CN108424949B - Method for rapidly screening electronic cigarette liquid based on streptococcus mutans - Google Patents
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Abstract
The invention relates to a method for rapidly screening electronic cigarette liquid based on streptococcus mutans, and belongs to the technical field of electronic cigarette research and development. The method combines an in-vitro bacteriostasis detection method and a tobacco juice blending technology, firstly prepares tobacco juice containing different plant extracts, and screens the prepared tobacco juice by adopting a trace dilution bacteriostasis detection technology to obtain the tobacco juice with bacteriostasis activity. The method of the invention is characterized in that: the streptococcus mutans is used as a test strain, a trace dilution bacteriostasis detection system suitable for the electronic cigarette liquid is determined, the screening detection efficiency is high, a cigarette liquid formula with bacteriostasis activity can be obtained in a short time, and effective technical support is provided for obtaining safe and high-quality electronic cigarette liquid.
Description
Technical Field
The invention relates to a method for rapidly screening electronic cigarette liquid based on streptococcus mutans, and belongs to the technical field of electronic cigarette research and development.
Background
The electronic cigarette, also called as an electric atomization cigarette, is a novel tobacco product which is close to the traditional cigarette in aspects of appearance, physiological feeling, psychological perception, smoking mode and the like. The electronic cigarette transfers nicotine to a respiratory system through an electronic heating means, and mainly comprises a cigarette rod, an atomizer, a suction nozzle, a charger, a cigarette bullet and the like. The tobacco juice is a main smoking raw material of an electronic cigarette product, volatilizes under low-temperature heating to generate smoke, and the formula of the tobacco juice is also one of the core technologies of the electronic cigarette. However, the flavor of the cigarette liquid smoke is greatly different from the flavor of the traditional cigarette, and the acceptance of the cigarette liquid by consumers is not high, so the formula of the cigarette liquid needs to be improved to meet the requirements of the consumers. For example, the invention patent CN104397871B discloses a formula of electronic cigarette liquid containing tea extract, which is added with tea extract, so as to improve the smoke of smoking cigarettes, enrich the cigarette fragrance, reduce the smoking harm as much as possible, and also play a role in promoting the production of body fluid, preserving the moisture, reducing the stimulation and improving the quality of electronic cigarettes. The invention patent CN105876867A discloses an electronic cigarette liquid formula containing eucommia ulmoides extracts, which has good fragrance and good taste, and also has the effects of resisting and preventing cancer, viruses, oxidation and aging, reducing blood pressure, diminishing inflammation and inhibiting bacteria, protecting gallbladder and benefiting liver, calming and hypnotizing, tonifying kidney and strengthening body, relieving pain and preventing miscarriage, losing weight, preventing senile dementia and the like. Therefore, the natural plant extract is a high-quality formula raw material of the electronic cigarette liquid.
In addition, the electronic cigarette is an electronic product which is repeatedly used, bacteria are easy to breed after the electronic cigarette is used for a long time, and the research and development of the cigarette liquid with the antibacterial activity become important contents of the research and development of the electronic cigarette. The bacteria are very diverse in variety, have great influence and effect difference on human health, have unobvious effect on some bacteria, have probiotic effect and are called probiotics and have harmful effect and are called pathogenic bacteria. A large number of researches show that the difference of the bacteriostatic action of the same active ingredient on different bacteria is obvious, so that the screening of the bacteriostatic active ingredients aiming at different test strains is very important. At present, no published report exists for screening the antibacterial activity of the electronic cigarette oil aiming at different bacteria.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for rapidly screening electronic cigarette liquid based on streptococcus mutans, and provides effective technical support for obtaining safe and high-quality electronic cigarette liquid.
Streptococcus mutans (Streptococcus mutans) Is a gram-positive coccus belonging to the genus Streptococcus (Streptococcus) Is an important population in the oral natural flora, is a main pathogenic bacterium of human caries and has more transmission modes. The method is based on streptococcus mutans, combines an in-vitro bacteriostasis detection method and a tobacco juice blending technology, firstly prepares tobacco juice containing different plant extracts, screens the prepared tobacco juice by adopting a trace dilution bacteriostasis detection technology to obtain the tobacco juice with better bacteriostatic activity, and provides effective technical support for obtaining safe and high-quality electronic tobacco juice.
