CN116426426B - Lactobacillus helveticus LH05 and application thereof in preparation of medicines for preventing or treating intestinal inflammation - Google Patents
Lactobacillus helveticus LH05 and application thereof in preparation of medicines for preventing or treating intestinal inflammation Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses lactobacillus helveticus LH05 and application thereof in preparing medicines for preventing or treating intestinal inflammation, and belongs to the technical field of microorganisms. The preservation number of the Lactobacillus helveticus LH05 disclosed by the invention is CGMCC No.23452. The Lactobacillus helveticus LH05 can inhibit DSS-induced neutrophil aggregation to the intestinal tract of the zebra fish, so that theoretical reference and guiding basis are provided for developing medicines for preventing or treating intestinal inflammation by using the Lactobacillus helveticus LH 05.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus helveticus LH05 and application thereof in preparing medicines for preventing or treating intestinal inflammation.
Background
The pathogenesis of enteritis is largely related to the unhygienic diet of patients. When the pathogens enter the intestinal tracts of patients along with food, the pathogens can be propagated in the intestinal tracts, and when the pathogens are propagated to a certain quantity, the pathogens can induce inflammation in the intestinal tracts, and inflammatory secretion can stimulate intestinal mucosa and nerves to induce enteritis symptoms. Patients easily show obvious clinical manifestations of diarrhea, abdominal pain, vomit, inappetence, fever and the like in a short time.
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.
Accordingly, providing lactobacillus helveticus LH05 and its use 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 this, the invention provides lactobacillus helveticus LH05 and its use in the preparation of a medicament 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:
A Lactobacillus helveticus (Lactobacillushelveticus) LH05 with the preservation number of CGMCC No.23452 is preserved in China general microbiological culture center, CGMCC for short, and is named as Lactobacillus helveticus Lactobacillus helveticus by classification, wherein the preservation date is 2021, 09 and 18 days, and the China academy of sciences of China, no. 3, north Star, qing dynasty area, beijing, and the like.
Further, the lactobacillus helveticus LH05 is applied to the preparation of medicines for preventing or treating intestinal inflammation.
Further, the application of the lactobacillus helveticus LH05 in preparing medicines for inhibiting DSS-induced neutrophil aggregation to zebra fish intestinal tracts.
Further, the lactobacillus helveticus LH05 is a bacterial suspension.
Compared with the prior art, the invention discloses the lactobacillus helveticus LH05 and the application thereof in preparing the medicine for preventing or treating the intestinal inflammation, wherein the lactobacillus helveticus LH05 is separated and screened from the feces of long-life old people in Jiangling county of plum-state, guangdong, and the lactobacillus helveticus LH05 can inhibit DSS-induced neutrophil aggregation at the intestinal tract of 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 helveticus LH 05.
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 helveticus LH05 of the present invention on MRS agar plates;
FIG. 2 is a visual diagram showing that Lactobacillus helveticus LH05 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 helveticus 55163; e: lactobacillus helveticus LH05;
FIG. 3 is a graph showing statistics of inhibiting DSS-induced neutrophil aggregation to zebra fish intestinal tract by Lactobacillus helveticus LH05 according to the present 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 helveticus 55163 (ATCC 55163) was purchased from bio-technology limited of beijing Bai-o-borg.
EXAMPLE 1 isolation, identification and preservation of Lactobacillus helveticus LH05
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) The samples were diluted to 10 -2、10-3、10-4、10-5、10-6、10-7 using a gradient dilution method.
4) 100 Mu L of sample diluent is sucked from a centrifuge tube filled with 10 -4 sample diluent and inoculated on MRS solid culture medium and BS solid culture medium respectively, 100 mu L of bacterial liquid is flattened and coated to be dry, the coating method is mild, the action is rapid, and the operation is required to be performed 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.
5) The above steps were repeated to complete the dilution coating at a dilution gradient of 10 -5、10-6、10-7.
6) After the coating, the dishes were cultured at 37℃under anaerobic conditions for 48 hours, and then were subjected to observation and recording.
7) 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.
