Marine fish meal culture medium and its application in antibiotic fermentation production and fermentation method

Abstract

The invention relates to the technical field of culture medium, and in particular to a marine fish meal culture medium and an application thereof in the fermentation production of antibiotics and a fermentation method thereof. The culture medium comprises the following components: 15-20 parts by weight of marine fish meal, 25-30 parts by weight of pea leaching liquid, 0.01-0.03 parts by weight of activated carbon, 5-7 parts by weight of lactose, 6-9 parts by weight of maltodextrin, 4-6 parts by weight of yeast extract powder, 2-5 parts by weight of soybean oil, 0.4-0.8 parts by weight of phenylalanine and 10-15 parts by weight of inorganic salts, wherein the pea leaching liquid is obtained by steaming fresh peas, hydrolyzing them with trypsin and filtering them; the culture medium comprises a carbon source, a nitrogen source, an inorganic salt, a growth factor, a defoamer and a promoter, and on the original basis, a quick nitrogen source pea leaching liquid and an anti-nutritional factor adsorbent activated carbon are added, thereby improving the efficiency of the culture process and the yield of the antibiotics.

Description

Technical Field

[0001] The invention relates to the technical field of culture medium, in particular to a marine fish meal culture medium and its application in antibiotic fermentation production and a fermentation method. Background Art

[0002] Marine fish meal is a high-protein product made by processing marine fish (usually small marine fish or fish by-products) through processes such as cooking, degreasing, drying and grinding. It is mainly used as an additive to animal feed, but in the fields of microbiology and biotechnology, marine fish meal is also used as one of the components of culture medium. The protein content of marine fish meal is usually between 50% and 70%, and the protein is of high quality, containing all essential amino acids, rich in B vitamins, calcium, phosphorus, etc., and in appropriate proportions. Therefore, using marine fish meal to make culture medium can provide microorganisms with a rich nitrogen source, as well as essential vitamins and minerals, which can meet the growth needs of microorganisms, and because marine fish meal is inexpensive, it can also reduce costs.

[0003] Streptomyces roseoflavus Men-myco-93-63 is an antagonistic Streptomyces isolated from the soil of the natural decline of potato scab, which has inhibitory effects on a variety of plant pathogens. Studies have shown that the fungus and its secondary metabolites have a strong inhibitory effect on cotton Verticillium wilt of different pathogenicity, which can cause deformation of Verticillium wilt hyphae and be accompanied by bacteriolysis. The results of greenhouse and field experiments show that its fermentation liquid has a good field control effect on cotton Verticillium wilt. Its significant biological control effect and potential antibiotic production capacity make Streptomyces roseoflavus Men-myco-93-63 an important research object in the field of biological control of plant diseases. However, the antibiotic fermentation yield of the existing culture medium is low. In view of this, we propose a marine fish meal culture medium and its application in antibiotic fermentation production and a fermentation method. Summary of the invention

[0004] The object of the present invention is to provide a marine fish meal culture medium and its application in antibiotic fermentation production and a fermentation method to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides a marine fish meal culture medium, comprising the following components: 15-20 parts by weight of marine fish meal, 25-30 parts by weight of pea leaching liquid, 0.01-0.03 parts by weight of activated carbon, 5-7 parts by weight of lactose, 6-9 parts by weight of maltodextrin, 4-6 parts by weight of yeast extract powder, 2-5 parts by weight of soybean oil, 0.4-0.8 parts by weight of phenylalanine and 10-15 parts by weight of inorganic salt, wherein the inorganic salt is 1-3 parts by weight of potassium dihydrogen phosphate, 2-3 parts by weight of magnesium sulfate heptahydrate, 1-2 parts by weight of calcium carbonate and 4-6 parts by weight of manganese dichloride, wherein the pea leaching liquid is obtained by steaming fresh peas, enzymolyzing with trypsin and then filtering.

[0006] Marine fish meal is used as a complex nitrogen source for the culture medium. Marine fish meal is a powder made from the residues of fish or other marine organisms. It contains a large amount of protein, fat, minerals and vitamins and is a high-quality nitrogen source. However, protein exists in fish meal in the form of complex peptides and proteins, which require proteases and peptidases secreted by microorganisms to break them down into smaller peptides and amino acids before they can be absorbed and utilized. Therefore, as a complex nitrogen source, it can provide a continuous and stable carbon source for microorganisms.

[0007] Pea juice is used as a simple nitrogen source for the culture medium. Since peas are rich in protein, the protein is hydrolyzed into peptides and free amino acids through the extraction process. These amino acids and peptides are ideal nitrogen sources for microbial growth and can be directly absorbed and utilized by microorganisms to promote their rapid growth and metabolic activities, and promote the rapid growth and cell division of bacteria. Adequate supply of amino acids and nutrients can activate or accelerate the metabolic pathways of antibiotic synthesis and increase the production of antibiotics.

[0008] Activated carbon is used to adsorb pigments, impurities and inhibitors in the culture medium, creating a cleaner growth environment for microorganisms, reducing toxic effects on microorganisms, and increasing the yield of antibiotics and the purity of the product.

[0009] Lactose, as a simple carbon source, is easily metabolized by microorganisms, providing a quick source of energy and promoting the initial growth of microorganisms. Maltodextrin, as a complex carbon source, needs to be hydrolyzed into monosaccharides by the microorganism's own enzyme system, which can provide a continuous energy supply for microorganisms and support bacterial growth and metabolic activities. Therefore, a suitable and sufficient carbon source is the material basis for the synthesis of antibiotics.

[0010] Yeast extract powder can be used as a growth factor. It is rich in B vitamins, such as vitamin B1, B2, B3, B5, B6, B7, B9 and B12. These vitamins are components of coenzymes and cofactors and are involved in a wide range of biochemical reactions such as energy metabolism, amino acid metabolism, and nucleic acid synthesis. The nucleotides in yeast extract powder are precursors to DNA and RNA synthesis and are essential for cell replication and the transmission of genetic information. Therefore, the addition of yeast extract powder can optimize the growth conditions of microorganisms, promote the growth and metabolism of microorganisms, and improve the yield and quality of target products.

[0011] Soybean oil can be used as a defoamer in culture medium fermentation production. During the fermentation process, a large amount of foam will form in the culture medium due to mechanical stirring, gas introduction or carbon dioxide produced by metabolic processes. These foams not only reduce the effective volume of the fermentation tank, but also may cause the fermentation liquid to overflow, resulting in waste of raw materials and microbial contamination, while affecting the oxygen transfer efficiency, thereby affecting the fermentation efficiency and product quality. Since soybean oil is hydrophobic, when it is introduced into the foam, it will diffuse to the foam interface and reduce the surface tension between the water phase and the air phase. This reduction in surface tension destroys the stability of the foam, causing the foam film to become thinner and then rupture. In addition, adding a small amount of soybean oil, because it forms a continuous oil layer on the liquid surface, reduces the capture of air, thereby reducing the probability of foam formation, and then inhibiting the formation of new foam.

