CN109718368B - Nattokinase and natto antibacterial peptide mixed preparation as well as preparation method and application thereof - Google Patents
Nattokinase and natto antibacterial peptide mixed preparation as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a nattokinase and natto antibacterial peptide mixed preparation, belonging to the technical field of biological medicine, and the method comprises the following steps: 1) mixing natto with water, grinding, and filtering to obtain ground filtrate; 2) adding solid ammonium sulfate into the ground filtrate, standing, taking the first supernatant, and sterilizing to obtain natto antibacterial peptide solution; 3) mixing natto and phosphate buffer solution, grinding, mixing the obtained ground substance and the phosphate buffer solution, leaching, centrifuging, and taking second supernatant; 4) adding ammonium sulfate aqueous solution into the second supernatant, mixing, standing, centrifuging, and collecting precipitate to obtain nattokinase; 5) mixing the natto antibacterial peptide solution with the natto kinase, filtering for sterilization, and adjusting the pH value to 7.0 to obtain the mixed preparation of the natto kinase and the natto antibacterial peptide. The mixed preparation prepared by the invention has the effects of inhibiting bacteria, improving the immunity of organisms and improving the immune effect of veterinary vaccines.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a nattokinase and natto antibacterial peptide mixed preparation as well as a preparation method and application thereof.
Background
The natto is a bean product prepared by fermenting soybeans with bacillus subtilis, has viscosity, smelly smell and slightly sweet taste, not only maintains the nutritive value of the soybeans, is rich in vitamin K2 and improves the digestibility of protein, but also generates various physiological active substances in the fermentation process, and has the health-care effects of dissolving fibrin in a body and regulating physiological functions.
Natto extract refers to effective substances extracted from natto, including natto kinase, natto isoflavone, saponin, vitamin K2, etc.; the saponin can improve constipation, reduce blood lipid, prevent carcinoma of large intestine, reduce cholesterol, soften blood vessel, prevent hypertension and arteriosclerosis, and inhibit HIV; the natto isoflavone has obvious effects of clearing carcinogenic substances in vivo, improving memory, protecting liver, caring skin, delaying aging, etc., and can improve the digestibility of food; nattokinase has effects of dissolving thrombi, reducing blood viscosity, improving blood circulation, softening and increasing blood vessel elasticity.
However, the development of natto extract in the prior art is generally to extract and utilize single components in natto extract, but lacks comprehensive utilization of natto extract.
Disclosure of Invention
The invention aims to provide a mixed preparation of nattokinase and natto antibacterial peptide, a preparation method and application thereof, and the mixed preparation can improve the immune effect of veterinary vaccines.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a nattokinase and natto antibacterial peptide mixed preparation, which comprises the following steps:
1) mixing natto with water, grinding, and filtering to obtain ground filtrate;
2) adding solid ammonium sulfate into the ground filtrate in the step 1), standing, taking a first supernatant, and sterilizing to obtain a natto antibacterial peptide solution;
3) mixing natto and phosphate buffer solution, grinding, mixing the obtained ground substance and the phosphate buffer solution, leaching, centrifuging, and taking second supernatant;
4) adding an ammonium sulfate aqueous solution into the second supernatant in the step 3), mixing, standing, centrifuging, and taking a precipitate to obtain nattokinase;
5) mixing the natto antibacterial peptide solution obtained in the step 2) with the natto kinase obtained in the step 4), filtering and sterilizing, and adjusting the pH value to 7.0 to obtain a mixed preparation of the natto kinase and the natto antibacterial peptide;
there is no chronological restriction between the steps 1) to 2) and the steps 3) to 4).
Preferably, the mass ratio of the natto and the water in the step 1) is 1-3: 1.
Preferably, the grinding temperature in the step 3) is 1-5 ℃.
Preferably, the temperature of the phosphate buffer solution in the step 3) is 1-5 ℃; the pH value of the phosphate buffer solution is 7.5-8.0.
The invention also provides the nattokinase and nattokinase antibacterial peptide mixed preparation prepared by the preparation method in the scheme.
The invention also provides application of the mixed preparation in the scheme in preparing a medicine for improving the immunity of the organism.
The invention also provides application of the mixed preparation in the scheme in inhibiting bacteria.
