Bifidobacterium animalis subsp. Lactis BT-G9 with growth promoting function and application of bifidobacterium animalis subsp. Lactis BT-G9

Abstract

The invention discloses a bifidobacterium animalis subsp. Lactis BT-G9 with a growth promoting function and application of the bifidobacterium animalis subsp. Lactis BT-G9, the bifidobacterium animalis subsp. Lactis BT-G9 is preserved in China Center for Type Culture Collection, the preservation date is November 20, 2025, and the preservation number is CCTCC NO: M 20252626. The brand new bifidobacterium animalis subsp. Lactis BT-G9 provided by the invention has good tolerance to gastric juice and intestinal juice, is suitable for survival in gastrointestinal tracts, can effectively inhibit bacteria, and particularly can effectively inhibit campylobacter kirscheri, streptococcus agalactiae and / or propionibacterium acnes. Experimental results show that the strain can promote animal growth and development and improve the autism, especially when the animal bifidobacterium subsp. Lactis BT-G9 and the lactobacillus acidophilus BT-C15 are combined for use, the animal bifidobacterium subsp. Lactis BT-G9 and the lactobacillus acidophilus BT-C15 have a synergistic effect, and the improvement effect on the autism can be remarkably improved. In addition, the extracellular vesicles prepared from the strain can effectively repair the skin barrier function.

Description

Technical Field

[0001] This invention belongs to the technical field of Bifidobacterium animalis subsp. lactis, and particularly relates to a Bifidobacterium animalis subsp. lactis BT-G9 with growth-promoting function and its application. Background Technology

[0002] Childhood growth retardation refers to a child's significant lag behind the normal developmental standards for their age and sex in areas such as height, weight, motor skills, language, or cognitive abilities. It may be related to genetics, nutrition, endocrine disorders, chronic diseases, or environmental factors. Common interventions include: 1) Nutritional support – adjusting diet and supplementing key nutrients (such as iron and vitamin D); 2) Disease treatment – ​​such as lifelong thyroid hormone therapy for hypothyroidism; 3) Growth hormone therapy – only applicable to those with confirmed hormone deficiency and must be strictly followed as prescribed by a doctor; 4) Rehabilitation training – language, motor, or cognitive training to improve functional delays; 5) Psychological support – family involvement and positive guidance to reduce children's psychological stress. However, existing interventions are often ineffective or involve medication with significant side effects, impacting children's physical health.

[0003] Autism is a congenital neurodevelopmental disorder characterized by difficulties in social communication, repetitive and stereotyped behaviors, and narrow interests. It typically manifests in infancy and early childhood and persists throughout life. It is a spectrum disorder encompassing multiple subtypes, with significant individual variations in symptoms and severity. The exact cause of autism is not fully understood, but research suggests a combination of genetic factors (such as gene mutations or chromosomal abnormalities) and environmental factors (such as infections during pregnancy and drug exposure). Neurobiological research has found that the information processing and neural connectivity patterns in the brains of autistic individuals differ from those of typical individuals, potentially leading to abnormal responses to sensory stimuli, language comprehension, and social interaction. Currently, there is no cure for autism, but early intervention can significantly improve function. Examples include behavioral interventions such as Behavior Analysis (ABA) and Floor Time (DIR) to enhance social and self-care abilities; Language and Occupational Therapy to improve communication, sensory regulation, and fine motor skills; Personalized Education Programs (IEPs) to aid school integration; and Medication to alleviate comorbidities such as anxiety and hyperactivity. Existing autism intervention methods are generally ineffective. Drug intervention not only has significant side effects, but also only relieves symptoms and cannot truly improve the condition.

