Preparation method and application of cannabis sativa peptide with effects of reducing blood fat and adjusting lipid metabolism

Abstract

The invention discloses a preparation method and application of cannabis sativa peptide with effects of reducing blood fat and adjusting lipid metabolism, belongs to the technical field of biology, and has a wide application prospect. The fructus cannabis peptide obtained by the technical scheme and the preparation method disclosed by the invention has the following effects: 1, the fructus cannabis peptide prepared by the preparation method disclosed by the invention can be used for remarkably improving pancreatic lipase activity (the highest inhibition rate can reach 65.0%), so that digestion and absorption of a human body on lipid substances such as animal and vegetable oil and cholesterol are blocked; 2, the cannabis sativa peptide prepared by the preparation method disclosed by the invention can be used for remarkably improving the abnormality of four items of blood fat, reducing the level of total cholesterol, triglyceride and low-density lipoprotein and remarkably improving the level of high-density lipoprotein; 3, the prepared cannabis sativa peptide can obviously relieve non-alcoholic fatty liver disease caused by obesity and reduce liver fat accumulation; 4, the cannabis sativa peptide prepared by the invention can effectively relieve the occurrence and progress of obesity, reduce the weight change rate and reduce the obesity index, and has good lipid-lowering and weight-losing effects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of hemp peptide with the effects of lowering blood lipids and regulating lipid metabolism. Background Art

[0002] In contemporary society, reducing blood lipids and losing weight has evolved from a purely aesthetic need to a national health concern. With the rising incidence of metabolic diseases such as hyperlipidemia, obesity, and cardiovascular and cerebrovascular diseases, controlling blood lipid levels has become a crucial line of defense in preventing chronic diseases. Unlike traditional dieting, modern health concepts emphasize the dual goals of nutritional balance and metabolic regulation, providing new opportunities for the development of functional foods.

[0003] Hemp, an ancient cash crop in my country, has long been documented for its medicinal value in the Compendium of Materia Medica. Modern research has revealed that hemp meal, a byproduct of hemp seed physical pressing, is rich in high-quality protein. Hemp peptides, extracted through bio-enzymatic hydrolysis, have an amino acid profile that closely matches human needs, particularly with a significantly higher arginine content than other plant proteins. This unique amino acid profile not only provides essential nutritional support but also offers unique advantages in metabolic regulation. During lipid-lowering and weight-loss efforts, hemp peptides not only meet the body's need for increased protein intake but also improve the utilization of minerals like iron and zinc. Adequate mineral intake contributes to maintaining a healthy state of health. Furthermore, hemp peptides' biological activity in the body includes scavenging free radicals, enhancing insulin sensitivity, and promoting calorie consumption. These activities can also block the absorption of ingested carbohydrates.

[0004] At present, there are no public hemp peptide products and research related to lipid-lowering and weight loss on the market. Therefore, hemp lipid-lowering and weight loss products have great development potential and considerable market prospects. Its excellent nutritional value and activity can provide new options in the field of lipid-lowering and weight loss. Summary of the Invention

[0005] The present invention provides a preparation method and application of a hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism. The hemp peptide has excellent effects of lowering blood lipids and regulating lipid metabolism.

[0006] The present invention solves its technical problems by adopting the following technical solutions: A method for preparing hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism comprises the following steps: (1) subjecting the hemp meal to low-temperature air flow crushing and wall-breaking to obtain hemp meal powder; (2) Reflux degreasing the hemp meal powder with a 60-85 wt% ethanol solution to obtain defatted hemp meal; (3) adding cellulase, β-glucanase, and defatted hemp meal into water, performing enzymatic hydrolysis, and centrifuging to obtain a first enzymatic hydrolyzate; (4) adding alkaline protease and the first enzymatic hydrolysis solution into water, performing enzymatic hydrolysis to obtain a second enzymatic hydrolysis solution; (5) Add ammonium sulfate to the second enzymatic hydrolysis solution, perform ultrasonic salting out, homogenize, centrifuge, and collect the precipitate; (6) adding the precipitate, papain, and trypsin to water for enzymatic hydrolysis to obtain a third enzymatic hydrolyzate; (7) adding naringinase and tannase to the third enzymatic hydrolysis solution, performing enzymatic hydrolysis, and centrifuging to obtain a fourth enzymatic hydrolysis solution; (8) The fourth enzymatic hydrolyzate is subjected to reverse osmosis treatment, filtered, dried, and sterilized to obtain hemp peptide.

