CN110093393B - High-yield antibacterial peptide bacillus subtilis culture medium and liquid fermentation method - Google Patents
High-yield antibacterial peptide bacillus subtilis culture medium and liquid fermentation method Download PDFInfo
- Publication number
- CN110093393B CN110093393B CN201910321647.7A CN201910321647A CN110093393B CN 110093393 B CN110093393 B CN 110093393B CN 201910321647 A CN201910321647 A CN 201910321647A CN 110093393 B CN110093393 B CN 110093393B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- culture medium
- antibacterial peptide
- seed
- powder
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 125
- 230000004151 fermentation Effects 0.000 title claims abstract description 125
- 239000001963 growth medium Substances 0.000 title claims abstract description 72
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 50
- 239000007788 liquid Substances 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 30
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 29
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 29
- 239000000843 powder Substances 0.000 claims abstract description 54
- 240000008042 Zea mays Species 0.000 claims abstract description 30
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 30
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 30
- 235000005822 corn Nutrition 0.000 claims abstract description 30
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 27
- 239000008103 glucose Substances 0.000 claims abstract description 27
- 235000019764 Soybean Meal Nutrition 0.000 claims abstract description 26
- 239000004455 soybean meal Substances 0.000 claims abstract description 26
- 238000011218 seed culture Methods 0.000 claims abstract description 23
- 229920002261 Corn starch Polymers 0.000 claims abstract description 21
- 239000008120 corn starch Substances 0.000 claims abstract description 21
- 235000019750 Crude protein Nutrition 0.000 claims abstract description 18
- 239000002609 medium Substances 0.000 claims description 30
- 238000012258 culturing Methods 0.000 claims description 19
- 238000001514 detection method Methods 0.000 claims description 10
- 239000002054 inoculum Substances 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 8
- 230000000694 effects Effects 0.000 abstract description 3
- 238000011081 inoculation Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 7
- 208000012788 shakes Diseases 0.000 description 7
- 239000001888 Peptone Substances 0.000 description 6
- 108010080698 Peptones Proteins 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 244000046052 Phaseolus vulgaris Species 0.000 description 4
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 244000005700 microbiome Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 206010010144 Completed suicide Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a high-yield antibacterial peptide bacillus subtilis culture medium, which comprises a seed culture medium and a fermentation culture medium and is characterized in that the components of the culture medium and the contents of the culture medium in fermentation liquid are respectively as follows: 3-10g/L glucose, 15-25g/L corn starch, 15-25g/L soybean meal powder and 3-10g/L corn steep liquor dry powder. The invention adopts a fermentation culture medium containing four components of specific crude protein (dry basis) and specific content of soybean meal powder and corn steep liquor dry powder, and specific content of glucose and corn starch, takes specific strains for inoculation, adopts a liquid fermentation method under specific culture conditions, and the antibacterial peptide titer of the prepared fermentation broth is more than or equal to 14000IU/ml, which is improved by more than 3 times compared with the fermentation activity 4100IU/ml of the initial process, and the cost of the fermentation culture medium is reduced to 1/4 of the initial process.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a fermentation medium of high-yield antibacterial peptide bacillus subtilis and a liquid fermentation method.
Background
The antibacterial peptide is a small molecular polypeptide with biological activity which is induced in organisms, and has the characteristics of wide antibacterial spectrum, acid and alkali resistance, thermal stability and the like. The antibacterial peptide has wide application prospect in medicine, agriculture and industry, and various antibacterial polypeptides are being subjected to preclinical feasibility research.
However, the source of the antimicrobial peptide is a key issue limiting its development and application. The content of the antibacterial peptide in the animal body is very small, the yield of the antibacterial peptide extracted from the animal body is low, the time is long, the process is complex, the cost is high, and the large-scale production cannot be realized. Chemical synthesis and genetic engineering become the main means for obtaining antibacterial peptide, however, chemical synthesis of peptide has high cost, and direct expression of antibacterial peptide gene in microorganism may cause suicide of host microorganism and failure to obtain expression product. Therefore, how to realize industrialized production is a problem that must be solved by the application of antibacterial peptides.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the primary purpose of the invention is to provide a bacillus subtilis culture medium for high-yield antibacterial peptide.
