Bacillus coagulans fermentation culture process

Abstract

The invention belongs to the technical field of microbial strain fermentation culture, and particularly relates to a bacillus coagulans fermentation culture process. The bacillus coagulans is fermented and cultured by the following method: (1) first-generation plate activation; (2) second-generation plate activation; (3) culturing spores; (4) seed tank culture; and (5) culturing in a fermentation tank. According to the invention, mainly starting from the fermentation culture medium, the material variety and proportion of the fermentation culture medium are changed, so that the viable count, the spore count and the spore rate of the bacillus coagulans obtained by final fermentation are far higher than those of the bacillus coagulans obtained before improvement. Specifically, the viable count before improvement is 1 billion, the viable count after improvement can reach 2 billion, and the maximum spore number can reach 230 billion.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial strain fermentation and cultivation, and in particular relates to a fermentation and cultivation process of Bacillus coagulans. Background Art

[0002] The traditional fermentation culture method of Bacillus coagulans involves steps such as strain activation, plate preparation and liquid shake flask inoculation. However, this method has significant disadvantages, especially in strain preparation. Due to the differences between batches of liquid shake flask strains, the fermentation cycle is inconsistent between different batches and has a large time span, which affects the stability and efficiency of the fermentation.

[0003] In addition, there are also deficiencies in the fermentation formula. The original fermentation formula has simple nutrients and low concentrations, and there are fewer types of inorganic salts, which is not conducive to the growth of bacteria and the production of spores. Specifically, due to insufficient nutrients, the lag period of the bacteria is long. At the same time, due to the reliance on the natural rise of pH to control the timing of spore production, there are large differences between batches, and bacteria autolysis is prone to occur during the culture process. These problems have led to low fermentation bacteria, and there is great uncertainty in process adjustment and scale-up production.

[0004] In summary, the traditional fermentation culture method of Bacillus coagulans needs to be improved. In particular, in terms of strain preparation and fermentation formula, optimization is needed to improve the stability and efficiency of fermentation. By improving these aspects, the fermentation cycle can be shortened, the bacterial quantity and spore rate can be increased, thereby meeting the needs of industrial production. At the same time, it is also necessary to strengthen the research on fermentation process control to further improve the stability and controllability of fermentation. Summary of the invention

[0005] In order to solve the above-mentioned technical problems, the present invention provides a fermentation culture process for Bacillus coagulans. Compared with the traditional process, the biggest feature of the process is that it adopts an optimized formula: (1) increasing the overall concentration of the culture medium to provide a sufficient nutritional basis for the growth of Bacillus coagulans; (2) selecting carbon and nitrogen sources that are more conducive to bacterial growth and spore production as the main components of the culture medium; (3) increasing the inorganic salt component and proportion, and increasing trace inorganic salts to promote various enzymatic reactions during bacterial growth, thereby accelerating growth metabolism.

[0006] Optimized process: (1) 20% sodium hydroxide is used to control the pH at 6.3-6.5, so that the bacteria grow at a constant pH. The pH of about 6.5 is also the most suitable pH for the growth of Bacillus coagulans, which can make the bacteria proliferate rapidly, quickly utilize the effective ingredients in the culture medium, consume a large amount of nutrients, and achieve rapid spore production after the energy is consumed. The spore rate and maturity can reach more than 90% within 8-10 hours, shortening the fermentation cycle and reducing production costs.

[0007] The amount of the coagulant Bacillus obtained by fermentation culture through the process of the present invention is increased from 1 billion before the improvement to 20 billion, and the number of spores is significantly increased.

[0008] A fermentation and cultivation process of Bacillus coagulans mainly comprises the following steps:

[0009] (1) Preparation of strains:

[0010] Activate the glycerol tube bacteria on a sterile clean bench and culture them on a first-generation plate at 40-45°C for 2-3 days until colonies grow;

[0011] Pick 3-5 single colonies from the first-generation plate and activate the second-generation plate in a sterile clean bench, culture at 40-45℃ for 2-3 days until the colonies grow;

[0012] Wash the bacterial lawn on the second-generation plate thoroughly with sterile water, inoculate it into spore culture medium, and culture it at 40-45℃ for 3-4 days until the bacteria grow and produce spores completely;

[0013] (2) Fermentation culture

[0014] First-stage seed tank culture: take the cultured spores in (1) and the culture medium at a ratio of 0.3-1% and inoculate the seed tank. After inoculation, control the temperature at 40-45°C and the initial speed at 180 rpm. When the bacteria grow up and OD ≥ 3, increase the speed to 220 rpm and the initial air volume to 0.3 vvm. When DO% < 40%, increase the air volume to 0.8-1 vvm.

[0015] During the process, samples were taken regularly to test pH and OD, and microscopic examination was performed. When pH was 6.5-7.0, OD ≥ 5, and ≥ 200 cells were observed per visual field under a 100x oil microscope, the cells were cultured for 8-12 hours, and seed solution was obtained.

