CN102505033A - Method for preparing lactoferricin and method for applying lactoferricin in bacterial inhibition of foods - Google Patents
Method for preparing lactoferricin and method for applying lactoferricin in bacterial inhibition of foods Download PDFInfo
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- CN102505033A CN102505033A CN2012100014630A CN201210001463A CN102505033A CN 102505033 A CN102505033 A CN 102505033A CN 2012100014630 A CN2012100014630 A CN 2012100014630A CN 201210001463 A CN201210001463 A CN 201210001463A CN 102505033 A CN102505033 A CN 102505033A
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Abstract
The invention relates to a method for preparing lactoferricin and a method for applying the lactoferricin in bacterial inhibition of foods. The method for preparing the lactoferricin comprises the following steps of: (i) activating genetic engineering bacteria; (ii) performing bacterial lysis; (iii) separating an inclusion body and performing lysis; (iv) extracting fusion protein and performing acid hydrolysis; and (v) separating by using a chromatographic column. The method is feasible, easy to operate and low in cost; and the lactoferricin prepared by the method can be used for bacterial inhibition of aquatic products. The method for applying the lactoferricin comprises the following steps of: cleaning an aquatic product, and soaking in a solution of the lactoferricin at normal temperature or at the temperature of between 0 and 4 DEG C for 0.5 to 10 hours.
Description
Technical field
The present invention relates to a kind of methods for making and using same of fungistat, in particular, relate to a kind of preparation of lactoferricin and the application method in food is antibacterial thereof.
Background technology
Food safety is matters vital to national well-being and the people's livelihood, and the exploitation of the green fungistat of All Pure Nature is the advanced subject in the China and even the world with using.(Lactoferrin, LF) be a kind of natural existence to Lf lactoferrin, the iron that is distributed widely in exocrine secretions such as people and domestic animal milk, saliva, tears combines gp, has safe and harmless characteristics.(Lactoferricin Lfcin) is LF little peptide after the acidol-pepsin hydrolysis in digestive tube to lactoferrin polypeptide.Big quantity research shows that Lfcin has wide spectrum antibacterium, antiviral, anti-mycotic activity, though they do not have the iron ion binding site, fungicidal activity is higher than LF, can be used for the protective foods and the green food additive of development of new.But exploitation Lfcin can run into many difficulties, and wherein, the greatest problem that runs into is that manufacturing cost is too high, and a large amount of difficulties of making are difficult to realize commercialization.Using gene engineering technique changes Bovinelactoferrin peptide (Lfcin B) gene in the other biological over to and expresses, and is the approach that solves the insufficient tool potentiality in industrial Lfcin B source.
Application number " CN200610166536.6 "; The Chinese patent (application) of denomination of invention " preparation method that a kind of Bovinelactoferrin engineering bacteria and antibacterial peptide Bovinelactoferrin are plain " discloses the preparation method who prepares antibacterial peptide Bovinelactoferrin element through the luminous bacillus genetic engineering bacterium; Application number " CN200810110996.6 "; The Chinese patent (application) of denomination of invention " lactoferrin antimicrobial peptide and preparation method thereof with their purposes ", disclose through Lf lactoferrin in the enzymic hydrolysis ox colostrum prepare the method for lactoferrin antimicrobial peptide and in milk powder, sour milk, beverage as the purposes of fungistat; Application number " CN200810116875.2 "; The Chinese patent (application) of denomination of invention " bovine lactoferrin antibacterial peptide fusion protein and encoding sox thereof and application ", the plant that discloses the coding of bovine lactoferrin antibacterial peptide fusion protein and utilized encoding sox to cultivate produces antibacterial peptide as bio-reactor method;
Above-mentioned these patents (application) all do not relate to antibacterial processing, particularly milk and the fishery products that utilize recombinant expressed Bovinelactoferrin peptide of intestinal bacteria and reorganization Bovinelactoferrin peptide to be used for food, particularly eat the edible safety of seafood raw to improve food.
Summary of the invention
The purpose of this invention is to provide a kind of preparation method of Bovinelactoferrin peptide, this is easy to implement the method, and is simple to operate, and colleges and universities express, and with low cost, the Bovinelactoferrin peptide that this method prepares gained has the high-efficiency broad spectrum anti-microbial activity.
