CN113218896A - Analytical method for judging food pest infestation degree - Google Patents
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Abstract
The invention relates to an analysis method for judging the degree of infestation of food by insect pests, which comprises the following steps: firstly, taking a plurality of groups of food samples to be detected with different weights, putting the food samples to be detected into a test tube, adding a sodium carbonate solution, and shaking to form a suspension; weight of food sample to be tested: taking the lightest group as a reference, and taking other groups as integral multiples of the lightest group; secondly, transferring each suspension to different centrifuge tubes, and centrifuging for a period of time; taking the supernatant to different volumetric flasks to obtain a sample stock solution containing uric acid; taking a proper amount of sample stock solution, adding the sample stock solution into different volumetric flasks, and diluting with distilled water to a constant volume to obtain a plurality of groups of uric acid-containing sample solutions with different solubilities; fourthly, preparing uric acid standard solution; fifthly, diluting the uric acid sample solution and the uric acid standard solution with different concentrations by using distilled water; sixthly, adding the diluted solution into potassium iron cyanide and ferric chloride respectively, and keeping for a period of time; and seventhly, respectively using an ultraviolet-visible spectrophotometer to obtain spectrophotometry readings of the developed solutions.
Description
Technical Field
The invention belongs to the technical field of food detection, and relates to an analysis method capable of quickly determining the content of uric acid in food.
Background
Often the food is stored for future consumption and may also be used for trade purposes. During storage, food may be affected by pests in addition to environmental factors. Insect attack occurs during storage of all kinds of food. Insect infestation has an effect on the damage of food particles both from the quality and quantity of the food. The effect of insect infestation on the stored product is a change in the chemical composition, the nutritional quality, ultimately to the use of the product, and also acts as a carrier for the toxigenic fungi. During insect growth, insects may also cause the spread and reproduction of microorganisms due to the increased moisture content in food.
The weight of the grain is reduced due to insect infestation. Insects produce eggs during the process of laying eggs and eating grains. One insect will consume approximately 35 mg of food as feed for the ovum to develop into an adult. The food is infested and the main contaminants are insect excretions: uric acid, exudates, insect debris, nets and secretions. Toxicological studies have shown that hyperuricemia is associated with renal disease induced by excessive dietary uric acid. Mealworms are the major insects in the storage of flour, grains and cereal products, and the secreted 2-ethyl-1, 4-benzoquinone, 2-methyl-1, 4-benzoquinone and 2-methoxy-1, 4-benzoquinone are known to be carcinogenic and can also contribute to the development of off-flavors in foods that are intolerable.
The main end product of insect nitrogen metabolism is uric acid, and excrement released by insects contains 80% of nitrogen, so that the uric acid content in food can be used as a good index of the infection degree and unsanitary conditions of infected food. Most of the current uric acid detection methods are based on chemicals, enzymes, fluorescence and biosensors, however, these detection methods take a long time to measure uric acid.
Disclosure of Invention
According to the existing method for measuring the content of uric acid in food, some methods take a long time to measure and some methods cannot analyze a large amount of samples. Therefore, it is necessary to develop a simple and fast method to solve the above problems. The invention provides a method for measuring the content of uric acid in insect food in the processing and storage processes by using an ultraviolet-visible spectrophotometry.
In order to achieve the purpose, the invention adopts the following technical scheme:
an analysis method for judging the degree of infestation of food by pests comprises the following steps:
taking a plurality of groups of food samples to be detected with different weights, respectively putting the plurality of groups of food samples to be detected into different test tubes, respectively adding sodium carbonate solution, and shaking up to form turbid liquid; the weight relationship of a plurality of groups of food samples to be detected is as follows: taking the lightest group as a reference, and taking other groups as integral multiples of the lightest group;
transferring each suspension to different centrifuge tubes, and centrifuging for a period of time; taking the supernatant to different first volumetric flasks to obtain a sample stock solution containing uric acid;
step three, taking a proper amount of the sample stock solutions of the groups obtained in the step two, adding the sample stock solutions into different second volumetric flasks, and diluting with distilled water to a constant volume to obtain a plurality of groups of uric acid-containing sample solutions with different solubilities;
step four, preparing uric acid standard solution and diluting with distilled water;
step five, diluting the uric acid-containing sample solutions with different concentrations prepared in the step three and the uric acid standard solution prepared in the step four respectively with distilled water;
step six, adding the diluted solution obtained in the step five into ferric potassium cyanide and ferric chloride respectively, and keeping for a period of time for color development;
and step seven, respectively using an ultraviolet-visible spectrophotometer to obtain spectrophotometry readings of the solutions developed in the step six.
