Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer and application of lactobacillus plantarum UJS001

Abstract

The invention relates to the field of lactobacillus plantarum, and particularly discloses lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer and application of the lactobacillus plantarum UJS001. The lactobacillus plantarum UJS001 is preserved in the China General Microbiological Culture Collection Center on October 31, 2023, and the preservation number of the lactobacillus plantarum UJS001 is CGMCC No: 28821. The lactobacillus plantarum UJS001 can be applied to prevention and / or treatment of colorectal cancer and / or adjustment of intestinal flora functions, and has the advantages that the application range of the lactobacillus plantarum is widened, the application value of the lactobacillus plantarum is improved, and a new choice is brought to daily treatment or health care of patients with colorectal cancer.

Description

Technical Field

[0001] The present application relates to the field of Lactobacillus plantarum, and more specifically, to a Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer and its application. Background Art

[0002] Colorectal cancer is the second leading cause of cancer death worldwide, and the number of people with colorectal cancer in China ranks second among all cancers. Colorectal cancer can be detected early through colonoscopy screening. Studies have shown that the 5-year survival rate of local colorectal cancer is 90%, while the survival rate of colorectal cancer that metastasizes to distant organs is only 14%. Early diagnosis and treatment of colorectal cancer are crucial to preventing and reducing colorectal mortality. The occurrence of colorectal cancer is related to gene mutations, diet, inflammation, lifestyle and intestinal flora. As the "second genome", intestinal microorganisms play a very important role in the occurrence and development of colorectal cancer. The researchers conducted fecal metagenomic and metabolomics studies on 616 participants who underwent colonoscopy, indicating that Fusobacterium nucleatum, anaerobic peptostreptococci, and enterotoxin-producing Bacteroides fragilis are related to the occurrence of colorectal cancer, while beneficial bacteria such as Trichoderma, Bifidobacterium, and Streptococcus thermophilus are often lacking in colorectal cancer. Intestinal microbial imbalance can induce epithelial cell proliferation, damage the intestinal barrier, interfere with host immunity and cause inflammatory response, thereby triggering colorectal cancer. Given the important role of intestinal flora in the occurrence and development of colorectal cancer, restoring the microecological stability of intestinal flora is a key link in the prevention and treatment of colorectal cancer.

[0003] Lactobacillus is a probiotic strain widely studied and developed in modern food and health industry. Wherein Lactobacillus plantarum (Lactobacillus Plantarum, L.plantarum) is a member of the genus Lactobacillus that is widely distributed, belongs to Gram-positive bacteria, is usually present in fermented foods, can ferment carbohydrates and produce a large amount of lactic acid, because of its superior fermentation performance and various functionality, it has received extensive attention, its fermented food has a long history of consumption in various parts of the world, and is also a GRAS edible fungus species. At present, Lactobacillus plantarum has been widely used in industries such as nutrition and health care, fermented foods, biomedicine, feed, animal husbandry and breeding. Lactobacillus plantarum is also a probiotic group of the human gastrointestinal tract, metabolism can produce a variety of natural antibacterial substances such as organic acids, bacteriocins, hydrogen peroxide, diacetyl, has the balance of regulating intestinal microbial flora, enhances the body's immunity, reduces cholesterol levels, alleviates lactose intolerance and inhibits the formation of tumor cells and other effects.

[0004] With regard to the above-mentioned related technologies, how to obtain a strain of Lactobacillus plantarum that can prevent colon cancer in order to develop drugs, foods or health foods that can prevent colon cancer, expand the application scope of Lactobacillus plantarum and improve its application value is a problem that needs to be solved urgently. Summary of the invention

[0005] In order to expand the application of Lactobacillus plantarum in the prevention and treatment of colorectal cancer and provide new ideas for the prevention and treatment of colorectal cancer, the present application provides a Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer and its application.

[0006] In a first aspect, the present application provides a Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer, using the following technical solution:

[0007] A Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer, wherein the Lactobacillus plantarum UJS001 was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with a deposit number of CGMCC No: 28821.

[0008] Preferably, the 16S rDNA sequence of the Lactobacillus plantarum UJS001 is shown as SEQ ID NO.1.

[0009] Preferably, the Lactobacillus plantarum is isolated and screened from sauerkraut samples in Zhenjiang City, Jiangsu Province.

[0010] In a second aspect, the present application provides a composition containing Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer, using the following technical solution:

[0011] A composition containing Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer, wherein the composition contains live bacteria and / or dead bacteria of Lactobacillus plantarum UJS001 and metabolites thereof.

