Bran Size Distribution at Milling and Mechanical and Biochemical
Characterization of Common Wheat Grain Outer Layers:
A Relationship Assessment
V. Greffeuille,1,2 J. Abecassis,1 C. Lapierre,3 and V. Lullien-Pellerin1,4
ABSTRACT
Mechanical properties of wheat grain outer layers from common wheat
(Triticum aestivum L.) cultivars known to display distinct milling behav-
ior were analyzed using uniaxial tension tests. Tensile modulus and strain
to rupture of the tissues distinguished between the wheat cultivars. Values
of strain to rupture were related to coarse bran size generated by grain
milling, a characteristic that distinguishes the two hardness classes. As
content of an aleurone marker in total or first break flour was also related
to coarse bran size, extensibility of wheat grain outer layers’ could be a
key parameter to explain the observed tissue mechanical behavior and thus
In the milling process, recovery of all the starchy endosperm
with the lowest contamination with peripheral tissues is required.
Efficiency of this process depends on differences in mechanical
properties, particularly elasticity, between the endosperm and the
other tissues and on tissue adhesion (Abecassis 1993). In this
context, friability of peripheral tissues appeared as an essential
factor for milling value because more friable peripheral tissues
would induce greater incorporation of fine bran particles in flours,
leading to lower flour purity. Recently, grains from distinct com-
mon wheat cultivars (Triticum aestivum L.) selected according to
their milling value differences displayed a distinct aleurone layer
fractionation behavior (Greffeuille et al 2005). This difference in
milling behavior was revealed at the first breaking step of the pro-
cess and could distinguish wheat cultivars according to their hard-
2
ness class. Furthermore, a significant negative relationship (R =
0.70) was found between the coarse bran production during milling
and the content of an aleurone marker (phytic acid) in the first
break roll flour (B1). Two hypotheses were suggested to explain
this distinct behavior within hardness classes of wheat and the
reduced size of bran particles from hard wheat. One involves the
grain mode of fracture and shearing forces applied by the endo-
sperm along the envelopes; the other suggests possible differences
in mechanical properties of the outer layers themselves. In plant
tissues, mechanical characteristics are determined at different org-
anization levels: tissue overall structure (cellular shape and density)
(Lucas et al 1995), intercellular adhesion (Waldron et al 1997;
Jarvis 2003), and cell wall architecture, which depends on nature,
proportion, and interactions between compounds (Whitney et al
1999; Jarvis and Mc Cann 2000). Indeed, a number of studies
underlined the involvement of cell wall biochemical composition
and structure to explain the mechanical properties of plant tissues
(Darley et al 2001; Shopfer et al 2001; MacAdam and Grabber
2002). Wheat outer layers are composed with several tissues dis-
tinct by their structure and biochemical composition and consist
mainly of cell walls (except the aleurone layer). Cereal cell walls
1 UMR Ingénierie des Agropolymères et Technologies Emergentes, INRA, 2 Place
Viala, 34060 Montpellier Cedex 01, France.
2 ARVALIS, Institut du Végétal, 16 rue Nicolas Fortin, 75013 Paris, France.
3 UMR Chimie Biologique, INA-PG, 78850 Thiverval-Grignon, France.
4 Corresponding author. Phone: 33 (0)4 99 61 31 05. Fax: 33 (0)4 99 61 30 76.
E-mail: lullien@ensam.inra.fr
DOI: 10.1094 / CC-83-0641
© 2006 AACC International, Inc.
Cereal Chem. 83(6):641–646
distribution of the aleurone layer content in flours. As tissue mechanical
properties are generally linked to the cell wall biochemical composition
and structure, analysis of the main wheat outer layers’ cell wall com-
pounds was undertaken to establish relationships with the differences ob-
served in mechanical properties. No clear correlation could be found with
one of the wheat outer layers’ component but involvement of the outer
layers’ cell wall structure in the tissues behavior at milling was con-
firmed.
are mainly composed of arabinoxylans (AX) and β-glucans as
well as lesser amounts of cellulose and lignin (Fincher and Stone
1986; Schwarz et al 1988; Mandalari et al 2005). Arabinoxylans
were esterified by ferulic acid which contributed to polymer
cross-linking (Hartley et al 1990; Ralph et al 1994). Modulation
of tissue mechanical properties by these polymer cross-links were
suggested in a number of cases as for example in Oryza (Tan et al
1991) and Avena coleoptiles (Kamisaka et al 1990) and also by
amounts of these polysaccharides in shoots of wheat seedlings
(Wakabayashi et al 2005). In durum wheat grains, relationship
between the outer layers’ mechanical properties and their cell wall
biochemical composition and structure was also demonstrated
(Peyron et al 2002a).
Therefore, the objective of this study was to explore the poten-
tial difference in mechanical properties of outer layers from each
of the wheat cultivars showing a distinct behavior during milling
and to relate them to the bran and flour characteristics. As tension
tests on hand-isolated outer layers were useful to explore the mech-
anical behavior of these tissues (Mabille et al 2001; Antoine et al
2003), they were used to compare each of the wheat cultivars. Fur-
thermore, the outer layers isolated from common wheat grains of
each cultivar were analyzed for their cell wall components as they
are generally related to the tissue mechanical properties. Finally,
relationships between the observed mechanical properties and the
cell wall biochemical composition were looked at and discussed.
