Application of indole-producing Chryseobacterium sp.

Abstract

The present invention provides cultures of the genus Chryseobacterium and active compounds isolated from the cultures, and uses and methods of use thereof for resisting the genus Chryseobacterium. The culture (including fermentation broth, fermentation supernatant, metabolite, extract and / or extract) of the indoleproducing Chryseobacterium sp. And the active compound separated from the culture can inhibit or kill the indoleproducing Chryseobacterium sp., can be used for preparing a product for practical application, and has huge application potential in the fields of medicines, pesticides and cleaning products.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to an application of indole-producing Chryseobacterium. Background Art

[0002] Natural products refer to the components, metabolites and / or endogenous chemical components in animals, plants, insects, marine organisms and microorganisms. Microbial natural products provide a rich source of chemical diversity and are one of the important sources of medicinal small molecules, food additives, insecticides, antibacterial agents, etc. The above-mentioned important chemical substances are usually derived from the secondary metabolites of microorganisms, which can be used alone or in combination with known compounds to achieve the purpose of insecticide and antibacterial.

[0003] Chryseobacterium belongs to the family Flavobacteria, phylum Firmicutes. Vandamme first described it in 1994, separating it from the genus Flavobacterium, and later Kampfer, Wu and others supplemented the characteristics of the genus. Chryseobacterium is widely found in nature, including soil, sewage, plant roots, fish, insect intestines, etc.

[0004] Chryseobacterium indologenes (CIN) is a non-fermenting Gram-negative bacillus that is widely present in natural environments such as soil, water, and plants, as well as in hospital environments. It is a conditional pathogen that can cause serious infections in humans, animals, and plants.

[0005] Chryseobacterium indologenes can cause a variety of diseases such as intraperitoneal infection, endocarditis, bacteremia, meningitis, etc. It is particularly common in patients who have been using broad-spectrum antibiotics and immunosuppressants for a long time, and multi-drug resistance is usually present. It is currently believed that the drug resistance mechanism of Chryseobacterium indologenes is mainly due to the metallo-β-lactamase (MBL) produced by the bacterium, which belongs to the Bush-Medeiros-Jacoby3 group and the molecular structure belongs to group B. It has a zinc ion active center and can hydrolyze β-lactam antibiotics such as carbapenems, cephalosporins and penicillins, but is insensitive to commonly used β-lactamase inhibitors such as clavulanic acid and sulbactam. According to Tang Manjuan's research, among the indologenic Chryseobacterium strains isolated clinically from January 2012 to December 2016, the resistance rate of CIN to sulfamethoxazole / trimethoprim was the lowest, at 3.6%, followed by levofloxacin, piperacillin / tazobactam, and ciprofloxacin, with resistance rates ranging from 30% to 40%, and the resistance rates to aminoglycosides, cephalosporins, carbapenems and other antimicrobial drugs were relatively high, all ranging from 80% to 100%. According to Huang Shaojun's research, among the indologenic Chryseobacterium strains isolated clinically from 2013 to 2017, 100% of them were resistant to amikacin, antralin, ampicillin, gentamicin, ceftriaxone, cefoxitin, cefepime, cefazolin, tobramycin, and imipenem, and the resistance rate to cotrimoxazole was 10.76%. In view of the broad-spectrum drug resistance of Chryseobacterium indologenes, it is necessary to develop new drugs for Chryseobacterium indologenes.

[0006] Chryseobacterium indologenes can also cause serious diseases in livestock, fish, frogs, Chinese soft-shelled turtles, river crabs and other aquatic animals. For example, in 2013, a large number of deaths occurred in the Channa argus breeding ponds in Zhejiang Province. Wang Xinyi and others believed that the pathogen causing the disease of the Channa argus was Chryseobacterium indologenes.

[0007] Indole-producing Chryseobacterium can also cause plant infection and death. For example, Zhang Jinhao and others found through research that the pathogen that causes root rot of Panax notoginseng in Yunnan is indole-producing Chryseobacterium. The root rot caused by this bacterium seriously harms the quality of Panax notoginseng and may even cause a total loss of Panax notoginseng harvest.

[0008] Due to the broad-spectrum drug resistance of Chryseobacterium indologenes and the harm to aquaculture and plants, there are currently few uses for Chryseobacterium indologenes. At present, there are reports on the uses of Chryseobacterium indologenes in the following aspects: (1) "Isolation, Identification and Enzymatic Properties of Chryseobacterium indologenes Producing a New Elastase" discloses a strain of Chryseobacterium indologenes with a strong ability to hydrolyze beef tendon powder. After liquid fermentation and other treatments, the strain can obtain elastase. The enzyme not only preferentially hydrolyzes elastin, but also has a strong hydrolysis effect on casein, gelatin and soy protein isolate. (2) YM5 is a strain of Chryseobacterium indologenes with high efficiency in feather keratin degradation. Its bacterial liquid and feather fermentation liquid are safe and non-toxic to mammals and can be used in industrial production. (3) Chryseobacterium indologenes (preservation number is CCTCC NO: M 2018651) can efficiently degrade organic pollutants in wastewater, thereby improving water quality. (4) Korean patent KR1020110092616A discloses a composition for preventing plant anthracnose and reducing the amount of pesticides used, which includes indoliferobacterium ISE14. (5) "Isolation, screening and identification of protein glutaminase producing bacteria" found that the fermentation enzyme activity of indoliferobacterium ZYF120413-7 was as high as 0.7168U / mL, which can be used to produce protein glutaminase. (6) Chinese patent ZL 202011064002.9 discloses indoliferobacterium strain EC200816 and the culture of the strain, and its use and method for resisting pine wood nematode (Bursaphelenchus xylophilus). Summary of the invention

