Compound casein peptide for assisting in improving sleep as well as preparation method and application of compound casein peptide

Abstract

The invention provides a compound casein peptide for assisting in improving sleep as well as a preparation method and application thereof, and belongs to the technical field of amino acid polypeptides, the compound casein peptide comprises the following raw materials in parts by weight: 32-36 parts of casein peptide, 28-32 parts of walnut peptide and 34-38 parts of wheat oligopeptide. The invention provides a compound casein peptide for assisting in improving sleep as well as a preparation method and application of the compound casein peptide, so that the effect of assisting in improving sleep of a compound product is good, and the effect of protecting the renal function is achieved.

Description

Technical Field

[0001] This invention relates to the field of amino acid peptide technology, specifically to a compound casein peptide for assisting in improving sleep, its preparation method, and its application. Background Technology

[0002] Insomnia, also known as sleep disorder insomnia, is a common health problem affecting the quality and quantity of sleep. It is often referred to as "sleeplessness" or "restless nights." Insomnia can be classified into several types based on clinical manifestations and causes, such as difficulty falling asleep, difficulty maintaining sleep, early awakening, and mixed types. Its causes are complex and diverse, including psychological factors, physiological factors, environmental factors, and lifestyle habits. The main clinical symptoms of insomnia include difficulty falling asleep, frequent awakenings during the night, early awakening with difficulty falling back asleep, and fatigue and poor concentration upon waking. Long-term insomnia can also lead to complications such as memory loss, mood swings, and weakened immunity.

[0003] Insomnia is a very common problem in today's society. Young people suffer from insomnia due to work pressure and the habit of staying up late; working professionals suffer from insomnia due to long hours and anxiety; and the elderly suffer from early awakening and fragmented sleep due to declining physiological functions. Treatment for insomnia usually involves a comprehensive approach, including psychotherapy (cognitive behavioral therapy), medication (such as short-term use of sleeping pills, but only as directed by a doctor), physical therapy (such as light therapy and music therapy), and traditional Chinese medicine. Medication options include estazolam, diazepam, amitriptyline, and agomelatine, but with the increasing emphasis on health in modern society, people are more inclined to use natural, safe products without side effects.

[0004] Chinese patent application CN119453495A, entitled "Composition Containing Casein Peptides for Improving Sleep, Preparation Method Thereof, and Application Thereof," discloses a composition containing casein peptides for improving sleep, its preparation method, and application. The composition includes the following components: casein peptide nanoparticles: prepared using ultrasonic nanotechnology; multilayer microencapsulation: comprising a bilayer structure formed by natural polysaccharides and lipid complexes to improve the stability of casein peptides in the gastrointestinal tract; and an enzyme inhibitor: used to temporarily inhibit the activity of pepsin to enhance the bioavailability of casein peptides. However, in the above-mentioned technical solution, only a single casein peptide provides the sleep-aiding effect, resulting in poor efficacy for patients with severe insomnia. Summary of the Invention

[0005] In view of this, the present invention provides a compound casein peptide for assisting in improving sleep, its preparation method and application, so that the compound product has a good effect in assisting in improving sleep and also plays a role in protecting kidney function.

[0006] To achieve the above objectives, the present invention provides a compound casein peptide to help improve sleep, comprising the following raw materials: casein peptide, walnut peptide and wheat oligopeptide.

[0007] Optional ingredients include the following parts by weight: 32-36 parts casein peptide, 28-32 parts walnut peptide, and 34-38 parts wheat oligopeptides.

[0008] Optional ingredients include the following parts by weight: 34 parts casein peptide, 30 parts walnut peptide, and 36 parts wheat oligopeptides.

[0009] Optionally, the casein peptide contains short peptides with amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0010] Optionally, the walnut peptide is a short peptide with an amino acid sequence as shown in SEQ ID NO: 3.

[0011] Optionally, the wheat oligopeptide contains a short peptide with an amino acid sequence as shown in SEQ ID No. 4 and SEQ ID No. 5.