[0016] Fermentation in the fermenter: transfer the seed liquid cultured in the first-level seed tank to the fermenter. During the transfer process, the pressure of the seed tank is controlled at 0.15-0.2MPa, and the pressure of the fermenter is controlled at 0.02-0.03MPa. After all the transfers, close the valve, introduce sterile air, turn on the stirring at 180rpm, the ventilation ratio is 0.3vvm, set the temperature at 40-45℃, start fermentation, the initial pH is 6.3-6.5, and the DO is calibrated to 100%. When the pH drops to 6.5, click the pH automatic control and control it at 6.3-6.5. When the bacteria grow and DO% drops to 40%, increase the speed to 220rpm, and then increase the ventilation ratio by 1.5vvm. The fermentation cycle is 24-30 hours.

[0017] Preferably, the steps for preparing the solid culture medium for the first-generation plate and the second-generation plate in (1) are as follows:

[0018] Take 0.5-1.5% peptone, 0.5-1.5% beef extract powder, 0.5-1.5% sodium chloride, add purified water, adjust the pH to 6.5-7.5, and finally add agar powder at a ratio of 1.5-2%, and sterilize at 121°C for 30 minutes; after sterilization is completed, take out the culture medium from the sterilizer, wait for the temperature to drop to 50-55°C, pour it into a plate under sterile conditions, and the liquid volume of each plate is 20-25mL; after solidification, culture at 40-45°C for 24-36 hours.

[0019] Preferably, (1) the mesospore culture medium is prepared as follows: 50%-60% bran, 0.3-0.8% potassium dihydrogen phosphate, 0.05-0.5% magnesium sulfate, 0.02-0.1% manganese sulfate, 40-50% water, sterilized at 121° C. for 30 min, and after cooling, sterilized again at 121° C. for 30 min;

[0020] The spore culture medium should be mixed every 16-24 hours after inoculation to make the bacteria uniform.

[0021] Preferably, the formula of the primary seed tank culture medium in (2) is: 1-2% peptone, 1-3% yeast extract, 1-3% glucose, 1-2% beef extract powder, 0.1-0.8% potassium dihydrogen phosphate, 0.1-0.8% magnesium sulfate, 0.1-0.7% urea, 0.05-0.5% ammonium sulfate, and 0.05-0.15% manganese sulfate.

[0022] Preferably, the formula of the fermentation tank culture medium in (2) is: 1.5-3.5% soybean flour or / and soybean protein powder, 1-3% yeast extract, 1-3% glucose, 1-2% white sugar, 0.1-0.8% potassium dihydrogen phosphate, 0.1-0.8% magnesium sulfate, 0.1-0.7% urea, 0.05-0.5% ammonium sulfate, 0.05-0.15% manganese sulfate, and 0.1-0.8% trace salt;

[0023] The percentage of each raw material in the trace salt is: ferrous sulfate 0.5%, zinc sulfate 0.08%, cobalt sulfate 0.1%, copper sulfate 0.3%, biotin 0.1%, and the balance is water.

[0024] Preferably, in (2), during the fermentation process in the fermenter, before the seed liquid is transferred to the fermenter, the transfer pipe is steam sterilized for 1 hour, and the pipe is cooled with sterile air before transferring the seeds.

[0025] Preferably, in (2), during the fermentation process in the fermenter, when the pH is automatically controlled, 20-30% sodium hydroxide sterilized in advance is used to adjust the pH.

[0026] Preferably, during the fermentation in the fermenter in (2), 20-30 wt % of a polyether defoamer is added according to the foaming conditions.

[0027] The beneficial effects of the present invention are:

[0028] (1) The present invention mainly starts from the fermentation medium, changes the material type of the fermentation medium, selects a carbon and nitrogen source that is more conducive to bacterial growth and spore production as the main component of the medium, increases the trace salt component and proportion (trace salt 0.1-0.8%, including ferrous sulfate 0.5%, zinc sulfate 0.08%, cobalt sulfate 0.1%, copper sulfate 0.3%, biotin 0.1%), and increases the overall concentration of the medium, adds soybean powder and / or soybean protein powder, white sugar and other nutrients, so that the coagulant Bacillus obtained by the final fermentation, whether it is the number of live bacteria or the number of spores, the spore rate far exceeds the number before the improvement;

[0029] (2) The process is optimized in the present invention, which can make the bacteria proliferate rapidly, quickly utilize the effective ingredients in the culture medium, consume a large amount of nutrients, and achieve rapid spore production after the energy is consumed. The spore rate and maturity are above 90% within 8-10 hours, shortening the fermentation cycle and reducing production costs;