The technical scheme of the invention is as follows: a method for rapidly screening electronic cigarette liquid based on streptococcus mutans comprises the following steps:
(1) preparation of test electronic tobacco juice samples
Weighing 10 g of basic electronic cigarette liquid sample, determining the addition amount of the plant extract according to the sensory evaluation effect, wherein the addition range is 0.01-0.5 g, and the control group is the basic electronic cigarette liquid;
(2) determination of the bacteriostatic Activity
1) Activating and culturing strains: taking out the frozen strain from the liquid nitrogen tank, and quickly melting the strain in a water bath at 37 ℃ to obtain a resuscitation bacteria liquid; inoculating the resuscitation bacteria liquid into a sterilized nutrient broth culture medium according to the proportion of 1.0% (v/v), placing the sterilized nutrient broth culture medium into an anaerobic incubator at 36 ℃, and standing and culturing for 24-30 h to obtain 1 st activated bacteria liquid; inoculating the 1 st activated bacterial liquid into a sterilized nutrient broth culture medium according to the proportion of 1.0% (v/v), and then placing the sterilized nutrient broth culture medium into an anaerobic incubator at 36 ℃, and standing and culturing for 24-30 h to obtain a 2 nd activated bacterial liquid;
2) and (3) measuring the concentration of the bacterial liquid: (a) taking 1mL of the 2 nd activated bacterium liquid, and adding 9mLPBS buffer solution to prepare 1:10 bacterium suspension; (b) preparing 10-fold serial diluted bacterial suspension according to the operation of (a); (c) selecting 3 dilutions in the 10-time serial diluted bacterial suspension, adding 1mL of the diluted bacterial suspension into an aseptic plate, adding 20mL of nutrient broth agar culture medium, uniformly mixing, placing the mixture in an anaerobic incubator for 24-30 h at 36 ℃, counting the number of bacterial colonies on the plate, and multiplying the number by the dilution times to calculate the concentration of the 2 nd activated bacterial liquid;
3) preparation of test bacterial suspension: calculating and adding the 2 nd activated bacterium liquid into the double-concentration nutrient broth culture medium according to the concentration of the 2 nd activated bacterium liquid to prepare a test bacterium suspension with the concentration of 106-108CFU/mL；
4) Sample adding: setting a control group, a blank group and an experimental group on a sterile 96-hole culture plate; adding 100 mu LPBS buffer solution into a control group and a blank group, and adding 100 mu L of electronic cigarette liquid with different dilution times into an experimental group; adding 100 μ L of double concentration nutrient broth medium to the blank group, adding 100 μ L of test bacterial suspension to the control group and the experimental group, and paralleling each group with 6 wells;
5) culturing: placing the 96-hole culture plate in an anaerobic incubator, and carrying out anaerobic constant-temperature culture at 36 ℃ for 24-30 h;
6) MTT incubation treatment: taking out the cultured 96-well culture plate, centrifuging at 10,000 rpm in a microplate centrifuge at room temperature for 5min, and removing the supernatant; adding 1.0-5.0 mg/mL MTT solution with 100 mu L/hole, placing on a microplate oscillator, oscillating for 5min at 36 ℃, and then placing in an anaerobic incubator, and incubating for 1.0-2.0 h at 36 ℃;
7) and (3) light absorption value determination: taking out the incubated 96-well culture plate, centrifuging at 10,000 rpm in a microplate centrifuge at room temperature for 5min, and removing the supernatant; adding DMSO (dimethylsulfoxide) at a concentration of 150 mu L/hole, and placing on a microplate oscillator for oscillation at 36 ℃ for 5 min; standing, and measuring light absorption values of a control group, a blank group and an experimental group at 570nm on an enzyme labeling instrument;
(3) calculation of bacteriostatic Activity
The bacteriostatic activity of the electronic cigarette liquid is expressed by bacteriostatic rate I, and is calculated according to a formula (1) according to a light absorption value, wherein the formula is as follows:
in the formula: i, bacteriostasis rate; ODn-average absorbance of the experimental group; ODo-average absorbance of the blank set; ODc-average absorbance of control group.
