CN115725461B - Bacillus amyloliquefaciens with helicobacter pylori resisting effect and application thereof - Google Patents
Bacillus amyloliquefaciens with helicobacter pylori resisting effect and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus amyloliquefaciens with helicobacter pylori resisting effect and application thereof, and relates to the technical field of microorganisms. The strain is preserved in the microorganism strain collection of Guangdong province at 8 months and 20 days of 2022, the preservation address is the building No. 59, the university of Mitsui No. 100 of Guangzhou City, and the preservation number is GDMCCNo:62718. the bacillus amyloliquefaciens JY022 is obtained through separation, and both the metabolite and the thalli of the bacillus amyloliquefaciens have strong inhibition capability on helicobacter pylori. The Lactobacillus paracasei JY062 has strong adhesion capability to prevent the colonization of helicobacter pylori, and the metabolites and thalli of the Lactobacillus paracasei JY062 and the Bacillus amyloliquefaciens JY022 are combined to have strong inhibition capability to helicobacter pylori, so that the Lactobacillus paracasei JY062 and the Bacillus amyloliquefaciens JY022 can jointly act to prevent and eliminate helicobacter pylori invaded into the body.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus amyloliquefaciens with helicobacter pylori resistance and application thereof.
Background
Helicobacter pylori (Helicobacter pylori, HP) is a microaerophilic gram-negative bacillus that is S-shaped or curved and survives at pH 4.0-8.5, causing gastrointestinal and various parenteral diseases. Helicobacter pylori infection is one of the most common infectious diseases worldwide. Helicobacter pylori was identified by the world health organization as a class I carcinogen, and the U.S. health and public service department of 2022 issued the latest edition, 15 th edition of carcinogen report, with an additional 8 carcinogens. In the latest reports, helicobacter pylori is listed as a definite carcinogen.
Research shows that Hp infection can cause digestive system diseases such as chronic gastritis, gastric cancer, peptic ulcer and the like, and can also cause other diseases except for digestive tracts such as cardiovascular diseases, diabetes and the like. Thus, although early helicobacter pylori infection is less harmful to humans, eradication thereof is still important in view of long-term risk. During the 21 st century, antibiotic resistance has become one of the ten major threats to global public health today. Among them, overuse of antibiotics accelerates antibiotic resistance. Currently, the four-way therapy of 10d-14d, i.e. PPI+bismuth+two antibiotics, is the first line treatment regimen. Tetrad therapy improves the eradication rate of HP, but for patients with multiple antibiotic resistances, antibiotic selection is very difficult, and bismuth agents are neurotoxic, limiting the use of the elderly and children.
With the vigorous development of the probiotic industry and the corresponding increase in research, the value of its role in anti-HP infection has also led to the study and attention of clinicians. At present, probiotics are reported from the aspects of preventing adhesion and colonization of helicobacter pylori, inhibiting growth of helicobacter pylori with strong acid-producing capability, inhibiting expression of helicobacter pylori urease, inhibiting growth of metabolites, assisting eradication of helicobacter pylori infection and the like.
Disclosure of Invention
The invention aims to provide a bacillus amyloliquefaciens with the function of resisting helicobacter pylori and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) with the effect of resisting helicobacter pylori, which is preserved in the microorganism strain collection of Guangdong province in 8 months and 20 days of 2022, wherein the preservation address is the microbiological institute of Guangdong province, building 5, national institute of sciences of Guangzhou, miao, 100 th Hill, guangzhou, and the preservation number is GDMCC No:62718.
the invention also provides application of the bacillus amyloliquefaciens in preparing products for preventing and/or treating helicobacter pylori infection.
The invention also provides a product for preventing and/or treating helicobacter pylori infection, which is characterized in that the active ingredients of the product comprise the bacillus amyloliquefaciens and/or the metabolites thereof.
Further, the active ingredient of the product also comprises Lactobacillus paracasei (Lactobacillus paracasei) JY062.
Further, the product is a pharmaceutical product.
Further, the medicine also comprises pharmaceutically acceptable carriers and/or auxiliary materials.
Further, the medicament is in the form of powder, granules, capsules, tablets, pills or oral liquid.
The invention also provides application of the bacillus amyloliquefaciens and/or the metabolite thereof in preparing helicobacter pylori inhibitors.
The invention also provides a helicobacter pylori inhibitor, and the active ingredients of the helicobacter pylori inhibitor comprise the bacillus amyloliquefaciens and/or the metabolites thereof.
Further, the active ingredient of the helicobacter pylori inhibitor also comprises lactobacillus paracasei JY062.
