Bifidobacterium longum subsp.infantis capable of promoting bone growth and metagen of bifidobacterium longum subsp.infantis

Abstract

The invention discloses bifidobacterium longum subsp.infantis capable of promoting bone growth and a metagen thereof, and belongs to the technical field of biology. The bifidobacterium longum subsp. Infantis CCFM1445 provided by the invention can promote the increase of bone volume fraction, femoral bone trabecula thickness and far-end growth plate thickness of mammals in a growth period, reduce the levels of serum bone resorption markers I-type collagen amino-terminal peptide and tartrate-resistant acid phosphatase, improve the levels of serum bone alkaline phosphatase and parathyroid hormone, and improve the clinical application value of the bifidobacterium longum subsp. Infantis CCFM1445. And the relative abundance of the other bacteria in the intestinal flora and the levels of creatine and indole-3-lactic acid are increased. The thermally inactivated bifidobacterium longum subsp. Infantis CCFM1445 can promote the increase of the volume fraction of femoral bones and the number of bone trabecula of malnourished mice and improve the N-terminal propeptide level of serum I-type procollagen. Therefore, the medicine or health care product prepared from the bifidobacterium longum subsp. Infantis CCFM1445 can be used for assisting in regulating bone metabolism and bone mass, and has a wide market prospect.

Description

Technical Field

[0001] This invention relates to a subspecies of Bifidobacterium longum that can promote bone growth and its postbiotics, belonging to the field of microbial technology. Background Technology

[0002] Early life is a crucial period for rapid bone mineral accumulation and the establishment of skeletal growth patterns, characterized by high bone growth rates and rapid bone turnover. Problems in bone development during this period can have irreversible negative effects on bone health in adulthood, potentially leading to short stature, osteoporosis, and an increased risk of fractures. Therefore, ensuring healthy bone growth in early life is of paramount importance.

[0003] Bifidobacteria are the dominant gut microbiota in early life and have a positive impact on the growth and development of infants and young children. Studies in bone metabolism-related disease models and human trials have found that Bifidobacteria can regulate bone metabolism through multiple pathways, including promoting calcium absorption, regulating gut microbiota structure, protecting the intestinal barrier, and immunomodulation, thereby improving osteoporosis, arthritis, and other bone metabolism-related diseases and maintaining bone health. The regulatory role of Bifidobacteria in bone metabolism gives them the potential to influence early life bone growth. Other Bifidobacteria participate in maintaining a healthy gut microenvironment; indole-3-lactic acid, an indole metabolite produced by Bifidobacteria from tryptophan metabolism, can regulate the body's immune balance. Current technology has not yet disclosed Bifidobacteria that, in both live and inactivated forms, can both promote early life bone growth, regulate the gut microbiota, and increase the levels of creatine and indole-3-lactic acid in gut microbiota metabolites. Summary of the Invention

[0004] This invention provides a strain of Bifidobacterium longum subsp. infantis CCFM1445 and its serotonin, wherein the Bifidobacterium longum subsp. infantis CCFM1445 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65607 and deposit date of December 11, 2024.

[0005] The *Bifidobacterium longum* subsp. *infantile* CCFM1445 was isolated from infant fecal samples from Jinhua, Zhejiang Province, and has the following characteristics:

[0006] (1) Colonies on MRS solid medium are milky white, smooth, and round and raised;

[0007] (2) It helps regulate bone metabolism and promote bone growth, especially the increase in bone mass.

[0008] The present invention also provides a microbial preparation containing the said Bifidobacterium longum subsp. infantis CCFM1445.

[0009] In one embodiment, the number of *Bifidobacterium longum* subsp. infantis CCFM1445 cells in the microbial preparation is not less than 1 × 10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

[0010] The present invention also provides a postbiotic prepared from the Bifidobacterium longum infant subspecies CCFM1445, wherein the postbiotic comprises dead cells of the Bifidobacterium longum infant subspecies CCFM1445.

[0011] The present invention also provides a medicine containing the said Bifidobacterium longum subsp. infantis CCFM1445 or the said microbial preparation or the said postbiotic.

[0012] In one embodiment, the drug contains the aforementioned Bifidobacterium longum subsp. infantis CCFM1445, a drug carrier, and / or pharmaceutical excipients.

[0013] The present invention also provides health products containing the said Bifidobacterium longum infantis subsp. CCFM1445 or the said microbial preparation or the said postbiotic.

[0014] In one embodiment, the health product helps improve bone density.

[0015] The present invention also provides the use of the aforementioned Bifidobacterium longum subsp. infantis CCFM1445 or a microbial preparation containing Bifidobacterium longum subsp. infantis CCFM1445 or the aforementioned postbiotic in the preparation of products that promote bone growth.

