A fermentation process for Bacillus subtilis

Abstract

This invention discloses a fermentation process for Bacillus subtilis, comprising: adding Bacillus subtilis seed culture to a fermenter containing a culture medium, and real-time collecting the real-time temperature, real-time dissolved oxygen content, and real-time pH of the culture medium; determining a first introduction rate of carbon dioxide gas in response to a real-time pH greater than 7; determining a second introduction rate of air based on the real-time dissolved oxygen content; determining a first introduction temperature of carbon dioxide gas and air based on the real-time temperature, the first introduction rate, and the second introduction rate; determining a first stirring speed of the stirrer based on the first and second introduction rates; introducing the first introduction rate of carbon dioxide gas and the second introduction rate of air into the culture medium after heating or cooling to the first introduction temperature, and controlling the stirrer to stir at the first stirring speed; determining that fermentation is complete when the content of the target product in the culture medium reaches a threshold. This invention simplifies the fermentation environment adjustment steps and reduces resource waste.

Description

Technical Field

[0001] This invention relates to the field of fermentation, and in particular to a fermentation process for Bacillus subtilis. Background Technology

[0002] Bacillus subtilis is a bacterium widely distributed in soil and decaying organic matter. During fermentation, Bacillus subtilis produces a variety of beneficial metabolites, such as enzymes (α-amylase, protease, lipase, cellulase, etc.), vitamins (B vitamins such as B1, B2, B6, and niacin), antibiotics (subtilisin, polymyxin, nystatin, bacitracin, lipopeptides, etc.), and organic acids. These products can be widely used in agriculture, animal feed, medicine, environmental protection, food, and other industries.

[0003] Bacillus subtilis requires specific environmental conditions during fermentation to achieve the desired fermentation efficiency, thereby increasing the yield and production efficiency of the desired metabolites. However, current technologies for altering the fermentation environment of Bacillus subtilis are generally one-to-one, unable to be serialized, cumbersome, and wasteful of resources. Summary of the Invention

[0004] In view of the aforementioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a fermentation process for Bacillus subtilis, which aims to simplify the fermentation environment adjustment steps of Bacillus subtilis and reduce resource waste.

[0005] To achieve the above objectives, the present invention discloses a fermentation process for Bacillus subtilis, the process comprising:

[0006] Step S1: Add the Bacillus subtilis seed liquid to a fermenter containing culture medium, and collect the real-time temperature, real-time dissolved oxygen content and real-time pH of the culture medium in the fermenter during the fermentation process of Bacillus subtilis.

[0007] Step S2: In response to the real-time pH being greater than 7, determine the first inlet flow rate of carbon dioxide gas; determine the second inlet flow rate of air based on the real-time dissolved oxygen content; determine the first inlet temperature of carbon dioxide gas and air based on the real-time temperature, the first inlet flow rate, and the second inlet flow rate; determine the first stirring speed of the stirrer in the fermenter based on the first inlet flow rate and the second inlet flow rate.

[0008] Step S3: The first amount of carbon dioxide gas and the second amount of air are heated or cooled to the first inlet temperature and then introduced into the culture medium. During the introduction process, the stirrer is controlled to stir at the first stirring speed so that the temperature of the culture medium 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: Detect the first content of the target product in the culture medium; in response to the first content reaching a threshold, determine that the fermentation of Bacillus subtilis is complete; wherein, the target product includes the metabolites of Bacillus subtilis and / or 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 outlet of the fermenter are shut off, and a vacuum operation is performed on the fermenter to bring the pressure inside the fermenter to 0.08-0.095 MPa, so as to reduce the solubility of carbon dioxide in the culture medium.

[0012] After maintaining the pressure inside the fermenter for 1-10 seconds, open the vent to release the carbon dioxide gas escaping from the culture medium; open the inlet and continue with step S2.

[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 injection rate of carbon dioxide gas is determined to be zero.

