Lactobacillus rhamnosus A21174 and products and applications thereof for inhibiting multi-drug resistant strains, clostridium difficile infection, antioxidant, anti-aging, improving cognition

Abstract

The application discloses a lactobacillus rhamnosus A21174 for inhibiting infection of multi-drug resistant strains and clostridium difficile, and application thereof. The lactobacillus rhamnosus A21174 has obvious inhibiting effect on drug-resistant staphylococcus aureus, pseudomonas aeruginosa, drug-resistant escherichia coli, drug-resistant acinetobacter baumannii and a model strain of clostridium difficile. Compared with a commercial strain of lactobacillus rhamnosus, the lactobacillus rhamnosus A21174 has better bacteriostatic effect on clostridium difficile. The lactobacillus rhamnosus A21174 also shows good effect in in-vivo and in-vitro experiments such as resistance to artificial gastric and intestinal fluid, resistance to bile salt, anti-aging, anti-oxidation, improvement of cognition and the like, and therefore, the strain can be used as a probiotic agent to prepare a product for regulating intestinal flora, and has important significance for preventing and / or treating diseases caused by infection of multi-drug resistant bacteria, clostridium difficile and pseudomonas aeruginosa.

Description

Technical Field

[0001] This invention relates to the field of functional strain technology, and in particular to a strain of Lactobacillus rhamnosus A21174 that inhibits multidrug-resistant strains, Clostridium difficile infection, and has antioxidant, anti-aging, and cognitive-enhancing effects, as well as its products and applications. Background Technology

[0002] Lactobacillus rhamnosus is an edible probiotic that regulates gut microbiota balance and enhances the host's intestinal resistance. It is widely used in infant formula and various dietary supplements and is a hot research topic in modern nutrition and medicine. Numerous studies have shown that it can prevent or alleviate diseases such as inflammatory bowel disease (IBD) and diarrhea by regulating gut microbiota composition, protecting the intestinal epithelial cell barrier, and modulating immune disorders.

[0003] Currently, the overuse of antibiotics has led to the emergence of numerous multidrug-resistant bacteria and even superbugs. Commonly seen multidrug-resistant bacteria in clinical practice include methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (such as Escherichia coli and Klebsiella pneumoniae), multidrug-resistant Pseudomonas aeruginosa (MDR-PA), and multidrug-resistant Acinetobacter baumannii (MDR-AB). Multidrug-resistant bacteria render conventional antibiotic treatment ineffective, significantly increasing the difficulty of treating clinical infections and raising patient risks. Furthermore, Clostridium difficile infection (CDI) is also a pressing public health threat, with main symptoms including abdominal pain, diarrhea, and fever. Improper or delayed treatment can even lead to pseudomyopathic enteritis and toxic megacolon, and it is one of the most common causes of nosocomial infections and death.

[0004] To combat multidrug-resistant bacterial infections, screening for new antibiotics is essential, but restoring gut balance by utilizing the antagonistic effect between probiotics and drug-resistant bacteria is a better and safer approach. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide *Lactobacillus rhamnosus* A21174, its products, and applications, which inhibit multidrug-resistant strains, *Clostridium difficile* infection, and have antioxidant, anti-aging, and cognitive-enhancing effects.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a Lactobacillus rhamnosus, which is Lactobacillus rhamnosus A21174, taxonomically named Lacticaseibacillus rhamnosus, and was deposited on January 13, 2025 at the Guangdong Microbial Culture Collection Center (GDMCC) (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), with accession number GDMCC No:65769.

[0008] In some embodiments of the present invention, the colony morphology of the Lactobacillus rhamnosus A21174 is round, raised, entire, and colorless.

[0009] In some embodiments of the present invention, the 16S sequence of Lactobacillus rhamnosus A21174 is shown in SEQ ID NO:3.

[0010] In some embodiments of the present invention, the screening and isolation method of Lactobacillus rhamnosus A21174 is as follows: take fecal samples from centenarians in Guangxi Zhuang Autonomous Region and put them into MRS solid culture medium. After spreading them evenly, place them in an anaerobic workstation for two days. Pick colonies with round, raised, entire, and colorless morphology from the plate and culture them in a minimum amount of MRS liquid culture medium. After PCR amplification and identification, Lactobacillus rhamnosus A21174 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 for the PCR amplification identification are shown in 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 as follows:

[0014] Components content 10 μM upstream primer 16S-F 1μL 10 μM downstream primer 16S-R 1μL PCR amplification solution 12.5μL DNA template 1μL <![CDATA[ddH2O]]> Add to a final volume of 25 μL.

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

[0016] A second aspect of the present invention provides a culture of Lactobacillus rhamnosus A21174.

