Culture medium for fermentation production of tylosin and preparation method thereof

Abstract

The invention discloses a culture medium for producing tylosin through fermentation and a preparation method of the culture medium, and relates to the technical field of microbial fermentation. The culture medium comprises jerusalem artichoke powder, corn starch, sunflower seed cake meal, rapeseed meal, cottonseed cake meal, pea meal, sodium chloride, monopotassium phosphate, disodium hydrogen phosphate, ammonium nitrate and saccharides. According to the culture medium for producing the tylosin through fermentation, traditional fish meal is replaced with the vegetable protein, so that the content of histamine in the prepared tylosin is obviously reduced, all the components in the fermentation culture medium have a synergistic effect, the content of the prepared tylosin and the content of a component A in the tylosin are obviously increased, and the content of the component A in the tylosin is increased; wide application prospects are realized.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a culture medium for producing tylosin by fermentation and a preparation method thereof. Background Art

[0002] Antibiotics are a group of chemical substances with antibacterial activity, represented by penicillin, streptomycin, and erythromycin. They are metabolites with anti-pathogen or other antibacterial activity produced by microorganisms including bacteria, fungi, and actinomycetes, or by certain higher animal and plant cells during their growth and metabolism. The classification of antibiotics mainly includes β-lactam antibiotics, macrolide antibiotics, aminoglycoside antibiotics, polypeptide antibiotics, tetracycline antibiotics, and anthracycline antibiotics.

[0003] Tylosin is a macrolide antibiotic and a polyketide compound. Its products include tartrate, phosphate, lactate, hydrochloride and sulfate. Tylosin is an effective non-residual antibacterial growth promoter for livestock and poultry. It has good therapeutic effects on swine epidemic pneumonia, sepsis and dysentery in chickens and is often used as a veterinary drug and feed additive. Tylosin contains four highly active components, namely tylosin A, decarboxylic acid tylosin B, macrocin C and ralomycin D. Among them, tylosin A is the main component and has the highest antibacterial properties.

[0004] Tylosin has a significant anti-mycoplasma effect, a broad antibacterial spectrum, and has the advantages of small blockage and low residual amount. Whether it is taken orally or injected, it can reach an effective antibacterial concentration in a short time and can be maintained for a long time. It is quickly excreted from the body after stopping taking it, and there is basically no residue in the tissue. Compared with other antibiotics, the obvious characteristics of tylosin are low toxicity and safe application.

[0005] Chinese patent CN103074402A discloses a culture medium for producing tylosin by fermentation of Streptozotocin freundii, a fermentation method and a method for producing tylosin using the culture medium. In the invention, the primary seed culture medium contains peanut meal and corn oil, and the secondary seed culture medium and the fermentation culture medium both contain mixed fish meal, peanut meal and corn oil. The present invention uses corn oil to replace soybean oil; mixed fish meal and peanut meal to replace fish meal, optimizes the culture medium formula, thereby solving the cost problem of raw and auxiliary materials, and minimizes the source of raw and auxiliary materials from being affected by the environment, ensuring their sufficient supply, and realizing stable and efficient production of tylosin.

[0006] Chinese patent CN112458135A discloses a tylosin fermentation formula, which mainly includes the following components by weight percentage: 35% fish meal, 20% corn gluten meal, 30% cottonseed meal, 11% peanut meal, and 4% potassium dihydrogen phosphate. The technical solution provided by the invention has the beneficial effect that the total protein content of the formula reaches 68.78% through the fish meal, corn gluten meal, cottonseed meal, and peanut meal with relatively high protein content, thereby reducing the types of tylosin fermentation raw materials, thereby reducing the impact of impurities in the raw materials on tylosin fermentation. However, since the above culture medium contains fish meal, histamine residues exist in the tylosin finished product.

[0007] Currently, it is a technical problem that needs to be solved urgently to provide a culture medium for producing tylosin by fermentation without histamine residue in the tylosin finished product. Summary of the invention

[0008] The purpose of the present invention is to provide a culture medium for fermenting and producing tylosin and a preparation method thereof.

