CN104360012A - Kit for simultaneously and rapidly measuring content of glucose, fructose and total sugar and application of kit - Google Patents
Kit for simultaneously and rapidly measuring content of glucose, fructose and total sugar and application of kit Download PDFInfo
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- CN104360012A CN104360012A CN201410725916.3A CN201410725916A CN104360012A CN 104360012 A CN104360012 A CN 104360012A CN 201410725916 A CN201410725916 A CN 201410725916A CN 104360012 A CN104360012 A CN 104360012A
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Abstract
The invention discloses a kit for simultaneously and rapidly measuring content of glucose, fructose and total sugar and an application method of the kit. The kit is developed based on a principle that carbohydrates are dehydrated to generate hydroxymethyl furfural under the acid action and the hydroxymethyl furfural and a developing agent are condensed to form a colored compound. The kit comprises four reagents, wherein the reagent I refers to a mixed standard substance of glucose, fructose and cane sugar; the reagent II refers to an ortho-toluidine reagent and is used for measuring the content of glucose; the reagent III refers to a resorcinol reagent and is used for measuring the content of fructose; and the reagent IV refers to a phenol reagent and is used for measuring the content of total sugar. The kit is used for simultaneously measuring the content of glucose, fructose and total sugar, has the characteristics of high specificity and high sensitivity, can realize high-flux and rapid detection of the glucose, fructose and total sugar, is large in sample treatment amount and relatively low in cost, is suitable for measuring the content of glucose, fructose and total sugar in honey, fruits and vegetables and other samples and is particularly suitable for large-scale field sample analysis.
Description
Technical field
The invention belongs to Active components of food and detect analysis field, be specifically related to kit and the application process thereof of a kind of Rapid Simultaneous Determination glucose, fructose and total sugar content.
Background technology
In honey, sugar content is one of Main Basis evaluating honey quality quality." national food safety standard honey " (GB14963-2011) defines the content requirement of sugar in honey, and it adopts liquid chromatography differential pulse polarograpll method to measure the content (GB/T 18932.22) of the sugar such as Fructose in Honey, glucose.In addition, about the detection method of sugar in honey also comprises titrimetry, polarimetry, UV-VIS spectrophotometry, near-infrared spectrum technique etc.But these methods now widely used exist that testing cost is high, complicated operation is loaded down with trivial details, experimental period is long or can not detect the problems such as multiple sugar simultaneously, be difficult to adapt to quick, large batch of detection demand.Therefore, this invention exploits a kind of easy, quick, advantage of lower cost, can the method for Simultaneously test glucose, fructose and total sugar content and kit, be applicable to the assay of glucose, fructose and total reducing sugar in the sample such as honey and fruits and vegetables.
Summary of the invention
The object of the present invention is to provide kit and the application process thereof of a kind of Rapid Simultaneous Determination glucose, fructose and total sugar content, this kit has the advantages that specificity is high, sensitivity is good, glucose, fructose and total reducing sugar high flux can be realized, detect fast, quantity of sample handling is large, cost is also relatively low, is specially adapted to on-the-spot large-scale sample analysis.
For achieving the above object, the present invention adopts following technical scheme:
A kit for Rapid Simultaneous Determination glucose, fructose and total sugar content, it comprises reagent I, reagent II, reagent III and reagent IV; Reagent I is the mixed solution of glucose, fructose and sucrose standard product; Reagent II is ortho-aminotoluene reagent, for measuring glucose content; Reagent III is resorcinol reagent, for measuring fructose content; Reagent IV is phenol reagent, for measuring total sugar content.
Reagent I is by glucose, fructose and each 0.001 ~ 10g of sucrose standard product, dissolves in facing used time adding distil water and is settled to 100mL; Reagent II is dissolved in 75mL glacial acetic acid by thiocarbamide 0.1 ~ 1g, then add ortho-aminotoluene 1 ~ 30mL, saturated boric acid 4mL, adds glacial acetic acid to 100mL, put in brown bottle and preserve after mixing; Reagent III is dissolved in 100mL hydrochloric acid solution by 0.1 ~ 2 g resorcinol, and used salt acid solution is that 2:1 is formulated by volume by water and concentrated hydrochloric acid; Reagent IV is that 0.1 ~ 10g phenol adding distil water is settled to 50mL.
