CN104004803B - Method for producing brevibacillus laterosporu antimicrobial peptide through activated carbon adsorption separation coupled method - Google Patents
Method for producing brevibacillus laterosporu antimicrobial peptide through activated carbon adsorption separation coupled method Download PDFInfo
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Abstract
The invention provides a method for producing brevibacillus laterosporu antimicrobial peptide through an activated carbon adsorption separation coupled method. In fermentation of the brevibacillus laterosporu, when the yield of the antimicrobial peptide is no longer increased, active carbon is directly added into the fermentation liquor; as the active carbon adsorbs the fermentation product, the inhibiting effect of the product is relieved, and the density of the thallus and the yield of the antimicrobial peptide are greatly improved. According to the method, the active carbon is taken as an adsorption stripping coupling agent, has low cost, is environmentally friendly, and can be directly added in the fermentation process without being activated in advance; the antimicrobial peptide can be adsorbed without regulating the pH value of the fermentation liquor; and the method is simple and easy to promote, and the active carbon can be regenerated through desorption after fermentation, thus being recycled repeatedly, and lowering the production cost.
Description
Technical field
The present invention relates to field of microbial fermentation, specifically, it is related to a kind of using the life of Activated Carbon Adsorption Separation coupled method
The method for producing Brevibacillus laterosporus antibacterial peptide.
Background technology
With the improvement of people ' s living standards with food-safe pay attention to day by day, customer demand reduction chemical preservative
Use and antibiotic residual, therefore develop safe and efficient biological preservative and turn into the important development direction of food industry,
Wherein antibacterial peptide (Antimicrobial peptide, AMP) due to its chemical nature be protein, the nothing in human or animal's body
Medicament residue, non-immunogenicity, nontoxicity, have potential application value in terms of chemical preservative and antibiotic is substituted.
Antibacterial peptide is a kind of micromolecule polypeptide being widely present in organism, and first separates the antibacterial peptide for obtaining and come from
In the silkworm chrysalis of giant silkworm, so far, the endogenic antibacterial peptide of kind more than 600 is had found in many animals, plant and microorganism,
But animal sources and antimicrobial peptide of plant origins limited source, it is difficult to accomplish scale production, and microbial source antibacterial peptide has fermentation week
Phase is short, low cost, the advantages of be easy to cultivate, be easily achieved industrialized production, it has also become the important directions of antibacterial peptide research.It is domestic
Existing more than the 50 years history of the outer research on microbial antibacterial peptide, most study be originating in lactic acid bacterium bacteriocin ---
Nisin, it has gone through to can be used for food industry, but the Nisin of originating in lactic acid bacterium has narrow antimicrobial spectrum, stability difference etc. mostly
Problem, therefore, new non-lactic acid bacteria antibacterial peptide is further developed as a urgent and long-term task.
Bacillus (Bacillus) has the applicating history of safety due to it in the food industry, and especially side spore is short
Bacillus, its antibacterial peptide for producing has the advantages that safe, has a broad antifungal spectrum, good stability, noresidue, is non-lactic acid bacteria
One important sources of bacteriocin, the focus as research in recent years has been widely used in food, feed and control of crop disease
Field.Research to Brevibacillus laterosporus antibacterial peptide in recent years finds, during fermentation of bacillus produces antibacterial peptide, when
Antibacterial peptide is not further added by after reaching certain yield, shows a kind of product inhibition phenomena, not only have impact on the growth of itself thalline,
The further synthesis of antibacterial peptide is further suppress simultaneously.Based on this, the yield of antibacterial peptide is improved, merely by supplemented medium
Continuous fermentation process does not reach production purpose, and should consider how to release this inhibitory action of antibacterial peptide, so as to from basic
It is upper to solve the bottleneck problem that limitation antibacterial peptide realizes high yield.
