Fructus cannabis cake meal functional peptide as well as preparation method and application thereof

Abstract

The invention discloses fructus cannabis cake meal functional peptide as well as a preparation method and application thereof, and belongs to the technical field of fructus cannabis processing. In order to solve the technical problems that in the prior art, the utilization rate, safety and digestibility of fructus cannabis cakes are low, the fructus cannabis cake functional peptide and the preparation method thereof are provided, through high-temperature extrusion puffing treatment and a two-step precipitation method, the extraction rate and digestibility of protein of the fructus cannabis cake functional peptide are increased, and the yield of the fructus cannabis cake functional peptide is increased. And the content of toxic substances is reduced. The fructus cannabis cake meal functional peptide provided by the invention has the functions of remarkably enhancing the phagocytic function of mouse mononuclear-macrophages, promoting spleen and thymus development of mice, improving the immune factor level in mice, reducing the inflammatory factor level in mice, promoting intestinal digestion and protecting intestinal tracts. The fructus cannabis cake meal functional peptide provided by the invention can be applied to immunosuppressive mouse in-vivo regulation and functional food preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of hemp seed processing, and particularly relates to a hemp seed cake functional peptide and a preparation method and application thereof. Background Art

[0002] Hemp seed is the fruit of hemp, which contains 20%-25% protein, 20%-35% oil, 20%-30% carbohydrates and rich minerals. At present, the use of hemp seed is mostly limited to the extraction of oil. After cold pressing hemp seed to extract oil, a large amount of by-products - hemp seed cake will be produced, which is usually used as animal feed or directly discarded, thus causing waste of resources and environmental pollution. Compared with other oils, hemp seed cake has a higher content of phytic acid. Phytic acid is an anti-nutrient that affects the digestion and absorption of peptides in hemp seed cake by animals by inhibiting the activity of proteases; in addition, the tetrahydrocannabinol contained in hemp can cause neurological disorders, unconsciousness, anxiety, depression, and vascular cell inflammation.

[0003] In order to solve the resource waste and environmental pollution caused by the discarding of a large amount of hemp seed cake in the prior art, the high content of phytic acid in hemp seed cake affects its digestion and absorption, and the technical problems that tetrahydrocannabinol in hemp can cause diseases; and the hemp seed polypeptides prepared by the existing process mostly only involve the effects of lowering blood pressure and inhibiting the activity of α-amylase and lipase, and are not disclosed to have the effect of regulating immunosuppression.

[0004] Those skilled in the art are eager to develop a hemp seed cake functional peptide with high digestibility and high safety and a preparation method thereof, so as to improve the utilization rate of hemp seed cake. Summary of the invention

[0005] The invention aims to solve the technical problems of low utilization rate, safety and digestion and absorption rate of hemp seed cake in the prior art, and provides a hemp seed cake functional peptide and a preparation method and application thereof.

[0006] One of the purposes of the present invention is to provide a method for preparing hemp seed cake functional peptide, the preparation method comprising the following steps:

[0007] S1: crushing hemp seed cake, adding organic solvent to defatting, and drying to obtain defatted hemp seed cake;

[0008] S2: The defatted hemp seed cake obtained in S1 is crushed, sieved, and then put into a twin-screw extruder, and water is added to perform high-temperature extrusion and expansion treatment to obtain an extrudate;

[0009] S3: Cooling, drying, crushing, and sieving the extrudate obtained in S2, and subjecting the extrudate obtained by the above-mentioned treatment to a two-step precipitation method to obtain hemp seed cake protein;

[0010] S4: Cooling, drying, crushing and sieving the hemp seed cake protein obtained in S3, enzymolysis treatment of the hemp seed cake protein powder obtained by the above treatment, and then performing enzyme inactivation treatment to obtain hemp seed cake functional peptide enzymolysis liquid.

[0011] In a preferred embodiment of the present invention, the organic solvent described in S1 is any one of n-hexane, isopropanol, petroleum ether or anhydrous ethanol.

[0012] In a preferred embodiment of the present invention, the extrusion parameters of the twin-screw extruder in S2 are: moisture content of 30%, screw speed of 220r / min, feeding rate of 10kg / h, and barrel temperature in the extrusion and puffing zone of 120°C, 140°C, 160°C, 180°C or 200°C.

[0013] In a preferred embodiment of the present invention, the steps of the two-step precipitation method in S3 are:

[0014] S11: mixing the treated extrudate and distilled water in a solid-liquid ratio of 1:10 (g / mL), adjusting the pH value to 6 with HCl solution, stirring and centrifuging, and collecting the precipitate;

[0015] S22: Use NaOH solution to adjust the pH value of the precipitate obtained in S11 to 10, then place it in a water bath at 50°C for 1 hour, centrifuge it at 4000 r / min for 10 minutes, and collect the supernatant;

[0016] S33: adjusting the pH value of the supernatant obtained in S22 to 4.5 using HCl solution, centrifuging at 4000 r / min for 10 min, and collecting the precipitate;

[0017] S44: adjusting the pH value of the supernatant obtained in S22 to 3.6 using HCl solution, centrifuging at 4000 r / min for 10 min, and collecting the precipitate;

[0018] S55: The precipitates obtained in S33 and S44 are mixed evenly, distilled water is added and stirred evenly, the pH value is adjusted to 7 with a NaOH solution, and the hemp seed cake protein is obtained after freeze-drying.

