wet milling properties of waxy wheat flours by two laboratory methods
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Journal of Food Engineering 72 (2006) 167–178
Wet-milling properties of waxy wheat flours by twolaboratory methods q
Abdulvahit Sayaslan a,*, Paul A. Seib b, Okkyung K. Chung c
a Department of Food Engineering, Gaziosmanpas�a University, 60240 Tokat, Turkeyb 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 DWmethod 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
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-
0260-8774/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2004.11.033
q Mention of firm names or trade products does not imply that they
are endorsed or recommended by the USDA, Kansas State University,
or Gaziosmanpas�a 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).
duction. Common or bread wheats (Triticum aestivum)
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 asthey 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
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 (stronggluten 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 hydrolysisproducts, 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 withflour 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 Martinand 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. Theadvantages 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 ofchromosomes, 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 forcold-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 wheatstarch 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) 167–178 169
(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 (NationalResearch 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 waxywheat 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 BaiHuo 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 theirtest weights measured with a test weight apparatus from
Burrows Equipment Co., Evanston, IL. Single kernel
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) weremeasured 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 InternationalIreland 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 NorthAmerica, 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 minon 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 56–70with 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 DD-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 DD-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-mlglass 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 % DD-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) wasadded 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 glutenhand-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-lmopening 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 waterfree of starch granules. The gluten fraction was placed in
a container, covered, and stored at room temperature
for 1h. 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 reducedpressure 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-lmopening sieve, and the mass remaining over the 75-lmopening 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 layerwas 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, primestarch, tailings, and water-solubles by essentially a
dough-dispersion and centrifugation (DD&C) method
A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178 171
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. Afterresting 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 smallgranular 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 Tukey�s 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 redwinter 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 wheatsamples were sound with no sprout damage; the a-amy-lase activities ranged from 0.07 to 0.20 Ceralpha units/g
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) eventhough 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-amylaseactivity 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) exceptfor 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) andthe 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, softwheats 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 theirelevated crude fat and pentosan contents (Table 1).
Fats provide cohesiveness to flours and reduce their
Table1
Compositionandproperties
ofstraight-gradeflours
roller-m
illedfrom
controlandwaxywheatsamples
Floursample
Kernel
hardness
index
(HI)
Flour
yield
(%)
Falling
number
(s)
Proteina
(N·5.7)(%
)
Ash
a
(%)
Crudea
fiber
(%)
Crudea
fat(%
)
Starcha
(%)
Starcha
damage
(%)
Amylose
(%)
IPP
(%)
SDS
(ml)
Gluten
index
(GI)
Pentosansa
(%DD-xylose)
Color
(L*)
Total
Soluble
Controlwhea
ts
Karl92(nonwaxy)
71
71.9
492
12.1
0.49
0.21
0.8
68.0
6.2
27.6
42.4
3.7
99
1.52
0.57
91.8
Ike(partialwaxy)
75
70.8
437
12.9
0.44
0.19
0.6
68.5
4.8
21.3
37.7
3.5
98
1.36
0.47
90.9
Waxy
whea
ts
Wx-A
77
66.1
ndb
12.0
0.46
0.25
0.9
63.3
5.8
1.2
38.3
2.8
nmc
2.30
0.47
91.3
Wx-B
68
67.1
nd
12.0
0.48
0.26
0.9
64.2
5.1
1.4
29.7
1.8
nm
2.08
0.60
91.4
Wx-C
31
66.8
nd
13.1
0.41
0.24
1.0
65.9
3.7
0.9
39.4
2.7
nm
1.97
0.60
93.6
Wx-1
nrd
nr
nd
14.2
0.48
0.30
1.2
61.9
4.5
1.6
24.7
1.3
nm
1.98
0.54
90.1
Wx-2
22
60.4
nd
10.9
0.37
0.20
1.0
64.6
3.2
0.9
28.8
2.2
nm
1.74
0.56
93.5
Wx-3
44
69.2
75
13.6
0.36
0.20
0.9
64.2
6.0
0.6
37.1
3.2
nm
1.74
0.60
91.4
Leona
48
67.2
nd
9.4
0.56
0.20
1.1
66.5
4.6
1.4
24.6
1.5
nm
1.86
0.44
93.0
a14%
flourmoisture
basis(flourmoisture
levelsranged
from
11.5to
13.5%).
bNotdetermined
aswaxywheatflours
giveunusuallylowfallingnumbersindependentoftheir
a-amylase
activities.
cNotmeasureddueto
blindingofGlutomaticwashingchamber
sieve(88-lm
opening)duringglutenwashing.
dNotreported
since
itwasreceived
asflour.
