Application of Kefir flora supernatant in preparation of drug-resistant bacteria inhibition preparation

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an application of a Kefir flora supernatant in preparation of a drug-resistant bacteria inhibition preparation, and a preparation method of the Kefir flora supernatant comprises the following steps: preparing a Kefir flora seed solution with the thallus concentration of 10 < 8 > CFU / mL to 10 < 9 > CFU / mL; inoculating the seed solution of the Kefir flora into an LB liquid culture medium, and culturing at 33-37 DEG C for 18-24 hours; and centrifuging at the temperature of 2-6 DEG C and 4000-6000 g for 3-8 minutes to remove thalli, and collecting the supernate, thereby obtaining the Kefir flora supernate. In the fermentation process of the Kefir flora supernate, through the synergistic effect of various natural metabolites, the growth of multi-drug-resistant pathogenic bacteria can be effectively inhibited, and an effective alternative scheme is provided for developing a new antibacterial therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the application of Kefir flora supernatant in the preparation of a preparation for inhibiting drug-resistant bacteria. Background Art

[0002] With the abuse of antibiotics and the rapid development of drug-resistant bacteria, many bacteria have gradually developed resistance to traditional antibiotics, leading to the failure of conventional treatment regimens. Multidrug-resistant bacteria, such as drug-resistant Pseudomonas aeruginosa, drug-resistant Escherichia coli, drug-resistant Candida albicans, etc., have extremely high resistance to conventional drugs. Therefore, there is an urgent need to find new treatment methods and alternatives.

[0003] The abuse of antibiotics promotes the proliferation of drug-resistant genes. After the recipient bacteria acquire the drug-resistant genes in the donor bacteria, drug resistance occurs. The horizontal transfer of drug-resistant genes includes conjugation, natural transformation, and transduction. Conjugation refers to the connection of donor and recipient bacteria through pili contact, enabling mobile genetic elements in the donor, such as plasmids or transposons, to be transferred to the recipient through pili; natural transformation refers to bacteria directly taking up free DNA from the environment and being able to acquire corresponding genetic traits; transduction refers to when phages proliferate and cut the DNA in the donor bacteria, accidentally carrying the bacterial DNA and infecting and transferring it to the recipient cell, resulting in the recipient cell obtaining resistance genes.

[0004] Currently, the technologies for inhibiting drug-resistant bacteria mainly rely on chemical bactericides and physical methods. Chemical bactericides include: chlorine preparations such as sodium hypochlorite and calcium hypochlorite, hydrogen peroxide, and quaternary ammonium salts such as cetyl dimethyl ammonium chloride, methylbenzyl dimethyl ammonium chloride, and trimethyl ammonium bromide; physical methods include high-temperature sterilization, steam disinfection, ultraviolet disinfection, etc. However, these technologies have some significant disadvantages and limitations in dealing with drug-resistant bacteria. Specifically, the disadvantages of current technologies are mainly manifested in the following aspects:

[0005] 1. Chemical bactericides are prone to cause the generation and spread of drug-resistant bacteria during long-term use. Drug-resistant bacteria can quickly develop drug resistance through mutation or gene transfer, rendering traditional antibacterial methods ineffective against them, thereby leading to a decline in treatment effects and increasing the difficulty of clinical treatment.

[0006] 2. Although chemical bactericides can effectively kill pathogenic bacteria, they often have a negative impact on beneficial bacteria in the human body or the environment. Long-term use may cause side effects such as immune system damage and intestinal flora imbalance, and even in some cases, have a toxic effect on human health.

[0007] 3. Existing antibacterial agents often target specific pathogenic bacteria or flora, while drug-resistant bacteria can quickly adapt and avoid existing antibacterial strategies through gene mutation or horizontal gene transfer. Therefore, the existing technologies face the problem of narrow antibacterial spectra and are difficult to deal with a wide variety of pathogenic bacteria.

