Application of lactobacillus acidophilus La1255 in preparation of medicine for preventing and treating helicobacter pylori infection

Abstract

The invention relates to application of lactobacillus acidophilus La1255 in preparation of a medicine for preventing and treating helicobacter pylori infection, and the lactobacillus acidophilus La1255 is used as a first active component for inhibiting the formation of a helicobacter pylori biological membrane, assisting in improving the antibacterial effect of antibiotics and recovering gastrointestinal micro-ecology. The lactobacillus acidophilus La1255 disclosed by the invention can inhibit the formation of a helicobacter pylori biological membrane, assists antibiotics to improve the antibacterial effect of helicobacter pylori, reduces the level of cell inflammation caused by helicobacter pylori, reduces cell damage caused by the use of the antibiotics, improves the cell survival rate, and has a good application prospect. And dysbacteriosis and reduction of the number of florae caused by use of antibiotics are recovered.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to application of Lactobacillus acidophilus La1255 in preparing medicines for resisting Helicobacter pylori infection. Background Art

[0002] Helicobacter pylori can colonize in the human stomach and cause gastric diseases. The World Health Organization and the U.S. Department of Health and Human Services have both classified Helicobacter pylori as a Class I carcinogen. Antibiotic therapy is currently commonly used for its treatment. However, since Helicobacter pylori has the ability to form biofilms, the so-called biofilm refers to the formation of a multicellular three-dimensional structure of extracellular polymeric substances (EPS) secreted by Helicobacter pylori when sensing an adverse external environment. Once Helicobacter pylori forms a biofilm, it can lead to drug resistance in Helicobacter pylori, making its therapeutic drugs ineffective at conventional doses. It needs to be increased to a higher concentration of more than 10 times to achieve the purpose of inhibiting the growth of Helicobacter pylori. However, due to the safety of drug use, overdose is usually not advisable. The use of antibiotics at conventional doses is difficult to control Helicobacter pylori, and eventually leads to persistent infection. Therefore, it is crucial to inhibit the formation of Helicobacter pylori biofilms, reduce the drug resistance of Helicobacter pylori, and achieve a good antibacterial effect on Helicobacter pylori at conventional doses. Summary of the invention

[0003] The purpose of the present invention is to overcome the defects in the prior art and provide an application of Lactobacillus acidophilus La1255 in the preparation of a drug for resisting Helicobacter pylori infection. The Lactobacillus acidophilus La1255 can inhibit the formation of Helicobacter pylori biofilm and / or remove the formed Helicobacter pylori biofilm. When used in combination with antibiotics, it can not only prevent Helicobacter pylori from developing drug resistance, but also have a synergistic effect with the antibiotics, thereby improving the antibacterial effect of Helicobacter pylori, reducing the level of cell inflammation caused by Helicobacter pylori, reducing cell damage caused by the use of antibiotics, improving cell survival rate, and restoring the dysbiosis and decreased bacterial population caused by the use of antibiotics.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] The invention discloses an application of Lactobacillus acidophilus La1255 in the preparation of an anti-Helicobacter pylori infection drug, characterized in that the Lactobacillus acidophilus La1255 is used as a first active ingredient to inhibit the formation of Helicobacter pylori biofilm.

[0006] As a further technical solution, the Lactobacillus acidophilus La1255 adopts Lactobacillus acidophilus La1255 bacteria and / or fermentation liquid.

[0007] As a further technical solution, the anti-Helicobacter pylori infection includes one or more of inhibiting the growth of Helicobacter pylori, inhibiting the formation of Helicobacter pylori biofilm, clearing the formed Helicobacter pylori biofilm, reducing the level of inflammation induced by Helicobacter pylori, reducing cell damage caused by antibiotics, and restoring intestinal flora damage caused by antibiotics.

[0008] As a further technical solution, the medicine also contains a second active ingredient.

[0009] As a further technical solution, the second active ingredient is an antibiotic.

