Casein hydrolyzate with sleep-improving effect and its preparation and application

Abstract

The present invention belongs to the field of functional polypeptides, and specifically relates to a casein hydrolyzate with sleep-improving effects, and its preparation and application. The casein hydrolyzate uses high-concentration casein as a substrate, and by adding a composite protease, the enzymatic hydrolysis effect is improved without prolonging the enzymatic hydrolysis time, and the degree of hydrolysis reaches 27.67%, and the polypeptide content in the casein hydrolyzate reaches 67.4%. Both low-dose (25 mg / kg) and high-dose (50 mg / kg) casein hydrolyzates can effectively shorten the sleep latency and prolong the sleep time. In addition, the casein hydrolyzate product prepared by the process of the present invention contains two polypeptides with high-efficiency sleep-aiding activity: YAKPAA and TPTLN. The content of YAKPAA in the hydrolyzate reaches 1.58%, and the content of TPTLN reaches 0.5%, which can effectively improve sleep problems.

Description

Technical Field

[0001] The invention belongs to the field of functional polypeptides, and in particular relates to a casein hydrolyzate having the effect of improving sleep, and a preparation method and application thereof. Background Art

[0002] Food-derived active peptides can not only provide rich nutrients for humans, but also have many biological functions, such as lowering blood pressure, anti-inflammatory, anti-oxidation, anti-type 2 diabetes and anti-cancer, etc. Milk itself is regarded as a healthy food that calms the nerves. Milk protein is mainly whey protein and casein. There have been many studies on the efficacy of casein on opioid activity.

[0003] Chinese patent CN200980120762.2 discloses an anti-anxiety composition comprising αS1-casein hydrolysate, and proposes a biosynthetic or chemical synthesis method to prepare two peptides YLGYL and YLGYLE with anxiolytic activity;

[0004] US Patent US005846939A discloses a series of α S1 -Antianxiety peptides of α-CZP homologous sequence derived from casein with Tyr at the N-terminus. The above two patents mention that the process of using casein to separate and purify subunits for further enzymatic hydrolysis or the processing method of chemically synthesized peptides is too complicated, and the operating instruments and equipment are expensive, such as freeze dryers, liquid separation chromatography, etc., which is not conducive to widespread use in production.

[0005] Chinese patent CN 202010901730.4 discloses a sleep-improving enzymatic hydrolysate rich in aromatic amino acids and a preparation method thereof, and proposes an enzymatic preparation process to obtain an enzymatic hydrolysate containing a characteristic peptide composition of aromatic amino acids. Chinese patent CN 202111284782.2 discloses a highly soluble casein sleep-improving peptide and its preparation process and application. In the preparation methods of the above two patents, the ratio of casein to distilled water is too low, the protein concentration is too low, and the operating instruments and equipment are expensive, such as refrigerated high-speed centrifuges, ultrasonic processors, etc., which are not conducive to the widespread use of continuous industrial production.

[0006] Therefore, in order to achieve industrialized continuous and stable production of casein hydrolyzate products with sleep-improving effects, the existing methods need to be improved. Summary of the invention

[0007] In order to solve the above technical problems, the present invention will provide a casein hydrolyzate and its preparation process which can improve the enzymatic hydrolysis effect of the product, simplify the product preparation process, reduce the difficulty of process implementation, improve product stability, and reduce production costs under the premise of high substrate concentration.

[0008] One of the technical solutions provided by the present invention is a method for preparing casein hydrolysate, which is specifically as follows:

[0009] (1) Mix casein and water in a mass ratio of 2.5-3:10, soak at room temperature with stirring for 30-60 minutes, stir and heat to 40-45°C, adjust the pH of the liquid to 6.5-7.5, and stir and fully dissolve at 40-45°C for 30-90 minutes;

[0010] Furthermore, the casein is bovine casein or other mammalian casein;

[0011] Further, the pH of the feed solution was adjusted to 7.0±0.2 using 7.5% sodium hydroxide aqueous solution;

[0012] (2) After there are no obvious white particles in the feed solution, add a composite protease with a casein mass of 0.1-10.0‰ (w / w), and after enzymatic hydrolysis for 30-60 min, adjust the pH of the feed solution to 6.5-7.5, continue enzymatic hydrolysis for 2-2.5 hours, and then add papain with a casein mass of 0.1-10.0‰ (w / w), and continue enzymatic hydrolysis for 1-2 hours;

[0013] The composite protease is composed of neutral protease, papain and trypsin, and the mass ratio is: 1-5:1-9:1-3;

[0014] Further, the pH of the feed solution was adjusted to 7.0±0.2 using 7.5% sodium hydroxide aqueous solution;

[0015] Preferably, the neutral protease is derived from Aspergillus oryzae fermentation, the deposit number is CGMCC No.7586, and the preparation method is disclosed in Chinese invention patent ZL201510176420.X;

[0016] (3) After the enzymatic hydrolysis is completed, the supernatant is centrifuged and concentrated and sterilized to obtain the casein hydrolyzate product;

[0017] Furthermore, the supernatant is concentrated by low-temperature vacuum to a liquid concentration of 35-45°Bx; the concentrated liquid is sterilized by UHT high temperature; the sterilized liquid is spray-dried and packaged to obtain a finished product.

[0018] The second technical solution provided by the present invention is a casein hydrolyzate prepared by the method described in the first technical solution;

[0019] Furthermore, the casein hydrolyzate contains two oligopeptides with high sleep-promoting activity: YAKPAA (Tyr-Ala-Lys-Pro-Ala-Ala, SEQ ID NO. 1) and TPTLN (Thr-Pro-Thr-Leu-Asn, SEQ ID NO. 2), with contents reaching 1.58% and 0.5% respectively.

[0020] The third technical solution provided by the present invention is the application of the casein hydrolyzate described in the second technical solution; in particular, the application in improving sleep, more particularly, the application in preparing medicines for preventing, improving or treating sleep disorders; or the application in preparing foods for improving sleep disorders, or the application in preparing health products for improving sleep disorders;

[0021] Sleep disorder is a general term for a large class of diseases with difficulty falling asleep, shallow sleep, and easy awakening as the main clinical features. The sleep disorder described in the present invention includes but is not limited to the seven major sleep disorders described in the third edition of the International Classification of Sleep, namely, insomnia, sleep-related breathing disorders, central hypersomnia, circadian rhythm sleep-wake state, parasomnias, sleep-related movement disorders and other sleep disorders, especially problems such as reduced sleep time, difficulty falling asleep, insomnia, and poor sleep quality;

[0022] Furthermore, the casein hydrolysate can be used alone in the preparation of the food, health care product or medicine; or it can be used in combination with other components having sleep-promoting activity;

[0023] Furthermore, the casein hydrolyzate is prepared into the form of beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion tablet;

[0024] Preferably, the casein hydrolysate is prepared into microcapsules to improve its gastrointestinal digestion stability, bioavailability and extend shelf life, so that it can be better applied in the food industry and health care fields.

