Content uploaded by David W Killilea
Author content
All content in this area was uploaded by David W Killilea on Feb 04, 2020
Content may be subject to copyright.
Health, Nutrition, & Food
Wheat germ agglutinin is a biomarker of whole
grain content in wheat flour and pasta
David W. Killilea , Rebecca McQueen, and Judi R. Abegania
Abstract: When consumed as whole grain, wheat has a high nutrient density that contributes to a healthy diet. Yet,
products labeled as whole wheat can still contain a substantial amount of refined grain leading to the confusion for
consumers, so a method was designed to determine the whole grain status within wheat-based foods. Wheat germ
agglutinin (WGA), a lectin found in the germ tissue of wheat kernels, was evaluated as a biomarker of whole grain wheat.
WGA content strongly correlated with the percentage of whole wheat within premade mixtures of whole and refined
(white) flours. Then, commercial flours labeled as whole wheat were tested for WGA content and found to contain up
to 40% less WGA compared to a whole grain standard. Commercial pasta products labeled as whole wheat were also
tested for WGA content and found to contain up to 90% less WGA compared to a whole grain standard. The differences
in WGA content were not likely due to varietal differences alone, as the WGA content in common varieties used in
domestic wheat flour production varied less than 25%. The levels of other constituents in wheat kernels, including starch,
mineral, phytate, and total protein, were not different among the commercial whole wheat flours and pasta products.
WGA is a unique biomarker that can identify wheat products with the highest whole grain content.
Keywords: biomarker, nutrition, wheat germ, whole grain, whole wheat
Practical Abstract: Whole grain wheat has a high nutrient density that can be part of a healthy diet, but products
labeled as whole wheat can still contain some refined grain. Wheat germ agglutinin (WGA) was tested as a biomarker
to measure whole grain status in wheat-based foods and revealed that some commercial whole wheat flour and pasta
contained unexpectedly lower levels of the WGA biomarker compared to a whole grain standard. WGA may therefore
be a useful way to test for whole grain wheat content.
1. INTRODUCTION
Whole grain wheat contains numerous essential micronutri-
ents and beneficial phytochemicals, particularly within the bran
and germ tissues of the wheat kernel (Jonnalagadda et al., 2010;
Okarter & Liu, 2010). Many epidemiological, cross-sectional, and
prospective studies have shown that higher intake of whole grain
wheat is associated with reduced risk of cardiovascular disease,
stroke, hypertension, hyperlipidemia, diabetes, obesity, and some
types of cancers (Aune et al., 2016; Bj ¨
orck et al., 2012, Cho, Qi,
Fahey, & Klurfeld, 2013; Jacobs, Andersen, & Blomhoff, 2007;
Jonnalagadda et al., 2010; Korczak et al., 2016; Kyrø, Tjønneland,
Overvad, Olsen, & Landberg, 2018; Li et al., 2016; Liu et al.,
1999; Mozaffarian, Appel, & Van Horn, 2011; Slavin, Jacobs,
& Marquart, 1997; Slavin, Tucker, Harriman, & Jonnalagadda,
2013; Wei et al., 2016; Wu et al., 2015). Yet, the majority of
wheat-based products consumed in the United States are made
from flour that has been “refined,” in which much of the bran
and germ have been separated and removed, leaving mostly the
white endosperm fraction (Albertson, Reicks, Joshi, & Gugger,
2016; Cleveland, Moshfegh, Albertson, & Goldman, 2000). This
process also removes the majority of the micronutrients and phy-
tochemicals within the wheat kernel, so consumption of refined
JFDS-2019-0946 Submitted 6/17/2019, Accepted 12/16/2019. Authors are with
Nutrition & Metabolism Center, Children’s Hospital Oakland Research Inst., 5700
Martin Luther King, Jr. Way, Oakland, CA 94609, U.S.A. Direct inquiries to
author Killilea (E-mail: address: dkillilea@chori.org).
Financial support: Community Grains, Oakland, CA, USA.
flour is not associated with the same health benefits as whole wheat
(Serra-Majem & Bautista-Casta ˜
no, 2015).
The demonstrated health benefits of whole wheat have
prompted recommendations for increased consumption, but it is
not always clear how much whole wheat is present in commercial
wheat products due to the lack of a standardized definition and
metrics for whole wheat content (American Association of Cereal
Chemists International, 2019; Korczak et al., 2016; Mozaffarian
et al., 2013; Ross et al., 2017). The U.S. Dept. of Agriculture
(USDA) and Federal Drug Administration (FDA) have adopted
the American Association of Cereal Chemists Intl. (AACCI) def-
inition of whole grain as the “intact, ground, cracked, or flaked
caryopsis of the grain whose principal components, the starchy
endosperm, germ, and bran, are present in the same relative pro-
portions as they exist in the intact grain” (American Association
of Cereal Chemists International, 1999; U.S. Food and Drug Ad-
ministration, 2006). However, this definition serves as an industry
guidance and does not confer regulatory authority (Whole Grains
Council, 2019a). For wheat-based foods, no definition has been
widely accepted. In the United States, manufacturers of wheat-
based foods can apply for a Whole Grain Health Claim from the
FDA if the product contains 51% or more whole grain content
by weight per reference amount customarily consumed (Korczak
et al., 2016; U.S. Food and Drug Administration, 2019). In fact,
products labeled as whole wheat may actually contain a substan-
tial fraction of refined wheat, which is not often reported on
food packaging. Certification programs can help consumers in
identifying whole grain products (Whole Grains Council, 2019b),
but many commercial products are not registered or based on
C2020 Institute of Food Technologists R
doi: 10.1111/1750-3841.15040 Vol. 0, Iss. 0, 2020 rJournal of Food Science 1
Further reproduction without permission is prohibited
Health, Nutrition, & Food
WGA is a biomarker of whole grain . . .
manufacturer-provided information. Dietary fiber has previously
been used as a biomarker for whole grains (Mozaffarian et al.,
2013), but the reliability of this measure has been questioned,
especially in products with added fiber (Curtain & Grafenauer,
2019). In fact, there are few options available for independent
evaluation of whole grain content in wheat flour and wheat-based
foods.
We attempted to assess the level of whole grains in wheat prod-
ucts by measuring specific constituents that correlate with dif-
ferent wheat tissues (Barron, Samson, Lullien-Pellerin, Abecassis,
& Rouau, 2011; Hemery et al., 2009; Hemery, Rouau, Lullien-
Pellerin, Barron, & Abecassis, 2007). We first investigated wheat
germ agglutinin (WGA), a small lectin protein expressed predomi-
nantly in germ tissue that functions as part of the immune system of
the wheat plant (Cammue, Raikhel, & Peumans, 1988; Mishkind,
Keegstra, & Palevitz, 1980; Raikhel, Mishkind, & Palevitz, 1984;
Smith & Raikhel, 1989). Barron and colleagues previously showed
that WGA levels varied with the proportions of germ from milling
streams, and proposed that WGA could be useful as a biomarker
(Barron et al., 2011; Hemery et al., 2009). Therefore, we tested
whether WGA could be an effective indicator of whole grain
content within commercial wheat flour and wheat-based pasta.
WGA levels were strongly correlated with whole grain content
in premade mixtures of whole wheat and refined (white) flours.
Then, commercial whole wheat flour and pasta were tested for
WGA content and compared to a whole grain standard. WGA
content could be useful for evaluating the whole grain content in
commercial wheat products.
2. MATERIALS AND METHODS
2.1 Materials
A convenience sampling of commercial wheat flours and pastas
was purchased between 2017 and 2018 from local grocery stores as
described in Table S1. Common wheat varieties used in domestic
flour production were chosen by reviewing the USDA Natl. Agri-
cultural Statistics Service state wheat production data (U.S. Food
and Drug Administration, 2018) and sourced from seed or compa-
nies as described in Table S2. Purified WGA, rabbit anti-Triticum
vulgaris WGA antibody, Immobilon-PSQ polyvinylidene fluoride
membrane, Luminata Crescendo Substrate, and OmniTrace ni-
tric acid, and other chemicals were obtained from MilliporeSigma
(St. Louis, MO, USA), unless otherwise specified. The mouse
anti-rabbit IgG-horseradish peroxidase (HRP) antibody was ob-
tained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
PAGEr 10% to 20% gradient Tris-glycine polyacrylamide gels were
obtained from Lonza (Morristown, NJ, USA). Precision Plus Pro-
tein Dual Color Standards were obtained from Bio-Rad (Hercules,
CA, USA). The Pierce Silver Stain Kit and Pierce BCA Protein
Assay Kit were obtained from Thermo Fisher Scientific (Waltham,
MA, USA). Seronorm Trace Element Serum Levels 1 and 2 were
obtained from Sero AS (Billingstad, Norway). The MegaZyme
Total Phytic Acid (Phytate)/Total Phosphorous and Starch HK
Assay Kits were obtained from Megazyme Intl. Ireland Limited
(Wicklow, Ireland).
