Lactobacillus acidophilus LAPerfecus 100 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus acidophilus LAPerfectus100 and application of the lactobacillus acidophilus LAPerfectus100. According to the present invention, the Lactobacillus acidophilus LAPerfect100 is separated from the intestinal tract of a healthy infant, and the preservation number of the Lactobacillus acidophilus LAPerfect100 is GDMCC No. 65439; the lactobacillus acidophilus LAPerfectus100 has no drug resistance, no hemolytic activity, no pathogenicity, no virulence gene and transferable drug-resistant gene, is high in safety, has better gastric acid resistance, cholate resistance and adhesiveness, not only can inhibit various pathogenic bacteria and improve colitis, but also has better effects of regulating immunity and intestinal flora, and can be used for preventing and treating colitis. The traditional Chinese medicine composition can be used for improving colitis and regulating immunity and intestinal flora.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Lactobacillus acidophilus LA Perfectus 100 and applications thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is an inflammatory disease that affects the gastrointestinal tract. Its incidence has gradually increased in recent years and has become a health issue of widespread concern worldwide. IBD has two main clinical manifestations: Crohn's disease (CD) and ulcerative colitis (UC). Common clinical symptoms of UC patients include diarrhea, abdominal pain, blood in the stool, and weight loss. Currently, UC cannot be completely cured, but can only relieve related symptoms. However, it is prone to relapse after relief, which seriously affects the quality of life.

[0003] UC treatment can be mainly divided into drug treatment and surgical treatment. Both treatment methods have certain side effects or limitations. 5-aminosalicylic acid (5-ASA), immunosuppressive drugs, biologics, and local or systemic steroids are the main drugs for the treatment of 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 limited by age. Surgical intervention is usually used to treat acute or more severe UC, but it may cause complications such as diarrhea, intestinal adhesions, and abdominal infections after surgery. In addition, for children, surgical treatment may affect their growth and development and reduce their quality of life. Therefore, it is necessary to demand safe and effective UC treatment methods.

[0004] Lactobacillus acidophilus is widely present in the human body and has been widely used in fermented foods and functional foods. It is a probiotic with important commercial significance. Studies have shown that Lactobacillus acidophilus has the effects of regulating intestinal flora, enhancing immunity, and inhibiting Helicobacter pylori. Phenotypic tests such as drug sensitivity, hemolysis, and cytotoxicity also show that it has certain safety. However, there are still deficiencies in the current research on Lactobacillus acidophilus. First, there is insufficient safety research: it only stays on the results of in vitro tests such as drug sensitivity, hemolysis, and cytotoxicity, and fails to comprehensively judge its safety by combining animal tests and genetic resistance and virulence gene analysis. The safety of strains is closely related to genes. Drug sensitivity, hemolysis, and cytotoxicity are only evaluated from phenotypic tests, which fail to fully reflect the safety of strains. The existing safety research on Lactobacillus acidophilus is not sufficient; second, there is insufficient industrial research: most strains only stay in laboratory research at the small and pilot levels, and have not been verified in industrial workshops. It is rare for strains to be successfully marketed commercially, and the functional application of strains cannot be realized.

[0005] The safety and beneficial properties of probiotics are highly strain-specific. The safety and efficacy properties of probiotics of the same species and different strains vary greatly. Researching new probiotics that can improve ulcerative colitis is still a research difficulty. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a Lactobacillus acidophilus (Lactobacillus acidophilus) LAPerfectus100 and its application in improving colitis, regulating immunity and intestinal flora.

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

[0008] In a first aspect, the present invention provides a Lactobacillus acidophilus (Lactobacillus acidophilus) LAPerfectus100; the Lactobacillus acidophilus (Lactobacillus acidophilus) LAPerfectus100 has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 8, 2024, with a deposit number of GDMCC No.65439.

[0009] The present invention screens and separates a Lactobacillus acidophilus LAPerfectus100 from the intestinal tract of infants and young children, which is classified as Lactobacillus acidophilus. The Lactobacillus acidophilus LAPerfectus100 has 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 the industry-recognized strongest adhesion strain Lactobacillus rhamnosus GG;

[0012] (3) It has good ability to inhibit Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, and Enterococcus hirae pathogens, and the present invention is the first to discover that Lactobacillus acidophilus has the effect of inhibiting Streptococcus pharyngitis and Enterococcus hirae pathogens;

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

[0014] (5) It has a good immune regulation effect. Its ability to regulate the proliferation of macrophages 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 levels and significantly increase the content of secretory immunoglobulin A (SIgA). In addition, it can also reduce the content of TNF-α, IL-6, and IL-1β pro-inflammatory factors in the colon of colitis mice, increase the content of IL-10 anti-inflammatory factors, and restore the content of TNF-α, IL-6, IL-1β, and IL-10 to normal levels.

