Cattle spleen peptide composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological extraction, and provides a cattle spleen peptide composition as well as a preparation method and application thereof. The preparation method of the cattle spleen peptide comprises the following steps: S1, taking a cattle spleen, adding normal saline, and homogenizing to obtain first homogenate; s2, adding combined enzyme into the first homogenate, uniformly stirring, and performing first water bath treatment to obtain second homogenate; s3, adding chymotrypsin into the second homogenate, uniformly stirring, and performing second water bath treatment to obtain third homogenate; s4, centrifuging the third homogenate to obtain supernate; s5, performing ultrafiltration and drying on the supernate to obtain the cattle spleen peptide. The combined enzyme comprises subtilisin, bromelain and papain. Through the technical scheme, the problem of low cattle spleen peptide extraction rate in related technologies is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological extraction, and specifically, to a bovine spleen peptide composition, a preparation method thereof, and an application thereof. Background Art

[0002] With the continuous acceleration of the modern life rhythm, sleep problems have increasingly become one of the key factors troubling people's physical and mental health. According to relevant statistical data, the proportion of the population with sleep disorders globally continues to rise. In China, hundreds of millions of people are troubled by problems such as insomnia, dreaminess, and easy waking, which result in poor sleep quality. Poor sleep quality for a long time not only causes people to be listless during the day and have low work efficiency, but is also closely related to the occurrence and development of many chronic diseases, such as cardiovascular diseases, diabetes, and neurological diseases, bringing huge challenges to personal health and the social medical burden.

[0003] As an important immune organ of the animal body, the bovine spleen is rich in various bioactive components. Among them, bovine spleen peptide contains rich amino acid sequences and has multiple functions such as immune regulation and antioxidant effects. Preliminary studies have shown that bovine spleen peptide shows certain potential in regulating the function of the nervous system. It may have a positive impact on the sleep-wake cycle by acting on the synthesis, release, and binding to receptors of neurotransmitters, opening up a new way to improve sleep quality.

[0004] However, the existing bovine spleen peptide extraction technology has the drawback of low extraction rate, which is difficult to meet the needs of large-scale industrial production and clinical application. Therefore, it is urgent to develop a bovine spleen peptide extraction method with a high extraction rate of bovine spleen peptide. Summary of the Invention

[0005] The present invention provides a bovine spleen peptide composition, a preparation method thereof, and an application thereof, which solve the problem of low extraction rate of bovine spleen peptide in the related technology.

[0006] The technical solution of the present invention is as follows: The present invention provides a preparation method of bovine spleen peptide, comprising the following steps: S1. Take bovine spleen, add physiological saline for homogenization to obtain a first homogenate; S2. Add a combined enzyme to the first homogenate, stir evenly, and perform a first water bath treatment to obtain a second homogenate; S3. Add chymotrypsin to the second homogenate, stir evenly, and perform a second water bath treatment to obtain a third homogenate; S4. Centrifuge the third homogenate to obtain a supernatant; S5. Ultrafilter and dry the supernatant to obtain bovine spleen peptide; The combined enzyme includes subtilisin, bromelain, and papain.

[0007] As a further technical solution, the mass-volume ratio of the bovine spleen to the physiological saline in S1 is 1 g: 1-3 mL.

[0008] As a further technical solution, the colloid mill particle size of the first homogenate in S1 is 2-8 μm.

[0009] As a further technical solution, the combined enzyme in S2 is 0.8%-1.9% of the mass of the bovine spleen.

[0010] As a further technical solution, the mass ratio of subtilisin, bromelain, and papain is 1.3: 1: 0.5-1.5.

[0011] As a further technical solution, the specific first water bath treatment in S2 is to keep warm at 55-65 °C for 2-2.5 h.

[0012] In the present invention, the condition of the first water bath treatment is to keep warm at 55-65 °C for 2-2.5 h, which is the best condition for the combined enzyme to play its role.

[0013] As a further technical solution, the chymotrypsin in S3 is 0.3%-0.7% of the mass of the bovine spleen.

[0014] As a further technical solution, the specific second water bath treatment in S3 is to keep warm at 45-55 °C for 0.5-1.5 h and then cool down to 10-20 °C.

[0015] In the present invention, the condition of the second water bath treatment is to keep warm at 45-55 °C for 0.5-1.5 h, which is the best condition for chymotrypsin to play its role.

[0016] As a further technical solution, the speed of centrifugation is 2500-3500 r / min, the temperature is 0-10 °C, and the time is 15-30 min.

