WO2021223366A1 - 一株抗生链霉菌及其代谢产物的制备以及其在抗病菌方面的应用 - Google Patents

一株抗生链霉菌及其代谢产物的制备以及其在抗病菌方面的应用 Download PDF

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WO2021223366A1
WO2021223366A1 PCT/CN2020/120625 CN2020120625W WO2021223366A1 WO 2021223366 A1 WO2021223366 A1 WO 2021223366A1 CN 2020120625 W CN2020120625 W CN 2020120625W WO 2021223366 A1 WO2021223366 A1 WO 2021223366A1
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streptomyces
streptomyces antibiotics
fermentation
culture
metabolites
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French (fr)
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杨祁云
林壁润
路征
张景欣
孙大元
吕永华
沈会芳
蒲小明
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广东省农业科学院植物保护研究所
中国烟草总公司广东省公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/28—Streptomyces
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N3/00—Spore forming or isolating processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces
    • C12R2001/48—Streptomyces antibioticus ; Actinomyces antibioticus

Definitions

  • the invention belongs to the field of biological pesticide research, and particularly relates to the preparation of a strain of Streptomyces antibiotics and its metabolites and its application in the field of disease resistance.
  • Candida albicans infection has increased by more than 40 times, and it has become one of the main causes of death from nosocomial infections (Liao Wanqing, 1985). Therefore, it is very meaningful to find new biologic agents for Candida.
  • Anthracnose of pepper and cucumber is a common and serious fungal disease in production, which can cause leaf rotten, rotten fruit, and plant death.
  • chemical agents such as carbendazim and mancozeb are mainly used in production for prevention and control, but the negative effects of pesticide residues and environmental pollution are gradually increasing. Therefore, it is necessary to find new biological agents for the prevention and treatment of anthracnose.
  • the primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of Streptomyces antibiotics.
  • Another object of the present invention is to provide a method for preparing the above-mentioned metabolite of Streptomyces antibiotics.
  • Another object of the present invention is to provide applications of the above-mentioned Streptomyces antibiotics and its metabolites.
  • the said Streptomyces antibiotics is inoculated on the culture medium and cultured at 28-30°C to obtain the spores of Streptomyces antibiotics.
  • composition of the culture medium is: soluble starch 1-5g, glucose 2-10g, yeast extract 1-3g, peptone 1-2g, KNO 3 0.5-1g, K 2 HPO 4 0.1-0.5g, NaCl 0.1 ⁇ 0.5g, FeSO 4 0.005 ⁇ 0.01g, MgSO 4 ⁇ 7H 2 O 0.01 ⁇ 0.05g, agar 10 ⁇ 15g, pH 7.2 ⁇ 7.4, add water to make up to 1000mL.
  • the soluble starch is preferably soluble corn starch.
  • the cultivation time is 3 to 5 days.
  • a metabolite of Streptomyces antibiotics is obtained by culturing the spores of the Streptomyces antibiotics and/or the Streptomyces antibiotics.
  • the preparation method of the metabolite of Streptomyces antibiotics includes the following steps:
  • the slant medium mentioned in step (1) is: soluble starch 1-5g, glucose 2-10g, yeast extract 1-3g, peptone 1-2g, KNO 3 0.5-1g, K 2 HPO 4 0.1-0.5g , NaCl 0.1 ⁇ 0.5g, FeSO 4 0.005 ⁇ 0.01g, MgSO 4 ⁇ 7H 2 O 0.01 ⁇ 0.05g, agar 10 ⁇ 15g, pH 7.2 ⁇ 7.4, add water to dilute to 1000mL.
  • the soluble starch is preferably soluble corn starch.
  • the culture time described in step (1) is 3 to 5 days.
  • the fermentation medium mentioned in step (2) is: KNO 3 0.5 ⁇ 1g, K 2 HPO 4 0.1 ⁇ 0.5g, MgSO 4 ⁇ 7H 2 O 0.1 ⁇ 0.5g, FeSO 4 0.005 ⁇ 0.01g, sea crystal 0.1 ⁇ 0.5g, soluble starch 2-10g, glucose 5-10g, yeast extract 15-20g, fixed volume to 1000mL, pH 7.2-7.4.
  • the soluble starch is preferably soluble corn starch.
  • step (2) The fermentation and culture conditions described in step (2) are: room temperature fermentation and culture for 5-7 days; preferably, room temperature fermentation and culture for 5 days.
  • the concentration described in step (2) is preferably concentrated by heating.
  • the concentration in step (2) is preferably concentrated to 1/20-3/1 of the volume of the fermentation broth.
  • the re-concentration in step (2) is preferably concentrated to 1/3 to 1/5 of the original volume.
  • the volume ratio of ethyl acetate to the concentrated solution in step (2) is 1:1.
  • the silica gel in the silica gel column described in step (3) is 200-300 mesh silica gel.
  • the method for preparing the metabolites of Streptomyces antibiotics further includes the following steps after step (3):
  • the metabolites of Streptomyces antibiotics obtained in step (3) are separated by high performance liquid chromatography to obtain Antimycin A1 (Antimycin A1); wherein the mobile phase of high performance liquid chromatography is methanol and 0.5% by volume formic acid aqueous solution The mixed solution obtained by volume ratio 4:1.
