Tracking polysaccharides through the brewing process

Carbohydrate Polymers 196 (2018) 465–473

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Tracking polysaccharides through the brewing process
a

a

c

d

Jonatan U. Fangel , Jens Eiken , Aafje Sierksma , Henk A. Schols , William G.T. Willats
⁎
Jesper Harholta,

b,⁎

T

,

a

Carlsberg Research Laboratory, J.C. Jacobsens Gade 4, DK-1799, Copenhagen V, Denmark
School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK
The Dutch Beer Institute, Lawickse Allee 11, 6701 AN, Wageningen, Netherlands

d
Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708WG, Wageningen, Netherlands
b
c

A R T I C LE I N FO

A B S T R A C T

Keywords:
Polysaccharides
Glycan arrays
Enzymes
Beer
Malt
Wort

Brewing is a highly complex stepwise process that starts with a mashing step during which starch is gelatinized
and converted into oligo- and/or monosaccharides by enzymes and heat. The starch is mostly degraded and
utilised during the fermentation process, but grains and hops both contain additional soluble and insoluble
complex polysaccharides within their cell walls that persist and can have beneficial or detrimental effects on the
brewing process. Previous studies have mostly been restricted to analysing the grain and/or malt prior to entering the brewing process, but here we track the fates of polysaccharides during the entire brewing process. To
do this, we utilised a novel approach based on carbohydrate microarray technology. We demonstrate the successful application of this technology to brewing science and show how it can be utilised to obtain an unprecedented level of knowledge about the underlying molecular mechanisms at work.

1. Introduction
Beer is the most popular alcoholic beverage and the third-most
consumed beverage in general after water and tea (Barth-Hass-Group,
2017). Despite of being a 6000–8000 years old practice (Nelson, 2005),
the high complexity of beer still proves a significant challenge when
identifying and quantifying the most important beer components.

Consequently, the brewing industry is constantly challenged to optimise practises in relation to product quality and cost effectiveness.
To produce beer, a stepwise batch process is most commonly used.
The malted barley is milled, mixed with water (called ‘mash’) and heated up (mashed) to release fermentable sugars and degrade the barley
cell wall (Gupta, Abu-Ghannam, & Gallaghar, 2010). The mash is separated into liquid, named ‘wort’ and the spent grains. The wort is then
boiled and hops are added. Afterwards the solids, referred to as the
‘trub’, are removed, and the wort is cooled, transferred to a fermentation tank and yeast is added. Following fermentation, the yeast is removed and beer is left to mature before being filtered and bottled.
Besides starch, many other complex polysaccharides are found in
the grain. In the case of barley, the cell walls consist of approximately
70% β-(1- > 3)(1- > 4)-glucan (referred to as β-glucan in brewing literature), 25% arabinoxylan, 2% cellulose, 2% mannan and arabinogalactan proteins (AGP) (Fincher, 1975; Lazaridou, Chornick, Biliaderis, &

⁎

Izydorczyk, 2008). Additionally, high molecular pectins and AGPs are
present in hops (Oosterveld, 2002). Part of these polysaccharides may
end up in the beer as such or as fragment after enzymatic degradation
during the brewing process.
One of the main challenges related to polysaccharides in brewing is
filtration, with high molecular weight β-(1- > 3)(1- > 4)-glucans as the
main culprit (Kumar, Kumar, Verma, Kharub, & Sharma, 2013). However, studies have also shown a negative correlation between filtration
efficiency and high molecular weight water extractable arabinoxylans
(Lu, Li, Gu, & Mao, 2005; Stewart et al., 1998). Besides these two
polymers, mannans have also been linked to filtration issues within the
food industry more widely. In coffee, they are known to effect viscosity
(Sachslehner, 2000) as well as initiate gel formation and can even
create crystalline structures similar to cellulose (Millane & Hendrixson,
1994). Although not yet reported, mannans may also affect the brewing
process.
Colloidal instability is another production issue partly related to
polysaccharide content, and one of the most important quality criteria
of beer related to long-term stability. Most common is chill haze, which

usually dissolves at higher temperatures, but may also result in covalently bound irreversible haze complexes (Steiner, Becker, & Gastl,
2010). The role of polysaccharides in haze formation is not well understood, but studies have shown both positive and negative effects of

Corresponding authors.
E-mail addresses: (J.U. Fangel), (J. Eiken), (A. Sierksma),
(H.A. Schols), (W.G.T. Willats), (J. Harholt).
/>Received 29 January 2018; Received in revised form 16 May 2018; Accepted 16 May 2018
Available online 22 May 2018
0144-8617/ © 2018 Published by Elsevier Ltd.


