A key preparation method of a bird's nest peptide and a bird's nest peptide for pregnant women

Abstract

The application provides a key preparation method of bird's nest peptide and bird's nest peptide for pregnant women, and the preparation method of the bird's nest peptide comprises the following steps: soaking dry bird's nest in pure water at 0-4 DEG C for 2 h at a material-to-liquid ratio of 1:10, beating and then drying to obtain soaked bird's nest; mixing the soaked bird's nest with pure water at a mass ratio of 1:15-20, sealing and then stewing at 121-123 DEG C for 12-15 min, repeating the stewing once after cooling to room temperature to obtain fresh stewed bird's nest; carrying out in-vitro digestion experiment on the fresh stewed bird's nest to simulate the digestion process of the fresh stewed bird's nest in the gastrointestinal tract, and obtaining digested bird's nest; centrifuging the digested bird's nest to obtain supernatant, treating the supernatant, and then carrying out protein sequencing; comparing the protein sequencing with an online database, screening out a peptide segment with high matching degree and a response value greater than 10 9 The bird's nest peptide can significantly improve the proliferation rate of nerve cells, and provides a cell basis for the development of the nervous system.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food processing, and particularly relates to a key preparation method of a bird's nest peptide and a bird's nest peptide for pregnant women. BACKGROUND

[0002] Bird's nest is a nest built by the saliva and down of various swallow species of the Apodidae family, mainly produced in Southeast Asian countries and the South China Sea islands. Edible bird's nest contains various nutrients, the main nutrients of which include protein, carbohydrates, lipids, vitamins, amino acids and various important inorganic elements, among which the protein content is as high as about 50%, and the main functional components are sialic acid and epidermal growth factor, which have physiological functions such as antioxidant, anti-aging and immune enhancement. Therefore, bird's nest has been used as a health food for nourishing, beautifying and health preservation.

[0003] Pregnant women eating bird's nest has multiple effects such as supplementing nutrition, enhancing immunity, promoting fetal growth and development, and beautifying the skin, among which promoting the development of nervous system function is an important function that attracts attention. However, bird's nest is relatively expensive, and usually needs to be taken for a long time to obtain the expected good effect, so for a long time, the promotion of bird's nest products has been limited.

[0004] The development of the nervous system needs various proteins to participate in the construction of the structure and function of nerve cells, and bird's nest protein provides necessary material support for the normal development of fetal nervous system. However, the mechanism of bird's nest protein after being ingested into the human body is still unclear, the research on peptide active ingredients in bird's nest digestion products is less, and the research on bird's nest peptides in promoting the proliferation of nerve cells and the development of nervous system is less.

[0005] In view of this, the application provides a bird's nest peptide and a key preparation method of a bird's nest peptide for pregnant women, which can significantly improve the proliferation rate of nerve cells and provide a cell basis for the development of the nervous system. SUMMARY

[0006] In view of the problems in the prior art that the mechanism of bird's nest protein in the human body is still unclear, and pregnant women need to take bird's nest for a long time to obtain the expected good effect, the application provides a bird's nest peptide and a key preparation method of a bird's nest peptide for pregnant women to solve the above technical defects.

[0007] The technical scheme adopted by the application to solve the technical problems is:

[0008] In a first aspect, the application provides a bird's nest peptide, and the amino acid sequence of the bird's nest peptide is PAAVPGI.

[0009] In a second aspect, the present application provides a bird's nest peptide pregnant woman bird's nest composition, which comprises the bird's nest peptide and fresh stewed bird's nest, and the mass ratio of the fresh stewed bird's nest to the bird's nest peptide is 100:0.2-1.

[0010] In a third aspect, the present application provides a method for preparing the bird's nest peptide, comprising the following steps:

[0011] S1, soaking dry bird's nest in pure water at 0-4℃ for 2h with a solid-liquid ratio of 1:10, then shaking and drying to obtain soaked bird's nest;

[0012] S2, mixing the soaked bird's nest with pure water at a mass ratio of 1:15-20, sealing and stewing at 121-123℃ for 12-15min, then repeating the stewing once after cooling to room temperature to obtain fresh stewed bird's nest;

[0013] S3, performing in vitro digestion experiment on the fresh stewed bird's nest to simulate the digestion process of the fresh stewed bird's nest in the gastrointestinal tract to obtain digested bird's nest;

[0014] S4, centrifuging the digested bird's nest to obtain supernatant, treating the supernatant, and then performing protein sequencing, and screening out peptide segments with high matching degree and response value greater than 10 9 to obtain the bird's nest peptide.

