Method for rapidly detecting sulfur dioxide in shrimps based on addition reaction fading technology

Abstract

The invention relates to the technical field of food quality supervision, in particular to a method for rapidly detecting sulfur dioxide in shrimps based on an addition reaction fading technology. The method comprises the following steps: S1, weighing a to-be-detected sample, adding pure water, homogenizing, and centrifuging to obtain a supernatant which is used as a sample extracting solution; s2, taking a test tube, adding V1 volume of the sample extracting solution and V2 volume of acetonitrile, uniformly shaking, performing centrifugal treatment to clarify the upper liquid, and taking the clarified upper liquid as a positive reference; v2 / (V1 + V2) is equal to 40-70%; and taking another test tube, synchronously operating to clarify the liquid at the upper part, dripping a malachite green reagent, slightly shaking, standing for 4-10 minutes, comparing with a positive reference, observing, and judging the sulfur dioxide content in the sample to be detected according to the color fading condition of lake blue in the test tube. A dynamic fading reaction is adopted to replace a static colorimetric card for colorimetric, the problems of subjectivity and matrix interference of a color card method are avoided, and the method is particularly suitable for rapid detection of shrimp samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food quality supervision, and particularly relates to a rapid detection method of sulfur dioxide in shrimps based on fading technology of addition reaction. BACKGROUND

[0002] Sulfur dioxide (including sulfite, metabisulfite, subsulfite, bisulfite, and the content is detected as sulfur dioxide) is used as a food additive and a processing aid, and can be used in specified food production. Because sulfur dioxide is harmful to human body, the ADI (Acceptable Daily Intake) value is 0-0.7 mg / kgbw, and therefore the maximum allowable residue of specified food is regulated in the relevant standards.

[0003] With the establishment of food safety regulations and the strengthening of food safety supervision, the innovation of detection technology and the application to the field of food safety rapid detection are increasingly strong. The characteristics of the food safety rapid detection method are as follows: allowing certain error (false negative and false positive rate), good sensitivity, and fast detection speed (time requirement). The existing sulfur dioxide determination methods mainly include spectrophotometry, distillation titration, and chromatography. The spectrophotometry needs to be equipped with a spectrophotometer, and the detection process is relatively complicated. The distillation titration needs to be equipped with a distillation instrument and other equipment, and at least 2 hours is required for detection, which cannot meet the demand of on-site rapid detection. The chromatography needs a high-priced chromatograph and high requirements for the detection personnel, and also cannot meet the demand of rapid detection.

[0004] At present, the rapid detection technologies applied to the field of food safety screening mainly include spectrophotometry (with instrument) and visual colorimetry derived from spectrophotometry. From the principle, the main methods include turmeric test paper method, pararosaniline colorimetry, and DTNB method. These methods also have some shortcomings, which lead to the failure to detect results or large deviation of results. For example, curcumin is photosensitive and is easy to decompose, thereby affecting the accuracy of the results. The color under different sulfur dioxide concentrations can only be measured by using standard samples to make a standard colorimetric card. During detection, the color of the sample (in a test tube) is compared with the standard colorimetric card (paper printing), and the approximate content is read, and the error is large. In the pararosaniline colorimetry, there are many factors affecting the color development, the blank value is high (purple red), and the color under different sulfur dioxide concentrations can only be measured by using standard samples to make a standard colorimetric card. During detection, the color of the sample (in a test tube) is compared with the standard colorimetric card (paper printing), and the approximate content is read, and the error is large.

[0005] In the DTNB method, the positive sample shows yellow color, and the color of the sample with low concentration of target molecule is also light, which is not easy to compare the color, and is not suitable for animal samples (the -SH material introduced by reduced glutathione and cysteine residues in animal / meat samples can make the reaction solution yellow, resulting in false positive results). This method can only use standard samples to measure the color under different sulfur dioxide concentrations to make a standard color card, and the actual sample color (in the test tube) is compared with the standard color card (paper printing) to read the approximate content, with a large error. The standard color card method relies on the comparison of the color depth of the sample and the static color card by the human eye, which is easily affected by subjective judgment (such as color blindness, light conditions) and printing color difference, and the error rate is usually 10-20%. In addition, the standard color card method cannot distinguish the difference between the sample color and the color development, especially for samples with deep color (containing the same color pigment), the error is more significant.

