Lactobacillus acidophilus LAPerfectus 100 and use thereof

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a lactobacillus acidophilus LAPerfectus 100 and application thereof.The present application isolates a lactobacillus acidophilus LAPerfectus 100 from the intestinal tract of healthy infants and young children, and the preservation number is GDMCC No.65439.The lactobacillus acidophilus LAPerfectus 100 is drug-resistant, hemolytic, pathogenic, virulence gene-free and transferable drug-resistant gene-free, is high in safety, has good gastric acid tolerance, bile salt tolerance and adhesion, can not only inhibit various pathogenic bacteria and improve colitis, but also has good effects of regulating immunity and intestinal flora, and can be used for improving colitis, regulating immunity and intestinal flora.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Lactobacillus acidophilus LAPerfectus100 and its applications. Background Technology

[0002] Inflammatory bowel disease (IBD) is an inflammatory disease affecting the gastrointestinal tract. Its incidence has been gradually increasing in recent years, becoming a global health concern. IBD mainly presents in two ways: Crohn's disease (CD) and ulcerative colitis (UC). Common clinical symptoms of UC include diarrhea, abdominal pain, rectal bleeding, and weight loss. Currently, UC cannot be completely cured; only symptoms can be relieved. However, it is prone to relapse after remission, severely impacting quality of life.

[0003] Treatment for ulcerative colitis (UC) can be mainly divided into drug therapy and surgical treatment. Both methods have certain side effects or limitations. 5-Aminosalicylic acid (5-ASA), immunosuppressants, biologics, and topical or systemic corticosteroids are the main drugs used to treat UC. Oral 5-ASA can cause gastrointestinal discomfort; long-term use of corticosteroids increases the risk of complications such as cataracts and glaucoma; long-term use of immunosuppressants can cause leukopenia and allergic reactions; and the use of biologics is age-restricted. Surgical intervention is usually used for acute or more severe UC, but postoperative complications may include diarrhea, intestinal adhesions, and abdominal infections. Furthermore, for children, surgery may affect their growth and development and reduce their quality of life. Therefore, the need for safe and effective treatments for UC is essential.

[0004] Lactobacillus acidophilus is ubiquitous in the human body and is widely used in fermented and functional foods. It is a probiotic of significant commercial importance, and studies have shown that it regulates intestinal flora, enhances immunity, and inhibits Helicobacter pylori. Phenotypic tests such as drug sensitivity, hemolysis, and cytotoxicity have also demonstrated a certain level of safety. However, current research on Lactobacillus acidophilus is insufficient. Firstly, safety studies are inadequate: they are limited to in vitro tests such as drug sensitivity, hemolysis, and cytotoxicity, failing to incorporate animal studies and genetic analysis of drug resistance and virulence genes to comprehensively assess safety. Strains are highly genetically related; drug sensitivity, hemolysis, and cytotoxicity only evaluate safety through phenotypic tests and do not fully reflect the overall safety profile of the strain. Existing safety research on Lactobacillus acidophilus is insufficient. Secondly, industrialization research is inadequate: most strains remain at the small-scale or pilot-scale laboratory level, failing to be validated in industrial production facilities. Very few strains have been successfully commercialized, hindering the realization of their functional applications.

[0005] The safety and probiotic properties of probiotics are highly strain-specific. Different strains of the same probiotic can have significant differences in safety and efficacy. Researching new probiotics that can improve ulcerative colitis remains a challenge in current research. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a Lactobacillus acidophilus LAPerfectus100 and its application in improving colitis, regulating immunity and intestinal flora.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a Lactobacillus acidophilus LAPerfectus100; the Lactobacillus acidophilus LAPerfectus100 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 8, 2024, with the accession number GDMCC No. 65439.

[0009] This invention isolates and screens a strain of Lactobacillus acidophilus, LAPerfectus100, from the intestinal tract of infants. This Lactobacillus acidophilus LAPerfectus100 exhibits the following effects:

[0010] (1) It has good resistance to gastric acid and bile salts, with a survival rate of over 90%;

[0011] (2) It has excellent adhesion, which is higher than that of Lactobacillus rhamnosus GG, which is recognized in the industry as the most adhesive strain;

[0012] (3) It has a good ability to inhibit Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, and Enterococcus haematobacterium. Moreover, this invention is the first to discover that Lactobacillus acidophilus has the effect of inhibiting Streptococcus pharyngitis and Enterococcus haematobacterium.

[0013] (4) It has a good effect on improving colitis, and can significantly inhibit weight loss and shorten the colon, thus improving colitis symptoms;

[0014] (5) It has a good immunomodulatory effect. Its ability to inhibit the proliferation of macrophage RAW264.7 is higher than that of the industry star strain Lactobacillus acidophilus NCFM. It can restore the spleen index of colitis mice to normal level and significantly increase the content of secretory immunoglobulin A (SIgA). In addition, it can reduce the content of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the colon of colitis mice, increase the content of anti-inflammatory factor IL-10, and restore the content of TNF-α, IL-6, IL-1β, and IL-10 to normal level.

[0015] (6) It has a good regulatory effect on intestinal flora, which can improve the richness and diversity of intestinal flora in mice, increase the beneficial bacteria Lactobacillus, Butyricicoccus, GCA_900066575, Coridextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001, and reduce the relative abundance of harmful bacteria Erysipelatoclostridium, Tuzzerella, and Escherichia_Shigella. Among them, it was discovered for the first time that Lactobacillus acidophilus has a regulatory effect on GCA_900066575, Coridextribacter, and Rikenellaceae_RC9_gut_group.

[0016] (7) This strain can be industrialized, and the number of live bacteria in the original powder of the live bacterial preparation is not less than 2×10⁻⁶. 11 CFU / g.

[0017] Based on the above characteristics, the Lactobacillus acidophilus LAPerfectus100 can be used to improve colitis, regulate immunity and gut microbiota.

[0018] Secondly, the present invention provides the use of the Lactobacillus acidophilus LAPerfectus100 in the preparation of products for improving colitis.

[0019] Preferably, the colitis is ulcerative colitis.

[0020] Thirdly, the present invention provides the application of the Lactobacillus acidophilus LAPerfectus100 in the preparation of immunomodulatory products.

[0021] Preferably, the Lactobacillus acidophilus LAPerfectus100 regulates immunity by promoting SIgA secretion and / or proliferation of macrophages RAW264.7.

[0022] Fourthly, the present invention provides the application of the Lactobacillus acidophilus LAPerfectus100 in the preparation of products for regulating intestinal flora.

