CN111500562A - Complex enzyme preparation for killing viruses and application thereof - Google Patents
Complex enzyme preparation for killing viruses and application thereof Download PDFInfo
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- CN111500562A CN111500562A CN202010466611.0A CN202010466611A CN111500562A CN 111500562 A CN111500562 A CN 111500562A CN 202010466611 A CN202010466611 A CN 202010466611A CN 111500562 A CN111500562 A CN 111500562A
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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Abstract
The invention discloses a complex enzyme preparation for killing viruses, which comprises keratinase, aminopeptidase, nuclease, lysozyme, acid protease, neutral protease and/or alkaline protease; wherein the nuclease and lysozyme are coated by an encapsulating material; also discloses the application of the compound enzyme preparation. The compound enzyme preparation of the invention is used for killing viruses of articles, air, sewage, garbage and excrement and urine, and has obvious killing effect.
Description
Technical Field
The invention belongs to the technical field of sanitary protection, and particularly relates to a complex enzyme preparation for killing viruses and application thereof.
Background
There are many ways in which viruses can be transmitted, and those susceptible to infection include contact transmission, droplet transmission, aerosol transmission, fecal oral transmission, and the like. In order to reduce various transmission risks, virus disinfection treatment is generally required for air environment, medical waste, domestic sewage of patients, excrement and urine and the like.
However, the existing disinfectants have poor stability and are easy to decompose, or have strong irritation and corrosiveness, or are not suitable for virus killing treatment of complex component backgrounds, or are easy to cause new environmental pollution, and the application range and the effect are limited.
Therefore, it is desired to provide a product that can be widely applied to various articles and environmental virus killing.
Disclosure of Invention
In view of the above, the invention provides a complex enzyme preparation for virus killing, which can be applied to virus killing treatment of complex component backgrounds such as articles, air, sewage, garbage, excrement and urine, and has good killing effect, safe use and environmental protection.
In order to achieve the purpose, the invention adopts the following technical scheme:
a complex enzyme preparation for killing viruses comprises keratinase, aminopeptidase, nuclease and lysozyme,
also included are acid proteases, neutral proteases and/or alkaline proteases;
wherein, the nuclease and the lysozyme are coated by an encapsulating material.
According to the invention, any one of acid protease, neutral protease and alkaline protease is used together with keratinase, aminopeptidase, nuclease and lysozyme, and the acid protease, the neutral protease or the alkaline protease can be selected according to different acid-base environments, so that the use of the complex enzyme preparation is wider; acid protease, neutral protease and alkaline protease can also be used in combination to make the killing action point more broad-spectrum. Protease firstly effectively decomposes outer membrane protein of the virus to expose nucleic acid thereof; furthermore, nuclease can carry out enzymolysis on nucleic acid into nucleotide, completely destroy the nucleic acid structure of the virus, and achieve the purpose of effectively killing the virus; lysozyme can hydrolyze the sugar chain of viral glycoprotein to destroy the viral protein shell, and can be directly combined with negatively charged viral protein to form double salt with DNA, RNA and coenzyme-removed protein to inactivate virus. The nuclease and the lysozyme are coated, so that the enzyme inactivation caused by improper storage or complex use environment can be avoided; the keratinase, aminopeptidase, acid protease, neutral protease and/or alkaline protease are used as main agents in the complex enzyme preparation and are not coated, so that the efficiency of killing the viruses on articles and the environment can be ensured; in the process of killing, nuclease and lysozyme are gradually released, so that the killing effect is further enhanced.
The complex enzyme preparation used in the invention is tasteless and nontoxic, can adapt to different pH environments, and has good killing effect on various viruses.
Preferably, the dosage ratio of (acid protease, neutral protease and/or alkaline protease), keratinase, aminopeptidase, nuclease and lysozyme is (10-1000) to (0.1-100) in terms of enzyme activity unit.
Preferably, the dosage ratio of (acid protease, neutral protease and/or alkaline protease), keratinase, aminopeptidase, nuclease and lysozyme is (100-.
