Application of BTS1 gene knockout saccharomyces cerevisiae in preparation of product for inhibiting growth of pathogenic bacteria

Abstract

The invention relates to an application of a BTS1 gene knockout saccharomyces cerevisiae in preparation of a product for inhibiting growth of pathogenic bacteria. The BTS1 gene with a nucleotide sequence shown as SEQ ID NO.1 is deleted from the saccharomyces cerevisiae. According to the invention, a single gene deletion strain taking BY4743 as a parent strain in a saccharomyces cerevisiae gene knockout library is screened and verified, and the BTS1 delta / delta saccharomyces cerevisiae is determined to have better antibacterial activity. After the BTS1 gene in the saccharomyces cerevisiae is deleted, the lactic acid content is increased, and an antibacterial effect can be generated; the BTS1 delta / delta saccharomyces cerevisiae cell-free supernatant shows broad-spectrum antibacterial activity in vivo and in vitro, especially for drug-resistant bacteria, the BTS1 delta / delta saccharomyces cerevisiae cell-free supernatant can obviously inhibit proliferation of pathogenic bacteria when co-cultured with the pathogenic bacteria, and shows low toxicity. Therefore, the BTS1 [delta] / [delta] saccharomyces cerevisiae and the fermentation liquor thereof have good application prospects in preparation of products for inhibiting growth of pathogenic bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of a strain of brewer's yeast with a BTS1 gene knocked out in preparing a product for inhibiting the growth of pathogenic bacteria. Background Art

[0002] At present, bacterial infection has become the second leading cause of death in the world, seriously threatening human health. Most deaths caused by bacterial infection are related to 33 common bacteria, among which 54.2% of deaths are caused by Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Klebsiella pneumoniae or Pseudomonas aeruginosa. Staphylococcus aureus causes the most deaths, followed by Escherichia coli.

[0003] When the human body is infected with bacteria, people tend to habitually take antibiotics, which can prevent bacteria from invading the body while the immune response clears infected cells and bacteria. However, with the abuse of antibiotics, bacteria will also evolve various resistance mechanisms through mutation and acquisition of antibiotic resistance genetic elements, thus producing multi-drug resistant "super bacteria". In recent years, bacterial resistance has become increasingly serious, posing a major challenge to anti-infection treatment. According to the "Antibiotic Resistance Research", by 2050, the number of deaths caused by antibiotic resistance is expected to be 10 million per year. Polymyxin is considered to be the last line of defense for the treatment of multi-drug resistant Gram-negative bacterial infections. However, in 2015, Chinese scholars first discovered a new plasmid-mediated polymyxin resistance gene mcr-1. The mcr-1 gene is carried very frequently in Escherichia coli in the intestines of animals and is transmitted between animals and humans, making the treatment of bacterial infections more difficult. In addition, methicillin-resistant Staphylococcus aureus (MRSA) is a common and highly toxic bacterium in clinical practice. It has broad-spectrum drug resistance and is resistant to commonly used clinical β-lactam and cephalosporin antibiotics. MRSA can also produce modified enzymes by changing the target of antibiotics, and develop varying degrees of resistance to aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, rifampicin and other antibiotics, making treatment extremely difficult. Therefore, there is an urgent need to develop novel, natural and effective drugs to fight bacterial infections.

[0004] Probiotics refer to "live microorganisms that can produce beneficial effects on the host when given in sufficient quantities". In recent years, more and more studies have shown that probiotics can play a probiotic role in intestinal health, immune development, nutritional metabolism, emotional management, liver disease, oral disease, gynecological diseases and skin health. Some intestinal probiotics can play a probiotic role by inhibiting the proliferation of pathogenic bacteria, enhancing the intestinal barrier, regulating intestinal flora, and synthesizing active metabolites. Currently, the widely recognized probiotics mainly include lactic acid bacteria, non-pathogenic Escherichia coli, Bacillus, Enterococcus and yeast, most of which come from the intestines or fermented foods. Compared with bacteria, yeast is larger in size and more resistant to antibiotics.

