Ochratoxin A enriching and purifying material as well as preparation method and application thereof

Abstract

The invention discloses an ochratoxin A enriching and purifying material and a preparation method and application thereof, the preparation method comprises the following steps: 1) mixing a carrier with a serum albumin solution, and carrying out a coupling reaction to obtain a carrier coupled with serum albumin; (2) sealing and cleaning the carrier coupled with the serum albumin, and treating by adopting any one of a pulsed electric field, heating, ultrasound and high pressure; wherein in the step (1), the carrier is superparamagnetic magnetic beads or agarose. According to the method, serum albumin which is low in cost and easy to obtain is used as a recognition element, superparamagnetic beads or agarose is used as a carrier, treatment is carried out by means of a pulsed electric field, heating, ultrasound and the like, and the enrichment and purification material which is high in binding force with the ochratoxin A is obtained and can be used for enriching and purifying the ochratoxin A in various samples such as vegetable oil and grains. Therefore, quantitative detection of the ochratoxin A in the sample in the later period is facilitated, and the detection cost of the ochratoxin A can be greatly reduced.

Description

technical field

[0001] The invention relates to the technical field of food safety detection. More specifically, it relates to an ochratoxin A enrichment and purification material and its preparation method and application. Background technique

[0002] Ochratoxin A (OTA) is a mycotoxin produced by certain species of Penicillin and Aspergillus, OTA can contaminate not only various agricultural products, but also various processed foods (such as wine, bread and meat products). OTA has various toxic effects such as neurotoxicity, liver and kidney toxicity, and is classified as a class 2B carcinogen by the International Agency for Research on Cancer (IARC). In addition, it acts as a reproductive toxin as well as an endocrine disruptor, causing damage to human reproductive health. Therefore, based on the multiple threats to human health caused by the toxic effects of OTA, in order to reduce the risk of ingesting OTA through food, many countries, regions and international orga...

Examples

Embodiment 1

[0057] Preparation of ochratoxin A enrichment and purification materials

[0058] (1) Prepare a human serum albumin solution with a concentration of 10 mg / mL, a solvent of 100 mmol / L, and a 2-(N-morpholine)ethanesulfonic acid buffer solution (MES) with pH=6.0.

[0059] (2) Take 1 mL of NHS magnetic beads (20% (v:v), 10-30 μm), and wash the NHS magnetic beads twice with absolute ethanol. According to the coupling ratio of 4 mg of human serum albumin and 1 mL of NHS magnetic beads, add human serum albumin solution to the NHS magnetic beads, and then supplement a certain amount of MES, so that the whole system is carried out in the MES environment, and slowly mix at room temperature. , incubate for 2 hours.

[0060] (3) After the incubation, place it in a magnetic separation rack, enrich the magnetic beads, and discard the supernatant. Then, add 1 mL of 0.1 M Tris-HCl buffer at pH 8.0, vortex for 30 s, mix slowly at room temperature, and incubate for 2 hours to block the remain...

Embodiment 2

[0069] The pretreatment method of enrichment and purification material based on embodiment 1 and its application in the detection of wheat OTA

[0070] (1) Prepare the sample extract to be tested

[0071] Weigh 5.0g of the pulverized wheat sample into a 50mL centrifuge tube, add 25.0mL of acetonitrile / water solution with a volume ratio of 60:40, and place it on a multi-tube vortex mixer at 2500rpm for 5min (or use a high-speed homogenizer). Homogenize at 11000 rpm for 3 min, or shake at 200 rpm on a shaker for 40 min), add 20 μL to the detection tube after vortexing, and then centrifuge at 7000 rpm for 5 min to obtain the sample extract to be tested;

[0072] (2) Sample pretreatment

[0073] 1) Prepare the OTA purification kit: including sample well 1, reaction well 2, cleaning wells 3 and 4, elution well 5 and magnetic bead storage well 6 (such as figure 1 shown).

[0074] Take 0.5mL-2mL of the sample extract to be tested and 0.45mL of 0.1% PBST buffer (containing 0.5% Twe...

Embodiment 3

[0083] The pretreatment method of enrichment and purification material based on Example 1 and its application in corn OTA detection

[0084] (1) Prepare the sample extract to be tested

[0085] Weigh 5.0 g of the pulverized sample to be tested in a 50 mL centrifuge tube, add 25.0 mL of acetonitrile / water solution with a volume ratio of 60:40, and place it on a multi-tube vortex mixer at 2500 rpm for 5 min (or use a high-speed homogenizer). Homogenize at 11000 rpm for 3 min on the device, or shake at 200 rpm on a shaker for 40 min), add 20 μL of the test sample extract to the detection tube, and then centrifuge at 7000 rpm for 5 min to obtain the test sample extract;

[0086] (2) Sample pretreatment

[0087] 1) Prepare OTA purification kit: including sample well 1, reaction well 2, cleaning wells 3 and 4, elution well 5 and magnetic bead storage well 6.

[0088] Take 0.5mL-2mL of the sample extract to be tested and 0.45mL of 0.1% PBST buffer (containing 0.5% Tween-20) and put...

technical field

[0001] The invention relates to the technical field of food safety detection. More specifically, it relates to an ochratoxin A enrichment and purification material and its preparation method and application. Background technique

[0002] Ochratoxin A (OTA) is a mycotoxin produced by certain species of Penicillin and Aspergillus, OTA can contaminate not only various agricultural products, but also various processed foods (such as wine, bread and meat products). OTA has various toxic effects such as neurotoxicity, liver and kidney toxicity, and is classified as a class 2B carcinogen by the International Agency for Research on Cancer (IARC). In addition, it acts as a reproductive toxin as well as an endocrine disruptor, causing damage to human reproductive health. Therefore, based on the multiple threats to human health caused by the toxic effects of OTA, in order to reduce the risk of ingesting OTA through food, many countries, regions and international orga...

Examples

Embodiment 1

[0057] Preparation of ochratoxin A enrichment and purification materials

[0058] (1) Prepare a human serum albumin solution with a concentration of 10 mg / mL, a solvent of 100 mmol / L, and a 2-(N-morpholine)ethanesulfonic acid buffer solution (MES) with pH=6.0.

[0059] (2) Take 1 mL of NHS magnetic beads (20% (v:v), 10-30 μm), and wash the NHS magnetic beads twice with absolute ethanol. According to the coupling ratio of 4 mg of human serum albumin and 1 mL of NHS magnetic beads, add human serum albumin solution to the NHS magnetic beads, and then supplement a certain amount of MES, so that the whole system is carried out in the MES environment, and slowly mix at room temperature. , incubate for 2 hours.

[0060] (3) After the incubation, place it in a magnetic separation rack, enrich the magnetic beads, and discard the supernatant. Then, add 1 mL of 0.1 M Tris-HCl buffer at pH 8.0, vortex for 30 s, mix slowly at room temperature, and incubate for 2 hours to block the remain...

Embodiment 2

[0069] The pretreatment method of enrichment and purification material based on embodiment 1 and its application in the detection of wheat OTA

[0070] (1) Prepare the sample extract to be tested

[0071] Weigh 5.0g of the pulverized wheat sample into a 50mL centrifuge tube, add 25.0mL of acetonitrile / water solution with a volume ratio of 60:40, and place it on a multi-tube vortex mixer at 2500rpm for 5min (or use a high-speed homogenizer). Homogenize at 11000 rpm for 3 min, or shake at 200 rpm on a shaker for 40 min), add 20 μL to the detection tube after vortexing, and then centrifuge at 7000 rpm for 5 min to obtain the sample extract to be tested;

[0072] (2) Sample pretreatment

[0073] 1) Prepare the OTA purification kit: including sample well 1, reaction well 2, cleaning wells 3 and 4, elution well 5 and magnetic bead storage well 6 (such as figure 1 shown).

[0074] Take 0.5mL-2mL of the sample extract to be tested and 0.45mL of 0.1% PBST buffer (containing 0.5% Twe...

Embodiment 3

[0083] The pretreatment method of enrichment and purification material based on Example 1 and its application in corn OTA detection

[0084] (1) Prepare the sample extract to be tested

[0085] Weigh 5.0 g of the pulverized sample to be tested in a 50 mL centrifuge tube, add 25.0 mL of acetonitrile / water solution with a volume ratio of 60:40, and place it on a multi-tube vortex mixer at 2500 rpm for 5 min (or use a high-speed homogenizer). Homogenize at 11000 rpm for 3 min on the device, or shake at 200 rpm on a shaker for 40 min), add 20 μL of the test sample extract to the detection tube, and then centrifuge at 7000 rpm for 5 min to obtain the test sample extract;

[0086] (2) Sample pretreatment

[0087] 1) Prepare OTA purification kit: including sample well 1, reaction well 2, cleaning wells 3 and 4, elution well 5 and magnetic bead storage well 6.

[0088] Take 0.5mL-2mL of the sample extract to be tested and 0.45mL of 0.1% PBST buffer (containing 0.5% Tween-20) and put...

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