A sleep-aiding composition containing gamma-aminobutyric acid and its use

Abstract

This invention belongs to the field of functional food technology, specifically relating to a sleep-aiding composition containing γ-aminobutyric acid (GABA) and its application. The sleep-aiding composition comprises: 2 to 8 parts of fermented soybean extract; 3 to 10 parts of oyster oligopeptide powder; 0.5 to 5 parts of GABA; 1 to 4 parts of Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex; and 1 to 4 parts of Acanthopanax senticosus extract. This composition is used in the preparation of products with sleep-improving effects. This invention utilizes a two-stage fermentation process to pre-convert soybean isoflavones to estrol and enrich GABA, combined with targeted enzymatic hydrolysis membrane separation to enrich taurine and zinc in oyster oligopeptides to maintain GABA-A receptor sensitivity. The addition of Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex improves water solubility and stability, achieving stable sleep-aiding effects and minimizing individual differences, while also promoting growth and development.

Description

Technical Field

[0001] This invention belongs to the field of functional food technology, specifically relating to a sleep-aiding composition containing γ-aminobutyric acid and its application. Background Technology

[0002] Sleep disorders manifest as difficulty falling asleep, difficulty maintaining sleep, or early awakening. Chronic insomnia can lead to impaired daytime function and inhibit growth hormone secretion, negatively impacting bone development and protein synthesis in children and adolescents. Since growth hormone is primarily secreted in pulses during deep sleep at night, decreased sleep quality directly reduces its total secretion. Therefore, simultaneously improving sleep depth and promoting growth hormone release is a core issue that needs to be addressed in the development of related functional foods.

[0003] Current clinical interventions mainly fall into two categories. One category consists of sedative-hypnotic drugs, represented by benzodiazepines, which shorten the time to fall asleep by enhancing inhibitory neurotransmission of GABA-A receptors. However, long-term use can easily lead to adverse reactions such as tolerance, dependence, and drowsiness the following day. The other category is recombinant human growth hormone injection therapy, which directly supplements exogenous growth hormone. However, it has drawbacks such as high cost, highly invasive administration methods, and poor patient compliance.

[0004] Gamma-aminobutyric acid (GABA) is an endogenous inhibitory neurotransmitter with physiological functions of regulating mood and inducing sleep. It can also promote the release of growth hormone through the hypothalamus-pituitary axis. However, oral GABA has two main drawbacks: first, its efficiency in crossing the blood-brain barrier is limited by various factors, and simply increasing the intake cannot linearly enhance the central effect; second, long-term high-dose supplementation with exogenous GABA can lead to decreased sensitivity of GABA-A receptors, and the sleep-aiding effect diminishes with the duration of use.

[0005] To overcome the limitations of GABA alone, existing technologies attempt to combine it with plant extracts. For example, a Chinese patent discloses a sleep tablet containing lily, γ-aminobutyric acid (GABA), tea theanine, and spearmint extract, which shortens sleep latency through the synergistic effect of multiple components. Another Chinese patent discloses a composition containing GABA, elderberry extract, lily extract, and human milk oligosaccharides, used to improve sleep and promote growth hormone secretion. While these solutions broaden the scope of formulations to some extent, they still have the following specific drawbacks: Firstly, existing compositions focus on increasing the absolute supply of GABA, neglecting the maintenance of GABA-A receptor sensitivity. Decreased GABA-A receptor sensitivity is a key reason for the diminished efficacy of oral GABA products. Taurine, as a positive allosteric regulator of GABA-A receptors, and zinc, as a cofactor of key enzymes in the melatonin and GABA synthesis pathways, both play crucial roles in maintaining receptor response levels. Oysters are naturally rich in taurine and zinc, but existing sleep aid compositions rarely utilize their active ingredients through targeted enrichment. Furthermore, there are no reports of using complex protease hydrolysis combined with multi-stage membrane separation technology to precisely control the molecular weight of oyster peptides to enrich taurine and zinc while ensuring batch-to-batch uniformity.

[0006] Secondly, equol, the active metabolite of soy isoflavones, has the potential to regulate the hypothalamic-pituitary-adrenal axis, alleviating anxiety, lowering cortisol levels, and creating favorable endocrine conditions for sleep. Some gut bacteria possess the dual metabolic capacity to produce equol and GABA. However, equol production is highly dependent on individual gut microbiota composition; a significant proportion of the population lacks specific transforming strains and cannot effectively produce equol, leading to significant individual differences in the efficacy of soy isoflavone products. Existing technologies do not report on combining the pre-fermentation process of equol-producing bacteria with the secondary fermentation process of GABA-producing bacteria to pre-complete the transformation at the raw material level to overcome individual differences.

[0007] Thirdly, most plant extracts in existing sleep-aid compositions are crude extracts obtained by direct pulverization or simple alcohol extraction, without targeted modification to address the physicochemical defects of each extract. This results in the synergistic effect between the active ingredients and GABA components being difficult to fully realize. Atractylodes macrocephala volatile oil is rich in lipid-soluble components such as atractylone and atractylodes lactone, possessing sedative and tranquilizing activities. However, its water solubility is extremely poor and it is easily oxidized, leading to uneven dispersion and low bioavailability in aqueous preparations. Existing technologies have not performed inclusion modification to improve its water solubility and stability. Therefore, there is a need to design a sleep-aid composition containing γ-aminobutyric acid and its application. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, a sleep-aiding composition containing γ-aminobutyric acid and its application are provided.

[0009] To achieve the above objectives, the present invention provides the following technical solution: A sleep-aid composition containing γ-aminobutyric acid (GABA), the sleep-aid composition comprising excipients and the following raw materials in parts by weight: Fermented soybean extract, 2 to 8 parts; Oyster oligopeptide powder, 3 to 10 parts; γ-Aminobutyric acid, 0.5 to 5 parts; Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex, 1 to 4 parts; Eleutherococcus senticosus extract, 1 to 4 parts.

[0010] The fermented soybean extract was prepared by the following steps: S1: Take soybeans, wash them, soak them in water at 25 to 30°C for 8 to 12 hours, drain them, steam them for 8 to 15 minutes, cool them, add water and grind them into a paste to obtain soy milk. The mass ratio of the material to the liquid is 1:5 to 1:8. S2: Inoculate the soy milk with isoflavone-converting Slack bacteria solution at an inoculation amount of 2% to 6% of the soy milk volume, and ferment at 35 to 38°C under anaerobic conditions for 20 to 36 hours to obtain a primary fermentation liquid. S3: Inoculate the primary fermentation broth with Lactobacillus plantarum culture at an inoculation amount of 2% to 5% of the volume of the primary fermentation broth, and ferment at 35 to 38°C for 12 to 24 hours to obtain the secondary fermentation broth. S4: The secondary fermentation broth is inactivated at 85 to 95°C for 10 to 20 minutes, cooled, and then vacuum concentrated to 20% of its original volume at 55 to 70°C and a vacuum degree of 0.08 to 0.095 MPa to obtain a concentrated broth; S5: Freeze-dry the concentrated liquid, pulverize it, and pass it through an 80 to 120 mesh sieve to obtain fermented soybean extract.

[0011] The isoflavone-converting Slack bacteria culture is prepared by the following method: inoculating the isoflavone-converting Slack bacteria into a modified GAM liquid medium containing 0.05% to 0.15% daidzein, and anaerobic culturing at 35 to 38°C for 24 to 48 hours until the OD600 value of the culture reaches 0.8 to 1.5, thus obtaining the isoflavone-converting Slack bacteria culture.

