Debranning of wheat prior to milling reduces xylanase but not xylanase

inhibitor activities in wholemeal and our

W. Gys
a,
*
, K. Gebruers
a
, J.F. Srensen
b
, C.M. Courtin
a
, J.A. Delcour
a
a
Laboratory of Food Chemistry, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium
b
Danisco, Enzyme Development, Edwin Rahrsvej 38, DK-8220 Brabrand, Denmark
Received 19 August 2003; revised 9 January 2004; accepted 23 January 2004
Abstract
Debranning of wheat to remove the outer 7% of the kernel, prior to grinding or milling reduced xylanase activity in wheat wholemeal and
wheat endosperm our by up to 80 and 60%, respectively, whereas there was no signicant reduction of xylanase inhibiting activity. Flours
obtained after debranning and milling showed no major differences in moisture content, whereas ash content decreased and protein and
arabinoxylan content decreased slightly with increasing debranning degree. Part of the xylanase activity in the our was lost on addition of
Triticum aestivum xylanase inhibitor (TAXI). Since TAXI specically inhibits glycosyl hydrolase family 11 xylanases and since endogenous
cereal xylanases belong exclusively to family 10, part of the xylanase activity in the our is most likely of microbial origin. Debranning also
signicantly reduced alpha-amylase activities in wheat wholemeal and wheat our. Debranning prior to milling can, therefore, impact on
our functionality.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Debranning; Wheat; Xylanase; Xylanase inhibitor; Exogenous enzymes
1. Introduction
Wheat milling has evolved from the use of pestle and
mortar to a fully automated process. Debranning of wheat
prior to milling enables the production of good-quality
milled products from lower quality wheat (Dexter and
Wood, 1996). Debranning improves the quality of wheat
infected with Karnal Bunt, a fungus causing undesirable
organoleptic properties, such as a shy odor (Sekhon et al.,
1992a). The use of ground, debranned wheat instead of
conventionally milled wheat our (North American Norkan
hard red winter wheat, 70% extraction rate) in the gluten-
starch separation improves gluten yield (Zhuge et al.,
1991). In addition, the removal of outer layers from
the sprouted wheat kernel reduces the alpha-amylase
content of the our (Henry et al., 1987; Liu et al., 1986;
Sekhon et al., 1992b).
Besides alpha-amylases (EC.3.2.1.1), xylanases
(EC.3.2.1.8) are also present in wheat (Bonnin et al.,
1998; Cleemput et al., 1995) and its our (Cleemput et al.,
1997). During germination, endogenous xylanases degrade
the walls of aleurone and endosperm cells, making starch
and storage proteins accessible to aleurone derived
amylases and proteases, respectively (Mares and Stone,
1973a,b). Xylanases of microbial origin are frequently used
industrially to improve cereal grain processing and/or the
quality of the end product. Based on genetic information,
structural analysis, hydrophobic cluster analysis and
sequence similarities (Henrissat, 1991), most xylanases
are classied in family 10 family or 11 glycosyl hydrolases.
Whereas fungi and bacteria produce xylanases from both
families, all plant xylanases so far identied belong to
family 10 (Simpson et al., 2003), implying that all
endogenous wheat xylanases most probably belong to
family 10.
Wheat and several other cereals contain xylanase inhi-
bitors. Triticum aestivum xylanase inhibitor (TAXI)-type
0733-5210/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2004.01.002
Journal of Cereal Science 39 (2004) 363369
www.elsevier.com/locate/jnlabr/yjcrs
* Corresponding author. Tel.: 32-16-32-16-34; fax: 32-16-32-19-97.
E-mail address: wouter.gys@agr.kuleuven.ac.be (W. Gys).
