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Cite this: DOI: 10.1039/d2fo03606a
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Trait stacking simultaneously enhances provitamin
A carotenoid and mineral bioaccessibility in
biofortified Sorghum bicolor†
Michael P. Dzakovich, *a,b Hawi Debelo, a Marc C. Albertsen, c Ping Che,
Todd J. Jones, c Marissa K. Simon, c Zuo-Yu Zhao,c Kimberly Glassmanc and
Mario G. Ferruzzi *a,d
c
Vitamin A, iron, and zinc deficiencies are major nutritional inadequacies in sub-Saharan Africa and disproportionately affect women and children. Biotechnology strategies have been tested to individually
improve provitamin A carotenoid or mineral content and/or bioaccessibility in staple crops including
sorghum (Sorghum bicolor). However, concurrent carotenoid and mineral enhancement has not been
thoroughly assessed and antagonism between these chemical classes has been reported. This work evaluated two genetically engineered constructs containing a suite of heterologous genes to increase carotenoid stability and pathway flux, as well as phytase to catabolize phytate and increase mineral bioaccessibility. Model porridges made from transgenic events were evaluated for carotenoid and mineral content as
well as bioaccessibility. Transgenic events produced markedly higher amounts of carotenoids (26.4 µg g−1
DW) compared to null segregants (4.2 µg g−1 DW) and wild-type control (Tx430; 3.7 µg g−1 DW). Phytase
activation by pre-steeping flour resulted in significant phytate reduction (9.4 to 4.2 mg g−1 DW), altered
the profile of inositol phosphate catabolites, and reduced molar ratios of phytate to iron (16.0 to 4.1), and
zinc (19.0 to 4.9) in engineered material, suggesting improved mineral bioaccessibility. Improved phytate :
mineral ratios did not significantly affect micellarization and bioaccessible provitamin A carotenoids were
Received 22nd November 2022,
Accepted 26th May 2023
DOI: 10.1039/d2fo03606a
rsc.li/food-function
1.
over 23 times greater in transgenic events compared to corresponding null segregants and wild-type
controls. A 200 g serving of porridge made with these transgenic events provide an estimated 53.7% of a
4–8-year-old child’s vitamin A estimated average requirement. These data suggest that combinatorial
approaches to enhance micronutrient content and bioaccessibility are feasible and warrant further assessment in human studies.
Introduction
Incidence of food insecurity is unusually high in sub-Saharan
Africa where diets are dominated by carbohydrate-rich cereal
grains.1,2 As a result of food scarcity and regional dietary patterns, vitamin A, iron, and zinc deficiencies remain
prominent.3,4 Chronic vitamin A deficiency can result in xer-
a
Plants for Human Health Institute, North Carolina State University, 600 Laureate
Way, Kannapolis, North Carolina 28081, USA
b
USDA-ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor
College of Medicine, 1100 Bates Ave., Houston, TX 77030, USA.
E-mail: Michael.Dzakovich@usda.gov
c
Corteva Agriscience, 8305 NW 62nd Ave., Johnston, IA 50131, USA
d
Arkansas Children’s Nutrition Center, Section of Developmental Nutrition,
University of Arkansas for Medical Sciences, 15 Children’s Way, Little Rock, AR
72202, USA. E-mail: MFerruzzi@uams.edu
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/
10.1039/d2fo03606a
This journal is © The Royal Society of Chemistry 2023
ophthalmia; the leading cause of preventable blindness in
children.5,6 Likewise, iron and zinc are required for normal
growth and development and their deficiencies are associated
with an increased risk of death from other diseases such as
diarrhea.7,8 Considering social, economic, and environmental
factors, food choice and availability are unlikely to markedly
change in sub-Saharan Africa in the near future. Improvement
of staple foods already predominant in the broader diet of atrisk populations is a viable strategy to ameliorate
malnutrition.
Sorghum (Sorghum bicolor (L.) Moench) is a close relative of
maize (Zea mays) and one of the most popular cereal grains
consumed in sub-Saharan Africa.9 Sorghum is often processed
into porridges (e.g. tô) by mixing flour with boiling water.10
However, sorghum and subsequent food products are generally low in micronutrients such as iron and zinc as well as provitamin A carotenoids.11,12 The nutritional impacts of low
mineral contents are exacerbated by the presence of high
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Food & Function
levels of phytate (inositol hexakisphosphate), a major phosphorous storage molecule that strongly chelates divalent
metals like iron and zinc, and substantially reduces their bioavailability in humans.13 Previous research using labeled
mineral elements estimate that molar ratios of phytate to iron
or zinc above 10–15 indicate poor bioavailability.14–17
Depending on sorghum genetic background and environmental conditions, reported molar mineral/phytate ratios are
overwhelmingly above these thresholds and vary between 6–55
for phytate to iron and 30–40 for phytate to zinc.11,18,19 Given
the low levels of carotenoids, high concentration of phytate,
and dietary significance of the crop, sorghum is an ideal candidate for biofortification efforts.
Biofortification strategies seeking to simultaneously
improve delivery of provitamin A and key shortfall minerals
such as iron and zinc are rare. Improvements in carotenoid
content, stability, and reducing phytate concentration have
been tested individually by ectopic expression of non-codon
optimized genes including homogentisate geranylgeranyl
transferase (HGGT ), phytoene synthase (PSY1), phytoene desaturase (CRTI), phytoene synthase (CRTB), deoxy-xylulose-phosphate synthase (DXS), and phytase (PhyA).20–24 These studies
demonstrated that not only could carotenoids concentrations
be significantly increased in sorghum grain (e.g. CRTB), but
their stability could be substantially enhanced by increasing
antioxidant tocopherol and tocotrienol species (e.g. HGGT ).
Through modern biotechnological approaches, it is theoretically possible to address both provitamin A carotenoid and
mineral content as well as minimizing factors that negatively
impact bioavailability. However, potential for antagonism
between carotenoid bioavailability and divalent minerals has
been reported in in vitro and in vivo studies.25–29 These
reported effects are concentration dependent and their
relationship in the context of sorghum biofortification
remains unexplored, but critical to define.
To investigate potential antagonism between carotenoid
bioavailability and divalent minerals released by enzymatic
phytate degradation, codon optimized vectors (ABS4-1 and
ABS4-2) were designed and used to develop multiple transgenic events (Table 1). These events were engineered to simul-
Table 1
Description of transgenic and non-transgenic material
Transgene combinationa
ABS4-1 = HGGT + PSY1 + CRTI + PhyA
ABS4-2 = HGGT + CRTB + CRTI + PhyA
NA
a
Event
identifier
Genetic
background
ABS4-1A
ABS4-1B
ABS4-1C
ABS4-2A
ABS4-2B
ABS4-2C
ABS4-2D
Control
Tx430
Tx430
Tx430
Enzyme abbreviations are defined as follows: PSY1, phytoene
synthase 1, CRTB, bacterial phytoene synthase, CRTI, bacterial phytoene desaturase, HGGT, homogentisate geranylgeranyl transferase,
PhyA, phytase, and PSY1, phytoene synthase 1.
Food Funct.
taneously improve provitamin A carotenoid biosynthetic
capacity, stability, and increased ability to degrade phytate
compared to previous efforts.21,22 Sorghum events were processed into model porridges and a three-stage in vitro digestion model was utilized to evaluate carotenoid and mineral
bioaccessibility. Due to the intrinsically low mineral content of
sorghum, we hypothesized that increased mineral release from
phytate degradation would not significantly counteract the
delivery of provitamin A carotenoids. Our results suggest that
these transgenic sorghum events provide dramatically higher
amounts of bioaccessible provitamin A carotenoids compared
to previous efforts and exhibit altered phytate : iron/zinc molar
ratios suggestive of enhanced mineral bioaccessibility.
2. Materials and methods
2.1
Chemicals and reagents
Reagents sourced from Sigma Aldrich (Sigma Chemical Co.,
St Louis, MO, USA) included mucin (M2378), α-amylase (A3176
(lot: SLCF0615)), pepsin (P7125 (lot: SLBW6671)), lipase (L3126
(lot: SLBX2124)), pancreatin (P7545 (lot: SLBV6830)), bile
(B8631 (lot: SLBX1760) and authentic standards of α-carotene,
β-carotene, lutein, lycopene, trans-β-apo-8′-carotenal, retinyl
palmitate, and zeaxanthin. Urea (U15-500) and uric acid
(A13346-14) as well as components for the oral phase base
solution ( potassium chloride, sodium phosphate, sodium
sulfate, sodium chloride, and sodium bicarbonate) were purchased from Fisher Scientific (Fisher Scientific, Waltham, MA,
USA). Reagents used for the extraction and analysis of carotenoids and inositol phosphates included LC-MS grade water,
methanol, and acetonitrile, HPLC grade ammonium acetate,
dihexylammonium acetate, ethyl acetate, and glacial acetic
acid, as well as ACS grade acetone, ethanol, hexanes, isopropanol, methyl tert-butyl ether, and petroleum ether purchased
from Fisher Scientific.
