Cubilose peptide and key preparation method of cubilose peptide cubilose for pregnant women

Abstract

The invention provides cubilose peptide and a key preparation method of cubilose peptide maternity cubilose, and the preparation method of the cubilose peptide comprises the following steps: soaking dry cubilose in pure water at 0-4 DEG C according to a material-liquid ratio of 1: 10 for 2 hours, scattering, and spin-drying to obtain soaked cubilose; mixing the soaked cubilose with pure water according to a mass ratio of 1: (15-20), sealing, stewing at 121-123 DEG C for 12-15 minutes, cooling to room temperature, and repeatedly stewing once to obtain fresh stewed cubilose; carrying out an in-vitro digestion experiment on the freshly stewed cubilose, and simulating the digestion process of the freshly stewed cubilose in gastrointestinal tracts to obtain digested cubilose; and centrifuging the digested cubilose to obtain a supernatant, treating the supernatant, carrying out protein sequencing, and carrying out on-line database comparison to screen out peptide fragments with high matching degree and response value greater than 109, thereby obtaining the cubilose peptide. The cubilose peptide can significantly improve the proliferation rate of nerve cells, and provides a cell basis for the development of a nervous system.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and in particular relates to a bird's nest peptide and a key preparation method of bird's nest peptide for pregnant women. Background Art

[0002] Bird's nests are made by the swiftlet (Apodidae) and various other swallow species using a mixture of saliva and down feathers. They are primarily produced in Southeast Asian countries and the islands of the South China Sea. Edible bird's nests contain a variety of nutrients, including protein, carbohydrates, lipids, vitamins, amino acids, and several important inorganic elements. Protein accounts for approximately 50%, and its main functional components are sialic acid and epidermal growth factor, which have antioxidant, anti-aging, and immune-boosting properties. Therefore, bird's nests have long been used as a health food for nourishment, beauty, and well-being.

[0003] Bird's nests are known for their multiple benefits for pregnant women, including nutritional supplements, immune system enhancement, fetal growth and development, and beauty enhancement. Stimulating nervous system function is a key benefit of particular interest. However, bird's nests are relatively expensive and typically require long-term consumption to achieve the desired effects. Therefore, the promotion of bird's nest products has long been limited.

[0004] The development of the nervous system requires the participation of multiple proteins in building the structure and function of nerve cells. Bird's nest protein provides the necessary material support for the normal development of the fetal nervous system. However, the mechanism by which bird's nest protein exerts its effects after ingestion by the human body is still unclear. There is little research on the active peptide components in bird's nest digestion products, and even less research on the role of bird's nest peptides in promoting nerve cell proliferation and nervous system development.

[0005] In view of this, the present invention proposes a bird's nest peptide and a key preparation method for bird's nest peptide for pregnant women, which can significantly increase the proliferation rate of nerve cells and provide a cellular basis for the development of the nervous system. Summary of the Invention

[0006] In view of the problems in the above-mentioned prior art that the mechanism of the efficacy 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 better effects, the present invention provides a bird's nest peptide and a key preparation method of bird's nest peptide for pregnant women to solve the above-mentioned technical defects.

[0007] The technical solution adopted by the present invention to solve the technical problem is:

[0008] In a first aspect, the present invention provides a bird's nest peptide having an amino acid sequence of PAAVPGI.

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

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

[0011] S1. Soak the dried bird's nest in pure water at 0-4°C at a material-to-liquid ratio of 1:10 for 2 h, break it up and spin dry to obtain the soaked bird's nest;

[0012] S2. Mix the soaked bird's nest with pure water in a mass ratio of 1:15-20, seal the mixture, and stew at 121-123° C. for 12-15 minutes. After cooling to room temperature, repeat the stewing once to obtain fresh stewed bird's nest;

[0013] S3, performing an 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. The digested bird's nest was centrifuged to obtain the supernatant, which was processed and then subjected to protein sequencing. After comparison with the online database, proteins with high matching degree and response value greater than 10 were selected. 9 peptide fragments to obtain bird's nest peptides.

