MBAA TQ doi:10.
1094 / TQ-47-1-0309-01
PEER-REVIEWED PAPER
The Enzymology of Cell Wall Breakdown
During Malting and Mashing: An Overview
C. W. Bamforth
Department of Food Science & Technology, University of California, Davis, CA.
ABSTRACT
Enzymolysis of the cell walls of the starchy endosperm of barley
can be considered a two-stage process: solubilization and polymer di-
gestion. Solubilase enzymes include not only carboxypeptidase acting
as an esterase, but also other enzymes, notably an early-developing xy-
lanase. It is supposed that pentosan is primarily located on the outside
of the cell wall. Nonetheless, pentosan is less efficiently degraded than
β-glucan, probably because the chief xylanases are blocked by an en-
dogenous inhibitor. Although there can be very efficient depolymeri-
zation of β-glucan, it appears that degradation is incomplete and that
the primary hydrolysis products are β-linked oligosaccharides. This is
likely because the exo-glucanases that hydrolyze them develop late in
germination and also have a very low affinity for their substrates. The
advantage of this for the brewer is that beer can be legitimately claimed
as a good source of putative prebiotics.
Keywords: arabinoxylan, cell wall, enzymes, fiber, β-glucan, prebi-
otics
Introduction
For the longest time, brewers have had great concerns about
barley β-glucans (3,4). Problems caused by these cell wall-de-
rived polysaccharides include reduced extract, reduced rates of
wort collection, impaired beer filtration, and hazes, gels, and pre-
cipitates. However, these polysaccharides are being championed
Corresponding author Charlie Bamforth is the Anheuser-Busch Endowed
Professor of Malting & Brewing Sciences at the University of California,
Davis. He has been part of the brewing industry for more than 30 years.
He was formerly deputy director-general of Brewing Research Inter-
national and research manager and quality assurance manager of Bass
Brewers. He is a special professor in the School of Biosciences at the
University of Nottingham, England, and was previously visiting professor
of brewing at Heriot-Watt University in Scotland. Bamforth is a Fellow
of the Institute of Brewing & Distilling, Institute of Biology, and Inter-
national Academy of Food Science and Technology. Bamforth is editor-
in-chief of the Journal of the American Society of Brewing Chemists, a
member of the editorial boards of several journals, including the MBAA
Technical Quarterly, and has published innumerable papers, articles, and
books on beer and brewing.
E-mail: cwbamforth@ucdavis.edu
Based on a paper presented at the 122nd Anniversary Convention of
the Master Brewers Association of the Americas, La Quinta, CA, Octo-
ber 2009.
This paper is dedicated to the memory of Gordon Allan.
© 2010 Master Brewers Association of the Americas
SÍNTESIS
La enzimólisis de las paredes de las células del endospermo fecu-
lento de la cebada puede ser considerada un proceso de dos etapas: la
solubilización y la digestión polimérica. Enzimas solubilasas no solo son
las carboxipeptidasas fungiendo de esterasas, sino también otras enzi-
mas, particularmente una xilanasa de desarrollo precoz. Si bien se su-
pone que el pentosano se encuentra en la parte externa de la pared de
la célula, el hecho es que este se degrada más lentamente que el β-glu-
cano, posiblemente porque las xilanasas principales son bloqueadas por
un inhibidor endovenoso. A pesar de que pueda haber una depolimeri-
zación muy eficiente de β-glucano, pareciera que su degradación es in-
completa y que sus productos primarios de la hidrólisis son oligosacári-
dos con lazos beta. Esto probablemente se debe a que las exo-glucanasas
que los hidroliza se desarrollan tardíamente durante la germinación y
también porque tienen una muy baja afinidad por sus sustratos. Esto per-
mite decir legítimamente que la cerveza es una buena fuente de prebió-
ticos putativos.
Palabras claves: arabioxilano, fibra, β-glucano, pared de célula, pre-
bióticos
within the food industry for their beneficial effects (1,20,25,26).
It is claimed that they can reduce reabsorption of cholesterol,
delay absorption of dietary fat, and enhance intestinal fermenta-
tion by microflora.
Might it be possible to regulate the breakdown of β-glucans
to get the best balance between positive and negative effects?
The prognosis is good: the problems caused by β-glucans are due
to high molecular weight molecules and those still bound into
the walls. It seems that beneficial β-glucans need only to be of a
low molecular weight (28)—the degradation products that do not
cause problems in brewing.
The cell walls of the barley starchy endosperm are composed
of 75% β(1-3,1-4)-glucan, 20% arabinoxylan (pentosan), and
traces of protein, ferulic acid (an antioxidant and precursor of
the clove-like flavor 4-vinylguiaicol), and acetic acid (13). Quan-
titatively, β-glucan is by far the most important component of the
cell walls, which explains the far greater attention paid to it com-
pared with arabinoxylan. The latter cannot be ignored, however,
and comprehensive reviews have recently been written about the
impact of malting and brewing on pentosans (12), as well as
on β-glucans (18,21). The narrower focus of this review is spe-
cifically on the enzymology of cell wall digestion.
Discussion
Solubilization
The tenets of the enzymolysis of cell wall digestion are sum-
marized in Figure 1. The initial release of cell wall polysaccha-
2 / MBAA TQ doi:10.1094 / TQ-47-1-0309-01 Enzymology of Cell Wall Breakdown