2.2 Whole grain standards
For wheat tissue standards for electrophoresis, hand-dissected
wheat tissue fractions (bran, endosperm, and germ) from intact
wheat kernels were kindly provided by Dr. Steve Jones (The Bread
Lab, Washington State University, WA, USA). Whole grain wheat
flour and wheat pasta standards were kindly provided by Com-
munity Grains (Oakland, CA, USA). For the whole wheat flour
standard, identity-preserved and traceable hard red winter wheat
(variety WB9229) from Fritz Durst Farming (Capay, CA, USA)
was milled and processed into flour. For the whole wheat pasta
standard, dry pasta product was made from identity-preserved and
traceable hard amber durum wheat (variety Durum Iraq) flour
from Full Belly Farms (Guinda, CA, USA) after milling and
processing. The milling was conducted completely at Bay State
Milling (Woodland, CA, USA) using an air-classifier mill without
sifting or separation, resulting in flour extraction of 100% whole
grain. The wheat kernels and final flours were stored in isolation
from all other grains. The flour was then analyzed and certified
as whole grain by external laboratory analysis (California Wheat
Commission, Woodland, CA, USA). The certificates of identity
and external laboratory analysis for both standards are provided as
Supplementary Material.
2.3 Flour and pasta preparation
Flour mixtures of varying whole wheat content were created by
mixing commercial whole grain hard red winter wheat (variety
WB9229) flour and all-purpose white flour to achieve desired ra-
tios. Flour from different varieties was created by in-house milling
of intact wheat kernels using a Mockmill Grain Mill Attachment
(Otzberg, Germany) for the KitchenAid mixer for 1 to 2 min on
extra fine setting according to manufacturer’s instructions. The
flour from each variety had a similar particle size. The mill was
cleaned and flushed between samples. Wheat flours from different
commercial sources were used directly from the original bag. Pasta
from different commercial sources was milled into flour using the
same procedure as the intact wheat kernels. The technical staff
performing the extractions and measurements was blinded to the
identity of the flour and pasta during the course of the analyses.
2.4 Protein extraction and quantification
Total protein was extracted from the commercial wheat flour
and pasta samples using 0.5 g portions submerged in 10 mL of
0.01N HCl for 1 hr at room temperature with constant stirring at
200 to 300 rpm. For isolated wheat tissue fractions with limited
mass, the sample amount was reduced to 0.05 g portions and
1 mL 0.01N HCl. For flour milled in-house, the sample size was
increased to 2.5 g portions and 50 mL of 0.01N HCl in order to
avoid bias from any heterogeneity in particle sizes. Protein content
in flour extracts was then quantified using the Pierce Bicinchoninic
Acid (BCA) Protein Assay modifications for a microplate format
according to manufacturer’s instructions. Bovine serum albumin
was used as the protein standard. Wheat proteins were separated by
SDS-PAGE on 10% to 20% gradient Tris-glycine polyacrylamide
gels followed by visualization with Pierce Silver Stain Kit according
to manufacturer’s instructions.
2.5 Phytate content analysis
The phytate content of wheat flour was determined by the
MegaZyme Total Phytic Acid (Phytate)/Total Phosphorous Kit ac-
cording to manufacturer’s instructions (McKie & McCleary, 2016).
Briefly, 1 g samples of flour were extracted in 20 mL of 0.66N
HCl for 12 to 16 hr at room temperature with constant stirring
at 200 to 300 rpm. The phytate content is then quantified using
a two-step enzyme assay with spectrophotometry (absorption at
655 nm) according to manufacturer’s instructions. Average intraas-
say precision was 6.0% and interassay precision was 10.9%; sample
values were compared to certified values for phytate in whole
2Journal of Food Science rVol. 0, Iss. 0, 2020
Health, Nutrition, & Food
WGA is a biomarker of whole grain . . .
wheat (0.10 g/100 g flour) and oat positive control (1.77 g/100 g
flour).
2.6 Starch content analysis
The starch content of flour was determined by the MegaZyme
Total Starch HK Assay Kit based on a modified American As-
sociation of Cereal Chemists method 76-13 (McCleary, Gibson,
& Mugford, 1997). Approximately 20 mg samples of wheat flour
were treated for starch extraction and glucose quantification by
spectrophotometry (absorption at 340 nm) according to manu-
facturer’s instructions. Average intraassay precision was 4.1% and
interassay precision was 15.8%; sample values were compared to
certified values for starch in whole wheat (69.6 g/100 g flour) and
maize starch positive control (86.7 g/100 g flour).
2.7 Mineral content analysis
The mineral content of the flours was determined by inductively
coupled plasma spectrometry (ICP-OES) as previously described
(Engle-Stone et al., 2017). Approximately 20 mg samples of wheat
flour were dissolved in 0.25 mL OmniTrace 70% HNO3and later
diluted to 5% HNO3. Mineral concentrations were then analyzed
with an Agilent 5100 SVDV ICP-OES calibrated with Natl. Inst.
of Standards and Technology–traceable elemental standards and
routinely validated with Seronorm Trace Element Serum Levels
1 and 2. The minerals calcium, potassium, magnesium, manganese,
phosphorous, sulfur, and zinc were added together as a combined
mineral content. Iron was not included because refined flour is
usually fortified with a substantial amount of iron; no other min-
erals are added in fortification of U.S. wheat flours. Average in-
traassay precision was 9.7% and interassay precision was 10.7% for
combined mineral content.
2.8 WGA content analysis
WGA content in protein samples was measured using im-
munoblot analysis. Protein samples were separated by conven-
tional SDS-based polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to Immobilon-PSQ membrane using a Bio-Rad
Trans-Blot SD unit. The membranes were hydrated in 1X TBST
(0.02M Tris base, 0.15M NaCl, 0.05% Tween 20, and pH 7.4) and
exposed to blocking solution (5% powdered milk in 1x TBST) for
12 to 16 hr at 5 °C. Then, the membranes were sequentially
incubated with rabbit anti-WGA antibody (1:1,000 in blocking
solution) for 3 hr at room temp followed by mouse anti-rabbit
IgG antibody labeled with HRP (1:1,000 in blocking solution)
for 1 hr at room temp. Immunolabeled protein was visualized by
chemiluminescence using Luminata Crescendo substrate accord-
ing to manufacturer’s instructions. Positive antibody signals were
quantified using Image J software. For each independent exper-
iment, distinct samples of wheat were extracted, protein content
was quantified, and immunoblot analysis was performed.
2.9 Statistics
Graphing, regression, and statistical analysis were conducted by
using Prism software, version 6 (GraphPad Software, Inc., San
Diego, CA, USA). For all tests, significance was defined as P<
0.05.
3. RESULTS
3.1 Quantification of WGA content in wheat tissues
Protein isolated from purified WGA or isolated wheat frac-
tions were separated by SDS-PAGE and quantified by immunoblot
(Figure 1A). Analysis of purified WGA protein demonstrated a
dominant band for WGA at approximately 18 kD, consistent with
the size for the monomeric WGA protein (Smith & Raikhel,
1989). At higher total protein levels, a band at approximately 36 kD
was visible, consistent with a dimeric form of WGA protein. Yet,
in the complete protein fraction from isolated whole fractions,
WGA protein could not be easily visualized by just SDS-PAGE
due to more abundant proteins that comigrated with WGA. Us-
ing an immunoblot, the antibody signal confirmed the dominant
WGA protein species at 18 kD with an apparent limit of detection
approaching 0.001 µg. Weak bands higher than 18 kD were also
detected but did not interfere with analysis. The 18 kD band of
monomeric WGA was mainly detectable in the germ, illustrat-
ing the selectivity of this protein. Interestingly, a strong band was
visible at approximately 30 kD in the bran. Since there should
be little WGA in the bran (Mishkind et al., 1980), this is likely
a protein with similar epitopes to WGA, but the identity of this
protein is unknown. Only the canonical 18 kD monomeric band
was used when quantifying the level of WGA protein within the
samples, avoiding the extraneous protein bands. Using a standard
curve, the WGA content of the germ was found to contribute
93 ±5% of the total WGA found in the wheat tissue (Figure 1B).
In the wheat tissue standards, WGA was calculated at 5.8 ±0.1 µg
WGA/mg germ protein.
3.2 Quantification of WGA content from mixed wheat
flours
WGA content was then tested for correlation with whole
wheat content in premade mixtures made with whole wheat and
endosperm-rich flours. The mixtures were created at 0%, 25%,
40%, 50%, 55%, 75%, and 100% total whole wheat content. Total
protein content in flour extracts was inversely correlated to whole
wheat content, measured as 1.87 ±0.03 mg/L, 1.76 ±0.02 mg/L,
1.52 ±0.02 mg/L, 1.50 ±0.01 mg/L, 1.14 ±0.04 mg/L,
0.98 ±0.02 mg/L, 0.81 ±0.02 mg/L, and 0.77 ±0.01 mg/L,
respectively (n=3). Although total protein decreased with
increasing whole grain, the fractional portion of WGA increased
(Figure 1C). WGA content was found to directly correlate with
whole grain content with r2=0.92 from n=5 independent
experiments (Figure S1). These results support the use of WGA as
a biomarker for whole grain content in commercial wheat flours.
