CN110923113A - Sucrose-glucose-fructose content determination kit and determination method thereof - Google Patents
Sucrose-glucose-fructose content determination kit and determination method thereof Download PDFInfo
- Publication number
- CN110923113A CN110923113A CN201910340851.3A CN201910340851A CN110923113A CN 110923113 A CN110923113 A CN 110923113A CN 201910340851 A CN201910340851 A CN 201910340851A CN 110923113 A CN110923113 A CN 110923113A
- Authority
- CN
- China
- Prior art keywords
- glucose
- sucrose
- fructose
- tube
- box body
- 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
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 40
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 36
- 239000008103 glucose Substances 0.000 claims abstract description 36
- 229930091371 Fructose Natural products 0.000 claims abstract description 33
- 239000005715 Fructose Substances 0.000 claims abstract description 33
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 33
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 30
- 229930006000 Sucrose Natural products 0.000 claims abstract description 30
- 239000005720 sucrose Substances 0.000 claims abstract description 30
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 238000007789 sealing Methods 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 6
- 238000003556 assay Methods 0.000 claims description 19
- 239000008363 phosphate buffer Substances 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 9
- 101710184309 Probable sucrose-6-phosphate hydrolase Proteins 0.000 claims description 7
- 102400000472 Sucrase Human genes 0.000 claims description 7
- 101710112652 Sucrose-6-phosphate hydrolase Proteins 0.000 claims description 7
- 235000011073 invertase Nutrition 0.000 claims description 7
- 238000002835 absorbance Methods 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 102000005548 Hexokinase Human genes 0.000 claims description 5
- 108700040460 Hexokinases Proteins 0.000 claims description 5
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000003149 assay kit Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000000287 crude extract Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 230000002035 prolonged effect Effects 0.000 claims description 3
- 239000010453 quartz Substances 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 claims 4
- 235000000346 sugar Nutrition 0.000 abstract description 7
- 239000000126 substance Substances 0.000 abstract description 3
- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 6
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 3
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Emergency Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of sugar content determination, and discloses a sucrose-glucose-fructose content determination kit and a determination method thereof. The sucrose-glucose-fructose content determination kit comprises a reagent box body, a reagent pipe, a movable connecting device, a sealing cover and a locking device, wherein the reagent pipe is fixedly arranged in the reagent box body, the movable connecting device is fixedly arranged on the right side of the reagent box body, and the sealing cover is fixedly arranged at the right end of the movable connecting device. According to the sucrose-glucose-fructose content determination kit and the determination method thereof, specific enzymes are utilized to respectively act on sucrose, glucose and fructose, background interference of other substances in a sample is eliminated, the detection result is more accurate, the three sugar data can be obtained through one-time detection, the experimental operation time is shortened, human errors caused by batch detection are reduced, and a time-saving, money-saving and non-toxic detection means is provided for detection of a large number of samples.
Description
Technical Field
The invention relates to the technical field of sugar content determination, in particular to a sucrose-glucose-fructose content determination kit and a determination method thereof.
Background
At present, most of biochemical methods for detecting sucrose, glucose and fructose in China are traditional acid hydrolysis methods, and finally have color reaction with chemical reagents such as anthrone or resorcinol, but the sensitivity and specificity of the common chemical color reagents are low, and high background values appear, so that the detected values are high.
Another method is detection by High Performance Liquid Chromatography (HPLC) and requires an expensive differential detector, which takes a long time from sample preparation to the three sugars (sucrose, glucose and fructose). The instrument used in the method has higher cost of consumables and reagents, is difficult to realize in a common laboratory, and is difficult to accept in the experimental screening of mass samples from both time and reagent cost.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a sucrose-glucose-fructose content determination kit and a determination method thereof, and the operation process is simple, convenient and environment-friendly; the experimental result is more accurate; the kit can obtain the contents of three sugars (sucrose, glucose and fructose), improves the experimental efficiency and the like, and solves the problems that the common detection method is easy to generate errors and has higher cost.
The technical scheme adopted by the invention for solving the technical problems is as follows: the utility model provides a sucrose-glucose-fructose assay kit, includes reagent box body, reagent pipe, swing joint device, sealed lid and locking device, the inside fixed mounting of reagent box body has the reagent pipe, the right side fixed mounting of reagent box body has swing joint device, swing joint device's right-hand member fixed mounting has sealed lid, the right-hand member surface of sealed lid and the fixed surface of reagent box body install locking device.
