Lactobacillus acidophilus BT-C15 with inflammation relieving and mucosal barrier repairing functions and application of lactobacillus acidophilus BT-C15

Abstract

The invention discloses lactobacillus acidophilus BT-C15 with inflammation relieving and mucosal barrier repairing functions and application of the lactobacillus acidophilus BT-C15, the lactobacillus acidophilus BT-C15 is preserved in the China Center for Type Culture Collection, the preservation date is November 20, 2025, and the preservation number is CCTCC NO: M 20252625. The invention provides a brand new lactobacillus acidophilus BT-C15 which mainly has the effects of relieving inflammation and whitening the skin. The lactobacillus acidophilus BT-C15 has good tolerance to gastric juice and intestinal juice, is high in cell adhesion capacity, is suitable for survival in gastrointestinal tracts, can effectively inhibit bacteria, and particularly can effectively inhibit campylobacter Korotkoff, streptococcus agalactiae and / or propionibacterium acnes. Experimental results show that the strain can improve colitis, fade spots, whiten skin and improve autism, especially when the lactobacillus acidophilus BT-C15 and the bifidobacterium animalis subsp. Lactis BT-G9 are used in a combined mode, the lactobacillus acidophilus BT-C15 and the bifidobacterium animalis subsp. Lactis BT-G9 have a synergistic effect, and the improvement effect on the autism can be remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of Lactobacillus acidophilus technology, and particularly relates to a strain of Lactobacillus acidophilus BT-C15 with functions of relieving inflammation and repairing mucosal barrier and its application. Background Technology

[0002] Colitis is an inflammation of the colonic mucosa, mainly manifesting as abdominal pain, diarrhea, and rectal bleeding. It can be caused by various factors such as infection, immune abnormalities, and drug irritation, requiring examination to determine the specific type before targeted treatment. Colitis can be classified according to its etiology into infectious colitis, ulcerative colitis, ischemic colitis, and drug- or radiation-induced colitis. Typical symptoms of colitis include persistent or intermittent abdominal pain, diarrhea, tenesmus, fever, fatigue, weight loss, and anemia. Long-term inflammation may increase the risk of colon cancer. Colitis is usually treated with antibiotics, but antibiotics have significant side effects and there is a certain probability of drug resistance.

[0003] In today's society, people pay more attention to their appearance, especially women, who have higher demands for their skin condition. Therefore, skin whitening and blemish removal are highly sought after by many women. Currently, skin whitening and blemish removal can be achieved through sun protection, topical whitening products, chemical peels, laser treatments, and oral medications. However, many problems remain. For example, daily sun protection and topical whitening products are not very effective; while chemical peels and laser treatments are highly effective, patients often have poor tolerance and they usually have some impact on the skin; in addition, oral medications have numerous side effects.

[0004] Lactobacillus acidophilus is a beneficial lactic acid bacterium, a Gram-positive bacterium, mainly found in the human intestine, oral cavity, and some fermented foods. It regulates the intestinal environment by producing organic acids such as lactic acid and acetic acid, helping to maintain intestinal flora balance and enhancing immunity and promoting nutrient absorption. Currently, the functions of existing Lactobacillus acidophilus are primarily limited to the intestinal tract, such as regulating intestinal flora, improving immune function, and promoting nutrient absorption; its scope and function are often relatively singular. Summary of the Invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a strain of Lactobacillus acidophilus BT-C15 with functions of relieving inflammation and repairing mucosal barrier 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 strain of Lactobacillus acidophilus with functions of relieving inflammation and repairing mucosal barrier, wherein the Lactobacillus acidophilus is Lactobacillus acidophilus BT-C15, which is deposited at the China Center for Type Culture Collection on November 20, 2025, with accession number CCTCC NO: M 20252625.

[0007] In a second aspect, the present invention provides a composition comprising Lactobacillus acidophilus BT-C15 as described in claim 1.

[0008] As a specific embodiment, the composition is a lyophilized powder containing *Lactobacillus acidophilus* BT-C15; preferably, the viable count of *Lactobacillus acidophilus* BT-C15 in the lyophilized powder is 1×10⁻⁶. 8 CFU / mL - 1×10 10 CFU / mL.

