CN101849608A - Rape-seed meal antibacterial peptide - Google Patents
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Abstract
The invention provides a new antibacterial peptide which plays a role in inhibiting molds in feed. The antibacterial peptide is derived from a rape-seed meal, has the advantages of high bacteriostatic activity, small molecular weight, high stability, safety and nontoxicity and shows better bacteriostatic effect than a single chemical mildew preventive in a feed rot test. The invention also provides a separation and purification method, a stability test method and an animal acute toxicity test method and an application method for preventing molds in the feed.
Description
One, technical field
The present invention relates to can be used for producing the rape-seed meal antibacterial peptide of feed and food preservative.
Two, background technology
Adding anticorrisive agent, mould inhibitor etc. in feed is one of effective measures that prolong the feed shelf-life, but these feed preservatives all are chemical agents, have residue problem, produces toxic and side effect, and the meeting remote-effects are to health.Antibiotic adds in the feed long-standing as antibiotic growth regulator.It can reduce the incidence of disease of animal; Suppress harmful microorganism in the enteron aisle, improve animal the nutrient absorbing rate is had tangible economic benefit.But, antibiotic in animal body with animal product in residual, and the resistance to the action of a drug problem that produces of pathogen has produced negative influence to human beings'health and environment.Along with antibiotic a large amount of, be widely used in the feed and people more and more higher to the requirement of food quality, people deepen day by day to the side effect understanding of its use, antibiotic had been faced with already at feed and had eliminated or the situation of forbidding.And chemical preservative, mould inhibitor and antibiotic arrive intestines and stomach along with feed, can influence normal microorganism species in the body, destroy balance therebetween, cause the disorders of digestion, and then cause disease of digestive tracts such as chronic diarrhea, seek novel, safe antiseptic and replace antibiotic, become an important content of current domestic and international feed subject.
Antibacterial peptide (antimicrobial peptides, AMPs) because of having the antibacterial activity of wide spectrum, the activity of antimycotic efficiently, virus and protozoon; Heat stability is good; Antibiotic mechanism uniqueness, characteristic such as be difficult for developing immunity to drugs is just causing people's attention, is expected to become substitute one of antibiotic product.Discover that antibacterial peptide extensively is present in prokaryotes, plant and the animal body, and in the natural defending system of biology, play an important role.Antibacterial peptide comprises antibacterium polypeptide (antibacterial peptides) and anti-fungus polypeptide (antifungal peptides).
At present, many researchers have done some relevant researchs to antibacterial peptide.Experimental studies have found that, the king crab element comprises that to the harmful microorganism in the multiple feed pathogenic and relative pathogenic feed bacterium, feed mould etc. all have bactericidal action efficiently, are expected to become a kind of green feed additive new and effective, environmental protection and are applied to the preservation and antisepsis of feed.Discovery chitterlings antibacterial peptides such as Ma Weiming all have in various degree inhibitory action to 11 strain bacteriums such as chicken, swine escherichia coli strain, white diarrhea salmonella strain, staphylococcus albus, staphylococcus aureus, fish pathogenic hydrophila gingivalis.People such as Wen Liufa find no matter to be in day growth speed, the relative weight gain rate, feed coefficient, survival rate, or the premunition aspect all is significantly increased by add antibacterial protein or antibacterial peptide in the feed of livestock and poultry fish.In addition, report abroad that antibacterial peptide also has certain effect to feedstuff mildew.
Domestic antibacterial peptide research mainly concentrates on the physicochemical property of animal and microbial source antibacterial peptide, and mechanism of action and genetic engineering application facet are less relatively to the research of plant-source antibacterial peptide.Sichuan University's resource microbiology and microorganism biological technology Sichuan Province key lab are all screening plant-source antibacterial albumen and peptide for a long time, confirm that by screening a lot of plants all contain antibacterial protein and peptide matters.Particularly the cabbage type rape of China's establishing in large scale is a rape big producing country, and also output is huge for byproduct rape cake afterwards and extract oil, for obtaining of antibacterial peptide provides rich in natural resources.
