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Journal of Food Engineering 72 (2006) 167–178

Wet-milling properties of waxy wheat flours by two

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

Department of Food Engineering, Gaziosmanpasßa University, 60240 Tokat, Turkey
Department of Grain Science and Industry, Kansas State University, Manhattan, 66506 KS, USA
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


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)

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

0260-8774/$ - see front matter  2004 Elsevier Ltd. All rights reserved.
168 A. Sayaslan et al. / Journal of Food Engineering 72 (2006) 167–178

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

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
(% D -xylose)



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
index (GI)

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 from
9.4% 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-

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



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-




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
fat (%)

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

fiber (%)

the variations in samples and methods of analysis used


in different studies.


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




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


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



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

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




Not reported since it was received as flour.

maintained their cohesiveness and elasticity during

yield (%)

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 cohesive

and elastic properties immediately upon starting to
index (HI)

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
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



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



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


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


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
recovery (%)

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 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


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



intermediate fraction (tailings) was found between the

Starch fraction

supernatant and the bottom prime starch layer. The

intermediate layer contained gluten protein particles
yield (%)

mixed with mostly small starch granules and insoluble



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.


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 gluten
(N · 5.7) (%)

agglomeration during wet-processing of wheat flours.


The reasons for the reduced rate and strength of gluten

Dry gluten fraction



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


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



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,



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

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

of a wheat flour and its relationship to flour protein




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


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

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.


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


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
recovery (%)

heavily on the gluten aggregation traits of flours. It is


therefore recommended that wet-milling of the waxy


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.


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



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 (%)

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



recovery (%)


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