Fermentation process of bacillus subtilis

Abstract

The invention discloses a bacillus subtilis fermentation process which comprises the following steps: adding a bacillus subtilis seed solution into a fermentation tank containing a culture solution, and collecting the real-time temperature, the real-time dissolved oxygen content and the real-time pH value of the culture solution in real time; in response to the real-time pH greater than 7, determining a first introduction amount of carbon dioxide gas; according to the real-time dissolved oxygen content, the second introduction amount of air is determined; determining a first introduction temperature of the carbon dioxide gas and the air according to the real-time temperature, the first introduction amount and the second introduction amount; determining a first stirring speed of the stirrer according to the first introduction amount and the second introduction amount; heating or cooling the first introduction amount of carbon dioxide gas and the second introduction amount of air to a first introduction temperature, introducing into the culture solution, and controlling the stirrer to stir at a first stirring speed; when the content of the target product in the culture solution is detected to reach a threshold value, judging that fermentation is completed. The method can simplify fermentation environment adjustment steps and reduce resource waste.

Description

Technical Field

[0001] The invention relates to the field of fermentation, in particular to a fermentation process of Bacillus subtilis. Background Art

[0002] Bacillus subtilis is a bacterium widely distributed in soil and decaying organic matter. Bacillus subtilis can produce a variety of beneficial metabolites during the fermentation process, such as enzymes (α-amylase, protease, lipase, cellulase, etc.), vitamins (B1, B2, B6, niacin and other B vitamins), antibiotics (subtilisin, polymyxin, nystatin, gramicidin, lipopeptide compounds, etc.) and organic acids, which can be widely used in agriculture, feed, medical treatment, environmental protection, food and other industries.

[0003] During the fermentation process of Bacillus subtilis, certain environmental conditions must be maintained to ensure that the fermentation efficiency reaches the required level, thereby increasing the yield and production efficiency of the required metabolites. However, the existing technology for changing the fermentation environment of Bacillus subtilis is generally one-to-one, and cannot be connected in series. The steps are cumbersome and wasteful of resources. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a fermentation process of Bacillus subtilis, aiming to simplify the steps of adjusting the fermentation environment of Bacillus subtilis and reduce resource waste.

[0005] To achieve the above object, the present invention provides a fermentation process of Bacillus subtilis, which comprises:

[0006] Step S1, adding Bacillus subtilis seed liquid to a fermentation tank containing culture liquid, and collecting the real-time temperature, real-time dissolved oxygen content and real-time pH of the culture liquid in the fermentation tank in real time during the fermentation process of the Bacillus subtilis;

[0007] Step S2, in response to the real-time pH being greater than 7, determining a first introduction amount of carbon dioxide gas; determining a second introduction amount of air according to the real-time dissolved oxygen content; determining a first introduction temperature of carbon dioxide gas and air according to the real-time temperature, the first introduction amount, and the second introduction amount; determining a first stirring speed of the stirrer in the fermentation tank according to the first introduction amount and the second introduction amount;

[0008] Step S3, heating or cooling the first amount of carbon dioxide gas and the second amount of air to the first introduction temperature and then introducing them into the culture solution, and controlling the stirrer to stir at the first stirring speed during the introduction process, so that the temperature of the culture solution is maintained between 34-37° C., the dissolved oxygen content is maintained between 10%-20%, and the pH is maintained between 6.5-7.5;

[0009] Step S4, detecting a first content of a target product in the culture solution; in response to the first content reaching a threshold, determining that the fermentation of the Bacillus subtilis is complete; wherein the target product includes a metabolite of the Bacillus subtilis and / or the Bacillus subtilis itself.

[0010] Optionally, after step S1, the process further includes:

[0011] In response to the real-time pH being less than 6.5, the air inlet and the air outlet of the fermenter are closed, and the fermenter is evacuated to a vacuum state so that the pressure in the fermenter reaches 0.08-0.095 MPa, so as to reduce the solubility of carbon dioxide in the culture solution;

[0012] After maintaining the pressure in the fermentation tank for 1-10 seconds, the gas outlet is opened to discharge the carbon dioxide gas escaping from the culture solution; the gas inlet is opened and step S2 is continued.

