CN109718368A - A kind of Nattokinase and natto antibacterial peptide mix preparation and its preparation method and application - Google Patents
A kind of Nattokinase and natto antibacterial peptide mix preparation and its preparation method and application Download PDFInfo
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Abstract
The present invention provides the preparation methods of a kind of Nattokinase and natto antibacterial peptide mix preparation, belong to biomedicine technical field, the described method comprises the following steps: 1) mixing natto and water, ground, filtered, obtain grinding filtrate；2) solid ammonium sulfate is added in grinding filtrate, stands, takes the first supernatant, sterilize, obtain natto antibacterial peptide solution；3) natto and phosphate buffer are mixed, is ground, obtained abrasive material and phosphate buffer mixing extracts, and centrifugation takes the second supernatant；4) ammonium sulfate solution is added in the second supernatant to be mixed, stands, centrifugation, taking precipitate obtains Nattokinase；5) natto antibacterial peptide solution is mixed, filtration sterilization with Nattokinase, adjusts pH value to 7.0, obtains the mix preparation of Nattokinase and natto antibacterial peptide.The mix preparation that the present invention prepares has the function of inhibiting bacterium, improve immunity of organisms and improving the immune effect of live vaccine.
Description
Technical field
The present invention relates to biomedicine technical field more particularly to a kind of Nattokinase and natto antibacterial peptide mix preparation and
Preparation method and application.
Background technique
Bean product are made by Bacillus natto (hay bacillus) fermentation by soya bean in natto, have stickiness, smell is more smelly, and taste is micro-
Sweet tea not only possesses the nutritive value of soya bean, the digestibility rich in farnoquinone, raising protein, it is often more important that fermentation
Process produces several physiological active substances, has the health-care effect for dissolving internal fibrin and other adjusting physiological functions.
Natto extract refers to the active principle extracted from natto, including Nattokinase, natto isoflavones, soap
Green element and farnoquinone etc.；The wherein green element of soap, can improve constipation, reduce blood lipid, and prevention colorectal cancer reduces cholesterol, softening blood
Pipe, preventing hypertension and artery sclerosis inhibit the functions such as AIDS virus；Natto isoflavones can remove internal carcinogen, mention
High memory, protect liver beauty, anti-aging etc. have positive effect, and the digestibility of food can be improved；Nattokinase has dissolution
The effects of thrombus reduces blood viscosity, improves blood circulation, softening and increase blood vessel elasticity.
But in the prior art to the exploitation of natto extract be usually to the extraction of single component in natto extract with
It utilizes, and lacks the comprehensive utilization to natto extract.
Summary of the invention
The purpose of the present invention is to provide a kind of Nattokinase and natto antibacterial peptide mix preparation and preparation method thereof and answer
With the mix preparation can be improved the immune effect of live vaccine.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides the preparation methods of a kind of Nattokinase and natto antibacterial peptide mix preparation, comprising the following steps:
1) natto and water are mixed, is ground, filtered, obtain grinding filtrate；
2) solid ammonium sulfate is added in step 1) the grinding filtrate, stands, takes the first supernatant, sterilize, received
Beans antibacterial peptide solution；
3) natto and phosphate buffer are mixed, is ground, obtained abrasive material and phosphate buffer mixing, leaching
It mentions, is centrifuged, takes the second supernatant；
4) ammonium sulfate solution is added in step 3) second supernatant to be mixed, stands, centrifugation takes precipitating
Object obtains Nattokinase；
5) step 2) the natto antibacterial peptide solution is mixed, filtration sterilization with Nattokinase described in step 4), is adjusted
PH value is saved to 7.0, obtains the mix preparation of Nattokinase and natto antibacterial peptide；
Step 1)~2) and step 3)~4) between there is no time sequencing limitation.
Preferably, the mass ratio of the step 1) natto and water is 1~3:1.
Preferably, the temperature of grinding described in step 3) is 1~5 DEG C.
Preferably, the temperature of phosphate buffer described in step 3) is 1~5 DEG C；The pH value of the phosphate buffer
It is 7.5~8.0.
Nattokinase and natto the antibacterial peptide mixing being prepared the present invention also provides preparation method described in above scheme
Preparation.
The present invention also provides application of the mix preparation described in above scheme in the drug that preparation improves immunity of organisms.
