QUALITY CHARACTERISTICS OF SNACKS PRODUCED FROM NIXTAMALIZED CORN FLOURS OF NEW DROUGHT-TOLERANT YELLOW CORN HYBRIDS
Abstract and keywords
Abstract (English):
Introduction. Producing new maize cultivars in areas with limited water resources is the main task of plant breeders. However, there is little information regarding their technological characteristics and industrial potential. Besides, snacks have gained worldwide acceptability and become part of modern food culture, especially among young people and children. Thus, our study aimed to produce corn snacks from new yellow corn hybrids planted under water stress in Delta region, Egypt. Study objects and methods. We investigated healthy processing techniques and used nixtamalization and baking instead of frying. We also evaluated the chemical composition and starch crystallinity of flour, the rheological properties of dough, as well as color attributes and sensory characteristics of baked snacks. Results and discussion. Significant differences (P ˂ 0.05) were found between all corn genotypes in their fat, protein, ash, crude fiber, and carbohydrate contents. The experimental drought conditions caused higher protein and fat contents compared to normal conditions. X-ray diffraction indicated that nixtamalization decreased starch crystallinity. Also, X-ray and rapid visco analysis showed that Y2 genotype exhibited the highest crystallinity and the lowest pasting properties, while Y3 and Y5 had the lowest crystallinity and the highest pasting properties. Baked snacks made from nixtamalized corn flour of genotypes planted under drought conditions had comparable quality characteristics in terms of color and sensory properties to the control snacks made from SC178 genotype planted under normal conditions. Conclusion. The new corn hybrids grown in limited water conditions and the developed snacks represent a healthy alternative to cornbased fried snacks.

Keywords:
Drought-tolerant plants, nixtamalization, X-ray, snacks, sensory evaluation, corn
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INTRODUCTION
In recent years, snack foods have gained importance
and popularity worldwide and become part of modern
food culture. However, they have a low nutritional
value due to high carbohydrate and fat contents and a
low protein content [1]. Moreover, the dependence on
convenience snacks has exposed consumers to a higher
risk of obesity, cardio-vascular disease, and cancer [2].
Recently, with the revolution in food marketing and the
consumer trend towards healthier low sodium, low oil,
and low calorie foods, it is necessary to develop new
products that offer quality, variety, cost-efficiency,
convenience, and nutritive value [3].
The current trend in the food industry is to develop
more nutritive snack foods, rather than eliminate
snacks from the diet, largely due to their economic
value [1]. Snacks has become very popular all over the
world, especially among children [4]. Tortilla chips
and corn chips are the most popular corn-based snack
products. Corn chips are fried products made from
corn flour, while tortilla chips are Mexican corn snacks
traditionally manufactured from nixtamalized corn
grains with or without frying [5]. The resulting snacks,
even fried after baking, have a lower oil content, firmer
texture, and a stronger alkaline flavor compared to
corn chips. They are convenient, ready-to eat, and
inexpensive corn products with digestive and dietary
principles of vital importance [6].
Nixtamalization is a process that involves alkaline
cooking and steeping of corn kernels, which are
then washed and ground to produce masa (soft and
moist dough). Corn masa is kneaded and molded, and
then baked on a hot griddle for tortilla chips [7−8].
Nixtamalization provides nutritional, technological, and safety benefits to corn grains. The nutritional benefits
include improved protein quality, increased calcium
and B-vitamins availability, and reduced phytic acid
and tannins contents [9]. Technologically, nixtamalized
grains are more easily ground due to softer pericarp and
endosperm, with gelatinized starch and improved aroma.
In addition, nixtamalization reduces mycotoxin contents
in corn grains [10].
Maize is a vital crop for both human food and
livestock feed, and the demand for maize and its
products grows day by day due to its versatile uses,
including medicine, textile, and biofuel production
[11−12]. By 2025, maize will be the most common crop
produced all over the world [13]. Water and productive
land limitation leads plant breeders to vertical expansion
through improving the efficiency of water use and
increasing unit area productivity [14−15].
In this regard, maize production programs are
continuously trying to increase yield, quality, and
stability under water deficit conditions [16]. While grain
yield is a commonly investigated parameter, quality
and technological parameters have less attention [17].
Therefore, we aimed to investigate the possibility of
using nixtamalized corn flours – obtained from the best
yellow corn hybrids based on grain yield under drought
conditions – in baked corn snacks production.