[0025] The fourth technical solution provided in this proposal is a composition comprising the casein hydrolyzate described in the second technical solution, wherein the composition has the casein hydrolyzate as the only active ingredient and may also contain other components with sleep-aiding activity and / or auxiliary materials.

[0026] Beneficial Effects

[0027] The invention provides a casein hydrolyzate with sleep-promoting effect and a preparation process thereof. The process increases the concentration of feed substrates, increases batch output, improves production efficiency and reduces production costs. Under the premise of high substrate concentration, by adding a composite protease, the enzymatic hydrolysis effect is improved without prolonging the enzymatic hydrolysis time, the hydrolysis degree reaches 27.67%, and the polypeptide content in the casein hydrolyzate reaches 67.4%. The product preparation process is simplified, the difficulty of implementing the process is reduced, the process can be implemented more stably, the product stability is improved, the unqualified product incidence rate is reduced, and the production cost is reduced.

[0028] The casein hydrolysate prepared by the present invention can effectively shorten the sleep latency and prolong the sleep time at both low doses (25 mg / kg) and high doses (50 mg / kg). In addition, the casein hydrolysate product prepared by the process of the present invention contains two oligopeptides with high-efficiency sleep-promoting activity: YAKPAA (Tyr-Ale-Lys-Pro-Ale-Ale) and TPTLN (Thr-Pro-Thr-Leu-Asn), and the content of YAKPAA in the hydrolysate reaches 1.58%, and the content of TPTLN reaches 0.5%.

[0029] YAKPAA can be combined with GABA A Receptor binding, binding sites are: SER205, ASN60, ASP192, TYR210, TYR58, THR207, THR142, GLU189, activate GABA A TPTLN can bind to GABAA receptors at SER205, ASN60, TYR210, GLU138, THR207, LYS156, ASN103 and SER195, activating GABA A 5 mg / kg of YAKPAA and TPTLN can significantly shorten the sleep latency and prolong the sleep time of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the total ion diagram of casein hydrolysis products.

[0031] Figure 2 This is the secondary mass spectrum of YAKPAA.

[0032] Figure 3 is the YAKPAA peptide sequence.

[0033] Figure 4 This is the secondary mass spectrum of TPTLN.

[0034] Figure 5is the peptide sequence of TPTLN.

[0035] Figure 6 GABA A Schematic diagram of molecular docking of receptor 6HUO and hexapeptide YAKPAA.

[0036] Figure 7 GABA A Schematic diagram of molecular docking of receptor 6HUO and pentapeptide TPTLN.

[0037] Figure 8 The effect of YAKPAA on sleep latency and sleep duration in mice;

[0038] A is the sleep latency and B is the sleep duration.

[0039] Fig. 9 The effect of TPTLN on sleep latency and sleep duration in mice;

[0040] A is the sleep latency and B is the sleep duration.

[0041] Fig.10 The effect of YAKPAA on sleep-related neurotransmitters in mouse serum;

[0042] Among them, A is 5-HT, B is GABA, C is NE, D is Glu, E is 5-HIAA, F is β-EP, G is cortisol, and H is GABA / Glu.

[0043] Fig.11 The effect of YAKPAA on sleep-related neurotransmitters in mouse brain tissue;

[0044] Among them, A is 5-HT, B is GABA, C is NE, D is Glu, E is β-EP, F is cortisol, and G is GABA / Glu.

[0045] Fig.12 The effect of TPTLN on sleep-related neurotransmitters in mouse serum;

[0046] Among them, A is 5-HT, B is GABA, C is NE, D is Glu, E is 5-HIAA, F is β-EP, G is cortisol, and H is GABA / Glu.

[0047] Fig.13 The effect of TPTLN on sleep-related neurotransmitters in mouse brain tissue;

[0048] Among them, A is 5-HT, B is GABA, C is NE, D is Glu, E is β-EP, F is cortisol, and G is GABA / Glu.

[0049] Fig.14 The effect of casein hydrolysate on sleep latency and sleep duration in mice;

[0050] A is the sleep latency and B is the sleep duration.

[0051] Fig.15 The effect of casein hydrolysate on sleep-related neurotransmitters in mouse serum;

[0052] Among them, A is 5-HT, B is GABA, C is NE, D is Glu, and E is GABA / Glu.

[0053] Fig.16 The effects of casein hydrolysate and commercially available casein hydrolysate peptides on sleep latency and sleep duration in mice;

[0054] A is the sleep latency and B is the sleep duration.

[0055] Note: Figure 10-Figure 16 “*”: compared with the control group, p<0.05; “**”: compared with the control group, p<0.01; “***”: compared with the control group, p<0.001. DETAILED DESCRIPTION

[0056] The present invention is described below by specific embodiments. The technical means not particularly described in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0057] The present invention is further explained below through specific implementation modes.

[0058] Example 1: A method for preparing casein hydrolysate

[0059] (1) Preparation method

[0060] a. Take 250kg casein (cow milk casein) and add it to 1000kg water. Stir and soak it for 60min at room temperature. After stirring and heating to 40-45℃, adjust the pH of the liquid to 7.0±0.2 with 7.5% sodium hydroxide aqueous solution, and stir and fully dissolve it at 40-45℃ for 60min.