The invention is realized by the following technical scheme:
the bacillus subtilis culture medium for high-yield antibacterial peptide comprises a seed culture medium and a fermentation culture medium, wherein the components of the culture medium and the contents of the culture medium in fermentation broth are respectively as follows: 3-10g/L glucose, 15-25g/L corn starch, 15-25g/L soybean meal powder and 3-10g/L corn steep liquor dry powder.
Wherein, the components of the culture medium and the contents of the culture medium in the fermentation broth are respectively as follows: glucose 5g/L, corn starch 20g/L, soybean meal 20g/L and corn steep liquor dry powder 5g/L.
Wherein, the crude protein (dry basis) of the soybean meal powder is larger than or equal to 50%; crude protein (dry basis) of the corn steep liquor dry powder is larger than or equal to 42%; adopting a culture medium containing high-content crude protein (dry basis) soybean meal, corn steep liquor dry powder and the like, wherein the antibacterial peptide titer of the prepared fermentation broth is more than or equal to 14000IU/ml; if the crude protein (dry basis) of the soybean meal and/or corn steep liquor dry powder is too low, the fermentation process is long in delay period and slow in thallus growth, and the activity of the metabolite antibacterial peptide is greatly affected.
A liquid fermentation method of a bacillus subtilis culture medium containing the high-yield antibacterial peptide comprises the following steps of:
1) Seed culture medium: 3-10g/L of glucose, 15-25g/L of corn starch, 15-25g/L of bean pulp powder and 3-10g/L of corn steep liquor dry powder, and regulating the pH to 6.8-7.2;
2) Preparing seed fermentation liquid: inoculating strain stored on solid culture medium into seed culture solution of 50ml/250ml (triangular flask), and culturing at 31-33deg.C and shaking table rotation speed of 150r/min for 12-15 hr;
3) Fermentation initial medium: 3-10g/L of glucose, 15-25g/L of corn starch, 15-25g/L of bean pulp powder and 3-10g/L of corn steep liquor dry powder, and regulating the pH to 6.8-7.2;
4) Fermentation culture process: inoculating the cultured seed fermentation broth into 100ml/500ml (triangular flask with baffle) fermentation initial culture medium, and culturing at 31-33deg.C and shaking table rotation speed 150r/min for 22-26 hr;
5) And (3) detection: and after fermentation, taking fermentation liquor, treating the fermentation liquor, and detecting antibacterial peptides in the fermentation liquor.
In the step 2), the strain is bacillus subtilis producing the antibacterial peptide, and is stored in a microorganism germplasm resource library in Guangdong province, and the strain number is GW-1-1601-1615-01.
Wherein the inoculation amount is 3-7% (v/v).
Wherein, in the step 5), the antibacterial peptide titer of the fermentation liquor is more than or equal to 14000 IU/ml.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, a fermentation medium containing four components of specific crude protein (dry basis) and specific content of soybean meal powder and corn steep liquor dry powder, and specific content of glucose and corn starch is adopted, specific strains are used for inoculation, and a liquid fermentation method with specific culture conditions is adopted, so that the antibacterial peptide titer of the prepared fermentation liquid is more than or equal to 14000IU/ml (the antibacterial peptide content is high, the optimized medium of the invention has better growth of bacillus subtilis and larger production of secondary metabolite thereof), the fermentation activity is improved by more than 3 times compared with 4100IU/ml of the initial process, and the cost of the fermentation medium is reduced to 1/4 of the initial process.
Detailed Description
The present invention will be further described by the following specific embodiments, which are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the following examples.
The method for measuring the potency of the antibacterial peptide comprises the following steps: a) Preparing a detection medium: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder, 5g/L of glucose, 20g/L of agar powder (added in the case of a solid culture medium) and pH value of 7.0; b) Preparation of an indication culture solution: coli (K) 12 D 31 ) Inoculating on inclined plane, culturing at 37deg.C for 16-24 hr, inoculating on LB liquid medium, shake culturing at 37deg.C for 16-18 hr, and regulating absorbance of cultured Escherichia coli bacteria liquid to OD with sterile water 600 Values of 2.00.+ -. 0.01 (with water as a blank); c) Melting detection culture medium (solid), cooling to 48-50deg.C, adding the above Escherichia coli bacterial liquid (100 ul per 100ml culture medium), shaking, sucking 10ml into culture dish (diameter of 90 mm), solidifying, and punching with puncher (diameter of 2.7 mm); d) Sample preparation: taking fermentation liquor to be detected, centrifuging, taking supernatant, adding 6 ul sample liquor to be detected into each hole of the perforated plate, repeating each sample for 3 times, culturing in a 28 ℃ incubator for 20 hours, measuring the diameter of a bacteriostasis ring by using a vernier caliper, and taking an average value; e) The calculation formula of the antibacterial peptide titer is as follows: IU (unit/ml) =2 X X1000X dilution x= (Y-2.7)/2.1, wherein Y: average diameter of inhibition zone (unit is millimeter); 2.7: a hole diameter constant; 2.1: the ratio constant of the concentration of the antibacterial peptide to the diameter of the inhibition zone.
