阅读说明：本技术 一种火麻仁蛋白制备抗氧化肽的方法 (Method for preparing antioxidant peptide from fructus cannabis protein ) 是由 孔令羽 刘胜贵 蒋永昌 李智高 薛红芬 张琼 于 2021-04-25 设计创作，主要内容包括：本发明公开了一种火麻仁蛋白制备抗氧化肽的方法,包括以下步骤：用超声波细胞粉碎器作用火麻仁得到火麻仁粕,用碱法提取火麻仁蛋白质；配制枯草芽孢杆菌液体发酵培养基,将火麻仁蛋白接种至发酵液中培养；发酵液离心取上清液,所得肽溶液以凝胶层析柱分离纯化,收集洗脱组分的第二峰,液质联用色谱分离鉴定,得到火麻仁抗氧化肽粗品；火麻仁抗氧化肽粗品经过二次纯化即得抗氧化纯品。有益效果为：本制备方法利用枯草芽孢杆菌进行发酵,具有酶产率和回收率高等优点,显著降低50～80%成本,提高10～20%得率,抗氧化肽纯品中火麻仁抗氧化肽的纯度较高,其对DPPH自由基和羟自由基的清除率达90%以上,且具有溶血栓和降血压的功效,有较高的经济价值。(The invention discloses a method for preparing antioxidant peptide from fructus cannabis protein, which comprises the following steps: using ultrasonic cell crusher to act fructus Cannabis to obtain fructus Cannabis dregs, and extracting fructus Cannabis protein with alkali method; preparing a bacillus subtilis liquid fermentation culture medium, and inoculating the hemp seed protein into the fermentation liquor for culture; centrifuging the fermentation liquor to obtain supernatant, separating and purifying the obtained peptide solution by using a gel chromatography column, collecting a second peak of an eluted component, and performing liquid chromatography-mass spectrometry to separate and identify to obtain a fructus cannabis antioxidant peptide crude product; and carrying out secondary purification on the hemp seed antioxidant peptide crude product to obtain an antioxidant pure product. The beneficial effects are that: the preparation method utilizes the bacillus subtilis to ferment, has the advantages of high enzyme yield and recovery rate and the like, obviously reduces the cost by 50-80%, improves the yield by 10-20%, has high purity of fructus cannabis antioxidant peptide in the pure antioxidant peptide product, has clearance rate of DPPH free radical and hydroxyl free radical of more than 90%, has the effects of dissolving thrombus and reducing blood pressure, and has high economic value.)

1. A method for preparing antioxidant peptides from fructus cannabis proteins is characterized in that the fructus cannabis protein antioxidant peptides are prepared by fermenting fructus cannabis proteins through bacillus subtilis, wherein the concentration of a substrate is 3% -5% of fructus cannabis proteins, the concentration of a fermentation product is 5% -15% of bacillus subtilis, the rotation speed of a shaking table is 180-.

2. The beer beverage containing cannabidiol as claimed in claim 1, wherein the method comprises the steps of;

step 1, preparation of hemp seed protein

Vacuum drying fructus Cannabis, removing shell, pulverizing, removing fructus Cannabis oil with ultrasonic cell pulverizer to obtain fructus Cannabis dregs, extracting protein with alkali method, and vacuum drying;

step 2, activation and purification of the strains

Preparing nutrient agar PDA culture medium, sterilizing; sterilizing, cooling, and pouring into a flat seed culture medium; inoculating strains for activation, and selecting strains with good growth for later use;

step 3, preparing antioxidant peptide

Preparing a fructus cannabis protein powder liquid fermentation culture medium, and sterilizing; after sterilization and temperature reduction, inoculating the strain into a culture medium for shaking table fermentation; centrifuging the fermentation liquor to obtain supernatant, separating and purifying the obtained peptide solution by using a gel chromatography column, collecting a second peak of an eluted component, and performing liquid chromatography-mass spectrometry to separate and identify to obtain a fructus cannabis antioxidant peptide crude product;

step 4, purifying the antioxidant peptide

And secondarily purifying the fructus cannabis antioxidant peptide crude product to obtain an antioxidant peptide pure product.

