CN103604674A - Method for extracting and purifying ochratoxin A from yeast for making hard liquor - Google Patents
Method for extracting and purifying ochratoxin A from yeast for making hard liquor Download PDFInfo
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- CN103604674A CN103604674A CN201310601670.4A CN201310601670A CN103604674A CN 103604674 A CN103604674 A CN 103604674A CN 201310601670 A CN201310601670 A CN 201310601670A CN 103604674 A CN103604674 A CN 103604674A
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Abstract
The invention belongs to the field of food processing processes, relates to methods for detecting toxins in solid-state food fermentation products and particularly relates to a method for extracting and purifying ochratoxin A from yeast for making hard liquor. The method comprises the following steps: (1) putting a certain amount of crushed yeast sample in a centrifuge tube, then, adding a certain volume of sample extracting solution, and violently oscillating on a vortex oscillator; (2) carrying out centrifugation by adopting a centrifuge; (3) obtaining supernatant, and filtering the supernatant through glass-fiber filter paper; (4) accurately measuring a certain volume of filtered liquid, putting the measured filtered liquid in a centrifuge tube, adding a chloroform reagent, violently oscillating on the vortex oscillator, and then, standing; (5) after delamination, putting a lower-layer chloroform phase in a centrifuge tube, and drying by blowing nitrogen gas at the temperature of 50 DEG C; (6) putting chromatographic-grade methanol in the centrifuge tube dried by blowing nitrogen gas, and violently oscillating to dissolve ochratoxin A. The method has the advantages of low cost, good effect, simplicity and easiness in operation and high extraction ratio.
Description
Technical field
The invention belongs to food processing technology field, relate to the detection method of toxin in solid-state food tunning, be specially extraction, the purification process of ochratoxin A in a kind of Daqu.
Background technology
Ochratoxin A is a kind of colourless crystallization compound.Dissolve in polar organic solvent and dilute sodium bicarbonate solution.Be slightly soluble in water.Ochratoxin A is to be produced by multiple aspergillus and the mould being grown on the crops such as grain (wheat, corn, barley, oat, rye, rice and broomcorn millet class etc.), peanut, vegetables (beans).Animal has been taken in after the feed going mouldy, and this toxin also may appear in the meat of pig and hen etc.Ochratoxin is mainly encroached on animal's liver and kidney.This toxin is mainly to cause kidney injury, and a large amount of toxin also may cause intestinal mucosa inflammation and the necrosis of animal.In animal experiment, also observe its teratogenesis.
In recent years, food security has caused that society pays close attention to widely.Employing ELASA(euzymelinked immunosorbent assay (ELISA)) ochratoxin content in detection testing sample that can be very fast.Yet how fast and effectively extraction and purified toxins have become the bottleneck of this technology application of restriction.Application present technique can fast, accurately be extracted the ochratoxin in food samples prepared by Daqu, wine unstrained spirits, solid state fermentation etc., for follow-up ELASA or liquid chromatographic detection.
Euzymelinked immunosorbent assay (ELISA) refers to utilizes the immune response of antigen and antibody and the efficient catalytic effect of enzyme, presents color and luster reaction under certain condition, by spectrophotometer, it is carried out to qualitative, quantitative mensuration.The method has the advantages such as high specificity, highly sensitive, analysis speed is fast, can for ochratoxin A content in testing sample, carry out qualitative and quantitative analysis fast.
ELASA method degree of accuracy not high when ochratoxin A content quantitatively detects in testing sample is the subject matter that long-term puzzlement the method is promoted the use of, traditional use immune affinity column extracts purifying, though effect is good, but high to equipment requirement, step is complicated, and expensive, cost is higher, therefore how simple and efficient, cost-effective extraction and purified toxins become the bottleneck of this technology application of restriction.
By the extraction effect of ochratoxin extraction effect and GB GBT25220-2010 immune affinity column method in comparison ochratoxin ELASA kit, find that the ochratoxin content that in existing ELASA kit, ochratoxin A extraction effect obtains than immune affinity column method of purification is low; And adopt ochratoxin immune affinity column to extract purifying ochratoxin, can greatly increase testing cost undoubtedly.Therefore very necessary by the improvement of ochratoxin A extraction, purifying process in solid finished product Daqu sample is solved to this problem.
