CN114164155A - Fermentation medium and fermentation process of bacillus - Google Patents

Fermentation medium and fermentation process of bacillus Download PDF

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CN114164155A
CN114164155A CN202111585874.4A CN202111585874A CN114164155A CN 114164155 A CN114164155 A CN 114164155A CN 202111585874 A CN202111585874 A CN 202111585874A CN 114164155 A CN114164155 A CN 114164155A
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fermentation
bacillus
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方文棋
谭文俊
黄钦耿
黄炜乾
唐谢芳
王珍珍
吴森雨
蒋顺进
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Qingyuan Yisheng Natural Biological Research Institute Co ltd
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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

Fermentation medium and fermentation process of bacillus
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)

1.一种芽孢杆菌的发酵培养基及其发酵工艺，其特征在于，包括以下步骤：1. a fermentation medium of bacillus and fermentation technique thereof, is characterized in that, comprises the following steps: S1：备好以下实验材料：S1: Prepare the following experimental materials: 1)菌种1) Bacteria 芽孢杆菌菌种；Bacillus species; 2)培养基2) Culture medium 种子培养基：酵母蛋白陈1％、酵母抽提物5％、氯化钠1％、PH值6.5；Seed medium: 1% yeast protein, 5% yeast extract, 1% sodium chloride, PH value 6.5; 发酵培养基：蔗糖1％、酵母蛋白陈1％、氯化钠0.5％、PH值7.0；Fermentation medium: 1% sucrose, 1% yeast protein, 0.5% sodium chloride, PH value 7.0; 3)主要试剂3) Main reagents 蔗糖、葡萄糖、玉米淀粉、麦芽糖、玉米浆粉、胰蛋白胨、酵母抽提物(膏)、牛肉膏、黄豆饼粉、尿素；Sucrose, glucose, corn starch, maltose, corn steep liquor powder, tryptone, yeast extract (paste), beef extract, soybean meal powder, urea; S2：培养方法S2: Cultivation method 1)菌种活化1) Strain activation 将保存的菌种转接到培养基内部，并将培养基放置在37℃恒温的培养箱内部培养24小时进行备用；Transfer the preserved strains to the inside of the medium, and place the medium in a 37°C constant temperature incubator for 24 hours for backup; 2)制备种子液2) Prepare the seed solution 取一环活化的菌种，接入体积为100ml种子培养基的250ml三角瓶中，放入37℃恒温的培养箱内部，转速160r/min，培养时间18小时；Take a ring of activated strains, insert it into a 250ml conical flask with a volume of 100ml seed medium, put it into an incubator with a constant temperature of 37°C, rotate speed 160r/min, and cultivate for 18 hours; 3)摇瓶培养3) Shake flask culture 种子液以1％的接种量接入发酵培养基(装液量100ml/250ml的三角瓶)中，温度37℃，转速160r/min，振荡培养24小时进行单因素的发酵条件研究。The seed liquid was inserted into the fermentation medium (conical flask with a liquid volume of 100ml/250ml) at 1% inoculum, the temperature was 37°C, the rotation speed was 160r/min, and the single-factor fermentation conditions were studied for 24 hours by shaking culture. S3：发酵培养基单因素实验S3: Fermentation medium single factor experiment 1)碳源1) Carbon source 分别用1％葡萄糖、玉米淀粉和麦芽糖等量替代发酵培养基中的蔗糖制成培养基，采用摇瓶发酵培养条件，24h后测定发酵液中的活菌含量以确定最佳碳源。The sucrose in the fermentation medium was replaced by 1% glucose, cornstarch and maltose in equal amounts to prepare the medium, and the culture medium was shaken. 2)氮源2) Nitrogen source 分别用1％酵母抽提物、牛肉膏、尿素、氯化铵替代基础发酵液1％的胰蛋白胨，采用摇瓶发酵培养条件，24h后测定发酵液中的活菌含量，以确定最佳氮源。1% yeast extract, beef extract, urea, and ammonium chloride were used to replace 1% tryptone in the basic fermentation broth respectively, and the culture conditions of shaking flasks were used. source. 3)无机盐3) Inorganic salts 分别用0.5％硫酸锰、硫酸镁、硫酸亚铁、氯化钙及不加无机盐的空白，替代等量的发酵培养基中氯化钠(0.5％)，采用摇瓶发酵培养条件，24h后测定发酵液中的活菌总数，以确定最佳无机盐。