[0015] Preferably, in step S3, the fresh stewed bird's nest is subjected to in vitro digestion experiment to simulate the digestion process of the fresh stewed bird's nest in the gastrointestinal tract to obtain digested bird's nest, which specifically comprises the following sub-steps:

[0016] S31, after shearing the fresh stewed bird's nest, adjusting the pH to 2.0, adding pepsin at 4% of the total mass of the fresh stewed bird's nest, simulating gastric digestion, and incubating in a 37℃ water bath for 4h to obtain gastric digestion products;

[0017] S32, first adjusting the pH of the gastric digestion products to 5.33, then adjusting the pH to 7.5, adding pancreatin at 4% of the total mass of the fresh stewed bird's nest, simulating intestinal digestion, and incubating in a 37℃ water bath for 4h to obtain intestinal digestion products;

[0018] S33, after incubation, placing the intestinal digestion products in boiling water for 10min to terminate digestion, and cooling to room temperature to obtain digested bird's nest.

[0019] Preferably, in step S31, the fresh stewed bird's nest is sheared for 30s using a high-speed shearing machine, and then the pH of the sheared bird's nest is adjusted to 2.0 using 0.1M HCl; in step S32, the pH of the gastric digestion products is adjusted to 5.33 using 0.9M NaHCO3, and then the pH is adjusted to 7.5 using 1M NaOH.

[0020] Preferably, in step S4, the centrifugation is performed at a temperature of 4℃, a centrifugal force of 8000xg for 20min, and the supernatant is stored at -20℃ after centrifugation.

[0021] Preferably, in step S4, the supernatant is sequenced by using a liquid chromatography-ionization tandem mass spectrometer, and an online database is used for comparison, and a peptide group with a high matching degree and a response value greater than 10 is screened out. 9 The peptide group contains a peptide with an amino acid sequence of PAAVPGI.

[0022] Further preferably, in step S4, the method further comprises: synthesizing the peptide by using a solid-phase synthesis technique, adding the peptide into the culture medium after the peptide is configured into a solution, culturing the neural cells, and observing the proliferation of the neural cells, and selecting a peptide with optimal activity, wherein the amino acid sequence of the peptide with optimal activity is PAAVPGI.

[0023] Preferably, in step S1, the soaking ratio is 4-5 times, and the spin-drying time is 2min.

[0024] Preferably, in step S2, the stewing temperature is 121-123℃, and the stewing time is 12-15min.

[0025] In summary, compared with the prior art, the key preparation method of the bird's nest peptide and the bird's nest for pregnant women provided by the application has the following beneficial effects:

[0026] (1) Promoting the proliferation of neural cells: the specific peptide (with an amino acid sequence of PAAVPGI) with the function of promoting the proliferation of neural cells is screened out from the bird's nest through in-vitro digestion simulation experiments. In the experiments, the bird's nest peptide can significantly improve the proliferation rate of neural cells, and provides a cell basis for the development of the nervous system.

[0027] (2) Promoting the development of the nervous system: the bird's nest peptide composition for pregnant women provided by the application can effectively promote the development of the nervous system and improve the function of the nervous system. This has important significance for pregnant women to supplement nutrients and promote the development of the nervous system of fetuses.

[0028] (3) Improving the utilization rate of bird's nest: the traditional edible method of bird's nest needs to be taken for a long time and has a limited utilization rate. The effective components in the bird's nest are converted into small molecular peptide segments by the specific preparation method, which are more easily absorbed and utilized by the human body, thereby improving the nutritional value and utilization efficiency of the bird's nest.

[0029] (4) Safe and reliable: the bird's nest peptide and the bird's nest composition for pregnant women provided by the application are derived from natural bird's nest, and the preparation process does not involve harmful chemical substances, thereby ensuring the safety and reliability of the product, and the product is suitable for long-term consumption by special groups such as pregnant women.

