A fermentation medium for producing trichotoxin and a fermentation method for increasing the yield of trichotoxin

Abstract

The present application relates to a kind of production of Fermentation Medium of trichotoxin and a kind of fermentation method for improving the yield of trichotoxin, belong to biological fermentation technical field.The present application uses pea starch, glucose as carbon source, uses soybean meal, corn serum as nitrogen source, with suitable inorganic salt (sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate, sodium selenite), metabolic auxiliary factor (pyridoxine) and amino acid, prepare a kind of suitable for bacillus subtilis production trichotoxin Fermentation Medium, the cost of the Fermentation Medium is low, and compared with the Fermentation Medium for producing trichotoxin in prior art, the yield of trichotoxin is significantly improved, provides new train of thought for the cost reduction and benefit increase in the process of large-scale production of trichotoxin.

Description

Technical Field

[0001] This invention relates to a fermentation culture medium for producing tahinitrofurantoin and a fermentation method for increasing the yield of tahinitrofurantoin, belonging to the field of biofermentation technology. Background Technology

[0002] Antimicrobial peptides (AMPs) are a class of small molecule polypeptides widely distributed in the biological world, possessing broad-spectrum antibacterial activity and unique immunomodulatory functions. As a key bridge connecting innate and adaptive immunity, they not only directly kill pathogens through physical membrane disruption mechanisms, but also play a central role in maintaining host homeostasis and controlling infection and inflammation through their complex immunomodulatory functions. Current research indicates that antimicrobial peptides possess broad-spectrum antibacterial, antiviral, antitumor, antiprotozoal, and immunomodulatory activities, and are not prone to inducing drug resistance. Antimicrobial peptides exhibit various immunomodulatory effects, including regulating inflammatory responses, chemotaxis of immune cells, promotion of cell differentiation, and activation of the innate and adaptive immune systems. These diverse immunomodulatory properties make antimicrobial peptides a promising candidate for treating infectious and immune diseases.

[0003] Snblancin is derived from Bacillus subtilis (Bacillus subtilis). Bacillus subtilis A novel antimicrobial peptide isolated and purified from fermentation products has been developed. Composed of 37 amino acid residues, its molecular weight is 3879.8 Da (based on 2018 international relative atomic mass). The structure of this peptide contains two disulfide bonds formed by five cysteine ​​residues, exhibiting structural stability and good resistance to high temperatures, acids, pepsin, and trypsin. Clinical trials have shown that this peptide can enhance the immune function of livestock, poultry, and aquatic animals, improve the effectiveness of vaccines, and has high safety with no drug residues. This peptide has been approved as a new feed additive and has received considerable attention in recent years, with demand continuously increasing. Therefore, increasing the production of this peptide to meet market demand is essential.

[0004] Chinese invention patent CN112029697B, published on September 21, 2021, discloses a recombinant Bacillus subtilis strain and its application. Specifically, it discloses the genetic engineering modification of Bacillus subtilis to obtain a high-yield recombinant Bacillus subtilis strain JY011802, which was deposited at the China General Microbiological Culture Collection Center on October 31, 2018, with accession number CGMCC NO.16667. The recombinant Bacillus subtilis strain JY011802 can produce 3100 mg / L of 3100 mg / L of 3100 mg / L of 3100 mg / L.

[0005] Chinese invention patent application CN115960982A, published on April 14, 2023, discloses a method for the industrial-scale production of tahinitrofurantoin through high-density fermentation. The method includes the following steps: revitalizing and activating the Bacillus subtilis fermentation strain for tahinitrofurantoin, using primary seed, secondary seed, a 30 L seed tank, and a 3 m³ [unclear text - likely a continuation of the previous sentence]. 3 Seed container, 30 m 3 Fermentation in a fermenter at 30 m 3 Feeding and tank emptying are carried out during the fermentation process. Fermentation in stages during the early stages of thiamphetapeptide production can prevent excessive growth of Bacillus subtilis and reduce the secretion of other metabolites; feeding in stages during the middle and late stages of thiamphetapeptide production can meet the nutritional needs of Bacillus subtilis for thiamphetapeptide production. Using this method for thiamphetapeptide production can increase the thiamphetapeptide content in the fermentation broth, shorten the fermentation time, and improve production efficiency.

[0006] Therefore, it can be seen that subtilisin is mainly produced through fermentation of Bacillus subtilis. Currently, the commonly used and effective methods are to obtain high-yielding strains through artificial screening, to modify Bacillus subtilis through genetic engineering, or to optimize the fermentation process. Considering the limitations of strain screening and genetic engineering of Bacillus subtilis, optimizing the fermentation process is a better breakthrough. Because different strains have different fermentation processes, current fermentation processes often cannot maximize yield, thus preventing the full realization of its high-yield performance. This is an objective problem that urgently needs to be addressed. Summary of the Invention

[0007] The first objective of this invention is to provide a fermentation medium for producing subtilisin, which provides a new fermentation medium with a significantly improved yield of the target product compared to the existing technology of using Bacillus subtilis to produce subtilisin.