[0012] Preferably, the composition contains live bacteria of Lactobacillus plantarum UJS001, the number of which is ≥ 1.0×10 10 CFU / mL.

[0013] Preferably, the composition is a pharmaceutical composition, a health product composition, and / or a food composition.

[0014] Preferably, the pharmaceutical composition is an antibacterial, anti-tumor, or inflammatory bowel disease preventing and treating pharmaceutical composition.

[0015] Preferably, the antibacterial pharmaceutical composition is a pharmaceutical composition against Gram-positive bacteria and / or Gram-negative bacteria;

[0016] And / or the anti-tumor pharmaceutical composition is a pharmaceutical composition for preventing or treating colorectal cancer;

[0017] And / or the drug for preventing and treating inflammatory bowel inflammation is a pharmaceutical composition for preventing and treating ulcerative colitis.

[0018] Preferably, the health care product composition is a health care product composition for assisting tumor treatment and / or a health care product composition for regulating the function of intestinal flora.

[0019] Preferably, the health care product composition for assisting tumor treatment is a health care product composition for assisting colorectal cancer treatment.

[0020] Preferably, the composition further comprises pharmaceutical excipients, health care product excipients, and / or food excipients.

[0021] In a third aspect, the present application provides an application of Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer, using the following technical solution:

[0022] An application of Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer, which is applied to preventing and / or treating colorectal cancer, and / or, to regulating the function of intestinal flora.

[0023] In summary, this application has the following beneficial effects:

[0024] 1. The Lactobacillus plantarum UJS001 of the present application was isolated and screened from sauerkraut samples in Zhenjiang City, Jiangsu Province, and has excellent application potential in anti-inflammatory colorectal cancer and maintaining intestinal homeostasis.

[0025] 2. The plant lactobacillus UJS001 of the present application can increase probiotics and reduce harmful bacteria to reshape the intestinal balance, which is beneficial to regulate the homeostasis of the intestinal environment, restore the intestinal barrier function, and has a protective effect on inflammatory colorectal cancer. It has strain specificity and can effectively inhibit the growth of subcutaneous transplanted colon cancer tumors.

[0026] 3. The Lactobacillus plantarum UJS001 of the present application can be applied to the fields of food, health products and medicine, which broadens the application scope of Lactobacillus plantarum, improves the application value of Lactobacillus plantarum, and provides a new option for the daily treatment and health care of colorectal cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a graph of the test results in Example 1 of the present application; wherein A is a photo of the colony morphology of the plate streak inoculation; B is the Gram staining result; C is the biochemical index identification result;

[0028] Figure 2 The figure is a graph of the test results in Example 1 of the present application; wherein A is the agarose gel electrophoresis test result; B is the phylogenetic tree; and C is the whole genome sequencing result;

[0029] Figure 3The figure is a test result diagram of the effect of Lactobacillus plantarum UJS001 on the length of the mouse colon in the AOM / DSS-induced inflammatory colon cancer mouse model in Example 2 of the present application; wherein A is a photo of the mouse colon test of the Control group, the AOM / DSS group, and the LP-UJS001 group; and B is a statistical graph of the length of the mouse colon in the Control group, the AOM / DSS group, and the LP-UJS001 group;

[0030] Figure 4 HE staining results of the colon tissues of mice in the Control group, AOM / DSS group, and LP-UJS001 group in Example 2 of the present application;

[0031] Figure 5 AB staining results of mouse colon tissues in the Control group, AOM / DSS group, and LP-UJS001 group in Example 2 of the present application;

[0032] Figure 6 This is a statistical graph of the content of diamine oxidase (DAO), histamine, and tight junction protein ZO-1 in the serum of mice in the Control group, AOM / DSS group, and LP-UJS001 group in Example 3 of the present application;

[0033] Figure 7 The results of the test on the effect of Lactobacillus plantarum UJS001 on subcutaneous transplanted tumors of MC38 colon cancer cells in Example 4 of the present application are shown in Figure A; wherein A is a test photograph of subcutaneous colon cancer transplanted tumors of mice in the Control group and the LP-UJS001 group; and B is a statistical relationship diagram between the volume of subcutaneous colon cancer transplanted tumors of the Control group and the LP-UJS001 group and the number of days of feeding of mice;

[0034] Figure 8 This is a diagram of the abundance of different bacterial species in the intestines of mice in the Control group, AOM / DSS group, and LP-UJS001 group in Example 5 of the present application;