[0005] Saccharomyces cerevisiae, belonging to the subphylum Ascomycota, class Bacillus, order Cryptococcales, family Cryptococcaceae, genus Saccharomyces, is also known as brewer's yeast or budding yeast. It is a eukaryotic model organism because of its advantages such as single cell, fast reproduction, and existence in haploid and diploid states. The completion of the whole genome sequencing of Saccharomyces cerevisiae and the research and exploration of its genome and proteomics have provided a basis for the research and exploration of the genome structure and function of other eukaryotic organisms, especially higher organisms and humans, and especially provided a model for the study of human diseases. However, it is not clear which gene changes in Saccharomyces cerevisiae can inhibit bacterial growth, and it is impossible to genetically modify Saccharomyces cerevisiae in a targeted manner to produce an antibacterial effect. Summary of the invention

[0006] The purpose of the present invention is to provide a strain of brewer's yeast with a knocked-out BTS1 gene for use in preparing a product for inhibiting the growth of pathogenic bacteria. After the BTS1 in the brewer's yeast is knocked out, the metabolic process of the brewer's yeast is affected, the lactic acid content is increased, the pH is reduced, and better antibacterial activity is produced.

[0007] To achieve the above objectives, the present invention provides a strain of Saccharomyces cerevisiae with a knockout BTS1 gene for use in preparing a product for inhibiting the growth of pathogenic bacteria. The Saccharomyces cerevisiae lacks the BTS1 gene with a nucleotide sequence as shown in SEQ ID NO.1, and the host of the Saccharomyces cerevisiae is Saccharomyces cerevisiae BY4743.

[0008] Preferably, the pathogenic bacteria include common drug-sensitive bacteria and drug-resistant bacteria.

[0009] Preferably, the drug-resistant bacteria include Escherichia coli carrying the polymyxin resistance gene mcr-1, methicillin-resistant Staphylococcus aureus MRSA, resistant strains of Pseudomonas aeruginosa, resistant strains of Klebsiella pneumoniae, and resistant strains of Acinetobacter baumannii.

[0010] Preferably, the common drug-sensitive bacteria include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella typhi.

[0011] Preferably, the product is used to increase the lactic acid content and reduce the pH value in the cell-free supernatant.

[0012] Preferably, the product is used to inhibit the proliferation of Escherichia coli and Staphylococcus aureus and their ability to adhere to solid surfaces.

[0013] Preferably, the product is used to inhibit the formation of biofilms of Escherichia coli and Staphylococcus aureus.

[0014] Preferably, the product is one of fermented food, health food, daily chemical products, feed additives, probiotic preparations, and medicines.

[0015] The present invention also provides a pharmaceutical preparation for inhibiting the growth of pathogenic bacteria, comprising (1) an effective amount of brewer's yeast with the BTS1 gene knocked out as an active ingredient, and (2) a selective pharmaceutically acceptable carrier.

[0016] Preferably, the pharmaceutical dosage form is capsule, tablet, powder, oral solution, pill, tincture, syrup or injection.

[0017] In one embodiment of the present invention, the BTS1 gene is involved in the metabolic process of Saccharomyces cerevisiae. When the BTS1 gene is deleted, the lactic acid content increases and the pH decreases, thereby producing better antibacterial activity.

[0018] In one embodiment of the present invention, starting from a diploid single gene deletion strain library of Saccharomyces cerevisiae purchased from Invitrogen Inc. of the United States, more than 4,600 single gene deletion strains in the library with BY4743 as the mother strain were screened by 96-well plate liquid culture: the strains were cultured for 24 hours on LB broth medium supplemented with Escherichia coli carrying the polymyxin resistance gene mcr-1, Escherichia coli standard strain ATCC25922, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus standard strain ATCC29213, and a gene deletion strain with good antibacterial activity was obtained, indicating that after the gene is deleted, Saccharomyces cerevisiae has a good antibacterial effect.

