A culture medium and fermentation method for improving tylosin fermentation unit

Abstract

The present invention relates to a culture medium and a fermentation method for improving the fermentation unit of tylosin, which is a culture medium for producing tylosin by fermenting Streptomyces freundii, comprising a primary seed culture medium, a secondary seed culture medium and a fermentation culture medium, wherein the primary seed culture medium, the secondary seed culture medium and the fermentation culture medium all contain palm oil and petroleum yeast powder, and the fermentation culture medium contains oxygen carriers and n-dodecane. The method adopts a new formula, which can greatly shorten the fermentation cycle by about 18 hours compared with the traditional process, reduce energy consumption and save the production cost of raw materials and auxiliary materials; the use of palm oil and corn starch milk can reduce the total cost by about 25%; the use of petroleum yeast powder and oxygen carriers and n-dodecane in a reasonable ratio in a tylosin fermentation tank can accelerate the production speed on the basis of increasing the palm oil consumption rate, so that the fermentation unit is greatly increased.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical fermentation, and in particular relates to a culture medium for improving tylosin fermentation units and a fermentation method. Background Art

[0002] Tylosin is a type of 16-membered macrolide compound produced by Streptomyces freundii, and is a special antibiotic for livestock and poultry. Tylosin is mainly composed of four biologically active components, namely tylosin A, B, C, and D. Among them, component A has the highest content and the strongest activity in animals. The Chinese Pharmacopoeia (Veterinary Drugs) and foreign pharmacopoeias stipulate that the content of component A in tylosin finished products is ≥80%, and A+B+C+D>95%.

[0003] At present, the domestic use of Streptomyces freundii as the strain, fish meal, corn gluten meal, corn flour, soybean oil and other raw materials as the main raw materials using three-stage culture medium fermentation to produce tylosin, the main problems are: 1. Since the price of soybean oil and fish meal, the raw materials of the traditional culture medium, accounts for the highest proportion of the total cost, it has become the primary problem restricting the cost of tylosin fermentation; 2. A large amount of oxygen is consumed in the process of bacterial anabolism, which increases the energy consumption for feeding. In the late stage of tylosin fermentation, due to the gradual decline of the bacteria and the slowdown of metabolism, the speed of synthesizing tylosin is gradually slowed down, and the culture medium is insufficient, so that the speed of converting tylosin component C into component A is gradually reduced; 3. The culture cycle in the conventional culture medium formula is relatively long, and it is difficult for the fermentation unit to break through the current domestic average level, and there is still a gap with the foreign average level. Summary of the invention

[0004] The purpose of the present invention is to provide a new culture medium and fermentation method for improving the tylosin fermentation unit, optimize the formula of the culture medium, achieve the goal of greatly shortening the fermentation cycle, reducing energy consumption and saving production costs, effectively increasing the content of tylosin supply component A, and improving the tylosin fermentation unit.

[0005] The first aspect of the present invention relates to a culture medium for producing tylosin by fermentation with Streptomyces freundii:

[0006] The invention comprises a primary seed culture medium, a secondary seed culture medium and a fermentation culture medium, wherein the primary seed culture medium, the secondary seed culture medium and the fermentation culture medium all contain palm oil and petroleum yeast powder, and the fermentation culture medium contains Aid-Plus ML-50D and n-dodecane.

[0007] Furthermore, the volume ratio of the fermentation medium oxygen carrier Aid-Plus ML-50D: n-dodecane is (0.1:1) (v:v).

[0008] In some embodiments of the first aspect of the present invention, the primary seed culture medium is composed of: 3.6-4.3%, petroleum yeast powder 1.2-1.9%, corn starch milk 6.5-7.4%, trimagnesium phosphate 0.002-0.005%, calcium carbonate 2.7-3.5%, and the rest is drinking water.

[0009] In some embodiments of the first aspect of the present invention, the secondary seed culture medium is composed of: 3.8-4.7% palm oil, 5.1-6.5% petroleum yeast powder, 6.9-7.8% corn starch milk, 0.1-0.2% sodium chloride, 0.3-0.5% calcium carbonate, and the rest is drinking water.

[0010] In some embodiments of the first aspect of the present invention, the fermentation medium consists of: 3.8-4.5% palm oil, 2.0-2.8% petroleum yeast powder, 3.7-4.3% corn starch milk, 0.10-0.14% sodium chloride, 0.12-0.15% trimagnesium phosphate, 0.19-0.23% calcium carbonate, oxygen carrier: n-dodecane (0.1:1) (v:v) 0.03-0.05%, betaine 0.6-0.8%, nickel sulfate 0.00034-0.00051%, and the rest is drinking water.