The calculation results of the costs of the fermentation media of examples 1 to 3 and comparative examples 1 to 2, application example 1 and application comparative example 1 are shown in Table 1:
TABLE 1
Example 1
The bacillus subtilis culture medium for high-yield antibacterial peptide comprises a seed culture medium and a fermentation culture medium, wherein the components of the culture medium and the contents of the culture medium in fermentation broth are respectively as follows: glucose 5g/L, corn starch 20g/L, soybean meal 20g/L and corn steep liquor dry powder 5g/L; wherein, the crude protein (dry basis) of the soybean meal powder is 55%; the crude protein (dry basis) of the corn steep liquor dry powder is 45%;
a liquid fermentation method of a bacillus subtilis culture medium containing the high-yield antibacterial peptide comprises the following steps of:
1) Seed culture medium: glucose 5g/L, corn starch 20g/L, soybean meal 20g/L, corn steep liquor dry powder 5g/L, and adjusting pH to 7.0;
2) Preparing seed fermentation liquid: inoculating bacillus subtilis with high yield of antibacterial peptide stored on a solid culture medium into seed culture solution of 50ml/250ml (triangular flask), and culturing at 32deg.C and shaking table rotation speed of 150r/min for 13 hr;
3) Fermentation initial medium: glucose 5g/L, corn starch 20g/L, soybean meal 20g/L, corn steep liquor dry powder 5g/L, and adjusting pH to 7.0;
4) Fermentation culture process: inoculating the cultured seed fermentation broth into sterilized fermentation initial medium of 100ml/500ml (triangular flask with baffle plate) according to inoculum size of 5% (v/v), and culturing at 32deg.C and shaking table rotation speed of 150r/min for 24 hr;
5) And (3) detection: after the fermentation, the fermentation broth was taken, the antibacterial peptide of the fermentation broth was detected, and the cost of the fermentation medium was calculated, and the results are shown in table 2.
Example 2
The bacillus subtilis culture medium for high-yield antibacterial peptide comprises a seed culture medium and a fermentation culture medium, wherein the components of the culture medium and the contents of the culture medium in fermentation broth are respectively as follows: glucose 10g/L, corn starch 15g/L, soybean meal 25g/L and corn steep liquor dry powder 3g/L; wherein, the crude protein (dry basis) of the soybean meal powder is 50%; the crude protein (dry basis) of the corn steep liquor dry powder is 50%;
a liquid fermentation method of a bacillus subtilis culture medium containing the high-yield antibacterial peptide comprises the following steps of:
1) Seed culture medium: 10g/L of glucose, 15g/L of corn starch, 25g/L of soybean meal powder and 3g/L of corn steep liquor dry powder, and regulating the pH to 7.0;
2) Preparing seed fermentation liquid: inoculating bacillus subtilis with high yield of antibacterial peptide stored on a solid culture medium into seed culture solution of 50ml/250ml (triangular flask), and culturing at 31deg.C and shaking table rotation speed of 150r/min for 15 hr;
3) Fermentation initial medium: 10g/L of glucose, 15g/L of corn starch, 25g/L of soybean meal powder and 3g/L of corn steep liquor dry powder, and regulating the pH to 7.0;
4) Fermentation culture process: inoculating the cultured seed fermentation broth into sterilized fermentation initial medium of 100ml/500ml (triangular flask with baffle plate) according to inoculum size of 5% (v/v), and culturing at 31deg.C and shaking table rotation speed of 150r/min for 22 hr;
5) And (3) detection: after the fermentation, the fermentation broth was taken, the antibacterial peptide of the fermentation broth was detected, and the cost of the fermentation medium was calculated, and the results are shown in table 2.
