Method for preventing and treating bacteriophage pollution in microbial industrial fermentation

Abstract

The invention discloses a method for preventing and treating bacteriophage pollution in microbial industrial fermentation, and belongs to the technical field of microbial fermentation culture. The prevention and control method comprises the following steps: carrying out disinfection treatment on a fermentation environment by using an alkaline protease solution to obtain a clean fermentation environment; adding the fermentation culture solution into a fermentation tank in a clean fermentation environment, then inoculating the seed culture solution, and carrying out fermentation culture to obtain an initial fermentation solution; and continuously adding the supplementary culture solution into the initial fermentation solution, and continuously carrying out fermentation culture to obtain a fermentation product. According to the method, the fermentation environment is killed by utilizing the strong degradation effect of the alkaline protease on the bacteriophage protein shell, then a large amount of ammonium salt is used for replacing part of organic nitrogen sources in the fermentation culture solution, and the pollution of the bacteriophage in the microbial industrial fermentation process is remarkably reduced and the success rate of fermentation is improved in an internal and external combination manner. Therefore, the control method has a good application prospect in microbial industrial fermentation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation and cultivation, and in particular relates to a method for preventing and controlling bacteriophage contamination in microbial industrial fermentation. Background Art

[0002] Phages, also known as bacterial viruses, are viruses that specifically infect bacteria, fungi, algae, and soil microorganisms. The structure of phages is similar to that of mammalian viruses, usually with a protein shell encapsulating genetic material, which can be DNA or RNA. There are many types of phages, and scientists estimate that there is at least one phage around each bacterium that can infect it.

[0003] The life cycle of bacteriophages can be divided into two types: lytic and lysogenic. After invading the host cell, the lytic phage will quickly replicate its genetic material and produce multiple protein shells, which will eventually cause the host cell to lyse and release multiple new phage particles to infect other cells. The genes of the lytic phage will be integrated into the chromosomes of the host cell. As the host cell reproduces, the phage genes will also be replicated, but the host cell will not lyse immediately. Under certain conditions, such as when exposed to ultraviolet light or other stresses, the phage will be released from the host chromosome and enter the lytic cycle.

[0004] During the microbial fermentation process, the presence of bacteriophages will have an impact on the growth of microorganisms, fermentation efficiency and product quality, mainly including the following aspects: 1) Phage infection of microorganisms may cause cell lysis and release intracellular components, which will not only reduce the number of microbial cells in the fermentation broth, but also cause the dissolved substances to affect the fermentation process. Some bacteriophages may carry genes that can cause bacteria to produce toxins or other harmful substances, which will have a negative impact on the purity and quality of the products in the fermentation process; 2) Phage contamination may also lead to instability in the fermentation process. When bacteriophages replicate in large quantities in the fermentation broth, it may cause a rapid decrease in the number of bacteria, thereby affecting the fermentation efficiency and the yield of the final product, thereby affecting the stability of the fermentation process; 3) Phage contamination also poses certain biosafety risks. If there is phage contamination in the fermentation broth, especially when the fermentation product is used in food, medicine and other fields, phages may pose a potential threat to human health.

[0005] Therefore, it is necessary to provide a method for preventing and controlling bacteriophage contamination in microbial industrial fermentation to solve the shortcomings of the prior art. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preventing and controlling bacteriophage contamination in microbial industrial fermentation. In the present invention, alkaline protease is used to treat the fermentation environment to reduce bacteriophage contamination, and the medium components in the fermentation process are optimized to further reduce bacteriophage contamination; through the synergistic effect of the above two aspects of treatment, bacteriophage contamination in microbial fermentation can be significantly reduced, and fermentation stability and the yield of fermentation products can be improved.

[0007] In a first aspect, the present invention provides a method for preventing and controlling bacteriophage contamination in microbial industrial fermentation, comprising the following steps: using an alkaline protease solution to disinfect the fermentation environment to obtain a clean fermentation environment; adding fermentation culture medium to a fermentation tank in the clean fermentation environment, and then accessing the seed culture medium to carry out fermentation culture to obtain an initial fermentation culture; continuing to add feed culture medium to the initial fermentation culture, continuing fermentation culture to obtain a fermentation product; wherein the fermentation culture medium includes 10-20 g / L of a carbon source, 10-20 g / L of an organic nitrogen source, 5-15 g / L of an ammonium salt, 4-8 g / L of potassium dihydrogen phosphate, 2-5 g / L of dipotassium hydrogen phosphate, 0.5-1 g / L of magnesium sulfate, 0.01-0.05 g / L of manganese sulfate, and a pH of 6.5-7.5.

