A neutral endoscope multi-enzyme cleaning solution, a preparation method and use thereof

Abstract

The present application relates to the field of medical cleaning agent, and specifically discloses a neutral endoscope multi-enzyme cleaning solution, a preparation method and an application. The cleaning solution is compounded with protease, amylase, lipase, cellulase, lysozyme and functional ingredients, and can fully decompose blood stains, fat, mucus and tissue residues attached to endoscopes and surgical instruments. Through detection, the bacterial removal rate of the product on blood bacterial mixed contaminants is greater than or equal to 99%, the ATP decline rate is greater than or equal to 99%, the artificial simulated contaminant removal rate is greater than or equal to 95%, and the removal rates of protein, starch and fat in the contaminants are not less than 98%, 94% and 80% respectively. The product has simple preparation process, low production cost, excellent decontamination effect in a neutral environment, almost no corrosion to stainless steel substrate, and can greatly reduce the risk of microbial residue and cross infection of instruments, and is suitable for large-scale popularization and application in medical institutions.

Description

Technical Field

[0001] This invention relates to the field of medical cleaning agents, specifically a neutral endoscopic multi-enzyme cleaning solution, its preparation method, and its uses. Background Technology

[0002] After use, surgical instruments such as endoscopes retain organic contaminants such as blood, fat, mucus, and tissue debris on their surfaces. These residues provide a nutrient substrate for bacterial colonization and reproduction. Once bacteria adhere to the instrument surface, they secrete extracellular polymeric substances (EPS). EPS, mainly composed of polysaccharides, proteins, and lipids, encapsulates the bacteria, forming a structured microbial community, or biofilm. If cleaning is incomplete, biofilms can persist on the instrument surface, exhibiting significantly enhanced resistance to antibiotics and disinfectants compared to planktonic bacteria, making them difficult to completely remove using routine disinfection procedures. Flexible endoscopes, due to their complex structure, narrow lumen, and materials susceptible to high temperatures and pressures, are particularly prone to biofilm formation, posing a significant potential risk factor for hospital-acquired infections.

[0003] It is estimated that approximately 65% ​​of human bacterial infections are associated with biofilms, and over 80% of chronic bacterial infections are caused by biofilms. Enterococci are among the most common pathogens causing nosocomial bloodstream infections and have a strong biofilm-forming ability; literature reports that approximately 68.68% of clinical isolates of Enterococcus faecalis exhibit biofilm-forming capability. Once enterococcal biofilms form, antibiotic resistance increases significantly, leading to treatment difficulties. Studies show that the 30-day mortality rate for nosocomial enterococcal bloodstream infections can reach 27.5%, with a mortality rate as high as 36.6% for those caused by vancomycin-resistant Enterococcus faecalis. Pseudomonas aeruginosa is a common pathogen causing nosocomial respiratory infections. A study involving 362 hospitalized Chinese patients showed that 54.7% of clinical isolates were strong biofilm-forming strains. Patients infected with strong biofilm-forming strains had longer hospital stays and significantly higher 30-day and 60-day mortality rates. Candida albicans has a strong biofilm-forming ability; approximately 66%–75.7% of clinical isolates can form biofilms in vitro, and biofilm formation is highly correlated with the incidence and mortality of hospital-acquired infections. Staphylococcus aureus is a common pathogen of implant-associated biofilm infections, and approximately 30%–40% of cases fail to respond to treatment even with combined surgical debridement and antibiotic therapy. Biofilm-associated infections have become the second leading cause of death worldwide, after cardiovascular disease. In the United States, there are approximately 1.96 million cases of biofilm-associated infections annually, resulting in approximately 268,000 deaths and direct treatment costs exceeding $18 billion.

