Fructus cannabis protein peptide and particle for inhibiting alpha-glucosidase as well as preparation method and application of fructus cannabis protein peptide and particle

Abstract

The invention discloses a fructus cannabis protein peptide and particle for inhibiting alpha-glucosidase as well as a preparation method and application of the fructus cannabis protein peptide and particle. The fructus cannabis protein peptide for inhibiting alpha-glucosidase comprises at least one of polypeptides of which the amino acid sequences are FY, SPVI, TGLGR and FR. The fructus cannabis protein peptide particles for assisting in reducing blood sugar comprise the following components: fructus cannabis protein peptide powder, a sugar-free auxiliary material, a flavoring agent and an adhesive, the mass ratio of the fructus cannabis protein peptide powder to the sugar-free auxiliary material is (1: 5)-(1: 9), and the addition amount of the flavoring agent is 0.2%-0.8% of the total mass of the fructus cannabis protein peptide powder and the sugar-free auxiliary material. The fructus cannabis protein peptide disclosed by the invention has a very high alpha-glucosidase inhibition rate; according to the particles prepared by screening proper auxiliary materials and proportioning, the stability of the fructus cannabis protein peptide in the storage process and the like can be ensured, and the fructus cannabis protein peptide is high in transfer rate, high in particle forming rate and yield and good in taking taste in the production process and has a good blood sugar reducing effect.

Description

Technical Field

[0001] The invention belongs to the field of hemp seed protein peptides, and in particular relates to a hemp seed protein peptide capable of inhibiting alpha-glucosidase, particles, and a preparation method and application thereof. Background Art

[0002] α-glucosidase (GAA) is mainly located on the brush border cell membrane of the small intestine. It is an enzyme that plays a key role in the biological sugar metabolism process. It can hydrolyze disaccharides, such as sucrose, maltose and other oligosaccharides, into monosaccharides that can be partially absorbed by the human small intestine. α-glucosidase inhibitors help reduce enzyme activity, reduce the rate at which oligosaccharides are decomposed into glucose, hinder the digestion and absorption of glucose in the intestine, reduce postprandial blood sugar peaks and blood sugar fluctuations, reduce the stimulation of high blood sugar on organs such as the pancreas, and maintain postprandial blood sugar concentrations at normal levels, thereby achieving the purpose of improving postprandial hyperglycemia or improving patients' impaired glucose tolerance.

[0003] At present, some blood sugar-lowering drugs are already on the market, such as acarbose, miglitol and voglibose, which work by inhibiting the activity of α-amylase and α-glucosidase, but the continuous use of these drugs usually brings adverse side effects, such as liver toxicity and symptoms of adverse reactions of the digestive system. In recent years, more and more studies at home and abroad have screened α-glucosidase inhibitors from natural products, such as bitter melon polypeptide, seaweed polysaccharide, quercetin, cinnamon polyphenols, etc. In recent years, more and more studies have screened α-glucosidase inhibitors from natural products.

[0004] Enzymatic hydrolysis technology is a relatively mild and green method, which is often used in the preparation of active peptides. For example, the Chinese patent with application number CN202011487041.X discloses a method for extracting hypoglycemic peptides from tea seed meal, including crushing tea seed meal, defatting tea seed meal, extracting tea seed meal protein, preparing tea seed meal crude peptides, separating and screening α-glucosidase inhibitory peptides, and structural identification of α-glucosidase inhibitory peptides. The Chinese patent with application number CN201910432490.5 discloses a method for preparing a hypoglycemic peony active peptide, using peony seed meal with seed coat as raw material, first extracting peony protein, adding a composite enzyme to the peony protein solution, the composite enzyme is neutral protease, trypsin, aminopeptidase, and the peony active peptide obtained after enzymolysis is embedded with cyclodextrin to remove the spicy taste, and the peptide bonds in the peony protein are hydrolyzed into polypeptides from the inside of the peptide chain under the action of two types of endopeptidases, neutral protease and trypsin. In the above technical solutions, plant protein is first extracted from plant seed meal, and then the plant protein is enzymatically hydrolyzed to obtain polypeptides.

[0005] Hemp, also known as hemp, is mainly planted in Guangxi, Yunnan, and the three northeastern provinces in my country. Hemp seeds, as the seeds of hemp, were listed as a resource of medicinal and edible origin in 2002. They are rich in nutrients such as protein, oil, and dietary fiber. In China, more research on hemp seeds focuses on hemp oil, tetrahydrocannabinol and other ingredients, while hemp seed meal, as a by-product after extracting hemp oil, is often used as feed, resulting in a waste of protein resources. Chinese patent application number 202110154677.0 discloses a method for producing a hemp seed meal protein polypeptide liquid and a polypeptide liquid with a uric acid-lowering effect and its application, comprising the following steps: pulping the hemp seed meal with water, ultrasonic treatment, enzyme inactivation, adjusting the pH value, adding alkaline protease, hydrolyzing and inactivating the enzyme to obtain a hemp seed one-step enzymatic hydrolyzate, and then obtaining a group of biologically active polypeptide liquids with uric acid-lowering function by enzymatic hydrolysis combined with fermentation or a two-step enzymatic hydrolysis method. This technology uses enzymatic hydrolysis to separate biologically active polypeptide liquid with uric acid lowering function from hemp seed meal.

[0006] In the prior art, there is no report on the preparation of active peptides that inhibit α-glucosidase from hemp seed meal, nor on the preparation of stable granular preparations containing such active peptides.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a hemp seed protein peptide, granules and a preparation method and application thereof for inhibiting α-glucosidase. The hemp seed protein peptide provided by the present invention has a very high α-glucosidase inhibition rate; the granules prepared by screening suitable auxiliary materials and proportions can ensure the stability of the hemp seed protein peptide during storage and the like, and the hemp seed protein peptide has a high transfer rate in the production process, a high granule forming rate and yield, a good taste when taken, and a good hypoglycemic effect.

[0009] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0010] The present invention provides a hemp seed protein peptide granule for assisting in lowering blood sugar, comprising the following components:

[0011] Hemp seed protein peptide powder, sugar-free auxiliary materials, flavoring agents and adhesives, wherein the mass ratio of the hemp seed protein peptide powder to the sugar-free auxiliary materials is 1:5-1:9, and the addition amount of the flavoring agent is 0.2%-0.8% of the total mass of the hemp seed protein peptide powder and the sugar-free auxiliary materials.

[0012] In a further embodiment, the mass ratio of the hemp seed protein peptide powder to the sugar-free auxiliary material is 1:6-1:8, preferably 1:7;

[0013] Preferably, the sugar-free auxiliary material is selected from one of isomalt, mannitol and maltitol, preferably maltitol.

[0014] In a further embodiment, the amount of the flavoring agent added is 0.4%-0.6% of the total mass of the hemp seed protein peptide powder and the sugar-free auxiliary material; preferably 0.5%;

[0015] Preferably, the flavoring agent is selected from one of strawberry flavor, lemon flavor and blueberry powder flavor, preferably blueberry powder flavor.

[0016] In a further embodiment, the amount of the adhesive added is 3.5%-4.5% of the total mass of the other components;

[0017] Preferably, the binder is water.

[0018] In a further embodiment, the preparation method of the hemp seed protein peptide powder comprises:

[0019] Take hemp seed meal, crush it, sieve it, add water to mix it, adjust the pH value, add protease, enzymolyze it at a certain temperature, inactivate the enzyme, centrifuge it, collect the supernatant, desalt it through a nanofiltration membrane, concentrate it through membrane filtration, and spray dry it to obtain hemp seed protein peptide powder.

[0020] In a further embodiment, the amount of the protease added is 0.1 wt% to 2.0 wt% of the mass of the hemp seed meal;

[0021] Preferably, the amount of protease added is 0.5wt% to 1.5wt% of the mass of the hemp seed meal; more preferably 1.0wt%;

[0022] Preferably, the protease is selected from one or more of alkaline protease 37071, alkaline protease AB8000, neutral protease AB7089, alkaline protease AY50C, trypsin, thermolysin, papain, bromelain, and composite protease;

[0023] Preferably, the protease is selected from one or more of composite protease, alkaline protease AB8000, alkaline protease AY50C, alkaline protease 37071, and neutral protease AB7089;

[0024] Preferably, the protease is selected from alkaline protease 37071.

[0025] In a further scheme, the pH value after adjustment is 7 to 11, the enzymatic hydrolysis temperature is 45 to 75°C, and the enzymatic hydrolysis time is 1 to 12 hours;

[0026] Preferably, the adjusted pH value is 7-9, the enzymolysis temperature is 45-65° C., and the enzymolysis time is 3-9 h.

