LWT - Food Science and Technology 65 (2016) 597e603

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LWT - Food Science and Technology

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Effects of baking conditions and dough formulations on phenolic

compound stability, antioxidant capacity and color of cookies made
from anthocyanin-rich corn flour


SlaCana Zili lı c, Jelena Van

c a, Tolgahan Kocadag cetovi € kmen c, *
c b, Vural Go
a
Department of Food Technology and Biochemistry, Maize Research Institute, Slobodana Bajica 1, 1185 Belgrade-Zemun, Serbia
b
Breeding Department, Maize Research Institute, Slobodana Bajica 1, 1185 Belgrade-Zemun, Serbia
c
Department of Food Engineering, Hacettepe University, 06800 Beytepe, Ankara, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: In this study, the effect of baking conditions and dough formulations on phenolic compounds, antioxi-
Received 17 June 2015 dant capacity and color of cookies made from anthocyanin-rich corn flour were investigated. The control
Received in revised form cookies were prepared from dark-red popping corn, blue popping corn and blue-standard corn flour
19 August 2015
according AACC method 10e54 and baked at 200
C for 7 and 10 min. In control corn cookies, the content
Accepted 22 August 2015
Available online 28 August 2015
of total flavonoids and anthocyanins, as free water-soluble phenolic compounds, was reduced by applied
baking conditions. Our results show that citric acid significantly increased the total flavonoids and an-
thocyanins content in the cookies prepared from blue popping corn and blue-standard corn. Compared
Keywords:
Corn cookies
with the control cookies baked at 200
C for 7 min, the cookies prepared with 0.5 g/100 g added citric
Baking conditions acid and baked at 150
C for 12 min had higher contents of total flavonoids and anthocyanins by 60 and
Dough formulations 70%, respectively. However, as a result of Maillard reaction inhibition at low pH, antioxidant capacity of
Phenolic compounds stability anthocyanins-rich blue popping corn and blue standard corn cookies with 0.5 and 1 g/100 g citric acid
Antioxidant capacity was lower by 56 and 39%, respectively, compared to antioxidant capacity of their control cookies baked at
200
C for 10 min.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction
From the market standpoint, attractive and stable color is the
important sensory property of foods. In recent years, the food in-
dustry has been increasingly using various natural colorants of
plant origin that have both coloring and antioxidant properties.
Contrary to the artificial additives, natural colorants have attracted
considerable interest due to their presumed safety, as well as po-
tential health effects. However, the incorporation of natural color-
ants to food systems is a certain challenge due to their low stability
regarding the factors such as light, oxygen, temperature and pH
(Ba˛ kowska-Barczak, 2005).
In addition to the carotenoids and betalains, flavonoids/antho-
cyanins are the main class of plant pigments (Tanaka, Sasaki, &
Ohmiya, 2008) that have found usage in functional food systems
due to their antioxidant properties and potential health benefits.
* Corresponding author.
€kmen).
E-mail address: vgokmen@hacettepe.edu.tr (V. Go
Summing up the results of many studies, He and Giusti (2010) re-
ported that anthocyanins possess anti-inflammatory and anti-
carcinogenic activity, cardiovascular disease prevention, obesity
control and diabetes alleviation properties. As a class of flavonoids,
anthocyanins are water-soluble glycosides with a flavylium nucleus
that provide a wide spectrum of colors ranging from red to purple/
blue. Anthocyanin pigments are labile compounds. Their structures,
stability and specific color of anthocyanins depend on co-pigments,
sugars, metal ions, oxygen, temperature and pH (Stintzing,
Stintzing, Carle, Frei, & Wrolstad, 2002). Generally, increasing hy-
droxylation of the B-ring shifts anthocyanins to bluer shades, while
the presence of many methoxyl groups shifts the color towards red
(Grotewold, 2006). At the same time, instability has the direct
relation with the number of hydroxyl groups and the indirect with
the number of methoxyl groups (Brouillard, 1982). The glycosyla-
tion level also affects stability. Diglucosides are more stable than
monoglucosides. Nevertheless, due to the presence of additional
sugar molecules, browning is more pronounced in the diglucosides
(Delgado-Vargas, Jime nez, & Paredes-Lo  pez, 2000). In addition,
color of foods rich in pelargonidin, cyanidin or delphinidin is less

