CN108048387B - Feeding and fermenting method for bacillus - Google Patents
Feeding and fermenting method for bacillus Download PDFInfo
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Abstract
The invention discloses a fed-batch fermentation method of bacillus, belonging to the technical field of microbial fermentation. The bacillus is inoculated into a fermentation culture medium for culture after primary and secondary seed amplification culture, during which the CFU of the bacillus per gram is not less than 1.0 multiplied by 10, which is obtained by the mixture of a fed-batch carbon source and glycine, methionine, tryptophan, threonine and proline relatively lacking in a basal culture medium through spray drying11And the unit yield is obviously improved, and the unit cost is reduced. The bacillus subtilis ANSB471 is used for degrading the vomitoxin, and after the reaction is carried out for 48 hours, the degradation rate of the vomitoxin reaches 95%.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a fed-batch fermentation method of bacillus.
Background
Deoxynivalenol (DON) is also called Deoxynivalenol or vomitoxin, and the pollution of the vomitoxin (DON) to grains is at the head of the fusarium toxin, thus seriously threatening the health of people and livestock. Cereals such as wheat, corn and the like and byproducts thereof are susceptible to vomitoxin, the incidence rate of foreign areas such as Canada, northern Europe area, United states and the like is high, and the grains can be generated from south to north in China. Research shows that the pollution rate and the content of vomitoxin in the diet of residents in the areas with high incidence of esophageal cancer and gastric cancer in China are quite high. In 1998, the International agency for research on cancer (IARC) published an evaluation report that lists vomitoxin as one of three carcinogens. A method for effectively controlling and solving the pollution of vomitoxin in grains and feeds is sought, so that the economic benefit of animal husbandry in China can be well improved, and the food safety level can be well improved. The traditional physicochemical method has some problems, such as low detoxification efficiency, nutrient loss in grain feed, influence on sensory quality of grain and feed, expensive equipment needed by some methods and the like, thereby limiting the application in practical production.
The invention patent with application publication number CN103243047A discloses a Bacillus subtilis ANSB471 (preservation number CGMCC No.7344) for efficiently degrading vomitoxin. The culture method is disclosed as that the concentration of viable bacteria is 109CFU/ml of Bacillus subtilis ANSB 4710.5-2.5 ml is inoculated into 50-100ml of culture medium, and subjected to shake flask fermentation culture at 30-40 ℃ for 16-24h, the rotation speed is 180-. Taking 900 mu l of fermentation liquor of the bacillus subtilis, adding 100 mu l of vomitoxin of 100 mu g/ml, adjusting the pH value of a reaction system to 7.2, reacting for 48h at 37 ℃, wherein the degradation rate of the bacillus subtilis ANSB471 to the vomitoxin reaches 80%.
The invention patent with application publication number CN104232526A discloses a preparation process of a bacillus subtilis live bacteria preparation, the preparation process of the bacillus subtilis ANSB471 live bacteria needs to give consideration to both the live bacteria number and the vomitoxin degradation activity, and the fermentation method provided in CN104232526A cannot meet the requirement that the bacillus subtilis ANSB01G can exert the optimal vomitoxin degradation characteristic.
Disclosure of Invention
The invention provides a fed-batch fermentation method of bacillus, aiming at solving the technical problems. The specific technical scheme is as follows:
a fed-batch fermentation method of bacillus comprises the following steps:
(1) preparing first-level seeds: inoculating the suspension of the viable bacillus strain into a first-stage seed culture medium, and then culturing;
(2) preparing secondary seeds: inoculating the first-stage seeds into a second-stage seed culture medium, and then culturing;
(3) fermentation: inoculating the secondary seeds into a fermentation culture medium, and then carrying out fermentation culture;
feeding a supplemented medium A when the fermentation time is 8-10h, and feeding a supplemented medium B when the fermentation time is 12-16 h;
the feed medium A comprises 5-15% of a carbon source by mass percent;
the feed medium B comprises 5-10% by mass of a carbon source and 2.1-11% by mass of strain-limiting amino acids lacking in the fermentation medium.
The bacillus is bacillus subtilis.
