Screening of microbial fermentation system composite bactericide and safe disinfection and killing of microbial fermentation system composite bactericide on space, surface, skin and like

Abstract

Screening of an amygdalin compound sterilizing agent and a safe sterilizing technology of the amygdalin compound sterilizing agent on space, surface, skin and the like are unique technical application after many years of exploration of fermentation conditions and culture media, screening of antibacterial substances, analysis of metabolome products and verification of a large number of experiments. A specific culture medium is prepared for pathogenic microorganisms, a reaction system containing functional molecules is prepared through stress fermentation, a target substance amygdalin is locked after bactericidal functional molecules are screened, and the target substance amygdalin is confirmed and utilized through safety and killing effect detection. In order to further improve the disinfecting and killing effect and flexibly meet the requirements of different scenes, the compound disinfecting and killing agent is formed by amygdalin, benzalkonium chloride, benzalkonium bromide, benzethonium chloride, chlorhexidine acetate, chlorine dioxide, coconut oil fatty acid diethanolamide, didecyl dimethyl ammonium bromide and other compounds, and stimulation and allergy experiments are carried out on hands, skin and mucosa. And an ideal and practical disinfection product is formed. The spray can be safely sprayed in a vehicle, an airplane and other spaces in which alcohol is forbidden, can efficiently kill pathogenic bacteria on air and surfaces in hospitals, waiting halls, waiting halls, schools, movie halls and other public places, can also be washed, wiped or soaked for disinfection, can disinfect food processing equipment and utensils, and can be used for sterilizing food processing equipment and utensils. And porous and non-porous or soft or hard surfaces can be safely disinfected.

Description

Technical Field

[0001] The present invention relates to the development of a composite amino acid disinfectant that is safe and has no toxic side effects by using biotechnology. This technology belongs to the field of disinfection and sterilization technology. Background Art

[0002] In response to the lack of indoor microenvironment disinfection technology for important biosafety barriers and the imperfect dynamic regulation mechanism, a common key technology for active prevention and control is constructed. Pathogenic microorganisms can seriously threaten human health. For example, after being poisoned and infected by Salmonella, Staphylococcus aureus, etc., symptoms such as vomiting, diarrhea, and abdominal pain often occur; after being infected by hemolytic streptococcus through droplet transmission in the air, it affects the upper respiratory tract. The most common pathogenic microorganisms are bacteria, which can cause upper respiratory tract infections, urinary tract infections, digestive system infections, or skin infections, and even enter the blood to cause bacteremia and septicemia. In addition, viruses, fungi, mycoplasmas, chlamydias, spirochetes, etc. can all cause diseases in humans or animals.

[0003] Disinfectants are an important part of health assurance. Since there are many pathogenic bacteria that cause upper respiratory tract infections, and most of them are Gram-positive bacteria, among which Staphylococcus aureus is relatively important, and its resistance to physical and chemical factors is relatively strong among many pathogenic bacteria [1], it is very important to prepare a series of effective disinfectants for pathogenic bacteria.

[0004] Upper respiratory tract infection is the most common disease affecting people's health. To prevent the occurrence of upper respiratory tract infections, clean air is one of the important measures. Disinfecting the air with ultraviolet lamps and air filters is difficult to widely promote for both families and public places. Although chemical disinfectants can achieve this purpose, long-term use will accumulate in the human body and pose a risk of poisoning [1 - 5]. The disinfectant we developed is completely made of natural substances, without using chemical substances or ethanol for preparation, and after meeting the requirements of the national standard for bactericidal drugs, it forms a highly effective disinfectant product that is safe for both the body and driving.

[0005] Experiments show that one kind of bacteria can be killed after being treated by another kind of bacteria [6]. This microbial mutual inhibition is widely used in biological control. For example, the compound agent of the metabolite of Bacillus licheniformis TFDY05 SJ1606 and a chemical fungicide has an inhibitory effect on Cercospora arachidicola; the combined use of the metabolite of the biocontrol bacterium SJ1606 with 6 chemical fungicides such as carbendazim and the compound use of antibacterial lipopeptides and chemical agents have inhibitory activity against pathogenic bacteria, and the combined use shows good inhibitory activity against Cercospora personata of peanut, among which the mixture of propiconazole and antibacterial lipopeptide has better inhibitory activity against Cercospora personata of peanut [7].