[0012] As an essential amino acid, phenylalanine is a direct precursor of microbial protein synthesis. Supplementing phenylalanine can increase the amino acid pool and promote protein synthesis, thereby accelerating the growth and metabolic activities of microorganisms. Phenylalanine also participates in the synthesis pathways of some antibiotics, affecting the structure and yield of antibiotics. It is a precursor of microbial anabolic antibiotic products and is particularly important for the biosynthesis of secondary metabolites such as antibiotics. Therefore, it can be used as a promoter participating in metabolic pathways.

[0013] Since phosphorus is an important element in the growth and metabolism of microorganisms, phosphorus and potassium are components of important biological molecules such as cell membranes, nucleic acids, and ATP, and are essential for cell division and metabolic processes, adequate phosphorus and potassium supply can optimize the synthetic pathway of antibiotics and increase production. Therefore, potassium dihydrogen phosphate is added to provide phosphorus and potassium for microorganisms and act as a buffer to help maintain the stability of the pH value of the culture medium; Ca 2+ It is mainly involved in regulating the physiological state of cells, can reduce the permeability of cell membranes, maintain the colloidal state of cells, etc. Studies have shown that CaCO 3 It is an indispensable component in the fermentation medium of most strains. 3 It has a great influence on the amount of bacteria after fermentation, so adding CaCO 3It promotes the formation of antibiotics, adjusts the pH value of the culture medium, neutralizes acidic metabolites, prevents the pH value from decreasing, maintains the stability of the pH value, and prevents the adverse effects of pH fluctuations on the growth of bacteria. Magnesium ions are essential for maintaining the activity of intracellular enzymes. Since magnesium is a cofactor of many enzymes and participates in the synthesis of nucleic acids and proteins, as well as energy metabolism, an adequate supply of magnesium ions can promote the activity of enzymes related to antibiotic synthesis, thereby possibly increasing the production of antibiotics. At the same time, magnesium sulfate can also regulate osmotic pressure. Therefore, magnesium sulfate heptahydrate is added to the culture medium to regulate osmotic pressure and promote the production of antibiotics. Manganese participates in the antioxidant defense system and plays an important role in the antioxidant capacity and metabolic process of cells. It can protect bacteria from oxidative damage and maintain normal metabolic functions. Appropriate manganese ion concentrations can enhance the antioxidant capacity of cells and reduce damage from free radicals.

[0014] Preferably, the preparation method of the pea juice is as follows:

[0015] Select fresh peas, add water, heat to boiling, then reduce the heat and continue heating until the peas become soft, the heating time is 30-50 minutes, use a sieve to separate the peas and water, gently squeeze the peas to help release more juice and nutrients, then add 3-5 parts by weight of trypsin, pour the filtered infusion into a sterile container, and then use a high-pressure steam sterilizer to sterilize at 120-125° C. for 20-30 minutes. After sterilization, let the infusion naturally cool to room temperature to obtain pea infusion.

[0016] Preferably, the mass ratio of the fresh peas to water is 1:3-4.

[0017] Preferably, the added amount of the trypsin is 0.5-0.8% of the mass of fresh peas.

[0018] Trypsin can specifically decompose anti-nutritional factors such as trypsin inhibitors, which can inhibit the activity of trypsin and affect the digestion of protein. Through the action of trypsin, more small molecule peptides and amino acids can be released, thereby improving the digestibility and nutritional availability of pea protein. In addition, trypsin can also promote the release of other nutrients in peas, such as vitamins and minerals, thereby enhancing the nutritional value of pea extract as a culture medium.

[0019] Preferably, the preparation method of the marine fish meal culture medium is as follows:

[0020] First, 100-110 parts by weight of distilled water is added into a container, and yeast extract powder, lactose, maltodextrin, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, manganese dichloride and phenylalanine are added in sequence, and stirred until completely dissolved. Then, pea extract and soybean oil are added, and then marine fish meal and activated carbon are added, and stirred fully until completely dispersed. Finally, calcium carbonate is added to adjust the pH value. The prepared culture medium is divided into fermentation tanks, and the containers are sealed. A high-pressure steam sterilizer is used for sterilization at 120-125° C. for 20-30 minutes, and the culture medium is naturally cooled to room temperature. Thus, a marine fish meal culture medium is prepared.

[0021] Preferably, the activated carbon is obtained by boiling in hot water for 3 minutes and then filtering and drying.

[0022] Preferably, the calcium carbonate is used to adjust the initial pH value of the culture medium to 6.8-7.2.

[0023] The initial pH value of the culture medium is an important parameter to ensure the normal reproduction and metabolism of microorganisms. The optimal initial pH value has a great influence on the yield and quality of the target product after fermentation of different types of microorganisms. The main reasons are: first, the pH value affects the charge of the protoplasmic membrane of microbial cells, thereby affecting the normal growth and metabolism of microorganisms; second, the pH value directly affects the enzyme activity in microbial cells, thereby affecting the growth, reproduction and metabolism of microorganisms; third, the pH value affects the dissociation of certain important nutrients and intermediate metabolites in the culture medium, thereby affecting the absorption and utilization of these substances by microorganisms.

[0024] The present invention provides a method for fermenting a marine fish meal culture medium, based on any one of the marine fish meal culture mediums described above, comprising the following steps:

[0025] Preparation of primary seed solution:

[0026] Use an inoculation loop to select a loop of antibiotic production fermentation bacteria and inoculate it into the primary seed culture medium, and culture it in a shake flask at 24-25°C and 200-220 rpm for 5-8 hours;

[0027] Preparation of secondary seed solution:

[0028] Take the first-level seed liquid and inoculate it into the second-level seed culture medium at a rate of 1-3%, and culture it in a shaking flask at 24-25°C and 200-220rpm for 18-22h;

[0029] Fermentation culture:

[0030] The secondary seeds are inoculated at a 10-12% (v / v) inoculation rate into a fermentation medium in a 5L fermenter, at 24-25°C, a liquid volume of 50-55% (v / v), an aeration volume of 0.8-1vvm, an initial pH value of 6.8-7.2, and pH is regulated with ammonia water and acetic acid. The fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30%-40% to maintain bacterial growth. When the fermentation time is 15-16h, the dissolved oxygen is controlled at 25-27%. When the fermentation time is 24h, feeding is stopped, and fermentation is stopped after the substrate is consumed.

