A probiotic microecological preparation for promoting the height growth of children and its application

Abstract

The application discloses a probiotic microecological preparation for promoting height growth of children and application thereof, and is characterized by comprising at least one of a fermented Lactobacillus mui ZGG35 strain with a preservation number of CGMCC No. 37997 and a plant Lactobacillus lactis ZGG180 strain with a preservation number of CGMCC No. 37998. The fermented Lactobacillus mui ZGG35 and the plant Lactobacillus lactis ZGG180 are inoculated into a fermentation culture medium at a proportion of 15% in volume ratio to obtain lactic acid bacteria culture, and the ZGG35 bacterial liquid and / or the ZGG180 bacterial liquid is added into a freeze-drying agent to prepare the probiotic microecological preparation. The probiotic microecological preparation has the advantages that the levels of growth hormone, insulin-like growth factor-1, insulin-like growth factor binding protein, alkaline phosphatase, osteocalcin, type I procollagen amino-terminal propeptide, insulin, leptin and vitamin D in serum can be improved, and the levels of IL-6 and CTX-I can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and functional foods, specifically relating to a probiotic microecological preparation that promotes height growth in children and adolescents and its application, particularly to a probiotic microecological preparation that promotes growth and development by regulating intestinal flora and growth hormone-related pathways and its application in promoting height growth in adolescents. Background Technology

[0002] Currently, exogenous growth hormone is mainly used in clinical practice to promote growth and development. However, this method has limitations due to its high cost, the need for long-term injections, and potential side effects. Therefore, developing safe and effective alternative or adjunctive interventions is of great significance. In recent years, the role of gut microbiota in regulating host metabolism, immunity, and endocrine function has gradually attracted attention, with its influence spanning multiple aspects such as nutrient absorption, immune regulation, endocrine balance, and mental health. Studies have shown that gut microbiota can directly affect linear growth in children by regulating the growth hormone / insulin-like growth factor 1 (GH / IGF-1) axis. Furthermore, gut microbiota can also influence bone development, promoting bone formation and reducing bone resorption. Therefore, developing a probiotic composition that can significantly promote growth and development with a clearly defined function and mechanism of action has significant application value and market potential. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a probiotic microecological preparation and its application that can effectively increase the levels of growth hormone (GH), insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein (IGFBP-3), alkaline phosphatase (ALP), osteocalcin (OCN), type I procollagen N-terminal propeptide (PINP), insulin (INS), leptin (LEP), and vitamin D (VD) in serum, and decrease the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and type I collagen C-terminal cross-linked peptide (CTX-Ⅰ) in children and adolescents.

[0004] The technical solution adopted by this invention to solve the above-mentioned technical problem is: a probiotic microecological preparation for promoting the height growth of children and adolescents, wherein the probiotic microecological preparation is *Lactobacillus fermentum* with preservation number CGMCC No. 37997. Limosilactobacillus fermentum ZGG35 strain and *Lactobacillus plantarum* with accession number CGMCC No. 37998 Lactiplantibacillus plantarum At least one component of the ZGG180 strain.

[0005] Furthermore, the microecological preparation is at least one of live bacteria and freeze-dried powder, and the number of live bacteria is 1×10⁻⁶. 6~1×10 12 CFU / mL.

[0006] Furthermore, the cell count ratio of the *Lactobacillus fermentans* ZGG35 strain to the *Lactobacillus plantarum* ZGG180 strain is (0.5–2):1.

[0007] Furthermore, the formulation also includes a pharmaceutically or food-grade carrier or excipient, and the dosage form of the formulation is a gavage, powder, capsule, tablet, or liquid beverage.

[0008] Another technical solution of the present invention also provides a method for preparing the above-mentioned probiotic microecological preparation, comprising the following steps: (1) Inoculate fermentation culture medium with Lactobacillus fermentum ZGG35 and / or Lactobacillus plantarum ZGG180 at a volume ratio of 1-5% and culture at 35-40 °C for 12-16 h to obtain lactic acid bacteria culture; (2) Centrifuge the lactic acid bacteria culture obtained in step (1) to obtain the lactic acid bacteria precipitate, add a freeze-drying protectant of the same volume as the lactic acid bacteria precipitate, mix well and place in a -70 to -90℃ refrigerator for overnight pre-freezing, and freeze-dry the pre-frozen sample to obtain a probiotic microecological preparation that promotes the height growth of children and adolescents.

[0009] Furthermore, the freeze-drying protectant is formulated with 110–125 g / L trehalose, 10–11 g / L sorbitol, 1–2 g / L sodium acetate, and 110–130 g / L skim milk powder, dissolved in pure water and sterilized at 120–125°C before use.

[0010] Another technical solution of the present invention also provides the application of the above-mentioned probiotic microecological preparation in the preparation of products that promote the height growth of children and adolescents, and the application of the probiotic microecological preparation in the preparation of products that increase body length and / or increase femur length, improve bone growth and development, improve bone metabolism, regulate growth-related factors, reduce inflammation levels, regulate metabolic indicators, or improve intestinal flora structure.

[0011] Furthermore, the improvement in bone growth and development includes increasing bone volume (BV), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and / or trabecular connectivity density (Conn.D); the improvement in bone metabolism includes increasing the levels of bone formation markers type I procollagen N-terminal propeptide (PINP), osteocalcin (OCN), and / or alkaline phosphatase (ALP), and / or decreasing the level of bone resorption marker type I collagen cross-linked C-terminal peptide (CTX-I).

[0012] Furthermore, the regulation of growth-related factors includes increasing serum levels of insulin-like growth factor-1 (IGF-1), growth hormone (GH), and / or insulin-like growth factor binding protein-3 (IGFBP-3); the regulation of metabolic indicators includes increasing serum levels of insulin (INS), vitamin D (VD), and / or leptin (LEP); and the reduction of inflammation levels includes reducing serum levels of interleukin-6 (IL-6).

[0013] Furthermore, the improvement of the gut microbiota structure includes: at the phylum level, increasing the relative abundance of Bacillota and / or decreasing the relative abundance of Bacteroidetes; at the family level, increasing the relative abundance of Lactobacillus, Lachnospiraceae, Akkermansiaceae, and / or Erysipelotrichaceae. Compared with the prior art, the advantages of this invention are: this invention discloses a probiotic microecological preparation that promotes height growth in children and adolescents and its application, which has been demonstrated through cell experiments to promote the secretion of growth hormone (GH) by GH3 cells, the secretion of insulin-like growth factor-1 (IGF-1) by HepG2 cells, and the differentiation and development of MC3T3-E1 osteoblasts. Mouse experiments have demonstrated that this product significantly improves the height and bone development of mice by increasing serum levels of: ① growth indicators (GH and IGF-1); ② bone development indicators (ALP, Osteocalcin (OCN), Type I procollagen N-terminal propeptide (PINP), and Type I collagen C-terminal cross-linked peptide (CTX-Ⅰ); ③ inflammatory indicators (TNF-α and IL-6); and ④ metabolic indicators (INS, leptin, and vitamin D). This fills the gap in growth-promoting products and addresses the shortcomings of traditional growth hormones, such as significant side effects.

