Fermentation medium and application thereof

Abstract

The invention discloses a fermentation medium and application thereof, and belongs to the technical field of microbial fermentation. The fermentation culture medium comprises a basic culture medium and cofactors, wherein the cofactor comprises at least one of the following components: salicylic acid with the mass concentration of 0.01-2 g / L, mercaptoethanol with the mass concentration of 0.01-2 g / L, betaine with the mass concentration of 0.01-2 g / L, spermidine with the mass concentration of 0.01-2 g / L and sodium butyrate with the mass concentration of 0.01-2 g / L. After the cofactor is added into the fermentation culture medium, when the fermentation culture medium is used for producing histidine through microbial fermentation, the cofactor is used for regulating cell metabolism, enhancing key enzyme activity and improving cell tolerance, so that the yield of histidine is remarkably increased, and the fermentation cost is remarkably reduced; therefore, the fermentation culture medium has a good application prospect in preparation of histidine through microbial fermentation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a fermentation medium and application thereof. Background Art

[0002] Histidine (L-histidine) is an essential amino acid with important physiological functions, such as participating in chemical signal transduction, regulating acid-base balance and synthesizing histamine. Its basic physical and chemical properties include: white crystalline powder, easily soluble in water, insoluble in ethanol and ether, melting point of 270-275℃, isoelectric point of 7.59. Histidine is widely used in the fields of food, medicine and biotechnology, such as nutritional supplements, drug intermediates and feed additives. The global histidine market will reach US$2 billion, and market demand continues to grow, especially in the fields of nutritional supplements and medicine.

[0003] Histidine is an essential amino acid that is widely used in the fields of food, medicine and biotechnology. Traditional methods for producing histidine mainly include chemical synthesis, chemical-enzymatic synthesis and microbial fermentation. The chemical synthesis method directly synthesizes histidine through chemical reactions, which has the advantages of stable yield, high purity and strong controllability, but it also has disadvantages such as serious environmental pollution, high cost and complex operation. The chemical-enzymatic synthesis method combines the advantages of chemical synthesis and enzyme catalysis, reduces the generation of pollutants, has high selectivity and moderate cost, but because the enzyme is easily inactivated in a chemical environment and the operation is complicated, the large-scale application of this method is limited. The microbial fermentation method converts carbon sources (such as glucose) into histidine through the metabolic activities of specific microorganisms. Commonly used microorganisms include Bacillus subtilis ( Bacillus subtilis ), Escherichia coli ( E. coli ) and yeast, etc. This method is environmentally friendly, low-cost, highly adjustable and sustainable, but the yield and conversion rate are affected by factors such as culture conditions and strain variation, the purity is low, and optimization is difficult.

[0004] In order to improve the efficiency of histidine production by microbial fermentation, researchers have adopted a variety of optimization strategies. For example, gene editing technology can be used to modify the metabolic pathways of production strains, enhance the activity of key enzymes, and reduce the generation of by-products. It is also possible to adjust the ratio of carbon and nitrogen sources, add cofactors and precursors, and provide a more suitable growth environment and nutritional conditions. Alternatively, the growth and metabolic environment of the strain can be optimized by adjusting parameters such as temperature, pH, dissolved oxygen, and stirring speed. However, existing optimization methods have problems such as low histidine production. Summary of the invention

[0005] The object of the present invention is to provide a fermentation medium and its application, which are used to solve the problem of low histidine yield in the existing optimization method.

[0006] In a first aspect, the present invention provides a fermentation medium, comprising a basal medium and a cofactor; wherein the cofactor comprises at least one of the following components: salicylic acid with a mass concentration of 0.01-2 g / L, mercaptoethanol with a mass concentration of 0.01-2 g / L, betaine with a mass concentration of 0.01-2 g / L, spermidine with a mass concentration of 0.01-2 g / L, and sodium butyrate with a mass concentration of 0.01-2 g / L.

[0007] In the present invention, the inventors have found that when cofactors are added to the fermentation medium and the fermentation medium is used for microbial fermentation to produce histidine, the cofactors significantly increase the yield of histidine by regulating cell metabolism, enhancing key enzyme activities and improving cell tolerance, and significantly reduce the fermentation cost.