MATERIALS AND METHODS
Wheat Samples
Six cultivars from common wheat (Triticum aestivum L.) with
different kernel hardness measured by NIRS analysis (Approved
Method 39-70A, AACC International 2000) were selected. Sois-
sons, Camp Rémy, and Caphorn are hard wheat cultivars with
hardness scores of 69, 64, and 81, respectively. Crousty, Scipion,
and Ornicar are soft wheat cultivars with hardness scores of 22,
22 and 14, respectively. All samples were harvested in France in
2002 from different locations and supplied by Arvalis-Institut du
Végétal (Paris, France) or Danone Vitapole (Palaiseau, France).
They were cleaned to remove impurities and stored at room temper-
ature before milling. Protein content of each batch was determined
by NIRS analysis (Approved Method 39-10, AACC International
2000) and had a range of 10.9 and 11.3% dry mass.
Standard molecules as phenolic acid monomers or sugars were
purchased from Sigma Chemical (St. Louis, MO).
Vol. 83, No. 6, 2006 641
Coarse Bran Fractions Production and Sieving
Cleaned wheat grains (10 kg) were tempered for 24 hr to 17.0%
moisture content before milling. Tempered grains were then fed
into a pilot mill (Siraga, ENSMIC, Paris, France) equipped with
four break rolls, four sizing rolls, and six reduction rolls. This
milling process led to 14 flour fractions and four bran or short
fractions from each wheat sample as described by Willm (1995).
Samples were collected and stored at 4°C. Coarse bran corresponds
to the collected fractions from the fourth breaking stage >1,000 μm
sieving. The coarse bran size distribution was further determined
by mechanical sieving (Rotachoc, Tripette et Renaud, Villeuneuve
La Garenne, France) into three fractions (a, b, c): 1,000 μm < a <
1,400 μm; 1,400 μm < b < 2,000; c < 2,000 μm.
Isolation of Wheat Grain Outer Layers for Mechanical Test
Wheat grains from which embryos and brush were removed by
cutting transversally with a razor blade were soaked overnight in
distilled water at 20°C. Then a crease incision was made and the
endosperm scraped from the outer layers using a scalpel.
After the careful removal of the endosperm, cleaned outer layer
strips were stored for 2 hr between two glass plates at 20°C to
impose a plane shape. Strips were then equilibrated at 17% mois-
ture content by leaving them at constant relative humidity (75%)
on a saturated NaCl solution at 25°C overnight. Dimensions of the
strips composed of the aleurone layer, nucellar layer, testa, and
pericarp were adjusted by cutting to 8 mm long and 2 mm wide
and their thicknesses were measured.
Isolation of Wheat Grain Outer Layers for Biochemical
Analysis
Cleaned outer layers strips were first dried at 20°C overnight
and ground with a steel ball mill (Dangoumeau, France). Ground
samples were then dried for 48 hr on phosphorus pentaoxide.
Mechanical Properties Measurements
Mechanical tests were performed on isolated outer layers using
dynamic mechanical thermal analysis (DMTA Mk III, Rheomet-
rics, Piscataway, NJ). The humidity of samples was fixed and con-
trolled with air bubbled through water at 27°C and flushed on the
furnace. This process allowed us to obtain a strip with moisture
content of 17% corresponding to the usual tempering condition
before milling. Sample equilibration was followed by a time sweep
test at imposed strain (0.05%), (total time = 10 min; frequency =
10 rad/sec, furnace temperature = 30°C). The stability of elastic
modulus (E) was used as an indicator of the sample equilibration.
Uniaxial tension tests were performed at a rate of 0.05 mm/sec
until disruption of the sample. Tension tests were conducted until
rupture of bran coat strips. Mechanical properties of the different
bran tissues were determined from standard average curves
expressing linear strength according to strain: linear strength to
elastic deformation and to rupture (fela, fmax); elastic strain (εela);
maximum tensile strain (εmax); tensile modulus (E); and rupture
energy (Wmax). Linear strength corresponds to the strength exerted
per unit of tissue width to cause elastic deformation (εela) then rup-
ture (εmax). This parameter allows us to compare tissues of differ-
ent thicknesses. Deformation was determined as ΔL/L0. Linear
modulus (E) is the initial maximum tangent and highlights the
sample stiffness. Finally, the energy to rupture (Wmax) corresponds
to the total work until rupture. Tension tests were performed on
6–10 strips (CV ≈15%).
Carbohydrate Content of Wheat Outer Layers
Ground samples (10 mg) were either treated first for 30 min
with aqueous 72% H2SO4 at room temperature as a prehydrolysis
step or hydrolyzed directly with sulfuric acid (1.0M, 2 hr, 100°C).
Sugars were converted into alditol acetates and analyzed by gas
liquid chromatography on an HP 225 column (50% CNPrph Me
siloxan, 30 m × 0.2 mm × 0.15 μm) at 225°C, using hydrogen as a
642 CEREAL CHEMISTRY
carrier gas and myoinositol (0.5 mg) as an internal standard.
Samples were analyzed in duplicate and results expressed as
mean values (CV < 5%). Quantities of arabinoxylans were calcu-
lated as the sum of anhydroarabinose and anhydroxylose. Cellu-
losic glucose was determined by the difference between values
obtained with and without the sulfuric acid prehydrolysis step; β-
glucans correspond to the glucose extracted without sulfuric acid
prehydrolysis.
