cofactors or pigments (400-405 nm, 497-500 nm), and the difference in chlorophyll residue (654-658 nm). The above four absorption peaks constitute the most important spectral characteristics of the protein product of the present invention.

[0089] The four peaks in the ultraviolet-visible spectrum of the Rumex patientia leaf protein powder prepared in Example 6 are located at 277 nm, 404 nm, 499 nm, and 653 nm. The four peaks in the ultraviolet-visible spectrum of the Rumex K-1 leaf protein powder prepared in Example 7 are located at 274 nm, 405 nm, 500 nm, and 654 nm.

[0090] 4) Non-target metabolomics detection The specific operations are as follows: (1) The sample is slowly thawed at 4°C. Weigh an appropriate amount of the sample (50 - 100 mg) accurately into a centrifuge tube, add 1 mL of extraction solution (water / acetonitrile / isopropanol, 1:1:1, v / v / v), vortex for 60 s, extract by low-temperature ultrasonic for 30 min, centrifuge at 12000 rpm for 10 min at 4°C, take the supernatant, place it at -20°C for 1 h to precipitate proteins, continue to centrifuge at 12000 rpm for 10 min at 4°C, take the supernatant for vacuum drying, add 200 μL of 30% acetonitrile solution for reconstitution, vortex, centrifuge at 14000 rpm for 15 min at 4°C, and take the supernatant for on-machine detection. (2) The data acquisition instrument system mainly includes an ultra-high performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and a high-resolution mass spectrometer (Q Exactive HFX, Thermo, USA). Chromatographic column: Waters HSS T3 (100×2.1 mm, 1.8 μm); Mobile phase: Phase A is an ultrapure water solution (containing 0.1% formic acid), and Phase B is an acetonitrile solution (containing 0.1% formic acid); Flow rate: 0.3 mL / min; Column temperature: 40°C; Injection volume: 2 μL; Elution gradient: At 0 min, Phase A / Phase B (100:0, v / v), at 1 min, Phase A / Phase B (100:0, v / v), at 4 min, Phase A / Phase B (40:60, v / v), at 6.5 min, Phase A / Phase B (5:95, v / v), at 6.6 min, Phase A / Phase B (100:0, v / v), at 8.0 min, Phase A / Phase B (100:0, v / v).

[0091] During the whole analysis process, the sample is placed in a 4°C autosampler. To avoid the influence caused by the signal fluctuation of the instrument detection, the samples are analyzed continuously in a random order. QC samples are evenly inserted into the sample analysis sequence to monitor and evaluate the stability of the system and the reliability of the experimental data. (3) The Q Exactive HFX high-resolution mass spectrometry system of Thermo Company in the United States is used to collect the first-level and second-level spectrograms. It is equipped with an electrospray ionization source (Electrospray ionization, ESI), the sheath gas is 40 arb, the auxiliary gas is 10 arb, the ion spray voltage is +3000 V / -2800 V, the temperature is 350°C, and the temperature of the ion transfer tube is 320°C. The scanning mode is Full-ms-ddMS2, and the scanning method is positive ion / negative ion. The first-level mass spectrometry scanning range (scan m / z range) is: 70 - 1050 Da, the first-level resolution is 70000, and the second-level resolution is 17500.

[0092] The non-targeted metabolomics detection results of the protein powder from the leaves of Rumex patientia L. var. sativa DC. prepared in Example 3 are as follows Figure 5 shown.

[0093] From Figure 5 analysis, among the concomitant compounds of the protein powder from the leaves of Rumex patientia L. var. sativa DC. prepared in Example 3, carboxylic acids and derivatives account for about 13%, organic oxygen compounds about 11%, pregnenolone lipids about 11%, fatty acyls about 10%, flavonoids about 10%, benzene and substituted derivatives 4.8%, steroids and steroid derivatives about 3%, isoflavones 2.4%, coumarins and derivatives 2.3%, glycerophospholipids 1.7%. Among them, flavonoids generally have various biological activities such as antioxidant, anti-inflammatory, neuroprotective, and anti-cancer. The flavonoids with relatively high proportions in the concomitant compounds are quercetin (and its glycosides), baicalin, and kaempferol (and its glycosides), accounting for about 4%, 1%, and 2% respectively in the above compounds.

