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CONCENTRATION OF BENEFICIAL PHYTOCHEMICALS IN HARVESTED GRAIN OF US YELLOW DENT MAIZE (Zea mays L.) GERMPLASM Carrie J. Butts-Wilmsmeyer, Rita H. Mumm, and Martin O. Bohn J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02034 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 9, 2017
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Journal of Agricultural and Food Chemistry
CONCENTRATION OF BENEFICIAL PHYTOCHEMICALS IN HARVESTED GRAIN OF US YELLOW DENT MAIZE (Zea mays L.) GERMPLASM
Carrie J. Butts-Wilmsmeyer, Rita H. Mumm, and Martin O. Bohn* Department of Crop Sciences, the University of Illinois at Urbana-Champaign, 1102 S. Goodwin Avenue, Urbana, IL 61801 *
Corresponding author:
[email protected] Phone: (217) 244 2536
Keywords: Maize, Phenolics, Tocopherols, Protein, Plant Breeding
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ABSTRACT
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Although previous studies have examined the concentration of various nutritional
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compounds in maize, little focus has been devoted to the study of commercial maize hybrids or
4
their inbred parents. In this study, a genetically and phenotypically diverse set of maize hybrids
5
and inbreds relevant to U.S. commercial maize germplasm was evaluated for its variability in
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phytochemical content. Total protein, unsaturated fatty acids, tocopherols, soluble phenolics, and
7
insoluble-bound phenolics were evaluated in this study. Of these compounds, only soluble and
8
insoluble-bound phenolic acids exhibited means and variances that were at least as large as the
9
means and variances reported for other sets of germplasm. This suggests that selection for high
10
phenolic acid content is possible in current U.S. commercial germplasm. In contrast, while the
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total protein, unsaturated fatty acid, or tocopherol content could possibly be improved using
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current U.S. commercial germplasm, the results of this study indicate that the incorporation of
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more diverse sources of germplasm would most likely result in quicker genetic gains.
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INTRODUCTION
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Over the last decade, consumers have become increasingly interested in the natural
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nutritional value of their food products.1 This increased interest is not surprising. High obesity
17
rates are common in affluent nations. The incidence of aging-related diseases in developed
18
countries is rising.2 Furthermore, there is greater public concern that processed foods are less
19
nutritious.3 Therefore, food companies must consider not only the calorie and vitamin content of
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their food products, but also the content of beneficial phytochemicals which occur naturally in
21
their food products.
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In 2016, 2.8 billion bushels (71.12 billion kg) of maize were devoted to human food
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consumption,4 thereby highlighting the importance of maize in the American diet. For this
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study, the variability in the concentration of total protein content, fatty acids, tocopherols, and
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phenolics was examined in a set of maize lines which are representative of the commercial maize
26
germplasm grown in the U.S. Corn Belt.5-6 All these phytochemicals are beneficial to human
27
health. Diets higher in total protein result in satiety and weight maintenance. Since satiety is
28
maintained for longer periods of time, people tend to eat less frequently.7 This reduced food
29
consumption contributes to a lower overall caloric intake and a decreased chance of developing
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an obesity-related disease. Unsaturated fatty acids play important roles in blood pressure
31
regulation, cell membrane integrity, and improving cholesterol levels.8-9 Oleic acid and linoleic
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acid are fatty acids important in the human diet and are found in maize.
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In contrast to the health benefits of protein and unsaturated fatty acids, tocopherols and
34
phenolics exhibit antioxidant activity, prebiotic activity, and other chemopreventive properties.10-
35
13
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Most studies have focused on α-tocopherol because it is the most efficient antioxidant of the
α-, γ-, and δ-Tocopherol are typically found in maize, whereas β-tocopherol is rarely present.14
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three tocopherols present in maize.13 Like the tocopherols, many of the soluble phenolics exhibit
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antioxidant activity in vitro, but, in vivo, they are more likely to activate gene cascades involved
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in immunoprotection and chemoprevention.11-12,
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ferulic acid and p-coumaric acid, both of which are hydroxycinnamic acids.16-17 In cereals, these
41
are mainly bound to the cell wall via ester bonds. Thus, insoluble-bound phenolics are not
42
readily available for the activation of gene cascades. However, in conjunction with the
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arabinoxylan fibers to which they are bound, the insoluble-bound phenolics act as prebiotics by
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creating an environment beneficial for gut bacteria, including Lactobacillus and Bifidobacterium
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species.18
15
The predominant phenolics in maize are
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A precursor to the phenolics in maize is the carboxylic acid cinnamic acid. Cinnamic acid
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is stored in the vacuoles of plants19 prior to being modified in, most likely, the Golgi bodies.