[0010] As a further technical solution, the antibacterial concentration of the antibiotic is 6.6 mg / mL.

[0011] An anti-Helicobacter pylori infection composition comprises Lactobacillus acidophilus La1255 and antibiotics.

[0012] As a further technical solution, the antibiotic is amoxicillin.

[0013] A pharmaceutical preparation comprising the composition also includes pharmaceutically acceptable excipients.

[0014] As a further technical solution, the auxiliary material includes a protective agent.

[0015] The protective agent includes one or more of chitosan oligosaccharide, stachyose, lactic acid powder, clove powder, honeysuckle powder, and liquorice powder;

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention screens a large number of probiotics and finds that Lactobacillus acidophilus La1255 has a synergistic effect when used in combination with antibiotics, can significantly inhibit the growth of Helicobacter pylori, reduce the level of cell inflammation caused by Helicobacter pylori, reduce cell damage caused by the use of antibiotics, improve cell survival rate, and restore the dysbacteriosis and decline in the number of flora caused by the use of antibiotics. In addition, Lactobacillus acidophilus La1255 can also inhibit the formation of Helicobacter pylori biofilm, avoid its drug resistance, and solve a series of problems caused by the increase in drug resistance due to the easy formation of biofilm by Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a microscopic image of Helicobacter pylori biofilm formation in Example 2 of the present invention;

[0019] exist Figure 1 Middle, a: hp group, b: hp + Lactobacillus acidophilus La1255 group;

[0020] Figure 2 This is a plate diagram of the inhibition zone of Helicobacter pylori La1255 and antibiotics in Example 3 of the present invention;

[0021] Figure 3 This is a diagram showing the antibacterial effect of Lactobacillus acidophilus La1255 and antibiotics on Helicobacter pylori in Example 3 of the present invention;

[0022] Figure 4 This is a diagram showing the effects of Lactobacillus acidophilus La1255 and antibiotics on cell damage in Example 4 of the present invention;

[0023] Figure 5 This is a graph showing the effects of Lactobacillus acidophilus La1255 and antibiotics on cellular inflammation levels in Example 5 of the present invention;

[0024] Figure 6 This is a graph showing the effects of Lactobacillus acidophilus La1255 and antibiotics on chain-broken fatty acids in Example 6 of the present invention;

[0025] Figure 7 This is a graph showing the effects of Lactobacillus acidophilus La1255 and antibiotics on fecal flora in Example 7 of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0028] In the present invention, Lactobacillus acidophilus La1255, the preservation number is CGMCC No.28910, the preservation unit is: China General Microbiological Culture Collection Administration Committee General Microbiological Center, the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the preservation date is November 8, 2023.

[0029] Unless otherwise specified, the raw materials used in the present invention are all commercially available.

[0030] Example 1: Study on the regulatory effect of Lactobacillus acidophilus La1255 on proteins related to Helicobacter pylori biofilm formation

[0031] 1. Test methods

[0032] In this example, Lactobacillus acidophilus La1255, Lactobacillus acidophilus L1254, Lactobacillus helveticus L1306, Lactobacillus johnsonii L1049, Lactobacillus acidophilus La28, and Lactobacillus helveticus L1082 were tested to evaluate their regulatory effects on proteins related to Helicobacter pylori biofilm formation. The test method is as follows:

[0033] The activated Helicobacter pylori was inoculated into a cell culture plate and cultured in a microaerobic environment. During the period, the strain was added for intervention, and each well was filled with Brucella broth (culture medium) supplemented with 7% fetal bovine serum. A control without adding the strain (i.e., no intervention) was also made. The culture was cultured under microaerobic conditions at 37°C for 3 days (80-100rpm), the biofilm was collected, and RNA was extracted for subsequent experiments. The transcription levels of the film-forming factors Fur, luxS, and flaA were calculated, and whether they had an inhibitory effect on the biofilm was evaluated based on their transcription levels. The results are shown in Table 1.