[0016] In one embodiment, promoting bone growth refers to regulating bone metabolism and increasing bone mass, including at least one of the following functions:

[0017] (1) In vitro production of indole-3-lactic acid;

[0018] (2) In vitro utilization of L-arabinose;

[0019] (3) Increase the bone volume fraction of individuals during the growth period;

[0020] (4) Increase the thickness of femoral trabecular bone in individuals during the growth period;

[0021] (5) Increase the thickness of the distal femoral growth plate in individuals during the growth period;

[0022] (6) Reduces serum N-terminal peptide levels of type I collagen in individuals during the growth period;

[0023] (7) Increase serum bone alkaline phosphatase levels in individuals during the growth period;

[0024] (8) Reduce the serum tartrate-resistant acid phosphatase level in individuals during the growth period;

[0025] (9) Increase serum parathyroid hormone levels in individuals during the growth period;

[0026] (10) Increase the relative abundance of Alternaria species in the gut microbiota of individuals during the growth period;

[0027] (11) Increase the concentration of creatine in the gut microbiota metabolites of individuals during the growth period;

[0028] (12) Increase the concentration of indole-3-lactic acid in the gut microbiota metabolites of individuals during the growth period;

[0029] (13) Under heat inactivation conditions, increase the bone volume fraction in malnourished individuals;

[0030] (14) Under heat inactivation conditions, the serum N-terminal propeptide level of type I procollagen in malnourished individuals was increased.

[0031] In one embodiment, the product includes pharmaceuticals or health products.

[0032] In one embodiment, the product contains live bacteria of Bifidobacterium longum subsp. infantis CCFM1445, fermentation broth, inactivated bacterial solution, and / or lyophilized powder.

[0033] In one embodiment, the product contains at least 1 × 10⁻⁶ live bacteria of *Bifidobacterium longum* subsp. *infantitidis*. 9 CFU / g or 1×10 9 CFU / mL.

[0034] Beneficial effects:

[0035] 1. The present invention relates to Bifidobacterium longum subsp. infantis CCFM1445, which is isolated from the intestinal flora of healthy infants. This strain has no toxic side effects on humans and can therefore be used to prepare products that regulate bone metabolism and promote bone growth, and has broad market prospects.

[0036] 2. The *Bifidobacterium longum* subsp. *infantis* CCFM1445 provided in this invention can regulate bone metabolism and promote bone growth, especially the increase in bone mass. Specific effects are as follows:

[0037] (1) In vitro production of indole-3-lactic acid;

[0038] (2) In vitro utilization of L-arabinose;

[0039] (3) Increase the bone volume fraction of individuals during the growth period;

[0040] (4) Increase the thickness of femoral trabecular bone in individuals during the growth period;

[0041] (5) Increase the thickness of the distal femoral growth plate in individuals during the growth period;

[0042] (6) Reduces serum N-terminal peptide levels of type I collagen in individuals during the growth period;

[0043] (7) Increase serum bone alkaline phosphatase levels in individuals during the growth period;

[0044] (8) Reduce the serum tartrate-resistant acid phosphatase level in individuals during the growth period;

[0045] (9) Increase serum parathyroid hormone levels in individuals during the growth period;

[0046] (10) Increase the relative abundance of Alternaria species in the gut microbiota of individuals during the growth period;

[0047] (11) Increase the concentration of creatine in the gut microbiota metabolites of individuals during the growth period;

[0048] (12) Increase the concentration of indole-3-lactic acid in the gut microbiota metabolites of individuals during the growth period;

[0049] (13) Under heat inactivation conditions, increase the bone volume fraction in malnourished individuals;

[0050] (14) Under heat inactivation conditions, it increases the level of N-terminal propeptide of type I procollagen in the serum of malnourished individuals and the effect is better than that of Bifidobacterium longum subsp. infantis CCFM1269 in patent CN116687980A.

[0051] Preservation of biological materials

[0052] A strain of *Bifidobacterium longum* subsp. *infantis*, CCFM1445, taxonomically named *Bifidobacterium longum* subsp. *infantis*, was deposited on December 11, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 65607), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0053] Figure 1 : In vitro indole-3-lactic acid production levels of different strains;

[0054] Figure 2 Micro-CT images of the distal femur of mice in different groups during their growth period;

[0055] Figure 3 The ratio of femoral bone volume fraction to body weight in mice of different groups during their growth period;

[0056] Figure 4 Thickness of femoral trabeculae in mice during different growth periods;

[0057] Figure 5 Toluidine blue staining of the distal femur of mice in different growth stages;

[0058] Figure 6 Thickness of the distal femoral growth plate in mice of different groups during their growth period;

[0059] Figure 7 Serum N-terminal peptide (NTX) levels of type I collagen in mice during different growth phases;

[0060] Figure 8 Serum bone alkaline phosphatase (BALP) levels in mice of different growth stages;

[0061] Figure 9 Serum tartrate-resistant acid phosphatase (TRACP) levels in mice of different growth stages;

[0062] Figure 10 Serum parathyroid hormone (PTH) levels in mice of different growth stages;

[0063] Figure 11 The relative abundance of Alistipes in the gut microbiota of mice in different groups during their growth period;

[0064] Figure 12 The relative abundance of creatine in the gut microbiota metabolites of mice in different groups during their growth period;

[0065] Figure 13 Concentration of indole-3-lactic acid in the intestinal flora metabolites of mice in different groups during their growth period;

[0066] Figure 14 Femoral bone volume fraction in malnourished mice of different groups;

[0067] Figure 15 Number of femoral trabeculae in malnourished mice in different groups;

[0068] Figure 16 Serum N-terminal propeptide (PINP) levels of type I procollagen in malnourished mice from different groups.

