A lactobacillus plantarum strain with the function of inhibiting the growth of multiple common pathogenic bacteria

Abstract

The present application is a plant lactobacillus with the function of inhibiting the growth of various common pathogenic bacteria, wherein the plant lactobacillus is plant lactobacillus TG008, which is preserved in the China Center for Type Culture Collection, and the preservation number is 【CCTCC M 20221458】 and the preservation time is 【September 20, 2022】. The present application has the function of significantly inhibiting the growth of various common pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a strain of *Lactobacillus plantarum* that has the effect of inhibiting the growth of a variety of common pathogenic bacteria. Background Technology

[0002] The balance of gut microbiota in the human intestine plays a crucial role in human health. If the number of harmful bacteria exceeds the dynamic balance of the microbiota, it will affect different organs, leading to various diseases and harming health. Different species of microorganisms have relationships of coexistence, mutualism, symbiosis, antagonism, competition, parasitism, and predation. Using probiotics to inhibit or kill harmful bacteria will have a significant impact on human health, and this method has no toxic side effects, making it a promising approach. In the near future, more and more probiotic drugs will be available on the market.

[0003] Lactic acid bacteria are the dominant bacteria in the normal intestinal flora of humans and animals. They have many physiological functions that are beneficial to human and animal health and are often used in fermented foods. They also have antibacterial effects. The antibacterial mechanism is mainly manifested in the following aspects: (1) Lactic acid bacteria produce organic acids such as acetic acid, lactic acid and propionic acid, which keep the environment acidic and inhibit the growth and reproduction of acid-sensitive harmful bacteria; (2) Lactic acid bacteria produce protein substances such as antibiotics or bacteriocins, which have the function of inhibiting or killing harmful bacteria; (3) The hydrogen peroxide produced by lactic acid bacteria can activate the catalase-thiocyanate system, which inhibits or kills harmful bacteria; (4) Lactic acid bacteria compete with harmful bacteria for nutrients through antagonism, thereby achieving antibacterial effect.

[0004] Lactobacillus plantarum is a bacterium with strong processing properties and has been widely used in food production. It has excellent properties in regulating the human immune system, having a strong adhesion ability to the intestinal mucosa and excellent resistance to gastrointestinal digestive juices. It can colonize and reproduce in the human intestine, improve the distribution of intestinal flora, antagonize the colonization of harmful bacteria, avoid intestinal diseases, regulate the human intestinal environment, and inhibit the growth of various pathogenic bacteria.

[0005] However, there are currently no reports of *Lactobacillus plantarum* inhibiting the growth of many common pathogenic bacteria. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a strain of *Lactobacillus plantarum* that inhibits the growth of various common pathogenic bacteria. This *Lactobacillus plantarum* significantly inhibits the growth of various common pathogenic bacteria in vitro, particularly *Bacteroides fragilis*, *Clostridium perfringens*, *Klebsiella pneumoniae*, *Shigella flexneri*, and *Shigella boydii*.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] A strain of *Lactiplantibacillus plantarum*, specifically *Lactiplantibacillus plantarum* TG008, which inhibits the growth of various common pathogenic bacteria, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20221458 on September 20, 2022.

[0009] The gene sequence of *Lactobacillus plantarum* TG008 is as follows:

[0010]

[0011] The aforementioned *Lactobacillus plantarum* TG008 strain was obtained from fecal samples of healthy individuals in China. It is Gram-positive, non-spore-forming, and its surface colonies are approximately 3 mm in diameter, raised, round, smooth, dense, and white. The optimal growth conditions are 37℃, pH 7, and an anaerobic environment.

[0012] The aforementioned strain of Lactobacillus plantarum TG008 exhibits significant inhibitory effects on the growth of various common pathogenic bacteria.

[0013] The aforementioned common pathogens include: Bacteroides fragilis, Clostridium perfringens, Klebsiella pneumoniae, Shigella flexneri, and Shigella boydii.

