Method for increasing acarbose fermentation unit

Abstract

The invention provides a method for increasing the fermentation unit of acarbose. The method mainly comprises the step of adding metal ions into a fermentation culture medium. According to the method, metal ions such as copper sulfate, zinc sulfate or manganese sulfate and the like are added into the fermentation culture medium, and ferric chloride is added in a matched manner, so that the fermentation titer of acarbose is increased to 8.83 mu g / mL under the condition of not changing strains and the fermentation culture medium, and the fermentation unit of acarbose is remarkably increased; in addition, the fermentation period is shortened, and the production cost is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a method for increasing acarbose fermentation units. Background Art

[0002] Acarbose is a secondary metabolite produced by Actinoplanes sp. It is a pseudotetrasaccharide substance of the C7N-aminocyclyl class. It can competitively inhibit α-glucosidase in the intestine and effectively reduce postprandial blood sugar in diabetic patients. Due to its good pharmacokinetic characteristics and low toxicity, it is widely used in the treatment of type 2 diabetes. In addition to its hypoglycemic effect, acarbose can also help diabetic patients lose weight, lower blood lipids, and improve intestinal function.

[0003] Acarbose is a secondary metabolite produced by microbial fermentation. Its biosynthetic pathway is complex and there are many structural analogs of metabolites. The increase in acarbose production is greatly affected by the physiological characteristics of the strain itself, but the optimization of various fermentation conditions is also crucial. The fermentation medium is a nutrient matrix for the growth, reproduction and metabolism of microorganisms, which contains carbon sources, nitrogen sources, inorganic salts and other growth factors. At present, the research on acarbose fermentation medium mainly focuses on the ratio of glucose and maltose in the fermentation broth, different nitrogen sources and their added concentrations. As the precursor and main nutrient for the synthesis of acarbose, the research on carbon and nitrogen sources has improved the fermentation level of acarbose to a certain extent, but there are still certain technical barriers to the fermentation process.

[0004] In addition to carbon and nitrogen sources, some inorganic ions have an important influence on microbial growth and product synthesis. The fermentation medium adapted to acarbose-producing strains usually contains several metal ions such as calcium, iron, potassium and sodium, but other trace elements, such as zinc, copper, cobalt, magnesium, copper, manganese, etc., as components or activators of enzymes, or their metal salts affect the pH of the medium, etc., and also have a certain regulatory effect on microbial metabolic activities and metabolic flow. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for increasing acarbose fermentation units, specifically, adding metal ions into the fermentation medium.

[0006] Furthermore, the metal ions are added separately into the prepared fermentation medium.

[0007] Furthermore, the metal ions are directly added as part of the fermentation medium.

[0008] Furthermore, the metal ions are one or more of copper ions, zinc ions and manganese ions.

[0009] Furthermore, the added concentration of the metal ions is 0.001 to 1.0 g / L; preferably, the added concentration of copper ions is 0.005 to 0.05 g / L, or the added concentration of zinc ions is 0.005 to 0.05 g / L, or the added concentration of manganese ions is 0.005 to 0.1 g / L; more preferably, the added concentration of copper ions is 0.03 g / L, or the added concentration of zinc ions is 0.03 g / L, or the added concentration of manganese ions is 0.05 g / L; the above added concentrations are the final concentrations of the metal ions after being added to the fermentation medium.

[0010] Furthermore, the metal ions are added in the form of sulfate or hydrochloride, preferably sulfate.

[0011] Furthermore, the copper ions, zinc ions or manganese ions are added together with iron ions.

[0012] Furthermore, the iron ions are added in the form of ferric chloride, and the added concentration of the iron ions is 0.2 to 2.5 g / L, preferably 0.5 to 2.0 g / L, and more preferably 1.5 g / L.

[0013] Furthermore, the metal ions are composed of ferric chloride with a concentration of 1.5 g / L, copper sulfate with a concentration of 0.03 g / L, zinc sulfate with a concentration of 0.03 g / L, or manganese sulfate with a concentration of 0.05 g / L.