172 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178
flowability (Neel & Hoseney, 1984). Yasui et al. (1999)
reported reduced (20%) flour yields from two waxy
wheats produced from Kanto 107 by mutagenesis. They
attributed the decrease in waxy flour yield to a decline in
their flowability that might be caused by their elevated
fat (�20%) and b-glucan (�30) levels. Kim et al.(2003) also found that waxy wheats yielded less flour
than partial waxy and nonwaxy wheats. However, the
elevated crude fat contents determined for waxy wheat
flours might be considered an artifact of the lipid extrac-
tion. With essentially no amylose present, there is noth-
ing to bind flour lipids, hence rendering the lipids more
accessible to the solvent and inflating the values.
The protein contents of the waxy flours ranged from9.4% to 14.2% compared to 12.1% to 12.9% for the con-
trols (Table 1). The lightness values (L*) of the flours
varied from 90.1 to 93.6. As compared to the control
flours, the waxy flours contained 20–40% more fat,
10–20% more total pentosans, and 10–30% more crude
fiber. Also, waxy wheat flours had �3–7% lower total
starch than the controls, perhaps because the flux of car-
bohydrates into starch was diminished due to the ab-sence of amylose biosynthesis. Amylose levels of the
waxy samples were higher than the expected levels, rang-
ing from 0.6% to 2.3%, whereas the controls gave the ex-
pected values (Table 1). Other investigators (Abdel-Aal
et al., 2002; Chakraborty et al., 2004; Kim et al., 2003)
also reported varying amylose levels (0.9–3.2%) for
waxy wheats. This discrepancy could be attributed to
the variations in samples and methods of analysis usedin different studies.
3.3. Wet-milling properties of flours by dough-washing
(DW) method
All wheat samples were separately roller-milled to
straight-grade flours, physically and/or chemically char-
acterized, and wet-milled to give wet gluten, primestarch, tailings, and water solubles fractions. The two
control flours and only two of the waxy wheat (Wx-1
and Wx-3) flours could be wet-milled by the original
DW method (Table 2). The doughs from those flours
maintained their cohesiveness and elasticity during
kneading and washing under water, thus enabling wash-
ing of the starch from the gluten fraction. The remaining
five waxy wheat flour doughs lost much of their cohesiveand elastic properties immediately upon starting to
knead under the stream of water, despite the fact that
cohesive stiff doughs were initially formed from all the
flours, including the waxy flour doughs whose gluten
agglomerates were broken apart into small pieces and
became sticky during washing. In order to wash the
starch away and isolate the gluten, those doughs were
wet-milled by hand-rubbing them on the 125-lm open-ing screen (modified DW method, MDW). Gentle rub-
bing and washing of the small dough pieces and gluten
Table2
Wet-m
illingdata
forwheatflours
bydough-washing(D
W)ormodified
dough-washing(M
DW)methodon100g(14%
mb)offlour
Floursample
Flour
Wetgluten
Dry
glutenfraction
Starchfraction
Solublea
solids(%
)
Unaccounteda
solids(%
)Proteinb
(N·5.7)
(%)
Yieldb(%
)Glutena
yield
(%)
Proteina
(N·5.7)(%
)
Protein
recovery(%
)
Starcha
yield
(%)
Proteina
(N·5.7)(%
)
Starch
recovery(%
)
Controlwhea
ts
Karl92(nonwaxy)(byDW)
12.1
30.4
11.9
92.0
77.9
63.2
0.24
79.9
3.8
21.1
Ike(partialwaxy)(byDW)
12.9
37.9
14.2
90.9
86.2
60.6
0.21
76.0
3.7
21.5
Waxy
whea
ts
Wx-A
(byMDW)
12.0
36.8
13.4
73.8
71.1
54.3
0.40
73.8
5.8
26.5
Wx-B
(byMDW)
12.0
48.9
15.9
70.3
80.0
55.1
0.49
73.9
6.2
22.8
Wx-C
(byMDW)
13.1
44.2
16.5
73.0
79.1
56.3
0.38
73.5
6.3
21.0
Wx-1
(byDW)
14.2
54.9
17.7
73.1
78.3
56.3
0.32
78.1
9.6
16.4
Wx-2
(byMDW)
10.9
33.0
12.3
82.2
79.9
58.7
0.20
78.2
8.3
20.7
Wx-3
(byDW)
13.6
45.7
16.5
81.6
85.0
53.9
0.20
72.1
9.5
20.2
Leona(byMDW)
9.4
25.9
9.0
77.6
63.6
52.4
0.35
67.8
9.9
28.8
Tukey�sHSD
(a=0.05)
–4.9
1.5
4.4
10.3
6.5
0.09
7.4
1.4
7.0
aDry
basis.
b14%
flourmoisture
basis.