[0035] Fig. 9 is a relative abundance diagram of potential functional pathways in Example 5 of the present application;

[0036] Fig.10 This is a heat map of amino acid-related differential metabolites of mice in the AOM / DSS group and the LP-UJS001 group in Example 6 of the present application;

[0037] Fig.11 This is the abundance diagram of essential amino acids and their intermediates of mice in the AOM / DSS group and the LP-UJS001 group in Example 6 of the present application;

[0038] Fig.12 This is a conversion diagram of tryptophan and its intermediates in Example 6 of the present application. DETAILED DESCRIPTION

[0039] To further help understand the technical solution of the present invention, the technical solution of the present invention is described in more detail below with reference to the accompanying drawings and by providing several specific implementation examples. All of these described embodiments are only partial embodiments of the present invention, not all;

[0040] The following specific embodiments may be combined with each other, and the same or similar concepts or processes may not be described in detail in some embodiments; and the reaction devices, monomer compounds, etc. involved in the following embodiments are all commercially available.

[0041] The following specific embodiments may be combined with each other, and the same or similar concepts or processes therein may not be described in detail in some embodiments.

[0042] The following examples are further explanations of the present invention, but the present invention is not limited thereto. Unless otherwise specified in the examples, the percentages are all by mass.

[0043] Example

[0044] Example 1

[0045] This embodiment discloses a Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer, which was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with a deposit number of CGMCC No: 28821, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] Lactobacillus plantarum UJS001 was isolated and screened from sauerkraut samples in Zhenjiang City, Jiangsu Province. The Lactobacillus plantarum strain isolated and purified from the sauerkraut samples was frozen for future use and was identified as follows:

[0047] (1) Observation of bacterial morphology

[0048] Observe the morphology of the colonies inoculated on the MRS plate by streaking;

[0049] Gram staining: add a drop of physiological saline on the slide, pick a single colony and spread it on the slide to dilute evenly, and quickly dry it to fix; add crystal violet to stain for 1 minute, rinse with tap water, cover and stain with iodine solution for 1 minute, rinse with tap water, decolorize with 95% alcohol for 30 seconds, rinse with tap water, re-stain with fuchsin for 1 minute, and observe under a microscope.

[0050] Reference Figure 1 A, it can be seen that the isolated strain forms smooth and moist white circular colonies with neat edges when inoculated on MRS agar medium by streaking; Figure 1B, Gram staining of a single colony showed a positive result, and the bacteria appeared to be long and thin or short rods under an optical microscope, which was consistent with the morphological characteristics of the genus Lactobacillus.

[0051] (2) Identification of biochemical indicators

[0052] According to the instructions of the Lactobacillus biochemical identification kit (Guangdong Huankai Microbiology Co., Ltd.), a single colony was picked from the plate and placed in a fermentation reagent bottle, cultured in a 37°C incubator for 24-48 hours, and the color change was observed.

[0053] Reference Figure 1 C. According to the results of biochemical identification, the isolated bacteria were found to be a plant lactobacillus strain that could ferment esculin, cellobiose, maltose, mannitol, salicin, sucrose, and raffinose, but could not ferment sorbitol.

[0054] (3) Identification of bacterial 16S rDNA

[0055] 16S rDNA sequencing primers 27F and 1492R were used to perform PCR amplification using the extracted genomic DNA of Lactobacillus plantarum UJS001 as a template. The PCR primer sequences are as follows:

[0056] 27F:5-AGAGTTTGATCCTGGCTCAG-3,

[0057] 1492R:5-CTACGGCTACCTTGTTACGA-3.

[0058] After the reaction is completed, agarose gel electrophoresis is performed and the results are as follows Figure 2 As shown in A, the agarose gel electrophoresis detection results show that a specific target band with a fragment length of about 1500 bp was amplified.

[0059] The PCR amplified fragments were confirmed and the gel with bands was cut out, recovered using a DNA recovery kit, and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were submitted to NCBI for comparison and construction of a phylogenetic tree.

[0060] The 16S rDNA sequence of the strain is shown in SEQ ID NO.1.

[0061] Reference Figure 2 B, The sequencing results of the amplified products were compared for sequence homology in NCBI and a phylogenetic tree was constructed. It can be seen that Lactobacillus plantarum UJS001 and Lactobacillus plantarum are located in the same branch.