The reagent box comprises a reagent box body, and is characterized in that the locking device comprises a limiting block, a fixing rod, a locking block, a movable clamping rod and a reset spring, the right end of the sealing cover is fixedly provided with the limiting block, the inside of the limiting block is movably connected with the fixing rod, the left side of the reagent box body is fixedly provided with the locking block, the inside of the locking block is movably connected with the movable clamping rod, and the reset spring is fixedly arranged on one side, opposite to the movable clamping rod.
Further, swing joint device includes pole fixed block, pole and rotatory piece, and reagent box body's right side fixed mounting has the pole fixed block, and one side fixed mounting that the pole fixed block is relative has the pole, and the surperficial swing joint of pole has rotatory piece.
Furthermore, the inside fixed mounting of locking block has the pin, and the activity kelly passes through pin and locking block swing joint.
Furthermore, the inside fixed mounting of locking block has the spring fixed block, and reset spring fixed mounting is in the both sides of spring fixed plate.
A sucrose-glucose-fructose content determination method comprises the following steps:
firstly, the method comprises the following steps: sample preparation: taking about 0.2g of sample, adding 1mL of distilled water for grinding, transferring all the crude extract into an EP tube, carrying out centrifugation at 12000rpm for 10min at normal temperature, and measuring the supernatant.
II, secondly: a detection step:
1) preheating the ultraviolet spectrophotometer for more than 30min, adjusting the wavelength to 340nm, and adjusting the distilled water to zero.
2) Taking four 1mL quartz cuvettes: sucrose assay tube: mu.L of sample, 25. mu.L of sucrase (10U), 25. mu.L of ADP were added in this order+(5mM) and 600. mu.L of phosphate buffer; and (3) sucrose control: mu.L sucrase (10U), 25. mu.L LNADP were added in succession+(5mM) and 625. mu.L of phosphate buffer; fructose and glucose assay tubes: add 25. mu.L of sample, 25. mu.L of LNADP in sequence+And 600. mu.L of phosphate buffer; fructose and glucose control tubes: 25 μ LNADP+And 625. mu.L of phosphate buffer; and after the reagents are sequentially added into the four tubes and are uniformly mixed, standing for 2min, and reading the A1 value of each tube at 340 nm.
3) 25 μ L of hexokinase and G6P enzyme mixture (10U) were added to each of the four operation tubes, mixed, reacted for 15min, and the A2 value of each tube was read at 340nm (if the A value continued to increase, the reaction time was extended until the absorbance remained unchanged for 2 minutes).
4) To the fructose and glucose assay tubes and control tubes were added 25. mu.L of phosphofructoisomerase (20U), respectively. Mixing, reacting for 15min, and reading A3 value at 340nm (if the A value continues to increase, the reaction time is prolonged until the absorbance value is kept unchanged within 2 min).
Thirdly, the method comprises the following steps: and (4) calculating a result:
Δ a glucose ═ (a2-a1) assay tube N- (a2-a1) control tube N;
Δ a fructose ═ assay tube N- (A3-a2) control tube N (A3-a 2);
Δ a sucrose ═ [ (a2-a1) assay tube M- (a2-a1) control tube M ] -. Δ a glucose;
1) and calculating according to the mass of the sample:
glucose content (mg/g fresh weight) ([ Δ a glucose ÷ (ε × d) ] × V2 × 103 × 180.16 ÷ (V1 ÷ V × W)
0.8 × Δ a glucose ÷ W × D
Fructose content (mg/g fresh weight) ([ Δ a fructose ÷ (ε × d) ] × V2 × 103 × 180.16 ÷ (V1 ÷ V × W)
0.8 × Δ a fructose ÷ W × D
Sucrose content (mg/g fresh weight) ([ Δ a sucrose ÷ (ε × d) ] × V2 × 103 × 342.3 ÷ (V1 ÷ V × W)
1.52 × Δ a sucrose ÷ W × D
Epsilon- -the molar absorptivity of NADPH is 6.3X 103L/mol/cm; d- -optical path distance, 1 cm;
v- - -volume of extract, 1 mL; v1 — sample volume, 25 μ L0.025 mL;
v2- -Total volume of reaction, 700. mu.L- -7X 10-4L; glucose molecular weight- -180.16;
the molecular weight of the fructose is-180.16; sucrose molecular weight- -342.3;
w-sample mass, g; d- - -dilution factor.
