CN116606781A - Lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof - Google Patents
Lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof Download PDFInfo
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- CN116606781A CN116606781A CN202310861908.0A CN202310861908A CN116606781A CN 116606781 A CN116606781 A CN 116606781A CN 202310861908 A CN202310861908 A CN 202310861908A CN 116606781 A CN116606781 A CN 116606781A
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- lactobacillus plantarum
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- 238000012809 post-inoculation Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229960002181 saccharomyces boulardii Drugs 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000011450 sequencing therapy Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- OSWPSODKJQKKLC-UHFFFAOYSA-M sodium ethanol 2-hydroxybenzoate Chemical compound [Na+].CCO.OC1=CC=CC=C1C([O-])=O OSWPSODKJQKKLC-UHFFFAOYSA-M 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
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Abstract
The invention relates to the technical field of functional microorganism screening and application, in particular to lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof. The strain has strong tolerance to gastrointestinal tract environment, can effectively degrade cholesterol, has remarkable antioxidation effect, and can obviously inhibit the growth and adhesion of helicobacter pylori. The strain is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan, china on 4 months and 10 days of 2023, and the preservation number is CCTCC NO: m2023501, can be widely applied to the fields of foods, health products or medicines.
Description
Technical Field
The invention relates to the technical field of functional microorganism screening and application, in particular to lactobacillus plantarum with antagonism to helicobacter pylori and application of the lactobacillus plantarum in foods, health-care products and medicines.
Background
Helicobacter pyloriHelicobacter pylori，Hp) Is present in the gastric epithelium and is able to penetrate the superficial mucus while also residing under the mucus and living on the surface of the gastric epithelium. The bacteria are mainly planted on the gastric, oral or intestinal mucosa and the like by flagella, are microaerophilic gram-negative bacillus, have great radical treatment difficulty after infection, are easy to repeatedly attack by patients, continuously develop peptic ulcer or atrophic gastritis in the course of disease, even develop gastric cancer, and cause great trouble to vast patients. Helicobacter pylori damages gastric mucosal epithelial cells by secreting catalase, lipase, proteolytic enzyme, toxins, etc. From 10 to 20% of people infected with helicobacter pylori may eventually develop peptic ulcers, eradicating helicobacter pylori, especially before cancerous changes, may be effective in reducing the risk of cancer occurrence.
Current methods for eradication of helicobacter pylori are triple therapy, quadruple therapy with or without bismuth (concomitant therapy, sequential therapy and mixed therapy) and double therapy. The standard mode of treatment is still a proton pump plus two antibiotics. However, as the use of antibiotics increases, the problem of antibiotic resistance is attracting more and more attention, and the disorder of antibiotics is also being reduced or avoided as much as possible. In addition, the recurrence rate of antibiotic treatment in clinical treatment is high, and the disease of most patients is repeated, so that the treatment difficulty is increased due to repeated infection of helicobacter pylori. And adverse reactions such as diarrhea, abdominal distension, nausea and vomiting often occur after the administration of the medicine to patients, so that the removal of helicobacter pylori is more difficult. Although there have been studies from the development of new antibiotics to replace the conventional antibiotics or to add strong antibacterial agents (bismuth compounds) in therapy or to use new proton pumps to increase the inhibition rate of helicobacter pylori, other side effects (especially in the presence of bismuth compounds) have been added. But the introduction of new antibiotics has also created new concerns. Thus, methods of acting against helicobacter pylori are of great importance for the treatment of helicobacter pylori.
In recent years, the clinical recommendation of the auxiliary treatment of gastritis by using probiotics is that good treatment effects are achieved. Probiotics are emerging alternatives to the treatment of gastrointestinal disorders, which act by modifying the microbiota as antibacterial agents or immunomodulators. The report shows that the probiotics can effectively kill helicobacter pylori for the helicobacter pylori infected patients, improve the clinical treatment effect and strengthen the gastritis rehabilitation of the patients. Domestic researches report that certain strains of lactic acid bacteria have the effect of relieving the side effects of antibiotics, and meanwhile, the external application also reports that certain lactic acid bacteria can be used in combination with antibiotics and proton pumps (mainly lactobacillus, bifidobacterium and saccharomyces boulardii), so that the eradication rate can be improved, and the side effects caused by the antibiotics can be reduced.
At present, a plurality of microbial products capable of inhibiting helicobacter pylori exist on the market, but the disadvantage of poor gastric juice tolerance exists generally, and probiotic bacterial strains must meet certain survival and vitality requirements to successfully reach gastric epithelium in a digestive tract to exert antibacterial effect. Therefore, screening of probiotics having a strong acid resistance and a remarkable inhibitory effect on helicobacter pylori is still the current research focus.
Disclosure of Invention
The invention aims at providing a novel lactobacillus plantarum strain with the helicobacter pylori antagonism functionLactiplantibacillus plantarum). The strain has strong tolerance to gastrointestinal tract environment, can effectively degrade cholesterol, has remarkable antioxidation effect, and can obviously inhibit helicobacter pyloriThe growth and adhesion effects can be widely applied to functional foods, health care products or medicines.
The invention provides a lactobacillus plantarum which is separated from sour dough and named as lactobacillus plantarumLactiplantibacillus plantarum) The VHProbi S26 strain is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan, china on the 4 th month 10 day 2023: m2023501.
