CN114164155A - Fermentation medium and fermentation process of bacillus - Google Patents
Fermentation medium and fermentation process of bacillus Download PDFInfo
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Abstract
The invention discloses a fermentation medium of bacillus and a fermentation process thereof, relating to the technical field of microbial fermentation, wherein 3 important factors of screened optimal carbon source, nitrogen source and inorganic salt are analyzed, counted and optimized, and optimal fermentation time, temperature and initial pH inoculation amount are optimized one by one in the optimization process. According to the fermentation medium and the fermentation process of the bacillus, provided by the invention, multiple groups of culture factors are used for screening, and a one-by-one optimized mode is used for further determination in the screening process, so that the required metering can be accurately known, the bacillus can be ensured to be fermented in the most favorable culture medium, and the fermentation speed and the total amount of the bacillus can be directly increased.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a fermentation medium of bacillus and a fermentation process thereof.
Background
Bacillus, a family of bacteria, a bacillus or a coccus capable of forming spores (endospores). Including Bacillus, Lactobacillus, Clostridium, Desulfoenterococcus, and Sporosarcina, etc. They have strong resistance to external harmful factors and wide distribution, and exist in soil, water, air, animal intestinal tracts and the like. A bacterium of the genus bacillus of the family bacillaceae.
Due to the wide distribution and important role of bacillus, more and more occasions need to be used by the bacillus, and therefore the bacillus needs to be fermented, so that the number of the bacillus is increased, but in the fermentation process of the existing bacillus, nutrients in a fermentation medium cannot meet the optimal fermentation environment of the bacillus, so that the fermentation speed of the bacillus is low, and the fermentation number of the bacillus is low.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a fermentation medium of bacillus and a fermentation process thereof.
The invention provides a fermentation medium of bacillus and a fermentation process thereof, comprising the following steps:
s1: the following experimental materials were prepared:
1) bacterial strain
A bacillus strain;
2) culture medium
Seed culture medium: 1% of yeast protein, 5% of yeast extract, 1% of sodium chloride and 6.5% of PH value;
fermentation medium: 1% of sucrose, 1% of yeast protein, 0.5% of sodium chloride and 7.0% of PH value;
3) primary reagent
Sucrose, glucose, corn starch, maltose, corn starch powder, tryptone, yeast extract (extract), beef extract, soybean cake powder and urea;
s2: culture method
1) Strain activation
Transferring the preserved strain into a culture medium, and placing the culture medium in an incubator with constant temperature of 37 ℃ for culturing for 24 hours for later use;
2) preparation of seed liquid
Taking a ring of activated strains, inoculating the strains into a 250ml triangular flask with the volume of 100ml seed culture medium, putting the flask into an incubator with the constant temperature of 37 ℃, rotating speed of 160r/min, and culturing for 18 hours;
3) shaking culture
Inoculating the seed solution into a fermentation medium (a triangular flask with the liquid loading amount of 100ml/250 ml) with the inoculation amount of 1%, performing shaking culture at 37 ℃ and the rotation speed of 160r/min for 24 hours, and performing single-factor fermentation condition research.
S3: fermentation Medium Single factor experiment
1) Carbon source
Respectively using 1% glucose, corn starch and maltose to equivalently replace sucrose in a fermentation medium to prepare culture media, adopting a shake flask fermentation culture condition, and measuring the viable bacteria content in the fermentation broth after 24h to determine the optimal carbon source.
2) Nitrogen source
1% yeast extract, beef extract, urea and ammonium chloride are respectively used for replacing 1% tryptone in the basic fermentation liquor, the shake flask fermentation culture condition is adopted, and the viable bacteria content in the fermentation liquor is measured after 24 hours so as to determine the optimal nitrogen source.
3) Inorganic salt
Respectively replacing equal amount of sodium chloride (0.5%) in the fermentation medium with 0.5% of manganese sulfate, magnesium sulfate, ferrous sulfate, calcium chloride and a blank without inorganic salt, and determining the total number of viable bacteria in the fermentation broth after 24h under shake flask fermentation culture conditions to determine the optimal inorganic salt.
