CN116004750B - Method for producing angustin by using erwinia persicae - Google Patents

Abstract

The invention discloses a method for producing anguillarum (animid) by using erwinia persicae, which comprises the following steps: 1) Inoculating erwinia persicae to a liquid culture medium for culture to obtain a fermentation broth, wherein in the culture, the fermentation temperature is 15-20 ℃, the fermentation time is 12-24 hours, the liquid culture medium comprises a carbon source, a nitrogen source and inorganic salts, the carbon source is sodium citrate and/or sodium gluconate, the nitrogen source is yeast extract, beef extract and/or tryptone, and the inorganic salts are sodium chloride; 2) And (3) collecting the angusticidin from the fermentation broth grown in the step (1). The purity of the crude extract of the angustifolia is 40% -60%, the yield is improved by 3.1 times compared with the original fermentation condition, the required cost is only 1/3 of that of the original fermentation condition, the operation process is simplified, and the manpower and material resources are greatly saved.

Description

Method for producing angustin by using erwinia persicae

Technical Field

The invention belongs to the technical fields of microbiology, biotechnology and biological control, and relates to a method for producing angustifolia by using erwinia persicae.

Background

Angicin (Andrimid) is a non-ribosomal peptide-polyketide antibiotic and has been studied over the last two decades to find secretion of a variety of different bacteria to produce Angicin, including Pantoea agglomerans @Pantoeaagglomerans),Serratia praecox(Serratia plymuthica)Vibrio corallolyticus is usedVibriocoralliilyticus)Etc. Anji is a nanomolar inhibitor of the enzyme acetyl-CoA carboxylase that catalyzes the first step of fatty acid biosynthesis in bacteria, and inhibition of this enzyme can block the step of prokaryotic fatty acid biosynthesis. In vitro experiments and computer simulation find that the medicine has higher medicine property score and no systemic toxicity, and is expected to be widely applied to the fields of biological pesticides, veterinary medicines, medicines and the like.

Currently, erwinia persicae is reported to produce angustifolia for the first time, and thus research on fermentation conditions thereof is still blank. However, matila et al have studied the carbon and nitrogen source and temperature in 2016 for the production of angustifolia by Serratia praecox strain A153 and found that angustifolia could be stably produced at 4-20deg.C but could not be detected at 30deg.C and was limited to the flask system. Giuubergia et al found that the addition of chitin increased the yield of anglerin in Vibrio corallolyticus strain S2052. However, the Erwinia persicae and the two strains have large differences, and the culture methods of the two strains cannot be directly used for reference.

Disclosure of Invention

It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below.

Thus, the fermentation conditions of erwinia persicae should be specifically optimized in combination with the characteristics of the species itself and the characteristics of the angustifolia gene cluster in an effort to obtain optimal yields of angustifolia.

It is an object of the present invention to provide an optimal way of fermentation for obtaining the highest yields of angustifolin, which can be performed in a shake flask system to a 5L fermenter system. The fermentation mode is suitable for the erwinia persicae strain with the function of producing the angustifolia.

For this purpose, the technical scheme provided by the invention is as follows:

the method for producing the angustin by the erwinia persicae comprises the following steps of:

1) The Erwinia persicae is treatedErwiniapersicina) Inoculating the strain into a liquid culture medium for culture to obtain a fermentation broth, wherein in the culture, the fermentation temperature is 15-20 ℃, the fermentation time is 9-45 hours, the liquid culture medium comprises a carbon source, a nitrogen source and inorganic salt, the carbon source is sodium citrate and/or sodium gluconate, the nitrogen source is yeast extract, beef extract and/or tryptone, and preferably, the inorganic salt is sodium chloride.

2) And (3) collecting the angusticidin from the fermentation broth grown in the step (1).

Preferably, in the method for producing the angustin by the erwinia persicae, the erwinia persicae adopts an erwinia persicae 2022TIBBST187 strain (BST 187 for short) and the erwinia persicae is prepared byClassification nomenclature of the erwinia persicae 2022TIBBST187 strain: erwinia persicaeErwiniapersicinaErwinia persicae strainErwiniapersicina2022TIBBST187 is deposited at the China Center for Type Culture Collection (CCTCC), and has the accession number: cctccc NO: m2022872, the preservation time is: 2022, 06, 13, deposit unit address: chinese university of Wuhan and Wuhan.