2.2 Sorghum transformation, plant material, and harvest
conditions
Immature embryo explants isolated from greenhouse grown
sorghum plants were transformed with Agrobacterium auxotrophic strain LBA4404 Thy-carrying a ternary vector transformation system to generate transgenic sorghum plants as
previously described.30 Transgenic grains used in this study
were generated from two transformation vectors: ABS4-1 and
ABS4-2 (Table 1). ABS4-1 carried the maize codon-optimized
phytoene desaturase CRTI22,31 gene from Pantoea ananatis to
increase provitamin A biosynthesis, the maize codon-optimized phytoene synthase PSY122,32 gene from Zea mays L. to
modulate flux through the carotenoid pathway, the homogentisate geranylgeranyl transferase HGGT22,33 gene from H. vulgare
to increase vitamin E accumulation, the maize codon optimized phytase PhyA34 gene from Aspergillus niger used for
phytate metabolism, and the phosphomannose isomerase
PMI30,35 gene from Escherichia coli as selectable marker. ABS42 carried all the identical genes as described in ABS4-1 except
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PSY1 was replaced by maize codon-optimized CRTB,36 another
phytoene synthase gene from Erwinia uredovora. In both constructs, CRTB, fused to a maize codon optimized delta-4-palmitoyl-ACP desaturase gene transit peptide (Cs-DPAD),37 and
PSY1 were driven with the same sorghum α-kafirin promoter.38
CRTI was fused to a maize codon optimized ribulose-1,5bisphosphate carboxylase small subunit transit peptide (PS
SSU TP)37,39 and was driven by the sorghum β-kafirin promoter.38 PhyA was fused to a H. vulgare alpha amylase signal
peptide (BAASS)40 and was driven by the endosperm-specific
ZM-LEG1A promoter. HGGT was driven by the Zm-WS1 whole
seed promoter (seed-specific KG86 promoter41), and PMI was
driven with the Zm-UBI1 promoter.30,35
Transgenic events derived from the same transformation
vector represent independent transgenic events (Table 1). All
transgenic events used in this study were homozygous with a
single T-DNA insertion. Both null segregants isolated through
segregation of corresponding transgenic events and wild-type
Tx430 were used as controls. Panicles were collected from
sorghum plants 45-day after pollination, air dried at 24 °C for
2 weeks before threshing, and then stored at −80 °C.
2.3 High-throughput sorghum flour and experimental
porridge preparation
Whole sorghum kernels were added to fill approximately half
of a 15 mL polycarbonate vial (SPEX Sample Prep, Metuchen,
NJ, USA; UX-04545-71) containing two 14″ 440C stainless steel
balls (Grainger, Minooka, IL, USA; 4RJJ1). Samples were milled
into flour using a Geno/Grinder® 2010-115 (SPEX Sample
Prep) operated at 1400 RPM for 2 cycles of 2.5 minutes with
samples cooled between cycles. Sorghum flour was stored at
−40 °C until analysis.
Steeped samples underwent the following pre-treatment:
sorghum flour (0.4 g) was suspended in 0.8 mL of doubly distilled water and incubated at 37 °C for 3 hours at 120 oscillations per minute (OPM). After incubation, an additional
0.8 mL of doubly distilled water was added to each tube and
samples were agitated by hand. Samples were then cooked and
processed as described below.
Porridges were formulated based on a traditional Burkina
Faso style tô containing 20% sorghum flour and 80% water by
weight10 and scaled to a 10 mL volume to align with the
cooking method outlined by Lipkie and others.21 Briefly, 0.4 g
of sorghum flour was weighed into 15 mL flip-cap tubes and
1.6 mL of doubly distilled water was added. Each tube was agitated by hand until flour was fully dispersed. Tubes were transferred with their lids open to metal racks and placed into an
induction heated stock pot submerging only the portion of the
tubes that contained suspended flour. The stockpot lid was
replaced to maintain temperature at 100 °C and high humidity
while the samples cooked for 10 minutes. After cooking, racks
were removed and 100 mg (± 5 mg) of canola oil was added to
each tube (5% lipid by porridge mass) to promote micellarization during the intestinal phase and incorporated by manual
mixing. After the addition of canola oil (∼30 min), sample
tubes were transferred to a −80 °C freezer and subjected to a
This journal is © The Royal Society of Chemistry 2023
Paper
simulated digestion within 12 hours. All sorghum samples
were processed in triplicate to account for experimental
variation.
2.4
High-throughput in vitro digestions of sorghum porridge
A previously described three-stage high-throughput in vitro
digestion method that utilizes a Tecan Freedom EVO 150
liquid handling system (Tecan; Mannedorf, Switzerland) was
adapted for sorghum porridge.21,42 Briefly, thawed porridge
samples (2 g FW) were mixed with 1.2 mL of oral phase solution (containing 31.8 mg mL−1 of α-amylase; 380 units) and
incubated at 37 °C for 10 minutes at 120 OPM. Samples were
diluted with a 0.4 mL of 10 mg mL−1 pepsin solution (in 0.1 M
HCl; 270 units) and 2.7 mL of saline (0.9% NaCl) solution and
then adjusted to pH 2.5 by addition of 1.0 M HCl. Samples
were diluted with additional saline to 8 mL, blanketed with
nitrogen gas, and incubated at 37 °C for 1 hour at 120 OPM.
Samples were then adjusted to pH 5.0 using 1.0 M NaHCO3.
Then, 0.4 mL of pancreatin-lipase solution (20 mg mL−1 of
each enzyme in 0.1 M NaHCO3; 3520 units lipase) and 0.6 mL
of bile extract (30 mg mL−1) in 0.1 M NaHCO3 were added to
each sample. Samples were adjusted to pH 7.0 using 1.0 M
NaHCO3 and saline was added to dilute samples to a final
volume of 10 mL. Nitrogen gas was added to each tube and
samples were incubated at 37 °C for 2 hours at 120 OPM. After
incubation, samples were hand-agitated and 4 mL of digesta
was removed, aliquoted, and stored at −80 °C for future analysis. The remaining samples were blanketed with nitrogen gas
and centrifuged at 3428 × g for 75 minutes in an Eppendorf
5920R centrifuge (Eppendorf, Hamburg, Germany) maintained
at 4 °C. Following centrifugation, 4 mL aliquots of the aqueous
fraction were filtered through 0.22 µm cellulose acetate filters.
Samples were blanketed with nitrogen gas and stored at
−80 °C until analysis.
2.5
High-throughput extraction and analysis of carotenoids
2.5.1 Extraction of carotenoids from sorghum flour. A
rapid extraction protocol was developed based on a previously
validated method for extracting carotenoids from sorghum
flour.21 Briefly, 300 mg of sorghum flour was weighed in
2.0 mL microfuge tubes. Two 1/8″ 440C stainless steel balls
(Grainger; 4RJH5) were added and 150 µL of HPLC grade water
was added into each tube to hydrate the sample matrix. After
resting for 5 minutes, 30 µL of 150 µM retinyl palmitate (RP)
dissolved in ethanol was spiked into each sample as an
internal standard. Samples rested for 10 minutes prior to
the addition of 1 mL of 3 : 2 acetone : ethyl acetate + 0.01%
BHT (w/v). Tubes were shaken at 1400 RPM for 45 seconds in a
Geno/Grinder® 2010-115. Samples were centrifuged at
20 000 × g for 3 minutes and the supernatant was collected
into borosilicate tubes. The remaining sample pellets were
twice extracted as outlined above with methyl tert-butyl
ether + 0.01% BHT (w/v) or until pellet was colorless. Combined
supernatants were dried using a RapidVap (Labconco, Kansas
City, MO, USA), blanketed with nitrogen, and stored at −80 °C.
Samples were redissolved in 2 mL of 1 : 1 ethyl acetate : methanol
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and filtered through 0.45 µm PTFE syringe filters prior to analysis. Extractions were conducted in triplicate and under low-light
to minimize photoisomerization.
2.5.2 Extraction of carotenoids from digesta and aqueous
fractions. Extraction from digesta and aqueous fractions was
based on a previously validated method and adapted for a
Tecan Freedom EVO 150 liquid handling unit.21,42 Briefly,
100 µL of RP was added to 2 mL of digesta (150 µM RP) or
4 mL aqueous samples (30 µM RP). Samples were extracted
three times with one volume of 1 : 3 acetone : petroleum ether
+ 0.01% BHT (w/v). Samples were vortexed for 1 minute, centrifuged for 2 minutes at 4000 RPM, and the supernatant was collected. Combined supernatants were dried in a RapidVap and
stored at −80 °C until analysis (<24 hours later). Digesta and
aqueous fraction samples were resolubilized in 200 or 100 µL
of 1 : 3 ethyl acetate : methanol + 0.01% BHT (w/v), respectively,
and filtered with a 0.22 µm PTFE filter prior to analysis.
