Journal of Cereal Science 39 (2004) 387–393

www.elsevier.com/locate/jnlabr/yjcrs

Wheat bran tissue fractionation using biochemical markers

Carole Antoine, Stéphane Peyron, Valérie Lullien-Pellerin, Joël Abecassis, Xavier Rouau*
UMR Agropolymer Engineering and Emerging Technologies, INRA, ENSA.M, UM II, CIRAD, 2, Place Pierre Viala, 34 060 Montpellier Cedex 1, France

Abstract
Phenolic acid analysis of hand-isolated outer grain layers and endosperm led to the identification of markers of pericarp and aleurone
layers, respectively. A new dehydrotrimer of ferulic acid (DHT) was found to be concentrated in the outer pericarp of wheat bran whereas
p-coumaric (p-CA) acid was mainly in the aleurone layer. Phytates were also used as a marker of aleurone layer and starch as a marker of
starchy endosperm. Biochemical markers constitute an original method for determining the histological composition of any technological
bran fractions. A pin milling process was applied to coarse bran produced by a conventional milling process. Three different fractions (B1, B2
and B3) were obtained by sieving the bran products and then the smallest bran particle fraction (B3) was air-classified to obtain two particle
size fractions (B3a and B3b with a D50 of 83 and 7 mm, respectively). The biochemical composition of these fractions was used to calculate
the distribution of tissues according to the sieving process. The dissociation behavior of individual bran tissues upon mechanical fractionation
was investigated in relation to particle size and discussed according to their mechanical properties.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Wheat bran; Aleurone layer; Pericarp; Phenolic acids; Phytates; Fractionation behavior

1. Introduction
The aim of the wheat milling process is to obtain the best
possible dissociation of the starchy endosperm from the
other parts of the grain to yield the white flour. Pericarp,
seed coats, nucellus and aleurone cells form the bran. The
wheat germ is partly eliminated during grain preparation
and the remainder is found as large flat particles in
middlings and bran fractions. Until recently, bran was
generally considered as a milling by-product, and previous
research was mostly focused on the separation of starchy
endosperm (white flour) from other botanical parts of the
grain to increase milling yields (Abecassis, 1991). However,
wheat bran is a nutritional raw material (Knudsen et al.,
1995; Betschart, 1988; Pomeranz, 1982), so the tissues of
this part must be efficiently disrupted in order to extract the
nutrients. Several molecules of nutritional interest were
reported to be concentrated in the contents of aleurone cells
(vitamins, minerals, etc.) (Amrein et al., 2003; Antoine et al.,
Abbreviations: p-CA, p-coumaric acid; DHD, dehydrodimers of ferulic
acid; DHT, dehydrotrimers of ferulic acid; TMCA, 2,3,5-trimethoxy-(E)-
cinnamic acid; RP-HPLC, reverse phase-high performance liquid
chromatography.
* Corresponding author. Tel.: þ33-4-99-61-22-02; fax: þ 33-4-67-52-
20-94.
E-mail address: rouau@ensam.inra.fr (X. Rouau).
2002), and others may be associated with aleurone cell walls
or other peripheral tissues (dietary fibers, phenolic acids,
etc.) (Buttrose, 1963; Morrison et al., 1975; Tanaka et al.,
1974). It is therefore of interest to be able to track bran tissue
distribution in response to a fractionation process. As shown
previously with durum wheat, histological markers rep-
resent an efficient tool to monitor the fate of botanical parts
through a milling process (Peyron et al., 2002a). The
complex distribution patterns of phenolic acids within
external parts of wheat grain (Onyeneho and Hettiarachchy,
1992; McCallum and Walker, 1991) constitute a suitable
starting point for investigating their potential use to identify
grain tissues.
This study was designed to characterize the dissociation
behavior of individual outer grain layers during re-milling
of coarse bran using biochemical markers. The phenolic
acid compositions of the hand-isolated aleurone layer,
intermediate layer and outer pericarp were determined to
identify possible markers. Phytate and starch contents were
also determined for specific quantification of aleurone cell
and starchy endosperm contents, respectively. Information
on the distribution of the markers in tissues was to bran
fractions obtained by pin milling and sieving of coarse
wheat bran. The relative distribution of outer grain layers in
relation to the particle sizes of fractions indicated marked

