KR100377539B1 - Method for Detection of Toxicity of Pesticide Residues in Foods Using Bioluminescent Bacteria - Google Patents

Abstract

The present invention relates to a method for detecting toxicity using luminescent bacteria after extracting the pesticide remaining in the food. More specifically, the pesticides remaining in the food are extracted with water and methanol and polyethylene glycol mixed solvent, and then the degree of toxicity is analyzed using luminescent bacteria genetically engineered to emit light for their specific toxicity. It is about a method.

The present invention is simpler than the pretreatment method that has been used to detect pesticides remaining in conventional foods, so that residual pesticides in foods can be efficiently extracted in a short time. In addition, since the extraction solvent has little toxicity, it is not only safe for the human body, but also has little effect on the toxicity detection ability of the luminescent bacteria, and thus only the toxicity of the extracted residual pesticides can be analyzed.

The present invention can reduce the damage caused by the toxicity of the pesticide when ingesting the food by providing a method for extracting and analyzing the pesticide components remaining in the food due to the use of indiscriminate pesticides.

Description

Method for Detection of Toxicity of Pesticide Residues in Foods Using Bioluminescent Bacteria}

The present invention relates to a toxicity detection method using luminescent microorganisms for pesticides remaining in food.

Methods for analyzing pesticide residues in food include acute toxicity test and genetic toxicity test using an analyzer such as GC, GC / MS, LC, LC / MS or laboratory animals. In order to analyze the toxicity of residual pesticides using this method, a complex extraction and pretreatment method is required, and it takes a lot of time, and after extracting it for a long time by a skilled technician, it also takes a long time to analyze it. . In addition, since the constituents of the solvent used in the extraction itself contains a lot of toxicity, it is difficult to verify the toxic effects of the extracted residual pesticides.

Therefore, in the present invention, after extracting the residual pesticide components in food simply with water and organic solvent, extracted using genetically recombined luminescent bacteria programmed to generate biological light when the influx of toxic substances damaged certain parts of the organism. This is to determine whether the pesticide residue is toxic.

An object of the present invention is to provide a convenient method for quickly identifying the toxicity due to pesticide components remaining in food. The present invention not only solves the complicated process and long time problem of extracting residual pesticides in the related art, but also consists of two stages in consideration of the physical and chemical properties of the pesticides, while harmless to the human body. The use of an extraction solvent that does not change the amount provides an efficient toxicity detection method.

1 is a detailed view of a method for extracting residual pesticides in food.

Figure 2 shows the effect on the degree of biological light and cell growth of luminescent bacteria for solvent selection.

Figure 3 is a result confirming the reactivity of the luminescent bacteria in various concentration ranges of pure methyl bromide.

4 is a result of artificially exposing methyl bromide to sesame leaves, and extracting to confirm the reactivity of the luminescent bacteria.

Figure 5 (a) (b) is a result of confirming the reactivity of the luminescent bacteria by artificial exposure to sesame leaves using DDT (a) and jurassic (b), and extracting it.

Figure 6 relates to a method for extracting residual pesticides in fruits containing a large amount of water in food.

7 is a result of confirming the reactivity of luminescent bacteria by extracting methyl bromide after exposure to orange

(a) is the result of checking the reactivity of the luminescent bacteria by extracting only the orange peel.

(b) is the result of checking the reactivity of the luminescent bacteria by extracting the orange kernel area.

(c) is the result of confirming the reactivity of the luminescent bacteria by extracting the orange completely after dividing with distilled water and extraction solvent.

