Postbiotic preparation and composition for improving activity of gamma-aminobutyric acid and promoting calcium absorption and application of postbiotic preparation and composition in sleep improvement and bone health

Abstract

The invention belongs to the technical field of microorganisms and compound nutrients, and particularly relates to a postbiotic preparation and composition for improving activity of gamma-aminobutyric acid and promoting calcium absorption and application of the postbiotic preparation and composition in sleep improvement and bone health. Through the synergistic effect of the specific metagen preparation and the gamma-aminobutyric acid, the multi-dimensional technical breakthrough is realized, and the physiological activity and the application value are remarkably improved. In the aspect of bioavailability, the intestinal barrier function is improved and the pH value of the intestinal tract is adjusted to be weakly acidic through the postbiotics, so that the concentration of serum GABA is increased by 2-3 times compared with that of single use, and the calcium absorption rate is increased from 30% to 55-60%. And on the aspect of function synergy, the limitation of single effect in the prior art is broken through, dual-function synergy of'sleep regulation-bone health 'is realized, meanwhile, the vicious circle of'poor sleep-calcium loss' is improved, and good practical application value is achieved.

Description

Technical Field

[0001] This invention belongs to the field of microbial and complex nutrient technology, specifically relating to a post-biotic preparation and composition that enhances γ-aminobutyric acid activity and promotes calcium absorption, and its application in sleep improvement and bone health. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] γ-Aminobutyric acid (GABA), as the main inhibitory neurotransmitter in the central nervous system, has important physiological activities such as sedation, anti-anxiety, and sleep improvement, and is widely used in functional foods, health products, and sleep aids. Calcium is a core nutrient for maintaining bone health, participating in key physiological processes such as bone density maintenance and neuromuscular transmission, and is a basic substance for preventing osteoporosis.

[0004] However, the application of both in the existing technologies has significant drawbacks. GABA has extremely low oral bioavailability and is easily affected by the gastrointestinal environment (such as gastric acid decomposition and intestinal flora metabolism) when supplemented exogenously. High doses (more than 500mg per day) are required to exert a sleep-regulating effect, which not only increases costs but may also cause side effects such as drowsiness and fatigue. Calcium preparations, on the other hand, face the problem of poor absorption efficiency. The absorption rate of inorganic calcium (such as calcium carbonate) is only about 30%, which can easily lead to gastrointestinal discomfort such as constipation and bloating. Although organic calcium (such as calcium citrate) is less irritating, it is expensive, and calcium supplementation alone cannot solve intestinal absorption disorders (such as intestinal function decline in the elderly and insufficient calcium absorption due to vitamin D deficiency).

[0005] More importantly, there is a significant gap in the synergistic application of GABA and calcium in existing technologies. On the one hand, research on their functions has long been limited to a single area (GABA focuses on neural regulation, while calcium focuses on bone health), failing to identify technical solutions linking "sleep improvement" with "bone health," and neglecting the bidirectional regulatory needs of "poor sleep quality leading to increased nighttime calcium loss" and "bone pain affecting sleep." On the other hand, regarding the bioavailability of GABA and calcium, existing technologies mostly employ single methods such as chemical modification and dosage form optimization, failing to recognize the regulatory role of the gut microbiota in their absorption. Although postbiotics have been proven to improve the bioavailability of active ingredients by enhancing intestinal barrier function, regulating intestinal pH, and promoting nutrient transport, there are currently no patents or literature reports on using postbiotics to simultaneously promote the absorption of GABA and calcium, and there is a lack of technical concepts for the synergistic effect of "postbiotic-GABA-calcium." This results in a lack of innovative products on the market that combine "highly effective sleep aid + highly absorbable calcium supplementation," failing to meet consumers' demand for "one-stop health management." Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a post-biotic preparation and composition that enhances γ-aminobutyric acid activity and promotes calcium absorption, and its application in sleep improvement and bone health. The technical problem to be solved by the present invention is achieved through the following technical solution: One aspect of the present invention provides a postbiotic preparation that enhances γ-aminobutyric acid (GABA) activity and promotes calcium absorption, said postbiotic preparation comprising at least (inactivated) Lactobacillus reuteri (…). Lactobacillus reuteri Nice-xt and Lactobacillus reuteri ( Lactobacillus reuteri Nice-06 and its fermentation metabolites.

[0007] Among them, Lactobacillus reuteri Nice-xt is deposited at the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province) on December 24, 2025, with the biological accession number CCTCC NO: M 20253001.

[0008] The Lactobacillus reuteri ( Lactobacillus reuteri Nice-06 is deposited at the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province) on June 29, 2023, with the biological accession number CCTCC NO: M20231123; this strain has been disclosed in Chinese patent CN116814503A.