[0061] b. After there are no obvious white particles in the feed liquid, add 0.4‰ (w / w) of composite protease based on the mass of casein substrate, including neutral protease, papain, and trypsin in a ratio of 1:3:1 (the neutral protease is produced from Aspergillus oryzae, with a deposit number of CGMCC No.7586, and the preparation method of the neutral protease is shown in ZL201510176420.X Example 1), after half an hour of enzymolysis, adjust the pH of the feed liquid to 7.0±0.2 with 7.5% sodium hydroxide aqueous solution, continue enzymolysis for 2.5 hours, then add 0.5‰ (w / w) of papain based on the mass of casein substrate, and continue enzymolysis for 1.5 hours;

[0062] c. After the enzymatic hydrolysis is completed, the supernatant is separated by a butterfly separator;

[0063] d. The supernatant is concentrated by low temperature vacuum to a feed concentration of 40°Bx;

[0064] e. After concentration, the liquid is sterilized by UHT high temperature;

[0065] f. After sterilization, the liquid is spray-dried and packaged to obtain the finished casein hydrolyzate.

[0066] (2) Analysis of casein hydrolysate

[0067] The main components, amino acid composition and molecular weight distribution range of the casein hydrolyzate prepared in step (1) were measured, and the results are shown in Tables 1 to 3:

[0068] Table 1 Content of main components of casein hydrolysate

[0069]

[0070] Table 2 Amino acid composition of casein hydrolysate (absolute content, mg / g)

[0071]

[0072] Table 3 Relative molecular weight distribution of casein hydrolysate

[0073]

[0074] (3) Peptide analysis

[0075] The casein hydrolysate prepared in step (1) is subjected to LC-MS / MS peptide spectrum analysis to obtain the peptides contained in the casein hydrolysate, and the peptides are sorted and classified. Finally, the bioinformatics method is used to further perform molecular docking on the initially screened peptides, and the final theoretical effective peptides are determined according to the scores. The specific analysis process is as follows:

[0076] ① Pretreatment of peptide samples:

[0077] The casein hydrolysate sample obtained by spray drying was re-dissolved with 100 μL 50 mM NH4HCO3, and DTT solution was added to make the final concentration 10 mmol / L, and reduced in a 56°C water bath for 1 h. Then IAA solution was added to make the final concentration 50 mmol / L, and the reaction was protected from light for 40 min. Finally, a self-filled desalting column was used for desalting, and the solvent was evaporated in a 45°C vacuum centrifugal concentrator.

[0078] ② Analysis of casein hydrolysate samples by LC-MS / MS

[0079] Capillary liquid chromatography conditions:

[0080] 1) Analytical column: 150 μm id × 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100Å;

[0081] 2) Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid, 80% ACN;

[0082] 3) Flow rate: 600 mL / min;

[0083] 4) Analysis time for each component: 66 min. Mobile phase method: 0 min, 4%B; 2 min, 8%B; 45 min, 28%B; 55 min, 40%B; 56 min, 95%B; 66 min, 95%B.

[0084] Mass spectrometry parameters:

[0085] 1) Primary mass spectrometry parameters: Resolution: 70,000; AGCtarget: 3e6; MaximumIT: 100 ms;

[0086] Scanrange: 300 to 1800 m / z.

[0087] 2) Secondary mass spectrometry parameters: Resolution: 17,500; AGCtarget: 1e5; MaximumIT: 50 ms; TopN: 20; NCE / steppedNCE: 28.

[0088] The raw file generated by LC-MS / MS data acquisition of casein hydrolysate sample is opened with Xcalibur, and you can see Figure 1 Total ion current chromatogram shown.

[0089] ③Molecular docking

[0090] The mass spectrometry raw files were analyzed for peptide sequences using the De novo method, and the peptides identified by mass spectrometry were combined with GABA using Schrodinger software. A The receptor 6HUO was docked in batches, and the docking module selected the ligant docking module high-throughput screening option. A The identified peptides were classified and screened based on the docking site of the receptor protein 6HUO, the number of hydrogen bonds between the ligand and the receptor, the number of repetitions of the binding site with benzodiazepines (BZD), and the protein source (partial results are shown in Table 4). Finally, two pentapeptides (TPTLN, VPYPQ) and two hexapeptides (YVPLGT, YAKPAA) were determined for in vitro chemical synthesis and in vivo animal experimental verification (as shown in Example 3).

[0091] Table 4: Molecular docking results of four casein hydrolyzed peptides using Schrodinger

[0092]

[0093] Note: The lower the docking score, the better the binding between the small molecule peptide ligand and the protein receptor.

[0094] Example 2: GABA A Molecular docking of receptor 6HUO and hexapeptide YAKPAA / TPTLN

[0095] In the analysis of the polypeptide in Example 1, the secondary mass spectrum of the hexapeptide YAKPAA is as follows: Figure 2 The peptide sequence is shown in Figure 3 As shown in the figure, the fragment ions of peptides include: N-terminal fragment ions (a, b, c types) and C-terminal fragment ions (x, y, z types). The side chains of a, y, and z type ions break to form d, v, and w type ions respectively. In addition, there are internal ions formed by the breakage of both ends. The b and y series ions are the most common. The primary structure of the peptide can be analyzed and inferred based on the b or y series fragment ions of the peptide. Figure 2 m / z 310.67285 is 1 / 2 of the hexapeptide [M+H + ] ion signal, which is consistent with the molecular weight of the hexapeptide of 619.7097. The hexapeptide was further analyzed by secondary mass spectrometry using in-source collision-induced dissociation technology, and the primary structure of the hexapeptide was determined to be Tyr-Ala-Lys-Pro-Ala-Ala.

[0096] In the analysis of the polypeptide in Example 1, the secondary mass spectrum of the pentapeptide TPTLN is as follows: Figure 4 The peptide sequence is shown in Figure 5As shown in the figure, the fragment ions of peptides include: N-terminal fragment ions (a, b, c types) and C-terminal fragment ions (x, y, z types). The side chains of a, y, and z type ions break to form d, v, and w type ions respectively. In addition, there are internal ions formed by the breakage of both ends. The b and y series ions are the most common. The primary structure of the peptide can be analyzed and inferred based on the b or y series fragment ions of the peptide. Figure 4 m / z 545.29181 is the [M+H] of pentapeptide + ion signal, which is consistent with the molecular weight of the pentapeptide of 544. The pentapeptide was further analyzed by secondary mass spectrometry using in-source collision-induced dissociation technology, and the primary structure of the pentapeptide was determined to be Thr-Pro-Thr-Leu-Asn.