[0023] Preferably, the regulation of intestinal flora includes increasing the richness and diversity of intestinal flora; and / or increasing the relative abundance of beneficial bacteria in the intestine; and / or decreasing the relative abundance of harmful bacteria in the intestine.

[0024] More preferably, the beneficial intestinal bacteria include at least one of Lactobacillus, Butyricicoccus, GCA_900066575, Coridextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001;

[0025] More preferably, the intestinal harmful bacteria include at least one of Erysipelatoclostridium, Tuzzerella, and Escherchia Shigella.

[0026] Fifthly, the present invention provides the application of the Lactobacillus acidophilus LAPerfectus100 in the preparation of antibacterial products.

[0027] Preferably, the antibacterial activity includes inhibition of at least one of Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, and Enterococcus haematobium.

[0028] The beneficial effects of this invention are as follows:

[0029] This invention obtained a *Lactobacillus acidophilus* LAPerfectus100 through screening and isolation; the *Lactobacillus acidophilus* LAPerfectus100 has the following effects:

[0030] (1) It has good resistance to gastric acid and bile salts, with a survival rate of over 90%;

[0031] (2) It has excellent adhesion, which is higher than that of Lactobacillus rhamnosus GG, which is recognized in the industry as the strain with the strongest adhesion.

[0032] (3) It has a good ability to antagonize pathogens such as Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, and Enterococcus haematobacterium. Moreover, this invention is the first to discover that Lactobacillus acidophilus has the effect of inhibiting Streptococcus pharyngitis and Enterococcus haematobacterium.

[0033] (4) It has a good effect on improving colitis, significantly inhibits colon shortening, improves colon symptoms, reduces colonic TNF-α, IL-6, IL-1β pro-inflammatory factors, increases IL-10 anti-inflammatory factor, and restores TNF-α, IL-6, IL-1β, IL-10 to normal levels. The effect is higher than the reported data.

[0034] (5) It has a good immunomodulatory effect. Its ability to inhibit the proliferation of macrophage RAW264.7 is higher than that of the industry star strain Lactobacillus acidophilus NCFM. It can restore the spleen index of colitis mice to normal level and significantly increase the content of secretory immunoglobulin A (SIgA).

[0035] (6) It has a good regulatory effect on intestinal flora, which can improve the richness and diversity of intestinal flora in mice, increase the abundance of beneficial bacteria such as Lactobacillus, Butyricicoccus, GCA_900066575, Coridextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001, and decrease the relative abundance of harmful bacteria such as Erysipelatoclostridium, Tuzzerella, and Escherchia_Shigella. Among them, it was discovered for the first time that Lactobacillus acidophilus has a regulatory effect on GCA_900066575, Coridextribacter, and Rikenellaceae_RC9_gut_group.

[0036] (7) The Lactobacillus acidophilus LAPerfectus100 of this application can be industrialized, and the number of live bacteria in the original powder of the live bacteria preparation is not less than 2×10⁻⁶. 11 The CFU / g concentration enables large-scale production, providing a solid and feasible foundation for its application in improving colitis, regulating immunity, and improving gut microbiota. Attached Figure Description

[0037] Figure 1 The images show the colony morphology and cell morphology of Lactobacillus acidophilus LAPerfectus100; Image A shows the colony morphology on an MRS plate, and Image B shows the cell morphology under a microscope.

[0038] Figure 2Phylogenetic tree of 16S rRNA for Lactobacillus acidophilus LAPerfectus100 and related species.

[0039] Figure 3 This is a complete chromosomal sequence diagram of the gene of Lactobacillus acidophilus LAPerfectus100.

[0040] Figure 4 The image shows the results of the hemolytic activity test for Lactobacillus acidophilus LAPerfectus100.

[0041] Figure 5 The results of the bioamine test for Lactobacillus acidophilus LAPerfectus100 are shown in Figure A; Figure B shows Lactobacillus acidophilus LAPerfectus100 and the control strain Escherichia coli.

[0042] Figure 6 The figures show the changes in mouse body weight and DAI score after different group treatments; Figure A shows the changes in mouse body weight, and Figure B shows the changes in DAI score.

[0043] Figure 7 The results show the colon length measurements of mice after different group treatments.

[0044] Figure 8 The images show the colon pathology of mice after different group treatments.

[0045] Figure 9 The results show the colonic myeloperoxidase (MPO) content of mice after different group treatments.

[0046] Figure 10 The results show the levels of colonic cytokines in mice after different group treatments.

[0047] Figure 11 The results of spleen index measurement in mice after different group treatments.

[0048] Figure 12 The results show the levels of secretory immunoglobulin A (SIgA) in the colon of mice after different group treatments.

[0049] Figure 13 The results show the α-diversity of the gut microbiota in mice after different group treatments.

[0050] Figure 14 The results show the relative abundance of beneficial bacteria in the gut microbiota of mice after different group treatments.

[0051] Figure 15 The results show the relative abundance of some harmful bacteria in the intestinal flora of mice after different group treatments. Detailed Implementation

[0052] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0053] The culture medium formulation used in this invention is as follows:

[0054] LBS medium (g / L): 5.0g yeast extract, 10.0g tryptone, 6.0g potassium dihydrogen phosphate, 0.034g ferrous sulfate, 0.575g magnesium sulfate, 20.0g glucose, 25.0g sodium acetate, 2.0g ammonium citrate, 0.12g manganese sulfate. Adjust the pH to 5.5, then add 1mL Tween-80 and 1.3mL glacial acetic acid. Dissolve in 1000mL distilled water by heating and stirring. Autoclave at 121℃ for 15min. For solid medium, add 15.0g agar.

[0055] The preparation method for MRS culture medium is in accordance with GB4789.35-2016, with 1.5% agar added to the solid culture medium.

[0056] The Caco-2 cells and RAW264.7 macrophages used in this invention were purchased from the Wuhan University Culture Collection Center; *Lactobacillus rhamnosus* GG, *Escherichia coli* ATCC25922, *Salmonella* ATCC14028, and *Staphylococcus aureus* ATCC25923 were purchased from the Guangdong Provincial Microbial Culture Collection Center; *Streptococcus pharyngitis* and *Enterococcus haematobacterium* are currently preserved in the applicant's laboratory. BALB / c male mice were purchased from Beijing Spefol Biotechnology Co., Ltd.

