CN101324509A - Method for determining acetic acid concentration and acetic acid determining reagent kit - Google Patents
Method for determining acetic acid concentration and acetic acid determining reagent kit Download PDFInfo
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- CN101324509A CN101324509A CNA2007100233622A CN200710023362A CN101324509A CN 101324509 A CN101324509 A CN 101324509A CN A2007100233622 A CNA2007100233622 A CN A2007100233622A CN 200710023362 A CN200710023362 A CN 200710023362A CN 101324509 A CN101324509 A CN 101324509A
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- ferricytochrome
- carbon dioxide
- stabilizing agent
- reduced coenzyme
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Abstract
The invention relates to a kit for diagnosing/mensurating acetic acid by utilizing the technologies of the enzymic colorimetry and the enzyme linked immunosorbent assay. The invention further relates to a method, a principle and the composition and the components of a reagent for mensurating the concentration of the acetic acid, and belongs to the technology field of food/environment inspection and measurement. The main components of the kit include a buffer solution, reduced coenzyme, carbon dioxide, ferricytochrome b1, pyruvate oxidase, lactate dehydrogenase and a stabilizer. Through mixing a sample and the reagent by a certain volume ratio, a series of enzymatic reactions occur, then the reactant is placed under an ultraviolet/visible light analyzer, and the degree/velocity of the decrease in absorbance at 340nm of the dominant wavelength is detected, thereby mensurating the concentration of the acetic acid.
Description
Technical field
The present invention relates to a kind of acetimetry kit, the invention still further relates to the method for measuring acetic acid concentration simultaneously, belong to food/environmental test determination techniques field.
Background technology
Along with the appearance of preparation of vinegar standard, whether the whether up-to-standard direct relation again of food additives acetate (acetic acid) people's health.Stipulate clearly that in clause compulsory standard preparation of vinegar G B10337-2000 the used acetate of preparation of vinegar should meet the regulation of G B 1903-1996, promptly should meet the requirement of food additives acetic acid.With industrial acetic acid personation " food additives acetic acid ", for example, use the industrial acetic acid of some secondary product or recovery to decolour, add hydrochloric acid, phosphoric acid etc. when concentration is not enough to increase acidity.This class acetic acid is very big to the harm of human body, because this class acetic acid not only contains poisonous, harmful inorganics of interpolation, also contains and is difficult to the harmful organic substance talked clearly a bit.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzymic colorimetric (EnzymaticColorimetric Method) and enzyme (even) united method (Couple Reaction) technology utilized, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for acetic acid concentration, simultaneously, the present invention also will provide in order to realize the acetimetry kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out acetic acid concentration on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Acetic acid concentration assay method principle of the present invention is as follows:
Acetate+carbon dioxide+ferricytochrome b1
Pyruvate oxidase
Pyruvic acid+ferricytochrome b1+ water
Pyruvic acid+reduced coenzyme
Lactic dehydrogenaseL-lactic acid+coenzyme
This method is used pyruvate oxidase (pyruvate oxidase; EC 1.2.2.2) enzyme (idol) connection lactic dehydrogenase (Lactate dehydrogenase EC 1.1.1.27; EC 1.1.1.28) enzymatic reaction continuous monitoring method/speed ratio color method.Pyruvate oxidase enzymolysis acetic acidreaction produces pyruvic acid, the effect of uniting lactic 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, can calculate the concentration of acetate.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, and the acetimetry kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Carbon dioxide 20mmol/L
Ferricytochrome b1 5mmol/L
Pyruvate oxidase 8000U/L
Lactic dehydrogenase 12000U/L
Acetimetry kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, carbon dioxide, ferricytochrome b1, pyruvate oxidase, lactic dehydrogenase.
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.
Also above-mentioned single agent reagent can be made into following pair of agent reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme, carbon dioxide, ferricytochrome b1.
Reagent 2
Damping fluid, stabilizing agent, pyruvate oxidase, lactic dehydrogenase.
Reduced coenzyme, carbon dioxide, ferricytochrome b1, pyruvate oxidase, the position of lactic dehydrogenase in reagent 1 or reagent 2 can not limit.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.
