CN108048370B - Fermentation process of bacillus subtilis - Google Patents
Fermentation process of bacillus subtilis Download PDFInfo
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Abstract
The invention relates to a fermentation process of bacillus subtilis, which belongs to the field of bacillus subtilis and aims to solve the problem of more mixed bacteria in the process of fermenting the bacillus subtilis, wherein the fermentation process of the bacillus subtilis comprises primary seed culture, secondary seed culture, tertiary seed culture and fermentation product generation, wherein the raw materials of a solid culture medium comprise straw, corn flour, bran, diethylaminoethyl cross-linked glucan gel, 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone.
Description
Technical Field
The invention relates to the field of bacillus subtilis, in particular to a fermentation process of bacillus subtilis.
Background
The utilization of the drug feed additive, especially the antibiotic feed additive, plays an important role in the aspects of preventing and treating diseases of animals, but simultaneously brings certain influence on the animal husbandry production and human beings, and the long-term use of the antibiotic can cause the problems of drug resistance, endogenous infection and the like of the animals, thereby causing the application of the antibiotic to be gradually limited.
The microbial feed additive is a live bacterial product containing beneficial microorganisms for animals, which is developed according to the micro-ecological theory, has multiple functions of promoting the growth and development of the animals and improving the immunity of the animal organisms by maintaining the ecological balance and volatilization of the microorganisms in the intestinal tracts of the animals, has no pollution, residue, drug resistance, no harm to the environment and the like, and is a novel green and environment-friendly feed additive. At present, the commonly used probiotic products for livestock and poultry comprise bacillus, lactobacillus, bifidobacterium and the like.
The bacillus has strong resistance to external adverse environment, can resist high temperature of over 60 ℃, has long storage time, is easy to culture, is convenient to process into various products, has stable effect and is widely used in animal husbandry. Various types of culture media for bacillus exist, and corn flour, bean cakes, bran and the like are prepared into solid culture media for fermentation on the basis of beef soup. However, in the process of fermentation on a solid medium, due to the complex components of the raw materials, the bacillus is easily infected by mixed bacteria through multi-stage culture and open fermentation, and the product quality is difficult to reach the standard.
Disclosure of Invention
The invention aims to provide a fermentation process of bacillus subtilis, which greatly reduces the pollution of mixed bacteria, and when the fermented bacillus subtilis is applied to poultry, the bacillus subtilis can greatly promote the growth of the poultry and is a feed additive with biological activity.
The above object of the present invention is achieved by the following technical solutions: a fermentation process of bacillus subtilis at least comprises the following steps:
s1, inoculating the bacillus subtilis into 20-30kg of liquid culture medium, adjusting the alkalinity of liquid culture amino acid to be neutral, and culturing for 12-18h under the aerobic condition at 28-36 ℃ to form first-grade seeds;
s2, inoculating the primary seeds into a liquid culture medium of 30-40kg, adjusting the alkalinity of the liquid culture amino acid to be neutral, and culturing for 24-32h under the aerobic condition at 28-36 ℃ to form secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium of 20-30kg for culture, culturing for 32-34h in a continuous fed-batch fermentation mode under the aerobic condition at 28-36 ℃, and standing for 12-16h at constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to 68-81.6kg of solid culture medium, fermenting and culturing for 32-36h, and crushing to obtain a fermentation product of the bacillus subtilis;
wherein the solid culture medium comprises the raw materials of straws, corn flour, bran, diethylaminoethyl crosslinked glucan gel, 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone.
Preferably, in the solid culture medium, the mass ratio of the straw, the corn flour, the bran, the diethylaminoethyl cross-linked glucan gel mixture, the 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and the D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone is 2:2:1:1:0.5: 0.3.
Preferably, in the solid fermentation step, diethylaminoethyl sephadex, 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone are uniformly mixed, then are added into water for soaking for 0.3 to 0.5h, then straws, corn flour and bran are added, and the solid culture medium is obtained after uniform stirring.
Preferably, the mass ratio of the diethylaminoethyl sephadex to the water is 1: 3.
Preferably, in step S1, the pH of the liquid medium is 7-8.
