A highly effective disinfectant capable of killing spores and a disinfection method thereof

Abstract

The invention discloses a preparation capable of killing spores efficiently and a disinfection method thereof. The preparation uses polyhexamethylene biguanide to kill spores of Bacillus subtilis var. niger, and its effective component is composed of polyhexamethylene biguanide with a certain concentration. The high-efficiency disinfectant can kill spores efficiently at normal temperature and pressure, and is non-toxic, non-irritating, and stable in nature. It can be used for disinfection operations in environments such as hospitals, places where people gather, confined spaces, public transportation, and livestock breeding, and has the characteristics of being green, safe, non-irritating, and stable in nature, and has great market application functional prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of disinfectants, and in particular to a highly effective disinfectant capable of killing spores and a disinfection method thereof. Background Art

[0002] Spores are metabolically dormant microorganisms that remain viable under a variety of environmental conditions. They are the most resistant organisms in the entire biological world and are particularly outstanding in terms of heat resistance, chemical resistance, and radiation resistance. Since they are different from vegetative cells in terms of structure and chemical composition, spores also have many characteristics different from vegetative cells. The most important feature of spores is their strong resistance to high temperature, ultraviolet rays, dryness, ionizing radiation, and many toxic chemicals. It takes 5 to 9.5 hours for the spores of Clostridium botulinum to be killed in boiling water; the radiation resistance of Bacillus megaterium spores is 36 times stronger than that of E. coli cells. The dormancy ability of spores is more outstanding. Under normal conditions, they can generally remain alive for several years to decades. According to literature records, some spores can even remain dormant for hundreds to thousands of years. Due to the stability of spores, spores are easily spread in polluted environments, especially in public places with large and dense traffic such as hospitals, cinemas, shopping malls, airplanes, trains, and ships, and often need to be disinfected. In addition, with the development of the economy, the import and export trade of goods has surged, and a large amount of goods need to be circulated. This requires disinfection of goods during import and export, especially for the import of goods. In order to prevent pathogens from invading with the goods and causing an epidemic, customs needs to effectively disinfect the goods.

[0003] The ethanol disinfectant used by medical staff cannot effectively kill spores. For the disinfection of some public places, oxygen-containing disinfectants, chlorine-containing disinfectants, aldehyde disinfectants or phenolic disinfectants are mostly used. Oxygen-containing disinfectants include peracetic acid, hydrogen peroxide, perglutaric acid, chlorine dioxide, etc. Peracetic acid and perglutaric acid are unstable and highly irritating. Long-term use is harmful to the eyes and respiratory mucosa of humans and animals, and has strong damage to the environment. Chlorine-containing disinfectant refers to a disinfectant that can produce hypochlorous acid with bactericidal activity in water. Its metabolite chloroform is highly carcinogenic, and most of them are highly irritating. Aldehyde disinfectants such as formaldehyde, glutaraldehyde, polyformaldehyde, etc. can produce free aldehyde groups and react with proteins and certain other components of microorganisms under appropriate conditions. The disadvantage is that the disinfection effect is very poor when organic pollutants are present, and formaldehyde and polyformaldehyde are highly irritating and carcinogenic. Glutaraldehyde is harmful to the eyes, skinIt has strong irritation and genetic toxicity to the mucous membrane. Phenolic disinfectants such as halogenated phenols (chlorocresol), cresol (coal phenol soap, also known as Lysol), xylenol, bisphenols, and compound phenols have strong carcinogenicity and cumulative toxicity, strong phenol odor, and are ineffective against spores. High concentrations of hydrogen peroxide (25-30%) have a killing effect on spores, but it takes a long time. However, high concentrations of hydrogen peroxide are highly toxic and can cause poisoning in humans simply by inhalation through the respiratory tract or skin contact; human beings are in danger of life if exposed to 100 mg / kg for a short time.