[0019] In a preferred embodiment of the present invention, the step of enzymolysis treatment described in S4 is: the processed hemp seed cake protein and distilled water are mixed according to the solid-liquid ratio of 1: 10 (g / mL), boiled in a boiling water bath for 5min, cooled to room temperature, NaOH solution is used to adjust the pH value to 10, alkaline protease is added to the above-mentioned hemp seed cake protein for enzymolysis treatment for 3h, and a single enzyme hydrolyzate is obtained; Flavourzyme composite flavor protease is added to the above-mentioned single enzyme hydrolyzate for enzymolysis treatment for 1h, and after the enzymolysis is completed, the enzyme is deactivated in a boiling water bath for 15min, and the supernatant is taken at 4000r / min for centrifugation for 15min to obtain a hemp seed cake functional peptide enzymolyzate.

[0020] In a preferred embodiment of the present invention, the added amount of the alkaline protease is 1000 U / g.

[0021] In a preferred embodiment of the present invention, the added amount of the Flavourzyme compound flavor protease is 750 U / g.

[0022] The second object of the present invention is to provide a hemp seed cake functional peptide, which is obtained by the above-mentioned preparation method.

[0023] The third object of the present invention is to provide the application of the above-mentioned hemp seed cake functional peptide in the regulation of immunosuppressive mice.

[0024] A fourth object of the present invention is to provide the application of the above-mentioned hemp seed cake functional peptide in food preparation.

[0025] Beneficial effects of the present invention: The present invention provides a hemp seed cake functional peptide and a preparation method thereof, which reduces the contents of phytic acid, cannabidiol and tetrahydrocannabinol in the hemp seed cake functional peptide by high-temperature extrusion and puffing treatment, improves the function of protein content, and improves the extraction rate and digestibility of hemp seed cake protein in the hemp seed cake functional peptide by a two-step precipitation method. Toxicity tests have shown that the hemp seed cake functional peptide provided by the present invention has little effect on the dietary activity, growth and weight of mice, indicating that the hemp seed cake functional peptide has low toxicity and high safety. The hemp seed cake functional peptide provided by the present invention has the function of significantly enhancing the phagocytic function of mouse monocytes and macrophages, significantly restoring and promoting the development of mouse spleen and thymus, significantly improving the levels of IgA, IgG and INF-γ immune factors in mice, significantly reducing the levels of IL-2 and TNF-α inflammatory factors in mice, and promoting intestinal digestion and protecting the intestines; wherein, the hemp seed cake functional peptide provided by the present invention, in an experimental group of mice administered with high-dose oral administration, showed a more stable ability to restore the in vivo regulation of immunosuppressed mice compared with soy protein isolate and hemp seed cake functional peptide that has not been subjected to high extrusion and puffing treatment, and can be applied to the in vivo regulation of immunosuppressed mice and the preparation of functional foods. DETAILED DESCRIPTION

[0026] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0027] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. The test methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0028] Example 1: Effect of high temperature extrusion puffing treatment temperature on the properties of hemp seed cake powder

[0029] The hemp seed cake is crushed and passed through a 100-mesh sieve, and n-hexane organic solvent is added for degreasing and drying to obtain defatted hemp seed cake; the defatted hemp seed cake is crushed, sieved, and then put into a twin-screw extruder, and water is added for high-temperature extrusion and expansion treatment, wherein the extrusion parameters of the twin-screw extruder are: the moisture content is 30%, the screw speed is 220r / min, and the barrel temperatures in the extrusion and expansion zone are 120°C, 140°C, 160°C, 180°C and 200°C respectively, to obtain an extrudate, and the extrudate is placed in a 50°C oven for drying, crushed and passed through a 100-mesh sieve to obtain hemp seed cake powder.

[0030] In this embodiment, the hemp seed cake powder subjected to high-temperature extrusion and puffing treatment at 120°C, 140°C, 160°C, 180°C and 200°C was tested for phytic acid content, and the hemp seed cake powder not subjected to high-temperature extrusion and puffing treatment was used as the control group. The results are shown in Table 1. The phytic acid content in the hemp seed cake powder subjected to high-temperature extrusion and puffing treatment is significantly lower than that in the control group, and the protein content is significantly higher than that in the control group. It can be seen that the high-temperature extrusion and puffing treatment adopted in the present invention has the function of reducing the phytic acid content in the hemp seed cake powder and increasing the protein content.

[0031] In this embodiment, the hemp seed cake powder subjected to high-temperature extrusion and puffing treatment at 120°C, 140°C, 160°C, 180°C and 200°C was tested for the content of cannabidiol (CBD) and tetrahydrocannabinol. The results are shown in Table 1. When the extrusion temperature is 200°C, the content of tetrahydrocannabinol and cannabidiol in the hemp seed cake powder is sharply reduced, and the content is below the detection line, and the safety is relatively high.

