Andrias davidianus peptide extract compound composition and its application in sleep promoting products

Abstract

This invention discloses a compound composition of giant salamander peptide extract and its application in sleep-promoting products. The giant salamander meat is hydrolyzed with papain at 45-55°C, followed by hydrolysis with a complex protease, and the enzymes are inactivated after hydrolysis. The mixture is then filtered through a ceramic membrane and adsorbed using activated carbon to obtain the giant salamander peptide extract. The giant salamander peptide extract is compounded with casein peptide, tea theanine, daylily extract, poria cocos extract, jujube seed extract, lily powder, and γ-aminobutyric acid (GABA) to obtain a compound composition. Applying this compound composition to sleep-promoting products can enhance their sleep-aiding effects.

Description

Technical Field

[0001] This invention belongs to the field of food and pharmaceutical technology, specifically relating to a compound composition of giant salamander peptide extract and its application in sleep-promoting products. Background Technology

[0002] Giant salamander peptides are small-molecule peptides extracted from the body tissues of giant salamanders and obtained through enzymatic hydrolysis. Rich in various amino acids, they possess characteristics such as small molecule size, easy absorption, low allergenicity, and multiple biological activities. They have been developed into functional foods or pharmaceuticals for purposes such as anti-oxidation, anti-fatigue, hypoglycemia, and anti-thrombosis. For example, patent application CN116803415A discloses the application of giant salamander collagen peptides in the preparation of drugs for anti-tumor purposes, while patent applications CN120436314A and CN118436766A disclose the anti-fatigue application of giant salamander peptides. However, there is very little research in the prior art on the application of giant salamander peptides to improve sleep. Summary of the Invention

[0003] The purpose of this invention is to provide a compound composition of giant salamander peptide extract and its application in sleep-promoting products, so as to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] The compound composition of giant salamander peptide extract includes giant salamander peptide extract, casein peptide, tea theanine, daylily extract, poria cocos extract, jujube seed extract, lily powder and γ-aminobutyric acid;

[0006] The giant salamander peptide extract is obtained by the following method: giant salamander meat is hydrolyzed with papain and complex protease at 45-55°C, and the enzymes are inactivated after hydrolysis; the extract is then filtered through a ceramic membrane and adsorbed with activated carbon to obtain the giant salamander peptide extract.

[0007] As a further embodiment of the present invention, the pH is controlled at 6.5~7.5 during the enzymatic hydrolysis process.

[0008] As a further embodiment of the present invention, the amount of papain added is 0.5~0.7wt%, preferably 0.6wt%.

[0009] As a further embodiment of the present invention, the amount of the compound protease added is 0.5~0.7wt%, preferably 0.6wt%.

[0010] As a further embodiment of the present invention: the ceramic membrane is a 0.1~0.3μm inorganic ceramic membrane, preferably a 0.2μm inorganic ceramic membrane.

[0011] As a further embodiment of the present invention, the amount of activated carbon added is 0.2~0.4wt%, preferably 0.2wt%.

[0012] As a further embodiment of the present invention: the activated carbon adsorption is carried out at 45~55℃, preferably 50℃.

[0013] As a further embodiment of the present invention: the compound composition comprises, by weight, 18-24 parts of giant salamander peptide extract, 3-7 parts of casein peptide, 6-10 parts of tea theanine, 9-13 parts of daylily extract, 5-9 parts of Poria cocos extract, 5-9 parts of jujube seed extract, 1-5 parts of lily powder and 0.5-1.5 parts of γ-aminobutyric acid.

[0014] As a further embodiment of the present invention: the daylily extract is obtained by water extraction of daylily.

[0015] As a further embodiment of the present invention: the water extraction process of the daylily extract includes: adding water to the raw material at a mass ratio of 1g:(10~20)ml, performing ultrasonic extraction at 40~60℃ and 100~300W for 20~40min, filtering, and spray drying the filtrate to obtain the daylily extract.

