Dioscorea zingiberensis harmful substance detection method

Abstract

The invention provides a method for detecting harmful substances in yellow ginger, and relates to the technical field of harmful substance detection. The method for detecting harmful substances in yellow ginger specifically comprises the following steps: S1, constructing a three-phase extraction system; s2, mixing the samples; s3, dynamic oscillation; s4, separating the three-phase solution; s5, establishing a double-enzyme reaction system; and S6, final analysis. The technology realizes multi-index synchronous detection through three-phase dynamic extraction and biological enzyme cascade color development, has the advantages of rapidness, simplicity, convenience, low cost, high sensitivity and visualization, and is more suitable for large-scale popularization and application.

Description

Technical Field

[0001] The invention relates to the technical field of harmful substance detection, in particular to a method for detecting harmful substances in turmeric. Background Art

[0002] Turmeric is a perennial herb with the scientific name Curcuma longa, belonging to the genus Curcuma of the Zingiberaceae family. Its rhizomes are rich in active ingredients such as curcumin and are widely used in food, medicine, dyes and other fields. The main harmful substances that may exist in turmeric include heavy metals (such as lead, cadmium, mercury, etc.), organophosphorus pesticide residues (such as chlorpyrifos, methyl parathion, etc.) and certain fungal toxins (such as aflatoxin). The reasons for conducting harmful substance testing include: ensuring food safety and consumer health, complying with relevant national quality standards and regulatory requirements, ensuring the quality and efficacy of turmeric products, preventing reduced efficacy or toxic side effects due to pollution, maintaining the reputation and market competitiveness of manufacturers, and providing necessary quality certification documents for international trade.

[0003] In the existing technology, the detection of harmful substances in turmeric mostly adopts a single indicator detection method, such as high performance liquid chromatography (HPLC) only for specific organic compounds, and atomic absorption spectroscopy (AAS) is limited to heavy metal detection. These methods have the following disadvantages: low detection efficiency, and multiple independent experiments are required to complete comprehensive detection; the equipment is expensive and the operation is complicated, which is difficult to implement quickly at the grassroots level or on-site; the reagent consumption is large and the detection cost is high; the sample pretreatment is cumbersome and time-consuming; it is impossible to achieve simultaneous detection of multiple indicators and lacks overall evaluation; the professional requirements for operators are high and it is difficult to popularize and apply; some methods are not sensitive enough to meet the needs of low-concentration pollutant detection; there is a lack of visual rapid interpretation means, and it is impossible to achieve instant result feedback; the traditional enzyme-linked immunosorbent assay has cross-reactions and insufficient specificity; and most of the existing technologies rely on large instruments and cannot meet the needs of rapid on-site screening. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a method for detecting harmful substances in turmeric, which solves the problems of low efficiency and high cost in the prior art.

[0005] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for detecting harmful substances in turmeric, specifically comprising the following steps:

[0006] S1. Construction of three-phase extraction system

[0007] Mix water, hexane, and dextran in a volume ratio of 5:3:2;

[0008] S2. Sample mixing

[0009] The crushed turmeric sample was mixed with the extraction system at a mass volume ratio of 1:10;

[0010] S3. Dynamic oscillation

[0011] The treatment was carried out with a 45° angle dynamic oscillation, an amplitude of 5 cm, a frequency of 120 rpm, and a rotation direction changed every 2 minutes for 15 minutes;

[0012] S4. Separation and treatment of three-phase solution

[0013] Separate the three phase solution and treat them separately:

[0014] a) Detection of organophosphorus pesticides after filtering the n-hexane phase through a nanofiber membrane;

[0015] b) Detection of heavy metal ions after EDTA dissociation of the dextran phase;

[0016] c) The aqueous phase is directly used for the determination of natural pigment content;

[0017] S5. Establishment of dual enzyme reaction system

[0018] Establishment of laccase-horseradish peroxidase dual enzyme reaction system:

[0019] 1) Laccase catalyzes the oxidation of organophosphorus pesticides to produce benzoquinone;

[0020] 2) Benzoquinone activates horseradish peroxidase to catalyze TMB color development;

[0021] S6. Final analysis

[0022] The organophosphorus pesticide content was semi-quantitatively analyzed by color intensity, the inhibition rate was calculated based on the competitive inhibition effect of heavy metal ions on laccase, and the curcumin content in the aqueous phase was determined by spectrophotometry to verify the integrity of the sample.

[0023] Preferably, the dextran in step S1 is carboxymethylated dextran with a molecular weight of 50 kDa and a concentration of 8% (w / v).

[0024] Preferably, the shaking treatment in step S3 is performed in a special magnetic stirring extraction tube, which has a spiral guide plate built in and a polytetrafluoroethylene coating on the tube wall.

[0025] Preferably, in step S5, the ratio of the dual enzyme system is 2.5 U / mL of laccase activity unit and 1.2 U / mL of horseradish peroxidase activity unit, and the reaction buffer is a citrate buffer with a pH of 5.8.

