Phytobacterium plantarum proliferation agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines and microorganisms, in particular to a plant lactobacillus proliferation agent as well as a preparation method and application thereof. The proliferation agent is prepared from the following components in percentage by mass: 0.02 to 5 percent of soybean peptide, 0.01 to 5 percent of glucose and a manganese source; on the basis of the total mass of the proliferation agent, the mass fraction of the Mn element in the manganese source is 0.0000001-0.005 wt%. The proliferation agent disclosed by the invention is high in safety, edible and capable of promoting proliferation of the plant lactobacillus; in addition, the compound also can promote the growth of targeted tumors of the plant lactobacillus and inhibit the growth of the tumors, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the fields of medicine and microbiology, specifically to a plant lactobacillus propagation agent, its preparation method, and its uses. Background Technology

[0002] Cancer treatment, due to its high cost, low cure rate, and short patient survival, has become a major challenge in the global public health field. Tumor cells exhibit abnormally active metabolism, and their rapid proliferation leads to disruption of the internal vascular system, creating a unique hypoxic microenvironment. This hypoxic region not only promotes tumor progression and drug resistance but also provides a unique ecological niche for the selective colonization of anaerobic microorganisms, thus opening up potential pathways for developing novel targeted cancer therapies.

[0003] Lactobacillus plantarum, as an anaerobic bacterium, can be designed as a living carrier for drug delivery or immunomodulation. However, its colonization and proliferation capabilities in animals, especially in tumor tissues, are weak, limiting its clinical translation. Existing technologies, such as patent CN117717600A, disclose a proliferation agent that promotes the growth of Lactobacillus plantarum in tumors, using dipeptides, cysteine ​​hydrochloride, uridine, xanthine, nicotinamide, manganese chloride, etc., to prepare the proliferation agent. However, this patent still has significant limitations: complex components, high cost, insufficient targeting specificity, and it is not edible, making it difficult to apply directly to humans, thus restricting its clinical applicability and promotion prospects.

[0004] Therefore, there is an urgent need for a microbial proliferation agent that is safe, highly specific, and cost-controllable, in order to efficiently and directionally increase the number of viable Lactobacillus plantarum in the tumor-specific microenvironment, thereby promoting the leap from laboratory to clinical application of tumor treatment. Summary of the Invention

[0005] Based on the above-mentioned technical problems, the present invention provides a plant lactobacillus propagation agent, its preparation method and uses.

[0006] To achieve the above and other related objectives, the present invention is obtained through the following technical solution.

[0007] The first aspect of the present invention provides a plant lactobacillus propagation agent, comprising the following components in mass fractions: 0.02-5 wt% soybean polypeptide, 0.01-5 wt% glucose and manganese source; based on the total mass of the propagation agent, the mass fraction of Mn element in the manganese source is 0.0000001-0.005 wt%.

[0008] A second aspect of the present invention provides a method for culturing *Lactobacillus plantarum*, comprising: inoculating *Lactobacillus plantarum* into a culture medium containing a proliferation agent as described above and culturing it.

[0009] A third aspect of the invention provides the use of the proliferator as described above in increasing the viable count of *Lactobacillus plantarum* and / or promoting *Lactobacillus plantarum*-targeted tumor growth.

[0010] A fourth aspect of the present invention provides a composition comprising the aforementioned proliferator and *Lactobacillus plantarum*.

[0011] The fifth aspect of the invention provides the use of the proliferator or composition as described above in at least one of the following:

[0012] 1) To assist in inhibiting tumor growth in vitro;

[0013] 2) Preparation of anti-tumor drugs.

[0014] As described above, the *Lactobacillus plantarum* propagation agent, its preparation method, and its uses according to the present invention have the following beneficial effects:

[0015] 1) The proliferator of the present invention can not only increase the number of viable bacteria of Lactobacillus plantarum, but also promote the targeted tumor growth of Lactobacillus plantarum.

[0016] 2) The raw materials of the growth promoter of the present invention include soybean peptone, glucose and manganese source. The raw materials are safe, convenient, edible and applicable to the human body.

[0017] 3) The proliferation agent of the present invention has strong specificity for Lactobacillus plantarum, and the number of viable bacteria of Lactobacillus plantarum is significantly higher after inoculation and culture in the proliferation agent.

[0018] 4) The proliferation agent of the present invention is low in cost and easy to control. Attached Figure Description

[0019] Figure 1 This is a graph showing the viable counts of different lactic acid bacteria grown in a growth promoter in Example 2 of the present invention.

