TWI750767B - Method for rapid detection of dioxin and/or PCBs in samples - Google Patents

Abstract

The present invention provides a method for rapid detection of dioxin and/or polychlorinated biphenyls in a sample, which can be used for rapid detection of dioxin, polychlorinated biphenyls or the congeners of the above compounds in wet-based samples or dry-based samples. Said, the method disclosed in the present invention is to mix a sample to be tested with a superabsorbent dispersant to obtain a processed sample, which can be directly subjected to procedures such as high-pressure extraction, automatic purification and mass spectrometry analysis; The disclosed method can simplify the conventional detection method, and directly extract dioxin and polychlorinated biphenyl congeners in the sample by means of high temperature solvent infiltration and pressure, so as to achieve the effect of greatly shortening the detection time.

Description

Method for rapid detection of dioxin and/or polychlorinated biphenyls in a sample

The present invention relates to a method for testing and processing toxic substances, particularly a method for rapidly testing dioxin and/or polychlorinated biphenyls in wet samples.

According to statistics, dioxins are by-products produced in industrial manufacturing processes or human activities. At present, more than 400 kinds of dioxin-related compounds have been analyzed. The vast majority of dioxins in the human body come from food. The reason is that when dioxins are produced, they will be scattered with the wind and pollute water or soil, causing pollution of agriculture, forestry and animal husbandry, and then causing dioxins to accumulate in fish, meat and eggs. Dioxin compounds are exposed to or accumulated in the human body, resulting in a variety of diseases, such as skin damage, fatigue, muscle soreness, difficulty breathing, liver damage, cancer, immune system diseases, etc., and even destroy human endocrine and cause gene mutation. , giving birth to deformed children, infant developmental delay, etc. Because the molecular structure of dioxin furan is flat, non-polar and has a very long half-life, it is difficult for the adipose tissue accumulated in the organism to be excreted from the body, so it is called "the poison of the century", and because dioxin compounds are difficult to degrade in the environment, With the long-distance transmission of atmospheric water flow without borders, countries have continued to invest human resources in environmental monitoring since the beginning of the last century.

In addition to controlling the pollution of dioxins in the environment, the toxic equivalents and levels accumulated in the human body through the food chain have been actively evaluated. In 1998, the World Health Organization (WHO) issued the first report on the toxic equivalents and maximum daily intake (TDI) of 17 dioxin furans and 12 dioxin-like polychlorinated biphenyls that must be paid attention to; the European Union continued in 2002. Announce the maximum allowable limits of various types of food and feed; accordingly, the Food and Drug Administration of the Ministry of Health and Welfare of Taiwan has also announced the inspection methods for dioxins in related foods. Since dioxins are mostly accumulated in oils and fats, the inspection process is necessary. The step is to extract the fat in the sample to the greatest extent, and then carry out the subsequent steps of acid washing, purification, and machine loading, and the current test method for dioxins in Taiwan is to use Soxhlet extraction (see December 1998, Republic of China). Announcement No. 0980113788 on the 17th, announcement method M801), liquid-liquid phase extraction (announcement method CNS14758) and the above two methods still have their shortcomings in the inspection schedule:

Soxhlet extraction samples must be vacuum freeze-dried to remove water to near dryness, so that the solvent can effectively penetrate into the internal tissue of the sample, and the time required for freeze-drying varies depending on the weight and water content of the sample.

Generally speaking, with a sample weight of 40 grams required for dioxin analysis, fresh milk and aquatic products with a moisture content higher than 80% need to be drained for 36 hours, and forage grass and egg yolks with a moisture content higher than 60% need to be drained for more than 24 hours. It takes more than 2 hours to test the sample: 18 to 24 hours with traditional Soxhlet extraction; 6 hours with rapid thermal Soxhlet extraction (such as Soxtech); Solvent extraction (eg Buchi instrument) for detection takes about 2 hours.

