NL2032962A

NL2032962A - Foam type long-acting citric acid-containing compound disinfectant, preparation method and use thereof

Info

Publication number: NL2032962A
Application number: NL2032962A
Authority: NL
Inventors: Xu Fei; Liu Huan; Zhang Mengmeng; Wang Huanhuan; Liu Yiming; Chen Xiaojie; Li Xiubo
Original assignee: Feed Res Institute Chinese Academy Of Agricultural Science
Priority date: 2022-05-10
Filing date: 2022-09-06
Publication date: 2023-11-16
Also published as: CN114916544A

Abstract

Disclosed are a foam type long-acting citric acid-containing compound disinfectant, and a preparation method and use thereof. The disinfectant contains the following components by 5 mass percentage: citric acid 0.5%-5.0%; benzalkonium bromide 0.5%-5.0%; decamethonium bromide 0.5%-5.0%; glutaraldehyde 0.25%-2.5%; polyhexamethylene biguanide 0.5%-5.0%; a cosolvent3.0%-15.0%; a penetrant 0.1% - 0.5%; a foam emulsifier 0.5% - 5.0%; a stabilizer, which, together With citric acid, forms a buffer system to maintain a pH value of 3.0-5.0; and sterilized water, Which is added to make up to 100%. The compounded disinfectant has a 10 synergistic or additive effect, and is capable of killing animal-derived pathogenic pathogenic microorganisms existing in breeding environments, including bacteria, viruses, mycoplasmas, fungi, etc., With microbicidal efficiency of 100%.

Description

FOAM TYPE LONG-ACTING CITRIC ACID-CONTAINING COMPOUND

DISINFECTANT, PREPARATION METHOD AND USE THEREOF

Field of the Invention

The present invention belongs to the field of prevention and control products for biosafety in livestock and poultry breeding, and particularly relates to a foam type long-acting citric acid- containing compound disinfectant, a preparation method thereof, and use thereof.

Background of the Invention

Major livestock and poultry epidemics not only seriously endanger the development of breeding industry, but also constantly threaten human health and public health security. Since 2018, the outbreak of African swine fever has brought a devastating blow to swine industry in

China, causing economic losses of trillions of RMB yuan. The continuous destructive power of

African swine fever makes breeding entrepreneurs turn pale at the mere mention of it.

Strengthening of prevention and control of major animal epidemics and exotic animal epidemics is thus a major demand for ensuring the safety of livestock and poultry product supply and national biosecurity.

Prevention and control products for biosafety play an extremely important role in epidemic prevention and control, and disinfectants are an important part of prevention and control products for biosafety. Existing prevention and control products for biosafety, however, have serious problems, such as low microbicidal efficiency, poor safety, poor persistence, and lack breakthrough technological innovation.

Problem 1: Low microbicidal efficiency

At present, animal-derived pathogenic microorganisms existing in breeding environments include bacteria, viruses, mycoplasmas, fungi, etc. It is often the case that a single disinfectant product cannot effectively kill all pathogenic microorganisms. Commonly used quaternary ammonium salt disinfectants are not very effective against bacterial spores. mycoplasmas, etc.

Therefore, it is often necessary to prepare compound formulations according to different bactericidal spectra of activity of disinfectants to solve the problem of low microbicidal efficiency.

Problem 2: Poor safety

Farm disinfection is a continuous process that consumes a large number of products. For the purpose of cost reduction, disinfection products with high levels of toxicity. such as caustic soda (with sodium hydroxide being an active component), aldehydes (formaldehyde,

glutaraldehyde), phenols (phenol), and the like are often used. Such disinfection products not only cause harm to animals in farm areas, but also have potential toxic effects on workers disinfecting the farms. There have been many reports of animal deaths due to overuse or inappropriate use of disinfectants. It is thus very necessary to develop green, environmentally friendly, and safe disinfectants.

Problem 3: Poor persistence

At present, disinfectants are generally used in the form of simple solution formulations, which are spraved on railings, troughs, and objects on the ground of animal houses. Due to their high fluidity. the solutions exhibit poor adhesion to and poor persistence on irregular surfaces such as railings, uneven ground, etc., and can hardly come into full contact with objects. This results in low microbicidal efficiency and thus the need to increase the frequency of disinfection, leading to excessive consumption of disinfectants. In order to solve this problem, it is desirable to develop a disinfection product with long-lasting foam to increase the adhesiveness and adhesion to exert a long-lasting killing effect.

It is therefore urgent to develop a green, efficient, safe, and low-cost prevention and control product for biosafety and realize industrialization and use thereof to promote green, sustainable and healthy development of the animal husbandry in China.

Summary of the Invention

Directed against the problems with the existing technologies, the present invention provides an environmentally-friendly, efficient. low-cost, residue-free, high-microbicidal efficiency, and convenient-to-transport foam type long-acting citric acid-containing compound disinfectant, and a preparation method thereof.

The foam type long-acting citric acid-containing compound disinfectant provided by the present invention comprises the following components by mass percentage: citric acid 0.5%-5.0%: benzalkonium bromide 0.5%-5.0%; decamethonium bromide 0.5%-5.0%;

Glutaraldehyde 0.25%-2.5%; polyhexamethylene biguanide 0.5%-5.0%; cosolvent 3.0%-15.0%; penetrant 0.1%-0.5%; foam emulsifier 0.5%-5.0%;

stabilizer, which, together with citric acid, forms a buffer system to maintain a pH value of 3.0-5.0; and surplus is sterilized water, which is added to make up to 100%.

Further, the foam type long-acting citric acid-containing compound disinfectant comprises the following components in mass percentage: citric acid 0.5%-4.0%; benzalkonium bromide 1.0%-2.5%; decamethonium bromide 2.0%-5.0%; glutaraldehyde 0.5%-1.0%; polyhexamethylene biguanide 0.5%-1.5%: cosolvent 5.0%-10.0%: penetrant 0.2%-0.4%: foam emulsifier 1.0%-3.0%; stabilizer, which, together with citric acid, forms a buffer system to maintain a pH value of 3.0-5.0: and surplus is sterilized water, which is added to make up to 100%.

