Gluten: properties and nonfood potential.
Gluten, the storage protein of wheat endosperm, was
reviewed at the annual meeting of the American Association of
Cereal Chemistry. Many studies have shown that gluten consists
mainly of two protein subclasses, gliadin and glutenin. Each
of these is a complex mixture of several protein types with unusual
structures. These proteins interact, in the kernel and upon subsequent
processing, through disulfide and noncovalent bonds to form complex
gluten polymers. Upon rehydration and mixing, gluten orients
and further associates into a unique viscoelastic network. These
properties explain gluten's critical importance in breadmaking:
the protein network expands to retain gas generated during fermentation
and stabilizes upon heating into a light, porous structure. A
major industry exists to isolate functional gluten. `Vital'
gluten is used primarily in foods, but has many other possible
industrial uses because of its elastic, viscous, adhesive, film-forming,
and thermosetting properties. Native or modified gluten can be
used in films, plastics, adhesives, graft polymers, and many other
products. However, most nonfood applications seem little used,
mostly because of competition from petrochemical-based alternatives.
Today, interest in such new uses is growing again, for many reasons.
Gluten is abundant, renewable, domestically available, easily
isolated, low in cost, `natural', and biodegradable.
Further research on gluten's applications and more fundamental
information about its properties are necessary to enhance gluten's
industrial utilization, benefiting everyone from producer to consumer.
Capillary electrophoresis: a state-of-the-art technique for wheat protein characterization.
Capillary electrophoresis (CE), a modern instrumental
method of protein analysis, now gives excellent separations of
wheat gluten proteins. Methods were developed first to analyze
gliadin. Variables examined include protein extractant; buffer
type, source, and pH; buffer additives (SDS, acetonitrile, reducing
agents, polymeric matrices); capillary length and diameter; voltage;
temperature; and injection mode. The best separations were at
22 kV with 0.1M phosphate buffer, pH 2.5, containing a linear
hydrophilic polymer, using a `20 cm x 20 µm'
i.d. uncoated silica capillary. Resolution in 10 min is as good
as or better than that of reversed-phase high-performance liquid
chromatography (RP-HPLC). However, the major advantage of CE
is that it complements RP-HPLC, as shown by analyzing isolated
RP-HPLC peaks. CE also is the first automated and accurately
quantifiable electrophoresis method. Excellent inter-laboratory
reproducibility can be achieved, but buffer composition is critical.
CE can readily differentiate wheat varieties, including sister
lines, and should be useful for selection during breeding and
in genetic studies. Modified ProSort(c) technology (Werner et
al. Cereal Chem 1994 91:397) also was used to reveal variation
among Glu-D1 high molecular weight glutenin subunits in
several cultivars. Observed heterogeneity paralleled that shown
by SDS-PAGE, but some differences in relative mobility of protein
bands were observed. These and other separation modes will make
CE an indispensable analytical tool for wheat protein analysis.
Two of the most important factors in optimizing reproducibility
and resolution of cereal proteins in uncoated capillaries are
rinsing (cleaning) protocols and buffer makeup. Various capillary
cleaning procedures were studied to improve migration time reproducibility:
water only; sodium hydroxide (0.1 M) only; phosphoric acid (1
M) only; and a combination of phosphoric acid, sodium hydroxide,
and water. The optimum reproducibility was obtained with a rinsing
protocol of 4 min with 1 M phosphoric acid. Resolution was improved
by the addition of certain organic solvents to the buffer. Various
concentrations of acetonitrile, dimethylformamide, dimethylsulfoxide,
ethylene glycol, methanol, and 2-propanol were studied: 20 %
acetonitrile produced the highest resolution (best baseline resolved
peaks). Separations on a `27 cm x 20 µm' (i.d.)
capillary were completed in 18 min with relative standard deviations
of 0.1 % on 15 peaks.
A fast method for wheat cultivar differentiation using capillary zone electrophoresis (CZE).
CZE conditions for the shortest analysis time include:
capillary inside diameter, 20 µm; shortest possible capillary
length, 27 cm (20 cm to detector); temperature, 45_C; voltage,
22 kV; and pressure injection for 4 sec (0.25 nL). Three alcohol-water-based
solvent systems were studied to improve extraction and analysis
of gliadins; 30 % ethanol-water was optimum. Gliadins were
extracted from cultivars representative of hard red winter, hard
red spring, and soft wheat classes and separated by CZE. Three
separate sets of cultivars that were not distinguished by PAGE
at pH 3.1 were differentiated in less than 10 min each by CZE.