The principle of the method of the invention is as follows: potassium ferricyanide reacts with uric acid in the infected sample and is converted to potassium ferrocyanide. The iron chloride reacts with potassium ferrocyanide to generate Prussian blue, and the potassium ferricyanide reacts with the iron or ferrous solution to form Prussian blue. The principle of the rapid assay method of the present invention is based on the formation of prussian blue.
Preferably, in the first step, the weight of the lightest group of food samples to be detected is 50-100 mg.
Preferably, in the first step, the sodium carbonate solution is 1-3 mL of 0.1% sodium carbonate solution.
Preferably, in the second step, the volume of the volumetric flask is 1L. The rotating speed of the centrifugal machine is 8000-12000 r/min, and the centrifugal time is 7-13 minutes.
Preferably, in the fourth step, the uric acid standard solution is 0.1-0.5 mmol/L.
Preferably, in the third step, the volume of the volumetric flask is 50 mL. The sample stock solution is 1-5 mL.
Preferably, in the fifth step, the volume of the uric acid solution with different concentrations is 0.5-2 mL. The number of dilutions with distilled water was 5.
Preferably, in the sixth step, the volume of the potassium ferric cyanide is 0.5-2 mL, and the volume of the ferric chloride is 0.5-2 mL. The holding time was 5 minutes.
Preferably, in the seventh step, the absorbance is measured at a wavelength of 290 to 690nm by using an ultraviolet-visible spectrophotometer.
The invention also discloses an analysis method for judging the degree of insect attack on food, which comprises the following steps:
step one, preparing uric acid standard solution and diluting with distilled water;
taking a plurality of groups of food samples to be detected with different weights, respectively putting the plurality of groups of food samples to be detected into different test tubes, respectively adding sodium carbonate solution, and shaking up to form turbid liquid; the weight relationship of a plurality of groups of food samples to be detected is as follows: taking the lightest group as a reference, and taking other groups as integral multiples of the lightest group;
transferring each group of turbid liquid to different centrifuge tubes, and centrifuging for a period of time; taking the supernatant to different first volumetric flasks to obtain a sample stock solution containing uric acid;
step four, taking a proper amount of each group of sample stock solution obtained in the step three, adding the sample stock solutions into different second volumetric flasks, and diluting with distilled water to a constant volume to obtain a plurality of groups of uric acid-containing sample solutions with different solubilities;
step five, diluting the uric acid-containing sample solutions with different concentrations prepared in the step four and the uric acid standard solution prepared in the step one with distilled water respectively;
step six, adding the diluted solution obtained in the step five into ferric potassium cyanide and ferric chloride respectively, and keeping for a period of time for color development;
and step seven, respectively using an ultraviolet-visible spectrophotometer to obtain spectrophotometry readings of the solutions developed in the step six.
The technical scheme is different from the technical scheme in that a step of preparing the uric acid standard solution is taken as a first step, and the technical scheme can be referred to for other contents.
The method can be used for quickly measuring the content of the uric acid, and has the characteristics of simplicity, convenience, rapidness, high accuracy and short measuring time. After the method disclosed by the invention is used for detecting the content of uric acid, the degree of the food infected by the insect pests can be judged according to the content of uric acid in the food per unit mass, and the more uric acid is contained in the food per unit mass, the more serious the food is infected by the insect pests. The method has important significance for judging the infection degree of the food and judging whether the food reaches the hygienic standard. The method of the invention is also helpful for the popularization of the disinsection method of the food storage system.