[0017] In some embodiments of the present invention, the culture comprises the supernatant obtained after culturing Lactobacillus rhamnosus A21174.

[0018] A third aspect of the present invention provides a microbial agent.

[0019] In some embodiments of the present invention, the microbial agent comprises *Lactobacillus rhamnosus* A21174 as described above and / or a culture of *Lactobacillus rhamnosus* A21174.

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

[0021] A fourth aspect of the present invention provides the use of the above-described Lactobacillus rhamnosus A21174 and / or the culture of said Lactobacillus rhamnosus A21174 and / or the bacterial agent in at least one of (I) to (IV);

[0022] (I) Preparation of antibacterial agents;

[0023] (II) To prepare products for the prevention and / or treatment of multidrug-resistant bacterial infections;

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

[0025] (IV) Prepare products that regulate gut microbiota.

[0026] In some embodiments of the present invention, the antibacterial agent is a reagent used to inhibit the number, activity and / or growth of viable bacteria in vitro, including but not limited to: any reagent used for surface sterilization, bacteriostasis or control of microbial reproduction.

[0027] In some embodiments of the present invention, the bacteria are Gram-positive or Gram-negative bacteria.

[0028] In some embodiments of the present invention, the bacteria include Clostridium difficile and Pseudomonas aeruginosa.

[0029] In some embodiments of the present invention, the bacteria also include multidrug-resistant bacteria; further including drug-resistant Escherichia coli and drug-resistant Acinetobacter baumannii.

[0030] In some embodiments of the present invention, the bacteria also include drug-resistant Staphylococcus aureus.

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

[0032] In some embodiments of the present invention, the multidrug-resistant bacteria described in (II) include bacteria resistant to β-lactam antibiotics.

[0033] In some embodiments of the present invention, the β-lactam antibiotics include carbapenem antibiotics, penicillin antibiotics, cephalosporin antibiotics, and monocyclic β-lactam antibiotics.

[0034] In some embodiments of the present invention, the penicillin antibiotics include methicillin.

[0035] In some embodiments of the present invention, the multidrug-resistant bacteria mentioned in (II) include drug-resistant Escherichia coli and drug-resistant Acinetobacter baumannii.

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

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

[0038] In some embodiments of the present invention, the multidrug-resistant bacteria in (II) also include drug-resistant Staphylococcus aureus.

[0039] In some embodiments of the present invention, the drug-resistant Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA).

[0040] A fifth aspect of the present invention provides a product comprising any one or more of the following: Lactobacillus rhamnosus A21174 as described above, a culture of Lactobacillus rhamnosus A21174, and the bacterial agent.

[0041] In some embodiments of the present invention, the product includes food, food additives, feed, feed additives, and pharmaceuticals.

[0042] In some embodiments of the present invention, the product also contains excipients.

[0043] In some embodiments of the present invention, the excipients include food additives or pharmaceutically acceptable excipients.

[0044] A sixth aspect of the present invention provides a non-therapeutic method for inhibiting the number and / or activity and / or growth of multidrug-resistant bacteria, Clostridium difficile, and Pseudomonas aeruginosa.

[0045] In some embodiments of the present invention, the method includes the steps of using *Lactobacillus rhamnosus* A21174 as described above, a culture of *Lactobacillus rhamnosus* A21174, and / or the bacterial agent.

[0046] In some embodiments of the present invention, the multidrug-resistant bacteria are Gram-positive or Gram-negative bacteria.

[0047] In some embodiments of the present invention, the multidrug-resistant bacteria include drug-resistant Escherichia coli and drug-resistant Acinetobacter baumannii.

[0048] In some embodiments of the present invention, the multidrug-resistant bacteria also include drug-resistant Staphylococcus aureus.

[0049] The beneficial effects of this invention are:

[0050] This invention provides *Lactobacillus rhamnosus* A21174, which inhibits multidrug-resistant strains and *Clostridium difficile* infections. It exhibits significant inhibitory effects against drug-resistant *Staphylococcus aureus*, *Pseudomonas aeruginosa*, drug-resistant *Escherichia coli*, drug-resistant *Acinetobacter baumannii*, and *Clostridium difficile* model strains. Compared with commercial strains... Compared to *Lactobacillus rhamnosus*, the *Lactobacillus rhamnosus* A21174 described in this invention exhibits superior antibacterial activity against *Clostridium difficile*. *Lactobacillus rhamnosus* A21174 also demonstrates good efficacy in in vivo and in vitro experiments on resistance to artificial gastrointestinal fluids, bile salt resistance, anti-aging, life-extending, antioxidant, and cognitive improvement. Therefore, this strain can be used as a probiotic agent to prepare products that regulate intestinal flora, which is of great significance for the prevention and / or treatment of diseases caused by multidrug-resistant bacteria, *Clostridium difficile*, and *Pseudomonas aeruginosa* infections. Attached Figure Description

[0051] Figure 1 This is the phylogenetic tree of Lactobacillus rhamnosus A21174.

