CN103913451A - Rapid qualitative detection method for activity of lipoxygenase - Google Patents
Rapid qualitative detection method for activity of lipoxygenase Download PDFInfo
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- 102000003820 Lipoxygenases Human genes 0.000 title claims abstract description 45
- 108090000128 Lipoxygenases Proteins 0.000 title claims abstract description 45
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 230000000694 effects Effects 0.000 title claims abstract description 21
- 102000004190 Enzymes Human genes 0.000 claims abstract description 44
- 108090000790 Enzymes Proteins 0.000 claims abstract description 44
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- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 claims abstract description 14
- 235000020778 linoleic acid Nutrition 0.000 claims abstract description 14
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 11
- 239000006172 buffering agent Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 52
- 230000002255 enzymatic effect Effects 0.000 claims description 35
- UORVGPXVDQYIDP-UHFFFAOYSA-N borane Chemical compound B UORVGPXVDQYIDP-UHFFFAOYSA-N 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 235000019197 fats Nutrition 0.000 claims description 14
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 claims description 13
- 229910000085 borane Inorganic materials 0.000 claims description 13
- BTIJJDXEELBZFS-UHFFFAOYSA-K hemin Chemical compound [Cl-].[Fe+3].[N-]1C(C=C2C(=C(C)C(C=C3C(=C(C)C(=C4)[N-]3)C=C)=N2)C=C)=C(C)C(CCC(O)=O)=C1C=C1C(CCC(O)=O)=C(C)C4=N1 BTIJJDXEELBZFS-UHFFFAOYSA-K 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 230000003647 oxidation Effects 0.000 claims description 8
- 238000007254 oxidation reaction Methods 0.000 claims description 8
- 239000000839 emulsion Substances 0.000 claims description 7
- 239000012723 sample buffer Substances 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000013016 damping Methods 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 150000002617 leukotrienes Chemical class 0.000 claims description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
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- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- IYXXQOGEFHAQGU-GPWRMYTLSA-N [(z)-(3-methyl-1,3-benzothiazol-2-ylidene)amino]azanium;chloride;hydrate Chemical compound O.Cl.C1=CC=C2S\C(=N/N)N(C)C2=C1 IYXXQOGEFHAQGU-GPWRMYTLSA-N 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 16
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 238000005259 measurement Methods 0.000 abstract description 2
- RJTANRZEWTUVMA-UHFFFAOYSA-N boron;n-methylmethanamine Chemical compound [B].CNC RJTANRZEWTUVMA-UHFFFAOYSA-N 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 19
- 239000000047 product Substances 0.000 description 16
- 244000068988 Glycine max Species 0.000 description 9
- 235000010469 Glycine max Nutrition 0.000 description 9
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- 102000004316 Oxidoreductases Human genes 0.000 description 7
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- 238000002798 spectrophotometry method Methods 0.000 description 4
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- 229920002472 Starch Polymers 0.000 description 3
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 3
- 241000209140 Triticum Species 0.000 description 3
- 235000021307 Triticum Nutrition 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
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- 239000008107 starch Substances 0.000 description 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
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- PHOLIFLKGONSGY-CSKARUKUSA-N (e)-(3-methyl-1,3-benzothiazol-2-ylidene)hydrazine Chemical compound C1=CC=C2S\C(=N\N)N(C)C2=C1 PHOLIFLKGONSGY-CSKARUKUSA-N 0.000 description 1
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- XMBWDFGMSWQBCA-UHFFFAOYSA-M iodide Chemical compound [I-] XMBWDFGMSWQBCA-UHFFFAOYSA-M 0.000 description 1
- 229940006461 iodide ion Drugs 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a rapid qualitative detection method for the activity of lipoxygenase enzyme, wherein, a solution A containing dimethylamine borane buffering agent and linoleic acid solution, a solution B containing phenol reagent and ferreme solution and an enzyme product solution containing lipoxygenase are mixed, and then are kept stand for color development; judging whether the enzyme product solution has the activity of the lipoxygenase according to whether the enzyme product solution is developed. The method for rapidly and qualitatively detecting the activity of the lipoxygenase has the characteristics of simple and convenient operation and rapid sample measurement, and is suitable for being applied in production practice.
Description
Technical field
The present invention relates to a kind of zymetology detection technique field, relate in particular to a kind of Quick qualitative detection method of lipoxidase enzymatic activity.