3. The method for preparing antioxidant peptides from hemp seed proteins as claimed in claim 1, wherein in step 1, the hemp seed is peeled off, pulverized by a medicine pulverizer, and sieved with a 0.45-1.2mm mesh screen; removing the hemp seed oil by using an ultrasonic cell crusher, wherein the action temperature is 25-27 ℃, the power is 200-: 20 (s/s), 60 min; extracting with alkali solution at 45 deg.C and pH of 8.5 for 60min at a ratio of 1:12 (g/mL).

4. The method for preparing antioxidant peptides from hemp seed proteins according to claim 1, wherein in the step 2, the nutrient agar PDA culture medium is prepared from the following components: 10g of peptone, 3g of beef extract powder, 5g of sodium chloride, 15g of agar and 1L of deionized water, adjusting the pH value to 7.0, stirring, heating, boiling until the pH value is completely dissolved, subpackaging in a test tube or a triangular flask, and autoclaving at 121 ℃ for 15 min.

5. The method for preparing antioxidant peptides from hemp seed protein according to claim 1, wherein in step 3, the liquid culture medium of hemp seed protein powder: fructus Cannabis protein powder 3-5%, testa Tritici 1% (w/v), brown sugar powder 0.5g, adjusting pH to 7.0, and autoclaving at 121 deg.C for 15 min; the strain inoculation amount is 5-15%, the highest fermentation liquid polypeptide yield is 32%; the rotating speed of the shaking table is 180r/min, and the fermentation time is 24 h; centrifuging the fermentation liquor at 8000r/min for 10 min; the gel column eluent potassium chloride is 0.025 mol/L-acetic acid is 0.2mol/L, and the flow rate is 1-3 ml/min.

6. The method for preparing antioxidant peptide from hemp seed protein as claimed in claim 1, wherein the second purification in step 4 comprises the following steps,

dissolving the hemp seed antioxidant peptide crude product in magnetized water, and filtering to obtain antioxidant peptide crude product solution;

b) adding the antioxidant peptide crude product solution into a semipermeable membrane, and dialyzing in buffer solution and magnetized water in sequence to obtain antioxidant peptide primary pure solution;

c) refining the antioxidant peptide primary pure solution by a reverse phase chromatography to obtain an antioxidant peptide refined pure solution;

d) transferring the pure antioxidant peptide solution into salt by an HPLC method to obtain an antioxidant peptide salt solution; decompressing, concentrating and drying to obtain the hemp seed antioxidant peptide pure product.

7. The method for preparing antioxidant peptide according to claim 5, characterized in that: the magnetized water in the secondary purification step contains 10-25 ppm of acetic acid; the process of the step b) is as follows: putting the crude antioxidant peptide solution into a semipermeable membrane, putting the semipermeable membrane into a buffer solution, and performing percolation at room temperature under stirring until the pH = 3.2-3.5 of the buffer solution; and then transferring the semipermeable membrane containing the antioxidant peptide solution into magnetized water, and performing percolation at room temperature under stirring until the pH = 4.6-5.0 of the solution in the semipermeable membrane.

8. The method for preparing antioxidant peptides from hemp seed proteins as claimed in claim 5 or 6, wherein in the reversed phase chromatography of step c), octadecylsilane chemically bonded silica is used as a stationary phase, ammonium sulfate solution with concentration of 5 mM-50 mM is used as phase A, and acetonitrile is used as phase B; regulating the pH value of the phase A to 2.0-3.5 by adopting sulfuric acid; and eluting the phase B by a gradient of 10% -40%, wherein the elution time is 60 min.

9. The method for preparing antioxidant peptides from hemp seed proteins in claim 5 or 6, wherein the salt transfer by HPLC method in step d) is ion exchange salt transfer by ammonium acetate and acetic acid; the drying in the step d) is realized by adopting a decompression freeze drying mode, and the operation process is as follows: pre-freezing to-50 to-10 ℃, keeping for 2 to 4 hours, keeping the vacuum degree constant at 0.01 to 1.00Mbar, and heating and drying for 12 to 36 hours.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for preparing antioxidant peptide from fructus cannabis protein.

Background

All active substances in a human body exist in the form of peptides, hundreds of peptides exist in the human body, the peptide of a brain accounts for about 70 percent of the peptide in the human body, and the peptide relates to hormone, nerve, endocrine, immunity, reproduction and the like of the human body. Almost all cells are subject to polypeptide regulation, such as cell growth, cell differentiation, synthesis and proliferation, regulation of neurohormonal transmitters, immunomodulation, and the like. These endogenous biological peptides exert strong physiological functions and biological activities in vivo, and are important physiological regulators in the human body. Insulin, auxin, glutathione, superoxide dismutase, and the like are peptides known to play a functional role in vivo.