[0069] Figures 1-16 In the table, "*" indicates a significant difference compared to the blank control group (p<0.05), "**" indicates a significant difference compared to the blank control group (p<0.01), "***" indicates a significant difference compared to the blank control group (p<0.001), and "****" indicates a significant difference compared to the blank control group (p<0.0001). The error is presented in the form of Mean±SEM. Detailed Implementation

[0070] The mice used in the following examples were SPF (Specific pathogen free) grade BALB / C mice, purchased from Vital River Laboratory Animal Technology Co., Ltd.; the ELISA kits for the indicators such as type I procollagen N-terminal propeptide, type I collagen N-terminal peptide, bone alkaline phosphatase, tartrate-resistant acid phosphatase, osteoprotegerin, and parathyroid hormone used in the following examples were purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.

[0071] The culture media involved in the following examples are as follows:

[0072] MRS liquid culture medium: tryptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, anhydrous sodium acetate 5 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate monohydrate 0.25 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate trihydrate 2.6 g / L, Tween 80 1 mL / L, cysteine ​​hydrochloride 0.5 g / L.

[0073] MRS solid culture medium: tryptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, anhydrous sodium acetate 5 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate monohydrate 0.25 g / L, diammonium citrate 2 g / L, dipotassium hydrogen phosphate trihydrate 2.6 g / L, Tween 80 1 mL / L, cysteine ​​hydrochloride 0.5 g / L, agar 20 g / L.

[0074] Example 1: Screening, identification, culture and preservation of Bifidobacterium longum subsp. infantis CCFM1445

[0075] (1) Screening

[0076] Take 0.5g of fecal samples from healthy infants in Jinhua, Zhejiang Province, preserved in 30% (v / v) glycerol. After serial dilution, spread the samples onto MRS solid medium (containing 0.5g / L cysteine) and anaerobic culture at 37℃ for 72h. Observe and record the colony morphology. Pick milky white, smooth, round, and convex colonies and streak them onto MRS solid medium (containing 0.5g / L cysteine). Purify the colonies under anaerobic conditions at 37℃. Repeat this operation 3 times to obtain purified single colonies. Pick purified colonies and inoculate them into MRS liquid medium (containing 0.5g / L cysteine). Incubate the colonies anaerobically at 37℃ for 24h to obtain strain CCFM1445.

[0077] (2) Identification

[0078] The genome of the selected strain was extracted, and the 16S rDNA of the strain was amplified and sequenced (the nucleotide sequence of the amplified 16S rDNA is shown in SEQ ID NO.1). The obtained sequence was compared with the nucleic acid sequence in NCBI-Blast. The results showed that the strain was Bifidobacterium longum subsp. infantis. After genome draft sequencing and phylogenetic analysis, it was further confirmed to be Bifidobacterium longum subsp. infantis and named it Bifidobacterium longum subsp. infantis CCFM1445.

[0079] (3)Save

[0080] Single colonies of *Bifidobacterium longum* subsp. infantis CCFM1445 were picked and inoculated into MRS liquid medium, and anaerobically cultured at 37°C for 24 h to obtain bacterial suspension. Under aseptic conditions, the bacterial suspension was placed in cryovials and centrifuged at 6000 rpm for 3 min to collect the bacterial cells. The cells were washed with sterile PBS buffer and centrifuged at 6000 rpm for 3 min to obtain the washed bacterial cells; this operation was repeated 3 times. The bacterial cells were resuspended in sterile 30% (v / v) glycerol, and the cryovials were stored at -80°C. *Bifidobacterium longum* subsp. infantis CCFM1445 has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 65607.

[0081] Example 2: In vitro in vitro indole-3-lactic acid production capacity of Bifidobacterium longum subsp. infantis CCFM1445

[0082] (1) Take a bacterial culture of Bifidobacterium longum subsp. infantis CCFM1445 from the cryopreservation tube and streak it on MRS solid medium. Incubate anaerobicly at 37°C for 48 h to obtain a single colony. Pick a single colony and inoculate it into MRS liquid medium. Incubate anaerobicly at 37°C for 36 h to obtain an activated solution. Inoculate the activated solution into MRS liquid medium at an inoculation rate of 1% (v / v). Incubate anaerobicly at 37°C for 24 h. Repeat the activation culture 3 times to obtain the activated bacterial culture.