2.5.3 Analysis of carotenoids. Carotenoids were analyzed
using a modified high performance liquid chromatography
photo diode array detector (HPLC-PDA) method developed by
Lipkie and colleagues.21 Briefly, filtered carotenoid extracts
were run on a Waters Alliance e2695 (Waters Corporation,
Milford, MA, USA) with mobile phases containing 98 : 2 HPLC
grade methanol: 1.0 M ammonium acetate adjusted to pH 4.6
(A) and ethyl acetate (B). Solvent flow rate was maintained at
0.45 mL per minute and carotenoids were separated on a
2.0 mm × 150 mm YMC C30 column with 3 µm particle size
(YMC America, Devens, MA) using the following gradient:
100% A to 20% A over 4.93 minutes, 20% A to 0% A over
1.65 minutes, 0% A held for 1.46 minutes, a return to 100% A
over 1.0 minute, and a hold on 100% A for 3.46 minutes to
recondition the column. The column compartment and autosampler were maintained at 35 and 15 °C, respectively.
Carotenoids were quantified at 450 nm using a Waters 2998
PDA detector based on response curves of authentic standards.
For analytes quantified without authentic standards, an
adjusted slope was calculated using a ratio of molar extinction
coefficients with the most structurally related carotenoid available. The internal standard RP was quantified at 325 nm.
Limits of detection determined by least squares regression for
lutein, zeaxanthin, α-carotene, β-carotene, and lycopene were
0.28, 0.03, 0.11, 0.09, and 0.21 picomoles on column,
respectively.
2.6
Extraction and analysis of minerals from sorghum flour
Minerals were hot acid extracted from 0.2 g of sorghum flour
using an open-vessel microwave system, then diluted and analyzed using ICP-OES. All extractions and analyses were conducted by A&L Great Lakes Laboratories (Fort Wayne, IN, USA).
2.7
Extraction and analysis of inositol phosphates
Extraction of inositol phosphates from raw and steeped
sorghum samples was carried as described previously with
minor modifications.43 Briefly, 50 mg of ground sorghum
samples were defatted using 1.5 mL of hexane. The hexane
layer was carefully removed after centrifuging (3600 rpm,
Food Funct.
Food & Function
4 min) and the pellet was dried under nitrogen. The dried
samples were then resuspended with 1 mL of HCl (0.4 M) and
sonicated for 1 h at room temperature. Samples were centrifuged, supernatants were transferred into culture tubes and
dried using RapidVap vacuum evaporator. Dried samples were
then resolubilized with 500 μl of 5% acetonitrile, filtered using
0.45 µm PES syringe filter prior to LC-MS analysis.
Inositol phosphate content was determined using a Waters
UPLC Acquity I Class system coupled with a Xevo TQ-S triple
quadrupole mass spectrometer. Separation was performed on
a BEH C18 column (2.1 mm × 50 mm, 1.7 µm) at a flow rate of
0.5 mL min−1 using an adapted gradient as follows: 70% A to
62% A over 2.04 minutes, 62% A to 60% A over 0.03 minutes,
60% A to 44% A over 0.72 minutes, 44% A to 20% A over
0.61 minutes, 20% A to 70% A over 0.03 minutes, and a hold of
70% A for an additional 1.67 minutes.43 Mobile phases consisted of (A) 5% aqueous acetonitrile containing ion pairing
agents dihexylammonium acetate (5 mM) and ammonium
acetate (5 mM) and (B) 100% acetonitrile. Calibration curves of
D-myo-inositol-1-monophosphate dipotassium salt (IP1), D-myoinositol-1,4-diphosphate sodium salt (IP2), D-myo-inositol-1,4,5triphosphate, sodium salt (IP3), D-myo-inositol-1,3,4,5-tetraphosphate, sodium salt (IP4), D-myo-inositol-1,3,4,5,6-pentaphosphate, sodium salt (IP5) and myo-inositol-hexakis (dihydrogen
phosphate) (IP6) were used to quantify inositol phosphate
species using optimized multiple reaction monitoring (MRM)
experiments. Details regarding MRM experiments can be found
in the ESI.† Analytical conditions were as follows: electrospray
ionization in negative mode; capillary voltage: 3.0 kV; probe
temp: 150 °C; source temp: 600 °C; desolvation gas flow: 1000 L
h−1; cone gas flow: 5 L h−1.
2.8
Statistical analysis and data visualization
Statistical analyses and data visualization were conducted
using R version 4.1.1.44 Box and whisker plots were generated
using ggplot2 using the Wes Anderson color palette
generator.45,46 Fixed-effect analysis of variance models were
generated to determine if genetic background, transgene state
of the transgenic construct, or phytase activation pre-treatment
significantly affected outcomes measured in our experiments.
For the analysis of raw material carotenoid, mineral, and
phytate content, (excluding pre-treated phytase activated
samples) the following model was used:
Y ijk ¼ μ þ Ei þ T j þ Rk ðEi Þ þ εijk
represents the estimate for a given analyte within ith event, jth
transgene state, and kth technical replicate. μ represents the
mean of a given analyte or metric, Ei represents the contribution due to genetic factors for ith event of the germplasm, Tj
represents the contribution due to the transgene state of the
transgenic construct for the jth state, Rk(Ei) represents the contribution of the kth technical replicate within transgenic event,
and can be interpreted as within event variation, and εijk represents the residual error. If significance was determined, a
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3. Results
3.1 Carotenoid content increased in transgenic sorghum
events
Sorghum flours were analyzed for several major carotenoids
including those with provitamin A activity (Table 2). A comprehensive summary and statistical analysis of all carotenoids
and their geometric isomers is detailed in Table S1.†
Carotenoid values are presented on a “per serving” basis to
reflect the amount present in a standard 200 g serving of porridge. Additionally, we report multiple aggregate values including xanthophylls (lutein, zeaxanthin, α-cryptoxanthin, and
β-cryptoxanthin),
carotenes
(α-carotene,
cis-β-carotene,
β-carotene, lycopene, and any monitored cis isomers), and provitamin A content (1/2(α-cryptoxanthin + β-cryptoxanthin +
α-carotene + any monitored cis isomers) + β-carotene).
Overwhelmingly, transgenic material outperformed null segregants and the wild-type control in terms of carotenoid content.
On average, transgenic events contained between 2.7 to 32
times more carotenoids than their non-transgenic counterparts and wild-type control, depending on the carotenoid. A
200 g porridge serving made with the null segregants or wild
type control used in this study contained on average 0.15 mg
of xanthophylls, 0.03 mg of carotenes, and 0.02 mg of provitamin A content while a serving of porridge made with transgenic events contained on average 0.35 mg of xanthophylls,
0.70 mg of carotenes, and 0.67 mg of provitamin A content. By
design, transgenic sorghum events were particularly high in
β-carotene with an average content of 0.63 mg per 200 g
serving of porridge.