[0016] As a further embodiment of the present invention: the Poria cocos extract is obtained by water extraction and alcohol extraction of Poria cocos.

[0017] As a further embodiment of the present invention: the water extraction process of Poria cocos includes adding water to the raw material at a material-to-liquid ratio of 1g:(10~20)ml, ultrasonically extracting for 20~40min at 40~60℃ and 100~300W, filtering, and obtaining filter residue and water extract.

[0018] As a further embodiment of the present invention: the alcohol extraction process of Poria cocos includes adding ethanol solution to the filter residue at a material-to-liquid ratio of 1g:(8~12)ml, ultrasonically extracting for 20~40min at 45~55℃ and 100~300W, filtering, and obtaining alcohol extract.

[0019] As a further embodiment of the present invention, the volume fraction of the ethanol solution is 50% to 70%.

[0020] As a further embodiment of the present invention: the jujube seed extract is obtained by defatting and enzymatically hydrolyzing jujube seeds, followed by alcohol extraction.

[0021] As a further embodiment of the present invention: the degreasing process of jujube kernels includes adding jujube kernel powder to an organic solvent at a material-to-liquid ratio of 1g:(8~12)ml, and degreasing at 60~90℃.

[0022] As a further embodiment of the present invention: the organic solvent is selected from at least one of n-hexane, petroleum ether, ethanol, and diethyl ether, preferably diethyl ether.

[0023] As a further embodiment of the present invention: the enzymatic hydrolysis process of jujube kernel uses β-glucosidase, with an addition amount of 0.8~1.2wt%, and the enzymatic hydrolysis temperature is 40~60℃.

[0024] Application of the compound composition described in any of the above-mentioned items in sleep-promoting products.

[0025] As a further embodiment of the present invention: the sleep-promoting product is prepared from the compound composition and raw material components including carboxymethyl chitosan, L-cysteine, genipin, calcium chloride and sodium carbonate.

[0026] As a further embodiment of the present invention, the weight parts of the raw material components are as follows: 70-85 parts of the compound composition, 95-105 parts of carboxymethyl chitosan, 3-5 parts of L-cysteine, 1-2 parts of genipin, 8-10 parts of calcium chloride, and 8-10 parts of sodium carbonate.

[0027] As a further embodiment of the present invention, the sleep-promoting product is prepared by the following method:

[0028] Carboxymethyl chitosan, L-cysteine, and genipin are mixed to form a cross-linking system;

[0029] The giant salamander peptide extract, casein peptide, and lily powder in the compound composition are added to the crosslinking system, and calcium chloride and sodium carbonate are added for curing. After wet milling, a dispersion is formed.

[0030] The remaining compound composition is added to the dispersion and mixed evenly to obtain the sleep-promoting product.

[0031] As a further embodiment of the present invention, the molecular weight of the carboxymethyl chitosan is 30,000 to 80,000, preferably 30,000 to 38,000.

[0032] As a further embodiment of the present invention, the degree of substitution of the carboxymethyl chitosan is 0.80~0.95, preferably 0.80~0.85.

[0033] As a further embodiment of the present invention, the sleep-promoting product is prepared by the following method:

[0034] S1. Dissolve carboxymethyl chitosan in water to make a solution, add L-cysteine, adjust the pH to 5-6 and stir; then add genipin and adjust the pH to 7.0-8.0, stir at 40-50°C to form a cross-linking system;

[0035] S2. Add giant salamander peptide, casein peptide and lily powder to the cross-linking system and stir. Then add calcium chloride and sodium carbonate and stir. After filtration, take the filter residue and wet grind it to obtain the dispersion.

[0036] S3. Add tea theanine, daylily extract, poria cocos extract, jujube seed extract and γ-aminobutyric acid to the dispersion and mix evenly to obtain a sleep-promoting product.

[0037] As a further embodiment of the present invention: the wet grinding process in step S2 includes grinding with water as the medium and 0.4~0.5mm zirconia beads at 1000~1500 rpm for 20~45 min.