The first-stage seed culture medium comprises the following raw materials: every 1000mL of the beef extract contains 8-12g of tryptone, 1.5-3g of yeast extract, 1.5-4g of glucose, 2-5g of beef extract, 3-6g of sodium chloride, 2.5-4g of disodium hydrogen phosphate, 0.5-1.5g of magnesium sulfate heptahydrate, 1000mL of distilled water and the pH value of 7.0-7.4.
The secondary seed culture medium comprises the following raw materials in percentage by mass: 0.5 to 3 percent of glucose, 0.5 to 3 percent of yeast extract, 0.5 to 5 percent of peptone, 0.02 to 0.05 percent of dipotassium phosphate, 0.5 to 1 percent of sodium chloride, 0.02 to 0.1 percent of magnesium sulfate and the pH value of a culture medium of 6.5 to 7.5.
The fermentation medium comprises the following raw materials in percentage by mass: the raw materials and dosage of the culture medium are 0.5-3.0 percent of glucose, 1-4 percent of bean cypress, 1-3 percent of corn steep liquor, 1-3 percent of yeast powder, 1-3 percent of peptone, 0.01-0.05 percent of dipotassium phosphate, 0.1-0.2 percent of magnesium sulfate and 0.002 percent of manganese sulfate, and the pH value of the culture medium is 6.5-7.5.
The concentration of the bacillus strain suspension is 1 multiplied by 1010-2×1011cfu/mL, inoculating the bacillus strain suspension into a primary seed culture medium according to the volume percentage of 1-5%;
inoculating a primary seed culture medium containing primary seeds into a secondary seed culture medium according to the volume percentage of 1-5%;
inoculating the secondary seed culture medium containing the secondary seeds into the fermentation culture medium according to the volume percentage of 1-5%.
The culture conditions in the step (1) are as follows: the temperature is 37-40 ℃, the time is 16-24h, and the rotating speed is 180-;
the culture conditions in the step (2) are as follows: the temperature is 30-37 ℃, the time is 8-10h, and the rotating speed is 180-;
the conditions of the fermentation culture in the step (3) are as follows: the temperature is 30-37 ℃, the time is 24-36h, the pressure of the fermentation tank is 0.02-0.05 Mpa, and the rotating speed is 180-200 r/min.
The feeding amount of the feeding culture medium A and the feeding culture medium B is 0.1-1.0 per mill of the volume of the fermentation culture medium; the carbon source is glucose, and the strain-limiting amino acids lacking in the fermentation medium are glycine, methionine, tryptophan, threonine, proline and tyrosine.
The feed medium A comprises the following raw materials in percentage by weight: 5 to 15 percent of glucose and 6.5 to 7.5 of culture medium pH;
the feed culture medium B comprises the following raw materials in parts by weight: 5 to 10 percent of glucose, 1.0 to 4.0 percent of glycine, 0.5 to 2.0 percent of methionine, 0.5 to 2.0 percent of tryptophan, 0.1 to 1.0 percent of threonine, 0.0 to 1.0 percent of proline, 0.0 to 1.0 percent of tyrosine, and the pH value of the culture medium is 6.2 to 7.5.
The loading amount of the fermentation medium is 50-70% of the effective volume of the fermentation tank.
The invention has the beneficial effects that:
(1) the bacillus subtilis ANSB471 of the invention is inoculated into a fermentation culture medium for culture after primary and secondary seed amplification culture, during which the mixture of a carbon source and relatively lacked restrictive amino acids glycine, methionine, tryptophan, threonine, proline and tyrosine in the fermentation culture medium is supplemented by a fed-batch technology, the number and spore conversion rate of the bacillus subtilis are increased, and the CFU of the bacillus subtilis is not less than 1.0 multiplied by 10 per gram of bacterial powder after spray drying11Compared with the original batch fermentation process, the number of live bacillus subtilis ANSB471 fermentation bacteria produced by the fed-batch process is increased by 1.76 orders of magnitude, the spore conversion rate is increased by 6.1 percent, the unit yield is obviously increased, and the unit cost is reduced; the prepared bacillus subtilis ANSB471 is used for degrading vomitoxin, and the degradation rate can reach 95% after 48 hours.