[0062] Reference Figure 2C, The whole genome sequencing results show the basic characteristics of Lactobacillus plantarum UJS001. The genome of Lactobacillus plantarum UJS001 consists of a chromosome with a length of 3085100bp and three plasmids with lengths of 8529bp, 7221bp, and 2021bp respectively. The genome circles from the inside to the outside, the first circle represents the scale; the second circle represents the GC offset; the third circle represents the GC content; the fourth and seventh circles represent the COG to which each CDS belongs; the fifth and sixth circles represent the positions of CDS, tRNA and rRNA on the genome. The total length of the whole genome is 2,037,216,978bp, the GC content is 44.55%, and a total of 389,466 coding sequences were identified.

[0063] In summary, based on the morphological characteristics, physiological and biochemical test results and molecular biological characteristics, it can be seen that the strain isolated from the Zhenjiang sauerkraut sample by the inventors is Lactobacillus plantarum, and it is deposited in the China General Microbiological Culture Collection and named Lactobacillus plantarum UJS001 (L. plantarum UJS001).

[0064] Example 2

[0065] This example studies the use of Lactobacillus plantarum UJS001 in alleviating AOM / DSS-induced inflammatory colorectal cancer, and conducts the following experiments:

[0066] (1) Preparation of Lactobacillus plantarum UJS001 powder

[0067] Lactobacillus plantarum UJS001 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 22-24 h. The bacterial solution was added with secondary activation medium (skim milk 100 g + yeast extract 1.0 g + double distilled water 1000 ml) and cultured again for 12 h (final concentration of about 1×10 11 CFU / mL); add 10% trehalose, 1% L-glutamic acid, 2% vitamin C, 2% proline, 0.5% glycine, 15% skim milk powder, and then freeze-dry; viable count ≥1.0×10 10 CFU / mL.

[0068] (2) Mouse preparation

[0069] 5-6 week old C57B / 6 male mice, weighing (18±2) g, were purchased from Changzhou Cavens Experimental Animal Co., Ltd. The mice were kept in an SPF laminar flow cabinet with a 12h:12h cycle, an ambient temperature of 23-25°C, and an ambient humidity of 40%-60%. The experiment was officially started after one week of adaptive feeding. 24 mice were randomly divided into 3 groups, 8 mice in each group, namely the normal group (Control group), the inflammatory colon cancer model group (AOM / DSS group), and the Lactobacillus plantarum group (LP-UJS001 group).

[0070] (3) Establishment of an in situ inflammatory colon cancer model in mice

[0071] The mice in the Control group were fed with regular drinking water; the mice in the AOM / DSS group and the LP-UJS001 group were treated as follows:

[0072] (1) Intraperitoneal injection of AOM (10 mg / kg); (2) feeding with regular drinking water for 4 days; (3) replacing drinking water with 2% DSS solution for one week; (4) feeding with regular drinking water for one week;

[0073] Step (3) and step (4) are repeated 3 times;

[0074] In addition, the LP-UJS001 group was fed with Lactobacillus plantarum UJS001 powder (1×10 10 CFU / g), the corresponding bacterial solution concentration is 10 10 CFU / mL was administered in 100 μL.

[0075] At the end of the experiment, the mice were fasted overnight, and the complete colon tissue was collected. The distal colon tissue of about 1 cm was fixed with formalin, and then HE staining and tissue morphology analysis were performed. HE staining was used to observe the integrity of the colon tissue structure; and AB staining was used to observe the colon mucus layer and goblet cells of the mice.

[0076] Reference Figure 3 A. Figure 3 B. Compared with the Control group, the colon length of mice in the AOM / DSS group was significantly shortened (P<0.001), indicating that the AOM / DSS-induced inflammatory colon cancer mouse model was successfully established; after the intervention of Lactobacillus plantarum UJS001, the colon length of mice in the LP-UJS001 group was significantly longer than that in the AOM / DSS group (P<0.05); it can be seen that Lactobacillus plantarum UJS001 can alleviate the shortening of the colon in the inflammatory colorectal cancer model.

[0077] Reference Figure 4 HE staining showed that Lactobacillus plantarum UJS001 could improve the integrity of colon tissue structure in AOM / DSS-induced inflammatory colon cancer mice.

[0078] Reference Figure 5 AB staining showed that the number of colonic goblet cells in the LP-UJS001 group increased significantly, and the apical exocytosis and secretion of mucin particles on the mucosal surface were also enhanced.

