Journal of Food Engineering 72 (2006) 167–178

www.elsevier.com/locate/jfoodeng

Wet-milling properties of waxy wheat flours by two

laboratory methods q
a,*
Abdulvahit Sayaslan , Paul A. Seib b, Okkyung K. Chung c

a
Department of Food Engineering, Gaziosmanpasßa University, 60240 Tokat, Turkey
b
Department of Grain Science and Industry, Kansas State University, Manhattan, 66506 KS, USA
c
USDA/ARS Grain Marketing and Production Research Center, Manhattan, 66502 KS, USA

Received 7 July 2004; accepted 18 November 2004

Available online 18 January 2005

Abstract

Straight-grade flours roller-milled from seven waxy and two control (nonwaxy and partial waxy) wheats (Triticum aestivum) were
wet-milled by dough-washing (DW) and dough-dispersion and centrifugation (DD&C) methods to give starch, gluten, tailings, and
water-solubles fractions. Compared to the controls, flour yields of the waxy wheats were lower. The waxy flours contained more
crude fat and pentosans and less total starch. Gluten proteins of the waxy flours appeared to be weaker. By the DW method, both
of the controls but only two of the seven waxy flours could be wet-milled. The remaining five waxy flours failed in the DW method as
their doughs lost cohesiveness and elasticity during kneading and washing under water. Those waxy flours were instead wet-milled
by the modified DW method, where the doughs were gently hand-rubbed on a 125-lm opening screen to facilitate the passage of the
starch and water-solubles through while retaining the gluten agglomerates on the screen. The prime starch and tailings of the waxy
flours were not separated as visibly as those of the controls during their centrifugal purification, which in turn led to a reduction in
yields and purities of their prime starch and gluten fractions. On the other hand, the wet-milling characteristics of the waxy flours by
the DD&C method were comparable to those of the controls because the DD&C method is apparently less dependent than the DW
method on the gluten aggregation traits of flours.

2004 Elsevier Ltd. All rights reserved.

Keywords: Waxy wheat flour; Wet-milling; Starch and Gluten separation

1. Introduction duction. Common or bread wheats (Triticum aestivum)

Wheat is among the leading cereal grains produced in
the world, and is used for food (67%), feed (20%), and
seed (7%). The industrial uses of wheat, which also in-
cludes its wet-milling to produce starch and vital gluten
as the major co-products, account for 6% of total pro-
q
Mention of firm names or trade products does not imply that they
are endorsed or recommended by the USDA, Kansas State University,
or Gaziosmanpasßa University over other firms or products not
mentioned.
*
Corresponding author. Tel.: +90 3562521616x3262; fax: +90 356
2521488.
E-mail address: sayaslan@gop.edu.tr (A. Sayaslan).
with hard or soft endosperm constitute about 95% of to-
tal production, whereas durum wheat (Triticum durum)
accounts for 5%. Durum and hard wheats contain
more protein (12–16%) than do soft wheats (8–10%)
(Oleson, 1994; Seib, 1997; USDA/NASS, 2001). Hard
and soft wheats are roller-milled to flour and commonly
used for producing yeast-leavened breads and rolls as
they form strong viscoelastic doughs when mixed with
water. Soft wheat flours, however, give weak doughs
and are thus preferred for cakes, cookies and crackers.
Durum wheat with the hardest endosperm among
wheats is milled to semolina and used in pasta and cous-
cous production due to its strong gluten elasticity and
desirable yellow color. Hard or soft wheat flours with