0733-5210/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2004.02.001
388 C. Antoine et al. / Journal of Cereal Science 39 (2004) 387–393
differences in behavior upon fractionation. These differ-
ences are discussed with regard to their known rheological
properties.
2. Experimental
2.1. Wheat samples
Grains of a hard common wheat (T. aestivum L., c. v.
Baroudeur) were harvested in France (Gascogne region)
in 2000.
2.2. Preparation of bran tissues
Wheat grains from which embryos were removed with a
razor blade were immersed in distilled water for 12 h at
room temperature. Grain ends (brush) were removed. The
remaining part was soaked again for 2 h in distilled water. A
crease incision was made and the endosperm removed using
a scalpel. Three bran tissues, the outer pericarp, an
intermediate layer and the aleurone layer were isolated by
insertion of a razor blade between them. The histological
composition of strips was assumed to be similar to that
determined by confocal laser scanning microscopy in a
previous study carried out on similar hand-isolated outer
grain layer tissues (Antoine et al., 2003). The intermediate
layer contained the inner pericarp, testa and nucellar tissue.
After rinsing, tissue fragments were air dried and ball-
milled (Dangoumeau, France) for 4 min, then freeze-dried.
2.3. Production and size reduction of bran
Bran was obtained from a conventional laboratory mill
(Bulher MLU-202, Uzwill, Switzerland) using 5 kg samples
of Baroudeur cultivars tempered to 17% moisture content.
The bran fraction was brushed (Bulher brush, MLU-302)
and reduced in size using a pin mill disposal unit (Alpine
160PZ, Hosokawa, Ausburg, Germany) in order to produce
fraction B0. To obtain a particle size less than that of the
aleurone cells, three successive passes through the waste
disposal unit were made and particles were separated using
two sieves of 450 and 200 mm, respectively, giving three
fractions: B1 (. 450 mm), B2 (450 mm . particle
size . 200 mm) and B3 (, 200 mm). B3 was then air-
classified into two fractions, i.e. B3a and B3b, with a 50
ATP selector (Hosokawa Alpine AG pilot, Ausburg,
Germany) to separate cell contents from walls by density
differences. The mean particle size (D50) of B3a and B3b,
determined by laser granulometry (Coulter, Beckman,
France), was 83 and 7 mm, respectively. Fig. 1 shows the
overall technological steps in the process leading from
wheat grains to bran fractions.
Fig. 1. Schematic representation of the process applied to coarse wheat
bran.
2.4. Phenolic acid analysis
Ground bran tissues (20 mg) or bran fractions
(100 mg) were treated for 2 h with 2.0 M sodium
hydroxide (10 ml) in the dark and under argon in order
to prevent hydroxycinnamate oxidation, at 35 8C and
under slow agitation. Internal standard, 2,3,5-triMethoxy-
(E)-cinnamic Acid (TMCA, T-4002, Sigma Chemical
Co., St Louis, USA) was added and the solution was
adjusted to pH 2.0 with 4 M hydrogen chloride. Phenolic
acids were extracted twice with diethyl ether (5 ml).
Ether phases were evaporated in the presence of argon.
The dry extract was dissolved in aqueous methanol
(MeOH/water: 50/50, v/v), filtered (0.45 mm) and injected
(20 ml) on an Alltima C18 column (5 mm, 250 £ 4.6 mm2,
Alltech, Deerfield, USA) for subsequent RP-HPLC
analysis. UV detection was carried out at 320 nm using
a 996 Waters photodiode array detector (Waters, Milford,
MA). Elution was performed in linear gradient of
acetonitrile and 0.05 M sodium acetate buffer, pH 4.6,
at 1 ml min21 at 35 8C, from 15/85 to 35/65 in 24 min,
from 35/65 to 60/40 in 0.5 min, from 60/40 to 15/85 in
4.5 min and maintained at 15/85 for 5 min. Response
factors of ferulic acid dehydrodimers determined by
Saulnier et al. (1999) were used. Products were identified
on the basis of their UV absorption spectra (Ralph et al.,
1994). 4-0-80 , 50 -500 Ferulic acid dehydrotrimer (DHT)
was identified and quantified according to Rouau et al
(Rouau et al., 2003).
2.5. Starch content
Starch content was determined according to the AOAC
996-11 method (P-8810, Megazyme kit, Sigma, Wicklow,
Ireland).
 C. Antoine et al. / Journal of Cereal Science 39 (2004) 387–393
2.6. Moisture content
Moisture content was determined according to the ISO
711-1978 method.
2.7. Determination of phytate content
Phytates from bran fractions or hand separated outer
layers were measured according to a colorimetric method
described by Latta and Eskin (1980) and modified by
Vaintraub (Vaintraub and Lapteva, 1988). A standard
curve was established with solutions of phytic acid
dodecasodium salt from corn (P-8810, Sigma).
3. Results