rides by enzymes referred to as solubilases enables hydrolysis

of the dissolved molecules by β-glucanases and pentosanases.
The first solubilase identified was the heat-tolerant carboxypep-
tidase, which works in an esterase mode (8) and is presumed to
act on glucan–protein linkages (15). It was later suggested that
there might be at least four different solubilases, one of them a

Figure 1. The tenets of cell wall degradation.

Figure 2. Enzyme-catalyzed release of β-glucan from purified cell walls

of barley (23).  = β(1→3,1→4)-glucanase;  = xylanase I;  = xy-
lanase II;  = xylanase III;  = feruloyl esterase;  = xyloacetyl es-
terase; and × = no enzyme.

Figure 3. Enzyme-catalyzed release of pentosan from purified cell walls

of barley (23).  = β(1→3,1→4)-glucanase;  = xylanase I;  = xy- Figure 4. Time course of enzyme development in germinating barley
lanase II;  = xylanase III;  = feruloyl esterase;  = xyloacetyl es- kernels (27). Black bars show activity in proximal end of barley ker-
terase; and × = no enzyme. nel; white bars show activity in distal end of barley kernel.
Enzymology of Cell Wall Breakdown MBAA TQ doi:10.1094 / TQ-47-1-0309-01 / 3

pentosanase (9). Kanauchi and Bamforth (23) showed that xy- There are a number of other β-glucanases found in barley.
lanases can dissolve β-glucans more effectively than can β-glu- Endo-β1-4-glucanase may play a role in digesting the cellulosic
canase (Fig. 2), whereas β-glucanase is incapable of solubilizing regions within glucans—those parts of the molecule that com-
pentosans (Fig. 3). This suggests that the access of enzymes to prise 10 or more successive β1-4 linkages (35). It has been sug-
β-glucans is hindered by pentosans. Kanauchi and Bamforth (22) gested that endo-β1-3-glucanase is a solubilase (10) or functions
also showed that when the fungus Trichoderma viride grows on as a defense against invading organisms (14). Perhaps the least
barley cell walls the first enzyme to develop is xylanase, which studied of the barley glucanases are the exo-glucanases (17) and
indicates the organism needs to first deal with pentosan. Endo- β-glucosidase (19), despite the fact that they would be expected
barley-β-glucanase is the last enzyme to develop. The first en- to play a primary role in completing the digestion of β-glucans
zyme to be “switched on” in barley at steeping is a xylanase (27), by catalyzing the hydrolysis of the aforementioned oligosaccha-
which is consistent with the initial need to degrade pentosan (Fig. rides.
4). This early elaboration of a xylanase is contrary to previous Kanauchi and Bamforth (24) studied the development of the
observations (30) that suggested endo-xylanases are developed various glucanases during malting (Fig. 8). In keeping with pre-
relatively late in germination. vious observations (7), it was found that there were very low
Based on their observations, Bamforth and Kanauchi (6) devel- levels of endo-barley-β-glucanase in raw barley and that the en-
oped a stylized model of the barley cell wall (Fig. 5). The model zyme developed during steeping and especially during germina-
explains why the stripping away of pentosan is key to enzyme tion. The most significant observation was the late development
access to β-glucan. The gaps in the pentosan layer represent the of exo-glucanase, which may be one of the reasons why sub-
observation that β-glucanase is able to gain some access to its stantial levels of β-linked oligosaccharides are found in beer
substrate. The enzymes that strip away acetate (32) and ferulate (5). Likely just as important, however, is the low affinity of the
(31) are capable of increasing access to β-glucan, but their im- exo-glucanases for β1-4–linked sugars compared with β1-3 link-
pact is much less substantial than that of xylanase (Figs. 2 and 3). ages (24) (Table 1). Because the enzyme’s mode of action is to
Further support for the model was found in studies on exoge- hydrolyze molecules starting at the nonreducing end, whereas
nous enzymes (29) (Fig. 6). When added to mashes with high the β1-3 bond is at the reducing end, it is unsurprising that the
levels of barley adjunct, low rates of xylanase cause an increase rate of hydrolysis of oligosaccharides is very slow.
in viscosity through the release of β-glucan. The ensuing decrease
in viscosity with higher rates of xylanase addition reflects the de-
crease in viscosity of the solubilized arabinoxylans. That β-glu-
canase has a much bigger impact on viscosity reflects, of course,
the much higher levels of glucans, but most significantly, it will
be seen that β-glucanase–xylanase mixtures have the biggest
benefit—we believe because of the role of xylanase in increasing
the access of β-glucanase to its substrate.
Hydrolysis
The key hydrolase responsible for digesting high-viscosity
β-glucans is endo-barley-β-glucanase. There are two isoenzymes
with very similar properties (33,34). Because their most signifi-
cant property is extreme heat sensitivity, important considerations
include 1) allowing the enzyme to act efficiently during germi-
nation in order to produce homogeneously well-modified malt
(2); 2) reduced temperature onsets to kilning to allow the en-
zyme to survive better into finished malt (11); and 3) low-tem-
perature mashing-in and mash rests (11). These enzymes spe- Figure 6. Impact of added enzymes on viscosity of worts from malt/bar-
cifically hydrolyze β1-4 bonds that are on the reducing side of ley (10:90) mashes (29).  = xylanase;  = β-glucanase; and  = xy-
β1-3 linkages. As there is for the most part one β1-3 bond every lanase plus β-glucanase.
third or fourth linkage, the products are trisaccharides and tet-
rasaccharides with a β1-3 bond at the reducing end (Fig. 7).

