Tyr-Pro-type casein peptide with sleep improving activity as well as preparation method and application of Tyr-Pro-type casein peptide

Abstract

The invention discloses a series of Tyr-Pro-type casein peptides with sleep improving activity as well as a preparation method and application of the Tyr-Pro-type casein peptides, and the casein peptides for improving sleep preferably refer to 5-7 peptides containing a Tyr-Pro-structure. The specific sequences of the fluorescent probe are as follows: Tyr-Pro-Ser-Tyr-Gly, Tyr-Pro-Ser-Gly-Ala, Tyr-Pro-Phe-Pro-Gly, Tyr-Pro-Glu-Leu-Phe, Tyr-Pro-Ser-Tyr-Gly-Leu-Asn, and Tyr-Pro-Ser-Gly-Ala-Trp-Tyr. The fluorescent probe can be used for preparing the fluorescent probe. Compared with other casein Tyr-type peptides or Tyr-Pro-type peptides with other lengths, the casein Tyr-Pro-type 5-7 peptides disclosed by the invention show stronger sleep improvement activity, and in the pentobarbital sodium induced mouse sleep behavior, the sleep time of a mouse can be obviously prolonged to 2526.28 + / -220.32 s to 3539.65 + / -835.41 s, the sleep time can reach 57.57%-78.24% of that of a positive control group, and the stability of the peptide is improved. The compound can be applied to functional foods or medicines for improving sleep.

Description

Technical Field

[0001] The present invention relates to bioactive peptides for improving anxiety-related sleep disorders, and in particular to a series of Tyr-Pro-type casein peptides with sleep-improving activity, and preparation methods and applications thereof. Background Art

[0002] Insomnia refers to a series of phenomena such as difficulty falling asleep, short sleep time, frequent dreams, and easy awakening. It affects more than 35% of the world's population and is an important risk factor for mood disorders, fatigue, memory impairment, neuroinflammation, brain dysfunction and decreased immunity.

[0003] At present, sleep assessment can be divided into two categories: objective sleep assessment and subjective sleep assessment. Due to the complexity of the mechanism or target of sleep disorders, multiple targets such as benzodiazepine receptors, serotonin, and melatonin receptors are involved. Therefore, there is currently no simple in vitro assessment standard that can represent the overall sleep structure. Sleep time is the most critical indicator for evaluating insomnia and the activity of bioactive substances in improving sleep in vivo, and is usually measured by sleep behavior test experiments in mice induced by pentobarbital. Therefore, by significantly prolonging the sleep time of mice induced by sodium pentobarbital, the sleep improvement effect of active substances can be intuitively reflected.

[0004] Casein is an animal protein rich in proline Pro and tyrosine Tyr. It has great advantages for the development of biologically active products due to its wide source, rich resources and high nutritional value. Although different casein hydrolysates have been shown to have different sleep-improving effects, the identification of sleep-improving peptides is still limited. The representative αs1-casein hydrolysate prepared with trypsin showed sleep-improving effects in the sodium pentobarbital-induced mouse sleep experiment, and a sleep-improving peptide YLGYLEQLLR was obtained after separation and identification. In addition, the casein hydrolysate obtained by enzymatic hydrolysis with other specific commercial proteases also has a strong effect on improving sleep (patents CN114181292B and CN112056453B). However, the casein hydrolysates obtained by different proteases showed different sleep-improving effects, and the underlying reasons are still unclear. Further research is needed to clarify the detailed structural characteristics of effective sleep-improving peptides from casein.

[0005] Therefore, analyzing the structural characteristics of casein peptides will help develop more natural and safe sleep-improving peptides. However, due to the limited data on sleep-improving peptides currently available, the prior art is still insufficient in studying the structural characteristics of sleep-improving peptides. A Chinese patent (CN112056453B) discloses a sleep-improving enzymatic hydrolysate rich in aromatic amino acids, and the casein hydrolysate has a strong sleep-improving effect. However, how to enhance the sleep-improving effect of casein peptides rich in aromatic amino acids is still unknown, and the existing reports on the sequence characteristics of sleep-improving peptides are still insufficient. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a series of Tyr-Pro-type casein peptides with strong sleep-improving activity and a preparation method and application thereof.