Ester-Linked Phenolic Acids Analysis
of Wheat Outer Layers
Ground hand-isolated tissues (20 mg) were treated at 35°C for
2 hr with 2.0M sodium hydroxide (10 mL) in the dark and under
argon to prevent hydroxycinnamate oxidation. After the addition
of an internal standard, 3,4,5-trimethoxy-(E)-cinnamic acid, the
solution was adjusted to pH 2.0 with 4M hydrogen chloride and
phenolic acids were extracted twice with diethyl ether. Ether
phases were evaporated in the presence of argon and the dry
extract was dissolved in aqueous methanol (50/50, v/v), filtered
(0.45 μm) and injected (20 μL) on an Alltima C18 column (5 μm,
250 × 4.6 mm, Alltech, Deerfield, IL) for RP-HPLC analysis.
Elution was obtained at 1 mL/min and 35°C by increasing
acetonitrile concentration in sodium acetate buffer (50 mM, pH
4.6). A linear gradient was performed with the following steps:
15/85 to 35/65 in 24 min, 35/65 to 60/40 in 0.5 min, 60/40 to
15/85 in 4.5 min, and 15/85 for 5 min. UV detection was con-
ducted at 320 nm with a photodiode array detector (996, Waters,
Milford, MA). Phenolic acids were identified by their absorption
spectra using reference molecules and quantified relative to the
internal standard and their response factors. Ferulic acid dehydro-
dimers (DHD: 8-5’-diFA, 8-5’-benzofuran, 8-O-4’ diFA and 5-5’-
diFA) and 4-O-8’, 5’-5” dehydrotrimer (DHT) were identified
and quantified, respectively, according to Saulnier et al (1999)
and Rouau et al (2003). Samples were analyzed in duplicate and
results expressed as mean values (observed CV < 10%).
Analysis of Lignins by Thioacidolysis
All the thioacidolysis experiments on bran tissue samples were
conducted according to a recently modified procedure (Desvignes
et al 2006) and in duplicate. Ground bran tissue samples (10–20 mg)
were treated with 10 mL of thioacidolysis reagent (dioxane/ethan-
ethiol 9/1, v/v, containing 0.1M HBF4 methyl ether) in a glass tube
closed with a Teflon-lined screw cap and 1 mL of internal standard
(docosane, 0.15 μg/mL in CH2Cl2) was added. Thioacidolysis was
performed at 100°C (oil bath) for 4 hr. The cooled reaction
mixture was diluted with 30 mL of water and adjusted to pH 3–4
(aq. 0.4M NaHCO3). The reaction mixture was extracted with
CH2Cl2 (3× 30 mL). Combined organic extracts were dried over
NaSO4 and then evaporated under reduced pressure at 40°C. The
final residue was dissolved in 0.5 mL of CH2Cl2 before silylation
and GC-MS analyses according to Lapierre et al (1995). This
quantitative determination was made from specific ion chromato-
grams reconstructed at m/z 269 for G monomers and m/z 299 for
the S monomers and m/z 239 for the H monomers after an appro-
priate calibration relative to the docosane internal standard.
Statistical Analysis
ANOVA was processed using the Statgraphics software package
(Manugistics, Rockville, MD).
RESULTS AND DISCUSSION
Milling Coarse Bran Size Distribution
Common wheat grains from six cultivars with different hard-
ness that display distinct milling behavior (Greffeuille et al 2005)
were used in this study. Indeed, differences in the outer layers’
content distribution were observed in milling flours and were possi-
bly related to differences in their friability. Therefore, analysis of
the coarse bran size obtained after milling from these different
cultivars was undertaken and summarized in Table I.
Coarse bran size distribution showed significant differences
between the two hardness classes as expected from previous
studies (Moss et al 1980; Willm, 1995). More than 40–50% of
coarse bran from wheat of the soft type showed a size >1,400 μm
whereas only 27–32% of the coarse bran fraction from the hard
type belong to this size fraction. Furthermore, inside each of the
hardness classes, ≈20% variability was observed for the propor-
tion of the highest size fraction (>2,000 μm), underlying differ-
ences in milling behavior between each of the wheat cultivars.
Wheat Outer Layers’ Mechanical Properties
To characterize the mechanical properties from grain outer
layers of each of the wheat cultivars, tension tests were performed
at controlled temperature and relative humidity on hand-isolated
tissues. Strength-strain curves displayed typical biphasic character-
istics of an elastoplastic material as commonly described for plant
tissues (Kölher 2000) and values were in the same range as already
published (Antoine et al 2003) for common wheat in the same
radial orientation. The first part of the curve (elastic stage) at 1.8–
6% strain corresponds to reversible deformation of the tissues.
The second part of the curve (plastic stage) corresponds to irre-
versible damage of the tissues until its rupture. Table II summar-
izes the main mean values measured from curves of each of the
wheat cultivars.
Differences between wheat cultivars were observed either at
the elastic or the plastic phase. Similar results were also found
previously with durum wheat (Peyron et al 2002a) suggesting influ-
ence of the wheat cultivar on the outer layers’ mechanical prop-
erties. Statistical comparison of data pointed out differences
between the two wheat hardness classes but also between cultivars
in the same class. Table II shows that tensile modulus (E) could
be used to distinguish Soissons, Camp Rémy, and Crousty
cultivars as the most rigid. These three cultivars had less exten-
sible outer layers than the other wheat cultivars in the elastic
phase (εelast). At the plastic phase, these cultivars display the
highest strength to rupture (fmax), resulting in high energy to
rupture (Wtot). However, high energy to rupture was also found in
Ornicar and Scipion tissues, which show the highest value of
maximum strain (εmax). Taking into account the tensile modulus
and maximum strain to rupture, data of all the samples from each
of the six cultivars were plotted in Fig. 1. For each cultivar,
relative data dispersion was observed between samples and should
correspond to natural intrinsic variability of the biological material.
