蔗糖/D-葡萄糖试剂盒(共5瓶) 250次 爱尔兰 型号:JKY/K-SUCGL/M245805
蔗糖/D-葡萄糖试剂盒（共5瓶） 250次 爱尔兰 型号:JKY/K-SUCGL/M245805
集团
外观 三玻璃瓶，1塑料瓶和一个1聚丙烯瓶。
比重 不适用
在水中的溶解度 易溶
pH值 7.0
气味 无
粉末和液体形式
稳定性 稳定的一个十年或以上的冷冻机
成分:
名称 比例
β-Fructosidase酶 1瓶（玻璃）
醋酸钠缓冲液 1瓶（玻璃）
葡萄糖测定试剂 1瓶（玻璃）
葡萄糖试剂缓冲 1瓶（玻璃）
葡萄糖标准溶液 1瓶（玻璃）
面粉控制 1瓶（塑料）
蔗糖/ D -葡萄糖试剂盒
测量的蔗糖和D -葡萄糖的果汁，饮料，蜂蜜和食品。
导言：
蔗糖和D -葡萄糖是两个常见的糖
在植物和食品，并严重影响人类
营养。 D -葡萄糖可方便地测量体液
使用商用套件基于葡萄糖
氧化酶/过氧化物酶或hexokinase/G6PDH
程序。然而， D -葡萄糖的植物提取物通常发生
连同麦芽糖， maltosaccharides ，淀粉，蔗糖和/或β -
名D -葡萄糖寡糖。因此，更严格的
要求放在纯度检测试剂。那个
试剂必须基本上没有淀粉降解酶，
蔗糖降解酶和β -葡萄糖苷酶，因为这些可能会导致
要么高估或 低估了自由D -葡萄糖
目前在提取或推导出具体的酶降解
葡萄糖含有低聚糖或多糖（如大麦
β -葡聚糖） 。
该Megazyme蔗糖/ D -葡萄糖试剂盒采用高
纯度葡萄糖氧化酶，过氧化物酶和β - fructosidase （蔗糖）和
可有信的具体测量
D -葡萄糖和蔗糖在植物提取物和食品。其中的颜色
形式是稳定在室温下至少两小时后
发育
For the measurement of sucrose and D-glucose in fruit juice, beverages, honey and food products.
The training video below demonstrates some general principles of wine analysis.
To view Parts 2-4 of this demonstration please visit our video channel by clicking on this link.
To purchase a copy of this dvd please click on this link.
Catalogue Number: K-SUCGL
Content: 250 assays per kit
Appearance Three glass vials, one plastic vial and one polypropylene vial.
Specific Gravity not applicable
Solubility in Water Readily soluble.
pH Value 7.0
Odour none
Form powders and liquids
Stability stable in a freezer for ten or more years
Ingredients Name CAS Proportion
b-Fructosidase enzyme 1 vial （glass）
Sodium acetate buffer 1 vial （glass）
Glucose Determination Reagent 1 vial （glass）
Glucose Reagent Buffer 1 vial （glass）
Glucose standard solution 1 vial （glass）
Flour Control 1 vial （plastic）
INTRODUCTION:
Sucrose and D-glucose are two of the most commonly occurring sugars
in plant and food products and have serious impacts on human
nutrition. D-Glucose can be conveniently measured in body fluids
using commercially available kits based on the glucose
oxidase/peroxidase or on the hexokinase/G6PDH enzymic
procedures. However, D-glucose in plant extracts usually occurs
together with maltose, maltosaccharides, starch, sucrose and/or b-
linked D-gluco-oligosaccharides. Consequently, more stringent
requirements are placed on the purity of the assay reagents. The
reagents must be essentially devoid of starch degrading enzymes,
sucrose degrading enzymes and b-glucosidase, as these can lead to
either an overestimation or an underestimation of free D-glucose
present in the extract or derived by specific enzymic degradation of
glucose containing oligosaccharides or polysaccharides （e.g. barley
b-glucan）. The Megazyme Sucrose/D-Glucose Test Kit employs high
purity glucose oxidase, peroxidase and b-fructosidase （invertase） and
can be used with confidence for the specific measurement of
D-glucose and sucrose in plant and food extracts. The colour which
forms is stable at room temperature for at least two hours after
development.