Preferably, the 3 selected important factors of the optimal carbon source, nitrogen source and inorganic salt are analyzed, counted and optimized, and the optimal fermentation time, temperature and initial pH inoculation amount are optimized one by one in the optimization process.
Preferably, in the process of optimizing the carbon source, 1% of glucose, sucrose, maltose and corn starch are respectively used as the carbon source, other components are fermented simultaneously, the total number of viable bacteria in the fermentation liquor is determined, wherein the growth of the bacillus is obviously better than that of the corn starch when the glucose, the sucrose and the maltose are used as the carbon source, after the fermentation by using the first 3 carbon sources, the viable bacteria number of the bacillus is more than 2.2x10CFU/ml, and the viable bacteria number of the bacillus is 0.9x10 when the corn starch is used10CFU/ml below, indicates that the ability of the Bacillus to utilize mono-and disaccharides is superior to starch, so the best carbon source for growth of the Bacillus is glucose, followed by sucrose and maltose.
Preferably, in the process of optimizing the nitrogen source, 1% of glucose is used as the carbon source, 1% of urea, ammonium chloride, beef extract, yeast extract and tryptone are used as the nitrogen source to prepare the fermentation culture medium, the effect of the organic nitrogen source is obviously superior to that of the inorganic nitrogen source, and after the organic nitrogen source is utilized for fermentation, the viable count of bacillus exceeds 2.2x1010CFU/ml, and the viable count when using inorganic nitrogen source is 1.0x1010CFU/ml is below, which shows that the capability of the bacillus to utilize the organic nitrogen source is better than that of the inorganic nitrogen source, and the most suitable nitrogen source for the growth of the bacillus is yeast extract, which is related to that the yeast extract is rich in growth factors such as amino acid, small peptide, nucleotide, B vitamins and the like.
Preferably, in the process of optimizing the inorganic salt, 1% of glucose and 1% of yeast extract are respectively used as a carbon source and a nitrogen source, and no inorganic salt is added as a blank, 0.5% of manganese sulfate, magnesium sulfate, ferrous sulfate, sodium chloride and calcium chloride are respectively added on the basis, wherein magnesium ions have a certain promotion effect on the growth of bacillus, the biomass of the bacillus is 1.2 times of that of a control group, iron ions, calcium ions, manganese ions and sodium ions have an inhibition effect on the growth of bacillus, and the growth amount of the bacillus is only 91.3%, 76.5%, 70.5% and 36.2% of that of the control group.
Preferably, each component in the culture medium has a certain internal relation, on the basis of a single-factor experiment, an orthogonal experiment is adopted to search for the optimal proportion of each component in the culture medium, the influence of factors on the viable bacteria content can be obtained as yeast extract > glucose > magnesium sulfate, wherein the significance of the yeast extract is higher than that of the glucose, the magnesium sulfate is an inconspicuous difference factor, and 3 parallel experiments are carried out for verification to obtain the total viable bacteria number of 4.49x1010CFU/ml, therefore, the optimal medium for Bacillus licheniformis was determined to be 1.5% glucose yeast extract 1.0% magnesium sulfate 0.1%.
A fermentation medium of bacillus and a fermentation process thereof are disclosed, wherein the fermentation process comprises the following steps:
(1) growth Curve determination
Inoculating into optimized culture medium (triangular flask with liquid content of 100ml/250 ml) at an inoculum size of 1%, placing into a incubator with constant temperature of 37 deg.C, rotating at 160r/min, culturing for 24 hr, measuring bacterial liquid biomass every 2 hr after self-inoculation, and using non-inoculated culture medium as blank control.
After fermentation culture for 0-2h, the growth of the bacillus is in a lag phase, the number of the bacteria is extremely small, the number of the bacteria is increased sharply when the bacillus enters a logarithmic growth phase after 2h, the growth peak phase is reached after 16h, the growth of the bacteria is slow when 12-15 h is a stable phase of the growth of the bacillus licheniformis, the number of the bacteria starts to decrease after 16h, and the bacillus enters a growth decay phase.
