Lactobacillus johnsonii A20083 for inhibiting infection of multi-drug-resistant strains and clostridium difficile and reducing fat as well as product and application of lactobacillus johnsonii A20083

Abstract

The invention discloses lactobacillus johnsonii A20083 for inhibiting infection of multiple drug-resistant strains and clostridium difficile and reducing fat and application of the lactobacillus johnsonii A20083. The lactobacillus johnsonii A20083 has an obvious inhibiting effect on drug-resistant staphylococcus aureus, pseudomonas aeruginosa, drug-resistant escherichia coli, drug-resistant acinetobacter baumannii and clostridium difficile type strains. Compared with a commercial strain 21076 of lactobacillus johnsonii, the lactobacillus johnsonii A20083 disclosed by the invention has a better antibacterial effect on clostridium difficile. The lactobacillus johnsonii A20083 has an obvious fat-reducing effect on a caenorhabditis elegans model, and also shows a good effect in in-vitro experiments of artificial gastrointestinal fluid resistance, cholate resistance and the like, so that the strain can be used as a probiotic agent for preparing products for reducing fat, losing weight and regulating intestinal flora, and has a good application prospect. The invention has important significance for preventing and / or treating diseases caused by multi-drug-resistant bacterium infection and clostridium difficile infection.

Description

Technical Field

[0001] The present invention relates to the technical field of functional strains, and in particular to a Lactobacillus johnsonii A20083 capable of inhibiting multidrug-resistant strains, Clostridium difficile infection and fat reduction, as well as products and applications thereof. Background Art

[0002] Clostridioides difficile infection (CDI) is one of the most pressing public health threats. Its main symptoms include abdominal pain, diarrhea, fever, etc. Improper or untimely treatment may even lead to pseudocolon and toxic megacolon. It is one of the most common causes of nosocomial infection and death.

[0003] Currently, antibiotics such as metronidazole, vancomycin, and rifampicin are the first-line treatments for CDI. However, antibiotic use can easily disrupt the host's microbiome, leading to recurrence of C. difficile infection or other diseases. Statistics show that the recurrence rate of CDI treated with antibiotics is as high as 25%-30%. In 1958, Dr. Eiseman first used fecal microbiota transplantation to treat CDI. Over 50 years of clinical application have demonstrated that fecal microbiota treatment for recurrent C. difficile infection is effective in up to 90% of patients. However, a small number of patients still experience CDI recurrence. Therefore, supplementing with functional probiotics can improve the treatment of CDI. Summary of the Invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems in the prior art. To this end, the present invention aims to provide a Lactobacillus johnsonii A20083 for inhibiting multidrug-resistant strains, Clostridium difficile infection and reducing fat, as well as a product and application thereof.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a Lactobacillus johnsonii, wherein the Lactobacillus johnsonii is Lactobacillus johnsonii A20083, taxonomically named Lactobacillus johnsonii, and deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on January 13, 2025 (the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), with a deposit number of GDMCC No: 65766.

[0007] In some embodiments of the present invention, the colony morphology of the Lactobacillus johnsonii A20083 is round, convex, entire and colorless.

[0008] In some embodiments of the present invention, the 16S sequence of the Lactobacillus johnsonii A20083 is shown as SEQ ID NO: 3.

[0009] In some embodiments of the present invention, the complete gene information of the Lactobacillus johnsonii A20083 is shown in Table 2.

[0010] In some embodiments of the present invention, the screening and isolation method of Lactobacillus johnsonii A20083 is as follows: a stool sample from a centenarian in Guangxi Zhuang Autonomous Region is taken to MRS solid culture medium, smeared evenly, and then cultured in an anaerobic workstation for two days. Colonies with round, raised, entire, and colorless colonies on the plate are picked and cultured in a small amount of MRS liquid culture medium. After PCR amplification and identification, Lactobacillus johnsonii A20083 is obtained.

[0011] In some embodiments of the present invention, the culture conditions in the anaerobic workstation are 37° C., 90% nitrogen + 10% carbon dioxide.

[0012] In some embodiments of the present invention, the primers used in the PCR amplification and identification are shown as SEQ ID NO: 1 and SEQ ID NO: 2.

[0013] In some embodiments of the present invention, the amplification system for PCR amplification identification is:

[0014] Components content 10 μM upstream primer 16S-F 1 μL 10 μM downstream primer 16S-R 1 μL PCR amplification solution 12.5μL DNA template 1 μL <![CDATA[ddH2O]]> Make up to 25 μL

[0015] In some embodiments of the present invention, the amplification program of the PCR amplification identification is 95°C for 3 min; 95°C for 40 s, 56°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 8 min.