STUDY OBJECTS AND METHODS
Raw materials. For this study, we used materials
planted under normal and water stress (drought)
conditions in the Experimental Farm of Agricultural
Research Centre (ARC), Delta region, EL-Kalyubia
Governorate, Egypt. We selected six of the best yellow
maize crosses (Y ̶Y6) according to their superiority
in grain yield under drought conditions in the field
experiment (yield and irrigation data published
in Esmail et al.) [14]. They were obtained from
hybridization between the imported CIMMYET parental
lines following the half-diallel crossing system. Single
cross Giza 178 was used as a chick variety. Chemicals
and other ingredients for ready-made snacks production
were purchased from the local market.
Chemical composition. Moisture, ash, fiber, protein,
and fat contents in corn hybrids were determined by
methods recommended by the Association of Official
Analytical Chemists [18]. Total carbohydrates were
calculated by difference.
Preparation of nixtamalized corn flour.
Nixtamalized corn flour was prepared according to
the method of Quintanar-Guzman et al. with some
modification [19]. In particular, corn kernels were
boiled in a 1% calcium hydroxide solution (percent
by grain weight) for 2 h, soaked in boiled water for
14 h, and washed with excess tap water followed by
decantation using a sieve. The washed nixtamalized
grains were dried for 8−10 h at 60°C and then cooled
to 25°C. The dried grains were milled in an analytical
mill (Brabender mill, Junior) to pass a 60 mesh
screen (0.0028 in sieve opening), and a minimum of
0.102 ± 0.06 cm of free space between the shaft and the
stationary body of the mill. The masa prepared from
grains was packed in polyethylene bags and stored in a
refrigerator (4°C) until use.
X-ray diffraction. Starch crystallinity was
evaluated by X-ray diffraction patterns of the samples
using monochromatic CuK radiation on a Philips
X-ray diffract meter at 35 kv and 15 mA (Central Lab,
National Research Centre, Egypt). Lyophilized samples
were placed on the l cm2 surface of a glass slide and
equilibrated overnight at * a relafive humidity of 91%
and run at 2–32 θ (diffraction angle 2 θ). The spacing
was computed according to Bragg’s law [20].
Pasting properties of flours. Pasting properties
of nixtamalized corn flours were determined using a
rapid visco analyzer starch master R&D pack V 3.0
(Newport Scientific Narrabeen, Australia) according to
the methods approved by the American Association of
Cereal Chemists [21]. The measured parameters were
pasting temperature, peak viscosity, trough viscosity,
final viscosity, breakdown and setback viscosity.
Preparation of snacks. Snacks were prepared
according to Agrahar-Murugkar et al. by mixing 100 g
NCF and 3 g salt in a planetary mixer for 2 min at a low
speed using a flat blade, then adding 15 mL sunflower oil
and mixing for another 6 min [2]. After this, we changed
the mixer blade to a hook type, added 50 mL water,
and mixed the dough for about 2 min at a low speed,
followed by a medium speed for 2−4 min until soft,
cohesive and pliable dough developed. The prepared
dough was covered with wet muslin cloth and left to
rest for 5 min at room temperature. Then, we sheeted
it manually, cut in a circular shape (1.50 mm thick) and
baked at 180°C for 8 min on one side and another 5 min
on the other side. The chips were then dried for 1 h at
70°C and cooled to room temperature.
Color quality of processed snacks. The color
parameters of snacks were evaluated using a Hunter
color meter (Hunter Associates Lab Inc. (Model No:
LabScan XE, USA). The instrument was calibrated
with a white standard tile of Hunter Lab color standard
(LX N o. 1 6379): x = 7 7.26, y = 8 1.94 a nd z = 8 8.14
(L* = 92.43, a* = −0.88, b* = 0.21). The results were
expressed in accordance with the CIELAB system for
L* (L* = 0 [black], L* = 100 [white]), a* (−a* = g reenness,
+a* = redness), and b* (−b* = blueness, +b* = yellowness).