[0008] The second objective of this invention is to provide a fermentation method for increasing the yield of bismuth subtilisin, thereby addressing the problem that the yield of bismuth subtilisin needs to be increased in the prior art.

[0009] To achieve the above objectives, the technical solution of the fermentation culture medium for producing kiwifruit peptides in this invention is as follows: A fermentation medium for producing tahinitroglycerin comprises the following components in the following amounts: pea starch 25-35 g / L, glucose 8-15 g / L, soybean meal 15-25 g / L, corn syrup 10-20 g / L, sodium chloride 1-3 g / L, dipotassium hydrogen phosphate 0.01-0.1 g / L, potassium dihydrogen phosphate 0.01-0.1 g / L, ammonium sulfate 1.5-2.5 g / L, magnesium sulfate 0.2-1.5 g / L, sodium selenite 1-5 mg / L, pyridoxine 0.1-0.5 g / L, and cysteine ​​0.05-0.3 g / L.

[0010] The beneficial effects of the above technical solution are as follows: The fermentation medium for producing subtilisin of the present invention is an improved invention. The present invention uses pea starch and glucose as carbon sources, soybean meal and corn syrup as nitrogen sources, supplemented with suitable inorganic salts (sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate, sodium selenite), metabolic cofactors (pyridoxine), and amino acids to prepare a fermentation medium suitable for Bacillus subtilis to produce subtilisin. This fermentation medium has low cost, and compared with the existing fermentation media used for producing subtilisin, the yield of subtilisin is significantly increased, providing a new approach to cost reduction and efficiency improvement in the large-scale production of subtilisin.

[0011] As a further improvement, the following components are included: pea starch 30-35 g / L, glucose 10-15 g / L, soybean meal 20-25 g / L, corn syrup 15-20 g / L, sodium chloride 2-3 g / L, dipotassium hydrogen phosphate 0.05-0.1 g / L, potassium dihydrogen phosphate 0.05-0.1 g / L, ammonium sulfate 2-2.5 g / L, magnesium sulfate 0.2-1 g / L, sodium selenite 3-5 mg / L, pyridoxine 0.1-0.5 g / L, and cysteine ​​0.2-0.3 g / L.

[0012] As a further improvement, the following components are included: pea starch 35g / L, glucose 10g / L, soybean meal 25g / L, corn syrup 15g / L, sodium chloride 3g / L, dipotassium hydrogen phosphate 0.05g / L, potassium dihydrogen phosphate 0.05g / L, ammonium sulfate 2.5g / L, magnesium sulfate 0.8~1g / L, sodium selenite 3~5mg / L, pyridoxine 0.1~0.5g / L, and cysteine ​​0.2g / L.

[0013] As a further improvement, the following components are included: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, ammonium sulfate 2.5 g / L, magnesium sulfate 0.85 g / L, sodium selenite 3 mg / L, pyridoxine 0.3 g / L, and cysteine ​​0.2 g / L.

[0014] As a further improvement, the pH of the fermentation medium is 7.0 to 7.5.

[0015] To achieve the above objectives, the technical solution of the fermentation method for increasing the yield of kiwifruit peptides in this invention is as follows: A fermentation method for increasing the yield of subtilisin involves inoculating Bacillus subtilis seed culture into the fermentation medium for producing subtilisin and then carrying out fermentation culture.

[0016] The beneficial effects of the above technical solution are as follows: Based on the fermentation medium for producing subtilisin, this invention provides a fermentation method that can effectively increase the yield of subtilisin. Using the fermentation method provided by this invention, the fermentation content of subtilisin reaches 3845.56 ug / mL in a 50L fermenter, which can significantly increase the fermentation yield of subtilisin and lay the foundation for large-scale fermentation production of subtilisin. Furthermore, the fermentation method of this invention is simple to operate and easy for large-scale fermentation production.

[0017] As a further improvement, the seed culture comprises 2%-5% of the fermentation medium volume.

[0018] As a further improvement, the fermentation culture temperature is 35-37°C.

[0019] As a further improvement, when the feed additive is used, the dissolved oxygen is controlled at 25%-45% during the fermentation and culture process, and alkaline water is added to control the pH to be no lower than 7.0.

