CN109490431B - Method for researching oral cavity release condition of flavor substance of oral smokeless tobacco product - Google Patents
Method for researching oral cavity release condition of flavor substance of oral smokeless tobacco product Download PDFInfo
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Abstract
The invention relates to a method for researching the oral release condition of a flavor substance of an oral smokeless tobacco product, belonging to the technical field of analysis of oral smokeless tobacco products. The method comprises the steps of screening oral smokeless tobacco products, determining simulated dissolution parameters, measuring the oral release condition of the flavor substances in vivo experiments, simulating in vitro experiments and the like. The device can completely simulate the state of smoking the smokeless tobacco product in the oral cavity of a human body, can regulate and control chewing strength, saliva flow rate, chewing frequency, saliva flow and oral cavity temperature, and has strong controllability, high stability and simple operation. The research method can well avoid the influence of subjective and objective factors caused by traditional manual taste, avoid ethical constraints of a large number of crowd experiments, save the experiment cost, reduce the harm to health caused by human experiments, and is suitable for batch research of samples.
Description
Technical Field
The invention belongs to the technical field of analysis of oral smokeless tobacco products, and particularly relates to a method for researching oral release conditions of flavor substances of oral smokeless tobacco products.
Background
Smokeless Tobacco products (STPs for short) refer to Tobacco products which are directly sucked through oral cavity or nasal cavity without combustion, and compared with traditional cigarettes, the Smokeless Tobacco products have less harmful components and less harm; meanwhile, the cigarette does not produce second-hand smoke, can relieve the contradiction between smoking ban and smoking in public places to a certain extent, and meets the physiological needs of smokers to a certain extent. The smokeless tobacco products have various forms such as bagged mouth tobacco, snuff, chewing tobacco and the like, wherein the consumption main body is an oral smokeless tobacco product which mainly comprises the bagged mouth tobacco, gum base type chewing tobacco and tobacco containing. The bagged buccal cigarette is prepared by mixing tobacco powder with certain granularity with proper amount of fragrant matter, corrective, water, humectant and pH regulator, heat treating, packing in non-woven fabric bag and consumption between lips and teeth. The gum base type smokeless Tobacco products (Tobacco chewing gum) were first developed successfully and put on the market in 2003 by the Swedish Match company (Swedish Match). The tobacco product is also called novel chewing tobacco, gum base type smokeless tobacco, tobacco chewing gum and gum base tobacco, and is a novel tobacco product which takes tobacco and/or tobacco extract as effective components, takes edible gum base as a carrier, and delivers fragrant substances to a human body in a chewing mode. In 2016, the sale of STPs in the world is more than 120 hundred million dollars, wherein the sale of bagged mouth tobacco is increased by more than 40 percent, and the sale of chewing tobacco is increased by about 10 percent. STPs cover 70 countries worldwide accounting for 73% of the general population. When the cigarette is consumed in the form of STPs, the tobacco is not combusted, and some peculiar harmful ingredients in cigarette smoke are not generated, more importantly, unpleasant 'second-hand smoke' is not generated, so that the cigarette is an important supplementary form for tobacco consumption in a smoking banned environment. Under the influence of the current world smoking control movement, tobacco products in this category are becoming an important development trend in the international tobacco field.
At present, most of smokeless tobacco is added with various flavor substances with different tastes to show the style characteristics of the product. For example, menthol is added to impart a cooling taste, citral is added to impart a lemon taste, etc. For better taste persistence, a sustained release of the flavor substance is of great importance. The main factor of formulation development is to achieve the sustained and slow release of the flavor substance to bring the continuous taste to the consumers, and the long-term off-spectrum. In order to perform in-vitro elution quantity rule research and quality evaluation on the fragrant substances of the products, people chewing experiments are generally adopted. However, the population experiment is limited by a plurality of factors such as ethics and society, and the main factors have large influence and are difficult to popularize. Some researchers in China have also conducted related researches on STPs simulation of oral dissolution. However, the influence of different influencing factors is researched by a fixed saliva flow rate, and the influence of a pressure factor (the using mode of the bagged mouth cigarette is generally put between a gum and a lip) and the influence of the saliva flow rate (different saliva release rates are different at different time when the oral cavity is stimulated) on the release of the flavor substances are not researched. The state of the human body when the mouth cavity sucks the smokeless tobacco product is not really and effectively simulated. Therefore, how to overcome the defects of the prior art is a problem which needs to be solved in the technical field of the analysis of the oral smokeless tobacco products.