[0006] During the process of producing spinosad by fermentation with Saccharopolyspora spinosa, it is affected by various factors. The concentration of various components in the culture medium also plays a crucial role in the growth and spinosad production of Saccharopolyspora spinosa. For the production strain, there is an appropriate concentration for any nutrient in the culture medium. In terms of increasing the yield per unit volume of the fermenter, the substrate concentration should be increased as much as possible. However, if the substrate concentration is too high, it will cause too high osmotic pressure in the culture medium, thus inhibiting the microorganisms. A high glucose concentration will also inhibit the growth of mycelia and the synthesis of spinosad [8].

[0007] This technology is unique and different from previous studies. It uses high-temperature fermentation stress to induce probiotic microorganisms to degrade proteins to form amino acid-peptide complexes with disinfection and sterilization functions. Moreover, the biodegradable amino acid peptides of this product do not contain any heavy metals and iodine, which is essentially different from others and is innovative.

[0008] References:

[0009] 【1】Lu Xianyu, Tao Kun, Li Huaping, etc. Study on the bactericidal and virucidal effects of traditional Chinese medicine air disinfectant and bactericide [J]. Journal of Chongqing Medical University, 1998(03): 301 - 303.

[0010] 【2】Zou Yuping, Zou Shigui, Xia Shiyin. Determination of total amino acids in compound amino acid bactericidal agents [J]. Pesticides, 1991(01): 22 - 23. DOI: 10.16820 / j.cnki.1006 - 0413.1991.01.009.

[0011] 【3】Kang Zhanying, Liu Yingli, Han Wenrui, etc. Bactericidal activity of mixed amino acid copper complex [J]. Journal of Harbin University of Science and Technology, 1996(01): 110 - 114.

[0012] 【4】Kang Zhanying, Liu Yingli, Han Wenrui, etc. Bactericidal activity of mixed amino acid copper complex [J]. Journal of Harbin University of Science and Technology, 1996(01): 110 - 114.

[0013] 【5】Du Lianxiang, Lu Fuping (eds.). Microbiology Experimental Techniques [M]. Beijing: China Light Industry Press, 2006.09. P140.

[0014] 【6】Zhang Kuanshen, Yu Yangyun. Experimental report on new discoveries of fungal bactericides [J]. Chinese Journal of Health Laboratory Technology, 2013, 23(04): 1045.

[0015] Journal, 2013, 23(04): 1045.

[0016] 【7】Yang Shengyu, Yang Fei, Duan Haiming, et al. Inhibitory activity of the metabolites of biocontrol bacteria combined with fungicides against Cercospora arachidicola [J]. Journal of Jianghan University (Natural Science Edition), 2023, 51(02): 61-67. DOI: 10.16389 / j.cnki.cn42-1737 / n.2023.02.007.

[0017] 【8】Yang Rong. Research progress of spinosad [J]. Chemical Industry Management, 2016, No.403(06)72-73. Summary of the Invention Summary of the Invention 1:

[0019] Search for a culture medium formula for antibacterial functional molecules of pathogenic microorganisms in the indoor environment.

[0020] Pathogenic microorganisms in the indoor environment pose risks to exposed populations. It is very important to prepare a culture medium for searching for antibacterial functional molecules. Culture medium formula:

[0021] Milk, soluble collagen, hydrolyzed collagen peptide, gingerol, soy polypeptide, carotenoid, DVc, sugar, salt, various vitamins, and the specific content can be between 0.5% and 25%.