[0020] As a further improvement, the fermentation process involves stirring and aeration, with the stirring speed at 100-300 rpm and the aeration rate at 200-400 m³ / h. 3 / h. Attached Figure Description

[0021] Figure 1 This is a liquid chromatogram of the content of 500mL flask-based horizontal thiamphetamine in Example 25 of the present invention; Figure 2 This is a liquid chromatogram of the content of 37-peptide in a 50L fermenter in Example 26 of the present invention. Figure 3 This is a liquid chromatogram of the content of 500mL flask-based triseptapeptide in Comparative Example 1 of the present invention. Figure 4 This is a liquid chromatogram of the content of 37-peptide of pyrimethanil in a 50L fermenter in Comparative Example 1 of the present invention. Figure 5 This is a liquid chromatogram of the content of 500mL shake flask of the present invention for the detection of 37-peptide of pyridamole. Figure 6 This is a liquid chromatogram of the content of 37-peptide of pyrimethanil in a 50L fermenter in Comparative Example 2 of the present invention. Figure 7 This is a liquid chromatogram of the content of 500 mL shake flask of culture medium from Comparative Example 1 in Comparative Example 4 of the present invention for the detection of thiamphetamine content. Figure 8This is a liquid chromatogram of the content of 500 mL of culture medium from Comparative Example 2 in Comparative Example 4 of the present invention for the detection of 37-peptide content of pyridostigmine in a shake flask. Figure 9 This is a liquid chromatogram of the content of 500 mL of culture medium in Example 1 of Comparative Example 4 of the present invention for the detection of 37-peptide content of pyridostigmine in a shake flask. Figure 10 This is a liquid chromatogram of the level cephalosporin content detected in a 500 mL shake flask of culture medium from Comparative Example 1 in Comparative Example 5 of the present invention. Figure 11 This is a liquid chromatogram of the level cephalosporin content detection in a 500 mL shake flask of culture medium from Comparative Example 2 in Comparative Example 5 of the present invention. Figure 12 This is a liquid chromatogram of the level cephalosporin content detected in a 500 mL shake flask of culture medium in Comparative Example 5 of the present invention. Detailed Implementation

[0022] Substantia nigra 37-peptide, composed of 37 amino acid residues, is stable and exhibits good resistance to high temperatures, acids, pepsin, and trypsin. It promotes growth, enhances the body's immune function, and improves the morphology and composition of the intestinal mucosa and gut microbiota. Furthermore, substantia nigra 37-peptide possesses unique advantages such as safety, high efficacy, no residue, and no withdrawal period, attracting significant attention and experiencing continuously increasing demand in recent years. Therefore, increasing the production of substantia nigra 37-peptide to meet market demand is essential.

[0023] Currently, thiamphetamine is mainly produced through fermentation by Bacillus subtilis. Common and effective ways to increase its yield are artificial screening of high-yielding strains and optimization of the fermentation process. Considering the limitations of strain screening, optimizing the fermentation process is a good starting point. The culture medium is composed of different nutrients in a certain proportion, providing a nutrient substrate for microbial growth and reproduction. The components of the fermentation medium generally include carbon sources, nitrogen sources, inorganic salts, growth factors, and water. Among them, the carbon source provides energy to the microorganisms and is the source of carbon in the cell structure and metabolic products; the nitrogen source is the source of nitrogen for microbial cell matter and nitrogenous metabolites, and also plays an important role in spore formation. Different microorganisms have different nutrient requirements, and different influencing substances have a significant impact on the yield of their fermentation products. Therefore, optimizing the nutrient composition of the fermentation medium is also one of the effective ways to increase the fermentation yield of thiamphetamine.

[0024] Based on this, the present invention first provides a fermentation medium for producing thiamphetamine, and then provides a fermentation method for increasing the yield of thiamphetamine based on the fermentation medium.

[0025] In the early stages of this invention, extensive research and experimentation were conducted to screen C and N sources and inorganic salts for fermentation media used in the production of thiamphetapeptide in existing technologies. However, analysis of specific experimental results revealed that the potential for increasing the content of thiamphetapeptide was very limited, reaching a bottleneck. The inventors then conducted an in-depth analysis of the amino acid composition and metabolic characteristics of thiamphetapeptide, combined with bottleneck analysis of peptide synthesis in microbial fermentation, and rationally added cysteine, an amino acid required for biological metabolism, to improve its biosynthetic efficiency. Furthermore, the addition of the metabolic cofactor pyridoxine not only maintained a stable fermentation environment and improved peptide synthesis efficiency but also regulated redox balance, delaying the aging stage of the microorganisms.

