阅读说明：本技术 一种绿豆低聚肽粉及其制备方法 (Mung bean oligopeptide powder and preparation method thereof ) 是由 王昭日 刘明川 杨胜杰 于 2019-10-06 设计创作，主要内容包括：本发明涉及一种绿豆低聚肽粉及其制备方法,所述绿豆低聚肽粉的肽含量在85wt％以上,其中98％以上绿豆肽的分子量小于1000Dalton。其制备方法包括蛋白提取、生物酶酶解、过滤、浓缩和干燥,操作工序简单、对环境无污染、对人体无伤害,产品率高、成本低,此工艺较为适合大规模生产。所述低聚肽粉具有较好的清除自由基活性等功效。(The invention relates to mung bean oligopeptide powder and a preparation method thereof, wherein the peptide content of the mung bean oligopeptide powder is more than 85 wt%, and the molecular weight of more than 98% of mung bean peptides is less than 1000 Dalton. The preparation method comprises the steps of protein extraction, biological enzyme enzymolysis, filtration, concentration and drying, has simple operation procedures, no pollution to the environment, no harm to human bodies, high product rate and low cost, and is suitable for large-scale production. The oligopeptide powder has the effects of better eliminating free radical activity and the like.)

1. The mung bean oligopeptide powder is characterized in that: the peptide content is more than 80 wt%, wherein the molecular weight of more than 80% of mung bean oligopeptides is less than 700Dalton, preferably, the molecular weight of more than 85% of mung bean oligopeptides is less than 700Dalton, the molecular weight of more than 90% of mung bean oligopeptides is less than 700Dalton, and the molecular weight of more than 95% of mung bean oligopeptides is less than 700 Dalton;

Molecular weight Dalton distribution

Preferably, the first and second electrodes are formed of a metal,

Molecular weight Dalton distribution

Preferably, the first and second electrodes are formed of a metal,

Molecular weight Dalton distribution

number average molecular weight range: 20-1100, weight average molecular weight range: 45-1300 parts; preferably, the number average molecular weight range: 100-1000, weight average molecular weight range: 100-1200; preferably, the number average molecular weight range: 200-900, weight average molecular weight range: 150 to 1100; preferably, the number average molecular weight range: 300-800, weight average molecular weight range: 200 to 1000 parts; preferably, the number average molecular weight range: 400-700, weight average molecular weight range: 300 to 900; the detection method of the peptide content is carried out by referring to the determination method of the peptide content described in GB/T22492-2008 appendix B and GB/T22729-; the molecular weight of the peptide is determined by high performance gel filtration chromatography (GPC) as described in GB/T22492-.

2. the method for preparing mung bean oligopeptide powder according to claim 1, which is characterized by comprising the following steps: adding water into the mung bean protein powder for redissolution, and sequentially carrying out enzymolysis, separation and purification, concentration and drying to obtain mung bean oligopeptide powder; the mung bean protein powder is commercially available mung bean protein powder.

3. The method of claim 2, wherein: the biological enzyme used for enzymolysis can be one of neutral protease (the enzyme activity is more than or equal to 30 ten thousand u/g), papain (the enzyme activity is more than or equal to 40 ten thousand u/g), bromelain (the enzyme activity is more than or equal to 30 ten thousand u/g), alkaline protease (the enzyme activity is more than or equal to 20 ten thousand u/g), acid protease, pepsin (the enzyme activity is more than or equal to 50 ten thousand u/g), pancreatin (the enzyme activity is more than or equal to 3000u/g), ficin (the enzyme activity is more than or equal to 5 ten thousand u/g) and momordica grosvenori protease (the enzyme activity is more than or equal to 5 ten thousand u/g) or a mixture thereof, and preferably food; the ratio of the enzyme mixture is 1: 1-1: 10.

4. The method of claim 2, wherein: the purification method can be a filter membrane filtration method and a resin separation method, and preferably a filter membrane filtration method is used; the concentration method can be vacuum concentration, normal pressure concentration, membrane concentration, preferably vacuum concentration; the drying method may be vacuum drying, heat drying, air drying, freeze drying and spray drying, and preferably spray drying is used.