If the sample is subjected to the oil extraction procedure, it will take at least 38 hours to obtain the oil and fat extract that can be used for purification and analysis. For example, fresh milk or aquatic products will take at least 38 hours. 26 hours; more than 90% of the time is freeze-drying;

In order to shorten the drying time, the liquid-liquid extraction method can be used instead. The sample is firstly pulverized and homogenized, and then the lipid of the micronized sample is dissolved with a non-polar solvent. Layering, most of the polar compounds in the biological matrix can be distributed in the aqueous layer, and a large amount of solvent can be used to extract lipids to the maximum extent. For example, a 40 g wet base sample needs to be added 200 mL of non-polar solvent for 2 to 3 times. The supernatant is collected by shaking and stratifying, and the total amount of solvent used is as high as 500 mL ~ 1500 mL; however, the above method uses a large amount of solvent, which is not only extremely unenvironmental, but also highly dependent on manpower, and must be removed by nitrogen blowing or concentration under reduced pressure after extraction. There is a lot of solvent in the bottle, so the total liquid-liquid extraction process of a single sample still takes more than 2 hours.

It can be seen from the above description that the existing extraction methods have disadvantages such as time-consuming, labor-consuming, and solvent-consuming. Activated carbon column) for more than 2 days, on-board analysis for half a day, etc., the duration of a single sample detection of dioxin is as long as 5 to 7 days, of which the extraction step has taken up 25% to 60% of the time. However, based on the existing extraction methods that require a lot of time and manpower, the cost of food dioxin testing is 20,000 to 30,000 yuan, which is much higher than that of pesticide testing (1 day/3,000 to 6,000) and heavy metal testing (1 day/2,000 to 3,000 yuan). Thousands), making it more difficult to control the promotion of dioxins in food.

The main purpose of the present invention is to provide a method for rapid detection of dioxins and/or polychlorinated biphenyls in samples, which can greatly shorten the detection time of dioxins in samples, and greatly reduce the use of detection solvents, so as to improve the Efficacy of testing performance.

Furthermore, the method for rapid detection of dioxin and/or polychlorinated biphenyls in samples disclosed in the present invention can be applied to wet-based samples and dry-based samples, such as fresh samples, feed (about 11% moisture content) or Soybean flour (about 13% water content) can be used. It can also greatly reduce the amount of solvent used without going through a lengthy drying process.

For example, the method for rapid detection of dioxins and/or polychlorinated biphenyls in a sample disclosed in the present invention is that after directly mixing a wet-based sample with a superabsorbent substance, the high-pressure extraction procedure can be directly performed, and the extraction time can be effectively shortened. From 2 to 48 hours in the past to 30 minutes, the detection time is greatly reduced by more than 75%.

The reason is that, in order to achieve the above-mentioned purpose, the present invention discloses a method for rapid detection of dioxin and/or polychlorinated biphenyls in a sample. Procedures such as automatic purification and mass spectrometry analysis to obtain the total toxic equivalents of dioxin and/or polychlorinated biphenyls in the sample, wherein the time spent on high pressure extraction is less than 30 minutes, and the time spent on automatic purification procedures is less than 90 minutes , the time for mass spectrometry analysis is about 120 minutes. With the method disclosed in the present invention, it is possible to complete the detection and analysis process of a single sample within 4 hours, and even a batch of 10 samples can complete the total toxic equivalent analysis within 24 hours.

Specifically, the method for rapid detection of dioxin and/or polychlorinated biphenyls in a sample disclosed by the present invention comprises the following steps:

Take a superabsorbent dispersant and mix it with a sample to be tested in a predetermined ratio to obtain a processed sample.

The treated sample is extracted under a high pressure environment to obtain an extract.

The extract was concentrated and the supernatant was collected.

The supernatant is taken for purification and then analyzed by mass spectrometry to obtain a detection result.

Wherein, the sample to be tested is a wet-based sample.

Wherein, the weight ratio between the superabsorbent dispersant and the sample to be tested is 0.1:1 to 1:1.

Wherein, the superabsorbent dispersant is water-absorbent resin, calcium carbonate, magnesium carbonate, or a mixture containing at least any two of the above-mentioned substances. For example, the superabsorbent dispersant is obtained by polymerizing acrylic acid or its salts. made of resin.

Wherein, step b is carried out in a high-pressure extraction system, and the used extraction solvent is n-hexane/dichloromethane, and the time required for step b is about 15-40 minutes per sample.

Wherein, in step d, the supernatant is processed with an automatic purifier before mass spectrometry analysis, and the processing time of the automatic purifier is about 70-180 minutes per sample.

Wherein, the mass spectrometry analysis in step d is performed by gas chromatography tandem mass spectrometer, which takes about 40 minutes per sample.

In the embodiment of the present invention, the sample to be tested is from milk, eggs, seafood (aquatic products), forage, feed (grains and beans), such as: milk, eggs, duck eggs, crabs (including hairy crabs), Pennisetum, pig feed, chicken feed, etc.

Referring to FIG. 1, a method for rapidly testing dioxin and/or polychlorinated biphenyls in a sample is provided in one embodiment of the present invention, which is to pre-treat a sample to be tested with a superabsorbent dispersant to obtain Once the treated sample is subjected to high-pressure extraction, automatic purification and mass spectrometry analysis in sequence, not only the results of the total toxic equivalents of dioxins in the sample to be tested can be obtained, but also the detection time can be greatly shortened, so that a single sample can be The test can be completed within 4 hours, and even a batch of 10 samples can complete the total toxic equivalent analysis within 24 hours.

Among them, the sample after the treatment is a uniformly dispersed granular material.

Wherein, the high-pressure extraction procedure is to extract the processed sample with a high-pressure solvent to obtain the oil and fat extract.

Among them, the super absorbent dispersant is a super absorbent resin, an inorganic mineral or a mixture of the above two components in any proportion, and the super absorbent resin can be an acrylate polymer resin, such as the commercially available Formosa Plastics (Formosa Plastics, Inc., Taiwan) or Q-Matrix (CEM corporation, US); inorganic minerals are magnesium carbonate, calcium carbonate, etc., such as commercially available Spe-ed ^TM matrix (Applied Separations Inc., US) is a mixture of magnesium carbonate and calcium carbonate ; As shown in Figure 2, the superabsorbent dispersant is a mixture of superabsorbent resin, magnesium carbonate and calcium carbonate.

More specifically, the superabsorbent dispersant and the sample to be tested are mixed in a predetermined ratio, and the weight ratio of the superabsorbent dispersant to the sample to be tested is 0.1:1 to 1:1.

For example, when the sample to be tested is egg yolk, and the superabsorbent dispersant is a ^{mixture of Q-Matrix and Spe-ed TM} matrix by equal weights, there is a gap between the superabsorbent dispersant and the sample to be tested. The weight ratio is 1:1; when the sample to be tested is milk and the superabsorbent dispersant is Q-Matrix, the weight ratio of the superabsorbent dispersant to the sample to be tested is 0.1:1.

The so-called "wet-based sample" in the present invention refers to a sample to be tested with a water content greater than or equal to 20%, such as fresh fruit.

The so-called "dry-based sample" in the present invention refers to a sample to be tested with a moisture content of less than 20%, such as feed (about 11% moisture content), soybean meal (about 13% moisture content), etc.

The so-called "superabsorbent dispersant" in the present invention refers to a substance with high water absorption capacity, such as water absorbent resin, polymer dispersant, nano powder and the like.

The so-called "water-absorbent resin" in the present invention refers to a water-absorbent resin produced by a chemical reaction, and is divided into starch-based superabsorbent resins, cellulose-based superabsorbent resins, and synthetic polymer-based superabsorbent resins, among which, Synthetic polymer superabsorbent resins include: polyacrylates, polyacrylonitrile hydrolyzates, vinyl acetate copolymers, modified polyvinyl alcohols; currently the most commonly used superabsorbent resins obtained by chemical synthesis are polyacrylic acid Salts are obtained by copolymerizing acrylic acid or its salts and monomers with difunctional groups. The preparation methods include solution polymerization followed by drying and pulverization and suspension polymerization.

The so-called "TEQ" in the present invention refers to the toxic equivalent of 2,3,7,8-tetrachlorodioxin.

Hereinafter, in order to further prove the effect of the present invention, a number of examples and diagrams will be given for detailed description as follows.

The announced detection method used in the following examples is based on the announced method of the Food and Drug Administration of the Ministry of Health and Welfare of my country: CNS14758 Soxhlet extraction multi-column purification method. After degreasing with concentrated sulfuric acid, acid silica gel column, acid silica gel activated carbon composite column, and purification treatment in sequence, and then analyzed by high-resolution gas chromatography mass spectrometer (HR-GCMS), the dioxin in the sample to be tested was obtained. The total toxic equivalent of the compound; in general, the time required for detection by traditional methods is 5-7 days/sample.

The superabsorbent dispersant in the following examples is a ^{mixture of Q-Matrix and Spe-ed TM} matrix by equal weight.

The "dioxin-like compounds" disclosed in the following examples include polychlorinated diphenyl dioxin furans (PCDDs/PCDFs) and dioxin-like polychlorinated biphenyls (DL-PCBs).

Example 1: Confirm the effect of the disclosed method of the present invention to detect dioxins in the sample

Take 6g of egg yolk sample and mix 6g of superabsorbent dispersant (a mixture of Q-Matrix and Spe-ed ^TM matrix by equal weight) into the extraction tube. ), polychlorinated biphenyls (DLPCBs and NDL-PCBs) internal standard solution, wherein, the extraction solvent is n-hexane/dichloromethane (1:1), and the extraction conditions are adding 30 ml of n-hexane/dichloromethane at the top, and wait for the temperature Raise the temperature to 120°C, add 10 ml of n-hexane/dichloromethane to the lower end, keep the temperature for 5 minutes, collect the extract, and continuously extract 2~3 times, the total extraction time is about 20~30 minutes; After the extract is concentrated by blowing nitrogen , and then add n-hexane to quantify to 10 ml; then analyze with automatic purification instrument and tandem mass spectrometer to obtain the recoveries of 17 kinds of dioxin furans and 12 kinds of polychlorinated biphenyls in egg yolk samples. The time is about 1.5 hours, and the analysis time of the tandem mass spectrometer is about 2 hours. The results of detection by the method disclosed in the present invention are compared with the recovery rate of the published detection method, as shown in Table 1 and Table 2 below. From the results in Tables 1 and 2, it can be seen that the recovery rates of the polychlorinated diphenyl dioxins and polychlorinated dibenzofurans tested by the method disclosed in the present invention are between 86% and 113%, which are in line with the dioxindol of the published detection method. The range of initial precision and recovery rate of chlorinated biphenyls shows that the detection of dioxins in the sample by the method disclosed in the present invention conforms to the specifications of the currently announced detection methods, and the time for detecting a single sample by the method disclosed in the present invention is short 4 hours, which means that the method disclosed in the present invention can effectively shorten the detection time.

Table 1: Dioxin and furan initial precision recovery rate of the disclosed method of the present invention (4 pieces of egg yolk samples) The method disclosed in the present invention Announcement Specification Compound name Starting precision (%) The relative standard deviation(%) Starting precision (%) The relative standard deviation(%) Analyte 2,3,7,8-TeCDF 94~103 4.1 86~136 14.7 1,2,3,7,8-PeCDF 97~107 4.3 84~124 11.9 2,3,4,7,8-PeCDF 101~111 4.4 72~148 11.6 1,2,3,4,7,8-HxCDF 99~111 5.4 80~116 14.8 1,2,3,6,7,8-HxCDF 100~108 3.3 92~120 11.0 2,3,4,6,7,8-HxCDF 101~112 5.2 72~148 10.0 1,2,3,7,8,9-HxCDF 96~107 5.0 84~120 10.7 1,2,3,4,6,7,8-HpCDF 100~111 4.8 88~112 11.1 1,2,3,4,7,8,9-HpCDF 99~109 4.7 84~124 12.9 OCDF 98~107 4.0 74~146 17.8 2,3,7,8-TeCDD 98~107 4.2 82~128 21.9 1,2,3,7,8-PeCDD 96~106 4.4 76~132 11.2 1,2,3,4,7,8-HxCDD 101~113 5.1 76~152 12.4 1,2,3,6,7,8-HxCDD 98~107 3.7 84~124 12.3 1,2,3,7,8,9-HxCDD 102~116 6.4 72~140 15.7 1,2,3,4,6,7,8-HpCDD 96~106 4.6 76~128 11.9 OCDD 103~114 5.1 88~126 15.1 Internal standard ¹³ C ₁₂ -2,3,7,8-TeCDF 53～59 4.1 31~113 31.0 ¹³ C ₁₂ -1,2,3,7,8-PeCDF 51～56 4.4 27~156 21.8 ¹³ C ₁₂ -2,3,4,7,8-PeCDF 48～51 2.8 16~279 13.6 ¹³ C ₁₂ -1,2,3,4,7,8-HxCDF 61～67 5.6 27~152 28.3 ¹³ C ₁₂ -1,2,3,6,7,8-HxCDF 54～58 3.1 30~122 28.7 ¹³ C ₁₂ -2,3,4,6,7,8-HxCDF 60～64 3.7 29~136 27.2 ¹³ C ₁₂ -1,2,3,7,8,9-HxCDF 62～67 4.2 24~157 25.5 ¹³ C ₁₂ -1,2,3,4,6,7,8-HpCDF 59～65 4.7 32~110 37.3 ¹³ C ₁₂ -1,2,3,4,7,8,9-HpCDF 55～60 4.0 28~141 28.4 ¹³ C ₁₂ -2,3,7,8-TeCDD 55～62 4.9 28~134 27.6 ¹³ C ₁₂ -1,2,3,7,8-PeCDD 51～54 3.1 27~184 21.2 ¹³ C ₁₂ -1,2,3,4,7,8-HxCDD 60～66 4.4 29~147 27.9 ¹³ C ₁₂ -1,2,3,6,7,8-HxCDD 60～69 5.6 34~122 31.3 ¹³ C ₁₂ -1,2,3,4,6,7,8-HpCDD 62～66 3.2 34~129 27.1 ^13C _12- OCDD 60～65 3.6 21~138 34.4 Purification standard ³⁷ Cl ₄ ^{-2,3,7,8-TeCDD} 64～76 8.0 39~154 23.4

Table 2: The initial precision recovery rate of PCBs of the disclosed method of the present invention (4 pieces of egg yolk samples) The method disclosed in the present invention Announcement Specification Compound name Starting precision (%) The relative standard deviation(%) Starting precision (%) The relative standard deviation(%) Analyte 3,4,4',5-TeCB 81 103~117 6.2 60~140 40 3,3',4,4'-TeCB 77 104~116 6.2 60~140 40 2',3,4,4',5-PeCB 123 112~124 6.2 60~140 40 2,3',4,4',5-PeCB 118 106~118 6.2 60~140 40 2,3,4,4',5-PeCB 114 97~109 6.2 60~140 40 2,3,3',4,4'-PeCB 105 106~116 6.2 60~140 40 3,3',4,4',5-PeCB 126 99~111 6.2 60~140 40 2,3',4,4',5,5'-HxCB 167 97~108 6.2 60~140 40 2,3,3',4,4',5-HxCB 156 99~109 6.2 60~140 40 2,3,3',4,4',5'-HxCB 157 97~106 6.2 60~140 40 3,3',4,4',5,5'-HxCB 169 100~117 6.2 60~140 40 2,3,3',4,4',5,5'-HpCB 189 102~115 6.2 60~140 40 Internal standard ¹³ C ₁₂ -3,4,4',5-TeCB 81L 57～68 8.0 35~135 50 ¹³ C ₁₂ -3,3',4,4'-TeCB 77L 61～73 7.9 35~135 50 ¹³ C ₁₂ -2', 3, 4, 4', 5-TeCB 123L 52～61 6.9 35~135 50 ¹³ C ₁₂ -2,3',4,4',5-PeCB 118L 53～62 6.7 35~135 50 ¹³ C ₁₂ -2,3,4,4',5-PeCB 114L 58～68 6.7 35~135 50 ¹³ C ₁₂ -2,3,3',4,4'-PeCB 105L 57～65 6.2 35~135 50 ¹³ C ₁₂ -3,3',4,4',5-PeCB 126L 61～69 5.3 35~135 50 ¹³ C ₁₂ -2,3',4,4',5,5'-HxCB 167L 66～83 11.3 35~135 50 ¹³ C ₁₂ -2,3,3',4,4',5-HxCB 156L 64～77 8.4 35~135 50 ¹³ C ₁₂ -2,3,3',4,4',5'-HxCB 157L 65～79 8.7 35~135 50 ¹³ C ₁₂ -3,3',4,4',5,5'-HxCB 169L 64～73 5.7 35~135 50 ¹³ C ₁₂ -2,3,3',4,4',5,5'-HpCB 189L 55～64 6.0 35~135 50

Example 2: Test results of real samples (wet-based samples)

Buy eggs from different sources (take their egg yolks respectively), hairy crabs, fresh milk, and Pennisetum collected around the pasture from the market, and then detect with the disclosed method and the announcement detection method of the present invention, and the results are shown in Table 3. . It can be seen from the results in Table 3 that the method disclosed in the present invention can accurately detect the total toxic equivalents of dioxins in eggs from different sources in a short time.

