Tyr-pro-type casein peptide with sleep improvement activity and preparation method and application thereof

Abstract

The application discloses a series of Tyr-Pro-type casein peptides with sleep improvement activity and a preparation method and application thereof. The casein sleep improvement peptide preferably refers to 5-7 peptides containing a Tyr-Pro structure. The specific sequences are 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 casein Tyr-Pro-type 5-7 peptides of the application exhibit stronger sleep improvement activity than other casein Tyr-type peptides or other length Tyr-Pro-type peptides. In a pentobarbital sodium-induced mouse sleep behavior experiment, the sleep time of the mice can be significantly prolonged to 2526.28+ / -220.32s-3539.65+ / -835.41s, and the sleep time can reach 57.57%-78.24% of that of a positive control group, so the casein Tyr-Pro-type 5-7 peptides can be applied to sleep improvement functional food or medicines.

Description

Technical Field

[0001] This invention relates to bioactive peptides for improving anxiety-related sleep disorders, specifically to a series of Tyr-Pro-type casein peptides with sleep-improving activity, their preparation methods, and applications. Background Technology

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

[0003] Currently, sleep assessment can be divided into two main categories: objective sleep assessment and subjective sleep assessment. Due to the complexity of the mechanisms and targets of sleep disorders, involving multiple targets such as benzodiazepine receptors, serotonin, and melatonin receptors, there is currently no simple in vitro assessment standard that can represent the overall sleep structure. Sleep duration is the most crucial indicator for assessing insomnia and the sleep-improving activity of bioactive substances in vivo, and it is usually measured using pentobarbital-induced sleep behavior testing experiments in mice. Therefore, significantly prolonging the sleep duration induced by sodium pentobarbital in mice can directly reflect the sleep-improving efficacy of the active substances.

[0004] Casein is an animal protein rich in proline (Pro) and tyrosine (Tyr). Due to its wide availability, abundant resources, and high nutritional value, it has significant advantages for the development of bioactive products. Although different casein hydrolysates have been shown to have different sleep-improving effects, the identification of sleep-improving peptides remains limited. A representative αs1-casein hydrolysate prepared with trypsin showed sleep-improving efficacy in a sodium pentobarbital-induced sleep experiment in mice, and a sleep-improving peptide, YLGYLEQLLR, was isolated and identified. Furthermore, casein hydrolysates obtained using other specific commercial proteases also exhibit strong sleep-improving effects (patents CN114181292B and CN112056453B). However, the different sleep-improving effects of casein hydrolysates obtained from different proteases remain unclear. Further research is needed to elucidate the detailed structural characteristics of effective sleep-improving peptides derived from casein.

[0005] Therefore, analyzing the structural characteristics of casein peptides can help develop more natural and safe sleep-improving peptides. However, due to the limited existing data on sleep-improving peptides, current research on their structural characteristics remains insufficient. Chinese patent (CN112056453B) discloses a sleep-improving enzymatic hydrolysate rich in aromatic amino acids, which exhibits strong sleep-improving effects. However, how to enhance the sleep-improving effects of casein peptides rich in aromatic amino acids remains unknown, and existing reports on the sequence characteristics of sleep-improving peptides are still insufficient. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and to provide a series of Tyr-Pro-type casein peptides with strong sleep-improving activity, their preparation methods and applications.

[0007] The present invention relates to the application of a Tyr-Pro-type casein peptide with sleep-improving activity in the preparation of functional foods or drugs for improving anxiety-related 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 powder, functional beverage, compressed candy, soft candy, or oral liquid; the drug is a powder, tablet, liquid, or capsule.

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

[0010] Furthermore, the excipients acceptable in the functional foods are milk powder, jujube seed, γ-aminobutyric acid, whey protein peptides, collagen peptides, soy peptides, or melatonin; the excipients acceptable in the drugs are sustained-release agents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, adsorbents, surfactants, or lubricants.