[0012] To achieve the above objectives, the present invention also provides a method for preparing a compound casein peptide to help improve sleep, wherein casein peptide, walnut peptide and wheat oligopeptide are uniformly mixed to obtain a compound casein peptide.

[0013] To achieve the above objectives, the present invention also provides the application of a compound casein peptide for assisting in improving sleep in a sleep-improving drug.

[0014] The above-described technical solution of the present invention has at least the following beneficial effects: 1. The technical solution of this invention combines casein peptides, walnut peptides, and wheat oligopeptides according to the recommended daily intake for humans to form a multi-target synergistic sleep aid product. Casein directly enhances GABA activity, walnut peptides regulate the sleep cycle through melatonin, and wheat oligopeptides reduce inflammatory interference. This avoids the side effects caused by excessive intake of casein peptide products or simultaneous use of GABA and melatonin combination products, and expands the target population (those with mild insomnia or stress-related sleep disorders; shift workers or travelers crossing time zones who need to regulate their biological clock; middle-aged and elderly people or those with reduced melatonin secretion). Mouse experiments have demonstrated that the compound product provided by this invention has an ideal effect on improving sleep, and the mechanism by which casein peptides, walnut peptides, and wheat oligopeptides improve sleep in mice has been preliminarily clarified.

[0015] 2. This invention, through animal experiments, showed that low, medium, and high doses of casein peptides and compound casein peptides reduced sodium barbital sleep latency to varying degrees and prolonged sodium pentobarbital-induced sleep time in mice, indicating that casein peptides have an ameliorative effect on mouse sleep. Open field experiments on different groups of mice revealed that low, medium, and high doses of casein peptides and compound casein peptides all increased the number of visits to the center, the time spent in the central region, and the total distance of activity to varying degrees, indicating that casein peptides and compound casein peptides improved sleep while increasing the mice's exploration and motor abilities. Changes in the levels of neurotransmitters 5-HT, DA, and NE in brain tissue showed that casein peptides and compound casein peptides regulate neurotransmitter pathways by modulating the levels of 5-HT, NE, and DA in the brain, maintaining neuroendocrine homeostasis, maintaining normal diurnal regulation, and ensuring normal sleep function. Furthermore, blood biochemical indicators showed that casein peptides and compound casein peptides did not produce toxic effects on blood biochemical indicators and had an inhibitory effect on the increase of serum creatinine. Compound casein peptides have an improving effect on sleep in mice and also have a protective effect on kidney function. Attached Figure Description

[0016] Figure 1 This is a graph showing the effect of compound casein peptide on the body weight of mice in the sodium barbital sleep latency experiment of this invention. Figure 2 This is a graph showing the effect of the compound casein peptide in this invention on the sleep latency of sodium barbital in mice. Figure 3 This is a graph showing the effect of compound casein peptide on the body weight of mice in the experiment of prolonging sodium pentobarbital sleep time in this invention. Figure 4 This is a graph showing the effect of the compound casein peptide in this invention on prolonging the sleep time of mice treated with sodium pentobarbital. Figure 5 This is a graph showing the effect of compound casein peptide on the body weight of mice in a subthreshold dose of sodium pentobarbital hypnotic experiment. Figure 6 This is a graph showing the effect of the compound casein peptide on the body weight of mice in the open field experiment. Figure 7 This is a graph showing the effect of the compound casein peptide in this invention on the mouse open field experiment; Figure 8 This is a graph showing the effect of the compound casein peptide on mouse creatinine levels in this invention. Figure 9 This is a diagram showing the effect of the compound casein peptide on neurotransmitters in the mouse brain in this invention. Figure 10 This is a graph showing the effect of the compound casein peptide in this invention on the insomnia time of zebrafish; Figure 11This is a graph showing the effect of the compound casein peptide in this invention on improving sleep in zebrafish. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the accompanying drawings of the embodiments of the present invention. Figures 1-11 The technical solutions of the embodiments of the present invention will be clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.

