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Phenolic Acids Profiles and Cellular Antioxidant Activity in Tortillas Produced

from Mexican Maize Landrace Processed by Nixtamalization and Lime
Extrusion Cooking

Article  in  Plant Foods for Human Nutrition · August 2017

DOI: 10.1007/s11130-017-0624-3

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Phenolic Acids Profiles and Cellular
Antioxidant Activity in Tortillas Produced
from Mexican Maize Landrace Processed
by Nixtamalization and Lime Extrusion
Cooking
Nallely Gaxiola-Cuevas, Saraid Mora-
Rochín, Edith Oliva Cuevas-Rodriguez,
Liliana León-López, Cuauhtémoc Reyes-
Moreno, et al.
Plant Foods for Human Nutrition

ISSN 0921-9668

Plant Foods Hum Nutr

DOI 10.1007/s11130-017-0624-3

1 23
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 Author's personal copy
Plant Foods Hum Nutr
DOI 10.1007/s11130-017-0624-3

ORIGINAL PAPER

Phenolic Acids Profiles and Cellular Antioxidant Activity

in Tortillas Produced from Mexican Maize Landrace Processed
by Nixtamalization and Lime Extrusion Cooking
Nallely Gaxiola-Cuevas 1 & Saraid Mora-Rochín 1,2 & Edith Oliva Cuevas-Rodriguez 1,2 &
Liliana León-López 1,2 & Cuauhtémoc Reyes-Moreno 1,2 & Alvaro Montoya-Rodríguez 1,2 &
Jorge Milán-Carrillo 1,2