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Cinnamic acid-4 hydroxylase acts on cinnamic acid, producing p-coumaric acid. p-Coumaric
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acid is acted upon by p-coumaric hydroxylase and then o-methyltransferase to produce ferulic
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acid. p-Coumaric acid and ferulic acid most likely are synthesized before esterification to
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arabinoxylans.20 It also appears that hydroxycinnamic acids are esterified to arabinoxylans in the
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Golgi bodies.21 Therefore, to understand the variability in the composition of the phenolics in
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maize, an understanding of cinnamic acid is useful.
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Our overall objective was to determine the potential for improving the nutritional content
55
of maize hybrids that are representative of U.S. Corn Belt germplasm. In this study, we
56
determined the average content and variability of total protein, unsaturated fatty acids, α-, γ-, and
57
δ-tocopherol, and phenolics in U.S. Corn Belt maize inbred parents (i.e., inbreds) and a subset of
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their hybrids. Using the estimated means and variability in the phytochemicals of interest, we
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determined if this germplasm is well suited for improving the nutritional quality of yellow dent
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maize hybrids or if the other germplasm sources must be tapped to access favorable alleles.
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Using the literature base, we also discuss the potential fate of these phytochemicals during maize
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food product processing and consider which phytochemicals may be the most feasible to
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improve using plant breeding techniques.
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MATERIALS AND METHODS
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65
Plant Materials
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The hybrids evaluated in this study were created from a genetically diverse set of inbreds
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that represent important heterotic subgroups in the current U.S. maize commercial germplasm
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base.5-6 A heterotic group is a group of parental genotypes whose observed combining ability and
69
heterosis are similar when crossed with parental genotypes from other groups.22 Heterotic
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groups can be broken into smaller sets called subgroups. Five of the inbreds (B73, LH1, PHG39,
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PHJ40, and 4676A) were derived from the Iowa Stiff Stalk Synthetic (SSS), which represents the
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female side of the heterotic pattern used in the U.S. Seven other inbreds (LH123HT, LH82,
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Mo17, PH207, PHG47, PHG84, and PHZ51) are of Non-Stiff Stalk (NSS) origin and are
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composed largely of Iodent, Minnesota 13, Oh07, Midland yellow Dent, Oh43, Lancaster, and
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broad-based subgroups which generally represent the male side of the heterotic pattern used in
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the U.S.6 Using a diallel mating design, 66 hybrids were created from the 12 inbreds used in this
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study. All inbreds and hybrids (NTotal = 78 entries) were grown at the University of Illinois Crop
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Sciences Research and Education Center in Urbana, IL in the summer seasons of 2009, 2010,
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and 2011.6 Plots were comprised of four rows with length of 5.3m and which were 0.76 m apart
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at a plant density of approximately 74,000 plants ha-1; only the center 2 rows were harvested for
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grain. Plots were managed in accordance with regional agronomic production practices (i.e. no
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irrigation, minimum tillage, and standard fertilizer regime). Three replications of the 78 entries
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were grown in each of the three years. Of the 78 entries, 25 of those entries were chosen for use
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in this study. Exactly 454 g of harvested grain from each plot was dried to a moisture content of
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approximately 8%, bagged, and placed in cold storage prior to nutritional analyses. From each
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bag, a 100 g grain sample was ground to a fine powder using a Foss Cyclone Mill (1 mm mesh
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screen). We used this powder in all wet-lab chemistry.
88 89
Subset Determination
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The available resources allowed us to analyze 25 entries, which amounted to nine
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samples per entry and a total number of 225 samples. For this study, we used all 12 parental
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inbreds and 13 hybrids. The set of 13 hybrids was composed of the five highest and the five
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lowest performing SSS×NSS hybrids in relation to their dry milling efficiency and three hybrids
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which have Iodent parent PH207 in common. Dry milling efficiency is the proportion by weight,
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on a dry basis, of flaking grits obtained from one kg of dry milled grain (for more information
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see Macke et al.6). Three hybrids with PH207 parentage were included because preliminary
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analyses (data not shown) indicated that the inbred PH207 displayed considerably high content
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of some of the phytochemicals of interest, yet none of the ten hybrids selected based on dry
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milling efficiency alone had PH207 as a parent. Considering that the content of many nutritional
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compounds in the maize grain, like starch, protein, and oil, is largely influenced by additive gene
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action,23 it was important that hybrids with PH207 as a parent were included as part of this study
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and that inferences were made on as diverse of a subset as possible. In addition, PH207
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represents an important heterotic subgroup in commercial maize breeding germplasm.
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Reagents
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Ethyl acetate, α-amylase, and methanol were purchased from Sigma-Aldrich (St. Louis,
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MO). Sodium hydroxide, sodim choloride, hydrochloric acid, and pyridine were purchased from
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Fisher Scientific (Pittsburgh, PA). N,O-Bis(tremethylsilyl)trifluoroacetamide (BSTFA) was
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obtained from Thermo Scientific (Waltham, MA). Ferulic acid, p-coumaric acid, sinapic acid,
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and cinnamic acid was purchased from both Sigma-Aldrich (St. Louis, MO) and Fisher Scientific
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(Pittsburgh, PA).