[0022] 5L culture medium formula:

[0023] Milk 0 - 200g

[0024] Soluble collagen 0 - 200g

[0025] Hydrolyzed collagen 0 - 200g

[0026] Gingerol 0 - 200ml

[0027] Soy polypeptide 0 - 200g

[0028] DVC 0 - 50g

[0029] Edible salt 0 - 50g

[0030] Sugar 0 - 50g

[0031] Additives such as various vitamins, nutritional additives, and trace elements 0 - 50g

[0032] Carotenoid 0 - 50g

[0033] Water 5L Summary of the Invention 2:

[0035] A high-salt environment stimulates microorganisms to ferment resistant substances, forming a fermentation reaction system that inhibits bacterial growth.

[0036] After adding 3 - 10% salt to the culture medium in 1, inoculate as many strains and bacterial communities as possible for fermentation to prepare a fermentation broth that can inhibit bacterial growth.

[0037] The fermentation temperature is 28°C - 37°C, with 33°C being preferred, and the fermentation time is 2 - 10 days. After fermentation, centrifuge at 1000 - 5000g and collect the supernatant for use in the antibacterial experiment. Summary of the Invention 3:

[0039] A method for finding antibacterial functional molecules.

[0040] Separate the supernatants fermented by different bacteria in 2 and apply them onto new culture medium plates respectively. At the same time, reserve a part of the supernatant for metabolome research.

[0041] Use the culture medium formula in 1 for the culture medium plates, add 2 - 10% (7% is optimal) agar, and prepare the plates with the culture medium. After applying the supernatant, place them in different places in the hospital that are easily contaminated by pathogenic bacteria, and select the plates without bacteria growth.

[0042] Perform metabolome analysis on the supernatants of the plates without bacteria growth or with antibacterial effects, analyze the metabolome data, and screen for the main antibacterial substances (see Figure 1) . Summary of the Invention 4:

[0044] Through metabolome analysis of the main antibacterial substances, the target substance is locked as amygdalin through antibacterial experiments.

[0045] Through metabolome analysis of the supernatants to determine the types of the main antibacterial substances, select the main antibacterial substances and conduct antibacterial experiments. It is found that amygdalin (Nitrilosides), with the molecular formula C20H27N011, has certain antibacterial effects ( Figure 2 ).

[0046] Further query the information on the discovered target substance amygdalin, indicating that in nature, amygdalin is obtained from the kernels and leaves of almonds, bitter almonds, peaches, nectarines, loquats, plums, apples, black cherries, etc. Its trihydrate is orthorhombic columnar crystals, with a melting point of 200°C, and the anhydrous form has a melting point of about 20°C. 1 g is soluble in 12 ml of water and is easily soluble in boiling water. Substances of the amygdalin type are non-toxic themselves, with chemically inactive properties, having little impact on healthy tissues, only invading and destroying cancer cells, being human metabolites, and only functioning in cancer cells.

[0047] After diluting the concentration to 1 - 10%, spray it on the skin behind the ears of 10 people in the technical team and on a larger area of the skin, and observe the allergic situation. There is no allergy. Experimental data and information query indicate that amygdalin can be used as an antibacterial agent. Summary of the Invention 5:

[0049] Combined use of amygdalin and benzalkonium chloride enhances antibacterial effect

[0050] To meet the requirements of disinfection and sterilization in different scenarios, amygdalin and benzalkonium chloride are compounded in a ratio of 0.5 - 2% amygdalin and 0.5 - 1% benzalkonium chloride, and the disinfection and sterilization effect is improved. Summary of the invention 6:

[0052] Combined use of amygdalin and benzalkonium bromide enhances antibacterial effect

[0053] To flexibly meet the needs of different scenarios, amygdalin and benzalkonium bromide are compounded in a ratio of 0.5 - 1% amygdalin and 0.5 - 1% benzalkonium bromide, and the disinfection and sterilization effect is improved. Summary of the invention 7:

[0055] Combined use of amygdalin and benzalkonium bromide enhances antibacterial effect

[0056] To flexibly meet the needs of different scenarios, amygdalin and enzethonium chloride are compounded in a ratio of 0.5 - 2% amygdalin and 0.5 - 1% enzethonium chloride, and the disinfection and sterilization effect is improved. Summary of the invention 8:

[0058] Amygdalin compounded with various other bactericides has good disinfection and sterilization effects.

