CN104962604A - Method for detecting bacterial reverse mutability of total particulate matters of cigarette smoke - Google Patents
Method for detecting bacterial reverse mutability of total particulate matters of cigarette smoke Download PDFInfo
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Abstract
The invention relates to a method for detecting the bacterial reverse mutability of the total particulate matters of cigarette smoke, belonging to the technical field of safety evaluation of tobacco and cigarette smoke. A system for detecting the bacterial reverse mutability of the total particulate matters of cigarette smoke adopts a combination of the following three important parameters: combination 1: the histidine concentration is 1mmol/L, the S9 concentration is 10% (V/V), and the light absorption value in the OD 600 of bacterial liquid is 2.0*10<9>/ml; and combination 2: the histidine concentration is 0.5mmol/L, the S9 concentration is 20% (V/V), and the light absorption value in the OD 600 of bacterial liquid is 2.0*10<9>/ml. The method provided by the invention has the following advantages: the synergistic effect of the three important parameters is comprehensively observed instead of adjustment of single parameter, so that the test sensitivity is guaranteed, and a relatively good linear dose-effect relationship can be realized in the detected dosage range.
Description
technical field:
The present invention relates to the detection method of mutagenicity of total particulate matter of cigarette smoke, belong to tobacco and tobacco smoke security biological assessment technical field.
background technology:
Now, chemical substance and pollutent thereof are more and more subject to common concern and the attention of society to the potential hazard of human body.B.N.Ames equals to set up for 1975 and the perfect salmonella reverse mutation assay (Salmonella reversion test) of development is widely adopted by countries in the world and organizing, current internationally recognized detection new drug, foodstuff additive, first-selection test [1-3] of the mutagenicity of packaging material for food and makeup etc.International tobacco scientific research Cooperation Centre CORESTA is organized in 2002 and has set up cigarette smoke external toxicity test job group, through a large amount of literature survey of working group and research work, this working group recommends to adopt Ames test (Salmonella reversion test) to detect the mutagenicity that tested material is potential respectively.This detection method is extensively adopted by domestic and international tobacco company at present.
Since Ames detection system is set up, constantly have scholar to carry out the improvement of each side to it, accuracy, the validity of Salmonella reversion test are also improved constantly.But still there is the uncertain factor of some in this test so far, such as, Histidine in detected system and precursor thereof are (as polypeptide, protein etc.) amount material impact is existed to Salmonella reversion test result, because they can support his-deficient cell to grow and division, increase final revertant colony number, thus cause false positive; Salmonella reversion test Salmonellas used belongs to prokaryotic organism and lacks metabolism activation system, therefore needs in Salmonella reversion test to add S9 mixed solution as metabolism activation system, and the power of this activation system activity may have an impact to test-results; In addition, because Salmonella reversion test counts the colony number producing reverse mutation, detect and use the too low tested material mutagenesis ability that may cause of the amount of bacterium to show not exclusively.Be set as 0.5mmol/L to histidine concentrations in [1] in toxicological evaluation of food safety procedure, S9 mixed liquid concentration is 10%(V/V), tested bacteria concentration is for being no less than 1 × 10
9individual/ml-2 × 10
9individual/ml, detects cigarette smoke condensates by above-mentioned reaction system, and when certainly beaming back change colony number and positive controls is all normal, the linear relationship that tested material detects gained dose-effect curve is not good.This may be due to above test system in the setting of Histidine, S9 mixed solution, these three important parameters of tested bacteria concentration to the given value determined of the working concentration of Histidine and S9 mixed solution, and only the lower limit of usage quantity is limited in the usage quantity to tested bacterium, may have an impact to detected result when tested bacteria concentration is too high.
Important references document:
1. Ministry of Health of the People's Republic of China. GB15193-2014 toxicological evaluation of food safety procedure [S].
2. Chinese food Drug Administration .YY/T0127.10-2009 oral cavity medical apparatus biological assessment the 2nd unit: test method salmonella typhimurium reverse mutation test (Salmonella reversion test) [S].