The invention provides isolation and purification method and stability test method, the animal acute toxicity test method of rape-seed meal antibacterial peptide and a kind of application process that prevents mould in the feed is provided.
Three, summary of the invention
The present invention relates to derive from the antibacterial peptide of rape cake (Rape-seed meal), suppress effect preferably causing in the feed that some fungi that goes mouldy has.
Feed causes feed quality because of microorganism especially mould contamination and descends, even influences the health of animal, brings enormous economic loss for the production of fodder enterprise and the herding producer.The ramp under suitable temperature, humidity and nutrient environment of the various moulds of feed mold causes, and consequently makes feed generate heat, lump, have musty, even changes the feed color and produce mycotoxin.Be subjected to the feed of mycotic infection,, and the constituent of feed is changed or decompose, cause the nutritive value of feed seriously to reduce because fungus growth consumes the nutriment in the feed.In addition, animal edible by the feed of mould contamination after, mycotoxin can remain in animal body and the metabolite thereof, thereby may cause animal food to pollute, and by food chain health is produced harm greatly again.Therefore, adopt the mould inhibitor of high-efficiency low-toxicity that mould in the feed and toxin thereof are prevented to handle, for guaranteeing feeding quality, the life security of protection people and animal has crucial meaning.
Recent study finds that antibacterial peptide not only has the broad-spectrum high efficacy bactericidal activity, also has good stability, good water solubility, antibiotic mechanism uniqueness, to characteristics such as the higher mammal normal cell are harmless.Antibacterial protein (peptide) comprises that in agricultural the research in the production of fodder is also deep gradually, but research focuses mostly in adding antibacterial peptide with the prevention Animal diseases in feed, thereby become aspects such as traditional antibiotic sub, but the research that its exploitation is become the Midew preventive for feed aspect but seldom.
The purpose of this invention is to provide a kind of new antibacterial peptide, can be used for the control of the rot fungi of feed even food.From the water-soluble substances of rape cake, be separated to little peptide with antifungal activity.This active peptide heat endurance is strong, and fungistatic effect is the most obvious in pH7～8.Animal acute toxicity test is the result show, the antibacterial peptide of extraction is an innocuous substance.By relatively finding mould in the feed is inhibiting with several anticorrisive agents, the fungicidal properties of rape grouts antibacterial peptide extract is better than grace and draws ancient cooking vessel (Enradin), fumaric acid, be only second to that composite type mouldproof agent is mould must to be restrained, can develop becomes a kind of plant-derived efficiently feed anticorrosion agent.
Another object of the present invention provides the isolation and purification method of a cover rape-seed meal antibacterial peptide.Antibacterial peptide of the present invention can with such as saltout, molecular sieve and multi-dimensional chromatograph technology etc. separate.Consider the cost problem, can be applied with the form of antibacterial peptide primary extract.
A further object of the present invention provides the application process of a kind of rape-seed meal antibacterial peptide in feedstuff mildew, comprises consumption and condition etc.
Step is:
1) medium preparation: PDA culture medium (potato potato culture)
Potato 200g, glucose 20g, agar 20g, water 1000mL, pH nature;
121 ℃ of sterilization 30min.
2) antifungal activity detection method
1. the preparation of spore suspension
Get the PDA flat board that covers with fungus colony (having formed conidium), add an amount of sterilized water, glass bar stirs the wash-out spore gently.Then this eluent is filtered, remove mycelia, add an amount of sterilized water and make 3～5 * 10
4The spore suspension that individual spore/a mL left side has is put under 4 ℃ of conditions and is preserved.Strains tested is oranges and tangerines green mold and aspergillus niger.
2. punch method
Make the PDA flat board, draw the spore suspension of 100 μ L for the examination bacterial classification, sterile working evenly is applied on the flat board, dries.With the card punch of 6mm diameter, vertically punching on PDA spore flat board.Add sample or sterilized water to flat full, the positive placement, 28 ℃ of incubators are cultivated 5d.Measure antibacterial circle diameter.
