Endophytic Streptomyces of Dabie Mountain Prunus truncatula producing nycthiome and preparation method thereof

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to an endophytic Streptomyces of Dabie Mountain Five-needle Pine that produces nystatin and its fermentation process. The present invention isolates an endophytic actinomycete WP-1 from the endangered plant Dabie Mountain Five-needle Pine, and its 16S rDNA contains the sequence shown in SEQ ID NO 1. The present invention also includes a fermentation process and application of the streptomycin to produce nystatin. The fermentation broth of the streptomycin of the present invention has a good activity against the plant pathogen Fusarium oxysporum, and the fermentation process has low cost and high yield, and large-scale industrial production can be achieved. The WP-1 Streptomyces and its fermentation metabolites disclosed in the present invention can be used to inhibit the mycelial growth and spore germination of various crop pathogenic fungi such as watermelon wilt fungus, avoid the use of chemical pesticides, and are promising biological pesticides.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to an endophytic streptomyces of Dabie Mountain pentaphyllum producing nystatin and a preparation method thereof. Background Art

[0002] During the planting process, crops are often threatened by bacterial, fungal and viral infections, especially fungal diseases, which are the most common, causing necrosis, rot, withering, discoloration, deformity, etc. during the growth of crops, seriously affecting the yield and quality of crops. At present, the main method for preventing and controlling fungal diseases of crops is chemical control, such as mancozeb, cymoxanil, Bordeaux, thiophanate-methyl, carbendazim, thiram, metalaxyl, tebuconazole, ethyl phosphine, etc., but similar chemical control methods often cause chemical residues in crops, environmental and food safety hazards, etc. Long-term use of chemical control will also accelerate the drug resistance of pathogenic bacteria in crops, thus creating a vicious cycle, which is not in line with the modern concept of green agriculture and sustainable development.

[0003] In recent years, biological control methods have been gradually used at home and abroad to control various crop diseases, which mainly include the use of beneficial microorganisms or fermentation metabolites of microorganisms to prevent and control various crop diseases. These beneficial microorganisms usually include bacteria, actinomycetes, yeasts and even molds. In practical applications, live cells or metabolites of beneficial microorganisms are often used to make powders, granules, liquids, suspensions or bacterial fertilizers to prevent and control fungal diseases of crops, and have achieved significant prevention and control effects and environmental improvement effects. The main principle is that beneficial microbial preparations can multiply in large quantities under natural conditions by spraying or watering, thereby forming a space occupation and nutritional competition with plant pathogens, and even secreting metabolites to directly kill plant pathogens or induce plants to develop resistance to pathogens; some beneficial microorganisms can also produce active substances such as plant growth stimulants and vitamins to promote plant growth and increase crop yields. The advantages of these microbial source products are low toxicity to humans, animals and beneficial insects, fast degradation in outdoor environments, small residues, and are conducive to green agriculture and sustainable development. At present, the products used in this field for the prevention and treatment of fungal diseases of crops mainly include live bacterial preparations of microorganisms such as Bacillus subtilis, Bacillus licheniformis, Bacillus mucilaginosus, Bacillus polymyxa, Bacillus cereus, Trichoderma, etc.; preparations of microbial metabolites are also widely promoted and used, such as Jinggangmycin aqueous solution (or wettable powder) for the prevention and treatment of rice sheath blight, Kasugamycin aqueous solution (or wettable powder) for the prevention and treatment of rice blast, Polyoxin aqueous solution (or wettable powder) for the prevention and treatment of wheat root rot, Wuyimycin aqueous solution (or wettable powder) for the prevention and treatment of cucumber powdery mildew, Zhongshengmycin aqueous solution (or wettable powder) for the prevention and treatment of apple rot, etc. Due to the wide variety of crop varieties and complex diseases, the number of varieties that can be used for biological control is still very limited, and there are also defects such as slow or unclear efficacy of some varieties. Summary of the invention

[0004] The purpose of the present invention is to provide a novel streptomyces for effectively preventing and controlling fungal diseases of crops.

[0005] Another object of the present invention is to provide an optimized fermentation process to increase the yield of the above-mentioned Streptomyces metabolites to achieve industrial production.

[0006] The present invention provides a strain of Streptomyces, and the morphology of the base hyphae, aerial hyphae, spores and spore hyphae of the strain on the insert is observed under an optical microscope after crystal violet staining. Figure 2 As shown, the colonies of actinomycetes are velvety in the early stage, powdery and dry in the later stage, with light yellow pigment deposited in concentric circles; the surface of the colonies is light grayish white, with a strong earthy smell; the hyphae in the base have no transverse septa and are multi-branched; the aerial hyphae are straight or curved, with transverse septa, and present obvious nodules.

