CN115725443A - Lactobacillus rhamnosus and application thereof - Google Patents
Lactobacillus rhamnosus and application thereof Download PDFInfo
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- CN115725443A CN115725443A CN202210919348.5A CN202210919348A CN115725443A CN 115725443 A CN115725443 A CN 115725443A CN 202210919348 A CN202210919348 A CN 202210919348A CN 115725443 A CN115725443 A CN 115725443A
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- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K35/74—Bacteria
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Abstract
The application discloses lactobacillus rhamnosus and application thereof, and research on mastitis prevention and treatment by taking a lactating sterile female mouse as an animal model shows that lactobacillus rhamnosus Probio-M9 can improve the immune response function of an organism, reduce the number of mammary inflammatory cells, relieve the mammary inflammation degree and have the effects of preventing and curing the mammary inflammation.
Description
The application is divisional application with the application number of 201911112479.7 and the application date of 2019.11.14, and the invention is named as lactobacillus rhamnosus for preventing and treating mastitis and application thereof.
Technical Field
The invention relates to the technical field of biology, and particularly relates to lactobacillus rhamnosus and application thereof.
Background
Mastitis is an inflammation of the mammary gland, usually caused by microbial infection of the mammary tissue, and lactation mastitis is a common disease of postpartum women, often resulting in the cessation of pure breast feeding. 25% of puerpera suffer from at least 1 mastitis, 4.0% -8.5% of puerpera suffer from repeated outbreaks, and the mastitis in lactation period is usually caused by pathogenic bacteria infection and milk stasis.
In the prior art, mastitis is treated by medicaments, so that medicament dependence and medicament resistance are easily caused.
Disclosure of Invention
The application aims to provide a strain of Lactobacillus rhamnosus Probio-M9 (Lactobacillus rhamnous Probio-M9).
The purpose of the application is realized by the following technical scheme:
the lactobacillus rhamnosus Probio-M9 is obtained by the following separation method:
a sample of the breast milk of healthy women in Chonghe Haoyet city is diluted moderately by sterile water, spread on a selective MRS solid culture medium and cultured for 72 hours in an anaerobic way at 37 ℃. Typical colonies are selected for gram stain microscopic selection, and then streaked on MRS solid medium for 2-3 times to obtain pure colonies. The combination of the physiological and biochemical identification and 16s rRNA molecules is identified as 1 strain of lactobacillus rhamnosus.
The strain is deposited by an agency approved for patent filing, and is deposited in the China general microbiological culture Collection center, wherein the microorganism preservation numbers are as follows: CGMCC No.18639, classification name: lactobacillus rhamnosus, deposit time: year 2019, month 10, day 08, deposit address: the detection result of the strain is survival in Xilu No.1 of Beijing, chaoyang, and institute of microbiology, national academy of sciences.
The lactobacillus rhamnosus Probio-M9 of the present application has the following biological properties: gram-positive bacteria, no flagellum, no movement, no spore formation and facultative anaerobism; the thallus is rod-shaped, and the bacterial colony is smooth and has complete edges and is milk white. The optimal growth temperature is 36-38 ℃; the optimum pH value is 6.0-7.0.
Another purpose of the application is to provide an application of Lactobacillus rhamnosus Probio-M9 in preventing and treating mastitis.
Another object of the application is to provide an application of Lactobacillus rhamnosus Probio-M9 in a starter culture, a fermented dairy product, a health food and animal breeding.
The application provides a lactobacillus rhamnosus strain for preventing and treating mastitis and application thereof, and a mastitis prevention research carried out by taking an aseptic mother mouse in a lactation period as an animal model finds that the lactobacillus rhamnosus Probio-M9 can improve the immune response function of an organism, reduce the number of mammary inflammation cells, relieve the mammary inflammation degree and have the effects of preventing and curing the mammary inflammation.
Detailed Description
The features and advantages of the present application will become more apparent and appreciated from the following detailed description of the application.
The application provides a Lactobacillus rhamnosus Probio-M9 (Lactobacillus rhamnous Probio-M9) for preventing and treating mastitis, which is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the microorganism preservation number as follows: CGMCC No.18639, classification name: lactobacillus rhamnosus, deposit time: year 2019, month 10, day 08, deposit address: beijing, chaoyang district, beichen Xilu No.1 institute, institute of microbiology, china academy of sciences.
The application of the lactobacillus rhamnosus Probio-M9 in preventing and treating mastitis is provided.
The application of the lactobacillus rhamnosus Probio-M9 in a starter, a fermented dairy product, a health food and animal breeding.