[0004] For the regulations of high-efficiency disinfectants or high-level disinfectants, whether they can eliminate spores is the most important indicator. In Table B.1 "Disinfectant Inspection Items" of the Health Industry Standard WS 628-2018 "Technical Requirements for Hygiene and Safety Evaluation of Disinfection Products" of the People's Republic of China, the killing test of Bacillus subtilis var. niger spores is a must-do item for high-level disinfectants. Table 1 of the national standard GB27949-2020 "General Requirements for Disinfectants for Medical Devices" points out that the killing or elimination logarithm of high-level disinfectants against Bacillus subtilis var. niger spores (ATCC9372) is not less than 3.00. Therefore, the killing of spores is an important indicator to measure whether it is a high-efficiency disinfectant or a high-level disinfectant.

[0005] Chinese patent CN113491709A discloses a multifunctional and efficient compound disinfectant and its preparation method, which is composed of the following main ingredients: polyhexamethylene biguanide (PHMB) 0.01-2.0%, water-soluble quaternary ammonium salt 0.05-0.2%, surfactant 0.1-2.0%, thickener 0.05-1.5%, moisturizer 0.05-2.0%, essence 0.001-0.1%, medical ethanol 0-40.0%, and the balance is pure water. The patent points out that the disinfectant has the effects of efficient sterilization and long-term antibacterial effect, and can kill Escherichia coli, Staphylococcus aureus, Candida albicans, etc. within 1 minute, but does not point out that it can kill spores. From the formula, it can be seen that polyhexamethylene biguanide and quaternary ammonium salts have bactericidal effects. As for polyhexamethylene biguanide, the patent points out in the background technology that the dosage of polyhexamethylene biguanide is only 200ppm, which can achieve a 100% bactericidal rate for Escherichia coli, Staphylococcus aureus, and Candida albicans, but it is not suitable for killing bacterial spores. Quaternary ammonium salt disinfectants are not suitable for killing bacterial spores and mycoplasmas, etc. Therefore, according to the cognition of those skilled in the art, the disinfectant cannot kill spores and cannot be called a high-efficiency disinfectant. Summary of the invention

[0006] The purpose of the present invention is to provide an environmentally friendly and safe high-efficiency disinfectant that can kill the black variant spores of Bacillus subtilis to solve the shortcomings of the prior art.

[0007] The technical solution adopted by the present invention is as follows: a highly effective disinfectant capable of killing spores, wherein the effective component of the highly effective disinfectant is polyhexamethylene biguanide.

[0008] Furthermore, the high-efficiency disinfectant has a logarithmic killing value of no less than 3.00 for Bacillus subtilis var. niger spores, which meets the requirements for high-level disinfectants in Table 1 of the national standard GB27949-2020 "General Requirements for Disinfectants for Medical Devices".

[0009] Furthermore, the highly effective disinfectant is in the form of a gel or a liquid.

[0010] Furthermore, the present invention has found through experiments that in terms of high-efficiency disinfection, polyhexamethylene biguanide cannot coexist with chelating agents (such as ethylenediaminetetraacetic acid and its salts). The presence of chelating agents will greatly reduce the bactericidal performance of polyhexamethylene biguanide. Therefore, high-efficiency disinfectants cannot contain chelating agents, such as ethylenediaminetetraacetic acid (EDTA) and its salts, N,N-bis(carboxymethyl)-L-glutamate tetrasodium (GLDA), iminodisuccinate tetrasodium, N-(1-carboxyethyl)iminodiacetic acid trisodium (MGDA), diethylenetriaminepentaacetic acid (DPTA), citrate, etc.

[0011] Furthermore, the present invention has found through experiments that in terms of high-efficiency disinfection, polyhexamethylene biguanide cannot coexist with oxidants. The presence of oxidants will greatly reduce the bactericidal performance of polyhexamethylene biguanide. Therefore, high-efficiency disinfectants cannot contain oxidants, such as hydrogen peroxide, sodium hypochlorite, bromochlorohydrin, etc.