Preferably, a ratio of a mass of glutaraldehyde to a sum of a mass of benzalkonium bromide and a mass of decamethonium bromide may be 1:2 or more, preferably 1: (2-4), specifically for example 1: 2 or 1:3 or 1:4.

Preferably, a ratio of the mass of benzalkonium bromide to the mass of decamethonium bromide may be 1: (1-4), preferably 1: (1-3). specifically for example 1:1 or 1:2 or 1: 3 or 1:4.

Preferably, the cosolvent may be one or more selected from alcoholic solvents containing 2-10 carbons, for example. one or more selected from alcoholic solvents such as ethanol, propylene glvcol, butanol, isobutanol, and octanol.

Preferably, the penetrant may be one or more selected from ethanol, glycerol trioleate, tricaprylin, fatty acid polyoxyethylene ether-9, fatty acid polyoxyethylene ether-7, and fatty acid polyoxyethylene ether-5.

Preferably, the foam emulsifier may be one or more selected from dodecyl dimethyl amine oxide, decyl glucoside, coconut oil diethanolamide. and cetyltrimethylammonium bromide.

Specifically, the foam emulsifier is decyl glucoside, and its mass content is preferably 1%; or, the foam emulsifier is coconut oil diethanolamide or cetyltrimethylammonium bromide, and its mass content is preferably 3%.

Preferably, the stabilizer may be selected from salts capable of forming a buffer system with citric acid, the salts being, for example, citrate, disodium hydrogen phosphate. dipotassium hydrogen phosphate, and sodium hydrogen phosphate.

The present invention also provides a preparation method of the above foam type long- acting citric acid-containing compound disinfectant.

The preparation method of the foam type long-acting citric acid-containing compound disinfectant provided by the present invention includes the following steps: (1) introducing nitrogen into part of the sterilized water: (2) adding the cosolvent to the sterilized water treated in step (1) and stirring; (3) adding decamethonium bromide and benzalkonium bromide to the system obtained in step (2) and stirring at a low speed (<150 rpm) until complete dissolution: (4) adding the penetrant and the foam emulsifier to the system obtained in step (3) and stirring at a low speed (<150 rpm) to form system A: (5) taking another part of the sterilized water, into which nitrogen is then introduced; (6) adding glutaraldehyde and polyhexamethylene biguanide to the sterilized water treated in step (5) and stirring at a low speed (<150 rpm) until complete dissolution; (7) adding citric acid and the stabilizer to the system obtained in step (6) and stirring at a low speed (<150 rpm) until complete dissolution to form system B; (8) transferring the system B to the system A, stirring uniformly at a low speed (<150 rpm), and adding the sterilized water to bring to 100% by mass, and maintaining a pH value of the resulting system to be 3.0-5.0.

In the above method. the stirring at a low speed each occurs at a rotation speed of less than 150 rpm.

In step (4) of the above method, the stirring lasts for 20-60 minutes.

The foam type long-acting citric acid-containing compound disinfectant provided by the present invention is capable of effectively killing pathogenic microorganisms that common on the ground. railings. and troughs in livestock breeding environments such as bacteria, viruses, mycoplasmas, spores, fungi. etc.

The viruses may specifically be swine fever virus (such as Thiveral stram of swine fever virus), chicken Newcastle disease virus (such as LaSota strain of chicken Newcastle disease virus).

The spores may specifically be bacillus subtilis.

The present invention also provides a disinfectant composition.

The disinfectant composition provided by the present invention comprises citric acid, benzalkonium bromide, decamethonium bromide, glutaraldehyde, and polyhexamethylene biguanide

A ratio of a mass of glutaraldehyde to a sum of a mass of benzalkonium bromide and a mass of decamethonium bromide is 1:2 or more, preferably 1: (2-4).

A ratio of the mass of benzalkonium bromide to the mass of decamethonium bromide is 1: (1-4), preferably 1: (1-3).

A ratio of a mass of polyhexamethylene biguanide to the mass of glutaraldehyde is 1: (0.5- 5), preferably 1: (1-3).

A ratio of a mass of citric acid to the mass of glutaraldehyde is 1: (1-10), preferably 1: (1- 8).

Directed against the problem of low microbicidal efficiency with the existing disinfectants, the present invention provides a compound disinfectant with a combination of components having a synergic effect. The dismfectant effectively solves the problem of low microbicidal efficiency of a disinfectant with a single component. The role of each component in the disinfectant is analyzed as follows. 1. Benzalkonium bromide and decylmethyl bromide are two cationic surfactants.

Enrichment of them on surfaces of cell walls can affect the filtration effect of cell membranes of microorganisms, thereby increasing cell permeability of bacteria, and causing water to penetrate into bacteria to lead to substance leakage. which affects the metabolism of bacteria and damages cell structures, and eventually leads to death of bacteria. Disinfectants of this type have low toxicity and strong decontamination ability, are non-corrosive to metal materials, and have a synergistic microbicidal effect. They are very efficient in killing animal-derived bacteria but are not very efficient in killing bacterial spores. 2. The killing effect of glutaraldehyde on microorganisms depends mainly on the aldehyde group, which acts primarily on the sulfhydryl, hydroxyl, carboxyl, and amino groups of bacterial protein and enable them to be alkylated. thereby causing the protein to coagulate and leading to death of bacteria. Glutaraldehyde has a good killing effect on microorganisms such as spores, fungi and spores thereof. 3. Polyhexamethylene biguanide is an environmentally friendly disinfectant. The guanidine group is highly active and can enable polymers to be electropositive and can therefore be easily adsorbed onto various electronegative bacteria, by way of which the division function of bacteria can be inhibited and bacteria can thus be caused to lose reproductive capacity. 4. Citric acid, the molecular formula of which is CsHsO:, is an important organic acid. It is a colorless, odorless crystal with a strong sour taste, and is easily soluble in water and can be used as a pH regulator. In the inventor 's previous experimental study, it is found that citric acid at a relatively low concentration (0.5%-1.0%) has a good killing effect on animal-derived mycoplasmas.