Disclosure of Invention
The invention provides a method for researching the oral cavity release condition of an oral smokeless tobacco product, aiming at the defects of the existing external simulation dissolution method of the aromatic substance, the method is a test method which is closer to the oral cavity condition of a human body, the method can be used for more accurately researching the oral smokeless tobacco product release condition in the oral cavity of the human body, and technical support and theoretical basis are provided for the processing and the quality control of the products.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method of studying the oral release of a flavorant from an oral smokeless tobacco product, comprising the steps of:
step (1), screening oral smokeless tobacco products: selecting a plurality of certain oral smokeless tobacco products to be researched for weight detection, and calculating the average value and variance of the single weight of the products; then, selecting a product with the weight range in the range of +/-1-3 times of the average value as a sample for subsequent measurement;
step (2), determination of simulated dissolution parameters: selecting a plurality of healthy volunteers to inhale the sample screened in the step (1) in a natural state, and collecting residual samples and saliva of the respective volunteers at predetermined time points, wherein one sample is used at each time point;
step (3), in vivo experiment of determination of oral cavity release condition of flavor substances: respectively testing the oral smokeless tobacco product to be researched, the residual sample and the saliva of the volunteer by utilizing gas chromatography to determine the release amount of the aroma substances of the sample sucked by the volunteer in each time period;
step (4), in-vitro test simulation: simulating to suck the oral smokeless tobacco product to be researched by adopting a full-simulation chewing machine, and setting several parameters of the pressing force, the saliva flow rate, the chewing frequency, the saliva amount and the oral cavity temperature parameter of the full-simulation chewing machine according to the release amount of the fragrant substances of the sample sucked by the volunteer obtained in the step (3) and the saliva amount obtained in the step (2);
step (5), gas chromatography analysis: respectively testing the oral smokeless tobacco product to be researched, the residual sample and artificial saliva in a full-simulation chewing machine by utilizing gas chromatography, and representing the oral cavity release condition of the flavor substance of the oral smokeless tobacco product to be researched by using the test result;
step (6), researching the oral release rule of the flavor substance: the full-simulation chewing machine and the set parameters are adopted to test different time points in the whole chewing process of the same oral smokeless tobacco product, and a flavor substance release curve is drawn to carry out the research on the oral release rule of the flavor substance.
When the parameters of the full-simulation chewing machine are set, the parameters of the full-simulation chewing machine can be set, and the parameters can be set according to the specific conditions of in vivo tests.
Further, it is preferred that in step (1), at least 50 certain oral smokeless tobacco products to be investigated are selected for weight measurement.
Further, in the step (1), the oral smokeless tobacco product is preferably selected from the group consisting of mouth-holding tobacco, chewing tobacco, buccal tobacco, paste-like buccal tobacco, dentifrice and soluble tobacco, wherein the mouth-holding tobacco is selected from the group consisting of bagged mouth-holding tobacco, tobacco chewing gum, lozenge and hard sugar.
Further, it is preferable that, in the step (2), the volunteer is aged 18 to 55 years.
Further, it is preferable that, in the step (2), the collection time point of the residual sample and the volunteer saliva is any point within 0-60min after the start of the experiment.
Further, it is preferable that the artificial saliva used in the in vitro test simulation is inorganic salt artificial saliva or protease artificial saliva.
Further, it is preferable that the method for analyzing the content of the fragrant substance in the product or the residual sample is: when in test, 1 product or residual sample is taken, packaged and cut into pieces, then placed into a container, added with 20mL of 1.00g/L ethanol solution of n-propyl benzoate, sealed and shaken at room temperature for 60min, filtered by a 0.45 mu m filter membrane, and then quantitatively analyzed by gas chromatography.
The size after cutting is not particularly limited in the present invention.