[0031] Preferably, the components of the primary seed culture medium are 1-2 parts by weight of glucose, 2-4 parts by weight of peptone, and 0.3-0.5 parts by weight of yeast powder, and 100 mL of water is added to adjust the pH value to 7.0-7.2; the components of the secondary seed culture medium are 15-18 parts by weight of glucose, 4-6 parts by weight of yeast powder, 3-5 parts by weight of dipotassium hydrogen phosphate, 2-3 parts by weight of ammonium sulfate, 0.1-0.3 parts by weight of magnesium sulfate, and the pH value is 6.7-6.9; the components of the fermentation medium are 15-20 parts by weight of marine fish meal, 25-30 parts by weight of pea extract, 0.01-0.03 parts by weight of activated carbon, 5-7 parts by weight of lactose, 6-9 parts by weight of maltodextrin, 4-6 parts by weight of yeast extract powder, 10-15 parts by weight of inorganic salt, 2-5 parts by weight of soybean oil, and 0.4-0.8 parts by weight of phenylalanine, and the pH value is 6.8-7.2.

[0032] The influence of temperature on fermentation is mainly manifested in two aspects: direct effect and indirect effect. Direct effects include affecting the growth rate, enzyme activity, cell composition and nutritional requirements of microorganisms, while indirect effects include affecting the solubility of solute molecules, the transport and diffusion of ions, the osmotic pressure and surface tension of cell membranes, etc. High temperature can denature or coagulate proteins in microbial cells, and also destroy the enzyme activity in microbial cells, thereby killing the microorganisms, while low temperature can inhibit the growth of microorganisms.

[0033] Dissolved oxygen refers to the amount of oxygen dissolved in the fermentation liquid. It is the basis for aerobic metabolism of bacteria and is another important factor in fermentation. Dissolved oxygen not only affects the synthesis pathway of secondary metabolites, but also affects the synthesis rate of metabolism. During the fermentation process, due to the high density of bacteria and the large oxygen intake during the fermentation process, it is necessary to ensure that the dissolved oxygen is within a certain range. Insufficient dissolved oxygen concentration will inhibit the growth of bacteria and the synthesis of secondary metabolites, resulting in a decrease in product yield. However, when the ventilation volume is too large, it will lead to excessive dissolved oxygen and autolysis of the bacteria, which is not conducive to the production of antibiotics.

[0034] The inoculum size refers to the number of bacteria initially invested. Too low an inoculum size will extend the delay period and increase the risk of contamination, while too high an inoculum size may lead to rapid consumption of nutrients, causing the bacteria to enter the stable period or decay period prematurely, affecting the yield and quality of the product. Therefore, an appropriate inoculum size helps to quickly start the fermentation process, shorten the delay period, and enable the bacteria to enter the logarithmic growth phase faster.

[0035] The liquid filling volume refers to the volume of the culture medium in the fermentation tank, which affects the liquid dynamics characteristics in the fermentation tank, such as mixing effect, mass transfer and heat transfer efficiency. Too high a liquid filling volume will reduce the solubility and transfer efficiency of oxygen, limit the growth of bacteria and the formation of products, while too low a liquid filling volume may lead to uneven distribution of nutrients, which will also affect the fermentation efficiency.

[0036] Ventilation refers to the volume of air or sterile gas supplied to the fermentation tank per unit time, which affects the oxygen supply in the fermentation system. Adequate ventilation ensures aerobic respiration of the bacteria, promotes growth and the synthesis of secondary metabolites, but insufficient ventilation will cause the bacteria to be hypoxic and enter an anaerobic metabolic state, inhibiting the production of products such as antibiotics that require oxygen to participate in synthesis.

[0037] The present invention also provides an application of a marine fish meal culture medium in the fermentation production of antibiotics. The marine fish meal culture medium fermentation method according to any one of the above-mentioned methods comprises the following steps:

[0038] S1.1. Activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into a primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 6 hours. The primary seed culture medium comprises 1 part by weight of glucose, 4 parts by weight of yeast powder, 3 parts by weight of dipotassium hydrogen phosphate, 2 parts by weight of ammonium sulfate, and 0.1 part by weight of magnesium sulfate, and the pH value is 6.8;

[0039] S1.2, take the first-level seed liquid and inoculate it into the second-level seed culture medium at an inoculum of 1-3%, and culture it at 25°C and 220rpm for 18h, wherein the components of the second-level seed culture medium are 15 parts by weight of glucose, 7 parts by weight of yeast powder, 5 parts by weight of dipotassium hydrogen phosphate, 3 parts by weight of ammonium sulfate, 0.3 parts by weight of magnesium sulfate, and the pH value is 6.9;

[0040] S1.3, inoculate the secondary seeds at 10% (v / v) inoculation amount into the fermentation medium in a 5L fermenter, at 25°C, the liquid volume is 55% (v / v), the ventilation volume is 1vvm, the initial pH value is 6.8, and the pH is regulated by ammonia and acetic acid. The fermentation starts at 0-14h, and the dissolved oxygen is controlled at 35% to maintain the growth of the bacteria. When the fermentation time is 16h, the dissolved oxygen is controlled at 27%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed. The components of the fermentation medium are 18 parts by weight of marine fish meal, 26 parts by weight of pea juice, 0.01 parts by weight of activated carbon, 5 parts by weight of lactose, 9 parts by weight of maltodextrin, 4 parts by weight of yeast extract powder, 12 parts by weight of inorganic salt, 1 part by weight of soybean oil, and 0.7 parts by weight of phenylalanine, and the pH value is 7.1;

[0041] S1.4. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes, remove the supernatant, wash the precipitate, and obtain the antibiotic.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The marine fish meal culture medium and its application in antibiotic fermentation production and fermentation method include a marine fish meal culture medium comprising a carbon source, a nitrogen source, an inorganic salt, a growth factor, a promoter, a defoaming agent and an anti-nutritional factor adsorbent. The marine fish meal is used as a complex nitrogen source to provide a stable nitrogen source for the growth of microorganisms, and pea extract is used as a simple nitrogen source to provide a quickly available nitrogen source for microorganisms. In the early stage of microbial culture, the delay period is shortened, and the microorganisms enter the logarithmic growth phase faster, thereby improving the efficiency of the entire culture process. Activated carbon is added as an anti-nutritional factor adsorbent to adsorb pigments, impurities and inhibitors in the culture medium, reduce toxic effects on microorganisms, and thus improve the yield of antibiotics and the purity of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the antibiotic standard curve of the present invention. DETAILED DESCRIPTION

[0045] 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 making creative work are within the scope of protection of the present invention.

[0046] The invention discloses a marine fish meal culture medium, which comprises the following components: 15-20 parts by weight of marine fish meal, 25-30 parts by weight of pea leaching liquid, 0.01-0.03 parts by weight of activated carbon, 5-7 parts by weight of lactose, 6-9 parts by weight of maltodextrin, 4-6 parts by weight of yeast extract powder, 2-5 parts by weight of soybean oil, 0.4-0.8 parts by weight of phenylalanine and 10-15 parts by weight of inorganic salt, wherein the inorganic salt is 1-3 parts by weight of potassium dihydrogen phosphate, 2-3 parts by weight of magnesium sulfate heptahydrate, 1-2 parts by weight of calcium carbonate and 4-6 parts by weight of manganese dichloride, and wherein the pea leaching liquid is obtained by steaming fresh peas, enzymolyzing them with trypsin and filtering them.

