Method for rapidly detecting mildew of tobacco leaves based on nitrite colorimetric probe
Through Ce-PCN-222 nanoenzyme as a nitrite colorimetric probe, the nitrite content changes during tobacco leaf mold was solved, and the problem of increased nitrite content caused by tobacco mold was achieved efficient and accurate Mold detection reduces economic losses and costs.
Patent Information
- Application Number
- CN202411486071.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2024-12-17
AI Technical Summary
The prior art is difficult to effectively solve the problem of increased nitrite content caused by tobacco mold, which affects tobacco quality and consumer health.
Using Ce-PCN-222 nanozyme as a nitrite colorimetric probe, the nitrite content changes during the moldy process of tobacco leaves was used to establish rapid detection by simulated peroxidase activity of Ce-PCN-222 nanozyme. The moldy method uses nitrite as a marker to improve the accuracy and efficiency of detection.
It has achieved an accuracy rate of up to 90% and quickly identified moldy tobacco leaves, reducing detection time and cost, reducing economic losses caused by moldy, and ensuring the quality of tobacco products and consumers' health.
Smart Images
Figure CN119147486A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of chemical analysis and detection, and in particular to a method for quickly detecting tobacco leaf mildew based on a nitrite colorimetric probe. Background Art
[0002] As an important cash crop, the process from field planting to final processing into cigarettes is complex and time-consuming, usually requiring multiple links such as harvesting, baking, purchasing, transportation, leaf threshing, re-baking, and aging. The entire cycle generally takes 2-3 years. In this process, tobacco quality maintenance and storage management are crucial, which is directly related to the quality of cigarette products and consumer acceptance.
[0003] Molds are fungal microorganisms that are widely found in nature. They are extremely sensitive to the temperature and humidity conditions of the growth environment. Once the conditions are suitable, they can reproduce rapidly, posing a serious threat to tobacco. Mildew not only damages the physical structure of tobacco leaves, but also causes the deterioration of chemical components, especially the increase in nitrite content, which not only affects the taste and aroma of tobacco, but also may pose a potential risk to human health. Therefore, exploring effective tobacco anti-mildew technology is of great significance to ensuring the sustainable development of the tobacco industry and maintaining consumer health.
[0004] At present, research on tobacco mold problems mainly focuses on exploring the mechanism of mold growth, developing new mold inhibitors, and optimizing storage conditions. However, these measures are often difficult to fundamentally solve the problem of increased nitrite content caused by mold growth. Studies have shown that mold activity can activate nitrate reductase and nitrite reductase in tobacco leaves. The increase in the activity of these two enzymes directly leads to nitrite accumulation, exacerbating the deterioration of tobacco quality.
[0005] Nanozymes, as an emerging enzyme-mimicking material, have shown broad application prospects in the fields of biosensing, environmental governance, and medical diagnosis due to their unique physicochemical properties and potential biocatalytic activity. Among them, metal-organic framework (MOFs) materials, especially mesoporous zirconium-porphyrin MOFs (such as PCN-222), are regarded as nanozyme candidates with great potential due to their porous structure, composition adjustability, and ease of chemical modification. However, the original PCN-222 nanoparticles still have shortcomings in simulating peroxidase activity, mainly manifested in limited electron transfer ability and weak enzyme activity, which limits its application in tobacco mildew prevention and nitrite control.
[0006] In view of this, the present invention is proposed. Summary of the invention
[0007] The purpose of the present invention is to provide a method for quickly detecting tobacco leaf mildew based on nitrite colorimetric probe. The present invention proposes for the first time to predict tobacco leaf mildew by utilizing the changing law of nitrite content in the process of tobacco leaf mildew, and establishes a new method for nitrite colorimetric probe determination by utilizing Ce-PCN-222 nanozyme. The identification process of the mildewed tobacco leaf is simple, rapid and accurate, and is easy to promote, which can effectively reduce the economic losses caused by tobacco leaf mildew.
[0008] The invention provides a method for quickly detecting tobacco leaf mildew based on a nitrite colorimetric probe. The method utilizes the property of Ce-PCN-222 nanozyme that oxidizes 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid to green oxidized 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid, and the effect of nitrite on sensitizing the enzyme of the Ce-PCN-222 nanozyme peroxidase oxidation system. Based on the change of nitrite concentration caused by the change of nitrate reductase caused by microorganisms in the process of tobacco leaf mildew, a method for quickly detecting tobacco leaf mildew with a nitrite colorimetric probe is established.