[0032] Table 1

[0033] Sample name Phytic acid (g / 100g) Protein content (%) CBD content (mg / g) Tetrahydrocannabinol content (%) Extrusion temperature 120℃ 5.81±0.01 58.94±0.02 25.84±0.16 0.15±0.02 Extrusion temperature 140℃ 5.19±0.02 53.53±0.05 26.91±0.25 0.12±0.06 Extrusion temperature 160℃ 5.06±0.04 46.82±0.09 26.68±0.22 0.08±0.08 Extrusion temperature 180℃ 4.84±0.06 51.05±0.1 15.4±0.51 0.05±0.02 Extrusion temperature 200℃ 4.61±0.06 56.8±0.11 9.4±0.3 Below the detection line Control group 6.79±0.05 38.91±1.81 22.6±0.52 0.2±0.05

[0034] Example 2: Preparation of hemp seed cake protein by two-step precipitation method

[0035] The hemp seed cake is crushed and passed through a 100-mesh sieve, and n-hexane organic solvent is added for degreasing, and drying is performed to obtain defatted hemp seed cake; the defatted hemp seed cake is crushed, sieved, and then put into a twin-screw extruder, and water is added for high-temperature extrusion expansion treatment, wherein the extrusion parameters of the twin-screw extruder are: a moisture content of 30%, a screw speed of 220 r / min, and a barrel temperature in the extrusion expansion zone of 200° C. to obtain an extrudate; the extrudate is cooled, dried, crushed, and sieved, and the extrudate obtained by the above-mentioned treatment is subjected to a two-step precipitation process to obtain hemp seed cake protein;

[0036] The steps of the two-step precipitation method are:

[0037] S11: the treated extrudate is mixed with distilled water at a solid-liquid ratio of 1:10 (g / mL), the pH value is adjusted to 6 using 1.0 mol / L HCl solution, the mixture is stirred and centrifuged, and the precipitate is collected;

[0038] S22: The pH value of the precipitate obtained in S11 was adjusted to 10 using 1.0 mol / L NaOH solution, and then placed in a water bath at 50°C for 1 h, centrifuged at 4 000 r / min for 10 min, and the supernatant was collected;

[0039] S33: adjusting the pH value of the supernatant obtained in S22 to 4.5 using 1.0 mol / L HCl solution, centrifuging at 4 000 r / min for 10 min, and collecting the precipitate;

[0040] S44: The pH value of the supernatant obtained in S22 was adjusted to 3.6 using 1.0 mol / L HCl solution, centrifuged at 4 000 r / min for 10 min, and the precipitate was collected;

[0041] S55: The precipitates obtained in S33 and S44 are mixed evenly, and then distilled water is added and stirred evenly. The pH value is adjusted to 7 using 1.0 mol / L NaOH solution, and the hemp seed cake protein is obtained after freeze-drying.

[0042] Example 3: Preparation of hemp seed cake functional peptides

[0043] The hemp seed cake is crushed and passed through a 100-mesh sieve, and n-hexane organic solvent is added for degreasing, and drying is performed to obtain defatted hemp seed cake; the defatted hemp seed cake is crushed, sieved, and then put into a twin-screw extruder, and water is added for high-temperature extrusion expansion treatment, wherein the extrusion parameters of the twin-screw extruder are: a moisture content of 30%, a screw speed of 220 r / min, and a barrel temperature in the extrusion expansion zone of 200° C. to obtain an extrudate; the extrudate is cooled, dried, crushed, and sieved, and the extrudate obtained by the above-mentioned treatment is subjected to a two-step precipitation process to obtain hemp seed cake protein;

[0044] The steps of the two-step precipitation method are:

[0045] S11: mixing the treated extrudate and distilled water in a solid-liquid ratio of 1:10 (g / mL), adjusting the pH value to 6 with HCl solution, stirring and centrifuging, and collecting the precipitate;

[0046] S22: Use NaOH solution to adjust the pH value of the precipitate obtained in S11 to 10, then place it in a water bath at 50°C for 1 hour, centrifuge it at 4000r / min for 10 minutes, and collect the supernatant;

[0047] S33: adjusting the pH value of the supernatant obtained in S22 to 4.5 using HCl solution, centrifuging at 4000 r / min for 10 min, and collecting the precipitate;

[0048] S44: adjusting the pH value of the supernatant obtained in S22 to 3.6 using HCl solution, centrifuging at 4000 r / min for 10 min, and collecting the precipitate;

[0049] S55: the precipitates obtained in S33 and S44 are mixed evenly, distilled water is added and stirred evenly, the pH value is adjusted to 7 with a NaOH solution, and the hemp seed cake protein is obtained after freeze-drying;

[0050] S4: The hemp seed cake protein obtained in S3 is cooled, dried, crushed, and sieved, and the treated hemp seed cake protein is mixed with distilled water at a solid-liquid ratio of 1:10 (g / mL), boiled in a boiling water bath for 5 minutes, cooled to room temperature, and the pH value is adjusted to 10 using a NaOH solution. 1000U / g of alkaline protease is added to the above hemp seed cake protein for enzymatic hydrolysis for 3 hours to obtain a single enzyme hydrolyzate; 750U / g of Flavourzyme composite flavor protease is added to the above single enzyme hydrolyzate for enzymatic hydrolysis for 1 hour. After the enzymatic hydrolysis is completed, the enzyme is inactivated in a boiling water bath for 15 minutes, and the mixture is centrifuged at 4,000r / min for 15 minutes. The supernatant is taken to obtain a hemp seed cake functional peptide enzymatic hydrolyzate.