[0094] 5) Protein functionality test (1) Protein solubility: The protein sample was prepared into a 5% (w / v) solution in a phosphate buffer solution (pH 7), and shaken in a constant temperature water bath (25 °C, 1 h), and then centrifuged for 30 min at a centrifugal force of 3836 g. The protein contents in the supernatant and the protein sample were determined by the bicinchoninic acid method. The solubility is equal to the percentage of the protein content in the supernatant to the total protein content.

[0095] The solubility results of the protein powder from the leaves of Rumex patientia L. var. sativa DC. prepared in Examples 1 to 5, the protein powder from the leaves of Rumex patientia L. prepared in Example 6, the protein powder from the leaves of Rumex patientia L. cv. Rumex K-1 prepared in Example 7, as well as the protein from Rumex patientia L. prepared in Comparative Examples 1 to 3 and the soy protein isolate in Comparative Example 4 are shown in Table 2.

[0096] Table 2 shows the solubility results of the protein powder from the leaves of Rumex patientia L. var. sativa DC. From the analysis in Table 2, it can be seen that the solubility of the protein powder from the leaves of Rumex patientia L. var. sativa DC. prepared in Examples 1 to 5, the protein powder from the leaves of Rumex patientia L. prepared in Example 6, and the protein powder from the leaves of Rumex patientia L. cv. Rumex K-1 prepared in Example 7 are all greater than or equal to 78%, showing good solubility.

[0097] (2) Emulsifying activity and emulsifying stability: A 2.5% protein sample solution was mixed with an equal volume of soybean oil and emulsified at 13500 rpm for 2 min in a high-speed emulsifying homogenizer to obtain an emulsion. Immediately, 20 μL of the emulsion was mixed evenly with 5 mL of 0.1% SDS solution, and the absorbance value of the diluted emulsion was measured at a wavelength of 500 nm, denoted as A0. 0.1% SDS was used as a blank control. The emulsifying activity index (EAI) was calculated using the following formula (Ⅰ): (I) Among them, in formula (I), T = 2.303, N = 250, c represents the protein concentration (g / mL) in the protein solution before the formation of the emulsion, φ represents the volume fraction of oil in the emulsion (0.25), represents the emulsifying activity index of the protein sample, with the unit of m 2 / g.

[0098] The emulsifying stability index is calculated by the following formula (II): (II) Among them, in formula (II), A0 represents the initial absorbance value, A t represents the absorbance value at 10 min, represents the emulsifying stability index of the protein sample.

[0099] The results of the emulsifying activity index and emulsifying stability index of the Portulaca oleracea leaf protein powder prepared in Examples 1 to 5, the Rumex patientia leaf protein powder prepared in Example 6, the Rumex K-1 leaf protein powder prepared in Example 7, the Portulaca oleracea protein prepared in Comparative Examples 1 to 3, and the soy protein isolate in Comparative Example 4 are shown in Table 3.

[0100] Table 3 shows the results of the emulsifying activity index and emulsifying stability index It can be analyzed from Table 3 that the Portulaca oleracea leaf protein powder prepared in Examples 1 to 5 all has good emulsifying activity index and emulsifying stability index.

[0101] (3) Foaming ability (foaming property): Place 35 mL of a 1% (w / v) protein sample solution in a 100 mL graduated cylinder (or a graduated cylinder of appropriate size), and homogenize it with a high-speed disperser at a speed of 15,000 r / min for 40 s, three times continuously for a total of 2 min. Record the total volume of the sample after homogenization, denoted as V0. The foaming ability is calculated by the following formula (III): (III) Among them, in formula (III), V0 represents the total volume of the sample after homogenization, with the unit of mL, represents the foaming ability of the protein sample.

[0102] The foaming ability results of the Portulaca oleracea leaf protein powder prepared in Examples 1 to 5, the Rumex patientia leaf protein powder prepared in Example 6, the Rumex K-1 leaf protein powder prepared in Example 7, the Portulaca oleracea protein prepared in Comparative Examples 1 to 3, and the soy protein isolate in Comparative Example 4 are shown in Table 4.

[0103] Table 4 shows the foaming ability results It can be analyzed from Table 4 that the protein powders from Rumex patientia leaves prepared in Examples 1 to 5, the protein powder from Rumex obtusifolius leaves prepared in Example 6, and the protein powder from Rumex K-1 leaves prepared in Example 7 all have good foaming ability.