Figure 1 shows the toxicity detection method of the present invention shows a schematic diagram for quickly and simply extracting the pesticide component from the food sample, to determine the degree of toxicity with luminescent bacteria. Methods for the extraction of residual pesticides in food can be roughly divided into primary washing with distilled water and secondary washing with extraction solvent. First, prepare a food sample in an appropriate size, and then first wash the food sample using 20 mL of distilled water, and the prepared extraction solvent is a solvent in the form of a mixture of 20% methanol and 15% polyethylene glycol (polyethylene glycol). 100 mL methanol and 60% polyethylene glycol are mixed so that the final concentration is 20% and 15%, respectively, and 20 mL of the second wash is performed for 90 seconds using a vortexer. In the case of the tube used for washing, in the present invention, 50mL Falcon tube was used, but other containers can be used, and the time required for washing is It takes 60 to 120 seconds, but more preferably 90 seconds. That is, when the washing time is more than 90 seconds, the extraction efficiency is increased, but the pigment is extracted together from the food. For example, after 90 seconds of washing, the final extracted sample became green for sesame leaves and orange for orange. The problem of adsorption of residual pesticides in the cleaning container by washing for 90 seconds to prevent the incidental stress caused by these pigments It did not occur. 2mL of the final sample thus extracted was taken to confirm the extraction efficiency using luminescent bacteria.

In addition, the method for extracting the residual pesticide in the fruit containing a large amount of water shown in Figure 6 is an additional process of completely crushing the food sample with a mixer to separate only the supernatant through centrifugation. Extraction samples obtained through the above process were all confirmed the extraction efficiency and toxicity by using genetically recombined luminescent bacteria. Among the many recombinant luminescent bacteria that detect toxic effects on specific parts of organisms, in particular, we use the toxic detection bacteria TV1061 (ATCC 69315), which is genetically recombined to emit light when toxic substances damage intracellular proteins. It was.

Genetically recombined bioluminescent bacteria are plasmids that artificially combine a promoter involved in recovering when a specific site is damaged and a lux gene that indicates whether the promoter is working as light. It contains. Thus, toxicity to a specific site triggers the corresponding promoter, which is represented by light through the expression of the luminescent gene. Thus, damage to certain parts of the organism due to the toxicity of the toxic substance will appear as an increase in the light of the recombinant luminescent bacteria. In the present invention, the degree of toxicity and the extraction efficiency of the pesticides extracted from food were confirmed through the extraction method developed using the recombinant bacterium TV1061 which is sensitive to protein damage in a specific damage site.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples do not limit the technical scope of the present invention, the biological light units described in the examples were limited to the following two. In the above formula, SBL (Specific Bioluminescence) is a unit indicating the amount of light generated per cell.

<Example 1>

The present invention provides a simple and rapid extraction of harmful pesticide components remaining in food, and extract samples obtained from them are compared with extraction efficiency and toxicity using their genetically modified luminescent bacteria. In order to increase the stress response of the bacteria due to the solvent used for the extraction is minimized, to determine only the toxic effect of the extracted pesticide component.

After preparing the food sample to an appropriate size, the first sample was washed first with 20 mL of purified water, and the prepared extraction solvent was a solvent of 100% mixed solvent of 100% methanol and 100% polyethylene glycol (100%). % Methanol and 60% polyethylene glycol were mixed so that the final concentration was 20% and 15%, respectively, and 20 mL of the second wash was performed for 90 seconds using a vortexer.

Therefore, the degree of stress response of the luminescent bacteria was confirmed using various solvents that have been used in the pretreatment of samples for analysis of pesticide components. In addition, methanol and polyetherene glycol, also known as hydrophobic carrier, are widely used to dissolve fat-soluble chemicals. Figure 2 shows the maximum amount of light (Max. SBL R; Maximum Specific Bioluminescence Ratio) and cell growth rate generated per one luminescent bacterial cell due to eight different solvents. The selected concentrations of the solvents used in FIG. 2 refer to those known to exhibit maximum efficiency in the conventional sample pretreatment process, and the specified concentrations refer to concentrations when inserted into the cell culture solution. In general, acetone and hexane, which are known to exhibit high efficiency in sample processing, have severe growth inhibition of luminescent bacteria and are not suitable for use in the extraction method of the present invention. Therefore, polyethylene glycol, ethanol, methanol, or a mixture of polyethylene glycol and polyethylene glycol, which have a minimum amount of biological light generated from luminescent bacteria and do not inhibit cell growth, are suitable.