[0020] Figure 2 The number of viable bacteria in each organ of the colon cancer mouse model in Example 3 of the present invention after administration of a proliferator and gavage with *Lactobacillus plantarum*.

[0021] Figure 3 This is a diagram showing the volume of dissected tumors in different groups of mouse models of colon cancer in Example 4 of the present invention. Detailed Implementation

[0022] *Lactobacillus plantarum* is an anaerobic bacterium that can survive under anaerobic conditions in the tumor microenvironment. Furthermore, the polysaccharide structures on the surface of *Lactobacillus plantarum* (such as capsular polysaccharides and lipoteichoic acid) can bind to receptors on the surface of tumor cells or stromal cells, enhancing their adhesion and colonization abilities. Therefore, *Lactobacillus plantarum* has been reported for anti-tumor applications. However, current reports indicate that *Lactobacillus plantarum* has a lower survival rate in tumor tissues than 5 × 10⁻⁶. 8The CFU / mL concentration did not show significant proliferation effect and lacked specificity.

[0023] The first aspect of the present invention provides a plant lactobacillus propagation agent, comprising the following components in mass fractions: 0.02-5 wt% soybean polypeptide, 0.01-5 wt% glucose and manganese source; based on the total mass of the propagation agent, the mass fraction of Mn element in the manganese source is 0.0000001-0.005 wt%.

[0024] In some embodiments, the weight percentage of the soybean peptone can be 0.02-5 wt%, 0.02-0.15 wt%, 0.1-1.5 wt%, 1-2.5 wt%, 2-3.5 wt%, 2.5-5 wt%, or 0.02 wt%, 0.05 wt%, 0.1 wt%, 0.5 wt%, 0.8 wt%, 1.1 wt%, 1.5 wt%, 1.8 wt%, 2.0 wt%, 2.2 wt%, 2.4 wt%, 2.6 wt%, 2.8 wt%, 3.0 wt%, 3.5 wt%, 3.8 wt%, 4 wt%, 4.2 wt%, 4.5 wt%, 4.8 wt%, or 5 wt%.

[0025] In some embodiments, the molecular weight of the soybean peptone peptide is 200-5000 Da, or it can be 200-500 Da, 400-1500 Da, 1000-2500 Da, or 2000-5000 Da. Soybean peptone with this molecular weight has a significant promoting effect on microbial growth.

[0026] In some embodiments, the glucose may be in the following weight proportions: 0.01-5 wt%, 0.02-0.15 wt%, 0.1-1.5 wt%, 1-2.5 wt%, 2-3.5 wt%, 2.5-5 wt%, or 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.1 wt%, 0.5 wt%, 0.8 wt%, 1.1 wt%, 1.5 wt%, 1.8 wt%, 2.0 wt%, 2.2 wt%, 2.4 wt%, 2.6 wt%, 2.8 wt%, 3.0 wt%, 3.5 wt%, 3.8 wt%, 4 wt%, or 5 wt%.

[0027] In some embodiments, based on the total mass of the proliferator, the mass fraction of Mn element in the manganese source is 0.0000001~0.005 wt%, or it can be 0.0000001~0.0006 wt%, or it can be 0.00005~0.003 wt%, or it can be 0.000001 wt%, 0.0000015 wt%, 0.00001 wt%, 0.00008 wt%, 0.0001 wt%, 0.0005 wt%, 0.0008 wt%, 0.001 wt%, 0.002 wt%, 0.003 wt%, or 0.005 wt%.

[0028] In some embodiments, the manganese source is selected from manganese salts or manganese-containing foods.

[0029] In some embodiments, the manganese salt is selected from one or more of manganese chloride, manganese sulfate, and manganese nitrate.

[0030] In some embodiments, the manganese-containing food is selected from one or more of oats, tea, and chickpeas.

[0031] In some specific embodiments, the oat grain size is 150μm~270μm, or it can be 150μm~200μm, 170~220μm, 200~250μm, or 250-270μm. According to the "Chinese Food Composition Table" (6th Standard Edition), 100 grams of raw oats contains 4.0~5.0 mg of manganese.

[0032] In some more specific embodiments, the oats are selected from one or both of oat flour or oat flakes.

[0033] In some embodiments, the tea leaves are tea powder. According to the "Chinese Food Composition Tables" (6th Standard Edition), 100 grams of dried tea leaves contain 30-50 mg of manganese.

[0034] In some embodiments, the particle size of the tea powder is 150μm~270μm, or it can be 150μm~200μm, or it can be 170~220μm, or it can be 200~250μm, or it can be 250~270μm.