112 113
Determination of Protein Content
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Whole grain samples (8% moisture) were measured using non-destructive NIR
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techniques. Calibration curves were built based on known standards. The remainder of the
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procedure was conducted following the direction of the DA 7200 Diode Array Analyzer
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Operation Manual24 and Bubert.25
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Determination of Fatty Acid Content
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The total oil content of ten random kernel maize samples and their corresponding ground
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samples was measured and these data were used for calibration of the Nuclear Magnetic
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Resonance (NMR) equipment.26 Ground samples underwent a hexane wash. The hexane layer
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was removed, placed in a separate vial, and evaporated, leaving only the oil extracted from a
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particular sample in the bottom of the vial. All vials were weighed before extraction and after
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evaporation. The difference in the mass is equal to the mass of oil extracted for that particular
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sample. Dividing this mass by the mass of ground maize used in the extraction gives the total oil
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content in g oil g-1 maize sample. Three technical replicates were used for each of the ten training
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samples, and the average content was calculated for the sample using these technical replicates.
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These values and their corresponding whole kernel samples were used to calibrate a prediction
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curve using the NMR. The calibration was performed by Dow AgroSciences. The total oil
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concentration of the 225 samples was estimated using NMR.
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Approximately 0.5 g of each sample was used for fatty acid quantification. The sample
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was placed in a vial, and 3 mL of hexane was added. The vials were capped, placed in a
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sonicator at 75°C for 10 min, and then put on a horizontal shaker for 10 min. Each vial was then
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vortexed before being centrifuged at 465 g for 5 min. The top hexane layer was removed and
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placed in a clean vial. The hexane wash was repeated two more times. The hexane extract was
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dried using a centrifugal evaporator. The oil remaining in the vial was reconstituted in 1 mL of
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heptane, and 300 L of the heptane/oil mixture was placed in a gas chromatography vial and
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derivitized using 20 L of sodium methoxide. Subsequently, the samples underwent a Fatty Acid
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Methyl Ester (FAME) analysis.
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The
proportion
of
total
oil
content
was
recorded
for
each
fatty
acid.
The following equation was used to calculate the concentration of each fatty acid: =
× 1000 × 1
143
where is the concentration of the ith fatty acid and the jth observation in mg per g of maize
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sample, is the proportion of total oil content for the ith fatty acid and the jth observation,
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is the total oil content, in g, for the jth observation, and is the recorded mass of the jth
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observation in grams.
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Determination of Tocopherol Content
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Following the removal of 300 L from the heptane aliquot for the FAME analysis, the
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heptane/oil mixture was evaporated to dryness. Gas chromatography was used for quantification
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of tocopherols in the oil extracts. The total mass of tocopherols (g) remaining was reported by
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the gas chromatography system. In order to calculate the concentration in the original sample, the
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following equation was used: 9 ACS Paragon Plus Environment
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1 × 10
= × 0.7 × 154
where is the actual tocopherol content of the ith tocopherol and the jth sample and
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is the reported content of the ith tocopherol in the jth sample before the correction. We applied
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a correction factor of 0.7, assuming that approximately 70% of the originally extracted
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tocopherols remained in the oil extract.
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Determination of Soluble Phenolics Content
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Following the hexane wash, the maize pellet was dried using a centrifugal evaporator.
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Then, 3 mL of 70% acetone was added to each vial. Vials were placed in a sonicator for 10 min
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at 75°C and then placed on a horizontal shaker for 10 min. Vials were then vortexed and
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centrifuged at 465 g for 5 min. The top acetone layer was removed and put in a separate vial. The
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acetone wash was completed two more times for a total of three acetone washes. The extracted
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acetone aliquot was then evaporated to dryness using a centrifugal evaporator. Soluble extracts
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were reconstituted in 2 mL of methanol, vortexted, and filtered using syringe filters. One mL of
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the filtered solution was then placed in an Ultra Performance Liquid Chromatography (UPLC)
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for analysis.
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A Waters Acquity UPLC H-Class with an Acquity BEH Shield RP18 (2.1×150 mm, 1.7
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m) column was used for the quantification of the soluble phenolics content. The column
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temperature was maintained at 25ºC. For the first 1.5 minutes, 60% acetonitrile with 0.1%
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formic acid and 40% methanol was used for the elution at a flow rate of 0.2 mL / min. For the
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time interval between 1.5 and 11 minutes, 50% acetonitrile with 0.1% formic acid and 50%
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methanol was used for the elution at a flow rate of 0.2 mL / min. For the time interval between
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11 and 12 minutes, 60% acetonitrile with 0.1% formic acid and 40% methanol was used for the 10 ACS Paragon Plus Environment
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elution at a flow rate of 0.2 mL / min. An Acquity UPLC LG 500 nm photodiode array detector
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was used for UV detection of analytes at 300 nm, 270 nm, and 245 nm. Data signals were
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recorded and processed using Empower 3 Software (Build 3471) (Waters Corp.).