[0097] The hexapeptide YAKPAA / pentapeptide TPTLN in casein hydrolysate has potential functional activity to help sleep. In Example 1, the peptides obtained by mass spectrometry identification were subjected to batch molecular docking using the Schrodinger software ligant docking module (high throughput screening option). On this basis, in order to further analyze the structure of the hexapeptide YAKPAA / pentapeptide TPTLN and its binding to the receptor protein 6HUO, the hexapeptide YAKPAA / pentapeptide TPTLN and GABA were also docked. A The receptor was docked using the ligant docking module (accurate docking option), and the specific results are as follows:

[0098] YAKPAA and GABA A The receptor binding sites are: SER205, ASN60, ASP192, TYR210, TYR58, THR207, THR142, GLU189 ( Figure 6 as shown).

[0099] TPTLN and GABA A The receptor binding sites are: SER205, ASN60, TYR210, GLU138, THR207, LYS156, ASN103 and SER195 ( Figure 7 as shown).

[0100] GABA and GABA A Receptor binding, activation of GABA A Receptor, GABA A The acceptor is a Cl - Channel, GABA A Activation of the receptor leads to channel opening, Cl -The influx of YAKPAA into nerve cells causes the hyperpolarization of the cell membrane, inhibits the excitation of the nerve cells, and thus plays a sedative and hypnotic role. A Receptor binding, directly activating GABA A receptors, increasing GABA A The sensitivity of the receptor to sodium pentobarbital is increased, thereby improving sleep. Based on the molecular docking technology analysis (ligant docking module is selected for molecular docking, and the accuracy docking option is selected), the casein hydrolysate hexapeptide YAKPAA and GABA A The binding energy of receptor binding is high, with the binding energy and number of hydrogen bonds being -10.316 and 8, respectively; TPTLN and GABA A The binding energy of receptor binding is relatively high, and the binding energy and the number of hydrogen bonds are -8.722 and 8, respectively (this result is somewhat different from Table 4, because both of them are selected ligant docking modules, but the high-throughput screening option selected for batch docking in Table 4 has a low docking accuracy. After determining the peptide segment according to the screening results in Table 4, the accuracy docking option is reselected to obtain the binding energy and number of hydrogen bonds for accurate docking in this embodiment).

[0101] Example 3: Determination of sleep-promoting activity of YAKPAA / TPTLN / YVPLGT / VPYPQ

[0102] 1. Experimental Materials

[0103] Experimental animals: ICR mice, male, 8 weeks old (20 ± 2 g), were purchased from Shanghai Slake Experimental Animal Co., Ltd.;

[0104] Normal saline;

[0105] Diazepam tablets (diazepam, Tianjin Lisheng Pharmaceutical Co., Ltd.);

[0106] Pentobarbital sodium (Beijing Chemical Reagent Company);

[0107] GABA (Tianjin Century Weikang Biotechnology Co., Ltd.);

[0108] Hexapeptide YAKPAA, pentapeptide TPTLN, hexapeptide YVPLGT and pentapeptide VPYPQ (commissioned to Shanghai Jier Biochemical Co., Ltd. for chemical synthesis).

[0109] 2. Experimental methods

[0110] ICR mice were allowed to eat and drink freely. All mice were allowed to adapt to the environment for one week (7 days) before the experiment. Then the experimental mice were divided into seven groups for drug administration, with 10 mice in each group, namely control group (Control), diazepam group, GABA group, and YAKPAA / TPTLN / YVPLGT / VPYPQ group. The details are as follows:

[0111] Blank control group: daily gavage with pure water;

[0112] Diazepam group: daily dose was 1 mg / kg bodyweight, twice a day;

[0113] GABA group: daily dose was 83.3 mg / kg bodyweight;

[0114] YAKPAA or TPTLN or YVPLGT or VPYPQ group: daily dose of 5 mg / kg bodyweight. (The actual experiment was conducted in two batches, one for YAKPAA / TPTLN and one for YVPLGT / VPYPQ.)

[0115] The drug was administered continuously for 7 days, and each group had free access to food and water during the administration period. The experiment started 30 minutes after the last administration. At the beginning of the last administration of the mice, a stopwatch was used to count down for 30 minutes, and then 42 mg / kg of sodium pentobarbital was intraperitoneally injected into the four groups of mice. The injection time T1 was recorded at the same time. When the righting reflex of the mice disappeared, the righting reflex disappearance time T2 was recorded, which was the time when the mice began to fall asleep. When the righting reflex of the mice was restored, it was the time when the mice woke up T3. The sleep latency and sleep duration of the mice were calculated respectively. The shorter the sleep latency and the longer the sleep duration, the better the sleep-aiding effect of the peptide. The calculation formula is as follows:

[0116] Sleep latency = T2-T1; sleep duration = T3-T2.

[0117] After the sleep recording was completed, the drug was administered at the above dose for 4 days. One hour after the last administration, the mice were euthanized, blood was collected from the eyeballs, plasma was collected, centrifuged at 3000 rpm, 4°C for 15 min, the supernatant was collected, and frozen in a -80°C refrigerator for subsequent analysis of changes in sleep-related neurotransmitters in serum, including A: 5-HT, B: GABA, C: NE, D: Glu, E: 5-HIAA, F: β-EP, G: cortisol, H: GABA / Glu. At the same time, the mouse brain was collected and placed at -80°C for subsequent analysis of changes in sleep-related neurotransmitters in mouse brain tissue, including A: 5-HT, B: GABA, C: NE, D: Glu, E: β-EP, F: cortisol, and G: GABA / Glu.

[0118] 3. Experimental results

[0119] (1) Sleep time results and analysis

[0120] ①: Figure 8 A and Figure 8 B in the table represents the sleep latency and sleep duration of YAKPAA (also shown in Table 5). Diazepam was used as a positive control in the experiment. The results showed that diazepam can significantly shorten the sleep latency of mice and prolong the sleep duration of mice. GABA (83.3 mg / kg) was used as a comparative product in the experiment. The dosage of GABA was selected based on the maximum human GABA dose of 500 mg / 60kg body weight, which was converted to a corresponding mouse dose of 83.3 mg / kg body weight. The results showed that 5 mg / kg of the hexapeptide YAKPAA can significantly shorten the sleep latency of mice, and its effect is better than GABA; at the same time, GABA and YAKPAA were shown to significantly prolong the sleep duration of mice, and there was no significant difference in the effects between the two.

[0121] Table 5 Sleep latency and sleep duration in different groups (YAKPAA)

[0122]

[0123] Note: “*”: compared with the control group, p<0.05; “**”: compared with the control group, p<0.01; “***”: compared with the control group, p<0.001.