[0027] The present invention also provides a preparation method of the hemp seed protein peptide particles as described above, comprising the steps of taking hemp seed protein peptide powder, sugar-free auxiliary materials and flavoring agents, crushing and sieving, mixing them in equal increasing amounts according to a certain proportion, adding them to a wet mixing granulator, mixing them evenly at a low speed, spraying an appropriate amount of adhesive, mixing at a high speed and stirring at a low speed to prepare a soft material, transferring them to a swing granulator to granulate, drying, and granulating to obtain formed particles.

[0028] The present invention also provides a hemp seed protein peptide for inhibiting α-glucosidase, comprising one or more polypeptides having amino acid sequences of FY, SPVI, TGLGR, and FR;

[0029] Preferably, it also includes one or more polypeptides having amino acid sequences of RL(I), FV, and AF.

[0030] The present invention also provides a use of the hemp seed protein peptide inhibiting α-glucosidase as described above in the preparation of health products or medicines for lowering blood sugar;

[0031] Preferably, the invention is used in the preparation of health products or medicines for assisting in lowering postprandial blood sugar.

[0032] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0033] 1. The present invention adopts an improved process, optimized protease, enzymolysis time, enzymolysis pH, enzymolysis temperature, etc., and the hemp seed protein peptide obtained by enzymolysis extraction of hemp seed meal has a very high α-glucosidase inhibition rate and has a significant blood sugar lowering effect.

[0034] 2. The hemp seed protein peptide particles prepared by the present invention can ensure the stability of the hemp seed protein peptide during storage and the like by screening suitable auxiliary materials and proportions, and the transfer rate of the hemp seed protein peptide in the production process is high, the loss of the hemp seed protein peptide is small, and all the enzymatic flavors of the hemp seed protein peptide itself can be well covered, the taste of taking is improved, and the compliance of the user is increased.

[0035] 3. The hemp seed protein peptide prepared by the present invention has most of the active polypeptides being small molecule peptides less than 1000Da, reaching more than 90%, which is more conducive to human digestion and absorption, and can be used in the preparation of health products or medicines for lowering blood sugar. Among them, a variety of polypeptides with high α-glucosidase inhibition rates cooperate with each other and work synergistically, making the hemp seed protein peptide have a significant blood sugar lowering effect.

[0036] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings are part of the present invention and are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings described below are only some embodiments. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the accompanying drawings:

[0038] Figure 1 This is a schematic diagram comparing the enzymatic hydrolysis effects of different proteases used in Experimental Example 2 of the present invention;

[0039] Figure 2 Schematic diagram of the effect of different enzymolysis times on the enzymolysis of hemp seed meal in Experimental Example 3 of the present invention;

[0040] Figure 3 Schematic diagram of the effect of different pH on the enzymatic hydrolysis of hemp seed meal in Experimental Example 3 of the present invention;

[0041] Figure 4 Schematic diagram of the effect of different temperatures on the enzymatic hydrolysis of hemp seed meal in Experimental Example 3 of the present invention;

[0042] Figure 5 Schematic diagram of the effect of different enzyme addition amounts on the enzymatic hydrolysis of hemp seed meal in Experimental Example 3 of the present invention;

[0043] Figure 6 is a schematic diagram of the molecular weight distribution of the enzymatic hydrolysis product in Experimental Example 5;

[0044] Figure 7 is the α-glucosidase inhibition curve of the enzymatic hydrolyzate in Experimental Example 5;

[0045] Figure 8 is the α-glucosidase inhibition rate of different components in Test Example 6;

[0046] Fig. 9 is the mass proportion of different components in Test Example 6;

[0047] Fig.10 This is the active peptide screening process in Experimental Example 6;

[0048] Fig.11 is an interaction diagram of α-glucosidase (2QMJ) and polypeptides (a) FY, (b) FR, (c) TGIGR, (d) IAF, (e) SPVI, and (f) INPLL in Experimental Example 6;

[0049] Fig.12 It is the α-glucosidase inhibition rate of the six peptides synthesized in vitro in Experimental Example 6.

[0050] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but are intended to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0052] In vitro α-glucosidase inhibitory activity assay

[0053] The α-glucosidase inhibition rate was determined on a 96-well microplate. First, 50 μL of 0.1M phosphate buffer (pH 6.8) was added, followed by 20 μL of enzymatic solution and 10 μL of α-glucosidase (1 U / mL), and incubated at 37°C for 10 min. After incubation, 20 μL of 5mM p-nitrobenzene-α-glucoside solution (pNPG) was added and incubated at 37°C for 20 min. Finally, 50 μL of 1M sodium carbonate was added to stop the reaction, and the absorbance was measured at 405 nm. All tests were performed three times and the average value was taken.

[0054] α-glucosidase inhibition rate = [1-(OD A -OD B ) / (OD0-OD C )]×100%

[0055] Among them, OD A OD B , OD0 and OD C Represent the absorbance value of the sample group, the absorbance value of the sample background, the absorbance value of the no sample group, and the absorbance value of the no sample blank group.

[0056] Example 1 Preparation of Hemp Seed Protein Peptide Powder

[0057] Preparation method:

[0058] The hot-pressed hemp seed meal was crushed and passed through a 60-mesh sieve, and then weighed to 50 g; 250 mL of water was added to the sieved hemp seed meal and mixed evenly, and the pH value was adjusted to 8, and then 0.25 g of alkaline protease 37071 was added, and the mixture was enzymolyzed at 45°C for 3.5 h, and the enzyme was inactivated in a 90°C water bath for 15 min. After centrifugation at 4°C and 8000 r / min for 15 min, the supernatant was taken, desalted by a nanofiltration membrane, concentrated by membrane filtration, and spray-dried to obtain hemp seed protein peptide powder.

[0059] Example 2 Preparation of Hemp Seed Protein Peptide Powder

[0060] Preparation method:

[0061] The hot-pressed hemp seed meal was crushed, passed through an 80-mesh sieve and weighed to 50 g; 1000 mL of water was added to the sieved hemp seed meal and mixed evenly, the pH value was adjusted to 9, 0.5 g of alkaline protease 37071 was added, and the mixture was enzymolyzed at 55° C. for 6 h, inactivated in a 100° C. water bath for 15 min, centrifuged at 4° C. and 8000 r / min for 10 min, and the supernatant was freeze-dried to obtain freeze-dried powder.

[0062] Example 3 Preparation of Hemp Seed Protein Peptide Powder

[0063] Preparation method:

[0064] The hot-pressed hemp seed meal was crushed, passed through an 80-mesh sieve and weighed to 50 g; 1000 mL of water was added to the sieved hemp seed meal and mixed evenly, the pH value was adjusted to 9, and 0.5 g of alkaline protease 37071 was added, and the mixture was enzymolyzed at 55°C for 6 h, inactivated in a 100°C water bath for 15 min, and centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant was taken, desalted by a nanofiltration membrane, concentrated by membrane filtration, and spray-dried to obtain hemp seed protein peptide powder.

[0065] Example 4 Preparation of Hemp Seed Protein Peptide Powder

[0066] Preparation method:

[0067] The hot-pressed hemp seed meal was crushed, passed through a 100-mesh sieve, and weighed to 50 g; 750 mL of water was added to the sieved hemp seed meal and mixed evenly, the pH value was adjusted to 10, and 0.75 g of alkaline protease 37071 was added, and the mixture was enzymolyzed at 65° C. for 1 h, inactivated in a 95° C. water bath for 15 min, and centrifuged at 4° C. and 8000 r / min for 10 min to obtain the supernatant, desalted through a nanofiltration membrane, concentrated by membrane filtration, and spray-dried to obtain hemp seed protein peptide powder.

[0068] Example 5 Preparation of Hemp Seed Protein Peptide Powder

[0069] Preparation method:

[0070] The hot-pressed hemp seed meal was crushed, passed through a 60-mesh sieve and weighed to 50 g; 500 mL of water was added to the sieved hemp seed meal and mixed evenly, the pH value was adjusted to 10, and 0.5 g of alkaline protease 37071 was added, and the mixture was enzymolyzed at 55° C. for 3 h, inactivated in a 100° C. water bath for 15 min, and centrifuged at 4° C. and 8000 r / min for 15 min to obtain the supernatant, desalted through a nanofiltration membrane, concentrated by membrane filtration, and spray-dried to obtain hemp seed protein peptide powder.