Preferably, in the method for producing the angusticium persicum from the erwinia persica, in the step 1), the liquid culture medium takes sodium citrate as a carbon source, beef extract as a nitrogen source and sodium chloride as an inorganic salt. The optimal fermentation medium is sodium citrate 20 g/L, beef extract 5g/L and sodium chloride 10g/L.

Preferably, in the method for producing the angusticium persicum from the Erwinia persicae, in the step 1), the initial pH of fermentation is 6-7, the pH is adjusted to be 6-7 in the fermentation process, and the proper final pH is 6.8-7.8. Preferably, the fermentation process is carried out with a pH of about 7.

Preferably, in the method for producing the angustin by the erwinia persicae, in the step 1), the culture is carried out in a 5L fermentation tank, and the DOI is maintained to be higher than 30% in the fermentation process. High dissolved oxygen can significantly increase the biomass of the cells of BST187.

Preferably, in the method for producing the angustin by the erwinia persicae, in the step 1), the seed solution of the erwinia persicae is inoculated in an amount of 1% -3%. Most preferably the inoculum size is 1%.

Preferably, the method for producing the angusticillin by the erwinia persicae further comprises the following steps: firstly, taking out the strain of the preserved erwinia persicae strain, streaking and activating on an LB solid medium, culturing overnight in a 28 ℃ incubator, and then inoculating the activated single colony into 5 mL of LB liquid medium, and shaking and culturing at 28 ℃ and 200 rpm for overnight to obtain the seed liquid. Of course, it is also preferable to use the optimal fermentation medium (sodium citrate 20 g/L, beef extract 5g/L and NaCl 10 g/L) and to carry out the production of seed liquid at 15-20deg.C.

The invention at least comprises the following beneficial effects:

the invention provides a fermentation medium of an erwinia persicae antibacterial substance anguillarum (anglimid) and an optimization method of fermentation conditions, and the invention adopts a liquid fermentation method, wherein the fermentation time is 12-24 h, the inoculum size is 1%, the initial and fermentation pH values are 6.5-7.5, the high dissolved oxygen level is maintained, and the temperature is 15-20 ℃. The method is characterized in that the fermentation production is carried out in a 5L fermentation tank under the optimal fermentation condition, the purity of the crude angustifolia extract obtained by precipitating the fermentation supernatant by 80% ammonium sulfate and dissolving the angustifolia extract by methanol is 40% -60%, the concentration is about 10.73 g/L, the fermentation process is improved by 3.1 times compared with the original fermentation condition, the required cost is only 1/3 of that of the original fermentation condition, the operation process is simplified, and the manpower and material resources are greatly saved.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

An erwinia persicae 2022TIBBST187 strain (BST 187 for short), the classification designation of the erwinia persicae 2022TIBBST187 strain: erwinia persicaeErwiniapersicinaErwinia persicae strainErwiniapersicina2022TIBBST187 is deposited at the China Center for Type Culture Collection (CCTCC), and has the accession number: cctccc NO: m2022872, the preservation time is: 2022, 06, 13, deposit unit address: chinese university of Wuhan and Wuhan.

FIG. 1 is a standard curve of an external standard method for calculating the concentration of anguillarum in an example of the present invention.

FIG. 2 is a graph showing the comparison of An Jijun element yields in both liquid and solid fermentation in the examples of the present invention-OD 297.

FIG. 3 is a graph showing the peak area of An Jijun element yield in both liquid and solid fermentation modes in accordance with the present invention.

FIG. 4 shows the effect of different culture media on BST187 biomass, with different letters representing a significant difference between the twoP<0.05 A) graph.

FIG. 5 shows the effect of different culture media on BST187 AnJijun production in the examples of the present invention, with different letters representing a significant difference between the twoP<0.05 A) graph.

FIG. 6 shows the effect of inoculum size on BST187 biomass in an example of the invention, with distinct letters representing a significant differenceP<0.05 A) graph.

FIG. 7 is a graph showing the effect of inoculum size on BST187 Anji production by an example of the invention, with distinct letters representing a significant difference between the twoP<0.05 A) graph.

FIG. 8 shows the effect of initial pH of the culture medium on BST187 biomass in the examples of the present invention, with different letters representing a significant differenceP<0.05 A) graph.

