CN117581869A - Disinfectant agent - Google Patents

Disinfectant agent Download PDF

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CN117581869A
CN117581869A CN202311003815.0A CN202311003815A CN117581869A CN 117581869 A CN117581869 A CN 117581869A CN 202311003815 A CN202311003815 A CN 202311003815A CN 117581869 A CN117581869 A CN 117581869A
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disinfectant
agent
cysteine
active agent
reducing agent
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熊小波
管涛
常国栋
张平
罗亮
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Protgen Ltd
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Protgen Ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N33/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic nitrogen compounds
    • A01N33/02—Amines; Quaternary ammonium compounds
    • A01N33/12—Quaternary ammonium compounds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/22—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing ingredients stabilising the active ingredients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides

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  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Engineering & Computer Science (AREA)
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  • Agronomy & Crop Science (AREA)
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  • General Chemical & Material Sciences (AREA)
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Abstract

The invention provides a novel disinfectant, comprising: an active agent, a reducing agent and a buffer. In a particularly preferred embodiment, the disinfectant consists of the following components: active agent, reducing agent, protective agent, chelating agent, buffer and auxiliary materials. The disinfectant has broad-spectrum disinfection effect, can be widely used for disinfecting various microorganisms such as bacteria, fungi, viruses and the like, and is especially suitable for disinfecting novel coronaviruses.

Description

Disinfectant agent
Cross-reference information
The present application claims priority from chinese patent application No. 202210957717.X, filed at 10 at 08 at 2022, which is incorporated herein by reference in its entirety.
Technical Field
The invention relates to the field of disinfectants, in particular to a disinfectant suitable for disinfecting a new coronavirus.
Background
The disinfectants on the market at present mainly comprise the following types. 1. Chlorine-containing disinfectants, such as 84 disinfectant, with the effective chlorine content of 5.5-6.5%, can kill various microorganisms, and is suitable for disinfection of object surfaces, environments and the like; however, the disinfectant needs to have good sterilization effect only by high concentration and long time, is unstable, and is easily decomposed into toxic and corrosive smoke at high temperature, thereby causing damage to human body. 2. Peroxide disinfectants, such as 3% hydrogen peroxide disinfectant, have strong oxidizing property, can effectively kill enteropathogenic bacteria, suppurative cocci and pathogenic microzyme, and are generally used for object surface disinfection; however, the disinfectant is corrosive to metals, and is not suitable for surface disinfection of ports, logistics and other articles. 3. Alcohol disinfectants, such as 75% ethanol, are suitable for disinfecting the surfaces of objects and human skin; however, the object can be sterilized by soaking or repeatedly wiping, and the sterilizing device is not suitable for sterilizing places with dense personnel due to inflammability and volatility. The novel sterilizing device is mainly used for medical care sterilization.
Quaternary ammonium salt disinfectant, a broad spectrum disinfectant. The disinfectant has strong sterilization capability and low effective use concentration, can effectively kill bacteria, fungi and viruses at the concentration of 0.01-0.1%, has stable property and no irritation and corrosiveness, can be directly used for disinfecting skin and object surfaces, and is suitable for large-scale use.
Benzalkonium chloride, an (N-alkyl-phenyl-dimethyl) ammonium chloride mixture, is a commonly used quaternary ammonium salt surfactant. The long-chain alkyl group can destroy the cell (wall) membrane of the microorganism, so that the microorganism dies, and the sterilization effect is achieved. Therefore, manufacturers develop various disinfectants containing benzalkonium chloride, and patent CN102065696B discloses a universal sterilizing disinfectant which takes benzalkonium chloride, sorbic acid, benzoic acid and the like as active agents and has high-efficiency sterilizing capability. But the activity pH value is 2.5-4.0, has weak acidity and is not suitable for popularization and use. Patent CN201611024695 discloses a composite disinfectant consisting of benzalkonium chloride and other surfactants, which can kill (log > 6) escherichia coli, staphylococcus aureus, streptococcus white, pseudomonas aeruginosa with high efficiency within 5 minutes. Patent CN201010289039 discloses a compound antibacterial agent and a preparation method thereof. The method takes benzalkonium chloride, didecyl dimethyl ammonium chloride and epsilon-polylysine as main antibacterial components, and can kill (log > 4) Escherichia coli, staphylococcus aureus and Streptococcus albus 100% within 2 minutes. Patent CN202010085304 discloses a formulation of a skin disinfection cleaning solution and a preparation method thereof, the cleaning solution is compounded with benzalkonium chloride and cetylpyridinium chloride, and can kill (log > 5) colibacillus 100% in 1 minute and 99.9% of skin surface bacteria in 10 minutes, the patent mentions that the method can kill novel coronaviruses, but no specific data is disclosed.