[0007] The present invention first performs low-temperature air flow wall breaking on hemp meal, reflux degreasing, then adopts cellulase and beta-glucanase as composite polysaccharide enzymes for hydrolysis, then uses bacterial protease (specifically alkaline protease) for hydrolysis, low-temperature salting out, homogenization, then hydrolyzes with composite protease (papain, trypsin), then removes bitterness with composite protease, reverse osmosis desalination, filtration, drying, and sterilization to obtain the hemp peptide. The present invention adopts a combination of multi-step enzymatic hydrolysis to obtain the hemp peptide with excellent blood lipid lowering and lipid metabolism regulating effects, and has broad application prospects.

[0008] As a preferred embodiment of the present invention, the hemp peptide has the following amino acid sequence: QQQPQPQPQQQQQQTGGR, TKTQQQLPPGPRPLPl, KGMTVGF, TQPAPAPVPVPVPVPVPVPVPFTSlPGDFY, AEMIPGK.

[0009] As a preferred embodiment of the present invention, the low-temperature airflow pulverizing and wall breaking is carried out under inert gas at a temperature of -22 to -18°C, and the pulverizing particle size is 100 to 600 meshes.

[0010] As a preferred embodiment of the present invention, the mass ratio of the hemp meal powder to the ethanol solution is 1:(4-10), the reflux time is 1-4 hours, and the reflux temperature is 70-80°C.

[0011] As a preferred embodiment of the present invention, the mass ratio of the cellulase, β-glucanase, defatted hemp meal and water is (0.5~2):(0.5~2):100:(400~1000).

[0012] As a preferred embodiment of the present invention, the mass ratio of the alkaline protease to the first enzymatic hydrolysis solution is (1-3):100.

[0013] As a preferred embodiment of the present invention, the mass ratio of ammonium sulfate to the second enzymatic hydrolysis solution is (2-4):100.

[0014] As a preferred embodiment of the present invention, the power of the ultrasound is 200-500W, the temperature of the salting-out is 2-6°C, and the time is 2-5h.

[0015] As a preferred embodiment of the present invention, the mass ratio of the precipitate, papain, trypsin, and water is 100: (0.5-1.2): (0.5-1.2): (400-1000); The mass ratio of the naringinase, tannase and the third enzymatic hydrolysate is (0.2-0.8): (0.2-0.8):100.

[0016] As a preferred embodiment of the present invention, the reverse osmosis treatment uses a CSM-RE2540-TE reverse osmosis membrane with a membrane outlet pressure of 1.8-2.2 MPa, a pump frequency of 40-60 Hz, and a material temperature of 10-25°C.

[0017] As a preferred embodiment of the present invention, the enzymatic hydrolysis temperature is 45-55° C. and the time is 2-6.

[0018] As a preferred embodiment of the present invention, the filtration in step (8) is carried out through a PVDF ultrafiltration membrane with a molecular weight cut-off of 1000 to 5000 Daltons.

[0019] As a preferred embodiment of the present invention, the sterilization temperature is 115-125° C. and the time is 20-40 minutes.

[0020] The present invention also provides a product having the effects of lowering blood lipids and regulating lipid metabolism, comprising the above-mentioned hemp peptide, and the product comprises a health food, a functional food or a medicine.

[0021] The beneficial effects of the present invention are as follows: the present invention first performs low-temperature air flow wall breaking on the hemp meal, reflux degreasing, then adopts cellulase and β-glucanase as composite polysaccharide enzymes for hydrolysis, then uses bacterial protease (specifically alkaline protease) for hydrolysis, low-temperature salting out, homogenization, then hydrolyzes with composite protease (papain, trypsin), then removes bitterness with composite protease, reverse osmosis desalination, filtration, drying, and sterilization to obtain the hemp peptide. The present invention adopts a multi-step enzymatic hydrolysis combination to obtain the hemp peptide with excellent lipid-lowering and lipid metabolism regulating effects, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 An indicator of obesity.

[0023] Figure 2 Four blood lipid tests.

[0024] Figure 3 Oil red O staining of the liver. DETAILED DESCRIPTION

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0026] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0027] In the present invention, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Furthermore, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0028] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0029] The reagents or instruments used in the present invention without indicating the manufacturer are all conventional products that can be obtained commercially. The raw materials used in the comparative examples and the raw materials used in the parallel experiments of the examples are the same commercially available products unless otherwise specified.

