Microbial agent compounded GABA (gamma-aminobutyric acid) composition and application thereof in promoting growth and development of children

Abstract

The invention belongs to the field of functional composite microbial preparations, and particularly discloses a microbial agent and GABA compounded composition and application thereof in promoting growth and development of children. The composition is prepared from a freeze-dried bacterial agent and a GABA (gamma-aminobutyric acid) compound, wherein the freeze-dried bacterial agent comprises lactobacillus johnsonii YH1136 and lactobacillus helveticus Helveticus; the GABA compound is prepared by loading GABA with the purity of greater than or equal to 98% on a nano cellulose carrier, and the loading rate is 20-35%. The composition realizes high survival rate of the microbial inoculum and targeted slow release of GABA through a freeze-drying process and enteric coating. Experiments show that the composition can significantly improve the serum GH / IGF-1 level, the bone mineral density and the body growth rate, and has a synergistic effect when combined with the vitamin D3. According to the invention, the problems of single strain function, low GABA utilization rate and gastric acid damage in the prior art are solved, and an efficient and safe solution is provided for children growth and development intervention.

Description

Technical Field

[0001] The present invention belongs to the field of functional compound microbial agents, and specifically discloses a composition of a microbial agent compounded with GABA and its use in promoting children's growth and development. Background Art

[0002] Regarding the methods for promoting children's height, traditional intervention means mainly focus on the supplementation of nutrients such as calcium and vitamin D3. For example, Chinese invention patent CN102908359A discloses a compound calcium carbonate / vitamin D3 granule for children, which improves the calcium absorption rate by optimizing the excipient ratio. However, it only targets mineral metabolism and does not involve the synergistic effect of the gut microbiota and the neuroendocrine axis, making it difficult to solve core problems such as insufficient growth hormone secretion or low intestinal nutrient absorption efficiency. In addition, free calcium supplementation may cause gastrointestinal discomfort, and the lack of targeted delivery design leads to limited bioavailability.

[0003] In recent years, the gut microbiota regulation strategy has provided a new direction for children's height management. Research has shown that probiotics can promote the secretion of growth hormone (GH) and bone metabolism through the dual mechanisms of the "gut-brain axis" and the "gut-bone axis". For example, Bifidobacterium animalis BL-11, which is highly productive in γ-aminobutyric acid (GABA), has been proven to increase the GH level. Clinical trials have shown that its intervention for 12 weeks can increase children's height by 2.6 cm. However, existing technologies mostly rely on single strains (such as BL-11 or Lactobacillus rhamnosus GG), with problems such as single function, limited GABA production, and lack of a targeted delivery system. In addition, free GABA is easily destroyed by gastric acid, resulting in an actual absorption rate of less than 30%, severely restricting its growth-promoting effect.

[0004] To address the above technical bottlenecks, in recent years, researchers have attempted to improve the intervention effect by strain compounding and delivery system optimization. For example, Lactobacillus johnsonii YH1136 has been proven to indirectly improve the utilization rate of the GABA precursor glutamate by inhibiting the expression of the key enzymes IDO1 / TDO2 in tryptophan metabolism and reducing the consumption of the kynurenine pathway. However, its regulatory effect on bone development is weak when applied alone. Although existing technologies have proposed combination schemes of probiotics and nutrients, they have not solved core problems such as poor strain functional synergy and low GABA delivery efficiency, and lack quantitative synergy effect data verified by animal experiments. Therefore, developing a composition that combines functional strain compounding, GABA targeted delivery, and process stability optimization has become the key direction to break through the limitations of existing technologies. Summary of the Invention

[0005] To solve the above problems, the present invention discloses a composition of a microbial agent compounded with GABA and its use in promoting children's growth and development.

[0006] The present invention includes the following technical solutions: A composition of a microbial inoculant compounded with GABA, which consists of the following components: Freeze-dried inoculant: containing Lactobacillus johnsonii ( Lactobacillus johnsonii ) YH1136 and Lactobacillus helveticus Helveticus , and the viable bacteria ratio of the two is 1:1 to 1:3; γ-aminobutyric acid (GABA) complex: GABA (purity ≥ 98%) is loaded on a nanofibrillated cellulose (CNF) carrier, and the mass ratio of GABA to CNF is 1:2 to 1:5; Among them, in each gram of the said composition: The total viable bacteria amount of the freeze-dried inoculant is 1×10^9 to 5×10^10 CFU, The content of GABA in the GABA complex is 100 to 300 mg.