[0052] Figure 2 The inhibitory effect of Lactobacillus rhamnosus A21174 on drug-resistant Staphylococcus aureus.

[0053] Figure 3 The inhibitory effect of Lactobacillus rhamnosus A21174 on Pseudomonas aeruginosa.

[0054] Figure 4 The inhibitory effect of Lactobacillus rhamnosus A21174 on drug-resistant Escherichia coli.

[0055] Figure 5 The inhibitory effect of Lactobacillus rhamnosus A21174 on drug-resistant Acinetobacter baumannii.

[0056] Figure 6 The antibacterial effect of Lactobacillus rhamnosus A21174 on Clostridium difficile.

[0057] Figure 7 The antibacterial effect of Lactobacillus rhamnosus A21174 culture supernatant on Clostridium difficile.

[0058] Figure 8 The effect of Lactobacillus rhamnosus A21174 on Clostridium difficile in a nematode model.

[0059] Figure 9 The colony of Lactobacillus rhamnosus A21174 grown on Columbia blood agar.

[0060] Figure 10 The antioxidant activity of Lactobacillus rhamnosus A21174.

[0061] Figure 11 The heat-resistant effect of Lactobacillus rhamnosus A21174.

[0062] Figure 12 The effect of Lactobacillus rhamnosus A21174 on the lifespan of nematodes.

[0063] Figure 13 The effect of Lactobacillus rhamnosus A21174 on the body bending of nematodes.

[0064] Figure 14 The effect of Lactobacillus rhamnosus A21174 on head movement in nematodes.

[0065] Figure 15 Commercial strains The antibacterial effect of Lactobacillus rhamnosus on Clostridium difficile.

[0066] Figure 16 Commercial strains The antibacterial effect of Lactobacillus rhamnosus culture supernatant on Clostridium difficile. Detailed Implementation

[0067] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0068] Example 1: Isolation and purification of Lactobacillus rhamnosus A21174

[0069] This embodiment provides a method for isolating and purifying Lactobacillus rhamnosus A21174. The specific experimental steps are as follows.

[0070] (1) Take fecal samples from centenarians in Guangxi Zhuang Autonomous Region, take an appropriate amount of fecal samples into MRS solid culture medium, spread them evenly, and place them in an anaerobic workstation for two days under the conditions of 37℃, 90% nitrogen + 10% carbon dioxide.

[0071] (2) After the culture is completed, pick out the colonies with the following morphology from the plate: round, raised, entire, and colorless. Add at least a small amount of MRS liquid medium (10 g / L peptone, 5 g / L beef extract, 4 g / L yeast extract, 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. Mix the above components, add 1 L of deionized water, adjust the pH to 6.2 ± 0.2, sterilize at 121℃ for 15 min, and then transfer to PCR tubes and incubate at 37℃ for 24 h.

[0072] (3) Add PCR amplification solution (2×Taq PCR StarMix with LoadingDye, Beijing Kangrun Chengye Biotechnology Co., Ltd.) to the cultured PCR tube and add 16S primers to perform PCR amplification (the amplification system is shown in Table 1 below).

[0073] The 16S primer is:

[0074] Upstream primer 16S-F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:1);

[0075] Downstream primer 16S-R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO:2);

[0076] Table 1. PCR amplification system of Lactobacillus rhamnosus

[0077]

[0078]

[0079] The amplification program was as follows: 95℃ for 3 min; 95℃ for 40 s, 56℃ for 30 s, 72℃ for 1 min, for 30 cycles; 72℃ for 8 min.

[0080] The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification.

[0081] Sequencing confirmed it to be *Lactobacillus rhamnosus*, with the following 16S sequence:

[0082]

[0083] (4) Perform two streak purifications on the confirmed culture medium, pick the purified single colonies for repeated culture to obtain bacterial solution, mix the bacterial solution with 40% glycerol preservation solution at a volume ratio of 1:1 in the strain cryopreservation tube, and store in a -80℃ freezer.

[0084] (5) The 16S sequence obtained in (3) was used to construct a phylogenetic tree on MEGA-X 10.2.2 software (https: / / www.megasoftware.net / ) using the neighbor-joining method.

[0085] The phylogenetic tree construction results are as follows Figure 1 As shown, the strain isolated and purified in this embodiment is Lactobacillus rhamnosus, and it is named Lactobacillus rhamnosus A21174.

[0086] The Lactobacillus rhamnosus A21174 obtained above was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC). Its taxonomic name is Lacticaseibacillus rhamnosus, the deposit date is January 13, 2025, the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No:65769.