Background technology
In the wheat flour of preparing food, due to the wheat place of production and the not equal factor of kind, cause the quality discrepancy of wheat flour very large, conventionally need to add flour improver to strengthen elasticity and the toughness of dough, and the rheological properties of improvement dough, so that the flour-made food texture making is even, regular, level and smooth, color and luster is pure white, springiness and delicate mouthfeel.
Traditional flour improver is taking chemical addition agent as main.Along with the raising day by day of people's living standard, food security and health diet more and more come into one's own.Therefore, the requirement to food material and adjuvant and monitoring are more and more stricter.Use safe biological enzyme formulation to substitute traditional chemicals and become a megatrend of food service industry.In order to cater to this industrial trend, and synchronous with world level, research and develop lipoxidase series of products.These series of products can increase dough volume well, and dough is had to certain bleaching function, have been subject to user's extensive expectation.
One of important quality index of biology enzyme product is the enzymatic activity of enzyme product.The size of quantitative measurment enzyme product activity accurately, all uses significance to the production and selling of biology enzyme.
" Wuxi Light Industry Univ.'s journal " (2001.01-0077-04) reported and three kinds of methods of measuring lipoxidase has been respectively piezometric method, spectrophotometric method and KI2 starch method.
1. piezometric method
Due to the effect of lipoxidase, substrate linoleic acid can be combined with oxygen, forms superoxide.Piezometric method is utilized this principle just, reduces the activity of indirect determination fat oxidation enzyme product by the amount of oxygen of measuring in enclosed system.
This assay method, to the not requirement of crude degree of lipoxidase sample, be applicable to comparatively elementary sample determination, but operation steps is comparatively complicated, can not adapt to the requirement of continuous sample determination, and need constantly vibrations when measuring, may affect enzyme activity, not accurate enough to micro-enzymatic activity monitoring, poor sensitivity.
2. spectrophotometric method
In the linoleic process of fat oxidation substrate for enzymatic activity, can produce the intermediate with conjugated diolefine key.A feature of conjugated diolefine key is to absorb 234nm light wave.Therefore by measuring 234nm light wave, can indirectly calculate enzyme activity.Spectrophotometric method is exactly according to this principle, measures the concentration of conjugated diolefine key, thereby measures the size of enzymatic activity.
The advantage of this method is continuously that Multi-example is measured, but instrument is had relatively high expectations, and owing to being to measure ultraviolet light wave, therefore comparatively responsive to the impurity in sample, therefore be only suitable for, in comparatively pure enzyme sample determination, being not suitable for being applied to the mensuration compared with crude samples in actual production.And owing to being the intermediate of directly measuring, there is instability, therefore temporal evolution, spectrophotometric value is also in continuous variation.Because sensitivity is low, be not suitable for the sample that enzymatic activity is lower.
3.KI2 starch method
Fat oxidation enzymatic linoleic acid forms superoxide, and superoxide can be iodine by iodide ion redox, and iodine can be combined with starch and develop the color.According to this principle, can indirect determination lipoxidase activity, be both KI2 method.
Similar to spectrophotometric method, this method is subject to impurity and time effects is larger.And owing to developing the color under acid condition, the impurity such as the protein in enzyme sample and fat also may flocculate under acid condition, the reliability of impact observation and result.Therefore this method also cannot adapt to enzyme sample condition complicated under working condition.
Above-mentioned three kinds of methods respectively have limitation, can not meet the needs of mensuration fast and accurately in actual production, have greatly limited their application.
Summary of the invention
The object of the invention is, for overcoming the deficiencies in the prior art, provide that a kind of method is easy, test sample is adapted at realizing flour being baked and banked up with earth to enzyme in light industry and food industrial processes rapidly---the Quick qualitative detection method of lipoxidase enzymatic activity.
According to general international standard, 1 unit fat oxidation enzyme activity is defined as: at 25 DEG C of temperature, in pH9.0 environment, enzyme per minute is oxidized the required vigor of 0.12 μ mole linoleic acid.And this vigor is equal at 25 DEG C of temperature, in pH9.0 environment, in 0.12 μ mole linoleic acid solution described in 3.0ml (1 centimetre of luminous range), shading value per minute on 234nm light wave changes 0.001 required enzyme activity.