5. The method according to claim 2, characterized by comprising the following operating steps:

Pretreatment of mung bean protein powder: mixing mung bean protein powder and water according to a weight ratio of 1: 20-1: 30, recording the volume V at the moment, adjusting the pH to 8-12, stirring for 20-30 min, adding acid to adjust the pH to 3-5, removing supernatant, adding water into the precipitate to the volume V, and stirring uniformly to obtain mung bean protein liquid;

enzymolysis: heating the mung bean protein liquid to 35-60 ℃, adjusting the pH value to be neutral, adding neutral protease which accounts for at least 0.1 wt% of the mung bean protein powder, stirring for enzymolysis for at least 3h, and boiling for inactivation for at least 2min to obtain a protease hydrolysate.

Separation and purification: sequentially treating the protein enzymolysis liquid with a microfiltration membrane with the aperture of 0.1-0.5 mu m and 2000-20000 Dalton;

Concentration and drying: concentrating at 40-80 ℃ until the solid content is 3-5 wt%, and spray-drying at inlet temperature of 140-160 ℃ and outlet temperature of 55-65 ℃ to obtain the mung bean oligopeptide powder with the yield of more than 50 wt%.

6. The mung bean oligopeptide powder has the peptide content of more than 70 wt%, wherein the molecular weight of more than 80% of mung bean oligopeptide is less than 700Dalton, and is characterized in that: prepared by the method of any one of claims 2 to 5; the detection method of the peptide content is carried out by referring to the determination method of the peptide content described in GB/T22492-; the molecular weight of the peptide is determined by high performance gel filtration chromatography (GPC) as described in GB/T22492-.

7. A composition characterized by: comprises the mung bean oligopeptide powder as claimed in claim 1 and pharmaceutically or food acceptable auxiliary agents; preferably, the dosage form is selected from plain tablets, film-coated tablets, sugar-coated tablets, enteric-coated tablets, dispersible tablets, capsules, granules, oral solutions or oral suspensions, and cosmetic dosage forms such as liquid, emulsion, cream, powder, block, etc.

8. Use of the mung bean oligopeptide powder according to claim 1 or the composition according to claim 7 for the preparation of a food, health food or pharmaceutical product for the treatment or prevention of symptoms caused by excess free radicals; the application of the preparation of food, health food or medicine for regulating intestinal beneficial flora, improving gastrointestinal motility and promoting nutrient absorption; the application of the composition in preparing food, health-care food or medicine for preventing, improving or treating related diseases caused by liver and lung injury; can be used for preparing food, health food or medicine for preventing muscle loss, repairing muscle injury, and increasing muscle tissue.

Technical Field

The invention belongs to the field of food processing, and relates to mung bean oligopeptide powder which is high in purity and good in activity. The invention also relates to a preparation process of the mung bean oligopeptide powder, which is simple and convenient to operate, green and environment-friendly and suitable for large-scale production.

Background

In recent years, with the continuous development of theories and technologies of pharmacy, medicine, medical immunology and molecular biology, people have more profound understanding on the biological activity of polypeptide substances, and the research on the substances also relates to a plurality of new fields. Active polypeptides from plants have attracted attention at present, and China obtains a plurality of active peptides from plants through enzymolysis, separation and purification and develops the active peptides into new drugs and health care products. As can be seen, plant peptides are becoming the focus of research.

Mung bean (Vigna radiata (Linn.) Wilczek.) is a mature seed of mung bean, a plant of the genus Vigna of the family leguminosae, also known as adzuki bean, beauty bean, bean-planting, etc. Mung beans are the main edible bean crops in China, have various edible modes, can be used for cooking mung bean porridge, grinding into powder and making cakes, and can be used as vegetables after sprouting. Mung beans are also traditional food materials used as both medicine and food in China, and are called as 'good food and good products in the middle and old world' and 'Jishichanggu'. The traditional Chinese medicine considers that the mung beans are sweet, cold and nontoxic. Enter heart and stomach meridians. Mainly used for treating erysipelas with dysphoria with smothery sensation, rubella, hot wind rushing to dolphin, raw ground into juice for oral administration, and also used for relieving swelling, descending qi, suppressing heat and removing toxicity. Mung beans are rich in protein, carbohydrate, dietary fiber, various vitamins and mineral substances. Each 100g of mung bean contains 21.6g of protein and 55.6g of carbohydrate. Mung bean protein is mostly globulin, and the amino acid composition of mung bean protein is rich in lysine, and less in methionine, tryptophan and tyrosine. For a long time, the processing and utilization of mung beans mainly use starch, and other nutritional ingredients such as protein in mung beans are not fully utilized. In recent years, as functional foods are popular, studies on physiological functions and product development of mung bean protein are increasing.

