Cationic antibacterial peptide compound preparation, anti-organic interference compound disinfectant and preparation method of cationic antibacterial peptide compound preparation and anti-organic interference compound disinfectant

Abstract

The invention provides a cationic antibacterial peptide compound preparation, an anti-organic interference compound disinfectant and a preparation method of the cationic antibacterial peptide compound preparation. The cationic antibacterial peptide compound preparation is obtained by culturing fermentation liquor through genetically engineered bacteria, purifying the fermentation liquor and adding a stabilizer. Effective disinfection components of the compound disinfectant resistant to organic interference are prepared by mixing the cationic antibacterial peptide compound preparation with an essential oil emulsifying preparation, a quaternary ammonium salt disinfectant and a guanidine disinfectant according to the mass ratio of the cationic antibacterial peptide compound preparation to the essential oil emulsifying preparation to the quaternary ammonium salt disinfectant to the guanidine disinfectant being (5-8): (4: 6): (4-5): (2-4). The prepared composite disinfectant can solve the problem that the titer of a conventional disinfectant is remarkably reduced in an organic matter highly-polluted environment, and compared with the conventional disinfectant, the composite disinfectant has the advantages that the influence of organic matter interference is remarkably reduced, the safety is high, and the composite disinfectant has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of disinfectants, and in particular relates to a composite disinfectant resistant to organic interference. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by the African swine fever virus (ASFV). It affects pigs of all breeds and ages, with morbidity and mortality rates reaching 100%. It is a contagious viral disease with a significant impact on domestic pigs.

[0003] Over the past few decades, numerous studies have been conducted worldwide on inactivated, live attenuated, and subunit vaccines for ASF. Despite significant progress, a safe and effective commercial ASF vaccine remains elusive. Several technical challenges remain in vaccine development: 1) ASFV is one of the largest known DNA viruses, and the identities and functions of most of its genes are unknown; 2) there are no readily available cell lines for cultivating ASFV for vaccine production; 3) ASFV has several genotypes with distinct phenotypic characteristics, and vaccines tested to date offer little or no cross-protection; 4) although African wild boars are known to possess considerable resistance to ASFV, a vaccine is still needed that can be administered orally to domestic pigs and wild boars. To date, attempts to induce protective immunity using various vaccine platforms have failed. Pigs that survive infection with ASFV are only resistant to the specific virus they were infected with. Therefore, establishing strict biosafety measures is paramount.

[0004] African swine fever virus is the only member of the genus Asfrigivirus in the family Assurviridae. It is a large (200nm), complex, enveloped, double-stranded DNA virus. The genome size is approximately 190Kb, and its genome structure and replication strategy are similar to those of other large double-stranded DNA viruses (including the Poxviridae, Iridoviridae, and Phycodnaviridae). ASFV has a wide temperature stability and is extremely resistant to corruption and drying. It is also highly resistant to certain chemicals (such as trypsin and EDTA) and physical treatments (such as freeze / thaw and ultrasound). It also has strong acid and alkali tolerance. Studies have shown that the virus has a wide pH adaptability range and can survive stably for more than 7 days at a pH of 3.9 to 13.4 (in a culture medium containing serum).

[0005] Due to the lack of vaccines and effective drugs, once farms are infected with ASF virus, large-scale pig deaths will occur. Therefore, using effective disinfectants to effectively disinfect infected places, vehicles, equipment and pollutants is an extremely important measure to prevent ASF contamination.

[0006] Multiple authoritative publications recommend highly effective, broad-spectrum, and convenient disinfectants for ASFV, including commercial disinfectants. These recommendations include chlorine-containing disinfectants, chlorine dioxide, peracetic acid, formaldehyde, potassium persulfate, iodine compounds (iodine tincture, iodine tincture), phenols, alcohol-based quaternary ammonium salts, and guanidines. Many commercial disinfectants based on these compounds are also recommended for ASFV. These disinfectants are suitable for disinfecting buildings, wooden structures, concrete surfaces, vehicles, and related facilities and equipment. For pig farms, it is crucial to follow strict disinfection procedures, including disinfection of vehicles, personnel, objects, empty sheds, occupied pens, roads and areas around sheds, carcass collection points, and pig delivery platforms. Highly effective disinfectants should also be used to eliminate areas of contact between personnel and animals. However, years of practical application have shown that many common disinfectants are ineffective, emphasizing the importance of using disinfectants specifically approved for ASFV use. Particular attention should be paid to the testing methods and scenarios for the effective concentrations of these disinfectants, ensuring that they are consistent with actual farming environments. It is also necessary to pay attention to whether the test data of these disinfectants contain complete information such as pollutants, action time, application range, and incompatibility.

[0007] In the actual clinical disinfection implementation process, even the use of disinfectants targeting ASF virus may deviate from the effect. One of the important influencing factors is that the presence of organic matter in the environment offsets the effect of the disinfectant. That is, in the presence of organic matter such as blood and feces, the virus is more stable and survives longer.

[0008] JIANG Cheng-gang et al. evaluated six commonly used disinfectants (iodic acid compound, compound potassium persulfate, citric acid, compound glutaraldehyde decanediamine bromide and decanediamine bromide). The effective concentrations used were almost the same as those recommended by the manufacturers, but the disinfectants did not show obvious effects in the case of organic contamination.