[0008] In some embodiments, the cofactors include the following components: salicylic acid with a mass concentration of 0.05-1 g / L, mercaptoethanol with a mass concentration of 0.05-1 g / L, betaine with a mass concentration of 0.05-1 g / L, spermidine with a mass concentration of 0.05-1 g / L, and sodium butyrate with a mass concentration of 0.05-1 g / L.

[0009] In the present invention, the inventors further studied and found that after salicylic acid, mercaptoethanol, betaine, spermidine and sodium butyrate were combined, the components acted synergistically to further increase the production of histidine.

[0010] In some embodiments, the basic culture medium includes the following components: sucrose 40-60 g / L, soybean meal 15-25 g / L, corn steep liquor 10-20 g / L, NaH2PO4 2-4 g / L, MgSO4 0.5-1.5 g / L, NaCl 2-4 g / L, and pH value is 6.5-7.5.

[0011] In some embodiments, the basic culture medium includes the following components: sucrose 50 g / L, soybean meal 20 g / L, corn steep liquor 15 g / L, NaH2PO4 3 g / L, MgSO4 1 g / L, NaCl 3 g / L, and pH value is 7.0.

[0012] It can be understood that the components in the basal culture medium can be routinely adjusted according to the type of fermentation microorganism. In the present invention, the basal culture medium preferably includes the following components: sucrose 50g / L, soybean meal 20g / L, corn steep liquor 15g / L, NaH2PO43g / L, MgSO41g / L, NaCl 3g / L, and pH value is 7.0.

[0013] In a second aspect, the present invention provides use of any of the above fermentation media in microbial fermentation to produce histidine.

[0014] In some embodiments, the microorganism comprises Bacillus subtilis.

[0015] It is understandable that the microorganism can be routinely selected according to the use requirements, as long as it can produce histidine. In the present invention, the microorganism preferably includes Bacillus subtilis.

[0016] In some embodiments, the production of histidine by microbial fermentation specifically includes the following steps: S1, inoculating the microorganism into a primary seed culture medium for culturing to obtain a primary seed solution; S2, inoculating the primary seed solution into a secondary seed culture medium for culturing to obtain a secondary seed solution; S3, inoculating the secondary seed solution into a basal culture medium for fermentation culture, then adding cofactors, and simultaneously feeding glucose, continuing the fermentation culture, and obtaining a fermentation liquid containing histidine.

[0017] The preparation method of histidine produced by microbial fermentation provided by the invention is simple, the raw materials used are cheap and easily available, the cost is low, and it is convenient for large-scale production and application.

[0018] In some embodiments, in step S1, the primary seed culture medium includes the following components: 3-10 g / L glucose, 5-15 g / L peptone, 3-10 g / L yeast extract powder, 5-15 g / L sodium chloride, and a pH value of 6.5-7.5; the culturing specifically includes: culturing for 8-16 hours at a temperature of 25-35°C and a rotation speed of 200-250 rpm.

[0019] In some preferred embodiments, the primary seed culture medium includes the following components: 5 g / L glucose, 10 g / L peptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, and a pH value of 7.0; the culture specifically includes: culturing for 12 hours at a temperature of 30°C and a rotation speed of 220 rpm.

[0020] In some embodiments, in step S2, the secondary seed culture medium includes the following components: 5-15 g / L starch, 15-25 g / L soybean meal, 5-15 g / L corn syrup, and a pH value of 7.0-8.0; the culturing specifically includes: culturing for 36-48 hours at a temperature of 25-35°C and a rotation speed of 200-250 rpm.

[0021] In some preferred embodiments, the secondary seed culture medium includes the following components: 10 g / L starch, 20 g / L soybean meal, 10 g / L corn steep liquor, and a pH value of 7.5; the cultivation specifically includes: culturing for 42 hours at a temperature of 30°C and a rotation speed of 220 rpm.

[0022] In some embodiments, in step S3, inoculating the secondary seed liquid into the basic culture medium for fermentation culture specifically includes: culturing for 8-16 hours at a temperature of 30-33°C, a rotation speed of 180-220rpm, and a dissolved oxygen of 10-30%; adding glucose at a mass concentration of 40-60%, and controlling the glucose content in the system to 1-2g / L; continuing the fermentation culture includes: culturing for 30-40 hours at a temperature of 30-33°C, a rotation speed of 180-220rpm, and a dissolved oxygen of 10-30%.

