CN109161574A - A kind of fermentation process improving bacillus subtilis antibacterial protein yield - Google Patents
A kind of fermentation process improving bacillus subtilis antibacterial protein yield Download PDFInfo
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- CN109161574A CN109161574A CN201811088861.4A CN201811088861A CN109161574A CN 109161574 A CN109161574 A CN 109161574A CN 201811088861 A CN201811088861 A CN 201811088861A CN 109161574 A CN109161574 A CN 109161574A
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Abstract
The invention discloses a kind of fermentation process for improving bacillus subtilis antibacterial protein yield, first obtain strain, then the strain that will acquire, which is successively inoculated into seed culture medium, carries out fermentation in fermentation medium obtains bacillus subtilis antibacterial protein, starts rear preceding 6~16 hours logarithmic growth phases according to thallus fermentation and carries out segmentation adjustment according to fermentation time to the stationary phase upgrowth situation of fermentation ends after thallus fermentation beginning 16 hours.Regulate and control strain inoculum concentration before 189983 antibacterial protein fermentation process of bacillus subtilis BNCC, the temperature of adjustment and control system, dissolved oxygen amount, pH value, shaking speed in fermentation process, it can enable the growth of earlier fermentation strain fast and stable by the adjustment of the above parameter, fermentation middle and later periods strain is capable of the production antibacterial protein of efficient stable, reduces the generation of other by-products.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of fermentation for improving bacillus subtilis antibacterial protein yield
Method.
Background technique
Bacillus can produce various bacteriostasis antibiosis substance antagonistic phytopathogens as a kind of important biocontrol microorganisms,
The main source of potentiality and biological control with controlling plant diseases.
Wherein bacillus subtilis is that a kind of nature is widely present, is nontoxic to people and animals, free from environmental pollution, to plant
Disease has the non-pathogenic bacillus of inhibiting effect.Currently, many scientific research personnel are to bacillus subtilis and other gemma
The cause of disease antagonism and its antibacterial Quality Research of bacillus are very extensive.Bacillus can generate a variety of antibacterial substance prevention and treatments
Disease, including bacteriocin, cell wall degradation enzyme, antibacterial protein, rouge skin class antibiotic, polyketides and more skin class chemical combination
Object etc..
Wherein antibacterial protein is the large molecular weight protein substance with antifungal activity, and amino acid residue is usually 50
More than a, molecular weight is greater than 5kDa.The antibacterial protein that bacillus category biocontrol bacteria generates is studied, on the one hand can be fermented
Production forms biological pesticide and is used for controlling plant diseases, and substitution or reduction chemical pesticide use；Another aspect bacillus subtilis
It is verified that the proliferation of colorectal cancer cells can be inhibited, cause cell-cycle arrest and promote Apoptosis, and it is expected to inciting somebody to action
To become a kind of probiotics for preventing intestinal canal tumour generation.The raw levulan of certain producing bacillus subtilis can also promote tumour thin
The growth of acetic acid, alanine, lactic acid and phosphoric acid in born of the same parents changes the biological energy source way of tumour cell by intracellular lactic acid accumulation
Diameter and cell homeostasis, to show antitumor activity.Also some researches show that bacillus subtilis ZA400 with very high
Plasmin activity and can in the expression system of Escherichia coli clonal expression, show that it may be applied in thrombus treatment.
Although there is also many problems in application for current bacillus subtilis, if industrial application is still immature, commodity
The drug of change is not promoted completely, and its numerous characteristics and mechanism of action are still not clear.But as people are to bacillus subtilis
Gradually deeply, it is more clear to understand it for the research of bacterium, it is believed that in the near future, the application of bacillus subtilis is fixed not to be terminated in
This.
Summary of the invention
Technical problem to be solved by the present invention lies in for the research predicament mentioned in above-mentioned background technique, one kind is provided
The fermentation process for improving bacillus subtilis antibacterial protein yield, it is anti-for efficiently producing bacillus subtilis BNCC 189983
Mycoprotein.
The invention adopts the following technical scheme:
A kind of fermentation process improving bacillus subtilis antibacterial protein yield, comprising the following steps:
Bacillus subtilis bacterial strain is first obtained, then strain inoculated is cultivated into seed culture medium, obtains seed liquor, then will
Seed liquor, which is inoculated into fermentation medium, ferments, and carries out segmentation regulation fermentation by logarithmic growth phase and stationary phase during the fermentation
Temperature, dissolved oxygen amount, pH value and the shaking speed of system, final post-processing obtain bacillus subtilis antibacterial protein product.