[0008] 4. The long-term use of chemical antibacterial agents and disinfectants may not only produce drug resistance but also pollute the ecological environment. Especially the accumulation in wastewater discharge and soil will further affect the health of the ecosystem and even trigger new environmental health problems.

[0009] Based on this, there is an urgent need to provide a new strategy to solve the drug resistance of drug-resistant bacteria. Summary of the Invention

[0010] To solve the problem that drug-resistant bacteria are resistant to traditional antibiotics, the present invention provides an application of Kefir flora supernatant in the preparation of a preparation for inhibiting drug-resistant bacteria.

[0011] The technical solution adopted by the present invention is as follows:

[0012] The present invention provides an application of Kefir flora supernatant in the preparation of a preparation for inhibiting drug-resistant bacteria. The preparation method of the Kefir flora supernatant includes the following steps:

[0013] Prepare a seed solution of Kefir flora with a cell concentration of 10 8 CFU / mL to 10 9 CFU / mL; inoculate the seed solution of Kefir flora into LB liquid medium and culture at 33°C to 37°C for 18 h to 24 h; centrifuge at 2°C to 6°C and 4000 g to 6000 g for 3 min to 8 min to remove the cells, and collect the supernatant to obtain the Kefir flora supernatant.

[0014] Preferably, the drug-resistant bacteria include at least one of drug-resistant Pseudomonas aeruginosa, drug-resistant Escherichia coli, and drug-resistant Candida albicans.

[0015] Preferably, the culture conditions are 35°C for 20 h; the conditions for collecting the supernatant are centrifuging at 4°C and 5000 g for 5 min to remove the cells.

[0016] Preferably, when inoculating the seed solution of Kefir flora into LB liquid medium, the inoculation amount of the seed solution of Kefir flora accounts for 1% of the volume of LB liquid medium.

[0017] Preferably, the Kefir flora supernatant reduces the resistance of drug-resistant bacteria to chemical bactericides.

[0018] Preferably, the chemical bactericides include at least one of chlorine preparations, hydrogen peroxide, and quaternary ammonium salts. The chlorine preparations include at least one of sodium hypochlorite and calcium hypochlorite; the quaternary ammonium salts include at least one of cetyl dimethyl ammonium chloride, methylbenzyl dimethyl ammonium chloride, and trimethyl ammonium bromide.

[0019] The present invention also provides a bactericide, which comprises the supernatant of the Kefir flora, a chemical bactericide and a pharmaceutically acceptable excipient.

[0020] Preferably, the pharmaceutically acceptable excipient comprises at least one of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickening agent, an emulsifier, a preservative and a stabilizer.

[0021] Preferably, the diluent comprises any one of starch, lactose, sucrose and mannitol.

[0022] Preferably, the disintegrant comprises any one of starch, microcrystalline cellulose and low-substituted hydroxypropyl cellulose.

[0023] Preferably, the precipitation inhibitor comprises any one of sodium dodecyl sulfate, Tween-80, polyvinylpyrrolidone and hydroxypropyl methylcellulose.

[0024] Preferably, the glidant comprises any one of cationic polyacrylamide, poly(diallyldimethylammonium chloride) and cationic starch.

[0025] Preferably, the binder comprises any one of starch paste, hydroxypropyl methylcellulose and polyvinylpyrrolidone.

[0026] Preferably, the dispersant comprises any one of sodium dodecyl sulfate, polyvinylpyrrolidone and sodium carboxymethylcellulose.

[0027] Preferably, the suspending agent comprises any one of gum arabic, tragacanth, sodium carboxymethylcellulose and hydroxypropyl methylcellulose.

[0028] Preferably, the isotonic agent comprises any one of sodium chloride, glucose and mannitol.

[0029] Preferably, the thickening agent comprises any one of gum arabic, xanthan gum and sodium carboxymethylcellulose.

[0030] Preferably, the emulsifier comprises any one of sodium dodecyl sulfate, benzalkonium chloride and sorbitan fatty acid esters.

[0031] Preferably, the preservative comprises any one of benzoic acid, sorbic acid, methyl p-hydroxybenzoate and benzalkonium bromide.

