CN110172415A - A kind of arctic nocardia and its tunning optimization method - Google Patents

Abstract

The invention discloses Nocardiopsis sp. Y469 and its fermentation and cultivation method, belonging to the field of biotechnology. The arctic Nocardiopsis sp.Y469 was isolated from the sediments of the 3,700-meter seabed of the Arctic Ocean. The strain was deposited in the Chinese Type Culture Collection Center of Wuhan, Wuhan University, China on August 13, 2018, and its preservation number is It is CCTCC NO: M 2018536. The present invention also provides a fermentation and cultivation method for Nocardiopsis sp.Y469 in the arctic. By comparing fermentation conditions such as different fermentation media, optimizing the fermentation conditions that can significantly increase the diversity of secondary metabolites is helpful to fully explore this The potential of the strain to produce new secondary metabolites.

Description

A kind of arctic Nocardia pseudocardiae and its fermentation product optimization method

technical field

The invention relates to the field of biotechnology, in particular to Nocardiopsis sp. Y469 bacterial strain and its fermentation process under five different medium culture conditions.

Background technique

The polar region has extreme environmental factors such as extreme low temperature, dryness, strong ultraviolet radiation, and high salinity, and has formed a unique ecosystem due to biogeographical isolation. Polar microorganisms have physiological, biochemical, genetic and metabolic characteristics to adapt to extreme environments, and are an important source of novel active substances. Among them, polar marine actinomycetes are important resources for mining new drug lead compounds and new antibiotics.

Nocardiopsis (Nocardiopsis) is a genus of Nocardiopsis in Actinomycetes Actinomycetes Actinomycetes Actinobacteriaceae, due to its ecological diversity and ability to produce rich biologically active secondary metabolites received widespread attention. Nocardioides are widely distributed in soil, marine environments or high-salt areas, and can produce a variety of enzymes, compatible solutes, surfactants, and natural products with various biological activities. Antibacterial compounds currently obtained from Nocardioides include polyketides, phenazines, quinine alkaloids, terphenyls, thiopeptides, and amines, and can synthesize anticancer compounds, tumor promoters, and P-glycoprotein inhibitors. compounds such as agents, immunomodulators and protein kinase inhibitors. Compounds derived from Nocardia spp. that are currently on the market include Apoptolidin and K-252a. Therefore, Nocardiopsis has great potential for active product synthesis and mining prospect.

The synthesis of secondary metabolites by microorganisms is affected by many factors, such as medium composition, fermentation temperature, fermentation time, fermentation method, etc. The fermentation products may vary greatly under different culture conditions, and the influence of the medium is particularly prominent. Therefore, how to maximize the type, diversity and yield of strain products requires exploration and optimization of fermentation conditions, especially the optimization of the medium is critical.

HPLC-TOF MS (high performance liquid chromatography/electrospray time-of-flight mass spectrometry) technology can be used to quickly detect the abundance of compounds in fermentation products and facilitate horizontal comparisons. HPLC-TOF MS combines the ability of liquid chromatography to effectively separate thermally unstable and high-boiling point compounds with the strong component identification ability of mass spectrometry, and is an effective means for separating and analyzing complex organic mixtures. Mass spectrometry can analyze the organic substances separated by high performance liquid chromatography one by one, and obtain the molecular weight. Combined with database search, the molecular formula of some compounds can be obtained, which provides information for inferring possible products. Therefore, HPLC-TOF MS analysis based on the crude extract of fermentation products can visually compare the richness of products under different conditions, which is beneficial to find the optimal fermentation conditions, and finally obtain the most abundant natural products, and maximize the excavation of polar actinomycetes Synthetic potential of natural products.

Contents of the invention

The purpose of the present invention is to provide a new species of Nocardiopsis arcticum, and optimize its fermentation and culture conditions to increase the diversity of secondary metabolites of the strain, and help fully explore the potential of the strain to produce new secondary metabolites.