Drawings
FIG. 1 is a calibration graph of the rapid method of the present invention.
Fig. 2 is a calibration graph of a prior art method (the former method).
FIG. 3 is a graph showing the results of comparing the standard uric acid in the rapid method of the present invention and the method of the prior art (the former method).
FIG. 4 is a graph comparing the linearity and precision of the method for measuring uric acid (standard substance) according to the present invention and the prior art method (former method).
FIG. 5 is a calibration graph of a prior art phosphotungstic acid reduction process.
FIG. 6 is a graph of a calibration curve for the rapid method of the present invention.
Detailed Description
The technical scheme of the invention is detailed by comparing the embodiment of the invention with the prior art.
The superiority of the ultraviolet-visible spectrophotometry is shown by comparing the colorimetric method of the present invention with the prior art in example 1.
This section was analyzed using a prior art colorimetric method (the pre-method) in comparison with the present UV-visible spectrophotometry method (the rapid method). The principle of the prior art colorimetric method is that uric acid is hydrolyzed at 100 ℃ using weak base. Converting uric acid into allantoin acid by using the activity of allantoin enzyme. Allantoin acid is degraded in acidic solution to urea and glyoxylic acid. Phenylhydrazine hydrochloride reacts with glyoxylic acid to generate phenyl. Potassium ferricyanide reacts with phenyl groups to form unstable chromophores.
Embodiment 1 of the present invention:
weighing five groups of flour samples to be tested, wherein the weight is respectively as follows: 70mg, 140mg,210mg,280mg and 350mg are respectively added into a test tube, 1mL of 0.1% sodium carbonate solution is added, and finally the mixture is shaken to form suspension. Then transferring the mixture into a centrifuge tube, and centrifuging the mixture for 10 minutes at the rotating speed of 10000r/min to obtain supernatant. The supernatant was then poured into a volumetric flask as a stock solution of the sample containing uric acid. 5mL of each stock solution was taken out, poured into a 50mL volumetric flask, and diluted with distilled water to a constant volume as a working standard. Five uric acid-containing sample solutions with different concentrations are obtained. Then 0.3mmol/L uric acid standard solution was used as a standard sample. 4 taking 5mL of the solution from the uric acid standard solution, pouring the solution into a 50mL volumetric flask, and diluting the solution with distilled water to a constant volume to obtain the standard solution. The following ultraviolet-visible spectrophotometry method is adopted, and the steps are as follows: uric acid-containing sample solutions of different concentrations (1mL) were taken, then diluted 5 times with distilled water, and then 1mL of iron potassium cyanide and iron chloride (1mL) were added and kept for 5 minutes for color development. This procedure was also repeated for uric acid standard sample solutions. The absorbance was measured at a wavelength of 520nm in an ultraviolet-visible spectrophotometer (Shimadzu; model: UV-1800).
The existing allantoin colorimetric method comprises the following steps: uric acid (1mL) was taken at various concentrations and placed in a boiling tube, to which was added 5mL of distilled water and 1mL of sodium hydroxide (0.05M). It was mixed well using a vortex (DLab; MX-S60HZ) and held in a water bath at 100 ℃ for 7 minutes. The boiling tube was cooled with water and the pH adjusted to 2.16 with 0.05M HCl. Then 1mL of phenylhydrazine hydrochloride (0.023M) was added and mixed with a vortex. The tube was again placed in a water bath at 100 ℃ for 7 minutes and then immediately poured into a bath of ice and alcohol (40% NaOH) for 7 minutes. Then 3mL of concentrated HCl and 1mL of potassium ferricyanide (0.05M) were added and held for 20 minutes. The standard solution also repeats these steps. The absorbance was measured at a wavelength of 522nm in an ultraviolet-visible spectrophotometer (Shimadzu; model: UV-1800).
