Cerebroprotein peptide and casein hydrolysate composition and application thereof in improving sleep and memory
By preparing a combination of brain protein peptides and casein hydrolysate, and modifying the initial casein hydrolysate using a specific enzymatic hydrolysis method and Maillard reaction, the problem of weak sleep-aiding effect of casein hydrolysate was solved, and the effects of improving sleep quality and memory function were achieved.
Patent Information
- Application Number
- CN202511125108.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-12
- Publication Date
- 2025-12-09
AI Technical Summary
Existing casein hydrolysates have a weak effect on improving sleep and are unlikely to effectively improve sleep quality and memory function.
By preparing a combination of brain protein peptides and casein hydrolysate, and modifying the initial product of casein hydrolysate using a specific enzymatic hydrolysis method and Maillard reaction, the reducing power, metal ion chelating ability and antioxidant capacity of casein hydrolysate are improved, thus forming a complex of brain protein peptides and casein hydrolysate.
It effectively shortens the sleep latency, increases the sleep onset rate, improves sleep quality, reduces the levels of dopamine and norepinephrine in the hypothalamus, and increases the levels of serotonin and GABA, thus having a good effect on relieving and treating sleep disorders.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a brain protein peptide and casein enzymatic hydrolysate composition and its application in improving sleep and memory, and belongs to the technical field of biological protein peptides. BACKGROUND
[0002] Casein is a kind of full-value protein with the largest content ratio in cow milk, and is a high-quality protein resource with excellent nutritional function. However, the solubility of casein is poor, which makes it difficult to exert the functional properties of casein and limits the development of casein in food processing. Meanwhile, casein is the parent protein of many bioactive peptides, and the bioactive peptide segments can be released by protease enzymolysis. The casein enzymatic hydrolysate produced after enzymolysis has more hydrophobic amino acids and smaller molecular weight, more reaction contact sites and higher digestibility compared with casein. After being ingested into the human body, the casein enzymatic hydrolysate can be quickly absorbed by the body, and has low allergenicity and has biological activities such as antioxidant activity, ACE inhibition activity and immune activity.
[0003] The research results show that the casein enzymatic hydrolysate of cow milk can prolong the sleep of mice induced by sodium pentobarbital, and the electroencephalogram recording results of rats show that the casein enzymatic hydrolysate can significantly enhance the slow wave sleep time of rats. However, compared with the conventional drug diazepam, the sleep-aiding effect of the casein enzymatic hydrolysate is weak. SUMMARY
[0004] The present application relates to a brain protein peptide and casein enzymatic hydrolysate composition and its application in improving sleep and memory, and belongs to the technical field of biological protein peptides.
[0005] The present application provides a brain protein peptide and casein enzymatic hydrolysate composition, which is composed of brain protein peptide and casein enzymatic hydrolysate; the preparation method of the brain protein peptide is as follows: the pig brain is first subjected to enzymolysis with pepsin and then subjected to enzymolysis with trypsin to obtain the brain protein peptide; the preparation method of the casein enzymatic hydrolysate is as follows: the casein of cow milk is subjected to enzymolysis with a protease composed of serratia peptidase and bromelain to obtain a casein enzymatic hydrolysate primary product; then, the casein enzymatic hydrolysate primary product is subjected to Maillard reaction with a carbonyl compound to obtain the casein enzymatic hydrolysate, and the carbonyl compound is composed of galactose and maltose.
[0006] Preferably, the mass ratio of the brain protein peptide to the casein enzymatic hydrolysate is 3-5:7-10.
[0007] Preferably, the method for enzymolysis of the pig brain with pepsin is as follows: fresh pig brain and water are homogenized to obtain a slurry, then the pH of the slurry is adjusted to 1.5-2, pepsin is added, and the enzymolysis is carried out at 45-50℃ for 2-4h.
[0008] Preferably, the mass ratio of the fresh pig brain and water is 2:3~3.5, the enzyme activity of pepsin is 10~20 thousand U / g, and the mass of pepsin is 2~3% of the mass of the fresh pig brain.