[0023] In some preferred embodiments, inoculating the secondary seed liquid into the basic culture medium for fermentation culture specifically includes: culturing for 12 hours at a temperature of 31°C, a rotation speed of 200 rpm, and a dissolved oxygen of 20%; adding glucose at a mass concentration of 50%, and controlling the glucose content in the system to be 1.5 g / L; continuing the fermentation culture includes: culturing for 38 hours at a temperature of 31°C, a rotation speed of 200 rpm, and a dissolved oxygen of 20%.

[0024] The beneficial effects of the present invention are as follows: different from the prior art, after adding cofactors to the fermentation medium, when the fermentation medium is used for microbial fermentation to produce histidine, the cofactors significantly increase the yield of histidine by regulating cell metabolism, enhancing key enzyme activities and improving cell tolerance, and significantly reduce the fermentation cost; therefore, the fermentation medium has good application prospects in the preparation of histidine by microbial fermentation. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0027] Example 1 A fermentation medium, comprising a basal medium and a cofactor; wherein the basal medium comprises the following components: 50 g / L sucrose, 20 g / L soybean meal, 15 g / L corn steep liquor, 3 g / L NaH2PO4, 1 g / L MgSO4, 3 g / L NaCl, and a pH value of 7.0; and the cofactor comprises salicylic acid with a mass concentration of 0.05 g / L.

[0028] A method using Bacillus subtilis ( Bacillus subtilis ) A method for producing histidine by fermentation, comprising the following steps: S1. Streak Bacillus subtilis onto an LB solid plate (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 2 g / L agar powder, pH 7.0), culture overnight at 30°C, then pick a single colony and inoculate it into a 250 mL shake flask containing 50 mL of primary seed culture medium (5 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0), culture at 30°C and 220 rpm for 12 h to obtain a primary seed solution; S2, inoculate the primary seed solution obtained in step S1 into a 1L shake flask containing 200mL of secondary seed culture medium (10g / L starch, 20g / L soybean meal, 10g / L corn steep liquor, pH value of 7.5) at a 10% inoculation amount, and culture for 42h at a temperature of 30°C and a rotation speed of 220rpm to obtain a secondary seed solution; S3, the secondary seed solution obtained in step S2 was inoculated into a 50L fermenter containing 30L basal medium (sucrose 50g / L, soybean meal 20g / L, corn steep liquor 15g / L, NaH2PO43g / L, MgSO41g / L, NaCl 3g / L, pH value 7.0) at an inoculum size of 0.1%, and the culture was carried out at a temperature of 31°C, a rotation speed of 200rpm, and a dissolved oxygen of 20% for 12h, and then salicylic acid was added, and glucose with a mass concentration of 50% was fed at the same time, and the content of glucose in the system was controlled to be 1.5g / L, and the culture was continued at a temperature of 31°C, a rotation speed of 200rpm, and a dissolved oxygen of 20% for 38h to obtain a fermentation liquid containing histidine.

[0029] Example 2 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis ) The method for producing histidine by fermentation is basically the same as that in Example 1, except that the cofactor includes mercaptoethanol with a mass concentration of 0.05 g / L.

[0030] Example 3 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis ) The method for producing histidine by fermentation is basically the same as that in Example 1, except that the cofactor includes betaine with a mass concentration of 0.05 g / L.

[0031] Example 4 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis) The method for producing histidine by fermentation is basically the same as that in Example 1, except that the cofactor includes spermidine at a mass concentration of 0.05 g / L.

[0032] Example 5 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis ) The method for producing histidine by fermentation is basically the same as that in Example 1, except that the cofactor includes sodium butyrate with a mass concentration of 0.05 g / L.

[0033] Example 6 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis ) The method for producing histidine by fermentation is basically the same as that in Example 1, except that the cofactor includes salicylic acid with a mass concentration of 2 g / L.

[0034] Example 7 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis ) The method for producing histidine by fermentation is basically the same as that in Example 1, except that the cofactor includes mercaptoethanol with a mass concentration of 2 g / L.