Preferably, the encapsulating material comprises maltodextrin and chitosan.
Preferably, the ratio of the maltodextrin to the chitosan is (2-3) to 1.
Preferably, the materials are weighed according to the weight ratio of (nuclease + lysozyme): (2-3): 1;
dispersing nuclease into water to prepare a solution A;
dispersing maltodextrin and chitosan in water to prepare solution B;
adding the solution B into the solution A, and uniformly stirring to obtain a solution C;
and (3) spray drying the solution C to obtain the nuclease and lysozyme coated by the encapsulating material.
The complex enzyme preparation for killing viruses is applied to killing of articles and environmental viruses; the environment includes an indoor environment and an outdoor environment.
The application in the environmental virus disinfection comprises garbage treatment, excrement treatment, air treatment or sewage treatment.
Further, the process of killing the environmental viruses needs to use a large dose of complex enzyme preparation, so that the human body needs to be prevented from directly contacting the complex enzyme preparation.
The complex enzyme preparation can be used for preparing air filter materials.
Furthermore, when the complex enzyme preparation is used for preparing the air filtering material, one or more dust filtering layers are arranged on two sides of the complex enzyme preparation, so that the complex enzyme preparation is prevented from escaping along with the filtered air when the complex enzyme preparation is used for a long time.
Further, the application of the complex enzyme preparation in air treatment is to dissolve the complex enzyme preparation into enzyme liquid, and use equipment to enable air to pass through the enzyme liquid for virus killing.
Furthermore, the complex enzyme preparation for killing the viruses can be dissolved into enzyme liquid, the enzyme liquid is sprayed downwards by using a spray tower, and air is convected from bottom to top, so that the viruses in the air are killed.
Furthermore, the complex enzyme preparation can be dissolved into enzyme liquid, the enzyme liquid is placed in a container or equipment, air is introduced into the container or equipment, and the virus killing can be realized through the enzyme liquid by the air.
Further, when the disinfectant is applied to disinfection of articles, the disinfectant can be diluted to a lower concentration properly so as to meet the virus disinfection requirements of articles such as clothes, medical instruments, articles for daily use and the like.
According to the technical scheme, the complex enzyme preparation can be directly used for killing viruses of various articles and environments, is safe and convenient to use, and has an obvious killing effect.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Example 1
A complex enzyme preparation for killing viruses comprises 30g of acid protease, 20g of keratinase, 10g of aminopeptidase, 20g of nuclease and 20g of lysozyme.
Wherein the content of the first and second substances,
the acid protease is acid protease, and the enzyme activity is 80 ten thousand U/g;
the keratinase is keratinase, and the enzyme activity is 20 ten thousand U/g;
the aminopeptidase is aminopeptidase with the enzyme activity of 5000U/g;
the nuclease is nuclease, and the enzyme activity is 10 ten thousand U/g;
the lysozyme is lysozyme, and the enzyme activity is 2 ten thousand U/g.
The nuclease and the lysozyme are coated by an encapsulating material, and the coating method is as follows:
dispersing 20g of nuclease and 20g of lysozyme in 1L water to prepare a solution A;
dispersing 25g of maltodextrin and 10g of chitosan in 1L water to prepare a solution B;
slowly adding the solution into the solution A while stirring uniformly to prepare a solution C;
and (3) spray drying the solution C to obtain the nuclease and lysozyme coated by the encapsulating material.
Mixing nuclease and lysozyme coated by the encapsulation material with acid protease, keratinase and aminopeptidase uniformly to obtain a complex enzyme preparation, and directly adding the complex enzyme preparation into garbage, sewage and excrement and urine for treatment or dissolving the complex enzyme preparation into a virus killing liquid for spraying treatment.
Further, verifying the virus killing capacity of the complex enzyme preparation; the tested viruses comprise influenza virus H5N3(MDCK cell is used as a vector) and hepatitis A virus (HepG2.2.15 cell is used as a vector).