[0009] The present invention discloses for the first time the use of nitrite as a tobacco leaf mildew marker, and the prediction of tobacco leaf mildew is carried out by using the changing law of nitrite content in the process of tobacco leaf mildew; at the same time, the Ce-PCN-222 nanozyme synthesized by the present invention has the simulated peroxidase property, in the presence of nitrite, hydroxyl radicals (·OH) excite NO2 - Produces NO2 - ·, thereby increasing the peroxidase activity of the system and establishing NO2 - A new method for colorimetric probe detection.
[0010] Specifically, Ce-PCN-222 nanozyme has the property of oxidizing 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to green oxidized 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid (ox-ABTS). At this time, the nitrite produced by tobacco leaf mildew will produce an enzyme sensitization effect on the Ce-PCN-222 nanozyme peroxidase oxidation system, thereby increasing the absorbance. During the mildew process of tobacco leaves, the nitrite content first gradually increases and then gradually decreases. When mildew occurs, the nitrite content reaches the maximum value, and its change rate is between 70% and 80%. Therefore, the change rate of nitrite content between 70% and 80% can be used as a prediction and identification standard for mildewed tobacco leaves.
[0011] Studies have shown that this method can quickly identify moldy tobacco leaves with an accuracy rate of over 90%, greatly improving the efficiency and accuracy of identifying moldy tobacco leaves.
[0012] As a preferred embodiment of the present technical solution, the method for rapidly detecting tobacco leaf mildew based on nitrite colorimetric probe of the present invention specifically comprises the following steps:
[0013] First, in the presence of 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid, H2O2, Ce-PCN-222 nanozyme and NO2 - In the standard detection system, add acetic acid-acetate buffer solution, react for 5-10 minutes, and establish the absorbance and NO2 - The quantitative relationship of concentration, draw the standard curve, and obtain the regression equation;
[0014] Then, normal tobacco leaf extract and moldy tobacco leaf extract were added to the sample detection system of 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid, H2O2 and Ce-PCN-222 nanozyme, and acetic acid-acetate buffer solution was added respectively. After reacting for 5-10 minutes, the absorbance was measured. According to the regression equation, the NO2 in the tobacco leaf extract can be obtained. - The content.
[0015] As a preferred embodiment of the present invention, in the standard detection system or the sample detection system, NO2 - The concentration range of the solution is 0.25-37.5 mg / L;
[0016] The concentration range of Ce-PCN-222 solution was 0.1 mg / mL, and the addition amount was 50-100 μL;
[0017] The concentration range of ABTS solution is 50mmol / L, and the addition amount is 20-50μL.
[0018] The concentration range of H2O2 is 50mmol / L, and the amount added is 20-50μL.
[0019] As a preferred embodiment of the present technical solution, the wavelength of the absorbance test of the present invention is 400-450nm, and preferably 415nm.
[0020] As a preferred embodiment of the present technical solution, the pH of the acetic acid-acetate buffer solution used in the present invention is 3.5-5, and preferably 4.
[0021] As a preferred embodiment of the present technical solution, the preparation method of the Ce-PCN-222 nanozyme comprises the following steps:
[0022] S1, take ZrCl4 and benzoic acid, add them to purified water and DMF, stir them evenly, add meso-tetrakis(4-carboxyphenyl)porphine, stir them evenly, and then add acetic acid to obtain a homogeneous solution;
[0023] S2, transferring the homogeneous solution to a high-pressure reactor, heating to 110-130°C for reaction for 4-5 hours, cooling to room temperature, and centrifuging, washing and vacuum drying the obtained product to obtain PCN-222;
[0024] S3. Disperse PCN-222 and Ce(NO3)3 in ethylene glycol, add deionized water, stir evenly, and then quickly add into the pre-cooled NaBH4 aqueous solution, continue stirring for 2-3 hours, and the obtained product is centrifuged, washed and vacuum dried in sequence to obtain Ce-PCN-222 nanozyme.