Further, it is preferred that the gas chromatography parameters are: a chromatographic column: an HP-innowax column of 30 m.times.0.25 mm.times.0.25 μm; carrier gas: helium, constant current 1.5 mL/min; sample introduction conditions are as follows: the temperature is 250 ℃, the sample injection amount is 1 mu L, and the split ratio is 1: 50; furnace temperature: keeping the temperature at 220 ℃; a detector: FID, 250 ℃.
The flavor substance is a volatile flavor substance which is added into the smokeless tobacco product and endows the smokeless tobacco product with taste and style characteristics, such as menthol, limonol, Maillard products and the like.
At present, artificial saliva is mainly divided into two types, one is inorganic salt artificial saliva, and the other is protease artificial saliva; the protease artificial saliva treatment comprises inorganic salt, protein and enzyme.
The inorganic salt artificial saliva may be shown in tables 1 and 2, but is not limited thereto.
Protease-based artificial saliva can be shown in table 3, but is not limited thereto.
TABLE 1 Artificial saliva formula I
TABLE 2 Artificial saliva formula II
TABLE 3 Artificial saliva formulation III (mmol/L)
Note: unit is units/mL
In the test process of the invention: aroma elution amount-total aroma content-residual sample aroma content.
The full-simulation chewing machine adopted by the invention can be a product ZL 201610554134.7, but is not limited to the product. The components used in the full simulation chew may be as shown in, but not limited to, ZL 201610555175.8, ZL 201610555228.6, ZL 201620743278.2 and ZL 201620742852.2.
The invention designs a method for researching the oral cavity release condition of an oral smokeless tobacco product flavor substance, which comprises the following steps: determining in vitro study parameters for oral cavity release of flavor substances of the oral smokeless tobacco product by in vivo experiments of volunteers; determining simulated dissolution parameters such as pressure force, chewing frequency, saliva flow rate, saliva amount, oral cavity temperature and the like; the release rules of the flavor substance release rate, the release amount and the like of the oral smokeless tobacco product are researched by utilizing a full-simulation chewing machine. The method has strong controllability and high stability, is simple to operate, can well avoid the influence of subjective and objective factors caused by traditional manual tasting, avoids ethical and moral restrictions of a large number of crowd experiments, saves the experiment cost, reduces the harm to health caused by human experiments, and is suitable for batch research of samples.
Meanwhile, firstly, the method adopts a weight detection method to screen the samples, so that the screening range is optimized, results can generate larger deviation if the screening range exceeds the screening range of the method, and the accuracy of quantification is reduced;
secondly, the in vitro experiment parameters are set based on the screened samples by taking the in vivo experiment parameters as the reference, so that the research deviation caused by the problem of setting the in vitro experiment parameters in the prior art is avoided.
Thirdly, the in-vitro test parameters set by the in-vivo test on the same type of products are adopted, the in-vivo test on the same type of products is not needed in the subsequent process, the set in-vitro test parameters can be directly adopted for testing, the oral cavity release rule of the flavor substance is researched, and the accuracy is excellent.
Fourthly, the method for analyzing the content of the fragrant substances is optimized, so that the fragrant substances are extracted as much as possible, and the deviation in research caused by the defects of the analysis method is avoided;
fifthly, when in-vitro test parameters are set, the sucking condition of a human body is researched, different pressing force and chewing frequency are set, different saliva adding amounts and chewing frequency are set according to different time points, the consumption conditions of the product in the oral cavity are simulated more truly, and the accuracy of results is greatly improved;
sixthly, during chromatographic analysis, chromatographic parameters are optimized, so that the separation condition of the fragrance substance peak is better, the interference of other substances to the fragrance substance peak is avoided, and the accuracy of the result is improved.
Compared with the prior art, the invention has the beneficial effects that:
the device can completely simulate the state of smoking the smokeless tobacco product in the oral cavity of a human body, can be regulated and controlled on the parameters of the pressing force, the saliva flow rate, the chewing frequency, the saliva amount and the oral cavity temperature, and has strong controllability, high stability and simple operation.