[0009] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0010] On the one hand, the present invention provides a culture medium for fermenting and producing tylosin, wherein the culture medium comprises Jerusalem artichoke powder, corn starch, sunflower seed cake, rapeseed meal, cottonseed cake powder, pea powder, sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, ammonium nitrate and sugars.

[0011] Specifically, the culture medium includes 5-15 g / L Jerusalem artichoke powder, 10-30 g / L corn starch, 0.01-2 g / L sunflower seed cake, 1-3 g / L rapeseed meal, 1-5 g / L cottonseed cake powder, 15-25 g / L pea powder, 0.01-0.05 g / L sodium chloride, 1-2 g / L potassium dihydrogen phosphate, 0.5-1 g / L disodium hydrogen phosphate, 0.5-1.5 g / L ammonium nitrate, and 0.1-10 g / L sugars.

[0012] Furthermore, the culture medium includes 5-10 g / L Jerusalem artichoke powder, 15-25 g / L corn starch, 0.05-1.5 g / L sunflower seed cake, 2-3 g / L rapeseed meal, 1-3 g / L cottonseed cake powder, 18-24 g / L pea powder, 0.03-0.05 g / L sodium chloride, 1.5-2 g / L potassium dihydrogen phosphate, 0.5-0.8 g / L disodium hydrogen phosphate, 0.8-1.2 g / L ammonium nitrate, and 5-10 g / L sugars.

[0013] Furthermore, the culture medium includes 8g / L Jerusalem artichoke powder, 16g / L corn starch, 1g / L sunflower seed cake, 2g / L rapeseed meal, 2g / L cottonseed cake powder, 20g / L pea powder, 0.04g / L sodium chloride, 1.5g / L potassium dihydrogen phosphate, 0.5g / L disodium hydrogen phosphate, 1g / L ammonium nitrate, and 8g / L sugars.

[0014] Specifically, the rapeseed meal is a product obtained by pressing and removing the oil from rapeseed oil.

[0015] Specifically, the sugar is selected from one or more of glucose, fructose, sucrose, lactose, galactose, maltose and mannose.

[0016] Furthermore, the sugar is fructose.

[0017] Specifically, the culture medium also contains amino acids.

[0018] Furthermore, the amino acid is selected from one or more of glycine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, alanine, aspartic acid, glutamic acid, proline, tryptophan, tyrosine, and valine.

[0019] Furthermore, the amino acid is tyrosine.

[0020] Furthermore, the culture medium contains 0.1-5 g / L of amino acids.

[0021] Furthermore, the culture medium contains 0.5 g / L of amino acids.

[0022] Specifically, the culture medium may also include amino acid salts.

[0023] Furthermore, the amino acid salt is selected from one or more of glycine hydrochloride, isoleucine hydrochloride, leucine hydrochloride, lysine hydrochloride, phenylalanine hydrochloride, serine hydrochloride, threonine hydrochloride, alanine hydrochloride, aspartic acid hydrochloride, glutamic acid hydrochloride, proline hydrochloride, tryptophan hydrochloride, tyrosine hydrochloride, and valine hydrochloride.

[0024] In another aspect, the present invention provides use of the above culture medium in fermentation production of tylosin.

[0025] In another aspect, the present invention provides a method for producing tylosin by fermentation, which uses the above-mentioned culture medium for fermentation production.

[0026] Specifically, the method for producing tylosin by fermentation comprises the following steps:

[0027] (1) Spread the Streptomyces freundii strains on the surface of the slant culture medium for cultivation, and inoculate the activated strains;

[0028] (2) taking fresh spores from the slant culture medium and inoculating them into the seed culture medium for cultivation;

[0029] (3) The seed liquid is inoculated into the fermentation medium and fermented for 5-10 days.

[0030] Furthermore, the slant culture medium in step (1) contains 0.1% peptone and 0.1% yeast powder.

[0031] Furthermore, the culture temperature in step (1) is 30° C. and the culture time is 7 days.

[0032] Furthermore, the culturing in step (2) is carried out at 25-30°C and 200-220 r / min.

[0033] Furthermore, the culturing in step (2) is carried out at 30° C. and 220 r / min.

[0034] Furthermore, the culturing time in step (2) is 24-48 hours; furthermore, the culturing time in step (2) is 48 hours.

