阅读说明：本技术 一株粘细菌及其在制备抑菌药物中的应用 (Myxobacteria and application thereof in preparation of antibacterial drugs ) 是由 朱红惠 周杨 姚青 蚁烁星 于 2021-07-20 设计创作，主要内容包括：本发明公开了一株粘细菌及其在制备抑菌药物中的应用。本发明采用大肠杆菌诱导法从菜地池塘沉积物中分离纯化出一株粘细菌Archangium violaceum 3-1,经形态学和分子生物学鉴定(16S rRNA基因和gyrB基因)为紫色原囊菌。本发明所述紫色原囊菌的发酵上清液粗浸膏溶液能够抑制白色念珠菌、耐甲氧西林金黄色葡萄球菌和植物病原真菌的生长,且抑菌活性大小表现出明显的剂量效应,显示出菌株3-1广谱的抑菌活性。本发明的粘细菌3-1在临床感染菌抑制药物开发和植物病原真菌生物防治菌剂、生物农药等方面具有较好的应用价值。(The invention discloses a myxobacteria and application thereof in preparing antibacterial drugs. The invention adopts an escherichia coli induction method to separate and purify a myxobacteria Archangium violacea 3-1 from the vegetable pond sediment, and the myxobacteria Archangium violacea is identified as ascomycete purpurea through morphology and molecular biology (16S rRNA gene and gyrB gene). The crude extract solution of the fermentation supernatant of the ascochyta purpurea can inhibit the growth of candida albicans, methicillin-resistant staphylococcus aureus and plant pathogenic fungi, and the antibacterial activity shows obvious dose effect and shows the broad-spectrum antibacterial activity of a strain 3-1. The myxobacteria 3-1 of the invention has better application value in the aspects of clinical infectious bacteria inhibition drug development, plant pathogenic fungi biological control microbial inoculum, biological pesticide and the like.)

1. The myxobacteria Archangium violacea 3-1, accession number: GDMCC No: 61803.

2. use of the myxobacteria Archangium violacea 3-1, or a culture, a bacterial solution, a fermentation broth, or an extract of a fermentation broth thereof according to claim 1, for the preparation of a bacteriostatic medicament.

3. The use according to claim 2, wherein the bacteriostatic agent is an agent that inhibits Candida albicans, methicillin-resistant Staphylococcus aureus or Fusarium oxysporum f.sp.cubense race 4.

4. The use of claim 2, wherein the application of the myxobacteria archaebacterium 3-1 in the preparation of antibacterial drugs is culturing the myxobacteria archaebacterium violacea 3-1 to obtain a fermentation broth, leaching the fermentation broth with methanol, concentrating and drying the methanol leaching solution to obtain an extract, and using the extract to prepare the antibacterial drugs.

5. The use of claim 4, wherein the macroporous resin is added to the fermentation broth for culturing the myxobacteria Archangium violacea 3-1, then the macroporous resin is soaked with methanol, and the methanol leaching liquor is concentrated and dried to obtain an extract for preparing the bacteriostatic drug.

6. A bacteriostatic agent comprising the myxobacteria Archangium violacea 3-1 according to claim 1, or a culture, a bacterial solution, a fermentation solution, or an extract of the fermentation solution thereof as an active ingredient.

7. A bacteriostatic agent according to claim 6, wherein the extract of the fermentation broth is obtained by culturing the myxobacteria Archangium violacea 3-1 to obtain a fermentation broth, leaching the fermentation broth with methanol, concentrating and drying the methanol leaching solution to obtain an extract.

8. The bacteriostatic agent according to claim 7, wherein macroporous resin is added to the fermentation broth of cultured myxobacteria Archangium violacea 3-1, then the macroporous resin is soaked with methanol, and the methanol extract is concentrated and dried to obtain extract.

The technical field is as follows:

the invention belongs to the field of microorganisms, and particularly relates to a myxobacteria strain and application thereof in preparation of antibacterial drugs.