Summary of The Invention
The invention provides a novel disinfectant, comprising: an active agent, a reducing agent and a buffer.
In a particularly preferred embodiment, the disinfectant consists of the following components: active agent, reducing agent, protective agent, chelating agent, buffer and auxiliary materials.
The disinfectant of the present invention is particularly suitable for disinfecting or inhibiting new coronaviruses. The disinfectant has the characteristics of short acting time, high sterilization effect, no pollution and the like, can be applied to the surface of a human body, such as a hand, and can also be applied to public places (such as hospitals, subways, schools and airports), logistics cold chains or physical surfaces, such as large containers in ports.
Detailed Description
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
"coronavirus" refers to a class of enveloped viruses that can cause disease in vertebrates, particularly mammals or birds, and has a single positive strand RNA genome. They belong to the order of the phylogenetic group of the order of the cage-viruses (Nidovirales), the family of the Coronaviridae (Coronaviridae). 7 coronaviruses have been found to be capable of infecting humans, including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. Some of the diseases they cause are milder, like common influenza, while others may be fatal, such as SARS, MERS and covd-19. The coronavirus responsible for the COVID-19 is SARS-CoV-2, also known as "New coronavirus".
Without being bound by any particular theory, the inventors have unexpectedly found that: the active agent, the reducing agent and the chelating agent in the disinfectant can play roles through different mechanisms, and the disinfectant can be used in combination, so that the disinfection effect can be well improved.
Based on the above findings, the inventor has developed a novel broad-spectrum disinfectant, which uses an active agent, a reducing agent and/or a chelating agent in combination, and uses their respective different action mechanisms, thereby greatly improving the disinfecting effect of the disinfectant.
The "active agent" may be various commonly used active agents suitable for the disinfection of microorganisms such as bacteria and viruses. Preferred surfactants are quaternary ammonium surfactants. For example, the preferred active agent may be one or more of the following: benzalkonium chloride, benzalkonium bromide, benzethonium chloride, cetylpyridinium chloride, and decylmethylammonium chloride. A particularly preferred active agent is benzalkonium chloride.
The active agents of the present invention may also comprise one or several other types of active agents, such as anionic or nonionic surfactants and the like.
The quaternary ammonium salt disinfectant has the advantages of low sterilization concentration, no irritation, no toxicity and no corrosiveness, and in addition, the quaternary ammonium salt disinfectant has the functions of surface adsorption of the surfactant, reducing surface tension and having a certain washing function.
The active agent in the disinfectant of the present invention may be one or more.
The "buffer" is used to provide a relatively stable solution environment, having the effect of buffering pH changes, avoiding the effect of extreme environments on the disinfectant's sanitizing effect. Preferably near neutral pH, e.g. 5-10. Various commonly used buffers may be used, such as Tris (hydroxymethyl) aminomethane (Tris), disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 3-morpholinopropane sulfonic acid (MOPS), and the like. A particularly preferred buffer is disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB).
The chelating agent can chelate heavy metal ions in water, so that the influence of the heavy metal ions on the disinfectant effect is reduced; in addition, the chelating agent has a coordination bond, and can form a stable ligand with metal ions, so that the metalloproteinase on the cell (virus) membrane is inactivated. Preferred chelating agents include one or more of the following: ethylenediamine tetraacetic acid (EDTA) and its metal salts, aminotriacetic acid (NTA) and its metal salts, ethylene glycol diethylether diamine tetraacetic acid (EGTA) and its metal salts, diethylenetriamine pentaacetic acid (DTPA) and its metal salts. Particularly preferred chelating agents of the invention are EDTA-Na 2 Etc. The chelating agent in the disinfectant of the present invention may be one or more.
The term "metal salt" as used herein refers to a soluble salt of an acid (e.g., EDTA) with a metal ion, such as sodium, potassium, etc. For example, "metal salts" of EDTA, such as sodium salts, may also be generally used as chelating agents.