[0014] Bacteroides fragilis is an opportunistic pathogen that primarily causes endogenous infections, leading to infections of the female reproductive system, pleural cavity, and intracranial cavity.

[0015] Clostridium perfringens is classified into five toxic types: A, B, C, D, and E. Of these five types, types A and C are the most pathogenic to humans. Type A is the most common, causing gas gangrene and gastroenteritis-type food poisoning; type C can cause necrotizing enteritis.

[0016] Klebsiella pneumoniae can cause acute lung inflammation, which is more common in the elderly, malnourished, chronically alcoholic, chronic bronchopulmonary disease, and patients with systemic failure.

[0017] Shigella flexneri causes gastrointestinal diseases such as dysentery, and complications usually include hemolytic uremic syndrome, manifested as bloody stools; kidney failure (usually due to the pathogen's toxins entering the kidneys through the bloodstream); and central nervous system disorders.

[0018] Shigella boydii primarily causes bacterial dysentery in humans. In recent years, its resistance to antibiotics has been increasing annually.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) The Lactobacillus plantarum TG008 of the present invention was obtained by screening from fecal samples of healthy human beings in China and has good application safety and functional adaptability.

[0021] (2) The *Lactobacillus plantarum* TG008 of the present invention has the effect of inhibiting the growth of a variety of common pathogenic bacteria. It can significantly inhibit the growth of a variety of common pathogenic bacteria in vitro, including: *Bacteroides fragilis*, *Clostridium perfringens*, *Klebsiella pneumoniae*, *Shigella flexneri*, and *Shigella boydii*.

[0022] (3) The plant lactobacillus that inhibits a variety of common pathogenic bacteria described in this invention is named Lactiplantibacillus plantarum TG008. This strain has been deposited in the China Center for Type Culture Collection, with accession number: [CCTCC NO: M 20221458] and deposit date: [September 20, 2022]. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0024] Figure 1 This is the plate phenotype of Lactobacillus plantarum TG008 in Example 1 of the present invention.

[0025] Figure 2 The image shows the Gram staining results of Lactobacillus plantarum TG008 in Example 1 of this invention.

[0026] Figure 3 This is the phylogenetic tree of Lactobacillus plantarum TG008 in Example 1 of the present invention.

[0027] Figure 4 This is the growth curve of Lactobacillus plantarum TG008 in Example 2 of the present invention.

[0028] Figure 5(1) shows the optimal temperature curve of Lactobacillus plantarum TG008 in Example 2 of the present invention, and Figure 5(2) shows the optimal pH curve of Lactobacillus plantarum TG008 in Example 2 of the present invention.

[0029] Figure 6 This is the Oxford Cup inhibition result of Lactobacillus plantarum TG008 against Bacteroides fragilis in Example 3 of the present invention.

[0030] Figure 7 This is the Oxford Cup inhibition result of Lactobacillus plantarum TG008 against Clostridium perfringens in Example 4 of the present invention.

[0031] Figure 8 This is the Oxford Cup inhibition result of Lactobacillus plantarum TG008 against Klebsiella pneumoniae in Example 5 of the present invention.

[0032] Figure 9 This is the Oxford Cup inhibition result of Lactobacillus plantarum TG008 against Shigella flexneri in Example 6 of the present invention.

[0033] Figure 10 This is the Oxford Cup inhibition result of Lactobacillus plantarum TG008 against Shigella boydii in Example 7 of the present invention.

[0034] Figure 11 This is the result of the inhibitory effect of Lactobacillus plantarum TG008 on HCT-8 cells in Example 8 of the present invention. Detailed Implementation

[0035] The embodiments of the present invention will be described in detail below with reference to the examples. These examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified can be commercially available conventional products.

[0036] This invention relates to a strain of *Lactiplantibacillus plantarum* that inhibits the growth of various common pathogenic bacteria. The *Lactiplantibacillus plantarum* strain is TG008, deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20221458 on September 20, 2022.