[0083] (2) The activated bacterial culture obtained in step (1) was inoculated into MRS liquid medium supplemented with 10 mmol / L tryptophan at an inoculation rate of 1% (v / v). After anaerobic culture at 37°C for 48 h, the fermentation broth was centrifuged and the supernatant was collected to obtain the bacterial culture supernatant. The indole-3-lactic acid level in the culture supernatant of Bifidobacterium longum subsp. infantis CCFM1445 was determined by high performance liquid chromatography. The commercially available strain Bifidobacterium longum subsp. infantis ATCC15697 was used as the control strain, and the culture method was the same as above.

[0084] The test results showed that ( Figure 1The concentration of indole-3-lactic acid in the culture supernatant of the commercial strain *Bifidobacterium longum* subsp. *infantii* ATCC15697 was 15.55 μg / mL, while the concentration of indole-3-lactic acid in the culture supernatant of *Bifidobacterium longum* subsp. *infantii* CCFM1445 was 27.08 μg / mL. The indole-3-lactic acid level in the culture supernatant of CCFM1445 was 1.74 times that of the culture supernatant of the commercial strain ATCC15697.

[0085] Example 3: In vitro L-arabinose utilization capacity of Bifidobacterium longum subsp. infantis CCFM1445

[0086] The *Bifidobacterium longum* subsp. *infantica* CCFM1269 used in this embodiment has been disclosed in patent number CN116687980A, with accession number GDMCC No: 62839.

[0087] The growth of three strains of *Bifidobacterium longum* subsp. *infantii* on a medium with six carbohydrates as the sole carbon source was determined. These included 2'-fucosylated lactose (2'-FL), 3'-sialylated lactose (3'-SL), lact-N-tetrasaccharide (LNT), L-arabinose, L-fucose, and galactooligosaccharides (GOS). A glucose-free mMRS medium was prepared, and bromocresol purple was added as an indicator. The carbohydrate solution was filtered through a 0.22 μm sterile membrane filter and added to the medium at a final concentration of 1% (w / w). 200 μL of the medium with different carbohydrates replacing glucose and the negative control medium were aliquoted into sterile 96-well plates. The strain activation procedure was the same as in Example 2. The activated bacterial solution was inoculated into 96 wells at a 1% (v / v) inoculation rate. Three parallel wells were measured for each strain and each carbohydrate. After anaerobic incubation at 37°C for 36–48 h, the color change of the medium in the wells was observed. The experiment was independently repeated three times.

[0088] *Bifidobacterium longum* subsp. *infantii* exhibits a preference for HMOs but a weaker ability to utilize plant-derived carbohydrates such as L-arabinose. Carbohydrate utilization experiments (Table 1) showed that all three *Bifidobacterium longum* subsp. *infantii* strains could grow in the presence of 2'-FL, 3'-SL, LNT, GOS, and L-fucose, but their ability to utilize L-arabinose differed. *Bifidobacterium longum* subsp. *infantii* ATCC15697 and CCFM1269 could not utilize L-arabinose, while *Bifidobacterium longum* subsp. *infantii* CCFM1445 could metabolize L-arabinose to produce acid. This result indicates that *Bifidobacterium longum* subsp. *infantii* CCFM1445 has a stronger ability to utilize plant-derived carbohydrates.

[0089] Table 1 Carbohydrate utilization profiles of different strains

[0090]

[0091] Note: "+" strains utilize this carbohydrate to produce acid; "-" strains do not utilize this carbohydrate to produce acid.

[0092] Example 4: Preparation of a suspension of Bifidobacterium longum subsp. infantis CCFM1445

[0093] (1) The activation culture steps are the same as in Example 2.

[0094] (2) The activated bacterial culture obtained in step (1) was inoculated into MRS liquid medium at an inoculation rate of 1% (v / v). After incubation at 37°C for 24 h, the fermentation broth was obtained. The fermentation broth was centrifuged to collect the bacterial cells. The cells were washed twice with PBS buffer at pH 7.4, resuspended in physiological saline, and adjusted to a viable count of 1×10⁻⁶. 9 CFU / mL was used to obtain a bacterial suspension for subsequent animal experiments.

[0095] Example 5: Regulatory effect of Bifidobacterium longum infant subsp. CCFM1445 on bone volume fraction in growing mice.

[0096] The treatment steps for Bifidobacterium longum infant subsp. *species* in growing mice are as follows:

[0097] The experimental subjects were young mice in their growth period. The young mice were given oral gavage starting at 1 week of age and were divided into a blank control group and a group receiving *Bifidobacterium longum* subsp. infantis CCFM1445. After weaning at 3 weeks of age, male and female young mice were separated and oral gavage continued until 5 weeks of age, at which point the young mice were sacrificed. Specific grouping and treatment details are as follows:

[0098] Control group: From day 8 to 21 after birth, the pups were allowed to freely consume breast milk and were also given 0.1 mL of physiological saline by gavage as a control; from day 22 to 35, the pups were allowed to freely eat and drink and were also given 0.1 mL of physiological saline by gavage as a control.