http://dx.doi.org/10.1016/j.lwt.2015.08.057
0023-6438/© 2015 Elsevier Ltd. All rights reserved.
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c et al. / LWT - Food Science and Technology 65 (2016) 597e603
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stable than that of food containing petunidin or malvidin aglycones
(von Elbe & Shwartz, 1996). Indeed, a major problem for the
food industry is the thermal degradation of anthocyanins whose
extent during heating depends on both the specific composition of
anthocyanins and the characteristics of the food matrix. The
presence of the sucrose (Nikkhan, Khaymay, Heidari, & Jamee,
2007), reducing sugars and Maillard reaction products (Debicki-
Pospi, Lovri, Trinajsti, & Sablji, 1983), as well as ascorbic acid (De
Rosso & Mercadante, 2007), and phenolic compounds (Galli &
Clemente, 2013), may alter degradation kinetics. According to Li,
Walker, and Faubion (2011) acylation of anthocyanins with
organic acids improves color and pigment stability.
As a gluten-free, corn is one of the grains suitable for celiac
consumption and, together with rice, the most cultivated cereal in
the world (de la Hera, Talego  n, Caballero, & Go mez, 2014). Among
cereals, pigmented corn is the most important sources of antho-
cyanins. Simple or acylated, anthocyanins are mainly located in the
aleurone layer or pericarp of the corn endosperm, greatly affecting
the color and antioxidant capacity of the kernels (Betran, Bocholt, &
Rooney, 2000). According to our previous research (Zili
c, Serpen,
lu, Go
Akıllıog €kmen, & Van
cetovi c, 2012), the corn kernels having
red and blue colors were found to contain a wide concentration
range of total anthocyanins with cyanidin 3-glucoside (Cy-3-Glu) as
the most dominant form. However, given that corn-based foods
have to be thermally processed prior to consumption, the thermal
degradation of anthocyanins and other phenolic compounds is a
major problem in the use of pigmented corn as raw materials in the
baking and confectionery industry. Among bakery products,
cookies could easily be fortified with polyphenols to promote
health and reduce disease risk. Recently, refined wheat flour is
being partially replaced by ingredients such as anthocyanins-rich
fruits or tea extract (Mildner-Szkudlarz, Zawirska-Woitasiak, Obu-
chowski, & Goslin ski, 2009; Pasqualone et al., 2014). In this study,
red and blue corn flours were used for functional cookie prepara-
tion. The objective was to determine the effect of baking conditions,
such as baking time and temperature, as well as dough formula-
tions on the content of phenolic compounds, antioxidant capacity
and color of cookies made from anthocyanin-rich corn flour. The
primary goal was to establish the effect of citric acid on the an-
thocyanins stability in corn cookies in order to assess the organic
acid usefulness as an ingredient of bakery foods.
in Belgrade, Serbia. Whole grain flours were produced at the lab-
oratory by Perten 120 lab miller (Perten Ins., Sweden) (particle
size < 500 mm). Sodium chloride, ammonium bicarbonate, sodium
bicarbonate, sucrose, nonfat dry milk and high fructose corn syrup
used in the formulation of cookies were obtained from local
producers.
2.3. Preparation of cookies
The cookies were prepared in four recipes according to the AACC
Method 10e54 (2000) with modifications as given in Table 1. All
ingredients were mixed thoroughly in accordance with the AACC
Method 10e54 procedure using the Kitchen Aid 5KSM150 dough
mixer. Dough was rolled out to disks with a diameter of 5 cm and a
height of 3 mm, and baked in the oven (Memmert, UNE 400) at
200
C for 7 and 10 min. The cookies prepared by the modified
recipes were baked at 150
C for 12 min. All baking experiments
were performed in duplicate. Dough and cookies are shown in
Fig. 1. During the research, several recipes have been developed and
various baking conditions were used. In order to keep the number
of recipes/samples affordable, analyzes were performed on samples
of cookies which, to visual assessments, had a satisfactory color and
textural properties.
2.4. Extraction of total phenolic compounds from cookies
Phenolic compounds in the corn cookies samples were extrac-
ted according to the procedure described by Antoine, Peyron,
Lullien-Pellerin, Abecassis, and Rouau (2004). Total phenolics in
500 mg of samples were released by alkaline hydrolysis for 4 h at
room temperature using 10 mL of 4 mol/L NaOH. After the pH was
adjusted to 2.0 by 6 mol/L HCl, 5 mL of all the hydrolyzates were
extracted with 5 mL of ethyl acetate and diethyl ether (1:1, mL:mL)
for four times. Five mL of combined extracts were evaporated under
N2 stream at 30
C to dryness. Final residues were redissolved in
1.5 mL of methanol. Such prepared methanolic solutions were used
for the analyses of total phenolic compounds, flavonoids and
phenolic acids. The extracts were kept at 70
C prior to analyses.
All extractions were performed in duplicate per each replications of
baking experiment.