The invention discloses the following technical effects:
the bacillus amyloliquefaciens JY022 is obtained through separation, and the metabolite and the thalli of the bacillus amyloliquefaciens have strong inhibition effect on helicobacter pylori.
The Lactobacillus paracasei JY062 has strong adhesion capability to prevent the colonization of helicobacter pylori, and the metabolites and thalli of the Lactobacillus paracasei JY062 and the Bacillus amyloliquefaciens JY022 are combined to have strong inhibition capability to helicobacter pylori, so that the Lactobacillus paracasei JY062 and the Bacillus amyloliquefaciens JY022 can jointly act to prevent and eliminate helicobacter pylori invaded into the body.
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 needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of adhesion testing of different probiotics;
FIG. 2 shows the results of the cross-coagulation ability test of different probiotics with helicobacter pylori.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
EXAMPLE 1 isolation, screening, identification and preservation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JY022
1. Bacterial strain isolation and screening
Sampling from red fermented bean curd of northeast traditional fermented dairy product, adding appropriate amount of PBS, homogenizing, mixing, gradient diluting, culturing in LB agar medium for 8 hr at 37deg.C, and activating and identifying single colony.
2. Identification of JY022 strain obtained by separation
(1) Morphological identification
Gram-positive staining, short rod (0.7-0.9 μm. Times.1.8-3.0 μm), and sporulation, mesogenic oval spores.
(2) Molecular biological identification
After extracting DNA and carrying out 16S sequencing, comparing the sequences, and determining that the JY022 strain is bacillus amyloliquefaciens.
Bacillus amyloliquefaciens JY02216S sequence (SEQ ID NO. 1):
CCGGGGCGGCGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAGGCATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCGAAGGTGACAGATGG。
3. preserving
Bacillus amyloliquefaciens JY022 was deposited at the microorganism strain collection center of Guangdong province at 8 months of 2022, with the deposit address of the institute of microorganisms of Guangdong province, hirship 100, guangzhou, building 5, and the deposit number of GDMCC No:62718.
example 2
The strains used in this example were Bacillus amyloliquefaciens JY022, lactobacillus paracasei (Lactobacillus paracasei) JY062 and standard strain of helicobacter pylori, wherein Lactobacillus paracasei JY062 was isolated from Tibetan traditional fermented milk (see "Zhang Yu for details: an adhesion and tolerance evaluation" of high-yield exopolysaccharide hypoglycemic Lactobacillus paracasei JY062 (TD 062 "); standard strains of H.pylori were purchased from North Biotechnology Co.Ltd.
1. Method of
1.1 cultivation of bacteria
The strains are all taken out from a refrigerator at the temperature of minus 80 ℃, wherein the lactobacillus paracasei JY062 is inoculated into an MRS broth culture medium at the content of 4 percent, and is cultured for 20 hours at the temperature of 37 ℃ to be activated for two generations; bacillus amyloliquefaciens JY022 is inoculated into LB liquid medium with 2 percent of inoculum size and is cultured for 8 hours under the condition of 180rpm/min at 37 ℃ for two generations of activation; the H.pylori standard strain was directly microaerophilically cultured in Columbia medium at 37℃for 72h.
1.2 measurement of Lactobacillus adhesion Rate fluorescence labelling was chosen. Mainly comprises the culture of Caco-2 cells, the fluorescent labeling of probiotics, the co-culture of lactobacillus and Caco-2 cells and the calculation of the adhesion rate.
Culturing Caco-2 cells: taking out Caco-2 cells frozen in a liquid nitrogen tank, rapidly thawing in a water bath at 37deg.C, adding into DMEM complete culture solution containing 20% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin, gently mixing, centrifuging (1000 rpm,5 min) to remove residual DMSO (dimethyl sulfoxide), collecting cells, transferring into a cell culture flask containing complete culture medium, and thawing at 37deg.C and 5% (v/v) CO 2 Incubate in incubator, change culture medium every 1 d. When incubated to the polarized state, they were washed with sterile PBS (ph=7.4) and digested with 0.25% (m/v) pancreatin-EDTA, then passaged 1:3.