[0009] In view of the current widespread drug resistance of indologenic Chryseobacterium to antibiotics and the imperfect development of its application, in order to find new compounds to control the indologenic Chryseobacterium and the imperfect development of the use of indologenic Chryseobacterium in the prior art, the present invention provides an application of indologenic Chryseobacterium, and uses it to prepare antibacterial preparations and products.

[0010] The present invention adopts the following technical solutions to achieve the above-mentioned purpose.

[0011] Use of a Chryseobacterium culture and / or an active compound separated from the culture in combating Chryseobacterium bacteria.

[0012] The Chryseobacterium culture is a fermentation broth, fermentation supernatant, metabolite, extract and / or extract of Chryseobacterium;

[0013] The active compound separated from the culture is one or more compounds separated from the fermentation liquid, fermentation supernatant, metabolite, extract and / or extract of Chryseobacterium and having activity against the bacteria of Chryseobacterium.

[0014] The fermentation liquid of Chryseobacterium is obtained by fermenting and culturing the strain of Chryseobacterium in a culture medium, and the bacterial cells are separated from the fermentation liquid by means of centrifugation, so as to obtain a fermentation supernatant.

[0015] The metabolites of Chryseobacterium are substances produced during the metabolism of Chryseobacterium strains. The supernatant, filtrate, fermentation broth, extracts, certain compounds, substances or by-products in the extracts of the strains can all be metabolites.

[0016] The extracts and / or extractants are all obtained by fermenting and culturing Chryseobacterium bacteria, and the resulting fermentation liquid is treated with water or an organic solvent.

[0017] The Chryseobacterium bacteria include but are not limited to: C. indologenes, C. indogens, C. balustinum, C. scophthalmum, C. shigense, C. jejuense, C. hominis, C. meningosepticum, C. lactis, C. hagamense, C. arothri, C. joostei, C. gleum, C. rhizoplanae, C. vietnamense, C. nakagawa, etc.

[0018] An application of Chryseobacterium indologenes, and an application of a culture of Chryseobacterium indologenes and / or an active compound separated from the culture in resisting Chryseobacterium indologenes;

[0019] The indole-producing Chryseobacterium was named EC200816 and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 24, 2020, with the collection number GDMCC No: 61309.

[0020] Furthermore, the indole-producing Chryseobacterium has the identification characteristics of the genus Chryseobacterium: the colonies are golden yellow, with round protrusions, translucent, smooth and moist surfaces, and complete edges; Gram staining is negative, non-motile, and does not form spores; it requires aerobic air and is suitable for growth at 30°C; and it is oxidase and catalase positive.

[0021] Furthermore, the indologenic Chryseobacterium is separated from pine wood nematodes and then obtained through culture.

[0022] Furthermore, the indologenic Chryseobacterium is isolated from pine wood nematode and then cultured in a culture medium at 25-37° C. and pH 7.0-8.0.

[0023] Application of the culture of Chryseobacterium indologenes and / or the active compounds isolated from the culture in antibacterial applications.

[0024] Use of a culture of Chryseobacterium indologenes and / or an active compound separated from the culture in the preparation of an antibacterial preparation.

[0025] The indologenic Chryseobacterium culture is the fermentation liquid, fermentation supernatant, metabolite, extract and / or extraction of indologenic Chryseobacterium;

[0026] The active compound separated from the culture is a compound with antibacterial activity obtained by separating one or more substances in the fermentation liquid, fermentation supernatant, metabolite, extract and / or extract of indologenic Chryseobacterium.

[0027] Furthermore, the fermentation supernatant of the indole-producing Chryseobacterium has antibacterial properties.

[0028] Furthermore, the extract of Chryseobacterium indologenes has antibacterial properties.

[0029] Furthermore, the extract of Chryseobacterium indologenes has antibacterial properties.

[0030] Furthermore, the metabolites of Chryseobacterium indologenes have antibacterial properties.

[0031] The fermentation liquid of the indologenic Chryseobacterium is obtained by fermenting and culturing the strain in a culture medium, and the bacterial cells are separated from the fermentation liquid by means of centrifugation, so as to obtain a fermentation supernatant.

[0032] The metabolites of the indologenic Chryseobacterium are substances produced during the metabolism of the strain. The supernatant, filtrate, fermentation broth, extract, certain compounds, substances or by-products in the extract of the strain can all be metabolites.

[0033] The metabolites of the indologenic bacterium of the present invention include indole.