[0004] Bifidobacterium animalis subsp. lactis is a Gram-positive, pleomorphic bacillus, appearing in Y-shaped, V-shaped, curved, or spoon-shaped forms. Its typical morphological characteristics include being a branched bacillus, not forming spores, non-motile, and obligately anaerobic. It is a probiotic belonging to the genus Bifidobacterium, widely distributed in the intestines of humans and animals. They play an important role in maintaining intestinal health and enhancing the immune system. Existing technologies disclose that Bifidobacterium animalis subsp. lactis primarily function to improve intestinal function, promote nutrient absorption, and regulate the immune system; however, they often suffer from limited functionality and poor efficacy. Summary of the Invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a Bifidobacterium lactis subspecies BT-G9 with growth-promoting function and its application.

[0006] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a subspecies of Bifidobacterium animalis lactis with growth-promoting function, wherein the subspecies of Bifidobacterium animalis lactis BT-G9 is deposited at the China Center for Type Culture Collection on November 20, 2025, with accession number CCTCC NO: M 20252626.

[0007] In a second aspect, the present invention provides a composition comprising the aforementioned Bifidobacterium lactis subsp. BT-G9.

[0008] Thirdly, the present invention provides the application of the aforementioned Bifidobacterium lactis subsp. BT-G9 and the aforementioned composition in the preparation of antibacterial agents.

[0009] Preferably, the antibacterial agent is capable of inhibiting Molybditis lappa, Streptococcus agalactiae, and / or Propionibacterium acnes.

[0010] Fourthly, the present invention provides the application of the aforementioned Lactobacillus curvature GG77 and the aforementioned composition in the preparation of products that promote animal growth and development.

[0011] Fifthly, the present invention provides the use of the aforementioned Lactobacillus curvature GG77 and the aforementioned composition in the preparation of products for improving autism.

[0012] As one implementation, the application includes the combined use of Lactobacillus curvature GG77 and Lactobacillus acidophilus BT-C15, wherein Lactobacillus acidophilus BT-C15 is deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2025, with accession number CCTCC NO: M 20252625.

[0013] In a sixth aspect, the present invention provides an extracellular vesicle prepared using the aforementioned Bifidobacterium lactis subsp. BT-G9.

[0014] In a seventh aspect, the present invention provides a method for preparing the aforementioned extracellular vesicles, comprising the following steps: (1) Centrifuge the fermentation broth of Bifidobacterium animalis BT-G9 cultured to the stable growth period to obtain metabolic supernatant and Bifidobacterium animalis BT-G9 cells. Filter the metabolic supernatant to further remove cell debris. (2) Add exosome extraction reagent to the filtrate obtained in step (1), mix well, let stand, then centrifuge to precipitate extracellular vesicles, resuspend the precipitated extracellular vesicles, centrifuge again, and take the supernatant, which is the crude extracted extracellular vesicles. (3) Finally, the crudely extracted extracellular vesicles were transferred into an EPF column, centrifuged, and cell debris and protein aggregates were removed to obtain purified Bifidobacterium animalis subsp. lactis BT-G9 extracellular vesicles.

[0015] Eighthly, the present invention provides the application of the aforementioned extracellular vesicles in the preparation of products for repairing skin barrier function.

[0016] Beneficial Effects: Compared with existing technologies, this invention provides a novel *Bifidobacterium animalis* subspecies *Lactobacillus* BT-G9, which primarily promotes animal growth and development and improves autism. This *Bifidobacterium animalis* subspecies *Lactobacillus* BT-G9 exhibits good tolerance to artificial gastric and intestinal fluids, is suitable for survival in the gastrointestinal tract, and can effectively inhibit bacteria, especially *Aerobiculatus koraiensis*, *Streptococcus agalactiae*, and / or *Propionibacterium acnes*. Experimental results show that this strain can promote animal growth and development and improve autism, especially when *Bifidobacterium animalis* subspecies *Lactobacillus* BT-G9 and *Lactobacillus acidophilus* BT-C15 are used together, the two have a synergistic effect, significantly improving the improvement of autism. Furthermore, extracellular vesicles prepared using this strain can effectively repair skin barrier function. Attached Figure Description

[0017] Figure 1 Gram staining results for Bifidobacterium lactis subsp. BT-G9.