The lactobacillus plantarum VHProbi S26 strain has a 16S rDNA sequence of SEQ ID NO:1.
the RAPD fingerprint of the Lactobacillus plantarum VHProbi S26 strain is shown in figure 4, the rep-PCR fingerprint is shown in figure 5, and the MALDI-TOF-MS protein fingerprint is shown in figure 6.
The invention also provides application of the lactobacillus plantarum VHProbi S26 strain in preparing foods.
The invention also provides application of the lactobacillus plantarum VHProbi S26 strain in preparation of health-care products.
The invention also provides application of the lactobacillus plantarum VHProbi S26 strain in preparing medicines with the function of preventing or treating gastritis.
The invention also provides a helicobacter pylori inhibitor, which comprises at least one of the live bacteria, the inactivated bacteria, the fermentation metabolite or the intracellular extract of the lactobacillus plantarum.
The invention also provides application of the helicobacter pylori inhibitor in preparing foods, health-care products or medicines.
The lactobacillus plantarum VHProbi S26 strain has strong acid resistance, and the viable count value reaches 8.01 Log CFU/mL after being subjected to rescreening by an acid-resistant culture medium; the plant can grow at 15 ℃ and 45 ℃, can grow at 1% -8% salt concentration, and has an optimal tolerance salt concentration of 5%. The degradation rate of the strain on cholesterol reaches 21.27%, and the degradation rate on salt-containing cholesterol also reaches 8.4%; the clearance rate of the bacterial suspension to DPPH free radical and HRS free radical reaches 23.73% and 41.41%, and the inhibition rate of lipid peroxidation reaches 48.63 +/-0.41%; the clearance rate of the fermentation supernatant to the HRS free radical is more up to 90.46%, and the antioxidation effect is remarkable.
The Lactobacillus plantarum VHProbi S26 strain can effectively inhibit the growth of helicobacter pylori, and the diameter of a bacteriostasis circle reaches 17.0+/-0.165 mm; the addition amount of the cell-free fermentation supernatant is positively correlated with the inhibition rate of helicobacter pylori, and when the addition amount reaches 15%, the inhibition rate of helicobacter pylori reaches 33.39%, so that unexpected technical effects are achieved. In addition, the strain can obviously reduce the adhesion effect of helicobacter pylori on human gastric adenocarcinoma cells (BGC-823), the adhesion inhibition rate is up to 29.30%, and the strain can be combined with helicobacter pylori to form obvious agglutinate and has stronger coagglutination capability.
The lactobacillus plantarum VHProbi S26 does not produce hemolysin, can not dissolve blood cells, and has good safety. Is sensitive to common antibiotics such as erythromycin, ampicillin, tetracycline, clindamycin and the like, has good biological safety, can be widely used in the production of functional foods or health care products, can be used for preparing medicines for preventing or treating diseases such as gastritis, gastric ulcer and the like caused by helicobacter pylori, and has broad prospect.
Drawings
FIG. 1 is a colony chart of strain S26;
FIG. 2 is a gram of strain S26;
FIG. 3 is a graph of API test results for strain S26;
FIG. 4 is a RAPD fingerprint of strain S26;
FIG. 5 is a rep-PCR fingerprint of strain S26;
FIG. 6 shows MALDI-TOF-MS fingerprint of strain S26;
FIG. 7 is a diagram showing the zone of inhibition of helicobacter pylori by strain S26;
FIG. 8 is a graph showing the inhibitory effect of the strain S26 on H.pylori adhesion to human gastric adenocarcinoma cells;
FIG. 9 is a coagglutination pattern of strain S26 against H.pylori.
Detailed Description
The lactobacillus plantarum VHProbi S26 provided by the invention meets the requirement of regulation, and is a newly discovered strain through multiphase taxonomy identification. The lactobacillus plantarum VHProbi S26 provided by the invention has the application of inhibiting the growth of helicobacter pylori, and has important application value for preventing or treating digestive tract diseases such as gastritis, gastric ulcer and the like caused by helicobacter pylori.
The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The invention will be further illustrated with reference to specific examples.
EXAMPLE 1 isolation screening of Lactobacillus plantarum VHProbi S26
MRS liquid medium: 1000mL of purified water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 1.0mL of Tween 80, 2.0g of citric acid diamine, 20g of calcium carbonate, 0.58 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate heptahydrate, 15g of agar, pH adjustment of 6.2-6.5 and high-pressure sterilization at 121 ℃ for 15min.
MRS solid medium: agar 15g was added to the liquid medium for sterilization.
1.1 Lactobacillus primary screening
Taking 1g of sour dough, flushing the sour dough by using sterile normal saline, putting the sour dough into a sterile sample bag, and beating and uniformly mixing the sour dough by using a homogenizer; and (3) taking 100 mu L of mixed solution, carrying out gradient dilution, coating the mixed solution on an MRS solid plate culture medium, and then culturing at 37 ℃ for 48 hours, and carrying out microscopic examination on a single colony after the plate grows. According to the microscopic examination result, the applicant screens out 50 potential lactobacillus strains, which are named S1, S2, … … and S50 respectively.