[0047] Example 1

[0048] A method for fermenting a marine fish meal culture medium comprises the following steps:

[0049] S3.1, primary seed culture medium: 1 part by weight of glucose, 2 parts by weight of peptone, 0.3 parts by weight of yeast powder, add 100 mL of water, and adjust the pH value to 7.0;

[0050] S3.2, secondary seed culture medium: 15 parts by weight of glucose, 4 parts by weight of yeast powder, 3 parts by weight of dipotassium hydrogen phosphate, 2 parts by weight of ammonium sulfate, 0.1 parts by weight of magnesium sulfate, pH value is 6.7;

[0051] S3.3, fermentation medium: 15 parts by weight of marine fish meal, 25 parts by weight of pea extract, 0.01 parts by weight of activated carbon, 5 parts by weight of lactose, 6 parts by weight of maltodextrin, 4 parts by weight of yeast extract powder, 1 part by weight of potassium dihydrogen phosphate, 2 parts by weight of magnesium sulfate heptahydrate, 1 part by weight of calcium carbonate, 4 parts by weight of manganese dichloride, 2 parts by weight of soybean oil, 0.4 parts by weight of phenylalanine, pH value is 7.0;

[0052] S3.4, prepare the primary seed solution: activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into the primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 5 hours;

[0053] S3.5, prepare the secondary seed solution: take the primary seed solution and inoculate it into the secondary seed medium at a rate of 3%, and culture it in a shaking flask at 25°C and 220 rpm for 20 h;

[0054] S3.6, fermentation culture: inoculate the secondary seeds at a 10% (v / v) inoculation rate into the fermentation medium in a 5L fermenter, at 25°C, with a liquid volume of 50% (v / v), an aeration volume of 1vvm, an initial pH value of 6.8, and adjust the pH with ammonia water and acetic acid. Fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30% to maintain bacterial growth. When the fermentation time is 15h, the dissolved oxygen is controlled at 25%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed.

[0055] S3.7. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes in a high-speed centrifuge, remove the supernatant, wash the precipitate, and obtain the antibiotic.

[0056] Example 2

[0057] A method for fermenting a marine fish meal culture medium comprises the following steps:

[0058] S3.1, primary seed culture medium: 1.5 parts by weight of glucose, 3 parts by weight of peptone, 0.4 parts by weight of yeast powder, add 100 mL of water, and adjust the pH value to 7.0;

[0059] S3.2, secondary seed culture medium: 16 parts by weight of glucose, 5 parts by weight of yeast powder, 4 parts by weight of dipotassium hydrogen phosphate, 2.5 parts by weight of ammonium sulfate, 0.2 parts by weight of magnesium sulfate, pH value is 6.7;

[0060] S3.3, fermentation medium: 15 parts by weight of marine fish meal, 25 parts by weight of pea extract, 0.01 parts by weight of activated carbon, 5 parts by weight of lactose, 6 parts by weight of maltodextrin, 4 parts by weight of yeast extract powder, 1 part by weight of potassium dihydrogen phosphate, 2 parts by weight of magnesium sulfate heptahydrate, 1 part by weight of calcium carbonate, 4 parts by weight of manganese dichloride, 2 parts by weight of soybean oil, 0.4 parts by weight of phenylalanine, pH value is 7.0;

[0061] S3.4, prepare the primary seed solution: activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into the primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 5 hours;

[0062] S3.5, prepare the secondary seed solution: take the primary seed solution and inoculate it into the secondary seed medium at a rate of 3%, and culture it in a shaking flask at 25°C and 220 rpm for 20 h;

[0063] S3.6, fermentation culture: inoculate the secondary seeds at a 10% (v / v) inoculation rate into the fermentation medium in a 5L fermenter, at 25°C, with a liquid volume of 50% (v / v), an aeration volume of 1vvm, an initial pH value of 6.8, and adjust the pH with ammonia water and acetic acid. Fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30% to maintain bacterial growth. When the fermentation time is 15h, the dissolved oxygen is controlled at 25%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed.

[0064] S3.7. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes in a high-speed centrifuge, remove the supernatant, wash the precipitate, and obtain the antibiotic.

[0065] Example 3

[0066] A marine fish meal culture medium and its application in antibiotic fermentation production and a fermentation method, comprising the following steps:

[0067] S3.1, primary seed culture medium: 2 parts by weight of glucose, 4 parts by weight of peptone, 0.5 parts by weight of yeast powder, add 100 mL of water, and adjust the pH value to 7.0;

[0068] S3.2, secondary seed culture medium: 18 parts by weight of glucose, 6 parts by weight of yeast powder, 5 parts by weight of dipotassium hydrogen phosphate, 3 parts by weight of ammonium sulfate, 0.3 parts by weight of magnesium sulfate, pH value is 6.7;

[0069] S3.3, fermentation medium: 15 parts by weight of marine fish meal, 25 parts by weight of pea extract, 0.01 parts by weight of activated carbon, 5 parts by weight of lactose, 6 parts by weight of maltodextrin, 4 parts by weight of yeast extract powder, 1 part by weight of potassium dihydrogen phosphate, 2 parts by weight of magnesium sulfate heptahydrate, 1 part by weight of calcium carbonate, 4 parts by weight of manganese dichloride, 2 parts by weight of soybean oil, 0.4 parts by weight of phenylalanine, pH value is 7.0;

[0070] S3.4, prepare the primary seed solution: activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into the primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 5 hours;

[0071] S3.5, prepare the secondary seed solution: take the primary seed solution and inoculate it into the secondary seed medium at a rate of 3%, and culture it in a shaking flask at 25°C and 220 rpm for 20 h;

[0072] S3.6, fermentation culture: inoculate the secondary seeds at a 10% (v / v) inoculation rate into the fermentation medium in a 5L fermenter, at 25°C, with a liquid volume of 50% (v / v), an aeration volume of 1vvm, an initial pH value of 6.8, and adjust the pH with ammonia water and acetic acid. Fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30% to maintain bacterial growth. When the fermentation time is 15h, the dissolved oxygen is controlled at 25%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed.

[0073] S3.7. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes, remove the supernatant, wash the precipitate, and obtain the antibiotic.