[0008] In the fermentation broth provided by the present invention, the carbon source may be, for example, 10 g / L, 12 g / L, 15 g / L, 18 g / L, 20 g / L or other values ​​within the range; the organic nitrogen source may be, for example, 10 g / L, 12 g / L, 15 g / L, 18 g / L, 20 g / L or other values ​​within the range; the ammonium salt may be, for example, 5 g / L, 7 g / L, 10 g / L, 13 g / L, 15 g / L or other values ​​within the range; potassium dihydrogen phosphate may be, for example, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L or other values ​​within the range. other values; dipotassium hydrogen phosphate can be, for example, 2g / L, 3g / L, 4g / L, 5g / L or other values ​​within this range; magnesium sulfate can be, for example, 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1g / L or other values ​​within this range; manganese sulfate can be, for example, 0.01g / L, 0.02g / L, 0.03g / L, 0.04g / L, 0.05g / L or other values ​​within this range; pH can be, for example, 6.5, 6.7, 7, 7.3, 7.5 or other values ​​within this range.

[0009] Furthermore, the carbon source in the fermentation culture medium provided by the present invention can be selected from conventional carbon sources in the prior art according to actual use needs, such as glycerol or corn starch; the organic nitrogen source can also be selected from conventional nitrogen sources in the prior art according to actual use needs, such as yeast extract powder, peptone, soybean meal or corn steep liquor.

[0010] In the present invention, the inventors have found that, in the process of microbial industrial fermentation, the fermentation environment is firstly disinfected by utilizing the strong degradation effect of alkaline protease on the phage protein shell, thereby ensuring that there is no phage residue in the external environment and air system of the fermentation tank, thereby reducing phage contamination from the outside; then, a large amount of ammonium salt is used to replace part of the organic nitrogen source in the fermentation culture medium, thereby reducing phage contamination from the inside, and improving the fermentation stability and the yield of the fermentation product. By combining the internal and external methods, the phage contamination in the process of microbial industrial fermentation is significantly reduced, and the success rate of the fermentation is improved.

[0011] Specifically, the ammonium salt in the fermentation culture medium provided by the present invention has the following effects: 1) ammonium salt can prevent bacteriophage from adsorbing and invading microorganisms; 2) competitive inhibition: ammonium salt, as an effective nitrogen source and / or phosphorus source, can promote the growth and reproduction of microorganisms and help competitively inhibit bacteriophages; 3) optimize the fermentation environment: ammonium salt can adjust the pH value of the culture medium and keep it in an appropriate range (usually 6.5-7.5), and the appropriate pH value helps to optimize the growth conditions of microorganisms, while also having a negative impact on the stability and infection ability of bacteriophages; 4) increase the stability of the culture medium: ammonium salt can improve the stability and uniformity of the culture medium, reduce the precipitation and uneven distribution of the culture medium components, and this stability helps to reduce the aggregation of bacteriophages in the culture medium, thereby reducing their chance of infection of microorganisms; 5) support microbial metabolism: ammonium salt can enhance the metabolic capacity of microorganisms and improve their resistance to external pressure. When the metabolic activity of microorganisms is enhanced, their resistance to bacteriophages will also be improved, further reducing the infection effect of bacteriophages.

[0012] In some embodiments, in the process of obtaining a clean fermentation environment, the concentration of the alkaline protease solution is 0.05-1 mg / ml, for example, it can be 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 1 mg / ml or other values ​​within this range; the step of disinfecting the fermentation environment includes: disinfecting the external environment of the fermentation and disinfecting the internal environment of the fermentation.

[0013] In some embodiments, the steps of disinfecting the external environment of fermentation specifically include: spraying alkaline protease solution on the floor, walls, fermentation tanks, and air duct surfaces of the fermentation workshop, then fumigating with formaldehyde, and finally rinsing with sterile water.

