CN101485408A - Method for degrading aflatoxin - Google Patents

Abstract

The invention discloses a method for degrading aflatoxin, which uses electron beams to radiate a sample containing the aflatoxin to obtain the sample with reduced aflatoxin content. The method can directly radiate finished products such as agricultural products, food, and feed, has simple operation and obvious effects, and makes the degradation rate of aflatoxin B1 reach 71.51 percent.

Description

A kind of method of aflatoxin degradation

Technical field

The present invention relates to a kind of method of aflatoxin degradation.

Background technology

(Aflatoxin AF), is the human and animal to be endangered one of big mycotoxin to aflatoxin.Great mass of data confirms that aflatoxin has very strong toxic action to the liver organization of people and animal, can cause liver cancer when serious, even dead.Aflatoxin contamination food is more serious, and is especially serious and general to the pollution of peanut, walnut, corn and goods thereof.In addition, in soybean, paddy, macaroni, milk and goods thereof, edible wet goods food, also often find aflatoxin.Aflatoxin not only reduces agricultural product and feed quality, and economy is taken a bath, but also can threaten for people's health by polluting or animal derived foods such as the meat of residual aflatoxin, breast enter human body.What wherein toxicity was the strongest is AFB ₁(C ₁₇H ₁₂O ₆, molecular weight 312), be 10 times of potassium cyanide, its chemical structural formula sees that (high bridge is controlled the man to formula I.Peanut aspertoxin pollution channel with prevent and kill off.Peanut science and technology.2000.1:37)。

(formula I)

At present, physical chemistry detoxification method all has the color of food and nutritional labeling such as vitamin, protein etc. and destroys in various degree and harmful effect.Biochemistry detoxification method cost is higher, is difficult in industrialized level and prepares high activity aflatoxin detoxicating activity material.

Summary of the invention

The method that the purpose of this invention is to provide a kind of aflatoxin degradation.

The method of aflatoxin degradation provided by the present invention is to obtain the sample that aflatoxin content reduces with the sample that electron beam irradiation contains aflatoxin.

In the described method, described irradiation dose is 2-10kGy, is preferably 6-10kGy, most preferably is 8-10kGy.

Described aflatoxin is an AFB ₁

The described sample that contains aflatoxin is to contain the agricultural product of aflatoxin or the goods that processing of farm products obtains, as finished products such as food, feeds.

Method of the present invention with electron beam direct to agricultural product, food, finished products such as feed carry out irradiation, method is simple to operate, effect is obvious, can make AFB ₁Degradation rate reaches 71.51%.

Description of drawings

Fig. 1 is an AFB ₁The selection chromatography of ions figure of standard items

Fig. 2 is an AFB ₁Calibration curve

The specific embodiment

Below in conjunction with instantiation the present invention is elaborated.Experimental technique among the following embodiment if no special instructions, is conventional method.

Reagent that following embodiment uses and instrument:

1) agents useful for same

Methyl alcohol (chromatographically pure Fisher); Formic acid (analyzing pure); N-hexane (chromatographically pure, Tianjin Da Mao chemical reagent factory); Sodium chloride; Ultra-pure water.

2) used instrument

Agilent1100 liquid phase systems (U.S. Agilent company);

Three grades of quadrupole rod mass spectrometer systems of API3000 (u.s.a. applied biosystem);

FW100 high speed Universalpulverizer (Tianjin Tai Site Instr Ltd.);

LD4-2 low speed centrifuge (Beijing Medical Centrifugal Machine Factory);

Solid-phase extraction device (U.S. Supelco company);

Oasis HLB solid-phase extraction column (3cc/60mg, Waters company).

AFB in embodiment 1, the reduction microbiological contamination peanut ₁Method

1, AFB in the microbiological contamination peanut ₁Determining of detection method

Now reported for AFB ₁Detection method mostly be high performance liquid chromatography-fluorescence detector detection method, many disturbing factors are arranged, and derive and easily cause experimental error.This experiment is measured AFB1 with high performance liquid chromatography-tandem mass method (being that HPLC-MS/MS detects), and the row method of going forward side by side is learned the validity of this method of checking.

1) selection of qualitative, quantitative determining instrument and condition: this experimental selection chromatogram (retention time is qualitative) and mass spectrum (the test substance characteristic ion is qualitative) double check (being that HPLC-MS/MS detects), can get rid of interference fully, qualitative results is accurately and reliably.Through the optimization of chromatographic condition, select for use retention time be 3.30min (as shown in Figure 1.When Mass Spectrometer Method, adopt the ESI positive ion source, select ion (313.0/241.1, the 269.1) conduct that stability is better and abundance is big to detect example, use the MRM scan mode, external standard method is quantitative.

2) selection of mobile phase: in the LC-MS technology, the selection of the phase that flows must be paid close attention to a separation component and enter mass spectrum thousand Ionized efficient except considering the chromatographic system separating effect, so that best resolution ratio and the highest sensitivity of acquisition.Because most of mycotoxins are soluble in methyl alcohol or acetonitrile, this experiment selects methyl alcohol mobile mutually with the acetonitrile conduct respectively.Experimental results show that, select acetonitrile to make the phase time that flows, the chromatographic system separating effect is better than methyl alcohol, but its degree of ionization is subjected to obvious inhibition, abundance of ions descends, and cause sensitivity to descend, and the degree of ionization of methyl alcohol is higher than acetonitrile, and abundance of ions is also higher, and this experiment adopts methyl alcohol as the phase that flows.

3) HPLC-MS/MS detection chromatogram and mass spectrum condition are as described below:

The selection of mass spectrum condition: this experiment adopts two kinds of different ionization modes of ESI+ and ESI-to AFB respectively for obtaining highly sensitive detection ₁Detect, find under the ESI+ condition AFB ₁Ionization Efficiency apparently higher than the ESI-condition, therefore adopt ESI+ ionization mode.To AFB ₁Under ESI+ ionization mode, carry out full ion scan, acquisition parent ion (M+H)+be 313.0, and determine its secondary ion, the fragment ion that abundance is the highest is 241.1,269.1.So this experimental selection ESI source, cation reaction detection pattern.Prime ion m/z=313.0, secondary ion m/z=241.1,269.1.Concrete chromatogram and mass spectrum condition are as described below:

1. chromatographic condition

The Agilent1100 liquid phase systems, G1312A binary pump system, G1367A type automatic sampler.

Chromatographic column: Agela Technologies Inc.Venusil ASB-C18, column internal diameter * column length 2.1 * 150mm, filler diameter 5 μ m.

Flow velocity: 200 μ l/min;

Phase flows: methyl alcohol: water (0.1% formic acid) (herein being that volumn concentration is 0.1% formic acid solution)=7: 3;

Sampling volume: 50 μ l.

2. mass spectrum condition

Three grades of quadrupole rod mass spectrometer systems of API3000;

Scan mode: multiple-reaction monitoring (MRM), cation analytical model;

Ion gun: Turbo Spray ion gun;

Prime ion: 313.0; Secondary ion: 241.1,269.1.

The mass spectrum parameter is provided with:

Atomization gas (Nebulizer Gas, NEB): nitrogen, 10ml/min;

Curtain gas (Curtain Gas, CUR): nitrogen, 12ml/min;

Ionization voltage (Ionspray Voltage, IS): 5500V;

Heating-up temperature (Temperature, TEM): 500 ℃;

Collision gas (Collision Gas, CAD): nitrogen, 8ml/min;

Go the collection bunch voltage (Declustering Potential, DP): 70V;

Focus voltage (Focusing Potential, FP): 300V;

Inlet voltage (Entrance Potential, EP): 10V;

Impact energy (Collision Energy, CE): 45V;

Collision pond outlet voltage (Collision Cell Exit Potential, CXP): 9V.

4) selection of sample constant volume liquid

Sample is decided solution component directly influences separation degree and the Ionization Efficiency of aflatoxin in chromatographic column, promptly mass spectral sensitivity.Confirm that by experiment methyl alcohol, acetonitrile all can disturb AFB as constant volume liquid ₁Go out peak-to-peak signal, separating degree is poor; And the mixed liquor of methyl alcohol and water (volume ratio of methyl alcohol and water is 5: 5) can make AFB as constant volume liquid ₁Abundance than other constant volume liquid increasing in various degree arranged all, and peak shape improves, separating degree improves.Therefore, select for use the mixed liquor (volume ratio of methyl alcohol and water is 5: 5) of methyl alcohol and water as constant volume liquid.

5) calibration curve

AFB ₁The preparation of standard reserving solution: with methyl alcohol with AFB ₁Standard items (purchasing the Sigma-Aldrich company in the U.S., purity 〉=97%) are mixed with the 100mg/L standard reserving solution, and 4 ℃ keep in Dark Place.

AFB ₁The preparation of standard operation liquid: mixed liquor (volume ratio of methyl alcohol and water is 5: 5) the dilution standard storing solution with methyl alcohol and water is mixed with 0.1,0.2,0.5,1,2,5,10,20,50 μ g/L AFB1 standard operation solution.

Accurately draw 0.1,0.2,0.5,1,2,5,10,20,50 μ g/L AFB1 standard operation solution, 50 μ l, HPLC-MS/MS analyzes, and obtains AFB ₁Peak area carries out linear regression to concentration, promptly gets calibration curve, as shown in Figure 2.

6) detectability and quantitative limit

By 3 times of signal-to-noise ratio computation, when the response of sample component equaled 3 times of baseline noise, the concentration of this sample promptly can be used as LDL.When the response of sample component equaled 10 times of baseline noise, the concentration of this sample promptly can be used as minimum quantitative limit.Work as AFB ₁When concentration was 0.03 μ g/kg, S/N=3.2 was so the LDL of method is about 0.03 μ g/kg; Work as AFB ₁When concentration was 0.1 μ g/kg, S/N=10.2 was so the minimum quantitative limit of method is about 0.1 μ g/kg.

7) rate of recovery and precision evaluation

Getting same not microbiological contamination peanut sample is background, adds 1 μ g/kg, 25 μ g/kg, 500 μ g/kg AFBs ₁(AFB ₁) after, its rate of recovery of HPLC-MS/MS assay determination, and calculate precision, the result is as shown in table 1.

Table 1. AFB ₁Add the rate of recovery and precision

Sample number	Addition (μ g/kg)	Detected value (μ g/kg)	The rate of recovery (%)	Precision RSD (%)
					1	1	1.05±0.03	105.32	4.26
2	25	23.8±0.11	95.22	2.13
					3	500	486.5±0.17	93.71	3.72

2, electron beam irradiation reduces AFB ₁And compliance test result

Aspergillus flavus (Aspergillus flavus) ATCC11498 (available from the ATCC of American Type Culture Collecti, No. 11498 bacterial strains) is inoculated in Czapek's medium ((sodium nitrate 2g, phosphoric acid cyanogen dipotassium 1g, potassium chloride 0.5g, magnesium sulfate 0.5g, ferrous sulfate 0.01g, sucrose 30g, agar 15-20g, water 1000ml) inclined-plane, 28 ℃ of temperature, humidity 50% was cultivated 7 days, treated that the inclined-plane covers with the yellow green mycelia, when slightly giving birth to spore on the mycelia, make spore suspension (10 with sterilized water ⁷Cfu/ml).With the 5g peanut after irradiation 30min sterilization under the uviol lamp, add the 5ml sterilized water (121 ℃, 20min), add spore suspension 1ml mixing then, 28 ℃ of temperature, humidity 50% is cultivated the peanut that obtained infecting Aspergillus flavus in 7 days.

Get the peanut sample of the infection Aspergillus flavus of 6 parts of above-mentioned acquisitions, (material is 99.9% biological grade polypropylene to place a 100ml polypropylene centrifuge tube respectively, radiation hardness) sealing in, put into electron beam irradiation field (SML5520 of Tsing-Hua University type electron linear accelerator) and carry out radiation treatment, irradiation dose is set at 0kGy (non-radiating contrast), 2kGy respectively, 4kGy, 6kGy, 8kGy, 10kGy.The beam energy that accelerator produces is 4.5MeV, and beam power is 2.5kW.Close rate is 0.1kGy/s.Above-mentioned experiment triplicate.