Carbohydrate Polymers 196 (2018) 465–473

J.U. Fangel et al.

Fig. 1. A diagram illustrating the brewing process and the steps where samples are collected. Modified after Die deutschen Brauer. Deutscher Brauer-Bund e.V (Die
deutschen Brauer and Deutscher Brauer-Bund, 2018). B shows the results of the carbohydrate analysis. The heatmap presents two separate analysis, one for solid
(green) and one for liquid (orange) samples. The highest value in each heatmap have been set to a 100 and the remaining values adjusted accordingly. A cut-off of five
have been introduced. CDTA: 50 mM diamino-cyclo-hexane-tetra-acetic acid, pH 7.5. NaOH: 1 ml 4 M NaOH with 0.1% v/v NaBH4. Homogalacturonan (HG),
Arabinogalactan protein (AGP). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

polysaccharides on haze formation (Siebert, Carrasco, & Lynn, 1996). A
second type of beer particles, known as ‘stringy floaters’, can be problematic within specific market sectors. These particles consist of

protein-carbohydrate complexes characterised by a high content of
cysteine residues, with the carbohydrate constituting 30–50% of the
complex (Vaag, Riis, & Outtrup, 2003). Polysaccharide-related
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J.U. Fangel et al.

2. Materials and methods

problems can be dealt with to some extent through alteration of the
mashing regime, use of enzyme-rich raw materials and/or addition of
external enzymes (Steiner, Auer, Becker, & Gastl, 2012). Consequently,
small adjustments to the enzyme combinations could lead to major
improvements on the aforementioned challenges – but a greater understanding of the polysaccharide content and transformation during
brewing is a prerequisite for achieving this. One of the newer techniques for plant cell wall polysaccharide analysis is based on plant carbohydrate microarrays, enabling the analysis of hundreds or thousands
of molecular interactions simultaneously using very small amounts of
analytes (Moller et al., 2007; Pedersen et al., 2012). The method provides information on the occurrence of glyco-epitopes, rather than
simply monosaccharide profiles. This is made possible through the use
of monoclonal antibodies (mAbs) selected to be specific to small (2–12
monosaccharide) motives (‘epitopes’) in polysaccharides. The technique
is already well established within the plant cell wall community and
have seen utilization within wine research, but has yet to be utilised for
brewing science (Gao, Fangel, Willats, Vivier, & Moore, 2015; Gao
et al., 2016).
Although carbohydrates in beer mostly are investigated in relation
to sensory or technological properties, a nutritional evaluation could
also be valuable for these compounds. Dietary fibre is routinely determined in non-liquid plant-based foods, but not in liquids like beer −
probably because the official methods for analysis are not readily applicable to beverages. Consequently, food composition tables often report zero dietary fibre content in beverages. However, drinks are
quantitatively important items in any diet and they may contain measurable amounts of soluble dietary fibre (SDF). According to a Spanish
study (Diaz-Rubio & Saura-Calixto, 2011), coffee and cocoa contain the
highest levels of SDF (around 8 g/L) of all beverages tested. Beer contains between 1 and 3.5 g/L SDF (depending on the type of beer), which
is comparable to fruit juices from apple, orange and peach. The amount
of SDF in wine depends on the type of wine making; red wine contains

around 2 g/L SDF whereas white wine only 0.2 g/L (Diaz-Rubio &
Saura-Calixto, 2011).
To put these amounts of fibre in beverages in perspective: according
to the European Food Safety Authority a food is regarded as a source of
fibre if it contains ≥ 3 g/100 g or ≥1.5 g/100 kcal and is deemed high
in fibre if it contains ≥6 g/100 g or ≥3 g/100 kcal. On average, fibre
intake for adult males in Europe range from 18 to 24 g/d and for females from 16 to 20 g/d, with little variation from country to country.
Recommendations for fibre intake for adults for most European countries are in the order of 30–35 g/d for men and 25–32 g/d for women
(Stephen et al., 2017). Since fibre intake in Europe is below recommended levels, it is important to report that beverages can contribute to the fibre intake. For example, in Spain, if beverages are taken
into account total fibre intake increases by 10.4%. A moderate consumption of beer of 500 ml per day could imply an intake of about 1 g
SDF (Diaz-Rubio & Saura-Calixto, 2011). Not only the total amount of
fibre is relevant, but also the composition and physiological and nutritional properties. Beer is made of barley and therefore contains
mainly water soluble arabinoxylans and β-(1- > 3)(1- > 4)-glucans –
with β-(1- > 3)(1- > 4)-glucans been shown to lower/reduce blood
cholesterol. In this study, we demonstrate the implementation of novel
high-throughput technology for tracking in detail the fate of polysaccharides throughout the brewing process – from grain to bottled
product. This allows the brewer to infer the occurrence of polysaccharides and the information indicates to the brewer which enzymes
will depolymerize this polysaccharide in the cases where adjustment is
required. In addition, we compare the microarray-based technique with
known quantitative methods for β-(1- > 3)(1- > 4)-glucans to compare
its potential for identifying beverages of particular nutritional interest.