FIG. 9 shows the effect of initial pH of the medium on BST187 AnJijun production in the examples of the present invention, with different letters representing a significant difference between the twoP<0.05 A) graph.

FIG. 10 is a graph showing the comparison of final pH after fermentation of media of different initial pH in the examples of the present invention, with different letters representing a significant difference between the twoP<0.05 A) graph.

FIG. 11 is a graph of the type of fermentor representing various amounts of dissolved oxygen in an embodiment of the present invention.

FIG. 12 shows the effect of dissolved oxygen on BST187 biomass in the examples of the present invention, with the different letters representing a significant difference between the twoP<0.05 A) graph.

FIG. 13 shows the effect of dissolved oxygen on BST187 Anji production in the examples of the present invention, with distinct letters representing a significant difference between the twoP<0.05 A) graph.

FIG. 14 shows the effect of temperature on BST187 biomass in an example of the present invention, with different letters representing a significant difference between the twoP<0.05 A) graph.

FIG. 15 shows the effect of temperature on BST187 An Jijun element yield in the examples of the present invention, with the difference between the two being represented by the different lettersP<0.05 A) graph.

FIG. 16 is a graph showing the variation of BST187 biomass and yield of Anji mushroom under the conditions of 5L fermenter in the examples of the present invention.

Fig. 17 is a verification of the phosphate solubilizing and bacteriostatic functions of BST187 in the examples of the present invention.

FIG. 18 is a liquid chromatography assay of crude extract of anguillarum in an example of the invention.

FIG. 19 is a first-order mass spectrum of An Jijun in an example of the invention.

FIG. 20 is a secondary mass spectrum of an anguillarum in an example of the invention.

Detailed Description

The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.

For a better understanding of the technical solution of the present invention, the following examples are now provided for illustration:

example 1 establishment of a standard curve for reversed-phase HPLC detection of An Jijun

The prior research uses preparative liquid chromatography to obtain pure product 10 mg of the angirudin. The present example establishes a standard curve using reverse phase high performance liquid chromatography and determines the fermentation yield of angustifolia by an external standard quantity. The specific method comprises the following steps: 1) Preparing a standard solution: dissolving 10 mg pure ansamitocin in methanol to prepare mother solution with the concentration of 10 mg/mL; 2) Gradient dilution: gradient diluting An Jijun element mother liquor by 10, 50, 100, 500, 1000, 5000, 10000 and 50000 times, and detecting by reverse phase high-performance liquid chromatography under the conditions of methanol: water: formic acid (30:70:0.1% vol/vol), ultraviolet detection wavelength 297nm; 3) Establishment of a standard curve: and calculating the linear relation between the peak area of the substance with the peak time of 9-11min and the substance concentration mass, and preparing a standard curve.

By the above method, the relation between the peak area (x) and the content of the angusticin (y: mg/mL) was calculated as y=57342x+62.829 （r ² =0.9994), the linear concentration range was detected to be 0.0005 mg/mL to 1mg/mL. The standard curve is shown in figure 1.

Example 2 fermentation mode

An erwinia persicae 2022TIBBST187 strain, classification designation of said erwinia persicae 2022TIBBST187 strain: erwinia persicaeErwiniapersicinaErwinia persicae strainErwiniapersicina2022TIBBST187 is deposited at the China Center for Type Culture Collection (CCTCC), and has the accession number: cctccc NO: m2022872, the preservation time is: 2022, 06, 13, deposit unit address: chinese university of Wuhan and Wuhan.