Patent CN 103409490A redissolves mung bean protein powder with content of more than 60%, and then obtains mung bean protein peptide by sequentially carrying out enzymolysis, filtration, ion exchange, concentration and drying. The ion exchange process is used in the process, the ion exchange process needs acid-base regeneration, the regeneration frequency is high, the acid-base dosage is high, and the influence on surrounding water and atmospheric environment is large. Patent CN 101979655B inventor uses ethanol and sodium sulfite to carry out the preliminary treatment with mung bean albumen powder before the proteolysis, and sodium sulfite can have the stimulation to eyes, skin, mucosa, pollutes the water source, receives the high thermolysis and produces poisonous sulphide flue gas, causes environmental pollution and health easily. The inventor of patent CN 103290086B prepares the mung bean protein peptide by two times of enzymolysis, activated carbon adsorption and filtration, and the method is complicated and the cost is increased. Therefore, the development of a simple, environment-friendly and low-cost preparation process of the mung bean protein peptide becomes a research hotspot.

Aiming at the defects of the prior art, the invention provides a preparation method of mung bean oligopeptide, and the high-purity mung bean oligopeptide can be obtained by one-step enzymolysis with single enzyme and a two-step filtration method without adding any organic solvent and harmful chemical substances in the preparation process. The method provided by the invention is simple and convenient, easy to operate, green and environment-friendly, low in cost, high in yield and particularly suitable for large-scale production.

disclosure of Invention

The invention provides mung bean oligopeptide powder with high purity and low molecular weight, and simultaneously provides a preparation process of mung bean oligopeptide powder, which is simple and convenient to operate, green and environment-friendly, low in cost and high in yield. The prepared mung bean oligopeptide powder has the effects of better eliminating free radical activity, improving sleep quality, improving fat metabolism of organisms, repairing muscle injury and promoting growth of the muscle injury, adjusting the pH value of intestinal tracts to improve beneficial flora, enhancing nutrient absorption, relaxing bowel, repairing liver injury and improving functions of the mung bean oligopeptide powder.

The mung bean oligopeptide powder is characterized in that: the peptide content is more than 85 wt%, wherein the molecular weight of more than 98% of the mung bean peptides is less than 1000 Dalton.

The method for detecting the peptide content of the mung bean oligopeptide powder is carried out by referring to the method for determining the peptide content in national standards GB/T22492-2008 appendix B and GB/T22729-2008 of the people's republic of China.

The method for measuring the peptide relative molecular weight distribution of the mung bean oligopeptide powder is carried out by high performance gel filtration chromatography (GPC) according to national standards GB/T22492 supplement 2008 appendix A and GB/T22729 supplement 2008 appendix A of the people's republic of China.

The preparation method of the mung bean oligopeptide powder comprises the following steps: and adding water into the mung bean protein powder for redissolution, and sequentially carrying out enzymolysis, purification, concentration and drying to obtain the mung bean oligopeptide powder.

The biological enzyme for proteolysis in the preparation method can be one or a mixture of neutral protease, papain, bromelain, alkaline protease, acid protease, pepsin, pancreatin, momordica grosvenori protease and ficin.

The purification method in the preparation method can be a filter membrane filtration method and a resin separation method, and the filter membrane filtration method is preferably used.

The concentration method in the preparation method can be vacuum concentration, normal pressure concentration and membrane concentration, and preferably the vacuum concentration method is used.

The drying method in the preparation method can be vacuum drying, heating drying, airing, air drying, freeze drying and spray drying.

The mung bean oligopeptide powder is prepared by the following method:

Pretreatment of mung bean protein powder: mixing mung bean protein powder and water according to a weight ratio of 1: 20-1: 30, recording the volume V at the moment, adjusting the pH to 8-12, stirring for 20-30 min, adding acid to adjust the pH to 3-5, removing supernatant, adding water into the precipitate to the volume V, and stirring uniformly to obtain mung bean protein liquid;

Enzymolysis: heating the mung bean protein liquid to 35-60 ℃, adjusting the pH value to be neutral, adding protease which is at least 0.1 wt% of the mung bean protein powder, stirring for enzymolysis for at least 3h, and boiling for inactivation for at least 2min to obtain a protease hydrolysate.

Separation and purification: treating the protease hydrolysate with microfiltration membrane, concentrating, and drying to obtain semen Phaseoli Radiati oligopeptide powder.