[0035] In some embodiments, the tea leaves are made from chickpeas or chickpea flour. Chickpeas (scientific name: *Cicerarietinum* L.) are the fruit of an annual or perennial climbing herbaceous plant belonging to the genus *Cicerarietinum* in the legume family. According to the *Chinese Food Composition Table* (6th standard edition), 100 grams of chickpeas contain 1.5-2.0 mg of manganese. Current reports indicate that chickpeas have effects such as regulating blood sugar, improving cardiovascular health, promoting development, delaying aging, and aiding weight loss.

[0036] In some embodiments, the particle size of the chickpea flour is 150μm~270μm, or it can be 150μm~200μm, or it can be 170~220μm, or it can be 200~250μm, or it can be 250~270μm.

[0037] Another aspect of the present invention provides a method for preparing the proliferation agent as described above, comprising: homogenizing the raw material with water, cooking, sterilizing, and obtaining the proliferation agent.

[0038] In some embodiments, the cooking time is 1 to 3 hours.

[0039] In some embodiments, the cooking time is 1 to 1.5 hours, or 2 to 3 hours, and can be 1 hour, 1.3 hours, 1.6 hours, 1.9 hours, 2.1 hours, 2.4 hours, 2.7 hours, or 3.0 hours.

[0040] In some embodiments, the sterilization temperature is 100~200°C.

[0041] In some embodiments, the sterilization temperature can be 100~160℃, 150~190℃, 160~190℃, or 100℃, 120℃, 130℃, 140℃, 150℃, or 160℃.

[0042] In some embodiments, the sterilization time is 10 to 60 minutes.

[0043] In some embodiments, the sterilization time is 10-30 min, or it can be 20-40 min, 30-50 min, 40-60 min, or 10 min, 20 min, 30 min, 40 min, 50 min, or 60 min.

[0044] Another aspect of the present invention provides a method for culturing *Lactobacillus plantarum*, comprising: inoculating *Lactobacillus plantarum* into a culture medium containing the proliferation agent described above and culturing it.

[0045] In some embodiments, the mass percentage of the proliferator is 10-30 wt%, preferably 20 wt%, based on the total mass of the culture medium.

[0046] In some embodiments, the inoculation concentration of the plant milk stem strain is 1~10×10⁻⁶. 7 CFU / mL can also be 1×10 7 CFU / mL, 2×10 7 CFU / mL, 4×10 7 CFU / mL, 6×107 CFU / mL, 8×10 7 CFU / mL, 9×10 7 CFU / mL, 10×10 7 CFU / mL.

[0047] In some embodiments, the culture temperature is 36-38°C. More preferably, the culture temperature can be 36-37.5°C or 37-38°C. Most preferably, it is 37°C.

[0048] In some embodiments, the culture time is 7-20 hours. More preferably, the culture time can be 7-20 hours, 7-15 hours, 8-16 hours, or 15-20 hours. Most preferably, it is 16 hours.

[0049] Another aspect of the present invention provides the use of the proliferator as described above in increasing the viable number of *Lactobacillus plantarum* and / or promoting *Lactobacillus plantarum*-targeted tumor growth.

[0050] In this invention, *Lactiplantibacillus plantarum* ST-III, *Lactobacillus rhamnosus* GG, *Limosilactobacillus fermentum* B44, *Lactobacillus helveticus* LH99, *Lactobacillus delbrueckii* ssp. Bulgaricus LB340, *Lactococcus lactis* subsp. lactis II1403, and *Streptococcus thermophilus* St-body 3 were inoculated into the proliferation agent described above and cultured. It was found that only *Lactiplantibacillus plantarum* ST-III achieved a viable count of 1.8 × 10⁻⁶. 9 CFU / mL, while the viable count of other lactic acid bacteria was less than 5 × 10⁻⁶. 8 The CFU / mL figure indicates that the proliferator of this invention can increase the number of viable Lactobacillus plantarum.

[0051] This invention used CT26 cells to construct a mouse model of colon cancer. It was found that after feeding the mouse with a proliferator and simultaneously administering *Lactobacillus plantarum* ST-III via gavage, *Lactobacillus plantarum* could specifically colonize colon cancer tissue, and the viable bacterial count could reach 1×10⁻⁶.7 A CFU / g or higher indicates that the proliferator of the present invention can promote the targeted tumor growth of Lactobacillus plantarum.

[0052] Another aspect of the present invention provides a composition comprising the aforementioned proliferator and *Lactobacillus plantarum*.

[0053] In some embodiments, the *Lactobacillus plantarum* is selected from *Lactobacillus plantarum* ST-III.