[0025] In the preparation process of Ce-PCN-222 nanozyme of the present invention, first, in step S1, ZrCl4 and benzoic acid are selected as the main raw materials, both of which have good solubility in water and DMF (dimethylformamide) solvent, providing a basis for the subsequent coordination reaction; meso-tetrakis (4-carboxyphenyl) porphine (TCPP) as an organic ligand has the ability to coordinate with metal ions. During the stirring process, TCPP and Zr 4+ The ions undergo coordination reaction to form a stable zirconium oxygen cluster (Zr6 cluster) structure; at the same time, benzoic acid, as an auxiliary ligand, also helps to stabilize this structure. Then, step S2 transfers the homogeneous solution after the coordination reaction to a high-pressure reactor for solvent thermal synthesis. Under high temperature and high pressure conditions, Zr6 cluster and TCPP are further assembled to form a MOF (metal organic framework) material with a porous structure, namely PCN-222. This MOF material has a large pore size and specific surface area, which is conducive to the subsequent catalytic reaction. Finally, in step S3, PCN-222 and Ce(NO3)3 are dispersed in ethylene glycol, and Ce is stirred to 3+ The ions are evenly distributed in the pores of PCN-222; ethylene glycol, as a solvent and a precursor of a reducing agent, is helpful for the subsequent reduction reaction; after further adding deionized water, a pre-cooled NaBH4 aqueous solution is quickly added. NaBH4, as a strong reducing agent, can reduce Ce at low temperatures. 3+ The ions are reduced to CeO2 nanoparticles or low-valent ions of Ce and uniformly deposited in the pores of PCN-222. In this process, ethylene glycol also plays a synergistic reduction role. Through the reduction reaction, CeO2 nanoparticles or low-valent ions of Ce are tightly combined with the framework structure of PCN-222 to form Ce-PCN-222 nanozymes.
[0026] Therefore, the Ce-PCN-222 nanozyme prepared by the present invention not only inherits the porous structure and stability of PCN-222, but also endows it with the catalytic activity of the Ce element. Since the Ce element has multiple valence states and excellent redox properties, the incorporation of the Ce element changes the electronic structure and surface properties of PCN-222 and endows it with the characteristics of simulating peroxidase activity.
[0027] As a preferred embodiment of the present technical solution, in step S1, the amounts of benzoic acid, purified water, DMF, meso-tetrakis(4-carboxyphenyl)porphine and acetic acid required for every 350-400 mg of ZrCl4 are 8-10 g, 8-10 mL, 80-100 mL, 350-400 mg and 5-6 mL respectively.
[0028] Specifically, step S1 comprises: weighing 350-400 mg ZrCl4 and 8-10 g benzoic acid, adding them to 8-10 mL purified water and 80-100 mL DMF, stirring for 5-10 min, adding 350-400 mg tetrakis (4-carboxyphenyl) porphine (TCPP), continuing to stir at room temperature for 5-10 min, and then adding 5-6 mL acetic acid, mixing evenly to obtain a homogeneous solution.
[0029] As a preferred embodiment of the present technical solution, step S2 includes: transferring the homogeneous solution to a polytetrafluoroethylene high-pressure reactor, heating to 110-130°C for reaction for 4-5 hours, cooling to room temperature, centrifuging the product at 8000-10000 rpm for 5-10 minutes, then washing with ethanol and acetone for 2-3 times respectively, and finally vacuum drying to obtain PCN-222.
[0030] As a preferred embodiment of the present technical solution, in step S3, the amounts of Ce(NO3)3, ethylene glycol, deionized water and NaBH4 aqueous solution corresponding to every 300-400 mg PCN-222 are 120-150 mg, 15-20 mL, 20-30 mL and 5-6 mL respectively; wherein, in the NaBH4 aqueous solution, the concentration of NaBH4 is 80-120 mM.
[0031] Specifically, step S3 includes: fully dispersing 300-400 mg PCN-222 and 120-150 mg Ce(NO3)3 in 15-20 mL ethylene glycol, then adding 20-30 mL deionized water, stirring at room temperature for 5-10 min, and then quickly adding 5-6 mL NaBH4 (100 mM, pre-cooled) aqueous solution, stirring for 2-3 h, and separating the product by centrifugation at 8000-10000 rpm for 5-10 min. The precipitate is washed alternately with deionized water and ethanol for 2-3 times, and finally vacuum dried to obtain Ce-PCN-222 nanozyme.