[0006] In summary, the existing sulfur dioxide detection technology has limitations and is not suitable for detecting sulfur dioxide in animal samples, especially in shrimp samples. SUMMARY

[0007] (I) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present application provides a rapid detection method for sulfur dioxide in shrimp based on addition reaction color fading technology, which takes shrimp meat slurry sample as a positive reference, and judges the content of sulfur dioxide in shrimp meat by the fading reaction of malachite green in shrimp meat slurry sample, to solve the limitations of the existing rapid detection technology for sulfur dioxide in shrimp samples.

[0008] (II) Technical solutions In the first aspect, the present application provides a rapid detection method for sulfur dioxide in shrimp based on addition reaction color fading technology, comprising the following steps: S1, preparing sample extract Weigh the sample to be tested, add pure water for homogenization, centrifuge the slurry to obtain supernatant, and use the supernatant directly as sample extract or filter it through a 0.45 μm membrane as sample extract; S2, detection Take a test tube, add V1 volume of sample extract and V2 volume of acetonitrile, shake well, centrifuge to make the upper liquid clear, and use this test tube as a positive reference; wherein V2 / (V1+V2)=40-70%; Take another test tube, add V1 volume of sample extract and V2 volume of acetonitrile, shake well, centrifuge, and add malachite green reagent to it, shake gently, and stand for 4-10 min. Compare and observe it with the positive reference, and judge the content of sulfur dioxide in the sample to be tested according to the fading of the lake color of malachite green reagent in the test tube.

[0009] According to the preferred embodiment of the present application, in S1, when preparing the sample extraction solution, the head part of the shrimp is removed, and the muscle part is weighed and taken as the sample to be tested.

[0010] According to the preferred embodiment of the present application, in S1, when preparing the sample extraction solution, the weighed shrimp meat is put into a food processor cup, pure water is added, and high-speed homogenization is performed for 20-30 seconds. After standing for 2-3 minutes, the homogenized solution is taken into a centrifuge tube, and microcentrifugation (5000 rpm) is performed for 2-3 minutes. The supernatant is directly used as the sample extraction solution or filtered through a 0.45 μm membrane to be used as the sample extraction solution.

[0011] According to the preferred embodiment of the present application, in S2, when the mass of malachite green contained in the malachite green reagent added dropwise into the test tube is 0.025 mg; at this time, if the total mass of sulfur dioxide contained in the test tube exceeds 0.01 mg, the lake blue of the malachite green reagent added dropwise into the test tube completely fades. Conversely, if the total mass of sulfur dioxide contained in the test tube does not exceed 0.01 mg, the lake blue of the malachite green reagent added dropwise into the test tube cannot completely fade.

[0012] According to the preferred embodiment of the present application, in S2, the total amount of V1+V2 is 1000 μL; wherein the mass of the shrimp meat weighed and used for preparing the sample extraction solution in step S1 is M, and the volume of the pure water added for preparing the sample extraction solution is V0; M and V0 satisfy the following formula: M×A×a×V1 / V0=0.01 mg; wherein A is the upper limit standard of the mass of sulfur dioxide in the shrimp meat, and a is the extraction rate (leaching rate) of sulfur dioxide when preparing the sample extraction solution. Preferably, the upper limit standard of the mass of sulfur dioxide in the shrimp sample is 100 mg / kg, i.e. A is 100 mg / kg.

[0013] According to the preferred embodiment of the present application, when a is 80%, the mass of the shrimp meat weighed and used for preparing the sample extraction solution in step S1 is 10-10.5 g, and the volume of the pure water added for preparing the sample extraction solution is 50 mL; in S2, V2 / (V1+V2)=40%, and the total amount of V1+V2 is 1000 μL; the mass of malachite green contained in the malachite green reagent added dropwise into the test tube is 0.025 mg. The way of judging the detection result in S2 is: the color development of the test tube to which the malachite green reagent is added is compared and observed with the positive reference, when the lake blue completely fades, it indicates that the content of sulfur dioxide in the shrimp sample exceeds the standard; otherwise, it indicates that the content of sulfur dioxide in the shrimp sample does not exceed the standard.