[0011] The second aspect of the present invention relates to a fermentation method for producing tylosin, comprising the following steps:

[0012] Step (1): inoculating Streptomyces freundii seed liquid into a primary seed culture medium to obtain a primary seed culture liquid, wherein the primary seed culture medium comprises: 3.6-4.3%, petroleum yeast powder 1.2-1.9%, corn starch milk 6.5-7.4%, trimagnesium phosphate 0.002-0.005%, calcium carbonate 2.7-3.5%, and the remainder is drinking water;

[0013] Step (2): inoculating the primary seed culture solution into a secondary seed culture medium to obtain a secondary seed culture solution, wherein the secondary seed culture medium comprises: 3.8-4.7% palm oil, 5.1-6.5% petroleum yeast powder, 6.9-7.8% corn starch milk, 0.1-0.2% sodium chloride, 0.3-0.5% calcium carbonate, and the remainder is drinking water;

[0014] Step (3): inoculating the secondary seed culture solution into a fermentation medium for fermentation to obtain a fermentation solution, wherein the fermentation medium comprises: 3.8-4.5% palm oil, 2.0-2.8% petroleum yeast powder, 3.7-4.3% corn starch milk, 0.10-0.14% sodium chloride, 0.12-0.15% trimagnesium phosphate, 0.19-0.23% calcium carbonate, oxygen carrier: n-dodecane (0.1:1) (v:v) 0.03-0.05%, betaine 0.6-0.8%, nickel sulfate 0.00034-0.00051%, and the rest is drinking water;

[0015] Step (4): extracting tylosin from the fermentation broth.

[0016] Some more specific embodiments of the second aspect of the present invention relate to a fermentation method for improving the unit culture medium of tylosin fermentation, the process steps of which include:

[0017] 1) Sterilize the primary seed culture medium, transfer the cultured Streptomyces freundii seed solution to the primary seed tank by pressure difference method, and culture when the pH value is 6.80-8.50 and the bacterial concentration reaches more than 15% and there are no foreign bacteria;

[0018] 2) Sterilize the secondary seed culture medium, transfer the primary seed liquid to the secondary seed tank by pressure difference method, and culture it at pH 6.80-8.50. Transfer the seed when the bacterial concentration reaches 25-50% and there are no foreign bacteria;

[0019] 3) Sterilize the fermentation medium, transfer the secondary seed liquid to the fermentation tank for cultivation using the pressure difference method, culture the fermentation liquid using variable temperature, control the temperature and air flow, and terminate the culture when the fermentation requirements are met.

[0020] The first-level seed culture conditions are: the tank temperature is controlled at 28.0-33.0℃ throughout the process, and the air flow rate is 0-15h: 30-85m 3 / h, 15h-transplantation: 160-200m 3 / h; the tank pressure is maintained at 0.030-0.040MPa. When the pH value is 6.80-8.50, the bacterial concentration is above 15% and there are no foreign bacteria, it can be moved into the secondary seed tank for cultivation. The cultivation cycle is 35-50h.

[0021] The conditions for secondary seed cultivation are: the tank temperature is controlled at 28.0-33.0℃ throughout the process, and the air flow rate is 0-15h: 50-100m 3 / h, 15h-transplantation: 100-200m 3 / h; the tank pressure is maintained at 0.030-0.100MPa. When the pH value is 6.80-8.50, the bacterial concentration reaches 25-50% and there are no foreign bacteria, it can be moved into the fermentation tank for culture. The culture cycle is 30-40h.

[0022] Fermentation culture conditions: use variable temperature culture, strictly control temperature and air flow, in 0-100h, the temperature before conversion is 28-33℃, the temperature during conversion is 36-38℃, the temperature after conversion to tank release is 28-32℃, tank pressure: 0.01-0.10MPa, air flow is 0-15h, 1000-1500m 3 / h; 15-2000-3000m before conversion 3 / h; 1500-2000m during conversion 3 / h, after conversion - tank discharge 500-800m 3 / h, stirring speed: 0-150r / min, fermentation cycle is 125-150h.

[0023] The feeding control conditions during the fermentation process are:

[0024] a Oil supplementation control: The fat content gradually decreases with the increase of culture time. When the fat content is lower than 1.5% during the conversion period, palm oil is added. From 45 hours to before conversion, the fat content is controlled at about 0.5-1.2%, and from the conversion period to tank release, it is about 0.5-0.8%. When releasing from the tank, it should be within 0.1-0.2%;

[0025] b. Control of drinking water supplement: The bacterial concentration will increase with the growth of culture time. When the bacterial concentration reaches 30%, drinking water supplementation begins. After transplantation and before transformation, when the bacterial concentration is greater than 35%, drinking water supplementation begins, supplementing 500-800L every 12h. When the bacterial concentration is greater than 40%, drinking water supplementation begins, supplementing 800-1200L every 12h.

[0026] c pH value control: 0-12h pH value is natural, 12h to conversion, pH value is controlled at 6.70-6.80, conversion period to release pH value is 6.50-6.70, if it is out of range, add 25% phosphoric acid solution or 15% sodium hydroxide solution for adjustment.

[0027] The third aspect of the present invention relates to the use of a fermentation medium in the production of tylosin by fermentation with Streptomyces freundii:

[0028] Furthermore, the fermentation medium is composed of: 3.8-4.5% palm oil, 2.0-2.8% petroleum yeast powder, 3.7-4.3% corn starch milk, 0.10-0.14% sodium chloride, 0.12-0.15% trimagnesium phosphate, 0.19-0.23% calcium carbonate, oxygen carrier: n-dodecane (0.1:1) (v:v) 0.03-0.05%, betaine 0.6-0.8%, nickel sulfate 0.00034-0.00051%, and the rest is drinking water.