Culturing each group of carrier cells respectively, inoculating virus when the cells grow into a monolayer, collecting the carrier cells when 3/4 cells have pathological changes, centrifuging after ultrasonic disruption, collecting supernatant, diluting with sterile water or feces-urine solution sterilized at high temperature, and making into about 1 × 104pfu/m L virus solution, and adjusting the pH of the virus solution to 6.0.
Dissolving the compound enzyme preparation in sterile water respectively to prepare enzyme liquid with the enzyme activity of acid protease about 800U/m L, mixing the enzyme liquid and virus liquid according to the volume ratio of 1: 10, processing at 25 +/-2 ℃ for 40min, diluting by 10 times of series gradient, inoculating cells, culturing at 37 ℃ for 48h, fixing by methanol, dyeing by crystal violet, cleaning, and counting the plaques (a round non-colored transparent area is a plaque). The virus content (pfu/m L) in each milliliter of the processing liquid is equal to the average plaque number × dilution times.
Simultaneously setting a blank control group, and mixing sterile water or feces and urine liquid subjected to high-temperature sterilization with virus liquid; the virus killing rate of the complex enzyme preparation is calculated according to the blank control group, and the test result is shown in table 1:
TABLE 1
Example 2
A complex enzyme preparation for killing viruses comprises 50g of neutral protease, 10g of keratinase, 10g of aminopeptidase, 10g of nuclease and 20g of lysozyme.
Wherein the content of the first and second substances,
the neutral protease is neutral protease 1398, and the enzyme activity is 15 ten thousand U/g;
the keratinase is keratinase, and the enzyme activity is 20 ten thousand U/g;
the aminopeptidase is aminopeptidase with the enzyme activity of 5000U/g;
the nuclease is nuclease, and the enzyme activity is 10 ten thousand U/g;
the lysozyme is lysozyme, and the enzyme activity is 2 ten thousand U/g.
The nuclease and the lysozyme are coated by an encapsulating material, and the coating method is as follows:
dispersing 10g of nuclease and 20g of lysozyme in 500m L of water to prepare a solution A;
taking 15g of maltodextrin and 6g of chitosan, and dispersing in 500m of L water to prepare a solution B;
slowly adding the solution into the solution A while stirring uniformly to prepare a solution C;
and (3) spray drying the solution C to obtain the nuclease and lysozyme coated by the encapsulating material.
Mixing nuclease and lysozyme coated by the encapsulation material with neutral protease and keratinase uniformly to obtain the complex enzyme preparation.
The complex enzyme preparation is suitable for killing viruses in the air environment, for example, the complex enzyme preparation is used as a filler, and is coated by a filter material to form a filter element, and the filter element is arranged in a fresh air system to complete virus killing when air in the environment is ventilated.
In addition, the complex enzyme preparation can be directly added into garbage, sewage and excrement and urine for treatment, and can also be dissolved to prepare virus killing liquid for spraying treatment.
Further, the complex enzyme preparation can be dissolved into enzyme liquid, the enzyme liquid is sprayed downwards by using a spray tower, and air is convected from bottom to top, so that virus in the air can be killed.
Furthermore, the complex enzyme preparation can be dissolved into enzyme liquid, the enzyme liquid is placed in a container or equipment, air is introduced into the container or equipment, and the virus killing can be realized through the enzyme liquid by the air.
Furthermore, the compound enzyme preparation can be dissolved into enzyme liquid for wiping and cleaning the objects to be disinfected.
The complex enzyme preparation is tested as follows:
1. skin irritation test:
selecting 4 healthy rabbits with intact skin, cutting hairs on two sides of a spinal column of the healthy rabbits 24 hours before test with the left and right sides of each rabbit being 3 × 3cm, preparing a neutral protease enzyme solution with sterile water, wherein the enzyme activity of the neutral protease enzyme solution is about 140U/m L enzyme solution, dripping 0.5m L solution on 4 layers of gauze (2 × 2cm), applying the gauze on the surface of the skin with hair removed on one side, covering the surface with a plastic film and fixing the gauze with a non-irritant adhesive plaster, dripping sterile physiological saline on the gauze on the other side, removing the gauze after applying for 4 hours, washing the applied part with warm water, and observing local reaction changes of the skin after removing the gauze for 1 hour, 24 hours and 48 hours.