[0014] In the present invention, the action time after spraying the alkaline protease solution can be conventionally adjusted according to actual use needs, for example, it can be 5-6 hours; and formaldehyde fumigation can kill bacteriophages in the air, creating a phage-free fermentation operation space, and using sterile water to rinse the treated alkaline protease solution can prevent the alkaline protease from affecting subsequent fermentation.

[0015] In some embodiments, the step of disinfecting the internal environment of the fermentation specifically includes: spraying an alkaline protease solution on the surface of the air filter element, and then using steam to sterilize the air filter element (including a coarse air filter element and a fine air filter element) and the air duct.

[0016] In the present invention, the action time of spraying the alkaline protease solution on the surface of the air filter element can be conventionally adjusted according to actual use needs, for example, it can be 4-6 hours; the sterilization treatment time can be conventionally adjusted according to actual use needs, for example, it can be 4-5 hours; steam sterilization is used to completely inactivate the alkaline protease on the surface of the air filter element to prevent the residual alkaline protease from entering the fermentation tank and affecting the microorganisms.

[0017] In some embodiments, during the preparation of the initial fermentation broth, the ammonium salt in the fermentation broth is selected from at least one of diammonium phosphate, ammonium chloride and ammonium sulfate.

[0018] In some embodiments, during the preparation of the initial fermentation broth, the inoculation amount of the seed culture broth is 5-10% (V / V), for example, 5%, 6%, 7%, 8%, 9%, 10% or other values ​​within this range; and the seed culture broth includes Escherichia coli seed culture broth and / or Bacillus subtilis seed culture broth.

[0019] In some embodiments, during the preparation of the initial fermentation broth, the fermentation culturing step specifically includes: fermenting and culturing under the conditions of a ventilation volume of 0.5-1.0 vvm (for example, 0.5 vvm, 0.6 vvm, 0.7 vvm, 0.8 vvm, 0.9 vvm, 1 vvm or other values ​​within the range), a rotation speed of 150-250 rpm (for example, 150 rpm, 170 rpm, 200 rpm, 230 rpm, 250 rpm or other values ​​within the range), and a temperature of 30-37° C. (for example, 30° C., 32° C., 34° C., 37° C. or other values ​​within the range); the OD of the bacteria in the initial fermentation broth is 600 The value 15-20 can be, for example, 15, 16, 17, 18, 19, 20 or other values ​​within this range.

[0020] In some embodiments, during the preparation of the fermentation product, the feed culture medium includes 150-400 g / L of carbon source, 30-100 g / L of organic nitrogen source and 30-100 g / L of ammonium salt, and the feeding time is 10-15 h; wherein the ammonium salt is selected from at least one of diammonium phosphate, ammonium chloride and ammonium sulfate.

[0021] In the feed culture medium provided by the present invention, the carbon source can be, for example, 150 g / L, 200 g / L, 250 g / L, 300 g / L, 350 g / L, 400 g / L or other values ​​within this range; the organic nitrogen source can be, for example, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L or other values ​​within this range; the ammonium salt can be, for example, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L or other values ​​within this range, and the feeding time can be, for example, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h or other values ​​within this range.

[0022] The carbon source in the feed culture medium provided by the present invention can be selected from conventional carbon sources in the prior art according to actual use needs, such as glycerol or glucose; the organic nitrogen source can also be selected from conventional nitrogen sources in the prior art according to actual use needs, such as peptone or fish peptone.

[0023] In the present invention, the inventors further studied and found that when ammonium salt is added to the feed culture medium, phage contamination can be further reduced.

[0024] In some embodiments, in the process of preparing the fermentation product, after continuing the fermentation culture step, an induction agent is added to induce culture to obtain the fermentation product; the induction culture step specifically includes: when the OD of the bacteria in the fermentation broth is 600 When the value is 30-35 (for example, it can be 30, 31, 32, 33, 34, 35 or other values ​​within the range), 0.3-0.8mM (for example, it can be 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM or other values ​​within the range) of inducer is added, and the induction culture is carried out for 8-20h, for example, 8h, 10h, 12h, 14h, 16h, 18h, 20h or other values ​​within the range, at a temperature of 20-30℃ (for example, it can be 20℃, 22℃, 25℃, 28℃, 30℃ or other values ​​within the range).

[0025] In the present invention, the inducer can be selected from conventional inducers in the prior art, such as the commonly used IPTG; and the induction time and temperature can be conventionally adjusted according to the characteristics of the fermentation product.