[0038] The present invention has at least the following technical effects:

[0039] 1. Optimize the extraction process of giant salamander peptide extract by using papain and compound enzymes for enzymatic hydrolysis, which can efficiently open the triple helix of giant salamander collagen, while reducing the fishy and bitter taste of giant salamander peptide extract.

[0040] 2. By combining giant salamander peptide extract with daylily extract, poria cocos extract, jujube seed extract, casein peptide and tea theanine to form a composition, zebrafish experiments have shown that this composition has the effect of improving sleep, providing a new application solution for giant salamander peptide extract;

[0041] 3. Using genipin-chitosan crosslinking and calcium carbonate mineralized microparticle structure as an oral formulation carrier, the preparation process is mild, which can retain the activity of peptides and plant components, and can also improve the bioavailability of giant salamander peptides and other active ingredients, thereby enhancing their sleep-promoting effect. Attached Figure Description

[0042] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0043] Figure 1 This is a schematic diagram of the movement trajectories of zebrafish in the blank group, model group, and experimental group in this embodiment of the invention;

[0044] Figure 2 This is a comparison of the relative expression levels of the gabra1 gene in zebrafish from the blank group, model group, and experimental group in this embodiment of the invention.

[0045] Figure 3 This is a comparison of the relative expression levels of the mtnr1aa gene in zebrafish from the blank group, model group, and experimental group in this embodiment of the invention. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0047] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0048] Papain: Provided by Shanghai Yuanye Biotechnology Co., Ltd., model number S10011.

[0049] Complex protease: provided by Novozymes (China) Biotechnology Co., Ltd., model Protamex 1.6, enzyme activity 1.6 AU-N / g, by mass fraction, composed of sodium chloride (CAS no. 7647-14-5) 88%, protease (neutral, CAS no. 9080-56-2) 9%, protease (subtilisin, CAS no. 7647-14-5) 2% and water (CAS no. 7732-18-5) 1%.

[0050] β-glucosidase: Provided by Shanghai Yuanye Biotechnology Co., Ltd., model number S10047, enzyme activity is 20~40U / mg.

[0051] Example 1: Preparation of giant salamander peptide extract

[0052] Giant salamander peptide extract is obtained through the following process:

[0053] S1. Crush the giant salamander meat, add 5 times the amount of water and boil for 2 hours. Let it cool and stand. Add 0.6% (w / w) of papain by weight of the giant salamander meat in a 50℃ water bath. Enzymatically hydrolyze at 50℃ for 2 hours at pH=6.5. Keep stirring during the enzymatic hydrolysis process and adjust the pH every 30 minutes.

[0054] S2. Maintain 50℃, adjust pH=7.0, and add 0.6% (w / w) of compound protease by weight of giant salamander meat. Enzymatically hydrolyze for 2 hours. During the enzymatic hydrolysis process, keep stirring and adjust the pH every 30 minutes. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95℃ for 30 minutes.

[0055] S3. Filter the solution through a 0.2 μm inorganic ceramic membrane and collect the filtrate. Add 0.2% (w / w) of activated carbon (by weight of giant salamander meat) to the filtrate and adsorb at 50 °C for 50 min. Filter the solution through a 0.2 μm inorganic ceramic membrane again, discard the filter residue, and freeze-dry at -50 °C for 24 h to obtain giant salamander peptide extract.

[0056] Example 2: Preparation of daylily extract

[0057] Daylily powder was added to deionized water at a mass ratio of 1g:20ml and ultrasonically extracted at 45℃ and 200W for 30min. The mixture was then filtered, and the filtrate was concentrated under reduced pressure at 55℃ and spray-dried (inlet air temperature 160℃, outlet air temperature 80℃, feed rate 3 mL / min) to obtain daylily extract.