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

[0016] The first amount of carbon dioxide gas introduced is determined based on the real-time pH, the volume of the culture medium, and the ideal pH. The introduced carbon dioxide gas is used to adjust the pH of the culture medium.

[0017] Optionally, the step S2 of determining the second air intake based on the real-time dissolved oxygen content includes:

[0018] The required oxygen amount is determined based on the real-time dissolved oxygen content, the volume of the culture medium, and the ideal dissolved oxygen content.

[0019] The second air intake is determined based on the required oxygen level and the oxygen content in the air.

[0020] Optionally, step S2, which involves determining the first inlet temperature of carbon dioxide gas and air based on the real-time temperature, the first inlet flow rate, and the second inlet flow rate, includes:

[0021] The first inlet temperature of carbon dioxide gas and air is determined based on the real-time temperature, the first inlet volume and the second inlet volume, the volume of the culture medium and the ideal temperature.

[0022] Optionally, in step S2, determining the first stirring speed of the agitator in the fermenter based on the first injection rate and the second injection rate includes:

[0023] The first stirring speed of the stirrer in the fermenter is determined based on the first dissolution rate of carbon dioxide gas and its corresponding first injection rate, and the second dissolution rate of oxygen in the air and its corresponding second injection rate; wherein the aeration rate of the fermenter is constant, and the first stirring is used to accelerate the dissolution rate of oxygen and carbon dioxide gas, thereby keeping the aeration rate and the dissolution rate of carbon dioxide and oxygen gas in balance.

[0024] Optionally, the process further includes:

[0025] During the fermentation process, the bacterial concentration of Bacillus subtilis is collected in real time. If the change in bacterial concentration exceeds the expected change, it is determined that the fermentation has become abnormal.

[0026] Optionally, step S1 includes:

[0027] The Bacillus subtilis seed culture was added to the fermenter containing 70% culture medium by volume at an inoculation rate of 0.5-5%.

[0028] The beneficial effects of this invention are as follows: 1. This invention can obtain adjustment parameters such as the amount and temperature of carbon dioxide and air introduced, as well as the stirring speed, based on the real-time temperature, dissolved oxygen content, and pH of the culture medium. Using these parameters, the real-time temperature, dissolved oxygen content, and pH of the culture medium in the fermenter can be adjusted in real time, ensuring that the fermentation environment of Bacillus subtilis is always in an ideal state during the fermentation process, thus improving fermentation efficiency. Existing technologies, if temperature increases are required, require separate heating of the entire fermenter, which is fragmented and wasteful of resources. Compared to existing technologies, this invention, through the introduction of air and carbon dioxide, can simultaneously adjust pH, temperature, and oxygen content, reducing resource waste. At the same time, the gas has more sufficient contact with the culture medium, resulting in a more balanced temperature rise or fall throughout the culture medium, ensuring the overall quality of fermentation. 2. This invention, by adapting the stirring speed to the gas introduction rate, maintains a balance between the gas introduction rate and the dissolution rate of carbon dioxide and oxygen. This avoids situations where excessive stirring leads to energy waste, or excessive stirring results in insufficient dissolution. 3. In this invention, when the real-time pH is less than 6.5, the inlet and outlet of the fermenter are shut off, and a vacuum operation is performed on the fermenter to bring the pressure inside to 0.08-0.095 MPa, thereby reducing the solubility of carbon dioxide in the culture medium. After maintaining the pressure inside the fermenter for 1-10 seconds, the outlet is opened to release the carbon dioxide gas escaping from the culture medium. This operation reduces the carbon dioxide and carbonic acid in the culture medium, thus achieving the effect of increasing the pH without the need for pH adjusters.

