Method for evaluating antibacterial ability of detergent

Abstract

The invention relates to the technical field of daily chemical products, and discloses a dishwashing liquid antibacterial ability evaluation method, which comprises: a1, taking 500 mL of a dishwashing liquid stock solution for standby application; a2, uniformly mixed liquid is obtained; step a3, selecting a test flat plate which is not polluted by infectious microbes to carry out a bacteriostasis experiment; step a4, adopting the third subculture as a test strain for a bacteriostatic test; a6, transferring the third subculture of the fourth ring into the sample bottle by using an inoculating loop, covering the sample bottle, uniformly mixing by using a mixer, and measuring and reading; step a7, observing strain growth distribution; and step a8, calculating the bacteriostatic effect of the detergent according to the following bacteriostatic rate calculation formula. The evaluation method for the antibacterial ability of the detergent provided by the invention is clear in operation, and makes up the blank of evaluation of the antibacterial ability of the detergent.

Description

Technical Field

[0001] The invention relates to the technical field of daily chemical products, in particular to a method for evaluating the antibacterial ability of detergent. Background Art

[0002] Antibacterial properties are widely used in people's daily lives for microbial treatment of skin, tableware, fruits and vegetables, fabrics and the environment. Antibacterial properties are achieved by killing or inhibiting microorganisms on the transmission medium, eliminating microorganisms outside the human body, effectively cutting off their transmission pathways, and controlling the pathogenicity of pathogenic bacteria. At the same time, with the improvement of living standards, consumers are paying more and more attention to family hygiene and safety, so the demand for antibacterial products is also increasing. There are a wide variety of daily chemical products on the market, such as laundry detergent, laundry powder, laundry soap, clothing care agents, etc. The various antibacterial claims of daily chemical products are dazzling, and consumers are becoming more and more rational in shopping at this stage, and they pay more attention to the actual ability of the products.

[0003] At present, the standards commonly used in evaluating the antibacterial and antibacterial capabilities of daily chemical products include "QB / T2738-2005 Evaluation Methods for Antibacterial and Antibacterial Effects of Daily Chemical Products", "QB / T2850-2007 Antibacterial and Antibacterial Detergents", "Disinfection Technical Specifications 2002 Edition", "GB15979-2002 Hygiene Standards for Disposable Sanitary Products", etc., which are relatively cumbersome.

[0004] Based on this, we proposed a method to evaluate the antibacterial ability of dishwashing liquid, hoping to solve the shortcomings of the existing technology. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the deficiencies of the prior art, the present invention provides a method for evaluating the antibacterial ability of dishwashing liquid.

[0007] (II) Technical solution

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0009] A method for evaluating the antibacterial ability of a dishwashing liquid comprises the following steps:

[0010] Step a1: Stir the detergent evenly in a sterile environment, take 500 mL of the detergent stock solution, and store it in a ground glass bottle for later use;

[0011] Step a2: Weigh the sample into a DTA solution melted at 50°C to simulate the state of dishwashing. Use a glass rod to fully stir the sample. Keep the temperature in a 50°C water bath to quickly and completely mix the sample and the DTA solution to obtain a mixed solution.

[0012] Wherein, the concentration of detergent in the mixed solution is 1000 mg / L;

[0013] Step a3: Pour the mixed solution into several sterile culture dishes, 20mL-25ml per dish, test plates, and mark accordingly. After solidification, invert and culture in a sterile environment for 48 hours. Select the test plates that are not contaminated by bacteria for antibacterial experiments;

[0014] Step a4: Use an inoculation loop to transfer the activated standard strain to a test tube containing TSB solution, mix well with a mixer, and then culture to obtain the first subculture;

[0015] Step a5: Use an inoculating loop to transfer several loops of the first subculture to a test tube containing 5 mL of TSB solution for culture to obtain the second subculture;

[0016] In the same way, the third subculture was obtained, and the third subculture was used as the test strain for the antibacterial test;