[0018] The short peptide shown in SEQ ID NO: 1 is KLPPVGPY; the short peptide shown in SEQ ID NO: 2 is APIASLLPPY; the short peptide shown in SEQ ID NO: 3 is GEEREERGLD; the short peptide shown in SEQ ID NO: 4 is APIASLLPP; and the short peptide shown in SEQ ID NO: 5 is KLPPVGPY.

[0019] Example 1 This invention provides a compound casein peptide for aiding sleep improvement, comprising the following raw materials in parts by weight: 34 parts casein peptide, 30 parts walnut peptide, and 36 parts wheat oligopeptide. The casein peptide contains short peptides with amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; the walnut peptide is a short peptide with an amino acid sequence as shown in SEQ ID NO: 3; and the wheat oligopeptide contains short peptides with amino acid sequences as shown in SEQ ID No. 4 and SEQ ID No. 5. The casein peptide, walnut peptide, and wheat oligopeptide are uniformly mixed at room temperature to obtain the compound casein peptide.

[0020] Example 2 This invention provides a compound casein peptide for assisting in improving sleep, comprising the following raw materials in parts by weight: 36 parts casein peptide, 29 parts walnut peptide, and 35 parts wheat oligopeptide. The casein peptide contains short peptides with amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; the walnut peptide is a short peptide with an amino acid sequence as shown in SEQ ID NO: 3; and the wheat oligopeptide contains short peptides with amino acid sequences as shown in SEQ ID No. 4 and SEQ ID No. 5. The casein peptide, walnut peptide, and wheat oligopeptide are uniformly mixed at room temperature to obtain the compound casein peptide.

[0021] Example 3 The present invention provides a compound casein peptide for assisting in improving sleep, comprising the following raw materials in parts by weight: 32 parts of casein peptide, 31 parts of walnut peptide, and 37 parts of wheat oligopeptide. The casein peptide contains short peptides with amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; the walnut peptide is a short peptide with an amino acid sequence as shown in SEQ ID NO: 3; the wheat oligopeptide contains short peptides with amino acid sequences as shown in SEQ ID No. 4 and SEQ ID No. 5. The casein peptide, walnut peptide, and wheat oligopeptide are uniformly mixed at room temperature to obtain the compound casein peptide.

[0022] Example 4 The present invention provides an application of a compound casein peptide for assisting in improving sleep in a drug for improving sleep.

[0023] Example 5 The present invention provides an application of a compound casein peptide for assisting in improving sleep in a drug for protecting renal function.

[0024] I. Mouse Experiment 1. Experimental Preparation: 1.1 Experimental Animals 200 SPF-grade male Kunming mice weighing 22 ± 2 g were selected and purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The experimental animal production license number is SCXK (Beijing) 2021-0006. The animals were housed in an SPF-grade constant temperature (22 ± 2) °C, constant humidity (55 ± 5) %, and light-dark cycle of 12 hours / 12 hours animal room, with free access to food and water. The experimental unit's use license number is SYXK (Shandong) 20200022.

[0025] 1.2 Grouping After one week of adaptive feeding of the mice, according to the principle of balanced mouse body weight, the mice were randomly divided into 4 large experimental groups: pentobarbital sleep latency experiment, experiment on prolonging the sleeping time of mice induced by pentobarbital sodium, pentobarbital sodium subthreshold dose hypnosis experiment, and open field experiment. Each large group was respectively set with a control group, low, medium, and high-dose casein peptide groups, and a compound casein peptide group, with 5 groups in total and 10 mice in each group.