"reducing agent" refers to a class of compounds that are capable of cleaving disulfide bonds of a protein or polypeptide. Preferred reducing agents include one or more of the following: dithiothreitol (DTT), reduced glutathione, beta-mercaptoethanol, mercaptoacetic acid, and metal salts thereof, L-Cysteine (Cysteine) or tris (2-carboxyethyl) phosphine (TCEP). A particularly preferred reducing agent is L-Cysteine (Cysteine). The reducing agent in the disinfectant of the present invention may be one or more.
"protectant" refers to a class of antioxidants that prevent or mitigate oxidation of a reducing agent in a disinfectant. Preferred protectants include one or more of the following: sodium sulfite (Na) 2 SO 3 ) Vitamin C, vitamin E. A particularly preferred protecting agent is sodium sulfite.
Preferably, a protective agent is added to the disinfectant to help prevent or mitigate oxidation of the reducing agent, which has a positive effect on improving the disinfectant's biocidal effect.
Those skilled in the art will appreciate that the protective agent may not be added if the reducing agent is relatively stable in nature or if the reducing agent is used in relatively large amounts.
Preferably, a chelating agent is added to the disinfectant to form a stable ligand with the metal ions, thereby inactivating the metalloprotease on the cell membrane (virus) and improving the disinfecting effect of the disinfectant.
The invention provides a novel disinfectant, comprising: an active agent, a reducing agent and a buffer. In some embodiments, the disinfectant consists of an active agent, a reducing agent, a buffer, and optionally an adjuvant.
In some embodiments, the disinfectant comprises an active agent, a reducing agent, a buffer, a protectant. In some embodiments, the disinfectant consists of an active agent, a reducing agent, a buffer, a protectant, and optionally an adjuvant.
In some embodiments, the disinfectant further comprises an active agent, a reducing agent, a buffer, a chelating agent. In some embodiments, the disinfectant consists of an active agent, a reducing agent, a buffer, a chelating agent, and optionally an adjuvant.
In some embodiments, the disinfectant comprises an active agent, a reducing agent, a buffer, a protectant, and a chelating agent.
In some embodiments, the disinfectant is composed of the following components: an active agent, a reducing agent, a protecting agent, a chelating agent, and a buffer.
In some preferred embodiments, the disinfectant consists of the following components: active agent, reducing agent, protective agent, chelating agent, buffer and auxiliary materials.
The term "adjuvant" refers to conventional additives known in the disinfectant art other than active agents, reducing agents, and the like, which do not significantly affect the disinfectant's disinfecting action, including but not limited to: solvents, fragrances, colorants, preservatives, wetting agents, and the like.
Those skilled in the art will appreciate that different types of adjuvants may be selected in order to prepare a disinfectant suitable for a particular use scenario. For example, for aqueous disinfectants, adjuvants such as sterile water may be added as a solvent; for disinfectants directly applied to the surface of the human body, such as disinfectants for the hands, adjuvants such as wetting agents (e.g. glycerol or hyaluronic acid) or fragrances (e.g. peppermint oil) may be added.
The term "optional" means that the object described is optional and may or may not be present. For example, "optional adjuvants" means that the disinfectant may or may not contain adjuvants, as may be determined by one skilled in the art according to the particular application scenario.
In some preferred embodiments, the disinfectant comprises: benzalkonium chloride as active agent, EDTA-Na as chelating agent 2 L-cysteine as a reducing agent, sodium sulfite as a protecting agent, and disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB) as a buffering agent.
In some particularly preferred embodiments, the disinfectant consists of the following components: benzalkonium chloride as active agent, EDTA-Na as chelating agent 2 L-cysteine as a reducing agent, sodium sulfite as a protecting agent, and disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB) as a buffering agent.
In some particularly preferred embodiments, the disinfectant consists of the following components: benzalkonium chloride as active agent, EDTA-Na as chelating agent 2 L-cysteine as a reducing agent, sodium sulfite as a protecting agent, disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB) as a buffering agent, and an auxiliary material.
In some embodiments, the concentration of quaternary ammonium surfactant as active agent is 0.001% to 1.0%
(W/V), for example, the concentration may be 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009% (W/V). In some embodiments, the concentration of quaternary ammonium surfactant as the active agent is from 0.01% to 0.1% (W/V). In some preferred embodiments, the concentration of benzalkonium chloride as the active agent is 0.01% to 0.1% (W/V).