[0019] The beneficial effects of the present invention are:

[0020] (1) The deletion of the BTS1 gene in the present invention affects the lactic acid metabolism process in Saccharomyces cerevisiae, increasing the lactic acid content in the cell-free supernatant (CFS) and decreasing the pH;

[0021] (2) The BTS1Δ / Δ Saccharomyces cerevisiae CFS screened by the present invention has good antibacterial activity, not only can it inhibit the proliferation of Escherichia coli and Staphylococcus aureus and their adhesion to solid surfaces, but also can inhibit their pathogenicity;

[0022] (3) Biofilm is an important virulence factor of bacteria and is closely related to the pathogenicity of bacteria; the BTS1Δ / Δ Saccharomyces cerevisiae CFS screened by the present invention can effectively inhibit the formation of biofilms of Escherichia coli and Staphylococcus aureus;

[0023] (4) The BTS1Δ / Δ Saccharomyces cerevisiae strain screened by the present invention can significantly inhibit the proliferation of pathogenic bacteria when co-cultured with Escherichia coli and Staphylococcus aureus;

[0024] (5) The BTS1Δ / Δ Saccharomyces cerevisiae CFS screened by the present invention has a broad-spectrum antibacterial activity, and has good antibacterial activity against common Gram-positive bacteria and Gram-negative bacteria and their resistant strains in clinical practice. Therefore, the Saccharomyces cerevisiae gene can be used as a research basis to develop products that inhibit the growth of pathogenic bacteria;

[0025] (6) The animal infection model of the present invention shows that BTS1Δ / Δ brewer's yeast has low toxicity and is not easy to destroy the normal intestinal flora, and is more ideal for preparing products that inhibit the growth of pathogenic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The graph is a graph of the growth rate of indicator bacteria after being treated with different doses of BTS1Δ / Δ Saccharomyces cerevisiae cell-free supernatant using the minimum inhibitory concentration (MIC) method;

[0027] Figure 2 The growth curves of the indicator bacteria under the treatment of BTS1Δ / Δ Saccharomyces cerevisiae cell-free supernatant; (A) Escherichia coli carrying the polymyxin resistance gene mcr-1; (B) Escherichia coli ATCC25922; (C) methicillin-resistant Staphylococcus aureus (MRSA); (D) Staphylococcus aureus ATCC29213; The results in (A) to (D) are expressed as mean ± SDs;

[0028] Figure 3 Statistical graphs of biofilm formation of indicator bacteria under treatment with BTS1Δ / Δ Saccharomyces cerevisiae cell-free supernatant; (A) Escherichia coli carrying the polymyxin resistance gene mcr-1; (B) Escherichia coli ATCC25922; (C) methicillin-resistant Staphylococcus aureus (MRSA); (D) Staphylococcus aureus ATCC29213; The results in (A) to (D) are expressed as mean ± SDs;

[0029] Figure 4Schematic diagram of the effect of co-culture of BTS1Δ / Δ Saccharomyces cerevisiae and indicator bacteria on the number of viable indicator bacteria by CFU determination; (A) BTS1Δ / Δ Saccharomyces cerevisiae and Escherichia coli ATCC25922; (B) BTS1Δ / Δ Saccharomyces cerevisiae and Staphylococcus aureus ATCC29213; The results in (A) and (B) are expressed as mean ± SDs;

[0030] Figure 5 The figure shows the survival curve of P. cerevisiae in the P. cerevisiae-E. coli model; (A) The cell-free supernatant can prolong the survival time of P. cerevisiae infected with E. coli; (B) BTS1Δ / Δ Saccharomyces cerevisiae can prolong the survival time of P. cerevisiae infected with E. coli;

[0031] In the above figures, * indicates a statistically significant difference (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001). DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific embodiments.

[0033] The test indicator bacteria involved in the following examples are Gram-positive bacteria and Gram-negative bacteria preserved in the laboratory.

