阅读说明：本技术 乳源活性肽ccl-1s在制备促睡眠产品中的应用 (Application of milk-derived active peptide CCL-1S in preparation of sleep-promoting product ) 是由 曹庸 梁曹雯 刘星雨 刘飞 于 2021-08-31 设计创作，主要内容包括：本发明属于促睡眠保健品领域,具体涉及乳源活性肽CCL-1S在制备促睡眠产品中的应用,本发明利用体外膜片钳技术从牛奶中分离鉴定出一种具有抑制神经细胞活性、能促进睡眠的乳源活性肽CCL-1S,该乳源活性肽具有如SEQ ID NO:1所示的氨基酸序列,分子量为934.11Da,本发明提供的促睡眠乳源活性肽安全无毒,可应用于制备促睡眠药品、促睡眠功能性食品等产品,可充分利用乳产品资源,为乳源多肽的高值化利用提供了新途径。(The invention belongs to the field of sleep-promoting health products, and particularly relates to application of milk-derived bioactive peptide CCL-1S in preparation of a sleep-promoting product, wherein the milk-derived bioactive peptide CCL-1S capable of inhibiting nerve cell activity and promoting sleep is separated and identified from milk by using an in-vitro patch clamp technology, has an amino acid sequence shown as SEQ ID NO 1 and has a molecular weight of 934.11 Da.)

1. The application of the milk-derived active peptide CCL-1S in preparing a sleep-promoting product is characterized in that the milk-derived active peptide CCL-1S has an amino acid sequence shown as SEQ ID NO. 1.

2. Use of the milk-derived active peptide CCL-1S according to claim 1 for the preparation of a sleep-promoting product, wherein the sleep-promoting product comprises a sleep-promoting drug, a sleep-promoting functional food.

3. Use of the milk-derived active peptide CCL-1S according to claim 1 for the preparation of a pro-sleep product, wherein the pro-sleep is an inhibition of the firing frequency of neuronal action potentials.

4. The sleep-promoting medicine is characterized by comprising a milk-derived active peptide CCL-1S, wherein the milk-derived active peptide CCL-1S has an amino acid sequence shown as SEQ ID NO. 1.

5. A sleep promoting pharmaceutical product according to claim 4, further comprising a pharmaceutically acceptable carrier and/or adjuvant.

6. A sleep promoting pharmaceutical product according to claim 4, wherein the dosage form of the sleep promoting pharmaceutical product comprises oral liquid, granule, capsule, tablet, granule, pill, paste and injection.

7. A functional food for promoting sleep, which is characterized by comprising milk-derived active peptide CCL-1S, wherein the milk-derived active peptide CCL-1S has an amino acid sequence shown as SEQ ID NO. 1.

8. A functional food for promoting sleep according to claim 7, further comprising a food-acceptable carrier and/or excipient.

9. The sleep-promoting functional food according to claim 8, wherein the sleep-promoting functional food is in the form of beverage, candy, pastry, noodles, meat product.

Technical Field

The invention belongs to the field of sleep-promoting health products, and particularly relates to application of milk-derived active peptide CCL-1S in preparation of sleep-promoting products.

Background

Insomnia is a common sleep disorder, the total sleep time of an insomnia patient does not reach the standard, the sleep quality is unqualified, and the sub-health state of the life and work of the patient is influenced. Long-term insomnia easily causes low work efficiency, amnesia, decreased thinking ability, depression, anxiety, mental stress and other emotions, and serious insomnia can lead to pessimism and boredom, psychosis, depression and the like. The sedative hypnotic drugs of chemical synthesis type, such as sedative hypnotic drugs mainly comprising benzodiazepines, are commonly used clinically, and can treat sleep disorder caused by insomnia and other diseases. However, the medicines have side effects of different degrees such as dizziness, hypodynamia, drowsiness, inattention and the like while prolonging the sleep time, and are easy to generate dependence, so that the effect of improving the sleep quality is not really achieved. Therefore, it is an important task in the medical and health field to provide a drug with significant efficacy, less side effects, and a safe sleep improvement effect.

The milk has sleep promoting effect, and the user can sleep more easily when drinking hot milk before sleeping. The cow milk contains various bioactive polypeptides, but the content of the active polypeptides in the natural cow milk is low, so that the requirements of sub-health people cannot be met. Bovine casein is a precursor of many potential bioactive substances, and small peptides with biological functions, namely milk-derived bioactive peptides, are obtained through the enzymolysis of protease. The small peptides generally consist of 2-20 amino acid residues, can positively influence multiple aspects of the digestive system, the cardiovascular system, the immune system and the like of an organism, and have the physiological activities of regulating the nervous system, resisting bacteria, resisting oxidation, resisting hypertension, regulating immunity and the like. The antibacterial, antioxidant, antihypertensive and immunoregulatory effects of milk-derived active peptides are more researched, and the research on the sleep condition improvement of the milk-derived active peptides in the nervous system is less. Therefore, the development of more hypnotic peptides with the effect of regulating the nervous system has important value for developing safe and efficient hypnotic products.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide the application of the milk-derived active peptide CCL-1S in preparing a sleep-promoting product.