[0009] Juszkiewicz studied eight disinfectants (formaldehyde, sodium hypochlorite, sodium hydroxide, glutaraldehyde, phenol, benzalkonium chloride, potassium peroxymonosulfate, and acetic acid) based on recommendations from the World Organization for Animal Health (OIE). The results showed that these disinfectants could inactivate viruses at recommended concentrations. However, cleaning prior to disinfection is crucial, as the effectiveness of disinfectants is significantly reduced in the presence of organic contamination. Therefore, the authors strongly recommend that pre-disinfection cleaning be performed to improve effectiveness.

[0010] M. Juszkiewicz evaluated four commercial disinfectants (sodium hypochlorite, oxidants, glutaraldehyde, and quaternary ammonium salts) using low and high concentrations of organic pollutants for testing. The low concentration of pollutants was 3.0 g / L of bovine serum, and the high concentration of pollutants was 10 g / L of bovine serum and 10 g / L of yeast extract. The results of the study again showed the importance of removing organic pollutants. Flushing can significantly improve the effectiveness of the commercial disinfectants evaluated.

[0011] Peter W. Kruga's research shows that the activity of disinfectants is greatly inhibited in the presence of blood, meat juice, and feces. This research also shows that blood strongly inhibits existing disinfectants, rendering them ineffective against ASFV or FMDV.

[0012] Studies in multiple academic literatures have shown that low- and high-concentration organic contamination conditions have a wide range of effects on various disinfectants. Most researchers recommend that effective cleaning should be performed before disinfection to remove residual organic pollutants.

[0013] Van Phan Le's disinfectant research employed the EU standard EN 14765:2015 for contaminant testing, using concentrations of 3-10g / L bovine serum albumin or 10g / L yeast extract as interfering substances (proteins). The results indicate that removing dirt or organic matter from surfaces during the pre-disinfection cleaning step is crucial for successful sanitation.

[0014] Maria Serena Beato listed the standards for testing the effectiveness of disinfectants, [UNI EN 14675:2015] and [ASTM E1053-20]. The concentration of organic interferents in the [UNI EN 14675:2015] standard disinfectant is 1% BSA + 1% YE or 0.3% BSA, and the concentration of organic interferents in the [ASTM E1053-20] standard disinfectant is 5% YE + 5% BSA + 5BM (note: YE yeast extract, BSA bovine serum, BM bovine mucin). These results show that the potency of common single-component disinfectants is significantly reduced by 50-90% in the presence of standard organic interferents.

[0015] Andrew D. Wales made a detailed classification of the contamination degree of organic interfering substances in disinfectant research. These classifications can serve as a good reference and basis for studying the true efficacy of disinfectants in actual application scenarios.

[0016] Some commonly used and successful commercial disinfectants target ASFV. The disclosed main ingredients include potassium persulfate, sodium chloride, organic acid, inorganic buffer, and anionic surfactant. It is primarily an oxidizing disinfectant. The mixture of bactericidal ingredients is acidic and enhanced by hypochlorous acid chemically generated within the product. Therefore, it can be assumed that in actual use, environmental disinfection after thorough cleaning can effectively kill ASFV. However, in the presence of high levels of organic matter, the mechanism of action of the main ingredients of this disinfectant determines that they will undergo a large amount of neutralization reaction with organic matter, thereby reducing the actual killing effect.

[0017] The phenomenon that the potency of most disinfectants decreases in the presence of organic matter interference is directly related to their disinfection mechanism and basic chemical properties: aldehyde disinfectants bind to proteins and inhibit transport mechanisms; hypochlorite disinfectants can penetrate cell membranes and oxidize proteins, interrupting cellular oxidative phosphorylation; peroxides can penetrate cell membranes and oxidize lipids, proteins and DNA; alkalis destroy cell membrane permeability and denature proteins.

[0018] From the analysis and summary of the above-mentioned literature research results and the mechanism of action of common disinfectants, it can be confirmed that most disinfectants will undergo neutralization reactions with organic matter in actual application scenarios, and only after the neutralization of the remaining part will they have a killing effect on viruses and bacteria. Therefore, effective anti-interference disinfectants should be considered to still be effective in the presence of organic matter, and effective anti-interference disinfectants should not react with proteins to the greatest extent and thus be consumed.

[0019] Fortunately, there are some drugs in the field of blood products and biomedicine that have both effects. They can, to a certain extent, not react with organic matter such as proteins, thereby allowing most of the ingredients of the drug to act directly on virus and bacterial cells, thereby inhibiting and killing bacteria and viruses. Typical chemical reagents include β-propiolactone, plant essential oils and antimicrobial peptides.

[0020] β-Propiolactone (CAS: 57-57-8) is a four-membered ring lactone organic compound with the molecular formula CH2CH2CO2. It is a colorless liquid that is highly soluble in water and organic solvents. It is highly reactive and readily hydrolyzes, reacting with hydroxyl, amino, carboxyl, sulfhydryl, and phenolic groups. β-Propiolactone is widely used in the inactivation of various vaccines. It does not interfere with the antigenicity of vaccines and has been used to inactivate influenza viruses. β-Propiolactone is also used to sterilize surgical instruments, plasma, tissue grafts, milk, water, nutrient solutions, and enzymes. Its sporicidal properties also make it useful against bacteria, pathogenic fungi, and viruses. β-Propiolactone's advantages over traditional virus inactivation agents include: 1) complete hydrolysis within 2 hours at 37°C; 2) the hydrolysis products are non-toxic and non-carcinogenic; and 3) it inactivates by interacting with DNA or RNA, rather than directly with proteins, maintaining high immunogenicity. Based on this, the International Serum Industry Association (ISIA) uses β-propiolactone as a viral inactivation method in combination with irradiation in fetal bovine serum products. The use of β-propiolactone does not compromise the potency of the active proteins in the serum. However, due to its high toxicity and the need for low-temperature storage, β-propiolactone is not suitable as a disinfectant raw material. Therefore, there is a need to identify other agents based on the same principle but with lower toxicity and better established properties that could serve as raw materials for disinfectants resistant to organic interference.