0260-8774/$ - see front matter
2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2004.11.033
168 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178
weak- to medium-strength gluten are suitable for the
Asian noodles, steam breads, and flat breads (Oleson,
1994; Posner, 2000; Seib, 1997). For wet-milling to pro-
duce wheat starch and vital wheat gluten, straight-grade
or high-yield flours from hard or soft wheats with high-
protein content (>11%), good mixing quality (strong
gluten aggregation), and low starch damage, ash, and
a-amylase activity are preferred (Maningat & Bassi,
1999). Wheat starch is used in a vast variety of food
and nonfood applications in unmodified or modified
forms; such as acid-thinned, bleached, oxidized, cross-
linked, substituted, cross-linked/substituted, and other
doubly modified starches (Maningat & Seib, 1997).
Wheat starch also is converted to starch hydrolysis
products, such as sweeteners (Schenck & Hebeda,
1992). Vital wheat gluten is a valuable co-product of
wheat wet-milling process that yields wheat starch
(Bergthaller, 1997; Cornell & Hoveling, 1998; Grace,
1988; Maningat & Bassi, 1999; Maningat, Bassi, & Hes-
ser, 1994). Vital wheat gluten is used in breakfast cereals
and snacks, meat and cheese analogs, breading and bat-
ter mixes, pizza, and in meat, fish, and poultry products
(Bergthaller, 1997; IWGA, 1989; Maningat & Bassi,
1999). Wheat gluten also is of great importance as an
industrial protein, and is converted to wheat protein iso-
late, hydrolyzed wheat protein, and texturized or deam-
idated wheat gluten (Bergthaller, 1997; Maningat et al.,
1994; Maningat, DeMeritt, Chinnaswamy, & Bassi,
1999).
Five industrial wet-milling processes that start with
flour rather than wheat kernel have been employed to
isolate wheat starch and vital wheat gluten (Maningat
& Bassi, 1999; Seib, 1994). Until the mid 1970s, wheat
starch had been produced primarily by the Martin pro-
cess that was invented in 1835 (Anderson, 1967; Barr,
1989; Fellers, 1973; Zwitserloot, 1989a) and rarely by
the Batter process developed in 1944 (Anderson, 1967,
1974; Fellers, 1973). Then, the Alfa-Laval/Raisio (Dahl-
berg, 1978; Kerkkonen, Laine, Alanen, & Renner, 1976;
Maijala, 1976) and Hydrocyclone (Verberne & Zwitser-
loot, 1978; Verberne, Zwitserloot, & Nauta, 1979) pro-
cesses were introduced. The most recent wet-milling
process commercialized in 1984 is the High-Pressure
Disintegration (HD) process (Bergthaller, Lindhauer,
& Zwingelberg, 1998; Meuser, Althoff, & Huster, 1989;
Witt, 1997; Zwitserloot, 1989a, 1989b). The Martin
and Batter processes are considered the traditional pro-
cesses, whereas the Alfa-Laval/Raisio, Hydrocyclone,
and HD processes are deemed the modern-day aqueous
dispersion processes. Currently, the Martin (modified
modern versions), Hydrocyclone, and Alfa-Laval/Raisio
processes are commonly used in North America, and the
HD process is popular in Europe (Maningat & Bassi,
1999). The Batter process was mostly abandoned. The
advantages of the aqueous dispersion processes include
a reduction in fresh water consumption, an increase in
prime starch yield, and less sensitivity to gluten protein
aggregation characteristics during wet processing (Zwit-
serloot, 1989a, 1989b). For details on the industrial wet-
milling processes for wheat flour, the reader is referred
to a recently published review by Sayaslan (2004).
Common wheats are hexaploid with three sets of
chromosomes, but they differ in the translation of gran-
ule-bound starch synthase isozymes in their kernels
(Yamamori & Quynh, 2000). Wild-type (nonwaxy or
typical) wheats contain all three possible waxy proteins
(Wx-A1, Wx-B1, and Wx-D1) and thus possess normal
level (28%) of amylose in the starch. Partial waxy
wheats are null for one or two of the three waxy pro-
teins, which results in reduced level of amylose (20–
26%). Waxy wheats are, however, triple-null for all the
three waxy proteins and their starch contains <1% amy-
lose (Kim, Johnson, Graybosch, & Gaines, 2003; Seib,
2000). Besides commonly grown nonwaxy and partial
waxy wheats, waxy wheats with hard or soft endosperm
have been developed recently and may find uses in wet-
milling for waxy starch production, extruded products
for volume expansion, frozen foods and doughs for
cold-temperature stability, bakery products for shelf-life
extension and fat replacement, instant food products for
easy-to-cook trait (Bhattacharya, Erazo-Castrejon,
Doehlert, & McMullen, 2002; Graybosch, 1998), and
for other purposes yet to be discovered. The low gelati-
nization temperature of waxy wheat starch compared to
waxy corn starch may be important in microwaved
foods. Hydroxypropylated and cross-linked waxy wheat
starch gave a thick paste with high-cold temperature
stability. Cross-linking waxy wheat starch, akin to
cross-linked waxy corn starch, imparts high and stable
viscosity to processed foods (Reddy & Seib, 2000).
Maltodextrins with dextrose equivalents of 1 and
10 were successfully produced from waxy wheat
starches because of their lower levels (<0.1%) of lipids
(Lumdubwong & Seib, 2001).
Waxy wheats were first developed in 1994 through
conventional plant breeding in Japan (Nakamura,
Yamamori, Hirano, Hidaka, & Nagamine, 1995), and
later in Canada (Chibbar, Baga, Demeke, & Hucl,
1997) and in the USA (Graybosch, 1998; Morris & Kon-
zak, 2001; Souza, 2001—personal communication). Waxy
wheats also have been produced through mutagenesis of
partial waxy wheats Kanto 107 or Ike (Kiribuchi-Otobe,
Nagamine, Yanagisawa, Ohnishi, & Yamaguchi,
1997; Yasui, Sasaki, Matsuki, & Yamamori, 1997; Kon-
zak, 1998—personal communication). Physical/chemical
properties of waxy wheat starches have been thoroughly
studied. Hexaploid waxy wheat starch was found to con-
tain 0.9 to 3.2% apparent amylose and 0.12–0.29% lipids
(Abdel-Aal, Hucl, Chibbar, Han, & Demeke, 2002; Cha-
kraborty et al., 2004; Kim et al., 2003; Yasui, Matsuki,
Sasaki, & Yamamori, 1996), in contrast with 22–25%
amylose and 0.8–1.2% lipids in nonwaxy wheat starch
 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178
(Morrison, Milligan, & Azudin, 1984). As expected,
waxy wheat starch showed the A-type X-ray pattern
and had an increased level of crystallinity compared to
wild types, thereby elevating the enthalpy of gelatiniza-
tion (Chakraborty et al., 2004; Fujita, Yamamoto,
Sugimoto, Morita, & Yamamori, 1998; Hayakawa, Ta-
naka, Nakamura, Endo, & Hoshino, 1997; Kim et al.,
2003; Sasaki, Yasui, & Matsuki, 2000). Tetraploid (dur-
um) waxy wheat starches also showed similar gelatiniza-
tion and pasting properties (Chakraborty et al., 2004;
Grant et al., 2001). Although physical and/or chemical
properties of waxy wheat starches have been the focus
of much research, wet-milling properties of waxy wheat
flours and their gluten fractions are yet to be studied.
The objective of this study was therefore to determine
the wet-milling properties of waxy wheat flours by two
laboratory methods that simulate the traditional Martin
and aqueous dispersion processes.
2. Materials and methods
2.1. Materials
Six experimental waxy wheat lines were obtained
from Dr. C.F. Konzak (Northwest Plant Breeding
Co., Pullman, WA), Dr. R.A. Graybosch (USDA/
ARS, University of Nebraska, Lincoln, NE), Dr. C.F.
Morris (USDA/ARS-WWQL, Washington State Uni-
versity, Pullman, WA), and Dr. R.N. Chibbar (National
Research Council, Plant Biotechnology Institute, Saska-
toon, SK, Canada). One of the samples was received as
flour. The waxy wheat samples were coded as follows:
Wx-A, Wx-B, Wx-C, Wx-1, Wx-2, and Wx-3. All of