[0007] The present invention provides an application of a Tyr-Pro-type casein peptide with sleep-improving activity in the preparation of a functional food or medicine for improving anxiety-type sleep disorders; the sequence of the Tyr-Pro-type casein peptide with sleep-improving activity is any one of Tyr-Pro, Tyr-Pro-Phe-Pro-Gly (SEQ.ID.NO.1), Tyr-Pro-Glu-Leu-Phe (SEQ.ID.NO.2), Tyr-Pro-Ser-Tyr-Gly-Leu-Asn (SEQ.ID.NO.3), Tyr-Pro-Ser-Gly-Ala (SEQ.ID.NO.4), Tyr-Pro-Ser-Tyr-Gly (SEQ.ID.NO.5), Tyr-Pro-Ser-Gly-Ala-Trp-Tyr (SEQ.ID.NO.6), and Tyr-Pro-Ser.

[0008] Furthermore, the functional food is a powdered granule, a functional drink, a compressed candy, a jelly candy or an oral liquid; and the medicine is in the form of a powder, a tablet, a liquid or a capsule.

[0009] Furthermore, the functional food or medicine also contains active ingredients and / or acceptable excipients that improve sleep in addition to the Tyr-Pro-type casein peptide.

[0010] Furthermore, the acceptable excipients in the functional food are milk powder, jujube seeds, γ-aminobutyric acid, whey protein peptides, collagen peptides, soybean peptides or melatonin; the acceptable excipients in the drug are sustained-release agents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, adsorption carriers, surfactants or lubricants.

[0011] Furthermore, the Tyr-Pro-type casein peptide with sleep-improving activity is a casein peptide based on Tyr-Pro- with the C-terminus extended by 5 to 7 amino acids, namely SEQ.ID.NO.4 to 6.

[0012] The present invention provides a series of Tyr-Pro-type casein peptides with sleep-improving activity, wherein the amino acid sequences of the Tyr-Pro-type casein peptides with sleep-improving activity are Tyr-Pro-Ser-Gly-Ala (SEQ.ID.NO.4), Tyr-Pro-Ser-Tyr-Gly (SEQ.ID.NO.5), Tyr-Pro-Ser-Gly-Ala-Trp-Tyr (SEQ.ID.NO.6), and Tyr-Pro-Ser.

[0013] The method for preparing the above-mentioned Tyr-Pro-type casein peptide having sleep-improving activity comprises the following steps:

[0014] (1) adding casein and water, stirring to dissolve, and adjusting the pH to 6-8 to prepare a casein dispersion;

[0015] (2) adding serine protease and mixed protease M to the casein dispersion of step (1), synergistically hydrolyzing, inactivating the enzymes, and cooling;

[0016] (3) Centrifuge at 8000-10000 rpm and 4-10° C. for 10-20 min, and collect the supernatant, which is a casein small molecule peptide solution containing SEQ.ID.NO.4-6 peptide segments.

[0017] Furthermore, in step (2), the serine protease is one of chymotrypsin and trypsin; the mixed protease M specifically includes aminopeptidase and carboxypeptidase (preferably in a mass ratio of 3:1); the temperature of the synergistic enzymolysis is 35-55°C; and the time of the synergistic enzymolysis is 1-8h.

[0018] Furthermore, the total amount of the serine protease and the mixed protease M added in step (2) is 0.2% to 2.0% of the mass of casein; the mass ratio of the serine protease to the mixed protease M is (1 to 10):(10 to 19); and the enzyme inactivation in step (2) is to heat the enzymatic solution to 90 to 100° C. and boil it for 15 to 30 minutes.

[0019] The casein enzymatic hydrolysis product obtained by the above preparation method, i.e., the casein small molecule peptide solution containing SEQ.ID.NO.4 to 6 peptide segments, is used in the preparation of drugs and health products for improving sleep. The casein enzymatic hydrolysis product can prolong sleep time by more than 2.37 times.

[0020] The Tyr-Pro-type casein peptides and the enzymatic hydrolyzates containing the Tyr-Pro-type casein peptides of the present invention can significantly prolong the sleep time of mice induced by sodium pentobarbital, thereby showing strong sleep improvement activity. At the same time, compared with other Tyr-type or Tyr-Pro-type peptides of different lengths, the sleep improvement time is longer.

