CN110093393A - A kind of high yield antibacterial peptide bacillus subtilis bacterium culture medium and liquid state fermentation method - Google Patents
A kind of high yield antibacterial peptide bacillus subtilis bacterium culture medium and liquid state fermentation method Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 109
- 230000004151 fermentation Effects 0.000 title claims abstract description 109
- 239000007788 liquid Substances 0.000 title claims abstract description 75
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 54
- 239000001963 growth medium Substances 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 33
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 32
- 239000000843 powder Substances 0.000 claims abstract description 69
- 239000002609 medium Substances 0.000 claims abstract description 45
- 244000046052 Phaseolus vulgaris Species 0.000 claims abstract description 31
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims abstract description 31
- 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 29
- 239000008103 glucose Substances 0.000 claims abstract description 29
- 238000011218 seed culture Methods 0.000 claims abstract description 26
- 229920002261 Corn starch Polymers 0.000 claims abstract description 23
- 239000008120 corn starch Substances 0.000 claims abstract description 23
- 229940099112 cornstarch Drugs 0.000 claims abstract description 23
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 16
- 238000011534 incubation Methods 0.000 claims abstract description 15
- 239000004615 ingredient Substances 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims description 12
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 10
- 239000002054 inoculum Substances 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 8
- 239000012531 culture fluid Substances 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 239000000243 solution Substances 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
- 230000000052 comparative effect Effects 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- 230000000844 anti-bacterial effect Effects 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 230000003519 ventilatory effect Effects 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010010144 Completed suicide Diseases 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- FFBHFFJDDLITSX-UHFFFAOYSA-N benzyl N-[2-hydroxy-4-(3-oxomorpholin-4-yl)phenyl]carbamate Chemical compound OC1=C(NC(=O)OCC2=CC=CC=C2)C=CC(=C1)N1CCOCC1=O FFBHFFJDDLITSX-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- 239000012895 dilution Substances 0.000 description 1
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- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000020265 peanut milk Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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Abstract
The invention discloses a kind of high yield antibacterial peptide bacillus subtilis bacterium culture mediums, including seed culture medium and fermentation medium, it is characterized in that, the ingredient of the culture medium and the content in fermentation liquid are respectively as follows: glucose 3-10g/L, cornstarch 15-25g/L, bean cake powder 15-25g/L, Dried Corn Steep Liquor Powder 3-10g/L.The present invention by using bean cake powder and Dried Corn Steep Liquor Powder containing specific thick protein (butt) and certain content, the glucose of certain content and cornstarch totally four kinds of components fermentation medium, specific strain is taken to be inoculated with, and using the liquid state fermentation method of Incubation Condition, antibacterial peptide potency >=the 14000IU/ml for the fermentation liquid being prepared, 3 times or more are improved compared with initial process fermentation activity 4100IU/ml, and the cost of fermentation medium drops to the 1/4 of initial process.
Description
Technical field
The present invention relates to technical field of microbial fermentation more particularly to a kind of fermentations of high yield antibacterial peptide bacillus subtilis
Culture medium and liquid state fermentation method.
Background technique
Antibacterial peptide is a kind of biologically active micromolecule polypeptide generated in organism through induction, has antimicrobial spectrum
Extensively, acid and alkali-resistance and the features such as thermal stability.Antibacterial peptide has broad application prospects in medical, agricultural and industrially, has
A variety of antibacterial polypeptides are carrying out preclinical feasibility study.
But the source of antibacterial peptide is a critical issue for restricting its development and application.Antibacterial peptide content in animal body
It is atomic, low yield of antibacterial peptides, time-consuming length, complex process, somewhat expensive are extracted out of animal body, cannot achieve large-scale production.
Chemical synthesis and genetic engineering become the main means for obtaining antibacterial peptide, but chemically synthesized peptides, higher cost pass through gene
Engineering technology directly expresses antibacterial peptide gene in microorganism, may cause host microorganism suicide and cannot obtain expression and produce
Object.Therefore, how to realize that industrialization production is that antibacterial peptide application must solve the problems, such as.