the samples were experimental waxy wheat lines, but
the Wx-1, Wx-2, and Wx-3 wheats were reportedly in
their advanced stages of field trials and selection for
agronomic traits. Leona waxy wheat sample, a waxy
wheat variety, was provided by Dr. E.J. Souza (Univer-
sity of Idaho, Aberdeen, ID). Control nonwaxy (Karl
92) and partial waxy (Ike) wheats were from Dr. J. Mar-
tin (Hays Branch of State Agricultural Experiment Sta-
tion, Kansas State University, Manhattan, KS). The
waxy wheats used in the study were reportedly produced
by either mutagenesis of Ike partial waxy wheat, or by
crossing Ike or Kanto 107 partial waxy wheat with Bai
Huo partial waxy Chinese wheat. Some of the waxy
wheats were further backcrossed with Klasic wheat.
2.2. Methods
2.2.1. General methods
Wheat samples were cleaned on a Carter-Day dock-
age tester (Carter-Day Co., Minneapolis, MN) and their
test weights measured with a test weight apparatus from
Burrows Equipment Co., Evanston, IL. Single kernel
169
characteristics of wheat samples were determined on
the Single Kernel Characterization System (SKCS
4100, Perten Instruments North America, Inc., Reno,
NV). All wheat samples were tempered at 15.5% mois-
ture for 20 h and roller-milled to straight-grade flours
on a Buhler experimental mill (Buhler Co., Uzwil, Swit-
zerland). Lightness values (L*) of flours were measured
using a Minolta Chroma Meter (Minolta Co., Osaka,
Japan).
Moisture, ash, crude fat, and crude fiber contents
were determined by the American Association of Cereal
Chemists (AACC, 2000) methods 44-15A, 08-01, 30-25,
and 32-10, respectively. Protein contents (N · 5.7) were
measured by combustion method on an FP Protein/
Nitrogen Analyzer (Leco Corp., St. Joseph, MI). Total
starch and starch damage were determined by the
AACC methods 76-13 and 76-31, respectively, using
assay kits from Megazyme International Ireland Ltd.,
Wicklow, Ireland. Amylose contents of flours and a-
amylase activities of wheat kernels were measured
using, respectively, amylose/amylopectin and Ceralpha
a-amylase assay kits from Megazyme International
Ireland Ltd., Wicklow, Ireland.
Mixograms of flours were obtained by the AACC
method 54-40A using a 10-g mixograph (National Man-
ufacturing Co., Lincoln, NE). The mixograms were
interpreted as described by Finney and Shogren
(1972). Gluten index (GI) and falling number of flours
were determined, respectively, on Glutomatic and Fall-
ing Number instruments (Perten Instruments North
America, Inc., Reno, NV) by the AACC methods
38-12 and 56-81B, respectively.
Insoluble polymeric protein (IPP) contents of flours
were determined essentially by the method of Bean,
Lyne, Tilley, Chung, and Lookhart (1998). Flour
(1.0 g, db) was mixed with 4 ml of 50% (v/v) aqueous
1-propanol and the mixture dispersed by hand with a
spatula. The sample was mixed continually for 5 min
on a vortex mixer, centrifuged at 8160g for 5 min, and
the supernatant discarded. This extraction procedure
was done a total of three times. After the extractions,
the pellet was freeze-dried and analyzed for protein
(N · 5.7). The IPP content was calculated as a percent-
age of total protein in a flour sample.
Sodium dodecyl sulfate (SDS) sedimentation volumes
of flours were determined by the AACC method 56–70
with modifications. Flour (2.0 g, 14% mb) was weighed
into a 100-ml graduated cylinder and 20.0 ml of water
containing 0.0004% bromophenol blue (w/v) was added.
The cylinder was capped and hand-shaken for 15 s, then
placed on a specially designed (USDA/ARS Grain Mar-
keting and Production Research Center, Manhattan,
KS) table-top wrist-action shaker for 225 s at room tem-
perature. Next, 20.0 ml of 2.5% (w/v) aqueous SDS solu-
tion was added. After gently shaking, the cylinders were
placed on the shaker for another 6 min. Finally, 10.0 ml
170 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178
of 0.47% (v/v) lactic acid solution was added and the cyl-
inder placed on the shaker for 6 min. The cylinder was
removed and placed on a rack with a back lighting for
20 min prior to reading sedimentation volume.
Total and soluble pentosans in flours were estimated
according to colorimetric method of Douglas (1981).
The concentration of pentose sugar D -xylose after
acid-catalyzed hydrolysis of pentosans in a flour (for to-
tal pentosan assay) or in its water extract (for soluble
pentosan assay) was estimated from a D -xylose standard
curve. Soluble pentosans were extracted from a flour
prior to colorimetric determination according to
Hashimoto, Shogren, and Pomeranz (1987). Flour
(100 mg, 14% mb) was weighed into a stoppered 15-ml
glass tube, 10.0 ml of distilled water added and the tube
shaken at 30 C in a reciprocal water bath (Precision
Instruments, Winchester, VA) at a medium speed for
2 h. The sample was then centrifuged at 2000g for
5 min and the supernatant collected for determination
of soluble pentosans. Soluble and total pentosans were
presented as % D -xylose (Douglas, 1981).
Granule size distributions of starches were deter-
mined on a laser-diffraction particle analyzer (Leco-
trac-LTS150, Leco Corp., St. Joseph, MI) equipped
with a recirculator, an on-line ultrasonic probe, and an
interfaced computer with Lecotrac software. Starch
(0.2 g) was mixed with distilled water (5 ml) and al-
lowed to stand at room temperature for 24 h. After vig-
orous agitation by hand, the suspension (1–2 ml) was
added to isotonic buffer solution (300 ml) (sodium chlo-
ride, 8.6 g/L; potassium chloride, 0.38 g/L; ethylenedia-
mine tetraacetic acid, 0.4 g/L; 2-phenoxyethanol, 0.2 g/
L; in deionized water) being circulated at 40 ml/s
through a standard cell. The addition of the starch/water
mixture was stopped when the particle analyzer pro-
duced a signal indicating that a sufficient number of par-
ticles were present for analysis. Then the ultrasonic
device was energized for 60 s prior to particle size mea-
surement. The software converted the diffracted light
data to particle diameters between 0.7 and 700 lm,
and the volume percentages of particles were calculated
assuming that the particles were spheres.
2.2.2. Wet-milling of flours by dough-washing (DW)
method
Wheat flours were fractionated by the AACC gluten
hand-washing method 38-10 with modifications, which
resembles the traditional Martin (dough-washing) pro-
cess, to give wet gluten, prime starch, tailings, and water
solubles. Flour (100 g, 14% mb) in a 100-g bowl pin mix-
er (National Manufacturing Co., Lincoln, NE) was
mixed with gradual addition of water (60–70 ml,
25 C) until the mixture became a cohesive, stiff
dough-ball and cleaned itself from the mixing bowl
(3–5 min). After resting the dough under a moist cover
for 1 h at room temperature, the dough was hand-
kneaded gently under a stream of water over a 125-lm
opening sieve until almost all starch and soluble materi-
als were removed. However, certain doughs failed to
maintain their cohesive and elastic properties during
hand-kneading under water. To facilitate the wet-
separation of starch and gluten, those doughs were in-
stead gently hand-rubbed on the screen under a spray
of water to allow the passage of the starch and water-
solubles while retaining the gluten protein agglomerates
on the screen. This method of wet-milling was in this
work called the modified dough-washing (MDW) meth-
od. Washing of the gluten mass by both the methods
(DW and MDW) was stopped when 1–2 drops of water
squeezed from the gluten into a beaker gave clear water
free of starch granules. The gluten fraction was placed in
a container, covered, and stored at room temperature