In some embodiments, the concentration of the chelating agent is in the range of 0.01-10.0mM, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1mM. In some preferred embodiments, EDTA-Na as chelating agent 2 The concentration of (C) is 0.05-10.0mM.
In some embodiments, the concentration of the reducing agent is in the range of 0.1-15.0mM, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5mM. In some preferred embodiments, the concentration of L-Cysteine (Cysteine) as a reducing agent is 1.0-15.0mM.
In some embodiments, the concentration of the protecting agent is in the range of 0.01-50.0mM, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0mM. In some preferred embodiments, sodium sulfite (Na 2 SO 3 ) The concentration of (C) is 1.0-50.0mM. .
It will be appreciated by those skilled in the art that as the concentration of the reducing agent and/or chelating agent is further increased, the concentration of the active agent may be further reduced due to the complementarity of the mechanism of action of the active agent, reducing agent and/or chelating agent. The same is true for the reducing agent and the chelating agent.
The disinfectant has broad-spectrum disinfection effect, and can be widely used for disinfecting various microorganisms such as bacteria, fungi, viruses and the like.
The disinfectant is especially suitable for killing new coronavirus. The novel coronavirus consists mainly of Envelope (Envelope) and nucleocapsid protein (Nucleocapsid Protein) -encapsulated RNA. Spike Protein (Spike Protein) on its envelope specifically recognizes ACE2 receptors on human cell membranes and thus mediates viral invasion. The disinfectant in the current market mainly changes the permeability of the viral envelope, coagulates and denatures membrane proteins so as to kill viruses, and the disinfection time is longer than 1 minute. However, protein denaturation is reversible to some extent, and the denatured viral proteins can still renature to proteins with biological activity under specific conditions, restoring infectivity to the virus. The disinfectant provided by the invention can permanently inactivate viruses within a few seconds. The disinfectant of the invention has the following virus killing principle: the active agent is utilized to break the virus envelope or denature nucleocapsid protein, and then under the action of the reducing agent, disulfide bonds of the protein are broken, so that the protein structure is changed irreversibly, thereby thoroughly breaking down the infectivity of the virus and achieving the purpose of permanently inactivating the virus.
Unless otherwise indicated, the methods and techniques used in this specification are generally performed according to methods well known and conventional in the art and described in the various references set forth or cited in this specification.
Example 1 preparation of disinfectant
1) Disinfectant component:
buffering agent: disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB);
active agent: benzalkonium chloride;
chelating agent: EDTA-Na 2 ；
Reducing agent: L-Cysteine;
and (3) a protective agent: na (Na) 2 SO 3 。
2) Reagent preparation
Control reagent: 1.0mM PB, pH 8.0.
Neutralizing agent: the agent can stop the killing effect of disinfectant (on microorganisms) in time, and the neutralizing agent has no inhibiting or killing effect on microorganisms and no adverse effect on a culture medium. 3.0g of Tween 80 was dissolved in 100mL of water, 0.3g of lecithin was weighed and added to the solution, and after complete dissolution, sterilization was performed at 121℃for 30 minutes.
Example 2 Sterilization experiments
1) Bacterial liquidPreparation: staphylococcus aureus (ATCC 6538) and Escherichia coli (CICC 10899) were inoculated on LB solid plates and cultured at 37 ℃. After single colony grows out, single colony is picked up to liquid LB culture medium and cultured at 37 ℃. When the bacterial liquid OD 600 Reaching 0.6 to 0.8 (bacterial log value)>8) And (3) centrifuging to collect thalli, discarding supernatant, re-suspending the thalli by using an equal volume of buffer solution, and placing the thalli on ice for later use.
2) Sterilization experiment: 10 mu L of bacterial liquid is taken into a 1.5mL EP tube, 90 mu L of disinfectant is added into the EP tube, the mixture is uniformly mixed, the reaction is carried out for 5 seconds at room temperature, and 900 mu L of neutralizer is immediately added to stop the sterilization reaction. The reaction product in the EP tube was then entirely spread on LB solid plates and incubated at 37℃for 16-24 hours.