[0074] Example 4

[0075] A method for fermenting a marine fish meal culture medium comprises the following steps:

[0076] S3.1, primary seed culture medium: 2 parts by weight of glucose, 4 parts by weight of peptone, 0.5 parts by weight of yeast powder, add 100 mL of water, and adjust the pH value to 7.0;

[0077] S3.2, secondary seed culture medium: 18 parts by weight of glucose, 6 parts by weight of yeast powder, 5 parts by weight of dipotassium hydrogen phosphate, 3 parts by weight of ammonium sulfate, 0.3 parts by weight of magnesium sulfate, pH value is 6.7;

[0078] S3.3, fermentation medium: 15 parts by weight of marine fish meal, 28 parts by weight of pea extract, 0.01 parts by weight of activated carbon, 5 parts by weight of lactose, 6 parts by weight of maltodextrin, 4 parts by weight of yeast extract powder, 1 part by weight of potassium dihydrogen phosphate, 2 parts by weight of magnesium sulfate heptahydrate, 1 part by weight of calcium carbonate, 4 parts by weight of manganese dichloride, 2 parts by weight of soybean oil, 0.4 parts by weight of phenylalanine, pH value is 7.0;

[0079] S3.4, prepare the primary seed solution: activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into the primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 5 hours;

[0080] S3.5, prepare the secondary seed solution: take the primary seed solution and inoculate it into the secondary seed medium at a rate of 3%, and culture it in a shaking flask at 25°C and 220 rpm for 20 h;

[0081] S3.6, fermentation culture: inoculate the secondary seeds at a 10% (v / v) inoculation rate into the fermentation medium in a 5L fermenter, at 25°C, with a liquid volume of 50% (v / v), an aeration volume of 1vvm, an initial pH value of 6.8, and adjust the pH with ammonia water and acetic acid. Fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30% to maintain bacterial growth. When the fermentation time is 15h, the dissolved oxygen is controlled at 25%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed.

[0082] S3.7. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes, remove the supernatant, wash the precipitate, and obtain the antibiotic.

[0083] Example 5

[0084] A method for fermenting a marine fish meal culture medium comprises the following steps:

[0085] S3.1, primary seed culture medium: 2 parts by weight of glucose, 4 parts by weight of peptone, 0.5 parts by weight of yeast powder, add 100 mL of water, and adjust the pH value to 7.0;

[0086] S3.2, secondary seed culture medium: 18 parts by weight of glucose, 6 parts by weight of yeast powder, 5 parts by weight of dipotassium hydrogen phosphate, 3 parts by weight of ammonium sulfate, 0.3 parts by weight of magnesium sulfate, pH value is 6.7;

[0087] S3.3, fermentation medium: 15 parts by weight of marine fish meal, 30 parts by weight of pea extract, 0.01 parts by weight of activated carbon, 5 parts by weight of lactose, 6 parts by weight of maltodextrin, 4 parts by weight of yeast extract powder, 1 part by weight of potassium dihydrogen phosphate, 2 parts by weight of magnesium sulfate heptahydrate, 1 part by weight of calcium carbonate, 4 parts by weight of manganese dichloride, 2 parts by weight of soybean oil, 0.4 parts by weight of phenylalanine, pH value is 7.0;

[0088] S3.4, prepare the primary seed solution: activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into the primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 5 hours;

[0089] S3.5, prepare the secondary seed solution: take the primary seed solution and inoculate it into the secondary seed medium at a rate of 3%, and culture it in a shaking flask at 25°C and 220 rpm for 20 h;

[0090] S3.6, fermentation culture: inoculate the secondary seeds at a 10% (v / v) inoculation rate into the fermentation medium in a 5L fermenter, at 25°C, with a liquid volume of 50% (v / v), an aeration volume of 1vvm, an initial pH value of 6.8, and adjust the pH with ammonia water and acetic acid. Fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30% to maintain bacterial growth. When the fermentation time is 15h, the dissolved oxygen is controlled at 25%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed.

[0091] S3.7. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes in a high-speed centrifuge, remove the supernatant, wash the precipitate, and obtain the antibiotic.

[0092] Example 6

[0093] A method for fermenting a marine fish meal culture medium comprises the following steps:

[0094] S3.1, primary seed culture medium: 2 parts by weight of glucose, 4 parts by weight of peptone, 0.5 parts by weight of yeast powder, add 100 mL of water, and adjust the pH value to 7.0;

[0095] S3.2, secondary seed culture medium: 18 parts by weight of glucose, 6 parts by weight of yeast powder, 5 parts by weight of dipotassium hydrogen phosphate, 3 parts by weight of ammonium sulfate, 0.3 parts by weight of magnesium sulfate, pH value is 6.7;

[0096] S3.3, fermentation medium: 15 parts by weight of marine fish meal, 30 parts by weight of pea extract, 0.02 parts by weight of activated carbon, 5 parts by weight of lactose, 6 parts by weight of maltodextrin, 4 parts by weight of yeast extract powder, 1 part by weight of potassium dihydrogen phosphate, 2 parts by weight of magnesium sulfate heptahydrate, 1 part by weight of calcium carbonate, 4 parts by weight of manganese dichloride, 2 parts by weight of soybean oil, 0.4 parts by weight of phenylalanine, pH value is 7.0;

[0097] S3.4, prepare the primary seed solution: activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into the primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 5 hours;

[0098] S3.5, prepare the secondary seed solution: take the primary seed solution and inoculate it into the secondary seed medium at a rate of 3%, and culture it in a shaking flask at 25°C and 220 rpm for 20 h;

[0099] S3.6, fermentation culture: inoculate the secondary seeds at a 10% (v / v) inoculation rate into the fermentation medium in a 5L fermenter, at 25°C, with a liquid volume of 50% (v / v), an aeration volume of 1vvm, an initial pH value of 6.8, and adjust the pH with ammonia water and acetic acid. Fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30% to maintain bacterial growth. When the fermentation time is 15h, the dissolved oxygen is controlled at 25%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed.

[0100] S3.7. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes in a high-speed centrifuge, remove the supernatant, wash the precipitate, and obtain the antibiotic.

[0101] Example 7

[0102] A method for fermenting a marine fish meal culture medium comprises the following steps:

[0103] S3.1, primary seed culture medium: 2 parts by weight of glucose, 4 parts by weight of peptone, 0.5 parts by weight of yeast powder, add 100 mL of water, and adjust the pH value to 7.0;

[0104] S3.2, secondary seed culture medium: 18 parts by weight of glucose, 6 parts by weight of yeast powder, 5 parts by weight of dipotassium hydrogen phosphate, 3 parts by weight of ammonium sulfate, 0.3 parts by weight of magnesium sulfate, pH value is 6.7;

[0105] S3.3, fermentation medium: 15 parts by weight of marine fish meal, 30 parts by weight of pea extract, 0.03 parts by weight of activated carbon, 5 parts by weight of lactose, 6 parts by weight of maltodextrin, 4 parts by weight of yeast extract powder, 1 part by weight of potassium dihydrogen phosphate, 2 parts by weight of magnesium sulfate heptahydrate, 1 part by weight of calcium carbonate, 4 parts by weight of manganese dichloride, 2 parts by weight of soybean oil, 0.4 parts by weight of phenylalanine, pH value is 7.0;

[0106] S3.4, prepare the primary seed solution: activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into the primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 5 hours;

[0107] S3.5, prepare the secondary seed solution: take the primary seed solution and inoculate it into the secondary seed medium at a rate of 3%, and culture it in a shaking flask at 25°C and 220 rpm for 20 h;

[0108] S3.6, fermentation culture: inoculate the secondary seeds at a 10% (v / v) inoculation rate into the fermentation medium in a 5L fermenter, at 25°C, with a liquid volume of 50% (v / v), an aeration volume of 1vvm, an initial pH value of 6.8, and adjust the pH with ammonia water and acetic acid. Fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30% to maintain bacterial growth. When the fermentation time is 15h, the dissolved oxygen is controlled at 25%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed.

