CN108277179B - High-density fermentation and spray drying method of bacillus subtilis - Google Patents
High-density fermentation and spray drying method of bacillus subtilis Download PDFInfo
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Abstract
The invention discloses a high-density fermentation and spray drying method of bacillus subtilis, which comprises the steps of inoculating bacillus subtilis seed liquid into a fermentation culture medium for fermentation culture, and improving the viable bacteria concentration of the bacillus subtilis by a double-stage temperature control regulation and control and fed-batch fermentation technology to obtain high-density bacterial liquid; and then adding a drying protective agent into the high-density bacterial liquid, uniformly mixing, and then carrying out spray drying to obtain the bacillus subtilis dry powder preparation. The method has the advantages of simple process, simple operation, easy control and low cost, and is suitable for large-scale industrial production. The dry powder preparation prepared by the invention has the advantages of high viable bacteria concentration, high spore rate, good reproductive performance and the like, and can effectively improve the micro-ecological environment of a water body, purify the water quality of the water body and protect the water environment.
Description
Technical Field
The invention belongs to the technical field of environmental microbial fermentation, and particularly relates to a high-density fermentation and spray drying method of bacillus subtilis.
Background
With the continuous expansion of the production scale of aquaculture in China, the culture intensification degree is improved year by year, and the water body environment is gradually deteriorated. A large amount of residual baits, feces and dead animals and plants accumulated in the culture process are deposited in water, so that the accumulation of harmful substances such as ammonia nitrogen, nitrate, nitrite, sulfide and the like in the water body is increased, and the eutrophication degree in the water body is higher and higher. The polluted water is seriously unbalanced in water ecology due to the self purification and regulation capability of the water body, so that a large amount of pathogenic bacteria are promoted to grow and reproduce, the water quality is seriously deteriorated, and the polluted water source causes eutrophication of water bodies in adjacent areas or even larger areas through the flow of the water body, thereby causing diseases and insect pests of the water bodies in large areas, seriously damaging the production activities of the water bodies and threatening the healthy life of human beings.
Bacillus subtilis, a species of Bacillus subtilis. It is an aerobic gram-positive bacterium, widely distributed in nature, and is non-toxic and harmless to human and livestock. After the bacillus subtilis is propagated in water, harmful microorganisms such as vibrio, escherichia coli, baculovirus and the like in water can be inhibited, and diseases such as enteritis, gill rot and the like of aquatic animals are effectively prevented; meanwhile, it secretes a large amount of extracellular enzymes such as chitinase which can decompose the cell walls of pathogenic fungi and toxic and harmful substances such as residual bait, excrement, organic matters and the like in water, thereby achieving the purposes of clearing garbage particles in water and improving the turbid water quality caused by the overflow of harmful blue-green algae. The bacillus subtilis degrades the residual organic matters in the water, so that the ammonia nitrogen and the nitro nitrogen in the water are reduced by more than 80 percent, various physical and chemical indexes in the water are effectively stabilized, and the water ecological balance is maintained. Therefore, the bacillus subtilis preparation is used for improving the micro-ecological environment of the water body and purifying the water quality of the water body, and is increasingly applied to water environment treatment.
In addition, the bacillus subtilis also has better protease, amylase and lipase activities, has strong biological oxygen-taking capacity, can improve the feed conversion rate and promote the growth of animals, and is also used for a microecological preparation for feed.
The fermentation of the bacillus subtilis is a complex physiological metabolic process, the quality of a fermentation result is related to the quality of strains and seed liquid, and the composition proportion and the metabolic regulation process of a fermentation culture medium are key factors. In the existing production method of the bacillus subtilis preparation, the fermentation medium has single component, the process control is too simple, the concentration of the obtained viable bacteria is lower, and the spore conversion rate is low. The existing drying technology generally adopts a freeze drying technology or a low-temperature drying technology, bacterial liquid or thallus needs to be concentrated by a centrifugal or filtering method before drying, the production period is long, the complexity and uncertainty of the preparation process are increased, the survival rate of the thallus in the dried bacterial powder preparation is further low, the actual using effect is not ideal, and the large-scale popularization and application are not facilitated. The spray drying method is a method for drying atomized materials by utilizing hot air, has simple and convenient process, simple operation and low cost, and is more applied to large-scale industrial production; however, the high temperatures generated during spray drying can cause irreversible damage to heat sensitive materials.
Disclosure of Invention
The invention provides a high-density fermentation and spray drying method of bacillus subtilis to overcome the problems in the prior art, the preparation process is simple and controllable, the production period is shortened, and the prepared bacillus subtilis has the advantages of high viable fermentation count, high spore conversion rate and high spray drying survival rate.