Then in the detection Limit (LOD), quantification Limit (LOQ), linearity, correlation coefficient (R)2) Comparative analyses were performed on the two methods described above in terms of Standard Deviation (SD) and Relative Standard Deviation (RSD). LOD is calculated as LOD of 3.3 σ/S, and LOQ is calculated as LOQ of 10 σ/S, where σ is expressed as a standard deviation and S is expressed as a slope of a calibration curve. The Relative Standard Deviation (RSD) is calculated by the formula
The primary criteria for reading accuracy and precision can be expressed in terms of RSD%. Uric acid concentration (μmol/L) — 100 (absorbance of uric acid-containing sample/absorbance of standard solution). Linearity is calculated by the least squares regression method. The final results are shown in fig. 1, fig. 2, fig. 3, fig. 4. The correlation coefficient R of the rapid method of the ultraviolet-visible spectrophotometer can be obtained by analysis20.9941 (FIG. 1), correlation coefficient R of the previous method20.9915 (FIG. 2), the accuracy of the two methods is similar; the linearity of the calibration curve of the rapid method is in the range of 20-100ppm (FIG. 3), but the linearity of the prior method is in the range of 60-100ppm, and the linearity of the standard curve shows higher accuracy in the rapid method than in the prior method; the LOD of the rapid method was 22ppm and the LOQ was 66ppm, while the LOD and LOQ of the previous method were 19ppm and 58ppm, respectively (FIG. 3). The results show that the rapid method is more sensitive than the previous method; the precision of the rapid and pre-methods is determined by the reproducibility of the samples. The reproducibility of the rapid method showed that the relative standard deviation (0.003) was small compared to the prior method (0.058) (FIG. 4), and the mean RSD (%) measured by the rapid method was 0.007(Rava), 0.008 (wheat flour) and 0.006 (wheat flour), respectively, and the mean RSD (%) measured by the prior method was 0.053, 0.030 and 0.057, respectively. The result shows that the rapid method has more accurate measurement result than the former methodAnd (8) determining.
The former method detects uric acid indirectly by converting uric acid into allantoin. Treating uric acid with weak alkaline solution, and converting uric acid into allantoin acid by using the activity of allantoin enzyme. As can be seen from the above steps, the rapid method of the present invention takes much less time than the previous method. R of the Rapid Process of the invention2(0.9941) is superior to the previous method (0.9915). The rapid method of the invention is linear, has high precision, and has more accurate accuracy, LOD and LOQ than the prior method.
In the method, the first-grade flour is used, then the first-grade flour is filtered by a 100-mesh sieve to ensure that the flour is clean, 70mg of the flour is weighed, then five eggs of the tribolium castaneum are put into the first-grade flour, and finally the uric acid concentration of the first-grade flour is measured to be 4.93ppm by the steps of the method. And judging the infection degree of the sample flour by taking the value as a reference. The uric acid concentration of 70mg of the sample flour is about 20ppm, and the infection degree of the sample flour is known to be more serious than that of the raw material first-grade flour.
The phosphotungstic acid reduction method in the prior art and the ultraviolet-visible spectrophotometry (rapid method) are used for comparative analysis. Uric acid is oxidized into allantoin and carbon dioxide by tungstic acid in an alkaline solution, phosphotungstic acid is reduced into tungstenic blue in the reaction, the generation amount of the tungstenic blue is in direct proportion to the uric acid content in a reaction solution, and colorimetric determination can be carried out.