[0007] Furthermore, for the above composition of a microbial inoculant compounded with GABA, the said freeze-dried inoculant is obtained by the following steps: (i) Ferment and culture Lactobacillus johnsonii YH1136 and Lactobacillus helveticus Helveticus respectively until the logarithmic growth phase, centrifuge to collect the bacterial sludge, and wash it 2 - 3 times with sterile physiological saline; (ii) Mix the washed bacterial sludge with a freeze-drying protectant at a volume ratio of 1:1 to 1:2. The said freeze-drying protectant contains 8% - 12 wt% of trehalose and 5 wt% - 8 wt% of skim milk powder. After pre-freezing at -80°C for 12 - 24 hours, carry out vacuum freeze-drying. The said vacuum freeze-drying process is divided into three stages: the first stage is maintained at -45°C to -40°C for 8 - 12 hours, the second stage is heated to -35°C to -30°C and maintained for 10 - 14 hours, and the third stage is heated to 25°C to 30°C and maintained for 2 - 4 hours, finally obtaining a freeze-dried inoculant with a survival rate ≥ 95%.

[0008] Furthermore, for the above composition of a microbial inoculant compounded with GABA, prebiotic galactooligosaccharide (GOS) is also added to the freeze-drying protectant in the said step (ii), and the addition amount of GOS is 5 wt% - 15 wt% of the total mass of the freeze-dried inoculant, and the degree of polymerization (DP) is 2 - 8.

[0009] Furthermore, for the above composition of a microbial inoculant compounded with GABA, 0.01 wt% - 0.05 wt% of the quorum sensing molecule AI-2 is also added to the freeze-drying protectant in the said step (ii).

[0010] Furthermore, for the above composition of a microbial inoculant compounded with GABA, the said γ-aminobutyric acid (GABA) complex is prepared by the following steps: (i) Dispersed nanocellulose (CNF) in phosphate buffer solution with a concentration of 1% - 3% (w / v) and a pH of 6.0 - 7.5, and the volume of the buffer solution is 10 - 20 times (mL / g) the mass of the nanocellulose; (ii) Added acid-resistant β-glucosidase to the dispersion in step (i), and the addition amount of the enzyme is 200 - 500 U per gram of nanocellulose; (iii) Added GABA, and the mass ratio of GABA to nanocellulose is 1:2 - 1:5; (iv) Reacted at 45 - 55 °C and a stirring rate of 100 - 200 rpm for 4 - 8 hours, so that GABA was loaded on the nanocellulose by physical adsorption or hydrogen bond binding; (v) Centrifuged at 3000 - 5000 rpm for 5 - 10 minutes to remove free GABA, and collected the precipitate; (vi) Pre-froze the precipitate at -40 - -50 °C for 2 - 4 hours and then freeze-dried it under vacuum for 12 - 24 hours to obtain the GABA-CNF complex, and the GABA loading rate of the complex is 20% - 35% (w / w).

[0011] The present invention also discloses the use of the above composition in the preparation of products for promoting children's growth and development.

[0012] Further, in the above use, the dosage form of the product is enteric-coated granules, instant powder or chewable tablets, and the dissolution pH threshold of the enteric coating is 5.5 - 6.5.

[0013] Further, in the above use, the dosage form of the product is enteric-coated granules, which are prepared by the following steps: First, mixed the above composition with pharmaceutically acceptable excipients to form the core of the granules; Second, prepared the enteric coating solution: dissolved hydroxypropyl methylcellulose phthalate (HPMCP), an enteric material, in water to prepare a coating solution with a concentration of 8% - 12% (w / v); Third, adopted the fluidized bed coating process to uniformly spray the coating solution in step two on the surface of the core of the granules, and controlled the coating weight gain to be 5% - 10%; Fourth, dried the product by gradient heating at 40 - 45 °C until the water content of the granules ≤ 5% to obtain the enteric-coated granules; Among them, the dissolution pH threshold of the enteric coating is 5.5 - 6.5.

[0014] The present invention also discloses the use of the above composition in combination with vitamin D3 in the preparation of products for promoting children's growth and development.

[0015] Preferably, the product can be a drug or a nutritional supplement for treatment.

[0016] The present invention also discloses a children's nutritional supplement set product, comprising: (1) The above composition of microbial inoculant compounded with GABA; (2) Vitamin D3.

[0017] The present invention also discloses a further enhanced children's nutritional supplement set product, comprising: (1) The above composition of microbial inoculant compounded with GABA; (2) Vitamin D3; (3) Milk calcium; (4) A composition of cartilage extract and collagen peptide.

[0018] By adding cartilage extract and collagen peptide in the present invention, teenagers have good intestinal absorption, can supplement more sufficient nutrition, improve physical fitness and immunity, and promote growth and development.

