CN116059259A - Application of lactobacillus fermentum E1 in preparation of medicines for treating or preventing allergic diseases - Google Patents
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- CN116059259A CN116059259A CN202310180248.XA CN202310180248A CN116059259A CN 116059259 A CN116059259 A CN 116059259A CN 202310180248 A CN202310180248 A CN 202310180248A CN 116059259 A CN116059259 A CN 116059259A
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Abstract
The invention discloses application of lactobacillus fermentum E1 in preparation of a medicament for treating or preventing allergic diseases, and belongs to the technical field of microorganisms. The lactobacillus fermentum E1 can obviously inhibit compound 48/80 from stimulating zebra fish to secrete Tryptase in vivo, can inhibit mast cells or basophils from degranulation in vivo, and has the potential of being applied to in vivo treatment or prevention of anaphylactic reaction. The lactobacillus fermentum E1 disclosed by the invention has great potential application prospect in the aspect of treating or preventing allergic reaction.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to application of lactobacillus fermentum E1 in preparation of medicines for treating or preventing allergic diseases.
Background
Allergy is that foreign antigens are read as harmful objects (such as bacteria, viruses, pollen, dust and the like) to generate allergy, phagocytes in immune cells can be activated, histamine and prostaglandin are released, the histamine and the prostaglandin can cause a series of effects such as capillary dilation, vascular permeability increase, itching, smooth muscle contraction and reflection, and the like, and the skin is little affected by erythema measles, and the skin is seriously affected by inflammation characterization such as swelling, heating and the like, which has a great influence on daily life of people. Allergic diseases are therefore listed as one of the three major diseases that are important to control in the 21 st century. Epidemiological surveys worldwide indicate that about 22% of the population suffers from different types of allergic diseases. At present, the means for dealing with allergic diseases is limited, only a few antiallergic drugs are taken when the diseases occur, and the occurrence of anaphylactic reaction cannot be avoided at all.
Probiotics are defined as viable microorganisms that improve the balance of intestinal flora and may benefit a variety of aspects of the host physiological response, including immune function. Several studies have shown that oral administration of bifidobacteria or lactobacilli can alleviate food allergy. Lactobacillus can attenuate the activation of mast cells and release inflammatory mediators associated with allergic reactions. Bifidobacterium BGN4 can inhibit the production of serum IgE and IgG1 in a food allergy mouse model, and in addition, lactobacillus casei Shirota can inhibit the production of antigen-specific IgE by stimulating the secretion of IL-12, so that the bifidobacterium BGN4 has good probiotic efficacy for preventing or treating allergic diseases. 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. 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 E1 for the preparation of a medicament for the treatment or prevention of allergic diseases is a problem to be solved by the person skilled in the art.
Disclosure of Invention
In view of this, the present invention provides the use of lactobacillus fermentum E1 in the manufacture of a medicament for the treatment or prevention of allergic diseases.
Mast cells are the main effector cells of the anaphylactoid reaction, the number of activated mast cells being related to the degree of allergy, the more activated mast cells, the more severe the degree of allergy. Tryptase (Tryptase) is a pre-synthesized neutral protease in mast cells, is the most abundant medium, is released outside cells through degranulation of mast cells, has high selectivity in storage and expression in mast cells, and can be used as a marker for activation and degranulation of mast cells. The Tryptase is normally secreted only in small amounts into the body fluid, and in large amounts into the body fluid when the hypersensitivity causes degranulation of mast cells. The predictive accuracy of the acute allergic positive and negative reactions of Tryptase was 92.6% and 54.3%, respectively, and the half-life of the Tryptase was 2 hours, so that the expression level of the Tryptase was frequently used as one of the detection markers of the allergic reaction.
Mast cells of zebra fish, which are structurally and functionally similar to mammals, are involved in immune and allergic reactions in the body. N-benzoyl-DL-arginine paranitroanilide hydrochloride (BAPNA) is a substrate specific to the Tryptase, and the expression level of the Tryptase in the zebra fish can be detected. The sensitization and antiallergic effects of a substance can be quantitatively analyzed by detecting the expression level of the Tryptase in the zebra fish body within 24 hours after the substance induces the zebra fish body.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
lactobacillus fermentum E1 with the preservation number of CGMCC No.21777 is preserved in China general microbiological culture Collection center, CGMCC for short, and has the preservation date of 2021, 02 month and 01 days, and is classified and named as Lactobacillus fermentum Lactobacillus fermentum.
Further, the application of the lactobacillus fermentum E1 in preparing medicaments for treating or preventing allergic diseases.
Further, the application of the lactobacillus fermentum E1 in preparing a medicament for inhibiting mast cells or basophils from degranulation.
Further, the lactobacillus fermentum E1 is a bacterial suspension.