Another object of the present invention provides above-mentioned Bovinelactoferrin peptide and is used for fishery products and milk bacteriostatic method of use.
For realizing the object of the invention, technical scheme of the present invention is:
A kind of preparation method of Bovinelactoferrin peptide is characterized in that, this method may further comprise the steps:
(i) genetic engineering bacterium activation: genetic engineering bacterium after activation on the LB of the sulfur acid kantlex substratum, in the fermention medium top fermentation, after 3～5 hours lactose-induced 6～10 hours;
(ii) bacterium cracking: the thalline after will inducing joins in the lysis buffer, 37 ℃ of incubated overnight behind the mixing, the centrifugal supernatant that goes adds equal-volume inclusion body lavation buffer solution, 37 ℃ hatch 2～3 hours after, the centrifugal again supernatant that goes;
(iii) inclusion bodies separating and cracking: the bacterium inclusion body is joined the washing 0.5～1.5 hour of vibrating in the zero(ppm) water; The centrifugal supernatant that goes; And then 10～30min is washed in vibration in inclusion bodies separating liquid; The centrifugal supernatant that goes joined in the inclusion body lysate continuous oscillation 18～22 hours, centrifugal collection supernatant (containing fusion rotein) at last again;
(iv) fusion rotein extracts and acid hydrolysis: precipitate the fusion rotein in the above-mentioned supernatant with 0.68 times of volume of ethanol;-20 ℃ of precoolings are after 10～30 minutes; Centrifugal collecting precipitation and supernatant, with the fusion rotein in 3.5 times of volume of ethanol deposition supernatants, twice ethanol sedimentation is fusion rotein again; With fusion rotein in hydrochloric acid soln, 40～56 ℃ of hydrolysis 25～35h;
(v) gel chromatography separation: fusion rotein acid hydrolysis products pH that regulating step (iv) obtains and elutriant basically identical; Behind membrane filtration, be splined on the CM52 ion exchange column that balance is crossed, the elutriant wash-out is collected the sample peak; The Sephadex G-25 chromatography column that adopts balance to cross carries out desalination to sample; The elutriant wash-out is collected the sample peak, and gained albumen carries out lyophilize and is the Bovinelactoferrin peptide.
In a preferred embodiment of the present invention, said genetic engineering bacterium is: E.coli-pED-Lfcin B BL21 secreting, expressing Bovinelactoferrin peptide fusion protein.
In a preferred embodiment of the present invention, in the step (i), said fermention medium is: Zulkovsky starch 7g/L, Carnis Bovis seu Bubali cream 30g/L, sodium-chlor 6.7g/L.
In a preferred embodiment of the present invention, step (ii), said cellular lysate damping fluid is: N,O-Diacetylmuramidase; Its mass percent is 0.02%, gathers the ethanol octyl phenyl ether, and its volume(tric)fraction is 0.5%; Tris-HCl, its pH value is 8.0, concentration is 50mmol/L; The consumption of said cellular lysate damping fluid is that every gram bacterium adds 4mL;
In a preferred embodiment of the present invention, step (ii) in, said inclusion body lavation buffer solution is: TritonX-100, its percent by volume is 0.2%, Tris-HCl, its pH value is 8.0, concentration is 50mmol/L.
In a preferred embodiment of the present invention, step (ii) in, said centrifugal speed is 8000r/min, the time is 20min.
In a preferred embodiment of the present invention, step (iii) in, the concentration of said urea soln is 2mol/L.
In a preferred embodiment of the present invention, step (iii) in, said inclusion body lysate contains: urea 4mol/L, Tris-HCl (pH8.0) 100mmol/L adds by the consumption of 1g inclusion body with the 4mL lysate.
In a preferred embodiment of the present invention, step (iv) in, said concentration of hydrochloric acid solution is 50mM, the mass content of fusion rotein is 5% in the hydrolytic process.
In a preferred embodiment of the present invention, (v), said filter membrane is 0.45 μ m to step; The balance liquid and the elutriant of balance and wash-out CM52 ion exchange column are respectively the 0.08mol/L ammonium acetate solution, and 0.24mol/L ammonium acetate solution, elution requirement are 0.5mL/min; The balance liquid and the elutriant of balance and wash-out Sephadex G-25 chromatography column are the 0.1M acetic acid soln, and elution requirement is 5mL/min.