It is a second object of the present invention to provide a sleep-promoting pharmaceutical product or a sleep-promoting functional food.

The purpose of the invention is realized by the following technical scheme:

application of milk-derived active peptide CCL-1S in preparing a sleep-promoting product, wherein the milk-derived active peptide CCL-1S has an amino acid sequence shown as SEQ ID NO: 1.

The amino acid sequence of the milk-derived active peptide CCL-1S is as follows: Asp-Met-Pro-Ile-Glu-Ala-Phe-Leu (DMPIQAFL) and 934.11, wherein a novel sleep-promoting milk-derived active peptide CCL-1S which can inhibit the action potential release frequency of nerve cells and promote sleep is separated and identified from milk by using an in-vitro patch clamp technology after removing impurities by using milk-derived casein zymolyte as a raw material and adopting basic means such as centrifugation, desalination and the like.

Preferably, the sleep-promoting product comprises a sleep-promoting medicine and a sleep-promoting functional food.

Preferably, the sleep promotion is the suppression of the firing frequency of nerve cell action potentials.

The invention also provides a sleep-promoting medicine which comprises milk-derived active peptide CCL-1S, wherein the milk-derived active peptide CCL-1S has an amino acid sequence shown as SEQ ID NO. 1.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier and/or an auxiliary material.

More preferably, the dosage form of the sleep-promoting medicine comprises oral liquid, granules, capsules, tablets, granules, pills, paste and injection.

The invention also provides a functional food for promoting sleep, which comprises milk-derived active peptide CCL-1S, wherein the milk-derived active peptide CCL-1S has an amino acid sequence shown as SEQ ID NO. 1.

Preferably, the functional food further comprises a food-acceptable carrier and/or an auxiliary material.

More preferably, the sleep-promoting functional food is in the form of beverage, candy, pastry, noodles and meat products.

Compared with the prior art, the invention has the beneficial effects that:

the invention utilizes the in vitro patch clamp technology to separate and identify a milk-derived bioactive peptide CCL-1S which has the functions of inhibiting the activity of nerve cells and promoting sleep from milk, the milk-derived bioactive peptide has an amino acid sequence shown as SEQ ID NO. 1, and the molecular weight is 934.11 Da.

Drawings

FIG. 1a is a sectional sample graph for preparing a liquid phase secondary separation, and FIG. 1b is AP inhibition rate of a prepared liquid phase secondary separation sample; FIG. 1c is a graph showing the rate of change of Vm in a sample prepared for liquid phase secondary separation;

FIG. 2 is a liquid phase diagram of sample analysis;

FIG. 3 shows AP induced by VLPO under physiological conditions on the left side, and inhibition of AP induced by monomer 1 on VLPO neurons on the right side of FIG. 3;

FIG. 4 is a CCL-1S amino acid sequencer profile.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Materials: milk casein hydrolysate produced by Guangzhou green extract biotechnology limited.

See table 1 for reagents.

TABLE 1

Example 1 isolation of sleep peptides

Firstly, raw material pretreatment: the method comprises the steps of removing ethanol from a milk casein zymolyte raw material of a company through rotary evaporation to obtain a milk polypeptide saturated water sample, and performing vacuum spray drying to obtain a polypeptide raw material.

Secondly, measuring the in vitro sleep promoting activity: according to the patent method with the application number of 201510915215.0 and the name of 'method for screening crude sleep-promoting peptide extract in milk source by using patch clamp technology', the in-vitro sleep-promoting activity is measured, a hypothalamus slice is manufactured by using the patch clamp technology, the slice is placed in a circulating perfusion system to prepare a microelectrode, and the microelectrode is sealed with VLPO neuron of the hypothalamus slice, so that a conventional whole cell mode is formed. Recording a section of spontaneous discharge as an experimental control, perfusing perfusate containing cow milk polypeptide to observe the change of the neuron discharge form, and evaluating the influence of VLPO neuron excitability inhibition rate by evaluating the change rate of Vm and the number of AP induced by depolarized current, thereby analyzing the in-vitro sleep-promoting activity of cow milk polypeptide.