Compared with the prior art, the invention discloses the application of the lactobacillus fermentum E1 in preparing the medicines for treating or preventing the allergic diseases, wherein the lactobacillus fermentum E1 is separated and screened from the faeces of long-life old people in the city of the abaca county of the cantonese, the lactobacillus fermentum E1 can obviously inhibit compound 48/80 from stimulating the zebra fish to secrete the Tryptase in vivo, can inhibit mast cells or basophils from degranulation in vivo, has the potential of being applied to treating or preventing the allergic reactions in vivo, and provides theoretical reference and guiding basis for developing a probiotic preparation for pre-treating or preventing the allergic reactions by using the lactobacillus fermentum E1.
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 drawing showing colony morphology of Lactobacillus fermentum E1 of the present invention on MRS agar plates;
FIG. 2 is a graph showing the effect of Lactobacillus fermentum E1 of the present invention on the expression level of compound 48/80 stimulated zebra fish to secrete Tryptase;
FIG. 3 is a graph showing the inhibition ratio of Lactobacillus fermentum E1 of the present invention to compound 48/80 stimulated zebra fish to secrete Tryptase.
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.
Sodium cromoglycate and N-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA) were purchased from Beijing Warce Shake chemical Co., ltd, compoud48/80 was purchased from Sigma Co., USA; lactobacillus fermentum 11739 (ATCC 11739) was purchased from bio-technology limited of beijing Bai-o-borg.
EXAMPLE 1 isolation, identification and preservation of Lactobacillus fermentum E1
(1) Separating:
1) The feces (about 0.1 g) of the elder with long life are dissolved in a 1.5mL centrifuge tube filled with 1mL sterile physiological saline, and are fully blown and evenly mixed by a 1mL sterile gun head for standby.
2) Into each of 6 sterile 1.5mL centrifuge tubes, 900. Mu.L of sterile physiological saline was added.
3) From 1 st is provided with 10 -1 Into a centrifuge tube for sample dilution, 100. Mu.L of the liquid was pipetted into a 2 nd centrifuge tube (10 -2 ) Diluted to 10 -2 ；
4) From 2 nd is provided with 10 -2 Into a centrifuge tube for sample dilution, 100. Mu.L of the liquid was pipetted into a 3 rd centrifuge tube (10 -3 ) Diluted to 10 -3 ；
5) Repeating the above steps until the dilution is 10 -4 、10 -5 、10 -6 、10 -7 。
6) From the package 10 -4 100 mu L of sample diluent is sucked into a centrifuge tube of the sample diluent and respectively inoculated on an MRS solid culture medium and a BHI solid culture medium, and 100 mu L of bacterial liquid is spread and dried, and the coating method is mild, fast in action and needs to be operated near the flame of an alcohol lamp. After coating, the side of the dish was marked, including information on name, sample number, medium name, incubation time, dilution gradient, incubation conditions (anaerobic/aerobic), etc.
7) Repeating the above steps to obtain 10 -5 、10 -6 、10 -7 Dilution coating of dilution gradient.
8) After the coating, the dishes were cultured at 37℃under anaerobic conditions for 48 hours, and then were subjected to observation and recording.
9) Single colony on the plate is picked up by an inoculating loop and streaked into MRS solid culture medium, anaerobic culture is carried out for 48 hours at 37 ℃, and pure colony is obtained by separation.
10 Inoculating pure bacterial colony on the flat plate into MRS liquid culture medium, anaerobic culturing at 37 deg.C for 12-16 hr, adding 20% glycerine, and storing in-80 deg.C refrigerator.
(2) Molecular biological identification of strains: genomic DNA was extracted from the obtained strain, and a full-length fragment of 16S rDNA was amplified by PCR technique using the universal primers 27F and 1492R of 16S rDNA, followed by sequencing to identify the species of the strain.
The primer sequences of the universal primers 27F and 1492R are as follows:
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；SEQ ID NO.1；
1492R：5’-GGTTACCTTGTTACGACTT-3’；SEQ ID NO.2。
experimental results: the strain E1 is identified as lactobacillus fermentum by morphological observation and 16S rDNA identification, wherein the 16S rDNA sequence of the strain is shown as SEQ ID NO. 3.
TACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGTCTCAGCGTCAGTTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCTGCACTCAAGTTATCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAGAAAACCGCCTGCACTCTCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTAAATACCGTCAACGTATGAACAGTTACTCTCATACGTGTTCTTCTTTAACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTATGGGCCGTGTCTCAGTCCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAGGCCGTTACCCCACCAACAAGCTAATGCACCGCAGGTCCATCCAGAAGTGATAGCGAGAAGCCATCTTTTAAGCGTTGTTCATGCGAACAACGCTGTTATGCGGTATTAGCATCTGTTTCCAAATGTTGTCCCCCGCTTCTGGGCAGGTTACCTACGTGTTACTCACCCGTCCGCCACTCGTTGG；SEQ ID NO.3。
The strain E1 single colony is inoculated on MRS solid culture medium, grows well under the aerobic condition at 37 ℃, and has milky white colony, regular edge, spherical shape and smooth surface (figure 1). The strain E1 is preserved in China general microbiological culture Collection center (CGMCC), and has a preservation date of 2021, 02 month and 01, and a classification name of Lactobacillus fermentum, and a preservation number of CGMCC No.21777.