Preparation of the nutrient broth culture medium: 9 g of nutrient broth culture medium powder was weighed, 500 mL of distilled water was added, and autoclaving was performed at 121 ℃ for 15 min to prepare a nutrient broth culture medium.
Preparation of the double concentration nutrient broth: 18g of nutrient broth powder was weighed, 500 mL of distilled water was added, and autoclaving was carried out at 121 ℃ for 15 min to prepare a double concentration nutrient broth.
The preparation of the nutrient broth agar culture medium comprises the following steps: 9.0 g of the nutrient broth agar medium powder was weighed, 6g of agar and 500 mL of distilled water were added, and autoclaved at 121 ℃ for 15 min to prepare a nutrient broth agar medium.
The invention is characterized in that:
1. adopting a trace dilution bacteriostasis detection system suitable for electronic cigarette liquid to screen the bacteriostasis activity of the electronic cigarette liquid, wherein a test strain adopts streptococcus mutans; 2. the detection system has high screening efficiency, and can obtain a tobacco juice formula with antibacterial activity in a short time; 3. the method can reduce the development cost and the period of the tobacco juice and provide effective technical support for obtaining safe and high-quality electronic tobacco juice.
Description of the drawings:
FIG. 1 is a graph of the bacteriostatic activity of tobacco juice containing mint extract and control tobacco juice on Streptococcus mutans.
FIG. 2 is a graph showing the bacteriostatic activity of tea extract-containing tobacco juice and control tobacco juice against Streptococcus mutans.
FIG. 3 is a graph showing the bacteriostatic activity of tobacco juice containing clove extract and control tobacco juice against Streptococcus mutans.
FIG. 4 is a graph showing the bacteriostatic activity of a tobacco juice containing an extract of Aucklandia lappa and a control tobacco juice against Streptococcus mutans.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of examples of the present invention, and not all examples. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
Example 1
1 materials and methods
1.1 test strains: streptococcus mutans (Streptococcus mutansATCC 25175) purchased from the collection of microorganisms of the guangdong province.
1.2 reagents and culture media: PBS buffer (pH 7.2-7.4), HyClone, USA; 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide (MTT), Sigma, USA; dimethylsulfoxide (DMSO), west longa chemical ltd; kanamycin, bio-engineering (shanghai) incorporated; nutrient broth medium, hangzhou microbial agents ltd.
1.3 instruments and devices: a secondary biosafety cabinet, Shanghai Likang, model HFsafe-1500; a microplate oscillator; inverted microscope, Nikon, Japan, model ECLIPSE TS 100-F-HMC; autoclave, available from ALP corporation of Japan, model CL-40M; electronic balance, Mettler Toledo, Switzerland, type XS204, sensitivity 0.1 mg; a pH meter, Mettler Toledo, Switzerland, Seven Esay S20K; a water-proof constant temperature incubator, Guangzhou Yongcheng company, model GHP-9270; a microplate centrifuge; a dual-beam ultraviolet spectrophotometer, model TU-1901, common instruments, beijing; microplate reader, Bio-Rad, USA, Model 680; constant temperature water bath shaker, VWS20, VIVO, Germany; anaerobic incubator, Shellab, USA, BACTRON type.
1.4 Experimental methods
1.4.1 preparation of the culture Medium
(1) Nutrient broth culture medium: weighing 9.0 g of nutrient broth culture medium powder, adding 500 mL of distilled water, and sterilizing with high pressure steam at 121 deg.C for 15 min to obtain nutrient broth culture medium;
(2) double concentration nutrient broth: weighing 18.0 g of nutrient broth culture medium powder, adding 500 mL of distilled water, and sterilizing with high pressure steam at 121 deg.C for 15 min to obtain double concentration nutrient broth culture medium;
(3) nutrient broth agar medium: 9.0 g of the nutrient broth agar medium powder was weighed, 6.0 g of agar and 500 mL of distilled water were added, and autoclaved at 121 ℃ for 15 min to prepare a nutrient broth agar medium.