[0009] Thirdly, the present invention provides a method for culturing the Lactobacillus acidophilus BT-C15, the method comprising inoculating the Lactobacillus acidophilus BT-C15 into a culture medium, fermenting it at 35℃-37℃ and pH 6.2-6.5, sealing the container during fermentation, maintaining pressure with an inert gas, stirring slowly, and culturing continuously for 12-20 hours.

[0010] As a preferred embodiment, the culture medium comprises the following components: Soybean peptone 8-12 g / L, yeast extract 5-7 g / L, hydrolyzed whey protein 4-6 g / L, tryptone 4-6 g / L, anhydrous glucose 15-25 g / L, sucrose 4-6 g / L, galactooligosaccharides 4-6 g / L, tomato pulp (solid content 20-30%) 1-5 g / L, dipotassium hydrogen phosphate 1-3 g / L, potassium dihydrogen phosphate 0.5-2.5 g / L, diamine citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.9 g / L, manganese sulfate 0.1-0.9 g / L, betaine 0.1-0.9 g / L, L-cysteine ​​0.5-1.5 g / L, Tween 80 0.5-1.5 ml / L.

[0011] Fourthly, the present invention provides the use of the aforementioned Lactobacillus acidophilus BT-C15 and the aforementioned composition in the preparation of antibacterial agents.

[0012] Preferably, the antibacterial agent is capable of inhibiting Molybditis lappa, Streptococcus agalactiae, and / or Propionibacterium acnes.

[0013] Fifthly, the present invention provides the use of the described Lactobacillus curvature GG77 and the composition in the preparation of products for improving colitis, lightening skin pigmentation and / or improving autism.

[0014] In a sixth aspect, the present invention provides a composition comprising the aforementioned *Lactobacillus acidophilus* BT-C15 and *Bifidobacterium animalis* subsp. *lactis* BT-G9, wherein *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: M 20252626.

[0015] In a seventh aspect, the present invention provides the use of the above-described composition comprising Lactobacillus acidophilus BT-C15 and Bifidobacterium animalis subsp. lactis BT-G9 in the preparation of products for improving autism.

[0016] Beneficial Effects: Compared with existing technologies, this invention provides a novel *Lactobacillus acidophilus* strain BT-C15, which primarily has anti-inflammatory and skin-whitening effects. This *Lactobacillus acidophilus* BT-C15 exhibits good tolerance to gastric and intestinal fluids, strong cell adhesion, and is well-suited for survival in the gastrointestinal tract. It effectively inhibits bacteria, particularly *Aggregatibacter korshinskii*, *Streptococcus agalactiae*, and / or *Propionibacterium acnes*. Experimental results show that this strain can improve colitis, lighten skin pigmentation, and improve autism. In particular, when *Lactobacillus acidophilus* BT-C15 is used in combination with *Bifidobacterium animalis* subsp. *lactobacter* BT-G9, the two have a synergistic effect, significantly enhancing the improvement of autism. Attached Figure Description

[0017] Figure 1 Gram staining results for Lactobacillus acidophilus BT-C15.

[0018] Figure 2 The fermentation production curve of Lactobacillus acidophilus BT-C15 is shown. Figure 3 The changes in body weight of mice in each group in Example 9.

[0019] Figure 4 The colon length of mice in each group in Example 9 is shown.

[0020] Figure 5 The effect of Lactobacillus acidophilus BT-C15 on the gene expression of intestinal mucosal tight junction proteins ZO-1 and Occludin in Example 10.

[0021] Figure 6 The content of melanin in each group of zebrafish in Example 11 is shown.

[0022] Figure 7 The activity of tyrosinase in zebrafish in each group in Example 11.

[0023] Figure 8 The distance traveled by the tbr1b zebrafish after exposure to probiotics in Example 12.

[0024] Figure 9 The migration rate of the tbr1b zebrafish after exposure to probiotics in Example 12 is shown.

[0025] Figure 10 The duration of agitation in the tbr1b zebrafish after exposure to probiotics in Example 12.