[0087] Example 2: Antibacterial test of Lactobacillus rhamnosus A21174 against drug-resistant Staphylococcus aureus

[0088] This embodiment provides a test of the antibacterial effect of Lactobacillus rhamnosus A21174 against methicillin-resistant Staphylococcus aureus (MRSA) (kindly provided by Researcher Huang Ying of the Institute of Microbiology, Chinese Academy of Sciences). The specific experimental steps are as follows.

[0089] (1) The Lactobacillus rhamnosus A21174 obtained in Example 1 was cultured in conventional MRS liquid medium and placed in a shaker at 37°C for 48 hours.

[0090] (2) Culture the drug-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℃-45℃, add the drug-resistant Staphylococcus aureus bacterial solution to the LB medium to a final concentration of 5% by volume. Pour the plates as soon as possible before the medium solidifies, and ensure that the volume of medium in each plate is consistent.

[0091] (3) Use a 9mm punch to punch holes in the plate, add 150μL of the above Lactobacillus rhamnosus A21174 bacterial suspension to the plate, set two parallel wells, and then incubate in a 37℃ biochemical incubator for 24 hours. Take a picture and record the size of the inhibition zone.

[0092] Experimental results are as follows Figure 2 As shown, the inhibition zone was 23 mm in size. The experimental results indicate that the drug-resistant Staphylococcus aureus strain is sensitive to Lactobacillus rhamnosus A21174, meaning that Lactobacillus rhamnosus A21174 has a significant inhibitory effect on drug-resistant Staphylococcus aureus.

[0093] Example 3: Antibacterial test of Lactobacillus rhamnosus A21174 against Pseudomonas aeruginosa

[0094] This embodiment provides a test of the antibacterial effect of Lactobacillus rhamnosus A21174 on Pseudomonas aeruginosa PAO1 (given by Researcher Huang Ying of the Institute of Microbiology, Chinese Academy of Sciences). The antibacterial test was conducted using Pseudomonas aeruginosa based on the experimental steps in Example 2.

[0095] Experimental results are as follows Figure 3 As shown, the average inner diameter of the inhibition zone was 16.5 mm. The experimental results indicate that Pseudomonas aeruginosa is sensitive to Lactobacillus rhamnosus A21174, meaning that Lactobacillus rhamnosus A21174 has a significant inhibitory effect on Pseudomonas aeruginosa.

[0096] Example 4: Antibacterial test of Lactobacillus rhamnosus A21174 against drug-resistant Escherichia coli

[0097] This embodiment provides a test of the antibacterial effect of Lactobacillus rhamnosus A21174 against drug-resistant Escherichia coli, wherein the drug-resistant Escherichia coli is ESBL-producing Escherichia coli (Gifted by Researcher Huang Ying of the Institute of Microbiology, Chinese Academy of Sciences). The test was conducted using drug-resistant Escherichia coli based on the experimental steps of Example 2.

[0098] Experimental results are as follows Figure 4 As shown, the average size of the inhibition zone was 19 mm. The experimental results indicate that the drug-resistant Escherichia coli strain is sensitive to Lactobacillus rhamnosus A21174, meaning that Lactobacillus rhamnosus A21174 has a significant inhibitory effect on drug-resistant Escherichia coli.

[0099] Example 5: Antibacterial test of Lactobacillus rhamnosus A21174 against drug-resistant Acinetobacter baumannii.

[0100] This embodiment provides a test of the antibacterial effect of *Lactobacillus rhamnosus* A21174 against drug-resistant *Acinetobacter baumannii*, specifically *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.) (provided by Researcher Huang Ying from the Institute of Microbiology, Chinese Academy of Sciences). The test was conducted using drug-resistant *Acinetobacter baumannii* based on the experimental steps of Example 2.

[0101] Experimental results are as follows Figure 5 As shown, the average size of the inhibition zone was 17 mm. The experimental results indicate that the drug-resistant Acinetobacter baumannii strain is sensitive to Lactobacillus rhamnosus A21174, meaning that Lactobacillus rhamnosus A21174 has a significant inhibitory effect on drug-resistant Acinetobacter baumannii.

[0102] Example 6: Antibacterial test of Lactobacillus rhamnosus A21174 against Clostridium difficile

[0103] This embodiment provides a test of the antibacterial effect of Lactobacillus rhamnosus A21174 against Clostridium difficile (ATCC 43255). The specific experimental steps are as follows.

[0104] (1) Lactobacillus rhamnosus A21174 was cultured in conventional MRS liquid medium and placed in a shaker at 37°C for 48 h.

[0105] (2) Take 1 mL of the cultured bacterial solution from (1), centrifuge at 5000 rpm for 1 min, wash once with 1×PBS, remove the supernatant, and keep the bacterial cells for later use.