[0007] The 16S rDNA of the Streptomyces contains the sequence shown as SEQ ID NO 1.

[0008] The present invention provides a new Streptomyces for effectively preventing and controlling fungal diseases of crops. The Streptomyces is isolated from the bark of the endangered plant Dabie Mountain pine, and has been deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on January 15, 2020, with the deposit number: CGMCC NO.19354. Combined with the morphological characteristics of strain growth and 16S rDNA sequence analysis, the strain belongs to Streptomyces sp, and the strain is named Streptomyces sp WP-1.

[0009] The present invention provides a method for preparing the streptomyces, comprising:

[0010] Clean and sterilize the bark of Dabie Mountain five-needle pine, cut it into small pieces and inoculate it on PDA medium;

[0011] After one week of cultivation, single colonies grown on the culture medium were picked out and inoculated onto PDA culture medium respectively. After one week of cultivation, bacterial cakes with a diameter of not less than 6 mm were taken for later use;

[0012] The plant pathogen Fusarium oxysporum was used as the target bacteria and the plate confrontation culture method was used to screen the biocontrol strains.

[0013] The PDA culture medium can be obtained through commercial channels.

[0014] On the other hand, the present invention provides an optimized fermentation process to increase the yield of the above-mentioned Streptomyces metabolites to achieve industrial production. According to preliminary studies, the basic fermentation process of the strain is: 2% glucose, 2% maltose, 5% cottonseed powder, and contains a small amount of inorganic salts such as copper sulfate, zinc sulfate, manganese sulfate, magnesium sulfate, etc., initial pH 6.5, liquid volume of 50ml / 250ml triangular flask, inoculation volume 8%, culture temperature 28°C, rotation speed 180r / min, culture for 6 days, and shake flask fermentation level can reach 1.3675g / L. The present invention optimizes the basic fermentation process in the early stage. Through carbon source regulation, addition of ammonia capture agent and inorganic phosphate and oxygen carrier, the shake flask fermentation level reaches more than 5.5g / L, the production cost can be greatly reduced, and it has the ability of industrial large-scale production. The streptomyces fermentation metabolite provided by the invention is a polyene macrolide substance, which is identified as fungichromin (FC), has a killing effect on most pathogenic fungi, and can be widely used for preventing and treating fungal diseases of crops, such as potato late blight caused by Phytophthora infestan, rice sheath blight caused by Rhizoctonia solani, watermelon wilt caused by Fusarium oxysporum, etc., and the substance is sensitive to light, easily decomposed under light conditions, and is not easy to cause environmental pollution and residues in crops. Taiwan Patent TWI338048B describes a method for preparing nystatin, which is to ferment Streptomyces padanus PMS-702 in a culture medium containing defatted soybean powder, glucose, calcium carbonate and trace divalent salt ions at 23.8°C for 6 days, and add a certain amount of corn oil during the fermentation process. The maximum yield of nystatin can reach about 2.05 g / L, and its fermentation level is far lower than that of the present invention.

[0015] The present invention provides a fermentation method of Streptomyces, comprising the following steps:

[0016] (1) Activation of Streptomyces WP-1;

[0017] (2) Liquid fermentation of Streptomyces WP-1, including preparation of seed liquid and fermentation of Streptomyces.

[0018] Preferably, the fermentation method uses an optimized sterile solid medium to culture streptomycin WP-1;

[0019] The optimized sterile solid culture medium contains, per 100 ml, 0.3-2.0 g of glucose, 0.3-1.0 g of yeast extract, 0.3-1.0 g of peptone, 0.08-0.30 g of beef extract, 0.02-0.30 g of magnesium sulfate, and 0-0.40 g of dipotassium hydrogen phosphate.

[0020] In the present invention, the fermentation conditions for Streptomyces WP-1 to produce nystatin include:

[0021] Basic process conditions for producing nystatin by liquid fermentation of Streptomyces WP-1:

[0022] Culture medium: 2% glucose, 2% maltose, 5% cottonseed meal, copper sulfate, zinc sulfate, manganese sulfate, magnesium sulfate, etc.; initial pH 6.5, liquid volume 50ml culture medium / 250ml Erlenmeyer flask; fermentation process: inoculation amount 8%, culture temperature 28℃, rotation speed 180r / min, continuous culture for 6 days, the shake flask fermentation level can reach 1.3675g / L.