[0030] (3) In the present invention, the bacterial strain is first activated on a first-generation plate, and then a single colony is taken out and activated on a second-generation plate, and then inoculated into a spore culture medium for cultivation. After spore production, a seed liquid is formed in a first-level seed tank, and then cultured in a fermentation tank. Compared with the previous improvement of "activating the bacterial strain on a plate and connecting the liquid shake bottle as the upper tank strain", the following disadvantages are overcome: there are differences between batches of liquid shake bottle strains, resulting in inconsistent fermentation cycles between batches (varying from 24h to 70h). Therefore, the operating steps of the present invention also greatly improve the stability of the fermentation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a microscopic picture of the fermentation of Bacillus coagulans at the end (100x oil objective). DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0033] Example 1

[0034] The fermentation process of Bacillus coagulans comprises the following steps:

[0035] 1. Preparation of bacterial strains

[0036] (1) Activate the glycerol tube bacteria on a sterile clean bench and place them on a first-generation plate. Incubate at 40°C for 2 days until colonies grow. They are milky white, have neat edges, are uniform in size, and are free of foreign bacteria.

[0037] (2) Pick 3 single colonies from the first-generation plate and activate the second-generation plate in a sterile clean bench; culture at 40°C for 2 days until the colonies grow. They are milky white, with neat edges, uniform size, and no foreign bacteria; the solid culture medium (%) in (1) and (2) is: 0.5% peptone, 0.5% beef extract powder, 0.5% sodium chloride, add purified water, adjust the pH to about 6.5, and finally add agar powder at a ratio of 1.5%, 121°C, and sterilize for 30 minutes; after sterilization is completed, take the culture medium out of the sterilizer, wait for the temperature to drop to about 50°C, and pour it into the plate (diameter 90mm) under sterile conditions. The liquid volume of each plate is approximately 20mL. After solidification, place it in an incubator at 40-45°C for 24 hours without foreign bacteria before use;

[0038] (3) Wash the bacterial lawn on the second-generation plate thoroughly with sterile water, inoculate it into spore culture medium, and culture it at 40°C for 3 days until the bacteria grow out and produce spores completely without any foreign bacteria;

[0039] The spore culture medium (%) is: 50% bran, 0.3% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.02% manganese sulfate, and about 40% water. Sterilize at 121°C for 30 minutes. After cooling, repeat the sterilization once at 121°C for 30 minutes.

[0040] The spore culture medium needs to be mixed every 16 hours after inoculation to make the bacteria uniform.

[0041] The strains prepared by the above method have uniform spore rate, more than 90% spore rate, and the spore number of the spore culture medium is about 5×10 8 / g, inoculated into seed culture medium for fermentation, the lag period of bacterial growth was shortened by about half, saving production costs.

[0042] 2. Fermentation culture process

[0043] 2.1 Primary seed tank culture

[0044] Table 1: Formula of culture medium for primary seed tank culture

[0045] Serial number raw material Ratio % (volume ratio) 1 Peptone 1 2 Yeast extract 1 3 glucose 1 4 Beef dip powder 1 5 Potassium dihydrogen phosphate 0.1 6 Magnesium sulfate 0.1 7 Urea 0.1 8 Ammonium sulfate 0.05 9 Manganese sulfate 0.05

[0046] 2.2 Fermentation process

[0047] Table 2 Key process parameters of fermentation in primary seed tank

[0048] Serial number content data 1 Tank inspection Check the sealing of the fermentation tank, replace the air filter, replace the sealing gasket, etc. 2 pH before digestion 6.5 3 pH after digestion 6.0 4 Volume before elimination Add water to about 600L 5 Post-dissipation volume 800L 6 Sterilization conditions 121℃,30min 7 Dissolved oxygen control ≥20％ 8 Defoamer dosage 0.1％ 9 Cultivation temperature 40℃ 10 Ventilation ratio 0.3VVM 11 Cultivation cycle 8h

[0049] 2.3 Inoculation

[0050] Inoculate the seed tank with the cultured spore culture medium at a ratio of 0.3% (using flame inoculation). After that, control the temperature at 40°C and the initial speed at 180rpm. When the bacteria grow up and OD ≥ 3, increase the speed to 220rpm. The initial air volume is 0.3vvm, and when DO% < 40%, increase the air volume to 0.8vvm.

[0051] During the process, samples were taken regularly to test pH, OD, and microscopic examination. When pH was about 6.5, OD: ≥5, and microscopic examination showed ≥200 cells per field of view (100x oil objective), the culture was continued for about 8 hours, and then the cells were transferred to the fermentation tank (the transfer pipes needed to be steam sterilized for about 1 hour, and the pipes were cooled with sterile air before transfer). All 900L of seed liquid was transferred to the fermentation tank.