[0015] (6) It has a good effect on regulating intestinal flora, improving the richness and diversity of intestinal flora in mice, increasing the number of beneficial intestinal bacteria Lactobacillus, Butyricicoccus, GCA_900066575, Colidextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001, and reducing the relative abundance of harmful bacteria Erysipelatoclostridium, Tuzzerella, and Escherichia_Shigella. For the first time, it was found that Lactobacillus acidophilus has a regulatory effect on GCA_900066575, Colidextribacter, and Rikenellaceae_RC9_gut_group.

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

[0017] Based on the above characteristics, the Lactobacillus acidophilus LAPerfectus 100 can be used to improve colitis, regulate immunity and intestinal flora.

[0018] In a second aspect, the present invention provides the use of Lactobacillus acidophilus LAPerfectus100 in preparing a product for improving colitis.

[0019] Preferably, the colitis is ulcerative colitis.

[0020] In a third aspect, the present invention provides the use of Lactobacillus acidophilus LAPerfectus100 in the preparation of an immune regulating product.

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

[0022] In a fourth aspect, the present invention provides the use of Lactobacillus acidophilus LAPerfectus100 in the preparation of a product for regulating intestinal flora.

[0023] Preferably, the regulating 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 reducing the relative abundance of harmful bacteria in the intestine.

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

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

[0026] In a fifth aspect, the present invention provides the use of Lactobacillus acidophilus LAPerfectus100 in the preparation of antibacterial products.

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

[0028] The beneficial effects of the present invention are:

[0029] The present invention obtains a Lactobacillus acidophilus LAPerfectus100 through screening and separation; 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 the industry-recognized strain with the strongest adhesion, Lactobacillus rhamnosus GG.

[0032] (3) It has good antagonism against pathogens such as Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, and Enterococcus hirae, and the present invention is the first to discover that Lactobacillus acidophilus has the effect of inhibiting Streptococcus pharyngitis and Enterococcus hirae;

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

[0034] (5) It has a good immune regulation effect. Its ability to regulate the proliferation of macrophages 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 levels and significantly increase the content of secretory immunoglobulin A (SIgA);

[0035] (6) It has a good effect on regulating intestinal flora, improving the richness and diversity of intestinal flora in mice, increasing the number of beneficial intestinal bacteria Lactobacillus, Butyricicoccus, GCA_900066575, Colidextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001, and reducing the relative abundance of harmful bacteria Erysipelatoclostridium, Tuzzerella, and Escherchia_Shigella. For the first time, it was found that Lactobacillus acidophilus has a regulatory effect on GCA_900066575, Colidextribacter, and Rikenellaceae_RC9_gut_group.

[0036] (7) The Lactobacillus acidophilus LA Perfectus 100 of the present application can be industrialized, and the number of live bacteria in the raw powder of the live bacteria preparation is not less than 2×10 11 CFU / g, and can achieve large-scale production, which provides a solid and feasible basis for its application in improving colitis, regulating immunity and intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Figure 2 shows the colony morphology and bacterial morphology of Lactobacillus acidophilus LA Perfectus 100; Figure A shows the colony morphology on an MRS plate, and Figure B shows the bacterial morphology under a microscope.

[0038] Figure 2This is the phylogenetic tree of 16s rRNA of Lactobacillus acidophilus LAPerfectus100 and related species.

[0039] Figure 3 This is the complete map of the genetic chromosome sequence of Lactobacillus acidophilus LAPerfectus100.

[0040] Figure 4 This is a graph showing the hemolytic test results of Lactobacillus acidophilus LAPerfectus100.

[0041] Figure 5 These are test results of bioamine production by Lactobacillus acidophilus LAPerfectus 100; Figure A is Lactobacillus acidophilus LAPerfectus 100, and Figure B is the control strain Escherichia coli.

[0042] Figure 6 These are the graphs showing the changes in mouse body weight and DAI scores after treatment in different groups; Figure A shows the changes in mouse body weight, and Figure B shows the changes in DAI scores.

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

[0044] Figure 8 The colon pathological images of mice after different group treatments.

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

[0046] Fig.10 The results show the content of cytokines in the colon of mice after treatment in different groups.

[0047] Fig.11 The spleen index of mice after different group treatments was measured.

[0048] Fig.12 These are the results of measuring the content of secretory immunoglobulin A (SIgA) in the colon of mice after different grouping treatments.

[0049] Fig.13 The results of α-diversity determination of intestinal flora of mice after different group treatments.

[0050] Fig.14 The results show the relative abundance of some beneficial bacteria in the intestinal flora of mice after different group treatments.

[0051] Fig.15 The results of relative abundance determination of some harmful bacteria in the intestinal flora of mice after different group treatments. DETAILED DESCRIPTION

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

[0053] The culture medium formula used in the present invention is:

[0054] LBS medium (g / L): 5.0 g yeast extract, 10.0 g tryptone, 6.0 g potassium dihydrogen phosphate, 0.034 g ferrous sulfate, 0.575 g magnesium sulfate, 20.0 g glucose, 25.0 g sodium acetate, 2.0 g ammonium citrate, 0.12 g manganese sulfate, adjust to pH 5.5, add 1 mL Tween-80 and 1.3 mL glacial acetic acid, heat and stir to dissolve in 1000 mL distilled water, and sterilize at 121°C for 15 min. Add 15.0 g agar to the solid medium.