[0021] Essential oils are volatile aromatic compounds composed of chemical molecules such as aldehydes, phenols, and alcohols. They are extracted from the flowers, leaves, roots, bark, fruits, seeds, and resins of herbal plants through distillation and pressing. Most essential oils possess antibacterial, antifungal, and antiviral properties, effectively inhibiting pathogens and reducing the number of enteric pathogens. Therefore, they are widely used as a novel feed additive to replace antibiotics. The antimicrobial activity of essential oils is determined by their chemical composition and concentration, with phenols, oxygenated terpenes, and terpenes exhibiting strong antimicrobial activity. The antimicrobial mechanisms of essential oils primarily involve alterations in the fatty acid envelope, damage to the cytoplasmic membrane, depletion of proton motive force, and metabolite and ion leakage. As natural plant antimicrobial agents with low toxicity and no residual residue, essential oils can be used as feed additives to maintain animal health and hold promise as an important alternative to antibiotics. Commonly used plant essential oils include cinnamon oil, clove oil, thyme oil, eucalyptus oil, oregano oil, and rosemary oil. Due to their unique mechanism of action, plant essential oils are also suitable as alternative raw materials for antibacterial and antiviral disinfectants because they contain a large amount of organic interfering substances in the environment where they work (on the surface of plants and animals or when taken orally).

[0022] Antimicrobial peptides, also known as antimicrobial peptides, are a class of small peptides that have the ability to defend against external microbial invasion and eliminate mutated cells in the body. They are widely present in organisms. They were first isolated from the pupae of the insect hyatophoracecropia by Swedish scientist Boman et al. in 1981. Since their discovery in the 1980s, over 3,000 antimicrobial peptides have been identified, dozens of which have entered clinical trials, and some have demonstrated promising therapeutic effects. These antimicrobial peptides share common characteristics: 1) a broad antimicrobial spectrum, targeting bacteria, fungi, parasites, some viruses, and tumor cells; 2) rapid action, killing microorganisms within 10 minutes; 3) most are non-toxic or low-toxic to eukaryotic cells, and most act through the bacterial cell membrane; 4) small molecular weight, mostly composed of 6-50 amino acid residues; 5) primarily cationic, with a small amount of anionic, exhibiting amphiphilic / lipophilic properties; 6) modified by glycosylation and carboxyl-terminal amidation, with a small portion formed by protease action; 7) secretory expression, rapid induction, and favorable for genetic engineering expression; 8) good thermal stability. The well-established mechanisms of action of antimicrobial peptides include disrupting bacterial membranes, interfering with bacterial metabolism, or directly acting on cytoplasmic components. Cationic antimicrobial peptides, in particular, exhibit membrane disruption mechanisms. Crucially, antimicrobial peptides possess a selective killing mechanism. This is due to the specificity of prokaryotic and eukaryotic cell membrane lipids, as well as differences in their composition and arrangement. Mammalian cell membrane lipids are neutrally charged amphoteric phospholipids, including phosphatidylcholine and sphingomyelin, while bacterial cell membranes contain a large number of negatively charged phospholipids. In short, this simple charge difference between prokaryotic and bacterial cell membranes is the key to the selective killing ability of antimicrobial peptides and their near-tolerance to normal eukaryotic cells. Antimicrobial peptides are not only used in medical applications but are also widely used in food as preservatives and as an alternative to antibiotics in animal feed. While there are few reports on the use of purified antimicrobial peptides as disinfectant raw materials, their structural characteristics and mechanism of action make them excellent raw materials for highly safe disinfectants with high resistance to organic interference.

[0023] Existing conventional single-ingredient disinfectants and commercial compound disinfectants are highly susceptible to organic pollutants, resulting in significant reductions in effectiveness in actual applications. The effectiveness of most disinfectants is incompletely validated, and actual efficacy data under highly contaminated conditions is not considered. Most existing conventional disinfectants are irritants or highly oxidizing, making them unsuitable for active direct disinfection. In particular, the range of options for direct contact disinfection of animal surfaces, feeding troughs, pipes, etc. is limited, resulting in poor safety. Summary of the Invention

[0024] The first object of the present invention is to provide a cationic antimicrobial peptide compound preparation suitable for use as an important reagent in an anti-organic interference compound disinfectant.

[0025] A cationic antimicrobial peptide compound preparation for preparing a composite disinfectant resistant to organic interference, wherein the cationic antimicrobial peptide is obtained by an Escherichia coli genetic engineering bacterial expression system, and the amino acid sequence of the cationic antimicrobial peptide is shown in Sequence 1:

[0026] Arg Arg Lys Asn Ser His His Thr Arg Met Gly Cys Tyr Gly Pro Leu GluTrp Ala Ile Asp Pro Ala Val Gln Phe,

[0027] The cationic antimicrobial peptide compound preparation is obtained by culturing a fermentation broth of genetically engineered bacteria, purifying it, and then adding a stabilizer. The purification process is sequentially carried out through pretreatment, centrifugal separation, ceramic membrane filtration, and reverse osmosis membrane concentration. The pretreatment adopts a high-pressure homogenization or aeration flocculation method.

