A hydrogen peroxide compound disinfectant and its application

Abstract

The present invention relates to the technical field of disinfection and protection, and specifically to a hydrogen peroxide compound disinfectant and its application, wherein the hydrogen peroxide compound disinfectant comprises hydrogen peroxide and a polypeptide, and may also comprise other adjuvants, carriers or auxiliary components. The present invention also discloses the application of the hydrogen peroxide compound disinfectant in a sterilization and disinfection product, and the hydrogen peroxide compound disinfectant can effectively improve the sterilization and disinfection effect and stability of hydrogen peroxide. Compared with the prior art, the hydrogen peroxide compound disinfectant has a good sterilization effect, especially for Candida albicans, good stability, and high safety, no hormones, no antibiotics, and has a broad-spectrum, high-efficiency, and safe sterilization activity, and can be further developed into various dosage forms of antibacterial disinfection products, so it has great potential to be used as a commonly used, safe, and efficient sterilization and disinfection agent, and has a good commercial application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of disinfection and protection, and in particular to a hydrogen peroxide compound disinfectant and application thereof. Background Art

[0002] With the development of society, the demand for disinfection products is growing. At present, the widely used liquid disinfectants in the market mainly include aldehyde disinfectants, iodine preparations, chlorine-containing disinfectants and peroxide disinfectants. Among them, aldehyde disinfectants are toxic in themselves and have certain hazards to the environment and personnel; iodine preparations have poor stability and single efficacy; chlorine-containing disinfectants are irritating and their decomposition products have certain hazards to the environment; peroxide disinfectants are a relatively prominent class of high-efficiency and broad-spectrum disinfectants among all biochemical disinfectants.

[0003] Hydrogen peroxide belongs to the peroxide disinfectant and is a colorless transparent liquid. Hydrogen peroxide is active in nature and kills bacteria by destroying cell structures. It is easily decomposed into oxygen and water, has no harmful residues, and does not cause secondary pollution to the environment. It is known as the cleanest disinfectant (Doll Michelle et al. Touchless Technologies for Decontamination in the Hospital: a Review of Hydrogen Peroxide and UV Devices. [J]. Current infectious disease reports, 2015, 17 (9): 498.). Hydrogen peroxide is often used for disinfection and sterilization of object surfaces, skin wound washing, food tools and equipment, and medical devices.

[0004] Hydrogen peroxide is a strong oxidant. High concentrations of hydrogen peroxide are corrosive to skin, mucous membranes, and metals (Su Weidong. Research progress on the application of hydrogen peroxide disinfectants [J]. Chinese Journal of Disinfection, 2019, 36(02): 150-153.). Low-concentration hydrogen peroxide disinfectants, such as the skin disinfection concentration of hydrogen peroxide (3%) commonly used in the market, are relatively safe and are practically non-toxic. They are non-irritating to the intact skin and eyes of rabbits, have no significant changes in the blood biochemical indicators of experimental animals, do not cause obvious pathological changes in major organs, and have no micronucleus-inducing effect on mouse bone marrow polychromatic erythrocytes (Huang Xiaobo, Yue Musheng, Peng Mingjun, Zheng Liguo. Toxicity study of hydrogen peroxide disinfectant [J]. Chinese Journal of Disinfection, 2006(04):296-298.). They have a good killing effect on bacterial propagules, but a poor killing effect on Candida albicans (Guo Di, Zhang Yani, Song Hui. Experimental study on some properties of hydrogen peroxide disinfectant [J]. Chinese Journal of Disinfection, 2007(01):81-82.). They are volatile, unstable, will slowly decompose into oxygen and water, and have a certain degree of corrosiveness to metals.