3) separation and purification of rape-seed meal antibacterial peptide
1. the preparation of rape protein crude extract
Take by weighing a certain amount of rape cake and grind, add the Tris-HCl buffer solution of the pH8.0 of 200mL0.05mol/L, behind 4 ℃ of placement 12h, 4 ℃, 4000r/min is centrifugal, and 30min gets supernatant.
2. ammonium sulfate precipitation crude protein
The supernatant saturation degree is 60 ± 5% ammonium sulfate precipitation, and 4 ℃ of placements are spent the night, 4 ℃, and 14, the centrifugal 20min of 000r/min gets precipitation, uses the Tris-HCl buffer solution dissolving of the pH8.0 of 1mL 0.05mol/L.
3. desalination
Crude extract is put into bag filter, and dislysate was selected the Tris-HCl buffer solution of the pH 8.0 of 0.05mol/L for use, changed one time dislysate every 2 hours.
4. molecular sieve column chromatography
Sephadex G-100 gets sample on the extract, uses the Tris-HCl buffer solution elution of the pH8.0 of 0.05mol/L, and flow velocity 0.15mL/min, every 3min collect 1 pipe, by the pipe collection, is the OD of the every pipe of blank determination with the eluent
280Value.And test the bacteriostatic activity of every pipe eluent.
5. (High Performance Liquid Chromatography, HPLC) purifying is further purified the collection liquid with fungistatic effect high performance liquid chromatography.Detect each peak bacteriostatic activity, collect the antibacterial activity part.
6. parting liquid concentrates
The parting liquid freeze drying that will have antibacterial activity, powder is standby with the Tris-HCl buffer solution dissolving of the pH8.0 of the 0.05mol/L of proper volume.
4) determination of activity of rape-seed meal antibacterial peptide
1. fungi is seeded on the PDA flat board, cultivated three days for 28 ℃, allow it form ripe spore.Strains tested Gibberella zeae, oranges and tangerines green mold, aspergillus niger, aspergillus flavus.
2. collude the spore that takes a morsel (sterile working) with transfer needle and sneak in the 2ml sterilized water, add in the PDA culture medium (acillin that has added 100 μ g/ml) of about 50 ℃ of about 200ml behind the concuss, mixing a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices cools off stand-by immediately.
3. during determination of activity, with 6mm diameter card punch, vertically punching on PDA spore flat board.Add sample to dull and stereotyped, the positive placement, 28 ℃ of incubators are cultivated 5d.Measure antibacterial circle diameter.
5) polyacrylamide gel electrophoresis
The Tricine-SDS-PAGE that separates little peptide, resolving gel concentration 4% concentrates gum concentration 15%, and sample adds reproducibility sample buffer (containing mercaptoethanol) and irreducibility sample buffer (not containing mercaptoethanol) respectively, in boiling water, boiled 5-10 minute, and cooled off and get sample on the 20 μ l after centrifugal.
6) stability analysis of antibacterial peptide
1. heat stability test
The antibacterial peptide parting liquid is handled 15min respectively under 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, 120 ℃, with the oranges and tangerines green mold serves as for the examination bacterium, with former antibacterial peptide parting liquid in contrast, repeat 3 times, measure its antibacterial circle diameter, each is handled and repeats 3 times, determines its heat endurance according to the antibacterial circle diameter size.
2. ph stability detects
It is 3,4,5,6,7 five processing that antibacterial peptide parting liquid 5mL is adjusted pH with 1mol/L HCl; Other gets 5mL antibacterial protein (peptide) parting liquid is 9,10 two processing with 1mol/LNaOH adjustment pH, with former antibacterial peptide parting liquid (pH8) in contrast, for being tried bacterium, each is handled and repeats 3 times, determines its stability according to the antibacterial circle diameter size with the oranges and tangerines green mold.