Embodiment 2 of the present invention: weighing five groups of wheat flour samples to be tested, wherein the weight is as follows: 80mg, 160mg,240mg, 320mg mg,400mg, added to a test tube, and 2mL of 0.1% sodium carbonate solution added, and finally shaken to form a suspension. Then, the mixture is transferred into a centrifuge tube and centrifuged for 12 minutes at the rotating speed of 11000r/min to obtain supernatant. The supernatant was then poured into a volumetric flask as a stock solution of the sample containing uric acid. 5mL of the solution was taken out of the stock solution, poured into a 50mL volumetric flask, and diluted with distilled water to a constant volume as a working standard. Five kinds of uric acid-containing sample solutions with different concentrations are obtained. Then 0.3mmol/L uric acid standard solution was used as a standard sample solution. 5mL of the solution was taken out of the uric acid standard solution, poured into a 50mL volumetric flask, and diluted with distilled water to a constant volume as a standard solution. The ultraviolet-visible spectrophotometry comprises the following steps: uric acid-containing sample solutions of different concentrations (2mL) were removed, then diluted 5 times with distilled water, and then 2mL of iron potassium cyanide and iron chloride (2mL) were added and held for 5 minutes for color development. 2mL of the solution was taken out of the uric acid standard solution, poured into a 50mL volumetric flask, and diluted with distilled water to a constant volume as a standard solution. The standard solution also repeats these steps. The absorbance was measured at a wavelength of 294nm in an ultraviolet-visible spectrophotometer (Shimadzu; model: UV-1800).
The prior art phosphotungstic acid reduction method comprises the following steps: taking out the uric acid-containing sample solutions with different concentrations (2mL) into a centrifuge tube, respectively adding 18mL of tungstic acid reagent, uniformly mixing, standing for 5 minutes, and then centrifuging for 5 minutes at 4000 r/min. And respectively adding 2mL of 100g/L sodium carbonate solution into the centrifuged supernatant with different concentrations, uniformly mixing, standing for 10 minutes, respectively adding 2mL of phosphotungstic acid application solution, uniformly mixing, standing for 20 minutes, and then using 0.3mmol/L uric acid standard solution as a standard sample. 5mL of the solution was taken out of the uric acid standard solution, poured into a 50mL volumetric flask, and diluted with distilled water to a constant volume as a standard solution. The standard solution also repeats these steps. The absorbance was measured at a wavelength of 690nm in an ultraviolet-visible spectrophotometer (Shimadzu; model: UV-1800). Uric acid concentration (μmol/L) — 100 (absorbance of uric acid-containing sample/absorbance of standard solution). Linearity is calculated by the least squares regression method. The results are shown in FIGS. 5 and 6. From analysis, the fast normal linearity ranged from 10 to 50 ppm. The phosphotungstic acid reduction method has poor linearity within 10-50ppm, thereby showing that the sensitivity of the phosphotungstic acid reduction method for determining the concentration of uric acid is not high. The comparison of the two shows that the linearity of the standard curve shows higher accuracy in a rapid method; the correlation coefficient of the rapid method is 0.998, while the correlation coefficient of the phosphotungstic acid reduction method is 0.980, which is smaller than that of the rapid method. And the rapid method is much shorter than the phosphotungstic acid reduction method in the whole process, so the rapid method is superior to the phosphotungstic acid reduction method.
In the embodiment, first-grade wheat flour is used, then the wheat flour is filtered by a 100-mesh sieve to ensure that the wheat flour is clean, 70mg of the wheat flour is weighed, then five eggs of the tribolium castaneum are put into the wheat flour, then the steps in the invention are used, and finally the uric acid concentration of the wheat flour is measured to be 4.73 ppm. Using this value as a reference, the degree of infestation in the sample wheat flour was judged. The uric acid concentration of 70mg of the sample wheat flour was about 10ppm, and it was found that the degree of infection was more serious than that of the starting first-grade wheat flour.
The invention provides a method for accurately and quickly measuring uric acid, which judges the degree of insect pest infection of food by the uric acid content in unit mass of food and is beneficial to popularization of the insect killing method of a food storage system.