[0026] 1.3 Dose Setting and Administration Method of Test Substances The recommended daily intake (DUI) of casein peptide for humans is 600 mg / 60 kg bw. Low, medium, and high doses of casein peptide were set at 5, 10, and 20 times the recommended DUI, respectively, resulting in daily intakes of 50 mg / kg bw, 100 mg / kg bw, and 200 mg / kg bw for each group of mice. The compound casein peptide was prepared as described in Example 1, with a casein peptide content of 34%. Based on the recommended DUI of 600 mg / 60 kg bw, the daily intake for mice in the compound casein peptide group was 294 mg / kg bw. Mice were administered the test substance orally once daily by gavage at a volume of 0.1 mL / 10 g bw. The concentrations of the casein peptide solutions in the low, medium, and high dose groups were 5 g / L, 10 g / L, and 20 g / L, respectively, while the concentration of the compound casein peptide solution was 29 g / L. The control group was administered an equal volume of deionized water instead of the test solution via gavage. Mice were weighed and their weight recorded weekly for 30 consecutive days. After the experiment, serum and mouse brain samples were collected to measure sleep function improvement.

[0027] 1.4 Index Detection Experiment 1.4.1 Direct Sleep Experiment: After the animals in each dose group were given the test substance for the last time, the control group animals were given an equal volume of deionized water. The disappearance of the righting reflex in mice was used as an indicator to observe whether the test substance group showed sleep phenomena.

[0028] Experimental results: After 30 days of administration, mice did not exhibit direct sleep phenomena in the low, medium, and high casein peptide dose groups and the compound casein peptide group.

[0029] 1.4.2 Sodium barbital sleep latency test: After the last administration of the test substance to each group of animals, and the administration of the same volume of deionized water to the control group animals, 15 minutes later, sodium barbital 200 mg / kg bw was injected intraperitoneally into each group of animals at a dose of 0.1 mL / 10 g bw. The effect of the test substance on the sodium barbital sleep latency was observed with the disappearance of the righting reflex as the indicator.

[0030] The effects of compound casein peptide on the body weight of mice in the sodium barbital sleep latency experiment are shown in Table 1. The graph showing the effect of compound casein peptide on the body weight of mice in the sodium barbital sleep latency experiment is shown in [Figure number missing]. Figure 1 .

[0031] Table 1. Effects of compound casein peptide on body weight of mice in the sodium barbital sleep latency experiment (Table 1) ±SEM, n=10)

[0032] From Table 1 and Figure 1 It can be seen that the weight of animals in each group continued to increase during the sodium barbital sleep latency experiment, and there was no significant difference in weight between the mice in each dose group and the control group.

[0033] The effect of compound casein peptide on the sleep latency of sodium barbital in mice is shown in Table 2, and the effect of compound casein peptide on the sleep latency of sodium barbital in mice is shown in the figure. Figure 2 .

[0034] Table 2. Effects of compound casein peptide on sodium barbital sleep latency in mice.

[0035] Note: The values ​​are described as mean ± standard error. *P<0.05 compared with the control group.

[0036] From Table 2 and Figure 2 It was found that, compared with the control group, the sleep latency of the low, medium and high casein peptide groups and the compound casein peptide group was significantly shortened (P<0.05).

[0037] 1.4.3 Experiment on prolonging sodium pentobarbital-induced sleep time in mice: After the last administration of the test substance to each dose group, and the administration of the same volume of deionized water to the control group, 15 minutes later, sodium pentobarbital 50 mg / kg was injected intraperitoneally at a dose of 0.1 mL / 10 g.bw.

[0038] The effects of compound casein peptide on the body weight of mice in the pentobarbital sodium sleep prolongation experiment are shown in Table 3. The graph showing the effect of compound casein peptide on the body weight of mice in the pentobarbital sodium sleep prolongation experiment is shown in [Figure number missing]. Figure 3 .

[0039] Table 3. Effects of compound casein peptide on body weight in mice undergoing prolonged pentobarbital sodium sleep (Table 3) ±SEM, n=10)

[0040] From Table 3 and Figure 3 It can be seen that during the experiment of prolonging the sleep time induced by sodium pentobarbital in mice, the body weight of the animals in each group continued to increase, and there was no significant difference in body weight between the mice in each dose group and the control group.