[0018] Figure 2 The hatching rate of each group in Example 5 after 72 hours is represented by the hatching rate.

[0019] Figure 3 The survival rate of each group after 10 days in Example 5 is shown.

[0020] Figure 4 The body length of each group in Example 5 after 10 days.

[0021] Figure 5 The weight of each group in Example 5 after 10 days is shown.

[0022] Figure 6 The distance traveled by the tbr1b zebrafish after exposure to probiotics in Example 6.

[0023] Figure 7 The migration rate of the tbr1b zebrafish after exposure to probiotics in Example 6 is shown.

[0024] Figure 8 The duration of agitation in the tbr1b zebrafish after exposure to probiotics in Example 6.

[0025] Figure 9 The duration of activity of tbr1b zebrafish after exposure to probiotics in Example 6.

[0026] Figure 10 The results show the changes in BMI before and after the experiment in Example 7. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0028] Example 1: Screening of beneficial human bacterial strains and construction of a strain library To obtain beneficial human strains for related functional evaluation experiments and screening of fermentable strains, a human beneficial strain library was first constructed. 152 samples of secretions / excretions and traditional fermented products from healthy individuals were collected. The screening criteria for healthy individuals were: priority was given to adults aged 20-45 years with normal physical examination results, a BMI controlled between 18.5 and 23.9, and exclusion of women from pregnancy and lactation; no history of chronic enteritis, gastritis, gastric ulcers, irritable bowel syndrome, or inflammatory bowel disease in the past year, and no persistent gastrointestinal discomfort such as constipation or diarrhea; no history of infectious diseases such as tuberculosis or hepatitis in the past 6 months, and no diabetes, hypertension, hyperlipidemia, or autoimmune diseases (such as rheumatoid arthritis or Sjögren's syndrome), and normal immune function; no use of oral, intravenous, or topical antibiotics in the past 3 months; no use of probiotic preparations, immunomodulators, hormones, or alcohol in the past month; regular and reasonable diet, no long-term dieting, no long-term smoking; signed informed consent, and voluntarily cooperated with sample collection. Three samples were collected using sterile cotton swabs for each sample, immediately placed in an anaerobic bag, and then stored in an ice box. The samples were promptly returned to the laboratory for further freezing. One sample from each case was selected, 5 ml of MRS medium was added, and the mixture was vortexed thoroughly before adding glycerol solution to achieve a final glycerol concentration of 15%-20%. The solution was then aliquoted into EP tubes, 1 ml per tube, and frozen at -80°C for subsequent strain screening.

[0029] Frozen glycerol-protected samples were serially diluted 1:10. After each serial dilution, the samples were spread onto MRS, TPY, Rogosa, and Columbia blood agar plates, with two spreads for each gradient and four parallel serial dilutions for each sample. The plates were incubated in anaerobic and 5% oxygen environments at 37°C. After 48 hours of incubation, single colonies showing good growth were selected from each gradient plate. MALDI-TOF mass spectrometry was used for rapid preliminary identification of the selected colonies. Known pathogens and opportunistic pathogens were discarded. Probiotics or second-generation probiotics were inoculated into 5 ml of MRS liquid medium for scale-up culture. After 48 hours of incubation at 35-37°C, the OD of the fermentation broth was measured. 600 The bacterial count in the fermentation broth was observed by comparison with a microscope. 2 ml of the strain with a fermentation OD value greater than 0.3 and good growth was added to 2 ml of 30%-40% glycerol solution, pre-cooled, and then frozen at -80℃ for further isolation, purification, and screening. Strains with poor fermentation effect or no obvious cell amplification were discarded. A candidate library of beneficial strains was constructed, and a total of 386 candidate strains were screened.