[0057] Lactobacillus acidophilus LAPerfectus100 was activated through three subcultures before the experiment. The specific activation methods are as follows: First generation activation: The strain was inoculated into MRS liquid medium at an inoculum of 2% (v / v) and cultured overnight at 37°C under anaerobic conditions; Second generation activation: The strain was inoculated into MRS liquid medium at an inoculum of 3% (v / v) and cultured overnight at 37°C under anaerobic conditions; Third generation activation: The strain was inoculated into MRS liquid medium at an inoculum of 2% (v / v) and cultured overnight at 37°C under anaerobic conditions.

[0058] All data in this invention are expressed as mean ± SD; where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and ns indicates P>0.05).

[0059] Example 1: Isolation and identification of Lactobacillus acidophilus LAPerfectus 100

[0060] 1. Isolation and screening of strains

[0061] Stool samples were collected from eligible infants (healthy children who had not consumed any probiotic products), placed in sterile sampling tubes, and transported in ice packs. The samples were then serially diluted with 0.85% physiological saline under sterile conditions. 10 samples were selected from each tube. -1 10 -2 10 -3 Dilution gradients were spread on LBS agar plates and anaerobically incubated at 37°C for 48 hours. Colony morphology was observed visually, and suspected single colonies were picked for Gram staining and microscopic examination to identify colony morphological characteristics, thus preliminarily screening for suspected *Lactobacillus* strains. The strains were purified by repeated streak plating on MRS solid medium, and the purified *Lactobacillus* strains were stored at -80°C.

[0062] 2. Morphological characteristics and molecular biological identification of the strain

[0063] (1) Morphological characteristics: After serially diluting the bacterial suspension and anaerobically incubating it on MRS plates at 36°C for 48 hours, the colony morphology was as follows: Figure 1 As shown in Figure A, single colonies of the strain on MRS solid plates are milky white, round, raised, with a smooth surface and neat edges.

[0064] Microscopic observation of bacterial cell morphology yielded the following results: Figure 1 As shown in B, the bacterial cell morphology is Gram-positive bacillus, which does not produce spores, is short rod-shaped, and is arranged singly or in pairs, with rounded ends.

[0065] (2) Molecular biological identification: The strain was cultured in liquid medium, and genomic DNA was extracted from the bacterial cells. 16S rRNA was detected, and a phylogenetic tree of *Lactobacillus acidophilus* LAPerfectus100 was constructed using MEGA software with 1000 replicates based on the 16S rRNA gene sequence. (See...) Figure 2 (Nodes display Bootstrap values ​​greater than 50%, with the superscript "T" indicating the type strain).

[0066] The results showed that the 16S rRNA gene sequence of *Lactobacillus acidophilus* LAPerfectus100 was 1364 bp. Nucleotide homology comparison with already registered sequences in Genebank using the Blast program showed that the 16S rRNA gene sequence of this strain had 100% homology with *Lactobacillus acidophilus*. Based on the molecular biological identification results, the obtained strain was confirmed as *Lactobacillus acidophilus*, and named *Lactobacillus acidophilus* LAPerfectus100.

[0067] The 16S rRNA gene sequencing sequence of Lactobacillus acidophilus LAPerfectus100 is shown in SEQ ID NO.1.

[0068] The Lactobacillus acidophilus LAPerfectus100 was deposited on November 8, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 65439.

[0069] 3. Whole genome sequencing

[0070] The whole genome sequence of Lactobacillus acidophilus LAPerfectus100 was detected using second-generation and third-generation high-throughput gene sequencing technology, and the whole genome map was drawn to identify the species at the genome level.

[0071] After amplification, the bacterial cells were collected by centrifugation at 8000 rpm for 5 min (4℃), the supernatant was discarded, and the cells were flash-frozen in liquid nitrogen. Once the cells were fully frozen, they were transferred to a -80℃ freezer and mailed to a testing institution for second-generation and third-generation sequencing. Second-generation sequencing was performed using the DNBSEQ platform, and third-generation sequencing was performed using the Nanopore platform. Library construction and sequencing were carried out according to the library construction requirements of each sequencing platform. After quality control and filtering, the third-generation data were calibrated using the second-generation data to complete the assembly of the bacterial whole genome map.

[0072] The results showed that the genomic DNA of *Lactobacillus acidophilus* LAPerfectus100 was circular, plasmid-free, with a genome size of 1.99 Mb and a GC content of 35.27%. A total of 12 ribosomal RNAs (rRNAs), 61 transfer RNAs (tRNAs), and 8 small RNAs (sRNAs) were detected. The complete genome chromosome sequence is shown in [image missing]. Figure 3 It was compared with the recognized NT database and identified as Lactobacillus acidophilus at the whole genome level.

[0073] Example 2: Food safety verification of Lactobacillus acidophilus LAPerfectus 100

[0074] 1. Drug sensitivity testing

[0075] According to EFSA 5206-2018 "Microbiological Characteristics of Feed Additives or Biofermented Products" 2.2.1 Antimicrobial Susceptibility Testing was conducted, and the results showed that Lactobacillus acidophilus LAPerfectus100 was sensitive to all antibiotics required for this item. The specific results are shown in Table 1.

[0076] Table 1. MIC values ​​and drug susceptibility test results of Lactobacillus acidophilus LAPerfectus 100.

[0077]

[0078]

[0079] 2. Hemolytic test

[0080] Using *Streptococcus pyogenes* as a control, the hemolytic activity of *Lactobacillus acidophilus* LAPerfectus 100 was tested. The experimental results are as follows: Figure 4 As shown, after Streptococcus pyogenes was inoculated onto Columbia agar plates, a clear hemolytic zone appeared around the colonies, indicating beta-hemolysis. However, after Lactobacillus acidophilus LAPerfectus100 was inoculated onto the same medium, no hemolytic zone appeared around the colonies. Therefore, it was determined that this strain is not hemolytic and will not cause hemolytic harm, making it a safe strain.

[0081] 3. Detection of biological amines

[0082] Culture media with amino acid decarboxylase activity can produce alkaline biogenic amines. The results can be preliminarily detected by observing the color change of an indicator.

[0083] The culture medium for the amino decarboxylase test was prepared as follows: 5g peptone, 3g yeast extract, 1g glucose, 1000mL distilled water, 1mL 1.6% bromocresol purple-ethanol solution, 1.8% agar, pH=6.8, and autoclaved at 115℃ for 20min. (The added amino acids were L-arginine, L-lysine, L-tryptophan, and L-histidine).

