A rapid detection method of sulfur dioxide in shrimp based on fading technology of addition reaction

Abstract

The present application relates to the technical field of food quality supervision, and particularly relates to a method for rapidly detecting sulfur dioxide in shrimps based on fading technology of addition reaction. The method comprises the following steps: S1, weighing the sample to be measured, adding pure water for homogenization, centrifuging, and obtaining supernatant, which is used as sample extract; S2, taking a test tube, adding V1 volume of sample extract and V2 volume of acetonitrile, shaking, centrifuging, making the upper liquid clear, and taking the upper liquid as positive reference; V2 / (V1+V2)=40-70%; taking another test tube for synchronous operation to make the upper liquid clear, then adding malachite green reagent dropwise, shaking gently, and standing for 4-10 min; comparing the test tube with the positive reference, and judging the content of sulfur dioxide in the sample to be measured according to the fading of the lake blue in the test tube. The present application uses dynamic fading reaction to replace static colorimetric card colorimetry, and avoids the subjectivity and matrix interference problems of color card method, and is especially suitable for rapid detection of shrimp samples.

Description

Technical Field

[0001] This invention relates to the field of food quality supervision technology, specifically to a rapid detection method for sulfur dioxide in shrimp based on addition reaction decolorization technology. Background Technology

[0002] Sulfur dioxide (including sulfites, metabisulfites, hyposulfites, and bisulfites, all measured as sulfur dioxide) can be used as a food additive and processing aid in the production of designated foods. However, due to its harmfulness to the human body, the ADI (Acceptable Daily Intake) is 0-0.7 mg / kgbw. Therefore, relevant standards specify the maximum allowable residue level for designated foods.

[0003] With the establishment of food safety regulations and the strengthening of food safety supervision, the demand for innovation in testing technologies and their application in the field of rapid food safety testing is becoming increasingly strong. Rapid food safety testing methods are characterized by: allowing for a certain degree of error (false negative and false positive rates), good sensitivity, and fast detection speed (time-sensitive requirements). Existing methods for determining sulfur dioxide mainly include spectrophotometry, distillation titration, and chromatography. Spectrophotometry requires a spectrophotometer, making the detection process relatively cumbersome; distillation titration requires at least 2 hours to complete and also requires equipment such as a distillation apparatus, which cannot meet the needs of rapid on-site testing; chromatographic methods require expensive chromatographs and demand highly skilled personnel, also failing to meet the needs of rapid testing.

[0004] Currently, the rapid testing technologies applied in food safety screening mainly include spectrophotometry (with instruments) and visual colorimetry, an extension of spectrophotometry. In principle, these include the turmeric test paper method, the pararosaniline colorimetric method, and the DTNB method. Each of these methods has its own drawbacks, leading to either undetectable results or significant deviations. For example, curcumin is photosensitized and easily decomposes, affecting the accuracy of the results; it can only measure the color at different sulfur dioxide concentrations using standard samples to create a standard colorimetric card, and during testing, the actual sample color (in a test tube) is compared with the standard color card (paper printout) to read the approximate content, resulting in significant errors. The pararosaniline colorimetric method is also problematic due to numerous factors affecting color development, resulting in a high blank value (purple-red). It can only measure the color at different sulfur dioxide concentrations using standard samples to create a standard colorimetric card, and during testing, the actual sample color (in a test tube) is compared with the standard color card (paper printout) to read the approximate content, leading to significant errors.

[0005] In the DTNB method, a positive result is yellow. The color is lighter when the concentration of the target molecule in the sample is low, making colorimetric comparison difficult and unsuitable for animal samples (the -SH substances introduced by reduced glutathione and cysteine ​​residues in animal / meat samples can cause the reaction solution to turn yellow, resulting in false positives). This method also requires pre-measuring the color of different sulfur dioxide concentrations using standard samples to create a standard color chart. During testing, the actual sample color (in a test tube) is compared with the standard color chart (printed paper) to obtain an approximate concentration, resulting in significant errors. The standard color chart method relies on the human eye to compare the color depth of the sample with the static color chart, making it susceptible to subjective judgment (such as color blindness, lighting conditions) and printing color differences, with an error rate typically reaching 10–20%. Furthermore, the standard color chart method cannot distinguish between the sample's base color and the developed color, especially for samples with inherently dark colors (containing the DTNB pigment), where the error is even more significant.