However, with the increase of age or various factors, endocrine disorders and nervous system disorders may be caused, and it is obvious that the regulation of physiological functions by means of the synthesis of various peptides in our body cannot meet the normal physiological needs of human body. Thus, the ingestion of bioactive peptides to maintain human health is gaining increased acceptance. The bioactive peptide is easy to absorb, has high nutrition, has physiological function, and can directly act on nerve transmission, stimulate the secretion of intestinal tract receptor hormone or kinase to exert physiological function. The active peptide promotes the health of human bodies by inhibiting the occurrence of chronic diseases and improving the immunity of the active peptide, so the application is more and more extensive, and the polypeptide product is applied as food base materials of infant food, old people food, beauty food, intestinal tract regulation functional food, immune food and the like and can be clinically used as auxiliary treatment food for patients with dyspepsia, wound and burn, encephalopathy and the like.

The antioxidant peptide is an important one of bioactive peptides and is a promising peptide in protein substances. Since some polypeptides and amino acids have been found to have high antioxidant activity in the last decade, the antioxidant activity of polypeptides has advantages over conventional antioxidants such as vitamin C and vitamin E in that its antioxidant duration is long, and the antioxidant peptides have a simple structure and are not allergic as compared to SOD enzymes. SOD can scavenge superoxide cations, but for superoxide cations produced by aging and unhealthy conditions, it is of interest to ingest antioxidant peptides to reduce the attack of these free radicals. The antioxidant properties of antioxidant peptides are superior to conventional antioxidants in some respects: good antioxidant activity, high safety, physiological regulation function and nutrition factors, so that the antioxidant peptide has good prospect in the fields of medicine and food and wide development and application prospect.

Cannabis is dry mature seed of Cannabis sativa L, also called Cannabis seed, Cannabis sativa L. China is the country with the most abundant hemp resources, is distributed in Yunnan, Heilongjiang, Liaoning, Guangxi, Sichuan and the like, and is collected in autumn when fruits are ripe, impurities are removed, and the fructus cannabis is obtained after being dried in the sun. Fructus cannabis is sweet in taste and neutral in nature, enters spleen, stomach and large intestine channels, and has the effects of relaxing bowel, moistening dryness and killing insects. The hemp seed is rich in beneficial substances, and especially unsaturated fatty acid and gamma-tocopherol bring strong effects of resisting oxidation, reducing blood fat, reducing blood pressure and delaying aging.

Fructus cannabis is rich in essential fatty acids which cannot be synthesized by the human body, especially gamma-linolenic acid, which is very beneficial to the health of the human body and is usually contained only in blue-green algae and blackcurrant seed oil; oleic acid, linoleic acid, arachidonic acid, etc. in fructus Cannabis are also beneficial to human body. 65-70% of the hemp seed protein is edestin and edestin, wherein the edestin is the most digestible protein of all proteins; the hemp seed protein does not contain tryptophan inhibitory factor, so that the absorption of the protein is not influenced, and the stomach distension and regurgitation are not caused. In addition, the content of vitamin E in fructus Cannabis is also higher. Through previous scientific research, the blood pressure of a normal rat is remarkably reduced by drenching the fructus cannabis extract; the control group is fed with high-fat feed, and 10% of fructus cannabis dry product is added to the administration group, and the result shows that fructus cannabis has the effect of preventing the serum cholesterol of rats from increasing. Therefore, the fructus cannabis has the effects of reducing blood pressure and blood fat. Moreover, with the progress of social economy and culture, more and more women focus on the healthy development of the women, and the requirements on oxidation resistance and aging resistance are gradually increased. Researches find that the hemp seed oil can delay aging by enhancing the anti-oxidation effect of organisms and the influence on immune system; the fructus cannabis tablets are used for intragastric administration to model mice once a day, and the defecation frequency of the mice can be obviously increased after continuous administration for 40 days. Analysis shows that the fructus cannabis has the effects of lubricating intestinal walls, slightly exciting intestinal canals, increasing peristalsis and reducing water absorption of large intestines.