[0030] (5) Innovative combination: the present application not only provides a bird's nest peptide with specific physiological functions, but also combines it with fresh stewed bird's nest to play a synergistic effect, providing a new idea and choice for the development of new maternal health care products or functional foods. BRIEF DESCRIPTION OF DRAWINGS

[0031] Other features, objects, and advantages of the application will become more apparent from the following detailed description when read in connection with the following drawings:

[0032] Figure 1 is a total ion chromatogram of the sample;

[0033] Figure 2 is a secondary mass spectrum of bird's nest peptide A (PAAVPGI);

[0034] Figure 3 is a secondary mass spectrum of bird's nest peptide B (HACASVD);

[0035] Figure 4 is a secondary mass spectrum of bird's nest peptide C (PAAMEGP);

[0036] Figure 5 is a secondary mass spectrum of bird's nest peptide D (TVPAAVP);

[0037] Figure 6 is a secondary mass spectrum of bird's nest peptide E (VEAGAGT);

[0038] Figure 7 is a comparison chart of SH-SY5Y cell proliferation rate;

[0039] Figure 8 is a zebrafish acetylcholinesterase (AChE) activity detection chart;

[0040] Figure 9 is a zebrafish dopamine (DA) content detection chart;

[0041] Figure 10 is a comparison chart of zebrafish average speed;

[0042] Figure 11 is a comparison chart of zebrafish moving distance. DETAILED DESCRIPTION

[0043] The present application will be further described in detail below in conjunction with the drawings and examples. It can be understood that the specific examples described herein are only used to explain the related application, and are not a limitation on the application. In addition, it should be noted that only the parts related to the application are shown in the drawings for ease of description.

[0044] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other in the case of no conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0045] In a first aspect, the present application provides a bird's nest peptide, the amino acid sequence of which is PAAVPGI.

[0046] In a second aspect, the present application provides a bird's nest peptide pregnant woman bird's nest composition, which comprises the bird's nest peptide and fresh stewed bird's nest, and the mass ratio of the fresh stewed bird's nest to the bird's nest peptide is 100:0.2-1.

[0047] In a third aspect, the present application provides a method for preparing the bird's nest peptide, comprising the following steps:

[0048] S1, soaking of bird's nest: dry bird's nest is soaked in pure water at 0-4℃ for 2h, the solid-liquid ratio is 1:10, the soaking ratio is controlled at 4-5 times, and then it is dispersed, placed in a filter bag, and placed in a centrifugal dryer for 2min to obtain soaked bird's nest.

[0049] S2, stewing of bird's nest: the soaked bird's nest in S1 is placed in a glass jar, mixed with pure water at a mass ratio of 1:15-20, and then sealed and stewed in a high-temperature sterilization pot, the stewing temperature is 121-123℃ each time, the stewing time is 12-15min, the second stewing is carried out after the first stewing is cooled to room temperature, and the fresh stewed bird's nest is obtained after the second stewing is completed and cooled to room temperature.

[0050] S3, in-vitro digestion: the fresh stewed bird's nest in S2 is subjected to in-vitro digestion experiment to simulate the digestion process of the fresh stewed bird's nest in the gastrointestinal tract, and the digested bird's nest is obtained.

[0051] The specific steps are as follows: the fresh stewed bird's nest after shaking is sheared for 30s using a high-speed shearing machine, the pH of the sheared bird's nest is adjusted to 2.0 using 0.1M HCl, 4% of the total mass of the fresh stewed bird's nest is added with pepsin, and the mixture is incubated for 4h under the condition of 37℃ water bath to simulate gastric digestion; then the pH of the above solution is adjusted to 5.33 using 0.9M NaHCO3, the pH is adjusted to 7.5 using 1M NaOH, 4% of the total mass of the fresh stewed bird's nest is added with trypsin, and the mixture is incubated for 4h under the condition of 37℃ water bath to simulate intestinal digestion, and the digestion is terminated by placing the mixture in boiling water for 10min, and the digestion solution is obtained after cooling to room temperature; the digestion solution is centrifuged under the condition of 4℃, 8000xg, 20min, and the supernatant is obtained after centrifugation, and the supernatant is stored in a refrigerator at -20℃.