The technical scheme of the invention is as follows:
the invention provides a high-density fermentation and spray drying method of bacillus subtilis, which comprises the following steps:
(a) and activating strains: streaking the bacillus subtilis stored on a slope at the temperature of 4 ℃ on an NA culture medium, and culturing for 20-30 h at the temperature of 30-37 ℃;
(b) and preparing seed liquid: selecting activated bacterial colonies, inoculating the bacterial colonies into a sterilized broth culture medium, and carrying out constant-temperature shaking table culture at the temperature of 30-37 ℃ and the rpm of 150-220 for 20-30 h to prepare a seed solution;
(c) and culturing in a fermentation tank: transferring the prepared bacillus subtilis seed solution into a fermentation tank filled with a fermentation culture medium according to the inoculation proportion of 1-10%; a fed-batch fermentation control method is adopted in the fermentation process: adding glucose solution before the 6h of fermentation to maintain the residual sugar concentration of 5-10 g/L; simultaneously, adding an acidic pH regulator or an alkaline pH regulator to maintain the pH of the fermentation liquor within the range of 6.5-8.0; other fermentation conditions were: 1VVM at the temperature of 30-37 ℃, 200-600 rpm at the stirring speed, 0.03-0.07 MPa in tank pressure and 20-30 h in fermentation time, wherein the ventilation quantity is 1-3; obtaining high-density zymophyte liquid;
(d) and (3) spray drying: adding 2-10% (w/v) of a protective agent into the high-density zymocyte liquid, uniformly stirring, and drying; the spray drying conditions were: the temperature of the air inlet is 150-180 ℃, the temperature of the air outlet is 70-90 ℃, and the feeding speed is 1-3L/h; and collecting the bacillus subtilis dry powder preparation.
Preferably, in the step (c), the temperature in the fermentation tank is controlled to be 34-37 ℃ from the initial state to the fermentation stage when the viable count in the fermentation tank reaches a stable state; the number of viable bacteria in the fermentation tank reaches a stable state until the tank is placed, and the temperature in the fermentation tank is controlled at 30-34 ℃.
Preferably, in the step (d), the protective agent is selected from at least one of corn starch, sucrose, dextrin, lactose, calcium carbonate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, polyaspartic acid, skim milk powder and trehalose.
Further preferably, in the step (d), the protective agent added into the zymocyte liquid is 1.8% of calcium carbonate, 3.2% of microcrystalline cellulose and 2% of polyaspartic acid.
Preferably, the components of the fermentation medium in step (c) are: 5-15 g/L of corn starch, 1-5 g/L of peptone, 1-5 g/L of yeast powder, 5-10 g/L of NaCl, 1-5 g/L of defoaming agent and the balance of water; the pH value of the fermentation medium after digestion is 6.5-7.5.
Preferably, the defoaming agent is at least one of polyethers, silicones and polyether modified silicon.
Preferably, in the step (c), the acidic pH regulator is selected from dilute hydrochloric acid or dilute sulfuric acid or dilute phosphoric acid with a concentration of 5-10 wt%, and the alkaline pH regulator is selected from ammonia water with a concentration of 5-10 wt% or 10-20 wt% sodium hydroxide.
Preferably, in the step (b), the sterilization condition of the broth culture medium is 115-121 ℃, the heat preservation time is 15-30 min, and the pH value before digestion is 6.5-7.5.
The viable count concentration of the bacillus subtilis of the zymocyte liquid obtained in the step (c) is more than or equal to 150 hundred million cfu/mL, and the spore conversion rate is more than or equal to 97 percent.
The viable count concentration of the bacillus subtilis dry powder preparation prepared by spray drying is more than or equal to 1000 hundred million cfu/g, and the survival rate of the strains is more than 85 percent.
In the preparation method, the count of the viable bacteria and spore dry powder is detected by adopting a flat plate dilution coating method.
The invention adopts a fed-batch fermentation control method in the fermentation process, and discovers a production method with simple and controllable process by adjusting and selecting the processes of strain activation and seed liquid preparation and various process parameters in the fermentation conditions. By controlling the fermentation process, a fed-batch fermentation method is adopted, particularly, the fermentation temperature and the concentration and time of fed-batch glucose are preferably and accurately controlled, and the high-density zymocyte liquid can be prepared by matching with proper ventilation and pressure, so that the prepared bacillus subtilis has high viable count and high spore conversion rate. The prepared bacillus subtilis dry powder preparation has the advantage of high survival rate of viable bacteria by adding a material protective agent during drying and adjusting and selecting a spray drying process; the process does not need centrifugation or concentration treatment before drying, simplifies the production process, shortens the production period, has small damage to spores, and the dried bacillus subtilis preparation exists in a uniform solid powder form, thereby being convenient for storage, transportation and use and effectively ensuring the stability and reliability of the product.
The invention optimizes the types and the addition amount of the protective agent, can greatly reduce the adverse effect of high temperature on materials during spray drying, improves the survival rate of viable bacteria, and can improve the stability of products.