EXAMPLE 2 preparation of Lactobacillus fermentum E1 suspension (thallus)
Inoculating lactobacillus fermentum E1 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 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 4 Effect of Lactobacillus fermentum E1 on Compound 48/80 stimulation of Tryptase secretion in zebra fish
Healthy wild-type AB-line zebra fish that developed to 5dpf (days post fertilization) were selected and placed in 96-well cell culture plates, 10 strips/well. The experiment set up was normal, model, intervention (positive control, lactobacillus fermentum 11739, lactobacillus fermentum E1), solvent zeroing, 6 replicate wells per set up. Adding PBS into normal group, adding PBS into model group, adding sodium cromoglycate solution (100 μg/mL) into positive control group, adding lactobacillus fermentum 11739 into 1×10 6 CFU/mL of Lactobacillus fermentum 11739, lactobacillus fermentum E1 added 1X 10 6 CFU/mL lactobacillus fermentum E1, adding PBS into a solvent zeroing group (zebra fish is not added), placing 100 mu L of the solvent zeroing group into a biochemical incubator at 28 ℃ for incubation, and replacing a new solution after 24 hours; after 48h incubation, 150. Mu.L PBS was added to the normal group, the model group, the positive control group, lactobacillus fermentum 11739, lactobacillus fermentum E1, and Compound 48/80 (8. Mu.L/mL) were added to each of the model group, the positive control group, lactobacillus fermentum E1, and the solvent zeroing groups, respectively, 150. Mu.L of each well was incubated at 28℃for 2h, 150. Mu.L of each well was placed in 2mL centrifuge tubes, 150. Mu.L of BAPNA solution (20 mg/mL) was added to each centrifuge tube, after 48h incubation at 37℃200. Mu.L of each centrifuge tube was placed in 96-well cell culture plates, and absorbance (OD) was measured at 405nm using an microplate reader. The expression level and inhibition rate of the Tryptase are expressed as follows:
SPSS 19.0 software is adopted for statistical data processing, and experimental data are all adopted
Data represent, analyzed by T-test, compared to normal group: #### p<0.001, compared to model group: * P (P)<0.05，****P<0.001。/>
The results of the expression level and the inhibition rate of the Tryptase are shown in fig. 2 and 3, and the results show that compared with the normal group (100.00+/-14.00%), the expression level of the Tryptase in the model group (339.07 +/-34.07%) is obviously increased, which indicates that the compound 48/80 induction allergy model is successfully established.
The positive control group (sodium cromolyn) showed a level of expression of Tryptase of 113.80.+ -. 7.10% and a rate of inhibition of Tryptase of 94.23.+ -. 2.97%, and showed significant differences compared to the model group (Tryptase expression level: 339.07.+ -. 34.07%) (P<0.001). Therefore, the cromolyn sodium has antiallergic effect, and is consistent with clinical results, which shows that the antiallergic test is effective. Lactobacillus fermentum 11739 group (1×10) 6 CFU/mL) the expression level of the Tryptase in the zebra fish was 294.88 ±12.78%, while the inhibition rate of the Tryptase was 18.48±5.35%, and the expression level of the Tryptase in the model group (expression level: 339.07 + -34.07%) is significantly different than the above (P)<0.05). In addition, lactobacillus fermentum E1 group (1X 10) 6 CFU/mL) the expression level of the Tryptase in the zebra fish was 147.75 ±25.91%, and the inhibition rate of the Tryptase was 80.03 ±10.84%, compared with the expression level of the Tryptase in the model group (expression level of the Tryptase: 339.07 + -34.07%) is significantly different than the above (P)<0.001 Shows good probiotic efficacy in inhibiting degranulation of mast cells or basophils. Thus, the above results indicate that at the same concentration, lactobacillus fermentum E1 has a stronger inhibitory effect on compound 48/80 to induce degranulation of mast cells or basophils in vivo than Lactobacillus fermentum 11739, and has the potential to treat or prevent allergic diseases.
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 (4)
1. Use of lactobacillus fermentum E1 in the manufacture of a medicament for the treatment or prevention of allergic diseases.
2. Use of lactobacillus fermentum E1 according to claim 1 for the preparation of a medicament for the treatment or prevention of allergic diseases, wherein the lactobacillus fermentum E1 is a bacterial suspension.
3. Use of lactobacillus fermentum E1 as claimed in claim 1 in the manufacture of a medicament for inhibiting degranulation of mast cells or basophils.
4. Use of lactobacillus fermentum E1 according to claim 3 for the manufacture of a medicament for inhibiting degranulation of mast cells or basophils, wherein said lactobacillus fermentum E1 is a bacterial suspension.
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