  • the fungus is plant anthracnose or Candida albicans.
  • the present invention screens and obtains a new strain (Streptomyces antibiotics PPI-16), which can produce new active compounds and can inhibit a variety of pathogenic fungi, such as inhibiting cucumber anthracnose pathogen, pepper anthracnose pathogen, tobacco anthracnose pathogen and Candida albicans
  • the growth and reproduction of bacteria provide new resources for the research and development of new antibiotics.
  • the present invention also provides fermentation technology for the active substances produced by the fermentation of Streptomyces antibiotics PPI-16.
  • Figure 1 is a photograph of the spores of Streptomyces antibiotics PPI-16.
  • Figure 2 is a photograph of spore filaments of Streptomyces antibiotics PPI-16.
  • Figure 3 is a gel electrophoresis image of PCR products of Streptomyces antibiotics PPI-16 16S rDNA (M: Marker; Lanes 1 and 2: 16S rDNA of Streptomyces antibiotics PPI-16).
  • Figure 4 is a crystal diagram of the active ingredient of Streptomyces antibiotics PPI-16.
  • the present invention will be further described in detail below in conjunction with examples, but the implementation of the present invention is not limited thereto.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the test methods that do not indicate specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions recommended by the manufacturer.
  • the reagents and raw materials used in the present invention are all commercially available.
  • Example 1 Separation and cultivation of marine actinomycetes
  • Biological materials and culture media The biological materials used to isolate marine actinomycetes are collected from mangroves in Enping City, Guangdong province, with a total of 792 samples.
  • the medium used for the isolation of actinomycetes is Gao's No. 1 medium (in order to ensure the number and types of isolated actinomycetes, the separation medium is prepared with artificial seawater.
  • the formula is: soluble corn starch 20g, KNO 3 1g, K 2 HPO 4 0.5g, MgSO 4 ⁇ 7H 2 O 0.5g, FeSO 4 ⁇ 7H 2 O 0.01g, NaCl 0.5g, agar 20g, artificial seawater 1000mL, pH 7.2 ⁇ 7.4; the artificial seawater formula is: NaCl 24.477g, MgCl 2 ⁇ 6H 2 O 4.981g, Na 2 SO 4 3.917g, CaCl 2 ⁇ 2H 2 O 1.102g, KCl 0.664g, NaHCO 3 0.192g, KBr 0.096g, H 3 BO 3 0.026g, SrCl 2 0.024g, NaF 0.0039g, distilled water 1000mL).
  • the media used for routine culture of actinomycetes were all prepared with distilled water.
  • Sample processing Put the above-mentioned freshly collected biological samples in a foam box with ice bags, and bring them back to the laboratory for processing as soon as possible. After the sample is weighed, it is washed with sterile sea water for 3 to 5 times to remove the impurities attached to the surface; the washed sample is placed in a sterile mortar, and sterile artificial sea water is added according to 1:8 (W/V).
  • the formula is the same as the above step 1) for homogenization; pipette 1 mL of the homogenate in a centrifuge tube and heat it in a water bath at 55°C for 6 minutes; the sample after pipetting the warm bath is diluted in multiples of 10, and 100 ⁇ L of each diluted solution is applied to the above separation culture. In the base, each treatment was repeated 3 times.
  • Actinomycete culture Place the separation medium coated with the homogenate in a 28°C constant temperature incubator and invert it for culture, and start to pick out single colonies growing on the medium for transfer purification about 7 days after culture. According to the morphological characteristics and culture characteristics of the strains, the repeated strains were removed, and a total of 792 different actinomycetes strains were isolated. The purified strains were inoculated into Gao's No. 1 slant medium, cultured in a constant temperature incubator at 28°C, and stored in a refrigerator at 4°C for later use.
  • Example 2 Screening, isolation and identification of antagonistic marine actinomycetes
  • Test strains and culture medium Using Candida albican (purchased from Guangdongzhou Microbiological Research) as the indicator bacteria, the isolated 792 strains of actinomycetes were tested for their inhibitory activity against Candida albicans to screen out the Candida has the strongest inhibitory activity.
  • the medium used for the culture of actinomycetes is Gao's No. 1 medium (the specific formula is as described in Example 1); the medium used for the culture of Candida albicans is Martin medium (the formula is: glucose 20g, peptone 5g , Yeast extract 4g, K 2 HPO 4 ⁇ 7H 2 O 0.63g, MgSO 4 ⁇ 7H 2 O 1.8g, agar 15g, add water to make the volume to 1000 mL).
  • 1.2 Strain activation Prepare the corresponding medium according to the above formula, heat and dissolve the medium in the Erlenmeyer flask and pour it into a petri dish with a diameter of 9 cm to prepare a medium plate. Pick the actinomycetes stored in the refrigerator at 4°C and inoculate them on the Gao's No. 1 medium plate, and place them in a constant temperature incubator at 28°C for 3 to 5 days.
  • Re-screening is to select strains with an inhibition zone diameter of 15mm or more.
  • strain PPI-16 has the best antibacterial effect on Candida albicans (see Table 2), and the inhibition zone diameter is 20.3mm.