The present invention also provides mix preparations described in above scheme to inhibit the application in bacterium.
Application the present invention also provides mix preparation described in above scheme as vaccine reinforcing agent.
Preferably, the vaccine is bird vaccine or domestic animals vaccine.
Preferably, the ratio of the mix preparation and domestic animals vaccine is 2~5mL:1~3 part；The mix preparation and fowl
The ratio of class vaccine is the plumage part of 0.1~1mL:1~3.
Beneficial effects of the present invention: the present invention provides the preparation sides of a kind of Nattokinase and natto antibacterial peptide mix preparation
Method, the Nattokinase and natto antibacterial peptide mix preparation that this method prepares have inhibit bacterium, improve immunity of organisms with
And improve the effect of the immune effect of live vaccine.By experimental verification, the Nattokinase and natto that the present invention prepares are anti-
The mix preparation of bacterium peptide is to Escherichia coli, salmonella is antibacterial, staphylococcus aureus and Pseudomonas aeruginosa all have fungistatic effect,
And compared with gentamicin, fungistatic effect is suitable in the test for inhibiting Escherichia coli and salmonella.Also, it is of the invention
The mix preparation of Nattokinase and natto antibacterial peptide, using safe and be conducive to enhance pig circular ring virus inactivated vaccine and chicken large intestine
The immune effect of bacillus inactivated vaccine.
Specific embodiment
The present invention provides the preparation methods of a kind of Nattokinase and natto antibacterial peptide mix preparation, comprising the following steps:
1) natto and water are mixed, is ground, filtered, obtain grinding filtrate；
2) solid ammonium sulfate is added in step 1) the grinding filtrate, stands, takes the first supernatant, sterilize, received
Beans antibacterial peptide solution；
3) natto and phosphate buffer are mixed, is ground, obtained abrasive material and phosphate buffer mixing, leaching
It mentions, is centrifuged, takes the second supernatant；
4) ammonium sulfate solution is added in step 3) second supernatant to be mixed, stands, centrifugation takes precipitating
Object obtains Nattokinase；
5) step 2) the natto antibacterial peptide solution is mixed, filtration sterilization with Nattokinase described in step 4), is adjusted
PH value is saved to 7.0, obtains the mix preparation of Nattokinase and natto antibacterial peptide；
Step 1)~2) and step 3)~4) between there is no time sequencing limitation.
The present invention mixes natto and water, is ground, and filters, and obtains grinding filtrate；The mass ratio of the natto and water
Preferably 1~3:1, more preferably 2:1；The time of the grinding is preferably 5~10min；Temperature of the present invention to the grinding
It is not particularly limited；The equipment of the grinding is preferably homogenizer or tissue grinder instrument；The homogenizer is preferably glass homogenate
Device, more preferably 50mL glass homogenizer.
In the present invention, the filtering preferably uses filtered through gauze；The filter opening aperture of the gauze is preferably 0.4~0.5 μ
M, more preferably 0.45 μm；The number of plies of the gauze is preferably 4~6 layers, and more preferably 5 layers.
The present invention after filtration, preferably further includes the filter residue that will be obtained by filtration and water mixing, extracts, and filtering is soaked
Merge after mentioning filtrate with grinding filtrate described in above scheme, obtains merging filtrate；The mass ratio of the filter residue and water is preferably 1~
3:1, more preferably 2:1；The temperature of the extraction is preferably 1~5 DEG C, and more preferably 4 DEG C；The time of the extraction is preferably
0.5~1.5h, more preferably 1h.
After obtaining merging filtrate, solid ammonium sulfate is added in the present invention in the merging filtrate, is stood, is taken the first supernatant
Liquid, sterilizing, obtains natto antibacterial peptide solution；The additional amount of the solid ammonium sulfate is preferably make ammonium sulfate in merging filtrate full
Reach 60%~65% with degree；The time of the standing is preferably 12~18h, more preferably 14~16h；The temperature of the standing
Preferably 1 DEG C~5 DEG C, more preferably 4 DEG C；The mode of the sterilizing is preferably high pressure sterilization and/or filtration sterilization；The high pressure
The temperature of sterilizing is preferably 115~121 DEG C；The autoclaved time is preferably 15~20min；The filter of the filtration sterilization
Membrane aperture is preferably 0.1 μm or 0.22 μm.