[0029] In summary, this invention simplifies the fermentation environment adjustment process and reduces resource waste. Attached Figure Description

[0030] Figure 1 This is a schematic flowchart of a fermentation process for Bacillus subtilis provided in a specific embodiment of the present invention;

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

[0032] Figure 3 This is a schematic diagram of the structure of a fermenter provided in a specific embodiment of the present invention. Detailed Implementation

[0033] This invention discloses a fermentation process for Bacillus subtilis. Those skilled in the art can refer to the content of this document and appropriately modify the technical details to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0034] The applicant's research revealed that Bacillus subtilis requires specific environmental conditions during fermentation to achieve the desired fermentation efficiency, thereby increasing the yield and production efficiency of the desired metabolites. However, existing technologies for altering the fermentation environment of Bacillus subtilis are generally one-to-one, unable to be serialized, cumbersome, and wasteful of resources.

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

[0036] Step S1: Add the Bacillus subtilis seed liquid to the fermenter containing the culture medium, and collect the real-time temperature, real-time dissolved oxygen content and real-time pH of the culture medium in the fermenter during the Bacillus subtilis fermentation process.

[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 element to promote bacterial growth and reproduction. Common nitrogen sources include soybean flour, peptone, beef extract, yeast extract powder, corn steep liquor powder, etc.

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

[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 medium can be obtained by collecting a small amount of sample solution.

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

[0044] Add 0.5-5% of Bacillus subtilis seed culture to a fermenter containing 70% culture medium by volume.

[0045] It should be noted that the ratio of Bacillus subtilis seed culture to culture medium must be appropriate in order to achieve the ideal fermentation rate.

[0046] Step S2: In response to a real-time pH greater than 7, determine the first inlet flow rate of carbon dioxide gas; determine the second inlet flow rate of air based on the real-time dissolved oxygen content; determine the first inlet temperature of carbon dioxide gas and air based on the real-time temperature, the first inlet flow rate, and the second inlet flow rate; determine the first stirring speed of the agitator in the fermenter based on the first inlet flow rate and the second inlet flow rate.

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

[0048] In response to a real-time pH value less than 6.5, the inlet and outlet of the fermenter are shut off, and a vacuum operation is performed on the fermenter to bring the pressure inside the fermenter to 0.08-0.095 MPa, in order to reduce the solubility of carbon dioxide in the culture medium.

[0049] After maintaining the pressure inside the fermenter for 1-10 seconds, open the vent to release the carbon dioxide gas escaping from the culture medium; then open the inlet and continue with step S2.

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

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

[0052] If the real-time pH is between 6.5 and 7.5, the initial carbon dioxide gas injection rate is determined to be zero.

[0053] It should be noted that when the real-time pH is between 6.5 and 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, step S2, in response to a real-time pH greater than 7, determines the first amount of carbon dioxide gas introduced, including:

[0055] The initial flow rate of carbon dioxide gas is determined based on the real-time pH, the volume of the culture medium, and the ideal pH. The introduced carbon dioxide gas is used to adjust the pH of the culture medium.

[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 to meet the requirements.

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

[0058] Step S201: Obtain the real-time pH of the culture medium and determine 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 outlet of the fermenter are shut off, and a vacuum operation is performed on the fermenter to bring the pressure inside the fermenter to 0.08-0.095 MPa, so as to reduce the solubility of carbon dioxide in the culture medium; after maintaining the pressure inside the fermenter for 1-10 seconds, the air outlet is opened to release the carbon dioxide gas escaping from the culture medium, and the air inlet is opened.

[0060] Step S203: In response to the real-time pH being between 6.5 and 7.5, carbon dioxide gas is not introduced.

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

[0062] In this specific embodiment, step S2, which determines the second air intake based on the real-time dissolved oxygen content, includes:

[0063] The required oxygen amount is determined based on the real-time dissolved oxygen content, the volume of the culture medium, and the ideal dissolved oxygen content.

[0064] The second air intake is determined based on the required oxygen level and the oxygen content in the air.

[0065] It should be noted that the purpose of introducing air is to introduce oxygen, as air is readily available and thus reduces costs. Carbon dioxide content in air is scarce and can therefore be ignored in this embodiment of the invention.