[0005] At the same time, the concentration of hydrogen peroxide is between 3.5% and 25%, which is a dangerous item for air transportation. Passengers cannot check it in or carry it with them. Therefore, in order to overcome the defects of a single component, improve stability and disinfection effect, reduce corrosiveness, and facilitate transportation, compound hydrogen peroxide disinfectants have become a research hotspot (Li Pengpeng, Zhou Degang, Bian Kui, Li Zhaobin. Research on the disinfection effect and performance of compound hydrogen peroxide solution [J]. Heilongjiang Animal Husbandry and Veterinary Medicine, 2018 (16): 188-190.).

[0006] Existing literature reports that hydrogen peroxide is used as a disinfectant after being compounded with other substances. For example, patent publication number CN202110193250.1 discloses a hydrogen peroxide and nanosilver compound hand disinfectant and a preparation method thereof, in which hydrogen peroxide and nanosilver are compounded; for example, patent number ZL 201380072149.4 discloses a disinfectant aqueous solution, which contains peracetic acid and hydrogen peroxide. Although hydrogen peroxide is a non-toxic, harmless and environmentally friendly disinfectant, the nanosilver and peracetic acid compounded with it in the above-mentioned patents have more or less hazards. For example, long-term use of nanosilver for antibacterial purposes will form silver deposits in organisms, which will have adverse effects on organisms and humans. Excessive deposition may even cause obvious poisoning. Long-term use will be harmful to the body, cause poisoning symptoms, and affect physical development. In addition, the volatilization of nanosilver into the environment will also have a certain impact on the ecology. Peracetic acid is a strong oxidant with certain toxicity and corrosiveness, and is extremely unstable. It can decompose and release oxygen at room temperature, and spontaneously ignite, burn or explode when exposed to open flames or high temperatures.

[0007] Therefore, it is necessary to seek environmentally friendly disinfectants and hydrogen peroxide to improve the bactericidal effect and stability of hydrogen peroxide. As a new type of biologically active ingredient, peptides are mild and non-irritating, and have incomparable advantages in drug resistance.

[0008] The inventors unexpectedly discovered during their research that the compounding of hydrogen peroxide with the polypeptide of the present invention can effectively improve the bactericidal effect and stability of hydrogen peroxide, and has good market application prospects. Summary of the invention

[0009] In view of the deficiencies in the prior art, one object of the present invention is to provide a hydrogen peroxide compound disinfectant, and a second object is to provide an application of a hydrogen peroxide compound disinfectant in the preparation of a sterilization and disinfection product, characterized in that the hydrogen peroxide compound disinfectant comprises hydrogen peroxide and a polypeptide having an amino acid sequence of SEQ ID NO.1.

[0010] Preferably, the compound disinfectant includes other auxiliary materials, carriers or auxiliary ingredients.

[0011] Preferably, the hydrogen peroxide compound disinfectant can be added to daily used products with bactericidal effects, including but not limited to skin disinfectants and surface disinfectants.

[0012] Preferably, the hydrogen peroxide compound disinfectant can be used on items that need to be disinfected, including but not limited to medical devices and product packaging.

[0013] Preferably, the hydrogen peroxide compound disinfectant is used in a sterile production process.

[0014] The invention provides a hydrogen peroxide compound disinfectant and an application thereof. The hydrogen peroxide compound disinfectant of the invention comprises hydrogen peroxide and a polypeptide, and may also comprise other auxiliary materials, carriers or auxiliary components. The hydrogen peroxide compound disinfectant can effectively improve the bactericidal and disinfecting effect and stability of hydrogen peroxide, especially improve the killing effect and stability of hydrogen peroxide on Candida albicans, and has high safety, is hormone-free, antibiotic-free, has broad-spectrum, high-efficiency and safe bactericidal activity, and has broad commercial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Results of cytotoxicity tests with different peptide concentrations. DETAILED DESCRIPTION

[0016] One object of the present invention is to provide a hydrogen peroxide compound disinfectant, and a second object is to provide an application of a hydrogen peroxide compound disinfectant in the preparation of a sterilization and disinfection product, characterized in that the hydrogen peroxide compound disinfectant comprises hydrogen peroxide and a polypeptide having an amino acid sequence of SEQ ID NO.1.

