Hemp seed oligopeptide, and preparation method and application thereof

Abstract

The present invention provides a hemp seed oligopeptide and a preparation method and application thereof. The preparation method of the hemp seed oligopeptide comprises: mixing hemp seed globulin powder with water to prepare a hemp seed globulin solution; then heating and ultrasonicating the hemp seed globulin solution and performing enzymatic hydrolysis; then ultrafiltration and desalting the resulting enzymatic hydrolysis product to obtain the hemp seed oligopeptide. The present invention can make the hemp seed globulin structure looser and improve the enzymatic hydrolysis efficiency by performing heating and ultrasonic pretreatment; performing two enzymatic hydrolysis reactions in sequence can significantly reduce the molecular weight of the product and alleviate the bitter taste of the protein; and can significantly improve the preparation yield. Finally, ultrafiltration and desalting the enzymatic hydrolysis product, the product has higher purity and better antioxidant activity, can be used in related foods and health products, and has value in actual production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of hemp seed polypeptide preparation, and particularly relates to a hemp seed oligopeptide and a preparation method and application thereof. BACKGROUND

[0002] Hemp belongs to industrial hemp, and its seeds, hemp seeds, have high edible value and can also be used as medicine. Hemp seeds contain rich hemp seed globulin (20-30%), and the hemp seed globulin is composed of more than 20 kinds of amino acids, including 8 kinds of essential amino acids for human body, and is a high-quality protein. The arginine content is higher than that of seed proteins of the same type, and has a good effect on maintaining cardiovascular health.

[0003] At present, the main method for producing hemp seed polypeptide in China is to use hemp seed protein powder as raw material, and then to prepare polypeptide through protease hydrolysis. CN110777183A discloses a hemp seed oligopeptide powder and a preparation method and application thereof. First, the raw material is screened, and then a slurry is obtained through dynamic extraction to obtain an extraction liquid. Then, enzyme hydrolysis is performed in steps to obtain an enzyme hydrolysis liquid. After the enzyme hydrolysis liquid is purified, powder processing is performed to obtain the hemp seed oligopeptide powder. This method is simple to operate, has low preparation cost, and is environmentally friendly and harmless. However, because hemp seed protein is mainly hemp seed globulin, the water solubility is poor, the structure is compact, and the protease is difficult to fully hydrolyze, the obtained polypeptide has a large molecular weight and a low yield.

[0004] At present, the extraction process of hemp seed polypeptide often has problems of large product molecular weight and low extraction rate. How to provide an extraction method of hemp seed polypeptide, which can improve the product yield and has smaller product molecular weight, is convenient to use and absorb, has become a problem to be solved. SUMMARY

[0005] In view of the deficiencies of the prior art and actual needs, the application provides a hemp seed oligopeptide and a preparation method and application thereof. Through heating and ultrasonic pretreatment, the enzyme hydrolysis effect can be improved, the extraction efficiency can be improved, the product molecular weight can be reduced, the impurities in the product can be reduced through ultrafiltration and desalination, the purity and activity of the product can be ensured, and the application value is higher.

[0006] To achieve the above purpose, the application adopts the following technical solutions:

[0007] In a first aspect, the application provides a preparation method of a hemp seed oligopeptide, which comprises the following steps:

[0008] The hemp seed globulin powder is mixed with water to prepare a hemp seed globulin solution, the hemp seed globulin solution is heated and ultrasonically pretreated, and then enzyme hydrolysis is performed, and the obtained enzyme hydrolysis product is subjected to ultrafiltration and desalination to obtain the hemp seed oligopeptide.

[0009] In the present application, by heat treatment of Cannabis seed globulin, the protein is denatured and inactivated, and the dense structure is destroyed, making it more susceptible to protease contact and enzymolysis. However, during heating, the protein may aggregate to form larger particles. Therefore, further ultrasonic treatment is used to disperse the protein and reduce the particle size, thereby further improving the efficiency of enzymolysis, increasing the yield of polypeptides, and reducing the molecular weight of the product. Finally, the extracted product is purified by ultrafiltration and desalination to improve the purity of the polypeptides, maintain the activity of the polypeptides, and well exert their biological functions.

[0010] Preferably, the mass concentration of Cannabis seed globulin in the Cannabis seed globulin solution is 1% to 15%, for example, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0011] Preferably, the preparation method of the Cannabis seed globulin powder is to extract Cannabis seed globulin from Cannabis seed powder using a salt solution, and then dry to obtain the Cannabis seed globulin powder.

[0012] Preferably, the method for extracting Cannabis seed globulin comprises: adding the Cannabis seed powder into the salt solution, extracting, centrifuging to take the supernatant, standing and cooling to precipitate the protein, and then centrifuging to obtain the Cannabis seed globulin.

[0013] Preferably, the ratio of the mass of the Cannabis seed powder to the volume of the salt solution is 1:(5-30) g / mL, for example, it can be 1:5 g / mL, 1:10 g / mL, 1:15 g / mL, 1:20 g / mL, 1:25 g / mL, or 1:30 g / mL.

[0014] Preferably, the salt solution comprises a sodium chloride solution.

[0015] Preferably, the mass concentration of the salt in the salt solution is 2% to 10%, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0016] Preferably, the extraction temperature is 40-60°C, for example, it can be 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C, preferably 55°C.

[0017] Preferably, the extraction time is 0.5-4 h, for example, it can be 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, or 4 h, preferably 2 h.

[0018] Preferably, the temperature of the standing cooling is 1-10℃, for example, it can be 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃ or 10℃, preferably 4℃.

[0019] Preferably, the time of the standing cooling is 1-12h, for example, it can be 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h or 12h, preferably 4h.

[0020] Preferably, the method further comprises the step of preparing the hemp seed powder before adding the hemp seed powder into the sodium chloride solution.

[0021] Preferably, the preparing the hemp seed powder comprises drying the hemp seed, defatting after crushing.

[0022] Preferably, the defatting comprises any one of physical pressing, solvent extraction or supercritical extraction.

[0023] Preferably, the solvent used in the solvent extraction comprises n-hexane and / or petroleum ether.

[0024] Preferably, the solvent used in the supercritical extraction comprises carbon dioxide.

[0025] Preferably, the oil content of the hemp seed powder is no more than 5%, for example, it can be 1%, 2%, 3%, 4% or 5%.

[0026] Preferably, the method for preparing the hemp seed globulin powder comprises:

[0027] drying the hemp seed, crushing, defatting by physical pressing, solvent extraction using n-hexane and / or petroleum ether as the solvent or supercritical extraction using carbon dioxide as the solvent, to obtain the hemp seed powder with oil content no more than 5%;

[0028] adding the hemp seed powder into the sodium chloride solution with mass fraction of 2%-10%, the ratio of the mass of the hemp seed powder to the volume of the sodium chloride solution is 1:(5-30)g / mL, extracting at 40-60℃ for 0.5-4h, taking the supernatant after centrifugation, standing cooling at 1-10℃ for 1-12h, precipitating the protein, centrifuging again, drying, to obtain the hemp seed globulin powder.

[0029] Preferably, the temperature of the heating is 80-100℃, for example, it can be 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃ or 100℃, preferably 95℃.