Abbreviations: AZCL-AX, azurine-cross-linked-arabinoxylan; AU,
alpha-amylase units; dm, dry matter; E
590
, extinction at 590 nm; InhU,
Inhibition Units; TAXI, Triticum aestivum xylanase inhibitor; TRIS, Tris
(hydroxymethyl)-aminomethane; XIP, xylanase inhibiting protein; XU,
xylanase units.
inhibitors (Debyser et al., 1999) exclusively inhibit bacterial
and fungal family 11 xylanases (Gebruers, 2002; Gebruers
et al., 2003), while a second type of inhibitor, xylanase-
inhibiting protein (XIP) (McLauchlan et al., 1999), inhibits
both family 10 and family 11 fungal xylanases but not
bacterial xylanases (Flatman et al., 2002).
Because of the importance of xylanases and their
inhibitors in industrial wheat processing we have examined
the occurrence of exogenous xylanases and their possible
interaction with endogenous xylanase inhibitors by compar-
ing their activities in wholemeals and ours prepared from
wheat that had been debranned to different extents prior to
milling.
2. Experimental
2.1. Materials
Meunier wheat (2001 harvest), of good breadmaking
quality and with a falling number of 418 s, was from Clovis
Matton (Kerkhove, Belgium).
All reagents were purchased from VWR International
(Haasrode, Belgium) and were of analytical grade. D-Allose
was from Sigma-Aldrich (Bornem, Belgium). Recombinant
Bacillus subtilis xylanase (Grindamyl H640) was from
Danisco (Brabrand, Denmark). Azurine-cross-linked arabi-
noxylan (AZCL-AX) tablets and Amylazyme tablets were
from Megazyme (Bray, Ireland).
2.2. Debranning
Wheat (1.4 kg) was debranned using a Satake Batch
debranner Type TM 05 (Satake, Bredbury, UK) in batches
of 175 g for 0, 4, 8, 12, 16, 20, 28 or 36 s. The bran, referred
to as Satake Bran, was separated from the kernels, referred
to as debranned kernels, and weighed.
2.3. Milling
Wholemeal was obtained from untreated and deb-
ranned wheat kernels by milling in an A-10 analytical
mill (IKA, Staufen, Germany) and sieving through a
400 mm sieve.
Wheat endosperm our, bran and shorts were obtained as
three separate fractions by milling the untreated and the
debranned kernels in a Chopin Laboratory mill CD1
(Chopin, Villeneuve-la-Garenne, France). Water (0.5%,
w/w) was added 30 min prior to milling. The resulting
bran is referred to as Chopin Bran.
Wholemeal and ours obtained from kernels after
debranning for 0, 4, 8, 12, 16, 20, 28 or 36 s are referred
to as 0S, 4S, 8S, 12S, 16S, 20S, 28S and 36S wholemeal and
our, respectively.
2.4. Analytical procedures
2.4.1. Moisture and ash
Moisture and ash contents were measured according to
AACC-methods 44-15a (AACC, 2000) and 08-12 (AACC,
2000), respectively.
2.4.2. Protein
Protein contents of the grain fractions were determined in
duplicate by the Dumas method, an adaptation of the AOAC
Ofcial Method (1995) for the automated Dumas protein
analysis system (EAS VarioMax N/CN, Elt, Gouda, The
Netherlands).
2.4.3. Monosaccharide analysis
The total monosaccharide composition of the ours was
determined by gasliquid chromatography of alditol acet-
ates. Flours were hydrolysed as described by Loosveld et al.
(1997) and the alditol acetates, prepared as described by
Englyst and Cummings (1984), were separated on a Supelco
SP-2380 column (30 m 0.32 mm I.D., 0.2 mm lm thick-
ness) (Supelco, Bellefonte, PA, USA) in an Agilent 6890
series chromatograph (Agilent, Wilmington, DE, USA)
equipped with autosampler, splitter injection port (split
ratio 1:20) and ame ionisation detector. The carrier gas was
helium. Separation was at 225 8C, with injection and
detection at 270 8C. The arabinoxylan content was calculated
as the sumof anhydro-xylose andanhydro-arabinose, usingthe
factor 0.88. Corrections were made for arabinose originating
from arabinogalactan-peptide (Loosveld et al., 1997).
