Lactobacillus plantarum L19 and application thereof
By using Lactobacillus plantarum L19, the problems of low safety, limited efficacy and high cost of existing liver injury treatment plans were solved, and effective relief and functional improvement of liver injury were achieved.
Patent Information
- Application Number
- CN202510380436.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-07-18
Smart Images
Figure CN120330083A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbiology, and specifically, to a Lactobacillus plantarum L19 and its application. Background Art
[0002] The liver is an important metabolic and detoxifying organ of the human body, undertaking various physiological functions such as synthesis, decomposition, storage, and excretion. However, in modern society, the incidence of liver diseases has been increasing year by year, and diseases such as hepatitis, fatty liver, liver cirrhosis, liver fibrosis, and liver cancer have become important threats to global public health. The inducing factors of liver injury are complex and diverse, including environmental toxins (such as heavy metals, pesticide residues), drug abuse (such as antibiotics, antipyretics and analgesics), excessive alcohol intake, viral infections (such as hepatitis B, hepatitis C), and metabolic disorders, etc. These factors lead to hepatocyte injury and even organ failure through mechanisms such as inducing oxidative stress, inflammatory response, and apoptosis.
[0003] Currently, the clinical treatment methods for liver injury mainly include hepatoprotective drugs (such as silymarin, glutathione), anti-inflammatory drugs (such as glucocorticoids), and artificial liver support systems. However, the existing treatment regimens have obvious deficiencies: low safety of long-term medication: some drugs may increase the metabolic burden of the liver and even cause secondary injury; limited efficacy: it is difficult to reverse advanced liver fibrosis or liver cirrhosis; high cost: such as the high cost of artificial liver treatment and dependence on medical resources.
[0004] In recent years, microbial therapy has received extensive attention due to its advantages such as high safety and low side effects. Therefore, developing a new microbial preparation that can directly act on the liver and repair damage through multiple mechanisms synergistically has important clinical value and social significance. Summary of the Invention
[0005] The purpose of the present invention is to provide a Lactobacillus plantarum L19 and its application, which can treat and / or prevent liver injury.
[0006] To achieve the above purpose, the present invention adopts the following technical solutions:
[0007] In the first aspect, the present invention provides a Lactobacillus plantarum L19, whose preservation number is CCTCC NO: M20242267.
[0008] In the second aspect, the present invention provides the application of the above-mentioned Lactobacillus plantarum L19 in the preparation of preventing and / or treating liver diseases.
[0009] The term "treatment" (also referred to as "treat" or "treating") refers to any administration of a therapeutic agent according to a therapeutic regimen that achieves the desired effect, namely, partially or completely alleviating, ameliorating, relieving, suppressing, delaying the onset, reducing the severity, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition (such as improving liver function, reducing liver histopathological changes, reducing inflammatory factors); in some embodiments, the administration of the therapeutic agent according to the therapeutic regimen is associated with the achievement of the desired effect. Such treatment can be directed to subjects who do not exhibit the relevant disease, disorder, and / or condition and / or to subjects who exhibit only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment can be directed to subjects who exhibit one or more identified signs of the relevant disease, disorder, and / or condition. In some embodiments, the treatment can be directed to subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, the treatment can be directed to subjects known to have one or more risk factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition.
[0010] In the present invention, the liver diseases include hepatitis, fatty liver, liver cirrhosis, liver fibrosis, hepatocellular carcinoma, drug-induced liver injury, alcoholic liver injury, and liver injury caused by the environment.
[0011] In some specific embodiments of the present invention, the product is a product for regulating the content of ALT and / or AST; further, the product is a product for reducing the content of ALT and / or AST.
[0012] In some specific embodiments of the present invention, the product is a product for reducing at least one of the inflammatory factors TNF-α, IL-1β, IL-6, and MPO.
[0013] In some specific embodiments of the present invention, the viable cell concentration of Lactobacillus plantarum L19 in the product is not less than 1×10 7 CFU / mL.