[0030] Example 1 A method for preparing hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism comprises the following steps: (1) Start the liquid nitrogen flow low-temperature pulverizer (M-250 deep-cold pulverizer) and pre-cool it to -20℃. Add the hemp meal into the liquid nitrogen flow low-temperature pulverizer and grind it to 400 mesh under liquid nitrogen.

[0031] (2) Hemp meal powder was refluxed and defatted using 80 wt% ethanol solution to obtain defatted hemp meal; the mass ratio of the hemp meal powder to the ethanol solution was 1:8, the reflux time was 3 h, and the reflux temperature was 75°C.

[0032] (3) Adding cellulase, β-glucanase, and defatted hemp meal to water, performing enzymatic hydrolysis at 48° C. for 4 h, and centrifuging at 5000 rpm for 10 min to obtain a first enzymatic hydrolyzate; the mass ratio of the cellulase, β-glucanase, defatted hemp meal, and water is 1:1:100:800.

[0033] (4) adding alkaline protease and the first enzymatic hydrolysis solution into water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a second enzymatic hydrolysis solution; the mass ratio of the alkaline protease, the first enzymatic hydrolysis solution, and water is 2.5:100:800.

[0034] (5) Add ammonium sulfate to the second enzymatic hydrolysate, perform salting out with 400W ultrasound, homogenize at 800 rpm for 10 min, centrifuge at 5000 rpm for 10 min, and collect the precipitate; the mass ratio of ammonium sulfate to the second enzymatic hydrolysate is 3:100.

[0035] (6) adding the precipitate, papain, and trypsin to water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a third enzymatic hydrolysis solution; the mass ratio of the precipitate, papain, trypsin, and water is 100:1.2:0.5:800; (7) Adding naringinase and tannase to the third enzymatic hydrolysis solution, enzymolyzing at 48°C for 4 hours, and centrifuging at 5000 rpm for 10 minutes to obtain a fourth enzymatic hydrolysis solution; the mass ratio of the naringinase, tannase, and the third enzymatic hydrolysis solution is 0.8:0.2:100.

[0036] (8) The fourth enzymatic hydrolyzate was subjected to reverse osmosis treatment, filtered through a PVDF ultrafiltration membrane with a molecular weight cutoff of 5000 Daltons (the filtrate was collected), dried to constant weight, and sterilized at 121°C for 30 min to obtain hemp peptide.

[0037] The reverse osmosis treatment used a CSM-RE2540-TE reverse osmosis membrane with a membrane outlet pressure of 2 MPa, a pump frequency of 50 Hz, a material temperature of 20° C., and concentrated to 30% of the original volume.

[0038] Example 2 A method for preparing hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism comprises the following steps: (1) Start the liquid nitrogen flow low-temperature pulverizer (M-250 deep-cold pulverizer) and pre-cool it to -20℃. Add the hemp meal into the liquid nitrogen flow low-temperature pulverizer and grind it to 400 mesh under liquid nitrogen.

[0039] (2) Hemp meal powder was refluxed and defatted using 80 wt% ethanol solution to obtain defatted hemp meal; the mass ratio of the hemp meal powder to the ethanol solution was 1:8, the reflux time was 3 h, and the reflux temperature was 75°C.

[0040] (3) Adding cellulase, β-glucanase, and defatted hemp meal into water, performing enzymatic hydrolysis at 48° C. for 4 h, and centrifuging at 5000 rpm for 10 min to obtain a first enzymatic hydrolyzate; the mass ratio of the cellulase, β-glucanase, defatted hemp meal, and water is 2:0.5:100:800.

[0041] (4) adding alkaline protease and the first enzymatic hydrolysis solution into water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a second enzymatic hydrolysis solution; the mass ratio of the alkaline protease, the first enzymatic hydrolysis solution, and water is 3:100:800.

[0042] (5) Add ammonium sulfate to the second enzymatic hydrolysate, perform salting out with 400W ultrasound, homogenize at 800 rpm for 10 min, centrifuge at 5000 rpm for 10 min, and collect the precipitate; the mass ratio of ammonium sulfate to the second enzymatic hydrolysate is 2:100.

[0043] (6) adding the precipitate, papain, and trypsin to water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a third enzymatic hydrolysis solution; the mass ratio of the precipitate, papain, trypsin, and water is 100:0.5:1.2:800; (7) Adding naringinase and tannase to the third enzymatic hydrolysis solution, enzymolyzing at 48°C for 4 hours, and centrifuging at 5000 rpm for 10 minutes to obtain a fourth enzymatic hydrolysis solution; the mass ratio of the naringinase, tannase, and the third enzymatic hydrolysis solution is 0.2:0.8:100.

