CN113234629A

CN113234629A - Streptomyces cmx-10-19 strain capable of producing natamycin by fermentation and application thereof

Info

Publication number: CN113234629A
Application number: CN202110528537.5A
Authority: CN
Inventors: 解云英; 何宁; 洪斌; 陈铭旭; 常珊珊
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2021-05-14
Filing date: 2021-05-14
Publication date: 2021-08-10
Anticipated expiration: 2041-05-14
Also published as: CN113234629B

Abstract

The invention relates to a streptomycete cmx-10-19(Streptomyces sp. cmx-10-19) strain capable of producing liitamycin by fermentation and application thereof, wherein the strain is preserved in China center for type culture Collection with the preservation number as follows: CCTCC M2021489. The invention also relates to a method for producing the natamycin by using the streptomycete cmx-10-19 strain through fermentation, which comprises the following steps: (1) seed culture to prepare seed liquid, (2) fermentation culture, and (3) extracting natamycin from fermentation liquid.

Description

Streptomyces cmx-10-19 strain capable of producing natamycin by fermentation and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a streptomycete cmx-10-19 strain capable of producing natamycin through fermentation and an application thereof.

Background

Since the first methicillin-resistant staphylococcus aureus (MRSA) infection disease was discovered in 1961, MRSA predominated in hospital and community-acquired infections. In recent years, MRSA infection has increased dramatically worldwide, posing a great threat to human health^[1]. Therefore, there is a great clinical need for drugs for the treatment of MRSA infections.

Yitacmycin (etamycin, viridogrisein) is an unconventional cyclic lipopeptide found in soil and marine streptomyces and belongs to the B-type antibiotics of streptogramins^[2]. It is composed of eight amino acids, namely 3-hydroxypyridic acid, L-threonine, D-leucine, 4-hydroxy-D-proline, sarcosine, N, beta-dimethyl-L-leucine, L-alanine and L-phenylsarcosine, and contains 5 unnatural amino acids. With the isolation of Yitadycin in 1954, the 4 amino acids 3-hydroxypyridic acid, 4-hydroxy-D-proline, L-phencyclamic acid and N, beta-dimethyl-L-leucine were first found in nature^[3]。

The natamycin has better in-vivo and in-vitro inhibitory activity on MRSA. In vitro experiments show that^[4]Which exhibit good antibacterial activity against a group of clinically relevant MRSA strains, including CA-MRSA strains (community-associated MRSA strains) and some HA-MRSA strains (hospital-associated MRSA strains) (MIC 1-2 mg/L), and HA-MRSA strains ATCC 33591 and Sanger 252(MIC 8-16 mg/L), and found that, compared to vancomycin, the most commonly used drug in MRSA treatment, the antibiotic effect of natamycin is faster than that of vancomycin, but the bactericidal effect is much less than that of vancomycin^[4]. The evaluation of the in vivo efficacy shows^[4]The betamycin has obvious protective effect on mice infected with MRSA, and the death rate is reduced by 55% in a monitoring period of 72 h. Furthermore, it did not show up to 128mg/L concentration for mammalian HeLa cellsShows low toxicity by presenting cell toxicity, and has the potential of becoming a medicament for treating MRSA infection^[4]。

The Yitamycin also showed inhibitory activity against other gram-positive bacteria, including Mycobacterium abscessus (MIC50 ═ 1.8-8.2. mu.M), Mycobacterium avium (MIC ═ 0.023-1.56 mg/L), Mycobacterium intracellulare (MIC ═ 0.023-1.56 mg/L), Streptococcus pyogenes (MIC ═ 8mg/L) and Streptococcus agalactiae (MIC ═ 8mg/L), and against vancomycin-resistant enterococcus faecalis (MIC ═ 16mg/L)^[4-6]. It is noteworthy that in vivo efficacy of the betamycin is superior to that of the core drug clarithromycin for the treatment of mycobacterium abscesses infection in a zebrafish infection model, and is a potential candidate drug^[6]. In addition, its antibacterial spectrum also extends to gram-negative bacteria and respiratory pathogens Moraxella catarrhalis (MIC ═ 1mg/L) and Haemophilus influenzae (MIC ═ 16mg/L)^[4]。

In conclusion, the Yitamycin has inhibition effect on various pathogens, especially MRSA and mycobacterium abscessus, and has low cytotoxicity, thus being a lead compound with great development prospect.