[0012] The *Lactobacillus plantarum* bacterial solution is prepared by the following method: *Lactobacillus plantarum* strain is inoculated into MRS liquid medium and cultured at 35 to 38°C for 18 to 30 hours until the OD600 value of the bacterial solution reaches 1.0 to 2.0, thus obtaining the *Lactobacillus plantarum* bacterial solution.

[0013] The oyster oligopeptide powder is prepared by the following steps: P1: Take fresh oyster meat, add purified water to homogenize, the material-to-liquid ratio is 1:3 to 1:5, treat at 90 to 100℃ for 15 to 20 minutes, and cool to 48 to 52℃; P2: Adjust the pH to 7.5 to 8.0, add complex protease, and add 2% to 4% of the total protein in the homogenate. Incubate at 48 to 52°C for 3 to 5 hours. P3: Inactivate enzymes at 85 to 95°C for 15 to 20 minutes, cool, centrifuge at 4 to 8°C and 6000 to 10000 r / min for 15 to 20 minutes, and collect the supernatant; P4: The supernatant is sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 3000 Daltons, and the permeate is collected. P5: The permeate is treated with a nanofiltration membrane with a molecular weight cutoff of 500 Daltons, and the retentate is collected; P6: Freeze-dry the retentate to obtain oyster oligopeptide powder. The obtained oyster oligopeptide powder contains more than 80% peptides with a molecular weight distribution in the range of 500 to 2000 Daltons, taurine content of not less than 45 mg / g, and zinc content of not less than 80 μg / g.

[0014] In step P2, the complex protease is composed of trypsin and alkaline protease in an activity ratio of 1:0.6 to 1:1.

[0015] The β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil was prepared by the following steps: B1: Take Atractylodes macrocephala slices, add purified water, with a material-to-liquid ratio of 1:6 to 1:10, and extract by steam distillation for 3 to 5 hours. Collect the volatile oil to obtain Atractylodes macrocephala volatile oil. B2: Dissolve β-cyclodextrin in purified water to prepare a 5% to 10% β-cyclodextrin solution, and stir at 40 to 50°C until completely dissolved; B3: Mix the volatile oil of Atractylodes macrocephala with anhydrous ethanol at a volume ratio of 1:1 to 1:2 to obtain a volatile oil ethanol solution. Under stirring conditions, slowly add the volatile oil ethanol solution to the β-cyclodextrin solution and continue stirring at 40 to 50°C for 2 to 4 hours to obtain an inclusion solution. B4: The inclusion solution is allowed to stand at 4 to 8°C for 12 to 24 hours, filtered, the precipitate is washed 2 to 3 times with purified water, and vacuum dried at 40 to 50°C and a vacuum degree of 0.08 to 0.095 MPa to obtain the β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil.

[0016] In step B3, the mass ratio of the Atractylodes macrocephala volatile oil to β-cyclodextrin is 1:4 to 1:8.

[0017] The raw materials also contain 1 to 3 parts of a compound extract, which is prepared by the following steps: F1: Take jujube seed, lily bulb and poria cocos in a mass ratio of 1:1:0.5 to 1:1:1.5, grind them separately to 40 to 60 mesh, and mix them to obtain a mixed powder; F2: Using 50% to 70% ethanol aqueous solution as solvent, with a material-to-liquid ratio of 1:8 to 1:12, ultrasonically assisted extraction was performed at 45 to 55°C and ultrasonic power of 150 to 300W for 30 to 60 minutes, and the extraction was repeated 2 to 3 times. The extracts were then combined. F3: The extract is concentrated under reduced pressure at 50 to 65°C and a vacuum of 0.08 to 0.095 MPa to obtain a concentrated solution; F4: The concentrate is reconstituted with water and brought to a volume of 2 to 3 times the original volume. It is then treated with an ultrafiltration membrane with a molecular weight cutoff of 100 kilodaltons, and the permeate is collected. F5: Freeze-dry the permeate to obtain the composite extract.

[0018] This composition is used in the preparation of products that have the effect of improving sleep.

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. In the sleep-aid composition provided by this invention, oyster oligopeptide powder is obtained through a complex protease hydrolysis and multi-stage membrane separation process. The molecular weight of the peptides is controlled within a relatively concentrated low molecular weight range, while taurine and zinc are directionally enriched. Taurine, as a positive allosteric regulator of GABA type A receptors, can enhance the receptor's response level to GABA. Zinc, as a cofactor of related enzymes in the melatonin and GABA synthesis pathways, participates in maintaining the normal metabolism of neurotransmitters. When these two components are used together with GABA, they help maintain the responsiveness of GABA type A receptors and alleviate the decline in receptor sensitivity that may be caused by long-term supplementation with exogenous GABA, thereby making it easier to maintain the sleep-aiding effect of the product.

[0020] 2. The fermented soybean extract of this invention is obtained through a two-stage fermentation process. First, *Synapticus slavantrum* converts soybean isoflavones into equol, and then *Lactobacillus plantarum* ferments to produce γ-aminobutyric acid (GABA). This pre-conversion of equol at the raw material stage eliminates the influence of individual gut microbiota differences on equol production. The resulting extract contains both equol and GABA. Equol participates in the functional regulation of the hypothalamus-pituitary-adrenal axis, helping to lower cortisol levels, relieve tension, and synergistically create suitable endocrine conditions for sleep, together with GABA.

[0021] 3. In this invention, after the Atractylodes macrocephala volatile oil is treated with β-cyclodextrin inclusion complex, its lipid-soluble components are encapsulated within the hydrophobic cavities of β-cyclodextrin, forming a β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil. Both water solubility and oxidative stability are improved. In aqueous formulations, this inclusion complex can be uniformly dispersed, reducing the risk of loss of Atractylodes macrocephala volatile oil due to precipitation or degradation. This facilitates the dissolution and absorption of sedative and tranquilizing components such as atractylone and atractylodes lactone in the Atractylodes macrocephala volatile oil in vivo, thereby better exerting its sedative activity.

[0022] 4. The Acanthopanax senticosus extract and the compound extract introduced into the composition of this invention work synergistically with the aforementioned oyster oligopeptide powder and fermented soybean extract. The saponins contained in the Acanthopanax senticosus extract can synergistically act with the oyster oligopeptide powder on the hypothalamus-pituitary axis, helping to promote growth hormone secretion. The compound extract, after ultrasonic extraction and ultrafiltration, removes macromolecular impurities while retaining sedative active ingredients such as jujube seed saponins and lily steroidal saponins, which help prolong deep sleep time. The above-mentioned raw materials intervene in the sleep regulation process through different pathways, simultaneously improving sleep depth and promoting growth hormone release. Attached Figure Description

[0023] Figure 1 These are the results of animal experimental indicator tests. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] In the specific embodiments of this application, the sources of various main raw materials are briefly described as follows: Daidzein was purchased from Hubei Jianchu Biomedical Co., Ltd., CAS No. 486-66-8.

[0026] The modified GAM liquid culture medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0027] Isoflavone-transformed Slackia strain (DSM 22006): purchased from Hangzhou Baosai Biotechnology Co., Ltd.

[0028] Lactobacillus plantarum strain, purchased from Wuhan Huanna Biotechnology Co., Ltd., product number SHBCC D73164.

[0029] MRS liquid culture medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0030] Fresh oyster meat, purchased from Rongcheng Ocean Oasis Biotechnology Co., Ltd.

[0031] Trypsin, purchased from Hefei Shengrun Biological Products Co., Ltd., CAS No. 9002-07-7, food grade.

[0032] Alkaline protease, purchased from Hefei Shengrun Biological Products Co., Ltd., is food grade.

[0033] The Atractylodes macrocephala slices were purchased from Guizhou Guangjitang Health Pharmaceutical Co., Ltd.