[0044] (8) The fourth enzymatic hydrolyzate was subjected to reverse osmosis treatment, filtered through a PVDF ultrafiltration membrane with a molecular weight cutoff of 5000 Daltons (the filtrate was collected), dried to constant weight, and sterilized at 121°C for 30 min to obtain hemp peptide.

[0045] The reverse osmosis treatment used a CSM-RE2540-TE reverse osmosis membrane with a membrane outlet pressure of 2 MPa, a pump frequency of 50 Hz, a material temperature of 20° C., and concentrated to 30% of the original volume.

[0046] Example 3 A method for preparing hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism comprises the following steps: (1) Start the liquid nitrogen flow low-temperature pulverizer (M-250 deep-cold pulverizer) and pre-cool it to -20℃. Add the hemp meal into the liquid nitrogen flow low-temperature pulverizer and grind it to 400 mesh under liquid nitrogen.

[0047] (2) Hemp meal powder was refluxed and defatted using 80 wt% ethanol solution to obtain defatted hemp meal; the mass ratio of the hemp meal powder to the ethanol solution was 1:8, the reflux time was 3 h, and the reflux temperature was 75°C.

[0048] (3) Adding cellulase, β-glucanase, and defatted hemp meal to water, performing enzymatic hydrolysis at 48°C for 4 hours, and centrifuging at 5000 rpm for 10 minutes to obtain a first enzymatic hydrolyzate; the mass ratio of the cellulase, β-glucanase, defatted hemp meal, and water is 0.5:2:100:800.

[0049] (4) adding alkaline protease and the first enzymatic hydrolysis solution into water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a second enzymatic hydrolysis solution; the mass ratio of the alkaline protease, the first enzymatic hydrolysis solution, and water is 1:100:800.

[0050] (5) Add ammonium sulfate to the second enzymatic hydrolysate, perform salting out with 400W ultrasound, homogenize at 800 rpm for 10 min, centrifuge at 5000 rpm for 10 min, and collect the precipitate; the mass ratio of ammonium sulfate to the second enzymatic hydrolysate is 4:100.

[0051] (6) adding the precipitate, papain, and trypsin to water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a third enzymatic hydrolysis solution; the mass ratio of the precipitate, papain, trypsin, and water is 100:0.5:1.2:800; (7) Adding naringinase and tannase to the third enzymatic hydrolysis solution, enzymolyzing at 48°C for 4 hours, and centrifuging at 5000 rpm for 10 minutes to obtain a fourth enzymatic hydrolysis solution; the mass ratio of the naringinase, tannase, and the third enzymatic hydrolysis solution is 0.2:0.8:100.

[0052] (8) The fourth enzymatic hydrolyzate was subjected to reverse osmosis treatment, filtered through a PVDF ultrafiltration membrane with a molecular weight cutoff of 5000 Daltons (the filtrate was collected), dried to constant weight, and sterilized at 121°C for 30 min to obtain hemp peptide.

[0053] The reverse osmosis treatment used a CSM-RE2540-TE reverse osmosis membrane with a membrane outlet pressure of 2 MPa, a pump frequency of 50 Hz, a material temperature of 20° C., and concentrated to 30% of the original volume.

[0054] Comparative Example 1

[0055] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 is not enzymatically hydrolyzed by cellulase or β-glucanase, and all other aspects are the same.

[0056] A method for preparing hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism comprises the following steps: (1) Start the liquid nitrogen flow low-temperature pulverizer (M-250 deep-cold pulverizer) and pre-cool it to -20℃. Add the hemp meal into the liquid nitrogen flow low-temperature pulverizer and grind it to 400 mesh under liquid nitrogen.

[0057] (2) Hemp meal powder was refluxed and defatted using 80 wt% ethanol solution to obtain defatted hemp meal; the mass ratio of the hemp meal powder to the ethanol solution was 1:8, the reflux time was 3 h, and the reflux temperature was 75°C.

[0058] (3) Adding alkaline protease and defatted hemp meal into water, performing enzymolysis at 48°C for 4 hours, and obtaining a second enzymatic hydrolyzate; the mass ratio of the alkaline protease, defatted hemp meal, and water is 2.5:100:800.

[0059] (4) Add ammonium sulfate to the second enzymatic hydrolysate, perform salting out with 400W ultrasound, homogenize at 800 rpm for 10 min, centrifuge at 5000 rpm for 10 min, and collect the precipitate; the mass ratio of ammonium sulfate to the second enzymatic hydrolysate is 3:100.

