Method for monitoring nicotine content in field fresh tobacco leaves

Abstract

The invention provides a method for monitoring nicotine content in field fresh tobacco leaves, which comprises the following steps: 1) collecting a fresh tobacco leaf sample, putting the sample into a grinding bag, adding water according to a liquid-material ratio of (6-15): 1, grinding, uniformly mixing, filtering and collecting filtrate; 2) taking a small amount of the filtrate, and diluting to obtain a sample diluent; (3) dropwise adding the sample diluent onto a nicotine test strip, standing, and measuring a T value and a C value on a portable fluorescence detector; and 4) collecting T values, C values and T / C values of all sample diluents, and combining the standard curve on mELISA software to obtain the nicotine content. The invention belongs to the technical field of agricultural detection, realizes rapid and quantitative detection of field fresh tobacco leaves, is suitable for whole-process monitoring from tobacco plant transplanting to tobacco leaf harvesting, facilitates regulation and control of tobacco leaf nicotine content, and provides a decision basis for production technical measures (such as fertilization) according to the accurate nicotine content.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of agricultural detection, and particularly relates to a method for monitoring the nicotine content in field fresh tobacco leaves. BACKGROUND

[0002] Nicotine, also known as nicotine, is the most prominent characteristic chemical component of tobacco and tobacco products, and is one of the important markers that distinguish tobacco and tobacco products from other genera. Nicotine is the main source of tobacco flavor, can stimulate the taste of smokers, and is an important factor in determining the sensory quality and industrial usability of tobacco leaves. At present, the nicotine content of high-quality tobacco leaves is about 1.5% to 3.5%, and the content is too low, which is insufficient, and the content is too high, which is too strong.

[0003] Based on the important influence of nicotine on tobacco leaves, the determination of its content has become an important content of the tobacco industry. There are many methods for determining the nicotine content in tobacco leaves, such as ultraviolet spectrophotometry, silicotungstic acid precipitation method, GC / MS method, high performance liquid chromatography, etc. These methods are complex in sample processing, require baking or killing dry leaves, take a long time, and have high requirements for instruments and equipment. Fresh tobacco leaves basically maintain the stability of nicotine content during subsequent processing such as baking after picking. On-line monitoring of nicotine content in fresh tobacco leaves can facilitate the regulation of nicotine content. Chinese patent application CN 113390810A discloses a method for determining the nicotine content of fresh tobacco leaves, which adopts acid extraction and release method, including the following steps: 1) taking fresh tobacco leaves, grinding with liquid nitrogen, and adding 16%-22% hydrochloric acid for extraction at room temperature; 2) high-speed centrifugation, taking the supernatant, diluting the supernatant with equal volume of water, and adding activated carbon with a final concentration of 5%, and mixing well; 3) high-speed centrifugation, taking the supernatant, adding 2mL of 8%-11% hydrochloric acid per 1mL of supernatant, and mixing well; 4) high-speed centrifugation, taking the supernatant, which is the nicotine extract; 5) measuring the absorbance of the nicotine extract at 259nm, 236nm and 282nm; setting the leaf moisture content to 90%, and calculating the nicotine content of the sample according to the formula. The determination method needs to use more organic solvents, and needs to be centrifuged for several times, which is not conducive to environmental protection and rapid detection.

[0004] Therefore, it is of great significance to provide a method for monitoring the nicotine content in field fresh tobacco leaves, which is easy to operate and reduces the use of organic solvents. SUMMARY

[0005] In order to solve the problems in the prior art, the present application provides a method for monitoring the nicotine content in field fresh tobacco leaves, which realizes rapid and quantitative detection of field fresh tobacco leaves, is suitable for monitoring the whole process from transplanting to harvesting of tobacco plants, and is convenient for regulating the nicotine content of tobacco leaves.

[0006] The object of the present application will be further illustrated by the following detailed description.

[0007] The application provides a monitoring method for nicotine content in field fresh tobacco leaves, comprising the following steps:

[0008] 1) collecting fresh tobacco leaf samples, placing the samples in grinding bags, adding water according to a liquid-to-material ratio of 6-15:1, grinding and mixing, filtering, and collecting the filtrate;

[0009] 2) taking a small amount of the filtrate, diluting to obtain a sample diluent;

[0010] 3) taking the sample diluent, dropping it on a nicotine test strip, standing, and determining T value and C value on a portable fluorescence detector;

[0011] 4) collecting T value, C value and T / C value of all sample diluents, combining with a standard curve on mELISA software, and obtaining nicotine content.