[0030] Example 2 Screening of in vitro antibacterial strains To screen for strains capable of inhibiting pathogenic bacteria, the strains screened in Example 1 were streaked and purified. Using *Bifidobacterium animalis* subsp. *lactobacter* HN019 as a control strain, both strains were inoculated into 50 ml of MRS liquid medium and cultured at 37°C for 16 h. The fermentation broth was then centrifuged at 10000 rpm for 10 min, and the supernatant and bacterial cells were collected separately. The bacterial cells were washed twice with 0.9% sterile saline, and then OD was prepared using 0.9% sterile saline. 600 The bacterial suspension was prepared at a concentration of 0.5-0.8 μL and stored at 4°C for future use. *Aeromonas coli* BNCC 377479, *Streptococcus agalactiae* BNCC 357545, and *Propionibacterium acnes* ATCC 6919 were selected as test strains for in vitro antibacterial experiments. The inhibition zone test was performed on the fermentation supernatant and bacterial suspension of each strain using the agar perforation method. Lactic acid was used to adjust the pH of the MRS culture medium to be the same as that of the supernatant as a blank control. A 0.05 mg / mL levofloxacin solution served as a positive control for inhibiting *Aeromonas coli* BNCC 377479, *Streptococcus agalactiae* BNCC 357545, and *Propionibacterium acnes* ATCC 6919. The size of the inhibition zone was measured; a zone radius larger than that of the MRS control was considered to have antibacterial activity. After excluding strains with insignificant antibacterial effects, the results of the evaluation of strains with significant antibacterial effects are shown in Table 1.

[0031] The bacterial suspension and supernatant of strain BT-G9 showed significant inhibitory effects on BNCC 377479, BNCC357545, and ATCC 6919, and the antibacterial ability was significantly better than that of Bifidobacterium animalis subsp. lactis HN019.

[0032] Table 1 Evaluation of antibacterial effect

[0033] Note: "-" indicates no obvious inhibition zone, "+" indicates inhibition zone diameter is between 3mm and 5mm, "++" indicates inhibition zone diameter is between 5mm and 10mm, and "+++" indicates inhibition zone diameter is >10mm. a is significantly different from BT-G32, b is significantly different from BT-212, and c is significantly different from HN019.

[0034] Example 3: Preservation, identification, and archiving of strains Based on the BT-G9 strain screened in Example 2, which has the ability to inhibit pathogenic bacteria, it was selected as the target strain.

[0035] (1) Preservation of strains After streaking the selected BT-G9 strain onto MRS agar medium for 48 hours, single colonies were selected and placed in 10 ml of liquid culture medium. This medium was then incubated at 37°C for 12-16 hours, and the OD of the culture medium was measured. 600 When the value is ≥1.2, add glycerol solution to the culture medium at a volume ratio of 1:1. The concentration of the glycerol solution is 30%-40%. Mix well by pipetting and dispensing into 2ml sterile cryovials. Pre-cool at 4℃ for 2h, then pre-freeze at -20℃ for 4h, and finally transfer to a -80℃ freezer or liquid nitrogen for storage.

[0036] (2) Further morphological identification Microscopic observation of the purified bacterial solution revealed that the bacteria were short rod-shaped, appearing singly or in pairs, or in short chains, and without spores. On agar medium, single colonies were round, grayish-white colonies with regular or slightly diffuse edges, smooth, translucent surfaces, and a raised center.

[0037] (3) Gram staining Gram staining result is Gram positive, such as Figure 1 As shown.

[0038] (4) Molecular biological identification of 16S rRNA The 16S rDNA gene sequence of strain BT-G9 was amplified and sequenced using published universal 16S primers (primer sequences: 8F: 5'-AGAFTTTGATCCTGGCTCA-3'; 1510R: 5'-GGTTACCTTGTTACGACTT-3'). The nucleotide sequence of the 16S rDNA of strain BT-G9 is sequence 1 in the sequence listing. After 16S rDNA gene alignment, the similarity rate with Bifidobacterium animalis subsp. lactis in Genebank reached 99%. Combined with microbial systematic identification, strain BT-G9 was identified as a subsp. lactis of Bifidobacterium animalis and named Bifidobacterium animalis subsp. lactis BT-G9, and its 16S rDNA is shown in SEQ ID NO.1.