[0034] The extracts and / or extractants are all obtained by fermenting and culturing Chryseobacterium indologenes, and the resulting fermentation liquid is treated with water or an organic solvent.

[0035] The preparation method of the extract comprises the following steps: fermenting and culturing indologenic Chryseobacterium to obtain a fermentation broth, collecting the bacterial bodies in the fermentation broth, adding water or an organic solvent for extraction, then removing the bacterial bodies, and evaporating the solvent to obtain a solution to obtain the extract.

[0036] The preparation method of the indologenic Chryseobacterium extract comprises the following steps: fermenting and culturing the indologenic Chryseobacterium to obtain a fermentation broth, removing the bacterial cells from the fermentation broth to obtain a fermentation supernatant, adding an organic solvent to the supernatant for extraction, and evaporating the solvent from the obtained organic solvent layer to obtain an extract.

[0037] The method for preparing the indologenic Chryseobacterium indologene extract comprises the following steps: the volume ratio of the fermentation supernatant to the organic solvent is 1:(0.5-5), and the extraction times are 1-5.

[0038] The method for preparing the extract of Chryseobacterium indologenes, wherein the organic solvent includes but is not limited to petroleum ether, ethyl acetate, dichloromethane, chloroform, ether, n-butanol, n-pentane, n-hexane and the like.

[0039] In the preparation method of the indologenic Chryseobacterium extract, the volume ratio of the fermentation supernatant to the organic solvent is 1:(0.5-5), and the active compound is obtained by extraction 1-5 times; the organic solvent is petroleum ether.

[0040] The active compound contains indole;

[0041] The active compound is further purified to obtain a single active compound, indole.

[0042] Use of the culture of Chryseobacterium indologenes and / or the active compounds separated from the culture in the preparation of antibacterial preparations;

[0043] The culture of Chryseobacterium indologenum is the fermentation liquid, fermentation supernatant, metabolite, extract and / or extraction of Chryseobacterium indologenum;

[0044] The active compound separated from the culture is one or more compounds with antibacterial activity separated from the fermentation liquid, fermentation supernatant, metabolites, extracts and / or extracts of Chryseobacterium indologenes.

[0045] An antibacterial preparation, wherein the active ingredient of the anti-indologenic Chryseobacterium preparation contains the indologenic Chryseobacterium culture and / or the active compound separated from the culture;

[0046] The indologenic Chryseobacterium culture is the fermentation liquid, fermentation supernatant, metabolite, extract and / or extraction of indologenic Chryseobacterium;

[0047] The active compound separated from the culture is one or more compounds with antibacterial activity separated from the fermentation liquid, fermentation supernatant, metabolites, extracts and / or extracts of Chryseobacterium indologenes.

[0048] The methods of obtaining the above-mentioned different active ingredients refer to the above records; the compounds obtained by extraction or extraction can also be synthesized by oneself or purchased from the market.

[0049] The antibacterial preparation comprises an active ingredient and an agriculturally acceptable carrier; wherein the active ingredient accounts for 0.001 to 99% of the product mass.

[0050] The active ingredients may also include insecticides, nematicides, miticides, bactericides, fungicides, herbicides, growth regulators, disinfectants, repellents, attractants and / or biopesticides.

[0051] The above antibacterial preparations may also include one or more of the following components: a wetting agent, a thickener, a viscosity modifier, a stabilizer, a surfactant and / or a dispersion medium (such as water, alcohols, edible oils, etc.).

[0052] An antibacterial composition, which includes the metabolites or extracts of the above-mentioned indologenic Chryseobacterium, and / or active compounds with antibacterial effects separated from the metabolites or extracts of the indologenic Chryseobacterium; and may also include other insecticides, nematicides, miticides, bactericides, fungicides, herbicides, growth promoters, growth regulators, disinfectants, repellents, attractants and / or biological pesticides.

[0053] The above antibacterial preparation or antibacterial composition can be used to prepare anti-indologenic Chryseobacterium indolegenum products.

[0054] Furthermore, the anti-indocyanobacterium indolegenicum product is an antibacterial drug, a pesticide or a cleaning product.

[0055] An antibacterial drug containing the above antibacterial preparation.

[0056] An antibacterial pesticide contains the above antibacterial preparation.

[0057] An antibacterial cleaning product contains the above antibacterial preparation.

[0058] An antibacterial method, comprising applying the above antibacterial preparation, product and / or composition to soil, water source, hospital and other places in need of antibacterial treatment infected by bacteria, so as to kill and / or inhibit unwanted bacteria.

[0059] The above-mentioned antimicrobial preparations and / or antimicrobial products can be applied to the soil. In some cases, the antimicrobial preparations or products are applied to the soil before planting; in some cases, the antimicrobial preparations or products are applied to the soil after planting; in some cases, the antimicrobial preparations or products are applied to the soil using a drip irrigation system; in some cases, the antimicrobial preparations or products are applied to the soil using a spraying system; in some cases, the antimicrobial preparations or products are tilled into the soil or applied in ditches.

[0060] The above-mentioned antibacterial preparations and / or antibacterial products can be applied to water sources that allow the application of the above-mentioned preparations and / or products, specifically, breeding ponds, etc.