Microbial fermentation is separated the technology being coupled effect in terms of Product inhibiton in solving fermentation process with product to show
Write, and it is easy to operate, this separation and fermentation technology in situ optionally can be continuously separated out from zymotic fluid to be had to thalline
Inhibition or unstable product, can both be such that fermentation process is carried out to the direction of Product formation, moreover it is possible to reduce product in complexity
The effect of natural degradation under environment.The isolation technics in situ being related at present mainly includes film fermentation method, electroosmose process, solvent extraction
Method and absorption method.From in terms of industrialized production angle, absorption method is with its low cost, selectivity is high, exchange capacity is big, operation letter
Singly, it is easy to the advantages of automatically controlling, as the effective way for realizing Brevibacillus laterosporus antibacterial peptide fermentation yield high.
In recent years, domestic and international researcher starts to explore the yield for improving small-molecular peptides using the adsorbing separation coupled method that ferments.
[Xu Hao, Wu Zhaoliang, Yin Hao, Yan Jin the fermentation adsorbing separation coupling production nisin technique macromolecule materials such as Wu Zhaoliang
Material scientific and engineering, 2010,26 (10):155-158] report, add cationic ion-exchange resin in the fermenting and producing of Nisin
D113 carries out adsorbing coupled fermentation as adsorbent, and when reaching fermentation termination, the potency of Nisin is improved by original 3350IU/mL
To 5189IU/mL, 54.9% is improve;But need to carry out activation process before resin high cost, and input zymotic fluid, operation is multiple
It is miscellaneous.[Pongtharangku T, the Demirci A.Online recovery of nisin during such as Pongtharangku
fermentation and its effect on nisin production in biofilm reactor.Applied
Microbiology and Biotechnology,2007,74(3):555-562] using microbial fermentation and UF membrane and suction
Fufen produces Nisin from coupled method, and the method makes Nisin potency improve nearly 4 times, effect is significant, but the production technology is multiple
It is miscellaneous, it is relatively costly, and the influence of zymotic fluid pH value is big, is not suitable for industrialized production.Therefore, find with low cost, simple to operate
Zymotechnique turns into problem demanding prompt solution.
The content of the invention
It is an object of the invention to provide a kind of product released using cheap adsorbing separation couplant in antibacterial peptide fermentation process
The method that thing suppresses and then improves cell density and yield of antibacterial peptides.
In order to realize the object of the invention, a kind of side of utilization Brevibacillus laterosporus fermenting and producing antibacterial peptide of the invention
Method, in Brevibacillus laterosporus (Brevibacillus laterosporu) fermentation process, treats that yield of antibacterial peptides is not further added by
When, to activated carbon is directly added in zymotic fluid, the absorption by activated carbon to tunning releases product inhibition, so that
Improve cell density and yield of antibacterial peptides.
Directly add a kind of adsorbing separation couplant during the fermentation --- activated carbon, activated carbon without early stage activation at
Reason, without preparing adsorption column, zymotic fluid need not adjust pH value, you can release product inhibition, improve the density of fermentation thalli
With the yield of antibacterial peptide.
The Brevibacillus laterosporus being related in the present invention including but not limited to B.laterosporu AS1.6343,
B.laterosporu AS1.2827、B.laterosporu AS1.2739、B.laterosporu AS1.864、
B.laterosporu ACCC06527, B.laterosporu ACCC05440, B.laterosporu ACCC10390 etc..
Concrete technology using Brevibacillus laterosporus fermenting and producing antibacterial peptide is as follows:
1) prepared by seed liquor:The inclined-plane of Brevibacillus laterosporus or glycerol tube are accessed the nutrient broth medium of sterilizing
In (NB culture mediums), 30-37 DEG C of cultivation temperature, incubation time 12-24h obtains primary seed solution;Then primary seed solution is pressed
3-5v/v% inoculum concentrations access nutrient broth medium, 30-37 DEG C, cultivate 6-18h, and it is 10 to obtain bacteria concentration7-108Cfu/mL's
Secondary seed solution;
2) fermented and cultured:To the fermentation medium for loading fermenter volume 60-80% in fermentation tank, accessed after sterilizing cooling
Step 1) prepare secondary seed solution, inoculum concentration 3-5v/v%;Fermentation condition is:30-34 DEG C of fermentation temperature, rotating speed 200-
400r/min, throughput 1:0.1-1:0.5v/v/min, dissolved oxygen 60-70%, incubation time 12-24h;
3) charcoal absorption:When 12-24h yield of antibacterial peptides of fermenting is not further added by, to activated carbon is added in zymotic fluid, together
When add Fermented Condensed culture medium, activated carbon addition 1-4w/v%, Fermented Condensed culture medium additional amount 1-3v/v% continue
Fermentation 12-24h;Fermentation condition is with step 2).
