Pretreatment method for extracting saxitoxin from aquatic products

Abstract

The invention relates to the technical field of aquatic product treatment, and particularly discloses a pretreatment method for extracting saxitoxin in an aquatic product, which comprises the following steps: step 1, preparing a sample of fat-soluble saxitoxin in the aquatic product to be extracted, cleaning and homogenizing the prepared sample, and measuring the moisture content of the sample; according to the present invention, the cleaned aquatic product is placed in the high-speed homogenizer, such that the sample can become fragile, and can be easily crushed during the homogenization process so as to improve the toxin release efficiency; through homogenization treatment, the aquatic products can be crushed into uniform and fine homogenate, so that toxin molecules are more easily released from cell structures, and the extraction efficiency is further improved; the low-temperature environment can slow down shearing force and friction force borne by a sample, damage to the sample in the homogenization process is reduced, the integrity of toxin molecules is protected, and degradation or inactivation of the toxin molecules due to physical action is avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic product processing, and specifically relates to a pre-treatment method for extracting shellfish toxins in aquatic products. Background Art

[0002] Shellfish toxins are produced by algae and enter shellfish products through the food chain. When people eat these toxic shellfish, they are easily poisoned. Shellfish toxins can be divided into water-soluble shellfish toxins and fat-soluble shellfish toxins according to their solubility. Common fat-soluble shellfish toxins include okadaic acid toxin (OA), diatomic toxin (DTX), yesso scallop toxin (YTX), scallop toxin (PTX), protozoan acid toxin (AZA) and gymnodinium mikimoto toxin (GYM). In early studies, the main symptom of poisoning after accidental ingestion of toxins such as okadaic acid toxin (OA) and diatomic toxin (DTX) was diarrhea, so they were also called diarrheal shellfish toxins. Studies have shown that the elimination half-life of fat-soluble shellfish toxins in shellfish is as long as dozens of days or even months, and it is not easy to remove and reduce during the processing and consumption process, posing potential health hazards to consumers.

[0003] After searching, the patent application document with the prior art publication number CN117969726A proposes a pretreatment method for extracting shellfish toxins from aquatic products, comprising the following steps: (1) washing the aquatic products and then homogenizing them; (2) taking the homogenized sample in step (1) and adding it to a solvent for extraction, and taking the supernatant, wherein the solvent is an acetonitrile aqueous solution; (3) freezing the supernatant of step (2), then taking the supernatant, removing the solvent, and testing; the shellfish toxins are fat-soluble shellfish toxins such as okadaic acid, spiromeric imine compounds, ichthyophthirius toxin 1, ichthyophthirius toxin 2 and / or protothecin 1; the method has simple steps, uses a small amount of organic reagents, has a low detection cost, and is suitable for pretreatment of large quantities of samples.

[0004] Compared with the above documents, although the method steps are simple, the amount of organic reagents used is small, the detection cost is low, and it is suitable for pre-treatment of large quantities of samples, in the above documents, the fat-soluble shellfish toxins extracted from aquatic products are not homogenized. In the absence of a homogenization step, the cell structure in the aquatic products may remain intact, making it difficult for the toxin molecules to be released from the cells; and although a low temperature environment helps to protect the integrity of the toxin molecules, if homogenization is not performed, the toxin molecules may be difficult to be effectively extracted due to the obstruction of the cell wall. For this reason, a pre-treatment method for extracting shellfish toxins from aquatic products is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a pretreatment method for extracting shellfish toxins from aquatic products to solve the problem that fat-soluble shellfish toxins in aquatic products are not homogenized and lack the homogenization step, the cell structure in the aquatic products may remain intact, making it difficult for toxin molecules to be released from the cells; and although a low temperature environment helps to protect the integrity of the toxin molecules, if no homogenization is performed, the toxin molecules may be difficult to be effectively extracted due to the obstruction of the cell wall.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A pretreatment method for extracting shellfish toxins from aquatic products, comprising:

[0008] Step 1: Prepare the sample for extracting fat-soluble shellfish toxins from aquatic products, wash and homogenize the prepared sample, and measure the water content of the sample;

[0009] 1. Rinse the aquatic products (such as scallops, clams, etc.) with tap water to remove the surface sand and impurities, and then rinse with ultrapure water (resistivity ≥ 18MΩ·cm) at least three times in a flowing state, with each rinse time not less than 5 minutes, to ensure that the impurities on the sample surface and in the gaps are completely removed;

[0010] 2. Place the cleaned aquatic products in a high-speed homogenizer and homogenize them at a low temperature of -4 to 4 degrees. The homogenization time should be no less than 5 minutes, and the homogenization speed should be controlled between 12000rpm and 15000rpm to ensure that the samples are fully broken into a uniform and fine homogenate.