[0061] The above antibacterial preparations and / or antibacterial products can be applied to places requiring antibacterial treatment, such as hospitals. In some cases, the antibacterial preparations or products are applied to hospitals using a spraying system; in some cases, the antibacterial preparations or products are applied by hand washing or the like.

[0062] The above-mentioned antibacterial preparations and / or antibacterial products can be applied to living bodies that require antibacterial treatment, such as human bodies, animals, etc.

[0063] The antibacterial effect includes bactericidal effect and bacteriostasis effect. Bactericidal effect refers to killing the vegetative and reproductive bodies of bacteria through a certain treatment. Bacteriostasis refers to reducing the growth and reproduction ability of bacteria, affecting the bacterial reproduction process or reducing the activity of bacteria through a certain treatment.

[0064] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0065] (1) The Chryseobacterium bacteria culture (including fermentation broth, fermentation supernatant, metabolites, extracts or extracts) and active compounds separated from the culture provided by the present invention have anti-Chryseobacterium bacteria effects, and can kill the bacteria or inhibit their development (including reducing their growth and reproduction ability, affecting the bacterial development and reproduction process or reducing the activity of the bacteria).

[0066] (2) The indocyanobacterium chrysenii provided by the present invention, its culture (including fermentation broth, fermentation supernatant, metabolites, extracts or extracts) and active compounds separated from the culture all have anti-bacterial effects and can kill indocyanobacterium chrysenii or the development of indocyanobacterium chrysenii (including reducing its growth and reproduction ability, affecting the bacterial development and reproduction process or reducing the activity of the bacteria).

[0067] (3) The active compound indole in the extract of Chryseobacterium indologenes provided by the present invention is an endogenous compound of Chryseobacterium indologenes. At an effective concentration for Chryseobacterium indologenes, it can not only inhibit Chryseobacterium indologenes, but also has little effect on the reproduction of other test bacteria. At the same time, Chryseobacterium indologenes does not produce tolerance to it, and it has no inhibitory activity on beneficial intestinal bacteria. Therefore, the endogenous compound indole has a great application prospect as a targeted drug in inhibiting Chryseobacterium indologenes.

[0068] (4) The indole-producing Chryseobacterium culture (including fermentation broth, fermentation supernatant, metabolites, extracts or extracts) and the active compounds separated from the culture provided by the present invention can be used to prepare corresponding preparations or products, and have great application potential in the fields of medicine, pesticides and cleaning products. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is a graph showing the antibacterial activity of the EC200816 fermentation supernatant in Example 3 of the present invention.

[0070] Figure 2 This is the HPLC chart of the active component PE01 separated from EC200816 in Example 3 of the present invention.

[0071] Figure 3 This is the HR-ESIMS spectrum of the purified sample PE01-1 in Example 3 of the present invention.

[0072] Figure 4 The purified sample PE01-1 in Example 3 of the present invention 1 H-NMR spectrum and 13 C-NMR spectrum. DETAILED DESCRIPTION

[0073] The technical solutions in the embodiments of the present invention are described clearly and completely below. The described embodiments are only a part of the present invention, not the whole of the invention. 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.

[0074] definition

[0075] As used herein, the terms "comprises," "comprising," "having," or any other variations thereof, are intended to cover a non-exclusive inclusion, subject to any limitation expressly stated. For example, a composition, mixture, process, or method that comprises a list of elements is not necessarily limited to those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.

[0076] The antibacterial effect of the present invention includes bactericidal effect and bacteriostasis effect. Bactericidal effect refers to killing the vegetative and reproductive bodies of bacteria through a certain treatment. Bacteriostasis refers to reducing the growth and reproduction ability of bacteria, affecting the development and reproduction process of bacteria, or reducing the activity of bacteria through a certain treatment.

[0077] The "resistance to drug" mentioned in the present invention is also called drug resistance, which refers to the tolerance of microorganisms to the effects of drugs. Once drug resistance occurs, the effect of the drug will be significantly reduced.

[0078] As used herein, "derived from" means: directly separated or obtained from a specified source, or as an alternative, having the identification characteristics of a substance or organism separated or obtained from a specified source; when the specified source is an organism, "derived from" means that it can be separated or obtained from the organism itself or from the culture medium used to culture or grow the organism.

[0079] The term "fermentation broth" as used herein refers to a liquid culture containing both cells and a culture medium. If the bacteria are grown on a culture plate, the cells can be collected in water or other liquids (whole culture).

[0080] The "supernatant" of the present invention refers to the liquid remaining when cells grown in a culture medium are collected and the cells are removed by centrifugation, filtration, sedimentation or other means in their own culture medium or another liquid.

[0081] The "isolated compound" of the present invention refers to a compound that is substantially free of other unwanted compounds or substances, or other unwanted compounds or substances are controlled within a desired content range; for example, at least about 10% pure, preferably about 20% pure, more preferably about 30% pure, more preferably about 40% pure, more preferably about 50% pure, more preferably about 60% pure, more preferably about 70% pure, more preferably about 80% pure, more preferably about 90% pure, more preferably about 95% pure, and most preferably about >97% pure; the analytical methods include but are not limited to conventional methods such as extraction, chromatography and / or electrophoresis.