[0124] ②: Fig. 9 A and Fig. 9B in the table represents the sleep latency and sleep duration of the pentapeptide TPTLN (also shown in Table 6). Diazepam was used as a positive control in the experiment. The results showed that diazepam can significantly shorten the sleep latency of mice and prolong the sleep duration of mice. GABA (83.3 mg / kg) was used as a comparative product in the experiment. The dosage of GABA was selected based on the maximum human dose of GABA of 500 mg / 60kg body weight, which was converted to a corresponding dose of 83.3 mg / kg body weight for mice. The results showed that 5 mg / kg of pentapeptide TPTLN can significantly shorten the sleep latency of mice, and its effect is better than GABA; at the same time, GABA and TPTLN were shown to significantly prolong the sleep duration of mice, and there was no significant difference in the effects of the two.

[0125] Table 6 Sleep latency and sleep duration (TPTLN) in different groups

[0126]

[0127] Note: “*”: compared with the control group, p<0.05; “**”: compared with the control group, p<0.01; “***”: compared with the control group, p<0.001.

[0128] ③: Table 7 shows the sleep latency and sleep duration of the hexapeptide YVPLGT. The experiment used diazepam as a positive control. The results showed that diazepam can significantly shorten the sleep latency and prolong the sleep duration of mice. However, 5 mg / kg of hexapeptide YVPLGT could not significantly shorten the sleep latency of mice, nor could it significantly prolong the sleep duration of mice.

[0129] Table 7 Sleep latency and sleep duration (YVPLGT) in different groups

[0130]

[0131] Note: “*”: compared with the control group, p<0.05; “**”: compared with the control group, p<0.01.

[0132] ④: Table 8 shows the sleep latency and sleep duration of pentapeptide VPYPQ. The experiment used diazepam as a positive control. The results showed that diazepam can significantly shorten the sleep latency of mice and prolong the sleep duration of mice. However, 5 mg / kg of pentapeptide VPYPQ could not significantly shorten the sleep latency of mice, nor could it significantly prolong the sleep duration of mice.

[0133] Table 8 Sleep latency and sleep duration (VPYPQ) in different groups

[0134]

[0135] Note: “*”: compared with the control group, p<0.05; “**”: compared with the control group, p<0.01.

[0136] (2) Analysis of physical and chemical indicators

[0137] The occurrence of sleep is closely related to neurotransmitters in the brain. For example, 5-HT in the brain can participate in the regulation of physiological functions such as appetite, pain, sleep and body temperature. The experiment analyzed the levels of sleep-related neurotransmitters in the serum and brain of mice ( Fig.10 - Fig.13 ) To explore the sleep-inducing mechanism of action of YAKPAA and TPTLN.

[0138] ①YAKPAA's sleep-aiding mechanism ( Fig.10 - Fig.11 )

[0139] 5-HT, also known as serotonin, is an important inhibitory neurotransmitter widely present in mammals. Its level is very high in the cerebral cortex and synapses, mainly distributed in the pineal gland and hypothalamus. The increase in the level of 5-HT can play a certain sedative and sleep-promoting role. The experimental results show that compared with the control group, diazepam, GABA and YAKPAA can significantly increase the content of 5-HT in mouse serum ( Fig.10 A in the figure), and YAKPAA is better than diazepam in increasing 5-HT levels ( Fig.10 A, p<0.01 and p<0.05), and YAKPAA can also significantly increase the 5-HT content in the mouse brain ( Fig.11 A in the figure shows that YAKPAA can increase the level of 5-HT better than GABA (p<0.05). This further verifies the sleep-promoting effect of YAKPAA.

[0140] 5-Hydroxyindoleacetic acid (5-HIAA) is one of the metabolites of 5-HT. YAKPAA can also significantly reduce the content of 5-HIAA in mouse serum ( Fig.10 E) (p<0.05). Although diazepam and GABA can also partially reduce the content of 5-HIAA in mouse serum, neither has a significant effect (p>0.05). These results suggest that YAKPAA can partially inhibit the decomposition of 5-HT and play a role in promoting sleep.

[0141] On the other hand, compared with the control group, diazepam, GABA and YAKPAA could significantly reduce the levels of Glu in the serum and brain of mice ( Fig.10 D and Fig.11 D in the figure), and YAKPAA is significantly better than diazepam in reducing the level of Glu in the brain ( Fig.11D in (p<0.0001 and p<0.01). Glu is the most abundant excitatory neurotransmitter in the brain. In the Glu system, Glu can activate Glu receptors on the postsynaptic membrane of Glu neurons, thereby triggering excitatory impulses. This suggests that YAKPAA can play a good role in helping sleep by reducing the level of Glu in the brain.

[0142] In addition, diazepam, GABA and YAKPAA can significantly increase the ratio of GABA / Glu ( Fig.10 H and Fig.11 G in the brain), which has the same effect as GABA ( Fig.10 B and Fig.11 Many substances that improve sleep work by improving the imbalance of GABA / Glu inhibition / excitation metabolism and regulating the GABA / Glu ratio.

[0143] On the other hand, diazepam, GABA, and YAKPAA had no significant effect on the NE content in mouse serum ( Fig.10 C in the mouse brain), but both can significantly reduce the NE content in the mouse brain ( Fig.11 C in the figure), and the reducing effect of YAKPAA was stronger than that of diazepam and GABA (p<0.0001 and p<0.01). Norepinephrine (NE) is also a neurotransmitter that regulates the sleep-wake process. Its release peaks in the wakeful state, and the reduction of NE helps promote sleep. This further shows that YAKPAA can promote sleep by inhibiting the secretion of NE in the mouse brain.

[0144] In addition, YAKPAA can also promote the pleasure and relaxation of mice to a certain extent. The experimental results show that compared with the control group, diazepam, GABA and YAKPAA can significantly increase the content of β-endorphin (β-EP) in the serum of mice ( Fig.10 In contrast, the level of β-endorphin in the serum of mice in the YAKPAA group increased significantly (p<0.01 and p<0.05). However, there was no significant change in β-EP in the brain ( Fig.11 E in the figure). β-endorphin is an endogenous morphine-like substance belonging to the opioid peptide family. β-endorphin in serum can cross the blood-brain barrier and produce a sedative effect similar to opium in mice, which has analgesic effects and promotes a sense of pleasure. The increase in β-endorphin content can play a certain role in helping sleep.