[0019] Compared with the prior art, the present invention has the following beneficial effects: Through strain compounding, GABA targeted delivery and process optimization, the present invention has achieved multi-dimensional technological breakthroughs, and the specific beneficial effects are as follows: 1. Synergistic effect of strains: Lactobacillus johnsonii YH1136 increases the supply of GABA precursor glutamate by inhibiting the expression of tryptophan metabolism enzymes IDO1 / TDO2, while Lactobacillus helveticus Helveticus uses its highly active glutamate decarboxylase to convert glutamate into GABA, forming a "precursor supply - conversion synergistic" closed loop. The endogenous GABA production is increased by 40% compared with a single strain (comparative example 1). At the same time, YH1136 enhances osteocalcin synthesis by regulating the intestinal flora, and together with Lactobacillus helveticus Helveticus activates the "gut - bone axis" together, resulting in a 25% increase in bone density (μCT detection data).

[0020] 2. Efficient delivery and stability improvement of GABA: By loading GABA with a nanocellulose (CNF) carrier and combining with acid-resistant β-glucosidase directional enzymatic hydrolysis, colon-targeted sustained release is achieved (release rate ≥ 82%), avoiding gastric acid destruction (the inactivation rate of free GABA in gastric juice is 45.6%, comparative example 2), and the bioavailability is increased by 2.1 times compared with traditional dosage forms; at the same time, the physical adsorption effect of CNF enables the GABA content retention rate to be ≥ 95% during the storage period (25°C / 12 months), which is significantly better than that of conventional powder preparations (retention rate ≤ 80%).

[0021] 3. Freeze-drying process innovation and enhanced colonization: Using a composite protective agent of trehalose (8% - 12%), skim milk (5% - 8%) and GOS (DP = 2 - 8), the freeze-drying survival rate of the bacterial agent is ≥95%; adding AI-2 quorum sensing molecule (0.01% - 0.05%) can activate the LuxS / AI-2 signaling pathway of the strain, promote the formation of intestinal biofilm, the colonization efficiency is 3.2 times higher than that of the group without AI-2 (Comparative Example 3), and the action time of the strain is extended.

[0022] 4. Precise release of enteric coating: Using HPMCP coating (pH threshold 5.5 - 6.5) and gradient drying process (weight gain 5% - 10%), the balance of mechanical strength and disintegration rate of the coating film is achieved without plasticizer. The particles are completely disintegrated within 25 minutes in the simulated duodenal environment (pH 5.5), and the absorption window matching degree is 60% higher than that of the colon-targeted preparation (Comparative Example 5), and there are no side effects of gastric irritation.

[0023] 5. Industrialization and safety advantages: The raw materials are all commercially available components (such as CCTCC preserved strains, food-grade GABA, pharmaceutical excipients), and the equipment is adapted to conventional production lines (fermenters, fluidized bed coating machines); long-term toxicity experiments (12 weeks) show that there are no abnormalities in liver and kidney functions at the recommended dose (ALT / AST / BUN has no difference from the blank group, p > 0.05), and only reversible intestinal inflammation is seen in the high-dose group. The safety is significantly better than that of chemically synthesized growth promoters. Brief Description of the Drawings

[0024] Figure 1 Shows the sustained release characteristics of the GABA complex; Figure 2 Shows the body length growth rate (%) of the experimental animals in Test Example 3; Figure 3 Shows the femoral density (g / cm³) of the experimental animals in Test Example 3; Figure 4 Shows the serum GH (ng / mL) of the experimental animals in Test Example 3. Detailed Embodiments

[0025] The technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0026] Table 1 shows the raw materials used in the embodiments of the present invention, and Table 2 shows the sources of the equipment in the embodiments.

[0027] Table 1 Raw Material Table Category Raw material name Specification / Model Product number / Deposit number Strain Lactobacillus johnsonii YH1136 Freeze-dried powder, viable bacteria ≥ 1×10¹¹ CFU / g CCTCC NO: M 20221116 Lactobacillus helveticus Freeze-dried powder, viable bacteria ≥ 1×10¹¹ CFU / g Lactobacillus helveticus R0052 GABA-related raw material γ-Aminobutyric acid (GABA) Food grade, purity ≥ 98% WB-GABA-F60 Cellulose nanofibers (CNF) Particle size 50 - 100 nm CEOLUS™ PH-F20 Acid-resistant β-glucosidase Enzyme activity ≥ 500 U / g NS-22118 Lyophilization protectant Trehalose Analytical pure T9531-100G Skim milk powder Food grade DM-980 Galactooligosaccharides (GOS) DP = 2 - 8, purity ≥ 95% FT-GOS200 Quorum sensing molecule AI-2 (DPD) Purity ≥ 98% BVT-AI2-10MG Enteric coating material Hydroxypropyl methylcellulose phthalate (HPMCP) HP-55 EUDRAGIT® L30D-55 Excipient Microcrystalline cellulose Pharmaceutical grade, particle size 90 μm VIVAPUR® 102 Phosphate buffer solution pH 7.0 ±0.1 B548119-0500 。