[0012] Preferably, the liquid-to-material ratio is 8-12:1. The liquid-to-material ratio refers to the mass ratio of added water to fresh tobacco leaves.

[0013] More preferably, the liquid-to-material ratio is 10:1.

[0014] Preferably, the dilution multiple is 180-220 times, and water is used for dilution.

[0015] More preferably, the dilution multiple is 200 times.

[0016] Preferably, the excitation wavelength of the fluorescence detector is 335-345 nm, and the emission wavelength is 610-620 nm.

[0017] More preferably, the excitation wavelength of the fluorescence detector is 340 nm, and the emission wavelength is 615 nm.

[0018] Preferably, the fresh tobacco leaves are selected from middle tobacco leaves. More preferably, when collecting fresh tobacco leaf samples, the samples are cut after avoiding main veins and branch veins.

[0019] Preferably, the standing time is 8-12 min.

[0020] More preferably, the standing time is 10 min.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] (1) The application applies existing nicotine test strips to monitoring nicotine content in field fresh tobacco leaves, and provides a monitoring method for nicotine content in field fresh tobacco leaves, which is simple in sample pretreatment, rapid in detection, high in accuracy of detection results, and very close to the detection results of the industry standard YC / T 246-2008 "Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatography".

[0023] (2) The monitoring method for nicotine content in field fresh tobacco leaves provided by the application is suitable for monitoring the whole process from transplanting to harvesting, is convenient for regulating and controlling the nicotine content of tobacco leaves, can detect all tobacco products or non-tobacco products containing nicotine, achieves qualitative and quantitative detection effects, and provides a decision basis for production technical measures (such as fertilization) for the accurate nicotine content. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The schematic diagram of the extract obtained by different solvents; the solvents from left to right are water, 5% acetic acid, methanol, methanol and 5% sodium hydroxide mixed solution with a volume ratio of 4:1.

[0025] Figure 2 The detection results of the method of the application are subjected to point-to-point operation function fitting. DETAILED DESCRIPTION

[0026] The application will be further described in detail below in combination with the drawings and examples.

[0027] In the application, the materials and reagents involved are all conventional commercially available products, or can be obtained by conventional technical means in the art. Unless otherwise specified, the % content refers to the mass percentage content.

[0028] Example 1: Optimization of extraction conditions of nicotine in fresh tobacco leaves

[0029] 1. Optimization of extraction solvent

[0030] Free and bound nicotine is contained in fresh tobacco leaves, and the proportion of the two is about 9:1, so water is considered as the solvent; according to the pretreatment method in the industry standard “Determination of total plant alkaloids in tobacco and tobacco products-continuous flow method” (YC / T 160-2002), acetic acid solution is considered for extraction; according to the pretreatment method in the industry standard “Determination of nicotine in tobacco and tobacco products-gas chromatography method” (YC / T 246-2008), methanol and sodium hydroxide solution are considered. Therefore, the extraction efficiency of the following four kinds of extraction solvents is investigated: including water, 5% acetic acid, methanol, and a mixed solution of methanol and 5% sodium hydroxide with a volume ratio of 4:1.

[0031] The middle fresh tobacco leaves are cut into leaf pieces in mirror symmetry with the main veins (mainly taking the middle part of the leaves) 5 g, which are divided into five equal parts of tobacco leaf samples, 1 g each. One part is used for the test of pure water extraction, one part is used for the test of 5% acetic acid extraction, one part is used for the test of methanol extraction, one part is used for the test of mixed solution of methanol and 5% sodium hydroxide with a volume ratio of 4:1, and one part is used as a comparison according to the gas phase standard method in YC / T246-2008. The tobacco leaf samples are respectively placed in grinding bags, and 10 mL of the corresponding solvent is respectively added. After being fully ground and crushed, the filtrate is collected after filtration. A small amount of the filtrate is taken, diluted 100 times with water, added to a commercial test strip, dropped into 4 drops, and left to stand for 10 minutes. The T value and C value are determined by using a portable fluorescence instrument with an excitation wavelength of 340 nm and an emission wavelength of 615 nm, and the nicotine concentration is calculated.