The invention also provides application of the mixed preparation in the scheme as a vaccine enhancer.
Preferably, the vaccine is an avian vaccine or a veterinary vaccine.
Preferably, the ratio of the mixed preparation to the livestock vaccine is 2-5 mL to 1-3 parts; the ratio of the mixed preparation to the poultry vaccine is 0.1-1 mL to 1-3 parts.
The invention has the beneficial effects that: the invention provides a preparation method of a nattokinase and natto antibacterial peptide mixed preparation, and the nattokinase and natto antibacterial peptide mixed preparation prepared by the method has the effects of inhibiting bacteria, improving the immunity of organisms and improving the immune effect of veterinary vaccines. Experiments prove that the mixed preparation of the nattokinase and the natto antibacterial peptide has antibacterial effects on escherichia coli, salmonella, staphylococcus aureus and pseudomonas aeruginosa, and compared with gentamicin, the mixed preparation has the antibacterial effect on escherichia coli and salmonella in experiments. In addition, the mixed preparation of the nattokinase and the natto antibacterial peptide is safe to use and is beneficial to enhancing the immune effect of the porcine circovirus inactivated vaccine and the chicken colibacillosis inactivated vaccine.
Detailed Description
The invention provides a preparation method of a nattokinase and natto antibacterial peptide mixed preparation, which comprises the following steps:
1) mixing natto with water, grinding, and filtering to obtain ground filtrate;
2) adding solid ammonium sulfate into the ground filtrate in the step 1), standing, taking a first supernatant, and sterilizing to obtain a natto antibacterial peptide solution;
3) mixing natto and phosphate buffer solution, grinding, mixing the obtained ground substance and the phosphate buffer solution, leaching, centrifuging, and taking second supernatant;
4) adding an ammonium sulfate aqueous solution into the second supernatant in the step 3), mixing, standing, centrifuging, and taking a precipitate to obtain nattokinase;
5) mixing the natto antibacterial peptide solution obtained in the step 2) with the natto kinase obtained in the step 4), filtering and sterilizing, and adjusting the pH value to 7.0 to obtain a mixed preparation of the natto kinase and the natto antibacterial peptide;
there is no chronological restriction between the steps 1) to 2) and the steps 3) to 4).
The invention mixes natto with water, grinds and filters to obtain ground filtrate; the mass ratio of the natto to the water is preferably 1-3: 1, and more preferably 2: 1; the grinding time is preferably 5-10 min; the temperature of the grinding is not particularly limited; the grinding device is preferably a homogenizer or a tissue grinder; the homogenizer is preferably a glass homogenizer, more preferably a 50mL glass homogenizer.
In the invention, the filtration is preferably carried out by adopting gauze; the aperture of the filter hole of the gauze is preferably 0.4-0.5 μm, and more preferably 0.45 μm; the number of layers of the gauze is preferably 4-6, and more preferably 5.
After filtering, the method preferably further comprises the steps of mixing the filter residue obtained by filtering with water, leaching, filtering to obtain leaching filtrate, and then combining the leaching filtrate with the grinding filtrate obtained by the scheme to obtain combined filtrate; the mass ratio of the filter residue to the water is preferably 1-3: 1, and more preferably 2: 1; the leaching temperature is preferably 1-5 ℃, and more preferably 4 ℃; the leaching time is preferably 0.5-1.5 h, and more preferably 1 h.
After a combined filtrate is obtained, adding solid ammonium sulfate into the combined filtrate, standing, taking a first supernatant, and sterilizing to obtain a natto antibacterial peptide solution; the addition amount of the solid ammonium sulfate is preferably selected to ensure that the saturation degree of the ammonium sulfate in the combined filtrate reaches 60-65%; the standing time is preferably 12-18 h, and more preferably 14-16 h; the temperature of the standing is preferably 1-5 ℃, and more preferably 4 ℃; the sterilization mode is preferably high-pressure sterilization and/or filtration sterilization; the temperature of the high-pressure sterilization is preferably 115-121 ℃; the time for autoclaving is preferably 15-20 min; the filter pore size of the filter for filter sterilization is preferably 0.1 μm or 0.22. mu.m.