To achieve the above object, the present invention adopts the following technical solutions:

The present invention provides a Nocardiopsis sp. Y469 in the Arctic named Nocardiopsis sp.Y469. The strain has been preserved in Wuhan, China and the Chinese Type Culture Collection Center of Wuhan University on August 13, 2018, and its preservation number is CCTCCNO : M2018536.

Arctic Nocardiopsis sp.Y469 was isolated from the sediments at 3,700 meters deep in the Arctic Ocean. The main morphology and biological characteristics of the strain are as follows: The colony morphology of the strain is shown in Figure 1, cultured on 2216E plates at 20°C In 2 to 3 days, the colony changed from smooth and colorless to white fluffy at the beginning, forming spores, and the edges of the colony began to appear irregular. In liquid medium, the strain grows as a granular pellet. Gram staining of the strain is purple, belonging to Gram-positive bacteria. The strain is a psychrotrophic bacterium, which can grow at a low temperature below 4°C, and the optimum growth temperature range is 25-30°C. The strain is salt-tolerant and can grow in the salinity range of 0%-12%.

The above strain was sequenced, and its 16S rRNA gene sequence is shown in SEQ ID NO.1. The sequence results were compared with known sequences for homology, and the molecular identification phylogenetic tree of the strain is shown in Figure 2. Combined with the culture characteristics and cell morphology of the bacteria, the strain was identified as a new strain of Nocardiopsis and named Nocardiopsissp.Y469.

Whole-genome sequencing showed that the strain has multiple gene clusters related to the synthesis of secondary metabolites, such as lantipeptide (lantipeptide compound), phenazine, ectoine, siderophile, terpenoid bacteriocin, polyketide, non- Biosynthetic gene clusters such as ribosomal polypeptides and lasso peptides. Therefore, this strain has good potential for natural product mining.

The present invention provides the fermentation culture method of above-mentioned arctic Nocardiopsis sp.Y469, comprising the following steps:

(1) inoculate the activated Nocardiopsis sp.Y469 bacterial strain in the seed medium, and cultivate to obtain the seed liquid;

(2) inoculating the seed liquid in the fermentation medium to carry out shake flask fermentation culture to obtain the fermentation liquid;

The fermentation medium uses old seawater as a solvent, and each liter of the medium includes: 4g of yeast extract, 10g of malt extract, 4g of glucose, and a pH of 7.5 (ISP2 liquid medium);

Or yeast extract 4g, malt extract 5g, glucose 4g, sodium chloride 50g, phenylalanine 1mg, alanine 0.3g, FeSO ₄ .7H ₂ O 2mg, ZnSO ₄ .7H ₂ O 1mg, MnCl ₂ . 4H ₂ O 1mg, and vitamin B1, riboflavin, niacin, vitamin B6, calcium pantothenate, inositol, β-aminobenzoic acid, biotin 0.5mg each, pH 7.5 (improved ISP2 liquid medium);

Or soybean flour 20g, peptone 2g, glucose 20g, starch 5g, yeast extract 2g, sodium chloride 4g, K ₂ HPO ₄ 0.5g, CaCO ₃ 2g, pH 7.5 (S3 liquid medium);

Or peptone 5g, glucose 10g, sucrose 340g, yeast extract 3g, malt extract 3g, MgCl ₂ .6H ₂ O 2ml (2.5M), glycine (20%) 25ml, pH 7.5 (YEME liquid medium);

Or casein hydrolyzate 2g, TES 5.73g, NaH ₂ PO ₄ 0.16g, K ₂ HPO ₄ 0.23g, MgSO ₄ 1.23g, glucose 9g, trace elements 0.2ml, pH 7.5 (SMMS liquid medium), in which trace The configuration method of the element solution is as follows: ZnSO ₄ 7H ₂ O, FeSO ₄ 7H ₂ O, MnC1 ₂ 4H ₂ O, CaCl ₂ 6H ₂ O and NaCl 0.1g each, ddH ₂ O to 1L, filter out bacteria.