2.1. Brewing samples
Samples were obtained from the Carlsberg Research Laboratory
pilot brewery (Carlsberg, Copenhagen, DK). A lager was brewed using
70% malt and 30% barley. Mashing was started at 52 °C for 20 min,
increased to 65 °C over 13 min and held for 60 min before increasing to
78 °C over 13 min and held for 5 min. The wort was subsequently boiled
for 60 min with addition of standard bitter hops. Saccharomyces
Cerevisiae was used for fermentation over 6 days at 15 °C. Samples of

15 ml were collected from 12 steps illustrated in Fig. 1A. Samples
containing both liquid and solids were separated by centrifugation at
4.000g for 10 min. All solid samples were lyophilised and grinded in a
Retsch mixer mill (Retsch GmbH, Hann, Germany) to a fine powder.

2.2. Comprehensive microarray polymer profiling (CoMPP)
All solid samples were analysed in triplicates of 20 mg material,
sequentially extracted with 1 ml 50 mM diamino-cyclo-hexane-tetraacetic acid, pH 7.5 (CDTA) and 1 ml 4 M NaOH with 0.1% v/v NaBH4
(NaOH). Each extraction was carried out in 2 ml eppendorf tubes at
1000 rpm for 2 h followed by 10 min centrifugation at 10.000g and the
supernatant collected. The liquid samples required no pre-treatment
and all samples were centrifuged at 10.000g before mixed with arrayjet
printing buffer (55.2% glycerol, 44% water, 0.8% Triton X-100) and
spotted unto a nitrocellulose membrane, pore size of 0.45 μm
(Whatman, Maidstone, UK) using an Arrayjet Sprint (Arrayjey, Roslin,
UK). Each sample was printed with four technical replicates and 4 dilutions and probed as described in Pedersen et al. (2012). See Table 1
for further information on monoclonal antibodies and please also refer
to Rydahl, Hansen, Kračun, and Mravec (2018). The arrays were
Table 1
Overview of monoclonal antibodies used throughout this study highlighting
epitope specificity and reference code. Homogalacturonan (HG),
Arabinogalactan protein (AGP).
Specificity

Code

Reference

HG partially/de-esterified

JIM5

HG partially esterified

JIM7

HG partially/de-esterified
HG partially/de-esterified
Backbone of rhamnogalacturonan
I
Backbone of rhamnogalacturonan
I
(1 → 4)-β-D-galactan
(1 → 5)-α-L-arabinan
Linearised (1 → 5)-α-L-arabinan

LM18
LM19
INRA-RU1

Clausen et al. (2003),
Verhertbruggen et al. (2009)
Clausen et al. (2003),
Verhertbruggen et al. (2009)
Verhertbruggen et al. (2009)
Verhertbruggen et al. (2009)
Ralet et al. (2010)

INRA-RU2

Ralet et al. (2010)

LM5
LM6
LM13
BS-400-4
LM21

Jones et al. (1997)
Willats et al. (1998)
Moller et al. (2007),
Verhertbruggen et al. (2009)
Pettolino et al. (2001)
Marcus et al. (2010)

LM22
BS-400-2
BS-400-3
LM15
LM25

Marcus et al. (2010)
Meikle et al. (1991)
Meikle et al. (1994)
Marcus et al. (2008)
Pedersen et al. (2012)

LM10
LM11
LM27

LM28
JIM20
JIM13
LM2

McCartney et al. (2005)
McCartney et al. (2005)
Cornuault et al. (2015)
Cornuault et al. (2015)
Smallwood et al. (1994)
Yates et al. (1996)
Smalwood, Yates, Willats,
Martin, and Knox (1996)