[0026] Preferably, the step S6 uses a three-stage colorimetric colorimetric card, including standard color scales (0.5ppm, 1ppm, 2ppm) and a blank control area, and the color development time is controlled at 8±0.5 minutes;

[0027] The threshold for heavy metal inhibition rate is:

[0028] 15%-30% → Slightly polluted

[0029] 30%-50% → Moderate pollution

[0030] >50% → severe pollution.

[0031] Preferably, the nanofiber membrane is a 3:1 composite membrane of nitrocellulose and chitosan, with a pore size distribution of 200-500 nm and a thickness of 150 μm.

[0032] The present invention provides a method for detecting harmful substances in turmeric, which has the following beneficial effects:

[0033] The invention provides a method for detecting harmful substances in yellow turmeric. The method adopts a three-phase dynamic extraction system of water, normal hexane and dextran, realizes efficient separation of pollutants of different properties in a sample, greatly improves detection efficiency, constructs a laccase-horseradish peroxidase double enzyme reaction system, realizes rapid visual detection of organophosphorus pesticides through enzymatic color development reaction, has simple and intuitive operation, utilizes the competitive inhibition effect of heavy metal ions on laccase, establishes a rapid evaluation method for heavy metal pollution degree, has high sensitivity, combines with spectrophotometry to determine curcumin content, can simultaneously verify sample integrity and authenticity, does not require expensive instruments in the entire detection process, significantly reduces detection costs, has high method sensitivity, can detect pollutants as low as ppb level, has short detection time, can complete the whole process from sample processing to result interpretation within about 40 minutes, has visualized detection results, can be quickly interpreted through a colorimetric card, does not require professional operation, has strong method specificity, avoids common cross-reaction problems in traditional methods, has a wide detection range, can simultaneously evaluate organic pollutants, heavy metal pollution and sample quality, provides comprehensive quality evaluation, and the entire method is environmentally friendly and safe, uses less reagents, generates less waste, and conforms to the concept of green chemistry. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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.

[0035] The embodiment of the present invention provides a method for detecting harmful substances in turmeric, which specifically comprises the following steps:

[0036] Construction of three-phase extraction system

[0037] Ultrapure water (water), n-hexane and 8% carboxymethylated dextran solution were injected into the extraction tube at a volume ratio of 5:3:2 to form a stable three-phase system, wherein the carboxymethylation modification of the dextran phase enhanced the heavy metal chelating ability.

[0038] Dynamic extraction process

[0039] Weigh 1.0g of turmeric powder and mix it with 10mL of the extraction system, and place it in a special magnetic stirring tube. Set the 45° bevel oscillation mode, amplitude 5cm, frequency 120rpm, and switch the rotation direction every 2 minutes through the automatic reversing device for 15 minutes. The spiral guide vane design creates a vortex effect at the three-phase interface to improve mass transfer efficiency.

[0040] Three-phase separation and treatment

[0041] After standing and stratification:

[0042] Hexane phase: Filtered through a nitrocellulose / chitosan (3:1) composite membrane to retain particles with a particle size of >200 nm, and the filtrate was collected for organophosphorus detection

[0043] Dextran phase: Add 5 mM EDTA solution to dissociate heavy metal complexes

[0044] Aqueous phase: directly used for determination of curcumin content

[0045] Construction of dual enzyme combined detection system

[0046] Take 2 mL of the n-hexane phase extract, add pH 5.8 citrate buffer, and inject laccase (2.5 U / mL) and horseradish peroxidase (1.2 U / mL) in sequence. Organophosphorus pesticides are oxidized to generate benzoquinone substances under the catalysis of laccase, which then activate HRP to catalyze TMB color development, and the blue-purple color development reaction is completed within 8 minutes.

[0047] Multi-parameter simultaneous detection

[0048] Organophosphorus pesticides: Compare the colorimetric solution with a three-step colorimetric card (0.5 / 1 / 2ppm) to achieve semi-quantitative analysis

[0049] Heavy metal detection: Take an equal amount of laccase solution, add dextran phase dissociation solution, and calculate the pollution level by the enzyme activity inhibition rate (inhibition rate> 50% is judged as heavy pollution)

[0050] Sample verification: The absorbance of the aqueous phase at 412 nm was determined by ultraviolet spectrophotometry. Curcumin content < 0.8 mg / g was considered an abnormal sample.

[0051] The entire detection process was completed within 40 minutes, which greatly shortened the detection time compared with traditional methods (such as HPLC-AAS combination method). The special magnetic stirring extraction tube and automatic reversing device achieved efficient dynamic oscillation extraction, reducing manual operation time and errors.

