CN112999337A - Biological enzyme disinfectant preparation and preparation method thereof - Google Patents
Biological enzyme disinfectant preparation and preparation method thereof Download PDFInfo
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- CN112999337A CN112999337A CN201911314816.0A CN201911314816A CN112999337A CN 112999337 A CN112999337 A CN 112999337A CN 201911314816 A CN201911314816 A CN 201911314816A CN 112999337 A CN112999337 A CN 112999337A
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- lysostaphin
- lysozyme
- biological enzyme
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0041—Mammary glands, e.g. breasts, udder; Intramammary administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24075—Lysostaphin (3.4.24.75)
Abstract
The invention discloses a biological enzyme disinfectant preparation for preventing cow mastitis and a preparation method thereof, and the biological enzyme disinfectant preparation comprises the following components in percentage by weight: 0.1 to 20 percent of lysostaphin; 2% -80% of lysozyme; 1% -50% of protein stabilizer; 1-20% of ionic compound; 0.1 to 30 percent of bactericide; the rest is distilled water; the sum of the weight percentages of the components is 100 percent. The biological enzyme disinfectant preparation of the invention overcomes the problems of chapped skin and damage caused by disinfectant mainly containing iodine and iodine residue in milk, has convenient use and obvious sterilization effect, and can be used for preventing mastitis of dairy cows.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a biological enzyme disinfectant preparation for preventing dairy cow mastitis and a preparation method thereof.
Background
Cow Mastitis (Mastitis) is a diverse inflammation of its mammary tissues, including occult Mastitis and clinical Mastitis. The disease is a frequently-occurring disease and frequently-occurring disease of dairy cows, about one third of the dairy cows all over the world are reported to have various types of mastitis, the incidence rate of the mastitis of the dairy cows in China is generally higher than that of the mastitis of the dairy cows in foreign countries due to the influence of a plurality of factors, particularly under the high-temperature and high-humidity conditions in the south of China, the incidence rate of the mastitis of the dairy cows is higher, and the incidence rate can reach more than 70% in rainy season. Mastitis can cause the reduction of milk yield, the reduction of the quality of fresh milk and the increase of replacement cost of cow herds, and is one of the most serious diseases causing the economic loss of cow production. The economic loss caused each year is about 1135 billion yuan. It has been reported that a scientific and rational teat dipping bath can reduce breast infection by 50%, i.e. reduce economic losses by 567 billion yuan.
Because of the long-term, high-dose use of antibiotics and chemosynthetic drugs to prevent and treat mastitis in cows, the amount of residual drug accumulated in the body and milk of cows has far exceeded the safety standards for human use. Although long-term scientific use of teat dip solutions can effectively prevent and reduce the incidence of mastitis in dairy cows, the iodine content of milk may increase if the teat is not thoroughly cleaned and wiped clean after long-term use or before milking. Recently, the sampling survey results of 135 parts of fresh milk and 60 parts of milk powder by the Ministry of health in China show that the detection rate of antibiotic residues in the fresh milk is 22 percent, and the detection rate of milk powder is 3.3 percent. The detection rate of antibiotic residues in fresh milk in local areas is even up to 60 percent. In China, the group with the highest milk consumption level focuses on infants and children, and a large amount of 'milk resistance' seriously harms the body health of people. With the continuous improvement of living standard, the demand of consumers for milk and dairy products is increasing day by day, and the quality and safety problems need to be paid attention and invested particularly by the nation, the society and the dairy industry. The chapped skin and the damage caused by the current disinfectant mainly containing iodine and the problem of iodine residue in milk are serious.
Disclosure of Invention
The invention aims to overcome the defects that the existing antibacterial preparation has strong irritation, has residual problems, is easy to generate drug resistance and is ineffective to drug-resistant bacterial infection, and provides a biological enzyme disinfectant preparation for preventing the mastitis of the dairy cows, which has the advantages of convenient use, strong activity exertion action time and obvious sterilization effect.
The invention also provides a preparation method of the biological enzyme disinfectant for preventing the cow mastitis.
The technical scheme of the invention is as follows:
a biological disinfectant preparation for preventing cow mastitis comprises effective bactericidal amount of lysostaphin, lysozyme and other auxiliary components, wherein the preparation comprises the following components in percentage by weight:
0.1 to 20 percent of lysostaphin;
2% -80% of lysozyme;
1% -50% of protein stabilizer;
1-20% of ionic compound;
0.1 to 30 percent of bactericide;
the rest is distilled water;
the sum of the weight percentages of the components is 100 percent.