Summary of the invention
In view of the defects existing in the prior art with deficiency, the primary purpose of the present invention is that it is withered to provide a kind of high yield antibacterial peptide
Careless bacillus culture medium.
The present invention is achieved by the following technical solutions:
A kind of high yield antibacterial peptide bacillus subtilis bacterium culture medium, including seed culture medium and fermentation medium, the culture medium
Ingredient and the content in fermentation liquid are respectively as follows: glucose 3-10g/L, cornstarch 15-25g/L, bean cake powder 15-25g/L, jade
Rice & peanut milk dry powder 3-10g/L.
Wherein, the ingredient of the culture medium and the content in fermentation liquid are respectively as follows: glucose 5g/L, cornstarch 20g/
L, bean cake powder 20g/L, Dried Corn Steep Liquor Powder 5g/L.
Wherein, thick protein (butt)≤50% of the bean cake powder；The Dried Corn Steep Liquor Powder thick protein (butt)≤
42%；Using the bean cake powder of the thick protein (butt) comprising high level and the culture medium of Dried Corn Steep Liquor Powder etc., it is prepared
Antibacterial peptide potency >=14000IU/ml of fermentation liquid；And if the bean cake powder used and/or the thick protein of Dried Corn Steep Liquor Powder are (dry
Base) it is too low, it will lead to that fermentation process lag phase is long, thalli growth is slow, the activity of metabolite antibacterial peptide is a greater impact.
A kind of liquid state fermentation method comprising above-mentioned high yield antibacterial peptide bacillus subtilis bacterium culture medium, includes the following steps:
1) seed culture medium: glucose 3-10g/L, cornstarch 15-25g/L, bean cake powder 15-25g/L, Dried Corn Steep Liquor Powder 3-
10g/L, adjusting pH are 6.8-7.2；
2) preparation of seed fermentation liquid: the strain being stored on solid medium of going bail for is seeded to 50ml/250ml(triangular flask)
In seed culture fluid, in 31-33 DEG C, shaking speed 150r/min, incubation time 12-15h；
3) ferment initial medium: glucose 3-10g/L, cornstarch 15-25g/L, bean cake powder 15-25g/L, Dried Corn Steep Liquor Powder
3-10g/L, adjusting pH are 6.8-7.2；
4) above-mentioned cultured seed fermentation liquid fermented and cultured process: is seeded to triangular flask of the 100ml/500ml(with baffle)
Fermentation initial medium in, in 31-33 DEG C, shaking speed 150r/min, incubation time 22-26h；
5) it detects: after fermentation, taking fermentation liquid, and handle fermentation liquid, then detect the antibacterial peptide in fermentation liquid.
Wherein, in step 2, the strain is the bacillus subtilis for producing antibacterial peptide, is stored in Guangdong Province's microorganism germ plasma
Resources bank, strain number GW-1-1601-1615-01.
Wherein, the inoculum concentration is 3-7%(v/v).
Wherein, in step 5), antibacterial peptide potency >=14000IU/ ml of the fermentation liquid.
Compared with prior art, the present invention having the following beneficial effects:
The present invention by using bean cake powder and Dried Corn Steep Liquor Powder containing specific thick protein (butt) and certain content, specific contain
The fermentation medium of the glucose and cornstarch of amount totally four kinds of components, takes specific strain to be inoculated with, and uses specific training
Antibacterial peptide potency >=14000IU/ml(antibacterial peptide content of the liquid state fermentation method for the condition of supporting, the fermentation liquid being prepared is high, says
Be illustrated the present invention optimization after culture medium to bacillus subtilis growth preferably and its cometabolism product yield compared with
Greatly), 3 times or more are improved compared with initial process fermentation activity 4100IU/ml, and the cost of fermentation medium drops to initial process
1/4。
Specific embodiment
Further illustrate that the present invention, following embodiment are the preferable embodiment party of the present invention below by specific embodiment
Formula, but embodiments of the present invention are not limited by following embodiments.
