CN101329266A - Saccharose determination reagent kit and method for determining saccharose concentration - Google Patents
Saccharose determination reagent kit and method for determining saccharose concentration Download PDFInfo
- Publication number
- CN101329266A CN101329266A CNA2007100247358A CN200710024735A CN101329266A CN 101329266 A CN101329266 A CN 101329266A CN A2007100247358 A CNA2007100247358 A CN A2007100247358A CN 200710024735 A CN200710024735 A CN 200710024735A CN 101329266 A CN101329266 A CN 101329266A
- Authority
- CN
- China
- Prior art keywords
- reagent
- sucrose
- coenzyme
- stabilizing agent
- phosphate buffer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a saccharose determination kit which uses the techniques of an enzyme colorimetry method and an enzyme-linked method; meanwhile, the invention also relates to a method principle used for determining saccharose consistency, reagent composition and component, belonging to the field of food detection determination technique. The main components of the kit of the invention comprise phosphoric acid buffer solution, coenzyme, sucrose phosphorylase, fructose dehydrogenase and stabilizer; samples and reagents are mixed according to a certain volume proportion so as to generate a series of enzymatic reactions; subsequently, reaction matters are arranged under an ultraviolet/visible light analyser so as to detect the degree/speed of the absorbency increment of the main wavelength at the 340nm position, thus calculating the consistency of the saccharose.
Description
Technical field
The present invention relates to a kind of sucrose-determination kits, the invention still further relates to the method for measuring sucrose concentration simultaneously, belong to Food Inspection determination techniques field.
Background technology
Current, the phenomenon that honey is mingled is serious, and according to investigations, in the honey of basic unit purchase, in the sample of mingling of appearance, the content of sucrose is many between 10-28%, have in addition up to more than 50%.In addition, in, low-grade tea adds and mixes sugar man-hour, the phenomenon of seeking undue profits to gain in weight is also commonplace.Sugarfree foods is found the problem that contains sucrose, and what is heard is arranged in the time of also, and the diabetic is worked the mischief.
The detection method of cane sugar content in a kind of sugar-cane juice of the number of applying for a patent 200510099310.4 discloses a kind of assay method of sucrose, and its method comprises the hydrolysis of sucrose, the reaction of the reducing sugar and the potassium ferricyanide, steps such as the potentiometric titration of potassium ferrocyanide.
Because loaded down with trivial details with conventional titration measuring cane sugar content process, poor accuracy also utilizes high performance liquid chromatograph that sample is detected.
State Standard of the People's Republic of China GB 5009.8-85 has announced the assay method of sucrose in the food, and its principle is: sample is after removing deproteinize, and wherein sucrose is converted into reducing sugar through hydrochloric acid hydrolysis, presses reducing sugar test again.The difference of reducing sugar is a cane sugar content before and after the hydrolysis.This national standard is proposed by food hygienic standard sub-committee of national hygienic standard technical committee, and is relevant by Food Hygiene Surveillance check institute of the Ministry of Public Health.This national standard is responsible for drafting by Ministry of Public Health's Food Hygiene Surveillance check.
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, metering/continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for sucrose concentration, simultaneously, the present invention also will provide in order to realize the sucrose-determination kits of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out sucrose concentration on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Sucrose concentration assay method principle of the present invention is as follows:
Sucrose+phosphate radical
Sucrose phosphorylaseD-fructose+alpha-D-glucose-1-phosphoric acid
Fructose+coenzyme
Fructose dehydrogenaseDehydrogenation fructose+reduced coenzyme
This method is used sucrose phosphorylase (Disaccharide phosphorylases; EC 2.4.1.7) coupling Fructose dehydrogenase (fructose dehydrogenase; EC 1.1.1.124; EC 1.1.99.11) enzyme ' s reaction speeding colourimetry/end-point method.The reaction of sucrose phosphorylase enzymolysis sucrose produces fructose, effect by the coupling Fructose dehydrogenase again, coenzyme (not having absorption peak at the 340nm place) reduces the most at last becomes reduced coenzyme (absorption peak being arranged at the 340nm place), thereby measured degree/speed that reduced coenzyme rises in 340nm place absorbance, by measuring degree/speed that 340nm place absorbance rises, can calculate the concentration of sucrose size.