[0014] Furthermore, the fermentation medium contains the following components in mass concentrations: per 100 mL, 3 to 6 g of glucose, 2 to 4 g of maltose, 1.5 to 3 g of soybean powder, 0.3 to 0.6 g of sodium glutamate, 0.05 to 0.2 g of dipotassium hydrogen phosphate, 0.03 to 0.15 g of ferric chloride, 0.2 to 0.5 g of calcium chloride, and 0.2 to 0.5 g of calcium carbonate.

[0015] Furthermore, the method for increasing acarbose fermentation units comprises the following steps:

[0016] 1) inoculating acarbose-producing bacteria into a seed culture medium to prepare a seed solution;

[0017] 2) Taking the seed liquid prepared above, inoculating it into a fermentation medium for fermentation, so as to obtain acarbose fermentation liquid. Before inoculation, adding metal ions into the fermentation medium.

[0018] Furthermore, the acarbose-producing bacteria in step 1) is Actinoplanes utahensis MYIH97, which is deposited in China Center for Type Culture Collection (CCTCC) in Wuhan, with a deposit number of CCTCC, NO: M2016237.

[0019] Furthermore, the step 1) is seed culture, the seed culture is shake flask seed culture or fermenter seed culture, and the corresponding seed liquid obtained is shake flask seed liquid or fermenter seed liquid.

[0020] The shake flask seed culture is as follows: a small amount of bacterial moss is shoveled into the shake flask seed culture medium, and shake-cultured at 250rpm for 44-48h to obtain the shake flask seed liquid; the fermenter seed culture is as follows: the cultured shake flask seed liquid is inoculated into the seed tank at an inoculation amount of 0.1-0.5% of the volume of the seed tank culture medium, and cultured at 120-200rpm for 44-48h to obtain the fermenter seed liquid.

[0021] Furthermore, the components and contents of the shake flask seed culture medium are: 20 g / L soybean powder, 10 g / L glycerol, 40 g / L corn starch, and 3 g / L calcium carbonate; the pH of the shake flask seed culture medium before sterilization is 6.8-7.0, the filling volume is 100 mL / 500 mL, the shaking culture speed is 250 rpm, the temperature is 26-30°C, preferably 27.5°C, and the culture is 44-48 hours.

[0022] Furthermore, the components and proportions of the seed tank culture medium are: 20 g / L soybean powder, 10 g / L glycerol, 40 g / L corn starch, 3 g / L calcium carbonate and 0.5 g / L folate, the pH of the seed tank culture medium before sterilization is 6.8-7.0; the inoculation amount of the fermentation tank seed liquid is 0.1-0.5%, preferably 0.3%, the tank pressure is 0.04-0.05 MPa, the stirring is 120 rpm-200 rpm, and the ventilation volume is 1:1-1:1.5 ( v / v ) The culture temperature is 26-30°C, preferably 28°C, and the culture time is 44-48h.

[0023] Furthermore, the step 2) is a fermentation culture, and the fermentation culture is a shake flask culture or a fermenter culture.

[0024] The shake flask culture comprises: inoculating the cultured shake flask seed liquid into the shake flask fermentation medium at an inoculation rate of 5-10%, and culturing at 250rpm for 120-168h; the fermenter culture comprises: inoculating the cultured fermenter seed liquid into the fermenter at an inoculation rate of 6-15% of the volume of the fermentation medium, and fermenting and culturing for 120-168h.

[0025] Furthermore, the components and proportions of the shake flask fermentation medium are: 40 g / L glucose, 20 g / L maltose, 20 g / L soybean powder, 3 g / L sodium glutamate, 2.5 g / L calcium carbonate, 1.5 g / L dipotassium hydrogen phosphate, 0.5 g / L ferric chloride, and 2 g / L calcium chloride; the shake flask fermentation medium has a pH of 6.8-7.0 before sterilization, a filling volume of 50 mL / 500 mL, an oscillating culture speed of 250 rpm, a temperature of 26-30° C., preferably 28° C., and a culture time of 144-168 h.