[0006] In summary, existing sulfur dioxide detection technologies have limitations and are not suitable for detecting sulfur dioxide in animal samples, especially shrimp samples. Summary of the Invention

[0007] (a) Technical problems to be solved

[0008] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a rapid detection method for sulfur dioxide in shrimp based on addition reaction bleaching technology. The method uses shrimp meat slurry sample as a positive reference and determines the content of sulfur dioxide in shrimp meat by the occurrence of malachite green bleaching reaction in the shrimp meat slurry sample, thereby overcoming the limitations of existing rapid detection technologies for sulfur dioxide in shrimp samples.

[0009] (II) Technical Solution

[0010] In a first aspect, the present invention provides a rapid detection method for sulfur dioxide in shrimp based on addition reaction decolorization technology, comprising the following steps:

[0011] S1. Preparation of sample extract

[0012] Weigh the sample to be tested, add pure water to homogenize, centrifuge the slurry to obtain the supernatant, and use the supernatant directly as the sample extract or filter it through a 0.45μm membrane as the sample extract;

[0013] S2, Detection

[0014] Take a test tube, add V1 volume of sample extract and V2 volume of acetonitrile, shake well, and centrifuge until the upper liquid becomes clear. Use this test tube as the positive reference; where V2 / (V1+V2) = 40-70%.

[0015] Take another test tube, add V1 volume of sample extract and V2 volume of acetonitrile, shake well, centrifuge, add malachite green reagent, shake gently and let stand for 4-10 minutes, compare it with the positive reference, and judge the presence of sulfur dioxide in the sample by the fading of the lake blue color of the malachite green reagent added to the test tube.

[0016] According to a preferred embodiment of the present invention, in S1, when preparing the sample extract, the shrimp head is removed first, and the muscle portion is weighed quantitatively as the sample to be tested.

[0017] According to a preferred embodiment of the present invention, in S1, when preparing the sample extract, a quantitative amount of shrimp meat is placed into a food processor cup, pure water is added, homogenized at high speed for 20-30 seconds, and allowed to stand for 2-3 minutes. Then, the homogenized solution is taken into a centrifuge tube and centrifuged for 2-3 minutes using a microcentrifuge (5000 rpm). The supernatant is used directly as the sample extract or filtered through a 0.45 μm membrane as the sample extract.

[0018] According to a preferred embodiment of the present invention, in S2, when the mass of malachite green contained in the malachite green reagent added to 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 color of the malachite green reagent added to the test tube will completely fade.

[0019] Conversely, if the total mass of sulfur dioxide in the test tube does not exceed 0.01 mg, the malachite green reagent added to the test tube will not completely fade its blue color.

[0020] According to a preferred embodiment of the present invention, in 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:

[0021] M×A×a×V1 / V0=0.01mg;

[0022] Wherein, A represents the upper limit standard for sulfur dioxide in shrimp meat, and a represents the extraction rate (leaching rate) of sulfur dioxide when preparing the sample extract. Preferably, the upper limit standard for sulfur dioxide in shrimp samples is 100 mg / kg, i.e., A is 100 mg / kg.

[0023] According to a preferred embodiment of the present invention, when a is 80%, the mass of shrimp meat 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;

[0024] The method for judging the test results in S2 is as follows: compare the color development of the test tube with the added malachite green reagent 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; otherwise, it indicates that the sulfur dioxide content in the shrimp sample does not exceed the standard.

[0025] 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).

[0026] According to a preferred embodiment of the present invention, in step S2, when comparing the color development of the test tube containing malachite green reagent with a 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.

[0027] Secondly, the present invention provides a rapid detection method for sulfur dioxide in shrimp based on addition reaction decolorization technology, comprising the following steps:

[0028] 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).