The bacillus subtilis is used as a species found and named earlier in bacillus, widely exists in nature, is non-toxic and harmless to people and livestock, does not pollute the environment, and has a wide peptide spectrum enzyme system. The bacillus subtilis is an important microorganism for manufacturing traditional food, the application of the bacillus subtilis can be traced back to more than one thousand years, the bacillus subtilis is used for producing the traditional food natto in the Japanese peace time, and the fermentate is characterized in that soybean protein is hydrolyzed into amino acid, polypeptide and ammonia, so that the function of a bean product is improved. In addition, the bacillus subtilis makes an important contribution to the prepared antioxidant peptide product, such as Japanese natto, and has the effects of dissolving thrombus and reducing blood pressure. The bacillus subtilis is particularly rich in enzyme system, secretes a large amount of extracellular proteases such as neutral protease, alkaline protease, carboxypeptidase and aminopeptidase in the growth and metabolism process, has strong enzyme production capacity, high enzymolysis capacity and short fermentation time, has more advantages than other beneficial microorganisms, and is widely valued for research and application.

Disclosure of Invention

The invention aims to provide a preparation method of antioxidant peptide, which can utilize bacillus subtilis to ferment by acting on a hemp seed protein preparation method, has the advantages of high enzyme yield and recovery rate, and the like, obviously reduces the cost by 50-80%, improves the yield by 10-20%, has high purity of hemp seed antioxidant peptide in pure antioxidant peptide products, has clearance rate of DPPH free radical and hydroxyl free radical of more than 90%, has the effects of dissolving thrombus and lowering blood pressure, and has high economic value.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a preparation method of antioxidant peptide specifically comprises the following steps:

step 1, preparation of hemp seed protein

Vacuum drying fructus Cannabis, removing shell, pulverizing, removing fructus Cannabis oil with ultrasonic cell pulverizer to obtain fructus Cannabis dregs, extracting protein with alkali method, and vacuum drying;

step 2, activation and purification of the strains

Preparing nutrient agar PDA culture medium, sterilizing; sterilizing, cooling, and pouring into a flat seed culture medium; inoculating strains for activation, and selecting strains with good growth for later use;

step 3, preparing antioxidant peptide

Preparing a fructus cannabis protein powder liquid fermentation culture medium, and sterilizing; after sterilization and temperature reduction, inoculating the strain into a culture medium for shaking table fermentation; centrifuging the fermentation liquor to obtain supernatant, separating and purifying the obtained peptide solution by using a gel chromatography column, collecting a second peak of an eluted component, and performing liquid chromatography-mass spectrometry to separate and identify to obtain a fructus cannabis antioxidant peptide crude product;

step 4, purifying the antioxidant peptide

And secondarily purifying the fructus cannabis antioxidant peptide crude product to obtain an antioxidant peptide pure product.

Further, in the step 1, after the outer skin of the fructus cannabis is removed, the fructus cannabis is crushed by a medicine crusher and passes through a 0.45-1.2mm mesh screen; removing the hemp seed oil by using an ultrasonic cell crusher, wherein the action temperature is 25-27 ℃, the power is 200-: 20 (s/s), 60-65 min; extracting with alkali solution at 45-47 deg.C and pH of 8.3-8.5 for 60-65min at a ratio of 1:12 (g/mL).

Further, in the step 2, the nutrient agar PDA culture medium comprises the following components in proportion: 10g of peptone, 3g of beef extract powder, 5g of sodium chloride, 15g of agar and 1L of deionized water, adjusting the pH value to 7.0, stirring, heating, boiling until the pH value is completely dissolved, subpackaging in a test tube or a triangular flask, and autoclaving at 121 ℃ for 15 min;

further, in the step 3, the fructus cannabis protein powder liquid culture medium: fructus Cannabis protein powder 3-5%, testa Tritici 1% (w/v), brown sugar powder 0.5g, adjusting pH to 7.0, and autoclaving at 121 deg.C for 15 min; the strain inoculation amount is 5-15%, the highest fermentation liquid polypeptide yield is 32%; the rotating speed of the shaking table is 180r/min, and the fermentation time is 24 h; centrifuging the fermentation liquor at 8000r/min for 10 min; gel column washing

Further, in the step 4, the secondary purification comprises the following steps,

a) dissolving the hemp seed antioxidant peptide crude product in magnetized water, and filtering to obtain antioxidant peptide crude product solution;

b) adding the antioxidant peptide crude product solution into a semipermeable membrane, and dialyzing in buffer solution and magnetized water in sequence to obtain antioxidant peptide primary pure solution;

c) refining the antioxidant peptide primary pure solution by a reverse phase chromatography to obtain an antioxidant peptide refined pure solution;

d) transferring the pure antioxidant peptide solution into salt by an HPLC method to obtain an antioxidant peptide salt solution; decompressing, concentrating and drying to obtain the hemp seed antioxidant peptide pure product.