Test results show that the above components can synergize with each other to kill animal- derived pathogenic microorganisms existing in breeding environments. including all pathogenic microorganisms such as bacteria, viruses. mycoplasmas, fungi. etc., with microbicidal efficiency of 100%.

With respect to the problem of poor safety in using the existing disinfectants for disinfection, in the present invention, only glutaraldehyde is an aldehyde product which is to some degree irritating and corrosive. However, as there is synergy between the components, the concentration of glutaraldehyde used in the compound disinfectant is reduced by more than 60%-80% compared with that of glutaraldehyde used in a disinfectant with a single component.

Toxicity test results show that the disinfectant product with the components of the present invention is safe, effective and non-irritating in use.

With respect to the problem of poor persistence of the existing disinfectants when used for disinfection, among the components of the disinfectant of the present invention, there is the SF penetrant, the cosolvent, the stabilizer, the foam-forming agent, and in particular, polyhexamethylene biguanide. Polyhexamethylene biguanide is a high molecular polymer and can help to form the product into a high molecular solution state. The product in such a state can be easily adsorbed onto all kinds of electronegative bacteria. When used, the product forms rich foam through a foaming gun or a foaming machine and adheres to surfaces of objects being disinfected. thereby exhibiting long-lasting microbicidal effects.

Compared with the existing technologies, the present invention has the following beneficial effects. (1) The disinfectant of the present invention exhibits a broad-spectrum activity against microorganisms. Due to the synergistic or additive effect, the compound disinfectant has enhanced total microbicidal efficacy due to the compounding. Generally, disinfectants with quaternary ammonium salts alone cannot kill bacterial spores, while aldehyde disinfectants have good killing effect on bacterial spores. The present invention synergizes quaternary ammonium salts, aldehydes. acids, and guanidine compounds and can kill animal-derived pathogenic microorganisms existing in breeding environments, including all pathogenic microorganisms such as bacteria. viruses. mycoplasmas, fungi, etc., with microbicidal efficiency of 100%. (2) The disinfectant of the present invention has long-lasting effects. Among the components of the disinfectant of the present invention, there is the SF penetrant, the cosolvent, the stabilizer, the foam-forming agent, and in particular, polyhexamethylene biguanide.

Polyhexamethylene biguanide is a high molecular polymer and can help to form the product into a high molecular solution state. The product in such a state can be easily adsorbed onto all kinds of electronegative bacteria. When used. the product forms rich foam through a foaming gun or a foaming machine and adheres to surfaces of objects being disinfected. thereby exhibiting long-lasting microbicidal effects. Compared with the existing technology, the disinfectant of the present invention can save on use amount of disinfectants. (3) The disinfectant of the present invention is highly safe. Aldehyde disinfectants are irritating to skin and have acute toxicity. In the present invention, through the efficient synergy between the components of the disinfectant, glutaraldehyde is used in a greatly reduced amount which is only 20% of the original amount, and therefore has no toxic and side effects on the human body. Animal toxicology tests show that the disinfectant with the components is efficient, safe and non-toxic. (4) The disinfectant of the present invention can effectively kill mycoplasmas.

Mycoplasmas. as a type of pathogens, are able to spread vertically. horizontally and mechanically, and infection routes thereof are also complex and diverse. Mycoplasmas live widely in breeding environments depending on organic biomass. Compared with the existing technology, the present invention can effectively kill mycoplasmas in breeding environments, and plays an important role in effectively controlling the spread of mycoplasma diseases in livestock and poultry farms. (5) The disinfectant of the present invention is simple to produce, stable and easy to produce on large-scale, and has remarkable on-site killing effects, and is worth promotion and use.

Specific Embodiments

The present invention will be further described below in conjunction with specific embodiments. The present invention, however, is not limited to the following embodiments.

Methods described are conventional methods unless otherwise specified. Raw materials used are commercially available unless otherwise specified.

Example 1 (1) Synergy screening for contents of glutaraldehyde and quaternary ammonium salts

Glutaraldehyde, decamethonium bromide. and benzalkonium bromide, as technical materials, were brought to room temperature, and prepared into different groups of disinfectants by using PBS solution as a solvent in accordance with what shown in Table 1, with decamethonium bromide and benzalkonium bromide being in a ratio (mass ratio) of 1:1.

Suspension quantitative tests were carried out for microbicidal effects of the disinfectants using bacillus subtilis CMCC (B) 63501 as indicator bacteria, and the screening was based on microbicidal rates.

Table 1 Results of the tests for ratios of glutaraldehyde to quaternary ammonium salts

Group No. Technical materials Microbicidal rate 1% glutaraldehyde+4%

Group 1 100% quaternary ammonium salts 1% glutaraldehyde+3%

Group 2 100% quaternary ammonium salts 1% glutaraldehyde+2%

Group 3 99.90% quaternary ammonium salts 1% glutaraldehyde+1%

Group 4 } 99.04% quaternary ammonium salts

Group 5 1% glutaraldehyde+0.5% 89.64%

quaternary ammonium salts 1% glutaraldehyde+0.1% Co

Group 6 i 85.35% quaternary ammonium salts

Group 7 1% glutaraldehyde 85.27%

Group 8 4% quaternary ammonium salts 7.8%

Notes: The above results are the average of results obtained after the tests are repeated three times. All the tests are carried out at conditions of a temperature of 20£2°C, with the disinfectants being diluted 1:2 and then enabled to act for 1 hour.

The results show that the quaternary ammonium salts used alone in Group 8 have a very low ability to kill bacillus subtilis (7.8%); however, when 1% glutaraldehyde is mixed with 0.1%-4% by mass concentration of a quaternary ammonium salts solution, the microbicidal rate increases with the increase of the content of the quaternary ammonium salts. The test results demonstrate that when glutaraldehyde and quaternary ammonium salts are mixed in a ratio of 1:2 or more, the microbicidal rate can be up to 99.90% or above, in which case the microbicidal effect meets the standard. (2) Synergy screening for contents of two quaternary ammonium salts

Glutaraldehvde. decamethonium bromide, and benzalkonium bromide, as technical materials, were brought to room temperature, and prepared into different groups of test disinfectant mixtures in accordance with what shown in Table 2. Suspension quantitative tests were carried out for microbicidal effects of the disinfectants using bacillus subtilis 63501 as indicator bacteria. Results are as follows.