7) to the inhibiting scanning electron microscopic observation of fungi
In the bacteriostatic experiment of oranges and tangerines green mold, fungistatic effect does not take place in the flat board that does not add the rape-seed meal antibacterial peptide extract, the flat board that adds the rape-seed meal antibacterial peptide extract has produced fungistatic effect, downcut the culture medium (A) of the former mycelia normal growth and the culture medium in the latter (B) inhibition zone respectively, carry out electron microscopic observation.
8) animal acute toxicity test of rape-seed meal antibacterial peptide extract
Select 20 of Kunming kind small white mouses, wherein female 10, male 10, body weight is 20 ± 2g.Animal used as test is divided into 2 groups at random, 10 every group, an administration group and a control group.Male and female are separately fed.Irritating stomach 0.4ml (having reached maximum volume) by the per 10 gram body weight of mouse is subjected to the reagent thing (the rape-seed meal antibacterial peptide extract 0.8g/mL), is administered once in one day.Observe immediately after the administration to 6 hours, observe later every day, and continuous 7 days, the 8th day anatomic observation.
9) rape cake protein extract is to mould inhibitory action in the feed.
1. bacteriostatic activity test
Adopt the plate inhibition zone method, with the variable concentrations for preparing the mildew-resistant material---rape-seed meal antibacterial peptide, grace draw ancient cooking vessel pre-mixing agent, fumaric acid, mould must the gram respectively of composite type mouldproof agent to be added to mixing in the quantitative mould medium that has dissolved quantitatively by 0.8%, 0.25%, 0.08%, 0.05%, pour in the culture dish of sterilization, treat that culture medium cools off the back and inoculates various test strain in the centre.Because grace draws ancient cooking vessel to be mainly used in the disease of digestive tract of control domestic animal, aspect mildew-proof function, do not appear in the newspapers, so its addition increases to 250 times of normal use amount when testing.Blank group is set simultaneously, places (28 ℃) cultivation in the climatic chamber, observe the growing state of mould.
2. feedstuff mildew experiment
Rape-seed meal antibacterial peptide extract, grace draw the mould addition that must restrain respectively by 0.8%, 0.25%, 0.08%, 0.05% of ancient cooking vessel, fumaric acid, composite type mouldproof agent to add in the powder, and establish blank group (CK).Above-mentioned each composition carries out strenuous test and routine test respectively.
Strenuous test: this method is to improve the feed moisture, worsens environment (high temperature, high humidity), impels feed mouldy as early as possible, and the order of severity and mensuration total number of molds are judged anti-mold effect according to going mouldy.Ratio in design in the powder of 18% water content is added the mildew-resistant material, puts incubator (36 ℃ of temperature, relative humidity is more than 95%) and cultivates.
Routine test: the ratio in design in normal feed (the natural moisture of powder is 12.58%) is added the mildew-resistant material, blank group is set simultaneously, store under natural environment that (the duration of test temperature is 25 ℃～30 ℃, relative air humidity is about 80%), each test specimen adopts independent packaging, is convenient to sampling.Regularly carry out the mensuration of moisture, total number of molds, every day time sight room temperature, material temperature, relative air humidity and feed mold situation etc.
3. assay method
The mensuration of mold colony sum: the assay method of pressing total number of molds in the GB/T 13092-2006 feed is measured.
The mensuration of moisture: the assay method of pressing GB/T 6435-1986 feed moisture is measured.
Four, the specific embodiment
1) medium preparation: PDA culture medium (potato potato culture)
Potato 200g, glucose 20g, agar 20g, water 1000mL, pH nature;
121 ℃ of sterilization 30min.
2) antifungal activity detection method
1. the preparation of spore suspension
Get the PDA flat board that covers with fungus colony (having formed conidium), add an amount of sterilized water, glass bar stirs the wash-out spore gently.Then this eluent is filtered with 4 layers of sterile gauze, remove mycelia, add an amount of sterilized water and make 3～5 * 10
4Spore suspension about individual spore/mL is put under 4 ℃ of conditions and is preserved.Strains tested is oranges and tangerines green mold and aspergillus niger.