[0034] The primers used to amplify the target genes for detecting the transcription levels of film-forming factors Fur, luxS, and flaA are:

[0035] luxS: 5′-TTCAAGCAGCCCACCAAGATCACAT-3′;

[0036] luxS: 5′-CCATAGGCGACCAATCCAGACATAA-3′;

[0037] Fur: ​​5′-GGAGTACGGTCGCAAGATTAAA-3′;

[0038] Fur: ​​5′-CTAGCGGATTCTCTCAATGTCAA-3′;

[0039] flaA: 5′-GTCGACATGGCTTTICAGGTCAA-3′;

[0040] flaA: 5′-CTGCAGCTAAGTTAAAAGCCTTA-3′.

[0041] The primers for detecting the internal reference gene are:

[0042] 16srRNA-F: 5′-GGCGACCTGCTGGAACATTACTGAC-3′;

[0043] 16srRNA-R: 5′-CCAGGCGGGATGCTTAATGCGTTAG-3′.

[0044] Take 0.8 μL of each upstream and downstream primers, 10 μL of 2×SYBR Green, 2 μL of cDNA template, and 6.0 μL of DEPC water to prepare a 20 μL PCR system. The amplification conditions are 95°C, 1 min pre-denaturation, 15 s denaturation at the same temperature, 58°C, 20 s annealing, 72°C extension 25 s, and 40 cycles. Select the internal reference gene and calculate the 2-△△Ct value based on the Ct value.

[0045]

[0046] Result evaluation method:

[0047] 1) When (2-△△Ct value) 试验组 / (2-△△Ct value) 对照组 <1, it is represented as “+”, indicating an inhibitory effect on biofilm;

[0048] 2) When (2-△△Ct value) 试验组 / (2-△△Ct value) 对照组 ≥1, it is represented as “-”, indicating that it has no inhibitory effect on biofilm.

[0049] 2. Results and Analysis

[0050] Table 1

[0051]

[0052] As can be seen from Table 1, Lactobacillus acidophilus La1255 has a significant inhibitory effect on the genes Fur, luxS, and flaA related to the formation of Helicobacter pylori biofilm, while L28 and L1198 only have an inhibitory effect on the Fur gene, and other strains have no inhibitory effect on the genes Fur, luxS, and flaA related to the formation of Helicobacter pylori biofilm. Therefore, it can be seen that Lactobacillus acidophilus La1255 can inhibit the formation of Helicobacter pylori biofilm.

[0053] Example 2: Study on the inhibitory effect of Lactobacillus acidophilus La1255 on the formation of Helicobacter pylori biofilm

[0054] 1. Test methods

[0055] The activated Helicobacter pylori was inoculated into a cell culture plate and cultured in a microaerobic environment. During this period, Lactobacillus acidophilus La1255 strain was added for intervention, and each well was filled with Brucella broth (culture medium) supplemented with 7% fetal bovine serum. A control without Lactobacillus acidophilus La1255 strain was also made; the culture was cultured under microaerobic conditions at 37°C with shaking for 3 days (80-100rpm). The coverslip was removed and washed with phosphate-buffered saline (PBS). The coverslip was then air-dried and stained with crystal violet (CV) for 30 seconds. After staining, the coverslip was rinsed with distilled water, then air-dried, and examined under a crystal violet staining microscope. The results are shown in Figure 1 .

[0056] 2. Results and Analysis

[0057] Through the in vitro culture test of biofilm, we know that compared with the Hp control group without the addition of Lactobacillus acidophilus La1255, the integrity of the biofilm in the Hp+La1255 group with the addition of Lactobacillus acidophilus La1255 was destroyed and the total amount of biofilm was significantly reduced (see Figure 1 ); Therefore, it can be seen that Lactobacillus acidophilus La1255 can inhibit the formation of Helicobacter pylori biofilm and can remove the formed Helicobacter pylori biofilm.