[0084] The cultured bacterial suspension was inoculated at 2% into 1.6 mL of amino decarboxylase culture medium without the addition of precursor amino acids and cultured overnight. Activated bacterial suspension was added dropwise to each filter paper disc to moisten it. The sterile filter paper discs were then placed on modified amino decarboxylase detection agar medium supplemented with precursor amino acids and incubated at 37°C for 2 days. The color change of the transparent ring around the filter paper disc was observed. If the ring turned purple, it indicated the production of biogenic amines; otherwise, it indicated the absence of biogenic amines.

[0085] The results are as follows Figure 5 As shown, no color change occurred around *Lactobacillus acidophilus* LAPerfectus 100, but *Escherichia coli* produced alkaline biogenic amines, causing the culture medium around its filter paper to change from yellow to purple. The results indicate that *Lactobacillus acidophilus* LAPerfectus 100 does not produce biogenic amines and has good safety.

[0086] 4. Detection of bacterial pathogenicity

[0087] Bacterial pathogenicity tests were conducted according to the methods in Appendix A of the "Technical Guidelines for Safety Testing and Evaluation of Microbial Strains Used in Health Food Raw Materials (2020 Edition)".

[0088] Table 2 Results of mouse bacterial pathogenicity (intraperitoneal injection) test of Lactobacillus acidophilus LAPerfectus 100

[0089]

[0090] The results of the mouse bacterial pathogenicity (intraperitoneal injection) test of Lactobacillus acidophilus LAPerfectus100 are shown in Table 2. Mice of both sexes were intraperitoneally injected once with a bacterial suspension of Lactobacillus acidophilus LAPerfectus100, with each mouse receiving 1.07 × 10⁻⁶ bacteria. 7 CFU (bacterial concentration of 5.34 × 10⁻⁶) 7 The animals were in normal condition during the 21-day observation period (CFU / mL, injection volume of 0.2 mL / animal), with no signs of poisoning or death.

[0091] Table 3. Results of the mouse bacterial pathogenicity test (oral administration) of Lactobacillus acidophilus LAPerfectus 100

[0092]

[0093] The results of the mouse bacterial pathogenicity (oral gavage) test of Lactobacillus acidophilus LAPerfectus100 are shown in Table 3. Mice of both sexes were administered Lactobacillus acidophilus LAPerfectus100 bacterial suspension by gavage for 3 consecutive days, with each gavage dose being 5.00 × 10⁻⁶. 9 CFU / kg·BW and 2.50×10 10 CFU / kg·BW (bacterial suspension concentration was 2.54×10⁻⁶) 8 CFU / mL and 1.25×10 9 (CFU / mL, gavage volume calculated based on the actual body weight of the mice at 20 mL / kg·BW). During the 21-day observation period, the animals were in normal condition, with no signs of poisoning or death.

[0094] According to the bacterial pathogenicity test method for raw materials used in health food, the mouse bacterial pathogenicity test result of Lactobacillus acidophilus LAPerfectus100 was negative, indicating that the strain is non-pathogenic.

[0095] 5. Analysis of drug resistance and virulence genes in Lactobacillus acidophilus LAPerfectus100

[0096] (1) The genomic sequences of the strains were compared with the latest version of the internationally recognized antibiotic resistance gene database (CARD) and sorted by identity. The top 3 results are shown in Table 4. Using sequence identity ≥85% as the criterion for judging drug resistance genes, the analysis results showed that the gene with the highest sequence identity was GL001221 (identity = 81.32%). Therefore, no drug resistance gene with sequence identity ≥85% was found in *Lactobacillus acidophilus* LAPerfectus100, and no drug resistance gene was predicted.

[0097] Table 4. Sequence alignment results of Lactobacillus acidophilus LAPerfectus100 with drug resistance gene database.

[0098] No. Gene_id Identity, % Align_length 1 GL001221 81.82 218 2 GL001721 72.73 143 3 GL000819 70.66 1190

[0099] (2) The genome sequence of the strain was compared with the latest version of the Virulence Gene Database (VFDB) and sorted by identity. The top 3 results are shown in Table 5. The gene with the highest sequence identity was GL000819 (identity = 73.146%). According to the "Technical Guidelines for Safety Inspection and Evaluation of Microbial Strains for Raw Materials of Health Food" (2020 edition), Lactobacillus acidophilus LAPerfectus100 does not have any known virulence factors or toxin metabolism-related genes with a sequence length coverage ≥ 60% and a sequence identity ≥ 85% that are clearly related to pathogenicity. No virulence genes were predicted.

[0100] Table 5. Sequence alignment results of Lactobacillus acidophilus LAPerfectus100 with the virulence gene database.

[0101] No. Gene_id Identity, % Coverage, % Align_length 1 GL000819 73.146 98.98734 1190 2 GL000672 69.634 96.46465 587 3 GL001650 69.595 97.36842 884

[0102] In summary, based on phenotypic results including drug susceptibility, hemolysis, and pathogenicity in mice, as well as virulence and resistance gene analysis, *Lactobacillus acidophilus* LAPerfectus 100 exhibits no resistance to antibiotics specified by EFSA, has a negative hemolytic reaction, shows no pathogenicity in animal studies, and gene analysis reveals the absence of virulence genes and transferable resistance genes. Therefore, *Lactobacillus acidophilus* LAPerfectus 100 is a safe strain that meets human safety requirements.

[0103] Example 3: Evaluation of gastric juice and bile salt tolerance of Lactobacillus acidophilus LAPerfectus 100

[0104] 1. Evaluation of tolerance to artificial gastric juice:

[0105] Experimental steps: ① Preparation of artificial gastric fluid: 0.5% NaCl (w / v), adjust pH to 2.5 with 1M HCl, add 0.3% pepsin (w / v), dissolve thoroughly, and filter through a 0.22μm sterile microporous membrane for sterilization; ② After the strain is activated, take 0.1mL of bacterial suspension and add it to 0.9mL of artificial gastric fluid, and incubate at 37℃ for 3h; after serial dilution at 0h and 3h, respectively, spread on solid plates for viable cell counting. ③ Calculate the survival rate of the strain according to formula (1).

[0106] Strain survival rate = N1 / N0 × 100% —— Equation (1)

[0107] In the above formula: N1 represents the number of viable bacteria (CFU / mL) in the strain system after treatment, and N0 represents the initial number of viable bacteria in the strain system, that is, the number of viable bacteria (CFU / mL) measured at 0h.