The present invention preferably further includes that solid ammonium sulfate is added in the first supernatant after obtaining the first supernatant, quiet
It sets, takes supernatant again, sterilize；The additional amount of the solid ammonium sulfate is preferably to make the saturation degree of ammonium sulfate in the first supernatant
Reach 60%~65%.
The present invention mixes natto and phosphate buffer, is ground, and obtained abrasive material and phosphate buffer is mixed
It closes, extracts, centrifugation takes the second supernatant；The pH value of the phosphate buffer is preferably 7.5~8.0, and more preferably 7.8；Institute
The temperature for stating phosphate buffer is preferably 1~5 DEG C, and more preferably 4 DEG C.In the present invention, the phosphate buffer is preferred
It is prepared by following methods: dipotassium hydrogen phosphate 5.59g, potassium dihydrogen phosphate 0.41g is dissolved in 1000mL purified water, successively filtered
Degerming, high pressure sterilization；The present invention is not particularly limited the filtration sterilization and autoclaved parameter, using this field routine
Method.
In the present invention, the natto and phosphate buffer mass ratio are preferably 1~3:1, more preferably 2:1；It is described to grind
The temperature of mill is preferably 1~5 DEG C, and more preferably 4 DEG C；The time of the grinding is preferably 5~10min, more preferably 8min；Institute
The equipment for stating grinding is preferably mortar or tissue grinder instrument；The mortar or tissue grinder instrument are preferably cooled to 4 DEG C in advance；The present invention
The effect that the temperature of middle setting grinding is 1~5 DEG C is that enzyme is avoided to inactivate.
In the present invention, the volume ratio of the abrasive material and phosphate buffer is preferably 0.5~1.5:2, and more preferably 1:
2；The temperature of the extraction is preferably 1~5 DEG C, and more preferably 4 DEG C；The time of the extraction is preferably 9~16h, more preferably
12~14h；The revolving speed of the centrifugation is preferably 4000~6000r/min, more preferably 5000r/min；The time of the centrifugation
Preferably 5~10min.
After obtaining the second supernatant, ammonium sulfate solution is added in the second supernatant and mixes by the present invention, stands, from
The heart, taking precipitate obtain Nattokinase；The saturation degree of ammonium sulfate is preferably 57%~63% in the ammonium sulfate solution, more
It is preferably 60%；The process that ammonium sulfate solution is added is preferably with stirring；The time of the standing is preferably 1~5
DEG C, more preferably 4 DEG C；The time of the standing is preferably 9~16h, more preferably 12~14h；The revolving speed of the centrifugation is preferred
For 4000~6000r/min, more preferably 5000r/min；The time of the centrifugation is preferably 5~10min, more preferably
8min。
After obtaining natto antibacterial peptide solution and Nattokinase, natto antibacterial peptide solution is mixed with Nattokinase, mistake
Bacterium is filtered out, pH value is adjusted to 7.0, obtains the mix preparation of Nattokinase and natto antibacterial peptide；The filter hole of the filtration sterilization
Diameter is preferably 0.1 μm or 0.22 μm；The reagent for adjusting pH value is preferably NaOH aqueous solution.
Nattokinase and natto the antibacterial peptide mixing being prepared the present invention also provides preparation method described in above scheme
Preparation.
The present invention also provides application of the mix preparation described in above scheme in the drug that preparation improves immunity of organisms.
The present invention also provides mix preparations described in above scheme to inhibit the application in bacterium；The bacterium is preferably wrapped
Include Escherichia coli and salmonella.
Application the present invention also provides mix preparation described in above scheme as vaccine reinforcing agent；The vaccine is birds
Vaccine or domestic animals vaccine；The ratio of the mix preparation and domestic animals vaccine is preferably 2~5mL:1~3 part；The mix preparation
Ratio with bird vaccine is preferably the plumage part of 0.1~1mL:1~3；The domestic animals vaccine is preferably pig circular ring virus inactivated vaccine；
The bird vaccine is preferably chicken colibacillosis inactivated vaccine.