[0030] In the present application, the heating temperature is in the range of 80-100℃, in which the protein can be denatured, the protein structure can be loose, the peptide bond of the protein can be exposed, and the enzymolysis of the protease is more favorable; when the temperature is lower than 80℃, the hemp seed globulin is difficult to be fully denatured and inactivated, and when the temperature is too high, the polypeptide can be completely inactivated, which affects the function.

[0031] Preferably, the heating time is 10-60min, for example, it can be 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min or 60min.

[0032] Preferably, the ultrasonic power is 100-1000W / L, for example, it can be 100W / L, 200W / L, 300W / L, 400W / L, 500W / L, 600W / L, 700W / L, 800W / L, 900W / L or 1000W / L, preferably 400W / L.

[0033] In the present application, the ultrasonic treatment can make the protein aggregated by heating re-disperse into small particles, increase the specific surface area of the protein, and improve the hydrolysis of the protein by the protease; when the ultrasonic power is lower than 100W / L, the effect of the protein depolymerization into small particles is poor; and when the ultrasonic power is greater than 1000W / L, the cost is increased and the effect is not obviously improved.

[0034] Preferably, the ultrasonic time is 10-60min, for example, it can be 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min or 60min.

[0035] Preferably, the enzymolysis includes primary enzymolysis and secondary enzymolysis.

[0036] In the present application, the hemp seed globulin can be hydrolyzed into polypeptides with smaller molecular weight by primary enzymolysis, which is convenient for subsequent enzymolysis treatment; the molecular weight of the polypeptides is further reduced by secondary enzymolysis, and the hydrophobic amino acids at the end of the polypeptide chain are removed, which eliminates the bitter taste, improves the yield of the polypeptides and optimizes the taste; the present application carries out two hydrolysis respectively, which can overcome the shortcomings of the product molecular weight distribution range being large and the product being complex when the two enzymolysis are carried out at the same time, and the obtained product has small molecular weight and is relatively single, which is convenient for subsequent processing and utilization.

[0037] Preferably, the primary enzymolysis includes adding protease to the obtained product after heating and ultrasonic treatment, and then carrying out enzyme inactivation after primary enzymolysis to obtain the product of primary enzymolysis.

[0038] Preferably, the protease is added in an amount of 0.5% to 5% of the mass of the hemp seed globulin, for example, it can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.

[0039] Preferably, the protease comprises any one or a combination of at least two of alkaline protease, papain, pepsin, trypsin or neutral protease, for example, it can be alkaline protease or a combination of papain and pepsin.

[0040] Preferably, the temperature of the first enzymolysis is 40 to 60°C, for example, it can be 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C, preferably 55°C.

[0041] Preferably, the time of the first enzymolysis is 2 to 4h, for example, it can be 2h, 2.5h, 3h, 3.5h or 4h.

[0042] Preferably, the temperature of the enzyme inactivation is not less than 80°C, for example, it can be 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or 90°C.

[0043] Preferably, the time of the enzyme inactivation is 10 to 30min, for example, it can be 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min or 30min.

[0044] Preferably, the method further comprises a step of cooling after the enzyme inactivation.

[0045] Preferably, the temperature of the cooling is 40 to 60°C, for example, it can be 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C.

[0046] Preferably, the second enzymolysis comprises adding a protease to the product of the first enzymolysis, and then inactivating the enzyme to obtain the product of the second enzymolysis.

[0047] Preferably, the protease is added in an amount of 0.5% to 10% by mass of the hemp seed globulin, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

[0048] Preferably, the protease comprises a flavour protease.

[0049] Preferably, the temperature of the secondary enzymolysis is 40 to 60°C, for example, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C.

[0050] Preferably, the time of the secondary enzymolysis is 2 to 4 hours, for example, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours.

[0051] Preferably, the temperature of the enzyme inactivation is not lower than 80°C, for example, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, or 90°C.

[0052] Preferably, the time of the enzyme inactivation is 10 to 30 minutes, for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, or 30 minutes.

[0053] Preferably, the method further comprises a step of cooling after the enzyme inactivation.

[0054] Preferably, the temperature of the cooling is 20 to 30°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.

[0055] Preferably, the enzymolysis comprises:

[0056] A protease is added to the obtained product after heating and ultrasonic treatment, the protease comprising any one or a combination of at least two of alkaline protease, papain, pepsin, trypsin, or neutral protease, the protease being used in an amount of 0.5% to 5% by mass of the hemp seed globulin, and the protease is subjected to a primary enzymolysis at 40 to 60°C for 2 to 4 hours, and then subjected to enzyme inactivation at not lower than 80°C for 10 to 30 minutes, and then cooled to 40 to 60°C to obtain a product of the primary enzymolysis.

[0057] Adding flavour protease to the product of the first enzymolysis, the flavour protease is used in an amount of 0.5% to 10% of the mass of the hemp seed globulin, and the second enzymolysis is carried out at 40 to 60℃ for 2 to 4 hours, and then the enzyme is inactivated at not less than 80℃ for 10 to 30 minutes, and then the temperature is reduced to 20 to 30℃, to obtain the product of the second enzymolysis.

[0058] Preferably, the molecular cut-off of the ultrafiltration membrane used in the ultrafiltration is 1000 to 10000 Da, for example, it can be 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da or 10000 Da, preferably 3000 Da.

[0059] In the present application, when the molecular cut-off of the ultrafiltration membrane is in the range of the molecular weight, the antioxidant activity of the hemp seed oligopeptide prepared is better.

[0060] Preferably, the step of ultrafiltration further comprises a centrifugation step.

[0061] Preferably, the desalination comprises column chromatography desalination.

[0062] In the present application, in order to maintain the activity of protease in the enzymolysis, it is necessary to adjust the pH by adding acid or alkali solution, so that more salt will be produced in the solution in the later stage of the enzymolysis, which will affect the purity and activity of the oligopeptide, and the column chromatography can remove the salt in the polypeptide to the maximum extent without losing the activity of the polypeptide itself.

[0063] Preferably, the elution solvent used in the column chromatography desalination comprises water.

[0064] Preferably, the column chromatography desalination uses dextran gel and / or agarose gel as the column filler.

[0065] Preferably, the step of desalination further comprises a drying step.

[0066] Preferably, the drying comprises freeze-drying and / or spray-drying.

[0067] As a preferred technical solution, the preparation method of the hemp seed oligopeptide provided by the present application comprises the following steps:

[0068] (1) drying the hemp seeds, crushing them, and then defatting them by physical pressing, solvent extraction using n-hexane and / or petroleum ether as the solvent, or supercritical extraction using carbon dioxide as the solvent, to obtain hemp seed powder with an oil content of not more than 5%;

[0069] (2) dissolving the hemp seed powder in a 2%-10% sodium chloride solution, the ratio of the mass of the hemp seed powder to the volume of the sodium chloride solution being 1:(5-30) g / mL, extracting at 40-60°C for 0.5-4 h, taking the supernatant after centrifugation, and standing to cool at 1-10°C for 1-12 h to precipitate the protein, and then centrifuging and drying to obtain hemp seed globulin powder; mixing the hemp seed globulin powder with water to obtain a hemp seed globulin solution with a mass concentration of 1%-15% of hemp seed globulin;

[0070] (3) heating the hemp seed globulin solution at 80-100°C for 10-60 min, and then ultrasonating at 100-1000 W / L for 10-60 min;