[0034] β-Cyclodextrin was purchased from Hubei Xinghengye Technology Co., Ltd., CAS No. 7585-39-9.

[0035] γ-Aminobutyric acid (GABA) was purchased from Guangdong Leercon Biotechnology Co., Ltd., CAS No. 56-12-2, and is classified as food grade.

[0036] The extract of Acanthopanax senticosus was purchased from Xi'an Sanjiang Bioengineering Co., Ltd., CAS No. 7374-79-0.

[0037] The jujube seed, lily bulb, and poria cocos were all purchased from Fufeng Sinote Biotechnology Co., Ltd.

[0038] The technical solution of this application is as follows: A sleep-aid composition containing γ-aminobutyric acid (GABA), the sleep-aid composition comprising excipients and the following raw materials in parts by weight: Fermented soybean extract, 2 to 8 parts; Oyster oligopeptide powder, 3 to 10 parts; γ-Aminobutyric acid, 0.5 to 5 parts; Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex, 1 to 4 parts; Eleutherococcus senticosus extract, 1 to 4 parts.

[0039] The fermented soybean extract was prepared by the following steps: S1: Take soybeans, wash them, soak them in water at 25 to 30°C for 8 to 12 hours, drain them, steam them for 8 to 15 minutes, cool them, add water and grind them into a paste to obtain soy milk. The mass ratio of the material to the liquid is 1:5 to 1:8. S2: Inoculate the soy milk with isoflavone-converting Slack bacteria solution at an inoculation amount of 2% to 6% of the soy milk volume, and ferment at 35 to 38°C under anaerobic conditions for 20 to 36 hours to obtain a primary fermentation liquid. S3: Inoculate the primary fermentation broth with Lactobacillus plantarum culture at an inoculation amount of 2% to 5% of the volume of the primary fermentation broth, and ferment at 35 to 38°C for 12 to 24 hours to obtain the secondary fermentation broth. S4: The secondary fermentation broth is inactivated at 85 to 95°C for 10 to 20 minutes, cooled, and then vacuum concentrated to 20% of its original volume at 55 to 70°C and a vacuum degree of 0.08 to 0.095 MPa to obtain a concentrated broth; S5: Freeze-dry the concentrated liquid, pulverize it, and pass it through an 80 to 120 mesh sieve to obtain fermented soybean extract.

[0040] The isoflavone-converting Slack bacteria culture is prepared by the following method: inoculating the isoflavone-converting Slack bacteria into a modified GAM liquid medium containing 0.05% to 0.15% daidzein, and anaerobic culturing at 35 to 38°C for 24 to 48 hours until the OD600 value of the culture reaches 0.8 to 1.5, thus obtaining the isoflavone-converting Slack bacteria culture.

[0041] The *Lactobacillus plantarum* bacterial solution is prepared by the following method: *Lactobacillus plantarum* strain is inoculated into MRS liquid medium and cultured at 35 to 38°C for 18 to 30 hours until the OD600 value of the bacterial solution reaches 1.0 to 2.0, thus obtaining the *Lactobacillus plantarum* bacterial solution.

[0042] The oyster oligopeptide powder is prepared by the following steps: P1: Take fresh oyster meat, add purified water to homogenize, the material-to-liquid ratio is 1:3 to 1:5, treat at 90 to 100℃ for 15 to 20 minutes, and cool to 48 to 52℃; P2: Adjust the pH to 7.5 to 8.0, add complex protease, and add 2% to 4% of the total protein in the homogenate. Incubate at 48 to 52°C for 3 to 5 hours. P3: Inactivate enzymes at 85 to 95°C for 15 to 20 minutes, cool, centrifuge at 4 to 8°C and 6000 to 10000 r / min for 15 to 20 minutes, and collect the supernatant; P4: The supernatant is sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 3000 Daltons, and the permeate is collected. P5: The permeate is treated with a nanofiltration membrane with a molecular weight cutoff of 500 Daltons, and the retentate is collected; P6: Freeze-dry the retentate to obtain oyster oligopeptide powder. The obtained oyster oligopeptide powder contains more than 80% peptides with a molecular weight distribution in the range of 500 to 2000 Daltons, taurine content of not less than 45 mg / g, and zinc content of not less than 80 μg / g.

[0043] In step P2, the complex protease is composed of trypsin and alkaline protease in an activity ratio of 1:0.6 to 1:1.

[0044] The β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil was prepared by the following steps: B1: Take Atractylodes macrocephala slices, add purified water, with a material-to-liquid ratio of 1:6 to 1:10, and extract by steam distillation for 3 to 5 hours. Collect the volatile oil to obtain Atractylodes macrocephala volatile oil. B2: Dissolve β-cyclodextrin in purified water to prepare a 5% to 10% β-cyclodextrin solution, and stir at 40 to 50°C until completely dissolved; B3: Mix the volatile oil of Atractylodes macrocephala with anhydrous ethanol at a volume ratio of 1:1 to 1:2 to obtain a volatile oil ethanol solution. Under stirring conditions, slowly add the volatile oil ethanol solution to the β-cyclodextrin solution and continue stirring at 40 to 50°C for 2 to 4 hours to obtain an inclusion solution. B4: The inclusion solution is allowed to stand at 4 to 8°C for 12 to 24 hours, filtered, the precipitate is washed 2 to 3 times with purified water, and vacuum dried at 40 to 50°C and a vacuum degree of 0.08 to 0.095 MPa to obtain the β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil.

[0045] In step B3, the mass ratio of the Atractylodes macrocephala volatile oil to β-cyclodextrin is 1:4 to 1:8.

[0046] The raw materials also contain 1 to 3 parts of a compound extract, which is prepared by the following steps: F1: Take jujube seed, lily bulb and poria cocos in a mass ratio of 1:1:0.5 to 1:1:1.5, grind them separately to 40 to 60 mesh, and mix them to obtain a mixed powder; F2: Using 50% to 70% ethanol aqueous solution as solvent, with a material-to-liquid ratio of 1:8 to 1:12, ultrasonically assisted extraction was performed at 45 to 55°C and ultrasonic power of 150 to 300W for 30 to 60 minutes, and the extraction was repeated 2 to 3 times. The extracts were then combined. F3: The extract is concentrated under reduced pressure at 50 to 65°C and a vacuum of 0.08 to 0.095 MPa to obtain a concentrated solution; F4: The concentrate is reconstituted with water and brought to a volume of 2 to 3 times the original volume. It is then treated with an ultrafiltration membrane with a molecular weight cutoff of 100 kilodaltons, and the permeate is collected. F5: Freeze-dry the permeate to obtain the composite extract.

[0047] This composition is used in the preparation of products that have the effect of improving sleep.

[0048] The present invention will be described in detail below through examples and comparative examples, but the scope of protection of the present invention is not limited to these examples. Unless otherwise specified, the chemical reagents and raw materials used in the following examples and comparative examples are all conventional commercially available products.

[0049] Example 1 A sleep aid composition containing γ-aminobutyric acid (GABA) comprises excipients and the following raw materials in parts by weight: 8 parts fermented soybean extract, 6.5 parts oyster oligopeptide powder, 0.5 parts GABA, 4 parts Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex, 2.5 parts Acanthopanax senticosus extract, and 1 part compound extract.