[0059] To meet the requirements of disinfection and sterilization in different scenarios, amygdalin is compounded with existing quaternary ammonium salt disinfectants such as chlorhexidine acetate, chlorine dioxide, coconut oil fatty acid diethanolamide, and didodecyldimethylammonium bromide in a ratio of 0.5 - 1% amygdalin and 0.5 - 3% quaternary ammonium salt, and the disinfection and sterilization effect is improved, flexibly meeting the needs of different scenarios. Summary of the invention 9:

[0061] The amygdalin compounded disinfectant has no irritation or allergic reaction to hands, skin, and mucous membranes, and because of its antioxidant ability, it is an ideal and practical disinfection and sterilization product. Summary of the invention 10:

[0063] The amygdalin compounded disinfectant is not prepared with ethanol and can be safely sprayed inside cars, airplanes, and indoors. It has a high killing rate for bacteria on surfaces and in the air. Therefore, this disinfectant has a disinfection effect on pathogenic bacteria in the air of public places such as hospitals, waiting rooms, and airport lounges, with high safety and technical originality. The amygdalin compounded disinfectant can be used for flushing, wiping, or soaking disinfection, for disinfecting food processing equipment and utensils, and can also disinfect porous and non-porous or soft or hard surfaces, with high safety. Description of the Drawings:

[0064] Figure 1: Use the supernatant of the plate without bacteria growth for metabolomics (left), analyze the metabolomics data (right), and screen out the main antibacterial substance amygdalin.

[0065] Figure 2 : Experiments show that amygdalin has a certain antibacterial effect. The red circle indicates an obvious antibacterial zone of amygdalin, the others are other substances analyzed by metabolomics, and the blue circle is the control.

[0066] Example 1:

[0067] Screening of Antibacterial Substances against Pathogenic Microorganisms

[0068] The nitrogen source is a high-concentration protein of 0.5 - 10 mg / ml, which can be milk, soluble collagen, hydrolyzed collagen peptide, gingerol, soy polypeptide, etc. Among them, 0.5 - 1 mg / ml soluble collagen is the preferred nitrogen source. The carbon source is sugar. Among them, 2 - 3% sugar is the preferred carbon source.

[0069] The carbon-nitrogen ratio can be within the range of 1:3 to 1:10. It can be used after sterilization.

[0070] Inoculate as many strains and bacterial communities as possible into the culture medium for fermentation. The fermentation temperature is 28°C - 37°C, among which 33°C is preferred, and the fermentation time can be 2 - 10 days. After fermentation, centrifuge at 1000 - 5000 g and collect the supernatant.

[0071] Apply the supernatant to new solid culture medium plates respectively, and leave a part of the supernatant for metabolomics research. The culture medium plate uses the culture medium formula in 1, add 2 - 10% (7% is the best) agar, and prepare the plate with the culture medium. After applying the supernatant, place it in different places in the hospital that are easily contaminated by germs, and select the plates without bacteria growth.

[0072] Use the supernatant of the plate without bacteria growth or with antibacterial effect for metabolomics, analyze the metabolomics data, and screen the main antibacterial substances (see Figure 1) .

[0073] The supernatant of the plate without bacteria growth or with antibacterial effect contains antibacterial substances against pathogenic microorganisms.

[0074] Example 2:

[0075] Antibacterial Detection Report of Amygdalin Mixture

[0076] Test Items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0077] Testing Basis: "Disinfection Technical Specification" 2002 Edition

[0078] Test conclusion: The amygdalin mixture can kill more than 80.9% of bacteria in 10 minutes. This disinfectant is effective in disinfecting indoor, outdoor, skin, surface and public place air, and preventing upper respiratory tract bacterial and viral infections.

[0079] The original solution of this sample has a bactericidal effect on Escherichia coli, Staphylococcus aureus and Candida albicans after 10 minutes of action. The bactericidal rates are: Escherichia coli > 80.91, Staphylococcus aureus > 80.92, Candida albicans > 81.91.