(2) The fed-batch fermentation method realizes the optimization of three indexes of the number of the fermentation viable bacteria, the transformation spore rate and the activity of degrading the vomitoxin, has the functions of cost reduction and synergy in industrial fermentation production, and inspires for other related technologies of bacillus fermentation processes.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The bacillus subtilis ANSB471 used in the following examples has been deposited in the china general microbiological culture collection management center at 21.3.2013, with the deposit number of CGMCC No.7344, and is disclosed in chinese patent document CN 103243047A.
Example 1: the invention relates to fermentation of bacillus subtilis ANSB471
The fermentation method of the bacillus subtilis ANSB471 comprises the following steps:
(1) preparing first-level seeds: and (3) filling a 250mL triangular flask with 100mL of primary seed culture medium, carrying out moist heat sterilization at 115 ℃ for 20min, inoculating 1mL of ANSB471 viable bacteria freeze-dried tube strain into the primary seed culture medium, and culturing for 16h at 37 ℃ and at the rotating speed of 200 r/min.
The formula (g/L) of the first-order seed culture medium is as follows: 8g of tryptone, 1.5g of yeast extract, 1.5g of glucose, 2g of beef extract, 3g of sodium chloride, 2.5g of disodium hydrogen phosphate and 0.5g of magnesium sulfate heptahydrate, and distilled water is added until the volume is 1000mL, and the pH value is 7.0.
(2) Preparing secondary seeds: placing 200L of secondary seed culture medium in 500L fermentation tank, sterilizing at 115 deg.C for 20min, inoculating the primary seed into the secondary seed culture medium at a ratio of 1:100, and culturing at 30 deg.C for 10 hr.
The formula of the secondary seed culture medium is as follows: 0.5% of glucose, 0.5% of yeast extract, 0.5% of peptone, 0.02% of dipotassium hydrogen phosphate, 0.5% of sodium chloride, 0.02% of magnesium sulfate and 6.5% of culture medium pH.
(3) Fermentation: at 10m3The fermentation tank is filled with 7m3Bacillus subtilisSterilizing the viable bacteria fermentation culture medium at 115 deg.C for 20min, inoculating 70L of secondary seeds into the fermentation culture medium, controlling the pressure of the tank at 0.02Mpa, and culturing at 30 deg.C for 24 hr.
The formula of the fermentation medium is as follows: 0.5% of glucose, 1% of bean curd, 1% of corn steep liquor, 1% of yeast powder, 1% of peptone, 0.01% of dipotassium hydrogen phosphate, 0.1% of magnesium sulfate, 0.002% of manganese sulfate and the pH value of a culture medium of 6.5.
(4) Feeding in a flowing mode: when the fermentation time is 8 hours, 0.7L of the feed medium A is fed, and when the fermentation time is 12 hours, 0.7L of the feed medium B is fed.
The formula of the feed medium A is as follows: 5% of glucose, 85% of water and 6.5% of pH of a culture medium;
the formula of the feed medium B is as follows: 5% of glucose, 1.0% of glycine, 0.5% of methionine, 0.5% of tryptophan, 0.1% of threonine and 0.0% of tyrosine, and the pH value of the culture medium is 7.5.
Example 2: fermentation of Bacillus subtilis ANSB471
The fermentation method of the bacillus subtilis ANSB471 comprises the following steps:
(1) preparing first-level seeds: and (3) filling a 250mL triangular flask with 100mL of primary seed culture medium, carrying out moist heat sterilization at 121 ℃ for 15min, inoculating 1mL of ANSB471 viable bacteria freeze-dried tube strain into the primary seed culture medium, and culturing for 20h at 40 ℃ and 180 r/min.
The formula (g/L) of the primary seed culture medium is as follows: 12g of tryptone, 3g of yeast extract, 4g of glucose, 5g of beef extract, 6g of sodium chloride, 4g of disodium hydrogen phosphate and 1.5g of magnesium sulfate heptahydrate, and distilled water is added until the volume is 1000mL, and the pH value is 7.0.
(2) Preparing secondary seeds: placing 300L of secondary seed culture medium in 500L fermentation tank, sterilizing at 121 deg.C for 15min, inoculating the primary seed into the secondary seed culture medium at a ratio of 1:100, and culturing at 35 deg.C for 8 hr.