However, using these two rheological parameters as axes clearly
allowed us to separate wheats into three groups. The first group
comprises Soissons, Camp Rémy, and CapHorn, the three hard
wheat cultivars, which display low extensibility as well as high
tensile modulus values for Soissons and Camp Rémy. The second
group is formed with Ornicar and Scipion, which show the lowest
values for tensile modulus but highest values for maximum strain.
The last cultivar, Crousty, as a soft wheat shows an intermediate
rheological behavior with a high tensile modulus and an interme-
diate maximum strain to rupture.
Figure 1 pointed out differences between hard and soft types of
grains. Indeed, outer layers from soft wheat grains appear more
extensible than tissues from hard wheat grains. These results are
in accordance with the behavior observed in wheat tissues at
milling, as soft wheat produces a higher percentage of bran with
large particle size.
Relationships Between Outer Layers’ Mechanical Properties
and Milling Coarse Bran Size
To analyze whether the observed differences in mechanical prop-
erties of the wheat outer layers could explain part of the milling
coarse bran size distribution, correlations between mean values
for the main parameters obtained from rheological tests on tissues
from each wheat cultivar and proportion of larger size (>2,000 μm)
coarse bran were looked at. Results summarized in Table III show
a very strong positive correlation between maximum extensibility
(εmax) of the wheat grain outer layers and the relative amount of
larger size (>2,000 μm) coarse bran obtained after milling. A
similar positive correlation was also found with tissue extensibility
at elastic stage. Consequently, a low negative correlation with
tensile modulus was also observed as rigidity of the material must
indeed lead to smaller bran particles. However, no correlation could
be found with other rheological parameters and notably strength
to rupture.
The same strong correlation between maximum strain to rup-
ture and proportion of large size coarse bran was also true with
the coarse bran fraction at 1,400–2,000 μm (R2 = 0.90). The poten-
tial relationship between the extensibility characteristics of isolated
tissues obtained from tension tests and size reduction of bran
particles was also suggested by studying durum wheat (Peyron et
al 2002b). These results first confirm the interest to analyze the

TABLE I
Coarse Bran Size Distributiona
Hard Cultivars Soft Cultivars
Size (μm) Soissons Camp Rémy Caphorn Crousty Ornicar Scipion
>2,000 6.1 5.1 4.2 11.7 15.3 18.4
1,400–2,000 25.6 24.0 22.4 29.1 30.2 30.9
1,250–1,400 19.5 18.0 21.9 17.9 17.7 16.7
<1,250 48.8 52.9 51.5 41.3 36.8 34.0
a % of total coarse bran mass.

TABLE II
Mechanical Properties of Common Wheat Outer Layers Isolated According to Radial Orientation and Measured Using Tension Testsa,b
Cultivar E (N.mm–1) felast (N.mm–1) εelast (%) fmax (N.mm–1) εmax (%) Wtot (N.mm)
Soissons 76.8a 2.1a 1.8d 3.2a 11.1c 0.3ab
Camp Rémy 68.6a 1.8ab 2.7c 2.6b 8.7d 0.2c
Caphorn 37.8b 1.1cd 3.0c 1.6d 9.7d 0.1d
Crousty 63.3a 1.7ab 2.7c 2.7ab 15.0b 0.3a
Ornicar 23.8c 1.4bc 4.6b 2.2bc 19.6a 0.3a
Scipion 14.8c 0.8d 6.0a 1.7cd 21.3a 0.2bc
a Linear strength to elastic deformation and to rupture (felast, fmax), elastic and maximum strain or extensibility (εelast, εmax), tensile modulus (E), and total energy to
rupture (Wtot). Mean values for n = 6–10, depending on cultivar.
b Values followed by the same letter in the same column are not significantly different (P < 0.05).

Vol. 83, No. 6, 2006 643

mechanical properties of wheat grain isolated tissues to charac-
terize their milling behavior and outline the maximum strain
value as a key rheological parameter to predict the coarse bran
large size fraction. This also points out the importance to control
this parameter as production of large bran particles should allow
easier separation from endosperm particles and thus reduction of
bran compounds contamination in flours (Abecassis 1993; Willm
1995). In a previous study, flour composition obtained after grain
milling of the same cultivars was studied, particularly the phytic
acid concentration, allowing the analysis of aleurone layer content
distribution and showing a distinct behavior between wheat culti-
vars (Greffeuille et al 2005). Analysis of relationships between
maximum strain and amount of phytic acid in first break and total
flours showed negative correlations with R2 = 0.88 and 0.75,
respectively. Thus, maximum extensibility of the peripheral tissues
appears as a determinant parameter to describe the wheat grain
behavior at milling and could explain part of the aleurone tissue
distribution in flours. A similar relationship was also reported in
durum wheat between maximum strain and coarse bran contami-
nation of semolina (Peyron et al 2002a).
Analysis of Wheat Grain Outer Layers’ Biochemical
Composition
A number of studies have already highlighted the significant
influence of the cell wall biochemical composition on mechanical
properties of the tissues and particularly the amount, structure, and
interactions of polysaccharides as the main compound (Whitney
et al 1999; Darley et al 2001). Composition in polysaccharides
from cell walls of isolated outer layers from each of the wheat
cultivars were analyzed and compared in Table IV.