[0026] Furthermore, the components and proportions of the fermentation tank culture medium are: 40 g / L glucose, 20 g / L maltose, 20 g / L soybean powder, 3 g / L sodium glutamate, 2.5 g / L calcium carbonate, 1.5 g / L dipotassium hydrogen phosphate, 0.5 g / L ferric chloride, 2 g / L calcium chloride and 0.5 g / L folate, the fermentation culture medium loading amount is preferably 50-60%, the culture medium pH is 6.8-7.0 before sterilization, the inoculation amount is 6-15%, preferably 10%; the fermentation tank control conditions are: tank pressure 0.04-0.06 MPa, preferably 0.05 MPa, stirring 150 rpm-200 rpm, ventilation 1:1-1:1.5 (v / v), culture temperature 26-30°C, preferably 28°C, dissolved oxygen control 40-80%, and culture 144-168 hours.

[0027] Furthermore, in the step 2), the fermentation medium is first prepared, and then metal ions are added separately after cooling, and then the seed liquid is inoculated for fermentation culture.

[0028] Furthermore, the copper, zinc or manganese ions are added to the fermentation medium in the form of sulfate alone, and ferric chloride is also added alone.

[0029] Furthermore, the metal ions are added in the form of a sulfate or hydrochloride aqueous solution, and the concentration of the sulfate or hydrochloride aqueous solution is 1 to 100 g / L, preferably 5 to 60 g / L.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1) The present invention increases the fermentation titer of acarbose to 8.83 ug / mL by adding metal ions such as copper sulfate, zinc sulfate or manganese sulfate to the fermentation medium and simultaneously adding ferric chloride without changing the strain and the fermentation medium, thereby significantly increasing the fermentation unit of acarbose;

[0032] 2) The fermentation cycle of the present invention is shortened, thereby reducing production costs. DETAILED DESCRIPTION

[0033] In order to further illustrate the content of the present invention, the present invention will be specifically described in conjunction with the following examples, but they should not be construed as limiting the scope of the present invention.

[0034] Example 1

[0035] Shake flask fermentation culture.

[0036] Scoop up 1x1cm-sized, fully grown bacterial moss into a shake flask seed culture medium, culture at 28°C, 250rpm for 48h, prepare the fermentation medium, and divide it evenly into 8 fermentation bottles after preparation. Sterilize the fermentation medium at 121°C for 30min. After cooling, add different volumes of 10g / L copper sulfate aqueous solution to the 8 fermentation bottles, respectively, with the volumes of 0mL, 0.005mL, 0.025mL, 0.05mL, 0.15mL, 0.25mL, 0.5mL and 2.5mL, and use the shake flask without copper sulfate as the control. Then, inoculate 7% of the cultured shake flask seed solution, and ferment the shake flask with 50mL / 500mL, culture at 28°C, 250rpm for 144h to obtain acarbose fermentation liquid.

[0037] Similar to the above steps, different volumes of 10 g / L zinc sulfate aqueous solution were added to 8 fermentation bottles, the volumes were 0 mL, 0.005 mL, 0.025 mL, 0.05 mL, 0.15 mL, 0.25 mL, 0.5 mL and 2.5 mL, respectively. The shake flask without zinc sulfate was used as a control. Then, the cultured shake flask seed solution was inoculated with 7% inoculation amount, and the shake flask fermentation volume was 50 mL / 500 mL. The shake flask was shaken and cultured at 28° C. and 250 rpm for 144 h to obtain acarbose fermentation broth.