[0071] (4) adding a protease to the obtained product after heating and ultrasonation, the protease including any one or a combination of at least two of alkaline protease, papain, pepsin, trypsin or neutral protease, the protease being used in an amount of 0.5%-5% of the mass of the hemp seed globulin, and then performing primary enzymolysis at 40-60°C for 2-4 h, and then inactivating the enzyme at not less than 80°C for 10-30 min, and then cooling to 40-60°C to obtain a primary enzymolysis product;

[0072] (5) adding flavour protease to the primary enzymolysis product, the flavour protease being used in an amount of 0.5%-10% of the mass of the hemp seed globulin, and then performing secondary enzymolysis at 40-60°C for 2-4 h, and then inactivating the enzyme at not less than 80°C for 10-30 min, and then cooling to 20-30°C to obtain a secondary enzymolysis product;

[0073] (6) centrifuging the secondary enzymolysis product, and then using an ultrafiltration membrane with a molecular cut-off of 1000-10000 Da to perform ultrafiltration on the obtained supernatant to obtain an ultrafiltration membrane permeate, and then performing column chromatography desalting using water as the elution solvent, and using a chromatography column filler including dextran gel and / or agarose gel to obtain a desalting membrane permeate;

[0074] (7) performing freeze drying and / or spray drying on the desalting membrane permeate to obtain the hemp seed oligopeptide.

[0075] A flow chart of the preparation method of the hemp seed oligopeptide is shown in Figure 1 .

[0076] In a second aspect, the present application provides a hemp seed oligopeptide prepared by the preparation method of the first aspect.

[0077] In the present application, by preparing the hemp seed globulin solution, then heating, ultrasonic and twice enzymolysis, then ultrafiltration and desalination, the hemp seed oligopeptide prepared has the advantages of small molecular weight, high purity, good activity and no bitter taste, and has practical application value.

[0078] Preferably, the peptide content in the hemp seed oligopeptide is not less than 90%, for example, it can be 90%, 91%, 92%, 93%, 94% or 95%, the oligopeptide content with a molecular weight below 3000Da is not less than 70%, for example, the molecular weight can be 2800Da, 2850Da, 2900Da, 2950Da or 3000Da, and the content can be 70%, 71%, 72%, 73%, 74% or 75%.

[0079] In a third aspect, the present application provides the use of the hemp seed oligopeptide according to the second aspect in the preparation of antioxidant food and / or health products.

[0080] In the present application, the hemp seed oligopeptide prepared is rich in various essential amino acids for the human body, has good antioxidant and anti-aging effects, can improve immunity, and the taste is improved by flavor protease, improving the use experience, and has very high application value.

[0081] Compared with the prior art, the present application has the following beneficial effects:

[0082] (1) In the present application, the hemp seed globulin solution is pretreated by heating and ultrasonic, the protein structure is more loose, and is dispersed into small particles, the protein hydrolysis rate is improved, the product yield is improved, the oligopeptide polypeptide content is above 94.5%, and the soluble polypeptide yield is above 75.2%, compared with no heating and ultrasonic treatment, the yield is increased by 10-25%; through twice enzymolysis reaction, the molecular weight of the product is reduced, the yield of oligopeptide with a molecular weight less than 3000Da is not less than 70.5%, compared with single enzymolysis or mixed enzymolysis, the yield is increased by 15%-25%; and the types of the product are relatively single, and the bitter taste is improved; through ultrafiltration and column chromatography, the impurities in the oligopeptide are removed, the product has higher purity and better activity, the EC 50 value of DPPH free radical scavenging is not more than 2.0mg / mL, and the EC 50 value of ABTS free radical scavenging is not more than 0.6mg / mL.

[0083] (2) The hemp seed oligopeptide prepared by the above method has high purity and low molecular weight, can better play its antioxidant and anti-aging functions, and the taste of the product is good, which can be made into food or health products, facilitating use and promoting the use and promotion of the product. BRIEF DESCRIPTION OF DRAWINGS

[0084] Figure 1 Flow chart of the preparation method of the hemp seed oligopeptide according to the present application. DETAILED DESCRIPTION

[0085] In order to further illustrate the technical means adopted by the present application and its effects, the present application is further described below in conjunction with examples and drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

[0086] Unless specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art, or according to the product instructions are used. Unless the manufacturer of the reagents or instruments is specified, they are all conventional products that can be commercially available through regular channels.

[0087] Raw materials:

[0088] Hemp seeds were purchased from Yushexian Tianhe Biological Technology Co., Ltd.;

[0089] Flavourzyme, alkaline protease, papain, trypsin, pepsin, sodium chloride, sodium hydroxide, hydrochloric acid, ethanol and potassium persulfate were purchased from Sinopharm Group;

[0090] DPPH and ABTS were purchased from Aladdin.

[0091] Preparation Example 1

[0092] The hemp seed powder was prepared by the following four methods, as follows:

[0093] 1-1: The hemp seeds were dried, crushed, and then solvent-extracted with n-hexane as the solvent to remove fat, to obtain the hemp seed powder.

[0094] 1-2: The difference from 1-1 is that petroleum ether is used instead of n-hexane, and the rest of the preparation method is the same.

[0095] 1-3: The hemp seeds were dried, crushed, and then physically pressed to remove fat, to obtain the hemp seed powder.

[0096] 1-4: The hemp seeds were dried, crushed, and then supercritical extracted with carbon dioxide as the solvent to remove fat, to obtain the hemp seed powder.

[0097] Determination of oil content

[0098] The hemp seed powder was dried to a water content of less than 10%, 2.0 g of the sample was weighed, and the Soxhlet extractor was used to extract the fat with n-hexane until the sample mass change value was less than 0.02 g, and the fat content was calculated.

[0099] Fat content = (decreased sample mass) / (original sample mass) × 100%.

[0100] The oil content in the hemp seed powder prepared by 1-1, 1-2, 1-3 and 1-4 is 3%, 4%, 5% and 3% respectively, which all meet the requirement of less than 5%.

[0101] In the subsequent preparation examples and embodiments, the hemp seed powder is prepared by the method of 1-1.

[0102] Preparation Example 2

[0103] In this preparation example, the hemp seed powder prepared by 1-1 in Preparation Example 1 is used to extract hemp seed globulin powder by the following 11 methods, as follows:

[0104] 2-1: The hemp seed powder is added into a 5% sodium chloride solution, the ratio of the mass of the hemp seed powder to the volume of the sodium chloride solution is 1:20 g / mL, and the extraction is carried out at 55°C for 2h. After centrifugation, the supernatant is taken and cooled at 4°C for 4h, and then the protein is precipitated. After centrifugation and drying, the hemp seed globulin powder is obtained.

[0105] 2-2: The difference from 2-1 is only that the mass fraction of the sodium chloride solution is 2%, and the rest of the preparation method is the same as that of 2-1.

[0106] 2-3: The difference from 2-1 is only that the mass fraction of the sodium chloride solution is 4%, and the rest of the preparation method is the same as that of 2-1.

[0107] 2-4: The difference from 2-1 is only that the mass fraction of the sodium chloride solution is 6%, and the rest of the preparation method is the same as that of 2-1.

[0108] 2-5: The difference from 2-1 is only that the mass fraction of the sodium chloride solution is 10%, and the rest of the preparation method is the same as that of 2-1.

[0109] 2-6: The difference from 2-1 is only that the extraction temperature is 40°C, and the rest of the preparation method is the same as that of 2-1.