3. Ministry of Health of the People's Republic of China .GB7919-87 safety evaluation of cosmetics progresses and methods [S].
summary of the invention:
The object of the invention is to overcome prior art Problems existing, a kind of detection method for cigarette smoke condensates reverse mutation is provided.
The present invention is directed to above-mentioned technical problem, synthetic study has been carried out to histidine concentrations, S9 concentration, bacteria concentration three important factors, result of study shows to there is synergy between three influence factors, is not within the scope of certain limitation, carry out arbitrary combination to three factors just can draw better result.
The present invention improves histidine concentrations, S9 concentration, bacteria concentration three important tests factor parameters combination in test system, is specially:
Combination 1: histidine concentrations is 1mmol/L, S9 concentration is 10%(V/V), bacterium liquid OD
600lower light absorption value is 2.0 × 10
9individual/ml.
Combination 2: histidine concentrations is 0.5mmol/L, S9 concentration is 20%(V/V), bacterium liquid OD
600lower light absorption value is 2.0 × 10
9individual/ml.
The inventive method is compared with art methods, advantage is: the synergy between integrated survey three important parameters, instead of single certain parameter is adjusted, namely ensure that the sensitivity of test, dose-effect relationship also can be made in institute's detection agent weight range to present good linear relationship.The mutagenicity of tested material is reflected accurately while reducing false positive rate.
embodiment:
Below in conjunction with specific examples, the present invention is described in detail, but is not limitation of the present invention.
embodiment one:
For the detection of Kentucky reference cigarette 3R4F cigarette smoke condensate mutagenicity
1. the preparation of cigarette smoke condensates (TPM)
Prepare sample with reference to National Standard of the People's Republic of China GB/T 19609-2004 " cigarette routine analysis smoking machine measures total particulate matter and tar ", sample concentration is 10mg/ml.
2. the preparation of bottom substratum
In accordance with Salmonella typhimurium touchstone preparation in GB15193.4-2014 " toxicological evaluation of food safety procedure and method ".
3. the preparation of top layer substratum
In accordance with Salmonella typhimurium touchstone preparation in GB15193.4-2014 toxicological evaluation of food safety procedure and method.
4. Zengjing Granule
Getting adequate nutrition broth culture joins in aseptic triangular flask, and by TA98 inoculation in nutrient broth medium, vibrate under 37 DEG C of conditions (100 times/min) cultivate 10h.
5. tested bacteria concentration is determined
Employing is smeared colony counting method and is determined tested bacteria concentration.
6.TPM dosage setting
TPM detects dosage and comprises 5 non-zero-doses: 25 μ g/ wares, 50 μ g/ wares, 100 μ g/ wares, 125 μ g/ wares, 200 μ g/ wares.
7. the setting of positive control, untreated fish group (certainly beaming back change) and solvent control
Select 2-aminofluorene as the positive thing of TA98, dosage is 10 μ g/ wares;
Untreated fish group only adds the fresh bacterium liquid of 0.1ml, measures its spontaneous recovery sudden change colony number.
Solvent control group adds dimethyl sulfoxide (DMSO), and dosage is 20 μ l/ wares;
The preparation of 8.S9 mixed solution
Be prepared in accordance with Salmonella typhimurium touchstone in GB15193.4-2014 toxicological evaluation of food safety procedure and method.