2. Materials and methods 2.5. Analysis of total phenolic compounds in cookies

2.1. Chemicals and consumables The total phenolic content was determined by the Folin-
Ciocalteu assay as described by Singleton, Orthofer, and Lamuela-
The chemicals of analytical grade such as Folin-Ciocalteau re- Raventos (1999) and expressed as mg of gallic acid equivalent
agent (2 N), gallic acid, 6-Hydroxy-2,5,7,8-tetramethylchroman-2- (GAE) per kg of dry matter (d.m.). One hundred mL of the extract
carboxylic acid (Trolox), 2,20 -azino-bis/3-ethyl-benzothiazoline- was transferred into test tubes and their volume made up to 500 mL
6-sulfonic acid (ABTS), ferulic acid, p-coumaric acid, as well as with distilled water. After addition of Folin-Ciocalteu reagent
methanol and acetonitrile for HPLC gradient grade were pur-
chased from SigmaeAldrich (Steinheim, Germany). Formic acid
Table 1
(reagent grade) was purchased from JT Baker (Deventer, Holland). The composition of recipes used to prepare cookies.
Sodium hydroxide, sodium carbonate, hydrochloric acid (12 mol/
Ingredients Amount in the dough (g)
L), pure ethyl acetate and pure diethyl ether were purchased from
Merck (Darmstadt, Germany). Potassium peroxydisulfate was Recipe 1 Recipe 2 Recipe 3 Recipe 4
purchased from Fluka Chemie AG (Buchs, Switzerland). Syringe Corn flour 40.0 40.0 40.0 40.0
filters (nylon, 0.45 mm) were supplied by Thermo Scientific (Wal- Shortening (refined palm oil) 16.0 16.0 16.0 16.0
tham, MA, USA). Sucrose powder 16.8 16.8 16.8 16.8
Nonfat dry milk 0.4 0.4 0.4 0.4
High-fructose corn syrup 0.6 e e e
2.2. Flour samples and cookie ingredients Sodium chloride 0.5 e e e
Sodium bicarbonate 0.4 0.4 0.4 0.4
Blue popping corn (Zea mays L. spp. Everta), dark-red popping Ammonium bicarbonate 0.2 e e 0.5
corn (Z. mays L. spp. Everta) and blue standard corn (Z. mays L.) Citric acid e 0.2 0.4 e
Water 8.8 8.8 8.8 8.8
genotypes were developed in the Maize Research Institute (MRIZP)