Fluorescent labeling of probiotics: 5mg of viable cell fluorescent tracer probe (cFDA-SE) was weighed and dissolved in 8.969mL of DMSO to give a 1mM fluorescent stock solution. Centrifuging to collect probiotics at the end of log phase, repeatedly washing bacterial mud with sterile PBS for three times, re-suspending bacterial mud in PBS, and adjusting bacterial liquid concentration to 10 9 cfu/mL. And (3) adding the prepared fluorescent stock solution into the probiotic suspension to ensure that the final concentration is 20 mu m/L, immediately transferring into a water bath at 37 ℃ and placing the bacterial strain in a dark place for 20min to ensure that the bacterial strain is fully marked, and then washing the bacterial strain with PBS buffer solution for 3 times to remove the residual fluorescent dye in the bacterial solution. First, the effect of fluorescent labeling is detected by using a flow cytometer, and the initial fluorescence intensity of the strain after fluorescent labeling is measured and recorded by using a fluorescence spectrophotometer. The marked bacteria are then re-grownSuspending in high sugar culture solution (containing 4500mg/L glucose, 584mg/L sodium glutamate, 110mg/L sodium pyruvate, 3700mg/L sodium bicarbonate, pH 7.2-7.4), and adjusting bacterial concentration to 10 9 cfu/mL for use.
Co-culturing lactobacillus and Caco-2 cells: caco-2 cells cultured to the polarized state were cultured at 10 from the cell culture flask 5 Inoculating the cells/holes into a six-hole plate, replacing the culture solution with a double-antibody-free high-sugar culture solution 24 hours before adding bacteria, and culturing until the single-layer cells are fully paved on the six-hole plate. The culture solution in the six-well plate was aspirated and washed twice with PBS, the bacterial suspension was added, and the culture was performed in a cell incubator for 2 hours, three replicates were set for each group, and the whole procedure was performed in the absence of light.
And (3) calculating an adhesion rate: bacterial suspension in six well plates incubated for 2h was aspirated, washed twice with PBS, and unbound lactic acid bacteria were removed. Then, 0.5mL of pancreatin was added to each well for 3min, and then 1.0mL of incomplete culture solution was added to terminate digestion, and the mixed solution of cells and lactic acid bacteria in the wells was collected, and the fluorescence intensity of the lactic acid bacteria suspension was measured with a fluorescence spectrophotometer. The fluorescence detection conditions are as follows: excitation wavelength is 490nm, emission wavelength is 520nm. The adhesion rate was calculated as follows:
adhesion (%) = fluorescence intensity after adhesion/strain initial fluorescence intensity x 100%.
1.3 determination of the in vitro helicobacter pylori inhibiting Capacity of the bacterial suspension and the supernatant
1.3.1 acquisition of bacterial suspension and supernatant
Centrifuging cultured probiotic liquid at 6000r/min for 10min, collecting supernatant, storing at-20deg.C, suspending the precipitate with small amount of sterile PBS, dispersing the thallus uniformly, measuring absorbance at 600nm, and dispersing with PBS to obtain OD 600 Adjusting to 0.4-0.6 to obtain bacterial suspension.
1.3.2 in vitro determination of helicobacter pylori inhibition Capacity
The bacterial colony of helicobacter pylori after two generations of activation on Columbia solid culture medium is picked and is prepared into bacterial suspension by using sterile PBS, and the bacterial suspension is quantified by using a Mitsubishi turbidimeter tube to be 10 8 cfu/mL, dripping 90 μL onto 90mm Columbia blood plate, and sterilizing the bacterial suspension on the plate with sterile cotton swabEvenly spreading, punching a flat plate by using a sterile oxford cup, wherein the aperture is 6mm, the hole depth is 5mm, 4-5 holes are formed in each plate, after the bottom of each hole is sealed by using 0.8% agar liquid, 120 mu L of JY 062-bacterial suspension, 120 mu L of JY 062-supernatant, 120 mu L of JY 022-bacterial suspension, 120 mu L of JY 022-supernatant, 60 mu L of JY 062-bacterial suspension+60 mu L of JY 022-bacterial suspension, 60 mu L of JY 062-supernatant+60 mu L of JY 022-supernatant and 60 mu L of JY 022-supernatant are respectively added into the holes, and PBS and normal saline are simultaneously added as blank control groups; metronidazole is added dropwise as positive control, the liquid level is as close as possible to the surface of the blood plate, the blood plate cannot overflow, and the plate is placed in a microaerophilic bag for culturing at the constant temperature of 37 ℃ for 72 hours. And measuring the diameter of the inhibition ring on the flat plate by using a caliper.
1.4 determination of the capability of the probiotics to cross-aggregate with helicobacter pylori
Respectively sucking 2mL of probiotic bacterial suspension and helicobacter pylori bacterial suspension in a test tube, uniformly mixing by vortex oscillation, respectively measuring the absorbance at 600nm at 0h, 6h, 12h, 18h and 24h, and calculating the interaction condensation rate between bacteria according to the following steps:
cross-agglomeration rate= (1-a) mix /A 0 )×100％
Wherein: a is that 0 The absorbance value of the mixed solution is 0h, A mix Absorbance values for each time point of the mixture.