[0017] Step a6: Pipette 100 mL of TSB solution into a turbidity meter sample bottle, use an inoculation loop to transfer 4 loops of the third subculture into the sample bottle, cover it, mix well with a mixer, and measure the reading;

[0018] Use PBS as diluent to dilute to the concentration of the bacterial suspension used to measure the concentration of the bacterial suspension;

[0019] The pour plate method was used to count the viable bacteria in the test bacterial suspension;

[0020] Step a7: Use a micropipette to transfer 0.1 ml of the bacterial suspension in step a6 to a sterile test plate, and evenly apply it on the surface of the plate with an L-shaped glass curved rod. After the plate is completely infiltrated, invert the plate in a constant temperature incubator to observe the growth distribution of the bacteria;

[0021] Step a8: Calculate the antibacterial effect of detergent according to the following antibacterial rate calculation formula:

[0022] Antibacterial rate (%) = ((I-II) / I)*100%;

[0023] Where: I: average colony count of control sample;

[0024] II: Average colony count of test samples.

[0025] As a further technical solution, the instruments used in steps a1-a7, including ground glass bottles, glass rods, culture dishes, inoculation loops, mixers, test tubes, turbidity meter sample bottles, and micropipettes, are all sterilized.

[0026] As a further technical solution, the sterilization treatment is performed by steam sterilization at 121°C;

[0027] Among them, the steam sterilization time is 15 minutes.

[0028] As a further technical solution: the preparation method of the TSB solution is:

[0029] Weigh 30-40 g of TSB powder, put it into a conical flask, add 1 L of ultrapure water, heat it in a water bath at 60°C to completely dissolve the powder in the water, and sterilize it with steam at 121°C in a pressure sterilizer for 15 min, and set aside.

[0030] As a further technical solution, the preparation method of the DTA solution is:

[0031] Weigh 30-35 g of DTA powder, put it into a conical flask, add 1 L of ultrapure water, heat in a 50°C water bath to completely dissolve the powder in water, steam sterilize at 121°C in a pressure sterilizer for 15 min, and set aside.

[0032] As a further technical solution, the activated standard strain is activated in a nutrient agar medium;

[0033] The activation temperature is 37°C and the activation time is 24 hours.

[0034] As a further technical solution: the standard strain is but not limited to Staphylococcus aureus and Escherichia coli.

[0035] As a further technical solution: the method for preparing the nutrient agar medium is:

[0036] Weigh 30-40g of nutrient agar medium powder, put it into a conical flask, add 1L of ultrapure water, heat it in a water bath at 55℃, stir it to completely dissolve it in water, steam sterilize it at 121℃ in a pressure sterilizer for 15min, and after it cools down, pour it into a sterile test tube in a sterile environment in a clean bench, and place it at a certain angle to the horizontal laboratory table. After it solidifies, place the nutrient agar slope in a 2-8℃ refrigerator for storage for later use.

[0037] As a further technical solution: the preparation method of the PBS is:

[0038] Weigh appropriate amounts of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and anhydrous potassium dihydrogen phosphate (KH2PO4) reagents into a conical flask, add ultrapure water and stir to dissolve, so that the reagents are completely dissolved in the ultrapure water to obtain a 0.1 mol / L PBS solution, adjust the pH value to 7.4, and steam sterilize at 121°C in a pressure sterilizer for 20 min for use.

[0039] (III) Beneficial effects

[0040] Compared with the prior art, the present invention provides a method for evaluating the antibacterial ability of detergent, which has the following beneficial effects:

[0041] The method for evaluating the antibacterial ability of detergent provided by the present invention is clear and straightforward to operate, thus filling the gap in the evaluation of the antibacterial ability of detergent.

[0042] In the present invention, under strict aseptic operation conditions, the test plate is pre-cultured for 24 hours to dry its surface before being used for antibacterial testing. If it is contaminated, colonies can be observed on the plate at this time, so there is no need to culture for 48 hours to determine that the plate is not contaminated by miscellaneous bacteria.