3.1. Identification of biochemical markers of wheat bran
tissues
To study the behavior and fate of the different parts of
wheat bran during mechanical processing, it is essential to
be able to specifically track these tissues in the products.
Levels of specific compounds were measured in dissected
outer grain tissues to identify potential specific markers for
each tissue.
3.2. Phytates and starch
It is well established (Betchel and Pomeranz, 1981;
Jacobsen et al., 1971) that phytates are located specifically
within aleurone grains in the aleurone layer and the germ
(Pomeranz, 1982). Thus, since coarse bran is devoid of
germ, phytates can be used as specific indicators of
aleurone cell contents. Whole wheat bran was found to
have a phytate content of 79 mg/mg (dry weight,
c.v. ¼ 1%) whereas the content in the aleurone layer
was 217.4 mg/mg (dry weight, c.v. ¼ 8%). Starch is
specifically concentrated in the starchy endosperm of
wheat grains. The starch content of the starchy endosperm
was 74% (dry basis). These data allowed us to specifically
determine the percentage of starchy endosperm in the
coarse bran fractions.
3.3. Phenolic acids
Table 1 gives the phenolic acid contents of the starchy
endosperm, and hand-isolated outer grain layers and each
of the tissues isolated from the outer grain layers. The outer
wheat layers were shown to contain a broad array of
phenolic acids, including p-coumaric acid (p-CA), sinapic
acid (SA), ferulic acid (FA) and dehydrodimers of ferulic
acid (DHD), in agreement with the compositions reported
for common wheat and durum wheat (Buttrose, 1963). As
previously noted in maize bran (Antoine et al., 2003),
wheat bran also contained a trimer of ferulic acid, namely
389
Table 1
Phenolic acid (2 samples analyzed, c.v. , 10%) mg/g contents of hand-
isolated parts of wheat grains
Wheat bran tissues AFa DHDa DHT SA p-CA
Bran 5.26 1.01 0.24 0.25 0.09
Endosperm 0.10 0.03 0.00 0.01 0.00
Aleurone layer 8.17 1.07 0.11 0.44 0.21
Intermediate layer 5.92 0.91 0.07 0.08 0.07
Pericarp 8.18 5.12 1.21 0.01 0.04
a
Values from Antoine et al. (2003), 2 samples analyzed, c.v. , 10%.
4-O-80 , 50 -500 dehydrotriferulic acid (DHT). FA is the major
phenolic acid of wheat bran (Peyron et al., 2002a) and
DHT was found to constitute 3.6% of the total FA. It was
principally concentrated in the outer pericarp (the outer
pericarp exhibited a DHT content 11- and 17-fold higher
than the aleurone and intermediate layers, respectively).
The SA concentration in wheat bran was similar to that of
DHT, whereas the p-CA content of the outer layers was
almost 2- and 3-fold lower.
The same phenolic acid compounds were found in the
hand-isolated, outer grain layers but they appeared to be
differentially distributed in the different histological parts of
the grain. These differences in phenolic contents among the
individual outer grain tissues appeared to be higher than
differences between wheat varieties or differences due to
environmental conditions, thus indicating that this method
could be used with any wheat bran fractions (Lempereur
et al., 1997; Peyron et al., 2002a). SA content was very low
in the starchy endosperm and outer pericarp and appeared to
be concentrated in the aleurone layer, i.e. 5.5-fold higher
concentration in this tissue than in the intermediate layer.
p-CA was also found to be concentrated in the aleurone
layer. The outer pericarp and intermediate layer exhibited
only trace amounts of p-CA (p-CA was 3- and 5-fold more
concentrated in the aleurone layer than in the intermediate
layer and outer pericarp, respectively). Finally, the starchy
endosperm contained only trace amounts of FA and DHD.
Therefore some of the phenolic acids appeared to be located
in specific grain tissues or compartments. DHT was shown
to be an indicator of the presence of outer pericarp and
p-CA, which is known to be covalently bound to cell wall
polysaccharides of wheat grain tissues (Smith and Hartley,
1983; Hartley and Ford, 1989), could be considered as a
characteristic indicator of aleurone cell walls. Phenolic
acids of the aleurone layer were previously shown to be
specifically located in aleurone cell walls (Piot et al., 2000;
Saadi et al., 1998). On the other hand, SA was also mainly
present in the aleurone layer. However, its concentration
was not correlated with the p-CA concentration or with the
phytate concentration. Therefore SA was likely not
concentrated in aleurone cell walls and its distribution
between aleurone cell walls and the aleurone cell content
remain to be determined.
390 C. Antoine et al. / Journal of Cereal Science 39 (2004) 387–393