[0023] In a preferred embodiment of the present invention, the HPLC detection conditions of nystatin (FC) are:

[0024] The content of nystatin in the fermentation broth was determined by HPLC using a C18 reverse phase column (Waters X-Bridge 5μm 4.6×250mm). The mobile phase was HPLC grade methanol: water = 60:40 (V / V), the flow rate was 0.8mL / min, the detection wavelength was 357nm, and the injection volume was 10μL.

[0025] Preferably, in the fermentation method, the reducing sugar concentration is not less than 2 g / ml.

[0026] Preferably, Ca3(PO4)2 is added to the fermentation broth to a final concentration of 10 to 100 mM, or Mg3(PO4)2 is added to a final concentration of 10 to 80 mM.

[0027] Preferably, an oxygen carrier is added to the fermentation broth, and the oxygen carrier is selected from n-dodecane, Tween 80, oleic acid or soybean oil.

[0028] Preferably, the fermentation method further comprises extraction, purification and identification of active metabolites obtained by fermentation;

[0029] The extraction of active products from the Streptomyces fermentation broth comprises:

[0030] The fermentation liquid of Streptomyces is centrifuged to obtain a supernatant and mycelium. After the supernatant is concentrated, an equal volume of ethyl acetate is added, and extraction is performed at room temperature for 1-5 times, and the extracts are combined to obtain an ethyl acetate phase I; the mycelium is soaked in an ethanol aqueous solution and centrifuged, and the process is repeated 1-5 times, and the soaking liquid is combined and concentrated until there is no ethanol, and an equal volume of ethyl acetate is extracted 3 times, and the ethyl acetate phase II is combined. The ethyl acetate phases I and II are combined and concentrated to dryness to obtain a total ethyl acetate extract;

[0031] The ethyl acetate extract is adsorbed by a macroporous resin, eluted by an ethanol aqueous solution, and the eluate is concentrated under reduced pressure to obtain a yellow paste, which is the crude extract of the Streptomyces fermentation broth;

[0032] The yellow paste crude extract was dissolved in methanol, separated by silica gel column chromatography, gradient eluted with dichloromethane and methanol solution, and the part with the largest absorption peak was collected, and concentrated under reduced pressure to obtain a preliminary pure product;

[0033] The preliminary pure product was dissolved in methanol and purified by HPLC. It was determined to be nystatin by electrospray ionization mass spectrometry nuclear magnetic resonance and analysis of the physicochemical properties of the compound.

[0034] The invention also provides an application of the fermentation method, that is, using the fermentation product for mycelium growth and spore germination of pathogenic fungi of crops.

[0035] Preferably, the application is to produce nystatin, and the fermentation product or metabolically active substance is mainly nystatin.

[0036] The present invention isolates an endophytic actinomycete WP-1 from the endangered plant Dabie Mountain five-needle pine plant, ferments it and screens the activity of the fermentation liquid, and finds that it has good activity against the plant pathogen Fusarium oxysporum, and identifies the main active product as nystatin. By optimizing the culture medium components and fermentation conditions, the antibacterial activity of the optimized fermentation liquid is significantly enhanced, and the output of the optimized active metabolites is greatly increased, which is about 4.2 times that before optimization, and its output can be increased to 5741.7 mg / L, which can realize large-scale industrial production. The WP-1 Streptomyces and its fermentation metabolites disclosed in the present invention can be used to inhibit the mycelial growth and spore germination of various crop pathogenic fungi such as watermelon wilt fungi, avoid the use of chemical pesticides, and are more promising biological pesticides.

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Antagonistic effect of Streptomyces WP-1 against Fusarium oxysporum of watermelon.

[0039] Figure 2 Slant growth and SEM morphology of Streptomyces WP-1.

[0040] Figure 3Effects of active metabolites on spore germination of Fusarium oxysporum: a control group; b FC (12.5 μg / ml)

[0041] Experimental group.

[0042] Figure 4 Structural analysis of active compounds: a hydrogen spectrum; b carbon spectrum; c DEPT spectrum.

[0043] Figure 5 Effects of different carbon sources supplemented at different times on FC production by Streptomyces WP-1. In 5B, 1, 4, and 7 are glucose groups, 2, 5, and 8 are sucrose groups, and 3, 6, and 9 are maltose groups.

[0044] Figure 6 Effects of various phosphates on FC production by Streptomyces WP-1.

[0045] Figure 7 Effects of different types of ammonium scavengers on FC production by Streptomyces WP-1 and ammonium ion concentration in the fermentation system.

[0046] Figure 8 Effects of various oxygen carriers at different addition concentrations on FC production by Streptomyces WP-1.

[0047] Fig. 9 Effects of various oxygen carriers added at different times on FC production by Streptomyces WP-1.