[0041] The effect of compound casein peptide on prolonging pentobarbital sodium sleep time in mice is shown in Table 4, and the effect of compound casein peptide on prolonging pentobarbital sodium sleep time in mice is shown in the figure. Figure 4 .

[0042] Table 4. Effects of compound casein peptides on prolonging pentobarbital sodium sleep time in mice. ±SEM)

[0043] Note: Mean ± standard error is used for description. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001.

[0044] From Table 4 and Figure 4 It was found that, compared with the control group, the sleep time of mice in the low, medium and high casein peptide dose groups and the compound casein peptide group was significantly prolonged (P<0.05).

[0045] 1.4.4 Subthreshold dose-induced hypnosis experiment with sodium pentobarbital: Fifteen minutes after the last administration of the test substance to animals in each dose group, and the administration of an equal volume of deionized water to the control group, animals in each group were intraperitoneally injected with the maximum subthreshold dose of sodium pentobarbital (30 mg / kg / bw) at 0.1 mL / 10 g / bw. The number of animals that fell asleep within 30 minutes after the righting reflex disappeared for more than 1 minute was recorded. The effect of compound casein peptide on the body weight of mice in the subthreshold dose-induced hypnosis experiment with sodium pentobarbital is shown in Table 5. The graph showing the effect of compound casein peptide on the body weight of mice in the subthreshold dose-induced hypnosis experiment with sodium pentobarbital is shown in [Figure number missing]. Figure 5 .

[0046] Table 5. Effects of compound casein peptide on body weight of mice in subthreshold pentobarbital sodium hypnotic experiments. ±SEM, n=10)

[0047] From Table 5 and Figure 5 It can be seen that during the subthreshold dose hypnotic experiment of sodium pentobarbital, the weight of animals in each group continued to increase, and there was no significant difference in weight between the mice in each dose group and the control group.

[0048] The effect of compound casein peptide on the incidence of sleep induced by subthreshold doses of sodium pentobarbital in mice is shown in Table 6.

[0049] Table 6. Effect of compound casein peptide on the incidence of sleep induced by subthreshold dose of pentobarbital sodium in mice. ±SEM, n=10)

[0050] Note: Mean ± standard error is used for description. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001.

[0051] As shown in Table 6, the number of mice falling asleep tended to increase compared with the control group, but there was no significant difference in the incidence of sleep among the groups (P>0.05).

[0052] 1.4.5 Open Field Experiment: The Open Field Test (OFT) is a commonly used experimental paradigm to assess the behavioral responses of laboratory animals (such as mice or rats) in novel environments, thereby inferring their psychological states, including anxiety, depression, exploratory behavior, and motor abilities. Total distance traveled is typically used to measure the animal's activity level and motor ability. The number of visits to the central area reflects the animal's curiosity and exploratory behavior towards the new environment, as well as its adaptability and anxiety level. Similar to the number of visits, the duration of activity in the central area also reflects the animal's curiosity and exploratory behavior, and its anxiety level.

[0053] Open field experimental method: The experiment was conducted in a square box (48 cm long × 48 cm wide × 36 cm high) made of compressed wood. The bottom was divided into 16 equal squares by a white grid, including an outer area (12 surrounding squares) and a central area (4 central squares). Mice were placed in the central area and allowed to explore freely for 10 minutes, tracked by a CCD camera. Data were analyzed using SMART 3.0, Panlab, Spain. Before starting the next test, the experimental apparatus was wiped with 75% ethanol to remove odor. The total distance the experimental animals moved within 10 minutes was used as a measure of spontaneous activity, while the time spent in the central area and the number of visits to the central area were used as measures of anxiety and exploratory behavior.

[0054] The effects of compound casein peptide on the body weight of mice in the open field experiment are shown in Table 7, and the graph showing the effect of compound casein peptide on the body weight of mice in the open field experiment is shown in [Figure number missing]. Figure 6 .