Background art:

candida albicans (Candida albicans) is a conditionally pathogenic fungus, and can cause a series of mucosal infections and systemic infections in the process of human body hypoimmunity or catheter intervention operations. About 4000 million cases of candida-induced infectious diseases annually in the global scope, wherein candida albicans is the most common and the most fatal, accounts for about 50-70% of candida infection cases, has a total death rate of 43%, and has become an important problem to be solved urgently in the fields of public health systems and medical care. In infected people, candida albicans mostly exists in the form of a biofilm, so that the drug resistance of the candida albicans is enhanced, and even the candida albicans can be prevented from being attacked by the immune system of a host, and the prevention difficulty is further increased.

The common use of various antibacterial drugs promotes the continuous enhancement of drug resistance of pathogenic bacteria, and particularly, the multiple drug-resistant strains seriously influence the progress of clinical treatment. Methicillin-resistant Staphylococcus aureus (MRSA), which is one of multiple drug-resistant strains, is an important pathogenic bacterium of current hospital infection, can cause various infectious diseases of human bodies such as blood, lower respiratory tract and skin, and the infection rate and the mortality rate of the methicillin-resistant Staphylococcus aureus (MRSA) are rising year by year according to clinical observation. On one hand, the difficulty of clinically treating drug-resistant bacteria infection is increased, and on the other hand, the difficulty of developing new antibiotics is also increased. Research and development of antibiotics and antibacterial drugs with new types of chemical structures, new action mechanisms or new action targets are effective ways for treating drug-resistant bacterial infection.

On the other hand, filamentous fungi are coated by a hard cell wall containing chitin, which is significantly different from bacteria and yeasts, and thus have different drug targets. The mould is not only an important pathogenic fungus for clinical infection, but also the most important pathogen causing diseases of food crops, and causes huge economic loss of agriculture. The accumulation of plant pathogenic fungi in soil is an important factor for the occurrence of soil-borne diseases and continuous cropping obstacles, and the difficulty in controlling the caused diseases is high. In addition, almost every crop has several to tens of fungal diseases, often resulting in the failure of a single control means. Therefore, broad-spectrum antibacterial drugs have important significance for controlling plant diseases.

Myxobacteria, as a novel drug source microorganism, can produce abundant and diverse secondary metabolites with novel structures and unique action mechanisms, and has become the second largest antibiotic producing strain after actinomycetes at present. Myxobacteria are a kind of predatory bacteria that can kill and lyse predatory bacteria including bacteria and fungi. During the predation process of myxobacteria, secondary metabolites play an important role. The broad predation spectrum of myxobacteria means that it can be used for the inhibition of a wide variety of pathogenic bacteria. However, the development and utilization of secondary metabolites of myxobacteria is far behind that of actinomycetes.

The invention content is as follows:

the first objective of the present invention is to provide a myxobacteria Archangium violacea 3-1 capable of producing bacteriostatic substances, which is deposited at 13.7.2021 in the culture Collection of microorganisms of Guangdong province (GDMCC), address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61803.

the second purpose of the invention is to provide the application of the myxobacteria Archangium violacea 3-1, or the culture, bacterial liquid, fermentation liquid or extract of the fermentation liquid in preparing bacteriostatic drugs.

The bacteriostatic drug is a drug for inhibiting Candida albicans, methicillin-resistant Staphylococcus aureus or Fusarium oxysporum f.sp.cubense race 4.

The application of the myxobacteria Archanium violacea 3-1 in preparing the antibacterial drug is to culture the myxobacteria Archanium violacea 3-1 to obtain fermentation liquor, then leach the fermentation liquor with methanol, concentrate and dry the methanol leaching liquor to obtain extract for preparing the antibacterial drug.

Preferably, the method comprises the steps of adding macroporous resin into fermentation liquor for culturing the myxobacteria Archangium violacea 3-1, soaking the macroporous resin with methanol, concentrating and drying methanol leaching liquor to obtain extract for preparing the antibacterial drug.

It is a third object of the present invention to provide a bacteriostatic agent containing Archangium violacea 3-1, or a culture, a bacterial solution, a fermentation broth or an extract of a fermentation broth thereof as an active ingredient.