This journal is © The Royal Society of Chemistry 2023
192.2 ± 3.6ab
201.3 ± 6.9a
193.6 ± 4.9ab
183.3 ± 8.6b
189.2 ± 6.6ab
144.0 ± 4.2c
133.5 ± 8.8c
72.6 ± 2.8d
73.6 ± 2.2d
59.9 ± 0.9d
61.6 ± 5.1d
65.2 ± 3.8d
73.7 ± 4.7d
58.2 ± 1.2d
64.9 ± 5.6d
194.2 ± 2.7a
152.7 ± 3.3bc
162.2 ± 7.0b
126.3 ± 4.2d
149.9 ± 0.4c
127.9 ± 5.7d
134.2 ± 2.3d
93.2 ± 3.6e
75.9 ± 2.3f
76.9 ± 2.4f
64.2 ± 5.2g
77.5 ± 3.4f
84.0 ± 1.4ef
80.5 ± 1.5f
60.9 ± 1.3g
18.1 ± 1.7abc
9.9 ± 2.2a 17.7 ± 2.0ab
19.5 ± 1.6a
11.5 ± 1.9a 16.1 ± 0.5b
10.6 ± 7.2abcde 9.4 ± 0.7a 15.5 ± 0.6b
20.6 ± 1.0a
8.4 ± 4.0a 36.7 ± 1.4a
18.4 ± 14.8ab 10.1 ± 7.9a 28.6 ± 24.9ab
15.9 ± 1.0abcd 8.2 ± 0.5a 22.3 ± 1.3ab
13.7 ± 0.9abcde 10.2 ± 0.6a 23.9 ± 0.3ab
nd
4.8 ± 3.0cde
nd f
5.0 ± 0.3bcde
nd
nd
3.7 ± 2.1de
nd
nd
3.9 ± 0.4de
nd
nd
4.7 ± 0.3cde
nd
nd
2.4 ± 1.8e
nd
nd
5.9 ± 0.5bcde
nd
nd
4.8 ± 0.5abc
nd
nd
α-Carotene
581.2 ± 6.1c
614.7 ± 13.6b
624.9 ± 22.6b
606.4 ± 20.9bc
850.7 ± 13.4a
505.5 ± 14.6d
599.5 ± 12.3bc
20.8 ± 0.4e
21.3 ± 0.5e
17.8 ± 2.4e
15.5 ± 1.4e
19.3 ± 2.6e
23.7 ± 2.8e
17.9 ± 1.7e
11.0 ± 0.9e
β-Carotene
36.70 ± 8.77a
37.35 ± 3.01a
37.23 ± 1.43a
26.88 ± 4.00ab
31.94 ± 15.13ab
16.75 ± 1.01bcd
22.67 ± 4.42abc
6.91 ± 0.88d
6.35 ± 0.52d
7.33 ± 4.70cd
5.43 ± 0.77d
5.89 ± 1.16d
7.10 ± 4.77cd
4.66 ± 1.48d
6.30 ± 1.15d
cis-β-Carotene
10.3 ± 1.1d
7.6 ± 0.7e
8.5 ± 1.1e
24.7 ± 0.6b
28.6 ± 0.7a
22.1 ± 0.2c
25.7 ± 1.0b
nd
nd
nd
nd
nd
nd
nd
nd
Lycopene
414.4 ± 9.4a
385.1 ± 6.7ab
375.8 ± 12.1b
338.7 ± 15.5c
366.7 ± 30.5bc
295.9 ± 10.0d
291.7 ± 12.5d
170.2 ± 8.3e
154.6 ± 3.8ef
140.3 ± 5.0ef
129.8 ± 10.6f
147.3 ± 6.3ef
159.2 ± 8.6ef
144.6 ± 3.0ef
130.6 ± 6.5f
Xanthoc
646.0 ± 6.8c
675.8 ± 17.0bc
686.1 ± 22.7bc
694.7 ± 24.4b
939.5 ± 42.1a
566.7 ± 16.9d
671.7 ± 7.7bc
28.1 ± 1.2e
28.2 ± 0.7e
25.4 ± 3.2e
21.1 ± 0.8e
25.5 ± 4.1e
31.2 ± 7.2e
23.0 ± 2.9e
18.0 ± 1.6e
Carotenesd
622.4 ± 5.3b
657.0 ± 13.8b
661.3 ± 22.4b
652.7 ± 24.0b
894.6 ± 32.7a
537.1 ± 15.9c
634.7 ± 10.6b
26.6 ± 1.1d
27.2 ± 0.7d
23.3 ± 1.2d
20.3 ± 1.2d
24.8 ± 3.4d
28.2 ± 4.3d
23.4 ± 2.3d
16.9 ± 1.0d
Provitamin
Ae
a
Transgene state indicates if a transgenic construct is present or absent (Null). b Sum of all-trans and cis isomers of α-cryptoxanthin. c Xantho represent the sum of xanthophyll carotenoids
(lutein, zeaxanthin, and α and β-cryptoxanthin). d Carotenes represent the sum of α and β-carotene, cis-β-carotene isomers, and lycopene. e Provitamin A was calculated as the sum of
β-carotene plus half of the sum of α and β-cryptoxanthin, α-carotene, 15-cis-β-carotene, 13-cis-β-carotene, and 9-cis-β-carotene. f Not detected. Values with unique letters are statistically significant as determined by a Tukey’s HSD (α = 0.05) post hoc test.
Present
Present
Present
Present
Present
Present
Present
Null
Null
Null
Null
Null
Null
Null
NA
ABS4-1A
ABS4-1B
ABS4-1C
ABS4-2A
ABS4-2B
ABS4-2C
ABS4-2D
ABS4-1A
ABS4-1B
ABS4-1C
ABS4-2A
ABS4-2B
ABS4-2C
ABS4-2D
Control
β-Cryp.
Y ij ¼ μ þ T i þ P j þ T i P j þ εij
α-Crypb
where model parameters are as previously defined and Pi indicates phytase activation pre-treatment. Tukey–Kramer post-hoc
tests (α = 0.05) were used to define significance between
groups within a given analyte.
To determine significance among the interactions between
transgenic construct transgene state and phytase activation
pre-treatment, the following model was used:
Zeaxanthin
Y i ¼ μ þ P i þ εi
Lutein
Y i ¼ μ þ T i þ εi
Transgene
statea
Tukey–Kramer post-hoc test (α = 0.05) was conducted to determine between which groups differences exist using the
package agricolae.47
To isolate the effect of transgenic construct transgene state
or phytase activation pre-treatment on relative and absolute
bioaccessibility, the following simplified models were used:
Event
identifier
Carotenoid profiles of raw material reported as means ± standard deviation (µg per 200 g porridge (40 g dry flour))
where model parameters are as previously defined and TiPj
indicates the interaction term between transgene state and
phytase activation pre-treatment.
Table 2
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3.2 Relative and absolute bioaccessibility of carotenoids
varied in transgenic and non-transgenic sorghum events
In our germplasm, relative bioaccessibility (micellarization
efficiency) was higher for almost all carotenoids in the non-transgenic material (Fig. 1A). On average, 23.5% of β-carotene in null
segregants and wild type control was bioaccessible compared to
18.8% in transgenic lines. A detailed breakdown of relative bioaccessibility values for all carotenoids and respective geometric
isomers quantified are reported in Table S2.† β-Cryptoxanthin,
α-carotene, and lycopene were low or not detectable in non-transgenic material (Fig. 1 and Table 2).
For all carotenoids measured in our study, transgenic
material had substantially higher absolute bioaccessibility
(quantity of carotenoids available for absorption; Fig. 1B). On
average, transgenic events released 0.118 mg of β-carotene
while non-transgenic material released 4.57 µg per 200 g
serving of porridge. This finding represents an approximate
26-fold increase in absorbable β-carotene from porridges made
using transgenic sorghum events. Individual transgenic and
null segregants exhibited differences in bioaccessibility within
their respective groups. Notably, ABS4-2A released the most
β-carotene, total xanthophylls, carotenes, and provitamin A
carotenoids (Table S2†).
3.3
Mineral profiles were similar in all sorghum lines studied
Mineral elements common to cereal grains were analyzed in
sorghum samples to better understand the potential interactions between their presence and carotenoid bioaccessibility
and to determine if specific events caused alterations in
mineral homeostasis (Table 3 and Table S3†). Mineral profiles
of all sorghum lines did not significantly vary regardless of
Food & Function
transgene state. It is important to emphasize that these constructs were not engineered to differentially accumulate minerals and all germplasm studied here were grown in a controlled environment.
3.4 Total inositol phosphate pools decreased as phytate (IP6)
was metabolized into other inositol phosphate forms (IP1–IP5)
in steeped transgenic sorghum events
The effects of activating both native and heterologous phytase
enzymes on inositol phosphate (IP1–IP6) profiles and carotenoid bioaccessibility were also examined in this study. Without
steeping prior to porridge production and digestion, transgenic and non-transgenic events did not substantially differ
from one another in terms of their phytate catabolite (IP1–IP5)
profiles (Table S4†). However, large statistically significant
differences were observed when flours were steeped prior to
cooking (Fig. 2 and Table S4†). Phytate catabolites (IP1–IP5)
were significantly higher in steeped transgenic material compared to their non-steeped counterparts as well as both
steeped and non-steeped null segregants and non-transgenic
controls (Table S4†). In non-steeped transgenic material, IP1–
IP3 represented 0.6% of the total inositol phosphate pool on
average whereas IP1–IP3 comprised 25.7% of the total inositol
phosphate pool in steeped transgenic events. In non-transgenic material, changes in inositol phosphate profiles due to
steeping were less pronounced, but statistical differences were
observed for IP2–IP6 (Table S4†). Additionally, a significant
reduction in total inositol phosphate species was observed in
transgenic material after steeping compared to non-steeped
counterparts as well as both steeped and non-steeped null segregants and wild-type control (Fig. 2 and Table S4†). More precisely, steeped transgenic material contained, on average,
Fig. 1 Relative (A) and absolute (B) bioaccessibility comparing transgenic to non-transgenic material. Transgene state indicates if a transgenic construct is present or absent (null). Lowercase letters above box and whisker plots that are different indicate statistical significance within a carotenoid
as determined by a post-hoc Tukey’s HSD test. Missing letters indicate that an analyte was not detectable in one of the groups and not subject to
statistical comparison.