[0050] The fermented soybean extract is prepared by the following steps: S1, soybeans are taken, washed, soaked in water at 30℃ for 10 hours, drained, steamed for 8 minutes, cooled, and ground with water to obtain soy milk, with a material-to-liquid mass ratio of 1:5; S2, isoflavone-converting Slack bacteria solution is added to the soy milk at an inoculation amount of 6% of the soy milk volume, and fermented at 35℃ under anaerobic conditions for 36 hours to obtain a primary fermentation liquid; S3, Lactobacillus plantarum solution is added to the primary fermentation liquid at an inoculation amount of 3.5% of the primary fermentation liquid volume, and fermented at 35℃ for 24 hours to obtain a secondary fermentation liquid; S4, the secondary fermentation liquid is inactivated at 90℃ for 10 minutes, cooled, and then vacuum concentrated to 20% of the original volume at 70℃ and a vacuum degree of 0.0875MPa to obtain a concentrated liquid; S5, the concentrated liquid is freeze-dried, pulverized, and passed through an 80-mesh sieve to obtain the fermented soybean extract.

[0051] The isoflavone-converting Slack bacteria culture was prepared by the following method: the isoflavone-converting Slack bacteria were inoculated into a modified GAM liquid medium containing 0.15% daidzein and anaerobic cultured at 36.5℃ for 24 hours until the OD600 value of the culture reached 1.5, thus obtaining the isoflavone-converting Slack bacteria culture.

[0052] The *Lactobacillus plantarum* bacterial solution is prepared by the following method: *Lactobacillus plantarum* strain is inoculated into MRS liquid medium and cultured at 36.5℃ for 18 hours until the OD600 value of the bacterial solution reaches 2.0, thus obtaining the *Lactobacillus plantarum* bacterial solution.

[0053] The above-mentioned isoflavone-converted Slack bacteria and Lactobacillus plantarum bacterial solutions can be prepared according to the methods described in this application, or commercial bacterial solutions that meet the corresponding requirements can be purchased directly.

[0054] The oyster oligopeptide powder is prepared by the following steps: P1, fresh oyster meat is taken, purified water is added and homogenized at a material-to-liquid ratio of 1:4, and the mixture is treated at 90℃ for 20 minutes and then cooled to 50℃; P2, the pH is adjusted to 7.5, a complex protease is added, and the amount of enzyme added is 4% of the total protein content in the homogenate, based on the total protein content in the homogenate, and enzymatic hydrolysis is performed at 50℃ for 3 hours; P3, the enzyme is inactivated at 95℃ for 17.5 minutes, cooled, and then subjected to hydrolysis at 4℃ and 10000... Centrifuge at r / min for 17.5 minutes and collect the supernatant; P4, treat the supernatant sequentially through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 3000 Daltons, and collect the permeate; P5, treat the permeate through a nanofiltration membrane with a molecular weight cutoff of 500 Daltons, and collect the retentate; P6, freeze-dry the retentate to obtain oyster oligopeptide powder. The obtained oyster oligopeptide powder contains peptides with a molecular weight distribution in the range of 500 to 2000 Daltons accounting for more than 80% of the total peptide mass, with a taurine content of not less than 45 mg / g and a zinc content of not less than 80 μg / g. In step P2, the complex protease is composed of trypsin and alkaline protease at an activity ratio of 1:1.

[0055] The β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil was prepared by the following steps: B1, Atractylodes macrocephala slices were taken, purified water was added, the material-to-liquid ratio was 1:6, and steam distillation was used to extract for 4 hours. The volatile oil was collected to obtain Atractylodes macrocephala volatile oil; B2, β-cyclodextrin was dissolved in purified water to prepare a 5% (w / w) β-cyclodextrin solution, and stirred at 50°C until completely dissolved; B3, the Atractylodes macrocephala volatile oil was mixed with anhydrous ethanol at a volume ratio of 1:1.5 to obtain a volatile oil ethanol solution. Under stirring conditions, the volatile oil ethanol solution is slowly added dropwise to the β-cyclodextrin solution, and stirring is continued at 40°C for 4 hours to obtain an inclusion solution; in step B3, the mass ratio of Atractylodes macrocephala volatile oil to β-cyclodextrin is 1:8; in step B4, the inclusion solution is allowed to stand at 6°C for 12 hours, filtered, the precipitate is washed 2 to 3 times with purified water, and vacuum dried at 50°C and a vacuum degree of 0.0875 MPa to obtain the Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex.

[0056] The composite extract was prepared by the following steps: F1, Ziziphus jujuba seed, lily bulb, and Poria cocos were taken in a mass ratio of 1:1:0.5, pulverized to 50 mesh, and mixed to obtain a mixed powder; F2, using a 50% ethanol aqueous solution as solvent, with a material-to-liquid ratio of 1:8, ultrasonic extraction was performed at 50℃ and 150W for 60 minutes, twice, and the extracts were combined; F3, the extract was concentrated under reduced pressure at 50℃ and 0.095MPa to obtain a concentrated solution; F4, the concentrated solution was reconstituted with water and brought to a final volume of 2.5 times the original volume, treated with an ultrafiltration membrane with a molecular weight cutoff of 100 kilodaltons, and the permeate was collected; F5, the permeate was freeze-dried to obtain the composite extract.

[0057] Example 2 In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows: A sleep aid composition containing γ-aminobutyric acid, comprising excipients and the following raw materials in parts by weight: 2 parts fermented soybean extract, 10 parts oyster oligopeptide powder, 2.75 parts γ-aminobutyric acid, 1 part Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex, 4 parts Acanthopanax senticosus extract, and 2 parts compound extract.

[0058] The fermented soybean extract is prepared by the following steps: S1, soybeans are taken, washed, soaked in water at 25°C for 12 hours, drained, steamed for 12 minutes, cooled, and ground with water to obtain soy milk, with a material-to-liquid mass ratio of 1:8; S2, isoflavone-converting Slack bacteria solution is added to the soy milk at an inoculation amount of 2% of the soy milk volume, and fermented at 36.5°C under anaerobic conditions for 20 hours to obtain a primary fermentation liquid; S3, Lactobacillus plantarum solution is added to the primary fermentation liquid at an inoculation amount of 5% of the primary fermentation liquid volume, and fermented at 36.5°C for 12 hours to obtain a secondary fermentation liquid; S4, the secondary fermentation liquid is inactivated at 95°C for 15 minutes, cooled, and then vacuum concentrated to 20% of the original volume at 55°C and a vacuum degree of 0.095MPa to obtain a concentrated liquid; S5, the concentrated liquid is freeze-dried, pulverized, and passed through a 100-mesh sieve to obtain the fermented soybean extract.

[0059] The isoflavone-converting Slack bacteria culture was prepared by the following method: the isoflavone-converting Slack bacteria were inoculated into a modified GAM liquid medium containing 0.05% daidzein and cultured anaerobically at 38°C for 36 hours until the OD600 value of the culture reached 0.8, thus obtaining the isoflavone-converting Slack bacteria culture.

[0060] The *Lactobacillus plantarum* bacterial solution is prepared by the following method: *Lactobacillus plantarum* strain is inoculated into MRS liquid medium and cultured at 38°C for 24 hours until the OD600 value of the bacterial solution reaches 1.0, thus obtaining the *Lactobacillus plantarum* bacterial solution.

[0061] The oyster oligopeptide powder is prepared by the following steps: P1, fresh oyster meat is taken, purified water is added to homogenize it, the material-to-liquid ratio is 1:3, and it is treated at 95℃ for 15 minutes, then cooled to 52℃; P2, the pH value is adjusted to 7.75, a complex protease is added, and the amount of enzyme added is 2% of the total protein content in the homogenate, based on the total protein content in the homogenate, and enzymatic hydrolysis is performed at 52℃ for 4 hours; P3, the enzyme is inactivated at 85℃ for 20 minutes, cooled, and then subjected to hydrolysis at 6℃ and 6000... Centrifuge at r / min for 20 minutes and collect the supernatant; P4, treat the supernatant sequentially through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 3000 Daltons, and collect the permeate; P5, treat the permeate through a nanofiltration membrane with a molecular weight cutoff of 500 Daltons, and collect the retentate; P6, freeze-dry the retentate to obtain oyster oligopeptide powder. The obtained oyster oligopeptide powder contains peptides with a molecular weight distribution in the range of 500 to 2000 Daltons accounting for more than 80% of the total peptide mass, with a taurine content of not less than 45 mg / g and a zinc content of not less than 80 μg / g. In step P2, the complex protease is composed of trypsin and alkaline protease at an activity ratio of 1:0.8.

