Evaluation method for antibacterial activity of chemical oral solution

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for evaluating the antibacterial activity of a chemical oral solution. The evaluation method comprises the following steps: preparing a bacterial suspension of a bacterial strain; drawing a test group growth curve; obtaining lambda, mumax and ODmax according to the growth curve of the test group; drawing a growth curve of a control group; obtaining lambda0, mumax0 and ODmax0 according to the growth curve of the control group; when at least one of the conditions of lambda / lambda 0 > 1, mumax / mumax 0 < 1 and ODmax / ODmax 0 < 1 is met, it is judged that the to-be-detected sample has the antibacterial activity, and the larger the lambda / lambda 0 value is, the smaller the mumax / mumax 0 value is, and the smaller the ODmax / ODmax 0 value is, the higher the antibacterial activity of the to-be-detected sample is. According to the method, high-flux and rapid evaluation of the antibacterial activity of the chemical oral solution is realized.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for evaluating the antibacterial property of a chemical drug oral solution. Background Art

[0002] Chemical oral solutions are non-sterile drugs with high water activity and most of them contain nutrients such as sugars and proteins. Microorganisms are easy to grow and reproduce, and the drugs themselves may have a certain level of bioburden. Therefore, this type of drug is at a higher risk of microbial contamination. In order to prevent microbial contamination and reproduction during the shelf life of the drug and during the use of the drug (especially multi-dose preparations), national pharmacopoeias and drug development guidelines, including the Chinese Pharmacopoeia, allow the addition of antibacterial agents or preservatives to ensure the safety and effectiveness of the drug throughout its life cycle. In order to evaluate whether the antibacterial properties of such products meet the requirements, the General Rules 1121 of the Fourth Part of the Chinese Pharmacopoeia <Antibacterial Efficacy Test Method> stipulates that "if the drug itself does not have sufficient antibacterial efficacy, then appropriate antibacterial agents should be added according to the characteristics of the preparation (such as water-soluble preparations)". Therefore, the selection of the type and concentration of antibacterial agents are an important part of the drug development stage.

[0003] Common types of antibacterial agents used in chemical oral solutions include sodium benzoate, potassium sorbate, parabens, benzyl alcohol, etc. Regarding the amount of antibacterial agents added, the Chinese Pharmacopoeia, the Dictionary of Pharmaceutical Additives, the FDA Inactive Database, the Manual of Pharmaceutical Excipients, the Joint Expert Committee on Food Additives of the World Health Organization (JECFA) and other standards all have regulations in different dimensions, including the maximum amount of addition, the maximum amount of addition per unit dose, the maximum daily exposure, the acceptable daily intake, etc., and the limits between different standards vary greatly, making it difficult for pharmaceutical companies to determine the amount of antibacterial agents added by consulting the standards. In addition, the pH value, water activity, osmotic pressure, excipients, packaging materials and other factors between different varieties of chemical oral solutions vary greatly, and will also affect the antibacterial activity. Therefore, the antibacterial system is a complex system affected by multiple factors. Based on the above, each variety of oral solution has a specific antibacterial system, and antibacterial evaluation needs to be carried out to determine whether the type and concentration of the antibacterial agent are appropriate.

[0004] At present, the general method for evaluating antibacterial properties is the antibacterial efficacy test method, which is specifically embodied in that the antibacterial substance is fully mixed with a high-concentration bacterial solution and then allowed to stand. After a period of interaction, the number of microorganisms is regularly measured, and the colony-forming units (cfu) are counted by naked eye. The logarithmic value of the decrease in microorganisms is used to characterize the strength of the antibacterial property. The advantages of this method are that the results are simple and intuitive, and can be directly compared with the pharmacopoeia standards to determine whether they meet the requirements. The disadvantages are that the operation is cumbersome, the experimental cycle is as long as 28 days, the manual counting by naked eye leads to large errors, and the judgment criteria are extensive. Especially for complex situations with multiple strains, multiple dilution levels, and multiple influencing factors, it is very labor-intensive, costly, time-consuming, and labor-intensive.