[ REFERENCE ] to

[1] Wang soldiers, medical examination and clinical week J. Staphylococcus aureus infection status analysis [ J ] 2013, (5):40-42,91.

[2]Xie Y,Wang B,Liu J,et al.Identification of the biosynthetic gene cluster and regulatory cascade for the synergistic antibacterial antibiotics griseoviridin and viridogrisein in Streptomyces griseoviridis[J].Chembiochem, 2012,13(18):2745-2757.

[3]Sheehan J C,Zachau H G,Lawson W B.The Structure of Etamycin[J].Journal of the American Chemical Society,1958,80(13):3349-3355.

[4]Haste N M,Perera V R,Maloney K N,et al.Activity of the streptogramin antibiotic etamycin against methicillin-resistant Staphylococcus aureus[J].J Antibiot(Tokyo),2010,63(5):219-224.

[5]Hosoda K,Koyama N,Kanamoto A,et al.Discovery of Nosiheptide,Griseoviridin,and Etamycin as Potent Anti-Mycobacterial Agents against Mycobacterium avium Complex[J].Molecules,2019,24(8).

[6]Hanh B T B,Kim T H,Park J W,et al.Etamycin as a Novel Mycobacterium abscessus Inhibitor[J].Int J Mol Sci,2020,21(18).

[7]Yoon S H,Ha S M,Lim J,et al.A large-scale evaluation of algorithms to calculate average nucleotide identity[J].Antonie Van Leeuwenhoek,2017,110(10):1281-1286.

[8]Nakashima T,Kimura T,Miyano R,et al.Nanaomycin H:A new nanaomycin analog[J].J Biosci Bioeng, 2017,123(6):765-770.

[9]Hashizume H,Sawa R,Yamashita K,et al.Structure and antibacterial activities of new cyclic peptide antibiotics,pargamicins B,C and D,from Amycolatopsis sp.ML1-hF4[J].J Antibiot(Tokyo),2017,70(5): 699-704.

[10]Guo Z K,Liu S B,Jiao R H,et al.Angucyclines from an insect-derived actinobacterium Amycolatopsis sp.HCa1 and their cytotoxic activity[J].Bioorg Med Chem Lett,2012,22(24):7490-7493.

[11]Serrill J D,Tan M,Fotso S,et al.Apoptolidins A and C activate AMPK in metabolically sensitive cell types and are mechanistically distinct from oligomycin A[J].Biochem Pharmacol,2015,93(3):251-265.

[12]Dasari V R,Muthyala M K,Nikku M Y,et al.Novel Pyridinium compound from marine actinomycete, Amycolatopsis alba var.nov.DVR D4 showing antimicrobial and cytotoxic activities in vitro[J].Microbiol Res, 2012,167(6):346-351.

Disclosure of Invention

The invention firstly relates to a streptomycete cmx-10-19 strain capable of producing the liitamycin by fermentation, wherein the strain is preserved in China center for type culture Collection with the preservation date of 2021, No. 4 and No. 29, and the biological classification is as follows: streptomyces cmx-10-19(Streptomyces sp. cmx-10-19) with the deposition number: CCTCC M2021489; the address of the preservation unit is as follows: wuhan university, post code: 430072, telephone: (027) -68754052.

The invention also relates to the application of the cmx-10-19 strain in fermentation production of the natamycin.