[0039] (5) Collection of microbial strains Bifidobacterium animalis subspecies Lactobacillus BT-G9, identified by biological analysis, was deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2025, with accession number CCTCC NO: M 20252626, and the deposit address is Wuhan University, Wuhan, Hubei Province, People's Republic of China.

[0040] Example 4: In vitro tolerance test of Bifidobacterium lactis subsp. BT-G9 Mix 0.5 mL of bacterial suspension with 4.5 mL of artificial gastric fluid (final bacterial suspension concentration is 1 × 10⁻⁶). 7 The bacterial suspension (CFU / mL) was incubated statically in a 37°C incubator. Samples were taken after 2 hours and 4 hours, diluted and plated, and then anaerobically incubated at 37°C for 24 hours. The colony count was determined, and the bacterial suspension without artificial gastric juice treatment was used as a control and the survival rate was calculated. Mix 0.5 mL of bacterial suspension with 4.5 mL of artificial intestinal fluid (final bacterial suspension concentration is 1 × 10⁻⁶). 7 The bacterial suspension (CFU / mL) was incubated statically in a 37℃ incubator. Samples were taken after 2h and 4h, diluted and plated, and then anaerobically incubated at 37℃ for 24h. The colony count was determined. The bacterial suspension without artificial intestinal fluid treatment was used as a control and the survival rate was calculated. The results of Bifidobacterium animalis subsp. lactis BT-G9's resistance to gastric and intestinal fluids are shown in Table 2.

[0041] Table 2 Results of acid resistance and artificial intestinal fluid resistance of Bifidobacterium lactis subsp. BT-G9

[0042] Note: Colony counts without artificial gastric or intestinal fluid treatment are compared to 5.0 × 10⁻⁶. 7 CFU / mL.

[0043] As shown in Table 2, the survival rate of Bifidobacterium animalis subsp. lactis BT-G9 remained above 68% after 4 hours of treatment with artificial gastric fluid, indicating its excellent acid resistance and ability to reach the intestines through the stomach. In artificial intestinal fluid, due to trypsin action, the survival rate of Bifidobacterium animalis subsp. lactis BT-G9 was 85%, indicating that it has good tolerance to artificial intestinal fluid and can therefore colonize and survive in the bile duct. This demonstrates that Bifidobacterium animalis subsp. lactis BT-G9 has good tolerance to both artificial gastric and intestinal fluids and is suitable for survival in the gastrointestinal tract.

[0044] Example 5: Effects of Bifidobacterium animalis subsp. Lactobacillus BT-G9 on the growth and development of AB zebrafish larvae. Zebrafish were grouped according to Table 3. The sterile zebrafish incubation method was as follows: AB zebrafish fertilized eggs were transferred to sterile wide-mouth bottles containing AB-GZM solution and incubated at 28℃ for 4.5 h. After rinsing, the fertilized eggs were gently immersed in a 0.4 g povidone-iodine aqueous solution for 1 min, rinsed three times with sterile GZM solution for approximately 10 min, then bleached with 0.02 g sodium hypochlorite for 15 min, rinsed three times with sterile GZM solution for approximately 10 min, and finally transferred to 6-well plates for culture at 28.5℃ under a 14 h light, 10 h dark photoperiod, with half of the culture medium changed daily. Bifidobacterium animalis subsp. lactis BT-G9 and Bifidobacterium lactis HN019 were prepared at a ratio of 1×10⁻⁶. 7 CFU / ml, after sterile AB zebrafish treatment, fertilized zebrafish eggs were placed in a culture medium and BT-G9 or HN019 bacterial solution was added. Half of the culture medium was changed daily. The eggs were hatched together with the control group. At 72 hpf, the hatching rate was calculated as the number of hatched zebrafish divided by the total number of eggs used in the experiment. At 10 dpf, the zebrafish body length, survival rate, and weight were recorded. Weight was recorded by placing 20 fish into 1.5 mL centrifuge tubes (the tube weight was zeroed after weighing), centrifuging, aerating, and weighing on a balance. The survival rate was calculated as the number of surviving zebrafish fry or eggs divided by the initial number of eggs used in the experiment. Results are as follows: Figures 2-5 As shown.