[0028] Table 2 Equipment List Equipment category Equipment name Model Usage Fermentation equipment Automatic fermenter BIOSTAT® B-DCU II Strain amplification culture (such as high-density fermentation of probiotics and engineered bacteria) Centrifugation and lyophilization equipment High-speed refrigerated centrifuge CR22N Bacterial cell collection and cell debris separation (rotation speed ≥ 20,000 rpm) Vacuum freeze dryer FreeZone 6L Preservation of freeze-dried bacteria agents, GABA complexes and active ingredients (-50℃ freeze-drying) Coating and preparation equipment Fluidized bed coater GPCG 2 Preparation of enteric-coated granules (control of HPMCP coating layer thickness) Wet granulator GHL-10 Preparation of particle core material (particle size 100 - 500 μm) Detection and analysis equipment High performance liquid chromatograph (HPLC) 1260 Infinity II GABA content detection (C18 column, mobile phase: methanol-water gradient elution) Laser particle size analyzer Mastersizer 3000 Analysis of particle size distribution (statistics of D50 and D90 particle sizes) UV-visible spectrophotometer UV-2600 <![CDATA[Measurement of OD value of bacterial solution (at a wavelength of 600 nm, OD 600 ).]]> Other equipment pH meter FE28 pH calibration of buffer solution (accuracy ±0.01, range 0 - 14) Constant temperature shaker IS-RDD3 Temperature control for enzymatic reaction (37℃ ±0.5℃, oscillation frequency 100 - 300 rpm) 。

[0029] Example 1 1. Composition Formula Lyophilized Bacterial Agent: Lactobacillus johnsonii YH1136: viable cell count 1×10^10 CFU / g Lactobacillus helveticus Helveticus : viable cell count 1×10^10 CFU / g Viable Cell Ratio of Strains: YH1136: Lactobacillus helveticus Helveticus = 1:1 Lyoprotectant: trehalose 10wt%, skim milk 6wt%, GOS 10wt% (DP = 4), AI-2 0.03wt% GABA Complex: GABA:CNF mass ratio = 1:3, GABA loading rate 28% (w / w) Ratio: The total viable cell count of the lyophilized bacterial agent in each gram of the composition is 2×10^10 CFU, and the GABA content is 200 mg.

[0030] 2. Preparation Method Lyophilization of Bacterial Agent: Fermentation and Culture: Lactobacillus johnsonii YH1136: MRS medium, anaerobic culture at 37°C for 18 h until OD600 = 3.2.

[0031] Lactobacillus helveticus Helveticus : MRS medium (containing 1% sodium glutamate), anaerobic culture at 37°C for 20 h until OD600 = 3.0.

[0032] Centrifugal Collection: Centrifuge at 5000 rpm for 10 min, collect the bacterial sludge separately, and wash it 2 - 3 times with sterile saline.

[0033] Preparation of Lyoprotectant: Dissolve 10% trehalose, 6% skim milk, 10% GOS (DP = 4), and 0.03% AI-2 in deionized water.

[0034] Precooling and Drying: The bacterial sludge and the protective agent are mixed at a ratio of 1:1 (w / w), precooled at -80°C for 16 hours, and then subjected to vacuum freeze-drying. The vacuum freeze-drying process is divided into three stages: the first stage is maintained at -45°C to -40°C for 10 hours, the second stage is heated to -35°C to -30°C and maintained for 12 hours, and the third stage is heated to 25°C to 30°C and maintained for 3 hours, finally obtaining a freeze-dried bacterial agent with a survival rate ≥ 95%.

[0035] Preparation of GABA Complex: Dispersion of CNF: 1.5 g of nanocellulose is dispersed in 22.5 mL of phosphate buffer solution with pH 7.0 (15 mL / g CNF).

[0036] Enzymatic Reaction: Add acid-resistant β-glucosidase (300 U / g CNF), add 0.5 g of GABA (GABA:CNF = 1:3), and react at 50°C for 6 h (150 rpm).

[0037] Centrifugation and Freeze-drying: Centrifuge at 4000 rpm for 8 min to remove free GABA, precool the precipitate at -45°C for 4 h, and then vacuum-dry for 16 h, with a loading rate of 28%.

[0038] 3. Enteric-coated Granules (Without Plasticizer): Preparation of Core Material: The composition (freeze-dried bacterial agent:GABA complex = 2:1) and microcrystalline cellulose (auxiliary material) are mixed at a ratio of 9:1 and granulated.

[0039] Coating Solution: 12% (w / v) of HPMCP is dissolved in water (increase the concentration to compensate for the effect of no plasticizer).

[0040] Coating Process: Fluidized bed spraying, with a coating weight gain of 10%, and gradient drying at 42°C until the water content ≤ 5%.

[0041] Examples 2 - 6

[0042] The parameters of Examples 2 - 6 are modified based on Example 1, as shown in Table 3 below.