# Springer Science+Business Media, LLC 2017

Abstract Phenolic acids profiles, chemical antioxidant activ- Abbreviations

ities (ABTS and ORAC), as well as cellular antioxidant activ- ABAP 2,2′-azobis (2-amidinopropane)
ity (CAA) of tortilla of Mexican native maize landraces elab- dihydrochloride
orated from nixtamalization and lime cooking extrusion pro- ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
cesses were studied. Both cooking procedures decreased total acid)
phenolics, chemicals antioxidant activity when compared to AOX Chemical antioxidant activity
raw grains. Extruded tortillas retained 79.6–83.5%, 74.1– DCFH-DA 2′,7′-dichlorofluorescin diacetate
77.6% and 79.8–80.5% of total phenolics, ABTS and GAE Gallic Acid equivalents
ORAC values, respectively, compared to 47.8–49.8%, 41.3– HBSS Hanks´ balanced salt solution
42.3% and 43.7–44.4% assayed in traditional tortillas, respec- HEPG2 Liver cancer cell lines
tively. Approximately 72.5–88.2% of ferulic acid in raw HPLC High performance liquid chromatography
grains and their tortillas were in the bound form. Regarding ORAC Oxygen radical absorbance capacity
of the CAA initially found in raw grains, the retained percent- PBS Phosphate-buffered saline
age for traditional and extruded tortillas ranged from 47.4 to
48.7% and 72.8 to 77.5%, respectively. These results suggest
that Mexican maize landrace used in this study could be con-
sidered for the elaboration of nixtamalized and extruded food Introduction
products with nutraceutical potential.
Northwestern Mexico has a broad diversity of genetic resources
of pigmented maize (Zea mays L.) landraces. Recent collec-
Keywords Maize landrace . Nixtamalization . Lime cooking tions of more than 300 maize accessions have revealed 13
extrusion . Tortilla . Cellular antioxidant activity assay maize landraces, being Tabloncillo and Elotero of Sinaloa the
distinctive native materials of this biogeographic region [1].
Native pigmented maize has been reported to possess a
valuable source of a broad range of phenolic compounds;
these bioactive compounds are mainly phenolic acids, antho-
cyanins, and carotenoids, amongst others [2, 3]. They could
* Jorge Milán-Carrillo play the role as a natural antioxidant and be able to function as
jmilanc@uas.edu.mx
scavengers for oxygen and hydroxyl radicals, as well as other
1
mechanisms such as anti-inflammatory properties, inhibition
Programa Regional en Biotecnología, Facultad de Ciencias Químico
Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa,
of enzymes, and induction of detoxification enzymes [4, 5].
C.P., Mexico However, pigmented maize needs to be subjected to an alka-
2
Progama de Posgrado en Ciencias y Tecnología de Alimentos,
line cooking process to produce tortilla for human consump-
Facultad de Ciencias Químico Biológicas, Universidad Autónoma de tion, which may modify or degrade its natural
Sinaloa, Culiacán, Sinaloa, C.P., Mexico phytochemicals.
 Author's personal copy
The utilization of tortillas and their snack foods (maize
chips, tortillas chips, and tacos) has grown considerably since
the increase popularity of Mexican food and awareness of
their health benefits [6]. Nixtamalization denotes to the alka-
line cooking process of converting maize into food such as
tortillas and their products. However, this traditional process
requires large soaking time, generating high liquid waste dis-
charges [7]. Also, lime-cooking extrusion has been used to
produce instant flours suitable for tortillas with similar char-
acteristics as theirs counterparts produced using
nixtamalization process. This process offers the advantage of
the null generation of wastewater (nejayote) [8]. The resulting
products of lime-cooking extrusion are considered as whole
grain foods due that the nutrients associated with the pericarp
tissues are retained [9].
Several studies concerning antioxidant activity in
pigmented maize and its derived products have been focused
mainly on the assessment of the chemical AOX activity
[10–12]. The CAA assay is a more suitable method than the
currently used assay because it provides a biological evalua-
tion of antioxidant activity. In fact, the CAA determines the
complexity of biological systems and for some aspects the
uptake, metabolism, and location of antioxidant compounds
within cells [13, 14].
Recently, other studies have reported the use of CAA assay