[0043] The present invention provides an evaluation method for the antibacterial ability of dishwashing liquid to evaluate the antibacterial ability of dishwashing liquid. The antibacterial performance detection steps of the dishwashing liquid are simple and the measurement accuracy is high. By adopting the evaluation method of the present invention, antibacterial monitoring can be performed on daily necessities including dishwashing liquid. Antibacterial is achieved by killing or inhibiting microorganisms on the transmission medium, eliminating the microorganisms outside the human body, effectively cutting off their transmission pathways, and achieving the purpose of controlling the pathogenicity of pathogenic bacteria. The evaluation method of the present invention can screen out products with high antibacterial performance, which can meet consumers' needs for household hygiene and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The figure is a flow chart of the evaluation method of the antibacterial ability of dishwashing liquid. DETAILED DESCRIPTION

[0045] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0046] All percentages, parts and ratios are calculated by weight of the total compositions of the present invention, unless otherwise specified. All weights pertaining to listed ingredients are given as active material, unless otherwise specified.

[0047] The term "test sample" of the present invention refers to detergent having antibacterial ability.

[0048] The term "standard strain" of the present invention is selected from but not limited to Staphylococcus aureus and Escherichia coli.

[0049] The term "antibacterial" in the present invention includes basic processes such as disinfection, sterilization, disinfection, antibacterial, mildew prevention and antiseptic, which refers to the process of killing bacteria or hindering bacterial growth, proliferation and activity by chemical or physical means. It is a general term for disinfection and antibacterial.

[0050] The graduated pipette (1.0 mL, 5.0 mL, 10.0 mL) involved in the present invention.

[0051] In the examples, the following designations and abbreviations will be used.

[0052] (1) Bacterial species

[0053] Staphylococcus aureus ATCC 6538,

[0054] Escherichia coli

[0055] (2) Reagents

[0056] Dextrose Tryptone Agar (DTA)

[0057] Nutrient agar (NA),

[0058] Tryptone Soy Broth (TSB),

[0059] Sabouraud agar (SDA),

[0060] Phosphate buffered saline (PBS),

[0061] The test sample is a dishwashing liquid with antibacterial ability, and the control is standard hard water.

[0062] The following are specific embodiments:

[0063] Example 1

[0064] A method for evaluating the antibacterial ability of a dishwashing liquid comprises the following steps:

[0065] Step a1: Stir the detergent evenly in a sterile environment, take 500 mL of the detergent stock solution, and store it in a ground glass bottle for later use;

[0066] Step a2: Weigh the sample into a DTA solution melted at 50°C to simulate the state of dishwashing. Use a glass rod to fully stir the sample. Keep the temperature in a 50°C water bath to quickly and completely mix the sample and the DTA solution to obtain a mixed solution.

[0067] Wherein, the concentration of detergent in the mixed solution is 1000 mg / L;

[0068] Step a3: Pour the mixed solution into several sterile culture dishes, 20 mL per dish, and test plates, and mark them accordingly. After solidification, invert and culture in a sterile environment for 48 hours, and select the test plates that are not contaminated by bacteria for antibacterial experiments;

[0069] Step a4: Use an inoculation loop to transfer the activated standard strain to a test tube containing TSB solution, mix well with a mixer, and then culture to obtain the first subculture;

[0070] Step a5: Use an inoculating loop to transfer several loops of the first subculture to a test tube containing 5 mL of TSB solution for culture to obtain the second subculture;

[0071] In the same way, the third subculture was obtained, and the third subculture was used as the test strain for the antibacterial test;

[0072] Step a6: Pipette 100 mL of TSB solution into a turbidity meter sample bottle, use an inoculation loop to transfer 4 loops of the third subculture into the sample bottle, cover it, mix well with a mixer, and measure the reading;

[0073] Use PBS as diluent to dilute to the concentration of the bacterial suspension used to measure the concentration of the bacterial suspension;

[0074] The pour plate method was used to count the viable bacteria in the test bacterial suspension;

[0075] Step a7: Use a micropipette to transfer 0.1 ml of the bacterial suspension in step a6 to a sterile test plate, and evenly apply it on the surface of the plate with an L-shaped glass curved rod. After the plate is completely infiltrated, invert the plate in a constant temperature incubator to observe the growth distribution of the bacteria;

[0076] Step a8: Calculate the antibacterial effect of detergent according to the following antibacterial rate calculation formula:

[0077] Antibacterial rate (%) = ((I-II) / I)*100%;

[0078] Where: I: average colony count of control sample;

[0079] II: Average colony count of test samples.