1.2 Lactobacillus re-screening
Taking 1L of MRS liquid culture medium, adding 3.2g of porcine mucosa pepsin, shaking to dissolve, and placing in a 37 ℃ water bath shaking table for water bath for 1h to prepare the acid-resistant culture medium. 50 strains of lactobacillus S1, S2, … … and S50 obtained by screening are respectively inoculated into the acid-resistant culture medium according to the inoculum size of 6 percent, and are subjected to stationary culture for 72 hours at 37 ℃, and fermentation liquor is taken for bacterial count.
The result shows that the S26 strain in the 50 lactobacillus fermentation liquid is subjected to acid-resistant culture medium rescreening, the viable count is the greatest, and the logarithmic value is as high as 8.01 Log CFU/mL.
Example 2 identification of strains
The inoculum preparation in this example was as follows: under the aseptic condition, a proper amount of fresh S26 bacterial liquid is taken, centrifuged for 5min at 5000rpm/min, washed for 2 times by PBS buffer, and then the bacterial cells are resuspended by the same volume of PBS buffer to be used as inoculation liquid.
2.1 Colony morphology identification
The S26 strain was inoculated on MRS agar medium and cultured at 37℃for 48 hours. As shown in FIG. 1, the colony pattern of the S26 strain is milky white, the colony diameter is about 1.5-2mm, and the surface is wet.
As shown in FIG. 2, the S26 strain has positive gram staining, short bar shape under microscope, and round two ends.
2.2 physiological Biochemical identification
2.2.1 salinity tolerance test
Under aseptic conditions, 190. Mu.L of MRS liquid culture medium with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% was added to the 96-well plate, 3 replicates of each salt concentration, and then 10. Mu.L of inoculation liquid was added, and wells without bacteria were used as controls. 50. Mu.L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37deg.C, and observing whether the culture medium becomes turbid.
The results show that the S26 strain can grow at a salt concentration of 1% -8%, and the optimal tolerance salt concentration is 5%.
2.2.2 Temperature tolerance test
Inoculating the inoculation liquid into 10mL MRS liquid culture medium according to 10% inoculation amount, taking 5mLMRS liquid culture medium without inoculating bacteria as control, respectively placing into 15 ℃ constant temperature incubator for 7 days, 45 ℃ constant temperature incubator for 2 days, and observing whether the bacterial liquid becomes turbid.
The results showed that S26 strain can reproduce at 15℃and 45 ℃.
2.2.3 Catalase experiment
The fresh bacterial liquid is taken, dropped on a clean glass slide, and then a drop of 3% hydrogen peroxide solution is dropped on the glass slide, and the S26 strain is observed to generate no bubbles and is a negative reaction.
2.2.4 Carbon source metabolism test
The carbon source metabolism experiment of strain S26 was validated using API 50CHL reagent. The API 50CHL reagent can be used to identify differences in the strain at the genus or species level. The experimental method and the result analysis are specifically described in the API 50CHL kit instruction.
The API test results are shown in FIG. 3, and the ID value of the S26 strain and the lactobacillus plantarum is 99.9%, which is an excellent identification result. The bacteria can be primarily identified as the lactobacillus plantarum according to the carbon source metabolism resultLactiplantibacillus plantarum）。
2.2.5 Glucose acidogenesis and gas production test
The formula of the culture medium is as follows: peptone 0.5g; 0.3g of yeast extract; tween 80.1 ml; salt solution a 0.5mL; salt solution B0.5 mL; 0.5g of sodium acetate; glucose 2.5g; 0.05mL of 2% bromocresol green (w/v); distilled water 100mL; the pH is 6.8-7.0. The prepared culture medium was dispensed into large tubes containing inverted small tubes, 3 mL/tube, and autoclaved at 121℃for 15min.
Salt solution A: KH (KH) 2 PO 4 10g、K 2 HPO 4 1.0g, dissolved in distilled water, was fixed to a volume of 100mL.
Salt solution B: mgSO (MgSO) 4 ·7H 2 O 11.5g、MnSO 4 ·2H 2 O 2.4g、FeSO 4 ·7H 2 O0.68 g was dissolved in distilled water to a volume of 100mL.
Under aseptic conditions, inoculating the culture medium with 10% of inoculation amount, taking the culture medium without inoculating bacteria as a control, sealing the top with 2mL of aseptic liquid paraffin, culturing at 37 ℃ for 24 hours, and observing whether the color of the culture medium changes.
The results show that: after 24h of culture at 37 ℃, the culture medium turns from green to yellow, and no gas exists in the small inverted tube, which indicates that the S26 strain produces acid and does not produce gas.
2.3 molecular biological identification
2.3.1 16s rDNA Gene sequence analysis
1. Genomic DNA extraction
Reference was made to the Tiangen bacterial genomic DNA extraction kit (catalog number: DP 302).
2. 16s rDNA Gene amplification
(1) Primer sequence:
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
(2) Reaction system (50. Mu.L)
TABLE 1 16s rDNA PCR amplification System
| Composition of components | Reaction volume |
| 10×PCR buffer | 5μL |
| dNTPs | 4μL |
| 27F | 2μL |
| 1492R | 2μL |
| DNA | 2.5μL |
| rTaq | 0.5μL |
| ddH 2 O | 34μL |
(3) Electrophoresis verifies that the PCR product meets the requirement when the nucleic acid electrophoresis result is about 1500 bp.
