1

-Glucan (mixed linkage), colorimetric method

AOAC method 955.16

Catalogue number: AK00271, 100 tests

Introduction

-1,3-1,4-Glucans are common constituents of cereals,

mainly barley and oats, bacteria, yeasts and mushrooms. In

common with other fibre components, dietary -1,3-1,4-

glucans have well recognized beneficial properties for human

health. However, solubility of -glucans is associated with

several anti-nutritive effects in animal nutrition and affect

different processes developed by the brewing industry.

Application

This accurate and convenient enzymatic method (as

described by AOAC method 955.16 and AACC method 32-23)

is used for the determination of -1,3-1,4-glucans in barley,

oats, fibre and other food products, even those containing

high levels of glucose.

Principles

Mixed-linkage -glucans are hydrolyzed to -gluco-

oligosaccharides through the action of a microbial lichenase.

Subsequently, -gluco-oligosaccharides are hydrolyzed to D-

glucose by a -glucosidase. Free D-glucose in the sample is

determined directly with GOD-POD Reagent by conversion to

a red coloured quinoneimine dye compound through the

combined action of glucose oxidase and peroxidase.

Kit composition

Suspension 1. Lichenase (1 mL). Stable for 2 years at 4 °C. Swirl bottle before use.

Dilute the contents of bottle 1 to 20.0 mL with Buffer A (not

provided). Divide into different aliquots and store in PP tubes

at -20 °C between uses (stable for 2 years). Keep refrigerated

during usage.

Suspension 2. -Glucosidase (1 mL). Stable for 2 years at 4

°C. Swirl bottle before use.

Dilute the contents of bottle 2 to 20.0 mL with Buffer B (not

provided). Divide into different aliquots and store in PP tubes

at -20 °C between uses (stable for 2 years). Keep refrigerated

during usage.

Solution 3. 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. On storage, salt crystals may form in

concentrated buffer. Be sure that all of these are completely

dissolved before dilution to 1 L with distilled water.

Mixture 4. 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 4 in approx. 20 mL of

solution 3 and quantitatively transfer this to the bottle

containing the remainder of solution 3. Cover this bottle with

aluminum foil to protect the enclosed reagent from light.

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

storage at -20 °C, this reagent should be divided in smaller

aliquots that should be freeze/thawed only once during use.

Solution 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. Use as supplied.

Powder 6. Control barley flour. -Glucan content shown on

vial label.

Use as supplied. Prepare as a sample (see Sample

preparation)

Hydrolysis of -glucans

-glucan + H2O -gluco-oligosaccharides

-gluco-oligosaccharides + H2O D-Glucose

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

2 H2O2 + p-hydroxybenzoic acid + 4-aminoantipirine

Quinoneimine dye + 4H2O

GOD

POD

Lichenase

pH 6.5

at -glucosidase

pH 4.0

at

2

Powder 7. Control oat flour. -Glucan content shown on vial

label.

Use as supplied. Prepare as a sample (see Sample

preparation)

Preparation of buffers (not provided)

Buffer A. 20 mM Sodium phosphate buffer, pH 6.5.

Dissolve 3.12 g of NaH2PO4.2H2O in 900 mL of distilled water

and adjust pH to 6.5 with 1M NaOH. Bring volume to 1 L.

Add 0.2 g of sodium azide. Stable for 2 months at 4 °C.

Buffer B. 50 mM Sodium acetate buffer, pH 4.0

Add 2.9 mL of glacial acetic acid to 900 mL of distilled water

and adjust pH to 4.0 with 1 M NaOH. Bring volume to 1 L.

Add 0.2 g of sodium azide. Stable for 2 months at 4 °C.

Buffer C. 200 mM Sodium acetate buffer, pH 4.0.

Add 11.6 mL of glacial acetic acid to 900 mL of distilled water

and adjust pH to 4.0 with 1 M NaOH. Bring volume to 1 L.

Add 0.2 g of sodium azide. Stable for 2 months at 4 °C.

Important notes

Include reagent blanks and D-glucose controls (100 g,

in quadruplicate) with each set of assays:

Reagent blank = 0.2 mL of 50 mM sodium acetate

buffer + 3.0 mL GOPOD Reagent.

D-Glucose

control

= 0.1 mL of D-glucose standard

solution (100 μg) + 0.1 mL of

distilled water + 3.0 mL GOPOD

Reagent.

Analyse at least one extract from the control barley

flour with each set of assays.

The time of incubation with GOD-POD 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 batch of

GOD-POD reagent (usually 15 min; See Figure 1). The

colour formed through the reaction is stable at room

temperature for at least 2 hours after development.

It is indispensable that lichenase preparation is not

cross-contaminated with the -glucosidase.

Figure 1. Colour formation following the incubation of 100 g of D-

glucose, at 40 ºC using 1 cm path length cuvettes, with GOD-POD

reagent. Time for maximum colour formation is usually 15 min.