[0029] The technical solution of the present invention has the following beneficial technical effects:

[0030] Cost, dissolved oxygen, pH value and temperature have always been the main factors restricting the tylosin fermentation unit. The culture medium of the present invention adopts a new formula and a variable fermentation temperature culture technology, which can greatly shorten the fermentation cycle by about 18 hours, reduce energy consumption and save the production cost of raw materials and auxiliary materials, and improve the fermentation unit.

[0031] 1. Palm oil and corn starch milk are used in the production of tylosin culture medium raw material components, which overcomes the high cost of traditional culture medium raw materials, reduces energy consumption during the fermentation process, and reduces the total cost by about 25%.

[0032] 2. A reasonable ratio of oxygen carrier Aid-Plus ML-50D and n-dodecane was used in the fermentation medium to overcome the problem of gradual death of bacteria, slow metabolism, gradual slowdown of tylosin synthesis, and insufficient stamina of the medium in the late fermentation period. In the tylosin fermentation tank, the addition of oxygen carrier can accelerate the production rate of tylosin on the basis of increasing the consumption rate of palm oil, stabilize the pH value, and efficiently and quickly convert tylosin component C into component A, greatly improving the fermentation unit.

[0033] 3. Petroleum yeast is a single-cell protein containing lecithin. Its lysine content is similar to that of fish meal. It contains a variety of trace elements required for mycelium growth, which is very different from yeast in my country that uses cake and fish meal as culture medium. Its energy and protein are much better than domestic yeast powder, and its price is relatively low. It is an excellent protein source. It greatly promotes the synthesis of tylosin, increases the content of total effective ingredients, improves the fermentation unit, and makes the properties of the fermentation liquid more conducive to the next extraction process.

[0034] 4. Palm oil is the vegetable oil variety with the largest production volume and the lowest price in the world. It has unique oxidation stability during storage, transportation and fermentation metabolism. It is used as a carbon source for tylosin fermentation and a precursor for synthesizing tylosin. During the fermentation production process, a large amount of carbon dioxide is produced. Carbon dioxide adsorbed on the liquid feed easily causes liquid expansion, making it difficult to control the liquid level in the fermentation tank. Palm oil can inhibit liquid expansion, lower the liquid level, better control the generation of fermentation foam, better improve the fermentation unit, and save costs during the implementation of the present invention. DETAILED DESCRIPTION

[0035] All other embodiments obtained belong to the scope of protection of the present invention. The following embodiments of the present invention will be clearly and completely described in conjunction with the examples. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, the gain effect is obvious in the following embodiments by comparison with conventional processes without creative work by ordinary technicians in this field.

[0036] The volume of the main fermentation production equipment is: first-level tank 2.5m 3 , secondary tank 6m 3 , fermentation tank 30m 3 .

[0037] Example 1

[0038] (1) Primary culture steps:

[0039] 40kg petroleum yeast powder, 180L corn starch milk, 2.4kg trimagnesium phosphate, 120L palm oil, 1300L water, 7.8kg calcium carbonate, the tank temperature is controlled at about 30.0℃ throughout the process, and the air flow is controlled at 0-15h:55m 3 / h, 15h-transplantation: 180m 3 / h; the tank pressure is maintained at about 0.045MPa, the bacterial concentration is 20%, and the culture cycle is 40h;

[0040] (2) Secondary culture steps:

[0041] 360kg of petroleum yeast powder, 450L of corn starch milk, 6.0kg of trimagnesium phosphate, 280L of palm oil, 3000L of water, 24kg of calcium carbonate, 8kg of sodium chloride, the tank temperature is controlled at 30.0℃ throughout the process, and the air flow is controlled at 0-15h:890m 3 / h, 15h-transplantation: 1150m 3 / h; the tank pressure was maintained at 0.053MPa, when the pH value was 7.02, the bacterial concentration was 33%, the mycelium growth was observed under the microscope to meet the transplanting standard and there were no foreign bacteria, and the culture cycle was 32h;

[0042] (3) Fermentation culture step:

[0043] 690kg petroleum yeast powder, 1200L corn starch milk, 40kg trimagnesium phosphate, oxygen carrier Aid-Plus ML-50D: n-dodecane volume ratio (8L, 80L), 40.0kg sodium chloride, 50.0kg betaine, 1050L palm oil, 16m 3, 65kg calcium carbonate, 0.028kg nickel sulfate, 0.030kg cobalt chloride. Use variable temperature culture, strictly control temperature and air flow, in 0-100h, the temperature before conversion is 31℃, the temperature during conversion is 38℃, the temperature after conversion to tank release is 30℃, tank pressure: 0.07MPa, air flow is 0-15h, 1350m 3 / h; 15-2570m before conversion 3 / h; 1600m during conversion 3 / h, after conversion - tank discharge 500m 3 / h, stirring speed: 120r / min, fermentation cycle is 148h, fat content is 0.14%, tylosin A component is 92.3%, and tank potency is 16509U / ml.

[0044] For the control of feeding during fermentation:

[0045] a Oil supplementation control: during the conversion period, when the fat content is lower than 1.5%, palm oil is added. The fat content before 45 hours of conversion is 1.0%. During the conversion period until the tank is put into storage, the fat content is 0.7%. When the tank is put into storage, the fat content is controlled within 0.1-0.2%.

