CN117603198A - Method and device for rapidly detecting patulin in fruit and vegetable juice and beverage thereof - Google Patents

Method and device for rapidly detecting patulin in fruit and vegetable juice and beverage thereof Download PDF

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CN117603198A
CN117603198A CN202410093018.4A CN202410093018A CN117603198A CN 117603198 A CN117603198 A CN 117603198A CN 202410093018 A CN202410093018 A CN 202410093018A CN 117603198 A CN117603198 A CN 117603198A
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patulin
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fruit
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antibody
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杨星星
王炳志
付辉
邓启福
李细清
肖昭理
莫秋华
王伟维
梁秋乙
覃冬梅
王水树
刘运旺
高荣健
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SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD
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Abstract

The invention discloses a rapid detection method and a rapid detection device for patulin in fruit and vegetable juice and beverage thereof, and relates to a detection device for patulin derivatives, antigens, antibodies and colloidal gold, and a rapid detection method for patulin in fruit and vegetable juice and beverage thereof. The invention provides an artificial antigen prepared by coupling a patulin derivative with a carrier protein, and an antibody which is specifically directed against the patulin derivative and is produced by a body of the animal after the animal is immunized, wherein the linear detection range of the antibody against the patulin derivative is 0.06-2.37ng/mL, and the sensitivity is 0.383 ng/mL. The invention can be used for detecting the patulin in fruit and vegetable juices and beverages, has the advantages of good specificity, high sensitivity, simple and convenient operation and the like, can better meet the requirements of self-checking of production enterprises and on-site quick detection of detection institutions, and can be popularized and used for large-scale quick and accurate screening of patulin in fruit and vegetable juices and beverages thereof.

Description

Method and device for rapidly detecting patulin in fruit and vegetable juice and beverage thereof
Technical Field
The invention relates to the technical field of food safety detection, in particular to a rapid detection method and a detection device for patulin in fruit and vegetable juices and beverages thereof.
Background
Patulin (PAT), also known as Patulin, is a class of polyacetyllactone mycotoxins produced mainly by fungi of the genera Penicillium (Penicillium), aspergillus (Aspergillus) and mycelial (Byssochlamys). Wherein, the penicillium expansum (P. Expansum) is probably the main toxigenic fungus of PAT, infects ripe fruits mainly through the route such as wound, and host scope includes apple, peach, pear, grape, kiwi fruit, hawthorn etc. is one of the most important postharvest pathogenic bacteria. The processed products such as fruit juice, jam, fruit puree and the like are main food categories polluted by the patulin. A number of studies have shown that excessive intake of PAT has a wide range of acute and chronic virulence to humans and animals, including liver and kidney injury, teratogenicity, carcinogenicity, DNA damage, and immunotoxicity. The maximum limit standard of patulin in fruit products, beverages and wines prepared by taking apples and hawthorns as raw materials in GB 2761-2017 "limit of mycotoxins in food safety national standard food" is 50 mug/kg.
At present, PAT detection methods commonly adopted at home and abroad mainly comprise Thin Layer Chromatography (TLC), high Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and the like. The TLC operation is required to be high, the sensitivity is low, and the interference of impurities such as 5-hydroxymethylfurfural and the like exists, so that the TLC operation is rarely adopted. The HPLC has the characteristics of high sensitivity, high accuracy and the like, and can selectively separate the interference of impurities such as 5-hydroxymethylfurfural and the like. However, there are problems in that the pretreatment process is complicated, the detection time is long, the cost is high, and the like, and it is difficult to satisfy the requirement of rapid detection. Since patulin itself has some characteristics of unsaturated lactone, patent documents with publication numbers CN 114716535B and CN116102569 a disclose a conventional method for preparing patulin hapten, i.e. retaining the main structure thereof, resulting in that the prepared antigen is easy to react with thiol (-SH) compound in animal body during immunization, and the problem of poor immune effect and low antibody sensitivity exists.
Therefore, the patulin is rapidly derivatized, converted into a derivative with stable structure, and then antigen and antibody of the derivative are prepared, so that the aim of indirectly detecting patulin is fulfilled, and a corresponding patulin rapid detection method is established, thereby realizing rapid detection of patulin in fruit and vegetable juices and drinks thereof by an immunological method, and solving the problem of low sensitivity of the existing antibody.
Disclosure of Invention
The invention aims to provide a rapid detection method and a detection device for patulin in fruit and vegetable juices and beverages thereof.
According to one aspect of the present invention, there is provided a patulin derivative having the structure shown in formula (I):
，
wherein: r is R 1 H, CH of a shape of H, CH 3 、SO 3 、SO 3 Na、Ph、C 6 H 5 CH 2 Or (CH) 2 ) n CH 3 Wherein n is an integer other than 0; r is R 2 H, CH of a shape of H, CH 3 、SO 3 、SO 3 Na, ph or C 6 H 5 CH 2 。
According to another aspect of the present invention, there is provided a method for preparing a patulin derivative, wherein patulin is reacted with an excess of an adjacent dimercapto compound in a buffer solution having a pH of 7-9 at room temperature or under heating conditions to prepare the patulin derivative, wherein the structure of the adjacent dimercapto compound is represented by formula (II), and the reaction process is represented by the following reaction formula:
，
wherein R is 1 H, CH of a shape of H, CH 3 、SO 3 、SO 3 Na、Ph、C 6 H 5 CH 2 Or (CH) 2 ) n CH 3 Wherein n is an integer other than 0; r is R 2 H, CH of a shape of H, CH 3 、SO 3 、SO 3 Na, ph or C 6 H 5 CH 2 。
In some embodiments, one of 1, 2-ethanedithiol, dimercaptopropanol, 2, 3-dimercaptosuccinic acid, sodium 2, 3-dimercaptopropanesulfonate, or 2, 3-butanedithiol.
In some embodiments, the molar ratio of patulin to adjacent dimercapto compound is 1: (2-5).
According to a further aspect of the present invention there is provided a conjugate of a patulin derivative antigen with a carrier protein, the carrier protein being bovine serum albumin, human serum albumin, chicken ovalbumin or hemocyanin.
According to a fourth aspect of the present invention there is provided the use of a patulin derivative or patulin derivative antigen for non-disease diagnosis in immunological detection of patulin and patulin derivatives.
According to a fifth aspect of the present invention there is provided the use of a patulin derivative as a hapten and as a standard in immunological detection of patulin and patulin derivatives.
According to a sixth aspect of the present invention there is provided a patulin antibody, the patulin antibody being prepared from a patulin derivative antigen by animal immunization, the patulin antibody being a patulin monoclonal antibody.
According to a seventh aspect of the present invention, there is provided a device for detecting patulin, comprising a test strip and a microporous reagent, the test strip being provided with a detection line, the detection line being coated with patulin derivative antigen, the microporous reagent comprising colloidal gold labelled patulin derivative antibody.
According to an eighth aspect of the present invention, there is provided a method for rapidly detecting patulin in fruit and vegetable juices and beverages thereof, wherein patulin in a sample to be detected is subjected to derivatization treatment by using a patulin detection device, the patulin derivative in the sample to be detected is detected by using a patulin detection device, the sample is fruit and vegetable juice or a beverage containing fruit and vegetable juice, and the fruit and vegetable juice is one of fruit juice, vegetable juice or a mixture of fruit juice and vegetable juice.
The invention has the beneficial effects that: the patulin derivative and the preparation method thereof provided by the invention have the advantages that the used chemical reagent is easy to obtain, the operation process is simple, the synthesis steps are simple and effective, and the reaction is carried outThe yield is higher, and the detection cost is lower. The present invention provides artificial antigen prepared with patulin derivative coupled carrier protein and antibody specific to patulin derivative produced by the body of the animal after immunization of test animal, and the antibody has sensitivity (IC) 50 ) Linear detection range (IC) for patulin derivatives at 0.383ng/mL 20 -IC 80 ) 0.06-2.37ng/mL. The invention applies the immunological detection method to carry out exclusive detection on the patulin in fruit and vegetable juices and beverages thereof, has the advantages of good specificity, high sensitivity, simple and convenient operation, capability of realizing on-site rapid detection and the like, and can better meet the requirements of food production enterprises on-site rapid detection.
Drawings
FIG. 1 is a synthetic scheme of a patulin derivative according to an embodiment of the invention.
Fig. 2 is a mass spectrum of a patulin derivative according to an embodiment of the invention.
FIG. 3 is a standard curve of a monoclonal antibody based on a patulin derivative according to an embodiment of the invention.
FIG. 4 is an ultraviolet scan of an immunizing antigen of a patulin derivative according to one embodiment of the invention.
FIG. 5 is an ultraviolet scan of antigens for coating of a patulin derivative according to one embodiment of the invention.
Detailed Description
The invention will be described in further detail with reference to specific embodiments thereof, it being understood that these embodiments are for purposes of illustration only and not for purposes of limiting the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the invention, and are defined in the claims appended hereto. Unless otherwise specified, all materials and reagents of the invention are those commercially available in the conventional market.
EXAMPLE 1 Synthesis and identification of patulin derivatives
A process for the preparation of a patulin derivative comprising the steps of:
weighing patulin 50 and mg, buffering and dissolving with 10mL 0.1M pH8.0 carbonate, and stirring at room temperature to obtain solution A; adding 10mL of methanol into 3 eq of 2, 3-butanedithiol to dissolve to obtain solution B, slowly adding the solution B into the solution A, continuously stirring at room temperature for reaction for 5 min after the solution B is added, evaporating to dryness after the reaction is finished to dissolve, adding distilled water and extracting with ethyl acetate, and purifying an organic phase by a column to obtain the patulin derivative with the structure shown in the formula (I).
The patulin derivative is subjected to structural analysis and nuclear magnetic resonance hydrogen spectrum identification to obtain: 1H NMR (500 MHz, CDCl 3) delta (ppm) 4.66 (s, 1H), 4.31 (t, J=5.5 Hz, 1H), 4.12 (dd, J=12.2, 5.3Hz, 1H), 3.97 (dd, J=12.4, 5.7Hz, 1H), 3.89 (dd, J=2.0, 1.1Hz, 1H), 3.86 (s, 1H), 3.75 (s, 1H), 3.72 (s, 2H), 3.53 and 3.39 (AB system, J=15.3 Hz, each 1H), 1.64 (s, 6H), 1.56 (s, 3H), 1.54 (s, 3H).
13C NMR (126 MHz, CDClδ (ppm) 174.45, 140.33, 138.02,66.78, 56.54,49.56, 48.95, 48.36, 48.34, 40.17, 19.33, 18.25, 17.90.
Identification of patulin derivatives by mass spectrometry, and the obtained mass spectrum is shown in figure 2 of the accompanying drawings. As can be seen from the mass spectrum, the molecular ion peak of the patulin derivative is EI-MS (negative) m/z:379.0[ M-H ] -and is the highest peak, which corresponds to the molecular weight 380.06 of the patulin derivative, indicating successful synthesis of the patulin derivative of formula (I).
EXAMPLE 2 Synthesis of patulin derivatives
A process for the preparation of a patulin derivative comprising the steps of:
weighing patulin 50 and mg, buffering and dissolving with 10mL 0.1M pH8.0 carbonate, and stirring at room temperature to obtain solution A; adding 10mL of methanol into 2eq dimercaptopropanol to dissolve to obtain solution B, slowly adding the solution B into the solution A, continuously stirring at room temperature for reaction for 5 min after the addition, evaporating to dryness to dissolve after the reaction is finished, adding distilled water, extracting with ethyl acetate, and purifying an organic phase by a column to obtain the patulin derivative with the structure shown in the formula (I).
EXAMPLE 3 Synthesis of patulin derivatives
Weighing patulin 50 and mg, buffering and dissolving with 10mL 0.1M pH8.0 carbonate, and stirring at room temperature to obtain solution A; and 5eq of 2, 3-dimercaptopropane sodium sulfonate is taken, 15mL of acetonitrile is added for dissolution, liquid B is obtained, the liquid B is slowly added into liquid A, stirring reaction is continued at room temperature for 5 min after the addition is finished, distilled water is added for extraction after the reaction is finished, and an organic phase is subjected to column purification, so that the patulin derivative with the structure shown in the formula (I) is obtained.
EXAMPLE 4 Synthesis of antigens for immunization and coating of patulin derivatives
An antigen for immunization and an antigen for coating of a patulin derivative were prepared by using the patulin derivative prepared in example 1 as a hapten.
Specifically, 0.1 mmol of the patulin derivative is dissolved in 2 mL of DMF, 0.2 mmol of DCC (dicyclohexylcarbodiimide) and 0.15 mmol of NHS (N-hydroxysuccinimide) are added with stirring, the mixture is magnetically stirred at 4 ℃ for reaction overnight, the supernatant after centrifugation is C solution, and bovine Lactoferrin (LF) 140 mg is weighed and dissolved in PBS (pH8.0) with the concentration of 10mL of 0.1 mol/L. DMF (1 mL) is added, stirred and dissolved to prepare solution D, and solution C is gradually dripped into solution D under magnetic stirring, and reacted at 4 ℃ for 12 h. After centrifugation, the supernatant was collected, dialyzed against physiological saline at 4℃for 3 days, and the dialysate was changed 3 times per day, and the obtained antigen for immunization against the patulin derivative was dispensed at a concentration of 1.1 mg/mL into a 0.5-mL centrifuge tube and frozen in a refrigerator at-20 ℃.
The same preparation procedure as described above for the preparation of the antigen for immunization against a patulin derivative was used to prepare the antigen for coating against a patulin derivative by substituting bovine Lactoferrin (LF) with Bovine Serum Albumin (BSA) in the above preparation method.
Under the ultraviolet light of 190-350 nm, the antigen for immunization of bovine Lactoferrin (LF), bovine Serum Albumin (BSA) and the antigen for coating of the derivatives of the patulin are subjected to ultraviolet scanning measurement.
The identification results are shown in figures 4 and 5, and the ultraviolet characteristic absorption peaks of the antigen for the immunization of the patulin derivative can be seen from figure 4, the ultraviolet characteristic absorption peaks are offset to different degrees relative to the patulin derivative and the carrier protein (LF), and the characteristic absorption peaks of the patulin derivative and the LF are found, which indicate that the patulin derivative and the LF are successfully coupled, and the antigen for the immunization of the patulin derivative is successfully prepared.
As can be seen from FIG. 4, the ultraviolet characteristic absorption peaks of the antigen coated with the patulin derivative are shifted to different degrees relative to both the patulin derivative and the carrier protein (BSA), and the patulin derivative coated antigen is found to have the characteristic absorption peaks of the patulin derivative and the BSA at the same time, which indicates that the patulin derivative and the BSA are successfully coupled, and the patulin derivative coated antigen is successfully prepared.
EXAMPLE 5 preparation and purification of monoclonal antibodies to patulin derivatives
5.1 Immunization of animals
Healthy BALB/c mice of 6 to 8 weeks of age were selected for immunization, and the patulin immunization antigen obtained in example 2 was mixed and emulsified with an equivalent amount of Freund's adjuvant, and then the BALB/c mice were subjected to subcutaneous multipoint injection of the back of the neck (except for sprint immunization). The first immunization is carried out by using complete Freund's adjuvant, and the dosage is 200 mug/dose; boosting is carried out after 4 weeks at a dose of 100 mug/dose, the mixture is emulsified by using incomplete Freund's adjuvant, and then the boosting is carried out for a plurality of times for 3 weeks; the dose was halved again during the sprint immunization, 50. Mu.g/mouse, and the complete antigen was diluted with physiological saline and injected intraperitoneally into mice. The tail-breaking blood sampling detection can be carried out after the third immunization of the mice, and the titer and IC of the serum of the mice can be detected by an indirect competition enzyme-linked immunosorbent assay (IC-ELISA) 50 High selective potency, IC 50 Low mice were fused;
5.2 Cell fusion and cloning
Spleen cells of immunized BALB/c mice were taken and fused by electrofusion according to 8:1 and SP2/0 myeloma cells, and screening to obtain a patulin derivative monoclonal hybridoma cell strain which stably secretes patulin monoclonal antibodies;
5.3 Cell cryopreservation and resuscitation
Freezing solution for preparing patulin derivative monoclonal hybridoma cells into 1×10 6 Cell suspensions of individual/mL were stored in liquid nitrogen for long periods. Taking out the freezing tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen solution, and transferring into a culture flask for culture;
5.4 Preparation and purification of monoclonal antibodies
BALB/c mice were intraperitoneally injected with sterilized paraffin oil 0.5. 0.5 mL/mouse, and after 7 days, stable monoclonal hybridoma cell lines were intraperitoneally injected 5X 10 5 Ascites were collected after 7 days. Purifying ascites by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody of patulin derivative, and preserving at-20deg.C;
5.5 preparation of goat anti-mouse antibody
Sheep is used as immune animals, and a murine antibody is used as immunogen to immunize pathogen-free sheep, so that the goat anti-mouse antibody is obtained.
Example 6 test for Performance measurement of patulin derivative antibodies
The test establishes ELISA standard curve of the monoclonal antibody of the patulin derivative prepared in example 5 by optimizing parameters such as coating concentration, antibody dilution times, primary antibody and secondary antibody reaction time and the like and utilizing an ICELISA program, and the IC of the patulin derivative antibody is established by referring to figure 3 of the specification and the attached drawing 50 And a linear range, sensitivity (IC) of detecting the patulin derivative antibody by ICELISA 50 ) At 0.383ng/mL, linear detection range (IC 20 -IC 80 ) Is 0.06-2.37ng/mL. And then the derivatization reagent 2, 3-Ding Erliu and the patulin are respectively subjected to cross reaction, and the cross reaction rate of the derivatization reagent 2, 3-Ding Erliu and the patulin is found to be 0, so that the patulin derivative antibody has higher specificity on the patulin derivative.
EXAMPLE 7 preparation of patulin colloidal gold chromatography detection device
7.1 Preparation of colloidal gold
Diluting 1% chloroauric acid into 0.01% (mass fraction) with double distilled deionized water, placing 100 mL into a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5 mL 1% trisodium citrate under continuous high temperature and continuous stirring, stopping stirring and heating until the solution is transparent red, cooling to room temperature, recovering original volume with deionized water, and storing at 4deg.C. The prepared colloidal gold has pure appearance, is transparent and has no sediment or floaters;
7.2 Preparation of colloidal gold-labeled monoclonal antibody
The pH value of the colloidal gold solution was adjusted to 7.0, and the mixture was stirred uniformly by a constant speed stirrer, and the monoclonal antibody of the patulin derivative prepared in example 5 was added dropwise at the same time, and after 1 hour, PEG having the same amount of antibody was added, and after the reaction was completed for 30 minutes, BSA having the same amount of antibody was added, and stirring was continued for 30 minutes. Centrifuging at 9000 rpm for 30 min to obtain homogeneous gold-labeled antibody precipitate, and adding PNPB to resuspend;
7.3 Preparation of microwell reagents
Adding 100 mu L of a patulin derivative monoclonal antibody-colloidal gold marker into a micropore reagent micropore plate, putting into a freeze dryer, pre-freezing for 3 hours at the cold trap temperature of minus 50 ℃, and then drying in vacuum for 15 h to obtain a micropore reagent freeze-dried with the patulin derivative monoclonal antibody-colloidal gold marker, and sealing and preserving;
7.4 Preparation of sample absorbent pad
Placing the sample absorption pad in phosphate buffer solution containing 0.5% bovine serum albumin and having pH of 7.2 and 0.1 mol/L, soaking for 2h, and drying at 37deg.C for 2 h;
7.5 Preparation of reaction film
The patulin derivative coating antigen is coated on the reaction membrane to form a detection line, and the goat anti-mouse antibody is coated on the reaction membrane to form a quality control line.
The coating process comprises the following steps: diluting the patulin derivative coating antigen to 1 mg/mL by using a phosphate buffer solution, coating the patulin derivative coating antigen on a detection line (T line) on a nitrocellulose membrane by using an isolow spot film tester, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse antibody was diluted to 200. Mu.g/mL with 0.01 mol/L phosphate buffer at pH7.4, and coated on a quality control line (C line) on a nitrocellulose membrane in an isolow spot film instrument in an amount of 1.0. Mu.L/cm. Drying the coated reaction film for 2 hours at 37 ℃ for standby;
7.6 Assembly of colloidal gold test strip
Sequentially adhering a sample absorption pad, a reaction film and a water absorption pad on a PVC bottom plate; the tail end of the sample absorption pad is connected with the starting end of the reaction film, the tail end of the reaction film is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate; the reaction film is provided with a detection line and a quality control line, and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are perpendicular to the length of the test paper; the detection line is positioned at one side close to the tail end of the sample absorption pad; the quality control line is positioned at one side far away from the tail end of the sample absorption pad; cutting the test paper into small strips with the width of 4 mm by a machine for standby;
7.7 preparation of colloidal gold detection device
And filling the colloidal gold test strip, the micropore reagent and the drying agent into a test paper tube to form the colloidal gold detection device.
Example 8A method for rapidly detecting patulin in fruit and vegetable juices and beverages thereof
8.1 Pretreatment of samples
Adding 5 parts of distilled water into a fruit sample to be detected, pulping, centrifuging, taking 1mL of supernatant as a fruit juice sample to be detected, adding PBS buffer solution 5mL with pH of 8.0 (when the fruit juice sample to be detected is fruit juice with strong acidity such as hawthorn, neutralizing with 1N NaOH solution to be neutral) to obtain solution A; 2, 3-butanedithiol is dissolved by acetonitrile to prepare 2mg/mL of acetonitrile solution of 2, 3-butanedithiol, 10 mu L of 2mg/mL of acetonitrile solution of 2, 3-butanedithiol is added into the solution A, after shaking for 10 seconds, standing for 20 minutes at room temperature (or heating for 10 minutes), derivatization treatment is completed, and liquid to be detected is obtained, namely sampling detection can be performed;
8.2 And (3) detection: vertically dripping 9-10 drops (about 200 mu L) of liquid to be detected into a reaction cup, pumping up and down for 10 times, uniformly mixing, starting the first-step reaction at 20-40 ℃, and timing for 3 min; inserting the test strip into a reaction cup, starting a second reaction step at 20-40 ℃ and timing for 3 min; taking out the test strip from the reaction cup, gently scraping the sample pad at the lower end of the test strip, and judging and reading the result;
8.3 Interpretation of results
1) Negative (undetected): the color of the T line is more than or equal to the color of the C line.
2) Positive (detected): the color of the T line is less than that of the C line; t line does not develop.
3) Invalidation: the T line developed C line does not develop color or the C, T line does not develop color.
Example 9 sample detection example of a patulin colloidal gold chromatography detection device
1) Detection limit
A blank juice sample was taken, and patulin was added thereto to a final concentration of 25. Mu.g/L, 50. Mu.g/L, 100. Mu.g/L, respectively, and the patulin colloidal gold chromatography detection apparatus prepared in example 7 was used for detection by the rapid detection method of example 8, and each sample was repeatedly assayed three times.
When the juice sample is detected by using the test strip, the test strip is negative when the addition concentration of the patulin is 25 mug/L; the test strip showed positive when the patulin addition concentration was 50, 100. Mu.g/L. The addition experiment proves that the detection limit of the patulin colloidal gold chromatography detection device in the invention on patulin is 50 mug/L.
2) False positive rate and false negative rate test
2 blank sample substrates of apple juice and fruit wine were taken, each substrate was measured for 4 concentration levels (including 50 times for blank sample, 50 times for 0.5 times for detection limit addition, 50 times for 1 times for detection limit addition, and 50 times for 2 times for detection limit addition), and the test results are shown in table 1.
TABLE 1 calculation of Performance indicators for patulin detection apparatus
As can be seen from Table 1, the patulin colloidal gold detection device and the rapid detection method of the invention have high sensitivity and good specificity for detecting patulin in fruit and vegetable juice and beverage thereof, can reach lower detection limit, have low false positive rate and false negative rate, and ensure that the detection result reaches high accuracy.
Example 10 cross-over rate test of patulin colloidal gold detection apparatus
Taking 4 parts of blank apple juice sample,adding appropriate amount of patulin standard solution, 5-hydroxy furfural standard solution, deoxynivalenol standard solution and aflatoxin B into each part 1 Standard solutions were prepared as 4 samples with final concentrations of 50 μg/L, 5000 μg/L and 5000 μg/L, respectively, and the patulin colloidal gold detection device prepared in example 7 was used, and 3 repeated experiments were performed on the 4 samples using the rapid detection method of example 8, and the detection results are shown in table 2.
TABLE 2 Cross-reaction test results Table
Note that: "+" indicates positive and "-" indicates negative;
as can be seen from Table 2, only the apple juice sample with patulin added was positive, while 5-hydroxyfurfural, deoxynivalenol and aflatoxin B were added 1 The apple juice sample is not detected, which shows that the detection device has better specificity to the patulin and low cross reaction rate.
The foregoing is merely illustrative of some embodiments of the invention, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the inventive concept.

Claims (10)

1. The patulin derivative is characterized in that the patulin derivative has a structure shown in a formula (I):
，
wherein: r is R 1 H, CH of a shape of H, CH 3 、SO 3 、SO 3 Na、Ph、C 6 H 5 CH 2 Or (CH) 2 ) n CH 3 Wherein n is an integer other than 0; r is R 2 H, CH of a shape of H, CH 3 、SO 3 、SO 3 Na, ph or C 6 H 5 CH 2 。
2. A process for preparing a patulin derivative according to claim 1, wherein the patulin derivative is prepared by reacting patulin with an excess of an adjacent dimercapto compound having a structure as shown in formula (ii) in a buffer having a pH of 7-9 at room temperature or under heating conditions, wherein the reaction process is as follows:
，
wherein R is 1 H, CH of a shape of H, CH 3 、SO 3 、SO 3 Na、Ph、C 6 H 5 CH 2 Or (CH) 2 ) n CH 3 Wherein n is an integer other than 0; r is R 2 H, CH of a shape of H, CH 3 、SO 3 、SO 3 Na, ph or C 6 H 5 CH 2 。
3. The method of claim 2, wherein the adjacent dimercapto compound is one of 1, 2-ethanedithiol, dimercaptopropanol, 2, 3-dimercaptosuccinic acid, sodium 2, 3-dimercaptopropanesulfonate, or 2, 3-butanedithiol.
4. The method according to claim 2, characterized in that the molar ratio of patulin to adjacent dimercapto compound is 1: (2-5).
5. A patulin derivative antigen, characterized in that the patulin derivative antigen is a conjugate of the patulin derivative of claim 1 and a carrier protein, wherein the carrier protein is bovine serum albumin, human serum albumin, chicken egg albumin or hemocyanin.
6. Use of a patulin derivative according to claim 1 or a patulin derivative antigen according to claim 5 for non-disease diagnosis in immunological detection of patulin or patulin derivatives.
7. Use of a patulin derivative according to claim 1 as hapten and standard substance in immunological detection of patulin and patulin derivatives.
8. The patulin derivative antibody is prepared from the patulin derivative antigen according to claim 5 through animal immunization, and the patulin derivative antibody is a patulin monoclonal antibody.
9. The device for detecting the patulin is characterized by comprising a test strip and a microporous reagent, wherein the test strip is provided with a detection line, the detection line is coated with the patulin derivative antigen as claimed in claim 5, and the microporous reagent contains the patulin derivative antibody as claimed in claim 8 marked by colloidal gold.
10. A method for rapidly detecting patulin in fruit and vegetable juices and beverages thereof is characterized in that the method is characterized in that patulin in a sample to be detected is subjected to derivatization treatment by adopting the method for preparing patulin derivatives according to claim 2, then the patulin derivatives in the sample to be detected are detected by using the patulin detection device according to claim 9, and the sample is fruit and vegetable juice or a beverage containing the fruit and vegetable juice, and the fruit and vegetable juice is fruit juice, vegetable juice or a mixture of fruit juice and vegetable juice.
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* Cited by examiner, † Cited by third party
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WO2012106794A1 (en) * 2011-02-10 2012-08-16 Universidade Federal Do Rio Grande Do Sul - Ufrgs Use of nucleophilic chalcogen or amino acid compounds as reagents for reducing or eliminating patulin from fruit-based beverages and processed foodstuffs
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