Other biochemical components within wheat kernels, includ-
ing starch, mineral, phytate, and total protein, were then tested
for correlation with whole wheat content (Figure S1). Starch is
mostly found in the endosperm tissue, so starch content was ex-
pected to decrease with higher whole grain content. In the test
flour mixtures, the starch content was inversely correlated to the
whole wheat content of the flour (r2=0.87, n=2 indepen-
dent experiments), with refined flour being approximately 10%
higher than whole grain flour. Minerals are highest in the bran
and germ tissues, so mineral content was expected to increase
with higher whole grain content. In the test flour mixtures, the
combined mineral content was directly correlated to the whole
wheat content of the flour (r2=0.99, n=3 independent exper-
iments), with refined flour being approximately 60% lower than
whole grain flour. Phytate is mostly found in the aleurone layer
just beneath the bran tissue, so starch content was expected to in-
crease with higher whole grain content. In the test flour mixtures,
the phytate content was directly correlated to the whole wheat
content of the flour (r2=0.94, n=2 independent experiments),
with refined flour being approximately 75% lower than whole
grain flour. Finally, protein is found throughout the kernel but is
Vol. 0, Iss. 0, 2020 rJournal of Food Science 3
Health, Nutrition, & Food
WGA is a biomarker of whole grain . . .
AB
C
Figure 1–Identification of WGA protein from wheat tissues. (A) Representative SDS-PAGE (top panel) and immunoblot (bottom panel) show WGA
standards (lanes 2 to 8) and isolated wheat kernel tissue fractions (lanes 9 to 11) probed by an anti-WGA antibody. The arrow indicates the major
WGA monomer band at 18 kD, as determined by molecular weight biomarkers (MWM). The content of the WGA standards is indicated. Five micrograms
of protein was loaded from isolated germ (GERM), endosperm (ENDO), and bran (BRAN) wheat tissues. (B) Quantification of the WGA protein content
indicated that 93% of the signal from the major WGA monomer band was found in the germ tissue. Mean ±SE are shown for three independent
experiments. Differences in WGA content between tissues were tested by one-way ANOVA followed by Tukey’s multiple-comparisons test. Asterisk
identifies the means significantly different from the germ tissue (P<0.05). (C) Quantification of the WGA protein content, normalized to 0–100% whole
wheat, indicated a positive correlation between WGA content and whole grain composition (r2=0.92). Mean ±SE are shown for five independent
experiments.
highest in the endosperm, so protein content was expected to de-
crease with higher whole grain content. In the test flour mixtures,
the total protein content was inversely correlated to the whole
wheat content of the flour (r2=0.91, n=3 independent exper-
iments), with refined flour being approximately 65% higher than
whole grain flour.
3.3 Quantification of WGA content from commercial
wheat flours
Whole wheat content in a group of commercial flours branded
as all-purpose white or whole wheat (Table S1) was then assessed
by measuring WGA content. The commercial flours were pur-
chased at a local grocery store, so it was not possible to control
for product characteristics, including storage history and time on
shelf. Therefore, the flours used in this analysis were not specif-
ically identified since they were a limited convenience sampling.
Protein was extracted from multiple samples of each commer-
cial flour and used to measure WGA content by immunoblot
(Figure 2A). WGA levels varied between the flour samples, with
the lowest from the all-purpose flour and the highest from the
whole wheat standard. When compared to the whole wheat flour
standard, some brands had only about 60% of the expected WGA
content (Figure 2B).
One potential reason for reduced WGA content in commercial
brands of whole wheat flour is if there are inherent differences
in WGA content between different wheat varieties. The variety
composition of commercial flour is not normally listed on the
packaging or online content for the products, and was not found
for most of the commercial flours tested. Therefore, a range of
wheat varieties were collected and analyzed for WGA content,
including hard red winter, hard red spring, and hard white winter
types common in domestic wheat-growing states Kansas, Mon-
tana, Oklahoma, and California (Table S2). Despite the diversity
of wheat types, WGA levels ranged from 2% to 24% of the WGA
level in the standard variety WB9229 (Figure S2). This variance in
WGA levels among the wheat varieties is substantially smaller than
differences in WGA content between flours, so variety differences
alone may not fully explain the variance in WGA levels among
the commercial flours.
The correlation of whole wheat content in the flour was then
tested with other biochemical components within wheat, in-
cluding starch, mineral, phytate, and total protein (Figure S3).
As expected, the levels of starch and total protein were higher
in the all-purpose flour relative to the whole wheat standard.
Also, the levels of mineral and phytate were lower in the all-
purpose flour relative to the whole wheat standard. Unlike the
4Journal of Food Science rVol. 0, Iss. 0, 2020
Health, Nutrition, & Food
WGA is a biomarker of whole grain . . .
A
C
B
D
Figure 2–Quantification of WGA protein in commercial wheat flour and pasta products. (A) Representative immunoblot shows WGA standards (lanes
2 to 5) and commercial flours (lanes 6 to 10) as refined (REFINED), brands A to C (A, B, and C), and whole grain whole wheat standard (STANDARD)
flour. The arrow indicates the major WGA monomer band at 18 kD, as determined by molecular weight biomarkers (MWM). (B) Quantification of
the WGA protein content showed substantial variance in difference in refined (REFINED) compared to whole wheat flours. Mean ±SE are shown for
four independent experiments. Differences in WGA content were tested by one-way ANOVA followed by Tukey’s multiple-comparisons test. Asterisks
identify the means significantly different from the whole wheat standard (P<0.05). (C) Representative immunoblot shows WGA standards (lanes 2
to 4) and commercial pasta (lanes 5 to 10) as brands A to D (A, B, C, and D), and whole grain whole wheat standard (STANDARD) pasta. The arrow
indicates the major WGA monomer band at 18 kD, as determined by MWM. (D) Quantification of the WGA protein content showed substantial variance
in whole wheat pasta. Mean ±SE are shown for three independent experiments. Differences in WGA content were tested by one-way ANOVA followed
by Tukey’s multiple-comparisons test. Asterisks identify the means significantly different from the whole wheat standard (P<0.05).
WGA content, the levels of starch, mineral, phytate, and to-
tal protein from the other commercial flours labeled as whole
wheat were not statistically different from the whole wheat
standard.
3.4 Quantification of WGA content from commercial
wheat pasta
Whole wheat content in a group of commercial pasta branded
as whole wheat (Table S1) was then assessed by measuring WGA
content. The commercial pastas were purchased at a local grocery
store, so it was not possible to control for product characteris-
tics, including storage history and time on shelf. Therefore, the
pastas used in this analysis were not specifically identified since
they were a limited convenience sampling. The pastas were milled
into powder and then protein was extracted from multiple sam-
ples of each commercial pasta for measurement of WGA con-
tent by immunoblot (Figure 2C). WGA levels varied between
the pasta samples, with the highest measured in the whole wheat
pasta standard. When compared to the whole wheat pasta stan-
dard, some brands had only 10% of the expected WGA content
(Figure 2D). Unlike WGA content, the levels of mineral and total
protein from the other commercial pasta labeled as whole wheat
were not statistically different from the whole wheat standard
(Figure S4).
4. DISCUSSION
We developed a method to assess the amount of whole grain
within wheat flour and pasta using the endogenous lectin WGA.
WGA content was found to strongly correlate with the percentage
of whole wheat in premade mixtures of whole and white flours.
Then, when applied to commercial flour and pasta branded as
whole wheat, WGA content was found to be unexpectedly low in
Vol. 0, Iss. 0, 2020 rJournal of Food Science 5
Health, Nutrition, & Food
WGA is a biomarker of whole grain . . .
some products compared to whole grain standards. WGA content
could discriminate between different flour and pasta products,
while the other endogenous indicators, such as starch, mineral,
phytate, and total protein, could not. Thus, WGA appears to be
useful for detecting differences in whole wheat content within
commercial wheat products.
Of the biomarkers tested, only WGA is found predominantly
in the germ tissue, while mineral, phytate, and protein are found
in multiple tissues within the wheat kernel (Barron et al., 2011;
Mishkind et al., 1980; Mishkind, Raikhel, Palevitz, & Keegstra,
1982). This specific localization of WGA to germ may account
for the effectiveness as an indicator of whole wheat. At first, bran-
specific biomarkers might seem like good candidates for biomark-
ers of whole wheat, but bran can be added to refined flour to
improve the fiber content or perception of the product as whole
wheat. This exogenous bran will also increase the mineral, phy-
tate, and protein levels in the flour, thereby reducing the utility
of those constituents as biomarkers of whole grain wheat. In con-
trast, germ contains oils and other lipids that are prone to oxidation
and rancidity (Galliard, 1986), making the addition of exogenous
germ unlikely due to negative effects on product shelf life. Thus,
germ-specific biomarkers like WGA may best reflect the pres-
ence of whole grain independent from downstream processing
and manufacturing modifications.