The invention has the beneficial effects that:

1) the mouthfeel is improved, and the nutritive value is improved: by adopting a microbial fermentation method, certain bitter peptide groups can be modified and recombined, so that small peptides and amino acids are grafted and rearranged, and the prepared antioxidant peptide has no bitter taste and peculiar smell; the prepared antioxidant peptide mixture is rich in amino acids and mineral substances,

2) the ultrasonic cell crusher is used for crushing cells and strengthening mass transfer, so that solvent molecules penetrate into tissue cells and are better contacted with the solvent molecules, soluble molecules in the cells are better released, the yield of crude protein is 82.4 percent, and is improved by 10.1 percent. The best yield of fructus cannabis extracted by alkali liquor is 84.6%, and the extraction time is shortened at the same time 1/3.

3) The antioxidant peptide has the strongest scavenging capacity on DPPH and HO < - >, the iron chelating capacity and the inhibition capacity of an oleic acid peroxidation system, the antioxidant peptide prepared within 48h has the strongest scavenging capacity on free radicals, the content of free amino acid, the content of polysaccharide and the protease activity are higher than those of other time periods, the content of amino acid nitrogen is 5.47 percent, the content of polysaccharide is 482.4mg/L, and the protease activity is 328U/mL.

4) After multiple times of purification, the purity of the hemp seed antioxidant peptide can be more than 99%, the effective rate of the effective components can exceed 60%, and the economic value is obvious; adding a trace amount of acetic acid into the magnetized water for dissolving and dialyzing the antioxidant peptide is helpful for improving the activity of the magnetized water. Therefore, the antioxidant peptide, the small molecular protein and the small peptide in the fructus cannabis antioxidant peptide crude product are more fully dissolved, and the loss of the antioxidant peptide in the dissolving and dialysis processes is avoided, so that the purity and the yield of the antioxidant peptide in the final product are reduced.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.

Example 1

1. Preparation of hemp seed protein

The fructus cannabis is dried for 4 hours in vacuum at 60 ℃, the moisture content is 4%, the fructus cannabis is shelled, the fructus cannabis is crushed by a fructus cannabis crusher and passes through a 0.45mm mesh screen, the fructus cannabis oil is removed by an ultrasonic cell crusher, the action temperature is 25 ℃, the power is 200w, the duty ratio is 20: 20 (s/s) and 60min to obtain fructus Cannabis meal; extracting with alkali solution at 45 deg.C and pH of 8.5 for 60min at a ratio of 1:12 (g/mL). Vacuum drying for use.

2. Activation and purification of the Strain

Preparing a nutrient agar PDA culture medium: 10g of peptone, 3g of beef extract powder, 5g of sodium chloride, 15g of agar and 1L of deionized water, adjusting the pH value to 7.0, stirring, heating, boiling until the pH value is completely dissolved, subpackaging in a test tube or a triangular flask, and autoclaving at 121 ℃ for 15 min; sterilizing, cooling to 40 deg.C, and adding into plate seed culture medium; inoculating 2 rings of Bacillus subtilis strains from original strains to a plate culture medium for activation, activating at 35 ℃ for 24h, selecting strains with better colony morphology, repeating the operation until strains with good growth (coarse, opaque, non-flashing, expanded, white or slightly yellow colonies) are selected and inoculated to the culture medium for later use.