Preferably, in step S2, the pH of the liquid medium is 7-8.
Preferably, the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of potassium dihydrogen phosphate, 4 parts of dipotassium hydrogen phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of potassium dihydrogen phosphate.
Preferably, the liquid medium and the solid medium are sterilized before use.
In conclusion, the invention has the following beneficial effects:
1. the pollution of mixed bacteria in the fermentation product of the bacillus subtilis is greatly reduced, and the prepared fermentation product of the bacillus subtilis is a feed additive with biological activity.
2. According to the invention, during solid fermentation, 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone in a solid culture medium are firstly adsorbed in a macromolecular chain of diethylaminoethyl cross-linked glucan gel, during the process of culturing bacillus subtilis, 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone are oxidized, and the generated oxidation product has a bacteriostatic effect and effectively inhibits the generation of mixed bacteria, and the bacteriostatic effect is optimal by the matched use of 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone, so that the bacillus subtilis- (2- (β -D-hydroxyphenyloxy) -4, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone and D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone are gradually released along with the culture of bacillus subtilis.
Detailed Description
All materials referred to in the examples of the present invention are commercially available.
First, the manufacturing embodiment
Example 1
S1, inoculating the bacillus subtilis into 25kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7.5, and culturing for 15h under an aerobic condition at 32 ℃ to obtain first-stage seeds; wherein the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of monopotassium phosphate, 4 parts of dipotassium phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of monopotassium phosphate;
s2, inoculating the primary seeds into 35kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7.5, and culturing for 28h under the aerobic condition of 32 ℃ to obtain secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium of 25kg for culture, culturing for 33h in a continuous fed-batch fermentation mode under the aerobic condition of 32 ℃, and standing for 14h at constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to a solid culture medium, fermenting and culturing for 33h, and crushing to obtain a fermentation product of the bacillus subtilis;
the preparation method of the solid culture medium comprises the steps of firstly uniformly mixing 11kg of diethylaminoethyl cross-linked dextran gel, 5.5kg of 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and 3.3kg of D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone, then adding 33kg of water for soaking for 0.4h, then adding 22kg of straw, 22kg of corn flour and 11kg of bran, and uniformly stirring to obtain the solid culture medium.
Example 2
S1, inoculating the bacillus subtilis into 20kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7, and culturing for 18h under the aerobic condition of 28 ℃ to obtain primary seeds; wherein the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of monopotassium phosphate, 4 parts of dipotassium phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of monopotassium phosphate;
s2, inoculating the primary seeds into 40kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7, and culturing for 32h under the aerobic condition of 32 ℃ to obtain secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium 20kg for culture, culturing for 32h in a continuous fed-batch fermentation mode under the aerobic condition at 28 ℃, and standing for 12h at constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to a solid culture medium, fermenting and culturing for 36h, and crushing to obtain a fermentation product of the bacillus subtilis;
the preparation method of the solid culture medium comprises the steps of uniformly mixing 10kg of diethylaminoethyl cross-linked dextran gel, 5kg of 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and 3kg of D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone, then adding 30kg of water for soaking for 0.3h, then adding 24kg of straws, 24kg of corn flour and 10kg of bran, and uniformly stirring to obtain the solid culture medium.
Example 3
S1, inoculating the bacillus subtilis into 30kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7, and culturing for 18h under the aerobic condition of 28 ℃ to obtain first-level seeds; wherein the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of monopotassium phosphate, 4 parts of dipotassium phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of monopotassium phosphate;
s2, inoculating the primary seeds into 30kg of liquid culture medium, adjusting the pH of the liquid culture medium to 8, and culturing for 32h under the aerobic condition of 36 ℃ to obtain secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium 20kg for culture, adopting a continuous fed-batch fermentation mode for culture for 32h under an aerobic condition at 36 ℃, and standing for 12h at a constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to a solid culture medium, fermenting and culturing for 36h, and crushing to obtain a fermentation product of the bacillus subtilis;
the preparation method of the solid culture medium comprises the steps of uniformly mixing 10kg of diethylaminoethyl cross-linked dextran gel, 6kg of 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and 3kg of D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone, then adding 25kg of water to soak for 0.3h, then adding 24kg of straws, 20kg of corn flour and 12kg of bran, and uniformly stirring to obtain the solid culture medium.