[0026] Specifically, the inventors first optimized the nitrogen source, nitrogen source, and basic inorganic salts, selecting pea starch and glucose as carbon sources, soybean meal and corn syrup as nitrogen sources, and sodium chloride, potassium dihydrogen phosphate, dipotassium bicarbonate, and ammonium sulfate as basic inorganic salts. Secondly, they optimized the dosage of the selected components to obtain the following basic nutritional formula: pea starch 25-35 g / L, glucose 8-15 g / L, soybean meal 15-25 g / L, corn syrup 10-20 g / L, sodium chloride 1-3 g / L, dipotassium hydrogen phosphate 0.01-0.1 g / L, potassium dihydrogen phosphate 0.01-0.1 g / L, and ammonium sulfate 1.5-2.5 g / L. Finally, after extensive research and experimentation, the inventors added cysteine, pyridoxine, sodium selenite, and magnesium sulfate to the basic nutritional formula.

[0027] The dosage of the aforementioned basic nutrients and the content of thiamphetamine produced by fermentation are shown in Table 1.

[0028] Table 1

[0029]

[0030]

[0031] Pea starch and glucose, as efficient carbon sources, and soybean meal and corn syrup, as efficient nitrogen sources, can promote the rapid proliferation of Bacillus subtilis cells and provide them with abundant nutrients, effectively promoting cell metabolism. The rational ratio of pea starch, glucose, soybean meal, and corn syrup ensures carbon-nitrogen balance, enabling the culture medium to achieve a high yield of subtilis-7-peptide at a relatively low cost. The rational formulation of various inorganic salts, such as sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate, and sodium selenite, effectively maintains the pH stability of the culture medium and provides essential trace elements. The supplementation of metabolic cofactors further enhances the metabolic capacity of Bacillus subtilis. All components interact to effectively promote the metabolic activity of Bacillus subtilis, ultimately increasing the content and quality of subtilis-7-peptide in the fermentation products of Bacillus subtilis.

[0032] The present invention will be further described below with reference to specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. The equipment and raw materials used are all commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0033] The basal culture medium formulation used in the following examples is as follows: Activation medium: 5 g / L soybean protein powder, 0.5 g / L potassium chloride, 0.5 g / L magnesium sulfate, 0.5 g / L dipotassium hydrogen phosphate, 0.5 g / L sodium chloride, 0.02 g / L ferrous sulfate, 10 g / L glucose, 15 g / L agar, with water as the solvent. Sterilize at 121℃ for 20 min.

[0034] Seed culture medium: 30 g / L corn extract, 5 g / L glucose, 20 g / L water-soluble starch, 5 g / L beef extract, 0.5 g / L ferrous sulfate, 0.5 g / L sodium chloride, with water as the solvent; after making up to volume, adjust the pH to 7.0-7.5 with 20 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min.

[0035] I. A specific embodiment of the fermentation culture medium for producing thiamphetamine of the present invention: Example 1 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0036] The preparation process is as follows: Dissolve the above components in water, and after making up the volume, adjust the pH to 7.0-7.5 with 20mol / L sodium hydroxide solution. Sterilize at 121℃ for 20min.

[0037] Example 2 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 5 mg / L, pyridoxine 0.5 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0038] The preparation process is as described in Example 1.

[0039] Example 3 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 5 mg / L, pyridoxine 0.1 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0040] The preparation process is as described in Example 1.

[0041] Example 4 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 5 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.3 g / L, and water as the solvent.

[0042] The preparation process is as described in Example 1.

[0043] Example 5 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.3 g / L, and water as the solvent.

[0044] The preparation process is as described in Example 1.

[0045] Example 6 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.5 g / L, cysteine ​​0.3 g / L, and water as the solvent.

[0046] The preparation process is as described in Example 1.

[0047] Example 7 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 1 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.1 g / L, and water as the solvent.

[0048] The preparation process is as described in Example 1.

[0049] Example 8 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 1 mg / L, pyridoxine 0.5 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0050] The preparation process is as described in Example 1.

[0051] Example 9 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 15 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.1 g / L, and water as the solvent.

[0052] The preparation process is as described in Example 1.

[0053] Example 10 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 1.5 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.1 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0054] The preparation process is as described in Example 1.

[0055] Example 11 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.1 g / L, and water as the solvent.

[0056] The preparation process is as described in Example 1.

[0057] Example 12 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 5 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.1 g / L, and water as the solvent.

[0058] The preparation process is as described in Example 1.

[0059] Example 13 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 1.5 g / L, ammonium sulfate 2.5 g / L, sodium selenite 1 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0060] The preparation process is as described in Example 1.

[0061] Example 14 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.1 g / L, cysteine ​​0.1 g / L, and water as the solvent.

[0062] The preparation process is as described in Example 1.

[0063] Example 15 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.5 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0064] The preparation process is as described in Example 1.