[0011] Furthermore, the Tyr-Pro-type casein peptide with sleep-improving activity is a casein peptide based on Tyr-Pro- with a 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 Tyr-Pro-type casein peptide with sleep-improving activity includes the following steps:

[0014] (1) Add casein and water, stir to dissolve, adjust pH to 6-8, and obtain casein dispersion;

[0015] (2) Add serine protease and mixed protease M to the casein dispersion from step (1) for synergistic enzymatic hydrolysis, enzyme inactivation, and cooling;

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

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

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

[0019] In the application of the casein hydrolysate obtained by the above preparation method, namely the casein small molecule peptide solution containing peptide segments SEQ.ID.NO.4-6, in the preparation of drugs and health products for improving sleep, the casein hydrolysate can prolong sleep time by more than 2.37 times.

[0020] The Tyr-Pro-type casein peptide and its enzymatic hydrolysate significantly prolong the sleep duration induced by sodium pentobarbital in mice, thus exhibiting strong sleep-improving activity. Furthermore, compared to other Tyr-type or Tyr-Pro-type peptides of different lengths, the sleep-improving effect lasts for a longer period.

[0021] The Tyr-Pro-type casein peptides in this invention, particularly the preferred Tyr-Pro-type 5-7 peptides, directly demonstrate their sleep-improving effects by prolonging the sodium pentobarbital-induced sleep time in mice. For example, the previously reported casein nonapeptide YPFPGPIPN (patent CN117659154A) prolonged the sodium pentobarbital-induced sleep time in mice to 3186.83±635.34 s, achieving a sleep-improving activity of 53.75% compared to the positive control group. The casein Tyr-Pro-type 5-7 peptides of this invention achieve an activity of 57.57%–78.24%, demonstrating a significantly enhanced efficacy.

[0022] In summary, appropriately increasing the amino acid content at the C-terminus of the Tyr-Pro monomer is an effective method for developing Tyr-type peptides with stronger sleep-improving activity. However, simply lengthening the sequence is not necessarily more effective; increasing the sequence length to 5-7 amino acids is more suitable. 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] The present invention has the following advantages and effects compared with the prior art:

[0024] The casein Tyr-Pro-type 5-7 peptide of the present invention exhibits stronger sleep-improving activity compared to other Tyr-type peptides or Tyr-Pro-type peptides of other lengths, and significantly prolongs the sleep time induced by sodium pentobarbital in mice. It can be used to prepare effective functional foods or drugs for improving sleep. Attached Figure Description

[0025] Figure 1 A comparative diagram showing the effects of Tyr-Pro-type 5-7 peptides, other Tyr-type peptides, and Tyr-Pro-type peptides of other lengths on pentobarbital-induced sleep time in mice.

[0026] Figure 2 This is a comparison of the ratios of Tyr-Pro-type 5-7 peptides, other Tyr-type peptides, and Tyr-Pro-type peptides of other lengths to the positive control drug diazepam in inducing pentobarbital-induced sleep time in mice.

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

[0028] Figure 4 BPC-MS images of different casein hydrolysis products in Examples 9-10 and Comparative Example 3.

[0029] Figures 5a-5cMass spectrometry images of Tyr-Pro-type 5-7 peptides (SEQ.ID.NO.4-6) in different casein hydrolysates of Examples 9-10 and Comparative Example 3. Detailed Implementation

[0030] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

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

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

[0033] One hundred and ninety-two Kunming mice (6-8 weeks old) were placed in a controlled environment (25±2℃, 12 / 12h light / dark cycle) with free access to food. Fourteenty-four of these mice underwent randomized chronic stress treatment (including tail clamping, behavioral restriction, water deprivation, food deprivation, tilted cages, reversed day / night cycles, and wet cages) for two weeks and were randomly assigned to one of the following groups (n=12): a normal group (normal mice, without stress stimulation), a model group (stress stimulation but without medication), a casein peptide group (each group received oral gavage of 1.8 mg / kg casein peptide during stress stimulation), and a casein protein hydrolysate group (each group received oral gavage of 75 mg / kg casein protein hydrolysate during stress stimulation). The casein peptide group received oral gavage for 7 days, while simultaneously receiving stress stimulation; the model group and the casein peptide / hydrolysate group received combined stimulation for 21 days. Sleep behavior was measured after the final gavage.