[0060] (5) adding the precipitate, papain, and trypsin to water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a third enzymatic hydrolysis solution; the mass ratio of the precipitate, papain, trypsin, and water is 100:1.2:0.5:800; (6) Adding naringinase and tannase to the third enzymatic hydrolysis solution, enzymolyzing at 48°C for 4 hours, and centrifuging at 5000 rpm for 10 minutes to obtain a fourth enzymatic hydrolysis solution; the mass ratio of the naringinase, tannase, and the third enzymatic hydrolysis solution is 0.8:0.2:100.

[0061] (7) The fourth enzymatic hydrolyzate was subjected to reverse osmosis treatment, filtered through a PVDF ultrafiltration membrane with a molecular weight cutoff of 5000 Daltons (the filtrate was collected), dried to constant weight, and sterilized at 121°C for 30 min to obtain hemp peptide.

[0062] The reverse osmosis treatment used a CSM-RE2540-TE reverse osmosis membrane with a membrane outlet pressure of 2 MPa, a pump frequency of 50 Hz, a material temperature of 20° C., and concentrated to 30% of the original volume.

[0063] Comparative Example 2

[0064] The difference between Comparative Example 2 and Example 1 is that pectinase and amylase are used in Comparative Example 2 to replace cellulase and β-glucanase, and the other ingredients are the same.

[0065] A method for preparing hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism comprises the following steps: (1) Start the liquid nitrogen flow low-temperature pulverizer (M-250 deep-cold pulverizer) and pre-cool it to -20℃. Add the hemp meal into the liquid nitrogen flow low-temperature pulverizer and grind it to 400 mesh under liquid nitrogen.

[0066] (2) Hemp meal powder was refluxed and defatted using 80 wt% ethanol solution to obtain defatted hemp meal; the mass ratio of the hemp meal powder to the ethanol solution was 1:8, the reflux time was 3 h, and the reflux temperature was 75°C.

[0067] (3) Pectinase, amylase, and defatted hemp meal were added to water, and the mixture was enzymolyzed at 48° C. for 4 h, and centrifuged at 5000 rpm for 10 min to obtain a first enzymatic hydrolyzate; the mass ratio of the pectinase, amylase, defatted hemp meal, and water was 1:1:100:800.

[0068] (4) adding alkaline protease and the first enzymatic hydrolysis solution into water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a second enzymatic hydrolysis solution; the mass ratio of the alkaline protease, the first enzymatic hydrolysis solution, and water is 2.5:100:800.

[0069] (5) Add ammonium sulfate to the second enzymatic hydrolysate, perform salting out with 400W ultrasound, homogenize at 800 rpm for 10 min, centrifuge at 5000 rpm for 10 min, and collect the precipitate; the mass ratio of ammonium sulfate to the second enzymatic hydrolysate is 3:100.

[0070] (6) adding the precipitate, papain, and trypsin to water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a third enzymatic hydrolysis solution; the mass ratio of the precipitate, papain, trypsin, and water is 100:1.2:0.5:800; (7) Adding naringinase and tannase to the third enzymatic hydrolysis solution, enzymolyzing at 48°C for 4 hours, and centrifuging at 5000 rpm for 10 minutes to obtain a fourth enzymatic hydrolysis solution; the mass ratio of the naringinase, tannase, and the third enzymatic hydrolysis solution is 0.8:0.2:100.

[0071] (8) The fourth enzymatic hydrolyzate was subjected to reverse osmosis treatment, filtered through a PVDF ultrafiltration membrane with a molecular weight cutoff of 5000 Daltons (the filtrate was collected), dried to constant weight, and sterilized at 121°C for 30 min to obtain hemp peptide.

[0072] The reverse osmosis treatment used a CSM-RE2540-TE reverse osmosis membrane with a membrane outlet pressure of 2 MPa, a pump frequency of 50 Hz, a material temperature of 20° C., and concentrated to 30% of the original volume.

[0073] Comparative Example 3

[0074] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 is not treated with alkaline protease, and all other aspects are the same.

[0075] A method for preparing hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism comprises the following steps: (1) Start the liquid nitrogen flow low-temperature pulverizer (M-250 deep-cold pulverizer) and pre-cool it to -20℃. Add the hemp meal into the liquid nitrogen flow low-temperature pulverizer and grind it to 400 mesh under liquid nitrogen.