[0065] Example 16 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 1.5 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.5 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0066] The preparation process is as described in Example 1.

[0067] Example 17 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 1.5 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.3 g / L, and water as the solvent.

[0068] The preparation process is as described in Example 1.

[0069] Example 18 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 1 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.3 g / L, and water as the solvent.

[0070] The preparation process is as described in Example 1.

[0071] Example 19 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.1 g / L, cysteine ​​0.3 g / L, and water as the solvent.

[0072] The preparation process is as described in Example 1.

[0073] Example 20 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 2.5 g / L, sodium selenite 5 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0074] The preparation process is as described in Example 1.

[0075] Example 21 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.1 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0076] The preparation process is as described in Example 1.

[0077] Example 22 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 1 mg / L, pyridoxine 0.1 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0078] The preparation process is as described in Example 1.

[0079] Example 23 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 2.5 g / L, sodium selenite 1 mg / L, pyridoxine 0.3 g / L, cysteine ​​0.2 g / L, and water as the solvent.

[0080] The preparation process is as described in Example 1.

[0081] Example 24 The components of the fermentation medium for producing subtilisin in this embodiment are as follows: The ingredients are: pea starch 35 g / L, glucose 10 g / L, soybean meal 25 g / L, corn syrup 15 g / L, sodium chloride 3 g / L, dipotassium hydrogen phosphate 0.05 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.85 g / L, ammonium sulfate 2.5 g / L, sodium selenite 3 mg / L, pyridoxine 0.5 g / L, cysteine ​​0.1 g / L, and water as the solvent.

[0082] The preparation process is as described in Example 1.

[0083] II. Specific embodiments of the fermentation method for increasing the yield of kiwifruit peptide according to the present invention: Example 25 The fermentation method for increasing the yield of basidipotassium taurateurin in this embodiment utilizes the fermentation medium for producing basidipotassium taurateurin in Examples 1-24, and the specific implementation is as follows: 1. Activation culture: Take the Bacillus subtilis strain (CGMCC 15404) preserved in glycerol tubes out of the -80℃ freezer, take 100μL and spread it on an activation medium plate, and incubate it in a 35℃ incubator for 5-7 days.

[0084] 2. Seed bottle culture: Remove the activated plates from the 35℃ constant temperature incubator, pick up colonies with an inoculation stick and transfer them to the seed culture medium, and incubate at 35℃ and 220rpm for 24-48 hours.

[0085] 3. Fermentation culture in 500mL shake flasks: Seed culture was extracted in a clean bench and inoculated into the fermentation medium at a 10% inoculum. The medium was cultured at 35-37℃ and 220rpm for 2 days. The content of tahinitropeptide in the fermentation broth was detected by high performance liquid chromatography.

[0086] The specific process for detecting quinacrine triseptide in fermentation broth using high performance liquid chromatography is as follows: Take a sample (mL), add an appropriate amount of extraction solution, shake to mix, sonicate for 15-30 min, and bring the volume to 10 mL. Shake well. Take an appropriate amount of sample solution into a centrifuge tube, centrifuge at 12000 r / min for 2 min, take the supernatant and filter it through a 0.22 μm filter membrane, discard 1 mL of filtrate, and then collect the sample in a sample vial for determination using high performance liquid chromatography.

[0087] Instrument debugging and condition setting: (1) Selection of chromatographic column: C8 column (4.6mm×150mm, 5μm) or C18 column (100mm×2.1mm, 1.7μm) can be used to ensure effective separation of the main peak of tahinitroseptapeptide from the impurity peak (in this example, C8 column (4.6mm×150mm, 5μm) is selected).

[0088] (2) Mobile phase preparation: Gradient elution mode is adopted. Mobile phase A is 0.1% trifluoroacetic acid aqueous solution and mobile phase B is 80% acetonitrile solution. The mobile phase is degassed by ultrasonication for 15 min in advance to remove air bubbles and avoid affecting the detection baseline.

[0089] (3) Detection conditions: column temperature 25~35℃ (normally 25℃), flow rate 1.0mL / min (can be adjusted to 0.3mL / min for complex matrices), detection wavelength 280nm, injection volume 10~20μL (normally 20μL); gradient elution program is adjusted according to sample matrix.

[0090] Sample determination: (1) Instrument balancing: Start the HPLC instrument, turn on the pump, detector and workstation in sequence, and rinse the column with the set mobile phase for more than 30 minutes until the baseline is stable and the column pressure is stable, so as to avoid baseline drift affecting the identification of the main peak.