[0035] Furthermore, the inoculation amount in step (3) is 5%-10%; furthermore, the inoculation amount in step (3) is 5%.

[0036] Furthermore, the fermentation culture conditions in step (3) are 25-30°C, 200-220r / min, and pH 6.0-7.0.

[0037] Furthermore, the fermentation culture conditions in step (3) are 30° C., 200 r / min, and pH 7.0.

[0038] Furthermore, the fermentation culture time in step (3) is 7 days.

[0039] The beneficial effects of the present invention are:

[0040] The culture medium for fermenting and producing tylosin provided by the present invention uses plant protein instead of traditional fish meal, so that the content of histamine in the prepared tylosin is significantly reduced, and the components in the fermentation medium act synergistically, which significantly increases the content of the prepared tylosin and the content of component A in the tylosin. At the same time, the method for fermenting and producing tylosin provided by the present invention is simple to operate, has high yield, and has broad application prospects. DETAILED DESCRIPTION

[0041] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0042] Example 1

[0043] Seed culture medium: 20 g / L water-soluble starch, 10 g / L peptone, 0.04 g / L sodium chloride, 1.5 g / L potassium dihydrogen phosphate, 0.5 g / L disodium hydrogen phosphate, adjust the pH to 7.0, and sterilize under high pressure at 121°C for 20 min.

[0044] Fermentation medium: Jerusalem artichoke powder 5g / L, corn starch 10g / L, sunflower seed meal 0.01g / L, rapeseed meal 1g / L, cottonseed meal 1g / L, pea meal 15g / L, sodium chloride 0.01g / L, potassium dihydrogen phosphate 1g / L, disodium hydrogen phosphate 0.5g / L, ammonium nitrate 0.5g / L, fructose 0.1g / L, tyrosine 0.1g / L, and the rest is water.

[0045] Fermentation culture of tylosin:

[0046] (1) S. freundii was spread on the surface of a slant culture medium containing 0.1% peptone and 0.1% yeast powder, and cultured at 30°C for 7 days. The activated strain was then used for inoculation.

[0047] (2) Take fresh spores from the slant culture medium and inoculate them into a flask containing seed culture medium. Inoculate 0.5 mL of the bacterial strain into 100 mL of seed culture medium and culture at 30°C and 220 r / min for 48 h.

[0048] (3) The seed liquid was inoculated into a triangular flask containing fermentation medium at an inoculation rate of 5%. The total volume of the fermentation medium was 100 mL. After inoculation, the fermentation culture was carried out at 30° C., 200 r / min, and pH 7.0 for 7 days.

[0049] Example 2

[0050] Seed culture medium: 20 g / L water-soluble starch, 10 g / L peptone, 0.04 g / L sodium chloride, 1.5 g / L potassium dihydrogen phosphate, 0.5 g / L disodium hydrogen phosphate, adjust the pH to 7.0, and sterilize under high pressure at 121°C for 20 min.

[0051] Fermentation medium: Jerusalem artichoke powder 5g / L, corn starch 15g / L, sunflower seed meal 0.05g / L, rapeseed meal 2g / L, cottonseed meal 1g / L, pea meal 18g / L, sodium chloride 0.03g / L, potassium dihydrogen phosphate 1.5g / L, disodium hydrogen phosphate 0.5g / L, ammonium nitrate 0.8g / L, fructose 5g / L, tyrosine 0.4g / L.

[0052] Fermentation culture of tylosin:

[0053] (1) S. freundii was spread on the surface of a slant culture medium containing 0.1% peptone and 0.1% yeast powder, and cultured at 30°C for 7 days. The activated strain was then used for inoculation.

[0054] (2) Take fresh spores from the slant culture medium and inoculate them into a flask containing seed culture medium. Inoculate 0.5 mL of the bacterial strain into 100 mL of seed culture medium and culture at 30°C and 200 r / min for 36 h.

[0055] (3) The seed liquid was inoculated into a triangular flask containing fermentation medium at an inoculation rate of 5%. The total volume of the fermentation medium was 100 mL. After inoculation, the fermentation culture was carried out at 30° C., 200 r / min, and pH 6.5 for 7 days.