c et al. / LWT - Food Science and Technology 65 (2016) 597e603
S. Zili 599

Fig. 1. Color of corn dough and cookies. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(250 mL) and 20 g/100 g aqueous Na2CO3 solution (1.25 mL), tubes
were vortexed. The absorbance of the mixture was measured at
750 nm after 40 min.
2.6. Analysis of total flavonoids in cookies
The flavonoid content was determined according to Eberhardt,
Lee, and Liu (2000), and expressed as mg of catechin equivalent
(CE) per kg of d.m. Briefly, 75 mL of 5 g/100 g NaNO2 solution was
mixed with 100 mL of the extract and 1.4 mL of distilled water. After
6 min, 150 mL of 10 g/100 g AlCl3 solution was added to form a
flavanoidealuminum complex. After 7 min, 500 mL of 1 mol/L NaOH
was added, and after 10 min the mixture was centrifuged at
9000g for 5 min. Absorbance of the supernatant was measured at
510 nm.
2.7. Analysis of total anthocyanins in cookies
Anthocyanins were extracted from 200 mg of colored corn
cookies by mixing with 10 mL of methanol acidified with 1 mol/L
HCl (85:15, mL:mL) and shaking for 30 min at ambient tempera-
ture. The crude extract was centrifuged at 14,000g for 5 min and
absorbances of the supernatant were measured at 535 and 700 nm
to detect anthocyanins. Using the molar extinction coefficient of
25,965 Abs/M  cm and a molecular weight of 449.2 g/mol the level
of anthocyanins was calculated and expressed as mg of cyanidin 3-
glucoside equivalent (CGE) per kg of d.m.
2.8. Analysis of individual phenolic acids
Chromatographic analyses were performed on a Thermo
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c et al. / LWT - Food Science and Technology 65 (2016) 597e603
S. Zili
Scientific Ultimate 3000 HPLC system consisting of photodiode
array detector, quaternary pump, autosampler and column oven.
Phenolic acids were separated on a Thermo Scientific Hypersil
GOLD aQ C18 column (150 mm  4.6 mm, i.d., 3 mm) using a linear
gradient elution program with a mobile phase containing solvent A
(formic acid/H2O, 1:99, mL:mL), and solvent B (methanol) at a flow
rate of 0.8 mL/min. The solvent gradient was programmed as