[0058] Example 3: Preparation of Poria cocos extract

[0059] Poria cocos granules were pulverized, and water was added at a material-to-liquid ratio of 1g:15ml. The mixture was ultrasonically extracted for 30 min at 45 ℃ and 200 W. The aqueous extract and residue were obtained by filtration. The residue was added to 70% ethanol solution at a material-to-liquid ratio of 1g:10ml. The mixture was ultrasonically extracted for 20 min at 50 ℃ and 200 W. The ethanol extract was obtained by filtration. The aqueous extract and ethanol extract were combined, concentrated under reduced pressure at 55 ℃, and spray-dried to obtain Poria cocos extract.

[0060] Example 4: Preparation of Jujube Seed Extract

[0061] Ziziphus jujuba seed powder was added to petroleum ether at a ratio of 1g:10ml and defatted twice at 70℃ for 2 hours each time using Soxhlet. The defatted ziziphus jujuba seed powder was added to citrate buffer at a ratio of 1g:10ml, and β-glucosidase was added at 1% (w / w). The mixture was enzymatically hydrolyzed at 50℃ and 200 rpm for 12 hours. After the enzymatic hydrolysis was completed, the enzyme was inactivated at 95℃ for 5 minutes and cooled to room temperature. The hydrolysate was directly added with 2 volumes of 70% ethanol, sonicated at 40℃ and 180 W for 30 minutes, centrifuged, and the supernatant was collected. The supernatant was concentrated under reduced pressure at 55℃ and spray-dried to obtain the ziziphus jujuba seed extract.

[0062] The citrate buffer solution is prepared by dissolving 10.5g of citric acid and 35.8g of disodium hydrogen phosphate in distilled water and bringing the volume to 500mL.

[0063] Example 5: Compound Composition

[0064] The compound composition, by weight, consists of 20 parts of giant salamander peptide extract, 5 parts of casein peptide, 8 parts of tea theanine, 11 parts of daylily extract, 7 parts of poria cocos extract, 7 parts of jujube seed extract, 3 parts of lily powder, and 1 part of γ-aminobutyric acid.

[0065] Example 6: Preparation of sleep-promoting products

[0066] A method for preparing a sleep-promoting product includes the following steps:

[0067] The raw materials shown in Example 5 were weighed and passed through an 80-mesh sieve, then mixed evenly to obtain a sleep-promoting product.

[0068] Example 7: Preparation of sleep-promoting products

[0069] A method for preparing a sleep-promoting product includes the following steps:

[0070] S1. Weigh the raw materials shown in Example 5 and pass them through an 80-mesh sieve. Mix them in dry powder form for 15 minutes.

[0071] S2. Add deionized water to the material at a solid-liquid ratio of 1g:8ml and stir for 20 minutes.

[0072] S3. Add 0.2% sodium carboxymethyl cellulose by mass of the material as a suspending agent and continue stirring for 10 minutes;

[0073] S4. Use a high-speed shearing machine to shear at 8000 rpm for 3 minutes to obtain the sleep-promoting product.

[0074] Example 8: Preparation of sleep-promoting products

[0075] A method for preparing a sleep-promoting product includes the following steps:

[0076] S1. Dissolve 100 parts by weight of carboxymethyl chitosan with a molecular weight of 38,000 and a degree of substitution of 0.80 in deionized water to prepare a solution of 50 mg / mL; add 4 parts by weight of L-cysteine, adjust the pH to 5.5 with 1 mol / L acetic acid, and stir the reaction at 25°C for 4 h; then add 1.5 parts by weight of genipin, adjust the pH to 7.0 with 0.5 mol / L sodium hydroxide solution, and stir the reaction at 40°C for 6 h to form a cross-linked system;

[0077] S2. Add 24 parts by weight of giant salamander peptide, 7 parts by weight of casein peptide, and 5 parts by weight of lily powder to the cross-linking system and stir for 10 min. Then add 8 parts by weight of calcium chloride to prepare a 1.0 mg / mL solution and stir at 25°C for 30 min. Then add 8 parts by weight of sodium carbonate to prepare a 1.0 mg / mL solution and stir at 25°C for 30 min. Microspheres are formed through the precipitation reaction of calcium ions and carbonate ions. Filter the reaction solution through a 250-mesh nylon sieve, collect the retained particles, wash them three times with deionized water, and then add an equal mass of deionized water. Grind the particles with 0.5 mm zirconia beads at 1500 rpm for 45 min to obtain a dispersion.