Example 3
The bacillus subtilis culture medium for high-yield antibacterial peptide comprises a seed culture medium and a fermentation culture medium, wherein the components of the culture medium and the contents of the culture medium in fermentation broth are respectively as follows: glucose 5g/L, corn starch 25g/L, soybean meal 15g/L and corn steep liquor dry powder 10g/L; wherein, the crude protein (dry basis) of the soybean meal powder is 60%; the crude protein (dry basis) of the corn steep liquor dry powder is 55%;
a liquid fermentation method of a bacillus subtilis culture medium containing the high-yield antibacterial peptide comprises the following steps of:
1) Seed culture medium: glucose 5g/L, corn starch 25g/L, soybean meal 15g/L and corn steep liquor dry powder 10g/L, and adjusting the pH to 7.0;
2) Preparing seed fermentation liquid: inoculating bacillus subtilis with high yield of antibacterial peptide stored on a solid culture medium into seed culture solution of 50ml/250ml (triangular flask), and culturing at 33deg.C and shaking table rotation speed of 150r/min for 12 hr;
3) Fermentation initial medium: glucose 5g/L, corn starch 25g/L, soybean meal 15g/L and corn steep liquor dry powder 10g/L, and adjusting the pH to 7.0;
4) Fermentation culture process: inoculating the cultured seed fermentation broth into sterilized fermentation initial medium of 100ml/500ml (triangular flask with baffle plate) according to inoculum size of 5% (v/v), and culturing at 33deg.C and shaking table rotation speed of 150r/min for 26 hr;
5) And (3) detection: after the fermentation, the fermentation broth was taken, the antibacterial peptide of the fermentation broth was detected, and the cost of the fermentation medium was calculated, and the results are shown in table 2.
Application example 1: liquid fermentation method using 50L fermentation tank
A high yield antibacterial peptide bacillus subtilis culture medium, the same as in example 1;
a liquid fermentation method of a bacillus subtilis culture medium containing the high-yield antibacterial peptide comprises the following steps of:
1) Shake flask seed medium: 10g/L of glucose, 20g/L of corn starch, 20g/L of bean pulp powder and 5g/L of corn steep liquor dry powder, and regulating the pH to 7.0;
2) Preparing first-stage shake flask seed liquid: inoculating 1 loop of slant strain (bacillus subtilis with high yield of antibacterial peptide) with good growth into primary seed culture solution of 50ml/250ml (triangular flask), and culturing under the following conditions: the culture temperature is 31 ℃, the rotation speed of the shaking table is 150r/min, and the culture time is 13h;
3) Preparing a secondary shake flask seed liquid: the seed solution was inoculated into 200ml/1000ml (triangular flask) secondary seed culture medium at an inoculum size of 5% (10 ml), and culture conditions were: culturing at 31deg.C with shaking table rotation speed of 150r/min for 8-12 hr;
3) Fermentation initial medium: 10g/L of glucose, 20g/L of corn starch, 20g/L of bean pulp powder and 5g/L of corn steep liquor dry powder, and regulating the pH to 7.0;
4) Fermentation and culture process of 50L tank: inoculating the cultured seed fermentation broth into a fermentation initial medium according to an inoculum size of 4% (namely 1000 ml), and filling the liquid with 25L, wherein the culture conditions are as follows: the culture temperature is 31 ℃, the rotating speed is 200r/min, the ventilation rate is 1500L/min, the culture time is 24h, and the pH value in the fermentation process is natural;
5) And (3) detection: after the fermentation, the fermentation broth was taken, the antibacterial peptide of the fermentation broth was detected, and the cost of the fermentation medium was calculated, and the results are shown in table 2.
Comparative example 1
The bacillus subtilis culture medium comprises a seed culture medium and a fermentation culture medium, wherein the components of the culture medium and the contents of the culture medium in fermentation broth are respectively as follows: 20g/L of peptone, 10g/L of yeast powder and 20g/L of glucose;
a liquid fermentation method comprising the bacillus subtilis culture medium, comprising the following steps:
1) Seed culture medium: 20g/L peptone, 10g/L yeast powder and 20g/L glucose, and adjusting the pH to 7.0;
2) Preparing seed fermentation liquid: inoculating bacillus subtilis producing antibacterial peptide stored on a solid culture medium into 50ml/250ml (triangular flask) seed culture solution, and culturing at 32deg.C and shaking table rotation speed of 150r/min for 13 hr;
3) Fermentation initial medium: 20g/L peptone, 10g/L yeast powder and 20g/L glucose, and adjusting the pH to 7.0;
4) Fermentation culture process: inoculating the cultured seed fermentation broth into sterilized fermentation initial medium of 100ml/500ml (triangular flask with baffle plate) according to inoculum size of 5% (v/v), and culturing at 32deg.C and shaking table rotation speed of 150r/min for 24 hr;
5) And (3) detection: after the fermentation, the fermentation broth was taken, the antibacterial peptide of the fermentation broth was detected, and the cost of the fermentation medium was calculated, and the results are shown in table 2.