[0014] In a third aspect, the present invention provides a product for preventing and / or treating liver injury and related diseases, and the active ingredient thereof contains Lactobacillus plantarum; the Lactobacillus plantarum is the above-mentioned Lactobacillus plantarum L19.
[0015] In the present invention, the product includes, but is not limited to, food, health products, drugs, or daily necessities.
[0016] The term "drug" covers drugs for both humans and animals in human medicine and veterinary medicine, and also covers drugs for incorporation into animal feed (such as livestock feed and / or pet food). In addition, the term "drug" as used herein means any substance that provides a therapeutic, prophylactic, and / or beneficial effect. The term "drug" as used herein is not necessarily limited to substances that require Marketing Approval, but includes substances that can be used in cosmetics, health products, food (including, for example, feed and beverages), probiotic cultures, and dietary supplements.
[0017] In a fourth aspect, the present invention provides a fermenting agent for relieving liver injury, which comprises the Lactobacillus plantarum L19 described above.
[0018] In a fifth aspect, the present invention provides a bacterial powder, which comprises the Lactobacillus plantarum L19 described above.
[0019] Advantageous effects compared with the prior art:
[0020] (1) Relieving liver injury: Lactobacillus plantarum L19 can significantly relieve liver injury, improve liver function, and reduce liver histopathological changes such as vacuolization, inflammatory cell accumulation, and hepatic sinusoid hemorrhage;
[0021] (2) Reducing the contents of serum ALT and AST: It is proved by experiments that Lactobacillus plantarum L19 can significantly reduce the contents of ALT and AST in the serum of liver injury mice, indicating its protective effect on the liver;
[0022] (3) Anti-inflammatory effect: Lactobacillus plantarum L19 can reduce the release of inflammatory factors (such as TNF-α, IL-1β, IL-6), reduce liver inflammatory response, and thus alleviate liver injury;
[0023] (4) The Lactobacillus plantarum L19 provided by the present invention can be used to prepare food, health products, drugs, or daily necessities, and has broad application prospects, especially in the prevention and / or treatment of liver injury and related diseases.
[0024] Strain preservation information:
[0025] Lactobacillus plantarum L19 was deposited at the China Center for Type Culture Collection on October 21, 2024. The deposit number of this strain is: CCTCC NO: M 20242267, address: Wuhan, China; the deposit name is Lactobacillus plantarum L19. Description of the drawings
[0026] Figure 1 Changes in serum ALT, AST and liver tissues of liver injury mice;
[0027] Figure 2 Effect of Lactobacillus plantarum L19 on food intake, water intake and body weight of liver injury mice;
[0028] Figure 3 Effect of Lactobacillus plantarum L19 on liver organ index and histopathological changes of liver injury mice;
[0029] Figure 4 Effect of Lactobacillus plantarum L19 on serum ALT and AST levels of liver injury mice;
[0030] Figure 5 Effect of Lactobacillus plantarum L19 on anti-inflammatory ability of liver in liver injury mice;
[0031] Figure 6 Figure of hepatocyte apoptosis. Detailed implementation manners
[0032] In order to enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood by those skilled in the art that this should not be construed as a limitation on the scope of the claims of the present invention. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. At the same time, it should be noted that the reagents or instruments in the present invention are all conventional biochemical reagents or instruments without special instructions and can be obtained through commercial purchase.
[0033] Strain preservation information:
[0034] Lactobacillus plantarum L19 was deposited at the China Center for Type Culture Collection on October 21, 2024. The deposit number of this strain is: CCTCC NO: M 20242267, address: Wuhan, China; the deposit name is Lactobacillus plantarum L19.
[0035] Example 1: Effect of Lactobacillus plantarum L19 on alleviating basic symptoms of liver injury mice
[0036] 1 Experimental method
[0037] 1.1 Establishment of mouse liver injury model
[0038] Twenty-five 7-week-old C57 mice were adaptively fed for one week. After that, they were placed in a constant temperature incubator at 40 ± 0.5 °C from 9:00 to 11:00 every morning for 2 hours for 9 consecutive days. Four hours after the end, 3 mice were randomly selected at different time points (1, 3, 5, 7, 9 days) for euthanasia. Before euthanasia, mouse serum was collected by orbital blood sampling for detecting the activities of ALT and AST. After euthanasia, mouse liver tissues were collected, fixed with 4% formaldehyde solution and then subjected to HE staining. The modeling conditions were determined by comprehensively analyzing the results of serum ALT, AST and HE staining.