The coefcient of variation, dened as the ratio of the
standard deviation to the mean of the analytical results, was
lower than 5%.
2.5. Xylanase activity
Xylanase activities were determined by the xylazyme-
AX method (Megazyme, Bray, Ireland) as follows: whole-
meal or our (1.0 g) was suspended in 10.0 ml sodium
acetate buffer (25.0 mM, pH 5.0), extracted for 1 h at room
temperature and centrifuged (10,000 g, 30 min, 6 8C) in a
Beckman J2-21 centrifuge (Beckman, Fullerton, CA, USA).
The extracts (1.0 ml) were equilibrated for 10 min at 50 8C
before adding an AZCL-AX tablet. Incubation was for 3 h
(wholemeal extracts) or 20 h (our extracts) at 50 8C. The
reaction was terminated by addition of 1.0% (w/v) TRIS
[tris (hydroxymethyl)-aminomethane] solution (10.0 ml)
and mixing on a vortex mixer. After 10 min at room
temperature, the tubes were shaken vigorously and the
contents ltered through a MN 615 Filter (Macherey-Nagel,
Duren, Germany). The E
590
values (extinction at 590 nm)
[Ultraspec III UV/Visible spectrophotometer (Pharmacia
Biotech, Uppsala, Sweden)] were measured against a
control, prepared by incubating the extracts without the
substrate tablet for 3 h (wholemeal) or 20 h (our) at 50 8C
and addition of the substrate tablet after adding 1.0% TRIS
W. Gys et al. / Journal of Cereal Science 39 (2004) 363369 364
solution to the extract. Correction was made for
non-enzymatic colour release from the AZCL-AX tablets.
Activities were expressed in units. One xylanase unit (XU)
corresponds to an increase in E
590
of 1.0 g sample h
21
,
under the assay conditions.
2.6. Xylanase inhibitor activity
Xylanase inhibitor activities were measured by the
xylazyme-AX method as described by Gebruers et al.
(2002). Bacillus subtilis xylanase solution, was prepared by
extracting Grindamyl H640 with a solution of bovine serum
albumin (0.5 mg/ml) in sodium acetate buffer (25.0 mM, pH
5.0). The solution contained 2.0 units per ml where 1.0 unit
corresponded to an increase in E
590
of 1.0 in the xylazyme-
AX method.
Xylanase solution (0.5 ml) was preincubated for 30 min
at room temperature with, either, an equal amount of sample
possibly containing xylanase inhibitor activity, or, with an
equal amount of buffer (referred to as the reference sample).
After preincubation, the mixtures were held at 30 8C and
after 10 min an AZCL-AX tablet was added. The suspen-
sions were then incubated for 60 min at 30 8C. The reaction
was terminated by addition of 1.0% (w/v) TRIS solution
(10 ml) and mixing on a vortex mixer. After 10 min at room
temperature, the tubes were shaken vigorously and their
contents ltered (MN 615 Filter). The E
590
values were
measured against a control prepared by incubating the
sample with buffer instead of enzyme solution. The
difference between the extinction values of the samples
and the reference was used as a measure of inhibitor
activity. Xylanase inhibitor activities were expressed as
inhibition units (InhU), where one InhU is the amount of
inhibitor per gram sample that inactivates 50% of a given
activity E
590
1:0 of xylanase under the conditions
described by Gebruers et al. (2002).