It is further improved as present aspect, control strain inoculum concentration is 2~6%, preferably 4%.
It is further improved as present aspect, the culture specific steps being inoculated into seed culture medium are as follows:
Bacillus subtilis bacterial strain is inoculated in inclined-plane, is cultivated in 36~38 DEG C of constant incubators, then by inclined-plane culture
Bacterial strain after activating on base is prepared into bacteria suspension, is inoculated in seed culture medium, in 36~38 DEG C, 200~220r/min
Obtain seed liquor within shake culture 12~16 hours on shaking table.
It is further improved as present aspect, according to mass percent meter, the seed culture medium includes glucose 1.0%,
Peptone 1.0%, NaCl 1.0%, KH2PO40.15%, MgSO4·7H2The water of O 0.15% and surplus；PH value is 7.0.
(being equivalent to water herein is 100 parts, and the accounting of various substances is mass ratio, remaining is water, similarly hereinafter.)
As present aspect be further improved, the fermentation specific steps are as follows: by seed liquor according to volume ratio be (4:100)~
(8:100) ratio is inoculated into fermentation medium, carries out the antibacterial protein fermentation of bacillus subtilis.
It is further improved as present aspect, according to mass percent meter, the fermentation medium includes glucose 1.0%,
Peptone 1.5%, yeast extract 1.5%, KH2PO40.1%, NaCl 0.5%, MgSO4·7H2O 0.15% and surplus
Water；Initial pH value is 7.0,0.1MPa high pressure sterilization 15min.
It is further improved as present aspect, fermentation system temperature is 36~38 DEG C in logarithmic growth phase, and dissolved oxygen amount is set as
60%~80%, pH value is set as 6.9~7.1, and shaking speed is set as 180r/min~240r/min.
It is further improved as present aspect, fermentation system temperature is adjusted to 34~36 DEG C in stationary phase, and dissolved oxygen amount is adjusted to
40%~50%, pH value is adjusted to 6.6~6.8, and shaking speed is adjusted to 220r/min~250r/min.
It is further improved as present aspect, the pH value of fermentation system is adjusted with lactic acid and NaOH.
It is further improved as present aspect, the bacillus subtilis is 18998 bacillus subtilis of BNCC.
By compared with prior art, the invention has the advantages that
The present invention passes through to the growth of fermentation of bacillus subtilis process and production bacillus subtilis antibacterial protein rule
Further investigation, propose the fermentation process for improving bacillus subtilis antibacterial protein yield, including obtain strain and withered grass bud
Spore bacillus antibacterial protein fermentation, wherein bacillus subtilis antibacterial protein fermentation process is that will cultivate the bacterium after 12~16 hours
Kind of culture solution is inoculated into fermentation medium according to the ratio that volume ratio is 4:100~8:100 to be made fermentation system and starts withered grass
The fermentation of bacillus antibacterial protein；Temporally segmentation regulates and controls fermentation system in the fermentation process of bacillus subtilis antibacterial protein
Temperature, dissolved oxygen, pH value and shaking speed, earlier fermentation strain fast and stable can be enable by the adjustment of the above parameter
Growth, fermentation middle and later periods strain are capable of the production bacillus subtilis antibacterial protein of efficient stable, reduce the generation of other by-products.
Further, seed culture based component described in present invention process includes glucose 1.0%, peptone 1.0%, NaCl
1.0%, KH2PO40.15%, MgSO4·7H2O 0.15%, the pH value of the seed culture medium is 7.0, in this nutritional ingredient
Under (according to mass percent meter) ratio, faster, the vigor of strain reaches the growth and breeding of strain in seed liquor used in the fermentation of preparation
To highest.
Further, present invention process according to the growth for producing bacillus subtilis antibacterial protein strain and produces element rule, mentions
Go out Discrete control Fermentation Process of Parameter, it is effective to produce element for strain early growth and later period and provide good condition, very greatly
The production level of bacillus subtilis antibacterial protein, and bacillus subtilis antibacterial protein stable yield are improved in degree,
Fermentation medium nutrient formulation primary raw material is cheap and easy to get, saves cost, is suitble to industrial scale production.