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Standards of the typical hydroxycinnamic acids in grains, i.e., ferulic acid, p-coumaric
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acid, sinapic acid, and cinnamic acid were first analyzed using UPLC to record the retention time
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of each of these compounds and to build a standard curve for the calculation of concentrations in
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the maize samples. Our maize samples were then analyzed using UPLC using the protocol as
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described, and the standard curve was used to calculate the concentration of the
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hydroxycinnamic acids in each sample using the equation 2 × =
185
where is the content of the ith soluble phenolic in the jth observation and is the recorded
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mass of the ith soluble phenolic in the jth observation as calculated from the standard curve, and
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is the recorded mass of the jth observation. was multiplied by a factor of 2 to account for
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the reconstitution of the soluble phenolics in 2 mL of methanol.
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Determination of Insoluble-Bound Phenolics Content
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The measurement of the insoluble-bound phenolics content took place using the protocol,
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including -amylase digestion, as described by Butts-Wilmsmeyer and Bohn27 with the
193
exception that standards of p-coumaric acid, cinnamic acid, and sinapic acid were also used in
194
creating the standard curve. Samples were analyzed using a gas chromatograph with a flame
195
ionization detector (Agilent 6890). A DB-200 megabore column (ID = 0.53 mm, length = 30 m)
196
was used. Hydrogen gas was used as the carrier gas with a split inlet and a split ratio of 1:10
197
(split vent flow = 10 mL / min, column flow = 1 mL / min). 11 ACS Paragon Plus Environment
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198 199
Statistical Analyses An Analysis of Variance (ANOVA) was performed based on the following linear mixed
200 201
model: = + + + + +
202
where is the observed phenotypic value corresponding to the ith year, the jth replication
203
within the ith year, and the kth genotype, is the grand population mean, is the random effect
204
of the ith year, is the random effect of the jth field replication nested within the ith year, is
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the fixed effect of the kth genotype, is the random interaction between the ith year and the kth
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genotype, and
207
the ith year, and the kth genotype. The random error effects
208
independently distributed with mean 0 and variance !"# .
is the random error term associated with the ith year, the jth replication within are
assumed to be normally and
209
Inbreds and hybrids were analyzed separately using this statistical model in SAS PROC
210
MIXED (version 9.3). Due to adverse field conditions in 2010 and 2011, a balanced set of inbred
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samples was not available for analysis. Therefore, the REML method was used to conduct the
212
ANOVA and calculate p-values associated with the genotypic effect as well as the standard error
213
of the differences between inbred means. The REML method was also used to conduct the
214
ANOVA for the tocopherols because several samples had tocopherol contents below the GC
215
detection level, leading to an unbalanced data set for the tocopherol analyses.
216
The assumptions of normality and homogenous variances were validated by conducting
217
Shapiro-Wilk’s test of normality on the residuals in PROC UNIVARIATE and by using the
218
Brown-Forsythe modification of the Levene test in the MEANS option of PROC GLM. If a
219
highly significant year-by-genotype interaction existed and the average concentration was high 12 ACS Paragon Plus Environment
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enough to warrant further investigation of the compound, multi-degree of freedom contrast
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statements were used to examine the genotype effect in each year. If phytochemicals displayed
222
either a mean or a variance that was at least as large as what is found in other germplasm sources
223
reported in the literature, the relationship between years was measured using Pearson correlation
224
coefficients, which were calculated in PROC CORR.
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RESULTS
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Total Protein Content
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The mean total protein content of hybrids and inbreds was found to be 9.12% and
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11.51%, respectively (Table 2). The protein content observed in this study ranged between
229
8.34% and 10.22% in the hybrids and between 9.86% and 13.24% in the inbreds.
230 231
Unsaturated Fatty Acid Content
232
In the hybrid group, oleic acid varied between 7.21 and 12.22 mg g-1, whereas linoleic
233
acid ranged from 17.38 to 21.71 mg g-1. In the inbred group, oleic acid ranged between 4.77 and
234
9.51 mg g-1, whereas linoleic acid ranged between 15.92 and 22.90 mg g-1. The hybrids
235
examined in this study varied between 21.2% and 33.4% for oleic acid and between 50.4% and
236
62.7% for linoleic acid content in the oil (Table 2).
237 238
Tocopherols The mean content of α-, γ-, and δ-tocopherol in the hybrids were 5.42 g g-1, 21.74 g g-
239 240
1
, and 1.47 g g-1, respectively (Table 2). The mean content of α-, γ-, and δ-tocopherol in the
241
parental inbreds was 5.70 g g-1, 18.68 g g-1, and 1.16 g g-1, respectively (Table 2). The
242
tocopherol values observed in the hybrids differed very little from the values observed in the
243
inbreds.