Results showed that the proportion of the different types of
polysaccharides are in accordance with previously published data
(Antoine et al 2003; Beaugrand et al 2004). Arabinoxylans (AX)
were the main compound in cell walls from the wheat grain outer
layers and corresponds to 60–70% of total polysaccharides with a
relatively high degree of substitution at ≈0.6 (Ara/Xyl). Other
polysaccharides corresponding to cellulose and β-glucans are found
in variable amounts of ≈10% each. They interact with AX through
noncovalent interactions (Izydorczyk and Mac Gregor 2000).
Analysis of released phenolic acids after alkaline hydrolysis of
covalent bonds with cell wall compounds (Table IV) also allowed
us to detect mainly ferulic acid and a little amount of p-coumaric
acid that are reported to be linked to AX or lignin, respectively,
another compound also found in wheat outer layers (Scalbert et al
1985; Hatfield et al 1999; Fry et al 2000). Furthermore, ferulic
acid was either found in free or dehydrodimer forms and even
trimeric forms, which were suggested to play roles in mechanical
properties or protection of the tissues (Arnason et al 1992; Grab-
ber et al 2004). Therefore, the molecular ratio between xylose and
dehydrodimer (Xyl/DHD) was also calculated as it could be con-
sidered an estimate of mean AX cross-linking (Hartley et al 1990).
To complete the analysis of cell wall biochemical composition
of the wheat kernel outer layers, thioacidolysis was used to study
lignification in the tissue samples. Thioacidolysis of plant tissues
coupled to gas chromatography-mass spectrometry (GC-MS) of
the lignin-derived products makes it possible to study very small
amounts of lignin without interference with p-coumaric and ferulic
acids. A slightly modified thioacidolysis reagent using HBF4,
instead of BF3, as the Lewis acid, allowed us to substantially in-
crease the recovery of thioacidolysis-derived monomers, probably
by providing better accessibility to lignin. The recovered thioethyl-
ated H, G, and S monomers originate from lignin units only
involved in labile β-O-4 ethers. Therefore, total yield of thioacido-
lysis monomers reflects the amount of lignin units involved in β-
O-4 ether bonds, known as uncondensed lignin as reported in
Table V. The total amount of lignin-derived monomers was 5–25
μmol/g of dry matter, a result which is in accordance with recent
data on similar samples (Desvignes et al 2006). With the assump-
tion that ≈1,000 μmol of monomers are released from 1 g of
wheat lignin, this suggests that the total lignin amount has a range
of 0.5–2.5% (by weight). This low lignin level in tissue fractions
available in small amounts precludes the use of the conventional
methods of gravimetric lignin determination. The two lowest thio-
acidolysis yields were obtained for the samples from Camp Rémy
and Scipion which display definitely distinct mechanical properties.

TABLE III
Linear Regression Analysis Valuesa
E εelast felast εmax fmax Wtot
R2 0.50 0.73 0.29 0.98 0.10 0.13 Fig. 1. Data of tensile modulus and strain-to-rupture from curves obtained
a Determination coefficients (R2) obtained from linear regression analysis of after uniaxial tension tests of each isolated sample of grain outer layers
relationships between the main mechanical parameters of hand-isolated wheat from six distinct wheat cultivars. Delimited area marks samples from hard
grain outer layers measured using uniaxial tension tests and proportion of wheat cultivars. Data were from Soissons (‘), Camp Rémy („), CapHorn
large size (>2,000 μm) coarse bran related to total amount. (U), Crousty (S), Ornicar (…), and Scipion (z).

TABLE IV
Wheat Grain Outer Layers’ Composition in Polysaccharides and Phenolic Acidsa,b
Cultivar Cellulose β-Glu AX Ara Ara/Xyl FA DHD DHT p-CA Xyl/DHD
Soissons 9.4a 8.6b 31.2ab 11.8bc 0.61b 0.46a 0.10a 0.018a 0.008a 473c
Camp Rémy 10.0ab 8.4b 32.8b 12.5c 0.62b 0.61e 0.13bc 0.027b 0.017d 395b
Caphorn 8.8a 9.9c 31.9ab 12.1bc 0.61b 0.50b 0.12b 0.026b 0.012b 402b
Crousty 11.0bc 7.3a 31.7ab 12.5c 0.64c 0.63e 0.14d 0.032c 0.015c 343a
Ornicar 9.9ab 9.2bc 30.1a 11.4ab 0.61b 0.54c 0.10a 0.018a 0.016c 470c
Scipion 11.6c 7.2a 30.8ab 10.9a 0.55a 0.58d 0.13cd 0.035c 0.008a 366ab
a Cellulose, β-glucans (β-glu), and arabinoxylans (AX) as sum of xylose (Xyl) and arabinose (Ara) resulting from total acid hydrolysis of the tissues; arabinose
(Ara) content, total ferulic acid (FA), sum of ferulic acid dehydrodimers (DHD), ferulic acid dehydrotrimer (DHT), and p-coumaric acid (p-CA) concentration
released after alkali hydrolysis. Ara/Xyl, ratio between arabinose and xylose; Xyl/DHD, molar ratio of xylose to ferulic acid dehydrodimers. Distinct letters in
the same column indicate distinct groups determined by ANOVA taking into account corresponding standard errors (P < 0.05).
b Mean values in % dm.