[0026] In some embodiments, the method further includes passing the tail gas generated during the fermentation process into a tail gas tank for treatment; wherein the tail gas tank contains an alkaline solution.

[0027] In some preferred embodiments, the concentration of the alkaline solution is 0.5-1 M, and the alkaline solution is selected from at least one of sodium hydroxide and potassium hydroxide.

[0028] The beneficial effects of the present invention are as follows: Different from the prior art, in the prevention method provided by the present invention, the fermentation environment is firstly disinfected by using the strong degradation effect of alkaline protease on the phage protein shell, thereby ensuring that the external environment and air system of the fermentation tank are free of phage residues, and reducing phage contamination from the outside; then a large amount of ammonium salt is used to replace part of the organic nitrogen source in the fermentation culture medium, which can reduce phage contamination from the inside, and improve the fermentation stability and the yield of the fermentation product. By combining the internal and external methods, the phage contamination in the microbial industrial fermentation process is significantly reduced, and the fermentation success rate is improved. Therefore, the prevention and control method has a good application prospect in microbial industrial fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Picture 1 The present invention is a flow chart of the method for preventing and controlling bacteriophage contamination in microbial industrial fermentation. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0032] See also Picture 1, which is a flow chart of the method for preventing and controlling bacteriophage contamination in microbial industrial fermentation in the present invention. Specifically, the method comprises the following steps: using an alkaline protease solution to disinfect the fermentation environment to obtain a clean fermentation environment; adding a fermentation culture medium to a fermentation tank in the clean fermentation environment, and then accessing the seed culture medium to carry out fermentation culture to obtain an initial fermentation culture; continuing to add a feed culture medium to the initial fermentation culture, continuing the fermentation culture, and then adding an inducer for induction culture to obtain a fermentation product; wherein the fermentation culture medium comprises a carbon source of 10-20 g / L, an organic nitrogen source of 10-20 g / L, an ammonium salt of 5-15 g / L, potassium dihydrogen phosphate of 4-8 g / L, potassium dihydrogen phosphate of 2-5 g / L, magnesium sulfate of 0.5-1 g / L, and manganese sulfate of 0.01-0.05 g / L, and a pH of 6.5-7.5.

[0033] Example 1 A target for Escherichia coli ( E. coli The method for preventing and controlling bacteriophage contamination in BL21(DE3) industrial fermentation (using a 50L fermenter) includes the following steps: 1) Use a sprayer or spraying equipment to evenly spray 0.5mg / ml alkaline protease solution everywhere in the fermentation workshop, including the ground, corners, fermentation tank surface, air and steam pipe surface. While spraying the alkaline protease solution, use formaldehyde fumigation equipment to fumigate the fermentation workshop. After the alkaline protease solution has fully acted for 5.5 hours, use clean water to rinse the fermentation workshop; then use a spray pot to spray 1mg / ml alkaline protease solution on the surface of the air filter element (air coarse filter element and air fine filter element), repeat the operation several times to ensure uniform coverage, and treat for more than 5 hours, then use steam to sterilize the air filter element (including air coarse filter element and air fine filter element) and air pipe for 4.5 hours, and finally get a clean fermentation environment; 2) Add fermentation medium (450 g glycerol, 240 g yeast extract powder, 240 g peptone, 360 g ammonium dihydrogen phosphate, 180 g potassium dihydrogen phosphate, 90 g dipotassium hydrogen phosphate, 24 g magnesium sulfate, 0.9 g manganese sulfate, add water to 30 L; adjust the medium pH to 7.0) to the fermentation tank in the clean fermentation environment obtained in step 1), sterilize at 121°C for 30 min, cool to 37°C, inoculate 3 L Escherichia coli seed culture medium, and ferment at a ventilation volume of 0.8 vvm, a rotation speed of 200 rpm, and a temperature of 37°C until the OD of the bacteria in the initial fermentation liquid reaches 0. 600 Value 18; 3) Feed culture medium (200 g diammonium phosphate, 225 g peptone, 450 g glycerol, 450 g glucose, and water to make a volume of 5 L) was added to the initial fermentation broth obtained in step 2) for 12 h. Fermentation was continued until the OD of the bacteria in the fermentation broth reached600 When the value was 30, 0.5 mM IPTG was then added for induction culture for 8 h to obtain mannanase; 4) The tail gas generated in the fermentation tank is passed into a tail gas tank containing 1M sodium hydroxide solution for treatment.

