CN110684064B - Compound with antibacterial activity and preparation method and application thereof - Google Patents

Abstract

The invention discloses a compound with antibacterial activity and a preparation method and application thereof. The compound with the bacteriostatic activity is obtained by separating and purifying a product obtained by performing specific fermentation culture on streptomyces diastatochromogenes D. The preservation number of the Streptomyces diastatochromogenes (Streptomyces diachromogenes) D is CGMCCNo.2060. The streptomyces diastatochromogenes D can be fermented and extracted to obtain an active compound TetramycinZ with an inhibiting effect on part of plant fungal disease pathogenic bacteria, and the compound has a strong inhibiting effect on common plant pathogenic fungi; the invention provides a new way for developing new agricultural fungicides in the future.

Description

Compound with antibacterial activity and preparation method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a novel compound with bacteriostatic activity, a preparation method and application thereof.

Background

Of the crop diseases caused by different pathogenic bacteria, about 70% are caused by fungi. Polyene macrolide antibiotics are antibiotics with a plurality of conjugated double bonds, and have finger-shaped ultraviolet absorption and very obvious characteristics. According to the number of conjugated double bonds, polyene antibiotics can be divided into 5 groups such as trienes, tetraenes, pentaenes, hexaenes, heptaenes, etc., generally have strong antifungal activity and a wide antifungal spectrum, and are effective against crop major pathogenic fungi such as rhizoctonia solani (rhizoctonia solani), Botrytis cinerea (Botrytis cinerea), early blight of tomato (alternaria solani), gibberella graminis (fusarium graminearum) and fusarium oxysporum (f. oxysporum); it is active against common fungi in food and feed, such as Aspergillus flavus, Aspergillus ochraceus and Aspergillus niger, and Saccharomyces cerevisiae. The antibiotics are reported to be mainly applied to medicines at present, and if polyene macrolide compounds are used as main active ingredients to be developed into broad-spectrum antifungal biological preparations, good biocontrol materials are provided for agricultural production.

At present, chemical pesticides are mainly used for preventing and treating plant diseases to kill pathogenic bacteria, but the problems of toxic and side effects and residues of the chemical pesticides on human and livestock are still difficult to effectively solve so far. The biological control of plant pathogenic fungi diseases by utilizing active substances of actinomycetes for resisting pathogenic fungi has the advantages of short period, easy research, convenient production, no toxicity, no harm and the like.

Disclosure of Invention

According to the invention, a biocontrol actinomycete is separated from Hangzhou soil, antibacterial components in a fermentation metabolite of the biocontrol actinomycete are separated to obtain main active components, and the structural formula of the biocontrol actinomycete is determined through four-large-spectrum analysis. The invention provides excellent starting strains and bioactive compounds for further development of microbial pesticides.

The invention aims to provide a novel anti-plant pathogenic fungi active compound aiming at the defects of unsafe chemical pesticide and less varieties of microbial pesticide; another object of the present invention is to provide a controlling effect of the active compound on plant diseases.

The separation, screening and identification of the streptomyces diastatochromogenes of the invention are as follows:

is obtained and stored by the steps of separation, culture, fermentation, activity determination, screening of effective strains for inhibiting plant fungal disease pathogenic bacteria and the like in a soil sample collected in Linan Tianmu mountain in Hangzhou, Zhejiang in 2006 for 8 months; identified as Streptomyces diastatochromogenes (s.diastatochromogenes) by microbial taxonomy, and named as Streptomyces diastatochromogenes (s.diastatochromogenes) D. The strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No. 1 of Beijing republic of Chaoyang, the preservation date is 5 months and 25 days in 2007, the preservation registration number is CGMCC No.2060, and the preservation information of the strain is published in China patent application CN 201410785316.6.

Microscopic morphological characteristics of streptomyces diastatochromogenes D: the spore has straight, hooked, loose spiral shape, and long, elliptic or oval shape.

The amylase of the inventionStreptomyces chromogenes D is fermented and extracted to obtain an active compound with an inhibiting effect on part of plant fungal disease pathogenic bacteria, and the molecular formula of the compound is C₃₅H₅₁NO₁₃The chemical structural formula is as follows:

is a novel tetraene macrolide compound which is not reported in the literature and is named as tetramycin Z with the name Tetramycin Z in England.

The purpose of the invention is realized by the following technical scheme:

the preparation method of the novel compound comprises the following steps:

(1) activating and culturing strains: transferring the separated and stored streptomyces diastatochromogenes D strain to a Gao's first inclined plane in a test tube, and culturing at the temperature of 28 +/-1 ℃ until the inclined plane of the test tube is full of spores;

(2) fermenting and culturing, wherein the fermentation culture solution contains glucose 5g, yeast extract 4g, malt extract 6g, CaCO per 1000m L₃5g and 2g of NaCl, sterilizing, cooling to 50 ℃, inoculating the streptomyces diastatochromogenes D into the fermentation culture solution under the aseptic condition, and culturing for 7 days under the condition of 28 +/-1 ℃;

(3) and (3) extraction of fermentation products: after fermentation, collecting the fermentation liquor, extracting and chromatographing by a conventional chemical method, and separating and purifying to obtain an active compound TetramycinZ

(4) The active compounds are used for the determination of the bacteriostatic activity.