[0106] (3) The Clostridium difficile model strain (ATCC 43255) was cultured in commercially available MYPG liquid medium and anaerobic cultured at 37°C for 48 hours;

[0107] (4) In the anaerobic workstation, take 300 μL of Clostridium difficile culture obtained in (3) onto a MYPG solid plate, spread it evenly, and then add 20 μL of Lactobacillus rhamnosus A21174 cells obtained in (2) onto the plate. Set up three parallel plates, and then anaerobic culture at 37℃ for 24 h. Take pictures and record the size of the inhibition zone.

[0108] Experimental results are as follows Figure 6 As shown, the average diameter of the inhibition zone was 14.5 mm. The experimental results indicate that Clostridium difficile is sensitive to Lactobacillus rhamnosus A21174, meaning that Lactobacillus rhamnosus A21174 cells have a significant inhibitory effect on Clostridium difficile.

[0109] Example 7: Antibacterial test of culture supernatant of Lactobacillus rhamnosus A21174 against Clostridium difficile

[0110] This embodiment provides a test of the antibacterial effect of Lactobacillus rhamnosus A21174 culture supernatant against Clostridium difficile (ATCC 43255). The specific experimental steps are as follows.

[0111] (1) The culture method of Lactobacillus rhamnosus A21174 is the same as the experimental steps (1) in Example 6;

[0112] (2) Take 1 mL of the cultured bacterial solution from (1), centrifuge at 5000 rpm for 1 min, and take the supernatant for later use;

[0113] (3) The culture method of Clostridium difficile model strain (ATCC 43255) is the same as the experimental steps (3) in Example 6;

[0114] (4) In the anaerobic workstation, take 300 μL of Clostridium difficile bacterial solution onto a MYPG solid plate, spread it evenly, punch holes in the plate with a 9 mm punch, add 150 μL of the culture supernatant of Lactobacillus rhamnosus A21174 prepared in (2) onto the plate, set two parallel wells, and then anaerobic culture at 37℃ for 24 hours, take pictures and record the size of the inhibition zone.

[0115] Experimental results are as follows Figure 7 As shown, the average diameter of the inhibition zone was 21.75 mm, indicating that Clostridium difficile is sensitive to the secondary metabolites of Lactobacillus rhamnosus A21174, and that the culture supernatant of Lactobacillus rhamnosus A21174 has a significant inhibitory effect on Clostridium difficile.

[0116] Example 8: Effects of *Lactobacillus rhamnosus* A21174 on *Clostridium difficile* in a nematode model.

[0117] This embodiment provides a test of the antibacterial effect of Lactobacillus rhamnosus A21174 against Clostridium difficile. The specific experimental steps are as follows.

[0118] (1) The culture steps of Lactobacillus rhamnosus A21174 and Clostridium difficile (ATCC 43255) are the same as those in (1) and (3) in Example 6;

[0119] (2) Escherichia coli OP50 was cultured in LB liquid medium under the following conditions: temperature 37℃, time 20 hours, rotation speed 200 rpm.

[0120] (3) Take Lactobacillus rhamnosus A21174 and Escherichia coli OP50 bacterial suspensions and plate them on nematode growth medium (NGM) and incubate overnight;

[0121] The NGM medium formula is as follows: 3g sodium chloride, 17g agar powder, 2.5g peptone, and 975mL deionized water. After thoroughly mixing the above components, seal the container with aluminum foil. Autoclave for 20 minutes, then cool the container to 55°C in a water bath. Under aseptic conditions, add the following sterile solutions: 1mL of 1M CaCl2, 1mL of 1M MgSO4, 25mL of 1M KPO4 buffer (108.3g KH2PO4 and 35.6g K2HPO4 dissolved in 1000mL distilled water, adjusted to pH 6.0), and 1mL of 5mg / mL cholesterol (dissolved in 95% ethanol).

[0122] (4) After centrifuging the thawed wild-type nematodes (Caenorhabditis elegans, kindly provided by Researcher Wang Bin of Guangxi Academy of Sciences), they were added to NGM medium containing Escherichia coli OP50 and incubated at 20°C for 3 days.

[0123] (5) Use M9 buffer (Na2HPO4: 6.0g; KH2PO4: 3.0g; NaCl: 5.0g; MgSO4·7H2O: 0.25g; dissolved in 1L distilled water and sterilized by high temperature and high pressure) to suspend the nematodes cultured in (4) and aspirate them into culture tubes. Add lysis buffer (5mol / L NaOH and 5% sodium hypochlorite solution by volume) to each tube and lyse for 6 minutes.