[0034] (2) Behavioral assay of pentobarbital sodium-induced sleep in mice

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

[0036] (3) Mass spectrometry identification method

[0037] Bioactive peptides were identified using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS). Gradient elution chromatography was employed for separation, followed by quantification using secondary mass spectrometry. 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, and mobile phase B was acetonitrile (chromatographic grade). 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, equilibration to initial state with 5% B. Mass spectrometry parameters: electron impact ion source energy 7 eV, capillary voltage 4500 V, ion source temperature 200 °C, dry gas flow rate 8.0 L / min, nebulizer pressure 1.5 bar, and mass scan range m / z 50–1500.

[0038] Example 1

[0039] When the pentapeptide Tyr-Pro-Ser-Tyr-Gly was administered orally at a dose of 1.8 mg / kg, its sodium pentobarbital induced a sleep duration of 2526.28 ± 220.32 s in mice.

[0040] Example 2

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

[0042] Example 3

[0043] When the pentapeptide Tyr-Pro-Phe-Pro-Gly was administered orally at a dose of 1.8 mg / kg, its sodium pentobarbital induced a sleep duration of 3055.25 ± 428.78 s in mice.

[0044] Example 4

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

[0046] Example 5

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

[0048] Example 6

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

[0050] Comparative Example 1

[0051] When the hexapeptide Tyr-Phe-Tyr-Pro-Glu-Leu was administered orally at a dose of 1.8 mg / kg, its sodium pentobarbital induced a sleep duration of 2024.0554 ± 672.99 s in mice.

[0052] Comparative Example 2

[0053] When the pentapeptide Tyr-Tyr-Val-Pro-Leu was administered orally at a dose of 1.8 mg / kg, its sodium pentobarbital induced a sleep duration of 1012.0277 ± 72.13 s in mice.

[0054] It is evident that the Tyr-Pro-type of this invention prolongs the sleep time induced by sodium pentobarbital in mice.

[0055] Example 7

[0056] When the dipeptide Tyr-Pro was administered orally at a dose of 1.8 mg / kg, the sodium pentobarbital induced a sleep duration of 1792.69 ± 259.75 s in mice.

[0057] Example 8

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

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

[0060] Example 9

[0061] (1) Weigh buffalo milk casein and distilled water in a mass ratio of 1:10, stir moderately to dissolve, and prepare casein dispersion. Adjust the pH of the reaction system to 8.0 using 4M NaOH solution.

[0062] (2) Add 0.5% trypsin and 1.5% mixed protease M (based on casein mass) to the reaction system of step (1), enzymatically hydrolyze at 55°C for 8 hours, inactivate enzyme at 100°C for 15 minutes, and cool; the mixed protease M includes aminopeptidase and carboxypeptidase (mass ratio 3:1).

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

[0064] Example 10

[0065] (1) Weigh buffalo milk casein and distilled water in a mass ratio of 1:10, stir moderately to dissolve, and prepare casein dispersion. Adjust the pH of the reaction system to 6.0 using 4M NaOH solution.

[0066] (2) Add 0.1% chymotrypsin and 0.1% mixed protease M (based on casein mass) to the reaction system of step (1), enzymatically hydrolyze at 35°C for 1 h, inactivate enzyme at 90°C for 30 min, and cool.

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

[0068] Comparative Example 3

[0069] (1) Weigh buffalo milk casein and distilled water in a mass ratio of 1:10, stir moderately to dissolve, and prepare casein dispersion. Adjust the pH of the reaction system to 7.0 using 4M NaOH solution.

[0070] (2) Add 0.2% trypsin (based on casein mass) to the reaction system of step (1), enzymatically hydrolyze at 55°C for 8 hours, enzymatically hydrolyze at 55°C for 8 hours, inactivate enzyme at 100°C for 15 minutes, and cool.