[0028] Specifically, the stabilizer added to the cationic antimicrobial peptide compound formulation includes a glycine-sodium hydroxide buffer solution, the concentration of which after addition in the cationic antimicrobial peptide compound formulation is 0.05 mol / L and the pH is 9.5. The stabilizer also includes arginine and lysine. After addition, the mass percentage concentration of arginine in the cationic antimicrobial peptide compound formulation is 0.5% to 2%, and the mass percentage concentration of lysine in the cationic antimicrobial peptide compound formulation is 0.5% to 2%.

[0029] Furthermore, the stabilizer also includes isopropyl alcohol. After addition, the mass percentage concentration of isopropyl alcohol in the cationic antimicrobial peptide compound preparation is 4% to 10%, preferably 5% to 6%.

[0030] The second purpose of the present invention is to make up for the shortcomings of existing disinfectants and provide a high-efficiency disinfectant with strong anti-organic interference ability and high safety against African swine fever virus.

[0031] In order to achieve the above object, the present invention adopts the following technical scheme: a composite disinfectant resistant to organic interference, wherein the disinfection active ingredient is prepared by mixing the cationic antimicrobial peptide composite preparation according to any one of claims 1 to 4, an essential oil emulsified preparation, a quaternary ammonium salt disinfectant, and a guanidine disinfectant in the following mass parts: cationic antimicrobial peptide composite preparation: essential oil emulsified preparation: quaternary ammonium salt disinfectant: guanidine disinfectant = (5-8): (4:6): (4-5): (2-4),

[0032] The essential oil emulsion preparation is prepared by emulsifying the essential oil with a selected emulsifier. The essential oil is a plant essential oil with a bactericidal function. The essential oil mixture is prepared by mixing eucalyptus oil, thyme and eugenol in the ratio of eucalyptus oil:thyme:eugenol in parts by weight of (3-4): (2.5-3): (1-2). The selected emulsifier is a composite emulsifier prepared by mixing one or both of cetyltrimethylammonium chloride and cetyltrimethylammonium bromide. The essential oil mixture is added to the composite emulsifier, and the solution is continuously stirred at a temperature of 25-30°C until it is completely emulsified. The mass percentage of the essential oil mixture accounts for 6%-10% of the essential oil emulsion preparation.

[0033] Preferably, the mixing ratio of cetyltrimethylammonium chloride and cetyltrimethylammonium bromide is preferably 6:4 in parts by mass.

[0034] Preferably, the preferred mixing ratio of eucalyptus oil, thyme and eugenol is, by mass, eucalyptus oil: thyme: eugenol = 4:2.5:1.5, and the preferred addition amount of the essential oil mixture and the compound emulsifier by mass percentage is, by mass, essential oil mixture: compound emulsifier = 8:92.

[0035] Preferably, the composite disinfectant mixed solution contains, by mass, 0.5% to 0.8% of carbamide, 0.8% to 1.0% of Triton X-100, 0.4% to 0.6% of ethylphenyl polyethylene glycol and 1.5% to 2.0% of polyethylene glycol.

[0036] Specifically, the quaternary ammonium salt disinfectant includes a mixture of one or more of benzyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, C14-C16 alkylbenzyldimethylammonium chloride, and didecyldimethylammonium bromide.

[0037] Specifically, the guanidine disinfectant includes polyhexamethylene biguanidine hydrochloride or polyhexamethylene guanidine hydrochloride, or a mixture of the two.

[0038] As a preferred embodiment, the composite disinfectant mixed solution is prepared by mixing the following reagents in parts by mass: 5% to 8% of a cationic antimicrobial peptide compound preparation, 4% to 6% of an essential oil emulsified preparation, 0 to 1.0% of benzyltrimethylammonium chloride, 0.5% to 1.5% of hexadecyltrimethylammonium bromide, 1.0% to 2.0% of C14 to C16 alkylbenzyldimethylammonium chloride, 1.0% to 2.0% of didecyldimethyl bromide. Ammonium, 1.0% to 2.5% of polyhexamethylene biguanide hydrochloride and 1.0% to 2.0% of polyhexamethylene guanidine hydrochloride, 0.5% to 0.8% of carbonamide, 0.8% to 1.0% of Triton X-100, 0.4% to 0.6% of ethylphenyl polyethylene glycol and 1.5% to 2.0% of polyethylene glycol, 15.0% to 20.0% of isopropyl alcohol, 5.0% to 10.0% of ethanol, and the balance is water.

[0039] The third object of the present invention is to provide a method for preparing a composite disinfectant resistant to organic interference, which comprises the following steps:

[0040] (1) Deionized water is injected into the reactor tank and the water temperature is controlled to 20-35°C; organic solvents such as isopropyl alcohol and ethanol are added and mixed thoroughly;

[0041] (2) preparing a cationic antimicrobial peptide preparation and adding it to the reactor tank, stirring thoroughly until mixed;

[0042] (3) preparing an essential oil emulsion and adding it to the reactor tank, stirring thoroughly until mixed;

[0043] (4) Add quaternary ammonium disinfectant and stir thoroughly until mixed;

[0044] (5) Add guanidine disinfectant and stir thoroughly until mixed;

[0045] (6) Adding additives and cracking agents;

[0046] (7) After the materials are fully stirred, they are aseptically packaged and poured into a sterile plastic barrel of a certain volume and sealed for storage.