[0071] Example 6 Preparation of Particles Containing Hemp Protein Peptide

[0072] Prescription: 0.5 Kg of hemp seed protein peptide powder (prepared in Example 1), 3.48 Kg of maltitol and 0.02 Kg of blueberry powder flavor.

[0073] Preparation method: blueberry powder essence, hemp seed protein peptide powder and maltitol are mixed in equal amounts until all materials are mixed completely, and then added to a LHSZ20 type granulation wet mixing granulator, mixed at a low speed of 100 rpm, and 143.5g (3.59%) purified water is slowly sprayed in 4 minutes as a binder, mixed at a high speed of 200 rpm, and stirred at a low speed of 100 rpm for 1 minute to obtain a soft material for standby use. The soft material is transferred to a Yk-160 type swing granulator, granulated with a 14-mesh screen, and wet granules are collected. The CT-C-0 type hot air circulation oven is used to dry at 60°C for 30 minutes, and the dry granules are collected, and granulated with a 10-mesh and 60-mesh screen to obtain molded granules.

[0074] Example 7 Preparation of Granules Containing Hemp Protein Peptide

[0075] Prescription: 0.5 Kg of hemp seed protein peptide powder (prepared in Example 3), 4.0 Kg of maltitol and 0.0225 Kg of blueberry powder flavor.

[0076] Preparation method: blueberry powder essence, hemp seed protein peptide powder and maltitol are mixed in equal increments until all materials are mixed completely, added to LHSZ20 type granulation wet mixing granulator, mixed at a low speed of 100 rev / min, slowly sprayed with 180.9g (4.0%) purified water as a binder within 4min, mixed at a high speed of 200 rev / min, stirred at a low speed of 100 rev / min for 1min, and soft material is obtained for standby use. The soft material is transferred to Yk-160 type swing granulator, granulated with a 14 mesh sieve, and wet granules are collected. Use CT-C-0 type hot air circulation oven to dry at 60°C for 30min, collect dry granules, granulate with 10 mesh and 60 mesh sieves, and obtain molded granules.

[0077] Experimental Example 1 Comparison of enzymatic hydrolysis effects of different raw materials

[0078] Using shelled hemp seed meal and protein extracted therefrom, dehulled hemp seed meal and protein extracted therefrom as raw materials, alkaline protease 37071 was used for enzymolysis, and the conditions were solid-liquid ratio of 1:10, pH 8.5, temperature 55°C, enzyme addition amount 1%, enzymolysis for 6 hours, and other conditions such as enzyme inactivation and centrifugation were referred to Example 1, and the protein recovery rate and inhibition rate of the enzymolysis solutions of different raw materials were compared. The enzymolysis results of different raw materials are shown in Table 1.

[0079] Table 1 Enzymatic hydrolysis effects of different raw materials

[0080]

[0081] As shown in Table 1, although the protein extracted from hemp seed meal as raw material has a higher protein content, the inhibitory activity of its hydrolyzate is not as high as that of the direct hemp seed meal hydrolyzate. The possible reasons are: on the one hand, the composition of the seed meal system is more complex, and polyphenols and polysaccharides may also play an inhibitory role; on the other hand, the shelled seed meal has undergone many reactions after high-temperature hot pressing, and its composition is more complex than that of the dehulled seed meal. Therefore, the shelled hemp seed meal was selected for the next step of enzymatic optimization research.

[0082] Experimental Example 2 Screening of different types of enzymes

[0083] Alkaline protease 37071, alkaline protease AB8000, neutral protease AB7089, alkaline protease AY50C, trypsin, thermolysin, papain, bromelain, composite protease and other enzymes were selected, and enzymolysis was carried out under the optimal conditions of substrate concentration of 10%, enzyme addition amount of 0.5%, pH and temperature for various enzymes. After 3 hours of enzymolysis, the samples were taken out, the enzyme was inactivated at 100°C for 10 to 15 minutes, and then the supernatant was obtained by centrifugation to determine the protein recovery rate and α-glucosidase inhibition rate of the enzymatic hydrolysis products.

[0084] The protein recovery rate and α-glucosidase inhibition rate of different protease hydrolysates are shown in Figure 1 .Depend on Figure 1 It can be seen that the protein recovery rate obtained after enzymatic hydrolysis by AY50C and 37071 was significantly higher (p<0.05) than that of other enzymes. From the perspective of α-glucosidase inhibition, alkaline protease 37071 had the best effect, reaching 80.90%. The inhibition rate was ranked from large to small as alkaline protease 37071>complex protease>AY50C>AB7089>AB8000>thermolysin>bromelain>papain>trypsin. Considering the protein recovery rate, α-glucosidase inhibition rate and production cost control, alkaline protease 37071 was selected as the best enzyme system, and its enzymatic hydrolysis process was further optimized.

[0085] Experimental Example 3 Single Factor Experiment on Enzymatic Hydrolysis of Hemp Seed Meal

[0086] In the present test examples, alkaline protease NS37071 was used for enzymatic hydrolysis, and the enzyme was inactivated at 100°C for 10-15 minutes and then centrifuged to obtain the supernatant.

[0087] (1) Effect of enzymatic hydrolysis time on protein recovery rate and α-glucosidase inhibition rate of enzymatic hydrolysis products

[0088] The substrate concentration was selected as 10%, the pH was adjusted to 8.0, the enzyme addition amount was 0.50%, and the hemp seed meal was enzymolyzed at a hydrolysis temperature of 55°C for 1h, 3h, 6h, 9h, and 12h, respectively. After the enzymolysis was completed, the protein recovery rate and α-glucosidase inhibition rate of the enzymolysis product were determined.

[0089] Effects of different enzymatic hydrolysis times on protein recovery and α-glucosidase inhibitory activity of enzymatic hydrolysates Figure 2 The effects of different enzymatic hydrolysis times on the protein recovery rate and α-glucosidase inhibitory activity of the enzymatic hydrolysates are shown in Figure 2 As shown in the figure, with the extension of enzymolysis time, both the α-glucosidase inhibition rate and the protein recovery rate have a trend of first rising and then falling. When the enzymolysis time is 6h, the protein recovery rate and the α-glucosidase inhibition rate both reach a high level, which are 55.46% and 82.51% respectively. When the enzymolysis time is greater than 6h, both key indicators have a trend of slowly decreasing. Therefore, considering the two α-glucosidase inhibition rates and protein recovery rates, it is preferred to select enzymolysis for 3h to 9h as the basis for the next step of optimization, and 6h is more preferred.

[0090] (2) Effect of enzymatic hydrolysis pH on protein recovery and α-glucosidase inhibition rate of enzymatic hydrolysis products

[0091] The substrate concentration was selected as 10%, the enzyme addition amount was 0.50%, the enzymatic hydrolysis temperature was 55°C, the pH was adjusted to 7.0, 8.0, 9.0, 10.0, and 11.0 respectively, and the hemp seed meal was enzymatically hydrolyzed for 3 hours. After the enzymatic hydrolysis was completed, the protein recovery rate and α-glucosidase inhibition rate of the hemp seed meal enzymatic hydrolysis product were determined.

[0092] like Figure 3 As shown in the figure, when the enzymatic pH is 7-9, the protein recovery rate of the enzymatic hydrolysate increases rapidly with the increase of the enzymatic pH. When the pH is 9.0, the protein recovery rate of the enzymatic hydrolysate reaches 68.59%. When the enzymatic pH is 9-11, the growth trend of the protein recovery rate slows down, which may be because the appropriate alkaline environment is conducive to the dissolution of protein and the effective contact of protease. The inhibitory activity of the enzymatic hydrolysate gradually increases with the increase of pH when the pH is <8.0. When the enzymatic pH is 8.0, the inhibition of α-glucosidase reaches 87.93%. When the enzymatic pH is 9-11, the inhibition rate of α-glucosidase shows a downward trend and has certain fluctuations. At the same time, considering that the increase of the enzymatic pH will deepen the color of the enzymatic hydrolysate and increase the sodium ion content in the enzymatic hydrolysate, it is not conducive to subsequent experiments. Therefore, the enzymatic pH of 7-9 is selected as the basis for the next step of optimization.

[0093] (3) Effect of enzymatic hydrolysis temperature on protein recovery rate and α-glucosidase inhibition rate of enzymatic hydrolysis products

[0094] The substrate concentration was selected as 10%, the pH was adjusted to 8.0, the enzyme addition amount was 0.50%, and the hemp seed meal was enzymolyzed at 37°C, 45°C, 55°C, 65°C and 75°C for 3 hours. After the enzymolysis was completed, the protein recovery rate and α-glucosidase inhibition rate of the hemp seed meal enzymolysis product were determined.