[0055] The preparation method of MRS culture medium refers to GB4789.35-2016, and 1.5% agar is added to the solid culture medium.

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

[0057] Lactobacillus acidophilus LAPerfectus100 was activated by three subcultures before the experiment, and the specific activation methods were as follows: first-generation activation: the strain was inoculated into MRS liquid culture medium at an inoculum size of 2% (v / v), and cultured at 37°C overnight under anaerobic conditions; second-generation activation: the strain was inoculated into MRS liquid culture medium at an inoculum size of 3% (v / v), and cultured at 37°C for 8-9h under anaerobic conditions; third-generation activation: the strain was inoculated into MRS liquid culture medium at an inoculum size of 2% (v / v), and cultured at 37°C overnight under anaerobic conditions.

[0058] All data in the present invention are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns means P > 0.05).

[0059] Example 1: Isolation and Identification of Lactobacillus acidophilus LAPerfectus100

[0060] 1. Isolation and screening of strains

[0061] Fecal samples of infants and young children who meet the requirements (healthy and have not consumed any probiotic products) were collected and placed in sterile sampling tubes for transportation in ice boxes. They were diluted with 0.85% saline in a gradient manner under sterile conditions and 10 samples were selected. -1 , 10 -2 , 10 -3 The dilution gradient was spread on LBS agar plates and cultured anaerobically at 37°C for 48h. The colony morphology on the plate was observed by naked eyes and suspected single colonies were picked for Gram staining. The morphological characteristics of the colonies were observed under a microscope to preliminarily screen suspected Lactobacillus strains. The strains were repeatedly streaked and purified on MRS solid medium to obtain purified Lactobacillus strains, which were stored in an ultra-low temperature refrigerator at -80°C.

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

[0063] (1) Morphological characteristics: The bacterial suspension was diluted in a gradient manner and cultured anaerobically at 36°C on an MRS plate for 48 h. The colony morphology was as follows: Figure 1 As shown in A. The single colony of the strain on the MRS solid plate is milky white, round, convex, with a smooth surface and neat edges.

[0064] The morphology of the bacteria was observed under a microscope. Figure 1 As shown in B, the bacterial body morphology is a Gram-positive bacillus, does not produce spores, is short rod-shaped, and is arranged singly or in pairs, with a rounded end.

[0065] (2) Molecular biological identification: The strain was cultured in liquid medium, the cells were collected to extract genomic DNA, and 16S rRNA was detected. The phylogenetic tree of Lactobacillus acidophilus LAPerfectus100 was constructed based on the 16S rRNA gene sequence using MEGA software and 1000 repetitions. Figure 2 (Nodes show Bootstrap values ​​greater than 50% and the superscript "T" indicates the model strain).

[0066] The result showed that the 16S rRNA gene sequence of Lactobacillus acidophilus LAPerfectus100 was 1364bp. The nucleotide homology comparison was carried out with the sequence registered in Genebank using the Blast program. The 16S rRNA gene sequence of the strain had 100% homology with Lactobacilus acidophilus. According to the molecular biological identification result, the obtained strain was determined to be Lactobacillus acidophilus, and the strain was named as 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 in the Guangdong Provincial Microbiological Culture Collection Center on November 8, 2024, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No.65439.

[0069] 3. Whole genome sequencing

[0070] The second and third generation high-throughput gene sequencing technologies were used to detect the full gene sequence of Lactobacillus acidophilus LAPerfectus100, draw a whole genome map and identify the species at the genome level.

[0071] After the strain was expanded, the cells were collected by centrifugation at 8000rpm for 5min (4℃), the supernatant was discarded, and the cells were quickly frozen in liquid nitrogen. After the cells were frozen through, they were transferred to a -80℃ refrigerator and mailed to the testing agency to complete the second-generation and third-generation sequencing. The second-generation sequencing platform was used, and the third-generation sequencing platform was used. The library was constructed and sequenced on the machine according to the library construction requirements of the sequencing platform. After the data was qualified after quality inspection and filtering, the third-generation data was calibrated with 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, without plasmid, with a genome size of 1.99Mb 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 chromosome sequence of the gene is shown in Figure 3 The strain was compared with the recognized NT database and identified as Lactobacillus acidophilus at the whole genome level.