[0079] In summary, Lactobacillus plantarum UJS001 can not only alleviate the colon shortening of the inflammatory colorectal cancer model, but also has the effect of restoring the intestinal barrier.

[0080] Example 3

[0081] This example studies the effect of lactobacillus UJS001 on the permeability of mice with inflammatory colorectal cancer, and conducts the following experiments:

[0082] The eyeball blood of the mice in the Control group, AOM / DSS group, and LP-UJS001 group of Example 2 was collected using EP tubes. The collected blood was allowed to stand at room temperature for two hours and then centrifuged at 4°C, 3000 g for 15 min. The upper serum was collected and packaged in a -80°C refrigerator for later use.

[0083] (1) ELISA to detect the content of diamine oxidase (DAO)

[0084] The double antibody sandwich method was used to determine the level of mouse diamine oxidase (DAO) in the specimen. The microplate was coated with purified mouse diamine oxidase (DAO) capture antibody to prepare a solid-phase antibody. Mouse diamine oxidase (DAO) was added to the coated microwells in sequence, and then combined with the HRP-labeled detection antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted into blue under the catalysis of HRP enzyme, and converted into the final yellow under the action of acid. The depth of color was positively correlated with the mouse diamine oxidase (DAO) in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using an enzyme reader, and the mouse diamine oxidase (DAO) content in the sample was calculated using a standard curve.

[0085] (2) ELISA to detect histamine content

[0086] The double antibody sandwich method was used to determine the mouse histamine level in the specimen. The microplate was coated with purified mouse histamine capture antibody to prepare a solid phase antibody. Mouse histamine was added to the coated microwells in sequence, and then combined with the HRP-labeled detection antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted into blue under the catalysis of HRP enzyme, and converted into the final yellow under the action of acid. The depth of color was positively correlated with the mouse histamine in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using an enzyme reader, and the mouse histamine content in the sample was calculated using the standard curve.

[0087] (3) ELISA detection of tight junction protein ZO-1 content

[0088] The double antibody sandwich method was used to determine the level of mouse tight junction protein ZO-1 in the specimen. The microplate was coated with purified mouse tight junction protein ZO-1 capture antibody to prepare a solid phase antibody. Mouse tight junction protein ZO-1 was added to the coated microwells in sequence, and then combined with the HRP-labeled detection antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted into blue under the catalysis of HRP enzyme, and converted into the final yellow under the action of acid. The depth of color was positively correlated with the small tight junction protein ZO-1 in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using an enzyme reader, and the content of mouse tight junction protein ZO-1 in the sample was calculated using a standard curve.

[0089] Reference Figure 6 It can be seen that compared with the Control group, the levels of diamine oxidase (DAO), histamine and tight junction protein ZO-1 in the serum of the AOM / DSS group mice were significantly increased, but after the administration of Lactobacillus plantarum UJS001 powder, the levels of DAO, Histamine and ZO-1 in the serum of the LP-UJS001 group were significantly decreased and dropped to the level of the Control group.

[0090] Example 4

[0091] This example studies the effect of Lactobacillus plantarum UJS001 on the growth inhibition of subcutaneous transplanted tumors of colon cancer MC38 cells. The test method is as follows:

[0092] (1) Preparation of Lactobacillus plantarum UJS001 powder

[0093] Lactobacillus plantarum UJS001 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 22-24 h. The bacterial solution was added with secondary activation medium (skim milk + yeast extract + double distilled water) and cultured again for 12 h (final concentration was about 1×10 11 CFU / mL); add 10% trehalose, 1% L-glutamic acid, 2% vitamin C, 2% proline, 0.5% glycine, 15% skim milk powder, and then freeze-dry; viable count ≥1.0×10 10 CFU / mL.

[0094] (2) Preparation of mice

[0095] 5-6 week old C57B / 6 male mice weighing (18±2) g were purchased from Changzhou Cavens Experimental Animal Co., Ltd. The mice were housed in an SPF laminar flow cabinet with a 12h:12h cycle, an ambient temperature of 23-25°C, and an ambient humidity of 40%-60%. The experiment was officially started after one week of adaptive feeding.

[0096] (3) Establishment of colorectal cancer transplant model

[0097] MC38 colorectal cancer cells were injected into the armpits of mice to establish a colorectal cancer xenograft model. When the xenograft grew to a volume of 50 mm 3 At the same time, 10 mice were randomly divided into 2 groups, 5 mice in each group, namely the model group (Control group) and the Lactobacillus plantarum group (LP-UJS001 group). The mice were fed with Lactobacillus plantarum UJS001 powder (1×10 10 CFU / g), the corresponding bacterial solution concentration is 10 10 CFU / mL was administered in 100 μL, and tumor size was measured in real time.