[0035] Example 8 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis ) The method for producing histidine by fermentation is basically the same as that in Example 1, except that the cofactor includes betaine with a mass concentration of 2 g / L.

[0036] Example 9 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis ) The method for producing histidine by fermentation is basically the same as that in Example 1, except that the cofactor includes spermidine at a mass concentration of 2 g / L.

[0037] Example 10 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis ) The method for producing histidine by fermentation is basically the same as that in Example 1, except that the cofactor includes sodium butyrate with a mass concentration of 2 g / L.

[0038] Embodiment 11 A fermentation medium, comprising a basal medium and cofactors; wherein the basal medium comprises the following components: 50 g / L sucrose, 20 g / L soybean meal, 15 g / L corn steep liquor, 3 g / L NaH2PO4, 1 g / L MgSO4, 3 g / L NaCl, and a pH value of 7.0; and the cofactors comprise salicylic acid with a mass concentration of 0.05 g / L, mercaptoethanol with a mass concentration of 0.05 g / L, betaine with a mass concentration of 0.05 g / L, spermidine with a mass concentration of 0.05 g / L, and sodium butyrate with a mass concentration of 0.05 g / L.

[0039] A method using Bacillus subtilis ( Bacillus subtilis ) A method for producing histidine by fermentation, comprising the following steps: S1. Streak Bacillus subtilis onto an LB solid plate (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 2 g / L agar powder, pH 7.0), culture overnight at 30°C, then pick a single colony and inoculate it into a 250 mL shake flask containing 50 mL of primary seed culture medium (5 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0), culture at 30°C and 220 rpm for 12 h to obtain a primary seed solution; S2, inoculate the primary seed solution obtained in step S1 into a 1L shake flask containing 200mL of secondary seed culture medium (10g / L starch, 20g / L soybean meal, 10g / L corn steep liquor, pH value of 7.5) at a 10% inoculation amount, and culture for 42h at a temperature of 30°C and a rotation speed of 220rpm to obtain a secondary seed solution; S3, the secondary seed solution obtained in step S2 was inoculated into a 50L fermenter containing 30L basal medium (sucrose 50g / L, soybean meal 20g / L, corn steep liquor 15g / L, NaH2PO43g / L, MgSO41g / L, NaCl 3g / L, pH value 7.0) at an inoculum size of 0.1%, and the culture was carried out at a temperature of 31°C, a rotation speed of 200rpm, and a dissolved oxygen of 20% for 12h, and then salicylic acid, mercaptoethanol, betaine, spermidine and sodium butyrate were added, and glucose with a mass concentration of 50% was added at the same time, and the content of glucose in the system was controlled to be 1.5g / L, and the culture was continued at a temperature of 31°C, a rotation speed of 200rpm, and a dissolved oxygen of 20% for 38h to obtain a fermentation liquid containing histidine.

[0040] Example 12 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis) The method for producing histidine by fermentation is basically the same as that in Example 11, except that the cofactors include salicylic acid with a mass concentration of 0.5 g / L, mercaptoethanol with a mass concentration of 0.5 g / L, betaine with a mass concentration of 0.5 g / L, spermidine with a mass concentration of 0.5 g / L, and sodium butyrate with a mass concentration of 0.5 g / L.

[0041] Example 13 In this embodiment, the fermentation medium is Bacillus subtilis ( Bacillus subtilis ) The method for producing histidine by fermentation is basically the same as that in Example 11, except that the cofactors include salicylic acid with a mass concentration of 1 g / L, mercaptoethanol with a mass concentration of 1 g / L, betaine with a mass concentration of 1 g / L, spermidine with a mass concentration of 1 g / L, and sodium butyrate with a mass concentration of 1 g / L.

[0042] Comparative Example 1 In this comparative example, the fermentation medium and the Bacillus subtilis ( Bacillus subtilis ) The method for producing histidine by fermentation is basically the same as that in Example 1, except that no cofactor is added.

[0043] Test of Histidine Content Produced by Bacillus subtilis The histidine content in the fermentation broth prepared in Examples 1-13 and Comparative Example 1 was tested, and the results are shown in Table 1 below.