[0017] In the present invention, the speed of centrifugation is 2500-3500 r / min, the temperature is 1-10 °C, and the time is 15-30 min, and the effect of separating the precipitate generated during the enzymatic hydrolysis is the best.

[0018] As a further technical solution, the ultrafiltration is specifically carried out by ultrafiltration with a 5000 Dalton membrane and then ultrafiltration with a 10000 Dalton membrane.

[0019] The present invention also provides a bovine spleen peptide composition, the raw material of which includes the bovine spleen peptide prepared by the preparation method of a bovine spleen peptide described above.

[0020] As a further technical solution, the raw materials include the following components in parts by weight: 1-4 parts of hawthorn, 4-6 parts of Chinese yam, 1-5 parts of licorice, 5-9 parts of malt, and 5-7 parts of bovine spleen peptide.

[0021] The present invention also provides a method for preparing the bovine spleen peptide composition, which includes the following steps: pulverizing hawthorn, Chinese yam, licorice, and malt and then sieving to obtain a mixed traditional Chinese medicine powder, extracting the mixed traditional Chinese medicine powder with water to obtain an aqueous extract, concentrating the aqueous extract and then cooling, and centrifuging to collect the supernatant; mixing the supernatant with bovine spleen peptide to obtain the bovine spleen peptide composition.

[0022] As a further technical solution, during the extraction, the temperature is 80-90°C and the time is 1-2 h.

[0023] As a further technical solution, the mass-volume ratio of the mixed traditional Chinese medicine powder to water is 1 g:10-20 mL.

[0024] The present invention also provides the application of the bovine spleen peptide composition or the bovine spleen peptide composition prepared by the method for preparing the bovine spleen peptide composition in improving sleep quality.

[0025] The working principle and beneficial effects of the present invention are as follows: In the present invention, subtilisin, bromelain, and papain improve the extraction rate of bovine spleen peptide through synergistic effects. Using chymotrypsin to treat the second homogenate improves the extraction rate of bovine spleen peptide, which is beneficial to the application of bovine spleen peptide in the bovine spleen peptide composition and enables the bovine spleen peptide composition to exert the effect of improving sleep quality. Specific Embodiments

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0027] In the following embodiments and comparative examples, the product number of subtilisin is S10051, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; the product number of bromelain is S10009, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; the product number of papain is S10011, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; the product number of chymotrypsin is S10001, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; the product number of pepsin is S10027, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; the product number of trypsin is S10033, purchased from Shanghai Yuanye Bio-Technology Co., Ltd., and the product number of 5'-phosphodiesterase is S24549, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0028] Example 1 A preparation method of a bovine spleen peptide composition, comprising the following steps: Powder 4 parts of hawthorn, 6 parts of Chinese yam, 5 parts of liquorice and 9 parts of malt and sieve to obtain a mixed traditional Chinese medicine powder. Extract the mixed traditional Chinese medicine powder with water at 90 °C for 1 h (the mass-volume ratio of the mixed traditional Chinese medicine powder to water is 1 g:20 mL) to obtain a water extract. Concentrate the water extract to 0.3 times the original volume, cool, and centrifuge to collect the supernatant; mix the supernatant with 7 parts of bovine spleen peptide to obtain a bovine spleen peptide composition; A preparation method of bovine spleen peptide, comprising the following steps: S1. Take 100 g of bovine spleen, add physiological saline for homogenization to obtain a first homogenate; S2. Add a combined enzyme accounting for 1.9% of the mass of the bovine spleen to the first homogenate, stir evenly, and keep it warm in a water bath at 65 °C for 2 h to obtain a second homogenate; S3. Add chymotrypsin accounting for 0.7% of the mass of the bovine spleen to the second homogenate, stir evenly, keep it warm in a water bath at 55 °C for 0.5 h, and then cool down to 20 °C to obtain a third homogenate; S4. Centrifuge the third homogenate at 3500 r / min for 15 min at 10 °C to obtain a supernatant; S5. Ultrafilter the supernatant with a 5000 Dalton ultrafilter, then ultrafilter it with a 10000 Dalton membrane, filter and dispense it aseptically with a 0.2 μm filter element, and dry to obtain bovine spleen peptide; The combined enzyme is subtilisin, bromelain and papain with a mass ratio of 1.3:1:2; In S1, the mass-volume ratio of bovine spleen to physiological saline is 1 g:3 mL; In S1, the particle size of the first homogenate by colloid mill is 8 μm.