[0052] The detection limit of organophosphorus pesticides is 0.3ppm, which is better than the 1ppm of the national standard (GB 2763-2021). The sensitivity of heavy metal detection is 10 times higher than that of atomic absorption spectrometry (AAS), and the IC50 value is 0.8ppm (based on Cd 2 + as an example). The intra-batch RSD is ≤4.2% and the inter-batch RSD is ≤6.8%, ensuring the stability and reliability of the test results.

[0053] It can simultaneously detect organophosphorus pesticides, heavy metal pollution and the content of natural pigments in samples, and realize multi-parameter comprehensive evaluation. The laccase-horseradish peroxidase dual enzyme system is not only used for the detection of organophosphorus pesticides, but also can calculate the degree of heavy metal pollution through inhibition rate.

[0054] The cost of a single test reagent is only ￥3.5 / sample, and the amount of n-hexane used is only 2mL / sample, which reduces the amount of organic solvent used by 80% compared with traditional methods. The amount of waste generated is less than 5mL / sample, there is no highly toxic waste liquid, and it meets environmental protection standards (HJ 168-2020).

[0055] The required equipment investment is less than ￥5000, which is much lower than the expensive instruments required by traditional methods (such as HPLC and AAS). The steps are clear and the operation is simple. It can be used without complex training and is suitable for rapid testing in laboratories and on-site. The cost of a single test is only 1 / 5 of that of traditional methods, which greatly reduces the cost of testing. The equipment is simple and the maintenance cost is low, which is suitable for large-scale promotion and application.

[0056] A stable three-phase system is constructed using water, n-hexane and carboxymethylated dextran to improve the extraction efficiency and selectivity. A nitrocellulose and chitosan composite membrane with a pore size distribution of 200-500nm is used to effectively intercept organophosphorus pesticides and improve detection accuracy. A built-in spiral guide plate and a polytetrafluoroethylene coating on the tube wall enhance the mass transfer efficiency and stability. The method for detecting harmful substances in turmeric of the present invention not only has significant advantages in detection speed, sensitivity, accuracy and environmental protection, but also is easy to operate, low in cost, suitable for rapid detection in laboratories and on-site, and has broad application prospects.

[0057] The following is the relevant experimental data table:

[0058]

[0059] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting harmful substances in turmeric, characterized in that: The specific steps include: S1. Construction of three-phase extraction system Mix water, hexane, and dextran in a volume ratio of 5:3:2; S2. Sample mixing The crushed turmeric sample was mixed with the extraction system at a mass volume ratio of 1:10; S3. Dynamic oscillation The treatment was carried out with a 45° angle dynamic oscillation, an amplitude of 5 cm, a frequency of 120 rpm, and a rotation direction changed every 2 minutes for 15 minutes; S4. Separation and treatment of three-phase solution Separate the three phase solution and treat them separately: a) Detection of organophosphorus pesticides after filtering the n-hexane phase through a nanofiber membrane; b) Detection of heavy metal ions after EDTA dissociation of the dextran phase; c) The aqueous phase is directly used for the determination of natural pigment content; S5. Establishment of dual enzyme reaction system Establishment of laccase-horseradish peroxidase dual enzyme reaction system: 1) Laccase catalyzes the oxidation of organophosphorus pesticides to produce benzoquinone; 2) Benzoquinone activates horseradish peroxidase to catalyze TMB color development; S6. Final analysis The organophosphorus pesticide content was semi-quantitatively analyzed by color intensity, the inhibition rate was calculated based on the competitive inhibition effect of heavy metal ions on laccase, and the curcumin content in the aqueous phase was determined by spectrophotometry to verify the integrity of the sample.

2. A method for detecting harmful substances in turmeric according to claim 1, characterized in that: The dextran in step S1 is carboxymethylated dextran with a molecular weight of 50 kDa and a concentration of 8% (w / v).

3. A method for detecting harmful substances in turmeric according to claim 1, characterized in that: The shaking treatment in step S3 is performed in a special magnetic stirring extraction tube, which has a spiral guide plate built in and a polytetrafluoroethylene coating on the tube wall.

4. A method for detecting harmful substances in turmeric according to claim 1, characterized in that: In step S5, the ratio of the dual enzyme system is 2.5 U / mL of laccase activity unit and 1.2 U / mL of horseradish peroxidase activity unit, and the reaction buffer is a citrate buffer with a pH of 5.

8.

5. A method for detecting harmful substances in turmeric according to claim 1, characterized in that: The step S6 uses a three-stage colorimetric colorimetric card, including standard color levels (0.5ppm, 1ppm, 2ppm) and a blank control area, and the color development time is controlled at 8±0.5 minutes; The threshold for heavy metal inhibition rate is: 15%-30% → Slightly polluted 30%-50% → Moderate pollution >50% → severe pollution.

6. A method for detecting harmful substances in turmeric according to claim 1, characterized in that: The nanofiber membrane is a composite membrane of nitrocellulose and chitosan in a ratio of 3:1, with a pore size distribution of 200-500nm and a thickness of 150μm.

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