[0017] Preferably, the hydrogen peroxide compound disinfectant can be added to daily-used products with bactericidal effects, including but not limited to skin disinfectants and surface disinfectants.

[0018] Preferably, the hydrogen peroxide compound disinfectant can be used on items that need to be disinfected, including but not limited to medical devices and product packaging.

[0019] Preferably, the hydrogen peroxide compound disinfectant is used in a sterile production process.

[0020] The other adjuvants, carriers or auxiliary ingredients of the present invention may contain formulation materials for changing, maintaining or preserving, for example, the pH, color, smell, stability of the composition.

[0021] Other auxiliary materials, carriers or auxiliary components are not listed one by one, and ordinary technicians in this field can make specific selections based on common knowledge.

[0022] Preferably, the primary vehicle or carrier of the composition may be aqueous or non-aqueous in nature, such as a suitable vehicle or carrier may be purified water.

[0023] A person skilled in the art can select suitable excipients according to the liquid dosage form to be prepared, according to the general technical knowledge and requirements of the dosage form in the art, add appropriate excipients and additives on the basis of the polypeptide, and prepare it according to conventional formulation technology.

[0024] The preparation of the present invention can be prepared by any suitable method known in the art, and can be deleted or adjusted according to the requirements of sterilization.

[0025] The preferred concentration for a particular situation can be determined routinely by one skilled in the art.

[0026] The polypeptide of the present invention can be obtained by chemical synthesis, expression, separation and purification through genetic engineering technology (for specific methods, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0027] As used in the present invention, the terms "compound disinfectant", "composition" and "preparation" are interchangeable unless otherwise specified and refer to a preparation comprising two active ingredients, hydrogen peroxide and the polypeptide of the present invention, and may also contain other excipients, carriers or auxiliary ingredients.

[0028] As used in the present invention, the term "other auxiliary materials, carriers or auxiliary components" refers to non-toxic liquid diluents or other preparation auxiliary materials and carriers, and the composition can be made into a preparation according to actual needs.

[0029] The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure which is defined only by the claims and the specification.

[0030] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. In the examples, the reagents added are all commercially available unless otherwise specified.

[0031] The embodiments of this part further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention.

[0032] Since hydrogen peroxide has a good killing effect on bacterial propagules, but a poor killing effect on Candida albicans, in order to highlight the improvement of the bactericidal effect of hydrogen peroxide after the combination of hydrogen peroxide and polypeptide, Candida albicans was selected as the bactericidal test strain.

[0033] Since the concentration of hydrogen peroxide skin disinfectant commonly used in the market is 3%, this concentration of hydrogen peroxide was selected for the Candida albicans killing test.

[0034] Example 1

[0035] Take 10 g of 30% hydrogen peroxide and add water to 100 g, the final concentration of hydrogen peroxide is 3%.

[0036] Example 2

[0037] Take 0.01 g of polypeptide powder and add water to 100 g, the final polypeptide concentration is 0.01%.

[0038] Example 3

[0039] Take 0.03 g of polypeptide powder and add water to 100 g, the final polypeptide concentration is 0.03%.

[0040] Example 4

[0041] Take 0.05 g of polypeptide powder and add water to 100 g, the final polypeptide concentration is 0.05%.

[0042] Example 5

[0043] A hydrogen peroxide compound disinfectant comprises, by mass fraction: 10 g of 30% hydrogen peroxide, 0.01 g of polypeptide powder, water is added to 100 g, the final concentration of hydrogen peroxide is 3%, and the final concentration of polypeptide is 0.01%.

[0044] Example 6

[0045] A hydrogen peroxide compound disinfectant comprises, by mass fraction: 10 g of 30% hydrogen peroxide, 0.03 g of polypeptide powder, water is added to 100 g, the final concentration of hydrogen peroxide is 3%, and the final concentration of polypeptide is 0.03%.