[0034] Table 1 Strains used in the experiment

[0035]

[0036] Example 1: 96-well plate screening

[0037] (1) Preparation of cell-free supernatant (CFS): Saccharomyces cerevisiae single gene deletion strain and BY4743 mother strain frozen at -80°C were streaked on plates and cultured at 30°C for 24 h; single clones were picked and inoculated into 5 mL YPD broth, and cultured at 30°C and 200 rpm for 16 h. After culture, the supernatant was centrifuged at 4000 × g for 12 min at 4°C; the supernatant was filtered through a syringe filter (pore size of 0.22 μm), and the pH value of the CFS was measured with a pH meter, and then stored at -80°C for future use. The CFS of BY4743 mother strain was used as a control.

[0038] YPD medium composition: 1% yeast extract, 2% peptone, 2% glucose; if making solid culture medium, add 2% agar powder.

[0039] (2) Escherichia coli carrying the polymyxin resistance gene mcr-1, Escherichia coli ATCC25922, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus ATCC29213 were selected as indicator strains. The indicator strains were cultured overnight until the exponential growth phase, and the bacterial suspension was diluted to 1×10 using LB medium. 8 cells / mL;

[0040] (3) Add 90 μL of CFS to each well of a 96-well plate, and then add 10 μL of the diluted bacterial suspension; the experimental group consisted of CFS of the deletion strain plus bacterial suspensions of various indicator strains (CFS+mcr-1, CFS+ATCC25922, CFS+MRSA, CFS+ATCC29213); the control group consisted of CFS of BY4743 plus bacterial suspensions of various indicator strains; each group was set up with three replicates;

[0041] (4) The 96-well plate was placed in a 37°C incubator and incubated for 24 h. The optical density at 600 nm was measured using a microplate reader. After deducting the absorbance of the blank culture medium, the ratio of the OD value of each well divided by the OD value of the control group was taken as its growth rate. Strains with an inhibition rate (1-growth rate) greater than 80% were considered to have antibacterial activity. It was found that the BTS1Δ / Δ brewer's yeast strain had antibacterial activity.

[0042] Example 2: CFS antibacterial activity determination

[0043] The minimum inhibitory concentration (MIC) method was used for determination. The MIC test was based on the microbroth dilution method developed by the Clinical and Laboratory Standards Institute (CLSI) of the United States. The specific steps are as follows:

[0044] In this example, Escherichia coli and Staphylococcus aureus were used as indicator bacteria to evaluate the antibacterial activity of CFS. Escherichia coli carrying the polymyxin resistance gene mcr-1, Escherichia coli ATCC25922, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus ATCC29213 were selected as indicator strains.

[0045] According to the microbroth dilution method established by the Clinical and Laboratory Standards Institute (CLSI), the indicator strain grown to the exponential growth phase was diluted with LB broth medium, and then 200 μL of BTS1Δ / Δ Saccharomyces cerevisiae strain CFSCFS was added to well A of each column of the 96-well plate, and 100 μL of an indicator strain was added to wells B to H. Then, starting from well A, the bacterial solution in the wells was diluted by the doubling dilution method, so that the final volume of each well was 100 μL, and the final concentration of the bacterial solution was 1×10 6cells / mL, and well H was not added with CFS as a negative control. Then the 96-well plate was placed in a 35°C constant temperature incubator for 24 hours. The minimum drug concentration at which no fungal growth was observed by naked eye, or the lowest concentration that could inhibit 80% fungal cell growth was the MIC value.

[0046] like Figure 1 As shown, the CFS of the BTS1Δ / Δ Saccharomyces cerevisiae strain has good antibacterial activity, and the maximum inhibition rate of bacterial growth can reach 97.5%. At the same time, it can be observed that as the amount of CFS added decreases, the antibacterial active substances decrease and the antibacterial activity gradually decreases.