[0052] S4, Protein sequencing: centrifuge the digested bird's nest described in step S3 to obtain supernatant, and send the supernatant to Beijing Baitai Piker Biotechnology Co., Ltd. for protein sequencing after treatment. After online database comparison, 5 peptide chains with high matching degree and response value greater than 10 are screened out, which are PAAVPGI, HACASVD, PAAMEGP, TVPAAVP and VEAGAGT. 9

[0053] S5, Function screening: use solid-phase synthesis technology (see below for details) to synthesize the polypeptide of step S4, add the polypeptide into the culture medium after being configured into a solution, culture the nerve cells, observe the proliferation of the nerve cells, and select the polypeptide fragment with the optimal activity, and the amino acid sequence is PAAVPGI.

[0054] S6, Animal experiment: mix the target polypeptide PAAVPGI of step S5 with fresh stewed bird's nest at a ratio of fresh stewed bird's nest: bird's nest peptide = 100:0.2-1, cultivate zebrafish larvae, detect the acetylcholinesterase activity and dopamine content in zebrafish larvae, and observe the behavior of zebrafish larvae, and evaluate the nervous system development of zebrafish larvae.

[0055] Example 1

[0056] Step one, preparation of fresh stewed bird's nest

[0057] Take 10g of dry bird's nest treated by picking hair, soak in 200g of pure water for 2h, the soaking temperature is 0-4℃; after soaking, put the soaked bird's nest into a filter bag, put the filter bag into a centrifugal dryer and dry for 2min to remove excess water; put the dried bird's nest into a glass jar, add 200g of water, cover, and cook for the first time, the cooking condition is 121℃, 14min; after the first cooking, cool to room temperature in a ventilated place, cook for the second time, the cooking condition is 123℃, 12min, after the second cooking, cool to room temperature in a ventilated place, shake evenly to obtain fresh stewed bird's nest.

[0058] Step two, in vitro simulation digestion of fresh stewed bird's nest

[0059] ​The fresh stewed bird's nest after shaking is sheared for 30s using a high-speed shearing machine, and then the pH of the sheared bird's nest is adjusted to 2.0 using 0.1M HCl, 4% of the total mass of the fresh stewed bird's nest is added with pepsin, and the mixture is incubated for 4h under the condition of 37℃ water bath to simulate gastric digestion; then the pH of the above solution is adjusted to 5.33 using 0.9M NaHCO3, and then the pH is adjusted to 7.5 using 1M NaOH, 4% of the total mass of the fresh stewed bird's nest is added with trypsin, and the mixture is incubated for 4h under the condition of 37℃ water bath to simulate intestinal digestion, and then the mixture is placed in boiling water for 10min to terminate the digestion, and then the mixture is cooled to room temperature to obtain a digestion solution; the digestion solution is centrifuged under the condition of 4℃, 8000xg, 20min, and then the supernatant is taken and stored in a refrigerator at -20℃.

[0060] Step three, polypeptide sequencing in the supernatant after digestion and peptide synthesis

[0061] The mixture after digestion is centrifuged to obtain a supernatant. The amino acid sequence of the peptide chain in the supernatant is sequenced and identified using an ultra-high performance liquid chromatography-ionization tandem mass spectrometer (UPLC-ESI-MS / MS). The specific chromatographic conditions are as follows:

[0062] Pre-column: 150μm i.d.×50mm, packing: Reprosil-Pur120C18-AQ 3μm, analysis column: 150μm i.d.×170mm, packing: Reprosil-Pur120C18-AQ 1.9μm

[0063] Mobile phase A: 0.1% FA;

[0064] Mobile phase B: 0.1% FA, 80% ACN;

[0065] Flow rate: 600nL / min;

[0066] Analysis time of each component: 66min;

[0067] The specific chromatographic conditions are as follows:

[0068] Time (min) B phase 0 4％ 2 8％ 45 28％ 55 40％ 56 95％ 66 95％

[0069] Mass spectrometry conditions:

[0070] Primary mass spectrometry parameters:

[0071] Resolution: 70,000

[0072] AGCtarget: 3e6

[0073] MaximumIT: 100ms

[0074] Scanrange: 100 to 1500 m / z

[0075] MS parameters:

[0076] Resolution: 17,500

[0077] AGCtarget: 1e5

[0078] MaximumIT: 50 ms

[0079] TopN: 20

[0080] NCE / steppedNCE: 28

[0081] Then, through the online system database comparison, 5 peptide segments with high matching degree and relative intensity greater than 10 9 are screened out, and the specific sequence is shown in Table 1.