[0047] The composite antimicrobial peptide preparation of the present invention can be used to prepare disinfectants after purification, avoiding the problem of precipitation and flocculation of impurities such as bacterial proteins, ensuring high potency of the disinfectant. Furthermore, the purified antimicrobial peptides are stabilized by the addition of stabilizers and buffer salts. The essential oil emulsified preparation of the present invention is prepared by emulsifying essential oils with a selected emulsifier that does not antagonize the antimicrobial peptides, quaternary ammonium salts, and biguanides in the disinfectant. The resulting composite disinfectant can address the significant reduction in potency of conventional disinfectants in environments highly contaminated by organic matter. Compared with conventional disinfectants, it is significantly less susceptible to organic interference and is safer, offering promising application prospects. DETAILED DESCRIPTION

[0048] The various strains used in the embodiments of this specification are commercially available, for example, but not limited to, purchased from the China National Institute for the Control of Biological Products.

[0049] Example 1

[0050] This example is an example of preparing a composite disinfectant that is resistant to organic interference, and it is carried out according to the following steps:

[0051] (1) Deionized water is injected into the reactor tank and the water temperature is controlled to 20-35°C; organic solvents such as isopropyl alcohol and ethanol are added until fully mixed; the reactor tank needs to be made of non-metallic materials, such as PE plastic or ceramic materials, and the stirring device in the tank needs to be made of polytetrafluoroethylene or ceramic materials. The reaction tank has heating and heat preservation functions.

[0052] (2) Preparation of cationic antimicrobial peptide preparations;

[0053] The cationic antimicrobial peptide of this embodiment is derived from an Escherichia coli genetically engineered bacterial expression system. The amino acid sequence of the cationic antimicrobial peptide is: Arg Arg Lys Asn Ser His His Thr Arg Met Gly Cys Tyr Gly Pro LeuGlu Trp Ala Ile Asp Pro Ala Val Gln Phe. The Escherichia coli genetically engineered bacteria are purchased commercially, for example, from the China National Institute for the Control of Biological Products.

[0054] (2.1) The genetically engineered bacteria are cultured in three stages after seed culture medium, fermentation tank culture medium, and bacterial activation, and then gradually expanded to a 5 cubic meter culture tank for cultivation.

[0055] (2.2) After fermentation is complete, separation and purification are performed to remove impurities such as bacterial proteins and organic solids to improve the purity, solubility, and potency of the antimicrobial peptides. The purification process is as follows: pretreatment - high-speed centrifugation - ceramic membrane filtration - reverse osmosis membrane concentration - and standby use.

[0056] The pretreatment is high-pressure homogenization, in which the antimicrobial peptide fermentation stock solution is pumped into a high-pressure homogenizer for wall breaking, with a homogenization pressure of more than 60 MPa. The pretreatment can also be aeration flocculation, in which the antimicrobial peptide fermentation stock solution is pumped into a tank equipped with an aeration device and aerated continuously, with the following parameters: the feed liquid temperature is 25-45°C, the gas is industrial-grade carbon dioxide, and the aeration time is 20-50 minutes.

[0057] The pre-treated material is separated into solid and liquid by a high-speed disc centrifuge, and the supernatant is collected. The centrifugal speed is 3000-8000rpm, and the equivalent separation factor of the disc centrifuge is 5000-12000ω2r / g.

[0058] Ceramic membrane filtration: The supernatant after centrifugation is further processed through a ceramic membrane device and the supernatant is collected.

[0059] Reverse osmosis membrane concentration: The material is pumped into the reverse osmosis membrane concentration equipment for concentration treatment to make the material concentration reach more than 10%.

[0060] The purified cationic antimicrobial peptide liquid was tested for antimicrobial potency using a 96-well plate format, using Escherichia coli as the standard test target. A potency exceeding the MIC of 8 μg / ml was considered acceptable. The cationic antimicrobial peptide of the present invention had a minimum inhibitory concentration (MIC) of 10 μg / ml against Staphylococcus aureus, 10.0 μg / ml against Escherichia coli, and 12 μg / ml against Salmonella. The purified liquid was free of precipitates and impurities.

[0061] Qualified antimicrobial peptide concentrates need to be added with buffer salt, which is 0.05 mol / L glycine-sodium hydroxide buffer, and the pH after adding the buffer is 9.5.

[0062] Arginine, lysine and isopropanol are added as stabilizers. After addition, the mass content of the cationic antimicrobial peptide preparation is 1.5% of arginine, 2.0% of lysine and 5% of isopropanol.

[0063] The prepared cationic antimicrobial peptide preparation has a concentration of 10%, is added into the reactor tank, and is fully stirred until mixed for use in preparing a disinfectant.

[0064] (3) Preparation of essential oil emulsified preparations

[0065] Plant essential oils can be extracted from plants or purchased commercially. Plant essential oil extraction methods can be conventional or known methods in the art. In this example, commercially available eucalyptus oil, thyme, and eugenol were selected as essential oil raw materials, and cetyltrimethylammonium chloride and cetyltrimethylammonium bromide were selected as composite emulsifiers.

[0066] The essential oil ratio is shown in Table 1 below (in parts by mass):

[0067] Table 1

[0068] Reagent name Recipe A Recipe B Recipe C Eucalyptus oil 4 4.0 3.0 Thyme scent 2.5 3.0 3.0 Eugenol 1.5 1.0 2.0

[0069] The proportions of the compound emulsifier are shown in Table 2 below (in parts by mass):

[0070] Table 2

[0071]

[0072] The cetyltrimethylammonium chloride solution and the cetyltrimethylammonium bromide solution were fully mixed in the proportions shown in Table 2 above to prepare a composite emulsifier, and the solvent was deionized water.

[0073] Gradually add a blend of eucalyptus oil, thyme, and eugenol to the compounded emulsifier, with the total amount of essential oils accounting for 8% of the total mass of the essential oil emulsion. Slowly add the essential oils to the emulsifier, stirring continuously and maintaining the solution at 25-30°C until fully emulsified. The prepared emulsified essential oil preparation is set aside for use in disinfectant preparation.