Food Funct.
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Table 3
Mineral concentrations in sorghum flour reported as means ± standard deviation (mg per 200 g porridge (40 g dry flour))
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Event identifier
ABS4-1A
ABS4-1B
ABS4-1C
ABS4-2A
ABS4-2B
ABS4-2C
ABS4-2D
ABS4-1A
ABS4-1B
ABS4-1C
ABS4-2A
ABS4-2B
ABS4-2C
ABS4-2D
Control
Paper
Transgene statea
Present
Present
Present
Present
Present
Present
Present
Null
Null
Null
Null
Null
Null
Null
NA
Zinc
b
1.3 ± 0.0abc
1.6 ± 0.3abc
1.5 ± 0.2abc
2.0 ± 0.2a
1.6 ± 0.4ab
1.6 ± 0.2abc
1.5 ± 0.1abc
1.4 ± 0.1abc
1.3 ± 0.2abc
1.3 ± 0.2abc
1.5 ± 0.2abc
1.4 ± 0.1abc
1.2 ± 0.1bc
1.8 ± 0.2ab
1.0 ± 0.3c
Iron
Calcium
Magnesium
1.4 ± 0.2b
1.5 ± 0.1b
1.8 ± 0.6ab
2.1 ± 0.7ab
1.3 ± 0.1b
1.7 ± 0.3ab
1.6 ± 0.2ab
1.3 ± 0.1b
1.5 ± 0.2b
1.4 ± 0.5b
2.5 ± 0.7a
1.5 ± 0.1b
1.4 ± 0.1b
2.0 ± 0.2ab
1.3 ± 0.2b
14.7 ± 2.3ab
16.0 ± 0.0ab
17.3 ± 2.3ab
17.3 ± 6.1ab
16.0 ± 0.0ab
16.0 ± 0.0ab
17.3 ± 2.3ab
13.3 ± 2.3b
21.3 ± 6.1ab
13.3 ± 2.3b
22.7 ± 2.3a
16.0 ± 0.0ab
13.3 ± 2.3b
17.3 ± 2.3ab
13.3 ± 2.3b
58.7 ± 6.1abc
68.0 ± 13.9ab
65.3 ± 6.1ab
73.3 ± 4.6a
68.0 ± 0.0ab
66.7 ± 12.9ab
70.7 ± 4.6ab
61.3 ± 2.3abc
62.7 ± 11.5ab
57.3 ± 6.1abc
68.0 ± 4.0ab
65.3 ± 2.3ab
56.0 ± 8.0bc
64.0 ± 8.0ab
45.3 ± 2.3c
a
Transgene state indicates if a transgenic construct is present or absent (null). b Values with unique letters are statistically significant as determined by a Tukey’s HSD (α = 0.05) post hoc test.
Fig. 2 Donut plots of phytate (IP6) and its catabolites (IP1–IP5) in transgenic events (ABS4-1 (A) and ABS4-2 (B) constructs, respectively) represented as a percentage of total inositol phosphates (IP1–IP6). The outer rings represent non-steeped porridges while the inner rings represent
steeped porridges. The average change in total inositol phosphates as a function of steeping for each event are displayed in the center of its corresponding figure. The first number represents total inositol phosphates in the non-steeped porridges while the latter number represents total inositol
phosphates after steeping in mg per 200 g porridge (40 g dry flour).
370.8 mg of total inositol phosphates whereas non-steeped
events contained 937.1 mg per 200 g of porridge.
3.5 The molar ratios of phytate to iron and zinc were reduced
in steeped transgenic sorghum events
We calculated molar ratios of phytate (IP6) to zinc and iron as
a proxy for mineral bioaccessibility. In this analysis, both
steeped and non-steeped transgenic and non-transgenic
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material were compared to determine phytate degradation
efficiency among wild-type, transgenic and corresponding
nulls. As shown in Fig. 3 and Table S5,† no significant differences in the phytate to zinc (21.6 and 21.2; transgenic and
non-transgenic) and phytate to iron (18.2 and 16.4; transgenic
and non-transgenic) ratios were observed for all the materials
tested without steeping. However, a significant reduction in
phytate to zinc (4.9 and 17.6; transgenic and non-transgenic)
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Fig. 3 Molar IP6 to mineral ratios of steeped ( phytase activated) or non-steeped ( phytase not activated) transgenic and non-transgenic (null)
sorghum porridges. The plot is separated by mineral class (A: zinc and B: iron) and lowercase letters above box and whisker plots that are different
indicate statistical significance within a mineral as determined by a post-hoc Tukey’s HSD test.
and phytate to iron (4.1 and 13.6; transgenic and non-transgenic) ratios was observed for all the sorghum lines after steeping, indicating that the steeping treatment was able to activate
not only the heterologous phytase, but also naturally occurring
phytase enzymes. Although steeping reduced the phytate to
iron and zinc ratios in both transgenic and non-transgenic
material, transgenic material exhibited the most dramatic
reduction falling below 5.0 for both minerals (Fig. 3 and
Table S5†).
3.6 Relative and absolute bioaccessibility of carotenoids were
not affected by phytase activation in transgenic sorghum events
We focused our remaining analysis on the transgenic events
given the notable differences in their total phytate, likely due
to the heterologous phytase activated by steeping (Fig. 2 and
Table S4†). Relative bioaccessibility was not significantly
impacted for any of the carotenoids measured in this study by
phytase activation (Fig. 4A). In terms of absolute bioaccessibility, cis-β-carotene isomers were statistically more bioaccessible
from non-steeped wild-type and null controls, but no other
individual or aggregate measurements of carotenoids were
affected (Fig. 4B and Table S2†). A modest and consistent
pattern can be seen in Fig. 4B and Table S2† suggesting a
slight downward trend in deliverable carotenoids as a function
of steeping and phytase activation, but these differences did
not reach a level of statistical significance. Therefore, delivery
of carotenoids and provitamin A carotenoids were found to be
similar between steeped and non-steeped transgenic material
and not significantly impacted by the presumed increase of
divalent cations liberated through phytase activation.
Food Funct.
4.
Discussion
Among the leading causes of death for children under 5 years
of age in sub-Saharan Africa are vitamin A and mineral
deficiencies.3 The sorghum germplasm used in this study were
specifically engineered to address how modifying the carotenoid biosynthetic pathway in endosperm tissue impacts carotenoid profiles and bioaccessibility. Secondarily, the interactions
among carotenoid bioaccessibility, phytase activation, and the
presence of liberated divalent cations was also studied by
including PhyA in these constructs. The two constructs used in
this study differed only in that ABS4-1 contained PSY1 while
ABS4-2 contained CRTB (Table 1). In both cases, carotenoid
profiles in the germplasm represent improvements over previous iterations with prominent increases in β-carotene.21–23
However, concentrations of xanthophylls such as lutein and
zeaxanthin were slightly lower in this germplasm likely due to
increased pathway flux towards β-carotene. Given that PSY1
and CRTB both encode phytoene synthase and catalyze the
same reaction through conserved prenyl transferase domains,
changes in gene expression through codon optimization
(codon optimized PSY1 was used in this study compared to
previous iterations22) and/or difference in the enzyme catalytic
activity differences may have contributed to variation in
xanthophyll content as well as provitamin A carotenoids in
different transgenic constructs.48 Regardless, xanthophyll concentrations in transgenic events were between ∼3–4× higher
than our non-transgenic control (Tx430) and may provide
greater long term benefits related to eye health (Table 2).49,50
Depending on factors such as age and sex, the estimated
average requirement (EAR) of retinol activity equivalents (RAE)
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Fig. 4 Relative (A) and absolute (B) bioaccessibility comparing steeped ( phytase activated) to non-steeped ( phytase not activated) porridges.