0.5% manganese sulfate, magnesium sulfate, ferrous sulfate, calcium chloride and blank without inorganic salt were used to replace the same amount of sodium chloride (0.5%) in the fermentation medium. Determine the total number of viable bacteria in the fermentation broth to determine the best inorganic salts. 2.根据权利要求1所述的一种芽孢杆菌的发酵培养基及其发酵工艺，其特征在于，所述将筛选出的最优碳源、氮源和无机盐的3个重要因素进行分析统计优化处理，并且在优化的过程中对最佳发酵时间、温度、初始PH值接种量进行逐一优化处理。2. the fermentation medium of a kind of bacillus according to claim 1 and fermentation technique thereof, it is characterized in that, the described 3 important factors of optimal carbon source, nitrogen source and inorganic salt that will be screened are analyzed and counted Optimize the treatment, and optimize the optimal fermentation time, temperature, and initial pH value inoculum one by one during the optimization process. 3.根据权利要求2所述的一种芽孢杆菌的发酵培养基及其发酵工艺，其特征在于，所述在对碳源进行优化过程中，分别以1％葡萄糖、蔗糖、麦芽糖、和玉米淀粉作为碳源，其他组分均相同进行发酵，测定发酵液中的活菌总数，其中葡萄糖、蔗糖和麦芽糖为碳源时芽孢杆菌的生长明显优于玉米淀粉，利用前3种碳源发酵后，芽孢杆菌活菌数超过2.2x10CFU/ml，而利用玉米淀粉时其活菌数在0.9x1010CFU/ml以下，说明芽孢杆菌利用单糖和二糖的能力优于淀粉，因此芽孢杆菌生长的最佳碳源是葡萄糖，其次是蔗糖和麦芽糖。3. the fermentation medium and fermentation process thereof of a kind of bacillus according to claim 2, is characterized in that, described in carbon source is carried out optimization process, respectively with 1% glucose, sucrose, maltose, and cornstarch As the carbon source, other components were fermented in the same way, and the total number of viable bacteria in the fermentation broth was determined. When glucose, sucrose and maltose were the carbon sources, the growth of Bacillus was significantly better than that of corn starch. The number of viable bacteria of Bacillus exceeds 2.2x10CFU/ml, while the number of viable bacteria when using corn starch is below 0.9x10 10 CFU/ml, indicating that the ability of Bacillus to utilize monosaccharides and disaccharides is better than that of starch, so the growth of Bacillus is the most efficient. The best carbon source is glucose, followed by sucrose and maltose. 4.根据权利要求1所述的一种芽孢杆菌的发酵培养基及其发酵工艺，其特征在于，所述在对氮源进行优化过程中，以1％葡萄糖为碳源，分别以1％尿素、氯化铵、牛肉膏、酵母抽提物、胰蛋白胨为氮源制成发酵培养基，可知有机氮源效果要明显优于无机氮源，利用有机氮源发酵后，芽孢杆菌活菌数都超过2.2x1010CFU/ml，而利用无机氮源时的活菌数在1.0x1010CFU/ml以下，说明该芽孢杆菌利用有机氮源的能力优于无机氮源，芽孢杆菌生长的最适氮源是酵母抽提物，这与酵母抽提物富含氨基酸、小肽、核苷酸、B族维生素等生长因子有关。4. the fermentation medium of a kind of bacillus according to claim 1 and fermentation process thereof, it is characterized in that, described in the nitrogen source is carried out optimization process, with 1% glucose as carbon source, respectively with 1% urea , ammonium chloride, beef extract, yeast extract, tryptone as nitrogen source to make fermentation medium, it can be seen that the effect of organic nitrogen source is obviously better than that of inorganic nitrogen source. More than 2.2x10 10 CFU/ml, and the number of viable bacteria when using inorganic nitrogen sources is less than 1.0x10 10 CFU/ml, indicating that the ability of Bacillus to use organic nitrogen sources is better than inorganic nitrogen sources, and the optimum nitrogen for Bacillus growth The source is yeast extract, which is related to the fact that yeast extract is rich in growth factors such as amino acids, small peptides, nucleotides, and B vitamins. 5.