[0080] Example 3:

[0081] Detection Report of 0.5% Amygdalin and 0.5% Benzalkonium Chloride Compound Disinfectant

[0082] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0083] Test basis: "Disinfection Technical Specification" 2002 edition

[0084] Test conclusion: The original solution of this sample has a bactericidal effect on Escherichia coli, Staphylococcus aureus and Candida albicans after 10 minutes of action. The bactericidal rates are: Escherichia coli > 99.01, Staphylococcus aureus > 99.05, Candida albicans > 98.9.

[0085] Example 4:

[0086] Detection Report of 2% Amygdalin and 1% Benzalkonium Chloride Compound Disinfectant

[0087] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0088] Test basis: "Disinfection Technical Specification" 2002 edition

[0089] Test conclusion: The original solution of this sample has a bactericidal effect on Escherichia coli, Staphylococcus aureus and Candida albicans after 2 minutes of action. The bactericidal rates are: Escherichia coli > 99.98, Staphylococcus aureus > 99.98,

[0090] Candida albicans > 99.98.

[0091] Example 5:

[0092] Disinfection Effect of 0.5% Amygdalin and 0.5% Benzalkonium Bromide

[0093] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0094] Test basis: "Disinfection Technical Specification" 2002 edition

[0095] Test conclusion: The original solution of this sample has bactericidal effect on Escherichia coli, Staphylococcus aureus and Candida albicans after acting for 2 minutes. The bactericidal rates are: Escherichia coli > 99.90, Staphylococcus aureus > 99.91, Candida albicans > 99.92.

[0096] Example 6:

[0097] Disinfection and sterilization effect of 1% amygdalin and 1% benzalkonium bromide

[0098] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0099] Test basis: "Disinfection Technical Specification" 2002 edition

[0100] Test conclusion: The original solution of this sample has bactericidal effect on Escherichia coli, Staphylococcus aureus and Candida albicans after acting for 2 minutes. The bactericidal rates are: Escherichia coli > 99.98, Staphylococcus aureus > 99.98, Candida albicans > 99.98.

[0101] Example 7:

[0102] Test report on disinfection and sterilization effect of 0.5% amygdalin and 0.5% benzethonium chloride:

[0103] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0104] Test basis: "Disinfection Technical Specification" 2002 edition

[0105] Test conclusion: The original solution of this sample has bactericidal effect on Escherichia coli, Staphylococcus aureus and Candida albicans after acting for 2 minutes. The bactericidal rates are: Escherichia coli > 99.91, Staphylococcus aureus > 99.90,

[0106] Candida albicans > 99.91.

[0107] Example 8

[0108] Test report on disinfection and sterilization effect of 2% amygdalin and 1% benzethonium chloride:

[0109] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0110] Test basis: "Disinfection Technical Specification" 2002 edition

[0111] Test conclusion: The original solution of this sample has bactericidal effect on Escherichia coli, Staphylococcus aureus and Candida albicans after acting for 2 minutes. The bactericidal rates are: Escherichia coli > 99.95, Staphylococcus aureus > 99.93,

[0112] Candida albicans > 99.92.

[0113] Example 9

[0114] The amygdalin compound disinfectant and insecticide is not prepared with ethanol and can be safely sprayed in spaces where alcohol is prohibited, such as inside cars and airplanes. It has a relatively high killing rate for pathogenic bacteria on various surfaces and in the air and is highly safe. Disinfectant test report:

[0115] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0116] Test basis: "Disinfection Technical Specification" 2002 edition

[0117] Test conclusion: The original solution of this sample has a bactericidal effect on Escherichia coli, Staphylococcus aureus, and Candida albicans after acting for 2 minutes. The bactericidal rates are: Escherichia coli > 99.94, Staphylococcus aureus > 99.98, Candida albicans > 99.97.