  • the morphological characteristics of the strains were observed by the plate insert method (Yan Xunchu, 1992).
  • the test strains were inoculated on 7 solid mediums including synthetic starch, Krebs, Chase, Glucose aspartin agar, potato pieces and Gao's No. 1, insert slices, and cultured at 28°C for 7-20 days. Observe and record the growth, the color of the intrabasal hyphae, aerial hyphae and soluble pigments, and at the same time take out the inserts, and observe the air filaments, the morphology of the basic filaments, and whether there are characteristics such as diaphragms, breaks, and swellings with an optical microscope. After the selected cultures are fixed and dehydrated, they are observed and photographed by scanning electron microscope.
  • Figure 1 shows the spores of Streptomyces antibiotics PPI-16
  • Figure 2 shows the spore filaments of Streptomyces antibiotics PPI-16.
  • the strain PPI-16 was identified according to the conventional identification method of Streptomyces, specifically: refer to Lechevalie's method (Lechevalier et al, 1980) to determine the strain’s gelatin liquefaction, starch hydrolysis, milk coagulation and peptonization, cellulose hydrolysis, and carbon source Utilizing other characteristics, and using S.antibioticus EF063450 and S. griseoruber AB184209 (both from Guangdong Institute of Microbiology) as controls.
  • Streptomyces antibiotics PPI-16 Streptomyces griseus glucose + + + Mannitol + + - D + + + sucrose - + - Raffinose - + - Inositol + + + Arabic candy + - + fructose + - + Xylose + - +
  • Microcrystalline cellulose thin-layer chromatography was used to analyze the glycoforms of whole-cell amino acids and whole-cell hydrolysate.
  • Strain PPI-16 whole cell hydrolysate contains L,L–DAP (L-) (diaminopimelic acid), glycine, alanine and aspartic acid, contains ribose and glucose, and has no characteristic sugars (glycotype C) ,
  • the cell wall component belongs to type I, which accords with the characteristics of the chemical classification of Streptomyces.
  • the 16S rDNA sequence of strain PPI-16 was amplified by PCR.
  • the electrophoresis result is shown in Figure 3. Show. The sequence was determined to be 1440bp long, and its G+C content was 59%.