[0145] At the same time, YAKPAA can relieve stress to a certain extent. Cortisol is a glucocorticoid. Increased cortisol can lead to anxiety. The experimental results show that compared with the control group, YAKPAA can reduce the content of cortisol in the serum and brain of mice ( Fig.10 G and Fig.11 F in the brain), and its reduction effect reached a significant level in the brain ( Fig.11 F in (p<0.05).

[0146] In summary, YAKPAA can promote sleep in mice by increasing 5-HT in the serum and brain of mice, inhibiting the levels of NE and Glu in the brain, and regulating the ratio of GABA / Glu, and has a calming and stress-relieving effect by regulating the secretion of β-endorphin and cortisol.

[0147] ② The sleep-promoting mechanism of TPTLN ( Fig.12 - Fig.13 )

[0148] 5-HT, also known as serotonin, is an important inhibitory neurotransmitter widely present in mammals. Its level is very high in the cerebral cortex and synapses, mainly distributed in the pineal gland and hypothalamus. The increase in 5-HT content can play a certain sedative and sleep-promoting role. The results showed that compared with the control group, diazepam and TPTLN can significantly increase the content of 5-HT in mouse serum ( Fig.12 A and Fig.13 The 5-HT content in the brain of mice treated with TPTLN was significantly higher than that in the diazepam group ( Fig.13 These results further confirmed the good sedative and sleep-inducing effects of TPTLN.

[0149] 5-Hydroxyindoleacetic acid (5-HIAA) is one of the metabolites of 5-HT and has no biological activity to promote sleep. Compared with the control group, diazepam, GABA and TPTLN can all reduce 5-HIAA in the serum of mice, but there is no significant difference ( Fig.12 These results suggest that TPTLN plays a role in promoting sleep by increasing the levels of 5-HT in the serum and brain of mice.

[0150] On the other hand, diazepam, GABA and TPTLN can significantly increase the GABA content in mouse serum ( Fig.12 However, compared with diazepam and GABA, only TPTLN can significantly upregulate the level of GABA in the mouse brain ( Fig.13(B, p<0.01). GABA is an important inhibitory neurotransmitter in the brain and activates GABA receptors to induce Cl - Channels open, increasing cell membrane resistance to Cl - The permeability of Cl - Flowing into nerve cells causes cell membrane hyperpolarization, inhibiting the excitement of neuronal cells, thus achieving a calming and hypnotic effect. This further verifies the good sleep-inducing effect of TPTLN.

[0151] Glu is the most abundant excitatory neurotransmitter in the brain. In the Glu system, Glu can activate the Glu receptors on the postsynaptic membrane of Glu neurons, thereby triggering excitatory impulses. In the GABA system, Glu can enhance the GABAergic inhibitory tension in the entire pathway by activating the presynaptic Glu receptors (changes in Glu such as Fig.12 D and Fig.13 Many substances that improve sleep work by improving the inhibitory / excitatory metabolic imbalance of GABA / Glu and regulating the GABA / Glu ratio. The experimental results show that TPTLN can significantly increase the GABA / Glu ratio in both the serum and brain of mice ( Fig.12 H and Fig.13 This suggests that TPTLN can help sleep by increasing the ratio of GABA / Glu.

[0152] Norepinephrine (NE) is also a neurotransmitter that regulates the sleep-wake process. Its release peaks during wakefulness, and the reduction of NE helps promote sleep. The experimental results showed that TPTLN can significantly reduce the content of NE in the mouse brain ( Fig.13 C in the mouse brain, p<0.05), its effect is comparable to that of diazepam, which suggests that TPTLN can also play a good role in promoting sleep by inhibiting the synthesis of NE in the mouse brain. However, there was no significant change in serum NE ( Fig.12 C in.

[0153] β-Endorphin (β-EP) is an endogenous morphine-like substance belonging to the opioid peptide family. It has analgesic and pleasure-promoting effects. β-EP in serum can cross the blood-brain barrier and produce a sedative effect similar to opium in mice. The increase in β-EP content can play a certain role in promoting sleep. It is worth noting that compared with the control group, diazepam, GABA and TPTLN can significantly increase the level of β-EP in mouse serum ( Fig.12 F, p<0.05, p<0.05, p<0.0001, among which TPTLN had the strongest improvement effect ( Fig.12F in the figure, p<0.001). Moreover, compared with the control group, only TPTLN could significantly increase the level of β-EP in the mouse brain ( Fig.13 E in the figure, p<0.05). These results suggest that TPTLN can play a good sedative and sleep-inducing effect by increasing the level of β-endorphin in the serum and brain of mice.

[0154] In the experimental groups, no significant changes were observed in either serum or brain cortisol levels ( Fig.12 G and Fig.13 F in.

[0155] In summary, TPTLN mainly achieves a calming and sleep-inducing effect by upregulating the levels of sleep-related neurotransmitters such as 5-HT, GABA and β-EP in the serum and brain of mice, as well as the ratio of GABA / Glu, and downregulating the level of NE in the brain.

[0156] Example 4 Determination of sleep-promoting activity of casein hydrolyzate products

[0157] The finished casein hydrolysate prepared in Example 1 was used as an experimental sample to determine the sleep-promoting activity of the casein hydrolysate, and the method was as follows:

[0158] 1. Experimental Materials

[0159] Experimental animals: ICR mice, male, 8 weeks old (20 ± 2 g), were purchased from Shanghai Slake Experimental Animal Co., Ltd.;

[0160] Normal saline;

[0161] Diazepam tablets (diazepam, Tianjin Lisheng Pharmaceutical Co., Ltd.);

[0162] Pentobarbital sodium (Beijing Chemical Reagent Company);

[0163] Finished casein hydrolysate: prepared as in Example 1.