[0009] Preferably, when the enzymolysis is performed by trypsin, the pH of the system after the pig brain is enzymolyzed by pepsin is adjusted to 8.5~9, trypsin is added, and the enzymolysis is performed at 45~50℃ for 4~7h.
[0010] Preferably, the enzyme activity of trypsin is 25~30 thousand U / g, and the mass of trypsin is 2~5% of the mass of the fresh pig brain.
[0011] Preferably, after the pig brain is enzymolyzed by pepsin and then by trypsin, the enzymolysis liquid is subjected to high-temperature inactivation to obtain an inactivated liquid, the inactivated liquid is then adjusted to neutral and subjected to centrifugation to separate supernatant, the supernatant is vacuum freeze-dried to obtain the brain protein peptide.
[0012] Preferably, the mass ratio of the serralysin and bromelain is 5~7:3~4, the enzyme activity of serralysin is 100~200 thousand U / g, and the enzyme activity of bromelain is 500~600 thousand U / g; when the cow milk casein is enzymolyzed by the protease composed of serralysin and bromelain, the enzymolysis temperature is 45~50℃, the enzymolysis pH is 6.5~7.2, the enzymolysis time is 5~7h, and the mass of the protease is 2~3% of the mass of the cow milk casein.
[0013] Preferably, the method for performing the Maillard reaction of the casein enzymolysis primary product and the carbonyl compound is as follows: the casein enzymolysis primary product and a carbonyl compound solution with a mass fraction of 5~7% are uniformly mixed, the pH is adjusted to 7.8~8.4, then heated to 92~98℃, and mixed for 5~8h; after the reaction, the system after the reaction is vacuum freeze-dried to obtain the casein enzymolysis product; the mass ratio of the galactose and maltose is 3~6:2~5, and the mass ratio of the casein enzymolysis primary product and the carbonyl compound solution is 1:1.2~1.5.
[0014] The application further provides a use of the brain protein peptide and casein enzymolysis product composition in improving sleep and memory.
[0015] Compared with the prior art, the brain protein peptide and casein enzymolysis product composition and the use thereof in improving sleep and memory have the following beneficial effects:
[0016] The application realizes the Maillard modification of casein enzymolysis primary product by screening the enzyme protease of bovine milk casein and by screening the carbonyl compound for Maillard reaction with the casein enzymolysis primary product, effectively improves the reducing power, metal ion chelating capacity and antioxidant capacity of the casein enzymolysis product, and further ensures the elimination of free radicals in the body, reduces the oxidative stress reaction, thereby laying a good foundation for improving the sleep quality. The brain protein peptide and the casein enzymolysis product are compounded in the application, which can effectively shorten the sleep latency, improve the sleep rate, effectively improve the sleep, and can reduce the content of dopamine and norepinephrine in the hypothalamus, improve the content of 5-hydroxytryptamine and aminobutyl adrenocortical hormone, and has the effects of relieving, treating sleep disorders and improving memory. DETAILED DESCRIPTION
[0017] The following examples are intended to further illustrate the content of the application, but not to limit the protection scope of the application.
[0018] The specific implementation of the brain protein peptide and casein enzymolysis product composition and its application in improving sleep and memory of the application is as follows:
[0019] Example 1
[0020] The brain protein peptide and casein enzymolysis product composition of the embodiment is composed of brain protein peptide and casein enzymolysis product with a mass ratio of 3:7.
[0021] The preparation method of the brain protein peptide is as follows: fresh pig brain and water with a mass ratio of 2:3 are added into a homogenizer for homogenization to obtain a slurry, then the slurry is added into a reaction kettle, the pH of the material in the reaction kettle is adjusted to 1.5, pepsin (the enzyme activity of pepsin is 10,000 U / g, and the mass of pepsin is 2% of the mass of fresh pig brain) is added, the temperature of the material in the reaction kettle is adjusted to 45℃, and the enzyme is stirred for 2h, then the pH of the material in the reaction kettle is adjusted to 8.5, trypsin (the enzyme activity of trypsin is 25,000 U / g, and the mass of trypsin is 2% of the mass of fresh pig brain) is added, and the enzyme is stirred for 4h, after the enzyme is digested, the enzyme solution is inactivated at high temperature to obtain an inactivated solution, then the inactivated solution is adjusted to neutral and centrifuged to separate the supernatant, the supernatant is vacuum freeze-dried to obtain the brain protein peptide.