[0005] There are 7 types of antibacterial agents commonly used in chemical oral solutions, but the physical and chemical properties (such as pH, water activity, viscosity, osmotic pressure, etc.), excipients, packaging materials and other factors of different types of oral solutions vary greatly, which may affect the strength of antibacterial properties. Therefore, it is necessary to conduct research on the selection of antibacterial agent types and concentration determination for each variety. As a pharmacopoeia method, the antibacterial efficacy test method of the Chinese Pharmacopoeia manually measures the number of microorganisms by the naked eye using CFU. It is necessary to calculate the logarithmic value of the decrease in the number of microorganisms after 28 days to judge the results. From the level of national pharmacopoeia standards, it guides enterprises to conduct experiments to determine the minimum effective amount of antibacterial agents, which is a reference standard for drug registration and declaration. However, in the process of drug research and development, quality control or inspection, the use of this method will face problems such as long experimental cycle, large workload, and extensive judgment standards. In particular, a series of concentrations of multiple antibacterial agents need to be screened in the research and development stage, which will also consume more manpower, material resources, financial resources and time costs. Summary of the invention

[0006] In order to solve at least one of the above technical problems, the present invention provides a method for evaluating the antibacterial activity of a chemical oral solution; the antibacterial activity of a chemical oral solution is evaluated in a high-throughput, rapid and precise manner by using a growth curve based on OD600.

[0007] As one aspect, the present invention provides a method for evaluating the antibacterial activity of a chemical oral solution, comprising the following steps: preparing a bacterial suspension of a strain; mixing a sample to be tested, the bacterial suspension and a liquid culture medium, culturing the mixture, collecting OD values ​​representing the number of strains, and drawing a growth curve of a test group; obtaining the hysteresis time λ and the maximum growth rate μ of the strain in the test group according to the growth curve of the test group; max and maximum optical density OD max ; Mix the bacterial suspension and liquid culture medium, culture them, collect the OD value of the data characterizing the number of strains, and draw the growth curve of the control group; according to the growth curve of the control group, obtain the hysteresis time λ0 and the maximum growth rate μ of the control group strain max0 and maximum optical density OD max0; When at least λ / λ0>1, μ max / μ max0 <1, OD max / OD max0 <1, the sample to be tested is judged to have antibacterial properties, and the larger the λ / λ0 value, the greater the μ max / μ max0 The smaller the value, the higher the OD max / OD max0 The smaller the value, the stronger the antibacterial activity of the sample to be tested.

[0008] In some embodiments of the present invention, the strain includes at least one of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans.

[0009] In some embodiments of the present invention, the liquid culture medium includes at least one of trypticase soy liquid culture medium and Sabouraud dextrose liquid culture medium.

[0010] In some embodiments of the present invention, the culture is carried out in a microplate; wherein the initial inoculum size M of the strain is: 0 cfu / well <M<10 6 cfu / well, for example, could be 10 2 cfu / hole, 10 3 cfu / hole, 10 4 cfu / hole, 3.6×10 4 cfu / hole, 9.7×10 4 cfu / hole, 4.8×10 5 cfu / hole, 6.2×10 5 cfu / hole, 7.8×10 5 cfu / hole, 9.7×10 5 cfu / well, etc.

[0011] In some embodiments of the present invention, the culture temperature is 30-35°C, for example, it can be 31.6°C, 32.9°C, 33.4°C, 34.1°C, 34.8°C, etc.; the rotation speed of the instrument is 600rpm-800rpm, for example, it can be 609rpm, 653rpm, 696rpm, 738rpm, 762rpm, 798rpm, etc.

[0012] In some embodiments of the invention, the maximum growth rate μ max is the maximum slope of the growth curve, which is calculated as follows: starting from the first collection point, define 7 collection points as a set of data for linear regression to evaluate the slope, and then repeat the above operation from the second collection point, and so on, until the maximum slope is obtained.

[0013] In some embodiments of the present invention, the hysteresis time λ is the time for the growth curve to slowly increase, defined as μ max The time intercept of the associated linear regression intersects the horizontal line of the first acquisition point.