[0032] As a preferred embodiment of the present technical solution, the preparation method of normal tobacco leaf extract or moldy tobacco leaf extract comprises:
[0033] Weigh tobacco leaf powder (accurate to 1 mg) and activated carbon and place them in a centrifuge tube, then add purified water and saturated borax solution in sequence, mix well, ultrasonicate for 10-15 minutes, heat and extract at 70-80°C for 15-20 minutes, centrifuge at 5000r / min for 10-15 minutes, take the supernatant and obtain the tobacco leaf extract.
[0034] As a preferred embodiment of the present technical solution, the amounts of activated carbon, purified water and saturated borax solution required per 1g of tobacco leaf powder are 1.5-2.0g, 50-60mL and 3-4mL respectively.
[0035] The method for preparing moldy tobacco leaves comprises the following steps:
[0036] The tobacco leaf samples were placed in a constant temperature and humidity chamber (temperature 25±2°C, humidity 60±5%) for a tobacco leaf mildew experiment. The samples were taken in the following manner with a 14-day cycle:
[0037] (1) On day 0, i.e., before being placed in a constant temperature and humidity chamber at 25°C and 65% humidity, the first sampling was performed;
[0038] (2) Days 1-14: sampling was conducted on days 1, 2, 4, 7, 9, 11, 12, 13, and 14, respectively.
[0039] According to the NO2 content of tobacco samples with different degrees of mildew - The content of NO2 - The correlation between the content change rate and tobacco leaf mildew was determined to identify the NO2 - Rate of change range.
[0040] The method for quickly detecting tobacco leaf mildew based on nitrite colorimetric probe of the present invention has at least the following beneficial effects:
[0041] 1. The present invention is based on the change of nitrite concentration caused by the change of nitrate reductase caused by microorganisms during the mildew process of tobacco leaves. The porphyrin-based metal-organic framework compound (Ce-PCN-222) with a mixed structure of nanocubes and sheets has the simulated oxidase nanozyme activity to oxidize 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to green oxidized ox-ABTS. Since the nitrite produced by mildew has an enzyme sensitization effect on the nanozyme peroxidase oxidation system, resulting in an increase in absorbance, a new method for nitrite detection is established. During the mildew process of tobacco leaves, the nitrite content gradually increases and then gradually decreases. When mildew occurs, the nitrite content reaches the maximum value, and its change rate is between 70% and 80%. Therefore, the change rate of nitrite content between 70% and 80% can be used as a prediction and identification standard for mildewed tobacco leaves. This method can quickly identify mildewed tobacco leaves with an accuracy rate of more than 90%, greatly improving the recognition efficiency and accuracy of mildewed tobacco leaves;
[0042] 2. Compared with traditional tobacco leaf mildew detection methods, such as sensory evaluation, chemical analysis or microbial culture, the method of the present invention is simpler and more direct, does not require complicated sample pretreatment or long experimental process, and can quickly determine the mildew state of tobacco leaves through colorimetric reaction, greatly shortening the detection time and improving work efficiency;
[0043] 3. The materials used in the present invention (such as Ce-PCN-222 nanozyme, ABTS, etc.) are relatively easy to obtain and low in cost. They are also easy to operate, which reduces the reliance on expensive testing equipment and professionals, reduces testing costs, and has significant economic advantages.
[0044] 4. The present invention can timely and accurately identify moldy tobacco leaves, which helps to take measures in the early stage of mold, such as adjusting storage conditions and timely handling of moldy tobacco leaves, thereby effectively reducing the quality decline and economic losses of tobacco leaves caused by mold. It is of great significance to ensure the quality stability of tobacco products, safeguard the rights and interests of consumers, and promote the sustainable development of the tobacco industry;
[0045] In summary, the method for quickly identifying tobacco leaf mold based on nitrite colorimetric probe provided by the present invention not only improves detection efficiency and accuracy, but also reduces costs, which is of great significance for ensuring the quality of tobacco products, reducing economic losses and promoting the healthy development of the tobacco industry. BRIEF DESCRIPTION OF THE DRAWINGS
[0046] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0047] Figure 1 Transmission electron microscopy (a) and scanning electron microscopy (b) images of Ce-PCN-222 nanozyme synthesized by the present invention;
[0048] Figure 2 In the embodiment of the present invention, NO2 - Linear UV-visible absorption spectrum;
[0049] Figure 3 In the embodiment of the present invention, NO2 - Linear regression equation;
[0050] Figure 4 and 5 The effects of different coexisting substances on NO2 in the embodiments of the present invention are respectively - The impact results. DETAILED DESCRIPTION
[0051] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.
