CN101849608B - Rape-seed meal antibacterial peptide - Google Patents
Rape-seed meal antibacterial peptide Download PDFInfo
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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 extend the feed shelf-life, but these feed preservatives are all chemical agents, have residue problem, produces toxic and side effect, and the meeting remote-effects are to health.Antibiotic adds in feed long-standing as antibiotic growth regulator.It can reduce the incidence of disease of animal; Suppress harmful microorganism in enteron aisle, the raising animal has obvious economic benefit to the absorptivity of nutrient.But, antibiotic in animal body with animal product in residual, and the resistance to the action of a drug problem that produces of pathogen, produced negative impact to the mankind's health and environment.Along with antibiotic a large amount of, be widely used in 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 already had been faced with and had eliminated or the situation of forbidding at feed.And chemical preservative, mould inhibitor and antibiotic arrive intestines and stomach along with feed, can affect normal microorganism species in body, destroy balance therebetween, cause the disorders of digestion, and then cause the disease of digestive tracts such as chronic diarrhea, find novel, safe antiseptic and replace antibiotic, become an important content of the outer feed subject of Present Domestic.
Antibacterial peptide (antimicrobial peptides, AMPs) is because having the antibacterial activity of wide spectrum, efficiently the activity of antimycotic, virus and protozoon; Heat stability is good; The Antibacterial Mechanism uniqueness, the characteristic such as be difficult for developing immunity to drugs, just causing people's concern, is expected to one of product become substitute antibiotics.Research finds that antibacterial peptide extensively is present in prokaryotes, plant and animal body, and plays an important role in biological natural defending system.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 multiple feed pathogenic and relative pathogenic feed bacterium, feed mould etc. all have efficient bactericidal action, 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.The discovery Antibacterial Peptide Extracted from Pig Small Intestines such as Ma Weiming all have inhibitory action in various degree to 11 strain bacteriums such as chicken, swine escherichia coli strain, white diarrhea salmonella strain, staphylococcus albus, staphylococcus aureus, fish pathogenic hydrophila gingivalis.The people such as Wen Liufa, by add antibacterial protein or antibacterial peptide in the feed of livestock and poultry fish, 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.In addition, abroad report, 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 relatively less to the research of antimicrobial peptide of plant origins.Sichuan University's resource microbiology and microbial biotechnology Key Laboratory of Sichuan Province are all being screened plant-source antibacterial albumen and peptide for a long time, by screening, confirm that a lot of plants all contain antibacterial protein and peptide matters.Particularly the cabbage type rape of China's establishing in large scale, be rape big producing country, and extract oil, also output is huge for byproduct rape cake afterwards, 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 feed is provided.
Three, summary of the invention
The present invention relates to derive from the antibacterial peptide of rape cake (Rape-seed meal), to causing some fungi gone mouldy in feed, inhibition is preferably arranged.
Feed causes feed quality because of microorganism especially mould contamination and descends, and even affects the health of animal, brings huge economic loss to Feedstuff Enterprises 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 produces mycotoxin.Be subject to the feed of mycotic infection, because fungus growth consumes the nutriment in feed, and under the effect of the contained enzyme of mould, make the constituent of feed change or decompose, cause the nutritive value of feed seriously to reduce.In addition, animal edible by the feed of mould contamination after, mycotoxin can remain in animal body and metabolite thereof, thereby may cause Contaminating Animal Foodstuff, then by food chain, health is produced to harm greatly.Therefore, adopt the mould inhibitor of high-efficiency low-toxicity to prevent to process to mould in feed and toxin thereof, for guaranteeing feeding quality, the life security tool of protection man and animal is of great significance.