[0014] The above-mentioned fermented Lactobacillus mucinus ( Limosilactobacillus fermentum ZGG35, strain preservation number CGMCC No. 37997, *Lactobacillus plantarum* ( Lactiplantibacillus plantarum ZGG180, strain preservation number CGMCC No. 37998, was deposited on March 23, 2026, at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0015] Figure 1 The amount of GH secreted by GH cells stimulated by lactic acid bacteria; Figure 2 The amount of IGF-1 secreted by HepG2 cells stimulated by lactic acid bacteria; Figure 3 In the figure, A represents the amount of GH secreted by GH cells stimulated by the compound bacteria, and B represents the amount of IGF-1 secreted by HepG2 cells stimulated by the compound bacteria. Figure 4 ALP staining results of MC3T3-E1 cells stimulated by different treatment groups, including (a) ZGG35, (b) ZGG180, (c) ZGG, (d) blank control, (e) positive control, and (f) negative control. Figure 5 Alizarin Red staining results of MC3T3-E1 cells stimulated by different treatment groups, including (a) ZGG35, (b) ZGG180, (c) ZGG, (d) blank control, (e) positive control, and (f) negative control. Figure 6 Growth curves for strains ZGG35 and ZGG180; Figure 7 Phylogenetic trees of strains ZGG35 and ZGG180; Figure 8 Figures showing the body weight and length of mice in week 3 under different treatment groups; Figure 9 Figures showing the body weight and body length of mice at week 6 under different treatment groups; Figure 10 Femur length of mice at week 3 and week 6 under different treatment groups; Figure 11 Results of bone volume (BV), bone volume fraction (BV / TV), and bone mineral density (BMD) of mouse femur; Figure 12 The results show the trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular connectivity density (Conn.D) of the mouse femur. Figure 13 The results of the detection of growth-related indicators in mouse serum, including insulin-like growth factor-1 (IGF-1), growth hormone (GH), and insulin-like growth factor binding protein-3 (IGFBP-3); Figure 14 The results of the detection of type I procollagen N-terminal propeptide (PINP), osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen cross-linked C-terminal peptide (CTX-I) in mouse serum are presented. Figure 15 The results of the detection of the inflammatory factor interleukin-6 (IL-6) in mouse serum; Figure 16The results of detection of relevant metabolic indicators such as insulin (INS), vitamin D and leptin (LEP) in mouse serum; Figure 17 The results of HE staining of mouse ileum are shown in Figure A, where HE staining image is shown, intestinal villus height is shown, and villus crypt depth is shown (V / C). Figure 18 The results of AB-PAS staining in mouse ileum are shown, where A is the AB-PAS staining pattern, B is the number of goblet cells, and C is the positive area ratio. Figure 19 This is a diagram showing the phylum and family composition of the gut microbiota in mice at week 3, where A represents the phylum level and B represents the family level. Figure 20 The graph shows the gut microbiota diversity analysis of mice in week 3, where A is the Shannon index, B is the Chao1 index, and C is the Simpson index. Figure 21 This is a diagram showing the phylum and family composition of the gut microbiota in mice at week 6, where A represents the phylum level and B represents the family level. Figure 22 The graph shows the gut microbiota diversity analysis of mice at week 6, where A is the Shannon index, B is the Chao1 index, and C is the Simpson index. Note: Con / Control group: control group; ZGG35 group: ZGG35 bacteria group; ZGG180 group: ZGG180 bacteria group; ZGGL group: low-dose combination group; ZGGH group: high-dose combination group; GH group: growth hormone group. In the figures and tables, different lowercase letters (a, b, c, etc.) are used to indicate the significance level of differences between groups: differences between groups with different letter labels are statistically significant (P<0.05), while differences between groups with the same letter labels are not statistically significant (P≥0.05). Detailed Implementation

[0016] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0017] Specific Implementation Example 1: Screening of probiotic strains with growth-promoting functions.

[0018] 1. Preparation of culture medium and solution MRS broth medium: Dissolve MRS liquid medium in 1000 mL of distilled water and sterilize at 121 °C for 15 min. The formula of the MRS liquid medium is 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 5 g sodium acetate, 20 g glucose, 80 mL Tween, 0.5 g magnesium sulfate and 0.25 g manganese sulfate.

[0019] MRS solid medium: Dissolve the MRS solid medium in 1000 mL of distilled water and sterilize at 121 °C for 15 min. The formula of the MRS solid medium is 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 5 g sodium acetate, 20 g glucose, 80 mL Tween, 0.5 g magnesium sulfate, 0.25 g manganese sulfate and 15 g agar powder.

[0020] Ham's F-12K complete medium contains 15% horse serum (HS), 2.5% fetal bovine serum (FBS), 81.5% F-12K basal medium, and 1% antibiotics.

[0021] DMEM high-glucose complete medium: contains 9% fetal bovine serum (FBS), 90% DMEM basal medium and 1% antibiotics.

[0022] α-MEM complete medium: contains 10% fetal bovine serum, 89% α-MEM and 1% penicillin-dextrose antibodies.

[0023] Osteogenic induction medium (200 mL): osteogenic differentiation basal medium (175 mL), fetal bovine serum (20 mL), penicillin and bismuth subsalicylate (2 mL), glutamine (2 mL), sodium β-glycerophosphate (2 mL), ascorbic acid (400 uL), dexamethasone (20 uL).

[0024] GH3 cells belong to the rat pituitary tumor cell line and are semi-suspended, semi-adherent cells. Depending on the cell growth status, they were digested and passaged every 3-4 days, with the cells used in the experiment reaching passages 5-10. The complete culture medium used for GH3 cells was Ham's F-12K complete medium.

[0025] HepG2 cells are a human liver cancer cell line and are adherent cells. Depending on the cell growth status, they are digested and passaged every 3-4 days. The cell passages used in the experiment are 5-10. The complete culture medium used for HepG2 cells is DMEM high-glucose complete medium.

[0026] MC3T3-E1 cells belong to the mouse osteogenic progenitor cell line and are adherent cells. Depending on the cell growth status, they were digested and passaged every 2-3 days, with 5-10 passages used in the experiment. The complete culture medium used for MC3T3-E1 cells was α-MEM complete medium.

[0027] 2. Activation of the strain Twelve acid- and bile-tolerant lactic acid bacteria strains were screened from the strain bank of the Institute of Animal Products Processing, Ningbo University. The results are shown in Table 1. Two mL of the strains, stored at -80℃, were inoculated into MRS broth medium and incubated at 37℃ for 12 h to obtain the seed culture, which was then stored for later use.

[0028] Table 1. Sources and Collection Locations of Lactic Acid Bacteria

[0029] 3. Preparation of OD-CFU curves for the strain After activating the above seed solution for three generations, the original solution was diluted to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 30, 1 / 50, 1 / 100, 1 / 200, 1 / 300, 1 / 400, and 1 / 500 of the original solution concentration. The OD values ​​of each diluted solution were then measured. 600nm The absorbance was measured, and each concentration of the diluted solution was diluted tenfold and plated. Then, the OD-CFU curve was plotted based on the OD and the corresponding bacterial count (CFU).