[0098] Reference Figure 7 It can be seen that Lactobacillus plantarum UJS001 can significantly inhibit the growth of subcutaneous transplanted tumors of colon cancer MC38 cells, with an inhibition efficiency of nearly 80%.

[0099] Example 5

[0100] This example studies the effect of Lactobacillus plantarum UJS001 on the homeostasis of intestinal flora in mice with inflammatory colorectal cancer. The experimental method is as follows:

[0101] The feces of mice in the Control group, AOM / DSS group, and LP-UJS001 group of the AOM / DSS-induced inflammatory colon cancer mouse model of Example 2 were collected into cryopreservation tubes, quickly frozen in liquid nitrogen, and stored at -80°C for later use.

[0102] (1) Extraction of total microbial DNA

[0103] Total DNA was extracted from mouse fecal samples according to the instructions of the DNA extraction kit and stored at -80°C.

[0104] (2) Sample PCR amplification

[0105] Nanodrop was used to quantify DNA, and agarose gel electrophoresis was used to test the quality of DNA extraction. PCR primers were synthesized based on the highly variable V3-V4 region of the bacterial 16S rRNA gene, and PCR amplification was performed on the target fragment.

[0106] The PCR primer sequences are as follows:

[0107] 338F: 5'-barcode+ACTCCTACGGGAGGCAGCA-3';

[0108] 806R: 5'-GGACTACHVGGGTWTCTAAT-3'.

[0109] The PCR reaction system is as follows: 0.25 μL of ultra-fidelity DNA polymerase, 5 μL each of 5×Reaction Buffer and 5×High GC Buffer, 2 μL each of dNTP (10 mM) and template DNA, 1 μL each of forward and reverse primers (10 μM), and 8.75 μL of DEPC water.

[0110] The PCR amplification program was set as follows: 98°C pre-denaturation for 30 s, 98°C denaturation for 15 s, 50°C annealing for 30 s, 72°C extension for 30 s, and the number of cycles was set to 25-27 times. Finally, the product was kept at 72°C for 5 min to complete the extension and stored at 4°C.

[0111] The amplified product was subjected to 2% agarose gel electrophoresis, and the target fragment was cut out and recovered and purified.

[0112] (3) Library construction and sequencing

[0113] The PCR products were subjected to fluorescence quantitative detection, and then the samples were mixed according to the data volume required for each sample to construct the library. The libraries that passed the quality inspection were sequenced with 2×250bp double-end sequencing using an Illumina NovaSeq sequencer.

[0114] Reference Figure 8 , by detecting the 16s sequencing of mouse feces, it can be seen that Lactobacillus plantarum UJS001 significantly increases probiotics such as Lactobacillus, Coprococcus, and Mucispirillum in the process of inducing inflammatory colorectal cancer, and significantly reduces harmful bacteria such as Bifidobacterium, Sutterella, and Turicibacter, remodeling intestinal balance, which is beneficial to regulating intestinal homeostasis and has the effect of restoring the intestinal barrier. This shows that Lactobacillus plantarum UJS001 inhibits "bad bacteria" and produces beneficial butyrate bacteria.

[0115] Reference Fig. 9 Further analysis by microbiome-related functional methods showed that the top five enriched pathways were amino acid biosynthesis, nucleoside and nucleotide biosynthesis, vitamin biosynthesis, fatty acid biosynthesis and carbohydrate biosynthesis, with amino acid biosynthesis being the most prominent.

[0116] In summary, Lactobacillus plantarum UJS001 can significantly restore the intestinal barrier function of inflammatory colorectal cancer and regulate the homeostasis of intestinal flora in mice with inflammatory colorectal cancer. Therefore, Lactobacillus plantarum UJS001 can be widely used to improve intestinal barrier function damage and related development diseases, prevent cancer occurrence, or be used in the daily health care of cancer patients.

[0117] Example 6

[0118] This example studies the effect of Lactobacillus plantarum UJS001 on fecal metabolites in colitis mice, and the test method is as follows:

[0119] (1) Preparation of Lactobacillus plantarum UJS001 powder

[0120] Lactobacillus plantarum UJS001 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 22-24 h. The bacterial solution was added with secondary activation medium (skim milk + yeast extract + double distilled water) and cultured again for 12 h (final concentration was about 1×10 11 CFU / mL); add 10% trehalose, 1% L-glutamic acid, 2% vitamin C, 2% proline, 0.5% glycine, 15% skim milk powder, and then freeze-dry; viable count ≥1.0×10 10 CFU / mL.