Wherein, step 2) described in the formula of fermentation medium be:Carbon source 0.5-3.0w/v%, nitrogen source 0.5-3.0w/
V%, inorganic salts 0.005-1.0w/v%, surfactant 0.1-1.0w/v%, adjust pH7.0-7.4, are prepared with water.
Step 3) described in the formula of Fermented Condensed culture medium be:Carbon source 5.0-30.0w/v%, nitrogen source 5.0-30.0w/
V%, inorganic salts 0.05-10.0w/v%, surfactant 1.0-10.0w/v%, adjust pH7.0-7.4, are prepared with water.
In above-mentioned antibacterial peptide zymotechnique, available carbon source is selected from glucose, sucrose, soluble starch, dextrin, molasses
Or one or more in glycerine etc..
In above-mentioned antibacterial peptide zymotechnique, available nitrogen source is selected from corn pulp, beef extract, peptone, dusty yeast or beans
One or more in dregs of rice powder etc..
In above-mentioned antibacterial peptide zymotechnique, available inorganic salts are selected from Ca2+、Zn2+、Mg2+、Mn2+、Fe2+Or Cu2+Salt etc.
In one or more.
In above-mentioned antibacterial peptide zymotechnique, available surfactant is selected from Tween-20, Tween-40, Tween-
60、Tween-80、Triton X-100、Triton X-114、Triton X-116、TX-4、TX-6、TX-7、TX-8、TX-9、
One or more in TX-10, AEO-3, AEO-7 or AEO-9 etc..
The activated carbon added in above-mentioned antibacterial peptide zymotechnique is powdered or granular bamboo charcoal, charcoal, fruit shell carbon or
Coconut husk charcoal.After charcoal absorption tunning, can be regenerated by means such as desorptions, realize recycling for activated carbon.
Using above-mentioned antibacterial peptide zymotechnique, fermenting and producing, compared with activated carbon is not added with, viable bacteria are carried out in fermentation tank
Number improves the 2-4 order of magnitude, reaches 1010-1012Cfu/mL, yield of antibacterial peptides improves 50-130%, and potency is 2200-
3500AU/mL。
Fermentation strain used by the present invention is Brevibacillus laterosporus, is also called bacillus laterosporus, its metabolite tool
There are suppression various bacteria, the function of fungi.In the food industry, it is applied to production functional food and food antiseptic is protected
It is fresh.In feed industry, feeding micro-ecological preparation can be used for by Ministry of Agriculture's approval, be applied to broiler chicken, meat duck, pig and shrimp
Cultivation, is a kind of potential probiotics with high security.
Adsorbing separation couplant used by the present invention is activated carbon, with low cost, environmentally friendly, in fermentation process directly
Addition, without carrying out activating pretreatment, and zymotic fluid need not adjust pH value, you can realize the absorption of antibacterial peptide.Process is simple, easily
In after popularization, fermentation ends regenerated carbon can be processed by desorption, being used repeatedly for activated carbon is realized, reduced
Production cost.
The antibacterial peptide zymotechnique that the present invention is provided relieves the inhibitory action of product in fermentation process, make fermentation thalli and
Yield of antibacterial peptides is greatly improved.
Using the antibacterial peptide of Brevibacillus laterosporus fermenting and producing, its chemical nature is protein, noresidue, security
Good, stability is high, has a broad antifungal spectrum, can suppress various bacteria and fungi, can be widely applied to food, medicine, livestock and poultry cultivation, life
The industries such as thing agricultural.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
In the conventional meanses that are well known to those skilled in the art of technological means used, it is raw materials used to be commercial goods.