[0048] Fig.10 HPLC chromatogram of FC in the fermentation product of Streptomyces WP-1. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present application are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0050] Example 1 Isolation, Screening, Identification and Preservation of Mycocyanin-Producing Bacteria

[0051] 1.1 Collect the bark of Dabie Mountain five-needle pine, clean its surface with running water, dry it indoors and disinfect it, rinse it quickly with 75% ethanol for about 1 minute, rinse it with sterile water 3 times, disinfect it with 1% sodium hypochlorite solution for 4 minutes, rinse it with sterile water 3 times, absorb the water with sterile filter paper, cut it into 0.5×0.5 small pieces and inoculate it on PDA culture medium, culture it at 28℃ for 5-7 days, pick out 26 single colonies grown on the culture medium, inoculate them on PDA culture medium respectively, culture them at 28℃ for 5-7 days, and take 7mm diameter cakes for use after the plates are basically full.

[0052] 1.2 The plant pathogen Fusarium oxysporum (F.oxysporum) was used as the target bacteria, and the biocontrol strains were screened by the plate confrontation culture method. Prepare PDA plate culture medium, draw a cross on the back of a 90mm diameter plate, the intersection of the cross is the center of the plate circle, and inoculate 3 strains of the above-mentioned 7mm diameter bacterial cakes at 3 points on the cross line 25mm away from the center of the circle, and inoculate the remaining point with a blank 7mm diameter PDA culture medium as a control. Inoculate the 7mm diameter Fusarium oxysporum bacterial cake in the center of the PDA plate and culture it in a 28℃ incubator for 5 to 7 days. Take it out and compare the antagonism of each strain to Fusarium oxysporum.

[0053] 1.3 From the multiple endophytic bacteria isolated from the bark of the five-needle pine in the Dabie Mountains, the plate confrontation culture method was used to screen out Streptomyces WP-1, which has a significant inhibitory effect on the plant pathogen Fusarium oxysporum. Figure 1 The strain was then classified and identified as follows:

[0054] (1) Morphological characteristics of strains

[0055] The strain WP-1 was inoculated into PDA medium and cultured in a constant temperature incubator at 28°C for 3 days. After crystal violet staining, the morphology of the basal hyphae, aerial hyphae, spores and spore hyphae of the strain on the insert was observed under an optical microscope. Figure 2 As shown, the colonies of actinomycetes are velvety in the early stage, powdery and dry in the later stage, with light yellow pigment deposited in concentric circles; the surface of the colonies is light grayish white, with a strong earthy smell; the hyphae in the base have no transverse septa and are multi-branched; the aerial hyphae are straight or curved, with transverse septa, and present obvious nodules.

[0056] (2) 16S rDNA sequence determination and analysis

[0057] The total length of the 16S rDNA nucleotide sequence of strain WP-1 is 1457 bp, and the sequence is as follows:

[0058]

[0059] The sequences were searched for homology on BLAST, and the strains with the highest homology were selected to construct a phylogenetic tree. It was inferred that strain WP-1 should be a branch of the actinomycete Streptomyces genus, but it was a different species from the selected known species. Combined with the phylogenetic tree and morphological characteristics, the strain was preliminarily identified as Streptomyces sp.

[0060] 1.4 On January 15, 2020, the strain WP-1 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number: CGMCC No.19354.

[0061] Example 2 Preparation and Analysis of Active Metabolites of Streptomyces WP-1

[0062] 2.1 Cultivation of Streptomyces WP-1

[0063] Use the optimized solid culture medium to prepare the test tube slant culture medium, pick the colonies from the preserved slant culture medium of the strain, inoculate them into the test tube slant culture medium, and culture them at 28°C for 4 days until the mycelium matures, the spores grow densely and turn bright white.

[0064] Among them, the optimized solid culture medium composition is: 1g glucose, 1g yeast extract, 0.5g peptone, 0.1g beef extract, a small amount of MgSO4, 2.0g agar, 100mL water, the pH value of the culture medium is adjusted to 7.4, and it is sterilized at 121℃ and 0.1Mpa for 20min.

[0065] 2.2 Liquid fermentation of Streptomyces WP-1

[0066] (1) Preparation of Streptomyces WP-1 seed solution: A uniform spore suspension was prepared, and the spore suspension was inoculated into an optimized seed culture medium and cultured at 28° C. and 180 rpm for 40 hours to obtain a seed solution.