[0046] Example 7

[0047] A hydrogen peroxide compound disinfectant comprises, by mass fraction: 10 g of 30% hydrogen peroxide, 0.05 g of polypeptide powder, water is added to 100 g, the final concentration of hydrogen peroxide is 3%, and the final concentration of polypeptide is 0.05%.

[0048] Example 8 Test on the anti-Candida albicans performance of the combined disinfectant of hydrogen peroxide and polypeptide The bactericidal experiment was carried out in accordance with the provisions of 2.1.1.9 of the Technical Specification for Disinfection (2002).

[0049] Preparation of fungal suspension: Continuously subculture Candida albicans ATCC 10231 as required, take the fresh culture of the 4th generation of Sandburg agar medium slant (18h~24h), use a 5.0ml pipette to draw 3.0ml~5.0ml of dilution into the slant test tube, blow and aspirate repeatedly to wash off the fungal lawn. Then, use a 5.0ml pipette to transfer the washing liquid to another sterile test tube, mix it with an electric mixer for 20s, and adjust the OD value so that the corresponding bacterial liquid concentration of Candida albicans is 1×10 7 cfu / mL~5×10 7 cfu / mL as the test bacterial suspension.

[0050] A sterile 7 mL centrifuge tube was used for disinfection test. 0.5 mL of the test bacterial suspension was first added, and then 0.5 mL of the organic interfering substance was added. The mixture was mixed. After being placed in a 20°C ± 1°C water bath for 5 min, 4.0 mL of the compound disinfectant of Examples 1-7 was drawn with a sterile pipette and injected thereinto. The tubes were respectively recorded as test groups 1-7. The mixture was quickly mixed and the time was recorded immediately.

[0051] After the test bacteria and disinfectant have interacted for 20 minutes, take 0.5 ml of the mixture of test bacteria and disinfectant of each test group and add it to 4.5 ml of sterilized neutralizer and mix well.

[0052] After the mixed solution of test bacteria and disinfectant in each tube is treated with neutralizer for 10 minutes, 1.0 ml of sample solution is taken out respectively, and the number of surviving bacteria is determined by the live bacteria culture counting method. Each tube of sample solution is inoculated into 2 plates, and the culture counting is performed. If the number of colonies growing on the plate is large, a series of 10-fold dilutions can be performed, and then live bacteria culture counting can be performed. At the same time, the diluent is used instead of the disinfectant to perform a parallel test as a positive control, which is recorded as the control group.

[0053] All test samples were cultured in a 37°C incubator, and the final results of Candida albicans were observed for 48 hours. The test was repeated 3 times, and the viable bacterial concentration (cfu / ml) of each group was calculated and converted into a logarithmic value (N), and then the killing logarithmic value was calculated according to the following formula:

[0054] Killing logarithm (KL) = logarithm of the average viable bacteria concentration in the control group (No) - logarithm of the viable bacteria concentration in the test group (Nx).

[0055] After averaging the number of surviving colonies in each test group, the killing logarithm was calculated. The specific values ​​are shown in Table 1.

[0056] According to the provisions of 2.1.1.9.6 of the Disinfection Technical Specifications (2002), if the logarithmic killing value of Candida albicans is ≥4.00, it can be judged that the product has qualified for the disinfection of fungal contaminants.

[0057] Table 1 Determination of the anti-Candida albicans activity of hydrogen peroxide compound disinfectant in each embodiment

[0058]

[0059] As shown in Table 1, 3% hydrogen peroxide, 0.01% polypeptide, 0.03% polypeptide, and 0.05% polypeptide were used alone to kill Candida albicans for 20 minutes, and the killing effects were all unqualified; when 3% hydrogen peroxide was compounded with 0.01% polypeptide, 0.03% polypeptide, and 0.05% polypeptide, Candida albicans was killed for 20 minutes, the number of surviving colonies was 0, and the killing effects were all qualified. It can be seen that after different concentrations of polypeptides are compounded with 3% hydrogen peroxide, the killing effect of 3% hydrogen peroxide on Candida albicans can be effectively improved.