[0022] The preparation method of the casein enzymolysis product is as follows:
[0023] (1) dissolve the casein in PBS buffer solution with pH=7 to obtain a casein solution with mass fraction of 4%; then adjust the temperature and pH value of the casein solution to the required temperature and pH value for enzyme hydrolysis, and then add protease (the mass of the protease is 2% of the mass of the casein) for enzyme hydrolysis; after the enzyme hydrolysis is completed, the enzyme hydrolysis solution is inactivated at high temperature to obtain an inactivated solution, and then the inactivated solution is adjusted to neutral and centrifuged to separate the supernatant, which is the casein enzyme hydrolysis primary product; the enzyme hydrolysis temperature is 45℃, the enzyme hydrolysis pH is 6.5, the protease is composed of serratia peptidase and bromelain with a mass ratio of 5:3, the enzyme activity of the serratia peptidase is 100,000 U / g, the enzyme activity of the bromelain is 500,000 U / g, and the enzyme hydrolysis time is 5 h.
[0024] (2) add the casein enzyme hydrolysis primary product and a carbonyl compound solution with mass fraction of 5% into a reaction kettle, adjust the pH of the materials in the reaction kettle to 7.8, then heat to 92℃, and stir for 5 h; after the reaction is completed, the system after the reaction is vacuum freeze-dried to obtain the casein enzyme hydrolysis product; the carbonyl compound solution is composed of carbonyl compound and water, the carbonyl compound is composed of galactose and maltose with a mass ratio of 3:2, and the mass ratio of the casein enzyme hydrolysis primary product to the carbonyl compound solution is 1:1.2.
[0025] Example 2
[0026] The brain protein peptide and casein enzyme hydrolysis product composition of the present embodiment is composed of brain protein peptide and casein enzyme hydrolysis product with a mass ratio of 4:9.
[0027] The preparation method of the brain protein peptide is as follows: add fresh pig brain and water with a mass ratio of 2:3.2 into a homogenizer for homogenization to obtain a slurry, then add the slurry into a reaction kettle, adjust the pH of the materials in the reaction kettle to 1.8, add pepsin (the enzyme activity of the pepsin is 150,000 U / g, and the mass of the pepsin is 2.5% of the mass of the fresh pig brain), adjust the temperature of the materials in the reaction kettle to 48℃, and stir for enzyme hydrolysis for 3 h; then adjust the pH of the materials in the reaction kettle to 8.8, add trypsin (the enzyme activity of the trypsin is 280,000 U / g, and the mass of the trypsin is 3% of the mass of the fresh pig brain), and stir for enzyme hydrolysis for 6 h; after the enzyme hydrolysis is completed, the enzyme hydrolysis solution is inactivated at high temperature to obtain an inactivated solution, and then the inactivated solution is adjusted to neutral and centrifuged to separate the supernatant; the supernatant is vacuum freeze-dried to obtain the brain protein peptide.
[0028] The preparation method of the casein enzyme hydrolysis product is as follows:
[0029] (1) dissolve the casein in PBS buffer solution with pH = 7 to obtain a casein solution with a mass fraction of 4.5%; then adjust the temperature and pH of the casein solution to the required temperature and pH for enzymolysis, and then add protease (the mass of the protease is 2.5% of the mass of the casein) for enzymolysis; after the enzymolysis is completed, inactivate the enzymolysis solution at high temperature to obtain an inactivated solution; then adjust the inactivated solution to neutral, and centrifuge to separate the supernatant, which is the initial product of casein enzymolysis; the enzymolysis temperature is 48℃, the enzymolysis pH is 6.9, the protease is composed of serratia peptidase and bromelain with a mass ratio of 6:3.5, the enzyme activity of the serratia peptidase is 150,000 U / g, the enzyme activity of the bromelain is 550,000 U / g, and the enzymolysis time is 6 h.