[0016] The second aspect of the present invention provides a culture of Lactobacillus johnsonii A20083.

[0017] In some embodiments of the present invention, the culture includes the supernatant obtained after culturing Lactobacillus johnsonii A20083.

[0018] The third aspect of the present invention provides a bacterial agent.

[0019] In some embodiments of the present invention, the bacterial agent comprises the Lactobacillus johnsonii A20083 described in the above aspects and / or a culture of the Lactobacillus johnsonii A20083.

[0020] In some embodiments of the present invention, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0021] A fourth aspect of the present invention provides the use of any one or more of the Lactobacillus johnsonii A20083, the culture of the Lactobacillus johnsonii A20083, and the bacterial agent described in the above aspects in at least one of the following (I) to (V);

[0022] (1) preparing an antibacterial agent;

[0023] (II) preparing products for preventing and / or treating multidrug-resistant bacterial infections;

[0024] (III) preparing products for the prevention and / or treatment of Clostridium difficile infection;

[0025] (IV) preparing products for regulating intestinal flora;

[0026] (V) preparing products for reducing blood lipids and weight.

[0027] In some embodiments of the present invention, the antibacterial agent is an agent used to inhibit the number, activity and / or growth of live bacteria in vitro, including but not limited to any agent used for surface sterilization, antibacterial or control of microbial reproduction.

[0028] In some embodiments of the present invention, the bacteria are Gram-positive bacteria or Gram-negative bacteria.

[0029] In some embodiments of the invention, the bacteria include Clostridium difficile and Pseudomonas aeruginosa.

[0030] In some embodiments of the present invention, the bacteria also include multidrug-resistant bacteria; further including drug-resistant Staphylococcus aureus, drug-resistant Escherichia coli and drug-resistant Acinetobacter baumannii.

[0031] In some embodiments of the present invention, the multidrug-resistant bacteria in (II) are Gram-positive bacteria or Gram-negative bacteria.

[0032] In some embodiments of the present invention, the multidrug-resistant bacteria in (II) include drug-resistant Staphylococcus aureus, drug-resistant Escherichia coli and drug-resistant Acinetobacter baumannii.

[0033] In some embodiments of the present invention, the multidrug-resistant bacteria include bacteria resistant to β-lactam antibiotics.

[0034] In some embodiments of the present invention, the β-lactam antibiotics include methicillin, carbapenem antibiotics, penicillin antibiotics, cephalosporin antibiotics and monobactam antibiotics.

[0035] In some embodiments of the present invention, the drug-resistant Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA).

[0036] In some embodiments of the present invention, the drug-resistant Escherichia coli is ESBLs-producing Escherichia coli (Extended-Spectrumβ-Lactamases (ESBL)-Producing Escherichia coli).

[0037] In some embodiments of the present invention, the drug-resistant Acinetobacter baumannii is drug-resistant Acinetobacter baumannii HRAB-85.

[0038] A fifth aspect of the present invention provides a product comprising any one or more of the Lactobacillus johnsonii A20083 described in the above aspects, a culture of the Lactobacillus johnsonii A20083, and the bacterial agent.

[0039] In some embodiments of the present invention, the products include food, food additives, feed, feed additives and medicines.

[0040] In some embodiments of the present invention, the product further contains auxiliary materials.

[0041] In some embodiments of the present invention, the excipient includes a food additive or a pharmaceutically acceptable excipient.

[0042] A sixth aspect of the present invention provides a non-therapeutic method for inhibiting the number and / or activity and / or growth of live multidrug-resistant bacteria, Clostridium difficile and Pseudomonas aeruginosa.

[0043] In some embodiments of the present invention, the method comprises the step of using the Lactobacillus johnsonii A20083, the culture of the Lactobacillus johnsonii A20083 and / or the bacterial agent described in the above aspects.

[0044] In some embodiments of the present invention, the multidrug-resistant bacteria are Gram-positive bacteria or Gram-negative bacteria.

[0045] In some embodiments of the present invention, the multidrug-resistant bacteria include drug-resistant Staphylococcus aureus, drug-resistant Escherichia coli and drug-resistant Acinetobacter baumannii.