The extraction steps are specifically as follows:

collecting fermentation liquor after fermentation, adding equal volume of ethyl acetate for extraction, taking the lower layer, extracting with equal volume of n-butanol, taking the upper layer, concentrating under vacuum condition to obtain extract, and performing subsequent column chromatography.

The steps of chromatography, separation and purification are as follows: dissolving the extract with methanol, centrifuging to obtain supernatant, then loading the supernatant on an ODS column, performing gradient elution by adopting 10-90% methanol water solution by volume ratio, and separating and purifying to obtain the active compound TetramycinZ.

The invention has the beneficial effects that:

the invention provides a novel tetraene macrolide compound Tetramycin Z (Tetramycin Z), which has stronger inhibition effect on common plant pathogenic fungi;

the invention also provides a preparation method of the novel tetraene macrolide compound tetramycin Z, which adds a new way for developing new agricultural fungicides in the future.

Drawings

FIG. 1 is an ultraviolet absorption spectrum of tetramycin Z;

FIG. 2 is an infrared spectrum of tetramycin Z;

FIG. 3 is a high resolution mass spectrum of tetramycin Z;

FIG. 4 is a hydrogen spectrum of tetramycin Z;

FIG. 5 is a carbon spectrum of tetramycin Z.

Detailed Description

Example 1: (isolation and screening of Streptomyces diastochromogenes D)

In the invention, Streptomyces diastatochromogenes (Streptomyces diachromogenes) D is obtained from a soil sample collected in Tianmu mountain at Lin' an Zhejiang at 2006 and stored through the steps of separation, culture, fermentation, activity determination, screening of pathogenic bacteria effective for inhibiting plant fungal diseases and the like.

A method for determining antibacterial activity of Streptomyces diastochromogenes (S.diastatochromogenes) D comprises the steps of putting 1m L of fermentation supernatant into a sterile culture dish, rapidly mixing with 9m L PDA culture medium cooled to 50 ℃, placing 1 test fungus cake such as cucumber rhizoctonia solani or tomato botrytis cinerea with the diameter of 4mm on each culture medium plane after cooling, inoculating the fungus cake to the center of the culture dish (the diameter of the culture dish is 9cm), culturing in an incubator at 28 ℃ for 36h, treating with sterile water as a control, repeating for 3 times, determining the diameter of a colony by a cross method, and calculating the inhibition rate by the following formula:

the measurement result shows that: the inhibition rates of the amylase chromophoric streptomycete D metabolite on rhizoctonia solani kuhn and botrytis cinerea are 100% and 78% respectively.

Example 2: (preparation method of Streptomyces diastochromogenes D fermentation metabolite)

The method comprises the following steps:

(1) activated culture of the slant of the strain test tube: the slant culture medium is a Gao's first culture medium and comprises the following components in percentage by weight: k₂HPO₄0.5g、KNO₃1g、MgSO₄0.5g of soluble starch, 20g of soluble starch, 0.5g of NaCl and FeSO₄0.01g of agar and 20g of agar, dissolving and then adding water to 1L, picking a little of spores of streptomyces diastatochromogenes D by using an inoculating loop under the aseptic condition, putting the spores into a sterilized Gao's first culture medium test tube (15 × 150mm), placing the test tube in an incubator at the temperature of 28 +/-1 ℃, and carrying out activation culture for 96 hours until the slant of the test tube is full of spores;

(2) fermenting and culturing, wherein the fermentation culture solution contains glucose 5g, yeast extract 4g, malt extract 6g, CaCO per 1000m L₃5g of NaCl2g and the balance of water, bottling 60m of L fermentation culture solution in each 300m L triangular flask, sterilizing at 121 ℃ for 20min after preparation, cooling to 50 ℃, selecting platinum-ring amylase chromogenes D spores under the aseptic condition, inoculating, and carrying out fermentation culture for 5 days at 28 +/-1 ℃;

(3) and (3) fermentation product extraction, namely collecting fermentation liquor after fermentation is finished, adding equal volume of ethyl acetate for extraction, taking a lower layer, extracting with equal volume of n-butanol, taking an upper layer, concentrating under vacuum condition to obtain an extract, dissolving the extract with methanol, centrifuging to obtain a supernatant, then loading the supernatant on an ODS column, eluting with methanol and water (water/methanol: V/V, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 4L), and further separating and purifying to obtain an active compound TetramycinZ (tetramycin Z).