to determine antioxidant activity of pigmented wheat, barley,
rice and flaxseed [15, 16]. However, there is no available
information on CAA of pigmented maize landrace and its
primary products (tortillas). Hence, this study evaluated the
effect of nixtamalization and extrusion cooking processes on
phenolic concentrations, phenolic acids profile and antioxi-
dant activities based on chemical assays (ABTS and
ORAC), as well as on CAA of tortillas produced from
Mexican pigmented maize.
Materials and Methods
Materials The study was performed on two contrasting (blue
and white) kernels corresponding to Elotero of Sinaloa and
Chapalote maize landraces. Both native maize accessions were
collected from open-pollinated areas maintained by traditional
farmers at their villages in the municipality of Concordia (lo-
cated at 23° 17′ 18″ N, 106° 4′ 3″ W), in the state of Sinaloa,
Mexico. All materials were grown and harvested during 2016.
Crops were managed following standard recommendations.
Maize samples were stored at 4 °C until use.
Production of Nixtamalized Maize Flour (NMF) NMF was
obtained with method described by Milán-Carrillo et al. [17].
Maize kernels (100 g lots) were cooked for 31 min with lime
solution [5.4 g Ca(OH)2/L water] at 85 °C using a 1:3 (w/v)
ratio of grain to cooking medium, followed by a steep time
Plant Foods Hum Nutr
of 8.1 h at room temperature. The cooking liquor (nejayote)
was drained and discarded, and the nixtamal washed with
running tap water for 60 s. Wet nixtamal was dried at 55 °C
for 12 h in a forced air oven and then cooled at room temper-
ature for 30 min. Finally, the dried nixtamal was milled to pass
an 80-US mesh (0.180 mm) screen, packed in plastic bags.
NMF was stored at 4 °C until use.
Production of Extruded Maize Flour (EMF) The procedure
recommended by Milán-Carrillo et al. [8] was used. Maize
grits (1 kg lots) were mixed with lime [0.21 g Ca(OH)2/
100 g maize] and conditioned with water until reached a mois-
ture content (28%, w/w). Extrusion cooking was done using a
single-screw laboratory extruder model 20 DN (CW
Brabender Instruments, Inc., NJ, USA) equipped with a
19 mm screw diameter, 20:1 length/diameter, 2:1 nominal
compression ratio and 2.4 mm die opening. Extrusion temper-
ature and screw speed were 85 °C and 240 rpm, respectively.
Extrudates were cooled and milled to pass an 80-US mesh
(0.180 mm) screen packed in plastic bags. EMF was stored
at 4 °C for further analysis.
Tortilla Preparation Tortillas were made according to
Cuevas-Rodríguez et al. [7]. Briefly, masa (30 g) was formed
into flat discs (15 cm), baked on a hot griddle at 270 °C for
30 s on one side, followed by 10 s on the other side, and then
again on the first side until puffing occurred. After baking, the
resulting tortillas were allowed to equilibrate to room temper-
ature for at least 30 min, and milled to pass an 80-US mesh
(0.180 mm) sieve. Tortillas of traditional nixtamalized flours
(NT) and extruded flours (ET) were stored at −20 °C for fur-
ther analysis.
Extraction of Free and Bound Phenolic Compounds Free
phenolic compounds were extracted using the method report-
ed by Adom and Liu [18]. Briefly, 1 g of maize flour was
blended with 10 mL of chilled ethanol-water (80:20, v/v) for
10 min in a shaker at 50 rpm. The mixture was then centri-
fuged at 2500x g for 10 min. The supernatant was removed,
and the remaining pellets were again extracted with similar
procedure two times. The supernatants were pooled and under
vacuum. The final extracts were stored at −75 °C until
analysis.
Bound phenolics of maize samples were extracted using
the method described by Adom and Liu [18]. Briefly, the
residue from the free extraction was first digested with
20 mL 2 M sodium hydroxide at room temperature for 1 h
with shaking under nitrogen. The mixture was then neutral-
ized with an appropriate amount of concentrated hydrochloric
acid. Hexanes were used to remove lipids in the mixture. The
remaining mixture was then extracted five times with ethyl
acetate. The ethyl acetate fraction was pooled and evaporated
at 45 °C to dryness under vacuum. The content of bound
 Author's personal copy
Plant Foods Hum Nutr
phenolics was reconstituted in 10 mL of 50% methanol and
stored at −75 °C until analysis.
Total Phenolic Content (TPC) Phenolics of sample extracts
were determined using the colorimetric method described by