[0032] The detection results of different solvent extractions are compared with the detection results of the gas phase standard method, and the results are shown in Table 1. The detection results of water as the solvent extraction are closest to the results of the gas phase standard method. Therefore, water is used as the extraction solvent.

[0033] Table 1 Comparison of detection results after extraction of different solvents

[0034]

[0035] 2. Optimization of liquid-to-material ratio

[0036] The middle fresh tobacco leaves are cut into leaf pieces in mirror symmetry with the main veins (mainly taking the middle part of the leaves) 4 g, which are divided into four equal parts of tobacco leaf samples, 1 g each. One part is used for the test of liquid-to-material ratio 10:1, one part is used for the test of liquid-to-material ratio 1:1, one part is used for the test of liquid-to-material ratio 1:10, and one part is used as a comparison according to the gas phase standard method in YC / T246-2008. The tobacco leaf samples are respectively placed in grinding bags, and the corresponding volume of solvent water is added. After being fully ground and crushed, the filtrate is collected after filtration. A small amount of the filtrate is taken, diluted 100 times with water, added to a commercial test strip, dropped into 4 drops, and left to stand for 10 minutes. The T value and C value are determined by using a portable fluorescence instrument with an excitation wavelength of 340 nm and an emission wavelength of 615 nm, and the nicotine concentration is calculated. The detection results of different liquid-to-material ratios after extraction are compared with the detection results of the gas phase standard method, and the results are shown in Table 2. The detection results of liquid-to-material ratio 10:1 extraction are closest to the results of the gas phase standard method. Therefore, the liquid-to-material ratio of 10:1 is used in the subsequent experiments.

[0037] Table 2 Comparison of detection results after extraction of different liquid-to-material ratios

[0038]

[0039] 3. Optimization of tobacco leaf sampling position

[0040] The middle fresh tobacco leaves are cut with mirror symmetry along the main veins, 2 g of leaf tip, middle and base are taken and divided into two equal parts, one part is tested by using test paper, and the other part is tested by using the gas phase standard method in YC / T246-2008. The tobacco leaf sample is placed in a grinding bag, the corresponding volume of solvent water is added, and the sample is fully ground and crushed. After filtration, the filtrate is collected. A small amount of filtrate is taken, diluted 100 times with water, added to a commercial test paper, 4 drops are dropped, and after 10 minutes of standing, a portable fluorescence instrument is used for determination. The excitation wavelength is 340 nm, the emission wavelength is 615 nm, the T value and the C value are determined, and the nicotine concentration is calculated. The detection results of different sampling parts of the tobacco leaves are compared with the detection results of the corresponding gas phase standard method, and the results are shown in Table 3. The detection results of the middle sampling part are closest to the results of the gas phase standard method, and therefore, the middle sampling part is used in the subsequent experiment.

[0041] Table 3 Comparison of detection results of different sampling parts

[0042] Sample Name Tobacco Leaf Part Test Strip Results (%) Gas Phase Results Sample 3 Leaf Tip 0.09 0.05％ Sample 3 Mid Leaf 0.12 0.15％ Sample 3 Leaf Base 0.13 0.20％

[0043] 4. Dilution multiple optimization

[0044] The middle fresh tobacco leaves are cut with mirror symmetry along the main veins (the middle part is taken) 5 g, which are divided into five equal parts of tobacco leaf samples. One part is used for 100 times dilution test, one part is used for 200 times dilution test, one part is used for 500 times dilution test, one part is used for 1000 times dilution test, and one part is used as a comparison according to the gas phase standard method in YC / T246-2008. The tobacco leaf sample is placed in a grinding bag, the corresponding volume of solvent water is added, and the sample is fully ground and crushed. After filtration, the filtrate is collected. A small amount of filtrate is taken, diluted by the corresponding multiple with water, added to a commercial test paper, 4 drops are dropped, and after 10 minutes of standing, a portable fluorescence instrument is used for determination. The excitation wavelength is 340 nm, the emission wavelength is 615 nm, the T value and the C value are determined, and the nicotine concentration is calculated. The detection results of different liquid-material ratios after extraction are compared with the detection results of the gas phase standard method, and the results are shown in Table 4. The detection results of 200 times dilution are closest to the results of the gas phase standard method, and therefore, 200 times dilution is used in the subsequent experiment.