[0055] Table 7. Effects of compound casein peptides on body weight of mice in open field experiments (Table 7) ±SEM, n=10)

[0056] From Table 7 and Figure 6 It can be seen that the weight of animals in each group continued to increase during the open field experiment, and there was no significant difference in weight between the mice in each dose group and the control group.

[0057] The effects of compound casein peptides on the mouse open field test are shown in Table 8, and the graph showing the effects of compound casein peptides on the mouse open field test is shown below. Figure 7 .

[0058] Table 8. Effects of compound casein peptides on mouse open field test ( ±SEM, n=10)

[0059] Note: The values ​​are described as mean ± standard error. Compared with the control group, *P<0.05, **P<0.01.

[0060] From Table 8 and Figure 7 The results showed that, compared with the control group, the number of visits to the central region was significantly increased in the medium-dose casein peptide group and the compound casein peptide group (P < 0.05), and the time spent visiting the central region and the total distance of activity were significantly increased in the low-dose, medium-dose casein peptide group and the compound casein peptide group (P < 0.05).

[0061] 1.4.6 Detection of blood biochemical indicators: To evaluate whether casein peptide and compound casein peptide have toxic effects on blood biochemical indicators in mice, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), total protein (TP), albumin (ALB), triglycerides (TG), total cholesterol (TCH), glucose (GLU), and creatinine (CREA) in mouse serum were detected.

[0062] The effects of compound casein peptide on mouse blood biochemical parameters are shown in Table 9, and the effects of compound casein peptide on mouse creatinine parameters are shown in the graph. Figure 8 .

[0063] Table 9. Effects of compound casein peptide on blood biochemical parameters in mice ( ±SEM, n=10)

[0064] Note: The values ​​are described as mean ± standard error. *P<0.05 compared with the control group.

[0065] Table 9 shows that, compared with the control group, there were no significant differences in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), total protein (TP), albumin (ALB), triglycerides (TG), total cholesterol (TCH), and glucose (GLU) in mice at various doses of casein peptide and complex casein peptide (P>0.05). Figure 8 The results showed that the serum creatinine (CREA) in mice in the low- and medium-dose casein peptide groups and the compound casein peptide group was significantly reduced (P<0.05), indicating that the compound casein peptide provided by the present invention has a protective effect on kidney function.

[0066] 1.4.7 Detection of neurotransmitter levels in the brain: The levels of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) in mouse brain tissue were detected by ELISA according to the kit instructions.

[0067] The effects of compound casein peptides on neurotransmitters in the mouse brain are shown in Table 10, and the graph showing the effects of compound casein peptides on neurotransmitters in the mouse brain is shown below. Figure 9.

[0068] Table 10 Effects of Compound Casein Peptide on Neurotransmitters in Mouse Brain

[0069] Note: The values ​​are described as mean ± standard error. Compared with the control group, *P<0.05, **P<0.01.

[0070] From Table 10 and Figure 9 The results showed that, compared with the control group, the levels of casein peptide 5-HT and DA were significantly increased in the low, medium and high dose groups (P<0.05), and the levels of NE were significantly increased in the medium and high dose casein peptide groups and the compound casein peptide group (P<0.05).

[0071] 5-HT (serotonin) is an important monoamine neurotransmitter in the brain, closely related to cognitive and sleep functions. When tryptophan levels in the brain increase, 5-HT levels also increase. When 5-HT levels reach a certain level, fatigue and drowsiness occur. Norepinephrine (NE) plays a crucial role in neuronal communication and is closely related to mood, attention, and sleep. When NE levels decrease, neurotransmitter pathways are disrupted, causing neuroendocrine imbalances and disrupting normal diurnal regulation, leading to sleep disorders. Dopamine (DA), as a monoamine neurotransmitter, has a regulatory effect on the central nervous system. Decreased DA levels indicate a disruption in the balance of neurotransmitters in the brain, resulting in insomnia and memory loss. Therefore, the compound casein peptide provided by this invention improves sleep quality by increasing the levels of 5-HT, DA, and NE in the body.