[0014] According to a preferred embodiment of the present invention, in S2, the malachite green content in the malachite green reagent is 500 ppm, and the volume of malachite green reagent added to the test tube is 50 μL; or the malachite green content in the malachite green reagent is 625 ppm, and the volume of malachite green reagent added to the test tube is 40 μL (exactly 2 drops).

[0015] According to a preferred embodiment of the present invention, in S2, when comparing the color development of the test tube containing malachite green reagent with the positive reference, a non-blue solid-color background can be placed behind the two test tubes to increase the accuracy of the human eye in judging whether the color has completely faded.

[0016] Secondly, the present invention provides a rapid detection method for sulfur dioxide in shrimp based on addition reaction decolorization technology, comprising the following steps: Step 1: Remove the shrimp heads and shells, take 10-10.5g of shrimp meat into the blender cup, add 50ml of pure water, blend on high speed for 20 seconds, let stand for 2 minutes, take about 2mL of the homogenized solution and transfer it to a 2mL centrifuge tube, centrifuge at 5000rpm for 2 minutes, and use the supernatant directly as the sample extraction solution or filter it through a 0.45μm membrane as the sample extraction solution (since membrane filtration results in some sulfur dioxide loss, it is best not to filter and use the supernatant directly as the sample extraction solution). Step 2: Take a test tube, add 600 μL of sample extraction solution and 400 μL of acetonitrile, shake well, and centrifuge for 15-20 seconds until the upper liquid becomes clear. Use this test tube as a positive reference. Take another test tube, add 600 μL of sample extract and 400 μL of acetonitrile, shake well, centrifuge for 15-20 seconds, add 50 μL of malachite green reagent containing 500 ppm malachite green, shake gently and let stand for 5 minutes: compare the color development of the test tube with the positive reference. When the lake blue color completely fades, it indicates that the sulfur dioxide content in the shrimp sample exceeds the standard (i.e., sulfur dioxide content ≥ 100 mg / kg); otherwise, it indicates that the sulfur dioxide content in the shrimp sample does not exceed the standard (i.e., sulfur dioxide content is less than 100 mg / kg or does not contain sulfur dioxide).

[0017] In step 1, take 10.4g of shrimp meat into the food processor cup to prepare the sample extract.

[0018] It should be noted that in this application, sulfur dioxide does not refer only to SO2 gas, but to sulfites, metabisulfites, hyposulfites, or bisulfites, all of which are measured as sulfur dioxide when their content is detected.

[0019] (III) Beneficial Effects The present application is based on the principle that sulfur dioxide can rapidly react with malachite green (MG, lake blue in neutral water system) at room temperature to generate a product without color, thereby fading the solution, and a new rapid detection method for the content of sulfur dioxide in shrimp samples is constructed. The method can be completed in 4 minutes (not more than 8 minutes), and the sample extract (without adding MG) is used as the actual reference, which can reduce the judgment error compared with the standard color card method.

[0020] The sample extract prepared from shrimp sample is very turbid, and must be treated to be clear for colorimetry. In order to solve this problem, a certain proportion (40-70%) of acetonitrile is added to the sample extract, which can change the hydrophilicity of proteins, part of inorganic substances and other organic substances (such as pigments) in the sample extract, reduce the solubility in water, and play a clarifying role. At the same time, acetonitrile and water are completely miscible, which can not only avoid the influence on the fading reaction, but also can clarify the sample extract, and avoid the colorimetric error and judgment error caused by turbid liquid.

[0021] The detection method of the present application can be completed in 4-8 minutes, which can avoid the side reaction (such as MG photodegradation) caused by long time placement by using the rapid kinetic characteristics (second-level reaction) of sulfur dioxide and MG. The standard color card method is unstable, and improper storage can affect the stability of the standard color card. The present application uses dynamic fading reaction instead of static colorimetry, which can avoid the subjective and matrix interference problems of the color card method in principle, and is especially suitable for rapid detection of complex biological samples (such as shrimp). BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The negative detection result picture of shrimp sample A.