[0029] 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.

[0030] 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).

[0031] In step 1, take 10.4g of shrimp meat into the food processor cup to prepare the sample extract.

[0032] 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.

[0033] (III) Beneficial Effects

[0034] This invention is 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 product is colorless, thus causing the solution to decolorize. A new rapid detection method for sulfur dioxide content in shrimp samples has been developed. This method can be completed in as little as 4 minutes (not exceeding 8 minutes). The sample extract (without MG added) processed in parallel is used as the actual reference. Compared with the standard color chart method, the results are less affected by ambient light and background color introduced by the sample, which can reduce judgment error.

[0035] Because the extract from shrimp samples is very turbid, it must be clarified for easy colorimetric analysis. To solve this problem, this invention adds a certain proportion (40-70%) of acetonitrile to the extract. Acetonitrile alters the hydrophilicity of proteins, some inorganic substances, and other organic substances (such as pigments) in the extract, reducing their solubility in water and thus clarifying the sample. Furthermore, acetonitrile and water are completely miscible, so this not only does not affect the fading reaction but also clarifies the extract, avoiding colorimetric and judgment errors caused by turbid liquids.

[0036] The detection method of this invention completes the reaction within 4-8 minutes, utilizing the rapid kinetic characteristics (second-level reaction) of sulfur dioxide and MG, avoiding side reactions (such as MG photodegradation) caused by prolonged storage. In contrast, the standard color chart method is unstable, and improper storage can affect the stability of the standard color chart. This invention uses a dynamic fading reaction instead of static colorimetry, fundamentally avoiding the subjectivity and matrix interference problems of the color chart method, making it particularly suitable for the rapid detection of more complex biological samples (such as shrimp). Attached Figure Description

[0037] Figure 1 Image showing the negative test result for shrimp meat sample A.

[0038] Figure 2 The image shows the positive test result for shrimp meat sample B. Detailed Implementation

[0039] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] The basic principle of this invention is as follows: Sulfur dioxide reacts rapidly with malachite green (MG, which appears as a lake blue color in neutral aqueous solutions) at room temperature (reaction process as shown below). The product is colorless, thus decolorizing the solution. The higher the concentration of sulfur dioxide, the greater the degree of decolorization. This reaction can occur under neutral conditions without the need for additional acid-base adjustments.

[0041]

[0042] To detect whether the sulfur dioxide content in shrimp samples exceeded the standard, the bleaching conditions were first optimized. After adding water to the shrimp meat, homogenizing and centrifuging the sample extract, the liquid was still turbid. The liquid still contained pigments such as astaxanthin from the shrimp meat, and even after centrifugation, a clear liquid could not be obtained. The turbid liquid could not be used for colorimetric analysis.

[0043] To address this problem, this invention adds acetonitrile as a clarifying agent to the sample extract. Acetonitrile is completely miscible with water and alters the hydrophilicity of proteins, some inorganic substances, and other organic substances (such as pigments), reducing their solubility in water. Combined with centrifugation, this effectively clarifies the sample extract. While other commonly used water-miscible organic solvents, such as methanol, ethanol, and acetone, can also be used to clarify sample extracts, their clarifying effect is inferior to that of acetonitrile. Acetonitrile is not only infinitely miscible with water, but its clarifying effect is also superior to several other organic solvents. Therefore, acetonitrile was ultimately chosen as the clarifying agent to cause the complex components in the sample extract to precipitate and separate into layers without affecting the addition reaction, resulting in a clear supernatant that facilitates colorimetric analysis.