Sample preparation

Mill approx. 50 g of sample (barley, oats or fibre) to pass a

0.5 mm sieve using a centrifugal mill. Homogenize sample by

shaking and inversion. Determine moisture content by

recording weight loss on storage of flour samples (0.5 g) at

103 ⁰C for 16 h or until weigth stabilization. Use this

information for the final calculations.

Procedure – Streamlined Enzymatic Method

Assay method for Barley and Oat flours and Fibre

Samples

1. Accurately weight milled sample (80-120 mg) to a glass

centrifuge tube. Tap the tube to ensure that all sample

particles fall to the bottom of the tube.

Note: When analysing samples containing high levels of

glucose pre-extract 80-100 mg milled sample 2x with 10 ml

aqueous ethanol (50% v/v). Centrifuge slurry at 1,000 g and

discard supernatant. Use resulting sediment for the analysis.

2. To wet sample add 0.2 mL of aqueous ethanol (50%

v/v). Add 4.0 mL of Buffer A (pH 6.5) and stir the

contents on a vortex mixer to assure complete

dispersion.

3. Immediately place the tube in a boiling water bath and

incubate for 1 min. Stir the mixture vigorously on a

vortex mixer and incubate in boiling water bath for a

further 2 min. Stir again.

4. Incubate the tube in a water bath at 50 °C. Equilibrate

for 5 min. Add 0.2 mL lichenase solution (Suspension 1,

diluted) and stir the tube contents on a vortex mixer.

Seal the tube and incubate for 1 h at 50 °C. Stir

vigorously on a vortex mixer 3-4 times during

incubation.

5. Add 5.0 mL Buffer C (pH 4.0) to each tube and

vigorously mix tube contents on a vortex mixer.

6. Cool tubes to room temperature (5 min) and centrifuge

(1,000 g, 10 min).

0.00

0.20

0.40

0.60

0.80

1.00

0 5 10 15 20 25

Incubation time

(min)

Maximum colour formation

3

7. Carefully and accurately dispense supernatant aliquots

(0.1 mL) into the bottom of three test tubes.

8. Add 0.1 mL of β-glucosidase solution (Suspension 2,

diluted) to two of these tubes (reaction solution). To the

third tube (reaction blank), add 0.1 mL of Buffer B (pH

4.0). Incubate all tubes at 50 °C for 10 min.

9. Add 3.0 mL of GOD-POD Reagent (Solution 3+4) to

each tube (reaction solutions, test and control flours,

reaction blanks, D-Glucose standards and reagent

blanks) and incubate at 50 °C for a further 20 min.

10. Remove the tubes from the water bath and measure the

absorbance at 510 nm against the reagent blank within

1 h.

Notes:

The amount of D-glucose present in the test tube should

range from 10 to 100 μg. Therefore, before Step 6 (β-

glucosidase treatment) the sample solution must be diluted

sufficiently with Buffer B (pH 4.0) to yield a sugar

concentration between 0.10 and 1.0 g/L, which is equivalent

to approx. between 0.85 to 8.5% of β-glucan in the original

sample. Include dilution factor when performing calculations.

Calculations

The concentration of -1,3-1,4-glucans in solid samples is

calculated as follows:

Where,

A = sample GOD-POD absorbance (after lichenase

and -glucosidase treatment) minus reaction

blank absorbance

F =

=

factor to convert absorbance to mol of

glucose

100 (𝑔 𝑜𝑓 𝐷 − 𝐺𝑙𝑢𝑐𝑜𝑠𝑒)

absorbance of 100 g of D − Glucose

94 = volume correction factor (0.1 of solution from

9.4 mL was analyzed)

1

1000

= conversion from g to mg

100

W = factor to express -1,3-1,4-glucans as a

percentage of sample weight

W = weight of the sample analyzed in mg (fresh

weight))

162

180

= factor to convert free D-glucose, as

determined, to anhydrous-D-glucose, as

occurs in -glucans

D = further dilution previous to incubation with -

glucosidase, if necessary

References

AOAC International (2000). AOAC Official method 995.16

McCleary, B. V. & Mugford, D. C. (1997). Determination of β-glucan in barley and oats by streamlined enzymic method: summary of collaborative study. Journal of AOAC International, 80(3), 580-583.

McCleary, B. V. & Codd, R. (1991). Measurement of (1-3) (1-4)-

β-D-glucan in barley and oats: A streamlined enzymic

procedure. J. Sci. Fd. Agric., 55, 303-312.

Released 02/17

-Glucans, % w/w (fresh weight basis)

= A x F x 94 x 1

1000 x

100

W x

162

180 x D

= A x 𝐹

W x 8.46 x D

-Glucans, % w/w (dry weight basis)

= -Glucans, % w/w (“as is”) x 100

100−moisture content (% w w)

4

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 Result

Enzyme purity Pass

Functional assay Pass

Approved by:

José Prates

Senior Manager, Quality Systems