Application the present invention also provides mix preparation described in above scheme as vaccine reinforcing agent；The birds are January
Age chicken；The domestic animals are 12~15 age in days pigs；It is preferably root that the mix preparation and birds or domestic animals vaccine, which are used cooperatively method,
According to the ratio, with mix preparation dilute birds freeze dried vaccine or domestic animals freeze dried vaccine or with birds aqueous vaccine or domestic animals
Aqueous vaccine passes through the approach vaccine inoculation such as injection, drinking-water, eye droppings collunarium after being sufficiently mixed.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
The preparation method of a kind of Nattokinase of embodiment 1 and natto antibacterial peptide mix preparation
1. commercially available 500g natto is added in 500mL distilled water, it is fully ground with tissue grinder instrument, filtered through gauze obtains
To grinding filtrate and filter residue；
2. pressing filter residue: distilled water=1:1 adds distilled water in filter residue, and extraction 2 times is repeated under conditions of 4 DEG C, is soaked every time
The time mentioned is 1h, obtains extraction filtrate；
3. grinding filtrate is merged with extraction filtrate, 50g solid ammonium sulfate is added, 4 DEG C of standing 12h collect the first supernatant
Liquid；50g solid ammonium sulfate is added in first supernatant again, 4 DEG C of standing 12h collect supernatant, in 121 DEG C of high pressure sterilizations
20min filters up to natto antibacterial peptide solution；
4. the phosphate buffer buffer that the pH=7.8 of 500mL pre-cooling is added in commercially available 500g natto is extracted, pre-
10min is ground in cold tissue grinder instrument, obtains abrasive material；
5. the phosphate buffer low temperature extraction of the pre-cooling pH=7.5 of 2 times of volumes is added in abrasive material, 4 DEG C of refrigerators are put
It sets overnight, 5000r/min is centrifuged 10min, abandons precipitating, takes the second supernatant；
6. the second supernatant to be added to the ammonium sulfate that saturation degree is 60% under 4 DEG C, stirring condition, after being sufficiently mixed
4 DEG C of refrigerators are placed, are settled, overnight, 4 DEG C of next day, 5000r/min is centrifuged 10min, removes supernatant, takes and precipitates up to Nattokinase；
7. Nattokinase is dissolved with resulting natto antibacterial peptide solution, sufficiently mix, then is sieved through 0.1 μm of aseptic filtration
Degerming, adjusting pH value is 7.0 to get Nattokinase and natto antibacterial peptide mix preparation.
The preparation method of a kind of Nattokinase of embodiment 2 and natto antibacterial peptide mix preparation
1. commercially available 1500g natto is added in 500mL distilled water, it is fully ground with tissue grinder instrument, filtered through gauze obtains
To grinding filtrate and filter residue；
2. pressing filter residue: distilled water=3:1 adds distilled water in filter residue, and extraction 2 times is repeated under conditions of 4 DEG C, is soaked every time
The time mentioned is 1h, obtains extraction filtrate；
3. grinding filtrate is merged with extraction filtrate, 80g solid ammonium sulfate is added, stands 15h, the first supernatant of collection；
80g solid ammonium sulfate is added in first supernatant again, stands 15h, supernatant is collected, in 121 DEG C of high pressure sterilization 20min, mistake
It filters up to natto antibacterial peptide solution；
4. the phosphate buffer buffer that the pH=7.8 of 500mL pre-cooling is added in commercially available 1500g natto is extracted,
10min is ground in the tissue grinder instrument of pre-cooling, obtains abrasive material；
5. the phosphate buffer low temperature extraction of the pre-cooling pH=7.8 of 2 times of volumes is added in abrasive material, 4 DEG C of refrigerators are put
It sets overnight, 5000r/min is centrifuged 10min, abandons precipitating, takes the second supernatant；
6. the second supernatant to be added to the ammonium sulfate that saturation degree is 60% under 4 DEG C, stirring condition, after being sufficiently mixed
4 DEG C of refrigerators are placed, are settled, overnight, 4 DEG C of next day, 5000r/min is centrifuged 10min, removes supernatant, takes and precipitates up to Nattokinase；
7. Nattokinase is dissolved with resulting natto antibacterial peptide solution, sufficiently mix, then is sieved through 0.1 μm of aseptic filtration
Degerming, adjusting pH value is 7.0 to get Nattokinase and natto antibacterial peptide mix preparation.
The Nattokinase and natto antibacterial peptide mix preparation that embodiment 3 prepares embodiment 1 carry out quality inspection
It directly visually observes 1. character is upchecked as faint yellow or yellowish transparency liquid.