3.4. Quantification of bran tissue contents using marker (30%) and outer pericarp (6%). When sieved, nearly 55% of
concentrations these bran particles passed through the 200 mm sieve (B1),
28% were between 200 and 450 mm (B2) and 17% were
Starch, phytates, p-CA and DHT were selected as retained on the 450 mm sieve (B3). The histological
markers to quantify the proportion of starchy endosperm composition of bran as determined by adding the amounts
(%e), aleurone cell content (%ac ), aleurone walls (%aw ), of individual bran tissues within the different fractions
intermediate layer (%i) and outer pericarp (%p) in bran (starchy endosperm 16%, aleurone layer 43%, intermediate
products (B). The relations were as follows: layer 34%, outer pericarp 6%) was similar to the histological
composition of the initial bran. The histological compo-
½p-CAB ¼ %aw ½p-CAaw þ %i½p-CAi þ %p½p-CAp sitions of conventional milling fractions obtained from
durum wheat (Ardente and Lloyd cultivars), were deter-
½DHTB ¼ %aw ½DHTaw þ %i½DHTi þ %p½DHTp mined previously (Buttrose, 1963). The proportions estab-
lished for the aleurone layer were in the same range as noted
½starchB ¼ %e½starche in the present study, but the pericarp ratio in the
corresponding durum fractions was equivalent to the ratio
½phytateB ¼ %ac ½phytateac of the sum of the intermediate layer plus outer pericarp in
the present study, since it was not possible to isolate an
%e þ %ac þ %aw þ %i þ %p ¼ 100 intermediate layer from durum grains.
Knowing the percentage of each wheat grain tissue in the
Where: [p-CA]: p-coumaric acid c; [DHT]: FA trimer
different bran fractions obtained by sieving and air
content; [starch]: starch concentration; [phytate]: phytate
classification allowed us to analyze each tissue during
concentrations in bran fraction (B), purified endosperm (e),
processing. The largest particle size fraction (B1,
aleurone cell content (ac ), aleurone cell walls (aw ),
. 450 mm, about 20% of the initial bran) was thus found
intermediate layer (i) and outer pericarp (p).
to be composed mainly of aleurone and intermediate layers.
By combining the measurement of phenolic acid, phytate
The two other pin-milled bran fractions, i.e. B2 and B3, with
and starch contents of the different outer grain tissues and
particle sizes comprised respectively between 200 and
technological fractions, the equations enabled calculations
450 mm (about 30% of the initial bran) or under 200 mm
to be made of the ratios of the four tissues within bran
(about 50% of the initial bran), exhibited a histological
fractions.
composition similar to the starting bran, except for the
Coarse bran from a conventional milling process was
starchy endosperm which was concentrated in the smaller
reprocessed in order to generate different reduction fractions
particle size fraction (B3). Two fractions differing in
varying in particle size. Table 2 reports the DHT, p-CA,
particle size were obtained from this B3 fraction by air
phytate and starch contents of the different bran fractions
classification: B3a (83 mm D50, 43% w/w initial bran) and
obtained by pin milling of the bran (B0) and after sieving
B3b (7 mm D50, 10% w/w initial bran). This fractionation
(B1, B2 and B3) and air classification (B3a and B3b).
allowed isolation of approximately 20% of the starting
Starchy endosperm, aleurone (cell walls and cell contents,
aleurone cell content. This was the main component of B3b
separately), intermediate layer and outer pericarp pro-
together with low amounts of starchy endosperm and
portions were calculated in each fraction from the relations
intermediate layer, whereas aleurone walls, starchy endo-
given above (Table 3).
sperm, intermediate layer and outer pericarp were found in
The pin-milled bran (B0) had a mean particle size of
the larger particle size fraction (B3a).
about 800 mm (D50). It was composed of starchy endo-
sperm (15%), aleurone layer (49%), intermediate layer
3.5. Distribution of individual tissues within processed
bran fractions
Table 2
The fate of separate bran tissues can be monitored
Phenolic acid (2 samples analyzed, c.v. , 10%, except for B0, c.v. ¼ 15%)
mg/g, phytate (2 samples analyzed, c.v. , 10%) mg/g and starch (2 samples during different processing steps (Fig. 1). The botanical
analyzed, c.v. , 10%) (%, dry mass) contents of wheat bran fractions parts of the grain behaved differently during milling and
sieving. The endosperm was found to mainly produce
Bran fractions DHT p-CA Phytate Starch
particles of 83 mm D50 (2/3 of the initial bran starchy
endosperm content) even though some . 200 mm particles
B0 0.21 0.11 73.0 10.7
B1 0.06 0.11 73.4 4.7 (18%) were also present in the fraction. The latter starchy
B2 0.21 0.11 66.1 4.5 endosperm fragments probably remained attached to the
B3 0.23 0.09 52.7 19.5 bran tissues due to high adhesiveness with the aleurone
B3a 0.26 0.10 45.6 19.0 layer. Sixty percent of the particles originating from
B3b 0.07 0.04 143.0 17.1
the aleurone layer passed through the 200 mm sieve and
the aleurone cell contents were distributed among all
 C. Antoine et al. / Journal of Cereal Science 39 (2004) 387–393 391