c et al. (2012). The chromatograms were recorded
described by Zili
at 280 nm by monitoring spectra within the wavelength range
190e400 nm. Identified phenolic acids peaks were confirmed and
quantified by data acquisition and spectral evaluation using the
Thermo Scientific Dionex Chromeleon 7.2. chromatographic
software.
2.9. Analysis of total antioxidant capacity of cookies
The total antioxidant capacity was measured by QUENCHER
method described by Serpen, Go € kmen, Pellegrini, and Fogliano
(2008). Stock solution of ABTSþ was prepared by reacting a
7 mmol/L aqueous solution of ABTS with 2.45 mmol/L K2O8S2 (final
concentration). The cookie sample (10 mg) was mixed with 20 mL
of ABTSþ working solution, which was obtained by diluting the
stock solution in water/ethanol mixture (50:50, mL:mL), and the
mixture was shaken for 25 min. After centrifugation at 9200g
for 5 min at 10
C the absorbance measurement was performed
at 734 nm. The total antioxidant capacity was expressed as Trolox
equivalent antioxidant capacity (TEAC) in mmol of Trolox per kg
of d.m.
2.10. Measurement of pH
Ground cookies (0.4 g) were mixed with 20 mL of deionized
water and vortexed for 3 min. The mixture was held at ambient
temperature for 1 h to separate solid and liquid phases. After
centrifugation the pH of supernatants was measured using a pH
meter (MeterLab PHM210, France).
2.11. Analysis of color
Color measurements (CIE L*a*b*) were performed using a
computer vision based image analysis technique (Go € kmen, Açar,
Arribas-Lorenzo, & Morales, 2008). A color image obtained by a
digital camera, under controlled and defined illumination condi-
tions, was analyzed using software developed for this purpose by
using Matlab 8.1 (The MathWorks Inc., USA). Illumination was
achieved with 2 Philips, Natural Daylight 18 W fluorescent lamps
with color temperature of 6500 K. Images were captured at a res-
olution of 5.1 megapixel and stored in a personal computer in jpeg
format without compression. The surface color of cookies were
given as average L* (lightness), a* (redness), b* (yellowness) values.
2.12. Statistical analysis
The analytical data are reported as mean ± standard deviation of
two independent extractions per each replications of baking
experiment. Significance of differences between cookies samples
were analyzed by the Fisher's least significant differences test
(LSD), after the analysis of variance for trials set up according to the
complete randomised block design. Differences with p < 0.05 were
considered significant.
3. Results and discussion
Several studies have indicated fortification of wheat cookies
with polyphenols from different ingredients such as grape,
blueberry, poppy seed flours, as well as pomegranate peel and tea
extract (Acun & Gül, 2014; Aksoylu, Çag indi, & Ko€ se, 2015; Ismail,
akhtar, Riaz, & Ismail, 2014; Mildner-Szkudlarz et al., 2009). In
this study, dark-red popping corn, blue popping corn and blue-
standard corn flours, which differ in the content of phenolic com-
pounds, were chosen as raw materials for cookie preparation. The
content of total phenolic compounds, flavonoids, anthocyanins, as
well as phenolic acids in both corn flours and cookies are given in
Tables 2 and 3. Dark-red popping corn flour had a considerably
higher total phenolic and flavonoid content (5293.9 mg GAE/kg
d.m. and 248.7 mg CE/kg d.m., respectively) than those from blue
popping corn, as well as blue-standard corn flour. On the other
hand, although not significantly different from red popping corn,
the highest content of anthocyanins was found in blue popping
corn flour (914.6 mg CGE/kg d.m.) (Table 1). In addition to total
flavonoids, phenolic acids had a high positive correlation with the
content of total phenolic compounds. Among the used corn flours,
the highest content of total ferulic and p-coumaric phenolic acids
was detected in dark-red popping corn flour (3314.1 and 337.3 mg/g
d.m., respectively) followed by blue popping corn and blue-
standard corn flour.
The control cookies were prepared from corn flours according to
the recipe described in AACC Methods 10e54 (2000) (Recipe 1) and
baked at commonly used temperature of 200
C for 7 and 10 min.
The content of phenolic compounds in the cookies was significantly
affected by temperature and baking time (Tables 2 and 3). The data
were given per kg of cookies and also according to flour basis in
cookies (per kg of flour) to compare with the raw flour. The content
of total flavonoids, as well as anthocyanins, as free water-soluble
flavonoids, was reduced by applied baking conditions. The con-
tent of anthocyanins in the dark-red popping corn, blue popping
corn and blue-standard corn cookies baked at 200
C for 10 min was
decreased by 4, 5 and 3.5-fold, respectively, in relation to the
content in their flour (Table 2). On the other hand, in comparison
with the raw flour, the content of ferulic acid was increased by 48
and 22% after baking at 200
C for 10 min in cookies made from blue
popping corn and blue-standard corn flours, respectively (Table 3).
Similarly p-coumaric acid was increased by 55 and 24% in the same
cookies on flour basis, respectively. Baking process increased con-
tent of total phenolic compounds by affecting the solubility of
bound forms of phenolic acids. It should be mentioned that Folin-
Ciocalteu reagent does not only measure phenolic compounds,
and it may react with any reducing substance. It therefore measures
the total reducing capacity of a sample, not just phenolic com-
pounds. Certain Maillard reaction products have also reductone
structures, which are Folin-Ciocalteu reactive substances. There-
fore, not only increase in free phenolic acids from bond forms but
also formation of Maillard reaction products contributed to in-
crease in total phenolic compounds measured by Folin-Ciocalteu
assay.
Recent studies have shown that the anthocyanins with acylating
substituents are more stable during processing and storage than
other natural pigments (Baublis, Spomer, & Berber-Jime nez, 1994;
Ba˛ kowska-Barczak, 2005). These anthocyanins retain more color
at the higher pH values and show an improved resistance to other
factors such as heat and light than their unacylated analogs
(Brouillard, Chassaing, & Fougerousse, 2003). The stable acylated
anthocyanins are present in large amounts in pigmented corn
(Betran et al., 2000) and can be suitable for the application not only
in food with a low pH but also for neutral and slightly alkaline
products. However, our results show that in the baking of food
system the anthocyanins have to be protected (Fig. 1).
In order to investigate the effect of citric acid on the stability of
anthocyanins from pigmented corn flour, 0.5 and 1 g/100 g of citric
acid, on flour basis, was added to dough of blue popping corn and