(4) Sequencing PCR products: the 16S rDNA sequence of the S26 strain is obtained by sequencing, and the sequence is compared in NCBI database, and the S26 strain is preliminarily determined to be lactobacillus plantarum @Lactiplantibacillus plantarum). SEQ ID NO: the sequence 1 is specifically as follows:
CCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTCAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACTGC。
2.3.2 RAPD and rep-PCR fingerprint identification
1. RAPD fingerprint identification
(1) Primer sequence: m13 (5'-GAGGGTGGCGGTTCT-3');
(2) RAPD reaction system
TABLE 2 RAPD reaction System
| Reaction components | Volume of |
| TaqDNA polymerase (5U/. Mu.L) | 0.2 μL |
| 10 XBuffer (containing Mg) 2+ ） | 2 μL |
| Primer (10 uM) | 1 μL |
| dNTPs（2.5 mM） | 0.8 μL |
| DNA template | 2 μL |
| Sterile double distilled water | 14 μL |
| Total volume of | 20 μL |
(3) Electrophoresis: 1.5% agarose gel plates were prepared, DL2000 DNA markers were used as a result control, 100V electrophoresis was performed for 80min at a constant pressure, and the electropherograms were detected using a gel imaging system. RAPD finger print of S26 strain is shown in FIG. 4.
2. rep-PCR fingerprint
(1) rep-PCR primer: CTACGGCAAGGCGACGCTGACG.
(2) reaction system of rep-PCR
TABLE 3 reaction System for rep-PCR
| Reaction components | Volume of |
| r TaqDNA polymerase | 0.2μL |
| 10X Ex Taq DNA Buffer (containing Mg) 2+ ） | 2μL |
| Primer (10 uM) | 1 μL |
| dNTPs（2.5 mM） | 2μL |
| DNA template | 2μL |
| Sterile double distilled water | 12.8 μL |
(3) Electrophoresis: the voltage is 100V, and the electrophoresis time is 80min to detect the amplification result. DL2000 DNA Marker served as a result control. The rep-PCR fingerprint of S26 strain is shown in FIG. 5.
2.3.3 MALDI-TOF-MS detection of strain ribosomal protein expression
S26, centrifuging fresh bacterial liquid at 5000rpm/min for 5min, washing with sterile water for 4 times, and airing surface moisture. And then a small amount of fresh thalli is uniformly coated on a target plate in a film form, 1 mu L of lysate is added to cover the sample, after the sample is dried, 1 mu L of matrix solution is added to cover the sample, after the sample is dried, the sample target is put into a mass spectrometer for identification. Protein fingerprint is obtained by utilizing Autofms 1000 analysis software Autof Analyzer v1.0, and main ion peaks of the strain S26 are as follows:m/z 2594.3893932.785, 5192.135, 7865.555, 9500.578, etc., and the results of the authentication are shown in fig. 6.
In conclusion, by combining the colony morphology, the physiological and biochemical characteristic results and the molecular biological identification results of the S26 strain, it can be concluded that the S26 strain is a novel lactobacillus plantarum, and is named as lactobacillus plantarum VHProbi S26%Lactiplantibacillus plantarum VHProbi S26）。
EXAMPLE 3 test of haemolytic and antibiotic resistance of Lactobacillus plantarum VHProbi S26
Preparation of lactobacillus plantarum VHProbi S26 bacterial suspension:
selecting purified lactobacillus plantarum VHProbi S26 colony, inoculating the colony into fresh MRS liquid culture medium, and culturing for 24 hours at 37 ℃; inoculating the strain into MRS liquid culture medium according to the inoculum size of 1% (V/V), and continuously culturing at 37 ℃ for 24-48 h; taking 1ml of lactobacillus plantarum VHProbi S26 fresh bacterial liquid, centrifuging at 5000rpm for 5min, and respectively collecting fermentation supernatant and thalli; washing thallus with PBS buffer solution of pH7.4 for 2 times, and regulating bacterial concentration to 5×10 with PBS buffer solution 7 CFU/mL (OD 600 absorbance value about 0.4) to give a bacterial suspension.
3.1 Hemolysis test
The components of TBS basal medium (tryptone 17.0 g, soytone 3.0 g, sodium chloride 5.0g, potassium dihydrogen phosphate (anhydrous) 2.5g, glucose 2.5g, distilled water 1000.0 ml) were weighed and dissolved, and then autoclaved at 121℃for 15min, and 5% of sterilized defibrinated sheep blood was added and mixed evenly when the medium was cooled to 50℃and poured into a plate. And (3) streaking and inoculating the test strain to a prepared blood cell plate, culturing in an incubator at 37 ℃ for 24-48 hours, and observing whether the test strain has a hemolysis phenomenon.
The results show that: the Lactobacillus plantarum VHProbi S26 was able to grow and the blood cell plates were unchanged, indicating that the Lactobacillus plantarum VHProbi S26 did not produce hemolysin and was unable to lyse blood cells.
3.2 Antibiotic resistance test
Preparing antibiotics: ampicillin, erythromycin, gentamicin, streptomycin and tetracycline are prepared into stock solution of 2048 mug/mL, and the stock solution is preserved at-20 ℃ for standby. When in use, the storage liquid is subjected to 2-time serial gradient dilution by using the MRS liquid culture medium to form a use liquid, wherein the gradient dilution concentration is 1-1024 mu g/mL and 11 gradients are total.