[0044] After determining acetonitrile as the clarifying agent, its addition ratio also needed optimization. Malachite green decolorizes rapidly in pure water, within seconds; however, it decolorizes very slowly in organic solvents (the slow reaction is easily affected by oxygen in the air). This is mainly because MG tends to dissolve more in the organic phase, while sulfur dioxide tends to dissolve more in the aqueous phase, resulting in a slow decolorization reaction of MG in pure organic solvents, which does not meet the requirements for rapid detection. To achieve both clarification and rapid, complete color change in the sample extract to meet the needs of rapid detection, the inventors optimized the addition ratio of acetonitrile through experiments, ultimately determining that the acetonitrile addition ratio is 40-70%, including: a mixture of 40% acetonitrile and 60% sample extract, or a mixture of 50% acetonitrile and 50% sample extract, a mixture of 60% acetonitrile and 40% sample extract, and a mixture of 70% acetonitrile and 30% sample extract, etc. At the aforementioned ratio, not only can the sample extract be clarified, but the decolorization reaction between MG and sulfur dioxide can also be completed within 4-7 minutes, meeting the requirements for rapid detection.

[0045] Furthermore, although sulfur dioxide and malachite green undergo an addition reaction in a 1:1 molar ratio, the actual fading process is not a complete reaction occurring in a 1:1 ratio; both reactants exhibit a certain conversion rate. Experiments have determined that in a liquid reaction system containing 0.025 mg MG, the amount of sulfur dioxide must be at least 0.01 mg to completely decolorize MG. For example, in a 1000 μL liquid reaction system, a sulfur dioxide concentration of at least 10 ppm is sufficient to completely decolorize 0.025 mg MG; conversely, if the sulfur dioxide concentration is below 10 ppm, it is difficult to completely decolorize the added 0.025 mg MG. Based on these research results, the technical solution of this invention was finally obtained.

[0046] Example 1

[0047] This example compares the decolorization reaction rate under different acetonitrile addition ratios. The experimental procedure is as follows:

[0048] (1) Prepare a mother liquor using sodium sulfite and pure water. The sulfite in the mother liquor is expressed as sulfur dioxide, and the sulfur dioxide concentration in the mother liquor is 1000 ppm. Prepare a malachite green reagent with a concentration of 500 ppm (prepared with pure water).

[0049] (2) Purchase high-quality wild shrimp and ensure that the wild shrimp are free of sulfur dioxide by HPLC testing. Remove the head of the shrimp sample, take 10 grams of the muscle part into the food processor cup, add 50 ml of purified water, homogenize at high speed for 20 seconds, let stand for 2 minutes to obtain a homogenized solution, take about 2 mL of the homogenized solution into a 2 mL centrifuge tube, centrifuge at 5000 rpm for 2 minutes using a micro centrifuge, and use the supernatant directly as the sample extraction solution or filter it through a 0.45 μm membrane as the sample extraction solution (free of sulfur dioxide).

[0050] (3) Measure acetonitrile, sample extract (excluding sulfur dioxide) and sulfur dioxide mother liquor according to the ratio in Table 1, mix them in a 2 mL centrifuge tube, shake well and centrifuge for 15 seconds. Then add 50 μL of malachite green reagent (MG reagent) to the centrifuge tube, shake gently and let stand. Observe the time required from the addition of malachite green reagent until the lake blue color completely fades and record it in Table 1.

[0051] Table 1: Acetonitrile addition ratio and reaction time

[0052]

[0053] As shown in the table above, acetonitrile to water volume ratios of 4:6, 5:5, 6:4, and 7:3 all rapidly clarified the turbid shrimp meat sample extract. Furthermore, the fading time was shorter at 4:6 and 5:5, requiring only 4 minutes. Therefore, in subsequent experiments, acetonitrile was added to the sample extract at a 4:6 acetonitrile to water volume ratio. The experiments also showed that when the amount of pure MG added was 0.025 mg, 10 ppm of sulfur dioxide in a total volume of 1000 μL was sufficient to completely decolorize the MG. However, once the amount of pure MG added to the reaction system exceeded 0.025 mg, 10 ppm of sulfur dioxide in a total volume of 1000 μL was insufficient to completely decolorize the MG.