[0082] Table 1: Sequences of screened peptide segments

[0083] Peptide segment number Peptide segment sequence Edible bird's nest peptide A PAAVPGI (the most effective peptide segment) Edible bird's nest peptide B HACASVD Edible bird's nest peptide C PAAMEGP Edible bird's nest peptide D TVPAAVP Edible bird's nest peptide E VEAGAGT

[0084] Solid phase synthesis process is adopted, and Yannest peptide A is synthesized in the direction from C terminal to N terminal, and the specific method is as follows:

[0085] 4.0 g of Fmoc-Wang Resin with a substitution degree of 0.4 mmol / g is weighed and added to a solid phase reaction column, 20 mL of DMF is added and washed twice, the solvent is removed, 60 mL of DMF is added and swelled for 30 min. Washed twice with DMF, add piperidine DMF mixed solution (volume ratio 1:3) and stir for 20 min, monitor the reaction completion by ninhydrin coloration method. Washed with DMF and DCM for 5 times respectively, 2.17 g (6.40 mmol) of Fmoc-Ile-OH and 1.04 g (7.60 mmol) of HOBt are dissolved in DMF, 1.20 mL (7.6 mmol) of DIC is added under ice bath condition, avoid light and stir for 8 min, then add the above solid phase reaction column which has removed the solvent, add 0.08 g (0.64 mmol) of DMAP, stir under nitrogen protection for 3 h, monitor the reaction completion by ninhydrin coloration method. Remove the solvent, wash with DMF for 5 times to obtain Fmoc-Ile-Wang Resin. According to the above coupling method, the Fmoc protected amino acids of Gly, Pro, Val, Ala, Ala, Pro are added and coupled in turn to extend the peptide chain. After the last coupling reaction is completed, the resin is washed with DCM, DMF and MeOH for 4 times respectively.

[0086] The obtained peptide was purified by high performance liquid chromatography to obtain Edible Bird's Nest Peptide A with a purity of 95%. Edible Bird's Nest Peptide B, C, D and E were obtained in turn by using a similar method. Figure 1 A total ion chromatogram of a sample is shown, Figures 2-6 Mass spectra of Edible Bird's Nest Peptide A, Edible Bird's Nest Peptide B, Edible Bird's Nest Peptide C, Edible Bird's Nest Peptide D and Edible Bird's Nest Peptide E are shown respectively.

[0087] Experiment 1: Screening of the function of peptide fragments in promoting nerve cell proliferation

[0088] To verify the biological activity of the peptide fragments in Table 1, the following cell experiments were performed:

[0089] Well-grown 3-4 generation SH-SY5Y cells were adjusted to a cell suspension concentration, and were dispensed into 96-well plates, 100 μL per well, 3 x 10 3 The well-grown 3-4 generation SH-SY5Y cells were adjusted to a cell suspension concentration, and were dispensed into 96-well plates, 100 μL per well, 3 x 10

[0090] After the treatment of the experimental groups 1-5, the model group and the control group, the cells were cultured for another 24 h, and the cell culture plates were taken out. 10 μL of CCK-8 solution was added to each well, and the cells were cultured for another 4 h. The absorbance value at 450 nm was detected by an enzyme label instrument. The cell proliferation rate was calculated according to the following formula.