[0066] In this specific embodiment, step S2, determining the first inlet temperature of carbon dioxide gas and air based on the real-time temperature, the first inlet flow rate, and the second inlet flow rate, includes:

[0067] The first inlet temperature of carbon dioxide gas and air is determined based on the real-time temperature, the first inlet volume and the second inlet volume, the volume of the culture medium and the ideal temperature.

[0068] It should be noted that because the infusion rate is constant, the time required to adjust the temperature of the culture medium based on the total infusion volume can be calculated, thereby determining the appropriate infusion temperature to maintain the culture medium temperature between 34-37℃.

[0069] In this specific embodiment, step S2, determining the first stirring speed of the agitator in the fermenter based on the first and second injection rates, includes:

[0070] The first stirring speed of the agitator in the fermenter is determined based on the first dissolution rate of carbon dioxide gas and its corresponding first injection rate, and the second dissolution rate of oxygen in the air and its corresponding second injection rate. The aeration rate of the fermenter is constant, and the first stirring is used to accelerate the dissolution rate of oxygen and carbon dioxide gas, thereby maintaining the balance between the aeration rate and the dissolution rate of carbon dioxide and oxygen.

[0071] It should be noted that maintaining a balance between the ventilation rate and dissolution rate of carbon dioxide and oxygen is essential to ensuring the effective regulation of pH and oxygen content.

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

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

[0074] It should be noted that, in order to avoid abnormal reproduction of Bacillus subtilis due to unknown factors, which would affect fermentation, the embodiments of the present invention monitor the bacterial concentration in real time, so that abnormalities can be detected and dealt with in a timely manner.

[0075] Step S3: The first amount of carbon dioxide gas and the second amount of air are heated or cooled to the first inlet temperature and then introduced into the culture medium. During the introduction process, the stirrer is controlled to stir at the first stirring speed so that the temperature of the culture medium 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: Detect the first content of the target product in the culture medium; in response to the first content reaching the threshold, determine that Bacillus subtilis fermentation is complete.

[0077] The target products include metabolites of Bacillus subtilis and / or Bacillus subtilis itself.

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

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

[0080] This invention allows for the determination of adjustment parameters such as the injection rate and temperature of carbon dioxide and air, as well as the stirring speed, based on the real-time temperature, dissolved oxygen content, and pH of the culture medium. These parameters enable real-time adjustment of the temperature, dissolved oxygen content, and pH of the culture medium in the fermenter, ensuring that the fermentation environment of Bacillus subtilis remains ideal during the fermentation process and improving fermentation efficiency. Existing technologies, such as those requiring temperature increases, necessitate separate heating of the entire fermenter, which is fragmented and wasteful of resources. In contrast, this invention, through the injection of air and carbon dioxide, can simultaneously adjust pH, temperature, and oxygen content, reducing resource waste. Furthermore, the gas has more thorough contact with the culture medium, resulting in a more even temperature rise or fall throughout the culture medium and ensuring the overall quality of fermentation.

[0081] In this embodiment of the invention, the stirring speed is adapted to the gas flow rate to maintain a balance between the gas flow rate and the dissolution rate of carbon dioxide and oxygen. This avoids situations where stirring is too fast, resulting in energy waste, or stirring is too slow, resulting in insufficient dissolution time.

[0082] In this embodiment of the invention, when the real-time pH is less than 6.5, the air inlet and outlet of the fermenter are shut off, and a vacuum operation is performed on the fermenter to bring the pressure inside to 0.08-0.095 MPa, thereby reducing the solubility of carbon dioxide in the culture medium. After maintaining the pressure inside the fermenter for 1-10 seconds, the air outlet is opened to release the carbon dioxide gas escaping from the culture medium. This operation reduces the carbon dioxide and carbonic acid in the culture medium, thus achieving the effect of increasing the pH without the need for pH adjusters.

[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 document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0085] The various embodiments in this specification are described in a related manner. Similar or identical parts between embodiments can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions of the method embodiments.