[0109] S3.7. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes in a high-speed centrifuge, remove the supernatant, wash the precipitate, and obtain the antibiotic.

[0110] Example 8

[0111] A method for fermenting a marine fish meal culture medium comprises the following steps:

[0112] S3.1, primary seed culture medium: 2 parts by weight of glucose, 4 parts by weight of peptone, 0.5 parts by weight of yeast powder, add 100 mL of water, and adjust the pH value to 7.0;

[0113] S3.2, secondary seed culture medium: 18 parts by weight of glucose, 6 parts by weight of yeast powder, 5 parts by weight of dipotassium hydrogen phosphate, 3 parts by weight of ammonium sulfate, 0.3 parts by weight of magnesium sulfate, pH value is 6.7;

[0114] S3.3, fermentation medium: 15 parts by weight of marine fish meal, 30 parts by weight of pea extract, 0.02 parts by weight of activated carbon, 5 parts by weight of lactose, 6 parts by weight of maltodextrin, 4 parts by weight of yeast extract powder, 1 part by weight of potassium dihydrogen phosphate, 2 parts by weight of magnesium sulfate heptahydrate, 1 part by weight of calcium carbonate, 4 parts by weight of manganese dichloride, 2 parts by weight of soybean oil, 0.4 parts by weight of phenylalanine, pH value is 6.8;

[0115] S3.4, prepare the primary seed solution: activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into the primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 5 hours;

[0116] S3.5, prepare the secondary seed solution: take the primary seed solution and inoculate it into the secondary seed medium at a rate of 3%, and culture it in a shaking flask at 25°C and 220 rpm for 20 h;

[0117] S3.6, fermentation culture: inoculate the secondary seeds at a 10% (v / v) inoculation rate into the fermentation medium in a 5L fermenter, at 25°C, with a liquid volume of 50% (v / v), an aeration volume of 1vvm, an initial pH value of 6.8, and adjust the pH with ammonia water and acetic acid. Fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30% to maintain bacterial growth. When the fermentation time is 15h, the dissolved oxygen is controlled at 25%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed.

[0118] S3.7. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes, remove the supernatant, wash the precipitate, and obtain the antibiotic.

[0119] Example 9

[0120] A method for fermenting a marine fish meal culture medium comprises the following steps:

[0121] S3.1, primary seed culture medium: 2 parts by weight of glucose, 4 parts by weight of peptone, 0.5 parts by weight of yeast powder, add 100 mL of water, and adjust the pH value to 7.0;

[0122] S3.2, secondary seed culture medium: 18 parts by weight of glucose, 6 parts by weight of yeast powder, 5 parts by weight of dipotassium hydrogen phosphate, 3 parts by weight of ammonium sulfate, 0.3 parts by weight of magnesium sulfate, pH value is 6.7;

[0123] S3.3, fermentation medium: 15 parts by weight of marine fish meal, 30 parts by weight of pea extract, 0.03 parts by weight of activated carbon, 5 parts by weight of lactose, 6 parts by weight of maltodextrin, 4 parts by weight of yeast extract powder, 1 part by weight of potassium dihydrogen phosphate, 2 parts by weight of magnesium sulfate heptahydrate, 1 part by weight of calcium carbonate, 4 parts by weight of manganese dichloride, 2 parts by weight of soybean oil, 0.4 parts by weight of phenylalanine, pH value is 7.2;

[0124] S3.4, prepare the primary seed solution: activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into the primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 5 hours;

[0125] S3.5, prepare the secondary seed solution: take the primary seed solution and inoculate it into the secondary seed medium at a rate of 3%, and culture it in a shaking flask at 25°C and 220 rpm for 20 h;

[0126] S3.6, fermentation culture: inoculate the secondary seeds at a 10% (v / v) inoculation rate into the fermentation medium in a 5L fermenter, at 25°C, with a liquid volume of 50% (v / v), an aeration volume of 1vvm, an initial pH value of 6.8, and adjust the pH with ammonia water and acetic acid. Fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30% to maintain bacterial growth. When the fermentation time is 15h, the dissolved oxygen is controlled at 25%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed.

[0127] S3.7. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes in a high-speed centrifuge, remove the supernatant, wash the precipitate, and obtain the antibiotic.

[0128] Comparative Example 1

[0129] Adopt the method of embodiment 6, do not add pea soaking juice.

[0130] Comparative Example 2

[0131] The method of Example 6 was adopted without using activated carbon.

[0132] Comparative Example 3

[0133] Adopt the method of embodiment 6, without enzymolysis of fresh peas.

[0134] The present invention adopts the rapid nitrogen source and anti-nutritional factor adsorbent components of the marine fish meal culture medium to prepare a marine fish meal culture medium, wherein the performance index inspection items and inspection standards of the marine fish meal culture medium and its application in the fermentation production of antibiotics and the fermentation method are as follows:

[0135] For the determination of antibiotic content, a standard curve was first established. 0.0100 g of antibiotic standard was accurately weighed, dissolved in 80% ethanol, and the volume was fixed to 50 mL in a volumetric flask to obtain an antibiotic solution with a concentration of 0.200 mg / mL. The solution was graded and diluted 8, 10, 12, 14, 16, 32, and 64 times to a concentration of 0.0250, 0.0200, 0.0167, 0.0143, 0.0125, 0.0063, and 0.0031 mg / mL, respectively. The solutions of different concentrations were measured at 363 nm. The standard curve is drawn based on the absorbance at 363 nm. Since the antibiotics produced by Streptomyces roseoflavus Men-myco-93-63 are insoluble in water, they exist in the precipitate of the fermentation broth. The fermentation broth is shaken, 5 mL of the fermentation broth is added with 20 mL of ethanol and oscillated to mix. At this time, the antibiotics are dissolved in the liquid phase (containing 80% ethanol). The mixture is centrifuged at 12000 r / min for 3 min, and the supernatant is diluted 40 times. The absorbance at 363 nm is measured by an ultraviolet spectrophotometer, which is the response value. The concentration is substituted into the standard curve to calculate the concentration.