[0110] 2-7: The difference from 2-1 is only that the extraction temperature is 50°C, and the rest of the preparation method is the same as that of 2-1.

[0111] 2-8: The difference from 2-1 is only that the extraction temperature is 60°C, and the rest of the preparation method is the same as that of 2-1.

[0112] 2-9: The difference from 2-1 is only that the cooling temperature is 1°C, and the rest of the preparation method is the same as that of 2-1.

[0113] 2-10: The difference from 2-1 is only that the cooling temperature is 7°C, and the rest of the preparation method is the same as that of 2-1.

[0114] 2-11: The difference from 2-1 is only that the temperature of standing cooling is 10℃, and the rest of the preparation method is the same as 2-1.

[0115] The hemp seed globulin powder yield = the mass of the hemp seed globulin powder obtained by drying / the mass of the hemp seed powder × 100%.

[0116] The yield of the hemp seed globulin powder prepared in Preparation Examples 2-1 to 2-11 is shown in Table 1.

[0117] Table 1

[0118]

[0119] From Table 1, it can be seen that, according to the results of Comparative Preparation Examples 2-1 to 2-5, with the increase of the mass fraction of sodium chloride solution, the globulin yield first increases and then decreases, and the mass fraction is 5% when the globulin yield is the highest; combined with the results of Preparation Examples 2-1 and 2-6 to 2-8, it can be seen that with the increase of the extraction temperature, the globulin yield first increases and then remains relatively stable; combined with the results of Preparation Examples 2-1 and 2-9 to 2-11, it can be seen that with the increase of the temperature of standing cooling, the globulin yield first increases and then decreases. According to the above results, it can be seen that when the mass fraction of sodium chloride solution is 5%, the extraction temperature is 55℃, and the standing cooling temperature is 4℃, the best extraction effect can be achieved.

[0120] Preparation Example 3

[0121] In this preparation example, the hemp seed globulin powder prepared in 2-1 of Preparation Example 2 is used, and the hemp seed globulin solution is pretreated by ultrasonic at different heating temperatures and 400W / L for 40min, and then the hemp seed oligopeptide is prepared by one-step enzymatic hydrolysis, as follows:

[0122] 3-1:

[0123] (1) The hemp seed globulin powder is mixed with water to obtain a hemp seed globulin solution with a mass concentration of 10% of the hemp seed globulin;

[0124] (2) The hemp seed globulin solution is heated at 25℃ for 40min, and then ultrasonic is performed at 400W / L for 40min;

[0125] (3) Alkaline protease is added to the obtained product after heating and ultrasonic, and the use amount of the alkaline protease is 2% of the mass of the hemp seed globulin, one-step enzymatic hydrolysis is performed at 55℃ for 4h, and then the enzyme is inactivated at 80℃ for 30min, and the temperature is reduced to 25℃ to obtain a one-step enzymatic hydrolysis product;

[0126] (4) The product of the first enzymolysis is centrifuged, and the supernatant is subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off of 3000 Da to obtain an ultrafiltration membrane permeate, and the ultrafiltration membrane permeate is subjected to column chromatography desalting using water as the eluent, and the column chromatography desalting uses a column packing including Sephadex G-50 to obtain a desalting membrane permeate;

[0127] (5) The desalting membrane permeate is subjected to freeze-drying to obtain the hemp seed oligopeptide.

[0128] 3-2: The difference from 3-1 is only that the heating temperature in step (2) is 70°C, and the rest of the preparation method is the same as that of 3-1.

[0129] 3-3: The difference from 3-1 is only that the heating temperature in step (2) is 75°C, and the rest of the preparation method is the same as that of 3-1.

[0130] 3-4: The difference from 3-1 is only that the heating temperature in step (2) is 80°C, and the rest of the preparation method is the same as that of 3-1.

[0131] 3-5: The difference from 3-1 is only that the heating temperature in step (2) is 85°C, and the rest of the preparation method is the same as that of 3-1.

[0132] 3-6: The difference from 3-1 is only that the heating temperature in step (2) is 90°C, and the rest of the preparation method is the same as that of 3-1.

[0133] 3-7: The difference from 3-1 is only that the heating temperature in step (2) is 95°C, and the rest of the preparation method is the same as that of 3-1.

[0134] 3-8: The difference from 3-1 is only that the heating temperature in step (2) is 98°C, and the rest of the preparation method is the same as that of 3-1.

[0135] Soluble polypeptide yield calculation

[0136] After the enzymolysis is completed, the supernatant is obtained by centrifugation, and the supernatant is freeze-dried to obtain the soluble polypeptide, and the yield of the soluble polypeptide is calculated according to the formula.

[0137]

[0138] Oligopeptide yield calculation

[0139] After ultrafiltration, the ultrafiltration membrane permeate is freeze-dried to obtain the oligopeptide, and the yield of the oligopeptide is calculated according to the formula.

[0140]

[0141] The calculation results of the soluble polypeptide yield and the oligopeptide yield of the preparation examples 3-1 to 3-8 are shown in Table 2.

[0142] Table 2

[0143]

[0144]

[0145] From Table 2, it can be seen that when the heat treatment temperature exceeds 80℃, the yield of soluble polypeptide is significantly improved, and there is no significant change when the temperature exceeds 95℃; when the temperature is higher than 90℃, the yield of oligopeptide has no significant change, therefore, it is comprehensively judged that the pretreatment at 95℃ can improve the yield of the product.

[0146] Preparation Example 4

[0147] This preparation example uses the hemp seed globulin powder prepared in 2-1 in Preparation Example 2, and the hemp seed globulin solution is pretreated by different ultrasonic powers under the condition of heating at 95℃ for 40min, and then enzymolysis is carried out once to prepare hemp seed oligopeptide, which is as follows:

[0148] 4-1:

[0149] (1) The hemp seed globulin powder is mixed with water to obtain a hemp seed globulin solution with a mass concentration of 10% of the hemp seed globulin;

[0150] (2) The hemp seed globulin solution is heated at 95℃ for 40min, and then ultrasonic is applied at 50W / L for 40min;

[0151] (3) Alkaline protease is added to the obtained product after heating and ultrasonic, and the use amount of the alkaline protease is 2% of the mass of the hemp seed globulin, and the first enzymolysis is carried out at 55℃ for 4h, and then the enzyme is inactivated at 80℃ for 30min, and then the temperature is reduced to 25℃ to obtain the product of the first enzymolysis;

[0152] (4) After centrifugation of the product of the first enzymolysis, the obtained supernatant is subjected to ultrafiltration using an ultrafiltration membrane with a molecular cut-off of 3000Da to obtain the ultrafiltration membrane permeate, and then column chromatography is carried out using water as the elution solvent to desalt, and the chromatography column filler includes Sephadex G-50 to obtain the desalted membrane permeate;

[0153] (5) The desalted membrane permeate is subjected to freeze-drying to obtain the hemp seed oligopeptide.

[0154] 4-2: The difference from 4-1 is only that the power of ultrasonic in step (2) is 100W / L, and the rest of the preparation method is the same as that of 4-1.

[0155] 4-3: The difference from 4-1 is only that the power of ultrasonic in step (2) is 200W / L, and the rest of the preparation method is the same as that of 4-1.

[0156] 4-4: The difference from 4-1 is only that the power of ultrasonic in step (2) is 400 W / L, and the rest of the preparation method is the same as 4-1.

