A sleep-promoting oligopeptide derived from sheep milk, its preparation method and application

Abstract

This invention belongs to the field of extraction technology of effective components from milk sources. It discloses a sleep-promoting oligopeptide derived from sheep milk, its preparation method, and its application. This invention uses sheep milk casein as raw material to isolate and purify an oligopeptide with sleep-promoting effects. This oligopeptide has the amino acid sequence Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Ile-Met and a molecular weight of 1796.9675 Da. The sleep-promoting oligopeptide prepared by this invention has good activity, is safe and non-toxic, and can significantly prolong the sleep duration of *C. elegans*. It can be applied in the field of sleep promotion and is beneficial to the resource utilization of sheep milk casein.

Description

Technical Field

[0001] This invention belongs to the field of extraction technology of effective components from milk sources, specifically relating to a sleep-promoting oligopeptide derived from sheep milk, its preparation method, and its application. Background Technology

[0002] Humans spend approximately one-third of their lives sleeping, and sleep quality is closely related to physical and mental health. In today's fast-paced life, environmental factors such as chronic stress, anxiety, depression, shift work, and malnutrition severely impact sleep quality. Sleep disorders can lead to various health problems, including obesity, diabetes, and hypertension. In my country, over 300 million people suffer from sleep disorders. The use of sleeping pills and sedative-antidepressants for the treatment of chronic insomnia is widely adopted. However, long-term use of these synthetic drugs often results in significant side effects such as tolerance and dependence, including poor sleep quality, increased awakenings, and impaired or suppressed REM sleep. Therefore, discovering novel sleep-aiding substances with fewer or no side effects has become a research hotspot and challenge in recent years.

[0003] Bioactive peptides are protein-derived fragments that perform various physiological functions in the human body. Milk protein contains abundant bioactive peptides, and sleep-promoting peptides have been isolated from milk protein. With increasing awareness of the quality and nutritional value of goat milk, goat milk products are becoming increasingly popular. The main goat milk products on the market include fresh goat milk, goat milk powder, yogurt, and goat cheese, but the in-depth development of goat milk resources is insufficient. Therefore, enzymatic hydrolysis of goat milk protein to extract bioactive peptides beneficial to human health meets the current needs of deep processing of goat milk products. The enzymatic hydrolysis of goat milk protein can produce polypeptides with antihypertensive, antioxidant, hypoglycemic, anti-inflammatory, antibacterial, opioid, or immunomodulatory activities. Sleep-promoting peptides derived from goat milk still await further development. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the above-mentioned problems existing in the prior art, and firstly, to provide a sleep-promoting oligopeptide derived from sheep milk.

[0005] A second objective of this invention is to provide a method for preparing the aforementioned oligopeptides.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A sleep-promoting oligopeptide derived from goat milk, the amino acid sequence of which is shown in SEQ ID NO: 1. The amino acid sequence of the oligopeptide is: Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Ile-Met, and the molecular weight is 1796.9675 Da.

[0008] This invention also provides the application of the oligopeptide in the preparation of functional products that prolong sleep duration. The oligopeptide, obtained by enzymatic hydrolysis of sheep milk casein and subsequent purification, can prolong the sleep time of *C. elegans*, providing a basis for the preparation of foods or drugs with sleep-promoting effects.

[0009] Preferably, the functional product is food, medicine, or health product.

[0010] The present invention also provides a method for preparing the oligopeptide, comprising the following steps:

[0011] S1. The sheep milk casein is hydrolyzed with trypsin. After hydrolysis, it is centrifuged and filtered, and then freeze-dried to obtain sheep milk casein hydrolysate powder.

[0012] S2. Prepare an aqueous solution of sheep milk casein hydrolysate with a concentration of 150 mg / ml. After filtration, perform preliminary separation by high performance liquid chromatography. Based on the peak time and peak shape similarity in the chromatogram, divide the sample into 6 segments for inoculation. Collect the components separately, concentrate and freeze-dry them under vacuum. Measure the sleep-promoting activity of each component using nematodes.

[0013] S3. The component with the best sleep-promoting activity obtained in the previous step was prepared into an aqueous solution with a concentration of 25 mg / ml. After filtration, it was separated a second time by preparative high performance liquid chromatography. According to the peak time and peak shape similarity in the spectrum, it was divided into 4 segments for sample collection. The components were collected separately, concentrated and freeze-dried under vacuum. The sleep-promoting activity of each component was measured by Caenorhabditis elegans.