1.4.2 Strain activation and concentration determination
(1) Activating and culturing strains: taking out the frozen strain from the liquid nitrogen tank, and quickly melting the strain in a water bath at 37 ℃ to obtain a resuscitation bacteria liquid; inoculating the resuscitation bacteria liquid into a sterilized nutrient broth culture medium according to the proportion of 1.0% (v/v), placing the sterilized nutrient broth culture medium into an anaerobic incubator at 36 ℃, and standing and culturing for 24-30 h to obtain 1 st activated bacteria liquid; inoculating the 1 st activated bacterial liquid into a sterilized nutrient broth culture medium according to the proportion of 1.0% (v/v), and then placing the sterilized nutrient broth culture medium into an anaerobic incubator at 36 ℃, and standing and culturing for 24-30 h to obtain a 2 nd activated bacterial liquid;
(2) and (3) measuring the concentration of the bacterial liquid: (a) taking 1mL of the 2 nd activated bacterium liquid, and adding 9mLPBS buffer solution to prepare 1:10 bacterium suspension; (b) preparing 10-fold serial diluted bacterial suspension according to the operation (a) in the step 1.4.2 (1); (c) and selecting 3 dilutions in the 10-time serial diluted bacterial suspension, adding 1mL of the diluted bacterial suspension into an aseptic plate, adding 20mL of nutrient broth agar culture medium, uniformly mixing, placing the mixture in an anaerobic incubator for culturing at 36 ℃ for 24-30 h, counting the number of bacterial colonies on the plate, and multiplying the number by the dilution times to calculate the concentration of the 2 nd activated bacterial liquid.
1.4.3 preparation of test electronic cigarette liquid samples
Weighing 10.0g of a basic electronic cigarette liquid sample, adding 0.01g of mint extract according to sensory evaluation results, and numbering YY 1; the control group is the basic electronic cigarette liquid with the number YY 0.
1.4.4 measurement of bacteriostatic Activity
(1) Preparation of test bacterial suspension: calculating the concentration of the 2 nd activated bacterium solution obtained according to 1.4.2, and adding the 2 nd activated bacterium solution into the double concentration nutrient broth culture medium to obtain a test bacterium suspension with the concentration of 106 CFU/mL；
(2) Sample adding: setting a control group, a blank group and an experimental group on a sterile 96-hole culture plate; adding 100 mu LPBS buffer solution into a control group and a blank group, and adding 100 mu L of electronic cigarette liquid with different dilution times into an experimental group; adding 100 μ L of double concentration nutrient broth medium to the blank group, adding 100 μ L of test bacterial suspension to the control group and the experimental group, and paralleling each group with 6 wells;
(3) culturing: placing the 96-hole culture plate in an anaerobic incubator, and carrying out anaerobic constant-temperature culture at 36 ℃ for 24 h;
(4) MTT incubation treatment: taking out the cultured 96-well culture plate, centrifuging at 10,000 rpm in a microplate centrifuge at room temperature for 5min, and removing the supernatant; adding 1.0 mg/mL MTT solution, 100 muL/hole, placing on a microplate oscillator, oscillating for 5min at 36 ℃, and then placing in an anaerobic incubator, and incubating for 1.0h at 36 ℃;
(5) and (3) light absorption value determination: taking out the incubated 96-well culture plate, centrifuging at 10,000 rpm in a microplate centrifuge at room temperature for 5min, and removing the supernatant; adding DMSO (dimethylsulfoxide) at a concentration of 150 mu L/hole, and placing on a microplate oscillator for oscillation at 36 ℃ for 5 min; after standing, the sample is placed on a microplate reader to measure the absorbance at 570 nm.
1.4.5 calculation of bacteriostatic activity:
the bacteriostatic activity of the electronic cigarette liquid is expressed by bacteriostatic rate I, and is calculated according to a formula (1) according to a light absorption value, wherein the formula is as follows:
in the formula: i, bacteriostasis rate; ODn-average absorbance of the experimental group; ODo-average absorbance of the blank set; ODc-average absorbance of control group.