2.7. Detection of exogenous xylanases on wheat grain
The presence of exogenous xylanase in wheat our
extracts was measured by adding TAXI to the extracts and
determining the decrease in xylanase activity. Wheat ours
(2.0 g), obtained after debranning for 0, 4, 8, 12, 16, 20, 28
and 36 s and milling, were extracted with 8.0 ml sodium
acetate buffer (25 mM, pH 5.0) at room temperature for 1 h
and centrifuged (10,000 g, 6 8C, 30 min). Supernatant
(800 ml) was mixed either with sodium acetate buffer
(380 ml) or with TAXI (100 ml of a 273 mg/ml solution) and
sodium acetate buffer (280 ml) and preincubated for 30 min
at room temperature. After equilibration for 10 min at 40 8C
an AZCL-AX tablet was added and incubation continued for
24 h. The remainder of the procedure was as described for
xylazyme-AX method.
2.8. alpha-Amylase activity
alpha-Amylase activity was measured by the Amyla-
zyme-method (Megazyme). Flour and wholemeal samples
(0.5 g) were weighed accurately into glass test tubes and
incubated at 60 8C with sodium maleate buffer (5.0 ml,
100 mM, pH 6.0, 5 mM CaCl
2
, pre-equilibrated to 60 8C).
After 5 min continuous stirring, an Amylazyme tablet was
added. The reaction was allowed to continue for exactly
5 min and was stopped with TRIS solution (6.0 ml, 2%, w/v,
pH 9.0) under vigorous stirring on a vortex mixer. After
ltration through a MN 615 Filter, the E
590
of the ltrate was
measured against a reaction blank, prepared by adding TRIS
solution before the Amylazyme tablet. alpha-Amylase
activities per g sample, expressed in units (AU), were
calculated from the standard curve supplied with the
Amylazyme kit (Megazyme, Bray, Ireland).
3. Results
3.1. Debranning
Debranning for 0, 4, 8, 12, 16, 20, 28 and 36 s resulted in
0, 4.1, 7.0, 9.8, 12.0, 14.4, 18.8 and 22.4% Satake Bran (%
of kernel weight), respectively (Fig. 1). Standard deviation
between different batches (175 g) was less than 0.3%.
According to Shetlar et al. (1947), the outer pericarp, the
inner pericarp, the testa and the aleurone layer represent 3.9,
0.9, 0.7 and 9.0% of the kernel weight, respectively. This is
more or less in agreement with values given by MacMasters
et al. (1971) who assigned approximately 7% of the kernel
weight to pericarp, testa and nucellar tissue, and approxi-
mately 7% to the aleurone layer. Because the thickness of
the pericarp-seed coat layers differs among wheat varieties
and debranning proceeds unevenly over the grain surface,
debranning for 4 s on average would remove most of the
outer pericarp while further debranning would remove the
other non-aleurone layers. The aleurone layer was pre-
sumably completely removed only after 20 s of debranning.
Fig. 1. Yields (% of total weight) of wheat fractions (Satake Bran, Chopin
Bran, shorts and our) after milling debranned kernels. Debranning was for
0, 4, 8, 12, 16, 20, 28 and 36 s.
W. Gys et al. / Journal of Cereal Science 39 (2004) 363369 365
3.2. Impact of debranning on wholemeal characteristics
The characteristics of wholemeal of debranned kernels
are given in Table 1.
No distinction could be made in the moisture (,13%) or
protein contents [,12% (dm)] between the different whole-
meals. Ash content gradually decreased with increasing
degree of debranning as observed by Sekhon et al. (1992a,b).
Debranning affected xylanase activities substantially.
The activity in the reference wholemeal (0S) amounted to
0.48 XU but removal of the outer 4% of the kernel (4S
wholemeal) lowered the xylanase activity to 0.33 XU.
Debranning for 8 s (removal of outer 7%, Satake Bran)
diminished the xylanase activity in the debranned kernel by
more than 80%, to a level of 0.09 XU. Further debranning
did not lead to a noticeable decrease in xylanase activity,
implying that over 80% of the xylanase activity in wheat
kernel is located in the outer 7% of the kernel.