4, AFB ₁The detection of content

To detect AFB through the peanut sample after step 3 is handled ₁Content, concrete grammar is as described below:

1) extract: the irradiation peanut sample places the 100ml centrifuge tube to the peanut sample after above-mentioned irradiation is housed with being equipped with not respectively, adding 50ml volumn concentration is 60% methanol in water, 5g NaCl, 10ml n-hexane, shake up, put into the centrifugal extraction of centrifuge 30min.After the extraction, filter, go into the separatory funnel standing demix, get 5ml lower floor solution and carry out following purification with quantitative filter paper.

2) purify: with OASIS HLB solid-phase extraction column (available from Waters, article No. WAT094226) place the vacuum solid-phase extraction device (available from Agela Technologies, article No. M0401001) on, add the activation of 2ml methyl alcohol, when treating methyl alcohol to OASIS pillar adsorbent (this adsorbent is to belong to solid-phase extraction column to carry) upper strata, add 2ml pure water balance pillar again, add the above-mentioned solution 5ml of lower floor that obtains then and cross post, flow velocity is 1ml/min, add 3ml pure water drip washing OASIS pillar twice, the back is 30% methanol in water with the 2ml volumn concentration, and drip washing OASIS pillar is drained with vavuum pump to remove impurity.With the 1ml methanol-eluted fractions, eluent is settled to 2ml with pure water at last, and is to be analyzed.

3) LC-MS detects

LC-MS analyzes: the AFB that injects 50 μ L debita spissitudos respectively ₁Standard liquid and step 2) sample that obtains of constant volume is respectively in the using high performance liquid chromatography tandem mass spectrum instrument (LC-MS), and 1 described analysis condition is analyzed set by step, the record peak area.The retention time of according to standard sample is qualitative, and external standard method is quantitative.

Through 2kGy, 4kGy, 6kGy, the microbiological contamination peanut sample of 8kGy or 10kGy absorbed dose of radiation irradiation, the AFB after extracting, purify, advance the LC-MS analysis in the sample ₁Peak area, the combined standard curve is converted into the concentration of sample solution, calculates the quality of solute, then divided by the quality of the peanut that takes by weighing, amounts to concentration after obtaining degrading.Degradation rate by under the different absorbents amount amount to concentration with irradiation not the time the concentration of amounting to compare and draw.AFB in the microbiological contamination peanut of calculated not irradiation ₁Pollution concentration be 64 μ g/kg.The aforementioned calculation data are three mean values that repeat.

The result is as shown in table 2, and the result shows, behind electron beam irradiation, and AFB ₁Degradation rate can reach 71.51%.China GB2761-2005 " mycotoxin is limited the quantity of in the food " stipulates AFB ₁Limit the quantity of and be＜20 μ g/kg; And in the world about the content of total aflatoxina generally all less than 15 μ g/kg, to AFB ₁Limit the quantity of require lower.Peanut aflatoxin B ₁Level of pollution is generally 6-400 μ g/kg, therefore, utilizes electron beam irradiation to handle 8kGy and just can effectively lower peanut aflatoxin B ₁About 54%, 10kGy's can effectively lower peanut aflatoxin B ₁About 71.51%, the export trade had the using value of reality.

AFB in the table 2. microbiological contamination peanut ₁Irradiation-induced degradation

Absorbed dose of radiation	Amount to concentration/μ g/kg	Degradation rate/%
			0	64	0
2	49.72	22.31
			4	37.85	40.86
6	34.23	46.51
			8	29.42	54.03
10	18.23	71.51

Claims (6)

1, a kind of method of aflatoxin degradation is to obtain the sample that aflatoxin content reduces with the sample that electron beam irradiation contains aflatoxin.

2, method according to claim 1 is characterized in that: described irradiation dose is 2-10kGy.

3, method according to claim 2 is characterized in that: described irradiation dose is 6-10kGy.

4, method according to claim 3 is characterized in that: described irradiation dose is 8-10kGy.

5, method according to claim 1 is characterized in that: described aflatoxin is an AFB ₁

6, according to any described method among the claim 1-5, it is characterized in that: the described sample that contains aflatoxin is to contain the agricultural product of aflatoxin and/or the goods that processing of farm products obtains.

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