[0026] Figure 11 The duration of activity of tbr1b zebrafish after exposure to probiotics in Example 12. 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 strains capable of inhibiting tyrosinase A screening medium for tyrosinase-inhibiting strains was constructed. Based on the strains obtained in Example 1, after streak purification, the 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 r / min for 10 min, and the supernatant and bacterial cells were collected. The bacterial cells were washed twice with 0.9% sterile saline, and then a bacterial suspension with an OD600 of 0.5-0.8 was prepared with 0.9% sterile saline and stored at 4°C for future use. Tyrosinase stock solution was prepared and diluted with PBS buffer to an activity unit of 400 U / mL. The substrate solution was 0.5 mmol / L tyrosine solution. Arbutin was used as a positive control. The control group received no inhibitor and was replaced with an equal volume of buffer. The blank group received no enzyme solution. Each experimental group was incubated with 5% of the supernatant and bacterial cells of the respective strains and incubated at 37°C for 1 h. The absorbance at 475 nm wavelength (OD600) was measured using a microplate reader. 475 The method for calculating the tyrosinase inhibition rate is as follows: Tyrosinase inhibition rate (%) = [(experimental group OD] 475 - Blank group OD 475 ) ÷ (control group OD) 475 - Blank group OD 475 )]×100% By screening strains and supernatants with inhibitory capabilities, strain BT-C15 was selected as having strong tyrosinase inhibitory ability. The supernatant of BT-C15 showed stronger tyrosinase inhibitory ability than the bacterial suspension.

[0031] Table 1 Evaluation of tyrosinase inhibitory capacity

[0032] Example 3: Preservation, identification, and archiving of strains The BT-C15 strain screened in Example 2 was identified and preserved.

[0033] (1) Preservation of strains After streaking the selected BT-C15 strain on MRS agar for 48 hours, single colonies were selected and placed in 10 ml of MRS liquid medium containing 4.5% soybean lecithin and 2% calcium carbonate. This medium was incubated at 37°C for 12-16 hours. When the OD600 value of the culture was ≥1.2, a glycerol solution (30%-40% concentration) was added to the culture at a 1:1 volume ratio. The mixture was then aliquoted into 2 ml sterile cryovials and pre-cooled at 4°C for 2 hours, followed by pre-freezing at -20°C for 4 hours. Finally, the cryovials were transferred to a -80°C freezer or stored in liquid nitrogen.

[0034] (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.

[0035] (3) Gram staining Gram staining result is Gram positive, such as Figure 1 As shown.

[0036] (4) Molecular biological identification of 16S rRNA The 16S rDNA gene sequence of strain BT-C15 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-C15 is sequence 1 in the sequence listing. After 16S rDNA gene alignment, the similarity rate with Lactobacillus acidophilus in Genebank reached 99%. Combined with microbial systematic identification, strain BT-C15 was identified as a Lactobacillus acidophilus strain and named Lactobacillus acidophilus BT-C15, and its 16S rDNA is shown in SEQ ID NO.1.

[0037] (5) Collection of microbial strains The biologically identified Lactobacillus acidophilus BT-C15 strain was deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2025, with accession number CCTCC NO: M 20252625, and the deposit address is Wuhan University, Wuhan, Hubei Province, People's Republic of China.

[0038] Example 4: In vitro tolerance test of Lactobacillus acidophilus BT-C15 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 Lactobacillus acidophilus BT-C15 resistance to gastric and intestinal fluids are shown in Table 2.

[0039] Table 2 Results of acid resistance and artificial intestinal fluid resistance of Lactobacillus acidophilus BT-C15

[0040] Note: Colony counts without artificial gastric or intestinal fluid treatment are compared to 5.0 × 10⁻⁶. 7 CFU / mL As shown in Table 2, the survival rate of Lactobacillus acidophilus BT-C15 remained above 70% after 2 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, the survival rate of Lactobacillus acidophilus BT-C15 was 54% due to trypsin action, indicating that Lactobacillus acidophilus BT-C15 has good tolerance to artificial intestinal fluid and can therefore colonize and survive in the bile duct and intestines. This demonstrates that Lactobacillus acidophilus BT-C15 has good tolerance to both artificial gastric and intestinal fluids and is suitable for survival in the gastrointestinal tract.