[0052] 3. Fermentation tank culture

[0053] 3.1 Medium formulation for fermentation tank culture

[0054] Table 3 Medium formula for fermentation tank culture

[0055] Serial number raw material Ratio % (volume ratio) 1 Soybean powder (soy protein powder) 1.5 2 Yeast extract 1 3 glucose 1 4 white sugar 1 5 Potassium dihydrogen phosphate 0.1 6 Magnesium sulfate 0.1 7 Urea 0.1 8 Ammonium sulfate 0.05 9 Manganese sulfate 0.05 10 Trace salt 0.1

[0056] Trace salts (%): ferrous sulfate 0.5%, zinc sulfate 0.08%, cobalt sulfate 0.1%, copper sulfate 0.3%, biotin 0.1%, and the balance is water.

[0057] 3.2 Fermentation process

[0058] Table 4 Key process parameters for fermentation in fermenter

[0059] Serial number content data 1 Tank inspection Check the sealing of the fermentation tank, replace the air filter, replace the sealing gasket, etc. 2 pH before digestion 6.5 3 pH after digestion 6.0 4 Volume before elimination Add water to 6T 5 Post-dissipation volume 8T 6 Sterilization conditions 121℃,30min 7 Dissolved oxygen control ≥20％ 8 Defoamer dosage 0.1％ 9 Cultivation temperature 40℃ 10 Ventilation ratio 0.3VVM 11 pH 20% sodium hydroxide to control pH 6.3 12 Cultivation cycle 26h

[0060] After the seed tank is grown, follow the operation and use the pressure difference method to press the seeds into the fermentation tank. The pressure of the seed tank is controlled at 0.15MPa, and the pressure of the fermentation tank is controlled at 0.02MPa. After all the transfers, close the valve, introduce sterile air, turn on the stirring at 180rpm, the ventilation ratio is 0.3vvm, set the temperature at 40℃, and start fermentation. The initial pH is about 6.3, and the DO is calibrated to 100%. When the pH drops to about 6.5, click on the pH automatic control and control it at 6.3 (using 20% ​​sodium hydroxide sterilized in advance). When the bacteria grow up, the DO% drops to about 40%, increase the speed to 220rpm, and then increase the ventilation ratio by 1.5vvm. During the process, according to the foam situation, the defoamer is added in a flow, and polyethers are used, configured to a concentration of 20%, and added as appropriate.

[0061] 4. Results Analysis

[0062] The fermentation cycle is about 26 hours. When the spore rate is ≥95% and the maturity is ≥95%, the tank can be stopped and the fermentation bacteria count can be tested. The total bacteria count is 24 billion and the spore count is 20 billion.

[0063] Example 2

[0064] The fermentation process of Bacillus coagulans comprises the following steps:

[0065] 1. Preparation of Bacteria

[0066] (1) Activate the glycerol tube bacteria on a sterile clean bench and culture them on a first-generation plate at 45°C for 3 days until colonies grow. They are milky white, have neat edges, are uniform in size, and are free of foreign bacteria;

[0067] (2) Pick 5 single colonies from the first-generation plate and activate the second-generation plate in a sterile clean bench; culture at 45°C for 3 days until the colonies grow. They are milky white, with neat edges, uniform size, and no foreign bacteria; the solid culture medium (%) in (1) and (2) is: 1.5% peptone, 1.5% beef extract powder, 1.5% sodium chloride, add purified water, adjust the pH to about 7.5, and finally add agar powder at a ratio of 2%, sterilize at 121°C for 30 minutes; after sterilization is completed, take the culture medium out of the sterilizer, wait for the temperature to drop to about 55°C, and pour it into the plate (diameter 90mm) under sterile conditions. The liquid volume of each plate is approximately 25mL. After solidification, place it in a 45°C incubator for 36 hours without foreign bacteria before use;

[0068] (3) Wash the bacterial lawn on the second-generation plate thoroughly with sterile water, inoculate it into spore culture medium, and culture it at 45°C for 4 days until the bacteria grow out, produce spores completely, and are free of foreign bacteria;

[0069] The spore culture medium (%) is: 60% bran, 0.8% potassium dihydrogen phosphate, 0.5% magnesium sulfate, 0.1% manganese sulfate, and about 50% water. Sterilize at 121°C for 30 minutes. After cooling, repeat the sterilization once at 121°C for 30 minutes.

[0070] The spore culture medium needs to be mixed every 24 hours after inoculation to make the bacteria uniform.

[0071] The strains prepared by the above method have uniform spore rate, more than 90% spore rate, and the spore number of spore culture medium is about 10×10 8 / g, inoculated into seed culture medium for fermentation, the lag period of bacterial growth was shortened by about half, saving production costs.

