CN104130318A - Post-extraction process for producing antibacterial peptide through bacillus subtilis - Google Patents
Post-extraction process for producing antibacterial peptide through bacillus subtilis Download PDFInfo
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Abstract
The invention relates to a post-extraction process for producing antibacterial peptide through bacillus subtilis and belongs to the technical field of biology. The process comprises the following steps: by taking bacillus subtilis as a production strain, preparing a finished antibacterial peptide product through centrifugation, membrane separation and spray drying treatment of fermentation liquor, centrifuging by adopting two continuous centrifugal machines, performing classification and membrane separation, removing the thallus and hybrid proteins to the greatest degree, thus obtaining the antibacterial peptide with high activity. By utilizing heat resistance of the antibacterial peptide, the antibacterial peptide is rapidly dried through a spray drying technology, the preparation efficiency of the antibacterial peptide is improved, and maltodextrin serving as an auxiliary material has the advantages of high solubility and high stability, is unsuitable for moisture absorption and deterioration and contains rich trace elements and mineral substances. The separated and purified antibacterial peptide is high in activity, low in cost, simple in process and low in post-treatment loss.
Description
Technical field
The present invention relates to a kind of extraction process, be applicable to the production of antibacterial peptide, be particularly useful for utilizing subtilis to produce antibacterial peptide, specifically, relate to a kind of rear extraction process of subtilis production antibacterial peptide, belong to biological technical field.
Background technology
Antibacterial peptide (Antimicrobial peptides, AMP) refers to that the class producing through induction in organism has the micromolecule polypeptide of anti-microbial activity, is a part for innate immune defence system.This proteinoid has that acid resistance is good conventionally, has a broad antifungal spectrum, good water solubility, safe and act on the features such as quick.Its mechanism of action is that antibacterial peptide, by protein coacervation effect, directly inserts bacterial cell membrane by its end tail chain, punches and forms a passage, bacterium content is leaked and cause bacterium death.Compared with microbiotic, antibacterial peptide have do not kill probiotic bacterium, improve human body immune function, have no side effect, noresidue, have no drug resistance, can life-time service etc. advantage.Because of the characteristic that antibacterial peptide has, can be widely used in multiple fields.Aspect medical, antibacterial peptide can balance and adjusting inflammatory response the specific adaptive immunity reaction of challenging antigen.Aspect food antiseptic, antibacterial peptide is as a kind of natural antiseptic agent, and toxic side effect is little, and degradable uses safety, even human body is had to health-care effect.Aspect fodder additives, antibiotic abuse earth effect to the utmost the development of China's livestock industry, and antibacterial peptide has exactly solved problems such as its resistance is residual, resistance, environmental pollution are a kind of environment-friendly highly efficient fodder additivess.
In above these fields, China also has no small gap in technical and universal aspect compared with some developed countries in the world, and due to the reason of each side, China not yet completely forbids feeding antibiotic at present, and most antibiotic feed additives are external eliminated or by disabled product, these problems will affect China's livestock product reputation in the world undoubtedly, and cause huge financial loss.Because microorganisms producing antibacterial peptide has that cost is low, toxicological harmless, the cycle obviously advantage such as short, the research of accelerating antibacterial peptide is imperative, but current research mainly concentrates on milk-acid bacteria, and the antibacterial peptide that milk-acid bacteria produces exists the defect such as poor heat resistance, narrow antimicrobial spectrum.The antibacterial peptide antimicrobial spectrum of producing bacillus subtilis is wide, stability is high, starts to cause domestic and international researchist's attention, and produces the suitability for industrialized production of antibacterial peptide for its fermentation, especially extraction process after, still face at present high cost, process complexity, the tire problems such as loss is excessive of product.
Summary of the invention
Technical problem solved by the invention is for the deficiencies in the prior art, taking subtilis as producing bacterial strain, by the post-processed of fermented liquid is processed into antibacterial peptide finished product, have that operating procedure is simple, step is less, the lower and product of the production cost higher advantage of tiring.
For solving above technical problem, the present invention is by the following technical solutions: a kind of subtilis produces the rear extraction process of antibacterial peptide, it is characterized in that: described rear extraction process comprise carry out successively put tank, centrifugal, membrane sepn, adjusting pH value and the drying step of spraying.