2) Initial pH
The pH mainly influences the membrane charge, the membrane permeability and the ionization degree of nutrient substances of thallus cells, so that the nutrient absorption of the thallus is influenced, the pH in the shaking flask fermentation process is difficult to control, and only the initial pH of the fermentation liquor can be controlled, so that the initial pH is respectively adjusted to 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, the biomass of the fermentation liquor is measured after the fermentation is carried out for 16 hours in a shaking flask, the bacillus can well grow within the range of the initial pH of 5.5-8, the growth is best when the pH is 6.5, the adaptability of the bacillus to the pH is wider, and the biomass is in a descending trend along with the increase of the pH.
(3) Temperature of
The temperature affects the growth of the cells mainly by changing the rate of the enzyme reaction. The temperature of a general culture medium is increased, the enzyme reaction rate is increased, the growth and metabolism are accelerated, the production phase is advanced, but the enzyme is easy to lose activity due to overheating, the phenomenon that thalli are easy to age is shown, the influence of the final product on the biomass of fermentation liquor is respectively measured by selecting 25 ℃, 30 ℃, 35 ℃ and 40 ℃ as the culture temperature, the biomass of the fermentation liquor is measured after the final product is cultured by a shake flask at l6h, the bacillus can well grow at 25-40 ℃, and the optimal growth temperature is 35 ℃.
(4) Amount of inoculation
1 percent, 3 percent, 5 percent, 7 percent and 4 inoculum sizes are respectively selected, the biomass of the bacillus is measured after the bacillus is cultured for 16 hours in a shake flask, the optimal inoculum size for the growth of the bacillus is 50, and when the bacillus grows in the range of 1 to 7 percent, the difference of the influence of the inoculum size on the biomass of the bacillus is very small.
Preferably, the fermentation medium of bacillus is optimized in a shake flask by adopting a single-factor and orthogonal experimental method, the total number of viable bacteria is used as an index for evaluating the quality of the culture medium, the optimized culture medium comprises 1.5% of glucose, 1% of yeast extract and 0.1% of magnesium sulfate, and the optimal culture condition is determined by the single-factor experimental method: the temperature is 35 ℃, the initial pH is 6.5, the inoculation amount is 5%, and the fermentation time is 16 h.
The invention has the beneficial effects that:
according to the fermentation medium and the fermentation process of the bacillus, provided by the invention, multiple groups of culture factors are used for screening, and a one-by-one optimized mode is used for further determination in the screening process, so that the required metering can be accurately known, the bacillus can be ensured to be fermented in the most favorable culture medium, and the fermentation speed and the total amount of the bacillus can be directly increased.
Drawings
FIG. 1 is a schematic view of a fermentation medium of Bacillus and a process for fermentation thereof.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1, referring to fig. 1, the present invention provides a fermentation medium of bacillus and a fermentation process thereof, comprising the following steps:
s1: the following experimental materials were prepared:
1) bacterial strain
A bacillus strain;
2) culture medium
Seed culture medium: 1% of yeast protein, 5% of yeast extract, 1% of sodium chloride and 6.5% of PH value;
fermentation medium: 1% of sucrose, 1% of yeast protein, 0.5% of sodium chloride and 7.0% of PH value;
3) primary reagent
Sucrose, glucose, corn starch, maltose, corn starch powder, tryptone, yeast extract (extract), beef extract, soybean cake powder and urea;
s2: culture method
1) Strain activation
Transferring the preserved strain into a culture medium, and placing the culture medium in an incubator with constant temperature of 37 ℃ for culturing for 24 hours for later use;
2) preparation of seed liquid
Taking a ring of activated strains, inoculating the strains into a 250ml triangular flask with the volume of 100ml seed culture medium, putting the flask into an incubator with the constant temperature of 37 ℃, rotating speed of 160r/min, and culturing for 18 hours;
3) shaking culture
Inoculating the seed solution into a fermentation medium (a triangular flask with the liquid loading amount of 100ml/250 ml) with the inoculation amount of 1%, performing shaking culture at 37 ℃ and the rotation speed of 160r/min for 24 hours, and performing single-factor fermentation condition research.