644 CEREAL CHEMISTRY

This result emphasizes the fact that such macroscopic properties
cannot be simply accounted for by one molecular property such
as lignin structure in the present case.
Syringyl (S) and guaiacyl (G) lignin-released monomers were
the main compounds with a relative proportion varying at ≈60%
or 30%, respectively, while proportion of p-hydroxyphenyl (H)
appears to be <10%.
Study of the outer layers’ biochemical composition underlines
the variability between wheats. Indeed, comparison of the poly-
saccharide content from cell walls of isolated outer layers from
each of the wheat cultivars showed that polysaccharides such as
cellulose and β-glucans vary at ≈10% between wheats. Moreover,
a higher variability is found in phenolic acid compounds from cell
walls of outer layers as ferulic acid dehydrotrimer and p-CA vary
at ≈30% between cultivars (Table IV). Total yield of thioacidolysis-
released monomers also depended on the wheat cultivar with an
observed variability of ≈40% and with Ornicar and Scipion culti-
vars showing the highest and the lowest values (Table V),
respectively. Furthermore, the molar ratio between the main lignin
monomers syringyl (S) and guaiacyl (G) was 1.5–2.5 and relative
proportion of p-hydroxyphenyl (H) varied by 20% depending on
the wheat cultivar.
As differences in biochemical composition of the outer layers
from the different wheat cultivars as well as differences in mech-
anical properties were found, correlations between the two are in-
teresting, particularly maximum strain which was correlated with
both bran size at milling and break and total flours composition.
However, comparison between independent groups defined by
studies of the mechanical properties of wheat outer layers and
those defined by analysis of their biochemical composition does
not show a clear direct relationships. Even if phenolic profile and
cross-linking structure were already involved in tissue property of
extensibility (Tan et al 1991; Waldron et al 1997), no relationship
could be found with these biochemical markers. As the outer layers
are composed with different tissues (outer and inner pericarp, testa,
nucellar, and aleurone layers) showing a distinct composition
(Bacic et al 1988; Antoine et al 2003), a significant modification
of composition of one of the tissues in relation to changes in the
mechanical properties could be diluted and masked in the global
biochemical composition analysis of the entire wheat grain outer
layers. Indeed variability of the biochemical composition of the
outer layers’ tissues between wheats was already pointed out. Cell-
ulose, DHT, β-glucans, and p-CA previously found mainly in the
pericarp and the aleurone layer (Antoine et al 2003, 2004) varied
by 10 or 30%. Furthermore, tissues between the aleurone layer and
the outer pericarp containing the major part of lignin polymer
(Schwarz et al 1988; Antoine et al 2003) also varied as difference
in yield and relative proportion of the monomers released by thio-
acidolysis was pointed out. Additionally, in each composing tis-
sues, other minor components may also contribute to the cell wall
structure (Rhodes and Stone 2002; Parker et al 2005).
TABLE V
Analysis of Lignin Structure from Wheat Grain Outer Layers
Obtained After Thioacidolysis of β-O-4 Linked Unitsa
Cultivar H+G+S %H %G %S S/G
Soissons 15.7b 5.9a 36.8d 57.3a 1.55a
Camp Rémy 10.3a 7.5b 31.8b 60.7b 1.91b
Caphorn 16.5b 5.1a 29.9ab 65.0cd 2.17c
Crousty 17.0b 8.6c 34.0c 57.4a 1.69a
Ornicar 21.8c 5.8a 28.2a 66.1d 2.35d
Scipion 6.8a 5.3a 31.3b 63.5c 2.03bc
a Total yield (H+G+S) in µmol/g of sample and relative distribution (molar
ratio) of the main guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H)
thioacidolysis monomers (mean value of duplicate analyses). S/G, ratio of
main monomers. Different letters in the same column indicate different
groups determined by ANOVA taking into account corresponding standard
errors (P < 0.05).
However, extensibility of the outer layers was negatively related
to the sum of cell wall arabinoxylans and β-glucans content (R2 =
0.76), suggesting a potential relationship between relative propor-
tion of cell walls and the mechanical properties of the tissues. A
similar influence of cell wall volume fraction on mechanical prop-
erties was also reported with other plant tissues (Lucas et al 1995;
Znudek and Umeda 2005) or in wheat shoots grown under hyper-
gravity conditions (Wakabayashi et al 2005). This correlation could
probably not solely explain variation of the outer layer mechanical
properties as variability of these compounds between wheats was
low. However, a little difference in quantity could mask other
effects due to the existence of polymers with distinct structure
(molecular orientation or interactions) or local distribution, leading
to important changes in rheological properties. Thus, these results
confirm the potential importance of the relative proportion of the
cell wall in the mechanical properties of the outer layers and also
highlight the potential involvement of each of the tissue in the
outer layers’ mechanical properties (Peyron et al 2002a; Antoine
et al 2003). Although, this relationship needs to be extended to
other wheat cultivars, it pointed out the close relationship between
composition and structure of the outer layers in their milling
behavior.
CONCLUSIONS
Relationship between size of wheat bran particles obtained
after milling and mechanical properties of the outer layers from
common wheat grains was demonstrated. This outlined for the
first time that common wheat grain behavior of external tissues at
milling could be mainly due to intrinsic mechanical properties
rather than differences in grain fracture between grains of distinct
hardness as suggested earlier (Moss et al 1980). Furthermore, size
of wheat bran particles was also correlated with phytic acid
content in total flours, which could be considered as a marker of
the cellular aleurone layer distribution. Attempts to explain bio-
chemical basis of these differences in outer layers’ mechanical
properties between wheats only pointed out differences in content
of the main cell wall polysaccharides, suggesting involvement of
the tissues cell walls proportion. Analysis of genetically charac-
terized cultivars differing by their milling behavior is in progress
to test this assessment.