[0051] Comparative Example 1: Preparation of hemp seed cake protein by conventional one-time precipitation method

[0052] The hemp seed cake is pulverized through a 100 mesh sieve, and n-hexane organic solvent is added for degreasing, and oven dry is performed to obtain defatted hemp seed cake; the defatted hemp seed cake is pulverized, sieved, and then dropped into a twin screw extruder, and water is added for high-temperature extrusion expansion, wherein the extrusion parameters of the twin screw extruder are: a moisture content of 30%, a screw speed of 220 r / min, and a barrel temperature in the extrusion expansion zone of 200 ℃ to obtain an extrudate; the extrudate is cooled, dried, pulverized, and sieved, and the extrudate obtained by the above-mentioned treatment is mixed with distilled water according to a solid-liquid ratio of 1: 10 (g / mL), and the boiling water bath is cooled to room temperature after 5 min, and a 1.0 mol / L NaOH solution is used to adjust the pH value to 10, and hemp seed cake protein is obtained after freeze drying.

[0053] Comparative Example 2: Preparation of functional peptides from hemp seed cake without extrusion and puffing treatment

[0054] The difference between this comparative example and Example 3 is that no extrusion puffing treatment is performed in S2, and the other steps and parameters are the same as those in Example 3.

[0055] Effect experiment:

[0056] (1) The present invention tests the physical and chemical properties of the hemp seed cake protein prepared by the two-step precipitation method in Example 2 and the hemp seed cake protein prepared by the conventional one-time precipitation method in Comparative Example 1. The results are shown in Table 2. The hemp seed cake protein prepared by the two-step precipitation method improves the protein extraction rate by 3.78% and the protein digestibility by 1.93% compared with the conventional one-time precipitation method. It can be seen that the two-step precipitation method adopted in the present invention has the function of improving the extraction rate and digestibility of hemp seed cake protein.

[0057] Table 2

[0058] Group Extraction rate (%) Digestibility (%) Comparative Example 1 33.09±0.21 67.02±0.24 Example 2 36.87±0.05 68.95±1.13

[0059] (2) The present invention selects mice purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. as test subjects, and divides them into two groups, each with 8 mice, and conducts acute toxicity experiments on the hemp seed cake functional peptide prepared in Example 3 and the hemp seed cake functional peptide prepared in Comparative Example 2. The mice that have not been treated with medication are used as the control group. The mice are gavaged once in the morning and evening at a maximum volume dose of 0.4mL / 10g, and the maximum dosage is 0.5g / mL, which is 187 times the human dosage. Continuous observation for 14 days begins immediately, and the physiological state, mental state, etc. of the mice are observed every day, and the body weight of the mice is measured on the first and 14th days of medication.

[0060] As shown in Table 3, it can be seen from continuous observation for 14 days that the mice administered with the hemp seed cake functional peptide prepared in Comparative Example 2 by gavage showed symptoms such as diarrhea, mental depression, and withered hair at the moment of administration, and continued until the 14th day of administration, the mice still maintained the above-mentioned adverse state; while the mice administered with the hemp seed cake functional peptide prepared in Example 3 by gavage were in the same state as the control group mice at the moment of administration, and all performed normally in terms of diet, drinking water, and activity, and continued until the 14th day of administration, the mice still maintained a normal state.

[0061] As shown in Table 4, there was no significant difference in body weight between mice administered with the hemp seed cake functional peptide prepared in Example 3 and the control group of mice, while the body weight of mice administered with the hemp seed cake functional peptide prepared in Comparative Example 2 was significantly lower than that of mice in the control group and mice administered with the hemp seed cake functional peptide prepared in Example 3.

[0062] The hemp seed cake functional peptide prepared in Example 3 has higher safety than the hemp seed cake functional peptide prepared in Comparative Example 2, and has less effect on the normal growth of mice; it can be seen that the high-temperature extrusion puffing treatment adopted in the present invention has the function of improving the safety of hemp seed cake functional peptide.

[0063] Table 3

[0064]

[0065] Table 4

[0066] Group 1 day medication 14 days of medication Control group 18.33±2.44 28.48±1.22 Comparative Example 2 18.46±2.17 27.13±1.92 Example 3 18.82±2.05 28.26±1.86

[0067] Example 4: Application of hemp seed cake functional peptides in in vivo regulation of immunosuppressed mice

[0068] (1) Establishment of immunosuppressive mouse model

[0069] First modeling: All mice except the control group were given compound cyclophosphamide tablets by gavage at a dosage of 80 mg / kg for 3 consecutive days;

[0070] Second modeling: The second administration was given 7 days before the end of the experiment. All mice except the control group were intragastrically administered with compound cyclophosphamide tablets at a dosage of 80 mg / kg.

[0071] The experimental groups and dosing regimens are as follows:

[0072] In this embodiment, 60 ICR clean-grade mice successfully modeled for the first time were evenly divided into 6 groups, each group had 10 mice, half of which were male and half were female. Untreated mice were used as the model group, mice administered with normal saline by gavage at a dose of 5.2g / kg were used as the control group, mice administered with soy protein isolate by gavage at a dose of 5.2g / kg were used as the positive control group, mice administered with hemp seed cake functional peptide prepared by comparative example 2 by gavage at a dose of 5.2g / kg were used as the comparison group, and mice administered with hemp seed cake functional peptide prepared by gavage in Example 3 were used as the test group; wherein, the test group with a gavage dose of 3.6g / kg was used as the low-dose test group, and the test group with a gavage dose of 5.2g / kg was used as the high-dose test group, and each group was gavaged according to a dose of 0.2mL / 10g as the gavage; each group of mice was normally raised under standard conditions, and each group was continuously supported with nutrition for 21 days, during which a total of 5 weighings were performed, and the food intake was calculated every day.