The percentage sign " % " being related in the present invention, refers to mass percent if not specified;But the percentage of solution
Than referring to the grams containing solute in 100mL solution unless otherwise specified.
The technique that embodiment 1 utilizes Brevibacillus laterosporus fermenting and producing antibacterial peptide
Concrete technology is as follows:
1st, seed culture
The glycerol tube that B.laterosporu AS1.2827 will be preserved accesses the NB culture mediums of sterilizing, 32 DEG C, during culture
Between 24h;5v/v% inoculum concentrations being pressed again and accessing NB culture mediums, 32 DEG C are cultivated 8h, it is 10 to obtain bacteria concentration7The secondary seed of cfu/mL
Liquid.
2nd, 5L fermentation tank cultures
The secondary seed solution obtained in step 1 is pressed into 5v/v% inoculum concentrations and accesses the 5L fermentation tanks equipped with 3L fermentation mediums
In, 32 DEG C, 300r/min, throughput 1:15h is cultivated under the conditions of 0.5v/v/min.
Wherein, fermentation medium (w/v):Glycerine 2.0%, bean cake powder 2.0%, CaCl20.005%th, Tween-
200.5%, pH7.2 is adjusted, prepared with water.
3rd, Activated Carbon Adsorption Separation couplant is added
In step 2, when culture 15h after fermentation liquid antibacterial peptide contents no longer increase, to the activity of addition 3% in zymotic fluid
Charcoal, while adding 3v/v% Fermented Condensed culture mediums, fermented and cultured 20h is continued by fermentation condition in step 2.
Wherein, Fermented Condensed culture medium is 10 times of concentrates of fermentation medium in step 2.
Viable count and antibacterial peptide potency are determined after fermentation ends, compared with the control group for being not added with activated carbon, viable count is carried
2 orders of magnitude high, yield of antibacterial peptides improves 80%.
The technique that embodiment 2 utilizes Brevibacillus laterosporus fermenting and producing antibacterial peptide
Concrete technology is as follows:
1st, seed culture
The inclined-plane that B.Laterosporu AS1.2738 will be preserved accesses the NB culture mediums of sterilizing, 34 DEG C, incubation time
24h, then NB seed culture mediums are accessed by 3v/v% inoculum concentrations, 34 DEG C are cultivated 12h, and it is 10 to obtain bacteria concentration8Two grades of cfu/mL
Seed liquor.
2nd, fermentation tank culture
The secondary seed solution obtained in step 1 is pressed into 8v/v% inoculum concentrations and accesses the 5L fermentation tanks equipped with 4L fermentation mediums
In, carry out fermented and cultured.
Fermentation medium (w/v):Soluble starch 1.5%, dusty yeast 1.5%, CaCl20.15%th, ZnCl20.008%th,
MnCl20.05%, Tween-202.0%, pH7.0, are prepared with water.
Fermentation condition:34 DEG C of temperature, throughput 1:0.5v/v/min, dissolved oxygen 60%, dissolved oxygen is associated with rotating speed, rotating speed
200-400r/min, cultivates 16h.
3rd, Activated Carbon Adsorption Separation couplant is added
In step 2, when cultivate 16h after to zymotic fluid in add 1.5% activated carbon, while add 2v/v% Fermented Condenseds training
Base is supported, fermented and cultured 24h is continued by fermentation condition in step 2.Fermented Condensed culture medium is 10 times of fermentation medium in step 2
Concentrate.
Viable count and antibacterial peptide potency are determined after fermentation ends, compared with the control group for being not added with activated carbon, viable count is carried
3 orders of magnitude high, yield of antibacterial peptides improves by about one time.
The activated carbon of embodiment 3 adds influence of the time to viable count and yield of antibacterial peptides
With Brevibacillus laterosporus (B.laterosporu) AS1.864 as fermentation strain, the glycerine of bacterial strain will be preserved first
Pipe accesses the NB culture mediums of sterilizing, 32 DEG C, cultivates 24h, and NB culture mediums are accessed by 3v/v% inoculum concentrations, and 32 DEG C of culture 12h are obtained
Bacteria concentration is 108The secondary seed solution of cfu/mL.