[0099] Bifidobacterium longum infant subspecies CCFM1445 group: pups were allowed free access to breast milk and were given 0.1 mL of bacterial suspension by gavage from day 8 to day 21 after birth; from day 22 to day 35, they were allowed free access to food and were given 0.1 mL of bacterial suspension by gavage.

[0100] The body weight of juvenile mice was recorded at 35 days of age. After sacrifice, whole blood was collected and serum obtained by centrifugation. The left femur was harvested, muscle removed, and fixed in 4% paraformaldehyde solution for 48 hours. Micro-computed tomography (micro-CT) was then used to scan the trabecular bone region of the distal femur and measure relevant parameters. The fixed left femur was used for tissue sectioning and staining. After decalcification with EDTA solution, the tissue was embedded and cut into 5 μm thick sections, which were then stained with toluidine blue.

[0101] Bone volume fraction represents the ratio of trabecular bone volume to bone tissue volume, directly reflecting changes in bone mass. The ratio of bone volume fraction to body weight eliminates the interference of body weight on the results. The measurement results are shown below. Figure 2-3 .like Figure 2 As shown, the femoral bone mass of both female and male juvenile mice in the CCFM1445 group was significantly greater than that in the blank control group. The results of the bone volume fraction to body weight ratio determination showed ( Figure 3 The ratio of bone volume fraction to body weight in female juvenile mice in the blank control group was 0.6490, while that in the CCFM1445 group was 0.6854. The ratio of bone volume fraction to body weight in male juvenile mice in the blank control group was 0.6414, while that in the CCFM1445 group was 0.6735. The ratios of bone volume fraction to body weight in both female and male juvenile mice in the CCFM1445 group were significantly higher than those in the blank control group.

[0102] Trabecular bone thickness refers to the average thickness of the trabecular bones, reflecting their fineness and thickness, and is one of the factors affecting bone strength. The results of trabecular bone thickness measurements show (...) Figure 4 The trabecular bone thickness of female juvenile mice in the blank control group was 0.1087 μm, while that of female juvenile mice in the CCFM1445 group was 0.1512 μm. The trabecular bone thickness of male juvenile mice in the blank control group was 0.1090 μm, while that of male juvenile mice in the CCFM1445 group was 0.1472 μm. The trabecular bone thickness of female and male juvenile mice in the CCFM1445 group was significantly higher than that of the blank control group by 1.39 times and 1.35 times, respectively.

[0103] The above results indicate that the present invention, *Bifidobacterium longum* subsp. *infantis* CCFM1445, can promote an increase in the femoral bone volume fraction in growing mice. This increase in bone volume fraction is achieved by increasing the thickness of the trabecular bone, and the thicker trabecular bone indicates that the femur of the CCFM1445 group of mice has higher bone strength and fracture resistance.

[0104] Example 6: Regulation of growth plate thickness in growing mice by Bifidobacterium longum infant subsp. CCFM1445

[0105] The growth plate is a specialized cartilage structure located at the distal end of the femur. It promotes longitudinal bone growth through chondrocyte proliferation, hypertrophy, and matrix mineralization, and is a crucial site for bone development during the growth period. Growth plate thickness reflects the proliferative and differentiation activity of chondrocytes. Toluidine blue staining, which stains chondrocytes and matrix blue-purple, can be used to measure growth plate thickness. Figure 5 As shown, the red area represents the growth plate. The growth plate thickness measurement results show ( Figure 6The average growth plate thickness of female pups in the blank control group was 186.2 μm, while that in the CCFM1445 group was 192.9 μm. The average growth plate thickness of male pups in the blank control group was 187.8 μm, while that in the CCFM1445 group was 199.1 μm. The growth plate thickness of both female and male pups in the CCFM1445 group was significantly higher than that in the blank control group, by 1.04 times and 1.06 times, respectively. This indicates that *Bifidobacterium longum* subsp. *infantis* CCFM1445 can significantly promote the proliferation and differentiation activity of chondrocytes in growing mice.

[0106] Example 7: Regulatory effect of Bifidobacterium longum subsp. infantis CCFM1445 on N-terminal peptide of type I collagen in the serum of growing mice.

[0107] Type I collagen N-terminal peptide (NTX) is associated with osteoclast-mediated bone resorption and reflects the level of bone resorption; it is a commonly used clinical marker of bone resorption. Figure 7 As shown, the NTX level in female pups in the blank control group was 7.40 nmol / L, while the NTX level in female pups in the CCFM1445 group was 6.53 nmol / L; the NTX level in male pups in the blank control group was 6.17 nmol / L, while the NTX level in male pups in the CCFM1445 group was 5.28 nmol / L. Compared with the blank control group, CCFM1445 intervention significantly reduced the serum NTX level, a marker of bone resorption, in female and male pups by 0.88-fold and 0.86-fold, respectively. This demonstrates that the intervention of Bifidobacterium longum infantis subsp. CCFM1445 in this invention can inhibit bone resorption, making bone formation metabolism higher than bone resorption metabolism to a certain extent, thereby promoting bone growth in growing mice.