[0004] Currently, among commercially available multi-enzyme cleaning solutions, leading companies such as Ruwolf in the United States have products with biofilm removal capabilities. However, most other products, both domestic and international, primarily focus on pretreatment for cleaning and disinfection, with relatively limited ability to actively remove bacteria and biofilms during the cleaning process. This invention, based on protease, amylase, and lipase, further introduces cellulase and lysozyme. Cellulase decomposes the cellulose components in the extracellular polymers of biofilms, disrupting the three-dimensional structure of the biofilm; lysozyme lyses the peptidoglycan in bacterial cell walls, causing bacterial rupture and death. The synergistic effect of these two enzymes can simultaneously decompose organic contaminants, assist in removing bacteria from instrument surfaces, disrupt biofilm structures, reduce microbial residues, and decrease the occurrence of instrument-related infections. Therefore, the purpose of this invention is to provide a neutral endoscopic multi-enzyme cleaning solution that combines cleaning power and sterilization functions to meet the higher infection control requirements of medical institutions. Summary of the Invention Summary of the Invention

[0005] This invention provides a neutral endoscope multi-enzyme cleaning solution with both cleaning and biofilm removal capabilities, and its preparation method. It introduces cellulase and lysozyme into the basis of protease, amylase, and lipase to solve the technical problem that the existing multi-enzyme cleaning solutions have limited ability to actively remove bacteria and biofilms during the cleaning process.

[0006] This invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a neutral endoscopic multi-enzyme washing solution, comprising the following components in addition to water:

[0008] Protease, amylase, lipase, cellulase, lysozyme, borax, sodium acetate, calcium chloride, triethanolamine, propylene glycol, ethylparaben, pH acidity regulator.

[0009] Preferably, the cleaning solution contains, by percentage mass concentration (g / L): 0.5%-2% protease, 0.5%-2% amylase, 0.1%-1% lipase, 0.1%-1% cellulase, 0.05%-0.2% lysozyme, 1%-5% borax, 1%-3% sodium acetate, 0.05%-0.2% calcium chloride, 2%-10% triethanolamine, 2%-10% propylene glycol, 0.05%-0.15% ethylparaben, and 3%-8% pH acidity adjuster.

[0010] More preferably, the cleaning solution contains, by percentage mass concentration, 1% protease, 1% amylase, 0.5% lipase, 0.5% cellulase, 0.1% lysozyme, 3% borax, 1.5% sodium acetate, 0.1% calcium chloride, 5% triethanolamine, 5% propylene glycol, 0.1% ethylparaben, and 5% pH acidity adjuster.

[0011] Preferably, the protease, amylase, lipase, cellulase, and lysozyme are all liquid enzyme preparations.

[0012] Preferably, the pH value of the cleaning solution is 6.5-7.5.

[0013] Preferably, the pH adjuster of the cleaning solution is an inorganic weak acid.

[0014] Preferably, the cleaning solution is used for cleaning endoscopes and surgical instruments.

[0015] In a second aspect, the present invention provides a method for preparing a neutral endoscopic multi-enzyme cleaning solution, using the cleaning solution described in any one of the first aspects above, comprising the following steps:

[0016] (1) Take half the total volume of water, borax and pH acidity adjuster of the formula and add them to the stirred jacketed kettle, heat and stir until completely dissolved;

[0017] (2) In a separate container, add the remaining half volume of water, sodium acetate and calcium chloride, and stir to dissolve.

[0018] (3) In a separate container, add propylene glycol and ethylparaben in sequence and stir to dissolve;

[0019] (4) At room temperature, slowly add the solutions obtained in steps (2) and (3) to the solution obtained in step (1) and stir to mix.

[0020] (5) Add protease, amylase, lipase, cellulase, lysozyme and triethanolamine to the solution obtained in step (4) in sequence, stir and mix well to obtain neutral endoscope multi-enzyme cleaning solution.

[0021] Preferably, the heating temperature in step (1) is 40-60°C.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention achieves synergistic cleaning and sterilization functions through a combination of five enzymes: protease, amylase, lipase, cellulase, and lysozyme. Lysozyme actively lyses bacterial cell walls during the cleaning process, causing bacterial rupture and death; cellulase decomposes the cellulose components in the extracellular polymers of biofilms, assisting in the disruption of biofilm structure. The synergistic effect of these two enzymes gives this invention both detergency and biofilm removal capabilities.