The invention has the beneficial effects that:
1. the invention provides a specific, accurate and rapid detection method, which utilizes specific enzymes to respectively act on sucrose, glucose and fructose, eliminates background interference of other substances in a sample, enables a detection result to be more accurate, can obtain three sugar data through one-time detection, shortens experimental operation time and human errors caused by batch detection, and provides a time-saving, money-saving and non-toxic detection means for detecting a large number of samples.
2. The sucrose-glucose-fructose content determination kit and the determination method thereof have the advantages of simple and environment-friendly operation process, good experimental repeatability and more accurate experimental data, can simultaneously obtain the contents of three sugars (sucrose, glucose and fructose) in one experiment, and improve the experimental efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic structural diagram of a kit according to the present invention;
FIG. 2 is a schematic view of the locking device of the present invention.
Description of reference numerals: 1-reagent box body, 2-reagent tube, 3-movable connecting device, 4-sealing cover, 5-locking device, 501-limiting block, 502-fixed rod, 503-locking block, 504-movable clamping rod and 505-reset spring.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
Referring to fig. 1-2, a sucrose-glucose-fructose assay kit includes a reagent box 1, a reagent tube 2, a movable connection device 3, a sealing cover 4 and a locking device 5, wherein: reagent box body 1's inside fixed mounting has reagent pipe 2, reagent box body 1's right side fixed mounting has swing joint device 3, swing joint device 3 includes the round bar fixed block, round bar and rotatory piece, reagent box body 1's right side fixed mounting has the round bar fixed block, the relative one side fixed mounting of round bar fixed block has the round bar, the surperficial swing joint of round bar has rotatory piece, swing joint device 3's right-hand member fixed mounting has sealed lid 4, the right-hand member surface of sealed lid 4 and reagent box body 1's fixed surface install locking device 5.
A sucrose-glucose-fructose content determination method comprises the following steps:
firstly, sample preparation: taking about 0.2g of sample, adding 1mL of distilled water for grinding, transferring all the crude extract into an EP tube, carrying out centrifugation at 12000rpm for 10min at normal temperature, and measuring the supernatant.
II, detection step:
1) preheating the ultraviolet spectrophotometer for more than 30min, adjusting the wavelength to 340nm, and adjusting the distilled water to zero.
2) Taking four 1mL quartz cuvettes: sucrose assay tube: mu.L of sample, 25. mu.L of sucrase (10U), 25. mu.L of ADP were added in this order+(5mM) and 600. mu.L of phosphate buffer; and (3) sucrose control: mu.L sucrase (10U), 25. mu.L LNADP were added in succession+(5mM) and 625. mu.L of phosphate buffer; fructose and glucose assay tubes: add 25. mu.L of sample, 25. mu.L of LNADP in sequence+And 600. mu.L of phosphate buffer; fructose and glucose control tubes: 25 μ LNADP+And 625. mu.L of phosphate buffer; and after the reagents are sequentially added into the four tubes and are uniformly mixed, standing for 2min, and reading the A1 value of each tube at 340 nm.
3) 25 μ L of hexokinase and G6P enzyme mixture (10U) were added to each of the four operation tubes, mixed, reacted for 15min, and the A2 value of each tube was read at 340nm (if the A value continued to increase, the reaction time was extended until the absorbance remained unchanged for 2 minutes).
4) To the fructose and glucose assay tubes and control tubes were added 25. mu.L of phosphofructoisomerase (20U), respectively. Mixing, reacting for 15min, and reading A3 value at 340nm (if the A value continues to increase, the reaction time is prolonged until the absorbance value is kept unchanged within 2 min).