Figure 5. A stylized model of the cell wall of the starchy endosperm of Figure 7. Mode of action of endo-barley-β-glucanase. – = β1→4; ~ =
barley. β1→3.
4 / MBAA TQ doi:10.1094 / TQ-47-1-0309-01
Figure 8. Development of endo-barley-β-glucanase and exo-β-glucan-
ase during malting (24).
Cell Wall Materials in Beer
Carbohydrates have been investigated in a range of beers us-
ing high-performance liquid chromatography (Kanauchi and
Bamforth, unpublished observations), which showed that there
were high levels of very high molecular weight pentosan in most
of the beers tested, whereas the β-glucans were of a much lower
molecular weight. It is clear that there must be some factor(s)
limiting pentosan degradation—most likely an endogenous xy-
lanase inhibitor (16).
In their study, Kanauchi and Bamforth (unpublished) found
that the average β-glucan level across the beers tested was 4.8 g/L,
whereas the average pentosan level was 1.1 g/L. The difference
is that the β-glucan represented primarily low molecular weight
oligosaccharides (for the reasons described earlier [24]), whereas
the pentosan was largely high molecular weight. To make a
claim for high soluble fiber, a product must contain more than
750 mg/L. Clearly beer can be categorized as high in fiber; how-
ever, it is still necessary to prove that the oligosaccharides con-
tained in beer truly are prebiotic (5).
Implications
The implications of the observations in this review are that
1) Pentosan may likely be a bigger downstream problem than
β-glucan with respect to high viscosity and less soluble ma-
terial.
2) Strategies for eliminating pentosan include finding a way
to eliminate the xylanase inhibitor and/or to use exogenous
xylanases.
3) If the brewer would prefer to sacrifice possible health claims
(β-linked oligosaccharides) for a higher yield of ferment-
able sugar, then use of a commercial exo-glucanase with a
Enzymology of Cell Wall Breakdown
Table 1. Kinetic constants (Km) of exo-glucanases from malted barleya
Exo-glucanase Km cellulose (mg/mL) Km laminarin (mg/mL)
I 162.3 0.78
II 3.01 1.03
III 36.64 1.10
a Kanauchi and Bamforth (24).
much higher affinity for β1-4 linkages could allow for sub-
stantially increased fermentable extract in the form of glu-
cose.
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(Accepted for publication December 12, 2009)