The composition comprises the mung bean oligopeptide powder and pharmaceutically or food acceptable auxiliary agents.

according to the prior art, the composition can be prepared into any dosage form, such as plain tablets, film-coated tablets, sugar-coated tablets, intestine-coated tablets, dispersible tablets, capsules, granules, oral solution or oral suspension, and liquid, emulsion, cream, powder, block and other cosmetic dosage forms.

The mung bean oligopeptide powder is used for preparing food, health-care food or medicine for preventing or treating symptoms caused by excessive free radicals; used for preparing food, health food or medicine for improving fat metabolism; is used for preparing food, health food or medicine for preventing muscle loss, repairing muscle injury, and increasing muscle tissue; used for preparing food, health food or medicine for regulating intestinal beneficial flora, improving gastrointestinal motility, and promoting nutrient absorption; can be used for preparing food, health food or medicine for preventing, improving or treating related diseases caused by liver injury.

Compared with the prior art, the invention has the following advantages:

(1) the mung bean oligopeptide powder provided by the invention has the characteristics of high purity and small molecular weight, is easier to absorb and has stronger efficacy.

(2) the mung bean oligopeptide powder provided by the invention has good water solubility, clear water solution and good taste, and is particularly suitable for being used as liquid beverage and the like.

(3) The preparation process of the mung bean oligopeptide powder provided by the invention only needs one-step enzymolysis and one enzyme, and multi-step hydrolysis of multiple enzymes is not needed. The more kinds of the used biological enzymes are, the larger the workload of enzyme activity detection at the early stage is, and the higher the purchase cost of the biological enzymes is, so that the cost is increased. The multi-step enzymolysis causes complex process steps, prolonged hydrolysis time and increased cost.

(4) The preparation process of the mung bean oligopeptide powder provided by the invention does not need any organic solvent or any toxic or harmful chemical substance, and the product does not have any organic solvent or any toxic or harmful chemical substance residue, is green and environment-friendly, is harmless to human body, and can be eaten at ease.

(5) The protease used in the invention is edible enzyme, and has wide source, low cost and small addition amount.

(6) The invention refers to the determination method of peptide content and relative molecular weight distribution described in the national standards GB/T22492-.

Drawings

FIG. 1: influence of growth of lactobacillus brevis in intestinal tracts of the zebra fish after the treatment of the mung bean peptides;

FIG. 2: a typical graph of the liver fat signal intensity of the zebra fish after the treatment of the mung bean peptide;

FIG. 3: h & E staining results of zebra fish muscle fibers after the treatment of the mung bean peptide.

Examples

The invention is further illustrated by the following examples. It should be understood that the method described in the examples is only for illustrating the present invention and not for limiting the present invention, and that simple modifications of the preparation method of the present invention based on the concept of the present invention are within the scope of the claimed invention. All the raw materials and solvents used in the examples are commercially available products unless otherwise specified.

Preparation of example 1

Adding 2000L of water into 100kg of mung bean protein powder (the protein content is more than 30 wt%), adding food-grade sodium hydroxide with the concentration of about 2mol/L to adjust the pH value to 8.5-9.5, stirring for 30min, adding food-grade hydrochloric acid with the concentration of about 1mol/L to adjust the pH value to 3.5-4.0 while stirring, standing until a precipitate is separated from a supernatant, removing the supernatant, adding water to 2000L, and uniformly stirring to obtain the mung bean protein solution. Heating the mung bean protein liquid to 45 ℃, adjusting the pH value to be neutral, adding 100g of food-grade neutral protease (the enzyme activity is 30 ten thousand u/g), stirring for enzymolysis for 3h, boiling and inactivating for 2min to obtain a protease hydrolysate. Treating the protein enzymolysis solution with microfiltration membrane with pore diameter of 0.1 μm, concentrating at 60 deg.C until solid content is 5 wt%, and spray drying to obtain 18.5kg semen Phaseoli Radiati oligopeptide powder (batch number L-1) with yield of 18.5 wt%. By adopting the detection method of GB/T22492-2008 appendix A and appendix B, the peptide content is 87.7%, and the molecular weight of less than 1000Dalton accounts for 98.1%.