  • the extract of the present invention can be obtained by extracting and separating from the ethyl acetate extract of the culture (fermentation broth) of marine actinomycetes PPI-16, and its specific preparation method includes the following steps:
  • PDA potato dextrose agar
  • the preparation method is: weigh 200g potatoes, wash, peel and chop, add water to 1000mL, boil for half an hour, filter with gauze, add 20g glucose and 20g agar, fully dissolve it while hot Filter them with gauze and distribute them into glass test tubes.
  • Example 5 Inhibition experiment of the metabolites of the present invention on Candida albicans

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Abstract

一株保藏编号为GDMCC NO：60970的抗生链霉菌Streptomyces antibioticus PPI-16，所产生的活性化合物可以抑制多种病原真菌，包括黄瓜炭疽病菌、辣椒炭疽病菌、烟草炭疽病菌和白色念珠菌。

Description

一株抗生链霉菌及其代谢产物的制备以及其在抗病菌方面的应用 技术领域
本发明属于生物农药研究领域，特别涉及一株抗生链霉菌及其代谢产物的制备以及其在抗病菌方面的应用。
背景技术
随着广谱抗生素及免疫抑制剂的广泛应用和侵入性治疗方法的开展，新的真菌病不断出现，一些古老的真菌病发病率开始上升，尤其是机会性真菌感染日益增多。真菌感染(特别是侵入性真菌感染)的发生率不断增大，对人类健康造成了严重危害。人类的真菌感染可分为3种：浅部真菌病、深部真菌病和系统性真菌病(Garber，2001)，其中最严重的、可危及生命的真菌感染是系统性真菌病(Meis，2001)。深部真菌病的病原菌主要是念珠菌属，尤其是白色念珠菌(Candida albicans)。它在念珠菌所有种中致病力最强，一旦机体的免疫力下降或者正常的菌群平衡被破坏，它就会在粘膜下大量繁殖，侵入深层组织，引起系统性念珠菌感染(Maertens et al.，2001)。医院真菌感染中占第一位的是念珠菌感染，感染后造成病死率达21％，念珠菌几乎可以侵犯全身所有脏器，各种念珠菌仍是造血干细胞移植最常见的真菌感染源(周绮等，2000)。尽管机体终生都被白色念珠菌这种机会性致病菌致敏，但是并没有因此发展成免疫耐受，所以念珠菌感染的发病率呈不断上升趋势，在免疫抑制患者中念珠菌的发病率列居第一位(Radentz，1989)。近20年来白色念珠菌感染增长了40倍以上，已成为医院感染的主要死亡原因之一(廖万青，1985)。因此，寻找念珠菌新生物药剂十分有意义。
辣椒和黄瓜炭疽病是生产上普遍发生且危害严重的真菌病害，可造成烂叶、烂果、植株死亡。目前生产上主要利用多菌灵、代森锰锌等化学药剂进行防治，但农药残留及环境污染等负面效应也逐步增长。因此，寻找防治炭疽病新生物药剂十分必要。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一株抗生链霉菌。
本发明的另一目的在于提供上述抗生链霉菌的代谢物的制备方法。
本发明的再一目的在于提供上述抗生链霉菌及其代谢物的应用。
本发明的目的通过下述技术方案实现：
一株抗生链霉菌，名称为Streptomyces antibioticus PPI-16(抗生链霉菌PPI-16)，保藏编号为GDMCC NO：60970，该菌株于2020年3月5日保藏于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心。
一种所述抗生链霉菌的孢子。
所述的抗生链霉菌的孢子的制备方法，将所述的抗生链霉菌接种到培养基上，于28～30℃条件下进行培养，得到抗生链霉菌的孢子。
所述的培养基的组成成分为：可溶性淀粉1～5g，葡萄糖2～10g，酵母提取物1～3g，蛋白胨1～2g，KNO 3 0.5～1g，K 2HPO 4 0.1～0.5g，NaCl 0.1～0.5g，FeSO 4 0.005～0.01g，MgSO 4·7H 2O 0.01～0.05g，琼脂10～15g，pH 7.2～7.4，加水定容至1000mL。
所述的可溶性淀粉优选为可溶性玉米淀粉。
所述的培养的时间为3～5天。
一种抗生链霉菌的代谢物，为通过培养所述的抗生链霉菌和/或所述的抗生链霉菌的孢子获得。
所述的抗生链霉菌的代谢物的制备方法，包括如下步骤：
(1)将所述的抗生链霉菌接种到斜面培养基上，于28～30℃条件下进行培养；
(2)将斜面培养基上的菌株接种到发酵培养基中进行发酵培养，过滤除去菌体，得到发酵液；然后将发酵液浓缩，得到浓缩液；浓缩液用乙酸乙酯萃取后再次浓缩，得到发酵浓缩物；
(3)将发酵浓缩物采用硅胶柱进行色谱分离，然后用体积比为3:20的乙酸乙酯与石油醚进行清洗，再用体积比为1:1的乙酸乙酯与石油醚进行洗脱，并收集洗脱液(棕色油状物)，得到抗生链霉菌的代谢物。