c et al. / LWT - Food Science and Technology 65 (2016) 597e603
S. Zili 601

Table 2
Content of total phenolic, flavonoids and anthocyanins in pigmented corn flour and their cookies.

Corn samples Recipe Baking conditions Total phenolics (mg GAE/kg) Total flavonoids (mg CE/kg) Total anthocyanins (mg CGE/kg)

Per kg of cookies Per kg of flour Per kg of cookies Per kg of flour Per kg of cookies Per kg of flour
c a
Dark-red popping corn Flour 5293.9 ± 169.3 248.7 ± 11.8 847.4 ± 28.5b
1 200
C/7 min 1820.7 ± 44.5g 4042.7 ± 98.9f 17.2 ± 0.6d 38.2 ± 1.4h 151.9 ± 2.6b 337.4 ± 5.7c
1 200
C/10 min 2443.4 ± 121.3c 5417.4 ± 246.8c 29.0 ± 2.8b 64.3 ± 6.5ef 93.3 ± 0.1c 206.9 ± 1.8d
Blue popping corn Flour 4384.0 ± 289.5de 114.9 ± 2.6c 914.6 ± 51.1b
1 200
C/7 min 2275.7 ± 141.6d 5131.1 ± 295.5c 29.7 ± 1.0b 66.9 ± 2.2e 138.1 ± 0.1b 311.3 ± 0.1c
1 200
C/10 min 2748.4 ± 92.5b 6227.3 ± 209.4b 30.2 ± 1.3b 68.4 ± 2.9e 77.2 ± 2.6c 174.9 ± 5.8d
2 150
C/12 min 3353.5 ± 129.1a 7368.2 ± 283.6a 74.0 ± 6.5a 162.7 ± 14.3b 461.5 ± 6.5a 1014.0 ± 14.3a
Blue-standard corn Flour 4001.0 ± 220.3f 94.2 ± 9.9d 358.6 ± 1.9c
1 200
C/7 min 1843.5 ± 115.3fg 4151.6 ± 259.6ef 22.7 ± 2.2c 51.1 ± 4.4g 83.9 ± 0.4c 188.9 ± 0.9d
1 200
C/10 min 2016.1 ± 163.8ef 4536.6 ± 259.5d 24.9 ± 2.4c 56.2 ± 5.7fg 45.7 ± 1.3d 102.9 ± 2.9e
3 150
C/12 min 2439.1 ± 117.9c 5363.4 ± 259.4c 27.9 ± 0.4b 61.3 ± 0.9ef 150.7 ± 8.1b 331.4 ± 17.7c
4 150
C/12 min 1809.0 ± 113.3g 3976.3 ± 248.8f 15.2 ± 1.3d 33.4 ± 2.9h 80.1 ± 14.3c 175.9 ± 31.5d

Superscript letter in each column indicate statistically significant difference (p < 0.05). The measurements were done in duplicate per each replications of baking experiment.

Table 3
Content of phenolic acids in pigmented corn flour and their cookies.