[0108] 2. Evaluation of bile salt tolerance:

[0109] Experimental steps: ① Preparation of bile salt solution: Add 0.2% (w / v) sodium thioglycolate to MRS liquid medium, then add bile salt to a final concentration of 0.3% (w / v), dissolve thoroughly, and filter with a 0.22μm sterile microporous membrane for sterilization; ② After the strain is activated, take 0.1mL of bacterial suspension and add it to 0.9mL of bile salt solution, and incubate at 37℃ for 3h; after serial dilution of the treatment solution at 0h and 3h, spread it on solid plates for viable count; ③ Calculate the survival rate of the strain according to formula (1).

[0110] 3. Experimental Results

[0111] This experiment also used Lactobacillus acidophilus LA79, LA82, LA86, and LA188 from the same batch for comparison.

[0112] The results of Lactobacillus acidophilus LAPerfectus100's tolerance to gastric juice and bile salts are shown in Table 6. After treatment with artificial gastric juice (pH=2.5) for 3 hours, the survival rate of Lactobacillus acidophilus LAPerfectus100 reached over 97%, and after treatment with bile salt solution (0.3%) for 3 hours, the survival rate reached over 93%, significantly higher than that of Lactobacillus acidophilus selected in the same batch.

[0113] Table 6. Tolerance of Lactobacillus acidophilus to gastric juice and bile salts

[0114]

[0115]

[0116] Example 4: Evaluation of the adhesion ability of Lactobacillus acidophilus LAPerfectus 100

[0117] 1. Caco-2 cell culture: Caco-2 cells were cultured in DMEM medium supplemented with 10% heat-inactivated (56℃, 30 min) fetal bovine serum (FBS), 1% penicillin and streptomycin, and cultured in an incubator at 37℃, 90% humidity and 5% CO2.

[0118] 2. Cell adhesion experiment: ① Liquid culture of experimental strains: Activated second-generation bacterial culture, 2% inoculum, 9 mL medium / 15 mL centrifuge tube, 37℃, static culture for 15 h. ② Preparation of monolayers: Caco-2 cells were inoculated into DMEM medium supplemented with 20% (v / v) fetal bovine serum, and transferred to 12-well cell culture plates, with a cell addition of 1 mL per well. 5 Cells were cultured at 37°C with 5% CO2, with the medium changed every other day, until a monolayer was obtained for use; ③ Preparation of bacterial suspension: At the same time, the cultured bacterial suspension was collected by centrifugation at 10000 r / min at room temperature for 1 min, and the cells were washed twice with sterile PBS, resuspended in DMEM medium, and adjusted to a bacterial concentration of 10. 8 CFU / mL; ④ Co-culture: After preparing a monolayer of Caco-2 cells, remove the culture medium, add PBS buffer and wash twice, remove the buffer, add 1 mL of the prepared bacterial suspension per well, mix well, and incubate at 37°C for 2 h with 5% CO2; ⑤ Carefully remove the culture supernatant, add sterile PBS and wash 5 times to remove unadhered bacteria; ⑥ Add 0.2 mL of trypsin cell digestion solution per well and digest for 5 min to allow the cells to elute from the wells of the culture plate, and collect the solution as the sample; ⑦ Perform serial dilution and viable cell count on the collected sample, and calculate the adhesion ability according to formula (2).

[0119] Adhesion capacity (CFU / cell) = Number of bacteria adhering to the cell (CFU) / Number of cells in the well (cells)

[0120] —Equation (2)

[0121] In this experiment, *Lactobacillus acidophilus* LA82, which had the second-highest gastric juice tolerance and bile salt tolerance screened in Example 3, and *Lactobacillus rhamnosus* GG, a commercially recognized strain with the strongest adhesion, were selected as control strains. Each experiment was performed in triplicate. The results are shown in Table 7. The adhesion ability of *Lactobacillus acidophilus* LAPerfectus100 was significantly higher than that of *Lactobacillus acidophilus* LA82 screened in the same batch and the commercially popular strain *Lactobacillus rhamnosus* GG.

[0122] Table 7. Adhesion ability of Lactobacillus acidophilus LAPerfectus100 to Caco-2 cells

[0123]

[0124]

[0125] Example 5: Evaluation of the proliferative capacity of Lactobacillus acidophilus LAPerfectus100 on RAW264.7 macrophages

[0126] Experimental methods:

[0127] 1. RAW264.7 Cell Culture: After resuscitation, RAW264.7 cells were cultured in DMEM complete medium containing 1% non-essential amino acids, 1% penicillin and streptomycin, and 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The medium was changed every other day. When the cell confluence reached 80% or higher, the cells were passaged. The cells were washed twice with sterile PBS, and 2 mL of medium was added. The cells were gently scraped off with a cell scraper, centrifuged, and the supernatant was discarded. The cells were resuspended in DMEM complete medium and passaged at a ratio of 1:3.

[0128] The RAW264.7 cell concentration was adjusted to 1×10⁻⁶. 4 Cells were seeded at 100 μL / well in 96-well plates and cultured for 24 h until the cells adhered and grew. Then, the supernatant in the wells was aspirated and the cells were washed twice with PBS.

[0129] 2. The experiment was divided into a blank group and a probiotic group. Blank group: 100 μL of DMEM medium was added to the wells of the plate and placed in a 5% CO2 incubator at 37°C for 24 h. Probiotic group: 100 μL of Lactobacillus acidophilus LAPerfectus100 bacterial suspension was added to the wells (the bacterial suspension was washed twice with PBS, resuspended in DMEM, and diluted at a bacterial volume: cell volume ratio of 100:1) and cultured for 24 h.

[0130] After culture, the supernatant in the well plate was aspirated, the plate was washed once with PBS, and 100 μL of DMEM medium containing 10% CNC-8 was added. The plate was incubated at 37°C in the dark for 1 h. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Cell viability (CV) was calculated according to formula (3).

[0131] Cell viability (%) = OD1 / OD2 × 100% —— Equation (3)

[0132] In the above formula, OD1: the OD value of each treatment group at a wavelength of 450nm, and OD2: the OD value of the blank group at a wavelength of 450nm.

[0133] This experiment also used the commercially available star strain Lactobacillus acidophilus NCFM for comparison.

[0134] Table 8. Proliferative effects of Lactobacillus acidophilus LAPerfectus 100 on macrophages

[0135]

[0136] The proliferation results of RAW264.7 macrophages induced by Lactobacillus acidophilus LAPerfectus100 are shown in Table 8. Compared with NC, the number of macrophages in the Lactobacillus acidophilus LAPerfectus100 treatment group was 148.95% of that in the control group, which significantly promoted macrophage proliferation and was higher than that of macrophages induced by Lactobacillus acidophilus NCFM, indicating that Lactobacillus acidophilus LAPerfectus100 has the potential for immunomodulation.