The preferable components and weight percentage are as follows:
0.1 to 10 percent of lysostaphin;
2% -40% of lysozyme;
1% -5% of a protein stabilizer;
1-20% of ionic compound;
0.1 to 2 percent of bactericide;
the rest is distilled water;
the sum of the weight percentages of the components is 100 percent.
The lysostaphin and lysozyme are 1: 20-400.
Wherein the lysostaphin is one or more of wild-type, recombinantly produced, mutated xenogeneic or any recombinant or related glycine-containing cross-linked enzyme that retains anti-staphylococcal cell wall peptidoglycan; the lysozyme is one or more of bacterial muralytic enzyme, yeast muralytic enzyme and mould muralytic enzyme. The lysostaphin is produced by gene recombination and secreted by Bacillus subtilis. The lysozyme is extracted from egg white or produced by gene recombination.
The protein stabilizer comprises polyethylene glycol substances or medicinal high molecular compounds such as chitosan, chitin, N-acetyl-D-amino and the like, and can wrap the protein or polypeptide to prevent the protein or polypeptide from being damaged by other substances, so that the stability of the protein or polypeptide is enhanced;
the ionic compound is K+、Na+、Mg2+、PO4 3-、HPO4 2-、H2PO4 -、CI-In combination of any two anions with cations, e.g. NaH2PO4KCI and the like can assist in enhancing the stability of the compound preparation, and can adjust the pH value of the compound preparation, so that the compound preparation is suitable for wound surfaces or mucous membranes of different parts.
The polyethylene glycol comprises one or more of polyethylene glycol 400-6000.
In order to enhance the bactericidal power of the compound preparation and expand the bactericidal spectrum, the compound preparation also comprises 0-30% of bactericide by the total weight of the preparation. The bactericide is chlorhexidine acetate, antibacterial peptide or Chinese medicinal extract with antibacterial effect, such as allicin, thymol, catechol, tomatidine, and emodin.
A method for preparing a formulation of the invention, the method comprising the steps of:
(1) weighing lysostaphin, lysozyme and a protein stabilizer according to the proportion to prepare a solution A;
(2) respectively weighing ionic compounds according to the proportion, stirring the solution A, and slowly adding the ionic compounds into the solution A to obtain a solution B;
(3) weighing the bactericide according to the proportion, stirring the solution B, slowly adding the bactericide into the solution B, and uniformly stirring to obtain a finished product.
The biological disinfectant preparation for preventing the cow mastitis is characterized in that:
the biological protein is very unstable at normal temperature, especially in a liquid preparation, because the components are relatively complex, the activity of the biological protein is more difficult to maintain, and the preparation contains a plurality of stabilizers and synergists which are screened by people, so that the stability is greatly enhanced, and the preparation can be suitable for industrial application.
Compared with stability experiments of the care solution preparation, the care solution preparation is stored for 3 months at 37 ℃ (equivalent to being stored for 2 years at normal temperature), and is shown in table 1.
TABLE 1 comparison of lysostaphin Activity before and after 3-month storage at 37 ℃ for the Care solution preparation
As can be seen from the table, the core component lysostaphin of the care solution preparation has high enzyme activity stability.
1. The bactericidal spectrum is wider than that of lysostaphin. The bactericidal composition can effectively kill staphylococcus, candida albicans, pseudomonas aeruginosa, escherichia coli, pneumococcus, enterococcus coli, tetragonococcus, listeria monocytogenes, streptococcus, anaerobe, stenotrophomonas maltophilia and other common pathogenic bacteria, and also has a strong killing effect on gonococcus and gastrohelospiral. In addition, the bactericidal composition also shows strong bactericidal effect on some drug-resistant bacteria, such as drug-resistant staphylococcus aureus, drug-resistant pneumococcus and the like.
2. The bactericidal activity and stability are much higher than those of lysostaphin. The lysostaphin compound enzyme is compounded with lysostaphin as main component and lysozyme, the small molecular compound, stabilizer, synergist and other components, and has higher stability than pure lysostaphin.