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned postbiotic preparation, the method comprising: S1. Strain activation: Lactobacillus reuteri Nice-xt and Lactobacillus reuteri Nice-06 were placed in activation medium to obtain pure strains; S2. Preparation of Primary Seed Culture: Select single colonies of *Lactobacillus reuteri* Nice-xt and *Lactobacillus reuteri* Nice-06 obtained in step S1 and place them separately in the first culture medium for static culture to obtain primary seed culture of *Lactobacillus reuteri* Nice-xt and *Lactobacillus reuteri* (… Lactobacillus reuteri Nice-06 Grade 1 Seed Solution; S3. Preparation of secondary seed culture: Combine the above-mentioned Lactobacillus reuteri Nice-xt primary seed culture with Lactobacillus reuteri ( Lactobacillus reuteri The secondary seed culture is obtained by co-inoculating the primary seed culture of Nice-06 into the secondary culture medium and allowing it to stand for culture. S4. Preparation of the third-level seed culture: The second-level seed culture prepared above is inoculated into the third culture medium for fermentation culture.

[0010] A third aspect of the present invention provides the application of the above-mentioned postbiotic preparation in enhancing γ-aminobutyric acid activity and promoting calcium absorption.

[0011] A fourth aspect of the present invention provides a composition comprising at least the above-described postbiotic preparation and γ-aminobutyric acid.

[0012] A fifth aspect of the invention provides the use of the above-described composition in the preparation of products for improving sleep and maintaining bone health.

[0013] Specifically, the improvement of sleep and maintenance of bone health includes sleep quality regulation, anti-anxiety and emotion management, nerve fatigue repair, bone density enhancement, and bone growth and repair.

[0014] The beneficial technical effects of one or more of the above technical solutions are as follows: The aforementioned technical solution achieves multi-dimensional technological breakthroughs through the synergistic effect of specific post-biotics and γ-aminobutyric acid (GABA), significantly enhancing physiological activity and application value. Regarding bioavailability, this invention improves intestinal barrier function and regulates intestinal pH to a slightly acidic level (pH 6.0-6.5) through post-biotics, resulting in a 2-3 times increase in serum GABA concentration compared to using GABA alone, and an increase in calcium absorption rate from 30% to 55%-60%, effectively addressing the industry pain point of "intake ≠ absorption." In terms of functional synergy, it overcomes the limitations of existing technologies with single-effect effects, achieving a dual-function synergy of "sleep improvement" and "bone health." GABA exerts a sedative effect, shortening sleep latency and increasing the proportion of deep sleep, while efficient calcium absorption enhances bone density and improves bone health, simultaneously mitigating the vicious cycle of "poor sleep-calcium loss," demonstrating significant industrialization value. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 This is the lethality curve of 4 dpf zebrafish after incubation of GABA (A) and post-genetic agent (B) samples of the present invention for 24 h.

[0017] Figure 2 This is a graph showing the CI-sleep time improvement rate of the GABA and post-biotic composition of the present invention.

[0018] Figure 3 This is a graph showing the rate of increase in CI-gabra1 expression in the GABA and post-biotic composition of the present invention.

[0019] Figure 4 The changes in body weight and length of mice in different groups according to the present invention.

[0020] Figure 5 The changes in femoral length, bone weight, and bone density in mice of different groups are shown in this invention.

[0021] Figure 6 The changes in serum GABA content in mice of different groups according to the present invention.

[0022] Figure 7 The changes in serum IGF-1 levels in mice of different groups according to the present invention.

[0023] Figure 8 This invention relates to the effect of different concentrations of calcium citrate on cell activity.

[0024] Figure 9 This invention relates to the effect of different concentrations of postbiotics on cell viability.

[0025] Figure 10 The mean fluorescence intensity of FITC-labeled calcium ions in MC3T3-E1 cells was detected by flow cytometry in this invention (**<0.01).

[0026] Figure 11 The fluorescence of Fluo-4 in MC3T3-E1 cells was detected by fluorescence microscopy in this invention (scale bar 20 μm). Detailed Implementation

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0029] In a typical embodiment of the present invention, a postbiotic preparation is provided to enhance γ-aminobutyric acid activity and promote calcium absorption, wherein the postbiotic preparation contains at least (inactivated) Lactobacillus reuteri (… Lactobacillus reuteri --xt and Lactobacillus reuteri ( Lactobacillus reuteri Nice-06 and its fermentation metabolites.

[0030] Among them, Lactobacillus reuteri ( Lactobacillus reuteri Nice-xt, deposited at the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province), on December 24, 2025, with accession number CCTCC NO: M 20253001.

[0031] The Lactobacillus reuteri ( Lactobacillus reuteri Nice-06 is deposited at the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province) on June 29, 2023, with the biological accession number CCTCC NO: M20231123; this strain has been disclosed in Chinese patent CN116814503A.