[0043] Table 3 Examples 2 - 6 Key differences Verification target Example 2 Viable bacteria ratio of strains = 1:3 (YH1136: Lactobacillus helveticus) Effect of strain ratio on GABA conversion Example 3 No GOS in the lyophilization protectant (only trehalose + skim milk powder) Contribution of GOS to the survival rate of the bacterial agent Example 4 GABA:CNF = 1:2 (loading rate 22%) Regulation of the sustained-release efficiency by the carrier ratio Example 5 AI-2 = 0.05 wt% in the lyoprotectant Optimization of the colonization efficiency by the AI-2 concentration Example 6 Coating weight gain = 8% (HPMCP 10% w / v) Effect of the coating thickness on the targeted release 。

[0044] Comparative Examples 1 - 6

[0045] The parameters of Comparative Examples 1 - 6 are modified based on Example 1, as shown in Table 4 below.

[0046] Table 4 Comparative Examples 1 - 6 Key differences Control 1 Containing only Lactobacillus johnsonii YH1136 (without Lactobacillus helveticus) Control 2 Using free GABA (not loaded on CNF) Control 3 No AI-2 in the lyoprotectant Control 4 GABA complex loading rate = 15% (excess CNF) Control 5 Enteric coating pH threshold = 7.0 (Eudragit® S100) Control 6 Replacing Lactobacillus helveticus with Lactobacillus plantarum()[[]]END]] 。

[0047] Test Example 1

[0048] Testing method Grouping design: Example 1: Freeze-dried bacterial agent containing AI-2 (0.03 wt%) (YH1136: Helveticus =1:1); Example 3: No GOS in the freeze-drying protectant; Comparative Example 3: No AI-2 in the freeze-drying protectant.

[0049] Treatment with simulated gastric juice: Prepare simulated gastric juice: 0.2% NaCl + 0.3% pepsin (Sigma P7000), pH 2.0 (adjusted with HCl), preheat at 37°C; Take 100 mg of the freeze-dried bacterial agent and add it to 10 mL of gastric juice, and shake and treat at 37°C and 200 rpm for 2 h.

[0050] Viable count: Gradient dilute the treatment solution (10^-1 to 10^-6), take 100 μL and coat it on MRS (YH1136) or MRS (Lactobacillus helveticus Helveticus ) plate; Anaerobic culture (YH1136) or anaerobic culture (Lactobacillus helveticus Helveticus ) at 37°C for 48 h, and count the colony forming units (CFU).

[0051] Specific results are shown in Table 5 Table 5 Survival rate and acid resistance of bacterial agents Group Initial viable bacteria count (CFU / g) Viable bacteria count after treatment (CFU / g) Survival rate (%) Example 1 2.0×10^10 1.78×10^10 89.0 Example 3 2.0×10^10 1.65×10^10 82.5 Control 3 2.0×10^10 1.44×10^10 72.0 .

[0052] The survival rate of Example 1 is the highest (89.0%): AI-2 promotes biofilm formation by activating the LuxS / AI-2 quorum sensing system of the strain, reduces the erosion of gastric acid and pepsin on the bacterial cells, and the survival rate is increased by 17.0% compared with Comparative Example 3 (without AI-2) (p<0.01).

[0053] The survival rate of Example 3 decreased (82.5%): The lack of GOS in the freeze-drying protectant caused damage to the bacterial cells due to insufficient osmotic pressure regulation during the pre-freezing stage, and the survival rate decreased by 6.5% compared with Example 1 (p<0.05).

[0054] Test Example 2

[0055] Sustained release characteristics of GABA complex Testing method Grouping design: Example 1: GABA:CNF = 1:3 (loading rate 28%); Example 4: GABA:CNF = 1:2 (loading rate 22%); Comparative Example 2: Free GABA was not loaded; Comparative Example 4: GABA:CNF = 1:5 (loading rate 15%).

[0056] In vitro simulated digestion: Gastric juice stage: 0.1 M HCl (pH 2.0) + 0.3% pepsin, oscillated at 37 °C (100 rpm) for 2 h; Intestinal juice stage: 0.05 M phosphate buffer (pH 6.8) + 0.1% trypsin, continued for 6 h.

[0057] HPLC detection: Chromatographic conditions: C18 column (4.6×250 mm, Agilent ZORBAX), mobile phase acetonitrile - water (20:80, containing 0.1% TFA), flow rate 1.0 mL / min, detection wavelength 210 nm; Specific results are shown in Table 6 and Figure 1 .

[0058] Table 6 Sustained - release characteristics of GABA complex Group Release rate in the gastric juice stage (%) Release rate in the intestinal juice stage (%) Total release rate (%) Example 1 5.2 76.8 82.0 Example 4 7.1 68.3 75.4 Control 2 45.6 32.1 77.7 Control 4 12.3 38.5 50.8 .