[0011] 3. After the homogenization treatment is completed, take a small amount of homogenate to measure the moisture content to ensure that the moisture content is within the predetermined range (such as ≤85%) to ensure the subsequent extraction efficiency;

[0012] Through the setting of the above technical scheme, the washed aquatic products are placed in a high-speed homogenizer and homogenized in a low-temperature environment of -4 to 4 degrees to extract fat-soluble shellfish toxins in the aquatic products, which has the following benefits:

[0013] Low temperature environment helps samples become more fragile and easier to be broken during homogenization, thereby improving the release efficiency of toxins; homogenization can break aquatic products into a uniform and fine homogenate, making it easier for toxin molecules to be released from the cell structure, thereby improving the extraction efficiency; low temperature environment can slow down the shear force and friction force on the sample, reduce the damage to the sample during the homogenization process, and help protect the integrity of toxin molecules to prevent them from being degraded or inactivated due to physical effects;

[0014] After low-temperature homogenization, the toxin molecules of the samples are more soluble in the extraction solvent, thereby optimizing the subsequent solvent extraction step, making the extraction efficiency higher and the interferences less; the uniform sample homogenate also helps the subsequent filtration, concentration and instrumental analysis steps, improving the efficiency and accuracy of the overall processing; the low-temperature environment can reduce the activity of microorganisms and reduce the risk of sample contamination during the homogenization process, thereby improving the safety of the extraction process.

[0015] Step 2: Select a solvent for extracting fat-soluble shellfish toxins from aquatic products, and perform extraction and centrifugation on the selected solvent;

[0016] 1. Select acetonitrile aqueous solution as the extraction solvent, wherein the volume fraction of acetonitrile is 20% to 25% to balance the extraction efficiency and the interference removal effect;

[0017] 2. Add the homogenate obtained in step 1 to the extraction solvent at a ratio of 1 g: 6 mL to 1 g: 8 mL of the sample to the solvent, and vortex mix for five to ten minutes to fully mix the sample and the solvent; then use an ultrasonic generator to perform ultrasonic extraction for 20 to 30 minutes at a temperature of 28 to 32 degrees, and control the ultrasonic frequency between 40 kHz and 60 kHz;

[0018] 3. Centrifuge at 4000 rpm to 5000 rpm for 10 to 15 minutes to separate the supernatant and solid residue, and take the supernatant for later use;

[0019] 4. To improve the extraction efficiency, the above extraction steps can be repeated 1 to 2 times. Centrifugation is required after each extraction, and the supernatant is combined;

[0020] Through the setting of the above technical scheme, extraction is the process of extracting target compounds from samples using solvents. When extracting fat-soluble shellfish toxins in aquatic products, the extraction process can ensure that the target toxins are effectively separated from the samples; by selecting suitable solvents and extraction conditions, the extraction efficiency of the target toxins can be maximized; during the extraction process, the target toxins will be selectively extracted by the solvent, while most of the impurities will remain in the sample, thereby reducing the interference of impurities on subsequent analysis.

[0021] When extracting fat-soluble shellfish toxins from aquatic products, the benefits of centrifugation are as follows:

[0022] Through centrifugation, the solid and liquid components in the sample can be effectively separated, which facilitates subsequent analysis and detection; centrifugation can remove suspended matter and sediment in the sample, further improving the purity of the sample; during the centrifugation process, the solvent in the sample will evaporate faster, thereby shortening the time for subsequent processing.

[0023] Step 3: placing the supernatant obtained by the extraction solvent in step 2 in a freezing device, thawing and drying it; freezing at a temperature of -40 degrees to -30 degrees for 16 hours to 24 hours, keeping the sample in a static state during the freezing process to avoid the formation of ice crystals that interfere with the extraction of toxins;

[0024] Thawing and drying: After freezing, slowly thaw the sample at room temperature (or use a freeze dryer for drying) to remove excess moisture and possible interferences; during the freeze-drying process, the vacuum degree and temperature must be controlled to avoid sample damage or toxin loss.