[0038] Similar to the above steps, different volumes of 10 g / L manganese sulfate aqueous solution were added to 8 fermentation bottles, the volumes were 0 mL, 0.005 mL, 0.025 mL, 0.05 mL, 0.25 mL, 0.5 mL, 2.5 mL and 5 mL, respectively, and the shake flask without adding manganese sulfate was used as the control. Then, the cultured shake flask seed liquid was inoculated with 7% inoculation amount, and the shake flask fermentation volume was 50 mL / 500 mL. The shake flask was shaken and cultured at 28 ° C and 250 rpm for 144 h to obtain acarbose fermentation liquid.

[0039] The fermentation medium contains the following components and mass concentrations: per 100 mL, 4 g glucose, 2 g maltose, 2 g soybean powder, 0.3 g sodium glutamate, 0.25 g calcium carbonate, 0.15 g dipotassium hydrogen phosphate, 0.05 g ferric chloride, 0.2 g calcium chloride, and the pH of the fermentation medium before sterilization is 6.8-7.0.

[0040] The fermentation broth was centrifuged and the supernatant was taken, and the acarbose content in the fermentation broth was detected by HPLC. The results are shown in Table 1:

[0041] Table 1 Adding different concentrations of copper sulfate, zinc sulfate or manganese sulfate to the fermentation medium improves the fermentation level of acarbose

[0042]

[0043]

[0044] The results showed that when copper sulfate was added alone to the fermentation medium and the added concentration of copper sulfate was 0.005-0.05g / L, the fermentation unit of acarbose could be effectively increased. When the added concentration of copper sulfate was 0.03g / L, the fermentation unit of acarbose was increased to 8402ug / mL; when zinc sulfate was added alone to the fermentation medium and the added concentration of zinc sulfate was 0.005-0.05g / L, the fermentation unit of acarbose could be effectively increased. When the added concentration of zinc sulfate was 0.03g / L, the fermentation unit of acarbose was increased to 8389ug / mL; when manganese sulfate was added alone to the fermentation medium and the added concentration of manganese sulfate was 0.005-0.1g / L, the fermentation unit of acarbose could be effectively increased. When the added concentration of manganese sulfate was 0.05g / L, the fermentation unit of acarbose was increased to 8368ug / mL.

[0045] Example 2

[0046] Try a small zoom.

[0047] Scoop out a 1x1cm-sized, fully grown bacterial moss and place it in a shake flask seed culture medium. Culture it at 28°C and 250rpm for 48 hours. Inoculate the cultured shake flask seed solution into the seed tank at an inoculation rate of 0.3% of the volume of the seed tank culture medium. Culture it at 28°C for 48 hours. 。 2.5t fermentation tank, feed volume 1.5t, fermentation medium prepared and sterilized, after the medium cooled, add 5L of 9g / L copper sulfate aqueous solution to the fermentation tank separately, then add the seed liquid in the seed tank to the fermentation tank at a 10% inoculation rate, and culture at 28℃ for 144h.

[0048] The seed tank culture medium includes the following components and mass concentrations: soybean powder 20g / L, glycerol 10g / L, corn starch 40g / L, calcium carbonate 3g / L and 0.5g / L of chloranil. The seed tank volume is 500L, the liquid loading volume is 250L, and the pH of the culture medium before sterilization is 6.8-7.0 。

[0049] The fermentation tank culture medium includes the following components and mass concentrations: glucose 40g / L, maltose 20g / L, soybean powder 20g / L, sodium glutamate 3g / L, calcium carbonate 2.5g / L, potassium dihydrogen phosphate 1.5g / L, ferric chloride 0.5g / L, calcium chloride 2g / L and bubo 0.5g / L. The fermentation tank volume is 2.5t, the fermentation medium loading volume is 60%, and the medium pH is 6.8-7.0 before sterilization. 。

[0050] The acarbose content in the fermentation broth was detected by HPLC. The fermentation method can increase the acarbose unit to 8.53 g / L.