[0012] Furthermore, the highly effective disinfectant further contains a carrier and / or an acceptable auxiliary material, wherein the carrier is a solvent compatible with polyhexamethylene biguanide, such as water, propylene glycol, glycerol, polyethylene glycol, etc. The auxiliary material is a synergist, thickener, etc. compatible with polyhexamethylene biguanide.

[0013] Furthermore, based on 100% by mass of the high-efficiency disinfectant, the content of polyhexamethylene biguanide is not less than 0.2%.

[0014] Further, when the content of polyhexamethylene biguanide is less than 0.5%, for example, 0.2-0.5%, it still has the ability to kill Bacillus subtilis black variant spores, but the required disinfecting time is relatively extended. The present invention finds that when chitosan oligosaccharide is added as a synergist, chitosan oligosaccharide can significantly enhance the disinfecting ability of polyhexamethylene biguanide. Therefore, when the content of polyhexamethylene biguanide is less than 0.5%, polyhexamethylene biguanide can be used in combination with chitosan oligosaccharide to achieve the purpose of efficient disinfection. At this time, the content of chitosan oligosaccharide is not less than 0.2%.

[0015] Furthermore, when it is not necessary to add a synergist, in order to achieve the purpose of high-efficiency disinfection, the content of polyhexamethylene biguanide is not less than 0.5% based on 100% by mass of the high-efficiency disinfectant.

[0016] Furthermore, the present invention also includes a disinfection method, when the content of the polyhexamethylene biguanide is not less than 0.5% based on 100% by mass of the high-efficiency disinfectant, the disinfection action time is not less than 10 minutes.

[0017] In the high-efficiency disinfectant of the present invention, when the content of polyhexamethylene biguanide is not less than 0.5%, under the premise of not containing a chelating agent, the killing rate of the spores of Bacillus subtilis black variant after acting for 10 minutes is greater than 99.999%; when the content of polyhexamethylene biguanide is 0.2-0.5%, chitosan oligosaccharide is added as a synergist, and the killing rate of the spores of Bacillus subtilis black variant after acting for 20 minutes is greater than 99.999%.

[0018] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0019] 1. The present invention has found through experiments that, at a certain concentration, polyhexamethylene biguanide can not only kill spores, but also kill spores efficiently, breaking the cognitive bias that polyhexamethylene biguanide, as a cationic disinfectant, itself does not have the ability to kill spores (also refer to the background technology of CN113491709A), and found that among the common cationic disinfectants, only polyhexamethylene biguanide has the ability to kill spores efficiently, providing unlimited possibilities for the application of polyhexamethylene biguanide in the field of disinfection;

[0020] 2. The high-efficiency disinfectant of the present invention can effectively kill spores at room temperature, is non-toxic, non-irritating, and has stable properties. Compared with the oxygen-containing disinfectants, chlorine-containing disinfectants, aldehyde disinfectants or phenol disinfectants used, it has the outstanding advantages of being green and safe, and is expected to replace the use of these disinfectants;

[0021] 3. The present invention also found through experiments that polyhexamethylene biguanide cannot coexist with chelating agents. The presence of chelating agents will greatly reduce the bactericidal performance of polyhexamethylene biguanide, which provides a technical reference for the application research of polyhexamethylene biguanide;

[0022] 4. The present invention also found through experiments that polyhexamethylene biguanide cannot coexist with oxidants. The presence of oxidants will greatly reduce the bactericidal performance of polyhexamethylene biguanide, which provides a technical reference for the application research of polyhexamethylene biguanide;

[0023] 5. The present invention also found through experiments that chitosan oligosaccharide can enhance the ability of polyhexamethylene biguanide to kill spores. Combining chitosan oligosaccharide with polyhexamethylene biguanide can help reduce the amount of polyhexamethylene biguanide used.