Preparation of example 2

adding 3000L of water into 100kg of mung bean protein powder (the protein content is more than 70 wt%), adding food-grade sodium hydroxide with the concentration of about 2mol/L to adjust the pH value to 10.5-11.0, stirring for 30min, adding food-grade hydrochloric acid with the concentration of about 1mol/L to adjust the pH value to 4.5-5.0 while stirring, standing until a precipitate is separated from a supernatant, removing the supernatant, adding water to 3000L, and uniformly stirring to obtain the mung bean protein solution. Heating the mung bean protein liquid to 45 ℃, adjusting the pH value to be neutral, adding 100g of food-grade papain (the enzyme activity is 40 ten thousand u/g), stirring for enzymolysis for 3h, and boiling for inactivation for 5min to obtain a protein enzymolysis liquid. Treating the protein enzymolysis solution with microfiltration membrane with pore diameter of 0.5 μm, concentrating at 50 deg.C until solid content is 5 wt%, and spray drying to obtain 42.2kg semen Phaseoli Radiati oligopeptide powder (batch number L-2) with yield of 42.2 wt%. By adopting the detection method of GB/T22492-2008 appendix A and appendix B, the peptide content is 85.8%, and the molecular weight of less than 1000Dalton accounts for 98.4%.

Preparation of example 3

Adding 3000L of water into 100kg of mung bean protein powder (protein content is more than 80 wt%), and stirring to obtain mung bean protein solution. Heating the mung bean protein liquid to 45 ℃, adjusting the pH value to be neutral, adding 100g of food-grade neutral protease (the enzyme activity is 30 ten thousand u/g), stirring for enzymolysis for 3h, boiling and inactivating for 5min to obtain a protein enzymolysis liquid. The protein enzymatic hydrolysate was treated with a microfiltration membrane having a pore size of 0.5 μm, concentrated at a temperature of about 80 ℃ to a solid content of 5 wt%, and spray-dried to obtain 63.4kg of mung bean oligopeptide powder (lot L-3) with a yield of 63.4 wt%. By adopting the detection method of GB/T22492-2008 appendix A and appendix B, the peptide content is 86.0%, and the molecular weight of less than 1000Dalton accounts for 99.0%.

Preparation of example 4

Adding 3000L of water into 100kg of mung bean protein powder (protein content is more than 80 wt%), and stirring to obtain mung bean protein solution. Heating the mung bean protein liquid to 55 ℃, adjusting the pH value to be neutral, adding 100g of food-grade bromelain (the enzyme activity is 30 ten thousand u/g), stirring for enzymolysis for 3h, boiling and inactivating for 5min to obtain a protein enzymolysis liquid. Treating the protein enzymolysis solution with microfiltration membrane with pore diameter of 0.5 μm, concentrating at 80 deg.C until solid content is 5 wt%, and vacuum drying to obtain 62kg semen Phaseoli Radiati oligopeptide powder (lot L-4) with yield of 62 wt%. By adopting the detection method of GB/T22492-2008 appendix A and appendix B, the peptide content is 85.1%, and the molecular weight of less than 1000Dalton accounts for 98.4%.

Preparation of example 5

Adding 3000L of water into 100kg of mung bean protein powder (protein content is more than 80 wt%), and stirring to obtain mung bean protein solution. Heating the mung bean protein liquid to 50 ℃, adjusting the pH value to 8.0-9.0, adding 100g of food-grade alkaline protease (the enzyme activity is 20 ten thousand u/g), stirring for enzymolysis for 3h, boiling and inactivating for 5min to obtain a protein enzymolysis liquid. Treating the protein enzymolysis solution with microfiltration membrane with pore diameter of 10 μm, concentrating at 80 deg.C until solid content is 5 wt%, and freeze drying to obtain 64.4kg semen Phaseoli Radiati oligopeptide powder (lot L-5) with yield of 64.4 wt%. The detection method of GB/T22492-.

Preparation of example 6

Adding 3000L of water into 100kg of mung bean protein powder (protein content is more than 80 wt%), and stirring to obtain mung bean protein solution. Heating the mung bean protein liquid to 45 ℃, adjusting the pH value to 8.5-9.0, adding 100g of food-grade trypsin (the enzyme activity is 20 ten thousand u/g), stirring for enzymolysis for 4 hours, boiling and inactivating for 2min to obtain a protease hydrolysate. The protein enzymolysis liquid is processed by a microfiltration membrane with the aperture of 5 mu m, concentrated at the temperature of about 60 ℃ until the solid content is 5 wt%, and dried under normal pressure to obtain 65.4kg of mung bean oligopeptide powder (batch number L-6), wherein the yield is 65.4 wt%. By adopting the detection method of GB/T22492-2008 appendix A and appendix B, the peptide content is 85.9%, and the molecular weight of less than 1000Dalton accounts for 98.8%.