[0030] 4. Preparation of lactic acid bacteria culture Lactic acid bacteria seed culture was inoculated into MRS broth medium and cultured at 37 °C for 24 hours until the late logarithmic growth phase was reached. This process was repeated three times to obtain a lactic acid bacteria culture. The culture was centrifuged at 8000 rpm for 7 min at 4 °C, the supernatant was discarded, and the culture was washed twice with sterile PBS. The bacterial pellet was then resuspended and cultured at OD0.05. 600nm Measure the absorbance at the specified wells. Based on the OD-CFU curve plotted above, determine the corresponding bacterial count. Before co-culturing with cells, adjust the bacterial concentration added to the 96-well plate to 5 × 10⁻⁶ cells / well. 8 For CFU / mL added to 12-well plates, the bacterial concentration needs to be adjusted to 5 × 10⁻⁶. 7 When adding CFU / mL to a 6-well plate, the bacterial concentration needs to be adjusted to 3 × 10⁻⁶. 8 CFU / mL.

[0031] 5. Co-culture of lactic acid bacteria and cells GH3 cells (rat pituitary tumor cells) at 5×10 5 Seeds were placed in 96-well plates at a density of 3 × 10⁶ cells / mL. 5Cells were seeded at a density of [number] cells / mL in 6-well plates and incubated for 48–72 h. The culture medium was then discarded, and the cells were washed 2–3 times with 100 μL (96-well plate) / 1 mL (6-well plate) of PBS buffer. The bacterial suspension was then transferred to 10 volumes of pure Ham's F-12K medium, followed by 100 μL (96-well plate) / 2 mL (6-well plate) of F-12K medium suspension containing live bacteria (the control group only received F-12K). The cells were incubated at 37°C for 12 h. The supernatant was collected, centrifuged at 4°C, 6000–10000 × g for 5 min to remove the precipitate, and the supernatant was used for subsequent assays.

[0032] HepG2 cells were used at a rate of 5 × 10 5 Seeds were placed in 96-well plates at a density of 3 × 10⁶ cells / mL. 5 Cells were seeded at a density of [number] cells / mL in 6-well plates and incubated for 48–72 h. The culture medium was then discarded, and the cells were washed 2–3 times with 100 μL (96-well plate) / 1 mL (6-well plate) of PBS buffer. The bacterial suspension was transferred to 10 volumes of pure DMEM high-glucose medium, followed by 100 μL (96-well plate) / 2 mL (6-well plate) of DMEM medium containing live bacteria (the control group received only DMEM). The cells were incubated at 37°C for 12 h. The supernatant was collected, centrifuged at 4°C, 6000–10000 × g for 5 min to remove the precipitate, and the supernatant was used for subsequent assays.

[0033] The collected cell supernatant was analyzed using a rat growth hormone (GH) ELISA kit and a human insulin-like growth factor (IGF-1) ELISA kit purchased from Jiangsu Kete Biotechnology Co., Ltd. The results are as follows: Figure 1 , 2 As shown, GH was detected in the supernatant of GH3 cells. The control group (Con) had a concentration of 10.01 ng / mL, the ZGG35 group had a concentration of 23.35 ng / mL, the 13-JB-35 group had a concentration of 31.62 ng / mL, and the ZGG180 group had a concentration of 31.46 ng / mL. The ZGG35 group showed a 1.33-fold increase compared to the Con group, the 13-JB-35 group showed a 2.15-fold increase compared to the Con group, and the ZGG180 group showed a 2.14-fold increase compared to the Con group, indicating that it can significantly stimulate GH3 cells to secrete GH. IGF-1 was detected in the supernatant of HepG2 cells. The concentration was 522.99 ng / mL in the Con group, 738.08 ng / mL in the ZGG35 group, and 719.55 ng / mL in the ZGG180 group. The ZGG35 group showed a 41.12% increase compared to the Con group, and the ZGG180 group showed a 37.58% increase compared to the Con group, indicating that they can significantly stimulate HepG2 cells to secrete IGF-1.

[0034] Considering both indicators, strains ZGG35 and ZGG180 were selected. After a combination treatment, the two selected strains were co-cultured with GH3 and HepG2 cells in 6-well plates for 12 hours. The supernatant was then collected, and the contents of GH and IGF-1 were measured. The results are as follows: Figure 3 As shown, the GH detection results were: 12.71 ng / mL in the Con group, and 23.09, 18.82, and 16.26 ng / mL in the ZGG35+ZGG180 mixtures at ratios of 1:1, 2:1, and 1:2, respectively. The results indicate that the 1:1 ratio of ZGG35+ZGG180 resulted in the best effect, improving the concentration by 81.66% compared to the control group. This mixture was named ZGG.

[0035] 6. Cell staining MC3T3-E1 cells at 2×10 5 Cells were seeded at a density of 10 cells / mL in 12-well plates and incubated for 24 h. The culture medium was then discarded, and the cells were washed 2-3 times with 1 mL of PBS buffer. The bacterial suspension was diluted 10 times with osteogenic induction medium, and then 1 mL of osteogenic induction medium suspension containing live bacteria was added (for the blank control group, only osteogenic induction medium was added; for the positive control, 0.5 mM sodium butyrate solution was added to the osteogenic induction medium; and for the negative control, an equal volume of PBS buffer was added to the osteogenic induction medium). The cells were then cultured at 37°C and 5% CO2. Fresh osteogenic induction medium was added every 2-3 days, and the corresponding treatments were replenished according to the above proportions.

[0036] On day 6 of MC3T3-E1 culture, the culture medium was discarded, and the cells were slowly washed twice with PBS. 4% paraformaldehyde fixative was added, and the cells were fixed at room temperature for 15 min. The fixative was then discarded, and the cells were washed twice with PBS. ALP staining was performed using a kit, and the cells were observed and photographed under a microscope. The results are as follows: Figure 4 As shown in (a)-(f), the groups are as follows: (a) ZGG35, (b) ZGG180, (c) ZGG, (d) blank control, (e) positive control, and (f) negative control. Compared with the blank control and negative control groups, each bacterial treatment group increased the alkaline phosphatase activity of MC3T3-E1 cells to varying degrees, as evidenced by a significant increase in intracellular purplish-blue precipitate. Both ZGG35 and ZGG180 promoted osteogenic differentiation, but the staining intensity of the ZGG group was significantly stronger than that of the single bacterial treatment groups, indicating that the combined bacteria have a better effect on promoting early osteogenic differentiation of MC3T3-E1 cells.

[0037] On day 10 of MC3T3-E1 culture, the culture medium was discarded, and the cells were slowly washed twice with PBS. 4% paraformaldehyde fixative was added, and the cells were fixed at room temperature for 15 min. The fixative was then discarded, and the cells were washed twice with distilled water. 2% Alizarin Red S staining solution was added, and the cells were stained at room temperature for 20-30 min. The cells were repeatedly rinsed with distilled water until the background was clear. The formation of calcium nodules was observed under a microscope, and photographs were taken and recorded. The results are as follows: Figure 5 As shown in (a)-(f), the groups were the same as above, and there were significant differences in mineralization levels among the experimental groups. The ZGG group showed more obvious orange-yellow calcium nodule deposition, with a higher staining intensity than the ZGG35 and ZGG180 single-bacterial treatment groups, indicating that the compound bacteria had a better effect on promoting MC3T3-E1 cell mineralization. Apart from the above three groups, no obvious orange-yellow crystal deposition was observed in the blank control group, positive control group, and negative control group. In summary, the results indicate that the compound lactic acid bacteria have a more significant effect on promoting MC3T3-E1 cell mineralization.