[0067] Among them, the composition of the optimized seed culture medium is: 0.5g glucose, 0.5g peptone, 0.5g yeast extract, 0.1g beef extract, 0.2g magnesium sulfate, 0.25g dipotassium hydrogen phosphate, 100mL water, the pH value of the culture medium is adjusted to 7.4, and sterilized at 121℃ and 0.1Mpa for 20min.

[0068] (2) Fermentation of Streptomyces WP-1: The seed liquid of Streptomyces WP-1 was inoculated into a liquid fermentation medium at an inoculum rate of 8% by volume and cultured at 28° C. and 180 rpm for 6 days to obtain a fermentation liquid of Streptomyces WP-1.

[0069] The liquid fermentation medium is composed of 2% glucose, 2% maltose, 5% cottonseed powder, 0.0001% copper sulfate, 0.002% zinc sulfate, 0.0006% manganese sulfate, and 0.00005% magnesium sulfate; the initial pH is 6.5, the liquid volume is 50ml culture medium / 250ml Erlenmeyer flask, and sterilized at 121°C and 0.1Mpa for 30min.

[0070] 2.3 Extraction, purification and structural analysis of active metabolites of Streptomyces WP-1

[0071] (1) Extraction of active products from the fermentation broth of Streptomyces WP-1: The fermentation broth of Streptomyces WP-1 was centrifuged to obtain a supernatant and mycelium. After the supernatant was concentrated, an equal volume of ethyl acetate was added, and the mixture was extracted three times at room temperature, and the ethyl acetate phase 1 was obtained. The mycelium was soaked in 95% ethanol, centrifuged at 3000 r / min, and the soaking was repeated three times. The soaking liquid was combined and concentrated to ethanol-free. A certain volume of distilled water was added, and the mixture was extracted three times with an equal volume of ethyl acetate, and the ethyl acetate phase 2 was obtained. The ethyl acetate phases 1 and 2 were combined and concentrated to dryness to obtain a total ethyl acetate extract.

[0072] (2) The ethyl acetate extract was adsorbed on macroporous resin D101 at a ratio of 1:20 (g / mL), eluted with water:ethanol (V:V50:50), and the eluate was concentrated under reduced pressure to obtain a yellow paste, which was the crude extract of Streptomyces WP-1 fermentation broth. It had a large absorption at 365nm ultraviolet light. At the same time, activity detection found that this component had a very obvious inhibitory effect on the growth of Fusarium oxysporum hyphae.

[0073] (3) The yellow paste crude extract was dissolved in methanol, separated by 200 mesh silica gel column chromatography in a ratio of 1:5 (g / mL), and gradient eluted with dichloromethane:methanol in a ratio of 20:1-1:5. The portion with the largest absorption peak at ultraviolet (365 nm) was collected and concentrated under reduced pressure to obtain a preliminary pure product.

[0074] (4) The preliminary pure product was dissolved in methanol and purified by HPLC. The chromatographic conditions were as follows: the chromatographic column was a reverse phase C18 semi-preparative column, the mobile phase was methanol: water = 60:40, the flow rate was 3 mL / min, the wavelength was 365 nm, the elution was isocratic, and the eluate was concentrated and placed at low temperature and away from light to obtain a yellow compound, which had a significant inhibitory effect on the growth of Fusarium oxysporum hyphae.

[0075] Under sterile conditions, the slant was washed with physiological saline to obtain a spore suspension of Fusarium oxysporum, which was diluted with PDB medium to adjust the spore concentration to 5×10 5 / mL, and then inoculated into a 96-well plate at 90μL / well for culture. The experimental group was given 10μL / well of active metabolite solution with concentrations of 5mg / L, 12.5mg / L, and 25mg / L, respectively, and the control group was given 10μL / well of normal saline as a blank control. Then, the culture was continued for 20h, and the morphology of hyphae and spores was observed under an optical microscope and photographed and recorded. Results Figure 3 The results showed that 12.5 mg / L of the active product had a significant inhibitory effect on spore germination, with a germination inhibition rate of 95%.

[0076] (5) The compound was identified as Fungichromin (FC) through electrospray ionization mass spectrometry (ESI-MS) nuclear magnetic resonance and analysis of the physicochemical properties of the compound. Figure 4 This is the spectrum of this compound, whose molecular formula is C 35 H 58 O 12 , molecular weight 670, structural formula is as follows:

[0077]

[0078] Example 3 Effect of carbon source regulation on fermentation of Streptomyces WP-1

[0079] According to the basic fermentation process of Streptomyces WP-1, shake flask experiments were carried out, and different carbon sources were added at different fermentation times to compare the effects on the fermentation of Streptomyces WP-1. Figure 5 -A shows that the content of reducing sugar in the fermentation bottle decreased significantly in the first two days, and when the reducing sugar concentration in the culture medium was lower than 2g / L, Streptomyces WP-1 no longer produced nystatin; now the carbon source is supplemented by feeding, and the second day is selected as the feeding reference point, the third day is selected backward, and the first day is selected forward as the feeding time. Glucose, sucrose and maltose are used as carbon sources respectively, that is, after the 1st, 2nd and 3rd days of culture, 1mL of each of the three carbon source solutions is added to increase the sugar concentration to 2g / L, and then the culture is continued to the 6th day to explore the production of nystatin by Streptomyces WP-1 after one-time feeding with different carbon sources at different sugar supplementation times.