The application provides a lactobacillus rhamnosus strain for preventing and treating mastitis and application thereof, and a mastitis prevention research carried out by taking an aseptic mother mouse in a lactation period as an animal model finds that the lactobacillus rhamnosus Probio-M9 can improve the immune response function of an organism, reduce the number of mammary inflammation cells, relieve the mammary inflammation degree and have the effects of preventing and curing the mammary inflammation.
Example 1: separating, screening and identifying lactobacillus rhamnosus Probio-M9.
1. Materials and methods
1.1 Strain origin, isolation and identification
Weighing 10g of sample from a healthy woman breast milk sample, diluting the sample with 90g of sterile normal saline, taking 0.1mL of the sample, coating the sample on a selective MRS solid culture medium, and carrying out anaerobic culture at 37 ℃ for 72h. Selecting typical bacterial colony for gram staining microscopy, selecting gram positive bacterial strain, streaking on MRS solid culture medium, and culturing for 2-3 times to obtain pure bacterial colony. The strain is in a gram-like bacterium shape under a microscope, is in a straight rod shape, and is single, sometimes in pairs or in chains; the diameter of a bacterial colony on an MRS solid culture medium is 1-3mm, and the bacterial colony is milky, convex, round, smooth in surface and free of movement characteristics.
The isolated strains were subjected to physiological and biochemical identification and 16s rRNA molecular identification, and according to Bergey's Manual of bacteria identification, a bacterial micro biochemical identification tube (Kyoto Loop-Kai microbial technology Co., ltd.) was used for identification, and the identification results are shown in Table 1. Extracting a DNA sample of the strain, carrying out 16s rRNA molecular identification on the sample by Shanghai Meiji biological medicine science and technology Limited company, comparing a molecular sequence by a NCBI database blastn, and confirming that Probio-M9 is lactobacillus rhamnosus, wherein the 16s rRNA sequence is shown in a sequence table.
TABLE 1 Biochemical identification of Lactobacillus rhamnosus Probio-M9
Example 2 Lactobacillus rhamnosus Probio-M9 (Lactobacillus rhamnous Probio-M9) gastrointestinal fluid tolerance.
1. Gastrointestinal fluid tolerance test method:
1.1. sterilized PBS (adjusted with 1mol/L HCl) at pH2.5, added with 3.5g/L pepsin, and sterilized by filtration through a 0.22 μm microporous membrane to prepare simulated gastric fluid.
1.2. Selecting strains subjected to acid resistance screening for culturing, performing centrifugal washing twice after culturing for two generations, collecting thallus, adding simulated gastric juice with pH of 2.5 and the same amount as that of the culture medium, culturing at 37 ℃ for 3h, and measuring the viable count of the thallus by using an MRS agar culture medium pouring method at 0h and 3 h.
1.3. A simulated intestinal fluid was prepared by adding 0.1% trypsin and 1.8% bovine bile salt to sterilized PBS (pH 8.0 adjusted with 0.1mol/L NaOH), and sterilizing with a 0.22 μm microporous membrane.
1.4. And (3) centrifuging the bacterial liquid treated in the simulated gastric juice for 3 hours, washing the bacteria twice, collecting thalli, adding simulated intestinal juice which is equal to the simulated gastric juice, continuously culturing at 37 ℃, and measuring the viable count by using an MRS agar culture medium pouring method at 0 hour, 4 hours and 8 hours.
Survival rate = [ N1/N0] x100%
N0-0h viable count; n1-number of viable bacteria after 3 hours or 8 hours of simulated digestion.
2. Tolerance effect: after the lactobacillus rhamnosus Probio-M9 is treated by simulated gastric juice and simulated intestinal juice, the lactobacillus rhamnosus Probio-M9 has good tolerance characteristic and the survival rate of 78.33 percent.
TABLE 2 Lactobacillus rhamnosus Probio-M9 simulation of gastrointestinal digestive fluid survival
According to the effect of gastrointestinal fluid tolerance in example 2, lactobacillus rhamnosus Probio-M9 has the characteristic of good gastrointestinal fluid tolerance in the gastrointestinal tract and has potential probiotic properties.
MRS synthetic medium: guangdong Huaqiao microbiological science and technology Co., ltd, for the detection of microorganisms.
Method for counting live bacteria
The pouring flat plate counting method is adopted, and the specific operation steps are as follows: the detection is carried out according to the method GB 4789.35-2016.
(II) culture medium for determining viable count of probiotics
In the experimental example of the application, the culture medium for determining the viable count of the probiotics is an MRS culture medium.