S3: fermentation Medium Single factor experiment
1) Carbon source
Respectively using 1% glucose, corn starch and maltose to equivalently replace sucrose in a fermentation medium to prepare culture media, adopting a shake flask fermentation culture condition, and measuring the viable bacteria content in the fermentation broth after 24h to determine the optimal carbon source.
2) Nitrogen source
1% yeast extract, beef extract, urea and ammonium chloride are respectively used for replacing 1% tryptone in the basic fermentation liquor, the shake flask fermentation culture condition is adopted, and the viable bacteria content in the fermentation liquor is measured after 24 hours so as to determine the optimal nitrogen source.
3) Inorganic salt
Respectively replacing equal amount of sodium chloride (0.5%) in the fermentation medium with 0.5% of manganese sulfate, magnesium sulfate, ferrous sulfate, calcium chloride and a blank without inorganic salt, and determining the total number of viable bacteria in the fermentation broth after 24h under shake flask fermentation culture conditions to determine the optimal inorganic salt.
3 important factors of the screened optimal carbon source, nitrogen source and inorganic salt are analyzed, counted and optimized, and optimal fermentation time, temperature and initial pH inoculation amount are optimized one by one in the optimization process.
In the process of optimizing the carbon source, 1 percent of glucose, sucrose, maltose and corn starch are respectively used as the carbon source, other components are fermented simultaneously, the total number of viable bacteria in the fermentation liquor is measured, wherein the growth of bacillus is obviously better than that of the corn starch when the glucose, the sucrose and the maltose are used as the carbon source, after the fermentation by using the first 3 carbon sources, the viable bacteria number of the bacillus is more than 2.2x10CFU/ml, and the viable bacteria number of the corn starch is 0.9x1010CFU/ml below, indicates that the ability of the Bacillus to utilize mono-and disaccharides is superior to starch, so the best carbon source for growth of the Bacillus is glucose, followed by sucrose and maltose.
In the process of optimizing the nitrogen source, 1 percent of glucose is used as the carbon source, 1 percent of urea, ammonium chloride, beef extract, yeast extract and tryptone are used as the nitrogen source to prepare the fermentation culture medium, the effect of the organic nitrogen source is obviously superior to that of the inorganic nitrogen source, and after the organic nitrogen source is utilized for fermentation, the viable count of bacillus exceeds that of the inorganic nitrogen source2.2x1010CFU/ml, and the viable count when using inorganic nitrogen source is 1.0x1010CFU/ml is below, which shows that the capability of the bacillus to utilize the organic nitrogen source is better than that of the inorganic nitrogen source, and the most suitable nitrogen source for the growth of the bacillus is yeast extract, which is related to that the yeast extract is rich in growth factors such as amino acid, small peptide, nucleotide, B vitamins and the like.
In the process of optimizing inorganic salt, 1% of glucose and 1% of yeast extract are respectively used as a carbon source and a nitrogen source, and no inorganic salt is added as a blank, 0.5% of manganese sulfate, magnesium sulfate, ferrous sulfate, sodium chloride and calcium chloride are respectively added on the basis, wherein magnesium ions have a certain promotion effect on the growth of bacillus, the biomass of the bacteria is 1.2 times of that of a control group, iron, calcium, manganese and sodium ions have an inhibition effect on the growth of bacillus, and the growth amounts of the bacteria are only 91.3%, 76.5%, 70.5% and 36.2% of that of the control group.
On the basis of a single-factor experiment, an orthogonal experiment is adopted to seek the optimal proportion of each component in the culture medium, the influence of factors on the viable bacteria content can be obtained, wherein the influence of the factors on the viable bacteria content is that yeast extract is greater than glucose and magnesium sulfate is greater than magnesium sulfate, in order to verify that 3 parallel experiments are carried out, the total number of the viable bacteria is 4.49x1010CFU/ml, therefore, the optimal medium for Bacillus licheniformis was determined to be 1.5% glucose yeast extract 1.0% magnesium sulfate 0.1%.