[0118] Example 10

[0119] The amygdalin compound disinfectant and insecticide has no irritation or allergic reaction to hands, skin, and mucous membranes, and has high disinfection and insecticide efficiency:

[0120] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0121] Test basis: "Disinfection Technical Specification" 2002 edition

[0122] Test conclusion: The original solution of this sample has a bactericidal effect on Escherichia coli, Staphylococcus aureus, and Candida albicans after acting for 2 minutes. The bactericidal rates are: Escherichia coli > 99.94, Staphylococcus aureus > 99.97, Candida albicans > 99.95.

[0123] Example 11

[0124] The amygdalin compound disinfectant and insecticide can efficiently disinfect and kill pathogenic bacteria in the air and on surfaces in crowded public places such as hospitals, waiting rooms, and airport lounges. Disinfectant test report:

[0125] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0126] Test basis: "Disinfection Technical Specification" 2002 edition

[0127] Test conclusion: The original solution of this sample has a bactericidal effect on Escherichia coli, Staphylococcus aureus, and Candida albicans after acting for 2 minutes. The bactericidal rates are: Escherichia coli > 99.9, Staphylococcus aureus > 99.93, Candida albicans > 99.92.

[0128] Example 12

[0129] The amygdalin compound disinfectant can be sprayed on porous and non-porous, soft or hard surfaces inside cars, airplanes, and indoors for efficient disinfection. Disinfectant test report:

[0130] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0131] Test basis: "Disinfection Technical Specification" 2002 edition

[0132] Test conclusion: The original solution of this sample has a bactericidal effect on Escherichia coli, Staphylococcus aureus, and Candida albicans after acting for 2 minutes. The bactericidal rates are: Escherichia coli > 99.90, Staphylococcus aureus > 99.97, Candida albicans > 99.96.

[0133] Example 13

[0134] The amygdalin compound disinfectant can be used for flushing, wiping, or soaking disinfection. Disinfectant test report for food processing equipment and utensils:

[0135] Test items: Escherichia coli, Staphylococcus aureus, Candida albicans

[0136] Test basis: "Disinfection Technical Specification" 2002 edition

[0137] Test conclusion: The original solution of this sample has a bactericidal effect on Escherichia coli, Staphylococcus aureus, and Candida albicans after acting for 2 minutes. The bactericidal rates are: Escherichia coli > 99.91, Staphylococcus aureus > 99.98, Candida albicans > 99.97.

Claims

1. Screening of amygdalin compound disinfectants and their safe disinfection technologies for space, surface, skin, etc.

2. Culture medium formula for preparing bactericidal substances for pathogenic microorganisms according to claim 1.

3. Reaction system and screening technology for preparing functional molecules by adversity fermentation according to claim 1.

4. Method for finding bactericidal functional molecules according to claim 1 and locking of the target substance amygdalin.

5. Safety and disinfection effect detection of amygdalin according to claim 1.

6. The amygdalin and benzalkonium chloride complex disinfectant according to claim 1 has no irritation or allergic reaction to hands, skin, and mucous membranes. Because of its antioxidant ability, it also has the effects of soothing sensitivity and anti-aging. While preventing skin disinfection, it also prevents upper respiratory tract infections. It is an ideal and practical disinfection product.

7. The disinfection effect of the compound application of amygdalin and benzalkonium bromide, benzethonium chloride, chlorhexidine acetate, chlorine dioxide, coconut oil fatty acid diethanolamide, didodecyldimethylammonium bromide, and quaternary ammonium salt compounds according to claim 1 is improved in a certain proportion respectively, which can flexibly meet the needs of different scenarios.

8. The amygdalin compound disinfectant according to claim 1 is not prepared with ethanol and can be safely sprayed into spaces where alcohol is prohibited, such as inside cars and airplanes. It has a high killing rate of pathogenic bacteria on various surfaces and in the air and high safety.

9. The amygdalin compound disinfectant according to claim 1 is highly efficient and can effectively disinfect pathogenic bacteria in the air and on the surface of crowded public places such as hospitals, waiting rooms, airport lounges, schools, and movie theaters.

10. The amygdalin compound disinfectant according to claim 1 can be rinsed, wiped, or soaked for disinfection. It can disinfect food processing equipment and utensils, and can also disinfect porous and non-porous or soft or hard surfaces with high safety.

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