[0080] The instruments used in steps a1-a7, including ground glass bottles, glass rods, culture dishes, inoculation loops, mixers, test tubes, turbidity meter sample bottles, and micropipettes, are all sterilized.

[0081] Sterilization treatment is done by steam sterilization at 121°C;

[0082] Among them, the steam sterilization time is 15 minutes.

[0083] The preparation method of TSB solution is:

[0084] Weigh 30 g of TSB powder, put it into a conical flask, add 1 L of ultrapure water, heat in a water bath at 60°C to completely dissolve the powder in the water, steam sterilize at 121°C in a pressure sterilizer for 15 min, and set aside.

[0085] The preparation method of DTA solution is:

[0086] Weigh 30 g of DTA powder, put it into a conical flask, add 1 L of ultrapure water, heat in a 50°C water bath to completely dissolve the powder in water, steam sterilize at 121°C in a pressure sterilizer for 15 min, and set aside.

[0087] The activated standard strain is activated in nutrient agar medium;

[0088] The activation temperature is 37°C and the activation time is 24 hours.

[0089] The standard strain was Staphylococcus aureus.

[0090] The preparation method of nutrient agar medium is as follows:

[0091] Weigh 35 g of nutrient agar medium powder, put it into a conical flask, add 1 L of ultrapure water, heat it in a water bath at 55°C, stir it to completely dissolve it in water, and steam sterilize it at 121°C in a pressure sterilizer for 15 min. After it cools, pour it into a sterile test tube in a sterile environment in a clean bench, and place it at an angle to the horizontal laboratory table. After it solidifies, place the nutrient agar slope in a 2-8°C refrigerator for storage for later use.

[0092] The preparation method of PBS is as follows:

[0093] Weigh appropriate amounts of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and anhydrous potassium dihydrogen phosphate (KH2PO4) reagents into a conical flask, add ultrapure water and stir to dissolve, so that the reagents are completely dissolved in the ultrapure water to obtain a 0.1 mol / L PBS solution, adjust the pH value to 7.4, and steam sterilize at 121°C in a pressure sterilizer for 20 min for use.

[0094] Example 2

[0095] A method for evaluating the antibacterial ability of a dishwashing liquid comprises the following steps:

[0096] Step a1: Stir the detergent evenly in a sterile environment, take 500 mL of the detergent stock solution, and store it in a ground glass bottle for later use;

[0097] Step a2: Weigh the sample into a DTA solution melted at 50°C to simulate the state of dishwashing. Use a glass rod to fully stir the sample. Keep the temperature in a 50°C water bath to quickly and completely mix the sample and the DTA solution to obtain a mixed solution.

[0098] Wherein, the concentration of detergent in the mixed solution is 1000 mg / L;

[0099] Step a3: Pour the mixed solution into several sterile culture dishes, 20mL-25ml per dish, test plates, and mark accordingly. After solidification, invert and culture in a sterile environment for 48 hours. Select the test plates that are not contaminated by bacteria for antibacterial experiments;

[0100] Step a4: Use an inoculation loop to transfer the activated standard strain to a test tube containing TSB solution, mix well with a mixer, and then culture to obtain the first subculture;

[0101] Step a5: Use an inoculating loop to transfer several loops of the first subculture to a test tube containing 5 mL of TSB solution for culture to obtain the second subculture;

[0102] In the same way, the third subculture was obtained, and the third subculture was used as the test strain for the antibacterial test;