PRINCIPLE:
The reactions involved are:
1
（3）
b-fructosidase
Sucrose + H2O D-Glucose + D-Fructose
（1）
Glucose oxidase
D-Glucose + O2 + H2O D-Gluconate + H2O2
（2）
2H2O2 + p-Hydroxybenzoic acid + 4 Aminoantipyrine
Peroxidase
Quinoneimine Dye + 4H2O
Free D-glucose in the sample extract is determined by conversion to a
red coloured quinoneimine dye compound through the action of
glucose oxidase （1） and peroxidase （2） at pH 7.4, and employing
p-hydroxybenzoic acid and 4-aminoantipyrine.
At pH 4.6, sucrose is hydrolysed by the enzyme b-fructosidase to
D-glucose and D-fructose （3）. The determination of D-glucose after
inversion （total D-glucose） is carried out simultaneously according to
the principle outlined above. The sucrose content is calculated from
the difference of the D-glucose concentrations before and after
enzymatic inversion.
ACCURACY:
Standard errors of less than 5% are achieved routinely.
KIT CONTENTS:
Kits suitable for performing 250 determinations are available from
Megazyme. The kits contain the full assay method plus:
Bottle 1: Sodium acetate buffer （20 mL, 2 M, pH 4.6）.
Stable for > 2 years at 4°C.
Bottle 2: b-Fructosidase （invertase） （yeast; 5 mL; 100 U/mL
on sucrose） in 50 % glycerol plus bovine serum
albumin （BSA; 1 mg/mL） and sodium azide （0.02 %
w/v）. Stable for > 2 years at 4°C.
Bottle 3: GOPOD Reagent Buffer. Potassium phosphate
buffer （1 M, pH 7.4）, p-hydroxybenzoic acid （0.22 M）
and sodium azide （0.4 % w/v）. Stable for > 3 years
at 4°C.
Bottle 4: GOPOD Reagent Enzymes. Glucose oxidase
（> 12,000 U） plus peroxidase （> 650 U） and
4-aminoantipyrine （80 mg）. Freeze-dried powder.
Stable for > 5 years at -20°C.
Bottle 5: D-Glucose standard solution （5 mL, 1.0 mg/mL）
in 0.2 % （w/v） benzoic acid.
Stable for > 5 years at room temperature.
Bottle 6: Control flour sample. Sucrose and D-glucose
contents shown on vial label.
Stable for > 5 years at room temperature.
2
3
PREPARATION OF REAGENT SOLUTIONS/SUSPENSIONS:
1. Dilute the contents of bottle 1 to 400 mL with distilled water
before use, to give a final concentration of 100 mM.
Stable for > 1 year at 4°C.
（To increase stability, store in a 500 mL Duran® bottle, and
overlay the solution with 2 drops of toluene）.
2. Dilute 1.0 mL of the contents of bottle 2 to 10 mL with
sodium acetate buffer （100 mM, pH 4.6）. This is Solution 2.
In dispensing this viscous liquid, a positive displacement
dispenser is recommended （however, this is not essential, as
the enzyme is in excess）. Stable for > 2 years at -20°C.
3. Dilute the contents of bottle 3 （GOPOD Reagent Buffer） to
1 L with distilled water （this is Solution 3）. Use immediay.
NOTE:
1. If the concentrated buffer is stored at -20°C, it will form salt
crystals that must be compley dissolved when this buffer is
diluted to 1 L with distilled water.
2. This concentrated buffer contains 0.4 % （w/v） sodium azide.
This is a poisonous chemical and should be treated accordingly.
4. Dissolve the contents of bottle 4 in 20 mL of solution 3
and quantitatively transfer this to the bottle containing the
remainder of solution 3. Cover this bottle with aluminium
foil to protect the enclosed reagent from light. This is
Glucose Determination Reagent （GOPOD Reagent）.
Stable for ~ 3 months at 2-5°C or > 12 months at -20°C.
If this reagent is to be stored in the frozen state, it should be
divided into aliquots that should be freeze/thawed only once
during use （e.g. 200 mL in polypropylene containers）.
When the reagent is freshly prepared it may be light yellow
or light pink in colour. It will develop a stronger pink
colour over 2-3 months at 4°C. The absorbance of this
solution should be less than 0.05 when read against distilled
water.