[0030] (2) add the initial product of casein enzymolysis and a carbonyl compound solution with a mass fraction of 6% into a reaction kettle, adjust the pH of the materials in the reaction kettle to 8.1, then heat to 96℃, and stir for 7 h; after the reaction is completed, vacuum freeze-dry the system after the reaction to obtain the casein enzymolysis product; the carbonyl compound solution is composed of carbonyl compounds and water, the carbonyl compounds are composed of galactose and maltose with a mass ratio of 5:4, and the mass ratio of the initial product of casein enzymolysis to the carbonyl compound solution is 1:1.4.
[0031] Example 3
[0032] The brain protein peptide and casein enzymolysis product composition of the present embodiment is composed of brain protein peptide and casein enzymolysis product with a mass ratio of 5:10.
[0033] The preparation method of the brain protein peptide is as follows: add fresh pig brain and water with a mass ratio of 2:3.5 into a homogenizer to obtain a slurry, then add the slurry into a reaction kettle, adjust the pH of the materials in the reaction kettle to 2, add pepsin (the enzyme activity of the pepsin is 20,000 U / g, and the mass of the pepsin is 3% of the mass of the fresh pig brain), adjust the temperature of the materials in the reaction kettle to 50℃, and stir for 4 h of enzymolysis; then adjust the pH of the materials in the reaction kettle to 9, add trypsin (the enzyme activity of the trypsin is 30,000 U / g, and the mass of the trypsin is 5% of the mass of the fresh pig brain), stir for 7 h of enzymolysis, inactivate the enzymolysis solution at high temperature to obtain an inactivated solution, then adjust the inactivated solution to neutral, centrifuge to separate the supernatant, and vacuum freeze-dry the supernatant to obtain the brain protein peptide.
[0034] The preparation method of the casein enzymolysis product is as follows:
[0035] (1) The casein protein in milk is dissolved in PBS buffer with pH = 7 to obtain a casein protein solution with a mass fraction of 5%; then the temperature and pH of the casein protein solution are adjusted to the required temperature and pH for enzymolysis, and protease is added (the mass of the protease is 3% of the mass of the casein protein in milk) for enzymolysis; after the enzymolysis is completed, the enzymolysis solution is inactivated at high temperature to obtain an inactivated solution; then the inactivated solution is adjusted to neutral and centrifuged to separate the supernatant, which is the initial product of casein protein enzymolysis; the enzymolysis temperature is 50℃, the enzymolysis pH is 7.2, the protease is composed of serratia peptidase and bromelain with a mass ratio of 7:4, the enzyme activity of the serratia peptidase is 200,000 U / g, the enzyme activity of the bromelain is 600,000 U / g, and the enzymolysis time is 7 h.
[0036] (2) The initial product of casein protein enzymolysis and a carbonyl compound solution with a mass fraction of 7% are added to a reaction kettle, the pH of the materials in the reaction kettle is adjusted to 8.4, and then the reaction kettle is heated to 98℃ and stirred for 8 h; after the reaction is completed, the system after the reaction is vacuum freeze-dried to obtain a casein protein enzymolysis product; the carbonyl compound solution is composed of carbonyl compounds and water, the carbonyl compounds are composed of galactose and maltose with a mass ratio of 6:5, and the mass ratio of the initial product of casein protein enzymolysis to the carbonyl compound solution is 1:1.5.
[0037] Comparative Example 1
[0038] The brain protein peptide and casein protein enzymolysis product composition of the present comparative example differs from the brain protein peptide and casein protein enzymolysis product composition of Example 1 only in that the preparation method of the brain protein peptide used in the brain protein peptide and casein protein enzymolysis product composition of the present comparative example is as follows: fresh pig brain and water with a mass ratio of 2:3 are added to a homogenizer for homogenization to obtain a slurry, and then the slurry is added to a reaction kettle, the pH of the materials in the reaction kettle is adjusted to 1.5, pepsin is added (the enzyme activity of the pepsin is 100,000 U / g, and the mass of the pepsin is 7% of the mass of the fresh pig brain), the temperature of the materials in the reaction kettle is adjusted to 45℃, and the enzymolysis is stirred for 6 h; after the enzymolysis is completed, the enzymolysis solution is inactivated at high temperature to obtain an inactivated solution; then the inactivated solution is adjusted to neutral and centrifuged to separate the supernatant, and the supernatant is vacuum freeze-dried to obtain a brain protein peptide.
