A kind of extraction method for removing histamine

Abstract

The invention discloses an extraction method for removing histamine, and relates to the technical field of histamine extraction. The extraction method for removing histamine has the following specific operation steps: S1, sample preparation: a certain amount of fish meat, trichloroacetic acid solution, sodium hydroxide solution, n-pentanol, hydrochloric acid, saturated borax solution, distilled water, filter membrane and modified activated carbon; S2, accurately weighing 10g of a fish meat sample after pounding and mixing, putting the fish meat into a 50ml small beaker, using 20ml of 40%-80% ethanol solution as an extractant, treating the sample at a temperature of 20-60℃ for 30-70min, filtering, and adding 20ml of trichloroacetic acid (10%) as an extractant. The extraction method for removing histamine uses the ethanol solution as the extractant, and according to the property of histamine, histamine is easily soluble in the ethanol solution, so that the extraction method of histamine is simple and fast, greatly shortens the extraction time, reduces the cumbersome operation process, and satisfies the qualitative and semi-quantitative determination of histamine by a colloidal gold method.

Description

Technical Field

[0001] The invention relates to the technical field of histamine extraction, in particular to an extraction method for removing histamine. Background Art

[0002] Histamine (HA) is chemically named 4(5)-(2-aminoethyl)imidazole, which is a biogenic amine. When histidine is contaminated by bacteria rich in histidine decarboxylase (such as Proteus morganii, Achromobacter histamineus, Escherichia coli, Streptococcus, Staphylococcus, etc.), the free histidine in fish meat can undergo amino acid decarboxylation reaction to produce histamine. As an endogenous active substance in the human body, histamine has important physiological functions in biological cells. However, when the human body ingests excessive histamine, it will cause food poisoning, and in severe cases, it will endanger life.

[0003] At present, some existing methods for the extraction and determination of histamine include the biological method of AOAC (954.04), chemical method, fluorescence method and national standard method. However, these methods require special instruments and equipment, use more reagents and instruments, are cumbersome and time-consuming, and are not easy to meet the requirements of on-site rapid extraction and detection, thus slowing down the extraction and determination of histamine and prolonging the extraction process. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide an extraction method for removing histamine, which can solve the problems that some existing methods for extracting histamine are complicated and time-consuming and difficult to meet the requirements of rapid on-site extraction and detection.

[0005] To achieve the above object, the present invention provides the following technical solution: an extraction method for removing histamine, the specific operation steps are as follows:

[0006] S1. Sample preparation: some fish meat, trichloroacetic acid solution, sodium hydroxide solution, n-pentanol, hydrochloric acid, saturated borax solution, distilled water, filter membrane, modified activated carbon;

[0007] S2. Accurately weigh 10 g of the mashed and mixed fish sample, put the fish into a 50 ml beaker, use 20 ml of 40%-80% ethanol solution as an extractant, treat the sample at a temperature of 20°C-60°C and 30-70 min, filter, and add 20 ml of trichloroacetic acid (10%) as an extractant;

[0008] S3. Add 3.0 ml of n-pentanol, shake for 5 minutes, extract three times, combine the filtrate and make up to 10.0 ml with n-pentanol, take 1.0 ml of n-pentanol extract in a separatory funnel and extract three times with 1 mol / L hydrochloric acid, combine the hydrochloric acid extracts and make up to 10.0 ml with hydrochloric acid solution.

[0009] S4, add 1ml hydrochloric acid solution, add 3ml 15% sodium carbonate solution and 3ml azo reagent to each standard tube, add water to 10.0ml, and mix well;

[0010] S5. Take 10.0 mL of the sample to be tested and place it in a beaker. Add 5 mL of saturated borax solution, stir evenly and drain into a 500 mL beaker. Wash the beaker 3 to 4 times with distilled water. Add 5 mL of 220 g / L zinc acetate solution and stir for 8 to 12 minutes. Take 10 mL of the supernatant and centrifuge it in a centrifuge for 10 minutes. Centrifuge.

[0011] S6. The supernatant after centrifugation is filtered through a 0.22 μm filter membrane to obtain a finished histamine solution.

[0012] Preferably, in S1, the fresh fish meat is washed and cut into pieces, and subjected to low-temperature preservation treatment, using an automatic fish washing machine with a water temperature of 4-10° C. and automatically spraying tap water on a conveyor belt.

[0013] Preferably, the process simultaneously selects out miscellaneous fish and debris, washes and removes inedible parts of the fish, and puts the fish into a clean container for mashing and mixing.

[0014] Preferably, the S2 is added with 20 mL of 10% trichloroacetic acid solution and soaked for 2 h to 3 h, and shaken for 2 min to mix.

[0015] Preferably, the solution is allowed to stand and mix until it falls onto filter paper through absorbent cotton, and then falls into the beaker through the filter paper.

[0016] Preferably, the S2 is extracted at room temperature for 2h-3h, filtered, 1.0 ml of the filtrate is taken into a separatory funnel, and the pH is adjusted to 9-10 with sodium hydroxide solution (250g / L).

[0017] Preferably, after the ultrasonication in S5 is completed, the mixture is cooled and allowed to stand at room temperature, and 10 mL of the supernatant after ultrasonication is taken and centrifuged in a centrifuge for 10 minutes at a speed of 8000 r / min.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The extraction method for removing histamine uses ethanol solution as an extractant and, based on the property of histamine, histamine is easily soluble in ethanol solution, so that the method for extracting histamine is simple and quick, greatly shortening the extraction time, reducing the cumbersome operation process, and meeting the qualitative and semi-quantitative determination of histamine by colloidal gold method.

[0020] (2) The method for removing histamine from the extraction process uses modified activated carbon to decolorize the solution, making the color change of the solution more obvious in the subsequent identification and detection, so that the staff can more conveniently detect the finished product in the later stage.

[0021] (3) The extraction method for removing histamine, through the cooperation between filter paper and absorbent cotton, utilizes the characteristics of absorbent cotton itself to increase the filtration speed, so that the filtration speed of the solution is improved to a certain extent, thereby accelerating the progress.

[0022] (4) The extraction method for removing histamine uses a sodium hydroxide solution (250 g / L) to adjust the pH of the solution to 9-10 to make the solution alkaline and eliminate the hydrochloric acid in the solution, thereby making the extraction of histamine smoother. DETAILED DESCRIPTION

[0023] In the description of the present invention, "greater than", "less than", "exceed" etc. are understood as not including the number itself, and "above", "below", "within" etc. are understood as including the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0024] In the description of the present invention, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention based on the specific content of the technical solution.

[0025] The present invention provides a technical solution: an extraction method for removing histamine, and the specific operation steps are as follows:

[0026] S1. Sample preparation: some fish meat, trichloroacetic acid solution, sodium hydroxide solution, n-pentanol, hydrochloric acid, saturated borax solution, distilled water, filter membrane, modified activated carbon;

[0027] S2. Accurately weigh 10 g of the mashed and mixed fish sample, put the fish into a 50 ml beaker, use 20 ml of 40% ethanol solution as an extractant, treat the sample at 20°C for 30 min, filter, and add 20 ml of trichloroacetic acid (10%) as an extractant;

[0028] By using ethanol solution as the extractant and based on the properties of histamine, which is easily soluble in ethanol solution, the method for extracting histamine is simple and quick, greatly shortening the extraction time, reducing the cumbersome operation process, and meeting the qualitative and semi-quantitative determination of histamine by colloidal gold method.

[0029] Further, the mixture was extracted at room temperature for 2 h to 3 h, filtered, 1.0 ml of the filtrate was placed in a separatory funnel, and the pH was adjusted to 9-10 with sodium hydroxide solution (250 g / L);

[0030] S3, add 3.0 ml of n-pentanol, shake for 5 min, extract three times, combine the filtrate and dilute to 10.0 ml with n-pentanol, take 1.0 ml of n-pentanol extract in a separatory funnel and extract three times with 1 mol / L hydrochloric acid, combine the hydrochloric acid extracts and dilute to 10.0 ml with hydrochloric acid solution;

[0031] S4, add 1ml hydrochloric acid solution, add 3ml 15% sodium carbonate solution and 3ml azo reagent to each standard tube, add water to 10.0ml, and mix well;

[0032] S5, take 10.0mL of the sample to be tested and place it in a beaker, add 5mL of saturated borax solution, stir evenly and drain into a 500mL beaker, wash the beaker 3 times with distilled water, add 5mL of 220g / L zinc acetate solution, stir for 8min, cool and stand to room temperature after ultrasonication, take 10mL of the supernatant after ultrasonication and centrifuge in a centrifuge for 10min, and centrifuge at a speed of 8000r / min;

[0033] S6. The supernatant after centrifugation is filtered through a 0.22 μm filter membrane to obtain a finished histamine solution.

[0034] Furthermore, the sample is decolorized by adding modified activated carbon to the sample solution prepared in S5, vortex mixing for 1 minute, centrifuging, taking out the supernatant, and preparing the sample measurement solution. The decolorization treatment of the solution by the modified activated carbon makes the color change of the solution more obvious in the later identification and detection, so that the staff can more conveniently test the finished product later.

[0035] Embodiment 2:

[0036] Based on Example 1:

[0037] S1. Sample preparation: some fish meat, trichloroacetic acid solution, sodium hydroxide solution, n-pentanol, hydrochloric acid, saturated borax solution, distilled water, filter membrane, modified activated carbon;

[0038] Furthermore, the inedible parts of the fish are removed, and the fish is placed in a clean container to be crushed and mixed.

[0039] S2. Accurately weigh 10 g of the mashed and mixed fish sample, put the fish into a 50 ml beaker, use 20 ml of 60% ethanol solution as an extractant, treat the sample at 60°C for 30 min, filter, and add 20 ml of trichloroacetic acid (10%) as an extractant;

[0040] Furthermore, 20 mL of 10% trichloroacetic acid solution was added and soaked for 3 h, and shaken for 2 min to mix well, so that the mixed solution was allowed to fall onto the filter paper through the absorbent cotton, and then fall into the beaker through the filter paper, so that the filter paper and the absorbent cotton could cooperate with each other and the characteristics of the absorbent cotton could be used to increase the filtration speed, so that the filtration speed of the solution was improved to a certain extent, thereby speeding up the progress.

[0041] By using ethanol solution as the extractant and based on the properties of histamine, which is easily soluble in ethanol solution, the method for extracting histamine is simple and quick, greatly shortening the extraction time, reducing the cumbersome operation process, and meeting the qualitative and semi-quantitative determination of histamine by colloidal gold method.

[0042] S3, add 3.0 ml of n-pentanol, shake for 5 min, extract three times, combine the filtrate and dilute to 10.0 ml with n-pentanol, take 1.0 ml of n-pentanol extract in a separatory funnel and extract three times with 1 mol / L hydrochloric acid, combine the hydrochloric acid extracts and dilute to 10.0 ml with hydrochloric acid solution, and set aside;

[0043] S4, add 1ml hydrochloric acid solution, add 3ml 15% sodium carbonate solution and 3ml azo reagent to each standard tube, add water to 10.0ml, and mix well;

[0044] S5, take 10.0mL of the sample to be tested and place it in a beaker, add 5mL of saturated borax solution, stir evenly and drain into a 500mL beaker, wash the beaker 4 times with distilled water, add 5mL of 220g / L zinc acetate solution, stir for 12min, cool and stand to room temperature after ultrasonication, take 10mL of the supernatant after ultrasonication and centrifuge in a centrifuge for 10min, and centrifuge at a speed of 8000r / min;

[0045] S6. The supernatant after centrifugation is filtered through a 0.22 μm filter membrane to obtain a finished histamine solution.

[0046] Furthermore, the sample is decolorized by adding modified activated carbon to the sample solution prepared in S5, vortex mixing for 1 to 2 minutes, centrifuging, taking out the supernatant, and preparing the sample measurement solution. The solution is decolorized by the modified activated carbon to make the color change of the solution more obvious in the later identification and detection, so that the staff can more conveniently test the finished product later.

[0047] Embodiment three:

[0048] Based on the first and second embodiments:

[0049] S1. Sample preparation: some fish meat, trichloroacetic acid solution, sodium hydroxide solution, n-pentanol, hydrochloric acid, saturated borax solution, distilled water, filter membrane, modified activated carbon;

[0050] Furthermore, the inedible parts of the fish are removed, and the fish is placed in a clean container to be crushed and mixed.

[0051] S2. Accurately weigh 10 g of the mashed and mixed fish sample, put the fish into a 50 ml beaker, use 20 ml of 80% ethanol solution as an extractant, treat the sample at 60°C for 70 min, filter, and add 20 ml of trichloroacetic acid (10%) as an extractant;

[0052] Furthermore, 20 mL of 10% trichloroacetic acid solution was added and soaked for 3 h, and shaken for 2 min to mix well, so that the mixed solution was allowed to fall onto the filter paper through the absorbent cotton, and then fall into the beaker through the filter paper, so that the filter paper and the absorbent cotton could cooperate with each other and the characteristics of the absorbent cotton could be used to increase the filtration speed, so that the filtration speed of the solution was improved to a certain extent, thereby speeding up the progress.

[0053] By using ethanol solution as the extractant and based on the properties of histamine, which is easily soluble in ethanol solution, the method for extracting histamine is simple and quick, greatly shortening the extraction time, reducing the cumbersome operation process, and meeting the qualitative and semi-quantitative determination of histamine by colloidal gold method.

[0054] Further, the mixture was extracted at room temperature for 3 h, filtered, 1.0 ml of the filtrate was placed in a separatory funnel, and the pH was adjusted to 10 with sodium hydroxide solution (250 g / L);

[0055] S3, add 3.0 ml of n-pentanol, shake for 5 min, extract three times, combine the filtrate and dilute to 10.0 ml with n-pentanol, take 1.0 ml of n-pentanol extract in a separatory funnel and extract three times with 1 mol / L hydrochloric acid, combine the hydrochloric acid extracts and dilute to 10.0 ml with hydrochloric acid solution, and set aside;

[0056] S4, add 1ml hydrochloric acid solution, add 3ml 15% sodium carbonate solution and 3ml azo reagent to each standard tube, add water to 10.0ml, and mix well;

[0057] S5, take 10.0mL of the sample to be tested and place it in a beaker, add 5mL of saturated borax solution, stir evenly and drain into a 500mL beaker, wash the beaker 4 times with distilled water, add 5mL of 220g / L zinc acetate solution, stir for 12min, cool and stand to room temperature after ultrasonication, take 10mL of the supernatant after ultrasonication and centrifuge in a centrifuge for 10min, and centrifuge at a speed of 8000r / min;

[0058] S6. The supernatant after centrifugation is filtered through a 0.22 μm filter membrane to obtain a finished histamine solution.

[0059] Furthermore, the sample is decolorized: modified activated carbon is added to the sample solution prepared in S5, vortex-mixed for 2 minutes, centrifuged, and the supernatant is taken out to prepare the sample measurement solution. The solution is decolorized by the modified activated carbon to make the color change of the solution more obvious in the later identification and detection, so that the staff can more conveniently test the finished product later.

Claims

1. An extraction method for removing histamine, characterized in that: The specific steps are as follows: S1. Sample preparation: some fish meat, trichloroacetic acid solution, sodium hydroxide solution, n-pentanol, hydrochloric acid, saturated borax solution, distilled water, filter membrane, modified activated carbon; S2. Accurately weigh 10 g of the mashed and mixed fish sample, put the fish into a 50 ml beaker, use 20 ml of 40%-80% ethanol solution as an extractant, treat the sample at a temperature of 20°C-60°C and 30-70 min, filter, and add 20 ml of trichloroacetic acid (10%) as an extractant; S3, add 3.0 ml of n-pentanol, shake for 5 min, extract three times, combine the filtrate and dilute to 10.0 ml with n-pentanol, take 1.0 ml of n-pentanol extract in a separatory funnel and extract three times with 1 mol / L hydrochloric acid, combine the hydrochloric acid extracts and dilute to 10.0 ml with hydrochloric acid solution; S4, add 1ml hydrochloric acid solution, add 3ml 15% sodium carbonate solution and 3ml azo reagent to each standard tube, add water to 10.0ml, and mix well; S5. Take 10.0 mL of the sample to be tested and place it in a beaker. Add 5 mL of saturated borax solution, stir evenly and drain into a 500 mL beaker. Wash the beaker 3 to 4 times with distilled water. Add 5 mL of 220 g / L zinc acetate solution and stir for 8 to 12 minutes. Take 10 mL of the supernatant and centrifuge it in a centrifuge for 10 minutes. Centrifuge. S6, filtering the supernatant after centrifugation through a 0.22 μm filter membrane to obtain a finished histamine solution; S7 and S1 clean the fresh fish meat, cut it into pieces, and keep it fresh at low temperature. Use an automatic fish washing machine with a water temperature of 4-10°C and automatically spray tap water on the conveyor belt; S8, picking out miscellaneous fish and debris, cleaning and removing inedible parts of the fish, and putting the fish into a clean container to mash and mix; Add 20 mL 10% trichloroacetic acid solution to S9 and S2 and soak for 2 h to 3 h, then shake for 2 min to mix. S10, allowing the mixed solution to fall onto filter paper through absorbent cotton, and then fall into the beaker through the filter paper.

2. A method for removing histamine according to claim 1, characterized in that: The S2 was extracted at room temperature for 2 h-3 h, filtered, 1.0 ml of the filtrate was put into a separatory funnel, and the pH was adjusted to 9-10 with sodium hydroxide solution (250 g / L).

3. A method for removing histamine according to claim 2, characterized in that: After the ultrasound in S5 is completed, the mixture is cooled and allowed to stand at room temperature. 10 mL of the supernatant after ultrasound is taken and centrifuged in a centrifuge for 10 minutes at a speed of 8000 r / min.