(1 → 4)-β-D-(galacto)mannan
(1 → 4)-β-D-(galacto)(gluco)
mannan
(1 → 4)-β-D-(gluco)mannan
(1 → 3)-β-D-glucan
(1 → 3)(1 → 4)-β-D-glucan
Xyloglucan (XXXG motif)
Xyloglucan/unsubstituted β-Dglucan
(1 → 4)-β-D-xylan
(1 → 4)-β-D-xylan/arabinoxylan
Grass xylan preperations
Glucuronoxylan
Extensin
AGP
AGP, β-linked GlcA

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scanned using a flatbed scanner (CanoScan 9000 Mark II, Canon,
Søborg, Denmark) at 2400 dpi and quantified using Array-Pro Analyzer
6,3 (Media Cybernetics, Rockville, MD, USA). For each sample, an
average was calculated based on the three sample replicates, the four
technical replicates and the four dilutions resulting in 48 measurements
per signal value. For multivariate data analysis, the raw value was used
and for heatmaps, the highest value was set to a 100 and the rest adjusted accordingly. For all heatmaps a cut-off five have been introduced.

presented in Fig. 1B. It is important to note that the heatmap values do
not represent absolute levels of polysaccharides, but the relative
abundance of each epitope across the sample set. Pre-germination, the
grain (1) shows a glycan profile with the main polysaccharides being
mannan (mAb BS-400-4), β-(1- > 3)(1- > 4)-glucan (mAb BS-400-3)
and arabinoxylan (mAb LM11) which is in accordance with previous
reports (Lazaridou et al., 2008). After germination, the malt sample (2)
showed no change for arabinoxylan (mAb LM11), but reduction for
mannan (mAb BS-400-4) and β-(1- > 3)(1- > 4)-glucan (mAb BS-4003), which could indicate a function as storage polymers utilised during
germination. This reduction in β-(1- > 3)(1- > 4)-glucan content
during malting is a direct quality parameter in malting varieties of
barley, facilitated by expression of glucanases and under redox control,
showing the potential of also using CoMPP in breeding efforts (Betts
et al., 2017; Singh et al., 2017) For both grain and malt, the NaOH
extract contained the highest amounts with particularly arabinoxylan

(mAb LM11). Additionally, a small signal for the presence of rhamnogalacturonan-I was detected in the grain (RG-I) (mAb INRA-RU1) with
increased detection in the malt, together with galactan (mAb LM5) and
arabinan (mAb LM6) known to form sidechains on the RG-I backbone
(Buffetto et al., 2015). With the addition of water to the malt in the
mashing tun (3) the sample now constitutes a kind of porridge and a
solid and liquid sample were taken at the three temperature steps
during mashing. For the liquid sample, the prime difference is observed
between 52 and 65 °C. Here feruloyate groups (mAb LM12) and β-(1> 3)(1- > 4)-glucan (mAb BS-400-3) increased, while arabinoxylan
(LM11) and AGP (mAb JIM13) were reduced which is consistent with βglucanases, xylanases and proteases being activated during these processing steps (Vivian, Aoyagui, de Oliveira, & Catharino, 2016). Most
notable in the CDTA extraction was the increased detection of homogalacturonan (HG) (mAb JIM5, JIM7, LM18) not observed in malt. This
suggests the occurrence and activation of pectinases during the mashing
and a pectin structure with HG with intermediate methyl esterification
as well as egg box domains together with RG-I domains. Although
traces of uronic acids have been found in barley grains (Englyst, 1989)
this is to the authors knowledge, the first description of complex pectin
structures in barley grains. Following the mash tun the wort was
transferred to the lauter tun to separate the husks and non-converted
material from the liquid. The first liquid to enter the wort kettle is referred to as the first wort. Extra water (sparging liquor) was added to
wash the remaining sugar out of the milled cereals and is referred to as
the weak wort. The first wort (4) showed a very similar profile to the
mash at 78 °C in the mash tun with the exception of mannan and β-(1> 3)(1- > 4)-glucan being greatly increased. This is an effect of the
grains used as a natural filter to separate the liquid and an increased
extraction takes place. The weak wort showed an identical, but diluted
profile as would be expected. The excess material, spent grains (5),
were very similar in glycan profile to that of the solid 78 °C samples
from the lauter tun though all signals were increased. In the wort kettle
(6) the first and weak wort have been mixed and a diluted glycan
profile is observed with the same relative amount of polysaccharides as
in the lauter tun. The addition of hops and subsequent boil did not
affect the glycan profile with the exception of reduction in HG (mAb