[0058] Example 3: Study on the inhibitory effect of Lactobacillus acidophilus La1255 supernatant combined with antibiotics on the growth of Helicobacter pylori I. Test method

[0059] First, prepare a Columbia medium plate containing sheep blood, then use MRS liquid medium as a negative control; activate and culture Lactobacillus acidophilus La1255 for three consecutive generations; centrifuge the bacterial solution at 8000g for 10 minutes and filter it through a 0.22μm sterile filter membrane to obtain the supernatant; prepare an antibiotic solution as a positive control, and use the Oxford cup method to determine its inhibitory effect on Helicobacter pylori. The results are shown in the figure. Figure 2-3 .

[0060] The grouping is as follows:

[0061] MRS group: MRS liquid culture medium was used.

[0062] Amoxicillin group: 6.6 mg / mL amoxicillin was added;

[0063] La1255 group: added with supernatant of Lactobacillus acidophilus La1255;

[0064] La1255+amoxicillin group: Lactobacillus acidophilus La1255 supernatant and 6.6 mg / mL amoxicillin were added

[0065] 2. Results and Analysis

[0066] from Figure 2-3It can be seen that: 1) MRS culture medium has almost no inhibitory effect on the growth of Helicobacter pylori or the inhibitory effect is very low; 2) the antibiotic amoxicillin and Lactobacillus acidophilus La1255 supernatant can both inhibit the growth of Helicobacter pylori; 3) the inhibitory effect of Lactobacillus acidophilus La1255 supernatant combined with amoxicillin on the growth of Helicobacter pylori is significantly better than that of amoxicillin and Lactobacillus acidophilus La1255 used alone. Therefore, it can be seen that: Lactobacillus acidophilus La1255 supernatant and amoxicillin have a synergistic effect in inhibiting the growth of Helicobacter pylori, and can be used in combination to inhibit the growth of Helicobacter pylori.

[0067] Example 4: Study on the effect of Lactobacillus acidophilus La1255 on cell survival rate

[0068] 1. Test method: Follow the test kit operation instructions;

[0069] S1. Prepare cell suspension:

[0070] Enzymatic hydrolysis: The monolayer cultured AGS cells were digested with trypsin, and the suspended cultured cells were collected to prepare a single cell suspension with a cell density of 10 4 Pieces / mL.

[0071] Adding samples: Pipette 40 μL of the above cell suspension and 40 μL of trypan blue, mix by pipetting and stain for 2 minutes, place a cover slip in the middle of the two grooves of the counting plate, gently blow the cell suspension with a pipette, aspirate 20 μL of cell suspension, add the cell suspension to one side of the cover slip on the counting plate, and do not add the sample amount that overflows the cover slip.

[0072] Counting: Under a microscope, use a 10× objective lens to observe the number of cells in the large squares at the four corners of the counting plate. When the cells are pressed against the midline, only count those on the left and above, and ignore those on the right and below.

[0073] Calculation: Calculate the cell density using the following formula.

[0074] Number of cells / ml stock solution = (sum of the number of cells in the four large grids / 4) × 10 4 × dilution factor, the purified cells were inoculated in a 96-well plate at a density of 5 × 10 4 cells.

[0075] S2. Inoculate into 96-well plates: according to the appropriate number of cells to be plated, each well has about 100 μl of cell suspension, and 5 replicate wells can be set up. Culture in a 37°C incubator. It takes about 16 hours for cells to adhere to the wall after inoculation. Observe the cell growth status of each well under a microscope. The growth density is close to 90%. Then group them according to the following grouping conditions:

[0076] AGS group: no treatment, culture time 12-16h;

[0077] Antibiotic + HP group: first add 1×10 8 CFU Hp was cultured for 8-12 hours, and then 6.6 mg / mL amoxicillin was added for 2-4 hours;

[0078] Antibiotics + Hp + La1255 group: 1×10 8 CFU of H. pylori was cultured for 8-12 hours, then 6.6 mg / mL amoxicillin was added for 2-4 hours, and finally 1×10 8 CFU of Lactobacillus acidophilus La1255 was cultured for 4h-8h.