[0023] Figure 2 The positive detection result picture of shrimp sample B. DETAILED DESCRIPTION

[0024] In order to better explain the present application, the present application is described in detail by specific embodiments in combination with the drawings.

[0025] The basic principle of the present application is that sulfur dioxide can rapidly react with malachite green (MG, lake blue in neutral water system) at room temperature to generate a product without color, thereby fading the solution. The higher the concentration of sulfur dioxide, the higher the fading degree. The reaction can occur in neutral environment without additional acid-base adjustment treatment.

[0026] In order to detect whether the content of sulfur dioxide in shrimp samples exceeds the standard, the fading conditions are first optimized. The sample extract obtained by homogenizing and centrifuging the shrimp meat after adding water is still turbid, and the liquid also contains astaxanthin and other pigment components contained in shrimp meat, that is, even after centrifugation, a clear liquid cannot be obtained, and the turbid liquid cannot be colorimetric.

[0027] To solve this problem, acetonitrile is added to the sample extract as a clarifying agent. Acetonitrile is completely miscible with water and can change the hydrophilicity of proteins, part of inorganic substances and other organic substances (such as pigments), thereby reducing their solubility in water. In combination with centrifugal treatment, the sample extract can be effectively clarified. Some other commonly used organic solvents that are miscible with water, such as methanol, ethanol and acetone, can also be used to clarify the sample extract, but the clarification effect is not as good as acetonitrile. Acetonitrile is not only completely miscible with water, but also has better clarification effect than other organic solvents. Therefore, acetonitrile is finally determined as the clarifying agent, which can separate the complex components in the sample extract without affecting the addition reaction, so that the supernatant becomes clear and colorimetric.

[0028] After determining acetonitrile as the clarifying agent, the addition ratio also needs to be optimized. The fading reaction of malachite green in pure water is very fast, which is a second-level reaction; but the fading reaction of malachite green in organic solvents is very slow (too slow reaction is easily affected by oxygen in the air). This is mainly because MG tends to dissolve in the organic phase, but sulfur dioxide tends to dissolve in the aqueous phase, which leads to a slow fading reaction of MG in pure organic solvents, which does not meet the fast detection requirement. In order to make the sample extract meet the clarification requirement and also have a fast and complete fading reaction to meet the fast detection requirement, the inventors optimize the addition ratio of acetonitrile through experiments, and finally determine that the addition ratio of acetonitrile is 40-70%, including: 40% acetonitrile and 60% sample extract mixture, or 50% acetonitrile and 50% sample extract mixture, 60% acetonitrile and 40% sample extract mixture, 70% acetonitrile and 30% sample extract mixture, etc. Under the above ratio, not only can the sample extract be clarified, but also the fading reaction of MG and sulfur dioxide can be completed within 4-7 min, which meets the fast detection requirement.

[0029] In addition, although the addition reaction of sulfur dioxide and malachite green occurs in a molar ratio of 1:1, the actual fading process is not a complete reaction in a 1:1 ratio, and there is a certain conversion rate of both reactants. It is determined through experiments that in a liquid reaction system of 0.025 mg of MG, the amount of sulfur dioxide needs to reach 0.01 mg or more to completely fade MG. For example, 1000 μL of liquid reaction system contains 10 ppm or more of sulfur dioxide, which can completely fade 0.025 mg of MG, otherwise if the concentration of sulfur dioxide is less than 10 ppm, it is difficult to completely fade the 0.025 mg of MG dropped. Based on the above research results, the technical scheme of the present application is finally obtained.

[0030] Example 1 This example compares the fading reaction speed under different acetonitrile addition ratios. The experimental process is as follows: (1) A mother liquor is prepared using sodium sulfite and pure water, and the concentration of sulfur dioxide in the mother liquor is 1000 ppm. A malachite green reagent with a concentration of 500 ppm is prepared (prepared with pure water).