[0046] The beneficial effects of the present invention are:

[0047] The present invention provides a Lactobacillus johnsonii A20083 for inhibiting multidrug-resistant bacterial strains, Clostridium difficile infection, and fat reduction. The strain has a significant inhibitory effect on drug-resistant strains of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and a model strain of Clostridium difficile. Compared with the standard strain of Lactobacillus johnsonii CICC6252, the Lactobacillus johnsonii A20083 of the present invention has a better antibacterial effect on Clostridium difficile. The Lactobacillus johnsonii A20083 has a significant fat-reducing effect on a Caenorhabditis elegans model and also shows good results in in vitro experiments such as resistance to artificial gastrointestinal fluid and bile salts. Therefore, the strain is of great significance for preventing and / or treating multidrug-resistant bacterial infections and Clostridium difficile infections. At the same time, it can also be used as a probiotic agent for preparing products for reducing fat, losing weight, and regulating intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the evolutionary tree of Lactobacillus johnsonii A20083.

[0049] Figure 2 The inhibitory effect of Lactobacillus johnsonii A20083 on drug-resistant Staphylococcus aureus.

[0050] Figure 3 This is the inhibitory effect of Lactobacillus johnsonii A20083 on Pseudomonas aeruginosa.

[0051] Figure 4 This is the inhibitory effect of Lactobacillus johnsonii A20083 on drug-resistant Escherichia coli.

[0052] Figure 5 The inhibitory effect of Lactobacillus johnsonii A20083 on drug-resistant Acinetobacter baumannii.

[0053] Figure 6 This is the antibacterial effect of Lactobacillus johnsonii A20083 on Clostridium difficile.

[0054] Figure 7 This is the antibacterial effect of the culture supernatant of Lactobacillus johnsonii A20083 on Clostridium difficile.

[0055] Figure 8 The effect of Lactobacillus johnsonii A20083 on Clostridium difficile in a nematode model.

[0056] Figure 9 This is a comparison chart of the fat granule effects of Lactobacillus johnsonii A20083 and the control group.

[0057] Figure 10 This is a bar chart comparing the fat-reducing effect of Lactobacillus johnsonii A20083 in the nematode model.

[0058] Figure 11 This is a colony of Lactobacillus johnsonii A20083 grown on Columbia blood agar.

[0059] Figure 12 This is the antibacterial effect of Lactobacillus johnsonii standard strain CICC6252 on Clostridium difficile.

[0060] Figure 13 This is the antibacterial effect of the culture supernatant of Lactobacillus johnsonii standard strain CICC6252 on Clostridium difficile. DETAILED DESCRIPTION

[0061] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0062] Example 1 Isolation and purification of Lactobacillus johnsonii A20083

[0063] This example provides a method for isolating and purifying Lactobacillus johnsonii A20083. The specific experimental steps are as follows.

[0064] (1) Fecal samples from centenarians in Guangxi Zhuang Autonomous Region were collected, and an appropriate amount of the fecal sample was added to MRS solid culture medium. After smearing evenly, the sample was placed in an anaerobic workstation and cultured at 37°C in 90% nitrogen + 10% carbon dioxide for two days.

[0065] (2) After the incubation, the colonies with round, raised, entire and colorless morphology were picked from the plate and placed in a minimum amount of MRS liquid medium (10 g / L peptone, 5 g / L beef extract powder, 4 g / L yeast extract powder, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L triammonium citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 15 g / L agar, 1 g / L Tween-80. After mixing the above components, 1 L deionized water was added, the pH value was adjusted to 6.2 ± 0.2, and sterilized at 121 ° C for 15 min. After mixing evenly, the mixture was transferred to a PCR tube and placed in a 37 ° C incubator for further incubation for 24 h.

[0066] (3) Add PCR amplification solution (2× Taq PCR StarMix with LoadingDye, Beijing Kangrun Chengye Biotechnology Co., Ltd.) to the incubated PCR tube and add 16S primers to perform PCR amplification (the amplification system is shown in Table 1 below);

[0067] Among them, the primers for 16S are:

[0068] Upstream primer 16S-F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO: 1);

[0069] Downstream primer 16S-R: 5′-TACGGCTACCTTGTTACGACTT-3′ (SEQ ID NO: 2);

[0070] Table 1 Lactobacillus johnsonii PCR amplification system

[0071] Components content 10 μM upstream primer 16S-F 1 μL 10 μM downstream primer 16S-R 1 μL PCR amplification solution 12.5μL DNA template 1 μL <![CDATA[ddH2O]]> Make up to 25 μL

[0072] The amplification program was as follows: 95°C for 3 min; 95°C for 40 s, 56°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 8 min;

[0073] The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification.