2 results and analysis
The stock solution of the control tobacco juice YY0 has certain inhibiting effect on streptococcus mutans, wherein the bacteriostatic rates of the stock solution and the 1/2 stock solution are 14.43 percent and 9.98 percent respectively, and the 1/4 stock solution has no inhibiting effect.
Compared with a control tobacco juice YY0, the tobacco juice YY1 added with the mint extract has obvious inhibition effect on streptococcus mutans, and the bacteriostasis rate of the stock solution is 30.50 percent and is obviously higher than 14.43 percent of the stock solution of the control tobacco juice. Therefore, compared with the control tobacco juice, the tobacco juice added with the mint extract has a more obvious inhibition effect on streptococcus mutans.
Example 2
Weighing 10.0g of a basic electronic cigarette liquid sample, adding 0.5g of tea extract according to sensory evaluation results, and numbering YY 2; the control group is the basic electronic cigarette liquid with the number YY 0.
The material and method of example 1 was followed for the bacteriostatic activity test, wherein the concentration of the Streptococcus mutans suspension was 107CFU/mL; loading the sample, placing the sample in an anaerobic incubator, and carrying out anaerobic culture at 36 ℃ for 28 h; the concentration of MTT test solution is 5.0mg/mL, and the solution is incubated for 1.5 h at 36 ℃ under anaerobic constant temperature.
The result shows that compared with the control tobacco juice YY0, the tobacco juice YY2 added with the tea extract has obvious inhibition effect on the streptococcus mutans, and the bacteriostasis rate of the stock solution is 35.32 percent and is obviously higher than 14.43 percent of the stock solution of the control tobacco juice. Therefore, compared with the control tobacco juice, the tobacco juice added with the tea extract has obvious inhibition effect on streptococcus mutans.
Example 3
Weighing 10.0g of a basic electronic cigarette liquid sample, adding 0.2g of clove extract according to sensory evaluation results, and numbering the sample YY 3; the control group is the basic electronic cigarette liquid with the number YY 0.
The material and method of example 1 was followed for the bacteriostatic activity test, wherein the concentration of the Streptococcus mutans suspension was 108CFU/mL; loading the sample, placing the sample in an anaerobic incubator, and carrying out anaerobic culture at 36 ℃ for 30 h; the concentration of MTT test solution is 3.0 mg/mL, and the solution is incubated for 1.5 h at 36 ℃ under anaerobic constant temperature.
The result shows that the tobacco juice YY3 added with the clove extract has an inhibiting effect on streptococcus mutans compared with the control tobacco juice YY0, the bacteriostatic rates of the stock solution and the 1/2 stock solution are 14.77% and 5.35% respectively, and are basically consistent with the bacteriostatic rate of the control tobacco juice. Therefore, the tobacco liquid added with the clove extract has lower inhibition effect on streptococcus mutans compared with the control tobacco liquid.
Example 4
Weighing 10.0g of basic electronic cigarette liquid sample, adding 0.1g of elecampane extract according to sensory evaluation result, and numbering YY 4; the control group is the basic electronic cigarette liquid with the number YY 0.
The material and method of example 1 was followed for the bacteriostatic activity test, wherein the concentration of the Streptococcus mutans suspension was 107CFU/mL; loading the sample, placing the sample in an anaerobic incubator, and carrying out anaerobic culture at 36 ℃ for 26 h; the concentration of MTT test solution is 4.0 mg/mL, and the solution is incubated for 2.0h at the constant anaerobic temperature of 36 ℃.
The result shows that compared with the control tobacco juice YY0, the tobacco juice YY4 added with the costus root extract has the inhibiting effect on streptococcus mutans, the bacteriostasis rates of the stock solution and the 1/2 stock solution are respectively 35.73% and 9.77%, and the bacteriostasis rate of the stock solution is obviously higher than that of the control tobacco juice. Therefore, compared with the control tobacco juice, the tobacco juice added with the costus root extract has obvious inhibition effect on streptococcus mutans.