Minimal inhibitory concentration MIC values of antibiotics against lactobacillus plantarum VHProbi S26 were determined by a micro broth dilution method.
Sequentially adding MRS liquid culture medium without antibiotics into the 1 st column of the 96-well plate as a negative control, sequentially adding 190 mu L of MRS liquid culture medium with antibiotics with different concentrations into the 2 nd-12 nd column, then respectively inoculating 10 mu L of the inoculation liquid, making 3 parallel wells, and taking 1 well of the non-added bacteria liquid as a blank.
(2) 50. Mu.L of paraffin oil was added to cover the water and prevent evaporation.
(3) The 96-well plate was incubated at 37℃for 24 hours, then removed, and OD was measured 600 Values, MIC values of antibiotics against strains were counted with 24h results, see table 4.
TABLE 4 antibiotic MIC values for Lactobacillus plantarum VHProbi S26
| Erythromycin MIC | Erythromycin R/S | Gentamicin MIC | Gentamicin R/S | Streptomycin MIC | Streptomycin R/S | Ampicillin MIC | Ampicillin R/S | Tetracycline MIC | Tetracycline R/S |
| 4 | / | 32 | / | 64 | / | 2 | / | 2 | / |
MIC units μg/mL.
From Table 4, the lactobacillus plantarum VHProbi S26 provided by the invention is sensitive to common antibiotics such as erythromycin, tetracycline, ampicillin and the like, and has good biological safety.
Example 4 gastric juice tolerance test of Lactobacillus plantarum VHProbi S26
9ml of artificial gastric juice was placed on a 37℃water bath shaker for 1h to simulate the human body temperature. 1mL of fresh bacterial liquid of Lactobacillus plantarum VHProbi S26 (10) 8 cfu/mL or so), added to 9mL of artificial gastric juice, and placed on a 37℃water bath shaker (200 rpm) for 2h. 1ml of sample was taken at 0h and 2h post inoculation, respectivelyAnd measuring the quantity of the living bacteria.
The result shows that the lactobacillus plantarum VHProbi S26 is digested by gastric juice for 2 hours, and the bacterial load is reduced by only 0.07 Log CFU/mL, so that the lactobacillus plantarum VHProbi S26 is proved to have strong gastric juice tolerance.
EXAMPLE 5 inhibitory Effect of Lactobacillus plantarum VHProbi S26 on helicobacter pylori
5.1 And (3) culturing pathogenic bacteria:
the frozen and preserved helicobacter pylori (ATCC 353909/ATCC 354364) is spread on Columbia blood agar medium and placed at 37 ℃ in 10% CO 2 Culturing for 72h under the condition; scraping bacterial mud into brain heart infusion liquid culture medium under aseptic condition, and culturing at 37deg.C for 24 hr under micro-oxygen condition (oxygen concentration 5%, carbon dioxide concentration 10%, nitrogen concentration 85%, which is volume%); taking 5mL of bacterial liquid, centrifuging at 5000r/min for 5min, re-suspending bacterial cells with phosphate buffer solution (PBS, pH 7.4), and adjusting helicobacter pylori bacterial liquid concentration to about 10 6 cfu/mL。
5.2 And (3) bacteriostasis circle test:
the method is carried out by adopting an oxford cup double-layer flat plate method.
In a clean workbench, taking a sterile flat plate, sterilizing nutrient agar, pouring the sterilized nutrient agar into the flat plate, and spreading the flat plate to serve as a lower culture medium; after the agar is solidified, uniformly spreading 10ml of Columbia blood agar culture medium on the upper layer to serve as an upper layer culture medium; after the upper layer culture medium is solidified, sucking 0.2mL of helicobacter pylori bacterial liquid on a solid culture medium plate, and uniformly coating; placing for 1h, and punching after bacterial liquid on the surface is fixed on the surface of the flat plate, wherein the diameter of the hole is 8mm, and the depth of the hole is 3mm; 0.1mL of the Lactobacillus plantarum VHProbi S26 strain suspension described in example 3 was injected into the well, and after culturing at 37℃for 8 hours, the results were observed and the diameter of the inhibition zone was measured.
As shown in FIG. 7, the diameter of the antibacterial circle of the Lactobacillus plantarum VHProbi S26 on helicobacter pylori reaches 17+/-0.16 mm, which shows that the strain has remarkable inhibition effect on helicobacter pylori.
EXAMPLE 6 inhibitory Effect of Lactobacillus plantarum VHProbi S26 fermentation supernatant on helicobacter pylori growth
(1) Experimental group:
the fresh bacterial solutions of helicobacter pylori were inoculated into brain heart infusion liquid culture medium to which 2.5% (v/v), 5% (v/v) and 10% (v/v) of the lactobacillus plantarum VHProbi S26 fermentation supernatant described in example 3 were added, respectively, at a volume ratio of 1%.
(2) Control group:
inoculating fresh helicobacter pylori bacterial liquid into brain heart infusion liquid culture medium according to a volume ratio of 1%; 37 ℃ and 10% CO 2 Culturing for 24h under the condition; the OD values of the culture solutions were measured at 600 nm wavelength, respectively. The growth inhibition ratio of Lactobacillus plantarum VHProbi S26 cell-free fermentation supernatant to helicobacter pylori was calculated as 100% of the OD600 value of the control culture broth. The specific results are shown in Table 5.