[0032] In another specific embodiment of the present invention, a method for preparing the above-mentioned postbiotic preparation is provided, the method comprising: S1. Strain activation: Lactobacillus reuteri Nice-xt and Lactobacillus reuteri Nice-06 were placed in activation medium to obtain pure strains; S2. Preparation of Primary Seed Culture: Select single colonies of *Lactobacillus reuteri* Nice-xt and *Lactobacillus reuteri* Nice-06 obtained in step S1 and place them separately in the first culture medium for static culture to obtain primary seed culture of *Lactobacillus reuteri* Nice-xt and *Lactobacillus reuteri* (… Lactobacillus reuteri Nice-06 Grade 1 Seed Solution; S3. Preparation of secondary seed culture: Combine the above-mentioned Lactobacillus reuteri Nice-xt primary seed culture with Lactobacillus reuteri ( Lactobacillus reuteri The secondary seed culture is obtained by co-inoculating the primary seed culture of Nice-06 into the secondary culture medium and allowing it to stand for culture. S4. Preparation of the third-level seed culture: The second-level seed culture prepared above is inoculated into the third culture medium for fermentation culture.

[0033] In step S1, the activation culture medium comprises the following components: yeast peptone 0.2%-1.0%, beef extract 0.3-1.0%, glucose 1.0%-2%, yeast extract 0.1-0.2%, sodium acetate 0.05-0.2%, γ-aminobutyric acid 0.05-0.2%, seaweed calcium 0.05-0.2%, potassium dihydrogen phosphate 0.05-0.2%, agar powder 0.15-0.2%, and pH adjusted to 6.5.

[0034] In step S2, the culture temperature is 28-30℃ and the culture time is 12-14 hours; The first culture medium consists of the following components: 1%-2% glucose, 0.1-0.2% yeast peptone, 0.3-1.0% beef extract, 0.1-0.2% yeast extract, 0.1-0.2% sodium acetate, 0.1-0.2% potassium dihydrogen phosphate, 0.05-0.2% γ-aminobutyric acid, 0.05-0.2% seaweed calcium, and pH adjusted to 6.5.

[0035] In step S3, *Lactobacillus reuteri* Nice-xt primary seed culture and *Lactobacillus reuteri* ( Lactobacillus reuteri The inoculation volume ratio of Nice-06 primary seed solution is 1:0.5-5 (preferably 1:2); the total inoculation amount is controlled at 0.1-5% (preferably 1%). v / v The incubation temperature is 28-35℃ (preferably 30℃), and the culture is carried out statically for 12-14 hours. The composition of the second culture medium is as follows: glucose 1-2%, yeast peptone 1-2%, beef extract 0.1-1%, yeast extract 0.1-1%, γ-aminobutyric acid 0.05-0.2%, Tween-80 0.1-0.2%, seaweed calcium 0.05-0.2%, sodium acetate 0.1-0.2%, potassium dihydrogen phosphate 0.1-0.2%, pH 6.5; In step S4, the inoculation amount is controlled at 0.5-8% (preferably 3%). The composition of the third culture medium is as follows: glucose 1-2%, yeast peptone 1-2%, beef extract 1.5-2%, yeast extract 1.5-2%, γ-aminobutyric acid 0.2-0.4%, Tween-80 0.1-0.2%, seaweed calcium 0.2-0.4%, sodium acetate 0.05-0.2%, potassium dihydrogen phosphate 0.05-0.2%, pH 6.5.

[0036] Specifically, the fermentation method in step S4 includes: culturing at 28 °C, 40-50 r / min (preferably 40 r / min) for 3-4 hours; after 3-4 hours, increasing the stirring speed to 60-80 r / min (preferably 70 r / min), raising the fermentation temperature to 30 °C, maintaining the tank pressure at 0.08 MPa, adjusting the pH to 6.5, and fermenting for 2-3 hours; after 5-7 hours, keeping the stirring speed and temperature constant, raising the tank pressure to 0.1 MPa, adjusting the pH to 6.5 again, and fermenting for 4-5 hours; after 9-12 hours, keeping the stirring speed constant, raising the temperature to 45 °C, adding 0.1-0.2% (preferably 0.1%) of glycerol by volume of the fermentation liquid to the fermenter, and raising the temperature to inactivate the bacterial strain (e.g., maintaining 85 °C for 30 min) to obtain the final product.

[0037] Furthermore, step S4 also includes a step of spray drying the obtained fermentation product.

[0038] In another specific embodiment of the present invention, the above-mentioned postbiotic preparation is provided for its application in improving γ-aminobutyric acid activity and promoting calcium absorption.

[0039] In another specific embodiment of the present invention, a composition is provided, the composition comprising at least the above-mentioned postbiotic preparation and γ-aminobutyric acid.

[0040] The mass ratio of the post-biotic preparation to γ-aminobutyric acid is 1:2 to 1:10, such as 1:1, 1:2, 1:5, 1:8 and 1:10.

[0041] In another specific embodiment of the present invention, the use of the above composition in the preparation of products for improving sleep and maintaining bone health is provided.

[0042] Specifically, the improvement of sleep and maintenance of bone health includes sleep quality regulation, anti-anxiety and emotion management, nerve fatigue repair, bone density enhancement, and bone growth and repair.

[0043] Furthermore, the product may be food or medicine.

[0044] More specifically, the medicine may also include at least one other inactive pharmaceutical ingredient.