[0078] S3. Add 10 parts by weight of tea theanine, 13 parts by weight of daylily extract, 9 parts by weight of poria cocos extract, 9 parts by weight of jujube seed extract and 1.5 parts by weight of γ-aminobutyric acid to the dispersion and mix evenly to obtain the sleep-promoting product.

[0079] Test Example 1

[0080] 1. Experimental Methods

[0081] Two hundred zebrafish were randomly divided into a control group, a model group, and experimental groups A, B, and C, with 40 fish in each group. The sleep deprivation model was established by continuously stimulating the zebrafish with LED lights for 3 days.

[0082] The blank group was placed in a normal light and dark alternation environment;

[0083] Models were created in both the model group and the experimental group under continuous 3D illumination.

[0084] After the model was established, neither the blank group nor the model group of zebrafish received any drug treatment.

[0085] Experimental group A was given 125 μg / mL of the sleep-promoting product obtained in Example 6;

[0086] Experimental group B was given 125 μg / mL of the sleep-promoting product obtained in Example 7;

[0087] Experimental group C was given 125 μg / mL of the sleep-promoting product obtained in Example 8;

[0088] The culture medium was changed every 24 hours, and the treatment was continued for 96 hours.

[0089] 2. Experimental Results

[0090] 2.1 Toxicity of Sleep-Promoting Products to Zebrafish

[0091] The survival status of zebrafish during the cultivation process is shown in Table 1. The results show that the zebrafish in each group survived well, indicating that the sleep-promoting products obtained in Examples 6-8 do not have significant toxicity to zebrafish.

[0092] Table 1

[0093]

[0094] 2.2 The sleep-improving effects of sleep-promoting products on zebrafish

[0095] Disruptions to the circadian rhythm profoundly affect the normal life and physiological state of zebrafish. When a zebrafish's circadian rhythm is disrupted, its behavior exhibits a series of abnormal changes, including irregular activity patterns, disordered feeding times and behaviors, impaired social interaction, chaotic sleep patterns, and abnormalities in physiological functions and the immune system. These changes not only affect the zebrafish's health but may also lead to increased stress responses and a decline in their overall adaptability.

[0096] The movement trajectories of each group of zebrafish are as follows Figure 1 As shown in the figure. The results showed that the zebrafish in the blank group had regular movement trajectories, while the zebrafish in the model group had irregular and disordered movement trajectories. The movement trajectories of the zebrafish in the experimental group became regular and orderly again, indicating that the sleep-promoting product provided by this invention has the effect of improving the sleep quality of zebrafish. Among the experimental groups, the movement trajectory of the zebrafish in experimental group C was the most regular, indicating that the sleep-promoting product obtained in Example 8 had the best effect on improving the disordered diurnal rhythm of zebrafish.

[0097] 2.3 Evaluation of the efficacy of sleep-promoting products in improving sleep in zebrafish from a genetic perspective

[0098] The gabra1 gene encodes a gamma-aminobutyric acid (GABA) receptor. GABA primarily functions as an inhibitory neurotransmitter in the human body, playing a crucial role by inhibiting the transmission of excitatory signals. GABA activity is mediated by its receptor, and activation of the GABA receptor has been shown to promote sleep.

[0099] Melatonin is a hormone secreted by the pineal gland in the brain, and its encoding gene is mtnr1aa. Melatonin can induce natural sleep, and its secretion exhibits a distinct circadian rhythm. At night, the activity of enzymes that synthesize melatonin in the pineal gland increases, leading to increased melatonin secretion levels. When melatonin binds to specific receptors, it initiates cell signal transduction, shortens sleep latency, and prolongs sleep duration, thereby overcoming sleep disorders, improving circadian rhythm disruption, and enhancing sleep quality. Melatonin receptor 1A is one of the receptors through which melatonin functions in sleep disorders.