Summary of the invention
The present invention, just for above technical matters, provides extraction, the purification process of ochratoxin A in a kind of Daqu, and the testing cost of the method is low, effective, low for equipment requirements, and step is simple and quick.
Concrete technical scheme of the present invention is as follows:
1. get the Daqu sample after a certain amount of pulverizing, be placed in centrifuge tube, then add a certain amount of sample extracting solution, concuss on vortex oscillator; Described sample extracting solution is the liquid that deionized water mixes for the ratio of 7:3 by volume with methyl alcohol, and methyl alcohol is chromatographically pure; The quality of Daqu sample: the volume=1:4 of sample extracting solution, the time of concuss is 10min on vortex oscillator.
2. adopt hydro-extractor to carry out centrifugal; The hydro-extractor adopting is 4000rpm, and centrifugation time is 5min.
3. get supernatant, by glass fiber filter paper, filter;
4. after accurately measuring a certain amount of filtration, liquid, in centrifuge tube, adds methenyl choloride reagent, standing again after concuss on vortex oscillator; Described methenyl choloride reagent is chromatographically pure, and the liquid after filtration and the volume ratio of methenyl choloride are 1:1.Standing again after concuss on vortex oscillator, the time of its concuss is 5min, standing 10min
5. after layering, taking off layer methenyl choloride in centrifuge tube, is with nitrogen, to dry up under the condition of 50 ℃ in temperature;
6. get in the centrifuge tube of hplc grade methanol after nitrogen dries up, concuss, dissolves ochratoxin A.In last 0.5mL methyl alcohol, the content of ochratoxin A equates with the content in 0.5g Daqu.
Good effect of the present invention is embodied in:
(1), extraction cost is low, effective, extract and purifying rate high;
(2), low for equipment requirements, step is simple and quick;
(3), the method for this extraction and purifying relatively saves time, National Standard Method, due to needs affinity column method of purification, at least need the time of 4-6, and this method only needs the time about half an hour.
Accompanying drawing explanation
Fig. 1 is the typical curve of drawing after ochratoxin A standard items gradient dilution.As can be seen from Figure 1, related coefficient reaches 0.9994, and aberration rate is less than 10%, meets requirement of experiment.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, the present invention is described in further detail, but this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following embodiment.
Embodiment 1:
The extraction of ochratoxin A, a purification process in Daqu, specifically comprise the following steps:
1. get the Daqu sample after 5g pulverizes, be placed in the centrifuge tube of 50ml, then add the sample extracting solution of 20ml, concuss 10min on vortex oscillator; Sample extracting solution is the liquid that deionized water mixes for the ratio of 7:3 by volume with methyl alcohol, and methyl alcohol is chromatographically pure, the quality of Daqu sample: the volume=1:4 of sample extracting solution.
2. adopt the hydro-extractor of 4000rpm to carry out centrifugal 5min;
3. the supernatant of getting 10ml, filters by glass fiber filter paper;
4. accurately measure 4ml filter after liquid in the centrifuge tube of 20ml, add 4ml methenyl choloride reagent, standing 10min again after concuss 5min on vortex oscillator;
5. after layering, taking off layer methenyl choloride phase 2ml in the centrifuge tube of 5ml, is with nitrogen, to dry up under the condition of 50 ℃ in temperature;
6. get in the centrifuge tube of 0.5ml hplc grade methanol after nitrogen dries up, concuss, dissolves ochratoxin A, and in 0.5mL methyl alcohol, the content of ochratoxin A equates with the content in 0.5g Daqu.
The drafting of typical curve
Standard items carry out obtaining working concentration gradient after 3 times of dilutions and are respectively 0ppb, 0.1ppb, and 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb (ppb is equivalent to μ g/kg), label is C1, C2, C3, C4, C5, C6.Microplate reader is set and in 450nm place, carries out the detection of OD value, detect testing result that data obtain after by RIDAWIN software analysis and typical curve if table 1 is with as shown in Fig. 1:
The testing result of table 1. ochratoxin A standard model
Comparative example 1: keep remaining parameter constant with embodiment 1, only change methyl alcohol in sample extracting solution (being called for short A) and the water volume ratio of (being called for short B), other steps are with this technique, and its testing result is as shown in the table:
Table 2. methyl alcohol and water different proportion extract testing result
As shown in Table 2: when methyl alcohol and water ratio reach after 7:3, extraction effect is best, and more stable, and effect reaches best during 7:3, and the optimal proportion of determining methyl alcohol and water is 7:3.