Lowercase letters above box and whisker plots that are different indicate statistical significance within a carotenoid as determined by a post-hoc
Tukey’s HSD test.
for vitamin A can range from 275 µg up to 885 µg for children
4–8 and lactating mothers, respectively.51 The reported conversion of dietary β-carotene to retinol is 12 : 1.7 However, biofortified cereals like maize and Golden rice have been shown to
have more effective conversion rates (6 : 1 and 3.8 : 1,
respectively).52,53 Given the similarity of sorghum flour’s composition to maize, retinol conversion rates may be closer to
those reported for other cereals. A recent report using a
Mongolian gerbil model suggests that the conversion
efficiency of β-carotene to retinol from similar transgenic
sorghum events is 4.5 : 1.54 Based on the 4.5 : 1 conversion
rate, a 200 g porridge serving made with the transgenic events
used in this study contains on average 665.69 µg of β-carotene
equivalents (147.93 RAE); fulfilling 53.7% of a 4–8-year-old
child’s vitamin A EAR (275 µg RAE). However, the efficiency of
provitamin A carotenoid conversion to retinol will be affected
by many other factors, such as porridge matrix, preparation
method, carotenoid bioaccessibility, and interindividual differences in absorption and metabolism capabilities.55
Consistent with previous reports, relative bioaccessibility
was lower in transgenic events with high concentrations of
carotenoids (Fig. 1A).21,56,57 This trend could not be observed
in β-cryptoxanthin, α-carotene, and lycopene as these carotenoids were low or not detectable in non-transgenic material. We
hypothesize that due to the prominently higher carotenoid
concentrations in transgenic events, and the process by which
carotenoids partition into water-soluble micelles may have
been approaching saturation. Regardless, the transgenic
events used in this study have the potential to deliver significantly more total and provitamin A carotenoids than their
non-transgenic counterparts (Fig. 1B), with a particular
emphasis on β-carotene. Despite the modestly lower relative
bioaccessibility in these transgenic sorghum events, higher
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absolute bioaccessibility indicates these lines would have
potential for public health impact to consumers of traditional
porridges.
It has been previously estimated that addressing zinc
deficiencies could prevent 4.4% of childhood deaths.8
Biofortification strategies have sought to improve mineral
content in crops through enhanced uptake and/or partitioning
in edible tissues.20,24,58 However, mineral content is poorly
associated with bioavailability.59 Mineral elements co-localize
in sorghum tissues (e.g. pericarp) with phytate which reduces
bioavailability due to its ability to chelate metal ions.60
We explored the effects of activating both naturally occurring and heterologous phytase enzymes on the profiles of inositol phosphates as well as carotenoid bioaccessibility. Phytase
has long been known to catabolize phytate into various inositol phosphate products in legumes61 as well as cereals including sorghum, corn, rice, and wheat.62 Similar trends were
observed in the current study as phytase activation shifted inositol phosphate profiles, normally dominated by phytate (IP6),
to smaller catabolites (IP1–5; Fig. 2 and Table S4†). Notably,
the heterologous PhyA in our transgenic events more effectively reduced total inositol phosphate pools in sorghum
flours. This finding was evident in significant shifts in calculated molar ratios of phytate to zinc and iron (Fig. 3). Lower
molar ratios of phytate to zinc and iron are associated with
higher mineral bioaccessibility.16,63 While estimates vary, critical values for molar phytate to zinc or iron are generally
believed to fall between 10–15.14–17 Above these thresholds,
bioaccessibility is impaired due to the chelating activity of
phytate. Our study demonstrated that phytase activated transgenic material exhibited the most drastic reduction of phytate
to iron ratio from 16.0 to 4.1 and phytate to zinc ratio from
19.0 to 4.9 (Fig. 3), far below reported critical thresholds.
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However, a potential challenge emerges due to the known
antagonistic relationship between carotenoid micellarization
and minerals.
Previous studies have shown that divalent cations in concentrations above certain thresholds are able to precipitate bile
salts64 as well as fatty acids.65 The resulting formation of insoluble soaps has been hypothesized to be a potential mechanism by which carotenoid bioaccessibility is negatively
impacted by minerals.26,27,66,67 In this study, total phytate
remaining in steeped, transgenic events was on average 22.9%
of the original amount and additional minerals were ostensibly released through phytate degradation. However, no significant effect was observed on relative or absolute carotenoid
bioaccessibility in our study as a function of pre-steeping and
phytase activation. This outcome may be due to the inherently
low concentrations of mineral elements in sorghum relative to
the treatments tested in other bodies of work in which concentrations of minerals tended to fall within the mmol
range.26,27,29,66,67 When prepared as a traditional porridge, an
average 200 g serving of porridge (40 g of flour) from all germplasm studied in this work contains 1.62 and 1.47 mg (29.01
and 22.48 µmol L−1) of iron and zinc, respectively. Given the
effects of divalent minerals on micellarization are concentration dependent,26,27,67 we hypothesize that the low mineral
content of sorghum may obscured any significant effects of
steeping, phytase activation, and subsequent liberation of minerals on micellarization efficiency. Critical values above which
micellarization is negatively affected for divalent cations such
as zinc, magnesium, and calcium have previously been
reported at 100, 300, and 500 mg L−1, respectively.28 While
iron and zinc both fell below 40 mg L−1 in the sorghum
material studied here, magnesium was present at 1584.4 mg
L−1 on average. The relatively higher concentrations of magnesium likely contributed to modest reductions observed.
However, the modest decreases in micellarization efficiency
observed in steeped transgenic sorghum porridges were not
statistically significant. This finding is consistent with studies
conducted by our group in other crops such as spinach.68
Regardless, the quantity of carotenoids available for absorption was not significantly affected by steeping and the subsequent release of divalent cations. Should future iterations of
this material generate transgenic events capable of delivering
larger quantities of minerals per serving, it would be prudent
to ensure micellarization efficiency and deliverable carotenoids
are not significantly impacted. Given the chemical aspects of
the sorghum germplasm in this study, our findings indicate
that the transgenic events reported here can effectively deliver
both carotenoids and divalent minerals simultaneously and
more efficiently than non-transgenic controls.
The present study sought to leverage biotechnology for the
purpose of generating nutritionally enhanced cultivars of
sorghum. We utilized these events to study interactions
between divalent minerals and carotenoid bioaccessibility
through the activation of native or heterologous phytase. Data
generated in this study indicate that transgenic events reported
here can simultaneously deliver significant quantities of provi-
Food Funct.
Food & Function
tamin A carotenoids, carotenoids associated with eye and
brain development, and divalent minerals required for normal
growth and development. Importantly, increasing the deliverability of mineral elements by enzymatically degrading phytate
did not significantly compromise carotenoid bioavailability.
While human trials are needed to determine the clinical
efficacy of these sorghum events, our data suggest that simultaneously addressing multiple (and competing) nutrient
deficiencies is feasible and warrants additional attention.
Disclaimer statement
The findings and conclusions in this publication are those of
the authors and should not be construed to represent any
official USDA or U.S. Government determination or policy.
Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement
by the U.S. Department of Agriculture. The USDA is an equal
opportunity provider and employer.
Data availability
Novel biological materials described in this publication may
be available to the academic community and other not-forprofit institutions solely for non-commercial research purposes upon acceptance and signing of a material transfer
agreement between the author’s institution and the requestor.
In some cases, such materials may contain genetic elements
described in the manuscript that were obtained from a third
party(s) (e.g., Zm-WS1 whole seed promoter41) and the authors
may not be able to provide materials including third party
genetic elements to the requestor because of certain thirdparty contractual restrictions placed on the author’s institution. In such cases, the requester will be required to obtain
such materials directly from the third party. The author’s and
authors’ institution do not make any express or implied permission(s) to the requester to make, use, sell, offer for sale, or
import third party proprietary materials. Obtaining any such
permission(s) will be the sole responsibility of the requestor.
Corteva Agriscience™ proprietary plant germplasm and any
transgenic material will not be made available except at the
discretion of the owner and then only in accordance with all
applicable governmental regulations.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
We thank Sydney Corbin, Jacob Fitzgerald, Micaela Hayes,
Zulfiqar Mohamedshah, and Candace Nunn for their assist-
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Food & Function
ance during in vitro digestion and carotenoid extraction experiments. This work was supported by a gift from the Pioneer
Foundation (to MGF) and in part by the U.S. Department of
Agriculture, Agricultural Research Service (USDA-ARS Project
6026-51000-012 and 3092-51000-061-000-D).
References
1 S. Fraval, J. Hammond, J. R. Bogard, M. Ng’endo, J. van Etten,
M. Herrero, S. J. Oosting, I. J. M. de Boer, M. Lannerstad,
N. Teufel, C. Lamanna, T. S. Rosenstock, T. Pagella,
B. Vanlauwe, P. M. Dontsop-Nguezet, D. Baines, P. Carpena,
P. Njingulula, C. Okafor, J. Wichern, A. Ayantunde, C. Bosire,
S. Chesterman, E. Kihoro, E. J. O. Rao, T. Skirrow, J. Steinke,
C. M. Stirling, V. Yameogo and M. T. van Wijk, Food Access
Deficiencies in Sub-saharan Africa: Prevalence and
Implications for Agricultural Interventions, Front. Sustain.
Food Syst., 2019, 3, 104.
2 M. Xu, Y. Luan, Z. Zhang and S. Jiang, Dietary pattern
changes over Africa and its implication for land requirements for food, Mitig Adapt Strateg Glob Change, 2021, vol.
26, p. 13.