[0076] (2) Hemp meal powder was refluxed and defatted using 80 wt% ethanol solution to obtain defatted hemp meal; the mass ratio of the hemp meal powder to the ethanol solution was 1:8, the reflux time was 3 h, and the reflux temperature was 75°C.

[0077] (3) Adding cellulase, β-glucanase, and defatted hemp meal to water, performing enzymatic hydrolysis at 48° C. for 4 h, and centrifuging at 5000 rpm for 10 min to obtain a first enzymatic hydrolyzate; the mass ratio of the cellulase, β-glucanase, defatted hemp meal, and water is 1:1:100:800.

[0078] (4) Add ammonium sulfate to the first enzymatic hydrolysate, perform salting out with 400W ultrasound, homogenize at 800 rpm for 10 min, centrifuge at 5000 rpm for 10 min, and collect the precipitate; the mass ratio of the ammonium sulfate to the first enzymatic hydrolysate is 3:100.

[0079] (5) adding the precipitate, papain, and trypsin to water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a third enzymatic hydrolysis solution; the mass ratio of the precipitate, papain, trypsin, and water is 100:1.2:0.5:800; (6) Adding naringinase and tannase to the third enzymatic hydrolysis solution, enzymolyzing at 48°C for 4 hours, and centrifuging at 5000 rpm for 10 minutes to obtain a fourth enzymatic hydrolysis solution; the mass ratio of the naringinase, tannase, and the third enzymatic hydrolysis solution is 0.8:0.2:100.

[0080] (7) The fourth enzymatic hydrolyzate was subjected to reverse osmosis treatment, filtered through a PVDF ultrafiltration membrane with a molecular weight cutoff of 5000 Daltons (the filtrate was collected), dried to constant weight, and sterilized at 121°C for 30 min to obtain hemp peptide.

[0081] The reverse osmosis treatment used a CSM-RE2540-TE reverse osmosis membrane with a membrane outlet pressure of 2 MPa, a pump frequency of 50 Hz, a material temperature of 20° C., and concentrated to 30% of the original volume.

[0082] Comparative Example 4

[0083] The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 is not treated with papain or trypsin, and all other aspects are the same.

[0084] A method for preparing hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism comprises the following steps: (1) Start the liquid nitrogen flow low-temperature pulverizer (M-250 deep-cold pulverizer) and pre-cool it to -20℃. Add the hemp meal into the liquid nitrogen flow low-temperature pulverizer and grind it to 400 mesh under liquid nitrogen.

[0085] (2) Hemp meal powder was refluxed and defatted using 80 wt% ethanol solution to obtain defatted hemp meal; the mass ratio of the hemp meal powder to the ethanol solution was 1:8, the reflux time was 3 h, and the reflux temperature was 75°C.

[0086] (3) Adding cellulase, β-glucanase, and defatted hemp meal to water, performing enzymatic hydrolysis at 48° C. for 4 h, and centrifuging at 5000 rpm for 10 min to obtain a first enzymatic hydrolyzate; the mass ratio of the cellulase, β-glucanase, defatted hemp meal, and water is 1:1:100:800.

[0087] (4) adding alkaline protease and the first enzymatic hydrolysis solution into water, and performing enzymatic hydrolysis at 48°C for 4 hours to obtain a second enzymatic hydrolysis solution; the mass ratio of the alkaline protease, the first enzymatic hydrolysis solution, and water is 2.5:100:800.

[0088] (5) Add ammonium sulfate to the second enzymatic hydrolysate, perform salting out with 400W ultrasound, homogenize at 800 rpm for 10 min, centrifuge at 5000 rpm for 10 min, and collect the precipitate; the mass ratio of ammonium sulfate to the second enzymatic hydrolysate is 3:100.

[0089] (6) The precipitate, naringinase, and tannase were added to the second enzymatic hydrolysis solution, and the enzymatic hydrolysis was carried out at 48°C for 4 hours, and the solution was centrifuged at 5000 rpm for 10 minutes to obtain a fourth enzymatic hydrolysis solution; the mass ratio of the precipitate, naringinase, tannase, and the second enzymatic hydrolysis solution was 100:0.8:0.2:800.

[0090] (7) The fourth enzymatic hydrolyzate was subjected to reverse osmosis treatment, filtered through a PVDF ultrafiltration membrane with a molecular weight cutoff of 5000 Daltons (the filtrate was collected), dried to constant weight, and sterilized at 121°C for 30 min to obtain hemp peptide.