Growth inhibition (%) = (control OD 600-experimental OD 600)/control OD600 x 100%.
TABLE 5 growth inhibition of Lactobacillus plantarum VHProbi S26 fermentation supernatant on helicobacter pylori
| Supernatant inoculum size | Inhibition rate | Standard deviation of |
| 2.5% | 4.31% | 0.19% |
| 5% | 12.10% | 0.31% |
| 10% | 23.51% | 0.71% |
| 15% | 33.39% | 0.54% |
From the results shown in Table 5, the addition of the non-cell fermentation supernatant of the Lactobacillus plantarum VHProbi S26 can significantly inhibit the growth of pathogenic helicobacter pylori, and the inhibition rate of helicobacter pylori is continuously improved along with the increase of the addition amount of the fermentation supernatant of the Lactobacillus plantarum VHProbi S26; when the addition amount reaches 15%, the inhibition rate of helicobacter pylori reaches 33.39%, and unexpected technical effects are achieved.
EXAMPLE 7 adhesion inhibition experiment of Lactobacillus plantarum VHProbi S26 on helicobacter pylori
(1) Culture of human gastric adenocarcinoma cells (BGC-823): human gastric adenocarcinoma cells (BGC-823) are taken out from the liquid nitrogen tank, resuscitated and subcultured, and the cells are diluted. Human gastric adenocarcinoma cells (BGC-823) are inoculated into a six-hole culture plate with built-in cell climbing plates and 10% fetal bovine serum (DMEM) culture solution, and the number of cell plates of each hole is about 2 multiplied by 10 6 cells, six well plates were placed in a carbon dioxide incubator for 24 hours.
(2) Adhesion inhibition test: the BGC-823 single cell layer adhered to the six-hole plate is washed 3 times by using PBS buffer solution, 1mL of lactobacillus plantarum VHProbi S26 and helicobacter pylori suspension described in the example 3 are respectively added, cells without the strain are used as blank control, and the blank control is placed into a carbon dioxide incubator for culturing for 2 hours. The cell slide was repeatedly washed 3 times with PBS buffer to remove non-adherent bacteria. Fixing with anhydrous methanol for 20 min, taking out the cell climbing sheet, air drying, gram staining, observing 20 random fields under a 100 times oil microscope, and calculating the number of helicobacter pylori adhered on each cell. The ability of Lactobacillus plantarum VHProbi S26 to inhibit the adhesion of helicobacter pylori to BGC-823 cells was evaluated by comparing the change in the adhesion of helicobacter pylori in the presence and absence of Lactobacillus plantarum VHProbi S26 and examining the decrease in the adhesion of helicobacter pylori in the presence of Lactobacillus plantarum VHProbi S26 with the adhesion of Lactobacillus plantarum VHProbi S26 as 100%.
As a result, as shown in FIG. 8, when treated with VHProbi S26, which was not added with Lactobacillus plantarum, the adhesion rate of H.pylori to human gastric adenocarcinoma cells was 1, and the adhesion inhibition rate of VHProbi S26, which was not added with Lactobacillus plantarum, to H.pylori was 29.30%. As is evident from the figure, the number of H.pylori adhering to the human gastric adenocarcinoma cells of the experimental group is significantly smaller than that of the control group. Thus, it was demonstrated that Lactobacillus plantarum VHProbi S26 significantly inhibited the adhesion of H.pylori to human gastric adenocarcinoma cells.
EXAMPLE 8 Lactobacillus plantarum VHProbi S26 agglutination adsorption assay
300. Mu.L of the Lactobacillus plantarum VHProbi S26 strain suspension described in example 3 was added to a 24-well plate, followed by 300. Mu.L of the helicobacter pylori strain suspension (10 6 cfu/mL) as experimental group; equal amounts of the Lactobacillus plantarum VHProbi S26 bacterial suspension and buffer were mixed as control groups, 2 replicates per control and experimental group. The 24-well plate was placed in a microplate thermostatted shaker at 400rpm/min, room temperature and incubated with shaking. And observing and photographing by a microscope, recording the initial orifice plate state and the orifice plate states at different times, and observing whether agglutination phenomenon occurs.
As a result, as shown in FIG. 9, the binding of Lactobacillus plantarum VHProbi S26 to helicobacter pylori showed a significant agglutinate.
Example 9 adhesion experiments of human intestinal epithelial cells (Caco-2)
Culture of human intestinal epithelial cells (Caco-2) cells:
and taking out Caco-2 cells from the liquid nitrogen tank, resuscitating, subculturing, and amplifying the number of the cultured cells to the required dosage. Subsequent experiments can be performed when the cell growth confluence is observed to be close to 80% under an inverted microscope. The original medium was discarded, rinsed twice with PBS buffer, and a suitable amount of pancreatin was added. After adding pancreatin, the cells were returned to the incubator and visually inspectedAfter the complete shedding of the cells is observed, adding a culture solution with the volume of 2-3 times of pancreatin to stop digestion, repeatedly blowing for about ten times, and observing under a mirror to form a single cell state as much as possible. The single cell suspension is sucked into a 15ml or 50ml centrifuge tube, centrifuged for 5 minutes at 1000 revolutions, the supernatant is discarded, the cell sediment is scattered slightly, and a proper amount of new culture medium is added for blowing and resuspension. The cell counting plate is used for counting cells, and a proper amount of PBS is used for diluting the cell suspension, and the cell dilution is recommended to be 20-50 cells per big cell. The number of plates per well cell in the six-well plate was 1.5X10 6 The amount of the culture medium added per well was 2ml. The six-hole plate is placed in a carbon dioxide incubator for 24 hours, and a subsequent cell adhesion experiment can be performed.
