CN101082575A - Method for measuring citric acid concentration and citric acid diagnose reagent kit - Google Patents
Method for measuring citric acid concentration and citric acid diagnose reagent kit Download PDFInfo
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- CN101082575A CN101082575A CN 200710024769 CN200710024769A CN101082575A CN 101082575 A CN101082575 A CN 101082575A CN 200710024769 CN200710024769 CN 200710024769 CN 200710024769 A CN200710024769 A CN 200710024769A CN 101082575 A CN101082575 A CN 101082575A
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Abstract
The invention relates to a kind of enzyme colorimetric method and a method of the coupling method measuring the density of citric acid and a diagnosis reagent box applying the citrate synthetase and coupling malic dehydrogenase enzymatic reaction continues monitoring method / ratio colorimetric method. The reaction of citrate synthetase enzymolysis citric acid generates oxaloacetic acid and then through the effect of coupling malic dehydrogenase at last oxidizes the reduced coenzyme (there is a absorption peak at the site of 340nm) becoming the coenzyme (there is a absorption peak at the site of 340nm) so we can assay the degree/speed of the fall-way absorbance at the place of 340nm. It can measure and calculate the density of citric acid by measuring the degree/speed of the fall-way absorbance at the place of 340nm. The method has high specificity and it is not polluted by the endogenous and exogenous object and the test result is precise and accurate. The invention can get the array result by the ultraviolet/visible light analytic instrument so it is convenience to extend and apply.
Description
Technical field
The present invention relates in the fields such as medicine, food, environment detection to citric acid concentration, relate in particular to enzymic colorimetric and coupling method and measure the method for citric acid concentration, and the citric acid diagnose reagent kit that makes thus, belong to the citric acid concentration technical field of analysis and detection.
Background technology
Citric acid is mainly used in food, beverage industry as acid, flavoring additives and antiseptic, antistaling agent, also is used to prepare medical freshener, also is used as antioxidant, plastifier, washing agent in chemical industry, cosmetic industry and washing industry.
Mandatory standard GB 17203-1998 " food additive citric acid calcium " regulation, calcium citrate content is 98.0～100.5%.State General Administration for Quality Supervision's tissue has carried out the government supervision selective examination to food additives agent citric acid its esters (citric acid, potassium citrate, sodium citrate, calcium citrate) product quality.The main Quality of finding in the selective examination is that calcium citrate content is defective, has individual product food additive citric acid calcium content to surpass the requirement of national Specification in the selective examination, mainly is that raw material lime carbonate transforms not exclusively, has impurity in the finished product.
Summary of the invention
The purpose of this invention is to provide a kind of method of measuring citric acid concentration, and use the formulated citric acid diagnose reagent kit of this method.
For realizing purpose of the present invention, a kind of enzymic colorimetric (Enzymatic Colorimetric Method) and coupling method (Couple Reaction) are measured the method for citric acid concentration, utilize citrate synthetase to be the colour developing enzyme for effect enzyme, malic dehydrogenase, citric acid in the testing sample is detected, adopts following steps to carry out:
With sample of measuring and the reagent mixing that contains coacetylase, citrate synthetase, reduced coenzyme, malic dehydrogenase, make it to take place following reaction,
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect degree/speed that predominant wavelength 340nm absorbance descends, calculate the size of citric acid concentration.
Above-mentioned enzymic colorimetric and coupling method are measured in the method for citric acid concentration, and described reduced coenzyme is a kind of among NADH, NADPH, thio-NADH or the thio-NADPH.
Realize that citric acid diagnose reagent kit of the present invention can be single agent, is grouped into by following one-tenth:
Damping fluid 20～500mmol/L,
Stabilizing agent 1～4000mmol/L,
Reduced coenzyme 0.1～0.35mmol/L,
Coacetylase 1～50mmol/L,
Citrate synthetase 1000～80000U/L,
Malic dehydrogenase 1000～80000U/L.
Kit can be a dry powder, also can be mixed with liquid reagent, directly uses.
Also the various compositions in above single agent can be carried out formulated in combination and become two agent, such as:
The prescription of two agent is not limited only in the above-mentioned table listed, wherein the composition of reagent I: reduced coenzyme, coacetylase can be placed on reagent II; Citrate synthetase among the reagent II, malic dehydrogenase also can be put into reagent I, so can form multiple formulations, enumerate no longer one by one.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
In addition, for reducing the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, more than add stabilizing agent usually in the middle of the reagent I/ reagent II of single agent, two agent, concentration is within 1～4000mmol/L or 0.1%～100% volume ratio scope.