Comparative example 2: keep all the other parameter constants with embodiment 1, only change in Daqu sample when ochratoxin A purifies, sample extracting solution supernatant (being called for short C): chloroform soln (being called for short D) volume ratio is respectively 3:1,2:1,1:1,1:2,1:3, and testing result is as shown in the table:
Table 3. sample extracting solution supernatant: chloroform soln different proportion purifies testing result
As shown in Table 3: when sample extraction supernatant and methenyl choloride ratio reach after 1:1, gained testing result tends towards stability, and considers clean-up effect and drug expenditure, determines that optimal proportion is 1:1.
Comparative example 3: the method for extraction and purification of the ochratoxin A that relatively ELASA immune reagent kit provides, the method for extraction and purification (being immune affinity chromatographic column method) of ochratoxin A that GB GBT25220-2010 provides and the difference of the method for extraction and purification of the ochratoxin A that this patent provides;
The extracting method that ELASA immune reagent kit provides is:
(1) preparation of sample extracting solution: with deionized water, methyl alcohol is diluted by 6:4 volume ratio, 6 parts of methyl alcohol add 4 parts of deionized waters, for extracting the ochratoxin A of sample;
(2) pulverize and get the representational sample of 5g in 50mL centrifuge tube, add 25mL sample extracting solution, add 2mL sherwood oil simultaneously, concuss 5min on vortex oscillator;
(3) on hydro-extractor, use the centrifugal 5min of rotating speed of 4000r/min;
(4) get middle layer and use distilled water two-fold dilution, after dilution, liquid is to be measured, and in sample, the content of ochratoxin A is equivalent to dilute 10 times.
The immune affinity chromatographic column method adopting in GB GBT25220-2010:
(1) preparation of sample extraction reagent:
1. sample extracting solution: methyl alcohol is diluted to (8 parts of methyl alcohol+2 part deionized waters) by 8:2 volume ratio with deionized water;
2. PBS damping fluid: take 8g sodium chloride, 1.2g sodium hydrogen phosphate, 0.2g potassium dihydrogen phosphate, 0.2g potassium chloride, dissolves with 990ml distilled water, then with concentrated hydrochloric acid, regulates pH value to 7.0, finally with distilled water diluting to 1000ml;
3. cleaning buffer solution: 25g sodium chloride, 5g sodium bicarbonate and 0.1ml Tween-20 with distilled water diluting to 1000ml;
(2) get 10g pulverizing sample and be dissolved in 20ml sample extracting solution, then add 1g sodium chloride with 2 minutes min of homogenizer high-speed stirred;
(3) on hydro-extractor, use the centrifugal 5min of rotating speed of 4000r/min;
(4) get 10ml supernatant and add 40ml PBS damping fluid, by glass fiber filter paper, filtering, obtaining filtrate;
(5) immune affinity column is connected under glass syringe, accurately pipettes in 10mL filtrate implantation glass syringe;
(6) air pressure pump is connected with glass syringe, regulates pressure, make solution pass through immune affinity column with the flow velocity of about 2mL/min, until air enters in affinity column;
(7) use successively 10mL cleaning buffer solution and 10mL distilled water drip washing immune affinity column, discard whole effluxes, and make 2ml-3ml air pass through cylinder;
(8) finally accurately add 1ml chromatogram methanol-eluted fractions, flow velocity 1ml/min, in 1ml eluent, the content of OTA is equivalent to the content in 1g sample, collects eluent, for detecting.
Extracting method, GB ochratoxin immune affinity column method of purification (GBT25220-2010) that method for extraction and purification used in embodiment 1 and ELASA immune reagent kit are provided compare, by ELASA method testing result relatively.The results are shown in Table 4:
The testing result of table 4 Different Extraction Method ochratoxin A
As seen from the results in Table 4: the testing result of the extraction and purification methods after improve in laboratory is compared yield and improved 23% with kit supplying method, compare with GB (GBT25220-2010) method, can reach the effect that in GB, immune-affinity chromatography reaches, the shortening time is more than 50%, cost-saving more than 90%.