[0104] (4)Hypoglycemic activity The hypoglycemic activity of the protein samples was determined by the following method, i.e., the inhibitory activities against α-amylase and α-glucosidase:[[]] 4.1) α-Amylase inhibitory activity: Mix 100 μL of the protein sample solution with 50 μL of α-amylase solution (10 U / mL) and incubate at 37 °C for 15 minutes. Subsequently, add 100 μL of soluble starch solution (10 mg / mL) and 50 μL of phosphate buffered saline (PBS, pH 6.8, 0.2 M), and incubate for another 10 minutes. Terminate the reaction with 200 μL of DNS reagent (containing 10 g / L 3,5-dinitrosalicylic acid, 10 g / L NaOH, 200 g / L potassium sodium tartrate, 2 g / L freshly distilled phenol, and 5 g / L anhydrous sodium sulfite), heat in a boiling water bath for 10 minutes, cool to room temperature, and dilute with 3.5 mL of distilled water. Measure the absorbance of the solution at 540 nm using a Synergy H1 microplate reader (BioTek, Vermont, USA). Calculate the inhibition percentage (PIAA) of the α-amylase activity of the protein sample using the following formula (Ⅳ).

[0105] (Ⅳ) Wherein, in formula (Ⅳ), A1 represents the absorbance value of the blank group (blank), A2 represents the absorbance value of the blank control group (blank control), A3 represents the absorbance value of the inhibitor group (inhibitor), and A4 represents the absorbance value of the background control group (background control), represents the inhibition percentage of the α-amylase activity of the protein sample. These test tubes only differ in the presence of enzyme, test protein sample, and PBS before the start of the reaction. Specifically: Group A1 does not contain the test protein sample, Group A2 does not contain enzyme and test protein sample, Group A3 contains both, and Group A4 does not contain enzyme. The missing solution is replaced with PBS. The protein powders from Rumex patientia leaves prepared in Examples 1 to 5, the protein powder from Rumex obtusifolius leaves prepared in Example 6, and the protein powder from Rumex K-1 leaves prepared in Example 7 obtained IC 50 through different protein concentration gradients and fitting, and the results are shown in Table 5 and Figure 6 as follows.

[0106] IC of α-amylase activity in protein samples in Table 5 50 As can be analyzed from Table 5 and Figure 6 it can be seen that the IC of α-amylase enzyme activity in the protein powder of Rumex patientia leaves prepared in Examples 1 to 5, the protein powder of Rumex obtusifolius leaves prepared in Example 6, and the protein powder of Rumex K-1 leaves prepared in Example 7 50 are all not higher than 6.11 mg / mL.

[0107] 4.2) α-glucosidase inhibitory activity: Mix 20 μL of the protein sample with 10 μL of α-glucosidase solution (0.2 U / mL) and incubate at 37 °C for 20 minutes. Then, add 40 μL of pNPG (10 mM) and 50 μL of PBS (0.2 M, pH 6.8), and incubate at 37 °C for another 30 minutes. Terminate the reaction with 100 μL of Na2CO3 (1 M). Read the absorbance at 405 nm. Calculate the inhibition percentage (PIGA) of α-glucosidase activity using the following formula (Ⅴ).

[0108] (Ⅴ) wherein, in formula (Ⅴ), A0 represents the absorbance of the control group (replacing the sample with PBS), A1 represents the absorbance of the protein sample group, and A2 represents the absorbance of the background control group (replacing α-glucosidase with PBS). The protein powder of Rumex patientia leaves prepared in Examples 1 to 5, the protein powder of Rumex obtusifolius leaves prepared in Example 6, and the protein powder of Rumex K-1 leaves prepared in Example 7 obtain IC 50 by different protein concentration gradients and fitting, and the results are shown in Table 6 and Figure 7 as follows.

[0109] Table 6 IC of α-glucosidase activity in protein samples 50 As can be analyzed from Table 6 and Figure 7 it can be seen that the IC of α-glucosidase activity in the protein powder of Rumex patientia leaves prepared in Examples 1 to 5, the protein powder of Rumex obtusifolius leaves prepared in Example 6, and the protein powder of Rumex K-1 leaves prepared in Example 7 50 are all not higher than 5.63 mg / mL.