The experimental result shows that the application parts of 4 rabbits have no red and swollen phenomenon, namely the enzyme solution of neutral protease prepared by the compound enzyme preparation, the enzyme activity of which is about 140U/m L, has no obvious skin irritation.
2. Eye irritation test
Selecting 4 normal rabbits, preparing enzyme solution of neutral protease with activity of about 140U/m L with sterile water, dripping 0.1m L into conjunctival sac of one side of the rabbit, using sterile normal saline as control, closing the eyes of the rabbit for 1s after dripping, washing the eyes with normal saline after 30s, and observing hyperemia and swelling conditions of cornea, iris and conjunctiva after dripping for 1h, 24h, 48h and 72 h.
The results of the experiment are shown in table 2:
TABLE 2
| Stimulation conditions | 1h (number) | 24h (number) | 48h (number) | 72h (number) |
| Corneal opacity | 0 | 0 | 0 | 0 |
| Iris damage | 0 | 0 | 0 | 0 |
| Conjunctival congestion or edema | 2 | 2 | 1 | 0 |
The experiments show that the conjunctival congestion or edema caused by enzyme solution of neutral protease with the activity of about 140U/m L can be recovered within 72h, and the use is safe, but for environmental virus disinfection, the concentration of the complex enzyme preparation can be diluted by pollutants, the dosage of the complex enzyme preparation is relatively increased, and if the complex enzyme preparation directly contacts skin and mucosa, irritation reaction can be caused, so that in the actual operation, the complex enzyme preparation is prevented from directly contacting human bodies.
Example 3
A complex enzyme preparation for killing viruses comprises 20g of alkaline protease, 30g of keratinase, 20g of aminopeptidase, 10g of nuclease and 20g of lysozyme.
Wherein the content of the first and second substances,
the alkaline protease is alkaline protease, and the enzyme activity is 45 ten thousand U/g;
the keratinase is keratinase, and the enzyme activity is 20 ten thousand U/g;
the aminopeptidase is aminopeptidase with the enzyme activity of 5000U/g;
the nuclease is nuclease, and the enzyme activity is 10 ten thousand U/g;
the lysozyme is lysozyme, and the enzyme activity is 2 ten thousand U/g.
The nuclease and the lysozyme are coated by an encapsulating material, and the coating method is as follows:
dispersing 10g of nuclease and 20g of lysozyme in 500m L of water to prepare a solution A;
taking 15g of maltodextrin and 7g of chitosan, and dispersing in 500m of L water to prepare a solution B;
slowly adding the solution into the solution A while stirring uniformly to prepare a solution C;
and (3) spray drying the solution C to obtain the nuclease and lysozyme coated by the encapsulating material.
Mixing nuclease and lysozyme coated by the encapsulation material with neutral protease and keratinase uniformly to obtain a complex enzyme preparation, and directly adding the complex enzyme preparation into garbage, sewage and excrement and urine for treatment or dissolving the complex enzyme preparation into a virus killing liquid for spraying treatment.
Further, verifying the virus killing capacity of the complex enzyme preparation; the tested viruses comprise influenza virus H5N3(MDCK cell is used as a vector) and hepatitis A virus (HepG2.2.15 cell is used as a vector).
Culturing each group of carrier cells respectively, inoculating virus when the cells grow into monolayer, collecting the carrier cells when 3/4 cells have pathological changes, centrifuging after ultrasonic disruption, collecting supernatant, diluting with sterile water or sterilized domestic sewage respectively, and making into 1 × 104pfu/m L virus solution, and adjusting the pH of the virus solution to 8.0.