3 R. E. Black, L. H. Allen, Z. A. Bhutta, L. E. Caulfield, M. de
Onis, M. Ezzati, C. Mathers and J. Rivera, Maternal and
child undernutrition: global and regional exposures and
health consequences, Lancet, 2008, 371, 243–260.
4 R. Harika, M. Faber, F. Samuel, J. Kimiywe, A. Mulugeta
and A. Eilander, Micronutrient Status and Dietary Intake of
Iron, Vitamin A, Iodine, Folate and Zinc in Women of
Reproductive Age and Pregnant Women in Ethiopia, Kenya,
Nigeria and South Africa: A Systematic Review of Data from
2005 to 2015, Nutrients, 2017, 9, 1096.
5 J. H. Humphrey, K. P. West and A. Sommer, Vitamin A
deficiency and attributable mortality among under-5-yearolds., Bull. W. H. O., 1992, 70, 225–232.
6 J. P. Whitcher, M. Srinivasan and M. P. Upadhyay, Corneal
blindness: a global perspective, Bull. W. H. O., 2001, 79,
214–221.
7 P. Trumbo, A. A. Yates, S. Schlicker and M. Poos, Dietary
Reference Intakes: Vitamin A, Vitamin K, Arsenic, Boron,
Chromium,
Copper,
Iodine,
Iron,
Manganese,
Molybdenum, Nickel, Silicon, Vanadium, and Zinc, J. Am.
Diet. Assoc., 2001, 101, 294–301.
8 C. L. Fischer Walker, M. Ezzati and R. E. Black, Global and
regional child mortality and burden of disease attributable
to zinc deficiency, Eur. J. Clin. Nutr., 2009, 63, 591–597.
9 Y. Peng, K. F. Schertz, S. Cartinhour and G. E. Hart,
Comparative genome mapping of Sorghum bicolor (L.)
Moench using an RFLP map constructed in a population of
recombinant inbred lines, Plant Breed., 1999, 118, 225–235.
10 S. Da, J. O. Akingbala, L. Rooney, J. F. Scheuring and
F. R. Miller, in Evaluation of Tô Quality in a Sorghum
Breeding Program, ed. L. W. Rooney and D. S. Murty,
International Crops Research Institute for the SemiArid
Tropics, Patancheru, A.P., India, 1981, pp. 11–23.
This journal is © The Royal Society of Chemistry 2023
Paper
11 A. P. P. Kayodé, A. R. Linnemann, J. D. Hounhouigan,
M. J. R. Nout and M. A. J. S. van Boekel, Genetic and
Environmental Impact on Iron, Zinc, and Phytate in Food
Sorghum Grown in Benin, J. Agric. Food Chem., 2006, 54,
256–262.
12 E. G. Kean, N. Bordenave, G. Ejeta, B. R. Hamaker and
M. G. Ferruzzi, Carotenoid bioaccessibility from whole
grain and decorticated yellow endosperm sorghum porridge, J. Cereal Sci., 2011, 54, 450–459.
13 Ael-M Afify, H. S. El-Beltagi, S. M. A. El-Salam and
A. A. Omran, Bioavailability of Iron, Zinc, Phytate and
Phytase Activity during Soaking and Germination of White
Sorghum Varieties, PLoS One, 2011, 6, e25512.
14 D. Oberleas and B. E. Harland, Phytate content of foods:
Effect on dietary zinc bioavailability, J. Am. Diet. Assoc.,
1981, 79, 433–436.
15 J. R. Turnlund, J. C. King, W. R. Keyes, B. Gong and
M. C. Michel, A stable isotope study of zinc absorption in
young men: effects of phytate and a-cellulose, Am. J. Clin.
Nutr., 1984, 40, 1071–1077.
16 E. R. Morris and R. Ellis, Usefulness of the dietary phytic
acid/zinc molar ratio as an index of zinc bioavailability to
rats and humans, Biol. Trace Elem. Res., 1989, 19, 107–117.
17 P. R. Saha, C. M. Weaver and A. C. Mason, Mineral
Bioavailability in Rats from Intrinsically Labeled Whole
Wheat Flour of Various Phytate Levels, J. Agric. Food Chem.,
1994, 42, 2531–2535.
18 A. P. Kayodé, A. R. Linnemann, M. J. Nout and M. A. Van
Boekel, Impact of sorghum processing on phytate, phenolic
compounds and in vitro solubility of iron and zinc in thick
porridges, J. Sci. Food Agric., 2007, 87, 832–838.
19 J. Kruger, A. Oelofse and J. R. N. Taylor, Effects of aqueous
soaking on the phytate and mineral contents and phytate:
mineral ratios of wholegrain normal sorghum and maize and
low phytate sorghum, Int. J. Food Sci. Nutr., 2014, 65, 539–546.
20 G. S. Khush, S. Lee, J.-I. Cho and J.-S. Jeon, Biofortification
of crops for reducing malnutrition, Plant Biotechnol. Rep.,
2012, 6, 195–202.
21 T. E. Lipkie, F. F. De Moura, Z.-Y. Zhao, M. C. Albertsen,
P. Che, K. Glassman and M. G. Ferruzzi, Bioaccessibility of
Carotenoids from Transgenic Provitamin A Biofortified
Sorghum, J. Agric. Food Chem., 2013, 61, 5764–5771.
22 P. Che, Z.-Y. Zhao, K. Glassman, D. Dolde, T. X. Hu, T. J. Jones,
D. F. Gruis, S. Obukosia, F. Wambugu and M. C. Albertsen,
Elevated vitamin E content improves all-trans β-carotene
accumulation and stability in biofortified sorghum, Proc. Natl.
Acad. Sci. U. S. A., 2016, 113, 11040–11045.
23 P. Che, Z.-Y. Zhao, M. Hinds, K. Rinehart, K. Glassman and
M. Albertsen, in Sorghum: Methods and Protocols, ed. Z.-Y.
Zhao and J. Dahlberg, Springer, New York, NY, 2019, pp.
209–220.
24 J. Díaz-Gómez, R. M. Twyman, C. Zhu, G. Farré,
J. C. Serrano, M. Portero-Otin, P. Muñoz, G. Sandmann,
T. Capell and P. Christou, Biofortification of crops with
nutrients: factors affecting utilization and storage, Curr.
Opin. Biotechnol., 2017, 44, 115–123.
Food Funct.
View Article Online
Open Access Article. Published on 14 July 2023. Downloaded on 7/16/2023 8:41:19 PM.
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
Paper
25 E. Biehler, A. Kaulmann, L. Hoffmann, E. Krause and
T. Bohn, Dietary and host-related factors influencing carotenoid bioaccessibility from spinach (Spinacia oleracea),
Food Chem., 2011, 125, 1328–1334.
26 E. Biehler, L. Hoffmann, E. Krause and T. Bohn, Divalent
Minerals Decrease Micellarization and Uptake of
Carotenoids and Digestion Products into Caco-2 Cells,
J. Nutr., 2011, 141, 1769–1776.
27 J. Corte-Real, M. Iddir, C. Soukoulis, E. Richling,
L. Hoffmann and T. Bohn, Effect of divalent minerals on
the bioaccessibility of pure carotenoids and on physical
properties of gastro-intestinal fluids, Food Chem., 2016,
197, 546–553.
28 J. Corte-Real, M. Bertucci, C. Soukoulis, C. Desmarchelier,
P. Borel, E. Richling, L. Hoffmann and T. Bohn, Negative
effects of divalent mineral cations on the bioaccessibility of
carotenoids from plant food matrices and related physical
properties of gastro-intestinal fluids, Food Funct., 2017, 8,
1008–1019.
29 R. E. Kopec, C. Caris-Veyrat, M. Nowicki, J.-P. Bernard,
S. Morange, C. Chitchumroonchokchai, B. Gleize and
P. Borel, The Effect of an Iron Supplement on Lycopene
Metabolism and Absorption During Digestion in Healthy
Humans, Mol. Nutr. Food Res., 2019, 63, 1900644.
30 P. Che, A. Anand, E. Wu, J. D. Sander, M. K. Simon,
W. Zhu, A. L. Sigmund, G. Zastrow-Hayes, M. Miller, D. Liu,
S. J. Lawit, Z.-Y. Zhao, M. C. Albertsen and T. J. Jones,
Developing a flexible, high-efficiency Agrobacteriummediated sorghum transformation system with broad
application, Plant Biotechnol. J., 2018, 16, 1388–1395.
31 X. Ye, S. Al-Babili, A. Klöti, J. Zhang, P. Lucca, P. Beyer and
I. Potrykus, Engineering the provitamin A (beta-carotene)
biosynthetic pathway into (carotenoid-free) rice endosperm,
Science, 2000, 287, 303–305.