3) Control test: 10. Mu.L of the bacterial liquid was taken in a 1.5mL EP tube, then 90. Mu.L of PB buffer was added to the EP tube, and the mixture was mixed well and reacted at room temperature for 5 seconds, followed by immediately adding 900. Mu.L of a neutralizing agent. The reaction product in the EP tube was then serially diluted to 10 -8 After doubling, the cells were spread on LB solid plates and incubated at 37℃for 16 to 24 hours.
Further, 10. Mu.L of the bacterial liquid was taken into a 1.5mL EP tube, and 990. Mu.L of a neutralizing agent was then added to the EP tube. The reaction was carried out at room temperature for 1 minute, and then the reaction product in the EP tube was gradually diluted to 10 -8 After doubling, the cells were spread on LB solid plates and incubated at 37℃for 16 to 24 hours.
The number of single colonies growing on the plates is taken as a quantitative basis, three plates are adopted for parallel test in each group of disinfection experiments, and the average value of the number of single colonies of the three plates is taken.
Example 3 evaluation of sterilizing Effect of disinfectant
The disinfectant is prepared according to the following concentration:
TABLE 1
The pH was adjusted to 8.0.
The bactericidal potency assessment of the disinfectant and the following components was performed according to the method of example 2:
1. buffering agent: 1.0mM PB
2. Active agent: 0.01% benzalkonium chloride
3. A mixed solution of a chelating agent, a reducing agent and a protecting agent: 0.05mM EDTA-Na 2 +1.5mM L-Cysteine+2.0mM Na 2 SO 3 。
The sterilization experiments were performed as described in example 2 using the above 3 groups of reagents and disinfectants, respectively, with the following results:
the active agent benzalkonium chloride has stronger bactericidal activity when used alone, but the bactericidal effect of 100% cannot be achieved in a short time. When benzalkonium chloride is used in combination with other components shown in table 1 (i.e., formulated into a disinfectant), a 100% quick-sterilization effect can be achieved. The results show that other components can improve the disinfection efficiency of the active agent, and the buffering agent has no disinfection effect.
Example 4 disinfectant optimum pH exploration
Disinfectants were formulated with the formulation shown in table 1 of example 3 and pH values were adjusted to 7.0, 7.4, 8.0, 8.5, 9.0, 9.5 using HCl or NaOH, respectively, and the disinfectants of different pH values were tested for killing of e.coli, the results are shown in the following table:
the result shows that the sterilizing activity is not affected when the pH value of the disinfectant is in the range of 7.0-9.5.
Example 5 active ingredient concentration discovery of disinfectants
With reference to the formulation shown in table 1 of example 3, under the condition that the concentrations of other components are the same as those in table 1, the concentrations of benzalkonium chloride are respectively adjusted to be 0.005%, 0.0075%, 0.01%, 0.05%, 0.075% and 0.1%, and the killing rate of the disinfectants with different benzalkonium chloride concentrations on escherichia coli is tested, and the results are shown in the following table:
the results show that the disinfectant at 0.1%, 0.075%, 0.05%, 0.01% active ingredient (benzalkonium chloride) was able to kill 100% of the test bacteria, while at 0.075% and 0.005% concentrations it was unable to kill 100% of the test bacteria. It was demonstrated that in this experiment the minimum effective concentration of benzalkonium chloride was 0.01%.
Example 6 disinfectant chelating agent concentration exploration
With reference to the formulation shown in Table 1 of example 3, the disinfectant was prepared, and EDTA-Na was prepared separately under the conditions that the concentrations of the other components were the same as in Table 1 2 Disinfectant at 0, 0.05, 0.1, 0.5, 5.0, 10.0mM and tested for killing of E.coli, the results are shown in the following Table:
the results indicate that the chelating agent component of the disinfectant (EDTA-Na 2 ) The minimum effective concentration is 0.05mM.
Example 7 disinfectant reductant concentration discovery
With reference to the formulation shown in Table 1 of example 3, disinfectants were prepared and tested for their killing rate against E.coli by preparing disinfectants having L-cysteine concentrations of 0, 0.5, 1.0, 1.5, 3.0, 15.0mM, respectively, at the same concentrations as in Table 1, as shown in the following table:
the results show that the reducing agent component (L-Cysteine) of the disinfectant has a minimum effective concentration of 1.0mM in this experiment.