[0032] Preferably, the stabilizer comprises any one of sodium sulfite, sodium bisulfite, tocopherol and disodium ethylenediaminetetraacetate.

[0033] Preferably, the acceptable dosage form of the bactericide comprises one of tablets, capsules, granules, pills, powders, ointments and oral liquids.

[0034] The present invention also provides a formulation of the bactericide, wherein the v / v of the Kefir flora supernatant is 15%, the w / v of sodium hypochlorite is 0.2%, the w / v of cetyl dimethyl ammonium chloride is 0.1%, the w / v of sodium benzoate is 0.1%, the w / v of polysorbate - 80 is 0.5%, the w / v of disodium ethylenediaminetetraacetate is 0.05%, and the balance is made up with water.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] The present invention provides an application of the Kefir flora supernatant in the preparation of a preparation for inhibiting drug - resistant bacteria. The preparation method of the Kefir flora supernatant comprises the following steps: preparing a seed solution of the Kefir flora with a cell concentration of 10 8 CFU / mL to 10 9 CFU / mL; inoculating the seed solution of the Kefir flora into an LB liquid medium and culturing at 33°C - 37°C for 18 h - 24 h; centrifuging at 2°C - 6°C and 4000 g - 6000 g for 3 min - 8 min to remove the bacteria, and collecting the supernatant, i.e., obtaining the Kefir flora supernatant. During the fermentation process of the Kefir flora supernatant, through the synergistic action of various natural metabolites, it can effectively inhibit the growth of various drug - resistant pathogenic bacteria and provide an effective alternative for the development of new antibacterial therapies. Specifically, the purposes include the following aspects:

[0037] 1. With the widespread spread of drug - resistant bacteria such as methicillin - resistant Staphylococcus aureus and carbapenem - resistant Escherichia coli, traditional antibiotic therapies are gradually becoming ineffective. The active ingredients in the Kefir flora supernatant of the present invention have a certain antibacterial effect, can inhibit the growth and spread of drug - resistant bacteria, and provide a new solution to the problem of drug resistance.

[0038] 2. The abuse and over - use of traditional antibiotics are the main reasons for the generation of drug - resistant bacteria. Therefore, using the Kefir flora supernatant as a natural antibacterial substance can effectively reduce the use of antibiotics, reduce the risk of generating drug - resistant bacteria, and reduce the potential harm of antibiotics to the human body and the environment.

[0039] 3. The Kefir flora supernatant, as a natural and non - antibiotic source of bacteriostatic substance, can provide important clues for the development of new antibacterial preparations. These antibacterial substances not only have a good inhibitory effect on common sensitive bacteria, but may also show unique antibacterial activities against some drug - resistant bacteria. Therefore, it can become a new direction for the research of anti - drug - resistant bacteria and promote the innovation and diversification of antibacterial drugs.

[0040] 4. As a natural fermentation product, the supernatant of the Kefir flora has a lower risk to humans and the environment compared to traditional chemical antibacterial agents or antibiotics. It is less toxic and biodegradable, meeting the green and environmental protection concept of modern biological agents.