[0046] b Control of drinking water supplementation: Start supplementing drinking water when the bacterial concentration reaches 35%. After transplanting and before transformation, the bacterial concentration is 28%, and 790L is supplemented every 12 hours; when the bacterial concentration is greater than 40%, 1180L is supplemented every 12 hours.

[0047] c pH value control: 0-12h pH value is natural, 12h to before conversion, pH value is 6.75, conversion period to release pH value is 6.58. If the fermentation liquid is not within the range during the culture process, 25% phosphoric acid solution or 15% sodium hydroxide solution can be added for adjustment.

[0048] Example 2

[0049] (1) Primary culture steps:

[0050] 63kg of petroleum yeast powder, 212L of corn starch milk, 2.61kg of trimagnesium phosphate, 142L of palm oil, 1320L of water, 8.1kg of calcium carbonate, the tank temperature is controlled at about 30.5℃ throughout the process, and the air flow rate is 0-15h: 55m 3 / h, 15h-transplantation: 200m 3 / h; the tank pressure is maintained at about 0.048MPa, the bacterial concentration is 20%, and the culture cycle is 41h;

[0051] (2) Secondary culture steps:

[0052] 380kg of petroleum yeast powder, 442L of corn starch milk, 6.2kg of trimagnesium phosphate, 294L of palm oil, 3010L of water, 27kg of calcium carbonate, 7.8kg of sodium chloride, the tank temperature is controlled at 30.0℃ throughout the process, and the air flow rate is 0-15h: 920m 3 / h, 15h-transplantation: 1100m 3 / h; the tank pressure was maintained at 0.048MPa, when the pH value was 6.85, the bacterial concentration was 33%, the mycelium growth met the transplanting standard and there were no foreign bacteria under microscopic examination, and the culture period was 33h;

[0053] (3) Fermentation culture step:

[0054] 680kg petroleum yeast powder, 1190L corn starch milk, 45kg trimagnesium phosphate, oxygen carrier Aid-Plus ML-50D: n-dodecane volume ratio (7L, 69L), 39.0kg sodium chloride, 52.0kg betaine, 1100L palm oil, 16m 3 , 70kg calcium carbonate, 0.031kg nickel sulfate, 0.028kg cobalt chloride. Use variable temperature culture, strictly control temperature and air flow, in 0-100h, the temperature before conversion is 30℃, the temperature during conversion is 37℃, the temperature after conversion to tank release is 30℃, tank pressure: 0.08MPa, air flow is 0-15h, 1200m 3 / h; 15-2600m before conversion 3 / h; 2000m during conversion 3 / h, after conversion - tank discharge 550m 3 / h, stirring speed: 130r / min, fermentation cycle: 152h, fat content 0.12%, tylosin A component 92.3%, tank potency 17361U / ml.

[0055] For the control of feeding during fermentation:

[0056] a Oil supplementation control: during the conversion period, when the fat content is lower than 1.5%, palm oil is added. The fat content is 1.1% before 45 hours of conversion. The fat content is 0.8% during the conversion period until the tank is released. When the tank is released, the fat content is controlled within 0.1-0.2%;

[0057] b. Control of drinking water supplement: When the bacterial concentration reaches 35%, drinking water supplement begins. From transplantation to transformation, when the bacterial concentration is 30%, 800L is supplemented every 12 hours; when the bacterial concentration is greater than 40%, 1180L is supplemented every 12 hours;

[0058] c pH value control: 0-12h pH value is natural, 12h to before conversion, pH value is 6.77, conversion period to release pH value is 6.53. If the fermentation liquid is not within the range during the culture process, 25% phosphoric acid solution or 15% sodium hydroxide solution can be added for adjustment.

[0059] Example 3

[0060] (1) Primary culture steps:

[0061] 63kg of petroleum yeast powder, 212L of corn starch milk, 2.61kg of trimagnesium phosphate, 142L of palm oil, 1320L of water, 8.1kg of calcium carbonate, the tank temperature is controlled at about 30.5℃ throughout the process, and the air flow rate is 0-15h: 55m 3 / h, 15h-transplantation: 190m 3 / h; the tank pressure is maintained at about 0.048MPa, the bacterial concentration is 20%, and the culture cycle is 41h;

[0062] (2) Secondary culture steps:

[0063] 395kg of petroleum yeast powder, 451L of corn starch milk, 6.9kg of trimagnesium phosphate, 303L of palm oil, 3220L of water, 30kg of calcium carbonate, 8.1kg of sodium chloride, the tank temperature is controlled at 29.0℃ throughout the process, and the air flow rate is 0-15h: 950m 3 / h, 15h-transplantation: 1180m 3 / h; the tank pressure was maintained at 0.042MPa, the pH value was 7.2, the bacterial concentration was 33%, and the mycelium growth was observed under the microscope to meet the transplanting standards and there were no foreign bacteria. The culture period was 34h;

[0064] (3) Fermentation culture step:

[0065] 700kg petroleum yeast powder, 1230L corn starch milk, 53kg trimagnesium phosphate, oxygen carrier Aid-Plus ML-50D: n-dodecane volume ratio (5.8L, 60L), 42.0kg sodium chloride, 57.0kg betaine, 1298L palm oil, 18m water 3 , 67kg calcium carbonate, 0.029kg nickel sulfate, 0.030kg cobalt chloride. Use variable temperature culture, strictly control temperature and air flow, in 0-100h, the temperature before conversion is 32℃, the temperature during conversion is 37℃, the temperature after conversion to tank release is 30℃, tank pressure: 0.09MPa, air flow is 0-15h, 1250m 3 / h; 15-2600m before conversion 3 / h; 1750m during conversion 3 / h, after conversion - tank discharge 600m 3 / h, stirring speed: 110r / min, fermentation cycle: 150h, fat content 0.18%, tylosin A component 93.1%, tank potency 17496U / ml.