5 & 6. Use the contents of bottles 5 and 6 as supplied.
Stable for > 5 years at room temperature.
SOLUTIONS FOR SAMPLE CLARIFICATION:
Carrez I solution.- Dissolve 3.60 g of potassium hexacyanoferrate （II）
{K4[Fe（CN）6].3H2O} （Sigma cat. no. P-9387） in 100 mL of distilled
water. Store at room temperature.
Carrez II solution.- Dissolve 7.20 g of zinc sulphate （ZnSO4.7H2O）
（Sigma cat. no. Z-4750） in 100 mL of distilled water. Store at room
temperature.
Sodium Hydroxide （100 mM）.- Dissolve 4 g of sodium hydroxide in
1 litre of distilled water. Store at room temperature.
EQUIPMENT （RECOMMENDED）:
1. Glass test tubes （round bottomed; 16 x 100mm and 18 x 150mm）.
2. Micro-pipettors, e.g. Gilson Pipetman® 100 μL, 200 μL and
500 μL.
3. Positive displacement pipettor e.g. Eppendorf Multipette®
- with 12.5 Combitip® [to dispense 1.0 mL aliquots of
b-fructosidase （in 50 % glycerol）].
- with 5.0 mL Combitip® （to dispense 0.2 mL aliquots of diluted
b-fructosidase and buffer）.
4. Analytical balance.
5. Spectrophotometer set at 510 nm.
6. Vortex mixer （e.g. IKA YellowLab Test Tube Shaker TTS）.
7. Thermostatted water bath （set at 50.0°C）.
8. Boiling water bath （set at 85-90oC）.
9. Stop clock.
10. Whatman No. 1 （9 cm） glass fibre filter papers.
CONTROLS AND PRECAUTIONS:
1. The time of incubation with GOPOD reagent is not critical, but
should be at least 20 min.
2. Include reagent blanks and D-glucose controls （100 μg
quadruplicate） with each set of determinations.
3. Analyse an extract from the control powder with each set of
determinations.
4
a. The reagent blank consists of 0.4 mL of distilled water
+ 3.0 mL GOPOD Reagent.
b. The D-glucose control consists of 0.1 mL of D-glucose
standard solution （1 mg/mL） + 0.3 mL of distilled water
+ 3.0 mL GOPOD Reagent.
5
4. With each new batch of GOPOD Reagent, the time for maximum
colour formation with 100 μg of D-glucose standard should be
checked. This is usually approx. 15 min.
ASSAY PROCEDURE:
Assay for Glucose and Sucrose:
1. Add 0.2 mL of sample extract （containing D-glucose + sucrose at a
concentration of 0.02 - 0.5 mg/mL） to the bottom of four
16 x 100 mm glass test tubes. Add either 100 mM sodium acetate
buffer or b-fructosidase （Solution 2） to duplicate tubes as follows:
- 0.2 mL of sample + 0.2 mL acetate buffer [D-Glucose] . . . . A
- 0.2 mL of sample + 0.2 mL b-fructosidase [Sucrose
+ D-Glucose] . . . . . B
2. Incubate all tubes, including the Reagent Blanks and D-glucose
controls at 50°C for 20 min.
3. Add 3.0 mL of GOPOD Reagent to all tubes and incubate these at
50ºC for 20 min.
4. Measure all absorbances at 510 nm against the reagent blank:
Absorbances: DA = GOPOD absorbance for A
DB = GOPOD absorbance for B
CALCULATIONS:
D-Glucose; g/L of sample solution:
= DA x F x 1 x 1000 x Dilution
0.2 1000 1000
= DA x F x 0.0050
Sucrose; g/L of sample solution:
= DB - DA x F x 1 x 1000 x 342 x Dilution
0.2 1000 1000 180
= （DB - DA）x F x Dilution x 0.0095.
where:
DA/0.2 and DB/0.2
= absorbances （510 nm） （GOPOD Reagent） for 0.2 mL of
sample treated with acetate buffer （DA） （free D-glucose）;
or b-fructosidase （DB）（free D-glucose plus D-glucose from
sucrose）.
F = factor to convert from absorbance to μg for 100 μg
of D-glucose （= 100/absorbance for 100 μg D-glucose）.
1/1000 = conversion from micrograms to milligrams.