The invention also relates to a method for producing natamycin by fermentation, comprising the following steps:

(1) seed culture is carried out to prepare seed liquid, and the steps are as follows:

after the streptomycete cmx-10-19 bacterial suspension is melted, inoculating the melted streptomycete cmx-10-19 bacterial suspension on an ISP2 culture medium plate, and culturing for 6-8 days at 28 ℃, preferably, for 7 days;

culturing again on an ISP2 culture medium plate at 28 ℃ for 6-8 days after passage, preferably, the culture time is 7 days;

about 2cm in length²Inoculating the solid culture with the size into ISP2 liquid culture medium, and culturing at 28 ℃ for 48 hours to obtain seed liquid;

(2) fermentation culture, which comprises the following steps:

mixing the seed liquid and a fermentation medium according to the ratio of 1: 15-25, preferably, the ratio of the seed liquid to the fermentation medium is 1: 20 (volume ratio);

culturing for 6-10 days at 28 ℃, preferably for 9 days;

the culture medium is

M10 liquid culture medium, its formulation is: 20.0g of glycerin, 10.0g of molasses, 5.0g of casein, CaCO₃4.0g, peptone 1.0g, sterile water 1.0 liter;

(3) extracting crude extract of the natamycin from fermentation liquor, which comprises the following steps:

freeze drying the liquid fermented liquid and extracting with methanol;

after centrifugation, the supernatant is loaded on an ODS solid phase extraction plate, and solid residues (mycelia and the like) are discarded;

then adding methanol with the volume of 1/3 fermentation liquor for two times of elution, merging effluents, and volatilizing the solvent to obtain a fermentation extract (crude extract) containing the natamycin;

preferably, the amount of methanol used for extracting the freeze-dried fermentation broth is about equal to the amount of the fermentation broth of step (2), and the amount of methanol used for elution is about 1/3 of the amount of the methanol used for extraction;

preferably, the centrifugation parameter is 4500rpm centrifugation for 10 minutes.

(4) Refining crude extract of natamycin by reversed phase chromatography cascade semi-preparative HPLC to obtain natamycin

Firstly, separating a crude extract of the natamycin by Flash reverse chromatography, using water (A) and acetonitrile (B) as mobile phases, adding 0.1 percent formic acid into the two phases to improve the chromatographic behavior, and eluting by a gradient elution method, wherein the elution procedure is as follows: 0-50min, 50% B-100% B; obtaining primary pure Yitamycin;

preferably, the chromatographic Column is RediSep Column, 12g C18, the flow rate is 5mL/min, and the elution procedure is as follows:

0min-10min,50％B；

10min-15min,50％B-60％B；

15min-25min,60％B；

25min-30min,60％B-70％B；

30min-40min,70％B；

40min-45min,70％B-100％B；

45min-50min,100％B；

then the primary pure natamycin was separated using HPLC using water (a) and acetonitrile (B) as mobile phase, both phases added with 0.1% formic acid to improve the chromatographic behavior, flow rate 2.5mL/min, elution procedure: 0-50min, 62% B-100% B; to obtain the Yitamycin;

preferably, the column incubator is 30 ℃ and the semi-preparative chromatography column is dr. maisch GmbH C185 μm 250 × 10mm,

the elution procedure was:

0min-45min,62％B；

45min-50min,62％B-100％B；

50min-60min,100％B；

60min-65min,100％B-62％B；

the peak retention time (Rt) of the active substance, natamycin, is: and (4) 41 min.

The invention has the beneficial effects that:

(1) the application discovers a streptomycete potential new strain cmx-10-19 which takes the natamycin as a main fermentation extract by screening the inhibitory activity of the staphylococcus aureus.

(2) The main fermentation extract of the strain cmx-10-19 is confirmed to be the natamycin through mass spectrum and bioinformatics;

(3) further screening the conditions of the target strain cmx-10-19 for fermenting the natamycin, determining that the fermentation culture medium with the most yield of the natamycin is M10 liquid culture medium, amplifying the fermentation and re-screening, and having good reproducibility.