The higher xylanase activity in wholemeal 20S (0.18
XU) was surprising. After debranning for 20 s, 14.4% of the
wheat kernel was removed, which is approximately the
amount of material, which comprises pericarp, seed coat and
(part of the) aleurone layer. Further work will be needed to
understand the observed increased level of xylanase.
All wholemeals had similar xylanase inhibition activities
(,190 InhU), indicating that in each debranned fraction of
the wheat kernel approximately the same level of xylanase
inhibitor was present.
alpha-Amylase activity also decreased on debranning.
The activity in debranned wholemeals compared to the
reference was reduced by 42% when 22.4% of the grain was
removed.
3.3. Milling
Milling of debranned wheat showed that as more Satake
Bran was removed in the debranning step, the less Chopin
Bran was obtained after milling (Fig. 1). The total amount of
bran (Satake Bran plus Chopin Bran) increased with the extent
of debranning, while the shorts and our yields decreased
slightly. Milling yields for fractions 0S, 4S, 8S, 12S, 16S, 20S,
28S and 36S were 68, 66, 66, 65, 65, 64, 62 and 59%,
respectively. The decreased our yield with increased degree
of debranning was in line with earlier observations for both
sound and sprouted wheats (Sekhon et al., 1992b).
3.4. Impact of debranning on wheat our characteristics
3.4.1. Moisture, protein and ash content
The different our samples had comparable moisture
contents (,13%) (Table 2). With increasing degree of
debranning, the protein content decreased slightly from
10.5% (dm) to 9.8% (dm), while ash content decreased
continuously from 0.68% for our 0S to 0.50% for our
36S, with the exception of our 4S, where the ash content
was lower than expected (0.58%).
Table 1
Analytical data of wheat wholemeals (0S, 4S, 8S, 12S, 16S, 20S, 28S and 36S) obtained after debranning wheat for 0, 4, 8, 12, 16, 20, 28 and 36 s, respectively,
followed by conventional milling
Wholemeal Moisture (%) Protein (% dm) Ash (% dm) alpha-amylase
activity (AU)
Xylanase activity
(XU)
Xylanase inhibition-
activity (InhU)
0S 13.1 ^ 0.2 12.1 ^ 0.2 1.70 ^ 0.08 0.12 ^ 0.00 0.48 ^ 0.02 183 ^ 12
4S 13.1 ^ 0.0 12.3 ^ 0.2 1.63 ^ 0.03 0.10 ^ 0.01 0.33 ^ 0.00 184 ^ 10
8S 13.1 ^ 0.2 12.1 ^ 0.1 1.47 ^ 0.03 0.10 ^ 0.01 0.09 ^ 0.00 207 ^ 3
12S 13.0 ^ 0.0 12.0 ^ 0.1 1.35 ^ 0.03 0.09 ^ 0.01 0.09 ^ 0.00 190 ^ 6
16S 12.9 ^ 0.2 12.0 ^ 0.1 1.23 ^ 0.02 0.10 ^ 0.01 0.08 ^ 0.00 188 ^ 10
20S 13.0 ^ 0.0 12.0 ^ 0.1 1.18 ^ 0.00 0.08 ^ 0.01 0.18 ^ 0.00 199 ^ 5
28S 13.0 ^ 0.0 11.9 ^ 0.1 1.04 ^ 0.00 0.09 ^ 0.00 0.09 ^ 0.00 194 ^ 12
36S 12.8 ^ 0.0 12.0 ^ 0.2 0.96 ^ 0.01 0.07 ^ 0.01 0.09 ^ 0.00 187 ^ 3
Table 2
Analytical data of wheat ours (0S, 4S, 8S, 12S, 16S, 20S, 28S and 36S) obtained after debranning wheat for 0, 4, 8, 12, 16, 20, 28 and 36 s, respectively,
followed by conventional milling
Flour Moisture (%) Protein (% dm) Ash (% dm) alpha-amylase
activity (AU)
Xylanase activity
(XU)
Xylanase inhibition-
activity (InhU)
0S 13.2 ^ 0.1 10.5 ^ 0.1 0.68 ^ 0.02 0.03 ^ 0.01 0.13 ^ 0.00 102 ^ 3