1000/1000 = conversion from mg/mL to g/litre.
342/180 = conversion from μg of D-glucose （as measured） to
μg of sucrose.
Dilution = dilution of the original sample solution.
When analyzing solid and semi-solid samples which are weighed out for
sample preparation, the result is calculated from the amount weighed:
Content of sucrose
= csucrose （g/L sample solution） x 100 [g/100 g]
weightsample （g/L sample solution）
SAMPLE DILUTION:
The amount of sucrose and D-glucose present in the cuvette should
range between 10 μg and 100 μg. The sample solution must
therefore be diluted sufficiently to yield a sugar concentration
between 0.02 and 0.5 g/L.
Dilution table
estimated amount of dilution dilution
sucrose + glucose per litre with water factor
< 0.5 g - 1
0.5 - 5.0 g 1 + 9 10
5.0 - 50 g 1 + 99 100
> 50 g 1 + 999 1000
SAMPLE PREPARATION:
1. Liquid foodstuffs
Use clear, colorless or slightly colored solutions directly or after
dilution according to the dilution table for the assay. Filter turbid
solutions （Whatman GF/C glass fibre filter papers） or clarify with
Carrez reagents. Strongly colored solutions which are used undiluted
for the assay because of their low sucrose and D-glucose
concentrations must be decolorized with polyvinylpolypyrrolidone
（PVPP）. Beverages containing gas should be degassed under vacuum.
6
7
Examples:
Determination of D-glucose and sucrose in fruit juices and
similar beverages
Filter turbid juices （alternatively clarify with Carrez reagents） and
dilute sufficiently to yield a sucrose and D-glucose concentration of
approx. 0.02 - 0.5 g/L. The diluted sample solution can be used for
assay even if slightly colored. With highly colored solutions,
decolorize as follows:
Mix 10 mL of juice and approx. 0.2 g of polyvinylpolypyrrolidone, stir
for 1 min and filter. Use the clear, slightly colored solution for the
assay.
Determination of sucrose in sweetened condensed milk and
ice-cream
Weigh approx. 1 g sample accuray into a 100 mL volumetric flask,
add approx. 60 mL water and incubate for 15 min at approx. 70°C;
shake from time to time. For protein precipitation, add 5 mL Carrez
1 solution, 5 mL of Carrez 2 solution and 10 mL of NaOH （100 mM）,
shake vigorously after each addition, adjust to room temperature and
fill up to the mark with water, filter. Use the clear, possibly slightly
opalescent solution diluted, according the dilution table for the assay.
2. Solid Foodstuffs
Mince solid and semi-solid foodstuffs （e.g. bread and pastries, fruit,
vegetables, meat and milk products） in an electric mixer, meat grinder
or mortar. Weigh out the well mixed sample and extract with water,
heated to 60°C if necessary. Transfer quantitatively into a volumetric
flask and fill up to the mark with water. Filter, clarify with Carrez
reagents, if necessary, and use the clear solution, diluted if necessary,
for the assay.
Examples:
Determination of sucrose in chocolate
Accuray weigh approx. 1 g of finely grated chocolate into a 100 mL
volumetric flask, add approx 70 mL water, and heat in a water bath at
60-65°C for 20 min. Shake from time to time. After the chocolate
has been compley suspended, allow to cool and fill up to the mark
with water. To separate the fat, place in a refrigerator for at least
20 min. Filter the cold solution through a glass fibre filter paper
（Whatman GF/A）. Use the clear filtrate diluted according to the
dilution table, if necessary, for the assay. Alternatively, clarify with the
Carrez reagents.
Determination of sucrose and D-glucose in （roast） coffee
Weigh approx. 1 g ground coffee into a 100 mL volumetric flask and
add 60 mL hot water （90°C）. Stir for 5 min on a magnetic stirrer.
Allow to cool to room temperature and remove the magnetic stirrer
bar. Clarify with Carrez reagents as for “sweetened condensed milk
and ice-cream” （as above）. Use the clear, possibly slightly colored
filtrate for the assay.
3. Pasty products
Homogenize semi-solid samples, extract with water or dissolve, filter
if necessary, clarify with Carrez reagents or decolorize.