The novel compounds were structurally characterized by Ultraviolet (UV), Infrared (IR), Mass (MS), and Nuclear Magnetic Resonance (NMR). The specific analysis conditions and analysis structures are as follows:

the pure product is entrusted with the analysis and test center of Zhejiang university of industry for spectrum analysis, the model of an ultraviolet absorption spectrum analyzer is UV-1800spectrophotometer (Shimadzu Corporation, Kyoto, Japan), the spectrum bandwidth is 1.00nm, the spectrum scanning range is 190.00 to 400.00nm, the infrared spectrum analysis adopts a Fourier infrared spectrometer Bruk A L PHA, the mass spectrum analysis adopts a L C-MSagint 1260 and 6130 mass spectrometer (Agilent company), the nuclear magnetic resonance spectrum analysis adopts BrukerAvance 400MHz (Bruker company), the active substance is dissolved by deuterated methanol, Tetramethylsilane (TMS) is an internal standard, and the structure of the compound is analyzed according to the UV, IR, MS and NMR spectrum data of the compound and the related documents.

The ultraviolet spectrum is shown in figure 1, and the compound has ultraviolet absorption at (291 +/-2) nm, (302 +/-2) nm and (320 +/-3) nm. Infrared spectrum shown in FIG. 2, infrared spectrum (IR, cm-1): 3423(-OH stretching vibration), 2933(-CH2 antisymmetric stretching), 1712 (conjugated ester C ═ O stretching vibration), 1630 (lactone C ═ O stretching vibration), 1571 (multiconjugated C ═ C stretching vibration), 1401 (multiconjugated C — H variable angle vibration). The high-resolution mass spectrum is shown in FIG. 3, and the mass spectrum conditions are as follows: ionization mode ESI +, capillary voltage 0.6kV, vaporization temperature: 450 ℃, scanning range m/z: 50-1000 amu. As can be seen, the molecular weight of tetramycin Z was determined to be about 693.34.

NMR data on Tetramycin Z

The compound is a new compound named as tetramycin Z, and the specific structural formula is shown in the following formula

Example 3: (Tetramycin Z inhibitory action against Rhizoctonia solani or Botrytis cinerea)

(1) Preparing tetramycin Z solutions with various concentrations;

(2) the bacteriostatic activity is measured by taking tetramycin Z solution 1m L with various concentrations in a sterile culture dish, rapidly mixing with PDA culture medium cooled to 50 ℃ by 9m L, respectively placing 1 cake of cucumber rhizoctonia solani or tomato botrytis cinerea with the diameter of 4mm on each culture medium plane after cooling, inoculating the cake in the center of the culture dish (the diameter of the culture dish is 9cm), placing the culture dish in an incubator for 36h at 28 ℃, using conventional chemical agents and sterile water treatment as controls, repeating for 3 times, measuring the diameter of a colony by adopting a cross method, and calculating the inhibition rate by the following formula:

the results are shown in Table 1. As seen from Table 1, the EC of tetramycin Z against Rhizoctonia solani and Botrytis cinerea₅₀The values are all lower than those of carbendazim, and the inhibition effect is stronger than that of the conventional chemical agent carbendazim.

TABLE 1 inhibition of Rhizoctonia solani and Botrytis cinerea by Tetramycin Z

TABLE 1 inhibition of Rhizoctonia solani and Botrytis cinerea by Tetramycin Z

Different letters in the same row represent significant differences at the 0.05 level.

Claims (4)

1. A compound tetramycin Z with bacteriostatic activity is characterized by the following structural formula:

2. a process for the preparation of TetramycinZ compound as claimed in claim 1, which comprises the steps of:

(1) activating and culturing strains: transferring the separated and stored streptomyces diastatochromogenes D strain to a Gao's first inclined plane in a test tube, and culturing at the temperature of 28 +/-1 ℃ until the inclined plane of the test tube is full of spores; the preservation registration number of the streptomyces diastatochromogenes D is CGMCC No. 2060;

(2)fermenting and culturing, wherein the fermentation culture solution contains glucose 5g, yeast extract 4g, malt extract 6g, CaCO per 1000m L₃5g, NaCl2 g; sterilizing, cooling to 45-50 deg.C, inoculating Streptomyces diastatochromogenes D into the fermentation culture solution under aseptic condition, and culturing at 28 + -1 deg.C for 5-7 days;

(3) and (3) extraction of fermentation products: extracting, carrying out chromatography, separating and purifying the fermentation product to obtain a compound TetramycinZ;

the extraction steps are specifically as follows:

collecting fermentation liquor after fermentation, adding equal volume of ethyl acetate for extraction, taking the lower layer, extracting with equal volume of n-butanol, taking the upper layer, concentrating under vacuum condition to obtain extract, and performing subsequent column chromatography;

the steps of chromatography, separation and purification are as follows: dissolving the extract with methanol, centrifuging to obtain supernatant, then loading the supernatant on an ODS column, performing gradient elution by adopting 10-90% methanol water solution by volume ratio, and separating and purifying to obtain the active compound TetramycinZ.

3. Use of the compound tetramycin z according to claim 1 for the preparation of a medicament active against phytopathogenic fungi.

4. The use according to claim 3, wherein the phytopathogenic fungus is Rhizoctonia solani or Botrytis cinerea.

Images