[0056] Example 3

[0057] Seed culture medium: 20 g / L water-soluble starch, 10 g / L peptone, 0.04 g / L sodium chloride, 1.5 g / L potassium dihydrogen phosphate, 0.5 g / L disodium hydrogen phosphate, adjust the pH to 7.0, and sterilize under high pressure at 121°C for 20 min.

[0058] Fermentation medium: Jerusalem artichoke powder 8g / L, corn starch 16g / L, sunflower seed meal 1g / L, rapeseed meal 2g / L, cottonseed meal 2g / L, pea meal 20g / L, sodium chloride 0.04g / L, potassium dihydrogen phosphate 1.5g / L, disodium hydrogen phosphate 0.5g / L, ammonium nitrate 1g / L, fructose 8g / L, tyrosine 0.5g / L.

[0059] The fermentation culture of tylosin is the same as that in Example 1.

[0060] Example 4

[0061] Seed culture medium: 20 g / L water-soluble starch, 10 g / L peptone, 0.04 g / L sodium chloride, 1.5 g / L potassium dihydrogen phosphate, 0.5 g / L disodium hydrogen phosphate, adjust the pH to 7.0, and sterilize under high pressure at 121°C for 20 min.

[0062] Fermentation medium: Jerusalem artichoke powder 10g / L, corn starch 25g / L, sunflower seed cake 1.5g / L, rapeseed meal 3g / L, cottonseed cake powder 3g / L, pea powder 24g / L, sodium chloride 0.05g / L, potassium dihydrogen phosphate 2g / L, disodium hydrogen phosphate 0.8g / L, ammonium nitrate 1.2g / L, fructose 10g / L, tyrosine 1g / L.

[0063] The fermentation culture of tylosin is the same as that in Example 1.

[0064] Comparative Example 1

[0065] Compared with Example 3, the only difference is the components in the fermentation medium. The fermentation medium in Comparative Example 1 is:

[0066] Jerusalem artichoke powder 8g / L, sunflower seed cake 1g / L, cottonseed cake powder 2g / L, sodium chloride 0.04g / L, disodium hydrogen phosphate 0.5g / L, fructose 8g / L.

[0067] Comparative Example 2

[0068] Compared with Example 3, the only difference is the components in the fermentation medium. The fermentation medium in Comparative Example 2 is:

[0069] Corn starch 16g / L, rapeseed meal 2g / L, pea flour 20g / L, potassium dihydrogen phosphate 1.5g / L, ammonium nitrate 1g / L, tyrosine 0.5g / L.

[0070] Comparative Example 3

[0071] Compared with Example 3, the only difference is that the components in the fermentation medium are different, the Jerusalem artichoke powder in the fermentation medium is replaced by fish meal, and the rest are the same.

[0072] Comparative Example 4

[0073] Compared with Example 3, the only difference is that the components in the fermentation medium are different, the pea flour in the fermentation medium is replaced by bean cake flour, and the rest are the same.

[0074] Comparative Example 5

[0075] Compared with Example 3, the only difference is that the components in the fermentation medium are different, and the tyrosine in the fermentation medium is replaced by glutamic acid, and the rest are the same.

[0076] Comparative Example 6

[0077] Compared with Example 3, the only difference is that the content of the components in the fermentation medium is different. The fermentation medium in Comparative Example 6 is: Jerusalem artichoke powder 20 g / L, corn starch 35 g / L, sunflower seed cake 2.5 g / L, rapeseed meal 4 g / L, cottonseed cake powder 6 g / L, pea powder 30 g / L, sodium chloride 0.1 g / L, potassium dihydrogen phosphate 3 g / L, disodium hydrogen phosphate 2 g / L, ammonium nitrate 2 g / L, fructose 12 g / L, tyrosine 6 g / L.

[0078] Experimental Example 1 Detection of histamine content in tylosin

[0079] Reagent preparation:

[0080] Histamine reference stock solution: Weigh 33 mg of histamine hydrochloride reference (purity > 99%) accurately into a 200 mL brown volumetric flask, dilute to the mark with 0.1 mol / L hydrochloric acid solution, shake well, and prepare a 100 mg / L histamine reference stock solution.