The components of the fermentation medium, including the components of the defoaming agent, are optimized, and the optimized fermentation medium can better provide nutrients such as carbon sources, nitrogen sources, inorganic salts and the like required by the bacillus subtilis, accelerate the growth speed of the bacillus subtilis, shorten the fermentation period and improve the spore conversion rate and the viable bacteria density; on the other hand, the fermentation medium and the protective agent are matched, so that the tolerance capacity of the bacillus subtilis to high temperature and pressure in the spray drying process can be improved, the survival rate of viable bacteria is further improved, and the storage stability of the product is better.
The invention also optimizes the temperature control in the fermentation process, and can improve the total viable count and the spore conversion rate of the bacillus subtilis in the fermentation liquid by adopting a two-stage temperature control technology combined with a fed-batch fermentation technology.
The invention also optimizes the type and concentration of the pH regulator and the sterilization condition of the broth culture medium, further ensures the stability and controllability of the production process, ensures the propagation speed of the bacillus subtilis, improves the spore conversion rate and shortens the production period.
Compared with the prior art, the invention has the beneficial effects that: (1) the centrifugal or concentration treatment is not needed, the production process is simplified, the production period is shortened, the process is simple, and the production cost is low; (2) the total viable count and spore transformation rate of the bacillus subtilis are high; (3) the protective agent is used in the spray drying process, the prepared bacillus subtilis dry powder preparation has uniform granularity, small spore damage, strong stability and good reproduction, and is more convenient to store and use.
The bacillus subtilis dry powder preparation is applied to polluted water, can effectively remove ammonia nitrogen, nitronitrogen, COD and the like in the water, and prevents water plant diseases and insect pests.
Detailed Description
The technical solutions of the present invention are described in detail and fully below with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without any inventive step, are within the scope of the present invention. Equivalent changes or substitutions of method, process route, function and the like by those skilled in the art according to the following embodiments are within the scope of the present invention.
For better comparison and explanation of experimental effects, the bacillus subtilis in the examples and the comparative examples adopts the same strain, the serial number is CGMCC NO.1.2172, and the bacillus subtilis is purchased from the China general microbiological culture Collection center (CGMCC) without limiting the protection scope of the invention, and the fermentation and spray drying method of the invention is also applicable to other bacillus subtilis strains.
The raw materials and reagents in the following experimental examples are all of analytical grade, and can be obtained from commercial sources unless otherwise specified. In addition, the unit controls in the examples are as follows: "DEG C" is temperature unit "centigrade", "h" is time unit "hour", "MPa" is pressure unit "megapascal", "rpm" is rotation speed unit "revolution/minute", "VVM" is aeration ratio unit "fermentation aeration volume/fermentation liquid volume", "g/L" is mass concentration unit "g/liter", "cfu/mL (g)" is colony total unit "colony total cultured in each milliliter or per gram of sample to be detected", and "wt%" is weight percentage unit.
Example 1
(a) And activating strains: streaking the bacillus subtilis stored on a slant at the temperature of 4 ℃ on an NA culture medium, and culturing for 24 hours at the temperature of 35 ℃;
(b) and preparing seed liquid: selecting activated bacterial colony, inoculating into broth culture medium (sterilization condition: 115 deg.C, 15min), and shake-culturing at 35 deg.C and 220rpm for 24 hr to obtain seed solution;
(c) and (3) putting the mixture into a tank for fermentation culture: transferring the bacillus subtilis seed solution prepared in the previous step into a 5L fermentation tank filled with 3L fermentation medium according to the inoculation proportion of 5%, and culturing for 20-30 h at the temperature of 30-37 ℃.
Initial fermentation conditions: the ventilation volume is 1:1VVM, the temperature is 35 ℃, the stirring speed is 300rpm, the pH value after digestion is 7.2, and the tank pressure is 0.03 MPa. In the fermentation process, before the viable count of the bacillus subtilis in the fermentation tank reaches a stable state, DO is associated with stirring, pH is associated and controlled to be 7.2 +/-0.1, and glucose solution is fed from the 5 th hour to maintain the concentration of residual sugar in the fermentation liquid at 10 g/L; and stopping supplementing glucose from the beginning of the transformation of the bacillus subtilis into a spore state, reducing the culture temperature to 33 ℃, and culturing for 24 hours to finish fermentation.
The fermentation medium comprises the following components: 12g/L of corn starch, 2g/L of peptone, 2g/L of yeast powder, 5g/L of NaCl, 3g/L of antifoaming agent and the balance of water, wherein the pH value is 7.2, and the sterilization conditions are as follows: keeping the temperature at 121 ℃ for 20 min.