步骤(1)中所述的斜面培养基为：可溶性淀粉1～5g，葡萄糖2～10g，酵母提取物1～3g，蛋白胨1～2g，KNO 3 0.5～1g，K 2HPO 4 0.1～0.5g，NaCl 0.1～0.5g，FeSO 4 0.005～0.01g，MgSO 4·7H 2O 0.01～0.05g，琼脂10～15g，pH 7.2～7.4，加水定容至1000mL。
所述的可溶性淀粉优选为可溶性玉米淀粉。
步骤(1)中所述的培养的时间为3～5天。
步骤(2)中所述的发酵培养基为：KNO 3 0.5～1g，K 2HPO 4 0.1～0.5g，MgSO 4·7H 2O 0.1～0.5g，FeSO 4 0.005～0.01g，海水晶0.1～0.5g，可溶性淀粉2～10g，葡萄糖5～10g，酵母膏15～20g，定容至1000mL，pH7.2～7.4。
所述的可溶性淀粉优选为可溶性玉米淀粉。
步骤(2)中所述的发酵培养的条件为：室温发酵培养5～7天；优选为：室温发酵培养5天。
所述的室温优选为28～30℃；更优选为28℃。
步骤(2)中所述的浓缩优选为采用加热的方式进行浓缩。
步骤(2)中所述的浓缩优选为浓缩至发酵液体积的1/20～3/1。
步骤(2)中所述的再次浓缩优选为浓缩至原体积的1/3～1/5。
步骤(2)中所述的乙酸乙酯与浓缩液的体积比为1:1。
步骤(3)中所述的硅胶柱中的硅胶为200～300目硅胶。
所述的抗生链霉菌的代谢物的制备方法，在步骤(3)之后还包括如下步骤：
将步骤(3)中得到的抗生链霉菌的代谢物通过高效液相色谱进行分离，得到抗霉素A1(AntimycinA1)；其中高效液相色谱的流动相为甲醇与体积百分数为0.5％的甲酸水溶液按体积比4:1得到的混合溶液。
所述的抗生链霉菌，抗生链霉菌的孢子，以及抗生链霉菌的代谢物中的至少一种在生产抗霉素A1(AntimycinA1)中的应用。
所述的抗生链霉菌，抗生链霉菌的孢子，以及抗生链霉菌的代谢物中的至少一种在防治真菌感染中的应用。
所述的真菌为植物炭疽病菌或白色念珠菌。
所述的植物炭疽病菌包括黄瓜炭疽病菌、辣椒炭疽病菌和烟草炭疽病菌等。
本发明相对于现有技术具有如下的优点及效果：
本发明筛选得到一株新的菌株(抗生链霉菌PPI-16)，该菌株能够产生新的活性化合物，可以抑制多种病原真菌，如抑制黄瓜炭疽病菌、辣椒炭疽病菌、烟草炭疽病菌和白色念珠菌生长繁殖等，为新抗生素研发提供新的资源，另外，本发明还提供了该抗生链霉菌PPI-16发酵产生的活性物质的发酵技术。
附图说明
图1是抗生链霉菌PPI-16的孢子照片图。
图2是抗生链霉菌PPI-16的孢子丝照片图。
图3是抗生链霉菌PPI-16 16S rDNA PCR产物的凝胶电泳图(M：Marker； 泳道1和2：抗生链霉菌PPI-16的16S rDNA)。
图4是抗生链霉菌PPI-16的有效成分结晶体图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件或按照制造厂所建议的实验条件。除非特别说明，本发明所用试剂和原材料均可通过市售获得。
实施例1：海洋放线菌的分离培养
1.生物材料及培养基：用于分离海洋放线菌的生物材料为采自广东省恩平市的红树林，共计792份样品。用于放线菌分离所用的培养基为高氏一号培养基(为保证分离放线菌的数量和种类，分离培养基以人工海水配制，配方为：可溶性玉米淀粉20g、KNO 3 1g、K 2HPO 4 0.5g、MgSO 4·7H 2O 0.5g、FeSO 4·7H 2O 0.01g、NaCl 0.5g、琼脂20g、人工海水1000mL，pH 7.2～7.4；其中人工海水配方为：NaCl 24.477g、MgCl 2·6H 2O 4.981g、Na 2SO 4 3.917g、CaCl 2·2H 2O 1.102g、KCl 0.664g、NaHCO 3 0.192g、KBr 0.096g、H 3BO 3 0.026g、SrCl 2 0.024g、NaF 0.0039g、蒸馏水1000mL)。以下试验中放线菌常规培养所用培养基均以蒸馏水配制。
2.样品处理：将上述新鲜采集的生物样品放于置有冰袋的泡沫箱内，带回实验室尽快处理。样品称重后，以无菌海水清洗3～5次，以去除表面附着的杂质；将清洗后的样品置于无菌研钵中，按1：8(W/V)加入无菌人工海水(配方同上述步骤1)进行匀浆；吸取1mL匀浆液于离心管中，55℃水浴加热6min；吸取温浴后的样品以10的倍数梯度稀释，分别取各稀释液100μL，涂布于上述分离培养基中，每个处理重复3次。
3.放线菌培养：将涂布匀浆液的分离培养基置于28℃恒温培养箱中倒置培养，培养7d左右开始挑取培养基上生长的单菌落进行转接纯化。根据菌株的形态特征和培养特征，去除重复的菌株，最后共计分离获得792株不同的放线菌菌株。对已纯化的菌株，接种于高氏一号斜面培养基中，于28℃恒温培养箱中培养后置于4℃冰箱保存备用。
实施例2：拮抗海洋放线菌的筛选、分离和鉴定
1、拮抗海洋放线菌的筛选和分离
1.1供试菌株及培养基：以白色念珠菌(Candida albican)(购于广东省微生物研究)作为指示菌，测定所分离的792株放线菌对白色念珠菌的抑制活性，以从中筛选出对念珠菌抑制活性最强的菌株。用于放线菌培养所用培养基为高氏一号培养基(具体配方如实施例1中所述)；用于白色念珠菌培养的培养基为马丁培养基(配方为：葡萄糖20g，蛋白胨5g，酵母浸膏4g，K 2HPO 4·7H 2O 0.63g，MgSO 4·7H 2O 1.8g，琼脂15g，加水定容至1000mL)。