Recent study finds, antibacterial peptide not only has the broad-spectrum high efficacy bactericidal activity, also have good stability, good water solubility, Antibacterial Mechanism unique, to characteristics such as the higher mammal normal cell are harmless.Antibacterial protein (peptide) comprises that in agricultural the research in Feed Manufacturing is also deep gradually, but research focuses mostly in adding antibacterial peptide with the prevention Animal diseases in feed, thereby become the aspects such as traditional antibiotic sub, still its exploitation is become to the research of 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 even control of the rot fungi of food of feed.Be separated to the little peptide with antifungal activity from the water-soluble substances of rape cake.This active peptide heat endurance is strong, and in pH7～8, fungistatic effect is the most obvious.The animal acute toxicity test result shows, the antibacterial peptide of extraction is innocuous substance.By relatively finding mould in feed is inhibiting with several anticorrisive agents, the fungicidal properties of rape grouts Antimicrobial Peptide Extracts is better than grace and draws ancient cooking vessel (Enradin), fumaric acid, being only second to that composite type mouldproof agent is mould must gram, can develop and become a kind of efficient plant-derived feed anticorrosion agent.
Another object of the present invention is to provide the isolation and purification method of a set of rape-seed meal antibacterial peptide.Antibacterial peptide of the present invention can with such as saltouing, molecular sieve and multi-dimensional chromatograph technology etc. separated.Consider Cost Problems, can be applied with the form of antibacterial peptide primary extract.
A further object of the present invention is to provide the application process of a kind of rape-seed meal antibacterial peptide in feedstuff mildew, comprises consumption and condition etc.
Step is:
1) culture medium preparation: PDA culture medium (potato potato culture)
Potato 200g, glucose 20g, agar 20g, water 1000mL, pH nature;
121 ℃ of sterilizing 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 appropriate sterilized water, glass bar stirs the wash-out spore gently.Then this eluent is filtered, remove mycelia, add appropriate sterilized water and make 3～5 * 10
4the spore suspension that individual spore/a mL left side has, put under 4 ℃ of conditions and preserve.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 be applied to flat board upper, dries.With the card punch of 6mm diameter, vertical perforating 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 a certain amount of rape cake and grind, add the Tris-HCl buffer solution of the pH8.0 of 200mL0.05mol/L, after 4 ℃ of placement 12h, 4 ℃, 4000r/min is centrifugal, and 30min gets supernatant.
2. ammonium sulfate precipitation crude protein
The ammonium sulfate precipitation that supernatant is 60 ± 5% by saturation degree, 4 ℃ of placements are spent the night, and 4 ℃, the centrifugal 20min of 14,000r/min, get precipitation, uses the Tris-HCl buffer solution of the pH8.0 of 1mL 0.05mol/L to dissolve.
3. desalination
Crude extract is put into to bag filter, and dislysate was selected the Tris-HCl buffer solution of the pH 8.0 of 0.05mol/L, changed one time dislysate every 2 hours.
4. molecular sieve column chromatography
Sephadex G-100, get the extract loading, by the Tris-HCl buffer solution elution of the pH8.0 of 0.05mol/L, and flow velocity 0.15mL/min, every 3min collects 1 pipe, by pipe, collects, and take the OD of eluent as the every pipe of blank determination
280value.And test the bacteriostatic activity of every pipe eluent.
5. high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) purifying is further purified the collection liquid with fungistatic effect.Detect each peak bacteriostatic activity, collect the antibacterial activity part.
6. parting liquid is concentrated
The parting liquid freeze drying that will have antibacterial activity, powder dissolves standby with the Tris-HCl buffer solution 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, cultivates three days, allow it form ripe spore for 28 ℃.Strains tested Gibberella zeae, oranges and tangerines green mold, aspergillus niger, aspergillus flavus.
2. hook with transfer needle the spore (sterile working) that takes a morsel and sneak in the 2ml sterilized water, add after concuss in the PDA culture medium (acillin that has added 100 μ g/ml) of 50 ℃ of left and right of about 200ml, mix immediately a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, cooling stand-by.
3. during determination of activity, with 6mm diameter card punch, vertical perforating 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%, concentrated gum concentration 15%, sample adds respectively reproducibility sample buffer (containing mercaptoethanol) and irreducibility sample buffer (not containing mercaptoethanol), boil 5-10 minute in boiling water, coolingly get 20 μ l loadings after centrifugal.