[0014] In some embodiments of the present invention, the maximum optical density value OD max To obtain the maximum value of the growth curve, define 7 collection points as a set of data and report the maximum value among the average values.

[0015] In some embodiments of the present invention, the number of strains in the bacterial suspension is 10 6 ~10 8 cfu / mL, for example, 1.3×10 6 cfu / mL, 2.6×10 6 cfu / mL, 4.2×10 6 cfu / mL, 8.6×10 6 cfu / mL, 1.8×10 7 cfu / mL, 3.7×10 7 cfu / mL, 6.2×10 7 cfu / mL, 9.8×10 7 cfu / mL, etc.

[0016] In some embodiments of the present invention, the conditions for collecting OD values ​​of data characterizing the number of strains are: the data collection time interval is 10 to 30 minutes, for example, it can be 12.1 minutes, 16.3 minutes, 19.6 minutes, 23.6 minutes, 26.2 minutes, 29.1 minutes, etc.; the wavelength is 600nm.

[0017] The technical solution provided by the embodiment of the present invention has the following advantages:

[0018] The method for evaluating the antibacterial activity of the chemical oral solution of the embodiment of the present invention utilizes the growth curve based on OD600 to evaluate the activity of the antibacterial agent in a high-throughput, rapid and precise manner, and is used for the screening of drug preparation prescriptions and the inspection and testing of finished preparations; it overcomes the problems of the antibacterial efficacy inspection method, such as long experimental cycle, cumbersome operation process, and heavy workload.

[0019] Specifically, the evaluation method of the embodiment of the present invention is based on the positive correlation between optical density OD600 and the number of microorganisms, which can distinguish the differences in growth behavior between different strains, characterized by the trend of the growth curve and three characteristic parameters (hysteresis time λ, maximum growth rate μ max and maximum optical density OD max ). The three characteristic parameters of the control group are set as λ0, μ max0 OD max0, the ratio of the characteristic parameters of the experimental group to the control group is recorded as the relative characteristic parameter. max / μ max0 <1 or OD max / OD max0 <1, indicating that the growth ability is inhibited and the antibacterial property is strong. Taking into account the measurement error, if the relative characteristic parameter difference exceeds 10% (i.e. |λ / λ0-1|>10%, |μ max / μ max0 -1|>10% or|OD max / OD max0 -1∣>10%), or there is a difference after statistical analysis, which indicates that there is a significant difference in antibacterial activity. This evaluation method can generally detect bacteria within 24 hours and fungi within 48 hours, and can achieve high-throughput and rapid evaluation.

[0020] In addition, because the growth curve method has been applied to the research of nanomaterials, plant active ingredients, microbial populations, drug activity and pharmacodynamics, laboratories with manual or automatic optical density detectors can apply the evaluation method of the embodiment of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0023] Figure 1 This is a growth curve diagram of four strains in the "strain specificity" experiment of an embodiment of the present invention;

[0024] Figure 2 The growth curves of the strains in the bacterial solution group and the group with antibacterial agent added in the "precision and repeatability" experiment of the embodiment of the present invention are shown;

[0025] Figure 3 The growth curves of the bacterial solution group of Example 2 of the present invention, the bacterial strains with added sodium benzoate, and the bacterial strains with added potassium sorbate;

[0026] Figure 4 This is a growth curve of Pseudomonas aeruginosa after adding oral liquids from different manufacturers in Example 3 of the present invention;

[0027] Figure 5 This is a growth curve of Candida albicans after adding oral liquids from different manufacturers in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0030] The typical method for evaluating antibacterial properties is the antibacterial efficacy test method. After a high-concentration bacterial solution is mixed with the antibacterial substance, samples are taken regularly and the CFU is counted manually by the naked eye. The logarithmic value of the decrease in the number of microorganisms is used as an evaluation indicator. The entire experimental cycle takes 28 days, which is time-consuming, cumbersome and has rough standards. It is difficult to screen the appropriate type of antibacterial agent and determine the minimum effective concentration of the antibacterial agent.