[0054] Example 2

[0055] In this embodiment, sulfur dioxide was extracted from shrimp samples using a water homogenization method. The extraction rate of sulfur dioxide from the shrimp samples by water extraction was determined using the iodine titration method (GB5009.34), and the extraction rate was found to be approximately 78-80%. The experimental methods are as follows:

[0056] 1. Sample pretreatment

[0057] Take about 100g of headless, shelled shrimp meat, rinse off any surface residue with pure water, pat dry, and then chop and mix well. Accurately weigh 10.00g of the shrimp meat sample (recorded as m=10.00g), place it in the food processor cup, add 50.0mL of pure water, homogenize on high speed for 30s, and let stand at room temperature for 3min.

[0058] 2. Water extraction method for SO2 extraction

[0059] Transfer the homogenized solution to a centrifuge tube. Take approximately 2 mL of the solution into a 2 mL centrifuge tube and centrifuge at 5,000 rpm for 2 minutes. Collect the supernatant as the "sample extraction solution" for subsequent measurements. (Note: The extraction solution should be prepared fresh and used immediately to avoid SO2 oxidation loss due to prolonged storage.)

[0060] 3. The SO2 content in the water extraction solution was determined by acid distillation-iodine titration.

[0061] The measurement process is as follows:

[0062] Accurately transfer 10.0 mL of the above sample extract into a 250 mL distillation flask. Add 10.0 mL of 25 wt% phosphoric acid solution, and immediately purge with high-purity nitrogen gas (flow rate of approximately 50 mL / min) for 10 min (GB 5009.34 recommends 10 min to ensure complete release of SO2 and removal of oxygen interference).

[0063] Insert the gas outlet tube into the receiving flask containing 10 mL of 3% H₂O₂ absorbent. Use the direct titration method: add 1 mL of starch indicator (10 g / L) to the distillation flask, and titrate with 0.01 mol / L iodine standard solution until the solution turns pale blue and does not fade within 30 seconds; record the volume of iodine solution consumed. Repeat the above operation three times and take the average value. Record the volume of iodine solution consumed as V. a (mL).

[0064] 4. Blank control determination

[0065] Replace the sample extract with 10.0 mL of deionized water, and perform the remaining operations as above. Record the volume of iodine solution consumed in the blank titration, denoted as V. b (mL).

[0066] 5. Calculate the SO2 content in shrimp meat samples determined by the water extraction method.

[0067] SO2 content (mg / kg) in shrimp meat determined by water extraction method = [(V a -V b [( )×C×32.03×1000] / m;

[0068] Among them, V a : Volume of iodine solution consumed in sample titration (mL);

[0069] V b : Volume of iodine solution consumed in blank titration (mL);

[0070] C: Actual concentration of iodine solution (mol / L);

[0071] 32.03 is the molar mass of SO2 (g / mol);

[0072] 1000 is the conversion of SO2 mass to mg;

[0073] m represents the shrimp sample mass (g).

[0074] 6. Spike recovery experiment (to verify the reliability of the method)

[0075] Take another 10.00g shrimp meat sample and add an appropriate amount of Na2SO3 standard solution before homogenization to make the concentration 10mg / kg (calculated as SO2, 10mg sulfur dioxide per kg of shrimp meat). Determine the SO2 content in the spiked sample according to the above water extraction method and acid distillation-iodine titration method.

[0076] Calculate the spiked recovery rate: Recovery rate (%) = [(C measured - C background) / C spiked] × 100%;

[0077] C test: SO2 content (mg / kg) was measured by adding the spiked sample;

[0078] Background (C): SO2 content (mg / kg) measured without standard.

[0079] C addition: Theoretical addition amount (10mg / kg)

[0080] The recovery rate was in the range of 90-100%, indicating that the method is reliable.

[0081] 7. Determine the theoretical SO2 content in shrimp meat according to GB 5009.34.