[0044] Table 1 Test results of histidine content in fermentation broth

[0045] It can be seen from the data in Table 1 that compared with the fermentation culture in Comparative Example 1 in which no cofactors were added, cofactors were added to the fermentation medium in Examples 1-13. It was found that the histidine content was significantly increased when Bacillus subtilis was fermented using the fermentation medium in Examples 1-13; and after combining several cofactors, the histidine content was further increased through the synergistic effect between the components.

[0046] In summary, the present invention adds cofactors to the fermentation medium, and when the fermentation medium is used for microbial fermentation to produce histidine, the cofactors significantly increase the yield of histidine by regulating cell metabolism, enhancing key enzyme activities and improving cell tolerance.

[0047] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0048] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A fermentation medium, characterized in that The fermentation medium includes a basal medium and cofactors; Wherein, the cofactor includes at least one of the following components: salicylic acid with a mass concentration of 0.01-2g / L, mercaptoethanol with a mass concentration of 0.01-2g / L, betaine with a mass concentration of 0.01-2g / L, spermidine with a mass concentration of 0.01-2g / L, and sodium butyrate with a mass concentration of 0.01-2g / L.

2. The fermentation medium according to claim 1, characterized in that The cofactor comprises the following components: salicylic acid with a mass concentration of 0.05-1g / L, mercaptoethanol with a mass concentration of 0.05-1g / L, betaine with a mass concentration of 0.05-1g / L, spermidine with a mass concentration of 0.05-1g / L, and sodium butyrate with a mass concentration of 0.05-1g / L.

3. The fermentation medium according to claim 1, characterized in that The basic culture medium comprises the following components: sucrose 40-60 g / L, soybean meal 15-25 g / L, corn steep liquor 10-20 g / L, NaH2PO4 2-4 g / L, MgSO4 0.5-1.5 g / L, NaCl 2-4 g / L, and a pH value of 6.5-7.

5.

4. The fermentation medium according to claim 3, characterized in that The basic culture medium comprises the following components: 50 g / L sucrose, 20 g / L soybean meal, 15 g / L corn steep liquor, 3 g / L NaH2PO4, 1 g / L MgSO4, 3 g / L NaCl, and a pH value of 7.

0.

5. Use of the fermentation medium according to any one of claims 1 to 4 in the production of histidine by microbial fermentation.

6. The use according to claim 5, characterized in that: The microorganism includes Bacillus subtilis.

7. The use according to claim 5, characterized in that: The microbial fermentation production of histidine specifically comprises the following steps: S1, inoculating the microorganism into a primary seed culture medium for culturing to obtain a primary seed solution; S2, inoculating the primary seed solution into a secondary seed culture medium for culturing to obtain a secondary seed solution; S3, inoculating the secondary seed liquid into the basic culture medium for fermentation culture, then adding cofactors, and simultaneously feeding glucose, and continuing the fermentation culture to obtain a fermentation liquid containing histidine.

8. The use according to claim 7, characterized in that: In step S1, the primary seed culture medium comprises the following components: 3-10 g / L glucose, 5-15 g / L peptone, 3-10 g / L yeast extract powder, 5-15 g / L sodium chloride, and a pH value of 6.5-7.5; The cultivation specifically includes: cultivating for 8-16 hours at a temperature of 25-35° C. and a rotation speed of 200-250 rpm.

9. The use according to claim 7, characterized in that: In step S2, the secondary seed culture medium comprises the following components: 5-15 g / L starch, 15-25 g / L soybean meal, 5-15 g / L corn steep liquor, and a pH value of 7.0-8.0; The cultivation specifically includes: cultivating for 36-48 hours at a temperature of 25-35° C. and a rotation speed of 200-250 rpm.

10. The use according to claim 7, characterized in that: In step S3, inoculating the secondary seed liquid into the basic culture medium for fermentation culture specifically includes: culturing for 8-16 hours at a temperature of 30-33° C., a rotation speed of 180-220 rpm, and a dissolved oxygen of 10-30%; The mass concentration of the glucose added is 40-60%, and the content of the glucose in the system is controlled to be 1-2 g / L; Continuing the fermentation culture includes: culturing for 30-40 hours at a temperature of 30-33° C., a rotation speed of 180-220 rpm, and a dissolved oxygen content of 10-30%.

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