The research method for the oral release of the flavor substances of the oral smokeless tobacco product can well avoid the influence of subjective and objective factors caused by traditional manual tasting, avoid ethical and moral restrictions of a large number of crowd experiments, save the experiment cost, reduce the harm to health caused by human body experiments, is suitable for batch research of samples, and provides technical support and theoretical basis for formula development, processing and quality control of the products.
Drawings
FIG. 1 is a chromatogram of menthol under examination; wherein A is a menthol standard substance; b is product No. 1 in Table 4; the internal standard is n-propyl benzoate;
FIG. 2 is a graph of the in vivo and in vitro dissolution profiles of menthol from gum based chewing tobacco;
FIG. 3 is a graph of the in vitro simulated dissolution trend of menthol from four bagged mouth tobacco of application example 1; wherein product 1 is product number 10 of table 4, purchased from usa; product 2 is product number 13 in table 4, purchased in canada; product 3 is product number 21 in table 4, a yunnan zhongyan homemade sample, and product 4 is product number 24 in table 4, a yunnan zhongyan homemade sample.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the 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 materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
Example 1
A method of studying the oral release of a flavorant from an oral smokeless tobacco product, comprising the steps of:
step (1), screening oral smokeless tobacco products: selecting a plurality of certain oral smokeless tobacco products to be researched for weight detection, and calculating the average value and variance of the single weight of the products; then, selecting a product with the weight range within the range of +/-2 times of variance of the average value as a sample for subsequent measurement;
the mouth type smokeless tobacco product is buccal tobacco;
step (2), determination of simulated dissolution parameters: selecting a plurality of healthy volunteers to inhale the sample screened in the step (1) in a natural state, and collecting residual samples and saliva of the respective volunteers at predetermined time points, wherein one sample is used at each time point;
step (3), in vivo experiment of determination of oral cavity release condition of flavor substances: respectively testing the oral smokeless tobacco product to be researched, the residual sample and the saliva of the volunteer by utilizing gas chromatography to determine the release amount of the aroma substances of the sample sucked by the volunteer in each time period;
step (4), in-vitro test simulation: simulating to suck the oral smokeless tobacco product to be researched by adopting a full-simulation chewing machine, and setting several parameters of the pressing force, the saliva flow rate, the chewing frequency, the saliva amount and the oral cavity temperature parameter of the full-simulation chewing machine according to the release amount of the fragrant substances of the sample sucked by the volunteer obtained in the step (3) and the saliva amount obtained in the step (2);
step (5), gas chromatography analysis: respectively testing the oral smokeless tobacco product to be researched, the residual sample and artificial saliva in a full-simulation chewing machine by utilizing gas chromatography, and representing the oral cavity release condition of the flavor substance of the oral smokeless tobacco product to be researched by using the test result;
step (6), researching the oral release rule of the flavor substance: the full-simulation chewing machine and the set parameters are adopted to test different time points in the whole chewing process of the same oral smokeless tobacco product, and a flavor substance release curve is drawn to carry out the research on the oral release rule of the flavor substance.
Example 2
A method of studying the oral release of a flavorant from an oral smokeless tobacco product, comprising the steps of:
step (1), screening oral smokeless tobacco products: selecting 50 certain oral smokeless tobacco products to be researched for weight detection, and calculating the average value and variance of the single weight of the products; then, selecting a product with the weight range within the range of +/-1 time of variance of the average value as a sample for subsequent measurement;
the oral smokeless tobacco product comprises the tobacco;
step (2), determination of simulated dissolution parameters: selecting a plurality of healthy volunteers aged 18-55 years to inhale the sample screened in the step (1) in a natural state, and collecting residual samples and saliva of the respective volunteers at predetermined time points, wherein one sample is used at each time point;
the collection time points of the residual sample and the saliva of the volunteer are any point within 0-60min after the experiment is started;
step (3), in vivo experiment of determination of oral cavity release condition of flavor substances: respectively testing the oral smokeless tobacco product to be researched, the residual sample and the saliva of the volunteer by utilizing gas chromatography to determine the release amount of the aroma substances of the sample sucked by the volunteer in each time period;
step (4), in-vitro test simulation: simulating to suck the oral smokeless tobacco product to be researched by adopting a full-simulation chewing machine, and setting several parameters of the pressing force, the saliva flow rate, the chewing frequency, the saliva amount and the oral cavity temperature parameter of the full-simulation chewing machine according to the release amount of the fragrant substances of the sample sucked by the volunteer obtained in the step (3) and the saliva amount obtained in the step (2);
the artificial saliva is inorganic salt artificial saliva;
step (5), gas chromatography analysis: respectively testing the oral smokeless tobacco product to be researched, the residual sample and artificial saliva in a full-simulation chewing machine by utilizing gas chromatography, and representing the oral cavity release condition of the flavor substance of the oral smokeless tobacco product to be researched by using the test result;
step (6), researching the oral release rule of the flavor substance: the full-simulation chewing machine and the set parameters are adopted to test different time points in the whole chewing process of the same oral smokeless tobacco product, and a flavor substance release curve is drawn to carry out the research on the oral release rule of the flavor substance.