[0034] Example 2 In this example, Escherichia coli ( E. coli The method for preventing and controlling bacteriophage contamination in industrial fermentation of BL21(DE3) (using a 50L fermenter) is basically the same as that in Example 1, except that: In step 2), the fermentation culture medium includes the following components: 450 g glycerol, 240 g yeast extract powder, 240 g peptone, 300 g ammonium chloride, 180 g potassium dihydrogen phosphate, 90 g dipotassium hydrogen phosphate, 24 g magnesium sulfate, 0.9 g manganese sulfate, add water to 30 L; adjust the pH of the culture medium to 7.0; In step 3), the feed culture medium includes the following components: 225 g of ammonium chloride, 225 g of peptone, 450 g of glycerol, and 450 g of glucose, and water is added to prepare a volume of 5 L.

[0035] Example 3 In this example, Escherichia coli ( E. coli The method for preventing and controlling bacteriophage contamination in industrial fermentation of BL21(DE3) (using a 50L fermenter) is basically the same as that in Example 1, except that: In step 2), the fermentation culture medium includes the following components: 450 g glycerol, 240 g yeast extract powder, 240 g peptone, 330 g ammonium sulfate, 180 g potassium dihydrogen phosphate, 90 g dipotassium hydrogen phosphate, 24 g magnesium sulfate, 0.9 g manganese sulfate, add water to 30 L; adjust the pH of the culture medium to 7.0; In step 3), the feed culture medium includes the following components: 250 g ammonium sulfate, 225 g peptone, 450 g glycerol, 450 g glucose, and water is added to prepare a volume of 5 L.

[0036] Example 4 In this example, Escherichia coli ( E. coli The method for preventing and controlling bacteriophage contamination in industrial fermentation of BL21(DE3) (using a 50L fermenter) is basically the same as that in Example 1, except that: In step 2), the fermentation culture medium includes the following components: 450 g glycerol, 240 g yeast extract powder, 240 g peptone, 300 g diammonium dihydrogen phosphate, 90 g ammonium chloride, 180 g dipotassium dihydrogen phosphate, 90 g dipotassium hydrogen phosphate, 24 g magnesium sulfate, 0.9 g manganese sulfate, and water is added to 30 L; the pH of the culture medium is adjusted to 7.0; In step 3), the feed culture medium includes the following components: 120 g of ammonium dihydrogen phosphate, 130 g of ammonium chloride, 225 g of peptone, 450 g of glycerol, and 450 g of glucose, and water is added to prepare a volume of 5 L.

[0037] Example 5 In this example, Escherichia coli ( E. coli The method for preventing and controlling bacteriophage contamination in BL21(DE3)) industrial fermentation (using a 500L fermenter) is basically the same as that in Example 1, except that: In step 2), the fermentation medium includes the following components: 4500g glycerol, 2500g yeast extract powder, 2000g peptone, 2700g ammonium chloride, 900g ammonium sulfate, 2000g potassium dihydrogen phosphate, 800g dipotassium hydrogen phosphate, 250g magnesium sulfate, 9g manganese sulfate, add water to 300L; adjust the medium pH to 7.0; the ventilation volume is 0.7vvm, and the OD of the bacteria in the initial fermentation liquid is 600 value 20; In step 3), the feed culture medium includes the following components: 1000 g ammonium chloride, 500 g ammonium sulfate, 1000 g peptone, 3500 g glycerol, 3500 g glucose, water is added to make a volume of 20 L, the feeding time is 15 h, and 1 mM IPTG is added.