2. pH value measurement is measured according to existing " Chinese veterinary pharmacopoeia " annex, pH value 7.0.
3. 20% sulfosalicylic acid 2.0mL is added by directly visually observing in protein qualitative reaction sampling 2.0mL, should be
Without turbidity and precipitation, react for feminine gender.
4. protein content determination is measured according to existing " Chinese veterinary pharmacopoeia " annex " Forint phenol method ", protein content
1.8mg/mL should be not less than.
5. Ribose concentration measurement is carried out according to " ribose measuring method " in existing " the transfer factor solution national drug standards "
Measurement, Ribose concentration should be not less than 40 μ g/mL.
6. steriling test is measured according to existing " Chinese veterinary pharmacopoeia " annex, asepsis growth is answered.
7. Bacterial endotoxin test is measured according to existing " Chinese veterinary pharmacopoeia " annex " detection of bacterial endotoxin method ", answer
No more than 10EU/mL.
8. mycoplasma inspection is measured according to existing " Chinese veterinary pharmacopoeia " annex " mycoplasma inspection ", fluid nutrient medium is answered
No PH variation, agar solid plate is without typical " fried egg " shape mycoplasma bacterium colony.
9. heat source inspection is tested according to existing " Chinese veterinary pharmacopoeia " annex " heat source check method ", rabbit auricular vein note
The mix preparation of Nattokinase and natto antibacterial peptide is penetrated, the body temperature raising of rabbit should not be 2 DEG C high.
10. abnormal toxicity test is tested according to existing " Chinese veterinary pharmacopoeia " annex " abnormal toxicity tests method ", mouse abdomen
Chamber injects the mix preparation of Nattokinase and natto antibacterial peptide, and whole mouse should be good for work in 48h.
The Nattokinase and natto antibacterial peptide mix preparation of preparation carry out quality test results, are shown in Table 1
Nattokinase and natto antibacterial peptide mix preparation prepared by table 1 carries out quality test results
| Inspection project | As a result |
| Character | Yellowish transparency liquid |
| PH value | 7.0 |
| Steriling test | Asepsis growth |
| Mycoplasma is examined | Fluid nutrient medium changes without PH, and solid plate is without typical " fried egg " shape mycoplasma bacterium colony, no mycoplasma contamination |
| Heat source is examined | It is negative |
| Abnormal toxicity test | Feminine gender, whole mouse are strong in 48h lives |
| Protein qualitative reaction | Feminine gender, naked eyes, which are seen, to be wiped without turbidity and precipitation |
| Protein content determination | 2.3mg/mL |
| Bacterial endotoxin test | 4EU/mL |
| Ribose concentration measurement | 49μg/mL |
As seen from the results in Table 1, the mix preparation character of Nattokinase and natto antibacterial peptide is yellowish transparency liquid；PH value
It is 7.0；Asepsis growth and without mycoplasma contamination；Heat source is examined, abnormal toxicity test and protein qualitative reaction are feminine gender；Egg
White matter assay is 2.3mg/mL；Bacterial endotoxin test is 4EU/mL；Ribose concentration measures 49 μ g/mL.In conclusion receiving
The mix preparation of beans kinases and natto antibacterial peptide meets regulation through completely examining.
The Nattokinase and natto antibacterial peptide mix preparation fungistatic effect of 4 embodiment 1 of embodiment preparation
1. the antibacterial effect of Nattokinase and natto antibacterial peptide mix preparation progress prepare using filter paper method to embodiment 1
Fruit measurement, indicator bacteria used are salmonella, Escherichia coli, staphylococcus aureus, Aspergillus, Candida albicans, green pus bar
Bacterium；It the results are shown in Table 2.In table 2: " ++++" indicating that fungistatic effect is fine, inhibition zone is in 25mm or more；" +++ " indicates fungistatic effect
Preferably, inhibition zone is in 15mm or more；" ++ " indicates that fungistatic effect is general, and inhibition zone is in 5mm or more；"+" indicates fungistatic effect not
Good, inhibition zone is less than 5mm, and "-" indicates no fungistatic effect, without inhibition zone.
The mix preparation of 2 Nattokinase of table and natto antibacterial peptide carries out fungistatic effect
As seen from the results in Table 2, the mix preparation of Nattokinase and natto antibacterial peptide to Escherichia coli, salmonella it is antibacterial,
Staphylococcus aureus and Pseudomonas aeruginosa all have fungistatic effect, and compared with gentamicin, are inhibiting Escherichia coli and sand
Fungistatic effect is suitable in the test of door Salmonella.