In addition, the total color difference (ΔE)
between the control snacks (made from SC178 planted
under normal irrigation conditions) and those made from
corn genotypes planted under drought conditions was
calculated as follows:
ΔE = [(ΔL)2 + (Δa)2 + (Δb)2] 0.5
Along with this, we calculated Hue angle, Chroma,
and Browning Index (BI) using the following expression:
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Chroma = [(a*)2 + (b*)2] 0.5
Hue angle = tan−1 (b*/a*)
Browning Index (BI) =
[100 (x − 0.31)]
0.17
Where, x =
(a* + 1.75L*)
(5.645L* + a* − 3.012b*)
Sensory evaluation. Snacks were evaluated for
their sensory characteristics by 15 trained panelists.
The tested characteristics included color, flavor, taste,
crispiness, appearance, and overall acceptability [22].
Statistical analysis. The obtained data were
statistically analyzed using the SAS Systems for
Windows software, version 6.12 TS020 (SAS, Statistical
Analysis System, Institute Inc., Cary, NC, 1996). We
performed analysis of variance (ANOVA) and the least
significant difference (LSD) test (P < 0.05) to determine
significant differences between the treatment means.
RESULTS AND DISCUSSION
Chemical composition of yellow corn hybrids.
The chemical composition of tested corn samples
planted under normal irrigation and drought conditions
is presented in Table 1. We found significant genotype
differences in moisture, protein, fat, fiber, ash, and
carbohydrates. The moisture contents of corn genotypes
varied in a narrow range from 11.33 to 12.70%. We
noticed a slight decrement in moisture among all corn
hybrids planted under drought conditions compared to
normal conditions. This decrement was insignificant
in some genotypes (SC178, Y1, Y2, and Y5) and
significant in others (Y3, Y4, and Y6). The protein
content, however, varied in a wide range: its highest
value (13.28%) was found in Y4 genotype planted
under drought conditions and the lowest (9.52%), in Y6
genotype planted under normal conditions. Also, the
fat content varied from 4.28 to 5.50% for Y6 and Y2
genotypes planted under normal conditions, respectively.
Generally, we found that the corn genotypes planted
under drought conditions had higher protein and fat
contents compared to those planted under normal
conditions. Each genotype showed higher protein and
fat contents under water stress conditions compared to
normal conditions. Carbohydrate contents, however,
showed a reverse trend. Similar results were reported
by Barutcular et al. for maize and Rharrabti et al. for
wheat [12, 23]. Mousavi et al. reported that water stress,
especially during the flowering stage, affected the
photosynthesis process and thus greatly decreased the
starch content while increasing protein and fat contents
in the grains [24].
The fiber contents of corn genotypes varied from
2.95 to 3.30% for Y2 planted under water stress and
SC178 planted under normal conditions, respectively.
At the varietal level, there were no significant
differences between the fiber contents of Y1, Y2, Y4,
and Y5 genotypes under both irrigation conditions. Fiber
contents of SC17 and Y6 genotypes showed a significant
decrement under drought conditions compared to
normal conditions. By contrast, Y3 genotype revealed a
significant increment in fiber under drought conditions.
Regarding ash, we found that SC178 showed the highest
Table 1 Chemical composition of yellow corn genotypes (% on dry weight basis)
Genotype Moisture Protein Fat Fiber Ash Carbohydrates
Yellow corn hybrids planted under normal conditions
SC178 11.82BCD 10.15F 4.39F 3.30A 1.65A 80.51AB
Y1 11.95BC 9.75G 5.20B 3.12BC 1.44BC 80.49AB
Y2 11.09E 10.90CD 5.21B 3.19ABC 1.30D 79.40BCD
Y3 12.22AB 10.65DE 4.80D 2.98EF 1.31CD 80.26AB
Y4 12.70A 10.50EF 4.50EF 3.10CDE 1.26DE 80.64AB
Y5 11.70CD 10.51E 4.30F 3.20ABC 1.32CD 80.67AB
Y6 12.50A 9.52G 4.28F 3.29A 1.19DEF 81.72A
Yellow corn hybrids planted under drought conditions
SC178 11.50CDE 11.80B 4.90CD 3.10CDE 1.12F 79.08BCD
Y1 11.56CDE 10.78CDE 5.10BC 3.10CDE 1.49B 79.53BCD
Y2 11.51CDE 11.69B 5.50A 2.95F 1.19DEF 78.67CD
Y3 11.40DE 13.01A 4.50EF 3.17ABC 1.25DE 78.17CD
Y4 11.33DE 13.28A 4.70DE 3.20ABC 1.22DEF 77.60D
Y5 11.65CD 11.05C 4.79E 3.25AB 1.30D 79.61BC
Y6 11.59CDE 10.89CD 4.80E 3.03DEF 1.15EF 80.13ABC
LSD 0.5103 0.3591 0.2536 0.1345 0.1397 1.9701
SC178 = Single Cross Giza 178, Y1–Y6 = new yellow corn hybrids
Means with the same letters in the same column are not significantly different
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value (1.65%) under normal irrigation and the lowest
value (1.12%) under drought conditions. However,
there were no significant differences between the ash
contents of the six new genotypes under both irrigation
conditions.