(d) And (3) spray drying: adding 5% (w/v) calcium carbonate into the zymocyte liquid, uniformly stirring, and drying under the spray drying conditions: the air inlet temperature is 180 ℃, the air outlet temperature is 90 ℃, and the feeding speed is 1.5L/h. Collecting to obtain Bacillus subtilis dry powder preparation, sealing and packaging to prevent dampness, and storing in shade.
Through the detection of a flat plate dilution coating method, the viable count concentration of the bacillus subtilis of the zymocyte liquid obtained in the step (c) of the embodiment is 200 hundred million cfu/mL, and the spore conversion rate is 98.2%; the viable count of the prepared bacillus subtilis dry powder preparation is 1200 hundred million cfu/g, and the survival rate of the strains is 88.3 percent.
Example 2
(a) And activating strains: streaking Bacillus subtilis (purchased from China general microbiological culture Collection center, number CGMCC NO.1.2172) stored at 4 deg.C on NA culture medium, and culturing at 37 deg.C for 24 hr;
(b) and preparing seed liquid: selecting activated bacterial colony, inoculating into broth culture medium (sterilization condition: 115 deg.C, 30min), and shake-culturing at constant temperature of 37 deg.C and 200rpm for 24 hr to obtain seed solution;
(c) and (3) putting the mixture into a tank for fermentation culture: transferring the bacillus subtilis seed solution prepared in the previous step into a 5L fermentation tank filled with 3L fermentation medium according to the inoculation proportion of 5%, and culturing for 20-30 h at the temperature of 30-37 ℃.
Initial fermentation conditions: the ventilation volume is 1:1VVM, the temperature is 37 ℃, the stirring speed is 300rpm, the pH value after digestion is 7.2, and the tank pressure is 0.03 MPa. In the fermentation process, before the viable count of the bacillus subtilis in the fermentation tank reaches a stable state, DO is associated with stirring, pH is associated and controlled to be 7.2 +/-0.1, and glucose solution is fed from the 4 th hour to maintain the concentration of residual sugar in the fermentation liquid at 10 g/L; and stopping supplementing glucose from the beginning of the transformation of the bacillus subtilis into a spore state, reducing the culture temperature to 33 ℃, and culturing for 22h to finish fermentation.
The fermentation medium comprises the following components: 10g/L of corn starch, 1g/L of peptone, 3g/L of yeast powder, 5g/L of NaCl, 4g/L of defoaming agent and the balance of water, wherein the pH value is 7.2, and the sterilization conditions are as follows: keeping the temperature at 121 ℃ for 20 min.
(d) And (3) spray drying: adding 10% (w/v) of corn starch into the zymocyte liquid, uniformly stirring, and then drying, wherein the spray drying conditions are as follows: the air inlet temperature is 160 ℃, the air outlet temperature is 90 ℃, and the feeding speed is 2L/h. Collecting to obtain Bacillus subtilis dry powder preparation, sealing and packaging to prevent dampness, and storing in shade.
Through the detection of a flat plate dilution coating method, the viable count concentration of the bacillus subtilis of the zymocyte liquid obtained in the step (c) of the embodiment is 190 hundred million cfu/mL, and the spore conversion rate is 97.9%; the viable count of the prepared bacillus subtilis dry powder preparation is 1100 hundred million cfu/g, and the survival rate of the strains is 85.2 percent.
Example 3
(a) And activating strains: streaking Bacillus subtilis (purchased from China general microbiological culture Collection center, number CGMCC NO.1.2172) stored at 4 deg.C on NA culture medium, and culturing at 30 deg.C for 30 h;
(b) and preparing seed liquid: selecting activated colony, inoculating into broth culture medium (sterilization condition: 121 deg.C, 15min), and shake-culturing at 30 deg.C and 150rpm for 30h to obtain seed solution;
(c) and (3) putting the mixture into a tank for fermentation culture: transferring the bacillus subtilis seed solution prepared in the previous step into a 5L fermentation tank filled with 3L fermentation medium according to the inoculation proportion of 1%, and culturing for 20-30 h at the temperature of 30-37 ℃.
Initial fermentation conditions: the ventilation volume is 2:1VVM, the temperature is 37 ℃, the stirring speed is 600rpm, the pH value is 7.0 after the stirring is finished, and the tank pressure is 0.05 MPa. In the fermentation process, before the viable count of the bacillus subtilis in the fermentation tank reaches a stable state, DO is associated with stirring, pH is associated and controlled to be 7.0 +/-0.1, and glucose solution is fed from the 2 nd hour to maintain the concentration of residual sugar in the fermentation liquid at 5 g/L; and stopping supplementing glucose from the beginning of the transformation of the bacillus subtilis into a spore state, keeping the culture temperature at 37 ℃, and culturing for 20 hours to finish fermentation.
The fermentation medium comprises the following components: 8g/L of corn starch, 5g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 5g/L of antifoaming agent, and the balance of water, pH 6.5, and sterilization conditions are as follows: keeping the temperature at 115 ℃ for 30 min.