Table 2 Results of the tests for ratios of decamethonium bromide to benzalkonium bromide

Content ratio of quaternary /

Group No. / Microbicidal rate ammonium salts decamethonium bromide: /

Group 1 / / 100% benzalkonium bromide=4:1 decamethonium bromide:

Group 2 i / 100% benzalkonium bromide=3:1 decamethonium bromide:

Group 3 i / 99.98% benzalkonium bromide=2:1 decamethonium bromide:

Group 4 / / 99.90% benzalkonium bromide=1:1

Group 5 decamethonium bromide: 96.52% benzalkonium bromide=1:2

Group 6 decamethonium bromide: 04.78% benzalkonium bromide=1:3

Group 7 decamethonium bromide: 89 57%, benzalkonium bromide=1:4

Notes: The above results are the average of results obtained after the tests are repeated three times. All the tests are carried out at conditions of a temperature of 20+1°C, with the disinfectants bemg diluted 1:2 and then enabled to act for 1 hour.

The results show that, with the total content of 1% glutaraldehyde and 2% quaternary ammonium salt being constant, the microbicidal rate increases with the increase of the proportion of decamethonium bromide. When the ratio of decamethonium bromide to benzalkonium bromide is 1:1, the microbicidal rate is 99.92%; when it is 2:1, the microbicidal rate is 99.98%; and when it is 3:1, the microbicidal rate is 100%. The microbicidal effect in each case meets the standard. (3) Synergy screening for contents of polyhexamethylene biguanide

Contents of polyhexamethylene biguanide were screened as follows. Different groups of test disinfectant mixtures were prepared in accordance with what shown in Table 3. Suspension quantitative tests were carried out for microbicidal effects of the disinfectants using bacillus subtilis 63501 as indicator bacteria. Results are as follows.

Table 3 Results of the tests for proportions of polyhexamethylene biguanide

Group No. Glutaraldehyde Polyhexamethylene biguanide Herons

Group 1 1.0 1.5 100%

Group 2 1.0 L.0 100%

Group 3 1.0 0.5 100%

Group 4 0.5 1.5 99.95%

Group 5 0.5 1.0 99.92%

Group 6 0.5 0.5 99.90%

The results show that when the content of polyhexamethylene biguanide is 0.5%-1.5%, the microbicidal rate of each group is more than 99.9%, in which case the microbicidal effect meets the standard.

(4) Synergy screening for contents of citric acid

Contents of citric acid were screened as follows. Different groups of test disinfectant mixtures were prepared in accordance with what shown in Table 4. Suspension quantitative tests were carried out for microbicidal effects of the disinfectants using 19 clinical strains of

Mycoplasma bovis (BS1738-BS1748) as indicator bacteria. Results are as follows.

Table 4 Results of screening for citric acid contents in systems (%o)

Group Glutaraldehyd Decamethoniu Benzalkoniu Polyhexamethy Citric Microbici

No. € m bromide ~~ m bromide lene biguanide acid dal rate

Group 1 0.3 0.5 0.5 0.5 4.0 100%

Group 2 0.5 0.5 0.5 0.5 3.0 100%

Group 3 0.5 0.3 0.5 0.5 2.0 100%

Group 4 0.5 0.3 0.5 0.5 1.0 100%

Group 5 0.3 0.5 0.5 0.5 0.5 99.9%

Group 6 0.5 0.5 0.5 0.5 0.25 82.5%

The results show that when the content of citric acid 1s 0.5%-4.0%, the microbicidal rate of each group is more than 99.9%, in which case the microbicidal effect meets the standard. (5) Screening for a foam emulsifier

Coconut oil diethanolamide. decyl glucoside. and cetyltrimethylammonium bromide, with a content of 1%-3%, were screened for an emulsifier. Test results are as follows. The 1% decyl glucoside produces rich and delicate foam which lasts for up to 60 minutes. The 1% coconut oil diethanolamide and 1% cetyltrimethylammonium bromide, however, produce less foam which lasts only for a short time, and the foam produced by the 1% coconut oil diethanolamide disappear completely at 60 minute. The 3% coconut oil diethanolamide and the 3% cetyltrimethylammonium bromide can meet the required lasting time of foam of up to 60 minutes.

Example2 Foam type long-acting citric acid-containing compound disinfectant

Composition of the disinfectant is shown in the table below.

Glutaraldehyde 0.5% 1.0 kg of the foam type long-acting compound disinfectant was prepared in accordance with the above components and contents (w/w). Specific steps are as follows. 3 (1) Nitrogen was introduced into part of the sterilized water. (2) Propylene glycol was added to the sterilized water and stirred. (3) Decamethonium bromide and benzalkonium bromide were added to the system obtained in step (2) and stirred at a low speed (<150 rpm) until complete dissolution. (4) Fatty acid polyoxyethylene ether-9 and decyl glucoside were added to the system obtained in step (3) and stirred at a low speed for 20 minutes to form system A. (5) Another part of the sterilized water was taken and nitrogen was introduced thereinto. (6) Glutaraldehyde and polyhexamethylene biguanide were added to the sterilized water and stirred at a low speed (<150 rpm) until complete dissolution. (7) Citric acid and sodium citrate were added to the system obtained in step (6) and stirred atalow speed (<150 rpm) until complete dissolution to form system B. (8) The system B was transferred to the system A, and stirred uniformly at a low speed (<150 rpm) for 20 minutes, followed by adding the sterilized water to bring to volume by mass, and a pH value of the system was measured to be 3.0-5.0.

Example 3 Foam type long-acting citric acid-containing compound disinfectant

Composition of the disinfectant is shown in the table below.