[0041] Example 5 Cell adhesion assay of Lactobacillus acidophilus BT-C15 Add 1.0 × 10⁻⁶ cells to each cell culture dish. 9Lactobacillus acidophilus BT-C15 (CFU) was cultured in serum- and antibiotic-free culture dishes for 4 hours under specific conditions. The cell membranes were then washed with saline solution to remove any non-adhering bacteria. The cells were then lysed, and the lysate was diluted and cultured for another 48-72 hours. Adhesion ability was expressed as the percentage of adherent bacteria (log CFU) relative to the total number of cells. For two intestinal cell types, HT-29 and LS174T, Lactobacillus acidophilus BT-C15 achieved adhesion rates of 93% and 95%, respectively, demonstrating strong adhesion ability.

[0042] Example 6 Screening of in vitro antibacterial strains Lactobacillus acidophilus BT-C15 was prepared into OD 600 The bacterial suspension was prepared at a concentration of 0.5-0.8, and the supernatant was collected after centrifugation and stored at 4℃ for later use. *Aeromonas curvaturei* 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 curvaturei* BNCC 377479, *Streptococcus agalactiae* BNCC 357545, and *Propionibacterium acnes* ATCC 6919. The size of the inhibition zone was measured; a zone with a radius larger than that of the MRS control was considered to have antibacterial activity. Excluding some strains with insignificant antibacterial effects, the evaluation results of strains with significant antibacterial effects are shown in Table 3.

[0043] The bacterial suspension and supernatant of strain BT-C15 showed significant inhibitory effects on *Aerobicus kohlii* BNCC 377479, *Streptococcus agalactiae* BNCC357545, and *Propionibacterium acnes* ATCC 6919.

[0044] Table 3 Evaluation of Antibacterial Effect

[0045] 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.

[0046] Example 7 Hemolysis test of Lactobacillus acidophilus BT-C15 Lactobacillus acidophilus BT-C15 was inoculated into 5 mL MRS at a 2% inoculum size. Enterococcus faecalis (β-hemolytic, CICC23658, purchased from the China Industrial Microbiological Culture Collection Center) was used as a positive control, and a blank medium was used as a negative control. All strains were anaerobically cultured in MRS medium at 37°C for 12 h to obtain activated strains. 2.5 μL of each activated strain was inoculated onto Columbia blood agar plates (Shanghai Komarga Microbial Technology Co., Ltd.), with three replicates per group. After 48 h of anaerobically culture at 37°C, observation was performed. The positive control strains showed a clearly defined, completely transparent hemolytic zone around the colonies, indicating β-hemolysis; the culture medium around the Lactobacillus acidophilus BT-C15 colonies remained unchanged, indicating γ-hemolysis, i.e., no hemolysis. Therefore, there is no risk of hemolysis for human use, meeting the safety requirements for use as a food or cosmetic ingredient.

[0047] Example 8: Preparation of high-yield industrial freeze-dried powder of Lactobacillus acidophilus BT-C15 (1) Culture medium preparation.

[0048] To achieve industrial-scale, high-density, low-cost fermentation of Lactobacillus acidophilus BT-C15, food-grade conventional raw materials were selected to prepare the culture medium, and the culture medium was optimized.

[0049] Culture medium scheme 1: Soybean peptone 10 g / L, yeast extract 6 g / L, hydrolyzed whey protein 5 g / L, tryptone 5 g / L, anhydrous glucose 20 g / L, sucrose 5 g / L, galactooligosaccharides 5 g / L, tomato pulp (25% solids) 3 g / L, dipotassium hydrogen phosphate 2 g / L, potassium dihydrogen phosphate 1.5 g / L, diamine citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.5 g / L, betaine 0.5 g / L, L-cysteine ​​sulfate 1.0 g / L, Tween 80 1 ml / L.

[0050] Culture medium scheme 2: Soy peptone 8 g / L, yeast extract 4 g / L, hydrolyzed whey protein 3 g / L, tryptone 3 g / L, anhydrous glucose 10 g / L, sucrose 2 g / L, galactooligosaccharides 2 g / L, dipotassium hydrogen phosphate 2 g / L, potassium dihydrogen phosphate 1.5 g / L, diamine citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.5 g / L, L-cysteine ​​sulfate 1.0 g / L, Tween 80 1 ml / L.