[0015] Preferably, in step S3, the fresh stewed bird's nest is subjected to an in vitro digestion experiment to simulate the digestion process of the fresh stewed bird's nest in the gastrointestinal tract to obtain the digested bird's nest, which specifically includes 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 to simulate gastric digestion and incubating in a 37°C water bath for 4 hours under shaking conditions to obtain gastric digestion products;

[0017] S32, first adjusting the pH of the gastric digestion product to 5.33, then adjusting the pH to 7.5, adding pancreatic enzyme at 4% of the total mass of the fresh stewed bird's nest, and incubating in a 37°C water bath with shaking for 4 hours to simulate intestinal digestion to obtain intestinal digestion products;

[0018] S33. After the incubation is completed, the intestinal digestion products are placed in boiling water for 10 minutes to terminate the digestion, and then cooled to room temperature to obtain the digested bird's nest.

[0019] Preferably, in step S31, the fresh stewed bird's nest is sheared for 30 seconds 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 product 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 operation is performed at 4°C and 8000xg centrifugal force for 20 min, and the supernatant is taken after centrifugation and stored at -20°C.

[0021] Preferably, in step S4, the supernatant is sequenced using a liquid chromatography ionization tandem mass spectrometer, and the samples with high matching degree and response value greater than 10 are screened out after comparison with the online database. 9 A peptide group, wherein the peptide group includes a peptide with an amino acid sequence of PAAVPGI.

[0022] Further preferably, in step S4, it also includes: synthesizing peptides using solid-phase synthesis technology, configuring the peptides into a solution and adding them into the 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.

[0023] Preferably, in step S1, the expansion ratio is 4 to 5 times, and the drying time is 2 minutes.

[0024] Preferably, in step S2, the stewing temperature for both times is 121-123° C., and the stewing time is 12-15 minutes.

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

[0026] (1) Promotes nerve cell proliferation: Through in vitro simulated digestion experiments, the present invention screened a specific peptide segment (amino acid sequence: PAAVPGI) from bird's nest that promotes nerve cell proliferation. In the experiment, the bird's nest peptide was able to significantly increase the proliferation rate of nerve cells, providing a cellular basis for the development of the nervous system.

[0027] (2) Promotes nervous system development: The bird's nest peptide composition for pregnant women provided by the present invention can effectively promote the development of the nervous system and enhance the function of the nervous system. This is of great significance for supplementing nutrition for pregnant women and promoting the development of the fetal nervous system.

[0028] (3) Improve the utilization rate of bird's nest: Traditional bird's nest consumption requires long-term consumption and has limited utilization rate. The present invention uses a specific preparation method to convert the active ingredients in bird's nest into small molecule peptides, which are easier for the human body to absorb and utilize, thereby improving the nutritional value and utilization efficiency of bird's nest.

[0029] (4) Safe and reliable: The bird's nest peptide and its bird's nest composition for pregnant women of the present invention are derived from natural bird's nests. The preparation process does not involve harmful chemicals, which ensures the safety and reliability of the product and is suitable for long-term consumption by special groups such as pregnant women.

[0030] (5) Innovative combination: The present invention not only provides a bird's nest peptide with specific physiological functions, but also combines it with fresh stewed bird's nest to exert a synergistic effect, providing new ideas and options for the development of new health products or functional foods for pregnant women. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Other features, objects and advantages of the present application will become more apparent upon reading the detailed description of non-limiting embodiments made with reference to the following drawings:

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

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

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

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

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

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

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

[0039] Figure 8 Zebrafish acetylcholinesterase (AChE) activity assay diagram;

[0040] Figure 9 This is a graph showing the detection of dopamine (DA) content in zebrafish;

[0041] Figure 10 This is a schematic diagram comparing the average speed of zebrafish;

[0042] Figure 11 This is a schematic diagram comparing the distances zebrafish move. DETAILED DESCRIPTION

[0043] The present application will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the relevant invention and are not intended to limit the invention. It should also be noted that, for ease of description, only portions relevant to the relevant invention are shown in the accompanying drawings.