[0045] The inactive pharmaceutical ingredient can be a carrier, excipient, or diluent commonly used in pharmaceuticals. Furthermore, it can be formulated into oral dosage forms such as powders, granules, suspensions, emulsions, syrups, and sprays using conventional methods. The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0046] In another specific embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil, etc.

[0047] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0048] In each embodiment, the composition of the activation culture medium is as follows: 0.5% yeast peptone, 1.0% beef extract, 2% glucose, 0.2% yeast extract, 0.05% sodium acetate, 0.2% γ-aminobutyric acid, 0.2% seaweed calcium, 0.05% potassium dihydrogen phosphate, 0.2% agar powder, and pH adjusted to 6.5.

[0049] The first culture medium consisted of: 2% glucose, 0.2% yeast peptone, 1.0% beef extract, 0.2% yeast extract, 0.2% sodium acetate, 0.2% potassium dihydrogen phosphate, 0.2% γ-aminobutyric acid, and 0.2% seaweed calcium, with the pH adjusted to 6.5.

[0050] The second culture medium consisted of: 2% glucose, 2% yeast peptone, 0.5% beef extract, 0.5% yeast extract powder, 0.1% γ-aminobutyric acid, 0.1% Tween-80, 0.1% seaweed calcium, 0.1% sodium acetate, 0.1% potassium dihydrogen phosphate, and pH 6.5.

[0051] The composition of the third culture medium is as follows: glucose 2%, yeast peptone 2%, beef extract 1.5%, yeast extract 1.5%, γ-aminobutyric acid 0.3%, Tween-80 0.2%, seaweed calcium 0.3%, sodium acetate 0.1%, potassium dihydrogen phosphate 0.2%, pH 6.5.

[0052] The percentage of each component in the culture medium is a mass-volume percentage, expressed in g / mL.

[0053] Example 1 A method for preparing an epigenetic agent includes the following steps: Lactobacillus reuteri Nice-xt and Lactobacillus reuteri Nice-06 were streaked onto the activation medium to obtain pure strains; single colonies of Lactobacillus reuteri were picked and placed in the first culture medium at 30°C for 14 hours to obtain primary seed culture. The primary seed culture was prepared at a ratio of 1:2 (Lactobacillus reuteri Nice-xt: Lactobacillus reuteri Nice-06, v / v The proportion of 1% of the total inoculum is inoculated into the second culture medium and incubated at 30 °C for 14 hours to obtain the secondary seed culture. The above-mentioned Lactobacillus reuteri secondary seed culture was inoculated into a fermenter containing sterilized third culture medium at an inoculation rate of 3%. The culture was incubated at 28 °C and 45 r / min for 3 hours. After 3 hours, the stirring speed was increased to 70 r / min, the fermentation temperature was increased to 30 °C, the tank pressure was maintained at 0.08 MPa, and the pH was adjusted to 6.5 using ammonia. Fermentation continued for 3 hours. After 6 hours, the stirring speed and temperature remained constant, the tank pressure was increased to 0.1 MPa, and the pH was adjusted to 6.5 using ammonia. Fermentation continued for 5 hours. After 11 hours, the stirring speed remained constant, the temperature was increased to 45 °C, and 0.1% of the fermentation liquid volume of glycerol was added to the fermenter. The temperature was increased to 85 °C and maintained for 30 min before spray drying to obtain the post-biotic preparation.

[0054] Example 2 A method for preparing a GABA postbiotic composition includes the following steps: Lactobacillus reuteri Nice-xt and Lactobacillus reuteri Nice-06 were streaked onto the activation medium to obtain pure strains; single colonies of Lactobacillus reuteri were picked and placed in the first culture medium at 30 ℃ for 14 hours to obtain the first seed culture. The primary seed culture was prepared at a ratio of 1:2 (Lactobacillus reuteri Nice-xt: Lactobacillus reuteri Nice-06, v / v The proportion of 1% of the total inoculum is inoculated into the second culture medium and incubated at 30 °C for 14 hours to obtain the secondary seed culture. The above-mentioned *Lactobacillus reuteri* was inoculated into a fermenter containing sterilized culture medium at an inoculation rate of 3%, and cultured at 28 °C and 45 r / min for 3 hours. After 3 hours, the stirring speed was increased to 70 r / min, the fermentation temperature was increased to 30 °C, the tank pressure was maintained at 0.08 MPa, and the pH was adjusted to 6.5 using ammonia water, and fermentation continued for 3 hours. After 6 hours, the stirring speed and temperature remained unchanged, the tank pressure was increased to 0.1 MPa, the pH was adjusted to 6.5 using ammonia water, and fermentation continued for 5 hours. After 11 hours, the stirring speed remained unchanged, the temperature was increased to 45 °C, 0.1% of the fermentation liquid volume of glycerol was added to the fermenter, the temperature was increased to 85 °C and maintained for 30 min, and then spray-dried to obtain the post-biotic preparation.

[0055] The post-biotic preparation was mixed with GABA powder and mixed in a three-dimensional mixer at 100 r / min for 60 minutes to obtain the GABA post-biotic composition.