[0073] Example 2: Verification of food safety of Lactobacillus acidophilus LAPerfectus100

[0074] 1. Drug sensitivity testing

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

[0076] Table 1 MIC values ​​and drug sensitivity test results of Lactobacillus acidophilus LAPerfectus100

[0077]

[0078]

[0079] 2. Hemolytic test

[0080] The hemolytic activity of Lactobacillus acidophilus LAPerfectus100 was tested with Streptococcus pyogenes as a control. Figure 4 As shown in the figure, after Streptococcus pyogenes was inoculated on Columbia agar plate for culture, transparent hemolytic zones appeared around the colonies, which was type B hemolysis (β hemolysis); while after Lactobacillus acidophilus LA Perfectus 100 was inoculated on the culture medium, no hemolytic zones appeared around the colonies. Therefore, it was judged that the strain was not hemolytic, would not cause hemolytic hazards, and was a safe strain.

[0081] 3. Detection of biogenic amines

[0082] The culture medium with amino acid decarboxylase activity can produce alkaline biogenic amines. The result can be displayed based on the color change of the indicator, which can be used to preliminarily detect whether biogenic amines are produced.

[0083] Aminodecarboxylase test medium ratio: peptone 5g, yeast extract 3g, glucose 1g, distilled water 1000mL, 1.6% bromocresol purple-ethanol solution 1mL, agar 1.8%, pH = 6.8, 115℃ high pressure sterilization for 20min. (Added amino acids are L-arginine, L-lysine, L-tryptophan, L-histidine).

[0084] Inoculate 2% of the cultured bacterial solution into 1.6 mL of aminodecarboxylase culture solution without the addition of precursor amino acids and culture overnight; drip the activated cultured bacterial solution onto each filter paper to soak the filter paper, place the sterile filter paper on the modified aminodecarboxylase detection agar medium with the addition of precursor amino acids, and culture at 37°C for 2 days. Observe whether the transparent circle around the filter paper changes color. If the color around the filter paper turns purple, it means that biogenic amines are produced, otherwise, it means that no biogenic amines are produced.

[0085] The results are as follows Figure 5 As shown, there was no color change around Lactobacillus acidophilus LAPerfectus100, but Escherichia coli produced alkaline biogenic amines, and the culture medium around its filter paper changed from yellow to purple. The results showed that Lactobacillus acidophilus LAPerfectus100 did not produce biogenic amines and had good safety.

[0086] 4. Bacterial pathogenicity detection

[0087] The bacterial pathogenicity test was carried out according to the method in Appendix A of the "Technical Guidelines for Safety Testing and Evaluation of Bacterial Species Used in Health Food Ingredients (2020 Edition)".

[0088] Table 2 Results of bacterial pathogenicity test (intraperitoneal injection) of Lactobacillus acidophilus LAPerfectus100 in mice

[0089]

[0090] The results of the mouse bacterial pathogenicity test (intraperitoneal injection) of Lactobacillus acidophilus LAPerfectus100 are shown in Table 2. The bacterial suspension of Lactobacillus acidophilus LAPerfectus100 was injected intraperitoneally once to mice of both sexes, and the amount of bacteria injected to each mouse was 1.07×10 7 CFU (bacterial concentration was 5.34×10 7 CFU / mL, injection volume was 0.2mL / animal). During the 21-day observation period, the animals were in normal condition, with no abnormal conditions such as poisoning signs and death.

[0091] Table 3 Results of the mouse bacterial pathogenicity test (oral gavage) of Lactobacillus acidophilus LAPerfectus100

[0092]

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

[0094] According to the bacterial pathogenicity test method used for health food raw materials, the mouse bacterial pathogenicity test result of Lactobacillus acidophilus LA Perfectus 100 was negative, indicating that the strain is non-pathogenic.

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

[0096] (1) The genome sequence of the strain was 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. Sequence identity ≥ 85% was used 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, there was no drug resistance gene with sequence identity ≥ 85% in Lactobacillus acidophilus LAPerfectus100, and no drug resistance gene was predicted.

[0097] Table 4 Sequence alignment results of Lactobacillus acidophilus LAPerfectus100 and 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 measured 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 (identity) is GL000819 (identity = 73.146%). According to the "Technical Guidelines for Safety Inspection and Evaluation of Bacteria for Health Food Ingredients" (2020 edition), Lactobacillus acidophilus LAPerfectus100 does not have known virulence factors and toxin metabolism-related genes that are clearly related to pathogenicity with sequence length coverage (coverage) ≥ 60% and sequence identity (identity) ≥ 85%, and no virulence genes were predicted.

[0100] Table 5 Sequence alignment results of Lactobacillus acidophilus LAPerfectus100 and 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, combined with the phenotypic results such as drug sensitivity, hemolysis, bacterial pathogenicity in mice, and virulence gene and resistance gene analysis, it can be seen that Lactobacillus acidophilus LAPerfectus100 has no resistance to the antibiotics specified by EFSA, the hemolysis reaction is negative, the animal test is non-pathogenic, and the gene analysis shows no virulence genes and transferable resistance genes. In summary, Lactobacillus acidophilus LAPerfectus100 is a safe strain that meets the safety requirements of the human body.