[0044] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0045] In a first aspect, the present invention provides a bird's nest peptide having an amino acid sequence of PAAVPGI.

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

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

[0048] S1. Soaking the bird's nest: Soak the dry bird's nest in pure water at 0-4℃ for 2 hours, with a material-liquid ratio of 1:10 and a soaking rate of 4-5 times. Then break it up and put it in a filter bag. Put the filter bag in a spin dryer and spin dry for 2 minutes to obtain the soaked bird's nest.

[0049] S2. Stewing bird's nest: Place the soaked bird's nest described in S1 in a canned glass bottle, mix it with pure water at a mass ratio of 1:15-20, seal the bottle, and stew it in a high-temperature sterilizer. The stewing temperature for each time is 121-123°C, and the stewing time is 12-15 minutes. After the first stewing, it needs to be cooled to room temperature before the second stewing. After the second stewing is completed, place it in a ventilated place to cool to room temperature, shake it well to obtain fresh stewed bird's nest.

[0050] S3. In vitro digestion: The fresh stewed bird's nest described in S2 is subjected to an 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.

[0051] The specific steps are as follows: the shaken fresh stewed bird's nest is sheared with a high-speed shearing machine for 30 seconds, and then the pH of the sheared bird's nest is adjusted to 2.0 with a concentration of 0.1M HCl, and pepsin is added at 4% of the total mass of the fresh stewed bird's nest, and the mixture is shaken and incubated in a 37°C water bath for 4 hours to simulate gastric digestion and enzymatic hydrolysis; then the pH of the above solution is adjusted to 5.33 with a concentration of 0.9M NaHCO3, and then the pH is adjusted to 7.5 with a concentration of 1M NaOH, and pancreatic enzyme is added at 4% of the total mass of the fresh stewed bird's nest, and the mixture is shaken and incubated in a 37°C water bath for 4 hours to simulate intestinal digestion. After the incubation, it is placed in boiling water for 10 minutes to terminate digestion, and cooled to room temperature to obtain a digestive fluid; the above digestive fluid is centrifuged at 4°C, 8000xg, and 20 minutes. The supernatant is taken after centrifugation and stored in a -20°C refrigerator.

[0052] S4, protein sequencing: The digested bird's nest described in step S3 was centrifuged to obtain the supernatant, which was processed and sent to Beijing Biotech Biotechnology Co., Ltd. for protein sequencing. After comparison with the online database, proteins with high matching degree and response value greater than 10 were selected. 9 A total of 5 peptide chains were screened out, namely PAAVPGI, HACASVD, PAAMEGP, TVPAAVP, and VEAGAGT.

[0053] S5. Functional screening: The polypeptide from step S4 was synthesized using solid-phase synthesis technology (see below for details). The polypeptide was prepared into a solution and added to the culture medium. Neuronal cells were cultured and their proliferation was observed. The polypeptide fragment with the best activity was selected, with the amino acid sequence of PAAVPGI.

[0054] S6. Animal experiment: The target polypeptide PAAVPGI of step S5 was mixed with fresh stewed bird's nest at a ratio of fresh stewed bird's nest: bird's nest peptide = 100:0.2-1, and zebrafish fry were cultured. The acetylcholinesterase activity and dopamine content in the zebrafish fry were detected. At the same time, the behavioral state of the zebrafish fry was observed to evaluate the development of the zebrafish fry's nervous system.

[0055] Example 1

[0056] Step 1: Preparation of fresh stewed bird's nest

[0057] Take 10g of dry bird's nest after hair removal, place it in 200g of pure water and soak it for 2h, and the soaking temperature is 0-4°C; after soaking, place the soaked bird's nest in a filter bag, put the filter bag into a spin dryer and spin dry for 2min to remove excess water; place the dried bird's nest in a canned glass bottle, add 200g of water, seal the bottle, and stew it for the first time, and the stewing conditions are 121°C and 14min; after the first stewing is completed, place it in a ventilated place to cool to room temperature, and stew it for the second time, and the stewing conditions are 123°C and 12min. After the second stewing is completed, place it in a ventilated place to cool to room temperature, and shake it well to obtain the freshly stewed bird's nest.