for 1 h. The wet gluten was then shaped into a sphere,
pressed as dry as possible between a pair of watch
glasses, and weighed. Wet gluten was partly frozen in
freezer (1 h), cut into 1-cm3 pieces, and placed in jars
that were attached to a freeze-dryer (Flexi-Dry/MP,
TMS Systems, Inc., Stone Ridge, NY). The reduced
pressure caused the gluten pieces to expand and created
a high-surface area for rapid freeze-drying. The freeze-
dried gluten, which typically contained <5% moisture,
was ground in a Thomas-Wiley intermediate mill (Tho-
mas Scientific Co., Philadelphia, PA) to pass through a
420-lm opening sieve.
The so-called starch milk, which passed through the
125-lm opening sieve, was resieved through a 75-lm
opening sieve, and the mass remaining over the 75-lm
opening sieve was washed with water (50 ml). The mate-
rial that remained atop the screen was collected. The
material passing through the 75-lm opening sieve was
centrifuged at 2500g for 20 min, and the supernatant
was collected. The upper-pigmented layer (tailings) atop
the white starch was carefully removed with a spatula.
The starch was resuspended by hand-shaking in water
(200 ml) and centrifuged at 2500g for 20 min. The super-
natant was collected, combined with the supernatant
collected in the first centrifugation step, and sampled
(100 ml) to determine water-soluble solids. The tailings
were carefully removed with a spatula. The remnants
of the 75-lm opening sieve and the tailings removed
after the first and second centrifugation steps were com-
bined and freeze-dried. The starch on the bottom layer
was collected and oven-dried at 40 C for two days.
The dried starch (8% moisture) was gently ground
with a mortar and pestle before being analyzed for mois-
ture and protein.
2.2.3. Wet-milling of flours by dough-dispersion and
centrifugation (DD&C) method
Wheat flours were fractionated into wet gluten, prime
starch, tailings, and water-solubles by essentially a
dough-dispersion and centrifugation (DD&C) method
 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178
reported by Czuchajowska and Pomeranz (1993, 1995),
which somewhat simulates the aqueous dispersion pro-
cesses. Dough preparation step was similar to that in
the DW method. The developed stiff dough-ball was
covered with water––a total of 200 ml of water including
that added to the flour in the dough mixing stage. After
resting for 30 min at room temperature, the dough and
liquid were transferred to a Waring blender and dis-
persed at high speed for 1 min. The dry solids concentra-
tion of the slurry was adjusted to 27% with additional
water prior to centrifugation at 2500g for 15 min. Upon
centrifugation, the supernatant was collected and the
top layer of sediment, which consisted mainly of gluten
plus insoluble pentosans, damaged starch, and small
granular starch, was carefully removed from the prime
starch layer (bottom). The top layer was gently manipu-
lated under a stream of water (3 · 200 ml) to obtain the
cohesive gluten fraction. The gluten fraction was treated
similarly to those in the DW method to obtain dry glu-
ten. The prime starch layer and the washings (starch
milk) of the gluten fraction were combined and purified
by washing with water and centrifugation twice as de-
scribed in the DW method.
2.2.4. Data analysis
Gluten and starch separation and the physical/chem-
ical analyses were carried out at least in two replications,
and the means were compared by the TukeyÕs HSD test
in one-way analysis of variance (ANOVA), using the
Statistical Analysis System Software, V. 6.12 (SAS Insti-
tute, Inc., Cary, NC).
3. Results and discussion
3.1. Wheat kernel properties
Karl 92 and Ike wheats are both the U.S. hard red
winter wheat varieties. Karl 92 is a nonwaxy wheat that
contains all three waxy proteins (Wx-A1, Wx-B1, and
Wx-D1) and thus possesses normal level (28%) of
amylose in its starch. Ike wheat is a partial waxy wheat,
double-null for the Wx-A1 and Wx-B1 proteins, with re-
duced amylose (21%). The waxy wheats are, however,
triple-null for all the three waxy proteins and their
starches contain <1% amylose (Graybosch et al., 1998;
Seib, 2000). The waxy wheats often contain Ike partial
waxy wheat in their genetic backgrounds.
The waxy wheat lines used in the study were pro-
duced by either mutagenesis of Ike, or by crossing Ike
or Kanto 107 wheats, both null for the Wx-A1 and
Wx-B1 proteins, with a Chinese wheat cultivar Bai
Huo null for the Wx-D1 protein. Some of the waxy lines
were further backcrossed with Klasic wheat. All wheat
samples were sound with no sprout damage; the a-amy-
lase activities ranged from 0.07 to 0.20 Ceralpha units/g
171
kernel and the falling numbers of the control flours were
well above 400 s (Table 1). The falling number of only
one waxy flour sample (Wx-3) was determined. In accor-
dance with the previous findings (Abdel-Aal et al., 2002;
Graybosch, Guo, & Shelton, 2000) that the Wx-3 waxy
flour gave an extremely low falling number (75 s) even
though it was milled from a sample of sound kernels
with a low a-amylase activity (0.12 Ceralpha units/g ker-
nel). In sound waxy and nonwaxy wheat flours, a-amy-
lase activities were reported to range from 0.04 to 0.16
Ceralpha units/g flour (Abdel-Aal et al., 2002; Gray-
bosch et al., 2000; Yasui, Sasaki, & Matsuki, 1999),
while a sprout-damaged wheat flour had an a-amylase
activity of 0.33 (Graybosch et al., 2000).
The waxy wheats possessed hard, soft, and mixed-
hardness endosperms, whereas the control wheats had
hard endosperms (Table 1). All waxy wheat lines and
the control wheats contained relatively high levels of
protein, ranging from 12.8% to 14.9% (14% mb). Leona
waxy wheat, however, contained the lowest protein
(10.6%). The test weights of the waxy wheats were with-
in the range of the control wheats (76–80 kg/hL) except
for the Wx-2 (74 kg/hL) and Wx-3 (73 kg/hL) lines. A
large variation was observed in terms of single kernel
weights (26–40 mg). Single kernel diameters of the
waxy wheats were in general close to those of the control
wheats (2.5 mm), with a minimum of 2.0 mm to a
maximum of 2.8 mm (data not shown).
3.2. Flour properties
Flour composition and properties are listed in Table
1. All waxy wheat samples gave less flour than the con-
trol hard wheats by roller-milling. It was noticed that
the fractions of shorts from the waxy wheats, specifically
from soft waxy wheats, were heavily contaminated with
flour. In order to recover the contaminated flour, the
shorts were sieved (150 lm opening sieve, 2 min) and
the recovered flours were combined with their respective
flours. Still, the flour yields from the waxy wheats were
3–7% lower than the two control wheats. The Wx-2
sample gave even less flour (15%), which was perhaps
caused by its quite small (2.0 mm) kernel size. The re-
duced flour yields from the waxy wheats with soft endo-
sperm may be explained by the typical inferior bolting
quality of soft wheats (Posner & Hibbs, 1997). Also, soft
wheats were milled on a Buhler experimental mill that
was set for hard wheat milling. Souza (2001—personal
communication) reported that soft waxy wheat endo-
sperm tended to flake on the smooth reduction rolls dur-
ing milling. The test weights of neither the controls nor
the waxy wheats correlated with their flour yields. The
reduced flour yields from the waxy wheats with hard
endosperm, however, might be explained by their
elevated crude fat and pentosan contents (Table 1).
Fats provide cohesiveness to flours and reduce their
172 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178