Singleton et al. [19]. The results were expressed as milligrams
of gallic acid equivalents (GAE) per 100 g of dry weight basis
(DW).
Determination of Phenolic Acid Composition The phenolic
composition was conducted using the HPLC method reported
previously [16]. Identification of each peak was confirmed
using retention time and the absorbance of each pure com-
pound. Final values of phenolics concentration were
expressed as mg phenolic acids/100 g grain (DW).
Antioxidant Activity Antioxidant activity of extracts was
evaluated by ABTS.+ radical scavenging activity measured
according to Re et al. [20]. Oxygen radical absorbance capac-
ity (ORAC) was measured as previously described by Ou
et al. [21]. The antioxidant activity in each case was expressed
as mg Trolox equivalents (TE) per 100 g DW.
Cytotoxicity Cytotoxicity effects were measured using the
methods as described previously [22]. In brief, HepG2 cells
were cultured at 4 × 104 cells/well on a 96-well microplate in
100 μL of growth medium for 24 h at 37 °C. Cytotoxicity was
determined by a 10% reduction of absorbance at 570 nm read-
ing for each concentration compared to the control.
Cellular Antioxidant Activity The CAA assay was reported
previously [14, 23]. Briefly, HepG2 human liver cells were
seeded at a density of 6 × 104 cells/well on a 96-well micro-
plate in 100 μL of growth medium/well for 24 h. Afterward,
the treatments medium was removed and managed with PBS
wash or no PBS wash protocols. Wells were treated in tripli-
cate for 1 h with different concentrations of maize extracts
Table 1 Total phenolic content and percent of contribution in different Mexican raw native maize types and their tortillas produced throughout
nixtamalization or lime cooking extrusion
Maize type Sample/ Product Phenolic content
[mg gallic acid equivalent (GAE)/100 g DW]
Free % Bound
B-512 Raw 41.2 ± 1.9a (16.9)
NT 21.7 ± 2.7c (17.9)
ET 20.8 ± 1.2c (10.2)
W-111 Raw 32.6 ± 1.8b (14.0)
NT 16.3 ± 1.2d (14.8)
ET 21.7 ± 0.4c (11.7)
1
Values within columns with no letters in common are significantly different (p < 0.05); NT, Tortillas produced from nixtamalized flour; ET, Tortillas
produced from extruded flour. Values in parentheses indicate percentage contribution of this fraction to the corresponding to the total phenolic content
plus 25 μM DCFH-DA. Then 600 μM ABAP was added to
the cells in 100 μL of HBSS, and the 96-well microplate was
placed into the reader (Victor™ X3 Multi-Label, Perkin
Elmer, Inc., MA, USA) at 37 °C. Emission at 535 nm was
measured after excitation at 485 nm every 5 min for 1 h. The
CAA values were calculated by dividing the EC50 of quercetin
standards by the EC50 of the sample, and the final value was
expressed as μmol quercetin equivalents (QE)/ 100 g grain
(DW).
Statistical Analysis Experimental results were performed in
triplicate and data are presented as mean ± S.D. Analysis of
variance and comparison of treatments means (Tukey’s tests,
p ≤ 0.05) were analyzed using statistical software Minitab
version 16.
Results and Discussion
Total Phenolic Content (TPC) Amongst raw pigmented
maize studied, the TPC varied from 233.1 to 244.0 mg
GAE/100 g DW in W-111 and B-512 maize genotypes, re-
spectively (Table 1). The amounts of TPC of native maize
tested herein were similar than those obtained on different
Chilean pigmented maize, and Mexican pigmented maize
populations reported earlier [24, 25]. However, López-
Martínez et al. [2], reported that TPC of 18 Mexican
pigmented maize showed higher amounts compared to all
the genotypes tested in our study. These differences could be
mainly ascribed to the variability in pigmented maize geno-
types concerning genetic background, geographical location,
and physical properties of grain [25].
Both cooking processes used to prepare tortillas affected
significantly (p < 0.05) the reduction of phenolic levels
when compared to raw grains. Interestingly, extruded torti-
llas contained about twice the total phenolics assayed in
counterparts prepared from nixtamalized flour. The higher
% Total Retention (%)
202.8 ± 3.1a (83.1) 244.0 ± 5.1a
99.7 ± 3.0d (82.1) 121.4 ± 2.6d 49.8 ± 1.6c
183.0 ± 1.7b (89.8) 203.8 ± 2.5c 83.5 ± 2.1a
200.5 ± 4.3a (86.0) 233.1 ± 3.9b
94.2 ± 3.8d (85.2) 110.5 ± 4.1e 47.8 ± 1.3c
163.9 ± 4.2c (88.3) 185.6 ± 3.8c 79.6 ± 3.1b
 Author's personal copy
Plant Foods Hum Nutr