2. punch method
Make the PDA flat board, draw the spore suspension of 100 μ L for the examination bacterial classification, sterile working evenly is applied on the flat board, dries.With the card punch of 6mm diameter, vertically punching on PDA spore flat board.Add sample or sterilized water to flat full, the positive placement, 28 ℃ of incubators are cultivated 5d.Measure antibacterial circle diameter.
3) separation and purification of rape-seed meal antibacterial peptide
1. the preparation of rape protein crude extract
Take by weighing rape cake 40g and grind, add the Tris-HCl buffer solution of the pH8.0 of 200mL 0.05mol/L, behind 4 ℃ of placement 12h, 4 ℃, 4000r/min is centrifugal, and 30min gets supernatant.
2. ammonium sulfate precipitation crude protein
Supernatant 200mL is divided into 5 parts, uses ammonium sulfate precipitation, and saturation degree is respectively 30%, 40%, and 50%, 60% and 70%4 ℃ of placement is spent the night, and 4 ℃, 14, the centrifugal 20min of 000r/min gets precipitation, uses the Tris-HCl buffer solution dissolving of the pH8.0 of 1mL 0.05mol/L.Detect its fungistatic effect, filter out saturation degree, saltout by this saturation degree with best fungistatic effect.
3. desalination
Crude extract is put into bag filter, and dislysate was selected the Tris-HCl buffer solution of the pH 8.0 of 0.05mol/L for use, changed one time dislysate every 2 hours.
4. molecular sieve column chromatography
Sephadex G-100 gets sample on the extract, uses the Tris-HCl buffer solution elution of the pH8.0 of 0.05mol/L, and flow velocity 0.15mL/min, every 3min collect 1 pipe, by the pipe collection, is the OD of the every pipe of blank determination with the eluent
280Value.And test the bacteriostatic activity of every pipe eluent.
5. high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) purifying
Collection liquid with fungistatic effect is further purified.Detect each peak bacteriostatic activity, collect the antibacterial activity part.
6. parting liquid concentrates
The parting liquid freeze drying that will have antibacterial activity, powder is standby with the Tris-HCl buffer solution dissolving of the pH8.0 of the 0.05mol/L of proper volume.
4) determination of activity of rape-seed meal antibacterial peptide
1. fungi is seeded on the PDA flat board, cultivated three days for 28 ℃, allow it form ripe spore.Strains tested Gibberella zeae, oranges and tangerines green mold, aspergillus niger, aspergillus flavus.
2. collude the spore that takes a morsel (sterile working) with transfer needle and sneak in the 2ml sterilized water, add in the PDA culture medium (acillin that has added 100 μ g/ml) of about 50 ℃ of about 200ml behind the concuss, mixing a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices cools off stand-by immediately.
3. during determination of activity, with 6mm diameter card punch, vertically punching on PDA spore flat board.Add sample to dull and stereotyped, the positive placement, 28 ℃ of incubators are cultivated 5d.Measure antibacterial circle diameter.
5) polyacrylamide gel electrophoresis
The Tricine-SDS-PAGE that separates little peptide, resolving gel concentration 4% concentrates gum concentration 15%, and sample adds reproducibility sample buffer (containing mercaptoethanol) and irreducibility sample buffer (not containing mercaptoethanol) respectively, in boiling water, boiled 5-10 minute, and cooled off and get sample on the 20 μ l after centrifugal.
6) stability analysis of antibacterial peptide
1. heat stability test
The antibacterial peptide parting liquid is handled 15min respectively under 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, 120 ℃, with the oranges and tangerines green mold serves as for the examination bacterium, with former antibacterial peptide parting liquid in contrast, repeat 3 times, measure its antibacterial circle diameter, each is handled and repeats 3 times, determines its heat endurance according to the antibacterial circle diameter size.