(4) According to the research of relative yield of fermentation, the yield is determined to be the highest at 9 days of fermentation.

In conclusion, the strain cmx-10-19 can stably produce the betamycin with patent drug value, and other products are relatively few, so that the strain has the potential of further industrial application and commercial transformation.

Drawings

FIG. 1, phylogenetic tree based on the sequence of strain cmx-10-1916S rRNA.

FIG. 2, phylogenetic tree based on strain cmx-10-19 multi-site sequence typing.

FIG. 3 shows the inhibitory rate of the fermentation extract of strain cmx-10-19 against Staphylococcus aureus.

FIG. 4 shows LC-MS spectra of the extract obtained by fermentation with strain cmx-10-19.

FIG. 5, the Yitamycin precursor ion M/z 879[ M + H ]]⁺MS/MS spectrogram and analysis.

FIG. 6, the Yitamycin precursor ion M/z 901[ M + Na ]]⁺MS/MS spectrum of (1).

FIG. 7, chemical structure of the natamycin.

FIG. 8, the natamycin biosynthesis gene cluster.

FIG. 9 shows the biosynthesis pathway of natamycin in strain cmx-10-19.

FIG. 10, LC-MS spectrum of strain cmx-10-19 rescreening.

FIG. 11 is a graph of the relative yield of natamycin versus fermentation time.

Detailed Description

Example 1: identification of potential new species of streptomycete strain cmx-10-19

Species identification is carried out on the strain cmx-10-19 based on 16S rRNA sequence, and the species is determinedIn Streptomyces (FIG. 1). Phylogenetically analyzed evolutionary relationships based on multi-site sequence typing (http:// automlst. ziemertlab. com), the results indicated that the Streptomyces strain, cmx-10-19, may be a potential new species (FIG. 2). The ANI (average nucleotide identity) values of 5 model strains having the strain cmx-10-19 in close evolutionary relationship were 89.9% (Streptomyces violacea NRRL-16381), 89.5% (Streptomyces ambofaciens ATCC 23877), 83.7% (Streptomyces calensis NRRL B-24567), 83.4% (Streptomyces calensis NRRL B-245667) and 83.0% (Streptomyces virdowochromogenes DSM 40736) (model strains having no available genomic information were replaced with strains having available genomes in their species), respectively, which were less than 95% of the minimum average nucleotide identity within the species^[7]The streptomyces strain cmx-10-19 is indicated as a potential new species.

Example 2: streptomyces strain cmx-10-19 culture condition screening for producing natamycin

1. After thawing the bacterial suspension of Streptomyces cmx-10-19 cryopreserved at-80 ℃, 100. mu.L of the bacterial suspension was inoculated on an ISP2 medium plate, cultured at 28 ℃ for 7 days, then transferred on an ISP2 medium plate again, and cultured at 28 ℃ for 7 days. Then about 2cm²The solid culture of the size was inoculated into 50mL of ISP2 liquid medium and cultured at 220rmp at 28 ℃ for 48 hours to obtain a seed solution.

The strain cmx-10-19 was fermented using 12 media in a microplate method. Each well was inoculated with 800. mu.L of medium, 40. mu.L. The liquid fermentation was cultured at 28 ℃ for 8 days with shaking at 900 rpm/min. Blank control group seed solution was replaced with equal amount of blank ISP2 medium and other steps were run in parallel.