[0124] (6) Then centrifuge the lysed liquid of (5) at 3500 r / min for 1 min, discard the supernatant, wash repeatedly with M9 buffer 4 times, centrifuge again and discard the supernatant, transfer the precipitate to NGM medium, and culture at 20℃ overnight to obtain L1 stage nematode larvae.

[0125] (7) After rinsing and centrifuging once with M9 buffer, the nematode larvae are transferred to NGM plates containing Escherichia coli OP50 and cultured at 20°C for 28-30 hours to obtain synchronized nematodes, namely L4 stage nematodes.

[0126] (8) The synchronized nematodes obtained in (7) were randomly divided into two groups (control group (Clostridium difficile) and experimental group (Lactobacillus rhamnosus A21174 + Clostridium difficile, mixed at a concentration of 1:1), with 150 nematodes in each group for culture. The day when L4 stage nematodes were picked was recorded as day 0. During the experiment, the NGM medium containing Clostridium difficile and Clostridium difficile A20083 + Clostridium difficile was changed every 2 days and the lifespan was recorded until all nematodes died. Nematodes that died abnormally were not included in the statistics. GraphPad Prism 5 was used for statistical analysis. One-way ANOVA was used to compare the significance of differences among multiple groups; t-test was used to analyze the differences between pairs of groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely significant.

[0127] Experimental results are as follows Figure 8 As shown, the median lifespan of nematodes in the experimental group was 22 days, while that in the control group was 18 days. The lifespan of nematodes in the experimental group was increased by 22.22% compared to that in the control group. The experimental results indicate that Lactobacillus rhamnosus A21174 has a significant buffering effect on alleviating the toxicity of Clostridium difficile to nematodes, and Lactobacillus rhamnosus A21174 can be used to regulate the intestinal microbiota of the host.

[0128] Example 9: Hemolysis test of Lactobacillus rhamnosus A21174

[0129] This embodiment provides a hemolysis test for Lactobacillus rhamnosus A21174. The specific experimental steps are as follows.

[0130] The *Lactobacillus rhamnosus* A21174 obtained in Example 1 was activated and cultured on Columbia agar plates containing 5% sheep blood, and then placed in an anaerobic workstation at 37°C for 48 hours. The presence of hemolytic zones around the colonies was observed.

[0131] Experimental results are as follows Figure 9 As shown, no hemolytic zone was observed around the Lactobacillus rhamnosus A21174 colony, indicating that Lactobacillus rhamnosus A21174 has a high safety profile.

[0132] Example 10: In vitro test of Lactobacillus rhamnosus A21174's resistance to artificial gastrointestinal fluid.

[0133] This embodiment provides an in vitro experiment demonstrating the resistance of Lactobacillus rhamnosus A21174 to artificial gastrointestinal fluid. The specific experimental steps are as follows.

[0134] (1) Take the Lactobacillus rhamnosus A21174 obtained in Example 1 and culture it overnight for activation. Inoculate the bacterial solution into artificial gastric juice (Legend Biotechnology) at a volume ratio of 10%.

[0135] (2) At 0 hours, 1 hour and 3 hours, 100 μL of the solution was spread on MRS plates and anaerobically incubated at 37°C for 48 hours before single colony counts were performed.

[0136] (3) Using the bacterial culture treated with gastric juice for 3 hours as the initial bacterial concentration for the intestinal fluid experiment, the bacterial culture was inoculated into artificial intestinal fluid (Regen Biotech) at pH=8. 100 μL of the solution was plated onto MRS plates at 2, 4, 6, and 8 hours, and then anaerobically cultured at 37°C for 48 hours before single colony counting. Based on the single colony counts from the above experiments, and using the 0h concentration of the gastric juice as the initial concentration, the survival rate of *Lactobacillus rhamnosus* A21174 in the artificial gastrointestinal fluid was calculated.

[0137] The experimental results are shown in Table 2 below. The survival rate of Lactobacillus rhamnosus A21174 in artificial gastric fluid was 97.17% after 3 hours, 20.91% after 6 hours in intestinal fluid, and 35.22% after 8 hours in intestinal fluid. Lactobacillus rhamnosus A21174 showed a gradual growth trend in intestinal fluid, indicating that Lactobacillus rhamnosus A21174 has a strong colonization ability in the gastrointestinal tract without a protectant.

[0138] Table 2. Resistance of Lactobacillus rhamnosus A21174 to artificial gastrointestinal fluid

[0139]

[0140] Example 11: In vitro experiment of Lactobacillus rhamnosus A21174 with bile salt tolerance

[0141] This embodiment provides an in vitro experiment demonstrating the bile salt tolerance of Lactobacillus rhamnosus A21174. The specific experimental steps are as follows.