[0041] During the fermentation process of the Kefir flora, the diversity of the microbial population and complex metabolic activities result in the production of a large number of bioactive metabolites. These metabolites mainly include organic acids, lipids, organic heterocyclic compounds, antimicrobial peptides, etc. These substances gradually accumulate in the culture medium to form the supernatant. This supernatant has a significant inhibitory effect on a variety of pathogenic microorganisms, including bacteria and fungi. During the fermentation of the Kefir flora, lactic acid bacteria such as Lactococcus lactis and Bifidobacterium are among the main fermenting microorganisms. These bacteria produce a large amount of lactic acid by metabolizing lactose. The accumulation of lactic acid causes the pH value of the culture medium to decrease, creating an acidic environment. Many pathogenic microorganisms, especially bacteria, cannot survive or reproduce in a low-pH environment. Therefore, the action of lactic acid gives the supernatant of the Kefir flora natural antibacterial properties. In addition to lactic acid, acetic acid is also a common organic acid in the supernatant of the Kefir flora. It is usually a metabolite of acetic acid bacteria such as Acetobacter. Acetic acid can not only further lower the pH but also inhibit the growth of certain pathogenic bacteria by disrupting the cell membrane and metabolic processes of microorganisms. The metabolites of yeasts in the Kefir flora are also part of its antibacterial properties. Some yeasts can produce antibacterial metabolites such as lipids and alkaloids. These metabolites damage the structure and function of the cell membrane and inhibit enzyme activity, having an inhibitory or killing effect on pathogenic microorganisms. The metabolites of yeasts can also enhance the inhibitory effect on pathogenic bacteria by improving the antioxidant capacity of the Kefir fermentation broth, further enhancing its natural antibacterial effect.

[0042] In addition, the Kefir microbial community and its metabolites also have a variety of bioactive properties, including anti-cancer, antibacterial, anti-inflammatory, cholesterol-lowering, wound healing, antioxidant, and gastrointestinal assistance properties. Therefore, the supernatant of the Kefir flora has the potential to become a new type of antibacterial agent. Description of the Drawings

[0043] Figure 1 It is a morphological diagram of the Kefir flora on the LB solid medium.

[0044] Figure 2 It is an antibacterial result diagram of the supernatant of the Kefir flora against drug-resistant Escherichia coli and drug-resistant Pseudomonas aeruginosa. A: Drug-resistant Escherichia coli; B: Drug-resistant Pseudomonas aeruginosa.

[0045] Figure 3This is the result graph of the inhibition of the transfer of resistant genes and natural transformation by the supernatant of the Kefir flora. A: The sediment environment of drug-resistant Escherichia coli; B: The supernatant environment of drug-resistant Escherichia coli. Detailed implementation manners

[0046] The present invention will be further described below through specific examples, but the scope of the present invention is not limited. Without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications or substitutions all fall within the protection scope of the present invention.

[0047] The inventive concept of the present invention is as follows:

[0048] With the abuse of antibiotics, bacteria have developed drug resistance, and new antibiotics are needed to kill drug-resistant bacteria. Small molecules derived from natural products have always been the main source of new antibiotics. The Kefir flora includes lactic acid bacteria, acetic acid bacteria and yeasts. Among them, lactic acid bacteria include Leuconostoc, Lactobacillus, Streptococcus, Lactococcus, Enterobacter, Acinetobacter, Enterococcus and Pseudomonas; yeasts include Kluyveromyces lactis, Candida, Torulopsis, Saccharomyces, Rhodotorula and Zygosaccharomyces. During the fermentation process of the Kefir flora, the diversity of the microbial population and complex metabolic activities result in the production of a large number of bioactive metabolites. The present invention discovers that the supernatant of the Kefir flora, as a natural antibacterial agent, can effectively inhibit the growth of drug-resistant bacteria, especially showing potential in the treatment of some multi-drug resistant bacterial infections. In addition, it has a wide source and no obvious toxic and side effects, providing a safer and greener antibacterial option for clinical use.

[0049] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with specific examples. In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0050] The drug-resistant Escherichia coli involved in the present invention is from the reference: Li Songwei, Zhou Xiaoqin, Zhao Meijuan, et al. Inactivation efficiency of tetracycline-resistant Escherichia coli by ultrasonic combined with UV-LED [J]. China Environmental Science, 2023, 43(12): 6313-6320. DOI: 10.19674 / j.cnki.issn1000-6923.20230817.003.

[0051] The non-drug-resistant Pseudomonas aeruginosa is from the reference: Zhan Xuli, Tang Jianhua, Pu Jiekun, et al. Clinical distribution, multilocus sequence typing and drug resistance analysis of Pseudomonas aeruginosa [J]. Pharmaceutical Biotechnology, 2025, 32(01): 58-63. DOI: 10.19526 / j.cnki.1005-8915.20250110.