[0080] The test results are as follows Figure 5 -B shows that the antibacterial activity of the fermentation broth after adding glucose did not increase with the increase of sugar content, but gradually decreased compared with the fermentation without glucose supplementation. The reason is that antibiotics are secondary metabolites of microorganisms. When nutrients are lacking and the growth rate decreases, microorganisms will begin to express related genes of the secondary metabolic pathway to facilitate the production of secondary metabolites. Therefore, when a large amount of glucose is supplemented at one time, the bacteria will use it for growth, and the production of nystatin will be inhibited, so that its yield cannot be increased.

[0081] Different carbon sources will provide the required carbon sources for microbial metabolism at different stages of microbial fermentation. After adding glucose, an accelerating carbon source, Streptomyces WP-1 showed sugar inhibition at different feeding times; when maltose or sucrose was added, it could not only maintain mycelial growth and prolong the production period, but also increase the fermentation titer of nystatin. On the second day of fermentation, after adding 1% sucrose solution, the fermentation titer of nystatin increased by up to 26% compared with the fermentation without feeding.

[0082] Example 4 Effects of Inorganic Phosphate and Ammonia Capture on Fermentation of Streptomyces WP-1

[0083] According to the basic fermentation process of Streptomyces WP-1, shake flask experiments were carried out, mainly using four phosphates, KH2PO4, K2HPO4, NaH2PO4 and Na2HPO4, and two phosphate ammonia scavengers, Ca3(PO4)2 and Mg3(PO4)2, as the research objects, and investigating the effect of adding concentrations ranging from 0.1 to 100mM on the fermentation synthesis of nystatin. Figure 6 and Figure 7 It can be seen that Na2HPO4, Mg3(PO4)2 and Ca3(PO4)2 have obvious effects on increasing yield, and the optimal concentrations are 50mmol / L, 40mmol / L and 100mmo / L, respectively, and the yields are increased by 84%, 410% and 360%, respectively. Mg3(PO4)2 and Ca3(PO4)2 have the strongest effects, and it is possible that the two, as ammonia scavengers, play a good role in promoting the formation of metabolites. KH2PO4, K2HPO4 and NaH2PO4 are not conducive to the synthesis of nystatin, and the addition of high concentrations of phosphates has an inhibitory effect on the synthesis of nystatin. In addition, Figure 7 It can be seen that after adding 10 to 100 mM Ca3(PO4)2 and 40 to 80 mM Mg3(PO4)2, the fermentation titer of nystatin increased and was concentration-dependent with ammonium ions. When the initial concentration of ammonium ions was reduced to 8 μg / mL, the growth of the bacteria was inhibited due to the excessively low ammonia concentration, which also led to a decrease in the production of nystatin.

[0084] According to the above phosphate fermentation results, the orthogonal experimental design table 1 was designed to explore the effect of the mixed addition of phosphate Na2HPO4 and two ammonia scavengers on fermentation. From the orthogonal experimental result table 2, it can be seen that the optimal condition should be A1B2C2, that is, 12.5mM disodium hydrogen phosphate dodecahydrate, 50mM calcium phosphate and 20mM magnesium phosphate. This part of the experiment is to reduce the dosage of ammonia scavengers by adding a variety of phosphates, while increasing the concentration of inorganic phosphates, thereby further increasing the yield of mycocyanin, and the cost is lower.

[0085] Example 5 Effect of oxygen carrier addition on fermentation of Streptomyces WP-1

[0086] According to the basic fermentation process of Streptomyces WP-1 mentioned above, a shake flask experiment was carried out. In order to solve the problem of dissolved oxygen limit of Streptomyces WP-1 in the fermentation process, the effect of adding different oxygen carriers at 0h in the initial stage of fermentation on bacterial growth and nystatin production in shake flask fermentation was investigated. The four oxygen carriers were n-dodecane, Tween 80, oleic acid and soybean oil, and each batch of fermentation contained different oil phase mass fractions and several parallel groups.