[0038] 7. Growth curve determination After activating the seed culture for three generations, it was inoculated and cultured at an inoculum rate of 2% (v / v). The OD value of the fermentation broth was measured every 2 hours. 600nm Measure the absorbance. Shake the culture medium thoroughly before each measurement, and perform three replicates. Results are as follows: Figure 6 As shown, both strains entered the logarithmic growth phase at around 3 hours and entered the stasis phase after 12 hours.

[0039] 8. Identification and evolutionary analysis of 16S rDNA of lactic acid bacteria Fresh bacterial culture was streaked and plated. The plates were then incubated at 37°C for 48 hours before being sent to the Guangdong Provincial Microbial Culture Collection Center for identification. ZGG35 was identified as *Lactobacillus fermentatus* (…). Limosilactobacillus fermentum ZGG180 is *Lactobacillus plantarum* ( Lactiplantibacillus plantarum The resulting phylogenetic tree is as follows: Figure 7 As shown.

[0040] The above-mentioned fermented Lactobacillus mucinus ( Limosilactobacillus fermentum ZGG35 strain, with accession number CGMCC No. 37997, was deposited on March 23, 2026, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0041] The above-mentioned Lactobacillus plantarum ( Lactiplantibacillus plantarumThe ZGG180 strain, with accession number CGMCC No. 37998, was deposited on March 23, 2026, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0042] Specific Implementation Example 2: Animal Experiment Design for Growth-Promoting Mice.

[0043] 1. Laboratory animals and their housing conditions One hundred and thirty-five healthy, three-week-old SPF-grade male mice, weighing 7–12 g, were selected and provided by a reputable laboratory animal center. The animals were housed in an SPF-grade animal facility with an ambient temperature controlled at 22±2℃, relative humidity at 50%±10%, and a 12-hour light-dark cycle. They had free access to food and water. An acclimatization period of 3 days was administered before the experiment.

[0044] 2. Grouping of experimental animals After acclimatization, the mice were randomly divided into the following 9 groups, with 3 cages in each group and 5 mice in each cage: (1) Normal control group (Con / Control): normal feed + equal volume of PBS buffer; (2) Group ZGG35 strain: ordinary feed + strain ZGG35 (1×10 9 CFU / d); (3) Group ZGG180 strain: ordinary feed + strain ZGG180 (1×10 9 CFU / d); (4) Low-dose group of strain ZGG35 + strain ZGG180 (ZGGL): ordinary feed + mixed microbial agent of strain ZGG35 and strain ZGG180 (total 1×10 9 CFU / d, strain ZGG35 and strain ZGG180 were mixed at a 1:1 ratio (CFU / d, strain ZGG35 and strain ZGG180 were mixed at a 1:1 ratio). (5) High-dose group of strain ZGG35 + strain ZGG180 (ZGGH): ordinary feed + mixed bacterial agent of strain ZGG35 and strain ZGG180 (total 1×10 10 CFU / d, strain ZGG35 and strain ZGG180 were mixed at a 1:1 ratio (CFU / d, strain ZGG35 and strain ZGG180 were mixed at a 1:1 ratio). (6) Growth hormone group (GH): ordinary feed + growth hormone injection.

[0045] 3. Administration method and dosage Strains ZGG35, ZGG180, and their combined intervention were administered via gavage once daily at a volume of 0.2 mL / 20 g body weight per animal. The bacterial concentration was diluted with PBS and adjusted to 1 × 10⁻⁶ according to the experimental groups. 9CFU / mL or 1×10 10 CFU / mL.

[0046] The growth hormone group and the combination group were administered the drug subcutaneously at a dose of 0.1 IU / kg / day (0.033 mg / kg / day) once daily.

[0047] The normal control group was given an equal volume of PBS buffer.

[0048] 4. Experimental period and sampling time The entire experiment lasted for 6 weeks. Mid-term sampling was conducted in week 3, with blood and tissue samples collected from a random sample of mice. Final sampling was conducted at the end of week 6, with blood, femur, and related tissue samples collected from all mice.

[0049] 5. Content of indicator testing The following indicators were tested on each group of mice: (1) Growth indicators: body weight, body length and femur length; (2) Bone metabolism indicators: bone volume fraction, trabecular bone thickness, etc. (micro-CT detection); (3) Serum growth-related factors: IGF-1, GH, IGFBP-3, etc.; (4) Bone formation and bone resorption indicators: PINP, OCN, ALP, CTX-Ⅰ, etc.; (5) Inflammatory factors: IL-6; (6) Relevant metabolic indicators: INS, VD, etc. (7) Intestinal tissue morphological indicators: villus height, crypt depth, etc. (8) Sequencing of fecal 16S rRNA.

[0050] Specific Example 3: Analysis of the growth-promoting effect of probiotic microecological preparations.

[0051] 1. Changes in weight and height During the experiment, the weight and length changes of mice in each group were measured and recorded weekly. Weight and length were measured again at the mid-treatment stage of the third week of the experiment, and the results are as follows: Figure 8 As shown, compared with the control group, mice treated with ZGG35, ZGG180, or their combined intervention all showed varying degrees of weight increase. In terms of length, the ZGG180 (8.92 cm), ZGGL (8.95 cm), and ZGGH (8.98 cm) mice were all longer than the Con group (8.75 cm) and slightly shorter than the GH group (9.09 cm). The weight and length results at week six are shown below. Figure 9As shown, the weight gain was similar to that of the third week, with all experimental groups showing varying degrees of increase compared to the Con group. In terms of length, the ZGGH group (9.76 cm) still showed the best effect, increasing by 4.7% compared to the Con group (9.32 cm). The other experimental groups also showed better results than the Con and GH groups (9.49 cm). The experimental results indicate that the strains described in this invention and their combined interventions exhibit superior effects in promoting weight and length growth, demonstrating a stronger growth-promoting effect.

[0052] 2. Analysis of bone growth and development indicators The length of the right femur of each mouse was measured in weeks 3 and 6 of the experiment. The measurement results are as follows: Figure 10 As shown, in week 3, mice treated with ZGG35, ZGG180, or their combined intervention all showed varying degrees of increase in femoral length. The ZGG180 (15.36 mm), ZGGL (15.42 mm), and ZGGH (15.22 mm) groups showed significantly higher femoral lengths than the Con group, with the GH group (14.48 mm) showing the highest. In week 6, the increase in femoral length was similar to that in week 3, with the ZGGH group (15.70 mm) showing the longest increase. The ZGG35 (15.48 mm), ZGG180 (15.50 mm), and ZGGL (15.65 mm) groups were all significantly higher than the Con group (14.38 mm) and also higher than the GH group (15.00 mm).

[0053] Femurs collected in the sixth week of the experiment were preserved by immersion in 4% paraformaldehyde. Then, micro-CT was used to scan and analyze the mouse femurs to detect indicators such as bone volume (BV), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), number of trabecular bones (Tb.N), bone mineral density (BMD), and trabecular connectivity density (Conn.D).