[0014] S4. The fraction with the best sleep-promoting activity obtained in the previous step is prepared into an aqueous solution with a concentration of 20 mg / ml. After filtration, it is separated for the third time by preparative high performance liquid chromatography. According to the peak time and peak shape similarity in the spectrum, it is divided into 7 segments for sample collection. The components are collected separately, concentrated and freeze-dried under vacuum. The sleep-promoting activity of each component is measured by nematode elegans. The fraction with the best sleep-promoting activity is the sleep-promoting oligopeptide described in this invention.

[0015] Preferably, the chromatographic column in step S2 is a self-packed glass column with a size of 20mm × 450mm and C18 packing; the mobile phase is: pump A contains ultrapure water with 0.1% TFA, and pump B contains acetonitrile with 0.1% TFA; the flow rate is 10mL / min; the injection volume is 5mL; the detection wavelength is 214nm and 280nm; the elution conditions are: 10%-48%, 0.01-66.00min, 48%-90%, 66.00-66.01min, 90%-90%, 66.01-71.00min, 90%-10%, 71.00-74.00min, and 10%-10%, 74.00-80.00min.

[0016] Preferably, the chromatographic column in step S3 is a PRC-ODS(K) column, the mobile phase A is primary water containing 0.1% TFA, the mobile phase B is acetonitrile containing 0.1% TFA, the flow rate is 10 mL / min, the injection volume is 4 mL, the monitoring is at 214 nm and 280 nm, and the elution program is 25%-35%, 0-70.00 min, 90%-90%, 70.00-75.00 min, 25%-25%, 75.00-80.00 min.

[0017] Preferably, the chromatographic column in step S4 is an ACCHROM C18 steel column, mobile phase A is primary water containing 0.1% TFA, mobile phase B is acetonitrile containing 0.1% TFA, the flow rate is 10 mL / min, the injection volume is 5 mL, and the elution is monitored at 214 nm and 280 nm. The elution program is as follows: 5%-30%, 0.01-5.00 min; 30%-40%, 5.01-65.00 min; 40%-90%, 65.01-70.00 min; 90%-90%, 70.01-80.00 min; 90%-10%, 80.01-90.00 min; 10%-10%, 90.01-95.00 min.

[0018] This invention also provides a method for tracking the activity of sleep-promoting oligopeptides, comprising the following steps:

[0019] S1. Prepare nematode production culture medium (NGM). Depending on the amount of peptone added, NGM is divided into conventional NGM, drug-treated NGM, and video recording NGM, with peptone addition amounts of 2.5 g / L, 0.25 g / L, and 0 g / L, respectively.

[0020] S2. Strain coating: The bacterial solution and sample are mixed at a ratio of 9:1. 100 μL of conventional NGM coating, 100 μL of drug-treated NGM coating, and 80 μL of video-coated NGM coating.

[0021] In a preferred embodiment, the area of ​​NGM that is coated in step S2 is a circle with a diameter of 5 cm.

[0022] S3. After synchronization of *C. elegans*, the eggs were inoculated on culture media with or without drug administration, and cultured at 20°C for 48 hours before being transferred to video recording medium.

[0023] In a preferred embodiment, step S3 involves selecting larvae in the middle L4 stage, with 15 larvae selected and evenly placed in the video culture medium coating area.

[0024] S4. Place the recording medium from S3 into the wormlab device to record behavioral videos of *C. elegans* at a frame rate of 10 seconds / frame for a recording duration of 11.5 hours.

[0025] S5. Use Wormlab software to analyze the video recorded in S4 to determine the sleep duration of *C. elegans*.