flowability (Neel & Hoseney, 1984). Yasui et al. (1999)

Color
reported reduced (20%) flour yields from two waxy
(L*)

91.8
90.9

91.3
91.4
93.6
90.1
93.5
91.4
93.0
wheats produced from Kanto 107 by mutagenesis. They
attributed the decrease in waxy flour yield to a decline in
Soluble
their flowability that might be caused by their elevated
(% D -xylose)

0.57
0.47

0.47
0.60
0.60
0.54
0.56
0.60
0.44
Pentosansa

fat (20%) and b-glucan (30) levels. Kim et al.

(2003) also found that waxy wheats yielded less flour
Total

1.52
1.36

2.30
2.08
1.97
1.98
1.74
1.74
1.86
than partial waxy and nonwaxy wheats. However, the
elevated crude fat contents determined for waxy wheat
index (GI)

flours might be considered an artifact of the lipid extrac-

Gluten

tion. With essentially no amylose present, there is noth-

nmc
nm
nm
nm
nm
nm
nm
99
98

ing to bind flour lipids, hence rendering the lipids more

accessible to the solvent and inflating the values.
SDS
(ml)

3.7
3.5

2.8
1.8
2.7
1.3
2.2
3.2
1.5
The protein contents of the waxy flours ranged from
9.4% to 14.2% compared to 12.1% to 12.9% for the con-
42.4
37.7

38.3
29.7
39.4
24.7
28.8
37.1
24.6
IPP
(%)

trols (Table 1). The lightness values (L*) of the flours

varied from 90.1 to 93.6. As compared to the control
Amylose

flours, the waxy flours contained 20–40% more fat,

27.6
21.3

1.2
1.4
0.9
1.6
0.9
0.6
1.4

10–20% more total pentosans, and 10–30% more crude

(%)

fiber. Also, waxy wheat flours had 3–7% lower total

damage

starch than the controls, perhaps because the flux of car-

Starcha

bohydrates into starch was diminished due to the ab-

Not determined as waxy wheat flours give unusually low falling numbers independent of their a-amylase activities.
(%)

6.2
4.8

5.8
5.1
3.7
4.5
3.2
6.0
4.6

sence of amylose biosynthesis. Amylose levels of the

Not measured due to blinding of Glutomatic washing chamber sieve (88-lm opening) during gluten washing.