According to this definition, the present invention has designed the scheme of indirect measurement linoleic acid concentration change.Linoleic acid concentration change, its oxidation intermediates dyes to it with dyestuff, thereby judges whether enzyme product has activity, and the speed and the depth that change from dye colour, also the activity size of enzyme product roughly relatively.
For reaching above technical purpose, the technical solution used in the present invention is as follows:
A kind of Quick qualitative detection method of lipoxidase enzymatic activity, wherein, the solution A that comprises dimethyamine borane buffering agent and linoleic acid solution, the solution B that comprises phenol reagent and ferriheme solution and fatty oxidasic enzyme product solution are mixed, leave standstill colour developing, judge according to whether developing the color whether described enzyme product solution has lipoxidase enzymatic activity.
Particularly, described solution A comprises the dimethyamine borane damping fluid that initial concentration is 20mM, linoleic acid solution and the water that initial concentration is 25mM, and three's volume ratio is 25:1:24.
Compound method: the dimethyamine borane damping fluid of described 20mM comprises dimethyamine borane solid, described dimethyamine borane solid is with after a certain amount of dissolving with hydrochloric acid, and water is settled to desired concn.Described linoleic acid solution is the emulsion of leukotrienes and water.Described emulsion fully stirs into emulsion fluid by appropriate leukotrienes and a certain amount of water and obtains.
Described solution B comprises the phenol reagent that initial concentration is 10mM, ferriheme solution and the water that initial concentration is 0.1mg/ml, and three's volume ratio is 1:1:48.
Compound method: described phenol reagent is the aqueous solution of 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate.The aqueous solution that described ferriheme solution is ferriheme.
Described enzyme product solution is the fat oxidation enzyme extract being dissolved in enzyme sample buffer, trishydroxymethylaminomethane, the sodium chloride of 8.766g/l and the glycerine of 100g/l that described enzyme sample buffer comprises 7.88g/l.
The Quick qualitative detection method of described lipoxidase enzymatic activity, described blend step is:
A. at normal temperatures, described solution A, solution B and enzyme product solution are mixed in 96 orifice plates according to the ratio of 4.5:4.5:1, leave standstill colour developing.
Selectively, also comprise step (B): if not colour developing of described step (A), by repeating step (A) after described enzyme product solution dilution certain multiple.
Selectively, also comprise: 96 orifice plates that are loaded with described mixed liquor in described steps A are detected under 590nm wavelength to the relative value of the colour developing of each mixed liquor by detecting instrument, with the relative activity size of the enzyme of more each enzyme product solution.
Compared with prior art, the present invention has following advantage:
(a) use phenol reagent and dimethyamine borane as developer, and adopt the catalyst of ferriheme as phenol reagent and dimethyamine borane oxidation, make final colour developing result for being easy to macroscopic blueness or darkviolet, therefore save loaded down with trivial details step and the dependence to detecting instrument, be more suitable for the qualitative determination to enzymatic activity under production environment.
(b) use 96 orifice plates and corresponding detecting instrument thereof to detect lipoxidase enzymatic activity, can disposable multiple samples be measured on the one hand, greatly improve detection efficiency; On the other hand, enzymatic activity size that can more multiple roughly samples by instrument, also greatly improves detection efficiency.
Brief description of the drawings
Fig. 1 is the lipoxidase qualitative detection result that adopts lipoxidase enzymatic activity Quick qualitative detection method of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
In the extract of detection soybean fat oxidase, whether have lipoxidase enzymatic activity, step is as follows:
(1) obtain solution A
Described solution A comprises 5ml20mM dimethyamine borane (DMAB) damping fluid, 0.2ml25mM linoleic acid solution and 4.8ml water, now with the current.
The preparation of described 20mM DMAB damping fluid: 165mg DMAB is dissolved in 2.5ml1N hydrochloric acid solution, then adds water and be settled to 50ml with the volumetric flask of 50ml.
The preparation of described 25mM linoleic acid solution: by 155 μ l(140mg) leukotrienes is dissolved in 5ml aqueous solution, fully stirs and makes solution whole milk change into emulsion fluid, and add water and be settled to 20ml with the volumetric flask of 20ml; The described solution preparing is divided into 1ml aliquot ,-20 DEG C of preservations.
(2) obtain solution B
Described solution B comprises 0.2ml10mM phenol reagent, 0.2ml0.1mg/ml ferriheme solution and 9.6ml water, now with the current.