[0023] The cleaning solution of this invention has a pH value of 6.5-7.5, is neutral, and has virtually no corrosive effect on stainless steel, making it suitable for cleaning precision endoscopes.

[0024] The cleaning solution of this invention is simple to prepare, the raw material cost is controllable, and the enzyme activity has a long shelf life, making it suitable for widespread use in medical institutions. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Preparation of Neutral Endoscopic Multi-Enzyme Washing Solution

[0027] This embodiment provides a neutral endoscopic multi-enzyme cleaning solution, which, by mass percentage concentration (g / L), consists of: 1% protease, 1% amylase, 0.5% lipase, 0.5% cellulase, 0.1% lysozyme, 3% borax, 1.5% sodium acetate, 0.1% calcium chloride, 5% triethanolamine, 5% propylene glycol, 0.1% ethylparaben, 5% pH acidity adjuster, and the balance being water.

[0028] The preparation method is as follows: Take half the total volume of water, borax, and inorganic weak acid of the formula and add them to a stirred jacketed kettle. Heat to 40-60℃ and stir until completely dissolved. In another container, add the other half volume of water, sodium acetate, and calcium chloride of the formula and stir to dissolve. In another container, add propylene glycol and ethylparaben in sequence and stir to dissolve. At room temperature, slowly add the above two solutions to the borax solution and stir to mix. Add protease, amylase, lipase, cellulase, and lysozyme in sequence and stir to mix. Finally, add triethanolamine and stir to mix to obtain the neutral endoscope multi-enzyme washing solution.

[0029] Example 2: Physical and chemical properties testing of the cleaning solution

[0030] In this embodiment, the physicochemical properties of the cleaning solution prepared in Example 1 were tested, and the results are shown in Table 1.

[0031] Test methods: pH value determination was carried out in accordance with 2.2.1.4 of the "Disinfection Technical Specifications" (2002 edition); arsenic content determination was carried out in accordance with Appendix F of GB / T 9985-2000; heavy metal content determination was carried out in accordance with Appendix G of GB / T 9985-2000; low temperature stability test was carried out in accordance with T / WSJD 002-2019. The sample was placed in a -10℃ low temperature constant temperature bath for 24 hours and then restored to room temperature. The appearance changes were observed.

[0032] Table 1. Results of Physicochemical Performance Tests Testing items Test results pH value (25℃) 7.24 Arsenic (calculated as arsenic in 1% solution), mg / kg <0.05 Heavy metals (calculated as lead in 1% solution), mg / kg <1 Low temperature test (-10℃, 24h) The appearance is free of layering and sediment.

[0033] Example 3: Test on the removal effect of cleaning solution on mixed contaminants of blood and bacteria

[0034] This embodiment tests the removal effect of the cleaning solution prepared in Example 1 on mixed contaminants of blood and bacteria. The results are shown in Tables 2 and 3.

[0035] The detection method was performed according to Appendix C of T / WSJD 002-2019. The test strain was Enterococcus (ATCC 6057), and the test carrier was a slotted cylindrical head screw. The test carrier was immersed in a bacterial blood suspension prepared by mixing fresh sheep blood with Enterococcus bacterial culture, then removed and dried for later use. Under simulated test conditions of washing at 40℃ and 60 rpm for 3.0 min, the sample was treated with a test solution prepared at a ratio of 1:300 with sterile distilled water. The bacterial removal test used plate counting, and the ATP content was determined using an ATP analyzer.