9. flat board mixes
Adopt 4 horizontal various combination conditions of orthogonal experiment study group histidine content, bacteria concentration, these 3 factors of S9 concentration on the impact of Salmonella reversion test, detect respectively under the test parameter combination condition of 16 in table 1:
Table 1 Salmonella reversion test influence factor orthogonal test detect parameters
| Test number | Histidine (mmol/L) | S9 concentration (V/V %) | Bacteria concentration (individual/ml) |
| 1 | 1 | 20 | 3.0×10 9 |
| 2 | 1 | 10 | 2.0×10 9 |
| 3 | 1 | 5 | 1.0×10 9 |
| 4 | 1 | 2.5 | 0.5×10 9 |
| 5 | 0.5 | 10 | 3.0×10 9 |
| 6 | 0.5 | 20 | 2.0×10 9 |
| 7 | 0.5 | 2.5 | 1.0×10 9 |
| 8 | 0.5 | 5 | 0.5×10 9 |
| 9 | 0.25 | 5 | 3.0×10 9 |
| 10 | 0.25 | 2.5 | 2.0×10 9 |
| 11 | 0.25 | 20 | 1.0×10 9 |
| 12 | 0.25 | 10 | 0.5×10 9 |
| 13 | 0.125 | 2.5 | 3.0×10 9 |
| 14 | 0.125 | 5 | 2.0×10 9 |
| 15 | 0.125 | 10 | 1.0×10 9 |
| 16 | 0.125 | 20 | 0.5×10 9 |
Adopt direct flat plate incorporation methods, fresh bacterium liquid 0.1ml, S9 mixed solution 0.5ml and the detected sample of respective concentration is added in the top layer substratum of about 2.0ml, turbine mixer mixes, pour on bottom substratum, rotating culture dish makes it be evenly distributed on bottom substratum, puts into 37 DEG C of incubators and cultivate 48h after it solidifies.Each dosage makees three parallel wares.
10. cultivate and observe
The reply colony number that substratum grows is counted.
11. results judge
From beam back become colony number should between 30-50 and positive controls colony number more than 1000, should show that test system is normal.Under the normal prerequisite of detection system, the recovery mutation colony number of tested material becomes colony number more than 2 times as being greater than from beaming back, and has dose-response relationship, then can think that this tested mutagenesis testing result is positive.
This laboratory is detected the mutagenicity of cigarette smoke condensates after improving above-mentioned reaction system, the results are shown in Table 2.
To cigarette smoke condensates Salmonella reversion test detected result under table 2 various combination test parameter
| Test number | Dose effect curve R 2Value | Produce positive findings lowest dose level (μ g/ ware) | From beaming back change group sudden change colony number | Positive controls sudden change colony number |
| 1 | 0.996 | 50 | 34.7±2.3 | 1883±24.3 |
| 2 | 0.879 | 25 | 32.0±5.3 | 1469±42.3 |
| 3 | 0.941 | 25 | 22.3±3.7 | 468±38.7 |
| 4 | 0.049 | Negative | 30.7±2.7 | 411±55.3 |
| 5 | 0.508 | 25 | 35.7±1.7 | 1814±46.7 |
| 6 | 0.87 | 25 | 32.3±9.7 | 1241±35.3 |
| 7 | 0.452 | Negative | 25.3±5.3 | 818±14.7 |
| 8 | 0.057 | Negative | 20.0±2.3 | 1770±87.3 |
| 9 | 0.00008 | Negative | 15.7±4.7 | 657±42.7 |
| 10 | 0.051 | Negative | 10.0±4.3 | 347±20.3 |
| 11 | 0.815 | 25 | 17.3±3.7 | 687±35.3 |
| 12 | 0.853 | 25 | 15.7±6.7 | 1854±75.7 |
| 13 | 0.523 | Negative | 9.3±4.3 | 608±40.3 |
| 14 | 0.07 | Negative | 23.7±6.3 | 1220±44.3 |
| 15 | 0.967 | 25 | 13.7±8.7 | 721±32.7 |
| 16 | 0.678 | 25 | 18.7±9.3 | 647±14.3 |
As can be seen from above test-results, meet in above 16 test system schemes from beam back become colony number should between 30-50 and positive controls colony number condition only should have 4 (scheme 1, scheme 2, scheme 5, schemes 6) more than 1000, wherein scheme 1 test system produces positive findings lowest dose level higher than scheme 2, scheme 5, scheme 6 test system detected result.Scheme 2, scheme 5, scheme 6 test system detected result dose-effect relationship are scheme 2 and scheme 6 preferably, and namely in detection system, histidine concentrations, S9 concentration, bacteria concentration three important tests factor parameters combination are:
Combination 1: histidine concentrations is 1mmol/L, S9 concentration is 10%(V/V), bacterium liquid OD
600lower light absorption value is 2.0 × 10
9individual/ml.