The preparation of described 10mM phenol reagent: 0.233g3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH) is dissolved in 10ml water, 4 DEG C of preservations.
The preparation of described 0.1mg/ml ferriheme solution: add water and be settled to 10ml with the volumetric flask of 10ml after the ferriheme of 2mg is dissolved in water.
(3) preparation enzyme product solution
A) preparation of enzyme sample buffer: 7.88g trishydroxymethylaminomethane (Tris), 8.766g sodium chloride (NaCl) and 100g glycerine (glycerol) are dissolved in water, then add water and be settled to 1L with the volumetric flask of 1L.
B) preparation of lipoxidase standard solution (be labeled as+, represent positive control): the lipoxidase powder of appropriate purchase (Cayman Chemical60700) is dissolved in appropriate described enzyme sample buffer.
C) preparation of soybean fat oxidase extract:
-soybeans soaking is spent the night.
-hand-held disintegrating machine for the soybean of steeping (the hand-held disintegrating machine of KitchenAid double speed) is smashed, and use 8 feet of stainless steel filtering nets (Good Cook filter screen) to filter, filtrate is supplemented 5 times to soybean dry weight of described enzyme sample buffer, obtains crude extract.
-by described hydro-extractor (Heckman J2-HS) centrifugal 20min under 10,500g centrifugal force for crude extract, get supernatant, obtain soybean fat oxidase extract (being labeled as S.E.).
(4) chromogenic reaction
At normal temperatures, in the each sample well of 96 orifice plate, add described in 90 μ l solution B described in solution A and 90 μ l, after mixing, in corresponding sample well, add respectively soybean fat oxidase extract (S..E.) described in 20 μ l, lipoxidase standard solution (+) or water (be labeled as-, represent negative contrast), leave standstill 5min.In visible-range, show blueness or darkviolet, prove that this sample has lipoxidase activity.
As shown in Figure 1, be loaded with in the sample well of wherein three 96 orifice plates of reaction mixture, "+" hole has demonstrated blueness, "-" hole is water white transparency, respectively as positive control and negative contrast, if other sample wells demonstrate blueness, show that this sample exists lipoxidase enzymatic activity using this, if be still water white transparency, show that this sample does not exist lipoxidase enzymatic activity." S.E. " hole is shown as blueness, shows that described soybean fat oxidase extract exists lipoxidase enzymatic activity.
Further, in the case of the concentration of known described lipoxidase standard solution, can carry out by the depth relatively developing the color the height of rough more different sample room enzymatic activitys.Continue with reference to figure 1, the colour developing of " S.E. " is darker than "+", shows that the enzymatic activity of activity ratio's lipoxidase standard items of soybean fat oxidase extract is high.
(5) dilution
If described step (4) is colour developing not, can be by described lipoxidase standard solution or soybean fat oxidase extract dilution certain multiple, as 4 times, 8 times or 10 times, then repeating step (4), further to verify whether this sample has lipoxidase activity.
(6) colorimetric
At normal temperatures, 96 orifice plates that are loaded with above-mentioned reaction mixture are put into orifice detector, colorimetric under the spectrum of 590nm wavelength, can compare the relative activity size (not showing result) of various kinds product.
In sum, easy and simple to handle, test sample feature rapidly that the Quick qualitative detection method of lipoxidase enzymatic activity of the present invention has, is adapted at producing in reality and applies.
Above-described embodiment is preferably embodiment of the present invention; but be not merely restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being all included in protection scope of the present invention.
Claims (12)
1. the Quick qualitative detection method of a lipoxidase enzymatic activity, it is characterized in that: the solution A that comprises dimethyamine borane buffering agent and linoleic acid solution, the solution B that comprises phenol reagent and ferriheme solution and fatty oxidasic enzyme product solution are mixed, leave standstill colour developing; Judge according to whether developing the color whether described enzyme product solution has lipoxidase enzymatic activity.
2. the Quick qualitative detection method of lipoxidase enzymatic activity as claimed in claim 1, it is characterized in that: described solution A comprises the dimethyamine borane damping fluid that initial concentration is 20mM, linoleic acid solution and the water that initial concentration is 25mM, and three's volume ratio is 25:1:24.