[0039] Comparative Example 2
[0040] The brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example differs from the brain protein peptide and casein enzymatic hydrolysate composition of Example 1 only in that the brain protein peptide used in the brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example is prepared as follows: fresh pig brain and water in a mass ratio of 2:3 are added to a homogenizer for homogenization to obtain a slurry, the slurry is then added to a reaction kettle, the pH of the material in the reaction kettle is adjusted to 8.5, trypsin (the enzyme activity of the trypsin is 250,000 U / g, and the mass of the trypsin is 2.8% of the mass of the fresh pig brain) is added, the temperature of the material in the reaction kettle is adjusted to 45°C, and the enzymatic hydrolysis is stirred for 6 h, after the enzymatic hydrolysis is completed, the enzymatic hydrolysate is inactivated at high temperature to obtain an inactivated solution, the inactivated solution is then adjusted to neutral and centrifuged to separate the supernatant, and the supernatant is vacuum freeze-dried to obtain the brain protein peptide.
[0041] Comparative Example 3
[0042] The brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example differs from the brain protein peptide and casein enzymatic hydrolysate composition of Example 1 only in that the brain protein peptide used in the brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example is prepared as follows: fresh pig brain and water in a mass ratio of 2:3 are added to a homogenizer for homogenization to obtain a slurry, the slurry is then added to a reaction kettle, the pH of the material in the reaction kettle is adjusted to 8.5, trypsin (the enzyme activity of the trypsin is 250,000 U / g, and the mass of the trypsin is 2.8% of the mass of the fresh pig brain) is added, the temperature of the material in the reaction kettle is adjusted to 45°C, and the enzymatic hydrolysis is stirred for 6 h, after the enzymatic hydrolysis is completed, the enzymatic hydrolysate is inactivated at high temperature to obtain an inactivated solution, the inactivated solution is then adjusted to neutral and centrifuged to separate the supernatant, and the supernatant is vacuum freeze-dried to obtain the brain protein peptide.
[0043] Comparative Example 4
[0044] The brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example differs from the brain protein peptide and casein enzymatic hydrolysate composition of Example 1 only in that the brain protein peptide used in the brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example is prepared as follows: fresh pig brain and water in a mass ratio of 2:3 are added to a homogenizer for homogenization to obtain a slurry, the slurry is then added to a reaction kettle, the pH of the material in the reaction kettle is adjusted to 8.5, trypsin (the enzyme activity of the trypsin is 250,000 U / g, and the mass of the trypsin is 2.8% of the mass of the fresh pig brain) is added, the temperature of the material in the reaction kettle is adjusted to 45°C, and the enzymatic hydrolysis is stirred for 6 h, after the enzymatic hydrolysis is completed, the enzymatic hydrolysate is inactivated at high temperature to obtain an inactivated solution, the inactivated solution is then adjusted to neutral and centrifuged to separate the supernatant, and the supernatant is vacuum freeze-dried to obtain the brain protein peptide.
[0045] Comparative Example 5
[0046] The brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example differs from the brain protein peptide and casein enzymatic hydrolysate composition of Example 1 only in that the protease used in step (1) of the preparation method of the casein enzymatic hydrolysate in the brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example is bromelain, the enzyme activity of the bromelain is 500,000 U / g, and the mass of the protease is 1% of the mass of the casein of the milk.
[0047] Comparative Example 6
[0048] The brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example differs from the brain protein peptide and casein enzymatic hydrolysate composition of Example 1 only in that the carbonyl compound used in step (2) of the preparation method of the casein enzymatic hydrolysate in the brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example is galactose.
[0049] Comparative Example 7
[0050] The brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example differs from the brain protein peptide and casein enzymatic hydrolysate composition of Example 1 only in that the carbonyl compound used in step (2) of the preparation method of the casein enzymatic hydrolysate in the brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example is maltose.