[0103] Example 3: Evaluation of gastric juice and bile salt tolerance of Lactobacillus acidophilus LAPerfectus100

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

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

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

[0107] In the above formula: N1 represents the number of viable bacteria in the strain system after treatment (CFU / mL), 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. Bile salt tolerance evaluation:

[0109] Experimental steps: ① Preparation of bile salt solution: add 0.2% (w / v) sodium thioglycolate to MRS liquid culture medium, then add bile salt with a final concentration of 0.3% (w / v), and after fully dissolving, filter and sterilize with a 0.22 μm sterile microporous filter membrane for use; ② After the strain is activated, take 0.1 mL of the bacterial suspension and add it to 0.9 mL of bile salt solution, and incubate at 37°C for 3 h; at 0 h and 3 h, the treated solution is gradiently diluted and spread on a solid plate for live bacteria counting; ③ Calculate the strain survival rate according to formula (1).

[0110] 3. Experimental results

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

[0112] The results of gastric juice and bile salt tolerance of Lactobacillus acidophilus LAPerfectus100 are shown in Table 6. The survival rate of Lactobacillus acidophilus LAPerfectus100 after being treated in artificial gastric juice (pH=2.5) for 3 hours reached more than 97%, and the survival rate after being treated in bile salt solution (0.3%) for 3 hours reached more than 93%, which was significantly higher than that of Lactobacillus acidophilus screened 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°C, 30 min) fetal bovine serum (FBS), 1% penicillin and streptomycin, and cultured in a 37°C, 90% humidity, 5% CO2 incubator.

[0118] 2. Cell adhesion experiment: ① Liquid culture of experimental strains: Activated second generation bacterial solution, 2% inoculation volume, 9 mL culture medium / 15 mL centrifuge tube, 37°C, static culture for 15 h. ② Preparation of monolayer: Caco-2 cells were inoculated into DMEM culture medium supplemented with 20% (v / v) fetal bovine serum, transferred to 12-well cell culture plates, and the cell addition volume was 10 1 mL per well. 5 cell / mL, 5% CO2, 37℃ constant temperature culture, change the medium every other day until a single cell layer is obtained for use; ③ Preparation of bacterial suspension: At the same time, take the cultured bacterial solution of the strain, centrifuge at 10000r / min for 1min at room temperature to collect the bacteria, wash twice with sterile PBS, resuspend in DMEM medium, and adjust the bacterial suspension concentration to 10 8 CFU / mL; ④ Co-culture: Prepare the monolayer of Caco-2 cells, remove the culture medium, add PBS buffer to rinse twice, remove the buffer, add 1mL / well of the prepared bacterial suspension, mix well, and incubate at 5% CO2 and 37°C for 2h; ⑤ Carefully remove the culture supernatant, add sterile PBS to rinse 5 times to remove non-adherent bacteria; ⑥ Add 0.2mL / well of trypsin cell digestion solution and digest for 5min to elute the cells from the culture plate wells, and the collected solution is the sample; ⑦ Perform gradient dilution and live bacteria count on the collected samples, and calculate the adhesion capacity according to formula (2).

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

[0120] ——Formula (2)

[0121] In this experiment, Lactobacillus acidophilus LA82 with the second best gastric juice tolerance and bile salt tolerance selected in Example 3 and the commercial strain Lactobacillus rhamnosus GG with the strongest adhesion recognized by the industry were selected as comparison strains, and 3 parallels were performed for each experiment. The results are shown in Table 7, and the adhesion ability of Lactobacillus acidophilus LAPerfectus100 is significantly higher than that of Lactobacillus acidophilus LA82 and the commercial star strain Lactobacillus rhamnosus GG selected from the same batch.

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

[0123]

[0124]

[0125] Example 5: Evaluation of the proliferation ability of Lactobacillus acidophilus LAPerfectus100 on macrophage RAW264.7

[0126] Experimental methods:

[0127] 1. RAW264.7 cell culture: After RAW264.7 cells were taken out for recovery, they were cultured in a 5% CO2 constant temperature incubator at 37°C with DMEM complete medium containing 1% non-essential amino acids, 1% penicillin-streptomycin and 10% fetal bovine serum. The medium was changed every other day, and when the cell confluence reached more than 80%, subculture was performed. Wash twice with sterile PBS, add 2 mL of culture medium, gently scrape the cells with a cell scraper, centrifuge and discard the supernatant, resuspend with DMEM complete culture medium, and subculture at a ratio of 1:3.

[0128] The concentration of RAW264.7 cells was adjusted to 1 × 10 4 cell / mL, inoculated into 96-well plates at 100 μL / well, cultured for 24 h until the cells adhered to the wall and grew, then aspirated the supernatant in the wells and washed twice with PBS.

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

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

[0131] Cell survival rate (%) = OD1 / OD2 × 100% —— Formula (3)

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

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

[0134] Table 8 Effect of Lactobacillus acidophilus LAPerfectus100 on the proliferation of macrophages

[0135]

[0136] The results of Lactobacillus acidophilus LAPerfectus100 on the proliferation of macrophages RAW264.7 are shown in Table 8. Compared with NC, the number of macrophages in the group treated with Lactobacillus acidophilus LAPerfectus100 was 148.95% of that in the blank group, which can significantly promote the proliferation of macrophages, and is higher than the proliferation rate of macrophages by Lactobacillus acidophilus NCFM, indicating that Lactobacillus acidophilus LAPerfectus100 has the potential for immunomodulation.