[0157] 4-5: The difference from 4-1 is only that the power of ultrasonic in step (2) is 600 W / L, and the rest of the preparation method is the same as 4-1.

[0158] 4-6: The difference from 4-1 is only that the power of ultrasonic in step (2) is 800 W / L, and the rest of the preparation method is the same as 4-1.

[0159] 4-7: The difference from 4-1 is only that the power of ultrasonic in step (2) is 1000 W / L, and the rest of the preparation method is the same as 4-1.

[0160] The calculation method of the yield of soluble polypeptide and the yield of oligopeptide is the same as that of Preparation Example 3.

[0161] The calculation results of the yield of soluble polypeptide and the yield of oligopeptide of Preparation Examples 4-1 to 4-7 are shown in Table 3.

[0162] Table 3

[0163]

[0164]

[0165] From Table 3, it can be seen that when the power of ultrasonic is more than 200 W / L, the yield of soluble polypeptide and oligopeptide is obviously improved, and when the power of ultrasonic is more than 400 W / L, the yield of soluble polypeptide and oligopeptide has no obvious change, and the production cost is improved, therefore, heating at 95℃ and ultrasonic at 400 W / L can achieve better pretreatment effect.

[0166] Preparation Example 5

[0167] In this preparation example, the hemp seed globulin powder prepared in 2-1 of Preparation Example 2 is used, and under the condition of heating at 95℃ for 40 min and ultrasonic at 400 W / L for 40 min, hemp seed oligopeptide is prepared by the following 10 methods, as follows:

[0168] 5-1:

[0169] (1) The hemp seed globulin powder is mixed with water to obtain a hemp seed globulin solution with a mass concentration of 10% of the hemp seed globulin powder;

[0170] (2) The hemp seed globulin solution is heated at 95℃ for 40 min, and then ultrasonic is applied at 400 W / L for 40 min;

[0171] (3) adding alkaline protease to the obtained heated and ultrasonicated product, wherein the amount of alkaline protease used is 2% of the mass of the hemp seed globulin, performing a primary enzymatic hydrolysis at 55° C. for 4 h, then deactivating the enzyme at 80° C. for 30 min, and cooling to 55° C. to obtain a primary enzymatic hydrolysis product;

[0172] (4) adding flavor protease to the product of the primary enzymatic hydrolysis, wherein the amount of the flavor protease used is 4% of the mass of the hemp seed globulin, performing secondary enzymatic hydrolysis at 50° C. for 4 h, then deactivating the enzyme at 80° C. for 30 min, and cooling to 25° C. to obtain a secondary enzymatic hydrolysis product;

[0173] (5) After the product of the secondary enzymatic hydrolysis is centrifuged, the resulting supernatant is ultrafiltered using an ultrafiltration membrane with a molecular cutoff of 3000 Da to obtain an ultrafiltration membrane permeate, which is then desalted by column chromatography using water as an elution solvent, wherein the chromatography column filler used includes Sephadex G-50 to obtain a desalted membrane permeate;

[0174] (6) The desalting membrane permeate is freeze-dried to obtain the hemp seed oligopeptide.

[0175] 5-2: The only difference from 5-1 is that the amount of alkaline protease used in step (3) is 1% of the mass of the hemp seed globulin, and the rest of the preparation method is the same as 5-1.

[0176] 5-3: The only difference from 5-1 is that the amount of alkaline protease used in step (3) is 3% of the mass of the hemp seed globulin, and the rest of the preparation method is the same as 5-1.

[0177] 5-4: The only difference from 5-1 is that the amount of alkaline protease used in step (3) is 5% of the mass of the hemp seed globulin, and the rest of the preparation method is the same as 5-1.

[0178] 5-5: The only difference from 5-1 is that the temperature of the first enzymatic hydrolysis in step (3) is 40°C, and the rest of the preparation method is the same as 5-1.

[0179] 5-6: The only difference from 5-1 is that the temperature of the first enzymatic hydrolysis in step (3) is 50°C, and the rest of the preparation method is the same as 5-1.

[0180] 5-7: The only difference from 5-1 is that the temperature of the first enzymatic hydrolysis in step (3) is 60°C, and the rest of the preparation method is the same as 5-1.

[0181] 5-8: The only difference from 5-1 is that the enzymatic hydrolysis time in step (3) is 2 hours. The rest of the preparation method is the same as 5-1.

[0182] 5-9: The difference from 5-1 is only that the time of one enzymolysis in step (3) is 3h, and the rest of the preparation method is the same as 5-1.

[0183] 5-10: The difference from 5-1 is only that the time of one enzymolysis in step (3) is 5h, and the rest of the preparation method is the same as 5-1.

[0184] The calculation method of the yield of soluble polypeptide and the yield of oligopeptide is the same as Preparation Example 3.

[0185] The calculation results of the yield of soluble polypeptide and the yield of oligopeptide of Preparation Examples 5-1 to 5-10 are shown in Table 4.

[0186] Table 4

[0187] Group Soluble polypeptide yield (%) Oligopeptide yield (%) 5-1 79.9 71.2 5-2 54.4 64.5 5-3 80.1 71.1 5-4 80.9 71.9 5-5 71.2 69.8 5-6 75.9 70.1 5-7 74.3 70.4 5-8 59.8 68.2 5-9 69.7 69.6 5-10 79.8 71.6

[0188] It can be seen from Table 4 that on the basis of heating and ultrasonic treatment, when the addition amount of alkaline protease is higher than 2% of the mass of hemp seed globulin, the yield of soluble polypeptide is basically stable, so that the addition amount of alkaline protease is selected as 2% to reduce the cost; the temperature of one enzymolysis has an influence on the yield of soluble polypeptide, and the yield increases first and then decreases with the increase of temperature, and the yield of soluble polypeptide is the highest when the temperature is 55℃; when the enzymolysis time is within 4h, the polypeptide yield increases with the increase of enzymolysis time, and the yield of soluble polypeptide is stable when the enzymolysis time is greater than 4h, so that 4h is a more optimal condition.

[0189] Preparation Example 6

[0190] This preparation example uses the hemp seed globulin powder prepared in 2-1 in Preparation Example 2, and the method of one enzymolysis is the same as 5-1 under the conditions of heating at 95℃ for 40min and ultrasonic treatment at 400W / L for 40min, and hemp seed oligopeptide is prepared by different methods of secondary enzymolysis, which is as follows:

[0191] 6-1:

[0192] (1) The hemp seed globulin powder is mixed with water to obtain a hemp seed globulin solution with a mass concentration of 10% of the hemp seed globulin;

[0193] (2) The hemp seed globulin solution is heated at 95℃ for 40min, and then ultrasonic treated at 400W / L for 40min;

[0194] (3) Alkaline protease is added to the obtained product after heating and ultrasonic treatment, and the use amount of the alkaline protease is 2% of the mass of the hemp seed globulin, and one enzymolysis is carried out at 55℃ for 4h, and then the enzyme is inactivated at 80℃ for 30min, and then cooled to 55℃ to obtain a product of one enzymolysis;

[0195] (4) adding flavor protease to the product of the first enzymolysis, the flavor protease is used in an amount of 4% of the mass of the hemp seed globulin, and the second enzymolysis is performed at 50℃ for 4h, and then the enzyme is inactivated at 80℃ for 30min, and then the temperature is reduced to 25℃, to obtain a product of the second enzymolysis;

[0196] (5) after centrifugation of the product of the second enzymolysis, the obtained supernatant is subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off of 3000Da, to obtain an ultrafiltration membrane permeate, and then the ultrafiltration membrane permeate is subjected to column chromatography desalting using water as an elution solvent, and the chromatography column filler includes Sephadex G-50, to obtain a desalting membrane permeate;

[0197] (6) after freeze-drying of the desalting membrane permeate, the hemp seed oligopeptide is obtained.