The extract of the fermentation liquor is obtained by culturing the myxobacteria Archangium violacea 3-1 to obtain the fermentation liquor, then leaching the fermentation liquor with methanol, and concentrating and drying the methanol leaching liquor to obtain extract.

Preferably, the method comprises the steps of adding macroporous resin into fermentation liquor for culturing the myxobacteria Archangium violacea 3-1, soaking the macroporous resin with methanol, and concentrating and drying methanol extract to obtain extract.

The invention adopts an escherichia coli induction method to separate and purify a myxobacteria Archangium violacea 3-1 from the vegetable pond sediment, and the myxobacteria Archangium violacea is identified as ascomycete purpurea through morphology and molecular biology (16S rRNA gene and gyrB gene). The crude extract solution of the fermentation supernatant of the ascochyta purpurea can inhibit the growth of candida albicans, methicillin-resistant staphylococcus aureus and plant pathogenic fungi, and the antibacterial activity shows obvious dose effect and shows the broad-spectrum antibacterial activity of a strain 3-1. The myxobacteria 3-1 of the invention has better application value in the aspects of clinical infectious bacteria inhibition drug development, plant pathogenic fungi biological control microbial inoculum, biological pesticide and the like.

The myxobacterium archangelium 3-1 was deposited at 13.7.2021 at the Guangdong province culture Collection (GDMCC) with the following addresses: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61803.

description of the drawings:

FIG. 1 is a morphological feature of the myxobacterium Archangium violacea 3-1; a: colony morphology of violaceum3-1 grown on VY/2 solid medium for 3 days; b: growth of violaceum3-1 on VY/2 solid medium for 10 days to form a morphology of large numbers of mature fruit bodies; c: observing the solid form of the A.violaceum 3-1 seed under a stereoscopic microscope; d: violaceum3-1 rod-shaped vegetative cell crystal violet staining; e: violaceum3-1 rod vegetative cells and spherical myxospore crystal violet staining.

FIG. 2 shows the inhibition of Candida albicans SC5314 by a crude extract solution of fermentation supernatant from the strain Archangium violacea 3-1.

FIG. 3 shows the inhibition of methicillin-resistant Staphylococcus aureus (Staphylococcus aureus) GDMCC 1.1263 by fermentation supernatant crude extract solution of a myxobacteria strain Archangium violaceum 3-1.

FIG. 4 shows the inhibition of Fusarium oxysporum f.sp.cubense race 4 by fermentation supernatant crude extract solution of a myxobacteria strain Archangium violacea 3-1.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1: isolation and purification of the strains

1.1 sample Collection and pretreatment

Collecting sediment deposits from rape pond of West village in pond of east town of Shantou Denghai region, filtering to remove water, and naturally air drying a part of the sediment deposits at room temperature; and adding another part of fresh sediment sample into sterile deionized water according to a ratio of 1:10(v/v), shaking for 1h, centrifuging to obtain supernatant, and filtering with a 0.22 mu m filter membrane to prepare a leaching solution. Utilizes the characteristic that myxobacteria can generate anti-adversity sporocarp, and inhibits interference of other bacteria, fungi and protists by air drying treatment.