[0051] Culture medium scheme 3: Soybean peptone 15 g / L, yeast extract 10 g / L, hydrolyzed whey protein 10 g / L, tryptone 10 g / L, anhydrous glucose 25 g / L, sucrose 8 g / L, galactooligosaccharides 8 g / L, tomato pulp (25% solids) 5 g / L, dipotassium hydrogen phosphate 2 g / L, potassium dihydrogen phosphate 1.5 g / L, diamine citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.5 g / L, betaine 1.5 g / L, L-cysteine ​​sulfate 1.0 g / L, Tween 80 1 ml / L.

[0052] (2) Liquid culture After preparing the culture medium, adjust the pH to 6.5, sterilize at 115℃ for 30 minutes, and cool to 38℃-39℃. Inoculate the fermenter at a rate of 3%-10%, preferably 8% in this example. The culture temperature is 35℃-37℃, and the pH is 6.2-6.5. During fermentation, the fermenter is sealed and gas-free, with nitrogen used to maintain pressure. Stir slowly. Culture continuously for 12-20 hours, and measure the viable cell count in the fermentation broth at 12 hours. The culture medium for Scheme 1 is 3.5 × 10⁻⁶ cells / mL. 9 CFU / mL, culture medium scheme 2: 1.6 × 10⁻⁶ 8 CFU / mL, culture medium scheme 3: 7.2 × 10⁻⁶ 8 The CFU / mL level of culture medium in Scheme 1 already represents a high level of viable cell count, meeting the requirements for industrial-scale high-density fermentation production. Its fermentation growth curve is shown below. Figure 2 As shown.

[0053] (3) Centrifugation To avoid prolonged fermentation leading to bacterial aging and affecting the activity of the freeze-dried powder, the viable cell count was increased to 10^6 microorganisms after 12 ± 1 hours of cultivation. 8 CFU / mL-10 9 CFU / mL, based on OD 600 After the growth curve reached the plateau phase, the fermentation broth was quickly cooled to below 15°C and centrifuged using a tubular centrifuge at 5000 rpm to harvest the mycelium sludge.

[0054] (4) Emulsification and freeze-drying of protective agents The harvested mushroom sludge was diluted to a moisture content of 60%-80%, and an equal volume of mushroom sludge protectant was added for emulsification. The mushroom sludge protectant consisted of: 70% water, 10% skim milk powder, 5% sucrose, 5% mannitol, 1% Tween-80, 3% betaine, 0.5% monosodium glutamate, and 5% soluble starch. The dried material was characterized by a moisture content of less than 5% and a water activity of 0.08-0.20 aw.

[0055] The bacterial powder from culture medium scheme 1, after fermentation, centrifugation, and freeze-drying of the mycelium sludge, achieved an activity of 4.6 × 10⁻⁶. 11With a CFU / g, it meets the high-yield requirements and can be used as a food additive for lactic acid bacteria powder or for the preparation of microecological intervention preparations.

[0056] Example 9: Interventional effect of Lactobacillus acidophilus BT-C15 on DSS-induced colitis Forty-eight male BALB / c mice were randomly divided into five groups: a control group (Control), a model group (DSS), and a Lactobacillus acidophilus BT-C15 intervention group (BT-C15), with 12 mice in each group. Mice in the model group (DSS) were administered 200 μL of skim milk by gavage daily from day 1 to 14, and had free access to distilled water. From day 14 to 21, the DSS group was administered 200 μL of skim milk by gavage daily, and had free access to 3% DSS solution. Mice in the BT-C15 intervention group were administered 5 × 10⁵ L of skim milk by gavage daily from day 1 to 14. 9 200 μL of Lactobacillus acidophilus BT-C15 bacterial suspension (CFU / mL) was administered to patients with free access to distilled water. From day 14 to day 21 of the experiment, patients were given the same amount of BT-C15 bacterial suspension by gavage and free access to 3% DSS solution. The control group was treated as follows: during the experiment, patients were given 200 μL of skim milk by gavage daily and free access to distilled water.