Previous studies have investigated the use of WGA in test-
ing wheat. Early studies measured the agglutinination response of
WGA, but this activity is diminished by high temperature and
so not suitable for heat-processed products like pasta (Matucci
et al., 2004). Other studies measured the amount of WGA in
wheat fractions using ELISA or dot blot methods that rely on
antibody selectively to identify WGA (Hemery et al., 2009; Vin-
cenzi et al., 2002). However, our immunoblot results demonstrated
that a commercial antibody marketed only as anti-WGA actually
identified multiple protein species in addition to the canonical
monomeric WGA, including an abundant protein in the bran
fraction that is unlikely to be WGA (Barron et al., 2011; Mishkind
et al., 1982). Our observations are consistent with earlier reports
that showed multiple proteins were identified by anti-WGA an-
tibodies in wheat germ lysates (Mansfield, Pewmans, & Raikhel,
1998; Raikhel & Wilkins, 1987; Smith & Raikhel, 1989). These
findings raise concern that ELISA and dot blot methods might be
prone to false-positive results, so antibody specificity should first
be tested in these types of studies.
Once our method was validated against flour of known whole
wheat content, we analyzed the WGA content in several com-
mercial wheat flours and pasta products branded as whole wheat
and found that some had unexpectedly low WGA content com-
pared to a whole grain wheat standard. The flours tested were
common commercial brands, which listed only wheat flour in the
ingredients. All flours seemed to be of a similar particle size and
were well mixed before analysis. For most pasta products, only
whole wheat flour and water were listed in the ingredients, but
were analyzed in the dry form, so hydration amount was not a
factor. One brand of pasta had an added ingredient inulin, al-
though inulin is a small polysaccharide that should not affect the
amount of detectable WGA. Pasta was ground up to a similar par-
ticle size before being analyzed. There were no apparent physical
differences in the wheat products that might bias the compar-
isons. Yet, when these products were tested for WGA compared
to a whole wheat standard, some flour products were up to 40%
lower and some pasta products were up to 90% lower in WGA
content.
One hypothesis to explain lower WGA levels in some wheat
products might be that the varieties of wheat in the flour and pasta
had less endogenous WGA than the variety used in the whole
wheat standard. Yet, manufacturers rarely list the varieties of wheat
used in production on the product packaging or online informa-
tion (other than broad classification, such as “hard red winter”),
so it was not possible to test WGA levels in the specific varieties
used in these products. Instead, several wheat varieties commonly
used for flour production in the United States were selected for
analysis for WGA content. The levels of WGA found among the
varieties were relatively similar, varying at most by 24%. This find-
ing was different from a previous study in which several European
wheat varieties were found to have a wide range of WGA content
(Barron et al., 2011). However, these varieties tested are not as
common in the United States and the WGA content was mea-
sured by ELISA with no antibody selectivity reported, making it
difficult to compare to our study. Although we cannot be certain
how much varietal differences contribute to WGA content in this
study, the variation in WGA from common domestic wheat va-
rieties was significantly less than observed in different commercial
wheat flours.
Another hypothesis to explain lower WGA content in some
wheat products could be effects of different environmental or
processing conditions on the wheat. For environmental effects,
studies in isolated wheat embryos or cell cultures have shown
that WGA expression is influenced by stress conditions, including
dehydration, oxidative stress, and osmotic imbalances, though the
magnitude of the response diminishes with later developmental
stages of the wheat (Bhaglal, Singh, Bhullar, & Kumar, 1998;
Morris, Maddock, Jones, & Bowles, 1985; Raikhel, Bednarek, &
Wilkins, 1988; Singh, Bhaglal, & Bhullar, 1996; Singh, Bhaglal,
& Bhullar, 2000; Triplett & Quatrano, 1982). More importantly,
it is not known if these changes are relevant in vivo, since few
studies have extended these investigations to wheat in field settings
(Bhaglal et al., 1998). For processing effects, no studies were found
that investigated relevant conditions on WGA expression, so it
was not possible to estimate the impact of processing variables on
WGA content. Moreover, manufacturers rarely provide details on
how the constituent wheat was grown or treated on the product
packaging or online information, so environmental and processing
variables could not even be defined for the commercial products
analyzed in this study. Since the few studies on environmental
conditions showed only minor effects on WGA levels in mature
wheat, we presume this is likely similar for processing conditions
too.
If varieties, environment, and processing do not fully explain the
differences in WGA content within commercial wheat flour and
pasta, then the best remaining explanation is that some of the prod-
ucts tested were not completely whole grain wheat as suggested
by branding. Industry observers have previously raised concerns
regarding the true levels of whole grain in wheat products (Center
for Science in the Public Interest, 2018) and several groups have
advocated for greater clarity in definition and regulation of guide-
lines for whole grain foods (Korczak et al., 2016; Ross et al., 2017;
Whole Grains Council, 2019a). Current governmental guidelines
on whole wheat are limited and do not specify how much of the
wheat tissues must be present to retain whole grain status (Whole
Grains Council, 2019a). The major guideline focuses on the use
of the FDA whole grain health claim, in which a product contains
51% or more whole grain ingredient(s) by weight per reference
amount customarily consumed (U.S. Food and Drug Administra-
tion, 2019). However, the exact whole wheat content is not often
6Journal of Food Science rVol. 0, Iss. 0, 2020
Health, Nutrition, & Food
WGA is a biomarker of whole grain . . .
indicated on the product packaging or available online, so con-
sumers may struggle to identify the products with highest whole
grain content.
Our findings suggest that WGA content can be a useful
biomarker for the determination of whole grain levels within
wheat flour and wheat-based pasta. Biochemical metrics like
WGA are important for the independent evaluation of whole
grain content within existing wheat products. Measurement of
WGA could ultimately become part of a panel of biomarkers to
assess and validate the whole grain status in certain foods. It should
be emphasized, however, that this analysis was conducted with a
convenience sample of commercial flour and pasta, so it was not
possible to control for all potential differences in processing, han-
dling, and storage conditions that might have affected the products
and influenced the levels of WGA. Future work should utilize
defined, identity-preserved wheat flours carried through the pro-
duction of pasta and other wheat products to reduce process and
matrix differences.
Whole grain wheat has an abundance of vitamins, minerals,
fiber, and other phytochemicals, many of which are known to be
shortfall nutrients within the U.S. diet (Papanikolaou & Fulgoni,
2017). The nutrient content is diminished, however, when whole
wheat flour is even partially substituted with refined flour. Having a
clear indication of the actual level of whole grain within commer-
cial wheat-based foods would provide consumers the transparency
to make the best choices for their health.
ACKNOWLEDGMENTS
The authors thank Dr. Steve Jones at The Bread Lab (Washing-
ton State University, WA, USA) for wheat tissue standards. The
authors thank Tai Holland, Kathy Schultz, June Jiao, Jacqueline
Chang, Hailey Zhou, Sophie Egan, and Teal Dudziak for technical
assistance.
AUTHOR CONTRIBUTIONS
D.W.K. designed the research project. R.M. and J.R.A. con-
ducted the research. D.W.K. analyzed the data and performed
statistical analysis. D.W.K. wrote the paper and took primary re-
sponsibility for final content. All authors read and approved the
final manuscript.
CONFLICTS OF INTEREST
J.R.A. reported no conflicts of interest related to the study.
R.M. was an employee at Community Grains during the time of
the research project. D.W.K. served on a scientific advisory board
at Community Grains during the time of the research project, but
did not receive any salary or honorarium. The funders had no role
in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
ABBREVIATIONS
AACCI American Association of Cereal Chemists Intl.
BCA bicinchoninic acid
EDTA ethylenediaminetetraacetic acid
ELISA enzyme-linked immunosorbent assay
FDA Federal Drug Administration
HRP horseradish peroxidase
PAGE polyacrylamide gel electrophoresis
SDS sodium dodecyl sulfate
WGA wheat germ agglutinin
REFERENCES
Albertson, A. M., Reicks, M., Joshi, N., & Gugger, C. K. (2016). Whole grain consumption
trends and associations with body weight measures in the United States: Results from the cross
sectional National Health and Nutrition Examination Survey 2001–2012. Nutrition Journal,
15,8.
American Association of Cereal Chemists International. (1999). Whole grain defini-
tion. Cereal Foods World. Retrieved from http://www.aaccnet.org/initiatives/definitions/
Pages/WholeGrain.aspx
American Association of Cereal Chemists International (AACCI). (2019). Whole grain defini-
tion. Cereal Foods World. Retrieved from https://www.aaccnet.org/initiatives/definitions/
Pages/WholeGrain.aspx
Aune, D., Keum, N., Giovannucci, E., Fadnes, L. T., Boffetta, P., Greenwood, D. C., . . . Norat,
T. (2016). Whole grain consumption and risk of cardiovascular disease, cancer, and all cause
and cause specific mortality: Systematic review and dose–response meta-analysis of prospective
studies. Bmj (Clinical Research Ed.),353, i2716.
Barron, C., Samson, M. F., Lullien-Pellerin, X., Abecassis, V., & Rouau, X. (2011). Wheat grain
tissue proportions in milling fractions using biochemical biomarker measurements: Application
to different wheat cultivars. Journal of Cereal Science,53, 306–311.
Bhaglal, P., Singh, P., Bhullar, S. S., & Kumar, S. (1998). Drought stress induced accumulation
of wheat germ agglutinin in the developing embryos of wheat (Triticum aestivum L.) and its
likely role in vivo.Journal of Plant Physiology,153, 163–166.