[0095] Effects of different enzymatic hydrolysis temperatures on protein recovery and inhibitory activity of enzymatic hydrolysis products Figure 4 As shown. The enzymatic hydrolysis temperature mainly affects the activity of the protease and the structure and aggregation state of the substrate protein. With the increase of the enzymatic hydrolysis temperature, the protein recovery rate tends to increase first and then decrease. The highest recovery rate is 54.61% at 55°C. From the perspective of α-glucosidase inhibition rate, when the enzymatic hydrolysis temperature is 45-75°C, the enzymatic hydrolysis products have high inhibitory activity. When the enzymatic hydrolysis temperature is 65°C, the α-glucosidase inhibition rate reaches 87.48%. However, there is no significant difference between different enzymatic hydrolysis temperatures (p>0.05). Therefore, considering the enzymatic hydrolysis effect and power consumption loss, 45°C to 65°C is preferred as the basis for the next step of enzymatic hydrolysis optimization.

[0096] (4) Effect of enzyme addition amount on protein recovery rate and α-glucosidase inhibition rate of enzymatic hydrolysis products

[0097] The substrate concentration was selected as 10%, the pH was adjusted to 8.0, the enzymatic hydrolysis temperature was 55°C, and the hemp seed meal was enzymatically hydrolyzed for 3 hours under the conditions of enzyme addition amounts of 0.10%, 0.60%, 1.00%, 1.50%, and 2.0%, respectively. After the enzymatic hydrolysis was completed, the protein recovery rate and α-glucosidase inhibition rate of the hemp seed meal enzymatic hydrolysis products were determined.

[0098] Depend on Figure 5It can be seen that the inhibitory activity of the enzymatic hydrolysate showed a trend of increasing first and then decreasing with the increase of the enzyme addition amount. When the enzyme addition amount was 1.00%, the α-glucosidase inhibition rate of the enzymatic hydrolysate was significantly higher (p<0.05) than other enzyme addition amounts, reaching 81.79%. This may be because with the increase of the enzyme addition amount, the degree of protein enzymatic hydrolysis continued to increase, and the active peptides were released. Therefore, moderate enzymatic hydrolysis can increase the inhibitory activity of the enzymatic hydrolysate, but excessive enzymatic hydrolysis causes the protein to be broken down into smaller peptides, and the physiological activity decreases instead. Ren et al. found that the hemp protein hydrolysate with a hydrolysis degree of 27.24% had a high α-glucosidase inhibitory activity, and the hydrolysate with a hydrolysis degree of 9.68% may be an activator of α-glucosidase. The protein recovery rate of the enzymatic hydrolysate continued to increase with the increase of the enzyme addition amount. When the enzyme addition amount was 0.10%, the protein recovery rate was 43.71%, and when the enzyme addition amount was increased to 2.00%, the protein recovery rate could reach 65.27%. Taking both into consideration, an enzyme addition amount of 0.50% to 1.50% was selected for further optimization.

[0099] Experimental Example 4 Orthogonal Optimization of Hemp Seed Meal Enzyme Hydrolysis Process

[0100] Taking protein recovery rate and α-glucosidase inhibition rate as indicators, the effects of pH value, temperature, time and enzyme addition amount were studied respectively. On the basis of single factor experiment, a four-factor three-level orthogonal experiment was designed. The factor-level orthogonal experiment table is shown in Table 2.

[0101] Table 2 Hemp seed meal enzymolysis factor-level orthogonal experiment table

[0102]

[0103] The orthogonal test results are shown in Table 3 below.

[0104] Table 3 Results of orthogonal experiment on enzymatic hydrolysis of hemp seed meal

[0105]

[0106]

[0107] It can be seen from the above table that the primary and secondary relationships of the factors affecting the enzymatic hydrolysis effect are: enzymatic hydrolysis pH > enzymatic hydrolysis temperature > enzyme addition > enzymatic hydrolysis time. The optimal scheme selected is A2B2C2D2. Among the experimental schemes that have been done, A2B1C2D3 and A3B3C2D1 have relatively high comprehensive scores. At the same time, scheme A2B2C2D2 is not involved in the 9 experimental schemes. Therefore, the three experimental schemes A2B1C2D3, A3B3C2D1 and A2B2C2D2 were repeated for experimental verification. The experimental results are shown in Table 4.

[0108] Table 4 Orthogonal experiment verification experimental results

[0109]

[0110] The protein recovery rate of scheme A2B1C2D3 was significantly lower (p<0.05) than that of the other two schemes, while there was no significant difference in the protein recovery rates of schemes A3B3C2D1 and A2B2C2D2. There was no significant difference in the α-glucosidase inhibition rate among the three enzymatic hydrolysis schemes (p>0.05). Taking into account the enzymatic hydrolysis effect and cost control, A2B2C2D2 was selected as the optimal enzymatic hydrolysis process, that is, the material-liquid ratio was 1:10, the enzymatic hydrolysis pH was 8.0, the enzymatic hydrolysis temperature was 55℃, the enzyme addition amount was 1.00%, and the enzymatic hydrolysis was 6h. At this time, the protein recovery rate was 57.71% and the α-glucosidase inhibition rate was 88.45%.

[0111] Experimental Example 5 Characterization and physicochemical properties analysis of hemp seed meal enzymatic hydrolysis products

[0112] The enzymatic hydrolyzate of hemp seed meal prepared in Example 2 was tested as follows:

[0113] (1) Determination of short peptide content in hemp seed meal enzymatic hydrolysis products

[0114] The trichloroacetic acid-soluble nitrogen method (TCA-NSI) was used to calculate the yield of short peptides. The specific method is as follows:

[0115] Accurately weigh about 0.5g of solid sample, place it in a small beaker, add 10mL of 10% trichloroacetic acid, stir magnetically, dissolve, dilute to a 50mL volumetric flask, let it stand for 30min, centrifuge at 4000rpm for 15min, take an appropriate amount of supernatant to determine the protein content, and calculate the short peptide yield of the hemp seed meal enzymatic hydrolysis product.

[0116]

[0117] (2) Calculation of protein content and degree of hydrolysis of hemp seed meal enzymatic hydrolysis products

[0118] The protein content of the enzymatic hydrolysis product was measured by referring to the Kjeldahl nitrogen determination method in the national standard.

[0119] The hydrolysis degree of the enzymatic solution was measured by formaldehyde titration method, and the calculation formula is as follows:

[0120] Degree of hydrolysis (DH) = ammonia nitrogen content in supernatant / total nitrogen content in raw material × 100%.

[0121] Results: The basic indicators of hemp seed meal enzymatic hydrolysis products such as protein recovery rate, hydrolysis degree, and short peptide yield are shown in Table 5.

[0122] Table 5 Basic indicators of enzymatic hydrolysis products

[0123]

[0124] As shown in Table 5, the yield of short peptides in the enzymatic hydrolysis products exceeds 90%, which indicates that most of the enzymatic hydrolysis products obtained after enzymatic hydrolysis are small molecular peptides, which is also confirmed by the results of peptide molecular weight distribution.

[0125] (3) Determination of molecular weight distribution of hemp seed meal enzymatic hydrolysis products

[0126] The molecular weight distribution of hemp seed meal enzymatic hydrolysate was determined by HPLC. The specific method is as follows: 5 mg / mL of hemp seed meal enzymatic hydrolysate was prepared and filtered through a 0.22 μm needle filter. The chromatographic column TSKgel 2000SWXL (7.8×300 mm, 250 mm) was selected, the detection wavelength was 220 nm, and the column temperature was 30°C. The mobile phase was configured as acetonitrile: water: trifluoroacetic acid = 20:80:0.01 (V / V), and the elution was isocratic at 0.5 mL / min. The standards are: bovine serum albumin (Mw 68000), cytochrome C (Mw 12384), aprotinin (Mw 6511.53), bacitracin (Mw 1422.69), glutathione (oxidized and reduced forms, Mw 612.63 and 307.32 respectively) and di-Gly (132.12). The molecular weight distribution of the enzymatic hydrolysis product was calculated by making a regression equation with the logarithm of the standard molecular weight as the ordinate and the elution time as the abscissa.