<Example 2>

Mixing form of methanol and polyethylene glycol used in Example 1 is shown in Table 1 (a, b) for selecting the concentration representing the extraction efficiency of the extraction solvent selected in Figure 2 according to the change in the concentration of methanol and polyethylene glycol It was selected through experiments to show the biological light of the bacteria and the degree of cell growth.

Table 1. Biological Light and Cell Growth of Luminescent Bacteria with Different Concentrations of Methanol and Polyethylene Glycol

(a) the amount of biological light produced by luminescent bacteria

Methanol Concentration (%) Polyethylene Glycol Concentration (%) 0 7.5 15 30 60 0 One 1.13 1.42 1.12 1.3 5 1.38 1.49 1.52 1.39 1.28 10 1.39 1.4 1.42 1.3 2.29 20 1.42 1.38 1.36 1.42 2.8 30 3.36 4.9 4.56 5.0 4.81 40 4.8 5.26 5.23 4.9 5.012 50 6.7 6.84 6.45 6.24 7.12 60 10 10.5 12 14.6 12.4

(b) Cell growth rate of luminescent bacteria (1 / hr)

Methanol Concentration (%) Polyethylene Glycol Concentration (%) 0 7.5 15 30 60 0 1.48 1.51 1.51 1.46 1.40 5 1.43 1.54 1.42 1.39 1.32 10 1.39 1.35 1.4 1.38 1.29 20 1.55 1.34 1.38 1.42 1.30 30 1.38 1.29 1.41 1.31 1.39 40 1.38 1.51 1.44 1.38 1.48 50 1.3 1.32 1.25 1.26 1.2 60 1.19 1.16 1.2 1.18 1.18

Table 1 shows the amount of biological light and cell growth rate of luminescent bacteria due to various mixing of methanol and polyethylene glycol at various concentrations. In this case, since the amount of biological light generated as described above is minimal and hardly affects the cell growth rate, it is appropriate to select the concentration of the block designation site. In addition, when these are used as the extraction solvent, the residual pesticide components in the food should be able to be extracted more efficiently, and thus the amount of light generated should be increased. For this purpose, the sesame leaves were artificially exposed to 10,000 ppm of methyl bromide component to perform extraction efficiency experiment.

Table 2 (a, b) shows the biological light and cell growth of luminescent bacteria after extraction of residual pesticides in food according to the concentration of methanol and polyethylene glycol. When mixing 20% methanol and 15% polyethylene glycol of Table 2, it was confirmed that the maximum toxic effect due to only pesticide components. At lower concentrations, the extraction efficiency is less than that. At higher concentrations, not only the toxic effect of the pesticide component but also the effect of the extraction solvent is shown to be mixed. Was found to be the most suitable for extracting residual pesticides.

Table 2. Biological Light and Cell Growth of Luminescent Bacteria after Extraction of Residual Pesticides in Foods with Different Methanol and Polyethylene Glycol Concentrations

(a) the amount of biological light generated by luminescent bacteria after extraction

Methanol Concentration (%) Polyethylene Glycol Concentration (%) 0 7.5 15 30 60 0 One 2.31 3.39 3.81 3.3 5 2.56 2.12 5.12 4.39 4.28 10 3.96 3.41 4.95 9.59 7.96 20 5.61 4.18 10.26 20.16 26.15 30 10.21 12.18 15.12 25 30.8 40 40.12 38.12 44.12 51.16 120.8 50 51.49 55.11 49.51 78.16 207.1 60 100.69 99.1 120.69 146.2 198.1

(b) the rate of cell growth of luminescent bacteria after extraction (1 / hr)

Methanol Concentration (%) Polyethylene Glycol Concentration (%) 0 7.5 15 30 60 0 1.48 1.42 1.44 1.52 1.45 5 1.39 1.30 1.302 1.245 1.21 10 1.29 1.201 1.17 1.18 1.46 20 1.18 1.18 1.22 1.24 1.31 30 1.05 1.028 1.12 1.14 1.09 40 1.08 0.95 0.91 0.83 0.84 50 0.75 0.81 0.90 0.83 0.89 60 0.62 0.611 0.72 0.77 0.71

<Example 3>

In order to confirm the effects of the present invention, until recently, a method of extracting by exposure to food artificially methyl bromide as a pesticide ingredient frequently sprayed on vegetables and fruits in farms. Therefore, Figure 3 shows the degree of response to the methyl bromide of the luminescent bacteria sensitive to protein damage. It was confirmed that the concentration of methyl bromide, the amount of light generated, and the rate of cell growth were consistently correlated.