Bj¨
orck, I., Ostman, E., Kristensen, M., Anson, N. M., Price, R. K., Haenen, G. R. M. M., . ..
Riccardi, G. (2012). Cereal grains for nutrition and health benefits: Overview of results from
in vitro, animal and human studies in the HEALTHGRAIN project. Trends in Food Science &
Te c h n o l o g y ,25, 87–100.
Cammue, B. P. A., Raikhel, N. V., & Peumans, W. J. (1988). Occurrence and synthesis of
isolectins in different tissues of wheat. Biochem Physiol Pflanzen,183, 379–387.
Center for Science in the Public Interest. (2018). Whole grain. Retrieved from https://cspinet.
org/topics/whole-grain
Cho, S. S., Qi, L., Fahey, G. C. J., & Klurfeld, D. M. (2013). Consumption of cereal fiber,
mixtures of whole grains and bran, and whole grains and risk reduction in type 2 diabetes,
obesity, and cardiovascular disease. American Journal of Clinical Nutrition,98, 594–619.
Cleveland, L. E., Moshfegh, A. J., Albertson, A. M., & Goldman, J. D. (2000). Dietary intake of
whole grains. Journal of the American College of Nutrition,19, S331–S338.
Curtain, F., & Grafenauer, S. (2019). Health Star Rating in grain foods—Does it adequately
differentiate refined and whole grain foods? Nutrients,11, 415–427.
Engle-Stone, R., Nankap, M., Ndjebayi, A. O., Allen, L. H., Shahab-Ferdows, S., Hampel,
D., .... Brown,K.H.(2017). Iron, zinc, folate, and vitamin B-12 status increased among
women and children in Yaound´
e and Douala, Cameroon, 1 year after introducing fortified
wheat flour. Journal of Nutrition,147, 1426–1436.
Galliard, T. (1986). Hydrolytic and oxidative degradation of lipids during storage of wholemeal
flour: Effects of bran and germ components. Journal of Cereal Science,4, 179–192.
Hemery, Y., Rouau, X., Lullien-Pellerin, V., Barron, C., & Abecassis, J. (2007). Dry processes
to develop wheat fractions and products with enhanced nutritional quality. Journal of Cereal
Science,46, 327–347.
Hemery, Y., Lullien-Pellerin, V., Rouau, X., Abecassis, J., Samson, M.-F., Aman, P., . . . Spo-
erndlic, C. (2009). Biochemical biomarkers: Efficient tools for the assessment of wheat grain
tissue proportions in milling fractions. Journal of Cereal Science,49, 55–64.
Jacobs, D. R., Jr., Andersen, L. F., & Blomhoff, R. (2007). Whole-grain consumption is asso-
ciated with a reduced risk of noncardiovascular, noncancer death attributed to inflammatory
diseases in the Iowa Women’s Health Study. American Journal of Clinical Nutrition,85, 1606–
1614.
Jonnalagadda, S. S., Harnack, L., Liu, R. H., McKeown, N., Seal, C., Liu, S., & Fahey, G.
C. (2010). Putting the whole grain puzzle together: Health benefits associated with whole
grains—Summary of American Society for Nutrition 2010 Satellite Symposium. Jour nal of
Nutrition,141, 1011S–1022S.
Korczak, R., Marquart, L., Slavin, J. L., Ringling, K., Chu, Y., O’Shea, M., . .. Unnevehr, L.
(2016). Thinking critically about whole-grain definitions: Summary report of an interdisci-
plinary roundtable discussion at the 2015 Whole Grains Summit. American Journal of Clinical
Nutrition,104, 1508–1514.
Kyrø, C., Tjønneland, A., Overvad, K., Olsen, A., & Landberg, R. (2018). Higher whole-
grain intake is associated with lower risk of type 2 diabetes among middle-aged men and
women: The Danish Diet, Cancer, and Health Cohort. Journal of Nutrition,148, 1434–
1444.
Li, B., Zhang, G., Tan, M., Zhao, L., Jin, L., Tang, X., . .. Zhong, K. (2016). Consumption
of whole grains in relation to mortality from all causes, cardiovascular disease, and diabetes:
Dose–response meta-analysis of prospective cohort studies. Medicine,95, e4229.
Liu, S., Stampfer, M. J., Hu, F. B., Giovannucci, E., Rimm, E., Manson, J. E., . . . Willett, W.
C. (1999). Whole-grain consumption and risk of coronary heart disease: Results from the
Nurses’ Health Study. American Journal of Clinical Nutrition,70, 412–419.
Mansfield, M. A., Pewmans,W.J., & Raikhel, N. V. (1998). Wheat-germ agglutinin is synthesized
as a glycosylated precursor. Planta,173, 482–489.
Matucci, A., Veneri, G., Pellegrin, C. D., Zoccatelli, G., Vincenzi, S., Chignola, R., . . . Rizzi,
C. (2004). Temperature-dependentdecay of wheat ger m agglutinin activityand its implications
for food processing and analysis. Food Control,15, 391–395.
McCleary, B. V., Gibson, T. S., & Mugford, D. C. (1997). Measurement of total starch in
cereal products by amyloglucosidase-α-amylase method: Collaborative study. Journal of AOAC
International,80, 571–579.
McKie, V. A., & McCleary, B. V. (2016). A novel and rapid colorimetric method for measur ing
total phosphorus and phytic acid in foods and animal feeds. Journal of AOAC International,99,
738–741.
Mishkind, M., Keegstra, K., & Palevitz, B. A. (1980). Distribution of wheat germ agglutinin in
young wheat plants. Plant Physiology,66, 950–955.
Mishkind, M., Raikhel, N. V., Palevitz, B. A., & Keegstra, K. (1982). Immunocytochemical
localization of wheat germ agglutinin in wheat. Journal of Cell Biology,92, 753–764.
Morris, P. C., Maddock, S. E., Jones, M. G., & Bowles, D. J. (1985). Changes in the levels
of wheat- and barley-germ agglutinin during embryogenesis in vivo, in vitro and during
germination. Planta,166, 407–413.
Mozaffarian, D., Appel, L. J., & Van Horn, L. (2011). Components of a cardioprotective diet:
New insights. Circulation,123, 2870–2891.
Vol. 0, Iss. 0, 2020 rJournal of Food Science 7
Health, Nutrition, & Food
WGA is a biomarker of whole grain . . .
Mozaffarian, R. S., Lee, R. M., Kennedy, M. A., Ludwig, D. S., Mozaffarian, D., & Gortmaker,
S. L. (2013). Identifying whole grain foods: A comparison of different approaches for selecting
more healthful whole grain products. Public Health Nutrition,16, 2255–2264.
Okarter, N., & Liu, R. H. (2010). Health benefits of whole grain phytochemicals. Critical Reviews
in Food Science and Nutrition,50, 193–208.
Papanikolaou, Y., & Fulgoni, V. L. (2017). Grain foods are contributors of nutrient density
for American adults and help close nutrient recommendation gaps: Data from the National
Health and Nutrition Examination Survey, 2009–2012. Nutrients,9, 873–887.
Raikhel, N. V., Mishkind, M. L., & Palevitz, B. A. (1984). Characterization of a wheat germ
agglutinin-like lectin from adult wheat plants. Planta,162, 55–61.
Raikhel, N. V., & Wilkins, T. A. (1987). Isolation and characterization of a cDNA clone encoding
wheat germ agglutinin. Proceedings of the National Academy of Sciences of the Usa,84, 6745–6749.
Raikhel, N. V., Bednarek, S. Y., & Wilkins, T. A. (1988). Cell-type-specific expression of a
wheat-germ agglutinin gene in embryos and young seedlings of Triticum aestivum.Planta,176,
406–414.
Ross, A. B., van der Kamp King, J. W., Lˆ
e, R., Mejborn, K. A., Seal, H., & Thielecke, C.
J.; Healthgrain Forum. (2017). Perspective: A definition for whole-grain food products—
Recommendations from the Healthgrain Forum. Advances in nutrition,8, 525–531.
Serra-Majem, L., & Bautista-Casta ˜
no, I. (2015). Relationship between bread and obesity. British
Journal of Nutrition,113, S29–S35.
Singh, P., Bhaglal, P., & Bhullar, S. S. (1996). Differential levels of wheat germ agglutinin
(WGA) in germinating embryos of different wheat cultivars in response to osmotic stress.
Plant Physiology and Biochemistry,34, 547–552.
Singh, P., Bhaglal, P., & Bhullar, S. S. (2000). Wheat germ agglutinin (WGA) gene expression
and ABA accumulation in the developing embryos of wheat (Tr iticum aestivum) in response to
drought. Plant Growth Regulation,30, 145–150.
Slavin, J., Jacobs, D., & Marquar t, L. (1997). Whole-grain consumption and chronic disease:
Protective mechanisms. Nutrition and Cancer,27, 14–21.
Slavin, J., Tucker, M., Harriman, C., & Jonnalagadda, S. S. (2013). Whole grains: Definition,
dietary recommendations, and health benefits. Cereal Foods World,58, 191–198.
Smith, J. J., & Raikhel, N. V. (1989). Production of an antibody specific for the propeptide of
wheat germ agglutinin. Plant Physiology,91, 473–476.