[0137] Example 6: Evaluation of the antagonistic ability of Lactobacillus acidophilus LAPerfectus100 against pathogens

[0138] 1. Preparation of pathogenic bacterial suspension: ① Escherichia coli ATCC25922, Salmonella ATCC14028, and Staphylococcus aureus ATCC25923 were inoculated into LB liquid medium at 2% (v / v) and incubated at 37℃ for 16-20 h. The bacterial suspension concentration was then adjusted to 10. 8 CFU / mL. ② Enterococcus hesperidin and Streptococcus pharyngitis were inoculated into BHI liquid medium at 5% (v / v), and cultured at 37°C for 16–20 h. The bacterial concentration was then adjusted to 10 CFU / mL. 8 CFU / mL.

[0139] 2. Antagonistic Bacterial Test: ① Cool MRS broth or BHI broth containing 1.5% (w / v) agar to approximately 55°C, and mix it with the indicator bacterial suspension in a certain proportion (using MRS broth for Escherichia coli, Salmonella, and Staphylococcus aureus; using BHI broth for Enterococcus hesei and Streptococcus pharyngitis), ensuring the viable count of the indicator bacteria is around 10. 6 The concentration was on the order of CFU / mL. The medium was then quickly poured into plates pre-placed with Oxford cups. After the medium cooled and solidified, the Oxford cups were removed, and 200 μL of Lactobacillus acidophilus LAPerfectus100 fermentation broth was injected into each well. After overnight incubation at 37°C, the diameter of the inhibition zone was measured.

[0140] 3. Experimental Results

[0141] Table 9. Inhibition zones of Lactobacillus acidophilus LAPerfectus 100 against different pathogens.

[0142] Pathogens Antibacterial zone (mm) Escherichia coli 39.83±0.29 salmonella 41.00±1.00 Staphylococcus aureus 20.83±0.76 Streptococcus pharyngitis 26.83±0.29 Enterococcus seais 14.25±0.29

[0143] This embodiment tested the inhibitory effect of Lactobacillus acidophilus on five common pathogens: Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, and Enterococcus haematobacterium. The antagonistic ability of Lactobacillus acidophilus LAPerfectus100 against the five pathogens is shown in Table 9. The results showed that Lactobacillus acidophilus LAPerfectus100 had a good inhibitory effect on Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, and Enterococcus haematobacterium.

[0144] Example 7: Effects of Lactobacillus acidophilus LAPerfectus100 on body weight and DAI score in colitis mice

[0145] Establishment of a colitis model: The animal model was induced using the DSS method. Twenty-four SPF BALB / c male mice (19–20 g) were used. Animals were housed at a room temperature of 23 ± 2℃, humidity of 50% ± 10%, with artificial light for 12 hours / day and free access to food. After one week of acclimatization, all mice were randomly divided into three groups of eight mice each: normal control group (NC), model group (DSS), and Lactobacillus acidophilus treatment group (LAPerfectus100). The experiment lasted for two weeks, and the specific experimental procedure is as follows:

[0146] Control group: Drink water normally throughout the experiment and receive 0.2 mL of phosphate-buffered saline (PBS) via gavage at regular intervals every day;

[0147] Model group: Mice had normal drinking water from day 1 to day 7. From day 8 onwards, mice had free access to 3.5% DSS solution for 7 days. The DSS solution was changed every 2 days to ensure the concentration and quality of the DSS solution. Throughout the experiment, mice were given 0.2 mL of PBS by gavage at regular intervals every day.

[0148] Lactobacillus acidophilus LAPerfectus100 intervention group: Mice had normal drinking water from day 1 to day 7. From day 8 onwards, mice had free access to 3.5% DSS solution for 7 days. The DSS solution was changed every 2 days to ensure its concentration and quality. Throughout the experiment, mice were administered 0.2 mL of Lactobacillus acidophilus LAPerfectus100 bacterial suspension (viable bacterial count concentration of 5 × 10⁻⁶) by gavage. 9 (CFU / mL).

[0149] After the experiment, all mice were fasted for 12 hours, then sacrificed and dissected to collect samples for subsequent indicator measurement and analysis.

[0150] Changes in mouse DAI score: Mice were weighed and recorded daily during the modeling period to track weight changes. Feces were collected daily during the modeling period to observe fecal characteristics and detect fecal bleeding using occult blood test strips. The DAI score was calculated according to formula (4), and the specific scoring criteria are shown in Table 10. The results are as follows: Figure 6As shown. The remission rate is calculated according to equation (5).

[0151] DAI score = (percentage of weight loss + stool viscosity + fecal occult blood) / 3 —— Equation (4)

[0152] Remission rate = (weight change rate in intervention group - weight change rate in model group) / (weight change rate in blank group - weight change rate in model group) — Equation (5)

[0153] Table 10 DAI Scoring Criteria

[0154]

[0155]

[0156] Depend on Figure 6 As shown in A, the body weight of mice in the model group continued to decrease during the modeling period. By the end of the experiment, the body weight change rates (body weight on day 7 of modeling / body weight on day 1 of modeling) of the blank group, model group, and Lactobacillus acidophilus LAPerfectus100 intervention group were 1.0918, 0.8258, and 0.9323, respectively. The body weight of mice in the model group was significantly lower than that in the control group (p < 0.0001), indicating successful model establishment. After intervention with Lactobacillus acidophilus LAPerfectus100, the remission rate reached over 40% (remission rate = (0.9323 - 0.8258) / (1.0918 - 0.8258) = 40.04%), effectively alleviating the decrease in mouse body weight (p < 0.01), which was higher than that of previously reported strains: Lactobacillus acidophilus CCFM1200 (remission rate = (0.94 - 0.91) / (1.02 - 0.91) = 27.27%); Lactobacillus acidophilus NCFM18.18% (remission rate = (0.93 - 0.91) / (1.02 - 0.91) = 18.18%), source: patent number CN114231470A).

[0157] The trend of DAI changes is opposite to that of weight changes. Figure 6 B showed that the DAI score of the model group continued to rise during the modeling period. At the end of the experiment, the DAI score of the model group mice was significantly higher than that of the blank group (p<0.0001), while the DAI score of the Lactobacillus acidophilus LAPerfectus100 group was significantly lower than that of the DSS group (p<0.001), indicating that Lactobacillus acidophilus LAPerfectus100 effectively relieved the symptoms of colitis.