Comparative example 2
The bacillus subtilis culture medium for high-yield antibacterial peptide comprises a seed culture medium and a fermentation culture medium, wherein the components of the culture medium and the contents of the culture medium in fermentation broth are respectively as follows: glucose 10g/L, corn starch 20g/L, soybean meal 20g/L and corn steep liquor dry powder 5g/L; wherein, the crude protein (dry basis) of the soybean meal powder is 40%; the crude protein (dry basis) of the corn steep liquor dry powder is 35%;
the liquid fermentation method was the same as in example 1, and then the antibacterial peptide in the fermentation broth was detected, and the cost of the fermentation medium was calculated, and the results are shown in Table 2.
Comparative example 1 was applied: liquid fermentation method using 50L fermentation tank
A bacillus subtilis culture medium, as in comparative example 1;
a liquid fermentation method comprising the bacillus subtilis culture medium, comprising the following steps:
1) Shake flask seed medium: 20g/L peptone, 10g/L yeast powder and 20g/L glucose, and adjusting the pH to 7.0;
2) Preparing first-stage shake flask seed liquid: inoculating 1 loop of slant strain (bacillus subtilis with high yield of antibacterial peptide) with good growth into primary seed culture solution of 50ml/250ml (triangular flask), and culturing under the following conditions: the culture temperature is 31 ℃, the rotation speed of the shaking table is 150r/min, and the culture time is 13h;
3) Preparing a secondary shake flask seed liquid: the seed solution was inoculated into 200ml/1000ml (triangular flask) secondary seed culture medium at an inoculum size of 5% (10 ml), and culture conditions were: culturing at 31deg.C with shaking table rotation speed of 150r/min for 8-12 hr;
3) Fermentation initial medium: 20g/L peptone, 10g/L yeast powder and 20g/L glucose, and adjusting the pH to 7.0;
4) Fermentation and culture process of 50L tank: inoculating the cultured seed fermentation broth into a fermentation initial medium according to an inoculum size of 4% (namely 1000 ml), and filling the liquid with 25L, wherein the culture conditions are as follows: the culture temperature is 31 ℃, the rotating speed is 200r/min, the ventilation rate is 1500L/min, the culture time is 24h, and the pH value in the fermentation process is natural;
5) And (3) detection: after the fermentation, the fermentation broth was taken, the antibacterial peptide of the fermentation broth was detected, and the cost of the fermentation medium was calculated, and the results are shown in table 2.
TABLE 2
| Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Application example 1 | Comparative example 1 was used | |
| Antibacterial peptide titer (IU/ml) | 17642 | 15604 | 18477 | 4723 | 9462 | 20603 | 6050 |
| Cost of fermentation Medium (Yuan/1 ton liquid) | 237 | 254.3 | 253.5 | 1020 | 235 | 257 | 1020 |
Claims (2)
1. The liquid state fermentation method of the bacillus subtilis culture medium for high yield of the antibacterial peptide is characterized by comprising the following steps of:
1) Seed culture medium: glucose 5g/L, corn starch 20g/L, soybean meal 20g/L, corn steep liquor dry powder 5g/L, and adjusting pH to 6.8-7.2; the dry basis of crude protein of the soybean meal powder is larger than or equal to 50%; the dry basis of crude protein of the corn steep liquor dry powder is larger than or equal to 42%;
2) Preparing seed fermentation liquid: inoculating strain stored on solid culture medium into seed culture solution of 50ml/250ml triangular flask, and culturing at 31-33deg.C and shaking table rotation speed of 150r/min for 12-15 hr;
3) Fermentation initial medium: glucose 5g/L, corn starch 20g/L, soybean meal 20g/L, corn steep liquor dry powder 5g/L, and adjusting pH to 6.8-7.2; the dry basis of crude protein of the soybean meal powder is larger than or equal to 50%; the dry basis of crude protein of the corn steep liquor dry powder is larger than or equal to 42%;
4) Fermentation culture process: inoculating the cultured seed fermentation broth into a fermentation initial culture medium of a 100ml/500ml triangular flask with a baffle, and culturing at 31-33 ℃ and a shaking table rotation speed of 150r/min for 22-26h;
5) And (3) detection: after fermentation, taking fermentation liquor, treating the fermentation liquor, and detecting antibacterial peptide in the fermentation liquor;
the titer of the antibacterial peptide of the fermentation liquor is more than or equal to 14000 IU/ml.