waxy samples were higher than the expected levels, rang-

Starcha

68.0
68.5

63.3
64.2
65.9
61.9
64.6
64.2
66.5

ing from 0.6% to 2.3%, whereas the controls gave the ex-
(%)

pected values (Table 1). Other investigators (Abdel-Aal

Composition and properties of straight-grade flours roller-milled from control and waxy wheat samples
Crudea
fat (%)

et al., 2002; Chakraborty et al., 2004; Kim et al., 2003)

also reported varying amylose levels (0.9–3.2%) for
0.8
0.6

0.9
0.9
1.0
1.2
1.0
0.9
1.1

waxy wheats. This discrepancy could be attributed to

fiber (%)

the variations in samples and methods of analysis used

Crudea

in different studies.
0.21
0.19

0.25
0.26
0.24
0.30
0.20
0.20
0.20

3.3. Wet-milling properties of flours by dough-washing

Asha

0.49
0.44

0.46
0.48
0.41
0.48
0.37
0.36
0.56
(%)

14% flour moisture basis (flour moisture levels ranged from 11.5 to 13.5%).

(DW) method
(N · 5.7) (%)

All wheat samples were separately roller-milled to

Proteina

straight-grade flours, physically and/or chemically char-

12.1
12.9

12.0
12.0
13.1
14.2
10.9
13.6
9.4

acterized, and wet-milled to give wet gluten, prime

starch, tailings, and water solubles fractions. The two
number (s)

control flours and only two of the waxy wheat (Wx-1

and Wx-3) flours could be wet-milled by the original
Falling

DW method (Table 2). The doughs from those flours

ndb
492
437

nd
nd
nd
nd

nd
75

Not reported since it was received as flour.

maintained their cohesiveness and elasticity during

yield (%)

kneading and washing under water, thus enabling wash-

Flour

ing of the starch from the gluten fraction. The remaining

71.9
70.8

66.1
67.1
66.8

60.4
69.2
67.2
nr

five waxy wheat flour doughs lost much of their cohesive

and elastic properties immediately upon starting to
index (HI)
hardness

knead under the stream of water, despite the fact that

Kernel

cohesive stiff doughs were initially formed from all the

nrd
71
75

77
68
31

22
44
48

flours, including the waxy flour doughs whose gluten

agglomerates were broken apart into small pieces and
Karl 92 (nonwaxy)
Ike (partial waxy)

became sticky during washing. In order to wash the

Control wheats
Flour sample

Waxy wheats

starch away and isolate the gluten, those doughs were

wet-milled by hand-rubbing them on the 125-lm open-
Table 1

Leona
Wx-A

Wx-C
Wx-B

ing screen (modified DW method, MDW). Gentle rub-

Wx-1
Wx-2
Wx-3

d
a

bing and washing of the small dough pieces and gluten

 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178 173

Unaccounteda agglomerates on the sieve retained gluten agglomerates

solids (%) on the sieve while allowing the starch granules and the
soluble solids to pass through the screen. Because of

21.1
21.5

26.5
22.8
21.0
16.4
20.7
20.2
28.8
7.0
the stickiness of their glutens, many of the screen open-
ings became plugged during washing, which increased
the time and water usage required to purify the gluten
solids (%)

fraction. Interestingly, the wet gluten fractions retained

Solublea

on the screen recovered their elasticity and cohesiveness

3.8
3.7

5.8
6.2
6.3
9.6
8.3
9.5
9.9
1.4
during washing at a point where about two-thirds or
more of the starch were washed away from the gluten
recovery (%)

fractions. This may be explained by the increase in the

concentration of the gluten proteins in the gluten frac-
Starch

tions and/or removal of an unknown component from

79.9
76.0

73.8
73.9
73.5
78.1
78.2
72.1
67.8
7.4
the dough mass, which enabled the gluten proteins to
interact with each other, agglomerate, and provide cohe-
siveness. Furthermore, when the starch milk passing
(N · 5.7) (%)

through the screen from the waxy flour dough washings

a
Protein

were centrifuged to recover the starch fraction, a thick

Wet-milling data for wheat flours by dough-washing (DW) or modified dough-washing (MDW) method on 100 g (14% mb) of flour

0.24
0.21

0.40
0.49
0.38
0.32
0.20
0.20
0.35
0.09

intermediate fraction (tailings) was found between the

Starch fraction

supernatant and the bottom prime starch layer. The

intermediate layer contained gluten protein particles
yield (%)
a
Starch

mixed with mostly small starch granules and insoluble

63.2
60.6

54.3
55.1
56.3
56.3
58.7
53.9
52.4
6.5

pentosans. In contrast, centrifugation of the starch milk

from the control flours gave a sharp interface between
the supernatant and sedimented starch with only a thin
recovery (%)

layer of tailings (darkened zone) atop the starch.

Protein

Other investigators (Hamer, Weegels, Marseille, &

77.9
86.2

71.1
80.0
79.1
78.3
79.9
85.0
63.6
10.3

Kelfkens, 1989; Meuser, 1994; Meuser et al., 1989; Wee-

gels, Marseille, & Hamer, 1988; Zwitserloot, 1989a)
have reported differences in the rate and extent of gluten
(N · 5.7) (%)

agglomeration during wet-processing of wheat flours.

a

The reasons for the reduced rate and strength of gluten

Protein
Dry gluten fraction

4.4
92.0
90.9

73.8
70.3
73.0
73.1
82.2
81.6
77.6

agglomeration in flour-water doughs and slurries have

not been fully understood. High molecular weight glute-
nins and glutenin macropolymer formed by the glutenin
yield (%)

particles have been shown in the last 10 years to add

a
Gluten

strength (elasticity) to a wheat flour dough (Antes &

11.9
14.2

13.4
15.9
16.5
17.7
12.3
16.5
9.0
1.5

Wieser, 2001; Bekkers, Lichtendonk, Graveland, & Plij-

ter, 2000; Don, Lichtendonk, Plijter, & Hamer, 2003a,
Wet gluten
Yield (%)

2003b; Schropp & Weiser, 1996; Singh & MacRitchie,

2001; Southan & MacRitchie, 1999; Uthayakumaran,
b

30.4
37.9

36.8
48.9
44.2
54.9
33.0
45.7
25.9
4.9

Stoddard, Gras, & Bekes, 2000; Veraverbeke, Verbrug-

gen, & Delcour, 1998; Wooding, Kavale, MacRitchie,
& Stoddard, 1999). However, the wet-processing quality
(N · 5.7)
b
Protein

of a wheat flour and its relationship to flour protein

Flour

12.1
12.9

12.0
12.0
13.1
14.2
10.9
13.6
9.4
(%)

quality remain mostly unknown.