4S 13.5 ^ 0.1 10.4 ^ 0.0 0.58 ^ 0.02 0.09 ^ 0.04 0.06 ^ 0.00 96 ^ 2
8S 13.2 ^ 0.3 10.4 ^ 0.0 0.64 ^ 0.00 0.01 ^ 0.01 0.05 ^ 0.00 106 ^ 2
12S 13.2 ^ 0.3 10.4 ^ 0.0 0.63 ^ 0.00 0.01 ^ 0.00 0.06 ^ 0.00 100 ^ 1
16S 13.1 ^ 0.1 10.3 ^ 0.0 0.59 ^ 0.00 0.01 ^ 0.00 0.04 ^ 0.00 111 ^ 1
20S 13.2 ^ 0.2 10.1 ^ 0.0 0.58 ^ 0.02 0.00 ^ 0.00 0.04 ^ 0.00 89 ^ 3
28S 13.1 ^ 0.2 10.0 ^ 0.1 0.53 ^ 0.01 0.01 ^ 0.00 0.04 ^ 0.00 104 ^ 4
36S 12.8 ^ 0.4 9.8 ^ 0.0 0.50 ^ 0.01 0.01 ^ 0.00 0.04 ^ 0.00 70 ^ 2
W. Gys et al. / Journal of Cereal Science 39 (2004) 363369 366
3.4.2. Monosaccharide composition
Total arabinose and xylose contents in the ours
decreased with increased debranning (Table 3). This was
reected in the calculated arabinoxylan content, which
decreased from 1.92% for reference our 0S to 1.72% in
our 36S. As expected, the arabinose to xylose ratio
remained fairly constant in all ours and amounted to 0.56
on average. Indeed, although variable degrees of debranning
must have removed AX of lower A/X, neither such AX nor
those still present in the Chopin Bran reach the A/X in our.
Together with the arabinose and xylose contents, galactose
and mannose levels decreased slightly when the outer layers
of wheat were removed before milling. The glucose content
ranged between 82.2 and 78.4%.
3.4.3. Xylanase and xylanase inhibitor activity
Xylanase activities (Table 2) in our as a function of
debranning followed the same trend as in wholemeal our.
Thus, a sharp decrease in activity between reference our 0S
(0.13 XU) and our 4S (0.06 XU) was observed: more than
50% of xylanase activity in our was removed when there
was a 4.1% loss of kernel weight by debranning before
milling. Debranning for 16 s or more (12% of kernel weight
removed) led to a 70% decrease in xylanase activity,
resulting in 0.04 XU. This suggests that at least part of the
xylanases in ours produced by conventional milling, arise
by contamination of the our with particles of bran.
In contrast to xylanases in wheat endosperm, which are
assumed to be endogenous, a large part of the xylanases in
the outer layers of grain are possibly from bacterial and/or
fungal origin (Noots et al., 1998). Because removal of bran
prior to milling reduces xylanase activity in our, this could
imply that part of the xylanases in our is exogenous, as
already suggested by Dorfer (2001) for rye our and for
cellulases present in barley (Hoy et al., 1981). To examine
this hypothesis, the presence of exogenous xylanases
in wheat our extracts was analysed making use of the
known specicity of TAXI for family 11 xylanases
(Gebruers, 2002; Gebruers et al., 2003), which are absent
in cereals. Indeed, endogenous cereal enzymes are family 10
glycosyl hydrolases (Simpson et al., 2003).
Xylanase inhibition activities (Table 2) uctuated around
100 InhU, but, except from our 36S (70 InhU), no major
differences were found.
3.4.4. Detection of exogenous xylanases from wheat
To detect the presence of bacterial and fungal family 11
xylanases, an excess of TAXI was added to our extracts.