[0072] 2. Fermentation culture process

[0073] 2.1 Primary seed tank culture

[0074] Table 5: Formulation of culture medium for primary seed tank culture

[0075] Serial number raw material Ratio % (volume ratio) 1 Peptone 2 2 Yeast extract 3 3 glucose 3 4 Beef dip powder 2 5 Potassium dihydrogen phosphate 0.8 6 Magnesium sulfate 0.8 7 Urea 0.7 8 Ammonium sulfate 0.5 9 Manganese sulfate 0.15

[0076] 2.2 Fermentation process

[0077] Table 6 Key process parameters of fermentation in primary seed tank

[0078]

[0079]

[0080] 2.3 Inoculation

[0081] Inoculate the seed tank with the cultured spore culture medium at a ratio of 1% (using flame inoculation). After that, control the temperature at 45°C and the initial speed at 180rpm. When the bacteria grow up and OD ≥ 3, increase the speed to 220rpm. The initial air volume is 0.3vvm, and when DO% < 40%, increase the air volume to 0.8vvm.

[0082] During the process, samples were taken regularly to test pH, OD, and microscopic examination. When pH was about 7.0, OD: ≥5, and microscopic examination showed ≥200 cells per field of view (100x oil objective), the culture was continued for about 12 hours and then transferred to the fermentation tank (the transfer pipe needed to be steam sterilized for about 1 hour, and the pipe was cooled with sterile air before transfer). All 900L of seed liquid was transferred to the fermentation tank.

[0083] 3. Fermentation tank culture

[0084] 3.1 Medium formulation for fermentation tank culture

[0085] Table 7 Medium formula for fermentation tank culture

[0086] Serial number raw material Ratio % (volume ratio) 1 Soybean powder (soy protein powder) 3.5 2 Yeast extract 3 3 glucose 3 4 white sugar 2 5 Potassium dihydrogen phosphate 0.8 6 Magnesium sulfate 0.8 7 Urea 0.7 8 Ammonium sulfate 0.5 9 Manganese sulfate 0.15 10 Trace salt 0.8

[0087] Trace salts (%): ferrous sulfate 0.5%, zinc sulfate 0.08%, cobalt sulfate 0.1%, copper sulfate 0.3%, biotin 0.1%, and the balance is water.

[0088] 3.2 Fermentation process

[0089] Table 8 Key process parameters for fermentation in fermenter

[0090] Serial number content data 1 Tank inspection Check the sealing of the fermentation tank, replace the air filter, replace the sealing gasket, etc. 2 pH before digestion 7.5 3 pH after digestion 7.0 4 Volume before elimination Add water to 6T 5 Post-dissipation volume 8T 6 Sterilization conditions 125℃,40min 7 Dissolved oxygen control ≥40％ 8 Defoamer dosage 0.1％ 9 Cultivation temperature 45℃ 10 Ventilation ratio 0.8VVM 11 pH 20% sodium hydroxide to control pH 6.5 12 Cultivation cycle 30h

[0091] After the seed tank is grown, follow the operation and use the pressure difference method to press the seeds into the fermentation tank. The pressure of the seed tank is controlled at 0.2MPa, and the pressure of the fermentation tank is controlled at 0.03MPa. After all the transfers, close the valve, introduce sterile air, turn on the stirring at 180rpm, the ventilation ratio is 0.3vvm, set the temperature at 45℃, and start fermentation. The initial pH is about 6.5, and the DO is calibrated to 100%. When the pH drops to about 6.5, click on the pH automatic control and control it at 6.5 (using 30% sodium hydroxide sterilized in advance). When the bacteria grow up, the DO% drops to about 40%, increase the speed to 220rpm, and then increase the ventilation ratio by 1.5vvm. During the process, according to the foam situation, the defoamer is added in a flow, and polyethers are used, configured to a concentration of 30%, and added as appropriate.

[0092] 4. Results Analysis

[0093] The fermentation cycle is about 30 hours. When the spore rate is ≥95% and the maturity is ≥95%, the tank can be stopped and the fermentation bacteria count can be tested. The total bacteria count is 23.1 billion and the spore count is 22.8 billion.

[0094] Example 3

[0095] The fermentation process of Bacillus coagulans comprises the following steps:

[0096] 1. Preparation of Bacteria

[0097] (1) Activate the glycerol tube bacteria on a sterile clean bench and culture them on a first-generation plate at 42°C for 3 days until colonies grow. They are milky white, have neat edges, are uniform in size, and are free of foreign bacteria;

[0098] (2) Pick 4 single colonies from the first-generation plate and activate the second-generation plate in a sterile clean bench; culture at 42°C for 3 days until the colonies grow. They are milky white, with neat edges, uniform size, and no foreign bacteria; the solid culture medium (%) in (1) and (2) is: 1.0% peptone, 1.0% beef extract powder, 1.0% sodium chloride, add purified water, adjust the pH to about 7.0, and finally add agar powder at a ratio of 1.7%, sterilize at 121°C for 30 minutes; after sterilization is completed, take the culture medium out of the sterilizer, wait for the temperature to drop to about 52°C, and pour it into the plate (diameter 90mm) under sterile conditions. The liquid volume of each plate is approximately 23mL. After solidification, place it in a 42°C incubator for 30 hours without foreign bacteria before use;

[0099] (3) Wash the bacterial lawn on the second-generation plate thoroughly with sterile water, inoculate it into spore culture medium, and culture it at 42°C for 4 days until the bacteria grow out, produce spores completely, and are free of foreign bacteria;

[0100] The spore culture medium (%) is: 55% bran, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate, 0.05% manganese sulfate, and about 45% water. Sterilize at 121°C for 30 minutes. After cooling, repeat the sterilization once at 121°C for 30 minutes.