[0110] In summary, the method for extracting leaf protein of the present invention has the following advantages: 1) Synergistic optimization of high purity and high yield: By adopting the "acid / alkali / alcohol gradient purification" process, through staged regulation of acidic pretreatment, alkaline redissolution, and gradient ethanol for selective precipitation, proteins are effectively separated from other components (such as phenolic substances, polysaccharides, cellulose, etc.), achieving a protein purity of 68% - 95% (dry basis) under mild conditions, far higher than the purity of traditional methods (mostly below 60%), and even superior to the purity of the high-temperature strong-alkali method (without considering protein activity guarantee). In particular, by controlling the extraction conditions, the ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit can be retained. This protein is an important nutritional and functional component in Rumex patientia L., and also provides a key indicator for product quality control.

[0111] The method for extracting leaf protein of the present invention has a protein yield of not less than 50% on the premise of ensuring high purity. Compared with the traditional alkali dissolution and acid precipitation method (yield often < 50%), the present invention significantly improves the yield on the premise of ensuring high purity, solving the contradiction of "high purity relying on extreme conditions" and "low yield under mild conditions" in the prior art. For example, by optimizing the ethanol concentration of 70 - 80%, efficient separation of impurities is achieved, avoiding the destruction of protein structure by high temperature or strong alkali, thus taking into account both purity and yield and reducing protein loss. The high yield reduces production costs, making Rumex patientia L. protein more competitive in the plant-based food market. At the same time, it improves the utilization rate of Rumex patientia L. resources and is conducive to the development of sustainable agriculture. 2) Comprehensive enhancement of functional properties: The method for extracting leaf protein of the present invention emphasizes mild extraction, avoiding the destruction of protein structure by extreme conditions such as high temperature and strong alkali, and retaining the natural functional properties of proteins to the greatest extent. High solubility (not less than 78%): Protein solubility is an important factor affecting its application. The protein prepared by the present invention has excellent solubility under neutral conditions, enabling it to be more easily added to various food formulations, improving the texture and taste of products. High emulsifying property (not less than 45 m 2 / g): Emulsifying property is the key for proteins to form stable emulsions in food processing. The protein prepared by the present invention has excellent emulsifying performance, especially having good emulsifying property (not less than 25 m 2 / g), enabling it to be widely used in the production of food products such as dairy product substitutes, salad dressings, margarine, etc. High foaming ability (not less than 170%) and good foam stability (stability not less than 30% after 30 minutes): Foaming ability and foam stability are important properties of proteins in applications such as baked goods and desserts. The protein prepared by the present invention has excellent foaming ability and foam stability, enabling it to improve the fluffiness and texture of products. These excellent functional properties enable the protein products prepared by the present invention to be widely used in various food fields, such as: ① Plant-based foods: As a key component of products such as plant-based meat, plant-based milk, and plant-based yogurt, providing necessary nutrition and improving texture; ② Functional foods: Added to products such as sports nutrition foods and dietary supplements to meet the nutritional needs of specific populations; ③ Baked goods: Improving the texture and taste of bread, cakes, etc. The above highlights the retention and enhancement of the natural functions of proteins by the method of the present invention.

[0112] 3) Retaining hypoglycemic activity and having potential health benefits: The protein products prepared by the present invention retain the phenolic substances naturally present in Atriplex hortensis L., which have significant hypoglycemic activity. By inhibiting the activities of α-glucosidase and α-amylase, they can delay the digestion and absorption of carbohydrates, thereby reducing postprandial blood glucose levels. This property gives the protein products prepared by the present invention potential health benefits and enables them to be applied to functional foods: developing foods for diabetic populations to help control blood glucose levels; dietary supplements: as a natural hypoglycemic component and added to health products.

[0113] 4) Comprehensive development of by-products to maximize resource utilization: When extracting protein by the present invention, the first by-product (the first supernatant after drying) and the second by-product (the third precipitate after drying) can be obtained. The by-product obtained after drying the first supernatant contains acid-soluble proteins, polypeptides, amino acids, soluble sugars, and organic acids. Fibers or secondary proteins can be extracted from the second precipitate, realizing the resource utilization of all components of Atriplex hortensis L. By further processing and utilizing the by-products, for example, using cellulose to produce biofuels or animal feeds, and extracting phenolic substances as natural antioxidants for use in the food or cosmetic fields, the comprehensive utilization of Atriplex hortensis L. resources can be achieved, improving economic and environmental benefits.