3. the Quick qualitative detection method of lipoxidase enzymatic activity as claimed in claim 2, it is characterized in that: the dimethyamine borane damping fluid of described 20mM comprises dimethyamine borane solid, described dimethyamine borane solid is with after a certain amount of dissolving with hydrochloric acid, and water is settled to desired concn.
4. the Quick qualitative detection method of lipoxidase enzymatic activity as claimed in claim 2, is characterized in that: described linoleic acid solution is the emulsion of leukotrienes and water.
5. the Quick qualitative detection method of lipoxidase enzymatic activity as claimed in claim 4, is characterized in that: described emulsion fully stirs into emulsion fluid by appropriate leukotrienes and a certain amount of water and obtains.
6. the Quick qualitative detection method of lipoxidase enzymatic activity as claimed in claim 1, it is characterized in that: described solution B comprises the phenol reagent that initial concentration is 10mM, ferriheme solution and the water that initial concentration is 0.1mg/ml, and three's volume ratio is 1:1:48.
7. the Quick qualitative detection method of lipoxidase enzymatic activity as claimed in claim 6, is characterized in that: described phenol reagent is the aqueous solution of 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate.
8. the Quick qualitative detection method of lipoxidase enzymatic activity as claimed in claim 6, is characterized in that: the aqueous solution that described ferriheme solution is ferriheme.
9. the Quick qualitative detection method of lipoxidase enzymatic activity as claimed in claim 1, it is characterized in that: described enzyme product solution is the fat oxidation enzyme extract being dissolved in enzyme sample buffer trishydroxymethylaminomethane, the sodium chloride of 8.766g/l and the glycerine of 100g/l that described enzyme sample buffer comprises 7.88g/l.
10. the Quick qualitative detection method of lipoxidase enzymatic activity as claimed in any one of claims 1 to 9 wherein, is characterized in that, described blend step is:
A. at normal temperatures, described solution A, solution B and enzyme product solution are mixed in 96 orifice plates according to the volume ratio of 4.5:4.5:1, leave standstill colour developing.
The Quick qualitative detection method of 11. lipoxidase enzymatic activitys as claimed in claim 10, is characterized in that, also comprises step (B): if not colour developing of described step (A), by repeating step (A) after described enzyme product solution dilution certain multiple.
The Quick qualitative detection method of 12. lipoxidase enzymatic activitys as described in claim 10 or 11, it is characterized in that, also comprise: 96 orifice plates that are loaded with described mixed liquor in described steps A are detected under 590nm wavelength to the relative value of the colour developing of each mixed liquor by detecting instrument, with the relative activity size of the enzyme of more each enzyme product solution.
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| CN106706610A (en) * | 2015-11-12 | 2017-05-24 | 北京鑫华乐源科技发展有限公司 | Rapid alpha-linolenic acid containing egg distinguishing technology |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1296080A (en) * | 2000-09-13 | 2001-05-23 | 安徽省农业科学院水稻研究所 | Method for detecting crop poxidase iso enzyme LOX-1, LOX-2 and LOX-3 |
| CN103114129A (en) * | 2012-11-08 | 2013-05-22 | 中国农业科学院作物科学研究所 | Polymorphism molecular marking method of barley lipoxygenase (LOX-1) synthesis defective gene |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1296080A (en) * | 2000-09-13 | 2001-05-23 | 安徽省农业科学院水稻研究所 | Method for detecting crop poxidase iso enzyme LOX-1, LOX-2 and LOX-3 |
| CN103114129A (en) * | 2012-11-08 | 2013-05-22 | 中国农业科学院作物科学研究所 | Polymorphism molecular marking method of barley lipoxygenase (LOX-1) synthesis defective gene |
Non-Patent Citations (2)
| Title |
|---|
| 张瑛等: "稻胚脂肪氧化酶Lox- 3 生化特性和作用机制研究", 《中国粮油学报》, vol. 18, no. 2, 30 April 2003 (2003-04-30), pages 1 - 4 * |
| 董德坤: "利用显色法快速筛选大豆脂肪氧化酶缺失突变体", 《中国粮油学报》, vol. 27, no. 7, 31 July 2012 (2012-07-31), pages 110 - 114 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106706610A (en) * | 2015-11-12 | 2017-05-24 | 北京鑫华乐源科技发展有限公司 | Rapid alpha-linolenic acid containing egg distinguishing technology |
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