The old sea water is taken from polar regions, and can also be replaced by artificial sea water, which is prepared according to the international standard ASTM D1141-98.

In the present invention, HPLC and HPLC-TOF MS analysis of the fermentation product is carried out according to the type of culture medium of the strain, and the expression of the product of the strain is preliminarily obtained. The results of HPLC spectrum reflect the richness of the compounds in the fermentation product, and HPLC-TOF MS can detect the molecular weight of the fermentation mixture. Studies have shown that Nocardiopsis sp.Y469 can be fermented and cultured in the above five mediums to produce secondary metabolites, but the types and molecular weights of the products are different, such as those obtained under the fermentation conditions of S3 medium There are more product peaks than other conditions; compared with other fermentation conditions, the molecular weight of the product fermented on SMMS medium is relatively large.

In step (1), the seed medium is TSB medium.

The conditions for culturing the seed solution are 20-30° C., 150-200 rpm and culturing until the OD ₆₀₀ value is 0.6-0.8. The medium used for the activation is 2216E medium, and the activation temperature is 20-30°C. According to research, the optimal growth temperature of the strain is in this temperature range, and the strain has the best vigor at this stage of cultivation, which is suitable for activation and subsequent expansion.

In the step (2), the inoculum amount of the seed liquid in the fermentation medium is 1-3% by volume percentage.

The conditions of the fermentation culture are 20-30° C., and the vibration culture is 7-10 days at a rotating speed of 150-200 rpm. Under this fermentation condition, the strain grows faster in the early stage and can reach a sufficient amount of bacteria, and prolonging the culture time is beneficial to the synthesis of secondary metabolites in the later stage.

The beneficial effect that the present invention possesses:

The present invention isolates a new arctic Nocardiopsis sp.Y469 from the sediments of the 3,700-meter seabed of the Arctic Ocean, and optimizes the fermentation conditions that can significantly increase the diversity of secondary metabolites by comparing fermentation conditions such as different fermentation media , help to fully explore the potential of the strain to produce new secondary metabolites.

Description of drawings

Figure 1 shows the morphology of Nocardiopsis sp.Y469 strain.

Figure 2 is the phylogenetic tree of Nocardiopsis sp.Y469 based on 16S rRNA.

Figure 3 is a representative illustration of the experimental results of the inhibition zone of Nocardiopsis sp.Y469 fermentation broth, wherein (A) is Rhizoctonia solani, (B) is the inhibition zone of Bacillus subtilis, and C in the figure is the test paper soaked in methanol Control, and 1-4 are the parallel repetitions of the experimental group respectively.

Fig. 4 is the operation flow chart of the fermentation process of five culture media of Nocardiopsis sp.Y469.

Fig. 5 is the HPLC spectrogram of the methanol extract of Nocardiopsis sp.Y469 under five medium conditions.

Fig. 6 is the HPLC spectrogram of the acetone extract of Nocardiopsis sp.Y469 under five culture medium conditions.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto. For the experimental methods without specific conditions indicated in the following examples, according to conventional operations or conditions recommended by the manufacturer, the materials of the present invention are commercial common reagents or chemical products.

The medium formula and configuration method involved in the following examples, and the pretreatment method of macroporous resin are as follows:

(1) Medium composition and configuration method for activating bacterial strains:

2216E liquid medium:

Ready-made medium mix 37.4g

After weighing, add 1L ddH ₂ O, adjust the pH to about 7.5, and autoclave at 120°C for 20 minutes.

2216E Plate Medium:

Ready-made medium mix 37.4g

Agar powder 15g

After weighing, add 1L ddH ₂ O, adjust the pH to about 7.5, and autoclave at 120°C for 20 minutes. cooled to

Around 55-60°C, pour plate.