The method for analyzing the content of the aroma substances in the product or the residual sample comprises the following steps: when in test, 1 product or residual sample is taken, packaged and cut into pieces, then placed into a container, added with 20mL of 1.00g/L ethanol solution of n-propyl benzoate, sealed and shaken at room temperature for 60min, filtered by a 0.45 mu m filter membrane, and then quantitatively analyzed by gas chromatography. There is no particular limitation on the size after cutting.
The gas chromatography parameters are as follows: a chromatographic column: an HP-innowax column of 30 m.times.0.25 mm.times.0.25 μm; carrier gas: helium, constant current 1.5 mL/min; sample introduction conditions are as follows: the temperature is 250 ℃, the sample injection amount is 1 mu L, and the split ratio is 1: 50; furnace temperature: keeping the temperature at 220 ℃; a detector: FID, 250 ℃.
Example 3
A method of studying the oral release of a flavorant from an oral smokeless tobacco product, comprising the steps of:
step (1), screening oral smokeless tobacco products: selecting 100 certain oral smokeless tobacco products to be researched for weight detection, and calculating the average value and variance of the single weight of the products; then, selecting a product with the weight range within the range of +/-3 times of the variance as a sample for subsequent measurement;
the mouth-use smokeless tobacco product is in the shape of paste containing smoke.
Step (2), determination of simulated dissolution parameters: selecting a plurality of healthy volunteers aged 18-55 years to inhale the sample screened in the step (1) in a natural state, and collecting residual samples and saliva of the respective volunteers at predetermined time points, wherein one sample is used at each time point;
the collection time points of the residual sample and the saliva of the volunteer are any point within 0-60min after the experiment is started;
step (3), in vivo experiment of determination of oral cavity release condition of flavor substances: respectively testing the oral smokeless tobacco product to be researched, the residual sample and the saliva of the volunteer by utilizing gas chromatography to determine the release amount of the aroma substances of the sample sucked by the volunteer in each time period;
step (4), in-vitro test simulation: simulating to suck the oral smokeless tobacco product to be researched by adopting a full-simulation chewing machine, and setting several parameters of the pressing force, the saliva flow rate, the chewing frequency, the saliva amount and the oral cavity temperature parameter of the full-simulation chewing machine according to the release amount of the fragrant substances of the sample sucked by the volunteer obtained in the step (3) and the saliva amount obtained in the step (2);
the adopted artificial saliva is protease artificial saliva;
step (5), gas chromatography analysis: respectively testing the oral smokeless tobacco product to be researched, the residual sample and artificial saliva in a full-simulation chewing machine by utilizing gas chromatography, and representing the oral cavity release condition of the flavor substance of the oral smokeless tobacco product to be researched by using the test result;
step (6), researching the oral release rule of the flavor substance: the full-simulation chewing machine and the set parameters are adopted to test different time points in the whole chewing process of the same oral smokeless tobacco product, and a flavor substance release curve is drawn to carry out the research on the oral release rule of the flavor substance.
The method for analyzing the content of the aroma substances in the product or the residual sample comprises the following steps: when in test, 1 product or residual sample is taken, packaged and cut into pieces, then placed into a container, added with 20mL of 1.00g/L ethanol solution of n-propyl benzoate, sealed and shaken at room temperature for 60min, filtered by a 0.45 mu m filter membrane, and then quantitatively analyzed by gas chromatography. There is no particular limitation on the size after cutting.