244 245
Soluble and Insoluble-Bound Phenolics
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Soluble ferulic acid and p-coumaric acid were consistently detected at low concentration
247
levels using UPLC. The average concentrations of these two compounds in hybrids were 1.44 g
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g-1 and 1.65 g g-1, respectively. In addition to the low mean concentration of both soluble
249
ferulic acid and soluble p-coumaric acid, these compounds exhibited a small range in
250
concentrations among both inbreds and hybrids (Table 2). Conversely, soluble cinnamic acid, a
251
carboxylic acid, was consistently more prevalent than either of the detected phenolic acids (Table
252
2). The average soluble cinnamic acid concentration was 67.09 g g-1 in the hybrids and 78.89
253
g g-1 in the inbreds. Additionally, the range in soluble cinnamic acid content was relatively
254
large in comparison to the soluble phenolics (Table 2).
255
Insoluble-bound ferulic acid and p-coumaric acid were consistently detected using gas
256
chromatography. Insoluble-bound ferulic acid was the most prevalent of all the phenolics. The
257
average concentration of insoluble-bound ferulic acid in hybrids and inbreds was 1,950.29 g g-1
258
and 1,952.49 g g-1, respectively. The average concentration of insoluble-bound p-coumaric acid
259
was 176.19 g g-1 in the hybrids and 222.94 g g-1 in the inbreds. The range observed in the
260
insoluble-bound phenolics content was the highest of all compounds examined in this research
261
(Table 2).
262 263
Year×Genotype Interactions
264
The three growing seasons used in this experiment were widely different with regard to
265
their temperature and rainfall totals.28 In general, 2009 was a very mild year. The year 2010 was
266
mild but wet during the first half of the growing season and hot and somewhat dry during the
267
second half of the growing season. Lastly, the year 2011 was very hot and dry during flowering,
268
pollination, and seed set. Not surprisingly, the Year and Year×Genotype interaction terms in the
269
model were often significant. However, while the mean concentration of a genotype for a
270
particular phytochemical may have changed depending on the specific environment, the relative
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ranking of the hybrid and inbred means for each specific beneficial phytochemical changed very
272
little. Interestingly, the beneficial phytochemicals which displayed relatively large means or
273
variances in this study, in comparison to the means and variances reported for other sets of
274
germplasm, were largely controlled by genotypic effects rather than by environmental effects
275
(Table 3). Furthermore, the correlation between the mean insoluble-bound ferulic acid content
276
for each hybrid across all three years ranged between 76% and 83%. The correlation between the
277
mean insoluble-bound p-coumaric acid content for each hybrid across all three years ranged
278
between 84% and 92%. Lastly, the correlation between the mean soluble cinnamic acid content
279
for each hybrid across all three years ranged between 73% and 87%.
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DISCUSSION
281
The ultimate goal of this research was to examine the potential for improving the
282
nutritional content and grain composition of maize hybrids adapted to the U.S. Corn Belt. As a
283
first step toward this goal, we determined the average content and variability of total protein,
284
unsaturated fatty acids, tocopherols, and phenolics in elite U.S. maize germplasm. This
285
information could prove beneficial to plant breeders, food scientists, and animal scientists who
286
have either an interest in improving the nutritional quality of maize grain for animal feed or an
287
interest in improving the nutritional quality of processed maize food products for human
288
consumption. If the research focus is to increase the quality of processed maize food products for
289
human consumption, then both breeding and processing considerations must be examined in
290
future studies. Here, we address the initial question of whether the U.S. maize germplasm alone
291
is well-suited for use in improving the nutritional quality of the whole grain.
292 293
Precision of Analytical Chemistry Analyses
294
The standard errors of all compounds analyzed were very small in comparison to the
295
averages of the respective traits (Table 2). This is indicative of very precise analytical chemistry
296
techniques, which is unquestionably favorable because it shows the high quality of the analyses
297
in this study. However, this also shows that even the slightest differences among hybrid or inbred
298
means could result in a significant P-value. Even if significant differences between hybrid means
299
or inbred means exist, as indicated by a significant P-value, the range in those differences might
300
be much smaller than what is already present in other germplasm collections. Additionally, as in
301
this study, the mean concentrations of certain phytochemicals were small in comparison to those
302
reported in other studies, even though significant P-values were detected for the differences in
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mean content. Thus, while plant improvement, as measured by the selection response, can be
304
accomplished if significant differences exist between genotypes, the rate and magnitude of the
305
selection response will be greater if plant breeders work with germplasm that already exhibits a
306
large mean and variance for the trait(s) of interest. Therefore, while significance levels for all
307
phytochemicals examined are reported, the magnitude of the mean and range of each beneficial
308
phytochemical should also be considered and compared to other sets of germplasm.