Adopt this technical scheme, the technique of described rear extraction process is simple, step is few, and extraction effect is good.
A kind of prioritization scheme, in described membrane sepn step, the supernatant liquor after centrifugal is carried out to classified filtering, 40 DEG C of filtration temperatures, the import and export pressure drop of ceramic membrane equipment is 0.1～0.15MPa, average flux is 0.5m3/h, remove thalline and foreign protein rate reaches 99.97%～99.99%, in the supernatant liquor after membrane sepn, the inhibitory potency of antibacterial peptide is 12689～12813.
Adopt this technical scheme, remove the effective of thalline and foreign protein, and effectively ensure the activity of antibacterial peptide.
Another kind of prioritization scheme, described classified filtering comprises level Four filtration;
The first step is filtered and is adopted 500,000 molecular weight ceramic membranes, the second stage to filter employing 350,000 molecular weight ceramic membranes, and third stage filtration adopts 150,000 molecular weight ceramic membranes, and the fourth stage filters and adopts 50,000 molecular weight ceramic membranes.
Adopt this technical scheme, utilize classified filtering, more effectively remove thalline and foreign protein in supernatant liquor.
Another prioritization scheme, in described adjusting pH value step: the clear liquid after collection membrane separates, adjust pH value to 3.5, leave standstill after 24h, filtering upper strata supernatant liquor obtains containing sedimentary concentrated solution, repeats filtering operation until concentrated solution volume is to adjust 50% of the front supernatant liquor volume of pH value.
Adopt this technical scheme, both ensured the content of effective constituent in concentrated solution, be beneficial to again the carrying out of subsequent handling, lay a good foundation for rapidly and efficiently obtaining antibacterial peptide dry powder.
Further prioritization scheme, after adding 25% maltodextrin to stir as excipient in described concentrated solution, spraying to be dried according to the treatment capacity of 50 L/h obtains antibacterial peptide dry powder.
Adopt this technical scheme, can effectively ensure the inhibitory potency of antibacterial peptide, and be convenient to rapid drying acquisition antibacterial peptide dry powder.
Further prioritization scheme, described spray-dired inlet temperature is 120～180 DEG C, air outlet temperature is 70～90 DEG C.
Adopt this technical scheme, the good and activity of not losing antibacterial peptide of drying effect.
Further prioritization scheme, described in to put the storage time of fermented liquid in storage tank in tank step be 2 hours.
Adopt this technical scheme, both ensured the inhibitory potency of antibacterial peptide, increased to a certain extent again output, reduced production cost, and shortened the production cycle.
A kind of prioritization scheme, adopts two continuous centrifuges of series connection centrifugal in described centrifugation step.
Adopt this technical scheme, can effectively remove a large amount of precipitations in fermented liquid, be conducive to the carrying out of subsequent handling.
Another kind of prioritization scheme, in described adjusting pH value step, conditioning agent is hydrochloric acid, phosphoric acid or citric acid.
Adopt this technical scheme, the good and activity of not losing antibacterial peptide of pH value regulating effect.
Another prioritization scheme, adopts described rear extraction process to extract compared with the inhibitory potency of the inhibitory potency of the antibacterial peptide dry powder obtaining and antibacterial peptide in putting tank primary fermentation liquid, and rate of descent is less than 15%.
The present invention adopts after above technical scheme, compared with prior art, have the following advantages: taking subtilis as producing bacterial strain, prepare antibacterial peptide finished product by treatment steps such as centrifugal, the membrane sepn to fermented liquid, spraying are dry, the present invention put after tank centrifugal through two continuous centrifuges, pass through again membrane sepn, can remove to greatest extent thalline and foreign protein, obtain active higher antibacterial peptide.In addition, when antibacterial peptide is processed into dry powder, utilize the thermotolerance of antibacterial peptide, pass through spray drying technology, make antibacterial peptide rapid drying, improved antibacterial peptide preparation efficiency, and maltodextrin has that solvability is good, stability is high, should not the moisture absorption go bad, contain and enrich the advantage such as trace element and mineral substance as auxiliary material.Separation and purification antibacterial peptide activity of the present invention is high, with low cost, and technique is simple, and post-processed loss is lower.