The gas chromatography parameters are as follows: a chromatographic column: an HP-innowax column of 30 m.times.0.25 mm.times.0.25 μm; carrier gas: helium, constant current 1.5 mL/min; sample introduction conditions are as follows: the temperature is 250 ℃, the sample injection amount is 1 mu L, and the split ratio is 1: 50; furnace temperature: keeping the temperature at 220 ℃; a detector: FID, 250 ℃.
Example 4
A method of studying the oral release of a flavorant from an oral smokeless tobacco product, comprising the steps of:
step (1), screening oral smokeless tobacco products: selecting 150 pieces of certain oral smokeless tobacco products to be researched for weight detection, and calculating the average value and variance of the single weight of the products; then, selecting a product with the weight range within the range of +/-2 times of variance of the average value as a sample for subsequent measurement;
the oral smokeless tobacco product is soluble tobacco;
step (2), determination of simulated dissolution parameters: selecting a plurality of healthy volunteers aged 18-55 years to inhale the sample screened in the step (1) in a natural state, and collecting residual samples and saliva of the respective volunteers at predetermined time points, wherein one sample is used at each time point;
the collection time points of the residual sample and the saliva of the volunteer are any point within 0-60min after the experiment is started;
step (3), in vivo experiment of determination of oral cavity release condition of flavor substances: respectively testing the oral smokeless tobacco product to be researched, the residual sample and the saliva of the volunteer by utilizing gas chromatography to determine the release amount of the aroma substances of the sample sucked by the volunteer in each time period;
step (4), in-vitro test simulation: simulating to suck the oral smokeless tobacco product to be researched by adopting a full-simulation chewing machine, and setting several parameters of the pressing force, the saliva flow rate, the chewing frequency, the saliva amount and the oral cavity temperature parameter of the full-simulation chewing machine according to the release amount of the fragrant substances of the sample sucked by the volunteer obtained in the step (3) and the saliva amount obtained in the step (2);
the artificial saliva is inorganic salt artificial saliva;
step (5), gas chromatography analysis: respectively testing the oral smokeless tobacco product to be researched, the residual sample and artificial saliva in a full-simulation chewing machine by utilizing gas chromatography, and representing the oral cavity release condition of the flavor substance of the oral smokeless tobacco product to be researched by using the test result;
step (6), researching the oral release rule of the flavor substance: the full-simulation chewing machine and the set parameters are adopted to test different time points in the whole chewing process of the same oral smokeless tobacco product, and a flavor substance release curve is drawn to carry out the research on the oral release rule of the flavor substance.
The method for analyzing the content of the aroma substances in the product or the residual sample comprises the following steps: when in test, 1 product or residual sample is taken, packaged and cut into pieces, then placed into a container, added with 20mL of 1.00g/L ethanol solution of n-propyl benzoate, sealed and shaken at room temperature for 60min, filtered by a 0.45 mu m filter membrane, and then quantitatively analyzed by gas chromatography. There is no particular limitation on the size after cutting.
The gas chromatography parameters are as follows: a chromatographic column: an HP-innowax column of 30 m.times.0.25 mm.times.0.25 μm; carrier gas: helium, constant current 1.5 mL/min; sample introduction conditions are as follows: the temperature is 250 ℃, the sample injection amount is 1 mu L, and the split ratio is 1: 50; furnace temperature: keeping the temperature at 220 ℃; a detector: FID, 250 ℃.
Comparative experiment 1
The oscillation times were 5min and 100min, respectively, and the rest was the same as in example 4. The results showed that the peak of the chromatographic flavor was very small and the extraction was insufficient at an oscillation time of 5min, as compared with example 4; when the oscillation time is 100min, the detection result is not obviously different from that of the example 4.
The extraction mode was examined and selected from leaching, sonication, shaking, and the rest was the same as in example 4. The results compared to example 4 show that the leaching, chromatographically fragrant material peaks are all small and should be under-extracted; ultrasonic treatment, disordered peaks on a chromatogram, difficult separation of fragrance material peaks and extraction of a plurality of redundant components; the oscillation extraction mode is optimal.