3. Drug resistance is not easy to generate. Lysostaphin and lysozyme as main active ingredients have a unique sterilization mechanism, and the lysostaphin achieves the effect of quickly killing pathogenic bacteria by specifically cutting off glycine bonds in muramyl peptidoglycan; lysozyme, on the other hand, acts on beta-1, 4-glucosidic bonds or beta-1, 3-glucan in the cell wall, breaking it. The two enzymes are compounded according to a certain proportion, and the bacterial cell walls are cracked doubly under the synergistic effect, so that the bacteria are not easy to generate drug resistance to the bacteria.
4. Has no toxic and side effects on organisms. Lysostaphin and lysozyme are protein products, which stimulate skin and mucosa, do not produce any toxic or side effect on organisms, can be degraded thoroughly through metabolism, and have no residue and no enrichment.
Detailed Description
The biological disinfectant preparation for preventing cow mastitis of the present invention is further described in detail with reference to the following embodiments.
Example 1
1. Composition and proportion
Lysostaphin 0.1%;
2% of lysozyme;
4001% of polyethylene glycol;
NaH2PO4 1％；
chlorhexidine acetate 0.1%;
the rest is distilled water;
the sum of the weight percentages of the components is 100 percent.
2. Preparation step
(1) Weighing lysostaphin, lysozyme and polyethylene glycol 400 according to the proportion to prepare a solution A;
(2) respectively weighing NaH according to the proportion2PO4Stirring solution A, slowly adding NaH to solution A2PO4Obtaining a solution B;
(3) and weighing chlorhexidine acetate according to the ratio, stirring the solution B, slowly adding the chlorhexidine acetate into the solution B, and uniformly stirring to obtain a finished product.
Example 2
1. Composition and proportion
Lysostaphin 10%;
80% of lysozyme;
polyethylene glycol 60005%;
KCI 20％；
chlorhexidine acetate 2%;
the rest is distilled water;
the sum of the weight percentages of the components is 100 percent.
2. Preparation step
(1) Weighing lysostaphin, lysozyme and polyethylene glycol 6000 according to the proportion to prepare a solution A;
(2) respectively weighing KCI according to the proportion, stirring the solution A, and slowly adding KCI into the solution A to obtain a solution B;
(3) and weighing chlorhexidine acetate according to the ratio, stirring the solution B, slowly adding the chlorhexidine acetate into the solution B, and uniformly stirring to obtain a finished product.
Example 3
1. Composition and proportion
1% of lysostaphin;
20% of lysozyme;
polyethylene glycol 200010%;
Na2HPO4 5％；
1% of allicin;
the rest is distilled water;
the sum of the weight percentages of the components is 100 percent.
2. Preparation step
(1) Weighing lysostaphin, lysozyme and polyethylene glycol 2000 according to the proportion to prepare a solution A;
(2) respectively weighing NaH according to the proportion2PO4Stirring solution A, slowly adding NaH to solution A2PO4Obtaining a solution B;
(3) weighing allicin according to the proportion, stirring the solution B, slowly adding the allicin into the solution B, and uniformly stirring to obtain a finished product.
The clinical trial report of the biological disinfectant preparation for preventing the cow mastitis shows that the biological disinfectant preparation has the following effects:
the first clinical test is as follows: sterilization experiment of biological disinfectant preparation for preventing cow mastitis on staphylococcus aureus and escherichia coli
First, equipment
1. Test bacteria: staphylococcus aureus generation 6 (supplied by Jun get medical academy of sciences, with flowing billy taste detection center), Escherichia coli generation 6 (supplied by Jun get medical academy of sciences, with disinfection detection center)
2. Sample preparation: biological disinfectant preparation for preventing cow mastitis
3. Neutralizing agent: D/E broth of 2% histidine, 8% lecithin wax, 10% tween-80
4. PBS: 0.03mol/L phosphate buffer solution
5. GNP-9160 type water-proof constant temperature incubator (Shanghai Jing hong experiment equipment Co., Ltd.)
6. Culture medium: common nutrient agar
7. Mechanical stopwatch (504 type)
8. Adjustable constant temperature water bath box (CBN28-3)
Second, method
1. The inspection basis is as follows: sanitary Standard for Disposable sanitary articles GB15979-2002 appendix C3
2. And (3) identification test of a neutralizer: test strains: concentration of staphylococcus aureus sample: 1: 20 diluent, action time: 0.5min
3. Quantitative sterilization test: sample concentration: 1: 20 dilution
4. The test temperature is 20 ℃, and the test is repeated for 3 times
Three, result in
1. The result of the neutralizer identification test shows that: the number of colonies grown in groups 1 and 2 was 0cfu/mL and 4.47 x 10, respectively2The error rates among cfu/mL, groups 3, 4 and 5 were 7.80%, 9.72% and 4.08%, respectively.