[0127] Results: The molecular weight distribution of hemp seed meal protein peptides is shown in Table 6 and Figure 6 As shown in the figure, after 6 hours of enzymatic hydrolysis, most of the large molecular weight fragments were hydrolyzed into small molecular weight peptides, with molecular weight <5kDa accounting for 99%, mainly concentrated in the <1kDa fragment, accounting for more than 90%.

[0128] Table 6 Molecular weight distribution of enzymatic hydrolysis products

[0129]

[0130] (4) Determination of amino acid composition of hemp seed meal enzymatic hydrolysis products

[0131] The amino acid composition of the enzymatic hydrolysate was determined according to the method of GB 5009.124-2016 "Food Safety Standard Determination of Amino Acids in Food". Results: The amino acid composition analysis of the enzymatic hydrolysate is shown in Table 7.

[0132] Table 7 Amino acid composition of hemp seed meal enzymatic hydrolysate

[0133]

[0134] As shown in Table 7, the amino acid composition of the hydrolyzed products is similar to that of the hemp protein mentioned earlier, among which Glu, Asp and Arg have the highest content, followed by Leu. The proportion of hydrophobic amino acids in the hydrolyzed products is 31.78%; correspondingly, the proportion of essential amino acids in the hydrolyzed products is 27.37%.

[0135] (5) IC50 of hemp seed meal enzymatic hydrolysis products 50 Calculation

[0136] The freeze-dried hemp seed meal hydrolysate obtained in Example 2 was used to prepare a series of sample solutions with a concentration gradient (40, 20, 10, 5, 2.5, 1.25, 0.625 mg / ml), and the inhibition rates were calculated. The IC values ​​of the hydrolysate were calculated by nonlinear curve fitting using GraphPad. 50 value.

[0137] Results: The results of α-glucosidase inhibition activity determination of hemp seed meal enzymatic hydrolysate were as follows Figure 7 The results showed that the α-glucosidase inhibitory activity increased with the increase of concentration, and there was a good dosing effect. The IC50 value obtained by computer fitting calculation was 2.755 mg / mL, which had a very good α-glucosidase inhibitory effect.

[0138] Experimental Example 6 Isolation, identification, activity screening and verification of hemp seed protein peptides

[0139] (1) Isolation of hemp seed protein peptides

[0140] Weigh an appropriate amount of hemp seed meal enzymatic hydrolyzate freeze-dried powder, prepare a sample solution with a mass concentration of 40 mg / mL, add an appropriate amount of food-grade cold anhydrous ethanol (4°C) dropwise to make the ethanol volume fraction 20%, mix well and place in a 4°C refrigerator for 1 hour, centrifuge at 8000rpm for 10 minutes at 4°C, and the precipitate component is recorded as E20. Add cold anhydrous ethanol to the supernatant component to make the ethanol volume fraction 40%, mix well and continue to place at 4°C for 1 hour and then centrifuge, and the resulting precipitate composition is E40. Similarly, 60% and 80% ethanol graded precipitation components E60, E80 and supernatant E supernatant are obtained respectively, and all components are concentrated at 55°C under reduced pressure to remove ethanol and freeze-dried for use.

[0141] The peptide component with the best α-glucosidase inhibition rate was screened out by ethanol classification, and the α-glucosidase inhibition rate of each component was analyzed. The results are as follows Figure 8 As shown, the mass proportion of different components is Fig. 9 shown.

[0142] from Figure 8The results show that with the increase of ethanol concentration, the inhibition rate of α-glucosidase of the components obtained after step-by-step separation first decreases and then increases. Among them, the inhibition rate of α-glucosidase of the E60 component is the lowest, only 29.22%; the α-glucosidase inhibition rate of the supernatant component is the highest, reaching 70.02%, and it is significantly different from other components (p<0.05), and by Fig. 9 It can be seen that the mass proportion of the supernatant component is also the highest, reaching more than 70%, and the supernatant component is selected as the best component for subsequent further mass spectrometry separation and identification.

[0143] (2) Mass spectrometry separation and identification of hemp seed protein peptides

[0144] The samples were separated and identified using Ultimate3000-QExative high-resolution liquid chromatography-mass spectrometry. The mobile phase consisted of 0.1% (v / v) formic acid aqueous solution (A) and acetonitrile (B), and the elution method was: 0-2min 95% A and 5% B, 2-32.00min 55% A and 45% B, 32-40.00min 10% A and 90% B, 40-50.00min 95% A and 5% B, the flow rate was 0.05ml / min, and the injection volume was 2μL. After the separated peptides were separated by liquid chromatography, they were split through the electrospray interface, and the ESI positive ion scanning mode was used, with a scanning range of 100-1500m / z. High-purity nitrogen was used as the nebulizer and drying gas, the drying temperature was 180℃, and the drying gas flow rate was 4.0L / min.

[0145] The content of a specific peptide was equal to the ratio of its peak area to the total peak area of ​​HPLC-MS × 100%.

[0146] After high-precision LC-MS identification of the supernatant components after classification, a total of 345 peptides were identified, of which 2-5 amino acids were the main peptides. According to the relative peak area of ​​each peptide, the amino acid characteristics of reported α-glucosidase inhibitory peptides, Peptide Ranker and ToxinPred scoring results, the open source software AutoDockVina was used to further screen peptides with α-glucosidase inhibitory activity. The screening process is as follows: Fig.10 The peptides with a relative content greater than 0.5% in the hemp seed protein peptides analyzed by mass spectrometry are shown in Table 8.

[0147] Table 8 Mass spectrometry analysis and identification results of active peptides

[0148]

[0149] From the analysis in Table 4, we can see that there are 49 short peptides with relative peak area greater than 0.5%, and the sum of peak area reaches 62%. Among them, RL(I), FV, and AF have the highest content, accounting for 5.17%, 4.43%, and 3.84%, respectively. Most of all peptides contain two amino acids, accounting for 61.2%, and there are 8 peptides containing three amino acids, accounting for 16.3%. From the perspective of amino acid composition, there are more peptides containing hydrophobic amino acids such as isoleucine, leucine, and phenylalanine.

[0150] (3) Screening of the activity of hemp seed protein peptides

[0151] The predicted scores of the peptide biological activities were ranked according to PeptideRaker, and the peptides with scores greater than 0.5 were screened out. The toxicity of the peptides was analyzed using the ToxinPred online tool.

[0152] Peptide Ranker is a free online tool developed by the Shields laboratory based on peptide and proteomics databases. It can predict the possibility of a peptide having biological activity. The full score is 1. The higher the score, the greater the possibility that the peptide has biological activity. It is generally believed that peptides with a prediction score greater than 0.5 have strong physiological activity. As shown in Table 9, there are 18 peptides with a prediction score greater than 0.5 among the 49 peptides, which were screened out for further analysis.

[0153] Table 9 Peptide Ranker prediction score table

[0154]

[0155]

[0156] So far, the structure-activity relationship of α-glucosidase inhibitory peptides is not completely clear, but through a large number of research statistical results, it is found that the α-glucosidase inhibitory effect of the peptide is closely related to the properties of the N-terminal and C-terminal amino acid residues of its peptide chain. The study found that the peptides with serine, threonine, tyrosine, lysine or arginine as the final N-terminal residue, proline at the second C-terminal, and alanine or methionine at the final C-terminal have good α-glucosidase inhibitory activity. Based on this, a statistical analysis was conducted on 49 relatively high-content peptides, and 11 peptides with the above structural characteristics, including SY, SL, IA, SPVI, SAG, TGLGR, KL, EA, TA, SR, and TLG, were screened for the next step of screening.

[0157] Peptides from natural sources may be toxic. It is important to evaluate their toxicity before studying the biological activity of peptides to ensure the safe use of active peptides. ToxinPred is an online tool for predicting or designing the toxicity of active peptides. It can also provide information such as the hydrophilicity, hydrophobicity and charge of peptides. The toxicity and other parameters of the 29 peptides screened above were further predicted, with the SVM value as an indicator, negative values ​​indicating non-toxicity and positive values ​​indicating toxicity. The prediction results are shown in the following table. As can be seen from Table 10, all 29 peptides are non-toxic and can be used in the food and pharmaceutical industries. At the same time, it can be seen from Table 10 that among all the peptides, FI has the highest hydrophobicity and SR has the highest hydrophilicity.