Example 4
S1, inoculating the bacillus subtilis into 20kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7, and culturing for 12h under the aerobic condition of 36 ℃ to obtain primary seeds; wherein the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of monopotassium phosphate, 4 parts of dipotassium phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of monopotassium phosphate;
s2, inoculating the primary seeds into 40kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7.5, and culturing for 28h under the aerobic condition of 28 ℃ to obtain secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium 30kg for culture, adopting a continuous fed-batch fermentation mode for culture for 32h under an aerobic condition at 28 ℃, and standing for 16h at a constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to a solid culture medium, fermenting and culturing for 32h, and crushing to obtain a fermentation product of the bacillus subtilis;
the preparation method of the solid culture medium comprises the steps of uniformly mixing 12kg of diethylaminoethyl cross-linked dextran gel, 5kg of 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and 3kg of D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone, then adding 36kg of water to soak for 0.5h, then adding 20kg of straws, 24kg of corn flour and 11kg of bran, and uniformly stirring to obtain the solid culture medium.
Example 5
S1, inoculating the bacillus subtilis into 30kg of liquid culture medium, adjusting the pH of the liquid culture medium to 8, and culturing for 12h under the aerobic condition of 36 ℃ to obtain first-level seeds; wherein the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of monopotassium phosphate, 4 parts of dipotassium phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of monopotassium phosphate;
s2, inoculating the primary seeds into 30kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7, and culturing for 28h under the aerobic condition of 32 ℃ to obtain secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium 30kg for culture, culturing for 34h in a continuous fed-batch fermentation mode under an aerobic condition at 36 ℃, and standing for 16h at a constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to a solid culture medium, fermenting and culturing for 32h, and crushing to obtain a fermentation product of the bacillus subtilis;
the preparation method of the solid culture medium comprises the steps of firstly uniformly mixing 12kg of diethylaminoethyl cross-linked dextran gel, 6kg of 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and 3.6kg of D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone, then adding 36kg of water for soaking for 0.5h, then adding 20kg of straws, 20kg of corn flour and 10kg of bran, and uniformly stirring to obtain the solid culture medium.
Example 6
S1, inoculating the bacillus subtilis into 20kg of liquid culture medium, adjusting the pH of the liquid culture medium to 8, and culturing for 12h under the aerobic condition of 28 ℃ to obtain primary seeds; wherein the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of monopotassium phosphate, 4 parts of dipotassium phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of monopotassium phosphate;
s2, inoculating the primary seeds into 40kg of liquid culture medium, adjusting the pH of the liquid culture medium to 8, and culturing for 24h under the aerobic condition of 36 ℃ to obtain secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium 30kg for culture, culturing for 34h in a continuous fed-batch fermentation mode under the aerobic condition at 28 ℃, and standing for 12h at constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to a solid culture medium, fermenting and culturing for 32h, and crushing to obtain a fermentation product of the bacillus subtilis;
the preparation method of the solid culture medium comprises the steps of firstly uniformly mixing 12kg of diethylaminoethyl cross-linked dextran gel, 5kg of 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and 3.6kg of D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone, then adding 36kg of water for soaking for 0.3h, then adding 20kg of straws, 24kg of corn flour and 12kg of bran, and uniformly stirring to obtain the solid culture medium.
Second, preparation of comparative example
Comparative example 1
S1, inoculating the bacillus subtilis into 25kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7.5, and culturing for 15h under an aerobic condition at 32 ℃ to obtain first-stage seeds; wherein the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of monopotassium phosphate, 4 parts of dipotassium phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of monopotassium phosphate;
s2, inoculating the primary seeds into 35kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7.5, and culturing for 28h under the aerobic condition of 32 ℃ to obtain secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium of 25kg for culture, culturing for 33h in a continuous fed-batch fermentation mode under the aerobic condition of 32 ℃, and standing for 14h at constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to a solid culture medium, fermenting and culturing for 33h, and crushing to obtain a fermentation product of the bacillus subtilis;
the preparation method of the solid culture medium comprises the following steps: taking 22kg of straws, 22kg of corn flour and 11kg of bran, and uniformly stirring to obtain the solid culture medium.