The kit of described Rapid Simultaneous Determination glucose, fructose and total sugar content can be used for the mensuration of glucose in honey, fructose and total sugar content, and its mensuration comprises the steps:
1) as shown in table 1, get microwell plate, the reagent I of decreasing volumes is added successively in the hole of A1 ~ A5, B1 ~ B5, C1 ~ C5, for drawing the typical curve of glucose, fructose and total reducing sugar, in A1, B1, C1 hole, the volume of reagent I is 10 ~ 200 μ L, in number hole of A6 ~ A8, B6 ~ B8, C6 ~ C8, separately adds the water of 10 ~ 200 μ L as blank;
2) all the other micropores are 1 determination unit with every 3 row 3 row, often arrange the honey sample solution S adding decreasing volumes successively
n;
3) in the micropore that reagent or sample solution are housed, every three rows add 5 ~ 150 μ L reagent II, 5 ~ 150 μ L reagent III, 5 ~ 150 μ L reagent IV+5 ~ 150 μ L concentrated sulphuric acids in order respectively;
4) after being added a cover by microwell plate, at 60-95 DEG C, water-bath, after 5 ~ 60 minutes, measures the absorbance in each hole with microplate reader or ultraviolet-visible spectrophotometer respectively at 630nm, 480nm and 490nm place;
5) absorbance of sample solution is substituted in obtained typical curve respectively, calculates the content of glucose in sample solution, fructose and total reducing sugar: as data that D1 ~ D3 hole is obtained substitute into A1 ~ A5 in the typical curve drawn, calculate sample S
2in glucose content be X1, X2, X3, then calculate its mean value; In the typical curve that the data substitution B1 ~ B5 obtained in E1 ~ E3 hole draws, calculate sample S
2in fructose content be Y1, Y2, Y3, then calculate its mean value; In the typical curve draw F1 ~ F3 hole data substitution C1 ~ C5, calculate sample S
2the content of middle total reducing sugar is Z1, Z2, Z3, then calculates its mean value.
Table 1 microwell plate application of sample table
Described microwell plate is 96 holes or 384 holes;
The concentration of described honey sample solution is 10mg/mL.
significant advantage of the present invention is:kit of the present invention dewaters under acid effect based on glucide to generate hydroxymethylfurfural, the principle being condensed into colored compound again with developer develops, mainly comprise carbohydrate standard items, ortho-aminotoluene reagent, resorcinol reagent and phenol reagent, can be used for measuring glucose, fructose and total sugar content in honey and fruits and vegetables sample.The inventive method is easy, quick, and quantity of sample handling is large, advantage of lower cost, required instrument and equipment is simple, microplate reader or general ultraviolet-visible spectrophotometer is adopted to measure, and it has specificity is high, sensitivity is good feature, be specially adapted to on-the-spot large-scale sample analysis.
Embodiment
More being convenient to make content of the present invention understand, below in conjunction with embodiment, technical solutions according to the invention are described further, but the present invention being not limited only to this.
The preparation of embodiment 1 carbohydrate standard items (reagent I), ortho-aminotoluene reagent (reagent II), resorcinol reagent (reagent III) and phenol reagent (reagent IV):
Reagent I: by each to glucose, fructose and sucrose standard product 0.0100 g, dissolves in facing used time adding distil water and is settled to 100mL, being mixed with the standard solution of 0.1mg/mL; Reagent II: thiocarbamide 0.1 g is dissolved in 75mL glacial acetic acid, then add ortho-aminotoluene 1mL, saturated boric acid 4mL, add glacial acetic acid after mixing to 100mL, put in brown bottle and preserve; Reagent III: 0.1 g resorcinol is dissolved in 100mL hydrochloric acid solution, used salt acid solution is that 2:1 is formulated by volume by water and concentrated hydrochloric acid; Reagent IV: 0.1g phenol adding distil water is settled to 50mL.
The preparation of embodiment 2 carbohydrate standard items (reagent I), ortho-aminotoluene reagent (reagent II), resorcinol reagent (reagent III) and phenol reagent (reagent IV):
Reagent I: by each to glucose, fructose and sucrose standard product 0.5 g, dissolves in facing used time adding distil water and is settled to 100mL, being mixed with the standard solution of 5mg/mL; Reagent II: thiocarbamide 0.5 g is dissolved in 75mL glacial acetic acid, then add ortho-aminotoluene 15mL, saturated boric acid 4mL, add glacial acetic acid after mixing to 100mL, put in brown bottle and preserve; Reagent III: 1.000 g resorcinols are dissolved in 100mL hydrochloric acid solution, used salt acid solution is that 2:1 is formulated by volume by water and concentrated hydrochloric acid; Reagent IV: 5.000 g phenol adding distil waters are settled to 50mL.
The preparation of embodiment 3 carbohydrate standard items (reagent I), ortho-aminotoluene reagent (reagent II), resorcinol reagent (reagent III) and phenol reagent (reagent IV):
Reagent I: by each to glucose, fructose and sucrose standard product 10 g, dissolves in facing used time adding distil water and is settled to 100mL, being mixed with the standard solution of 100mg/mL; Reagent II: thiocarbamide 1 g is dissolved in 75mL glacial acetic acid, then add ortho-aminotoluene 30mL, saturated boric acid 4mL, add glacial acetic acid after mixing to 100mL, put in brown bottle and preserve; Reagent III: 2g resorcinol is dissolved in 100mL hydrochloric acid solution, used salt acid solution is that 2:1 is formulated by volume by water and concentrated hydrochloric acid; Reagent IV: 10 g phenol adding distil waters are settled to 50mL.