[0158] Example 8: Effects of Lactobacillus acidophilus LAPerfectus100 on mouse colon tissue

[0159] The grouping and modeling of mice were the same as in Example 7. Mice were sacrificed, and their colons were harvested. The length of the colons was measured, and 0.5 cm of ileal tissue was collected. The tissue was fixed by soaking in 10% formaldehyde solution, then stained with hematoxylin and eosin (HE), and finally observed under an optical microscope.

[0160] Colon length as Figure 7 As shown, the colon lengths of the blank group, model group, and Lactobacillus acidophilus LAPerfectus100 intervention group were 9.30, 5.02, and 6.92 cm, respectively. Compared with the blank group, the colon length of mice in the model group was significantly shortened (p < 0.0001), while Lactobacillus acidophilus LAPerfectus100 intervention significantly inhibited colon shortening (p < 0.01), and the colon length was 137.8% of that in the model group, which is higher than previously reported data (115% for Lactobacillus acidophilus CCFM1200, which is superior to Lactobacillus acidophilus NCFM, source: patent number CN114231470A).

[0161] Colon pathology results as follows Figure 8 As shown, the colonic mucosa and crypts of mice in the blank control group were intact, with normal morphology and structure, abundant goblet cells, and no obvious lesions. In the model group, the integrity of the colonic structure was disrupted, with mucosal damage, significant loss of crypts and goblet cells, and inflammatory cell infiltration. Compared with the model group, the intestinal damage in mice in the Lactobacillus acidophilus LAPerfectus100 group was restored, and the intestinal morphology and structure were more intact, indicating that Lactobacillus acidophilus LAPerfectus100 can effectively alleviate colonic damage and reduce the degree of colonic lesions in UC mice.

[0162] Example 9: Effect of Lactobacillus acidophilus LAPerfectus 100 on the content of myeloperoxidase (MPO) in the colon of mice with colitis

[0163] Infiltrating inflammatory cells such as neutrophils, monocytes and lymphocytes into the colonic mucosa is an important pathological manifestation of inflammatory colitis. Myeloperoxidase (MPO) in the colon is a marker enzyme for neutrophil function and activation, which can reflect the degree of neutrophil infiltration and the level of cellular oxidative stress in colonic tissue.

[0164] This example describes the effect of Lactobacillus acidophilus LAPerfectus 100 on MPO in the colon of mice with colitis. The grouping and modeling of mice were the same as in Example 7. After the experiment, mice were sacrificed, their colons were dissected, placed in phosphate buffered saline solution, and homogenized. The MPO content in the colon homogenate of each group of mice was determined according to the detection method specified in the ELISA kit manufacturer's instructions.

[0165] The results are as follows Figure 9As shown, compared with the control group, the MPO content in the colon of mice in the model group was significantly increased (p < 0.001), while the MPO level in the colon of mice in the Lactobacillus acidophilus LAPerfectus100 group was decreased compared with the model group (p < 0.01). This indicates that Lactobacillus acidophilus LAPerfectus100 can significantly reduce the MPO level in the colon of mice with colitis, suggesting that Lactobacillus acidophilus LAPerfectus100 can reduce neutrophil aggregation and thus alleviate intestinal inflammation.

[0166] Example 10: Effects of Lactobacillus acidophilus LAPerfectus 100 on cytokines in the colon of colitis-affected mice

[0167] The grouping and modeling of mice were the same as in Example 7. After the experiment, the mice were euthanized and dissected. The colons were placed in phosphate buffer solution and homogenized. The levels of TNF-α, IL-6, L-1β, and IL-10 in the colon homogenate of each group of mice were determined according to the detection method in the ELISA kit manufacturer's instructions.

[0168] The results are as follows Figure 10 As shown, compared with the control group, the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the model group were significantly increased (p < 0.05), while the level of anti-inflammatory cytokine IL-10 was significantly decreased (p < 0.01), indicating a strong inflammatory response in colitis mice. After intervention with Lactobacillus acidophilus LAPerfectus100, the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the colon of mice were significantly reduced (p < 0.05) and returned to normal levels (no significant difference compared with the control group, P > 0.05). Lactobacillus acidophilus LAPerfectus100 also promoted the secretion of IL-10 in the colon of colitis mice, and the level was significantly higher than that in the model group (p < 0.01). This indicates that Lactobacillus acidophilus LAPerfectus100 can effectively reduce the increase of pro-inflammatory factors in mice, while promoting the secretion of anti-inflammatory factors and inhibiting the degree of inflammation.

[0169] Example 11: Effect of Lactobacillus acidophilus LAPerfectus 100 on spleen index in colitis mice

[0170] The grouping and modeling of mice were the same as in Example 7. Spleens were collected from dissected mice, weighed, and the spleen index was calculated. The spleen is an important immune organ and a site for the growth and proliferation of immune cells.

[0171] The results are as follows Figure 11As shown, DSS-induced acute enteritis mice showed splenomegaly. Compared with the control group, the spleen index in the model group was significantly increased (p < 0.0001), while the spleen index in the Lactobacillus acidophilus LAPerfectus100 group was significantly lower than that in the model group (p < 0.001), and there was no significant difference compared with the control group (p > 0.05). This indicates that oral administration of Lactobacillus acidophilus LAPerfectus100 can improve spleen damage in colitis mice, restore spleen weight to normal levels, and improve the impact of colitis on the development of important immune organs.

[0172] Example 12: Effect of Lactobacillus acidophilus LAPerfectus100 on the level of secretory immunoglobulin A (SIgA) in the colon of colitis mice

[0173] The grouping and modeling of mice were the same as in Example 7. After the experiment, the mice were euthanized, and the colons were dissected, placed in phosphate buffered saline solution, and homogenized. The SIgA content in the colon homogenate of each group of mice was measured according to the detection method in the ELISA kit manufacturer's instructions (SIgA is an important immunoglobulin that can enhance humoral immunity).

[0174] The results are as follows Figure 12 As shown, intervention with Lactobacillus acidophilus LAPerfectus100 can promote the secretion of SIgA in the colon. Compared with the model group, the SIgA concentration in the Lactobacillus acidophilus LAPerfectus100 intervention group was significantly increased (p<0.001), and its content was about 1.7 times that of the model group and 1.4 times that of the blank group. This indicates that Lactobacillus acidophilus LAPerfectus100 can enhance immunity by stimulating SIgA. Combined with the results of Examples 10 and 11, this shows that Lactobacillus acidophilus LAPerfectus100 has the effect of regulating the immunity of colitis mice.