Claims (10)
1. An analytical method for judging the degree of infestation of food by pests is characterized by comprising the following steps:
taking a plurality of groups of food samples to be detected with different weights, respectively putting the plurality of groups of food samples to be detected into different test tubes, respectively adding sodium carbonate solution, and shaking up to form turbid liquid; the weight relationship of a plurality of groups of food samples to be detected is as follows: taking the lightest group as a reference, and taking other groups as integral multiples of the lightest group;
transferring each suspension to different centrifuge tubes, and centrifuging for a period of time; taking the supernatant to different first volumetric flasks to obtain a sample stock solution containing uric acid;
step three, taking a proper amount of the sample stock solutions of the groups obtained in the step two, adding the sample stock solutions into different second volumetric flasks, and diluting with distilled water to a constant volume to obtain a plurality of groups of uric acid-containing sample solutions with different solubilities;
step four, preparing uric acid standard solution and diluting with distilled water;
step five, taking the uric acid-containing sample solutions with different concentrations prepared in the step three and the uric acid standard solution prepared in the step four, and diluting the uric acid-containing sample solutions and the uric acid standard solution with distilled water respectively;
step six, adding the diluted solution obtained in the step five into ferric potassium cyanide and ferric chloride respectively, and keeping for a period of time for color development;
and step seven, respectively using an ultraviolet-visible spectrophotometer to obtain spectrophotometry readings of the solutions developed in the step six.
2. The analytical method for determining the degree of infestation of food products by pests according to claim 1, wherein in the first step, the weight of the lightest group of food samples to be tested is 50 to 100 mg.
3. The analytical method for judging the degree of infestation of food by insect pests according to claim 1 or 2, wherein in the first step, the sodium carbonate solution is 1 to 3mL of 0.1% sodium carbonate solution.
4. The analytical method for determining the infestation degree of the food by the pests according to claim 1, wherein in the second step, the volume of the first volumetric flask is 1L; the centrifugal speed is 8000-12000 r/min, and the centrifugal time is 7-13 minutes.
5. The analytical method for determining the degree of infestation of food by pests according to claim 1, wherein in the fourth step, the uric acid standard solution is 0.1-0.5 mmol/L.
6. The analytical method for determining the infestation degree of the pests on the food according to claim 1, wherein in the third step, the volume of the second volumetric flask is 50 mL; 1-5 mL of sample stock solution.
7. The analytical method for judging the degree of infestation of food by pests according to claim 1, wherein in the fifth step, the volume of the uric acid solution with different concentrations is 0.5-2 mL; the number of dilutions with distilled water was 5.
8. The analytical method for judging the degree of infestation of food by pests according to claim 1, wherein in the sixth step, the volume of the ferric potassium cyanide is 0.5 to 2mL, and the volume of the ferric chloride is 0.5 to 2 mL; the holding time was 5 minutes.
9. An assay method according to any one of claims 1 or 2 or 4 to 8 for determining the infestation degree of a food product by pests, wherein in step seven, the absorbance is measured with a UV-Vis spectrophotometer at a wavelength of 290 to 690 nm.
10. An analytical method for judging the degree of infestation of food by pests is characterized by comprising the following steps:
step one, preparing uric acid standard solution and diluting with distilled water;
taking a plurality of groups of food samples to be detected with different weights, respectively putting the plurality of groups of food samples to be detected into different test tubes, respectively adding sodium carbonate solution, and shaking up to form turbid liquid; the weight relationship of a plurality of groups of food samples to be detected is as follows: taking the lightest group as a reference, and taking other groups as integral multiples of the lightest group;
transferring each group of turbid liquid to different centrifuge tubes, and centrifuging for a period of time; taking the supernatant to different first volumetric flasks to obtain a sample stock solution containing uric acid;
step four, taking a proper amount of each group of sample stock solution obtained in the step three, adding the sample stock solutions into different second volumetric flasks, and diluting with distilled water to a constant volume to obtain a plurality of groups of uric acid-containing sample solutions with different solubilities;
step five, taking the uric acid-containing sample solutions with different concentrations prepared in the step four and the uric acid standard solution prepared in the step one, and diluting the uric acid-containing sample solutions and the uric acid standard solution with distilled water respectively;
step six, adding the diluted solution obtained in the step five into ferric potassium cyanide and ferric chloride respectively, and keeping for a period of time for color development;
and step seven, respectively using an ultraviolet-visible spectrophotometer to obtain spectrophotometry readings of the solutions developed in the step six.
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