Triplett, B. A., & Quatrano, R. S. (1982). Timing, localization, and control of wheat germ
agglutinin synthesis in developing wheat embryos. Developmental Biology,91, 491–496.
U.S. Food and Drug Administration. (2006). Guidance for industry and FDA staff whole grain
label statements. Retrieved from https://www.fda.gov/regulatory-information/search-fda-
guidance-documents/draft-guidance-industry-and-fda-staff-whole-grain-label-statements
U.S. Food and Drug Administration. (2018). National Agricultural Statistics Service. Retrieved from
https://www.nass.usda.gov/Statistics_by_State
U.S. Food and Drug Administration. (2019). Health claim notification for whole grain foods.Re-
trieved from https://www.fda.gov/food/food-labeling-nutrition/health-claim-notification-
whole-grain-foods
Vincenzi, S., Zoccatelli, G., Perbellini, F., Rizzi, C., Chignola, R., Curloni, A., & Peruffo,
A. D. B. (2002). Quantitative determination of dietary lectin activities by enzyme-linked
immunosorbent assay using specific glycoproteins immobilized on microtiter plates. Journal of
Agricultural and Food Chemistry,50, 6266–6270.
Wei, H., Gao, Z., Liang, R., Li, Z., Hao, H., & Liu, X. (2016). Whole-grain consumption and
the risk of all-cause, CVD and cancer mortality: A meta-analysis of prospective cohort studies.
British Journal of Nutrition,116, 514–525.
Whole Grains Council. (2019a). Government guidance. Retrieved from http://wholegrainscouncil.
org/resources/government-guidance
Whole Grains Council. (2019b). While grains stamp. Retrieved from http://wholegrainscouncil.
org/whole-grain-stamp
Wu,H., Flint, A. J., Qi, Q., van Dam, R. M., Sampson, L. A., Rimm, E. B., .. . Sun, Q. (2015).
Association between dietary whole grain intake and risk of mortality: Two large prospective
studies in US men and women. JAMA Internal Medicine,175, 373–384.
Supporting Information
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
Figure S1. Quantification of WGA protein and other biomarkers
in wheat flour from varying whole grain content.
Figure S2. Effect of wheat cultivar on WGA content in wheat
flour.
Figure S3. Quantification of mineral, total protein, starch, and
phytate in commercial wheat flour.
Figure S4. Quantification of mineral and total protein in com-
mercial pasta products.
Table S 1 . Information on commercial wheat flour and commer-
cial wheat pasta products used in these studies.
Table S 2 . Information on wheat varieties used in this study.
Material S1. Certificate of identity and external laboratory testing
for whole grain flour standard.
Material S2. Certificate of identity and external laboratory testing
for whole grain pasta standard.
8Journal of Food Science rVol. 0, Iss. 0, 2020
wheat
tissue
WGA standards
GERM
ENDO
flour (% whole grain)
25%
50%
0%
40%
55%
75%
60%
0.25
0.1
0.025
0.01
A B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
100%
SUPPLEMENTAL DATA
Supplemental Figure 1: Quantification of WGA protein and other biomarkers in wheat flour from varying whole grain content. (A)
Representative immunoblot shows WGA standards (lanes 2-5), wheat kernel tissue fractions (lanes 14-15), and mixed flours probed for
WGA content (lanes 6-13). The arrow indicates the major WGA monomer band at 18kD, as determined by molecular weight biomarkers
(MWM). (B) WGA content (black) was compared to the concentrations of starch (green), mineral (red), phytate (blue), and total protein
(pink), normalized to 0% whole wheat (starch and protein) or 100% whole wheat (WGA, mineral, and phytate), correlated with whole
wheat composition of mixed flours (r2>0.87-0.99). Mean ±SE are shown for 2-5 independent experiments.
wheat
tissue
B
wheat cultivar
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
WGA standards wheat cultivar
B
D
A
C
E
G
F
0.25
0.1
0.025
0.01
H
GERM
ENDO
SUPPLEMENTAL DATA
A
Supplemental Figure 2: Effect of wheat cultivar on WGA content in wheat flour. (A) Representative immunoblot showing WGA standards
(lanes 2-5), different cultivars of whole wheat flours (lanes 6-13) and isolated wheat kernel tissues (lanes 14-15) probed for WGA content.
The arrow indicates the major WGA monomer band at 18kD, as determined by molecular weight markers (MWM). The cultivars used are
listed in Supplemental Table 2. Quantification of the WGA protein content relative to cultivar A (WB9229) reference. Mean ±SE are shown
for 3 independent experiments. Differences in WGA content between cultivars were tested by one-way ANOVA followed by Tukey’s
multiple-comparisons test; no cultivar was significantly different from the cultivar A (WB9229) reference (p<0.05).
SUPPLEMENTAL DATA
C D
commercial flour brands
A B
*
*
*
commercial flour brands
commercial flour brands commercial flour brands
SUPPLEMENTAL DATA
Supplemental Figure 3: Quantification of mineral, total protein, starch, and phytate in commercial wheat flour. (A) Quantification of the
combined mineral content in refined (REFINED), brands A-C (A, B, and C), and whole grain whole wheat standard (STANDARD) flour. Mean ±
SE are shown for 3 independent experiments. Differences in mineral content were tested by one-way ANOVA followed by Tukey’s multiple-
comparisons test. Asterisk identifies the means significantly different from the whole wheat standard (p<0.05). (B) Quantification of total
protein content in refined (REFINED), brands A-C (A, B, and C), and whole grain whole wheat standard (STANDARD) flour. Mean ±SE are
shown for 4 independent experiments. Differences in protein content were tested by one-way ANOVA followed by Tukey’s multiple-
comparisons test. Asterisk identifies the means significantly different from the whole wheat standard (p<0.05). (C) Quantification of starch
content in refined (REFINED), brands A-C (A, B, and C), and whole grain whole wheat standard (STANDARD) flour. Mean ±SE are shown for 2
independent experiments. Differences in starch content were tested by one-way ANOVA followed by Tukey’s multiple-comparisons test; no
flour brand was significantly different from the whole wheat standard (p<0.05). Asterisk identifies the means significantly different from the
whole wheat standard (p<0.05). (D) Quantification of phytate content in refined (REFINED), brands A-C (A, B, and C), and whole grain whole
wheat standard (STANDARD) flour. Mean ±SE are shown for 2 independent experiments. Differences in phytate content were tested by
one-way ANOVA followed by Tukey ’s multiple-comparisons test. Asterisk identifies the means significantly different from the whole wheat
standard (p<0.05).
SUPPLEMENTAL DATA
commercial flour brands
A B
commercial pasta brands
**
*
Supplemental Figure 4: Quantification of mineral and total protein in commercial pasta products. (A) Quantification of the combined
mineral content in brands A-E (A, B, C, D, and E) and whole grain whole wheat standard (STANDARD) flour. Mean ±SE are shown for 3
independent experiments. Differences in mineral content were tested by one-way ANOVA followed by Tuke y ’s multiple-comparisons test.
Asterisk identifies the means significantly different from the whole wheat standard (p<0.05). (B) Quantification of total protein content in
brands A-E (A, B, C, D, and E) and whole grain whole wheat standard (STANDARD) flour. Mean ±SE are shown for 3 independent
experiments. Differences in mineral content were tested by one-way ANOVA followed by Tukey’s multiple-comparisons test; no flour brand
was significantly different from the whole wheat standard (p<0.05).
SUPPLEMENTAL DATA
Supplemental Table 1: Information on commercial wheat flour and commercial wheat pasta products used in these studies. Product
brand, description, and SKU/UPC number are shown.
variety ID
variety name
variety type
source
A
WB9229
hard red spring
Fritz Durst Farming & Tule Farms
B
Vida
hard red spring
Montana State Seed Laboratory
C
Judee
hard red winter
Montana State Seed Laboratory
D
TAM111
hard red winter
Albers Enterprises, Inc.
E
Everest
hard red winter
Kansas Wheat Alliance, Inc.
F
Duster
hard red winter
Oklahoma Foundation Seed Stocks, Oklahoma Genetics, Inc.
G
Yell owsto ne
hard red winter
Montana State Seed Laboratory
H
Patwin 515HP
hard white winter
Fritz Durst Farming & Tule Farms
SUPPLEMENTAL DATA
Supplemental Table 2: Information on wheat varieties used in this study. Wheat variety name, type, and source are shown.
The varieties were chosen to reflect a range of common domestic wheats, according to USDA National Agricultural Statistics
Service data (https://www.nass.usda.gov). Based on seeded acreages from 2016, Vida was the 1st most abundant spring
variety in Montana, Judee was the 2nd most abundant winter variety in Montana, TAM111 was the 3rd most abundant winter
variety in Kansas, Everest was the 1st most abundant winter variety in Kansas, Duster was the 2nd most abundant winter
variety in Oklahoma, and Yellowstone was the 1st most abundant winter variety in Montana. These were compared to
WB9229 (spring variety) and Patwin 515HP (winter variety) which are popular in California.