[0074] After sequencing, it was confirmed to be Lactobacillus johnsonii, and its 16S sequence was:

[0075]

[0076] (4) The confirmed culture fluid was streaked twice for purification, and a single colony was picked for repeated culture to obtain a bacterial solution. The bacterial solution was mixed with 40% glycerol preservation solution at a volume ratio of 1:1 in a strain freezing tube and stored in a -80°C refrigerator.

[0077] (5) The 16S sequences obtained in (3) were used to construct a phylogenetic tree using the neighbor-joining method using MEGA-X 10.2.2 software (https: / / www.megasoftware.net / ).

[0078] The results of constructing the phylogenetic tree are as follows Figure 1 As shown, it can be seen that the strain isolated and purified in this example is Lactobacillus johnsonii, and it is named Lactobacillus johnsonii A20083.

[0079] The Lactobacillus johnsonii A20083 obtained above was sent to the Guangdong Provincial Microbial Culture Collection Center (GDMCC) for preservation, and its taxonomic name was Lactobacillus johnsonii. The preservation date was January 13, 2025, and the preservation address was 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number was GDMCC No: 65766.

[0080] Example 2 Detection of virulence factors and pathogenicity genes of Lactobacillus johnsonii A20083

[0081] This example provides the detection of virulence factors and pathogenic genes of Lactobacillus johnsonii A20083. The specific experimental steps are as follows.

[0082] Lactobacillus johnsonii A20083 obtained in Example 1 was cultured and activated overnight, and the genome was extracted using a bacterial DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). The sample was then sent for whole genome sequencing (Annuoroad Gene Technology (Beijing) Co., Ltd.) and analyzed to detect the presence of virulence factors and pathogenic genes.

[0083] The sequencing results are shown in Table 2 below. -5 Under the conditions of , a search was conducted in the existing VFDB (Virulence Factor Database) virulence factor database, and the results did not match any virulence factors and pathogenic genes, indicating that Lactobacillus johnsonii A20083 has the potential to be used as a functional probiotic supplement.

[0084] Table 2 Whole genome analysis of Lactobacillus johnsonii A20083

[0085]

[0086] Example 3 Antibacterial test of Lactobacillus johnsonii A20083 against drug-resistant Staphylococcus aureus

[0087] This example provides a test of the antibacterial effect of Lactobacillus johnsonii A20083 on methicillin-resistant Staphylococcus aureus (MRSA) (kindly provided by Professor Huang Ying of the Institute of Microbiology, Chinese Academy of Sciences). The specific experimental steps are as follows.

[0088] (1) Lactobacillus johnsonii A20083 obtained in Example 1 was cultured in a conventional MRS liquid medium and incubated in a shaker at 37°C for 48 hours;

[0089] (2) Cultivate methicillin-resistant Staphylococcus aureus in LB liquid medium (tryptone, 10 g / L; yeast extract, 5 g / L; sodium chloride, 10 g / L). Prepare LB solid medium. Before pouring the medium, when the medium temperature is about 42°C-45°C, add the resistant Staphylococcus aureus liquid to the LB to a final concentration of 5% by volume. Pour the medium into the plate as soon as possible before it solidifies, and keep the volume of medium consistent on each plate.

[0090] (3) Punch a hole in the plate using a 9 mm hole puncher, add 150 μL of the above-mentioned Lactobacillus johnsonii A20083 bacterial solution onto the plate, set up two parallel wells, and then culture in a 37°C biochemical incubator for 24 hours. Take pictures and record the size of the inhibition zone.

[0091] The experimental results are as follows Figure 2 As shown, the size of the inhibition zone is 21 mm. The experimental results show that the resistant strain Staphylococcus aureus is sensitive to Lactobacillus johnsonii A20083, that is, Lactobacillus johnsonii A20083 has a significant inhibitory effect on resistant Staphylococcus aureus.

[0092] Example 4 Antibacterial test of Lactobacillus johnsonii A20083 against Pseudomonas aeruginosa

[0093] This example provides a test of the antibacterial effect of Lactobacillus johnsonii A20083 on Pseudomonas aeruginosa PAO1 (donated by Researcher Huang Ying of the Institute of Microbiology, Chinese Academy of Sciences). The antibacterial test was performed using Pseudomonas aeruginosa based on the experimental steps of Example 3.

[0094] The experimental results are as follows Figure 3 As shown, the average inner diameter of the inhibition zone is 17.5 mm, and the average outer diameter is 23 mm. The experimental results show that Pseudomonas aeruginosa PAO1 is sensitive to Lactobacillus johnsonii A20083, that is, Lactobacillus johnsonii A20083 has a significant inhibitory effect on Pseudomonas aeruginosa PAO1.