[0056] Effect verification The composition that synergistically enhances the sleep-improving effect of GABA is validated by the following method: (1) Determination of MTC Wild AB zebrafish with normal development (4 dpf) were used as experimental animals. Different concentrations of post-genetic agents and GABA were added to the samples, and the samples were cultured in a constant temperature incubator at 28℃ for 24 h. The LC10 and LC50 values ​​of the two samples were calculated.

[0057] like Figure 1 As shown, the LC10 and LC50 of GABA were 0.84% ​​and 1.74% of the original sample concentration, respectively, which translates to a final concentration below the maximum non-toxic dose of 400 μg / mL. The LC10 and LC50 of post-biotics were 1.91% and 3.87% of the original sample concentration, respectively, which translates to a final concentration below the maximum non-toxic dose of 500 μg / mL.

[0058] (2) Effects of combined use of post-biotics and GABA on arousal activity in zebrafish Normal zebrafish were selected under a stereomicroscope, and a blank control group was randomly set up. Then, they were placed in a light incubator and continuously illuminated at 4000 lux for 24 hours to build a model. They were then randomly divided into a model group and a GABA and post-biotic combination group (2+2, 4+2, 10+2, 40+5, 100+10 μg / mL, i.e., mass ratios of 1:1, 2:1, 5:1, 8:1 and 10:1), with 8 fish in each group and 3 replicates.

[0059] Zebrafish juveniles treated with drugs at 5 dpf were subjected to a behavioral experiment on arousal activity level at 10:00 AM at 6 dpf. Each group of juveniles was placed in a 48-well plate, one fish per well, with 8 fish per group, for a total of 5 groups. The 48-well plate was placed in a zebrafish behavior analyzer (Viewpoint Zebralab). Arousal activity time, swimming distance, and sleep latency were detected using the Zeblalab zebrafish behavior analysis system. The method is as follows: after placing the juveniles in the dark chamber of the behavior analyzer, they were allowed to adapt for 15 minutes. Data was collected in a quiet environment using the behavior analyzer. The experiment lasted until 9:00 AM on the third day, with darkness provided from 11:00 PM to 9:00 AM and 50% light from 9:00 AM to 11:00 PM. Data was collected every 10 minutes. The maximum detection threshold (2000) and the minimum detection threshold (2) were set. After setting the behavioral parameters, the data were collected and output after 48 hours of continuous monitoring.

[0060] The test results showed that the daytime activity levels of zebrafish in each group were significantly higher than at night, exhibiting a clear diurnal rhythm. On the first day, the model group, due to light deprivation, had significantly lower daytime activity levels than the normal control group. The activity levels of the metabiotic-GABA group and the combined group were significantly higher than the model group. On the first night, the model group, due to sleep deprivation, had significantly higher nighttime activity levels than the control group. The activity levels of the metabiotic-GABA combined group were significantly lower than the model group, with the optimal ratio of GABA:metabiotic being 2:1. On the second day, the model group's activity levels remained significantly lower than the control group, while the metabiotic-GABA combined group showed an increase compared to the model group. On the second night, the model group's nighttime activity levels remained significantly higher than the control group, while the activity levels of the metabiotic-GABA combined group were significantly lower than the model group, with the optimal ratio of GABA:metabiotic being 2:1. The results in summary indicate that the combined use of metagenerol and GABA has a certain effect on improving sleep in zebrafish, and no drowsiness was observed as a side effect. Further studies will select a GABA:metagen ratio of 2:1 to further evaluate the synergistic effect.

[0061] (3) Effects of the synergistic effect of metagenin and GABA on the level of arousal activity in zebrafish Normal zebrafish were selected under a stereomicroscope, and a blank control group was randomly set up. They were then placed in a light incubator and continuously illuminated at 4000 lux for 24 hours to construct a model. Afterward, they were randomly divided into a model group, a GABA group, a metatrophic factor group, and a GABA + metatrophic factor group. Normal group: normal zebrafish, without GABA or metatrophic factor; Model group: zebrafish with sleep disorders, without GABA or metatrophic factor; GABA group: final mass and concentration gradients were set to 300, 200, 150, 100, and 20 μg / mL; Metatrophic factor final mass and concentration gradients were set to 150, 100, 75, 50, and 10 μg / mL; GABA + metatrophic factor group: final mass and concentration gradients were set to 300+150, 200+100, 150+75, 100+50, and 20+10 μg / mL. Eight fish were used in each group, with three replicates, according to the drug requirements of each experiment. Based on the above method, the wakefulness time, swimming distance, and sleep latency were detected using the Zeblab zebrafish behavior analysis system, and the sleep improvement rate was calculated. Sleep improvement rate = (sleep time in the drug-treated group - sleep time in the model group) / sleep time in the model group × 100%. Then, Real-time qPCR was used to detect the expression of gabral sleep-related genes, and the gene expression improvement was calculated. Gene expression improvement rate = (relative mRNA expression level in the drug-treated group - relative mRNA expression level in the model group) / relative mRNA expression level in the model group × 100%. Finally, the synergistic effect was evaluated by calculating the synergistic index (CI) based on the Chou-Talalay synergistic index method.

