Raffinose/Sucrose/D-Glucose, colorimetric method

Catalogue number: AK00261, 100 tests of each analyte

Application

This rapid and simple specific enzymatic method is used for

the simultaneous determination of sucrose, D-glucose and

galactosyl-sucrose oligosaccharides (raffinose, stachyose and

verbascose) in flour mixtures, seeds, seed meal as well as

other matrices.

Introduction

Leguminous are significant component of both human and

livestock diets. Galactosyl-sucrose or raffinose-series

oligosaccharides (RSO) are major components in many food

legumes. The anti-nutricional activity of grain legumes is

frequently attributed to these oligosaccharides. Due to the

absence of -galactosidase, RSO are not hydrolysed in the

upper part of digestive tract. In the lower intestine,

metabolization of RSO by bacteria leads to flatulence and

diarrhoea. Thus, RSO limit the use of grain legumes in both

human and animal diets.

Principles

Free D-glucose in the sample is determined directly with

GOPOD Reagent by conversion to a red coloured

quinoneimine dye compound through the combined action

of glucose oxidase and peroxidase.

Galactosyl-sucrose oligosaccharides or raffinose-series

oligosaccharides (RSO) are hydrolysed to sucrose and D-

galactose using -galactosidase. Sucrose is hydrolysed to D-

glucose and D-fructose with -fructosidase, and measured as

D-glucose. This method does not differentiate between

raffinose, stachyose and verbascose, nevertheless measure

these as a group (RSO). Since one mole of raffinose-series

oligosaccharides contains one mole of D-glucose, the

concentrations are presented on a molar basis.

Linearity and precision

Linearity of the determination exists from 10 to 100 μg D-

glucose, raffinose or sucrose per assay (see Figure 1).

Standard errors below 5% are reached routinely.

Kit composition

Solution 1. Buffer (20 mL, pH 4.6). Stable for 2 years at 4 °C.

Dilute the content of bottle 1 to 100 mL with distilled water

before use. Stable for > 1 year at 4ºC.

Suspension 2. -Fructosidase (2.4 mL). Stable for 2 years at 4

°C. Swirl bottle before use.

Add 0.02 ml of Suspension 2 plus 0.180 ml of Solution 1, per

assay, to a test tube and homogenise (Solution 1+2). This

solution should be prepared for each assay day.

Suspension 3. -Galactosidase (2.2 mL). Stable for 2 years at

4 °C. Swirl bottle before use.

Add 0.005 of Suspension 3 plus 0.02 ml of Suspension 2 plus

0.175 ml of Solution 1, per assay, to a test tube and

homogenise (Solution 1+2+3). This solution should be

prepared for each assay day.

Solution 4. GOD-POD reagent buffer (30 mL, pH 7.4), p-

hydroxybenzoic acid and sodium azide (0.64% w/v) as a

preservative. Stable for 3 years at 4 °C.

Dilute the contents of the bottle to 1.0 L with distilled water

and use immediately.

Mixture 5. GOD-POD reagent enzymes. Freeze-dried powder

of glucose oxidase (GOD), peroxidase (POD) and 4-

aminoantipyrine. Stable for 5 years at -20 °C.

Dissolve the contents of one bottle 5 in approx. 20 mL of

solution 4 and quantitatively transfer this to the bottle

containing the remainder of solution 4. Cover this bottle with

D-glucose + O2 + H2O D-Gluconate + H2O2

2 H2O2 + p-hydroxybenzoic acid + 4-aminoantipirine

Quinoneimine dye + 4 H2O

Hydrolysis of raffinose (RSO) (at pH 4.6)

Raffinose Sucrose + D-Galactose

Hydrolysis of sucrose (at pH 4.6)

Sucrose + H2O D-Glucose + D-Fructose

GOx

POD

-galactosidase

-fructosidase

aluminum foil to protect the enclosed reagent from light.

Stable for 3 months at 2-5 °C or 12 months at -20 °C.

Solution 6. D-Glucose standard solution (5 mL, 1.0 mg or

0.556 mol/mL) in 0.2% (w/v) benzoic acid. Stable for 5 years

at room temperature.