Preparation of example 7

Adding 3000L of water into 100kg of mung bean powder, adding food-grade sodium hydroxide with the concentration of about 2mol/L to adjust the pH value to 9.5-10.0, stirring for 60min, adding food-grade hydrochloric acid with the concentration of about 1mol/L to adjust the pH value to 3.5-4.0 while stirring, standing until a precipitate is separated from a supernatant, removing the supernatant, adding water to 3000L, and uniformly stirring to obtain the mung bean protein liquid. Heating the mung bean protein liquid to 45 ℃, adjusting the pH value to be neutral, adding 100g of food-grade neutral protease (the enzyme activity is 30 ten thousand u/g), stirring for enzymolysis for 5h, boiling and inactivating for 20min to obtain a protein enzymolysis liquid. The protein enzymatic hydrolysate was treated with a microfiltration membrane having a pore size of 0.5 μm, concentrated at a temperature of about 50 ℃ to a solid content of 5 wt%, and spray-dried to obtain 11.1kg of mung bean oligopeptide powder (lot L-7) with a yield of 11.1 wt%. The detection method of GB/T22492-. Biological Activity example 1 (evaluation of antioxidant Activity)

1.ABTS⁺Free radical scavenging experiments

Preparation of PBS buffer: weighing 8g of sodium chloride, 0.2g of potassium chloride, 0.24g of potassium dihydrogen phosphate and 3.62g of disodium hydrogen phosphate dodecahydrate, putting the materials into a 1000mL beaker, adding 800mL of distilled water, stirring to dissolve the materials, adjusting the pH value to 7.4 by using hydrochloric acid or sodium hydroxide, transferring the materials into a 1000mL volumetric flask, adding distilled water to dilute the materials to a scale, and shaking the materials uniformly for later use.

ABTS⁺Preparation of a storage solution: ABTS is weighed to precision⁺Placing the mixture into a 20mL brown volumetric flask, adding 15mL distilled water, carrying out ultrasonic treatment for 5min, fixing the volume to the scale with the distilled water, and shaking up. Accurately weighing about 76mg of potassium persulfate, placing the potassium persulfate in a 2mL brown volumetric flask, adding 1mL of distilled water, performing ultrasonic dissolution, fixing the volume to the scale with the distilled water, and shaking up. Add 352. mu.L of potassium persulfate solution to the ABTS solution with precision, shake up, and stand overnight.

ABTS⁺Preparing a working solution: accurately pipette 1mL of the storage solution, add about 65mL of PBS buffer, and shake well.

preparing a test solution: precisely weighing an appropriate amount of mung bean peptide powder (L-1), placing in a 20mL brown volumetric flask, adding 15mL PBS buffer, performing ultrasonic treatment for 5min, fixing the volume to the scale with the PBS buffer, and shaking up to obtain the final product.

The method comprises the following operation steps: accurately sucking 0.5mL of test solution and 5mL of ABTS working solution, and uniformly mixing; accurately sucking 0.5mL of test solution and 5mL of PBS buffer solution, and uniformly mixing; accurately pipette 5mL of ABTS working solution and 0.5mL of PBS buffer, mix well, immediately measure absorbance at 734nm, and calculate the radical clearance according to the following formula:

IR％＝[1-(Ai-Aj)/A0]*100％；

wherein Ai represents the absorbance of the solution after the solution to be tested and the ABTS are mixed;

aj represents the absorbance of the solution after the solution to be detected and the solvent are mixed;

A0 represents the absorbance of the solution after mixing ABTS and solvent.

SRSA superoxide anion radical scavenging experiments

Preparation of 0.1moL/L PBS buffer (pH 7.4): weighing 80g of sodium chloride, 2g of potassium chloride, 2.4g of potassium dihydrogen phosphate and 23.1g of dipotassium hydrogen phosphate trihydrate, putting the mixture into a 1000mL beaker, adding 600mL of distilled water, stirring to dissolve the mixture, adjusting the pH to 7.2 by using hydrochloric acid or sodium hydroxide, transferring the mixture into a 1000mL volumetric flask, adding distilled water to dilute the mixture to a scale, and shaking the mixture uniformly for later use.

Preparation of 150. mu. moL/L NBT solution: accurately weighing 12.5mg of NBT, placing the NBT in a 100mL brown volumetric flask, adding distilled water, dissolving the NBT by ultrasonic treatment, fixing the volume to a scale by using the distilled water, and shaking up to obtain the NBT-NBT.

