ENZYMATIC YEAST

β-GLUCAN
ASSAY PROTOCOL

K-EBHLG
07/23

(50 Assays per Kit)

© 2023, Neogen Corporation; © 2023, Megazyme. All rights reserved.

Neogen is a registered trademark of Neogen Corporation. Megazyme is a registered trademark of Megazyme Ltd.
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INTRODUCTION:
(1-3)-β-Glucans are widely distributed in nature, especially in algae, fungi and yeast, but
also in higher plants. They form the major structural components of cell walls, they act
as storage carbohydrates and they sometimes play a protective role by forming at
specific sites in response to particular stimuli such as wounding.1
Yeast β-glucan substantially enhances the function of the immune system by
activating macrophages. Literature indicates that linear 6-branched (1-3)-β-glucan
extracted from the mushroom Grifola frondosa (also known as Maitake) can be
linked to potent anti-tumour properties.2-4 The use of yeast β-glucan in animal feed,
especially broiler, has been demonstrated to be beneficial against some bacterial
infections with comparable results to those displayed using antibiotics. Yeast β-
glucan is therefore a promising replacement for antibiotics use in animal nutrition.5
Some β-glucan forms (e.g. high molecular weight (~ 800 KDa) β-glucan produced by
the fungus Botrytis cinerea (grey rot) can however pose technological challenges in
winemaking and are linked to clarification and filtration problems.6
An enzymic method for yeast β-Glucan measurement has been published by
Danielson et al.7 which is known as the ‘GEM’ assay. The method employs an exo-1,3-
β- glucanase/β-glucosidase (E-EXBGOS) from Megazyme and the enzymes lyticase
and pustulanase. This method is suitable for yeast β-glucan measurement, but its
widespread adoption is limited by the high cost of lyticase and the potential over-
estimation due to contaminating enzyme activities in the enzyme mix. In this assay
protocol, we describe the K-EBHLG method, an alternate enzymic procedure for the
measurement of (1-3)(1-6)-β-glucan in commercial extracted yeast β-glucan
preparations. The K-EBHLG method is significantly simpler to perform than the GEM
assay and the recombinant nature of the enzymes present in K-EBHLG provides
excellent reproducibility while also removing the potential for enzymatic side
activities (from impure enzymes) that can artificially inflate the β-glucan content in
samples. Indeed, hydrolysis of α-glucans (starch, mutan, glycogen and sucrose) and
cellulose by the Glucamix enzyme in K-EBHLG is minor (< 2%, see Table 1, page 7).
The K-EBHLG method described in this assay protocol does not give quantitative
measurement of β-glucan in mushrooms. For these samples, we recommend the use
of Neogen’s Megazyme brand ‘β-Glucan Assay Kit (Yeast and Mushroom)’ (K-YBGL)
assay kit. This kit is based on an acid hydrolysis/enzymic procedure for the
determination of β-glucan in yeast and mushroom.8 The acid hydrolysis measures the
Total Glucan content (α-Glucan + β-Glucan) while the enzymic treatment is specific
for α-glucan. The β-Glucan content in this method is determined by difference (i.e.
Total Glucan- α-Glucan).
PRINCIPLE:
(1-3)(1-6)-β-D-Glucan, (1-3)(1-4)-β-D-glucan and (1-3)-β-D-glucans are
solubilised/hydrated in 2 M potassium hydroxide with stirring and the solution is
subsequently adjusted to pH 4.0-4.5 with 1.2 M sodium acetate buffer.9 The slurry is
incubated with the Glucamix enzyme mixture (highly specific yeast β-glucan hydrolytic
enzyme suspension) for 16 h at 40°C. After dilution and centrifugation, an aliquot is

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removed for determination of glucose with GOPOD reagent.

ACCURACY:
Standard errors of < 3% are achieved routinely (see Table 2, page 7).