Use as supplied.

Powder 7. Control flour sample. RSO, sucrose and D-glucose

contents shown on vial label.

Use as supplied. Prepare as a sample (see Examples of

sample preparation)

Figure 1. Linearity of the assay. Standard curves relating D-Glucose

and Raffinose concentration (g/test) to absorbance at 510 nm (40

ºC)

Safety

The general safety measures that apply to all chemical

substances should be followed. For more information

regarding the safe usage of this kit please refer to MSDS

available at www.nzytech.com.

Precautions and controls

Include reagent blanks and D-glucose controls 0.556 moles

(i.e. 100 g) in quadruplicate with each set of assays.

Analyse an extract from the control powder with each set of

assays.

The time of incubation with GOPOD reagent is not critical,

but should be at least 20 min. However, the time for

maximum colour formation with 100 μg of D-glucose

standard should be checked, for each new batch of GOPOD

Reagent (usually: 15 min).

The colour formed through the reaction is stable at room

temperature for at least 2 hours after development.

Procedure (endpoint analysis)

Wavelength: 510 nm

Cuvette: 1 cm light path (glass or plastic)

Temperature: 40 °C

Final volume: 3.3 mL

Sample solution: 10-100 μg of total RSO, sucrose and D-

glucose per cuvette

Read against reagent blank

Mixtures can be obtained with a plastic spatula or by gentle inversion after sealing with a cuvette cap or Parafilm

®.

*Pipette both Solution 1,1+2, 1+2+3 and sample into the bottom of the cuvette and mix by gentle swirling

0.00

0.20

0.40

0.60

0.80

1.00

0 20 40 60 80 100

Pipette into cuvettes (mL) Blank D-Glucose

Standard

For each sample

D-Glucose

assay (A)

Sucrose + D-Glucose

assay (B)

RSO + Sucrose +

D-Glucose assay (C)

Solution 1

Solution 1+2*

Solution 1+2+3*

Sample solution

H2O

-

-

-

-

0.30

-

-

-

0.10

0.20

0.20

-

-

0.10

-

-

0.20

-

0.10

-

-

-

0.20

0.10

Mix. Incubate for 20 min at 40 ºC. Then add:

GOD-POD reagent (4+5) 3.00 3.00 3.00 3.00 3.00

Mix, incubate at 40 °C for 20 min and read absorbances at 510 nm against the reagent blank to obtain ΔAD-glucose standard, Δ A, ΔB

and ΔC

Ab

sorb

ance

@ 5

10

nm

g/test

D-Glucose

Raffinose

Calculation

The concentration of D-glucose, sucrose and RSO

(mmol/100g) are calculated as follows:

Where,

A = GODPOD absorbances for assay A

B = GODPOD absorbances for assay B

C = GODPOD absorbances for assay C

F = factor to convert from absorbances to mol of glucose

500 = conversion to 50 mL extract (i.e., 0.5g sample)

200 = conversion from 0.5 to 100g sample

1/1000 = conversion from moles to millimoles

The concentrations of sucrose and D-Glucose can be

represented as millimoles/100g or determined as g/100g of

flour sample, as proposed bellow. However, since RSO are a

mixture of raffinose, stachyose and verbascose, is not

possible to express RSO as g/100g. If the main component of

a sample is known, then is possible to calculate an

approximate value in g/100g of flour by using the molecular

weight of this component.

Where,

0.180 = MW of D-Glucose (180)/1000 mg of D-Glucose

0.342 = MW of Sucrose (342)/1000 mg of Sucrose

MW/1000 = MW of main RSO/1000 mg of RSO

If the sample has been diluted or a different sample volume

was used during the reaction, the result must be multiplied

by the corresponding dilution/concentration factor.

Interferences

An internal standard should be included during sample

analysis if the presence of interfering substances is

suspected. A quantitative recovery of this standard should be

expected.

With each new batch of GOD-POD Reagent, the time of

maximum colour formation with 100 µg of D-Glucose

standard should be checked. This is approximately 15 min.

(See Figure 2).