Preparation of 60. mu. moL/L PMS solution: accurately weighing 18.8mg of PMS, placing the PMS in a volumetric flask of 1000mL, adding distilled water, dissolving the PMS by ultrasonic treatment, fixing the volume to a scale by using the distilled water, and shaking up to obtain the PMS.

preparation of 468. mu. moL/L NADH solution: accurately weighing 33.9mg of NADH, placing in a 100mL volumetric flask, adding distilled water, dissolving by ultrasonic treatment, fixing the volume to the scale with distilled water, and shaking up to obtain the final product.

Preparing a test solution: precisely weighing appropriate amount of semen Phaseoli Radiati peptide powder (L-1), adding water, ultrasonic dissolving, mixing, and testing.

Preparing a working solution: 1mL of 0.1moL/L PBS buffer solution (pH 7.4) is put into a volumetric flask, 1mL of 150. mu. moL/L NBT solution is added, 2mL of 468. mu. moL/L NADH solution is added, 1mL of 60. mu. moL/L PMS solution is added, the mixture is stirred uniformly, the mixture reacts for 5min at 25 ℃, and the absorbance value is measured at the wavelength of 560 nm.

the method comprises the following operation steps: accurately sucking 0.5mL of test solution and 5mL of the working solution, and uniformly mixing; accurately sucking 0.5mL of test solution and 5mL of distilled water, and uniformly mixing; accurately sucking 5mL of the above working solution and 0.5mL of distilled water, uniformly mixing, immediately measuring the absorbance at 560nm, and calculating the radical clearance according to the following formula:

IR％＝[1-(Ai-Aj)/A0]*100％；

Wherein Ai represents the absorbance of the solution after the solution to be tested and the SRSA are mixed;

Aj represents the absorbance of the solution after the solution to be detected and the solvent are mixed;

a0 represents the absorbance of the solution after mixing the SRSA and solvent.

Mung bean peptide powder (L-1) is prepared at the concentration of 500 mug/mL and 1000 mug/mL, vitamin C is used as a positive control (the concentration is 100 mug/mL), and the test results are shown in Table 1:

TABLE 1 mung bean peptide powder scavenging free radical test results

As can be seen from Table 1, the mung bean peptide powder prepared by the method of the invention shows good scavenging effect on ABTS and SRSA free radicals and has good antioxidant activity.

Biological Activity example 2 (evaluation of intestinal flora regulating Effect)

Labeling of Lactobacillus brevis with CM-DiI at 6X 10⁶Feeding 5dpf of sterile wild AB strain zebra fish by using lactobacillus brevis with per mL concentration, and establishing a zebra fish intestinal symbiotic bacteria model; the sterile zebrafish fed with Lactobacillus brevis were incubated at 35 ℃ to 6 dpf. At 6dpf, the Lactobacillus brevis was removed and randomly distributed into 6-well plates with 30 tails per well and a fish farming water volume of 3mL per well. The concentration of the water-soluble mung bean peptide powder (L-1) is 2000 mug/mL, and a model control group and a normal control group are arranged at the same time. Continuously culturing zebra fish of each experimental group at 35 ℃ for 6h, randomly selecting 10 zebra fish of each experimental group, collecting pictures under a fluorescence microscope, calculating the fluorescence intensity of the lactobacillus brevis in intestinal tracts of the zebra fish (S, the fluorescence intensity represents the number of the lactobacillus brevis in the intestinal tracts), and calculating the fluorescence intensityAnd (4) evaluating the regulation effect of the mung bean peptide powder on the intestinal flora according to the statistical analysis result. The formula is as follows: intestinal flora regulating effect (%) - (S)_{Test article}-S_{model set})/S_{model set}X 100%, statistical analysis using one-way analysis of variance and T test, p<0.05 showed significant differences, and the results are shown in table 2.