[0062] The β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil was prepared by the following steps: B1, Atractylodes macrocephala slices were taken, purified water was added, the material-to-liquid ratio was 1:10, and steam distillation was used to extract for 5 hours. The volatile oil was collected to obtain Atractylodes macrocephala volatile oil; B2, β-cyclodextrin was dissolved in purified water to prepare a 7.5% (w / w) β-cyclodextrin solution, and stirred at 40°C until completely dissolved; B3, the Atractylodes macrocephala volatile oil was mixed with anhydrous ethanol at a volume ratio of 1:1 to obtain an ethanol solution of volatile oil. In step B3, the volatile oil ethanol solution is slowly added dropwise to the β-cyclodextrin solution under stirring conditions, and the mixture is stirred at 45°C for 2 hours to obtain an inclusion solution; in step B4, the mass ratio of Atractylodes macrocephala volatile oil to β-cyclodextrin is 1:6; in step B5, the inclusion solution is allowed to stand at 8°C for 18 hours, filtered, the precipitate is washed 2 to 3 times with purified water, and vacuum dried at 40°C and a vacuum degree of 0.095 MPa to obtain the Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex.

[0063] The composite extract was prepared by the following steps: F1, Ziziphus jujuba seed, lily bulb, and Poria cocos were taken in a mass ratio of 1:1:1.5, pulverized to 60 mesh, and mixed to obtain a mixed powder; F2, using a 60% ethanol aqueous solution as solvent, with a material-to-liquid ratio of 1:12, ultrasonic extraction was performed at 55℃ and 225W for 30 minutes, and the extraction was repeated 3 times, and the extracts were combined; F3, the extract was concentrated under reduced pressure at 57.5℃ and 0.08MPa to obtain a concentrated solution; F4, the concentrated solution was reconstituted with water and brought to a final volume of 3 times the original volume, treated with an ultrafiltration membrane with a molecular weight cutoff of 100 kilodaltons, and the permeate was collected; F5, the permeate was freeze-dried to obtain the composite extract.

[0064] Example 3 In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows: A sleep aid composition containing γ-aminobutyric acid, comprising excipients and the following raw materials in parts by weight: 5 parts fermented soybean extract, 3 parts oyster oligopeptide powder, 5 parts γ-aminobutyric acid, 2.5 parts Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex, 1 part Acanthopanax senticosus extract, and 3 parts compound extract.

[0065] The fermented soybean extract is prepared by the following steps: S1, soybeans are taken, washed, soaked in water at 27.5℃ for 8 hours, drained, steamed for 15 minutes, cooled, and ground with water to obtain soy milk, with a material-to-liquid mass ratio of 1:6.5; S2, isoflavone-converting Slack bacteria solution is added to the soy milk at an inoculation amount of 4% of the soy milk volume, and fermented at 38℃ under anaerobic conditions for 28 hours to obtain a primary fermentation liquid; S3, Lactobacillus plantarum solution is added to the primary fermentation liquid at an inoculation amount of 2% of the primary fermentation liquid volume, and fermented at 38℃ for 18 hours to obtain a secondary fermentation liquid; S4, the secondary fermentation liquid is inactivated at 95℃ for 20 minutes, cooled, and then vacuum concentrated to 20% of the original volume at 62.5℃ and a vacuum degree of 0.08MPa to obtain a concentrated liquid; S5, the concentrated liquid is freeze-dried, pulverized, and passed through a 120-mesh sieve to obtain the fermented soybean extract.

[0066] The isoflavone-converting Slack bacteria culture was prepared by the following method: the isoflavone-converting Slack bacteria were inoculated into a modified GAM liquid medium containing 0.1% daidzein and cultured anaerobically at 35°C for 48 hours until the OD600 value of the culture reached 1.15, thus obtaining the isoflavone-converting Slack bacteria culture.

[0067] The *Lactobacillus plantarum* bacterial solution is prepared by the following method: *Lactobacillus plantarum* strain is inoculated into MRS liquid medium and cultured at 35°C for 30 hours until the OD600 value of the bacterial solution reaches 1.5, thus obtaining the *Lactobacillus plantarum* bacterial solution.

[0068] The oyster oligopeptide powder is prepared by the following steps: P1, fresh oyster meat is taken, purified water is added to homogenize it, the material-to-liquid ratio is 1:5, and it is treated at 100℃ for 17.5 minutes, then cooled to 48℃; P2, the pH value is adjusted to 8.0, a complex protease is added, and the amount of enzyme added is 3% of the total protein content in the homogenate, based on the total protein content in the homogenate, and enzymatic hydrolysis is performed at 48℃ for 5 hours; P3, the enzyme is inactivated at 90℃ for 15 minutes, cooled, and then subjected to hydrolysis at 8℃ and 8000... Centrifuge at r / min for 15 minutes and collect the supernatant; P4, treat the supernatant sequentially through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 3000 Daltons, and collect the permeate; P5, treat the permeate through a nanofiltration membrane with a molecular weight cutoff of 500 Daltons, and collect the retentate; P6, freeze-dry the retentate to obtain oyster oligopeptide powder. The obtained oyster oligopeptide powder contains peptides with a molecular weight distribution in the range of 500 to 2000 Daltons accounting for more than 80% of the total peptide mass, with a taurine content of not less than 45 mg / g and a zinc content of not less than 80 μg / g. In step P2, the complex protease is composed of trypsin and alkaline protease at an activity ratio of 1:0.6.

[0069] The β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil was prepared by the following steps: B1, Atractylodes macrocephala slices were taken, purified water was added, the material-to-liquid ratio was 1:8, and steam distillation was used to extract for 3 hours. The volatile oil was collected to obtain Atractylodes macrocephala volatile oil; B2, β-cyclodextrin was dissolved in purified water to prepare a 10% (w / w) β-cyclodextrin solution, and stirred at 45°C until completely dissolved; B3, the Atractylodes macrocephala volatile oil was mixed with anhydrous ethanol at a volume ratio of 1:2 to obtain a volatile oil ethanol solution. Under stirring conditions, the volatile oil ethanol solution is slowly added dropwise to the β-cyclodextrin solution, and stirring is continued at 50°C for 3 hours to obtain an inclusion solution; in step B3, the mass ratio of Atractylodes macrocephala volatile oil to β-cyclodextrin is 1:4; in step B4, the inclusion solution is allowed to stand at 4°C for 24 hours, filtered, the precipitate is washed 2 to 3 times with purified water, and vacuum dried at 45°C and a vacuum degree of 0.08 MPa to obtain the Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex.

[0070] The composite extract was prepared by the following steps: F1, Ziziphus jujuba seed, lily bulb, and Poria cocos were taken in a mass ratio of 1:1:1, pulverized to 40 mesh, and mixed to obtain a mixed powder; F2, using a 70% ethanol aqueous solution as solvent, with a material-to-liquid ratio of 1:10, ultrasonic extraction was performed at 45℃ and 300W for 45 minutes, twice, and the extracts were combined; F3, the extract was concentrated under reduced pressure at 65℃ and 0.0875MPa to obtain a concentrated solution; F4, the concentrated solution was redissolved in water and brought to twice the original volume, treated with an ultrafiltration membrane with a molecular weight cutoff of 100 kilodaltons, and the permeate was collected; F5, the permeate was freeze-dried to obtain the composite extract.