The measuring method of antibacterial peptide potency: A) detection culture medium preparation: sodium chloride 10g/L, peptone 10g/L, yeast powder
Added when 5g/L, glucose 5g/L, agar powder 20g/L(solid medium), pH value 7.0；B) instruction culture solution preparation: by large intestine
Bacillus (K12D31) be inoculated on inclined-plane, 37 degree of culture 16-24h choose a ring with oese and are seeded in LB liquid medium, and 37
DEG C shaking table culture 16-18 hours, cultured Escherichia coli bacteria liquid light absorption value is adjusted to OD with sterile water600Value for 2.00 ±
0.01(does blank control with water)；C it) will test after culture medium (solid) melts when it is cooled to 48-50 DEG C, addition is above-mentioned big
(100ul is added in every 100ml culture medium) in enterobacteria bacterium solution, draws in 10ml to culture dish (plate diameter 90mm) after shaking up,
Solidification is punched with punch (diameter 2.7mm)；D) sample preparation: fermentation liquid centrifuging and taking supernatant to be measured is taken, in above-mentioned punching
The sample liquid to be tested of 6 ul is added in every hole in plate, and each sample is repeated 3 times, and after cultivating 20h in 28 DEG C of incubators, uses vernier calliper
Ruler measures antibacterial circle diameter, is averaged；E) antibacterial peptide potency calculation formula: IU(unit/ml)=2X× 1000 × dilution times
Number, X=(y -2.7)/2.1, wherein Y: antibacterial circle diameter average value (unit is millimeter)；2.7: hole diameter constant；2.1: anti-
The ratio numerical constant of bacterium peptide concentration and antibacterial circle diameter.
Embodiment 1-3 and comparative example 1-2, Application Example 1 and Comparison study example 1 fermentation medium cost calculating
The results are shown in Table 1:
Table 1
Embodiment 1
A kind of high yield antibacterial peptide bacillus subtilis bacterium culture medium, including seed culture medium and fermentation medium, the culture medium
Ingredient and the content in fermentation liquid are respectively as follows: glucose 5g/L, cornstarch 20g/L, bean cake powder 20g/L, Dried Corn Steep Liquor Powder
5g/L；Wherein, the thick protein (butt) of the bean cake powder is 55%；The thick protein (butt) of the Dried Corn Steep Liquor Powder is
45%；
A kind of liquid state fermentation method comprising above-mentioned high yield antibacterial peptide bacillus subtilis bacterium culture medium, includes the following steps:
1) seed culture medium: glucose 5g/L, cornstarch 20g/L, bean cake powder 20g/L, Dried Corn Steep Liquor Powder 5g/L, adjusting pH is
7.0；
2) preparation of seed fermentation liquid: the bacillus subtilis for the high yield antibacterial peptide being stored on solid medium of going bail for is inoculated in
50ml/250ml(triangular flask) seed culture fluid in, in 32 DEG C, shaking speed 150r/min, incubation time 13h；
3) ferment initial medium: glucose 5g/L, cornstarch 20g/L, bean cake powder 20g/L, Dried Corn Steep Liquor Powder 5g/L are adjusted
PH is 7.0；
4) fermented and cultured process: by above-mentioned cultured seed fermentation liquid by 5%(v/v) inoculum concentration be seeded to and sterilized
Triangular flask of the 100ml/500ml(with baffle) fermentation initial medium in, in 32 DEG C, shaking speed 150r/min, culture when
Between for 24 hours；
5) it detects: after fermentation, taking fermentation liquid, detect the antibacterial peptide of fermentation liquid, and calculate the cost of fermentation medium, as a result
As shown in table 2.