[0059] From the data in Table 6, it can be seen that the total release rate of Example 1 is 82.0%: The CNF carrier is specifically degraded by β - glucosidase in intestinal juice, and the GABA sustained - release effect is significant. The loss in the gastric juice stage is only 5.2%, which is significantly better than that of Comparative Example 2 (the gastric juice loss of free GABA is 45.6%, p < 0.01).

[0060] The release rate of Example 4 decreased (75.4%): When GABA:CNF = 1:2, the carrier loading rate was insufficient, resulting in some GABA not being effectively encapsulated during the preparation stage (p < 0.05 vs Example 1).

[0061] The release of Comparative Example 4 was blocked (50.8%): Excessive CNF (GABA:CNF = 1:5) led to a decrease in the porosity of the carrier and a decrease in enzymatic hydrolysis efficiency (p < 0.01).

[0062] Test Example 3

[0063] Animal growth indicators Test method Group design: Blank control: Intragastric administration of normal saline; Example 1: 50 mg / kg of the composition per day; Example 1 + VD3: 50 mg / kg composition + vitamin D3 (600 IU / kg); Comparative Example 6: Replace Lactobacillus helveticus Helveticus with Lactobacillus plantarum, 50 mg / kg per day.

[0064] Animal model: SD rats (4 weeks old, male, body weight 80 ± 10 g), n = 10 / group, raised in a SPF-level environment.

[0065] Detection indexes: Body length growth rate: Measure the length from the tip of the nose to the base of the tail after 4 weeks of intervention; Serum GH / IGF-1: ELISA kit (Abcam ab100545 / ab100695); Femur density: μCT scan (Skyscan 1276, resolution 10 μm).

[0066] The specific results are shown in Table 7 and Figure 2 - Figure 4 .

[0067] Table 7 Growth indexes Group Body length growth rate (%) Femur density (g / cm³) Serum GH (ng / mL) Blank control 100.0 ± 2.1 0.28 ± 0.03 5.2 ± 0.8 Example 1 118.5 ± 3.2* 0.35 ± 0.04* 6.9 ± 1.1* Example 1 + VD3 122.3 ± 3.5* 0.41 ± 0.05* 7.1 ± 1.2* Control 6 105.6 ± 2.3 0.29 ± 0.03 5.4 ± 0.7 .

[0068] As can be seen from the results in Table 7, the body length increase in Example 1 was +18.5%: YH1136 promoted the conversion of tryptophan to 5-HT by inhibiting IDO1 / TDO2, and stimulated the vagus nerve to promote GH secretion; Lactobacillus helveticus Helveticus converted glutamate to GABA and directly activated the release of pituitary GH (p < 0.01 vs blank).

[0069] The combination with vitamin D3 had a synergistic effect: VD3 promoted calcium absorption and synergistically enhanced the activity of osteoblasts with GABA, and the femur density increased by 46% (p < 0.01 vs Example 1).

[0070] The effect of Comparative Example 6 was weak: After replacing Lactobacillus helveticus Helveticus with Lactobacillus plantarum, the ability to convert GABA was lost, and the growth indexes were not different from those of the blank (p > 0.05).

[0071] Test Example 4

[0072] Intestinal flora and GABA metabolism Test method Animal grouping and intervention Thirty-six SPF-level male SD rats (4 weeks old, body weight 80 ± 10 g) were selected and randomly divided into 3 groups (n = 12 / group): Example 1 group: gavaged daily with a composition containing AI-2 (0.03 wt%) (50 mg / kg); Example 5 group: the concentration of AI-2 was increased to 0.05 wt%, and the rest was the same as in Example 1, (50 mg / kg); Control group 3: AI-2 was not added to the lyoprotectant (50 mg / kg).

[0073] Intervention period: 4 consecutive weeks, administered at a fixed time every day, and fed a standard diet ad libitum.

[0074] Sample collection and preservation On the 28th day of intervention, fecal samples from the terminal colon (≥0.5 g per animal) were collected after fasting for 12 h, snap-frozen in liquid nitrogen and stored at -80 °C.

[0075] Detection method 16S rRNA sequencing: Fecal microbial DNA was extracted using the QIAamp DNA Stool Mini Kit, amplified in the V3-V4 region and sequenced by Illumina MiSeq, and the microbial abundance was analyzed by QIIME2.

[0076] GABA concentration detection: LC-MS method (HILIC column 2.1×100mm, 1.7μm; mobile phase: acetonitrile - 0.1% formic acid water, volume ratio 10:90, flow rate 0.3 mL / min), mass spectrometry parameters ESI+ (m / z 104.1→87.1), ion source temperature 500 °C.

[0077] The specific experimental results are shown in Table 8.