[0198] 6-2: the difference from 6-1 is only that the amount of flavor protease used in step (4) is 1% of the mass of the hemp seed globulin, and the rest of the preparation method is the same as that of 6-1.

[0199] 6-3: the difference from 6-1 is only that the amount of flavor protease used in step (4) is 2% of the mass of the hemp seed globulin, and the rest of the preparation method is the same as that of 6-1.

[0200] 6-4: the difference from 6-1 is only that the amount of flavor protease used in step (4) is 6% of the mass of the hemp seed globulin, and the rest of the preparation method is the same as that of 6-1.

[0201] 6-5: the difference from 6-1 is only that the amount of flavor protease used in step (4) is 10% of the mass of the hemp seed globulin, and the rest of the preparation method is the same as that of 6-1.

[0202] 6-6: the difference from 6-1 is only that the temperature of the second enzymolysis in step (4) is 40℃, and the rest of the preparation method is the same as that of 6-1.

[0203] 6-7: the difference from 6-1 is only that the temperature of the second enzymolysis in step (4) is 60℃, and the rest of the preparation method is the same as that of 6-1.

[0204] 6-8: the difference from 6-1 is only that the time of the second enzymolysis in step (4) is 2h, and the rest of the preparation method is the same as that of 6-1.

[0205] 6-9: the difference from 6-1 is only that the time of the second enzymolysis in step (4) is 3h, and the rest of the preparation method is the same as that of 6-1.

[0206] 6-10: the difference from 6-1 is only that the time of the second enzymolysis in step (4) is 5h, and the rest of the preparation method is the same as that of 6-1.

[0207] The calculation method of soluble polypeptide yield and oligopeptide yield is the same as that of Preparation Example 3.

[0208] The calculation results of soluble polypeptide yield and oligopeptide yield of Preparation Examples 6-1 to 6-10 are shown in Table 5.

[0209] Table 5

[0210]

[0211]

[0212] From Table 5, it can be seen that, by comparing Preparation Examples 6-1 to 6-5, it can be seen that, with the increase of the amount of flavor protease, the yield of soluble polypeptide and oligopeptide is increased, but when the amount is higher than 4%, the effect of yield improvement is not obvious, and the utilization rate of flavor protease is low; by comparing Preparation Examples 6-1 and 6-6 to 6-7, it can be seen that, with the increase of the enzyme hydrolysis temperature, the yield of soluble polypeptide is decreased, the yield of oligopeptide is first increased and then decreased, and the yield at 50°C is the highest; by comparing Preparation Examples 6-1 and 6-8 to 6-10, it can be seen that, when the secondary enzyme hydrolysis time is within 4h, the longer the time, the higher the yield of the product; when the time is more than 4h, the yield does not change obviously, so the enzyme hydrolysis time of 4h is more appropriate. According to the above results, when the amount of flavor protease is 4% of the mass of hemp seed globulin, the secondary enzyme hydrolysis temperature is 50°C, and the secondary enzyme hydrolysis time is 4h, a better secondary enzyme hydrolysis effect can be achieved.

[0213] Preparation Example 7

[0214] In this preparation example, the secondary enzyme hydrolysis product prepared in 6-1 of Preparation Example 6 is used to prepare hemp seed oligopeptide by different ultrafiltration methods, as follows:

[0215] 7-1:

[0216] (1) After centrifugation of the secondary enzyme hydrolysis product, the obtained supernatant is subjected to ultrafiltration using an ultrafiltration membrane with a molecular cut-off of 3000Da, to obtain an ultrafiltration membrane permeate, which is then subjected to column chromatography desalting using water as the elution solvent, and the chromatography column filler includes Sephadex G-50, to obtain a desalting membrane permeate;

[0217] (2) After freeze-drying of the desalting membrane permeate, the hemp seed oligopeptide is obtained.

[0218] 7-2: The difference from 7-1 is only that the ultrafiltration membrane used has a molecular cut-off of 1000Da, and the rest of the preparation method is the same as that of 7-1.

[0219] 7-3: The difference from 7-1 is only that the ultrafiltration membrane used has a molecular cut-off of 5000Da, and the rest of the preparation method is the same as that of 7-1.

[0220] 7-4: The difference from 7-1 is only that the molecular cut-off of the ultrafiltration membrane used is 10000 Da, and the rest of the preparation method is the same as 7-1.

[0221] DPPH free radical scavenging detection

[0222] (1) Prepare a 0.2 mmol / L DPPH (2,2-di(4-tert-octylphenyl)-1-hydrazone)-ethanol solution, and prepare a gradient solution of hemp seed globulin oligopeptide solution with a concentration of (0.5-5 mg / mL), and the gradient concentration is 0.1 mg / mL;

[0223] (2) Take 1 mL of oligopeptide solution with different concentrations and 1 mL of DPPH-ethanol solution, respectively, mix them, and react in the dark for 30 min. Measure the absorbance at 517 nm as the experimental group. Take 1 mL of ethanol and perform the same operation as the control group. Take 1 mL of deionized water and perform the same operation as the blank group;

[0224] (3) Calculate the DPPH clearance rate according to the formula, and record the concentration of hemp seed oligopeptide solution when the clearance rate is 50% (EC 50 ).

[0225] DPPH clearance rate (%) = [1-(A experiment-A control) / A blank] x 100%.

[0226] ABTS free radical scavenging detection

[0227] (1) Prepare a 7 mmol / L ABTS (2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) solution and a 140 mmol / L potassium persulfate solution. Prepare a gradient solution of hemp seed globulin oligopeptide solution with a concentration of (0.5-2 mg / mL), and the gradient concentration is 0.1 mg / mL;

[0228] (2) Mix 176 μL of potassium persulfate solution and 10 mL of ABTS solution, and react in the dark for 12 h to obtain an ABTS working solution;

[0229] (3) Add (50-80) times the volume of deionized water to the ABTS working solution, and the absorbance at 734 nm at room temperature is 0.7±0.02;

[0230] (4) Mix 0.7 mL of oligopeptide solution and 2.8 mL of ABTS solution, and react in the dark for 6 min. Measure the absorbance at 734 nm as the experimental group. Take 0.7 mL of deionized water and perform the same operation as the control group;

[0231] (5) Calculate the ABTS clearance rate according to the formula, and record the concentration of the cannabis seed oligopeptide solution when the clearance rate is 50% (EC 50 ).

[0232] ABTS clearance rate (%) = (1-A experiment / A blank) x 100%.

[0233] The calculation method of the oligopeptide yield is the same as that of Preparation Example 3.

[0234] The results of the oligopeptide yield, EC 50 value of DPPH free radical scavenging, and EC 50 value of ABTS free radical scavenging of Preparation Examples 7-1 to 7-4 are shown in Table 6.