[0052] The drug-resistant Pseudomonas aeruginosa is from the reference: Lu Zan, Zhao Hongyan, Li Chunfu, et al. Experimental study on the expression of 10 membrane protein-encoding genes in carbapenem-resistant Pseudomonas aeruginosa in Kunming area [J]. Journal of Modern Laboratory Medicine, 2025, 40(01): 7-12.

[0053] Example 1

[0054] The application of Kefir flora supernatant in the preparation of antibacterial agents against drug-resistant bacteria is as follows:

[0055] 1. Observation on the morphology of Kefir flora.

[0056] 1.1 Source of Kefir flora.

[0057] The Kefir flora was purchased from Bioprox in France, with the product name of Vegi-Prox K01 kefir strain and the product number of Di-Prox K013

[0058] 1.2 Preparation of culture medium.

[0059] Each liter of Pseudomonas agar basal medium contains: gelatin peptone 16 g, acid hydrolysate of casein 10 g, anhydrous potassium sulfate 10 g, anhydrous magnesium chloride 1.4 g, cetrimonium bromide 0.2 g, nalidixic acid 0.015 g, agar 14 g, and the rest is made up with distilled water. The pH is adjusted to 7.0 at 25 °C.

[0060] Each liter of Pseudomonas solid medium contains: weigh 50.6 g of Pseudomonas agar basal medium, take another 10 mL of glycerol, and make up the rest with distilled water. Heat and dissolve, dispense, sterilize at 121 °C for 15 min by high-pressure steam sterilization. After sterilization, cool to 50 °C and place it in the ultra-clean bench for standby.

[0061] Each liter of LB liquid medium contains: tryptone 10 g, yeast extract powder 5 g, sodium chloride 10 g, and the rest is made up with distilled water. The pH is adjusted to 7.0 at 25 °C, and sterilized at 121 °C for 20 min by high-pressure steam sterilization, then placed in the ultra-clean bench for standby.

[0062] Each liter of LB solid medium contains: 10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, 15 g of agar, and the rest is made up with distilled water. The pH is adjusted to 7.0 at 25 °C, and it is autoclaved at 121 °C for 20 min and then placed in a laminar flow hood for standby.

[0063] 1.3 Preparation of Kefir flora seed liquid.

[0064] In a 100 mL Erlenmeyer flask, add 0.1 g of Kefir powder and 50 mL of ultrapure water, and shake well on a vortex mixer to ensure uniform dispersion of the sample. Inoculate the sample into 100 mL of LB liquid medium at an inoculation amount of 1% by volume, and culture at 35 °C for 24 h. Then inoculate it into 100 mL of LB liquid medium at an inoculation amount of 1% by volume and ferment for 24 h. After that, centrifuge the fermentation broth at 4 °C and 4000 g for 10 min, discard the supernatant, and wash the bacterial pellet twice with sterile PBS under the same centrifugation conditions. Finally, resuspend the bacterial pellet with sterile PBS of the same volume as the fermentation broth, and the resulting bacterial suspension is used as the seed liquid for subsequent fermentation. The seed liquid is stored at 4 °C for standby.

[0065] 1.4 Morphological observation.

[0066] Take 100 μL of the seed liquid and spread it on the LB solid medium, then invert it and culture it in an incubator at 35 °C for 24 h. Small colonies about 5 μm - 10 μm in size, with smooth edges, round, and milky white appear on the medium; and small colonies about 0.5 μm - 1.0 μm in size, with smooth edges, spherical, and milky white, see Figure 1 .

[0067] 2. Antibacterial activity of Kefir supernatant.

[0068] 2.1 Preparation of Kefir supernatant.

[0069] Inoculate the above-mentioned Kefir seed liquid into a 100 mL Erlenmeyer flask containing 100 mL of LB liquid medium at an inoculation amount of 1% by volume and culture it in a constant temperature biochemical incubator at 35 °C for 20 h. Centrifuge at 4 °C and 5000 g for 5 min to remove the bacteria, and store the supernatant at -20 °C for subsequent determination.