[0071] Example 4 In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows: A sleep aid composition containing γ-aminobutyric acid (GABA) comprises excipients and the following raw materials in parts by weight: 5 parts fermented soybean extract, 6.5 parts oyster oligopeptide powder, 2.75 parts GABA, 2.5 parts Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex, 2.5 parts Acanthopanax senticosus extract, and 2 parts compound extract.

[0072] The fermented soybean extract is prepared by the following steps: S1, soybeans are taken, washed, soaked in water at 27.5℃ for 10 hours, drained, steamed for 11.5 minutes, cooled, and ground with water to obtain soybean milk, with a material-to-liquid mass ratio of 1:6.5; S2, isoflavone-converting Slack bacteria are added to the soybean milk at an inoculation amount of 4% of the soybean milk volume, and fermented at 36.5℃ under anaerobic conditions for 28 hours to obtain a primary fermentation broth; S3, Lactobacillus plantarum is added to the primary fermentation broth at an inoculation amount of 3.5% of the primary fermentation broth volume, and fermented at 36.5℃ for 18 hours to obtain a secondary fermentation broth; S4, the secondary fermentation broth is inactivated at 90℃ for 15 minutes, cooled, and then vacuum concentrated to 20% of its original volume at 62.5℃ and a vacuum degree of 0.0875MPa to obtain a concentrated liquid; S5, the concentrated liquid is freeze-dried, pulverized, and passed through a 100-mesh sieve to obtain the fermented soybean extract.

[0073] The isoflavone-converting Slack bacteria culture was prepared by the following method: the isoflavone-converting Slack bacteria were inoculated into a modified GAM liquid medium containing 0.1% daidzein, and anaerobic cultured at 36.5℃ for 36 hours until the OD600 value of the culture reached 1.15, thus obtaining the isoflavone-converting Slack bacteria culture.

[0074] The *Lactobacillus plantarum* bacterial solution is prepared by the following method: *Lactobacillus plantarum* strain is inoculated into MRS liquid medium and cultured at 36.5℃ for 24 hours until the OD600 value of the bacterial solution reaches 1.5, thus obtaining the *Lactobacillus plantarum* bacterial solution.

[0075] The oyster oligopeptide powder is prepared by the following steps: P1, fresh oyster meat is taken, purified water is added to homogenize it, the material-to-liquid ratio is 1:4, and it is treated at 95℃ for 17.5 minutes, then cooled to 50℃; P2, the pH value is adjusted to 7.75, a complex protease is added, and the amount of enzyme added is 3% of the total protein content in the homogenate, based on the total protein content in the homogenate, and enzymatic hydrolysis is performed at 50℃ for 4 hours; P3, the enzyme is inactivated at 90℃ for 17.5 minutes, cooled, and then subjected to hydrolysis at 6℃ and 8000... Centrifuge at r / min for 17.5 minutes and collect the supernatant; P4, treat the supernatant sequentially through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 3000 Daltons, and collect the permeate; P5, treat the permeate through a nanofiltration membrane with a molecular weight cutoff of 500 Daltons, and collect the retentate; P6, freeze-dry the retentate to obtain oyster oligopeptide powder. The obtained oyster oligopeptide powder contains peptides with a molecular weight distribution in the range of 500 to 2000 Daltons accounting for more than 80% of the total peptide mass, with a taurine content of not less than 45 mg / g and a zinc content of not less than 80 μg / g. In step P2, the complex protease is composed of trypsin and alkaline protease at an activity ratio of 1:0.8.

[0076] The β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil was prepared by the following steps: B1, Atractylodes macrocephala slices were taken, purified water was added, the material-to-liquid ratio was 1:8, and steam distillation was used to extract for 4 hours. The volatile oil was collected to obtain Atractylodes macrocephala volatile oil; B2, β-cyclodextrin was dissolved in purified water to prepare a 7.5% (w / w) β-cyclodextrin solution, and stirred at 45°C until completely dissolved; B3, the Atractylodes macrocephala volatile oil was mixed with anhydrous ethanol at a volume ratio of 1:1.5 to obtain an ethanol solution of volatile oil. In step B3, the volatile oil ethanol solution is slowly added dropwise to the β-cyclodextrin solution under stirring conditions, and the mixture is stirred at 45°C for 3 hours to obtain an inclusion solution; in step B4, the mass ratio of Atractylodes macrocephala volatile oil to β-cyclodextrin is 1:6; in step B5, the inclusion solution is allowed to stand at 6°C for 18 hours, filtered, the precipitate is washed 2 to 3 times with purified water, and vacuum dried at 45°C and a vacuum degree of 0.0875 MPa to obtain the Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex.

[0077] The composite extract was prepared by the following steps: F1, Ziziphus jujuba seed, lily bulb, and Poria cocos were taken in a mass ratio of 1:1:1, pulverized to 50 mesh, and mixed to obtain a mixed powder; F2, using a 60% ethanol aqueous solution as solvent, with a material-to-liquid ratio of 1:10, ultrasonic extraction was performed at 50℃ and 225W for 45 minutes, twice, and the extracts were combined; F3, the extract was concentrated under reduced pressure at 57.5℃ and 0.0875MPa to obtain a concentrated solution; F4, the concentrated solution was redissolved in water and brought to a volume of 2.5 times the original volume, treated with an ultrafiltration membrane with a molecular weight cutoff of 100 kilodaltons, and the permeate was collected; F5, the permeate was freeze-dried to obtain the composite extract.

[0078] Compared to Example 1, the oyster oligopeptide powder was replaced with commercially available ordinary oyster protein powder. This ordinary oyster protein powder was not subjected to complex protease hydrolysis and multi-stage membrane separation treatment, and its molecular weight distribution range was wide, with taurine content below 20 mg / g and zinc content below 40 μg / g. The types and amounts of other raw materials were exactly the same as in Example 1, namely 8 parts fermented soybean extract, 0.5 parts γ-aminobutyric acid, 4 parts Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex, 2.5 parts Acanthopanax senticosus extract, and 1 part compound extract. The types and amounts of excipients were also consistent with those in Example 1.

[0079] Compared to Example 1, the fermented soybean extract was replaced with unfermented crude soybean isoflavone extract, which was obtained by reflux extraction of soybeans with a 70% (v / v) ethanol aqueous solution, followed by concentration and drying. This crude extract was not subjected to two-stage fermentation with *S. plantarum* and *Lactobacillus plantarum*, and its equol content was below the detection limit, while its γ-aminobutyric acid (GABA) content was below 5 mg / g. The types and amounts of other raw materials were exactly the same as in Example 1.

[0080] Compared to Example 1, the β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil was replaced with crude Atractylodes macrocephala volatile oil obtained by steam distillation of Atractylodes macrocephala slices. This crude oil was not treated with β-cyclodextrin inclusion, had poor water solubility, and was difficult to disperse evenly in the formulation. The types and amounts of other raw materials were exactly the same as in Example 1.

[0081] Compared with Example 1, the compound extract was omitted, and the types and amounts of the remaining raw materials were exactly the same as in Example 1, namely 8 parts of fermented soybean extract, 6.5 parts of oyster oligopeptide powder, 0.5 parts of γ-aminobutyric acid, 4 parts of Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex, and 2.5 parts of Acanthopanax senticosus extract. The types and amounts of excipients were also consistent with those in Example 1.

