CN113234629B

CN113234629B - Streptomyces cmx-10-19 strain capable of producing natamycin by fermentation and application thereof

Info

Publication number: CN113234629B
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: 2023-03-31
Anticipated expiration: 2041-05-14
Also published as: CN113234629A

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: the method comprises the steps of (1) seed culture to prepare seed liquid, (2) fermentation culture, and (3) extraction of natamycin from fermentation products of fermentation liquor.

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 dominates 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.

Yitamycin (etamycin, viridogrisein) is an unconventional cyclic lipopeptide found in soil and marine streptomyces and belongs to B-type antibiotics in streptograminesVegetable extract ^[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 showed 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, compared to vancomycin, the most commonly used drug in MRSA treatment, it was found that the bacteriostatic action of natamycin was faster than that of vancomycin, but the bactericidal action was much smaller 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. In addition, the compound shows no cytotoxicity to HeLa cells of mammals at the concentration of up to 128mg/L, which shows that the toxicity is low, and the compound has the potential of becoming a therapeutic drug for MRSA infection ^[4] 。

The pentamycin also showed inhibitory activity against other gram-positive bacteria, including Mycobacterium abscessus (MIC 50= 1.8-8.2. Mu.M), mycobacterium avium (MIC =0.023-1.56 mg/L), M.intracellulare (MIC =0.023-1.56 mg/L), streptococcus pyogenes (MIC =8 mg/L) and Streptococcus agalactiae (MIC =8 mg/L), and against vancomycin-resistant enterococcus faecalis (MIC =16 mg/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 =1 mg/L) and Haemophilus influenzae (MIC =16 mg/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 Xiaobing, medical test 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 Amycolatopsissp.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 depository 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, the bacterial suspension is inoculated on an ISP2 culture medium plate and cultured for 6 to 8 days at the temperature of 28 ℃, and the culture time is preferably 7 days;

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

about 2cm in length ² Inoculating the solid culture with the size into ISP2 liquid culture medium, and culturing for 48 hours 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, 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

The M10 liquid culture medium comprises the following formula: 20.0g of glycerin, 10.0g of molasses, 5.0g of casein ₃ 4.0g, peptone 1.0g, sterile water 1.0 liter;

(3) Extracting crude extract of the Yitamycin 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 of the fermentation liquid for two times of elution, merging the effluents, and volatilizing the solvent to obtain a fermentation extract (crude extract) containing the natamycin;

preferably, the amount of methanol used for extracting the fermentation liquid after freeze drying is approximately equal to that of the fermentation liquid in the step (2), and the amount of methanol used for elution is about 1/3 of that 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%; obtaining primary pure Yitamycin;

preferably, the chromatographic Column is RediSep Column,12g C18, the flow rate is 5mL/min, and the elution program 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, 62-100% by weight B; to obtain the Yitamycin;

preferably, the column incubator is 30 ℃ and the semi-preparative chromatography column is dr. Maisch GmbH C18 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) 41min.

The invention has the beneficial effects that:

(1) The application discovers a streptomycete potential new strain cmx-10-19 taking 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 an 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-10-19 1696 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.

Figure 5 Yitamycin precursor ion m/z 879[ 2 ], [ 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 the strain, cmx-10-19, for the natamycin.

FIG. 10, LC-MS spectrogram 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

The strain cmx-10-19 was species-identified based on the 16S rRNA sequence and was determined to belong to the genus 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 an intraspecific genome) each less than the minimum value of the average nucleotide identity within the species of 95% ^[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 ℃, inoculating 100 μ L on ISP2 medium plate, culturing at 28 ℃ for 7 days, then transferring on ISP2 medium plate again, and culturing at 28 ℃ for 7 days. Then about 2cm ² The solid culture of the size was inoculated into 50mL of ISP2 liquid medium and cultured at 220rmp,28 ℃ for 48 hours to obtain a seed liquid.