Embodiment 2
A kind of high yield antibacterial peptide bacillus subtilis bacterium culture medium, including seed culture medium and fermentation medium, the culture medium
Ingredient and the content in fermentation liquid are respectively as follows: glucose 10g/L, cornstarch 15g/L, bean cake powder 25g/L, Dried Corn Steep Liquor Powder
3g/L；Wherein, the thick protein (butt) of the bean cake powder is 50%；The thick protein (butt) of the Dried Corn Steep Liquor Powder is
50%；
A kind of liquid state fermentation method comprising above-mentioned high yield antibacterial peptide bacillus subtilis bacterium culture medium, includes the following steps:
1) seed culture medium: glucose 10g/L, cornstarch 15g/L, bean cake powder 25g/L, Dried Corn Steep Liquor Powder 3g/L adjust pH
It is 7.0；
2) preparation of seed fermentation liquid: the bacillus subtilis for the high yield antibacterial peptide being stored on solid medium of going bail for is inoculated in
50ml/250ml(triangular flask) seed culture fluid in, in 31 DEG C, shaking speed 150r/min, incubation time 15h；
3) ferment initial medium: glucose 10g/L, cornstarch 15g/L, bean cake powder 25g/L, Dried Corn Steep Liquor Powder 3g/L are adjusted
Saving pH is 7.0；
4) fermented and cultured process: by above-mentioned cultured seed fermentation liquid by 5%(v/v) inoculum concentration be seeded to and sterilized
Triangular flask of the 100ml/500ml(with baffle) fermentation initial medium in, in 31 DEG C, shaking speed 150r/min, culture when
Between 22h；
5) it detects: after fermentation, taking fermentation liquid, detect the antibacterial peptide of fermentation liquid, and calculate the cost of fermentation medium, as a result
As shown in table 2.
Embodiment 3
A kind of high yield antibacterial peptide bacillus subtilis bacterium culture medium, including seed culture medium and fermentation medium, the culture medium
Ingredient and the content in fermentation liquid are respectively as follows: glucose 5g/L, cornstarch 25g/L, bean cake powder 15g/L, Dried Corn Steep Liquor Powder
10g/L；Wherein, the thick protein (butt) of the bean cake powder is 60%；The thick protein (butt) of the Dried Corn Steep Liquor Powder is
55%；
A kind of liquid state fermentation method comprising above-mentioned high yield antibacterial peptide bacillus subtilis bacterium culture medium, includes the following steps:
1) seed culture medium: glucose 5g/L, cornstarch 25g/L, bean cake powder 15g/L, Dried Corn Steep Liquor Powder 10g/L adjust pH
It is 7.0；
2) preparation of seed fermentation liquid: the bacillus subtilis for the high yield antibacterial peptide being stored on solid medium of going bail for is inoculated in
50ml/250ml(triangular flask) seed culture fluid in, in 33 DEG C, shaking speed 150r/min, incubation time 12h；
3) ferment initial medium: glucose 5g/L, cornstarch 25g/L, bean cake powder 15g/L, Dried Corn Steep Liquor Powder 10g/L are adjusted
Saving pH is 7.0；
4) fermented and cultured process: by above-mentioned cultured seed fermentation liquid by 5%(v/v) inoculum concentration be seeded to and sterilized
Triangular flask of the 100ml/500ml(with baffle) fermentation initial medium in, in 33 DEG C, shaking speed 150r/min, culture when
Between 26h；
5) it detects: after fermentation, taking fermentation liquid, detect the antibacterial peptide of fermentation liquid, and calculate the cost of fermentation medium, as a result
As shown in table 2.