Further, the ingredient of present invention process strain fermentation culture medium is glucose 1.0%, peptone 1.5%, yeast
Soak powder 1.5%, KH2PO40.1%, NaCl 0.5%, MgSO4·7H2O 0.15%；Initial pH value 7.0,0.1MPa high pressure are gone out
Bacterium 15min.Under this nutritional ingredient (according to mass percent meter) ratio, bacillus subtilis metabolic secretion bacillus subtilis
The vigor of 189983 antibacterial protein of BNCC is stronger, and yield is higher.
Detailed description of the invention
Fig. 1 is that the product bacillus subtilis antibacterial protein to be fermented using this method causes staphylococcus aureus growth
The schematic diagram one of influence；
Fig. 2 is that the product bacillus subtilis antibacterial protein to be fermented using this method causes staphylococcus aureus growth
The schematic diagram two of influence.
Specific embodiment
A kind of fermentation process for improving bacillus subtilis antibacterial protein yield of the present invention, including step are as follows:
Strain is first obtained, the strain that then will acquire, which is inoculated into fermentation medium, carries out fermentation acquisition bacillus subtilis
Antibacterial protein starts rear preceding 6~16 hours logarithmic growth phases according to thallus fermentation and extremely sends out after thallus fermentation beginning 16 hours
The stationary phase upgrowth situation that ferment terminates carries out segmentation adjustment according to fermentation time: in bacillus subtilis antibacterial protein fermentation process
Preceding regulation strain inoculum concentration, temperature, dissolved oxygen amount, pH value, the shaking speed of adjustment and control system, pass through the above parameter in fermentation process
Adjustment can enable the growth of earlier fermentation strain fast and stable, and fermentation middle and later periods strain is capable of the production antibacterial egg of efficient stable
It is white, reduce the generation of other by-products.
Specific fermentation process are as follows:
S1, purchase Bacillus subtilis strain, number is BNCC 189983, creates the connection limited public affairs of biotechnology in Bei Na
Department (BNCC) biology is saved.
S2,189983 bacillus subtilis of fermenting microbe BNCC is inoculated into nutritional ingredient (according to mass percent meter)
For glucose 1.0%, peptone 1.0%, NaCl 1.0%, KH2PO40.15%, MgSO4·7H2O 0.15%, surplus
Water cultivates 12~16 hours to obtain seed liquor in seed culture medium, and cultivation temperature is 37 DEG C, initial pH value of medium 7.0.
S3, by produce 189983 antibacterial protein of bacillus subtilis BNCC strain seed liquor according to volume ratio be 4:100~
8:100 ratio, which is inoculated into fermentation medium, carries out the fermentation of 189983 antibacterial protein of bacillus subtilis BNCC.The wherein hair
Ferment medium nutrient content be (according to mass percent meter) glucose 1.0%, peptone 1.5%, yeast extract 1.5%,
KH2PO4 0.1%, NaCl 0.5%, MgSO4·7H2O 0.15%；Initial pH value 7.0,0.1MPa high pressure sterilization 15min.
The temperature of adjustment and control system, dissolved oxygen amount, pH value, shaking speed in fermentation process specifically:
(1) fermentation start after before dissolved oxygen amount is set as 60%~80% in fermentation system in 6~16 hours, fermentation is opened
After beginning 16 hours into fermentation ends fermentation system dissolved oxygen amount be adjusted to 40%~50%.
(2) after fermentation starts before pH value is set as 6.9~7.1 in fermentation system in 6~16 hours, after fermentation starts
16 hours into fermentation ends fermentation system pH value be adjusted to 6.6~6.8, (adjust fermentation system with lactic acid and 5mol/L NaOH
PH value).
(3) after fermentation starts before in 6~16 hours shaking speed be set as 180~220r/min, after fermentation starts
220~240r/min is adjusted to fermentation ends shaking speed within 16 hours.
(4) after fermentation starts before in 6~16 hours fermentation system temperature be adjusted to 36~38 DEG C, fermentation start after 16
Hour to fermentation ends fermentation system temperature is adjusted to 34~36 DEG C.