[0060] Example 9 Stability test of hydrogen peroxide and polypeptide compound disinfectant

[0061] The stability experiment was conducted by using a microbial assay method, and the compound disinfectants of Example 1, Example 2, and Example 5 were placed at room temperature for an initial time, 1 month, 3 months, and 6 months to determine their killing effects on Staphylococcus aureus.

[0062] Preparation of bacterial suspension: Staphylococcus aureus ATCC 6538 was cultured according to the preparation method of bacterial vegetative suspension in 2.1.1.2.3 of the Disinfection Technical Specifications (2002). The preliminary bacterial suspension was diluted to a concentration of 1×10 8 ~5×10 8 cfu / ml.

[0063] A 7 mL centrifuge tube was used for disinfection test. First, 0.5 mL of the test bacterial suspension was added, and then 0.5 mL of the organic interfering substance was added. The mixture was mixed. After being placed in a 20°C ± 1°C water bath for 5 min, 4.0 mL of the compound disinfectant of Example 1, Example 2, and Example 5 was drawn with a sterile pipette and injected thereinto. The tubes were recorded as Test Group 1, Test Group 2, and Test Group 3. The mixture was mixed quickly and the time was recorded immediately.

[0064] After the test bacteria and disinfectant have interacted for 20 minutes, take 0.5 ml of the mixture of test bacteria and disinfectant of each test group and add it to 4.5 ml of sterilized neutralizer and mix well.

[0065] After the mixed solution of test bacteria and disinfectant in each tube is treated with neutralizer for 10 minutes, 1.0 ml of sample solution is taken out respectively, and the number of surviving bacteria is determined by the live bacteria culture counting method. Each tube of sample solution is inoculated on 2 plates. If the number of colonies growing on the plate is large, a series of 10-fold dilutions can be performed, and then live bacteria culture counting can be performed. At the same time, the diluent is used instead of the disinfectant to conduct parallel tests as a positive control, which is recorded as the control group.

[0066] All test samples were cultured in a 37°C incubator, and the bacterial propagules were cultured for 48 hours to observe the final results. The test was repeated 3 times, and the viable bacterial concentration (cfu / ml) of each group was calculated and converted into a logarithmic value (N), and then the killing logarithmic value was calculated according to the following formula:

[0067] Killing logarithm (KL) = logarithm of the average viable bacteria concentration in the control group (No) - logarithm of the viable bacteria concentration in the test group (Nx).

[0068] When calculating the log kill value, take two decimal places and round off the numbers. See Table 2 for specific values.

[0069] According to the provisions of 2.1.1.7.7 of the Disinfection Technical Specifications (2002), if the logarithmic killing value of Staphylococcus aureus is ≥5.00, the disinfection can be judged as qualified.

[0070] Table 2 Determination of the stability of hydrogen peroxide compound disinfectant in each embodiment

[0071]

[0072]

[0073] As shown in Table 2, when 3% hydrogen peroxide is placed at room temperature for 6 months or more, the killing effect on Staphylococcus aureus is significantly reduced, and the killing effect is unqualified. The killing effect of 0.01% polypeptide alone on Staphylococcus aureus at room temperature is unqualified at different times; when 3% hydrogen peroxide is compounded with 0.01% polypeptide, the killing effect on Staphylococcus aureus is qualified at room temperature for 6 months, and the number of surviving colonies is 0, which is consistent with the initial bactericidal effect of 3% hydrogen peroxide. It can be seen that after 3% hydrogen peroxide is compounded with polypeptide, the stability of 3% hydrogen peroxide can be effectively improved.