[0047] Example 3: Detection of lactic acid content in CFS by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS)

[0048] Metabolite extraction: BTS1Δ / Δ Saccharomyces cerevisiae CFS, BY4743 mother strain CFS and YPD broth medium were taken out of the freezer and thawed in a 4°C refrigerator. 5 μL of the supernatant of each sample to be tested was taken into a centrifuge tube, and 35 μL of the prepared derivatization reagent 2-aminomethylpyridine (CAS No.: 3731-51-9) and 35 μL of the condensation agent O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (CAS No.: 148893-10-1) were added; after vortex mixing for 20 minutes, 100 μL of PBS and 300 μL methyl tert-butyl ether, cover the centrifuge tube tightly and vortex for 10 minutes; immediately after vortexing, place the centrifuge tube in a centrifuge at 4℃6000rpm for 5 minutes; carefully take out 50 μL of supernatant and put it into a new centrifuge tube, then put the centrifuge tube containing the supernatant into a vacuum concentrator and centrifuge and concentrate at 40℃ for about 20 minutes; then add 100 μL of 10% acetonitrile water reconstitution solution to the evaporated centrifuge tube for reconstitution; after vortexing for 5 minutes, immediately centrifuge at 4℃12000rpm for 15 minutes, then carefully take out 40 μL of supernatant and transfer it to the injection vial for testing.

[0049] Preparation of standard curve: Take 5 μL of supernatant (2 mL of standard stock solution prepared with acetonitrile) from each point of the standard working curve into a new centrifuge tube, add 35 μL of the prepared derivatization reagent 2-aminomethylpyridine (CAS No.: 3731-51-9) and 35 μL of the condensation agent O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (CAS No.: 148893-10-1); vortex mix for 20 minutes, then add 100 μL of PBS and 300 μL of methyl tert-butyl ether, cover the centrifuge tube tightly and vortex for 10 minutes; after vortexing, place the centrifuge tube in a centrifuge at 4°C and 6000 rpm for 5 minutes; carefully take out 50 μL of supernatant into a new centrifuge tube, then place the centrifuge tube containing the supernatant into a vacuum concentrator at 40°C and centrifuge for about 20 minutes; then add 100 μL of Reconstitute with 10% acetonitrile water solution; vortex for 5 minutes, then centrifuge at 4°C 12000rpm for 15 minutes, then carefully remove 80μL of supernatant and transfer to the injection vial. Perform LC-MS / MS analysis on the calibration solution. y represents the peak area of ​​the target compound, and x represents the concentration of the target compound (ng / mL). The least squares method is used for linear regression. When the weight is set to none, the calibration solution recovery (accuracy) and linear coefficient (R2) are the best, and both R and R2 are greater than 0.99. If the signal-to-noise ratio (S / N) of a calibration concentration is close to or less than 10, the calibration point of that concentration is excluded. The final calibration curve has at least 5 calibration points.

[0050] As shown in Table 2, compared with the wild-type Saccharomyces cerevisiae BY4743, the lactic acid content in the CFS of BTS1Δ / Δ Saccharomyces cerevisiae is about 35 times that of the wild-type Saccharomyces cerevisiae BY4743. Lactic acid can reduce the pH of CFS and make the environment acidic, which is not conducive to the growth of Gram-positive and Gram-negative bacteria.

[0051] Table 2 pH value and lactic acid content of CFS

[0052]

[0053] Example 4: Growth Curve

[0054] Escherichia coli, Escherichia coli ATCC25922, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus ATCC29213 carrying the polymyxin resistance gene mcr-1 were selected as indicator strains.

[0055] (1) Dilute the indicator strain culture solution activated overnight with LB broth to the corresponding concentration;

[0056] (2) Add the diluted indicator strain solution, CFS, YPD broth and sterile water to a 10 mL EP tube at the same time to ensure that the final ratio is 90% CFS, 50% CFS, and 25% CFS. The group without CFS is used as the negative control, and the group with sterile water is used as the blank control;

[0057] (3) After measuring the original absorbance at 600 nm using an enzyme reader, the culture was maintained at 37°C and shaken at 200 rpm;

[0058] (4) 100 μL of samples were collected every 2 h and the absorbance at 600 nm was measured using an ELISA reader. Three replicates were set up for each group.