(d) And (3) spray drying: adding 2% (w/v) of corn starch into the zymocyte liquid, uniformly stirring, and then drying, wherein the spray drying conditions are as follows: the air inlet temperature is 150 ℃, the air outlet temperature is 70 ℃, and the feeding speed is 1L/h. Collecting to obtain Bacillus subtilis dry powder preparation, sealing and packaging to prevent dampness, and storing in shade.
Through the detection of a flat plate dilution coating method, the viable count concentration of the bacillus subtilis of the zymocyte liquid obtained in the step (c) of the embodiment is 170 hundred million cfu/mL, and the spore conversion rate is 97 percent; the viable count of the prepared bacillus subtilis dry powder preparation is 1000 hundred million cfu/g, and the survival rate of the strains is 85 percent.
Example 4
(a) And activating strains: streaking Bacillus subtilis (purchased from China general microbiological culture Collection center, number CGMCC NO.1.2172) stored at 4 deg.C on NA culture medium, and culturing at 37 deg.C for 20 hr;
(b) and preparing seed liquid: selecting activated bacterial colony, inoculating into broth culture medium (sterilization condition: 118 deg.C, 20min), and shake-culturing at constant temperature of 37 deg.C and 220rpm for 20 hr to obtain seed solution;
(c) and (3) putting the mixture into a tank for fermentation culture: transferring the bacillus subtilis seed solution prepared in the previous step into a 5L fermentation tank filled with 3L fermentation medium according to the inoculation proportion of 10%, and culturing for 20-30 h at the temperature of 30-37 ℃.
Initial fermentation conditions: the ventilation volume is 3:1VVM, the temperature is 34 ℃, the stirring speed is 200rpm, the pH value is 7.8 after digestion, and the tank pressure is 0.04 MPa. In the fermentation process, before the viable count of the bacillus subtilis in the fermentation tank reaches a stable state, DO is associated with stirring, pH is associated and controlled to be 7.8 +/-0.1, and glucose solution is fed from the 3 rd hour to maintain the concentration of residual sugar in the fermentation liquid at 8 g/L; and stopping supplementing glucose from the beginning of the transformation of the bacillus subtilis into a spore state, reducing the culture temperature to 30 ℃, and culturing for 20 hours to finish fermentation.
The fermentation medium comprises the following components: 10g/L of corn starch, 2g/L of peptone, 2g/L of yeast powder, 7g/L of NaCl, 1g/L of antifoaming agent and the balance of water, wherein the pH value is 6.5, and the sterilization conditions are as follows: keeping the temperature at 115 ℃ for 30 min.
(d) And (3) spray drying: adding 5% (w/v) of microcrystalline cellulose into the zymocyte liquid, uniformly stirring, and drying under the spray drying conditions: the air inlet temperature is 150 ℃, the air outlet temperature is 70 ℃, and the feeding speed is 1L/h. Collecting to obtain Bacillus subtilis dry powder preparation, sealing and packaging to prevent dampness, and storing in shade.
Through the detection of a flat plate dilution coating method, the viable count concentration of the bacillus subtilis of the zymocyte liquid obtained in the step (c) of the embodiment is 200 hundred million cfu/mL, and the spore conversion rate is 98.9%; the viable count of the prepared bacillus subtilis dry powder preparation is 1300 hundred million cfu/g, and the survival rate of the strains is 89.9 percent.
Example 5
(a) And activating strains: streaking Bacillus subtilis (purchased from China general microbiological culture Collection center, number CGMCC NO.1.2172) stored at 4 deg.C on NA culture medium, and culturing at 37 deg.C for 20 hr;
(b) and preparing seed liquid: selecting activated bacterial colony, inoculating into broth culture medium (sterilization condition: 118 deg.C, 20min), and shake-culturing at constant temperature of 37 deg.C and 220rpm for 20 hr to obtain seed solution;
(c) and (3) putting the mixture into a tank for fermentation culture: transferring the bacillus subtilis seed solution prepared in the previous step into a 5L fermentation tank filled with 3L fermentation medium according to the inoculation proportion of 5%, and culturing for 20-30 h at the temperature of 30-37 ℃.
Initial fermentation conditions: the air flow is 3:1VVM, the temperature is 36 ℃, the stirring speed is 200rpm, the pH value after digestion is 7.2, and the tank pressure is 0.04 MPa. In the fermentation process, before the viable count of the bacillus subtilis in the fermentation tank reaches a stable state, DO is associated with stirring, pH is associated and controlled to be 7.2 +/-0.1, and glucose solution is fed from the 4 th hour to maintain the concentration of residual sugar in the fermentation liquid at 8 g/L; and stopping supplementing glucose from the beginning of the transformation of the bacillus subtilis into a spore state, reducing the culture temperature to 33 ℃, and culturing for 20 hours to finish fermentation.