Table 3: Announcement detection method and the disclosed method of the present invention carry out the analysis result comparison of egg yolk from different sources sample water content Total toxic equivalent concentration of dioxins (pg WHO-TEQ/g fat) The relative value of the detected concentration of the two methods Evaluation of the difference between the two methods Announcement inspection method (CNS14758) The method disclosed in the present invention This disclosure method/announcement method The relative deviation of the detected concentration of the two methods RPD(%) Egg 1 (red fresh egg) 75% 0.422 0.493 1.2 15.5% Egg 2 (Diamond Egg) 76% 0.435 0.533 1.2 20.2% hairy crab 82% 2.777 3.260 1.2 16.0% fresh milk 87% 0.019 0.019 1.0 0% Pennisetum 86% 0.030 0.043 1.4 35.6%

Example 3: Test results of real samples (dry samples)

Corn kernels and soybean meal were purchased from the market, and pig feed and chicken feed were collected from animal husbandry farms. It can be seen from the results in Table 4 that the method disclosed in the present invention can accurately detect the total toxic equivalent of dioxins in hairy crabs in a short time.

Table 4: Comparison of the analysis results of the announced detection method and the method disclosed in the present invention to detect corn kernels, soybean meal, chicken feed and pig feed sample water content Total toxic equivalent concentration of dioxins (pg WHO-TEQ/g fat) The relative value of the detected concentration of the two methods Evaluation of the difference between the two methods Announcement inspection method (CNS14758) The method disclosed in the present invention This disclosure method/announcement method The relative deviation of the detected concentration of the two methods RPD(%) corn kernels 14% 0.054 0.040 0.7 29.8% soy flour 10% 0.577 0.579 1.0 0.3% chicken feed 4% 0.026 0.022 0.8 16.7% Food for pig 4% 0.018 0.015 0.8 18.1%

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Fig. 1 shows the operation flow of the method for rapidly testing dioxin and/or polychlorinated biphenyls in a sample disclosed in an embodiment of the present invention and the estimated time of each step. Figure 2 shows a superabsorbent resin dispersant disclosed in one embodiment of the present invention.

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Claims (7)

A method for rapid detection of dioxins and/or polychlorinated biphenyls in a sample, which is to complete the detection of dioxins in a single sample within 4 hours, comprising the following steps: a. Take a superabsorbent dispersant, which is combined with a The sample to be tested is mixed and processed in a predetermined ratio to obtain a processed sample, wherein the superabsorbent dispersant is selected from the group consisting of the following substances: resins formed by polymerizing acrylic acid, resins formed by polymerizing acrylic acid salts. The obtained resin and acrylate polymer resin, magnesium carbonate and calcium carbonate are mixed in any proportion; b. The treated sample is extracted under high pressure to obtain an extract; c. The extract is concentrated and Collect the supernatant; d. take the supernatant for purification and then analyze by mass spectrometry to obtain a detection result. The method for rapidly testing dioxin and/or polychlorinated biphenyls in a sample as described in claim 1, wherein the sample to be tested is a wet-based sample. The method for rapidly testing dioxin and/or polychlorinated biphenyls in a sample as described in claim 1, wherein the weight ratio between the superabsorbent dispersant and the sample to be tested is 0.1:1 to 1:1. The method for rapidly testing dioxin and/or polychlorinated biphenyls in a sample as described in claim 1, wherein the extraction solvent in step b is n-hexane/dichloromethane. The method for rapidly detecting dioxin and/or polychlorinated biphenyls in a sample as described in claim 1, wherein, in step d, the supernatant is processed with an automatic purifier before mass spectrometry analysis. The method for rapidly detecting dioxin and/or polychlorinated biphenyls in a sample as described in claim 1, wherein the mass spectrometry analysis in step d is performed by gas chromatography tandem mass spectrometer. The method for rapidly testing dioxin and/or polychlorinated biphenyls in a sample as described in claim 1, wherein the sample to be tested is from eggs, milk, seafood, cereals, beans, grains, meat or feed.

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