1.2菌株的活化：按以上配方制备相应的培养基，将锥形瓶中的培养基加热溶解后倒入直径为9cm培养皿中，制备得到培养基平板。分别挑取保存于4℃冰箱的放线菌接种于高氏一号培养基平板上，置于28℃恒温培养箱中培养3～5d。
1.3菌株筛选
1.3.1初筛：采用纸片扩散法(Kirby Bauer，KB法)(Hunfeld，2001)测定样品对念珠菌抑制活性。马丁固体培养基倒平板，接种0.1mL(浓度为1*10 6cfu/ml)白色念珠菌液，均匀涂布。用无菌镊子将含放线菌液的滤纸片(直径6mm)平整的贴在平板表面。37℃培养1d后，测量抑菌圈直径，三次重复。初筛结果如表1所示(表中菌株名称均为自命名)。
表1抗白色念珠菌的活性菌株的初筛结果
Figure PCTCN2020120625-appb-000001
Figure PCTCN2020120625-appb-000002
1.3.2复筛：选取初筛效果好的放线菌株(对念珠菌抑菌圈直径＞15mm)(见表1)，将菌种在高氏一号固体培养基复壮培养。培养2代以后，用无菌打孔器打取直径6mm的菌块，置与涂布有白色念珠菌的马丁培养基中。37℃培养1d后，测量抑菌圈直径，三次重复。
复筛是选取抑菌圈直径15mm以上的菌株，其中以菌株PPI-16对白色念珠菌抑菌效果最好(见表2)，对念珠菌抑菌圈直径为20.3mm。
表2抗白色念珠菌的活性菌株的复筛结果
菌株名称 抑菌圈直径(mm) 菌株名称 抑菌圈直径(mm)
H122-01 19.3±0.12b H75-11 15.1±0.12f
H74-21 16.4±0.09d H122-02 15.9±0.09e
200-09 17.5±0.21c PPI-16 20.3±0.13a
注：表内数据为3次重复平均值±SE，采用DPS进行方差分析，相同字母者表示在5％水平差异上不显著。
通过以上方法，从分离获得的6株(对念珠菌抑菌圈直径15mm以上的菌株)放线菌中复筛出一株对白色念珠菌抑菌能力较强的菌株PPI-16。
2、菌株PPI-16的鉴定
2.1形态观察
采用平皿插片法观察菌株的形态特征(阎逊初，1992)。将试验菌株接种在合成淀粉、克氏、察氏、葡萄糖天门冬素琼脂和马铃薯块及高氏一号等7种固 体培养基上，插片，28℃培养7～20d。观察并记录生长情况、基内菌丝、气生菌丝和可溶性色素的颜色，同时取出插片，用光学显微镜观察气丝、基丝形态以及是否有横隔、断裂、膨大等特征。选取培养物经固定、脱水等处理后，扫描电镜观察并照相。
结果如图1和2所示：图1为抗生链霉菌PPI-16的孢子，图2为抗生链霉菌PPI-16的孢子丝。该菌株在高氏一号琼脂培养7～10d后，基内菌丝和气生菌丝丰茂，基丝不断裂，孢子丝着生在气生菌丝上，孢子丝柔曲，孢子光滑呈圆柱形，成熟的孢子链孢子个数在8个左右，没有观察到孢囊、孢核等结构。
2.2生理生化特征
按照链霉菌常规鉴定方法对菌株PPI-16进行鉴定，具体为：参考Lechevalie的方法(Lechevalier et al，1980)测定菌株的明胶液化、淀粉水解、牛奶的凝固与胨化、纤维素水解、碳源利用等特性，同时以抗生链霉菌(S.antibioticus EF063450)和灰红链霉菌(S.griseoruber AB184209)(均来源于广东省微生物研究所)为对照。
结果如表3所示：结果表明，PPI-16能使明胶液化，能水解淀粉，在纤维素上不生长，牛奶不胨化，也不液化，不产黑色素，能利用6种碳源。
表3 PPI-16与抗生链霉菌和灰红链霉菌碳源利用比较
特征 抗生链霉菌 PPI-16 灰红链霉菌
葡萄糖 + + +
甘露醇 + + -
鼠李糖 + + +
蔗糖 - + -
棉子糖 - + -
肌醇 + + +
阿拉伯糖 + - +
果糖 + - +
木糖 + - +
注：+表示能利用该碳源，-表示不能利用该碳源。
2.3细胞壁化学组成分析
采用微晶纤维素薄层层析法进行全细胞氨基酸及全细胞水解液糖型分析。菌株PPI-16全细胞水解液含L,L–DAP(左旋)(二氨基庚二酸)、甘氨酸，丙氨酸和天门冬氨酸，含有核糖和葡萄糖，无特征性糖(糖型C)，细胞壁组份属Ⅰ型，符合链霉菌属的化学分类特征。
2.4 16S rDNA序列测定及分析
以正向引物PA(5’-AGAGTTTGATCATGGCTCAG-3’)、反向引物PB为引物(5’-AAGGAGGTGATCCAGCCGCA-3’)，PCR扩增得到菌株PPI-16的16S rDNA序列，电泳结果如图3所示。经测定获得其序列长1440bp，G+C含量为59％。
Figure PCTCN2020120625-appb-000003
将该序列与GenBank中相关数据进行BLAST相似性分析，搜索得到相似性序列84个，选取其中与PPI-16的16S rDNA序列相似性较高的9个典型菌株序列进行比对。这些菌株如表4所示：
表4 GenBank中与PPI-16相似的菌株
Subject ID Strain Identity％
EF063450 S.antibioticus 99
AB184209 S.griseoruber 99
AB184534 S.spinichromogenes 98
AB184266 S.cinnabarinus 98
AB184192 S.cellostaticus 98
AB045860 S.panayensis 98
AB184461 S.roseogriseus 98
AB184536 S.viridochromogenes 98