Cultivars that were related closely (sister lines or intercrossings)
were differentiated readily, and cultivars that were not genetically
close exhibited quite different CZE patterns.
Wheat protein fractions, separated by the Osborne
Solvent Fractionation Procedure, were characterized by HPCE. Each
fraction was separated on a 27-cm fused-glass capillary (20 µm,
i.d.) using 0.1 M phosphate buffer (pH 2.5) containing hydroxypropylmethylcellulose,
a polymer additive, at 45_C and 22 kV constant voltage. Albumins
and globulins migrated in the first 4 min, whereas gliadins and
glutenins migrated after 4 min. Individual alpha, beta, gamma,
and omega gliadin proteins, which were collected from RP-HPLC
separations, also were separated by HPCE. Combined results of
this study and our previous studies provide a catalog of individual
gliadin information from HPCE, HPLC, acid (A)-PAGE, and SDS-PAGE,
relating class, relative molecular size, hydrophobicity, relative
charge, and separation times of each gliadin subclass by HPLC
and HPCE. The main advantages of HPCE are: 1) the complementing
of other electrophoretic and chromatographic protein separation
methods and 2) safety, because no toxic acrylamides and only minute
amounts of organic solvents and buffers are used.
Differentiation of 1AL-1RS from 1BL-1RS wheat-rye translocation lines by capillary electrophoresis.
Wheat-rye translocations, 1BL-1RS and
1AL-1RS, have been used to transfer desirable genes from
rye to wheat. Both beneficial and deleterious influences on wheat
end-use quality have been identified. The most commonly cited
difficulties encountered with 1BL-1RS are reduced gluten
strength (diminished mixing tolerance) and dough `stickiness'.
CE was used to differentiate 1AL-1RS from 1BL-1RS wheat
lines in less than 15 min by analyzing 30 % ethanol or water extracts.
Samples analyzed included both near-isogenic lines and a large
number of advanced breeding lines from USDA-ARS regional
testing nurseries, with and without the rye chromosome segment.
Differences in the omega secalins were found. The CE pattern
of the 1AL-1RS lines exhibited a doublet with equal peak
heights, whereas the pattern of the 1BL-1RS lines had a doublet
consisting of a larger peak followed by a one-half size peak,
both around 13 min. However, the 1AL-1RS doublet migrated
slightly slower than the 1BL-1RS doublet. Sample size ranged
from bulk flour to one-half of single wheat kernel.
A cooperative study on starch lipids (SL) was conducted
between Kansas State University (KSU) and the ARS. A commercial
wheat starch (Type I, unmodified, Sigma Chem. Co.) was purified
to remove starch surface lipids by stirring for 4 hr at room temperature
with a mixture of 1-propanol and water (3:1 v/v). SL were extracted
by the SFE method using an ISCO two pump modifier SFE system:
the optimum SFE method developed for SL was to use a carbon dioxide
SFE at 9,950 psi at 100 C with 30 % 1-propanol-water (3:1, v/v)
as a modifier and to decompress just before the dynamic stage.
SL were analyzed by a HPLC method, which was developed by us
for quantifying lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines
(LPE) in SL. The optimum HPLC method was to use a silica column
(200 x 4.6 mm, si 100 with a 10 µm bead size), and a gradient
elution with Phase A (hexane-2-propanol-water, 48:50:2 v/v/v)
and Phase B (35:50:15), starting with 100 % A, going to 25 % B
in 7 min, then to 100 % B in 2 min, and holding at 100 % B for
6 min. We reduced SL extraction time from 12 to 2 hr by using
an optimum SFE method and HPLC analysis time from 80 to 15 min.
Chromatographic separation of wheat flour glycolipids.
Free lipids from wheat flour, extracted with petroleum
ether on a Soxhlet, were fractionated into nonpolar, glyco-, and
phospho-lipids by open column silicic acid chromatography. Nonpolar
lipids were eluted first by a mixture of chloroform and acetone
(4:1 v/v), instead of 100 % chloroform with which the elution
of all nonpolar lipid components, especially free fatty acid,
was often incomplete. Two main glycolipids, monogalactosyldiglycerides
(MGDG) and digalactosyldiglycerides (DGDG), were separated by
normal-phase HPLC using a silica column (250 x 4 mm with 5 µm
particle size) within 7 min. We could decrease substantially
the retention time and flow rate by using a mini column (10 x
4 mm with 5 µm particle size). The separation of MGDG and
DGDG was attained in 3 min at a flow rate of 2.6 ml/min and in
5 min at 1.5 ml/min. Because the intensity of absorbance at a
low range of UV spectra was affected by the degree of unsaturation
in fatty acids, quantifying lipids containing different unsaturations
by a UV detector is difficult. The eluted lipids were detected
by an evaporative light scattering detector (ELSD) and UV multidiode
array detector (UV-MDAD). The chromatograms obtained by ELSD
showed no fluctuation on baseline whereas those obtained by UV-MDAD
did show baseline fluctuation.