The made antibacterial peptide dry powder of the present invention not only can suppress gram positive bacterium, multiple gram negative bacterium and fungi is all had to stronger restraining effect simultaneously, and ph stability is strong, better heat stability.
The made antibacterial peptide dry powder of the present invention belongs to natural antibacterial agent, has no side effect, and can be used as food preservatives, fodder additives, agricultural biological control agent and animal diseases prevention agent, has advantages of nontoxic, noresidue, does not produce resistance, safe.
Below in conjunction with drawings and Examples, the present invention is described in detail.
Brief description of the drawings
Accompanying drawing 1 is the schematic flow sheet of rear extraction process in the embodiment of the present invention;
Accompanying drawing 2 is bioactivities of antibacterial peptide in the embodiment of the present invention.
Embodiment
Below by specific embodiment narration the present invention.Unless stated otherwise, in the present invention, technique means used is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claims.To those skilled in the art, do not deviating under the prerequisite of essence of the present invention and scope various changes that the material component in these embodiments and consumption are carried out or change and also belong to protection scope of the present invention.
Embodiment 1, a kind of subtilis produces the rear extraction process of antibacterial peptide, as shown in Figure 1, comprises the steps:
1) put tank
Utilize pressurized air that fermentor tank pressure is adjusted to 0.05MPa left and right, storage tank pressure is zero, the fermented liquid that utilizes the pressure difference between fermentor tank and storage tank, by stainless steel material pipe, subtilis is produced to antibacterial peptide is pressed onto in storage tank, stores and within 2 hours, carries out centrifugal treating.
Through verification experimental verification, the storage time, when long can there is certain loss in antibacterial peptide, and inhibitory potency can decline to a great extent, and storage time when too short will be unfavorable in thalline that the further metabolism of antibacterial peptide discharges, and then affect product and tire.The storage time of fermented liquid in storage tank is 2 hours, both ensure the inhibitory potency of antibacterial peptide, increased to a certain extent again output, reduced production cost, and shortened the production cycle, the storage time impact of the inhibitory potency on antibacterial peptide in fermented liquid is as shown in table 1.
The impact that table 1 storage time tires on antibacterial peptide
2) centrifugal
Fermented liquid is centrifugal by two continuous centrifuges of series connection, discards precipitation, stores supernatant liquor.
The GQ-105 type tubular-bowl centrifuge that described whizzer selects " Liaoyang Long Da pharmaceutical machine company limited " to produce, the rotating speed of whizzer is 12000rpm, the treatment capacity of whizzer is 1 m
3/ h, the supernatant liquor after centrifugal is received in relay tank and is entered membrane separation program by whizzer drain pipe, after the solid impurity in rotary drum scrapes, after the processing such as sterilizing, discards.
Through double centrifugal treating, can effectively remove a large amount of precipitations in fermented liquid, be conducive to the carrying out of subsequent handling.
3) membrane sepn
Supernatant liquor carries out classified filtering by 50,000～500,000 molecular weight ceramic membranes, to remove thalline and foreign protein, and fully keeps the activity of antibacterial peptide.
First select 500,000 molecular weight ceramic membranes to carry out first step filtration, the macromole that is greater than 500,000 molecular weight is trapped, be less than the material permeance ceramic membrane of 500,000 molecular weight, adopt 350,000 molecular weight ceramic membranes to carry out the second stage filtration clear liquid filtering through the first step, clear liquid after filtration adopts 150,000 molecular weight ceramic membranes to carry out third stage filtration again, finally selects 50,000 molecular weight ceramic membranes to carry out fourth stage filtration again.
Filtration temperature: 40 DEG C, working pressure: intake pressure is 0.2MPa; Top hole pressure 0.1MPa, import and export pressure drop is 0.1MPa, supernatant liquor is with 0.5m
3the flow velocity of/h flows through along the surface of ceramic membrane, described ceramic membrane carries out cross flow filter to supernatant liquor, under the driving of pressure-acting, supernatant liquor flows in film pipe, small-molecule substance permeation ceramic membrane, contain the liquid of macromolecular components by ceramic membrane interception, thereby make supernatant liquor reach the object of separation, purifying.