The formula of the secondary seed culture medium is as follows: 3% of glucose, 3% of yeast extract, 3% of peptone, 0.05% of dipotassium phosphate, 1% of sodium chloride, 0.1% of magnesium sulfate and 6.5% of the pH value of the culture medium.
(3) Fermentation: at 40m3The fermentation tank is filled with 30m3Bacillus subtilis live bacteria fermentationSterilizing the culture medium at 121 deg.C for 15min, inoculating 300L of secondary seeds into the fermentation culture medium, controlling the pressure of the tank at 0.02Mpa, and culturing at 35 deg.C for 36 h.
The formula of the fermentation medium is as follows: 3% of glucose, 4% of bean curd, 3% of corn steep liquor, 3% of yeast powder, 3% of peptone, 0.05% of dipotassium hydrogen phosphate, 0.2% of magnesium sulfate, 0.002% of manganese sulfate and 6.5% of culture medium pH.
(4) Feeding in a flowing mode: when the fermentation time is 10h, 3.0L of the feed medium A is fed, and the fermentation time is 16h3.0L of the feed medium B is fed.
The formula of the feed medium A is as follows: glucose 15%, water 95%, medium pH 6.5.
The formula of the feed medium B is as follows: 10% of glucose, 4.0% of glycine, 2.0% of methionine, 2.0% of tryptophan, 1.0% of threonine, 1.0% of proline, 1.0% of tyrosine and 6.2% of pH of a culture medium.
Example 3: preparation of bacillus subtilis ANSB471 bacterial powder
The preparation method of the bacillus subtilis ANSB471 bacterial powder comprises the following specific operations: heating the fermentation liquor of the bacillus subtilis in the embodiments 1 and 2 to 55 ℃, respectively, feeding the fermentation liquor into a concentrator, concentrating the fermentation liquor under the vacuum degree of 85kpa, and starting to discharge when the concentration is carried out until the solid content reaches 15-30%; drying and granulating the concentrated fermentation liquor by using a multistage low-temperature spray fluidized drying device, sorting the granules, directly discharging the granules out of the tower according with the granule requirements to obtain bacterial powder bacillus subtilis CFU not less than 1.0 multiplied by 1011And (4) respectively. The inlet air temperature is 170 ℃, and the outlet temperature is 60-80 ℃.
Example 4: degradation effect of bacillus subtilis ANSB471 fermentation liquor on vomitoxin
Sucking 900. mu.l of Bacillus subtilis ANSB471 fermentation broth prepared in example 2, and reacting with 100. mu.l of vomitoxin (100. mu.g/mL); the control group was 900. mu.l of Bacillus subtilis ANSB471 fermented broth, reacted with 100. mu.l of vomitoxin (100. mu.g/mL); blank control group 900. mu.l 0.01mol/L PBS buffer with 100. mu.l vomitoxin (100. mu.g/mL); the pH value of the reaction system is adjusted to 7.2, and the treatment group, the control group and the blank control group are respectively reacted at 37 ℃ for 48 h.
Bacillus subtilis A in control groupThe NSB471 culture method comprises the following steps: taking viable bacteria concentration as 109CFU/ml Bacillus subtilis ANSB 4711 ml is inoculated into 100ml culture medium, and subjected to shake flask fermentation culture at 37 deg.C for 24h, rotation speed of 220r/min, and pH value of 7.0.
Adding 1ml methanol to terminate reaction, centrifuging each sample obtained after reaction of the treated group, the control group and the blank control group for 5min, filtering with 0.22 μm filter membrane, and detecting vomitoxin content by high performance liquid chromatography.
Detection conditions are as follows: a chromatographic column: clovertil C18, 150mm × 4.6mm × 5 μm; the mobile phase is methanol-water (10: 90); the flow rate is 1 ml/min; the column temperature is 35 ℃; detecting the wavelength lambda 218 nm; the amount of the sample was 20. mu.l.
And (3) detection results: the degradation rate of bacillus subtilis ANSB471 to vomitoxin is 95%, and the degradation rate of bacillus subtilis to vomitoxin in the control group is 80%.