retention and stability of the bioactive phenolics in extrud-
ed tortillas (average 81.6%) could be probably attributed to
the short thermal processing time of the whole meal; more-
over, during the lime cooking extrusion process, there were
not physical losses of the anatomical component of maize
grain [11]. Nevertheless, the lower percentage of retention
of phenolics compounds in nixtamalized tortillas (47.4 to
49.8%) could be related to the combined effect of high
alkaline pH and thermal processing during traditional
nixtamalization process, as well as physical losses of the
pericarp and leaching of phenolics into the cooking liquor
[11]. Other studies have also found total phenolics reduc-
tion in nixtamalized tortillas [10, 11].
Our study evidently confirmed that majority of the phe-
nolics associated with the raw kernel and nixtamalized, and
extruded tortillas were present in bound rather than in free
forms. The percentage contribution of bound phenolics to
the total varied from 83.1 to 86.0% in raw maize, whereas
bound phenolics contributed for nixtamalized and extruded
tortillas from 82.1 to 85.2% and 88.3 to 89.8%, respective-
ly (Table 1).
Phenolic Acid Composition Recently, there is interest in the
antioxidant activities and potential health benefits associated
with individual phenolic compounds [16]. Phenolic acid pro-
files observed in raw pigmented maize genotypes and their
tortillas were very similar, with the presence of six peaks.
Six major phenolic acids compounds, including p-
hydroxybenzoic, vanillic, syringic, p-coumaric, sinapic and
ferulic acid, were identified and quantified in the free and
bound extracts of the samples tested (Table 2). Results indi-
cated that vanillic and syringic acids were only distributed in
the bound fraction; all other phenolic compounds were present
in both free and bound fractions. Ferulic acid and p-coumaric
acid were the predominant phenolic compound in both raw
maize types tested, which ranged from 177.9 to 185.9 and
15.6 to 21.4 mg phenolic acid /100 g DW, respectively.
Interestingly, extruded tortillas had 3.6-fold high total
ferulic acid compared with nixtamalized tortillas (Table 2).
Extruded tortillas from pigmented maize retained from 94.1
to 96.1% of total ferulic acid initially assayed in raw kernels.
Mora-Rochin et al. [11] reported similar results with 96.7%
retention of ferulic acid in blue maize tortillas produced from