[0142] The *Lactobacillus rhamnosus* A21174 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 taken from each of the following hours: 0, 1, 2, 3, and 4 hours and spread onto MRS plates. After anaerobic culture at 37°C for 48 hours, the number of single colonies was counted.

[0143] The experimental results are shown in Table 3 below. The survival rates of Lactobacillus rhamnosus A21174 in bile salts for 1 hour, 2 hours, 3 hours and 4 hours were 137.33%, 87.33%, 152% and 188%, respectively, indicating that Lactobacillus rhamnosus A21174 can survive and even proliferate in 0.1 g / L bovine bile salts.

[0144] Table 3. Bile salt tolerance of Lactobacillus rhamnosus A21174

[0145]

[0146] Example 12 Antioxidant activity of Lactobacillus rhamnosus A21174

[0147] This embodiment provides the detection of the antioxidant activity of Lactobacillus rhamnosus A21174. The specific experimental steps are as follows.

[0148] (1) Synchronized L4-stage hermaphroditic nematodes were cultured according to the experimental steps in Example 8.

[0149] (2) Synchronized L4-stage hermaphroditic nematodes of similar size and viability were randomly divided into two groups: a control group (fed with Escherichia coli OP50) and an experimental group (fed with experimental strain A21174). Both groups were cultured in a 20°C incubator. The day L4-stage nematodes were picked was designated as day 0. On day 5, the nematodes were transferred to NGM medium containing 3.375 mM H2O2 (30 nematodes per group). The number of dead nematodes was counted under a microscope every hour according to the nematode mortality criteria until all nematodes had died.

[0150] Experimental results are as follows Figure 10 As shown, A21174 can increase the antioxidant capacity of nematodes by 75%.

[0151] Example 13: Heat stress at 37°C on Lactobacillus rhamnosus A21174

[0152] This embodiment provides the detection of the 37℃ heat stress effect of Lactobacillus rhamnosus A21174. The specific experimental steps are as follows.

[0153] (1) Synchronized L4-stage hermaphroditic nematodes were cultured according to the experimental steps (1)-(7) in Example 8.

[0154] (2) Synchronized L4-stage hermaphroditic nematodes of similar size and viability were randomly divided into two groups: a control group (fed with Escherichia coli OP50) and an experimental group (fed with experimental strain A21174). Both groups were incubated at 20°C. The day L4-stage nematodes were picked was designated as day 0. On day 5, the nematode plate was transferred to a 37°C incubator for heat shock experiments (30 nematodes per group). The number of dead nematodes was counted under a microscope every hour according to the nematode mortality criteria until all nematodes had died.

[0155] Experimental results are as follows Figure 11 As shown, A21174 can increase the heat stress resistance of nematodes by 25%, thereby improving the host's ability to cope with environmental stress.

[0156] Example 14: Effect of Lactobacillus rhamnosus A21174 on the lifespan of Caenorhabditis elegans

[0157] This embodiment provides the detection of the 37℃ heat stress effect of Lactobacillus rhamnosus A21174. The specific experimental steps are as follows.

[0158] (1) Synchronized L4-stage hermaphroditic nematodes were cultured according to the experimental steps in Example 8.

[0159] (2) Synchronized L4-stage hermaphroditic nematodes of similar size and viability were randomly divided into two groups: a control group (fed with E. coli OP50) and an experimental group (fed with experimental strain A21174). The nematodes were cultured in a 20℃ incubator, and the day the L4-stage nematodes were picked was designated as day 0. During the experiment, nematodes were transferred to new NGM plates coated with the corresponding bacterial solution every 48 hours. The number of surviving and dead nematodes was observed and counted daily under a stereomicroscope until all nematodes died. Accidental loss or abnormal death of nematodes was not included in the lifespan statistics. Nematodes were considered dead when they stopped pharyngeal suction or when they did not respond to gentle mechanical stimulation with a platinum wire picker. Abnormal deaths included death due to physical intervention (poking, scalding, drying on a picker needle); unexplained death within the first 5 days; transparent bodies; death due to internal hatching; and inactivity within the first 5 days with a large number of eggs nearby.

[0160] Experimental results are as follows Figure 12 As shown, Lactobacillus rhamnosus A21174 can extend the lifespan of nematodes by 13.33%.

[0161] Example 15 Anti-aging test of Lactobacillus rhamnosus A21174

[0162] This embodiment provides an anti-aging test of Lactobacillus rhamnosus A21174. The specific experimental steps are as follows.

[0163] Following the experimental steps of Example 8, L4-stage hermaphroditic nematodes synchronized with *Escherichia coli* OP50 and *Lactobacillus rhamnosus* A21174 were prepared. Thirty nematodes were randomly selected from each group, and their body bending rates were measured using a stereomicroscope on days 5, 10, and 15 of culture. The test nematodes were placed on clean NGM plates and allowed to move freely for 30 seconds to acclimatize before testing began. A single sine / cosine movement was recorded as one instance of spontaneous and / or regular sine / cosine motion, and the duration was recorded for 30 seconds.