[0045] Compared with the control group, the zebrafish in the sterile group showed poor growth in terms of hatching rate, body length, survival rate, and weight. Under exposure to BT-G9 and HN019, the zebrafish showed significant improvement in growth, with even better growth under exposure to Bifidobacterium animalis subsp. lactis BT-G9. Compared with the sterile group, zebrafish exposed to BT-G9 showed a 17.1% increase in hatching rate at 72 hours, a 43.6% increase in survival rate at 10 days, a 22.3% increase in body length, and a 58.7% increase in weight. This demonstrates the safety and growth-promoting effect of Bifidobacterium animalis subsp. lactis BT-G9 on zebrafish growth, suggesting its potential application in children's growth and development.

[0046] Table 3 Experimental Groups

[0047] Example 6: Intervention of Bifidobacterium animalis subsp. Lactobacillus BT-G9 on autism The autistic TBR1B strain of zebrafish was used as an animal model. Five days after 5 days of gestation, the juvenile TBR1B zebrafish were divided into four groups: the TBR1B group, the *Bifidobacterium lactis* subsp. laccoside BT-G9 intervention group (BT-G9), the *Lactobacillus acidophilus* BT-C15 intervention group (BT-C15), and the 1:1 ratio of *Bifidobacterium lactis* subsp. laccoside BT-G9 to *Lactobacillus acidophilus* BT-C15 intervention group (BT-G9-C15). The control group consisted of AB zebrafish (CK). Each group contained 24 zebrafish. The *Lactobacillus acidophilus* BT-C15 used was deposited on November 20, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCCNO: M 20252625, located at Wuhan University, Wuhan, Hubei Province, People's Republic of China. The intervention groups were treated with 1×10⁻⁶ TBR1B strain zebrafish juveniles at 5 days of gestation. 7 Expose the fish eggs to a probiotic solution at CFU / ml concentration. Place 5 eggs per well in a 6-well plate and perform a half-life change of medium daily, adding 1×10⁻⁶ eggs. 7 CFU / ml corresponds to probiotics. After 10 days of probiotic exposure, 24 juvenile fish were randomly selected from each group and placed in a 48-well plate, one fish per well, for simultaneous testing of both groups. In complete darkness, the movement of the juvenile fish was recorded for 15 minutes using a high-speed infrared camera at a sampling rate of 157 times / s, obtaining video data of movement distance (mm), speed (mm / s), cumulative duration of agitation (s), and cumulative duration of activity (s). Finally, the video data was analyzed using computer tracking software, and the results are as follows: Figures 6-9 As shown.

[0048] Compared with the control group, autistic TBR1B zebrafish showed significantly lower movement distance, speed, cumulative duration of agitation, and cumulative duration of activity. Exposure to probiotics improved these indicators in autistic TBR1B zebrafish. However, exposure to either *Bifidobacterium lactis* subsp. 9 or *Lactobacillus acidophilus* BT-C15 alone did not significantly improve these indicators. Exposure to a 1:1 ratio of *Bifidobacterium lactis* subsp. 9 and *Lactobacillus acidophilus* BT-C15 significantly improved these indicators, with no significant difference compared to the control group. This indicates that *Bifidobacterium lactis* subsp. 9 or *Lactobacillus acidophilus* BT-C15 can help improve the activity of live juvenile autistic TBR1B zebrafish and reduce their anxiety. The 1:1 combination of *Bifidobacterium lactis* subsp. 9 and *Lactobacillus acidophilus* BT-C15, in particular, showed a more significant intervention effect on the activity level and anxiety relief of autistic juvenile zebrafish. Furthermore, a 1:1 combination of Bifidobacterium animalis subsp. lactis BT-G9 and Lactobacillus acidophilus BT-C15 can be used in microecological intervention preparations for children with autism.