[0013] Optionally, after step S1, the process further includes:

[0014] In response to the real-time pH being between 6.5 and 7.5, the first introduction amount of carbon dioxide gas is determined to be zero.

[0015] Optionally, in step S2, in response to the real-time pH being greater than 7, determining a first introduction amount of carbon dioxide gas includes:

[0016] The first introduction amount of carbon dioxide gas is obtained according to the real-time pH, the volume of the culture solution and the ideal pH, and the introduced carbon dioxide gas is used to adjust the pH of the culture solution.

[0017] Optionally, determining the second air intake amount according to the real-time dissolved oxygen content in step S2 includes:

[0018] Determining the required oxygen amount according to the real-time dissolved oxygen content, the volume of the culture solution and the ideal dissolved oxygen content;

[0019] The second intake amount of air is determined according to the required oxygen amount and the oxygen content in the air.

[0020] Optionally, determining the first incoming temperature of the carbon dioxide gas and the air according to the real-time temperature, the first incoming amount, and the second incoming amount in step S2 includes:

[0021] The first introduction temperature of carbon dioxide gas and air is determined according to the real-time temperature, the first introduction amount and the second introduction amount, the volume of the culture solution and the ideal temperature.

[0022] Optionally, in step S2, determining a first stirring speed of a stirrer in the fermenter according to the first feed-in amount and the second feed-in amount comprises:

[0023] The first stirring speed of the agitator in the fermentation tank is determined according to a first dissolution rate of carbon dioxide gas and its corresponding first introduction amount and a second dissolution rate of oxygen in the air and its corresponding second introduction amount; wherein the ventilation speed of the fermentation tank is constant, and the first stirring is used to accelerate the dissolution rate of oxygen and carbon dioxide gas, thereby keeping the ventilation speed and the dissolution speed of carbon dioxide gas and oxygen balanced.

[0024] Optionally, the process further comprises:

[0025] During the fermentation process, the bacterial concentration of the Bacillus subtilis is collected in real time. When the bacterial concentration changes beyond an expected change, it is determined that the fermentation is abnormal.

[0026] Optionally, the step S1 includes:

[0027] The Bacillus subtilis seed solution is added into the fermentation tank containing 70% culture solution by volume at an inoculation rate of 0.5-5%.

[0028] Beneficial effects of the present invention: 1. The present invention can obtain the amount of carbon dioxide gas and air, the temperature of the introduction, and the stirring speed according to the real-time temperature, real-time dissolved oxygen content, and real-time pH of the culture solution. The real-time temperature, real-time dissolved oxygen content, and real-time pH of the culture solution in the fermenter can be adjusted in real time by using these adjustment parameters, so as to ensure that the fermentation environment of Bacillus subtilis during the fermentation process is always in an ideal state, thereby improving the fermentation efficiency. If the temperature needs to be increased in the prior art, the entire fermenter needs to be heated separately, etc. These steps are fragmented and waste resources. Compared with the prior art, the present invention can adjust the pH, temperature, and oxygen content at the same time by introducing air and carbon dioxide, thereby reducing the waste of resources. At the same time, the gas is more fully in contact with the culture solution, so that the temperature of the culture solution rises or falls evenly at various places, thereby ensuring the overall quality of the fermentation. 2. The present invention makes the ventilation speed and dissolution speed of carbon dioxide gas and oxygen balanced by adapting the stirring speed to the amount of gas introduction, thereby avoiding the situation where energy waste is caused by stirring too fast and dissolving too slowly due to stirring too slowly. 3. When the real-time pH is less than 6.5, the present invention closes the air inlet and outlet of the fermenter, and performs a vacuum operation on the fermenter to make the pressure in the fermenter reach 0.08-0.095Mpa to reduce the solubility of carbon dioxide in the culture solution; after maintaining the pressure in the fermenter for 1-10S, the air outlet is opened to discharge the carbon dioxide gas escaping from the culture solution. Such an operation can reduce the carbon dioxide in the culture solution and reduce the presence of carbonic acid, thereby achieving the effect of increasing the pH without a pH regulator.