The subsequent fermentation mode was determined by comparing the content of angstatin in the metabolites of solid fermentation and liquid fermentation by strain 2022TIBBST187 (hereinafter abbreviated as BST 187). The specific method comprises the following steps: 100 mL of LB solid medium (yeast extract 5g/L, tryptone 10g/L, naCl 10g/L, agar 15 g/L) and 100 mL of LB liquid medium (yeast extract 5g/L, tryptone 10g/L, naCl 10 g/L) were prepared, respectively, and sterilized at 121℃for 20min under high temperature and high pressure. Wherein 100 mL solid media was prepared as LB solid plates of about 6 90 mm petri dishes, and 100 mL liquid media was contained in 250 mL flasks. Activating and propagating the strain BST187 to prepare seed liquid, which comprises the following specific steps: firstly, BST187 strains are taken out from a refrigerator at the temperature of-80 ℃, streaked and activated on LB solid medium, and cultured overnight in an incubator at the temperature of 28 ℃. The next day, the activated single colony was inoculated into 5 mL LB liquid medium, after shaking at 28℃and 200 rpm overnight, the bacterial suspension was centrifuged at 10000rpm for 5min, the supernatant was removed, and the precipitated bacterial cells were subjected to 0.85% sterile physiological saline to adjust the OD of the seed solution ₆₀₀ 0.6-1.0, and a concentration of about 10 is obtained ⁹ CFU/mL of bacterial suspension; uniformly coating 1mL bacterial suspension on 6 LB solid plates, and inoculating 1mL of bacterial suspension into 100 mL of LB liquid medium; the inoculated liquid LB culture was shake cultured at 28℃and 200 rpm for 12-24 h, and the inoculated solid LB culture was dark cultured at 28℃in an incubator. Culturing 12-24 h, centrifuging the liquid fermentation product at 10000rpm for 10 min to obtain supernatant, and removing thallus with 0.22 μm filter membrane; mixing the solid ferment with sterile water according to the volume ratio of 1:1After the mixture is fully crushed by a juicer, the mixture is extracted for 30min at room temperature, the mixture is centrifuged at 10000rpm for 10 min to obtain a supernatant, and the supernatant is filtered by a filter membrane with the diameter of 0.22 mu m to remove thalli. The maximum absorption wavelength of the metabolite was measured as a control of 100 mL liquid LB medium and 100 mL solid LB medium without inoculation after the same treatment as described above. Meanwhile, the quantitative detection of the angustifolia is carried out by reverse phase high performance liquid chromatography (RP-HPLC). HPLC conditions: using Agela&Phenomenex Innoval C18 (4.6 mm ×250mm, 5 μm) column; the mobile phase is methanol containing 0.1% formic acid and water according to the following steps: methanol is 30:70, and the methanol is eluted at the flow rate of 0.6 mL/min for 15 min at equal temperature; the sample injection amount is 10 mu L; the detection wavelength is 297nm; the column temperature was 25 ℃.

As shown in FIG. 2 and FIG. 3, the fermented product obtained by the two methods has maximum absorption peak at about 297 and nm of ultraviolet absorption wavelength, wherein the solid fermented product OD ₂₉₇ And the value of the HPLC detection peak area was about 3 times that of the liquid fermentation product. However, compared with solid fermentation, the liquid fermentation and extraction process is simpler, high-density cell fermentation can be performed, and the potential for large-scale production and application is provided, so that a liquid fermentation system is selected in the subsequent method.

EXAMPLE 3 fermentation time

Seed solutions were prepared as in example 1, added to 100 mL of LB liquid medium at a ratio of 1:100, shake-cultured at 28℃and 200 rpm, sampled at 2, 4, 6, 8, 10, 12, 18, 20, 24, 36, 48 and h, respectively, and OD was measured ₆₀₀ For characterization of biomass, peak areas were measured by HPLC for characterization of the angstatin concentration. The specific method comprises the following steps: taking out shake flask from shake table, sucking 1mL bacterial suspension in ultra clean bench, centrifuging at 13000 rpm for 5min, filtering supernatant with 0.22 μm filter, detecting with liquid phase bottle by HPLC, and re-suspending precipitate with 1mL of 0.85% sterile physiological saline to measure OD ₆₀₀ 。

The results showed that the yield of angustifolia increased gradually over time, but after a certain fermentation time, the yield of angustifolia reached a maximum and then began to decrease. Therefore, to achieve maximum yield of angustifolia, the fermentation time is controlled well, with an optimal fermentation time range of 12-24 hours.

Example 4 Medium optimization

Basic fermentation media (glucose 20 g/L, yeast extract 5g/L, naCl 10 g/L) were selected and BST187 best fermentation media were screened using a one-factor method.

In the early experiments, the carbon source in the basal fermentation medium was replaced with glycerol, sucrose, glucose, sodium citrate, water-soluble starch, mannitol, malt extract, xylose, fructose, sorbitol, corn starch, molasses and sodium gluconate, respectively. The nitrogen source was replaced with ammonium sulfate, urea, ammonium chloride, tryptone, beef extract, yeast extract, casein peptone and soybean meal powder, respectively, to prepare a medium, a seed solution was prepared according to example 1, the seed solution was inoculated into 30 mL liquid medium in an amount of 1%, shaking culture was performed at 28℃and 200 rpm for 12-24 h, and the angustifolin was quantitatively detected by HPLC. As a result, it was found that the optimal carbon sources were sodium citrate and sodium gluconate, and the optimal nitrogen sources were yeast extract, beef extract and tryptone.