[0038] Example 6 A method for preventing and controlling bacteriophage contamination in industrial fermentation of Bacillus subtilis 168 (using a 500L fermentation tank), comprising the following steps: 1) Use a sprayer or spraying equipment to evenly spray 0.5mg / ml alkaline protease solution everywhere in the fermentation workshop, including the ground, corners, fermentation tank surface, air and steam pipe surface. While spraying the alkaline protease solution, use formaldehyde fumigation equipment to fumigate the fermentation workshop. After the alkaline protease solution has fully acted for 5.5 hours, use clean water to rinse the fermentation workshop; then use a spray pot to spray 1mg / ml alkaline protease solution on the surface of the air filter element (air coarse filter element and air fine filter element), repeat the operation several times to ensure uniform coverage, and treat for more than 5 hours, then use steam to sterilize the air filter element (including air coarse filter element and air fine filter element) and air pipe for 4.5 hours, and finally get a clean fermentation environment; 2) Add fermentation culture solution (3000 g corn starch, 2500 g soybean meal, 2500 g corn steep liquor, 2000 g ammonium dihydrogen phosphate, 1500 g potassium dihydrogen phosphate, 1000 g dipotassium hydrogen phosphate, 200 g magnesium sulfate, 5 g manganese sulfate, add water to 300 L; adjust the pH of the culture medium to 7.0) to the fermentation tank in the clean fermentation environment obtained in step 1), sterilize at 121°C for 30 min, cool to 37°C, inoculate 30 L of Bacillus subtilis seed culture solution, and ferment under the conditions of ventilation volume of 0.7 vvm, rotation speed of 200 rpm, and temperature of 30°C until the OD of the bacteria in the initial fermentation solution reaches 0.5. 600 Value 18; 3) Adding feed culture solution (1000 g of diammonium phosphate, 1000 g of fish peptone, 6000 g of glucose, and adding water to make a volume of 20 L) to the initial fermentation liquid obtained in step 2) for 10 h, and continuing the fermentation culture. When the feeding is finished, smears are made every 2 h and observed under a microscope. When spores are formed by about 90%, the spores are placed in a tank for treatment to obtain spores; 4) The tail gas generated in the fermentation tank is passed into a tail gas tank containing 1M sodium hydroxide solution for treatment.

[0039] Example 7 The method for preventing and controlling bacteriophage contamination in industrial fermentation of Bacillus subtilis 168 (using a 500L fermenter) in this example is basically the same as that in Example 6, except that: In step 2), the fermentation culture medium includes the following components: 3000 g corn starch, 2500 g soybean meal, 2500 g corn steep liquor, 1800 g ammonium sulfate, 1500 g potassium dihydrogen phosphate, 1000 g dipotassium hydrogen phosphate, 200 g magnesium sulfate, 5 g manganese sulfate, and water is added to 300 L; the pH of the culture medium is adjusted to 7.0; In step 3), the feed culture medium includes the following components: 1200 g of ammonium sulfate, 1000 g of fish peptone, 6000 g of glucose, and water is added to prepare a volume of 20 L.

[0040] Example 8 The method for preventing and controlling bacteriophage contamination in industrial fermentation of Bacillus subtilis 168 (using a 500L fermenter) in this example is basically the same as that in Example 6, except that: In step 2), the fermentation culture medium includes the following components: 3000 g corn starch, 2500 g soybean meal, 2500 g corn steep liquor, 1480 g ammonium chloride, 1500 g potassium dihydrogen phosphate, 1000 g dipotassium hydrogen phosphate, 200 g magnesium sulfate, 5 g manganese sulfate, and water is added to 300 L; the pH of the culture medium is adjusted to 7.0; In step 3), the feed culture medium includes the following components: 1000 g of ammonium chloride, 1000 g of fish peptone, 6000 g of glucose, and water is added to prepare a volume of 20 L.

[0041] Example 9 The method for preventing and controlling bacteriophage contamination in industrial fermentation of Bacillus subtilis 168 (using a 500L fermenter) in this example is basically the same as that in Example 6, except that: In step 2), the fermentation culture medium includes the following components: 3000g corn starch, 2500g soybean meal, 2500g corn steep liquor, 2400g ammonium dihydrogen phosphate, 2400g ammonium sulfate, 1500g potassium dihydrogen phosphate, 1000g dipotassium hydrogen phosphate, 200g magnesium sulfate, 5g manganese sulfate, and water is added to 300L; the pH of the culture medium is adjusted to 7.0; In step 3), the feed culture medium includes the following components: 1000 g of ammonium dihydrogen phosphate, 500 g of ammonium sulfate, 1000 g of fish peptone, and 6000 g of glucose, and water is added to prepare a volume of 20 L.