[0086] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A fermentation process for Bacillus subtilis, characterized in that, The process includes: Step S1: Add the Bacillus subtilis seed liquid to a fermenter containing culture medium, and collect the real-time temperature, real-time dissolved oxygen content and real-time pH of the culture medium in the fermenter during the fermentation process of Bacillus subtilis. Step S2: In response to the real-time pH being less than 6.5, the air inlet and outlet of the fermenter are shut off, and a vacuum operation is performed on the fermenter to bring the pressure inside the fermenter to 0.08-0.095 MPa, so as to reduce the solubility of carbon dioxide in the culture medium; after maintaining the pressure inside the fermenter for 1-10 seconds, the air outlet is opened to release the carbon dioxide gas escaping from the culture medium. Open the air intake; In response to the real-time pH being between 6.5 and 7, the first introduction of carbon dioxide gas is determined to be zero; In response to the real-time pH being greater than 7, a first intake of carbon dioxide gas is determined; a second intake of air is determined based on the real-time dissolved oxygen content. Based on the real-time temperature, the first injection rate, and the second injection rate, the first injection temperature of carbon dioxide gas and air is determined; based on the first injection rate and the second injection rate, the first stirring speed of the stirrer in the fermenter is determined; wherein, if the real-time pH is between 7 and 7.5, the first injection rate of carbon dioxide gas is determined to be zero. Step S3: The first amount of carbon dioxide gas and the second amount of air are heated or cooled to the first inlet temperature and then introduced into the culture medium. During the introduction process, the stirrer is controlled to stir at the first stirring speed so that the temperature of the culture medium 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: Detect the first content of the target product in the culture medium; in response to the first content reaching a threshold, determine that the fermentation of Bacillus subtilis is complete; wherein, the target product includes the metabolites of Bacillus subtilis and / or Bacillus subtilis itself.

2. 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 the first amount of carbon dioxide gas introduced includes: The first amount of carbon dioxide gas introduced is determined based on the real-time pH, the volume of the culture medium, and the ideal pH. The introduced carbon dioxide gas is used to adjust the pH of the culture medium.

3. The fermentation process of Bacillus subtilis according to claim 1, characterized in that, The step S2, which involves determining the second air intake based on the real-time dissolved oxygen content, includes: The required oxygen amount is determined based on the real-time dissolved oxygen content, the volume of the culture medium, and the ideal dissolved oxygen content. The second air intake is determined based on the required oxygen level and the oxygen content in the air.

4. The fermentation process of Bacillus subtilis according to claim 1, characterized in that, The step S2, which involves determining the first inlet temperature of carbon dioxide gas and air based on the real-time temperature, the first inlet flow rate, and the second inlet flow rate, includes: The first inlet temperature of carbon dioxide gas and air is determined based on the real-time temperature, the first inlet volume and the second inlet volume, the volume of the culture medium and the ideal temperature.

5. The fermentation process of Bacillus subtilis according to claim 1, characterized in that, In step S2, determining the first stirring speed of the agitator in the fermenter based on the first injection rate and the second injection rate includes: The first stirring speed of the stirrer in the fermenter is determined based on the first dissolution rate of carbon dioxide gas and its corresponding first injection rate, and the second dissolution rate of oxygen in the air and its corresponding second injection rate; wherein the aeration rate of the fermenter is constant, and the first stirring speed is used to accelerate the dissolution rate of oxygen and carbon dioxide gas, thereby keeping the aeration rate and the dissolution rate of carbon dioxide and oxygen gas in balance.

6. 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 Bacillus subtilis is collected in real time. If the change in bacterial concentration exceeds the expected change, it is determined that the fermentation has become abnormal.

7. The fermentation process of Bacillus subtilis according to claim 1, characterized in that, Step S1 includes: The Bacillus subtilis seed culture was added to the fermenter containing 70% culture medium by volume at an inoculation rate of 0.5-5%.

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