Comparative example 2
S1, inoculating the bacillus subtilis into 25kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7.5, and culturing for 15h under an aerobic condition at 32 ℃ to obtain first-stage seeds; wherein the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of monopotassium phosphate, 4 parts of dipotassium phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of monopotassium phosphate;
s2, inoculating the primary seeds into 35kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7.5, and culturing for 28h under the aerobic condition of 32 ℃ to obtain secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium of 25kg for culture, culturing for 33h in a continuous fed-batch fermentation mode under the aerobic condition of 32 ℃, and standing for 14h at constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to a solid culture medium, fermenting and culturing for 33h, and crushing to obtain a fermentation product of the bacillus subtilis;
the preparation method of the solid culture medium comprises the steps of uniformly mixing 11kg of diethylaminoethyl crosslinked glucan gel and 5.5kg of 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone, then adding 33kg of water, soaking for 0.4h, then adding 22kg of straws, 22kg of corn flour and 11kg of bran, and uniformly stirring to obtain the solid culture medium.
Comparative example 3
S1, inoculating the bacillus subtilis into 25kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7.5, and culturing for 15h under an aerobic condition at 32 ℃ to obtain first-stage seeds; wherein the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of monopotassium phosphate, 4 parts of dipotassium phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of monopotassium phosphate;
s2, inoculating the primary seeds into 35kg of liquid culture medium, adjusting the pH of the liquid culture medium to 7.5, and culturing for 28h under the aerobic condition of 32 ℃ to obtain secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium of 25kg for culture, culturing for 33h in a continuous fed-batch fermentation mode under the aerobic condition of 32 ℃, and standing for 14h at constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to a solid culture medium, fermenting and culturing for 33h, and crushing to obtain a fermentation product of the bacillus subtilis;
the preparation method of the solid culture medium comprises the following steps: firstly, uniformly mixing 11kg of diethylaminoethyl cross-linked dextran gel and 3.3kg of D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone, then adding 33kg of water to soak for 0.4h, then adding 22kg of straws, 22kg of corn flour and 11kg of bran, and uniformly stirring to obtain a solid culture medium.
The liquid medium and the solid medium used in each of the above examples and comparative examples were sterilized before use.
Third, testing the Performance results
Test subjects: 405 egg chicks were selected and randomly divided into 9 treatment groups, each group was assigned 3 replicates, each replicate 15 chicks, with a test period of 8 weeks. Of these, 9 treatment groups added 0.5% of the bacillus subtilis fermentation preparations prepared in examples 1 to 6 and comparative examples 1 to 3 to the basal diet, respectively. The feed is manually fed three times a day, and the water is freely drunk to ensure the cleanness and sanitation of water, materials, a groove and the house.
And (3) testing the production performance: and (3) respectively weighing on an empty stomach by taking the repetition as a unit at the beginning and the end of the test period, accurately recording the material consumption of each weight, and calculating the average daily gain, the material consumption and the feed weight gain ratio. The test results are shown in table 1.
TABLE 1 results of the performance tests of the examples and comparative examples of Bacillus subtilis
| Daily consumption (g/only) | Average daily gain (g/only) | Feed/weight gain | |
| Example 1 | 17.17 | 10.09 | 1.70 |
| Example 2 | 16.73 | 9.65 | 1.73 |
| Example 3 | 16.19 | 9.10 | 1.78 |
| Example 4 | 16.94 | 9.87 | 1.72 |
| Example 5 | 16.54 | 9.46 | 1.75 |
| Example 6 | 16.70 | 9.62 | 1.74 |
| Comparative example 1 | 15.68 | 7.37 | 2.13 |
| Comparative example 2 | 16.45 | 8.34 | 1.97 |
| Comparative example 3 | 16.63 | 8.51 | 1.96 |
As can be seen from the table, the number of mixed bacteria in the prepared bacillus subtilis fermentation product is greatly reduced, the product performance of the prepared bacillus subtilis fermentation product is greatly improved, the bacillus subtilis fermentation product has a good promotion effect on the growth of chicks, and the bacillus subtilis fermentation product is a feed additive with biological activity.
