CN101210877A - Method for quantitative determination for crop seeds lipoxidase activity - Google Patents
Method for quantitative determination for crop seeds lipoxidase activity Download PDFInfo
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Abstract
The invention relates to a method for performing single grain quantitative measurement of lipoxidase activity in crop seed, belonging to biochemistry field. The method comprises the following steps of: extracting crude lipoxidase, preparing linoleic acid substrate solution, preparing starch solution, preparing potassium iodide solution, preparing citric acid solution, and measuring the activity of the crude lipoxidase. The invention has the advantages of single grain seed measurement, simple equipment, easy operation, rapid measurement, high throughput and accurate measurement results.
Description
Technical field
The invention belongs to biochemical field, relate to a kind of method of lipoxidase activity in the crop seed being carried out simple grain, quantitative measurement.
Background technology
Wheat is one of human topmost food source, and it can be preserved and utilize for many years.But all the time, the research of wheat mainly concentrated on improve output, protein quality and resistance aspect, and do not pay close attention to its storage quality, wheat is as China's important food strategic reserve material, its storage quality and output no less important.Wheat beginning ageing in 1 year under general holding conditions is rotten, and the time that the bad change of ageing takes place in the storage of hot and humid area is then shorter, and the rotten quality that will cause of wheat ageing reduces, commodity descends, add and go mouldy, influences such as storage pest harm, the storage safety of grain in serious threat.
Lipoxidase (Lipoxygenase, EC 1.13.11.12) is a kind of oxidoreducing enzyme of iron content, that its catalysis contains is suitable-and suitable-1, the polyunsaturated fatty acid of 4-pentadiene and ester thereof form hydroperoxides.Lipoxidase extensively is present in nature, finds in plant, animal and fungi, has found the isodynamic enzyme of multiple lipoxidase in plant, as: in soya seeds, found to have at least three kinds of isodynamic enzymes by the different genes coding.The substrate of lipoxidase is linoleic acid and arachidonic acid, and linoleic acid is the highest fatty acid of content in many vegetable seedss.Linoleic acid forms C6 aldehydes, alcohol compound through the fat oxidation enzymatic, and these volatile matter can produce old flavor, and the quality that causes processing food reduces.Studies show that: the degraded of fatty acid is to cause quality deterioration in the seed preservation process, produces the root of old flavor and bad change, and lipoxidase disappearance or its active reduction can reduce oxidation deterioration, prolong the storage of seeds phase.
The assay method of wheat lipoxidase activity has been reported nearly following 3 kinds.The most normal application is the content of measuring the direct product-hydroperoxides of 234nm place fat oxidation enzyme reaction, and this method is very suitable to the enzyme of having purified, but is not suitable for measuring thick enzymatic activity, understands interference measurement because contain other material that absorbs ultraviolet light in the thick enzyme; Simultaneously, therefore the method complex operation has limited its application.Another kind method is oxygen-electrode method, and it is very accurate that this method is measured, but because oxygen electrode costs an arm and a leg, needs the professional simultaneously, measures loaded down with trivial detailsly, promotes very difficult.The third method is a monoclonal antibody method, and this method need prepare single-minded antibody, measure accurately, but the cycle is long, and very high to the experimental facilities requirement, common lab is difficult to carry out.More than 3 kinds of methods all need the above analytic samples of number gram, can not realize the simple grain quantitative measurement.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of quick, accurate, easy crop single seed lipoxidase activity method for quantitatively determining is provided.
Crop of the present invention comprises crops such as wheat, corn, soybean, paddy rice, peanut, buckwheat, barley.
The cardinal principle of lipoxidase activity method for quantitatively determining in the einkorn seed of the present invention: within a certain period of time, the proportional example of amount of the hydroperoxides that active size and its catalytic reaction of lipoxidase generates, hydroperoxides are under acid condition, can make potassium iodide be oxidized to iodine, iodine and starch reaction generate blue material, and this colour substance has characteristic absorption peak at the 470nm place.Therefore, the amount of mensuration blue material just can correspondingly calculate the activity of lipoxidase.The reaction principle of this experiment is as follows:
Hydroperoxides+I
-+ H
+→ I
2+ linoleic acid alcohol
I
2+ starch → blue material
The present invention measures the method for crop seeds lipoxidase activity, may further comprise the steps:
(1), extracts thick enzyme
Get the simple grain crop seed, (wheat, paddy rice, barley, buckwheat add 0.6ml, and corn adds 1.0ml to add 0.6～3.0ml PH6.0～7.0 phosphate buffers behind the abrasive dust, soybean adds 2.0ml, and peanut adds 3.0ml), left standstill 1 hour in 0 ℃, vibrate each 30 seconds therebetween 2 times; Centrifugal, 12,000 rev/mins, 10 minutes, 4 ℃, supernatant was crossed three layers of garrha and is promptly got crude enzyme liquid, put into-20 ℃ of preservations immediately; Adopt the Coomassie brilliant blue method to measure the content of soluble protein in the extract, typical curve is set up by bovine serum albumin (concentration 10-60 μ g/ml).