[0095] Example 5 Antibacterial test of Lactobacillus johnsonii A20083 against drug-resistant Escherichia coli

[0096] This example provides a test of the antibacterial effect of Lactobacillus johnsonii A20083 on drug-resistant Escherichia coli. The drug-resistant Escherichia coli is ESBLs-producing Escherichia coli (kindly donated by Researcher Huang Ying of the Institute of Microbiology, Chinese Academy of Sciences). The test was performed using drug-resistant Escherichia coli based on the experimental procedures of Example 3.

[0097] The experimental results are as follows Figure 4 As shown, the average size of the inhibition zone is 19 mm. The experimental results show that the resistant strain Escherichia coli is sensitive to Lactobacillus johnsonii A20083, that is, Lactobacillus johnsonii A20083 has a significant inhibitory effect on resistant Escherichia coli.

[0098] Example 6 Antibacterial test of Lactobacillus johnsonii A20083 against Acinetobacter baumannii

[0099] This example provides a test of the antibacterial effect of Lactobacillus johnsonii A20083 on drug-resistant Acinetobacter baumannii, wherein the drug-resistant Acinetobacter baumannii is Acinetobacter baumannii HRAB-85 (heteroresistant Acinetobacter baumannii strain HRAB-85, reference: Li P, Huang Y, Yu L, Liu Y, Niu W, Zou D, Liu H, Zheng J, Yin X, Yuan J, Yuan X, Bai C. Isolation and Whole-genome Sequence Analysis of the Imipenem Heteroresistant Acinetobacter baumannii Clinical Isolate HRAB-85. Int J Infect Dis. 2017 Sep, 62: 94-101.) (donated by Researcher Huang Ying, Institute of Microbiology, Chinese Academy of Sciences). The test was performed using Acinetobacter baumannii based on the experimental procedures of Example 3.

[0100] The experimental results are as follows Figure 5 As shown, the average size of the inhibition zone is 17 mm. The experimental results show that the resistant strain Acinetobacter baumannii is sensitive to Lactobacillus johnsonii A20083, that is, Lactobacillus johnsonii A20083 has a significant inhibitory effect on the resistant Acinetobacter baumannii.

[0101] Example 7 Antibacterial test of Lactobacillus johnsonii A20083 against Clostridium difficile

[0102] This example provides a test of the antibacterial effect of Lactobacillus johnsonii A20083 on the model strain of Clostridium difficile (ATCC 43255). The specific experimental steps are as follows.

[0103] (1) Lactobacillus johnsonii A20083 was cultured in conventional MRS liquid medium and incubated in a shaking incubator at 37°C for 48 h;

[0104] (2) Take 1 mL of the cultured bacterial solution in (1), centrifuge at 5000 rpm for 1 min, wash once with 1× PBS, remove the supernatant, and keep the bacterial cells for later use;

[0105] (3) The model strain of Clostridium difficile (ATCC 43255) was cultured in commercially available MYPG liquid medium and anaerobically cultured at 37°C for 48 h;

[0106] (4) In the anaerobic workstation, take 300 μL of the Clostridium difficile culture obtained in (3) onto the MYPG solid plate, spread it evenly, and then add 20 μL of the Lactobacillus johnsonii A20083 obtained in (2) onto the plate. Set up three parallel plates, then culture anaerobically at 37°C for 24 h, take pictures and record the size of the inhibition zone.

[0107] The experimental results are as follows Figure 6 As shown, the average diameter of the inhibition zone is 14.5 mm. The experimental results show that Clostridium difficile is sensitive to Lactobacillus johnsonii A20083, that is, Lactobacillus johnsonii A20083 bacteria have a significant inhibitory effect on Clostridium difficile.

[0108] Example 8 Antibacterial test of the culture supernatant of Lactobacillus johnsonii A20083 against Clostridium difficile

[0109] This example provides a test of the antibacterial effect of the culture supernatant of Lactobacillus johnsonii A20083 on the model strain of Clostridium difficile (ATCC 43255). The specific experimental steps are as follows.