[0024] 6. The present invention has found the minimum effective concentration and minimum action time of polyhexamethylene biguanide for effectively killing spores, providing data reference for the research and development of high-efficiency disinfectant products;

[0025] 7. The high-efficiency disinfectant of the present invention can be used for disinfection operations in hospitals, places where people gather, confined spaces, public transportation vehicles, and livestock and poultry breeding environments. It has the characteristics of being green, safe, non-irritating, and stable in nature, and has great market application prospects. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] In the field of disinfectants, polyhexamethylene biguanide is usually used as a cationic guanidine disinfectant. It is a broad-spectrum antibacterial agent that has a killing effect on Gram-positive bacteria, Gram-negative bacteria, fungi and yeasts. Its bactericidal mechanism is as follows: (1) Guanidine groups have high activity and polymers are positively charged. Since various bacteria and viruses are generally negatively charged, guanidine salts are easily adsorbed, which prevents bacteria and viruses from dividing and reproducing, and thus loses their activity; (2) It causes the cell membrane structure to collapse, forming transmembrane pores, rupturing the cell membrane, destroying the energy metabolism of microorganisms, and making bacteria and viruses lose their activity; (3) It can form a thin film to block the respiratory tract of microorganisms, causing them to suffocate. In medicine, polyhexamethylene biguanide is used for the disinfection of contact lenses, eye drops and surgical operations. It is also widely used in the fields of cosmetics, personal care products, textiles, food industry, animal husbandry, swimming pools, etc. for sterilization, disinfection and algae removal. Public data show that the bactericidal rate of 0.02% polyhexamethylene guanidine against Escherichia coli, Staphylococcus aureus, Candida albicans, and gonococci is 100%, the score of 0.5% polyhexamethylene guanidine eye irritation test is 3.0 points, and the score of acute skin irritation test is 0 points, which is non-irritating, and 1% polyhexamethylene guanidine is greater than 5000mg / kgBW, which is actually non-toxic. For those skilled in the art, polyhexamethylene biguanide has always been classified as an inefficient disinfectant because it belongs to cationic guanidine disinfectants, so it is not used to kill spores. Based on this cognition, there is currently no research record on polyhexamethylene biguanide in killing spores. However, the present invention unexpectedly discovered through experiments that at a certain concentration, polyhexamethylene biguanide can efficiently kill spores, thereby breaking this cognitive bias. Based on this, the present invention has developed a highly efficient disinfectant that can kill spores, and all indicators meet the corresponding standards.

[0028] In order to verify the innovative achievements of the present invention, the following verification experiments are used to illustrate the invention.

[0029] 1. Quantitative killing test of Bacillus subtilis var. niger spore suspension

[0030] 1. Main Materials

[0031] ①. Strain: Bacillus subtilis var. niger spore ATCC9372.

[0032] ②. Samples to be tested: Prepare the samples to be tested in advance according to 1 to 13 recipes in Table 2, a total of 13 recipes.

[0033] 2. Test methods

[0034] ①. The test shall be carried out in accordance with 2.1.1.7.4 “Quantitative Bactericidal Test for Suspension” of the Technical Specifications for Disinfection 2002 Edition.

[0035] ②. The quantitative identification test of the neutralizer suspension shall be carried out in accordance with the "Technical Specifications for Disinfection" 2002 edition - 2.1.1.5.5 "Quantitative Identification Test of Neutralizer Suspension".

[0036] 3. Neutralizer

[0037] ①.D / E broth.

[0038] 3. Test results

[0039] See Table 1 below and Table 2 for the formula.

[0040] Table 1. Detection results of Bacillus subtilis var. niger spores

[0041]

[0042]

[0043]

[0044] 4. Experimental conclusion

[0045] 4.1 From the experimental results of formulas 1, 2, 4, 5, 6, and 9, it was found that when the minimum dosage of polyhexamethylene biguanide was 0.5%, it could effectively kill the black variant spores of Bacillus subtilis. After 10 minutes of action, the killing rate was > 99.999%. When the dosage of polyhexamethylene biguanide was less than 0.5%, in the absence of a synergist, the killing effect of the black variant spores of Bacillus subtilis was poor.