The high protein and fat yielding genotypes and
the comparable fiber and ash contents under drought
conditions may be due to the drought tolerance of the
new hybrids. The chemical composition of yellow
maize (on a dry weight basis) was previously reported
by Watson as 71.7% starch, 9.5% protein, 4.3% fat,
and 1.4% ash [25]. Compared to these data, all the corn
genotypes in our study had high protein and fat contents.
Similar values for these macronutrients were also found
among 1245 corn samples from different locations
all over the world [26]. Also, the reported values for
moisture and fat contents of yellow corn are close to
those reported by Yaseen et al. and Hussein et al.,
being 12.50 and 5.15%, respectively [27, 28]. However,
they reported lower values for crude protein (7.88%),
ash (0.5%), crude fiber (2.5%), and total carbohydrates
(76.0%).
Starch crystallinity of yellow corn genotypes.
The X-ray diffraction pattern diagrams for raw and
nixtamalized corn samples are shown in Fig. 1a and
B, respectively), and the respective crystallinities are
illustrated in Fig. 1c. All raw corn genotypes planted
under normal and drought conditions showed A-type
diffraction peaks around 9.9, 5.8, 5.1 and 3.8 Å at 8.8°,
15.0°, 17.4° and 22.9° (at 2θ), respectively. There were no
clear differences between the diffractograms of yellow
corn genotypes.
Similar results were previously reported in [29–32].
They stated that X-ray diffractions of native cereal
starches showed pure “A” type peaks. In addition, Abd-
Allah et al. mentioned that the calculated “d” spacing
of yellow corn starch ranged between 5.4004 and
3.4767 Å [29]. Also, they assumed that symmetric X-ray
diffraction patterns of the tested samples could be due
to the fact that cereal starch is a homogeneous material
mainly composed of amylose and amylopectin. On the
other hand, the specified diffracting angle (at 2θ) for
each peak in each starch type could be explained by the
molecular weight and the amylose/amylopectin ratio
variations.
As we can see in Fig. 1b, a diffraction peak at about
4.4 Å was developed in the nixtamalized samples.
It is also clear that the specified peaks in the NCF
diffractograms were characterized by decreased
intensity and broad background compared to those
in the raw samples (Fig. 1a). The peak at 4.4 is the
first indication of a V-type amylose-lipid complex
pattern [33].
Arambula et al. revealed that an amylose–lipid
complex developed as a result of starch gelatinization
during extrusion or nixtamalization of corn flour [31].
Besides, Mondragon et al. mentioned that amylose–lipid
complexes might develop during alkali steeping [34].
Finally, Agrahar-Murugkar et al. noted that the location
of this peak was slightly displaced from the strong 4.4 Å
Figure 1 X-ray diffraction diagrams for raw corn samples (a) and nixtamalized corn samples (b), and crystallinity values for raw
corn samples (c). NSC178 = Single Cross Giza 178 planted under normal conditions, DSC178 = Single Cross Giza 178 planted
under drought conditions, Y1–Y6 = new yellow corn hybrids planted under drought conditions
(a) (b) (c)
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to around 4.5–4.7 Å in the X-ray pattern of fried tortilla
chips [2].
Beside the transition from pure “A” pattern in raw
corn flour to “A + V” pattern in NCF, the decreased
peak intensity and its broad background indicated
the transition from the semi-crystalline phase to the
amorphous phase resulting in a partial disruption of
the crystalline starch structure [31, 34]. As we can see
in Fig. 1c, Y2 genotype had the highest crystallinity
value (87.11%), followed by Y6 and Y4 genotypes
(64 and 60%, respectively). Y3 genotype had a lower
crystallinity value (24.58%), with the lowest recorded for
Y5 (21%). In general, starch crystallinity in corn flours
may be affected by mechanical (milling process) and
amylolytic activity, as it decreases with damage caused
to starch granules [32, 35].