[0039] 1.2 Intervention of Lactobacillus plantarum L19 on mouse liver injury model
[0040] After determining the model conditions, the mouse liver injury model was intervened with Lactobacillus plantarum L19: Thirty-six male C57BL / 6 mice were adaptively fed for one week and then randomly divided into 6 groups of 6 mice each. They were the blank group (Control), the model group (Model / M), the Lactobacillus plantarum intervention groups (high dose (1×109 CFU / mL, LP-H), medium dose (1×10 8 CFU / mL, LP-M), low dose (1×10 7 CFU / mL)), and the positive drug N-acetylcysteine control group (NAC). Each group of mice was given 200 μL of the corresponding drug by gavage at a fixed time every day: PBS (Control, Model / M), the Lactobacillus plantarum L19 bacterial suspension at the corresponding concentration (LP-H, LP-M, LP-L), and N-acetylcysteine (NAC) at a concentration of 150 mg / kg. After 3 days of gavage, the treatment was carried out according to 1.1, and the gavage treatment was continued during this period.
[0041] 2 Result analysis
[0042] 2.1 Establishment of mouse liver injury model
[0043] As Figure 1 shown; the results showed that: the ALT value in mouse serum gradually increased from the 3rd day, showed a highly significant difference from the control group at the 7th day, and there was no significant difference between the 9th day and the 7th day; the AST value started to increase from the 3rd day, there was no significant difference between the 5th day and the 3rd day, showed a highly significant difference from the control group at the 7th day, which was consistent with the change of the ALT value; the AST value on the 9th day was slightly lower than that on the 7th day ( Figure 1 -A, B).
[0044] The HE staining results showed that pathological changes occurred in the liver tissue; on the 1st and 3rd days, the main manifestations of tissue lesions were inflammatory cell infiltration. Central vein congestion, hepatic cord congestion, and vacuolar degeneration began to appear on the 5th day. Obvious lesions occurred in the liver tissue on the 7th and 9th days, with an increase in vacuolization, and fatty degeneration and hepatic sinus congestion were also observed. Figure 1 -C).
[0045] Based on the comprehensive detection of serum ALT and AST and the HE staining results of the liver, in this invention, the mice were placed in a constant temperature incubator at 40 ± 0.5 °C from 9:00 to 11:00 every morning for 2 hours, continuously for 7 days as the modeling conditions for mouse liver injury.
[0046] 2.2 Intervention of Lactobacillus plantarum L19 on the mouse liver injury model
[0047] During the intervention of the model with L19, the food intake, water intake, and body weight changes of each group of mice were weighed regularly every day, as Figure 2 shown: The intake of Lactobacillus plantarum L19 increased the food intake of mice with liver injury and decreased their water intake. The body weight of the model mice decreased significantly. After intervention with Lactobacillus plantarum, although it could not significantly reduce the decrease in body weight, it could slow down the degree of body weight decrease, and was better than the positive control drug NAC. It is shown that Lactobacillus plantarum L19 of this invention has the basic symptoms of alleviating the reduced food intake, increased water intake, and decreased body weight of mice with liver injury.
[0048] Example 2: Effects of Lactobacillus plantarum L19 on the liver organ index and histopathological changes of mice with liver injury
[0049] 1. Experimental method
[0050] The establishment of the mouse liver injury model and the grouping and treatment of mice were the same as in Example 1.
[0051] After the treatment ended on the 7th day, the body weights of each group of mice were weighed. After the mice were euthanized, the livers were collected, the weights of the livers were weighed, and the liver organ indices of each group of mice were calculated: (liver weight / body weight) × 100%; then, part of the liver tissue was taken and fixed in 4% formaldehyde solution for liver tissue pathological analysis.