Application Example 1: liquid state fermentation method is carried out using 50L fermentor
A kind of high yield antibacterial peptide bacillus subtilis bacterium culture medium, with embodiment 1；
A kind of liquid state fermentation method comprising above-mentioned high yield antibacterial peptide bacillus subtilis bacterium culture medium, includes the following steps:
1) shake-flask seed culture medium: glucose 10g/L, cornstarch 20g/L, bean cake powder 20g/L, Dried Corn Steep Liquor Powder 5g/L are adjusted
Saving pH is 7.0；
2) preparation of level-one shake-flask seed liquid: taking well-grown slant strains (bacillus subtilis of high yield antibacterial peptide) 1 ring,
Be inoculated in 50ml/250ml(triangular flask) first order seed culture solution in, condition of culture: 31 DEG C of cultivation temperature, shaking speed
150r/min, incubation time 13h；
3) preparation of second-level shake flask seed liquor: taking above-mentioned seed liquor, is seeded to 200ml/1000ml(tri- by 5% inoculum concentration (10ml)
Angle bottle) secondary seed culture solution in, condition of culture: 31 DEG C of cultivation temperature, shaking speed 150r/min, incubation time 8-12h；
3) ferment initial medium: glucose 10g/L, cornstarch 20g/L, bean cake powder 20g/L, Dried Corn Steep Liquor Powder 5g/L are adjusted
Saving pH is 7.0；
4) above-mentioned cultured seed fermentation liquid 50L tank fermented and cultured process: is seeded to hair by 4% inoculum concentration (i.e. 1000ml)
In ferment initial medium, liquid amount 25L, condition of culture: 31 DEG C of cultivation temperature, revolving speed 200r/min, ventilatory capacity 1500L/min,
Incubation time for 24 hours, fermentation process pH it is natural；
5) it detects: after fermentation, taking fermentation liquid, detect the antibacterial peptide of fermentation liquid, and calculate the cost of fermentation medium, as a result
As shown in table 2.
Comparative example 1
A kind of bacillus subtilis bacterium culture medium, including seed culture medium and fermentation medium, the ingredient of the culture medium and are sending out
Content in zymotic fluid is respectively as follows: peptone 20g/L, yeast powder 10g/L, glucose 20g/L；
A kind of liquid state fermentation method comprising above-mentioned bacillus subtilis bacterium culture medium, includes the following steps:
1) seed culture medium: peptone 20g/L, yeast powder 10g/L, glucose 20g/L, adjusting pH is 7.0；
2) preparation of seed fermentation liquid: the bacillus subtilis for the production antibacterial peptide being stored on solid medium of going bail for is inoculated in
50ml/250ml(triangular flask) seed culture fluid in, in 32 DEG C, shaking speed 150r/min, incubation time 13h；
3) ferment initial medium: peptone 20g/L, yeast powder 10g/L, glucose 20g/L, adjusting pH is 7.0；
4) fermented and cultured process: by above-mentioned cultured seed fermentation liquid by 5%(v/v) inoculum concentration be seeded to and sterilized
Triangular flask of the 100ml/500ml(with baffle) fermentation initial medium in, in 32 DEG C, shaking speed 150r/min, culture when
Between for 24 hours；
5) it detects: after fermentation, taking fermentation liquid, detect the antibacterial peptide of fermentation liquid, and calculate the cost of fermentation medium, as a result
As shown in table 2.
Comparative example 2
A kind of high yield antibacterial peptide bacillus subtilis bacterium culture medium, including seed culture medium and fermentation medium, the culture medium
Ingredient and the content in fermentation liquid are respectively as follows: glucose 10g/L, cornstarch 20g/L, bean cake powder 20g/L, Dried Corn Steep Liquor Powder
5g/L；Wherein, the thick protein (butt) of the bean cake powder is 40%；The thick protein (butt) of the Dried Corn Steep Liquor Powder is
35%；
Then liquid state fermentation method detects the antibacterial peptide in fermentation liquid with embodiment 1, and calculate the cost of fermentation medium, ties
Fruit is as shown in table 2.
Comparison study example 1: liquid state fermentation method is carried out using 50L fermentor
A kind of bacillus subtilis bacterium culture medium, with comparative example 1；
A kind of liquid state fermentation method comprising above-mentioned bacillus subtilis bacterium culture medium, includes the following steps:
1) shake-flask seed culture medium: peptone 20g/L, yeast powder 10g/L, glucose 20g/L, adjusting pH is 7.0；
2) preparation of level-one shake-flask seed liquid: taking well-grown slant strains (bacillus subtilis of high yield antibacterial peptide) 1 ring,
Be inoculated in 50ml/250ml(triangular flask) first order seed culture solution in, condition of culture: 31 DEG C of cultivation temperature, shaking speed
150r/min, incubation time 13h；
3) preparation of second-level shake flask seed liquor: taking above-mentioned seed liquor, is seeded to 200ml/1000ml(tri- by 5% inoculum concentration (10ml)
Angle bottle) secondary seed culture solution in, condition of culture: 31 DEG C of cultivation temperature, shaking speed 150r/min, incubation time 8-12h；
3) ferment initial medium: peptone 20g/L, yeast powder 10g/L, glucose 20g/L, adjusting pH is 7.0；
4) above-mentioned cultured seed fermentation liquid 50L tank fermented and cultured process: is seeded to hair by 4% inoculum concentration (i.e. 1000ml)
In ferment initial medium, liquid amount 25L, condition of culture: 31 DEG C of cultivation temperature, revolving speed 200r/min, ventilatory capacity 1500L/min,
Incubation time for 24 hours, fermentation process pH it is natural；
5) it detects: after fermentation, taking fermentation liquid, detect the antibacterial peptide of fermentation liquid, and calculate the cost of fermentation medium, as a result
As shown in table 2.