ACKNOWLEDGMENTS
This work is part of a French research project between INRA (National
Institute of Agronomic Research, Paris) and AFSA (French Association
of the Small Grain Cereal Breeders, Paris), ANMF (National Association
of French Flour-milling, Paris), ARVALIS-Institut du Végétal (Paris),
IRTAC (Paris), Danone Vitapole (Palaiseau), Tripette and Renaud –
Chopin (Villeneuve la Garenne) and ULICE (Riom). We gratefully thank
B. Pollet for thioacidolysis analysis and GC-MS analyses as well as A.
Surget and C. Olivé for their technical assistance in phenolics and
polysaccharide determination. We also thank particularly J. Stragliati
from Limagrain for her careful reading and English corrections.
LITERATURE CITED
Abecassis, J. 1993. Nouvelles possibilités d’apprécier la valeur meunière
et la valeur semoulière des blés. Ind. Céréales 81:25-37.
Antoine, C., Peyron, S., Mabille, F., Lapierre, C., Bouchet, B., Abecassis,
J., and Rouau, X. 2003. Individual contribution of grain outer layers
and their cell wall structure to the mechanical properties of wheat bran.
J. Agric Food Chem. 51:2026-2033.
Antoine, C., Peyron, S., Lullien-Pellerin, V., Abecassis, J., and Rouau, X.
2004. Wheat bran tissue fractionation using biochemical markers. J.
Cereal Sci. 39:387-393.
Arnason, J. T., Gale, J., Conilh de Beyssac, B., Sen, A., Miller, S. S.,
Philogene, B. J. R., Lambert, J. D. H., Fulcher, R. G., Serratos, A., and
Mihm, J. 1992. Role of phenolics in resistance of maize grain to the
stored grain insects, Prostephanus truncatus (Horn) and Sitophilus
zeamais (Motsch). J. Stored Prod. Res. 28:119-126.
Vol. 83, No. 6, 2006 645
Bacic, A., Harris, P. J., and Stone, B. A. 1988. Structure and function of
plant cell wall. Pages 297-371 in: The Biochemistry of Plant. Aca-
demic Press: Cambridge.
Beaugrand, J., Crônier, D., Debeire, P., and Chabbert, B. 2004. Arabin-
oxylan and hydroxycinnamate content of wheat bran in relation to
endoxylanase susceptibility. J. Cereal Sci. 40:223-230.
Darley, C. P., Forrester, A. M., and McQueen-Mason, S. J. 2001. The
molecular basis of plant cell wall extension. Plant Mol. Biol. 47:179-195.
Desvignes, C., Olivé, C., Lapierre, C., Rouau, X., Pollet, B., and Lullien-
Pellerin, V. 2006. Effects of calcium chloride treatments on wheat
grain peroxidase activity and outer layers mechanical properties. J. Sci.
Food Agric. 86:1596-1603.
Fincher, G. B., and Stone, B. A. 1986. Cell walls and their components in
cereal grain technology. Pages 207-295 in: Advances in Cereal
Sciences. Vol 8. Y. Pomeranz, ed. AACC International: St. Paul, MN.
Fry, S. C., Willis, S. C., and Paterson, A. E. J. 2000. Intraprotoplasmic
and wall-localised formation of arabinoxylan-bound diferulates and
larger ferulate coupling products in maize cell-suspension cultures.
Planta 211:679-692.
Grabber, J. H., Ralph, J., Lapierre, C., and Barrière, Y. 2004. Genetic and
molecular basis of grass cell-wall degradability. I. Lignin-cell wall
matrix interactions. C. R. Biologies 327:455-465.
Greffeuille, V., Abecassis, J., Bar L’Helgouac’h, C., and Lullien-Pellerin,
V. 2005. Differences in the aleurone layer fate between hard and soft
common wheats at grain milling. Cereal Chem. 82:138-143.
Hartley, R. D., Morrison, W. H., Himmelsbach, D. S., and Borneman, W.
S. 1990. Cross-linking of cell wall phenolic arabinoxylans in gramina-
ceous plants. Phytochemistry 29:3705-3709.
Hatfield, R. D., Ralph, J., and Grabber, J. H. 1999. Cell wall cross-linking
by ferulate in grasses. J. Sci. Food Agric. 79:403-407.
Izydorczyk, M. S., and MacGregor, A. W. 2000. Evidence of intermolec-
ular interactions of β-glucans and arabinoxylans. Carbohydr. Polym.
41:417-420.
Jarvis, M. C., and McCann, M. C. 2000. Macromolecular biophysics of
the plant cell wall: Concepts and methodology. Plant Physiol. Biochem.
38:1-13.
Jarvis, M. C., Briggs, S. P. H., and Knox, J. P. 2003. Intercellular
adhesion and cell separation in plants. Plant Cell Environ. 26:977-989.
Kamisaka, S., Takeda, S., Takahashi, K., and Shibata, K. 1990. Diferulic
and ferulic acid in the cell wall of Avena coleoptiles. Their relation-
ships to the mechanical properties of the cell wall. Physiol. Plantarum
78:1-7.