A fermentation medium of bacillus and a fermentation process thereof are disclosed, wherein the fermentation process comprises the following steps:
(1) growth Curve determination
Inoculating into optimized culture medium (triangular flask with liquid content of 100ml/250 ml) at an inoculum size of 1%, placing into a incubator with constant temperature of 37 deg.C, rotating at 160r/min, culturing for 24 hr, measuring bacterial liquid biomass every 2 hr after self-inoculation, and using non-inoculated culture medium as blank control.
After fermentation culture for 0-2h, the growth of the bacillus is in a lag phase, the number of the bacteria is extremely small, the number of the bacteria is increased sharply when the bacillus enters a logarithmic growth phase after 2h, the growth peak phase is reached after 16h, the growth of the bacteria is slow when 12-15 h is a stable phase of the growth of the bacillus licheniformis, the number of the bacteria starts to decrease after 16h, and the bacillus enters a growth decay phase.
2) Initial pH
The pH mainly influences the membrane charge, the membrane permeability and the ionization degree of nutrient substances of thallus cells, so that the nutrient absorption of the thallus is influenced, the pH in the shaking flask fermentation process is difficult to control, and only the initial pH of the fermentation liquor can be controlled, so that the initial pH is respectively adjusted to 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, the biomass of the fermentation liquor is measured after the fermentation is carried out for 16 hours in a shaking flask, the bacillus can well grow within the range of the initial pH of 5.5-8, the growth is best when the pH is 6.5, the adaptability of the bacillus to the pH is wider, and the biomass is in a descending trend along with the increase of the pH.
(3) Temperature of
The temperature affects the growth of the cells mainly by changing the rate of the enzyme reaction. The temperature of a general culture medium is increased, the enzyme reaction rate is increased, the growth and metabolism are accelerated, the production period is advanced, but the enzyme is easy to lose activity due to overheating, the phenomenon that thalli are easy to age is shown, the influence of the final product on the biomass of fermentation liquor is respectively measured by selecting 25 ℃, 30 ℃, 35 ℃ and 40 ℃ as the culture temperature, the biomass of the fermentation liquor is measured after the fermentation is carried out for 16h in a shaking flask, the bacillus can well grow at the temperature of 25-40 ℃, and the optimal growth temperature is 35 ℃.
(4) Amount of inoculation
1 percent, 3 percent, 5 percent, 7 percent and 4 inoculum sizes are respectively selected, the biomass of the bacillus is measured after the bacillus is cultured for 16 hours in a shake flask, the optimal inoculum size for the growth of the bacillus is 50, and when the bacillus grows in the range of 1 to 7 percent, the difference of the influence of the inoculum size on the biomass of the bacillus is very small.
A single-factor and orthogonal experimental method is adopted to optimize a fermentation culture medium of bacillus in a shake flask, the total number of viable bacteria is used as an index for evaluating the quality of the culture medium, the optimized culture medium comprises 1.5 percent of glucose, 1 percent of yeast extract and 0.1 percent of magnesium sulfate, and the optimal culture condition is determined by the single-factor experimental method: the temperature is 35 ℃, the initial pH is 6.5, the inoculation amount is 5%, and the fermentation time is 16 h.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (8)
1. A fermentation medium of bacillus and a fermentation process thereof are characterized by comprising the following steps:
s1: the following experimental materials were prepared:
1) bacterial strain
A bacillus strain;
2) culture medium
Seed culture medium: 1% of yeast protein, 5% of yeast extract, 1% of sodium chloride and 6.5% of PH value;
fermentation medium: 1% of sucrose, 1% of yeast protein, 0.5% of sodium chloride and 7.0% of PH value;
3) primary reagent
Sucrose, glucose, corn starch, maltose, corn starch powder, tryptone, yeast extract (extract), beef extract, soybean cake powder and urea;
s2: culture method
1) Strain activation
Transferring the preserved strain into a culture medium, and placing the culture medium in an incubator with constant temperature of 37 ℃ for culturing for 24 hours for later use;
2) preparation of seed liquid
Taking a ring of activated strains, inoculating the strains into a 250ml triangular flask with the volume of 100ml seed culture medium, putting the flask into an incubator with the constant temperature of 37 ℃, rotating speed of 160r/min, and culturing for 18 hours;
3) shaking culture
Inoculating the seed solution into a fermentation medium (a triangular flask with the liquid loading amount of 100ml/250 ml) with the inoculation amount of 1%, performing shaking culture at 37 ℃ and the rotation speed of 160r/min for 24 hours, and performing single-factor fermentation condition research.