[0066] For the control of feeding during fermentation:

[0067] a Oil supplementation control: during the conversion period, when the fat content is lower than 1.5%, palm oil is added. The fat content before 45 hours of conversion is 1.09%. During the conversion period until the tank is released, the fat content is 0.92%. When releasing the tank, the fat content is controlled within 0.1-0.2%;

[0068] b. Control of drinking water supplement: When the bacterial concentration reaches 35%, drinking water supplement begins. After transplantation and before transformation, when the bacterial concentration is 30%, 787L of drinking water is supplemented every 12 hours; when the bacterial concentration is greater than 40%, 1196L of drinking water is supplemented every 12 hours;

[0069] c pH value control: 0-12h pH value is natural and does not need to be controlled artificially. From 12h to before conversion, the pH value is 6.77, and the pH value from the conversion period to the tank release is 6.65. If the fermentation liquid is not within the range during the culture process, 25% phosphoric acid solution or 15% sodium hydroxide solution can be added for adjustment.

[0070] Comparative Example 1

[0071] Using fish meal, soybean oil, corn gluten meal and peanut meal as the main raw materials, according to the public Tylos fermentation process, Streptomyces freundii is used to produce Tylosin through three-stage fermentation. The volume of the main fermentation production equipment is: the first tank is 1m 3 , secondary tank 10m 3 , fermentation tank 100m 3 .

[0072] First-level seed culture medium: 18L soybean oil, 12kg corn protein powder, 13kg peanut cake powder, 2.5L corn syrup, 0.35kg diammonium phosphate, 1.5kg light calcium carbonate, and the rest is drinking water. After the culture medium is sterilized, the fermentation liquid of Streptomyces freundii that meets the inoculation conditions is inoculated into the culture medium using the flame method. The tank temperature is controlled at about 30.0℃ throughout the process, and the air flow rate is 0-15h: 50m 3 / h, 15h-transplantation: 148m 3 / h; the tank pressure is maintained at about 0.04MPa, the bacterial concentration is 20%, and the culture cycle is 35h;

[0073] Secondary seed culture medium: 191L ​​soybean oil, 120kg corn protein powder, 130kg peanut cake powder, 25L corn steep liquor, 3.5kg diammonium phosphate, 15kg light calcium carbonate, and the rest is drinking water. Move the primary seed liquid that meets the transplanting requirements into the disinfected secondary seed culture medium. The tank pressure is controlled at 0.028MPa, the tank temperature is 30℃, and the air is 1100m 3 / h, stirring at 140r / min, the end point of culture is pH 7.4, bacterial concentration 38%, no foreign bacteria under microscopic examination, and the culture period is 36h;

[0074] Fermentation tank culture medium: 2950L soybean oil, 720kg fish meal, 1100kg peanut meal, 1800kg corn gluten meal, 63kg betaine hydrochloride, 89kg potassium chloride, 11kg diammonium phosphate, 0.39kg nickel sulfate, 0.52kg cobalt chloride, 260kg light calcium carbonate, and the rest is drinking water. The secondary seed liquid that meets the transplanting requirements is transferred to the fermentation tank, the tank pressure is 0.045MPa, the transplanting is 125h, the tank temperature is 28℃, the stirring is 200r / min, and the fermentation is completed after 125h, the temperature is 28℃, the speed is 280r / min, and the air flow is 1600m 3 / h, pH 6.8, fat content when released from the tank was 0.3%, tylosin A content was 85.2%, fermentation time was 162h, and potency was 13845U / ml.

[0075] Comparative Example 2

[0076] With cottonseed meal, soybean oil, corn gluten meal and corn flour as the main raw materials, tylosin is produced by three-stage fermentation with Streptomyces freundii according to the public tylosin fermentation process. The volume of the main fermentation production equipment is: the first tank is 1m 3 , secondary tank 10m 3 , fermentation tank 100m 3 .