[0078] Table 8 Intestinal flora and GABA metabolism Group Abundance of Lactobacillus helveticus (relative %) Colonic GABA concentration (μg / g) Example 1 3.8 ± 0.5 12.3 ± 1.2 Example 5 4.2 ± 0.6 13.1 ± 1.4 Control 3 1.2 ± 0.3 8.7 ± 0.9 。

[0079] From the data in Table 8, it can be seen that AI-2 promotes the colonization of Lactobacillus helveticus Helveticus Colonization: In Example 1, AI-2 activated quorum sensing, and the abundance of Lactobacillus helveticus Helveticus was increased by 3.2 times compared with Control group 3 (without AI-2) (p<0.01), and the GABA concentration in the colon was simultaneously increased by about 40%.

[0080] Optimization effect of Example 5: After the concentration of AI-2 was increased to 0.05 wt%, the abundance of Lactobacillus helveticus Helveticus further increased to 4.2% (p<0.05 vs Example 1), proving the dose-dependent effect of AI-2.

[0081] Test Example 5

[0082] Verification of enteric coating performance Testing Method Group Design: Example 1: The weight gain of HPMCP coating is 8%; Example 6: The weight gain of HPMCP coating is 10%; Control Example 5: Coated with Eudragit® S100 (pH threshold 7.0).

[0083] Disintegration Experiment: Dissolution Medium: pH 5.5 (0.05 M acetate buffer), pH 6.0 / 7.0 (phosphate buffer); Condition: 37°C, 50 rpm, visually observe the complete disintegration time of the particles.

[0084] The results are shown in Table 9.

[0085] Table 9 Verification of Enteric Coating Performance Group Disintegration time at pH 5.5 (min) Disintegration time at pH 6.0 (min) Disintegration time at pH 7.0 (min) Example 1 25 ± 3 18 ± 2 - Example 6 35 ± 4 22 ± 3 - Control 5 - - 65 ± 5 。

[0086] Precision Release of Example 1: HPMCP coating (pH threshold 5.5) rapidly disintegrates in the duodenum (25 min), ensuring the release of GABA during the peak window of GH secretion, and the absorption efficiency is increased by 60% compared with Control Example 5 (pH 7.0 colon release) (p<0.01).

[0087] Influence of Coating Weight Gain: A 10% weight gain in Example 6 results in an overly thick coating film and the disintegration time is extended to 35 min (p<0.05 vs Example 1).

[0088] Test Example 6

[0089] Long-term Safety Assessment Testing Procedures: Animal Model: SD rats (4 weeks old, n = 10 / group), Blank Control: Gastric gavage with normal saline; Example 1 (Recommended Dose): 50 mg / kg; Example 1 (High Dose): 100 mg / kg (2 times the recommended dose).

[0090] Intervention Period: Administer the drug continuously for 12 weeks.

[0091] Detection Indicators: Blood Biochemistry (Liver and Kidney Functions: ALT, AST, BUN); Body Weight and Food Intake.

[0092] The results are shown in Table 10.

[0093] Table 10 Long-term Safety Assessment Group ALT (U / L) AST (U / L) BUN (mmol / L) Blank control 35 ± 5 38 ± 5 6.2 ± 0.8 Example 1 (recommended) 38 ± 6 42 ± 6 6.5 ± 0.7 Example 1 (high) 42 ± 7 45 ± 7 6.8 ± 0.9 。

[0094] As can be seen from the results in Table 10, the safety at the recommended dose: there was no significant difference in ALT, AST, and BUN compared with the blank group (p > 0.05), demonstrating the safety of long-term use.

[0095] In summary, as can be seen from the above test examples: Acid resistance of the bacterial agent: The survival rate of Example 1 (containing AI-2) was 89%, significantly better than that of Comparative Example 3 (72%); GABA sustained release: The gastric juice release rate of Example 1 was only 5.2%, and the intestinal juice release rate was 76.8%, superior to free GABA (Comparative Example 2); Animal growth: The body length increase in Example 1 was +18.5%, and the bone mineral density increased by 46% after combined use with vitamin D3; Intestinal flora: AI-2 significantly increased the colonization of Lactobacillus helveticus Helveticus (3.8% vs 1.2% in Comparative Example 3); Enteric coating performance: The HPMCP coating disintegrated in 25 minutes at pH 5.5, precisely targeting the duodenum.

[0096] The present invention solves the problems of single function of strains, low utilization rate of GABA, and gastric acid destruction in the prior art, and provides an efficient and safe solution for the intervention of children's growth and development.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Therefore, based on the innovative concept of the present invention, any changes and modifications made to the embodiments described herein, or equivalent structural or equivalent process transformations made using the content of the specification of the present invention, and directly or indirectly applying the above technical solutions to other related technical fields, are all included within the protection scope of the present invention patent.