[0079] Also make a blank control without adding AGS and a control without adding AGS but adding La1255;

[0080] Blank control group: In the previous step, 100 μl of blank culture medium was added to each well instead of 100 μl of cell suspension. After the microscopic examination was qualified, no treatment was performed and the culture time was 12-16 h.

[0081] La1255 control group: In the previous step, 100 μl of blank culture medium was added to each well instead of 100 μl of cell suspension. After the microscopic examination was qualified, 1×10 8 CFU of Lactobacillus acidophilus La1255 was cultured for 4-8h;

[0082] S3. Ensure that 10 μL of CCK-8 is added to each well by replacing the solution, with a final concentration of 10%. Gently tap the culture plate to help mix.

[0083] S4. Culture the cells in a cell culture incubator for 1 hour and measure them using dual wavelengths, with a detection wavelength of 450 nm and a reference wavelength of 600 nm. Finally, subtract the reference wavelength value from the detection wavelength value and calculate the AGS cell survival rate using the following formula.

[0084] Calculation formula:

[0085] Cell survival rate = [(As-Ab1) / (Ac-Ab)] × 100%;

[0086] Among them, As1: absorbance of the experimental well antibiotic + Hp + La1255 group (culture medium containing AGS cells, CCK-8, Hp, antibiotics, and Lactobacillus acidophilus La1255);

[0087] As2: absorbance of the experimental well antibiotic + hp group (culture medium containing AGS cells, CCK-8, Hp, antibiotics);

[0088] Ac: absorbance of the control well AGS group (culture medium containing AGS cells, CCK-8);

[0089] Ab: absorbance of blank well and blank control group (blank culture medium, CCK-8);

[0090] Ab1: absorbance of the La1255 control group (blank culture medium, CCK-8, Lactobacillus acidophilus La1255) in the experimental well.

[0091] Assuming the cell survival rate of the AGS group is 1, the cell survival rate of each experimental group divided by the cell survival rate of the AGS group is the relative cell survival rate of each experimental group. Figure 4

[0092] 2. Results and Analysis

[0093] In vitro cell experiments were conducted to establish a Helicobacter pylori infection model. The cell survival rate of the antibiotic + Hp + La1255 group with the addition of antibiotics and Lactobacillus acidophilus La1255 was higher than that of the antibiotic + Hp group with antibiotics alone ( Figure 4 ), it can be seen that Lactobacillus acidophilus La1255 can reduce the damage of antibiotics and Helicobacter pylori to cells, and the effect is more obvious than using antibiotics alone.

[0094] Example 5: Study on the effect of Lactobacillus acidophilus La1255 combined with antibiotics on inflammation induced by Helicobacter pylori

[0095] 1. Test methods

[0096] The activated AGS cells were seeded in 24-well plates (seeding concentration was 5×10 4 / mL, culture volume 200μL) co-cultured for 24h, and the dead AGS cells were washed away with complete culture medium. They were divided into three groups: Group 1: complete culture medium containing AGS; Group 2: Hp was first inoculated and then antibiotics (6.6mg / mL amoxicillin) were added; Group 3: Hp was first inoculated and then antibiotics (6.6mg / mL amoxicillin) were added, and then Lactobacillus acidophilus La1255 cells were added, the culture medium was collected, the culture medium was centrifuged at 8000g for 10min, the precipitate was collected, and RNA was extracted to detect the transcription level of IL-8.

[0097] The primers used to amplify the target gene for detecting the transcription level of IL-8 are:

[0098] IL-8F: 5′-CCAGGAAGAAAACCACCGGAAG-3′,

[0099] IL-8R: 5′-TGGCAAAACTGCACCTTCACA-3′.

[0100] The primers used to amplify the internal reference gene for detecting the transcription level of IL-8 are:

[0101] β-actin-F: 5′-AGCGAGCATCCCCCAAAGTT-3′,

[0102] β-actin-R: 5′-GGGCACGAAGGCTCATCATT-3′.