[0029] In summary, the present invention simplifies the fermentation environment adjustment steps and reduces resource waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic flow diagram of a fermentation process of Bacillus subtilis provided by a specific embodiment of the present invention;

[0031] Figure 2 It is a schematic diagram of a pH adjustment process provided by a specific embodiment of the present invention;

[0032] Figure 3 It is a schematic diagram of the structure of a fermentation tank provided in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0033] The present invention discloses a fermentation process of Bacillus subtilis. Those skilled in the art can refer to the content of this article and appropriately improve the technical details to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0034] The applicant has found that Bacillus subtilis needs to maintain certain environmental conditions during the fermentation process in order to achieve the required fermentation efficiency and thus improve the yield and production efficiency of the required metabolites. However, the existing technology generally changes the fermentation environment of Bacillus subtilis one-to-one, cannot be connected in series, has cumbersome steps, and wastes resources.

[0035] Therefore, the present invention provides a fermentation process of Bacillus subtilis, such as Figure 1 As shown, the process includes:

[0036] Step S1, adding Bacillus subtilis seed liquid into a fermentation tank containing culture liquid, and collecting the real-time temperature, real-time dissolved oxygen content and real-time pH of the culture liquid in the fermentation tank in real time during the fermentation of Bacillus subtilis.

[0037] It should be noted that the culture medium generally requires the following four components:

[0038] Carbon source: provides energy and carbon elements. Common carbon sources include glucose, brown sugar, soluble starch, corn starch, etc.

[0039] Nitrogen source: Provides nitrogen to promote bacterial growth and reproduction. Common nitrogen sources include soybean powder, peptone, beef extract, yeast extract powder, corn steep liquor powder, etc.

[0040] Inorganic salts: provide mineral elements required for microbial growth, such as dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, sodium chloride, etc.

[0041] Trace elements: such as manganese sulfate, play an important role in the growth and metabolism of microorganisms.

[0042] It is worth mentioning that the real-time dissolved oxygen content and real-time pH of the culture solution can be obtained by collecting a small amount of sample solution.

[0043] In this specific embodiment, step S1 includes:

[0044] The Bacillus subtilis seed solution is added into a fermentation tank containing 70% culture solution by volume at an inoculation rate of 0.5-5%.

[0045] It should be noted that the ratio of Bacillus subtilis seed solution and culture solution must be appropriate in order for the fermentation speed to reach the ideal efficiency.

[0046] Step S2, in response to the real-time pH being greater than 7, determining a first introduction amount of carbon dioxide gas; determining a second introduction amount of air according to the real-time dissolved oxygen content; determining a first introduction temperature of carbon dioxide gas and air according to the real-time temperature, the first introduction amount and the second introduction amount; determining a first stirring speed of the agitator in the fermentation tank according to the first introduction amount and the second introduction amount.

[0047] In this specific embodiment, after step S1, the process further includes:

[0048] In response to the real-time pH being less than 6.5, the air inlet and the air outlet of the fermentation tank are closed, and the fermentation tank is evacuated to a pressure of 0.08-0.095 MPa to reduce the solubility of carbon dioxide in the culture solution;

[0049] After maintaining the pressure in the fermentation tank for 1-10 seconds, the gas outlet is opened to discharge the carbon dioxide gas escaping from the culture solution; the gas inlet is opened and step S2 is continued.

[0050] It should be noted that by vacuuming the medium to reduce the solubility of carbon dioxide, the carbon dioxide in the culture medium is converted into gas and escapes, thereby achieving the effect of increasing the pH.

[0051] In this specific embodiment, after step S1, the process further includes:

[0052] In response to the real-time pH being between 6.5 and 7.5, the first injection amount of carbon dioxide gas is determined to be zero.