[0037] The gene sequence of *Lactobacillus plantarum* TG008 is as follows:

[0038] CCGGGGGGGGGCGTGCCTATACATGCAAGTCGAACGAACTCTGGTATTG

[0039] ATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAG

[0040] TAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACA

[0041] GATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAG

[0042] ATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATG

[0043] GTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGG

[0044] TAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGC

[0045] AGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCC

[0046] GCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAG

[0047] AACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAA

[0048] GCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAG

[0049] CGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGT

[0050] CTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGG

[0051] AAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAA

[0052] TGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCT

[0053] GTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATA

[0054] CCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTC

[0055] CGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTAC

[0056] GGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG

[0057] GTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTC

[0058] TTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATG

[0059] GATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT

[0060] TAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTT

[0061] GGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT

[0062] GACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAA

[0063] TGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAA

[0064] AGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGG

[0065] AATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGG

[0066] CCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGAACCCAAA.

[0067] The aforementioned *Lactobacillus plantarum* strain was obtained from fecal samples of healthy individuals in China. It is Gram-positive, non-spore-forming, and its surface colonies are approximately 3 mm in diameter, raised, round, smooth, dense, and white. The optimal growth conditions are 37°C, pH 7, and an anaerobic environment.

[0068] The aforementioned strain of Lactobacillus plantarum TG008 exhibits significant inhibitory effects on the growth of various common pathogenic bacteria.

[0069] Example 1: Screening and identification of Lactobacillus plantarum TG008.

[0070] Using fecal samples from healthy individuals in Xiamen, Fujian Province, the samples were inoculated into human gut microbiota serum culture flasks for enrichment culture. On days 1, 3, 6, and 9, the enriched culture was aspirated and serially diluted with sterile physiological saline and plated onto BHI agar plates (BHI medium formulation: 200 g / L calf brain (extract powder); 250 g / L calf heart (extract powder); 2.0 g / L glucose; 5 g / L sodium chloride; 10 g / L peptone; 2.5 g / L disodium hydrogen phosphate; final pH 5.7±0.2, sterilized at 115℃ for 25 min). After incubation at 37℃ in an anaerobic incubator for 48 h, single colonies were picked and streaked onto BHI agar plates for purification in three rounds. The purified single colonies were inoculated into BHI liquid medium and incubated at 37℃ and 200 rpm on a shaker for 24–48 h. Genomic DNA was then extracted using a bacterial genomic DNA rapid extraction kit (T5 Direct PCR Kit).

[0071] Using the extracted genomic DNA as a template, PCR amplification was performed using the full-length 16S rDNA primer pair: 27F: 5'AGAGTTTGATCCTGGCTCAG 3'; 1492R: 5'TACGGCTACCTTGTTACGACTT 3'. The PCR product was then sequenced, and the sequenced sequence was compared with BLAST on the NCBI website. The results showed that the bacterium was a strain of *Lactiplantibacillus plantarum*, and its 16S rDNA sequence is shown in SEQ ID No. 1. The plate morphology of this strain is as follows. Figure 1 As shown, the colony surface has a diameter of about 3mm, is raised, round, smooth, dense, and white.

[0072] The strain stained blue-purple after Gram staining, indicating that it is a Gram-positive bacterium. The Gram staining results are as follows: Figure 2 As shown.

[0073] The genetic relationship between this strain and commercially available Lactobacillus plantarum is as follows: Figure 3 As shown in the phylogenetic tree diagram, this strain of *Lactobacillus plantarum* is named *Lactobacillus plantarum* TG008.