[0054] In some embodiments, the *Lactobacillus plantarum* strain is obtained by inoculating *Lactobacillus plantarum* strains onto the MRS liquid medium (purchased from Merck Co., Germany). Inoculation with *Lactobacillus plantarum* is a procedure well known to those skilled in the art, for example, by inoculating the appropriate amount required for the experiment under suitable culture conditions.

[0055] In some specific embodiments, the inoculum concentration of *Eriocaulon buergerianum* strain is 0.1–200 × 10⁻⁶. 6 CFU / g. More preferably, the inoculum concentration of *Lactobacillus plantarum* can be 1~200×10⁻⁶. 6 CFU / g, or 10×10 6 CFU / g, 15×10 6 CFU / g, 20×10 6 CFU / g, 25×10 6 CFU / g, 30×10 6 CFU / g, 35×10 6 CFU / g, 40×10 6 CFU / g, 45×10 6 CFU / g, 50×10 6 CFU / g, 55×10 6 CFU / g, 60×10 6 CFU / g, 65×10 6 CFU / g, 70×10 6 CFU / g, 75×10 6 CFU / g, 80×10 6 CFU / g, 85×10 6 CFU / g, 90×10 6 CFU / g, 95×10 6 CFU / g, 100×10 6 CFU / g. Preferably, it is 1×10⁻⁶. 6 CFU / g.

[0056] In some specific embodiments, the culture temperature is 36~38℃. More preferably, the culture temperature can be 36~37.5℃ or 37~38℃. Most preferably, it is 37℃.

[0057] In some specific embodiments, the culture time is 7-40 hours. More preferably, the culture time can be 7-20 hours, 7-15 hours, 8-16 hours, 15-25 hours, 20-35 hours, or 30-40 hours. Most preferably, it is 32 hours.

[0058] In one specific embodiment, a single colony of *Lactobacillus plantarum* was inoculated into 5 mL of MRS liquid medium and cultured in an anaerobic incubator at 37°C for 16 h. Then, it was inoculated into 150 mL of MRS liquid medium at a 3% (v / v) inoculation rate and cultured at 37°C for another 16 h. The bacterial cells were obtained by solid-liquid separation and resuspended in PBS.

[0059] In some embodiments, the viable count of *Lactobacillus plantarum* in the composition is not less than 1 × 10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g, for example 1×10 8 CFU / g, 1×10 9 CFU / g, 2×10 9 CFU / g, 5×10 9 CFU / g, 8×10 9 CFU / g, 1×10 10 CFU / g, 5×10 10 CFU / g, 1×10 11 CFU / g, etc.

[0060] In some embodiments, the composition further includes a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable means that when the drug is appropriately administered to animals or humans, it does not produce adverse, allergic, or other adverse reactions. A "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, i.e., miscible with it without significantly reducing the drug's effectiveness under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to aid in the stability of the formulation or to contribute to its activity or bioavailability, or to produce an acceptable taste or aroma when taken orally.

[0061] In some embodiments, the composition is one or more of the following: patches, solutions, injections, sprays, nasal drops, aerosols, powders, tablets, capsules, and granules. The pharmaceutical compositions in all of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0062] In some embodiments, the pharmaceutical composition may be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, via injection, spray, nasal drops, eye drops, infiltration, absorption, or physical or chemical mediated methods; or it may be introduced into the body after being mixed with or encapsulated with other substances. The composition may also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.

[0063] In some embodiments, the proliferator is a powder administered orally; the *Lactobacillus plantarum* is a solution administered by gavage.

[0064] Another aspect of the present invention provides the use of the proliferator or composition as described above in at least one of the following:

[0065] 1) To assist in inhibiting tumor growth in vitro;

[0066] 2) Preparation of anti-tumor drugs.

[0067] In some embodiments, the tumor is selected from one or more of the following: bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, or thyroid cancer. Preferably, it is colon cancer.

[0068] The present invention further provides a method for treating tumors, comprising: administering an effective amount of the proliferator or the composition described above to a target subject.

[0069] The target organism is a mammal, including but not limited to humans, primates, livestock, pets, laboratory test animals, or captured wild animals. Primates are preferred. Humans are the most preferred target organism. The target organism can be a cancer patient or an individual seeking cancer prevention. The proliferator or the composition can be administered to the target organism before, during, or after cancer treatment.

[0070] This invention used CT26 cells to construct a mouse model of colon cancer and found that after feeding a proliferator and simultaneously administering *Lactobacillus plantarum* via gavage, *Lactobacillus plantarum* could specifically colonize colon cancer tissue, and the viable bacterial count could reach 1×10⁻⁶. 7 CFU / g or higher. The proliferation agent of this invention successfully solves the problem of insufficient colonization of *Lactobacillus plantarum* in the hypoxic microenvironment of tumors.