Köhler, L. 2000. Biphasic mechanical behaviour of plant tissues. Mater.
Sci. Eng. C11:51-56.
Lapierre, C., Pollet, B., and Rolando, C. 1995. New insights into the
molecular architecture of hardwood lignins by chemical degradative
methods. Res. Chem. Intermed. 21:397-412.
Lucas, P. W., Darvell, B. W., Lee, P. K. D., Yuen, T. D. B., and Choong,
M. F. 1995. The toughness of plant cells. Phil. Trans. R. Soc. Lond. B.
348:363-372.
MacAdam, J. W., and Grabber, J. H. 2002. Relationship of growth cessa-
tion with the formation of diferulate cross-links and p-coumaroylated
lignins in tall fescue leaf blades. Planta 215:785-793.
Mabille, F., Gril, J., and Abecassis, J. 2001. Mechanical properties of
wheat seed coats. Cereal Chem. 78:231-235.
Mandalari, G., Faulds, C. B., Sancho, A. I., Saija, A., Bisignano, G.,
LoCurto, R., and Waldron, K. W. 2005. Fractionation and characterisa-
tion of arabinoxylans from brewers’ spent grain and wheat bran. J.
[Received December 1, 2005. Accepted April 27, 2006.]
646 CEREAL CHEMISTRY
Cereal Sci. 42:205-212.
Moss, R., Stenvert, N. L., Kingswood, K., and Pointing, G. 1980. The
relationship between wheat microstructure and flour milling. Scanning
Electron Microsc. 3:613-620.
Parker, M. L., Ng, A., and Waldron, K. W. 2005. The phenolic acid and
polysaccharide composition of cell walls of bran layers of mature wheat
(Triticum aestivum L. cv. Avalon) grains. J. Sci. Food Agric. 85:2539-
3545.
Peyron, S., Chaurand, M., Rouau, X., and Abecassis, J. 2002a. Relation-
ship between bran mechanical properties and milling behaviour of
durum wheat (Triticum durum Desf.). Influence of tissue thickness and
cell wall structure. J. Cereal Sci. 36:377-386.
Peyron, S., Abecassis, J., Autran, J. C., and Rouau, X. 2002b. Influence of
UV exposure on phenolic acid content, mechanical properties of bran,
and milling behavior of durum wheat (Triticum durum Desf.). Cereal
Chem. 79:726-731.
Ralph, J., Quideau, S., Grabber, J. H., and Hatfield, R. D. 1994.
Identification and synthesis of new ferulic acid dehydrodimers present
in grass cell walls. J. Chem. Soc. Perkin Trans. 3485-3498.
Rhodes, D. I., and Stone, B. A. 2002. Proteins in cell walls of wheat
aleurone cells. J. Cereal Sci. 36:83-101.
Rouau, X., Cheynier, V., Surget, A., Gloux, D., Barron, C., Meudec, E.,
Louis-Montejo, J., and Criton, M. 2003. A dehydrotrimer of ferulic
acid from maize bran. Phytochemistry 63:899-903.
Saulnier, L., Crépeau, M. J., Lahaye, M., Thibault, J. F., Garcia Conesa,
M. T., Kroon, P., and Williamson, G. 1999. Isolation and structural
determination of two 5-5’-diferuloyl oligosaccharides indicate that
maize heteroxylans are covalently cross-linked by oxidatively coupled
ferulates. Carbohydr. Res. 320:82-92.
Scalbert, A., Monties, B., Lallemand, J.-Y., Guittet, E., and Rolando, C.
1985. Ether linkage between phenolic acids and lignin fractions from
wheat straw. Phytochemistry 24:1359-1362.
Schwarz, P. B., Kunerth, W. H., and Youngs, V. L. 1988. The distribution
of lignin and other fiber components within hard red spring wheat
bran. Cereal Chem. 65:59-64.
Shopfer, P., Lapierre, C., and Nolte, T. 2001. Light-controlled growth of
the maize seedling mesocotyl: Mechanical cell-wall changes in the elon-
gation zone and related changes in lignification. Physiol. Plantarum
111:83-92.
Tan, K. S., Hoson, T., Masuda, Y., and Kamisaka, S. 1991. Correlation
between cell wall extensibility and the content of diferulic and ferulic
acids in cell walls of Oryza sativa coleoptiles grain under water and in
air. Plant Cell Physiol. 33:103-108.
Wakabayashi, K., Soga, K., Kamisaka, S., and Hoson, T. 2005. Increase
in the level of arabinoxylan-hydroxycinnamate network in cell walls of
wheat coleoptiles grown under continuous hypergravity conditions.
Physiol. Plantarum 125:127-134.
Waldron, K. W., Ng, A., Parker, M. L., and Parr, A. J. 1997. Ferulic acid
dehydrodimers in the cell walls of Beta vulgaris and their possible role
in texture. J. Sci. Food Agric. 74:221-228.
Whitney, S. E. C., Gothard, M. G. E., Mitchell, J. T., and Gidly, M. J.
1999. Roles of cellulose and xyloglucan in determining the mechanical
properties of primary cell walls. Plant Physiol. 121:657-663.
Willm, C. 1995. Comportement en mouture de 9 variétés de blés tendre,
I: influence de la dureté et de l’apport d’azote. Ind. Céréales 92:18-29.
Zdunek, A., and Umeda, M. 2005. Influence of cell size and cell wall
volume fraction on failure properties of potato and carrot tissue. J.
Texture Stud. 36:25-43.