[0072] The above experimental data are statistically analyzed as follows: Sleep duration, weight, and biochemical indicators are all numerical variables. All data were analyzed using SPSS statistical software for ANOVA. If the variances were unequal, the rank-sum test was used for statistical analysis. The incidence of sleep disturbances was analyzed using the chi-square test.

[0073] II. Zebrafish Experiment 1. Experimental preparation: 1.1 Laboratory Animals Wild-type AB strain zebrafish were bred through natural pair mating. A total of 330 fish, aged 5 dpf, were used, with 30 fish in each experimental group (for experimentation to determine the concentration of casein peptide compound).

[0074] Wild-type AB strain zebrafish were bred through natural pair mating. A total of 210 fish, aged 5 dpf, were used, with 30 fish in each experimental group (to evaluate the sleep-improving effect of the test product on caffeine-induced insomnia in zebrafish).

[0075] Zebrafish were raised in fish culture water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 480 - 510 μS / cm; pH was 6.9 - 7.2; hardness was 53.7 - 71.6 mg / L CaCO3). The license number for the use of experimental animals was: SYXK (Zhe) 2012 - 0171. The feeding management complied with the requirements of international AAALAC accreditation.

[0076] 1.2 Experimental samples The pure peptide compound of casein, a yellow powder, is the compound casein peptide prepared in Example 2, provided by Zhongshi Duqing (Shandong) Biotechnology Co., Ltd. on August 16, 2019, and stored in a cool cabinet at 20 °C.

[0077] Pure casein peptide, a white powder, is a single casein peptide, provided by Zhongshi Duqing (Shandong) Biotechnology Co., Ltd. on August 16, 2019, and stored in a cool cabinet at 20 °C.

[0078] Caffeine, a white powder, was purchased from Aladdin Reagent (Shanghai) Co., Ltd., batch number 404 - 520 - 2. Immediately before use, it was prepared into a stock solution with a concentration of 50 mM using DMSO.

[0079] 1.3 Instruments and reagents Dissecting microscope (SZX7, OLYMPUS, Japan); 6 - well plate (Fisher Scientific, China); 96 - well plate (Fisher Scientific, China); precision electronic balance (CP214, Ohaus, China); behavior analyzer (V3.11, ViewPoint Life Sciences, France).

[0080] 1.4 Testing to determine the maximum tolerance (MTC) of zebrafish to the pure peptide compound of casein Six groups were set for comparison. The experimental group 1 was the control group, and zebrafish were treated with fish culture water; the concentrations of the pure peptide compound of casein in experimental groups 2 - 6 were 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, and 2000 μg / mL respectively.

[0081] The experimental results are shown in Table 11.

[0082] Table 11 Statistical table of the number of dead zebrafish at the tolerance concentration of the pure peptide compound of casein (n = 30)

[0083] As shown in Table 11, the mortality rate of zebrafish was 73% (22 / 30) at a concentration of 2000 μg / mL for the casein pure peptide compound; the mortality rate was 7% (2 / 30) at a concentration of 1000 μg / mL; and no obvious abnormalities were observed in zebrafish within the concentration range of 125-500 μg / mL. Therefore, the MTC of the casein pure peptide compound is 500 μg / mL.

[0084] 1.5 Evaluation of the sleep-improving effect of casein pure peptide compound on zebrafish Five groups were set up for comparison. Experiment 1 was the control group, in which zebrafish were treated with fish farming water. Experiment 2 was the model control group, in which wild-type AB zebrafish were treated with caffeine at 5 dpf to establish a zebrafish insomnia model. Experiment 3 was the positive control drug lithium chloride at 500 μM. Experiment 4 was a casein pure peptide compound at 500 μg / mL. Experiment 5 was casein pure peptide at 177 μg / mL.