Above-mentioned single agent reagent can also be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme, carbon dioxide, ferricytochrome b1.
Reagent 2
Damping fluid, stabilizing agent, lactic dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, pyruvate oxidase.
Reduced coenzyme, carbon dioxide, ferricytochrome b1, pyruvate oxidase, the position of lactic dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.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.
No matter be single agent, two agent or three doses, the present invention measures the method for acetic acid concentration, and its reduced coenzyme can be a kind of among NADPH, NADH or the thio-NADH.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The acetimetry reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Carbon dioxide 20mmol/L
Ferricytochrome b1 5mmol/L
Pyruvate oxidase 8000U/L
Lactic 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 acetic acid sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), about about 0 minute of time delay is 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 acetate.
Embodiment two
The acetimetry reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Carbon dioxide 20mmol/L
Ferricytochrome b1 5mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Pyruvate oxidase 8000U/L
Lactic 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 acetic acid sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), about about 0 minute of time delay is 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 acetate.
Embodiment three
The acetimetry reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Carbon dioxide 20mmol/L
Ferricytochrome b1 5mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Lactic dehydrogenase 12000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Pyruvate oxidase 8000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring acetic 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 acetic acid sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is negative reaction (reaction descends), and about about 0 minute of time delay is 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 acetate.
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, and is easy to utilize.
Claims (6)
1. acetic acid concentration assay method that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Acetate+carbon dioxide+ferricytochrome b1
Pyruvate oxidase
Pyruvic acid+ferricytochrome b1+ water
Pyruvic acid+reduced coenzyme
Lactic dehydrogenaseL-lactic acid+coenzyme
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 concentration measurement result of acetate.
2. acetimetry kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-4000mmol/L
Reduced coenzyme 0.1-0.35mmol/L
Carbon dioxide 1-50mmol/L
Ferricytochrome b1 1-50mmol/L
Pyruvate oxidase 1000-80000U/L
Lactic dehydrogenase 10 00-80000U/L
It is characterized in that: 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.Carbon dioxide replaces with supercarbonate, for example sodium bicarbonate or saleratus.
3. according to the described acetimetry kit of claim 2, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, carbon dioxide, ferricytochrome b1, pyruvate oxidase, lactic dehydrogenase.
4. according to the described acetimetry kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, carbon dioxide, ferricytochrome b1, pyruvate oxidase, lactic dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, carbon dioxide, ferricytochrome b1; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvate oxidase, lactic dehydrogenase.Reduced coenzyme, carbon dioxide, ferricytochrome b1, pyruvate oxidase, the position of lactic dehydrogenase in reagent 1 or reagent 2 can not limit.
5. according to the described acetimetry kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, carbon dioxide, ferricytochrome b1, pyruvate oxidase, lactic dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, carbon dioxide, ferricytochrome b1; Reagent 2 is made up of damping fluid, stabilizing agent, lactic dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, pyruvate oxidase.Reduced coenzyme, carbon dioxide, ferricytochrome b1, pyruvate oxidase, the position of lactic dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described acetimetry kit of claim 2, it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100233622A CN101324509A (en) | 2007-06-13 | 2007-06-13 | Method for determining acetic acid concentration and acetic acid determining reagent kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100233622A CN101324509A (en) | 2007-06-13 | 2007-06-13 | Method for determining acetic acid concentration and acetic acid determining reagent kit |
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| CNA2007100233622A Pending CN101324509A (en) | 2007-06-13 | 2007-06-13 | Method for determining acetic acid concentration and acetic acid determining reagent kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN113340834A (en) * | 2021-06-11 | 2021-09-03 | 成都市排水有限责任公司 | Method for rapidly determining content of sodium acetate in industrial-grade sodium acetate solution |
-
2007
- 2007-06-13 CN CNA2007100233622A patent/CN101324509A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN113340834A (en) * | 2021-06-11 | 2021-09-03 | 成都市排水有限责任公司 | Method for rapidly determining content of sodium acetate in industrial-grade sodium acetate solution |
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Open date: 20081217 |