[0087] According to the experimental results Figure 8 It can be seen that the activity of mycocyanin produced by fermentation with added oxygen carriers is higher than that without added oxygen carriers, and the production capacity of mycocyanin is highest when 2% oleic acid, 4% Tween 80, 1% n-dodecane and 3% soybean oil are added; it can also be seen from the changes in biomass in the figure that the addition of oleic acid and soybean oil is beneficial to the growth of bacteria, and their biomass is higher than that of the blank control group; finally, compared with the blank fermentation results, we can also find that the pH change of the fermentation liquid is not very obvious. These seem to indicate that the addition of different oxygen carriers can effectively improve the oxygen transfer in the fermentation liquid system, thereby facilitating the growth of bacteria and the production of secondary metabolites.

[0088] Dissolved oxygen has an obvious turning point in the fermentation process, but since the above oxygen carrier addition experiments were all added at the beginning of fermentation, it is necessary to examine the effect of adding oxygen carriers at different fermentation times on the synthesis of nystatin. The optimal concentration of each oxygen carrier was selected for the experiment, and the addition time was 0d, 1d, 2d, 3d, and 4d.

[0089] According to the experimental results Fig. 9 It can be seen that the effects of the four oxygen carriers at different addition times are different. The effects of n-dodecane, Tween 80, oleic acid and soybean oil added on the 0th, 2nd, 2nd and 3rd days of fermentation are the most obvious. Among them, the addition of 3% soybean oil at the beginning of fermentation has the strongest ability to produce nystatin.

[0090] According to the basic process conditions of liquid fermentation of Streptomyces WP-1 to produce nystatin, on the second day of fermentation, adding 1% sucrose solution can increase the fermentation titer of nystatin by about 1.26 times; in the shake flask medium, adding 50mmol / L inorganic phosphate Na2HPO4 can increase it by 1.84 times, and combining two phosphate ammonia scavengers can increase the yield of nystatin by 3.8 times. When adding oxygen carriers to the shake flask medium, it is best to add 3% soybean oil at the beginning of fermentation, and its yield can be increased by about 2.5 times.

[0091] Based on the results of the above embodiments, the optimized process conditions for the liquid fermentation of Streptomyces WP-1 to produce nystatin were determined: 2% glucose, 2% maltose, 5% cottonseed powder, copper sulfate, zinc sulfate, manganese sulfate, magnesium sulfate, etc.; initial pH 6.5, liquid volume 50 ml culture medium / 250 ml Erlenmeyer flask, inoculation amount 8%, culture temperature 28 ° C, rotation speed 180 r / min, the pH value was not controlled during the entire fermentation process. On this basis, 3% soybean oil, 12.5mM disodium hydrogen phosphate dodecahydrate, 50mM calcium phosphate and 20mM magnesium phosphate were added to the fermentation culture, and 1% sucrose solution was added on the second day of fermentation culture. When cultured to the sixth day, the yield of nystatin reached 5741.7 mg / L, which was about 4.2 times before optimization.

[0092] Table 1 Orthogonal design table of phosphate and ammonia capture agent addition test

[0093]

[0094] Table 2 Orthogonal test results of phosphate and ammonia capture agent addition

[0095]

[0096]