[0054] Bone volume (BV) represents the absolute volume of bone tissue, while bone volume fraction (BV / TV) reflects the percentage of bone density per unit space. An increase in either indicates a significant increase in bone mass. Figure 11 As shown, in terms of bone volume (BV), the ZGG35 group (1.28 mm) 3 ZGG180 group (1.18 mm) 3 ZGGL group (1.16 mm) 3 ) and ZGGH group (1.33 mm) 3 Both were lower than the Con group (0.73 mm). 3 The level was significantly higher in the GH group (0.57 mm). 3 ). At the same time, such as Figure 11As shown, the same trend was observed in bone volume fraction (BV / TV), with the ZGG35 group (0.32), ZGG180 group (0.33), ZGGL group (0.37), and ZGGH group (0.32) all significantly higher than the Con group (0.20) and GH group (0.20).

[0055] Bone mineral density (BMD) represents the amount of minerals in a unit volume of bone; an increase indicates enhanced bone mineralization and stronger bone. Figure 11 As shown, the ZGG35 group (244.59 mg / cm³) 3 ZGG180 group (261.40 mg / cm) 3 ZGGL group (255.70 mg / cm³) 3 ) and ZGGH group (261.58 mg / cm) 3 Both were lower than those in the Con group (180.97 mg / cm²). 3 ) and GH group (187.14 mg / cm 3 () significantly increased.

[0056] Trabecular thickness (Tb.Th) represents the average thickness of a single trabecular bone, reflecting the robustness of the bone structure; an increase indicates improved bone quality. For example... Figure 12 As shown, the values ​​of ZGG35 (0.090 mm), ZGG180 (0.089 mm), ZGGL (0.091 mm), and ZGGH (0.091 mm) were all higher than those of Con (0.074 mm) and GH (0.079 mm).

[0057] The number of trabeculae (Tb.N) represents the number of trabeculae per unit length; an increase indicates an increase in the number of trabeculae and increased new bone formation. For example... Figure 12 As shown, ZGG35 group (3.85 mm) -1 ZGG180 group (4.00 mm) -1 ZGGL group (3.85 mm) -1 ) and ZGGH group (3.91 mm) -1 Both were lower than the Con group (3.57 mm). -1 ) and GH group (3.52 mm) -1 () significantly increased.

[0058] Trabecular connectivity density (Conn.D) represents the number of connections between trabeculae per unit volume; an increase indicates more trabecular connections and a more stable structure. For example... Figure 12 As shown, group ZGG35 (177.17 mm) -3 ZGG180 group (171.82 mm) -3ZGGL group (170.37 mm) -3 ) and ZGGH group (175.73 mm) -3 Both were lower than the Con group (139.54 mm). -3 ) and GH group (123.97 mm) -3 () significantly increased.

[0059] The above results indicate that the probiotics and their microecological preparations screened in this invention can significantly improve bone structure parameters and promote bone growth and development.

[0060] 3. Analysis of serum growth-related indicators The levels of growth-related factors in mouse serum, including insulin-like growth factor-1 (IGF-1), growth hormone (GH), and insulin-like growth factor binding protein-3 (IGFBP-3), were detected using enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 13 As shown.

[0061] Insulin-like growth factor-1 (IGF-1) is the main effector molecule of GH, promoting chondrocyte proliferation and bone matrix deposition, and is a core indicator for evaluating its growth-promoting effect. Compared with the Con group (354.75 pg / mL), the ZGG35 group (816.20 pg / mL), ZGG180 group (1203.44 pg / mL), ZGGL group (1071.65 pg / mL), and ZGGH group (1365.28 pg / mL) all showed highly significant differences. P <0.0001), with the ZGGH group having the highest content, 2.85 times higher than the Con group, and significantly higher than the GH group (1010.47 pg / mL) ( P <0.05). This indicates that ZGG35, ZGG180, and their combined intervention have a promoting effect on IGF-1 secretion, and the combined use of the two has a better effect.

[0062] Growth hormone (GH) is secreted by the anterior pituitary gland and can promote bone growth and protein synthesis by stimulating the liver to produce IGF-1, making it a key regulator of longitudinal growth. Compared with the Con group (277.76 pg / mL), the ZGG35 group (601.42 pg / mL), ZGG180 group (1050.30 pg / mL), ZGGL group (1148.20 pg / mL), and ZGGH group (1291.96 pg / mL) all showed highly significant differences. P <0.0001), with the ZGGH group having the highest content, 3.65 times higher than the Con group, and significantly higher than the GH group (1048.89 pg / mL) ( P<0.05), indicating that the injected growth hormone had a regulatory effect on the GH level in mice, but it was still lower than ZGGL and ZGGH, indicating that the probiotic microecological preparation had a stronger regulatory effect on the GH level in mice.

[0063] Insulin-like growth factor binding protein-3 (IGFBP-3) is the most abundant IGF-1 carrier protein in the blood, prolonging the half-life of IGF-1 and regulating its biological activity. Its elevation typically reflects enhanced growth axis activity. Compared with the Con group (51.83 ng / mL), the ZGG35 group (89.96 ng / mL), ZGG180 group (112.24 ng / mL), ZGGL group (108.89 ng / mL), and ZGGH group (122.29 ng / mL) all showed highly significant differences. P <0.0001), with the ZGGH group showing a similar pattern to the two indicators mentioned above, exhibiting the highest content, 1.36 times higher than the Con group, and significantly higher than the GH group (92.12 ng / mL). P <0.05).

[0064] In summary, compared with the Con group, the levels of IGF-1, GH, and IGFBP-3 in each experimental group were significantly increased, with the ZGGH group showing the largest increase. This indicates that the probiotic microecological preparation described in this invention can promote growth and development by regulating the GH / IGF-1 axis.

[0065] 4. Analysis of serum bone metabolism-related indicators Enzyme-linked immunosorbent assay (ELISA) was used to detect serum markers related to bone formation and bone resorption, including type I procollagen N-terminal propeptide (PINP), osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen C-terminal crosslinked peptide (CTX-I). The results are as follows: Figure 14 As shown.

[0066] PINP is a precursor peptide released during type I collagen synthesis and is one of the most sensitive indicators for evaluating bone formation; its elevation indicates increased collagen synthesis and active bone formation. Compared with the Con group (75.23 ng / mL), the ZGG35 group (125.72 ng / mL), ZGG180 group (143.31 ng / mL), ZGGL group (134.35 ng / mL), and ZGGH group (147.55 ng / mL) all showed highly significant differences. P <0.0001), with the ZGG180 group having the highest content, exceeding the Con group by 87.84%, and significantly exceeding the GH group (127.13 ng / mL) ( P <0.01).

[0067] OCN, secreted by osteoblasts, is a specific indicator of bone formation activity; increased expression suggests active osteoblasts and enhanced bone matrix synthesis. Compared with the Con group (0.80 ng / mL), the ZGG35 group (1.39 ng / mL), ZGG180 group (1.78 ng / mL), ZGGL group (1.48 ng / mL), and ZGGH group (1.50 ng / mL) all showed highly significant differences. P <0.0001), with the ZGG180 group having the highest content, 1.23 times higher than the Con group, and significantly higher than the GH group (1.38 ng / mL) ( P <0.05).