(2) Medium composition and configuration method for cultivating seed liquid:

TSB liquid medium:

Tryptone Soy Broth 30g

Yeast Extract 5g

Sucrose 340g

After weighing, add 1L ddH ₂ O, adjust the pH to about 7.5, and autoclave at 120°C for 20 minutes.

(3) Components and configuration methods of five different mediums used for fermentation culture:

ISP 2 Broth:

Yeast Extract 4g

Malt Extract 10g

Glucose 4g

After weighing, add 1L of old sea water, adjust the pH to about 7.5, and autoclave at 120°C for 20 minutes.

Modified ISP2 liquid medium:

Multivitamins (vitamin B1, riboflavin, niacin, vitamin B6, calcium pantothenate, inositol, β-aminobenzoic acid, biotin each 0.5mg;

After weighing, add 1L of old sea water, adjust the pH to about 7.5, and autoclave at 120°C for 20 minutes.

S3 liquid medium:

After weighing, add 1L of old sea water, adjust the pH to about 7.5, and autoclave at 120°C for 20 minutes.

YEME liquid medium:

After weighing, add 1L of old sea water, adjust the pH to about 7.5, and autoclave at 120°C for 20 minutes. After autoclaving, 2ml _MgCl.6H2O (2.5M); 25ml Glycine (20%) were added.

SMMS liquid medium:

Trace elements solution configuration method: ZnSO ₄ 7H ₂ O, FeSO ₄ 7H ₂ O, MnC1 ₂ 4H ₂ O, CaC1 ₂ 6H ₂ O and NaCl each 0.1g, ddH ₂ O to 1L, filter out bacteria.

After weighing, dissolve in 1L ddH ₂ O and adjust the pH value to 7.5. Autoclave at 120°C for 20 minutes.

(4) Pretreatment of XAD-16 nonionic macroporous resin:

① Suspend 250g XAD160 resin in 1.5L deionized water, adjust the pH to 7.0-7.5;

② Pour off the supernatant and rinse with 2L deionized water for 3-4 times (stir briefly when rinsing, after the resin settles to the bottom, pour out the supernatant, and remove as much water as possible after the last washing);

③ Suspend XAD in a Soxhlet extractor (80% volume), cool in cold water, rinse with 100ml acetone, that is, pour out after soaking in 100ml acetone for 1 hour, repeat three times;

④Soak the resin in methanol for 3h;

⑤ Pour out the methanol, rinse with deionized water 3-4 times, and finally store in about 600ml of deionized water, use small bottles, 200ml blue cap reagent bottles, each bottle contains 150ml, and sterilize for later use.

Example 1

1. Isolation and identification of strains

Strain Y469 was isolated from a sediment sample at 3,700 meters in the Arctic Ocean, and was isolated and purified from a 2216E plate by a gradient dilution method. The colony morphology of this strain is shown in Figure 1. When cultured on 2216E plates at 20°C for 2 to 3 days, the colonies changed from smooth and colorless to white fluffy at first, forming spores, and the edges of the colonies began to appear irregular.

Aseptically pick a single colony on the plate, inoculate it into 2216E liquid medium for expansion, extract genomic DNA with QIAGEN kit (Cat.No.51304), and use primer pair 27F/1492R to perform PCR amplification of 16S rRNA gene And sequenced, the resulting nucleotide sequence is shown in SEQ ID NO.1. The NCBI database and EzBioCloud platform were submitted for identification. The comparison results showed that the most similar strain was the Nocardiopsis genus, and the closest standard strain was Nocardiopsis dassonvillei subsp.albirubida NBRC 13392 ^T (the 16S rRNA gene sequence similarity was 99.7%). Based on the 16S rRNA gene sequence of the closely related standard strain, the phylogenetic tree was constructed using the neighbor-joining method of the MEGA V6.0 software, as shown in Figure 2. Consistent with the above comparison results, Y469 and Nocardiopsis dassonvilleisubsp.albirubida NBRC 13392 ^T belong to the same branch on the evolutionary tree, indicating that the relationship between them is the closest.