[0058] Step 2: Simulate in vitro digestion of fresh stewed bird’s nest

[0059] The shaken fresh stewed bird's nest was sheared with a high-speed shearing machine for 30 seconds, and then the pH of the sheared bird's nest was adjusted to 2.0 with a concentration of 0.1M HCl, and pepsin was added at 4% of the total mass of the fresh stewed bird's nest. The mixture was shaken and incubated in a water bath at 37°C for 4 hours to simulate gastric digestion and enzymatic hydrolysis; then the pH of the above solution was adjusted to 5.33 with a concentration of 0.9M NaHCO3, and then the pH was adjusted to 7.5 with a concentration of 1M NaOH, and pancreatic enzyme was added at 4% of the total mass of the fresh stewed bird's nest. The mixture was shaken and incubated in a water bath at 37°C for 4 hours to simulate intestinal digestion. After the incubation, it was placed in boiling water for 10 minutes to terminate the digestion, and cooled to room temperature to obtain a digestive fluid; the above digestive fluid was centrifuged at 4°C, 8000xg, and 20 minutes. The supernatant was taken after centrifugation and stored in a refrigerator at -20°C.

[0060] Step 3: Peptide sequencing and peptide synthesis in the digestion supernatant

[0061] The digested bird's nest mixture was centrifuged to obtain the supernatant. The peptide chain amino acid sequence in the supernatant was sequenced and identified using ultra-high performance liquid chromatography ionization tandem mass spectrometry (UPLC-ESI-MS / MS). Specific chromatographic conditions:

[0062] Pre-column: 150μm i.d. × 50mm, packing: Reprosil-Pur120C18-AQ 3μm, analytical 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: 600 nL / min;

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

[0067] The specific chromatographic conditions are:

[0068] Time (min) Phase B 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] Scan range: 100 to 1500 m / z

[0075] Secondary mass spectrometry parameters:

[0076] Resolution: 17,500

[0077] AGCtarget: 1e5

[0078] MaximumIT: 50ms

[0079] TopN:20

[0080] NCE / steppedNCE: 28

[0081] Then, the database is compared through the online system to screen out the products with high matching degree and relative intensity greater than 10 9 The specific sequences of the five peptides are shown in Table 1.

[0082] Table 1 Screened peptide sequences

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

[0084] The bird's nest peptide A was synthesized from the C-terminus to the N-terminus using a solid-phase synthesis process. The specific method is as follows:

[0085] 4.0 g of Fmoc-Wang Resin (with a substitution degree of 0.4 mmol / g) was weighed and added to a solid-phase reaction column. The column was washed twice with 20 mL of DMF, the solvent removed, and 60 mL of DMF was added to swell for 30 minutes. The column was washed twice with DMF, and a mixture of piperidine and DMF (1:3 by volume) was added and stirred for 20 minutes. Completion of the reaction was monitored using the ninhydrin colorimeter. The column was washed five times with DMF and five times with DCM. 2.17 g (6.40 mmol) of Fmoc-Ile-OH and 1.04 g (7.60 mmol) of HOBt were dissolved in DMF. 1.20 mL (7.6 mmol) of DIC was added in an ice bath. The column was stirred in the dark for 8 minutes and then added to the solid-phase reaction column from which the solvent had been removed. 0.08 g (0.64 mmol) of DMAP was added, and the reaction was stirred under nitrogen for 3 hours. Completion of the reaction was monitored using the ninhydrin colorimeter. The solvent was removed and the resin was washed five times with DMF to obtain Fmoc-Ile-Wang Resin. Following the above coupling method, Fmoc-protected amino acids (Gly, Pro, Val, Ala, Ala, and Pro) were added sequentially for condensation coupling to extend the peptide chain. After the final coupling reaction, the resin was washed four times each with DCM, DMF, and MeOH.

[0086] The obtained peptide was purified by high performance liquid chromatography to obtain bird's nest peptide A with a purity of 95%. Bird's nest peptides B, C, D, and E were obtained in sequence using a similar method. Figure 1 shows the total ion current of the sample, Figure 2-Figure 6 The mass spectra of bird's nest peptide A, bird's nest peptide B, bird's nest peptide C, bird's nest peptide D and bird's nest peptide E are shown respectively.