Ceramic membrane equipment used is that " Anhui Plum Membrane Technology Co., Ltd. " produces.
Described membrane sepn control parameter is as shown in table 2 on removing the impact of thalline and foreign protein effect and antibacterial peptide inhibitory potency.
Table 2 membrane sepn control parameter is on removing the impact of thalline and foreign protein effect and antibacterial peptide inhibitory potency
In table 2, effect refers to the effect of removing thalline and foreign protein, adopt orthogonal experiment, through lot of experiments checking, adopt the membrane sepn control parameter in the present embodiment, remove thalline and foreign protein rate reaches 99.98%, and in the supernatant liquor obtaining, the inhibitory potency of antibacterial peptide reaches 12765.
4) regulate pH value
Clear liquid after collection membrane separates, adds therein hydrochloric acid pH value is adjusted to 3.5, leaves standstill lower floor after 24h and occurs precipitation, then obtain containing sedimentary concentrated solution through membrane sepn elimination part clear liquid, and repetition aforesaid operations is until concentrated solution volume is original 50%.
Through verification experimental verification, through repeatedly leaving standstill, membrane filtration is when obtaining concentrated solution volume and being original 50%, both ensured the content of effective constituent in concentrated solution, is beneficial to again the carrying out of subsequent handling, lay a good foundation for rapidly and efficiently obtaining antibacterial peptide dry powder.
5) spraying is dry
After adding 25% maltodextrin to stir as excipient in concentrated solution, spraying to be dried according to the treatment capacity of 50 L/h obtains antibacterial peptide dry powder, and described spray-dired inlet temperature is 120 DEG C, and air outlet temperature is 70 DEG C.
Spray-drier used is that " Wuxi City Neng Da drying plant company limited " produces.
Through verification experimental verification, select 25% maltodextrin as vehicle, can effectively ensure the inhibitory potency of antibacterial peptide, and be convenient to rapid drying and obtain antibacterial peptide dry powder.
Embodiment 2, a kind of rear extraction process of fermentation of bacillus subtilis liquid, as shown in Figure 1, comprises the steps:
1) put tank
Utilize pressurized air that fermentor tank pressure is adjusted in to 0.07MPa left and right, storage tank pressure is zero, the fermented liquid that utilizes the pressure difference between fermentor tank and storage tank, by stainless steel material pipe, subtilis is produced to antibacterial peptide is pressed onto in storage tank, and storage is no more than 2 hours and carries out centrifugal treating.
Through verification experimental verification, the storage time, when long can there is certain loss in antibacterial peptide, and inhibitory potency can decline to a great extent, and storage time when too short will be unfavorable in thalline that the further metabolism of antibacterial peptide discharges, and then affect product and tire.The storage time of fermented liquid in storage tank is 2 hours, both ensured the inhibitory potency of antibacterial peptide, increased to a certain extent again output, reduced production cost, and shortened the production cycle.
2) centrifugal
Fermented liquid is centrifugal by two continuous centrifuges of series connection, discards precipitation, stores supernatant liquor.
The GQ-105 type tubular-bowl centrifuge that described whizzer selects " Liaoyang Long Da pharmaceutical machine company limited " to produce, the rotating speed of whizzer is 12000rpm, the treatment capacity of whizzer is 1 m
3/ h, the supernatant liquor after centrifugal is received in relay tank and is entered membrane separation program by whizzer drain pipe, after the solid impurity in rotary drum scrapes, after the processing such as sterilizing, discards.
Through double centrifugal treating, can effectively remove a large amount of precipitations in fermented liquid, be conducive to the carrying out of subsequent handling.
3) membrane sepn
Supernatant liquor carries out classified filtering by 50,000～500,000 molecular weight ceramic membranes, to remove thalline and foreign protein, and fully keeps the activity of antibacterial peptide.