KITS:
Kits suitable for carrying out 50 assays are available. The kits contain the full assay
method plus:
Bottle 1: Glucamix preparation (Yeast β-glucan hydrolytic enzyme
suspension), 2.2 mL.
Store at 4°C. See individual label for expiry date.
Bottle 2: GOPOD Reagent Buffer. Buffer (50 mL, pH 7.4), p-hydroxybenzoic acid
and sodium azide (0.09% w/v).
Store at 4°C. See individual label for expiry date.
Bottle 3: GOPOD Reagent Enzymes. Glucose oxidase plus peroxidase and 4-
aminoantipyrine. Freeze dried powder.
Store below -10°C. See individual label for expiry date.
Bottle 4: D-Glucose standard solution (5 mL, 1.5 mg/mL) in 0.2% w/v benzoic
acid. Store sealed at room temperature. See individual label for expiry
date.
Bottle 5: Control fungal β-glucan preparation (~ 2 g, β-glucan content stated on
the bottle label).
Store sealed at room temperature. See individual label for expiry
date.
Bottle 6: Control starch preparation (~ 2 g, 96% starch dwb).
Store sealed at room temperature. See individual label for expiry
date.

PREPARATION OF REAGENT SOLUTIONS/SUSPENSIONS:

1. Use bottle 1 as supplied. Swirl the container contents before removing
aliquots. Stand the bottle in an upright position between use.
2. Dilute the contents of GOPOD Reagent Buffer bottle to 1.0 L with distilled or
deionised water. This is Solution 1. Use immediately.
NOTE:
1. On storage, salt crystals may form in the concentrated GOPOD Reagent Buffer.
These must be completely dissolved when this buffer is diluted to 1 L with
distilled water.
2. This buffer contains 0.09% (w/v) sodium azide. This is a poisonous chemical and
should be handled accordingly.

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3. Dissolve the contents of GOPOD Reagent Enzyme bottle in approx. 20 mL of
Solution 1 and quantitatively transfer this to the bottle containing the
remainder of Solution 1. Cover this bottle with aluminium foil to protect the
enclosed reagent from light. This is Glucose Determination Reagent (GOPOD
Reagent).
Stable for ≥ 1 month at 4°C or ≥ 12 months below -10°C.

If this reagent is to be stored in the frozen state, preferably it should be divided

into aliquots. Do not freeze/thaw more than once.
When the reagent is freshly prepared it may be light yellow or light pink in colour.
The solution may develop a stronger pink colour upon storage at 4°C. The
absorbance of this solution should be less than 0.05 when read against distilled
water.
4, 5 Use bottles 4, 5 & 6 as supplied.
& 6.
REQUIRED REAGENTS (NOT SUPPLIED):
1. Sodium acetate buffer (200 mM, pH 5.0).
Add 11.6 mL of glacial acetic acid (1.05 g/mL) to 900 mL of distilled water and
adjust to pH 5.0 using 4 M (16 g/100 mL) sodium hydroxide solution. Adjust the
volume to 1 L.
2. Sodium acetate buffer (1.2 M, pH 3.8).
Add 68.6 mL of glacial acetic acid (1.05 g/mL) to 800 mL of distilled water and
adjust to pH 3.8 using 4 M sodium hydroxide. Adjust the volume to 1 L with
distilled water.
3. Potassium Hydroxide (2 M).
Add 112 g of KOH to 800 mL of distilled water and dissolve by stirring. Adjust
the volume to 1 L.

EQUIPMENT (RECOMMENDED):
1. Glass test tubes (16 x 100 mm, 14 mL capacity).
2. Glass culture tubes with screw cap (16 x 125 mm).
3. Micro-pipettors, 100 μL (e.g. Gilson Pipetman).
4. Positive displacement pipettor, (e.g. Eppendorf Multipette®).
5. Magnetic stirrer plus stirrer bars (5 x 15 mm).
6. Analytical balance.
7. Spectrophotometer set at 510 nm.
8. Vortex mixer.
9. Thermostated water bath set at 40°C.

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10. Bench centrifuge (required speed 3,000 rpm; i.e. approx. 1,500 g), with tube holders
large enough to fully accommodate tubes.
11. Centrifugal mill, with 12-tooth rotor and 0.5 mm sieve.