[0091] (2) Standard curve plotting: Inject the series of standard working solutions into the chromatograph in order of increasing concentration, repeating the injection three times for each concentration, and record the retention time and peak area of ​​the main peak; plot the standard curve with the standard concentration as the abscissa (x) and the peak area as the ordinate (y), and require the correlation coefficient R. 2 ≥0.999, to ensure accurate quantification.

[0092] (3) Test sample determination: Inject the pretreated test sample solution into the chromatograph and repeat the injection 3 times under the same conditions. Record the retention time and peak area of ​​the main peak. At the same time, set up a blank control (solvent only) and a negative control (blank sample without thiamphetamine) to eliminate interference from solvent, matrix and other bacteria.

[0093] Record the area of ​​the target chromatographic peak and substitute it into the standard curve formula to calculate the concentration (μg / mL) of tahinitrophenylephrine in the sample. Then calculate the content of tahinitrophenylephrine according to the following formula.

[0094]

[0095] Then, the content of 37-peptide of calciferol is converted. The conversion example is as follows: when the content of 37-peptide of calciferol calculated according to the above formula is 0.1%, the content of 37-peptide of calciferol in the fermentation culture medium is 1000 μg / mL.

[0096] The specific results are shown in Table 2, where the high-performance liquid chromatogram of Example 1 is shown in Table 2. Figure 1 As shown.

[0097] Table 2

[0098] Example 26 The fermentation method for increasing the yield of basidipotassium taurateurin in this embodiment utilizes the fermentation medium for producing basidipotassium taurateurin in Example 1, and the specific implementation is as follows: 1. Activation culture: Take the Bacillus subtilis strain (CGMCC 15404) preserved in glycerol tubes out of the -80℃ freezer, take 100μL and spread it on an activation medium plate, and incubate it in a 35℃ incubator for 5-7 days.

[0099] 2. Seed bottle culture: Remove the activated plates from the 35℃ constant temperature incubator, pick up colonies with an inoculation stick and transfer them to the seed culture medium, and incubate at 35℃ and 220rpm for 24-48 hours.

[0100] 3. Fermentation in a 50L bioreactor: The fermentation medium in the 50L bioreactor was 10L, with 0.5g / L of antifoaming agent added during preparation. The fermentation seed was cultured using the same method as shake-flask fermentation and inoculated using flame inoculation, with an inoculation volume of 250mL. The reactor stirring speed was set to 100-300rpm, and the aeration rate was 200-400m³ / h. 3 The fermentation process involves maintaining a constant temperature of 35-37℃ and a tank pressure of 0.02-0.08MPa. Dissolved oxygen is controlled at 25-45% during fermentation, and alkaline water is added to maintain the pH at no less than 7.0.

[0101] Note: During fermentation, feeding strategies can be formulated based on feedback from parameters such as residual sugar concentration, ammonia nitrogen concentration, and dissolved oxygen concentration (OD) in the bioreactor. Specifically, in this embodiment, an online glucose sensor is used to detect the residual sugar concentration. Feeding is initiated when the residual sugar concentration drops to a threshold (e.g., 0.5 g / L) and stopped when the residual sugar concentration recovers to a set value (e.g., 0.2 g / L).

[0102] The content of kilotonine in the fermentation broth (fermentation for 48 h) was determined by high performance liquid chromatography (HPLC). The procedure was as described in Example 25 and will not be repeated here. The specific results are as follows: Figure 2 As shown.

[0103] As shown in the figure, when fermented in a 50L fermenter using the fermentation medium of Example 1, the fermentation content of 3845.56 μg / mL of kiwifruit 37 peptides reached.

[0104] III. Comparative Example Comparative Example 1 This comparative example uses a commonly used fermentation medium for the fermentation production of tahinitroglycerin, with the following components: The ingredients are: soybean protein powder 15g / L, glucose 5g / L, sodium chloride 2g / L, dipotassium hydrogen phosphate 0.05g / L, potassium dihydrogen phosphate 0.05g / L, magnesium sulfate 0.55g / L, ammonium sulfate 2.5g / L, sodium selenite 3mg / L, tyrosine 0.2g / L, glycine 0.23g / L, and the remainder is water.

[0105] The preparation process is as follows: Dissolve the above components in water, and after making up the volume, adjust the pH to 7.0-7.5 with 20mol / L sodium hydroxide solution. Sterilize at 121℃ for 20min.

[0106] Bacillus subtilis (CGMCC 15404) was cultured in shake flasks and fermented in a 50L bioreactor using the fermentation medium of this comparative example. The content of subtilis-37-peptide in the fermentation broth was detected by high-performance liquid chromatography (HPLC). The specific procedures are as described in Examples 25 and 26, and will not be repeated here. Specific results are as follows: Figure 3 and Figure 4 As shown.