[0097] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims. Sequence Listing <110> Fudan University <120> Endophytic Streptomyces of Dabie Mountain Prunus truncatula producing nycthiome and preparation method thereof <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1458 <212> DNA <213> Artificial Sequence <400> 1 tcaggacgaa cgctggcggc gtgcttaaca catgcaagtc gaacgatgaa gcccttcggg 60 gtggattagt ggcgaacggg tgagtaacac gtgggcaatc tgccctgcac tctgggacaa 120 gccctggaaa cggggtctaa taccggatat gaccgtcttg ggcatccttg acggtgtaaa 180 gctccggcgg tgcaggatga gcccgcggcc tatcagcttg ttggtgaggt aacggctcac 240 caaggcgacg acgggtagcc ggcctgagag ggcgaccggc cacactggga ctgagacacg 300 gcccagactc ctacgggagg cagcagtggg gaatattgca caatgggcga aagcctgatg 360 cagcgacgcc gcgtgaggga tgacggcctt cgggttgtaa acctctttca gcagggaaga 420 agcgaaagtg acggtacctg cagaagaagc gccggctaac tacgtgccag cagccgcggt 480 aatacgtagg gcgcaagcgt tgtccggaat tattgggcgt aaagagctcg taggcggctt 540 gtcacgtcgg ttgtgaaagc ccggggctta accccgggtc tgcagtcgat acgggcaggc 600 tagagttcgg taggggagat cggaattcct ggtgtagcgg tgaaatgcgc agatatcagg 660 aggaacaccg gtggcgaagg cggatctctg ggccgatact gacgctgagg agcgaaagcg 720 tggggagcga acaggattag ataccctggt agtccacgcc gtaaacggtg ggcactaggt 780 gtgggcaaca ttccacgttg tccgtgccgc agctaacgca ttaagtgccc cgcctgggga 840 gtacggccgc aaggctaaaa ctcaaaggaa ttgacggggg cccgcacaag cggcggagca 900 tgtggcttaa ttcgacgcaa cgcgaagaac cttaccaagg cttgacatac accggaaagc 960 attagagata gtgcccccct tgtggtcggt gtacaggtgg tgcatggctg tcgtcagctc 1020 gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgtccc gtgttgccag 1080 caggcccttg tggtgctggg gactcacggg agaccgccgg ggtcaactcg gaggaaggtg 1140 gggacgacgt caagtcatca tgccccttat gtcttgggct gcacacgtgc tacaatggcc 1200 ggtacaatga gctgcgatac cgtgaggtgg agcgaatctc aaaaagccgg tctcagttcg 1260 gattggggtc tgcaactcga ccccatgaag tcggagtcgc tagtaatcgc agatcagcat 1320 tgctgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacgtca cgaaagtcgg 1380 taacacccga agccggtggc ccaacccctt gtgggaggga gctgtcgaag gtgggaccag 1440 cgattgggac gaagtcgt 1458

Claims

1. A fermentation method of Streptomyces, characterized in that: The following steps are involved: (1) Activation of Streptomyces; (2) The liquid fermentation of Streptomyces, including the preparation of seed liquid and fermentation of Streptomyces; In the fermentation method, streptomycin is cultured using a sterile solid culture medium; The composition of the sterile solid culture medium is as follows: glucose 0.3-2.0 g, yeast extract 0.3-1.0 g, peptone 0.3-1.0 g, beef extract 0.08-0.30 g, magnesium sulfate 0.02-0.30 g, and dipotassium hydrogen phosphate 0-0.40 g per 100 ml; The fermentation of Streptomyces uses a liquid culture medium; The composition of the liquid culture medium is: 2% glucose, 2% maltose, 5% cottonseed powder, 0.0001% copper sulfate, 0.002% zinc sulfate, 0.0006% manganese sulfate, and 0.00005% magnesium sulfate; The fermentation of Streptomyces also includes adding inorganic phosphate, ammonia scavenger, oxygen carrier during fermentation culture, and adding carbon source on the second day of fermentation culture; The inorganic phosphate is: 12.5 mM disodium hydrogen phosphate dodecahydrate; The ammonia capture agent is: 50mM calcium phosphate and 20mM magnesium phosphate; The oxygen carrier is: 3% soybean oil; The carbon source is: 1% sucrose solution; The Streptomyces is deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration, with the deposit number: CGMCC NO. 19354. Streptomyces sp .) The strain was named WP-1.

2. The fermentation method according to claim 1, characterized in that The fermentation method also includes extraction, purification and identification of active metabolites obtained by fermentation; The extraction of active products from the Streptomyces fermentation broth comprises: The fermentation liquid of Streptomyces is centrifuged to obtain a supernatant and mycelium, and an equal volume of ethyl acetate is added to the supernatant after concentration, and extracted 1 to 5 times at room temperature, and the extracts are combined to obtain an ethyl acetate phase I; the mycelium is soaked in an ethanol aqueous solution and centrifuged, and the process is repeated 1 to 5 times, and the soaking liquids are combined and concentrated until there is no ethanol, and an equal volume of ethyl acetate is extracted 3 times, and the ethyl acetate phase II is combined; the ethyl acetate phases I and II are combined, and concentrated to dryness to obtain a total ethyl acetate extract; The ethyl acetate extract is adsorbed by a macroporous resin, eluted by an ethanol aqueous solution, and the eluate is concentrated under reduced pressure to obtain a yellow paste, which is the crude extract of the Streptomyces fermentation broth; The yellow paste crude extract was dissolved in methanol, separated by silica gel column chromatography, gradient eluted with dichloromethane and methanol solution, and the part with the largest absorption peak was collected, and concentrated under reduced pressure to obtain a preliminary pure product; The preliminary pure product was dissolved in methanol and purified by HPLC. It was determined to be nystatin by electrospray ionization mass spectrometry nuclear magnetic resonance and analysis of the physicochemical properties of the compound.

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