[0071] Further tumor volume data showed that the proliferator of this invention could also significantly inhibit tumor growth. Compared with the PBS group, the average tumor volume of mice decreased by 94.0% when administered *Lactobacillus plantarum* by gavage and fed with a diet containing the proliferator. Compared with the PBS group, the average tumor volume of mice decreased by 75.0% when administered *Lactobacillus plantarum* by gavage alone; the average tumor volume of mice decreased by 2.0% when administered PBS by gavage and fed with a diet containing the proliferator. This demonstrates that the proliferator not only increases the viable count of *Lactobacillus plantarum*, but also synergistically enhances the therapeutic effect of *Lactobacillus plantarum* by strengthening the targeted colonization ability of the strain.

[0072] Therefore, the proliferator of this invention regulates the colonization of *Lactobacillus plantarum* in specific lesions (such as tumors and sites of intestinal inflammation) in vivo by oral administration or feeding, laying a solid foundation for the development of next-generation in vivo biopharmaceuticals, tumor-targeted delivery systems, and microecological regulation therapies, and has broad scientific value and market transformation potential.

[0073] To make the inventive objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. Those skilled in the art can easily understand other advantages and effects of this invention from the content disclosed in this specification.

[0074] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0075] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0076] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0077] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0078] Lactiplantibacillus plantarum ST-III strain (Bright Dairy Industry Co., Ltd.), Lactobacillus rhamnosus GG strain (Chr. Hansen, Denmark), Limosilactobacillus fermentum B44 strain (Bright Dairy Industry Co., Ltd.), Lactobacillus helveticus strain LH99 strain (Bright Dairy Industry Co., Ltd.), Lactobacillus delbrueckii subsp. bulgaricus LB340 strain (Chr. Hansen, Denmark), Lactococcus lactis subsp. lactis II 1403 strain (Chr. Hansen, Denmark), Streptococcus thermophilus St-body 3 strain (Chr. Hansen, Denmark), soybean peptone (Shanghai Sangon Biotech Co., Ltd., A600214).

[0079] In the following embodiments of this application, the mouse feed was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. (XTI01WC-008) and consisted of: corn, wheat, imported fish meal, chicken meal, soybean meal, soybean oil, sodium chloride, limestone powder, dicalcium phosphate, choline chloride, methionine, vitamin A, vitamin D3, dL-α-tocopherol acetate, sodium menadione bisulfite, thiamine nitrate, riboflavin, pyridoxine hydrochloride, cyanocobalamin, nicotinamide, D-calcium pantothenate, folic acid, D-biotin, basic copper chloride, ferrous sulfate, manganese sulfate, yeast selenium, etc.

[0080] In the following embodiments of this application, the oats are oat flour with a particle size of 200 μm; the tea leaves and chickpeas are commercially available dried tea leaves and dried chickpeas, ground into powder with a particle size of 200 μm.

[0081] Examples 1-4: Proliferating Agents and Their Preparation Methods

[0082] The formulations of the proliferation agents in Examples 1-4 are shown in Table 1. The proliferation agents obtained in each example are labeled as proliferation agent 1, proliferation agent 2, proliferation agent 3, and proliferation agent 4, respectively.

[0083] Preparation method of the growth agent: Mix all raw materials and water according to the formula, homogenize, cook at 100℃ for 1 hour, sterilize at 121℃, and cool to obtain the growth agent, which is a liquid concentrated growth agent (labeled as 5× growth agent). Based on the total mass of the growth agent, the concentration of manganese chloride is 0.005 wt%, and the mass fraction of Mn is 0.0022 wt%; based on the total mass of the growth agent, the concentration of oats is 10 wt%, and the mass fraction of Mn is 0.0004-0.0005 wt%; based on the total mass of the growth agent, the concentration of tea is 10 wt%, and the mass fraction of Mn is 0.003-0.005 wt%; based on the total mass of the growth agent, the concentration of chickpeas is 10 wt%, and the mass fraction of Mn is 0.00015-0.0002 wt%.

[0084] Dilute to 1× proliferation agent before application.

[0085] Meanwhile, control groups 1-3 were established. Control group 1 did not contain a manganese source, control group 2 did not contain glucose, and control group 3 did not contain soybean peptone. The formulas are shown in Table 1, and the preparation method is the same as above, thus obtaining the control group growth agent.

[0086] Table 1 (wt%)

[0087]

[0088] — indicates that no addition has been made.