[0235] Table 6

[0236]

[0237] From Table 6, it can be seen that, by comparing the results of 7-1 to 7-4, as the molecular cut-off of the ultrafiltration membrane increases, the oligopeptide yield increases, and when ultrafiltration is performed using an ultrafiltration membrane with a molecular cut-off of 3000 Da, the values of EC 50 and EC 50 of DPPH free radical scavenging and ABTS free radical scavenging are both smaller, indicating that the antioxidant activity of the oligopeptide is better, and therefore, a molecular cut-off of 3000 Da can achieve better ultrafiltration effect.

[0238] Example 1

[0239] The present example provides a preparation method of cannabis seed oligopeptide, comprising the following steps:

[0240] (1) The cannabis seed powder is added into a 5% sodium chloride solution, the ratio of the mass of the cannabis seed powder to the volume of the sodium chloride solution is 1:20 g / mL, and the mixture is extracted at 55°C for 2 h. After centrifugation, the supernatant is taken and cooled at 4°C for 4 h, and the protein is precipitated. After centrifugation and drying, the cannabis seed globulin powder is obtained. The cannabis seed globulin powder is mixed with water to obtain a cannabis seed globulin solution with a mass concentration of 10% of the cannabis seed globulin;

[0241] (2) The cannabis seed globulin solution is heated at 95°C for 40 min, and then ultrasonicated at 400 W / L for 40 min;

[0242] (3) Papain is added to the obtained product after heating and ultrasonication, the use amount of the papain is 2% of the mass of the cannabis seed globulin, and the mixture is once-enzymolyzed at 55°C for 4 h. After enzyme inactivation at 80°C for 30 min, the temperature is lowered to 55°C to obtain the once-enzymolyzed product;

[0243] (4) adding flavor protease to the product of the first enzymolysis, the flavor protease is used in an amount of 4% of the mass of the hemp seed globulin, and the second enzymolysis is performed at 50°C for 4h, and then the enzyme is inactivated at 80°C for 30min, and then the temperature is reduced to 25°C to obtain a product of the second enzymolysis;

[0244] (5) after centrifugation of the product of the second enzymolysis, the obtained supernatant is subjected to ultrafiltration using an ultrafiltration membrane with a molecular cut-off of 3000Da to obtain an ultrafiltration membrane permeate, and then the ultrafiltration membrane permeate is subjected to column chromatography desalting using water as an elution solvent, and the chromatography column filler includes dextran gel G-50 to obtain a desalting membrane permeate;

[0245] (6) after freeze-drying of the desalting membrane permeate, the hemp seed oligopeptide is obtained.

[0246] Example 2

[0247] The difference from Example 1 is that in this example, pepsin is used instead of papain, and the other raw materials and preparation methods are the same as those in Example 1.

[0248] Example 3

[0249] The difference from Example 1 is that in this example, trypsin is used instead of papain, and the other raw materials and preparation methods are the same as those in Example 1.

[0250] Comparative Example 1

[0251] The difference from Example 1 is that in this comparative example, step (4) is not performed, and the other raw materials and preparation methods are the same as those in Example 1.

[0252] Comparative Example 2

[0253] The difference from Example 1 is that in this comparative example, step (2) is not performed, and the other raw materials and preparation methods are the same as those in Example 1.

[0254] Comparative Example 3

[0255] The difference from Example 1 is that in this comparative example, pepsin is used instead of papain, and steps (2) and (5) are not performed, and the other raw materials and preparation methods are the same as those in Example 1.

[0256] Comparative Example 4

[0257] The difference from Example 1 is that in this comparative example, papain and flavor protease are added together, and the first enzymolysis is performed at 55°C for 40min, and the second enzymolysis is not performed, and the other raw materials and preparation methods are the same as those in Example 1.

[0258] Determination of peptide content in oligopeptide

[0259] Determination of the nitrogen content of the oligopeptide sample by Kjeldahl method, and then convert the nitrogen content to the peptide content by multiplying the conversion factor F = 6.25. The formula is as follows:

[0260] Peptide content (%) of the oligopeptide = [nitrogen content × F] / oligopeptide mass × 100%

[0261] The soluble polypeptide yield, oligopeptide yield, peptide content of the prepared Cannabis sativa L. oligopeptide, DPPH radical scavenging EC 50 value and ABTS radical scavenging EC 50 value of the oligopeptides prepared in Examples 1-3 and Comparative Examples 1-4 were calculated, and the bitterness of the prepared products was evaluated. The results are shown in Table 7.

[0262] Table 7

[0263]

[0264]

[0265] As can be seen from Table 7, Examples 1-3 were subjected to heating, ultrasonic pretreatment, two-step enzymatic hydrolysis, ultrafiltration and desalination during preparation, and the yield of the product was high, the soluble polypeptide yield was above 75.2%, the oligopeptide yield was above 70.5%, the peptide content of the oligopeptide was above 94.5%, and the activity of the prepared oligopeptide was also good, the DPPH radical scavenging EC 50 value was not more than 2.0 mg / mL, the ABTS radical scavenging EC 50 value was not more than 0.6 mg / mL; the bitterness of the product was not obvious, and the use experience was improved.

[0266] Comparing with Examples 1-3, it can be seen that the yield of the Cannabis sativa L. oligopeptide prepared in Comparative Examples 1-3 was slightly lower, and the activity was slightly worse, and the bitterness of the oligopeptide obtained by two-step enzymatic hydrolysis with no wind flavor protease was more obvious. In Comparative Example 1, no two-step enzymatic hydrolysis was performed, so the yield was lower, and the bitterness was more obvious. In Comparative Example 2, the Cannabis sativa L. globulin solution was not heated and ultrasonicated, and the structure of the protein was relatively dense, which affected the hydrolysis of the protease. In Comparative Example 3, no pretreatment and ultrafiltration were performed, the lack of pretreatment affected the hydrolysis efficiency of the protease, and the lack of ultrafiltration could not separate the oligopeptide from the peptide with a larger molecular weight, so the oligopeptide yield and content could not be calculated, and the activity of the obtained product was also lower. The above results show that heating, ultrasonic pretreatment, two-step enzymatic hydrolysis and ultrafiltration play an important role in improving the yield and activity of the product. Only when the above operations are performed at the same time, can a better extraction effect be achieved.

[0267] Comparing the comparative example 4 with the example 1, it can be seen that the soluble polypeptide and oligopeptide prepared by carrying out the enzymolysis twice and only once respectively have higher content and better activity, which indicates that the effect of the enzymolysis carried out by the two proteases respectively is better than that of the enzymolysis carried out once at the same time.

[0268] In addition, comparing the results of the comparative examples 1-3, the papain is used in the example 1, the pepsin is used in the example 2, and the trypsin is used in the example 3, and the yield of the hemp seed oligopeptide prepared in the example 3 is the highest and the activity is the best, which indicates that compared with the papain and the pepsin, the trypsin has better hydrolysis effect on the pretreated hemp seed globulin.

[0269] In summary, the application provides a preparation method of hemp seed oligopeptide, the hydrolysis area of the substrate and the protease can be increased and the hydrolysis efficiency can be improved by heating and ultrasonic pretreatment of the raw material, the molecular weight of the product can be significantly reduced by sequentially carrying out the enzymolysis twice, the substance type of the product is equivalent to single, and the bitter taste of the product can be improved, the purity of the hemp seed oligopeptide prepared by the ultrafiltration and desalination of the enzymolysis product is higher, and the biological activity is better, the hemp seed oligopeptide prepared by the above method has good antioxidant capacity, can be prepared into corresponding food or health care product for direct consumption, and has wide application value.