After the first supernatant is obtained, preferably adding solid ammonium sulfate into the first supernatant, standing, taking the supernatant again, and sterilizing; the solid ammonium sulfate is preferably added in an amount such that the saturation degree of the ammonium sulfate in the first supernatant reaches 60-65%.
Mixing natto and a phosphate buffer solution, grinding, mixing the obtained ground substance and the phosphate buffer solution, leaching, centrifuging, and taking a second supernatant; the pH value of the phosphate buffer solution is preferably 7.5-8.0, and more preferably 7.8; the temperature of the phosphate buffer solution is preferably 1-5 ℃, and more preferably 4 ℃. In the present invention, the phosphate buffer is preferably prepared by the following method: dissolving 5.59g of dipotassium hydrogen phosphate and 0.41g of monopotassium phosphate in 1000mL of purified water, sequentially filtering, sterilizing and autoclaving; the present invention is not particularly limited with respect to the parameters of the filter sterilization and the autoclaving, and may be performed by a method which is conventional in the art.
In the invention, the mass ratio of the natto to the phosphate buffer solution is preferably 1-3: 1, and more preferably 2: 1; the grinding temperature is preferably 1-5 ℃, and more preferably 4 ℃; the grinding time is preferably 5-10 min, and more preferably 8 min; the grinding equipment is preferably a mortar or a tissue grinder; the mortar or tissue grinder is preferably precooled to 4 ℃; the grinding temperature is set to be 1-5 ℃ so as to avoid enzyme inactivation.
In the invention, the volume ratio of the ground material to the phosphate buffer solution is preferably 0.5-1.5: 2, and more preferably 1: 2; the leaching temperature is preferably 1-5 ℃, and more preferably 4 ℃; the leaching time is preferably 9-16 h, and more preferably 12-14 h; the rotation speed of the centrifugation is preferably 4000-6000 r/min, and more preferably 5000 r/min; the time for centrifugation is preferably 5-10 min.
After the second supernatant is obtained, adding an ammonium sulfate aqueous solution into the second supernatant for mixing, standing, centrifuging, and taking a precipitate to obtain nattokinase; the saturation degree of ammonium sulfate in the ammonium sulfate aqueous solution is preferably 57-63%, and more preferably 60%; the process of adding the ammonium sulfate aqueous solution is preferably accompanied by stirring; the standing time is preferably 1-5 ℃, and more preferably 4 ℃; the standing time is preferably 9-16 h, and more preferably 12-14 h; the rotation speed of the centrifugation is preferably 4000-6000 r/min, and more preferably 5000 r/min; the time of centrifugation is preferably 5-10 min, and more preferably 8 min.
After the natto antibacterial peptide solution and the natto kinase are obtained, mixing the natto antibacterial peptide solution and the natto kinase, filtering and sterilizing, and adjusting the pH value to 7.0 to obtain a mixed preparation of the natto kinase and the natto antibacterial peptide; the pore size of the filter membrane for filtration sterilization is preferably 0.1 μm or 0.22 μm; the pH adjusting agent is preferably NaOH aqueous solution.
The invention also provides the nattokinase and nattokinase antibacterial peptide mixed preparation prepared by the preparation method in the scheme.
The invention also provides application of the mixed preparation in the scheme in preparing a medicine for improving the immunity of the organism.
The invention also provides the application of the mixed preparation in the scheme in inhibiting bacteria; the bacteria preferably include Escherichia coli and Salmonella.
The invention also provides the application of the mixed preparation in the scheme as a vaccine enhancer; the vaccine is an avian vaccine or an livestock vaccine; the proportion of the mixed preparation to the livestock vaccine is preferably 2-5 mL to 1-3 parts; the proportion of the mixed preparation to the poultry vaccine is preferably 0.1-1 mL to 1-3 feathers; the livestock vaccine is preferably porcine circovirus inactivated vaccine; the avian vaccine is preferably inactivated chicken colibacillosis vaccine.