[0036] Table 2. Removal effect on bacteria in bacterial blood suspension Group Colony count in the experimental group (CFU / sample) Colony count in the positive control group (CFU / sample) Bacterial removal rate (%) 1 <![CDATA[1.87×10 1 ]]> <![CDATA[1.07×10 6 ]]> 99.83 2 <![CDATA[1.54×10 1 ]]> <![CDATA[9.30×10 6 ]]> 99.83 3 <![CDATA[1.93×10 1 ]]> <![CDATA[1.24×10 6 ]]> 99.84 average value — — 99.83

[0037] Table 3. Effects on reducing ATP content in bacterial blood suspension Group ATP levels measured in the experimental group (amol) ATP levels (amol) in the positive control group ATP content decrease rate (%) 1 <![CDATA[1.97×10 3 ]]> <![CDATA[1.42×10 6 ]]> 99.86 2 <![CDATA[1.64×10 3 ]]> <![CDATA[1.07×10 6 ]]> 99.85 3 <![CDATA[2.08×10 3 ]]> <![CDATA[1.55×10 6 ]]> 99.87 average value — — 99.86

[0038] After three repeated tests, under simulated test conditions of 40℃, 60r / min washing for 3.0min, the test solution prepared by mixing the cleaning solution from Example 1 with sterile distilled water at a ratio of 1:300 showed a bacterial removal rate of >99% and an ATP content reduction rate of >99% in the bacterial blood suspension, which meets the requirements of T / WSJD 002-2019 for effective cleaning of mixed contaminants of blood and bacteria.

[0039] Example 4: Test on the removal effect of cleaning solution on artificially simulated pollutants

[0040] This embodiment tests the removal effect of the cleaning solution prepared in Example 1 on artificially simulated pollutants, and the results are shown in Table 4.

[0041] The detection method was performed according to Appendix D of T / WSJD 002-2019. The test carrier was an STF cleaning effect test card, which was prepared by coating with artificial simulated contaminants and drying. Under simulated test conditions of washing at 40℃ and 60r / min for 2.0min, the sample was treated with a test solution prepared at a ratio of 1:300 with sterile distilled water. The removal rate was calculated by weighing the carrier before and after cleaning, and the residue on the carrier surface was visually observed.

[0042] Table 4. Artificial Simulation Pollutant Removal Effect Group <![CDATA[Average weight m0 (g) of contaminants before cleaning]]> <![CDATA[Average weight m1 (g) of contaminants after cleaning]]> <![CDATA[Average weight m2 (g) of contaminants after washing]]> Removal rate (%) 1 1.2257 1.2089 1.2081 95.45 2 1.2327 1.2156 1.2148 95.53 3 1.2219 1.2043 1.2034 95.14 average value — — — 95.37

[0043] After three repeated tests, under simulated test conditions of 40℃, 60r / min washing for 2.0min, the test solution prepared by mixing the cleaning solution from Example 1 with sterile distilled water at a ratio of 1:300 completely dissolved and detached the artificial simulated pollutants by visual inspection. The surface was clean and bright with no residue, and the removal rate was >95%, which meets the requirements of T / WSJD 002-2019 for the cleaning effect of simulated pollutants.

[0044] Example 5: Test on the removal effect of cleaning solution on protein, starch and fat

[0045] This embodiment tests the removal effect of the cleaning solution prepared in Example 1 on protein, starch and fat, and the results are shown in Tables 5-7.

[0046] The detection method was performed according to Appendix E of T / WSJD 002-2019. The test carrier was a polytetrafluoroethylene sheet (10cm×5cm, each weighing approximately 1.200g±0.010g). Protein-simulated contaminants containing bovine serum albumin (purity ≥90%), starch-simulated contaminants containing starch (purity ≥90%) and wallpaper paste, and fat-simulated contaminants containing lard (food grade) were prepared, coated onto the carrier surface, and dried. Under simulated test conditions of washing at 40℃ and 60r / min for 2.0min, the sample was treated with a test solution prepared at a ratio of 1:300 with sterile distilled water. The removal rate of each component was calculated by weighing the carrier before and after washing.