[0270] The applicant declares that the application is illustrated by the above examples, but the application is not limited to the above detailed method, that is, it does not mean that the application must depend on the above detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the application, equivalent replacement of each raw material of the product of the application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the application.

Claims

1. Hemp seed powder with an oil content of 3%-5% The method for preparing hemp seed oligopeptide is characterized in that: The preparation method of the hemp seed oligopeptide comprises: Drying hemp seeds, crushing and defatting to obtain hemp seed powder, wherein the oil content of the hemp seed powder is 3%-5%; The hemp seed powder is added to a sodium chloride solution with a mass concentration of 5%-10%, extracted at 50-60° C. for 0.5-4 h, centrifuged, and the supernatant is collected. After standing and cooling at 1-4° C. for 1-12 h, protein is precipitated, and centrifuged again to obtain hemp seed globulin, which is then dried to prepare hemp seed globulin powder; the hemp seed globulin powder is mixed with water to prepare a hemp seed globulin solution; the hemp seed globulin solution is then heated, ultrasonicated, and then enzymatically hydrolyzed, and the resulting enzymatic hydrolysis product is ultrafiltered and desalted to obtain the hemp seed oligopeptide; The heating temperature is 80-100° C., and the heating time is 10-60 min; The ultrasonic power is 400-800 W / L, and the ultrasonic time is 10-60 min; The molecular cut-off capacity of the ultrafiltration membrane used in the ultrafiltration is 3000~10000 Da; The enzymatic hydrolysis includes primary enzymatic hydrolysis and secondary enzymatic hydrolysis; The primary enzymatic hydrolysis comprises adding a protease to the obtained heated and ultrasonicated product, and then inactivating the enzyme to obtain a primary enzymatic hydrolysis product, wherein the protease comprises papain, and the amount of the protease added is 0.5% to 5% of the mass of the hemp seed globulin; The secondary enzymatic hydrolysis comprises adding protease to the product of the primary enzymatic hydrolysis, and then inactivating the enzyme after the secondary enzymatic hydrolysis to obtain the product of the secondary enzymatic hydrolysis. The protease comprises flavor protease, and the added amount of the protease is 0.5% to 10% of the mass of the hemp seed globulin.

2. the preparation method of hemp seed oligopeptide according to claim 1, is characterized in that, The mass concentration of hemp protein in the hemp protein solution is 1% to 15%.

3. the preparation method of hemp seed oligopeptide according to claim 1, is characterized in that, The ratio of the mass of the hemp seed powder to the volume of the sodium chloride solution is 1:(5-30) g / mL.

4. the preparation method of hemp seed oligopeptide according to claim 1, is characterized in that, The extraction temperature was 55°C.

5. the preparation method of hemp seed oligopeptide according to claim 1, is characterized in that, The extraction time is 2 hours.

6. the preparation method of hemp seed oligopeptide according to claim 1, is characterized in that, The temperature of the static cooling is 4°C.

7. the preparation method of hemp seed oligopeptide according to claim 1 is characterized in that, The cooling time is 4 h.

8. the preparation method of hemp seed oligopeptide according to claim 1 is characterized in that, The degreasing includes any one of physical pressing, solvent extraction or supercritical extraction.

9. the preparation method of hemp seed oligopeptide according to claim 8 is characterized in that, The solvent used for the solvent extraction includes n-hexane and / or petroleum ether.

10. The preparation method of hemp seed oligopeptide according to claim 8, wherein The solvent used in the supercritical extraction includes carbon dioxide.

11. The method for preparing the hemp seed oligopeptide according to claim 1, wherein The heating temperature is 95°C.

12. The preparation method of hemp seed oligopeptide according to claim 1, wherein The power of the ultrasound is 400 W / L.

13. The preparation method of hemp seed oligopeptide according to claim 1, wherein The temperature of the primary enzymatic hydrolysis is 40-60°C.

14. The method for preparing the hemp seed oligopeptide according to claim 13, wherein The temperature of the primary enzymatic hydrolysis is 55°C.

15. The preparation method of hemp seed oligopeptide according to claim 1, wherein The time of the enzymatic hydrolysis is 2 to 4 hours.

16. The method for preparing the hemp seed oligopeptide according to claim 1, wherein In the primary enzymatic hydrolysis, the temperature for inactivating the enzyme is not lower than 80°C.

17. The method for preparing the hemp seed oligopeptide according to claim 1, wherein In the primary enzymatic hydrolysis, the enzyme inactivation time is 10 to 30 minutes.

18. The method for preparing the hemp seed oligopeptide according to claim 1, wherein In the primary enzymatic hydrolysis, the step of cooling the temperature is further included after the enzyme is inactivated.

19. The method for preparing the hemp seed oligopeptide according to claim 18, wherein The cooling temperature is 40-60°C.

20. The method for preparing hemp seed oligopeptide according to claim 1, wherein The temperature of the secondary enzymatic hydrolysis is 40-60°C.

21. The method for preparing the hemp seed oligopeptide according to claim 1, wherein The secondary enzymatic hydrolysis time is 2 to 4 h.

22. The method for preparing the hemp seed oligopeptide according to claim 1, wherein In the secondary enzymolysis, the temperature for inactivating the enzyme is not lower than 80°C.

23. The method for preparing the hemp seed oligopeptide according to claim 1, wherein In the secondary enzymatic hydrolysis, the enzyme inactivation time is 10 to 30 minutes.

24. The method for preparing the hemp seed oligopeptide according to claim 1, wherein In the secondary enzymatic hydrolysis, the step of cooling the temperature is also included after the enzyme is inactivated.

25. The method for preparing the hemp seed oligopeptide according to claim 24, wherein The cooling temperature is 20-30°C.

26. The method for preparing the hemp seed oligopeptide according to claim 1, wherein The molecular cut-off capacity of the ultrafiltration membrane used in the ultrafiltration is 3000 Da.

27. The method for preparing the hemp seed oligopeptide according to claim 1, wherein The method further comprises a centrifugation step before the ultrafiltration.

28. The method for preparing the hemp seed oligopeptide according to claim 1, wherein The desalting includes column chromatography desalting.

29. The method for preparing the hemp seed oligopeptide according to claim 28, wherein The elution solvent used in the column chromatography desalting includes water.

30. The method for preparing the hemp seed oligopeptide according to claim 28, wherein The chromatography column filler used for the column chromatography desalting comprises dextran gel and / or agarose gel.

31. The method for preparing the hemp seed oligopeptide according to claim 28, wherein The method further comprises a drying step after the desalination.

32. The method for preparing the hemp seed oligopeptide according to claim 31, wherein The drying includes freeze drying and / or spray drying.

33. A hemp seed oligopeptide prepared by the preparation method according to any one of claims 1 to 32, characterized in that: The peptide content in the hemp seed oligopeptide is not less than 95%, and the content of oligopeptides with a molecular weight below 3000 Da is not less than 70%.

34. Use of the hemp seed oligopeptide as claimed in claim 33 in preparing antioxidant foods.

35. Use of the hemp seed oligopeptide as claimed in claim 33 in the preparation of antioxidant health products.

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