[0026] In a preferred embodiment, the method for determining the sleep duration of *C. elegans* in step S5 is as follows: During the analysis, the circular area coated with bacterial solution is selected and converted into a binary image. The activity of *C. elegans* is quantified by its movement speed. A movement speed of less than 1 μm / s for three consecutive frames is defined as a stationary state, and the interval between two consecutive stationary states is less than 10 minutes (otherwise it is considered a non-stationary state). For each recording, the start of the first four consecutive stationary states is taken as the time point of entering sleep, and the end of the last four consecutive stationary states is taken as the time point of awakening. The total sleep time refers to the time from entering sleep to awakening.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention uses sheep milk casein as raw material to isolate and purify an oligopeptide with sleep-promoting effects. The oligopeptide has the amino acid sequence Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Ile-Met and a molecular weight of 1796.9675 Da. The sleep-promoting oligopeptide prepared by this invention has good activity, is safe and non-toxic, and can significantly prolong the sleep duration of *C. elegans*. It can be applied in the field of sleep promotion and is beneficial to the resource utilization of sheep milk casein. Attached Figure Description

[0029] Figure 1 This is a diagram illustrating the NGM coating process.

[0030] Figure 2 The vulvar morphology of *C. elegans* at different substages of L4;

[0031] Figure 3 The effect of GMCH on total sleep duration in *C. elegans*;

[0032] Figure 4 To prepare a sample collection diagram for single-stage liquid phase separation;

[0033] Figure 5 To study the effect of single-phase liquid chromatography separation on the total sleep duration of *C. elegans*;

[0034] Figure 6 To prepare a sample collection diagram for secondary liquid phase separation;

[0035] Figure 7 To study the effect of preparing liquid-phase secondary separation samples on the total sleep duration of *C. elegans*;

[0036] Figure 8To prepare a sample collection diagram for three-stage liquid phase separation;

[0037] Figure 9 To study the effect of three-stage liquid phase separation on the total sleep duration of *C. elegans*;

[0038] Figure 10 RP-HCLP chromatogram of sleep-promoting oligopeptide F4;

[0039] Figure 11 The first-order mass spectrum of the sleep-promoting oligopeptide F4;

[0040] Figure 12 The secondary mass spectrum of the sleep-promoting oligopeptide F4;

[0041] Figure 13 PPSQ sequencing image of sleep-promoting oligopeptide F4. Detailed Implementation

[0042] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] Unless otherwise specified, the experimental methods used in the following embodiments and experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified; and the equipment used are conventional experimental equipment unless otherwise specified.

[0044] Example 1: Determination of the sleep-promoting activity of sheep milk casein hydrolysate

[0045] (1) Materials and reagents

[0046] Materials: Goat micellar casein concentrate 60-63 (gMCC60-63) provided by Ausnutria Dairy (China) Co., Ltd., which is goat milk casein with a protein content of 60-63%.

[0047] Reagents: Sodium chloride, potassium phosphate, disodium hydrogen phosphate, sodium hydroxide, technical agar powder, streptomycin sulfate, tryptone, trypsin. All reagents were of analytical grade.

[0048] (2) Casein hydrolysis process of sheep milk

[0049] A certain mass of casein was weighed and placed in an Erlenmeyer flask. First-grade deionized water was added at a material-to-liquid ratio of 1:10. Trypsin was added at a concentration of 0.4%. Enzymatic hydrolysis was carried out for 2.5 hours. During the hydrolysis process, the pH of the hydrolysate was adjusted to 8.0 using a 2 mol / L NaOH solution, and the hydrolysis temperature was controlled at 40℃. After hydrolysis, the enzyme was inactivated at 95℃ for 10 minutes. The mixture was then cooled to room temperature and centrifuged at 10000 rpm for 15 minutes to remove the enzyme and undigested proteins and other macromolecules. The resulting product was then freeze-dried to obtain Goat milk casein hydrolysate (GMCH) powder.

[0050] (3) Assay of sleep-promoting activity of sheep milk casein hydrolysate

[0051] 3.1 Preparation of nematode growth medium

[0052] Weigh 17g of agar powder, 2.5g of tryptone, 3g of sodium chloride, and 0.2g of streptomycin and dissolve them in 1L of ultrapure water. Place the solution together with 1mol / L CaCl2, MgSO4, and K2HPO4-KH2PO4 buffer solution in an autoclave for sterilization. The sterilization conditions are: temperature 121℃ and time 20min. After cooling to 80-90℃, remove the solution and add 1ml of CaCl2 and MgSO4, 25ml of K2HPO4-KH2PO4 solution, and 1ml of 5mg / ml cholesterol ethanol filtered through a 0.22μm filter to the culture medium solution in a clean bench. Mix well and transfer to a sterilized culture dish to obtain solid nematode growth medium (NGM). After the NGM cools and solidifies, store it in a refrigerator for later use. Depending on the amount of peptone added, NGM is divided into conventional NGM, drug-eluting NGM, and video recording NGM, with peptone addition amounts of 2.5 g / L, 0.25 g / L, and 0 g / L, respectively.