Thirdly, the method comprises the following steps: and (4) calculating a result:
Δ a glucose ═ (a2-a1) assay tube N- (a2-a1) control tube N;
Δ a fructose ═ assay tube N- (A3-a2) control tube N (A3-a 2);
Δ a sucrose ═ [ (a2-a1) assay tube M- (a2-a1) control tube M ] -. Δ a glucose;
1) and calculating according to the mass of the sample:
glucose content (mg/g fresh weight) ([ Δ a glucose ÷ (ε × d) ] × V2 × 103 × 180.16 ÷ (V1 ÷ V × W)
0.8 × Δ a glucose ÷ W × D
Fructose content (mg/g fresh weight) ([ Δ a fructose ÷ (ε × d) ] × V2 × 103 × 180.16 ÷ (V1 ÷ V × W)
0.8 × Δ a fructose ÷ W × D
Sucrose content (mg/g fresh weight) ([ Δ a sucrose ÷ (ε × d) ] × V2 × 103 × 342.3 ÷ (V1 ÷ V × W)
1.52 × Δ a sucrose ÷ W × D
Epsilon- -the molar absorptivity of NADPH is 6.3X 103L/mol/cm; d- -optical path distance, 1 cm;
v- - -volume of extract, 1 mL; v1 — sample volume, 25 μ L0.025 mL;
v2- -Total volume of reaction, 700. mu.L- -7X 10-4L; glucose molecular weight- -180.16;
the molecular weight of the fructose is-180.16; sucrose molecular weight- -342.3;
w-sample mass, g; d- - -dilution factor.
When in use, the invention provides a simple, sensitive and rapid determination method, sucrose is hydrolyzed into glucose under the specific action of sucrase, the glucose is converted into glucose-6-phosphate under the action of hexokinase, fructose is converted into glucose-6-phosphate under the specific action of hexokinase and fructose phosphate isomerase, all generated glucose-6-phosphate is reduced into NADPH under the specific action of G6P enzyme, and the contents of sucrose, glucose and fructose are respectively calculated by detecting the amount of NADH.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A sucrose-glucose-fructose assay kit and an assay method thereof comprise a reagent box body (1), a reagent tube (2), a movable connecting device (3), a sealing cover (4) and a locking device (5), and are characterized in that: a reagent tube (2) is fixedly arranged in the reagent box body (1), a movable connecting device (3) is fixedly arranged on the right side of the reagent box body (1), a sealing cover (4) is fixedly arranged at the right end of the movable connecting device (3), and a locking device (5) is fixedly arranged on the surface of the right end of the sealing cover (4) and the surface of the reagent box body (1);
locking device (5) include stopper (501), dead lever (502), locking block (503), activity kelly (504) and reset spring (505), the right-hand member fixed mounting of sealed lid (4) has stopper (501), the inside swing joint of stopper (501) has dead lever (502), the left side fixed mounting of reagent box body (1) has locking block (503), the inside swing joint of locking block (503) has activity kelly (504), the equal fixed mounting in one side that activity kelly (504) is relative has reset spring (505).
2. The sucrose-glucose-fructose content determination kit according to claim 1, characterized in that: the movable connecting device (3) comprises a round bar fixing block, a round bar and a rotating block, the round bar fixing block is fixedly mounted on the right side of the reagent box body (1), the round bar is fixedly mounted on one side, opposite to the round bar fixing block, of the round bar, and the rotating block is movably connected to the surface of the round bar.
3. The sucrose-glucose-fructose content determination kit according to claim 1, characterized in that: the inside fixed mounting of locking block (503) has the pin, and activity kelly (504) is through pin and locking block (503) swing joint.
4. The sucrose-glucose-fructose content determination kit according to claim 1, characterized in that: the spring fixing block is fixedly arranged in the locking block (503), and the return springs (505) are fixedly arranged on two sides of the spring fixing plate.
5. A method for measuring the content of sucrose-glucose-fructose is characterized by comprising the following steps: the method comprises the following steps:
firstly, sample preparation: taking about 0.2g of sample, adding 1mL of distilled water for grinding, transferring all the crude extract into an EP tube, carrying out centrifugation at 12000rpm for 10min at normal temperature, and measuring the supernatant.
II, detection step:
1) preheating the ultraviolet spectrophotometer for more than 30min, adjusting the wavelength to 340nm, and adjusting the distilled water to zero.