NOTE:
With each set of determinations, include at least one control fungal preparation (analyse
bottle 5 as described in the procedure below).
A sample blank is also recommended for each sample in order to account for any
free glucose that may be present. Prepare the sample blank by including a duplicate
of the sample analysis as outlined in the procedure below. At Step 4 in the
procedure, add deionised water in place of Glucamix enzyme. Subtract the
absorbance value for the sample blank from the result obtained for the sample
prior to calculation of the β-glucan content.
Also include reagent blanks and glucose standards of 150 μg (in quadruplicate).
The reagent blank consists of 0.1 mL of sodium acetate buffer (200 mM, pH 5.0) +
4.0 mL glucose oxidase/peroxidase reagent.
The D-glucose standard consists of 0.1 mL D-glucose standard (1.5 mg/mL) + 4.0 mL
glucose oxidase/peroxidase reagent.

MEASUREMENT OF 1,3:1,6-β-GLUCAN IN YEAST PREPARATIONS:

1. Mill yeast sample or other material to pass a 0.5 mm screen using a centrifugal mill.
2. Add milled sample (approx. 20 mg, weighed accurately to the nearest 0.1 mg) to a
glass culture tube. Record the weight. Tap the tube to ensure that all of the sample
falls to the bottom of the tube.
3. Add 0.4 mL of 2 M KOH and a 5 x 15 mm stirring bar. Stir the contents for 30 min in
an ice water bath over a magnetic stirrer (Figure 1, page 6).
4. Add 1.6 mL of 1.2 M sodium acetate buffer (pH 3.8), mix well and then add 40 μL of
Glucamix enzyme mixture and cap the tubes. Continue mixing in the ice water bath
for 2 min and then transfer the tubes to a water bath set at 40°C and incubate
(without stirring) overnight (~ 16 h).
5. Add 10 mL of distilled water to each tube and mix the contents thoroughly.
Centrifuge the tubes at 3,000 rpm for 10 min in a bench centrifuge. As an
alternative, the sample can be filtered through the Whatman Type I filter paper (or
equivalent).
6. Carefully transfer 0.1 mL aliquots of the sample in duplicate to the bottom of
glass test tubes.
7. Add 4 mL of GOPOD reagent to each of the reaction tubes, the controls, the
standards and reagent blanks, and incubate the tubes for 20 min at 40°C.

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8. Read the absorbance at 510 nm of each solution against a reagent blank.
CALCULATIONS:
NOTE: These calculations can be simplified by using the Megazyme Mega-Calc™,
downloadable from where the product appears on the Megazyme website
(www.megazyme.com).

β-Glucan (% w/w)

= ∆A x F x 12.04 x 100 x 1 x 162

0.1 W 1000 180

= ∆A x F/W x 10.836.

where:
∆A = Absorbance (reaction) - Absorbance (sample blank)
F = Conversion from absorbance to μg (150 μg of D-glucose) standard
divided by GOPOD absorbance of this 150 μg).

12.04/0.1 = Volume correction (0.1 mL taken from 12.04 mL).

100/W = Factor to present β-glucan as a percentage of sample

weight.
1/1000 = Conversion from μg to mg.
W = Weight of sample analysed in mg.
162/180 = Factor to convert from free D-glucose to anhydro-D-glucose as occurs
in β-glucan.

REFERENCES:
1. Stone, B. A. & Clarke, A. E. (1992). "Chemistry and Biology of (1-3)-β-
Glucans." La Trobe University Press, Victoria, Australia.
2. Ohno, N., Suzuki, I., Oikawa, S., Sato, K., Miyazaki, T. & Yadonne,
T. (1984). Antitumour Activity and Structural Characterisation
of Glucans Extracted from Cultured Fruit Bodies of Grifola frondosa. Chem.
Pharm. Bull., 32, 1142-1151.
3. Ohno, N., Lino, K., Takahiro, T., Suzuki, I., Sato, K., Oikawa, S., Miyazaki, T. &
Yadonne, T. (1985). Structural Characterisation and Antitumour Activity of the
Extracts from Matted Mycelium of Cultured Grifola frondosa. Chem. Pharm. Bull.,
33, 3395-3401.
4. Lino, K., Ohno, N., Suzuki, I., Miyazaki, T. & Yadomae, T. (1985). Structural
Characterisation of a Neutral Antitumour β-D-Glucan Extracted with Hot Sodium
Hydroxide from Cultured Fruit Bodies of Grifola frondosa. Carbohydrate