[0077] Primary seed culture medium: 17L soybean oil, 13kg corn protein powder, 12kg corn flour, 2.4L corn steep liquor, 0.36kg diammonium phosphate, 1.6kg light calcium carbonate, and the rest is drinking water. After the culture medium is sterilized, the fermentation liquid of Streptomyces freundii that meets the inoculation conditions is inoculated into the culture medium using the flame method. The tank temperature is controlled at about 30.0℃ throughout the process, and the air flow rate is 0-15h: 52m 3 / h, 15h-transplantation: 153m 3 / h; the tank pressure is maintained at about 0.043MPa, the bacterial concentration is 25%, and the culture cycle is 40h;

[0078] Secondary seed culture medium: 179L soybean oil, 125kg corn protein powder, 130kg corn flour, 28L corn steep liquor, 3.6kg diammonium phosphate, 16kg light calcium carbonate, and the rest is drinking water. The primary seed liquid that meets the transplanting requirements is transferred into the disinfected secondary seed culture medium. The tank pressure is controlled at 0.028MPa, the tank temperature is 30℃, and the air is 1210m 3 / h, stirring 140r / min, the culture end point is pH 7.5, bacterial concentration 39%, no foreign bacteria under microscopic examination, and the culture period is 39h;

[0079] Fermentation tank culture medium: 2831L soybean oil, 718kg cottonseed meal, 1096kg corn flour, 1914kg corn gluten meal, 65kg betaine hydrochloride, 88kg potassium chloride, 13kg diammonium phosphate, 0.38kg nickel sulfate, 0.51kg cobalt chloride, 249kg light calcium carbonate, and the rest is drinking water. The secondary seed liquid that meets the transplanting requirements is transferred to the fermentation tank, the tank pressure is 0.045MPa, the transplanting is 125h, the tank temperature is 28℃, the stirring is 210r / min, and the fermentation is completed in 125h, the temperature is 28℃, the speed is 290r / min, and the air flow is 1800m 3 / h, pH value 6.9, fat content when released from the tank is 0.41%, tylosin A content is 86.9%, fermentation time is 168h, and potency is 13762U / ml.

[0080] Comparative Example 3

[0081] Using fish meal, soybean oil, corn gluten meal and cottonseed meal as the main raw materials, according to the public Tylos fermentation process, Streptomyces freundii is used to produce tylosin through three-stage fermentation. The volume of the main fermentation production equipment is: the first tank is 1m 3 , secondary tank 10m 3 , fermentation tank 100m 3 .

[0082] Primary seed culture medium: 20L soybean oil, 14kg corn protein powder, 13kg cottonseed cake powder, 2.6L corn steep liquor, 0.38kg diammonium phosphate, 1.4kg light calcium carbonate, and the rest is drinking water. After the culture medium is sterilized, the fermentation liquid of Streptomyces freundii that meets the inoculation conditions is inoculated into the culture medium using the flame method. The tank temperature is controlled at about 30.0℃ throughout the process, and the air flow rate is 0-15h: 57m 3 / h, 15h-transplantation: 160m 3 / h; the tank pressure is maintained at about 0.046MPa, the bacterial concentration is 27%, and the culture cycle is 42h;

[0083] Secondary seed culture medium: 198L soybean oil, 132kg corn protein powder, 129kg cottonseed cake powder, 31L corn steep liquor, 4.0kg diammonium phosphate, 19kg light calcium carbonate, and the rest is drinking water. Move the primary seed liquid that meets the transplanting requirements into the disinfected secondary seed culture medium. The tank pressure is controlled at 0.030MPa, the tank temperature is 30℃, and the air is 1312m 3 / h, stirring 170r / min, the culture end point is pH 7.6, bacterial concentration 38%, no foreign bacteria under microscopic examination, and the culture period is 40h;

[0084] Fermentation tank culture medium: 2731L soybean oil, 740kg fish meal, 1121kg cottonseed meal, 1898kg corn gluten meal, 73kg betaine hydrochloride, 86kg potassium chloride, 14kg diammonium phosphate, 0.44kg nickel sulfate, 0.63kg cobalt chloride, 267kg light calcium carbonate, and the rest is drinking water. The secondary seed liquid that meets the transplanting requirements is transferred to the fermentation tank, the tank pressure is 0.045MPa, the transplanting is 125h, the tank temperature is 28.5℃, the stirring is 220r / min, and the fermentation is completed in 125h, the temperature is 28℃, the speed is 285r / min, and the air flow is 1930m 3 / h, pH 6.8, fat content when released from the tank was 0.35%, tylosin A content was 87.1%, fermentation time was 165h, and potency was 14897U / ml.

[0085] The control method of oil replenishment, water replenishment and pH adjustment in the above fermentation process is as follows:

[0086] When the feed method is used, soybean oil is added when the fat content is lower than 1.5%, the fat content of the fermentation liquid is tested every 8 hours, and the oil addition is stopped when the fat content is controlled at 1.2%; when the fermentation time is 60 hours, the bacterial concentration is greater than 40%, and sterilized drinking water is added to control it at 35-40%. The pH value is 6.3-7.5 before the component conversion after 30 hours of fermentation, and the pH value is 6.3-6.8 during the component conversion period until the end of fermentation. When the pH value is not within the range, it can be adjusted with 20-30% ammonium sulfate solution or 20% sodium hydroxide solution as needed.