[0049] Example 7: Effects of Bifidobacterium animalis subsp. lactis BT-G9 on children's growth and development Sixty-four children aged 5-12 years with anorexia and low body weight (BMI < 15) were randomly divided into an experimental group and a control group. The experimental group was given lyophilized Bifidobacterium animalis subsp. lactis BT-G9 powder (2g per sachet, containing 2.0 × 10⁻⁶ Bifidobacterium animalis subsp. lactis BT-G9) that met food safety requirements. 10 (CFU / g, other excipients were maltodextrin); the control group received a placebo (2g maltodextrin per packet) that met food safety requirements, taken with warm water, one packet each time, twice a day, morning and evening, for 180 days. Changes in weight and height were measured before and after the experiment, and the corresponding BMI changes are shown below. Figure 10 As shown in the figure. According to the statistical results, after supplementing with Bifidobacterium animalis subsp. lactis BT-G9, the experimental group showed a significant increase in BMI compared to the control group, with the average BMI reaching the normal weight range. This indicates that Bifidobacterium animalis subsp. lactis BT-G9 can be used in probiotic intervention preparations for children's development.

[0050] Example 8: Effects of Bifidobacterium animalis subsp. lactis BT-G9 extracellular vesicles (BT-G9 Baevs) on skin barrier function Sensitive skin is characterized by impaired skin barrier function, leading to increased transepidermal water loss, decreased stratum corneum moisture content, and increased susceptibility to inflammation and redness, accompanied by discomfort such as stinging, burning, and itching. Extracellular vesicles of Bifidobacterium lactis subsp. 9 (BT-G9) were prepared to test its intervention on the skin barrier repair function of sensitive skin.

[0051] Bifidobacterium animalis BT-G9 fermentation broth cultured in liquid MRS medium to the stable growth phase (37℃ for 36 h) was centrifuged at 4500×g for 10 min at 4℃ to obtain metabolic supernatant and Bifidobacterium animalis BT-G9 cells. The metabolic supernatant was then filtered through a 0.45 μm filter to further remove cell debris. Extracellular vesicles were extracted from the filtrate using an exosome purification kit: the filtrate was transferred to a new centrifuge tube, ESC (Exosome Concentration Solution) was added, and the mixture was vortexed for 10 s. The mixture was then incubated at 4℃ for 20 h, followed by centrifugation at 10000×g at 4℃ for 1 h to precipitate the extracellular vesicles. The precipitated extracellular vesicles were resuspended in pre-chilled PBS at 4℃, centrifuged at 12000×g at 4℃ for 2 min, and the supernatant was collected as the crude extracted extracellular vesicles. Finally, the crudely extracted extracellular vesicles were transferred to an EPF (Exosome Purification Filter) column and centrifuged at 3000×g for 10 min at 4 ℃ to further remove cell debris and protein aggregates, yielding purified extracellular vesicles of Bifidobacterium animalis subsp. lactis BT-G9 (BT-G9 Baevs), which were then stored at -80℃. 10 μL of the extracted extracellular vesicles was diluted to 30 μL and analyzed using a NanoFCMN30E nanoparticle tracer. The results showed that the average diameter of the extracellular vesicle particles was approximately 191.5 nm, and the total particle concentration was 1.2 × 10⁻⁶. 10 Particles / mL. The prepared extracellular vesicles were diluted to 1.0 × 10⁻⁶. 8 Particles / mL, 5.0×10 8 Particles / mL and 1.0×10 9 The particle count / mL was used for testing, while the animal Bifidobacterium BT-G9 cell suspension obtained after centrifugation was used as a control.