SUPPLEMENTAL MATERIAL
Supplemental Material 1: Certificate of identity and external laboratory testing for whole grain flour standard
Supplemental Material 2: Certificate of identity and external laboratory testing for whole grain pasta standard
Explore the Harvests
← Go back
BATCH 0613D002
FARM
FARMER(S) Fritz Durst Farming
To put it simply: The farmer is the one that feeds you. We want you to know where
and how your food is being grown. It just tastes better that way, doesn't it?
ABOUT THE FARM Fritz Durst is a sixth generation grain farmer in the Sacramento Valley. He grows
dryland crops, organic vegetables, safflower, rice, wine grapes, and sunflowers for
seed at Tule Farms, his 6,000 acre family farm outside of Woodland, CA. On his
certified organic plot, Fritz grows organic wheat and rye for Community Grains.
In the mid-eighties, Fritz and his father began planting wheat and barley directly
into the residue of the previous crop without tilling, in order to prevent erosion
gullies from forming during winter rains. This no-till method helped reduce soil
loss from 6 tons to 2 tons in just one year.
Without enough water from his irrigation district for all of his property, Fritz has
come to understand that dry-farming a large part of his property simply makes
economic sense. By following a special rotation of crops to help retain water,
4,000 acres of wheat, garbanzo beans, and safflower are able to rely solely on rain.
Fritz's work has earned him a Resource Conservation District “Cooperator of the
Year Award” in 1986, and the Conservation Tillage Farmer Innovator Award from
the USDA’s Natural Resources Conservation Service in 2011.
CERTIFICATIONS Cer tifie d O rga nic by CC OF
Non-GMO Project Verified
Certifications set a minimum standard for good farming practices and food
production, including land stewardship and conservation, chemical use, ecological
diversity, labor practices, and food safety. We aim to go above and beyond these
standards.
LABOR Fritz employs 8 people year round and can add an additional 15 individuals for
pruning, weeding and harvesting. He says his employees are the corner stone of
the farm and operate as a team, tending to crops, machinery and to each other.
Attuned to the crops and animals, they often collaborate on new alternatives to
farming practices. One employee has been with Fritz for over 14 years and three
others with 8 years each. He says, "I am very proud of all of them and their
honesty, integrity, and productivity. I am committed to providing a safe work
environment and providing living wages for my people."
Labor is a complex issue, and we've found that Organic Certification doesn't address it
very well. Grain farming is done primarily by a few skilled workers operating
machines, requiring far less labor than other crops.
SEED
CLASS Hard Red Winter Wheat
Class (hard red, etc.) is primarily applicable to wheat. Designated by color, hardness
and growing season (e.g., Hard Red Spring Wheat), there are a range of quality
characteristics within classes, giving customers some indication of how to use a given
flour. We like to challenge common assumptions about how to use each grain!
VARIETY WB9229
SEED SOURCE Untreated from Adams Grain
Seed source and supply is a complicated, and somewhat political, issue. We are
actively engaged in developing a steady source of publicly available seed in farmer
quantities. The source of a seed can signal the intent of breeding, as some modern
breeds were developed for high yield and to withstand modern chemical fertilizers.
YIELD 30 acres; 4848 lbs. per acre
Yield is important before and after planting - from selecting seed for a particular field
to the ultimate price of the grain. The yield of a particular variety does have to work
for the farmer economically, wherein low yielding grains - primarily heirlooms - can
signal a higher priced product.
HARVEST DATE 06/21/2016
As a dry good, grains maintain freshness for several years in their whole kernel form.
We harvest yearly, and store in a chemical-free environment. If the grains were held
for several years in a fumigated environment, you'd really want to know about it.
SOIL
LAND QUALITY This is beautiful soil, a rich alluvial deposit from the Sacramento River. It has
been leveled to a perfect slope for irrigation and is very fertile due to natural
capacities and years of good farming practices.
Land quality, categorized by the USDA, is the jumping off point - it helps farmers
determine what can be grown and how best to manage the soil. Characteristics, like
depth, slope, uniformity, and organic matter, impact the soil's ability to retain
nutrients and water. Most of our grains are grown on Class 1 or 2 soils.
SOIL DEVELOPMENT All of last year's corn fodder was incorporated into the soil and we coupled that
with 3 tons/acre of poultry compost.
Organic matter, soil carbon accumulation and active microbial communities are
primary indicators of soil quality. Regenerative soil management practices, such as
conservation tillage, cover cropping, crop rotations, etc., can enhance the soil while
simultaneously restoring the environment, generating resilience, and improving
human health.
We're drawing attention to this particular soil management practice as an area ripe
for experimentation. Here we learn how farmers may use no till or conservation tillage
in combination with soil-enhancing rotations to increase biological activity and
diversity.
ECOLOGY
BIODIVERSITY We manage the banks of the water ways to keep vegetation year round. We have
also planted valley oaks and sycamores to foster birdlife.
Organic Certification underscores a number of ways to increase biodiversity (or
wildlife) on farms. Here we look at how farms are going beyond that standard to
include avian, insect and pollinator ecology.
WATER USE This crop was irrigated once using flood irrigation. The irrigation helped to
mitigate the dry spring and to keep the soil biology thriving.
A major goal of regenerative soil management is to help soil hold onto water longer,
thereby needing less. The decision to irrigate depends on a number of factors,
including land quality, rain, and wheat variety. Tall, lanky heirloom wheats, for
example, do not hold up well when irrigated.
MILL
STORAGE METHOD Unfumigated Farm Storage
Storage is an overlooked aspect of grain farming, where the kernels may be held for
years. Methods to keep bugs and mold at bay can involve fumigation. Organic grains
are stores without the use of chemicals.
MILLER Bay State Milling | www.baystatemilling.com
There is so much unseen in a flour mill. Who they are and what they stand for is
immensely important.
TYPE OF MILL Our innovative mill is central to the high functionality of our flour. It’s an air-
classifier mill that creates exceptionally fine, uniformly granulated 100% whole
grain flour that works just as well for baked goods as it does for our pastas, and
produces wonderfully creamy polentas.
Nothing is sifted out in the process of milling — whole kernels enter the mill, and
100% whole grain flour comes out. The mill agitates whole grain kernels at
extremely high speeds so that the grains shatter against each other and the
rotating grinder surfaces, until all the particles of the grain, whether they be
from the germ, bran, or endosperm, are all the same size — this is why our flour’s
texture is so special.
The surface texture created by our mill, called “damaged” or “activated” starch,
allows it to absorb water extremely well. In the case of wheat, this
high absorption of water benefits the baking properties and flavor of breads and
pastries. Moreover, because the process requires very low heat, the grains’
proteins and other nutrients don’t break down in the process — they’re kept
fresh and wholesome.
The milling method is the key determinant of flour's functionality, flavor, and nutrient
density. The invention of the steel roller mill was a major turning point in history,
enabling the mass production of refined white flour. High-speed mills can generate
enough heat to destroy vital nutrients (like protein and vitamin E) and create
rancidity. Air-classifier mills have more control over their drying and grinding
elements.
MILLING DATE 12/10/2017
Milling date can impact flavor and shelf-life. Our flour is, with rare exception, shelf-
stable for over a year in cool, dry storage - and best refrigerated. That said, we can't
deny that freshly milled flour has a wonderful, enhanced fragrance.
FLOUR EXTRACTION 100% Whole-Wheat
Extraction describes the amount of wheat that is retained after milling. Whole Wheat
Flour, for example, is described as having 100% extraction, while white flours
typically have extraction rates of between 67% and 78% (predominantly the bran and
germ are sifted out). The FDA doesn't require food manufacturers to disclose the exact
quantity of whole grain. When we say 100% Whole Grain, we mean the whole thing.
FLOUR
PROTEIN 9.44
The protein content of wheat can vary from as low as 6% to as high as 20%. Protein in
bread dough traps gases formed in the dough, allowing it to lighten and rise. The
protein's elasticity, stability, tenacity, and plasticity are also extremely important in
determining the flour’s baking characteristics.
MOISTURE 8.57
This figure indicates the percentage of natural moisture, by weight, in relation to the
overall weight of a given sample. Beyond a certain point—sometimes pegged at
around 15%— content of the flour can compromise its storability.
Ash is the mineral content in the wheat (primarily from the bran), so high ash content
produces darker hued flour that may ferments more quickly. Small kernels have a
higher proportion of bran and therefore more crude fiber than large, plump kernels.
INDUSTRY ANALYSIS WB9229
Industry analyses are the standard tests that help determine the best and/or baking
qualities of flour. Principal tests include protein, ash, moisture, farinograph, falling
number, and alveogram.
Copyright 2016 Community Grains | All Rights Reserved.
Explore the Harvests
← Go back
BATCH 9142017
FARM
FARMER(S) Full Belly Farm
To put it simply: The farmer is the one that feeds you. We want you to know where
and how your food is being grown. It just tastes better that way, doesn't it?