JIM7) and arabinan (mAb LM6) likely due to thermal degradation of
corresponding epitopes (Fraeye et al., 2007). The transfer to the
whirlpool (7), and separation of the trub (8), yielded no change to the
liquid glycomic profile. The trub showed small traces of arabinan (mAb
LM13), feruloyate groups (mAb LM12) and β-(1- > 3)(1- > 4)-glucan
(mAb BS-400-3) probably originating for the liquid trapped in the trub
which predominately consists of high molar weight protein (Mathias,
Alexandre, Cammarota, de Mello, & Servulo, 2015). Yeast (9) used for
fermentation has a cell wall mainly constructed of β-(1- > 3)-glucans
and mannoproteins. Β-(1- > 3)-glucan was clearly detected (BS-400-2)
while mannan in the proteoglycans are α linked and not detectable by
plant cell wall antibodies (Lipke & Ovalle, 1998). The addition of yeast
showed no alteration of the glycomic profile as seen at the start of

2.3. Multivariate data analysis
Patterns within certain microarray datasets were investigated with
principal component analysis (PCA) using SIMCA 12 (MKS Data
Analytics Solutions, Umeå, Sweden). The data were prior to PCA
modelling scaled to unit variance (UV).
2.4. β-(1- > 3)(1- > 4)-glucan samples
Samples were prepared in triplicates from five commercially available beers having varying β-(1- > 3)(1- > 4)-glucan amount. The
samples were analysed using the β-Glucan Assay Kit (Megazyme
International, Ireland) following preparation procedure C and E which
differs in the isolation of polysaccharides before enzymatic degradation. For procedure C, 300 μl of beer is boiled for 5 min and 300 μl 95%
ethanol is added and vortexed. Additional 500 μl of 95% ethanol is
added and the solution is centrifuged for 10 min at 1800g. The supernatant is discarded, and the pellet is suspended in 800 μl 50% ethanol
and vortexed before centrifugation for 10 min at 1800g and supernatant
discarded. For procedure E 500 ul of beer was de-gassed by heating to
approx. 80 °C in a boiling water bath. 250 mg of finely milled ammonium sulphate crystals was added and dissolved before incubation for
approx. 20 h at 4 °C. The sample was centrifuged at 1000g for 10 min

and the supernatant discarded. The pellet was washed twice by dissolving the pellet in 100 ul of 50% ethanol followed by additional 1 ml
of 50% ethanol and centrifuged for 5 min at 1000g. A variation of
procedure C was also carried out with containing a single 70% precipitation step centrifuged at 10.000g for 10 min and the supernatant
discarded and will be referred to as C*. All three pellets were then
dissolved in sodium phosphate buffer (20 mM, pH 6.5) and subjected to
lichenase and glucanase treatment as described in the Megazyme protocol. For each procedure, a sample was taken prior to the addition βglucosidase for oligosaccharide fragment analysis.
2.5. High-performance anion exchange chromatography with pulsed
amperometric detection (HPAEC-PAD)
Glc-β-(1- > 4)-Glc-β-(1- > 3)-Glc (DP3) and Glc-β-(1- > 4)-Glc-β(1- > 4)-Glc-β-(1- > 3)-Glc (DP4) oligomers were quantified with
HPAEC-PAD using a Dionex ICS 5000+ DC system equipped with a
4 μm SA-10 column with 2 × 250 mm dimensions and a guard column.
Run conditions were 0.4 ml/min, column temperature 40 °C, isocratic
100 mM NaOH eluent for 15 min. Standard for quantification were
produced by 1 U/ml lichenase (Megazyme International, Ireland) digestion of known quantities of water soluble medium viscosity β-glucan
(Megazyme International, Ireland) in 50 mM MES pH 6.5 assuming an
equal molecular PAD response ratio between DP3 and DP4.
3. Results and discussion
3.1. Carbohydrate microarrays
To illustrate the capability of the experimental procedure during the
entire brewing process it was divided into 12 key steps as seen on
Fig. 1A. Liquid and/or solid material were acquired from each step and
analysed with CoMPP. The polysaccharide profiles of each step are
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Fig. 2. Carbohydrate microarray analysis of 60 commercial beer samples. The heatmap shows the relative abundance of cell wall related epitopes with the highest

value set to a 100 and the rest adjusted accordingly. A cut-off of five have been introduced. Arabinogalactan protein (AGP).