Polyhexamethylene biguanide

Disodium hydrogen phosphate-citric acid buffer _

Sterilized water 1.0 kg of the foam type long-acting compound disinfectant was prepared in accordance with the above components and contents (w/w). Specific steps are as follows. (1) Nitrogen was introduced into part of the sterilized water. (2) Propylene glycol and ethanol were added to the sterilized water and stirred. (3) Decamethonium bromide and benzalkonium bromide were added to the system obtained in step (2) and stirred at a low speed (<150 rpm) until complete dissolution. (4) Fatty acid polyoxyethylene ether-9 and cetyltrimethy lammonium bromide were added to the system obtained in step (3) and stirred at a low speed for 20 minutes to form system A. (5) Another part of the sterilized water was taken and nitrogen was introduced thereinto. (6) Glutaraldehyde and polyhexamethylene biguanide were added to the sterilized water and stirred at a low speed (<150 rpm) until complete dissolution. (7) Citric acid and disodium hydrogen phosphate were added to the system obtained in step (6) and stirred at a low speed (<150 rpm) until complete dissolution to form system B. (8) The system B was transferred to the system A, and stirred uniformly at a low speed (<150 rpm) for 20 minutes, followed by adding the sterilized water to bring to volume by mass, and a pH value of the system was measured to be 3.0-5.0.

Example 4 Foam tvpe long-acting citric acid-containing compound disinfectant

Composition of the disinfectant is shown in the table below.

1.0 kg of the foam type long-acting compound disinfectant was prepared in accordance with the above components and contents (w/w). Specific steps are as follows. 3 (1) Nitrogen was introduced into part of the sterilized water. (2) Propylene glycol was added to the sterilized water and stirred. (3) Decamethonium bromide and benzalkonium bromide were added to the system obtained in step (2) and stirred at a low speed (<150 rpm) until complete dissolution. (4) Fatty acid polyoxyethylene ether-9 and decyl glucoside were added to the system obtained in step (3) and stirred at a low speed for 20 minutes to form system A. (5) Another part of the sterilized water was taken and nitrogen was introduced thereinto. (6) Glutaraldehyde and polyhexamethylene biguanide were added to the sterilized water and stirred at a low speed (<150 rpm) until complete dissolution. (7) Citric acid and sodium citrate were added to the system obtained in step (6) and stirred at alow speed (<150 rpm) until complete dissolution to form system B. (8) The system B was transferred to the system A, and stirred uniformly at a low speed (<150 rpm) for 20 minutes, followed by adding the sterilized water to bring to volume by mass, and a pH value of the system was measured to be 3.0-5.0.

Example S Foam type long-acting citric acid-containing compound disinfectant

Composition of the disinfectant is shown in the table below.

Fatty acid polyoxyethylene ether-7

Dipotassium hydrogen phosphate-citric acid | pH 3.0-5.0

HE PR SE

Sterilized water

1.0 kg of the foam type long-acting compound disinfectant was prepared in accordance with the above components and contents (w/w). Specific steps are as follows. (1) Nitrogen was introduced into part of the sterilized water. 3 (2) Propylene glycol and ethanol were added to the sterilized water and stirred. (3) Decamethonium bromide and benzalkonium bromide were added to the system obtained in step (2) and stirred at a low speed (<150 rpm} until complete dissolution. (4) Fatty acid polyoxyethylene ether-7 and decyl glucoside were added to the system obtained in step (3) and stirred at a low speed for 20 minutes to form system A. (3) Another part of the sterilized water was taken and nitrogen was introduced thereinto. (6) Glutaraldehyde and polyhexamethylene biguanide were added to the sterilized water and stirred at a low speed (<150 rpm) until complete dissolution. (7) Citric acid and dipotassium hydrogen phosphate were added to the system obtained in step (6) and stirred at a low speed (<150 rpm) until complete dissolution to form system B. (8) The system B was transferred to the system A, and stirred uniformly at a low speed (<150 rpm) for 20 minutes, followed by adding the sterilized water to bring to volume by mass, and a pH value of the system was measured to be 3.0-5.0.

Example 6 Effects of the foam type long-acting citric acid-containing compound disinfectant (1) Tests of virucidal effects on swine fever virus

Suspension quantitative method was used to measure the rate of activation of Thiveral strain of swine fever virus by the disinfectant at different concentrations after acting for 3 minutes. 5 minutes, 7 minutes. 10 minutes. and 15 minutes. The results show that the foam type long-acting compound disinfectant prepared in Example 4, when diluted 1:600, can achieve a rate of inactivation of Thiveral strain of swine fever virus of up to 100% after acting thereon for 10 minutes. The foam type long-acting compound disinfectant, when diluted 1:500, can achieve arate of inactivation of Thiveral strain of swine fever virus of up to 70.4% after acting thereon for 3 minutes, can achieve an increased rate of inactivation of Thiveral strain of swine fever virus of up to 95.2% after acting thereon for an extended period of time namely 7 minutes, and can achieve a further increased rate of inactivation of Thiveral strain of swine fever virus of up to 100% after acting thereon for a further extended period of time namely 10 minutes, in which case the inactivation effect meets the standard. The disinfectant, when diluted 1:400, can achieve a rate of inactivation of 7Thiveral strain of swine fever virus of up to 100% after acting thereon for only 3 minutes. The foam type long-acting compound disinfectant therefore has a good virucidal effect on the Thiveral strain of swine fever virus, and exhibits good virucidal performance.

Table 5 Inactivation effect of disinfectants on 7hiveral strain of swine fever virus by which the (%) disinfectant is | / / | _ diluted 3min Smin 7min 10min 15min 200 100 100 100 100 100 300 100 100 100 100 100 400 100 100 100 100 100 300 70.4 80.1 95.2 100 100 600 66.2 71.8 83.1 100 100

Notes: The above results are the average of results obtained after the tests are repeated three times. All the tests are carried out at conditions of a temperature of 20+2°C. The diluent with which the disinfectant is diluted is standard hard water (WS/T 798-2022). (2) Tests of virucidal effects on chicken Newcastle disease virus

With chicken embryos being infected, suspension quantitative tests were used to measure the rate of inactivation of LaSota strain of chicken Newcastle disease virus by the disinfectant at different concentrations after acting for 5 minutes, 10 minutes, and 15 minutes. The results demonstrate that the disinfectant prepared in Example 4, when diluted 1:400, achieves a rate of inactivation of LaSota strain of chicken Newcastle disease virus of 100% after acting thereon for 5 minutes, and achieves a rate of inactivation maintained at 100% after extended action time such as 10 minutes and 15 minutes, and that when the disinfectant is diluted to a concentration lower than the above, the virucidal effect thereof is not good. The foam type long-acting compound disinfectant has a good virucidal effect on LaSofa strain of chicken Newcastle disease virus when diluted 1:400.