The composition of 12 culture media:

(1) PDB culture medium: 15g of glucose, 5g of potato extract powder, 10g of peptone, 5g of NaCl and 1L of sterile water;

(2) ISP2 medium: 4g of yeast extract powder, 4g of glucose, 10g of maltose and 1L of sterile water, and adjusting the pH value to 7.2;

(3) m3 medium: 20.0g of soluble starch, 5.0g of glycerol, 10.0g of defatted wheat germ, 3.0g of meat extract, 3.0g of dry yeast, 33.0 g of CaCO, 1.0L of sterile water,adjusting the pH to 7.0^[8]；

(4) M4 medium: galactose 3.3g, dextrin 3.3g, glycerol 1.7g, soytone 1.7g, corn steep liquor 0.83g, (NH)₄)₂SO₄ 0.33 g，CaCO₃2.0g of 1.0L of sterile water, pH adjusted to 7.0^[9]；

(5) M5 medium: 10.0g of glucose, 10.0g of beef powder, 1.0g of peptone, 5.0g of NaCl and 1.0L of sterile water, and adjusting the pH to 7.0^[10]；

(6) M331 medium: 20g of glucose, 5g of common starch, 6g of peptone (NH)₄)₂SO₄ 7g，CaCO₃2g, 1L of sterile water;

(7) f1 medium: 20g of common starch, 20g of glucose, 3g of peptone, 3g of beef extract and CaCO₃2.5g, 1mL of trace elements (trace elements preparation/0.1L: FeSO)₄ 0.1g，MnCl₂ 0.1g，ZnSO₄0.1g of CuSO, 0.1g of CoCl, 0.1g of peanut powder cake and 1L of sterile water, and the pH value is adjusted to 7.2;

(8) m2 medium: 40g of mannitol, 40g of malt extract powder, 10g of yeast extract powder and K₂HPO₄ 2g，MgSO₄·7H₂O 0.5g， FeSO₄·7H₂O0.01 g, 1L of sterile water, and adjusting the pH value to 7.2;

(9) cazpeck medium: k₂HPO₄ 1g，NaNO₃ 0.3g，KCl 0.005g，MgSO₄·7H₂O 0.005g，FeSO₄0.001g, 30g of cane sugar and 1L of sterile water, and adjusting the pH value to 7.0;

(10) m10 medium: 20.0g of glycerin, 10.0g of molasses, 5.0g of casein, CaCO₃4.0g, peptone 1.0g, sterile water 1.0 liter^[11]；

(11) M11 medium: glucose 20.0g, malt extract powder 40.0g, yeast extract powder 4.0g, K₂HPO₄ 5.0g，NaCl 2.5g， ZnSO₄ 0.04g，CaCO₃0.4g of 1.0L of sterile water, pH adjusted to 6.0^[12]；

(12) YMS medium: 4g of yeast extract powder, 10g of malt extract powder, 4g of soluble starch and 1L of sterile water.

2. Freeze-drying the liquid fermentation product obtained in the step 1, extracting for 30 minutes by shaking with 900 mu L/hole methanol, centrifuging for 10 minutes at 4500rmp, loading the supernatant of each hole to an ODS solid phase extraction column (100mg ODS/column), eluting with 300 mu L/column methanol, absorbing 300 mu L of effluent, volatilizing the solvent, and redissolving in 20 mu L DMSO for antibacterial activity determination and metabolite analysis.

3. 0.5MCF (Mycoplasma turbidimetric Unit) (1-2X 10) using MHB medium⁸CFU/mL) of Staphylococcus aureus suspension to 5X 10⁵CFU/mL. MHB medium composition: 2.0g of beef powder, 17.5g of acid hydrolyzed casein, 1.5g of soluble starch and 1.0L of sterile water, and the pH value is 7.2. Staphylococcus aureus suspension dilutions (assay bacteria) were added to 96-well plates at 100. mu.L/well. Then, 1. mu.L of the sample was added, cultured at 30 ℃ for 24 hours, and the OD value of the activity result was read by a microplate reader to calculate the inhibition rate.

Several fermented extracts showed strong inhibitory activity against staphylococcus aureus, with 4 of them showing an inhibition rate of more than 80% (fig. 3).