[0031] Growth curves are curves that reflect the changes in the number of microorganisms over the culture time, usually including the hysteresis period, logarithmic growth period, plateau period and decay period. The drawing of growth curves has evolved from manual sampling at fixed points to manually counting microorganisms with the naked eye using CFU, to automatically and high-throughput measurement of the optical density (OD) value at around 600nm to characterize the number of microorganisms. It has the advantages of short cycle, high throughput, easy operation, and higher time resolution. The degree to which the number of microorganisms is affected by variables can be judged by analyzing the changes in the trend / characteristic parameters of the growth curve, which has been applied to food, medicine, materials and other fields.

[0032] According to an embodiment of one aspect of the present invention, a method for evaluating the antibacterial activity of a chemical oral solution is provided, comprising the following steps: preparing a bacterial suspension of a strain; mixing a sample to be tested, the bacterial suspension and a liquid culture medium, culturing the mixture, collecting OD values ​​representing the number of strains, and drawing a growth curve of a test group; obtaining a hysteresis time λ and a maximum growth rate μ of the strain in the test group according to the growth curve of the test group; max and maximum optical density OD max ; Mix the bacterial suspension and liquid culture medium, culture them, collect the OD value of the data characterizing the number of strains, and draw the growth curve of the control group; according to the growth curve of the control group, obtain the hysteresis time λ0 and the maximum growth rate μ of the control group strain max0 and maximum optical density OD max0 ; When at least λ / λ0>1, μ max / μ max0 <1, OD max / OD max0 <1, the sample to be tested is judged to have antibacterial properties, and the larger the λ / λ0 value, the greater the μ max / μ max0 The smaller the value, the higher the OD max / OD max0 The smaller the value, the stronger the antibacterial activity of the sample to be tested.

[0033] In some embodiments of the present invention, a method for evaluating the antibacterial activity of antibacterial agents (including acids, parabens, alcohols, etc.) commonly used in chemical oral solutions comprises the following steps: using an automatic growth curve analyzer based on OD600 to evaluate the ability of the antibacterial agent to inhibit / kill microorganisms in a microplate in a high-throughput manner, which is manifested as changing the growth rate and growth behavior of the microorganisms, that is, affecting the trend of the growth curve, which can be specifically digitized into three characteristic parameters (including hysteresis time λ, maximum growth rate μ max and maximum optical density OD max ) changes, and establish a method for rapid evaluation of antibacterial activity.

[0034] In order to further illustrate the effect of the present invention, specific embodiments are used for illustration below.

[0035] 1. Preparation of experimental strains and samples

[0036] 1.1 Selection and preparation of experimental strains

[0037] Referring to the relevant content of the antibacterial efficacy test method in Part IV of the Chinese Pharmacopoeia, 1121, we selected five experimental strains: Staphylococcus aureus [CMCC(B)26003], Escherichia coli [CMCC(B)44102], Pseudomonas aeruginosa [CMCC(B)10104], Candida albicans [CMCC(F)98001], and Aspergillus niger [CMCC(F)98003]. The first four strains were adjusted to 10 6 ~10 8 cfu / mL, Aspergillus niger will not be discussed because the number of microorganisms is not proportional to the OD value.

[0038] 1.2 Sample preparation

[0039] Accurately weigh 0.2560 g of benzalkonium bromide, place it in a 25 mL volumetric flask, add sterile purified water to dissolve and dilute to the mark, shake well, and prepare a 1024 μg / mL stock solution for immediate use.

[0040] 2. Mixed culture of components and data collection

[0041] 2.1 Addition of components to the microplate

[0042] First, add 100 μL of liquid culture medium to each well, tryptic soy broth (TSB) for bacterial culture or Sabouraud dextrose broth (SDB) for fungal culture. Then, add 20 μL of the prepared antibacterial sample and 80 μL of TSB / SDB to the wells in the first column, and draw 100 μL from each well in the first column and add it to the wells in the second column for 2-fold dilution. Finally, add 100 μL of experimental bacterial solution to each well, and the total volume of each well is 200 μL. Set up a positive control in which TSB / SDB replaces the antibacterial agent (i.e., the culture medium in the positive control group is not added with an antibacterial agent, and the composition of the culture medium is a liquid culture medium and an experimental bacterial solution; wherein the liquid culture medium is TSB or SDB), and a negative control in which TSB / SDB replaces the bacterial solution (i.e., the culture medium in the negative control group is not added with an experimental bacterial solution, and the composition of the nutrient solution is a liquid culture medium and an antibacterial agent; wherein the liquid culture medium is TSB or SDB).