[0091] The reverse osmosis treatment used a CSM-RE2540-TE reverse osmosis membrane with a membrane outlet pressure of 2 MPa, a pump frequency of 50 Hz, a material temperature of 20° C., and concentrated to 30% of the original volume.

[0092] Test Case

[0093] 1. Enzyme inhibition test Dissolve 150 mg of p-nitrophenyl palmitate in 0.5 mL of DMSO and dilute to 15 mg / mL with PBS buffer (pH 7.4). Dissolve pancreatic lipase in PBS buffer to 20 mg / mL, centrifuge at 4000 rpm for 5 minutes, and remove the supernatant. Sample solutions of varying concentrations were prepared using PBS buffer. The entire reaction system was performed in a 96-well microplate with a total volume of 150 μL, consisting of 50 μL of sample solution (the hemp peptides from the Examples and Comparative Examples, respectively), 50 μL of enzyme solution, and 50 μL of substrate solution. After incubation at 37°C in a water bath for 40 minutes, the absorbance at 405 nm was measured (A1). A blank sample control group used buffer instead of the lipase solution, measuring the absorbance at A2. A control group used buffer instead of the test solution, measuring the absorbance at B1. A blank control group used buffer instead of the sample and lipase solutions, measuring the absorbance at B2. Calculate the inhibition rate of lipase: inhibition rate = [1-(A1-A2) / (B1-B2)] × 100%.

[0094] 2. Antioxidant performance: The scavenging rate of DPPH is in accordance with T / SHRH 006-2018 "Cosmetics - Free Radical (DPPH) Scavenging Test Method".

[0095] Table 1

[0096] As can be seen from Table 1, the hemp peptide of the present invention has excellent lipase inhibitory activity and anti-inflammatory effect, and can effectively block the digestion and absorption of lipid substances such as animal and vegetable oils and cholesterol by the human body.

[0097] 3. Animal Testing Forty-eight eight-week-old C57BL / 6 male mice were randomly divided into two groups, 12 in the first group and 36 in the second, based on weight balance. The first group, the control group (NC group), was fed a standard diet, while the second group was fed a high-fat diet for 8 consecutive weeks, until the weight difference between the control and high-fat groups was greater than 20%. The high-fat group was then randomly divided into three groups, 12 in each group: the model group (HFD group) (high-fat diet plus normal saline), the low-dose group (LHeP group) (high-fat diet plus 500 mg / kg hemp seed peptide), and the high-dose group (HHeP group) (high-fat diet plus 1000 mg / kg hemp seed peptide). The mice were gavaged daily with different concentrations of hemp seed peptide solution or normal saline for 6 consecutive weeks.

[0098] Analysis of obesity characteristics: Before sampling, body length and weight were measured, and Lee's and BMI indexes were selected. Figure 1As shown in the results, the Lee's index, BMI index, liver index, body weight and weight gain of the mice in the HFD group were significantly higher than those in the NC group (p < 0.01); compared with the HFD group, the Lee's index, BMI index and liver index of the mice in the LHeP group were significantly reduced (p < 0.05 or p < 0.01); the Lee's index, BMI index, liver index, body weight and weight gain of the mice in the HHeP group were significantly reduced (p < 0.05 or p < 0.01), which shows that hemp peptide has a good lipid-lowering and weight-loss effect.

[0099] Four blood lipid tests: Take the serum of each group of mice after centrifugation and measure serum total cholesterol (TC), serum total triglyceride (TG), serum low-density lipoprotein (LDL-C), and serum high-density lipoprotein (HDL-C). Figure 2 As shown in the results, the serum TC, TG, and LDL-C levels of mice in the HFD group were significantly increased compared with those in the NC group (p < 0.01), and the HDL-C level was significantly decreased (p < 0.01), indicating that mice fed a high-fat diet showed obvious symptoms of hyperlipidemia and lipid metabolism disorders; after intervention with hemp peptide, the serum TC, TG, and LDL-C levels of mice in the LHeP group and HHeP group were significantly decreased compared with those in the HFD group (p < 0.01); the HDL-C level was significantly higher than that in the HFD group (p < 0.01), which further indicates that hemp peptide can alleviate dyslipidemia and protect cardiovascular health by regulating lipid metabolism.