S4, fermentation liquid is concentrated using Rotary Evaporators under conditions of 0.1MPa, 45 DEG C after fermentation, 80% ethyl alcohol
Precipitating is dialysed desalination 24 hours, and Sephadex G-50 gel-purified is freeze-dried 20 hours, obtains powdered bacillus subtilis
189983 antibacterial protein product of BNCC.
S5, the quality for weighing antibacterial protein, pass through the antibacterial activity of biocidal property experimental verification antibacterial protein.
Method of the invention is illustrated below in conjunction with specific embodiment:
Embodiment 1
S1, purchase Bacillus subtilis strain, number is BNCC 189983, for the Bei Na wound connection limited public affairs of biotechnology
Department (BNCC) biology is saved.Bacillus subtilis bacterial strain is inoculated in inclined-plane, is cultivated in 37 DEG C of constant incubators, it then will be oblique
On the culture medium of face activate after bacterial strain be prepared into concentration be 5000000/ml bacteria suspension, according to 4% inoculum concentration be inoculated in
In fresh seed culture medium, in shake culture on 36 DEG C, the shaking table of 180r/min.
S2,189983 bacillus subtilis of fermenting microbe BNCC is inoculated into nutritional ingredient (according to mass percent meter)
For glucose 1.0%, peptone 1.0%, NaCl 1.0%, KH2PO40.15%, MgSO4·7H2O 0.15%, the water of surplus
Seed culture medium in cultivate 12 hours seed liquor, cultivation temperature be 36 DEG C, initial pH value of medium 7.0.
S3, by produce 189983 antibacterial protein of bacillus subtilis BNCC strain seed liquor according to volume ratio be 4:100 ratio
Example, which is inoculated into fermentation medium, carries out the fermentation of 189983 antibacterial protein of bacillus subtilis BNCC.The wherein fermented and cultured
Base nutritional ingredient (according to mass percent meter) is glucose 1.0%, peptone 1.5%, yeast extract 1.5%, KH2PO4
0.1%, NaCl 0.5%, MgSO4·7H2O 0.15%；Initial pH value 7.0,0.1MPa high pressure sterilization 15min.
(1) after fermentation starts before dissolved oxygen amount is set as 60% in fermentation system in 6~16 hours, fermentation start after 16
Hour dissolved oxygen amount into fermentation ends fermentation system is adjusted to 40%.
(2) after fermentation starts before pH value is set as 6.9 in fermentation system in 6~16 hours, 16 is small after fermentation starts
6.6 are adjusted to up to pH value in fermentation ends fermentation system.
(3) after fermentation starts before in 6~16 hours shaking speed be set as 180r/min, fermentation start after 16 hours
220r/min is adjusted to fermentation ends shaking speed.
(4) after fermentation starts before in 6~16 hours fermentation system temperature be adjusted to 36 DEG C, fermentation start after 16 hours
34 DEG C are adjusted to fermentation ends fermentation system temperature.
S4, fermentation liquid is concentrated under conditions of 0.1MPa, 45 DEG C using Rotary Evaporators after fermentation within 54 hours,
80% ethanol precipitation is dialysed desalination 24 hours, and Sephadex G-50 gel-purified is freeze-dried 20 hours, obtains powdered withered grass
189983 antibacterial protein product of bacillus BNCC.
S5, the quality for weighing antibacterial protein, calculating 189983 antibacterial protein yield of bacillus subtilis BNCC is
14.26g/L passes through the antibacterial activity of biocidal property experimental verification antibacterial protein.
Embodiment 2
S1, purchase Bacillus subtilis strain, number is BNCC 189983, for the Bei Na wound connection limited public affairs of biotechnology
Department (BNCC) biology is saved.Bacillus subtilis bacterial strain is inoculated in inclined-plane, is cultivated in 37 DEG C of constant incubators, it then will be oblique
On the culture medium of face activate after bacterial strain be prepared into concentration be 5000000/ml bacteria suspension, according to 4% inoculum concentration be inoculated in
In fresh seed culture medium, in shake culture on 37 DEG C, the shaking table of 200r/min.
S2,189983 bacillus subtilis of fermenting microbe BNCC is inoculated into nutritional ingredient (according to mass percent meter)
For glucose 1.0%, peptone 1.0%, NaCl 1.0%, KH2PO40.15%, MgSO4·7H2O 0.15%, the water of surplus
Seed culture medium in cultivate 14 hours seed liquor, cultivation temperature be 37 DEG C, initial pH value of medium 7.0.