Table 6 Inactivation effect of the disinfectant on chicken Newcastle disease virus

Dilution factor by HA/ Rate of inactivation of the which the Action time (min) virus % disinfectant is I I mn ded 100 5 0/100 0/100 0/100 10 0/100 0/100 0/100 15 0/100 0/100 0/100 200 5 0/100 0/100 0/100

10 0/100 0/100 0/100 15 0/100 0/100 6/100 400 5 0/100 0/100 0/100 10 0/100 0/100 0/100 15 0/100 0/100 0/100 500 5 2°70 25/0 21/0 10 2°20 2°/0 27820 15 2/60 276120 23220 600 5 2°%0 2°/0 2/0 10 2527/20 2°/0 2°10 15 25540 2°10 2535/20 800 5 2°%0 210 29/0 10 29210 2°/0 2%0 15 278/20 2°10 28870) 1000 5 219270 21/0 2°%/0 10 2730 2°40 2°50 15 2°7/0 2°/0 2740

Notes: The diluent with which the disinfectant is diluted is standard hard water (WS/T 798-2022). (3) Tests of effects of on-site disinfection of surfaces with clinical natural bacteria

In accordance with the requirements of “Technical Specifications for Identification of

Veterinary Disinfectants”, more than four animal houses of two livestock and poultry farms for which no disinfectant had been used over the past 1 month, with the average area of the houses not being less than 50 m2, were selected to carry out the tests of effects of on-site disinfection by the disinfectant.

Table 7 Clinical test grouping

Groups Concentration of the disinfectant

Test disinfectant high-dose group Diluted 1:100

Test disinfectant moderate-dose group Diluted 1:200

Test disinfectant low-dose group Diluted 1:400

Recommended dose specified in the

Control disinfectant group (Baidusha) instruction manual

Parallel test using tap water instead of the

Blank control group disinfectant

Three sampling sites (5x5 cm’ ground, railings, and troughs) were pre-determined in each test house. Each sampling site was sampled three times. The sampling sites were adjacent but not the same. A sterile cotton swab was infiltrated with a neutralizer and then used to wipe a 5x5 cm? area horizontally and straightly for 10 times, with a wiping surface of the swab being constantly changed. A cotton end of the swab was cut into a 10 ml sterile centrifuge tube, which was then sealed and labeled. Samples were collected before disinfection, and 30 minutes, 60 minutes, and 90 minutes after the disinfection, respectively. After the samples were brought back to a laboratory, 2 ml of sterile physiological saline was added to each of the samples. The samples each were then vortexed for 1 minute, eluted as thoroughly as possible, serially diluted with physiological saline to an appropriate concentration. and placed in an LB petri dish containing 5% calf serum for bacteria culture. The number of colonies was counted, and microbicidal rates were calculated.

Table 8 Microbicidal effect of the disinfectant on clinical natural bacteria

Groups disinfection (%) 30 min 60 min 90 min

Test group high concentration 986 942 97.7

Test group moderate

Ground concentration 98.4 92.2 93.2

Test group low concentration 869 77.6 83.1

Control group 79.8 852 82.0

Test group high concentration 925 95,5 928

Test group moderate

Trough concentration 96.7 93.9 97.0

Test group low concentration 598 81.9 76.8

Control group 68.3 90.2 81.7

Test group high concentration 918 92.9 96.6

Test group moderate

Railing concentration 93.3 92.7 91.7

Test group low concentration 546 591 822

The results show that the microbicidal effect of the foam type long-acting compound dismfectant prepared in Example 4 on the ground, the troughs and the railings in livestock and poultry houses increases with the increase of its concentration, and the moderate concentration can meet the clinically required microbicidal rate of 90%. Compared with the control group, the disinfectant has a longer-lasting and more outstanding microbicidal effect.

Other examples provided in the present invention can all achieve the above microbicidal effect. (4) Evaluation test of safety of the disinfectants

Safety evaluation of the disinfectants prepared in the examples was carried out in a GLP laboratory. The evaluation involved oral acute toxicity tests in rats, acute skin irritation tests in rabbits. and acute eve irritation tests in rabbits.

Oral acute toxicity of the disinfectants prepared in the examples was evaluated using SD rats in accordance with “Guidelines for Acute Toxicity Testing (LD50 determination) for

Veterinary Drugs”. 10 SD rats (half male and half male) were exposed to the dismfectant by intragastric administration via the oral route at doses of 1009 mg/kg b.w.. 1453 mg/kg b.w.. 2093 mg/kg b.w., 3014 mg/kg b.w.. 4340 mg/kg b.w.. 6250 mg/kg b.w., 9000 mg/kg b.w,, respectively. Body weights were recorded before the exposure on the day of the exposure (0 day). and on the 7 day and the 14" day after the exposure. Detailed symptoms were observed before the exposure on the 0" day, and about 20 minutes, 2 hours, and 4 hours after the exposure, and symptoms were observed once a day from the 1¥ day to the 14" day. Gross dissection was performed to all animals exposed to the disinfectant.

Acute skin irritation/corrosion tests of the disinfectants in rabbits were conducted using

Japanese white rabbits as test animals in accordance with “Technical Specifications for

Disinfection (edition 2002)”. Three male rabbits were used for the tests. 8.5 mL of a stock solution of a test sample was evenly applied each day to the skin on the right side of the back of each of the three animals, and the skin on the left side, without the test sample, was used as a control; and the animals were exposed for 4 hours. The disinfectant was applied once a day, for 14 consecutive days. On the day before the application and on the 1* day of the application, clinical observation was performed once a day. and after that, clinical observation was performed 24 hours after each application. Skin responses at sites where the test sample was applied were observed each day 24 hours after application, and scored according to the skin irritation response scoring standard.