[0074] (4) Add benzyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, C14-C16 alkylbenzyldimethylammonium chloride, and didecyldimethylammonium bromide, and stir thoroughly until mixed;

[0075] (5) Add polyhexamethylene biguanidine hydrochloride and polyhexamethylene guanidine hydrochloride and stir thoroughly until mixed;

[0076] (6) adding the cleavage agent (Triton X-100, ethylphenyl polyethylene glycol) and the auxiliary agent (carbonamide, polyethylene glycol) in sequence;

[0077] (7) After the materials are evenly mixed, they are aseptically packaged and poured into a sterile plastic barrel of a certain volume and sealed for storage.

[0078] The compound disinfectant is prepared according to the proportions in Table 3:

[0079] Table 3

[0080]

[0081]

[0082] Example 2

[0083] In this example, the modified broth microdilution method was used to test the bactericidal activity of the disinfectant to evaluate the bactericidal activity of the composite disinfectant of the present invention. The composite disinfectant in this example was prepared using a composite cationic antimicrobial peptide formulation A and a composite emulsifier formulation B according to composite disinfectant formulation 1. The bactericidal activity of the disinfectant was determined according to the following steps:

[0084] The strain frozen at -80°C was thawed in ice, streaked onto MH agar plates with an inoculation loop, and cultured at 37°C to form single colonies; the single colonies were inoculated into fresh MH broth medium and cultured overnight at 37°C for activation; the overnight culture suspension was transferred to fresh MH broth medium at a ratio of 1:100, and cultured at 37°C with constant temperature and shaking at 250 rpm until OD600nm = 0.5; 10 μL of the bacterial suspension with OD600nm = 0.5 was added to 10 mL of fresh MH broth medium and vortexed to mix thoroughly, and the bacterial count was adjusted to 5×105 ~1×10 6 CFU / mL, used for determination of minimum inhibitory concentration (MIC).

[0085] Add 90 μL of the prepared bacterial suspension to each of the first through eighth wells of a sterile 96-well round-bottom culture plate. Add 100 μL of the bacterial suspension to the eleventh well. Add 100 μL of MH broth to the twelfth well as a negative control. Add 10 μL of serially diluted disinfectant to each of the first through eighth wells. The eleventh well serves as a positive control and does not receive disinfectant. The plate is incubated at 37°C for 18–24 hours to maintain humidity. After incubation, observe the bottom of each well for bacterial precipitation. The minimum concentration at which no bacterial precipitation is visible is the MIC of the antimicrobial peptide. The MIC is the lowest concentration of an antimicrobial substance that completely inhibits bacterial growth. 10 μL of the contents from each well without bacterial growth is plated onto MH agar plates in triplicate. After complete absorption, the plate is incubated at 37°C for 18–24 hours. The presence of bacterial growth is then used to determine the minimum bactericidal concentration (MBC). The MBC is the lowest concentration of an antimicrobial substance that prevents the formation of colonies by the remaining bacteria. The test results are shown in Table 2. In the table, the smaller the MIC and MBC values, the stronger the antibacterial ability.

[0086] Table 4 below shows the measured minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the diluted disinfectant against several bacteria.

[0087] Table 4

[0088]

[0089]

[0090] Note: The disinfectant in this example is diluted 200 times

[0091] As can be seen from the above table, this disinfectant can kill common harmful bacteria in the aquaculture field and has basic bactericidal ability.

[0092] Example 3

[0093] This example is a quantitative killing test of the disinfectant of the present invention on a multi-drug resistant Pseudomonas aeruginosa suspension.

[0094] This example tests the killing effect of the disinfectant prepared in the example on multidrug-resistant Pseudomonas aeruginosa in accordance with the 2002 edition of the Technical Specification for Disinfection.

[0095] 1. Neutralizer identification test:

[0096] At a constant temperature of 20°C, D / E neutralizing broth was used as a neutralizing agent. The disinfectant prepared according to the composite cationic antimicrobial peptide preparation formula A of Example 1 and the composite emulsifier formula B according to the composite disinfectant formula 1 was tested three times with an action time of 0.5 minutes. The identification results are shown in Table 5:

[0097] Table 5 Neutralizer identification test results

[0098]

[0099] Group 1: Microorganisms + disinfectant, no colonies, indicating that the disinfectant is effective in killing bacteria

[0100] Group 2: Microorganisms + neutralizers, showing low levels of growth, indicating that the neutralizer is non-toxic to the bacteria

[0101] Group 3: Microorganisms + disinfectant + neutralizer, showing growth, indicating that the neutralizer effectively neutralized the disinfectant

[0102] Group 4: Microorganisms + culture medium (positive control), showing a large amount of growth in the positive control group

[0103] Group 5: Neutralizer + Disinfectant, indicating that the neutralizer effectively neutralized the disinfectant

[0104] Group 6: Culture medium blank (negative control), showing sterile state, ensuring no contamination. Conclusion: From the above test results, it can be seen that under a constant temperature of 20°C, the D / E neutralization broth neutralizer can effectively neutralize the residual effect of the example disinfectant on multidrug-resistant Pseudomonas aeruginosa, and the neutralizer and its neutralization product solution have basically no effect on the growth of multidrug-resistant Pseudomonas aeruginosa.