[0031] (2) High-quality wild shrimps are purchased, and HPLC detection is performed to ensure that the wild shrimps do not contain sulfur dioxide. The shrimps are decapitated, and 10 grams of muscle part is taken into a food processor cup, 50 ml of pure water is added, and the mixture is homogenized at high speed for 20 seconds. After standing for 2 min, a homogenate solution is obtained, and about 2 mL of the homogenate solution is taken into a 2 mL centrifuge tube and centrifuged at 5000 rpm for 2 min. The supernatant is directly used as a sample extract or filtered through a 0.45 μm membrane as a sample extract (without sulfur dioxide).

[0032] (3) Acetonitrile, sample extract (without sulfur dioxide) and sulfur dioxide mother liquor are taken according to the ratio in Table 1, mixed in a 2 mL centrifuge tube, shaken well, and then centrifuged for 15 seconds. Then 50 μL of malachite green reagent (MG reagent) is added to the centrifuge tube, shaken gently, and then observed for the time required for the lake blue to completely fade after the addition of the malachite green reagent and recorded in Table 1.

[0033] Table 1: Acetonitrile addition ratio and reaction time From the above table, acetonitrile and water phase volume ratio of 4:6, 5:5, 6:4, 7:3, can make the turbid shrimp sample extract liquid quickly clear, and when the acetonitrile and water phase volume ratio is 4:6, 5:5, the discoloration time is shorter, only 4 min. Therefore, in the subsequent experiment, acetonitrile and water phase volume ratio of 4:6 is adopted to add acetonitrile to the sample extract liquid. The above experiment also shows that when the pure MG added to the MG reagent is 0.025 mg, 10 ppm of sulfur dioxide in a total volume of 1000 μL can make the MG completely fade, and once the pure MG added to the reaction system exceeds 0.025 mg, 10 ppm of sulfur dioxide in a total volume of 1000 μL cannot make the MG completely fade.

[0034] Example 2 In this example, the water homogenization method is used for sulfur dioxide extraction of shrimp samples, and the iodine titration method (GB5009.34) is used to determine the extraction rate of sulfur dioxide in shrimp samples by water extraction method, and the extraction rate is about 78-80%. The experimental method is as follows: 1. Sample pretreatment Take about 100 g of headless and shelled shrimp meat, rinse the surface residues with pure water, and cut and mix after absorbing the water. Accurately weigh 10.00 g of shrimp sample (recorded as m = 10.00 g), and put it into the food processor cup. Add 50.0 mL of pure water, and homogenize at high speed for 30 s, and stand at room temperature for 3 min.

[0035] 2. Water extraction of SO2 Transfer the homogenate to a centrifuge tube, take about 2 mL in a 2 mL centrifuge tube, centrifuge at 5,000 rpm for 2 min, and take the supernatant as "sample extract", which is used for subsequent determination. (Note: the extract should be prepared and used immediately to avoid oxidation loss of SO2 due to long storage) 3. Determination of SO2 content in water extraction solution by acid distillation-iodine titration method The determination process is as follows: Accurately transfer 10.0 mL of the above sample extract to a 250 mL distillation flask. Add 10.0 mL of 25wt% phosphoric acid solution, immediately introduce high-purity nitrogen gas (flow rate about 50 mL / min), and continue for 10 min (GB 5009.34 recommends 10 min to ensure complete release of SO2 and removal of oxygen interference).

[0036] Insert the gas outlet conduit into the receiving bottle containing 10 mL of 3% H2O2 absorption liquid. Use direct titration method: add 1 mL of starch indicator (10 g / L) in the distillation flask, and titrate with 0.01 mol / L iodine standard solution until the solution is light blue and does not fade for 30 seconds; record the iodine consumption volume, repeat the above operation 3 times, take the average value, and the iodine consumption volume is recorded as V a (mL).

[0037] 4. Blank determination Take 10.0 mL of deionized water instead of sample extract, and follow the above procedures. Record the volume of iodine solution consumed in the blank titration, denoted as V b (mL).

[0038] 5. Calculation of SO2 content in shrimp samples determined by water extraction method SO2 content in shrimp samples determined by water extraction method (mg / kg) = [(V a -V b ) x C x 32.03 x 1000] / m; wherein, V a : volume of iodine solution consumed in sample titration (mL); V b : volume of iodine solution consumed in blank titration (mL); C: actual concentration of iodine solution (mol / L); 32.03 is the molar mass of SO2 (g / mol); 1000 is for converting the mass of SO2 into mg; m is the mass of shrimp sample (g).