The fermentation medium comprises the following components: 8g/L of corn starch, 2g/L of peptone, 3g/L of yeast powder, 5g/L of NaCl, 5g/L of antifoaming agent, and the balance of water, pH7.0, and sterilization conditions are as follows: keeping the temperature at 115 ℃ for 20 min.
(d) And (3) spray drying: adding 1.8% of calcium carbonate, 2.2% of microcrystalline cellulose and 1% of polyaspartic acid into the zymocyte liquid, uniformly stirring, and drying, wherein the spray drying conditions are as follows: the air inlet temperature is 160 ℃, the air outlet temperature is 80 ℃, and the feeding speed is 2L/h. Collecting to obtain Bacillus subtilis dry powder preparation, sealing and packaging to prevent dampness, and storing in shade.
Through the detection of a flat plate dilution coating method, the viable count concentration of the bacillus subtilis of the zymocyte liquid obtained in the step (c) of the embodiment is 260 hundred million cfu/mL, and the spore conversion rate is 99.0 percent; the viable count of the prepared bacillus subtilis dry powder preparation is 1820 hundred million cfu/g, and the survival rate of the strains is 95.2 percent.
Comparative example 1
(a) And activating strains: streaking Bacillus subtilis (purchased from China general microbiological culture Collection center, number CGMCC NO.1.2172) stored at 4 deg.C on NA culture medium, and culturing at 35 deg.C for 24 hr;
(b) and preparing seed liquid: selecting activated bacterial colony, inoculating into broth culture medium (sterilization condition: 115 deg.C, 15min), and shake-culturing at 35 deg.C and 220rpm for 24 hr to obtain seed solution;
(c) and (3) putting the mixture into a tank for fermentation culture: transferring the bacillus subtilis seed solution prepared in the previous step into a 5L fermentation tank filled with 3L fermentation medium according to the inoculation proportion of 5%, and culturing for 20-30 h at the temperature of 30-37 ℃.
Initial fermentation conditions: the ventilation volume is 0.5:1VVM, the temperature is 35 ℃, the stirring speed is 150rpm, the pH value is 7.2 after the digestion, and the tank pressure is 0.03 MPa. In the fermentation process, before the viable count of the bacillus subtilis in the fermentation tank reaches a stable state, DO is associated with stirring, pH is associated and controlled to be 7.2 +/-0.1, and glucose solution is fed from 12h to maintain the concentration of residual sugar in the fermentation liquid at 20 g/L; and stopping supplementing glucose from the beginning of the transformation of the bacillus subtilis into a spore state, reducing the culture temperature to 33 ℃, and culturing for 24 hours to finish fermentation.
The fermentation medium comprises the following components: 12g/L of corn starch, 2g/L of peptone, 2g/L of yeast powder, 5g/L of NaCl, 3g/L of antifoaming agent and the balance of water, wherein the pH value is 7.2, and the sterilization conditions are as follows: keeping the temperature at 121 ℃ for 20 min.
(d) And (3) spray drying: adding 5% (w/v) calcium carbonate into the zymocyte liquid, uniformly stirring, and drying under the spray drying conditions: the air inlet temperature is 180 ℃, the air outlet temperature is 90 ℃, and the feeding speed is 1.5L/h. Collecting to obtain Bacillus subtilis dry powder preparation, sealing and packaging to prevent dampness, and storing in shade.
That is, only the fermentation conditions in step (c) as well as the glucose addition time and residual sugar concentration were changed compared to example 1.
The viable count of the bacillus subtilis of the zymocyte liquid obtained in the step (c) of the comparative example is 80 hundred million cfu/mL and the spore conversion rate is 72 percent through the detection of a plate dilution coating method.
Comparative example 2
(a) And activating strains: streaking Bacillus subtilis (purchased from China general microbiological culture Collection center, number CGMCC NO.1.2172) stored at 4 deg.C on NA culture medium, and culturing at 35 deg.C for 24 hr;
(b) and preparing seed liquid: selecting activated bacterial colony, inoculating into broth culture medium (sterilization condition: 115 deg.C, 15min), and shake-culturing at 35 deg.C and 220rpm for 24 hr to obtain seed solution;
(c) and (3) putting the mixture into a tank for fermentation culture: transferring the bacillus subtilis seed solution prepared in the previous step into a 5L fermentation tank filled with 3L fermentation medium according to the inoculation proportion of 5%, and culturing for 20-30 h at 38 ℃.
Initial fermentation conditions: the ventilation volume is 1:1VVM, the temperature is 38 ℃, the stirring speed is 300rpm, the pH value after digestion is 7.2, and the tank pressure is 0.03 MPa. In the fermentation process, before the viable count of the bacillus subtilis in the fermentation tank reaches a stable state, DO is associated with stirring, pH is associated and controlled to be 7.2 +/-0.1, and glucose solution is fed from the 5 th hour to maintain the concentration of residual sugar in the fermentation liquid at 10 g/L; and stopping supplementing glucose from the beginning of the transformation of the bacillus subtilis into a spore state, culturing at 38 ℃, and finishing fermentation after culturing for 24 hours.