2. ph stability detects
It is 3,4,5,6,7 five processing that antibacterial peptide parting liquid 5mL is adjusted pH with 1mol/L HCl; Other gets 5mL antibacterial protein (peptide) parting liquid is 9,10 two processing with 1mol/LNaOH adjustment pH, with former antibacterial peptide parting liquid (pH8) in contrast, for being tried bacterium, each is handled and repeats 3 times, determines its stability according to the antibacterial circle diameter size with the oranges and tangerines green mold.
7) to the inhibiting scanning electron microscopic observation of fungi
In the bacteriostatic experiment of oranges and tangerines green mold, fungistatic effect does not take place in the flat board that does not add the rape-seed meal antibacterial peptide extract, the flat board that adds the rape-seed meal antibacterial peptide extract has produced fungistatic effect, downcut the culture medium (A) of the former mycelia normal growth and the culture medium in the latter (B) inhibition zone respectively, carry out electron microscopic observation.
8) animal acute toxicity test of rape-seed meal antibacterial peptide extract
Select 20 of Kunming kind small white mouses, wherein female 10, male 10, body weight is 20 ± 2g.Animal used as test is divided into 2 groups at random, 10 every group, an administration group and a control group.Male and female are separately fed.Irritating stomach 0.4ml (having reached maximum volume) by the per 10 gram body weight of mouse is subjected to the reagent thing (the rape-seed meal antibacterial peptide extract 0.8g/mL), is administered once in one day.Observe immediately after the administration to 6 hours, observe later every day, and continuous 7 days, the 8th day anatomic observation.
9) rape cake protein extract is to mould inhibitory action in the feed.
1. bacteriostatic activity test
Adopt the plate inhibition zone method, with the variable concentrations for preparing the mildew-resistant material---rape-seed meal antibacterial peptide, grace draw ancient cooking vessel pre-mixing agent, fumaric acid, mould must the gram respectively of composite type mouldproof agent to be added to mixing in the quantitative mould medium that has dissolved quantitatively by 0.8%, 0.25%, 0.08%, 0.05%, pour in the culture dish of sterilization, treat that culture medium cools off the back and inoculates various test strain in the centre.Because grace draws ancient cooking vessel to be mainly used in the disease of digestive tract of control domestic animal, aspect mildew-proof function, do not appear in the newspapers, so its addition increases to 250 times of normal use amount when testing.Blank group is set simultaneously, places (28 ℃) cultivation in the climatic chamber, observe the growing state of mould.
Antibacterial vigor (%)=(contrast bacterium dish expansion diameter-contain the antibacterial material bacterium to coil the expansion diameter) * 100%/contrast bacterium dish expansion diameter.
2. feedstuff mildew experiment
Rape-seed meal antibacterial peptide extract, grace draw the mould addition that must restrain respectively by 0.8%, 0.25%, 0.08%, 0.05% of ancient cooking vessel, fumaric acid, composite type mouldproof agent to add in the powder, and establish blank group (CK).Above-mentioned each composition carries out strenuous test and routine test respectively.
Strenuous test: this method is to improve the feed moisture, worsens environment (high temperature, high humidity), impels feed mouldy as early as possible, and the order of severity and mensuration total number of molds are judged anti-mold effect according to going mouldy.Ratio in design in the powder of 18% water content is added the mildew-resistant material, puts incubator (36 ℃ of temperature, relative humidity is more than 95%) and cultivates.
Routine test: the ratio in design in normal feed (the natural moisture of powder is 12.58%) is added the mildew-resistant material, blank group is set simultaneously, store under natural environment that (the duration of test temperature is 25 ℃～30 ℃, relative air humidity is about 80%), each test specimen adopts independent packaging, is convenient to sampling.Regularly carry out the mensuration of moisture, total number of molds, every day time sight room temperature, material temperature, relative air humidity and feed mold situation etc.