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 Research, 141, 111-119.
5. Ott, C. P., Omara, I. I., Persia, M. E. & Dalloul, R. A. (2018). The Impact of Beta-
Glucans on Performance and Response of Broiler Chickens During a Coccidiosis
challenge, Poultry Sci, 97, 2713-2721.
6. Villettaz, J. C., Steiner, D. & Trogus, H. (1984). The use of a Beta Glucanase as an
Enzyme in Wine Clarification and Filtration. Am. J. Enol. Vitic., 35, 253-256.
7. Danielson, M. E., Dauth, R., Elmasry, N. A., Langeslay, R. R., Magee, A.
S. & Will, P. M. (2010). Enzymatic method to measure β-1,3-β-1,6- glucan content
in extracts and formulated products (GEM assay).
J. Agric. Food Chem., 58, 10305-10308.
8. McCleary, B. V. & Draga, A. (2016). Measurement of beta-Glucan in Mushroom
and Mycelial Products. J. AOAC International, 99, 364-373.
9. McCleary, B. V. & Monaghan, D. A. (2002). Measurement of resistant starch. J.
AOAC International, 85, 665-675.

APPENDICES:

Figure 1. Arrangement of ice-water bath over a magnetic stirrer for dissolution/hydration

of β-glucan in KOH.

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Table 1. β-glucan content is measured using the enzymic procedure, K-EBHLG, for a
range of standards and enzyme contamination screenings. The undesired hydrolysis of
α-glucans and oligosaccharides (mutan, starch, glycogen and sucrose) and cellulose
by the Glucamix enzyme mixture is minor (< 2 %) which is indicative of the purity of the
enzyme components.
Percentage hydrolysis,
Sample Description reported as β-glucan (%,
w/w, as-is)
Fungal β-glucan control (K-EBHLG, Lot 211101a) 34.0
USP Yeast β-glucan, (Ref cat 1048288) 65.6*
Alpha-Cellulose (Avicel, FMC) 2.1
Soluble starch (Sigma, S9765) 0.1
Glycogen (Sigma, G8751) 1.1
Mutan (P-AGLU13, Lot 210401) 1.0
Sucrose (Sigma, S7903) 1.7
* The content on the label states ~ 78% glucose on a dried weight basis
(dwb). The K-EBHLG analysis is expressed as % w/w of β-glucan (as-is basis).
When the reference sample moisture and the conversion factor between
β-glucan and glucose are taken into account, the result obtained
with K-EBHLG and is 80% glucose (dwb).

Table 2. The repeatability of the assay for yeast samples was determined by analysis of
10 yeast samples over a period of 3 days, with 6 separate extractions of β-glucan,
analysed in duplicate as per the standard procedure.
β-Glucan (% w/w)
Day 1 Day 2 Day 3 Average St. Dev n %CV
Sample 1 68.28 67.53 67.80 67.87 0.38 6 0.57
Sample 2 67.99 67.14 67.96 67.70 0.48 6 0.71
Sample 3 21.16 20.58 20.67 20.80 0.31 6 1.50
Sample 4 16.17 15.95 15.82 15.98 0.17 6 1.09
Sample 5 66.70 65.09 65.03 65.61 0.94 6 1.44
Sample 6 13.67 13.37 13.00 13.35 0.33 6 2.48
Sample 7 22.20 21.71 21.85 21.92 0.26 6 1.17
Sample 8 31.70 31.58 31.75 31.68 0.09 6 0.27
Sample 9 24.96 24.83 24.79 24.86 0.09 6 0.36
Sample 10 23.68 23.49 22.78 23.31 0.47 6 2.02

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Without guarantee
The information contained in this assay protocol is, to the best of our knowledge, true and accurate, but
since the conditions of use are beyond our control, no warranty is given or is implied in respect of any
recommendation or suggestions which may be made or that any use will not infringe any patents. It is the
user’s responsibility to perform in-house matrix validation work prior to routine use.

© 2023, Neogen Corporation; © 2023, Megazyme. All rights reserved.

Neogen is a registered trademark of Neogen Corporation. Megazyme is a registered trademark of Megazyme Ltd.