[0059] (5) Record the values ​​and draw a growth curve.

[0060] like Figure 2 As shown, compared with the negative control group, the CFS of the BTS1Δ / Δ Saccharomyces cerevisiae strain can significantly inhibit the growth of the indicator strain and is dose-dependent; 90% CFS can completely inhibit the growth of pathogenic bacteria; compared with the standard strains ATCC25922 and ATCC29213, the antibacterial effect on drug-resistant bacteria is better, which may be because the toxicity of drug-resistant bacteria is lower than that of their standard strains. In addition, a blank control group was set up in this embodiment, and sterile water was used instead of YPD broth to examine the effect of nutritional differences on the experimental group. There was no significant difference in the growth of indicator bacteria between the blank control (representing the minimum amount of additional nutrients) and the YPD broth control (representing the maximum amount of additional nutrients), indicating that the indicator bacteria culture is rich in nutrients, so nutritional depletion is not a factor leading to growth changes.

[0061] Example 5: Biofilm inhibition test

[0062] The inhibition of CFS on biofilm was studied by crystal violet (CV) method. Escherichia coli carrying polymyxin resistance gene mcr-1, Escherichia coli ATCC25922, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus ATCC29213 were selected as indicator strains.

[0063] (1) 100 μL of bacterial suspension treated with 90% CFS, 50% CFS, and 25% CFS were transferred to a 96-well plate and cultured at 37°C for 24 h; the control group was treated with YPD broth;

[0064] (2) After the culture was completed, the 96-well plate was removed, the cell suspension was removed, and the biofilm cells were washed twice with 150 μL of sterile distilled water. The biofilm cells were dried at 37°C for 30 min.

[0065] (3) Take out the 96-well plate, fix the biofilm cells with 200 μL of anhydrous ethanol for 20 min, and then dry them at 37°C;

[0066] (4) Stain with 150 μL of 0.1% crystal violet (CV) solution for 30 min; remove the CV solution and wash twice with sterile distilled water to remove excess CV;

[0067] (5) Dry at 37°C and treat the biofilm cells with 150 μL of a dissolving solution (30% methanol and 10% acetic acid). Use an ELISA reader to measure the absorbance at 595 nm. Each group has three replicates.

[0068] Biofilms can make pathogens have drug resistance and immune escape ability, so the formation of biofilms is also an important virulence factor of pathogens. The crystal violet staining method was used to determine the biomass of biofilms in the culture medium containing different amounts of CFS in 96-well plates. Figure 3 As shown in the figure, with the increase of CFS addition amount of BTS1Δ / Δ Saccharomyces cerevisiae strain, the ability to inhibit biofilm gradually increased, showing a dose-dependency. For Escherichia coli, when the addition amount was 50%, the inhibition rate could reach more than 80%; for Staphylococcus aureus resistant bacteria MRSA, the inhibition rate could reach more than 60% when the addition amount was 25%. However, for the standard strain of Staphylococcus aureus, because its virulence may be higher than that of resistant bacteria, the inhibition rate only reached 60% when the addition amount was 50%.

[0069] Example 6: CFS antibacterial spectrum determination

[0070] The antibacterial activity of CFS against several common bacteria was determined by the MIC method, and the results are shown in Table 3: CFS has a significant inhibitory effect on both Gram-positive and Gram-negative bacteria. At the same time, the pH value of CFS decreases (Table 2), and the acidic environment is not conducive to bacterial growth.

[0071] Table 3 Antibacterial spectrum of cell-free supernatant of BTS1Δ / Δ Saccharomyces cerevisiae strain

[0072]

[0073] Example 7: Co-cultivation experiment of BTS1Δ / Δ cerevisiae and indicator strains

[0074] Samples were taken at specific times, and then the effect of BTS1Δ / Δ Saccharomyces cerevisiae on the indicator strains was determined by the spot plate counting method. Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 were selected as indicator strains.