–

Gluten proteins in the waxy wheat flours appeared to

14% flour moisture basis.

be weaker than those of the controls as indicated by

Karl 92 (nonwaxy) (by DW)
Ike (partial waxy) (by DW)

their lower SDS sedimentation volumes and IPP con-

TukeyÕs HSD (a = 0.05)

tents (Table 1) and shorter mixograph mixing times

and lower mixing tolerances (Fig. 1). The IPP contents
Leona (by MDW)
Wx-A (by MDW)

Wx-C (by MDW)

Wx-B (by MDW)

Wx-2 (by MDW)

of flours were reported to correlate strongly with dough

Wx-1 (by DW)

Wx-3 (by DW)

Dry basis.
Control wheats
Flour sample

Waxy wheats

mixing time and mixing tolerance (Bean et al., 1998).

Also, elevated insoluble pentosans of the waxy flours
Table 2

(Table 1) may be the reason for the loss of their

b
a

cohesiveness and elasticity during kneading. Pentosans

174 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178
Fig. 1. Mixograms of flours from nonwaxy and partial waxy control wheats (top row) and waxy wheats (middle and bottom rows) at optimum water
absorption levels (protein contents of flours given in parenthesis on 14% mb).
(2–3%) of wheat flours (Hashimoto et al., 1987; Line-
back & Rasper, 1988) were reported to bind or hold 5-
to 10-fold water by their mass (Bushuk, 1966; Courtin
& Delcour, 2002) and to increase the viscosity of the li-
quid phase during wet-milling. This in turn caused a
reduction in the sedimentation rate of small B-starch
granules in a centrifugal field, translating to a reduced
starch yield (Witt, 1997). Pentosans also interfered with
gluten formation and aggregation during dough mixing
through viscosity-related interference of pentosans on
gluten aggregation (McCleary, 1986; Wang, Hamer,
Vliet, & Oudgenoeg, 2002), steric hindrance of gluten
aggregation due to formation of a pentosan network
during dough mixing (Rouau, El-Hayek, & Moreau,
1994; Labat, Rouau, & Morel, 2002; Weegels, Marseille,
& Voorpostel, 1991), and through direct interaction of
pentosans with glutenin particles during dough mixing
(Wang et al., 2002; Wang, 2003; Wang, Hamer, et al.,
2003a; Wang, Oudgenoeg, Vliet, & Hamer, 2003b), all
of which are expected to affect the wet-milling properties
of flours. In waxy wheats, genetic backgrounds, not the
waxy trait per se, are more likely the cause of weak
glutens.
Other methods of wet-milling were reported to be less
dependent on agglomeration traits of gluten in a dough
(Sayaslan, 2004; Zwitserloot, 1989a, 1989b), and such
methods may work better for the separation of vital glu-
ten and starch from flours with poor gluten strength/
elasticity. According to Zwitserloot (1989a), some weak
flour doughs that weaken excessively in the Martin pro-
cess can be wet-milled by the HD process. In that pro-
cess and possibly in the other dispersion processes
(Hydrocyclone and Alfa-Laval/Raisio), the sensitivity
to gluten agglomeration quality exhibited in the Martin
process may be marginalized because density differences
of the flour components appear to be the primary sepa-
ration factor in those processes. Also, concentrating the
gluten proteins and removing the viscosity-building sol-
uble pentosans early in the wet-milling steps through
centrifugation, which is practiced in the dispersion pro-
cesses, may facilitate the wet-milling of waxy wheat
flours. In fact, this approach proved to be viable for
the wet-milling of the waxy wheat flours that failed by
the DW method (see results in the DD&C method below).
As expected, the yields of dry gluten fractions were
closely associated with the protein contents of their
flours. With the exception of Wx-2 and Leona, the waxy
wheat flours mostly yielded more dry glutens than the
control flours, but their protein contents (purity) were
7–20% lower (Table 2). This particular Leona waxy
wheat sample gave the least dry gluten because of its ex-
tremely low protein content (9.4%). A high-protein Leo-
na sample is likely to give improved gluten yield and
wet-milling quality. The difficulty encountered during
washing the starch away from the gluten reduced the
purities of the waxy flour gluten fractions. The protein
contents of the glutens isolated in this work were all
>73%, meeting the minimum 73% protein (N · 5.7, db)
required by the Codex Alimentarius of the Food and
Agriculture Organization (FAO, 2002). Commercial
glutens have been found to contain 73–80% protein on
dry solids basis (Miller & Hoseney, 1996). Dreese, Fau-
bion, and Hoseney (1988) showed that starch accounted
for about 8% of the impurities in the glutens from differ-
ent flours.
As listed in Table 2, the starch recoveries from the
waxy wheat flours (72–78%) were statistically similar
to the recoveries (76–80%) from the control flours, ex-
cept for the reduced recovery (68%) from Leona.
However, some of the waxy starches were contaminated
with more proteins (0.4–0.5%) than the starches from
the controls (<0.3%). Granule size distributions were
 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178 175

12 12
Karl 92 (nonwaxy) Ike (partialwaxy)
10 10

8 8

Volume (%)