[0052] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.
[0053] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0054] Example
[0055] The preparation method of Ce-PCN-222 nanozyme is as follows:
[0056] S1. Weigh 350 mg ZrCl4 and 9 g benzoic acid, add 10 mL purified water and 100 mL DMF, stir for 10 min, add 400 mg tetrakis(4-carboxyphenyl)porphine (TCPP), continue stirring at room temperature for 5-10 min, add 6 mL acetic acid, mix well, and obtain a homogeneous solution;
[0057] S2, the homogeneous solution was transferred to a polytetrafluoroethylene autoclave, heated to 120°C for 4-5 hours, cooled to room temperature, and the product was separated by centrifugation at 10000 rpm for 5 minutes, washed with ethanol and acetone for 2-3 times respectively, and vacuum dried to obtain PCN-222;
[0058] S3. Disperse 300 mg PCN-222 and 120 mg Ce(NO3)3 in 15 mL ethylene glycol, add 30 mL deionized water, stir at room temperature for 5 min, then quickly add 5 mL NaBH4 (100 mM, pre-cooled) aqueous solution, continue stirring for 2-3 h, separate the product by centrifugation at 8000 rpm for 10 min, wash the precipitate alternately with deionized water and ethanol 2-3 times, and vacuum dry to obtain Ce-PCN-222 nanozyme.
[0059] Figure 1 The transmission electron microscopy image (a) and scanning electron microscopy image (b) of the Ce-PCN-222 nanozyme synthesized by the present invention.
[0060] Depend on Figure 1 It can be seen that the Ce-PCN-222 nanozyme synthesized in the present invention has a mixed structure of nanocubes and sheets.
[0061] The method for evaluating the peroxidase activity of Ce-PCN-222 nanozyme is as follows:
[0062] Using ABTS and H2O2 as substrates, by adding NO2 - Or without adding NO2 - , the mimetic enzyme activity of peroxidase was determined.
[0063] Depend on Figure 2 It can be seen that in NO2 - In the presence of NO2, the absorption peak at 415 nm was significantly enhanced, indicating that - It has a sensitizing effect on the simulated peroxidase activity of the system.
[0064] NO2 - The method for making the standard working curve is as follows:
[0065] Add 100 μL of 0.1 mg / mL Ce-PCN-222 nanozyme, 100 μL of 50 mmol / L ABTS, 50 μL of 50 mmol / L H2O2, and NO2 in the range of 0.25-37.5 mg / L into a 5 mL stoppered colorimetric tube. - Standard solution, add acetic acid-acetate buffer solution with pH 4.0 to 3mL, react for 5-10min, test the absorbance at 415nm wavelength, and use NO2 - With concentration as the horizontal axis and absorbance A as the vertical axis, a standard curve is drawn to obtain the regression equation.
[0066] Figure 3 NO2 - The standard curve and linear regression equation are shown in Table 1.
[0067] Table 1 Regression equation, correlation coefficient, relative standard deviation, linear range
[0068]
[0069] The present invention further investigates the influence of coexisting substances that may exist in flue-cured tobacco samples on the colorimetric probe system, and the specific method is as follows:
[0070] like Figure 4 and 5 As shown, in NO2 - When the concentration is 12.5 mg / L, add 100 times the concentration of Mg 2+ Cr 3+ 、Ni 2+ , Mn 2+ 、Co 2+ 、NO3 - 、SO4 2- , Cl - 、F - ; 2 times Pb 2+ 、Cd 2+ , Hg 2+ ; At the same time, add 5 times the concentration of glucose, ascorbic acid, nicotine, leucine, lysine, tyrosine, and arginine.