[0091] Cell proliferation rate (%) = [OD (experimental group) - OD (blank group)] / [OD (control group) - OD (blank group)] * 100%

[0092] Table 2: Treatment methods of each experimental group

[0093]

[0094] SH-SY5Y cells, a subclone of the human bone marrow-derived neuroblastoma cell line SK-N-SH, are widely used for screening and toxicity testing of neuroactive substances due to their high similarity in morphology, physiology and biochemical functions to human cells. SH-SY5Y cells can serve as an effective testing platform to help researchers better evaluate the effects of various active substances on nerve cells. Figure 7 A comparison chart of SH-SY5Y cell proliferation rates is shown as follows, Figure 7As shown, the SH-SY5Y cell proliferation rate of the bird's nest peptide A provided by the present application is 110.53%, which indicates that the bird's nest peptide A has the effect of promoting the proliferation of SH-SY5Y cells, which indicates that the bird's nest peptide A provided by the present application has the effect of promoting the proliferation of nerve cells to a certain extent. Although the bird's nest peptides B and E have a certain effect of promoting the proliferation of SH-SY5Y cells, they are obviously weaker than the bird's nest peptide A, and the bird's nest peptides C and D cannot promote the proliferation of SH-SY5Y cells, which may be related to the amino acid sequence and spatial structure of the peptide segment itself.

[0095] Experiment two: Effect of the composition on the neural development of zebrafish

[0096] Based on the above findings, the promoting effect of the bird's nest peptide A and the fresh stewed bird's nest composition on neural development was further evaluated. The experimental grouping design is shown in Table 3.

[0097] Table 3 Composition of the bird's nest peptide composition in different treatment groups

[0098]

[0099]

[0100] Zebrafish breeding environment: room temperature: 28℃±1; light: 12h light / 12h dark cycle; fish water pH value: about 7.0; fish water salinity: 0.25-0.75‰; fish water conductivity: 400-450μS / cm.

[0101] Healthy zebrafish larvae of uniform size 5 days after fertilization were selected, and then the larvae were placed in a 6-well cell culture plate containing 0.5mg / L bird's nest peptide composition culture solution, and the control group was PBS solution, and cultured for 24h. After the culture was completed, the culture solution was aspirated, and washed with PBS solution for 3 times to wash away the culture solution.

[0102] Acetylcholinesterase (AchE) activity detection:

[0103] 20 zebrafish larvae were added to each tube, 300μl pre-cooled buffer lysis solution (0.1M PBS, 1% Triton X-100) was added, and ultrasonic crushing was performed on ice for 3 times, 10s each time, and 30s interval each time. After crushing, centrifugation was performed at 12000rpm for 15min at 4℃, and the supernatant was detected.

[0104] In the 96-well plate, 50μl supernatant, 50μl color developing agent (DTNB) and 50μl substrate working solution (ATCHI) were added to each well of the experimental group and the control group, 50μl supernatant, 50μl color developing agent (DTNB) and 50μl PBS solution were added to the blank group, and the reaction was carried out at 37℃ for 25min in the dark. After the reaction was completed, 50μl 1% SDS solution was added to terminate the reaction, and the absorbance (OD) was detected by the enzyme marker at 412nm.

[0105] Enzymatic activity (nmol / min / mg protein) = (OD of experimental group - OD of blank group) / (13600 M -1 cm -1 x 0.6 cm x 25 min x 6)

[0106] Dopamine (DA) content detection:

[0107] Add 20 zebrafish larvae to each tube, add 200 μΐ of 0.1 M HC104, and freeze-thaw for 3 times, each time for 10 s, with 30 s interval, 4 °C, 12,000 rpm, 15 min, and take the supernatant.

[0108] Detect the DA content according to the steps of the ELISA kit.

[0109] Acetylcholinesterase is the degradation enzyme of acetylcholine, and its activity reflects the maturity and functional state of cholinergic neurons. In zebrafish, AChE activity is closely related to the development of motor neurons, neuromuscular junctions and cholinergic pathways in the brain. The AChE activity of zebrafish larvae increases significantly at 5-7 days, and the AChE activity of zebrafish larvae at this time is generally in the range of 80-120 nmol / min / mg protein. DA is a key neurotransmitter for regulating movement, reward behavior and cognition, and its content reflects the development state of dopaminergic neurons (such as the hindbrain and hypothalamus). At 5 days, DA neurons have been initially formed but are still in the process of maturation, and the DA content of zebrafish larvae at this time is generally 60-250 pg. The combination of these two indicators can better evaluate the development of the nervous system of zebrafish larvae.