[0068] ALP, especially bone-specific ALP, plays an important role in bone mineralization. Increased ALP activity suggests enhanced osteoblast differentiation and improved bone mineralization capacity. Compared with the Con group (0.40 ng / mL), the ZGG35 group (0.57 ng / mL), ZGG180 group (0.75 ng / mL), ZGGL group (0.74 ng / mL), and ZGGH group (0.80 ng / mL) all showed highly significant differences. P <0.0001), with the ZGGH group having the highest content, more than twice that of the Con group, and significantly higher than the GH group (0.58 ng / mL) ( P <0.01).

[0069] CTX-I is a classic biomarker reflecting bone resorption levels. It originates from the degradation of type I collagen and directly reflects the degree of osteoclast-mediated bone matrix breakdown. Elevated CTX-I indicates enhanced bone resorption, which is detrimental to bone mass accumulation. The Con group (0.55 ng / mL) showed the highest level among all groups, significantly higher than the ZGG35 group (0.46 ng / mL). P <0.01), which was significantly higher than that of the ZGG180 group (0.35 ng / mL), the ZGGL group (0.29 ng / mL), and the ZGGH group (0.37 ng / mL). P <0.0001), with the ZGGL group having the lowest content, 47.27% lower than the Con group, and the ZGGL group significantly lower than the GH group (0.48 ng / mL). P <0.01).

[0070] The above results indicate that, compared with the Con group, the levels of PINP, OCN, and ALP in each experimental group were significantly increased, while the level of CTX-I showed a decreasing trend, suggesting enhanced bone formation and inhibited bone resorption. This demonstrates that the probiotic microecological preparation described in this invention has the effect of promoting bone formation and inhibiting bone resorption.

[0071] 5. Serum inflammatory factor analysis The level of the inflammatory cytokine interleukin-6 (IL-6) in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA), and the results are as follows: Figure 15 As shown.

[0072] IL-6, as an important regulator of inflammatory responses, can promote osteoclastogenesis and interfere with the growth hormone axis function. Its sustained elevation is closely related to osteopenia and growth retardation. The GH group (54.99 pg / mL) and the Con group (54.19 pg / mL) showed the highest levels among all groups, significantly higher than the ZGG35 group (43.24 pg / mL), ZGG180 group (36.39 pg / mL), ZGGL group (42.55 pg / mL), and ZGGH group (38.70 pg / mL). P <0.05).

[0073] The results showed that, compared with the Con group, the IL-6 levels in all probiotic groups were significantly lower, indicating that the inflammatory response was alleviated. This demonstrates that the probiotic microecological preparation described in this invention can improve the internal environment of the body by reducing inflammation levels, thereby promoting growth and development.

[0074] 6. Analysis of other relevant metabolic indicators The levels of insulin (INS), vitamin D, and leptin (LEP) in mouse serum were detected using enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 16 As shown.

[0075] INS is an important hormone regulating glucose metabolism, promoting glucose uptake and protein synthesis, and participating in growth regulation to some extent. A moderate increase in INS is beneficial for anabolism. Compared with the Con group (0.62 ng / mL), the ZGG35 group (1.79 ng / mL), ZGG180 group (2.42 ng / mL), ZGGL group (2.39 ng / mL), and ZGGH group (2.21 ng / mL) all showed highly significant differences. P <0.0001), with the ZGGL group having the highest content, 2.85 times higher than the Con group, and significantly higher than the GH group (2.08 ng / mL). P <0.05) LEP is secreted by adipose tissue and participates in regulating energy metabolism and neuroendocrine function. It can indirectly regulate the growth axis by acting on the hypothalamus, and a moderate increase in LEP usually reflects improved nutritional status and is beneficial to growth and development. Compared with the Con group (98.48 pg / mL), the ZGG35 group (148.67 pg / mL), ZGG180 group (200.63 pg / mL), ZGGL group (200.86 pg / mL), and ZGGH group (201.94 pg / mL) all showed highly significant differences. P <0.0001), among which the ZGG180 group, ZGGL group and ZGGH group all showed significantly higher levels than the GH group (177.54 pg / mL). P <0.01).

[0076] Vitamin D promotes intestinal calcium absorption and bone mineralization, and is an important hormone for maintaining bone health. Elevated levels contribute to bone formation and increased bone density. Compared with the Con group (9.39 ng / mL), the ZGG35 group (22.27 ng / mL), ZGG180 group (23.98 ng / mL), ZGGL group (26.42 ng / mL), and ZGGH group (28.88 ng / mL) all showed highly significant differences. P <0.0001), with the ZGGH group having the highest content, 2.07 times higher than the Con group, and significantly higher than the GH group (21.17 ng / mL) ( P <0.01).

[0077] The results showed that, compared with the Con group, the levels of INS, VD, and LEP in each experimental group were significantly increased, suggesting that the probiotic treatment affected multiple aspects of the mouse's metabolism, thereby promoting growth. This indicates that the probiotic microecological preparation described in this invention can improve the metabolic environment in the body, thus benefiting growth and development.

[0078] 7. Morphological analysis of intestinal tissue The ileum tissue of mice was stained with HE and AB-PAS to observe the intestinal villus height, crypt depth, goblet cell number and positive area ratio.

[0079] HE staining results of the ileum are as follows Figure 17 As shown in Figure A, the groups in the figure are (a) Con, (b) ZGG35, (c) ZGG180, (d) ZGGL, (e) ZGGH, and (f) GH. The villous morphology in the figure roughly shows that the villous morphology of each experimental group is superior to that of the Con group. Figure 17As shown in B, the pile height data shows that the pile height of the ZGG180 group (0.21 mm), ZGGL group (0.20 mm), and ZGGH group (0.20 mm) is significantly higher than that of the Con group (0.17 mm). P <0.01), with the ZGG180 group having the highest fluff height, significantly higher than the GH group (0.18 mm). P <0.0001). For example... Figure 17 As shown in C, the ratio of villus height to crypt depth (V / C) shows that all experimental groups are superior to the Con group (2.13), with the ZGGH group (2.76) having the largest ratio, which is higher than the GH group (2.05).

[0080] AB-PAS staining results of the ileum are as follows Figure 18 As shown in A, the groups are as above. Staining results showed that in the groups treated with the probiotics of this invention, goblet cells significantly increased, and the Alsin blue positive area significantly enlarged, indicating an increase in mucus secretion, which helps enhance intestinal barrier function and may promote nutrient absorption. Further, as... Figure 18 As shown in B, at the goblet cell level, the ZGGL group (38.96 cells / mm) and the ZGGH group (40.18 cells / mm) showed the best performance, significantly higher than the GH group (35.73 cells / mm). P <0.05). For example... Figure 18 As shown in C, the ZGGH group (6.41%) had the highest positive area ratio, which was significantly higher than that of the GH group (3.77%). P <0.001). Based on the above HE and AB-PAS results, it can be concluded that ZGG35, ZGG180 and their combined intervention can improve ileal structure and promote nutrient absorption.