6) stability analysis of antibacterial peptide
1. heat stability test
The antibacterial peptide parting liquid is processed respectively under 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, 120 ℃ to 15min, take the oranges and tangerines green mold as supplying the examination bacterium, with former antibacterial peptide parting liquid in contrast, repeat 3 times, measure its antibacterial circle diameter, each is processed and repeats 3 times, according to the antibacterial circle diameter size, determines its heat endurance.
2. ph stability detects
It is 3,4,5,6,7 five processing that antibacterial peptide parting liquid 5mL is adjusted to pH with 1mol/L HCl; Separately getting 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, take the oranges and tangerines green mold as tested bacterium, and each is processed and repeats 3 times, according to the antibacterial circle diameter size, determines its stability.
7) to the inhibiting scanning electron microscopic observation of fungi
In the bacteriostatic experiment of oranges and tangerines green mold, do not add the flat board of rape-seed meal antibacterial peptide extract that fungistatic effect does not occur, add the flat board of rape-seed meal antibacterial peptide extract to produce fungistatic effect, cut respectively the culture medium (A) of the former mycelia normal growth and the culture medium in the latter (B) inhibition zone, 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 to 2 groups at random, 10 every group, an administration group and a control group.Male and female are separately fed.By the tested medicine of the every 10 gram body weight gavage 0.4ml (having reached maximum volume) of mouse, (the rape-seed meal antibacterial peptide extract 0.8g/mL), is administered once in one day.After administration, observe immediately 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 feed.
1. bacteriostatic activity test
Adopt the plate inhibition zone method, by the variable concentrations for preparing the mildew-resistant material---rape-seed meal antibacterial peptide, grace are drawn ancient cooking vessel pre-mixing agent, fumaric acid, composite type mouldproof agent is mould must be added in the quantitative mould medium dissolved and mix by 0.8%, 0.25%, 0.08%, 0.05% respectively by gram quantitatively, pour in the culture dish of sterilizing, inoculate various test strain in centre after culture medium is cooling.Draw ancient cooking vessel and be mainly used in preventing and treating the disease of digestive tract of domestic animal due to grace, aspect mildew-proof function, have no report, therefore its addition increases to 250 times of normal use amount during test.Arrange blank group simultaneously, be placed in (28 ℃) in climatic chamber and cultivate, observe the growing state of mould.
2. feedstuff mildew experiment
Rape-seed meal antibacterial peptide extract, grace draw that ancient cooking vessel, fumaric acid, composite type mouldproof agent are mould must gram to be added in powder by 0.8%, 0.25%, 0.08%, 0.05% addition respectively, and establish blank group (CK).Above-mentioned each composition carries out respectively strenuous test and routine test.
Strenuous test: the method is to improve Moisture in Feed content, worsens environment (high temperature, high humidity), impels feed mouldy as early as possible, and according to going mouldy, the order of severity and mensuration total number of molds judge anti-mold effect.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 (Natural Water of powder is divided into 12.58%) is added the mildew-resistant material, arrange blank group simultaneously, store under natural environment that (the duration of test temperature is 25 ℃～30 ℃, relative air humidity is 80% left and right), 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 GB/T 13092-2006 feed is measured.
The mensuration of moisture: the assay method of pressing GB/T 6435-1986 Moisture in Feed is measured.
Four, the specific embodiment
1) culture medium preparation: PDA culture medium (potato potato culture)
Potato 200g, glucose 20g, agar 20g, water 1000mL, pH nature;
121 ℃ of sterilizing 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 appropriate sterilized water, glass bar stirs the wash-out spore gently.Then this eluent is filtered with 4 layers of sterile gauze, remove mycelia, add appropriate sterilized water and make 3～5 * 10
4the spore suspension of individual spore/mL left and right, put under 4 ℃ of conditions and preserve.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 be applied to flat board upper, dries.With the card punch of 6mm diameter, vertical perforating 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 rape cake 40g and grind, add the Tris-HCl buffer solution of the pH8.0 of 200mL 0.05mol/L, after 4 ℃ of placement 12h, 4 ℃, 4000r/min is centrifugal, and 30min gets supernatant.