S3, by produce 189983 antibacterial protein of bacillus subtilis BNCC strain seed liquor according to volume ratio be 6:100 ratio
Example, which is inoculated into fermentation medium, carries out the fermentation of 189983 antibacterial protein of bacillus subtilis BNCC.The wherein fermented and cultured
Base nutritional ingredient (according to mass percent meter) is glucose 1.0%, peptone 1.5%, yeast extract 1.5%, KH2PO4
0.1%, NaCl 0.5%, MgSO4·7H2O 0.15%, the water of surplus；Initial pH7.0,0.1MPa high pressure sterilization 15min.
(1) after fermentation starts before dissolved oxygen amount is set as 70% in fermentation system in 6~16 hours, fermentation start after 16
Hour dissolved oxygen amount into fermentation ends fermentation system is adjusted to 45%.
(2) after fermentation starts before pH value is set as 7.0 in fermentation system in 6~16 hours, 16 is small after fermentation starts
6.7 are adjusted to up to pH value in fermentation ends fermentation system.
(3) after fermentation starts before in 6~16 hours shaking speed be set as 200r/min, fermentation start after 16 hours
230r/min is adjusted to fermentation ends shaking speed.
(4) after fermentation starts before in 6~16 hours fermentation system temperature be adjusted to 37 DEG C, fermentation start after 16 hours
35 DEG C are adjusted to fermentation ends fermentation system temperature.
S4, fermentation liquid is concentrated under conditions of 0.1MPa, 45 DEG C using Rotary Evaporators after fermentation within 56 hours,
80% ethanol precipitation is dialysed desalination 24 hours, and Sephadex G-50 gel-purified is freeze-dried 20 hours, obtains powdered withered grass
189983 antibacterial protein product of bacillus BNCC.
S5, the quality for weighing antibacterial protein, calculating 189983 antibacterial protein Product yields of bacillus subtilis BNCC is
15.51g/L passes through the antibacterial activity of biocidal property experimental verification antibacterial protein.
Embodiment 3
S1, purchase Bacillus subtilis strain, number is BNCC 189983, for the Bei Na wound connection limited public affairs of biotechnology
Department (BNCC) biology is saved.Bacillus subtilis bacterial strain is inoculated in inclined-plane, is cultivated in 37 DEG C of constant incubators, it then will be oblique
On the culture medium of face activate after bacterial strain be prepared into concentration be 5000000/ml bacteria suspension, according to 4% inoculum concentration be inoculated in
In fresh seed culture medium, in shake culture on 38 DEG C, the shaking table of 220r/min.
S2,189983 bacillus subtilis of fermenting microbe BNCC is inoculated into nutritional ingredient (according to mass percent meter)
For glucose 1.0%, peptone 1.0%, NaCl 1.0%, KH2PO40.15%, MgSO4·7H2O 0.15%, the water of surplus
Seed culture medium in cultivate 16 hours seed liquor, cultivation temperature be 37 DEG C, initial pH value of medium 7.0.
S3, by produce 189983 antibacterial protein of bacillus subtilis BNCC strain seed liquor according to volume ratio be 8:100 ratio
Example, which is inoculated into fermentation medium, carries out the fermentation of 189983 antibacterial protein of bacillus subtilis BNCC.The wherein fermented and cultured
Base nutritional ingredient (according to mass percent meter) is glucose 1.0%, peptone 1.5%, yeast extract 1.5%, KH2PO4
0.1%, NaCl 0.5%, MgSO4·7H2O 0.15%, the water of surplus；Initial pH value 7.0,0.1MPa high pressure sterilization 15min.
(1) after fermentation starts before dissolved oxygen amount is set as 80% in fermentation system in 6~16 hours, fermentation start after 16
Hour dissolved oxygen amount into fermentation ends fermentation system is adjusted to 50%.
(2) after fermentation starts before pH value is set as 7.1 in fermentation system in 6~16 hours, 16 is small after fermentation starts
6.8 are adjusted to up to pH value in fermentation ends fermentation system.