[0021] The Tyr-Pro-type casein peptides in the present invention, preferably Tyr-Pro-type 5 to 7 peptides, intuitively embody the effect of improving sleep by prolonging the time of sodium pentobarbital-induced mice. For example, the casein nonapeptide YPFPGPIPN (patent CN117659154A) reported previously prolongs the time of sodium pentobarbital-induced mice to 3186.83±635.34s, and compared with the positive drug group, the sleep improvement activity reaches 53.75%, while the casein Tyr-Pro-type 5 to 7 peptides of the present invention can reach 57.57% to 78.24%, and the efficacy is significantly improved.

[0022] In summary, appropriately adding amino acids to the C-terminus of the Tyr-Pro-monomer is an effective method for developing Tyr-type peptides with stronger sleep-improving activity, but blindly extending is not necessarily more effective, and increasing the sequence length to 5 to 7 amino acids is more appropriate. Based on this, the Tyr-Pro-type casein peptide of the present invention can be used to prepare functional foods or drugs with sleep-improving effects.

[0023] Compared with the prior art, the present invention has the following advantages and effects:

[0024] The casein Tyr-Pro-type 5-7 peptides of the present invention show stronger sleep-improving activity compared with other Tyr-type peptides or Tyr-Pro-type peptides of other lengths, significantly prolong the sleep time of mice induced by sodium pentobarbital, and can be used to prepare effective functional foods or drugs for improving sleep. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure compares the effects of Tyr-Pro-type 5-7 peptides, other Tyr-type peptides, and Tyr-Pro-type peptides of other lengths on the sleep time of mice induced by sodium pentobarbital.

[0026] Figure 2 It is a comparison chart of the ratio of the effects of Tyr-Pro-type 5-7 peptides, other Tyr-type peptides, and Tyr-Pro-type peptides of other lengths and the positive drug diazepam on the sleep time of mice induced by sodium pentobarbital.

[0027] Figure 3 This is a comparison chart of the effects of different casein enzymatic hydrolysates in Examples 9-10 and Comparative Example 3 on the sleep time of mice induced by sodium pentobarbital.

[0028] Figure 4 BPC-MS diagram for mass spectrometric identification of different casein hydrolysis products in Examples 9-10 and Comparative Example 3.

[0029] Figure 5a to Figure 5cIt is the mass spectrometry identification diagram of Tyr-Pro-type 5-7 peptides (SEQ.ID.NO.4-6) in different casein enzymatic hydrolysis products in Examples 9-10 and Comparative Example 3. DETAILED DESCRIPTION

[0030] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0031] The experimental indicators and methods involved in the embodiments of the present invention are as follows:

[0032] (1) Drug administration and treatment of mice

[0033] 192 Kunming mice (6-8 weeks old) were placed in a controlled environment (25±2℃, 12 / 12h light / dark cycle) with free access to food. Among them, 144 mice were subjected to random chronic stress (including tail clamping, restricted behavior, water deprivation, fasting, tilted cage, day and night reversal, wet cage, etc.) for 2 weeks and randomly divided into groups (n=12): normal group (normal mice, no stress stimulation), model group (stress stimulation but no drug administration), casein peptide group (each group was orally gavaged with casein peptide 1.8mg / kg at the same time as stress stimulation) and casein enzymatic hydrolysate (each group was orally gavaged with casein enzymatic hydrolysate 75mg / kg at the same time as stress stimulation). The casein peptide group mice were treated with oral gavage for 7 days, and stress stimulation, that is, the model group and casein peptide / enzymatic hydrolysate group were stimulated for 21 days. After the last gavage, sleep behavior was measured.

[0034] (2) Pentobarbital sodium-induced sleep behavior in mice

[0035] Refer to Chapter 10 of the Technical Specifications for Inspection and Evaluation of Health Foods, "Test Methods for Improving Sleep Function" for determination. Mice were injected with sodium pentobarbital (42 mg / kg, intraperitoneal injection) 30 minutes after the last oral administration. Then the number of mice that fell asleep after the righting reflex disappeared within 15 minutes was recorded. The sleep latency and sleep duration of mice within 2 hours were recorded with a stopwatch.