S3: fermentation Medium Single factor experiment
1) Carbon source
Respectively using 1% glucose, corn starch and maltose to equivalently replace sucrose in a fermentation medium to prepare culture media, adopting a shake flask fermentation culture condition, and measuring the viable bacteria content in the fermentation broth after 24h to determine the optimal carbon source.
2) Nitrogen source
1% yeast extract, beef extract, urea and ammonium chloride are respectively used for replacing 1% tryptone in the basic fermentation liquor, the shake flask fermentation culture condition is adopted, and the viable bacteria content in the fermentation liquor is measured after 24 hours so as to determine the optimal nitrogen source.
3) Inorganic salt
Respectively replacing equal amount of sodium chloride (0.5%) in the fermentation medium with 0.5% of manganese sulfate, magnesium sulfate, ferrous sulfate, calcium chloride and a blank without inorganic salt, and determining the total number of viable bacteria in the fermentation broth after 24h under shake flask fermentation culture conditions to determine the optimal inorganic salt.
2. The fermentation medium and the fermentation process of bacillus according to claim 1, wherein 3 important factors of the selected optimal carbon source, nitrogen source and inorganic salt are analyzed, statistically optimized and processed, and the optimal fermentation time, temperature and initial PH inoculation amount are optimized one by one in the optimization process.
3. The fermentation medium and process of claim 2, wherein in the process of optimizing the carbon source, 1% of glucose, sucrose, maltose and corn starch are used as the carbon source, and other components are fermented together to determine the total viable count in the fermentation broth, wherein the growth of bacillus is significantly better than that of corn starch when glucose, sucrose and maltose are used as the carbon source, the viable count of bacillus is more than 2.2x10CFU/ml after fermentation by using the first 3 carbon sources, and the viable count of corn starch is 0.9x1010CFU/ml below, indicating sporesThe ability of bacilli to utilize mono-and disaccharides is superior to starch, so the best carbon source for growth of bacilli is glucose, followed by sucrose and maltose.
4. The fermentation medium and the fermentation process of bacillus according to claim 1, wherein in the process of optimizing the nitrogen source, 1% of glucose is used as the carbon source, and 1% of urea, ammonium chloride, beef extract, yeast extract and tryptone are used as the nitrogen source to prepare the fermentation medium, so that the effect of the organic nitrogen source is obviously better than that of the inorganic nitrogen source, and after the organic nitrogen source is utilized for fermentation, the viable count of bacillus exceeds 2.2x1010CFU/ml, and the viable count when using inorganic nitrogen source is 1.0x1010CFU/ml is below, which shows that the capability of the bacillus to utilize the organic nitrogen source is better than that of the inorganic nitrogen source, and the most suitable nitrogen source for the growth of the bacillus is yeast extract, which is related to that the yeast extract is rich in growth factors such as amino acid, small peptide, nucleotide, B vitamins and the like.
5. The fermentation medium and the fermentation process of bacillus according to claim 1, wherein in the process of optimizing inorganic salt, 1% of glucose and 1% of yeast extract are used as a carbon source and a nitrogen source respectively, and no inorganic salt is added as a blank, 0.5% of manganese sulfate, magnesium sulfate, ferrous sulfate, sodium chloride and calcium chloride are added respectively, wherein magnesium ions have certain promotion effect on the growth of bacillus, the biomass of the bacillus is 1.2 times of that of a control group, iron, calcium, manganese and sodium ions have inhibition effect on the growth of bacillus, and the growth amount of the bacillus is only 91.3%, 76.5%, 70.5% and 36.2% of that of the control group.