[0043] 2.2 Data collection and analysis

[0044] The microplate used in this experiment is a 96-well microplate, and the automatic microbial monitor is an instrument produced by BioTek in the United States. The culture temperature is 33°C, the rotation speed is 600rpm, and the data collection time interval is 20min. Based on the growth curve and data analysis software Gen5, three characteristic parameters can be calculated: hysteresis time (λ) (h), maximum growth rate (μ max )(h -1 ) and maximum OD value (OD max ). max The maximum slope of the growth curve is defined as μ. Starting from the first collection point, the program defines 7 collection points as a set of data for linear regression to evaluate the slope, and then repeats the above operation from the second collection point, and so on to report the maximum slope. λ is the time for the curve to grow slowly, which is defined by the program as μ max The time at which the associated linear regression intersects the horizontal line of the first acquisition point. max To obtain the maximum value of the growth curve, the program defines 7 collection points as a set of data and reports the maximum value among the average values.

[0045] 3. Result judgment

[0046] The three characteristic parameters of the control group without adding antibacterial agent are set as λ0, μ max0 OD max0 , the three characteristic parameters under other conditions are the ratios relative to the control group, recorded as relative characteristic parameters. Antibacterial activity can be reflected by the simultaneous change of one, two or three of the three relative characteristic parameters, and the strength of antibacterial activity can be judged according to the change trend and size. Specifically, the increase of λ value means that it takes longer for microorganisms to grow, the growth ability is inhibited, and the antibacterial activity is stronger; μ maxThe value decreases, indicating that the microbial growth rate slows down, the growth ability is inhibited, and the antibacterial property is strong; OD max The value decreases, which means that the total number of microorganisms is reduced, the growth ability is inhibited, and the antibacterial property is strong. Taking into account factors such as instrument measurement errors, in order to ensure the accuracy and reliability of the results, the three relative characteristic parameters of the experimental group are compared with those of the control group. max0 OD max0 If the difference exceeds 10%, or is proven to be significant by statistical tools, it indicates that there is a significant difference in antibacterial activity.

[0047] 4. Method Validation

[0048] 4.1 Strain specificity

[0049] At the same initial inoculum size of 10 4 Under the condition of 100 cfu / well, the growth curves of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans all showed S-shaped growth curves, and the three characteristic parameters λ, μ max OD max All of them are specific and can be used to distinguish different strains.

[0050] 4.2 Precision and repeatability

[0051] Taking Candida albicans as an example, set up the bacterial liquid group (10 4 cfu / well of Candida albicans) and the group with antibacterial agent (10 4 cfu / well of Candida albicans + 12 μg / mL benzalkonium bromide, of which 10 4 cfu / well represents the initial inoculation level of Candida albicans, and 12 μg / mL represents the initial concentration of the antibacterial agent benzalkonium bromide in the microplate). Each group has 6 parallel wells, and the growth curve is drawn ( Figure 2 ), and calculated three characteristic parameters (Table 2). The results showed that the growth curves of the six parallel wells basically overlapped, and the CV% of the three characteristic parameters of the six parallel wells were all less than 10%, which was within the acceptable range.