[0107] As shown in the figure, the fermentation content of 37-peptide of kiwifruit in the shake flask level of this comparative example was 886.54 μg / mL, while the fermentation content of 37-peptide of kiwifruit in the 50L fermenter level was 2204.54 μg / mL.

[0108] This demonstrates that the fermentation production of thiamphetamine using the fermentation medium of Example 1 of this invention is 49.4% higher at the shake flask level and 74.4% higher at the 50L fermenter level compared to Comparative Example 1.

[0109] Comparative Example 2 This comparative example uses a commonly used fermentation medium for the fermentation production of tahinitroglycerin, with the following components: The ingredients are: 20 g / L pea starch, 5 g / L yeast extract, 1.5 g / L sodium chloride, 0.05 g / L dipotassium hydrogen phosphate, 0.05 g / L potassium dihydrogen phosphate, 0.75 g / L ferrous sulfate, 2 g / L ammonium sulfate, 0.25 g / L pyridoxine, 0.2 g / L cysteine, and the remainder is water.

[0110] The preparation process is as follows: Dissolve the above components in water, and after making up the volume, adjust the pH to 7.0-7.5 with 20mol / L sodium hydroxide solution. Sterilize at 121℃ for 20min.

[0111] Bacillus subtilis (CGMCC 15404) was cultured in shake flasks and fermented in a 50L bioreactor using the fermentation medium of this comparative example. The content of subtilis-37-peptide in the fermentation broth was detected by high-performance liquid chromatography (HPLC). The specific procedures are as described in Examples 25 and 26, and will not be repeated here. Specific results are as follows: Figure 5 and Figure 6 As shown.

[0112] As shown in the figure, the fermentation content of 37-peptide of kiwifruit in the shake flask level of this comparative example was 796.35 μg / mL, while the fermentation content of 37-peptide of kiwifruit in the 50L fermenter level was 1827.32 μg / mL.

[0113] This demonstrates that the fermentation production of thiamphetamine using the fermentation medium of Example 1 of this invention is 66.3% higher at the shake flask level and 110.4% higher at the 50L fermenter level compared to Comparative Example 2.

[0114] Comparative Example 3 This comparative study examined the effects of adding different amino acids to the fermentation medium on the expression level of subtilisin.

[0115] Based on the fermentation medium of Example 1, two separate cultures were prepared: one without cysteine ​​(basic control group), and the other with cysteine ​​replaced by glycine and tyrosine, respectively. Both cultures were then used to culture Bacillus subtilis (CGMCC 15404) in shake flasks. The content of subtilis thiamphetamine in the fermentation broth was detected using high-performance liquid chromatography (HPLC), as described in Example 25. The expression of subtilis thiamphetamine in cultures containing different amino acids was compared with that in the basic control group. The specific results are shown in Table 3.

[0116] Table 3. Increase in the content of trihexyphenidyl subtilis peptide in culture media containing different amino acids compared with the basal control group. amino acid categories Compared with the baseline control group (%) Cysteine Increase by 23% glycine Increase by 5% Tyrosine Increase by 2% The results in the table show that, when the amount of each of the three amino acids is the same, cysteine ​​can significantly increase the content of 37-peptide.

[0117] Comparative Example 4 This comparative example uses the culture medium of Example 1 and Comparative Examples 1-2 as the fermentation medium to ferment wild-type Bacillus subtilis (Bacillus subtilis collected in the wild that can express subtilis 37-peptide) to compare the content of subtilis 37-peptide in the fermentation broth. The specific operation process is as described in Example 25.

[0118] The specific results are shown in Table 4, and the high-performance liquid chromatograms are shown below. Figures 7-9 As shown.

[0119] Table 4. Expression content of Bacillus subtilis thiamphetamine in various culture media.

[0120] Whether wild Bacillus subtilis can also increase the expression of subtilis 37-peptide in the culture medium provided by the present invention, the results above show that both Bacillus subtilis (CGMCC 15404) and wild strains can significantly increase the expression of subtilis 37-peptide in the culture medium provided by the present invention during fermentation culture.

[0121] Comparative Example 5 This comparative example uses the culture medium of Example 1 and Comparative Examples 1-2 as the fermentation medium to ferment Bacillus subtilis (CGMCC 15404) to compare the content of cephalosporin in the fermentation broth. The specific operation process is as described in Example 25.

[0122] The specific results are shown in Table 5, and the high-performance liquid chromatograms are shown below. Figures 10-12 As shown.

[0123] Table 5. Cephalosporin expression levels of Bacillus subtilis in various culture media.

[0124] Whether the fermentation medium provided by the present invention can increase the expression level of other metabolites of Bacillus subtilis. The results show that, compared with the culture medium of the prior art, the fermentation medium provided by the present invention does not significantly increase the content of cephalosporin.