[0121] (2) Construction of mouse model

[0122] C57B / 6 male mice aged 5-6 weeks, weighing (18±2) g, were purchased from Changzhou Cavens Experimental Animal Co., Ltd. The mice were kept in an SPF-grade laminar flow cabinet with a 12h:12h cycle, an ambient temperature of 23-25°C, and an ambient humidity of 40%-60%. The experiment was officially started after one week of adaptive feeding. Ten mice were randomly divided into two groups, 5 in each group, namely the colitis model group (AOM / DSS group) and the Lactobacillus plantarum group (LP-UJS001 group).

[0123] The AOM / DSS group and the LP-UJS001 group were treated as follows:

[0124] a. Intraperitoneal injection of AOM (10 mg / kg); b. Regular drinking water feeding for 4 days; c. 2% DSS solution replaced drinking water for one week; d. Regular drinking water feeding for one week; Steps c and d were repeated 3 times;

[0125] In addition, the LP-UJS001 group was fed with Lactobacillus plantarum UJS001 powder (1×1010 CFU / g) throughout the whole process, and the corresponding bacterial liquid concentration was 1010 CFU / mL, with 100 μL given.

[0126] (3) Metabolomics testing

[0127] a. Metabolite Extraction

[0128] Appropriate amounts of mouse metabolite samples were added to precooled methanol / acetonitrile / water solution (2:2:1, v / v), subjected to low-temperature ultrasound for 30 min, allowed to stand at -20°C for 10 min, centrifuged at 14000g and 4°C for 20 min, and the supernatant was vacuum dried.

[0129] b. High performance liquid chromatography-mass spectrometry (LC-MS / MS)

[0130] 100 μL of acetonitrile solution (acetonitrile: water = 1:1, v / v) was added, centrifuged at 4°C for 15 min, and the supernatant was sampled for analysis. Agilent 1290 Infinity LC HILIC column was used for separation, mobile phase A: water, 25 mM ammonium acetate and 25 mM ammonia, mobile phase B: acetonitrile.

[0131] The gradient elution process was: 95% B, 0.5 min; 95%-65% B, 6.5 min; 65%-40% B, 1 min; 40% B, 1 min; 40%-95% B, 0.1 min; 95% B, 2.9 min. The sample was automatically injected at 4°C with a volume of 2 μL.

[0132] (2) Non-targeted metabolomics analysis

[0133] SIMCA-P 14.1 software was used for pattern recognition. After Pareto-scaling preprocessing, P < 0.05 and VIP > 1 were used to screen differential metabolites.

[0134] Reference Fig.10Through the analysis of amino acid metabolites in mouse feces, the results of heat map enrichment analysis showed that after intervention with Lactobacillus plantarum UJS001 powder, the LP-UJS001 group had significantly increased levels of lysine (L-Lysine), methionine (L-Methionine(S)-S-oxide), arginine (L-Arginine), valine (L-Valine), and histidine (L-Histidinal) compared with the model control group (AOS / DSS group), while threonine (L-Threonine), arginine's intermediate metabolite citrulline (Citrulline) and N2-succinyl-L-ornithine The levels of N2-succinyl-L-ornithine, 5-hydroxylysine and beta-alanyl-L-lysine, the intermediate products of lysine, N-formyl-L-methionine, the intermediate products of methionine, L-kynurenine and L-formylkynurenine, the intermediate metabolites of tryptophan, and L-histidinol, the intermediate metabolite of histidine, were significantly reduced, indicating that the composition of fecal amino acid metabolites of inflammatory colorectal cancer changed after intervention with Lactobacillus plantarum UJS001.

[0135] Reference Fig.11 By studying the abundance diagram of essential amino acids, further metabolomics detection revealed that after the action of Lactobacillus plantarum UJS001, a variety of essential amino acids and their intermediates were enriched, including lysine, tryptophan, methionine, threonine, valine and histidine. Combined with the above bacterial flora composition analysis results, Lactobacillus plantarum UJS001 significantly increased the content of essential amino acids such as lysine, tryptophan, methionine, threonine, valine and histidine. Among them, the intermediate products of tryptophan, kynurenine and N-formylkynurenine, decreased significantly. Fig.12 , tryptophan generates N-formylkynurenine under the action of oxygenase, and further generates kynurenine and formic acid through the hydrolysis of formylase, which is the tryptophan-kynurenine metabolic pathway. This shows that Lactobacillus plantarum UJS001 may play a role in treating or preventing colorectal cancer through tryptophan metabolism driven by intestinal flora.