[0062] The simulation equations for the effect of GABA on sleep duration improvement were: y = 0.0921x + 5.2162 (R² = 0.9915); for the effect of post-biotics on sleep duration improvement, y = 0.1894x + 4.0131 (R² = 0.9961); and for the effect of the GABA + post-biotic combination on sleep duration improvement, y = 0.1304x + 2.481 (R² = 0.9965). Based on the Chou-Talalay equation CI = D1 / D1x + D2 / D2x, the synergistic index CI of GABA and post-biotics was calculated, where D1 and D2 are the concentrations of GABA and post-biotics respectively when the sleep duration improvement reaches a certain level, and D1x and D2x are the concentrations of GABA and post-biotics used alone to achieve the above effect. The synergistic inhibitory effect of the two (mass ratio 2:1) on sleep duration improvement was evaluated. When CI < 1, the combined effect of GABA and post-genetic agents is a synergistic effect; when CI = 1, the combined effect of GABA and post-genetic agents is an additive effect; when CI > 1, the combined effect of GABA and post-genetic agents is an antagonistic effect.

[0063] (4) Effects of post-genetic synergy on the expression of sleep-related genes gabra1 The gene encodes the α1 subunit of the GABAA receptor, a key inhibitory neurotransmitter receptor component in the central nervous system. It mediates chloride ion influx by binding to GABA, reducing neuronal excitability and playing a central role in maintaining the excitation-inhibition balance, a crucial foundation for regulating sleep. The normality of its expression level directly affects the normal rhythm and quality of sleep. Therefore, we further investigated the effects of combined use of post-biotics and GABA on... gabra1 The impact on gene expression.

[0064] Compared with the control group, the zebrafish tissue in the model group had... gabra1 The relative expression levels of mRNA were significantly reduced in the GABA group, post-genetic group, and combined treatment group compared to the model group. gabra1 The relative expression levels of GABA mRNA increased in all treatment groups, with a significant increase observed in the combined treatment group. These results indicate that each treatment group improved sleep in zebrafish by upregulating the expression level of GABA receptor mRNA. Based on these results, GABA's effect on... gabra1 The simulation equation for the rate of increase in expression level is: y = 0.0861x + 8.5474 (R² = 0.9982); postgenetic pairs gabra1 The simulation equation for the increase in expression level is: y = 0.1357x + 6.148 (R² = 0.9949); GABA + post-biotic combination on gabra1The simulation equation for the increase in expression level is: y = 0.1215x + 2.745 (R² = 0.996). Based on the Chou-Talalay equation CI = D1 / D1x + D2 / D2x, the synergistic index CI of GABA and its post-synergist is calculated, where D1 and D2 represent the synergistic effect of the GABA and post-synergist combination. gabra1 The concentrations of GABA and its derivatives when expression levels increase to a certain extent are represented. D1x and D2x represent the concentrations of GABA and its derivative used alone to achieve the above effects. The synergistic inhibitory effect of the two (mass ratio 2:1) on the increase of gabra1 expression levels was evaluated. When CI < 1, the combined effect of GABA and its derivative was synergistic; when CI = 1, the combined effect of GABA and its derivative was additive; when CI > 1, the combined effect of GABA and its derivative was antagonistic.

[0065] The verification method for its application in the field of bone health is as follows: Forty one-month-old SPF-grade male and female mice were randomly divided into a blank control group and three experimental groups (n=10 per group). After acclimatization for 5 days, gavage treatment began. The blank control group was administered physiological saline by gavage, while the experimental groups were administered post-biotic, GABA, and GABA+post-biotic by gavage, respectively, once daily. The gavage doses were post-biotic 10 mg / kg, GABA 20 mg / kg, and GABA+post-biotic (20+10) mg / kg, respectively, for 4 weeks. During the feeding period, the mice's body weight and body length were monitored weekly. At the end of the experiment, femur length, bone weight, and bone mineral density levels were measured. Serum insulin-like growth factor-1 (IGF-1) and GABA levels were also investigated.

[0066] GABA and metagenerogenes, both alone and in synergy, can promote body length growth. The synergistic effect of GABA and metagenerogenes results in the highest increase in body length. GABA and metagenerogenes, both alone and in synergistic effect, have no significant effect on mouse body weight. Figure 4 Further increases in femoral length, bone weight, and bone mineral density were observed in both the GABA and metatrophic factor groups alone and in the GABA and metatrophic factor synergistic group, with the most significant increases observed in the GABA and metatrophic factor synergistic group. Figure 5 Furthermore, metabiotics can synergistically promote GABA absorption, with the absorption rate of GABA being 2-3 times higher than that of the GABA-only group. Figure 6 ).