A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178 173
agglomerates on the sieve retained gluten agglomerates
on the sieve while allowing the starch granules and the
soluble solids to pass through the screen. Because of
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 glutenfraction. Interestingly, the wet gluten fractions retained
on the screen recovered their elasticity and cohesiveness
during washing at a point where about two-thirds or
more of the starch were washed away from the gluten
fractions. This may be explained by the increase in the
concentration of the gluten proteins in the gluten frac-
tions and/or removal of an unknown component from
the dough mass, which enabled the gluten proteins tointeract with each other, agglomerate, and provide cohe-
siveness. Furthermore, when the starch milk passing
through the screen from the waxy flour dough washings
were centrifuged to recover the starch fraction, a thick
intermediate fraction (tailings) was found between the
supernatant and the bottom prime starch layer. The
intermediate layer contained gluten protein particles
mixed with mostly small starch granules and insolublepentosans. 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
layer of tailings (darkened zone) atop the starch.
Other investigators (Hamer, Weegels, Marseille, &
Kelfkens, 1989; Meuser, 1994; Meuser et al., 1989; Wee-
gels, Marseille, & Hamer, 1988; Zwitserloot, 1989a)
have reported differences in the rate and extent of glutenagglomeration during wet-processing of wheat flours.
The reasons for the reduced rate and strength of gluten
agglomeration in flour-water doughs and slurries have
not been fully understood. High molecular weight glute-
nins and glutenin macropolymer formed by the glutenin
particles have been shown in the last 10 years to add
strength (elasticity) to a wheat flour dough (Antes &
Wieser, 2001; Bekkers, Lichtendonk, Graveland, & Plij-ter, 2000; Don, Lichtendonk, Plijter, & Hamer, 2003a,
2003b; Schropp & Weiser, 1996; Singh & MacRitchie,
2001; Southan & MacRitchie, 1999; Uthayakumaran,
Stoddard, Gras, & Bekes, 2000; Veraverbeke, Verbrug-
gen, & Delcour, 1998; Wooding, Kavale, MacRitchie,
& Stoddard, 1999). However, the wet-processing quality
of a wheat flour and its relationship to flour protein
quality remain mostly unknown.Gluten proteins in the waxy wheat flours appeared to
be weaker than those of the controls as indicated by
their lower SDS sedimentation volumes and IPP con-
tents (Table 1) and shorter mixograph mixing times
and lower mixing tolerances (Fig. 1). The IPP contents
of flours were reported to correlate strongly with dough
mixing time and mixing tolerance (Bean et al., 1998).
Also, elevated insoluble pentosans of the waxy flours(Table 1) may be the reason for the loss of their
cohesiveness and elasticity during kneading. Pentosans
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).
174 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178
(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 ongluten 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 suchmethods 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 Martinprocess 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 theirflours. 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 duringwashing 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 contaminatedwith more proteins (0.4–0.5%) than the starches from
the controls (<0.3%). Granule size distributions were
Wx-A
0
2
4
6
8
10
12
804020105310
Vol
ume
(%)
Wx-B
0
2
4
6
8
10
12
804020105310
Vol
ume
(%)
Wx-C
0
2
4
6
8
10
12
804020105310
Vol
ume
(%)
Karl 92 (nonwaxy)
0
2
4
6
8
10
12
804020105310
Granule Size (µm) Granule Size (µm)
Granule Size (µm)Granule Size (µm) Granule Size (µm)
Ike (partialwaxy)
0
2
4
6
8
10
12
804020105310
Vol
ume
(%)
Vol
ume
(%)
Fig. 2. Typical bimodal size distributions of starch granules isolated from control wheats (top row) and several selected waxy wheats (bottom row).
A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178 175
determined for selected waxy starches (Fig. 2); as ex-
pected they showed a typical bimodal size distribution
with �25–30% by weight of the granules below 10 lm.In other words, granule size distributions of the waxy
and control starches isolated by the two methods of
wet-milling were comparable, which is consistent with
the results of Yoo and Jane (2002) and Abdel-Aal etal. (2002). The soluble flour solids varied from 5.8% to
9.9% in the waxy flours (Table 2), which is within the
range of 5–15% water-solubles found in wheat flours
(Hamer et al., 1989; Maijala, 1976; Witt, 1997). About
one-fifth of the flour weight for all the control and waxy
wheat flours was unaccounted for in the gluten, starch,
and water solubles fractions (Table 2). The unaccounted
solids for Leona flour were even higher (�29%).Those unaccounted solids were mostly in the tailings re-
moved during centrifugal purification of the starch
fractions.