Table 2 Phenolic acid composition of two different Mexican raw native maize types and their tortillas produced throughout nixtamalization or lime
cooking extrusion

Maize/ products Phenolic Acid

(mg phenolic acid/100 g DW)

Fractions Syringic Vanillic p-Hydroxybenzoic p-Coumaric Synapic Ferulic % Retention Total

ferulic acid

B-512
Raw Total 0.9 ± 0.0b 2.1 ± 0.0d 0.9 ± 0.0b 15.6 ± 0.2d 4.2 ± 0.3c 168.4 ± 4.2c (88.2) 201.5 ± 4.3c
Free ND ND 0.4 ± 0.0d 1.1 ± 0.0f 0.3 ± 0.0g 0.4 ± 0.0g 2.2 ± 0.2h
Bound 0.9 ± 0.0b 2.1 ± 0.0d 0.5 ± 0.0c 14.5 ± 0.2d 3.8 ± 0.3b 168.0 ± 4.2c 199.83 ± 3.2c
NT Total 0.8 ± 0.1b 1.9 ± 0.1d 1.0 ± 0.0b 8.2 ± 0.2e 6.0 ± 0.3b 47.4 ± 2.0d (72.5) 26.6 ± 1.1b 65.3 ± 2.6e
Free ND ND 0.5 ± 0.0c 0.7 ± 0.0f 0.5 ± 0.0f 2.7 ± 0.2e 4.4 ± 0.3f
Bound 0.8 ± 0.1b 1.9 ± 0.1d 0.5 ± 0.0c 7.5 ± 0.3e 5.5 ± 0.3b 44.7 ± 1.8d 60.9 ± 4.9e
ET Total 1.5 ± 0.0b 3.0 ± 0.0b 1.3 ± 0.0a 18.0 ± 0.9c 8.8 ± 0.5a 167.4 ± 5.6c (83.7) 94.1 ± 2.6a 199.9 ± 4.6c
Free ND ND 0.7 ± 0.0c 0.8 ± 0.0f 0.5 ± 0.0f 1.6 ± 0.6f 3.6 ± 0.3g
Bound 1.5 ± 0.0b 3.0 ± 0.0b 0.6 ± 0.0c 17.2 ± 0.9c 8.2 ± 0.5a 165.8 ± 5.2c 196.3 ± 4.3d
W-111
Raw Total 1.6 ± 0.0b 3.1 ± 0.0a 0.8 ± 0.0b 21.4 ± 0.7a 2.4 ± 0.0d 185.9 ± 1.1a (86.4) 215.3 ± 1.8a
Free ND ND 0.4 ± 0.0d 0.9 ± 0.0f 0.4 ± 0.0f 0.2 ± 0.0h 1.9 ± 0.3h
Bound 1.6 ± 0.0b 3.1 ± 0.0a 0.4 ± 0.0d 20.5 ± 0.7b 2.1 ± 0.0d 185.7 ± 1.1a 213.4 ± 4.3a
NT Total 1.5 ± 0.1b 2.5 ± 0.1c 0.9 ± 0.0b 8.9 ± 0.0e 2.4 ± 0.3d 49.4 ± 1.6d (75.3) 26.6 ± 1.6b 65.6 ± 1.8e
Free ND ND 0.4 ± 0.0d 0.6 ± 0.0f 0.3 ± 0.0g 2.3 ± 0.2e 3.6 ± 0.3g
Bound 1.5 ± 0.1b 2.5 ± 0.1c 0.5 ± 0.0c 8.3 ± 0.0e 2.1 ± 0.0d 47.1 ± 1.4d 62.0 ± 4.3e
ET Total 2.2 ± 0.0a 3.2 ± 0.0a 0.8 ± 0.0b 21.3 ± 0.0a 6.6 ± 0.0b 178.7 ± 0.5b (84.0) 96.1 ± 3.1a 212.8 ± 1.3a
Free ND ND 0.3 ± 0.0d 0.6 ± 0.0f 0.3 ± 0.1g 0.5 ± 0.1g 1.7 ± 0.3h
Bound 2.2 ± 0.0a 3.2 ± 0.0a 0.5 ± 0.0c 20.7 ± 0.0b 6.3 ± 1.1b 178.2 ± 2.4b 211.1 ± 2.3a
1
Values within columns with no letters in common are significantly different (p < 0.05); NT, Tortillas produced from nixtamalized flour; ET, Tortillas
produced from extruded flour. Percent contribution to total phenolic acid is in parentheses; ND, not detected
 Author's personal copy
Plant Foods Hum Nutr
extruded flour. Our study showed that up to 70% of the phe-
nolic acid is degraded when subjected to nixtamalization com-
pared with the raw native maize. To our knowledge, this is the
first time that phenolic acids profiles in bound form have been
reported for extruded and nixtamalized tortillas.
Chemical Antioxidant Activity Chemical antioxidant capac-
ity assays are based on different mechanisms: one approach is
based on single electron transfer and involves reduction of a
colored oxidant such as ABTS, the other method involves a
hydrogen atom transfer, like ORAC assay, in which antioxi-
dant and substrate compete for thermally generated peroxyl
radicals [26]. The total antioxidant activities of raw pigmented
maize using ABTS and ORAC tests ranged from 7481.6–
7,661.1 and 10,320.6–10,878.6 μmol TE/100 g DW, respec-
tively (Fig. 1). Bound phytochemicals of raw kernels were the
primary contributors (83.9 to 91.8%) to the total antioxidant
activity (Fig. 1). Researchers have also reported that bound
polyphenols are vital contributors to the antioxidant content
and this fraction represents approximately 85% of the total
antioxidant content in maize [11, 12].
The total antioxidant activity decreased as result of thermal
processing in all Mexican maize landraces types. The ABTS
and ORAC values for extruded tortillas ranged from 5543.9 to
5945.1 and 8235.9 to 8800.8 μmol TE/100 g DW, respective-
ly (Fig. 1). The retention of antioxidant activity levels in
pigmented maize extruded tortillas was significantly higher
(p < 0.05) compared to counterparts traditionally produced.
Most tortillas manufactured from extruded flours retained
84.4% of the ORAC and 75.9% of the ABTS values observed
in raw kernels. Our results indicated that the proposed lime-
cooking extrusion strategy was instrumental in retaining high
levels of phytochemicals (total phenolics and phenolic acids)
and antioxidants associated with raw grains.
Fig. 1 ABTS and ORAC values
of free, bound and total fractions
of Mexican native maize kernels
and their corresponding tortillas
manufactured from nixtamalized
or extruded flours. Bars with no
letters in common are
significantly different (p < 0.05).
NT = tortillas from nixtamalized
flour; ET = tortillas from extruded
flour
Cellular Antioxidant Activity The more informative CAA
assay has the advantage over traditional chemical antioxidant
tests because it takes into consideration the cellular uptake,
distribution, bio-accessibility, and metabolism of bioactive
components and therefore predicts better the antioxidant abil-
ity of bioactive compounds [13, 14].
The CAA was measured using two protocols (with and
without a PBS wash) cell after incubation. Both protocols
were used owing to the difference that they may provide in-
formation on the degree of uptake and membrane association
of the phytochemicals compounds present in the extracts.
Hence, washing the cell with PBS remove compounds that
are only loosely associated with the cell membrane and in-
creased the consistency of the results [14].
The results of CAA for free, bound and total phenolics in
PBS wash protocol are presented in Table 3. Among all raw
maize, total CAA values indicated that the blue maize had the
greatest cellular antioxidant activity (18.7 μmol QE/100 g
DW) followed by white maize (11.4 μmol QE/100 g DW).
The bound fraction of raw maize exhibited the higher CAA
values compared to the free fraction. The contributions of
bound phenolics to the total CAA values were 79.8 to
84.0%. These phenolics are physiologically essential because
some fractions can survive the human stomach and small in-
testines digestion and reach the colon intact, where they are
released and exert in situ antioxidant effects [27].
Both traditional and lime extrusion cooking processes used
for the production of tortillas caused significant (p < 0.05)
reductions of CAA when compared to raw kernels. The re-
maining total CAA values for traditional and extruded tortillas
prepared from Mexican native maize ranged from 5.4 to
9.1 μmol QE/100 g DW.
The total CAA values of tortillas prepared with
nixtamalized flour were significantly lower (p < 0.05) com-
pared to counterparts made with extruded flours (Table 3).
 Author's personal copy
Plant Foods Hum Nutr