[0089] By examining the proliferation of *Lactobacillus plantarum* in Examples 1-4 and Control Groups 1-3, agents that can promote the proliferation of viable *Lactobacillus plantarum* were screened.

[0090] Since the composition of bovine serum is close to that of the tumor microenvironment, a culture medium containing 80% bovine serum was used to simulate the tumor microenvironment to investigate the proliferation of *Lactobacillus plantarum* in the culture medium.

[0091] The culture medium contains: 80 wt% bovine serum and 20 wt% diluted to 1× proliferation agent 1 (1× proliferation agent 1 is obtained by diluting the liquid concentrated proliferation agent obtained in Example 1).

[0092] Lactiplantibacillus plantarum ST-III was inoculated at a concentration of 1×10⁻⁶. 7 CFU / mL was inoculated into the culture media prepared in Examples 1-4 and Control Groups 1-3, and then incubated at 37°C for 16 h. After incubation, the media were diluted and plated on MRS agar plates, and incubated at 37°C for 2-3 days for counting. The results are shown in Table 2.

[0093] Table 2

[0094] Group MRS viable cell count (CFU / mL) Example 1 <![CDATA[1.5×10 9 ]]> Example 2 <![CDATA[1.3×10 9 ]]> Example 3 <![CDATA[1.2×10 9 ]]> Example 4 <![CDATA[1.4×10 9 ]]> Control group 1 <![CDATA[1.7×10 7 ]]> Control group 2 <![CDATA[1.1×10 7 <!-- 7 -->]]> Control group 3 <![CDATA[1.0×10 7 ]]>

[0095] As shown in Table 2, Lactiplantibacillus plantarum ST-Ⅲ survived well in the proliferation agents prepared in Examples 1-4, and its viable bacterial count was twice that of the control group, indicating that soybean peptone, glucose and manganese source synergistically promoted the proliferation of Lactiplantibacillus plantarum in this invention.

[0096] Example 5: Specificity of the proliferation agent against different strains

[0097] In Example 5, the specificity of the proliferation agent of the present invention for different strains was examined, including the following:

[0098] 5.1 Investigation of different bacteria in growth promoters

[0099] Using the culture medium obtained in Example 1 diluted to 1× proliferation agent, and then inoculating at a concentration of 1×10⁻⁶... 7 The proliferation effect of different lactic acid bacteria strains in the proliferation agent was investigated by measuring the viable cell count. After inoculation, the culture was carried out at 37℃ for 16 h. The inoculated strains were then diluted and plated onto MRS or M17 agar plates (Lactococcus lactis subsp. lactis and Streptococcus thermophilus strains were plated on M17 plates, and other strains were plated on MRS plates). The plates were incubated at 37℃ for 2-3 days for counting. The results are shown below. Figure 1 .

[0100] The different lactic acid bacteria were Lactiplantibacillus plantarum ST-III strain, Lactobacillus rhamnosus GG strain, Limosilactobacillus fermentum B44 strain, Lactobacillus helveticus strain LH99 strain, Lactobacillus delbrueckii ssp. Bulgaricus LB340 strain, Lactococcus lactis subsp. lactis II1403 strain, and Streptococcus thermophilus St-body 3 strain.

[0101] from Figure 1 It can be seen that only Lactiplantibacillus plantarum ST-Ⅲ grew well in the proliferation agent, with a viable count reaching 1.8 × 10⁻⁶. 9 CFU / mL, while the viable count of other lactic acid bacteria was less than 5 × 10⁻⁶.8 CFU / mL.

[0102] 5.2 Effects of different soybean peptones, glucose, and manganese chloride on viable bacterial counts

[0103] The concentrations of soybean peptone, glucose, and manganese chloride in the growth promoter obtained in Example 1 were adjusted, and then diluted to 1× growth promoter. A culture medium was formed with 80 wt% bovine serum, and then inoculated at a concentration of 1×10⁻⁶. 7 Lactiplantibacillus plantarum ST-III at CFU / mL was incubated at 37℃ for 16 h. After incubation, the culture was diluted and plated on MRS agar plates, and incubated at 37℃ for 2-3 days for counting. The results are shown in Table 3.

[0104] Table 3

[0105] Soy peptone (wt%) Glucose (wt%) <![CDATA[MnCl2(wt%)]]> viable bacteria count (CFU / mL) 0.01 0.04 0.002 1.00E+07 0.8 0.8 0.00015 1.26E+09 0.4 0.1 0.00015 5.65E+08 0.4 0.4 0.00015 7.60E+08 0.04 0.1 0.0000015 2.05E+08 0.04 0.1 0.000015 2.65E+08 0.1 0.1 0.0000015 3.00E+08 0.1 0.1 0.000015 4.85E+08

[0106] Table 3 shows that the viable count of *Lactobacillus plantarum* can be controlled at 1×10⁻⁶ by adjusting the concentration of each component of the growth promoter. 7 -1×10 9 CFU / mL.

