CN116270763A

CN116270763A - Application of lactobacillus fermentum E15 in preparation of medicines for preventing or treating intestinal inflammation

Info

Publication number: CN116270763A
Application number: CN202310351402.5A
Authority: CN
Inventors: 陈涛; 郑康帝; 张召
Original assignee: Guangdong Yiyuan Lanxin Biotechnology Co ltd; Guangdong Longsee Medical Technology Co ltd
Current assignee: Guangdong Yiyuan Lanxin Biotechnology Co ltd; Guangdong Longsee Medical Technology Co ltd
Priority date: 2023-04-04
Filing date: 2023-04-04
Publication date: 2023-06-23

Abstract

The invention discloses application of lactobacillus fermentum E15 in preparation of a medicine for preventing or treating intestinal inflammation, and belongs to the technical field of microorganisms. The application of the lactobacillus fermentum E15 in preparing the medicine for preventing or treating the intestinal inflammation disclosed by the invention can inhibit DSS (direct sequence) induced neutrophil aggregation to the intestinal tract of the zebra fish, so that theoretical reference and guiding basis are provided for developing the medicine for preventing or treating the intestinal inflammation by utilizing the lactobacillus fermentum E15.

Description

Application of lactobacillus fermentum E15 in preparation of medicines for preventing or treating intestinal inflammation

Technical Field

The invention relates to the technical field of microorganisms, in particular to application of lactobacillus fermentum E15 in preparation of medicines for preventing or treating intestinal inflammation.

Background

Enteritis is an inflammatory disorder of the small intestine or colon caused by infection with bacteria, viruses, fungi or parasites, etc. The main clinical symptoms are abdominal pain, watery stool and mucopurulent bloody stool, and part of patients can be accompanied with fever or tenesmus. According to the duration of the disease, enteritis can be classified into acute enteritis and chronic enteritis, which is commonly seen clinically. The chronic enteritis generally has a disease course of more than two months, and is commonly caused by chronic bacillary dysentery, chronic amebic dysentery, schistosomiasis, nonspecific ulcerative colitis, localized enteritis and the like.

Traditional methods of treating enteritis can be classified into modern medical treatment and traditional Chinese medicine treatment methods. The probiotics, serving as endogenous and immune defensive barriers of intestinal tracts, can antagonize pathogenic bacteria, has the characteristics of safety, controllability, effectiveness and small side effect, and is an ideal method for treating enteritis. The current international probiotics patent application is concentrated on the traditional research and development of America, japanese and Russia, and China lacks functional strains with independent intellectual property rights. The probiotics strain used by domestic production enterprises depends on import for a long time, and foreign strains are not necessarily suitable for gastrointestinal tract physiological conditions of residents in China. In addition, the lack of powerful scientific research evidence of the function of probiotics seriously affects the popularization of the probiotics and products thereof. Based on the method, aiming at the function deep excavation of strain resources, the novel probiotic bacterial strain which has independent intellectual property, specific functional property and physiological property suitable for Chinese crowd is screened out, and the method is particularly important for improving the core competitiveness of Chinese probiotic production enterprises and promoting the development of Chinese probiotic products.

Therefore, providing the use of lactobacillus fermentum E15 in the preparation of a medicament for preventing or treating intestinal inflammation is a problem to be solved by the person skilled in the art.

Disclosure of Invention

In view of the above, the invention provides application of lactobacillus fermentum E15 in preparing medicines for preventing or treating intestinal inflammation.

Inflammation is a reaction of the immune system to tissue injury and infection and is mainly characterized by the accumulation of leukocytes (granulocytes, macrophages) around the infected tissue. The immune system of zebra fish is very similar to that of mammal, and when wound happens, neutrophils and macrophages respond to traumatic inflammation almost simultaneously, the migration speed of neutrophils is high, the neutrophils are firstly recruited to the damaged part, and then the macrophages reach the damaged part. After several hours, the inflammation began to subside, and macrophages and neutrophils left the injury site. The inflammation of the intestinal tract of the zebra fish is induced by Dextran Sodium Sulfate (DSS) to promote the immune response of the zebra fish neutrophils.