Claims (7)
1. A fed-batch fermentation method of bacillus is characterized by comprising the following steps:
(1) preparing first-level seeds: inoculating the bacillus strain suspension into a first-stage seed culture medium, and then culturing;
(2) preparing secondary seeds: inoculating the first-stage seeds into a second-stage seed culture medium, and then culturing;
(3) fermentation: inoculating the secondary seeds into a fermentation culture medium, and then carrying out fermentation culture;
feeding a supplemented medium A when the fermentation time is 8-10h, and feeding a supplemented medium B when the fermentation time is 12-16 h;
the feed medium A comprises 5-15% of a carbon source by mass percent;
the feed medium B comprises 5-10% of carbon source by mass and 2.1-11% of strain-limiting amino acid lacking in the fermentation medium by mass;
the feeding amount of the feeding culture medium A and the feeding culture medium B is 0.1-1.0 per mill of the volume of the fermentation culture medium; the carbon source is glucose, and the strain-limiting amino acids lacking in the fermentation medium are glycine, methionine, tryptophan, threonine, proline and tyrosine;
the feed medium A comprises the following raw materials in percentage by weight: 5 to 15 percent of glucose and 6.5 to 7.5 of culture medium pH;
the feed culture medium B comprises the following raw materials in parts by weight: 5 to 10 percent of glucose, 1.0 to 4.0 percent of glycine, 0.5 to 2.0 percent of methionine, 0.5 to 2.0 percent of tryptophan, 0.1 to 1.0 percent of threonine, 0.0 to 1.0 percent of proline, 0.0 to 1.0 percent of tyrosine, and the pH value of the culture medium is 6.2 to 7.5;
the bacillus is bacillus subtilis; the bacillus is capable of degrading emetic toxins.
2. The fermentation process of claim 1, wherein the primary seed medium comprises the following raw materials: every 1000mL of the composition comprises 8-12g of tryptone, 1.5-3g of yeast extract, 1.5-4g of glucose, 2-5g of beef extract, 3-6g of sodium chloride, 2.5-4g of disodium hydrogen phosphate, 0.5-1.5g of magnesium sulfate heptahydrate, 1000mL of distilled water and the pH value of 7.0-7.4.
3. The fermentation method according to claim 1, wherein the secondary seed culture medium comprises the following raw materials in percentage by mass: 0.5 to 3 percent of glucose, 0.5 to 3 percent of yeast extract, 0.5 to 5 percent of peptone, 0.02 to 0.05 percent of dipotassium phosphate, 0.5 to 1 percent of sodium chloride, 0.02 to 0.1 percent of magnesium sulfate and the pH value of a culture medium of 6.5 to 7.5.
4. The fermentation method according to claim 1, wherein the fermentation medium comprises the following raw materials in percentage by mass: 0.5-3.0% of glucose, 1-4% of soybean meal, 1-3% of corn steep liquor, 1-3% of yeast powder, 1-3% of peptone, 0.01-0.05% of dipotassium phosphate, 0.1-0.2% of magnesium sulfate, 0.002% of manganese sulfate and 6.5-7.5% of the pH value of a culture medium.
5. The fermentation method according to claim 1, wherein the concentration of the bacillus strain suspension is 1 x 1010-2×1011cfu/mL, inoculating the bacillus strain suspension to the first-class strain according to the volume percentage of 1-5 percentIn a seed culture medium;
inoculating a primary seed culture medium containing primary seeds into a secondary seed culture medium according to the volume percentage of 1-5%;
inoculating the secondary seed culture medium containing the secondary seeds into the fermentation culture medium according to the volume percentage of 1-5%.
6. The fermentation process of claim 1, wherein the culture conditions in step (1) are: the temperature is 37-40 ℃, the time is 16-24h, and the rotating speed is 180-;
the culture conditions in the step (2) are as follows: the temperature is 30-37 ℃, the time is 8-10h, and the rotating speed is 180-;
the fermentation culture conditions in the step (3) are as follows: the fermentation temperature is 30-37 ℃, the fermentation time is 24-36h, the pressure of the fermentation tank is 0.02-0.05 Mpa, and the rotating speed is 180-200 r/min.
7. The fermentation process of claim 1, wherein the fermentation medium is charged in an amount of 50% to 70% of the available volume of the fermentor.
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