[0052] Table 2 Repeatability and CV values ​​of three characteristic parameters in the bacterial solution group and the group with antibacterial agent

[0053] Grouping Bacterial liquid group Add antibacterial agent group λ(h) <![CDATA[7.4241±0.1744(λ0)]]> <![CDATA[12.0741±0.6836(1.6λ0)]]> CV% 2.35 5.66 <![CDATA[μ max (h -1 )]]> <![CDATA[0.1435±0.0049(μ max0 )]]> <![CDATA[0.16535±0.0082(1.1μ max0 )]]> CV% 3.38 4.94 <![CDATA[OD max ]]> <![CDATA[1.349±0.01(OD max0 )]]> <![CDATA[1.659±0.008(1.2OD max0 )]]> CV% 0.75 0.48

[0054] 4.3 Investigation of influencing factors

[0055] (1) Investigation of initial inoculation volume of microplate

[0056] The measurement range of the OD value of the microbial growth curve monitor is 0.000~4.000. If the concentration of the initial inoculum is too high during the experiment, the initial OD value will be too large, thus affecting the accuracy of the lambda value calculation result. The experiment shows that the initial inoculum range should be less than 10 6 cfu / hole.

[0057] (2) Investigation of the total volume of liquid in each well of a microplate

[0058] The total volume of liquid in each well was set to 150μL and 200μL, and the trend differences of the growth curves of the four strains were compared. The results showed that when the total volume of liquid in each well was different, the growth curve trend showed a typical S-shaped curve. The 150μL curve moved slightly to the right compared to the 200μL curve, and the peak of the curve was slightly lower. For the convenience of calculation, the total volume of liquid in each well of the microplate was determined to be 200μL.

[0059] (3) Effect of culture medium dilution

[0060] Liquid culture medium stock solution and 2-fold diluted culture medium were prepared, and the growth curve trends of the four strains were compared with or without dilution. The results showed that the growth curve trends showed a typical S-shaped curve before and after the culture medium was diluted. Compared with the undiluted culture medium, the hysteresis time and maximum growth rate of the curve did not change significantly after dilution, and the peak value of the curve was slightly lower, which proved that whether the culture medium was diluted or not basically did not affect the trend of the growth curve and the calculation of the three characteristic parameters.

[0061] (4) Influence of instrument rotation speed

[0062] The growth curve needs to set a certain speed during the data collection stage to make the microorganisms dispersed as evenly as possible in the microplate. The speed of the instrument was set to 600rpm and 800rpm, and the trend differences of the growth curves of the four strains at different speeds were compared. The results showed that Escherichia coli and Candida albicans would adhere to the wall at 800rpm, and the adhesion phenomenon disappeared when the speed was adjusted to 600rpm. Therefore, the speed of the instrument was determined to be 600rpm.

[0063] Example 1: Rapid evaluation of pH on microbial antibacterial activity

[0064] Preparation of culture media with different pH values: Use dilute hydrochloric acid and sodium carbonate solution as regulators and measure with a pH meter. Prepare TSB and SDB with different pH values ​​of 3.0, 4.0, 5.0, 6.0, and 7.0. Place the culture media in a pressure sterilizer and sterilize at 121°C for 15 min. Use the culture media after cooling after sterilization.

[0065] Addition of components to the microplate and data collection: Add bacterial suspensions in different pH media to each well to make the inoculation level of the strain 10 4cfu / well, the instrument culture temperature was set to 33°C, the OD value data collection cycle was 20 min / time, the total measurement time for Staphylococcus aureus and Escherichia coli was 24 h, the total measurement time for Pseudomonas aeruginosa and Candida albicans was 48 h, and the measurement was repeated 3 times.

[0066] As shown in Table 3, overall, as the pH value gradually decreases from 7.0, the λ value of the three bacteria increases, and μ max Reduce, OD max The growth ability of Staphylococcus aureus was gradually inhibited, and under the conditions of pH 3.0 and 4.0, the growth curve had no growth trend and characteristic parameters could not be obtained. max OD max All of them have changed, especially μ max OD max More obvious; λ, μ of Escherichia coli max All of them have changed, especially λ, OD max There is almost no change; the λ and μ of Pseudomonas aeruginosa max All changes occurred, OD max The growth of Candida albicans was promoted at pH 4.0-6.0 compared with pH 7.0, but was inhibited at pH 3.0 compared with pH 7.0. max All changes occurred, OD max There is almost no change, which is consistent with the literature report that the optimal growth pH of Candida albicans is 4-6.