[0085] 1.6 Experimental Methods Wild-type AB zebrafish (5 dpf) were randomly selected and placed in 6-well plates, 30 fish per well. An insomnia model was established by inducing caffeine in the zebrafish. Casein peptide compound and casein peptide at concentrations of 500 μg / mL and 177 μg / mL were administered in water, respectively. Lithium chloride at a positive control concentration of 500 μM was also used. A normal control group (zebrafish treated with aquaculture water) and a model control group were also included. Each well (experimental group) contained 3 mL of liquid. After 18 h of treatment, the zebrafish were transferred to 96-well plates, one fish per well (i.e., per concentration group). Ten zebrafish were randomly selected from each experimental group, and the insomnia time was measured using a behavioral analyzer. The insomnia time was used to evaluate the sleep-improving effect of the test product on caffeine-induced insomnia in zebrafish. The formula for calculating the sleep-improving effect of the test product on insomniac zebrafish is as follows: Statistical analysis was performed using ANOVA and Dunnett's T-test. A p-value < 0.05 indicated a significant difference, providing a representative experimental profile.

[0086] The experimental results are shown in Table 12. Figure 10 for, Figure 11 .

[0087] Table 12 Quantitative data on the sleep-improving effect of the test sample on zebrafish (n=10)

[0088] From Table 12 and Figure 10 and Figure 11The insomnia time of zebrafish in the model control group (9272 s) was significantly shorter than that of the normal control group (6400 s), indicating that the model was successfully established. The insomnia time of zebrafish in the positive control group (lithium chloride 500 μM concentration group) was 6465 s, which was significantly shorter than that of the model control group, with a sleep improvement rate of 98%. This indicates that lithium chloride has a significant sleep improvement effect on caffeine-induced insomnia in zebrafish.

[0089] At concentrations of 500 μg / mL and 177 μg / mL, the insomnia duration in zebrafish was 5848 s and 6793 s, respectively, with sleep improvement effects of 119% and 86%, respectively. Compared with the model control group, p<0.01 & p<0.05. This suggests that both the casein peptide compound and casein peptide have significant sleep-improving effects on caffeine-induced insomnia in zebrafish at the experimental concentrations.

[0090] In summary, the compound casein peptide provided by this invention has a significant sleep-improving effect on caffeine-induced insomnia in zebrafish.

[0091] Based on mouse and zebrafish experiments, the compound casein peptide provided by this invention has a significant sleep-improving effect on organisms and a protective effect on renal function.

[0092] The above are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound casein peptide for aiding sleep improvement, characterized in that, Including the following raw materials: Casein peptides, walnut peptides, and wheat oligopeptides.

2. The compound casein peptide for aiding sleep improvement according to claim 1, characterized in that, The ingredients include the following parts by weight: 32-36 parts casein peptide, 28-32 parts walnut peptide, and 34-38 parts wheat oligopeptides.

3. The compound casein peptide for aiding sleep improvement according to claim 2, characterized in that, The ingredients include the following parts by weight: 34 parts casein peptide, 30 parts walnut peptide, and 36 parts wheat oligopeptides.

4. The compound casein peptide for aiding sleep improvement according to claim 1, characterized in that, The casein peptide contains short peptides with amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO:

2.

5. The compound casein peptide for aiding sleep improvement according to claim 1, characterized in that, The walnut peptide is a short peptide with an amino acid sequence as shown in SEQ ID NO:

3.

6. The compound casein peptide for aiding sleep improvement according to claim 1, characterized in that, The wheat oligopeptide contains short peptides with amino acid sequences as shown in SEQ ID No. 4 and SEQ ID No.

5.

7. A method for preparing a compound casein peptide for aiding sleep improvement as described in any one of claims 1 to 6, characterized in that, Casein peptides, walnut peptides, and wheat oligopeptides are mixed evenly to obtain compound casein peptides.

8. The use of a compound casein peptide as described in any one of claims 1 to 6 in a sleep-improving medicament.

9. The use of a compound casein peptide as described in any one of claims 1 to 6 in a medicament for protecting renal function.

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