Because actinomycetes often have very high 16S sequence similarity, but they are still different species, that is, the resolution of 16S in the division of species is not enough, so the whole genome sequences of the two were further compared and analyzed, and the DDH based on the whole genome sequence The (DNA-DNA hybridization) value was 50.3%, lower than the standard value of 70% belonging to the same species, indicating that the two may belong to different species, and Y469 may represent a new species of the genus Nocardiopsis. Therefore, Y469 was preliminarily identified as a new strain of Nocardiopsis sp. Y469 belonging to the Nocardiopsis family Actinomycetes Actinomycetes Actinomycetes Actinomycetes.

2. Analysis of the potential of strains to synthesize natural products

The strain Nocardiopsis sp.Y469 was preliminarily fermented in S3 and SMMS medium, and the fermented crude extract was extracted with methanol and acetone, respectively, and dissolved in methanol for preliminary screening of antibacterial tests. It was found that the methanol extract had the ability to inhibit Bacillus subtilis, The activities of Rhizoctonia solani and Fusarium moniliforme were the strongest against Fusarium moniliforme, followed by Rhizoctonia solani and Bacillus subtilis, and the methanol extract of S3 culture strain had the most obvious antibacterial activity. Therefore, the potential for synthesizing active products is demonstrated. The representative results of the inhibition zones are shown in Figure 3, which are respectively the inhibition zones of the methanol crude extract of Nocardiopsis sp.Y469 after S3 medium fermentation against Rhizoctonia solani (A) and Bacillus subtilis (B), where C It is the test paper soaked in methanol as the control, and 1 to 4 are the parallel repetitions of the experimental groups.

Moreover, based on the bioinformatics analysis of the whole genome sequence of the strain, it was found that at least 16 natural product synthesis gene clusters were encoded in the genome of the strain, see attached table 1 for details. These include various types of synthetic gene clusters such as lantipeptide, phenazine, ectoine, polyketide, non-ribosomal polypeptides, siderophiles, bacteriocins, terpenoids, lasso peptides, oligosaccharides, etc., and most of the synthetic gene clusters are related to The known gene clusters are very different, and there are even no similar gene clusters, indicating that strain Y469 can synthesize abundant and novel natural products.

Table 1 Secondary metabolite synthesis gene cluster encoded in Nocardiopsis sp.Y469 genome

3. Strain preservation

The above-mentioned strain was named Nocardiopsis sp.Y469, and it was preserved in Wuhan, China and the Chinese Type Culture Collection Center of Wuhan University on August 13, 2018. The preservation number is CCTCC NO: M 2018536, and it was identified as alive on August 28, 2018. .

Example 2

1. Preparation of seed solution

Pick a single colony of Nocardiopsis sp.Y469 that has been verified in Example 1 and put it in a 1L Erlenmeyer flask containing 250ml TSB medium, culture it at 30°C and 200rpm for 2-3 days, the _OD600 value is about 0.6-0.8, and the seed solution Preparation is complete.

2. Shake flask fermentation culture

The above-mentioned seed solution was respectively inoculated with 2L ISP2 medium, improved ISP2 medium, S3 medium, YEME medium and SMMS medium according to the inoculum amount of 1% (V/V), and placed at 30°C and cultured on a shaker at 200rpm. When fermenting for 3-4 days, add 2-4% pretreated XAD-16 non-ionic macroporous resin, and continue fermenting for 3-4 days.

3. Extraction of fermentation products

Use a 500ml centrifuge tube, centrifuge at 8000rpm for 15-20min to collect the cells and macroporous resin, filter through gauze to remove the supernatant; then freeze at -80°C overnight and then thaw to break the wall. Extract with 300ml methanol, ultrasonic for 30min, then shake the table for 30-60min to further improve the extraction effect; repeat the above steps 4 times; use a vacuum pump to obtain a mixed solution of methanol and product, remove the methanol by rotary evaporation, and weigh and record the crude extraction weight; dissolved in 2ml methanol. Repeat the extraction 4 times with the same volume of acetone solution and dissolve in 2 ml of methanol. See Figure 4 for the specific operation process.