[0100] Liver Oil Red O staining: First, take the frozen sections out of the -20℃ refrigerator, place them at room temperature for a while, then fix them with 4% paraformaldehyde for 10 minutes, and then wash them 3 times with PBS. Then, soak the sections in 60% isopropanol for 5 minutes, and then stain them in Oil Red O working solution for 10-15 minutes, shaking them gently from time to time. After that, differentiate them with 60% isopropanol for a few seconds until the background is clear, and then wash away the isopropanol with PBS. Finally, counterstain the cell nuclei with hematoxylin for 3-5 minutes, wash with PBS, seal the sections with glycerol gelatin, let them dry, and then observe and photograph them under a microscope. Figure 3 As shown, the livers of mice in the high-fat group showed severe hepatocellular fatty degeneration, with a large number of lipid droplets of varying sizes accumulating in the hepatocytes (orange-red areas). Due to the accumulation of fat, the hepatocyte volume increased. In contrast, the liver fat vacuoles of mice in the low- and high-dose hemp peptide groups were significantly reduced, and the size of hepatocytes was significantly reduced. This suggests that hemp peptide can alleviate non-alcoholic fatty liver disease by reducing the abnormal accumulation of lipids in the liver.

[0101] Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing hemp peptide with the effects of lowering blood lipids and regulating lipid metabolism, characterized in that: The following steps are involved: (1) subjecting the hemp meal to low-temperature air flow crushing and wall-breaking to obtain hemp meal powder; (2) Reflux degreasing the hemp meal powder with a 60-85 wt% ethanol solution to obtain defatted hemp meal; (3) adding cellulase, β-glucanase, and defatted hemp meal into water, performing enzymatic hydrolysis, and centrifuging to obtain a first enzymatic hydrolyzate; (4) adding alkaline protease and the first enzymatic hydrolysis solution into water, performing enzymatic hydrolysis to obtain a second enzymatic hydrolysis solution; (5) Add ammonium sulfate to the second enzymatic hydrolysis solution, perform ultrasonic salting out, homogenize, centrifuge, and collect the precipitate; (6) adding the precipitate, papain, and trypsin to water for enzymatic hydrolysis to obtain a third enzymatic hydrolyzate; (7) adding naringinase and tannase to the third enzymatic hydrolysis solution, performing enzymatic hydrolysis, and centrifuging to obtain a fourth enzymatic hydrolysis solution; (8) The fourth enzymatic hydrolyzate is subjected to reverse osmosis treatment, filtered, dried, and sterilized to obtain hemp peptide.

2. The method for preparing the hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism according to claim 1, wherein: The hemp peptide has the following amino acid sequence: QQQPQPQPQQQQQQTGGR, TKTQQQLPPGPRPLPl, KGMTVGF, TQPAPAPVPVPVPVPVPVPVPFTSlPGDFY, AEMIPGK.

3. The method for preparing the hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism according to claim 1, wherein: The low-temperature airflow crushing and wall breaking is carried out under inert gas at a temperature of -22 to -18°C, and the crushing particle size is 100 to 600 meshes; The mass ratio of the hemp meal powder to the ethanol solution is 1:(4-10), the reflux time is 1-4 hours, and the reflux temperature is 70-80°C.

4. The method for preparing the hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism according to claim 1, wherein: The mass ratio of the cellulase, beta-glucanase, defatted hemp meal and water is (0.5-2): (0.5-2): 100: (400-1000).

5. The method for preparing the hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism according to claim 1, wherein: The mass ratio of the alkaline protease to the first enzymatic hydrolysis solution is (1-3):

100.

6. The method for preparing the hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism according to claim 1, wherein: The mass ratio of the ammonium sulfate and the second enzymatic hydrolysis solution is (2-4):

100.

7. The method for preparing the hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism according to claim 1, wherein: The power of the ultrasound is 200-500W, the temperature of the salting-out is 2-6°C, and the time is 2-5h.

8. The method for preparing the hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism according to claim 1, wherein: The mass ratio of the precipitate, papain, trypsin and water is 100: (0.5-1.2): (0.5-1.2): (400-1000); The mass ratio of the naringinase, tannase and the third enzymatic hydrolysate is (0.2-0.8):(0.2-0.8):

100.

9. The method for preparing the hemp peptide having the effects of lowering blood lipids and regulating lipid metabolism according to claim 1, wherein: The reverse osmosis treatment uses a CSM-RE2540-TE reverse osmosis membrane with a membrane outlet pressure of 1.8-2.2 MPa, a pump frequency of 40-60 Hz, and a material temperature of 10-25°C.

10. A product with the effects of lowering blood lipids and regulating lipid metabolism, characterized in that: The product comprises the hemp peptide according to any one of claims 1 to 9, wherein the product comprises a health food, a functional food or a medicine.

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