<Example 4>

The extraction experiment of the residual pesticide component from the actual food sample was performed in the same manner as in FIG. 1 using the solvents selected as suitable as extraction solvents in Example 1. As described above, 10,000 ppm of methyl bromide was artificially exposed to the sesame leaves, followed by extraction experiments to obtain results as shown in FIG. 4. The control of Figure 4 is the reactivity of the luminescent bacteria without any stress, sesame leaf control is extracted by using the extraction method sesame leaves without artificially added pesticides. When the degree of extraction from various extraction solvents was compared, the mixed solvent of methanol and polyethylene glycol generated the highest amount of light, and thus the extraction method using the same was the most effective in the extraction. In addition, comparing the extraction degree of the sample after the first wash with distilled water and the sample after the second wash at the maximum SBL rate, it was confirmed that up to the second washing step using the extraction solvent to extract the pesticide component.

<Example 5>

Also in the extraction experiments for DDT and Juram, other pesticide components other than methyl bromide, it was confirmed that a large amount of pesticide was extracted only after the second step using the extraction solvent in the same manner as described above.

<Example 6>

In the case of sesame leaves, it was a food with little moisture content and little thickness. Therefore, in the present invention, in order to confirm the toxicity of residual pesticides in fruits with a large amount of water and a large volume of oranges, an extraction experiment was performed by exposing 10,000 ppm of methyl bromide as in the case described above. In the case of orange, the peel and kernel parts were clearly distinguished and the bulk was large, and after the pesticide exposure, the skin parts and the kernel parts were extracted and extracted, and the extraction efficiency after complete crushing using a mixer was also compared. Referring to Figure 7, in the case of (a) is extracted using only the shell, (b) in the case of extraction using kernels, (c) in the case of crushing the orange completely with distilled water and extraction solvent to extract the extraction efficiency It is confirmed. In the case of orange, unlike the sesame leaf, the stress response due to the components of the sample itself was slightly observed, and when exposed to pesticides and completely crushed using a mixer, a large amount of methyl bromide was extracted due to the inhibition of cell growth. In particular, when crushed with the extraction solvent, it was confirmed that the amount of methyl bromide is extracted more than with distilled water.

Since the present invention can easily extract the residual pesticide components in food with a mixed solvent of methanol and polyethylene glycol, there is no toxicity due to the solvent and the extraction method is simple and can be easily measured, and does not require ancillary devices and consumables. As a very economical method, it is possible to extract residual pesticide components more efficiently by applying different extraction methods for each type of food. In addition, these solvents have two steps in consideration of the chemical and physical properties of the final extraction pesticides, which is why they are more efficient than the conventional methods that do not change the toxicity. And even a highly skilled person can easily extract residual pesticides and determine the toxicity in foods at home.

Claims (5)

In the toxicity detection method for pesticides remaining in food, Cutting and washing a sample of the foodstuff to be examined in an appropriate size; Adding a mixed solvent of methanol and polyethylene glycol to the sample to be tested to obtain an extract, and Detecting the toxicity level of the pesticide residue in the foodstuff by measuring the amount of light generated by exposing the extract to the extract genetically recombined luminescent bacteria to emit light by the stress response according to the toxicity of the pesticide. Toxicity detection method delete The method of claim 1, Foods with high moisture content are toxic detection methods characterized in that the mixed solution of methanol and polyethylene glycol is added to the sample, completely crushed in a mixer, and the supernatant obtained by centrifugation as an extract. The method of claim 1, wherein the mixed solvent is a mixed solvent of 20% methanol and 15% polyethylene glycol. delete