[0164] Experimental results are as follows Figure 13 and Figure 14 As shown, Lactobacillus rhamnosus A21174 can significantly improve the motility of nematodes in the early, middle and late stages.

[0165] Example 16: Effect of Lactobacillus rhamnosus A21174 on a Parkinson's nematode model

[0166] This embodiment provides an evaluation of the effect of Lactobacillus rhamnosus A21174 on a Parkinson's nematode model. The specific experimental steps are as follows.

[0167] (1) Preparation of Parkinson's worm model using 6-OHDA

[0168] BZ555:egIs1[dat-1p::GFP];NL5901:pkIs2386[unc-54p::a-synuclein::YFP+unc-119] (hereinafter referred to as BZ555, purchased from the Caenorhabditis Genetics Center (CGC) in the United States) is a transgenic Parkinson's disease model nematode. Under normal circumstances, the nematodes need to be induced with the neurotoxin 6-OHDA (6-hydroxydopamine) to induce Parkinson's symptoms. Following the same treatment method as wild-type nematodes in Example 8, BZ555 was synchronized. The obtained nematode eggs were placed on NGM medium plates until the nematodes reached the L1-L2 age group. The nematodes were collected and exposed to 50mM 6-OHDA solution for 1 hour. The 6-OHDA was washed away by gently shaking M9 every 10 minutes, and the washing was repeated 3-5 times. The plates were centrifuged at 3000 rpm until the supernatant was clear. Then, an appropriate volume of the nematode solution was placed on plates containing Lactobacillus rhamnosus and normal plates and grown for 48 hours. The plates were divided into Lactobacillus rhamnosus A21174 experimental group and Parkinson's model experimental group. Wild-type nematodes were used as the control group and grown on normal plates for 48 hours.

[0169] (2) Observation of food perception behavior

[0170] The nematodes prepared in step (1) were picked and placed on sterile NGM medium containing E. coli OP50. After waiting for 2 minutes, the number of movements (body bending times) of the nematodes in different groups within 30 seconds was observed. Normal nematodes will move slowly on sterile plates when consuming food (E. coli OP50), and move faster on sterile plates when searching for food (E. coli OP50). The rate of decrease in food perception of nematodes was calculated using the following formula:

[0171] The rate of decrease = (number of times N-nematodes move under sterile conditions - number of times N-nematodes move under conditions with E. coli OP50) / number of times N-nematodes move under sterile conditions * 100%.

[0172] The experimental results showed that the rate of decrease in food perception was 73.08% in the normal control group and -58.33% in the model group, proving that the nematodes in the model group were induced to develop Parkinson's symptoms and cognitive confusion under the treatment of neurotoxin 6-OHDA; the rate of decrease in the A21174 experimental group was 51.96%, indicating that Lactobacillus rhamnosus A21174 has a very significant effect on inhibiting the pathogenesis of Parkinson's nematodes.

[0173] Comparative Example 1: Commercial strain Lactobacillus rhamnosus Inhibitory effect against Clostridium difficile

[0174] This embodiment provides a commercial strain. In vitro experiments on Clostridium difficile using Lactobacillus rhamnosus (hereinafter referred to as LGG). The specific experimental steps are as follows.

[0175] (1) Based on the experimental steps in Example 6, LGG was used to conduct antibacterial tests on Clostridium difficile;

[0176] (2) Based on the experimental steps in Example 7, the culture supernatant of the LGG was used to conduct an antibacterial test on Clostridium difficile.

[0177] Experimental results are as follows Figure 15-16 As shown, commercial strains The inhibition zone diameter of *Lactobacillus rhamnosus* was 11 mm. Figure 15 The diameter of the inhibition zone in the supernatant was 21 mm. Figure 16 The experimental results showed that Clostridium difficile was sensitive to the commercial strain LGG of Lactobacillus rhamnosus and its culture supernatant. At the same time, it was found that Lactobacillus rhamnosus A21174 had a better inhibitory effect on Clostridium difficile than LGG.

[0178] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of any one or several of Lactobacillus rhamnosus, a culture of Lactobacillus rhamnosus and a microbial agent comprising Lactobacillus rhamnosus for the preparation of a product for impeding the progression of the pathogenic process in Parkinson's nematodes, characterised in that, The Lactobacillus rhamnosus is Lactobacillus rhamnosus A21174, taxonomically named as Lacticaseibacillus rhamnosus , and preserved in the Guangdong Microbial Culture Collection Center on January 13, 2025, with a preservation number of GDMCC No:65769.

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