Comparative experiment 3
The extraction solvent is inspected, and compared with 20mL of ethanol, the extraction effect of the method is far better than that of the prior art; the chromatographic analysis of 20mL of ethanol is adopted, the chromatographic peak of the flavor substances is small, and the flavor substances are difficult to separate.
Application example 1
A method of studying the oral release of a flavorant from an oral smokeless tobacco product, comprising the steps of:
step (1), screening oral smokeless tobacco products: selecting 100 certain oral smokeless tobacco products to be researched for weight detection, and calculating the average value and variance of the single weight of the products; then, selecting a product with the weight range within the range of +/-2 times of variance of the average value as a sample for subsequent measurement;
the oral smokeless tobacco product is chewing tobacco; the selected sample information is shown in table 4;
TABLE 4
Note: the products with the serial numbers of 21-25 are products of tobacco industry responsibility limited company in Yunnan.
Step (2), determination of simulated dissolution parameters: selecting 10 healthy volunteers aged to 55 years old to inhale the sample screened in the step (1) in a natural state, and collecting residual samples and saliva of the respective volunteers at predetermined time points, wherein one sample is used at each time point;
the collection time points of the residual sample and the volunteer saliva are 0.5, 1, 2, 5, 10, 15, 20min after the start of the experiment;
step (3), in vivo experiment of determination of oral cavity release condition of flavor substances: respectively testing the oral smokeless tobacco product to be researched, the residual sample and the saliva of the volunteer by utilizing gas chromatography to determine the menthol dissolving amount of the sample sucked by the volunteer in each time period;
the gas chromatography parameters were: a chromatographic column: an HP-innowax column of 30 m.times.0.25 mm.times.0.25 μm; carrier gas: helium, constant current 1.5 mL/min; sample introduction conditions are as follows: the temperature is 250 ℃, the sample injection amount is 1 mu L, and the split ratio is 1: 50; furnace temperature: keeping the temperature at 220 ℃; a detector: FID, 250 ℃.
Step (4), in-vitro test simulation: simulating to suck the oral smokeless tobacco product to be researched by adopting a full-simulation chewing machine, and setting the pressing force, chewing frequency, saliva amount and oral cavity temperature parameters of the full-simulation chewing machine according to the aroma substance dissolution amount and saliva amount of the sample sucked by the volunteer obtained in the step (3); the artificial saliva is inorganic salt artificial saliva shown in table 1;
specific parameters of the full-simulation chewing machine including the pressure force, chewing frequency, saliva amount and oral cavity temperature are set as shown in table 5.
TABLE 5
Step (5), gas chromatography analysis: respectively testing the oral smokeless tobacco product to be researched, the residual sample and artificial saliva in a full-simulation chewing machine by utilizing gas chromatography, and representing the menthol oral cavity release condition of the oral smokeless tobacco product to be researched by using the test result;
step (6), researching the oral release rule of the flavor substance: the full-simulation chewing machine and the set parameters are adopted to test different time points in the whole chewing process of the same oral smokeless tobacco product, a menthol release curve is drawn, and the research on the menthol oral release rule is carried out.