[0051] Similar to the above method, after the fermentation tank culture medium was sterilized and cooled, 5L of 9g / L zinc sulfate aqueous solution was added to the fermentation tank. Then the seed solution was added and cultured at 28°C for 144h. The acarbose content in the fermentation broth was detected by HPLC, and the content reached 8.62g / L 。

[0052] Similar to the above method, after the fermentation tank culture medium was sterilized and cooled, 5L of 15g / L manganese sulfate aqueous solution was added to the fermentation tank. Then the seed solution was added and cultured at 28°C for 144h. The acarbose content in the fermentation broth was detected by HPLC, and the content reached 8.55g / L 。

[0053] Example 3

[0054] Pilot scale-up.

[0055] Shovel out 1x1cm-sized, fully grown bacterial moss into the shake flask seed culture medium, and culture at 28°C, 250rpm for 48 hours. Inoculate the cultured shake flask seed liquid into the seed tank at an inoculation rate of 0.3% of the volume of the seed tank culture medium, and culture at 28°C for 48 hours. Three 10t fermentation tanks, each with a liquid volume of 6t, prepare and sterilize the fermentation medium. After the culture medium is cooled, add 5L of 36g / L copper sulfate aqueous solution separately, and then add 50g / L ferric chloride aqueous solution separately to the fermentation tank. The volumes of ferric chloride aqueous solution added to the three fermentation tanks are 60L for tank No. 1, 180L for tank No. 2, and 240L for tank No. 3, respectively. Then, the seed liquid in the seed tank is connected to the fermentation tank at an inoculation rate of 10%, and cultured at 28°C for 144 hours.

[0056] Similar to the above method, after the fermentation tank culture medium was sterilized and cooled, 5 L of 36 g / L zinc sulfate aqueous solution was added to each of the three fermentation tanks, and then 60 L of tank 4, 180 L of tank 5, and 240 L of tank 6 were added to the fermentation tanks, respectively. Then, the seed solution was added and cultured at 28°C for 144 h.

[0057] Similar to the above method, after the fermentation tank culture medium is sterilized and cooled, 5L of 60g / L manganese sulfate aqueous solution is added to each of the three fermentation tanks, and then 60L of tank 7, 180L of tank 8, and 240L of tank 9 are added to the fermentation tanks. Then, the seed solution is added and cultured at 28°C for 144h. 。

[0058] The seed tank culture medium includes the following components and mass concentrations: soybean powder 20g / L, glycerol 10g / L, corn starch 40g / L, calcium carbonate 3g / L and 0.5g / L of chloranil. The seed tank volume is 1t, the liquid volume is 600L, and the pH of the culture medium before sterilization is 6.8-7.0 。

[0059] The fermentation tank culture medium also includes the following components and mass concentrations: glucose 40g / L, maltose 20g / L, soybean powder 20g / L, sodium glutamate 3g / L, calcium carbonate 2.5g / L, potassium dihydrogen phosphate 1.5g / L, calcium chloride 2g / L and 0.5g / L of folate. The fermentation tank volume is 10t, and the culture medium pH is 6.8-7.0 before sterilization.

[0060] The acarbose content in the fermentation broth was detected by HPLC. The fermentation method of Example 3 can increase the acarbose unit to 8.83 g / L, and the fermentation period is short, which is 144 h. The results in Table 2 show the effect of different ferric chloride concentrations on the acarbose yield.

[0061] Table 2 Effect of different ferric chloride concentrations on acarbose content

[0062]

[0063]

[0064] The results showed that compared with directly preparing it into fermentation medium, the fermentation unit of acarbose could be increased by adding ferric chloride alone.

[0065] Example 4

[0066] Try a small zoom.