[0053] It should be noted that when the real-time pH is between 6.5-7.5, it indicates that the pH environment of the culture medium meets the requirements, so there is no need to adjust the pH.

[0054] In this specific embodiment, in step S2, in response to the real-time pH being greater than 7, determining a first amount of carbon dioxide gas to be introduced includes:

[0055] According to the real-time pH, the volume of the culture solution and the ideal pH, a first introduction amount of carbon dioxide gas is obtained, and the introduced carbon dioxide gas is used to adjust the pH of the culture solution.

[0056] It should be noted that the ideal pH is 6.5-7.5, and the purpose of introducing carbon dioxide is to lower the pH so that the pH value meets the requirements.

[0057] In this specific embodiment, Figure 2 As shown, Figure 2 This is a schematic diagram of the pH adjustment process, including:

[0058] Step S201, obtaining the real-time pH of the culture solution, and determining whether the real-time pH is between 6.5 and 7.5;

[0059] Step S202, in response to the real-time pH being less than 6.5, the air inlet and the air outlet of the fermentation tank are closed, and the fermentation tank is evacuated to make the pressure in the fermentation tank reach 0.08-0.095Mpa to reduce the solubility of carbon dioxide in the culture solution; after maintaining the pressure in the fermentation tank for 1-10S, the air outlet is opened to discharge the carbon dioxide gas escaping from the culture solution, and the air inlet is opened.

[0060] Step S203: In response to the real-time pH being between 6.5 and 7.5, no carbon dioxide gas is introduced.

[0061] Step S204: In response to the real-time pH being greater than 7, a first intake amount of carbon dioxide gas is obtained according to the real-time pH, the volume of the culture solution and the ideal pH.

[0062] In this specific embodiment, determining the second air intake amount according to the real-time dissolved oxygen content in step S2 includes:

[0063] Determine the required oxygen amount based on the real-time dissolved oxygen content, the volume of the culture solution and the ideal dissolved oxygen content;

[0064] The second air intake amount is determined according to the required oxygen amount and the oxygen content in the air.

[0065] It should be noted that the purpose of introducing air is to introduce oxygen, because air is convenient and easily available, so introducing air can reduce costs. The carbon dioxide content in the air is rare, so it can be ignored in the embodiment of the present invention.

[0066] In this specific embodiment, in step S2, determining the first incoming temperature of carbon dioxide gas and air according to the real-time temperature, the first incoming amount, and the second incoming amount includes:

[0067] The first introduction temperature of carbon dioxide gas and air is determined according to the real-time temperature, the first introduction amount and the second introduction amount, the volume of the culture solution and the ideal temperature.

[0068] It should be noted that, because the introduction rate is constant, the total introduction amount and the temperature adjustment time of the culture solution can be calculated, and then it can be determined what introduction temperature can achieve the effect of maintaining the temperature of the culture solution between 34-37°C.

[0069] In this specific embodiment, determining a first stirring speed of a stirrer in the fermentation tank according to the first feed rate and the second feed rate in step S2 includes:

[0070] The first stirring speed of the agitator in the fermentation tank is determined according to the first dissolution rate of carbon dioxide gas and its corresponding first introduction amount and the second dissolution rate of oxygen in the air and its corresponding second introduction amount; wherein the ventilation speed of the fermentation tank is constant, and the first stirring is used to accelerate the dissolution rate of oxygen and carbon dioxide gas, thereby keeping the ventilation speed and dissolution speed of carbon dioxide gas and oxygen balanced.

[0071] It should be noted that the pH and oxygen content regulation effect can only be guaranteed by ensuring that the ventilation rate and dissolution rate of carbon dioxide gas and oxygen are balanced.

[0072] In this specific embodiment, the process further comprises:

[0073] During the fermentation process, the bacterial concentration of Bacillus subtilis is collected in real time. When the change in bacterial concentration exceeds the expected change, it is judged that the fermentation is abnormal.