[0103] Step a6: Pipette 100 mL of TSB solution into a turbidity meter sample bottle, use an inoculation loop to transfer 4 loops of the third subculture into the sample bottle, cover it, mix well with a mixer, and measure the reading;

[0104] Use PBS as diluent to dilute to the concentration of the bacterial suspension used to measure the concentration of the bacterial suspension;

[0105] The pour plate method was used to count the viable bacteria in the test bacterial suspension;

[0106] Step a7: Use a micropipette to transfer 0.1 ml of the bacterial suspension in step a6 to a sterile test plate, and evenly apply it on the surface of the plate with an L-shaped glass curved rod. After the plate is completely infiltrated, invert the plate in a constant temperature incubator to observe the growth distribution of the bacteria;

[0107] Step a8: Calculate the antibacterial effect of detergent according to the following antibacterial rate calculation formula:

[0108] Antibacterial rate (%) = ((I-II) / I)*100%;

[0109] Where: I: average colony count of control sample;

[0110] II: Average colony count of test samples.

[0111] The instruments used in steps a1-a7, including ground glass bottles, glass rods, culture dishes, inoculation loops, mixers, test tubes, turbidity meter sample bottles, and micropipettes, are all sterilized.

[0112] Sterilization treatment is done by steam sterilization at 121°C;

[0113] Among them, the steam sterilization time is 15 minutes.

[0114] The preparation method of TSB solution is:

[0115] Weigh 35 g of TSB powder, put it into a conical flask, add 1 L of ultrapure water, heat in a water bath at 60°C to completely dissolve the powder in the water, steam sterilize at 121°C in a pressure sterilizer for 15 min, and set aside.

[0116] The preparation method of DTA solution is:

[0117] Weigh 30 g of DTA powder, put it into a conical flask, add 1 L of ultrapure water, heat in a 50°C water bath to completely dissolve the powder in water, steam sterilize at 121°C in a pressure sterilizer for 15 min, and set aside.

[0118] The activated standard strain is activated in nutrient agar medium;

[0119] The activation temperature is 37°C and the activation time is 24 hours.

[0120] The standard strain was Escherichia coli.

[0121] The preparation method of nutrient agar medium is as follows:

[0122] Weigh 30 g of nutrient agar medium powder, put it into a conical flask, add 1 L of ultrapure water, heat it in a water bath at 55°C, stir it to completely dissolve it in water, and steam sterilize it at 121°C in a pressure sterilizer for 15 min. After it cools, pour it into a sterile test tube in a sterile environment in a clean bench, and place it at an angle to the horizontal laboratory table. After it solidifies, place the nutrient agar slope in a 2-8°C refrigerator for storage for later use.

[0123] The preparation method of PBS is as follows:

[0124] Weigh appropriate amounts of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and anhydrous potassium dihydrogen phosphate (KH2PO4) reagents into a conical flask, add ultrapure water and stir to dissolve, so that the reagents are completely dissolved in the ultrapure water to obtain a 0.1 mol / L PBS solution, adjust the pH value to 7.4, and steam sterilize at 121°C in a pressure sterilizer for 20 min for use.