[0052] Thirty-two healthy female subjects aged 25-50 years were recruited. A lactic acid stinging test was used to screen for those meeting the criteria for sensitive skin. These subjects were randomly divided into four groups (A, B, C, and D). After washing their faces morning and evening, groups A, B, and C applied 1 ml of extracellular vesicle solution evenly to their faces, while group D applied 1 ml of washed Bifidobacterium animalis BT-G9 cell suspension (containing 5.0 × 10⁻⁶ viable BT-G9 cells). 8 The CFU / mL solution was evenly applied to the face. The changes in skin TEWL values ​​were tested before use, on day 7 and day 28. The results are shown in Table 4.

[0053] Table 4. Changes in skin TEEWL values ​​before and after sample use.

[0054] Table 4 shows that after 7 and 28 days of use of *Bifidobacterium animalis* subsp. *lactobiformis* BT-G9 extracellular vesicles (BT-G9 Baevs), the skin TEWL (transepidermal water loss) of subjects in groups A, B, and C decreased. After 28 days of intervention, the mean change rates compared to before use were -28.8%, -44.8%, and -33.7%, respectively. Group D showed no significant change after 7 and 28 days of intervention. This indicates that the use of *Bifidobacterium animalis* subsp. *lactobiformis* BT-G9 extracellular vesicles (BT-G9 Baevs) enhanced skin barrier function and significantly reduced transepidermal water loss, demonstrating a good repair effect on sensitive skin. Furthermore, regarding the dosage, group B used 5.0 × 10⁻⁶ ppm. 8 After 28 days of intervention with particles / mL, the TEWL value was significantly lower than before treatment, and the effect was better than that of groups A and C. Therefore, the preferred dosage is 5.0 × 10⁻⁶. 8 Products for intervening in the repair of allergic skin barriers were prepared using extracellular vesicles of Bifidobacterium lactis subsp. BT-G9 (BT-G9 Baevs) at a concentration of particles / mL.

[0055] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A strain of Bifidobacterium animalis (Lactobacillus subsp.) with growth-promoting function, characterized in that, The Bifidobacterium animalis subsp. lactis BT-G9 is deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2025, with accession number CCTCC NO: M20252626.

2. A composition, characterized in that, The composition comprises Bifidobacterium lactis subsp. BT-G9 as described in claim 1.

3. The use of Bifidobacterium lactis subsp. BT-G9 of claim 1 and the composition of claim 2 in the preparation of antibacterial agents.

4. The application according to claim 3, characterized in that, The antibacterial agent can inhibit Molybditis lappa, Streptococcus agalactiae, and / or Propionibacterium acnes.

5. The use of the Lactobacillus curvature GG77 of claim 1 and the composition of claim 2 in the preparation of products that promote animal growth and development.

6. The use of the Lactobacillus curvature GG77 of claim 1 and the composition of claim 2 in the preparation of products for improving autism.

7. The application according to claim 6, characterized in that, The application involves the combined use of Lactobacillus curvature GG77 and Lactobacillus acidophilus BT-C15, which is deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2025, with accession number CCTCC NO: M 20252625.

8. An extracellular vesicle, characterized in that, The extracellular vesicles were prepared using Bifidobacterium lactis subsp. BT-G9 as described in claim 1.

9. The method for preparing extracellular vesicles according to claim 8, characterized in that, Includes the following steps: (1) Centrifuge the fermentation broth of Bifidobacterium animalis BT-G9 cultured to the stable growth period to obtain metabolic supernatant and Bifidobacterium animalis BT-G9 cells. Filter the metabolic supernatant to further remove cell debris. (2) Add exosome extraction reagent to the filtrate obtained in step (1), mix well, let stand, then centrifuge to precipitate extracellular vesicles, resuspend the precipitated extracellular vesicles, centrifuge again, and take the supernatant, which is the crude extracted extracellular vesicles. (3) Finally, the crudely extracted extracellular vesicles were transferred into an EPF column, centrifuged, and cell debris and protein aggregates were removed to obtain purified Bifidobacterium animalis subsp. lactis BT-G9 extracellular vesicles.

10. The use of the extracellular vesicles according to claim 8 in the preparation of products for repairing skin barrier function.

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