[0042] Example 10 The method for preventing and controlling bacteriophage contamination in industrial fermentation of Bacillus subtilis 168 (using a 500L fermenter) in this example is basically the same as that in Example 6, except that: In step 2), the fermentation culture medium includes the following components: 3000g corn starch, 2500g soybean meal, 2500g corn steep liquor, 1200g diammonium dihydrogen phosphate, 1200g ammonium chloride, 1500g potassium dihydrogen phosphate, 1000g dipotassium hydrogen phosphate, 200g magnesium sulfate, 5g manganese sulfate, and water is added to 300L; the pH of the culture medium is adjusted to 7.0; In step 3), the feed culture medium includes the following components: 800 g of ammonium dihydrogen phosphate, 800 g of ammonium chloride, 1000 g of fish peptone, and 6000 g of glucose, and water is added to prepare a volume of 20 L.

[0043] Embodiment 11 The method for preventing and controlling bacteriophage contamination in industrial fermentation of Bacillus subtilis 168 (using a 500L fermenter) in this example is basically the same as that in Example 6, except that: In step 2), the fermentation culture medium includes the following components: 3000g corn starch, 2500g soybean meal, 2500g corn steep liquor, 1500g ammonium sulfate, 1300g ammonium chloride, 1500g potassium dihydrogen phosphate, 1000g dipotassium hydrogen phosphate, 200g magnesium sulfate, 5g manganese sulfate, and water is added to 300L; the pH of the culture medium is adjusted to 7.0; In step 3), the feed culture medium includes the following components: 900 g of ammonium sulfate, 900 g of ammonium chloride, 1000 g of fish peptone, and 6000 g of glucose, and water is added to prepare a volume of 20 L.

[0044] Comparative Example 1 In this comparative example, Escherichia coli ( E. coli The method for preventing and controlling bacteriophage contamination in industrial fermentation of BL21(DE3) (using a 50L fermenter) is basically the same as that in Example 1, except that: In step 2), the fermentation culture medium includes the following components: 450 g glycerol, 500 g yeast extract powder, 500 g peptone, 180 g potassium dihydrogen phosphate, 90 g dipotassium hydrogen phosphate, 2.4 g magnesium sulfate, 0.9 g manganese sulfate, add water to 30 L; adjust the pH of the culture medium to 7.0; In step 3), the feed culture medium includes the following components: 250 g of peptone, 450 g of glycerol, and 450 g of glucose, and water is added to prepare a volume of 5 L.

[0045] Comparative Example 2 In this comparative example, Escherichia coli ( E. coli The method for preventing and controlling bacteriophage contamination in BL21(DE3)) industrial fermentation (using a 50 L fermenter) is basically the same as that in Example 1, except that the treatment in step 1) is not performed.

[0046] Comparative Example 3 The method for preventing and controlling bacteriophage contamination in industrial fermentation of Bacillus subtilis 168 (using a 500L fermenter) in this comparative example is basically the same as that in Example 6, except that: In step 2), the fermentation culture medium includes the following components: 3000 g corn starch, 2500 g soybean meal, 4500 g corn steep liquor, 1500 g potassium dihydrogen phosphate, 1000 g dipotassium hydrogen phosphate, 200 g magnesium sulfate, 5 g manganese sulfate, and water is added to 300 L; the pH of the culture medium is adjusted to 7.0; In step 3), the feed culture medium includes the following components: 2000 g of fish peptone, 6000 g of glucose, and water is added to prepare a volume of 20 L.

[0047] Comparative Example 4 The method for preventing and controlling bacteriophage contamination in industrial fermentation of Bacillus subtilis 168 (using a 500 L fermenter) in this comparative example is basically the same as that in Example 6, except that the treatment in step 1) is not performed.

[0048] 20 batches of fermentation were carried out continuously using the control methods in Examples 1-5 and Comparative Examples 1 and 2, respectively. The fermentation products and fermentation success rate during the fermentation process were statistically analyzed. The results are shown in Table 1 below.

[0049] Table 1 Fermentation products and fermentation success rate in industrial fermentation of Escherichia coli

[0050] It can be seen from the results in Table 1 that, compared with the comparative example, the prevention and control method of the present invention can significantly reduce the phage contamination in the fermentation process and improve the fermentation success rate.