2. Adhesion test:
washing the adhered Caco-2 single cell layer in the six-hole plate with PBS for 2 times; 1mL of the cell culture solution without the resistance is respectively added into the experiment, 1mL of the lactobacillus plantarum VHProbi S26 bacterial suspension described in the example 3 is put into a carbon dioxide incubator for 2h of culture; repeatedly washing with PBS for 5 times to remove non-adhering bacteria; digestion was stopped by adding 500ul pancreatin for 3 min, then 1.5ml cell culture medium was added, repeated pipetting was performed, and the resulting solution was collected into sterile EP tubes and the collected solution was subjected to 10-fold, 100-fold, 1000-fold, 10000-fold gradient dilution, plating to count the bacterial load.
Meanwhile, lactobacillus rhamnosus LGG strain having strong cell adhesion was used as a control group, and the procedure was performed with reference to the above steps.
The adhesion ability of lactobacillus plantarum VHProbi S26 was calculated according to the following formula:
adhesion capacity (CFU/cells) =total number of bacteria adhered per culture well/total number of cells per culture well.
TABLE 6 adhesion of Lactobacillus plantarum VHProbi S26 to human intestinal epithelial cells (Caco-2)
| Strain | Adhesion capability | Standard deviation of |
| Lactobacillus plantarum VHProbi S26 | 2.08 | 0.1% |
| Lactobacillus rhamnosus LGG | 0.06 | 0.01% |
The adhesion test results are shown in Table 6, and the adhesion with lactobacillus rhamnosus LGG strain is used as a positive control, and the adhesion capability of lactobacillus plantarum VHProbi S26 to human intestinal epithelial cells is 34.6 times that of lactobacillus rhamnosus LGG to cells, which indicates that the adhesion capability of lactobacillus plantarum VHProbi S26 to cells is stronger, and provides a basis for the positive effect of lactobacillus plantarum VHProbi S26 in intestinal tracts.
EXAMPLE 10 cytotoxicity test of Lactobacillus plantarum VHProbi S26
The lactobacillus plantarum VHProbi S26 bacterial suspension described in example 3 was inactivated in a water bath at 70 ℃ for 20 minutes for later use.
Culture of human gastric adenocarcinoma cells (BGC-823): human gastric adenocarcinoma cells (BGC-823) are taken out from the liquid nitrogen tank, resuscitated and subcultured, and the cells are diluted. Human gastric adenocarcinoma cells (BGC-823) are inoculated into a six-hole culture plate with built-in cell climbing plates and 10% fetal bovine serum (DMEM) culture solution, and the number of cell plates of each hole is about 2 multiplied by 10 6 cells, six well plates were placed in a carbon dioxide incubator for 24 hours.
Cytotoxicity test: resuscitates human gastric adenocarcinoma cells BGC-823, inoculates into 24-hole culture plate containing 10% calf serum cell culture solution, inoculation density is 2×10 5 cells/well, cell culture for 24h. The inactivated lactobacillus plantarum VHProbi S26 was subjected to MOI (infection recovery)Number) of 10, and a blank group without bacteria was set and cultured for 24 hours. MTT solution was added to each cell culture well to be tested at a final concentration of 0.3mg/ml and incubated for 3h in a carbon dioxide incubator. The supernatant was carefully discarded, and 500ul of DMSO was added to each 24-well plate cell culture well and incubated at 37℃for 30min to allow the purple crystals to dissolve well. Absorbance at 490nm was measured.
The detection result shows that the lactobacillus plantarum VHProbi S26 has no obvious effect on the proliferation activity of human gastric epithelial cells BGC-823, has no cytotoxicity on human gastric cells, and has good safety.
EXAMPLE 11 measurement of antioxidant Capacity of Lactobacillus plantarum VHProbi S26
11.1 DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) scavenging ability determination
1mL of the Lactobacillus plantarum VHProbi S26 bacterial suspension described in example 3 was taken, 1mL of 0.4 mM DPPH free radical solution was added, the mixture was mixed uniformly, then the mixture was subjected to shading reaction at room temperature for 30min, and then the absorbance A sample of the sample at 517nm was measured for 3 times. The control group sample is equal volume PBS solution and DPPH-ethanol mixed solution, and blank zeroing is carried out by equal volume bacterial suspension and ethanol mixed solution. The clearance is calculated according to the following formula: clearance% = [1- (a sample-a blank)/a control ] ×100%.
The specific results are shown in Table 7.