[0107] Example 6: Investigation of Lactobacillus plantarum targeting tumor growth

[0108] Construction of a mouse model of colon cancer: 1×10 6 CT26 cells were resuspended in 100 µL PBS and subcutaneously injected into the right back of 6-8 week old BALB / c mice; tumors were palpable after 7 days, and the tumor volume reached 100-300 mm² after 10-14 days. 3 A tumor-bearing mouse model of colon cancer was successfully constructed. A total of 6 mice were used.

[0109] The preparation method of *Lactobacillus plantarum* ST-III solution for gavage is as follows: *Lactobacillus plantarum* ST-III is inoculated into MRS liquid culture overnight, centrifuged, counted, and then diluted. Details are as follows:

[0110] Single colonies of *Lactobacillus plantarum* ST-III were selected using an inoculation loop and placed in a 10 mL sterile tube containing 5 mL of MRS liquid medium (purchased from Merck Co., Germany). The culture was incubated anaerobicly at 37°C for 16 h. Then, a 3% (v / v) inoculation was performed into 150 mL of MRS liquid medium, and the culture was continued at 37°C for another 16 h. The cells were obtained by centrifugation at 6000g for 5 minutes, washed twice with PBS, and resuspended in PBS to obtain the bacterial suspension. The suspension was serially diluted tenfold, and the appropriate dilution was plated onto MRS agar plates. The plates were then anaerobically incubated at 37°C for 2-3 days, and the viable cell count was determined. The bacterial suspension was then prepared with PBS to a concentration of 5 × 10⁶ cells / mL. 8CFU / mL (200 μL per mouse by gavage, which is 1 × 10⁻⁶) 8 CFU / animal) to obtain Lactobacillus plantarum ST-III solution.

[0111] The 5× growth promoter obtained in Example 1 was mixed with the feed at a weight ratio of 1:4 and then dried at 60°C to obtain a feed with a growth promoter content of 20wt% (abbreviated as experimental feed).

[0112] Each mouse was fed the experimental group diet and simultaneously administered 200 μL of *Lactobacillus plantarum* ST-III solution (1×10⁻⁶) by gavage every 3 days. 8 CFU / mouse). Mice were allowed free access to feed without food restrictions.

[0113] Two weeks later, the mice were sacrificed, and tissues such as tumor, heart, liver, spleen, lung, and kidney were removed, homogenized, diluted, and counted.

[0114] Results of viable bacterial counts in each tissue are shown below. Figure 2 .

[0115] from Figure 2 It is known that the number of viable bacteria in colon cancer tissue can reach 1×10⁻⁶. 7 The CFU / g level was above 1, while no viable bacteria were detected in other tissues. This indicates that the proliferation agent of the present invention can specifically promote the colonization and growth of *Lactobacillus plantarum* within the tumor.

[0116] Example 7: Oral administration of a proliferator inhibits antitumor activity in mice

[0117] 1×10 6 CT26 cells were resuspended in 100 µL PBS and subcutaneously injected into the right back of 6-8 week old BALB / c mice; tumors were palpable after 7 days, and the tumor volume reached 100–300 mm² after 10-14 days. 3 A successful tumor-bearing mouse model of colon cancer was constructed. The mice were randomly divided into 4 groups of 6 mice each.

[0118] (1) PBS group: 200 μL of PBS was administered by gavage every 3 days, and the mice were fed a diet without any added substances. The mice were not restricted in their food intake.

[0119] (2) PBS + proliferation agent group: 200 μL of PBS was administered by gavage every 3 days, and the mice were fed experimental feed (the experimental feed was the same as in Example 6). The mice were not restricted in their food intake.

[0120] (3) *Lactobacillus plantarum* group: 200 μL of *Lactobacillus plantarum* ST-III solution (1×10⁻⁶) was administered by gavage every 3 days. 8 CFU / animal, fed with feed without any added substances.

[0121] (4) *Lactobacillus plantarum* + proliferation agent group: 200 μL of *Lactobacillus plantarum* ST-III solution (1×10⁻⁶) was administered by gavage every 3 days. 8 CFU / mouse), fed with experimental feed (same as in Example 6), mice were not restricted in their food intake.

[0122] The mice were euthanized after 28 days, and tumor tissue was collected.

[0123] See results Figure 3 Table 4.