[0051] Comparative Example 8
[0052] The brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example differs from the brain protein peptide and casein enzymatic hydrolysate composition of Example 1 only in that the carbonyl compound used in step (2) of the preparation method of the casein enzymatic hydrolysate in the brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example is composed of glucose and maltose in a mass ratio of 3:2.
[0053] Comparative Example 9
[0054] The brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example differs from the brain protein peptide and casein enzymatic hydrolysate composition of Example 1 only in that the carbonyl compound used in step (2) of the preparation method of the casein enzymatic hydrolysate in the brain protein peptide and casein enzymatic hydrolysate composition of the present comparative example is composed of galactose and glucose in a mass ratio of 3:2.
[0055] Experimental Example
[0056] The present experimental example is used to evaluate the reducing power, metal ion chelating ability and antioxidant capacity of the casein enzymatic hydrolysates prepared in each example and comparative examples 4-9. The method for determining the reducing power is as follows: the casein enzymatic hydrolysate is dissolved with deionized water to obtain a diluent with a mass fraction of 0.2%, then equal volumes of the diluent, PBS buffer (pH=6.5) and potassium ferricyanide solution (mass fraction of 1%) are stirred uniformly to obtain a mixed solution, then the mixed solution is heated to 50°C and stirred for 30 min, then an equal volume of trichloroacetic acid solution (mass fraction of 10%) is added, after stirring uniformly, centrifugation is performed, the supernatant obtained by centrifugation is mixed with distilled water in equal volume, and then mixed uniformly with ferric chloride solution (mass fraction of 0.1%, volume of 1 / 4 of the volume of the supernatant), to obtain a test solution, then the absorbance of the test solution at a wavelength of 700 nm is tested.
[0057] The method for testing the metal ion chelating ability is as follows: the casein enzymatic hydrolysate is dissolved with deionized water to obtain a diluent with a certain concentration, then 1 mL of the diluent, 3.7 mL of distilled water, 0.1 mL of ferrous chloride solution with a concentration of 2 mmol / L and 0.2 mL of phenanthroline solution with a concentration of 5 mmol / L are added to a test tube, stirred uniformly to obtain a mixed solution, and placed at room temperature for 20 min, the absorbance A at 562 nm is tested, and the absorbance B at 562 nm of distilled water is tested at the same time, the metal ion chelating ability is calculated, and the metal ion chelating ability=(1-A / B)×100%.
[0058] The method for testing the antioxidant capacity is as follows: the casein enzymatic hydrolysate is dissolved with distilled water to obtain a diluent with a mass fraction of 0.1%; 30 mg of lecithin is dissolved in PBS solution with a concentration of 0.01 mol / L and pH=7.4 to obtain a lecithin solution; 15 g of trichloroacetic acid, 0.37 g of thiobarbituric acid and 2 mL of concentrated hydrochloric acid are stirred uniformly, and then distilled water is added to a total volume of 100 mL to obtain a trichloroacetic acid solution; 1 mL of the lecithin solution, 1 mL of ferric chloride solution with a concentration of 400 μmol / L and 1 mL of the diluent are stirred uniformly, and then placed in a dark environment at room temperature for 1 h, 2 mL of the trichloroacetic acid solution is then added, heated to boiling, and cooled to room temperature after 15 min, centrifuged, and then the absorbance A of the supernatant obtained by centrifugation at a wavelength of 532 nm is tested, and the absorbance B of distilled water at a wavelength of 532 nm is tested at the same time, the antioxidant capacity is calculated, and the antioxidant capacity=(B-A) / B×100%.
[0059] The reducing power, metal ion chelating ability and antioxidant capacity of the casein enzymatic hydrolysates prepared in each example and comparative examples 4-9 are shown in Table 1.
[0060] Table 1 Casein enzymatic hydrolysates prepared in each example and comparative examples 4-9
[0061] Reducing power, metal ion chelating ability and antioxidant capacity of the enzymatic hydrolysate
[0062]
[0063] As can be seen from Table 2, the casein enzymatic hydrolysate used in the embodiments 1-3 of the present application has higher reducing power, metal ion chelating ability and antioxidant capacity compared to the comparative examples 4-9, thereby ensuring the removal of free radicals in the body, reducing oxidative stress, and thus laying a good foundation for improving sleep quality.