[0074] Example 2: Physiological characteristics of Lactobacillus plantarum TG008

[0075] Growth curve determination method: Strain activation: *Lactobacillus plantarum* TG008 was removed from a -80℃ freezer, streaked onto BHI agar plates for activation, and incubated at 37℃ in an anaerobic incubator until single colonies appeared. Inoculation solution: Using a disposable inoculation loop (1 μL), a single colony from the BHI agar plate was inoculated into BHI liquid medium, shaken well, and incubated at 37℃ and 200 rpm on a shaker until OD reached. 600 The inoculum concentration (OD) should reach the range of 0.6-1.0. Inoculation with culture medium: Inoculate 1-5% of the culture medium into BHI liquid medium at different time points. After inoculation, incubate at 37℃ and 200rpm on a shaker. Take a sample every 2 hours for OD measurement. 600 This continued until the 24-hour experimental group was fully measured. The growth curve of *Lactobacillus plantarum* TG008 is shown below. Figure 4 As shown.

[0076] Optimal Temperature Determination: Strain Activation: *Lactobacillus plantarum* TG008 was removed from a -80℃ freezer, streaked onto BHI agar plates for activation, and incubated at 37℃ in an anaerobic incubator until single colonies appeared. Inoculation: Single colonies from the BHI agar were collected using an inoculation loop and inoculated into BHI liquid culture medium. The medium was shaken well and incubated at 37℃ on a shaker at 200 rpm until OD (October Expiratory Time) was reached. 600 The inoculum should reach the range of 0.6-1.0. Inoculation with culture medium: Inoculate 1-5% into BHI liquid medium and place in a shaker at different temperature gradients (27, 32, 37, 42, 47℃) for anaerobic culture. OD values ​​should be measured at appropriate time points. 600 The optimal temperature for Lactobacillus plantarum TG008 is shown in Figure 5(1).

[0077] Optimal pH determination: Strain activation: *Lactobacillus plantarum* TG008 was removed from the -80℃ freezer, streaked onto BHI agar plates for activation, and incubated in a 37℃ anaerobic incubator until single colonies grew. Inoculation: Single colonies from the BHI agar were collected using an inoculation loop and inoculated into BHI liquid culture medium. The medium was shaken well and incubated at 37℃ on a shaker at 200 rpm until OD (October Expiratory Time) was reached. 600The inoculum should reach the range of 0.6-1.0. Inoculation with culture medium: Inoculate 1-5% of the culture medium into BHI liquid culture medium at different pH gradients (pH 5, 6, 7, 8, 9), place on a shaker, and anaerobically incubate at 37°C and 200 rpm. OD values ​​should be measured at appropriate time points. 600 The optimal pH for Lactobacillus plantarum TG008 is shown in Figure 5(2).

[0078] Example 3: Oxford Cup inhibition results of Lactobacillus plantarum TG008 against Bacteroides fragilis.

[0079] Activation: *Lactobacillus plantarum* TG008 and *Bacteroides fragilis* were removed from the -80℃ freezer and streaked onto BHI agar plates for activation. They were then incubated in an anaerobic incubator at 37℃ until single colonies appeared. Fermentation Culture: Single colonies of *Lactobacillus plantarum* TG008 and *Bacteroides fragilis* grown on BHI agar plates were inoculated into BHI fermentation medium and incubated at 37℃ on a shaker at 200 rpm / min for the appropriate time. Oxford Cup Experiment: Positive control (antibiotic), negative control (medium), and *Lactobacillus plantarum* were marked on the back of the plates with a marker, and Oxford cups were placed in the corresponding positions. The pathogenic bacteria were mixed with melted solid medium (agar concentration should be 1.4%), resulting in a pathogenic bacteria concentration of 1%, and the final concentration of pathogenic bacteria in the medium was 10. 6 Mix cfu / ml in a 15ml centrifuge tube, then pour the mixture onto plates, each plate containing 20ml of bacteria. After the plates have dried completely, use tweezers to remove Oxford cups, creating wells. Add the corresponding reagent to each well, including a positive control antibiotic (e.g., 200μl 50mg / ml Kan), a negative control (e.g., 200μl BHI medium), and an experimental control (e.g., 200μl of *Lactobacillus plantarum* culture or centrifuged supernatant). Seal the plates with centrifuge sealing film to prevent moisture evaporation and incubate them upright in a strictly anaerobic incubator at 37°C. Observe the growth of pathogenic bacteria and the size of the inhibition zone daily.