[0125] In summary, this invention first provides a fermentation medium for producing thiamphetamine, and then, based on this fermentation medium, provides a fermentation method for increasing the yield of thiamphetamine. The fermentation medium provided by this invention has low cost, and compared with the fermentation medium used in the prior art for producing thiamphetamine, the yield of thiamphetamine is significantly increased, providing a new approach to cost reduction and efficiency improvement in the large-scale production of thiamphetamine.

[0126] To further clarify whether the optimized culture medium enhances the expression level by improving the performance of the strain itself or has a positive effect solely on the content of subtilisin, this invention further experimentally verified: 1. The expression level of subtilisin in wild-type strains was compared with that in the control culture medium and the culture medium provided by this invention; 2. The expression level of cephalosporin in screened strains was compared with that in the control culture medium and the culture medium provided by this invention. The results showed that the optimized culture medium of this invention improved the expression level of subtilisin in wild-type strains, while the cephalosporin content did not change significantly. These results indicate that the fermentation culture medium provided by this invention is targeted at the specific fermentation product subtilisin.

[0127] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. A fermentation medium for producing kilotonine, characterized in that: The components include the following: pea starch 25-35 g / L, glucose 8-15 g / L, soybean meal 15-25 g / L, corn syrup 10-20 g / L, sodium chloride 1-3 g / L, dipotassium hydrogen phosphate 0.01-0.1 g / L, potassium dihydrogen phosphate 0.01-0.1 g / L, ammonium sulfate 1.5-2.5 g / L, magnesium sulfate 0.2-1.5 g / L, sodium selenite 1-5 mg / L, pyridoxine 0.1-0.5 g / L, and cysteine ​​0.05-0.3 g / L.

2. The fermentation medium for producing kilotonine according to claim 1, characterized in that: The components include the following: pea starch 30-35 g / L, glucose 10-15 g / L, soybean meal 20-25 g / L, corn syrup 15-20 g / L, sodium chloride 2-3 g / L, dipotassium hydrogen phosphate 0.05-0.1 g / L, potassium dihydrogen phosphate 0.05-0.1 g / L, ammonium sulfate 2-2.5 g / L, magnesium sulfate 0.2-1 g / L, sodium selenite 3-5 mg / L, pyridoxine 0.1-0.5 g / L, and cysteine ​​0.2-0.3 g / L.

3. The fermentation medium for producing kilotonine according to claim 2, characterized in that: The components include the following: pea starch 35g / L, glucose 10g / L, soybean meal 25g / L, corn syrup 15g / L, sodium chloride 3g / L, dipotassium hydrogen phosphate 0.05g / L, potassium dihydrogen phosphate 0.05g / L, ammonium sulfate 2.5g / L, magnesium sulfate 0.8~1g / L, sodium selenite 3~5mg / L, pyridoxine 0.1~0.5g / L, and cysteine ​​0.2g / L.

4. The fermentation medium for producing kilotonine according to claim 3, characterized in that: The components include the following: pea starch 35g / L, glucose 10g / L, soybean meal 25g / L, corn syrup 15g / L, sodium chloride 3g / L, dipotassium hydrogen phosphate 0.05g / L, potassium dihydrogen phosphate 0.05g / L, ammonium sulfate 2.5g / L, magnesium sulfate 0.85g / L, sodium selenite 3mg / L, pyridoxine 0.3g / L, and cysteine ​​0.2g / L.

5. The fermentation medium for producing kilotonine according to any one of claims 1 to 4, characterized in that: The pH of the fermentation medium is 7.0~7.

5.

6. A fermentation method for increasing the yield of kiwifruit peptides, characterized in that: Bacillus subtilis seed culture is inoculated into the fermentation medium for producing subtilis 37-peptide as described in any one of claims 1 to 5, and fermentation is carried out.

7. The fermentation method for increasing the yield of kiwifruit peptide according to claim 6, characterized in that: The seed culture comprises 2%-5% of the fermentation medium volume.

8. The fermentation method for increasing the yield of kiwifruit peptide according to claim 6 or 7, characterized in that: The fermentation culture temperature is 35-37℃.

9. The fermentation method for increasing the yield of kiwifruit peptide according to claim 6 or 7, characterized in that: During the fermentation process, dissolved oxygen is controlled at 25%-45%, and alkaline water is added to control the pH to be no lower than 7.

0.

10. The fermentation method for increasing the yield of kiwifruit peptide according to claim 6 or 7, characterized in that: During the fermentation process, stirring and aeration are carried out at a speed of 100-300 rpm and an aeration rate of 200-400 m³ / h. 3 / h.

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