[0053] 3.2 Strain Culture

[0054] Uracil-deficient Escherichia coli (E. coli) OP50 was streaked onto solid agar plates and incubated for 1-2 days. Single colonies were then picked and transferred to 5 mL of sterilized LB (Luria-Bertani) liquid medium. E. coli OP50 was then incubated at 37°C and 200 rpm for 12 hours using a shaker. When the OD... 600 When the concentration is 0.4, it can be inoculated onto nematode growth medium (NGM) plates for feeding nematodes.

[0055] 3.3 Culture of *C. elegans*

[0056] E. coli OP50 was spread onto NGM, and then the nematodes were transferred to plates containing E. coli OP50. All nematodes were cultured in a constant temperature and humidity incubator at 20°C.

[0057] 3.4 Synchronization of *C. elegans*

[0058] Young adult nematodes were rinsed into sterile EP tubes using M9 buffer solution. Primary water, NaOH, and 10% NaClO were mixed in a volume ratio of 1:1:1 to prepare the lysis buffer for *C. elegans*. The lysate was mixed with the sterile EP tubes, shaken, and centrifuged at 3000 rpm for 1 min. The supernatant was discarded. The nematodes were rinsed twice with M9 solution, centrifuged again, and the supernatant was discarded. The eggs at the bottom of the EP tubes were then pipetted onto the cultured NGM medium. After about 48 hours, the fertilized eggs in the lysed nematodes had basically developed into L4 stage larvae, completing the synchronization.

[0059] 3.5 Route of administration

[0060] Sheep milk casein hydrolysate was prepared into aqueous solutions with concentrations of 0.5, 1.0, and 1.5 mg / ml, respectively. These solutions were sterilized by filtration through a 0.22 μm pore size sterile filter. The sterilized sample solutions were then thoroughly mixed with E. coli OP50 solution at a 1:9 ratio and coated onto the surface of NGM culture medium, resulting in final drug concentrations of 50, 100, and 150 mg / ml, respectively. The control group sample solution was replaced with an equal volume of sterile water. 100 μL of conventional NGM was used for bacterial coating, 100 μL of drug-grade NGM was used, and 80 μL of video-coated NGM was used. The coated area of ​​video-coated NGM was limited to a circle with a diameter of 5 cm (e.g., ...). Figure 1 As shown in the image, this area represents the maximum recording range within which the video recording system can clearly capture the movement of nematodes. Covering this area with bacterial solution is intended to allow the nematodes to move within the recording range.

[0061] 3.6 Developmental Sleep Assay of *C. elegans*

[0062] Based on the vulvar morphology of *C. elegans*, select larvae in the mid-L4 stage (e.g., *C. elegans*). Figure 2(As shown), 15 L4 mid-stage larvae were placed in an NGM bacterial solution area. Using a video recording device equipped with a Basler acA2440-35um USB 3.0 camera and a Nikon AF Micro 60mm f / 2.8D lens, the nematodes were imaged every 10 seconds for 11.5 hours. Image acquisition and subsequent analysis were performed using Wormlab behavioral analysis software (Version 3.1.0, MBF Bio-science, Williston, VT). During analysis, circular areas coated with the bacterial solution were selected and converted into binary images. The activity of *C. elegans* was quantified by movement speed. A movement speed of less than 1 μm / s for three consecutive frames was defined as a stationary state, with an interval of less than 10 minutes between two consecutive stationary states (otherwise considered a non-stationary state). For each recording, the start of the first four consecutive stationary states was taken as the time point of entering sleep, and the end of the last four consecutive stationary states was taken as the time point of awakening. Total sleep time refers to the time from entering sleep to awakening. A nematode's movement speed exceeding 1 μm / s is defined as an active event. Recordings containing images during sleep periods where the nematode's location cannot be resolved (escapes from the recording range or become entangled) are excluded from analysis.