(2), preparation linoleic acid substrate solution
Add 15.6 μ l polysorbas20s, 15.6 μ l linoleic acid in 800 μ l distilled water, shake up the NaOH that the back adds 100 μ l, 1M, adding distil water is settled to 5ml.
(3), preparation starch fluid
In 1.0 gram soluble starches, add 100ml distilled water, in the boiling water bath heating transparent up to solution, use the cooling back.
(4), preparation liquor kalii iodide
Take by weighing 1.5 gram KI and add 1.0ml distilled water, slowly heating for dissolving has the KI precipitation after the cooling.
(5), preparation citric acid solution
Take by weighing 52.5 gram citric acids, add distilled water and be settled to 250ml.
(6), the mensuration of thick enzymatic activity
Get the thick zyme extract of 0.1ml, add 0.2ml linoleic acid substrate solution, add the 0.1ml starch fluid after shaking up again, 0.2ml potassium iodide liquid and 2.4ml citric acid solution, 30 ℃ leave standstill the light absorption value of measuring the 470nm place after 5 minutes; Other gets the thick zyme extract of 0.1ml as blank, after in boiling water bath, boiling 10 minutes, add 0.2ml linoleic acid substrate solution, add the 0.1ml starch fluid after shaking up again, 0.2ml potassium iodide liquid and 2.4ml citric acid solution, 30 ℃ leave standstill the light absorption value of measuring the 470nm place after 5 minutes.Under above-mentioned reaction conditions, the enzyme amount that every milligram of total protein of per minute catalysis is required is defined as 1 unit of activity (U), draws the enzyme activity computing formula thus and is: U=(light absorption value * 1000)/(protein content mg * time min).
The purposes of the inventive method is as follows:
1., be used to carry out the genetics research of control lipoxidase activity related gene.
2., the assisted Selection index that is used for the storage endurance crop breeding.
3., screen lipoxidase disappearance or highly active material for the breeding scholar, in parent's resource of storage endurance crop breeding.
4., as the medium-term and long-term seed bank of grain storage department, and peasant's grain storage rich and influential family detects the foundation of its keeping quality, food processing quality.
5., as one of index of planting subdivision identification of species keeping quality.
The present invention has following advantage:
1., directly utilize crop seeds such as wheat to do material, only need single seed to get final product, solved the problem that to carry out the mensuration of lipoxidase activity to the single seed of crop;
2., utilize spectrophotometer that the change color of starch-kalium iodide is measured, do like this and not only have easy, advantage fast, and can carry out quantitative measurement lipoxidase activity;
3., do not need complicated experiment condition and high-accuracy experimental apparatus.This method only needs visible spectrophotometer and hydro-extractor to get final product, and the instrument and equipment expense is low, can satisfy domestic most laboratory conditions, is easy to promote;
4., this method has time weak point, the characteristics that flux is high.The mensuration of finishing single sample only needs 2-3 hour, and the mensuration of finishing up to a hundred samples needed get final product in 1-2 days, can satisfy in the crop breeding processes such as storage endurance wheat the screening to a large amount of separation offsprings;
5., the present invention proposes to measure the method for lipoxidase activity, and is easy and simple to handle, rapid.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1: the simple grain of wheat breed lipoxidase activity is measured
The extraction of thick enzyme: get one in No. 23 seeds in wheat breed Rikaze, add 0.6ml PH7.0 phosphate buffer behind the abrasive dust, left standstill 1 hour in 0 ℃, vibrate each 30 seconds therebetween 2 times; Centrifugal after 1 hour, 12,000 rev/mins, 10 minutes, 4 ℃, supernatant was crude enzyme liquid after crossing three layers of garrha, put into-20 ℃ of preservations immediately.The mensuration of soluble protein content is used the Coomassie brilliant blue method in the extract, and typical curve is set up by bovine serum albumin (concentration 10-60 μ g/ml).
The preparation of linoleic acid substrate solution: add 15.6 μ l polysorbas20s in 800 μ l distilled water, 15.6 μ l linoleic acid shake up the back and add 100 μ l, the NaOH of 1M, and adding distil water is settled to 5ml.
The preparation of starch fluid: in 1.0 gram soluble starches, add 100ml distilled water, in the boiling water bath heating transparent up to solution, use the cooling back.