2. using 96 hole reaction plates, Nattokinase and natto antibacterial peptide are measured to tested bacterium with micro meat soup doubling dilution
MIC value；The Nattokinase and natto antibacterial peptide progress Combination susceptibility testing using broth dilution chessboard method prepared by embodiment 1
FIC assessment of indices and result judgement, according to measuring Nattokinase and natto antibacterial peptide to the MIC value of tested bacterium, with 4 times, 2 times, 1
Times, 0.5 times, 0.25 times of concentration combined respectively, and set Nattokinase, natto antibacterial peptide control and bacterial controls, measurement connection
Medicine is shared to the antibacterial effect of salmonella, Escherichia coli, staphylococcus aureus and Pseudomonas aeruginosa.With part Mlc
(FIC) index is the judgment basis of Combination susceptibility testing,
Judgment criteria: FIC≤0.5 is synergistic effect, and 5 < FIC≤1 is summation action, and 1 < FIC≤2 are unrelated effect, FIC
> 2 is antagonism.FIC=(first medicine is combined MIC/ first prescription MIC)+(second medicine is combined MIC/ second prescription MIC).As a result see
Table 3.
The drug sensitive test of 3 Nattokinase of table and natto antibacterial peptide to the MIC value and drug combination of Quality Control bacterium and tested bacterium
As shown in Table 3, Nattokinase and natto antibacterial peptide mix preparation are to salmonella, Escherichia coli, golden yellow grape
The FIC index of coccus and Pseudomonas aeruginosa is respectively 0.375,0.375,0.25 and 0.5, that is, generates synergistic effect.
Influence of the mix preparation of 5 Nattokinase of embodiment and natto antibacterial peptide to live vaccine immune effect
1. the mix preparation of Nattokinase and natto antibacterial peptide prepared by embodiment 1 and pig circular ring virus inactivated vaccine are connect
The susceptible pig of kind health, if individually immune pig circular ring virus inactivated vaccine group, injects 2 part/heads of pig circular ring virus inactivated vaccine；It is single
The solely mix preparation group of inoculation Nattokinase and natto antibacterial peptide, injects the mix preparation 2mL/ of Nattokinase and natto antibacterial peptide
Head；It is inoculated with the mix preparation and pig circular ring virus inactivated vaccine group of Nattokinase and natto antibacterial peptide simultaneously, injects Nattokinase
With 1 part/head of mix preparation 1mL/ and pig circular ring virus inactivated vaccine of natto antibacterial peptide and nonimmune control group.
After injection after 14d, 21d, 28d respectively blood sampling detection serum neutralize antibody titers, with this evaluate Nattokinase and
Influence of the mix preparation of natto antibacterial peptide to pig circular ring virus inactivated vaccine immune effect.Test result is referring to table 4.
4 pig circular ring virus serum antibody titer measurement result of table
Note: compared with independent immune pig circular ring virus inactivated vaccine group: △ P < 0.05, △ △ P < 0.01；With it is nonimmune
Control group is compared: * P < 0.05, * * P < 0.01.
Table 4 is the results show that the inoculation of the mix preparation and pig circular ring virus inactivated vaccine of Nattokinase and natto antibacterial peptide is strong
The susceptible pig of health is strong to live；Single injecting immune pig circular ring virus inactivated vaccine serum neutralizing antibody is between 1.4~1.9, simultaneously
The mix preparation and pig circular ring virus inactivated vaccine serum neutralizing antibody of Nattokinase and natto antibacterial peptide are inoculated with 2.1~2.5
Between.The mix preparation and pig circular ring virus inactivated vaccine for illustrating while being inoculated with Nattokinase and natto antibacterial peptide significantly improve
The potency of pig circular ring virus serum antibody.Nonimmune control serum neutralizing antibody is individually inoculated with natto and swashs between 0.3~0.8
The mix preparation serum neutralizing antibody of enzyme and natto antibacterial peptide illustrates to be inoculated with Nattokinase and natto antibacterial between 1.1~1.4
The mix preparation of peptide can slow down the abatement of serum neutralizing antibody in pig body.