[0074] Example 10 Cytotoxicity test of polypeptides on Vero cells Since the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous thiazolyl blue (MTT) to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its light absorption value can be measured at a suitable wavelength using an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells.

[0075] Collect cells in the logarithmic phase, adjust the concentration of the cell suspension, add 100 μL to each well, and plate the cells to be tested to a density of 1000-10000 / well. Incubate at 5% CO2, 37°C until the cell monolayer covers the bottom of the well (96-well flat-bottom plate), add 1 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM of peptides in different concentration gradients, and set up 5 replicate wells.

[0076] 5% CO2, incubate at 37℃ for 24 hours, and observe under an inverted microscope. Add 10μL MTT solution (5mg / mL, i.e. 0.5% (w / v) MTT) to each well, continue to culture for 4 hours, and carefully remove the culture medium in the well. Add 150μL DMSO to each well, and place it on an oscillator for 10 minutes at a low speed to fully dissolve the crystals. 490nm Measure the absorbance of each well.

[0077] At the same time, zero adjustment wells (culture medium, MTT and DMSO) and control wells (cells, peptide dissolution medium of the same concentration, culture medium, MTT, DMSO) were set.

[0078] The results are as follows Figure 1 As shown, at the concentrations of 1 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM, the relative cell proliferation rates were all greater than 80%.

[0079] Referring to the literature published by Wang Chen et al. (Wang Chen, Liao Xin, Wang Yanting, Fan Yuan, Liang Ruizhen. Biosafety evaluation of four disinfectants for DUWLs microbial contamination control [J]. Stomatology, 2019, 39(07): 596-600.), the 5-level toxicity evaluation standard grading table (Table 3) shows that the peptide has no cytotoxicity to Vero cells at a concentration of 100 μM.

[0080] Table 3 Cytotoxicity evaluation criteria

[0081] Relative cell proliferation rate / % (RGR / %) Cytotoxicity score 80-100 0 60-80 1 40-60 2 20-40 3 0-20 4

[0082] Comprehensively considered, after the hydrogen peroxide of the present invention is compounded with the polypeptide, the killing effect of hydrogen peroxide on Candida albicans is improved, and the stability of hydrogen peroxide is also improved. In addition, since hydrogen peroxide is an environmentally friendly disinfectant, and the polypeptide of the present invention is composed of 19 amino acids, it is non-cytotoxic and non-irritating, and the degradation product is a natural amino acid, and no drug residue is produced, and there is no irritation. The disinfectant compounded by the two is highly safe and biodegradable, which is conducive to the further expansion of the scope of application of hydrogen peroxide and has broad commercial application prospects.

[0083] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention. Sequence Listing <110> Jiangsu Jirui Biotechnology Co., Ltd. <120> A hydrogen peroxide compound disinfectant and its application <141> 2021-10-29 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Arg Phe Lys Arg Phe Arg Lys Lys Phe Lys Lys Leu Phe Lys 1 5 10 15 Lys Leu Ser

Claims

1. A hydrogen peroxide compound disinfectant, characterized in that The invention comprises: hydrogen peroxide and a polypeptide, wherein the amino acid sequence of the polypeptide is SEQ ID NO.

1.

2. The compound disinfectant according to claim 1, characterized in that The compound disinfectant includes other auxiliary materials.

3. The use of the hydrogen peroxide compound disinfectant according to claim 1 or 2 in disinfection, characterized in that The amino acid sequence of the polypeptide is SEQ ID NO.

1.

4. The use according to claim 3, characterized in that The hydrogen peroxide compound disinfectant can be added to daily-used products with bactericidal effects, including skin disinfectants and surface disinfectants.

5. The use according to claim 3, characterized in that The hydrogen peroxide compound disinfectant can be used on items that need to be disinfected, including medical devices and product packaging.

6. The use according to claim 3, characterized in that Application of the hydrogen peroxide compound disinfectant in the aseptic production process.

Images