First select 500,000 molecular weight ceramic membranes to carry out first step filtration, the macromole that is greater than 500,000 molecular weight is trapped, be less than the material permeance ceramic membrane of 500,000 molecular weight, adopt 350,000 molecular weight ceramic membranes to carry out the second stage filtration clear liquid filtering through the first step, clear liquid after filtration adopts 150,000 molecular weight ceramic membranes to carry out third stage filtration again, finally selects 50,000 molecular weight ceramic membranes to carry out fourth stage filtration again.
Filtration temperature: 40 DEG C, working pressure: intake pressure is 0.28MPa; Top hole pressure 0.15MPa, imports and exports pressure drop 0.13MPa, and supernatant liquor is with 0.5m
3the flow velocity of/h flows through along the surface of ceramic membrane, described ceramic membrane carries out cross flow filter to supernatant liquor, under the driving of pressure-acting, supernatant liquor flows in film pipe, small-molecule substance permeation ceramic membrane, contain the liquid of macromolecular components by ceramic membrane interception, thereby make supernatant liquor reach the object of separation, purifying.
Ceramic membrane equipment used is that " Anhui Plum Membrane Technology Co., Ltd. " produces.
Described membrane sepn control parameter is as shown in table 2 on removing the impact of thalline and foreign protein effect and antibacterial peptide inhibitory potency.
Through lot of experiments checking, as shown in Table 2, the membrane sepn control parameter in employing the present embodiment, removes thalline and foreign protein rate reaches 99.97%, and in the supernatant liquor obtaining, the inhibitory potency of antibacterial peptide reaches 12689.
4) regulate pH value
Supernatant liquor after collection membrane separates, adds therein phosphoric acid pH value is adjusted to 3.5, leaves standstill lower floor after 24h and occurs precipitation, then obtain containing sedimentary concentrated solution through membrane sepn elimination part supernatant liquor, and repetition aforesaid operations is until concentrated solution volume is original 50%.
Through verification experimental verification, when obtaining concentrated solution volume and be original 50% through membrane sepn repeatedly, both ensured the content of effective constituent in concentrated solution, be beneficial to again the carrying out of subsequent handling, lay a good foundation for rapidly and efficiently obtaining antibacterial peptide dry powder.
5) spraying is dry
After adding 25% maltodextrin to stir as excipient in concentrated solution, spraying to be dried according to the treatment capacity of 50 L/h obtains antibacterial peptide dry powder, and described spray-dired inlet temperature is 150 DEG C, and air outlet temperature is 80 DEG C.
Spray-drier used is that " Wuxi City Neng Da drying plant company limited " produces.
Through verification experimental verification, select 25% maltodextrin as vehicle, can effectively ensure the inhibitory potency of antibacterial peptide, and be convenient to rapid drying and obtain antibacterial peptide dry powder.
Embodiment 3, a kind of rear extraction process of fermentation of bacillus subtilis liquid, as shown in Figure 1, comprises the steps:
1) put tank
Utilize pressurized air that fermentor tank pressure is adjusted in to 0.1MPa left and right, storage tank pressure is zero, the fermented liquid that utilizes the pressure difference between fermentor tank and storage tank, by stainless steel material pipe, subtilis is produced to antibacterial peptide is pressed onto in storage tank, and storage is no more than 2 hours and carries out centrifugal treating.
Through verification experimental verification, the storage time, when long can there is certain loss in antibacterial peptide, and inhibitory potency can decline to a great extent, and storage time when too short will be unfavorable in thalline that the further metabolism of antibacterial peptide discharges, and then affect product and tire.The storage time of fermented liquid in storage tank is 2 hours, both ensured the inhibitory potency of antibacterial peptide, increased to a certain extent again output, reduced production cost, and shortened the production cycle.
2) centrifugal
Fermented liquid is centrifugal by two continuous centrifuges of series connection, discards precipitation, stores supernatant liquor.
The GQ-105 type tubular-bowl centrifuge that described whizzer selects " Liaoyang Long Da pharmaceutical machine company limited " to produce, the rotating speed of whizzer is 12000rpm, the treatment capacity of whizzer is 1 m
3/ h, the supernatant liquor after centrifugal is received in relay tank and is entered membrane separation program by whizzer drain pipe, after the solid impurity in rotary drum scrapes, after the processing such as sterilizing, discards.
Through double centrifugal treating, can effectively remove a large amount of precipitations in fermented liquid, be conducive to the carrying out of subsequent handling.