The invention also provides the application of the mixed preparation in the scheme as a vaccine enhancer; the poultry are 1 month old chickens; the domestic animals are pigs of 12-15 days old; the method for using the mixed preparation and the poultry or livestock vaccine together is preferably that the poultry freeze-dried vaccine or the livestock freeze-dried vaccine is diluted by the mixed preparation according to the proportion, or the poultry freeze-dried vaccine or the livestock freeze-dried vaccine is fully mixed with the poultry liquid vaccine or the livestock liquid vaccine and then the vaccine is inoculated by the ways of injection, drinking water, eye drop, nose drop and the like.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 preparation of a Mixed preparation of Nattokinase and Natto antibacterial peptide
1. Adding commercially available natto 500g into 500mL of distilled water, fully grinding with a tissue grinder, and filtering with gauze to obtain ground filtrate and filter residue;
2. filtering residues: adding distilled water into the filter residue, and repeatedly leaching at 4 ℃ for 2 times, wherein the leaching time is 1h each time, so as to obtain leaching filtrate;
3. mixing the ground filtrate and the leaching filtrate, adding 50g of solid ammonium sulfate, standing at 4 ℃ for 12h, and collecting a first supernatant; adding 50g solid ammonium sulfate into the first supernatant, standing at 4 deg.C for 12 hr, collecting supernatant, autoclaving at 121 deg.C for 20min, and filtering to obtain antibacterial peptide solution of semen Sojae Preparatum;
4. adding commercially available natto 500g into 500mL of precooled phosphate buffer solution with pH 7.8, leaching, and grinding in a precooled tissue grinder for 10min to obtain ground substance;
5. adding 2 times volume of phosphate buffer solution with precooled pH value of 7.5 into the ground material for low-temperature leaching, standing overnight in a refrigerator at 4 ℃, centrifuging at 5000r/min for 10min, discarding the precipitate, and taking a second supernatant;
6. adding the second supernatant into 60% ammonium sulfate solution at 4 deg.C under stirring, mixing, standing in 4 deg.C refrigerator, settling, standing overnight, centrifuging at 4 deg.C for 10min at 5000r/min the next day, removing supernatant, and collecting precipitate to obtain nattokinase;
7. dissolving nattokinase in the obtained natto antibacterial peptide solution, mixing completely, sterilizing with a 0.1 μm sterilizing filter sieve, and adjusting pH to 7.0 to obtain the natto kinase and natto antibacterial peptide mixed preparation.
Example 2 preparation method of Nattokinase and Natto antibacterial peptide Mixed preparation
1. Adding 1500g of commercially available natto into 500mL of distilled water, fully grinding with a tissue grinder, and filtering with gauze to obtain ground filtrate and filter residue;
2. filtering residues: adding distilled water into the filter residue, and repeatedly leaching at 4 ℃ for 2 times, wherein the leaching time is 1h each time, so as to obtain leaching filtrate;
3. mixing the ground filtrate and the leaching filtrate, adding 80g of solid ammonium sulfate, standing for 15h, and collecting a first supernatant; adding 80g solid ammonium sulfate into the first supernatant, standing for 15 hr, collecting supernatant, autoclaving at 121 deg.C for 20min, and filtering to obtain antibacterial peptide solution of semen Sojae Preparatum;
4. adding 1500g commercially available natto into 500mL of precooled phosphate buffer solution with pH 7.8, leaching, and grinding in precooled tissue grinder for 10min to obtain ground substance;
5. adding 2 times volume of phosphate buffer solution with precooled pH value of 7.8 into the ground material for low-temperature leaching, standing overnight in a refrigerator at 4 ℃, centrifuging at 5000r/min for 10min, discarding the precipitate, and taking a second supernatant;
6. adding the second supernatant into 60% ammonium sulfate solution at 4 deg.C under stirring, mixing, standing in 4 deg.C refrigerator, settling, standing overnight, centrifuging at 4 deg.C for 10min at 5000r/min the next day, removing supernatant, and collecting precipitate to obtain nattokinase;
7. dissolving nattokinase in the obtained natto antibacterial peptide solution, mixing completely, sterilizing with a 0.1 μm sterilizing filter sieve, and adjusting pH to 7.0 to obtain the natto kinase and natto antibacterial peptide mixed preparation.
Example 3 quality control of the mixed preparation of Nattokinase and Natto antimicrobial peptide prepared in example 1
The character is detected to be light yellow or yellowish transparent liquid through direct visual observation.
② the pH value is measured according to the appendix of the current Chinese animal pharmacopoeia, and the pH value is 7.0.