[0052] 2. Result analysis
[0053] The experimental results are as Figure 3 shown. Compared with the Control group, the liver organ index of the Model group mice decreased extremely significantly, while after 1×10 9After the intervention of Lactobacillus plantarum L19 at CFU / mL, the liver organ index of mice was significantly increased, while the positive control drug NAC had no obvious effect on the liver organ index of liver injury mice. The results of liver HE staining showed that after the treatment of mice, vacuolization, inflammatory cell accumulation and hepatic sinus hemorrhage appeared in the liver tissue. After the intervention of Lactobacillus plantarum L19 at different concentrations and the positive drug NAC, the pathological changes of the liver tissue of mice were effectively alleviated.
[0054] Example 3: Effect of Lactobacillus plantarum L19 on the contents of serum ALT and AST in liver injury mice
[0055] 1. Experimental method
[0056] The establishment of the mouse liver injury model and the grouping and treatment of mice were the same as in Example 1.
[0057] After the treatment ended on the 7th day, the blood of each group of mice was collected by orbital blood collection to obtain mouse serum. The contents of ALT and AST in the mouse serum were detected using ALT and AST detection kits (Nanjing Jiancheng).
[0058] 2. Result analysis
[0059] The test results were as Figure 4 shown. After the intervention of high-dose and medium-dose Lactobacillus plantarum L19, the serum ALT level of liver injury mice could be extremely significantly reduced, except for the low-dose group; while high-dose, medium-dose and low-dose Lactobacillus plantarum L19 could all significantly reduce the serum AST content of liver injury mice.
[0060] Example 4: Effect of Lactobacillus plantarum L19 on improving the anti-inflammatory ability of the liver in liver injury mice
[0061] 1. Experimental method
[0062] The establishment of the mouse liver injury model and the grouping and treatment of mice were the same as in Example 1.
[0063] MPO is an important index reflecting the body's inflammatory response. After the treatment ended on the 7th day, an appropriate amount of liver tissue of each group of mice was collected, added with normal saline to make a 10% liver tissue homogenate, centrifuged at 5000 r / min for 15 min, and the supernatant was taken to determine the protein concentration by the BCA method. The content detection kit of Nanjing Jiancheng was used to detect the level of liver inflammation in mice.
[0064] In addition, RT-qPCR method was also used to detect the contents of liver inflammatory factors IL-6, IL-1β, and TNF-α.
[0065] 2. Result analysis
[0066] The results were shown as Figure 5: The contents of MPO and inflammatory factors in the livers of model mice increased significantly, but were significantly reversed after intervention with Lactobacillus plantarum L19.
[0067] Example 5: Effect of Lactobacillus plantarum L19 on apoptosis of hepatocytes in mice with liver injury
[0068] 1. Experimental method
[0069] The establishment of the mouse liver injury model and the grouping and treatment of mice were the same as in Example 1.
[0070] The Tunel cell apoptosis detection kit can be used to explore the level of cell apoptosis, and apoptotic cells will be stained dark brown. Take the prepared paraffin sections of liver tissue, perform dewaxing and hydration treatment, incubate with the diluent, and then add to the Tunel working solution, and detect strictly according to the kit instructions. After staining, mount the slides and observe the apoptosis of cells with an optical microscope.
[0071] 2. Result analysis
[0072] The results of Tunel staining showed that the liver cells of the Model group mice were densely stained dark brown, and a large number of cells underwent apoptosis; after intervention with LP-19, the content of apoptotic cells decreased, and there was a certain concentration dependence with the increase in concentration, indicating that Lactobacillus plantarum L19 can reduce the apoptosis of hepatocytes during liver injury ( Figure 6 ).