[0073] (2) Detection of phagocytic function of monocytes and macrophages

[0074] In this example, 30 minutes after the last oral administration of the corresponding experimental sample to each group, the weight of each group of mice in (1) was weighed, and after weighing, each group of mice was injected with 20% Indian ink-containing physiological saline (injection dose of 0.1 mL / 10 g) through the tail vein. At 2 minutes and 20 minutes after the tail vein injection, 20 μL of blood was collected from the orbital venous plexus, and then added to 2 mL of 0.1% NaCO3 solution and mixed evenly. The above mixed solution was placed in a spectrophotometer at a wavelength of 680 nm, zeroed with 0.1% NaCO3, and the absorbance OD2 and OD20 values ​​were measured respectively. The clearance index K and phagocytosis index α are as follows:

[0075]

[0076] Where: t20 and t2 represent the minutes after intravenous injection of India ink, logOD2 and logOD20 represent

[0077] Table 2 and 20 minutes of blood sample optical density.

[0078]

[0079] The results are shown in Table 5. Compared with the control group, the clearance index and phagocytosis index of the mice in the model group were significantly reduced, indicating that cyclophosphamide caused a decrease in the immune function of the mice. Compared with the model group, the clearance index and phagocytosis index of the mice in the positive control group, the high-dose test group and the low-dose test group were restored to the level of the control group, and the clearance index and phagocytosis index of the mice in the above three groups were higher than those in the control group and the model group (P<0.05). Among them, the high-dose test group had the most significant effect in restoring the phagocytic function of mononuclear macrophages in immunocompromised mice. The clearance index and phagocytosis index of the mice in the control group were restored compared with those in the model group, but they did not return to the level of the control group.

[0080] It can be seen that the hemp seed cake functional peptide provided by the present invention has the ability to significantly enhance the phagocytic function of mouse monocytes and macrophages, and enhance the in vivo regulation ability of immunosuppressed mice, compared with soy protein isolate and hemp seed cake functional peptide that has not been extruded and expanded.

[0081] Table 5

[0082] Group Clearance index (K) Phagocytic index (α) Control group 0.039±0.012 5.06±1.54 Model Group <![CDATA[0.023±0.007 * ]]> <![CDATA[3.72±1.14 * ]]> Positive control group <![CDATA[0.052±0.014 # ]]> <![CDATA[5.47±1.36 * ]]> High dose test group <![CDATA[0.066±0.013 #* ]]> <![CDATA[6.74±1.29 * ]]> Low dose test group <![CDATA[0.049±0.014 # ]]> <![CDATA[6.01±1.47 * ]]> Comparison group 0.032±0.012 3.92±1.25

[0083] Note: Compared with the control group, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05.

[0084] (3) Detection of immune organ index

[0085] The immune organ index can reflect the state of the body's immune function to a certain extent, and the important immune organs of the body are the thymus and spleen, which are the sites for the generation, proliferation, differentiation and maturation of immune cells, and the sites for immune response. In this example, the mice in each group in (1) were killed by cervical dislocation, and the thymus and spleen of each group of mice were removed. The thymus index and spleen index are as follows:

[0086]

[0087] The results are shown in Table 6. Compared with the control group, the chest spleen index and spleen index of the mice in the model group were significantly decreased (P<0.01), indicating that cyclophosphamide caused damage to the immune organs of the mice; compared with the model group, the chest spleen index and spleen index of the mice in the high-dose test group and the low-dose test group were increased to the level of the control group, and the chest spleen index and spleen index of the above two groups of mice were higher than those in the control group, and significantly higher than those in the model group (P<0.01); while the chest spleen index and spleen index of the mice in the positive control group and the comparison group were increased compared with those in the model group, but not increased to the level of the control group.

[0088] It can be seen that the hemp seed cake functional peptide provided by the present invention has better inhibition of cyclophosphamide damage to immune organs, and has the ability to significantly restore and promote the development of mouse spleen and thymus, compared with soy protein isolate and hemp seed cake functional peptide that has not been extruded and expanded.

[0089] Table 6

[0090] Group Thymus index Spleen index Control group 0.181±0.058 0.373±0.116 Model Group <![CDATA[0.059±0.021 ** ]]> 0.296±0.106 Positive control group <![CDATA[0.112±0.033 ## ]]> 0.304±0.068 High dose test group <![CDATA[0.204±0.049 ## ]]> 0.375±0.067 Low dose test group <![CDATA[0.222±0.083 ## ]]> 0.390±0.128 Comparison group <![CDATA[0.146±0.044 ## ]]> 0.332±0.069

[0091] Note: Compared with the control group, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05.

[0092] (4) Detection of immune factors

[0093] In this example, 30 minutes after the last oral administration of the corresponding experimental sample to each group, the eyeballs of the mice in each group in (1) were removed for blood sampling, and the blood was centrifuged at 4°C and 2800 r / min for 10 minutes. The supernatant was collected and stored at -80°C for later use. The levels of immunoglobulins IgA, IgG, and INF-γ in the serum of each group of mice were measured according to the instructions of the ELISA kit (Shanghai ELISA Biotechnology Co., Ltd.).