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 sucrose-determination kits of the present invention of following composition relation is comparatively desirable:
Phosphate buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Sucrose phosphorylase 10000U/L
Fructose dehydrogenase 12000U/L
The phosphate radical substrate can use phosphate buffer to replace, if use other damping fluids, then needs to add the phosphate radical substrate.
Sucrose-determination kits of the present invention can be single agent, comprising:
Phosphate buffer, stabilizing agent, coenzyme, sucrose phosphorylase, Fructose 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
Phosphate buffer, stabilizing agent, coenzyme.
Reagent 2
Phosphate buffer, stabilizing agent, sucrose phosphorylase, Fructose dehydrogenase.
Coenzyme, sucrose phosphorylase, the position of Fructose 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
Phosphate buffer, stabilizing agent, coenzyme.
Reagent 2
Phosphate buffer, stabilizing agent, Fructose dehydrogenase.
Reagent 3
Phosphate buffer, stabilizing agent, sucrose phosphorylase.
Coenzyme, sucrose phosphorylase, the position of Fructose 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 sucrose concentration, and its coenzyme can be NADP
+, NAD
+Or thio-NAD
+In a kind of.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The sucrose-determination reagent of present embodiment is single reagent, comprising:
Phosphate buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Sucrose phosphorylase 10000U/L
Fructose 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≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested sucrose sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), 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 rises, thereby calculates the concentration of sucrose size.
Embodiment two
The sucrose-determination reagent of present embodiment is double reagent, comprising:
Reagent 1
Phosphate buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Reagent 2
Phosphate buffer 100mmol/L
Stabilizing agent 500mmol/L
Sucrose phosphorylase 10000U/L
Fructose 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≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested sucrose sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), 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 rises, thereby calculates the concentration of sucrose size.
Embodiment three
The sucrose-determination reagent of present embodiment is three reagent, comprising:
Reagent 1
Phosphate buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Reagent 2
Phosphate buffer 100mmol/L
Stabilizing agent 500mmol/L
Fructose dehydrogenase 12000U/L
Reagent 3
Phosphate buffer 100mmol/L
Stabilizing agent 500mmol/L
Sucrose phosphorylase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring sucrose concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested sucrose sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is positive reaction (reaction of rising), 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 rises, thereby calculates the concentration of sucrose size.
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. the concentration of sucrose assay method of enzymic colorimetric and enzyme-linked method, its method principle is as follows:
Sucrose+phosphate radical
Sucrose phosphorylaseD-fructose+alpha-D-glucose-1-phosphoric acid
Fructose+coenzyme
Fructose dehydrogenaseDehydrogenation fructose+reduced 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 rises, calculate concentration of sucrose size measurement result.
2. sucrose-determination kits, principal ingredient comprises:
Phosphate buffer 20-500mmol/L
Stabilizing agent 1-4000mmol/L
DPN diphosphopyridine nucleotide-6mmol/L
Sucrose phosphorylase 1000-80000U/L
Fructose dehydrogenase 1000-80000U/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.The phosphate radical substrate can use phosphate buffer to replace, if use other damping fluids, then needs to add the phosphate radical substrate.
3. according to the described sucrose-determination kits of claim 2, it is characterized in that:
Form single agent reagent by phosphate buffer, stabilizing agent, coenzyme, sucrose phosphorylase, Fructose dehydrogenase.
4. according to the described sucrose-determination kits of claim 2, it is characterized in that:
Form two agent reagent by phosphate buffer, stabilizing agent, coenzyme, sucrose phosphorylase, Fructose dehydrogenase; Reagent 1 is made up of phosphate buffer, stabilizing agent, coenzyme; Reagent 2 is made up of phosphate buffer, stabilizing agent, sucrose phosphorylase, Fructose dehydrogenase.Coenzyme, sucrose phosphorylase, the position of Fructose dehydrogenase in reagent 1 or reagent 2 can not limit.