[0082] Example 1 Source and activation of EC200816 strain

[0083] Prepare MHA agar plate culture medium: 6 g / L beef powder, 1.5 g / L soluble starch, 17.5 g / L acid hydrolyzed casein, and 17 g / L agar.

[0084] The indologenic Chryseobacterium indologenum EC200816 strain (Chinese patent ZL 202011064002.9) was streaked onto MHA agar plate medium for activation and inverted culture at 28°C for 24 hours to obtain the activated EC200816 strain.

[0085] The above strain isolation process is detailed in Chinese patent ZL 202011064002.9. The strain is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 61309.

[0086] Example 2 Drug sensitivity test of Chryseobacterium indologenes EC200816

[0087] 1. Drug treatment

[0088] Streptomycin (STR): Streptomycin sulfate was used to prepare a 500 μg / mL solution.

[0089] Gentamicin (GEN): Use gentamicin sulfate to prepare a 2560 μg / mL stock solution, and use the two-fold dilution method to gradually dilute it into 1280, 640, 320, 160, 80, 40, 20, 10, and 5 μg / mL solutions.

[0090] Enrofloxacin (ENR): Use enrofloxacin to prepare a 2560 μg / mL stock solution, and use the two-fold dilution method to gradually dilute it into 1280, 640, 320, 160, 80, 40, 20, 10, and 5 μg / mL solutions.

[0091] All drugs were sterilized by filtration through a 0.22 μm membrane before use.

[0092] 2. Drug sensitivity test

[0093] Prepare MHA agar plate culture medium (6 g / L beef powder, 1.5 g / L soluble starch, 17.5 g / L acid hydrolyzed casein, 17 g / L agar) and MH broth culture medium (6 g / L beef powder, 1.5 g / L soluble starch, 17.5 g / L acid hydrolyzed casein).

[0094] The drug sensitivity was tested by disk diffusion and broth dilution methods.

[0095] Preparation of bacterial suspension: Pick a single colony of EC200816 on the above MHA agar plate, inoculate it into MH broth medium, and culture it in a shaking incubator at 35°C for 6-8h to obtain a logarithmic phase EC200816 bacterial suspension, which is used for drug sensitivity testing after being calibrated to 0.5 McFarland turbidity using a turbidimetric tube.

[0096] Paper diffusion method: Take a sterile blank drug-sensitive paper (6mm in diameter) and place it on a sterile and clean culture dish. Take 20μL of 500μg / mL streptomycin stock solution and drop it on the drug-sensitive paper to make the drug content of the drug-sensitive paper 10μg. Let it air dry naturally in the clean bench, and use the solvent as a blank control. Dip a sterile cotton swab in the above-mentioned logarithmic EC200816 bacterial suspension (0.5 McFarland turbidity), evenly spread it on the MHA plate, and dry it slightly in the clean bench. Gently stick the air-dried drug-sensitive paper on the MHA plate containing the EC200816 bacterial suspension. The paper cannot be moved after it is pasted. The distance between each drug-sensitive paper is >2.4cm. After inverted culture at 35℃ for 16-18h, measure the diameter of the inhibition zone (including the paper).

[0097] Broth dilution method: Add the twice diluted antibiotic solution to MH broth at a ratio of 1:9, mix the broth and the drug thoroughly, use the solvent as a blank control, and dispense into glass test tubes at 1 mL / tube. Prepare the EC200816 bacterial suspension in the logarithmic phase (0.5 McFarland turbidity, about 10 8 CFU / mL), and then diluted to 10 6 CFU / mL bacterial solution, 1mL inoculated per glass test tube 10 6 CFU / mL bacterial solution, so that the final concentration of the bacterial solution is 5×10 5CFU / mL, the final antibiotic concentrations were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0 μg / mL. After culturing in a shaking incubator at 35°C for 16 to 20 h, the turbidity of the glass tube was observed, and the lowest concentration that inhibited the visible growth of bacteria was the MIC value.

[0098] 3. Experimental results

[0099] There was no inhibition zone for streptomycin, MIC for gentamicin was 128 μg / mL, and MIC for enrofloxacin was 0.5 μg / mL.

[0100] The drug sensitivity of the strain was determined according to CLSI standards. EC200816 was resistant to streptomycin and gentamicin, and was intermediate to enrofloxacin.

[0101] Table 1 Bacterial drug sensitivity determination standard (CLSI)

[0102]

[0103] Example 3 Preparation of active components from the fermentation supernatant of Chryseobacterium indologenes EC200816 and identification of active compounds

[0104] 1. Preparation of EC200816 fermentation supernatant

[0105] LB liquid medium: tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L;

[0106] EC200816 was fermented and cultured using the above LB liquid medium at 28°C and 180 r / min shaking for 5 days to obtain EC200816 fermentation broth, which was centrifuged at 12,000 rpm / min for 10 min, and the supernatants were collected and combined, i.e., EC200816 fermentation supernatant.