1.2 isolation, purification and preservation of myxobacterial Strain 3-1

Taking 10g of air-dried sediment sample, adding 15mL of 100 mu g mL^-1Filtering sterilized cycloheximide, soaking overnight, centrifuging, and removing supernatant. Water agar medium (i.e., WCX medium, containing CaCl) using Escherichia coli as substrate₂·2H₂O1 g/L and agar 15g/L,the balance of water; pH 7.2), separating myxobacteria, drawing a 'field' shape on the surface of a WCX culture medium by using escherichia coli thalli, and respectively inoculating sediment samples with the sizes of soybean grains in four blank spaces of the 'field' shape. Culturing the above separation plate at 30 deg.C for 7 days, observing with a stereo microscope every day, separating and selecting fruiting body for purification (VY/2 culture medium containing yeast 5g/L, CaCl)₂·2H₂O 1g/L，VB₁₂0.5mg/L and 15g/L of agar, and the balance of water; pH 7.2). Picking up the fruiting body or colony edge and repeatedly transferring by using VY/2 until the pure strain is observed by naked eyes. The obtained pure strain is inoculated in a nutrient broth culture medium (containing 10g/L of peptone, 3g/L of beef extract powder and 5g/L of NaCl, the balance being water; pH 7.2), and the myxobacteria is tested to be pure after overnight culture at 30 ℃, if the culture solution is clear, the myxobacteria is the pure strain. Transferring the purified myxobacteria strain to VY/2 solid culture medium, scraping off after the fruiting body is mature, and making into glycerol seed and lyophilized seed, storing glycerol seed at-80 deg.C, and storing lyophilized seed at 4 deg.C. Thus, Strain 3-1 was obtained.

Example 2: identification of the Strain Archangium violacea 3-1

2.1 morphological and cultural characteristic observations of the Strain

The strain 3-1 shows a sliding expanded transparent colony on a VY/2 solid culture medium, and the whole colony is radial; aggregate formation was observed starting around 5 days of growth on VY/2 solid medium, and the mature fruiting bodies were spherical, dark purple brown in color. Gram staining was negative (fig. 1). The above morphological characteristics indicate that the strain is consistent with the basic characteristics of myxobacteria.

2.2 Strain identification based on 16S rRNA Gene and gyrB Gene sequences

Scraping fresh thallus 3-1 growing on VY/2 solid culture medium for 3 days, adding a small amount of sterile water, treating at 90 ℃ for 10min, centrifuging and taking supernatant as a template of PCR reaction. PCR reaction system (25. mu.L) includes buffer 12.5. mu.L, upstream and downstream primers 1. mu.L, template 1. mu.L, and sterile deionized water 9.5. mu.L. Reaction procedure: 5min at 95 ℃,30 s at 56 ℃, 90s at 72 ℃ for 30 cycles, 10min at 72 ℃. The PCR products were subjected to agarose electrophoresis to determine error-free and then sequenced by Jinzhi Biotechnology Ltd.

The 16S rRNA gene and the gyrB gene sequences were aligned with BLAST homology on EzBioCloud (https:// www.ezbiocloud.net /), respectively. The results showed that strain 3-1 has the highest similarity to the model strain Archangium violacea DSM 14727, with 99.28% and 98.16% sequence similarity for the 16S rRNA gene and the gyrB gene, respectively. The 16S rRNA gene and the gyrB gene are respectively shown in SEQ ID NO.1 and SEQ ID NO. 2.

Through the above morphological and molecular biological identification, the strain was identified as Archangium violaceum, numbered 3-1, deposited at the Guangdong province culture Collection (GDMCC) at 7/13/2020, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61803.

example 3: liquid fermentation of strain Archangium violacea 3-1 and preparation of crude extract of fermentation supernatant

The fermentation medium is MD1 liquid (calculated by mass fraction, casein peptone 0.6%, soluble starch 0.2%, MgSO 2)₄·7H₂O0.2％，CaCl₂·2H₂O0.04%, pH 7.2), adding 2% macroporous resin XAD-16 before inoculation, and inoculating Archangium violaceum3-1 seed solution according to the inoculation amount of 2% by volume. Shaking and culturing at 140rpm and 30 deg.C for 7d, filtering with gauze to collect resin, adding 2 times volume of methanol, shaking and leaching at 30 deg.C for 1h, filtering and collecting methanol; the leaching process was repeated 3-4 times until the methanol was colorless. The collected methanol was combined on a rotary evaporator at 35 ℃ for 40r min^-1And (5) decompressing and evaporating to dryness. Fermenting 4.5L of the strain to obtain 2.5873g of crude extract. Dissolving the above crude extract with small amount of Dimethyl Sulfoxide (DSMO), and diluting with sterile deionized water to concentration of 100, 50, 25, 12.5, 6.25 and 3.125mg ml^-1The crude extract solution of (1).