[0025] Step 4: removing and concentrating the solvent used in extracting fat-soluble shellfish toxins from aquatic products, and placing the supernatant after treatment in step 3 in a nitrogen drying device;

[0026] The supernatant after the treatment in step 3 is placed in a nitrogen drying device, and is dried with nitrogen at a temperature of 37 to 39 degrees until the solvent is completely evaporated to obtain a concentrated toxin extract; during the drying process, the nitrogen flow rate must be maintained at a moderate level (e.g., 20 L / min to 30 L / min) to avoid overheating or splashing of the sample;

[0027] After the drying is completed, a small amount of concentrated toxin extract is taken for weight measurement to ensure that the concentration efficiency is within the predetermined range (e.g. ≥80%);

[0028] Step 5: adding the concentrated toxin extract obtained in step 4 into methanol to remove solid impurities and particulate matter therein, and injecting the filtered sample into a high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS / MS) instrument for determination and analysis;

[0029] Dissolution and filtration: Add the concentrated toxin extract obtained in step (4) into methanol (the amount of methanol added is 3 to 4 times the volume of the concentrated toxin extract), and after sufficient dissolution, filter it using a microporous filter membrane with a pore size of 0.22 μm to remove possible solid impurities and particulate matter;

[0030] Instrumental analysis: The filtered sample is injected into a high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS / MS) instrument for measurement and analysis; during the measurement process, it is necessary to select appropriate chromatographic columns (such as C18 columns), mobile phases (such as methanol-water systems), detection wavelengths (such as ultraviolet detection wavelengths of 220nm to 254nm) and other parameters according to the properties of the target toxin; at the same time, method validation and quality control (such as linear range, sensitivity, precision, recovery rate, etc.) are also required to ensure the accuracy and reliability of the measurement results.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The cleaned aquatic products are placed in a high-speed homogenizer for homogenization at a low temperature of -4 to 4 degrees to extract fat-soluble shellfish toxins from the aquatic products, which has the following benefits:

[0033] Low temperature environment helps samples become more fragile and easier to be broken during homogenization, thereby improving the release efficiency of toxins; homogenization can break aquatic products into a uniform and fine homogenate, making it easier for toxin molecules to be released from the cell structure, thereby improving the extraction efficiency; low temperature environment can slow down the shear force and friction force on the sample, reduce the damage to the sample during the homogenization process, and help protect the integrity of toxin molecules to prevent them from being degraded or inactivated due to physical effects;

[0034] After low-temperature homogenization, the toxin molecules of the samples are more soluble in the extraction solvent, thereby optimizing the subsequent solvent extraction step, making the extraction efficiency higher and the interferences less; the uniform sample homogenate also helps the subsequent filtration, concentration and instrumental analysis steps, improving the efficiency and accuracy of the overall processing; the low-temperature environment can reduce the activity of microorganisms and reduce the risk of sample contamination during the homogenization process, thereby improving the safety of the extraction process. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a flow chart of the steps of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] like Figure 1 As shown, a pre-treatment method for extracting shellfish toxins from aquatic products comprises:

[0038] Step 1: Prepare the sample for extracting fat-soluble shellfish toxins from aquatic products, wash and homogenize the prepared sample, and measure the water content of the sample;

[0039] 1. Rinse the aquatic products (such as scallops, clams, etc.) with tap water to remove the surface sand and impurities, and then rinse with ultrapure water (resistivity ≥ 18MΩ·cm) at least three times in a flowing state, with each rinse time not less than 5 minutes, to ensure that the impurities on the sample surface and in the gaps are completely removed;

[0040] 2. Place the cleaned aquatic products in a high-speed homogenizer and homogenize them at a low temperature of -4 to 4 degrees. The homogenization time should be no less than 5 minutes, and the homogenization speed should be controlled between 12000rpm and 15000rpm to ensure that the samples are fully broken into a uniform and fine homogenate.

[0041] 3. After the homogenization treatment is completed, take a small amount of homogenate to measure the moisture content to ensure that the moisture content is within the predetermined range (such as ≤85%) to ensure the subsequent extraction efficiency;

[0042] Through the setting of the above technical scheme, the washed aquatic products are placed in a high-speed homogenizer and homogenized in a low-temperature environment of -4 to 4 degrees to extract fat-soluble shellfish toxins in the aquatic products, which has the following benefits:

[0043] Low temperature environment helps samples become more fragile and easier to be broken during homogenization, thereby improving the release efficiency of toxins; homogenization can break aquatic products into a uniform and fine homogenate, making it easier for toxin molecules to be released from the cell structure, thereby improving the extraction efficiency; low temperature environment can slow down the shear force and friction force on the sample, reduce the damage to the sample during the homogenization process, and help protect the integrity of toxin molecules to prevent them from being degraded or inactivated due to physical effects;

[0044] After low-temperature homogenization, the toxin molecules of the samples are more soluble in the extraction solvent, thereby optimizing the subsequent solvent extraction step, making the extraction efficiency higher and the interferences less; the uniform sample homogenate also helps the subsequent filtration, concentration and instrumental analysis steps, improving the efficiency and accuracy of the overall processing; the low-temperature environment can reduce the activity of microorganisms and reduce the risk of sample contamination during the homogenization process, thereby improving the safety of the extraction process.