[0081] Reference substance working solution: dilute the above-mentioned histamine reference substance stock solution to a concentration of 5.0 mg / L and prepare it freshly before use.

[0082] Extract: Weigh about 25g of trichloroacetic acid into a 250mL beaker, dissolve it completely with purified water, transfer it to a 500mL volumetric flask, and dilute to the mark.

[0083] Derivatization reagent: Weigh 0.25 g of dansyl chloride into a 25 mL brown volumetric flask and dissolve and dilute to the mark with acetone.

[0084] 0.01 mol / L ammonium acetate solution containing 1% formic acid: Weigh 0.77 g of ammonium acetate and dissolve it in purified water. Transfer it to a 1000 mL volumetric flask, add 10 mL of formic acid, and dilute to the mark with purified water.

[0085] Mobile phase A: Measure 100 mL of 0.01 mol / L ammonium acetate solution containing 1% formic acid and add 900 mL of acetonitrile.

[0086] Mobile phase B: Measure 900 mL of 0.01 mol / L ammonium acetate solution containing 1% formic acid and add 100 mL of acetonitrile.

[0087] Experimental steps:

[0088] The culture solution after fermentation in Example 1-4 and Comparative Example 1-6 was dried to obtain tylosin, 1.5 g was placed in a 20 mL centrifuge tube, 15 mL of the extract was added, and 10 mL of the supernatant was taken by centrifugation. Sodium chloride was added to the supernatant to completely dissolve it, 10 mL of n-hexane was added, vortexed and allowed to stand, and the lower layer of the sample solution was taken after stratification, and n-butane was added.

[0089] Pipette 5mL of the above sample solution into a centrifuge tube, adjust the pH to 12 with 5mol / L sodium hydroxide solution, add 5mL of n-butanol / chloroform mixed solution (ratio is 1:1), vortex and centrifuge, transfer the upper organic phase to a brown volumetric flask, extract the lower sample solution, combine the two extracts, and dilute to 10mL with n-butanol / chloroform mixed solution. Take another 5mL of the extract and add it to 200μL of 1mol / L hydrochloric acid, mix well, blow dry with nitrogen at 40℃, add 2.5mL of 0.1mol / L hydrochloric acid and vortex until the residue is completely dissolved.

[0090] Derivatization of reference substance and sample: Take 1mL reference substance working solution and 1mL test sample purified solution in 20mL test tubes respectively, add derivatization reagents (including 1mL saturated sodium bicarbonate solution, 100μL 1mol / L sodium hydroxide solution, and 1mL derivatization reagent) in sequence, vortex mix and place in a 60℃ constant temperature water bath for derivatization for 15min, add 100μL sodium glutamate solution after derivatization, oscillate and mix, and react at 60℃ for 15min. Take out the reaction solution, cool to room temperature, add 1mL purified water to the test tube, vortex mix for 1min, blow nitrogen at 40℃ to remove acetone, add about 0.5g sodium chloride, vortex and oscillate until sodium chloride is completely dissolved. Add 5mL ether, vortex and oscillate for 2min and let stand for stratification, absorb the upper organic phase, extract the remaining liquid again, combine the ether extracts and blow dry with nitrogen at 40℃. Add 10 mL of acetonitrile and vortex to completely dissolve, filter through a 0.22 μm filter membrane into an injection vial to prepare a histamine reference solution with a concentration of 0.5 mg / L and a test solution for HPLC analysis.

[0091] Chromatographic conditions:

[0092] Chromatographic column: C18 column (250 mm × 4.6 mm, 5 μm); flow rate: 0.8 mL / min; column temperature: 35°C; injection volume: 20 μL; detection wavelength: 254 nm; chromatograph: Waters high performance liquid chromatograph, gradient elution program as shown in Table 1.