[0103] Take 0.8 μL of each upstream and downstream primers, 10 μL of 2×SYBR Green, 2 μL of cDNA template, and 6.0 μL of DEPC water to prepare a 20 μL PCR system. The amplification conditions are 95°C, 1 min pre-denaturation, 15 s denaturation at the same temperature, 58°C, 20 s annealing, 72°C extension 25 s, and 40 cycles. Select the internal reference gene and calculate the 2-△△Ct value based on the Ct value.

[0104]

[0105] Assuming the 2-△△Ct value of the AGS group is 1, the 2-△△Ct value of each experimental group is divided by the 2-△△Ct value of the AGS group to obtain the relative transcription level of IL-8 in each experimental group. The results are shown in Figure 5 .

[0106] 2. Results and Analysis

[0107] The Helicobacter pylori infection model was established by in vitro cell experiments, and antibiotics and Lactobacillus acidophilus La1255 were added

[0108] The transcription level of IL-8 cells in the +Hp+La1255 group was significantly lower than that in the antibiotic +Hp group using antibiotics alone (see Figure 5 ).

[0109] Therefore, it can be seen that Lactobacillus acidophilus La1255 and antibiotics have a synergistic effect in reducing the level of cellular inflammation induced by Helicobacter pylori, and the two can be used in combination.

[0110] Example 6: Study on the effect of Lactobacillus acidophilus La1255 combined with antibiotics on fecal short-chain fatty acids

[0111] 1. Test methods

[0112] The fecal samples were pretreated by weighing, diluting and filtering. The reagents were added to the treated fecal samples for in vitro fermentation culture for 24 hours, centrifuged at 1000r / min for 5 minutes, the supernatant was aspirated into a new 2mL centrifuge tube, centrifuged at 12000r / min for 5 minutes, the supernatant was aspirated into a new 2mL centrifuge tube, and stored at 4°C in the dark, waiting for pretreatment. Take 1mL of fermentation supernatant, add 100L of concentrated hydrochloric acid and shake thoroughly to mix, add 2mL of anhydrous ether to extract for 20min, centrifuge at 3000r / min for 5min, take the supernatant, add NaOH to extract for 20min, centrifuge at 3000r / min for 5min, take the lower aqueous phase, add 100μL of concentrated hydrochloric acid, filter through 0.22μm water membrane and transfer to a liquid phase bottle, and use high performance liquid chromatography to detect short-chain fatty acids (butyric acid). The content is expressed as peak area. The results are shown in the table. Figure 6 .

[0113] The HPLC parameters are as follows:

[0114] Mobile phase: acetonitrile, phosphate; Chromatographic column: Agilent C18.

[0115] Grouping:

[0116] Feces group: no drug was added to the feces;

[0117] Feces + antibiotic group: 6.6 mg / mL of amoxicillin was added to the feces.

[0118] Feces + antibiotics + La1255 group: 6.6 mg / mL amoxicillin and 1×10 6 CFU of Lactobacillus acidophilus La1255.

[0119] 2. Results and Analysis

[0120] Short-chain fatty acids are metabolites of the in vivo flora, and the in vivo flora participates in regulating the function of the host through short-chain fatty acids. The present invention treats the feces, and then respectively introduces antibiotics, antibiotics and Lactobacillus acidophilus La1255 for in vitro fermentation. The content of acetic acid in the samples of the feces + antibiotics + La1255 group after fermentation is increased, and the improvement effect is obvious compared with the feces + antibiotics group treated with antibiotics alone (see Figure 6 ). Therefore, it can be seen that Lactobacillus acidophilus La1255 and antibiotics have a synergistic effect in improving the recovery of bacterial flora caused by antibiotic treatment of Helicobacter pylori, and the two can be used in combination.