[0067] Prepare the seed solution according to the method of Example 3, scoop out 1x1cm-sized, fully grown bacterial moss into the shake flask seed culture medium, and culture at 28°C, 250rpm for 48 hours. Inoculate the cultured shake flask seed solution into the seed tank at an inoculation rate of 0.3% of the volume of the seed tank culture medium, and culture at 28°C for 48 hours. Prepare the fermentation medium in 3 fermentation tanks, add copper sulfate, zinc sulfate and manganese sulfate to the culture medium, respectively, with final concentrations of 0.03g / L, 0.03g / L and 0.05g / L ，Then the fermentation medium was sterilized at 121° C. for 30 min, cooled, inoculated with the cultured seed solution at a 10% inoculation rate, and cultured for 144 h to obtain acarbose fermentation solution.

[0068] The fermentation tank culture medium also includes the following components and mass concentrations: glucose 40g / L, maltose 20g / L, soybean powder 20g / L, sodium glutamate 3g / L, calcium carbonate 2.5g / L, potassium dihydrogen phosphate 1.5g / L, ferric chloride 0.5g / L, calcium chloride 2g / L and bubo 0.5g / L. The fermentation tank volume is 2.5t, the feed volume is 1.5t, and the culture medium pH is 6.8-7.0 before sterilization 。

[0069] The fermentation broth was tested by HPLC. The acarbose production in the fermentation broth with copper sulfate added at 0.03 g / L, zinc sulfate added at 0.03 g / L, and manganese sulfate added at 0.05 g / L was 8.31 g / L, 8.19 g / L, and 8.27 g / L, respectively. 。

[0070] The results showed that the fermentation unit of acarbose can be increased by preparing the fermentation medium with metal ions such as copper sulfate, zinc sulfate and manganese sulfate together with ferric chloride and other materials, and the fermentation unit is slightly lower than that when the metal ions are added alone.

[0071] Comparative Example 1

[0072] Prepare the shake flask seed liquid according to the method of Example 1, prepare the fermentation medium, and divide it into 3 fermentation bottles after preparation. The shake flask seed liquid is connected to a 500mL fermentation bottle at a 7% inoculation amount, and the amount is 50mL, 250rpm, and cultured at 28°C for 144h. Different volumes of 10g / L cobalt chloride aqueous solution were added to the 3 fermentation bottles, respectively, with the volumes of 0mL, 0.05mL and 0.25mL, respectively, and the fermentation bottle without cobalt chloride was used as a control. The fermentation medium contains the following components and mass concentrations: per 100mL, 4g glucose, 2g maltose, 2g soybean powder, 0.3g sodium glutamate, 0.25g calcium carbonate, 0.15g dipotassium hydrogen phosphate, 0.05g ferric chloride, 0.2g calcium chloride, and the pH of the fermentation medium before sterilization is 6.8-7.0.

[0073] The fermentation broth was tested by HPLC, and the acarbose fermentation units in the fermentation broth with cobalt chloride added in an amount of 0.01 g / L and 0.05 g / L were 6.83 g / L and 5.65 g / L, respectively.

[0074] The results showed that the addition of cobalt chloride could not increase the fermentation unit of acarbose.

[0075] Comparative Example 2

[0076] Prepare the shake flask seed liquid according to the method of Example 1, prepare the fermentation medium, and divide it into 4 fermentation bottles after preparation. The seed liquid is connected to a 500mL fermentation bottle at a 7% inoculation amount, and the amount is 50mL, 250rpm, and cultured at 28°C for 144h. Different volumes of 10g / L magnesium chloride aqueous solution were added to 4 fermentation bottles, respectively, with the volumes of 0mL, 0.25mL, 0.5mL and 2.5mL, respectively, and the shake flask without magnesium chloride was used as a control. The fermentation medium contains the following components and mass concentrations: per 100mL, 4g glucose, 2g maltose, 2g soybean powder, 0.3g sodium glutamate, 0.25g calcium carbonate, 0.15g dipotassium hydrogen phosphate, 0.05g ferric chloride, 0.2g calcium chloride, and the fermentation medium pH is 6.8-7.0 before sterilization.