Since the addition of TAXI reduced the xylanase-activity
in each our (Fig. 2), at least part of the xylanases present in
wheat our are from family 11, and are thus exogenous in
origin. Except for our 0S, where only 16% of the xylanase
activity was inhibited, TAXI addition resulted in more than
25% inhibition. In contrast to expectation, 25% of the
xylanase activity was inhibited by TAXI in our 36S, i.e.
after the outer 22% of the kernel was removed by
debranning before milling. Although we had expected the
removal of the outer layers to be accompanied by a
reduction in the microbial xylanase activity in the our and
hence by a reduction in the level of TAXI-inhibition, this
was not seen. Again, this indicates that a high proportion of
Table 3
Monosaccharide composition (% our dm), arabinoxylan content (% our dm) and A/X ratio of wheat ours (0S, 4S, 8S, 12S, 16S, 20S, 28S and 36S) obtained
after debranning wheat for 0, 4, 8, 12, 16, 20, 28 and 36 s, respectively, followed by conventional milling
Flour Ara
a
(% dm) Xyl
a
(% dm) Gal
a
(% dm) Glc
a
(% dm) AX
b,c
(% dm) A/X
d,e
0S 0.93 ^ 0.01 1.40 ^ 0.01 0.36 ^ 0.01 82.2 ^ 1.1 1.92 ^ 0.02 0.56 ^ 0.00
4S 0.89 ^ 0.01 1.32 ^ 0.02 0.35 ^ 0.01 80.2 ^ 1.0 1.82 ^ 0.03 0.56 ^ 0.00
8S 0.87 ^ 0.03 1.32 ^ 0.06 0.34 ^ 0.00 79.5 ^ 0.8 1.80 ^ 0.08 0.55 ^ 0.01
12S 0.85 ^ 0.01 1.28 ^ 0.02 0.34 ^ 0.00 79.1 ^ 0.2 1.75 ^ 0.03 0.55 ^ 0.00
16S 0.84 ^ 0.01 1.28 ^ 0.03 0.33 ^ 0.00 78.4 ^ 0.1 1.74 ^ 0.03 0.55 ^ 0.01
20S 0.84 ^ 0.02 1.27 ^ 0.04 0.33 ^ 0.01 79.0 ^ 1.2 1.73 ^ 0.05 0.55 ^ 0.00
28S 0.84 ^ 0.01 1.28 ^ 0.02 0.33 ^ 0.00 79.2 ^ 0.1 1.75 ^ 0.02 0.55 ^ 0.00
36S 0.84 ^ 0.03 1.27 ^ 0.04 0.34 ^ 0.01 80.7 ^ 2.0 1.72 ^ 0.06 0.55 ^ 0.00
a
Ara: arabinose, Xyl: xylose, Gal: galactose, Glc: glucose, Man: mannose.
b
AX: arabinoxylan.
c
%AX (Ara 2 0.7 Gal Xyl)0.88.
d
A/X: arabinose to xylose ratio.
e
A/X (Ara 2 0.7 Gal)/Xyl.
Fig. 2. Xylanase activity in wheat our extracts and wheat our extracts
with added TAXI. The xylanase activity in wheat our extract (0S) was
used as a reference (100%).
W. Gys et al. / Journal of Cereal Science 39 (2004) 363369 367
the xylanase in wheat our is of family 11 origin. However,
it is almost certain that the xylanase activity resulting from
exogenous xylanases in our is much higher than the 25%
measured since family 10 microbial xylanases (which are
not inhibited by TAXI) are probably also present in the
our. Moreover, as TAXI and XIP are natural wheat
constituents, a large part of the exogenous xylanase in the
our is already inhibited by TAXI and XIP, making it
impossible to measure them in the present assay. Thus the
measured xylanase activity is more exactly referred to as
apparent xylanase activity.