[0137] Example 6: Evaluation of the ability of Lactobacillus acidophilus LAPerfectus100 to antagonize pathogens

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

[0139] 2. Antagonistic pathogen experiment: ① Cool the MRS broth medium or BHI broth medium containing 1.5% (w / v) agar to about 55°C, and mix it with the indicator bacteria suspension in a certain proportion (Mix with MRS broth medium for Escherichia coli, Salmonella, and Staphylococcus aureus, and mix with BHI broth medium for Enterococcus hirae and Streptococcus pharyngitis) until the number of viable indicator bacteria is 10 6 CFU / mL order of magnitude, and then quickly poured into a plate with an Oxford cup placed in advance. After the culture medium cooled and solidified, the Oxford cup was taken out, and 200 μL of Lactobacillus acidophilus LAPerfectus100 fermentation liquid was injected into each well. After culturing at 37°C overnight, the diameter of the inhibition zone was measured.

[0140] 3. Experimental results

[0141] Table 9 Inhibition zones of Lactobacillus acidophilus LAPerfectus100 against different pathogens

[0142] Pathogens Inhibition 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] In this example, the inhibitory effect of Lactobacillus acidophilus on five common pathogens, including Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, and Enterococcus hirae, was detected. The antagonistic ability of Lactobacillus acidophilus LAPerfectus100 against the five pathogens is shown in Table 9. The results show that Lactobacillus acidophilus LAPerfectus100 has a good inhibitory effect on Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, and Enterococcus hirae.

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

[0145] Establishment of colitis model: The animal model was induced by DSS. Twenty-four SPF BALB / c male mice (19-20g) were used in the experiment. The room temperature of the animals was 23±2℃, the humidity was 50%±10%, the artificial light was 12h / d, and they had free diet. After one week of adaptive feeding, all mice were randomly divided into 3 groups, 8 in each group, namely the normal group (NC), the model group (DSS), and the Lactobacillus acidophilus treatment group (LAPerfectus100). The experiment lasted for 2 weeks, and the specific experimental process is as follows:

[0146] Blank group: The mice drank water normally during the whole experiment, and were given 0.2 mL phosphate buffered saline (PBS) in the stomach at regular intervals every day;

[0147] Model group: mice were allowed to drink water normally from day 1 to day 7. From day 8, mice were allowed to drink 3.5% DSS solution freely for 7 days. The DSS solution was changed every 2 days to ensure the concentration and quality of the DSS solution. During the whole experiment, mice were gavaged with 0.2 mL PBS at regular intervals every day.

[0148] Lactobacillus acidophilus LAPerfectus100 intervention group: mice drank water normally from day 1 to day 7, and from day 8 onwards, mice drank 3.5% DSS solution freely for 7 days. The DSS solution was changed every 2 days to ensure the concentration and quality of the DSS solution. During the whole experiment, 0.2 mL of Lactobacillus acidophilus LAPerfectus100 suspension (viable cell count concentration of 5×10 9 CFU / mL).

[0149] After the experiment, all mice were fasted for 12 h, killed and dissected, and samples were collected for subsequent indicator measurement and analysis.

[0150] Changes in mouse DAI scores: During the modeling period, mice were weighed and recorded every day to count the changes in mouse weight. During the modeling period, mouse feces were collected every day, the fecal properties were observed, and the blood in the feces of mice was detected using occult blood test paper. The DAI score was calculated according to formula (4). The specific scoring criteria are shown in Table 10. The results are shown in Table 10. Figure 6The remission rate was calculated according to formula (5).

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

[0152] Remission rate = (weight change rate of intervention group - weight change rate of model group) / (weight change rate of blank group - weight change rate of model group) - Formula (5)

[0153] Table 10DAI scoring criteria

[0154]

[0155]

[0156] Depend on Figure 6 A shows that the body weight of mice in the model group continued to decrease during the modeling period. At the end of the experiment, the body weight change rates of the blank group, model group, and Lactobacillus acidophilus LAPerfectus100 intervention group (body weight on the 7th day of modeling / body weight on the 1st day of modeling) 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 that the model was successfully established. After the intervention of Lactobacillus acidophilus LAPerfectus100, the remission rate reached more than 40% (remission rate = (0.9323-0.8258) / (1.0918-0.8258) = 40.04%), which effectively alleviated the weight loss of mice (p < 0.01), which was higher than the 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 No. CN114231470A).