8) Pure bacterial colony on the flat plate is inoculated in MRS liquid culture medium, anaerobic culture is carried out for 12-16 h at 37 ℃, 20% glycerol is added, and the flat plate is placed in a refrigerator at-80 ℃ for preservation.
(2) Molecular biological identification of strains: genomic DNA was extracted from the obtained strain, and a full-length fragment of 16SrDNA was amplified by PCR technique using the 16SrDNA universal primers 27F and 1492R, 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 screened from feces of the elderly in Jiangling county of plum state of Guangdong province is identified by morphological observation and 16SrDNA, wherein the strain LH05 is identified as lactobacillus helveticus, and the 16SrDNA sequence is shown as SEQ ID NO. 3.
CTTAGACGGCTCCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAATTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTATAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCGTCCCCAGCGTCATTACCGAAGTAAATCTGCTGGTTCTGCTCGCTCGACTGCA;SEQ ID NO.3.
The strain LH05 single colony was inoculated onto MRS solid medium, and grown well anaerobically at 37℃with milky white, spherical and smooth surface (FIG. 1). The strain LH05 is preserved in China general microbiological culture Collection center (CGMCC), china No. 3 national academy of sciences of China, the Korean area North Star, the Beijing city, with a preservation date of 2021, 09 months and 18 days, and is classified and named as Lactobacillus helveticus Lactobacillushelveticus with a preservation number of CGMCC No.23452.
EXAMPLE 2 preparation of Lactobacillus helveticus LH05 bacterial suspension (thallus)
Inoculating lactobacillus helveticus LH05 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 cell pellet was washed twice with PBS, the cell was resuspended in PBS, and the cell concentration was adjusted to 1X 10 6 CFU/mL to obtain a cell suspension (cell).
EXAMPLE 3 preparation of Lactobacillus helveticus 55163 bacterial suspension (thallus)
Inoculating lactobacillus helveticus 55163 after activation culture in MRS liquid culture medium, culturing at 37deg.C for 24 hr, and centrifuging at 4deg.C at 6000r/min for 10min to obtain thallus precipitate; after the cell pellet was washed twice with PBS, the cell was resuspended in PBS, and the cell concentration was adjusted to 1X 10 6 CFU/mL to obtain a cell suspension (cell).
Example 4 Effect of Lactobacillus helveticus LH05 on DSS-induced neutrophil aggregation at the intestinal tract of zebra fish
Healthy Tg (mpx: EGFP) zebra fish (3 dpf) are selected and placed in a 6-hole cell culture plate, 1 hole is formed in each group, 15 holes are formed in each group (15 fish in one hole, the number of neutrophils in the intestinal tract of each fish is counted), 5mLPBS is added to a normal group, 5mL is added to a model group, a positive control group (mesalazine), a Swiss lactobacillus 55163 intervention group (1X 10 6 CFU/mL) and a Swiss lactobacillus LH05 intervention group (1X 10 6 CFU/mL), 5mL of 0.5% DSS solution (w/w) is added to each group, after the group is placed in a biochemical incubator for incubation for 72 hours at 28 ℃, PBS is used for washing each group of zebra fish for 3 times, and then 5mLPBS is added to the normal group and the model group; the positive control group was added with 5mL of 50. Mu.g/mL mesalamine solution; lactobacillus helveticus 55163 intervention group 1X 10 6 CFU/mL Lactobacillus helveticus 55163 was added, 5mL per well; the interference group of the Lactobacillus helveticus LH05 is added with 1X 10 6 CFU/mL of the Lactobacillus helveticus LH05, 5mL of each hole is placed in a biochemical incubator at 28 ℃ for further incubation for 48 hours, and then the mixture is placed under a fluorescence microscope to observe the aggregation condition of neutrophils at the intestinal tracts of zebra fish and recorded 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 <0.05, P <0.001.
The experimental results of the inhibition of DSS-induced neutrophil aggregation to the intestinal tract of zebra fish by lactobacillus helveticus LH05 are shown in fig. 2 and 3.