The fermentation medium comprises the following components: 12g/L of corn starch, 2g/L of peptone, 2g/L of yeast powder, 5g/L of NaCl, 3g/L of antifoaming agent and the balance of water, wherein the pH value is 7.2, and the sterilization conditions are as follows: keeping the temperature at 121 ℃ for 20 min.
(d) And (3) spray drying: adding 5% (w/v) calcium carbonate into the zymocyte liquid, uniformly stirring, and drying under the spray drying conditions: the air inlet temperature is 180 ℃, the air outlet temperature is 90 ℃, and the feeding speed is 1.5L/h. Collecting to obtain Bacillus subtilis dry powder preparation, sealing and packaging to prevent dampness, and storing in shade.
I.e.only the temperature of the fermentation was changed compared to example 1.
And (3) detecting by a flat plate dilution coating method, wherein the viable count concentration of the bacillus subtilis of the zymocyte liquid obtained in the step (c) of the comparative example is 70 hundred million cfu/mL, and the spore conversion rate is 36 percent.
Comparative example 3
(a) And activating strains: streaking Bacillus subtilis (purchased from China general microbiological culture Collection center, number CGMCC NO.1.2172) stored at 4 deg.C on NA culture medium, and culturing at 35 deg.C for 24 hr;
(b) and preparing seed liquid: selecting activated bacterial colony, inoculating into broth culture medium (sterilization condition: 115 deg.C, 15min), and shake-culturing at 35 deg.C and 220rpm for 24 hr to obtain seed solution;
(c) and (3) putting the mixture into a tank for fermentation culture: transferring the bacillus subtilis seed solution prepared in the previous step into a 5L fermentation tank filled with 3L fermentation medium according to the inoculation proportion of 5%, and culturing for 20-30 h at the temperature of 30-37 ℃.
Initial fermentation conditions: the ventilation volume is 1:1VVM, the temperature is 35 ℃, the stirring speed is 300rpm, the pH value after digestion is 7.2, and the tank pressure is 0.03 MPa. In the fermentation process, before the viable count of the bacillus subtilis in the fermentation tank reaches a stable state, DO is associated with stirring, pH is associated and controlled to be 7.2 +/-0.1, and glucose solution is fed from the 5 th hour to maintain the concentration of residual sugar in the fermentation liquid at 10 g/L; and stopping supplementing glucose from the beginning of the transformation of the bacillus subtilis into a spore state, reducing the culture temperature to 33 ℃, and culturing for 24 hours to finish fermentation.
The fermentation medium comprises the following components: 12g/L of corn starch, 2g/L of peptone, 2g/L of yeast powder, 5g/L of NaCl, 3g/L of antifoaming agent and the balance of water, wherein the pH value is 7.2, and the sterilization conditions are as follows: keeping the temperature at 121 ℃ for 20 min.
(d) And (3) spray drying: drying without adding a protective agent, wherein spray drying conditions are as follows: the air inlet temperature is 180 ℃, the air outlet temperature is 90 ℃, and the feeding speed is 1.5L/h. Collecting to obtain Bacillus subtilis dry powder preparation, sealing and packaging to prevent dampness, and storing in shade.
That is, compared with example 1, only the protecting agent in step (d) was removed and spray-dried directly.
The viable count of the bacillus subtilis dry powder preparation prepared by the comparative example is 530 hundred million cfu/g and the survival rate of the strains is 39.0 percent through the detection of a plate dilution coating method.
Comparative example 4
(a) And activating strains: streaking Bacillus subtilis (purchased from China general microbiological culture Collection center, number CGMCC NO.1.2172) stored at 4 deg.C on NA culture medium, and culturing at 35 deg.C for 24 hr;
(b) and preparing seed liquid: selecting activated bacterial colony, inoculating into broth culture medium (sterilization condition: 115 deg.C, 15min), and shake-culturing at 35 deg.C and 220rpm for 24 hr to obtain seed solution;
(c) and (3) putting the mixture into a tank for fermentation culture: transferring the prepared Bacillus subtilis seed solution into a 5L fermentation tank filled with 3L fermentation medium at an inoculation ratio of 5%, and culturing at 30-37 deg.C for 24 h.
Initial fermentation conditions: the ventilation volume is 1:1VVM, the temperature is 30 ℃, the stirring speed is 300rpm, the pH value after digestion is 7.2, and the tank pressure is 0.03 MPa. In the fermentation process, before the viable count of the bacillus subtilis in the fermentation tank reaches a stable state, DO is associated with stirring, the pH is associated and controlled to be 7.2 +/-0.1, and glucose solution is not supplemented; and (3) from the beginning of the transformation of the bacillus subtilis into a spore state, raising the culture temperature to 37 ℃, and culturing for 24 hours to finish the fermentation.