Example 4: inhibition of Candida albicans SC5314 by crude extract solution of fermentation supernatant of strain Archangium violacea 3-1

Scraping C.albicans SC5314 cultured overnight on YPD solid medium (glucose 20g/L, yeast extract 10g/L, peptone 20g/L, agar 2%), adding sterile deionized water to make into bacterial suspension, and dipping with cotton swabThe bacterial suspension was spread on fresh YPD solid medium. After the plate is dried in the air, 3 sterile filter paper sheets with the diameter of 0.5cm are laid, and 20 mul of crude extract solution with each concentration is added on each filter paper sheet. Geneticin G418 (100. mu.g/ml) and aqueous DSMO solutions were also set as positive and negative controls, respectively. After the filter paper is dried, the culture is carried out at 30 ℃, and the size of the inhibition zone is observed and measured after 12 hours. The result shows that the extract of the fermentation liquor of the strain 3-1 obviously inhibits the growth of C.albicans SC5314, and the inhibition effect has a dose effect. When the concentration of the fermentation liquid extract is 100, 50, 25, 12.5, 6.25 and 3.125mg ml^-1The diameters of the inhibition zones are 29.3mm, 24.6mm, 22.5mm, 21mm, 15.8mm and 12.6mm respectively (figure 2).

Example 5: inhibition of crude extract solution of fermentation supernatant of Archanium violacea 3-1 strain on GDMCC 1.1263 of methicillin-resistant Staphylococcus aureus

S.aureus GDMCC 1.1263 cultured overnight on TSA solid medium (tryptone 15g/L, soytone 5g/L, NaCl 5g/L, agar 1.5%, pH 7.3) was scraped, and sterile deionized water was added to prepare a bacterial suspension, which was then applied to fresh TSA solid medium with a cotton swab. After the plate is dried in the air, 3 sterile filter paper sheets with the diameter of 0.5cm are laid, and 20 mul of crude extract solution with each concentration is added on each filter paper sheet. Vancomycin (100. mu.g/ml) and a DSMO aqueous solution were also set as positive and negative controls, respectively. After the filter paper is dried, the culture is carried out at 30 ℃, and the size of the inhibition zone is observed and measured after 12 hours. The result shows that the fermentation liquor extract of the strain 3-1 with higher concentration obviously inhibits the growth of MRSA bacteria to be tested, and the inhibition effect has a dosage effect. When the concentration of the fermentation liquid extract is 100, 50, 25 and 12.5mg ml^-1The diameters of the inhibition zones are respectively 8.2mm, 10.5mm, 12.6mm and 13.5mm (figure 3).

Example 6: inhibition of strain Archangium violacea 3-1 fermented supernatant crude extract solution on filamentous plant pathogenic fungus Fusarium oxysporum f.sp.cubense race 4

Selecting F.oxysporum block, inoculating to PDA solid culture medium (peeled potato 200g/L, glucose 20g/L, agar 2%), and crossing when the diameter of fungus bacterial plaque reaches 2cm4 holes with the diameter of 0.5mm are punched at the position 1cm away from the edge of the colony in the crossing direction by a puncher, 20 mu L of crude extract solutions with different concentrations are added into each hole, and meanwhile, geneticin G418(100 mu G/ml) and a DSMO aqueous solution are respectively arranged as positive and negative controls. Culturing at 30 deg.C, observing and measuring the size of inhibition zone after 12 hr, and calculating inhibition rate. The results show that the 3-1 fermentation liquid extract can inhibit the growth of filamentous fungus fusarium oxysporum, and the inhibition effect has a dosage effect. When the concentration of the fermentation liquid extract is 100, 50, 25, 12.5, 6.25 and 3.125mg ml^-1The inhibition rates were 14.1%, 23.3%, 29.4%, 35.6%, 41.7% and 44.8%, respectively (fig. 4).

Sequence listing

<110> institute of microbiology, academy of sciences of Guangdong province (center for microbiological analysis and detection of Guangdong province)

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