2) Taking four 1mL quartz cuvettes: sucrose assay tube: mu.L of sample, 25. mu.L of sucrase (10U), 25. mu.L of ADP were added in this order+(5mM) and 600. mu.L of phosphate buffer; and (3) sucrose control: add in sequence 25. mu.L sucrase (10U), 25. mu.L NADP+(5mM) and 625. mu.L of phosphate buffer; fructose and glucose assay tubes: add 25. mu.L of the sample, 25. mu.L of NADP in sequence+And 600. mu.L of phosphate buffer; fructose and glucose control tubes: 25 μ L NADP+And 625. mu.L of phosphate buffer; and after the reagents are sequentially added into the four tubes and are uniformly mixed, standing for 2min, and reading the A1 value of each tube at 340 nm.
3) 25 μ L of hexokinase and G6P enzyme mixture (10U) were added to each of the four operation tubes, mixed, reacted for 15min, and the A2 value of each tube was read at 340nm (if the A value continued to increase, the reaction time was extended until the absorbance remained unchanged for 2 minutes).
4) To the fructose and glucose assay tubes and control tubes were added 25. mu.L of phosphofructoisomerase (20U), respectively. Mixing, reacting for 15min, and reading A3 value at 340nm (if the A value continues to increase, the reaction time is prolonged until the absorbance value is kept unchanged within 2 min).
Thirdly, the method comprises the following steps: and (4) calculating a result:
Δ a glucose ═ (a2-a1) assay tube N- (a2-a1) control tube N;
Δ a fructose ═ assay tube N- (A3-a2) control tube N (A3-a 2);
Δ a sucrose ═ [ (a2-a1) assay tube M- (a2-a1) control tube M ] -. Δ a glucose;
1) and calculating according to the mass of the sample:
glucose content (mg/g fresh weight) ([ Δ a glucose ÷ (∈ × D) ] × V2 × 103 × 180.16 ÷ (V1 ÷ V × W) ═ 0.8 × Δ a glucose ÷ W × D
Fructose content (mg/g fresh weight) ([ Δ a fructose ÷ (∈ xd) ] × V2 × 103 × 180.16 ÷ (V1 ÷ V × W) ═ 0.8 × Δ a fructose ÷ W × D ÷ f
Sucrose content (mg/g fresh weight) ([ Δ a sucrose ÷ (∈ xd) ] × V2 × 103 × 342.3 ÷ (V1 ÷ V × W) ═ 1.52 × Δ a sucrose ÷ W × D ÷ 1.52 × Δ a sucrose ÷
Epsilon- -the molar absorptivity of NADPH is 6.3X 103L/mol/cm; d- -optical path distance, 1 cm;
v- - -volume of extract, 1 mL; v1 — sample volume, 25 μ L0.025 mL;
v2- -Total volume of reaction, 700. mu.L- -7X 10-4L; glucose molecular weight- -180.16;
the molecular weight of the fructose is-180.16; sucrose molecular weight- -342.3;
w-sample mass, g; d- - -dilution factor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910340851.3A CN110923113A (en) | 2019-04-25 | 2019-04-25 | Sucrose-glucose-fructose content determination kit and determination method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910340851.3A CN110923113A (en) | 2019-04-25 | 2019-04-25 | Sucrose-glucose-fructose content determination kit and determination method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110923113A true CN110923113A (en) | 2020-03-27 |
Family
ID=69855618
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910340851.3A Pending CN110923113A (en) | 2019-04-25 | 2019-04-25 | Sucrose-glucose-fructose content determination kit and determination method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110923113A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA859419A (en) * | 1970-12-29 | Lauer Karl | Process for the determination of glucose and fructose | |
| JPH07280804A (en) * | 1994-02-17 | 1995-10-27 | Kureha Chem Ind Co Ltd | Measuring method for organismic sample |
| CN108254337A (en) * | 2017-12-04 | 2018-07-06 | 佛山科学技术学院 | It is a kind of to be used to predict the NIRS model building methods of glucose and fructose content in corn |
| CN208217282U (en) * | 2018-04-30 | 2018-12-11 | 惠安易成机电设备工程有限公司 | A kind of kit with fixed function |
| CN109596550A (en) * | 2018-12-24 | 2019-04-09 | 苏州科铭生物技术有限公司 | A kind of content of trehalose assay kit and its method based on spectrophotometry |
| CN109609594A (en) * | 2018-12-24 | 2019-04-12 | 苏州科铭生物技术有限公司 | A kind of glucose 6-phosphate assay kit and method based on micromethod |
-
2019