The transgenic neutrophil fluorescent fish (green) is adopted, and the DSS-induced zebra fish intestinal neutrophil quantity can be obviously increased compared with the normal zebra fish under a fluorescent microscope.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

use of lactobacillus fermentum E15 for the manufacture of a medicament for the prevention or treatment of intestinal inflammation, said lactobacillus fermentum E15 having a preservation number of cgmccno.21772 (see patent No. 202210216190.5).

Further, the application of the lactobacillus fermentum E15 in preparing medicines for inhibiting DSS-induced neutrophil aggregation to zebra fish intestinal tracts.

Further, the lactobacillus fermentum E15 is a bacterial suspension.

Compared with the prior art, the application of the lactobacillus fermentum E15 in preparing the medicine for preventing or treating the intestinal inflammation is disclosed, wherein the lactobacillus fermentum E15 is obtained by separating and screening the excrement of long-life old people in the city of the abaca county of the Guangdong province, and the lactobacillus fermentum E15 can inhibit DSS-induced neutrophil aggregation at the intestinal tract of the zebra fish, so that theoretical reference and guiding basis are provided for developing the medicine for preventing or treating the intestinal inflammation by utilizing the lactobacillus fermentum E15.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a visual diagram showing that lactobacillus fermentum E15 inhibits DSS-induced neutrophil aggregation to zebra fish intestinal tracts;

wherein A: normal group; b: a model group; c: a positive control group; d: lactobacillus fermentum 11739; e: lactobacillus fermentum E15;

FIG. 2 is a graph showing statistics of inhibiting DSS-induced neutrophil aggregation to zebra fish intestinal tracts by lactobacillus fermentum E15 according to the invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Zebra fish are green fluorescent zebra fish and AB-series zebra fish with Tg (mpx: EGFP) centromere cells.

Dextran Sodium Sulfate (DSS) (MedChemExpress); mesalamine (MedChemExpress); lactobacillus fermentum 11739 (ATCC 11739) was purchased from bio-technology limited of beijing Bai-o-borg.

EXAMPLE 1 preparation of Lactobacillus fermentum E15 suspension (thallus)

Inoculating lactobacillus fermentum E15 after activation culture in MRS liquid culture medium, culturing at 37 ℃ for 24 hours, and centrifuging at 4 ℃ for 10 minutes at 6000r/min to obtain bacterial precipitate; after the bacterial cell precipitate is washed twice by PBS, the bacterial cell is resuspended by PBS, and the cell concentration is regulated to be 1 multiplied by 10 ⁶ CFU/mL gave a bacterial suspension (cell).

EXAMPLE 2 preparation of Lactobacillus fermentum 11739 suspension (thallus)

Inoculating lactobacillus fermentum 11739 after activation culture in MRS liquid culture medium, culturing at 37deg.C for 24 hr, and centrifuging at 4deg.C for 10min at 6000r/min to obtain thallus precipitate; after the bacterial cell precipitate is washed twice by PBS, the bacterial cell is resuspended by PBS, and the cell concentration is regulated to be 1 multiplied by 10 ⁶ CFU/mL gave a bacterial suspension (cell).

Example 3 Effect of Lactobacillus fermentum E15 on DSS-induced neutrophil aggregation at the intestinal tract of zebra fish

Selecting healthy Tg (mpx: EGFP) zebra fish (3 dpf), placing in 6-well cell culture plate, 1 well per group, 15 wells per well (15 fish per well, counting neutrophil count in intestinal tract of each fish), adding 5mLPBS into normal group, model group, positive control group (mesalamine), and Lactobacillus fermentum 11739 intervention group (1×10) ⁶ CFU/mL), lactobacillus fermentum E15 intervention group (1×10) ⁶ CFU/mL) were added with 5mL of 0.5% dss solution (w/w), incubated at 28 ℃ in a biochemical incubator for 72h, each group of zebra fish was washed 3 times with PBS, and then 5mL PBS was added to the normal and model groups; the positive control group was added with 5mL of 50. Mu.g/mL mesalamine solution; lactobacillus fermentum 11739 intervention group addition 1×10 ⁶ CFU/mL Lactobacillus fermentum 11739, 5mL per well; lactobacillus fermentum E15 intervention group addition 1X 10 ⁶ CFU/mL lactobacillus fermentum E15, 5mL per well, was placed in a biochemical incubator at 28 ℃ for further incubation for 48 hours, and then placed under a fluorescence microscope to observe the aggregation of neutrophils at the intestinal tract of zebra fish and record by photographing.