The application method of Bovinelactoferrin peptide in fishery products are antibacterial that the present invention prepares gained is: after fishery products are cleaned up; Be immersed in the freshly prepared Bovinelactoferrin peptide solution (after the antibacterial liquid preparation to using the pitch time should not be above 1 hour; Cryopreservation can prolong its validity period); Normal temperature bubble or 0-4 ℃ of condition under, soaked 0.5-10 hour;
In a preferred embodiment of the present invention, the concentration of said Bovinelactoferrin peptide solution is 1000 μ g/mL, and the mass ratio of aquatic product quality and Bovinelactoferrin peptide solution is 1: 1～10.
The preparation method of Bovinelactoferrin peptide of the present invention has following advantage:
1, simple, the safety of the method for preparing the Bovinelactoferrin peptide involved in the present invention; Solve the natural Bovinelactoferrin peptide insufficient problem of originating thereby can be mass-produced; The Bovinelactoferrin peptide that is produced not only expression amount is high, and purity is high, and has anti-microbial activity efficiently.
2, related Bovinelactoferrin peptide is that edible natural is used composition; Harmless, the fishery products pathogenic bacterium are had good inhibition effect, can't develop immunity to drugs; It is residual in treating processes, also can not to produce objectionable impurities, can reach safety, health, green and effect efficiently.In addition, can not have a negative impact, possess the prospect of actual Application and Development the organoleptics propertys such as local flavor of fishery products itself.
3, the method for compound antibacterial liquid treating water product of the present invention is easy to operate, and equipment requirements is low, and good antimicrobial effect is safe in utilization; But present method applied range is suitable for fishery products production and processing link, also can directly use in circulation consumption link.
Description of drawings
Fig. 1 is that Lfcin B is to the fungistatic effect of Vibrio parahemolyticus under the different reserve temperatures, and wherein, concentration of treatment is 1000 μ g/mL; N=3.
Fig. 2 be under the different reserve temperatures Lfcin B to the fungistatic effect of Listeria monocytogenes, wherein,
Concentration of treatment is 1000 μ g/mL; N=3.
Embodiment
In order to make technique means of the present invention, creation characteristic, to reach purpose and effect and be easy to understand and understand, the present invention is done further elaboration below in conjunction with embodiment.
The preparation method of lactoferricin:
(i) genetic engineering bacterium activation: adopt transfering loop to inoculate a prf gene engineering bacteria in the LB liquid nutrient medium of 5mL sulfur acid kantlex after the activation, be seeded in the fermention medium in the ratio of 1.5% (v/v) again and ferment, after 4 hours lactose-induced 7 hours;
Wherein, fermention medium is: Zulkovsky starch 7g/L, Carnis Bovis seu Bubali cream 30g/L, sodium-chlor 6.7g/L
(ii) bacterium cracking: add about 80mL lysis buffer in the thalline after 20g induces; 37 ℃ of incubated overnight behind the mixing, the centrifugal supernatant (8000r/min, 20min) that goes adds equal-volume inclusion body lavation buffer solution; 37 ℃ hatch 2～3 hours after, the centrifugal again supernatant (8000r/min, 20min) that goes;
Wherein, the cellular lysate damping fluid is: N,O-Diacetylmuramidase (Lysozyme), and its mass percent is 0.02%, gathers ethanol octyl phenyl ether (Triton X-100), its percent by volume is 0.5%, Tris-HCl (pH8.0), its concentration is 50mmol/L; The inclusion body lavation buffer solution is that its percent by volume of Triton X-100 is 0.2%, and its concentration of Tris-HCl (pH8.0) is 50mmol/L.
(iii) inclusion bodies separating and cracking: add about 100mL zero(ppm) water in the 10g bacterium inclusion body; Vibration washing centrifugal supernatant (8000r/min, 20min) that goes after 1 hour; And then adding 100mL 2mol/L urea soln vibration washing 20min; The centrifugal supernatant (8000r/min, 20min) that goes adds 40mL inclusion body lysate, continuous oscillation 20 hours at last again, and centrifugal (8000r/min, 20min) collects supernatant (containing fusion rotein);
Wherein, the inclusion body lysate contains: urea 4mol/L, Tris-HCl (pH8.0) 100mmol/L.