[0136] In summary, this application established an AOM / DSS-induced inflammatory colon cancer mouse model to evaluate the effects of Lactobacillus plantarum UJS001 on relevant indicators in serum, colon tissue and intestinal flora. The results showed that Lactobacillus plantarum UJS001 could significantly reduce the levels of diamine oxidase (DAO), histamine and tight junction protein ZO-1 in serum, restoring them to normal levels; and hematoxylin-eosin (H&E) staining and Alcian blue (A&B) staining showed that LP-UJS001 could improve the integrity of colon tissue structure, increase the number of colonic goblet cells, and enhance the secretion of mucin.

[0137] In the subcutaneous model of colorectal cancer, Lactobacillus plantarum UJS001 showed an effective inhibitory effect on the growth of MC38 cell transplanted tumors; 16S rRNA sequencing analysis of mouse feces revealed that Lactobacillus plantarum UJS001 could significantly increase the number of beneficial bacteria such as Lactobacillus plantarum, Coprococcus and Mucispirillum, and affect the metabolic pathway of amino acid synthesis. Metabolomics detection further revealed that after the action of Lactobacillus plantarum UJS001, a variety of essential amino acids and their intermediates were enriched, especially the significant decrease of kynurenine and N-formylkynurenine in the tryptophan metabolic pathway, indicating that Lactobacillus plantarum UJS001 may play its role in treating or preventing colorectal cancer through tryptophan metabolism driven by intestinal flora.

[0138] The Lactobacillus plantarum UJS001 disclosed in the present application can significantly restore the intestinal barrier function of inflammatory colorectal cancer; in addition, Lactobacillus plantarum UJS001 has the effect of regulating the homeostasis of intestinal flora in mice with inflammatory colorectal cancer. Therefore, Lactobacillus plantarum UJS001 can be widely used to improve intestinal barrier function damage and related development diseases, as well as to prevent the occurrence of cancer or be used in the daily health care of cancer patients, which not only broadens the application scope of Lactobacillus plantarum and improves its application value, but also provides new options and hopes for the daily treatment or health care of colorectal cancer patients.

[0139] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer, characterized in that: The plant lactobacillus UJS001 was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with the deposit number CGMCC No: 28821.

2. The Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer according to claim 1, characterized in that The 16S rDNA sequence of the Lactobacillus plantarum UJS001 is shown in SEQ ID NO.

1.

3. A composition containing Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer as claimed in any one of claims 1 to 2, characterized in that: The composition comprises live bacteria and / or dead bacteria of Lactobacillus plantarum UJS001 and metabolites thereof.

4. The composition containing Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer according to claim 3, characterized in that: The composition contains live bacteria of Lactobacillus plantarum UJS001, the number of which is ≥1.0×10 10 CFU / mL.

5. The composition containing Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer according to claim 3, characterized in that: The composition is a pharmaceutical composition, a health product composition, and / or a food composition.

6. The composition containing Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer according to claim 5, characterized in that: The pharmaceutical composition is an antibacterial, anti-tumor and inflammatory bowel disease prevention and treatment pharmaceutical composition.

7. The composition containing Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer according to claim 6, characterized in that: The antibacterial drug composition is a drug composition against Gram-positive bacteria and / or Gram-negative bacteria; And / or the anti-tumor pharmaceutical composition is a pharmaceutical composition for preventing or treating colorectal cancer; And / or the drug for preventing and treating inflammatory bowel inflammation is a pharmaceutical composition for preventing and treating ulcerative colitis.

8. The composition containing Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer according to claim 5, characterized in that: The health care product composition is a health care product composition for assisting tumor treatment and / or a health care product composition for regulating the function of intestinal flora.

9. The composition containing Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer according to claim 8, characterized in that: The health care product composition for assisting tumor treatment is a health care product composition for assisting colorectal cancer treatment.

10. A use of Lactobacillus plantarum UJS001 for preventing and / or treating colorectal cancer according to any one of claims 1 to 2, characterized in that: Application in the prevention and / or treatment of colorectal cancer, and / or, application in regulating the function of intestinal flora.

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