SPSS19.0 software was used to statistically process the data, experimental data were all expressed as x+ -SEM data, analyzed by T-test, compared to normal group: ^#### p<0.001, compared to model group: * P<0.01，****P<0.001。

The experimental results of inhibiting DSS-induced neutrophil aggregation to zebra fish intestinal tract by lactobacillus fermentum E15 are shown in fig. 1 and 2.

As can be seen from fig. 1 and 2, there is a small number of neutrophil aggregates in the intestinal tract of the normal group of zebra fish (16.13±1.19), while there is a large number of neutrophil aggregates in the intestinal tract of the model group of zebra fish. Meanwhile, the number of neutrophils in the intestinal tract of the zebra fish in the model group is 35.87 +/-2.64, and compared with the normal group (16.13+/-1.19), the model group has obvious dissimilarity (P < 0.001), which indicates that the DSS-induced zebra fish intestinal inflammation model is successfully established.

As can be seen from fig. 1 and 2, only a small amount of neutrophils aggregated in the intestinal tract of zebra fish body spot fish of the positive control group (mesalamine). The number of neutrophils in the intestinal tract of zebra fish of the positive control group (mesalamine) is 20.60 +/-1.96, and the difference is compared with the model group (35.87 +/-2.64)Significant (P)<0.001). Therefore, the mesalamine has the effect of relieving the intestinal inflammation, and is consistent with clinical results, so that the test for evaluating the efficacy of relieving the intestinal inflammation is effective. Lactobacillus fermentum 11739 intervention group (1×10) ⁶ CFU/mL) of zebra fish with a large number of neutrophil aggregates in the intestine, similar to the model group; lactobacillus simultaneous fermentation 11739 intervention group (1×10) ⁶ The number of neutrophils in the intestinal tract of the zebra fish (CFU/mL) was 31.67+ -2.97, and there was no significant difference (P) compared with the model group (35.87 + -2.64)>0.05). In addition, only a small amount of neutrophils aggregated in the intestinal tract of zebra fish of lactobacillus fermentum E15 group, similar to the positive control group (mesalamine); lactobacillus simultaneous fermentation E15 intervention group (1×10) ⁶ The neutrophil count at the intestinal tract of the zebra fish (CFU/mL) was 24.13+ -1.95, and the difference was significant (P) compared with the model group (35.87 + -2.64)<0.01). Therefore, the results show that at the same concentration, the inhibition effect of the lactobacillus fermentum E15 on DSS-induced neutrophil aggregation to the intestinal tract of the zebra fish is stronger than that of the lactobacillus fermentum 11739, and the lactobacillus fermentum E15 has good potential for relieving intestinal inflammation.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of the lactobacillus fermentum E15 in preparing the medicine for preventing or treating the intestinal inflammation is characterized in that the preservation number of the lactobacillus fermentum E15 is CGMCC No. 2172.

2. Use of lactobacillus fermentum E15 according to claim 1 for the preparation of a medicament for the prevention or treatment of intestinal inflammation, wherein lactobacillus fermentum E15 is a bacterial suspension.

3. Use of lactobacillus fermentum E15 as claimed in claim 1 for the manufacture of a medicament for inhibiting DSS-induced neutrophil aggregation to the intestinal tract of zebra fish.

4. Use of lactobacillus fermentum E15 according to claim 3 for the preparation of a medicament for inhibiting DSS-induced neutrophil aggregation to the intestinal tract of zebra fish, wherein lactobacillus fermentum E15 is a bacterial suspension.