[0082] First, accurately weigh 10.00g of shrimp meat sample and place it in a 250mL distillation flask. Add 75mL of deionized water and stir with a glass rod to disperse it evenly. Connect the condenser to the distillation flask, and use a 250mL Erlenmeyer flask as the receiving flask, adding 10.0mL of 3% H2O2 solution. Insert the end of the gas outlet tube below the H2O2 liquid surface (ensuring complete absorption of SO2), ensuring all connections are sealed to prevent SO2 leakage. Add 10.0mL of 25% phosphoric acid solution to the distillation flask and immediately tighten the stopper. Turn on the nitrogen gas and adjust the flow rate to 50mL / min for 10 minutes. Turn on the heating device and control the distillation rate at 2-3mL / min (avoiding bumping), distilling for 35 minutes until the volume of the receiving liquid no longer increases and there is no irritating odor. After distillation, turn off the heating and nitrogen gas. Remove the receiving flask, rinse the end of the tube with a small amount of deionized water, and add the washings to the receiving flask. Add 2-3 drops of phenolphthalein. Titrate with 0.01M NaOH standard solution until the solution changes from colorless to pink (or from grayish-green to pink) and the color does not fade within 30 seconds. Record the volume of NaOH consumed. Perform three parallel samples and take the average value. Record the volume of NaOH consumed as V. c (mL).

[0083] Take another 10.00g of deionized water to replace the shrimp meat sample, and perform the same operation as before. Record the volume of NaOH consumed in the blank titration as V. d (mL).

[0084] Calculate according to the formula in step 5, and then use V. c Replace V in the formula of step 5 a V d Replace V in the formula of step 6 b The theoretical SO2 content in the shrimp meat sample was obtained.

[0085] 8. Calculation of extraction rate of sulfur dioxide by water extraction method

[0086] Extraction rate (%) = (SO2 content measured by water extraction / theoretical SO2 content in shrimp meat × 100%); where the SO2 content measured by water extraction is obtained from step 6. The theoretical SO2 content in shrimp meat is obtained from step 7.

[0087] The extraction rate of SO2 from shrimp meat samples using water extraction was determined to be approximately 77-80%. This indicates that water extraction has good applicability and a simple extraction process, meeting the rapid detection requirements for food quality supervision. For simplicity, the extraction rate will be assumed to be 80% in subsequent calculations.

[0088] It should be noted that the SO2 extraction rate for each sample only needs to be measured once, and will be used as a constant in subsequent routine testing. The SO2 extraction rates for different shrimp meat samples using the water extraction method are similar but not entirely equal.

[0089] Example 3

[0090] According to Example 1, a sulfur dioxide concentration of 10 ppm (total content of 0.01 mg) in a 1000 μL liquid-phase reaction system was sufficient to completely decolorize the added 0.025 mg of MG. To ensure complete decolorization of the added MG reagent, and considering the upper limit requirement of 100 mg / kg for sulfur dioxide in shrimp samples, the required mass of shrimp sample to be weighed was calculated.

[0091] The required mass of shrimp meat to be weighed and used to prepare the sample extract is M, where M satisfies: M × A × a × V1 / V0 = 0.01 mg; where A is the upper limit standard for sulfur dioxide in shrimp meat, 100 mg / kg (this standard may vary depending on specific circumstances), and a is the extraction rate of sulfur dioxide during sample extract preparation, taken as 80%. The volume of pure water added to prepare the sample extract is V0, which can be taken as 50 mL. Based on the determination in Example 1 of "adding acetonitrile to the sample extract at a volume ratio of 4:6 of acetonitrile to water", the amount of acetonitrile added is 400 μL, therefore the amount of shrimp meat sample extract added is 600 μL, i.e., V1 = 600 μL. Based on the aforementioned values, M is 10.4 g.

[0092] The method used in this embodiment to determine whether sulfur dioxide levels in shrimp meat samples exceed the standard is as follows:

[0093] (1) Prepare tools and consumables

[0094] Miniature centrifuge: ≤5000rpm

[0095] Pipettes and tips: 5-50μL, 10-1000μL, 5ml

[0096] Electronic scale: weighing range 200g, accuracy: 0.01g

[0097] Sample preparation machine (or food processor)

[0098] Sulfur dioxide rapid detection kit (reagent A, reagent B), reagent A: acetonitrile; reagent B: malachite green (MG) reagent, concentration 500ppm.