Figure 2. Colour formation on incubation of 100 g of D-glucose,

performed with NZYTech kit at 40 ºC using 1 cm pathlength

cuvettes. Time for maximum colour formation: 15 min.

General information on sample preparation

The total amount of RSO, sucrose and D-glucose present in

the cuvette should range between 10 and 100 μg. Thus, if a

sample volume of 0.10 mL is used the sample solution must

be diluted to yield a sugar concentration between 100 and

1000 mg/L.

To implement this assay use clear, colourless and practically

neutral liquid samples directly, or after dilution; filter turbid

solutions; degas samples containing carbon dioxide (e.g. by

filtration; measure "coloured" samples (against a sample

blank; treat "strongly coloured" samples that are used

undiluted or with a higher sample volume with PVPP (ad 0.2

g of PVPP/10 mL sample, shake vigorously for 5 min and

filter through Whatman nº1 filter paper); crush or

homogenize solid or semi-solid samples, extract with water

or dissolve; extract samples containing fat with hot water.

Sample dilution buffer (Sodium acetate buffer (50 mM, pH

4.5) Add 2.9 mL of glacial acetic acid (1.05 g/mL) to 900 mL

of distilled water. Adjust the pH to 4.5 by careful addition of

1 M (4 g/100 mL) sodium hydroxide solution. Adjust the

0.00

0.20

0.40

0.60

0.80

1.00

0 5 10 15 20 25

Incubation time

(min)

D-Glucose, millimoles/100g

= A x F x 500 x 200 x 1

1000

= A x F x 100

Sucrose, millimoles/100g

= (B-A) x F x 500 x 200 x 1

1000

= (B-A) x F x 100

Raffinose-series oligosaccharides, millimoles/100g

= (C-B) x F x 500 x 200 x 1

1000

= (C-B) x F x 100

D-Glucose, g/100g

= D-Glucose (mmoles/100g) x 0.180

Sucrose, g/100g

= Sucrose (mmoles/100g) x 0.342

RSO, g/100g

= RSO (mmoles/100g) x MW/1000

Ab

sorb

ance

@ 5

10

nm

Maximum colour formation

volume to 1 litre and store the buffer at 4°C. Sodium azide

(0.2 g) can be added as a preservative. Stable for > 6 months

at 4°C.

Examples of sample preparation

Determination of raffinose-series oligosaccharides,

sucrose and D-glucose in flour mixtures (Enzyme

Inactivation and Sugar Extraction)

Accurately weigh 0.5 g of milled sample into a glass test-tube

and add 5 mL of ethanol (95% v/v). Incubate the tube in a

water bath at 85-90°C and allow to reflux for 5 min (This

inactivates endogenous enzymes). Quantitatively transfer the

tube contents to a 50 mL volumetric flask using Sample

dilution buffer from a wash bottle to ensure complete

transfer. Adjust to volume (50 mL) with Sample dilution

buffer. Allow the sample to extract over 15 min. and then mix

thoroughly. Transfer 5 mL of this solution/suspension to a

glass test tube (suitable fort centrifugation at 1000 g). Add 2

mL of chloroform, mix vigorously on a vortex mixer for 15 sec

and centrifuge (1000 g) for 10 min. Use the upper aqueous

solution directly for analysis. (Sample will be at 10 mg solid

matter/ml).

References

McCleary, B. V., Charnock, S. J., Rossiter, P. C., O’Shea, M. F.,

Power, A. M. and Lloyd, R. M. (2006). Measurement of

carbohydrates in grain, feed and food. J. Sci. Food Agric., 86:

1648-1661.

Released 06/16

Please enquire info@nzytech.com to obtain any additional information about this kit,

including additional specific applications.

Estrada do Paço do Lumiar, 22 Campus do Lumiar - Edifício E, R/C

1649-038 Lisboa, Portugal Tel.:+351.213643514

Fax: +351.217151168

www.nzytech.com

Certificate of Analysis

Test Criteria Result

Test Performance Reaction completed within time stated Meets specification

Target value for recommended standard material +/- 10% Meets specification

Blank reaction absorbance +/- 10% of the blank value Meets specification

Approved by:

José Prates

Senior Manager, Quality Systems