[0094] As shown in Table 7, the IgA level in the serum of mice in the model group was significantly lower than that in the control group (P<0.01). Compared with the model group, the IgA levels in the serum of mice in the positive control group, high-dose test group and low-dose test group were all increased, but only the IgA level in the serum of mice in the high-dose test group was significantly higher than that in the control group, while the IgA levels in the serum of mice in the positive control group and the low-dose test group did not reach the level of the control group; the IgA level in the serum of mice in the comparison group was significantly lower than that in the control group, and significantly lower than that in the model group.

[0095] As shown in Table 7, the IgG level in the serum of mice in the model group was significantly lower than that in the control group (P<0.01). Compared with the model group, the IgG levels in the serum of mice in the positive control group, high-dose test group and low-dose test group were increased. The IgG levels in the serum of mice in the positive control group and high-dose test group were increased to the level of the control group. Among them, the IgG level in the serum of mice in the high-dose test group was significantly higher than that in the control group (P<0.05), while the IgG level in the serum of mice in the low-dose test group did not reach the level of the control group. The IgG level in the serum of mice in the comparison group was significantly lower than that in the control group and significantly lower than that in the model group.

[0096] As shown in Table 7, the INF-γ level in the serum of mice in the model group was significantly lower than that in the control group (P<0.01). Compared with the model group, the INF-γ levels in the serum of mice in the positive control group, high-dose test group and low-dose test group were increased. The INF-γ levels in the serum of mice in the positive control group and high-dose test group were increased. Among them, the INF-γ level in the serum of mice in the high-dose test group was closest to that in the control group (P<0.05), while the INF-γ levels in the serum of mice in the positive control group and low-dose test group were only higher than that in the model group, but significantly lower than that in the control group; the INF-γ level in the serum of mice in the comparison group was significantly lower than that in the control group and the model group.

[0097] It can be seen that the hemp seed cake functional peptide provided by the present invention has the ability to significantly increase the levels of IgA, IgG and INF-γ immune factors in mice under the condition of high-dose oral administration, and has the ability to restore the regulation of immunosuppressed mice.

[0098] Table 7

[0099] Group IgA (ng / μL) IgG (μg / μL) INF-γ (pg / nL) Control group 11.17±2.79 4.12±1.21 55.17±9.78 Model Group <![CDATA[4.15±1.02 ** ]]> <![CDATA[2.36±0.57 ** ]]> <![CDATA[30.90±9.87 ** ]]> Positive control group 4.61±1.15 <![CDATA[4.14±0.93 ## ]]> 35.58±9.25 High dose test group <![CDATA[14.96±4.50 ## ]]> <![CDATA[5.27±1.02 ## ]]> <![CDATA[51.21±15.34 # ]]> Low dose test group 5.04±1.30 <![CDATA[3.31±0.92 # ]]> 33.50±8.92 Comparison group 3.44±0.69 2.01±0.46 24.64±7.19

[0100] Note: Compared with the control group, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05.

[0101] (5) Antioxidant testing

[0102] In this example, 30 minutes after the last oral administration of the corresponding experimental sample to each group, the eyeballs of the mice in each group in (1) were removed for blood sampling, and the blood was centrifuged at 4°C and 2800 r / min for 10 minutes. The supernatant was collected and stored at -80°C for later use. The SOD and MDA levels in the serum of each group of mice were measured according to the instructions of the SOD and MDA kit (Nanjing Jiancheng Bioengineering Institute).

[0103] SOD is an important enzyme in the body's defense system. When the SOD content in the body decreases, the body is very susceptible to the invasion of oxygen free radicals. As shown in Table 8, the SOD content in the serum of the mice in the model group was significantly lower than that in the control group (P<0.01), indicating that cyclophosphamide caused an imbalance in the defense system in the mice, resulting in a decrease in the SOD content. Compared with the model group, the SOD content in the serum of the mice in the positive control group, the comparison group, the high-dose test group and the low-dose test group was increased to the level of the control group, and the SOD content was higher than that in the control group, among which the SOD content in the serum of the mice in the high-dose test group was significantly higher than that in the control group (P<0.01).

[0104] MDA is also an important enzyme in the body's defense system. When the MDA content in the body increases abnormally, it indicates that the lipids in the body are over-decomposed and the cells are threatened. As shown in Table 8, the MDA content in the serum of mice in the model group was significantly higher than that in the control group, indicating that cyclophosphamide caused an increase in MDA in the body, resulting in a decrease in the body's antioxidant capacity. Compared with the model group, the MDA content in the serum of mice in the positive control group, the high-dose test group, and the low-dose test group was reduced to the level of the control group, and the MDA content was lower than that in the control group. Among them, the MDA content in the serum of mice in the high-dose test group was significantly lower than that in the control group, and was extremely significantly lower than that in the model group (P<0.01).

[0105] It can be seen that the hemp seed cake functional peptide provided by the present invention has strong antioxidant properties, can better protect the cell membrane in mice from damage by free radicals, and has the ability to restore the regulation of immunosuppressed mice.

[0106] Table 8

[0107] Group SOD (U / mg prot) MDA (nmol / mL) Control group 202.02±7.68 8.64±2.2 Model Group <![CDATA[192.54±11.30 ** ]]> 13.10±3.52 Positive control group <![CDATA[230.44±14.64 ## ]]> <![CDATA[8.15±2.11 # ]]> High dose test group <![CDATA[237.21±6.60 ## ]]> <![CDATA[7.55±2.48 ## ]]> Low dose test group <![CDATA[221.3±15.78 ## ]]> <![CDATA[8.72±1.94 # ]]> Comparison group <![CDATA[223.67±3.73 ## ]]> 9.34±3.73

[0108] Note: Compared with the control group, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05.