2. ammonium sulfate precipitation crude protein
Supernatant 200mL, be 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 ℃, the centrifugal 20min of 14,000r/min, get precipitation, uses the Tris-HCl buffer solution of the pH8.0 of 1mL 0.05mol/L to dissolve.Detect its fungistatic effect, filter out the saturation degree with best fungistatic effect, saltoutd by this saturation degree.
3. desalination
Crude extract is put into to bag filter, and dislysate was selected the Tris-HCl buffer solution of the pH 8.0 of 0.05mol/L, changed one time dislysate every 2 hours.
4. molecular sieve column chromatography
Sephadex G-100, get the extract loading, by the Tris-HCl buffer solution elution of the pH8.0 of 0.05mol/L, and flow velocity 0.15mL/min, every 3min collects 1 pipe, by pipe, collects, and take the OD of eluent as the every pipe of blank determination
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 is concentrated
The parting liquid freeze drying that will have antibacterial activity, powder dissolves standby with the Tris-HCl buffer solution 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, cultivates three days, allow it form ripe spore for 28 ℃.Strains tested Gibberella zeae, oranges and tangerines green mold, aspergillus niger, aspergillus flavus.
2. hook with transfer needle the spore (sterile working) that takes a morsel and sneak in the 2ml sterilized water, add after concuss in the PDA culture medium (acillin that has added 100 μ g/ml) of 50 ℃ of left and right of about 200ml, mix immediately a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, cooling stand-by.
3. during determination of activity, with 6mm diameter card punch, vertical perforating 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%, concentrated gum concentration 15%, sample adds respectively reproducibility sample buffer (containing mercaptoethanol) and irreducibility sample buffer (not containing mercaptoethanol), boil 5-10 minute in boiling water, coolingly get 20 μ l loadings after centrifugal.
6) stability analysis of antibacterial peptide
1. heat stability test
The antibacterial peptide parting liquid is processed respectively under 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, 120 ℃ to 15min, take the oranges and tangerines green mold as supplying the examination bacterium, with former antibacterial peptide parting liquid in contrast, repeat 3 times, measure its antibacterial circle diameter, each is processed and repeats 3 times, according to the antibacterial circle diameter size, determines its heat endurance.
2. ph stability detects
It is 3,4,5,6,7 five processing that antibacterial peptide parting liquid 5mL is adjusted to pH with 1mol/L HCl; Separately getting 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, take the oranges and tangerines green mold as tested bacterium, and each is processed and repeats 3 times, according to the antibacterial circle diameter size, determines its stability.
7) to the inhibiting scanning electron microscopic observation of fungi
In the bacteriostatic experiment of oranges and tangerines green mold, do not add the flat board of rape-seed meal antibacterial peptide extract that fungistatic effect does not occur, add the flat board of rape-seed meal antibacterial peptide extract to produce fungistatic effect, cut respectively the culture medium (A) of the former mycelia normal growth and the culture medium in the latter (B) inhibition zone, 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 to 2 groups at random, 10 every group, an administration group and a control group.Male and female are separately fed.By the tested medicine of the every 10 gram body weight gavage 0.4ml (having reached maximum volume) of mouse, (the rape-seed meal antibacterial peptide extract 0.8g/mL), is administered once in one day.After administration, observe immediately 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 feed.