4. Fermentation extracts were analyzed using a Waters ACQUITY UPLC H-Class chromatography system in tandem with a Waters Xevo G2-XS QTof mass spectrometer. Chromatographic conditions are as follows: the chromatographic column is ACQUITY

CSHTM C18(1.7 μm, 2.1X 100mm), flow rate of 0.3mL/min, column oven 30 ℃; 10% acetonitrile (A) and acetonitrile (B) were used as mobile phases, with 0.1% formic acid added to both phases to improve the chromatographic behavior, under the following elution conditions: 0min-9.5min, 0% B-100% B; 9.5min-11.0min, 100% B; 11.0min-11.5min, 100% B-0% B; 11.5-13 min, 0% B. Mass spectrum conditions: fast DDA and a cation collection mode, and leucine-enkephalin is used as an internal reference; the scanning ranges of MS and MS/MS are respectively m/z 200-2000 and m/z 50-2000, and the acquisition time is 0.0-12.0 minutes; the desolvation temperature is 450.0 ℃, the desolvation gas flow rate is 800.0L/H, the taper hole gas flow rate is 30.0L/H, the capillary voltage is 3kV, and the sample taper hole voltage is 120V.

The results showed that in 8 fermentation extracts the addition ions M/z 879[ M + H ] + and 901[ M + Na ] +, Rt 7.6 min of the natamycin were detected. Among them, the natamycin was detected in the M2, M3, M4, M6, M9, M10, M11 and M12 fermented extracts, wherein the M10 liquid fermented extract was high in content and the other products were relatively less, so M10 was selected as the optimal fermentation medium for stability and optimal fermentation time examination.

Example 3: structure confirmation of Yitamycin

High resolution mass spectrometry gives the M/z of the target compound 879.4567[ M + H [ ]]⁺(calcd for C₄₄H₆₃N₈O₁₁879.4616), thus the molecular weight is 878.4539 and the molecular formula is C₄₄H₆₂N₈O₁₁The unsaturation degree was 14. Under different collision energies, MS/MS spectrums of hydrogenation (m/z 879) and sodium ion addition (m/z 901) are respectively obtained. Wherein the MS/MS spectra of the hydride ion (m/z 879) produced the ion peaks of amino acid fragments in the target compound, including m/z 74 (L-threonine), 86 (4-hydroxy-D-proline), 86 (D-leucine), 114(N, β -dimethyl-L-leucine), 120 (L-phenylsarcosine), and 122 (3-hydroxypyridinic acid) (FIG. 5). The MS/MS spectrum of the sodium ions (m/z 901) produces fragment ions of the sequential fragmentation products in the target compound, including two series of fragment ions: m/

z

901, 754, 683, 542, 471, 358 and m/

z

755, 633, 544, 403, 332, 350 (FIG. 6), it can be inferred from the mass difference of their adjacent fragment ions that the linking order of the residues is: l-phenylsarcosine (147) -L-alanine (71) -N, β -dimethyl-L-leucine (141) -sarcosine (71) -4-hydroxy-D-proline (113) and 3-hydroxypyridinic acid (122) -L-alanine (71) -water (18) -N, β -dimethyl-L-leucine (141) -sarcosine (71) -water (18), confirming the attachment sequence of 3-hydroxypyridinic acid to the exocyclic and L-phenylsarcosine-L-alanine-N, β -dimethyl-L-leucine-sarcosine-4-hydroxy-D-proline. The above mass spectra show structural features consistent with the chemical structure of the natamycin (fig. 7).

Furthermore, the complete biosynthesis gene cluster of the natamycin was found in the genome of the strain cmx-10-19^[2]It is located in region25.1, of the NRPS type (fig. 8 and 9), which further confirms its structure.

In conclusion, the target compound, i.e. the natamycin, is determined by combining the mass spectrum characteristics and the biosynthesis gene cluster.

Example 4: streptomyces strain cmx-10-19 for producing natamycin culture condition re-screening

The procedure from recovery of the seed culture to seed liquid was as in example 2.

The fermentation method comprises the following steps: 3 bottles were fermented using 500mL glass Erlenmeyer flasks, to which 100mL of M10 liquid medium was added, in an inoculum size of 5 mL. After 8 days of incubation at 28 ℃ and 220rpm, 0.8mL of fermentation product per bottle was placed in a 96-well plate and the subsequent processing was carried out in accordance with the method of example 2.