3) membrane sepn
Supernatant liquor carries out classified filtering by 50,000～500,000 molecular weight ceramic membranes, to remove thalline and foreign protein, and fully keeps the activity of antibacterial peptide.
First select 500,000 molecular weight ceramic membranes to carry out first step filtration, the macromole that is greater than 500,000 molecular weight is trapped, be less than the material permeance ceramic membrane of 500,000 molecular weight, adopt 350,000 molecular weight ceramic membranes to carry out the second stage filtration clear liquid filtering through the first step, clear liquid after filtration adopts 150,000 molecular weight ceramic membranes to carry out third stage filtration again, finally selects 50,000 molecular weight ceramic membranes to carry out fourth stage filtration again.
Filtration temperature: 40 DEG C, working pressure: intake pressure is 0.35MPa; Top hole pressure 0.2MPa, imports and exports pressure drop and is no more than 0.15MPa, and supernatant liquor is with 0.5m
3the flow velocity of/h flows through along the surface of ceramic membrane, described ceramic membrane carries out cross flow filter to supernatant liquor, under the driving of pressure-acting, supernatant liquor flows in film pipe, small-molecule substance permeation ceramic membrane, contain the concentrated solution of macromolecular components by ceramic membrane interception, thereby make supernatant liquor reach the object of separation, purifying.
Ceramic membrane equipment used is that " Anhui Plum Membrane Technology Co., Ltd. " produces.
Described membrane sepn control parameter is as shown in table 2 on removing the impact of thalline and foreign protein effect and antibacterial peptide inhibitory potency.
Through lot of experiments checking, as shown in Table 2, the membrane sepn control parameter in employing the present embodiment, removes thalline and foreign protein rate reaches 99.99%, and in the supernatant liquor obtaining, the inhibitory potency of antibacterial peptide reaches 12813.
4) regulate pH value
Supernatant liquor after collection membrane separates, adding citric acid is adjusted to 3.5 by pH value therein, leaves standstill lower floor after 24h and occurs precipitation, then obtain containing sedimentary concentrated solution through membrane sepn elimination part supernatant liquor, and repetition aforesaid operations is until concentrated solution volume is original 50%.
Through verification experimental verification, when obtaining concentrated solution volume and be original 50% through membrane sepn repeatedly, both ensured the content of effective constituent in concentrated solution, be beneficial to again the carrying out of subsequent handling, lay a good foundation for rapidly and efficiently obtaining antibacterial peptide dry powder.
5) spraying is dry
After adding 25% maltodextrin to stir as excipient in concentrated solution, spraying to be dried according to the treatment capacity of 50 L/h obtains antibacterial peptide dry powder, and described spray-dired inlet temperature is 180 DEG C, and air outlet temperature is 90 DEG C.
Spray-drier used is that " Wuxi City Neng Da drying plant company limited " produces.
Through verification experimental verification, select 25% maltodextrin as vehicle, can effectively ensure the inhibitory potency of antibacterial peptide, and be convenient to rapid drying and obtain antibacterial peptide dry powder.
Drug sensitive test: fermented liquid is carried out centrifugal, get supernatant liquor as test group 1, get the dry powder obtaining in embodiment 1 and be dissolved in purified water under aseptic condition, as test group 2, get the dry powder obtaining in embodiment 2 and be dissolved in purified water under aseptic condition, as test group 3, get the dry powder obtaining in embodiment 3 and be dissolved in purified water under aseptic condition, as test group 4, ferment YPD inoculation of medium subtilis, gained fermented liquid carries out centrifugal, get supernatant liquor as a control group, the effective constituent of described YPD substratum comprises: yeast extract paste, peptone and glucose, the preparation of described YPD substratum comprises the following steps: (1) gets yeast extract paste 10g and peptone 20g is dissolved in 900mL purified water, 121 DEG C of sterilizing 20min, (2) get glucose 20g and be dissolved in 100mL purified water, after 0.22 μ m filter filtration sterilization, sneak in the solution of step (1) gained.
Drug sensitive test detects substratum, and its effective constituent comprises: peptone, yeast extract paste, sodium-chlor, glucose and agar.