[0158] Table 10 Toxicity evaluation in ToxinPred online database

[0159]

[0160]

[0161] The molecular docking was performed using AutoDock Vina1.1.2 software. Before docking, the structure of hemp seed protein peptide was first drawn using ChemBiodraw and Chembio3D to minimize the energy of the peptide structure, and then molecular docking was performed using this structure. The X-ray crystal structure of α-glucosidase (PDB ID: 2QMJ) was downloaded from the PDB database, and the dehydration, hydrogenation, and charge balance operations of the receptor protein and ligand were completed in Auto Dock Tools (MGLTools-1.5.6). The docking results were analyzed using PyMol 1.7.2.1.

[0162] After molecular docking analysis, the binding energy of the positive control acarbose to α-glucosidase is -7.8kcal / moL. The peptides with a binding energy closer to -7.8kcal / moL are peptides with higher α-glucosidase inhibitory activity similar to acarbose. Molecular docking analysis was performed on the 29 peptides screened above, and their binding energies are shown in Table 11. Among them, 9 have a binding energy less than -6.9Kcal / moL, and 6 have a binding energy less than -7.0Kcal / moL, namely INPLL, SPVI, IAF, TGLGR, FR and FY. These peptides have similar hypoglycemic activity as acarbose.

[0163] Table 11 Results of further screening of molecular docking

[0164]

[0165] The molecular docking site analysis of the interaction between these six peptides and α-glucosidase was further performed. Fig.11, the binding sites and binding energies are shown in Table 12.

[0166] Table 12 Analysis of peptide binding sites to α-glucosidase

[0167]

[0168] As shown in Table 9, each peptide with a binding energy lower than -7.0 has at least three hydrogen bonds with α-glucosidase, and the bond length is short, the bond energy is large, and the binding is relatively tight. Therefore, these peptides were synthesized in vitro to verify their biological activity.

[0169] (4) Verification of 6 types of hemp seed peptides

[0170] The six hemp active peptides identified and screened were commissioned to a biological reagent company for solid phase synthesis and their α-glucosidase inhibitory activity was determined. The α-glucosidase inhibition rate of each synthetic peptide at different concentrations is shown in the figure below: Fig.12 shown.

[0171] Depend on Fig.12 The analysis shows that although 6 peptides with high α-glucosidase inhibitory activity were obtained through computer virtual screening, in vitro synthesis verification found that the inhibitory activities of INPLL and IAF were basically negligible, and among the remaining four peptides, FY had the best inhibitory activity, with an α-glucosidase inhibition rate of 30.66% at a concentration of 10 mg / mL, followed by SPVI, with an activity inhibition rate of 28.09% at a concentration of 10 mg / mL. The inhibitory activities of FR and TGLGR were relatively close, and their α-glucosidase inhibition rates were around 20% at a concentration of 10 mg / mL.

[0172] Test Example 7 Evaluation of the biological activity of hemp seed protein peptide

[0173] 1. Evaluation of the hypoglycemic activity of hemp seed protein peptides using cell models

[0174] (1) Construction of cell model and preparation of hemp seed protein peptide

[0175] The Caco-2 cells were revived, the medium was changed, and the cells were subcultured to the 5th to 15th generation, and the cell concentration was prepared to 2×10 5Cell / mL cell suspension, add 0.5mL of cell suspension to the upper chamber (Apical side, AP side) of the Transwell plate, add 1mL of cell culture medium to the lower chamber (Basolateral side, BL side), and the liquid levels of the inner and outer wells are roughly the same height. The Caco-2 cells were inoculated and the medium was changed once 72 hours later, and then changed once every 2 days, and the culture was continued for 21 days, so that the cells grew on the porous and permeable polycarbonate membrane to achieve fusion and juxtaposition, forming a polar cell monolayer, and expressing structural and functional characteristics similar to intestinal epithelial cells.

[0176] Preparation method of hemp seed protein peptide: hot-pressed hemp seed meal is crushed, passed through an 80-mesh sieve and weighed to 500 g; 5000 mL of water is added to the sieved hemp seed meal and mixed evenly, the pH value is adjusted to 8, 5 g of alkaline protease 37071 is added, constant temperature enzymolysis is performed at 55° C. for 3 h, the enzyme is inactivated in a 100° C. water bath for 15 min, and the supernatant is taken and freeze-dried to obtain freeze-dried powder after centrifugation at 4° C. and 8000 r / min for 10 min.

[0177] (2) Study on the inhibition of hemp seed protein peptide on the activities of sucrase and maltase in Caco-2 cells

[0178] Take the constructed cell model Transwell plate, discard the old culture medium of the cell plate, wash the cells repeatedly with PBS solution, add 250μL and 1000μL of PBS solution to the top side (AP side) and the bottom side (BL side), respectively, incubate at 37°C for 30min, and discard all the solution before conducting the experiment. The experiment set up a blank group, a negative control group, an experimental group (using the 28mmol / L sucrose solution of the negative control group to configure the hemp seed protein peptide to 5mg / mL, 2.5mg / mL, and 1mg / mL respectively), and a positive control group (using the sucrose solution of the negative control group to configure acarbose to 1mg / mL). Each group added solutions according to the following table:

[0179] Table 13

[0180] Group AP side BL side Blank group 250 μL PBS solution 1000 μL PBS solution Negative control group 250μL 28mmol / L sucrose solution 1000 μL PBS solution Experimental Group 250μL hemp seed protein peptide-sucrose solution of different concentrations 1000 μL PBS solution Positive control group 250 μL acarbose-glucose solution 1000 μL PBS solution

[0181] After the solution was added, it was placed in a 37°C incubator for incubation. At 120 minutes, receiving solutions were taken from the AP side and the BL side for testing, and the glucose content was tested using a glucose content detection kit to calculate the inhibition rate of hemp seed protein peptide on sucrase on Caco-2 cells.

[0182] The calculation formula is as follows:

[0183]

[0184] The 28mmol / L sucrose solution in the reaction was replaced with 28mmol / L maltose solution to carry out the experiment and calculate the inhibition rate of hemp seed protein peptide on maltase on Caco-2 cells.

[0185] Table 14 Inhibition rate of hemp seed protein peptide on sucrase and maltase on Caco-2 cells

[0186]

[0187] The experiment showed that hemp seed protein peptide has an inhibitory effect on sucrase on Caco-2 cells and can inhibit the conversion of sucrose on Caco-2 cells into glucose. The effect is dose-dependent, and the inhibitory effect on maltase is relatively weak. The inhibition rate of hemp seed protein peptide on sucrase and maltase is lower than that of the positive control acarbose.

[0188] (3) Study on the inhibition of glucose transport in Caco-2 cells by hemp seed peptide

[0189] Take the constructed cell model Transwell plate, discard the old culture medium of the cell plate, wash the cells repeatedly with PBS solution, add 250μL and 1000μL of PBS solution to the top side (AP side) and the bottom side (BL side), respectively, incubate at 37°C for 30min, and discard all the solution. The experiment set up a control group, an experimental group (the hemp seed protein peptide was configured to 5mg / mL, 2.5mg / mL, and 1mg / mL respectively with the 25mmol / L glucose solution of the control group) and a positive control group (acarbose was configured to 1mg / mL with the 25mmol / L glucose solution of the control group).

[0190] Each group added solutions according to the following table:

[0191] Table 15

[0192] Group AP side BL side Control group 250 μL 25 mmol / L glucose solution 1000 μL PBS solution Experimental Group 250μL hemp seed protein peptide-glucose solution of different concentrations 1000 μL PBS solution Positive control group 250 μL acarbose-glucose solution 1000 μL PBS solution

[0193] After the solution was added, it was placed in a constant temperature shaker at 37°C for incubation. At 120 minutes, the receiving solution was taken from the BL side for testing, and the glucose content was tested using a glucose content detection kit to calculate the inhibition rate of hemp seed protein peptide on the transport of high concentration glucose in Caco-2 cells.

[0194] The calculation formula is as follows:

[0195]

[0196] The 25mmol / L glucose solution in the reaction was replaced with 5mmol / L glucose solution to carry out the experiment, and the inhibition rate of hemp seed protein peptide on the transport of low concentration glucose in Caco-2 cells was calculated.

[0197] Table 16 Inhibition rate of hemp seed protein peptide on glucose transport in Caco-2 cells

[0198]

[0199] Experiments have shown that hemp seed protein peptide can inhibit the transport of glucose in Caco-2 cells in a dose-dependent manner under high glucose concentration conditions. The inhibitory effect of low-concentration hemp seed protein peptide is stronger under low glucose concentration conditions. The inhibition rate of hemp seed protein peptide on glucose transport under high and low glucose concentration conditions is better than that of the positive control acarbose.