[0105] 2. Test on the killing effect of multidrug-resistant Pseudomonas aeruginosa: The test temperature was constant at 20°C. The disinfectant prepared according to the composite cationic antimicrobial peptide preparation formula A of Example 1 and the composite emulsifier formula B was used. The action time was 2.5 minutes, 5.0 minutes and 7.5 minutes. The test was repeated 3 times. The test results are shown in Table 6 below:

[0106] Table 6 Killing effect on multi-drug resistant Pseudomonas aeruginosa

[0107]

[0108] Note: No bacteria grew in the negative control group. Conclusion: From the above test results, we can see that the average log kill values ​​of the disinfectant prepared using Formula 1 of Example 1 at different time points (2.5, 5.0, and 7.5 minutes) were all ">5.00", indicating that the bactericidal effect reached or exceeded the log kill value of 5.0 (i.e., 99.999% kill rate) in the shortest time of 2.5 minutes, indicating that the killing effect was completed at an early stage. In other words, the disinfectant was "very effective" at 2.5 minutes.

[0109] Example 4

[0110] This example is a test example of the disinfection effect of different disinfectants on ASF virus under organic interference.

[0111] There are a large number of organic interfering substances (such as feed, blood, feces, etc.) in the actual application environment. The use of standardized and scientific organic interference tests can effectively evaluate the actual application effect of disinfectants. The EU EN 14765:2015 standard is used to evaluate the virucidal activity of chemical disinfectants and preservatives in the veterinary field to test the effectiveness of disinfectants in feeding and production environments. This method uses standard concentrations of organic interfering substances to test the potency of disinfectants. The specific operation of this method is to add the disinfectant sample to the solution of virus suspension and interfering substances, and interact within a specified contact time. The reaction is terminated with a neutralization solution, and the TCID50 assay is used to measure the virus infectivity.

[0112] Aiming at the actual breeding and production environment of African swine fever virus, the present invention improves the test method for organic interference of African swine fever virus disinfectants based on the EN 14765 standard method, which is used to evaluate the disinfection effect of different disinfectants under the condition of a certain concentration of African swine fever positive organic interferents. The most commonly used fluorescent quantitative PCR in China is used as a detection tool for African swine fever virus in blood samples to replace the TCID50 method to improve accuracy and shorten the detection cycle. The specific operation is as follows:

[0113] 1. Preparation of Positive Swine Serum: Two positive pig blood samples were collected from a slaughterhouse known to have tested positive for ASFV. The ASFV Ct values ​​were determined using fluorescent PCR to be 26.31 and 26.31, respectively. The red blood cells and fibrinogen were removed to obtain the standard positive pig serum samples, which were then stored for future use.

[0114] 2. Preparation of positive low-level pollutants: Dilute the positive pig serum sample with a Ct value of 26.31 with deionized water to a serum dry matter concentration of 3.0 g / L and store for later use.

[0115] 3. Preparation of high-level pollutants: Prepare a mixture of positive pig serum and yeast extract with deionized water to a concentration of 3.0 g / L pig serum and 10.0 g / L yeast extract, and store it for later use.

[0116] 4. The non-swine fever disinfectant prepared according to the composite cationic antimicrobial peptide preparation formula A and the composite emulsifier formula B of Example 1 of this patent according to the composite disinfectant formula 1 is diluted with commercially available disinfectants (chlorine dioxide, glutaraldehyde, potassium persulfate, guanidine disinfectants) using deionized water to 0.10% according to the effective concentration of the disinfectant and set aside.

[0117] 5. Using the quantitative suspension method, place 10.0 ml of the organic interfering substance and 5.0 ml of the diluted test disinfectant in a culture dish. Incubate for 30 minutes, then collect the centrifuged supernatant to determine the sample Ct value. Compare the results with the initial positive sample. Each test group is replicated in two replicates. The test results are shown in Table 7 below:

[0118] Table 7 Fluorescence PCR results of composite disinfectants

[0119]

[0120] Low organic interference group: positive pig serum with a serum dry matter concentration of 3.0 g / L

[0121] High organic interference group: a mixture of positive pig serum with a serum dry matter concentration of 3.0 g / L and 10.0 g / L yeast extract

[0122] analyze:

[0123] The measured data showed that the commonly used disinfectants chlorine dioxide, glutaraldehyde, potassium persulfate, and polyhexamethylene biguanide hydrochloride were unable to effectively kill the African swine fever virus under low and high concentrations of organic interference. These disinfectants will produce a strong binding reaction with proteins, resulting in flocculation and coagulation of organic interference substances. The disinfectant components are consumed by a large amount of neutralization, resulting in a significant reduction in the components that disinfect the African swine fever virus. From the results of fluorescent PCR, the composite disinfectant prepared by Formula 1 in Example 1 showed complete inhibition of NoCt in the low organic interference group, and the Ct value in the high organic interference group increased significantly, indicating that the disinfectant has excellent viral nucleic acid destruction ability and resistance to organic interference. Compared with conventional disinfectants, the reason why the disinfectant of the present invention can effectively resist organic interference is that it uses materials that do not react violently with organic substances such as proteins as the main components of the disinfectant. Therefore, in actual application, the disinfectant of this patent can effectively kill African swine fever virus under the interference conditions of a large amount of organic matter (feces, feed, blood, etc.).