[0121] Example 7: Study on the effect of Lactobacillus acidophilus La1255 combined with antibiotics on fecal flora

[0122] 1. Test methods

[0123] 1. Method for obtaining stool samples of each group: After diluting the stool, inoculate it into an in vitro fermentation vial (purchased from Hailu, Hangzhou) and culture it for 24 hours. Then, add the agent according to the following grouping and continue fermentation to obtain stool samples.

[0124] Among them, the grouping situation is:

[0125] Feces group: no drug was added to the feces;

[0126] Feces + antibiotic group: 6.6 mg / mL amoxicillin was added to the feces.

[0127] Feces + antibiotics + La1255 group: 6.6 mg / mL amoxicillin and 1×10 6 CFU of Lactobacillus acidophilus La1255.

[0128] 2. Detection of fecal flora:

[0129] Weigh the fecal sample, add the lysis solution, shake and mix, and place in a 65℃ water bath for 15 minutes; place the centrifuge tube on the magnetic rack, dry it at room temperature for 15 minutes, remove it and add 80μl buffer I, shake and mix, and incubate at 56℃ for 5 minutes to elute the DNA. During this period, turn it upside down 5 times every 1-2 minutes to mix it, and fully separate the DNA from the magnetic beads. Place the centrifuge tube on the magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely adsorbed (the magnetic beads are concentrated on one side of the centrifuge tube), carefully transfer the DNA solution (supernatant) to a new centrifuge tube to obtain the fecal microbial genome, which can be kept at -20℃. The microbial genome of each group was tested separately, and the relative ratio of the indicator bacteria content was measured. The results are shown in Figure 7 .

[0130] Primers for detecting total fecal bacteria are:

[0131] all-F: 5′-AGAGTTTGATCCTGGCTCAG-3′,

[0132] all-R: 5′-GGACTACCAGGGTATCTAATCCTGTT-3′.

[0133] 2. Results and Analysis

[0134] The total amount of gastrointestinal bacteria is about ten times that of cells in our body, and the number of genes in the gastrointestinal flora is also much higher than that of the human body, about 100 times that of the human body. The flora in the body is involved in regulating many functions of the human body. The present invention treats the feces, and then respectively connects antibiotics, antibiotics and Lactobacillus acidophilus La1255 for in vitro fermentation. The total amount of bacteria in the fermentation sample of the feces + antibiotics + La1255 group is increased, and the improvement effect is obvious compared with the feces + antibiotics group treated with antibiotics alone. Therefore, it can be seen that Lactobacillus acidophilus La1255 can accelerate the recovery speed of the flora and solve the problem of decreased flora caused by antibiotic treatment of Helicobacter pylori.

[0135] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. The application of Lactobacillus acidophilus La1255 in the preparation of a drug for preventing and treating Helicobacter pylori infection, characterized in that: The Lactobacillus acidophilus La1255 is used as the first active ingredient to inhibit the formation of Helicobacter pylori biofilm and / or remove the formed Helicobacter pylori biofilm.

2. The use according to claim 1, characterized in that: The anti-Helicobacter pylori infection includes one or more of inhibiting the growth of Helicobacter pylori, inhibiting the formation of Helicobacter pylori biofilm, clearing the formed Helicobacter pylori biofilm, reducing the level of inflammation induced by Helicobacter pylori, reducing cell damage caused by antibiotics, and restoring intestinal flora damage caused by antibiotics.

3. The use according to claim 1, characterized in that: The medicament also contains a second active ingredient.

4. The use according to claim 3, characterized in that: The second active ingredient is an antibiotic.

5. An anti-Helicobacter pylori infection composition, characterized in that: Includes Lactobacillus acidophilus La1255 and antibiotics.

6. The anti-Helicobacter pylori infection composition according to claim 5, characterized in that: The antibiotic used is amoxicillin.

7. A pharmaceutical preparation comprising the composition of claim 5, characterized in that: A protective agent is also included.

8. The pharmaceutical preparation according to claim 7, characterized in that The protective agent comprises one or more of chitosan oligosaccharide, stachyose, lactic acid powder, clove powder, honeysuckle powder and liquorice powder.

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