6. The fermentation medium and process of claim 1, wherein the components in the medium have a certain internal relationship, and based on a single factor experiment, an orthogonal experiment is performed to find the optimal ratio of the components in the medium, so as to obtain the effect of the factor on the viable bacteria content, i.e., yeast extract > glucose >Magnesium sulfate, wherein the yeast extract is more significant than glucose, the magnesium sulfate is an insignificant difference factor, and for verification, 3 parallel tests are carried out to determine that the total viable count is 4.49x1010CFU/ml, therefore, the optimal medium for Bacillus licheniformis was determined to be 1.5% glucose yeast extract 1.0% magnesium sulfate 0.1%.
7. The fermentation medium of bacillus and the fermentation process thereof according to claim 1, wherein the fermentation process comprises:
(1) growth Curve determination
Inoculating into optimized culture medium (triangular flask with liquid content of 100ml/250 ml) at an inoculum size of 1%, placing into a incubator with constant temperature of 37 deg.C, rotating at 160r/min, culturing for 24 hr, measuring bacterial liquid biomass every 2 hr after self-inoculation, and using non-inoculated culture medium as blank control.
After fermentation culture for 0-2h, the growth of the bacillus is in a lag phase, the number of the bacteria is extremely small, the number of the bacteria is increased sharply when the bacillus enters a logarithmic growth phase after 2h, the growth peak phase is reached after 16h, the growth of the bacteria is slow when 12-15 h is a stable phase of the growth of the bacillus licheniformis, the number of the bacteria starts to decrease after 16h, and the bacillus enters a growth decay phase.
2) Initial pH
The pH mainly influences the membrane charge, the membrane permeability and the ionization degree of nutrient substances of thallus cells, so that the nutrient absorption of the thallus is influenced, the pH in the shaking flask fermentation process is difficult to control, and only the initial pH of the fermentation liquor can be controlled, so that the initial pH is respectively adjusted to 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, the biomass of the fermentation liquor is measured after the fermentation is carried out for 16 hours in a shaking flask, the bacillus can well grow within the range of the initial pH of 5.5-8, the growth is best when the pH is 6.5, the adaptability of the bacillus to the pH is wider, and the biomass is in a descending trend along with the increase of the pH.
(3) Temperature of
The temperature affects the growth of the cells mainly by changing the rate of the enzyme reaction. The temperature of a general culture medium is increased, the enzyme reaction rate is increased, the growth and metabolism are accelerated, the production period is advanced, but the enzyme is easy to lose activity due to overheating, the phenomenon that thalli are easy to age is shown, the influence of the final product on the biomass of fermentation liquor is respectively measured by selecting 25 ℃, 30 ℃, 35 ℃ and 40 ℃ as the culture temperature, the biomass of the fermentation liquor is measured after the fermentation is carried out for 16h in a shaking flask, the bacillus can well grow at the temperature of 25-40 ℃, and the optimal growth temperature is 35 ℃.
(4) Amount of inoculation
1 percent, 3 percent, 5 percent, 7 percent and 4 inoculum sizes are respectively selected, the biomass of the bacillus is measured after the bacillus is cultured for 16 hours in a shake flask, the optimal inoculum size for the growth of the bacillus is 50, and when the bacillus grows in the range of 1 to 7 percent, the difference of the influence of the inoculum size on the biomass of the bacillus is very small.
8. The fermentation medium and the fermentation process of bacillus according to claim 7, wherein the fermentation medium of bacillus is optimized in a shake flask by adopting a single-factor and orthogonal experimental method, the total number of viable bacteria is used as an index for evaluating the quality of the medium, the optimized medium is 1.5% of glucose, 1% of yeast extract and 0.1% of magnesium sulfate, and the optimal culture conditions are determined by the single-factor experimental method: the temperature is 35 ℃, the initial pH is 6.5, the inoculation amount is 5%, and the fermentation time is 16 h.
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