4. HPLC and LC/MS analysis

The crude extract dissolved in methanol was filtered through a 0.22 μm filter membrane and used for HPLC and LC/MS analysis. The HPLC analytical instrument used in the present invention is Waters e2695HPLC, and LC/MS analytical instrument is Agilent 6224TOF LC/MS, and the parameter that uses is as follows:

(1) HPLC analysis

The chromatographic column used is a C18 column (4.6×150mm, 3.5μm); mobile phase: aqueous phase A: H ₂ O+0.1% trifluoroacetic acid (TFA) and organic phase B: CH ₃ CN+0.1% TFA; flow rate: 1ml/min; injection volume: 10μl

Gradient elution conditions:

(2) LC/MS analysis

The chromatographic column used is C18 column (4.6×75mm, 3.5 μm); mobile phase: aqueous phase A: H ₂ O+0.1% HCOOH and organic phase B: CH ₃ CN+0.1% HCOOH; flow rate: 0.4ml/min; Injection volume: 10μl

Gradient elution conditions:

5. Result analysis

The HPLC spectrogram of its fermentation product extracted with methanol is shown in accompanying drawing 5. The HPLC spectrogram of its fermentation product extracted with acetone is shown in accompanying drawing 6. From the comparison of HPLC spectra, it can be seen that Nocardiopsis sp.Y469 has more product peaks under S3 medium fermentation conditions than other conditions, and the product amount is higher, indicating that the compounds fermented under this condition are more abundant than other conditions. The molecular weight with a peak height greater than or equal to 1000 points was extracted from the LC/MS results, and the results are shown in Table 2.

Table 2 List of molecular weights extracted by LC/MS of fermentation products.

It can be seen from the above table that the methanol extraction part of the fermentation product of S3 medium has the highest molecular weight detected, which is far more than that of other media. There is little difference in the number of molecular weights detected by the base. According to the comprehensive analysis, the molecular weight of the fermentation product obtained by S3 medium was the most abundant, but the molecular weight was relatively small (from 102.04 to 764.15), while the molecular weight of the fermentation product obtained by SMMS medium covered a large range and contained high molecular weight compounds up to 1270.55. The results show that the fermentation products of different media are different in abundance, diversity and type. By combining HPLC, LC/MS and other multispectral analysis, it is possible to obtain the most abundant products or products with a specific molecular weight range. Providing a basis for judgment is conducive to further digging into the natural products synthesized by the strain.

sequence listing

<110> China Polar Research Center

<120> An arctic Nocardia bacterium and its fermentation product optimization method

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<170> SIPOSequenceListing 1.0

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cctgactccg ggataagcgg tggaaacgcc gtctaatacc ggatacgacc cgccacctca 180

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ggggtaacgg cctaccaagg cgattacggg tagccggcct gagagggcga ccggccaacac 300

tgggactgag acacggccca gactcctgcg ggaggcagca ggggaatatt gcgcaatggg 360

cgaaagcctg acgcagcgac gccgcgtggg ggatgacggc cttcgggttg taaacctctt 420

ttaccaccaa cgcaggctcg aagttctctt cgggttgacg gtaggtgggg aataaggacc 480

ggctaactac gtgccagcag ccgcggtaat acgtaggggtc cgagcgttgt ccggaattat 540

tgggcgtaaa gagctcgtag gcggcgtgtc gcgtctgctg tgaaagaccg gggcttaact 600

ccggttctgc agtggatacg ggcatgctag aggtaggtag gggagactgg aattcctggt 660

gtagcggtga aatgcgcaga tatcaggagg aacaccggtg gcgaaggcgg gtctctgggc 720

cttacctgac gctgaggagc gaaagcatgg ggagcgaaca ggattagata ccctggtagt 780

ccatgccgta aacgttgggc gctaggtgtg gggactttcc acggtttccg cgccgtagct 840

aacgcattaa gcgccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga 900

cggggggcccg cacaagcggc ggagcatgtt gcttaattcg acgcaacgcg aagaacctta 960

ccaaggtttg acatcacccg tggactcgca gagatgtgag gtcatttagt tggcgggtga 1020

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gctagtaatc gcggatcagc aacgccgcgg tgaatacgtt cccgggcctt gtacacaccg 1380