③ 2.0mL of the protein qualitative inspection sample is added with 2.0mL of 20 percent sulfosalicylic acid, and the reaction is negative if turbid sediment is not generated through direct visual observation.
And fourthly, measuring the protein content according to the welfare phenol method in the appendix of the current Chinese veterinary pharmacopoeia, wherein the protein content is not lower than 1.8 mg/mL.
Measuring the ribose content according to the ribose measuring method in the current transfer factor solution national drug standard, wherein the ribose content is not lower than 40 mug/mL.
Sixthly, the sterility test is carried out according to the appendix of the current Chinese animal pharmacopoeia, and the growth is aseptic.
Seventhly, the detection of the bacterial endotoxin is determined according to the appendix of the current Chinese veterinary pharmacopoeia, namely the detection method of the bacterial endotoxin, and the concentration of the bacterial endotoxin is not more than 10 EU/mL.
The Mycoplasma generator test is determined according to Mycoplasma test in appendix of the current Chinese veterinary pharmacopoeia, the liquid culture medium has no PH change, and the agar solid plate has no typical Mycoplasma colony in the shape of fried egg.
Ninthly, heat source inspection is carried out according to the heat source inspection method in the appendix of the current Chinese veterinary pharmacopoeia, and the temperature rise of the rabbits should not be higher than 2 ℃ when the rabbits are subjected to intravenous injection of the mixed preparation of the nattokinase and the natto antibacterial peptide.
The abnormal toxicity test of the R is carried out according to the abnormal toxicity test method in the appendix of the current Chinese veterinary pharmacopoeia, the mice are injected with the mixed preparation of the nattokinase and the natto antibacterial peptide in the abdominal cavity, and all the mice are healthy and alive within 48 hours.
The quality test results of the prepared nattokinase and natto antibacterial peptide mixed preparation are shown in Table 1
Quality test results of the mixed preparation of nattokinase and natto antibacterial peptide prepared in Table 1
| Inspection item | Results |
| Traits | Yellowish transparent liquid |
| pH value of | 7.0 |
| Sterility testing | Sterile growth |
| Mycoplasma assay | The liquid culture medium has no PH change, and the solid plate has no typical 'fried egg' -shaped mycoplasma colony and no mycoplasma pollutionDyeing process |
| Inspection of heat source | Negative of |
| Abnormal toxicity test | Negative, all mice were healthy within 48h |
| Qualitative protein assay | Negative, visual rubbing without turbid precipitate |
| Protein content determination | 2.3mg/mL |
| Bacterial endotoxin test | 4EU/mL |
| Ribose assay | 49μg/mL |
As can be seen from the results in Table 1, the nature of the mixed preparation of the nattokinase and the natto antibacterial peptide is yellowish transparent liquid; the pH value is 7.0; sterile growth and no mycoplasma contamination; the heat source test, the abnormal toxicity test and the protein qualitative test are all negative; the protein content was determined to be 2.3 mg/mL; the bacterial endotoxin test is 4 EU/mL; ribose content was measured at 49. mu.g/mL. In conclusion, the mixed preparation of the nattokinase and the natto antibacterial peptide meets the regulation through comprehensive inspection.
EXAMPLE 4 bacteriostatic Effect of the Nattokinase/Natto antibacterial peptide mixture preparation prepared in example 1
1. The bacteriostatic effect of the natto kinase and natto antibacterial peptide mixed preparation prepared in the example 1 is measured by adopting a filter paper method, and the used indicator bacteria are salmonella, escherichia coli, staphylococcus aureus, aspergillus, candida albicans and pseudomonas aeruginosa; the results are shown in Table 2. In table 2: the "+++" shows that the bacteriostatic effect is good, and the bacteriostatic circle is more than 25 mm; the "+++" shows that the bacteriostatic effect is better, and the bacteriostatic circle is more than 15 mm; the "+ +" shows that the bacteriostatic effect is general, and the bacteriostatic zone is more than 5 mm; the "+" indicates that the bacteriostatic effect is poor, the bacteriostatic circle is less than 5mm, and the "-" indicates that no bacteriostatic effect and no bacteriostatic circle exist.