[0100] Therefore, studying the expression levels of the gabra1 and mtnr1aa genes can indirectly reflect the sleep-improving effects of sleep-promoting products on zebrafish.

[0101] The expression of the gabra1 gene in zebrafish from the control group, model group, and experimental groups A, B, and C is as follows: Figure 2 As shown, the expression of the mtnr1aa gene is as follows: Figure 3 As shown, the relative expression levels of the gabra1 gene in the blank group, model group, and experimental groups A, B, and C zebrafish were 1.00, 0.66, 0.83, 0.95, and 2.24, respectively, and the relative expression levels of the mtnr1aa gene were 1.01, 0.75, 0.84, 0.92, and 2.09, respectively.

[0102] The results showed that the expression levels of the gabra1 and mtnr1aa genes in the model group zebrafish were significantly lower than those in the control group, indicating successful model establishment. Compared with the model group, the expression levels of the gabra1 and mtnr1aa genes in experimental groups A, B, and C zebrafish all increased to some extent, indicating that the sleep-promoting product prepared in this invention can improve the decreased expression of the gabra1 and mtnr1aa genes caused by sleep deprivation. Among the experimental groups, experimental group C showed the most significant improvement effect, indicating that the sleep-promoting product obtained in Example 8 had the best effect on improving sleep in zebrafish.

[0103] The above description is merely an example and illustration of the structure of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. The application of a compound composition of giant salamander peptide extract in the preparation of sleep-promoting products, characterized in that, The weight composition of the sleep-promoting product is as follows: 70-85 parts of giant salamander peptide extract compound composition, 95-105 parts of carboxymethyl chitosan, 3-5 parts of L-cysteine, 1-2 parts of genipin, 8-10 parts of calcium chloride and 8-10 parts of sodium carbonate. The weight components of the giant salamander peptide extract compound composition include: 18-24 parts giant salamander peptide extract, 3-7 parts casein peptide, 6-10 parts tea theanine, 9-13 parts daylily extract, 5-9 parts Poria cocos extract, 5-9 parts jujube seed extract, 1-5 parts lily powder, and 0.5-1.5 parts γ-aminobutyric acid. The sleep-promoting product is prepared by the following method: S1. Dissolve carboxymethyl chitosan in water to make a solution, add L-cysteine, adjust the pH to 5.0~6.0 and stir; then add genipin and adjust the pH to 7.0~8.0, stir at 40~50℃ to form a cross-linking system; S2. Add the giant salamander peptide extract, casein peptide and lily powder to the cross-linking system and stir. Then add calcium chloride and sodium carbonate and stir. After filtration, take the filter residue and wet grind it. The wet grinding process uses water as a medium and grinds it with 0.4~0.5 mm zirconia beads at 1000~1500 rpm for 20~45 min to obtain a dispersion. S3. Add tea theanine, daylily extract, poria cocos extract, jujube seed extract and γ-aminobutyric acid to the dispersion and mix evenly to obtain a sleep-promoting product; The giant salamander peptide extract was obtained by the following method: giant salamander meat was hydrolyzed with papain and Protamex 1.6 complex protease at 45-55℃, and the enzymes were inactivated after hydrolysis; the extract was then filtered through a ceramic membrane and adsorbed with activated carbon to obtain the giant salamander peptide extract.

2. The application according to claim 1, characterized in that, The amount of papain added is 0.5~0.7wt%; And / or, the amount of the complex protease Protamex 1.6 added is 0.5~0.7wt%.

3. The application according to claim 1, characterized in that, The daylily extract was obtained by water extraction from daylilies.

4. The application according to claim 1, characterized in that, The Poria cocos extract was obtained by water extraction and alcohol extraction of Poria cocos.

5. The application according to claim 1, characterized in that, The jujube seed extract is obtained by defatting, enzymatic hydrolysis and alcohol extraction of jujube seeds.

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