[0046] 4.2 From the experimental results of formulas 5, 7, and 8, it can be concluded that when the dosage of polyhexamethylene biguanide is 1.0%, the killing rate of polyhexamethylene biguanide on spores is greatly reduced by adding chelating agents (disodium ethylenediaminetetraacetic acid and tetrasodium ethylenediaminetetraacetic acid) and / or oxidants (bromochlorohydrin) when the dosage is 20 minutes. The killing rate of spores of formula 5 (20 minutes) is greater than 99.9999%, while the killing rates of formulas 7 and 8 (20 minutes) on spores are only 16.53% and 8.12%;

[0047] 4.3 From the experimental results of formulas 3 and 11, it can be concluded that when the usage of benzalkonium chloride is 0.2% and 0.5%, respectively, it cannot effectively kill the spores of Bacillus subtilis var. niger. The killing rates of spores are 85.6032% and 95.8889% after 60 minutes and 20 minutes respectively.

[0048] 4.4 The experimental results of formula 10 show that when the usage of chlorhexidine acetate is 0.5%, it cannot effectively kill the spores of Bacillus subtilis var. niger. After 20 minutes of action, the killing rate of spores is 30.1488%;

[0049] 4.5 From the experimental results of formulas 12 and 13, it can be concluded that when the usage of polyhexamethylene biguanide is 0.2%, 0.2% chitosan oligosaccharide can enhance the killing ability of polyhexamethylene biguanide on spores. After 20 minutes of action, the killing rates of spores are 99.5426% and 99.9992% respectively;

[0050] 5. Disinfectant formula

[0051] Example 1: The product is a liquid preparation, prepared with the following raw materials in percentage by weight: 0.5% polyhexamethylene biguanide and the balance deionized water.

[0052] Preparation: ①. Take the above raw materials according to the mass ratio and put them into deionized water; ②. Stir them thoroughly and fill them into suitable containers using a filling machine to obtain the finished product.

[0053] The preparation of the present invention may further include one or more of the following components (including auxiliary materials) by mass percentage:

[0054] Chitosan oligosaccharide: 0.1-1.0%;

[0055] Chitosan: 1.0-10.0%;

[0056] Hydroxypropyl methylcellulose: 1.0-5.0%.

[0057] Example 2: The product is a gel preparation, which is prepared with the following raw materials in percentage by weight: polyhexamethylene biguanide 0.3%, chitosan: 5.0%, and deionized water as the balance.

[0058] Preparation: ①. Take the above raw materials according to the mass ratio, first add chitosan oligosaccharide into an appropriate amount of warm water at about 30°C, fully swell it, and obtain colloid A; ②. Then add the remaining raw materials into the remaining water, fully dissolve it, and mix it with solution A and stir it evenly; ③. Use a filling machine to pack it into suitable containers to obtain the finished product.

[0059] The preparation of the present invention may further be added with the following synergists and / or adjuvants by mass percentage:

[0060] Chitosan oligosaccharide: 0.1-1.0%;

[0061] Hydroxypropyl methylcellulose: 1.0-5.0%.

[0062] Example 3: The following table lists the raw materials of 15 formulations and their mass percentages (%). For each formulation, the raw materials were taken according to the mass percentage to prepare the corresponding preparations.

[0063] Table 2. Raw materials and mass percentage (%) of the formula

[0064]

[0065]

[0066] Note: In the table, PHMB: polyhexamethylene biguanide; CHX: chlorhexidine acetate; BZK: benzalkonium chloride; D2: ethylenediaminetetraacetic acid-2Na; D4: ethylenediaminetetraacetic acid-4Na; BCDM: bromochlorohydantoin; COS: chitosan oligosaccharide; CTS: chitosan; HPMC: hydroxypropyl methylcellulose.