[0071] (3) Centrifuge at 10000 rpm and 4℃ for 10 min, and take the supernatant, which is a casein small molecule peptide solution.

[0072] Figures 5a-5c The mass spectrometry images show the Tyr-Pro-type 5-7 peptides (SEQ.ID.NO.5-7) in different casein hydrolysates of Examples 9-10 and Comparative Example 3.

[0073] Depend on Figure 1It was found that the sleep time induced by sodium pentobarbital in mice under chronic stress (model group) was drastically shortened to only 735.80±172.83s. However, after oral administration of Tyr-Pro-type 5-7 peptide casein, the sleep time induced by sodium pentobarbital in mice under chronic stress was significantly prolonged compared to the model group, ranging from 2526.28±220.32s to 3539.65±835.41s. Furthermore, treatment with dipeptide Tyr-Pro, tripeptide Tyr-Pro-Ser, hexapeptide Tyr-Phe-Tyr-Pro-Glu-Leu, and pentapeptide Tyr-Tyr-Val-Pro-Leu resulted in sleep times of only 1012.0277±72.13s to 2024.0554±672.99s. This indicates that casein Tyr-Pro-type 5-7 peptide can more effectively prolong the sleep time induced by sodium pentobarbital in mice, and has the potential to improve sleep.

[0074] Depend on Figure 2 It can be seen that comparing the sleep time of the oral gavage sample group and the positive drug group provides a more direct comparison of sleep improvement efficacy. The results show that the sleep improvement effect of casein Tyr-Pro-type 5-7 peptide reached 57.57%–78.24% of the positive control group, while the sleep improvement effects of Tyr-Pro and Tyr-Pro-Ser were 40.86% and 42.08% of the positive control group, respectively. In contrast, the sleep improvement effects of the comparative pentapeptides and hexapeptides were only 23.06% and 46.13%, respectively.

[0075] Depend on Figure 3 It was found that the sleep time induced by sodium pentobarbital in mice under chronic stress (model group) was drastically shortened to only 735.80±172.83 s. However, after oral administration of the casein hydrolysates of Examples 9 and 10, the sleep time induced by sodium pentobarbital in mice under chronic stress was significantly prolonged to 1745.52±435.3 s and 1965.93±558.13 s, respectively, compared to the model group. In contrast, the sleep time after treatment in Comparative Example 3 was only 1312.06±427.06 s. This indicates that the casein hydrolysate containing Tyr-Pro-type 5-7 peptide can more effectively prolong the sleep time induced by sodium pentobarbital in mice, and has the potential to improve sleep.

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

Claims

1. An application of Tyr-Pro-type casein peptides that improve sleep, characterized in that... The application is in the preparation of functional foods or drugs that improve sleep; the sequence of the Tyr-Pro-type casein peptide with sleep-improving activity is Tyr-Pro-Ser-Gly-Ala-Trp-Tyr (SEQ. ID. NO. 6); the Tyr-Pro-type casein peptide can prolong sleep time.

2. The application according to claim 1, characterized in that, The functional food is a powder, functional beverage, compressed candy, soft candy, or oral liquid; the drug is a powder, tablet, liquid, or capsule.

3. The application according to claim 1, characterized in that, The functional food or drug also contains, in addition to the Tyr-Pro-type casein peptide, active ingredients that improve sleep and / or acceptable excipients.

4. The application according to claim 3, characterized in that, The excipients acceptable in the functional foods are milk powder, jujube seed, γ-aminobutyric acid, whey protein peptide, collagen peptide, soy peptide, or melatonin; the excipients acceptable in the drugs are sustained-release agents, fillers, binders, humectants, disintegrants, absorption promoters, adsorbents, or lubricants.

5. The application according to claim 3, characterized in that, The excipients acceptable in the drug are excipients or surfactants.

6. In the application of a casein small molecule peptide solution containing the Tyr-Pro-Ser-Gly-Ala-Trp-Tyr (SEQ. ID. NO. 6) peptide fragment in the preparation of drugs and health products for improving sleep, the casein small molecule peptide solution can prolong sleep time by more than 2.37 times.

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