Third, C18 column one-time separation

Preparing milk polypeptide powder (namely polypeptide raw material containing a large amount of milk hypnotize peptides) into a solution of 200mg/ml with ultrapure water, extracting with a syringe, passing through a 0.22um filter membrane, and primarily separating the milk hypnotize peptides by means of preparative high performance liquid chromatography (LC-8A, Shimadzu).

Chromatographic conditions are as follows: the column was a self-contained glass column [20mm × 450mm, C18 packing (10 μm,Macherey Nagel,France)](ii) a Mobile phase: pump a was ultrapure water (containing 0.1% TFA) and pump B was acetonitrile (containing 0.1% TFA); flow rate: 10 mL/min; sample introduction amount: 5 mL; detection wavelength: 214nm, 280 nm; elution conditions: 10% -20% (0.01-30min), 20% -35% (30-90min), 35% -52.5% (90-110min), 52.5% -90% (110-125 min). According to the condition of primary separation, the extract is divided into six segments named as P1, P2, P3, P4, P5 and P6, and is subjected to rotary concentration and freeze drying at 60 ℃ for later use.

As a result: the spectrum of the separation and detection of the raw material in the preparation liquid phase is shown in figure 1 a. The polypeptide is generally divided into 6 sections for sample connection under the detection wavelength of 214nm according to the peak emergence time and the similarity of peak shapes. The 6 fractions were collected separately, concentrated as soon as possible and lyophilized in vacuo to reduce polypeptide degradation. Their in vitro hypnotic activity was then measured and the results are shown in figure 1 b. Because the P5 and P6 samples are insoluble in cerebrospinal fluid, the P1-4 component is analyzed to find that the P3 component has the best effect of inhibiting the excitability of VLPO neurons, and the inhibition effect of other components is not obvious, so that the P3 component is selected for next separation and analysis.

Fourth, C18 column secondary separation

The P3 was subjected to preliminary activity evaluation using a patch clamp model, and the P3 sample was divided into three sections according to the activity peak.

Chromatographic conditions are as follows: the column was a Welch C18 column (4.6 mm. times.250 mm,5 μm, Ultimate); mobile phase: pump a was ultrapure water (containing 0.1% TFA) and pump B was acetonitrile (containing 0.1% TFA); sample introduction amount: 20 mu L of the solution; flow rate: 1 mL/min; detection wavelength: 214nm, 280 nm; elution conditions: 10-20% (0.01-10min), 20-30% (10-40min) and 30-90% (40-48 min).

As a result: according to the research, P3 has better sleep promoting activity, the P3 component is further separated into a front section, a middle section and a rear section, and the in-vitro sleep promoting activity is continuously evaluated. The anterior segment was found to have better sleep-promoting activity by further isolating the P3 component (fig. 1 c).

Fifthly, third separation of HCLP

The second part of the patch clamp model P3 was used for a preliminary activity assessment, which was divided into 4 single peaks.

Chromatographic conditions are as follows: the column was a Welch C18 column (4.6 mm. times.250 mm,5 μm, Ultimate); mobile phase: pump a was ultrapure water (containing 0.1% TFA) and pump B was acetonitrile (containing 0.1% TFA); sample introduction amount: 20 mu L of the solution; flow rate: 1 mL/min; detection wavelength: 214nm, 280 nm; elution conditions: 10-20% (0.01-10min), 20-30% (10-40min) and 30-90% (40-48 min).

As a result: the mid-section of P3 is explored, and the change rate of Vm of the monomer P1 is 11.46 +/-4.68%, the inhibition rate of AP is 7.5 +/-6.25%, and the compound has good sleep-promoting activity.

Table 2 effect of monomer 1 on the excitability of VLPO neurons (n ═ 9)

Note: p <0.05, p <0.01, compared to normal control group

Example 2 determination of sleep peptide Structure

The amino acid composition of the peptide is determined by using an amino acid sequencer (PPSQ), and the absolute sequence of the N terminal of the polypeptide can be determined by the PPSQ. According to the analysis chart of an amino acid sequencer (figure 4), the sequence of the polypeptide is determined to be Asp-Met-Pro-Ile-Glu-Ala-Phe-Leu (DMPIQAFL) by comparing the sequences of cow milk casein proteins in a Uniprot database, and the polypeptide is derived from beta casein in cow milk casein, and the sequence is firstly separated from natural products of food sources to form the hypnotic peptide.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Sequence listing

<110> southern China university of agriculture

Application of <120> milk-derived active peptide CCL-1S in preparation of sleep-promoting product

<130> ZM211206CS

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 1

Asp Met Pro Ile Gln Ala Phe Leu

1 5