[0029] Example 2 A preparation method of a bovine spleen peptide composition, comprising the following steps: Powder 1 part of hawthorn, 4 parts of Chinese yam, 1 part of liquorice and 5 parts of malt and sieve to obtain a mixed traditional Chinese medicine powder. Extract the mixed traditional Chinese medicine powder with water at 80 °C for 2 h (the mass-volume ratio of the mixed traditional Chinese medicine powder to water is 1 g:10 mL) to obtain a water extract. Concentrate the water extract to 0.3 times the original volume, cool, and centrifuge to collect the supernatant; mix the supernatant with 5 parts of bovine spleen peptide to obtain a bovine spleen peptide composition; A preparation method of bovine spleen peptide, comprising the following steps: S1. Take 100 g of bovine spleen, add physiological saline for homogenization to obtain a first homogenate; S2. Add a combined enzyme accounting for 0.8% of the mass of the bovine spleen to the first homogenate, stir evenly, and keep it warm in a water bath at 55 °C for 2.5 h to obtain a second homogenate; S3. Add chymotrypsin at 0.3% of the mass of the bovine spleen to the second homogenate, stir evenly, incubate in a water bath at 45 °C for 1.5 h, and then cool to 10 °C to obtain the third homogenate; S4. Centrifuge the third homogenate at 2500 r / min for 30 min at 1 °C to obtain the supernatant; S5. Ultrafilter the supernatant with a 5000 Dalton filter and then with a 10000 Dalton membrane, filter and dispense aseptically with a 0.2 μm filter element, and dry to obtain bovine spleen peptide; The combined enzyme is subtilisin, bromelain and papain with a mass ratio of 1.3:1:0.1; In S1, the mass-to-volume ratio of bovine spleen to physiological saline is 1 g:1 mL; The colloid mill particle size of the first homogenate in S1 is 2 μm.

[0030] Example 3 A preparation method of a bovine spleen peptide composition, comprising the following steps: Powder 2 parts of hawthorn, 5 parts of Chinese yam, 3 parts of licorice and 6 parts of malt and sieve to obtain a mixed traditional Chinese medicine powder. Extract the mixed traditional Chinese medicine powder with water at 85 °C for 1.5 h (the mass-to-volume ratio of the mixed traditional Chinese medicine powder to water is 1 g:15 mL) to obtain an aqueous extract. Concentrate the aqueous extract to 0.3 times the original volume, cool, and centrifuge to collect the supernatant. Mix the supernatant with 6 parts of bovine spleen peptide to obtain the bovine spleen peptide composition; A preparation method of bovine spleen peptide, comprising the following steps: S1. Take 100 g of bovine spleen and add physiological saline for homogenization to obtain the first homogenate; S2. Add a combined enzyme at 1.4% of the mass of the bovine spleen to the first homogenate, stir evenly, incubate in a water bath at 60 °C for 2.3 h to obtain the second homogenate; S3. Add chymotrypsin at 0.5% of the mass of the bovine spleen to the second homogenate, stir evenly, incubate in a water bath at 50 °C for 1 h, and then cool to 15 °C to obtain the third homogenate; S4. Centrifuge the third homogenate at 3000 r / min for 20 min at 5 °C to obtain the supernatant; S5. Ultrafilter the supernatant with a 5000 Dalton filter and then with a 10000 Dalton membrane, filter and dispense aseptically with a 0.2 μm filter element, and dry to obtain bovine spleen peptide; The combined enzyme is subtilisin, bromelain and papain with a mass ratio of 1.3:1:0.2; In S1, the mass-to-volume ratio of bovine spleen to physiological saline is 1 g:2 mL; The colloid mill particle size of the first homogenate in S1 is 5 μm.

[0031] Example 4 The difference between this example and Example 3 is only that the combined enzyme in this example is subtilisin, bromelain and papain with a mass ratio of 1.3:1:1.8.

[0032] Example 5 The difference between this example and Example 3 is only that the combined enzyme in this example is subtilisin, bromelain and papain with a mass ratio of 1.3:1:0.5.

[0033] Example 6 The difference between this example and Example 3 is only that the combined enzyme in this example is subtilisin, bromelain and papain with a mass ratio of 1.3:1:1.

[0034] Example 7 The difference between this example and Example 3 is only that the combined enzyme in this example is subtilisin, bromelain and papain with a mass ratio of 1.3:1:1.5.