- 2019-04-25 CN CN201910340851.3A patent/CN110923113A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA859419A (en) * | 1970-12-29 | Lauer Karl | Process for the determination of glucose and fructose | |
| JPH07280804A (en) * | 1994-02-17 | 1995-10-27 | Kureha Chem Ind Co Ltd | Measuring method for organismic sample |
| CN108254337A (en) * | 2017-12-04 | 2018-07-06 | 佛山科学技术学院 | It is a kind of to be used to predict the NIRS model building methods of glucose and fructose content in corn |
| CN208217282U (en) * | 2018-04-30 | 2018-12-11 | 惠安易成机电设备工程有限公司 | A kind of kit with fixed function |
| CN109596550A (en) * | 2018-12-24 | 2019-04-09 | 苏州科铭生物技术有限公司 | A kind of content of trehalose assay kit and its method based on spectrophotometry |
| CN109609594A (en) * | 2018-12-24 | 2019-04-12 | 苏州科铭生物技术有限公司 | A kind of glucose 6-phosphate assay kit and method based on micromethod |
Non-Patent Citations (3)
| Title |
|---|
| 张利奋: "国际饮料分析方法", 中国轻工业出版社, pages: 55 * |
| 杨泉女等: "甜玉米葡萄糖、果糖和蔗糖含量近红外反射光谱模型构建", 《中国农业科技导报》, no. 01, 15 January 2018 (2018-01-15) * |
| 杨泉女等: "甜玉米葡萄糖、果糖和蔗糖含量近红外反射光谱模型构建", 中国农业科技导报, vol. 20, no. 1, pages 137 - 146 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shapiro et al. | Regulation of glutamine synthetase. VII. Adenylyl glutamine synthetase: a new form of the enzyme with altered regulatory and kinetic properties. | |
| Silvestri et al. | Analysis of sulfate in complex carbohydrates | |
| Wise et al. | Determination of easily hydrolyzable fructose units in dextran preparations | |
| US5650334A (en) | Fluorescent labelling compositions and methods for their use | |
| Viola et al. | A microplate reader assay for rapid enzymatic quantification of sugars in potato tubers | |
| US6048707A (en) | Fluorophore assisted derivatization analysis of carbohydrates | |
| Patterson et al. | Rapid colorimetric assays to qualitatively distinguish RNA and DNA in biomolecular samples | |
| CN100487137C (en) | Multiple channel surface plasma resonant image sensor based on-chip PCR | |
| CN110923113A (en) | Sucrose-glucose-fructose content determination kit and determination method thereof | |
| Krystal | A silver-binding assay for measuring nanogram amounts of protein in solution | |
| Schachter | [1] Enzymic microassays for d-mannose, d-glucose, d-galactose, l-fucose, and d-glucosamine | |
| Che et al. | Characterization of derivatization of sialic acid with 2‐aminoacridone and determination of sialic acid content in glycoproteins by capillary electrophoresis and high performance liquid chromatography‐ion trap mass spectrometry | |
| CN102313785A (en) | Analysis method of citric acid fermentation aqueous solution | |
| US5869275A (en) | Affinity ultrafiltration assay for transferase activity | |
| JPH0720055A (en) | Method for measuring cis-diol, solid supporting body and measuring kit | |
| US6190522B1 (en) | Analysis of carbohydrates derivatized with visible dye by high-resolution polyacrylamide gel electrophoresis | |
| Huber et al. | Capillary electrophoretic determination of the component monosaccharides in hemicelluloses | |
| Brownlee et al. | On the determination of galactosamine-uronic acid | |
| Vacik et al. | Separation and measurement of isoenzymes and other proteins by high-performance liquid chromatography | |
| CN103149198B (en) | Process for rapidly and quantitatively detecting heparosan through enzymatic method | |
| CN109596550A (en) | A kind of content of trehalose assay kit and its method based on spectrophotometry | |
| Xu et al. | Direct conjugation of bacterial polysaccharides to proteins by squaric acid chemistry | |
| Coquet et al. | Determination of sugars by liquid chromatography with post-column catalytic derivatization and fluorescence detection | |
| Westfall et al. | High-resolution polyacrylamide gel electrophoresis of carbohydrates derivatized with a visible dye | |
| CN109632986A (en) | A kind of sugared and glycitols compound post-column derivation detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200327 |