3.4. Wet-milling properties of flours by dough-dispersion
and centrifugation (DD&C) method
Because of the difficulties encountered during thewet-milling of the waxy wheat flours by the DW method
as discussed above, which requires a strongly agglomer-
ated gluten in dough for efficient separation of the starch
and gluten fractions, the method described by Czucha-
jowska and Pomeranz (1993, 1995) was tested. In that
method, a stiff dough was mixed to optimum and rested
under �1 parts of water for 30 min. Then the dough wassheared with additional water in a blender to form a dis-persion. The dispersion is centrifuged to give mainly
three layers; a top supernatant, a middle protein-rich
layer, and a bottom layer of practically pure starch. It
appears that the gluten proteins were agglomerated,
mostly during dough mixing, and the protein strands
in the agglomerates were dislodged from starch granules
during high-shear dispersion step. During the centrifu-
gation, the dispersed gluten is thought to form a fine
mesh network which allows the passage of the rapidly
sedimenting large starch granules. After centrifugation,
the viscous supernatant containing the soluble pento-
sans and the sedimented starch layer (�50% of total
starch) was easily removed from the middle protein-richlayer. The middle layer in the centrifuged mixture was
sufficiently cohesive even at the beginning of kneading
so that the remaining starch was easily removed by
kneading and washing under water.
Leona waxy flour gave a similar wet-milling trend by
both the DW and DD&C methods; a lower quantity of
dry gluten, more protein in starch, and elevated water-
solubles (Tables 2 and 3) probably because of its lowprotein quantity and quality. However, when wet-milled
by the DD&C method, Leona waxy wheat gave even
lower dry gluten and protein recovery. The DD&C
method, as compared to the DW method, employed
more shear during the wet-processing, which was likely
to damage already weak gluten aggregates in Leona
sample. In contrast to the DW method, the separation
of starch and gluten fractions from all other waxy wheatflour doughs by the DD&C method was accomplished
with similar ease to that of the controls. In other words,
waxy flour doughs that failed for wet-milling in the ori-
ginal DW method could be wet-processed by the DD&C
method.
The waxy wheat flours wet-milled by the DD&C
method gave similar or elevated yields of gluten with re-
duced purities as compared to the control flours (Table3). Thus, both wet-milling methods gave reduced protein
levels in the gluten fractions from the waxy wheats.
However, the protein contamination in the waxy starch
fractions was almost cut in half to <0.3% by the DD&C
method of wet-milling (Table 3) compared to the DW
method (Table 2).
Table3
Wet-m
illingdata
forwheatfloursbydough-dispersionandcentrifugation(D
D&C)methodon100g(14%
mb)offlour
Floursample
Flour
Wetgluten
Dry
glutenfraction
Starchfraction
Solublea
solids(%
)
Unaccounteda
solids(%
)Proteinb
(N·5.7)
Yieldb(%
)Glutena
yield
(%)
Proteina
(N·5.7)(%
)
Protein
recovery(%
)
Starcha
yield
(%)
Proteina
(N·5.7)(%
)
Starch
recovery(%
)
Controlwhea
ts
Karl92(nonwaxy)
12.1
30.9
12.3
88.7
77.9
63.4
0.26
80.1
6.3
18.0
Ike(partialwaxy)
12.9
37.6
13.9
89.0
82.8
61.5
0.23
77.1
5.7
19.0
Waxy
whea
ts
Wx-A
12.0
39.3
13.9
80.5
79.9
55.3
0.23
75.1
10.1
20.8
Wx-B
12.0
43.4
16.2
72.2
84.0
57.0
0.15
76.3
8.4
18.4
Wx-C
13.1
43.6
16.1
80.0
84.4
56.9
0.19
74.3
7.7
19.3
Wx-1
14.2
52.9
18.2
80.4
88.6
57.7
0.26
80.1
9.7
14.5
Wx-2
10.9
30.3
11.9
83.8
78.5
59.8
0.16
79.6
9.0
19.3
Wx-3
13.6
41.6
16.0
85.7
86.9
54.5
0.22
73.0
11.0
18.5
Leona
9.4
11.0
3.7
79.6
27.1
56.0
0.37
72.4
11.2
29.1
Tukey�sHSD
(a=0.05)
–7.8
2.3
4.5
11.6
6.8
0.10
NSc
2.3
6.8
aDry
basis.
b14%
flourmoisture
basis.
cNosignificantdifference
ata=0.05(P
>0.05).
176 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178
4. Conclusions
Waxy wheats gave reduced flour yields, and the flours
contained less starch but more crude fat and pentosans.
The gluten proteins of waxy wheats were weaker than
those of the nonwaxy and partial waxy control wheatsas indicated by their poor mixograms, decreased IPP
contents, and lower SDS sedimentation volumes. It is
more likely the genetic backgrounds of the waxy wheats,
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
heavily on the gluten aggregation traits of flours. It is
therefore recommended that wet-milling of the waxywheat 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-
ence of pentosans on gluten formation and aggregation.
In this regard, the DD&C aqueous dispersion method of
wet-milling used in this study facilitated the wet-milling
of the waxy flour doughs and gave �80% recovery of
flour protein in the gluten fraction with �80% protein,and �75% waxy starch recovery with <0.3% protein.
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