[0087] Comparative Example 4

[0088] (1) Primary culture steps:

[0089] 40kg petroleum yeast powder, 180L corn starch milk, 2.4kg trimagnesium phosphate, 120L palm oil, 1300L water, 7.8kg calcium carbonate, the tank temperature is controlled at about 30.0℃ throughout the process, and the air flow is controlled at 0-15h:55m 3 / h, 15h-transplantation: 180m 3 / h; the tank pressure is maintained at about 0.045MPa, the bacterial concentration is 20%, and the culture cycle is 40h;

[0090] (2) Secondary culture steps:

[0091] 360kg of petroleum yeast powder, 450L of corn starch milk, 6.0kg of trimagnesium phosphate, 280L of palm oil, 3000L of water, 24kg of calcium carbonate, 8kg of sodium chloride, the tank temperature is controlled at 30.0℃ throughout the process, and the air flow is controlled at 0-15h:890m 3 / h, 15h-transplantation: 1150m 3 / h; the tank pressure was maintained at 0.053MPa, when the pH value was 7.02, the bacterial concentration was 33%, the mycelium growth was observed under the microscope to meet the transplanting standard and there were no foreign bacteria, and the culture cycle was 32h;

[0092] (3) Fermentation culture step:

[0093] 690kg of petroleum yeast powder, 1200L of corn starch milk, 40kg of trimagnesium phosphate, 40.0kg of sodium chloride, 50.0kg of betaine, 1050L of palm oil, 16m 3 , 65kg calcium carbonate, 0.028kg nickel sulfate, 0.030kg cobalt chloride. Use variable temperature culture, strictly control temperature and air flow, in 0-125h, the temperature before conversion is 32℃, the temperature during conversion is 38℃, the temperature after conversion to tank release is 30℃, tank pressure: 0.07MPa, air flow is 0-15h, 1350m 3 / h; 15-2570m before conversion 3 / h; 1600m during conversion 3 / h, after conversion - tank discharge 500m 3 / h, stirring speed: 120r / min, fermentation period is 158h, fat content is 0.31%, tylosin A component is 88.2%, and tank potency is 15218U / ml.

[0094] Comparative Example 5

[0095] (1) Primary culture steps:

[0096] 40kg petroleum yeast powder, 180L corn starch milk, 2.4kg trimagnesium phosphate, 120L palm oil, 1300L water, 7.8kg calcium carbonate, the tank temperature is controlled at about 30.0℃ throughout the process, and the air flow is controlled at 0-15h:55m 3 / h, 15h-transplantation: 180m 3 / h; the tank pressure is maintained at about 0.045MPa, the bacterial concentration is 20%, and the culture cycle is 40h;

[0097] (2) Secondary culture steps:

[0098] 360kg of petroleum yeast powder, 450L of corn starch milk, 6.0kg of trimagnesium phosphate, 280L of palm oil, 3000L of water, 24kg of calcium carbonate, 8kg of sodium chloride, the tank temperature is controlled at 28.0-32.0℃, and the air flow is controlled at 0-15h:890m 3 / h, 15h-transplantation: 1150m 3 / h; the tank pressure was maintained at 0.053MPa, when the pH value was 7.02, the bacterial concentration was 33%, the mycelium growth was observed under the microscope to meet the transplanting standard and there were no foreign bacteria, and the culture cycle was 32h;

[0099] (3) Fermentation culture step:

[0100] 690kg petroleum yeast powder, 1200L corn starch milk, 40kg trimagnesium phosphate, 8L oxygen carrier Aid-Plus ML-50D, 40.0kg sodium chloride, 50.0kg betaine, 1050L palm oil, 16m3 water 3 , 65kg calcium carbonate, 0.028kg nickel sulfate, 0.030kg cobalt chloride. Use variable temperature culture, strictly control temperature and air flow, in 0-125h, the temperature before conversion is 32℃, the temperature during conversion is 38℃, the temperature after conversion to tank release is 30℃, tank pressure: 0.07MPa,, air flow is 0-15h, 1350m 3 / h; 15-2570m before conversion 3 / h; 1600m during conversion 3 / h, after conversion - tank discharge 500m 3 / h, stirring speed: 120r / min, fermentation cycle is 148h, fat content is 0.18%, tylosin A component is 91.1%, and tank potency is 16002U / ml.

[0101] Comparative Example 6

[0102] (1) Primary culture steps:

[0103] 40kg petroleum yeast powder, 180L corn starch milk, 2.4kg trimagnesium phosphate, 120L palm oil, 1300L water, 7.8kg calcium carbonate, the tank temperature is controlled at about 30.0℃ throughout the process, and the air flow is controlled at 0-15h:55m 3 / h, 15h-transplantation: 180m 3 / h; the tank pressure is maintained at about 0.045MPa, the bacterial concentration is 20%, and the culture cycle is 40h;

[0104] (2) Secondary culture steps:

[0105] 360kg of petroleum yeast powder, 450L of corn starch milk, 6.0kg of trimagnesium phosphate, 280L of palm oil, 3000L of water, 24kg of calcium carbonate, 8kg of sodium chloride, the tank temperature is controlled at 28.0-32.0℃, and the air flow is controlled at 0-15h:890m 3 / h, 15h-transplantation: 1150m 3 / h; the tank pressure was maintained at 0.053MPa, when the pH value was 7.02, the bacterial concentration was 33%, the mycelium growth was observed under the microscope to meet the transplanting standard and there were no foreign bacteria, and the culture cycle was 32h;

[0106] (3) Fermentation culture step:

[0107] 690kg petroleum yeast powder, 1200L corn starch milk, 40kg trimagnesium phosphate, 80L n-dodecane by volume, 40.0kg sodium chloride, 50.0kg betaine, 1050L palm oil, 16m 3 , 65kg calcium carbonate, 0.028kg nickel sulfate, 0.030kg cobalt chloride. Use variable temperature culture, strictly control temperature and air flow, in 0-125h, the temperature before conversion is 32℃, the temperature during conversion is 38℃, the temperature after conversion to tank release is 30℃, tank pressure: 0.07MPa, air flow is 0-15h, 1350m 3 / h; 15-2570m before conversion 3 / h; 1600m during conversion 3 / h, after conversion - tank discharge 500m 3 / h, stirring speed: 120r / min, fermentation cycle is 150h, fat content is 0.17%, tylosin A component is 90.3%, and tank potency is 15034U / ml.