[0071] Studies have shown that the inorganic ion Mg 2+ Cr 3+ 、Ni 2+ , Mn 2+ 、Co 2+ 、NO3 - 、SO4 2- , Cl - 、F - ; 2 times Pb 2+ 、Cd2+ , Hg 2+ It has almost no effect on the catalytic oxABTS of the detection system, so there is no interference in the measurement results; the coexisting organic substances such as glucose, nicotine, leucine, lysine, tyrosine, and arginine have almost no effect on the activity of Ce-PCN-222 nanozyme; only ascorbic acid can reduce the activity of Ce-PCN-222 nanozyme.
[0072] The above results show that this method has good selectivity and anti-interference effect and can be selectively used for NO2 - Determination of.
[0073] Test example
[0074] The preparation method of moldy tobacco leaves is as follows:
[0075] The tobacco leaf samples were placed in a constant temperature and humidity chamber (temperature 25±2°C, humidity 60±5%) for tobacco leaf mildew experiment. The samples were taken in the following manner with a cycle of 14 days:
[0076] (1) On day 0, i.e., before being placed in a constant temperature and humidity chamber at 25°C and 65% humidity, the first sampling was performed;
[0077] (2) Days 1-14: sampling was conducted on days 1, 2, 4, 7, 9, 10, 11, 13, and 14, respectively.
[0078] The preparation method of tobacco leaf extract is as follows:
[0079] Twenty samples of first-cured tobacco leaves from central Pu'er, Baoshan, Lijiang and Dali were taken as the research objects. 1g (accurate to 1mg) of normal tobacco leaves and tobacco powder from different days of mildew test and 1.5-20g of activated carbon were weighed and placed in a 100mL centrifuge tube. 50-60mL of purified water and 3-4mL of saturated borax solution were added. After mixing, the mixture was ultrasonically treated for 10-15min, and then heated and extracted at 70-80℃ for 15-20min. The mixture was centrifuged at 5000r / min for 10-15min, and the supernatant was taken to obtain the tobacco extract.
[0080] The prediction method of tobacco leaf mildew is as follows:
[0081] In a 5mL stoppered colorimetric tube, add 100μL of 0.1mg / mL Ce-PCN-222 nanozyme, 100μL of 50mmol / L ABTS, 50μL of 50mmol / L H2O2, normal tobacco leaf extract or moldy tobacco leaf extract, add acetic acid-acetate buffer solution with a pH of 4.0 to 3mL, react for 5-10min, measure the absorbance at a wavelength of 415nm, and then substitute the absorbance into the above regression equation to obtain NO2 -The concentration was adjusted and the change rate of nitrite content was calculated according to the following formula. The results are shown in Table 2.
[0082] Nitrite content change rate x% = (nitrite content on the nth day of the mildew experiment - nitrite content on the 0th day of the mildew experiment) / nitrite content on the nth day of the mildew experiment × 100
[0083] Table 2 NO2 in the 14-day experimental period of the first-cured tobacco leaf samples - Content (μg / g)
[0084]
[0085]
[0086] As shown in Table 2, the nitrite content reached the maximum value on the 10th day of the mildew experiment, and mildew spots were visible to the naked eye. The change rate of nitrite content on the 10th day was calculated to be x%. The change range of nitrite content in the 20 samples was 70%-80%. Based on this, the change of nitrite content by 70%-80% was used as the judgment range for the beginning of tobacco leaf mildew.
[0087] In summary, the present invention utilizes the newly established nitrite determination method to measure nitrite in different moldy tobacco leaves, and uses this as a tobacco leaf mold marker to predict tobacco leaf mold. The method has the advantages of being simple, fast, accurate, and easy to promote. This method can effectively reduce the economic losses caused by tobacco leaf mold.
[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for rapidly detecting tobacco leaf mildew based on nitrite colorimetric probe, characterized in that: Taking advantage of the property of Ce-PCN-222 nanozyme to oxidize 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid to green oxidized 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid, and the effect of nitrite on the sensitization of the Ce-PCN-222 nanozyme peroxidase oxidation system, a method for rapid detection of tobacco leaf mildew with nitrite colorimetric probe was established based on the change of nitrite concentration caused by changes in nitrate reductase caused by microorganisms during tobacco leaf mildew.