[0077] The fermentation broth was tested by HPLC, and the acarbose production in the fermentation broth with magnesium chloride addition of 0.05 g / L, 0.1 g / L, and 0.5 g / L was 6.52 g / L, 6.13 g / L, and 4.86 g / L, respectively.

[0078] The results showed that the addition of magnesium chloride could not increase the fermentation unit of acarbose.

[0079] Comparative Example 3

[0080] Pilot scale-up.

[0081] Prepare the seed solution according to the method of Example 3, scoop out 1x1cm-sized, fully grown bacterial moss into the shake flask seed culture medium, and shake and culture at 28°C, 250rpm for 48h. Inoculate the cultured shake flask seed solution into the seed tank at an inoculation rate of 0.3% of the volume of the seed tank culture medium, and culture at 28°C for 48h. Three 10t fermentation tanks, each with a liquid volume of 6t, prepare and sterilize the fermentation medium. After the culture medium is cooled, copper sulfate aqueous solution, zinc sulfate aqueous solution, and manganese sulfate aqueous solution are added to the three tanks separately, and the mass concentrations in the culture medium are 0.1g / L, 0.1g / L, and 0.5g / L, respectively. Inoculate the cultured seed solution with an inoculation rate of 10%, culture for 144h, and obtain acarbose fermentation liquid.

[0082] The fermentation tank culture medium also includes the following components and mass concentrations: glucose 40g / L, maltose 20g / L, soybean powder 20g / L, sodium glutamate 3g / L, calcium carbonate 2.5g / L, potassium dihydrogen phosphate 1.5g / L, ferric chloride 0.5g / L, calcium chloride 2g / L and bubo 0.5g / L. The fermentation tank volume is 10t, the feed volume is 6t, and the pH of the culture medium before sterilization is 6.8-7.0.

[0083] According to this fermentation process, the acarbose contents in the fermentation broth to which 0.1 g / L copper sulfate, 0.1 g / L zinc sulfate, and 0.5 g / L manganese sulfate were added were 5.86 g / L, 6.23 g / L, and 6.02 g / L, respectively.

Claims

1. A method for increasing acarbose fermentation units, characterized in that: The metal ions are added to the fermentation medium. The metal ions are added to the prepared fermentation medium in a separate manner.

2. The method according to claim 1, characterized in that: The metal ions are one or more of copper ions, zinc ions and manganese ions.

3. The method according to claim 2, characterized in that: The metal ions are added together with iron ions.

4. The method according to claim 3, characterized in that: The iron ions are added in the form of ferric chloride.

5. The method according to claim 4, characterized in that: The metal ions are added in the form of sulfate and / or hydrochloride; After adding the copper salt, the concentration in the fermentation medium is 0.005-0.05 g / L, or 0.03 g / L; After adding the zinc salt, the concentration in the fermentation medium is 0.005-0.05 g / L, or 0.03 g / L; after adding the manganese salt, the concentration in the fermentation medium is 0.005-0.1 g / L, or 0.05 g / L.

6. The method according to claim 5, characterized in that: After the ferric chloride is added, the concentration of the ferric chloride in the fermentation medium is 0.2-2.5 g / L, or 0.5-2.0 g / L, or 1.5 g / L.

7. The method according to claim 6, characterized in that: The metal ions are added in the form of sulfate and / or hydrochloride aqueous solution.

8. The method according to claim 7, characterized in that: The concentration of the sulfate or hydrochloride aqueous solution is 1 to 100 g / L, or 5 to 60 g / L.

9. The method according to any one of claims 1 to 8, characterized in that: The fermentation medium contains the following components in mass concentrations: per 100 mL, 3-6 g of glucose, 2-4 g of maltose, 1.5-3 g of soybean powder, 0.3-0.6 g of sodium glutamate, 0.05-0.2 g of dipotassium hydrogen phosphate, 0.03-0.15 g of ferric chloride, 0.2-0.5 g of calcium chloride, and 0.2-0.5 g of calcium carbonate.

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