[0110] (1) The culture method of Lactobacillus johnsonii A20083 was the same as that of Experimental Step (1) in Example 7;

[0111] (2) Take 1 mL of the cultured bacterial solution in (1), centrifuge at 5000 rpm for 1 min, and take the supernatant for later use;

[0112] (3) The culture method of the model strain of Clostridium difficile (ATCC 43255) is the same as that of experimental step (3) of Example 7;

[0113] (4) In the anaerobic workstation, 300 μL of Clostridium difficile bacterial solution was taken to the MYPG solid plate, and after evenly spreading, a hole was punched on the plate with a 9 mm hole puncher. 150 μL of the culture supernatant of Lactobacillus johnsonii A20083 prepared in (2) was added to the plate, two parallel wells were set, and then anaerobically cultured at 37°C for 24 hours. Pictures were taken and the size of the inhibition zone was recorded.

[0114] The experimental results are as follows Figure 7 As shown, the average diameter of the inhibition zone was 22.75 mm, indicating that Clostridium difficile was sensitive to the secondary metabolites of Lactobacillus johnsonii A20083, and the culture supernatant of Lactobacillus johnsonii A20083 had a significant inhibitory effect on Clostridium difficile.

[0115] Example 9 Effect of Lactobacillus johnsonii A20083 on Clostridium difficile in a nematode model

[0116] This example provides a test of the antibacterial effect of Lactobacillus johnsonii A20083 on Clostridium difficile. The specific experimental steps are as follows.

[0117] (1) The culture steps of Lactobacillus johnsonii A20083 and Clostridium difficile (ATCC 43255) were the same as (1) and (3) in Example 7;

[0118] (2) Escherichia coli OP50 was cultured in LB liquid medium at 37°C for 20 hours at 200 rpm.

[0119] (3) Lactobacillus johnsonii A20083 and Escherichia coli OP50 were plated on nematode culture medium (NGM) and cultured overnight;

[0120] The NGM culture medium formula is: 3g sodium chloride, 17g agar powder, 2.5g peptone, 975mL deionized water. After fully mixing the above components, seal with tin foil. After high-pressure steam sterilization for 20 minutes, cool the water bath to 55°C. Add the following sterilized solutions under sterile conditions: 1mL of 1M CaCl2, 1mL of 1M MgSO4, 25mL of 1M KPO4 buffer (108.3gKH2PO4 and 35.6gK2HPO4, dissolved in 1000mL distilled water, adjust the pH to 6.0), 1mL of 5mg / mL cholesterol (dissolved in 95% ethanol);

[0121] (4) Thawed nematodes (C. elegans) were centrifuged and added to NGM medium containing E. coli OP50 and cultured in a 20°C incubator for 3 days;

[0122] (5) Use M9 buffer (Na2HPO4: 6.0 g; KH2PO4: 3.0 g; NaCl: 5.0 g; MgSO4·7H2O: 0.25 g; dissolved in 1 L of distilled water and sterilized by high temperature and high pressure) to suspend the nematodes cultured in (4) and transfer them to culture tubes. Add lysis solution (5 mol / L NaOH and 5% sodium hypochlorite solution by volume) to each tube and lyse for 6 minutes;

[0123] (6) The lysed liquid from step (5) was then centrifuged at 3500 rpm for 1 min, the supernatant was discarded, and the mixture was washed four times with M9 buffer, centrifuged again, and the supernatant was discarded. The precipitate was transferred to NGM medium and cultured at 20°C overnight to obtain L1 nematode larvae.

[0124] (7) After washing with M9 buffer and centrifuging once, the nematode larvae were transferred to NGM plates containing E. coli OP50 and cultured at 20°C for 28-30 hours to obtain synchronized nematodes, i.e., L4 stage nematodes;

[0125] (8) The synchronized nematodes obtained in (7) were randomly divided into two groups (control group (Clostridium difficile) and experimental group (Lactobacillus johnsonii A20083 + Clostridium difficile, mixed at a concentration of 1:1), with 150 nematodes in each group for culture. The day when the L4 nematodes were picked was recorded as day 0. During the experiment, the NGM culture medium containing Clostridium difficile and A20083 + Clostridium difficile was replaced every 2 days and the lifespan was counted until all nematodes died. Nematodes that died abnormally were not counted in the statistics. Statistical analysis was performed using GraphPad Prism 5. One-way analysis of variance was used to compare the significance of differences between multiple groups; the t-test was used to analyze the differences between two groups. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0126] The experimental results are as follows Figure 8 As shown, the median lifespan of nematodes in the experimental group was 19 days, and the median lifespan of nematodes in the control group was 18 days. The lifespan of nematodes in the experimental group was increased by 5.56% compared with that in the control group. The experimental results showed that Lactobacillus johnsonii A20083 had a significant buffering effect on alleviating the toxicity of Clostridium difficile to nematodes, and Lactobacillus johnsonii A20083 had the potential to regulate the host's intestinal microorganisms.