[0067] Table 3 Three relative characteristic parameters of four strains under different pH conditions

[0068]

[0069]

[0070] Note: “*” indicates that characteristic parameters, λ, μ, cannot be obtained. max OD max The numbers 1, 2, 3, and 4 represent the experimental strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans, respectively.

[0071] Example 2: Rapid evaluation of the antibacterial activity of different types of compounds

[0072] The results of the determination of the minimum inhibitory concentration (MIC) showed that the MIC values ​​of sodium benzoate and potassium sorbate against Candida albicans at pH 4.0 were the same, both 0.25 mg / mL. In order to further compare the differences in their antibacterial activities, the growth curve characteristic parameter method was used for analysis.

[0073] Sample preparation: Accurately weigh 0.0625 g of sodium benzoate and potassium sorbate, place in a 25 mL volumetric flask, add sterile purified water to dissolve and dilute to the mark, shake well, and prepare a 2.5 mg / mL antibacterial agent stock solution for immediate use.

[0074] Addition of components to the microplate and data collection: Set the pH of the culture condition to 4.0. Add 100 μL of bacterial suspension, 80 μL of culture medium, and 20 μL of antibacterial agent sample to each well to make the inoculation level of the strain 10 4 cfu / well. Set the instrument culture temperature to 33°C, the OD value data collection cycle to 20 min / time, the total measurement time of Candida albicans to 48 h, repeat 3 times, draw the growth curve, and analyze the 3 relative characteristic parameters.

[0075] Depend on Figure 3 As shown in Table 4, the growth curves of microorganisms change in different trends after adding two antibacterial agents. max All of them changed, μ max More obvious; adding potassium sorbate λ, μ max Both of them changed, λ was more obvious, and OD max There was a slight increase.

[0076] Table 4 Three relative characteristic parameters of sodium benzoate and potassium sorbate against Candida albicans at pH 4.0

[0077] Grouping λ <![CDATA[μ max ]]> <![CDATA[OD max ]]> Bacterial liquid group <![CDATA[λ0]]> <![CDATA[μ max 0]]> <![CDATA[OD max0 ]]> Sodium Benzoate <![CDATA[1.3λ0]]> <![CDATA[0.5μ max 0]]> <![CDATA[1.0OD max0 ]]> Potassium Sorbate <![CDATA[2.1λ0]]> <![CDATA[0.6μ max 0]]> <![CDATA[1.1OD max0 ]]>

[0078] Example 3: Rapid evaluation of antibacterial properties of finished formulations

[0079] Sample preparation: Take an oral solution of the same variety produced by different manufacturers, and record the 8 manufacturers as A, B, C, D, E, F, G, and H.

[0080] Addition of components to the microplate and data collection: Pseudomonas aeruginosa was used as an example for bacteria, and Candida albicans was used as an example for fungi. 100 μL of bacterial suspension and 100 μL of sample were added to each well to make the inoculation level of the strain 10 6 cfu / well. Set the instrument culture temperature to 33°C, the OD value data collection cycle to 20 min / time, the total measurement time to 24 h, and repeat 3 times to draw the growth curve.

[0081] Depend on Figure 4 , Figure 5 It can be seen that there are certain differences in the trends of the growth curves of samples produced by different companies, which can be used for preliminary qualitative comparison of the strength of antibacterial activity. The stronger the growth ability shown by the growth curve, the weaker the antibacterial activity. According to the trend of the growth curve, the antibacterial activity of samples produced by different companies was ranked, and compared with the antibacterial efficacy test method of the pharmacopoeia method in the previous study, as shown in Table 5. Because the general method (antibacterial efficacy test method) samples and measures at two time points of 14 days and 28 days, when the logarithmic values ​​of the microbial decline are equivalent, the strength of antibacterial activity cannot be compared. For Pseudomonas aeruginosa, the antibacterial efficacy test method can determine the strength of antibacterial activity of samples produced by 0 companies, and the growth curve method can determine the strength of antibacterial activity of samples produced by 4 companies; for Candida albicans, the antibacterial efficacy test method can determine the strength of antibacterial activity of samples produced by 4 companies, and the growth curve method can determine the strength of antibacterial activity of samples produced by 6 companies. That is to say, when the antibacterial efficacy test method cannot distinguish the strength of antibacterial activity, the growth curve method can further distinguish, and this method is more sensitive and convenient. It can be used for preliminary method applicability research and quality monitoring throughout the product life cycle.