In comparative example 1, 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone, D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone and diethylaminoethyl cross-linked dextran gel are not added, and even if the solid culture medium is sterilized before use in the preparation process of the fermentation product of the bacillus subtilis, the amount of mixed bacteria is increased due to environment suitability in the culture process of the bacillus subtilis, so that the prepared fermentation product of the bacillus subtilis carries a large amount of mixed bacteria, and the product performance of the fermentation product of the bacillus subtilis is seriously influenced.
In comparative example 2 and comparative example 3, D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone and 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone are not added respectively, and the inhibition effect on mixed bacteria in the fermentation product of the bacillus subtilis is lower than that of the mixed bacteria in the fermentation product of the bacillus subtilis, so that the prepared fermentation product of the bacillus subtilis carries mixed bacteria and the product performance of the fermentation product of the bacillus subtilis is influenced.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (7)
1. A fermentation process of bacillus subtilis is characterized by at least comprising the following steps:
s1, inoculating the bacillus subtilis into 20-30kg of liquid culture medium, adjusting the alkalinity of liquid culture amino acid to be neutral, and culturing for 12-18h under the aerobic condition at 28-36 ℃ to form first-grade seeds; the liquid culture medium comprises 0.2 part of magnesium sulfate, 2 parts of ammonium sulfate, 1 part of sodium citrate, 6 parts of monopotassium phosphate, 4 parts of dipotassium phosphate, 10 parts of peptone, 1 part of sodium chloride and 1 part of monopotassium phosphate;
s2, inoculating the primary seeds into a liquid culture medium of 30-40kg, adjusting the alkalinity of the liquid culture amino acid to be neutral, and culturing for 24-32h under the aerobic condition at 28-36 ℃ to form secondary seeds;
s3, inoculating the secondary seeds into a liquid culture medium of 20-30kg for culture, culturing for 32-34h in a continuous fed-batch fermentation mode under the aerobic condition at 28-36 ℃, and standing for 12-16h at constant temperature after the culture is finished to form tertiary seeds;
s4, inoculating the third-level seeds to a solid culture medium of 68-81.6kg, fermenting and culturing for 32-36h, and crushing to obtain a fermentation product of the bacillus subtilis, wherein the solid culture medium comprises straw, corn flour, bran, diethylaminoethyl cross-linked dextran gel, 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone, and the addition ratio of the diethylaminoethyl cross-linked dextran gel, the 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and the D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone is 10-12:5-6: 3-3.6.
2. The fermentation process of the bacillus subtilis according to claim 1, wherein in the solid culture medium, the mass ratio of straw, corn flour, bran, diethylaminoethyl sephadex mixture, 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone is 2:2:1:1:0.5: 0.3.
3. The fermentation process of bacillus subtilis according to claim 2, wherein in the step of solid fermentation, diethylaminoethyl cross-linked dextran gel, 1- (2- (β -D-glucopyranosyloxy) -4, 6-dihydroxyphenyl) -3- (4-hydroxyphenyl) -acetone and D-2,3,5, 6-tetrahydroxy-2-hexenoic acid-gamma-lactone are uniformly mixed, then added into water for soaking for 0.3-0.5h, then added with straw, corn flour and bran, and uniformly stirred to obtain the solid culture medium.
4. The fermentation process of bacillus subtilis according to claim 3, wherein the mass ratio of the diethylaminoethyl sephadex to the water is 1: 3.
5. The fermentation process of Bacillus subtilis according to claim 1, wherein the pH of the liquid culture medium in step S1 is 7-8.
6. The fermentation process of Bacillus subtilis according to claim 1, wherein the pH of the liquid culture medium in step S2 is 7-8.
7. The process of claim 1, wherein the liquid medium and the solid medium are sterilized before use.
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