[0136] According to the above standards, a marine fish meal culture medium prepared in the above Examples 1-9 and Comparative Examples 1-3 and its application in antibiotic fermentation production and fermentation method were tested, and the obtained data are shown in Table 1:

[0137] Table 1 Performance data of Examples 1-9 and Comparative Examples 1-3

[0138]

[0139] The above data fully show that compared with Comparative Examples 1-3, Examples 1-9 can fully show the role of the rapid nitrogen source and anti-nutritional factor adsorbent components of the marine fish meal culture medium in improving the antibiotic yield of a marine fish meal culture medium and its application in antibiotic fermentation production and fermentation method.

[0140] Since the present invention adopts the rapid nitrogen source and anti-nutritional factor adsorbent components of the marine fish meal culture medium to prepare a marine fish meal culture medium and its application in antibiotic fermentation production and fermentation method, the yield of antibiotics is greatly improved through the effective ingredients, as follows:

[0141] It can be seen from Examples 1-3 that the composition of the primary seed culture medium and the seed culture medium has little effect on the antibiotic yield. This is because the growth of microorganisms on the primary seed culture medium and the seed culture medium mainly plays a role in starting and amplifying, that is, starting from a small amount of strains until a sufficient number of healthy bacteria are obtained for large-scale fermentation. Therefore, the microorganisms are universal on the primary seed culture medium and the seed culture medium, and the composition of the fermentation medium is crucial for the synthesis of antibiotics. It provides all the nutrients required for microbial growth and metabolism, including carbon sources, nitrogen sources, trace elements, vitamins, etc. The composition of the fermentation medium directly determines the growth rate, metabolic pathways and synthesis efficiency of antibiotics of the microorganisms, and therefore indirectly reflects the dominant role of the fermentation medium and the auxiliary role of the primary seed culture medium and the seed culture medium.

[0142] It can be seen from Examples 3-5 that with the increase of the content of pea juice, the yield of antibiotics is greatly increased. This is because pea juice is rich in amino acids and peptides. These small molecular nitrogen sources can be quickly absorbed and utilized by microorganisms to provide direct raw materials for the synthesis of proteins and nucleic acids. This quickly available nitrogen source can accelerate the growth of microorganisms, especially in the early growth stage, which helps to shorten the delay period and promote the microorganisms to quickly enter the logarithmic growth phase, thereby increasing the synthesis rate of antibiotics. In addition to nitrogen sources, pea juice also contains other nutrients, such as carbohydrates, vitamins and trace elements, which can support the comprehensive growth and metabolic activities of microorganisms. This comprehensive nutritional supplement helps to improve the metabolic efficiency of microorganisms and the synthesis capacity of antibiotics, so the yield of antibiotics is greatly increased.

[0143] It can be seen from Examples 5-7 that with the increase of the content of activated carbon as the anti-nutritional factor adsorbent, the antibiotic yield first increases and then decreases. This is because activated carbon can effectively adsorb anti-nutritional factors in the fermentation broth. The presence of these anti-nutritional factors will inhibit the growth and metabolic activity of microorganisms and affect the synthesis of antibiotics. When the activated carbon content is low, it can effectively remove these inhibitors, thereby improving the fermentation environment and increasing the yield of antibiotics. However, when the activated carbon content is too high, it will not only adsorb anti-nutritional factors, but also nutrients in the fermentation broth, including amino acids, peptides, vitamins and trace elements necessary for microbial growth and antibiotic synthesis. This excessive adsorption will lead to a lack of nutrients in the fermentation broth, thereby inhibiting the growth of microorganisms and the synthesis of antibiotics, and ultimately leading to a decrease in the yield of antibiotics.

[0144] It can be seen from Examples 7-9 that the initial pH value of the fermentation medium has a significant effect on the antibiotic yield. This is because the pH value is an extremely important environmental parameter in the growth and metabolic activities of microorganisms. The suitability of the pH value directly affects the physiological state of the microorganisms, the activity of the metabolic pathways and the synthesis of antibiotics. Since each enzyme has an optimal pH range, within this range, the enzyme activity is the highest. If the pH value of the culture medium deviates from this range, the activity of the enzyme involved in the synthesis of antibiotics will decrease, thereby affecting the synthesis efficiency of the antibiotics. In addition, the pH value affects the charge state and permeability of the cell membrane, and affects the transmembrane transport of nutrients and metabolites. An inappropriate pH value will lead to a reduction in the absorption of nutrients by the strains and obstruction of the excretion of metabolites (including antibiotics), thereby affecting the yield of antibiotics. Therefore, a suitable pH value should be selected.

[0145] According to the above test experiments, the marine fish meal culture medium prepared according to Example 6 and its application in antibiotic fermentation production and fermentation method have the best performance, so Example 6 is taken as the best embodiment;

[0146] By comparing Example 6 with Comparative Examples 1-3, it can be seen that:

[0147] In Comparative Example 1, no pea soaking liquid was added, and the antibiotic yield was greatly reduced. This is because pea soaking liquid is rich in free amino acids and small peptides. These nitrogen sources can be quickly absorbed and utilized by microorganisms without going through a complex decomposition process and can be directly used for the synthesis of proteins and nucleic acids, thereby accelerating the growth and reproduction of microorganisms. In Comparative Example 1, no pea soaking liquid was added. Due to the lack of support from quickly available nitrogen sources and other nutrients, the growth of microorganisms was slow, the activation of metabolic pathways was limited, and ultimately the antibiotic yield was greatly reduced.

[0148] In Comparative Example 2, activated carbon as an anti-nutritional factor adsorbent is not used, and the yield of antibiotics is greatly reduced. This is because activated carbon as an anti-nutritional factor adsorbent can adsorb suspended particles, pigments and other impurities in the fermentation broth, improve the clarity of the fermentation broth, which is not only beneficial to the subsequent product extraction and purification process, but also can reduce the adverse effects on microorganisms and promote the synthesis of antibiotics. At the same time, activated carbon has a high adsorption capacity and can effectively adsorb and remove these anti-nutritional factors. Anti-nutritional factors are a type of substance that can interfere with the normal growth and metabolism of microorganisms. Therefore, adding an anti-nutritional factor adsorbent increases the yield of antibiotics. When activated carbon is not used, the presence of anti-nutritional factors and other adverse factors will inhibit the growth of microorganisms and the synthesis of antibiotics, resulting in a decrease in yield.

[0149] In Comparative Example 3, fresh peas were not enzymatically hydrolyzed, and the antibiotic yield was greatly reduced. This is because trypsin can decompose the protein in peas and convert it into smaller peptides and amino acids, which are necessary for microbial growth and antibiotic synthesis. Without the enzymatic action of trypsin, some proteins cannot be effectively decomposed, thereby reducing the amount of available amino acids and peptides, reducing the nutrients that can be directly used by microorganisms, and failing to accelerate the growth of microorganisms and shorten the growth cycle, thereby resulting in a decrease in antibiotic production.