The fermentation medium comprises the following components: 28.84g/L of rice flour, 18.21g/L of corn starch, 27.92g/L of yeast powder, 30.05g/L of corn steep liquor, 3.88g/L of soybean meal, 0.5g/L of monopotassium phosphate, 0.06g/L of magnesium sulfate, 3g/L of sodium chloride, 1g/L of calcium carbonate, pH 7.2 and sterilization conditions: keeping the temperature at 121 ℃ for 20 min.
(d) And (3) spray drying: adding 5% (w/v) calcium carbonate into the zymocyte liquid, uniformly stirring, and drying under the spray drying conditions: the air inlet temperature is 180 ℃, the air outlet temperature is 90 ℃, and the feeding speed is 1.5L/h. Collecting to obtain Bacillus subtilis dry powder preparation, sealing and packaging to prevent dampness, and storing in shade.
That is, compared to example 1, a fermentation medium with a higher starch content was used, glucose was not supplemented, and the fermentation temperature was changed.
The viable count of the bacillus subtilis of the zymocyte liquid obtained in the step (c) of the comparative example is 60 hundred million cfu/mL and the spore conversion rate is 20 percent through the detection of a flat plate dilution coating method. The viable count of the bacillus subtilis dry powder preparation prepared by the comparative example is 150 hundred million cfu/g, and the survival rate of the strains is 30 percent.
Having shown and described the basic principles, essential features and advantages of the invention, while embodiments of the invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (2)
1. Bacillus subtilis (B.subtilis)Bacillus subtilis) The high-density fermentation and spray drying method is characterized by comprising the following steps:
(a) and activating strains: streaking the bacillus subtilis stored on a slope at the temperature of 4 ℃ on an NA culture medium, and culturing for 20-30 h at the temperature of 30-37 ℃; the bacillus subtilis is purchased from the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the serial number of CGMCC NO. 1.2172; (b) and preparing seed liquid: selecting activated bacterial colonies, inoculating the bacterial colonies into a sterilized broth culture medium, and carrying out constant-temperature shaking table culture at the temperature of 30-37 ℃ and the rpm of 150-220 for 20-30 h to prepare a seed solution;
(c) and culturing in a fermentation tank: transferring the prepared bacillus subtilis seed solution into a fermentation tank filled with a fermentation culture medium according to the inoculation proportion of 1-10%; a fed-batch fermentation control method is adopted in the fermentation process: adding glucose solution before the 6h of fermentation to maintain the residual sugar concentration of 5-10 g/L; simultaneously, adding an acidic pH regulator or an alkaline pH regulator to maintain the pH of the fermentation liquor within the range of 6.5-8.0; other fermentation conditions were: 1VVM of ventilation amount of 1-3, 200-600 rpm of stirring rotation speed, 0.03-0.07 MPa of tank pressure and 20-30 h of fermentation time; obtaining high-density zymophyte liquid;
in the step (c), the temperature in the fermentation tank is controlled to be 34-37 ℃ from the initial state to the fermentation stage when the viable count in the fermentation tank reaches a stable state; the number of viable bacteria in the fermentation tank reaches a stable state until the tank is placed, and the temperature in the fermentation tank is controlled to be 30-34 ℃;
the fermentation medium comprises the following components: 5-15 g/L of corn starch, 1-5 g/L of peptone, 1-5 g/L of yeast powder, 5-10 g/L of NaCl, 1-5 g/L of defoaming agent and the balance of water; the pH value of the fermentation medium after digestion is 6.5-7.5, and the defoaming agent is at least one of polyether, organic silicon and polyether modified silicon;
(d) and (3) spray drying: adding 2-10% w/v of protective agent into the high-density zymophyte liquid, uniformly stirring, and drying; the spray drying conditions were: the temperature of the air inlet is 150-180 ℃, the temperature of the air outlet is 70-90 ℃, and the feeding speed is 1-3L/h; collecting to obtain a bacillus subtilis dry powder preparation; in the step (d), the protective agent is 1.8% of calcium carbonate, 3.2% of microcrystalline cellulose and 2% of polyaspartic acid.
2. According to claimA Bacillus subtilis of claim 1 (A)Bacillus subtilis) The high-density fermentation and spray drying method is characterized in that in the step (c), the acidic pH regulator is selected from dilute hydrochloric acid or dilute sulfuric acid or dilute phosphoric acid with the concentration of 5-10 wt%, and the alkaline pH regulator is selected from ammonia water with the concentration of 5-10 wt% or sodium hydroxide with the concentration of 10-20 wt%.
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