[0036] (3) Mass spectrometry identification method

[0037] Ultra-high performance liquid chromatography coupled to mass spectrometry (UPLC-MS / MS) was used to identify bioactive peptides. Gradient elution chromatography was used for separation, coupled with secondary mass spectrometry for identification and quantification. The elution rate was 0.2 mL / min and the injection volume was 2 μL. Mobile phase A was 0.1% formic acid in water, B was acetonitrile (chromatographic grade), and the elution program was: 0-10 min, 22%-47% B; 10-12 min, 47%-90% B; 12-14 min, 90% B; 14-15 min, 90%-5% B; 15-18 min, 5% B was balanced to the initial state. Mass spectrometry parameters: electron bombardment ion source energy 7 eV, capillary voltage 4500 V, ion source temperature 200 ° C, drying gas flow 8.0 L / min, nebulizer pressure 1.5 bar, mass scanning range m / z 50-1500.

[0038] Example 1

[0039] When the oral dose of pentapeptide Tyr-Pro-Ser-Tyr-Gly was 1.8 mg / kg, the sodium pentobarbital-induced sleep time in mice was 2526.28±220.32 s.

[0040] Example 2

[0041] When the pentapeptide Tyr-Pro-Ser-Gly-Ala was orally administered at a dose of 1.8 mg / kg, the sodium pentobarbital-induced sleep time in mice was 3433±613.69 s.

[0042] Example 3

[0043] When the oral dose of the pentapeptide Tyr-Pro-Phe-Pro-Gly was 1.8 mg / kg, the sodium pentobarbital-induced sleep time in mice was 3055.25±428.78 s.

[0044] Example 4

[0045] When the pentapeptide Tyr-Pro-Glu-Leu-Phe was orally administered at a dose of 1.8 mg / kg, sodium pentobarbital induced a sleep time of 3326.14±323.47 s in mice.

[0046] Example 5

[0047] When the oral dose of the heptapeptide Tyr-Pro-Ser-Gly-Ala-Trp-Tyr was 1.8 mg / kg, the sodium pentobarbital-induced sleep time in mice was 3219.38±658.62 s.

[0048] Example 6

[0049] When the oral dose of the heptapeptide Tyr-Pro-Ser-Tyr-Gly-Leu-Asn was 1.8 mg / kg, the sodium pentobarbital-induced sleep time in mice was 3251.38±878.04 s.

[0050] Comparative Example 1

[0051] When the oral dose of the hexapeptide Tyr-Phe-Tyr-Pro-Glu-Leu was 1.8 mg / kg, the sodium pentobarbital-induced sleep time in mice was 2024.0554±672.99 s.

[0052] Comparative Example 2

[0053] When the oral dose of the pentapeptide Tyr-Tyr-Val-Pro-Leu was 1.8 mg / kg, the sodium pentobarbital-induced sleep time in mice was 1012.0277±72.13 s.

[0054] It can be seen that the Tyr-Pro-type of the present invention prolongs the sleep time of mice induced by sodium pentobarbital.

[0055] Example 7

[0056] When the oral dose of dipeptide Tyr-Pro was 1.8 mg / kg, the pentobarbital sodium induced sleep time in mice to be 1792.69±259.75 s.

[0057] Example 8

[0058] When the tripeptide Tyr-Pro-Ser was administered orally at a dose of 1.8 mg / kg, sodium pentobarbital induced a sleep time of 1846.29±398.16 s in mice.

[0059] It can be seen that the Tyr-Pro-type casein peptides of the present invention can effectively improve the sleep duration, and the more preferred 5-7 peptides significantly prolong the sleep time of mice induced by sodium pentobarbital.