Pasting properties of hybrid nixtamalized corn
flour. The pasting properties of NCF dough were
rheologically evaluated by a rapid visco analyzer (Table
2). The results showed wide variations in peak viscosity,
trough value, breakdown, final, and setback viscosity of
yellow corn hybrids planted under drought conditions.
However, all corn hybrids showed the same peak time.
For instance, the peak and final viscosity values ranged
from 151 cp to 660 cp and from 250 cp to 1186 cp for
Y2 and Y3 genotypes, respectively. The trough value,
breakdown and final viscosity ranged from 128 cp to 541
cp, from 23 cp to 119 cp, and from 122 cp to 645 cp for
the same genotypes, respectively. All parameters were
greater for Y3 genotype, while Y2 genotype had lower
parameters.
Pasting properties are measurements of starch
behavior (gelatinization and retrogradation) during
processing [36]. These properties could be affected by
the molecular structure of amylopectin (branch chain
length and distribution) [37] and the granule size [38].
Amylopectin contributes to swelling and pasting of
starch during heating. Amylose contributes to starch
retrogradation during the cooling stage through its
aggregation by hydrogen bonds [39].
It was indicated that the presence of lipids restricts
the swelling of starch granules and amylose leaching,
resulting in reduced viscosity of the corn flour paste
during gelatinization, whereas amylose and lipids inhibit
the swelling [40, 41]. In our study, the lower viscosity
values of Y2 hybrid could be due to its high fat content
(Table 1) and a higher crystallinity degree (Fig. 1c).
On the other hand, Sefa-Dedeh et al. reported a drastic
reduction in the pasting properties of NCF compared to
raw flour [9]. They attributed the reduction in viscosity,
especially during the cooling stage, to the saturation of
hydroxyl groups on the starch molecules with calcium
ions (Ca2 ). The resulting Ca(OH) ions prevent any
further association of the starch molecules in the cooked
paste viscosity.
Color attributes of corn snacks. The color of
nixtamalized corn flour-based products is an important
quality parameter which directly influences the
consumer’s acceptability of the product. Table 3 and Fig.
2 show the color quality of snacks manufactured from
NCF of SC178 genotype planted under normal and water
stress conditions, as well as the new hybrids (Y1–Y6)
planted under water stress conditions. We found a wide
range of significant differences for all color parameters
of the snacks: 61.58 ̶ 69.91, 3.35 ̶ 9.17 and 25.02–32.12
for lightness (L*), redness (a*) and yellowness (b*),
respectively.
Noteworthily, the snacks produced from SC178
genotype planted under normal irrigation conditions
showed the lowest L* and the highest a* and b* values.
The highest L* was recorded for snacks produced from
Y1, while the lowest a* and b* values were recorded
for snacks produced from Y4 genotype. The total
color differences (ΔE), chroma (C*), hue angle (H*)
and browning index (B.I.) varied between 8.23 ̶ 11.96,
25.24 ̶ 33.40, 74.05 ̶ 83.32 and 47.36 ̶ 82.16, respectively.
The snacks produced from corn hybrids planted
under drought conditions tended to have higher L* and
H* values and lower a*, b*, C* and BI values, compared
to those produced from SC178 planted under normal
conditions. Similar previous studies stated that the color
of NCF ranged from white to dark yellow, depending on
the alkali concentration, processing conditions, and corn
type [2, 42, 43]. In addition, Sefa-Dedeh et al. stated that
the yellowish color in NCF-based products, even when
produced from white corn, was closely related to the
Table 2 Pasting properties of nixtamalized corn flour
Sample Peak Time, min Peak Viscosity, c.p. Trough, c.p. Breakdown, c.p. Final Viscosity, c.p. Setback, c.p.
NSC178 7.0 535 430 93 932 502
DSC178 7.0 569 471 98 952 481
Y1 7.0 430 354 76 784 430
Y2 6.9 151 128 23 250 122
Y3 7.0 660 541 119 1186 645
Y4 7.0 214 172 42 344 172
Y5 7.0 577 482 95 980 498
Y6 7.0 177 145 32 287 142
NSC178 = Single Cross Giza 178 planted under normal conditions, DSC178 = Single Cross Giza 178 planted under drought conditions,
Y1–Y6 = new yellow corn hybrids planted under drought conditions
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Table 3 Color attributes of snacks from drought-tolerant corn genotypes
Samples Lightness (L*) Redness
(a*)
Yellowness
(b*)
Total color
differences (ΔE)
Chroma
(C*)
Hue angle
(H*)
Browning
index (B.I.)