[0063] like Figure 3 As shown, the total sleep duration of *C. elegans* in the control group was 122.41 ± 21.56 min. Compared with the control group, the total sleep duration of *C. elegans* was significantly prolonged at drug concentrations of 100 mg / L and 150 mg / L, by 23.14% and 17.16%, respectively. The total sleep duration of nematodes in the GMCH (50 mg / L) group was comparable to that in the control group. The results indicate that GMCH promotes the total sleep duration of *C. elegans*, with an optimal drug concentration of 100 mg / L.

[0064] Example 2: Screening and Identification of Sleep-Promoting Peptides from Goat Milk

[0065] (1) Materials and reagents

[0066] Materials: Sheep milk casein hydrolysate, prepared as shown in Example 1.

[0067] Reagents: Acetonitrile, analytical grade; acetonitrile and trifluoroacetic acid, chromatographic grade; acetonitrile and formic acid, mass spectrometric grade; horse myoglobin, Sigma-Aldrich (USA).

[0068] (2) Preparation of liquid phase separation

[0069] Sheep milk casein hydrolysate was prepared into an aqueous solution with a concentration of 150 mg / ml. After passing through a 0.45 μm filter membrane, it was initially separated by preparative high performance liquid chromatography (LC-8A, Shimadzu). Based on the peak time and peak shape similarity in the chromatogram, it was divided into 6 segments for sample collection. The components were collected separately, concentrated (65°C rotary evaporation), and then freeze-dried under vacuum. The sleep-promoting activity of each component was measured using *C. elegans*.

[0070] Chromatographic conditions: The chromatographic column was a self-packed glass column (20 mm × 450 mm), and C18 packing material (10 μm). Macherey Nagel, France); Mobile phase: Pump A is ultrapure water (containing 0.1% TFA), Pump B is acetonitrile (containing 0.1% TFA); Flow rate: 10 mL / min; Injection volume: 5 mL; Detection wavelength: 214 nm, 280 nm; Elution conditions: 10%-48% (0.01-66.00 min), 48%-90% (66.00-66.01 min), 90%-90% (66.01-71.00 min), 90%-10% (71.00-74.00 min), 10%-10% (74.00-80.00 min).

[0071] like Figure 4 As shown, under 214nm wavelength detection conditions, the sheep milk casein hydrolysate could be divided into 6 segments based on peak elution time and peak shape similarity, named Y1, Y2, Y3, Y4, Y5, and Y6, respectively. These 6 components were collected separately, then immediately concentrated and freeze-dried under vacuum. Subsequently, the effects of the 6 components on the total sleep duration of *C. elegans* were measured. Figure 5 The results showed that the total sleep duration of nematodes in the CK group was 144.31 ± 20.61 min, and that of nematodes in the GMCH group was 162.65 ± 20.78 min. The total sleep duration of nematodes in the Y4 group was 24.89% longer than that of the CK group and 10.81% longer than that of the GMCH group. These results indicate that the Y4 subfraction has the best sleep-inducing effect. The next step is to separate the Y4 subfraction.

[0072] (3) Preparation of liquid phase secondary separation

[0073] Y4 group was prepared into an aqueous solution with a concentration of 25 mg / ml. After passing through a 0.22 μm filter membrane, it was initially separated by preparative high performance liquid chromatography (LC-8A, Shimadzu). Based on the peak time and peak shape similarity in the chromatogram, it was divided into 4 segments for sample collection. The components were collected separately, concentrated and freeze-dried under vacuum. The sleep-promoting activity of each component was measured by Caenorhabditis elegans.

[0074] Chromatographic conditions: The column was a Shimadzu PRC-ODS(K) column (30mm×250mm, 15μm, Shimadzu). Mobile phase A was primary water (containing 0.1% TFA), and mobile phase B was acetonitrile (containing 0.1% TFA). The flow rate was 10mL / min, and the injection volume was 4mL. The elution was monitored at 214nm and 280nm. The elution program (concentration of mobile phase B) was 25%-35% (0-70.00min), 90%-90% (70.00-75.00min), and 25%-25% (75.00-80.00min).