(iv) fusion rotein extracts and acid hydrolysis: with the (iii) fusion rotein in the supernatant of 0.68 times of volume of ethanol settling step;-20 ℃ of precoolings are after 20 minutes; 9000r/min centrifugal collecting precipitation and supernatant, with the fusion rotein in 3.5 times of volume ethanol deposition supernatants, twice ethanol sedimentation is fusion rotein again; With the 5g fusion rotein in 100mL 50mM hydrochloric acid soln, 48 ℃ of hydrolysis 30h;
(v) gel chromatographic columns separates: fusion rotein acid hydrolysis products pH that regulating step (iv) obtains and elutriant basically identical, behind 0.45 μ m membrane filtration, be splined on the CM52 ion exchange column of crossing through 150mL 0.08mol/L ammonium acetate balance, and the 0.24mol/L ammonium acetate carries out wash-out; Elution requirement is 0.5mL/min; Collect the sample peak, the Sephadex G-25 chromatography column that adopts 100mL 0.1M acetic acid soln balance to cross carries out desalination to sample, the elutriant wash-out; Elutriant is the 0.1M acetic acid soln; Elution requirement is 5mL/min, collects the sample peak, and gained albumen carries out lyophilize and is the Bovinelactoferrin peptide.
The Bovinelactoferrin peptide of embodiment 1 preparation gained is shown that through extracorporeal bacteria inhibitor test it has good bacteriostasis property to Vibrio parahemolyticus and Listeria monocytogenes, and the result sees Fig. 1 and Fig. 2.
Embodiment 2
To inoculate Vibrio parahemolyticus Octopus meat 25g places 225mL antibacterial liquid [embodiment 1 isolating Bovinelactoferrin peptide: 2000 μ g/mL] to handle 4 ℃ of following storages 3 days of low temperature after 30 minutes; Adopt National Standard Method to measure Vibrio parahemolyticus and total plate count, and with saline water as blank.The result shows, compares with control group, and Vibrio parahemolyticus content has reduced by 4.1 * 10 in the Octopus meat that antibacterial liquid is handled
5CFU/g.
Embodiment 3
To inoculate Vibrio parahemolyticus Octopus meat 25g places 225mL antibacterial liquid [embodiment 1 isolating Bovinelactoferrin peptide concentration: 3000 μ g/mL] to handle 4 ℃ of following storages 3 days of low temperature after 30 minutes; Adopt National Standard Method to measure Vibrio parahemolyticus and total plate count, and with saline water as blank.The result shows, compares with control group, and Vibrio parahemolyticus content has reduced by 5.1 * 10 in the Octopus meat that antibacterial liquid is handled
5CFU/g.
Embodiment 4
To inoculate Vibrio parahemolyticus Octopus meat 25g places 225mL antibacterial liquid [embodiment 1 isolating Bovinelactoferrin peptide concentration: 4000 μ g/mL] to handle 4 ℃ of following storages 3 days of low temperature after 30 minutes; Adopt National Standard Method to measure Vibrio parahemolyticus and total plate count, and with saline water as blank.The result shows, compares with control group, and Vibrio parahemolyticus content has reduced by 5.4 * 10 in the Octopus meat that antibacterial liquid is handled
5CFU/g.
Embodiment 5
Add isolating Bovinelactoferrin peptide (Lfcin B final concentration 2mg/mL) among the 40mg embodiment 1 in the 20mL pure milk, room temperature held 5 days is calculated total plate count with reference to GB4789.2-2010.The result shows that control group total plate count increment is 2.7 * 10
9CFU/g, treatment group is 1.6 * 10
5CFU/g.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification sheets just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.