[0087] Experiment 1: Screening of peptides for promoting neuronal proliferation

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

[0089] The SH-SY5Y cells of passage 3 to 4 that have grown well were adjusted to the concentration of cell suspension and dispensed into 96-well plates, with 100 μL per well and 3×10 3 Cells were cultured overnight in a 37°C, 5% CO2 incubator to allow attachment. The cells were then randomly divided into a control group, a model group, and experimental groups 1 to 5. A blank group, supplemented with culture medium only and no cells, was also designed, with three replicates per group. The model group and experimental groups 1 to 5 were treated with 0.8 mmol / L glutamate to create a glutamate injury model. Experimental groups 1 to 5 were treated with 400 μg / L of bird's nest peptides A, B, C, D, and E, respectively. Specific treatments for each experimental group are shown in Table 2.

[0090] After treatment, cells in experimental groups 1 to 5, the model group, and the control group were cultured for another 24 hours. The cell culture plates were removed, and 10 μL of CCK-8 solution was added to each well. The cells were cultured for another 4 hours. The absorbance at 450 nm was measured with a microplate reader, and 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 neuroblastoma cell line SK-N-SH, derived from human bone marrow, are widely used for screening and toxicity testing of neuroactive substances due to their high similarity to human cells in morphology, physiology, and biochemistry. SH-SY5Y cells serve as an effective testing platform, helping researchers better evaluate the effects of various active substances on neural cells. Figure 7 A comparison of SH-SY5Y cell proliferation rates is shown. Figure 7As shown, the SH-SY5Y cell proliferation rate of the bird's nest peptide A provided by the present invention is 110.53%, indicating that the bird's nest peptide A has the effect of promoting the proliferation of SH-SY5Y cells. This shows that the bird's nest peptide A provided by the present invention has the effect of promoting the proliferation of nerve cells to a certain extent. Although bird's nest peptides B and E have a certain effect of promoting the proliferation of SH-SY5Y cells, they are significantly weaker than bird's nest peptide A. Bird's nest peptides C and D are unable to promote the proliferation of SH-SY5Y cells. This may be related to the amino acid sequence and spatial structure of the peptide itself.

[0095] Experiment 2: Effects of the composition on zebrafish neural development

[0096] Based on the above findings, we further evaluated the neurodevelopmental effects of the combination of bird's nest peptide A and fresh stewed bird's nest. The experimental group design is shown in Table 3.

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

[0098]

[0099]

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

[0101] Healthy zebrafish larvae of uniform size, 5 days post-fertilization, were selected and placed in a 6-well cell culture plate containing a 0.5 mg / L bird's nest peptide composition. A control group was placed in PBS and cultured for 24 hours. After the culture was complete, the culture medium was aspirated and the cells were washed three times with PBS to remove the culture medium.

[0102] Acetylcholinesterase (AchE) activity detection:

[0103] Twenty zebrafish larvae were added to each tube, and 300 μl of pre-cooled lysis buffer (0.1 M PBS, 1% Triton X-100) was added. The cells were ultrasonically disrupted on ice three times, each time for 10 s, with an interval of 30 s between each time. After disruption, the cells were centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was taken for detection.

[0104] In a 96-well plate, 50 μl of supernatant, 50 μl of color developer (DTNB) and 50 μl of substrate working solution (ATCHI) were added to each well of the experimental group and the control group, and 50 μl of supernatant, 50 μl of color developer (DTNB) and 50 μl of PBS solution were added to the blank group. The reaction was incubated at 37°C in the dark for 25 min. After the reaction was completed, 50 μl of 1% SDS solution was added to terminate the reaction, and the absorbance (OD) was measured at 412 nm by a microplate reader.

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

[0106] Dopamine (DA) content detection:

[0107] Twenty zebrafish larvae were added to each tube, and 200 μl of 0.1 M HClO 4 was added. The tube was ultrasonically disrupted three times in an ice bath, each time for 10 s, with an interval of 30 s between each times, at 4°C, 12,000 rpm, for 15 min, and the supernatant was collected.