The preparation of liquor kalii iodide: take by weighing 1.5 gram KI and add 1.0ml distilled water, slowly heating for dissolving.
The preparation of citric acid solution: take by weighing 52.5 gram citric acids, add dissolved in distilled water, be settled to 250ml.
The mensuration of thick enzymatic activity: in the time of 30 ℃, in the thick zyme extract of 0.1ml, add 0.2ml linoleic acid substrate solution, add the 0.1ml starch fluid after shaking up again, 0.2ml potassium iodide liquid and 2.4ml citric acid solution (are got the thick zyme extract of 0.1ml in addition and are boiled 10 minutes as blank in boiling water bath, other operation is the same), leave standstill the light absorption value of measuring the 470nm place after 5 minutes, the activity of measuring No. 23 lipoxidases in Rikaze thus is 179.9U.
Embodiment 2: the simple grain of wheat breed lipoxidase activity is measured
Get one in No. 54 seeds in wheat breed Rikaze, other method is with embodiment 1, and the activity of measuring No. 54 lipoxidases in Rikaze is 177.3U.
Embodiment 3: the simple grain of corn variety lipoxidase activity is measured
Get one in corn variety S37 seed, add 1.0ml PH7.0 phosphate buffer behind the abrasive dust, other method is with embodiment 1, and the activity of measuring corn variety S37 lipoxidase is 149.8U.
Embodiment 4: the simple grain of buckwheat kind lipoxidase activity is measured:
Get one in Heisui River sweet buckwheat local varieties seed, other method is with embodiment 1, and the activity of measuring Heisui River sweet buckwheat local varieties lipoxidase is 83.8U.
Embodiment 5: the simple grain of rice varieties lipoxidase activity is measured
One in bright extensive 63 seeds of water intaking rice varieties, other method is with embodiment 1, and the activity of measuring bright extensive 63 lipoxidases is 126.6U.
Embodiment 6: the simple grain of peanut varieties lipoxidase activity is measured
Get one of peanut varieties self-sufficient and strategically located region tenth-seeded, add 3.0ml PH7.0 phosphate buffer behind the abrasive dust, other method is with embodiment 1, and the activity of measuring the lipoxidase of No. 10 peanuts in the self-sufficient and strategically located region is 188.6U.
Embodiment 7: the simple grain of soybean varieties lipoxidase activity is measured
Get soybean varieties and become one in No. 1 seed of beans, add 2.0ml PH6.0 phosphate buffer behind the abrasive dust, other method is with embodiment 1, and the activity of measuring into No. 1 lipoxidase of beans is 583.6U.
Embodiment 8: the simple grain of barley variety lipoxidase activity is measured
Get one in dark blue 311 seeds of barley variety, add 0.6ml PH7.0 phosphate buffer behind the abrasive dust, other method is with embodiment 1, and the activity of measuring dark blue 311 lipoxidases of barley is 174.1U.
Claims (2)
1. the method for a quantitative determination for crop seeds lipoxidase activity is characterized in that may further comprise the steps:
(1), extracts thick enzyme: get the simple grain crop seed, add 0.6～3.0ml PH6.0～7.0 phosphate buffers behind the abrasive dust, left standstill 1 hour in 0 ℃, vibrate each 30 seconds therebetween 2 times, 12000 rev/mins centrifugal 10 minutes, 4 ℃, supernatant is crossed three layers of garrha and is promptly got crude enzyme liquid, puts into-20 ℃ of preservations immediately, adopt the Coomassie brilliant blue method to measure the content of soluble protein in the extract, typical curve is the bovine serum albumin of 10-60 μ g/ml by concentration;
(2), preparation linoleic acid substrate solution: add 15.6 μ l polysorbas20s in 800 μ l distilled water, 15.6 μ l linoleic acid shake up the NaOH that the back adds 100 μ l, 1M, and adding distil water is settled to 5ml;
(3), the preparation starch solution: in 1.0 gram soluble starches, add 100ml distilled water, in the boiling water bath heating transparent up to solution, use the cooling back;
(4), preparation liquor kalii iodide: take by weighing 1.5 gram KI, add 1.0ml distilled water, slowly heating has the KI precipitation after the cooling;
(5), preparation citric acid solution: take by weighing 52.5 gram citric acids, add distilled water and be settled to 250ml;
(6), the mensuration of thick enzymatic activity: get the thick zyme extract of 0.1ml, add 0.2ml linoleic acid substrate solution, add 0.1ml starch fluid, 0.2ml potassium iodide liquid and 2.4ml citric acid solution after shaking up again, 30 ℃ leave standstill the light absorption value of measuring the 470nm place after 5 minutes; Other gets the thick zyme extract of 0.1ml as blank, after in boiling water bath, boiling 10 minutes, add 0.2ml linoleic acid substrate solution, add the 0.1ml starch fluid after shaking up again, 0.2ml potassium iodide liquid and 2.4ml citric acid solution, 30 ℃ leave standstill the light absorption value of measuring the 470nm place after 5 minutes; Under above-mentioned reaction conditions, the enzyme amount that every milligram of total protein of per minute catalysis is required is defined as 1 unit of activity (U), draws the enzyme activity computing formula thus and is: U=(light absorption value * 1000)/(protein content mg * time min).