3. assay method
The mensuration of mold colony sum: the assay method of pressing total number of molds in the GB/T 13092-2006 feed is measured.
The mensuration of moisture: the assay method of pressing GB/T 6435-1986 feed moisture is measured.
10) result: the suitableeest saturation degree of saltouing in the rape cake crude protein purification process with ammonium sulfate is 60%, separates the extract that obtains little peptide from the water soluble ingredient of rape cake, and its molecular weight is between 6.2～8.2KD.And be elevated to 90 ℃ in temperature and still keep active, fungistatic effect is the most obvious in pH7～8.This little peptide material is had the obvious suppression effect to trying fungi.This active peptide can clearly influence the normal physiological form of hypha,hyphae and spore, but mouse is had no adverse effects.By finding relatively that to mould in the feed is inhibiting the fungicidal properties of rape-seed meal antibacterial peptide extract is better than grace and draws ancient cooking vessel, fumaric acid, be only second to that composite type mouldproof agent is mould must to be restrained with several anticorrisive agents.
Claims (6)
1. the separation and purification of a rape-seed meal antibacterial peptide, stability and method for testing security comprise: (1) medium preparation; (2) antifungal activity check; (3) a certain amount of rape cake is ground, add the Tris-HCl buffer solution of the pH8.0 of 200mL 0.05mol/L, behind 4 ℃ of placement 12h, 4 ℃, 4000r/min is centrifugal, and 30min gets supernatant; Be 60 ± 5% ammonium sulfate precipitation, desalination (dislysate is selected the Tris-HCl buffer solution of the pH 8.0 of 0.05mol/L for use), molecular sieve column chromatography (Sephadex G-100 again by saturation degree, get sample on the extract, Tris-HCl buffer solution elution with the pH8.0 of 0.05mol/L) and high performance liquid chromatography crude protein is carried out purifying, with the parting liquid freeze drying powdered behind the purifying, the Tris-HCl buffer solution dissolving of the pH8.0 of the 0.05mol/L of usefulness proper volume is standby; (4) rape-seed meal antibacterial peptide that extracts is carried out determination of activity; (5) polyacrylamide gel electrophoresis is measured the molecular size of rape-seed meal antibacterial peptide; (6) rape-seed meal antibacterial peptide that extracts is carried out heat endurance, ph stability analysis; (7) the scanning electron microscopic observation rape-seed meal antibacterial peptide is to the inhibitory action of fungi; (8) select Kunming kind small white mouse, do the animal acute toxicity test of rape-seed meal antibacterial peptide extract; (9) by bacteriostatic activity test and feedstuff mildew experimental check rape-seed meal antibacterial peptide extract inhibitory action to mould in the feed.
2. the crude protein of claim 1 extracts material, is the rape grouts.
3. the purifying crude protein method of claim 1, the required ammonium sulfate saturation degree of described ammonium sulfate precipitation is 60 ± 5%.
4. the purifying crude protein method of claim 1, the required dislysate of described desalination is the Tris-HCl buffer solution of the pH 8.0 of 0.05mol/L.
5. the purifying crude protein method of claim 1, described column chromatography material is Sephadex G-100, eluent is the Tris-HCl buffer solution of the pH8.0 of 0.05mol/L.
6. the strains tested of claim 1 is Gibberella zeae, oranges and tangerines green mold, aspergillus niger, aspergillus flavus.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107455455A (en) * | 2017-09-07 | 2017-12-12 | 西南大学 | A kind of method for adopting rear infectious disease with antibacterial peptide PAF56 control citrusfruits |
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| CN104530204B (en) * | 2014-12-19 | 2017-11-10 | 中国农业科学院油料作物研究所 | A kind of rape cecropin B gene nPRP1 and its application |
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| CN107455455A (en) * | 2017-09-07 | 2017-12-12 | 西南大学 | A kind of method for adopting rear infectious disease with antibacterial peptide PAF56 control citrusfruits |
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