[0107] 2. Determination of antibacterial activity of EC200816 fermentation supernatant-1

[0108] Experimental method: The EC200816 fermentation supernatant was evaporated to dryness at 35°C in a vacuum rotary evaporator to obtain the EC200816 fermentation supernatant dry matter, which was then added with water to prepare 100 g / L EC200816 fermentation supernatant-1 (sterilized by 0.22 μm membrane filtration before use).

[0109] Prepare LB plate (10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, 15 g / L agar), raise one end of the LB plate in the clean bench to form a slope, add fermentation supernatant-1 so that the liquid surface covers 1 / 2 area of ​​the LB plate, let it stand overnight, pour out excess fermentation supernatant-1, and air dry naturally in the clean bench. Use a sterile cotton swab to dip into the logarithmic phase EC200816 bacterial suspension (0.5 McFarland turbidity), evenly spread it on the LB plate after the above treatment, dry it slightly in the clean bench, and invert and culture at 30°C for 48 h.

[0110] Experimental results: EC200816 growth status see Figure 1 It can be clearly seen from the figure that in the LB plate part soaked by the fermentation supernatant-1, only a small number of EC200816 colonies grew; while in the control LB liquid culture medium part, EC200816 bacterial lawn grew. This shows that the EC200816 fermentation supernatant-1 contains active substances that inhibit the growth of EC200816, that is, the EC200816 fermentation supernatant contains active substances that inhibit the growth of EC200816.

[0111] 3.Petroleum ether extraction of EC200816 fermentation supernatant

[0112] Add 1.5 times the volume of petroleum ether to the EC200816 fermentation supernatant, shake thoroughly to mix, let stand to separate, collect the upper petroleum ether layer, extract three times, combine the petroleum ether layers, and perform vacuum rotary evaporation at 35°C to evaporate the petroleum ether solvent to obtain the EC200816 fermentation supernatant petroleum ether extract PE01.

[0113] 4. Determination of the antibacterial activity of PE01 against EC200816

[0114] Experimental procedures: refer to the drug sensitivity test-disk diffusion method in Example 2.

[0115] PE01 sample preparation: DMSO was used to prepare 40g / L PE01 stock solution, which was diluted to 30, 20, and 10g / L sample solutions and sterilized by 0.22μm membrane filtration before use. The sample amounts of the drug-sensitive paper sheets were 0.8, 0.6, 0.4, and 0.2mg, respectively.

[0116] Experimental results: The diameter of the inhibition zone was measured, and the results are shown in the table below. When the paper contained more than 0.6 mg of PE01, it had antibacterial activity against EC200816, indicating that PE01 is an active component with inhibitory activity against indologenic Chryseobacterium indolegenes, and contains active compounds that inhibit indologenic Chryseobacterium indolegenes EC200816.

[0117] Table 2 Antibacterial effect of PE01 on EC200816

[0118] Drug content (mg) Diameter of inhibition zone (mm) 0 0 0.2 0 0.4 0 0.6 15 0.8 20

[0119] 5. Analysis and identification of PE01 active compounds

[0120] Prepare methanol solution of PE01 sample and perform HPLC analysis to obtain PE01 HPLC chart ( Figure 2 ). Liquid phase conditions: mobile phase A-0.1% formic acid water, mobile phase B-chromatographic methanol; chromatographic column ZORBOX Eclipse plus C18 (4.6*150mm, 5μm); chromatographic program: 50% B isocratic 19min; wavelength: 210nm; flow rate: 1mL / min; column temperature: 30℃.

[0121] Reference Figure 2 The main peak (retention time: 5.290 min) was collected by preparative liquid phase according to the PE01 HPLC spectrum and liquid phase conditions. After merging, the solvent was evaporated to obtain the purified sample PE01-1, which was then subjected to high-resolution mass spectrometry and nuclear magnetic resonance identification and analysis.

[0122] PE01-1 is white crystal, HR-ESIMS spectrum ( Figure 3 ) gives a quasi-molecular ion peak m / z 118.0651[M+H] + (calcd for C8H8N, 118.0657), the molecular weight of the compound is determined to be 117, the molecular formula is C8H7N, and the degree of unsaturation is calculated to be 6. 1 H-NMR (600MHz, CD3OD) spectrum is shown in Table 3. Figure 4 As shown, δ7.55 (1H, d, J = 7.8 Hz, H-4), 7.37 (1H, d, J = 7.8 Hz, H-7), 7.09 (1H, t, J = 7.8 Hz, H-6), 7.00 (1H, t, J = 7.8 Hz, H-5) indicate a group of hydrogen signals on the ortho-disubstituted benzene ring, and δ7.18 (1H, d, J = 2.8 Hz, H-1) and 6.44 (1H, d, J = 2.8 Hz, H-2) indicate a group of hydrogen signals on the double bond. 13 C-NMR (150MHz, CD3OD) spectrum is shown in Table 3. Figure 4 As shown, δ137.5 (C-8), 129.3 (C-3), 122.1 (C-5), 121.1 (C-4), 119.9 (C-6), 112.0 (C-7) indicate carbon signals on the benzene ring, and δ125.3 (C-1) and 102.2 (C-2) indicate a group of carbon signals on the ortho-disubstituted benzene ring. 1 H-NMR and 13The C-NMR data were compared with the indole reported in the literature and were basically consistent, so PE01-1 was identified as indole. The compound number and chemical structure are as follows:

[0123]

[0124] Table 3PE01-1 1 H(600MHz)and 13 C (150 MHz) NMR data

[0125]

[0126] 4. Indole antibacterial experiment

[0127] Experimental procedures: refer to the drug sensitivity test-disk diffusion method in Example 2.