TABLE 2 antibacterial effect of the mixture of nattokinase and antibacterial peptide of natto
As can be seen from the results in Table 2, the mixed preparation of nattokinase and natto antibacterial peptide has antibacterial effects on Escherichia coli, salmonella, Staphylococcus aureus and Pseudomonas aeruginosa, and compared with gentamicin, the antibacterial effects are equivalent in the test of inhibiting Escherichia coli and salmonella.
2. Determining the MIC values of the nattokinase and the natto antibacterial peptide to the tested bacteria by a two-fold dilution method of trace broth by adopting a 96-hole reaction plate; the method comprises the steps of performing FIC index measurement and result judgment on the nattokinase and the natto antibacterial peptide prepared in example 1 by adopting a broth dilution chessboard method, combining the nattokinase and the natto antibacterial peptide respectively in concentrations of 4 times, 2 times, 1 time, 0.5 time and 0.25 time according to the measured MIC values of the nattokinase and the natto antibacterial peptide to tested bacteria, setting nattokinase, natto antibacterial peptide control and bacteria control, and measuring the antibacterial effect of the combined medicine to salmonella, escherichia coli, staphylococcus aureus and pseudomonas aeruginosa. Taking part of the index of the antibacterial concentration (FIC) as the judgment basis of the combined drug sensitivity test,
and (4) judging the standard: the FIC is less than or equal to 0.5, the FIC is less than or equal to 1, the FIC is less than or equal to 5, the FIC is additive, the FIC is less than or equal to 1, the FIC is irrelevant, and the FIC is more than 2, the FIC is antagonistic. FIC (MIC for drug A/MIC for drug A) plus (MIC for drug B/MIC for drug B). The results are shown in Table 3.
TABLE 3 MIC values of nattokinase and natto antimicrobial peptides against quality control bacteria and test bacteria and drug sensitivity test of combined drugs
As can be seen from Table 3, the FIC indexes of the nattokinase and nattokinase mixed preparation for salmonella, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were 0.375, 0.25 and 0.5, respectively, which resulted in synergistic effects.
Example 5 Effect of Mixed preparation of Nattokinase and Natto antimicrobial peptide on the immunization Effect of veterinary vaccine
1. Inoculating the mixed preparation of the nattokinase and the natto antibacterial peptide prepared in the example 1 and the porcine circovirus inactivated vaccine to healthy susceptible pigs, setting a single immune porcine circovirus inactivated vaccine group, and injecting 2 heads of the porcine circovirus inactivated vaccine per head; inoculating a mixed preparation group of nattokinase and natto antibacterial peptide independently, and injecting 2 mL/head of the mixed preparation of nattokinase and natto antibacterial peptide; simultaneously inoculating a mixed preparation of natto kinase and natto antibacterial peptide and a porcine circovirus inactivated vaccine group, and injecting 1 mL/head of the mixed preparation of natto kinase and natto antibacterial peptide and 1 head/head of the porcine circovirus inactivated vaccine and a non-immune control group.
Blood is respectively collected 14d, 21d and 28d after injection to detect the titer of neutralizing antibodies in serum, so as to evaluate the influence of the mixed preparation of the nattokinase and the natto antibacterial peptide on the immune effect of the porcine circovirus inactivated vaccine. The test results are shown in Table 4.
TABLE 4 porcine circovirus serum antibody titer assay results
Note: compared with a group of inactivated vaccines for separately immunizing porcine circovirus: delta P is less than 0.05, delta P is less than 0.01; compared to the non-immune control group: p < 0.05, P < 0.01.
The results in Table 4 show that the mixed preparation of nattokinase and natto antibacterial peptide and the inactivated porcine circovirus vaccine inoculated healthy susceptible pigs are healthy and alive; the neutralizing antibody of the serum of the single injection immune porcine circovirus inactivated vaccine is between 1.4 and 1.9, and the neutralizing antibody of the serum of the single injection immune porcine circovirus inactivated vaccine is between 2.1 and 2.5 after the mixed preparation of the nattokinase and the natto antibacterial peptide and the neutralizing antibody of the serum of the porcine circovirus inactivated vaccine are inoculated. The result shows that the titer of the porcine circovirus serum antibody is obviously improved by simultaneously inoculating the mixed preparation of the nattokinase and the natto antibacterial peptide and the porcine circovirus inactivated vaccine. The neutralizing antibody of the nonimmune control serum is between 0.3 and 0.8, and the neutralizing antibody of the serum of the mixed preparation which is singly inoculated with the nattokinase and the natto antibacterial peptide is between 1.1 and 1.4, which shows that the mixed preparation which is inoculated with the nattokinase and the natto antibacterial peptide can slow down the reduction of the neutralizing antibody of the serum in a pig body.