TABLE 7 DPPH radical scavenging Rate
| Strain | Clearance rate% | Standard deviation of |
| Lactobacillus plantarum VHProbi S26 | 23.73% | 3.27% |
11.2 determination of Hydroxy Radical (HRS) scavenging Capacity
200ul of the bacterial suspension, 100ul of 5mM sodium salicylate-ethanol solution, 100ul of 5mM ferrous sulfate and 500ul of deionized water are uniformly mixed, 100ul of hydrogen peroxide solution (3 mM) is added, the absorbance of the bacterial suspension is measured at the wavelength of 510nm after 15min in a water bath at 37 ℃, and the clearance rate of the bacterial suspension to HRS is calculated. In addition, experiments were performed with equal doses of fermentation supernatant instead of bacterial suspension, and the clearance of the supernatant to HRS was determined. The hydroxyl radical scavenging rate was calculated according to the following formula. Clearance% = (a Sample of -A Control of ）/（A Blank space -A Control of ) X 100%, where A Control of Is the absorbance of the mixed solution of ferrous sulfate, hydrogen peroxide and sodium salicylate, A Blank space Is the absorbance of the mixed solution of ferrous sulfate and sodium salicylate. The specific results are shown in Table 8.
TABLE 8 scavenging of HRS free radicals by Lactobacillus plantarum VHProbi S26
| Lactobacillus plantarum VHProbi S26 | Clearance rate% | Standard deviation of |
| Thallus | 41.41% | 10.16% |
| Fermentation supernatant | 90.46% | 0.10% |
11.3 measurement of lipid peroxidation resistance
Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.
0.5 Adding 1mL linoleic acid emulsion and 1mL FAFESO into PBS solution (pH 7.4) 4 (1%) and 0.5mL sample, 37 ℃ water bath 1.5 h, 0.2mL TCA (4%), 2mL TBA (0.8%) and 100 ℃ water bath 30min, rapid cooling, 4000 rpm/min centrifugation 15min, collecting supernatant, and measuring absorbance at 532 nm to obtain A; the control group replaced the sample, A0, with 0.5mL distilled water. Inhibition rate/% = (A0-a)/a0×100%.
Note that: a is absorbance of a sample group; a0 is absorbance of the control group. The specific results are shown in Table 9.
TABLE 9 anti-lipid peroxidation inhibition rate tables
| Lactobacillus plantarum VHProbi S26 | Inhibition rate | Standard deviation of |
| Thallus | 48.63% | 0.41% |
| Fermentation supernatant | 14.69% | 0.65% |
| Intracellular extracts | 5.39% | 0.06% |
The results show that the lactobacillus plantarum VHProbi S26 provided by the invention has strong antioxidation capability, can effectively remove DPPH and HRS free radicals, and has remarkable lipid peroxidation resistance.
Example 12 in vitro cholesterol degradation experiments with Lactobacillus plantarum VHProbi S26
1g of cholesterol is accurately weighed, dissolved in absolute ethyl alcohol, and is fixed to a volume of 100mL, and is subjected to filtration sterilization by a microporous filter membrane of 0.22 mu m under the aseptic condition. Cholesterol measurement method according to GB/T5009.128-2003 < measurement of cholesterol in food >. Inoculating fresh bacterial liquid according to 0.1% of inoculation amount, standing at 37 ℃ for 48 hours, taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, uniformly mixing, standing for 10 minutes, centrifuging at 3000 r for 5 minutes, and taking supernatant for measuring cholesterol content.
The results show that: the degradation rate of the lactobacillus plantarum VHProbi S26 provided by the invention on cholesterol reaches 21.27%, and the degradation rate on salt-containing cholesterol also reaches 8.4%.
In conclusion, the lactobacillus plantarum obtained by separation in the inventionLacticaseibacillus paracasei) The VHProbi S26 strain has no hemolysis, is sensitive to antibiotics and strong in acid resistance, can effectively inhibit the growth of helicobacter pylori, degrade cholesterol and has remarkable antioxidation effect. In addition, the strain has certain adhesion capability to gastric cells and intestinal cells, can be aggregated with helicobacter pylori, increases the possibility that the helicobacter pylori is discharged out of the body along with gastrointestinal peristalsis, and can be widely applied to the fields of foods, health-care products or medicines. The strain is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan, china 4 months 10 years 2023, and the preservation number is CCTCC NO: m2023501.
Claims (8)
1. Lactobacillus plantarum strainLactiplantibacillus plantarum) Characterized in that the lactobacillus plantarum is preservedThe collection number is CCTCC NO: m2023501.
2. The lactobacillus plantarum of claim 1, wherein the RAPD fingerprint of lactobacillus plantarum is shown in figure 4; the rep-PCR fingerprint is shown in FIG. 5; the MALDI-TOF-MS protein fingerprint is shown in FIG. 6.
3. The lactobacillus plantarum of claim 1, wherein the 16s rDNA sequence of the lactobacillus plantarum is SEQ ID NO. 1.
4. Use of lactobacillus plantarum according to claim 1 for the preparation of a food product.
5. The use of lactobacillus plantarum according to claim 1 for the preparation of health products.
6. Use of lactobacillus plantarum according to claim 1 for the preparation of a medicament with the function of preventing or treating gastritis.
7. A helicobacter pylori inhibitor, characterized in that the inhibitor comprises at least one of live bacterial cells, inactivated bacterial cells, fermentation metabolites or intracellular extracts of Lactobacillus plantarum according to claim 1.
8. The use of the helicobacter pylori inhibitor according to claim 7 for the preparation of a food, a health product or a pharmaceutical product.
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