Acute eye irritation/corrosion tests of the disinfectants in rabbits were conducted using

Disinfection (edition 2002)”. Three male rabbits were used for the tests. Each of the animals was administered 0.1 mL of a test sample to the right eye, and the left eye was used as a control.

Clinical observation was performed once a day after the administration. Eye examinations were conducted at 1 hour, 24 hour, 48 hour, and 72 hour after the administration, and an “average score” for each of four aspects, namely corneal damage. iris damage, conjunctival hyperemia, and conjunctival edema, of each animal at different observations times, was calculated (the “average score” being a sum of scores for each aspect of the animal at 24 hour, 48 hour, and 72 hour divided by the number of times of the observation namely 3). Irritation intensity of the test sample to the eyes was judged on the basis of the average scores for the corneal damage, iris damage, conjunctival hyperemia, and conjunctival edema of the animals and recovery time therefrom in accordance with the eye irritation response grading standard.

Results are as follows.

Table 9 Results of evaluation tests of safety of the disinfectants

Example 2 Example 3 Example 4 Example 5

Female rats < >5000 >5000 >5000 >5000

LDs

Male rats LDso >5000 >5000 >5000 >5000

Skin irritation None None None None

Eye irritation None None None None

Note: LD is the median lethal dose, and its unit is mg/kg b.w.

In the oral acute toxicity tests, for the first to fifth groups of animals, none of the disinfectants prepared in the examples causes death to the animals; and starting from the sixth group (6250 mg/kg b.w.). the disinfectant prepared in some of the examples causes death to the animals. Calculated according to the 95% confidence interval, the median lethal dose for the disinfectants prepared in Examples 2-5 in female and male rats is greater than 5000 mg/kg b.w., indicating that the disinfectants are non-toxic.

In the skin irritation tests, none of the test animals show skin damage after 14 days of continuous exposure. indicating that the disinfectants do not irritate skin.

In the eye irritation tests, none of the test animals has corneal damage, iris damage, conjunctival hyperemia and conjunctival edema in its right eye after being exposed to the disinfectants for 24 hours, 48 hours, and 72 hours, indicating that the disinfectants do not irritate eyes.

In the following sections, clauses will be provided:

1. A foam type long-acting citric acid-containing compound disinfectant, comprising the following components by mass percentage: citric acid 0.5%-5.0%: benzalkonium bromide 0.5%-3.0%; 3 decamethonium bromide 0.5%-5.0%: glutaraldehyde 0.25%-2.5%: polyhexamethylene biguanide 0.5%-5.0%; a cosolvent 3.0%-15.0%; a penetrant 0. 1%-0.5%; a foam emulsifier 0.5%-5.0%. a stabilizer, which, together with citric acid, forms a buffer system to maintain a pH value of 3.0-5.0; and surplus is sterilized water, which is added to make up to 100%. 2. The foam type long-acting citric acid-containing compound disinfectant according to clause 1, comprising the following components by mass percentage: citric acid 0.5%-4.0%; benzalkonium bromide 1.0%-2.5%; decamethonium bromide 2.0%-5.0%; glutaraldehyde 0.5%-1.0%; polyhexamethylene biguanide 0.5%-1.5%; a cosolvent 5.0%-10.0%; a penetrant 0.2%-0.4%; a foam emulsifier 1.0%-3.0%: a stabilizer, which, together with citric acid. forms a buffer system to maintain a pH value of 3.0-5.0; and surplus is sterilized water, which is added to make up to 100%. 3. The foam type long-acting citric acid-containing compound disinfectant according to clause 1 or 2, wherein: a ratio of a mass of glutaraldehyde to a sum of a mass of benzalkonium bromide and a mass of decamethonium bromide is 1:2 or more, preferably 1: (2-4); a ratio of the mass of benzalkonium bromide to the mass of decamethonium bromide is 1: (1-4), preferably 1: (1-3): the cosolvent is one or more selected from alcoholic solvents containing 2-10 carbons: the penetrant is one or more selected from ethanol, glycerol trioleate, tricapry lin, fatty acid polyoxyethylene ether-9, fatty acid polyoxyethylene ether-7, and fatty acid polyoxyethylene ether-5; the foam emulsifier is one or more selected from dodecyl dimethyl amine oxide, decyl glucoside, coconut oil diethanolamide, and cetyltrimethylammonium bromide; and the stabilizer is selected from salts capable of forming a buffer system with citric acid, the salts including citrate, disodium hydrogen phosphate. dipotassium hydrogen phosphate, and sodium hydrogen phosphate.

4. The foam type long-acting citric acid-containing compound disinfectant according to any one of clauses 1 to 3, being capable of killing pathogenic microorganisms in animal husbandry and breeding environments, including bacteria, viruses, mycoplasmas, spores, and fungi. 5. The preparation method of the foam type long-acting citric acid-containing compound disinfectant according to any one of clauses 1 to 4, comprising the following steps: (1) introducing nitrogen into part of the sterilized water; (2) adding the cosolvent to the sterilized water treated in step (1) and stirring; (3) adding decamethonium bromide and benzalkonium bromide to the system obtained in step (2) and stirring at a low speed until complete dissolution; (4) adding the penetrant and the foam emulsifier to the system obtained in step (3) and stirring at a low speed to form system A; (5) taking another part of the sterilized water, into which nitrogen is then introduced; (6) adding glutaraldehyde and polyhexamethylene biguanide to the sterilized water treated in step (3) and stirring at a low speed until complete dissolution; (7) adding citric acid and the stabilizer to the system obtained in step (6) and stirring at a low speed (<150 rpm) until complete dissolution to form system B; (8) transferring the system B to the system A, stirring uniformly at a low speed, and adding the sterilized water to bring to 100% by mass, and maintaining a pH value of the resulting system to be 3.0-3.0. 6. The preparation method according to clause 5. wherein the stirring at a low speed each occurs at a rotation speed of less than 150 rpm, and in step (4), the stirring lasts for 20-60 minutes.