[0110] Figure 8 AChE activity detection of zebrafish is shown in FIG. 1, Figure 9 DA content detection of zebrafish is shown in FIG. 2, as Figure 8 and Figure 9 As shown in FIGS. 1-2, the AChE activity and DA content of zebrafish larvae can be significantly enhanced by the bird's nest peptide composition provided by the present application, and the values are still within the normal range of zebrafish larvae at this stage, indicating that the present application can effectively promote the development of the nervous system of zebrafish larvae without damaging the nervous system of zebrafish larvae.

[0111] Experiment three: behavioral analysis

[0112] To verify the promotion of nervous system function, behavioral observation was performed on zebrafish (grouping as shown in Table 3).

[0113] Zebrafish breeding environment: room temperature: 28℃±1; light: 12h light / 12h dark cycle; fish water pH: about 7.0; fish water salinity: 0.25-0.75‰; fish water conductivity: 400-450μS / cm.

[0114] According to the experimental needs, prepare the fish in advance, and after the fish is successfully prepared, observe the 4hpf fertilized and hatched larvae under a body microscope, pick healthy and fertilized larvae, clean the larvae twice with HoltBuffer larval culture solution, and then place the larvae in a 6-well cell culture plate containing 0.5mg / L bird's nest peptide composition culture solution, and the control group is HoltBuffer. Put 20 larvae in each well and add 5mL liquid, and each 12h light / 12h dark cycle, and update about 80% of the culture solution every 24h, and the whole culture time lasts for 6 days.

[0115] After the 6-day culture of the larvae is completed, the room temperature is maintained at 28℃±1, and the time is selected between 12:30-15:30, which meets the movement platform period of the larvae and the suitable temperature. Randomly select 12 larvae from each group, place the larvae in a 48-well cell culture plate, put one larva in each well, and add 1mL of HoltBuffer larval culture solution to each well, and then place the 48-well cell culture plate under the zebrafish movement behavior instrument to record the movement behavior. After 5 minutes of adaptation, set the movement cycle as 20min dark→1min light→5min dark→20min light, and record the data by Ethovision software and export and analyze the data after recording, and analyze and record the movement data for 46min.

[0116] Figure 10 The average speed of zebrafish is shown in the comparison diagram, Figure 11 The moving distance of zebrafish is shown in the comparison diagram, Figures 10-11 The results show that the experimental groups 1-5 added with the bird's nest peptide composition all accelerate the average moving speed and moving distance of the zebrafish to different extents, which shows that the bird's nest peptide composition provided by the application can enhance the movement ability and activity level of the zebrafish larvae, while the addition of bird's nest peptide A or fresh stewed bird's nest alone cannot significantly enhance the movement ability and activity level of the zebrafish larvae, which may be due to the synergistic effect of the two.

[0117] In the zebrafish experiment, the average speed in the dark environment is usually faster than that in the light environment. Zebrafish show a higher activity level in the dark environment, which may be because the dark environment makes them feel uneasy or need to actively explore the environment to find a safe area. In the light environment, the activity of zebrafish is relatively reduced, and the speed is slower, because the light makes them feel safer and the activity tends to be stable.

[0118] The behavior analysis of animals is relatively simple, and the nervous system function development can be conveniently found, so the behavior analysis is a sensitive index of the nervous system function development of the juvenile fish. Overall, compared with the control group, the average speed and the moving distance of the juvenile fish in each treatment group have a gradually increasing trend, whether in the dark period or in the light period, with the increase of the proportion of the bird's nest peptide in the composition. This shows that the bird's nest peptide composition can promote the nervous system function development of the zebrafish to a certain extent, which may be caused by the promotion of the bird's nest peptide composition to the development of the nervous system of the zebrafish.

[0119] In conclusion, the bird's nest peptide and the bird's nest peptide composition provided by the application have the effects of promoting the proliferation of nerve cells, promoting the development of the nervous system and improving the nervous system function development to a certain extent, which provides a certain basis for developing various products suitable for pregnant women or early infants.