2. The liquid fermentation process according to claim 1, wherein in step 4), the inoculum size is 3-7% (v/v).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910321647.7A CN110093393B (en) | 2019-04-22 | 2019-04-22 | High-yield antibacterial peptide bacillus subtilis culture medium and liquid fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910321647.7A CN110093393B (en) | 2019-04-22 | 2019-04-22 | High-yield antibacterial peptide bacillus subtilis culture medium and liquid fermentation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110093393A CN110093393A (en) | 2019-08-06 |
| CN110093393B true CN110093393B (en) | 2023-11-03 |
Family
ID=67445304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910321647.7A Active CN110093393B (en) | 2019-04-22 | 2019-04-22 | High-yield antibacterial peptide bacillus subtilis culture medium and liquid fermentation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110093393B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111607627A (en) * | 2020-05-12 | 2020-09-01 | 南京大方生物工程有限公司 | Fermentation process for producing antibacterial peptide bacillus subtilis and application of fermentation process in disease-resistant and yield-increasing of livestock and poultry |
| CN113430245A (en) * | 2021-08-03 | 2021-09-24 | 广州百仕肽生物科技有限公司 | Quantitative determination method for iturin antibacterial activity |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07267962A (en) * | 1994-03-30 | 1995-10-17 | Meiji Seika Kaisha Ltd | Novel antifungal substance pf-1140 and method for producing the same |
| CN103333937A (en) * | 2013-06-06 | 2013-10-02 | 徐州工程学院 | Technique for preparing antimicrobial peptide by using Bacillus subtilis |
| CN103865901A (en) * | 2014-04-08 | 2014-06-18 | 大连工业大学 | Fermentation culture medium for glucoamylase and glucoamylase fermentation method |
| CN103911396A (en) * | 2014-04-25 | 2014-07-09 | 山东仙普爱瑞科技股份有限公司 | Bacillus subtilis fermentation medium |
| CN104250660A (en) * | 2014-09-19 | 2014-12-31 | 中农颖泰林州生物科园有限公司 | Antibacterial peptide fermentation culture medium formula |
| CN104878060A (en) * | 2015-06-25 | 2015-09-02 | 湖南农业大学 | Bacillus subtilis culture medium for producing anti-microbial peptide and application thereof |
| CN106148457A (en) * | 2016-08-30 | 2016-11-23 | 林州中农颖泰生物肽有限公司 | A kind of antibacterial peptide fermentation medium prescription |
| CN107815431A (en) * | 2017-11-22 | 2018-03-20 | 山东京青农业科技有限公司 | A kind of method with water-soluble raw material culture bacillus subtilis |
| CN108821828A (en) * | 2018-06-26 | 2018-11-16 | 河南爵利生物技术有限公司 | A kind of preparation method using the fermentation tailing liquor production material of liquid fertilizer containing polyglutamic acid |
-
2019
- 2019-04-22 CN CN201910321647.7A patent/CN110093393B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07267962A (en) * | 1994-03-30 | 1995-10-17 | Meiji Seika Kaisha Ltd | Novel antifungal substance pf-1140 and method for producing the same |
| CN103333937A (en) * | 2013-06-06 | 2013-10-02 | 徐州工程学院 | Technique for preparing antimicrobial peptide by using Bacillus subtilis |
| CN103865901A (en) * | 2014-04-08 | 2014-06-18 | 大连工业大学 | Fermentation culture medium for glucoamylase and glucoamylase fermentation method |
| CN103911396A (en) * | 2014-04-25 | 2014-07-09 | 山东仙普爱瑞科技股份有限公司 | Bacillus subtilis fermentation medium |
| CN104250660A (en) * | 2014-09-19 | 2014-12-31 | 中农颖泰林州生物科园有限公司 | Antibacterial peptide fermentation culture medium formula |
| CN104878060A (en) * | 2015-06-25 | 2015-09-02 | 湖南农业大学 | Bacillus subtilis culture medium for producing anti-microbial peptide and application thereof |
| CN106148457A (en) * | 2016-08-30 | 2016-11-23 | 林州中农颖泰生物肽有限公司 | A kind of antibacterial peptide fermentation medium prescription |
| CN107815431A (en) * | 2017-11-22 | 2018-03-20 | 山东京青农业科技有限公司 | A kind of method with water-soluble raw material culture bacillus subtilis |