[0108] Example 8: Regulatory effect of Bifidobacterium longum infantis subsp. CCFM1445 on serum bone alkaline phosphatase in growing mice.

[0109] Bone alkaline phosphatase is an important biomarker for bone formation and osteoblast activity, participating in the mineralization process of the bone matrix. Serum bone alkaline phosphatase measurement results show ( Figure 8 In the control group, the serum BALP level in female pups was 3.31 ng / mL, while in the CCFM1445 group it was 3.74 ng / mL. In the control group, the serum BALP level in male pups was 3.81 ng / mL, while in the CCFM1445 group it was 4.36 ng / mL. The serum BALP levels in the CCFM1445 group were significantly higher than those in the control group (1.13 times and 1.15 times, respectively). These results indicate that intervention with *Bifidobacterium longum* subsp. infantis CCFM1445 can effectively increase osteoblast activity and promote bone formation to a certain extent.

[0110] Example 9: Regulatory effect of Bifidobacterium longum subsp. infantis CCFM1445 on tartrate-resistant acid phosphatase in growing mice

[0111] Tartrate-resistant acid phosphatase is an enzyme mainly secreted by osteoclasts and can directly reflect osteoclast activity. Serum TRACP assay results show ( Figure 9 In the control group, the serum TRACP level in female pups was 4.50 ng / mL, while in the CCFM1445 group it was 3.53 ng / mL. In the control group, the serum TRACP level in male pups was 4.72 ng / mL, while in the CCFM1445 group it was 3.57 ng / mL. The serum TRACP levels in the CCFM1445 group were significantly lower than those in the control group (0.78-fold and 0.76-fold, respectively). These results indicate that intervention with *Bifidobacterium longum* subsp. infantis CCFM1445 can effectively reduce osteoclast activity and inhibit bone resorption to some extent.

[0112] Example 10: Regulatory effect of Bifidobacterium longum infantis subsp. CCFM1445 on serum parathyroid hormone in growing mice.

[0113] Short-term, low-dose increases in parathyroid hormone levels can promote osteoblast proliferation and differentiation, increasing bone formation. Serum parathyroid hormone measurements show ( Figure 10 In the control group, the serum PTH level in female pups was 1183.12 pg / mL, while the serum OPG level in the CCFM1445 group was 1280.71 pg / mL. In the control group, the serum PTH level in male pups was 1073.84 pg / mL, while the serum PTH level in the CCFM1445 group was 1394.00 pg / mL. The serum PTH levels in the CCFM1445 group were significantly higher than those in the control group (1.08 times and 1.30 times, respectively). *Bifidobacterium longum* subsp. infantis CCFM1445 can activate osteoblasts and promote bone formation by upregulating serum parathyroid hormone levels.

[0114] Example 11: Regulatory effect of Bifidobacterium longum infantis subsp. CCFM1445 on gut microbiota in growing mice

[0115] The grouping for the animal experiments was the same as in Example 5. After the experiment, mouse feces were collected, and genomic DNA was extracted from the feces using a FastDNA Spin Kit (MP Biomedical, USA). Specific PCR amplification of the V3-V4 region of the extracted genomic DNA was performed, followed by 16S rDNA sequencing. Changes in fecal microbiota were analyzed, and the results are shown below. Figure 11After gavage administration of CCFM1445, the relative abundance of Alistipes in the feces of male and female mice was significantly higher than that in the control group by 1.42 times and 1.40 times, respectively. The relative abundance of Alistipes in the control group of female mice was 0.1102, while that in the CCFM1445 group was 0.1567; the relative abundance of Alistipes in the control group of male mice was 0.1421, while that in the CCFM1445 group was 0.1985.

[0116] Example 12: Regulatory effect of Bifidobacterium longum infantis subsp. CCFM1445 on intestinal creatine in growing mice

[0117] The grouping for the animal experiments was the same as in Example 5. After the experiment, mouse feces were collected and analyzed using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) for non-targeted metabolomics. The results are shown in [Figure 5]. Figure 12 . Figure 12 The relative abundance in the table represents the peak area after sample weight normalization and Log10 transformation. The peak area is positively correlated with the metabolite concentration. After gavage with CCFM1445, the concentration of fecal creatine in both male and female mice was significantly higher than that in the blank control group. The relative abundance of creatine in the blank control group of female mice was 7.20, while that in the CCFM1445 group was 7.79. The relative abundance of creatine in the blank control group of male mice was 7.16, while that in the CCFM1445 group was 7.60.

[0118] Example 13: Regulatory effect of Bifidobacterium longum infantis subsp. CCFM1445 on indole-3-lactic acid in the intestine of growing mice

[0119] The grouping for the animal experiments was the same as in Example 5. Feces were collected from mice on day 28 after birth, and the level of indole-3-lactic acid in the feces was determined by high-performance liquid chromatography. The results are shown below. Figure 13 The concentration of indole-3-lactic acid in the female mouse control group was 0.1627 μg / mL, while that in the CCFM1445 group was 0.2006 μg / mL. In the male mouse control group, the concentration of indole-3-lactic acid was 0.1090 μg / mL, while that in the CCFM1445 group was 0.1472 μg / mL. The concentrations of indole-3-lactic acid in the feces of female and male juvenile mice in the CCFM1445 group were 1.23 times and 1.35 times higher than those in the control group, respectively.