[0075] The cells of Saccharomyces cerevisiae and indicator strains cultured overnight were washed twice with phosphate buffered saline (PBS) and diluted to 1×10 7cells / mL. Saccharomyces cerevisiae and indicator bacteria were mixed at a concentration ratio of 1:1, 1 mL each, as the co-culture group; indicator bacteria culture was mixed with 1 mL of YPD broth, as the indicator bacteria single culture group; indicator bacteria culture was mixed with 1 mL of sterile water, as the indicator bacteria blank control group. The three experimental groups were cultured at 37°C and shaken at 200 rpm.

[0076] Take samples every 4 hours, dilute them 10 times, and spot 2 μL of the dilution on a LB solid plate with 10 μg / mL amphotericin B. Set up three replicates for each group. Place the plate with the bacterial solution at 37°C for more than 24 hours until a monoclonal colony grows. Record the number of colonies grown on the plate, calculate the number of viable bacteria in the original bacterial solution, and draw a graph.

[0077] This example explores the effect of co-culturing BTS1Δ / Δ Saccharomyces cerevisiae with pathogenic bacteria on the pathogenic bacteria. Figure 4 , the number of pathogenic bacteria cells after 16h and 24h of culture was evaluated by CFU count. Compared with the pathogenic bacteria monoculture group, the number of surviving pathogens in the co-culture group of Saccharomyces cerevisiae and pathogenic bacteria was significantly reduced, indicating that Saccharomyces cerevisiae can significantly inhibit the growth of pathogenic bacteria. In addition, sterile water was used instead of YPD broth as a blank control group to investigate the effect of nutritional differences on the experimental groups. There was no significant difference in the growth of indicator bacteria between the blank control (representing the minimum amount of additional nutrients) and the YPD broth control (representing the maximum amount of additional nutrients) (P>0.05), indicating that the indicator bacteria culture was nutrient-rich, so nutrient depletion was not a factor leading to growth changes.

[0078] Example 8: Greater wax nightshade-Escherichia coli infection model

[0079] (1) When purchasing wax moth larvae, make sure they are milky white, have no gray markings on their cuticles, and are in good health;

[0080] (2) Saccharomyces cerevisiae was inoculated into 5 mL of YPD broth and cultured at 30°C and 200 rpm for 16 h. The supernatant was removed and the Saccharomyces cerevisiae cells were collected. A tube of Saccharomyces cerevisiae cells was sterilized at 121°C for 15 min and then used for later use.

[0081] (3) Dilute the E. coli incubated overnight with PBS to 1×10 8 cells / mL; dilute the live and sterilized Saccharomyces cerevisiae to 1×10 cells / mL with PBS. 4cells / mL; PBS group, CFS group, Saccharomyces cerevisiae (live bacteria) group, Saccharomyces cerevisiae (inactivated) group, Escherichia coli group, CFS+Escherichia coli group, Saccharomyces cerevisiae (live bacteria)+Escherichia coli group, Saccharomyces cerevisiae (inactivated)+Escherichia coli group, Saccharomyces cerevisiae (live bacteria) pretreatment group were set up, and all groups were injected with wax nightshade intrahemocoel (10 μL) with a microsyringe, with 10 wax nightshade larvae in each group;

[0082] (4) Place the injected wax larvae in a clean culture dish and culture them at 37°C in the dark, and record the number of surviving and dead larvae every day.

[0083] In this example, a wax moth-Candida albicans infection model was established to explore the therapeutic effect of BTS1Δ / Δ cerevisiae yeast on Escherichia coli infection in vivo. After daily observation and recording of the survival status of infected wax moths, it was found that all wax moth larvae infected with Escherichia coli treated with PBS or inactivated cerevisiae yeast died within 48 hours. Figure 5 As can be observed in A, BTS1Δ / Δ Saccharomyces cerevisiae CFS can significantly reduce the pathogenicity of Escherichia coli and prolong the survival time of G. mellonella infected with E. coli (P<0.05), and injection of CFS alone will not cause the death of G. mellonella.