Examples:
Determination of sucrose and D-glucose in jam
Homogenize approx. 10 g of jam in an electric mixer. Weigh
approximay 0.5 g of the homogenized jam accuray into a 100 mL
volumetric flask, mix with water, and fill up to the mark. Filter
through glass fibre filter paper. Use the clear filtrate diluted according
to the dilution table, if necessary, for the assay.
Determination of sucrose and D-glucose in honey
Thoroughly stir the honey with a spatula. Take approx. 10 g of the
viscous or crystalline honey, heat in a beaker for 15 min at approx.
60°C, and stir occasionally with a spatula （there is no need to heat
liquid honey）. Allow to cool. Weigh approx. 1 g of the liquid sample
accuray into a 100 mL volumetric flask. Dissolve at first with only a
small portion of water, and then fill to the mark.
a） Determination of D-glucose
Dilute the 1 % v/v honey solution in a ratio of 1:20 （1+19） and use
for the assay.
b） Determination of sucrose
If the estimated sucrose content in the honey lies between 5 and
10 %, dilute the 1 % solution in a ratio of 1:5 （1+4） and use for the
assay. If the estimated sucrose content in the honey lies between 0.5
and 5 %, the excess D-glucose should be removed as much as
possible before sucrose is determined. D-Glucose is oxidised to
D-gluconate in the presence of the enzymes glucose oxidase （GOD）
and catalase.
（GOD）
D-glucose + H2O + O2 D-gluconate + H2O2
The hydrogen peroxide is destroyed by catalase:
（catalase）
2 H2O2 2 H2O + O2
8
9
Reagents.
1. Sodium phosphate buffer （300 mM, pH 7.6） plus 5 mM
MgCl2.7H2O.
Add 53.4 g of di-sodium hydrogen phosphate dihydrate
（Na2HPO4.2H2O） to 900 mL of distilled water and dissolve by
stirring. Add 1.11 g of MgCl2.7H2O and dissolve. Adjust the pH to
7.6 with 1 M NaOH （40 g/L） and adjust the volume to 1 L with
distilled water. Store at 4°C in a well sealed Duran® bottle. To
prevent microbial contamination, overlay the solution with 2 drops of
toluene.
2. Glucose oxidase （12,000 U） plus Catalase （300,000 U）.
（Megazyme cat. no. E-GOXCA）.
Dissolve the contents of 1 vial in 20 mL of 300 mM sodium
phosphate buffer （pH 7.6） plus 5 mM MgCl2.2H2O. Divide this
solution into 2.0 mL aliquots. Stable for > 3 years at -20°C.
Procedure for D-glucose oxidation
Pipette into a 25 mL volumetric flask Volume
300 mM phosphate buffer solution （1） 5.0 mL
Sample solution （up to approx. 5 mg/mL D-glucose） 5.0 mL
Enzyme solution （2） 0.2 mL
Incubate the flask at ~ 25°C and pass a current of air （O2） through
the mixture for 1 h （see Figure 1）. While this oxidation could
theoretically lead to a decrease in pH, no significant changes are
observed in solutions containing D-glucose at concentrations of up
to 5 mg/mL （due to the buffering capacity of the phosphate buffer
used）.
To inactivate the glucose oxidase plus catalase, incubate the
volumetric flask in a boiling water bath for 15 min, allow it to cool to
room temperature and dilute the contents to the mark with distilled
water. Mix and filter. Use 0.5 mL of the clear solution for the
determination of D-fructose. Determine the residual D-glucose as
usual.
10
Figure 1. Arrangement for the oxidation of glucose by glucose
oxidase plus catalase in the presence of a stream of air.
Clamp to control
air flow rate
Sample in 25 mL
volumetric flask Fish tank
air pump
REFERENCES:
1. Outlaw,W.H. Jr. and Tarczynski, M.C., （1988） in Methods of
Enzymatic Analysis （Bergmeyer, H.U. ed） 3rd ed., vol. 6,
pp 96-103. VCH Verlagsgesellschaft mbH,Weinheim,
Germany.
2. Kunst,A., Draeger, B. & Ziegenhorn, J. （1988） in Methods of
Enzymatic Analysis （Bergmeyer, H.U. ed） 3rd ed., vol. 6,
pp. 163-172. VCH Verlagsgesellschaft mbH,Weinheim,
Germany.
用途:食品
分类:囯际直购