[0120] Example 14: Regulation of bone volume fraction in malnourished mice by heat-inactivated Bifidobacterium longum subsp. infantis CCFM1445

[0121] The *Bifidobacterium longum* subsp. *infantica* CCFM1269 used in this embodiment has been disclosed in patent number CN116687980A, with accession number GDMCC No: 62839.

[0122] (1) Preparation of heat-inactivated strains

[0123] The steps for activating and culturing the bacterial strain are the same as in Example 2. The activated bacterial solution was inoculated into MRS liquid medium at an inoculum rate of 1% (v / v) and cultured at 37°C for 24 hours to obtain the fermentation broth. The fermentation broth was centrifuged to collect the bacterial cells, washed twice with PBS buffer (pH 7.4), and then heat-inactivated into a suspension at 65°C for 30 minutes. The supernatant was removed by centrifugation, and the suspension was resuspended in physiological saline and freeze-dried for storage. The cells were then reconstituted in physiological saline and adjusted to a cell count of 1 × 10⁻⁶. 9 The cell / mL data is used for subsequent animal experiments.

[0124] (2) Intervention of heat-inactivated strains in malnourished growing mice

[0125] A malnutrition model was established using a low-fat, low-protein diet (4% protein, 2% fat). The experimental subjects were young mice in their growth period. Starting at one week of age, the mice were administered the malnutrition model via gavage and divided into a blank control group, a model group, a heat-inactivated *Bifidobacterium longum* subsp. infantis CCFM1445 group (HK CCFM1445), and a heat-inactivated *Bifidobacterium longum* subsp. infantis CCFM1269 group (HK CCFM1269). After weaning at three weeks of age, male and female mice were separated and gavage continued until six weeks of age, at which point the mice were sacrificed. Specific grouping and treatment details are as follows:

[0126] Control group: From day 8 to 21 after birth, the pups were allowed to freely consume breast milk and were also given 0.1 mL of physiological saline by gavage as a control; from day 22 to 42, the pups were allowed to freely consume the control group feed (AIN-93G) and were also given 0.1 mL of physiological saline by gavage as a control.

[0127] Model group: From day 8 to 21 after birth, the young mice were allowed to freely consume breast milk and were also given 0.1 mL of physiological saline by gavage as a control; from day 22 to 42, they were allowed to freely consume a low-fat, low-protein diet and were also given 0.1 mL of physiological saline by gavage as a control.

[0128] Heat-inactivated Bifidobacterium longum infant subspecies CCFM1445 group: From day 8 to 21 after birth, the pups were allowed to freely consume breast milk and were given 0.1 mL of heat-inactivated bacterial suspension by gavage; from day 22 to 42, they were allowed to freely consume low-fat, low-protein diet and were given 0.1 mL of heat-inactivated bacterial suspension by gavage.

[0129] Heat-inactivated Bifidobacterium longum infant subspecies CCFM1269 group: From day 8 to 21 after birth, the pups were allowed to freely consume breast milk and were given 0.1 mL of heat-inactivated bacterial suspension by gavage; from day 22 to 42, they were allowed to freely consume low-fat, low-protein diet and were given 0.1 mL of heat-inactivated bacterial suspension by gavage.

[0130] The results of the determination of the trabecular bone volume fraction of the femur showed that ( Figure 14A low-fat, low-protein diet significantly reduced bone volume fraction in young mice. Compared to the model group, intervention with heat-inactivated CCFM1445 significantly increased bone volume fraction by 1.34 times, while intervention with heat-inactivated CCFM1269 increased it by 1.25 times. To further eliminate the interference of body weight, the ratio of bone volume fraction to body weight was statistically analyzed. The ratio of bone volume fraction to body weight in the model group was 0.3502, in the heat-inactivated CCFM1445 group it was 0.4667, and in the heat-inactivated CCFM1269 group it was 0.4474. The ratio of bone volume fraction to body weight in the heat-inactivated CCFM1445 group was 4.31% higher than that in the heat-inactivated CCFM1269 group.

[0131] Trabecular bone count represents the average number of intersections between bone and non-bone tissue per millimeter, reflecting the spatial distribution density of trabecular bone. The results of trabecular bone count measurements show ( Figure 15 The number of trabeculae in the model group was 0.42571 / mm, the number of trabeculae in the heat-inactivated CCFM1445 group was 0.46671 / mm, and the number of trabeculae in the heat-inactivated CCFM1269 group was 0.44741 / mm. The number of trabeculae in the heat-inactivated CCFM1445 group was 4.74% higher than that in the heat-inactivated CCFM1269 group.