[0075] like Figure 6 As shown, under 214 nm wavelength detection conditions, the sheep milk casein hydrolysate could be divided into four segments based on peak elution time and peak shape similarity, named P1, P2, P3, and P4, respectively. These four components were collected separately, then immediately concentrated and freeze-dried under vacuum. Subsequently, the effects of the four components on the total sleep duration of *C. elegans* were measured. Figure 7 The results showed that the total sleep duration of nematodes in the CK group was 117.48±18.88 min, the total sleep duration of nematodes in the Y4 group was 147.50±17.73 min, and the total sleep duration of nematodes in the P4 group was 27.42% longer than that of the CK group, but there was no significant difference between the P4 group and the Y4 group. In summary, the P4 subfraction had the best sleep-inducing effect, and the next step is to separate the P4 subfraction.

[0076] (4) Preparation of liquid phase three-stage separation

[0077] The P4 group was prepared into an aqueous solution with a concentration of 20 mg / ml. After passing through a 0.22 μm filter membrane, it was initially separated by preparative high performance liquid chromatography (LC-8A, Shimadzu). Based on the peak time and peak shape similarity in the chromatogram, it was divided into 7 segments for sample collection. The components were collected separately, concentrated, and freeze-dried under vacuum. The sleep-promoting activity of each component was measured by Caenorhabditis elegans.

[0078] The chromatographic column was an ACCHROM C18 steel column (20mm × 250mm, 10μm). The mobile phase A was primary water (containing 0.1% TFA), and the mobile phase B was acetonitrile (containing 0.1% TFA). The flow rate was 10 mL / min, the injection volume was 5 mL, and the elution was monitored at 214 nm and 280 nm. The elution program (concentration of mobile phase B) was as follows: 5%-30% (0.01-5.00 min), 30%-40% (5.01-65.00 min), 40%-90% (65.01-70.00 min), 90%-90% (70.01-80.00 min), 90%-10% (80.01-90.00 min), and 10%-10% (90.01-95.00 min).

[0079] like Figure 8 As shown, under 214nm wavelength detection conditions, the sheep milk casein hydrolysate could be divided into 7 segments based on peak elution time and peak shape similarity, named F1, F2, F3, F4, F5, F6, and F7, respectively. These 7 components were collected separately, then immediately concentrated and freeze-dried under vacuum. Subsequently, the effects of the 7 components on the total sleep duration of *C. elegans* were measured. Figure 9 The total sleep duration of nematodes in the CK group was 146.79 ± 20.72 min, while that in the P4 group was 171.06 ± 22.47 min. The total sleep duration of nematodes in the F4 group was 24.58% longer than that in the CK group and 6.91% longer than that in the P4 group. Furthermore, the purity of this component was determined using RP-HCLP, and its chromatogram (…) Figure 10 The result showed that this component had only a single peak, therefore F4 is a highly active polypeptide monomer, and further research was conducted on oligopeptide F4. This indicates that the separation and purification conditions can achieve excellent separation and purification of polypeptide components in sheep milk-derived polypeptides, and can also screen for highly active sleep-promoting peptides.

[0080] (5) Determination of the structure of sleep-promoting oligopeptides

[0081] To investigate the composition of the F4 sleep-promoting peptide, the molecular weight and structural information of the purified peptide were identified using an LTQ ORBITRAP VELOS PRO high-resolution mass spectrometer. The results were based on the primary mass spectrum (see attached image). Figure 11 The relative molecular mass of the sleep-promoting peptide F4 was determined to be 1796.9675 Da, suggesting that F4 is an oligopeptide composed of 13-19 amino acids. Based on this, the operating parameters of the amino acid sequencer were set to determine the N-segment amino acid sequence of F4. The sequence was obtained from the secondary mass spectrometry (see attached image). Figure 12 ) and amino acid sequencer chromatograms (attached) Figure 13 The final sequence of the sleep-promoting peptide F4 was determined to be Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Ile-Met.

[0082] (TQTPVVVPPFLQPEIM). By searching the UniProt protein database, the peptide sequence was determined to be derived from β-casein in sheep milk, and the sleep-promoting activity of the sequence TQTPVVVPPFLQPEIM is reported for the first time.

[0083] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of oligopeptides in the preparation of functional products that prolong sleep duration; characterized in that, The functional products are drugs or health products; the amino acid sequence of the oligopeptide is shown in SEQ ID NO: 1.

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