2. The method for rapid detection of tobacco leaf mildew based on nitrite colorimetric probe according to claim 1, characterized in that: The following steps are involved: In the presence of 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid, H2O2, Ce-PCN-222 nanozyme and NO2 - In the standard detection system, add acetic acid-acetate buffer solution, react for 5-10 minutes, and establish the absorbance and NO2 - The quantitative relationship of concentration, draw the standard curve and obtain the regression equation; In the sample detection system of 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid, H2O2 and Ce-PCN-222 nanozyme, normal tobacco leaf extract and moldy tobacco leaf extract were added respectively, and acetic acid-acetate buffer solution was added respectively. After reacting for 5-10 minutes, the absorbance was measured, and the NO2 in the tobacco leaf extract was obtained according to the regression equation. - The content.
3. The method for rapid detection of tobacco leaf mildew based on nitrite colorimetric probe according to claim 2, characterized in that: In the standard test system or sample test system, NO2 - The concentration of the solution is 0.25-37.5 mg / L; The concentration of Ce-PCN-222 solution was 0.1 mg / mL, and the amount added was 50-100 μL; The concentration of ABTS solution is 50mmol / L, and the amount added is 20-50μL. The concentration of H2O2 is 50mmol / L, and the amount added is 20-50μL.
4. The method for rapid detection of tobacco leaf mildew based on nitrite colorimetric probe according to claim 2, characterized in that: The wavelength of absorbance test is 400-450nm.
5. The method for rapid detection of tobacco leaf mildew based on nitrite colorimetric probe according to claim 2, characterized in that: The pH of the acetic acid-acetate buffer solution is 3.5-5.
6. The method for rapid detection of tobacco leaf mildew based on nitrite colorimetric probe according to claim 2, characterized in that: The preparation method of the Ce-PCN-222 nanozyme comprises the following steps: S1, take ZrCl4 and benzoic acid, add them to purified water and DMF, stir them evenly, add meso-tetrakis(4-carboxyphenyl)porphine, stir them evenly, and then add acetic acid to obtain a homogeneous solution; S2, transferring the homogeneous solution to a high-pressure reactor, heating to 110-130°C for reaction for 4-5 hours, cooling to room temperature, and centrifuging, washing and vacuum drying the obtained product to obtain PCN-222; S3. Disperse PCN-222 and Ce(NO3)3 in ethylene glycol, add deionized water, stir evenly, and then quickly add into the pre-cooled NaBH4 aqueous solution, continue stirring for 2-3 hours, and the obtained product is centrifuged, washed and vacuum dried in sequence to obtain Ce-PCN-222 nanozyme.
7. The method for rapid detection of tobacco leaf mildew based on nitrite colorimetric probe according to claim 6, characterized in that: In step S1, the amounts of benzoic acid, purified water, DMF, meso-tetrakis(4-carboxyphenyl)porphine and acetic acid required for every 350-400 mg ZrCl4 are 8-10 g, 8-10 mL, 80-100 mL, 350-400 mg and 5-6 mL, respectively.
8. The method for rapid detection of tobacco leaf mildew based on nitrite colorimetric probe according to claim 6, characterized in that: In step S3, the amounts of Ce(NO3)3, ethylene glycol, deionized water and NaBH4 aqueous solution required for every 300-400 mg PCN-222 are 120-150 mg, 15-20 mL, 20-30 mL and 5-6 mL respectively; Among them, the concentration of NaBH4 in the NaBH4 aqueous solution is 80-120mM.
9. The method for rapid detection of tobacco leaf mildew based on nitrite colorimetric probe according to claim 2, characterized in that: The preparation method of normal tobacco leaf extract or moldy tobacco leaf extract comprises: Weigh tobacco leaf powder and activated carbon and place them in a centrifuge tube, then add purified water and saturated borax solution in sequence, mix well and perform ultrasonic treatment for 10-15 minutes, then heat and extract at 70-80°C for 15-20 minutes, centrifuge and take the supernatant to obtain a tobacco leaf extract.
10. The method for rapid detection of tobacco leaf mildew based on nitrite colorimetric probe according to claim 9, characterized in that: For every 1g of tobacco leaf powder, the required amounts of activated carbon, purified water and saturated borax solution are 1.5-2.0g, 50-60mL and 3-4mL respectively.