[0051] 20 batches of fermentation were carried out continuously using the control methods in Examples 6-11 and Comparative Examples 3 and 4, respectively. The fermentation products and fermentation success rate during the fermentation process were statistically analyzed. The results are shown in Table 2 below.

[0052] Table 2 Fermentation products and fermentation success rate in industrial fermentation of Bacillus subtilis

[0053] It can be seen from the results in Table 2 that, compared with the comparative example, the prevention and control method of the present invention can significantly reduce the phage contamination in the fermentation process and improve the fermentation success rate.

[0054] In summary, the present invention utilizes the strong degradation effect of alkaline protease on the phage protein shell to disinfect the fermentation environment, and then uses a large amount of ammonium salt to replace part of the organic nitrogen source in the fermentation culture medium. Through the combination of internal and external methods, the phage contamination in the microbial industrial fermentation process is significantly reduced, and the success rate of fermentation is improved.

[0055] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0056] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preventing and controlling bacteriophage contamination in microbial industrial fermentation. It is characterized in that The steps include: Use alkaline protease solution to disinfect the fermentation environment to obtain a clean fermentation environment; Adding fermentation culture liquid to the fermentation tank in the clean fermentation environment, then adding seed culture liquid, and performing fermentation culture to obtain initial fermentation liquid; continuing to add feed culture solution to the initial fermentation solution, continuing fermentation culture, and obtaining a fermentation product; The fermentation culture medium includes 10-20 g / L of carbon source, 10-20 g / L of organic nitrogen source, 5-15 g / L of ammonium salt, 4-8 g / L of potassium dihydrogen phosphate, 2-5 g / L of dipotassium hydrogen phosphate, 0.5-1 g / L of magnesium sulfate, 0.01-0.05 g / L of manganese sulfate, and the pH is 6.5-7.

5.

2. The control method according to claim 1, It is characterized in that In the process of obtaining the clean fermentation environment, the concentration of the alkaline protease solution is 0.05-1 mg / ml, and the step of disinfecting the fermentation environment includes: disinfecting the external environment of the fermentation and disinfecting the internal environment of the fermentation.

3. The control method according to claim 2, It is characterized in that The steps of disinfecting the external environment of the fermentation specifically include: spraying the alkaline protease solution on the floor, walls, fermentation tanks and air duct surfaces of the fermentation workshop, then fumigating with formaldehyde, and finally rinsing with sterile water.

4. The control method according to claim 2, It is characterized in that The step of disinfecting the internal environment of the fermentation specifically includes: spraying the alkaline protease solution on the surface of the air filter element, and then using steam to sterilize the air filter element and the air duct.

5. The control method according to claim 1, It is characterized in that In the process of preparing the initial fermentation broth, the ammonium salt in the fermentation broth is selected from at least one of diammonium phosphate, ammonium chloride and ammonium sulfate.

6. The control method according to claim 1, It is characterized in that During the preparation of the initial fermentation broth, the inoculation amount of the seed culture broth is 5-10%, and the seed culture broth includes Escherichia coli seed culture broth and / or Bacillus subtilis seed culture broth.

7. The control method according to claim 1, It is characterized in that In the preparation process of the initial fermentation liquid, the fermentation culture step specifically includes: fermentation culture under the conditions of ventilation volume of 0.5-1.0vvm, rotation speed of 150-250rpm, and temperature of 30-37°C; OD of the bacteria in the initial fermentation liquid 600 Value 15-20.

8. The control method according to claim 1, It is characterized in that In the preparation process of the fermentation product, the feed culture solution includes 150-400 g / L of carbon source, 30-100 g / L of organic nitrogen source and 30-100 g / L of ammonium salt, and the feeding time is 10-15 h; Wherein, the ammonium salt is selected from at least one of diammonium phosphate, ammonium chloride and ammonium sulfate.

9. The control method according to claim 1, It is characterized in that In the preparation process of the fermentation product, after continuing the fermentation culture step, an induction agent is added to induce culture to obtain the fermentation product; the induction culture step specifically includes: when the OD of the bacteria in the fermentation liquid is 600 When the value is 30-35, add 0.3-0.8mM inducer and induce culture for 8-20h at a temperature of 20-30℃.

10. The control method according to claim 1, It is characterized in that The step also includes passing tail gas generated during the fermentation process into a tail gas tank for treatment; Wherein, the tail gas tank contains an alkaline solution.

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