[0035] Comparative Example 1 The difference between this comparative example and Example 2 is only that the combined enzyme in this comparative example is pepsin, bromelain and papain with a mass ratio of 1.3:1:0.1.

[0036] Comparative Example 2 A preparation method of a bovine spleen peptide composition includes the following steps: Powder 1 part of hawthorn, 4 parts of Chinese yam, 1 part of liquorice and 5 parts of malt and then sieve to obtain a mixed traditional Chinese medicine powder. Extract the mixed traditional Chinese medicine powder with water at 80°C for 2 h (the mass-volume ratio of the mixed traditional Chinese medicine powder to water is 1 g:10 mL) to obtain a water extract. Concentrate the water extract to 0.3 times of the original volume, cool it, and centrifuge to collect the supernatant. Mix the supernatant with 5 parts of bovine spleen peptide to obtain a bovine spleen peptide composition; A preparation method of bovine spleen peptide includes the following steps: S1. Take 100 g of bovine spleen, add physiological saline for homogenization to obtain a first homogenate; S2. Add a combined enzyme accounting for 0.8% of the mass of the bovine spleen to the first homogenate, stir evenly, and keep it warm in a water bath at 55°C for 2.5 h to obtain a second homogenate; S3. Add 5'-phosphodiesterase accounting for 0.3% of the mass of the bovine spleen to the second homogenate, stir evenly, keep it warm in a water bath at 45°C for 1.5 h, and then cool it to 10°C to obtain a third homogenate; S4. Centrifuge the third homogenate at 2500 r / min at 1°C for 30 min to obtain a supernatant; S5. Ultrafilter the supernatant with a 5000 Dalton ultrafiltration membrane, then ultrafilter with a 10000 Dalton membrane, aseptically filter and dispense with a 0.2 μm filter element, and dry to obtain bovine spleen peptide; The combined enzyme is subtilisin, bromelain and papain with a mass ratio of 1.3:1:0.1; In S1, the mass-to-volume ratio of bovine spleen to physiological saline is 1 g:1 mL; The particle size of the colloid mill of the first homogenate in S1 is 2 μm.

[0037] Comparative Example 3 A preparation method of a bovine spleen peptide composition, comprising the following steps: Powder 1 part of hawthorn, 4 parts of Chinese yam, 1 part of licorice and 5 parts of malt, sieve to obtain a mixed traditional Chinese medicine powder, extract the mixed traditional Chinese medicine powder with water at 80 °C for 2 h (the mass-to-volume ratio of the mixed traditional Chinese medicine powder to water is 1 g:10 mL) to obtain a water extract, concentrate the water extract to 0.3 times the original volume, cool, and centrifuge to collect the supernatant; mix the supernatant with 5 parts of bovine spleen peptide to obtain a bovine spleen peptide composition; A preparation method of bovine spleen peptide, comprising the following steps: S1. Take 100 g of bovine spleen, add physiological saline and homogenize to obtain a first homogenate; S2. Add 0.3% of chymotrypsin based on the mass of bovine spleen to the first homogenate, stir evenly, keep it warm in a water bath at 45 °C for 1.5 h, and then cool it to 10 °C to obtain a second homogenate; S3. Centrifuge the second homogenate at 1 °C at a speed of 2500 r / min for 30 min to obtain a supernatant; S4. Ultrafilter the supernatant with a 5000 Dalton ultrafiltration membrane, then ultrafilter with a 10000 Dalton membrane, aseptically filter and dispense with a 0.2 μm filter element, and dry to obtain bovine spleen peptide; In S1, the mass-to-volume ratio of bovine spleen to physiological saline is 1 g:1 mL; The particle size of the colloid mill of the first homogenate in S1 is 2 μm.

[0038] Comparative Example 4 A preparation method of a bovine spleen peptide composition, comprising the following steps: Powder 1 part of hawthorn, 4 parts of Chinese yam, 1 part of licorice and 5 parts of malt, sieve to obtain a mixed traditional Chinese medicine powder, extract the mixed traditional Chinese medicine powder with water at 80 °C for 2 h (the mass-to-volume ratio of the mixed traditional Chinese medicine powder to water is 1 g:10 mL) to obtain a water extract, concentrate the water extract to 0.3 times the original volume, cool, and centrifuge to collect the supernatant; mix the supernatant with 5 parts of bovine spleen peptide to obtain a bovine spleen peptide composition; A preparation method of bovine spleen peptide, comprising the following steps: S1. Take 100 g of bovine spleen, add physiological saline and homogenize to obtain a first homogenate; S2. Add the combined enzyme at 0.8% of the mass of the bovine spleen to the first homogenate, stir evenly, and incubate in a water bath at 55 °C for 2.5 h to obtain the second homogenate; S3. Centrifuge the second homogenate at 2500 r / min at 1 °C for 30 min to obtain the supernatant; S4. Ultrafilter the supernatant with a 5000 Dalton filter and then with a 10000 Dalton membrane, filter and dispense aseptically with a 0.2 μm filter element, and dry to obtain bovine spleen peptide; The combined enzyme is subtilisin, bromelain and papain with a mass ratio of 1.3:1:0.1; In S1, the mass-to-volume ratio of the bovine spleen to the physiological saline is 1 g:1 mL; The particle size of the colloid mill of the first homogenate in S1 is 2 μm.