[0081] The above results indicate that the probiotic microecological preparation of the present invention can improve intestinal structure, enhance nutrient absorption capacity, and thus promote body growth.

[0082] Specific Example 4: The regulatory effect of probiotic microecological preparations on the intestinal flora of mice.

[0083] 1. Fecal sample collection and testing Before euthanizing mice at mid-week 3 and end-week 6 of the experiment, fresh fecal samples were collected from each group of mice, flash-frozen in liquid nitrogen, and stored at -80 ℃. These samples were then sent to Beijing Novogene Technology Co., Ltd. for amplicon sequencing analysis of the microbial community. Specific primers were used to amplify the target region: the V3-V4 region of the bacterial 16S rRNA gene.

[0084] 2. Analysis of the composition and diversity of gut microbiota at the phylum level in week 3 At the phylum level, such as Figure 19 As shown in Figure A, the gut microbiota of mice in each group was mainly composed of Bacteroidota and Bacillota, which were the dominant phyla. In the control group, Bacteroidota accounted for 70.53% and Bacillota for 18.86%. Compared with the normal control group, different probiotic interventions had varying degrees of regulatory effects on the microbiota structure. In the ZGG180 group, the relative abundance of Bacillota increased to 51.48% while that of Bacteroidota decreased to 34.00%, suggesting that this strain may affect the energy metabolism-related structural composition of the gut microbiota. In contrast, the microbiota structure of the ZGG35 group was closer to that of the control group, indicating that its regulatory effect was relatively mild.

[0085] Regarding the combined bacterial intervention, both the ZGGL and ZGGH groups showed a trend of decreasing Bacteroidota and increasing Bacillota, with a more balanced relative proportion compared to the single-bacterial groups, indicating that high-dose combined bacteria have a more significant regulatory effect in maintaining the stability of the gut microbiota structure. Furthermore, the combined bacterial intervention also affected the distribution of low-abundance phyla (such as Actinomycetota and Verrucomicrobiota) to some extent, suggesting its broad regulatory potential on the gut microbiota. Specifically, the growth hormone-treated GH group showed an increased proportion of Bacteroidota (75%) and a decreased proportion of Bacillota (14.06%).

[0086] At the family level, such as Figure 19As shown in Figure B, the dominant bacterial communities mainly include Muribacillus, Lactobacillaceae, Lachnospiraceae, and Bifidobacteriaceae. In the Control group, Muribacillus (61.09%) was the absolutely dominant bacterial community. Compared with the Control group, the relative abundance of Lactobacillaceae increased in the ZGG180 group (37.56%), while the relative abundance of Muribacillus decreased (32.68%), suggesting that it may play a regulatory role by promoting the enrichment of lactic acid bacteria-related bacterial communities. The relative abundance of Lachnospiraceae in the ZGG35 group was significantly higher than in other groups. Notably, Lachnospiraceae is a typical short-chain fatty acid (SCFA) producing bacterium, and its increase may be related to improved host energy metabolism and nutrient utilization.

[0087] The combined bacterial intervention further influenced the distribution of key functional flora. The abundance of Lactobacillaceae and Lachnospiraceae was increased in both the ZGGL and ZGGH groups compared to the control group. Simultaneously, the ZGGH group also showed a trend of increased abundance of Erysipelotrichaceae, a bacterium involved in food breakdown and energy metabolism, and playing a crucial role in lipid metabolism, bile acid cycling, and immune regulation. This reveals that high-dose combined bacterial intervention has a more comprehensive regulatory effect in promoting the enrichment of multiple beneficial flora. The GH group was dominated by Muribaculalaceae, while other beneficial flora were relatively low.

[0088] AlPha diversity assessment results are as follows Figure 20 As shown, where Figure 20 In this context, A represents the Shannon index. Figure 20 B in the text represents the Chao1 exponent. Figure 20In the figure, C represents the Simpson index. There was a statistically significant trend in community diversity among the intervention groups. Regarding the Shannon index, the Kruskal-Wallis test showed significant differences among the groups. The box plot distribution trend showed that some probiotic intervention groups, such as the ZGG35 and ZGGL groups, exhibited higher or more stable Shannon scores compared to the Control group, indicating that candidate strains and combined interventions may have a more direct regulatory effect on community diversity. The Chao1 index reflects community richness, and the results showed differences in Chao1 scores among the groups, suggesting that intervention may affect community structure by altering the richness level of detectable bacteria. The Simpson index measures community dominance / evenness. The results indicate differences in the distribution of Simpson indices among the groups, suggesting that intervention not only affects community richness but may also alter the relative concentration of dominant bacteria and community evenness.

[0089] In summary, ZGG35, ZGG180, and their combined interventions with ZGGL and ZGGH can reshape the composition of gut microbiota at the phylum and family levels and significantly alter the AlPha diversity indices of gut microbiota (Shannon, Chao1, and Simpson).

[0090] 3. Analysis of the composition and diversity of gut microbiota at the phylum level in week 6 At the phylum level, such as Figure 21 As shown in Figure A, the gut microbiota of mice in each group mainly consisted of Verrucomicrobiota, Bacteroidota, and Bacillota. Compared with the mid-stage structure where Bacteroidota was the absolutely dominant phylum, the composition of the microbiota underwent significant remodeling. In the control group, the distribution of the three major phyla was relatively balanced, with Verrucomicrobiota accounting for 35.28%, Bacteroidota for 33.27%, and Bacillota for 11.90%.

[0091] Compared with the normal control group, different probiotic interventions had varying degrees of regulatory effects on the bacterial community structure. Specifically, in the ZGG35 group, the relative abundance of Bacillota increased (36.70%), while Bacteroidota decreased (9.12%), suggesting that this strain may promote the enrichment of Firmicutes-related flora. In contrast, in the ZGG180 group, Bacteroidota (25.40%) and Verrucomicrobiota (24.82%) decreased, while Bacillota (29.57%) increased, indicating that this strain had a significant remodeling effect on the bacterial community structure.

[0092] Regarding the combined bacterial intervention, both the ZGGL and ZGGH groups showed a trend of decreasing Bacteroidota and increasing Bacillota. Specifically, the ZGGL group had a relatively high proportion of Bacteroidota (31.72%), while the ZGGH group had the highest proportion of Verrucomicrobiota (40.28%), suggesting that high-dose combined bacterial intervention may have a promoting effect on this phylum. Furthermore, the combined bacterial intervention also affected the distribution of low-abundance phyla (such as Actinomycetota and Patescibacteria) to some extent, indicating its broad regulatory capacity on the gut microbiota. The GH group showed an increased proportion of Bacillota (27.71%), while Bacteroidota remained at a low level (16.55%).

[0093] At the family level, such as Figure 21 As shown in B, the dominant bacterial communities in each group mainly included Akkermansiaceae, Muribacillus Laceae, Lactobacillaceae, Bifidobacteriaceae, Erysipelotrichaceae, and Lachnospiraceae. In the control group, Akkermansiaceae (approximately 35%) and Muribacillus Laceae (approximately 30%) were the main dominant bacterial communities.