Fermentation media (table 1) were prepared by pairing with each other with the optimal carbon and nitrogen source, respectively, and the volume of the media was 100 mL with LB as a control. Inoculating 1% seed solution into the fermentation medium of Table 1, shake culturing at 28deg.C and 200 rpm for 12-24 h, and measuring OD ₆₀₀ For characterization of biomass, the area of the angusticin peak was measured by HPLC.

As a result, the biomass of the cells in the LB medium was the highest as shown in FIG. 4. As shown in fig. 5, the biomass difference between sodium citrate and sodium gluconate was smaller under the same nitrogen source conditions, and the beef extract and yeast extract biomass were significantly higher than tryptone under the same carbon source conditions. But the yield of the angustifolia of sodium citrate is obviously higher than that of sodium gluconate by taking beef extract as a nitrogen source, and the yield of the angustifolia of the beef extract as the nitrogen source is also obviously higher than that of tryptone and yeast extract under the same carbon source condition and is superior to that of the traditional LB culture medium. The optimal fermentation medium is therefore sodium citrate (2%), beef extract (0.5%) and NaCl (1%).

EXAMPLE 5 inoculum size

And taking the selected optimal carbon source and nitrogen source as culture mediums. Inoculating the seed solution into 100 mL liquid culture medium according to 0.1%, 0.5%, 1%, 2%, and 3%, shake culturing at 28deg.C and 200 rpm for 12-24 h, and measuring OD ₆₀₀ For characterization of biomass, the angusticin concentration was measured by HPLC.

As shown in fig. 6 and 7, the biomass of BST187 tended to increase with increasing inoculum size. Maximum was reached at 3% inoculum size, but there was no significant difference compared to 1%. While the yield of angstatin is maximized at 1% inoculum size, significantly higher than 0.1% inoculum size, and thus BST187 is suitably at about 1% -3% inoculum size.

EXAMPLE 6 initial pH

And taking the selected optimal carbon source and nitrogen source as culture mediums. The initial pH of the medium was adjusted to 6.0, 7, 7.8, 8.6. Inoculating the seed solution into 100 mL liquid culture medium at 28deg.C under shaking at 200 rpm for 12-24 h, and measuring OD ₆₀₀ For characterization of biomass, the angusticin concentration was measured by HPLC. And after fermentation is finished, detecting the final pH of BST187 fermentation liquid with different initial pH values.

As a result, as shown in FIGS. 8 and 9, the biomass of the cells of BST187 was highest at the initial pH of 7, but there was no significant difference, whereas the yield of angustifolia was significantly higher at pH 6-7 than at pH 7.8 and 8.6. Thus, the optimum initial pH is 6 to 7. After the fermentation was completed (FIG. 10), the BST187 broth at low initial pH (pH 6, pH 7) had an increase in pH of about 0.8. Thus, a suitable final pH is between 6.8 and 7.8, and the subsequent fermentation process requires a pH stabilization around 7.

Example 7 dissolved oxygen

The selected optimal carbon source and nitrogen source are used as culture mediums, and the grooves at the bottom of the triangular flask are designed to form vortex by utilizing the baffle effect of the triangular flask, so that the violent flow of the culture solution can be accelerated, the exchange between the surface of the liquid and oxygen is increased, and the dissolved oxygen is increased. 100 mL was added to four baffle, three baffle and no baffle 250 mL flasks (fig. 11) respectively, representing high, medium and low dissolved oxygen conditions. Inoculating the seed solution into 100 mL liquid culture medium according to 1% of inoculation amountIn the above steps, after shaking culture at 28 ℃ at 200 rpm in a shaking table for 12-24 h, OD is measured ₆₀₀ For characterization of biomass, the angusticin concentration was measured by HPLC.

As shown in fig. 12 and 13, the high dissolved oxygen significantly increased the biomass of BST187 cells (2.5-fold) and the yield of angustifolia (3.6-fold). Furthermore, there is no significant difference between the three baffles and the four baffles. The subsequent fermentation process therefore requires that dissolved oxygen be maintained at a certain level.