(3) after fermentation starts before in 6~16 hours shaking speed be set as 240r/min, fermentation start after 16 hours
240r/min is adjusted to fermentation ends shaking speed.
(4) after fermentation starts before in 6~16 hours fermentation system temperature be adjusted to 38 DEG C, fermentation start after 16 hours
36 DEG C are adjusted to fermentation ends fermentation system temperature.
S4, fermentation liquid is concentrated under conditions of 0.1MPa, 45 DEG C using Rotary Evaporators after fermentation within 58 hours,
80% ethanol precipitation is dialysed desalination 24 hours, and Sephadex G-50 gel-purified is freeze-dried 20 hours, obtains powdered withered grass
189983 antibacterial protein product of bacillus BNCC.
S5, the quality for weighing antibacterial protein, calculating 189983 antibacterial protein yield of bacillus subtilis BNCC is
13.96g/L passes through the antibacterial activity of biocidal property experimental verification antibacterial protein.
According to three above embodiment, the following table 1 is obtained:
1 fermentation process yield of table and traditional fermentation methods Yield comparison
This measurement method is that the fermentation liquid of fermentation ends is dense under conditions of 0.1MPa, 45 DEG C using Rotary Evaporators
Contracting, 80% ethanol precipitation are dialysed desalination 24 hours, and Sephadex G-50 gel-purified is freeze-dried 20 hours, obtains powdered
189983 antibacterial protein product of bacillus subtilis BNCC, weighs and calculates yield.Comparing can obtain, this fermentation process can be effective
The yield of ground raising bacillus subtilis BNCC189983 antibacterial protein.
The antibacterial effect of 2 tunning antibacterial proteins of table
This measurement uses staphylococcus aureus as experimental subjects, using LB culture medium as assay medium, to beat
The bacteriostatic diameter of hole method measurement antibacterial protein.LB culture medium is by peptone, yeast extract, NaCl composition.The advantage is that: peptone
It is by the processed albumen of pancreatin, after processing, the molecular weight of albumen of macromolecular becomes smaller, and bacterium is easier to utilize；In addition, also containing
There is purine, growth factor is provided.Peptone provides nitrogen source for the growth of bacterium.Secondly, the yeast extract yeast in culture medium extracts
Object main component is amino acid, and small peptide, nucleic acid, it is thin that b family vitamin, predominantly bacterium, which provide vitamin and microelement,
Necessary to bacterium growth.The effect of sodium chloride in the medium is to maintain osmotic pressure, and can provide inorganic salts.
The bacteriostatic diameter of antibacterial protein compares under 3 different pH condition of table
| PH value | Antibacterial circle diameter (mm) |
| 3 | 8.36 |
| 4 | 15.42 |
| 5 | 17.55 |
| 6 | 19.00 |
| 7(CK) | 20.17 |
| 8 | 18..43 |
| 9 | 15.59 |
| 10 | 13.27. |
| 11 | 6.48 |
By tunning antibacterial protein solution lactic acid and 5mol/L sodium hydroxide be adjusted to respectively pH value 3.0,4.0,5.0,
6.0,7.0,8.0,9.0,10.0,11.0, set 4 C overnights, with lactic acid and sodium hydroxide by each pH value recall to after neutrality with
Staphylococcus aureus is indicator bacteria, detects product bacteriostatic activity using punch method.
Experimental result is as shown in table 3, and pH value is between 4~10, active kept stable；When being 7, bacteriostatic activity
Highest, its bacteriostatic activity is higher when being 6~8.The most suitable range of 189983 antibacterial protein of bacillus subtilis BNCC be 6.5~
7.5.189983 antibacterial protein of bacillus subtilis BNCC is wider to the tolerance range of soda acid.
The expression activitiy of antibacterial protein after the different Protease Treatments of table 4
| Antibacterial circle diameter (mm) | |
| Blank control | 20.17 |
| Pepsin | 7.42 |
| Trypsase | 18.47 |
| Alkali protease | 17.65 |
| Chymotrypsin | 9.12 |
189983 antibacterial protein of bacillus subtilis BNCC that the fermentation of embodiment 2 generates is used into pepsin, pancreas egg respectively
It is instruction with staphylococcus aureus after white enzyme, alkali protease, chymotrypsin are handled under the conditions of respectively most suitable enzymatic
Bacterium detects product bacteriostatic activity using punch method.