[0094] Compared with the control group, the MuribacuLaceae level in the ZGG35 group was significantly reduced (8.20%), while the unclassified_Clostridia_UCG-014 level was significantly increased (17.78%). In contrast, the MuribacuLaceae level in the ZGG180 group increased to 23.04%, while the Lactobacillaceae level (13.96%) increased, and the Akkermansiaceae level decreased relatively (24.82%). This indicates that the strain has a certain promoting effect on multiple potential beneficial bacterial groups.

[0095] In terms of combined bacterial intervention, both the ZGGL and ZGGH groups showed a synergistic regulatory effect on the dominant bacterial flora. In the ZGGL group, Muribacula laceae had the highest proportion (28.97%), close to or even slightly higher than the control group, while Akkermansiaceae remained at a high level (34.62%), indicating a relatively balanced overall structure. In the ZGGH group, Akkermansiaceae increased to 40.28% (the highest among all groups), and Muribacula laceae also remained at a high level (23.62%), suggesting that high-dose combined bacterial intervention has a more significant enrichment effect in regulating the dominant bacterial flora. In the growth hormone-treated GH group, Muribacula laceae decreased (15.71%), while Akkermansiaceae remained at 33.26%, and Lactobacillaceae increased (14.69%).

[0096] From a temporal dynamic perspective, the gut microbiota structure of each treatment group exhibited distinct phased regulatory characteristics during the intervention process. Compared to the mid-term, the distribution of dominant microbiota in each group tended to stabilize by the end of the 6-week period. This was evident at the phylum level, where the initial fluctuating state gradually transitioned to a relatively balanced structure dominated by Bacteroidota, Bacillota, and Verrucomicrobiota. Simultaneously, at the family level, the relative abundance differences of key microbiota (such as Akkermansiaceae, Muribaculalaceae, and Lactobacillaceae) further widened and tended to stabilize. Specifically, some intervention groups (such as ZGG35 and ZGG180) showed directional changes in the mid-term, exhibiting a more stable microbiota structure in the final stage. The combined microbiota groups (especially ZGGH) showed a gradually strengthening regulatory trend over time, suggesting a cumulative effect on the gut microbiota. Furthermore, the microbiota distribution in the GH group shifted further in the final stage compared to the mid-term, indicating that probiotic intervention can sustainably influence microbiota composition over time. Overall, the gut microbiota response to different treatments showed a clear time dependence, with its structure gradually transitioning from early fluctuations to a relatively stable state in the later stages, suggesting that the intervention effect has the characteristics of gradualness and persistence.

[0097] AlPha diversity assessment results are as follows Figure 22As shown in AC, the Shannon, Chao1, and Simpson indices all exhibited certain differences among the different groups. Regarding the Shannon index, the ZGGL group was generally higher than the Control group, indicating an increase in gut microbiota diversity; the ZGGL group showed a more pronounced upward trend, while the ZGG35 and ZGG180 groups showed relatively smaller changes compared to the Control group. The Chao1 index results showed that the gut microbiota richness of the ZGGL and ZGGH groups was generally higher than the Control group, with larger fluctuations in the ZGGL and ZGGH groups, suggesting a more significant increase in gut microbiota species richness after intervention. Regarding the Simpson index, the overall differences among the groups were relatively small, but the ZGGL group showed a higher index value, indicating better community evenness, while the ZGGH group had a relatively lower Simpson index, indicating a slight increase in gut microbiota dominance in this group. Overall, the combined intervention groups of the candidate strains (ZGGL and ZGGH) had a significant impact on the diversity and richness of the mouse gut microbiota, suggesting that these treatments may have a certain regulatory effect on gut microbiota homeostasis.

[0098] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A probiotic microecological preparation for promoting height growth in children and adolescents, characterized in that: The probiotic microecological preparation mentioned is *Lactobacillus fermentum* with preservation number CGMCC No. 37997. Limosilactobacillus fermentum ZGG35 strain and *Lactobacillus plantarum* with accession number CGMCC No. 37998 Lactiplantibacillus plantarum At least one component of the ZGG180 strain.

2. The probiotic microecological preparation according to claim 1, characterized in that: The microecological preparation is at least one of live bacteria and freeze-dried powder, and the number of live bacteria is 1×10⁻⁶. 6 ~1×10 12 CFU / mL.

3. The probiotic microecological preparation according to claim 1, characterized in that: The cell count ratio of the *Lactobacillus fermentatus* ZGG35 strain to the *Lactobacillus plantarum* ZGG180 strain is (0.5–2):

1.

4. The probiotic microecological preparation according to claim 1, characterized in that: The formulation also includes a pharmaceutically or food-grade carrier or excipient, and the dosage form of the formulation is a gavage, powder, capsule, tablet or liquid beverage.

5. A method for preparing a probiotic microecological preparation according to any one of claims 1 to 4, characterized in that... Includes the following steps: (1) Inoculate fermentation culture medium with Lactobacillus fermentum ZGG35 and / or Lactobacillus plantarum ZGG180 at a volume ratio of 1-5% and culture at 35-40 °C for 12-16 h to obtain lactic acid bacteria culture; (2) Centrifuge the lactic acid bacteria culture obtained in step (1) to obtain the lactic acid bacteria precipitate, add a freeze-drying protectant of the same volume as the lactic acid bacteria precipitate, mix well and place in a -70 to -90℃ refrigerator for overnight pre-freezing, and freeze-dry the pre-frozen sample to obtain a probiotic microecological preparation that promotes the height growth of children and adolescents.

6. The preparation method according to claim 5, characterized in that: The freeze-drying protectant is formulated with 110-125 g / L trehalose, 10-11 g / L sorbitol, 1-2 g / L sodium acetate and 110-130 g / L skim milk powder, dissolved in pure water and sterilized at 120-125°C before use.

7. The use of the probiotic microecological preparation according to any one of claims 1 to 4 in the preparation of products that promote the height growth of children and adolescents, characterized in that: The application of the probiotic microecological preparation in the preparation of products that increase body length and / or increase femur length, improve bone growth and development, improve bone metabolism, regulate growth-related factors, reduce inflammation levels, regulate metabolic indicators, or improve intestinal flora structure.

8. The application according to claim 7, characterized in that: The improvement of bone growth and development includes increasing bone volume, bone volume fraction, trabecular thickness, number of trabecular bones, bone mineral density, and / or trabecular connectivity density. The improvement in bone metabolism includes increasing the levels of bone formation markers such as type I procollagen N-terminal propeptide, osteocalcin, and / or alkaline phosphatase, and / or decreasing the level of bone resorption markers such as type I collagen cross-linked C-terminal peptide.

9. The application according to claim 7, characterized in that: The regulation of growth-related factors includes increasing serum levels of insulin-like growth factor-1, growth hormone, and / or insulin-like growth factor binding protein-3; the regulation of metabolic indicators includes increasing serum levels of insulin, vitamin D, and / or leptin; and the reduction of inflammation levels includes reducing serum levels of interleukin-6.

10. The application according to claim 7, characterized in that... The improvement of gut microbiota structure includes: at the phylum level, increasing the relative abundance of Firmicutes and / or decreasing the relative abundance of Bacteroidetes; at the family level, increasing the relative abundance of Lactobacillus, Trichophyton, Akkermania and / or Erysipelothrix.

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