Table 3 Cellular antioxidant activity of free, bound and total fractions in Mexican raw native maize types and their tortillas produced throughout
nixtamalization or lime cooking extrusion

Maize type Sample/ Product Cellular antioxidant activitya

[micromole quercertin acid equivalent (μmol QE)/100 g dry weight (DW)]

Free % Bound % Total Retention (%)

B-512 Raw 3.0 ± 0.0b (16.0) 15.7 ± 0.9ab (84.0) 18.7 ± 1.5a
NT 2.6 ± 0.1d (28.6) 6.5 ± 0.3f (71.4) 9.1 ± 0.6dd 48.7 ± 1.5b
ET 3.7 ± 0.1d (25.5) 10.8 ± 0.7c (74.5) 14.5 ± 0.8b 77.5 ± 2.5a
W-111 Raw 2.3 ± 0.2b (20.2) 9.1 ± 0.9a (79.8) 11.4 ± 0.2cc
NT 2.1 ± 0.2b (38.9) 3.3 ± 0.2g (61.1) 5.4 ± 0.6ee 47.4 ± 1.5c
ET 2.5 ± 0.2b (30.1) 5.8 ± 0.7d (69.9) 8.3 ± 0.7dd 72.8 ± 1.7a
a
Values (means ± SD, n = 3) within columns with no letters in common are significantly different (p < 0.05)
b
NT, Tortillas produced from nixtamalized flour; ET, Tortillas produced from extruded flour
c
Values in parentheses indicate percentage contribution of this fraction to the corresponding to the total CAA

Overall, the reduction of the CAA of native maize samples
during the elaboration of tortillas could be attributed to the
combined effects of alkaline and thermal processing, which
facilitated the breakdown of certain antioxidant compounds
and reduced total phenolic as indicated previously (Table 1).
A remarkable retention of CAA values was observed in
extruded tortillas (72.8 to 77.5%). A substantial retention of
CAA values compared to raw grains was observed in B-512
(77.5%) maize extruded tortillas. Higher concentration and
stability of the cellular antioxidant activity in those tortillas
is mainly attributed to the short-time lime cooking extrusion
process of whole grains. The CAA values in the free and
bound phytochemicals from Mexican native maize genotypes
and their corresponding tortillas were determined for the first
time. When compared to CAA values of other cereals like
proso millet [28] and whole feijoada meal [29], the phyto-
chemicals extracted from Mexican maize genotypes and their
tortillas exhibited higher CAA.
Conclusions
The present research revealed for the first time cellular anti-
oxidant activity (CAA) of Mexican native maize and its
tortillas. Lime cooking extrusion appeared to be the most
promising process for keeping the bioactive compounds,
whereas overall, traditional nixtamalization had the most un-
favorable effects. It is noteworthy that tortillas obtained from
extruded native maize flour had high retention of bioactive
phytochemicals, including phenolic acid content, as well as
AOX and CAA. A major portion of ferulic acid, AOX, and
CAA of raw maize genotypes and their tortillas were found in
the bound fractions. However, a more detailed research on the
individual phenolic compounds present in different Mexican
native maize types and their tortillas needs to be carried out in
order to provide a complete characterization of their health
benefits.
Acknowledgements This research was partially supported by
Universidad Autónoma de Sinaloa (Project PROPAPI-UAS 2015) and
Fondo CONACyT (Project 168279).
Compliance with Ethical Standards
Conflict of Interest The authors declare that there are no conflicts of
interest.
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