2. Results
2.1 adhesion results for different probiotics are shown in table 1 and figure 1.
TABLE 1 adhesion Rate of different probiotics
| Strain name | Adhesion rate/% |
| Lactobacillus paracasei JY062 | 25.13 |
| Bacillus amyloliquefaciens JY022 | 12.5 |
2.2 results of the detection of the cross-agglomerating ability of different probiotics with helicobacter pylori are shown in Table 2 and FIG. 2.
TABLE 2 detection of the Cross-agglomeration Capacity of different probiotics and helicobacter pylori
| Time/h | Lactobacillus paracasei JY062 | Bacillus amyloliquefaciens JY022 |
| 6 | 45.3 | 45.6 |
| 12 | 52.6 | 49.3 |
| 18 | 55.7 | 51.3 |
| 24 | 58.7 | 56 |
2.3 the inhibitory effect of different probiotic bacterial suspensions and supernatants on helicobacter pylori in vitro is shown in Table 3.
TABLE 3 inhibition of helicobacter pylori in vitro by different probiotic suspensions and supernatants
In summary, the Lactobacillus paracasei JY062 has strong adhesion capability to prevent the colonization of helicobacter pylori, the Lactobacillus paracasei JY062 and the Bacillus amyloliquefaciens JY022 have strong cross-coagulation capability with helicobacter pylori, and the metabolites and the thalli of the Lactobacillus paracasei JY062 and the Bacillus amyloliquefaciens JY022 are combined to have strong inhibition capability to the helicobacter pylori, so that the Lactobacillus paracasei JY062 and the Bacillus amyloliquefaciens JY022 can jointly act to prevent and eliminate the helicobacter pylori invaded into the body.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. Bacillus amyloliquefaciens with helicobacter pylori resisting effectBacillus amyloliquefaciens) The method is characterized in that the microorganism strain is preserved in the microorganism strain preservation center of Guangdong province at 8-month 20 of 2022, the preservation address is the microbiological institute of Guangdong province, building 5 building university, hirship 100, guangzhou City, and the preservation number is GDMCC No:62718.
2. use of a strain of bacillus amyloliquefaciens according to claim 1 for the preparation of a product for the prevention and/or treatment of helicobacter pylori infection.
3. A product for preventing and/or treating helicobacter pylori infection, characterized in that an active ingredient of the product comprises the bacillus amyloliquefaciens of claim 1.
4. A product according to claim 3, wherein the active ingredients of the product further comprise lactobacillus paracasei @Lactobacillusparacasei)JY062。
5. The product of claim 4, wherein the product is a pharmaceutical product.
6. The product of claim 5, wherein the pharmaceutical product further comprises a pharmaceutically acceptable carrier and/or adjuvant.
7. The product according to claim 6, wherein the pharmaceutical product is in the form of powder, granule, capsule, tablet, pill or oral liquid.
8. Use of a strain of bacillus amyloliquefaciens according to claim 1 for the preparation of a helicobacter pylori inhibitor.
9. A helicobacter pylori inhibitor, characterized in that an active ingredient of the helicobacter pylori inhibitor comprises the bacillus amyloliquefaciens of claim 1.
10. The helicobacter pylori inhibitor according to claim 9, characterized in that the active ingredient of the helicobacter pylori inhibitor further comprises lactobacillus paracasei JY062.
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| WO2007064741A2 (en) * | 2005-11-29 | 2007-06-07 | Kemin Industries, Inc. | The use of bacillus pb6 for the prophylaxis or treatment of gastrointestinal and immuno-related diseases |
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| CN114686396A (en) * | 2021-12-23 | 2022-07-01 | 微康益生菌(苏州)股份有限公司 | Helicobacter pylori-resistant bacillus coagulans BC99 and application thereof |
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| WO2007064741A2 (en) * | 2005-11-29 | 2007-06-07 | Kemin Industries, Inc. | The use of bacillus pb6 for the prophylaxis or treatment of gastrointestinal and immuno-related diseases |
| KR20200040984A (en) * | 2018-10-10 | 2020-04-21 | 경북대학교 산학협력단 | Pharmaceutical composition for preventing or treating gastrointestinal disorder comprising as an active ingredient a compound isolated from Bacillus amyloliquefaciens RWL-1 culture medium |
| CN114686396A (en) * | 2021-12-23 | 2022-07-01 | 微康益生菌(苏州)股份有限公司 | Helicobacter pylori-resistant bacillus coagulans BC99 and application thereof |
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