Using untreated antibacterial protein as control, is shown according to table and show bacillus subtilis BNCC 189983
Antibacterial protein has good tolerance to trypsase, alkali protease, quick to chymotrypsin stomach function regulating protease part
Sense.
As shown in Figure 1 and Figure 2,189983 antibacterial protein of bacillus subtilis BNCC is (antibacterial to S. aureus L-forms growth effect figure
Circle size can indicate bacteriostatic activity power).
The above content is merely illustrative of the invention's technical idea, and this does not limit the scope of protection of the present invention, all to press
According to technical idea proposed by the present invention, any changes made on the basis of the technical scheme each falls within claims of the present invention
Protection scope within.
Claims (10)
1. a kind of fermentation process for improving bacillus subtilis antibacterial protein yield, which comprises the following steps:
Bacillus subtilis bacterial strain is first obtained, then strain inoculated is cultivated into seed culture medium, seed liquor is obtained, then by seed
Liquid, which is inoculated into fermentation medium, to ferment, and carries out segmentation regulation fermentation system by logarithmic growth phase and stationary phase during the fermentation
Temperature, dissolved oxygen amount, pH value and shaking speed, final post-processing obtains bacillus subtilis antibacterial protein product.
2. a kind of fermentation process for improving bacillus subtilis antibacterial protein yield according to claim 1, feature exist
In control strain inoculum concentration is 2~6%.
3. a kind of fermentation process for improving bacillus subtilis antibacterial protein yield according to claim 1, feature exist
In the culture specific steps being inoculated into seed culture medium are as follows:
Bacillus subtilis bacterial strain is inoculated in inclined-plane, is cultivated in 36~38 DEG C of constant incubators, it then will be on slant medium
Bacterial strain after activation is prepared into bacteria suspension, is inoculated in seed culture medium, in 36~38 DEG C, the shaking table of 200~220r/min
Obtain seed liquor within upper shake culture 12~16 hours.
4. a kind of fermentation process for improving bacillus subtilis antibacterial protein yield according to claim 3, feature exist
In, according to mass percent meter, the seed culture medium includes glucose 1.0%, peptone 1.0%, NaCl 1.0%,
KH2PO40.15%, MgSO4·7H2The water of O 0.15% and surplus；PH value is 7.0.
5. requiring a kind of fermentation process of raising bacillus subtilis antibacterial protein yield according to right 1, feature exists
In the fermentation specific steps are as follows: seed liquor is inoculated into fermented and cultured according to volume ratio for (4:100)~(8:100) ratio
In base, the antibacterial protein fermentation of bacillus subtilis is carried out.
6. a kind of fermentation process for improving bacillus subtilis antibacterial protein yield according to claim 5, feature exist
In, according to mass percent meter, the fermentation medium includes glucose 1.0%, peptone 1.5%, yeast extract 1.5%,
KH2PO40.1%, NaCl 0.5%, MgSO4·7H2The water of O 0.15% and surplus；Initial pH value is 7.0,0.1MPa high pressure
Sterilize 15min.
7. a kind of fermentation process for improving bacillus subtilis antibacterial protein yield according to claim 1, feature exist
In, fermentation system temperature is 36~38 DEG C in logarithmic growth phase, and dissolved oxygen amount is set as 60%~80%, pH value is set as 6.9~
7.1, shaking speed is set as 180r/min~240r/min.
8. a kind of fermentation process for improving bacillus subtilis antibacterial protein yield according to claim 1, feature exist
In, fermentation system temperature is adjusted to 34~36 DEG C in stationary phase, and dissolved oxygen amount is adjusted to 40%~50%, and pH value is adjusted to 6.6~
6.8, shaking speed is adjusted to 220r/min~250r/min.
9. a kind of fermentation process for improving bacillus subtilis antibacterial protein yield according to claim 7 or 8, feature
It is, the pH value of fermentation system is adjusted with lactic acid and NaOH.
10. a kind of fermentation process for improving bacillus subtilis antibacterial protein yield according to claim 1, feature exist
In the bacillus subtilis is 18998 bacillus subtilis of BNCC.
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| CN201811088861.4A CN109161574A (en) | 2018-09-18 | 2018-09-18 | A kind of fermentation process improving bacillus subtilis antibacterial protein yield |
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