2. the method for quantitative determination for crop seeds lipoxidase activity according to claim 1, it is characterized in that: the addition of phosphate buffer is different because of the difference of crop species, wherein the addition of wheat, paddy rice, barley, buckwheat is 0.6ml, the addition of corn is 1.0ml, the addition of soybean is 2.0ml, and the addition of peanut is 3.0ml.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102279181A (en) * | 2011-04-18 | 2011-12-14 | 中国农业大学 | Method for testing activity of corn seed embryo lipoxidase |
| CN102323229A (en) * | 2011-08-09 | 2012-01-18 | 丹阳市江南面粉有限公司 | Method for quickly measuring activity of lipoxidase in grain processing byproduct |
| CN102507488A (en) * | 2011-10-25 | 2012-06-20 | 长沙理工大学 | Quantitative measurement method for activity of lipoxygenases of animal tissue |
| CN103913450A (en) * | 2014-03-21 | 2014-07-09 | 中山市南方新元食品生物工程有限公司 | Accurate quantitative detection method for activity of lipoxygenase |
| CN106018311A (en) * | 2016-05-20 | 2016-10-12 | 浙江省农业科学院 | Method for quickly detecting anti-inflammatory activity of plant water extracts |
| CN106405024A (en) * | 2016-08-25 | 2017-02-15 | 青岛啤酒股份有限公司 | Method for evaluating malt lipid oxidation degree |
| CN106124640B (en) * | 2016-04-14 | 2018-06-15 | 中国水稻研究所 | A kind of method for measuring amylose in rice and amylopectin content with half |
| CN108624569A (en) * | 2018-05-08 | 2018-10-09 | 河南农业大学 | Wheat seed lipoxidase new gene and its application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1102022C (en) * | 2000-11-14 | 2003-02-26 | 南京农业大学 | Storage burable rice variety developing method |
| CN1680587A (en) * | 2005-01-27 | 2005-10-12 | 中国科学院等离子体物理研究所 | Rapid detection of lipase activity of crop seed |
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2006
- 2006-12-25 CN CNB2006100226380A patent/CN100547386C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102279181A (en) * | 2011-04-18 | 2011-12-14 | 中国农业大学 | Method for testing activity of corn seed embryo lipoxidase |
| CN102323229A (en) * | 2011-08-09 | 2012-01-18 | 丹阳市江南面粉有限公司 | Method for quickly measuring activity of lipoxidase in grain processing byproduct |
| CN102507488A (en) * | 2011-10-25 | 2012-06-20 | 长沙理工大学 | Quantitative measurement method for activity of lipoxygenases of animal tissue |
| CN102507488B (en) * | 2011-10-25 | 2013-08-14 | 长沙理工大学 | Quantitative measurement method for activity of lipoxygenases of animal tissue |
| CN103913450A (en) * | 2014-03-21 | 2014-07-09 | 中山市南方新元食品生物工程有限公司 | Accurate quantitative detection method for activity of lipoxygenase |
| CN106124640B (en) * | 2016-04-14 | 2018-06-15 | 中国水稻研究所 | A kind of method for measuring amylose in rice and amylopectin content with half |
| CN106018311A (en) * | 2016-05-20 | 2016-10-12 | 浙江省农业科学院 | Method for quickly detecting anti-inflammatory activity of plant water extracts |
| CN106018311B (en) * | 2016-05-20 | 2018-10-30 | 浙江省农业科学院 | A kind of method of quick detection low cost vegetable plant water extract anti-inflammatory activity |
| CN106405024A (en) * | 2016-08-25 | 2017-02-15 | 青岛啤酒股份有限公司 | Method for evaluating malt lipid oxidation degree |
| CN106405024B (en) * | 2016-08-25 | 2018-08-24 | 青岛啤酒股份有限公司 | A method of evaluation malt lipid oxidation degree |
| CN108624569A (en) * | 2018-05-08 | 2018-10-09 | 河南农业大学 | Wheat seed lipoxidase new gene and its application |
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