The mensuration of glucose, fructose and total sugar content in embodiment 4 honey:
Glucose content assay method: the reagent I getting 1mL variable concentrations respectively, in small test tube, respectively adds 1mL reagent II, shakes up, boiling water bath 30min.Negate should in microcolorimetric ware, be reference with reagent blank, with the absorbance of ultraviolet-spectrophotometric determination standard solution, draw out standard working curve by solution afterwards under 630nm wavelength, then according to the content of glucose in absorbance calculation sample;
Fructose content assay method: the reagent I getting 1mL variable concentrations respectively, in small test tube, respectively adds 1mL reagent III, shakes up, boiling water bath 30min.Negate should in microcolorimetric ware, be reference with reagent blank, with the absorbance of ultraviolet-spectrophotometric determination standard solution, draw out standard working curve by solution afterwards under 480nm wavelength, then according to the content of fructose in absorbance calculation sample;
Total sugar content assay method: get the reagent I of 1mL variable concentrations respectively in small test tube, add 1mL reagent IV and the 0.9mL concentrated sulphuric acid, react 30min under room temperature after, be reference with reagent blank under 490nm wavelength, the absorbance of bioassay standard solution, draw out standard working curve, then according to the content of total reducing sugar in absorbance calculation sample.
Embodiment 5 honey sample microplate reader is analyzed:
Get honey sample a certain amount of, 1 mg/mL solution is mixed with distilled water, get 96 hole microplates, selected 36 micropores, the reagent I of 100 μ L, 50 μ L, 25 μ L, 16.67 μ L and 12.5 μ L is added successively in the hole of A1 ~ A5, B1 ~ B5, C1 ~ C5, for drawing the typical curve of glucose, fructose and total reducing sugar, in number hole of A6 ~ A8, B6 ~ B8, C6 ~ C8, separately add the water of 100 μ L as blank; All the other micropores are 1 determination unit with every 3 row 3 row, often arrange the honey sample solution adding 100 μ L, 50 μ L, 25 μ L successively; In the micropore that reagent or sample solution are housed, every three rows add 100 μ L reagent II, 100 μ L reagent III, 100 μ L reagent IV+100 μ L concentrated sulphuric acids in order respectively; Then after being added a cover by microwell plate, at 90 DEG C, water-bath, after 30 minutes, measures the absorbance in each hole respectively at 630nm, 480nm and 490nm place by microplate reader; The absorbance of sample solution is substituted in obtained typical curve respectively, calculates the content of glucose in sample solution, fructose and total reducing sugar.Its measurement result is in table 2.
Table 2 measurement result
The typical curve of gained glucose, fructose and total reducing sugar is respectively:
y=1.8667
x-0.0193(
r 2 =0.9994),
y=0.5221
x-0.0185(
r 2 =0.9970),
y=2.2663
x-0.0858(
r 2 =0.9977); The result recorded by three groups of honey sample solution substitutes in the typical curve of glucose, fructose, total reducing sugar respectively, and the actual content calculating glucose in honey sample, fructose and total reducing sugar is respectively 31.14%, 36.79%, 70.93%.
Embodiment 6 stability test:
Reagent II, reagent III and reagent IV are deposited in 4 DEG C of refrigerators, take out after one month, the method described according to embodiment 5 carries out Stability Determination to same honey sample.Result shows, and in honey sample, the actual content of glucose, fructose and total reducing sugar is respectively 30.27%, 36.10%, 70.39%, close with embodiment 5 result, and having good stability of three kinds of reagent is described.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (5)
1. a kit for Rapid Simultaneous Determination glucose, fructose and total sugar content, is characterized in that: described kit comprises reagent I, reagent II, reagent III and reagent IV; Reagent I is the mixed solution of glucose, fructose and sucrose standard product; Reagent II is ortho-aminotoluene reagent, for measuring glucose content; Reagent III is resorcinol reagent, for measuring fructose content; Reagent IV is phenol reagent, for measuring total sugar content.
2. the kit of Rapid Simultaneous Determination glucose, fructose and total sugar content according to claim 1, is characterized in that: reagent I is by glucose, fructose and each 0.01 ~ 10g of sucrose standard product, dissolves in facing used time adding distil water and is settled to 100mL; Reagent II is dissolved in 75mL glacial acetic acid by thiocarbamide 0.1 ~ 1g, then add ortho-aminotoluene 1 ~ 30mL, saturated boric acid 4mL, adds glacial acetic acid to 100mL, put in brown bottle and preserve after mixing; Reagent III is dissolved in 100mL hydrochloric acid solution by 0.1 ~ 2 g resorcinol, and used salt acid solution is that 2:1 is formulated by volume by water and concentrated hydrochloric acid; Reagent IV is that 0.1 ~ 10g phenol adding distil water is settled to 50mL.