[0080] Lactobacillus plantarum TG008 exhibits significant inhibitory activity against Bacteroides fragilis; however, some antibiotics show stronger inhibitory effects. For example... Figure 6 As shown.

[0081] Example 4: Oxford cup inhibition results of Lactobacillus plantarum TG008 against Clostridium perfringens.

[0082] Activation: *Lactobacillus plantarum* TG008 and *Clostridium perfringens* were removed from the -80℃ freezer and streaked onto BHI agar plates for activation. They were then incubated in an anaerobic incubator at 37℃ until single colonies grew. Fermentation Culture: Single colonies of *Lactobacillus plantarum* TG008 and *Clostridium perfringens* grown on BHI agar plates were inoculated into BHI fermentation medium and incubated at 37℃ for the appropriate time using a shaker at 200 rpm / min. Oxford Cup Experiment: Positive control (antibiotic), negative control (medium), and *Lactobacillus plantarum* were marked on the back of the plates using a marker, and Oxford cups were placed in the corresponding positions. The pathogenic bacteria were mixed with melted solid medium (agar concentration should be 1.4%), resulting in a pathogenic bacteria concentration of 1%, and the final concentration of pathogenic bacteria in the medium was 10⁶ CFU / ml. The mixture was thoroughly mixed in 15 ml centrifuge tubes and poured onto plates, with each plate containing 20 ml of bacteria. After the plates have dried completely, use tweezers to remove the Oxford cups, creating holes. Add the corresponding reagent to each hole, including a positive control antibiotic (e.g., 200 μl 50 mg / ml Kan), a negative control (e.g., 200 μl BHI medium), and an experimental control (e.g., 200 μl of Lactobacillus plantarum culture or centrifuged supernatant). Seal the plates with centrifugation sealing film to prevent moisture evaporation and incubate them upright in a strictly anaerobic incubator at 37°C. Observe the growth of pathogenic bacteria and the size of the inhibition zone on the plates daily.

[0083] Lactobacillus plantarum TG008 exhibits significant inhibitory activity against Clostridium perfringens, approximately twice as potent as antibiotics. Figure 7 As shown.

[0084] Example 5: Oxford cup inhibition results of Lactobacillus plantarum TG008 against Klebsiella pneumoniae.

[0085] Activation: *Lactobacillus plantarum* TG008 and *Klebsiella pneumoniae* were removed from the -80℃ freezer and streaked onto BHI agar plates for activation. They were then incubated in an anaerobic incubator at 37℃ until single colonies grew. Fermentation Culture: Single colonies of *Lactobacillus plantarum* TG008 and *Klebsiella pneumoniae* grown on BHI agar plates were inoculated separately into BHI fermentation medium and incubated at 37℃ on a shaker at 200 rpm / min for the appropriate time. Oxford Cup Experiment: Positive control (antibiotic), negative control (medium), and *Lactobacillus plantarum* were marked on the back of the plates using a marker, and Oxford cups were placed in the corresponding positions. The pathogenic bacteria were mixed with melted solid medium (agar concentration should be 1.4%), resulting in a pathogenic bacteria concentration of 1%, and a final concentration of pathogenic bacteria in the medium of 10⁶ CFU / ml. The mixture was thoroughly mixed in 15 ml centrifuge tubes and poured onto plates, with each plate containing 20 ml of bacteria. After the plates have dried completely, use tweezers to remove the Oxford cups, creating holes. Add the corresponding reagent to each hole, including a positive control antibiotic (e.g., 200 μl 50 mg / ml Kan), a negative control (e.g., 200 μl BHI medium), and an experimental control (e.g., 200 μl of Lactobacillus plantarum culture or centrifuged supernatant). Seal the plates with centrifugation sealing film to prevent moisture evaporation and incubate them upright in a strictly anaerobic incubator at 37°C. Observe the growth of pathogenic bacteria and the size of the inhibition zone on the plates daily.