[0127] Example 10 Fat-reducing effect of Lactobacillus johnsonii A20083

[0128] This example provides a test of the fat-reducing effect of Lactobacillus johnsonii A20083. The specific experimental steps are as follows.

[0129] (1) Lactobacillus johnsonii A20083, Escherichia coli OP50, and Caenorhabditis elegans were cultured according to the experimental procedures (1)-(7) in Example 9, and the nematodes were synchronized;

[0130] (2) 100 synchronized nematodes were transferred to plates coated with Escherichia coli OP50 and Lactobacillus johnsonii A20083 for culture for 7 days, with the plates changed every other day;

[0131] (3) On the 7th day, the nematodes cultured in (2) were rinsed three times with cooled M9 buffer, resuspended in 4% paraformaldehyde, and gently shaken at room temperature for 1 hour;

[0132] (4) The mixture was then centrifuged at 3000–4000 rpm for 1 min, the supernatant was removed, and the nematodes were washed twice with M9 buffer;

[0133] (5) The nematodes were then resuspended in a PBS solution containing 60% isopropanol and 0.01% Triton X-100 by volume and incubated for 15 min.

[0134] (6) After the nematodes settle, remove the isopropanol, add 1 mL of 40% Oil Red O dye, and incubate on a shaker at 25°C for 1–2 h.

[0135] (7) The dye was removed, and the cells were washed twice with M9 buffer. 200 μL of M9 buffer was added, and each nematode was photographed under an inverted fluorescence microscope. Image data were analyzed using Image J, and the lipid granule degradation rate data were analyzed using GraphPad Prism v9.5. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference.

[0136] The experimental results are as follows Figure 9-10 As shown, compared with the E. coli OP50 control group, the fat particles of nematodes in the experimental group fed with Lactobacillus johnsonii A20083 were obviously lighter in color and smaller in size ( Figure 9 ), indicating that Lactobacillus johnsonii A20083 has a significant inhibitory effect on nematode fat particles, with an inhibition rate of 38.92% (P < 0.01) ( Figure 10 ).

[0137] Example 11 Hemolysis test of Lactobacillus johnsonii A20083

[0138] This example provides a hemolysis test for Lactobacillus johnsonii A20083. The specific experimental steps are as follows.

[0139] The Lactobacillus johnsonii A20083 obtained in Example 1 was activated and cultured on Columbia agar plates containing 5% sheep blood, and then cultured in an anaerobic workstation at 37° C. for 48 hours to observe whether there was a hemolytic zone around the colonies.

[0140] The experimental results are as follows Figure 11As shown, no hemolytic zone appeared around the colonies of Lactobacillus johnsonii A20083, indicating that Lactobacillus johnsonii A20083 has a high safety.

[0141] Example 12 In vitro test on resistance of Lactobacillus johnsonii A20083 to artificial gastrointestinal fluid

[0142] This example provides an in vitro experiment on the resistance of Lactobacillus johnsonii A20083 to artificial gastrointestinal fluid. The specific experimental steps are as follows.

[0143] (1) Lactobacillus johnsonii A20083 obtained in Example 1 was cultured and activated overnight, and the bacterial solution was inoculated into artificial gastric fluid (Legen Bio) at a volume ratio of 10%;

[0144] (2) 100 μL of the solution was spread on an MRS plate at 0, 1, and 3 hours, and the number of single colonies was counted after anaerobically incubating at 37°C for 48 hours.

[0145] (3) The initial concentration of the bacterial solution for the intestinal fluid experiment was 3 hours after treatment with gastric fluid. The bacterial solution was inoculated into artificial intestinal fluid (Legen Biotech) at pH 8. 100 μL of the solution was plated onto MRS plates after 2, 4, 6, and 8 hours, and the number of single colonies was counted after anaerobic incubation at 37°C for 48 hours. The survival rate of L. johnsonii A20083 in artificial intestinal fluid was calculated based on the number of single colonies obtained in the above experiment.

[0146] The experimental results showed that the survival rate of Lactobacillus johnsonii A20083 in artificial gastric juice for 3 hours was 81.37%, the survival rate in intestinal juice for 6 hours was 39.58%, and the survival rate in intestinal juice for 8 hours was 28.5%, indicating that Lactobacillus johnsonii A20083 has a certain colonization ability in the gastrointestinal tract in the absence of a protective agent.