3. an application process for Rapid Simultaneous Determination glucose, fructose and total sugar content kit as claimed in claim 1, is characterized in that: for the mensuration of glucose, fructose and total sugar content in honey.
4. the application process of Rapid Simultaneous Determination glucose, fructose and total sugar content kit according to claim 3, is characterized in that: comprise the steps:
1) microwell plate is got, the reagent I of decreasing volumes is added successively in 1 ~ No. 5 hole of first three row, for drawing the typical curve of glucose, fructose and total reducing sugar, in No. 1 hole, the volume of reagent I is 10 ~ 200 μ L, separately in 6 ~ No. 8 holes of first three row, adds the water of 10 ~ 200 μ L as blank;
2) all the other micropores are 1 determination unit with every 3 row 3 row, often arrange the honey sample solution adding decreasing volumes successively;
3) in the micropore that reagent or sample solution are housed, every three rows add 5 ~ 150 μ L reagent II, 5 ~ 150 μ L reagent III, 5 ~ 150 μ L reagent IV+5 ~ 150 μ L concentrated sulphuric acids in order respectively;
4) after being added a cover by microwell plate, at 60-95 DEG C, water-bath, after 5 ~ 60 minutes, measures the absorbance in each hole with microplate reader or ultraviolet-visible spectrophotometer respectively at 630nm, 480nm and 490nm place;
5) absorbance of sample solution is substituted in obtained typical curve respectively, calculate the content of glucose in sample solution, fructose and total reducing sugar.
5. the application process of Rapid Simultaneous Determination glucose, fructose and total sugar content kit according to claim 4, is characterized in that: described microwell plate is 96 holes or 384 holes;
The concentration of described honey sample solution is 10mg/mL.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106970073A (en) * | 2017-02-28 | 2017-07-21 | 中国农业科学院茶叶研究所 | The quick determination method of doping sucrose in a kind of tealeaves |
| CN108037087A (en) * | 2017-12-29 | 2018-05-15 | 广东省肇庆市质量计量监督检测所 | A kind of test method of Pachyrhizua angulatus polysaccharide composition |
| CN108387573A (en) * | 2018-02-08 | 2018-08-10 | 智锐达仪器科技南通有限公司 | Hydroxymethylfurfural quick detection reagent, kit and detection method in a kind of honey |
| CN109883968A (en) * | 2019-03-07 | 2019-06-14 | 长江水利委员会水文局 | The method for quickly testing and analyzing phosphorus in water using microplate reader microcolorimetry high-volume |
| WO2020006940A1 (en) * | 2018-07-03 | 2020-01-09 | 中国农业科学院北京畜牧兽医研究所 | Method and kit thereof for quantitatively detecting lactulose in liquid milk by using microplate reader enzymatic method |
| CN114739931A (en) * | 2022-05-10 | 2022-07-12 | 安徽农业大学 | Method for judging maturity of black-boned vegetables by using sugar ratio |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106970073A (en) * | 2017-02-28 | 2017-07-21 | 中国农业科学院茶叶研究所 | The quick determination method of doping sucrose in a kind of tealeaves |
| CN106970073B (en) * | 2017-02-28 | 2020-06-16 | 中国农业科学院茶叶研究所 | Rapid detection method for sucrose doped in tea |
| CN108037087A (en) * | 2017-12-29 | 2018-05-15 | 广东省肇庆市质量计量监督检测所 | A kind of test method of Pachyrhizua angulatus polysaccharide composition |
| CN108387573A (en) * | 2018-02-08 | 2018-08-10 | 智锐达仪器科技南通有限公司 | Hydroxymethylfurfural quick detection reagent, kit and detection method in a kind of honey |
| WO2020006940A1 (en) * | 2018-07-03 | 2020-01-09 | 中国农业科学院北京畜牧兽医研究所 | Method and kit thereof for quantitatively detecting lactulose in liquid milk by using microplate reader enzymatic method |
| CN109883968A (en) * | 2019-03-07 | 2019-06-14 | 长江水利委员会水文局 | The method for quickly testing and analyzing phosphorus in water using microplate reader microcolorimetry high-volume |
| CN114739931A (en) * | 2022-05-10 | 2022-07-12 | 安徽农业大学 | Method for judging maturity of black-boned vegetables by using sugar ratio |
| CN114739931B (en) * | 2022-05-10 | 2024-05-24 | 安徽农业大学 | Method for judging maturity of black vegetables by utilizing sugar ratio |
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