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J.U. Fangel et al.

fermentation (10).
After end fermentation, subtle changes were observed for most antibodies, mostly attributable the rising alcohol percentage changing the
solubility of the polymers in the liquids. The following filtration (11)
and study of filter content showed yeast to be the dominant remaining
component since the analysis yields were identical to that of pure yeast
samples. The final beer (12) was diluted with water to reach the desired
alcohol percentage giving the same profile as observed for the finalised
fermentation although reduced. The only polysaccharide not following
this pattern was β-(1- > 3)(1- > 4)-glucan, which have slightly increased. This is most likely due to increased solubility as the alcohol
percentage were decreased.

the other by pectin and glycoprotein. Interestingly, the mannan, RG-I,
and glycoprotein mAbs are located in the same groups, while the xylan
mAbs are divided between them with mAb LM10 + LM11 and mAb
LM27 + LM28 separated. This indicates a separation based on xylan
substructures linked to the specific polysaccharide fingerprint of the
beer with mAb LM27 + LM28 characteristic for barley and mAb
LM10 + LM11 for wheat. Comparing the six categories the alcohol free
beers and to some extent the bottom-fermented beers under 6% of alcohol seems to be lower in fibres compared to the other four. Additionally, they seem to be characterised by the two mannan antibodies
(mAb LM21 and BS-400-4). The bottom-fermented beers over 6% together with top fermented beers show no obvious difference between
them and contains a general mixture of all the polysaccharides detected. As seen before the wheat beers are separated on the presence of

RG-I domains as well as the presence of arabinoxylan.
In conclusion, our work has shown that carbohydrate microarray
technology is a powerful addition to the analytical toolbox for the detailed analysis of polysaccharides during the brewing process, including
end products. The methods is highly sensitive, high-throughput and
requires none to very little sample preparation. Microarray are especially valuable for rapidly obtaining semi-quantitative glycan profiles
across large numbers of samples and for informing subsequent more
quantitative analysis of sub-sets of those samples by slower more conventional biochemical techniques.

3.2. Beer analysis
In addition to tracking polysaccharides over a single brewing process, we also analysed polysaccharide profiles in a variety of commercial beers and interpreted these data in the context of brewing style and
specific conditions. Fig. 2 shows the analysis of 60 store bought beers,
grouped into six categories based on alcohol percentage, top-fermenting or bottom-fermenting yeast, and beers classified as wheat
beers. Overall, the same polysaccharides observed in the final sample of
our brewed beer (Fig. 1B) are detected again. However it is noteworthy
that while most polysaccharides are present in all samples in varying
amounts, mannan (mAb LM21, BS-400-4), β-(1- > 3)(1- > 4)-glucan
(mAb BS-400-3) and arabinoxylan (mAb LM11) are not detected in
several beers. While these three polysaccharides showed no obvious
pattern across the defined categories, the RG-I signal yielded higher
signal strength in the wheat beers compared to the rest of the data set.
As stated above, we report here the presence of complex pectin structures in barley, and pectic polymers have been reported in wheat grains
recently (Chateigner-Boutin et al., 2014). Although both barley and
wheat contain pectin, this analysis indicates either a higher amount in
the wheat kernels or a different structure leaving the pectin more prone
to water extraction compared to that of barley. It is possible that the
extra RG-I could come from citrus fruit peel, often added to wheat
beers, but since the sample set contains wheat beers both with and
without this addition this seems unlikely. In any event, an increased
RG-I signal do seem to be a consistent characteristic of the polysaccharide fingerprints of wheat beers. It should be noted that sample 7
which is the only sample with high RG-I detection not located in the

wheat category, is the alcohol free version of sample 55 and would have
been placed next to it if only based on its polysaccharide fingerprint and
is indeed a wheat beer. The detection of pectin in most samples also
highlight the sensitivity of the analysis as it only constitutes minor
amount of the total complex polysaccharide amount in grains. The
substantially reduced relative abundance of mannan and β-(1- > 3)(1> 4)-glucan in some beers most likely reflects the alternation of
mashing profiles, use of enzyme rich raw materials or the addition of
external enzymes. Addition of enzymes during mashing is a well know
practice when brewing with un-malted adjuncts and looking at the
grain information for samples showing little to no detection of mannan
and β-(1- > 3)(1- > 4)-glucan (sample 6, 7, 8, 9, 12, 28, 37, 51, 54 and
55) an overrepresentation of beers with un-malted adjuncts are observed. This also explains why the pattern is not related to any beer
style but to the brewing method in general.
In an attempt to reveal underlying patterns in more detail, the microarray raw data was subjected to principal component analysis (PCA)
(Fig. 3). Looking at the score plot the wheat group is clearly distinguishable from the other samples, but while any difference was very
difficult to see in the heatmap between the remaining categories, some
separation became apparent. The samples are in general spread out
based on fibre richness in the liquids indicated by the two arrows with
PC1 accounting for 35,5% of the variance and PC2 21,4%. As shown by
the loading plot the fibres detected seems to cluster together in two
groups. One dominated by mannan and β-(1- > 3)(1- > 4)-glucan and