[0039] 6. Standard addition recovery experiment (verification of the reliability of the method) Take another 10.00 g of shrimp sample, and add an appropriate amount of Na2SO3 standard solution before homogenization to make the added concentration 10 mg / kg (calculated as SO2, 10 mg of sulfur dioxide per kg of shrimp). Determine the SO2 content in the spiked sample by the above water extraction method and acid distillation-iodine titration method.

[0040] Calculate the recovery rate: recovery rate (%) = [(Cmeasured-Cbackground) / Cadded] x 100%; Cmeasured: SO2 content in the spiked sample (mg / kg); Cbackground: SO2 content in the unspiked sample (mg / kg) Cadded: theoretical added amount (10 mg / kg) The recovery rate is within the range of 90-100%, indicating that the method is reliable.

[0041] 7. Determination of the theoretical SO2 content in shrimp according to GB 5009.34 Similarly, accurately weigh 10.00 g of shrimp sample into a 250 mL distillation flask. Add 75 mL of deionized water, and stir with a glass rod to disperse evenly. Connect the distillation flask with the condenser, take a 250 mL conical flask as the receiving flask, and add 10.0 mL of 3% H2O2 solution. Insert the end of the gas outlet conduit below the H2O2 liquid surface (ensure that SO2 is completely absorbed), and all interfaces should be sealed well to prevent SO2 leakage. Add 10.0 mL of 25% phosphoric acid solution to the distillation flask, and immediately tighten the stopper. Turn on the nitrogen, and adjust the flow rate to 50 mL / min for 10 min. Turn on the heating device, and control the distillation rate to be 2-3 mL / min (avoid violent boiling), distill for 35 min, and the receiving liquid volume no longer increases, and there is no irritating odor. After distillation, turn off the heating and nitrogen. Remove the receiving flask, and rinse the end of the conduit with a small amount of deionized water, and the wash liquid is added to the receiving flask. Add 2-3 drops of phenolphthalein. Titrate with 0.01 M NaOH standard solution until the solution changes from colorless to pink (or gray-green to pink), and does not fade for 30 s. Record the NaOH consumption volume, take the average of three parallel samples, and the NaOH consumption volume is recorded as V c (mL).

[0042] Take another 10.00 g of deionized water instead of the shrimp sample, and the rest of the operations are exactly the same. Record the blank titration consumption NaOH volume, which is recorded as V d (mL).

[0043] Calculate according to the formula in step 5, and replace V c in the formula in step 5 with V a , replace V d in the formula in step 6 with V b , and the theoretical content of SO2 in the shrimp sample is obtained.

[0044] 8. Calculation of extraction rate of sulfur dioxide by water extraction method Extraction rate (%) = (SO2 content measured by water extraction method / theoretical SO2 content in shrimp × 100%; wherein, the SO2 content measured by water extraction method is obtained from step 6. The theoretical SO2 content in shrimp is obtained from step 7.

[0045] It is determined that the extraction rate of SO2 by water extraction method in the shrimp sample is about 77-80%, which indicates that the water extraction method has good applicability, and the extraction process is simple, which meets the rapid detection needs of food quality supervision. Subsequently, 80% is used as the extraction rate for simple calculation.

[0046] It should be noted that the extraction rate of SO2 by water extraction method in each sample to be tested only needs to be determined once, and it will be used as a constant in the subsequent daily detection process. The extraction rates of SO2 by water extraction method in different shrimp samples are close but not exactly the same.

[0047] Example 3 According to Example 1, the concentration of sulfur dioxide in the 1000 μL liquid reaction system is 10 ppm (total content is 0.01 mg), which can just make the 0.025 mg of MG added dropwise completely fade. In order to make the MG reagent added dropwise completely fade, combined with the upper limit value of sulfur dioxide in shrimp sample, which is 100 mg / kg, the mass of shrimp sample which needs to be weighed and used to make sample extraction solution is calculated.