根据权利要求1所述的一种芽孢杆菌的发酵培养基及其发酵工艺，其特征在于，所述在对无机盐进行优化过程中，分别以1％葡萄糖和1％酵母抽提物为碳源和氮源，以不加无机盐为空白，在此基础上分别加入0.5％的硫酸锰、硫酸镁、硫酸亚铁、氯化钠及氯化钙，其中镁离子对芽孢杆菌的生长有一定的促进作用，其菌的生物量为对照组的1.2倍，铁、钙、锰和钠离子对芽孢杆菌的生长有抑制作用，其菌的生长量仅为对照的91.3％、76.5％、70.5％、36.2％。5. the fermentation medium of a kind of bacillus according to claim 1 and fermentation process thereof, it is characterized in that, described in carrying out optimization process to inorganic salt, respectively with 1% glucose and 1% yeast extract as Carbon source and nitrogen source are blank without inorganic salts, and 0.5% manganese sulfate, magnesium sulfate, ferrous sulfate, sodium chloride and calcium chloride are added respectively on this basis. Among them, magnesium ions have a positive effect on the growth of Bacillus. It has a certain promoting effect, and its bacterial biomass is 1.2 times that of the control group. Iron, calcium, manganese and sodium ions have inhibitory effects on the growth of Bacillus, and its bacterial growth is only 91.3%, 76.5%, 70.5% of the control group. %, 36.2%. 6.根据权利要求1所述的一种芽孢杆菌的发酵培养基及其发酵工艺，其特征在于，所述培养基中各组分之间有一定的内在关系，在单因素实验的基础上，采用正交实验以寻求培养基中各组分的最佳配比，可以得出因子对活菌含量影响大小为酵母抽提物＞葡萄糖＞硫酸镁，其中酵母抽提物的显著性要比葡萄糖要高，硫酸镁为差异性不显著因素，为验证，进行3次平行试验，测得活菌总数为4.49x1010CFU/ml，因此确定地衣芽孢杆菌最佳培养基为葡萄糖1.5％酵母抽提物1.0％硫酸镁0.1％。6. the fermentation substratum of a kind of bacillus according to claim 1 and fermentation technique thereof, is characterized in that, there is a certain intrinsic relationship between each component in the described substratum, on the basis of single factor experiment, Orthogonal experiments were used to find the best ratio of each component in the medium. It can be concluded that the influence of factors on the content of viable bacteria is yeast extract>glucose>magnesium sulfate, and the significance of yeast extract is higher than that of glucose. For verification, three parallel experiments were carried out, and the total number of viable bacteria was 4.49x10 10 CFU/ml, so it was determined that the best medium for Bacillus licheniformis was 1.5% glucose yeast extraction 1.0% magnesium sulfate 0.1%. 7.根据权利要求1所述的一种芽孢杆菌的发酵培养基及其发酵工艺，其特征在于，所述发酵工艺为：7. the fermentation medium of a kind of Bacillus according to claim 1 and fermentation technique thereof, is characterized in that, described fermentation technique is: (1)生长曲线测定(1) Growth curve determination 按1％的接种量接入优化培养基(装液量100ml/250ml的三角瓶)中，放入37℃恒温的培养箱内部，转速160r/min，培养时间24小时，自接种后每2h测定菌液的生物量，并用未接种的培养基作为空白对照。1% of the inoculum was put into the optimized medium (conical flask with a liquid volume of 100ml/250ml), put into an incubator with a constant temperature of 37°C, the rotation speed was 160r/min, the incubation time was 24 hours, and the measurement was performed every 2 hours after inoculation. The biomass of bacterial liquid, and the uninoculated medium was used as blank control. 发酵培养0-2h后芽孢杆菌的生长停滞期，菌体数量极少，自2h以后进入对数生长期，菌体数量急剧增多，在16h达到生长高峰期，12～15h为地衣芽孢杆菌生长的稳定期，菌体生长缓慢，自16h后菌体数量开始减少，芽孢杆菌进入生长衰亡期，因此，采用12-16h时的菌液作为种子液较合适，此时芽孢杆菌为对数生长末期，既可保持高的细胞活力，又可获得尽可能多的细胞数。After 0-2 hours of fermentation and culture, the growth of Bacillus stagnates, and the number of bacteria is very small. After 2 hours, it enters the logarithmic growth phase, and the number of bacteria increases sharply. It reaches the peak growth period at 16 hours. In the stable period, the growth of the bacteria is slow, the number of bacteria begins to decrease after 16 hours, and the Bacillus enters the growth and decline period. Therefore, it is more appropriate to use the bacteria solution at 12-16 hours as the seed solution. At this time, the Bacillus is in the logarithmic growth stage. It is possible to maintain high cell viability while obtaining as many cell numbers as possible. 