| CN108821828A (en) * | 2018-06-26 | 2018-11-16 | 河南爵利生物技术有限公司 | A kind of preparation method using the fermentation tailing liquor production material of liquid fertilizer containing polyglutamic acid |
Non-Patent Citations (3)
| Title |
|---|
| 曹丁 ; 黄魁英 ; 林创伟 ; 夏枫耿 ; 张高贤 ; 张玲华 ; .产抗菌肽芽孢杆菌的发酵工艺研究.安徽农业科学.2015,第43卷(第13期),参见第1页左栏第2段-第3页右栏第4段. * |
| 朱晓立 ; 俞巍蔚 ; .枯草芽孢杆菌产芽孢发酵培养基的优化研究.化工设计通讯.2016,第42卷(第06期),全文. * |
| 枯草芽孢杆菌ZC1高密度培养的条件优化;管国强;《中国调味品》;20170131;第42卷(第1期);摘要、第16页左栏2.2.3-2.2.4、图4-5、第17页2.5、图8、图10 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110093393A (en) | 2019-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xiao et al. | Statistical optimization of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate | |
| Adinarayana et al. | Optimization of process parameters for cephalosporin C production under solid state fermentation from Acremonium chrysogenum | |
| Wu et al. | Production of L-lactic acid by Rhizopus oryzae using semicontinuous fermentation in bioreactor | |
| El-Enshasy et al. | Improvement of erythromycin production by Saccharopolyspora erythraea in molasses based medium through cultivation medium optimization | |
| CN101831414B (en) | Process for extracellularly producing recombinant alpha-cyclodextrin glucosyltransferase | |
| CN110093393B (en) | High-yield antibacterial peptide bacillus subtilis culture medium and liquid fermentation method | |
| CN104928202A (en) | Fermentation culture method of bacillus | |
| WO2022262384A1 (en) | Method for increasing yield of spinosyns of saccharopolyspora spinosa | |
| CN103717746B (en) | Manufacture the method for isopropyl alcohol by Continuous Cultivation | |
| Meng et al. | Production of mannitol from a high concentration of glucose by Candida parapsilosis SK26. 001 | |
| CN111394280A (en) | Culture medium suitable for growth of bacillus licheniformis and application thereof | |
| CN101851591B (en) | Fermentation method for producing epothilone B by sorangium cellulosum and fermentation medium | |
| CN103525881B (en) | Method for producing L-aspartic acid converted from fumaric acid through Escherichia coli undergoing fermentation culture in large pot with volume of 20m<3> | |
| CN109234215A (en) | A kind of Lactobacillus rhamnosus less salt culture medium and cultural method | |
| CN103266160A (en) | Method for lowering down proportion of by-product produced in fermentation of demethylchlortetracycline | |
| CN102943101A (en) | Method for producing enramycin by fermenting | |
| CN111733119A (en) | High-density fermentation method of bacillus subtilis | |
| CN104450571A (en) | Bacillus thuringiensis strain for efficient degradation of fly larvae protein | |
| CN113293110B (en) | Preparation method of antibacterial lipopeptid compound | |
| CN109112171A (en) | A kind of preparation method of the antibacterial substance based on marine microorganism | |
| CN107723325B (en) | Fermentation production method of doramectin based on pH control | |
| CN110777096A (en) | Streptomyces capable of producing trypsin with high yield and application thereof | |
| CN102660493A (en) | Trichoderma sporulation medium and sporulation method | |
| CN112300954B (en) | Bacillus amyloliquefaciens and application thereof in fermentation production of glutaminase | |
| CN116004750B (en) | Method for producing angustin by using erwinia persicae |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 510663 1 Pinnacle Road, Luogang District, Guangzhou, Guangdong Applicant after: Guangzhou Institute of Microbiology Group Co.,Ltd. Address before: 510663 1 Pinnacle Road, Luogang District, Guangzhou, Guangdong Applicant before: GUANGDONG INSTITUTE OF MICROBIOLOGY |
|
| CB02 | Change of applicant information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |