CN108535376A - The detection method of aflatoxin in a kind of vegetable protein beverage - Google Patents
The detection method of aflatoxin in a kind of vegetable protein beverage Download PDFInfo
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- CN108535376A CN108535376A CN201810294462.7A CN201810294462A CN108535376A CN 108535376 A CN108535376 A CN 108535376A CN 201810294462 A CN201810294462 A CN 201810294462A CN 108535376 A CN108535376 A CN 108535376A
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- aflatoxin
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The present invention relates to aflatoxin to test and analyze field, and in particular to the detection method of aflatoxin in a kind of vegetable protein beverage.Include the following steps：Vegetable protein beverage sample is taken, the methanol aqueous solution extraction and cleaning liquid containing trichloroacetic acid is added, ultrasonic extraction, centrifugation take clear solution to be diluted, and through affine in immunity column purification, are measured to aflatoxin content with high performance liquid chromatography.The method of the present invention can synchronize Aflatoxins M1, B1, B2, G1 and G2 in detection vegetable protein beverage, accurate, quick, safety, strong antijamming capability.Detection is limited to 0.05 μ g/kg, and specificity is high, repeats, the rate of recovery 96.00~104.80%.Manufacturing enterprise, government's detection and supervision demand are disclosure satisfy that, to ensureing that consumption safety is of great significance.
Description
Technical field
The present invention relates to aflatoxin to test and analyze field, and in particular to aflatoxin in a kind of vegetable protein beverage
Detection method.
Background technology
Aflatoxin (Aflatoxins, AFT) is toxic by one kind of aspergillus flavus and the generation of aspergillus parasiticus fungal metabolite
Mycotoxin, be that the mankind have found the strongest a kind of mycotoxin of pollution agricultural product toxicity so far.Vegetable protein beverage is mainly
It is primary raw material (such as soybean, peanut, almond, walnut kernel, coconut, ear of maize) or addition dairy products tune with plant kernel, pulp etc.
Milk beverage made of system.The raw material of the vegetable protein beverages such as peanut, walnut is highly prone to Huang in plantation, storage and process
Aspertoxin pollutes, since aflatoxin B1, B2, G1, G2 property are stablized, the raw material being contaminated meeting after being processed into beverage
Toxin is brought into finished product；In addition, Aflatoxins M1 is the main harm factor in milk and milk products product, through dairy products
The vegetable protein beverage of modulation, it is also possible to contain Aflatoxins M1 toxin.
If taking no action to monitor the aflatoxin contamination problem of vegetable protein beverage, in vegetable protein beverage
Aflatoxin contamination problem will become security-hidden trouble, it is serious that restrict vegetable protein beverage industry fast-developing.
To ensure vegetable protein beverage quality safety and consumption safety, need to study aspergillus flavus in a kind of vegetable protein beverage
Toxin detection technology is quickly measured with realization, is easy to operate, answered convenient for being promoted in manufacturing enterprise and primary monitor mechanism of government
Purpose, it is horizontal to promote manufacturing enterprise's product quality monitoring ability and the supervision of primary monitor mechanism of government, reduce finished product
Pollution ratio promotes industry healthy development, ensures vegetable protein beverage quality safety.
Invention content
Technical problem to be solved by the present invention lies in provide a kind of to meet plant egg in view of the deficiencies of the prior art
Aflatoxin detection demand, step are simple in white beverage, the method that aflatoxin quickly detects can be achieved.
To achieve the above object, the present invention takes following technical scheme：
The detection method for providing aflatoxin in a kind of vegetable protein beverage, includes the following steps：Vegetable protein is taken to drink
To expect sample, the methanol aqueous solution extraction and cleaning liquid containing trichloroacetic acid is added, ultrasonic extraction, centrifugation take clear solution to be diluted,
Through affine in immunity column purification, aflatoxin content is measured with high performance liquid chromatography.
By said program, the dilution is PBS solution with solution, and solution crosses qualitative filter paper after dilution, collects filtrate, then
Filtrate is subjected to purification concentration with Aspergillus flavus toxin immuno-affinity column, is eluted with methanol, eluent is collected, detection is spare；
The PBS solution is pH 7.4, the PBS solution of 0.01mol/L.
By said program, the aflatoxin is aflatoxin B1, B2, G1, G2 and M1；Aflatoxin content is surveyed
Surely the detection method used is Post-column photochemical derivatization high performance liquid chromatography.
By said program, the ultrasonic extraction conditions are 45~60 DEG C, and ultrasonic time is 5min or more.
By said program, the centrifugation is that 8000rpm/min centrifuges 5min.
Said program is connect, the Aspergillus flavus toxin immuno-affinity column is even as needed by the Ago-Gel that CNBr is activated
Join aflatoxin monoclonal antibody in being loaded in column tube, the aflatoxin monoclonal antibody can be selected as needed
Select the aflatoxin general purpose single gram generated by the hybridoma cell strain 1C11 secretions that deposit number is CCTCC NO.C201013
Grand antibody, the Aflatoxins M1 generated by the hybridoma cell strain 2C9 secretions that deposit number is CCTCC NO.C C201018
Monoclonal antibody etc..