It can be seen that the mix preparation of Nattokinase and natto antibacterial peptide of the invention, using safe and be conducive to enhance
Pig circular ring virus inactivated vaccine immune effect.
2. the mix preparation and chicken colibacillosis inactivated vaccine of Nattokinase of the invention and natto antibacterial peptide are inoculated with
The susceptible chick of health, if individually immune chicken colibacillosis inactivated vaccine group, injection 2 plumage part of chicken colibacillosis inactivated vaccine/
Plumage；It is inoculated with the mix preparation and pig circular ring virus inactivated vaccine group of Nattokinase and natto antibacterial peptide simultaneously, injects Nattokinase
With mix preparation 1mL/ plumage and pig circular ring virus inactivated vaccine 1 plumage part/plumage of natto antibacterial peptide and nonimmune control group.
Different time blood sampling detection serum neutralize antibody titers after injection, two exempt from after a week, to carry out attacking poison, observe it and attack
Malicious protecting effect evaluates the mix preparation of Nattokinase and natto antibacterial peptide to the immune effect of chicken colibacillosis inactivated vaccine with this
The influence of fruit.Test result is referring to table 5.
5 chicken colibacillosis antibody titer measurement result of table protects result with poison is attacked
Table 5 inactivates epidemic disease the results show that attacking the mix preparation and chicken colibacillosis of Nattokinase and natto antibacterial peptide before poison
Seedling is inoculated with the strong work of healthy susceptible chicken；It is inoculated with the mix preparation of Nattokinase and natto antibacterial peptide simultaneously and chicken colibacillosis goes out
Live vaccine serum neutralizing antibody is higher than individually immune chicken colibacillosis inactivated vaccine serum neutralizing antibody.
From the point of view of attacking poison protection result, while being inoculated with the mix preparation and chicken colibacillosis of Nattokinase and natto antibacterial peptide
Inactivated vaccine protective rate is higher than individually immune chicken colibacillosis inactivated vaccine protective rate.Thus it is clear that Nattokinase of the invention and
The mix preparation of natto antibacterial peptide, using it is safe and be conducive to enhance chicken colibacillosis inactivated vaccine immune effect.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. the preparation method of a kind of Nattokinase and natto antibacterial peptide mix preparation, comprising the following steps:
1) natto and water are mixed, is ground, filtered, obtain grinding filtrate；
2) solid ammonium sulfate is added in step 1) the grinding filtrate, stands, takes the first supernatant, sterilize, it is anti-to obtain natto
Bacterium peptide solution；
3) natto and phosphate buffer are mixed, is ground, obtained abrasive material and phosphate buffer mixing, extraction,
Centrifugation, takes the second supernatant；
4) ammonium sulfate solution is added in step 3) second supernatant to be mixed, stands, centrifugation, taking precipitate obtains
To Nattokinase；
5) step 2) the natto antibacterial peptide solution is mixed, filtration sterilization with Nattokinase described in step 4), adjusts pH
Value obtains the mix preparation of Nattokinase and natto antibacterial peptide to 7.0；
Step 1)~2) and step 3)~4) between there is no time sequencing limitation.
2. preparation method according to claim 1, which is characterized in that the mass ratio of the step 1) natto and water be 1~
3:1。
3. preparation method according to claim 1, which is characterized in that the temperature of grinding described in step 3) is 1~5 DEG C.
4. preparation method according to claim 1 or 3, which is characterized in that the temperature of phosphate buffer described in step 3)
Degree is 1~5 DEG C；The pH value of the phosphate buffer is 7.5~8.0.
5. Nattokinase and natto antibacterial peptide mix preparation that preparation method described in Claims 1 to 4 any one is prepared.
6. application of the mix preparation described in claim 5 in the drug that preparation improves immunity of organisms.
7. mix preparation described in claim 5 is inhibiting the application in bacterium.
8. application of the mix preparation described in claim 5 as vaccine reinforcing agent.
9. application according to claim 8, which is characterized in that the vaccine is bird vaccine or domestic animals vaccine.
10. application according to claim 9, which is characterized in that the ratio of the mix preparation and domestic animals vaccine be 2~
5mL:1~3 part；The ratio of the mix preparation and bird vaccine is the plumage part of 0.1~1mL:1~3.
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| Publication number | Priority date | Publication date | Assignee | Title |
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