3. Preparation of antioxidant peptides

Preparing a fructus cannabis protein powder liquid fermentation culture medium: fructus Cannabis protein powder 3%, testa Tritici 1% (w/v), brown sugar powder 0.5g, adjusting pH to 7.0, and autoclaving at 121 deg.C for 15 min; sterilizing, cooling to 40 ℃, inoculating 5% of bacillus subtilis into the culture medium, and fermenting in a shaking table at the rotating speed of 180r/min for 24 times; centrifuging the fermentation liquor to obtain supernatant, concentrating the filtrate with molecular weight less than 2kDa at low temperature to obtain concentrated extract, and ultrafiltering to obtain solution with molecular weight less than 2kDa and rich in antioxidant peptide; purifying and separating the obtained peptide solution by using a gel chromatography column, wherein the eluent is 0.025mol/L of potassium chloride-0.2 mol/L of acetic acid, and the flow rate is 3 ml/min; and collecting a second peak of the eluted components, and performing liquid chromatography-mass spectrometry chromatography separation and identification to obtain a fructus cannabis antioxidant peptide crude product.

4. Purified antioxidant peptides

Dissolving fructus cannabis antioxidant peptide crude product in magnetized water, and filtering to obtain antioxidant peptide crude product solution, wherein the magnetized water contains 10ppm of acetic acid; adding the antioxidant peptide crude product solution into a semipermeable membrane, placing the semipermeable membrane into a buffer solution, stirring and permeating the buffer solution at room temperature until the pH =3.2, then transferring the semipermeable membrane of the antioxidant peptide solution into magnetized water, and stirring and permeating the semipermeable membrane at room temperature until the pH = 4.6; the antioxidant peptide primary pure solution adopts reversed phase chromatography: octadecylsilane chemically bonded silica is used as a stationary phase, ammonium sulfate aqueous solution with the concentration of 10mM is used as a phase A, and acetonitrile is used as a phase B; regulating the pH value of the phase A to 2.0 by adopting sulfuric acid; eluting phase B with gradient of 10% for 60min to obtain pure antioxidant peptide solution; the salt conversion by an HPLC method is to obtain an antioxidant peptide salt solution by ion exchange salt conversion under the condition of ammonium acetate and acetic acid; the method is realized by decompression freeze drying, and the operation process is as follows: pre-freezing to-50 deg.C, maintaining for 2 hr to keep the vacuum degree constant at 0.01Mbar, heating and drying for 12 hr to obtain fructus Cannabis antioxidant pure product.

Example 2

1. Preparation of hemp seed protein

The fructus cannabis is dried for 4 hours in vacuum at 60 ℃, the moisture content is 4%, the fructus cannabis is shelled, the fructus cannabis is crushed by a fructus cannabis crusher and passes through a 0.7mm mesh screen, the fructus cannabis oil is removed by an ultrasonic cell crusher, the action temperature is 26 ℃, the power is 210..0w, the duty ratio is 20: 20 (s/s) and 60min to obtain fructus Cannabis meal; extracting with alkali solution at 45 deg.C and pH of 8.5 for 60min at a ratio of 1:12 (g/mL). Vacuum drying for use.

2. Activation and purification of the Strain

Preparing a nutrient agar PDA culture medium: 10g of peptone, 3g of beef extract powder, 5g of sodium chloride, 15g of agar and 1L of deionized water, adjusting the pH value to 7.0, stirring, heating, boiling until the pH value is completely dissolved, subpackaging in a test tube or a triangular flask, and autoclaving at 121 ℃ for 15 min; sterilizing, cooling to 40 deg.C, and adding into plate seed culture medium; inoculating 2-ring Bacillus subtilis strain from the original strain to a plate culture medium for activation, activating at 35 deg.C for 24 hr, and inoculating the strain to the culture medium.

3. Preparation of antioxidant peptides

Preparing a fructus cannabis protein powder liquid fermentation culture medium: fructus Cannabis protein powder 5%, testa Tritici 1% (w/v), brown sugar powder 0.5g, adjusting pH to 7.0, and autoclaving at 121 deg.C for 15 min; sterilizing, cooling to 40 ℃, inoculating 7% of bacillus subtilis into the culture medium, and fermenting in a shaking table at the rotating speed of 180r/min for 24 h; centrifuging the fermentation liquor to obtain supernatant, concentrating the filtrate with molecular weight less than 2kDa at low temperature to obtain concentrated extract, and ultrafiltering to obtain solution with molecular weight less than 2kDa and rich in antioxidant peptide; purifying and separating the obtained peptide solution by using a gel chromatography column, wherein the eluent is 0.025mol/L of potassium chloride-0.2 mol/L of acetic acid, and the flow rate is 2 ml/min; and collecting a second peak of the eluted components, and performing liquid chromatography-mass spectrometry chromatography separation and identification to obtain a fructus cannabis antioxidant peptide crude product.