3.3. β-(1- > 3)(1- > 4)-glucan quantification
β-(1- > 3)(1- > 4)-glucan in beer was quantitatively investigated
with several methods and sample preparations in order to assess the
effectiveness and precision of the methods and compare them to the
carbohydrate microarrays. High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used
as well as the commercially available ‘β-Glucan Assay Kit (Mixed
Linkage)’. Five beers with varying amounts of β-(1- > 3)(1- > 4)glucan based on carbohydrate profiling were analysed using three different sample preparations all taken from the β-Glucan Assay Kit
(Mixed Linkage) protocol.

Fig. 4 shows the comparison of the two detection methods with
three preparations for each of the five beers tested. The megazyme kit
has a procedure for liquids through ethanol precipitation (C) and a
procedure for beer approved by the European brewing convention (E).
In addition a simple 70% ethanol precipitation, commonly used to
precipitate polysaccharides, were used as well (C*). Since the Megazyme kit utilises a glucose oxidase/peroxidase reagent, HPAEC-PAD
was used as well to analyse the fragments. This method is able to distinguish oligosaccharides from β-(1- > 3)(1- > 4)-glucan and β-(1> 4)-glucan and can reveal the precision of the three preparation
methods C*, C and E.
Overall, the Megazyme kit, yields a higher or equal signal to that of
HPAEC-PAD for all samples. The E samples gives the most comparable
result between the two analysis methods while C* samples yields the
most diverse results. However it is the only method able to detect β-(1> 3)(1- > 4)-glucan in samples 37 and 8 containing the lowest β-(1> 3)(1- > 4)-glucan amounts.
Since the megazyme kit detects glucose of any form, fragments from
glucose containing polysaccharides such as starch cellulose and xyloglucan can interfere with the final result if present in the sample.
Since the HPAEC-PAD can separate the fragments we can measure the
presence of β-glucans nor originating from β-(1- > 3)(1- > 4)-glucan of
one to five sugars (Table 2). Looking at the maltooligosaccharides
present in the samples it is possible to determine how effective the three
sample preparations were at removing these. Amounts between 157
and 389 mg/L are detected in C* samples where the C samples only
contains 12–42 mg/L and Megazyme E less than 2 mg/L. This suggests
that glucose from sources other than β-(1- > 3)(1- > 4)-glucan may be
detected in the Megazyme assay, increasing the final result explaining
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J.U. Fangel et al.

Fig. 3. PCA score plot (A) and loading plot (B) of the raw carbohydrate microarray values from 60 commercial beers. The colour is according to beer categories in the
score plot and polysaccharide categories in the loading plot. Principal component (PC), Rhamnogalacturonan I (RG-I), Top or bottom fermented (Top/Bottom) 6%
refers to beverage alcohol level.

4. Conclusion

the increased amount seen in Fig. 4. In contrast the Megazyme E procedure appears to be more effective at eliminating maltooligosaccharides, yielding a result close to that of HPAEC-PAD using
the same procedure for beer 17 and 60, however little to no detection
was observed in beer 23, 37 and 8 suggesting that the increased purification induces a loss of β-(1- > 3)(1- > 4)-glucan. Conclusively the
C* preparation together with HPAEC-PAD is the fastest preparation
protocol and most precise detection method.
In order to compare the quantitative numbers with the semi-quantitative data from the carbohydrate microarrays, the highest number in
both datasets (beer 17) was set to the same amount and the rest adjusted accordingly (Fig. 5). Beer 60 is measured to the same level for
both assays while 23, 37 and 8 are below the HPAEC-PAD numbers. The
relative differences between the 5 samples are however correct and the
method is thus suitable to rank the beers on β-(1- > 3)(1- > 4)-glucan
content. To get a precise measurement on β-(1- > 3)(1- > 4)-glucan a
second approach has to be utilised.
In relation to nutritional value this is the first study estimating the
amount of β-(1- > 3)(1- > 4)-glucan in beer, measuring values up to
200 mg/L. Despite one of the ingredients of beer is a source of β-(1> 3)(1- > 4)-glucan (barley), probably filtration (or possibly the use of
enzymes) during the brewing process significantly reduces the β-glucan
levels in the beverage (Larsen, Wichmann, Sørensen, & Miller, 2015).