[0047] Table 5 Protein Removal Efficacy Group <![CDATA[Average weight m0 (g) of contaminants before cleaning]]> <![CDATA[Average weight m1 (g) of contaminants after cleaning]]> <![CDATA[Average weight m2 (g) of contaminants after washing]]> Removal rate (%) 1 1.2026 1.2315 1.2031 98.27 2 1.2061 1.2370 1.2065 98.71 3 1.2052 1.2361 1.2055 99.03 average value — — — 98.67

[0048] Group <![CDATA[Average weight m0 (g) of contaminants before cleaning]]> <![CDATA[Average weight m1 (g) of contaminants after cleaning]]> <![CDATA[Average weight m2 (g) of contaminants after washing]]> Removal rate (%) 1 1.2033 1.2328 1.2042 96.95 2 1.2018 1.2306 1.2033 94.79 3 1.1992 1.2275 1.1999 97.53 average value — — — 96.42

[0049] Table 7 Fat Removal Effect Group <![CDATA[Average weight m0 (g) of contaminants before cleaning]]> <![CDATA[Average weight m1 (g) of contaminants after cleaning]]> <![CDATA[Average weight m2 (g) of contaminants after washing]]> Removal rate (%) 1 1.2048 1.2336 1.2099 81.94 2 1.2065 1.2378 1.2112 82.91 3 1.1997 1.2286 1.2040 84.43 average value — — — 83.09

[0050] After three repeated tests, under simulated test conditions of 40℃, 60r / min washing for 2.0min, the test solution prepared by mixing the cleaning solution from Example 1 with sterile distilled water at a ratio of 1:300 showed a removal rate of >98% for protein, >94% for starch, and >80% for fat in the simulated pollutants, which meets the requirements of T / WSJD 002-2019 for qualified cleaning effects of protein, starch, and fat.

[0051] Example 6: Metal Corrosion Test of Cleaning Fluid

[0052] In this embodiment, the metal corrosivity of the cleaning solution prepared in Example 1 was tested, and the results are shown in Table 8.

[0053] The testing method is in accordance with Appendix A of T / WSJD 002-2019. The test metal sheets are stainless steel (GB / T 1220), carbon steel (GB / T 700), copper (GB / T 2059), and aluminum (GB / T 1173). After weighing, the metal sheets are immersed in the test solution, placed under specified conditions, removed, cleaned, dried, and weighed again. The corrosion rate is calculated, and the corrosion level is determined.

[0054] Table 8 Results of Metal Corrosion Test Metal types Weigh (g / tablet) before testing. Weigh the tablet after the test (g / tablet) Weight loss (g / tablet) Corrosion rate R (mm / a) Stainless steel 3.1803 3.1803 0.0000 0.0000 carbon steel 3.2845 3.2789 0.0056 0.0888 copper 3.5251 3.5234 0.0017 0.0246 aluminum 1.1303 1.1299 0.0004 0.0184

[0055] Testing showed that the cleaning solution in Example 1 had virtually no corrosive effect on stainless steel sheets (corrosion rate 0.0000 mm / a), and caused slight corrosion to carbon steel, copper, and aluminum sheets; all types of metal sheets showed no shape change, and stainless steel sheets showed no color change or rust.

[0056] Example 7 Enzyme activity stability test of the washing solution

[0057] This embodiment tests the enzyme activity stability of the cleaning solution prepared in Example 1.

[0058] The detection method followed T / WSJD 002-2019. After the samples were placed at room temperature for 90 days, the residual activities of protease, lipase, and amylase were measured, and the activity reduction rate was calculated. The results showed that the activity reduction rates of protease, lipase, and amylase were all ≤20% after 90 days at room temperature, indicating that the enzyme activity of the cleaning solution is stable at room temperature, and the product has a shelf life of up to 24 months.