Using 5L fermentation tanks, accessed by 5v/v% inoculum concentrations and be equipped with the system of 3L fermentation mediums, in 32 DEG C, throughput
1:0.5v/v/min, carries out fermented and cultured under the conditions of rotating speed 300r/min, respectively at the 12nd for fermenting, 16,20,24h, addition
2% powdered active carbon and 2v/v% concentrate feed culture mediums, are further continued for the 24h that ferments;Using be not added with activated carbon as control, it is living
Bacterium number and yield of antibacterial peptides are shown in Table 1.Activated carbon effect is added after fermentation 16h most obvious, viable count is improved compared with control group will
Nearly 3 orders of magnitude, antibacterial peptide potency improves 80%.
NB seed culture mediums (w/v):Peptone 1.0%, beef extract 0.3%, NaCl0.5%, adjust pH7.2, are prepared with water.
In 121 DEG C, high pressure steam sterilization 20min.
Fermentation medium (w/v):Glucose 1.5%, corn pulp 1.8%, CaCl20.15%th, ZnCl20.008%th,
Tween-800.1%, adjusts pH7.0, is prepared with water.
Fermented Condensed culture medium:10 times of concentrates of fermentation medium.
The activated carbon of table 1 adds influence of the time to viable count and antibacterial peptide potency
Influence of the activated carbon addition of embodiment 4 to viable count and yield of antibacterial peptides
Secondary seed solution system, as fermentation strain, is carried out with Brevibacillus laterosporus (B.laterosporu) AS1.864 first
Standby, seed culture medium is NB culture mediums, and 32 DEG C of cultivation temperature, acquisition bacteria concentration is 108The secondary seed solution of cfu/mL.
Using 5L fermentation tanks, 3L fermentation mediums are accessed by the inoculum concentration of 5v/v%, in 32 DEG C, throughput 1:0.5v/v/
Min, rotating speed 300r/min, after fermentation 16h, add 1%, 2%, 3% and 4% powdered active carbon, while adding respectively
2v/v% Fermented Condensed culture mediums, continue the 24h that ferments;Using be not added with activated carbon as control, viable count and yield of antibacterial peptides
It is shown in Table 2.The raising effect of cell density and yield of antibacterial peptides is most obvious when adding 2.0% powdered active carbon, viable count with it is right
Photograph reaches 10 than improving 3 orders of magnitude11Cfu/mL, antibacterial peptide potency is doubled.
Fermentation medium (w/v):Glucose 1.5%, corn pulp 1.8%, CaCl20.15%th, ZnCl20.008%th,
Tween-800.1%, adjusts pH7.0, is prepared with water.
Fermented Condensed culture medium:10 times of concentrates of fermentation medium.
Influence of the activated carbon addition of table 2 to viable count and antibacterial peptide potency
Influence of the Fermented Condensed material culture medium additional amount of embodiment 5 to viable count and yield of antibacterial peptides
Secondary seed solution system, as fermentation strain, is carried out with Brevibacillus laterosporus (B.laterosporu) AS1.864 first
Standby, it is 10 to obtain bacteria concentration8The secondary seed solution of cfu/mL;Using 5L fermentation tanks, 3L fermentation trainings are accessed by 5v/v% inoculum concentrations
Base is supported, in 32 DEG C, throughput 1:0.5v/v/min, under the conditions of rotating speed 300r/min, after fermentation 16h, the powdered work of addition 2%
Property charcoal, while add the Fermented Condensed culture medium of 1v/v%, 2v/v% and 3v/v% respectively, continue the 24h that ferments, to be not added with living
Property charcoal conduct control, viable count and yield of antibacterial peptides are shown in Table 3.The Fermented Condensed culture medium for adding 2v/v% improves effect to potency
Fruit significantly, about improves 90%.
Fermentation medium (w/v):Glucose 1.5%, corn pulp 1.8%, CaCl20.15%th, ZnCl20.008%th,
Tween-200.1%, adjusts pH7.0, is prepared with water.
Fermented Condensed material culture medium:10 times of concentrates of fermentation medium.