[0048] In the formula, the mass of shrimp meat which needs to be weighed and used to make sample extraction solution is M, M satisfies: M x A x a x V1 / V0 = 0.01 mg; wherein A is the upper limit standard of sulfur dioxide in shrimp meat, 100 mg / kg (the standard can be changed according to the specific situation), a is the extraction rate of sulfur dioxide when making sample extraction solution, which is 80%. The volume of pure water added to make sample extraction solution is V0, V0 can be 50 mL. According to the determination of "adding acetonitrile to sample extraction solution at a ratio of 4:6 of acetonitrile to water phase" in Example 1, the amount of acetonitrile added is 400 μL, and then the amount of shrimp sample extraction solution added is 600 μL, i.e. V1 = 600 μL. According to the above values, M is 10.4 g.

[0049] The method for determining whether the sulfur dioxide in shrimp sample exceeds the standard in this example is as follows: (1) Prepare tools and consumables Microcentrifuge: ≤5000 rpm Pipette and gun head: 5-50 μL, 10-1000 μL, 5 ml Electronic scale: range is 200 g, accuracy is 0.01 g Sample preparation machine (or food processor) Sulfur dioxide rapid detection kit (reagent A, reagent B), reagent A: acetonitrile; reagent B: malachite green (MG) reagent, concentration is 500 ppm.

[0050] (2) Make sample extraction solution Take two kinds of shrimp samples A and B respectively, remove the head and shell of the shrimp samples, and take the meat part for use. Accurately weigh 10.4 g of shrimp meat into the food processor cup, add 50 ml of distilled water, and homogenize at high speed for 20 seconds with the food processor, and stand for 2 min; take about 2 mL of homogenized solution into a 2 mL centrifuge tube, and centrifuge at 5000 rpm for 2 min with a microcentrifuge. The supernatant is the sample extraction solution.

[0051] (3) Take two 2mL test tubes, add 600μL of sample extract and 400μL of reagent A (acetonitrile) to each tube in sequence, shake well and centrifuge for 15 seconds. After taking out the two test tubes, add 50μL of reagent B (malachite green) to one of the test tubes, shake gently and let stand, and count down for 5 minutes. After the timer expires, use the test tube without reagent B as the positive reference and observe whether the lake blue color of the liquid in the test tube with reagent B has completely faded.

[0052] like Figure 1 As shown, if the lake blue color of the liquid in the test tube with reagent B added does not fade, it indicates that the corresponding shrimp meat sample (sample A) is a negative sample, meaning that the shrimp meat sample does not contain sulfur dioxide or the sulfur dioxide content does not exceed the limit of 100 mg / kg. The precipitate at the bottom of the test tube in the figure consists of proteins, some inorganic substances, and other organic substances (such as pigments) that have settled in the shrimp meat sample extract.

[0053] like Figure 2 As shown, if the lake blue color of the liquid in the test tube with reagent B completely fades, it indicates that the corresponding shrimp meat sample (sample B) is a positive sample, meaning that the sulfur dioxide content in the shrimp meat sample exceeds the limit of 100 mg / kg. Figure 2 The SO2 content in the shrimp meat of the positive sample (sample B) was determined according to GB 5009.34, and the result reached 150 mg / kg, which is consistent with the result of the test tube colorimetric method of this invention. This shows that the test tube colorimetric method of this invention can quickly and accurately determine whether sulfur dioxide in shrimp meat samples exceeds the standard.