[0082] Performance Test Results and Analysis Compositions were obtained and tested according to the parameters of the examples and comparative examples. The sleep-aiding effect was evaluated using a mouse pentobarbital-induced sleep synergistic experiment. Mice in each group were administered the drug by gavage for 14 consecutive days before the experiment. After the last administration, a subthreshold dose of pentobarbital sodium was injected intraperitoneally, and the sleep latency and sleep duration were recorded. Serum growth hormone and cortisol levels in mice were determined using enzyme-linked immunosorbent assay (ELISA). The contents of equol and γ-aminobutyric acid in fermented soybean extract or its substitutes were determined using high-performance liquid chromatography (HPLC). The taurine content in oyster oligopeptide powder or its substitutes was determined using an amino acid analyzer, and the zinc content was determined using atomic absorption spectrometry. The inclusion rate of volatile oil in the β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil or its substitutes was determined using ultraviolet spectrophotometry.

[0083] Table 1 shows the test results of the raw material quality indicators. Figure 1 These are the test results for animal experimental indicators, among which, Figure 1-1 This is the result of a sleep latency test. Figure 1 -2 represents the sleep duration test result. Figure 1 -3 represents the serum growth hormone test result. Figure 1 -4 represents the serum cortisol test result.

[0084] From Table 1 and Figure 1 As can be seen, the equol content in the fermented soybean extracts of Examples 1 to 4 was at a high level, indicating that the two-stage fermentation process of *S. lactis* and *Lactobacillus plantarum* can effectively convert soybean isoflavones into equol and accumulate γ-aminobutyric acid (GABA) during fermentation. In Comparative Example 2, the equol content was only 0.03 mg / g and the GABA content was only 4.2 mg / g, indicating that the unfermented crude soybean isoflavone extract contained almost no equol and lacked the GABA produced during fermentation. This directly led to the poor performance of Comparative Example 2 in subsequent animal experiments. The taurine and zinc contents in the oyster oligopeptide powders of Examples 1 to 4 remained at a high level, with the molecular weight distribution concentrated in the oligomeric range. This was due to the precise control of the directional enzymatic hydrolysis of the complex protease and the multi-stage membrane separation. In Comparative Example 1, the taurine content decreased to 16.5 mg / g and the zinc content decreased to 32.0 μg / g, indicating that the ordinary commercially available oyster protein powder had not undergone directional enrichment treatment, resulting in a significantly insufficient content of active ingredients and making it difficult to exert its receptor regulatory effect. The inclusion rates of the β-cyclodextrin inclusion complexes of Atractylodes macrocephala volatile oil in Examples 1 to 4 were all above 73%, while the inclusion rate of the unincluded volatile oil in Comparative Example 3 was 0, indicating that the inclusion process is a necessary step to obtain stable water-soluble inclusion complexes.

[0085] Table 1. Test Results of Raw Material Quality Indicators Group Estrol content (mg / g) γ-aminobutyric acid content (mg / g) Taurine content (mg / g) Zinc content (μg / g) Inclusion rate % Example 1 2.85 38.5 48.2 86.5 76.5 Example 2 2.55 35.2 45.8 82.0 73.0 Example 3 2.95 40.1 50.5 91.2 79.2 Example 4 3.15 42.8 53.0 95.0 82.5 Comparative Example 1 2.85 38.5 16.5 32.0 76.5 Comparative Example 2 0.03 4.2 48.2 86.5 76.5 Comparative Example 3 2.85 38.5 48.2 86.5 0 Comparative Example 4 2.85 38.5 48.2 86.5 76.5 Regarding sleep latency, Examples 1 through 4 all had relatively short sleep latencies, with Example 4 being the shortest at 4.2 min, followed by Example 1 at 5.2 min, Example 3 at 5.0 min, and Example 2 at 5.8 min. While there were fluctuations among the four examples, all were significantly better than the comparative examples. Comparative Example 1 had a sleep latency of 8.6 min, Comparative Example 2 9.2 min, Comparative Example 3 8.8 min, and Comparative Example 4 9.0 min. The prolonged sleep latency in Comparative Example 1 was due to the lack of targeted enrichment of taurine and zinc in the ordinary oyster protein powder used, which failed to effectively maintain the response level of GABA-A receptors. Furthermore, the insufficient receptor sensitivity of γ-aminobutyric acid (GABA) alone resulted in a slower onset of sedation. Comparative Example 2 had the longest sleep latency because its fermented soybean extract was not transformed by the bacterial strain, lacking equol and fermented GABA, thus failing to alleviate anxiety through endocrine regulation and making it difficult to supplement inhibitory neurotransmitters from the raw material source. In Comparative Example 3, the volatile oil of Atractylodes macrocephala was not encapsulated, resulting in uneven dispersion of fat-soluble components, low bioavailability, and insufficient sedative and tranquilizing activity. In Comparative Example 4, omitting the compound extract resulted in the loss of the rapid sedative effect of components such as jujube seed saponins contained in the compound extract, and the sleep latency was also significantly prolonged.

[0086] Regarding sleep duration, Example 4 achieved 62.0 min, while Examples 1 to 3 achieved 54.5 min, 51.0 min, and 55.2 min, respectively, maintaining a relatively long sleep maintenance time. Comparative Examples 1 to 4 achieved 37.5 min, 35.0 min, 36.2 min, and 36.8 min, respectively, all significantly shorter than the Examples. This difference mainly stems from the synergistic temporal effect of various technical features. In the Examples, γ-aminobutyric acid (GABA) and GABA from fermented soybean extract jointly provide the basis for inhibitory neurotransmitters. Taurine from oyster oligopeptide powder enhances GABA-A receptor sensitivity through positive allosteric regulation, and zinc ions promote endogenous GABA synthesis, thus maintaining nerve impulse inhibition throughout the sleep cycle. The β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil prolongs the release time of the lipid-soluble active ingredient through the sustained-release effect of the cyclodextrin cavity, working with the complex extract to maintain nerve tone during sleep maintenance. The Comparative Examples, lacking one or more of the above technical features, exhibit shortcomings in neurotransmitter supply or receptor regulation, resulting in a correspondingly shorter sleep duration.

[0087] The serum growth hormone secretion level in the examples was higher than that in the control examples, which was directly related to the improvement in deep sleep duration. Growth hormone is mainly secreted in a pulsatile manner during deep sleep. The examples increased the proportion of deep sleep by prolonging sleep duration, thereby promoting growth hormone release. In addition, the active peptides and zinc in oyster oligopeptide powder provided nutritional support for growth hormone synthesis. The extract of Acanthopanax senticosus and oyster oligopeptide powder worked synergistically on the hypothalamus-pituitary axis, further promoting growth hormone secretion. Control example 1 lacked sufficient zinc and active peptides, resulting in insufficient nutritional support for the pituitary axis; Control example 2 lacked equol, leading to an imbalance in the regulation of the hypothalamus-pituitary-adrenal axis and high cortisol levels, which inhibited growth hormone secretion; Control examples 3 and 4 had shortened sleep duration and reduced deep sleep stages, thus limiting the pulsatile secretion of growth hormone.

[0088] The serum cortisol level in Example 4 was 12.5 ng / mL, while in Examples 1 to 3 it was 15.2 ng / mL, 16.5 ng / mL, and 14.8 ng / mL, respectively, all remaining at low levels. Comparative Examples 1 to 4 were all higher than the Examples. Equol reduces cortisol levels by regulating the hypothalamic-pituitary-adrenal axis; in the Examples, the equol provided by the fermented soybean extract played a key role in this process. Comparative Example 2, lacking equol, had the highest cortisol level. The active ingredients in the compound extract also participated in alleviating anxiety and reducing stress response; in Comparative Example 4, omitting this component also significantly increased cortisol levels. The synergistic effect of oyster oligopeptide powder and Acanthopanax senticosus extract helped improve the body's metabolic stress state; in Comparative Example 1, due to insufficient active ingredients, the cortisol-reducing effect was weakened.