By said program, the extraction and cleaning liquid is that trichloroacetic acid and methanol aqueous solution are 0.95 in mass ratio:100~
1.05:The mixed solution of 100 compositions, preparation method：Methanol aqueous solution, ultrasonic dissolution is added in trichloroacetic acid by dosage according to the ratio
It obtains.
It is pressed by above-mentioned side, a concentration of the 45~55% of methanol aqueous solution.
It is pressed by above-mentioned side, the optimum amount of the extraction and cleaning liquid is that 0.5mL-2.0mL extraction and cleanings are added in every gram of sample
Liquid.
Said program is connect, the high performance liquid chromatography detection condition is：Chromatographic column：C184.6 × 250mm, 5 μm；Column
Temperature：30 DEG C, mobile phase：45% methanol aqueous solution；Flow velocity：0.8ml/min；Sample size：20 μ L, excitation wavelength 360nm, transmitting
Wavelength 440nm.
Beneficial effects of the present invention：
The present invention carries out pre-treatment using autogamy extraction and cleaning liquid, and pre-treating method is simple, and trifluoroacetic acid can quickly sink
Protein in the vegetable protein beverage of shallow lake reduces matrix interference, improves extraction efficiency, reduces interference；By coordinating aspergillus flavus poison
Plain affine in immunity column separating purification can be improved specificity and the sensitivity of aflatoxin detection, shorten detection time, aspergillus flavus
Toxin component separation is complete, and the rate of recovery is high, is suitable for the Quantitative detection of vegetable protein beverage, and then coordinate high-efficient liquid phase color
Spectral technology is detected, and can synchronize Aflatoxins M1, B1, B2, G1 and G2 in detection vegetable protein beverage, accurate, quick, peace
Entirely, strong antijamming capability.The detection of the method for the present invention is limited to 0.05 μ g/kg, and specificity is high, repeats, the rate of recovery
96.00~104.80% disclosure satisfy that manufacturing enterprise, government's detection and supervision demand, to ensureing that consumption safety has
Important meaning.
Overcome that traditional detection method processing is cumbersome, the problem of matrix interference and disadvantage that testing result is inaccurate.
Description of the drawings
Fig. 1 is 1HPLC separation of embodiment of the present invention aflatoxin B1, B2, G1, G2 and M1 spectrogram.Ordinate in figure
Fluorescence intensity, unit FU are represented, abscissa represents retention time, and unit is minute.1 it is AFM1 in figure, 2 be AFG2,3 is
AFG1,4 be AFB2,5 be AFB1.
Specific implementation mode
Below in conjunction with specific embodiment, the present invention is further explained.
Embodiment 1
Concentrate is extracted to prepare：The trichloroacetic acid of 1g ± 0.01g is weighed in beaker, the methanol of 100ml 50% is added
Water, ultrasonic dissolution are spare.
The Ago-Gel 4FF couplings that Aspergillus flavus toxin immuno-affinity column is activated by CNBr are CCTCC by deposit number
The hybridoma cell strain 1C11 of NO.C201013 secretes the aflatoxin monoclonal antibody generated and by deposit number
The Aflatoxins M1 monoclonal antibody that the hybridoma cell strain 2C9 secretion of CCTCC NO.C201018 generates in column tube in loading
It forms.
Weigh 20.0g peanut protein beverages, soy protein beverages, coconut milk protein beverage, walnut dairy protein drinks and apricot
Benevolence dairy protein drinks sample, is added 10.0ml extraction and cleaning liquid, and 50 DEG C of ultrasonic extraction 5min, 8000rpm/min centrifugation 5min take
Clear solution 10.0ml is diluted with 7.4 0.01mol/LPBS solution of 20.0ml pH, crosses qualitative filter paper, is collected filtrate, is taken
15.0ml sample extracting solutions, are concentrated and purified with Aspergillus flavus toxin immuno-affinity column, are eluted with methanol, with Post-column photochemical derivatization height
Effect liquid phase chromatogram method detects, testing conditions：Chromatographic column：C18 4.6 × 250mm, 5 μm；Column temperature：30 DEG C, mobile phase：45% first
Alcohol solution, flow velocity：0.8ml/min；Sample size：20 μ L, excitation wavelength 360nm, launch wavelength 440nm.
Prepare aflatoxin B1, B2, G1, G2 and M1 mixed solution simultaneously, concentration is respectively 0.1,0.25,0.5,1.0,
2.0, the series standard solution of 5.0,10.0,20.0 μ g/kg, sample introduction measures peak area under above-mentioned chromatographic condition, with external standard method
It is quantitative.Using peak area Y as ordinate, concentration X is that abscissa carries out linear regression, as a result such as table 1.
1 aflatoxin retention time of table, linear equation, detection limit and related coefficient
Peanut protein beverage, soy protein beverages, coconut milk protein beverage, walnut dairy protein drinks and almond lactoprotein drink
The testing result of material sample is shown in Table 2.
Aflatoxin B1, B2, G1, G2, M1 content in 2 variety classes sample of table
Note：ND is to be not detected.
Embodiment 2
Accurately weigh confirmed negative peanut protein sample, add respectively the aflatoxin B1 of various concentration, B2,
G1, G2 standard working solution are (specific a concentration of：0 μ g/kg, 2.5 μ g/kg and 5 μ g/kg), the Aflatoxins M1 of various concentration
Standard working solution (specific concentration：0 μ g/kg, 0.5 μ g/kg and 1 μ g/kg), each concentration carries out 5 replications.By table 3
It is found that aflatoxin B1, the relative standard deviation of B2, G1, G2 and M1 object are 1.01~6.42% in Duplicate Samples, sample
Recovery of standard addition 96.00~104.80%.
3 peanut protein beverage of table subscripts the rate of recovery and relative standard deviation
Note：ND is to be not detected.
Claims (10)
1. the detection method of aflatoxin in a kind of vegetable protein beverage, it is characterised in that：Include the following steps：Take plant egg
The methanol aqueous solution extraction and cleaning liquid containing trichloroacetic acid is added in white drink sample, and ultrasonic extraction, centrifugation take clear solution to carry out
Dilution, through affine in immunity column purification, is measured aflatoxin content with high performance liquid chromatography.
2. the detection method of aflatoxin in vegetable protein beverage according to claim 1, it is characterised in that：It is described dilute
It is PBS solution to release with solution, and solution crosses qualitative filter paper after dilution, collects filtrate, then that filtrate is close with Aspergillus flavus toxin immuno
Purification concentration is carried out with column, is eluted with methanol, eluent is collected, detection is spare.
3. the detection method of aflatoxin in vegetable protein beverage according to claim 1, it is characterised in that：The Huang
Aspertoxin is aflatoxin B1, B2, G1, G2 and M1；Light after aflatoxin content measures the detection method used as column
Chemical derivatization high performance liquid chromatography.
4. the detection method of aflatoxin in vegetable protein beverage according to claim 1, it is characterised in that：Described
Ultrasonic extraction conditions are 45~60 DEG C, and ultrasonic time is 5min or more.
5. the detection method of aflatoxin in vegetable protein beverage according to claim 1, it is characterised in that：Described
Centrifugation is that 8000rpm/min centrifuges 5min.
6. the detection method of aflatoxin in vegetable protein beverage according to claim 1, it is characterised in that：Described
The Ago-Gel that Aspergillus flavus toxin immuno-affinity column is activated by CNBr be coupled as needed aflatoxin monoclonal antibody in
It is loaded in column tube.
7. the detection method of aflatoxin in vegetable protein beverage according to claim 1, it is characterised in that：Described
Extraction and cleaning liquid is that trichloroacetic acid and methanol aqueous solution are 0.95 in mass ratio:100~1.05:The mixed solution of 100 compositions.
8. the detection method of aflatoxin in vegetable protein beverage according to claim 1 or claim 7, it is characterised in that：First
A concentration of the 45~55% of alcohol solution.
9. the detection method of aflatoxin in vegetable protein beverage according to claim 1, it is characterised in that：It is described to carry
It is that 0.5mL-2.0mL extraction and cleaning liquid is added in every gram of sample to take the dosage of scavenging solution.
10. the detection method of aflatoxin in vegetable protein beverage according to claim 1, it is characterised in that：It is described
High performance liquid chromatography detection condition be：Chromatographic column：C184.6 × 250mm, 5 μm；Column temperature：30 DEG C, mobile phase：45% methanol-water
Solution；Flow velocity：0.8ml/min；Sample size：20 μ L, excitation wavelength 360nm, launch wavelength 440nm.
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Cited By (4)
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| CN109324129A (en) * | 2018-10-22 | 2019-02-12 | 国家烟草质量监督检验中心 | A kind of method of aflatoxin in measurement electronic cigarette liquid |
| CN109468367A (en) * | 2018-12-07 | 2019-03-15 | 中国农业科学院油料作物研究所 | Aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount and its detection method |
| CN109557314A (en) * | 2018-12-07 | 2019-04-02 | 中国农业科学院油料作物研究所 | A method of virulence is produced for characterization and evaluation aflatoxicogenic strain |
| CN114166973A (en) * | 2021-12-06 | 2022-03-11 | 北京市粮食科学研究院有限公司 | Rapid quantitative detection method for aflatoxin B1 in vegetable oil |
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| CN109324129A (en) * | 2018-10-22 | 2019-02-12 | 国家烟草质量监督检验中心 | A kind of method of aflatoxin in measurement electronic cigarette liquid |
| CN109468367A (en) * | 2018-12-07 | 2019-03-15 | 中国农业科学院油料作物研究所 | Aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount and its detection method |
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| CN114166973A (en) * | 2021-12-06 | 2022-03-11 | 北京市粮食科学研究院有限公司 | Rapid quantitative detection method for aflatoxin B1 in vegetable oil |
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