[0108] The DA content was detected according to the ELISA kit procedure.

[0109] Acetylcholinesterase (AChE) is an acetylcholine-degrading enzyme, and its activity reflects the maturity and functional status of cholinergic neurons. In zebrafish, AChE activity is closely associated with the development of motor neurons, neuromuscular junctions, and brain cholinergic pathways. AChE activity increases significantly between 5 and 7 days old, typically ranging from 80–120 nmol / min / mg protein. DA is a key neurotransmitter regulating movement, reward behavior, and cognition, and its content reflects the developmental status of dopaminergic neurons (e.g., in the hindbrain and hypothalamus). By 5 days old, DA neurons have initially formed but are still maturing. At this time, DA levels typically range from 60 to 250 pg. Combining these two indicators can effectively assess the development of the zebrafish nervous system.

[0110] Figure 8 Shown is a diagram of zebrafish acetylcholinesterase (AChE) activity detection. Figure 9 Shown is a zebrafish dopamine (DA) content detection diagram, such as Figure 8 and Figure 9 As shown, the bird's nest peptide composition provided by the present invention can significantly enhance the AChE activity and DA content of zebrafish fry, while still being within the normal range of zebrafish fry at this stage, indicating that the present invention can effectively promote the development of the nervous system of zebrafish fry without damaging the nervous system of zebrafish fry.

[0111] Experiment 3: Behavioral Analysis

[0112] To verify the improvement of nervous system function, behavioral observations were performed on zebrafish (grouping is the same as in Table 3).

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

[0114] Prepare the fish for breeding in advance according to the experimental needs. After successful breeding, observe the 4hpf fertilized and hatched larvae under a stereomicroscope. Pick healthy and successfully fertilized larvae, clean them twice with HoltBuffer larvae culture medium, and then place them in a 6-well cell culture plate containing a 0.5mg / L bird's nest peptide composition culture medium. The control group is HoltBuffer. Place 20 larvae in each well and add 5mL of liquid. Every 12h light / 12h dark cycle, about 80% of the culture medium is refreshed every 24h. The entire culture period lasts for 6 days.

[0115] After the 6-day culture period, the room temperature was maintained at 28°C ± 1°C. The time was selected between 12:30 PM and 3:30 PM, which is consistent with the larval movement plateau and the optimal temperature. Twelve larvae were randomly selected from each group and placed in a 48-well cell culture plate, with one larvae per well. 1 mL of Holt Buffer larval culture medium was added to each well. The 48-well cell culture plate was then placed in a zebrafish locomotor behavior instrument to record locomotor behavior. After a 5-minute acclimation period, a 20-minute dark cycle, 1-minute light cycle, 5-minute dark cycle, and 20-minute light cycle were established. Data were recorded using Ethovision software and exported and analyzed after recording. A total of 46 minutes of locomotor data were analyzed.

[0116] Figure 10 Shows a schematic diagram of the average speed comparison of zebrafish, Figure 11 Shows a schematic diagram of the comparison of zebrafish movement distance, Figure 10-11 The results showed that experimental groups 1 to 5 to which the bird's nest peptide composition was added all accelerated the average movement speed and movement distance of the zebrafish to varying degrees. This indicates that the bird's nest peptide composition provided by the present invention can enhance the locomotion ability and activity level of zebrafish fry, while the addition of bird's nest peptide A or freshly stewed bird's nest alone cannot significantly enhance the locomotion ability and activity level of zebrafish fry. This may be due to a certain synergistic effect between the two.

[0117] In zebrafish experiments, average speeds in the dark are typically faster than in the light. Zebrafish exhibit higher activity levels in the dark, perhaps because they feel uneasy or need to actively explore their environment to find a safe area. In the light, however, zebrafish are less active and slower because the light makes them feel safer and their activity levels stabilize.