[0045] Step 2: Select a solvent for extracting fat-soluble shellfish toxins from aquatic products, and perform extraction and centrifugation on the selected solvent;

[0046] 1. Select acetonitrile aqueous solution as the extraction solvent, wherein the volume fraction of acetonitrile is 20% to 25% to balance the extraction efficiency and the interference removal effect;

[0047] 2. Add the homogenate obtained in step 1 to the extraction solvent at a ratio of 1 g: 6 mL to 1 g: 8 mL of the sample to the solvent, and vortex mix for five to ten minutes to fully mix the sample and the solvent; then use an ultrasonic generator to perform ultrasonic extraction for 20 to 30 minutes at a temperature of 28 to 32 degrees, and control the ultrasonic frequency between 40 kHz and 60 kHz;

[0048] 3. Centrifuge at 4000 rpm to 5000 rpm for 10 to 15 minutes to separate the supernatant and solid residue, and take the supernatant for later use;

[0049] 4. To improve the extraction efficiency, the above extraction steps can be repeated 1 to 2 times. Centrifugation is required after each extraction, and the supernatant is combined;

[0050] Through the setting of the above technical scheme, extraction is the process of extracting target compounds from samples using solvents. When extracting fat-soluble shellfish toxins in aquatic products, the extraction process can ensure that the target toxins are effectively separated from the samples; by selecting suitable solvents and extraction conditions, the extraction efficiency of the target toxins can be maximized; during the extraction process, the target toxins will be selectively extracted by the solvent, while most of the impurities will remain in the sample, thereby reducing the interference of impurities on subsequent analysis.

[0051] When extracting fat-soluble shellfish toxins from aquatic products, the benefits of centrifugation are as follows:

[0052] Through centrifugation, the solid and liquid components in the sample can be effectively separated, which facilitates subsequent analysis and detection; centrifugation can remove suspended matter and sediment in the sample, further improving the purity of the sample; during the centrifugation process, the solvent in the sample will evaporate faster, thereby shortening the time for subsequent processing.

[0053] Step 3: placing the supernatant obtained by the extraction solvent in step 2 in a freezing device, thawing and drying it; freezing at a temperature of -40 degrees to -30 degrees for 16 hours to 24 hours, keeping the sample in a static state during the freezing process to avoid the formation of ice crystals that interfere with the extraction of toxins;

[0054] Thawing and drying: After freezing, slowly thaw the sample at room temperature (or use a freeze dryer for drying) to remove excess moisture and possible interferences; during the freeze-drying process, the vacuum degree and temperature must be controlled to avoid sample damage or toxin loss.

[0055] Step 4: removing and concentrating the solvent used in extracting fat-soluble shellfish toxins from aquatic products, and placing the supernatant after treatment in step 3 in a nitrogen drying device;

[0056] The supernatant after the treatment in step 3 is placed in a nitrogen drying device, and is dried with nitrogen at a temperature of 37 to 39 degrees until the solvent is completely evaporated to obtain a concentrated toxin extract; during the drying process, the nitrogen flow rate must be maintained at a moderate level (e.g., 20 L / min to 30 L / min) to avoid overheating or splashing of the sample;

[0057] After the drying is completed, a small amount of concentrated toxin extract is taken for weight measurement to ensure that the concentration efficiency is within the predetermined range (e.g. ≥80%);

[0058] Step 5: adding the concentrated toxin extract obtained in step 4 into methanol to remove solid impurities and particulate matter therein, and injecting the filtered sample into a high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS / MS) instrument for determination and analysis;

[0059] Dissolution and filtration: Add the concentrated toxin extract obtained in step (4) into methanol (the amount of methanol added is 3 to 4 times the volume of the concentrated toxin extract), and after sufficient dissolution, filter it using a microporous filter membrane with a pore size of 0.22 μm to remove possible solid impurities and particulate matter;

[0060] Instrumental analysis: The filtered sample is injected into a high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS / MS) instrument for measurement and analysis; during the measurement process, it is necessary to select appropriate chromatographic columns (such as C18 columns), mobile phases (such as methanol-water systems), detection wavelengths (such as ultraviolet detection wavelengths of 220nm to 254nm) and other parameters according to the properties of the target toxin; at the same time, method validation and quality control (such as linear range, sensitivity, precision, recovery rate, etc.) are also required to ensure the accuracy and reliability of the measurement results.