Table 3
Histological composition of bran fractions (%, dry mass) obtained by calculation from their composition using the biochemical markers and equations
described in the text

Bran fractions Particle sizea Yieldb Endosperm Aleurone cells Aleurone walls Intermediate layer Pericarp

B0 800 97 15 (100)c 34 (100) 15 (100) 30 (100) 6 (100)

B1 839 17 7 (7) 34 (19) 15 (20) 45 (23) 0 (0)
B2 297 27 7 (11) 30 (28) 18 (39) 37 (30) 7 (35)
B3a 83 43 26 (68) 21 (31) 32 (43) 14 (43) 8 (67)
B3b 7 10 23 (14) 66 (22) 0 (0) 15 (4) 0 (0)
a
D50: 50% of the overall bran particles showed a smaller size (mm).
b
Dry mass (mg).
c
Bran tissue distribution (%, dry mass).

the fractions. However, the aleurone cell contents were
remarkable in that they produced the smallest particles as
compared to the other bran tissues (22% of the aleurone
cell content particles had a D50 of 7 mm). Aleurone cell
wall particles, on the other hand, were found to be larger
(1/4 exhibited a D50 of 83 mm and 1/4 were between 200
and 450 mm). The aleurone wall tissue behaved like the
intermediate layer, maybe due to the high adhesiveness
between the two tissues.
The intermediate layer is a heterogeneous and complex
material. Its particles were widely distributed, with a major
concentration in the larger fractions (B1 and B2). Most of
the smallest intermediate layer particles were concentrated
in B3a, as also observed for the aleurone cell walls. Particles
obtained from the outer pericarp, a single-layered brittle
tissue, were found exclusively in the B2 (1/3) and B3a (2/3)
fractions, with a mean particle size of approximately
160 mm.
4. Discussion
The tissue composition of bran obtained by milling
wheat grains (starchy endosperm 15%, aleurone layer 49%,
intermediate layer 30%, and outer pericarp 6%) was in the
same range as that noted in a previous study carried out on
the tissue composition of mill bran fraction (Peyron et al.,
2002a). DHT, p-CA and phytate contents could thus be
considered as efficient markers for the quantification of the
outer pericarp, intermediate layer, aleurone cell walls and
aleurone cell contents in wheat grain fractions. The validity
of this approach was confirmed by the total recovery of each
botanical part in each bran fraction. These biochemical
markers applied to sieving and air classification fractions,
analyzed in relation to particle size distribution, allowed us
to characterize the milling behavior of each bran tissue. The
distinct identification of the aleurone cell content from
aleurone cell walls provided further insight into the
dissociation of this interesting tissue during processing.
A study carried out on common and durum wheat showed
that the bran particle size, obtained from wheat grains by
the milling process, is correlated with the extensibility of
the outer grain layers (Peyron et al., 2002b). Thus, the
grinding behavior of the individual bran tissues during the
bran grinding process is discussed here with regard to their
previously characterized rheological properties (Antoine
et al., 2002). The outer pericarp, which surrounds the outer
grain layers, is a thin tissue weakly attached to the
intermediate layer (Peyron et al., 2002a). Its fragile
mechanical nature and low extensibility could explain its
high friability and marked particle size reduction during
grinding.