Example 8 temperature

LB was used as a medium. The seed solution is inoculated into 100 mL liquid culture medium according to the inoculation amount of 1%, and the fermentation temperature of 15-20 ℃, 25-28 ℃ and 30-37 ℃ is respectively used for shaking culture at 200 rpm for 12-24 h. Measuring OD ₆₀₀ For characterization of biomass, the angusticin concentration was measured by HPLC.

As shown in FIG. 14 and FIG. 15, the biomass and the yield of the angustifolia are remarkably improved at 15-20 ℃ compared with 25-28 ℃ by 1.3 times and 3.4 times respectively. Accordingly, BST187 is suitably fermented at a temperature of 15-20 ℃.

Example 9 5L fermenter pilot plant

According to the experiment, the optimal fermentation medium, inoculum size, initial pH and fermentation temperature of the BST187 producing the angustifolia are determined, and oxygen is required to be supplemented in the fermentation process. Thus, under optimal conditions, a certain dissolved oxygen level was maintained through a 5L fermenter, the temperature and pH were controlled for pilot scale, samples were taken at 9, 12, 15, 18, 21, 24, 32, 36 and 45 h, respectively, OD600 was measured for the characterization of biomass, and the angustifolia concentration was measured by HPLC.

As a result, as shown in FIG. 16, the maximum value of the peak area of the angustifolia can reach 2527.7, and the yield is improved by 3.1 times compared with the initial fermentation condition.

Example 10 isolation screening and functional identification of Strain BST187

Strain BST187 was isolated from tomato rhizosphere soil harvested in the eastern region of the body of the year 2022. The specific method comprises the following steps: mixing 1g collected soil with 99 mL 1 XPBS buffer solution (dilution ratio 10) ² ) Shaking culture is carried out for 30min at 200 rpm and 28 ℃. Standing for 20min, collecting supernatant of the soil suspension, and performing gradientDiluting the mixture to a certain degree, and respectively taking dilution factors of 10 ² 、10 ³ 、10 ⁴ The diluted solution 100 mu L of (2) is uniformly coated on an organophosphorus decomposing medium (glucose 10g/L, ammonium sulfate 0.5g/L, yeast extract 0.5g/L, sodium chloride 0.3 g/L, potassium chloride 0.3 g/L, magnesium sulfate 0.3 g/L, ferrous sulfate 0.03g/L, manganese sulfate 0.023 g/L, lecithin 0.2 g/L, and calcium carbonate 1g /), and the culture after coating is performed on the basis of a 28 ℃ incubator for inversion culture 24 h-48 h. And (3) selecting colonies which generate phosphate solubilizing rings to be streaked and purified on an LB solid culture medium plate until the colony forms are single, then expanding and breeding the single colonies in an LB liquid culture medium, mixing bacterial suspension and sterilizing glycerol (working concentration is 25%), and storing the bacterial suspension in a refrigerator at-80 ℃ for later use.

Through the above-mentioned process of separating and screening the phosphate-solubilizing bacteria, we separated a strain of highly efficient phosphate-solubilizing bacteria from the medium for organic phosphate-solubilizing bacteria, and the result is shown in FIG. 17 (left), and designated 2022TIBBST187. The similarity of the 1468 bp length 16S rRNA sequence in strain BST187 was found by molecular biology methods to E.persicae strain B64 in the National Center for Biotechnology Information (NCBI) database to 99.65%, thus combining the morphological characteristics of the cells with molecular biology to identify strain BST187 as E.persicae.

Other functions of strain BST187, including bacteriostatic activity, etc., were subsequently validated. The specific method comprises the following steps: pathogenic bacteria of rape single cell capsicum spot disease pathotypeXanthomonascampestris pv. vesicatoria) After activation, the strain is propagated in LB liquid medium until the concentration of the bacterial suspension is 10 ⁹ CFU/mL is evenly mixed into an uncured LB solid culture medium according to the proportion of 1:100, and a plate is poured to prepare a bacteria-carrying culture medium; 2 μl of bacterial strain BST187 suspension (concentration 10 ⁹ CFU/mL) was added dropwise to the solid medium plates with LB, three drops per plate, and after incubation at 28 ℃ for 24 h, it was observed whether or not a zone of inhibition was generated. As a result, strain BST187 has an inhibitory activity against bacteria as shown in FIG. 17 (right). To identify bacteriostatic actives in BST187, we found by analysis of secondary metabolite gene clusters that the entire genome of BST187 contained NRP: beta-lactam+Polykeyide (non-nucleotide: macrolide/Polyketide) polypeptide Angicin andrimid。

Example 11 extraction and identification of An Jijun element

The angustifolia belongs to non-ribosomal peptide-polyketide antibiotics and contains part of active amino acids, so that the extraction method can be carried out by referring to proteins, and the specific method is as follows.