[0057] During the experiment, the changes in mouse body weight were measured and recorded, and the results are as follows: Figure 3 As shown, on the day of sacrifice after the event, the colonic lesions of mice in each group were observed and recorded, and the colonic length of mice in each group was measured. The results are as follows. Figure 4 As shown, starting with free access to 3% DSS aqueous solution, the model group mice exhibited decreased food intake and weight loss over time, with the condition gradually worsening. The weight loss in the Lactobacillus acidophilus BT-C15 intervention group was slower than that in the model group, and their diarrhea and bloody stool symptoms were also alleviated to varying degrees. Similarly, compared to the model group, the colon length of the mice was slightly shorter than the control group, but the rate of decrease had slowed. This indicates that Lactobacillus acidophilus BT-C15 has a good ameliorative effect on DSS-induced colitis in mice.

[0058] Example 10: The repair effect of Lactobacillus acidophilus BT-C15 on the physical barrier of colonic mucosa. Based on the colons of mice collected in Example 9, real-time quantitative PCR was used to analyze the gene expression of colonic mucosal tight junction proteins ZO-1 and Occludin as indicators of physical barrier function. The results are as follows: Figure 5 As shown.

[0059] The results showed that, compared with the control group, the expression levels of ZO-1 and Occludin genes in the model group mice were significantly reduced after drinking DSS solution, indicating a decrease in the expression of genes related to tight junction proteins and damage to the mucosal mechanical barrier. In contrast, mice treated with Lactobacillus acidophilus BT-C15, which also drank DSS solution, did not show a significant decrease in the expression levels of ZO-1 and Occludin genes. Combined with the changes in body weight, colon length, and other physiological changes observed in each group of mice in Example 9, this indicates that Lactobacillus acidophilus BT-C15 can prevent or slow down the damage to the physical barrier of the mouse colonic mucosa caused by DSS, and can be used to repair physical barrier damage caused by colitis.

[0060] Example 11 Effects of Lactobacillus acidophilus BT-C15 on melanin content and tyrosinase activity in zebrafish Healthy wild-type AB strain zebrafish that had developed to 4 dpf were selected and placed in 6-well cell culture plates, with 20 fish per well. The experiment included a blank control group (CK-1), a ursolic acid positive control group (CK-2), an *Lactobacillus acidophilus* BT-C15 bacterial suspension intervention group (BT-C15), an *Lactobacillus acidophilus* BT-C15 fermentation metabolite intervention group (BT-C15-M), and *Lactobacillus acidophilus* NCFM bacterial suspension (NCFM) and metabolites (NCFM-M) as parallel controls. The fermentation metabolites were prepared by centrifuging BT-C15 at 12000×g for 10 min after 25 h of fermentation, and the supernatant (dry matter content ≥2.2%) was collected. Each group had 6 replicates. The blank control group was given PBS, while the intervention group was given 8% bacterial suspension or fermentation metabolites. After incubation at 28°C for 48 hours, zebrafish pigmentation was observed using a stereomicroscope. After rinsing twice with PBS, zebrafish from each well were collected and placed in 1.5 mL centrifuge tubes. The water in the centrifuge tubes was removed, and 150 μL of sodium deoxycholate solution (5 mg / mL) was added. The centrifuge tubes were then crushed using an ultrasonic homogenizer in an ice bath, centrifuged at 12000 × g at 4°C for 10 min, and the supernatant was collected to detect melanin content. The melanin content was detected by adding 150 μL of NaOH solution to the centrifuge tube with precipitate, heating in a 100°C water bath for 10 min, and shaking to fully dissolve the melanin. 100 μL of the solution was placed in a 96-well plate, and the absorbance (OD) was measured at 405 nm using a microplate reader. 405 Set up a solvent zeroing group (no zebrafish). Calculate the melanin content in the zebrafish using the following formula: Melanin content (%) = [(Experimental group OD] 405 - Blank group OD 405 ) ÷ (control group OD) 405 - Blank group OD 405 )]×100% The method for detecting tyrosinase activity is the same as in Example 2, and the results for each group are as follows: Figure 6 , Figure 7 As shown in the figure. The results showed that the fermentation metabolites of Lactobacillus acidophilus BT-C15 had a clearing effect on melanin deposition in zebrafish, and significantly reduced the activity of tyrosinase in zebrafish. This indicates that the fermentation metabolites of Lactobacillus acidophilus BT-C15 can significantly reduce the melanin content and tyrosinase activity in zebrafish, exhibiting a good skin-lightening and whitening effect, and can be used in oral or topical preparations for skin lightening and whitening.