[0132] The above results indicate that the present invention, Bifidobacterium longum subsp. infantis CCFM1445, can promote the increase of femoral bone volume fraction and trabecular bone number in malnourished mice under heat inactivation conditions, and its effect is better than that of heat-inactivated Bifidobacterium longum subsp. infantis CCFM1269.

[0133] Example 15: Regulatory effect of heat-inactivated Bifidobacterium longum subsp. infantis CCFM1445 on N-terminal propeptide of serum type I procollagen.

[0134] Type I procollagen N-terminal propeptide (PINP) is a specific marker of bone formation, reflecting osteoblast activity, and is one of the commonly used bone metabolism markers in clinical practice. Figure 16As shown, the PINP level in female pups in the model group was 93.60 ng / mL, the PINP level in female pups in the heat-inactivated CCFM1445 group was 118.95 ng / mL, and the PINP level in female pups in the heat-inactivated CCFM1269 group was 93.80 ng / mL; the PINP level in female pups in the heat-inactivated CCFM1445 group was 26.76% higher than that in female pups in the heat-inactivated CCFM1269 group. The PINP level in male pups in the model group was 73.35 ng / mL, the PINP level in male pups in the heat-inactivated CCFM1445 group was 96.39 ng / mL, and the PINP level in male pups in the heat-inactivated CCFM1269 group was 67.50 ng / mL; the PINP level in male pups in the heat-inactivated CCFM1445 group was 42.80% higher than that in male pups in the heat-inactivated CCFM1269 group. The results showed that the heat-inactivated Bifidobacterium longum subsp. infantis CCFM1445 of the present invention could significantly promote the increase of serum type I procollagen N-terminal propeptide level in malnourished mice, while heat-inactivated Bifidobacterium longum subsp. infantis CCFM1269 had no significant effect on serum type I procollagen N-terminal propeptide level in malnourished mice.

[0135] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. *Bifidobacterium longum* subsp. *infantis* CCFM1445, characterized by: The described *Bifidobacterium longum* subspecies *CCFM1445* is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65607, and the deposit date is December 11, 2024.

2. A microbial preparation containing the Bifidobacterium longum subsp. infantis CCFM1445 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, In the aforementioned microbial preparation, the bacterial count of *Bifidobacterium longum* subsp. infantis CCFM1445 is not less than 1 × 10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

4. The postbiotic prepared from *Bifidobacterium longum* subsp. *infantii* CCFM1445 according to claim 1, characterized in that, The metabiotic includes dead cells of the Bifidobacterium longum infant subspecies CCFM1445.

5. A medicine containing the Bifidobacterium longum subsp. infantis CCFM1445 of claim 1, or the microbial preparation of claim 2 or 3, or the postbiotic of claim 4.

6. The pharmaceutical product according to claim 5, characterized in that, The drug contains the aforementioned Bifidobacterium longum infantis subsp. CCFM1445, a drug carrier, and / or pharmaceutical excipients.

7. A health product for improving bone density, characterized in that, Contains the Bifidobacterium longum infantis subsp. CCFM1445 as described in claim 1, or the microbial preparation as described in claim 2 or 3, or the postbiotic as described in claim 4.

8. The use of the *Bifidobacterium longum* subsp. infantis CCFM1445 as described in claim 1, or the microbial preparation as described in claim 2 or 3, or the metabiotic as described in claim 4, in the preparation of products promoting bone growth, characterized in that... The promotion of bone growth refers to regulating bone metabolism and increasing bone mass, including at least one of the following functions: (1) In vitro production of indole-3-lactic acid; (2) Increase the bone volume fraction of individuals during the growth period; (3) Increase the thickness of femoral trabecular bone in individuals during the growth period; (4) Increase the thickness of the distal femoral growth plate in individuals during the growth period; (5) Reduce the level of type I collagen N-terminal peptide in the serum of individuals during the growth period; (6) Increase serum bone alkaline phosphatase levels in individuals during the growth period; (7) Reduces the serum tartrate-resistant acid phosphatase level in individuals during the growth period; (8) Increase serum parathyroid hormone levels in individuals during the growth period; (9) Increase the relative abundance of Alternaria species in the gut microbiota of individuals during the growth period; (10) Increase the concentration of creatine in the gut microbiota metabolites of individuals during the growth period; (11) Increase the concentration of indole-3-lactic acid in the gut microbiota metabolites of individuals during the growth period; (12) Increase bone volume fraction in malnourished individuals; (13) Increase serum N-terminal propeptide levels of type I procollagen in malnourished individuals.

9. The application according to claim 8, characterized in that, The product contains live bacteria of Bifidobacterium longum subsp. infantis CCFM1445, fermentation broth, inactivated bacterial solution and / or lyophilized powder.

10. The application according to claim 8 or 9, characterized in that, The product contains at least 1 × 10⁻⁶ cells of *Bifidobacterium longum* subsp. *infantitidis*. 9 CFU / g or 1×10 9 CFU / mL.

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