[0101] The spore culture medium needs to be mixed every 20 hours after inoculation to make the bacteria uniform.

[0102] The strains prepared by the above method have uniform spore rate, more than 90% spore rate, and the spore number of the spore culture medium is about 8×10 8 / g, inoculated into seed culture medium for fermentation, the lag period of bacterial growth was shortened by about half, saving production costs.

[0103] 2. Fermentation culture process

[0104] 2.1 Primary seed tank culture

[0105] Table 9: Formulation of culture medium for primary seed tank culture

[0106] Serial number raw material Example 1 Ratio % (volume ratio) 1 Peptone 1.5 2 Yeast extract 2 3 glucose 2 4 Beef dip powder 1.5 5 Potassium dihydrogen phosphate 0.5 6 Magnesium sulfate 0.5 7 Urea 0.5 8 Ammonium sulfate 0.25 9 Manganese sulfate 0.10

[0107] 2.2 Fermentation process

[0108] Table 10 Key process parameters of fermentation in primary seed tank

[0109] Serial number content data 1 Tank inspection Check the sealing of the fermentation tank, replace the air filter, replace the sealing gasket, etc. 2 pH before digestion 7.0 3 pH after digestion 6.5 4 Volume before elimination Add water to about 600L 5 Post-dissipation volume 850L 6 Sterilization conditions 122℃,35min 7 Dissolved oxygen control ≥30％ 8 Defoamer dosage 0.1％ 9 Cultivation temperature 42℃ 10 Ventilation ratio 0.6VVM 11 Cultivation cycle 10h

[0110] 2.3 Inoculation

[0111] Inoculate the seed tank with the cultured spore culture medium at a ratio of 0.5% (using flame inoculation). After that, control the temperature at 42°C and the initial speed at 180rpm. When the bacteria grow up and OD ≥ 3, increase the speed to 220rpm. The initial air volume is 0.3vvm, and when DO% < 40%, increase the air volume to 0.8vvm.

[0112] During the process, samples were taken regularly to test pH, OD, and microscopic examination. When pH was about 6.8, OD: ≥5, and microscopic examination showed ≥200 cells per field of view (100x oil objective), the cells were cultured for about 10 hours and transferred to the fermentation tank (the transfer pipes needed to be steam sterilized for about 1 hour, and the pipes were cooled with sterile air before transfer). All 900L of seed liquid was transferred to the fermentation tank.

[0113] 3. Fermentation tank culture

[0114] 3.1 Medium formulation for fermentation tank culture

[0115] Table 11 Medium formula for fermentation tank culture

[0116] Serial number raw material Example 1 Ratio % (volume ratio) 1 Soybean powder (soy protein powder) 2.5 2 Yeast extract 2 3 glucose 2 4 white sugar 1.5 5 Potassium dihydrogen phosphate 0.5 6 Magnesium sulfate 0.5 7 Urea 0.5 8 Ammonium sulfate 0.25 9 Manganese sulfate 0.1 10 Trace salt 0.5

[0117] Trace salts (%): ferrous sulfate 0.5%, zinc sulfate 0.08%, cobalt sulfate 0.1%, copper sulfate 0.3%, biotin 0.1%, and the balance is water.

[0118] 3.2 Fermentation process

[0119] Table 12 Key process parameters for fermentation in fermenter

[0120]

[0121]

[0122] After the seed tank is grown, follow the operation and use the pressure difference method to press the seeds into the fermentation tank. The pressure of the seed tank is controlled at 0.18MPa, and the pressure of the fermentation tank is controlled at 0.02MPa. After all the transfers, close the valve, introduce sterile air, turn on the stirring at 180rpm, the ventilation ratio is 0.3vvm, set the temperature at 42°C, and start fermentation. The initial pH is about 6.4, and the DO is calibrated to 100%. When the pH drops to about 6.5, click on the pH automatic control and control it at 6.4 (using 25% sodium hydroxide sterilized in advance). When the bacteria grow up, the DO% drops to about 40%, increase the speed to 220rpm, and then increase the ventilation ratio by 1.5vvm. During the process, according to the foam situation, the defoamer is added in a flow, and polyethers are used, configured to a concentration of 25%, and added as appropriate.