[0074] It should be noted that in order to prevent Bacillus subtilis from growing abnormally due to unknown factors, thereby affecting fermentation, the embodiment of the present invention detects the bacterial concentration in real time, and abnormalities can be discovered and processed in time.

[0075] Step S3, heating or cooling the first amount of carbon dioxide gas and the second amount of air to a first introduction temperature and then introducing them into the culture solution, and controlling the agitator to stir at a first stirring speed during the introduction process, so that the temperature of the culture solution is maintained between 34-37°C, the dissolved oxygen content is maintained between 10%-20%, and the pH is maintained between 6.5-7.5.

[0076] Step S4, detecting a first content of the target product in the culture solution; in response to the first content reaching a threshold value, determining that the fermentation of Bacillus subtilis is complete.

[0077] The target product includes the metabolites of Bacillus subtilis and / or Bacillus subtilis itself.

[0078] It should be noted that the target product may be a metabolite or may be Bacillus subtilis itself.

[0079] In this specific embodiment, the structure of the fermentation tank can be as follows Figure 3 As shown, it includes: a fermentation tank body 301, an air inlet 302 is arranged at the bottom of the fermentation tank body 301, and an air outlet 303 is arranged at the top, the air inlet 302 is connected to the air inlet pipe 304, the air inlet pipe 304 is outer-connected with a temperature regulating device 305 for regulating the temperature of the gas in the air inlet pipe 304, and the air inlet pipe 304 is respectively connected to the air inlet pipe 306 and the carbon dioxide inlet pipe 307.

[0080] The embodiment of the present invention can obtain the adjustment parameters such as the amount of carbon dioxide gas and air introduced and the introduction temperature and the stirring speed according to the real-time temperature, real-time dissolved oxygen content and real-time pH of the culture solution. The real-time temperature, real-time dissolved oxygen content and real-time pH of the culture solution in the fermenter can be adjusted in real time by using these adjustment parameters, so as to ensure that the fermentation environment of Bacillus subtilis during the fermentation process is always in an ideal state, thereby improving the fermentation efficiency. The prior art requires heating the entire fermenter separately if the temperature needs to be increased, and these steps are fragmented and waste resources. Compared with the prior art, the embodiment of the present invention can adjust the pH, temperature and oxygen content at the same time by introducing air and carbon dioxide, thereby reducing the waste of resources. At the same time, the gas is in more sufficient contact with the culture solution, so that the temperature of the culture solution rises or falls evenly at various places, thereby ensuring the overall quality of the fermentation.

[0081] In the embodiment of the present invention, the stirring speed is adapted to the gas introduction amount, so that the ventilation speed and dissolution speed of carbon dioxide gas and oxygen are balanced, thereby avoiding the situation where energy waste is caused by stirring too fast and dissolution is not possible due to stirring too slowly.

[0082] In the embodiment of the present invention, when the real-time pH is less than 6.5, the air inlet and outlet of the fermenter are closed, and the fermenter is vacuumed to make the pressure in the fermenter reach 0.08-0.095Mpa to reduce the solubility of carbon dioxide in the culture solution; after maintaining the pressure in the fermenter for 1-10S, the air outlet is opened to discharge the carbon dioxide gas escaping from the culture solution. Such operation can reduce the carbon dioxide in the culture solution and reduce the presence of carbonic acid, thereby achieving the effect of increasing the pH without a pH regulator.

[0083] In summary, the embodiments of the present invention simplify the fermentation environment adjustment steps and reduce resource waste.

[0084] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0085] Each embodiment in this specification is described in a related manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.