NSC178 61.58D 9.17A 32.12A 0.00E 33.40A 74.05D 82.16A
DSC178 69.53AB 3.33G 28.45D 10.53B 28.64D 83.32A 54.75D
Y1 69.91A 3.82E 29.53C 10.23B 29.78C 82.63A 57.42C
Y2 69.03AB 4.99B 26.24F 10.37B 26.71F 79.24C 52.09E
Y3 66.93C 3.55F 27.26E 9.16C 27.49E 82.58A 54.85D
Y4 69.25AB 3.35G 25.02G 11.96A 25.24G 82.38A 47.36F
Y5 68.58B 4.88C 30.06B 8.46D 30.45B 80.78B 61.34B
Y6 66.37C 4.49D 27.34E 8.23D 27.71E 80.68B 56.74C
LSD 1.1735 0.0859 0.4883 0.6242 0.4982 1.3986 1.0263
NSC178 = Single Cross Giza 178 planted under normal conditions, DSC178 = Single Cross Giza 178 planted under drought conditions,
Y1–Y6 = new yellow corn hybrids planted under drought conditions
Figure 2 Snacks processed from drought-tolerant corn genotypes. NSC178 = Single Cross Giza 178 planted under normal
conditions, DSC178 = Single Cross Giza 178 planted under drought conditions, Y1–Y6 = new yellow corn hybrids planted
under drought conditions
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lime concentration [9]. This observation could be due to
the varietal performance of yellow corn hybrids (yellow
pigments content) under drought conditions.
Browning index (BI) is the most important color
attribute in baked products because it affects their
final quality [44]. With respect to the yellow pigments
content, browning coloration could be due to both
enzymatic and non-enzymatic reactions. During
nixtamalization, once cell walls and cellular membranes
lose their integrity, enzymatic oxidation of phenolic
compounds rapidly takes place by polyphenols
oxidase [45]. However, the non-enzymatic Maillard
reaction takes place between reducing sugars and
proteins during the baking process.
Sensory evaluation of corn snacks. The mean
scores of sensory characteristics (Table 4) showed
significant differences (P ≤ 0.05) between the genotypes
for color, crispiness, odor, taste, appearance, and overall
acceptability. The snacks produced from SC178 planted
under normal conditions and Y5 planted under drought
conditions were rated highest in all sensory attributes,
while those produced from Y2 were rated lowest. As we
said above, color is a very important quality parameter
of baked products that reflects raw material formulation
and processing.
The brown-yellow color measured by the Hunter
instrument (Table 3) for the snack samples manufactured
from SC178 and Y5 NCF confirmed the results of
sensory analysis. The favorable taste and aroma of these
samples could be due to the Millard reaction that takes
place during baking. In a similar work by Agrahar-
Murugkar et al., nixtamalization improved the sensory
properties of chips [2]. Further, in a study to identify the
market demand for corn-based snacks, Menis-Henrique
et al. found a need for snacks with a lower fat content
and a better nutritional value [46]. Therefore, we can
conclude that nixtamalized corn flour is organoleptically
superior and this technology could be used on a
commercial scale.
CONCLUSION
We found that Y3 and Y5 genotypes grown under
water stress conditions provide corn grains with superior
quality that can be used in snack production. Also, we
can conclude that baked snacks made from nixtamalized
corn flour are a healthy alternative to fried snacks.
Finally, these findings could contribute to achieve both
food and nutritional security, especially in water scarce
areas.
CONTRIBUTION
The authors were equally involved in designing the
research plan. Prof. Ramadan Esmail was involved in the
production and cultivation of new yellow corn hybrids.
Ahmed Hussein and Ayman Mohammad took part in
the production of NCF, as well as in the manufacture
and evaluation of snacks. Attia Yaseen and Ayman
Mohammad were involved in writing the manuscript,
and Ayman Mohammad checked it for plagiarism.
CONFLICT OF INTEREST
The authors declare no conflict of interest.

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