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. The blank control group replaces the seed solution with an equal amount of blank ISP2 culture medium, and other steps are tested 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 culture 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, 3.0g of CaCO 3.0g and 1.0L of sterile water, and 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 ) ₂ SO ₄ 0.33g，CaCO ₃ 2.0g of sterile water (1.0L), and the pH was adjusted to 7.0 ^[9] ；

(5) M5 culture medium: 10.0g of glucose, 10.0g of beef powder, 1.0g of peptone, 5.0g of NaCl, 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 ) ₂ SO ₄ 7g，CaCO ₃ 2g,1L sterile water;

(7) F1 culture medium: 20g of common starch, 20g of glucose, 3g of peptone, 3g of beef extract ₃ 2.5g, 1mL of trace elements (trace elements preparation/0.1L ₄ 0.1g，MnCl ₂ 0.1g，ZnSO ₄ 0.1g，CuSO ₄ 0.1g，CoCl ₂ 0.1 g), 10g of peanut meal cake and 10L of sterile water, and the pH value is adjusted to 7.2;

(8) M2 culture medium: 40g of mannitol, 40g of malt extract powder, 10g of yeast extract powder ₂ HPO ₄ 2g，MgSO ₄ ·7H ₂ O 0.5g，FeSO ₄ ·7H ₂ O0.01g, 1L sterile water, pH adjusted to 7.2;

(9) Cazpeck medium: k is ₂ HPO ₄ 1g，NaNO ₃ 0.3g，KCl 0.005g，MgSO ₄ ·7H ₂ O 0.005g，FeSO ₄ 0.001g, 30g sucrose, 1L sterile water, pH adjusted to 7.0;

(10) M10 medium: 20.0g of glycerin, 10.0g of molasses, 5.0g of casein ₃ 4.0g, 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 ₂ HPO ₄ 5.0g，NaCl 2.5g，ZnSO ₄ 0.04g，CaCO ₃ 0.4g of 1.0L sterile water, and the pH value is 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 using 900 mu L/Kong Jiachun with shaking, centrifuging for 10 minutes at 4500rmp, respectively loading the supernatant of each hole to an ODS solid phase extraction column (100 mg ODS/column), eluting by using 300 mu L/column methanol, then sucking 300 mu L of effluent, volatilizing the solvent, redissolving in 20 mu L DMSO, and using for antibacterial activity determination and metabolite analysis.

3. 0.5MCF (Mycoplasma turbidimetric Unit) (1-2X 10) using MHB medium ⁸ CFU/mL) of Staphylococcus aureus suspension to 5X 10 ⁵ CFU/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 broth) were added to 96-well plates at 100. Mu.L/well. Then, 1. Mu.L of the sample was added, incubated at 30 ℃ for 24 hours, and the inhibition ratio was calculated by reading the OD value of the activity result with a microplate reader.

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 100 mm), 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 to 9.5min,0% by weight B to 100% by weight B;9.5min-11.0min,100 percent; 11.0-11.5min, 100% B-0%B;11.5min-13min,0% by weight 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 show that the addition ions m/z 879[ deg. ] M + H ] + and 901[ deg. ] M + Na ] +, rt =7.6min of the Yitamycin were detected in the 8 fermented extracts. Wherein, the natamycin is detected in the M2, M3, M4, M6, M9, M10, M11 and M12 fermentation extracts, wherein the content of M10 liquid fermentation extract is high, and other products are relatively less, therefore, M10 is selected as the optimal fermentation medium for the investigation of stability and optimal fermentation time.

Example 3: structure confirmation of Yitamycin

High resolution mass spectrometry gives the m/z of the target compound of 879.4567[ 2 ], [ M + H ]] ⁺ (calcd for C ₄₄ H ₆₃ N ₈ O ₁₁ 879.4616), therefore molecular weight 878.4539, molecular formula C ₄₄ H ₆₂ N ₈ O ₁₁ The 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 spectrum of the hydrogenating ion (m/z 879) produces the target compoundThe ion peaks of amino acid fragments of (1) include m/z 74 (L-threonine), 86 (4-hydroxy-D-proline), 86 (D-leucine), 114 (N, β -dimethyl-L-leucine), 120 (L-phenysteine), 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 culture condition rescreening for producing Yitamycin

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 of the M10 liquid culture medium were fermented in 500mL glass Erlenmeyer flasks, and 100mL of the M10 liquid culture medium was added to each bottle in an inoculum size of 5mL. After culturing at 28 ℃ and 220rpm for 8 days, 0.8mL of the fermentation product per bottle was placed in a 96-well plate and the subsequent treatment was carried out in the same manner as in 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 2.5mL.