[0124] Table 4

[0125]

[0126] from Figure 3 As shown in Table 4, only the group that received oral administration of *Lactobacillus plantarum* along with a growth promoter not only promoted the growth of *Lactobacillus plantarum* in the tumor and thus inhibited tumor growth, but also showed a 94.0% decrease in average tumor volume compared to the PBS group. Compared to the PBS group, the group that received oral administration of *Lactobacillus plantarum* alone showed a 75.0% decrease in average tumor volume; and the group that received oral administration of PBS along with a growth promoter showed a 2.0% decrease in average tumor volume.

[0127] Comparative Example 1

[0128] The difference between Comparative Example 1 and Example 1 is that tryptone (Shanghai Sangon Biotech Co., Ltd., A650217) was used instead of soybean peptone; otherwise, the process was the same as in Example 1. Furthermore, a culture medium was prepared by mixing the medium with 80 wt% bovine serum, and *Lactiplantibacillus plantarum* ST-III was inoculated. Proliferation was then assessed, as in Example 1.

[0129] Comparative Example 2

[0130] The difference between Comparative Example 2 and Example 1 is that fish peptone (Shanghai Guoyao Reagent Group, 10014963) was used instead of soybean peptone. Furthermore, it was mixed with 80wt% bovine serum to form a culture medium, inoculated with *Lactiplantibacillus plantarum* ST-III, and the proliferation was observed, as in Example 1.

[0131] The results are shown in Table 5.

[0132] Table 5

[0133] Group MRS viable cell count (CFU / mL) Example 1 <![CDATA[1.4×10 9 ]]> Comparative Example 1 <![CDATA[1.9×10 8 ]]> Comparative Example 2 <![CDATA[1.5×10 8 ]]>

[0134] As shown in Table 5, when tryptone and fish peptone were used as nitrogen sources, the proliferation effect of Lactiplantibacillus plantarum ST-Ⅲ was poor, with a difference of one order of magnitude in the number of viable bacteria.

[0135] In summary, the proliferator of this invention not only promotes the proliferation of *Lactobacillus plantarum* in vitro, but also promotes the growth and proliferation of *Lactobacillus plantarum* in tumor tissues after administration to animals. In addition, it can regulate the colonization of *Lactobacillus plantarum* in specific lesions (such as tumors and sites of intestinal inflammation) in vivo, laying a solid foundation for the development of next-generation in vivo biopharmaceuticals, tumor-targeted delivery systems, and microecological regulation therapies, and has broad scientific value and market transformation potential.

[0136] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A plant lactobacillus proliferation agent, characterized in that, It includes the following components by mass fraction: 0.02-5 wt% soybean polypeptide, 0.01-5 wt% glucose and manganese source; based on the total mass of the proliferator, the mass fraction of Mn element in the manganese source is 0.0000001-0.005 wt%.

2. The proliferation agent as described in claim 1, characterized in that, The molecular weight of the peptide fragments of the soybean peptone is 200~5000 Da; And / , the manganese source is selected from manganese salts or manganese-containing foods.

3. The proliferation agent as described in claim 2, characterized in that, The manganese salt is selected from one or more of manganese chloride, manganese sulfate, and manganese nitrate; And / or, the manganese-containing food is selected from one or more of oats, tea and chickpeas.

4. The proliferation agent as described in claim 3, characterized in that, The oats are selected from one or both of oat flour and oat flakes; And / or, the oat grain size is 150~270μm; And / or, the tea is tea powder; preferably, the particle size of the tea powder is 150~270μm; And / or, the chickpeas are chickpea flour; preferably, the particle size of the chickpea flour is 150~270μm.

5. A method for culturing *Lactobacillus plantarum*, characterized in that, include: Lactobacillus plantarum was inoculated into a culture medium containing the proliferation agent as described in any one of claims 1 to 4 and cultured.

6. Use of the proliferator as described in any one of claims 1 to 4 in increasing the viable count of *Lactobacillus plantarum* and / or promoting *Lactobacillus plantarum*-targeted tumor growth.

7. A composition, characterized in that, Includes the proliferator as described in any one of claims 1 to 4 and Lactobacillus plantarum.

8. The composition according to claim 7, characterized in that, The plant lactobacillus was selected from Lactobacillus plantarum ST-III; And / or, may also include pharmaceutically acceptable carriers or excipients.

9. Use of the proliferating agent according to claims 1-4 and / or the composition according to claim 7 or 8 in at least one of the following: 1) To assist in inhibiting tumor growth in vitro; 2) Preparation of anti-tumor drugs.

10. The use as described in claim 9, characterized in that, The tumor is selected from one or more of the following: bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

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