1. bacteriostatic activity test
Adopt the plate inhibition zone method, by the variable concentrations for preparing the mildew-resistant material---rape-seed meal antibacterial peptide, grace are drawn ancient cooking vessel pre-mixing agent, fumaric acid, composite type mouldproof agent is mould must be added in the quantitative mould medium dissolved and mix by 0.8%, 0.25%, 0.08%, 0.05% respectively by gram quantitatively, pour in the culture dish of sterilizing, inoculate various test strain in centre after culture medium is cooling.Draw ancient cooking vessel and be mainly used in preventing and treating the disease of digestive tract of domestic animal due to grace, aspect mildew-proof function, have no report, therefore its addition increases to 250 times of normal use amount during test.Arrange blank group simultaneously, be placed in (28 ℃) in climatic chamber and cultivate, observe the growing state of mould.
Antibacterial Activity (%)=(contrast bacterium dish expansion diameter-containing antibacterial material bacterium dish expansion diameter) * 100%/contrast bacterium dish expansion diameter.
2. feedstuff mildew experiment
Rape-seed meal antibacterial peptide extract, grace draw that ancient cooking vessel, fumaric acid, composite type mouldproof agent are mould must gram to be added in powder by 0.8%, 0.25%, 0.08%, 0.05% addition respectively, and establish blank group (CK).Above-mentioned each composition carries out respectively strenuous test and routine test.
Strenuous test: the method is to improve Moisture in Feed content, worsens environment (high temperature, high humidity), impels feed mouldy as early as possible, and according to going mouldy, the order of severity and mensuration total number of molds judge anti-mold effect.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 (Natural Water of powder is divided into 12.58%) is added the mildew-resistant material, arrange blank group simultaneously, store under natural environment that (the duration of test temperature is 25 ℃～30 ℃, relative air humidity is 80% left and right), 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 GB/T 13092-2006 feed is measured.
The mensuration of moisture: the assay method of pressing GB/T 6435-1986 Moisture in Feed is measured.
10) result: in rape cake crude protein purification process, saltouing by the suitableeest saturation degree of 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, in pH7～8, fungistatic effect is the most obvious.This little peptide material has obvious inhibitory action to tested fungi.This active peptide can clearly affect the normal physiological form of hypha,hyphae and spore, but mouse is had no adverse effects.By relatively finding mould in feed is inhibiting with several anticorrisive agents, the fungicidal properties of rape-seed meal antibacterial peptide extract is better than grace and draws ancient cooking vessel, fumaric acid, and being only second to that composite type mouldproof agent is mould must gram.
Claims (1)
1. the isolation and purification method of a rape-seed meal antibacterial peptide and stability, security and active assay method, it is characterized in that after Rapeseed meal is broken being dissolved in buffer solution preparing protein crude extract, again by ammonium sulfate precipitation crude protein, crude protein desalination, molecular sieve column chromatography and high-efficient liquid phase chromatogram purification, a series of separation and purification operations of freeze drying, obtain the rape-seed meal antibacterial peptide after purifying; Detect antibacterial activity, heat endurance, ph stability and the security of antibacterial peptide, and the check antibacterial peptide is to the inhibition of mould in feed simultaneously; Step is:
1) culture medium preparation:
PDA culture medium: potato 200g, glucose 20g, agar 20g, water 1000mL, pH nature, 121 ℃ of sterilizing 30min;
2) antifungal activity check:
1. the preparation of spore suspension: get to cover with and form conidial fungus colony PDA flat board, add appropriate sterilized water, glass bar stirs the wash-out spore gently; Then this eluent is filtered with 4 layers of sterile gauze, remove mycelia, add appropriate sterilized water to make 3～5 * 10