[0067] Serum insulin-like growth factor-1 (IGF-1) is a growth-promoting polypeptide hormone, also known as growth mediator C. Its core functions include acting as a major mediator of growth hormone, promoting linear bone growth and soft tissue proliferation in children and adolescents, maintaining bone mineral density and muscle mass in adults, lowering blood sugar by promoting glucose uptake and utilization, promoting protein synthesis and inhibiting breakdown, accelerating fat breakdown, and participating in cell proliferation, differentiation, and apoptosis, playing a role in tissue repair and tumorigenesis. Further analysis of serum IGF-1 levels revealed that both GABA and post-adrenergic receptors, individually and synergistically, increased serum IGF-1 levels, promoting bone growth and bone mineral density in mice. The synergistic group of GABA and post-adrenergic receptors showed the best effect in promoting height, bone weight, and upregulating IGF-1. Figure 7 ).

[0068] The verification method for the composition that synergistically enhances calcium absorption is as follows: Mouse embryonic osteoblast precursor cells MC3T3-E1 were selected as an in vitro model and cultured in a specific medium at 37℃ and 5% CO2. First, the well-tolerated drug concentrations were screened using the CCK-8 assay: calcium citrate (10, 30, 60, 80, 100 μg / mL) and post-genetic inhibitors (2, 4, 6, 8, 10 μg / mL). Then, the cells were divided into groups: a blank control group (medium only), calcium citrate groups (30 μg / mL, 60 μg / mL), and calcium citrate + post-genetic inhibitor groups (30 + 6 μg / mL, 60 + 6 μg / mL). Each group was cultured at 5.0 × 10⁻⁶ cells / mL. 5 Cells were seeded into 6-well plates and cultured for 21 days after reaching 80% confluence (with medium changes every 2-3 days). After culture, the following indicators were measured: ① cell viability (CCK-8); ② expression of osteogenic differentiation-related genes (Q-PCR detection of RUNX2, Col1a1, and Osterix mRNA); ③ calcium uptake capacity (fluo-4 AM fluorescent probe combined with flow cytometry and fluorescence microscopy to detect intracellular calcium ion fluorescence intensity).

[0069] Experimental results show that ( Figure 8 When the concentration of calcium citrate was 0-100 μg / mL, the effect on the activity of MC3T3-E1 cells was not significant; the activity of MC3T3-E1 cells was highest when the concentration of calcium citrate was 60 μg / mL; when the concentration of metagenerogen was 4-10 μg / mL, the activity of MC3T3-E1 cells tended to increase with increasing concentration, indicating that the cells tolerated the above different drug concentrations well. Figure 9 ). Further gene results are shown in Table 1. Compared with the calcium citrate group, the calcium citrate + post-biotic group showed highly significant differences in promoting the gene expression of RUNX2, Col1a1, and Osterix. When the calcium citrate + post-biotic group was 30 ± 6 μg / mL, the expression levels of RUNX2, Col1a1, and Osterix increased by 19.9%, 22.9%, and 26.5%, respectively. The results indicate that post-biotic intervention can significantly increase the expression of osteogenic differentiation-related genes in MC3T3-E1 cells.

[0070] Table 1. Results of RT-qPCR detection of mRNA expression of RUNX2, Col1a1, and Osterix

[0071] Note: Experimental groups A1 and A2 refer to 30 μg / mL calcium citrate and 60 μg / mL calcium citrate, respectively; Experimental groups B1 and B2 refer to 30 μg / mL calcium citrate + 6 μg / mL post-biotic and 60 μg / mL calcium citrate + 6 μg / mL post-biotic, respectively. Further analysis of the calcium uptake capacity of the 30±6 μg / mL combination was conducted. Fluo-4 AM fluorescent probe was added to MC3T3-E1 cells cultured for 21 days, and the fluorescence intensity of calcium ions in the cells was detected by flow cytometry and fluorescence microscopy. Target cell population analysis was performed after gating by flow cytometry, and the experimental results are as follows: Figure 10 As shown in the figure. Compared with the control group and the calcium citrate group, the number of cells and the average fluorescence intensity of the calcium citrate + post-genetic group were significantly increased, and the average fluorescence intensity of intracellular calcium ions was significantly increased by 22.4% compared with the calcium citrate group (P<0.01).

[0072] Figure 11 Further fluorescence microscopy images revealed weak, scattered fluorescence signals in the control group; moderate fluorescence intensity in the calcium citrate group; and strong, uniformly distributed fluorescence signals throughout the cells in the calcium citrate + metatrophic agent group, indicating increased calcium uptake and intracellular calcium reserves. These results suggest that metatrophic agents promote calcium uptake by pre-osteoblasts.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A post-biotic preparation that enhances γ-aminobutyric acid activity and promotes calcium absorption, characterized in that, The postbiotic preparation contains at least (inactivated) Lactobacillus reuteri (…). Lactobacillus reuteri Nice-xt and Lactobacillus reuteri ( Lactobacillus reuteri Nice-06 and its fermentation metabolites; The Lactobacillus reuteri ( Lactobacillus reuteri Nice-xt, deposited at the China Center for Type Culture Collection on December 24, 2025, with accession number CCTCC NO: M 20253001.