[0120] The above description is only the preferred embodiment of the present application and the explanation of the applied technical principles. It should be understood by those skilled in the art that the scope of the application involved in the present application is not limited to the technical solutions formed by the specific combination of the above technical features, and also covers other technical solutions formed by the combination of the above technical features or equivalent features without departing from the above inventive concept. For example, the technical solutions formed by the mutual replacement of the above features and the technical features disclosed in the present application (but not limited to) having similar functions.

Claims

1. A bird's nest peptide, characterized in that, The amino acid sequence of the bird's nest peptide is PAAVPGI.

2. A bird's nest peptide composition for pregnant women, characterized in that, The composition comprises the bird's nest peptide of claim 1 and freshly stewed bird's nest, wherein the mass ratio of the freshly stewed bird's nest to the bird's nest peptide is 100:0.2~1.

3. A method for preparing the bird's nest peptide of claim 1, characterized in that, Includes the following steps: S1. Soak the dried bird's nest in pure water at 0~4℃ at a material-to-liquid ratio of 1:10 for 2 hours, then break it up and shake it dry to obtain the soaked bird's nest. S2. Mix the soaked bird's nest with pure water at a mass ratio of 1:15~20, seal it, and stew it at 121~123℃ for 12~15 minutes. After cooling to room temperature, repeat the stewing process once to obtain fresh stewed bird's nest. S3. Conduct an in vitro digestion experiment on the freshly stewed bird's nest to simulate the digestion process of freshly stewed bird's nest in the gastrointestinal tract and obtain digested bird's nest. This includes the following sub-steps: S31. After cutting the fresh stewed bird's nest, adjust the pH to 2.0, add pepsin at 4% of the total mass of the fresh stewed bird's nest to simulate gastric digestion and incubate with shaking in a 37°C water bath for 4 hours to obtain gastric digestion products. S32. First, adjust the pH of the gastric digestion products to 5.33, then adjust the pH to 7.5, add pancreatic enzyme at 4% of the total mass of the fresh stewed bird's nest, and incubate with shaking in a 37°C water bath for 4 hours to simulate intestinal digestion and obtain intestinal digestion products. S33. After incubation, the intestinal digestion product is placed in boiling water for 10 minutes to stop digestion, and then cooled to room temperature to obtain the digested bird's nest. S4. Centrifuge the digested bird's nest to obtain the supernatant. Process the supernatant and perform protein sequencing. Compare the protein with an online database to screen for high-matching proteins with a response value greater than 10. 9 The peptide segments were used to obtain bird's nest peptides.

4. The method according to claim 3, characterized in that, In step S31, the freshly stewed bird's nest is cut using a high-speed shearing machine for 30 seconds, and then the pH of the cut bird's nest is adjusted to 2.0 using 0.1M HCl. In step S32, the pH of the gastric digestion products is adjusted to 5.33 using 0.9M NaHCO3, and then the pH is adjusted to 7.5 using 1M NaOH.

5. The method according to claim 3, characterized in that, In step S4, the centrifugation operation is carried out at a temperature of 4°C and a centrifugal force of 8000xg for 20 minutes. After centrifugation, the supernatant is collected and stored at -20°C.

6. The method according to claim 3, characterized in that, In step S4, the supernatant is sequenced using liquid chromatography-ionization tandem mass spectrometry. After comparison with an online database, sequences with high matching scores and response values ​​greater than 10 are selected. 9 The peptide set, wherein the peptide set comprises a peptide with the amino acid sequence PAAVPGI.

7. The method according to claim 6, characterized in that, Step S4 further includes: synthesizing the peptide using solid-phase synthesis technology, preparing the peptide into a solution and adding it to a culture medium, culturing nerve cells, observing the proliferation of nerve cells, and selecting the peptide with the best activity, wherein the amino acid sequence of the peptide with the best activity is PAAVPGI.

8. The method according to claim 3, characterized in that, In step S1, the soaking ratio is 4 to 5 times, and the spin-drying time is 2 minutes.

9. The method according to claim 3, characterized in that, In step S2, the temperature for both stewing processes is 121~123℃, and the time is 12~15min.

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