[0123] 4. Results Analysis

[0124] The fermentation cycle is about 26 hours. When the spore rate is ≥95% and the maturity is ≥95%, the tank can be stopped. At this time, the spore rate is 96.9%. The fermentation bacteria count is detected, the total bacteria count is 22.8 billion, and the spore count is 22.1 billion.

[0125] Example 4

[0126] Different from Example 3, the fermentation cycle of this example is 25 hours, the spore rate is ≥95%, and the maturity is ≥95%, and the tank can be stopped to detect the amount of fermented bacteria. The total number of bacteria is 22.5 billion, and the number of spores is 22.1 billion.

[0127] Example 5

[0128] Different from Example 3, the fermentation cycle of this example is 27 hours, the spore rate is ≥95%, and the maturity is ≥95%, and the tank can be stopped to detect the amount of fermented bacteria. The total number of bacteria is 23.5 billion, and the number of spores is 23 billion.

[0129] Example 6

[0130] (1) According to the above formula and process, 5 batches of 15m 3 The production of the tank, the following are the production data:

[0131] Table 13 Comparison of fermentation bacteria between new formula and process

[0132] batch volume content Fermentation cycle Total bacteria count 1 <![CDATA[15m 3 ]]> Original formula and process 65h 1.5 billion 2 <![CDATA[15m 3 ]]> Example 1 26h 24 billion 3 <![CDATA[15m 3 ]]> Example 2 30h 23.1 billion 4 <![CDATA[15m 3 ]]> Example 3 26h 22.8 billion 5 <![CDATA[15m 3 ]]> Example 4 25h 22.5 billion 6 <![CDATA[15m 3 ]]> Example 5 27h 23.5 billion

[0133] (2) After adjusting the formula, the spore rate and maturity of the bacteria were greatly improved, so that the yield of the fermentation liquid during spray drying was increased from the previous 50% to 95%.

[0134] Here are the relevant data:

[0135] Table 14 Comparison of spray drying data under new and old formulas

[0136] Serial number content Total bacteria count Spore count Spore rate Spray dry powder amount Spray dried powder yield 1 Original recipe 3 billion 1.5 billion 50％ 10.7 billion 50％ 2 Example 3 22.8 billion 22.1 billion 96.9％ 140 billion 95％

[0137] The culture process of the original formula in Table 14 is:

[0138] 1. Preparation of strains: Inoculate 0.5% of the strain in the glycerol tube into the shake flask medium, culture at 40-45℃ for 40-48h, and wait until the bacteria grow up and OD600≥3. Microscopic examination shows that there are no foreign bacteria and opposite stems. Inoculate 0.3-0.5% of the strain into the first-level seed tank;

[0139] 2. Cultivation conditions of the first-level seed tank: 40-45℃, rotation speed 220rpm, ventilation ratio 0.8vvm, tank pressure 0.05MPa, culture for about 24-30h, OD600≥3.5, normal under microscopic examination: 150 long rods, mostly opposite, transferred to the fermentation tank at 3%;

[0140] 3. Fermentation tank culture conditions: 40-45℃, rotation speed 200rpm, ventilation ratio 0.8vvm, tank pressure 0.05MPa, culture for about 40-60h.

[0141] In the above original culture process, the culture medium in the fermentation tank is as follows: 1.5% glucose, 1% yeast extract, 0.1% ammonium sulfate, 0.08% magnesium sulfate, and 0.1% defoaming agent.

[0142] In Table 13, the detection method of total bacterial count is as follows: the fermentation liquid and bacterial powder are diluted to a certain multiple by the stepwise dilution method, and then 1 mL is added to an empty culture dish, and the detection culture medium is poured onto the plate. After the culture medium solidifies, it is placed in a 40-45°C incubator and cultured for about 40 hours. Colonies are grown and the bacterial count is counted. The correct gradient is 30-100 colonies per plate.

[0143] In Table 13, the method for detecting the number of spores is as follows: the fermentation liquid is treated in a water bath at 80°C for 10 minutes, then cooled to room temperature, and then detected according to the above detection method.

[0144] Spore rate = number of spores in fermentation liquid / total number of bacteria in fermentation liquid × 100%

[0145] In summary, the fermentation culture process provided by the present invention mainly starts from the fermentation medium, by first activating the strain on the first-generation plate, then taking out a single colony and activating it on the second-generation plate, and then inoculating it into the spore culture medium for culture, forming a seed liquid in the first-level seed tank after spore production, and then culturing it in the fermentation tank, which greatly improves the stability of the fermentation process, so that the coagulant Bacillus obtained by the final fermentation, whether it is the number of live bacteria or the number of spores, the spore rate is far greater than the number before the improvement.