[0147] Example 13 In vitro bile salt tolerance test of Lactobacillus johnsonii A20083

[0148] This example provides an in vitro experiment on bile salt tolerance of Lactobacillus johnsonii A20083. The specific experimental steps are as follows.

[0149] Lactobacillus johnsonii A20083 obtained in Example 1 was cultured and activated overnight. The bacterial solution was inoculated into MRS liquid medium containing 0.1 g / L ox bile salt at a volume ratio of 10%. 100 μL of the solution was spread on an MRS plate after 0 hour, 1 hour, 2 hours, and 3 hours, and the number of single colonies was counted after anaerobically culturing at 37° C. for 48 hours.

[0150] The experimental results showed that the survival rates of Lactobacillus johnsonii A20083 in bile salts for 1 hour, 2 hours and 3 hours were 109%, 186.36% and 104.55%, respectively, indicating that Lactobacillus johnsonii A20083 can survive and even proliferate in 0.1 g / L ox bile salts.

[0151] Comparative Example 1 Inhibitory effect of standard strain Lactobacillus johnsonii CICC6252 on Clostridium difficile

[0152] This example provides an in vitro experiment on Lactobacillus johnsonii CICC6252 against Clostridium difficile. The specific experimental steps are as follows.

[0153] (1) Lactobacillus johnsonii standard strain CICC6252 was purchased from the Culture Collection Center, and the antibacterial test against Clostridium difficile was performed using the Lactobacillus johnsonii standard strain CICC6252 based on the experimental steps of Example 8;

[0154] (2) Based on the experimental steps of Example 9, the culture supernatant of the Lactobacillus johnsonii standard strain CICC6252 was used to perform an antibacterial test on Clostridium difficile.

[0155] The experimental results are as follows Figure 12-13 As shown, the diameter of the inhibition zone of the Lactobacillus johnsonii standard strain CICC6252 is 12.17 mm ( Figure 12 ), the diameter of the inhibition zone of the supernatant was 21 mm ( Figure 13 ), the experimental results showed that Clostridium difficile was sensitive to the standard strain of Lactobacillus johnsonii CICC6252 and its culture supernatant. At the same time, it can be seen that Lactobacillus johnsonii A20083 had a better antibacterial effect on Clostridium difficile than the standard strain CICC6252.

[0156] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A Lactobacillus johnsonii, characterized in that The Lactobacillus johnsonii is Lactobacillus johnsonii A20083, taxonomically named Lactobacillus johnsonii, and was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 13, 2025, with a deposit number of GDMCC No: 65766.

2. The culture of Lactobacillus johnsonii according to claim 1.

3. A bacterial agent, characterized in that The bacterial agent comprises the Lactobacillus johnsonii according to claim 1 and / or the culture according to claim 2.

4. The bacterial agent according to claim 3, characterized in that The bacterial agent is a solid bacterial agent or a liquid bacterial agent.

5. Use of any one or more of the Lactobacillus johnsonii according to claim 1, the culture of Lactobacillus johnsonii according to claim 2, and the bacterial agent according to claim 3 in at least one of the following (I) to (V); (1) preparing an antibacterial agent; (II) preparing products for preventing and / or treating multidrug-resistant bacterial infections; (III) preparing products for the prevention and / or treatment of Clostridium difficile infection; (IV) preparing products for regulating intestinal flora; (V) preparing products for reducing blood lipids and weight.

6. The use according to claim 5, characterized in that The multidrug-resistant bacteria include bacteria resistant to β-lactam antibiotics.

7. The use according to claim 5, characterized in that The multidrug-resistant bacteria include drug-resistant Staphylococcus aureus, drug-resistant Escherichia coli and drug-resistant Acinetobacter baumannii.

8. A product comprising any one or more of the Lactobacillus johnsonii according to claim 1, the culture of Lactobacillus johnsonii according to claim 2, and the bacterial agent according to claim 3, characterized in that: The products include food, food additives, feed, feed additives and medicines.

9. The product according to claim 8, characterized in that The product also contains auxiliary materials; the auxiliary materials include food additives or pharmaceutically acceptable auxiliary agents.

10. A method for inhibiting the number and / or activity and / or growth of multidrug-resistant bacteria, Clostridium difficile and Pseudomonas aeruginosa viable bacteria, characterized in that: The method comprises the steps of using a culture comprising the Lactobacillus johnsonii according to claim 1, the Lactobacillus johnsonii according to claim 2 and / or the bacterial agent according to claim 3; the multidrug-resistant bacteria are Gram-positive bacteria or Gram-negative bacteria.

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