5. according to the described sucrose-determination kits of claim 2, it is characterized in that:
Form multi-agent reagent by phosphate buffer, stabilizing agent, coenzyme, sucrose phosphorylase, Fructose dehydrogenase; Reagent 1 is made up of phosphate buffer, stabilizing agent, coenzyme; Reagent 2 is made up of phosphate buffer, stabilizing agent, Fructose dehydrogenase; Reagent 3 is made up of phosphate buffer, stabilizing agent, sucrose phosphorylase.Coenzyme, sucrose phosphorylase, the position of Fructose dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described sucrose-determination kits 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 (AmmoniaSulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethyleneglycol) and at least one of the preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100247358A CN101329266A (en) | 2007-06-21 | 2007-06-21 | Saccharose determination reagent kit and method for determining saccharose concentration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100247358A CN101329266A (en) | 2007-06-21 | 2007-06-21 | Saccharose determination reagent kit and method for determining saccharose concentration |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101329266A true CN101329266A (en) | 2008-12-24 |
Family
ID=40205185
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100247358A Pending CN101329266A (en) | 2007-06-21 | 2007-06-21 | Saccharose determination reagent kit and method for determining saccharose concentration |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101329266A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113640237A (en) * | 2021-08-11 | 2021-11-12 | 云南省农业科学院甘蔗研究所 | Method for rapidly detecting sucrose and reducing sugar in sugar solution |
-
2007
- 2007-06-21 CN CNA2007100247358A patent/CN101329266A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113640237A (en) * | 2021-08-11 | 2021-11-12 | 云南省农业科学院甘蔗研究所 | Method for rapidly detecting sucrose and reducing sugar in sugar solution |
| CN113640237B (en) * | 2021-08-11 | 2024-03-22 | 云南省农业科学院甘蔗研究所 | Method for rapidly detecting sucrose and reducing sugar in sugar solution |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101329265A (en) | Saccharose determination reagent kit and method for determining saccharose concentration | |
| CN101329266A (en) | Saccharose determination reagent kit and method for determining saccharose concentration | |
| CN101329268A (en) | Saccharose determination reagent kit and method for determining saccharose concentration | |
| CN101329258A (en) | Maltose determination reagent kit and method for determining maltose concentration | |
| CN101329267A (en) | Saccharose determination reagent kit and method for determining saccharose concentration | |
| CN101329328A (en) | Oxalic acid diagnosis / determination reagent kit and method for measuring oxalic acid concentration | |
| CN101329336A (en) | Glycerol diagnosis / determination reagent kit and method for determining glycerol concentration | |
| CN101329264A (en) | Sucrase diagnosis / determination reagent kit and method for determining sucrase active concentration | |
| CN101464347A (en) | Ammonia ion diagnosis/measuring reagent kit and ammonia ion concentration determination method | |
| CN101324572A (en) | Glutamic acid diagnosis/determination reagent kit and method for determining glutamic acid concentration | |
| CN101329335A (en) | Glycerol diagnosis / determination reagent kit and method for determining glycerol concentration | |
| CN101324571A (en) | Glutamic acid diagnosis/determination reagent kit and method for determining glutamic acid concentration | |
| CN101324508A (en) | Method for determining fruit sugar concentration and fruit sugar diagnosis / determination reagent kit | |
| CN101329259A (en) | Maltose determination reagent kit and method for determining maltose concentration | |
| CN101329332A (en) | Oxalic acid diagnosis / determination reagent kit and method for measuring oxalic acid concentration | |
| CN101329329A (en) | Oxalic acid diagnosis / determination reagent kit and method for measuring oxalic acid concentration | |
| CN101464349A (en) | Ammonia ion diagnosis/measuring reagent kit and ammonia ion concentration determination method | |
| CN101620141A (en) | Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration | |
| CN101620130A (en) | Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration | |
| CN101324515A (en) | Method for determining formic acid concentration and formic acid diagnosis/determination reagent kit | |
| CN101329260A (en) | Maltose determination reagent kit and method for determining maltose concentration | |
| CN101324517A (en) | Method for determining formic acid concentration and formic acid diagnosis/determination reagent kit | |
| CN101173941A (en) | Inorganic phosphorus diagnosis/measuring reagent kit and method for measuring inorganic phosphorus concentration | |
| CN101324583A (en) | Method for determining amino acid concentration and amino acid diagnosis/determination reagent kit | |
| CN101324518A (en) | Method for determining formic acid concentration and formic acid diagnosis/determination reagent kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Open date: 20081224 |