Claims

1. A culture medium for producing tylosin by fermentation with Streptomyces freundii, comprising a primary seed culture medium, a secondary seed culture medium and a fermentation culture medium, characterized in that The primary seed culture medium, the secondary seed culture medium and the fermentation culture medium all contain palm oil and petroleum yeast powder. The fermentation culture medium contains oxygen carriers such as Aid-Plus ML-50D and n-dodecane. The volume ratio of Aid-Plus ML-50D to n-dodecane is 0.1:1, wherein: The primary seed culture medium is composed of: 3.6-4.3% palm oil, 1.2-1.9% petroleum yeast powder, 6.5-7.4% corn starch milk, 0.002-0.005% trimagnesium phosphate, 2.7-3.5% calcium carbonate, and the rest is drinking water; The secondary seed culture medium is composed of: 3.8-4.7% palm oil, 5.1-6.5% petroleum yeast powder, 6.9-7.8% corn starch milk, 0.1-0.2% sodium chloride, 0.3-0.5% calcium carbonate, and the rest is drinking water; The fermentation medium comprises: 3.8-4.5% palm oil, 2.0-2.8% petroleum yeast powder, 3.7-4.3% corn starch milk, 0.10-0.14% sodium chloride, 0.12-0.15% trimagnesium phosphate, 0.19-0.23% calcium carbonate, 0.03-0.05% oxygen carrier, 0.6-0.8% betaine, 0.00034-0.00051% nickel sulfate, and the rest is drinking water.

2. A fermentation method for producing tylosin by fermentation of the culture medium according to claim 1, characterized in that The following steps are involved: Step (1): inoculating Streptomyces freundii seed liquid into a primary seed culture medium to obtain a primary seed culture liquid, wherein the primary seed culture medium comprises: 3.6-4.3% palm oil, 1.2-1.9% petroleum yeast powder, 6.5-7.4% corn starch milk, 0.002-0.005% trimagnesium phosphate, 2.7-3.5% calcium carbonate, and the remainder is drinking water; Step (2): inoculating the primary seed culture solution into a secondary seed culture medium to obtain a secondary seed culture solution, wherein the secondary seed culture medium comprises: 3.8-4.7% palm oil, 5.1-6.5% petroleum yeast powder, 6.9-7.8% corn starch milk, 0.1-0.2% sodium chloride, 0.3-0.5% calcium carbonate, and the remainder is drinking water; Step (3): inoculating the secondary seed culture solution into a fermentation medium to obtain a fermentation solution, wherein the fermentation medium comprises: 3.8-4.5% palm oil, 2.0-2.8% petroleum yeast powder, 3.7-4.3% corn starch milk, 0.10-0.14% sodium chloride, 0.12-0.15% trimagnesium phosphate, 0.19-0.23% calcium carbonate, 0.03-0.05% oxygen carrier, 0.6-0.8% betaine, 0.00034-0.00051% nickel sulfate, and the rest is drinking water; Step (4): extracting tylosin from the fermentation broth.

3. Use of the culture medium according to claim 1 in the production of tylosin by fermentation with Streptomyces freundii, characterized in that The culture medium includes a primary seed culture medium, a secondary seed culture medium and a fermentation culture medium, wherein the primary seed culture medium, the secondary seed culture medium and the fermentation culture medium all contain palm oil and petroleum yeast powder, and the fermentation culture medium contains oxygen carriers such as Aid-Plus ML-50D and n-dodecane, wherein the volume ratio of Aid-Plus ML-50D to n-dodecane is 0.1:1, wherein: The primary seed culture medium is composed of: 3.6-4.3% palm oil, 1.2-1.9% petroleum yeast powder, 6.5-7.4% corn starch milk, 0.002-0.005% trimagnesium phosphate, 2.7-3.5% calcium carbonate, and the rest is drinking water; The secondary seed culture medium is composed of: 3.8-4.7% palm oil, 5.1-6.5% petroleum yeast powder, 6.9-7.8% corn starch milk, 0.1-0.2% sodium chloride, 0.3-0.5% calcium carbonate, and the rest is drinking water; The fermentation medium comprises: 3.8-4.5% palm oil, 2.0-2.8% petroleum yeast powder, 3.7-4.3% corn starch milk, 0.10-0.14% sodium chloride, 0.12-0.15% trimagnesium phosphate, 0.19-0.23% calcium carbonate, 0.03-0.05% oxygen carrier, 0.6-0.8% betaine, 0.00034-0.00051% nickel sulfate, and the rest is drinking water.