Combination 2: histidine concentrations is 0.5mmol/L, S9 concentration is 20%(V/V), bacterium liquid OD
600lower light absorption value is 2.0 × 10
9individual/ml.
Under the histidine concentrations optimized in the inventive method, the combination condition of S9 mixed liquid concentration and tested bacteria concentration, smoke's total particulate matter, in the detection agent weight range of 25-200 μ g/ ware, presents good dose-response relationship.Although it be 0.5mmol/L, S9 mixed liquid concentration is 10%(V/V that the testing conditions of scheme 5 meets histidine concentrations in GB15193.4-2014 standard), tested bacteria concentration is for being no less than 1 × 10
9individual/ml-2 × 10
9the condition setting of individual/ml, but the linear degree detecting the dose-effect curve obtained is lower.
Method synthesis of the present invention has investigated the synergy between three important parameters, instead of single adjusting certain parameter, namely ensure that the sensitivity of test, and dose-effect relationship also can be made in institute's detection agent weight range to present good linear relationship.
Claims (1)
1. for a detection method for cigarette smoke condensates reverse mutation, it is characterized in that: carrying out, in cigarette smoke condensates Ames test test system, adopting the combination of following three important parameters:
Combination 1: histidine concentrations is 1mmol/L, S9 concentration is 10%(V/V), bacterium liquid OD
600lower light absorption value is 2.0 × 10
9individual/ml;
Or, combination 2: histidine concentrations is 0.5mmol/L, S9 concentration is 20%(V/V), bacterium liquid OD
600lower light absorption value is 2.0 × 10
9individual/ml.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108132222A (en) * | 2017-12-14 | 2018-06-08 | 云南中烟工业有限责任公司 | It is a kind of to be used to detect the method that gum base type chewing tobacco influences reverse mutation number |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101255456A (en) * | 2008-04-01 | 2008-09-03 | 中国烟草总公司郑州烟草研究院 | Toxicology neasuring evaluation method of cigarette smoke mutagenicity |
| CN101671735A (en) * | 2009-09-24 | 2010-03-17 | 中国烟草总公司郑州烟草研究院 | Improved cigarette smoke condensate Ames test |
| CN101851659A (en) * | 2010-04-23 | 2010-10-06 | 云南烟草科学研究院 | Ames experiment micro fluctuation method of mutagenicity detection for water bodies |
| CN102980913A (en) * | 2012-11-13 | 2013-03-20 | 湖北中烟工业有限责任公司 | Biological thermochemistry method for safety evaluation and screening of cigarette auxiliary material |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101255456A (en) * | 2008-04-01 | 2008-09-03 | 中国烟草总公司郑州烟草研究院 | Toxicology neasuring evaluation method of cigarette smoke mutagenicity |
| CN101671735A (en) * | 2009-09-24 | 2010-03-17 | 中国烟草总公司郑州烟草研究院 | Improved cigarette smoke condensate Ames test |
| CN101851659A (en) * | 2010-04-23 | 2010-10-06 | 云南烟草科学研究院 | Ames experiment micro fluctuation method of mutagenicity detection for water bodies |
| CN102980913A (en) * | 2012-11-13 | 2013-03-20 | 湖北中烟工业有限责任公司 | Biological thermochemistry method for safety evaluation and screening of cigarette auxiliary material |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108132222A (en) * | 2017-12-14 | 2018-06-08 | 云南中烟工业有限责任公司 | It is a kind of to be used to detect the method that gum base type chewing tobacco influences reverse mutation number |
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