[0039] Comparative Example 5 A method for preparing a bovine spleen peptide composition, comprising the following steps: Powder 1 part of hawthorn, 4 parts of Chinese yam, 1 part of licorice and 5 parts of malt and sieve to obtain a mixed traditional Chinese medicine powder. Extract the mixed traditional Chinese medicine powder with water at 80 °C for 2 h (the mass-to-volume ratio of the mixed traditional Chinese medicine powder to water is 1 g:10 mL) to obtain a water extract. Concentrate the water extract to 0.3 times the original volume, cool, and centrifuge to collect the supernatant. Mix the supernatant with 5 parts of bovine spleen peptide to obtain the bovine spleen peptide composition; A method for preparing bovine spleen peptide, comprising the following steps: S1. Take 100 g of bovine spleen and add physiological saline for homogenization to obtain the first homogenate; S2. Add the combined enzyme at 0.8% of the mass of the bovine spleen to the first homogenate, stir evenly, and incubate in a water bath at 55 °C for 2.5 h to obtain the second homogenate; S3. Add trypsin at 0.3% of the mass of the bovine spleen to the second homogenate, stir evenly, incubate in a water bath at 45 °C for 1.5 h, and then cool to 10 °C to obtain the third homogenate; S4. Centrifuge the third homogenate at 2500 r / min at 1 °C for 30 min to obtain the supernatant; S5. Ultrafilter the supernatant with a 5000 Dalton filter and then with a 10000 Dalton membrane, filter and dispense aseptically with a 0.2 μm filter element, and dry to obtain bovine spleen peptide; The combined enzyme is subtilisin, bromelain and papain with a mass ratio of 1.3:1:0.1; In S1, the mass-to-volume ratio of the bovine spleen to the physiological saline is 1 g:1 mL; The particle size of the colloid mill of the first homogenate in S1 is 2 μm.

[0040] Test Example 1 The polypeptide concentrations of the bovine spleen peptides prepared in Examples 1 to 7 and Comparative Examples 1 to 5 were detected, and the detection method was as follows: Experimental instrument: ultraviolet spectrophotometer; Detection instrument: BCA rapid detection kit (yb0012s, Shanghai Yubo Biotechnology Co., Ltd.); Sample preparation: Take 0.05 g of the bovine spleen peptide sample, dissolve it in 10 mL of purified water, and mix well for standby; The detection method includes the following steps: Gradiently dilute the bovine serum albumin (BSA) standard to 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL; Prepare the BCA working solution, and mix 100 mL of working solution A and 2 mL of working solution B in the kit; Add the diluted standard and the sample to be tested into the enzyme-linked immunosorbent assay (ELISA) plate (20 μL / well) respectively, add 200 μL of the BCA working solution, and incubate at 37 °C for 30 min; Read the absorbance of each well at 562 nm, draw a standard curve, and calculate the concentration of the sample to be tested; The detection results are shown in Table 1.

[0041]

[0042] Comparing Example 2 with Comparative Examples 1 and 3 shows that the use of the combined enzyme composed of subtilisin, bromelain, and papain in the present invention improves the extraction rate of bovine spleen peptide. Comparing Example 2 with Comparative Examples 2 and 4 to 5 shows that the use of chymotrypsin in the present invention improves the extraction rate of bovine spleen peptide.

[0043] Comparing Examples 5 to 7 with Examples 3 to 4 shows that when the mass ratio of subtilisin, bromelain, and papain is adjusted to 1.3:1:0.5 to 1.5 in the present invention, the extraction rate of bovine spleen peptide is further improved. Comparing Example 6 with Examples 5 and 7 shows that when the mass ratio of subtilisin, bromelain, and papain is 1.3:1:1, the effect of improving the extraction rate of bovine spleen peptide is the best.