From FIGS. 2 and 3, it can be seen that there is a small number of neutrophil aggregates in the intestinal tract of the normal group of zebra fish (13.07.+ -. 1.29), 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 34.40+/-2.34, and compared with the normal group (13.07+/-1.29), 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. 2 and 3, only a small amount of neutrophils aggregated in the intestinal tract of zebra fish body spot fish of the positive control group (mesalamine). Meanwhile, the number of neutrophils in the intestinal tract of the zebra fish of the positive control group (mesalazine) is 15.20+/-1.22, and the difference is obvious (P < 0.001) compared with the model group (34.40+/-2.34). 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. The number of neutrophils at the intestinal tract of zebra fish in the Lactobacillus helveticus 55163 intervention group (1×10 6 CFU/mL) was 25.87+ -2.06, and the variability was significant (P < 0.05) compared with the model group (34.40+ -2.34). In addition, only a small amount of neutrophils aggregated in the intestinal tract of zebra fish in lactobacillus helveticus LH05 group, similar to the positive control group (mesalamine); meanwhile, the number of neutrophils in the zebra fish intestinal tract of the Lactobacillus helveticus LH05 intervention group (1X 10 6 CFU/mL) is 17.53+/-1.24, and the variability is obvious (P < 0.001) compared with the model group (34.40+/-2.34). Therefore, the results show that at the same concentration, the inhibition effect of the lactobacillus helveticus LH05 on DSS-induced neutrophil aggregation to the intestinal tract of the zebra fish is stronger than that of the lactobacillus helveticus 55163, and the potential of relieving intestinal inflammation is good.
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. The application of lactobacillus helveticus (Lactobacillus helveticus) LH05 in preparing a medicament for preventing or treating intestinal inflammation is characterized in that the preservation number of the lactobacillus helveticus LH05 is CGMCC No.23452.
2. Use of lactobacillus helveticus LH05 according to claim 1 for the preparation of a medicament for the prevention or treatment of intestinal inflammation, wherein said lactobacillus helveticus LH05 is a bacterial suspension.
3. Use of lactobacillus helveticus LH05 as claimed in claim 1 for the preparation of a medicament for inhibiting DSS-induced neutrophil aggregation to the intestinal tract of zebra fish.
4. Use of lactobacillus helveticus LH05 according to claim 3 for the preparation of a medicament for inhibiting DSS-induced neutrophil aggregation to the intestinal tract of zebra fish, wherein said lactobacillus helveticus LH05 is a bacterial suspension.
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Citations (5)
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|---|---|---|---|---|
| WO2005067708A2 (en) * | 2004-01-14 | 2005-07-28 | Daniolabs Limited | Zebrafish model for autoimmune diseases |
| KR20130045511A (en) * | 2011-10-26 | 2013-05-06 | 주식회사한국야쿠르트 | Lactobacillus helveticus hy7801 having anti- rheumatoid arthritis functions, and products containing thereof as effective component |
| CN112877233A (en) * | 2021-01-23 | 2021-06-01 | 江苏斯卡露生物科技有限公司 | Lactobacillus helveticus strain and application thereof |
| CN114410514A (en) * | 2021-12-31 | 2022-04-29 | 上海新溢生物科技工程研究中心(有限合伙) | Bacillus smithii and uses thereof |
| CN115551990A (en) * | 2020-02-26 | 2022-12-30 | Gi生物群系公司 | Lactobacillus helveticus strain and composition for preventing or treating inflammatory diseases containing the same |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005067708A2 (en) * | 2004-01-14 | 2005-07-28 | Daniolabs Limited | Zebrafish model for autoimmune diseases |
| KR20130045511A (en) * | 2011-10-26 | 2013-05-06 | 주식회사한국야쿠르트 | Lactobacillus helveticus hy7801 having anti- rheumatoid arthritis functions, and products containing thereof as effective component |
| CN115551990A (en) * | 2020-02-26 | 2022-12-30 | Gi生物群系公司 | Lactobacillus helveticus strain and composition for preventing or treating inflammatory diseases containing the same |
| CN112877233A (en) * | 2021-01-23 | 2021-06-01 | 江苏斯卡露生物科技有限公司 | Lactobacillus helveticus strain and application thereof |
| CN114410514A (en) * | 2021-12-31 | 2022-04-29 | 上海新溢生物科技工程研究中心(有限合伙) | Bacillus smithii and uses thereof |
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