The preparation that described drug sensitive test detects substratum comprises the following steps: get peptone 1g, yeast extract paste 0.5g, sodium-chlor 1g, glucose 0.5g and agar 1.5g, distilled water is settled to 100mL, fully dissolves 115 DEG C of autoclaving 30min.
Test method: the drug sensitive test after autoclaving is detected to the water-bath insulation that substratum is placed in 50～55 DEG C;
In Bechtop, get the aseptic drug sensitive test of 100mL and detect substratum, add 100uL to detect bacterium bacterium liquid, described detection bacterium is intestinal bacteria, shakes up, and obtains detecting substratum containing bacterium;
What absorption mixed detects substratum 10mL containing bacterium, evenly spreads out cloth at the bottom of 90mm culture dish, is placed on Bechtop it is solidified.
After detecting culture medium solidifying containing bacterium, use the punch tool punching of sterilizing, the aperture of described punch tool is 2.7mm, on culture dish bottom, makes a call to seven loading holes uniformly, by choosing containing bacterium detection substratum in loading hole.
With marking pen be that mark is carried out in seven loading holes in culture dish bottom, described loading hole comprises a negative control loading hole, a positive microbiotic contrast loading hole and five testing sample loading holes, in described negative control loading hole, place sterilized water, described positive microbiotic contrast is placed positive microbiotic in loading hole, described testing sample loading is placed respectively test group 1 in hole, test group 2, test group 3, the testing sample of test group 4 and control group, drawing respectively 5uL testing sample with micropipet adds in each self-corresponding loading hole, and culture dish is placed in to 37 DEG C of incubators cultivates 16 ~ 18 h.
Cultivation results shows to have good bacteriostatic action by the antibacterial peptide finished product preparation of rear extraction process gained provided by the present invention.Measure antibacterial circle diameter with vernier callipers, calculate inhibitory potency.Result as shown in Figure 2, dry powder in visible 3 embodiment of the present invention all has higher inhibitory potency, compared with control group, it is tired and has improved respectively 35%, 31% and 41%, compared with test group 1, tire and declined respectively 12%, 15%, 8%, illustrate that antibacterial peptide still has high tiring after extracting, loss is less.In rear extraction process provided by the present invention, antibacterial peptide loss is lower, ensureing that bacterial metabolism product still has on the basis of high inhibitory potency, simplified leaching process, shortened the production time, reduced fermentation costs.
Claims (10)
1. subtilis produces the rear extraction process of antibacterial peptide, it is characterized in that: described rear extraction process comprise carry out successively put tank, centrifugal, membrane sepn, adjusting pH value and the drying step of spraying.
2. the rear extraction process that a kind of subtilis as claimed in claim 1 produces antibacterial peptide, it is characterized in that: in described membrane sepn step, the supernatant liquor after centrifugal is carried out to classified filtering, 40 DEG C of filtration temperatures, the import and export pressure drop of ceramic membrane equipment is 0.1～0.15MPa, average flux is 0.5m
3/ h, removes thalline and foreign protein rate reaches 99.97%～99.99%, and in the supernatant liquor after membrane sepn, the inhibitory potency of antibacterial peptide is 12689～12813.
3. the rear extraction process that a kind of subtilis as claimed in claim 2 produces antibacterial peptide, is characterized in that: described classified filtering comprises level Four filtration;
The first step is filtered and is adopted 500,000 molecular weight ceramic membranes, the second stage to filter employing 350,000 molecular weight ceramic membranes, and third stage filtration adopts 150,000 molecular weight ceramic membranes, and the fourth stage filters and adopts 50,000 molecular weight ceramic membranes.
4. the rear extraction process that a kind of subtilis as claimed in claim 3 produces antibacterial peptide, it is characterized in that: in described adjusting pH value step: the clear liquid after collection membrane separates, adjust pH value to 3.5, leave standstill after 24h, filtering upper strata supernatant liquor obtains containing sedimentary concentrated solution, repeats filtering operation until concentrated solution volume is to adjust 50% of the front supernatant liquor volume of pH value.