Volume (%)
6 6

4 4

2 2
0 0
0 1 3 5 10 20 40 80 0 1 3 5 10 20 40 80
Granule Size (µm) Granule Size (µm)

12 12 12
Wx-A Wx-B Wx-C
10 10 10

8
Volume (%)

8 8

Volume (%)
Volume (%)
6 6 6

4 4 4

2 2 2

0 0 0
0 1 3 5 10 20 40 80 0 1 3 5 10 20 40 80 0 1 3 5 10 20 40 80
Granule Size (µm) Granule Size (µm) Granule Size (µm)

Fig. 2. Typical bimodal size distributions of starch granules isolated from control wheats (top row) and several selected waxy wheats (bottom row).

determined for selected waxy starches (Fig. 2); as ex- during high-shear dispersion step. During the centrifu-
pected they showed a typical bimodal size distribution gation, the dispersed gluten is thought to form a fine
with 25–30% by weight of the granules below 10 lm. mesh network which allows the passage of the rapidly
In other words, granule size distributions of the waxy sedimenting large starch granules. After centrifugation,
and control starches isolated by the two methods of the viscous supernatant containing the soluble pento-
wet-milling were comparable, which is consistent with sans and the sedimented starch layer (50% of total
the results of Yoo and Jane (2002) and Abdel-Aal et starch) was easily removed from the middle protein-rich
al. (2002). The soluble flour solids varied from 5.8% to layer. The middle layer in the centrifuged mixture was
9.9% in the waxy flours (Table 2), which is within the sufficiently cohesive even at the beginning of kneading
range of 5–15% water-solubles found in wheat flours so that the remaining starch was easily removed by
(Hamer et al., 1989; Maijala, 1976; Witt, 1997). About kneading and washing under water.
one-fifth of the flour weight for all the control and waxy Leona waxy flour gave a similar wet-milling trend by
wheat flours was unaccounted for in the gluten, starch, both the DW and DD&C methods; a lower quantity of
and water solubles fractions (Table 2). The unaccounted dry gluten, more protein in starch, and elevated water-
solids for Leona flour were even higher (29%). solubles (Tables 2 and 3) probably because of its low
Those unaccounted solids were mostly in the tailings re- protein quantity and quality. However, when wet-milled
moved during centrifugal purification of the starch by the DD&C method, Leona waxy wheat gave even
fractions. lower dry gluten and protein recovery. The DD&C
method, as compared to the DW method, employed
3.4. Wet-milling properties of flours by dough-dispersion more shear during the wet-processing, which was likely
and centrifugation (DD&C) method to damage already weak gluten aggregates in Leona
sample. In contrast to the DW method, the separation
Because of the difficulties encountered during the of starch and gluten fractions from all other waxy wheat
wet-milling of the waxy wheat flours by the DW method flour doughs by the DD&C method was accomplished
as discussed above, which requires a strongly agglomer- with similar ease to that of the controls. In other words,
ated gluten in dough for efficient separation of the starch waxy flour doughs that failed for wet-milling in the ori-
and gluten fractions, the method described by Czucha- ginal DW method could be wet-processed by the DD&C
jowska and Pomeranz (1993, 1995) was tested. In that method.
method, a stiff dough was mixed to optimum and rested The waxy wheat flours wet-milled by the DD&C
under 1 parts of water for 30 min. Then the dough was method gave similar or elevated yields of gluten with re-
sheared with additional water in a blender to form a dis- duced purities as compared to the control flours (Table
persion. The dispersion is centrifuged to give mainly 3). Thus, both wet-milling methods gave reduced protein
three layers; a top supernatant, a middle protein-rich levels in the gluten fractions from the waxy wheats.
layer, and a bottom layer of practically pure starch. It However, the protein contamination in the waxy starch
appears that the gluten proteins were agglomerated, fractions was almost cut in half to <0.3% by the DD&C
mostly during dough mixing, and the protein strands method of wet-milling (Table 3) compared to the DW
in the agglomerates were dislodged from starch granules method (Table 2).
 176 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178

Unaccounteda 4. Conclusions

Waxy wheats gave reduced flour yields, and the flours

solids (%)

contained less starch but more crude fat and pentosans.

18.0
19.0

20.8
18.4
19.3
14.5
19.3
18.5
29.1
6.8
The gluten proteins of waxy wheats were weaker than
those of the nonwaxy and partial waxy control wheats
as indicated by their poor mixograms, decreased IPP
solids (%)

contents, and lower SDS sedimentation volumes. It is

Solublea

more likely the genetic backgrounds of the waxy wheats,

10.1

11.0
11.2
6.3
5.7

8.4
7.7
9.7
9.0

2.3
not the waxy trait per se, are the causes of their weak
glutens. Some of the waxy flour doughs failed in the
wet-milling by the DW method as the DW method relies
recovery (%)

heavily on the gluten aggregation traits of flours. It is

Starch

therefore recommended that wet-milling of the waxy

80.1
77.1

75.1
76.3
74.3
80.1
79.6
73.0
72.4
NSc wheat flours with weak gluten be done by an aqueous
dispersion process that is less dependent on gluten
strength and less affected from the deleterious interfer-
(N · 5.7) (%)

ence of pentosans on gluten formation and aggregation.

a
Protein

In this regard, the DD&C aqueous dispersion method of

0.26
0.23

0.23
0.15
0.19
0.26
0.16
0.22
0.37
0.10

wet-milling used in this study facilitated the wet-milling

Starch fraction

of the waxy flour doughs and gave 80% recovery of

flour protein in the gluten fraction with 80% protein,
Wet-milling data for wheat flours by dough-dispersion and centrifugation (DD&C) method on 100 g (14% mb) of flour

yield (%)
a

and 75% waxy starch recovery with <0.3% protein.

Starch

63.4
61.5

55.3
57.0
56.9
57.7
59.8
54.5
56.0
6.8
recovery (%)

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11.6

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