Ammonium sulfate was added to the liquid fermentation broth of strain BST187 to 80% saturation concentration, slowly stirred during the addition to dissolve, during which a brown solid began to precipitate, and the solution with ammonium sulfate added was placed overnight at 4 ℃. Centrifuging at 10000rpm and 4deg.C for 10 min the next day, removing supernatant, weighing precipitate, adding methanol for dissolving to give concentration of 0.1g/mL, centrifuging at 10000rpm and 4deg.C for 5min after dissolving precipitate, and collecting supernatant to obtain crude extract of angirudin. The method for precipitating the anguillarum by using the ammonium sulfate has the advantages of low cost of consumed reagents, simple operation process and great saving of manpower and material resources.

The crude extract of the angustifolia is subjected to further qualitative analysis by using ultra-high performance liquid chromatography-mass spectrometry, the separation result of the high performance liquid chromatography is shown in figure 18, the identification result of the mass spectrometry is shown in figures 19 and 20, and the substance with the peak time of 9.701 min can be qualitatively called angustifolia andriid (C27H 33N3O 5) by molecular formula searching and secondary fragment ion structure analysis. The content of the anguillarum in the fermented crude extract is 40% -60% calculated according to an area normalization method.

The number of modules and the scale of processing described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art.

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

Claims (8)

1. The method for producing the anguillarum by the erwinia persica is characterized by comprising the following steps of:

1) The Erwinia persicae is treatedErwinia persicina) Inoculating the strain to a liquid culture medium for culture to obtain a fermentation broth, wherein in the culture, the liquid culture medium comprises a carbon source, a nitrogen source and inorganic salts, wherein the carbon source is sodium citrate and/or sodium gluconate, the nitrogen source is yeast extract, beef extract and/or tryptone, the erwinia persicae adopts an erwinia persicae 2022TIBBST187 strain, and the erwinia persicae 2022TIBBST187 strain is named after classification: erwinia persicaeErwinia persicinaErwinia persicae strainErwinia persicina2022TIBBST187 is deposited at the China Center for Type Culture Collection (CCTCC), and has the accession number: cctccc NO: m2022872, the preservation time is: 2022, 06, 13, deposit unit address: chinese university of Wuhan;

2) And (3) collecting the angusticidin from the fermentation broth grown in the step (1).

2. The method for producing angustin by erwinia persicae according to claim 1, wherein in the step 1), the liquid culture medium uses sodium citrate as a carbon source, beef extract as a nitrogen source and sodium chloride as an inorganic salt.

3. The method for producing angustin by erwinia persicae according to claim 1, wherein in the step 1), the fermentation temperature is 15-20 ℃.

4. The method for producing angustin by erwinia persicae according to claim 1, wherein in the step 1), the fermentation time is 12-24 hours.

5. The method for producing angustin by erwinia persicae according to claim 1, wherein in the step 1), the fermentation initiation pH is 6 to 7, and the pH is adjusted to be 6 to 7 during the fermentation.

6. The method for producing angustin by erwinia persicae according to claim 1, wherein in the step 1), the cultivation is performed in a 5L fermenter and oxygen is supplemented during the fermentation to maintain the dissolved oxygen DO at 30% v/v or more.

7. The method for producing angustin by erwinia persicae according to claim 1, wherein in the step 1), the seed solution of erwinia persicae is inoculated in an amount of 1% -3%.

8. The method for producing angustin by erwinia persicae according to claim 7, further comprising the steps of: firstly, taking out the strain of the preserved erwinia persicae strain, streaking and activating on an LB solid medium, culturing overnight in a 28 ℃ incubator, and then inoculating the activated single colony into 5 mL of LB liquid medium, and shaking and culturing at 28 ℃ and 200 rpm for overnight to obtain the seed liquid.

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