AB184387 S.griseochromogenes 98
根据放线菌和链霉菌的分类鉴定手册，菌株PPI-16的形态特征、培养特征(孢子丝短而直、孢子是长圆柱形和表面光滑)及生理生化特性表现出链霉菌属的典型特征，归于金色类群。与9株同源率最高的链霉菌属菌株的16S rDNA进行构建系统发育树，菌株PPI-16与S.antibioticus EF063450处于同一分枝，亲缘关系最近，同源性高达99.9％，但其在培养特征、生理生化特征上与Streptomyces antibioticus有较大差异，故鉴定PPI-16为链霉菌金色类群新种抗白色念珠菌链霉菌Streptomyces antialbonostocticus。
表5抗白色念珠菌链霉菌与抗生链霉菌和灰红链霉菌的比较
Figure PCTCN2020120625-appb-000004
本发明将筛选得到的菌株PPI-16命名为抗生链霉菌(Streptomyces antibioticus)PPI-16，该菌株于2020年3月5日保藏于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心，保藏编号为GDMCC NO：60970。
实施例3代谢产物的制备
本发明提取物可以从海洋放线菌PPI-16的培养物(发酵液)的乙酸乙酯提取物中提取分离而得到，其具体制备方法包括以下步骤：
(1)海洋放线菌PPI-16的种子培养：
挑取菌株接入斜面培养基，于28～30℃培养3～5天；其中，
培养基(按重量比)：可溶性玉米淀粉5g，葡萄糖10g，酵母提取物3g，蛋白胨2g，KNO 3 1g，K 2HPO 4 0.5g，NaCl 0.5g，FeSO 4 0.01g，MgSO 4·7H 2O 0.05g，琼脂15g，pH7.4，用水定容至1000mL；制成试管斜面。
(2)海洋放线菌PPI-16的发酵培养：
将斜面中培养好的菌株(接种量为5％)挑入发酵培养基，于室温28℃发酵培养5天；其中，发酵培养基(按重量比)：KNO 3 1g，K 2HPO 4 0.5g，MgSO 4·7H 2O 0.5g，FeSO 4 0.01g，海水晶0.5g，可溶性玉米淀粉10g，葡萄糖10g,酵母膏20g，用水定容至1000mL，pH7.4。
(3)将上述培养好的发酵液过滤除去菌体；
(4)将发酵液加热浓缩至原液体积的1/20，然后用等体积乙酸乙酯萃取3次，浓缩乙酸乙酯萃取液(浓缩至原体积的1/3)，得到15g浓缩物，在硅胶柱(200～300目)中进行色谱分离，以乙酸乙酯/石油醚＝15％(体积百分数)清洗，然后用乙酸乙酯/石油醚＝50％(体积百分数)为洗脱剂洗脱，流速10ml/min。
(5)收集50％(体积百分数)的乙酸乙酯/石油醚洗脱液，浓缩得到棕色油状物(5g)，即为本发明所需的提取物(代谢产物)，在蔡司光学显微镜下观察到的有效成分结晶体如图4所示。
实施例4本发明代谢产物对黄瓜和辣椒炭疽病的防治试验
(1)配制供试样品溶液
取本发明提取物1mg(实施例3制备的红棕色油状物)超声溶解于1.0mL10％(v/v)甲醇溶液中。
(2)配制黄瓜和辣椒炭疽病菌的孢子悬浮液
用马铃薯葡萄糖琼脂(PDA)培养基(配制方法为：称取200g马铃薯，洗净去皮切碎，加水至1000mL煮沸半个小时，纱布过滤，再加20g葡萄糖和20g琼脂，充分溶解后趁热纱布过滤，分装至玻璃试管中，121℃、高压灭菌20min)28℃培养黄瓜炭疽病菌(Colletorichum cucumisativus)和辣椒炭疽病菌 (Colletotrichum capsici)，加灭菌蒸馏水刮取菌丝，分别制成孢子悬浮液(浓度为1*10 6cfu/ml)。
(3)抑菌实验
吸取5μL供试样品溶液加在灭菌的滤纸片(6mm)上，置于涂有指示菌(黄瓜炭疽病菌、辣椒炭疽病菌)的琼脂固体平板上，在恒温培养箱中培养48h后观察结果，根据抑菌圈大小判别抑菌活性。结果如下：
表6 PPI-16菌丝提取物的抗菌活性
供试病源菌 抑菌圈直径(mm)
黄瓜炭疽病(Colletorichum cucumisativus) 21.1±0.51a
辣椒炭疽病菌(Colletotrichum capsici) 15.5±.0.25d
注：表内数据为3次重复平均值±SE，采用DPS进行方差分析，相同字母者表示在5％水平差异上不显著。
实施例5本发明代谢产物对白色念珠菌的抑制实验
采用纸片扩散法(Kirby Bauer，KB法)(Hunfeld，2001)测定样品的抑对念珠菌抑制活性，具体步骤如下：
(1)配制供试样品溶液
取本发明提取物1mg(实施例2制备的红棕色油状物)超声溶解于1.0mL10％(v/v)甲醇溶液中。
(2)抑菌实验
配制马丁固体培养基(配方为：葡萄糖20g，蛋白胨5g，酵母浸膏4g，K 2HPO 4·7H 2O 0.63g，MgSO 4·7H 2O 1.8g，琼脂15g，水1000mL)，并倒培养皿中，接种0.1mL白色念珠菌(浓度为1*10 6cfu/ml)(白色念珠菌株在广东省微生物研究所购买)培养液，均匀涂布。然后用无菌镊子将含有供试样品溶液(5μL)的滤纸片(直径6mm)平整的贴在固体平板表面，以清水为对照。37℃培养1d后测量对念珠菌抑菌圈直径。
表7提取物抗念珠菌的活性
提取物 抑菌圈直径(mm)
PPI-16 21.3±0.10a
对照 0
注：表内数据为3次重复平均值±SE，采用DPS进行方差分析，相同字母者表示在5％水平差异上不显著。
实施例6