Claims

1. A composition of a microbial inoculant compounded with GABA, characterized in that, Composed of the following components: Lyophilized bacterial agent: containing Lactobacillus johnsonii YH1136 and Lactobacillus helveticus Helveticus , and the viable bacteria ratio of the two is 1:1 to 1:3; γ-aminobutyric acid GABA complex: GABA is loaded on the nano-cellulose CNF carrier, and the mass ratio of GABA to CNF is 1:2 to 1:5; Among them, in each gram of the said composition: The total viable count of the freeze-dried bacterial agent is 1×10^9 to 5×10^10 CFU, The content of GABA in the GABA complex is 100 to 300 mg.

2. The composition of a microbial inoculant compounded with GABA according to claim 1, wherein The said freeze-dried bacterial agent is obtained by the following steps: (i)Ferment and culture Lactobacillus johnsonii YH1136 and Lactobacillus helveticus separately Helveticus until the logarithmic growth phase, centrifuge to collect the bacterial sludge, and wash it 2-3 times with sterile normal saline; (ii) Mix the washed bacterial sludge with the freeze-drying protectant at a volume ratio of 1:1 to 1:

2. The freeze-drying protectant contains 8% to 12 wt% of trehalose and 5 wt% to 8 wt% of skim milk powder. After pre-freezing at -80°C for 12 - 24 hours, carry out vacuum freeze-drying. The vacuum freeze-drying process is divided into three stages: the first stage is maintained at -45°C to -40°C for 8 - 12 hours, the second stage is heated to -35°C to -30°C and maintained for 10 - 14 hours, and the third stage is heated to 25°C to 30°C and maintained for 2 - 4 hours, finally obtaining a freeze-dried bacterial agent with a survival rate ≥ 95%.

3. The composition of a microbial inoculant compounded with GABA according to claim 2, characterized in that, In the freeze-drying protectant in the said step (ii), prebiotic galactooligosaccharide GOS is also added, and the addition amount of GOS is 5 wt% to 15 wt% of the total mass of the freeze-dried bacterial agent, and the degree of polymerization DP is 2 to 8.

4. A composition of a microbial inoculant compounded with GABA according to claim 2, characterized in that, In the freeze-drying protectant in the said step (ii), 0.01 wt% to 0.05 wt% of the quorum sensing molecule AI-2 is also added.

5. A composition of a microbial inoculant compounded with GABA according to claim 1, characterized in that, The said γ-aminobutyric acid GABA complex is prepared by the following steps: (i) Disperse nano-cellulose CNF in a phosphate buffer solution with a pH of 6.0 to 7.

5. The volume of the buffer solution is 10 to 20 times the mass of the nano-cellulose, mL / g; (ii) Add acid-resistant β-glucosidase to the dispersion solution in step (i), and the addition amount of the enzyme is 200 to 500 U per g of nano-cellulose; (iii) Add GABA, and the mass ratio of GABA to nano-cellulose is 1:2 to 1:5; (iv) React at 45 to 55°C and a stirring rate of 100 to 200 rpm for 4 to 8 hours, so that GABA is loaded on nano-cellulose through physical adsorption or hydrogen bond binding; (v) Centrifuge at 3000 to 5000 rpm for 5 to 10 min to remove free GABA, and collect the precipitate; (vi) After pre-freezing the precipitate at -40 to -50°C for 2 to 4 h, carry out vacuum freeze-drying for 12 to 24 h to obtain the GABA-CNF complex, and the GABA loading rate of the complex is 20 wt% to 35 wt%.

6. Use of a composition of a microbial agent compounded with GABA as claimed in any one of claims 1 - 5 in the preparation of a product for promoting children's growth and development.

7. Use of a composition of a microbial inoculant and GABA compounded according to claim 6 in the preparation of a product for promoting the growth and development of children, characterized in that, The dosage form of the product is enteric-coated granules, and is prepared by the following steps: I. Mix the composition as claimed in any one of claims 1 - 5 with pharmaceutically acceptable excipients to form a granule core; II. Preparation of enteric coating solution: Dissolve hypromellose phthalate (HPMCP), an enteric material, in water to prepare a coating solution with a concentration of 8% - 12% (w / v). III. Adopt the fluidized bed coating process to evenly spray the coating solution in Step II onto the surface of the granular core material, and control the coating weight gain to be 5wt% - 10wt%. IV. Gradually increase the temperature for drying at 40 - 45°C until the water content of the granules ≤ 5wt% to obtain enteric-coated granules. Among them, the dissolution pH threshold of the enteric coating is 5.5 - 6.

5.

8. A children's nutritional supplement set product, characterized in that, Comprising: (1) A composition of the microbial inoculant compounded with GABA according to any one of claims 1 - 5; (2) Vitamin D3.

9. A children's nutritional supplement set product, characterized in that, Comprising: (1) A composition of the microbial inoculant compounded with GABA according to any one of claims 1 - 5; (2) Vitamin D3; (3) Milk calcium; (4) A composition of cartilage extract and collagen peptide.

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