[0059] in conclusion

[0060] This invention utilizes a five-enzyme combination of protease, amylase, lipase, cellulase, and lysozyme to construct a neutral multi-enzyme cleaning solution for endoscopes, possessing both highly efficient decontamination and active sterilization functions. Verified by a third-party testing institution according to T / WSJD002-2019 "Hygienic Requirements for Medical Cleaning Agents," under simulated cleaning conditions of 40℃, 60r / min, and a high dilution ratio of 1:300, the cleaning solution achieved a bacterial removal rate >99% and an ATP content reduction rate >99% in mixed blood and bacterial contaminants; a removal rate >95% for artificially simulated contaminants; and removal rates >98%, 94%, and 80% for proteins, starches, and fats in simulated contaminants, respectively. All indicators meet the requirements of medical cleaning agent standards, demonstrating its highly efficient removal capability for organic contaminants and bacteria.

[0061] The test strain was Enterococcus. The cleaning solution of this invention achieved a removal rate of >99% for Enterococcus at a high dilution ratio of 1:300, indicating that it can effectively remove bacteria attached to the surface of instruments during the cleaning process, reduce the amount of microbial residue, and reduce the risk of biofilm formation. It is suitable for scenarios such as endoscopy centers, sterilization supply centers, and operating rooms, providing a solution for the cleaning and treatment of medical devices after use that combines cleaning power and sterilization function.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A neutral endoscopic multi-enzyme washing solution, characterized in that, In addition to water, it also includes the following components: Protease, amylase, lipase, cellulase, lysozyme, borax, sodium acetate, calcium chloride, triethanolamine, propylene glycol, ethylparaben, pH acidity regulator.

2. The neutral endoscopic multi-enzyme washing solution according to claim 1, characterized in that, The product, by percentage mass concentration (g / L), includes: 0.5-2% protease, 0.5-2% amylase, 0.1-1% lipase, 0.1-1% cellulase, 0.05-0.2% lysozyme, 1-5% borax, 1-3% sodium acetate, 0.05-0.2% calcium chloride, 2-10% triethanolamine, 2-10% propylene glycol, 0.05-0.15% ethylparaben, and 3%-8% pH acidity adjuster.

3. The neutral endoscopic multi-enzyme washing solution according to claim 2, characterized in that, The product contains, by percentage mass concentration: 1% protease, 1% amylase, 0.5% lipase, 0.5% cellulase, 0.1% lysozyme, 3% borax, 1.5% sodium acetate, 0.1% calcium chloride, 5% triethanolamine, 5% propylene glycol, 0.1% ethylparaben, and 5% pH acidity adjuster.

4. The neutral endoscopic multi-enzyme washing solution according to any one of claims 1-3, characterized in that, The protease, amylase, lipase, cellulase, and lysozyme mentioned are all liquid enzyme preparations.

5. The neutral endoscopic multi-enzyme washing solution according to any one of claims 1-3, characterized in that, The pH value of the cleaning solution is between 6.5 and 7.

5.

6. The neutral endoscopic multi-enzyme washing solution according to any one of claims 1-3, characterized in that, The pH adjuster of the cleaning solution is an inorganic weak acid.

7. The neutral endoscopic multi-enzyme washing solution according to any one of claims 1-3, characterized in that, The cleaning solution is used for cleaning endoscopes and surgical instruments.

8. A method for preparing the neutral endoscopic multi-enzyme washing solution according to any one of claims 1-5, characterized in that, Includes the following steps: Add half the total volume of water, borax, and pH acidity adjuster to a stirred jacketed kettle, and heat and stir until completely dissolved; In a separate container, add half the total volume of water, sodium acetate, and calcium chloride, and stir to dissolve. In a separate container, add propylene glycol and ethylparaben in sequence, and stir to dissolve; At room temperature, slowly add the solutions obtained in steps (2) and (3) to the solution obtained in step (1) and stir to mix. In the solution obtained in step (4), protease, amylase, lipase, cellulase, lysozyme and triethanolamine are added in sequence and stirred until well mixed to obtain neutral endoscope multi-enzyme cleaning solution.

9. The preparation method according to claim 6, characterized in that, The heating temperature in step (1) is 40-60°C.

10. A neutral endoscopic multi-enzyme washing solution, characterized in that, Prepared using the method described in claims 8-9.