[0089] Test results show that this invention pre-converts soybean isoflavones into estrol and enriches γ-aminobutyric acid through a two-stage fermentation process, combines targeted enzymatic hydrolysis membrane separation to enrich taurine and zinc in oyster oligopeptides to maintain GABA-A receptor sensitivity, and supplements it with β-cyclodextrin inclusion complexation of Atractylodes macrocephala volatile oil to improve water solubility and stability, thereby stabilizing the sleep-aiding effect and minimizing individual differences, while also promoting growth and development.

[0090] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A sleep-aiding composition containing γ-aminobutyric acid, characterized in that, This sleep-aid composition includes excipients and the following raw materials in parts by weight: Fermented soybean extract, 2 to 8 parts; Oyster oligopeptide powder, 3 to 10 parts; γ-Aminobutyric acid, 0.5 to 5 parts; Atractylodes macrocephala volatile oil β-cyclodextrin inclusion complex, 1 to 4 parts; Eleutherococcus senticosus extract, 1 to 4 parts.

2. The sleep-aid composition containing γ-aminobutyric acid according to claim 1, characterized in that, The fermented soybean extract was prepared by the following steps: S1: Take soybeans, wash them, soak them in water at 25 to 30°C for 8 to 12 hours, drain them, steam them for 8 to 15 minutes, cool them, add water and grind them into a paste to obtain soy milk. The mass ratio of the material to the liquid is 1:5 to 1:

8. S2: Inoculate the soy milk with isoflavone-converting Slack bacteria solution at an inoculation amount of 2% to 6% of the soy milk volume, and ferment at 35 to 38°C under anaerobic conditions for 20 to 36 hours to obtain a primary fermentation liquid. S3: Inoculate the primary fermentation broth with Lactobacillus plantarum culture at an inoculation amount of 2% to 5% of the volume of the primary fermentation broth, and ferment at 35 to 38°C for 12 to 24 hours to obtain the secondary fermentation broth. S4: The secondary fermentation broth is inactivated at 85 to 95°C for 10 to 20 minutes, cooled, and then vacuum concentrated to 20% of its original volume at 55 to 70°C and a vacuum degree of 0.08 to 0.095 MPa to obtain a concentrated broth; S5: Freeze-dry the concentrated liquid, pulverize it, and pass it through an 80 to 120 mesh sieve to obtain fermented soybean extract.

3. The sleep-aid composition containing γ-aminobutyric acid according to claim 2, characterized in that, The isoflavone-converting Slack bacteria culture is prepared by the following method: inoculating the isoflavone-converting Slack bacteria into a modified GAM liquid medium containing 0.05% to 0.15% daidzein, and anaerobic culturing at 35 to 38°C for 24 to 48 hours until the OD600 value of the culture reaches 0.8 to 1.5, thus obtaining the isoflavone-converting Slack bacteria culture.

4. The sleep-aid composition containing γ-aminobutyric acid according to claim 2, characterized in that, The *Lactobacillus plantarum* bacterial solution is prepared by the following method: *Lactobacillus plantarum* strain is inoculated into MRS liquid medium and cultured at 35 to 38°C for 18 to 30 hours until the OD600 value of the bacterial solution reaches 1.0 to 2.0, thus obtaining the *Lactobacillus plantarum* bacterial solution.

5. The sleep-aid composition containing γ-aminobutyric acid according to claim 1, characterized in that, The oyster oligopeptide powder is prepared by the following steps: P1: Take fresh oyster meat, add purified water to homogenize, the material-to-liquid ratio is 1:3 to 1:5, treat at 90 to 100℃ for 15 to 20 minutes, and cool to 48 to 52℃; P2: Adjust the pH to 7.5 to 8.0, add complex protease, and add 2% to 4% of the total protein in the homogenate. Incubate at 48 to 52°C for 3 to 5 hours. P3: Inactivate enzymes at 85 to 95°C for 15 to 20 minutes, cool, centrifuge at 4 to 8°C and 6000 to 10000 r / min for 15 to 20 minutes, and collect the supernatant; P4: The supernatant is sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 3000 Daltons, and the permeate is collected. P5: The permeate is treated with a nanofiltration membrane with a molecular weight cutoff of 500 Daltons, and the retentate is collected; P6: Freeze-dry the retentate to obtain oyster oligopeptide powder. The obtained oyster oligopeptide powder contains more than 80% peptides with a molecular weight distribution in the range of 500 to 2000 Daltons, taurine content of not less than 45 mg / g, and zinc content of not less than 80 μg / g.

6. The sleep-aid composition containing γ-aminobutyric acid according to claim 5, characterized in that, In step P2, the complex protease is composed of trypsin and alkaline protease in an activity ratio of 1:0.6 to 1:

1.

7. The sleep-aid composition containing γ-aminobutyric acid according to claim 1, characterized in that, The β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil was prepared by the following steps: B1: Take Atractylodes macrocephala slices, add purified water, with a material-to-liquid ratio of 1:6 to 1:10, and extract by steam distillation for 3 to 5 hours. Collect the volatile oil to obtain Atractylodes macrocephala volatile oil. B2: Dissolve β-cyclodextrin in purified water to prepare a 5% to 10% β-cyclodextrin solution, and stir at 40 to 50°C until completely dissolved; B3: Mix the volatile oil of Atractylodes macrocephala with anhydrous ethanol at a volume ratio of 1:1 to 1:2 to obtain a volatile oil ethanol solution. Under stirring conditions, slowly add the volatile oil ethanol solution to the β-cyclodextrin solution and continue stirring at 40 to 50°C for 2 to 4 hours to obtain an inclusion solution. B4: The inclusion solution is allowed to stand at 4 to 8°C for 12 to 24 hours, filtered, the precipitate is washed 2 to 3 times with purified water, and vacuum dried at 40 to 50°C and a vacuum degree of 0.08 to 0.095 MPa to obtain the β-cyclodextrin inclusion complex of Atractylodes macrocephala volatile oil.

8. A sleep-aid composition containing γ-aminobutyric acid according to claim 7, characterized in that, In step B3, the mass ratio of Atractylodes macrocephala volatile oil to β-cyclodextrin is 1:4 to 1:

8.

9. A sleep-aid composition containing γ-aminobutyric acid according to claim 1, characterized in that, The raw materials also contain 1 to 3 parts of a compound extract, which is prepared by the following steps: F1: Take jujube seed, lily bulb and poria cocos in a mass ratio of 1:1:0.5 to 1:1:1.5, grind them separately to 40 to 60 mesh, and mix them to obtain a mixed powder; F2: Using 50% to 70% ethanol aqueous solution as solvent, with a material-to-liquid ratio of 1:8 to 1:12, ultrasonically assisted extraction was performed at 45 to 55°C and ultrasonic power of 150 to 300W for 30 to 60 minutes, 2 to 3 times, and the extracts were combined. F3: The extract is concentrated under reduced pressure at 50 to 65°C and a vacuum of 0.08 to 0.095 MPa to obtain a concentrated solution; F4: The concentrate is reconstituted with water and brought to a volume of 2 to 3 times the original volume. It is then treated with an ultrafiltration membrane with a molecular weight cutoff of 100 kilodaltons, and the permeate is collected. F5: Freeze-dry the permeate to obtain the composite extract.

10. The use of a sleep-aiding composition containing γ-aminobutyric acid as described in any one of claims 1-9, characterized in that, This composition is used in the preparation of products that have the effect of improving sleep.

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