[0157] The trend of DAI changes is opposite to that of weight changes. Figure 6 B shows that the DAI score of the model group continued to increase during the modeling period. At the end of the experiment, the DAI score of the mice in the model group was significantly higher than that in the blank group (p < 0.0001), while the DAI score of the Lactobacillus acidophilus LAPerfectus100 group was significantly lower than that in the DSS group (p < 0.001), indicating that Lactobacillus acidophilus LAPerfectus100 effectively alleviates 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. The mice were killed and their colons were taken to measure the length of the colon. 0.5 cm of mouse ileum tissue was collected and fixed with 10% formaldehyde solution, and then stained with hematoxylin-eosin (HE), and finally observed under an optical microscope.

[0160] Colon length 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 the shortening of the colon (p < 0.01), and the colon length was 137.8% of the colon length of the modeling group, which was higher than the reported data (Lactobacillus acidophilus CCFM1200 was 115%, better than Lactobacillus acidophilus NCFM, source: Patent No. CN114231470A).

[0161] Colon pathology results Figure 8 As shown in the figure, the colon mucosa and crypts of the blank group mice were intact, with normal morphology and structure, abundant goblet cells, and no obvious lesions. The colon structure integrity of the model group was destroyed, the mucosa was damaged, the crypts and goblet cells were lost in large quantities, and inflammatory cell infiltration was visible. Compared with the model group, the intestinal damage of the mice in the Lactobacillus acidophilus LAPerfectus100 group was restored, and the intestinal morphology and structure were relatively complete, indicating that Lactobacillus acidophilus LAPerfectus100 can effectively reduce the colon damage of UC mice and reduce the degree of colon lesions.

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

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

[0164] This example determines the effect of Lactobacillus acidophilus LAPerfectus100 on colonic MPO in colitis mice. The grouping and modeling of mice are the same as in Example 7. After the experiment, the mice were killed and the colons were dissected and placed in phosphate buffered saline and homogenized. The MPO content in the colon homogenate of each group of mice was determined according to the detection method in the ELISA kit manufacturer's instructions.

[0165] The results are as follows Fig. 9As shown, compared with the control group, the MPO content in the colon of the model group mice increased significantly (p < 0.001), and compared with the model group, the MPO level in the colon of the Lactobacillus acidophilus LAPerfectus100 group mice decreased (p < 0.01). It can be seen that Lactobacillus acidophilus LAPerfectus100 can significantly reduce the level of MPO in the colon of colitis mice, indicating that Lactobacillus acidophilus LAPerfectus100 has the effect of reducing neutrophil aggregation, thereby alleviating intestinal inflammatory response.

[0166] Example 10: Effect of Lactobacillus acidophilus LAPerfectus100 on cytokines in the colon of colitis mice

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

[0168] The results are as follows Fig.10 As shown in the results, compared with the blank group, the content of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the model group was significantly increased (p < 0.05), and the content of anti-inflammatory cytokine IL-10 was extremely significantly decreased (p < 0.01), indicating that the inflammatory response of colitis mice was strong. After intervention with Lactobacillus acidophilus LAPerfectus100, the pro-inflammatory factors 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 blank group, P > 0.05). Lactobacillus acidophilus LAPerfectus100 also promoted the secretion of IL-10 in the colon of colitis mice, and the content was extremely significantly higher than that of the model group (p < 0.01). This shows that Lactobacillus acidophilus LAPerfectus100 can effectively reduce the increase of pro-inflammatory factors in mice, promote the secretion of anti-inflammatory factors, and inhibit the degree of inflammation.

[0169] Example 11: Effect of Lactobacillus acidophilus LAPerfectus100 on spleen index of colitis mice

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

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

[0172] Example 12: Effect of Lactobacillus acidophilus LAPerfectus 100 on the Content 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 killed and the colons were dissected and homogenized in phosphate buffered saline. The SIgA content in the colon homogenate of each group of mice was determined according to the detection method in the manufacturer's instructions of the ELISA kit (SIgA is an important immunoglobulin that can enhance humoral immunity).

[0174] The results are as follows Fig.12 As shown, Lactobacillus acidophilus LAPerfectus100 intervention can promote the secretion of colon SIgA. 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, indicating that Lactobacillus acidophilus LAPerfectus100 can improve immunity by stimulating SIgA. Combined with the results of Examples 10 and 11, it is shown that Lactobacillus acidophilus LAPerfectus100 has the effect of regulating the immunity of mice with colitis.

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

[0176] α-diversity is an important indicator of the diversity of microbial communities, and highly diverse microbial communities are generally considered to be more beneficial to health. This example explores the effect of Lactobacillus acidophilus LAPerfectus100 on the intestinal flora of mice. The grouping and modeling of mice are the same as in Example 7. After the experiment, the mice were killed and dissected, the cecal contents were collected, and the 16S rRNA sequencing and analysis of the cecal contents of the mice were performed.