[0070] 2.2 Oxford cup plate diffusion method.

[0071] Adopt the Oxford cup plate diffusion method. The lower layer of double-layer nutrient agar is 10 mL and the upper layer is 10 mL. The amount of agar in the upper layer is 0.7%. Mix 0.2 mL of the bacterial amount of 10 -6Escherichia coli and Pseudomonas aeruginosa with drug resistance to the concentration. Respectively, 100 μL of Kefir supernatant, SJ1, SJ2, yeast supernatant and deionized water were added to the Oxford cups and cultured at 35 °C for 24 h. The diameter of the inhibition zone was measured with a vernier caliper, and the antibacterial activity of the Kefir supernatant was represented by the diameter of the inhibition zone. The results are shown in Figure 2 , and the specific results are shown in Table 1.

[0072] In the early stage of this invention, different bacteria were grown by spreading the Kefir flora seed liquid on the plate, and two single bacteria were selected and named SJ1 and SJ2. The supernatants of these two single bacteria were obtained by culturing them. The inhibition zone experiment was carried out with the supernatants of these two single bacteria to compare the antibacterial abilities of the flora and the single bacteria, and it was found that the flora had stronger antibacterial ability.

[0073] Table 1 Antibacterial results of Kefir supernatant

[0074]

[0075]

[0076] In Table 1, " / " indicates that there is no such item.

[0077] 2.3. Conjugation and natural transformation culture.

[0078] The bacterial solution of drug-resistant Escherichia coli with tetracycline resistance cultured with anhydrous sodium sulfate was centrifuged after growing for 12 h. The supernatant after centrifugation was filtered through a 0.22 μm organic filter membrane into a sterile cryogenic tube. At the same time, the bacterial precipitate after the above centrifugation operation was washed 3 times with PBS, and PBS was added after washing to the original bacterial solution concentration to obtain a bacterial precipitate solution.

[0079] 0.5 mL of the supernatant of drug-resistant Escherichia coli and the bacterial precipitate solution were respectively taken and contacted and cultured with non-drug-resistant Pseudomonas aeruginosa without antibiotic resistance cultured under the same sulfur form and sulfur concentration in a new liquid medium under the same sulfur form and sulfur concentration, that is, divided into two groups for culture. One group was the supernatant of drug-resistant Escherichia coli + non-drug-resistant Pseudomonas aeruginosa; the other group was the precipitate of drug-resistant Escherichia coli + non-drug-resistant Pseudomonas aeruginosa. Subsequently, the Kefir flora supernatant was added to the two groups, and the addition amount of the Kefir flora supernatant was set at 4 gradients, which were 0 mL, 1 mL, 2 mL and 4 mL respectively. After culturing to the logarithmic phase, 0.1 mL of the bacterial solution of the two groups was sucked with a pipette and respectively inoculated on the CN solid medium containing 20 μg / mL tetracycline, spread evenly with a sterile spreading rod and then cultured upside down for 20 h, see Figure 3 .

[0080] Figure 3Among them, the added amounts of 0 mL, 1 mL, 2 mL, and 4 mL of Kefir flora supernatant were denoted as S0, S1, S2, and S3 in sequence.

[0081] It can be seen from the results in Table 1 that Kefir supernatant can produce substances with strong antibacterial activity and has obvious inhibitory effects on drug-resistant Escherichia coli and Pseudomonas aeruginosa. According to Figure 3 it is known that Pseudomonas aeruginosa can produce a large amount of pyocyanin in the CN solid medium, while drug-resistant Escherichia coli cannot. Through pyocyanin, drug-resistant Escherichia coli colonies and Pseudomonas aeruginosa colonies can be distinguished. Whether in the environment of drug-resistant Escherichia coli bacterial precipitate or in the environment of drug-resistant Escherichia coli supernatant, as the added amount of the flora supernatant increases, the number of Pseudomonas aeruginosa decreases, indicating that Kefir supernatant can inhibit the conjugation and natural transformation of resistance genes. Therefore, the Kefir supernatant of the present invention has potential application prospects in antibacterial drugs.