[0118] Although animal behavioral analysis is relatively simple, it can easily reveal the development of nervous system functions. Therefore, behavioral analysis is a sensitive indicator of the development of nervous system functions in juvenile fish. Overall, whether in the dark or light period, compared with the control group, as the proportion of bird's nest peptides in the composition increases, the average speed and movement distance of the juvenile fish in each treatment group tend to gradually increase. This shows that the bird's nest peptide composition can promote the development of the nervous system function of zebrafish to a certain extent, which may be due to the bird's nest peptide composition promoting the development of the zebrafish nervous system itself.

[0119] In summary, the bird's nest peptide and bird's nest peptide composition provided by the present invention have the effects of promoting nerve cell proliferation, promoting nervous system development, and enhancing the development of nervous system functions to a certain extent, providing a certain basis for the development of a variety of products suitable for pregnant women or early infants.

[0120] The above description is merely a preferred embodiment of the present application and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to the technical solutions formed by the specific combination of the above-mentioned technical features, but also encompasses other technical solutions formed by any combination of the above-mentioned technical features or their equivalents without departing from the above-mentioned inventive concept. For example, a technical solution formed by replacing the above-mentioned features with (but not limited to) technical features with similar functions disclosed in this application.

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 pregnant woman bird's nest composition, characterized in that, The composition comprises the bird's nest peptide according to claim 1 and fresh-stewed bird's nest, wherein the mass ratio of the fresh-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 according to claim 1, characterized in that: The following steps are involved: S1. Soak the dried bird's nest in pure water at 0-4°C at a material-liquid ratio of 1:10 for 2 h, break it up and spin dry to obtain the soaked bird's nest; S2, mixing the foamed bird's nest with pure water in a mass ratio of 1:15-20, sealing and stewing at 121-123° C. for 12-15 minutes, cooling to room temperature and repeating the stewing once to obtain fresh stewed bird's nest; S3, performing an 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; S4, centrifuging the digested bird's nest to obtain a supernatant, processing the supernatant for protein sequencing, and screening out proteins with high matching degree and response value greater than 10 after comparison with an online database. 9 peptide fragments to obtain bird's nest peptides.

4. The method according to claim 3, characterized in that In step S3, the fresh stewed bird's nest is subjected to an in vitro digestion experiment to simulate the digestion process of the fresh stewed bird's nest in the gastrointestinal tract to obtain the digested bird's nest, which specifically includes the following sub-steps: S31, after shearing the fresh stewed bird's nest, adjusting the pH to 2.0, adding pepsin according to 4% of the total mass of the fresh stewed bird's nest to simulate gastric digestion and enzymatic hydrolysis, and incubating in a water bath at 37° C. for 4 h with shaking to obtain gastric digestion products; S32, first adjusting the pH of the gastric digestion product to 5.33, then adjusting the pH to 7.5, adding pancreatic enzyme according to 4% of the total mass of the fresh stewed bird's nest, and incubating in a 37° C. water bath with shaking for 4 hours to simulate intestinal digestion to obtain intestinal digestion products; S33. After the incubation is completed, the intestinal digestion product is placed in boiling water for 10 minutes to terminate the digestion, and then cooled to room temperature to obtain the digested bird's nest.

5. The method according to claim 4, characterized in that In step S31, the fresh stewed bird's nest is sheared for 30 seconds using a high-speed shearing machine, and 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 product is adjusted to 5.33 using 0.9M NaHCO3, and then the pH is adjusted to 7.5 using 1M NaOH.

6. The method according to claim 3, characterized in that In step S4, the centrifugation operation is performed at 4°C and 8000×g for 20 minutes, and the supernatant is stored at -20°C after centrifugation.

7. The method according to claim 3, characterized in that In step S4, the supernatant was sequenced using a liquid chromatography ionization tandem mass spectrometer, and the samples with high matching degree and response value greater than 10 were screened out after comparison with the online database. 9 A peptide segment group, wherein the peptide segment group comprises a peptide segment with an amino acid sequence of PAAVPGI.

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

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

10. The method according to claim 3, characterized in that In step S2, the stewing temperature is 121-123° C. for both times, and the stewing time is 12-15 minutes.

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