The rescreening results showed that Streptomyces cmx-10-19 could stably produce natamycin (FIG. 10).

Example 5: investigation of optimal fermentation time for Streptomyces cmx-10-19 to produce natamycin

The fermentation method comprises the following steps: a250 mL glass Erlenmeyer flask was used to ferment 15 flasks, 50mL of medium was added to each flask, and the inoculum size was 2.5 mL.

The culture was carried out at 28 ℃ and 220 rpm. From day 6 to

day

10, 3 bottles were taken daily, and 1mL of fermentation product was taken from each bottle into a 96-deep well plate.

Freeze-drying the liquid fermentation product, extracting with 2 mL/hole methanol for 30min, centrifuging at 4500rmp for 10min, filtering with 0.2 μm filter membrane, and analyzing with LC-MS.

Fermentation extracts were analyzed using a Waters ACQUITY UPLC H-Class chromatography system in tandem with a Waters Xevo G2-XS QTof mass spectrometer in a 1.0 μ L aliquot and repeated 3 times for each aliquot. Chromatographic conditions are as follows: the chromatographic column is

C18 column (2.7 μm,2.1 × 50mm), flow rate of 0.3mL/min, column oven of 30 deg.C; water (a) and acetonitrile (B) were used as mobile phases, with 0.1% formic acid added to both phases to improve the chromatographic behavior, under the following elution conditions: 0min-4.5min, 56% B; 4.5min-5min, 56% B-100% B; 5min-6.5min100% B; 6.5min-7.0min, 100% B-56% B; 7.0min-9.0min 56% B. Mass spectrum conditions: fast DDA and a cation collection mode, and leucine-enkephalin is used as an internal reference; the scanning ranges of MS and MS/MS are respectively m/z 200-2000 and m/z 800-1000, and the acquisition time is 2.0-5.0 minutes; the desolvation temperature is 450.0 ℃, the desolvation gas flow rate is 800.0L/H, the taper hole gas flow rate is 30.0L/H, the capillary voltage is 3kV, and the sample taper hole voltage is 120V.

LC-MS data showed the presence of betamycin in the fermented extract for 6 to 10 days, Rt 3.3 min. The extraction peak areas were integrated, and a line graph (FIG. 11) was constructed using the average values as a function of the relative yields versus the number of days of fermentation, and the results showed that the yield of natamycin rose overall from day 6 to day 9, the yield was the highest at day 9, and declined at day 10. Thus indicating that the optimal flask fermentation time is 9 days.

Example 6: preparation of natamycin

The 750mL fermentation broths from example 5 in 15 flasks were combined, centrifuged at 4500rmp for 10min, and the supernatant was separated from the mycelia. The mycelium was extracted 3 times with 100mL methanol by sonication, combined with the supernatant and the solvent was evaporated under reduced pressure.

And (3) carrying out primary separation on the combined samples by using HW-40C resin, using methanol as an eluent and using MRSA as an identifying bacterium to obtain an active component Fr.1. Flash reverse chromatography was used to separate fraction fr.1, Column RediSep Column, 12g C18, cat No: SO130012-0, flow rate 5mL/min, using water (A) and acetonitrile (B) as mobile phase, both phases added 0.1% formic acid to improve the chromatographic behavior, the elution conditions were: 0min-10min, 50% B; 10min-15min, 50% B-60% B; 15min-25min, 60% B; 25min-30min, 60% B-70% B; 30-40 min, 70% B; 40min-45min, 70% B-100% B; 45-50 min, 100% B, to obtain active component Fr.1-1.