Example 3: the lactobacillus rhamnosus Probio-M9 has the function of preventing and treating mastitis of mice.
1 method of experiment
1.1 preparation of Experimental Strain
The lactobacillus rhamnosus Probio-M9 bacterial powder preparation has the viable bacteria content of 2x1011CFU/g and is provided by Beijing Ketuhengtong biotechnology GmbH.
1.2 Experimental animals
The experiment adopts a C57BL/6J sterile pregnant mouse with the age of 4 months, is provided by third-military medical science and is used for establishing a mastitis animal model.
1.3 Main kit for experiment
Abcam company MPO (Myeloperoxoxidase), IL-10 (InterIeukin-10) mouse ELISA kit; biolegend IFN-y, IL-17, IL-1-beta, TNF-alpha, IL-6 mouse ELISA kits; shanghai mice endotoxin, IL-4, lysozyme ELISA kit; anti-NF-kB P65 antibody, anti-IkB beta antibody, anti-P38 antibody, anti-JNK1+ JNK2+ JNK3 antibody, anti-ERK1+ ERK2 antibody, anti-CD45 antibody, anti-CLC4 antibody, anti-STAT3 antibody, anti-AKT antibody, and Anti-TGR antibody.
1.4 design of the experiment
The mice are randomly divided into 3 groups, mastitis molding treatment is carried out 3 days before the farrowing of the female mice, and the experimental method comprises the following steps: in group I healthy group, 50. Mu.L of sterile physiological saline was slowly injected into the milk duct. In group II mastitis group, 50. Mu.L of LPS was drawn by syringe and slowly injected into the milk duct. Group III treatment group, 50. Mu.L LPS was slowly injected into the milk duct by syringe, and Lactobacillus rhamnosus Probio-M9 was administered at an intervention dose of 2X10 10 And (5) performing intragastric perfusion in CFU/d until the experiment is finished in 25 days.
Respectively randomly selecting 5 mice from each group of mice, taking a mammary gland tissue pathological section of the mice to perform HE (human immunodeficiency Virus) staining and immunohistochemical staining, detecting whether the mammary gland tissue of the mastitis mice has inflammation, detecting whether the mammary gland tissue of the treatment group of mice is different from that of the mastitis group of mice after probiotic intervention, detecting whether the inflammation occurs and the inflammation degree, and detecting whether the mammary gland tissue of the health group of mice is healthy and has no inflammation.
2 results and analysis
2.1 mouse mammary tissue pathological section results and analysis
And (3) carrying out inflammation degree pathological statistical analysis on the pathological section of the mammary tissue, and digitizing the inflammation reflected by the pathological section. The cells in the acinar section of the control group are regular, and no obvious inflammatory cell infiltration exists in the acinar; in the mastitis group, acinar injury and epithelial cell desquamation can be obviously seen in the mammary gland section, wherein acinar cells are disordered, and obvious inflammatory cell infiltration is formed between the inside of the acinar and the acinar space, so that mammary gland inflammation is obvious; mastitis occurs in the treatment group, clear and tidy acinus can be obviously observed from the section, few cast-off cells exist in the acinus, the number of inflammatory cells is small, the mastitis degree is greatly reduced compared with the mastitis group, and the prevention and treatment effect on mastitis can be realized by probiotic intervention, and the result is shown in table 3.
TABLE 3 pathological statistics of mammary tissue injury
2.2 mouse mammary tissue western blot (protein imprinting method) for detecting inflammatory protein expression
The mammary tissue of 2 mice of each group was randomly selected to detect the expression of inflammatory proteins, and the results are shown in table 4. Viral infection, lipopolysaccharide, host immune factor release, drugs, stress and the like can stimulate NF-Kb signal pathways. The JNK pathway plays an important role in various physiological activities and pathological processes such as cell stress, cell cycle, reproduction and apoptosis. P38 is often expressed in glandular cells, and the expression level of P38 is significantly increased during the period of inflammation and canceration of mammary tissues. The expression level of JNK in the control group is high, the expression level of JNK in the treatment group is very low, and the expression level of JNK in the mastitis group is almost zero. JNK has high expression level of mammary tissue under the influence of Lactobacillus rhamnosus Probio-M9, and has direct effect on preventing and resisting mastitis. Other proteins such as NF-KB are expressed in higher amount in mastitis group, and the treatment group is second, the trend is the same as the trend of the inflammatory condition of mammary tissue, and the result corresponds to the pathological section result.
Table 4 inflammatory protein expression based on grey value statistics
| Inflammatory proteins | Mastitis group | Treatment group | Control group |
| Nuclear transcription factor kB (NF-Kb) | 3500 | 1200 | 300 |
| Signal transducer and activator of transcription 3 (STAT 3) | 1900 | 600 | 450 |
| Chlorine channel protein-4 (C1C 4) | 700 | 750 | 980 |
| Extracellular signal regulated kinase (BRK) | 2100 | 720 | 100 |
| Protein kinase B (AKT) | 2800 | 810 | 187 |
| Nuclear factor kappa B inhibitor protein (IKB) | 2700 | 300 | 50 |
| Mitogen-activated protein kinase p38 (p 38) | 2600 | 900 | 109 |
| G protein-coupled bile acid receptor (TGR) | 2000 | 1500 | 62 |
| Stress activated protein kinase (JNK) | 17 | 60 | 1125 |
2.3 detection of immunological indicators in serum and tissue by ELISA
7 mice were randomly selected from each group, and their mammary glands were tested by ELISA method, the content of individual immune factors in serum was measured, and 1ml of physiological saline was added to 0.1g of mammary tissue, after grinding and homogenization, the supernatant was centrifuged at 12000rpm for 15min and used for ELISA test, and the serum was diluted 5-fold and then subjected to ELISA test, and the results are shown in Table 5.
TABLE 5 mammary tissue immunological indices
| Index of immune factor | Mastitis group | Treatment group | Control group |
| Mammary tissue IL-6 (pg/mL) | 480 | 900 | 200 |
| Mammary gland tissue MPO (ng/mL) | 180 | 170 | 50 |
| Mammary tissue TNF-a (ng/mL) | 700 | 900 | 300 |
| Serum IFN-y (ng/mL) | 600 | 420 | 410 |
| Serum IL-4 (pg/mL) | 120 | 113 | 105 |
| Serum IL-10 (pg/mL) | 410 | 402 | 387 |
| Serum IL-17 (pg/mL) | 800 | 621 | 407 |
| Serum LZM (ng/mL) | 18 | 14 | 11 |
| Serum endotoxin (EU/mL) | 4.8 | 4.3 | 4.1 |
And detecting the content of IL-6, MPO and TNF-a in the mammary tissue. The three factors in the control group are obviously lower than those in the other two groups, while the IL-6 and TNF-a contents in the treatment group are obviously higher than those in the M group, and P is less than 0.001. The intervention of probiotics improves the immune response intensity of organisms, particularly obviously improves the content of IL-6, thereby having the capacity of preventing and curing the mastitis.
IFN-y, IL-4,L-10, IL-17, lysozyme and endotoxin contents in serum are all mastitis groups > treatment group > control group, wherein endotoxin mastitis groups are higher than other two groups, and due to damage of normal structures of intestinal tracts, the endotoxin in blood is increased, so that mastitis is caused by stimulation.
The research on preventing and treating mastitis by taking an aseptic mother mouse in a lactation period as an animal model discovers that Lactobacillus rhamnosus Probio-M9 (Lactobacillus rhamnous Probio-M9) can improve the immune response function of an organism, reduce the number of mammary inflammation cells, relieve the mammary inflammation degree and have the effects of preventing and curing the mammary inflammation.
Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.
It will be understood that the present application is not limited to the precise arrangements that have been described above and that various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.
Claims (9)
1. A strain of Lactobacillus rhamnosus Probio-M9 (Lactobacillus rhamnous Probio-M9) is characterized in that the strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the microorganism preservation number as follows: CGMCC No.18639.
2. Use of lactobacillus rhamnosus Probio-M9 according to claim 1 in food products.
3. Use according to claim 2, wherein the food product is a health food, a health food or a fermented dairy product.
4. Use of lactobacillus rhamnosus Probio-M9 according to claim 1 in a starter.
5. Use of lactobacillus rhamnosus Probio-M9 according to claim 1 in animal farming.
6. Use of lactobacillus rhamnosus Probio-M9 according to claim 1 for the preparation of a medicament.
7. The use of claim 6, wherein the medicament is a medicament for enhancing the immune response function of the body.
8. The use according to claim 7, wherein the medicament is based on the use of Probio-M9 to increase the immune factor index of IL-6 and/or TNF-a in order to increase the intensity of the immune response in the body.
9. The use according to claim 7, wherein the medicament is based on the use of Probio-M9 to reduce the immune factor index of IL-17, IL-10, IL-4, LZM, endotoxin and IFN-y to increase the intensity of the immune response in the body.
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