[0150] In summary, the key strategies to increase the yield of antibiotics are to add pea juice as a fast nitrogen source, accelerate the growth of microorganisms, shorten the delay period, promote the microorganisms to enter the logarithmic growth phase, rationally use activated carbon as an adsorbent of anti-nutritional factors, improve the fermentation environment, and control the fermentation conditions.

[0151] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only preferred examples of the present invention, and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A marine fish meal culture medium, characterized in that: The invention comprises the following components: 15-20 parts by weight of marine fish meal, 25-30 parts by weight of pea extract, 0.01-0.03 parts by weight of activated carbon, 5-7 parts by weight of lactose, 6-9 parts by weight of maltodextrin, 4-6 parts by weight of yeast extract powder, 2-5 parts by weight of soybean oil, 0.4-0.8 parts by weight of phenylalanine and 10-15 parts by weight of inorganic salt; Wherein, the inorganic salt is 1-3 parts by weight of potassium dihydrogen phosphate, 2-3 parts by weight of magnesium sulfate heptahydrate, 1-2 parts by weight of calcium carbonate and 4-6 parts by weight of manganese dichloride; The preparation method of the marine fish meal culture medium is as follows: First, 100-110 parts by weight of distilled water is added to a container, and yeast extract powder, lactose, maltodextrin, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, manganese dichloride and phenylalanine are added in sequence, and stirred until completely dissolved, and then pea extract and soybean oil are added, and then marine fish meal and activated carbon are added, and stirred fully until completely dispersed, and finally calcium carbonate is added to adjust the pH value, and the prepared culture medium is divided into fermentation tanks, and the container is sealed, and sterilized at 120-125° C. for 20-30 minutes using a high-pressure steam sterilizer, and naturally cooled to room temperature, thereby preparing a marine fish meal culture medium; The preparation method of the pea juice is as follows: Select fresh peas, add water, heat to boiling, then reduce the heat and continue heating until the peas become soft, the heating time is 30-50 minutes, use a sieve to separate the peas and water, gently squeeze the peas to help release more juice and nutrients, then add 3-5 parts by weight of trypsin, pour the filtered infusion into a sterile container, and then use a high-pressure steam sterilizer to sterilize at 120-125°C for 20-30 minutes. After sterilization, let the infusion naturally cool to room temperature to obtain pea infusion; the mass ratio of the fresh peas to water is 1:3-4; the addition amount of the trypsin is 0.5-0.8% of the mass of the fresh peas.

2. A marine fish meal culture medium according to claim 1, characterized in that: The activated carbon is obtained by boiling in hot water for 3 minutes and then filtering and drying.

3. A marine fish meal culture medium according to claim 1, characterized in that: The calcium carbonate is used to adjust the initial pH value of the culture medium to 6.8-7.

2.

4. A fermentation method based on the marine fish meal culture medium according to any one of claims 1 to 3, characterized in that: The steps include: Preparation of primary seed solution: Use an inoculation loop to select a loop of antibiotic production fermentation bacteria and inoculate it into the primary seed culture medium, and culture it in a shake flask at 24-25°C and 200-220 rpm for 5-8 hours; Preparation of secondary seed solution: Take the first-level seed liquid and inoculate it into the second-level seed culture medium at a rate of 1-3%, and culture it in a shaking flask at 24-25°C and 200-220rpm for 18-22h; Fermentation culture: The secondary seeds are inoculated at an inoculation rate of 10-12% (v / v) into a fermentation medium in a 5L fermenter, at 24-25°C, a liquid volume of 50-55% (v / v), an aeration rate of 0.8-1vvm, an initial pH value of 6.8-7.2, and pH is regulated by ammonia water and acetic acid. The fermentation starts at 0-14h, and the dissolved oxygen is controlled at 30%-40% to maintain bacterial growth. When the fermentation time is 15-16h, the dissolved oxygen is controlled at 25-27%. When the fermentation time is 24h, feeding is stopped, and fermentation is stopped after the substrate is consumed. The fermentation medium is the marine fish meal medium described in any one of claims 1-3.

5. The fermentation method according to claim 4, characterized in that: The components of the primary seed culture medium are 1-2 parts by weight of glucose, 2-4 parts by weight of peptone, and 0.3-0.5 parts by weight of yeast powder. 100 mL of water is added to adjust the pH value to 7.0-7.2; the components of the secondary seed culture medium are 15-18 parts by weight of glucose, 4-6 parts by weight of yeast powder, 3-5 parts by weight of dipotassium hydrogen phosphate, 2-3 parts by weight of ammonium sulfate, and 0.1-0.3 parts by weight of magnesium sulfate, and the pH value is 6.7-6.9; the pH value of the marine fish meal culture medium is 6.8-7.

2.

6. An application of the marine fish meal culture medium according to any one of claims 1 to 3 in antibiotic fermentation production, characterized in that: S1.

1. Activate Streptomyces roseoflavus Men-myco-93-63, select a loop with an inoculation loop and inoculate it into a primary seed culture medium, and culture it at 25°C and 220 rpm in a shake flask for 6 hours. The primary seed culture medium comprises 1 part by weight of glucose, 4 parts by weight of yeast powder, 3 parts by weight of dipotassium hydrogen phosphate, 2 parts by weight of ammonium sulfate, and 0.1 part by weight of magnesium sulfate, and the pH value is 6.8; S1.2, take the first-level seed liquid and inoculate it into the second-level seed culture medium at an inoculum of 1-3%, and culture it in a shake flask at 25°C and 220rpm for 18 hours, wherein the components of the second-level seed culture medium are 15 parts by weight of glucose, 7 parts by weight of yeast powder, 5 parts by weight of dipotassium hydrogen phosphate, 3 parts by weight of ammonium sulfate, 0.3 parts by weight of magnesium sulfate, and the pH value is 6.9; S1.3, inoculate the secondary seeds at 10% (v / v) inoculation amount into the fermentation medium in a 5L fermenter, at 25°C, with a liquid volume of 55% (v / v), aeration volume of 1vvm, an initial pH value of 6.8, and use ammonia and acetic acid to adjust the pH. The fermentation starts at 0-14h, and the dissolved oxygen is controlled at 35% to maintain bacterial growth. When the fermentation time is 16h, the dissolved oxygen is controlled at 27%. When the fermentation time is 24h, stop feeding, and stop fermentation after the substrate is consumed; wherein the fermentation medium is the marine fish meal medium according to any one of claims 1-3 and the pH value is 7.1; S1.

4. After the fermentation is completed, centrifuge at 12000r / min for 3 minutes, remove the supernatant, wash the precipitate, and obtain the antibiotic.

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