Table 2
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example 1 | Comparative example 2 | Application Example 1 | Comparison study example 1 | |
| Antibacterial peptide potency (IU/ml) | 17642 | 15604 | 18477 | 4723 | 9462 | 20603 | 6050 |
| Fermentation medium cost (member/1 ton of liquid) | 237 | 254.3 | 253.5 | 1020 | 235 | 257 | 1020 |
Claims (7)
1. a kind of high yield antibacterial peptide bacillus subtilis bacterium culture medium, including seed culture medium and fermentation medium, which is characterized in that
The ingredient of the culture medium and the content in fermentation liquid are respectively as follows: glucose 3-10g/L, cornstarch 15-25g/L, dregs of beans
Powder 15-25g/L, Dried Corn Steep Liquor Powder 3-10g/L.
2. the high yield antibacterial peptide bacillus subtilis bacterium culture medium according to claim 1, which is characterized in that the culture medium
Ingredient and the content in fermentation liquid to be respectively as follows: glucose 5g/L, cornstarch 20g/L, bean cake powder 20g/L, corn pulp dry
Powder 5g/L.
3. according to claim 1 or high yield antibacterial peptide bacillus subtilis bacterium culture medium described in 2, which is characterized in that the dregs of beans
Thick protein (butt)≤50% of powder；Thick protein (butt)≤42% of the Dried Corn Steep Liquor Powder.
4. a kind of liquid state fermentation side comprising the described in any item high yield antibacterial peptide bacillus subtilis bacterium culture mediums of claim 1-3
Method, which comprises the steps of:
1) seed culture medium: glucose 3-10g/L, cornstarch 15-25g/L, bean cake powder 15-25g/L, Dried Corn Steep Liquor Powder 3-
10g/L, adjusting pH are 6.8-7.2；
2) preparation of seed fermentation liquid: the strain being stored on solid medium of going bail for is seeded to 50ml/250ml(triangular flask)
In seed culture fluid, in 31-33 DEG C, shaking speed 150r/min, incubation time 12-15h；
3) ferment initial medium: glucose 3-10g/L, cornstarch 15-25g/L, bean cake powder 15-25g/L, Dried Corn Steep Liquor Powder
3-10g/L, adjusting pH are 6.8-7.2；
4) above-mentioned cultured seed fermentation liquid fermented and cultured process: is seeded to triangular flask of the 100ml/500ml(with baffle)
Fermentation initial medium in, in 31-33 DEG C, shaking speed 150r/min, incubation time 22-26h；
5) it detects: after fermentation, taking fermentation liquid, and handle fermentation liquid, then detect the antibacterial peptide in fermentation liquid.
5. the liquid state fermentation method according to claim 4, which is characterized in that in step 2, the strain is to produce antibacterial peptide
Bacillus subtilis, be stored in Guangdong Province's microorganism germ plasma resources bank, strain number GW-1-1601-1615-01.
6. the liquid state fermentation method according to claim 4, which is characterized in that in step 4), the inoculum concentration is 3-7%
(v/v).
7. the liquid state fermentation method according to claim 4, which is characterized in that in step 5), the antibacterial peptide of the fermentation liquid
Potency >=14000IU/ ml.
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