[0128] Preparation of indole solution: DMSO was used to prepare 40g / L indole mother solution, which was diluted to 30, 20, and 10g / L solution and sterilized by 0.22μm membrane filtration before use. The drug content of the paper was 0.8, 0.6, 0.4, and 0.2mg respectively.

[0129] Experimental results: The diameter of the inhibition zone was measured. The results are shown in the table below. Indole has an inhibitory effect on EC200816. At the same drug content, its antibacterial activity is stronger than that of the fermentation supernatant petroleum ether extract PE01.

[0130] Table 4 Antibacterial effect of indole on EC200816

[0131] Drug content (mg) Diameter of inhibition zone (mm) 0 0 0.2 23.5 0.4 28.3 0.6 36.5 0.8 40.0

[0132] Example 4 Drug resistance and targeting experiments

[0133] 1. Enrofloxacin induces EC200816 resistance

[0134] One-step method: prepare an MHA culture plate containing 2 μg / mL ENR, inoculate it with the logarithmic phase EC200816 bacterial suspension in Example 2, invert and culture at 30°C, pick the grown colonies at 24, 48, 72, 96, and 120 hours and inoculate them on the same concentration of drug-containing plates for purification, and then inoculate them on drug-free culture plates for stable passage 10 times to obtain the EC200816-Enr2 strain, and detect its MIC value by referring to the drug sensitivity test-broth dilution method in Example 2.

[0135] Multi-step method: Prepare MH broth medium containing 16, 8, 4, 2, 1, 1 / 2, 1 / 4 MIC, i.e., 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / mL ENR, inoculate the logarithmic phase EC200816 bacterial suspension in Example 2 in MH broth medium with a concentration of 1 / 4 MIC, induction culture at 30°C, and then inoculate in MH broth medium with a concentration of 1 / 2 MIC, and so on, induce culture for 3 to 5 generations at each concentration, and pass one generation every 24 hours until the strain cannot grow normally. After the strain that can grow normally in the drug-containing medium is inoculated into the drug-free MH broth medium for 10 stable passages, the EC200816-Enr8 strain is obtained, and its MIC value is detected by referring to the drug sensitivity test-broth dilution method in Example 2.

[0136] Experimental results: The results are shown in the table below. Both methods can induce EC200816 strain to develop resistance to enrofloxacin, and its MIC values ​​are increased by 8 and 16 times, respectively.

[0137] Table 5 MIC determination of enrofloxacin-induced EC200816 resistant strain

[0138] Strain name MIC (μg / mL) Drug sensitivity EC200816 (original strain) 0.5 intermediary EC200816-Enr2 (one-step method) 4 Drug resistance EC200816-Enr8 (multi-step method) 8 Drug resistance

[0139] 2. Indole induces tolerance in Escherichia coli and EC200816

[0140] Referring to the above multi-step method, indole was used to induce the tolerance of Escherichia coli (E. coli) EC200816 to obtain the tolerant strain E. coli-YD.

[0141] The experimental results are shown in the table below. The MIC value of E. coli can be doubled by inducing the strain from low to high concentrations of indole. However, when the concentration of EC200816 increased from 1 / 4 MIC to MIC, EC200816 could not grow normally. This means that using the same method, indole did not induce EC200816 to develop tolerance to it.

[0142] Table 6 MIC determination of indole-induced E. coli tolerant strains

[0143] Strain name MIC (μg / mL) E.coli (original strain) 512 E.coli-YD (multi-step method) 1,024

[0144] 3. Indole antibacterial test

[0145] According to the drug sensitivity test-paper diffusion method in Example 2, the antibacterial experiment of indole on the following strains was carried out, and the results are shown in the following table. When the drug content was 0.6 mg, indole had the strongest inhibitory effect on Chryseobacterium indologenes EC200816, indicating that indole has a high targeting effect on Chryseobacterium indologenes EC200816.

[0146] Table 7 Antibacterial effect of indole on various strains

[0147]

[0148] Broth dilution microplate method: 1,600 μg / mL indole mother solution was prepared using MH broth as solvent, and diluted to 800, 400, 200, 100, 50, 25, 12.5, and 6.25 μg / mL by two-fold dilution method. 50 μL of the solution was added to the middle position of a 96-well microplate (blank culture medium was added to the peripheral wells), and each concentration was repeated three times, with the solvent as a blank control. Take the EC200816 bacterial suspension in the logarithmic phase of Example 2 (0.5 McFarland turbidity, about 10 8 CFU / mL), and then diluted to 10 6 CFU / mL bacterial solution, 50 μL inoculated into each well of 96-well microplate 10 6 CFU / mL bacterial solution, so that the final concentration of the bacterial solution is 5×10 5 CFU / mL, the final indole concentration was 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 0 μg / mL. After culturing at 35°C for 16-20 h, the turbidity of the 96-well microplate was observed, and the lowest concentration that inhibited the visible growth of bacteria was the MIC value.