[0054] In summary, this invention provides a novel rapid detection method for sulfur dioxide content in shrimp samples based on the principle that sulfur dioxide can rapidly undergo an addition reaction with malachite green (MG, which appears as a lake blue color in neutral water) at room temperature, and the resulting product is colorless. Compared with the standard color chart method, this method is less affected by ambient light and background color introduced by the sample, thus reducing judgment errors. Furthermore, it is simple to operate and allows for rapid detection.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations 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 rapid detection of sulfur dioxide in shrimp based on addition reaction discoloration technique, characterized in that, The method comprises the following steps: S1, preparing sample extract S2, detecting S1, in the preparation of sample extract, the head of the shrimp is removed, and the muscle part is quantitatively weighed as the sample to be tested. S1, in the preparation of sample extract, the shrimp meat is placed in a food processor cup, pure water is added, and high-speed homogenization is performed for 20-30s, and then the homogenized solution is taken to a centrifuge tube and centrifuged for 2-3min, and the supernatant is directly used as the sample extract or filtered through a 0.45μm membrane as the sample extract. S2, when the mass of malachite green contained in the malachite green reagent added to the test tube is 0.025mg; 2. The rapid detection method according to claim 1, characterized in that, At this time, if the total mass of sulfur dioxide contained in the test tube exceeds 0.01mg, the lake blue color of the malachite green reagent added to the test tube will completely fade; 3. The rapid detection method according to claim 2, characterized in that, On the contrary, if the total mass of sulfur dioxide contained in the test tube does not exceed 0.01mg, the lake blue color of the malachite green reagent added to the test tube cannot completely fade.

4. The rapid detection method according to claim 2, characterized in that, S2, the total amount of V1+V2 is 1000μL; wherein the mass of shrimp meat quantitatively weighed and used to prepare the sample extract in step S1 is M, and the volume of pure water added to prepare the sample extract is V0; M and V0 satisfy the following formula: M×A×a×V1 / V0=0.01mg; Wherein, A is the upper limit standard of the mass of sulfur dioxide in shrimp meat, and a is the extraction rate of sulfur dioxide when preparing the sample extract.

5. The rapid detection method according to claim 4, characterized in that, A is 100mg / kg. When a is 80%, the mass of shrimp meat quantitatively weighed and used to prepare the sample extract in step S1 is 10-10.5g, and the volume of pure water added to prepare the sample extract is 50mL; in S2, V2 / (V1+V2)=40%, and the total amount of V1+V2 is 1000μL; the mass of malachite green contained in the malachite green reagent added to the test tube is 0.025mg; The way to judge the detection result in S2 is: comparing the color development of the test tube with the positive reference, when the lake blue color completely fades, it indicates that the content of sulfur dioxide in the shrimp sample exceeds the standard; otherwise, it indicates that the content of sulfur dioxide in the shrimp sample does not exceed the standard.

6. The rapid detection method according to claim 5, characterized in that, ​ 7. The rapid detection method according to claim 6, characterized in that, ​ ​ 8. The rapid detection method according to claim 4, characterized in that, In S2, the content of malachite green in the malachite green reagent is 500 ppm, and the volume of the malachite green reagent added to the test tube is 50 μL; or the content of malachite green in the malachite green reagent is 625 ppm, and the volume of the malachite green reagent added to the test tube is 40 μL.

9. The rapid detection method of claim 1, wherein, In S2, when observing the color development of the test tube to which the malachite green reagent is added and the positive reference, a non-colored pure color background is placed behind the test tube to increase the accuracy of the human eye in judging whether the color is completely faded.

10. A method for rapid detection of sulfur dioxide in shrimp based on the addition reaction discoloration technique, characterized in that, The method comprises the following steps: Step 1: remove the shrimp head and shell, take 10-10.5 g of shrimp meat into a food processor cup, add 50 ml of pure water, homogenize at high speed for 20 seconds, stand for 2 min, take 2 mL of the homogenized solution into a 2 mL centrifuge tube, centrifuge for 2 min with a microcentrifuge, and directly use the supernatant as the sample extraction liquid or filter it through a 0.45 μm membrane to obtain the sample extraction liquid; Step 2: take a test tube, add 600 μL of the sample extraction liquid and 400 μL of acetonitrile, shake well, centrifuge for 15-20 seconds to make the upper liquid clear, and use this test tube as the positive reference; Take another test tube, add 600 μL of the sample extraction liquid and 400 μL of acetonitrile, shake well, centrifuge for 15-20 seconds, add 50 μL of malachite green reagent containing 500 ppm of malachite green to the test tube, shake gently, stand for 5 min, compare and observe the color development of the test tube to which the malachite green reagent is added with the positive reference, when the lake blue color is completely faded, it indicates that the content of sulfur dioxide in the shrimp sample exceeds the standard; otherwise, it indicates that the content of sulfur dioxide in the shrimp sample does not exceed the standard.

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