[0061] Furthermore, this design application is applied to extract fat-soluble shellfish toxins from aquatic products. The cleaned aquatic products are placed in a high-speed homogenizer and homogenized in a low-temperature environment of -4 to 4 degrees to extract fat-soluble shellfish toxins from aquatic products. The advantages are as follows:

[0062] A low temperature environment helps make samples more fragile and easier to break during the homogenization process, thereby improving the efficiency of toxin release; homogenization can break aquatic products into a uniform and fine homogenate, making it easier for toxin molecules to be released from the cell structure, thereby improving extraction efficiency; a low temperature environment can slow down the shear force and friction exerted on the sample, reduce damage to the sample during the homogenization process, and help protect the integrity of the toxin molecules and prevent them from being degraded or inactivated due to physical effects.

[0063] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pretreatment method for extracting shellfish toxins from aquatic products, characterized in that: include: Step 1: Prepare the sample for extracting fat-soluble shellfish toxins from aquatic products, wash and homogenize the prepared sample, and measure the water content of the sample; Step 2: Select a solvent for extracting fat-soluble shellfish toxins from aquatic products, and perform extraction and centrifugation on the selected solvent; Step 3: placing the supernatant obtained by the extraction solvent in step 2 in a freezing device, thawing and drying it; freezing at a temperature of -40 degrees to -30 degrees for 16 hours to 24 hours, keeping the sample in a static state during the freezing process to avoid the formation of ice crystals that interfere with the extraction of toxins; Step 4: removing and concentrating the solvent used in extracting fat-soluble shellfish toxins from aquatic products, and placing the supernatant after treatment in step 3 in a nitrogen drying device; Step 5: The concentrated toxin extract obtained in step 4 is added to methanol to remove solid impurities and particulate matter therein, and the filtered sample is injected into a high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS / MS) instrument for measurement and analysis.

2. A pretreatment method for extracting shellfish toxins from aquatic products according to claim 1, characterized in that: Ultrapure water is used to clean the samples of fat-soluble shellfish toxins in the aquatic products, and the resistivity of the ultrapure water is in the range of greater than or equal to 18 MΩ·cm. The number of cleaning times for cleaning the samples of fat-soluble shellfish toxins in the aquatic products is greater than or equal to three times, and the cleaning time is not less than five minutes.

3. A pretreatment method for extracting shellfish toxins from aquatic products according to claim 1, characterized in that: The machine for homogenizing the sample of fat-soluble shellfish toxins in the aquatic product is a high-speed homogenizer, the temperature range of the high-speed homogenizer is -4 degrees to 4 degrees, the homogenization time of the high-speed homogenizer is not less than five minutes, and the homogenization speed of the high-speed homogenizer is 12000rpm to 15000rpm.

4. A pretreatment method for extracting shellfish toxins from aquatic products according to claim 1, characterized in that: The extraction solvent used for the fat-soluble shellfish toxin sample in the aquatic product is an acetonitrile aqueous solution, the volume fraction of the acetonitrile aqueous solution is 20% to 25%, the ratio of the homogenate obtained in the step one to the solvent is 1g:6mL to 1g:8mL, the time for mixing the homogenate and the solvent is five minutes to ten minutes, the rotation speed of the solvent centrifugal treatment is 4000rpm to 5000rpm, and the number of times of solvent extraction is one to two times.

5. The pretreatment method for extracting shellfish toxins from aquatic products according to claim 1, characterized in that: The freezing temperature and freezing time of the supernatant obtained by the extraction solvent are respectively -40 degrees to -30 degrees and 16 hours to 24 hours.

6. A pretreatment method for extracting shellfish toxins from aquatic products according to claim 1, characterized in that: The temperature of the supernatant after the treatment in step 3 is placed in a nitrogen drying device at 37 to 39 degrees, and the flow rate of the nitrogen drying device for drying is 20 L / min to 30 L / min.

7. A pretreatment method for extracting shellfish toxins from aquatic products according to claim 1, characterized in that: The amount of methanol added is 3 to 4 times the volume of the concentrated toxin extract.

8. The pretreatment method for extracting shellfish toxins from aquatic products according to claim 1, characterized in that: The pore size of the sample filtration was 0.22 μm.

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