TABLE 2 Regulation of intestinal Lactobacillus brevis by mung bean peptide powder (n 10)

compared with the model control group,^*p<0.05

As shown in Table 2, the fluorescence intensity of the intestinal tract of the zebra fish in the model control group is 4189048 pixels, and compared with the normal control group (1517330 pixels), p is less than 0.001, so that the survival of the lactobacillus brevis in the intestinal tract is shown, and the model is successfully established. When the concentration of the mung bean peptide powder is 2000 mug/mL, the fluorescence intensity of the zebra fish intestinal tract is 5479110 pixels, p is less than 0.05 compared with 418904 pixels of a model control group, and the intestinal flora regulating effect is 31%. The mung bean peptide powder can promote the growth of lactobacillus brevis in intestinal tracts and has obvious intestinal flora regulating effect.

biological Activity example 3 (evaluation of hepatoprotective Effect)

And randomly selecting the melanin allele mutant type semitransparent Albino strain zebra fish 5 days (5dpf) after 180-tail fertilization into a six-hole plate, treating 30 zebra fish in each hole (experimental group), and inducing the zebra fish by using absolute ethyl alcohol to establish an alcoholic fatty liver model. The mung bean peptide was dissolved in water to give a concentration of 62.5. mu.g/mL and the positive control "RU 21" was dissolved in water to give a concentration of 100. mu.g/mL, and a normal control group (water-treated zebrafish for fish farming) and a model control group were set up at the same time, and the volume of the solution in each well was 3 mL. The other experimental groups except the normal control group are respectively treated with absolute ethyl alcohol for 30 hours. After treatment, the zebra fish is placed into 4% paraformaldehyde for fixation, taken out after overnight at 4 ℃, washed for 3 times by PBS, subjected to gradient dehydration by propylene glycol, dyed overnight in 0.5% oil red O working solution which is freshly prepared and filtered, and then 10 zebra fish of each group are randomly taken out after dyeing and observed under a dissecting microscopeLiver, photograph and save pictures. Carrying out image analysis by NIS-Elements D3.10 advanced image processing software, collecting data, analyzing and counting the intensity (S) of a fat signal in the liver of the zebra fish, and evaluating the protective effect of the test sample on the alcohol fatty liver induced by the absolute ethyl alcohol according to the statistical analysis result of the intensity of the liver fat signal, wherein the statistical processing result is expressed by mean +/-SE. The formula for the calculation of the hepatoprotective effect is as follows: hepatoprotective effect (%) - (S)_{Model (model)}-S_{test article})/(S_{Model (model)}-S_{Is normal}) X 100%, statistical analysis by T-test, p<0.05 showed significant differences, and the results are shown in Table 3.

TABLE 3 liver protecting action of the peptide powder of mung bean on zebrafish (n 10)

compared with the normal control group,^***p<0.001; compared with the model control group,^△△△p<0.001

as shown in Table 3, the comparison of the intensity of the liver fat signal of the zebrafish in the model control group (17863 pixels) with the intensity of the liver fat signal of the normal control group (11883 pixels) indicates that the model is successfully established, wherein p is less than 0.001. The positive control drug RU21 shows that the concentration of 100 mug/mL of the zebra fish liver fat signal intensity is 12742 pixels, compared with the model control group, p is less than 0.001, the liver protection effect is 86%, and the result shows that RU21 has the protection effect on the zebra fish alcoholic fatty liver. The fat signal intensity of the zebra fish liver with the concentration of the mung bean peptide of 62.5 mu g/mL is 12901 pixels, p is less than 0.001 compared with a model control group, and the liver protection effect is 83 percent respectively. The mung bean peptide is suggested to have an obvious protective effect on the alcoholic fatty liver of the zebra fish under the condition of the experimental concentration.

Biological Activity example 4 (evaluation of muscle fiber-enhancing action)

Randomly selecting 150 fertilized wild AB strain zebra fishes 2 days (2dpf) in a six-hole plate, treating 30 zebra fishes in each hole, and inducing the zebra fishes by using absolute ethyl alcohol to establish an alcoholic muscle injury model. The concentration of the mung bean peptide (L-1) was 62.5. mu.g/mL, and the volume of the drug solution in each well was 3 mL. The other experimental groups except the normal control group are respectively treated with absolute ethyl alcohol for 30 hours. After the test article treatment was completed, H & E staining (fixation-dehydration-embedding-sectioning-staining) was performed on zebrafish, and pathological analysis was performed, as shown in FIG. 3.

As can be seen from FIG. 3, the zebra fish skeletal muscle cells of the normal control group are uniform and long-striped, and the transverse striations are clear; the model control group had myofiber relaxation with inflammatory cell infiltration, but no necrosis. The mung bean peptide group muscle fibers are uniform and long-strip-shaped, have clear transverse striations and do not have myositis or necrosis, and are similar to a normal control group, so that the mung bean peptide group muscle fibers have the muscle fiber protection effect under the condition of the experimental concentration.