[0067] 2. Comparative Analysis

[0068] 1. From the experimental results of formula 12, it can be concluded that when the dosage of polyhexamethylene biguanide is 0.2%, the killing rates of spores are 99.5426% (K=2.373) and 91.3704 (K=1.063) after 20 minutes and 10 minutes of action respectively. If the dosage of polyhexamethylene biguanide is further reduced to 0.1%, the killing rate of spores will be even lower;

[0069] 2. From the experimental results of formula 9, formula 12 and formula 10, it is found that when the usage of polyhexamethylene biguanide is 0.5% (formula 9) and 0.2% (formula 12), and the usage of chlorhexidine acetate is 0.5% (formula 10), the killing rates of spores after 20 minutes of action are respectively >99.9999% (K>5.00), 99.5426% (K=2.373) and 30.1488% (K=0.153). It can be seen that as cationic guanidine disinfectants, polyhexamethylene biguanide can kill spores efficiently, while chlorhexidine acetate is a weaker disinfectant and cannot kill spores. When 0.1% polyhexamethylene biguanide and 0.1% chlorhexidine are combined, the killing effect of spores is much lower than that of directly using 0.2% polyhexamethylene biguanide;

[0070] 3. From the experimental results of Formula 9, Formula 12, Formula 11 and Formula 3, it is obtained that when the usage amount of polyhexamethylene biguanide is 0.5% (Formula 9) and 0.2% (Formula 12), and the usage amount of benzalkonium chloride (cationic quaternary ammonium salt) is 0.5% (Formula 11) and 0.2% (Formula 3), Formula 9, Formula 12 and Formula 11 act for 20 minutes, the killing rates of spores are respectively >99.9999% (K>5.00), 99.5426% (K=2.373) and 95.8889% (K=1.400), and Formula 3 acts for 60 minutes, and the killing rate of spores is 85.6032% (K=0.850). It can be seen that cationic quaternary ammonium salt disinfectants are weaker disinfectants and cannot kill spores. Only polyhexamethylene biguanide can effectively kill spores. When 0.1% polyhexamethylene biguanide and 0.1% cationic quaternary ammonium salts are combined, the sporicidal effect is much lower than that of directly using 0.2% polyhexamethylene biguanide.

[0071] The above descriptions are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of polyhexamethylene biguanide in the preparation of a highly effective disinfectant, characterized in that: The killing logarithmic value of the high-efficiency disinfectant against the black variant spores of Bacillus subtilis is not less than 3.00, the effective ingredient of the high-efficiency disinfectant is polyhexamethylene biguanide, and the high-efficiency disinfectant also contains chitosan oligosaccharides. Based on the mass percentage of the high-efficiency disinfectant being 100%, the content of polyhexamethylene biguanide is not less than 0.2%, and the content of chitosan oligosaccharides is not less than 0.2%.

2. The use of polyhexamethylene biguanide in the preparation of a highly effective disinfectant, characterized in that: The high-efficiency disinfectant has a killing logarithmic value of no less than 3.00 for Bacillus subtilis var. niger spores. The effective ingredient of the high-efficiency disinfectant is polyhexamethylene biguanide. Based on 100% by mass of the high-efficiency disinfectant, the content of polyhexamethylene biguanide is no less than 0.5%.

3. The use according to claim 1 or 2, characterized in that: The dosage form of the highly effective disinfectant is gel or liquid.

4. The use according to claim 1 or 2, characterized in that: The high-efficiency disinfectant does not contain a chelating agent and / or an oxidizing agent.

5. The use according to claim 1 or 2, characterized in that: The high-efficiency disinfectant also contains a carrier and / or acceptable auxiliary materials, wherein the carrier is a solvent compatible with polyhexamethylene biguanide; and the auxiliary materials are a synergist and a thickener compatible with polyhexamethylene biguanide.

6. The use according to claim 1, characterized in that The content of the polyhexamethylene biguanide is 0.2-0.5%.

7. The use according to claim 2, characterized in that The disinfection time of high-efficiency disinfectants shall not be less than 10 minutes.

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