4the spore suspension of individual spore/mL, put under 4 ℃ of conditions and preserve; 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 strains testeds, sterile working, evenly be applied to dull and stereotyped above, dries; With the card punch of 6mm diameter, vertical perforating on PDA spore flat board, then add sample or sterilized water to flat full, the positive placement, and 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 cake protein crude extract: 40g rape cake is ground, add the Tris-HCl buffer solution of the pH8.0 of 200mL 0.05mol/L, after 4 ℃ of placement 12h, at 4 ℃, under 4000r/min, centrifugal 30min, get supernatant;
2. ammonium sulfate precipitation crude protein: the ammonium sulfate precipitation that supernatant is 60 ± 5% by saturation degree, place under 4 ℃ and spend the night, then at 4 ℃, under 14000r/min, centrifugal 20min, get precipitation, with the Tris-HCl buffer solution of the pH8.0 of 1mL 0.05mol/L, dissolves;
3. desalination: crude extract is put into to bag filter, and dislysate was selected the Tris-HCl buffer solution of the pH8.0 of 0.05mol/L, changed one time dislysate every 2 hours;
4. molecular sieve column chromatography: choosing Sephadex G-100 is filler, gets the extract loading, by the Tris-HCl buffer solution elution of the pH8.0 of 0.05mol/L, and flow velocity 0.15mL/min, every 3min collects 1 pipe, by pipe, collects, and take the OD of eluent as the every pipe of blank determination
280be worth, and test the bacteriostatic activity of every pipe eluent;
5. high performance liquid chromatography purifying: the collection liquid that has fungistatic effect in molecular sieve column chromatography is further purified, detects the bacteriostatic activity of each peak eluent, collect the antibacterial activity part;
6. parting liquid is concentrated: will have the parting liquid freeze drying of antibacterial activity, powder dissolves standby with the Tris-HCl buffer solution 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, cultivates three days under 28 ℃, allow it form ripe spore, strains tested is Gibberella zeae, oranges and tangerines green mold, aspergillus niger, aspergillus flavus;
2. hook with transfer needle the spore that takes a morsel under sterile working and sneak in the 2ml sterilized water, added after concuss adding of 50 ℃ of 200mL in the PDA culture medium of 100 μ g/mL ampicillins, mix immediately a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, cooling stand-by;
3. during determination of activity, with 6mm diameter card punch, vertical perforating on PDA spore flat board, add sample to dull and stereotyped, the positive placement cultivated 5d in 28 ℃ of incubators, then measures antibacterial circle diameter;
5) molecular size of rape-seed meal antibacterial peptide is measured: adopt the Tricine-SDS-PAGE that separates little peptide to measure, resolving gel concentration 4%, concentrated gum concentration 15%, sample adds respectively containing the reproducibility sample buffer of mercaptoethanol with not containing the irreducibility sample buffer of mercaptoethanol, boil 5-10 minute in boiling water, coolingly get 20 μ L loadings after centrifugal;
6) heat endurance of rape-seed meal antibacterial peptide, ph stability detect:
1. heat stability test: the antibacterial peptide parting liquid is processed respectively under 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, 120 ℃ to 15min, take the oranges and tangerines green mold as supplying the examination bacterium, with former antibacterial peptide parting liquid in contrast, measure its antibacterial circle diameter, each is processed and repeats 3 times, according to the antibacterial circle diameter size, determines its heat endurance;
2. ph stability detects: it is 3,4,5,6,7 five processing that antibacterial peptide parting liquid 5mL is adjusted to pH with 1mol/L HCl; Separately getting 5mL antibacterial peptide parting liquid is 9,10 two processing with 1mol/L NaOH adjustment pH, with former antibacterial peptide parting liquid pH 8 in contrast, take the oranges and tangerines green mold as tested bacterium, and each is processed and repeats 3 times, according to the antibacterial circle diameter size, determines its ph stability;
7) inhibitory action of scanning electron microscopic observation rape-seed meal antibacterial peptide to fungi:
In the bacteriostatic experiment of oranges and tangerines green mold, do not add the flat board of rape-seed meal antibacterial peptide extract that fungistatic effect does not occur, add the flat board of the rape-seed meal antibacterial peptide extract after high performance liquid chromatography purification and separation liquid is concentrated to produce fungistatic effect, cut respectively the culture medium of the former mycelia normal growth and the culture medium in latter's inhibition zone, 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 to 2 groups at random, 10 every group, an administration group and a control group, male and female are separately fed; Rape-seed meal antibacterial peptide extract by the every 10 gram body weight gavage 0.4mL 0.8g/mL of mouse after high performance liquid chromatography purification and separation liquid is concentrated, be administered once in one day, observe immediately after administration to 6 hours, observe later every day, continuous 7 days, the 8th day anatomic observation;
9) rape-seed meal antibacterial peptide is to the inhibiting check of mould in feed:
1. bacteriostatic activity test: adopt the plate inhibition zone method, by the variable concentrations for preparing the mildew-resistant material--rape-seed meal antibacterial peptide, grace are drawn ancient cooking vessel pre-mixing agent, fumaric acid, composite type mouldproof agent is mould must be added in the quantitative mould medium dissolved and mix by 0.8%, 0.25%, 0.08%, 0.05% respectively by gram quantitatively, pour in the culture dish of sterilizing, inoculate the oranges and tangerines green mold in centre after culture medium is cooling, aspergillus niger, Gibberella zeae, aspergillus flavus test strain; Draw ancient cooking vessel and be mainly used in preventing and treating the disease of digestive tract of domestic animal due to grace, aspect mildew-proof function, have no report, therefore its addition increases to 250 times of normal use amount during test; Arrange blank group simultaneously, be placed in 28 ℃ of climatic chambers and cultivate, observe the growing state of mould;
Antibacterial Activity (%)=(contrast bacterium dish expansion diameter-containing antibacterial material bacterium dish expansion diameter) * 100%/contrast bacterium dish expansion diameter;
2. feedstuff mildew experiment: rape-seed meal antibacterial peptide extract, grace draw that ancient cooking vessel, fumaric acid, composite type mouldproof agent are mould must gram to be added in powder by 0.8%, 0.25%, 0.08%, 0.05% addition respectively, and establish blank group, above-mentioned each composition carries out respectively strenuous test and routine test;
Strenuous test: the method is to improve Moisture in Feed content, in the deterioration environment of high temperature, high humidity, impels feed mouldy as early as possible, and according to going mouldy, the order of severity and mensuration total number of molds judge anti-mold effect; Ratio in design in the powder of 18% water content is added the mildew-resistant material, is placed in 36 ℃, in the incubator of relative humidity more than 95%, cultivates;
Routine test: be divided into the ratio in design in 12.58% normal powder feed at Natural Water and add the mildew-resistant material, arrange blank group simultaneously, at 25 ℃～30 ℃, store under the natural environment that relative air humidity is 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;
3. assay method:
The mensuration of mold colony sum: the assay method of pressing total number of molds in GB/T 13092-2006 feed is measured;
The mensuration of moisture: the assay method of pressing GB/T 6435-1986 Moisture in Feed is measured.
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| CN104530204A (en) * | 2014-12-19 | 2015-04-22 | 中国农业科学院油料作物研究所 | Rape antibacterial peptide BnPRP1 and application thereof |
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| CN1360833A (en) * | 2000-12-29 | 2002-07-31 | 中国农业大学 | Prepn for earthworm antibacterial peptide |
| CN101280333A (en) * | 2008-02-15 | 2008-10-08 | 山东大学 | Method for preparing penicillium antibacterial peptide from grey rose penicillium |
| CN101602799A (en) * | 2009-07-21 | 2009-12-16 | 中国科学院新疆理化技术研究所 | The fast preparation method of primary antimicrobial polypeptide in a kind of garbanzo |
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| CN1360833A (en) * | 2000-12-29 | 2002-07-31 | 中国农业大学 | Prepn for earthworm antibacterial peptide |
| CN101280333A (en) * | 2008-02-15 | 2008-10-08 | 山东大学 | Method for preparing penicillium antibacterial peptide from grey rose penicillium |
| CN101602799A (en) * | 2009-07-21 | 2009-12-16 | 中国科学院新疆理化技术研究所 | The fast preparation method of primary antimicrobial polypeptide in a kind of garbanzo |
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| CN104530204A (en) * | 2014-12-19 | 2015-04-22 | 中国农业科学院油料作物研究所 | Rape antibacterial peptide BnPRP1 and application thereof |
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