Example 8 disinfectant protectant concentration discovery
With reference to the formulation shown in Table 1 of example 3, the disinfectant was prepared, and Na was prepared separately under the conditions that the concentrations of the other components were the same as those in Table 1 2 SO 3 Disinfectant at 0, 1.0, 2.0, 20.0, 50.0mM and tested for killing of E.coliThe results are shown in the following table:
the results show that in this experiment, the protectant component (Na 2 SO 3 ) The minimum effective concentration is 1.0mM.
EXAMPLE 9 evaluation of sterilizing Effect of disinfectants on Staphylococcus aureus (gram-Positive bacteria)
The disinfectant is prepared according to the following concentration:
TABLE 2
The pH was adjusted to 8.0.
The staphylococcus aureus was evaluated for its sterilizing effect as described in example 2, with the following results:
the results show that the disinfectant can kill about 4.8X10 at 100% in 5 seconds 9 And staphylococcus aureus. The disinfectant can not only effectively kill gram-negative bacteria (colibacillus) but also effectively kill gram-positive bacteria.
EXAMPLE 10 evaluation of sterilizing Effect of disinfectant on Yeast (fungi)
Disinfectants were formulated as in example 9, table 2.
Activated Pichia pastoris GS115 is inoculated in YPD (1% Yeast Extract,2% Peptone,2% glucose) liquid medium and cultured at 28℃for 16-24 hours. Taking 1mL of bacterial liquid, centrifuging and removing supernatant. Then 1mL of control reagent (1 mM PB, pH 8.0) was added and resuspended on ice for use.
450. Mu.L of disinfectant was placed in the EP tube, and then 50. Mu.L of the bacterial liquid to be tested was added thereto, and immediately timed. 100. Mu.L of the disinfection product was taken into 900. Mu.L of the neutralizer at 5 seconds, 10 seconds, 30 seconds and 60 seconds, and 1000. Mu.L of the neutralization product was then all coated onto YPD plates. Culturing at 28 deg.c for 2-3 days, counting Shan Junla growth number and calculating the killing rate. The results were as follows:
the results show that the disinfectant can kill about 8.2×10 at 100% in 30 seconds 9 And (3) yeast. The disinfectant can effectively kill fungus microorganisms.
EXAMPLE 11 evaluation of the inactivation Effect of disinfectants on novel coronaviruses (SARS-CoV-2) (this experiment was performed in the P3 laboratory)
Disinfectants were formulated as in example 9, table 2.
Preparing SARS-CoV-2 virus stock solution (logarithmic value > 4) to be tested.
450. Mu.L of disinfectant was placed in the EP tube at room temperature, and then 50. Mu.L of virus stock was added and immediately timed. 100. Mu.L of the disinfection product was taken into 900. Mu.L of the neutralizing agent at 5 seconds, 10 seconds, 30 seconds, 60 seconds. Then, 100 mu L of the neutralization product is taken to infect Vero-E6 cells, the culture is carried out for 2 to 3 days at 37 ℃, the fixed dyeing is carried out, the blank spot number is counted, and the inactivation rate of the disinfectant to the novel coronavirus is calculated. The results were as follows:
the results show that in this experiment, the disinfectant was able to kill about 1.5X10% at 100% in 5 seconds 4 New coronaviruses.

Claims (32)

1. A disinfectant, comprising: an active agent, a reducing agent and a buffer.
2. The disinfectant of claim 1, further comprising a protectant.
3. The disinfectant as set forth in claim 1, further comprising a chelating agent.
4. The disinfectant of claim 1, further comprising a protectant and a chelating agent.
5. A disinfectant as claimed in claim 1, which consists of the following components: an active agent, a reducing agent, a protecting agent, a chelating agent, and a buffer.
6. A disinfectant as claimed in claim 1, which consists of the following components: active agent, reducing agent, protective agent, chelating agent, buffer and auxiliary materials.
7. A disinfectant as claimed in any one of claims 1 to 6 wherein the active agent is a quaternary ammonium surfactant.
8. A disinfectant as claimed in any one of claims 1 to 7, wherein the active agent comprises one or more of the following: benzalkonium chloride, benzalkonium bromide, benzethonium chloride, cetylpyridinium chloride, and decylmethylammonium chloride.