[0099] (2) Preparation of sample extract

[0100] Take two types of shrimp samples, A and B, respectively. Remove the heads and shells from the shrimp samples and keep the meat. Accurately weigh 10.4g of shrimp meat into the blender cup, add 50ml of distilled water, and homogenize on high speed for 20 seconds. Let it stand for 2 minutes. Take about 2mL of the homogenized solution into a 2mL centrifuge tube and centrifuge at 5000rpm for 2 minutes. The supernatant is the sample extract.

[0101] (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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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 rapid detection method for sulfur dioxide in shrimp based on addition reaction decolorization technology, characterized in that, include: S1. Preparation of sample extract Weigh the sample to be tested, add pure water to homogenize it, centrifuge the slurry to obtain the supernatant, and use the supernatant directly as the sample extract or filter it through a 0.45μm membrane as the sample extract; S2, Detection Take a test tube, add V1 volume of sample extract and V2 volume of acetonitrile, shake well, and centrifuge until the upper liquid becomes clear. Use this test tube as a positive reference. V2 / (V1+V2) = 40% or 50%; the total amount of V1+V2 is 1000 μL. In step S1, the mass of shrimp meat used to prepare the sample extract 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 sulfur dioxide in shrimp meat, and a is the extraction rate of sulfur dioxide when preparing the sample extract. Take another test tube, add V1 volume of sample extract and V2 volume of acetonitrile, shake well, centrifuge, add malachite green reagent, shake gently, and let stand for 4-10 minutes. Compare it with the positive reference. Determine the sulfur dioxide content in the sample based on the fading of the lake blue color of the malachite green reagent in the test tube. The method for judging the test result is: 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; otherwise, it indicates that the sulfur dioxide content in the shrimp sample does not exceed the standard.

2. The rapid detection method according to claim 1, characterized in that, In S1, when preparing the sample extract, the shrimp head is removed first, and the muscle portion is weighed quantitatively as the sample to be tested.

3. The rapid detection method according to claim 2, characterized in that, In S1, when preparing the sample extract, a quantitative amount of shrimp meat is placed into the food processor cup, pure water is added, and the mixture is homogenized at high speed for 20-30 seconds. After standing for 2-3 minutes, the homogenized solution is transferred to a centrifuge tube and centrifuged for 2-3 minutes using a microcentrifuge. The supernatant is used directly as the sample extract or filtered through a 0.45μm membrane as the sample extract.

4. The rapid detection method according to claim 2, characterized in that, In S2, when the mass of malachite green in the reagent added to the test tube is 0.025 mg: At this point, if the total mass of sulfur dioxide in the test tube exceeds 0.01 mg, the lake blue color of the malachite green reagent added to the test tube will completely fade. Conversely, if the total mass of sulfur dioxide in the test tube does not exceed 0.01 mg, the lake blue color of the malachite green reagent added to the test tube will not completely fade.

5. The rapid detection method according to claim 1, characterized in that, A is 100 mg / kg.

6. The rapid detection method according to claim 5, characterized in that, When a is 80%, the shrimp meat weighed quantitatively in step S1 and used to prepare the sample extract 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.

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

8. The rapid detection method according to claim 1, characterized in that, 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 is placed behind the test tube to increase the accuracy of the human eye in judging whether the color has completely faded.

9. A rapid detection method for sulfur dioxide in shrimp based on addition reaction decolorization technology, characterized in that, It includes the following steps: Step 1: Remove the shrimp head and shell, take 10-10.5g of shrimp meat into the food processor cup, add 50ml of pure water, homogenize on high speed for 20 seconds, let stand for 2 minutes, take 2mL of homogenized solution into a 2mL centrifuge tube, centrifuge for 2 minutes using a micro centrifuge, and use the supernatant directly as the sample extraction solution or filter it through a 0.45μm membrane 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; otherwise, it indicates that the sulfur dioxide content in the shrimp sample does not exceed the standard.

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