[0093] Table 1 Gradient elution conditions

[0094] time Mobile phase A / % Mobile phase B / % 0 60 40 22 75 25 25 100 0 32 100 0 32.01 60 40 37 60 40

[0095] The test results are shown in the following table:

[0096] Table 2

[0097] Histamine content Example 1 2.5ppm Example 2 2.8ppm Example 3 Not detected Example 4 3.0ppm Comparative Example 1 9.1ppm Comparative Example 2 9.3ppm Comparative Example 3 17.5ppm Comparative Example 4 8.7ppm Comparative Example 5 8.5ppm Comparative Example 6 8.2ppm

[0098] It can be seen from the above table that the histamine content in the tylosin prepared in Examples 1-4 of the present invention is significantly lower than that in Comparative Examples 1-6. It can be seen from Comparative Example 3 that the addition of fish meal will lead to a significant increase in the histamine content in tylosin.

[0099] Experimental Example 2 Detection of Tylosin Yield and Component Content

[0100] Tylosin in the filtrate was detected by HPLC, and the chromatographic conditions were as follows:

[0101] Chromatographic column: C18 column (5 μm, 46 × 150 mm);

[0102] Mobile phase: acetonitrile: methanol: 5 mM ammonium dihydrogen phosphate (volume ratio) = 60:30:10;

[0103] Flow rate: 1 mL / min;

[0104] Detection wavelength: 290nm.

[0105] The test results are shown in the following table:

[0106] Table 3

[0107] Total component yield Tylosin component A / total components Example 1 7.81g / L 96.8％ Example 2 7.65g / L 96.6％ Example 3 8.55g / L 97.4％ Example 4 8.17g / L 97.1％ Comparative Example 1 3.26g / L 65.3％ Comparative Example 2 3.90g / L 64.7％ Comparative Example 3 6.72g / L 84.1％ Comparative Example 4 6.05g / L 84.2％ Comparative Example 5 5.83g / L 84.8％ Comparative Example 6 6.92g / L 85.5％

[0108] As can be seen from the above table, the yield of tylosin and the content of component A prepared in Examples 1-4 of the present invention are significantly higher than those in Comparative Examples 1-6, indicating that the synergistic effect between the components in the fermentation medium provided by the present invention can significantly increase the yield of tylosin and the content of component A, and has good application prospects.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A culture medium for fermenting and producing tylosin, characterized in that: The culture medium comprises Jerusalem artichoke powder, corn starch, sunflower seed cake, rapeseed meal, cottonseed cake powder, pea powder, sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, ammonium nitrate and sugar.

2. The culture medium according to claim 1, characterized in that The culture medium comprises 5-15 g / L Jerusalem artichoke powder, 10-30 g / L corn starch, 0.01-2 g / L sunflower seed meal, 1-3 g / L rapeseed meal, 1-5 g / L cottonseed meal, 15-25 g / L pea meal, 0.01-0.05 g / L sodium chloride, 1-2 g / L potassium dihydrogen phosphate, 0.5-1 g / L disodium hydrogen phosphate, 0.5-1.5 g / L ammonium nitrate and 0.1-10 g / L sugar.

3. The culture medium according to claim 1, characterized in that The sugar is selected from one or more of glucose, fructose, sucrose, lactose, galactose, maltose and mannose.

4. The culture medium according to claim 1, characterized in that The culture medium also contains amino acids.

5. The culture medium according to claim 4, characterized in that The amino acids are selected from one or more of glycine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, alanine, aspartic acid, glutamic acid, proline, tryptophan, tyrosine and valine.

6. The culture medium according to claim 4, characterized in that The culture medium contains 0.1-5 g / L of amino acids.

7. Use of the culture medium according to any one of claims 1 to 6 in the fermentation production of tylosin.

8. A method for producing tylosin by fermentation, characterized in that: The method comprises using the culture medium according to any one of claims 1 to 6 for fermentation production.

9. The method according to claim 8, characterized in that The following steps are involved: (1) Spread the Streptomyces freundii strains on the surface of the slant culture medium for cultivation, and inoculate the activated strains; (2) taking spores from the slant culture medium and inoculating them into the seed culture medium for cultivation; (3) The seed liquid is inoculated into the fermentation medium and fermented for 5-10 days.

10. The method according to claim 9, characterized in that The culture in step (2) is carried out at 25-30° C., 200-220 r / min, and the culture time in step (2) is 24-48 h; the inoculation amount in step (3) is 5%-10%; the fermentation culture conditions in step (3) are 25-30° C., 200-220 r / min, and pH 6.0-7.0.