[0082] Table 5 Comparison of the growth curve method and the antibacterial efficacy test method in evaluating the antibacterial strength of the same variety from different companies

[0083] Bacteria Growth curve method-ranking of antibacterial properties of different companies Antibacterial efficacy test method-antibacterial ranking of different companies Pseudomonas aeruginosa D<B<G≈A<C≈E≈F≈H A≈B≈C≈D≈E≈F≈G≈H Candida albicans D<G<B<A≈F≈H<C<E D<G<B<A≈C≈E≈F≈H

[0084] In summary, the method for evaluating the antibacterial properties of the chemical oral solution of the embodiment of the present invention overcomes the disadvantages of the existing antibacterial agent evaluation method, namely the antibacterial efficacy inspection method, such as long experimental cycle, cumbersome operation process, and large workload. It uses the relatively mature technology based on the OD600 growth curve to evaluate the activity of the antibacterial agent in a high-throughput, rapid, and precise manner, and is used for the screening of drug preparation prescriptions and the inspection and detection of finished preparations.

[0085] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0086] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for evaluating the antibacterial activity of a chemical oral solution, comprising the following steps: Prepare bacterial suspension of the strain; The sample to be tested, the bacterial suspension and the liquid culture medium are mixed and cultured, the OD value representing the number of strains is collected, and the growth curve of the test group is drawn; According to the growth curve of the test group, the hysteresis time λ and the maximum growth rate μ of the strain in the test group are obtained. max and maximum optical density OD max ; The bacterial suspension and liquid culture medium were mixed and cultured, and the OD value representing the number of strains was collected to draw a growth curve of the control group; The hysteresis time λ0 and the maximum growth rate μ of the control group strain were obtained according to the growth curve of the control group. max0 and maximum optical density OD max0 ; When at least λ / λ0>1, μ max / μ max0 <1, OD max / OD max0 <1, the sample to be tested is judged to have antibacterial properties, and the larger the λ / λ0 value, the greater the μ max / μ max0 The smaller the value, the higher the OD max / OD max0 The smaller the value, the stronger the antibacterial activity of the sample to be tested.

2. The evaluation method according to claim 1, characterized in that: The strain includes at least one of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans.

3. The evaluation method according to claim 1, characterized in that: The liquid culture medium includes at least one of a trypticase soy liquid culture medium and a Sabouraud dextrose liquid culture medium.

4. The evaluation method according to claim 1, characterized in that: The culturing is carried out in a microplate; Among them, the initial inoculum size M of the strain is: 0 cfu / well <M<10 6 cfu / hole.

5. The evaluation method according to claim 1, characterized in that: The culture temperature is 30-35°C, and the rotation speed of the instrument is 600rpm-800rpm.

6. The evaluation method according to claim 1, characterized in that: The maximum growth rate μ max is the maximum slope of the growth curve, which is calculated as follows: starting from the first collection point, define 7 collection points as a set of data for linear regression to evaluate the slope, and then repeat the above operation from the second collection point, and so on, until the maximum slope is obtained.

7. The evaluation method according to claim 1, characterized in that: The hysteresis time λ is the time for the growth curve to slowly increase, defined as μ max The time intercept of the associated linear regression intersects the horizontal line of the first acquisition point.

8. The evaluation method according to claim 1, characterized in that: The maximum optical density value OD max To obtain the maximum value of the growth curve, define 7 collection points as a set of data and report the maximum value among the average values.

9. The evaluation method according to claim 1, characterized in that: In the bacterial suspension, the number of strains is 10 6 ~10 8 cfu / mL.

10. The evaluation method according to claim 1, characterized in that: The conditions for collecting the OD value of the data characterizing the number of strains are: The data collection time interval is 10 to 30 minutes, and the wavelength is 600nm.

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