[0177] The results of α-diversity of intestinal flora in each group are as follows Fig.13As shown in the figure, compared with the blank group, the ACE index, Chao1 index, Simpson index and Shannon index in the intestinal microbial α-diversity of the model group were all reduced, among which the ACE index, Chao1 index and Smpson index were significantly reduced (p < 0.05), indicating that the richness and diversity of the intestinal flora of the colon mice decreased; compared with the model group, the ACE index, Chao1 index, Simpson index and Shannon index of the mice in the Lactobacillus acidophilus LAPerfectus100 intervention group were all increased, among which the ACE index, Chao1 index and Shannon index were significantly increased (p < 0.05), especially the Shannon index was not significantly different from the blank group (p > 0.05), indicating that Lactobacillus acidophilus LAPerfectus100 can restore it to normal levels. In summary, Lactobacillus acidophilus LAPerfectus100 can improve the richness and community diversity of intestinal microbial flora.

[0178] Differences in intestinal flora at the genus level Fig.14 and Fig.15 Compared with the blank group, the relative contents of beneficial bacteria such as Lactobacillus, Butyricicoccus, GCA_900066575, Colidextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001 in the intestine of the model group mice were reduced, and the harmful bacteria 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, Colidextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001 were significantly increased (P < 0.05), especially Oscillibacter, which could be restored to normal levels (no significant difference compared with the blank group, P > 0.05). The levels of harmful bacteria Erysipelatoclostridium, Tuzzerella, and Escherichia_Shigella were significantly reduced and could be reduced to normal levels (no significant difference compared with the blank group, P > 0.05).

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

[0180] The Lactobacillus acidophilus LAPerfectus100 seeds were amplified by primary culture and secondary culture to obtain fermented seed liquid, and the fermented seed liquid was inoculated into a 6-ton fermentation tank containing 5.5 tons of fermented liquid and fermented at 37±2°C for 8-12 hours, and the bacteria were centrifuged, emulsified by adding a protective agent, and the emulsified bacterial mud was freeze-dried to obtain Lactobacillus acidophilus LAPerfectus100 bacterial powder. After production, the moisture content, water activity and viable count were tested, and the results are shown in Table 11.

[0181] Table 11 Lactobacillus acidophilus LAPerfectus100 powder production results

[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 show that Lactobacillus acidophilus LAPerfectus 100 can be industrialized, which provides a solid and feasible foundation for its application in improving colitis, regulating immunity and intestinal flora.

[0184] The above experimental results show that the strain Lactobacillus acidophilus LAPerfectus100 of the present invention has excellent gastric acid and bile salt resistance, and its adhesion is higher than that of Lactobacillus rhamnosus GG, which is recognized by the industry as the strain with the strongest adhesion. The strain Lactobacillus acidophilus LAPerfectus100 of the present invention can not only significantly inhibit pathogenic bacteria such as Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis, Enterococcus hirae, etc., but also improve colitis, regulate immunity and intestinal flora, which is specifically manifested in that it can inhibit weight loss and shortening of the colon, improve symptoms of colitis, reduce the content of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β in the colon, increase the content of anti-inflammatory factors such as IL-10, restore the spleen of colitis mice to normal levels, significantly increase the content of secretory immunoglobulin A (SIgA), and improve the richness of intestinal flora in mice. The results showed that the relative abundance of beneficial intestinal bacteria Lactobacillus, Butyricicoccus, GCA_900066575, Colidextribacter, Rikenellaceae_RC9_gut_group, Oscillibacter, and Prevotellaceae_UCG-001 increased, and the relative abundance of harmful bacteria Erysipelatoclostridium, Tuzzerella, and Escherchia_Shigella decreased. For the first time, it was found that Lactobacillus acidophilus had a regulatory effect on GCA_900066575, Colidextribacter, and Rikenellaceae_RC9_gut_group.

[0185] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A Lactobacillus acidophilus LAPerfectus100, characterized in that: The Lactobacillus acidophilus LAPerfectus100 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 8, 2024, with the deposit number GDMCC No.65439.

2. Use of Lactobacillus acidophilus LAPerfectus100 as claimed in claim 1 in preparing a product for improving colitis.

3. The use according to claim 2, characterized in that The colitis is ulcerative colitis.

4. Use of Lactobacillus acidophilus LAPerfectus100 as claimed in claim 1 in the preparation of immune regulating products.

5. Use of Lactobacillus acidophilus LAPerfectus100 as claimed in claim 1 in preparing a product for regulating intestinal flora.

6. The use according to claim 5, characterized in that The regulating of intestinal flora includes improving the richness and diversity of intestinal flora; and / or increasing the relative abundance of beneficial bacteria in the intestine; and / or reducing the relative abundance of harmful bacteria in the intestine.

7. The use according to claim 6, characterized in that The beneficial intestinal bacteria include at least one of Lactobacillus, Butyricicoccus, GCA_900066575, Colidextribacter, 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. Use of Lactobacillus acidophilus LAPerfectus100 as claimed in claim 1 in the preparation of antibacterial products.

9. The use according to claim 8, characterized in that The antibacterial agent includes inhibiting at least one of Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus pharyngitis and Enterococcus hirae.

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