[0061] Example 12: Intervention of Lactobacillus acidophilus BT-C15 in autism Autistic zebrafish of strain tbr1b were used as an animal model. Five days after 5 days of growth (dpf) of tbr1b zebrafish juveniles, they were divided into four groups: the tbr1b group, the *Bifidobacterium lactis* subsp. 'Lactobacillus' intervention group (BT-G9), the *Lactobacillus acidophilus* BT-C15 intervention group (BT-C15), and the 1:1 ratio of *Bifidobacterium lactis* subsp. 'Lactobacillus acidophilus* BT-G9 to *Lactobacillus acidophilus* BT-C15 intervention group (BT-G9-C15). The control group was AB zebrafish (CK). Each group contained 24 fish. The *Bifidobacterium lactis* subsp. 'Lactobacillus' was deposited on November 20, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252626, located at Wuhan University, Wuhan, Hubei Province, People's Republic of China. The intervention groups were treated with 1×10⁻⁶ *Bifidobacterium lactis* 1×10⁻⁶ *Bifidobacterium lactis* at 5 days after 5 days of growth. 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 8-11 As shown.

[0062] 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.

[0063] 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 Lactobacillus acidophilus with functions of relieving inflammation and repairing the mucosal barrier, characterized in that, The Lactobacillus acidophilus mentioned is Lactobacillus acidophilus BT-C15, which is deposited at the China Center for Type Culture Collection on November 20, 2025, with accession number CCTCC NO: M 20252625.

2. A composition, characterized in that, The composition comprises Lactobacillus acidophilus BT-C15 as described in claim 1.

3. The composition according to claim 2, characterized in that, The composition is a lyophilized powder containing *Lactobacillus acidophilus* BT-C15; preferably, the viable count of *Lactobacillus acidophilus* BT-C15 in the lyophilized powder is 1 × 10⁻⁶. 8 CFU / mL - 1×10 10 CFU / mL.

4. The method for culturing Lactobacillus acidophilus BT-C15 according to claim 1, characterized in that, The method includes inoculating the Lactobacillus acidophilus BT-C15 into a culture medium, fermenting it at 35℃-37℃ and pH 6.2-6.5, sealing the container during fermentation, maintaining pressure with inert gas, stirring slowly, and culturing continuously for 12-20 hours.

5. The method for culturing Lactobacillus acidophilus BT-C15 according to claim 4, characterized in that, The culture medium comprises the following components: Soybean peptone 8-12 g / L, yeast extract 5-7 g / L, hydrolyzed whey protein 4-6 g / L, tryptone 4-6 g / L, anhydrous glucose 15-25 g / L, sucrose 4-6 g / L, galactooligosaccharides 4-6 g / L, tomato pulp (solid content 20-30%) 1-5 g / L, dipotassium hydrogen phosphate 1-3 g / L, potassium dihydrogen phosphate 0.5-2.5 g / L, diamine citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.9 g / L, manganese sulfate 0.1-0.9 g / L, betaine 0.1-0.9 g / L, L-cysteine ​​0.5-1.5 g / L, Tween 80 0.5-1.5 ml / L.

6. The use of Lactobacillus acidophilus BT-C15 according to claim 1, or the composition according to claim 2 or 3, in the preparation of antibacterial agents.

7. The application according to claim 6, characterized in that, The antibacterial agent can inhibit Molybditis lappa, Streptococcus agalactiae, and / or Propionibacterium acnes.

8. The use of the Lactobacillus curvature GG77 of claim 1, or the composition of claim 2 or 3, in the preparation of products for improving colitis, lightening skin pigmentation and / or improving autism.

9. A composition, characterized in that, The composition comprises Lactobacillus acidophilus BT-C15 as described in claim 1 and Bifidobacterium animalis subsp. lactis BT-G9, wherein Bifidobacterium animalis subsp. lactis BT-G9 is deposited at the China Center for Type Culture Collection on November 20, 2025, with accession number CCTCC NO: M 20252626.

10. Use of the composition of claim 9 in the preparation of a product for improving autism.

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