2)初始PH2) Initial PH PH主要通过影响菌体细胞膜电荷、膜渗透性及营养物质离子化程度，从而影响菌体对养分的吸收，由于摇瓶发酵过程中的PH难以控制，只能控制发酵液的初始pH，因此，将初始pH分别调至5.5、6.0、6.5、7.0、7.5和8.0，摇瓶培养16h后测其生物量，在初始pH5.5～8范围内芽孢杆菌均可良好生长，PH为6.5时生长最好，说明芽孢杆菌对pH的适应性较宽，但随pH的增大，生物量呈下降趋势。PH mainly affects the absorption of nutrients by the bacteria by affecting the cell membrane charge, membrane permeability and the degree of ionization of nutrients. Because the pH in the shake flask fermentation process is difficult to control, only the initial pH of the fermentation broth can be controlled. Therefore, The initial pH was adjusted to 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, respectively, and the biomass was measured after culturing in the shake flask for 16 hours. Bacillus can grow well within the initial pH range of 5.5 to 8, and the best growth is when the pH is 6.5. Good, indicating that Bacillus has a wide adaptability to pH, but with the increase of pH, the biomass shows a downward trend. (3)温度(3) Temperature 温度主要通过改变酶反应速率来影响菌体的生长。一般培养基温度升高，酶反应速率增大，生长代谢加快，生产期提前，但酶很容易因过热而失去活性，表现为菌体容易衰老，影响终产物选取25℃、30℃、35℃和40℃作为培养温度，分别测定不同温度对发酵液生物量的影响，摇瓶培养16h后测其生物量，得出芽孢杆菌在25℃～40℃均可良好生长，其生长的最适温度为35℃。Temperature mainly affects the growth of bacteria by changing the rate of enzymatic reaction. Generally, the temperature of the medium increases, the enzyme reaction rate increases, the growth and metabolism accelerates, and the production period is advanced, but the enzyme is easily lost due to overheating, which means that the bacteria are easily senescent, which affects the selection of 25°C, 30°C, and 35°C for the final product. and 40 °C as the culture temperature, respectively, to determine the effect of different temperatures on the biomass of the fermentation broth, and to measure the biomass after culturing in the shake flask for 16 hours. is 35°C. (4)接种量(4) Inoculum amount 分别选取1％、3％、5％、7％和4个接种量，摇瓶培养16h后测其生物量，芽孢杆菌生长的最适接种量为50，在1％～7％范围内时，接种量对芽孢杆菌生物量的影响差异很小。1%, 3%, 5%, 7% and 4 inoculum doses were selected respectively, and the biomass was measured after culturing in shake flasks for 16 hours. The optimal inoculum dose for Bacillus growth was 50. The effect of inoculum size on Bacillus biomass varies little. 8.根据权利要求7所述的一种芽孢杆菌的发酵培养基及其发酵工艺，其特征在于，所述采用单因素及正交实验法对芽孢杆菌的发酵培养基在摇瓶中进行优化，以活菌总数的高低作为评价培养基优劣的指标，优化后的培养基为葡萄糖1.5％，酵母抽提物1％，硫酸镁0.1％，并通过单因素实验法确定了最佳培养条件：温度35℃，初始PH为6.5，接种量5％，发酵时间为16h。8. the fermentation medium of a kind of bacillus according to claim 7 and fermentation process thereof, it is characterized in that, described adopting single factor and orthogonal experiment method to optimize the fermentation medium of bacillus in shake flask, The total number of viable bacteria was used as an index to evaluate the quality of the medium. The optimized medium was glucose 1.5%, yeast extract 1%, magnesium sulfate 0.1%, and the optimal culture conditions were determined by single factor experiment method: The temperature was 35°C, the initial pH was 6.5, the inoculum amount was 5%, and the fermentation time was 16h.
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Application publication date: 20220311