将实施例3获得的结晶体经DionexP680分析型高效液相色谱仪(美国戴安Dionex公司)产品，检测器为PDA-100PhotodiodeArrayDetector)分析为混合物，通过制备型高效液相色谱(美国Agilent公司1100型高效液相色谱仪)(甲醇/0.5％甲酸水溶液，体积比80:20)进行分离制备，得到化合物A，再通过LC-MS联用(美国Agilent公司AB 4000Q Trap质谱联用仪)，色谱分析条件：Agilent Eclipse Plus C 18(5μm)色谱柱(2.1mm x 150mm)；流动相：A(100％甲醇)：B(0.5％(v/v)甲酸水溶液)＝80:20；流速:300μL/min；检测波长:240nm。技术分析其化学结构。结果表明，化合物A为抗霉素A1(AntimycinA1)，其化学结构如下：
Figure PCTCN2020120625-appb-000005
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Figure PCTCN2020120625-appb-000006
Figure PCTCN2020120625-appb-000007
Figure PCTCN2020120625-appb-000008

Claims (10)

  1. 一株抗生链霉菌，其特征在于：名称为Streptomyces antibioticus PPI-16，保藏编号为GDMCC NO：60970，该菌株于2020年3月5日保藏于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心。
  2. 一种权利要求1所述的抗生链霉菌的孢子。
  3. 权利要求2所述的抗生链霉菌的孢子的制备方法，其特征在于：将权利要求1所述的抗生链霉菌接种到培养基上，于28～30℃条件下进行培养，得到抗生链霉菌的孢子；
    所述的培养基的组成成分为：可溶性淀粉1～5g，葡萄糖2～10g，酵母提取物1～3g，蛋白胨1～2g，KNO 30.5～1g，K 2HPO 40.1～0.5g，NaCl 0.1～0.5g，FeSO 40.005～0.01g，MgSO 4·7H 2O 0.01～0.05g，琼脂10～15g，pH 7.2～7.4，加水定容至1000mL；
    所述的培养的时间为3～5天。
  4. 一种抗生链霉菌的代谢物，其特征在于：为通过培养权利要求1所述的抗生链霉菌和/或权利要求2所述的抗生链霉菌的孢子获得。
  5. 权利要求4所述的抗生链霉菌的代谢物的制备方法，其特征在于，包括如下步骤：
    (1)将权利要求1所述的抗生链霉菌接种到斜面培养基上，于28～30℃条件下进行培养；
    (2)将斜面培养基上的菌株接种到发酵培养基中进行发酵培养，过滤除去菌体，得到发酵液；然后将发酵液浓缩，得到浓缩液；浓缩液用乙酸乙酯萃取后再次浓缩，得到发酵浓缩物；
    (3)将发酵浓缩物采用硅胶柱进行色谱分离，然后用体积比为3:20的乙酸乙酯与石油醚进行清洗，再用体积比为1:1的乙酸乙酯与石油醚进行洗脱，并收集洗脱液，得到抗生链霉菌的代谢物。
  6. 根据权利要求5所述的抗生链霉菌的代谢物的制备方法，其特征在于：
    步骤(1)中所述的斜面培养基为：可溶性淀粉1～5g，葡萄糖2～10g，酵母提取物1～3g，蛋白胨1～2g，KNO 30.5～1g，K 2HPO 40.1～0.5g，NaCl 0.1～0.5g，FeSO 40.005～0.01g，MgSO 4·7H 2O 0.01～0.05g，琼脂10～15g，pH 7.2～7.4，加水定容至1000mL；
    步骤(1)中所述的培养的时间为3～5天；
    步骤(2)中所述的发酵培养基为：KNO 30.5～1g，K 2HPO 40.1～0.5g，MgSO 4·7H 2O 0.1～0.5g，FeSO 40.005～0.01g，海水晶0.1～0.5g，可溶性淀粉2～10g，葡萄糖5～10g，酵母膏15～20g，定容至1000mL，pH 7.2～7.4；
    步骤(2)中所述的发酵培养的条件为：室温发酵培养5～7天；
    步骤(2)中所述的浓缩为采用加热的方式进行浓缩；
    步骤(2)中所述的浓缩为浓缩至发酵液体积的1/20～3/1；
    步骤(2)中所述的再次浓缩为浓缩至原体积的1/3～1/5；
    步骤(2)中所述的乙酸乙酯与浓缩液的体积比为1:1；
    步骤(3)中所述的硅胶柱中的硅胶为200～300目硅胶。
  7. 根据权利要求5所述的抗生链霉菌的代谢物的制备方法，其特征在于，在步骤(3)之后还包括如下步骤：
    将步骤(3)中得到的抗生链霉菌的代谢物通过高效液相色谱进行分离，得到抗霉素A1；其中高效液相色谱的流动相为甲醇与体积百分数为0.5％的甲酸水溶液按体积比4:1得到的混合溶液。
  8. 权利要求1所述的抗生链霉菌，权利要求2所述的抗生链霉菌的孢子，以及权利要求4所述的抗生链霉菌的代谢物中的至少一种在生产抗霉素A1中的应用。
  9. 权利要求1所述的抗生链霉菌，权利要求2所述的抗生链霉菌的孢子，以及权利要求4所述的抗生链霉菌的代谢物中的至少一种在防治真菌感染中的应用。
  10. 根据权利要求9所述的应用，其特征在于：
    所述的真菌为植物炭疽病菌或白色念珠菌；
    所述的植物炭疽病菌包括黄瓜炭疽病菌、辣椒炭疽病菌和烟草炭疽病菌。
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