9. A disinfectant as claimed in any one of claims 1 to 8 wherein the active agent is benzalkonium chloride.
10. A disinfectant as claimed in any one of claims 1 to 9, wherein the chelating agent comprises one or more of the following: ethylenediamine tetraacetic acid (EDTA) and its metal salts, aminotriacetic acid (NTA) and its metal salts, ethylene glycol diethylether diamine tetraacetic acid (EGTA) and its metal salts, diethylenetriamine pentaacetic acid (DTPA) and its metal salts.
11. The disinfectant as set forth in any one of claims 1 to 10, wherein said chelating agent is ethylenediamine tetraacetic acid (EDTA) or ethylenediamine tetraacetic acid sodium salt (EDTA-Na) 2 )。
12. A disinfectant as claimed in any one of claims 1 to 6, wherein the reducing agent comprises one or more of the following: dithiothreitol (DTT), reduced glutathione, beta-mercaptoethanol, mercaptoacetic acid, metal salts thereof, L-Cysteine (Cysteine), tris (2-carboxyethyl) phosphine (TCEP).
13. The disinfectant of any one of claims 1 to 12, wherein said reducing agent is L-Cysteine (Cysteine).
14. A disinfectant as claimed in any one of claims 1 to 13, wherein the protective agent comprises one or more of the following: sodium sulfite (Na) 2 SO 3 ) Vitamin C, vitamin E.
15. A disinfectant as claimed in any one of claims 1 to 14, wherein the protective agent is sodium sulfite.
16. A disinfectant as claimed in any one of claims 1 to 15, wherein the buffer comprises one or more of the following: tris (hydroxymethyl) aminomethane (Tris), disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 3-morpholinopropanesulfonic acid (MOPS).
17. The disinfectant of any one of claims 1 to 16, wherein the buffer is disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB).
18. The disinfectant of any one of claims 1 to 17, wherein the disinfectant comprises: benzalkonium chloride as active agent, EDTA-Na as chelating agent 2 L-cysteine as a reducing agent, sodium sulfite as a protecting agent, and disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB) as a buffering agent.
19. A disinfectant as claimed in any one of claims 1 to 18, wherein the disinfectant consists of: benzalkonium chloride as active agent, EDTA-Na as chelating agent 2 L-cysteine as a reducing agent, sodium sulfite as a protecting agent, and disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB) as a buffering agent.
20. A disinfectant as claimed in any one of claims 1 to 18, wherein the disinfectant consists of: benzalkonium chloride as active agent, EDTA-Na as chelating agent 2 L-cysteine as a reducing agent, sodium sulfite as a protecting agent, disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB) as a buffering agent, and an auxiliary material.
21. A disinfectant as claimed in any one of claims 1 to 20 wherein the concentration of quaternary ammonium surfactant as the active agent is from 0.001% to 1.0% (W/V).
22. A disinfectant as claimed in any one of claims 1 to 20 wherein the concentration of quaternary ammonium surfactant as the active agent is from 0.01% to 0.1% (W/V).
23. A disinfectant as claimed in any one of claims 1 to 20 wherein the concentration of benzalkonium chloride as the active agent is from 0.01% to 0.1% (W/V).
24. A disinfectant as claimed in any one of claims 1 to 23, wherein the concentration of the chelating agent is in the range 0.01 to 10 mM.
25. The disinfectant as set forth in claim 24, wherein the chelating agent is EDTA-Na 2 。
26. A disinfectant as claimed in any one of claims 1 to 25, wherein the concentration of the reducing agent is in the range 0.1 to 15.0mM.
27. A disinfectant as set forth in claim 26, wherein the reducing agent is L-Cysteine (Cysteine).
28. The disinfectant as set forth in any one of claims 1 to 27, wherein the concentration of the protecting agent is in the range of 0.01 to 50.0mM.
29. A disinfectant according to claim 28 wherein the protectant is sodium sulfite (Na 2 SO 3 )。
30. Use of a disinfectant as claimed in any one of claims 1 to 29 for killing or inhibiting a new coronavirus.
31. The use of claim 30, wherein the disinfectant is applied to a human surface.
32. The use of claim 31, wherein the disinfectant is applied to the surface of the object.
CN202311003815.0A 2022-08-10 2023-08-10 Disinfectant agent Pending CN117581869A (en)

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CN202210957717 2022-08-10

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