[0086] Lactobacillus plantarum TG008 exhibits significant inhibitory activity against Klebsiella pneumoniae, with effects comparable to those of antibiotics. Figure 8 As shown.

[0087] Example 6: Oxford cup inhibition results of Lactobacillus plantarum TG008 against Shigella flexneri.

[0088] Activation: *Lactobacillus plantarum* TG008 and *Shigella flexneri* were removed from the -80℃ freezer and streaked onto BHI agar plates for activation. They were then incubated in an anaerobic incubator at 37℃ until single colonies grew. Fermentation Culture: Single colonies of *Lactobacillus plantarum* TG008 and *Shigella flexneri* grown on BHI agar plates were inoculated separately into BHI fermentation medium and incubated at 37℃ on a shaker at 200 rpm / min for the appropriate time. Oxford Cup Experiment: Positive control (antibiotic), negative control (medium), and *Lactobacillus plantarum* were marked on the back of the plates using a marker, and Oxford cups were placed in the corresponding positions. Pathogenic bacteria were mixed with melted solid medium (agar concentration should be 1.4%), resulting in a pathogenic bacteria concentration of 1%, with a final concentration of 10⁶ CFU / ml. The mixture was thoroughly mixed in 15 ml centrifuge tubes and poured onto plates, with each plate containing 20 ml of bacteria. After the plates have dried completely, use tweezers to remove the Oxford cups, creating holes. Add the corresponding reagent to each hole, including a positive control antibiotic (e.g., 200 μl 50 mg / ml Kan), a negative control (e.g., 200 μl BHI medium), and an experimental control (e.g., 200 μl of Lactobacillus plantarum culture or centrifuged supernatant). Seal the plates with centrifugation sealing film to prevent moisture evaporation and incubate them upright in a strictly anaerobic incubator at 37°C. Observe the growth of pathogenic bacteria and the size of the inhibition zone on the plates daily.

[0089] Lactobacillus plantarum TG008 exhibits significant inhibitory effects against Shigella flexneri, exceeding the inhibitory effects of antibiotics. For example... Figure 9 As shown.

[0090] Example 7: Results of the Oxford Cup inhibition of Shigella boydii by Lactobacillus plantarum TG008.

[0091] Activation: *Lactobacillus plantarum* TG008 and *Shigella boydii* were removed from the -80℃ freezer and streaked onto BHI agar plates for activation. They were then incubated in an anaerobic incubator at 37℃ until single colonies grew. Fermentation Culture: Single colonies of *Lactobacillus plantarum* TG008 and *Shigella boydii* grown on BHI agar plates were inoculated separately into BHI fermentation medium and incubated at 37℃ on a shaker at 200 rpm / min for the appropriate time. Oxford Cup Experiment: Positive control (antibiotic), negative control (medium), and *Lactobacillus plantarum* were marked on the back of the plates with a marker, and Oxford cups were placed in the corresponding positions. The pathogenic bacteria were mixed with melted solid medium (agar concentration should be 1.4%), resulting in a pathogenic bacteria concentration of 1%, and a final concentration of pathogenic bacteria in the medium of 10⁶ CFU / ml. The mixture was thoroughly mixed in 15 ml centrifuge tubes and poured onto plates, with each plate containing 20 ml of bacteria. After the plates had completely dried, the Oxford cups were removed with tweezers, creating holes. Add the corresponding reagents to each well, including a positive control antibiotic (e.g., 200 μl 50 mg / ml Kan), a negative control (e.g., 200 μl BHI medium), and an experimental control (e.g., 200 μl of Lactobacillus plantarum culture or centrifuged supernatant). Seal the plates with centrifugation sealing film to prevent moisture evaporation, and incubate them upright in a strictly anaerobic incubator at 37°C. Observe the growth of pathogenic bacteria and the size of the inhibition zone on the plates daily.