[0175] Example 13: Effects of Lactobacillus acidophilus LAPerfectus100 on the intestinal flora of mice

[0176] Alpha diversity is an important indicator of microbial community diversity, and highly diverse microbiota are generally considered to be more beneficial to health. This example investigates the effects of Lactobacillus acidophilus LAPerfectus100 on the gut microbiota of mice. Mouse grouping and model establishment were the same as in Example 7. After the experiment, mice were sacrificed and dissected, cecal contents were collected, and 16S rRNA sequencing and analysis were performed on the cecal contents.

[0177] Results of α-diversity of gut microbiota in each group are as follows: Figure 13As shown, compared with the control group, the ACE, Chao1, Simpson, and Shannon indices of gut microbiota α-diversity in the model group were all decreased, with the ACE, Chao1, and Simpson indices being significantly decreased (p < 0.05), indicating a decline in the richness and diversity of gut microbiota in colonic mice. Compared with the model group, the ACE, Chao1, Simpson, and Shannon indices of mice in the *Lactobacillus acidophilus* LAPerfectus100 intervention group were all increased, with the ACE, Chao1, and Shannon indices being significantly increased (p < 0.05). In particular, the Shannon index showed no significant difference compared with the control group (p > 0.05), indicating that *Lactobacillus acidophilus* LAPerfectus100 could restore it to normal levels. In conclusion, *Lactobacillus acidophilus* LAPerfectus100 can improve the richness and diversity of gut microbiota.

[0178] Gut microbiota with differences at the genus level, such as Figure 14 and Figure 15 As shown in the figure, compared with the control group, the relative contents of beneficial bacteria such as Lactobacillus, Butyricicoccus, GCA_900066575, Coridextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001 in the intestines of mice in the model group were all decreased, while the contents of harmful bacteria such as Erysipelatoclostridium, Tuzzerella, and Escherichia_Shigella were significantly increased (P < 0.05). In the Lactobacillus acidophilus LAPerfectus100 intervention group, the levels of beneficial bacteria Lactobacillus, Butyricicoccus, GCA_900066575, Coridextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001 were significantly increased (P<0.05), especially Oscillibacter, which could recover to normal levels (no significant difference compared with the control group, P>0.05). The levels of harmful bacteria Erysipelatoclostridium, Tuzzerella, and Escherichia_Shigella were significantly decreased and could be reduced to normal levels (no significant difference compared with the control group, P>0.05).

[0179] Example 14: Preparation of Lactobacillus acidophilus LAPerfectus 100 bacterial powder

[0180] Lactobacillus acidophilus LAPerfectus100 seed culture was amplified through primary and secondary cultures to obtain fermentation seed liquid. This seed liquid was inoculated into a 6-ton fermenter containing 5.5 tons of fermentation broth and fermented at 37±2℃ for 8-12 hours. The bacterial cells were collected by centrifugation, emulsified with a protectant, and then freeze-dried to obtain Lactobacillus acidophilus LAPerfectus100 bacterial powder. After production, the moisture content, water activity, and viable cell count were measured, and the results are shown in Table 11.

[0181] Table 11 Production Results of Lactobacillus acidophilus LAPerfectus 100 Factant Powder

[0182] batch <![CDATA[Viable bacteria in bacterial powder (×10 11 CFU / g)]]> Moisture content (%) water activity 24G0395W 2.9 2.15 0.15

[0183] The production results demonstrate that Lactobacillus acidophilus LAPerfectus100 can be industrialized, providing a solid and feasible foundation for its application in improving colitis, regulating immunity, and intestinal flora.

[0184] The above experimental results demonstrate that the *Lactobacillus acidophilus* strain LAPerfectus100 of this invention possesses excellent resistance to gastric acid and bile salts, and its adhesiveness is higher than that of *Lactobacillus rhamnosus* GG, which is recognized in the industry as having the strongest adhesiveness. This strain, *Lactobacillus acidophilus* LAPerfectus100, not only significantly inhibits pathogenic bacteria such as *Escherichia coli*, *Salmonella*, *Staphylococcus aureus*, *Streptococcus pharyngitis*, and *Enterococcus haematobacterium*, but also improves colitis, regulates immunity and intestinal flora. Specifically, it can inhibit weight loss and colonic shortening, improve colitis symptoms, reduce the levels of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β in the colon, increase the levels of anti-inflammatory factor IL-10, restore the spleen of colitis-affected mice to normal levels, significantly increase the level of secretory immunoglobulin A (SIgA), and improve the richness of intestinal flora in mice. It enhanced community diversity, increased the abundance of beneficial intestinal bacteria such as Lactobacillus, Butyricicoccus, GCA_900066575, Coridextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001, and decreased the relative abundance of harmful bacteria such as Erysipelatoclostridium, Tuzzerella, and Escherchia_Shigella. Notably, it was discovered for the first time that Lactobacillus acidophilus had a regulatory effect on GCA_900066575, Coridextribacter, and Rikenellaceae_RC9_gut_group.

[0185] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A type of Lactobacillus acidophilus LAPerfectus100, characterized in that, The Lactobacillus acidophilus LAPerfectus100 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 8, 2024, with accession number GDMCC No. 65439.

2. The use of Lactobacillus acidophilus LAPerfectus100 as described in claim 1 in the preparation of a medicament for improving colitis.

3. The application as described in claim 2, characterized in that, The colitis mentioned is ulcerative colitis.

4. The use of Lactobacillus acidophilus LAPerfectus100 as described in claim 1 in the preparation of immunomodulatory drugs.

5. The use of Lactobacillus acidophilus LAPerfectus100 as described in claim 1 in the preparation of a drug for regulating intestinal flora.

6. The application as described in claim 5, characterized in that, The regulation of gut microbiota includes increasing gut microbiota richness and community diversity; and / or increasing the relative abundance of beneficial bacteria in the gut; and / or decreasing the relative abundance of harmful bacteria in the gut.

7. The application as described in claim 6, characterized in that, The beneficial intestinal bacteria include at least one of Lactobacillus, Butyricicoccus, GCA_900066575, Coridextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001; and / or, the harmful intestinal bacteria include at least one of Erysipelatoclostridium, Tuzzerella, and Escherchia_Shigella.

8. The use of Lactobacillus acidophilus LAPerfectus 100 as described in claim 1 in the preparation of an antibacterial drug, characterized in that, The antibacterial effect includes inhibition of at least one of Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, and Enterococcus haematobacterium.

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