Fraction fr.1-1 was separated using HPLC with a semi-preparative column dr. maisch GmbH C185 μm 250 × 10mm, water (a) and acetonitrile (B) as mobile phase, 0.1% formic acid was added to both phases to improve the chromatographic behavior, flow rate 2.5mL/min, column oven 30 ℃, elution conditions: 0min-45min, 62% B; 45min-50min, 62% B-100% B; 50min-60min, 100% B; 60min-65min, 100% B-62% B, Rt 41min to give 7.2mg of ethacrycin.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to limit the protection scope of the present invention.

Claims

1. A streptomycete cmx-10-19 strain capable of producing the natamycin by fermentation is preserved in China center for type culture Collection with the preservation number: CCTCC M2021489.

2. Use of the strain cmx-10-19 according to claim 1 for the fermentative production of natamycin.

3. A process for the fermentative production of a natamycin using the streptomyces cmx-10-19 strain according to claim 1, comprising the following steps:

after the streptomycete cmx-10-19 bacterial suspension is melted, inoculating the melted streptomycete cmx-10-19 bacterial suspension on an ISP2 culture medium plate, and culturing for 6-8 days at 28 ℃;

culturing for 6-8 days at 28 ℃ on an ISP2 culture medium plate after passage;

about 2cm in length²Inoculating the solid culture with the size into ISP2 liquid culture medium, and culturing at 28 ℃ to obtain seed liquid;

(2) fermentation culture, which comprises the following steps:

mixing the seed liquid and a fermentation medium according to the ratio of 1: 15-25, inoculating;

culturing for 6-10 days at 28 ℃;

the culture medium is M10 liquid culture medium, and the formula is as follows: 20.0g of glycerin, 10.0g of molasses, 5.0g of casein, CaCO₃4.0g, peptone 1.0g, sterile water 1.0 liter;

(3) extracting crude extract of the natamycin from fermentation liquor or the medium, which comprises the following steps:

after the liquid fermentation liquor is frozen and dried, firstly, methanol is used for extraction;

centrifuging the extract, and then loading the supernatant to an ODS solid phase extraction plate;

then adding methanol for two times of elution, merging effluents, and volatilizing the solvent to obtain crude extract containing the natamycin;

(4) refining a crude extract of the natamycin by reverse phase chromatography cascade semi-preparative HPLC to obtain the natamycin, which comprises the following steps:

firstly, separating a crude extract of the natamycin by Flash reverse chromatography, using water (A) and acetonitrile (B) as mobile phases, and eluting by a gradient elution method, wherein the elution procedure is as follows: 0-50min, 50% B-100% B; obtaining primary pure Yitamycin;

then the primary pure natamycin was separated using HPLC using water (a) and acetonitrile (B) as mobile phase, both phases added with 0.1% formic acid to improve the chromatographic behavior, flow rate 2.5mL/min, elution procedure: 0-50min, 62% B-100% B; to obtain the betamycin.

4. The method of claim 3,

in the step of preparing the seed solution in the step (1), the time for culturing on the ISP2 culture medium plate is 7 days each time, and the time for culturing in the liquid culture medium is 48 hours.

5. The method according to claim 3 or 4,

in the fermentation culture in the step (2),

the proportion of the seed liquid to the fermentation medium is 1: 20, the fermentation culture time is 9 days.

6. The method according to any one of claims 3 to 5,

in the step of extracting the natamycin from the fermentation broth in step (3),

the amount of methanol used for extracting the fermentation liquor after freeze drying is about equal to that of the fermentation liquor in the step (2), and the amount of methanol used for elution is about 1/3 of the amount of methanol used for extraction; the centrifugation parameters were 4500rpm for 10 minutes.

7. The method according to any one of claims 3 to 5,

refining crude extract of the natamycin by the reversed phase chromatography cascade semi-preparative HPLC described in the step (4) to obtain the natamycin,

when Flash reverse chromatography is used to separate the crude extract of the natamycin,

0.1% formic acid was added to both phases to improve the chromatographic behavior, the Column was a RediSep Column, 12g C18, flow rate 5mL/min, elution procedure was: