阅读说明：本技术 一种肉类食品中二噁英含量的检测方法 (Method for detecting content of dioxin in meat food ) 是由 张坤 于 2018-07-24 设计创作，主要内容包括：本发明涉及一种肉类食品中二噁英含量的检测方法,其特征在于包括以下步骤:取待检畜、禽类产品油脂样品；将样品溶于戊烷中,得到混合溶液；将混合溶液加热,依次使混合液中的戊烷和大部分油脂挥发,得到高含量的二噁英油脂；采用淋洗法将高含量的二噁英油脂净化提纯；将通过净化提纯得到的二噁英进行荧光分析,得到畜、禽类产品中二噁英含量的结果。本发明的优点：使用本发明所提供的检测方法,不用再将样品送入实验室进行检测,而且检测省时、快速、便捷、便宜、操作容易,给检测机构减少了大量的人力、物力、财力,节约了检测成本,提高了检测效率。(The invention relates to a method for detecting dioxin content in meat products, which is characterized by comprising the following steps of taking oil samples of livestock and poultry products to be detected; dissolving a sample in pentane to obtain a mixed solution; heating the mixed solution, and volatilizing pentane and most of grease in the mixed solution in sequence to obtain high-content dioxin grease; purifying and purifying high-content dioxin grease by adopting a leaching method; and performing fluorescence analysis on the dioxin obtained through purification to obtain the result of the dioxin content in livestock and poultry products. The invention has the advantages that: by using the detection method provided by the invention, a sample does not need to be sent into a laboratory for detection, the detection is time-saving, quick, convenient, cheap and easy to operate, a large amount of manpower, material resources and financial resources are reduced for a detection mechanism, the detection cost is saved, and the detection efficiency is improved.)

1. A method for detecting the content of dioxin in food is characterized by comprising the following steps:

a. taking solid fat of livestock and poultry products to be detected, heating and melting the solid fat into liquid grease, and sampling from the liquid grease;

b. completely dissolving the sampled liquid grease in pentane to obtain a mixed solution;

c. heating the mixed solution, keeping the heating temperature between the boiling point of pentane and the boiling point of grease, and keeping for a period of time to completely volatilize pentane in the mixed solution;

d. continuing to heat the solution without pentane at the temperature of 300 ℃ for 5-1Omin to volatilize most of the grease in the solution to obtain high-content dioxin grease;

e. purifying, namely filtering and purifying high-content dioxin grease by an elution method through an acidic silica gel column and an activated carbon column in sequence;

f. and performing fluorescence analysis on the dioxin obtained through purification to obtain the result of the dioxin content in livestock and poultry products.

2. The detection method according to claim 1, wherein the elution method comprises the steps of placing the acidic silica gel column and the activated carbon column in a pipeline to form a serial column, eluting the serial column with n-pentane, taking the acidic silica gel column out of the pipeline, eluting the activated carbon column with toluene alone, placing the acidic silica gel column back into the pipeline, and introducing high-content dioxin oil for elution and purification.

3. The detection method according to claim 1 or 2, wherein the acidic silica gel column is filled with glass fiber, 17 parts of anhydrous sodium sulfate and 30 parts of 33% sulfuric acid silica gel in sequence from bottom to top, and is rinsed by n-pentane after being filled.

4. The detection method according to claim 1 or 2, wherein the activated carbon column is sequentially filled with glass wool, 5 parts of anhydrous sodium sulfate, 3 parts of 1% X-CARB/kieselguhr and 10 parts of anhydrous sodium sulfate from bottom to top, and is sequentially rinsed with 1 part of acetone, 4 parts of toluene and 2 parts of n-pentane after being filled.

5. The detection method according to claim 1, wherein the fluorescence analysis comprises the steps of adding 400. mu.l of PMI1640 to the purified dioxin oil and fat, and concentrating until n-pentane is completely volatilized; adding culture solution containing dioxin luciferase cells and uniformly mixing; thirdly, uniformly adding the mixed solution to a 96-hole pore plate in which the cells H1L6.1c2 are placed, and culturing for 20-24 hours; and finally, irradiating the culture solution by infrared light, projecting the culture solution onto a fluorescence identification and analysis plate, extracting data by a fluorescence spectrophotometer, connecting the obtained data to a computer for data processing, obtaining a dioxin content result, and displaying the dioxin content result on a screen.

Technical Field

The invention relates to a food quality detection method, in particular to a detection method of dioxin content in meat food.

Background

With the development of society and the continuous improvement of living standard of people, food safety is more and more concerned by a plurality of national governments and people, the concept that people eat food as the day and eat food as the first is stronger and stronger in the mind of consumers, and particularly, the food safety events such as bird flu, clenbuterol, artificial honey, dioxin and the like which occur in recent years cause people to pay more attention to the food safety problem. With the development of times and economy, people do not worry about the problem of food shortage, the requirement on food quality safety is gradually higher and higher, especially on the food safety of poultry meat, and the demand of people on poultry meat products begins to change from quantity type to quality safety type.

Dioxins widely and persistently exist in the environment and are concentrated in pollutants of a food chain, and main sources of the dioxins are volcanic eruptions, forest fires, chemical production, waste combustion, metal smelting, paper making and the like. Dioxin-like substances exist not only in soil, sediments and water, but also in wild animals, livestock and human tissues. The dioxin substances in the food are not easily degraded biologically and chemically, so the food has biological enrichment and biological amplification, can retain the dioxin substances in organisms and in the environment for a long time, has lipophilicity, and after entering the bodies, people and animals mainly store fat mainly contact the dioxin substances through food, wherein most of the dioxin substances come from animal food! The device can quickly detect the content of dioxin in poultry meat, improve the safety quality of food and enable people to eat green pollution-free food.

At present, only a few laboratories in China have the capability of detecting the content of dioxin in meat products, and the detection cost is high, the operation method is complicated, the time consumption is long, and the requirement for detecting the dioxin can not be met. Therefore, a method capable of rapidly detecting the content of dioxin in meat products is urgently needed to achieve the purpose of rapidly detecting samples on site, and an ideal technical scheme is not found through wide retrieval.

Disclosure of Invention

The invention aims to solve the defects of high price, complicated operation method, long time consumption and the like of the detection of the content of the dioxin in meat food at the present stage, and provides a method for detecting the content of the dioxin in the food.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting the content of dioxin in food is characterized by comprising the following steps:

a. taking solid fat of livestock and poultry products to be detected, heating and melting the solid fat into liquid grease, and sampling from the liquid grease;

b. completely dissolving the sampled liquid grease in pentane to obtain a mixed solution;

c. heating the mixed solution, keeping the heating temperature between the boiling point of pentane and the boiling point of grease, and keeping for a period of time to completely volatilize pentane in the mixed solution;

d. then continuing to heat the solution without pentane, wherein the heating temperature is preferably 300 ℃, the heat preservation time is preferably 5-10min,

volatilizing most of grease in the solution to obtain high-content dioxin grease;

e. purifying, namely filtering and purifying high-content dioxin grease by an elution method through an acidic silica gel column and an activated carbon column in sequence;

f. and performing fluorescence analysis on the dioxin obtained through purification to obtain the result of the dioxin content in livestock and poultry products.

On the basis of the technical scheme, the following further technical scheme can be provided:

the leaching method comprises the following steps of placing the acidic silica gel column and the activated carbon column in a pipeline to form a serial column, leaching the serial column by using n-pentane, taking the acidic silica gel column out of the pipeline, leaching the activated carbon column by using toluene, placing the acidic silica gel column back to the pipeline, and introducing high-content dioxin grease for leaching and purification.

The acidic silica gel column is sequentially filled with glass fiber, 17 parts of anhydrous sodium sulfate and 30 parts of 33% sulfuric acid silica gel from bottom to top, and is leached by n-pentane after being filled.

The activated carbon column is sequentially filled with glass wool, 5 parts of anhydrous sodium sulfate, 3 parts of 1% X-CARB/kieselguhr and 10 parts of anhydrous sodium sulfate from bottom to top, and is sequentially leached by 1 part of acetone, 4 parts of toluene and 2 parts of n-pentane after being filled.

The fluorescence analysis comprises the following steps of firstly adding 400 mu 1PMI1640 to purified dioxin grease and concentrating until n-pentane is completely volatilized; adding culture solution containing dioxin luciferase cells and uniformly mixing; thirdly, uniformly adding the mixed solution to a 96-hole pore plate with cells H116.1c2 placed therein, and culturing for 20-24 hours; and finally, irradiating the culture solution by infrared light, projecting the culture solution onto a fluorescence identification and analysis plate, extracting data by a fluorescence spectrophotometer, connecting the obtained data to a computer for data processing, obtaining a dioxin content result, and displaying the dioxin content result on a screen.

In meat products such as livestock and poultry, because the content of dioxin in fat is higher than that of other tissues, when the content of dioxin in meat products is detected, grease of the meat products is taken for quality detection.

The detection method mainly comprises the steps of sample collection, dioxin substance extraction, purification, drying, cell treatment, cell lysis, fluorescence spectrophotometer analysis and data processing, the specific operation method is different due to different materials, the main difference between different samples is in the operation of extraction and purification of the dioxin pollutants contained in the samples, and the detection method mainly detects the content of the dioxin in meat foods such as livestock, poultry and the like.

The method has the advantages that the detection method provided by the invention is used, a sample does not need to be sent into a laboratory for detection, the detection is time-saving, quick, convenient and fast, cheap and easy to operate, a large amount of manpower, material resources and financial resources are reduced for a detection mechanism, the detection cost is saved, and the detection efficiency is improved.

Detailed Description

The invention provides a method for detecting the content of dioxin in meat products, which is characterized by comprising the following steps:

heating to obtain high-content dioxin grease, which comprises the following steps:

a. taking solid fat of meat food such as livestock and poultry to be detected, heating and melting into liquid oil, and taking out 50g of the obtained liquid oil as a sample.

b. Firstly, filling pentane into a heating device for concentration, adding 50g of obtained liquid grease into the heating device for concentration, wherein the pentane and the liquid grease are mixed according to the proportion of 10:1, and shaking up the mixed solution after mixing to ensure that the liquid grease is completely dissolved in the pentane to obtain the mixed solution.

c. And the heating device is used for heating the mixed solution, the heating temperature is kept between the boiling point of pentane and the boiling point of grease, and is kept for a period of time, wherein the heating temperature is preferably 40 ℃, and the heat preservation time is preferably 5-10min, so that all pentane in the mixed solution is volatilized.

d. And continuously heating the mixed solution without pentane, wherein the heating temperature is preferably 300 ℃, and the heat preservation time is preferably 5-10min, so that most of the grease in the solution is volatilized, and the high-content dioxin grease is obtained.

Secondly, purifying and purifying the high-content dioxin grease obtained in the first step by adopting a leaching method, wherein the method comprises the following steps:

e. and purifying the high-content dioxin-containing grease obtained in the first step by a 1Oml acid silica gel column and a 5ml activated carbon column which are connected in series.

The 10ml acidic silica gel column is prepared by sequentially filling glass fiber, 107g of anhydrous sodium sulfate and 3.0g of 33% sulfuric acid silica gel from bottom to top, and leaching with 30ml of n-pentane after filling.

The 5ml activated carbon column is prepared by sequentially filling glass wool, 0.5g of anhydrous sodium sulfate, 0.3g of 1% X-CARB/tylophora soil and 1.0g of anhydrous sodium sulfate from bottom to top, and then sequentially eluting with 5ml of acetone, 20ml of toluene and 1Oml of n-pentane after filling.

After preparing a 10ml acidic silica gel column and a 5ml activated carbon column, connecting the acidic silica gel column and the 5ml activated carbon column in series in a pipeline to form a series column, firstly leaching the series column by using n-pentane, and removing impurities in the two columns; taking the acidic silica gel column out of the pipeline, leaching the activated carbon column by using toluene to separate PCDD/Fs, and leaching the activated carbon column by using toluene to increase the passing rate of dioxin during filtering; and finally, putting the acidic silica gel column back to the pipeline, introducing high-content dioxin grease, carrying out rotary evaporation concentration on the eluent, and then using n-pentane to fix the volume of the concentrated solution to 4ml of sample.