[0045] Table 4 Comparison of detection results of different dilution multiples

[0046]

[0047] Monitoring method for nicotine content in field fresh tobacco leaves

[0048] The monitoring method for nicotine content in field fresh tobacco leaves comprises the following steps:

[0049] 1) Collect fresh tobacco leaf sample (take middle part of leaf), place in grinding bag, add water according to liquid material ratio 10:1, grind and mix, filter, collect filtrate;

[0050] 2) Take 10 μL of the filtrate, dilute 200 times to obtain sample diluent;

[0051] 3) Take the sample diluent, add 4 drops on nicotine test paper strip, stand for 10 min, measure T value and C value on portable fluorescence detector; excitation wavelength is 340 nm, emission wavelength is 615 nm;

[0052] 4) Collect T value, C value and T / C value of all sample diluents, combine with standard curve on mELISA software to obtain nicotine content.

[0053] According to the above method, 21 fresh tobacco leaf samples are detected, and at the same time, the gas phase standard method in YC / T 246-2008 is used as comparison. The results are shown in Table 5, and the standard curve of point-to-point operation is shown in Figure 2 From Table 5, it can be seen that the detection results of other samples except sample No. 10 are close to the gas phase standard method, indicating that the accuracy of the monitoring method provided by the present application is high.

[0054] Table 5 Detection results of 21 fresh tobacco leaf samples

[0055]

[0056] The above content is a further detailed description of the present application in combination with specific preferred embodiments, and the specific implementation of the present application cannot be limited to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, a number of simple deductions or substitutions can be made without departing from the concept of the present application, and all of them should be regarded as falling within the protection scope of the present application.

Claims

1. A method for monitoring nicotine content in fresh tobacco leaves grown in the field, characterized in that: The steps include: 1) Collect fresh tobacco leaf samples, place them in a grinding bag, add water at a liquid-to-solid ratio of 6-15:1, grind and mix, filter, and collect the filtrate; 2) taking a small amount of the filtrate and diluting it to obtain a sample dilution solution; 3) taking the sample dilution, adding it dropwise onto a nicotine test strip, letting it stand, and measuring the T value and C value on a portable fluorescence detector; 4) Collect T values, C values, and T / C values ​​of all sample dilutions and combine them with the standard curve on the mELISA software to obtain the nicotine content.

2. The method for monitoring nicotine content in fresh tobacco leaves grown in the field according to claim 1, wherein: The liquid-to-solid ratio is 8-12:

1.

3. The method for monitoring nicotine content in fresh tobacco leaves grown in the field according to claim 2, wherein: The liquid-to-material ratio is 10:

1.

4. The method for monitoring nicotine content in fresh tobacco leaves grown in the field according to claim 1, wherein: The dilution multiple is 180-220 times, and water is used for dilution.

5. The method for monitoring nicotine content in fresh tobacco leaves grown in the field according to claim 4, wherein: The dilution multiple is 200 times.

6. The method for monitoring nicotine content in fresh tobacco leaves grown in the field according to any one of claims 1 to 5, characterized in that: The excitation wavelength of the fluorescence detection instrument is 335-345 nm, and the emission wavelength is 610-620 nm.

7. The method for monitoring nicotine content in fresh tobacco leaves grown in the field according to claim 6, wherein: The excitation wavelength of the fluorescence detection instrument is 340 nm, and the emission wavelength is 615 nm.

8. The method for monitoring nicotine content in fresh tobacco leaves grown in the field according to any one of claims 1 to 5, characterized in that: The fresh tobacco leaves are selected from middle tobacco leaves.

9. The method for monitoring nicotine content in fresh tobacco leaves grown in the field according to any one of claims 1 to 5, characterized in that: The standing time is 8-12 minutes.

10. The method for monitoring nicotine content in fresh tobacco leaves grown in the field according to claim 9, characterized in that: The standing time is 10 min.

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