[0125] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for evaluating the antibacterial ability of detergent, characterized in that: The following steps are involved: Step a1: Stir the detergent evenly in a sterile environment, take 500 mL of the detergent stock solution, and store it in a ground glass bottle for later use; Step a2: Weigh the sample into a DTA solution melted at 50°C to simulate the state of dishwashing. Use a glass rod to fully stir the sample. Keep the temperature in a 50°C water bath to quickly and completely mix the sample and the DTA solution to obtain a mixed solution. Wherein, the concentration of detergent in the mixed solution is 1000 mg / L; Step a3: Pour the mixed solution into several sterile culture dishes, 20mL-25ml per dish, test plates, and mark accordingly. After solidification, invert and culture in a sterile environment for 48 hours. Select the test plates that are not contaminated by bacteria for antibacterial experiments; Step a4: Use an inoculation loop to transfer the activated standard strain to a test tube containing TSB solution, mix well with a mixer, and then culture to obtain the first subculture; Step a5: Use an inoculating loop to transfer several loops of the first subculture to a test tube containing 5 mL of TSB solution for culture to obtain the second subculture; In the same way, the third subculture was obtained, and the third subculture was used as the test strain for the antibacterial test; Step a6: Pipette 100 mL of TSB solution into a turbidity meter sample bottle, use an inoculation loop to transfer 4 loops of the third subculture into the sample bottle, cover it, mix well with a mixer, and measure the reading; Use PBS as diluent to dilute to the concentration of the bacterial suspension used to measure the concentration of the bacterial suspension; The pour plate method was used to count the viable bacteria in the test bacterial suspension; Step a7: Use a micropipette to transfer 0.1 ml of the bacterial suspension in step a6 to a sterile test plate, and evenly apply it on the surface of the plate with an L-shaped glass curved rod. After the plate is completely infiltrated, invert the plate in a constant temperature incubator to observe the growth distribution of the bacteria; Step a8: Calculate the antibacterial effect of detergent according to the following antibacterial rate calculation formula: Antibacterial rate (%) = ((I-II) / I)*100%; Where: I: average colony count of control sample; II: Average colony count of test samples.

2. The method for evaluating the antibacterial ability of detergent according to claim 1, characterized in that: The instruments used in steps a1-a7, including ground glass bottles, glass rods, culture dishes, inoculation loops, mixers, test tubes, turbidity meter sample bottles, and micropipettes, are all sterilized.

3. The method for evaluating the antibacterial ability of a dishwashing liquid according to claim 2, characterized in that: The sterilization treatment is performed by steam sterilization at 121°C; Among them, the steam sterilization time is 15 minutes.

4. The method for evaluating the antibacterial ability of a detergent according to claim 1, characterized in that: The preparation method of the TSB solution is: Weigh 30-40 g of TSB powder, put it into a conical flask, add 1 L of ultrapure water, heat it in a water bath at 60°C to completely dissolve the powder in the water, and sterilize it with steam at 121°C in a pressure sterilizer for 15 min, and set aside.

5. The method for evaluating the antibacterial ability of detergent according to claim 1, characterized in that: The preparation method of the DTA solution is: Weigh 30-35 g of DTA powder, put it into a conical flask, add 1 L of ultrapure water, heat in a 50°C water bath to completely dissolve the powder in water, steam sterilize at 121°C in a pressure sterilizer for 15 min, and set aside.

6. The method for evaluating the antibacterial ability of detergent according to claim 1, characterized in that: The activated standard strain is activated in a nutrient agar medium; The activation temperature is 37°C and the activation time is 24 hours.

7. The method for evaluating the antibacterial ability of detergent according to claim 6, characterized in that: The standard strains include but are not limited to Staphylococcus aureus and Escherichia coli.

8. The method for evaluating the antibacterial ability of detergent according to claim 6, characterized in that: The nutrient agar medium preparation method is: Weigh 30-40g of nutrient agar medium powder, put it into a conical flask, add 1L of ultrapure water, heat it in a water bath at 55℃, stir it to completely dissolve it in water, steam sterilize it at 121℃ in a pressure sterilizer for 15min, and after it cools down, pour it into a sterile test tube in a sterile environment in a clean bench, and place it at a certain angle to the horizontal laboratory table. After it solidifies, place the nutrient agar slope in a 2-8℃ refrigerator for storage for later use.

9. The method for evaluating the antibacterial ability of detergent according to claim 1, characterized in that: The preparation method of the PBS is: Weigh appropriate amounts of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and anhydrous potassium dihydrogen phosphate (KH2PO4) reagents into a conical flask, add ultrapure water and stir to dissolve, so that the reagents are completely dissolved in the ultrapure water to obtain a 0.1 mol / L PBS solution, adjust the pH value to 7.4, and steam sterilize at 121°C in a pressure sterilizer for 20 min for use.

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