[0146] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A fermentation and cultivation process of Bacillus coagulans, characterized in that: The steps include: (1) Preparation of bacterial strains: Activate the glycerol tube bacteria on a sterile clean bench and culture them on a first-generation plate at 40-45°C for 2-3 days until colonies grow; Pick 3-5 single colonies from the first-generation plate and activate the second-generation plate in a sterile clean bench. Culture at 40-45℃ for 2-3 days until bacterial moss grows; Wash the bacterial lawn on the second-generation plate thoroughly with sterile water, inoculate it into spore culture medium, and culture it at 40-45℃ for 3-4 days until the bacteria grow and produce spores completely; (2) Fermentation culture First-stage seed tank culture: Take the spores cultured in (1) and the culture medium at a ratio of 0.3-1% and inoculate them into the seed tank. After that, incubate at 40-45°C and 180 rpm until the bacteria grow up and OD ≥ 3, then increase the speed to 220 rpm, with an initial air volume of 0.3 vvm. When DO% < 40%, increase the air volume to 0.8-1 vvm, and culture for 8-12 hours to obtain the seed solution. Fermentation in fermenter: transfer the seed liquid cultured in the first-level seed tank to the fermenter, introduce sterile air, stir at 180rpm, ventilation ratio 0.3vvm, set the temperature at 40-45℃, start fermentation, initial pH 6.3-6.5, calibrate DO at 100%, when pH drops to 6.5, click pH automatic control, control it at 6.3-6.5, wait for the bacteria to grow, when DO% drops to 40%, increase the speed to 220rpm, increase the ventilation ratio to 1.5vvm, and the fermentation cycle is 24-30h.

2. The fermentation and cultivation process of Bacillus coagulans according to claim 1, characterized in that: (1) The steps for preparing the solid culture medium for the first-generation plate and the second-generation plate are as follows: Take 0.5-1.5% peptone, 0.5-1.5% beef extract powder, 0.5-1.5% sodium chloride, add purified water, adjust the pH to 6.5-7.5, and finally add 1.5-2% agar powder, sterilize at 121°C for 30 minutes; after sterilization is completed, take out the culture medium from the sterilizer, wait for the temperature to drop to 50-55°C, pour it into the plate under sterile conditions, and the liquid volume of each plate is 20-25mL; after solidification, culture at 40-45°C for 24-36 hours.

3. The fermentation and cultivation process of Bacillus coagulans according to claim 1, characterized in that: (1) The steps for preparing the mesospore culture medium are as follows: 50%-60% bran, 0.3-0.8% potassium dihydrogen phosphate, 0.05-0.5% magnesium sulfate, 0.02-0.1% manganese sulfate, and 40-50% water are sterilized at 121°C for 30 minutes, and after cooling, the mixture is sterilized again at 121°C for 30 minutes; The spore culture medium should be mixed every 16-24 hours after inoculation to make the bacteria uniform.

4. The fermentation and cultivation process of Bacillus coagulans according to claim 1, characterized in that: (2) The formula of the medium for the first-level seed tank is: peptone 1-2%, yeast extract 1-3%, glucose 1-3%, beef extract powder 1-2%, potassium dihydrogen phosphate 0.1-0.8%, magnesium sulfate 0.1-0.8%, urea 0.1-0.7%, ammonium sulfate 0.05-0.5%, and manganese sulfate 0.05-0.15%.

5. The fermentation and cultivation process of Bacillus coagulans according to claim 1, characterized in that: (2) The formula of the medium for the fermentation tank is: 1.5-3.5% soybean powder or / and soybean protein powder, 1-3% yeast extract, 1-3% glucose, 1-2% white sugar, 0.1-0.8% potassium dihydrogen phosphate, 0.1-0.8% magnesium sulfate, 0.1-0.7% urea, 0.05-0.5% ammonium sulfate, 0.05-0.15% manganese sulfate, and 0.1-0.8% trace salt; The percentage of each raw material in the trace salt is: ferrous sulfate 0.5%, zinc sulfate 0.08%, cobalt sulfate 0.1%, copper sulfate 0.3%, biotin 0.1%, and the balance is water.

6. The fermentation and cultivation process of Bacillus coagulans according to claim 1, characterized in that: (2) During the fermentation process in the fermenter, before transferring the seed liquid to the fermenter, the transfer pipe should be steam sterilized for 1 hour. When transferring the seeds, the pipe should be cooled with sterile air.

7. The fermentation and cultivation process of Bacillus coagulans according to claim 1, characterized in that: (2) During the fermentation process in the fermenter, when the pH is automatically controlled, 20-30% sodium hydroxide that has been sterilized in advance is used to adjust the pH.

8. The fermentation and cultivation process of Bacillus coagulans according to claim 1, characterized in that: (2) During the fermentation process in the fermenter, 20-30 wt% of a polyether defoamer is added according to the foam situation.

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