TABLE 2 neutralizer identification test results
2. When the test temperature is 20 ℃, the sterilization rate of the sample on staphylococcus aureus and escherichia coli is 100% after the sample is acted by the diluent with the ratio of 1: 2 for 2 minutes, 5 minutes, 10 minutes and 20 minutes.
TABLE 3 Sterilization effect on the test bacteria
And (2) clinical trial II: experiment of biological disinfectant preparation for preventing cow mastitis on candida albicans sterilization
First, equipment
1. Test bacteria: candida albicans generation 6 (supplied by the Chongyi medical science institute billy flow detection center)
2. Sample preparation: biological disinfectant preparation for preventing cow mastitis
3. Neutralizing agent: D/E broth of 2% histidine, 8% lecithin wax, 10% tween-80
4. PBS: 0.03mol/L phosphate buffer solution
5. GNP-9160 type water-proof constant temperature incubator (Shanghai Jing hong experiment equipment Co., Ltd.)
6. Culture medium: sabouraud's medium
7. Mechanical stopwatch (504 type)
8. Adjustable constant temperature water bath box (CBN28-3)
Second, method
1. The inspection basis is as follows: sanitary Standard for Disposable sanitary articles GB15979-2002 appendix C3
2. And (3) identification test of a neutralizer: sample concentration: stock solution, action time: 0.5min
3. Quantitative sterilization test: sample concentration: stock solution
4. The test temperature is 20 ℃, and the test is repeated for 3 times
Three, result in
1. The result of the neutralizer identification test shows that: the number of colonies grown in groups 1 and 2 were 0cfu/mL and 1.06 x 10, respectively3The error rates among cfu/mL, groups 3, 4 and 5 were 7.78%, 13.83% and 9.08%, respectively.
TABLE 4 neutralizer identification test results
2. When the test temperature is 20 ℃, the sterilization rate of the sample on candida albicans is 100% after the sample is acted by the 1: 2 diluent for 2 minutes, 5 minutes, 10 minutes and 20 minutes.
TABLE 5 Sterilization effect on the test bacteria
Claims (2)
1. A biological enzyme disinfectant solution preparation is characterized by comprising lysostaphin and lysozyme, and the biological enzyme disinfectant solution comprises the following components in percentage by weight:
0.1 to 20 percent of lysostaphin;
2% -80% of lysozyme;
1% -50% of protein stabilizer;
1-20% of ionic compound;
0.1 to 30 percent of bactericide;
the rest is distilled water;
the sum of the weight percentages of the components is 100 percent.
2. The preferred composition and weight percent as claimed in claim 1 is:
0.1 to 10 percent of lysostaphin;
2% -40% of lysozyme;
1% -5% of a protein stabilizer;
1-20% of ionic compound;
0.1 to 2 percent of bactericide;
the rest is distilled water;
the sum of the weight percentages of the components is 100 percent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911314816.0A CN112999337A (en) | 2019-12-19 | 2019-12-19 | Biological enzyme disinfectant preparation and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911314816.0A CN112999337A (en) | 2019-12-19 | 2019-12-19 | Biological enzyme disinfectant preparation and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112999337A true CN112999337A (en) | 2021-06-22 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911314816.0A Pending CN112999337A (en) | 2019-12-19 | 2019-12-19 | Biological enzyme disinfectant preparation and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113440645A (en) * | 2021-06-29 | 2021-09-28 | 西安汇朴成医疗科技有限公司 | Composite lysozyme liquid dressing for wound surface and preparation method thereof |
-
2019
- 2019-12-19 CN CN201911314816.0A patent/CN112999337A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113440645A (en) * | 2021-06-29 | 2021-09-28 | 西安汇朴成医疗科技有限公司 | Composite lysozyme liquid dressing for wound surface and preparation method thereof |
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