Influence of the Fermented Condensed material culture medium addition of table 3 to viable count and antibacterial peptide potency
Embodiment 630L fermentation tanks amplify scale fermentation test
With Brevibacillus laterosporus (B.laterosporu) AS1.864 as fermentation strain, in 32 DEG C, NB culture mediums are carried out
Prepared by secondary seed solution, it is 10 to obtain bacteria concentration8The secondary seed solution of cfu/mL.
Using 30L fermentation tanks, 20L fermentation mediums are accessed by 5v/v% inoculum concentrations, in 32 DEG C, throughput 1:0.5v/v/
Min, rotating speed 300r/min, add 2% powdered active carbon and 2v/v% Fermented Condensed culture mediums, followed by supervention after fermentation 15h
Ferment 20h.
Fermentation medium (w/v):Glucose 1.5%, corn pulp 1.8%, MnCl20.05%th, ZnCl20.005%th,
CaCl20.15%, pH7.0 is adjusted, prepared with water.
Fermented Condensed culture medium:10 times of concentrates of fermentation medium.
After fermentation ends, viable count reaches 5.0 × 1011Cfu/mL, antibacterial peptide potency 2916AU/mL.Be not added with activity
Charcoal is compared, and viable count increases by 3 orders of magnitude, and yield of antibacterial peptides improves by about one time.
The Brevibacillus laterosporus antibacterial peptide antimicrobial spectrum of embodiment 7 is tested
Brevibacillus laterosporus antibacterial peptide is obtained using activated carbon fermentation adsorbing separation coupling method, using Oxford cup inhibition zone
The antimicrobial spectrum of method research antibacterial peptide.
The result of table 4 shows that the antibacterial peptide has certain inhibitory action, including leather to various food-bornes and avian pathogenic bacteria
Lan Shi positive bacterias:Listeria monocytogenes, staphylococcus aureus, Streptococcus suis, bacillus cereus, bacillus subtilis
Bacterium, bacillus megaterium etc..Gram-negative bacteria:Escherichia coli, salmonella typhi, shigella flexneri, P. aeruginosa
Bacterium, Caused by Yersinia enterocolitica, Enterobacter sakazakii etc..It can be seen that, by the antibacterial peptide of Brevibacillus laterosporus generation in biological food
Anti-corrosion and livestock and poultry nonreactive cultivation field have broad application prospects.
The antimicrobial spectrum of the antibacterial peptide of table 4
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (1)
1. a kind of method of utilization Brevibacillus laterosporus fermenting and producing antibacterial peptide, it is characterised in that with Brevibacillus laterosporus
(B.laterosporu) AS 1.864 is fermentation strain, and in 32 DEG C, NB culture mediums carry out secondary seed solution preparation, obtain bacterium dense
Spend is 108The secondary seed solution of cfu/mL;
Using 30L fermentation tanks, 20L fermentation mediums are accessed by 5v/v% inoculum concentrations, in 32 DEG C, throughput 1:0.5v/v/min,
Rotating speed 300r/min, 2% powdered active carbon and 2v/v% Fermented Condensed culture mediums are added after fermentation 15h, are further continued for fermentation
20h;
Fermentation medium:Glucose 1.5%, corn pulp 1.8%, MnCl20.05%th, ZnCl20.005%th, CaCl20.15%,
PH 7.0 is adjusted, is prepared with water;
Fermented Condensed culture medium:10 times of concentrates of fermentation medium.
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CN107936087B (en) * | 2017-11-20 | 2021-02-02 | 河北科技大学 | Green and efficient extraction and preparation method of microbial source antibacterial peptide additive |
CN108795805A (en) * | 2018-06-08 | 2018-11-13 | 山东宸青生物科技有限公司 | Brevibacillus laterosporus fermentation modification method |
CN110577910B (en) * | 2019-09-17 | 2022-02-01 | 南京农业大学 | Brevibacillus laterosporus, antibacterial lipopeptide and application of antibacterial lipopeptide in agriculture and food |
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CN111269865B (en) * | 2020-04-01 | 2020-12-01 | 北京工商大学 | Brevibacillus laterosporus strain S62-9 and application thereof |
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