Thirdly, the dioxin obtained by purification in the second step is subjected to fluorescence analysis

f. Taking 1ml of the sample with the volume constant of 4ml out, placing the sample into a test tube, adding 400 mu 1PMI1640 into the test tube, concentrating, adding 400 mu 1 culture solution of the dioxin luciferase cell after n-pentane in the sample is completely volatilized, and mixing uniformly to obtain a mixed solution, wherein the dioxin incinerator luciferase cell is 1% of penilin-streptomycin + 8% of FBS in the prior art.

g. 190U1 of the mixed solution is added into each well of a 96-well plate on which the cells H116.1c2 are placed for culturing for 20-24 h, wherein the culturing temperature is set at 37 ℃, and the culture solution contains 5% of C02 by volume fraction.

i. After the culture solution in each hole is collected into the test tube after the culture, the test tube is irradiated by infrared light, light is projected to a fluorescence identification analysis plate after the irradiation, data is extracted by a fluorescence spectrophotometer and is connected to a computer for data processing, a dioxin content result is obtained and displayed on a screen, wherein, a chemiluminescence enzyme-labeling instrument is adopted to measure the luminosity, and the concentration of P ⑶ D is calculated by the luminosity.

Principle of detection

The principle of detecting dioxin substances in food by adopting the CA1UX method is based on the principle of biological action generated after the dioxin substances enter biological organisms. After Dioxin enters cells, the Dioxin is firstly combined with aromatic in cytoplasm through a receptor AhR to form a Dioxin-AhR combination, the combination is transferred into a cell nucleus, the aromatic is separated from 2 heat shock protein subunit Hsp90, 11 p23 and 11 XAP2 protein ligands combined with the aromatic through the receptor in the cell nucleus and is gathered in the cell nucleus, then the Dioxin-AhR mixture is combined with aromatic hydrocarbon receptor nuclear translocator protein in the cell nucleus to form Dioxin-AhR-ARNT protein dimer with high binding force with DNA, and the dimer is specifically combined with Dioxin reaction element DRE in an upstream enhancer of Dioxin reaction genes, so that the transcription expression of the target genes is started, and a series of biochemical changes and toxic effects related to the expression of the genes are caused.

The CA1UX method is a biological analysis method for detecting dioxin-like substances in food by using recombinant cells containing luciferase reporter genes. When the method is used, firstly, a recombinant plasmid containing a luciferase reporter gene is constructed, then the recombinant plasmid is transfected into animal cells to prepare a stably transfected recombinant cell line, and finally, the recombinant cells are used for detecting dioxin substances in food extracts. Theoretically, many different cell lines can be used for the CA1UX assay, as long as the cells contain a functionally sound aromatic hydrocarbon receptor signaling system, but in practice the response of different cell lines to various dioxins will vary. At present, cell strains commonly used include rat hepatoma cell strains, mouse hepatoma cell strains and human hepatoma cell strains.

When constructing plasmid, different numbers of dioxin reaction elements DREs are inserted into the plasmid containing luciferase reporter gene

The region regulated by MMTV promoter in the plasmid is made into recombinant plasmid, and then the recombinant plasmid is transferred into animal cell to make into stable transfected cell line. When the recombinant cells are treated by food extracts containing dioxin-like substances in detection, aromatic hydrocarbon receptors in the cells are activated after the cells are contacted with the dioxin-like substances, and further, luciferase reporter genes are expressed. The fluorescence generated by the expression of the luciferase gene can be detected by a fluorescence spectrophotometer. Since the amount of fluorescence of the luciferase expressed by the recombinant cells after contacting the dioxin-like substances is in direct proportion to the content of the dioxin-like substances within a certain range, the amount of the dioxin-like compounds can be calculated by detecting the amount of fluorescence.

In addition, transient transfection of cells with recombinant plasmids can be used to detect dioxins in food extracts, which saves the time required to produce stably transfected cells. However, the reproducibility of the transient transfection assay is not as good as that of the stably transfected cell line, and the gene transiently transferred into the cells is usually lost after 72 hours, so that the assay is only suitable for short-term studies.