Therefore, the mixed preparation of the nattokinase and the natto antibacterial peptide is safe to use and is beneficial to enhancing the immune effect of the porcine circovirus inactivated vaccine.
2. Inoculating the mixed preparation of the nattokinase and the natto antibacterial peptide and the inactivated vaccine for chicken colibacillosis to healthy susceptible chicks, setting a single inactivated vaccine group for immunizing chicken colibacillosis, and injecting the inactivated vaccine for chicken colibacillosis into the chicks with 2 feather parts/feather parts; simultaneously inoculating a mixed preparation of the nattokinase and the natto antibacterial peptide and a porcine circovirus inactivated vaccine group, and injecting 1 mL/feather of the mixed preparation of the nattokinase and the natto antibacterial peptide and 1 feather/feather of the porcine circovirus inactivated vaccine and a non-immune control group.
Blood is collected at different time after injection to detect the titer of the neutralizing antibody of serum, after one week of secondary immunization, the virus attack is carried out, the virus attack protection effect is observed, and the influence of the mixed preparation of the nattokinase and the natto antibacterial peptide on the immune effect of the inactivated vaccine for the chicken colibacillosis is evaluated. The test results are shown in Table 5.
TABLE 5 Chicken colibacillosis antibody titer determination results and challenge protection results
The results in Table 5 show that the mixed preparation of nattokinase and natto antibacterial peptide before challenge and the inactivated vaccine for chicken colibacillosis inoculated to healthy susceptible chicken are healthy and alive; the serum neutralizing antibody of the inactivated vaccine for chicken colibacillosis and the mixed preparation inoculated with the nattokinase and the natto antibacterial peptide are higher than the serum neutralizing antibody of the inactivated vaccine for chicken colibacillosis by single immunization.
From the result of virus attack protection, the protection rate of the inactivated vaccine for chicken colibacillosis and the mixed preparation inoculated with the nattokinase and the natto antibacterial peptide is higher than that of the inactivated vaccine for chicken colibacillosis by single immunization. Therefore, the mixed preparation of the nattokinase and the natto antibacterial peptide is safe to use and is beneficial to enhancing the immune effect of the chicken colibacillosis inactivated vaccine.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. The preparation method of the nattokinase mixed preparation comprises the following steps:
1) mixing natto with water, grinding, and filtering to obtain ground filtrate;
2) adding solid ammonium sulfate into the grinding filtrate in the step 1), standing, taking a first supernatant, and sterilizing to obtain a solution containing natto antibacterial peptide;
3) mixing natto and phosphate buffer solution, grinding, mixing the obtained ground substance and the phosphate buffer solution, leaching, centrifuging, and taking second supernatant;
4) adding an ammonium sulfate aqueous solution into the second supernatant in the step 3), mixing, standing, centrifuging, and taking a precipitate to obtain nattokinase;
5) mixing the natto antibacterial peptide solution obtained in the step 2) with the natto kinase obtained in the step 4), filtering and sterilizing, and adjusting the pH value to 7.0 to obtain a natto kinase mixed preparation;
no time sequence limitation exists between the steps 1) to 2) and the steps 3) to 4);
the mass ratio of the natto to the water in the step 1) is 1-3: 1.
2. The use according to claim 1, wherein the temperature of the grinding in step 3) is 1-5 ℃.
3. The use according to claim 1 or 2, wherein the temperature of the phosphate buffer solution in step 3) is 1 to 5 ℃; the pH value of the phosphate buffer solution is 7.5-8.0.
4. Use according to claim 1, wherein the vaccine is an avian vaccine or a veterinary vaccine.
5. The use according to claim 4, wherein the ratio of the nattokinase mixed preparation to the livestock vaccine is 2-5 mL: 1-3 parts; the ratio of the mixed preparation to the poultry vaccine is 0.1-1 mL to 1-3 parts.
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