309 310
Protein
311
The protein content measured in our study corresponds closely to the typical average of
312
8-11% in commercial maize germplasm.29 Also, the range in the average protein content in this
313
study is small in comparison to other studies, particularly the Illinois Long-Term Selection
314
Experiments. For instance, Goldman, et al.30 evaluated germplasm that ranged between 4% and
315
25% protein content, and Dudley and Lambert31 reported protein contents that ranged between
316
5.2% and 25.2%.
317
Furthermore, the mean protein content observed in this study is lower than what is
318
recorded for other germplasm sets. As another example, Singh et al.32 evaluated a set of 49
319
accessions used in the Germplasm Enhancement of Maize (GEM) project. These accessions had
320
protein contents ranging between 12 and 14%. However, most breeding efforts for commercial
321
germplasm have focused primarily on the improvement of grain yield. Grain yield is chiefly
322
governed by the starch content, and the starch content and grain yield significantly decrease if
323
protein content is increased beyond 11 to 12%.29 It also appears that as breeders have selected for
324
higher yield (and an inherently higher starch content), indirect selection has caused protein
325
contents to decrease in commercial maize germplasm in the U.S. The DuPont Pioneer Hi-Bred
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326
Era study found that generations of plant breeding and selection caused protein content to
327
decrease from approximately 13.4% in 1920 to the lower percentages seen in the adapted
328
germplasm grown today.33 Therefore, the relatively low protein content and the small range in
329
protein content observed in this study are not surprising. Thus, while it is possible to improve
330
protein content using typical commercial maize germplasm, it appears that the rate of
331
improvement would be greater if other germplasm sources, such as those found in the Illinois
332
High Protein experiments or the accessions used in the GEM project, were used in addition to
333
commercial maize germplasm. It should be noted that this statement is in reference to the
334
improvement of total protein, not the protein quality. Other studies, such as those making use of
335
the Opaque-2 gene in maize, have had considerable success in improving the quality of maize
336
protein. However, that is not the focus of this research.
337 338
Unsaturated Fatty Acids and Tocopherols
339
In this study, neither the oleic acid content nor the linoleic acid content displayed a large
340
mean or a wide range, particularly in comparison to studies such as the Illinois Long-Term
341
Selection experiments. This is true of both the inbreds and the hybrids (Table 2). In comparison
342
to the results presented here, a study conducted by Poneleit and Davis34 reported that oleic acid
343
accounts for approximately 17% to 39% of the total extracted oil, whereas linoleic acid
344
constituted approximately 44% to 69%. Another study which used lines derived from Cycle 90
345
of the Illinois High Oil population and Cycle 19 of the Illinois Low Oil population (Early
346
Maturity) as well as other maize lines with slightly less extreme phenotypes showed that the
347
oleic acid content of the oil ranged between approximately 14% and 38% and that the linoleic
348
acid content ranged between approximately 47% and 72%.35 This suggests that other germplasm
19 ACS Paragon Plus Environment
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Page 20 of 35
349
resources show more diversity not only for the total amount of unsaturated fatty acids but also
350
for the overall composition of these fatty acids in the oil. Given that corn yield is correlated with
351
starch content in the grain, it is not surprising that there is little variability in the unsaturated fatty
352
acid content and composition among the elite hybrids and inbreds used in this study. Breeders
353
have traditionally prioritized yield, and the grain yield of high oil lines tends to be reduced.29
354
As expected, γ-tocopherol was the most prevalent tocopherol, followed by α-tocopherol
355
and then δ-tocopherol (Table 2). The small ranges observed for both α- and δ-tocopherol (Table
356
2) and the fact that the concentrations of these two phytochemicals were so small that they were
357
barely detectable using gas chromatography make these two phytochemicals unlikely candidates
358
for future maize improvements studies using our germplasm alone without tapping other sources.
359
However, γ-tocopherol did exhibit some variability, which might present potential for continued
360
study and selection.
361
Interestingly, the tocopherol concentration found in our study and those reported by
362
Egesel et al.36 and Lipka et al.37 were considerably different. These discrepancies can be
363
explained by the different germplasm sets used in each study. Egesel et al.36 chose high-oil lines,
364
high tocopherol-containing lines, and inbreds which were known to have either a high or low
365
ratio of α- to γ-tocopherol. As a result, Egesel et al.36 reported ranges of 11.3 to 66.3 µg g-1 for α-
366
tocopherol and 22.8 to 238.7 µg g-1 for γ-tocopherol. They also reported means of 32.9 µg g-1
367
and 120.5 µg g-1 for α-tocopherol and γ-tocopherol, respectively. The concentration of δ-
368
tocopherol was not explicitly reported in that study. In Lipka et al,37 281 maize lines which
369
collectively represent a significant portion of the variation in both temperate and tropical maize
370
breeding programs were used, but these did not include as extreme phenotypes as those
371
evaluated in Egesel, et al.36 Lipka et al.37 reported ranges of 0.70 to 31.35 µg g-1, 5.04 to 85.94
20 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
372
µg g-1, and 0.22 to 3.32 µg g-1 for α-, γ-, and δ-tocopherol, respectively. Our results more closely
373
resembled those of Lipka, et al.37 because, like Lipka et al,37 we did not use as extreme
374
phenotypes as Egesel, et al.36 However, the range observed in our study is less than that observed
375
in Lipka, et al.37 because we did not measure as diverse of a germplasm set. Additionally, our
376
results indicate that for all three tocopherols, the environmental factor was the greatest source of
377
variability, whereas genotype×environment interactions were not significant. This could explain
378
why the means and ranges observed in our study differ from those reported in other studies, but it
379
is still apparent that the means and ranges of the materials used in our study would most likely
380
not be as extreme even under different environmental conditions.