4. Purified antioxidant peptides

Dissolving fructus cannabis antioxidant peptide crude product in magnetized water, and filtering to obtain antioxidant peptide crude product solution, wherein the magnetized water contains 10ppm of acetic acid; adding the antioxidant peptide crude product solution into a semipermeable membrane, placing the semipermeable membrane into a buffer solution, stirring and permeating the buffer solution at room temperature until the pH =3.2, then transferring the semipermeable membrane of the antioxidant peptide solution into magnetized water, and stirring and permeating the semipermeable membrane at room temperature until the pH = 4.6; the antioxidant peptide primary pure solution adopts reversed phase chromatography: octadecylsilane chemically bonded silica is used as a stationary phase, ammonium sulfate aqueous solution with the concentration of 20mM is used as a phase A, and acetonitrile is used as a phase B; regulating the pH value of the phase A to 3.0 by adopting sulfuric acid; eluting phase B with gradient of 20% for 60min to obtain pure antioxidant peptide solution; the salt conversion by an HPLC method is to obtain an antioxidant peptide salt solution by ion exchange salt conversion under the condition of ammonium acetate and acetic acid; the method is realized by decompression freeze drying, and the operation process is as follows: pre-freezing to-50 deg.C, maintaining for 2 hr to keep the vacuum degree constant at 0.01Mbar, heating and drying for 12 hr to obtain fructus Cannabis antioxidant pure product.

Example 3

1. Preparation of hemp seed protein

The fructus cannabis is dried for 4 hours in vacuum at 60 ℃, the moisture content is 4%, the fructus cannabis is shelled, the fructus cannabis is crushed by a fructus cannabis crusher and passes through a 1.2mm mesh screen, the fructus cannabis oil is removed by an ultrasonic cell crusher, the action temperature is 27 ℃, the power is 220w, the duty ratio is 20: 20 (s/s) and 60min to obtain fructus Cannabis meal; extracting with alkali solution at 45 deg.C and pH of 8.5 for 60min at a ratio of 1:12 (g/mL). Vacuum drying for use.

2. Activation and purification of the Strain

Preparing a nutrient agar PDA culture medium: 10g of peptone, 3g of beef extract powder, 5g of sodium chloride, 15g of agar and 1L of deionized water, adjusting the pH value to 7.0, stirring, heating, boiling until the pH value is completely dissolved, subpackaging in a test tube or a triangular flask, and autoclaving at 121 ℃ for 15 min; sterilizing, cooling to 40 deg.C, and adding into plate seed culture medium; inoculating 2 rings of Bacillus subtilis strains from original strains to a plate culture medium for activation, activating at 35 ℃ for 24h, selecting strains with better colony morphology, repeating the operation until strains with good growth (coarse, opaque, non-flashing, expanded, white or slightly yellow colonies) are selected and inoculated to the culture medium for later use.

3. Preparation of antioxidant peptides

Preparing a fructus cannabis protein powder liquid fermentation culture medium: fructus Cannabis protein powder 4%, testa Tritici 1% (w/v), brown sugar powder 0.5g, adjusting pH to 7.0, and autoclaving at 121 deg.C for 15 min; sterilizing, cooling to 40 ℃, inoculating 15% of bacillus subtilis into the culture medium, and fermenting in a shaking table at the rotating speed of 180r/min for 24 times; centrifuging the fermentation liquor to obtain supernatant, concentrating the filtrate with molecular weight less than 2kDa at low temperature to obtain concentrated extract, and ultrafiltering to obtain solution with molecular weight less than 2kDa and rich in antioxidant peptide; purifying and separating the obtained peptide solution by using a gel chromatography column, wherein the eluent is 0.025mol/L of potassium chloride-0.2 mol/L of acetic acid, and the flow rate is 1 ml/min; and collecting a second peak of the eluted components, and performing liquid chromatography-mass spectrometry chromatography separation and identification to obtain a fructus cannabis antioxidant peptide crude product.