The intermediate layer, containing different tissues
(nucellar epidermis, testa and inner pericarp), has a complex
and heterogeneous structure. The testa exhibited high
plasticity, which could explain the high proportion of
intermediate layer in the B1 and B2 fractions. In contrast,
the fact that particles containing the intermediate layer were
noted in the B3 fraction could be explained by the presence
of inner pericarp whose friability was similar to that of the
outer pericarp.
The strong association between the aleurone layer and
the extensible intermediate layer (Peyron et al., 2002a) was
partially responsible for the presence of aleurone layer in
particles above 450 mm. The aleurone cell contents were
mostly composed of aleurone grains, with a mean size of
about 4 mm. Aleurone cells have a mean diameter of about
50 mm (McMasters et al., 1971; Bradbury et al., 1956).
Therefore, in particles above 200 mm, the aleurone cell
contents were either enclosed within intact cells or
remained attached to cell wall fragments, thus explaining
their dispersion throughout the different size fractions.
Since cell contents are organized in small discrete granules
(aleurone grains), the aleurone layer has a substantial
ability to produce small particles. The mass ratio results
showed that the aleurone layer was composed of 70% cell
contents and 30% cell walls (dry mass) which is consistent
with data in the literature and microscopy observations
(Stevens, 1973).
The particle size classification of pin milled bran allowed
us to characterize two histological fractionations. First,
upon sieving, the pericarp was entirely eliminated from
392 C. Antoine et al. / Journal of Cereal Science 39 (2004) 387–393
the largest particles (B1) and then, upon air classification, a
part of the aleurone cell content, which is rich in minerals
and vitamins (B3b), was separated from the aleurone cell
walls, intermediate layer and outer pericarp (B3a), which
constitutes a fiber-rich fraction. Nevertheless, the aleurone
and intermediate layers which exhibit similar behavior
during the grinding process, are difficult to separate by size
classification. Future investigations concerning the impact
of moisture content on their mechanical properties could
allow us to determine if specific conditions could be found
for their dissociation and separation.
5. Conclusion
The technological behavior of different wheat bran
tissues has to be accurately characterized to be able to
produce bran fractions of high nutritional value. The
identification and quantification of efficient tissue markers
of wheat grain tissues allowed us: (i) to determine the tissue
composition of milling fractions, and (ii) to monitor the fate
of individual tissues of the material submitted for proces-
sing. The results obtained are in close agreement with the
known mechanical properties of the tissues. In particular,
the particle size reduction of the different tissues appeared to
be related to their intrinsic extensibility. Components
involved in controlling the rheological properties of bran
tissues must be identified in order to improve dry
fractionation processes.
Acknowledgements
We thank Cyrille Piantoni for running the milling and
fractionation process and Anne Surget and Georges Maraval
for technical assistance.
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