[0092] Lactobacillus plantarum TG008 exhibits significant inhibitory effects against Shigella boydii, approximately twice as strong as that of antibiotics. Figure 10 As shown.

[0093] Example 8: Growth inhibition of colorectal cancer cells HCT-8 by the metabolic supernatant of Lactobacillus plantarum TG008.

[0094] Strain culture: 500 μl of cultured *Lactobacillus plantarum* TG008 was inoculated into 10 mL of BHI liquid medium and cultured in an anaerobic incubator for 24-48 h. The OD600 value was measured, and 1 mL of the strain suspension with an OD600 value of approximately 1.5-2.5 was centrifuged (4℃, 5000 rpm, 8 min). The supernatant was carefully transferred to a sterile EP tube. Supernatant filtration: The centrifuged supernatant and BHI were transferred to a new EP tube through a 0.22 μm sterile filter. The bacterial supernatant / BHI liquid medium was diluted with cell culture medium at ratios of 0%, 0.3%, 0.6%, 1.2%, 2.5%, 5%, 10%, 20%, 30%, and 40%, and FBS-1640 medium was diluted with PBS buffer. The solutions were prepared and ready for use. Observation: Observe the growth status of HCT-8 cells under a microscope. After the cells are in good condition, remove the old culture medium and wash the cells once or twice with PBS or balanced salt solution. Add 2 mL of trypsin solution to a T25 culture dish and incubate at 37°C for 1-2 minutes. Observe under an inverted microscope. When the cells are about to separate and appear granular, gently tap the culture flask to allow the cells to detach from the flask wall. Add an appropriate amount of fresh culture medium containing serum to stop the trypsin reaction. Centrifuge and then remove the supernatant. Prepare cell suspension: Add an appropriate amount of fresh culture medium and use a pipette to aspirate and displace the cell clumps several times to break them up and mix thoroughly. Seedling: Seed the cells into 96-well plates. Adjust the cell density to 2 × 10⁶ cells / well during plating. 4 Cells were inoculated per well, with edge wells filled with sterile PBS (for moisture retention). Three replicates were performed for each concentration point. After inoculation, the cells were incubated at 37°C in a 5% CO2 incubator for 6 hours to allow cell adhesion. The old culture medium was discarded in the 96-well plate, and 150 μL of the supernatant of the test strain was added. The plate was incubated in the incubator for 48 hours. Then, 10 μL of CCK8 solution was added to each well (avoiding air bubbles). The plate was incubated at 37°C in a 5% CO2 incubator for 2 hours. The absorbance at 450 nm was measured using a microplate reader.

[0095] The inhibitory effect of Lactobacillus plantarum TG008 bacterial supernatant on the growth of HCT-8 colorectal cancer cells, such as Figure 11 As shown, the supernatant of *Lactobacillus plantarum* TG008 bacterial culture has an inhibitory effect on HCT-8 colorectal cancer cells. The higher the concentration of the bacterial supernatant, the stronger the inhibitory effect on the growth of HCT-8 colorectal cancer cells. When the bacterial supernatant concentration is 40%, the growth inhibition rate of HCT-8 colorectal cancer cells is approximately 50%. Figure 11 As shown.

[0096] The antibacterial properties of *Lactobacillus plantarum* can be utilized to target some pathogenic bacteria in the human body. This is because antibiotics not only affect pathogenic bacteria but also kill beneficial bacteria, disrupting the body's flora balance and harming health. The supernatant of *Lactobacillus plantarum* TG008 has an inhibitory effect on HCT-8 colorectal cancer cells. This strain possesses the potential to inhibit the growth of some pathogenic bacteria and can serve as a microecological drug resource for treating colorectal cancer. Using probiotics to treat related diseases can reduce antibiotic use, prevent the emergence of drug-resistant pathogens, improve people's health index, and reduce healthcare costs.

[0097] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Images