[0149] The MIC of indole against the following strains was determined according to the above-mentioned broth dilution microplate method, and the results are shown in the table below. It can be seen from the table that the MIC of indole against enrofloxacin-resistant indologenic Chryseobacterium strains EC200816-Enr2 and EC200816-Enr8 is the same as that of the original indologenic Chryseobacterium strain EC200816, indicating that indole also has an inhibitory effect on enrofloxacin-resistant EC200816 strains; the MIC of indole against common pathogenic bacteria Staphylococcus aureus and Escherichia coli is higher than that of indologenic Chryseobacterium, indicating that indole has a higher targeted inhibitory activity against indologenic Chryseobacterium; at the same time, indole has no inhibitory effect on the probiotic Bifidobacterium.

[0150] Table 8 MIC determination of indole against various strains

[0151] Strain name MIC (μg / mL) EC200816 200 EC200816-Enr2 200 EC200816-Enr8 200 Staphylococcus aureus 400 Escherichia coli 400 Bifidobacterium sp. —(No inhibitory effect)

Claims

1. An application of Chryseobacterium, characterized in that: The use of the Chryseobacterium culture and / or the active compound separated from the culture in combating the Chryseobacterium.

2. An application of Chryseobacterium indologenes, characterized in that: Application of the indocyanobacterium indocyane culture and / or active compounds separated from the culture in resisting indocyanobacterium indocyane.

3. The use of Chryseobacterium indolegenes according to claim 2, characterized in that: The indologenic Chryseobacterium culture is: fermentation liquid, fermentation supernatant, metabolite, extract and / or extraction of indologenic Chryseobacterium; The active compound separated from the culture is: one or more compounds with antibacterial activity separated from the fermentation liquid, fermentation supernatant, metabolites, extracts and / or extracts of Chryseobacterium indologenes.

4. The use of Chryseobacterium indolegenes according to claim 3, characterized in that: The fermentation liquid of the indologenic Chryseobacterium is obtained by fermenting and culturing the strain, and the fermentation liquid is separated from the bacteria to obtain the fermentation supernatant.

5. The use of Chryseobacterium indolegenes according to claim 4, characterized in that: The extracts and / or extractants are all obtained by fermenting and culturing Chryseobacterium indologenes, and the resulting fermentation liquid is treated with water or an organic solvent.

6. The use of Chryseobacterium indolenicum according to claim 5, characterized in that: The preparation method of the extract comprises the following steps: fermenting and culturing indologenic Chryseobacterium to obtain a fermentation broth, collecting the bacterial bodies in the fermentation broth, adding water or an organic solvent for extraction, and then removing the bacterial bodies, and the obtained solution is the extract.

7. The use of Chryseobacterium indolegenes according to claim 5, characterized in that: The preparation method of the indologenic Chryseobacterium extract comprises the following steps: fermenting and culturing the indologenic Chryseobacterium to obtain a fermentation broth, removing the bacterial cells from the fermentation broth to obtain a fermentation supernatant, adding an organic solvent to the supernatant for extraction, and evaporating the solvent from the obtained organic solvent layer to obtain an extract.

8. The use of Chryseobacterium indolegenes according to claim 7, characterized in that: In the preparation method of the extract, the volume ratio of the fermentation supernatant to the organic solvent is 1:(0.5-5), the number of extractions is 1-5 times; the organic solvent is one or more of petroleum ether, ethyl acetate, dichloromethane, chloroform, ether, n-butanol, n-pentane, and n-hexane.

9. The use of Chryseobacterium indolegenes according to claim 8, characterized in that: In the preparation method of the extract, the volume ratio of the fermentation supernatant to the organic solvent is 1:(0.5-5), and the active compound is obtained by extraction 1-5 times; the organic solvent is petroleum ether.

10. The use of Chryseobacterium indolegenes according to claim 3, characterized in that: The metabolites of Chryseobacterium indologenes are substances produced during the metabolism of the strain.

11. An anti-indologenic Chryseobacterium preparation, characterized in that: The active ingredients of the anti-indologenic Chryseobacterium preparation contain the indologenic Chryseobacterium culture and / or the active compounds separated from the culture according to claim 2; The indologenic Chryseobacterium culture is the fermentation liquid, fermentation supernatant, metabolite, extract and / or extraction of indologenic Chryseobacterium; The active compound separated from the culture is one or more compounds with antibacterial activity separated from the fermentation liquid, fermentation supernatant, metabolites, extracts and / or extracts of Chryseobacterium indologenes.

12. The anti-Chryseobacterium indolegenicum preparation according to claim 11, characterized in that: The anti-indolyogenic Chryseobacterium preparation comprises an active ingredient and an agriculturally acceptable carrier; wherein the active ingredient accounts for 0.001 to 99% of the mass of the anti-indolyogenic Chryseobacterium preparation.

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