ABOUT THE FARM Farm Partners: Dru Rivers, Andrew Brait, Judith Redmond, Amon Muller, Jenna
Muller and Paul Muller
Location: Guinda, CA
Full Belly Farm is a prominent and much loved certified organic 250-acre farm
located in the Capay Valley. Known for popularizing heirloom varieties and
supplying some of our area’s best restaurants, it grows over 80 kinds of flowers,
vegetables, grains, nuts and fruit year-round. The farm adopts a whole system
approach in which every action must be made with purpose, thought, and
consideration of the impact it will have on the long-term sustainability of the farm.
www.fullbellyfarm.com
CERTIFICATIONS Cer tifie d O rga nic by CC OF
Certifications set a minimum standard for good farming practices and food
production, including land stewardship and conservation, chemical use, ecological
diversity, labor practices, and food safety. We aim to go above and beyond these
standards.
LABOR Full Belly Farm focuses on providing a healthy safe work environment for all who
work and all who support the work of the farm. We focus on creating a farm
system design that can offer year round employment for the majority of our crew.
Many of our workers here have been employed here for over 20 years and we
often provide jobs for families (and youth aged 16 and above). Full Belly
recognizes farm work as dignified and skilled, requiring fair pay above minimum
wage and health benefits for that commitment to the farm. We provide bonuses,
shared by all workers on the farm, when we're financially capable. We are proud
that our crew can eat the food that is produced on the farm by taking produce
home in the evenings.
Labor is a complex issue, and we've found that Organic Certification doesn't address it
very well. Grain farming is done primarily by a few skilled workers operating
machines, requiring far less labor than other crops.
SEED
CLASS Wheat | Hard Amber Durum
Class (hard red, etc.) is primarily applicable to wheat. Designated by color, hardness
and growing season (e.g., Hard Red Spring Wheat), there are a range of quality
characteristics within classes, giving customers some indication of how to use a given
flour. We like to challenge common assumptions about how to use each grain!
VARIETY Durum Iraq | A landrace from Iraq that was popularized in California by Monica
Spiller, founder of The Whole Grain Connection. We are enamored of the golden
color and mild sweet character it lends to pastas and breads.
SEED SOURCE Saved seed. Originally sourced from Fritz Durst and Bob Klein.
Seed source and supply is a complicated, and somewhat political, issue. We are
actively engaged in developing a steady source of publicly available seed in farmer
quantities. The source of a seed can signal the intent of breeding, as some modern
breeds were developed for high yield and to withstand modern chemical fertilizers.
YIELD 1500/acre | Even though the yield was low because of the lodging, the wheat
berries were plump and had good color.
Yield is important before and after planting - from selecting seed for a particular field
to the ultimate price of the grain. The yield of a particular variety does have to work
for the farmer economically, wherein low yielding grains - primarily heirlooms - can
signal a higher priced product.
HARVEST DATE 06/15/2015
As a dry good, grains maintain freshness for several years in their whole kernel form.
We harvest yearly, and store in a chemical-free environment. If the grains were held
for several years in a fumigated environment, you'd really want to know about it.
SOIL
LAND QUALITY Tehama soils consist of deep, well-drained loam soils, formed in alluvium from
sedimentary rock sources. Slopes range from 0 to 5 percent. Elevation ranges
from 10 to 300 feet. Annual temperature is 62F, annual rainfall is 16-20 inches.
Silty clay loam that extends to a depth of more than 60 inches. Capay soils are
very fertile, and often used for irrigated row crops, field crops, dry-farmed grain,
and wildlife habitat.
Clay content ranges from 40-55%; neutral to moderately alkaline. Land
Capability is 2e (irrigated) and 4e (non irrigated), meaning that soil must be well-
managed to prevent erosion and runoff. Moderate water storage. Natural
fertility is high.
Land quality, categorized by the USDA, is the jumping off point - it helps farmers
determine what can be grown and how best to manage the soil. Characteristics, like
depth, slope, uniformity, and organic matter, impact the soil's ability to retain
nutrients and water. Most of our grains are grown on Class 1 or 2 soils.
SOIL DEVELOPMENT This field's tillage history has been minimal and the sequence of rainfall this year
created a few weed issues that required extra cleaning. Both purple vetch and
star thistle needed extra attention to be cleaned out thoroughly.
For fertilizer, we used 10 tons of green waste compost per acre.
There were no pest problems. The crop did lodge, meaning that it laid over in
May and wasn’t able to straighten up. It dried laying down so that harvest was
more difficult for all of these varieties.
Organic matter, soil carbon accumulation and active microbial communities are
primary indicators of soil quality. Regenerative soil management practices, such as
conservation tillage, cover cropping, crop rotations, etc., can enhance the soil while
simultaneously restoring the environment, generating resilience, and improving
human health.
ROTATIONS 2013: Vetch planted to restore nutrients.
2014: Oat hay planted. Both the oat hay and the Wheat were dry farmed.
We're drawing attention to this particular soil management practice as an area ripe
for experimentation. Here we learn how farmers may use no till or conservation tillage
in combination with soil-enhancing rotations to increase biological activity and
diversity.
ECOLOGY
BIODIVERSITY Full Belly produces over 80 different crops that includes stone fruits, nuts,
vegetable crops and grapes - this crop diversity facilitates year round
production and harvest. We have integrated some 200 sheep into the farm
system to forage and reduce crop waste by converting it into meat, wool and
fertilizer. Biodiversity is integrated into all aspects of the farm design, making it
a creative and interesting environment to work and live.
Organic Certification underscores a number of ways to increase biodiversity (or
wildlife) on farms. Here we look at how farms are going beyond that standard to
include avian, insect and pollinator ecology.
WATER USE This is a dry-farmed field with out a history of irrigation. This grain was rain
germinated with a December 2014 storm. It then had the driest January on
record for California, and a very dry February. Late February and March rainfall
made the crop.
A major goal of regenerative soil management is to help soil hold onto water longer,
thereby needing less. The decision to irrigate depends on a number of factors,
including land quality, rain, and wheat variety. Tall, lanky heirloom wheats, for
example, do not hold up well when irrigated.
MILL
STORAGE METHOD Unfumigated farm storage.
Storage is an overlooked aspect of grain farming, where the kernels may be held for
years. Methods to keep bugs and mold at bay can involve fumigation. Organic grains
are stores without the use of chemicals.
MILLER Bay State Milling | www.baystatemilling.com
There is so much unseen in a flour mill. Who they are and what they stand for is
immensely important.
TYPE OF MILL Our innovative mill is central to the high functionality of our flour. It’s an air-
classifier mill that creates exceptionally fine, uniformly granulated 100% whole
grain flour that works just as well for baked goods as it does for our pastas, and
produces wonderfully creamy polentas.
Nothing is sifted out in the process of milling — whole kernels enter the mill, and
100% whole grain flour comes out. The mill agitates whole grain kernels at
extremely high speeds so that the grains shatter against each other and the
rotating grinder surfaces, until all the particles of the grain, whether they be
from the germ, bran, or endosperm, are all the same size — this is why our flour’s
texture is so special.
The surface texture created by our mill, called “damaged” or “activated” starch,
allows it to absorb water extremely well. In the case of wheat, this
high absorption of water benefits the baking properties and flavor of breads and
pastries. Moreover, because the process requires very low heat, the grains’
proteins and other nutrients don’t break down in the process — they’re kept
fresh and wholesome.
The milling method is the key determinant of flour's functionality, flavor, and nutrient
density. The invention of the steel roller mill was a major turning point in history,
enabling the mass production of refined white flour. High-speed mills can generate
enough heat to destroy vital nutrients (like protein and vitamin E) and create
rancidity. Air-classifier mills have more control over their drying and grinding
elements.
MILLING DATE 02/29/2016
Milling date can impact flavor and shelf-life. Our flour is, with rare exception, shelf-
stable for over a year in cool, dry storage - and best refrigerated. That said, we can't
deny that freshly milled flour has a wonderful, enhanced fragrance.
FLOUR EXTRACTION 100% Whole-Wheat
Extraction describes the amount of wheat that is retained after milling. Whole Wheat
Flour, for example, is described as having 100% extraction, while white flours
typically have extraction rates of between 67% and 78% (predominantly the bran and
germ are sifted out). The FDA doesn't require food manufacturers to disclose the exact
quantity of whole grain. When we say 100% Whole Grain, we mean the whole thing.
FLOUR
PROTEIN 12.4%
The protein content of wheat can vary from as low as 6% to as high as 20%. Protein in
bread dough traps gases formed in the dough, allowing it to lighten and rise. The
protein's elasticity, stability, tenacity, and plasticity are also extremely important in
determining the flour’s baking characteristics.
MOISTURE 10.44%
This figure indicates the percentage of natural moisture, by weight, in relation to the
overall weight of a given sample. Beyond a certain point—sometimes pegged at
around 15%— content of the flour can compromise its storability.
Ash is the mineral content in the wheat (primarily from the bran), so high ash content
produces darker hued flour that may ferments more quickly. Small kernels have a
higher proportion of bran and therefore more crude fiber than large, plump kernels.
INDUSTRY ANALYSIS Iraqi Durum Lab Analyses from Full Belly Farm
Industry analyses are the standard tests that help determine the best and/or baking
qualities of flour. Principal tests include protein, ash, moisture, farinograph, falling
number, and alveogram.
Copyright 2016 Community Grains | All Rights Reserved.
A preview of this full-text is provided by Wiley.
Content available from Journal of Food Science
This content is subject to copyright. Terms and conditions apply.