Claims (1)
1. A method for rapidly screening electronic cigarette liquid based on streptococcus mutans is characterized by comprising the following steps: the method combines an in-vitro bacteriostasis detection method and a tobacco juice preparation technology, adopts streptococcus mutans as a test strain, and is used for rapidly screening the electronic tobacco juice, and comprises the following specific steps:
(1) preparation of test electronic tobacco juice samples
Weighing 10.0g of a basic electronic cigarette liquid sample, determining the addition amount of the plant extract according to the sensory evaluation effect, wherein the addition amount is 0.01-0.5 g, and the control group is the basic electronic cigarette liquid;
(2) determination of the bacteriostatic Activity
1) Activating and culturing strains: taking out the frozen strain from the liquid nitrogen tank, and quickly melting the strain in a water bath at 37 ℃ to obtain a resuscitation bacteria liquid; inoculating the resuscitation bacteria liquid into a sterilized nutrient broth culture medium according to the proportion of 1.0% (v/v), placing the sterilized nutrient broth culture medium into an anaerobic incubator at 36 ℃, and standing and culturing for 24-30 h to obtain 1 st activated bacteria liquid; inoculating the 1 st activated bacterial liquid into a sterilized nutrient broth culture medium according to the proportion of 1.0% (v/v), and then placing the sterilized nutrient broth culture medium into an anaerobic incubator at 36 ℃, and standing and culturing for 24-30 h to obtain a 2 nd activated bacterial liquid;
2) and (3) measuring the concentration of the bacterial liquid: (a) taking 1mL of the 2 nd activated bacterium liquid, and adding 9mLPBS buffer solution to prepare 1:10 bacterium suspension; (b) preparing 10-fold serial diluted bacterial suspension according to the operation of (a); (c) selecting 3 dilutions in the 10-time serial diluted bacterial suspension, adding 1mL of the diluted bacterial suspension into an aseptic plate, adding 20mL of nutrient broth agar culture medium, uniformly mixing, placing the mixture in an anaerobic incubator for 24-30 h at 36 ℃, counting the number of bacterial colonies on the plate, and multiplying the number by the dilution times to calculate the concentration of the 2 nd activated bacterial liquid;
3) preparation of test bacterial suspension: calculating and adding the 2 nd activated bacterium liquid into the double-concentration nutrient broth culture medium according to the concentration of the 2 nd activated bacterium liquid to prepare a test bacterium suspension with the concentration of 106-108CFU/mL；
4) Sample adding: setting a control group, a blank group and an experimental group on a sterile 96-hole culture plate; adding 100 mu LPBS buffer solution into a control group and a blank group, and adding 100 mu L of electronic cigarette liquid with different dilution times into an experimental group; adding 100 μ L of double concentration nutrient broth medium to the blank group, adding 100 μ L of test bacterial suspension to the control group and the experimental group, and paralleling each group with 6 wells;
5) culturing: placing the 96-hole culture plate in an anaerobic incubator, and carrying out anaerobic constant-temperature culture at 36 ℃ for 24-30 h;
6) MTT incubation treatment: taking out the cultured 96-well culture plate, centrifuging at 10,000 rpm in a microplate centrifuge at room temperature for 5min, and removing the supernatant; adding 1.0-5.0 mg/mL MTT solution with 100 mu L/hole, placing on a microplate oscillator, oscillating for 5min at 36 ℃, and then placing in an anaerobic incubator, and incubating for 1.0-2.0 h at 36 ℃;
7) and (3) light absorption value determination: taking out the incubated 96-well culture plate, centrifuging at 10,000 rpm in a microplate centrifuge at room temperature for 5min, and removing the supernatant; adding DMSO (dimethylsulfoxide) at a concentration of 150 mu L/hole, and placing on a microplate oscillator for oscillation at 36 ℃ for 5 min; standing, and measuring light absorption values of a control group, a blank group and an experimental group at 570nm on an enzyme labeling instrument;
(3) analysis of results
The bacteriostatic activity of the electronic cigarette liquid is expressed by bacteriostatic rate (I), and is calculated according to a formula (1) according to a light absorption value, wherein the formula is as follows:
in the formula: i, bacteriostasis rate; ODn-average absorbance of the experimental group; ODo-average absorbance of the blank set; ODc-average absorbance of control group.
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