[0082] Drug resistance genes can be horizontally transferred, and the ways of horizontal transfer include natural transformation and conjugation. The tetracycline resistance gene in drug-resistant Escherichia coli can enter drug-free Pseudomonas aeruginosa, enabling the originally drug-free Pseudomonas aeruginosa to acquire the drug resistance gene and become drug-resistant. The supernatant of drug-resistant Escherichia coli simulates natural transformation, and the bacterial precipitate simulates conjugation. In the invention, adding Kefir supernatant achieved the inhibition of the transfer of drug resistance genes.

[0083] The obtained Kefir flora supernatant was transferred to a 1.5 mL sterile cryotube and sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for metabolomics sequencing, and Excel 2023 was used to statistically analyze the metabolite data of Kefir flora supernatant.

[0084] As can be seen from Table 2, the main components of Kefir flora supernatant include organic acids and their derivatives, lipids and lipid-like molecules, organic heterocyclic compounds, organic oxygen compounds, benzene ring-type compounds, saccharides and polyketide compounds, nucleosides, nucleotides and their analogs, alkaloids and their derivatives, organic nitrogen compounds, lignans, neolignans and related compounds, homogeneous non-metal compounds, organic sulfur compounds, hydrocarbon derivatives, hydrocarbons, alkenes, 1,3-dipolar organic compounds, and organic phosphorus compounds.

[0085] Table 2 Classification of secondary identified metabolites in Kefir flora supernatant

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as falling within the scope described in this specification.

[0093] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Use of kefir flora supernatant in the preparation of a preparation for inhibiting drug-resistant bacteria, characterized in that, The preparation method of the Kefir flora supernatant comprises the following steps: Prepare a seed solution of Kefir flora with a cell concentration of 10 8 CFU / mL to 10 9 CFU / mL; Inoculate the seed liquid of the Kefir flora into LB liquid medium and culture at 33°C - 37°C for 18h - 24h; Centrifuge at 2°C - 6°C and 4000g - 6000g for 3min - 8min to remove the bacteria bodies, and collect the supernatant, namely the Kefir flora supernatant.

2. The application according to claim 1, characterized in that, The drug-resistant bacteria include at least one of drug-resistant Pseudomonas aeruginosa, drug-resistant Escherichia coli, and drug-resistant Candida albicans.

3. The application according to claim 1, characterized in that, The culture conditions are 35°C and 20h; The conditions for collecting the supernatant are centrifuging at 4°C and 5000g for 5min to remove the bacteria bodies.

4. The application according to claim 1, characterized in that, When inoculating the seed liquid of the Kefir flora into LB liquid medium, the inoculation amount of the seed liquid of the Kefir flora accounts for 1% of the volume of the LB liquid medium.

5. The application according to claim 1, characterized in that The Kefir flora supernatant reduces the resistance of drug-resistant bacteria to chemical bactericides.

6. The application according to claim 5, wherein The chemical bactericides include at least one of chlorine preparations, hydrogen peroxide, and quaternary ammonium salts.

7. The application according to claim 6, wherein, The chlorine preparations include at least one of sodium hypochlorite and calcium hypochlorite; The quaternary ammonium salts include at least one of cetyl dimethyl ammonium chloride, methylbenzyl dimethyl ammonium chloride, and trimethyl ammonium bromide.

8. A fungicide, characterized in that, The bactericide comprises the Kefir flora supernatant as claimed in claim 5, a chemical bactericide, and a pharmaceutically acceptable excipient.

9. The fungicide according to claim 8, wherein, The pharmaceutically acceptable excipient includes at least one of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickening agent, an emulsifier, a preservative, and a stabilizer.

10. The fungicide according to claim 8, characterized in that, The acceptable dosage forms of the bactericide include one of tablets, capsules, granules, pills, powders, ointments, and oral liquids.

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