[0064] Effect example
[0065] In order to investigate the application prospect of the brain protein peptide and casein enzymatic hydrolysate composition in improving sleep and memory, mice were used as experimental objects to study the sleep and memory improvement effect of the brain protein peptide and casein enzymatic hydrolysate composition. The experimental method is as follows: male experimental mice with the same breed, weight, age and physical condition were divided into 3 batches for experiments, the first batch of mice was used for sleep onset rate determination experiment, the second batch of mice was used for sleep latency determination experiment, and the third batch of mice was used for neurotransmitter content determination experiment, several mice in each batch were randomly divided into 14 groups, namely blank group, experimental groups 1-12 and diazepam group, 10 mice in each group, and the mice in each group were given intragastrical administration, the intragastrical volume was 0.2 mL / 20 g, the blank group was given distilled water, the experimental groups 1-12 were given brain protein peptide and casein enzymatic hydrolysate composition of the embodiments 1-3 and comparative examples 1-9 in turn, the dosage was 300 mg / kg, the diazepam group was given diazepam, the dosage was 2 mg / kg, and each mouse was given intragastrical administration once a day, continuously for 4 weeks.
[0066] The sleep onset rate determination experimental method is as follows: 1 hour after the last administration of the mice in each group, the maximum threshold hypnotic dose of sodium pentobarbital was injected intraperitoneally, the injection amount was 0.2 mL, and the disappearance of the righting reflex for more than 1 min was used as the judgment standard for entering the sleep state, the number of mice falling asleep within 30 min after injection was recorded, and the sleep onset rate of the blank group, the experimental group and the diazepam positive group was calculated.
[0067] The sleep latency determination experimental method is as follows: 1 hour after the last administration of the mice in each group, sodium barbital was injected intraperitoneally, the injection amount was 0.2 mL, and the period from injection completion to the disappearance of the righting reflex was the sleep latency, the sleep latency of each mouse in each group was recorded, and finally the average sleep latency of the mice in each group was calculated.
[0068] The neurotransmitter content determination experiment method is as follows: after the mice are gavaged for 4 weeks, the mice are anesthetized, blood is taken from the heart, the brain is taken out after ice bath craniotomy, the hypothalamus is separated, weighed and then put into liquid nitrogen for preservation, then physiological saline is added in a ratio of 1:9, homogenized at 4 DEG C, the homogenate is centrifuged at 3000r / min, 4 DEG C for 15min, then the contents of 5-hydroxytryptamine, butylamine, dopamine and norepinephrine in the supernatant are determined, and finally the average content of 5-hydroxytryptamine, butylamine, dopamine and norepinephrine in the hypothalamus of the mice in each group is calculated. The experimental results are shown in Table 2.
[0069] Table 2 sleep rate, average sleep latency and the average content of 5-hydroxytryptamine, butylamine, dopamine and norepinephrine in the hypothalamus of the mice in each group
[0070] 5-hydroxytryptamine, butylamine, dopamine and norepinephrine
[0071]
[0072] It can be seen from Table 2 that the brain protein peptide and casein enzymatic product composition of the present application can effectively shorten the sleep latency, improve the sleep rate, effectively improve the sleep, and reduce the content of dopamine and norepinephrine in the hypothalamus, and increase the content of 5-hydroxytryptamine and butylamine, which again proves that the brain protein peptide and casein enzymatic product composition of the present application has good application prospect for relieving and treating sleep disorders.
[0073] In summary, the present application realizes the Maillard modification of the casein enzymatic primary product by screening the enzyme protease for the enzymatic hydrolysis of casein in bovine milk and screening the carbonyl compound for the Maillard reaction with the casein enzymatic primary product, effectively improves the reducing power, metal ion chelating ability and antioxidant capacity of the casein enzymatic product, and further ensures the elimination of free radicals in the body and the reduction of oxidative stress, thereby laying a good foundation for improving the sleep quality. The experimental results show that the brain protein peptide and casein enzymatic product composition of the present application can effectively shorten the sleep latency, improve the sleep rate, effectively improve the sleep, and reduce the content of dopamine and norepinephrine in the hypothalamus, and increase the content of 5-hydroxytryptamine and butylamine, which has good effect of relieving, treating sleep disorders and improving memory.