By the repeatability of the method for extraction and purification in this technique and recovering effect check, result is as shown in the table:
Table 5 precision detects
Table 6 Recovery test
Test findings from table 5 and table 6 is known: new extraction and purification methods has that good reappearance is good and new method is good to the recovering effect of mark product, meets testing requirement completely.
Claims (6)
1. the extraction of ochratoxin A, a purification process in Daqu, is characterized in that comprising the following steps:
1. get the Daqu sample after a certain amount of pulverizing, be placed in centrifuge tube, then add a certain amount of sample extracting solution, concuss on vortex oscillator;
2. adopt hydro-extractor to carry out centrifugal;
3. get supernatant, by glass fiber filter paper, filter;
4. after accurately measuring a certain amount of filtration, liquid, in centrifuge tube, adds methenyl choloride reagent, standing again after concuss on vortex oscillator;
5. after layering, taking off layer methenyl choloride in centrifuge tube, is with nitrogen, to dry up under the condition of 50 ℃ in temperature;
6. get in the centrifuge tube of hplc grade methanol after nitrogen dries up, concuss, dissolves ochratoxin A.
2. extraction, the purification process of ochratoxin A in described Daqu according to claim 1, it is characterized in that: the sample extracting solution of step described in is 1. the liquid that deionization waters methyl alcohol mixes for the ratio of 7:3 by volume, and methyl alcohol is chromatographically pure.
3. extraction, the purification process of ochratoxin A in Daqu according to claim 1, is characterized in that: step 4. described methenyl choloride reagent is chromatographically pure, and the liquid after filtration and the volume ratio of methenyl choloride are 1:1.
4. extraction, the purification process of ochratoxin A in Daqu according to claim 1, is characterized in that: the quality of the Daqu sample of step described in 1.: the volume=1:4 of sample extracting solution, the time of concuss is 10min on vortex oscillator.
5. extraction, the purification process of ochratoxin A in Daqu according to claim 1, is characterized in that: the employing hydro-extractor of step described in 2. carries out centrifugal, and the hydro-extractor adopting is 4000rpm, and centrifugation time is 5min.
6. extraction, the purification process of ochratoxin A in Daqu according to claim 1, is characterized in that: step is standing again after concuss on vortex oscillator in 4., and the time of its concuss is 5min, standing 10min.
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Cited By (5)
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| CN104034835A (en) * | 2014-05-27 | 2014-09-10 | 中华人民共和国苏州出入境检验检疫局 | Method for detecting multiple biotoxins in fermented wine |
| CN104178535A (en) * | 2014-04-09 | 2014-12-03 | 上海市农业科学院 | Method for preparing and purifying ochratoxin A and ochratoxin B |
| CN104388318A (en) * | 2014-10-24 | 2015-03-04 | 天津科技大学 | Producing strain for OTA (Ochratoxin A) |
| CN105651915A (en) * | 2015-12-24 | 2016-06-08 | 宁波大学 | Method for detecting ochratoxin A in wine |
| CN113804803A (en) * | 2021-06-28 | 2021-12-17 | 乌鲁木齐海关技术中心 | Rapid determination method suitable for glycyrrhiza uralensis paste ochratoxin A |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104178535A (en) * | 2014-04-09 | 2014-12-03 | 上海市农业科学院 | Method for preparing and purifying ochratoxin A and ochratoxin B |
| CN104034835A (en) * | 2014-05-27 | 2014-09-10 | 中华人民共和国苏州出入境检验检疫局 | Method for detecting multiple biotoxins in fermented wine |
| CN104388318A (en) * | 2014-10-24 | 2015-03-04 | 天津科技大学 | Producing strain for OTA (Ochratoxin A) |
| CN105651915A (en) * | 2015-12-24 | 2016-06-08 | 宁波大学 | Method for detecting ochratoxin A in wine |
| CN113804803A (en) * | 2021-06-28 | 2021-12-17 | 乌鲁木齐海关技术中心 | Rapid determination method suitable for glycyrrhiza uralensis paste ochratoxin A |
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Application publication date: 20140226 |