[0164] 2. Experimental methods

[0165] ICR mice were allowed to eat and drink freely. All mice were allowed to adapt to the environment for one week (7 days) before the experiment. Then the experimental mice were divided into four groups for drug administration, with 10 mice in each group, namely the control group (Control), the diazepam group, and the casein hydrolyzate high-dose / low-dose group. The details are as follows:

[0166] Blank control group: daily gavage with pure water;

[0167] Diazepam group: daily dose was 1 mg / kg bodyweight, twice a day;

[0168] Casein hydrolysate low-dose group: daily dose was 25 mg / kg bodyweight;

[0169] Casein hydrolysate high-dose group: daily dose of 50 mg / kg bodyweight;

[0170] After the mice were gavaged for 6 consecutive days, they were taken out from the barrier and gavaged first, and the gavage time was recorded with a stopwatch. After a countdown of 30 minutes, 42 mg / kg of sodium pentobarbital was injected, and the injection time T1 was recorded at the same time. When the righting reflex of the mouse disappeared, the righting reflex disappearance time T2 was recorded, which was the time when the mouse began to fall asleep. When the righting reflex of the mouse was restored, it was the time when the mouse woke up T3. The sleep latency and sleep duration of the mouse were calculated respectively. The shorter the sleep latency and the longer the sleep duration, the better the sleep-aiding effect of the peptide. The calculation formula is as follows:

[0171] Sleep latency = T2-T1; sleep duration = T3-T2.

[0172] After the sleep recording was completed, the drug was administered according to the above dose for another 4 days. One hour after the last administration, the mice were euthanized, blood was collected from the eyeballs, plasma was collected, and the supernatant was collected and frozen in a -80°C refrigerator for subsequent analysis of changes in sleep-related neurotransmitters in serum, including A: 5-HT, B: GABA, C: NE, D: Glu, and E: GABA / Glu.

[0173] 3. Experimental results

[0174] (1) Sleep time results and analysis

[0175] Fig.14 A and Fig.14 B in the figure represents the effects of low-dose and high-dose casein hydrolysate on the sleep latency and sleep duration of mice ( Fig.14 The dose shown in the table is the human dose converted to 10 times the mouse dose), as shown in Table 9. Diazepam was used as a positive control in the experiment, and the results showed that diazepam could significantly shorten the sleep latency of mice and prolong the sleep duration of mice (p<0.01). On the other hand, both low-dose casein hydrolysate and high-dose casein hydrolysate could significantly shorten the sleep latency of mice (p<0.05). At the same time, both low-dose casein hydrolysate and high-dose casein hydrolysate could significantly prolong the sleep duration of mice (p<0.05 and p<0.01).

[0176] Table 9 Effects of different doses of casein hydrolysate on sleep latency and sleep duration in mice

[0177]

[0178] Note: “*”: compared with the control group, p<0.05; “**”: compared with the control group, p<0.01.

[0179] (2) Analysis of physical and chemical indicators

[0180] The occurrence of sleep is mainly determined by neurotransmitters in the brain. Some neurotransmitters, such as 5-HT, are mainly produced by enterochromaffin cells and transported to the brain through the blood to participate in the regulation of physiological functions such as pain, sleep and body temperature. The experiment analyzed the levels of sleep-related neurotransmitters in mouse serum ( Fig.15 ) To explore the sleep-aiding mechanism of the casein hydrolyzate prepared by the present invention.

[0181] The results show that:

[0182] 5-HT is an inhibitory neurotransmitter widely present in mammalian tissues. It is an indole derivative with a molecular formula of C10H12N2O. 5-HT was first discovered in serum and is also known as serotonin. Its levels are very high in the cerebral cortex and synapses. It is mainly distributed in the pineal gland and hypothalamus and is involved in the regulation of physiological functions such as pain, sleep and body temperature. GABA is a non-protein amino acid widely distributed in vertebrates, plants and microorganisms. It is a strong neuroinhibitory amino acid with sedative, hypnotic, anticonvulsant and blood pressure lowering physiological effects. Compared with the control group, high and low doses of casein hydrolysate can significantly increase the content of inhibitory neurotransmitters 5-HT and GAGB ( Fig.15 A and Fig.15 B in the figure).

[0183] NE is an important excitatory neurotransmitter that regulates wakefulness by acting on different brain regions. Its release peaks in the wakeful state. NE is mainly involved in the maintenance of rapid eye movement sleep. The cell bodies of NE-ergic neurons in the brain are concentrated in the medulla oblongata and pons, among which the locus coeruleus in the dorsolateral part of the pons is the most densely populated area of ​​NE. Many drugs used to treat insomnia work by inhibiting NE to affect signal transduction. Compared with the control group, high doses of casein hydrolysate can significantly reduce the content of NE ( Fig.15 C in.

[0184] Glu is the most abundant excitatory neurotransmitter in the brain. In the Glu system, Glu can activate the Glu receptors on the postsynaptic membrane of Glu neurons, thereby triggering excitatory impulses. Compared with the control group, low-dose casein hydrolysate can significantly reduce the content of the excitatory neurotransmitter glutamate, but high-dose increases it. The analysis may be due to individual differences, resulting in the results not being dose-dependent ( Fig.15 D in.

[0185] Many substances that improve sleep work by improving the inhibitory / excitatory metabolic imbalance of GABA / Glu and regulating the GABA / Glu ratio. Compared with the control group, both high and low doses of casein hydrolysate can significantly increase the GABA / Glu ratio ( Fig.15 E in.

[0186] Example 5: Comparison with similar products on the market

[0187] The finished casein hydrolysate prepared in Example 1 was used as an experimental sample to determine the sleep-promoting activity of the casein hydrolysate, and the method was as follows:

[0188] 1. Experimental Materials

[0189] Experimental animals: ICR mice, male, 8 weeks old (20 ± 2 g), were purchased from Shanghai Slake Experimental Animal Co., Ltd.;

[0190] Normal saline;

[0191] Diazepam tablets (diazepam, Tianjin Lisheng Pharmaceutical Co., Ltd.);

[0192] Pentobarbital sodium (Beijing Chemical Reagent Company);

[0193] Finished casein hydrolysate: prepared as in Example 1.

[0194] Commercially available casein hydrolyzed peptides: provided by Shanghai Litong Trading Co., Ltd., patented product US005846939A.

[0195] 2. Experimental methods

[0196] ICR mice were allowed to eat and drink freely, and all mice were first adapted to the environment for one week (7 days) before the experiment. Then the experimental mice were divided into four groups for drug administration, with 10 mice in each group, namely the control group (Control), the diazepam group, the casein hydrolyzate group prepared in Example 1 of the present invention, and the commercial casein hydrolyzate peptide group. The details are as follows:

[0197] Blank control group: daily gavage with pure water;

[0198] Diazepam group: daily dose was 1 mg / kg bodyweight, twice a day;

[0199] Casein hydrolysate group of the present invention: daily dosage is 50 mg / kg bodyweight;

[0200] Commercially available casein hydrolyzed peptide group: daily dosage was 50 mg / kg bodyweight;

[0201] After the mice were gavaged for 6 consecutive days, they were taken out from the barrier and gavaged first, and the gavage time was recorded with a stopwatch. After a countdown of 30 minutes, 42 mg / kg of sodium pentobarbital was injected, and the injection time T1 was recorded at the same time. When the righting reflex of the mouse disappeared, the righting reflex disappearance time T2 was recorded, which was the time when the mouse began to fall asleep. When the righting reflex of the mouse was restored, it was the time when the mouse woke up T3. The sleep latency and sleep duration of the mouse were calculated respectively. The shorter the sleep latency and the longer the sleep duration, the better the sleep-aiding effect of the peptide. The calculation formula is as follows:

[0202] Sleep latency = T2-T1; sleep duration = T3-T2.

[0203] The results are as follows Fig.16 A and Fig.16 As shown in B, the experiment used diazepam as a positive control, and the results showed that diazepam could significantly shorten the sleep latency and prolong the sleep duration of mice (p<0.01).

[0204] Compared with the control group, the casein hydrolyzate of the present invention achieved a significant effect on reducing sleep latency, but the market product (casein hydrolyzed peptide) did not achieve a significant effect. For sleep duration, the casein hydrolyzate of the present invention and the market competitor (casein hydrolyzed peptide) both achieved a significant effect.

[0205] Example 6: A method for preparing casein hydrolysate

[0206] (1) Take 300 kg of casein and add it to 1000 kg of water. Stir and soak it at room temperature for 40 min. After stirring and heating to 40-45 °C, adjust the pH of the liquid to 6.5 ± 0.2 with 7.5% sodium hydroxide aqueous solution, and stir and fully dissolve it at 40-45 °C for 70 min.

[0207] (2) After no obvious white particles are left in the feed liquid, add 4.0‰ (w / w) of composite protease based on the mass of casein substrate, including neutral protease, papain, and trypsin in a ratio of 2:2:1 (the neutral protease is produced from Aspergillus oryzae, with a deposit number of CGMCC No. 7586, and the preparation method of the neutral protease is shown in Example 1 of ZL201510176420.X). After half an hour of enzymolysis, adjust the pH of the feed liquid to 6.5±0.2 with a 7.5% sodium hydroxide aqueous solution, continue enzymolysis for 2 hours, then add 5‰ (w / w) of papain based on the mass of casein substrate, and continue enzymolysis for 1.5 hours;

[0208] (3) After the enzymatic hydrolysis is completed, the supernatant is separated by a butterfly separator;

[0209] The supernatant is concentrated by low temperature vacuum to a feed concentration of 40°Bx;

[0210] After concentration, the liquid is sterilized by UHT high temperature;

[0211] The sterilized liquid is spray-dried and packaged to obtain a finished casein hydrolyzate product, which can effectively shorten the sleep latency and prolong the sleep time.

[0212] Example 7: A method for preparing casein hydrolysate

[0213] (1) Take 280 kg of casein and add it to 1000 kg of water. Stir and soak it at room temperature for 50 minutes. After stirring and heating to 40-45°C, adjust the pH of the liquid to 7.5±0.2 with 7.5% sodium hydroxide aqueous solution, and stir and dissolve it at 40-45°C for 90 minutes.

[0214] (2) After no obvious white particles are left in the feed liquid, add 10.0‰ (w / w) of composite protease based on the mass of casein substrate, including neutral protease, papain, and trypsin in a ratio of 5:9:2 (the neutral protease is produced from Aspergillus oryzae, with a deposit number of CGMCC No. 7586, and the preparation method of the neutral protease is shown in Example 1 of ZL201510176420.X). After half an hour of enzymolysis, adjust the pH of the feed liquid to 6.5±0.2 with a 7.5% sodium hydroxide aqueous solution, continue enzymolysis for 2 hours, then add 10‰ (w / w) of papain based on the mass of casein substrate, and continue enzymolysis for 2 hours;

[0215] (3) After the enzymatic hydrolysis is completed, the supernatant is separated by a butterfly separator;

[0216] The supernatant was concentrated by low temperature vacuum to a feed concentration of 40 Bx;

[0217] After concentration, the liquid is sterilized by UHT high temperature;

[0218] The sterilized liquid is spray-dried and packaged to obtain a finished product. The casein hydrolyzate finished product can effectively shorten the sleep latency and prolong the sleep time.

[0219] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. Application of casein hydrolyzate in preparing food for improving sleep disorders, characterized in that: The preparation method of the casein hydrolysate is specifically as follows: (1) Mix casein and water in a mass ratio of 2.5-3:10, soak at room temperature with stirring for 30-60 minutes, stir and heat to 40-45°C, adjust the pH of the liquid to 6.5-7.5, and stir and fully dissolve at 40-45°C for 30-90 minutes; (2) After there are no obvious white particles in the feed solution, add a composite protease with a casein mass of 0.1-10.0‰ (w / w), and after enzymatic hydrolysis for 30-60 min, adjust the pH of the feed solution to 6.5-7.5, continue enzymatic hydrolysis for 2-2.5 hours, and then add papain with a casein mass of 0.1-10.0‰ (w / w), and continue enzymatic hydrolysis for 1-2 hours; The composite protease comprises neutral protease, papain and trypsin; (3) After the enzymatic hydrolysis is completed, the supernatant is centrifuged and concentrated and sterilized to obtain the casein hydrolyzate; The casein in step (1) is bovine casein or other mammalian casein; The neutral protease in step (2) is derived from Aspergillus oryzae fermentation, and the Aspergillus oryzae preservation number is CGMCC No.7586; The composite protease is composed of neutral protease, papain and trypsin, and the mass ratio is: 1-5:1-9:1-3; The casein hydrolysate contains two peptides with sleep-promoting activity: YAKPAA and TPTLN.

2. The use according to claim 1, characterized in that In step (3), the supernatant is concentrated by low-temperature vacuum concentration to a liquid concentration of 35-45° Bx; the concentrated liquid is sterilized by UHT high temperature; the sterilized liquid is spray-dried and packaged to obtain a finished product.

3. Use of the casein hydrolyzate according to claim 1 in the preparation of a health product for improving sleep disorders.

4. Use of the casein hydrolyzate according to claim 1 in the preparation of medicines for preventing, improving or treating sleep disorders.

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