381
The location of the fatty acids and tocopherols in maize kernels makes the improvement
382
of their concentrations in maize-based food products challenging. Fatty acids and tocopherols are
383
almost exclusively found in the germ.36 The germ is typically removed during food product
384
processing and used in the production of corn oil. However, oil-containing foods tend to have
385
relatively short shelf lives because oils can become rancid.36, 38 Therefore, many other maize-
386
based processed food products, including breakfast cereals and snack products, are often derived
387
from the endosperm. Thus, the small amount of oleic acid, linoleic acid, and tocopherols present
388
in the whole kernel will not be present in many maize-based processed food products.
389
γ- and δ-Tocopherol have much lower antioxidant activity than α-tocopherol. Since γ-
390
tocopherol is by far the most prevalent of the tocopherols and α-tocopherol was only found in
391
small quantities in the materials examined, this set of germplasm is not well suited for
392
improvement of tocopherol concentration if improved antioxidant activity is the main objective.
393
It should be noted, however, that the results of Egesel et al.36 and Lipka et al.37 indicate that other
394
germplasm resources have considerably higher mean tocopherol contents and more variable
21 ACS Paragon Plus Environment
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Page 22 of 35
395
tocopherol contents than the germplasm examined in our study. These studies suggest that it may
396
be possible to improve the tocopherol content of whole maize kernels. However, the yellow dent
397
maize genotypes adapted and commercialized in the U.S. Corn Belt do not appear to be well
398
suited for use in such a breeding program without the incorporation of other germplasm sources
399
into this collection of genotypes.
400 401
Soluble and Insoluble-Bound Phenolics
402
The concentrations reported in this study correspond closely to those reported in other
403
studies.17, 39 All phenolics detected were phenolic acids; furthermore, all of the phenolic acids
404
were hydroxycinnamic acids. Of these, ferulic acid was the most prevalent, followed by p-
405
coumaric acid. Most of the hydroxycinnamic acids were in the insoluble-bound state, which is in
406
agreement with Adom and Liu.17 In contrast to the hydroxycinnamic acids, cinnamic acid was
407
found primarily in the soluble state.
408
In comparison to other studies which examined the concentration of ferulic acid in
409
different maize genotypes, the amount of insoluble-bound ferulic acid extracted from maize
410
germplasm typical of the U.S. Corn Belt is moderate to high and variable between genotypes.
411
For instance, Lopez-Martinez et al.40 studied the amount and range of insoluble-bound ferulic
412
acid in 18 maize lines from Mexico, yellow dent maize, and more darkly pigmented maize
413
varieties. In that study, the extractable insoluble-bound ferulic acid content ranged between
414
1,380 g g-1 and 1,610 g g-1. In our study, the insoluble-bound ferulic acid content ranged from
415
1,567 g g-1 to 2,375 g g-1. Furthermore, Garcia-Lara and Bergvinson41 reported that P84, a
416
maize line bred specifically for resistance to insect feeding, largely due to an increased
417
concentration of insoluble-bound hydroxycinnamic acids, had an average ferulic acid
22 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
418
concentration of 2,200 g g-1. This is comparable to some of the hybrids and inbreds used in our
419
study.
420
The finding that maize germplasm from the U.S. Corn Belt contains relatively high levels
421
of insoluble-bound hydroxycinnamic acids, particularly ferulic acid, is not surprising. Insoluble-
422
bound hydroxycinnamic acid content is positively correlated with insect and pathogen resistance
423
in maize.42-47 Therefore, while plant breeders have not been directly selecting for higher
424
insoluble-bound hydroxycinnamic acid content, they have been selecting for improved resistance
425
to insect pests and diseases. However, insect and pathogen resistance are not solely influenced by
426
the insoluble-bound hydroxycinnamic acid content. Therefore, the observed variability in the
427
insoluble-bound hydroxycinnamic acid concentrations in our germplasm is not unexpected. It
428
should also be noted that Garcia-Lara and Bergvinson41 further increased P84’s insoluble-bound
429
phenolic acid content by incorporating new germplasm in their breeding program. Therefore,
430
while the adapted germplasm typical of the U.S. Corn Belt appears to be useful in improving the
431
phenolic acid content in maize, the gradual incorporation of other germplasm sources into the
432
plant breeding scheme may help improve the phenolic acid content in maize more rapidly.