Dissolving the complex enzyme preparation in sterile water respectively to prepare an alkaline protease enzyme solution with the enzyme activity of about 540U/m L, mixing the enzyme solution and a virus solution according to the volume ratio of 1: 10, treating at 25 +/-2 ℃ for 40min, diluting by 10 times of series gradient, inoculating cells, culturing at 37 ℃ for 48h, fixing by methanol, dyeing by crystal violet, cleaning, and counting the plaques (a round non-colored transparent area is one plaque). The virus content (pfu/m L) in each milliliter of the treatment solution is equal to the average plaque number × dilution times.
Simultaneously setting a blank control group, and mixing sterile water or sterilized domestic sewage with the virus solution; the virus killing rate of the complex enzyme preparation is calculated according to the blank control group, and the test result is shown in table 3:
TABLE 3
Further, the coating effect of the nuclease and the lysozyme in the compound enzyme preparation is verified:
wherein, the experimental group adopts the complex enzyme preparation of the embodiment 3, the complex enzyme preparation of the control group 1 comprises 20g of alkaline protease, 30g of keratinase, 20g of aminopeptidase, 10g of nuclease and 20g of lysozyme, and the complex enzyme preparation is not coated; the control group 2 complex enzyme preparation comprises 20g of alkaline protease, 30g of keratinase, 20g of aminopeptidase, 10g of nuclease and 20g of lysozyme, and all enzyme components are coated by maltodextrin and chitosan; after being prepared, the complex enzyme preparations of all groups are placed at room temperature for 7 days, and then the killing effect of the complex enzyme preparations of all groups is verified.
The test virus is influenza virus H5N3(MDCK cell is vector), the vector cell is cultured, the virus is inoculated when the cell grows full of monolayer, when 3/4 cell has pathological changes, the vector cell is collected, the carrier cell is centrifuged after being crushed by ultrasound, supernatant is taken out and diluted by sterilized domestic sewage respectively to prepare about 1 × 105pfu/m L virus solution, and adjusting the pH of the virus solution to 8.0.
Dissolving the above-mentioned composite enzyme preparations in sterile water respectively to obtain alkaline protease enzyme solution with enzyme activity of about 540U/m L, mixing the enzyme solution and virus solution according to the volume ratio of 1: 10, treating at 25 +/-2 deg.C for 40min, 10-fold serial gradient dilution, inoculating cell, culturing at 37 deg.C for 48 hr, fixing with methanol, crystal violet staining, cleaning and counting the plaque (round non-coloured transparent zone is a plaque), and the virus content (pfu/m L) in every ml of treatment solution is equal to average plaque × dilution times.
Meanwhile, setting a blank control group, and mixing sterilized domestic sewage with the virus liquid; the virus killing rate of the complex enzyme preparation is calculated according to the blank control group, and the test result is shown in table 4:
TABLE 4
| Group of | Ratio of virus killing (%) |
| Experimental group | 98.2 |
| Control group 1 | 82.0 |
| Control group 2 | 89.7 |
The embodiments described above are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A complex enzyme preparation for killing viruses is characterized in that,
including keratinase, aminopeptidase, nuclease and lysozyme,
also included are acid proteases, neutral proteases and/or alkaline proteases;
wherein the nuclease and the lysozyme are coated with an encapsulating material.
2. A complex enzyme preparation for viral eradication according to claim 1,
the dosage ratio of (acid protease, neutral protease and/or alkaline protease), keratinase, aminopeptidase, nuclease and lysozyme is (10-1000) to (0.1-100) in terms of enzyme activity unit.
3. A complex enzyme preparation for viral eradication according to claim 1,
the dosage ratio of (acid protease, neutral protease and/or alkaline protease), keratinase, aminopeptidase, nuclease and lysozyme is (100-) - (500-) - (20-200): (0.5-20) based on enzyme activity unit.