[0086] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. A fermentation process of Bacillus subtilis, characterized in that: The process comprises: Step S1, adding Bacillus subtilis seed liquid to a fermentation tank containing culture liquid, and collecting the real-time temperature, real-time dissolved oxygen content and real-time pH of the culture liquid in the fermentation tank in real time during the fermentation process of the Bacillus subtilis; Step S2, in response to the real-time pH being greater than 7, determining a first intake amount of carbon dioxide gas; and determining a second intake amount of air according to the real-time dissolved oxygen content; Determining first input temperatures of carbon dioxide gas and air according to the real-time temperature, the first input amount, and the second input amount; Determining a first stirring speed of a stirrer in the fermentation tank according to the first feeding amount and the second feeding amount; Step S3, heating or cooling the first amount of carbon dioxide gas and the second amount of air to the first introduction temperature and then introducing them into the culture solution, and controlling the stirrer to stir at the first stirring speed during the introduction process, so that the temperature of the culture solution is maintained between 34-37° C., the dissolved oxygen content is maintained between 10%-20%, and the pH is maintained between 6.5-7.5; Step S4, detecting a first content of a target product in the culture solution; in response to the first content reaching a threshold, determining that the fermentation of the Bacillus subtilis is complete; wherein the target product includes a metabolite of the Bacillus subtilis and / or the Bacillus subtilis itself.

2. The fermentation process of Bacillus subtilis according to claim 1, characterized in that: After step S1, the process further comprises: In response to the real-time pH being less than 6.5, the air inlet and the air outlet of the fermenter are closed, and the fermenter is evacuated to a vacuum state so that the pressure in the fermenter reaches 0.08-0.095 MPa, so as to reduce the solubility of carbon dioxide in the culture solution; After maintaining the pressure in the fermentation tank for 1-10 seconds, the gas outlet is opened to discharge the carbon dioxide gas escaping from the culture solution; the gas inlet is opened and step S2 is continued.

3. The fermentation process of Bacillus subtilis according to claim 1, characterized in that: After step S1, the process further comprises: In response to the real-time pH being between 6.5 and 7.5, the first introduction amount of carbon dioxide gas is determined to be zero.

4. The fermentation process of Bacillus subtilis according to claim 1, characterized in that: In step S2, in response to the real-time pH being greater than 7, determining a first amount of carbon dioxide gas to be introduced comprises: The first introduction amount of carbon dioxide gas is obtained according to the real-time pH, the volume of the culture solution and the ideal pH, and the introduced carbon dioxide gas is used to adjust the pH of the culture solution.

5. The fermentation process of Bacillus subtilis according to claim 1, characterized in that: Determining the second air intake amount according to the real-time dissolved oxygen content in step S2 includes: Determining the required oxygen amount according to the real-time dissolved oxygen content, the volume of the culture solution and the ideal dissolved oxygen content; The second intake amount of air is determined according to the required oxygen amount and the oxygen content in the air.

6. The fermentation process of Bacillus subtilis according to claim 1, characterized in that: Determining the first input temperature of the carbon dioxide gas and the air according to the real-time temperature, the first input amount, and the second input amount in step S2 includes: The first introduction temperature of carbon dioxide gas and air is determined according to the real-time temperature, the first introduction amount and the second introduction amount, the volume of the culture solution and the ideal temperature.

7. The fermentation process of Bacillus subtilis according to claim 1, characterized in that: Determining a first stirring speed of a stirrer in the fermenter according to the first feed-in amount and the second feed-in amount in step S2 includes: The first stirring speed of the agitator in the fermentation tank is determined according to a first dissolution rate of carbon dioxide gas and its corresponding first introduction amount and a second dissolution rate of oxygen in the air and its corresponding second introduction amount; wherein the ventilation speed of the fermentation tank is constant, and the first stirring is used to accelerate the dissolution rate of oxygen and carbon dioxide gas, thereby keeping the ventilation speed and the dissolution speed of carbon dioxide gas and oxygen balanced.

8. The fermentation process of Bacillus subtilis according to claim 1, characterized in that: The process also includes: During the fermentation process, the bacterial concentration of the Bacillus subtilis is collected in real time. When the bacterial concentration changes beyond an expected change, it is determined that the fermentation is abnormal.

9. The fermentation process of Bacillus subtilis according to claim 1, characterized in that: The step S1 comprises: The Bacillus subtilis seed solution is added into the fermentation tank containing 70% culture solution by volume at an inoculation rate of 0.5-5%.

Images