[0084] In addition, live saccharomyces cerevisiae, inactivated saccharomyces cerevisiae, and Escherichia coli treated with live saccharomyces cerevisiae were injected into the blood cavity of wax moths. The results showed that saccharomyces cerevisiae itself would not cause the death of wax moths and could prolong the survival time of wax moths infected with Escherichia coli (P < 0.05). In addition, live saccharomyces cerevisiae was injected into wax moths in advance, cultured at 37°C for 2 hours, and then injected with Escherichia coli. It can be observed that the mortality rate of wax moths was reduced and the survival time was prolonged ( Figure 5 B).

Claims

1. Use of a strain of Saccharomyces cerevisiae with a knockout BTS1 gene in the preparation of a product for inhibiting the growth of pathogenic bacteria, characterized in that: The Saccharomyces cerevisiae lacks the BTS1 gene with a nucleotide sequence as shown in SEQ ID NO.1, and the Saccharomyces cerevisiae parent strain is Saccharomyces cerevisiae BY4743.

2. Use of a strain of Saccharomyces cerevisiae with a knockout BTS1 gene according to claim 1 in preparing a product for inhibiting the growth of pathogenic bacteria, characterized in that: The pathogenic bacteria include common drug-sensitive bacteria and drug-resistant bacteria.

3. Use of a strain of Saccharomyces cerevisiae with a knockout BTS1 gene according to claim 2 in preparing a product for inhibiting the growth of pathogenic bacteria, characterized in that: The drug-resistant bacteria include Escherichia coli carrying the polymyxin resistance gene mcr-1, methicillin-resistant Staphylococcus aureus MRSA, resistant strains of Pseudomonas aeruginosa, resistant strains of Klebsiella pneumoniae, and resistant strains of Acinetobacter baumannii.

4. Use of a strain of Saccharomyces cerevisiae with a knockout BTS1 gene according to claim 2 in preparing a product for inhibiting the growth of pathogenic bacteria, characterized in that: The common drug-sensitive bacteria include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella typhi.

5. Use of a strain of Saccharomyces cerevisiae with a knockout BTS1 gene according to claim 1 or 2 in preparing a product for inhibiting the growth of pathogenic bacteria, characterized in that: The product is used for increasing the lactic acid content in the cell-free supernatant and lowering the pH value.

6. Use of a strain of Saccharomyces cerevisiae with a knockout BTS1 gene according to claim 1 or 2 in preparing a product for inhibiting the growth of pathogenic bacteria, characterized in that: The product is used to inhibit the proliferation of Escherichia coli and Staphylococcus aureus and their adhesion to solid surfaces.

7. Use of a strain of Saccharomyces cerevisiae with a knockout BTS1 gene according to claim 1 or 2 in preparing a product for inhibiting the growth of pathogenic bacteria, characterized in that: The product is used to inhibit the formation of biofilms of Escherichia coli and Staphylococcus aureus.

8. Use of a strain of Saccharomyces cerevisiae with a knockout BTS1 gene according to claim 1 or 2 in preparing a product for inhibiting the growth of pathogenic bacteria, characterized in that: The product is one of fermented food, health food, daily chemical products, feed additives, probiotic preparations and medicines.

9. A pharmaceutical preparation for inhibiting the growth of pathogenic bacteria, characterized in that: The invention comprises (1) an effective amount of brewer's yeast with BTS1 gene knocked out as an active ingredient, and (2) a selective pharmaceutically acceptable carrier.

10. A pharmaceutical preparation for inhibiting the growth of pathogenic bacteria according to claim 9, characterized in that: The drug dosage form is capsule, tablet, powder, oral liquid, pill, tincture, syrup or injection.

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