cccgtcacgt catgaaagtc ggcaacaccc gaaacttgcg gcctaaccccc ttgtgggagg 1440

gagtgagtga aggtggggct ggcgattggg acgaagtcgt aacaaggtag ccgtaccgga 1500

aggtgcggct ggatcacct 1519

Claims (7)

1. A Nocardiopsis arcticum, characterized in that the Nocardiopsis arctica is named Nocardiopsissp.Y469, and its preservation number is CCTCC NO: M 2018536. 2. a method for fermenting and cultivating arctic Nocardiopsis sp.Y469 as claimed in claim 1, is characterized in that, comprises the following steps: (1) Inoculate the activated Nocardiopsis sp.Y469 bacterial strain in the seed medium, and cultivate to obtain the seed liquid; (2) inoculating the seed solution in the fermentation medium for fermentation and culturing to obtain a fermentation solution; The fermentation medium uses old seawater as a solvent, and each liter of the medium includes: 4g of yeast extract, 10g of malt extract, 4g of glucose, and a pH of 7.5; Or yeast extract 4g, malt extract 5g, glucose 4g, sodium chloride 50g, phenylalanine 1mg, alanine 0.3g, FeSO ₄ .7H ₂ O 2mg, ZnSO ₄ .7H ₂ O 1mg, MnCl ₂ .4H ₂ O 1mg, and vitamin B1, riboflavin, niacin, vitamin B6, calcium pantothenate, inositol, β-aminobenzoic acid, biotin 0.5mg each, pH 7.5; Or soybean flour 20g, peptone 2g, glucose 20g, starch 5g, yeast extract 2g, sodium chloride 4g, K ₂ HPO ₄ 0.5g, CaCO ₃ 2g, pH 7.5; Or peptone 5g, glucose 10g, sucrose 340g, yeast extract 3g, malt extract 3g, MgCl ₂ .6H ₂ O 2ml, glycine 25ml, pH 7.5; Or casein hydrolyzate 2g, TES5.73g, NaH ₂ PO ₄ 0.16g, K ₂ HPO ₄ 0.23g, MgSO ₄ 1.23g, glucose 9g, trace elements 0.2ml, pH is 7.5, the configuration method of the trace elements solution: ZnSO ₄ ·7H ₂ O, FeSO ₄ ·7H ₂ O, MnC1 ₂ ·4H ₂ O, CaC1 ₂ ·6H ₂ O and NaCl 0.1g each, dilute to 1L with ddH ₂ O, filter through a 0.2μm filter to sterilize. 3. The fermentative culture method according to claim 2, characterized in that, in step (1), the seed culture medium is a TSB medium. 4. The fermentation culture method according to claim 2, characterized in that, in step (1), the conditions for seed liquid culture are 20-30° C., 150-200 rpm until the _OD600 value is 0.6-0.8. 5. The fermentation culture method according to claim 2, characterized in that, in step (1), the medium used for the activation is 2216E medium, and the activation temperature is 20-30°C. 6. The fermentation culture method according to claim 2, characterized in that, in step (2), the inoculum amount of the seed liquid in the fermentation medium is 1-3% by volume percentage. 7. The fermentation culture method according to claim 2, characterized in that, in step (2), the conditions of the fermentation culture are 20-30° C., and the vibration culture is 7-10 days at a rotation speed of 150-200 rpm.