5. the rear extraction process that a kind of subtilis as claimed in claim 4 produces antibacterial peptide, it is characterized in that: after adding 25% maltodextrin to stir as excipient in described concentrated solution, spraying to be dried according to the treatment capacity of 50 L/h obtains antibacterial peptide dry powder.
6. the rear extraction process that a kind of subtilis as claimed in claim 5 produces antibacterial peptide, is characterized in that: described spray-dired inlet temperature is 120～180 DEG C, air outlet temperature is 70～90 DEG C.
7. a kind of subtilis as claimed in claim 1 produces the rear extraction process of antibacterial peptide, it is characterized in that: described in to put the storage time of fermented liquid in storage tank in tank step be 2 hours.
8. the rear extraction process that a kind of subtilis as claimed in claim 1 produces antibacterial peptide, is characterized in that: in described centrifugation step, adopt two continuous centrifuges of series connection centrifugal.
9. the rear extraction process that a kind of subtilis as claimed in claim 3 produces antibacterial peptide, is characterized in that: in described adjusting pH value step, conditioning agent is hydrochloric acid, phosphoric acid or citric acid.
10. the rear extraction process of producing antibacterial peptide as a kind of subtilis of claim 1-9 as described in one of them, it is characterized in that: adopt described rear extraction process to extract compared with the inhibitory potency of the inhibitory potency of the antibacterial peptide dry powder obtaining and antibacterial peptide in putting tank primary fermentation liquid, rate of descent is less than 15%.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104910262A (en) * | 2014-11-27 | 2015-09-16 | 沈阳化工研究院有限公司 | Bacillus subtilis antibacterial peptide, separation and purification method, and application thereof |
| CN106146608A (en) * | 2015-03-16 | 2016-11-23 | 江西科诺生物科技有限公司 | A kind of extraction preparation method of apidaecin |
| CN107674895A (en) * | 2017-10-31 | 2018-02-09 | 李爽 | A kind of production of bacillus production immunomodulatory peptides and extracting method |
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| 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 |
| CN115553402A (en) * | 2022-10-25 | 2023-01-03 | 山东深海生物科技股份有限公司 | Biological polypeptide preparation capable of improving nonspecific immunity function of haliotis discus hannai and application thereof |
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| CN104910262A (en) * | 2014-11-27 | 2015-09-16 | 沈阳化工研究院有限公司 | Bacillus subtilis antibacterial peptide, separation and purification method, and application thereof |
| CN104910262B (en) * | 2014-11-27 | 2019-04-30 | 沈阳化工研究院有限公司 | Antisepsis peptide of wilted hay bacilli and its isolation and purification method, application |
| CN106146608A (en) * | 2015-03-16 | 2016-11-23 | 江西科诺生物科技有限公司 | A kind of extraction preparation method of apidaecin |
| CN107674895A (en) * | 2017-10-31 | 2018-02-09 | 李爽 | A kind of production of bacillus production immunomodulatory peptides and extracting method |
| CN110627881A (en) * | 2019-10-17 | 2019-12-31 | 中国烟草总公司陕西省公司 | Preparation and application of hypersensitive protein for biological prevention and treatment of tobacco mosaic virus disease |
| CN110627881B (en) * | 2019-10-17 | 2022-12-27 | 中国烟草总公司陕西省公司 | Preparation and application of hypersensitive protein for biological prevention and treatment of tobacco mosaic virus disease |
| 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 |
| CN115553402A (en) * | 2022-10-25 | 2023-01-03 | 山东深海生物科技股份有限公司 | Biological polypeptide preparation capable of improving nonspecific immunity function of haliotis discus hannai and application thereof |
| CN115553402B (en) * | 2022-10-25 | 2024-04-05 | 山东深海生物科技股份有限公司 | Biological polypeptide preparation capable of improving nonspecific immunity function of Haliotis discus hand-Mazz and application thereof |
| CN115957148A (en) * | 2023-01-09 | 2023-04-14 | 广州美思生物技术有限公司 | Nutrient containing composite plant antibacterial peptide and microcapsule thereof |
| CN115957148B (en) * | 2023-01-09 | 2023-10-27 | 广州美思生物技术有限公司 | Nutrient containing composite plant antibacterial peptide and microcapsule thereof |
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