Claims

1. A cationic antimicrobial peptide compound preparation for preparing a composite disinfectant resistant to organic interference, wherein the cationic antimicrobial peptide is obtained by an Escherichia coli genetic engineering bacterial expression system, and the amino acid sequence of the cationic antimicrobial peptide is shown in Sequence 1, Sequence 1: Arg Arg Lys Asn Ser His His Thr Arg Met Gly Cys Tyr Gly Pro Leu Glu TrpAla Ile Asp Pro Ala Val Gln Phe, The cationic antimicrobial peptide compound preparation is obtained by culturing a fermentation broth of genetically engineered bacteria, purifying it, and then adding a stabilizer. The purification process is sequentially carried out through pretreatment, centrifugal separation, ceramic membrane filtration, and reverse osmosis membrane concentration. The pretreatment adopts a high-pressure homogenization or aeration flocculation method.

2. The cationic antimicrobial peptide compound formulation according to claim 1, characterized in that: The stabilizer added to the cationic antimicrobial peptide compound preparation includes a glycine-sodium hydroxide buffer solution. After addition, the concentration in the cationic antimicrobial peptide compound preparation is 0.05 mol / L and the pH is 9.

5. The added stabilizer also includes arginine and lysine. After addition, the mass percentage concentration of arginine in the cationic antimicrobial peptide compound preparation is 0.5% to 2%, and the mass percentage concentration of lysine in the cationic antimicrobial peptide compound preparation is 0.5% to 2%.

3. A composite disinfectant resistant to organic interference, wherein the disinfectant active ingredient is prepared by mixing the cationic antimicrobial peptide composite preparation according to claim 1 or 2, an essential oil emulsified preparation, a quaternary ammonium salt disinfectant, and a guanidine disinfectant in the following mass parts: cationic antimicrobial peptide composite preparation: essential oil emulsified preparation: quaternary ammonium salt disinfectant: guanidine disinfectant = (5-8): (4:6): (4-5): (2-4), The essential oil emulsion preparation is prepared by emulsifying the essential oil with a selected emulsifier. The essential oil is a plant essential oil with a bactericidal function. The essential oil mixture is prepared by mixing eucalyptus oil, thyme and eugenol in the mass ratio of eucalyptus oil:thyme:eugenol = (3-4): (2.5-3): (1-2). The selected emulsifier is a composite emulsifier prepared by mixing one or both of cetyltrimethylammonium chloride and cetyltrimethylammonium bromide. The essential oil mixture is added to the composite emulsifier, and the solution is continuously stirred at a temperature of 25-30°C until it is completely emulsified. The mass percentage of the essential oil mixture in the essential oil emulsion preparation is 6%-10%.

4. The composite disinfectant resistant to organic interference according to claim 3, characterized in that: The mixing ratio of cetyltrimethylammonium chloride and cetyltrimethylammonium bromide is preferably 6:4 in parts by mass.

5. The composite disinfectant resistant to organic interference according to claim 3, characterized in that: The preferred mixing ratio of eucalyptus oil, thyme and eugenol is eucalyptus oil: thyme: eugenol = 4:2.5:1.5 in parts by mass, and the preferred addition amount of the essential oil mixture and the compound emulsifier in parts by mass is 8:92 in parts by mass.

6. The composite disinfectant resistant to organic interference according to claim 3, characterized in that: The composite disinfectant mixed solution contains, by mass, 0.5% to 0.8% of carbonamide, 0.8% to 1.0% of Triton X-100, 0.4% to 0.6% of ethylphenyl polyethylene glycol and 1.5% to 2.0% of polyethylene glycol.

7. The composite disinfectant resistant to organic interference according to claim 3, characterized in that: The quaternary ammonium salt disinfectant includes a mixture of one or more of benzyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, C14-C16 alkylbenzyldimethylammonium chloride, and didecyldimethylammonium bromide.

8. The composite disinfectant resistant to organic interference according to claim 3, characterized in that: The guanidine disinfectant includes polyhexamethylene biguanidine hydrochloride or polyhexamethylene guanidine hydrochloride or a mixture of the two.

9. The composite disinfectant resistant to organic interference according to claim 3, characterized in that: The composite disinfectant mixed solution is mixed by the following reagents in parts by mass: 5% to 8% of a cationic antimicrobial peptide compound preparation, 4% to 6% of an essential oil emulsified preparation, 0 to 1.0% of benzyltrimethylammonium chloride, 0.5% to 1.5% of hexadecyltrimethylammonium bromide, 1.0% to 2.0% of C14 to C16 alkylbenzyldimethylammonium chloride, 1.0% to 2.0% of didecyldimethylammonium bromide, 1.0% to 2.5% of polyhexamethylene biguanide hydrochloride and 1.0% to 2.0% of polyhexamethyleneguanidine hydrochloride, 0.5% to 0.8% of carbonamide, 0.8% to 1.0% of Triton X-100, 0.4% to 0.6% of ethylphenyl polyethylene glycol and 1.5% to 2.0% of polyethylene glycol, 15.0% to 20.0% isopropyl alcohol, 5.0% to 10.0% ethanol, and the balance is water.

10. A method for preparing a composite disinfectant resistant to organic interference according to any one of claims 3 to 9, characterized in that The following steps are involved: (1) Deionized water is injected into the reactor tank and the water temperature is controlled to 20-35°C; organic solvents such as isopropyl alcohol and ethanol are added and mixed thoroughly; (2) preparing a cationic antimicrobial peptide preparation and adding it to the reactor tank, stirring thoroughly until mixed; (3) preparing an essential oil emulsion and adding it to the reactor tank, stirring thoroughly until the mixture is evenly mixed; (4) Add quaternary ammonium disinfectant and stir thoroughly until mixed; (5) Add guanidine disinfectant and stir thoroughly until mixed; (6) Adding additives and cracking agents; (7) After the materials are fully stirred, they are aseptically packaged and poured into a sterile plastic barrel of a certain volume and sealed for storage.

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