End-use quality evaluation of wheat: past, present, and future.
(Presented at the ICC Meeting at Vienna, Austria.)
Differences between U.S. wheat classes are generally use-oriented,
and different wheat classes grow in different regions. By Congressional
mandate, four ARS Regional Wheat Quality Laboratories (RWQL) were
established for improvement of U.S. wheat quality based on their
end uses by evaluating breeding lines. In 1936, the Soft Wheat
Quality Laboratory was established at Wooster, OH; in 1937, the
Hard Winter Wheat Quality Laboratory at Manhattan, KS; in 1946,
the Western Wheat Quality Laboratory at Pullman, WA; and in 1963,
the Hard Red Spring and Durum Wheat Quality Laboratory at Fargo,
ND. All four RWQL evaluate physical and chemical characteristics
of grains; experimental milling; flour quality; dough properties;
and baking or other end-product, processing, quality parameters
for cookies, cakes, breads, spaghetti with durum wheat semolina,
and/or noodles, etc. Based on quality data accumulated by the
four RWQL during the past 30-60 years, quality-prediction
methods for functional properties have been developed and used
to predict certain end-use quality parameters by each lab.
All four labs replaced the Kjeldahl Method with the Leco N2 Analyzer;
use the USGMRL-developed Single-Kernel Wheat Characterization
System for grain characteristics; and use the NIRSystem 6500 for
chemical parameters of both wheats and flours and for a complete
spectral scanning of whole wheats, meals, and flours over 400-2,500
nm. Knowing that biochemical components are responsible for certain
functional properties of end products and experimental processing
data, we are working toward developing a quality prediction system.
The potential of using end-use quality to estimate whole wheat
kernels and as a basis of the wheat marketing system is approaching.
Status of the USGMRL single-kernel wheat characterization system (SKWCS).
(Reviewed at the ICC Meeting at Vienna, Austria.)
The need for and development of an objective wheat classification
system to be used by the USDA, Federal Grain Inspection Service
(FGIS) are reviewed with a historical perspective. This presentation
includes a brief description of three single kernel hardness determination
methods tested by the FGIS before selecting the USGMRL-SKWCS.
The methods described are: grinding as developed by the ARS,
Beltsville Instrumentation Laboratory; cutting as developed by
Kansas State University; and crushing as developed by the U.S.
Grain Marketing Research Laboratory (USGMRL). The presentation
primarily describes the following: procedures for single-kernel
physical measurements of weight, size moisture and hardness; computer-controlled
operation of the system; procedures used to establish calibrations
as developed by the USGMRL; classification results obtained by
FGIS after sampling the U.S. common market wheats; system performance
as determined by the USGMRL; recent steps toward commercialization
and evaluation of the system; and several on-going studies to
relate single-kernel properties of wheat to milling performance.
Varietal and environmental effects on wheat and flour quality parameters.
Wheat and flour quality parameters were determined
using 54 hard winter wheat samples from the Wheat Quality Council,
consisting of nine wheat varieties grown at six locations including
Akron, CO; Colby and Hutchinson, KS; Clay Center, NE; Lahoma,
OK; and Vernon, TX. Nine lines were grown and harvested in 1994
by each of five Agricultural Experiment Station sites. Two-way
analysis of variance showed significant effects of both varieties
(V) and growing locations (environment, E) on most quality parameters,
except for no significant V effects on mixograph and bake water
absorptions (WA), dough weights, and dough proof heights and no
significant E effects on mixograph mix time (MT). Three broad
groups of quality parameters were identified: (A) mixograph and
bake MT and mixograph mixing tolerances affected more by V; (B)
wheat physical parameters, ash and protein contents, flour yields,
and mixograph and bake WA affected more by E; and (C) NIR-hardness,
% large kernels, flour color values, and bread volumes and crumb
grains affected similarly by both E & V.