[0200] Two experiments, the study on the inhibition of sucrase and maltase activity on Caco-2 cells by hemp seed protein peptides and the inhibition of glucose transport in Caco-2 cells, proved that hemp seed protein peptides can inhibit the activity of sucrase and maltase, reduce the decomposition of sucrose and maltose into glucose, and at the same time inhibit the transport of glucose in cells and reduce the absorption of glucose. Hemp seed protein peptides can comprehensively control the concentration of glucose in cells from two aspects, while the positive control acarbose mainly inhibits the activity of sucrase and maltase, has a stronger effect, and has no inhibitory effect on the transport of glucose in cells.

[0201] 2. Animal experimental model to evaluate the hypoglycemic activity of hemp seed protein peptide

[0202] (1) Animal model construction

[0203] Healthy male rats were given ordinary maintenance feed for 5 days of adaptation and fasting for 4 hours. Blood was collected from the inner canthus of the eye to measure the blood sugar value before glucose administration (i.e., 0 hour), and the blood sugar value at 0.5 and 2 hours after 2.5g / kgBW glucose administration as the basal value of the batch of animals. Five groups were divided according to the blood sugar levels at 0 and 0.5 hours, namely, 1 blank control group, 1 model control group and 3 dose groups, with 11 rats in each group. The blank control group was not treated, and the 3 dose groups were gavaged with different concentrations of the test samples (the test samples were the hemp seed protein peptides prepared in 1 (1) of Test Example 7), and the model control group was given the same volume of pure water. Each group was fed with maintenance feed. After 1 week, the model control group and the 3 dose groups were replaced with high-energy feed. After feeding for 3 weeks, the model control group and the 3 dose groups were fasted for 24 hours (water was not allowed), and 104mg / kgBW of alloxan was intraperitoneally injected, with an injection volume of 1ml / 100g body weight. After the injection, the high-energy feed was continued for 5 days. At the end of the experiment, the animals in each group fasted for 4 hours, and their fasting blood glucose, glucose tolerance, serum pancreatic islet, cholesterol and triglyceride levels were tested.

[0204] Glucose tolerance index: Under the premise of model establishment, when the test sample dosage group is compared with the model control group, there is a statistically significant decrease in blood sugar (or an increase in the percentage of blood sugar decrease) at any time point of 0.5 or 2 hours after the administration of glucose or medical starch, or there is a statistically significant decrease in the area under the blood sugar curve at 0, 0.5, and 2 hours, and the test sample glucose tolerance index result is determined to be positive.

[0205] Blood lipid index: Under the premise of model establishment, the serum cholesterol or triglyceride of the test sample dosage group decreased significantly compared with the model control group, and the blood lipid lowering index of the test sample can be determined to be positive.

[0206] Normal healthy rats: 20 healthy adult rats were selected and divided into test sample group (high dose) and blank control group according to the blood glucose level after fasting for 4 hours, with 10 rats in each group. The sample group was given 25.0 mg / mL concentration sample solution, with an intragastric volume of 1 mL / 100 g BW, and the blank control group was given an equal volume of pure water, intragastricly once a day for 33 consecutive days, and fed with normal maintenance feed. The fasting blood glucose of the rats was measured before and after the experiment (fasting for 4 hours).

[0207] (2) Effect of hemp seed protein peptide on rat body weight

[0208] The initial body weight, mid-term body weight, final body weight and weight gain of rats in each dose group of the hyperglycemia model animal sample were compared with those in the model control group, and there were no significant differences (P>0.05), as shown in Table 17; the initial body weight, mid-term body weight, final body weight and weight gain of rats in the normal animal experimental group were compared with those in the blank control group, and there were no significant differences (P>0.05), as shown in Table 18. The results showed that hemp seed protein peptide had no significant effect on the weight gain of hyperglycemia model rats and normal rats.

[0209] Table 17 Effect of hemp seed protein peptide on body weight of hyperglycemia model rats

[0210]

[0211] Table 18 Effect of hemp seed protein peptide powder on body weight of normal rats

[0212]

[0213] (3) Effect of hemp seed protein peptide on blood glucose level in hyperglycemic rats

[0214] As can be seen from Table 19, there were no significant differences in the 0h (fasting), 0.5h, and 2h blood glucose and the area under the blood glucose curve among the rats in each group before the experiment (P>0.05).

[0215] Table 19 Results of glucose tolerance test of rats in each group before the test

[0216]

[0217] As can be seen from Table 20, when the experiment was finished, the 0h (fasting), 0.5h, 2h blood sugar and blood sugar area under the curve of the model control group were higher than each dosage group and the blank group, and 0.5h and 2h blood sugar values>10mmol / L, 0, 0.5h, 2h blood sugar and blood sugar area under the curve and the blank control group had a very significant difference (P<0.01), indicating that the hyperglycemia model was established. The 0h (fasting), 0.5h, 2h blood sugar and blood sugar area under the curve values ​​of each dosage group of the sample were all less than the model control group, and there were significant differences (P<0.05 or P<0.01), indicating that the glucose tolerance test result of hemp seed protein peptide was positive.

[0218] Table 20 Results of glucose tolerance test of rats in each group at the end of the experiment

[0219]

[0220] (4) Effects of hemp seed protein peptide on blood lipids and insulin levels in hyperglycemic rats

[0221] As shown in Table 21, at the end of the experiment, the serum TG and TC of the model control group were higher than those of the blank control group, and the difference between the two groups of TC was significant (P<0.05), indicating that the lipid metabolism disorder model was established; the serum TG and TC of each dose group of the sample were lower than those of the model control group, and the difference between the serum TC of two dose groups and the model control group was significant (P<0.05 or P<0.01), indicating that hemp seed protein peptide has the effect of reducing blood lipids in hyperglycemic rats. There was no significant difference in insulin between each dose group and the model control group (P>0.05), while the insulin resistance index values ​​of the model control group were higher than those of each dose group and the blank control group, and the difference was significant (P<0.01), indicating that the insulin resistance sugar\lipid metabolism disorder model was established, and suggesting that hemp seed protein peptide can inhibit the production of insulin resistance in hyperglycemic rats.

[0222] Table 21 Blood lipid and insulin content test results of each group of rats at the end of the experiment

[0223]

[0224] (5) Effect of hemp seed protein peptide on fasting blood glucose in normal rats

[0225] As can be seen from Table 22, when the sample was orally administered to normal rats at a dose of 250 mg / kg BW for 33 days, there was no significant difference in the fasting blood glucose values ​​of the rats compared with the blank control group (P>0.05), indicating that the sample had no significant effect on the fasting blood glucose of normal rats.

[0226] Table 22 Fasting blood glucose results of normal rats

[0227]

[0228] (6) Summary of Animal Experiments

[0229] Hemp seed protein peptide was given to hyperglycemic model rats by oral gavage at 50, 125, and 250 mg / kg BW for 33 consecutive days. After glucose was given, the 0h (fasting), 0.5h, and 2h blood glucose and the area under the blood glucose curve of the rats were all lower than those of the model control group, and there were significant differences (P<0.05 or P<0.01), indicating that the glucose tolerance test results of hemp seed protein peptide were positive. The serum TC of rats was lower than that of the model control group (P<0.05); it had no effect on the weight gain of rats and could inhibit the production of insulin resistance; the sample with a dose of 250 mg / kg BW was given to normal rats by oral gavage for 33 consecutive days, which had no effect on the weight gain and fasting blood glucose values ​​of rats. In summary, hemp seed protein peptide has the effect of assisting in lowering blood sugar.

[0230] Experimental Example 8 Screening of conditions for the preparation of hemp seed protein peptide particles

[0231] The hemp seed protein peptide powder used in this test example was prepared according to the method in 1(1) of Test Example 7.

[0232] 1. Selection of auxiliary materials

[0233] Hemp seed protein peptide powder is an extract made by spray drying after enzymatic hydrolysis of hemp seed eggs. It has high solubility and is very hygroscopic, so auxiliary materials must be added to make granules to ensure its stability during storage. The function of hemp seed protein peptide granules is to assist in lowering blood sugar. The auxiliary materials should be easily soluble in water and suitable for people with high blood sugar. Sugar-free auxiliary materials such as isomalt, mannitol, and maltitol are selected. Take 1 part of hemp seed protein peptide and mix it with 9 parts of auxiliary materials, spray an appropriate amount of adhesive to make a soft material, and then granulate it. Inspect the soft material and granule formability and granule yield to select the best auxiliary materials.