Carbohydrate microarrays were established as a novel highthroughput method for polysaccharide fingerprinting during all stages
of the brewing process for both the solid and liquid fractions. Liquids in
particular are rapid to analyse, as no sample preparation is required
since brewing is already an extraction procedure of polysaccharides in
itself. The technique is an attractive addition to the brewing analysis
toolbox and offers a great level of detail compared to established

methods. Besides giving valuable input to understand polysaccharide
related issues regarding filtration and colloidal instability, the results
can potentially give information regarding enzyme and grain usages in
a given beer as this seems to be revealed on the polysaccharide profiles
of the 60 shelf beer analysis. This information can prove very valuable
on how to optimise the addition of enzymes during mashing resulting in
a more cost effective brewing with increased long-term stability.
Furthermore, the analysis can rank the beers according to their levels of
dietary fibre matching that of established methods resulting of the selection of samples with potential of higher nutritional value.

Conflict of interest
Aafje Sierksma was employed by the Dutch Beer Institute during the
Fig. 4. Levels of β-(1- > 3)(1- > 4)-glucan as analysed with
HPAEC-PAD and “β-Glucan Assay Kit (Mixed Linkage)” using
three different sample preparations C*, C and E. High-performance anion exchange chromatography (HPAEC) detect oligomers using pulsed amperometric detection (PAD) detector
while the β-Glucan Assay Kit converts all mixed link glucan
specifically to glucose which is determined by an enzymatic
colour assay using glucose oxidase plus peroxidase and 4aminoantipyrine (GOPOD).

471


Carbohydrate Polymers 196 (2018) 465–473

J.U. Fangel et al.

Table 2
Glucose and maltodextrin levels (mg/L) in five different beers across three different preparation protocols as measured by HPAEC-PAD. All numbers are mg/L.
Glucose

Maltose

(mg/ml)
C*

C

E

Beer
Beer
Beer
Beer
Beer
Beer
Beer
Beer
Beer
Beer
Beer
Beer
Beer
Beer
Beer

17
60
23
37
8

17
60
23
37
8
17
60
23
37
8

69,3
67,7
74,8
70,7
67,9
1,9
3,0
1,7
1,9
1,4
0,0
0,0
0,0
0,0
0,0

±
±
±

±
±
±
±
±
±
±
±
±
±
±
±

Maltotriose

(mg/ml)
3,3
2,9
15,1
11,3
27,3
0,8
2,0
0,7
1,5
0,4
0,0
0,0
0,0
0,0

0,0

24,1
14,6
9,1
13,1
7,4
8,0
4,6
1,6
0,7
0,7
0,0
0,0
0,0
0,0
0,0

±
±
±
±
±
±
±
±
±
±
±
±

±
±
±

Maltotetraose

(mg/ml)
1,2
2,0
0,3
2,3
1,6
0,8
0,5
0,5
0,3
0,2
0,0
0,0
0,0
0,0
0,0

28,1
8,9
13,5
8,1
15,5
2,9
4,6

1,7
4,3
3,8
0,0
0,0
0,0
0,0
0,0

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

Maltopentose

(mg/ml)
20,2
10,9

5,9
3,7
3,9
0,8
1,6
0,5
2,3
1,0
0,0
0,0
0,0
0,0
0,0

58,7
20,3
44,0
214,2
30,8
4,2
4,4
2,4
24,9
3,3
0,3
0,3
0,2
0,7
0,2

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

Total

(mg/ml)
10,4
8,4
5,9
18,0
6,8
0,6
1,2
0,3
3,7
0,7
0,1

0,0
0,0
0,3
0,0

87,4
104,6
132,7
83,3
35,2
14,7
12,4
8,4
10,3
2,5
1,3
0,8
0,0
0,4
0,0

±
±
±
±
±
±
±
±
±

±
±
±
±
±
±

(mg/ml)
4,2
10,2
6,1
7,5
9,0
1,5
1,7
2,6
0,9
1,2
0,2
0,2
0,0
0,2
0,0

267,5
216,1
274,1
389,3
156,7
31,8

29,1
15,8
42,2
11,6
1,6
1,1
0,2
1,2
0,2

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

25,8
28,0
7,5
21,2

39,7
3,9
5,9
3,6
8,7
2,3
0,2
0,2
0,0
0,4
0,0

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study and during writing the manuscript. She had no role in the design
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organisation of the ten largest beer brewers in The Netherlands.
Acknowledgements
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[grant number 5112-00006B]; The Carlsberg Foundation; and by The
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