3.4.5. alpha-Amylase activity
An increase in alpha-amylase activity (Table 2) in wheat
our was observed when the outer 4% of the parent wheat
was removed by debranning. A similar observation was
made by Henry et al. (1987) who also observed a slight
increase in our alpha-amylase activity after debranning of
sound wheat. Further debranning resulted in a large
decrease in alpha-amylase activity. After 36 s debranning,
less than 30% of the original alpha-amylase activity (0.03
AU in our 0S) was still present.
Previous research showed that debranning sprouted wheat
greatly decreased alpha-amylase activity (Henry et al., 1987;
Liu et al., 1986; Sekhon et al., 1992b) because sprouting
produces high alpha-amylase levels in the scutellum and the
aleurone layer adjacent to the embryo of the grain. Removal of
the sprouted grain tissues containing the highest levels of
alpha-amylase activity reduced alpha-amylase levels in our
(Henry et al., 1987). However, by analogy with the reduction
in our xylanase, which in a large part is exogenous in origin,
we assume that the reductioninalpha-amylase activity in fully
debranned wheat was caused partially by reduction in
exogenous alpha-amylases.
4. Discussion
Debranning of wheat kernels prior to milling decreased
xylanase and alpha-amylase activities in the resultant wheat
our, whereas xylanase inhibition activities remained fairly
constant. Xylanases are important in the food (Christopher-
sen et al., 1997; Courtin et al., 2001; Courtin and Delcour,
2002), feed (Bedford, 2003) and beverage industries. A
reduction in their activity in wheat and wheat our can lead
to major positive or negative effects in processing
technology. For example in breadmaking, xylanase action
benecially hydrolyses water unextractable AX that are
known to have a negative effect on bread volume, while
largely leaving the WE-AX population intact. A positive
effect of debranning may be that it ultimately reduces the
levels in our of microbial xylanases with a putative
preference for water extractable AX, known to increase
bread volume. In contrast, removal of xylanases with
preference for water unextractable AX by debranning, may
have a negative effect on bread characteristics.
It was, however, surprising to nd a large decrease in
xylanase activity, while xylanase inhibition activities remained
fairly constant. This was probably due to the fact that xylanase
inhibitors are predominantly present in the endosperm (Geb-
ruers et al., 2002). Furthermore, because of the presence of
xylanase inhibitors, it became clear that xylanase activity in
wheat has been underestimated. Indeed, a variable proportion of
xylanases is inactivated by the inhibitors during aqueous
extraction, and is not measured in any assay. Thus, measured
xylanase activities are actually apparent xylanase activities.
The same is true for xylanase inhibitors, for which only an
apparent inhibition level can be measured. By implication,
probably more xylanases were removed than measured in the
assay. It further follows that duringabatter or doughpreparation,
increased mobility of the xylanase inhibitors could result in
some of the exogenous xylanases being inhibited. In other
words, addition of water to our would not only increase
xylanase activity as more substrate is accessed (increased
mobility of xylanases), but also increases the inhibition of
xylanase-activity (increased mobility of xylanase inhibitors).
Both effects probably occur during the preparation of a dough
or batter.
In terms of our yield it is signicant that under the
experimental conditions used, debranning for 8 s prior to
milling led to a large decrease in xylanase activity in our
but the milling yield decreased only slightly from 68 to
66%. Thus, debranning before milling could be economi-
cally feasible for cereal processing industries where
xylanase activities in our are undesirable, for example, in
the production of ours for refrigerated dough systems
where xylanase activity has been associated with the
development of dough syruping (Gys et al., 2003).
Acknowledgements
Masterfoods (Olen, Belgium) is thanked for supplying
the Satake Batch Debranner. We would also like to thank
Luc Van den Ende for excellent technical assistance in
preliminary experiments. C.M. Courtin is a postdoctoral
fellow of the Fund for Scientic Research-Flanders (Fonds
voor Wetenschappelijk Onderzoek-Vlaanderen, Brussels,
Belgium).
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