433
Due to their high concentrations and variability, the insoluble-bound hydroxycinnamic
434
acids present opportunity for further study and possible improvement in maize. Furthermore, the
435
lack of a change of rank in the hybrids and the inbreds, even when the Year and Year×Genotype
436
terms were significant, suggests that selection implemented in a breeding program would result
437
in genetic gain for these traits. Collectively, these observations suggest that breeding maize for
438
improved insoluble-bound hydroxycinnamic acid content in the whole grain is feasible.
439
However, because maize-based food products must be processed before human
440
consumption, special care should be taken to monitor the fate of the insoluble-bound
23 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 24 of 35
441
hydroxycinnamic acids during food product processing. There is some evidence that thermal
442
stresses encountered in processing may free insoluble-bound hydroxycinnamic acids from the
443
arabinoxylans of the cell wall.48 If this observation holds true during maize food product
444
processing, it is possible that the concentration of the soluble ferulic acid and p-coumaric acid
445
may increase while the concentration of the insoluble-bound hydroxycinnamic acids decreases. It
446
is also possible that processing and thermal stresses may degrade the hydroxycinnamic acids,
447
leading to higher concentrations of cinnamic acid. Due to the fact that most phenolic acids in
448
maize are located in the bran, it is also possible that many of the phenolic acids may be “lost” if
449
the bran is removed during food product processing. Future studies may examine the fate of both
450
soluble and insoluble-bound hydroxycinnamic acids as well as cinnamic acid throughout food
451
product processing. Such studies may prove beneficial in increasing the concentrations of these
452
phytochemicals in processed maize-based food products and point the way for new uses of
453
maize.
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Journal of Agricultural and Food Chemistry
ABBREVIATIONS USED
454 455
FAME – Fatty Acid Methyl Ester
456
NIR – Near-Infrared Spectroscopy
457
NMR – Nuclear Magnetic Resonance
458
NSS – Non-Stiff Stalk
459
SSS – Stiff Stalk Synthetic
460
UPLC – Ultra Performance Liquid Chromatography
461 462
ACKNOWLEDGEMENTS
463
The authors would like to thank Tom Patterson and the Analytical Technologies Team at Dow
464
AgroSciences for their assistance in developing these protocols and for their mentorship. We
465
would also like to thank Nicole Yana for her invaluable help in coordinating research logistics.
466
FUNDING SOURCES
467 468
This work was funded in part through gifts from the Kellogg Company and Dow AgroSciences
469
and through USDA Hatch Grant, award ILLU-802-354. Student support was provided by the
470
Illinois Distinguished Fellowship and the William B. and Nancy L. Ambrose Fellowship in Crop
471
Sciences.
472 473 474 475
CONFLICT OF INTEREST The authors declare no competing financial interest.
25 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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31 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
TABLES Table 1. Inbred maize parents used in this study and additional information regarding their pedigree background Line
Group
Assignee
Background
B73
SSS
None (Public)
Iowa Stiff Stalk Synthetic
LH1
SSS
Holden Foundation Seeds
Iowa Stiff Stalk Synthetic; B37 type
PHG39
SSS
Pioneer Hi-Bred International
Maiz Amargo/Iowa Stiff Stalk Synthetic; B37/B14 type
PHJ40
SSS
Pioneer Hi-Bred International
Iowa Stiff Stalk Synthetic
4676A
SSS
Dekalb Genetics Corporation
1067-1 / B-Line Composite; B14 type
LH123HT
NSS
Holden Foundation Seeds
Pioneer Hybrid 3535
LH82
NSS
Holden Foundation Seeds
Krug /W153
Mo17
NSS
None (Public)
Lancaster
PH207
NSS
Pioneer Hi-Bred International
Iodent/Long Ear OPV/Minn13
PHG47
NSS
Pioneer Hi-Bred International
Oh43/Iodent*Wf9/MKSDTA C10 Synthetic
PHG84
NSS
Pioneer Hi-Bred International
Oh07-Midland/Minn13/Iodent/Reid YD/Osterland YD/Lancaster/Pioneer Female Composite OPV
PHZ51
NSS
Pioneer Hi-Bred International
Minn13/Iodent/Reid YD/Osterland YD/Lancaster/South US Land Race Synthetic/Funks G4949/ Midland
31 ACS Paragon Plus Environment
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Page 33 of 35
Journal of Agricultural and Food Chemistry
Table 2. Summary statistics of protein, unsaturated fatty acids, tocopherols, phenolics, and cinnamic acid by generation. The minimum, maximum, and average values reported are based off of the LSMEANS of the hybrids and inbreds.
HYBRIDS Average Min Max Std. Errora P-Valueb
Protein
Unsaturated Fatty Acids
Total Protein
Oleic Acid
—%—
—— mg g-1 ——
9.12 8.34 10.22 0.26