[0109] (6) Detection of ear swelling inhibition rate

[0110] In this example, 20 μL of xylene was precisely aspirated and evenly applied to the right ears of each group of mice in (1) to construct an acute inflammation model of mouse ear swelling. After 30 minutes of treatment, each group of mice was killed, both ears were cut off, and the weight of each mouse ear was weighed. The ear swelling rate was:

[0111]

[0112] As shown in Table 9, the ear swelling inhibition rate of mice in the model group was significantly higher than that in the control group (P<0.05). Cyclophosphamide caused the mice to be in an immunosuppressive state, which in turn caused a more serious degree of inflammation. The ear swelling inhibition rate of mice in the positive control group was significantly lower than that in the model group, but significantly higher than that in the control group. The ear swelling inhibition rate of mice in the control group was significantly higher than that in both the model group and the control group. The ear swelling inhibition rate of mice in the high-dose test group and the low-dose test group was significantly lower than that in the model group, among which the ear swelling inhibition rate of mice in the low-dose test group was significantly higher than that in the control group, while the ear swelling inhibition rate of mice in the high-dose test group was significantly lower than that in the control group.

[0113] It can be seen that the hemp seed cake functional peptide provided by the present invention has a good ability to restore the body regulation of immunosuppressed mice, so the degree of inflammation caused is relatively low, and when a high dose of the hemp seed cake functional peptide provided by the present invention is administered by gavage, it has a certain anti-inflammatory ability and has the ability to restore the body regulation of immunosuppressed mice. At the same time, high-temperature extrusion and puffing treatment has the effect of reducing the toxicity of the hemp seed cake functional peptide.

[0114] Table 9

[0115] Group Ear swelling inhibition rate (%) Control group 16.26±3.34 Model Group 35.78±6.71* Positive control group <![CDATA[32.67±4.52 # ]]> High dose test group 25.50±4.2 Low dose test group <![CDATA[8.45±1.46 # ]]> Comparison group 45.88±5.26

[0116] Note: Compared with the control group, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05.

[0117] (7) Detection of inflammatory factors

[0118] In this example, 30 minutes after the last oral administration of the corresponding experimental sample to each group, the eyeballs of the mice in each group in (1) were removed for blood sampling, and the blood was centrifuged at 4°C and 2800 r / min for 10 minutes. The supernatant was collected and stored at -80°C for later use. The TNF-α and IL-2 levels in the serum of each group of mice were measured according to the instructions of the ELISA kit (Shaanxi White Shark Biotechnology Co., Ltd.).

[0119] As shown in Table 10, the IL-2 level in the serum of mice in the model group was significantly higher than that in the control group (P<0.05). Compared with the model group, the IL-2 levels in the serum of mice in the control group, high-dose test group and low-dose test group were all reduced. Among them, the IL-2 level in the serum of mice in the high-dose test group was extremely significantly lower than that in the control group (P<0.01), and the IL-2 level in the serum of mice in the low-dose test group was significantly lower than that in the control group (P<0.05). The IL-2 level in the serum of mice in the control group was lower than that in the model group, but significantly higher than that in the control group; the IL-2 level in the serum of mice in the positive control group was significantly higher than that in the control group, and significantly higher than that in the model group.

[0120] As shown in Table 10, the TNF-α level in the serum of mice in the model group was significantly lower than that in the control group. Compared with the model group, the TNF-α levels in the serum of mice in the positive control group, high-dose test group and low-dose test group were reduced. Among them, the TNF-α levels in the serum of mice in the high-dose test group and low-dose test group were significantly lower than those in the model group and control group (P<0.05). The TNF-α level in the serum of mice in the control group was significantly lower than that in the control group, but higher than that in the model group.

[0121] It can be seen that the hemp seed cake functional peptide provided by the present invention has the ability to significantly reduce the levels of IL-2 and TNF-α inflammatory factors in mice under the condition of high-dose oral administration, and has the ability to restore the regulation of immunosuppressed mice.

[0122] Table 10

[0123] Group IL-2 (pg / nL) TNF-α(pg / nL) Control group 38.94±11.87 17.46±5.04 Model Group <![CDATA[57.38±16.3 * ]]> 12.89±3.59 Positive control group 70.66±21.94 11.34±3.64 High dose test group <![CDATA[11.58±3.25 ## ]]> <![CDATA[6.07±1.68 # ]]> Low dose test group <![CDATA[21.42±6.86 # ]]> <![CDATA[6.69±1.23 # ]]> Comparison group 45.78±10.85 13.67±4.31

[0124] Note: Compared with the control group, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05.

[0125] (8) Detection of small intestinal propulsion rate

[0126] In this example, 30 minutes after the last oral administration of the corresponding experimental sample in each group, 0.5 mL of 2 mg / mL phenol red solution was administered to each group of mice in (1). After 20 minutes of treatment, the mice in each group were killed and the distance from the pylorus to the cecum of the dye was checked. The small intestinal propulsion rate was:

[0127]

[0128] As shown in Table 11, the small intestinal propulsion rate of mice in the model group was significantly lower than that of the control group (P<0.01). Since cyclophosphamide caused the mice to be in an immunosuppressive state, which led to the rhythmic disorder of the small intestinal activity of the mice, the small intestinal propulsion rate was significantly reduced. Compared with the model group, the small intestinal propulsion rate of mice in the positive control group, the comparison group, the high-dose test group and the low-dose test group increased, among which the small intestinal propulsion rate of mice in the high-dose test group and the low-dose test group was significantly higher than that in the model group, and significantly higher than that in the control group; while the small intestinal propulsion rate of mice in the positive control group and the comparison group was significantly higher than that in the model group, but still lower than that in the control group.