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

day

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

After freeze-drying the liquid fermentation product, extracting with 2 mL/Kong Jiachun for 30 minutes under shaking, centrifuging at 4500rmp for 10 minutes, filtering with 0.2 μm filter membrane, and analyzing with LC-MS.

The 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. Mu.L sample size, with 3 replicates per sample. Chromatographic conditions are as follows: the chromatographic column is

C18column (2.7 μm, 2.1X 50 mm), 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% by volume B;4.5min-5min,56% B-100%; 5min-6.5min,100% by weight B;6.5min-7.0min,100% by weight, B-56% by weight; 7.0min-9.0min 56% by weight. 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 taper hole voltage of the sample is 120V.

LC-MS data showed the presence of natamycin in the fermented extract from 6 to 10 days with 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 an optimal shake flask fermentation time of 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 performing primary separation on the combined sample by using HW-40C resin, using methanol as an eluent and using MRSA as an identification bacterium to obtain an active component Fr.1. Fraction Fr.1 was separated using Flash reverse chromatography on a RediSep Column,12g C18, cat No: SO130012-0 with flow rate of 5mL/min, using water (A) and acetonitrile (B) as mobile phases, both phases were added with 0.1% formic acid to improve the chromatographic behavior, with elution conditions: 0min-10min,50% B;10min-15min,50% by weight B-60% by weight B;15min-25min,60% B;25min-30min,60% by weight B-70% by weight B;30min-40min,70% by weight B;40min-45min,70% by weight B-100% by weight B;45min-50min,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 C18 μm 250 × 10mm, water (a) and acetonitrile (B) as mobile phases, 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, 62-100% by weight B;50min-60min,100 percent B;60min-65min,100% B-62% B, rt =41 min, yielding 7.2mg of the natamycin.

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 bacterial suspension on an ISP2 culture medium plate, and culturing for 6-8 days at 28 ℃;

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

2cm in length ² Inoculating 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 an M10 liquid culture medium, and the formula of the culture medium is as follows: 20.0g of glycerin, 10.0g of molasses, 5.0g of casein ₃ 4.0g, peptone 1.0g, sterile water 1.0 liter;

(3) Extracting crude extract of the natamycin from fermentation liquor, 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 elution twice, merging the effluents and volatilizing the solvent to obtain crude extract containing the Yitamycin;

(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, a Flash reverse chromatography is used for separating a crude extract of the natamycin,

elution was performed using a gradient elution method using water as phase a and acetonitrile as phase B, with the following elution procedure: 0-50min, 50-100% by weight B; to obtain primary pure Yitamycin;

the initially pure natamycin is then separated using HPLC,

water was used as phase a and acetonitrile as phase B, both phases were supplemented with 0.1% formic acid to improve the chromatographic behavior at a flow rate of 2.5mL/min, the elution procedure was: 0-50min, 62-100% by weight 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 of each culture on the ISP2 culture medium plate is 7 days, and the time of each culture in the liquid culture medium is 48 hours.

5. The method according to claim 3 or 4,

in the fermentation culture of 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 claim 3 or 4,

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

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

7. The method of claim 3 or 4,

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 a Flash reverse chromatography was 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 RediSep Column,12g C18, flow rate 5mL/min, elution procedure was:

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。

8. the method according to claim 3 or 4,

in the step (4) of refining crude extract of natamycin by reverse phase chromatography cascade semi-preparative HPLC to obtain natamycin, when primary pure natamycin is separated by HPLC,

a semi-preparative chromatographic column is Dr.Maisch GmbH C18 μm 250 × 10mm, a column incubator is 30 ℃,

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 retention time (Rt) of the natamycin peak is: and 41min.