[0060] Example 9

[0061] (1) adding buffalo milk casein and distilled water in a mass ratio of 1:10, stirring and dissolving to prepare a casein dispersion, and adjusting the pH value of the reaction system to 8.0 using a 4M NaOH solution;

[0062] (2) adding 0.5% by weight of trypsin and 1.5% by weight of mixed protease M (based on casein mass) to the reaction system of step (1), performing enzymatic hydrolysis at 55° C. for 8 h, inactivating the enzyme at 100° C. for 15 min, and cooling; the mixed protease M comprises aminopeptidase and carboxypeptidase (mass ratio 3:1);

[0063] (3) Centrifuge at 10,000 rpm and 4°C for 10 min and collect the supernatant, which is a casein small molecule peptide solution containing peptide segments of SEQ.ID.NO.5 to 7.

[0064] Example 10

[0065] (1) adding buffalo milk casein and distilled water in a mass ratio of 1:10, stirring and dissolving to prepare a casein dispersion, and adjusting the pH value of the reaction system to 6.0 using a 4M NaOH solution;

[0066] (2) adding 0.1% by weight of chymotrypsin and 0.1% of mixed protease M (based on casein mass) to the reaction system of step (1), performing enzymatic hydrolysis at 35° C. for 1 h, inactivating the enzyme at 90° C. for 30 min, and cooling;

[0067] (3) Centrifuge at 8000 rpm and 4°C for 20 min and collect the supernatant, which is a casein small molecule peptide solution containing peptide segments of SEQ.ID.NO.5 to 7.

[0068] Comparative Example 3

[0069] (1) adding buffalo milk casein and distilled water in a mass ratio of 1:10, stirring and dissolving to prepare a casein dispersion, and adjusting the pH value of the reaction system to 7.0 using a 4M NaOH solution;

[0070] (2) adding 0.2% by mass of trypsin (based on casein mass) to the reaction system of step (1), performing enzymatic hydrolysis at 55° C. for 8 h, performing enzymatic hydrolysis at 55° C. for 8 h, performing enzyme inactivation at 100° C. for 15 min, and cooling;

[0071] (3) Centrifuge at 10,000 rpm and 4°C for 10 min and collect the supernatant, which is the casein small molecule peptide solution.

[0072] Figure 5a to Figure 5c It is the mass spectrometry identification diagram of Tyr-Pro-type 5-7 peptides (SEQ.ID.NO.5-7) in different casein enzymatic hydrolysis products in Examples 9-10 and Comparative Example 3.

[0073] Depend on Figure 1It can be seen that the sleep time of mice stimulated by chronic stress (model group) induced by sodium pentobarbital was sharply shortened to only 735.80±172.83s, while after oral gavage of casein Tyr-Pro-type 5-7 peptide, the sleep time of mice stimulated by chronic stress induced by sodium pentobarbital was significantly extended to 2526.28±220.32s~3539.65±835.41s compared with the model group. After treatment with dipeptide Tyr-Pro, tripeptide Tyr-Pro-Ser, hexapeptide Tyr-Phe-Tyr-Pro-Glu-Leu, and pentapeptide Tyr-Tyr-Val-Pro-Leu, the sleep time was only 1012.0277±72.13s~2024.0554±672.99s. This indicates that casein Tyr-Pro-type 5-7 peptide can more effectively prolong the sleep time of sodium pentobarbital-induced mice and has the potential to improve sleep.

[0074] Depend on Figure 2 It can be seen that by comparing the sleep time of the oral gavage sample group with the positive drug group, the sleep improvement effect can be more intuitively compared. The results show that the casein Tyr-Pro-type 5-7 peptide improves sleep by 57.57% to 78.24% of the positive control group, and the sleep improvement effect of Tyr-Pro and Tyr-Pro-Ser is 40.86% and 42.08% of the positive control group, while the sleep improvement effect of the pentapeptide and hexapeptide in the comparative example is only 23.06% and 46.13%.

[0075] Depend on Figure 3 It can be seen that the sleep time of the mice stimulated by chronic stress (model group) induced by sodium pentobarbital was sharply shortened to only 735.80±172.83s, while after oral gavage of the casein hydrolysate of Examples 9 and 10, the sleep time of the mice stimulated by chronic stress induced by sodium pentobarbital was significantly extended to 1745.52±435.3s and 1965.93±558.13s compared with the model group. After the treatment of Comparative Example 3, the sleep time was only 1312.06±427.06s. It shows that the casein hydrolysate containing Tyr-Pro-type 5-7 peptide can more effectively prolong the sleep time of mice induced by sodium pentobarbital, and has the potential to improve sleep.