Claims
1. A composition of brain protein peptides and casein hydrolysate, characterized in that, It is composed of brain protein peptides and casein hydrolysate; the preparation method of the brain protein peptides is as follows: pig brain is first enzymatically hydrolyzed with pepsin and then with trypsin to obtain brain protein peptides; the preparation method of the casein hydrolysate is as follows: bovine milk casein is enzymatically hydrolyzed with a protease composed of Serratia marcescens and bromelain to obtain casein hydrolysate; then the casein hydrolysate and carbonyl compound are subjected to Maillard reaction to obtain casein hydrolysate, wherein the carbonyl compound is composed of galactose and maltose.
2. The brain protein peptide and casein hydrolysate composition as described in claim 1, characterized in that, The mass ratio of the brain protein peptides to the casein hydrolysate is 3~5:7~10.
3. The brain protein peptide and casein hydrolysate composition as described in claim 1, characterized in that, The method for enzymatically hydrolyzing pig brain with pepsin is as follows: Homogenize fresh pig brain with water to obtain a slurry, then adjust the pH of the slurry to 1.5~2, add pepsin, and enzymatically hydrolyze at 45~50℃ for 2~4 hours.
4. The brain protein peptide and casein hydrolysate composition as described in claim 3, characterized in that, The mass ratio of fresh pig brain to water is 2:3 to 3.5, the enzyme activity of pepsin is 10,000 to 20,000 U / g, and the mass of pepsin is 2 to 3% of the mass of fresh pig brain.
5. The brain protein peptide and casein hydrolysate composition as described in claim 3, characterized in that, When using trypsin for enzymatic hydrolysis, adjust the pH of the system after digesting pig brain with pepsin to 8.5-9, add trypsin, and hydrolyze at 45-50℃ for 4-7 hours.
6. The brain protein peptide and casein hydrolysate composition as described in claim 5, characterized in that, The trypsin has an enzyme activity of 25,000 to 30,000 U / g, and the mass of the trypsin is 2 to 5% of the mass of fresh pig brain.
7. The brain protein peptide and casein hydrolysate composition as described in claim 1, characterized in that, Pig brains were first hydrolyzed with pepsin and then with trypsin. The hydrolysate was then inactivated at high temperature to obtain an inactivated solution. The inactivated solution was then adjusted to neutral and centrifuged to separate the supernatant. The supernatant was then freeze-dried under vacuum to obtain brain protein peptides.
8. The brain protein peptide and casein hydrolysate composition as described in claim 1, characterized in that, The mass ratio of serratiopeptidase to bromelain is 5-7:3-4, the enzyme activity of serratiopeptidase is 100,000-200,000 U / g, and the enzyme activity of bromelain is 500,000-600,000 U / g. When enzymatically hydrolyzing bovine casein using a protease composed of serratiopeptidase and bromelain, the enzymatic hydrolysis temperature is 45-50℃, the enzymatic hydrolysis pH is 6.5-7.2, the enzymatic hydrolysis time is 5-7 hours, and the mass of the protease is 2-3% of the bovine casein protein mass.
9. The brain protein peptide and casein hydrolysate composition as described in claim 1, characterized in that, The Maillard reaction of casein hydrolysate and carbonyl compound is carried out as follows: the casein hydrolysate and a carbonyl compound solution with a mass fraction of 5-7% are mixed and the pH is adjusted to 7.8-8.
4. Then the mixture is heated to 92-98℃ and reacted for 5-8 hours. After the reaction is completed, the system is freeze-dried under vacuum to obtain casein hydrolysate. The mass ratio of galactose to maltose is 3-6:2-5, and the mass ratio of casein hydrolysate to carbonyl compound solution is 1:1.2-1.
5.
10. The use of a brain protein peptide and casein hydrolysate composition as described in any one of claims 1-9 in improving sleep and memory.