Claims (12)
1. the preparation method of a Bovinelactoferrin peptide is characterized in that, this method may further comprise the steps:
(i) genetic engineering bacterium activation: genetic engineering bacterium after activation on the LB of the sulfur acid kantlex substratum, in the fermention medium top fermentation, after 3～5 hours lactose-induced 6～10 hours;
(ii) bacterium cracking: the thalline after will inducing joins in the lysis buffer, 37 ℃ of incubated overnight behind the mixing, the centrifugal supernatant that goes adds equal-volume inclusion body lavation buffer solution, 37 ℃ hatch 2～3 hours after, the centrifugal again supernatant that goes;
(iii) inclusion bodies separating and cracking: the bacterium inclusion body is joined the washing 0.5～1.5 hour of vibrating in the zero(ppm) water; The centrifugal supernatant that goes; And then 10～30min is washed in vibration in inclusion bodies separating liquid; The centrifugal supernatant that goes joined in the inclusion body lysate continuous oscillation 18～22 hours, centrifugal collection supernatant (containing fusion rotein) at last again;
(iv) fusion rotein extracts and acid hydrolysis: precipitate the fusion rotein in the above-mentioned supernatant with 0.68 times of volume of ethanol;-20 ℃ of precoolings are after 10～30 minutes; Centrifugal collecting precipitation and supernatant, with the fusion rotein in 3.5 times of volume of ethanol deposition supernatants, twice ethanol sedimentation is fusion rotein again; With fusion rotein in hydrochloric acid soln, 40～56 ℃ of hydrolysis 25～35h;
(v) chromatographic column is separated: fusion rotein acid hydrolysis products pH that regulating step (iv) obtains and elutriant basically identical; Behind membrane filtration, be splined on the CM52 ion exchange column that balance is crossed, the elutriant wash-out is collected the sample peak; The Sephadex G-25 chromatography column that adopts balance to cross carries out desalination to sample; The elutriant wash-out is collected the sample peak, and gained albumen carries out lyophilize and is the Bovinelactoferrin peptide.
2. method according to claim 1 is characterized in that, in the step (i), said genetic engineering bacterium is: E.coli-pED-Lfcin B BL21 secreting, expressing Bovinelactoferrin peptide fusion protein.
3. method according to claim 1 is characterized in that, in the step (i), said fermention medium is Zulkovsky starch 7g/L, Carnis Bovis seu Bubali cream 30g/L, sodium-chlor 6.7g/L.
4. method according to claim 1 is characterized in that step (ii); Said cellular lysate damping fluid is: N,O-Diacetylmuramidase, and its mass percent is 0.02%, gathers the ethanol octyl phenyl ether; Its volume(tric)fraction is 0.5%, Tris-HCl, and its pH value is 8.0; Concentration is 50mmol/L, and the consumption of said cellular lysate damping fluid is that every gram bacterium adds 4mL;
5. method according to claim 1 is characterized in that, step (ii) in, said inclusion body lavation buffer solution is: Triton X-100, its percent by volume is 0.2%, Tris-HCl, its pH value is 8.0, concentration is 50mmol/L.
6. method according to claim 1 is characterized in that, step (ii) in, said centrifugal speed is 8000r/min, the time is 20min.
7. method according to claim 1 is characterized in that, step (iii) in, said inclusion bodies separating liquid is the urea soln of 2mol/L.
8. method according to claim 1; It is characterized in that; Step (iii) in, said inclusion body lysate is urea-Tris-HCl solution (pH8.0), wherein the concentration of urea is 4mol/L; The concentration of Tris-HCl is that 100mmol/L, pH are 8.0, adds the ratio of 4mL lysate in the 1g inclusion body and carries out.
9. method according to claim 1 is characterized in that, step (iv) in, concentration of hydrochloric acid solution is 50mM, the mass content of fusion rotein is 5% in the hydrolytic process.
10. method according to claim 1 is characterized in that, (v), said filter membrane is 0.45 μ m to step; The balance liquid and the elutriant of balance and wash-out CM52 ion exchange column are respectively the 0.08mol/L ammonium acetate solution, and 0.24mol/L ammonium acetate solution, elution requirement are 0.5mL/min; The balance liquid and the elutriant of balance and wash-out Sephadex G-25 chromatography column are the 0.1M acetic acid soln, and elution requirement is 5mL/min.
11. each described method of claim 1-10 prepares the application method of Bovinelactoferrin peptide in food is antibacterial of gained; After fishery products are cleaned up, be immersed in the freshly prepared Bovinelactoferrin peptide solution, the normal temperature bubble or 0-4 ℃ of condition under; Soaked 0.5-10 hour
12. method according to claim 11 is characterized in that, the concentration of said Bovinelactoferrin peptide solution is 1000 μ g/mL, and the mass ratio of aquatic product quality and Bovinelactoferrin peptide solution is 1: 1～10.
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| CN108341866A (en) * | 2018-04-03 | 2018-07-31 | 安徽农业大学 | A method of the extraction separation ferritin from silkworm blood |
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Application publication date: 20120620 |