32 J. A. Paine, C. A. Shipton, S. Chaggar, R. M. Howells,
M. J. Kennedy, G. Vernon, S. Y. Wright, E. Hinchliffe,
J. L. Adams, A. L. Silverstone and R. Drake, Improving the
nutritional value of Golden Rice through increased provitamin A content, Nat. Biotechnol., 2005, 23, 482–487.
33 E. B. Cahoon, S. E. Hall, K. G. Ripp, T. S. Ganzke,
W. D. Hitz and S. J. Coughlan, Metabolic redesign of
vitamin E biosynthesis in plants for tocotrienol production
and increased antioxidant content, Nat. Biotechnol., 2003,
21, 1082–1087.
34 Y. Han, D. B. Wilson and X. G. Lei, Expression of an
Aspergillus niger phytase gene ( phyA) in Saccharomyces
cerevisiae, Appl. Environ. Microbiol., 1999, 65, 1915–1918.
35 D. Negrotto, M. Jolley, S. Beer, A. R. Wenck and G. Hansen,
The use of phosphomannose-isomerase as a selectable
marker to recover transgenic maize plants (Zea mays L.) via
Agrobacterium transformation, Plant Cell Rep., 2000, 19,
798–803.
36 U. Neudert, I. M. Martínez-Férez, P. D. Fraser and
G. Sandmann, Expression of an active phytoene synthase
from Erwinia uredovora and biochemical properties of the
enzyme, Biochim. Biophys. Acta, 1998, 1392, 51–58.
Food Funct.
Food & Function
37 World
Intellectual
Property
Organization,
WO2014070646A1, 2014.
38 United States, US20090049571A1, 2009.
39 G. Coruzzi, R. Broglie, A. Cashmore and N. H. Chua,
Nucleotide sequences of two pea cDNA clones encoding
the small subunit of ribulose 1,5-bisphosphate carboxylase
and the major chlorophyll a/b-binding thylakoid polypeptide., J. Biol. Chem., 1983, 258, 1399–1402.
40 S. Park, R. G. Ong and M. Sticklen, Strategies for the production of cell wall–deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production, Plant Biotechnol. J., 2016, 14, 1329–1344.
41 World
Intellectual
Property
Organization,
WO2010122110A1, 2010.
42 M. Hayes, M. Pottorff, C. Kay, A. Van Deynze, J. OsorioMarin, M. A. Lila, M. Iorrizo and M. G. Ferruzzi, In Vitro
Bioaccessibility of Carotenoids and Chlorophylls in a
Diverse Collection of Spinach Accessions and Commercial
Cultivars, J. Agric. Food Chem., 2020, 68, 3495–3505.
43 S. Zhang, W. Yang, Q. Zhao, X. Zhou, Y. Fan and R. Chen,
Rapid Method for Simultaneous Determination of Inositol
Phosphates by IPC-ESI–MS/MS and Its Application in Nutrition
and Genetic Research, Chromatographia, 2017, 2, 275–286.
44 R Development Core Team, R: A language and environment
for statistical computing, 2018.
45 K. Ram, H. Wickham, C. Richards and A. Baggett, wesanderson: A Wes Anderson Palette Generator, 2018.
46 H. Wickham, ggplot2 Elegant Graphics for Data Analysis,
Springer-Verlag, New York, NY, 2016.
47 F. de Mendiburu and M. Yanseen, Agricolae: Statistical
Procedures for Agricultural Research, 2021.
48 F. X. Cunningham and E. Gantt, Genes and Enzymes of
Carotenoid Biosynthesis in Plants, Annu. Rev. Plant Physiol.
Plant Mol. Biol., 1998, 49, 557–583.
49 J. Mares, Lutein and Zeaxanthin Isomers in Eye Health and
Disease, Annu. Rev. Nutr., 2016, 36, 571–602.
50 S. E. Thomas and E. J. Johnson, Xanthophylls, Adv. Nutr.,
2018, 9, 160–162.
51 Institute of Medicine (US) Committee on Nutrition
Standards for National School Lunch and Breakfast
Programs, Nutrition Standards and Meal Requirements for
National School Lunch and Breakfast Programs: Phase
I. Proposed Approach for Recommending Revisions, National
Academies Press (US), Washington (DC), 2008.
52 G. Tang, J. Qin, G. G. Dolnikowski, R. M. Russell and
M. A. Grusak, Golden Rice is an effective source of vitamin
A, Am. J. Clin. Nutr., 2009, 89, 1776–1783.
53 S. Li, A. Nugroho, T. Rocheford and W. S. White, Vitamin A
equivalence of the β-carotene in β-carotene–biofortified
maize porridge consumed by women, Am. J. Clin. Nutr.,
2010, 92, 1105–1112.
54 H. You, Doctor of Philosophy, Iowa State University, Digital
Repository, 2016.
55 D. Weber and T. Grune, The contribution of β-carotene to
vitamin A supply of humans, Mol. Nutr. Food Res., 2012, 56,
251–258.
This journal is © The Royal Society of Chemistry 2023
View Article Online
Open Access Article. Published on 14 July 2023. Downloaded on 7/16/2023 8:41:19 PM.
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
Food & Function
56 M. L. Failla, C. Chitchumroonchokchai, D. Siritunga,
F. F. De Moura, M. Fregene, M. J. Manary and R. T. Sayre,
Retention during processing and bioaccessibility of
β-carotene in high β-carotene transgenic cassava root,
J. Agric. Food Chem., 2012, 60, 3861–3866.
57 I. J. Aragón, H. Ceballos, D. Dufour and M. G. Ferruzzi, Provitamin A carotenoids stability and bioaccessibility from
elite selection of biofortified cassava roots (Manihot esculenta, Crantz) processed to traditional flours and porridges,
Food Funct., 2018, 9, 4822–4835.
58 H. Debelo, M. Albertsen, M. Simon, P. Che and
M. Ferruzzi, Identification and Characterization of
Carotenoids, Vitamin E and Minerals of Biofortified
Sorghum, Curr. Dev. Nutr., 2020, 4, 1792.
59 R. P. Glahn and H. Noh, Redefining Bean Iron
Biofortification: A Review of the Evidence for Moving to a
High Fe Bioavailability Approach, Front. Sustain. Food Syst.,
2021, 5, 682130.
60 M. G. Ferruzzi, J. Kruger, Z. Mohamedshah, H. Debelo and
J. R. N. Taylor, Insights from in vitro exploration of factors
influencing iron, zinc and provitamin A carotenoid bioaccessibility and intestinal absorption from cereals, J. Cereal
Sci., 2020, 96, 103126.
61 J. Frias, R. Doblado, J. R. Antezana and C. Vidal-Valverde,
Inositol phosphate degradation by the action of phytase
enzyme in legume seeds, Food Chem., 2003, 81, 233–239.
62 M. A. Azeke, S. J. Egielewa, M. U. Eigbogbo and
I. G. Ihimire, Effect of germination on the phytase activity,
This journal is © The Royal Society of Chemistry 2023
Paper
63
64
65
66
67
68
phytate and total phosphorus contents of rice (Oryza
sativa), maize (Zea mays), millet (Panicum miliaceum),
sorghum (Sorghum bicolor) and wheat (Triticum aestivum), J. Food Sci. Technol., 2011, 48, 724–729.
G. Ma, Y. Li, Y. Jin, F. Zhai, F. J. Kok and X. Yang, Phytate
intake and molar ratios of phytate to zinc, iron and
calcium in the diets of people in China, Eur. J. Clin. Nutr.,
2007, 61, 368–374.
J. Gu, A. Hofmann, H. Ton-Nu, C. Schteingart and K. Mysels,
Solubility of calcium salts of unconjugated and conjugated
natural bile acids., J. Lipid Res., 1992, 33, 635–646.
J. O. Atteh and S. Leeson, Influence of Age, Dietary Cholic
Acid, and Calcium Levels on Performance, Utilization of
Free Fatty Acids, and Bone Mineralization in Broilers,
Poult. Sci., 1985, 64, 1959–1971.
D. Y. Graham and J. W. Sackman, Solubility of calcium
soaps of long-chain fatty acids in simulated intestinal
environment, Dig. Dis. Sci., 1983, 28, 733–736.
J. Corte-Real, C. Desmarchelier, P. Borel, E. Richling,
L. Hoffmann and T. Bohn, Magnesium affects spinach
carotenoid bioaccessibility in vitro depending on intestinal
bile and pancreatic enzyme concentrations, Food Chem.,
2018, 239, 751–759.
M. Hayes, S. Corbin, C. Nunn, M. Pottorff, C. D. Kay,
M. A. Lila, M. Iorrizo and M. G. Ferruzzi, Influence of simulated food and oral processing on carotenoid and chlorophyll in vitro bioaccessibility among six spinach genotypes,
Food Funct., 2021, 12, 7001–7016.
Food Funct.