[0234] Table 23 Screening of excipient types of hemp seed protein peptide granules

[0235]

[0236] The experimental results show that when maltitol is selected as the auxiliary material of hemp seed protein peptide granules, the soft material and granule forming properties and granule yield are better than isomalt and mannitol. Maltitol is the optimal auxiliary material for hemp seed protein peptide granules.

[0237] 2. Screening of auxiliary material ratio

[0238] Use maltitol as an auxiliary material, mix hemp seed protein peptide powder: auxiliary material ratio of 1:5-1:9, add appropriate amount of purified water as a binder to make a soft material, then granulate, examine the particle yield and molding rate, taste the sweetness of the obtained particles, and select the optimal auxiliary material ratio.

[0239] Table 24 Screening of auxiliary material ratios of hemp seed protein peptide granules

[0240]

[0241] During the screening of auxiliary material ratios, when the ratio is 1:5, if the amount of adhesive added is too much, the soft material will be too wet to be granulated, and if the amount of adhesive added is insufficient, the soft material will be loose and not formed. The amount of adhesive added ranges from 3.34% to 3.64%, which is extremely difficult to control. When the ratio is 1:6-1:9, the amount of adhesive added can be adjusted to make the soft material easy to form, and the prepared granules have good formability, are compact, and are not easy to break. When the ratio is 1:7, the amount of adhesive added ranges from 3.5% to 4.5%, and the adjustable range is large, which is easier to control in the industrial production process. Combined with the screening of particle yield, forming rate and taste sweetness, 1:7 is preferably selected as the auxiliary material ratio of hemp seed protein peptide granules.

[0242] 3. Selection of flavoring agent types and proportions

[0243] Hemp seed protein peptide has an enzymatic flavor, and the flavor is not good after being made into granules and dissolved. A flavoring agent must be added to improve the flavor. Strawberry flavor, lemon flavor, and blueberry powder flavor are selected, and the flavor is dissolved in water at a concentration of 0.5% and 1%. The lemon flavor is pungent, the strawberry flavor is strong, and the experience is not good. The blueberry powder flavor has the best flavor, and the blueberry powder flavor is selected to investigate the addition amount. Take the blueberry powder flavor, prepare hemp seed protein peptide granules at a concentration of 0.05%, 0.5%, and 1%, dissolve it in 150mL hot water, and smell its flavor.

[0244] The sample with 0.05% blueberry powder flavor has a very light fragrance, and the fragrance is lost too much during the drying process. The sample with 1% blueberry powder flavor has a strong fragrance and a slight aroma stimulation. The sample with 0.5% blueberry powder flavor has a fresh and pleasant fragrance without stimulation, which can well cover up all the enzymatic flavors of the hemp seed protein peptide itself. Therefore, the preferred amount of blueberry powder flavor added is 0.5%.

[0245] Table 25 Fragrance ratio screening

[0246]

[0247] 4. Process scale-up

[0248] Weigh 0.5Kg of hemp seed protein peptide powder, 3.48Kg of maltitol and 0.02Kg of blueberry powder essence, pulverize, and pass 80 mesh sieves for standby use.Blueberry powder essence, hemp seed protein peptide powder, maltitol are mixed in equal increments, until all materials are mixed completely, join LHSZ20 type granulation wet mixing granulator, with 100 rev / min low speed mixing, slowly spray 143.5g (3.59%) purified water as adhesive in 4min, with 200 rev / min high speed mixing, 100 rev / min low speed stirring 1min, make soft material, standby use.Soft material is transferred to Yk-160 type swing granulator, granulate with 14 mesh sieves, collect wet granules.Dry with CT-C-0 type hot air circulation oven with 60 ℃ of drying for 30min, collect dry granules, granulate with 10 mesh and 60 mesh sieves, make molded particles.

[0249] The protein content in the prepared 6 batches of hemp seed protein peptide particles was detected, and the transfer rate of hemp seed protein peptide was calculated. The results are shown in Table 26.

[0250] Table 26 Transfer rate of hemp seed protein peptide in production process

[0251]

[0252] It can be seen from the results in Table 26 that in the production process, the transfer rate of hemp seed protein peptide is between 98% and 103%, the loss of hemp seed protein peptide is small, and the hemp seed protein peptide granule preparation process is very stable.

[0253] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with the present invention can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A hemp seed protein peptide granule for assisting in lowering blood sugar, characterized in that: Includes the following ingredients: Hemp seed protein peptide powder, sugar-free auxiliary materials, flavoring agents and adhesives, wherein the mass ratio of the hemp seed protein peptide powder to the sugar-free auxiliary materials is 1:5-1:9, and the addition amount of the flavoring agent is 0.2%-0.8% of the total mass of the hemp seed protein peptide powder and the sugar-free auxiliary materials.

2. The hemp seed protein peptide granules according to claim 1, characterized in that The mass ratio of the hemp seed protein peptide powder to the sugar-free auxiliary material is 1:6-1:8, preferably 1:7; Preferably, the sugar-free auxiliary material is selected from one of isomalt, mannitol and maltitol, preferably maltitol.

3. The hemp seed protein peptide granules according to claim 1 or 2, characterized in that The amount of the flavoring agent added is 0.4%-0.6% of the total mass of the hemp seed protein peptide powder and the sugar-free auxiliary material; preferably 0.5%; Preferably, the flavoring agent is selected from one of strawberry flavor, lemon flavor and blueberry powder flavor, preferably blueberry powder flavor.

4. The hemp seed protein peptide granules according to any one of claims 1 to 3, characterized in that The amount of the adhesive added is 3.5%-4.5% of the total mass of the other ingredients; Preferably, the binder is water.

5. The hemp seed protein peptide granules according to any one of claims 1 to 4, characterized in that The preparation method of the hemp seed protein peptide powder comprises: Take hemp seed meal, crush it, sieve it, add water to mix it, adjust the pH value, add protease, enzymolyze it at a certain temperature, inactivate the enzyme, centrifuge it, collect the supernatant, desalt it through a nanofiltration membrane, concentrate it through membrane filtration, and spray dry it to obtain hemp seed protein peptide powder.

6. The hemp seed protein peptide granules according to claim 5, characterized in that The added amount of the protease is 0.1wt% to 2.0wt% of the mass of the hemp seed meal; Preferably, the amount of protease added is 0.5wt% to 1.5wt% of the mass of the hemp seed meal; more preferably 1.0wt%; Preferably, the protease is selected from one or more of alkaline protease 37071, alkaline protease AB8000, neutral protease AB7089, alkaline protease AY50C, trypsin, thermolysin, papain, bromelain, and composite protease; Preferably, the protease is selected from one or more of composite protease, alkaline protease AB8000, alkaline protease AY50C, alkaline protease 37071, and neutral protease AB7089; Preferably, the protease is selected from alkaline protease 37071.

7. The hemp seed protein peptide granules according to claim 5 or 6, characterized in that After adjustment, the pH value is 7-11, the enzymatic hydrolysis temperature is 45-75°C, and the enzymatic hydrolysis time is 1-12h; Preferably, the adjusted pH value is 7-9, the enzymolysis temperature is 45-65° C., and the enzymolysis time is 3-9 h.

8. A method for preparing hemp seed protein peptide particles as described in any one of claims 1 to 7, characterized in that: Take hemp seed protein peptide powder, sugar-free auxiliary materials and flavoring agents, crush and sieve, mix them in a certain proportion in equal increasing amounts, add them to a wet mixing granulator, mix them evenly at a low speed, spray an appropriate amount of adhesive, mix them at a high speed and stir them at a low speed to make a soft material, transfer them to a swing granulator to granulate, dry them, and granulate them to obtain formed particles.

9. A hemp seed protein peptide that inhibits α-glucosidase, characterized in that: One or more polypeptides having amino acid sequences of FY, SPVI, TGLGR, and FR; Preferably, it also includes one or more polypeptides having amino acid sequences of RL(I), FV, and AF; Preferably, the preparation method of the hemp seed protein peptide powder comprises: Take hemp seed meal, crush it, sieve it, add water to mix it, adjust the pH value, add protease, enzymolyze it at a certain temperature, inactivate the enzyme, centrifuge it, collect the supernatant, desalt it through a nanofiltration membrane, concentrate it through membrane filtration, and spray dry it to obtain hemp seed protein peptide powder.

10. Use of the hemp seed protein peptide that inhibits α-glucosidase as claimed in claim 9 in the preparation of health products or medicines for lowering blood sugar; Preferably, the invention is used in the preparation of health products or medicines for assisting in lowering postprandial blood sugar.

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