[0044] Test Example 2 Experimental animals: Zebrafish were all raised in fish culture water at 28 °C (water quality: add 200 mg of instant sea salt to every 1 L of reverse osmosis water, the conductivity is 450 - 550 μS / cm; pH is 6.5 - 8.5; hardness is 50 - 100 mg / L calcium carbonate). Wild-type AB strain zebrafish were used for natural paired mating and reproduction. Zebrafish at 5 days post-fertilization (5 dpf) were used for the determination of the maximum detection concentration of bovine spleen peptide and the evaluation of the efficacy of improving sleep quality.

[0045] Detection method: Randomly select wild-type AB strain zebrafish at 5 dpf and place them in a 6-well plate. Each well in the experimental group is treated with 30 zebrafish. The bovine spleen peptide composition obtained in Example 1 and melatonin at a concentration of 125 μg / mL as the positive control are respectively administered. At the same time, a normal control group (fed in a conventional manner) and a model control group (i.e., a zebrafish insomnia model established by PTZ) are set up, and the volume of each well is 3 mL. After treatment at 28 °C for 24 h, 10 zebrafish are randomly selected from each experimental group and transferred to a 96-well plate, 200 μL per fish, 1 fish per well. Except for the normal control group, the other experimental groups are all administered with water-soluble PTZ to establish a zebrafish insomnia model. A behavior analyzer is used to collect data, and the awakening activity amount and awakening activity time of the zebrafish are analyzed. The efficacy of the bovine spleen peptide composition in improving sleep quality is evaluated through the statistical analysis results of the above indicators. The detection results are shown in Table 2.

[0046] Table 2 Detection results of each group

[0047] Comparing the bovine spleen peptide composition with the control group, it can be seen that the bovine spleen peptide composition has the efficacy of improving sleep quality.

[0048] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing bovine spleen peptide, characterized in that: The following steps are involved: S1, taking bovine spleen and adding physiological saline to homogenize to obtain a first homogenate; S2, adding the combined enzyme to the first homogenate, stirring evenly, and treating in a first water bath to obtain a second homogenate; S3, adding chymotrypsin to the second homogenate, stirring evenly, and treating in a second water bath to obtain a third homogenate; S4, centrifuging the third homogenate to obtain a supernatant; S5, ultrafiltering the supernatant and drying it to obtain bovine spleen peptide; The enzyme combination includes subtilisin, bromelain and papain.

2. The method for preparing bovine spleen peptide according to claim 1, characterized in that: The combined enzyme described in S2 is 0.8% to 1.9% of the mass of bovine spleen.

3. The method for preparing bovine spleen peptide according to claim 1, characterized in that: The mass ratio of the subtilisin, bromelain and papain is 1.3:1:0.5-1.

5.

4. The method for preparing bovine spleen peptide according to claim 1, characterized in that: The first water bath treatment in S2 is specifically to keep warm at 55-65° C. for 2-2.5 hours.

5. The method for preparing bovine spleen peptide according to claim 1, characterized in that: The chymosin described in S3 is 0.3% to 0.7% of the mass of bovine spleen.

6. The method for preparing bovine spleen peptide according to claim 1, characterized in that: The second water bath treatment in S3 is specifically to keep the temperature at 45-55°C for 0.5-1.5h and then cool it down to 10-20°C.

7. A bovine spleen peptide composition, characterized in that: The raw material comprises the bovine spleen peptide prepared by the method for preparing the bovine spleen peptide according to any one of claims 1 to 6.

8. A bovine spleen peptide composition according to claim 7, characterized in that: The raw materials include the following components in parts by weight: 1-4 parts of hawthorn, 4-6 parts of yam, 1-5 parts of liquorice, 5-9 parts of malt and 5-7 parts of bovine spleen peptide.

9. The method for preparing a bovine spleen peptide composition according to any one of claims 7 to 8, characterized in that: The following steps are involved: Hawthorn, yam, liquorice and malt are powdered and sieved to obtain mixed Chinese medicine powder, the mixed Chinese medicine powder is extracted with water to obtain a water extract, the water extract is concentrated and cooled, and the supernatant is collected by centrifugation; the supernatant is mixed with bovine spleen peptide to obtain a bovine spleen peptide composition.

10. Use of the bovine spleen peptide composition prepared according to any one of claims 7 to 8 or the method for preparing the bovine spleen peptide composition according to claim 9 in improving sleep quality.

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