Reagent can also be made into following three reagent:
Similar with two agent, three doses prescription also is not limited only to above-mentioned prescription, wherein the reduced coenzyme among the reagent I, coacetylase can be placed among reagent II or the reagent III, malic dehydrogenase among the reagent II can be placed among reagent I or the reagent III, citrate synthetase among the reagent III also can be put among reagent I or the reagent II, so can form multiple formulations, enumerate no longer one by one.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
In addition, for reducing the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stabilizing agent in the middle of the reagent I/ reagent II of above single agent, two agent, three doses the reagent I/ reagent H/ reagent III, active concentration is within 1～4000mmol/L or 0.1%～100% volume ratio scope.
Material with used as stabilizers can be: at least a in ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), the ethylene glycol (Ethylene glycol).
No matter studies show that, take all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, be single agent or two agent, and the diagnosis/detection kit of following formula components relation is comparatively desirable, also is preferred version of the present invention:
Damping fluid 100mmol/L,
Stabilizing agent 500mmol/L,
Reduced coenzyme 0.25mmol/L,
Coacetylase 5mmol/L,
Citrate synthetase 10000U/L,
Malic dehydrogenase 12000U/L.
Utilize enzymic colorimetric (Enzymatic Colorimetric Method) and coupling method (CoupleReaction) technology, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for citric acid concentration, simultaneously, the present invention also will provide in order to the citric acid of realizing this method and measure kit, adopting this reagent not only can carry out citric acid concentration on ultraviolet analyser or half, automatic clinical chemistry analyzer measures, and finding speed is fast, accuracy is high, thereby can obtain practical applying.
The outstanding substantive distinguishing features and the obvious improvement of technical solution of the present invention mainly shows:
(1) the present invention utilizes enzymic colorimetric and coupling method to measure citric acid concentration, and test result is accurate;
(2) composition of participation reaction all adds, and is not subjected to the pollution of inside and outside source material, test process degree of accuracy height;
(3) this method is easy, easy to operate, can obtain testing result fast, and the reaction be under buffer conditions, to carry out, do not pollute the environment;
(4) but this method just fast detecting on general ultraviolet/visible light analysis instrument or semi-automatic/automatic clinical chemistry analyzer does not need special or additional instruments, testing cost is cheap, is convenient to apply in the industry;
(5) use the reagent that assay method provided by the invention can be made various ways such as liquid reagent, powdered reagent, be used for measuring the size of various sample citric acid concentrations;
(6) liquid citric acid diagnosis/detection kit provided by the invention, good stability has guaranteed the application testing effect well.Be made into after two agent, can further reduce the cross influence between the various compositions, testing result is more credible, and reagent is more stable, can store for a long time.
Embodiment
The assay method of a kind of citric acid concentration of the present invention and citric acid diagnose reagent kit, utilization citrate synthetase (citrate synthase; EC 2.3.3.1; EC 2.3.3.3) coupling malic dehydrogenase (Malatedehydrogenase; EC 1.1.1.37) enzymatic reaction continuous monitoring method/speed ratio color method.Citrate synthetase enzymolysis citric acid reactions produces oxaloacetic acid, the effect of uniting malic dehydrogenase again by (idol), reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place) the most at last, thereby measured degree/speed that reduced coenzyme descends in 340nm place absorbance, by measuring degree/speed that 340nm place absorbance descends, the concentration of measuring and calculating citric acid.
Below in conjunction with specific embodiment technical solution of the present invention is described further.These examples only are some exemplary applications, can not be interpreted as a kind of restriction to claim protection domain of the present invention.
Embodiment one (single agent)
Prepare citric acid diagnose reagent kit by following composition and consumption:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ammonium sulfate 500mmol/L,
NADH 0.25mmol/L，
Coacetylase 5mmol/L,
Citrate synthetase 10000U/L,
Malic dehydrogenase 12000U/L;
Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested citric acid sample and reagent is 1: 25, and the Direction of Reaction is negative reaction (reaction descends), about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of citric acid.