4. A complex enzyme preparation for viral eradication according to claim 1,
the encapsulating material includes maltodextrin and chitosan.
5. A complex enzyme preparation for viral eradication according to claim 4,
the ratio of the maltodextrin to the chitosan is (2-3) to 1.
6. A complex enzyme preparation for viral eradication according to claim 5,
weighing the materials according to the weight ratio of (nuclease + lysozyme) to maltodextrin to chitosan of (4-5) to (2-3) to 1;
dispersing nuclease into water to prepare a solution A;
dispersing maltodextrin and chitosan in water to prepare solution B;
adding the solution B into the solution A, and uniformly stirring to obtain a solution C;
and (3) spray drying the solution C to obtain the nuclease and lysozyme coated by the encapsulating material.
7. Use of a complex enzyme preparation for viral eradication according to any one of claims 1 to 6 in the eradication of an article or the environment; the environment includes an indoor environment and an outdoor environment.
8. The use according to claim 7,
the application in the disinfection of environmental viruses comprises garbage treatment, excrement treatment, air treatment or sewage treatment.
9. Use of a complex enzyme preparation for viral eradication according to any one of claims 1 to 6 in the preparation of an air filter material.
10. The use of the complex enzyme preparation for virus killing of any one of claims 1-6 in air treatment, characterized in that the complex enzyme preparation is dissolved into enzyme solution, and the virus killing is carried out by passing air through the enzyme solution by using equipment; including but not limited to spray towers.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010466611.0A CN111500562A (en) | 2020-05-27 | 2020-05-27 | Complex enzyme preparation for killing viruses and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112641532A (en) * | 2020-12-22 | 2021-04-13 | 江苏瞪羚科技有限公司 | Application method of compound enzyme disinfectant |
| CN113151228A (en) * | 2021-03-16 | 2021-07-23 | 李明达 | Complex enzyme preparation for killing viruses and preparation method and application thereof |
| CN114306584A (en) * | 2022-01-19 | 2022-04-12 | 百川开泰生物技术(北京)有限公司 | Application of proteinase K and compound enzyme thereof in preparation of virus inhibitor |
| CN114984746A (en) * | 2022-08-05 | 2022-09-02 | 山东建筑大学 | System method and device for treating VOCs waste gas in disinfectant and aquaculture wastewater pool |
-
2020
- 2020-05-27 CN CN202010466611.0A patent/CN111500562A/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112641532A (en) * | 2020-12-22 | 2021-04-13 | 江苏瞪羚科技有限公司 | Application method of compound enzyme disinfectant |
| CN113151228A (en) * | 2021-03-16 | 2021-07-23 | 李明达 | Complex enzyme preparation for killing viruses and preparation method and application thereof |
| CN114306584A (en) * | 2022-01-19 | 2022-04-12 | 百川开泰生物技术(北京)有限公司 | Application of proteinase K and compound enzyme thereof in preparation of virus inhibitor |
| CN114306584B (en) * | 2022-01-19 | 2024-03-15 | 百川开泰生物技术(北京)有限公司 | Application of proteinase K and complex enzyme thereof in preparation of virus inhibitor |
| CN114984746A (en) * | 2022-08-05 | 2022-09-02 | 山东建筑大学 | System method and device for treating VOCs waste gas in disinfectant and aquaculture wastewater pool |
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Effective date of registration: 20210618 Address after: 061000 No. 16 Jiuhe East Road, Cangzhou Economic Development Zone, Cangzhou City, Hebei Province Applicant after: Cangzhou Xiasheng Enzyme Biotechnology Co.,Ltd. Applicant after: Hunan Xiasheng Enzyme Technology Co.,Ltd. Applicant after: NINGXIA SUNSON INDUSTRY GROUP Co.,Ltd. Address before: 061000 No. 16 Jiuhe East Road, Cangzhou Economic Development Zone, Cangzhou City, Hebei Province Applicant before: Cangzhou Xiasheng Enzyme Biotechnology Co.,Ltd. |
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