[0129] It can be seen that the hemp seed cake functional peptide provided by the present invention has the function of promoting intestinal digestion and protecting the intestine.

[0130] Table 11

[0131] Group Small intestinal propulsion rate (%) Control group 60.27±7.03 Model Group <![CDATA[39.45±4.83 ** ]]> Positive control group 52.64±3.68 High dose test group <![CDATA[68.01±3.97 # ]]> Low dose test group <![CDATA[73.21±5.16 # ]]> Comparison group 45.43±3.25

[0132] Note: Compared with the control group, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05.

[0133] In summary, the hemp seed cake functional peptide provided by the present invention has low toxicity, high safety and strong antioxidant property. The hemp seed cake functional peptide provided by the present invention has the function of significantly enhancing the phagocytic function of mouse monocytes and macrophages, significantly restoring and promoting the development of mouse spleen and thymus, significantly improving the levels of IgA, IgG and INF-γ immune factors in mice, significantly reducing the levels of IL-2 and TNF-α inflammatory factors in mice and anti-inflammatory function, promoting intestinal digestion and protecting the intestine, and having the ability to restore the regulation of immunosuppressed mice.

[0134] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a functional peptide from hemp seed cake, characterized in that: The preparation method comprises the following steps: S1: crushing hemp seed cake, adding organic solvent to defatting, and drying to obtain defatted hemp seed cake; S2: The defatted hemp seed cake obtained in S1 is crushed, sieved, and then put into a twin-screw extruder, and water is added to perform high-temperature extrusion and expansion treatment to obtain an extrudate; S3: Cooling, drying, crushing, and sieving the extrudate obtained in S2, and subjecting the extrudate obtained by the above-mentioned treatment to a two-step precipitation method to obtain hemp seed cake protein; S4: Cooling, drying, crushing and sieving the hemp seed cake protein obtained in S3, enzymolysis treatment of the hemp seed cake protein powder obtained by the above treatment, and then performing enzyme inactivation treatment to obtain hemp seed cake functional peptide enzymolysis liquid.

2. The preparation method according to claim 1, characterized in that: The organic solvent described in S1 is any one of n-hexane, isopropanol, petroleum ether or anhydrous ethanol.

3. The preparation method according to claim 1, characterized in that: The extrusion parameters of the twin-screw extruder described in S2 are: moisture content of 30%, screw speed of 220r / min, feed rate of 10kg / h, and barrel temperature in the extrusion and puffing area of ​​120℃, 140℃, 160℃, 180℃ or 200℃.

4. The preparation method according to claim 1, characterized in that: The steps of the two-step precipitation method described in S3 are: S11: mixing the treated extrudate and distilled water in a solid-liquid ratio of 1:10 (g / mL), adjusting the pH value to 6 with HCl solution, stirring and centrifuging, and collecting the precipitate; S22: Use NaOH solution to adjust the pH value of the precipitate obtained in S11 to 10, then place it in a water bath at 50°C for 1 hour, centrifuge it at 4000 r / min for 10 minutes, and collect the supernatant; S33: adjusting the pH value of the supernatant obtained in S22 to 4.5 using HCl solution, centrifuging at 4000 r / min for 10 min, and collecting the precipitate; S44: adjusting the pH value of the supernatant obtained in S22 to 3.6 using HCl solution, centrifuging at 4000 r / min for 10 min, and collecting the precipitate; S55: The precipitates obtained in S33 and S44 are mixed evenly, distilled water is added and stirred evenly, the pH value is adjusted to 7 with a NaOH solution, and the hemp seed cake protein is obtained after freeze-drying.

5. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis step described in S4 is: uniformly mixing the treated hemp seed cake protein and distilled water at a solid-liquid ratio of 1:10 (g / mL), boiling in a water bath for 5 minutes, cooling to room temperature, adjusting the pH value to 10 using a NaOH solution, adding alkaline protease to the hemp seed cake protein for enzymatic hydrolysis for 3 hours to obtain a single enzyme hydrolyzate; adding Flavourzyme composite flavor protease to the single enzyme hydrolyzate for 1 hour, after the enzymatic hydrolysis is completed, inactivating the enzyme in a boiling water bath for 15 minutes, centrifuging at 4,000 r / min for 15 minutes, taking the supernatant, and obtaining a hemp seed cake functional peptide enzymatic hydrolyzate.

6. The preparation method according to claim 5, characterized in that: The added amount of the alkaline protease is 1000 U / g.

7. The preparation method according to claim 5, characterized in that: The added amount of the Flavourzyme compound flavour protease is 750 U / g.

8. A hemp seed cake functional peptide, characterized in that: The hemp seed cake functional peptide is obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the hemp seed cake functional peptide according to claim 8 in in vivo regulation of immunosuppressive mice.

10. Use of the hemp seed cake functional peptide according to claim 8 in food preparation.

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