[0073] Example 6: Preparation of a Lactobacillus plantarum L19 ferment starter with the efficacy of alleviating liver injury
[0074] Inoculate the original strain of Lactobacillus plantarum L19 into 11% skim milk (sterilized at 115 °C for 20 min), and culture at 37 °C for 18 - 24 h until curdling. Activate continuously for two generations as the mother ferment starter. Inoculate the mother ferment starter at an inoculation amount of 3% - 5% into 11% skim milk (sterilized at 115 °C for 20 min), and culture at 37 °C for 18 - 24 h until curdling. At this time, the viable cell count can reach 1×10 9 -10 10 CFU / mL, and thus the Lactobacillus plantarum L19 ferment starter is obtained.
[0075] Example 7: Preparation of bacterial powder using Lactobacillus plantarum L19 with the efficacy of alleviating liver injury
[0076] Use an inoculation loop to inoculate Lactobacillus plantarum L19 into 50mL MRS liquid culture medium, and place it in a 37℃ incubator for 18h. Then transfer 5% of the inoculation volume to a sterilized 250mL MRS liquid culture medium. Place it in a bacterial incubator at 37℃ and culture it for 24h. Centrifuge the resulting fermentation liquid (5000r / min, 10 minutes), then discard the supernatant and collect the bacterial precipitate. Rinse the bacteria twice with sterile phosphate buffer (PBS), 10 minutes each time, and the centrifugation speed is 5000r / min. The bacterial precipitate of Lactobacillus plantarum L19 can be obtained.
[0077] Weigh 4.1g of trehalose, 2.8g of sodium glutamate, and 8g of sucrose respectively and dissolve them in 10ml of 40℃ distilled water. Use 0.22u microporous filter membrane to filter and sterilize, then add them into 90ml sterile water to obtain solution 1 for later use. Dissolve 15g of skim milk powder in distilled water to obtain 100mL of 15% skim milk powder solution, sterilize at 110℃ to obtain solution II for later use. Mix solution 1 and solution II in a volume ratio of 1:1 to obtain a protective agent solution.
[0078] The prepared Lactobacillus plantarum L19 bacterial precipitate is fully mixed with the protective agent solution at a ratio of 1:5 (g / ml) to obtain a bacterial suspension. 5 ml of the bacterial suspension is divided into 10 ml sterile stoppered glass bottles, pre-frozen at -70°C, taken out after 2 hours and freeze-dried in a vacuum freeze dryer. The freeze-drying conditions are vacuum degree 5Pa, partition heating temperature 20°C, cold trap temperature -55°C, and freeze-dried for 30 hours before breaking to obtain Lactobacillus plantarum L19 bacterial powder.
[0079] It should be understood by those skilled in the art that the embodiments of the present invention described above and shown in the accompanying drawings are only examples and do not limit the present invention. The purpose of the present invention has been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and explained in the embodiments, and the embodiments of the present invention may be deformed or modified in any way without departing from the principles.
Claims
1. A Lactobacillus plantarum L19, characterized in that, The preservation number is CCTCC NO: M 20242267.
2. Use of Lactobacillus plantarum L19 according to claim 1 in the preparation of a product for preventing and / or treating liver diseases.
3. The application according to claim 2, wherein, The liver diseases include hepatitis, fatty liver, liver cirrhosis, liver fibrosis, hepatocellular carcinoma, drug-induced liver injury, alcoholic liver injury, and liver injury caused by the environment.
4. The application according to claim 2, wherein The product is a product for regulating the content of ALT and / or AST.
5. The application according to claim 2, wherein The product is a product for reducing the content of ALT and / or AST.
6. The application according to claim 2, wherein The product is a product for reducing at least one inflammatory factor of TNF-α, IL-1β, IL-6, and MPO.
7. The application according to claim 2, characterized in that, The viable cell concentration of Lactobacillus plantarum L19 in the said product is not less than 1×10 7 CFU / mL.
8. A product for preventing and / or treating liver injury and related diseases, characterized in that, Its active ingredient contains Lactobacillus plantarum; the Lactobacillus plantarum is Lactobacillus plantarum L19 according to claim 1.
9. A ferment for relieving liver injury, characterized in that, It contains Lactobacillus plantarum L19 according to claim 1.
10. A bacterial powder, characterized in that, It contains Lactobacillus plantarum L19 according to claim 1.