7. Use of the foam type long-acting citric acid-containing compound disinfectant according to any one of clauses 1 to 4. wherein the foam type long-acting citric acid-containing compound disinfectant is diluted with water by 100-200 times, for disinfection of environments or objects. 8. The use according to clause 7, wherein the environments are livestock and poultry breeding environments, and the objects are objects in the livestock and poultry breeding environments.

9. A disinfectant composition, comprising citric acid. benzalkonium bromide, decamethonium bromide, glutaraldehyde, and polyhexamethylene biguanide. 10. The disinfectant composition according to clause 9, wherein a ratio of a mass of glutaraldehyde to a sum of a mass of benzalkonium bromide and a mass of decamethonium bromide is 1:2 or 1: 2 or more, preferably 1: (2-4);

a ratio of the mass of benzalkonium bromide to the mass of decamethonium bromide is 1: (1-4), preferably 1: (1-3):

a ratio of a mass of polyhexamethylene biguanide to the mass of glutaraldehyde is 1: (0.5-

5), preferably 1: (1-3); and a ratio of a mass of citric acid to the mass of glutaraldehyde is 1: (1-10), preferably 1: (1-

8).

Claims

Conclusions

1. A long-acting disinfectant of a foam type with a citric acid-containing composition, comprising the following ingredients in percentages by mass: citric acid 0.5% -5.0%; benzalkonium bromide 0.5%-5.0%: decamethonium bromide 0.5%-5.0%; glutaraldehyde 0.25%-2.5%; polyhexamethylene biguanide 0.5%-5.0%: a cosolvent 3.0%-15.0%; a penetrant 0.1%-0.5%; a foam emulsifier 0.5%-5.0%; a stabilizer, which together with citric acid forms a buffer system to maintain a pH value of 3.0-5.0; and a surplus of sterilized water, which is added to make up to 100%.

The foam-type long-lasting disinfectant with a citric acid-containing composition according to claim 1, comprising the following components by mass percentage: citric acid 0.5%-4.0%: benzalkonium bromide 1.0%-2.5%; decamethonium bromide 2.0%-5.0%; glutaraldehyde 0.5%-1.0%; polyhexamethylene biguanide 0.5%-1.5%; a cosolvent 5.0%-10.0%; a penetrant 0.2%-0.4%; a foam emulsifier 1.0%-3.0%:; a stabilizer, which together with citric acid forms a buffer system to maintain a pH value of 3.0-5.0; and a surplus of sterilized water, which is added to make up to 100%.

The long-lasting foam-type disinfectant with a citric acid-containing composition according to claims 1 or 2, wherein: the ratio of a mass of glutaraldehyde to a sum of a mass of benzalkonium bromide and a mass of decamethonium bromide is 1:2 or more, preferably 1: ( 2-4): the ratio between a mass of benzalkonium bromide and a mass of decamethonium bromide is 1: (1-4), preferably 1: (1-3); the cosolvent is one or more alcoholic solvents with 2-10 carbons; the penetrant is one or more of the following substances: ethanol, glycerol trioleate, tricaprylin, fatty acid polyoxyethylene ether-9, fatty acid polyoxyethylene ether-7, and fatty acid polvoxyethylene ether-5; the foam emulsifier is one or more of the following substances: dodecyl-dimethylammonium oxide, decyl glucoside, coconut oil diethanolamide and cetyltrimethylammonium bromide; and the stabilizer is selected from salts that can form a buffer system with citric acid, including the salts citrate. disodium hydrogen phosphate, dipotassium hydrogen phosphate and sodium hydrogen phosphate.

4. The long-lasting foam-type disinfectant with a citric acid-containing composition according to any one of claims 1 to 3, capable of killing pathogenic microorganisms in livestock and breeding environments, including bacteria, viruses, mycoplasmas, spores and fungi.

A method of preparing the long-acting foam-type disinfectant with a citric acid-containing composition according to any one of claims 1 to 4, consisting of the following steps: (1) introducing nitrogen into a portion of the sterilized water; (2) adding the cosolvent to the sterilized water treated in step (1) and stirring: (3) adding decamethonium bromide and benzalkonium bromide to the system obtained in step (2) and stirring at low speed until completely dissolved ; (4) adding the penetrant and foam emulsifier to the system obtained in step (3) and stirring at a low speed to form system A; (3) taking another part of the sterilized water, into which nitrogen is then introduced: (6) adding glutaraldehyde and polyhexamethylene biguanide to the sterilized water treated in step (5) and stirring at a low speed until complete dissolution; (7) adding citric acid and the stabilizer to the system obtained in step (6) and stirring at low speed (<150 rpm) until complete dissolution to form system B (8) transferring from system B to system A, stirring at slow speed and add sterilized water to make up to 100% mass and maintain a pH of 3.0-5.0 for the resulting system.

The preparation method according to claim 5, wherein the stirring at a low speed always takes place at a rotation speed of less than 150 rpm, and wherein in step (4) the stirring lasts 20-60 minutes.

Use of the long-acting foam-type disinfectant with a citric acid-containing composition according to any one of claims 1 to 4, wherein the long-acting foam-type disinfectant with a citric acid-containing composition is diluted 100-200 times with water before disinfecting of environments or objects.

The use according to claim 7, wherein the environments are livestock and poultry breeding environments, and the objects are objects in the livestock and poultry breeding environments.

9. A disinfectant composition comprising citric acid, benzalkonium bromide, decamethonium bromide, glutaraldehyde and polyhexamethylene biguanide.

The disinfectant composition according to claim 9, wherein a ratio of a mass of glutaraldehyde to a sum of a mass of benzalkonium bromide and a mass of decamethonium bromide is 1:2 or 1:2 or more, preferably 1:(2-4); the ratio of a mass of benzalkonium bromide to a mass of decamethonium bromide is 1: (1-4), preferably 1: (1-3) the ratio of a mass of polyhexamethylene biguanide to a mass of glutaraldehyde is 1: 0.3-5, preferably 1 : 1-3: and the ratio between a mass of citric acid and a mass of glutaraldehyde is 1: (1-10), preferably 1: (1-8).