Fig. 1 shows that the method can better perform qualitative and quantitative determination on the oral cavity release condition of the flavor substance of the oral smokeless tobacco product, and the method is quick, accurate, simple and operable. As can be seen from figure 2, the fit degree of the simulated oral in vitro experimental data and the human in vivo experimental data is higher, and the trend of the in vivo and in vitro dissolution curve of the menthol is basically consistent, which indicates that the method can better simulate the in vivo dissolution behavior of the menthol in the gum-based chewing tobacco. The oral dissolution of the menthol in the oral smokeless tobacco product self-made by the Yunnan Chinese tobacco by using the method is compared with the foreign products, and the taste characteristics and dissolution rules of the self-made product and the American product are basically consistent, and the taste persistence can be comparable with the American product. However, the taste of the product purchased in canada differs significantly from the other three, which may be associated with regional taste preferences. In a word, the research method for the oral release of the flavor substances of the oral smokeless tobacco product can well avoid the influence of subjective and objective factors caused by traditional manual tasting, avoid ethical and moral constraints of a large number of crowd experiments, save the experiment cost, reduce the harm to health caused by human body experiments, is suitable for batch research of samples, and provides technical support and theoretical basis for formula development, processing and quality control of the products.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. A method of studying the oral release of a flavorant from an oral smokeless tobacco product, comprising the steps of:
step (1), screening oral smokeless tobacco products: selecting a plurality of certain oral smokeless tobacco products to be researched for weight detection, and calculating the average value and variance of the single weight of the products; then, selecting a product with the weight range in the range of +/-1-3 times of the average value as a sample for subsequent measurement;
step (2), determination of simulated dissolution parameters: selecting a plurality of healthy volunteers to inhale the sample screened in the step (1) in a natural state, and collecting residual samples and saliva of the respective volunteers at predetermined time points, wherein one sample is used at each time point;
step (3), in vivo experiment of determination of oral cavity release condition of flavor substances: respectively testing the oral smokeless tobacco product to be researched, the residual sample and the saliva of the volunteer by utilizing gas chromatography to determine the release amount of the aroma substances of the sample sucked by the volunteer in each time period;
step (4), in-vitro test simulation: simulating to suck the oral smokeless tobacco product to be researched by adopting a full-simulation chewing machine, and setting the pressing force, chewing frequency, saliva amount and oral cavity temperature parameters of the full-simulation chewing machine according to the release amount of the fragrant substances of the sample sucked by the volunteer obtained in the step (3) and the saliva amount obtained in the step (2);
step (5), gas chromatography analysis: respectively testing the oral smokeless tobacco product to be researched, the residual sample and artificial saliva in a full-simulation chewing machine by utilizing gas chromatography, and representing the oral cavity release condition of the flavor substance of the oral smokeless tobacco product to be researched by using the test result;
step (6), researching the oral release rule of the flavor substance: testing different time points of the same oral smokeless tobacco product in the whole chewing process by adopting the full-simulation chewing machine and set parameters, drawing a flavor substance release curve, and carrying out research on the flavor substance oral release rule;
the method for analyzing the content of the aroma substances in the product or the residual sample comprises the following steps: during testing, 1 product or residual sample is taken, packaged and cut into pieces, then placed into a container, added with 20mL of 1.00g/L ethanol solution of n-propyl benzoate, hermetically oscillated at room temperature for 60min, filtered by using a 0.45-micron filter membrane, and then quantitatively analyzed by using gas chromatography;
the gas chromatography parameters are as follows: a chromatographic column: an HP-innowax column of 30 m.times.0.25 mm.times.0.25 μm; carrier gas: helium, constant current 1.5 mL/min; sample introduction conditions are as follows: the temperature is 250 ℃, the sample injection amount is 1 mu L, and the split ratio is 1: 50; furnace temperature: keeping the temperature at 220 ℃; a detector: FID, 250 ℃;
wherein, specific parameters of the pressing force, chewing frequency, saliva amount and oral cavity temperature of the full-simulation chewing machine are set as shown in table 5;
TABLE 5
2. The method of studying the oral release of a flavor substance from an oral smokeless tobacco formulation of claim 1, wherein in step (1), at least 50 oral smokeless tobacco formulations to be studied are selected for weight measurement.
3. The method of studying the oral release of a flavor substance from an oral smokeless tobacco product according to claim 1, wherein in step (2), said volunteer is aged 18 to 55 years.
4. The method of claim 1, wherein in step (2), the time point of collection of the residual sample and the volunteer's saliva is any point within 0-60min after the start of the experiment.
5. The method of claim 1, wherein the artificial saliva used in the simulation of the in vitro test is an inorganic salt artificial saliva or a protease artificial saliva.
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| CN102353736A (en) * | 2011-07-14 | 2012-02-15 | 浙江中烟工业有限责任公司 | Method for determining menthol in cigarette filter rod |
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| CN102353736A (en) * | 2011-07-14 | 2012-02-15 | 浙江中烟工业有限责任公司 | Method for determining menthol in cigarette filter rod |
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