4. Purified antioxidant peptides

Dissolving fructus cannabis antioxidant peptide crude product in magnetized water, and filtering to obtain antioxidant peptide crude product solution, wherein the magnetized water contains 10ppm of acetic acid; adding the antioxidant peptide crude product solution into a semipermeable membrane, placing the semipermeable membrane into a buffer solution, stirring and permeating the buffer solution at room temperature until the pH =3.2, then transferring the semipermeable membrane of the antioxidant peptide solution into magnetized water, and stirring and permeating the semipermeable membrane at room temperature until the pH = 4.6; the antioxidant peptide primary pure solution adopts reversed phase chromatography: octadecylsilane chemically bonded silica is used as a stationary phase, ammonium sulfate aqueous solution with the concentration of 10mM is used as a phase A, and acetonitrile is used as a phase B; regulating the pH value of the phase A to 2.0 by adopting sulfuric acid; eluting phase B with gradient of 10% for 60min to obtain pure antioxidant peptide solution; the salt conversion by an HPLC method is to obtain an antioxidant peptide salt solution by ion exchange salt conversion under the condition of ammonium acetate and acetic acid; the method is realized by decompression freeze drying, and the operation process is as follows: pre-freezing to-50 deg.C, maintaining for 2 hr to keep the vacuum degree constant at 0.01Mbar, heating and drying for 12 hr to obtain fructus Cannabis antioxidant pure product.

Test examples

1. Determination of antioxidant Activity

The effect of the fructus cannabis antioxidant peptide on scavenging free radicals is determined through an in vitro antioxidant test, so that the antioxidant capacity of the fructus cannabis antioxidant peptide is verified.

DPPH is a stable free radical, and when a free radical scavenger is added to a DPPH solution, the solution becomes lighter in color, the absorbance at 517nm decreases, and the degree of decrease in absorbance is linear with the degree to which the free radical is scavenged. Therefore, the method can be used for detecting the scavenging condition of free radicals so as to evaluate the antioxidant capacity of a certain substance, and the capacity is expressed by the Scavenging Rate (SR), and the larger the scavenging rate is, the stronger the antioxidant capacity is.

Test materials: examples 1-3, commercially available antioxidant peptides.

The experimental method comprises the following steps: dissolving 1mL of fructus Cannabis antioxidant peptide in 2mL of absolute ethanol, completely mixing, adding 2mL of 60 mu mol/L DPPH solution, mixing, standing for 30min, adjusting to zero with absolute ethanol, and measuring absorbance at 517nm to be Ai; in the same method, 2mL of absolute ethyl alcohol solvent and 2mL of DPPH solution are uniformly mixed, and the absorbance is measured to be Ac; dissolving 1mL of fructus cannabis antioxidant peptide in 2mL of absolute ethyl alcohol, mixing uniformly, adding 2mL of absolute ethyl alcohol solvent, mixing uniformly, and determining the absorbance Aj. The radical scavenging rate was calculated by the following formula.

The clearance (%) [1- (Ai-Aj)/Ac ] x 100%

Wherein Aj reflects the contribution of the sample itself to absorbance; absorbance values of Ai samples after action on DPPH; ac is the absorption of DPPH itself at the measurement wavelength.

The results of the experiment are shown in table 1:

TABLE 1 Effect of the hemp seed antioxidant peptides of the present invention on scavenging free radicals (DPPH)

Sample (I)	Clearance (%)
		Example 1	89.3
Example 2	92.0
		Example 3	90.5
Commercially available fructus cannabis antioxidant peptides	64.3

The results in table 1 show that the clearance rate of free radical DPPH in examples 1 to 3 is significantly higher than that of the commercially available antioxidant peptide, which indicates that the water-soluble cannabidiol has better clearance capability to free radical DPPH, and indicates that the fructus cannabis antioxidant peptide has better antioxidant effect.

Stability test

High temperature, low temperature experiments: the hemp seed antioxidant peptides prepared in examples 1 to 3 were hermetically packaged and then placed in an oven (40. + -. 1 ℃ C.), room temperature (25. + -. 1 ℃ C.) and a refrigerator (4. + -. 1 ℃ C.) for 60 days, and the appearance color, properties and presence or absence of mold phenomenon were observed. The result shows that the color of the fructus cannabis antioxidant peptide is not changed, no impurity precipitation and no mildew flocculation phenomenon exist, and the character is kept stable in high-temperature, normal-temperature and low-temperature environments.

The above examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way. For process parameters or conditions not specifically mentioned, it can be carried out with reference to conventional techniques. It will be apparent to those skilled in the art that equivalent embodiments or modifications without departing from the technical spirit of the present invention are within the scope of the present invention.