[0114] 5) Simple and controllable process, easy for industrial production: The "acid / alkali / alcohol gradient purification" method adopted by the present invention is simple to operate and easy to control, and is suitable for industrial production. By optimizing the parameters of each step, such as solid-liquid ratio, temperature, pH value, ethanol concentration, centrifugation, etc., precise control of protein purity, yield, and functional properties can be achieved.

[0115] 6) Environmentally friendly and in line with the concept of sustainable development: The present invention uses water and ethanol as the main solvents, avoiding the use of toxic and harmful chemicals and reducing environmental pollution. The high yield and comprehensive utilization of by-products maximize the utilization of Rumex patientia resources and reduce waste. Rumex patientia itself has characteristics such as a short growth cycle and strong adaptability, and can grow on barren land, which is beneficial to improving the ecological environment. It has the value of popularization and application in the field of food processing technology.

[0116] The above embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are within the protection scope of the present invention.

Claims

1. A method for extracting leaf protein, characterized in that, It includes the following steps: S1. Pulverize plant leaves to obtain a first slurry, and centrifuge to obtain a first precipitate and a first supernatant; S2. Add water to the first precipitate, adjust the pH value to be greater than or equal to 7, and perform heat treatment to obtain a first precipitate dispersion; S3. Add an organic solvent to the first precipitate dispersion, and centrifuge to obtain a second precipitate and a second supernatant; S4. Recover the residual organic solvent in the second precipitate and the second supernatant respectively to obtain a third precipitate and a third supernatant; S5. Refine the third supernatant to obtain leaf protein.

2. The method for extracting leaf protein according to claim 1, wherein The plant leaves are selected from at least one of Rumex patientia L., Rumex tianschanicus A. Los., Rumex K-1, Medicago sativa L., and Morus alba L.

3. The extraction method of leaf protein according to claim 1, characterized in that, In S1, before centrifugation, it further includes: adjusting the pH value of the first slurry to acidic; and / or, the mass ratio of the solids to water in the first slurry is 1:5 to 1:30; and / or, centrifuge under the condition that the centrifugal force is 2000 - 4000 g to obtain the first precipitate and the first supernatant.

4. The extraction method of leaf protein according to claim 1, characterized in that, The mass ratio of the first precipitate to water is 1:5 to 1:30; and / or, the temperature of the heat treatment is 40 - 70 °C; and / or, in S2, an alkaline substance is used to adjust the pH value to be greater than or equal to 7; Preferably, the alkaline substance is selected from at least one of alkali metal hydroxides, alkaline earth metal hydroxides, alkali metal carbonates, alkali metal bicarbonates, and alkali metal phosphates; and / or, the pH value of the first precipitate dispersion is 7 - 12.

5. The method for extracting leaf protein according to claim 1, wherein, The organic solvent is selected from at least one of ethanol, methanol, isopropanol, and acetone; and / or, the concentration of the organic solvent in the mixture after adding the organic solvent to the first precipitate dispersion is 65 - 85% (v / v); and / or, centrifuge under the condition that the centrifugal force is 2000 - 4000 g to obtain the second precipitate and the second supernatant.

6. The method for extracting leaf protein according to claim 1, wherein, Use vacuum evaporation to recover the residual organic solvent in the second precipitate and the second supernatant respectively; The pressure of the vacuum evaporation is 20 - 40 kPa, and the temperature is 30 - 55 °C.

7. The method for extracting leaf protein according to claim 1, wherein The refining method is selected from at least three combinations of pH adjustment, centrifugation, membrane separation, adding water for modulation, and evaporation concentration.

8. The extraction method of leaf protein according to claim 1, characterized in that, The first supernatant is used as a beverage raw material; or, dry the first supernatant to obtain a first by-product, and the first by-product contains acid-soluble protein, polypeptide, amino acid, soluble sugar, and organic acid, and the first by-product is used as a raw material for solid beverages; and / or, dry the third precipitate to obtain a second by-product, and the second by-product contains fiber or secondary protein, and the second by-product is used as a raw material for dietary fiber foods or feed.

9. A leaf protein obtained by using the extraction method according to any one of claims 1 to 8.

10. An application of a leaf protein obtained by using the extraction method according to any one of claims 1 to 8 in the preparation of hypoglycemic products and / or products for improving insulin secretion function and / or products for improving food fluffiness.

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