2. The method for preparing the post-biotic preparation according to claim 1, characterized in that, The preparation method includes: S1. Strain activation: Lactobacillus reuteri Nice-xt and Lactobacillus reuteri Nice-06 were placed in activation medium to obtain pure strains; S2. Preparation of Primary Seed Culture: Select single colonies of *Lactobacillus reuteri* Nice-xt and *Lactobacillus reuteri* Nice-06 obtained in step S1 and place them separately in the first culture medium for static culture to obtain primary seed culture of *Lactobacillus reuteri* Nice-xt and *Lactobacillus reuteri* (… Lactobacillus reuteri Nice-06 Grade 1 Seed Solution; S3. Preparation of secondary seed culture: Combine the above-mentioned Lactobacillus reuteri Nice-xt primary seed culture with Lactobacillus reuteri ( Lactobacillus reuteri The secondary seed culture is obtained by co-inoculating the primary seed culture of Nice-06 into the secondary culture medium and allowing it to stand for culture. S4. Preparation of the third-level seed culture: The second-level seed culture prepared above is inoculated into the third culture medium for fermentation culture.

3. The preparation method according to claim 2, characterized in that, In step S1, the composition of the activation culture medium is as follows: yeast peptone 0.2%-1.0%, beef extract 0.3-1.0%, glucose 1.0%-2%, yeast extract 0.1-0.2%, sodium acetate 0.05-0.2%, γ-aminobutyric acid 0.05-0.2%, seaweed calcium 0.05-0.2%, potassium dihydrogen phosphate 0.05-0.2%, agar powder 0.15-0.2%, and pH adjusted to 6.

5.

4. The preparation method according to claim 2, characterized in that, In step S2, the culture temperature is 28-30℃ and the culture time is 12-14 hours; The first culture medium consists of the following components: glucose 1.0%-2%, yeast peptone 0.1-0.2%, beef extract 0.3-1.0%, yeast extract 0.1-0.2%, sodium acetate 0.1-0.2%, potassium dihydrogen phosphate 0.1-0.2%, γ-aminobutyric acid 0.05-0.2%, seaweed calcium 0.05-0.2%, and pH adjusted to 6.

5.

5. The preparation method according to claim 2, characterized in that, In step S3, *Lactobacillus reuteri* Nice-xt primary seed culture and *Lactobacillus reuteri* ( Lactobacillus reuteri The inoculation volume ratio of Nice-06 primary seed culture is 1:0.5-5; the total inoculation amount is controlled at 0.1-5%; the culture temperature is 28-35 ℃ (preferably 30 ℃), and the culture is allowed to stand for 12-14 hours. The composition of the second culture medium is as follows: glucose 1-2%, yeast peptone 1-2%, beef extract 0.1-1%, yeast extract 0.1-1%, γ-aminobutyric acid 0.05-0.2%, Tween-80 0.1-0.2%, seaweed calcium 0.05-0.2%, sodium acetate 0.1-0.2%, potassium dihydrogen phosphate 0.1-0.2%, pH 6.

5.

6. The preparation method according to claim 2, characterized in that, In step S4, the inoculation amount is controlled at 0.5-8%; The composition of the third culture medium is as follows: glucose 1-2%, yeast peptone 1-2%, beef extract 1.5-2%, yeast extract 1.5-2%, γ-aminobutyric acid 0.2-0.4%, Tween-80 0.1-0.2%, seaweed calcium 0.2-0.4%, sodium acetate 0.05-0.2%, potassium dihydrogen phosphate 0.05-0.2%, pH 6.5; Further, the fermentation method in step S4 includes: culturing at 28 °C and 40-50 r / min for 3-4 hours; after 3-4 hours, increasing the stirring speed to 60-80 r / min, raising the fermentation temperature to 30 °C, maintaining the tank pressure at 0.08 MPa, adjusting the pH to 6.5, and fermenting for 2-3 hours; after 5-7 hours, keeping the stirring speed and temperature constant, raising the tank pressure to 0.1 MPa, adjusting the pH to 6.5 again, and fermenting for 4-5 hours; after 9-12 hours, keeping the stirring speed constant, raising the temperature to 45 °C, adding 0.1-0.2% of the fermentation liquid volume of glycerol to the fermenter, and raising the temperature to inactivate the bacterial strain to obtain the final product.

7. The preparation method according to claim 2, characterized in that, Step S4 further includes spray drying the obtained fermentation product.

8. The application of the post-biotic preparation according to claim 1 in improving γ-aminobutyric acid activity and promoting calcium absorption.

9. A composition, characterized in that, The composition comprises at least the post-biotic preparation of claim 1 and γ-aminobutyric acid; Furthermore, the mass ratio of the post-biotic preparation to γ-aminobutyric acid is 1:2 to 1:

10.

10. The use of the composition of claim 9 in the preparation of products for improving sleep and maintaining bone health; Furthermore, the improvement of sleep and maintenance of bone health specifically includes sleep quality regulation, anti-anxiety and mood management, nerve fatigue repair, bone density enhancement, and bone growth and repair.

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