[0076] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. Application of Tyr-Pro-type casein peptide with sleep-improving activity, characterized in that The application is an application in the preparation of functional food or medicine for improving anxiety-type sleep disorders; the sequence of the Tyr-Pro-type casein peptide with sleep-improving activity is any one of Tyr-Pro, Tyr-Pro-Phe-Pro-Gly (SEQ.ID.NO.1), Tyr-Pro-Glu-Leu-Phe (SEQ.ID.NO.2), Tyr-Pro-Ser-Tyr-Gly-Leu-Asn (SEQ.ID.NO.3), Tyr-Pro-Ser-Gly-Ala (SEQ.ID.NO.4), Tyr-Pro-Ser-Tyr-Gly (SEQ.ID.NO.5), Tyr-Pro-Ser-Gly-Ala-Trp-Tyr (SEQ.ID.NO.6), and Tyr-Pro-Ser.

2. The use according to claim 1, characterized in that: The functional food is powdered granules, functional drinks, compressed candies, jelly candies or oral liquids; the medicine is in the form of powder, tablets, liquids or capsules.

3. The use according to claim 1, characterized in that: The functional food or medicine further contains active ingredients and / or acceptable auxiliary materials having the function of improving sleep in addition to the Tyr-Pro-type casein peptide.

4. The use according to claim 3, characterized in that: The acceptable excipients in the functional food are milk powder, jujube seeds, γ-aminobutyric acid, whey protein peptide, collagen peptide, soybean peptide or melatonin; the acceptable excipients in the medicine are sustained-release agents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, adsorption carriers, surfactants or lubricants.

5. The use according to claim 1, characterized in that The Tyr-Pro-type casein peptide with sleep-improving activity is a casein peptide based on Tyr-Pro- with the C-terminus extended by 5 to 7 amino acids, namely, SEQ.ID.NO.4 to 6.

6. A series of Tyr-Pro-type casein peptides with sleep-improving activity, characterized in that: The amino acid sequence of the Tyr-Pro-type casein peptide with sleep-improving activity is Tyr-Pro-Ser-Gly-Ala (SEQ.ID.NO.4), Tyr-Pro-Ser-Tyr-Gly (SEQ.ID.NO.5), Tyr-Pro-Ser-Gly-Ala-Trp-Tyr (SEQ.ID.NO.6), Tyr-Pro-Ser.

7. A method for preparing a Tyr-Pro-type casein peptide having sleep-improving activity as claimed in claim 6, characterized in that: The steps include: (1) adding casein and water, stirring to dissolve, and adjusting the pH to 6-8 to prepare a casein dispersion; (2) adding serine protease and mixed protease M to the casein dispersion of step (1), synergistically hydrolyzing, inactivating the enzymes, and cooling; (3) Centrifuge at 8000-10000 rpm and 4-10° C. for 10-20 min, and collect the supernatant, which is a casein small molecule peptide solution containing SEQ.ID.NO.4-6 peptide segments.

8. The method for preparing a Tyr-Pro-type casein peptide having sleep-improving activity according to claim 7, characterized in that: In step (2), the serine protease is one of chymotrypsin and trypsin; the mixed protease M specifically includes aminopeptidase and carboxypeptidase; the temperature of the synergistic enzymolysis is 35-55°C; and the time of the synergistic enzymolysis is 1-8h.

9. The method for preparing a Tyr-Pro-type casein peptide having sleep-improving activity according to claim 7, characterized in that: The total amount of the serine protease and the mixed protease M added in step (2) is 0.2% to 2.0% of the casein mass; the mass ratio of the serine protease to the mixed protease M is (1 to 10):(10 to 19); the enzyme inactivation in step (2) is to heat the enzymatic solution to 90 to 100° C. and boil it for 15 to 30 minutes.

10. The casein enzymatic hydrolysis product obtained by the preparation method of claim 7, i.e., the casein small molecule peptide solution containing SEQ.ID.NO.4 to 6 peptide segments, is used in the preparation of drugs and health products for improving sleep. The casein enzymatic hydrolysis product can prolong sleep time by more than 2.37 times.

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