Embodiment two (two agent)
Prepare citric acid diagnose reagent kit by following composition and consumption:
Reagent I---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Glycerine 50mmol/L,
Reduced coenzyme 0.25mmol/L,
Coacetylase 5mmol/L;
Reagent II---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Propylene glycol 500mmol/L,
NADPH 0.25mmol/L，
Coacetylase 5mmol/L,
Citrate synthetase 10000U/L,
Malic dehydrogenase 12000U/L.
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested citric acid sample and reagent I, reagent II is 2: 20: 5, and the Direction of Reaction is negative reaction (reaction descends), about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of citric acid.
Embodiment three (three doses)
Prepare citric acid diagnose reagent kit by following composition and consumption:
Reagent I---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ethylene glycol 50mmol/L,
thio-NADH 0.25mmol/L，
Coacetylase 5mmol/L;
Reagent II---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ammonium sulfate 500mmol/L,
Malic dehydrogenase 12000U/L;
Reagent III---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Glycerine 500mmol/L,
Citrate synthetase 10000U/L;
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring citric acid concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested citric acid sample and reagent I, reagent II, reagent III is 4: 40: 5: 5, the Direction of Reaction is negative reaction (reaction descends), about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of citric acid.
The applicant adopts other various reduced form chromogens combinations of putting down in writing in the above summary of the invention all can reach purpose of the present invention through experimental verification, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, be not subjected in the pollution of allogenic material, easy to utilize.
Claims (6)
1. the assay method of citric acid concentration is characterized in that may further comprise the steps
3) the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect degree/speed that predominant wavelength 340nm absorbance descends, calculate the size of citric acid concentration.
2. citric acid diagnose reagent kit, reagent is grouped into by following one-tenth in the box:
Damping fluid 20～500mmol/L,
Stabilizing agent 1～4000mmol/L,
Reduced coenzyme 0.1～0.35mmol/L,
Coacetylase 1～50mmol/L,
Citrate synthetase 1000～80000U/L,
Malic dehydrogenase 1000～80000U/L.
3. citric acid diagnose reagent kit according to claim 2 is characterized in that: described stabilizing agent is at least a in ammonium sulfate, glycerine, propylene glycol, the ethylene glycol.
4. citric acid diagnose reagent kit according to claim 2 is characterized in that: described reduced coenzyme is a kind of among NADH, NADPH, thio-NADH or the thio-NADPH.
5. any one citric acid diagnose reagent kit in the claim 2～4 is characterized in that: described reagent is made into single agent or two agent or three doses.
6. any one citric acid diagnose reagent kit in the claim 2～4, it is characterized in that: described kit is powdered reagent box or liquid reagent box.
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| CN 200710024769 CN101082575A (en) | 2007-06-28 | 2007-06-28 | Method for measuring citric acid concentration and citric acid diagnose reagent kit |
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| CN 200710024769 CN101082575A (en) | 2007-06-28 | 2007-06-28 | Method for measuring citric acid concentration and citric acid diagnose reagent kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680415A (en) * | 2011-03-16 | 2012-09-19 | 中国农业科学院作物科学研究所 | Colorimetric method for measuring the ketosuccinic acid content of the biological tissue |
| CN103674941A (en) * | 2013-07-19 | 2014-03-26 | 山东省科学院中日友好生物技术研究中心 | Method and device for dynamically detecting concentration of acid or alkali |
| CN108956500A (en) * | 2018-08-08 | 2018-12-07 | 大连大学 | A kind of low temperature malic dehydrogenase acetate concentration detection kit and its detection method |
-
2007
- 2007-06-28 CN CN 200710024769 patent/CN101082575A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680415A (en) * | 2011-03-16 | 2012-09-19 | 中国农业科学院作物科学研究所 | Colorimetric method for measuring the ketosuccinic acid content of the biological tissue |
| CN103674941A (en) * | 2013-07-19 | 2014-03-26 | 山东省科学院中日友好生物技术研究中心 | Method and device for dynamically detecting concentration of acid or alkali |
| CN103674941B (en) * | 2013-07-19 | 2016-02-03 | 山东省科学院中日友好生物技术研究中心 | The method of detection of dynamic acid or alkali concn and device |
| CN108956500A (en) * | 2018-08-08 | 2018-12-07 | 大连大学 | A kind of low temperature malic dehydrogenase acetate concentration detection kit and its detection method |
| CN108956500B (en) * | 2018-08-08 | 2021-08-03 | 大连大学 | Low-temperature malate dehydrogenase acetic acid concentration detection kit and detection method thereof |
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