Corn samples Recipe Baking conditions Ferulic acid (mg/g) p-Coumaric acid (mg/g)

Per g of cookies Per g of flour Per g of cookies Per g of flour

e
Dark-red popping corn Flour 3314.1 ± 1.7 337.3 ± 3.0ab
1 200
C/7 min 1287.2 ± 1.7g 2858.0 ± 3.9i 136.7 ± 2.1b 303.6 ± 4.7b
1 200
C/10 min 1515.3 ± 0.6d 3359.6 ± 1.3d 156.5 ± 2.0a 347.1 ± 4.5a
Blue popping corn Flour 2990.7 ± 0.8gh 233.9 ± 0.6cd
1 200
C/7 min 1652.8 ± 0.1b 3726.5 ± 1.0c 138.6 ± 0.7b 312.5 ± 1.5b
1 200
C/10 min 1959.3 ± 0.7b 4439.4 ± 1.5b 160.0 ± 0.1a 362.5 ± 0.1a
2 150
C/12 min 2042.2 ± 0.0a 4487.2 ± 0.0a 158.8 ± 9.3a 348.9 ± 20.5a
Blue-standard corn Flour 2635.7 ± 0.9j 214.5 ± 0.9d
1 200
C/7 min 1337.5 ± 0.1f 3012.0 ± 0.3g 118.7 ± 1.7c 267.3 ± 3.9c
1 200
C/10 min 1429.2 ± 0.4e 3215.9 ± 0.9f 118.3 ± 0.2c 266.2 ± 0.6c
3 150
C/12 min 1350.7 ± 0.9f 2970.3 ± 2.0h 120.5 ± 0.7c 265.0 ± 1.6c
4 150
C/12 min 1293.9 ± 5.8g 2842.5 ± 12.8i 108.1 ± 0.5c 237.6 ± 1.1cd

Superscript letter in each column indicate statistically significant difference (p < 0.05). The measurements were done in duplicate per each replications of baking experiment.

blue-standard corn, respectively. At the same time, high-fructose
corn syrup, sodium chloride and ammonium bicarbonate as in-
gredients enhancing the Maillard reaction and caramelization
(Go€kmen, Serpen, Çetinkaya Açar, & Morales, 2008), were skipped
from the formulation. Furthermore, in order to obtain light and
purple color development on the surface and milieu, Recipe 2 and 3
were baked at a lower temperature than of control cookies. The
baking temperature above 180
C provides browning through the
decomposition of sugar and Maillard reaction (Go € kmen, Açar,
Serpen, & Morales, 2008), while the baking time of 10 min or less
was found insufficient in terms of wheat cookies texture at tem-
peratures of 180e220
C (Go €kmen, Serpen, et al., 2008). Therefore,
the baking conditions of 150
C for 12 min was used for cookies
prepared using Recipes 2 and 3. A lower baking temperature
generally had the same effect on the content of total phenolics in
cookies prepared by the modified recipes as a high temperature of
200
C had. Compared with the control cookies baked at 200
C for
7 min, the cookies prepared according to the Recipe 2 with 0.5 g/
100 g added citric acid and baked at 150
C for 12 min had higher
contents of total flavonoids and anthocyanins by 2.5 and 3.4-fold,
respectively. Our results show that in addition to the stabilizing
effect, citric acid improved accessibility of the anthocyanins which
is in accordance with the research of Delgado-Vargas et al. (2000).
Expressed per kg of flour, the content of anthocyanins in the blue
popping corn cookies with 0.5 g/100 g citric acid was increased by
1.11-fold in relation to that in their flour (Table 2). However, 1 g/
100 g of citric acid added to the cookies prepared by the Recipe 3
and baked at 150
C for 12 min had no effect on the anthocyanins
extraction. Li et al. (2011) showed that the content of total
anthocyanins was increased in baked corn cookies with the addi-
tion of citric acid, as well as that their retention reached an
apparent plateau at about 1.5e2 g/100 g of acid. Citric acid affected
total anthocyanins in corn cookie dough and final cookies by
lowering their pH in the dough system and by acylation of their
sugar residues or flavylium cation. pH values of blue popping corn
and blue-standard corn cookie dough were 7.86 and 8.05, respec-
tively, while with the addition of 0.5 and 1 g/100 g citric acid, pH
values of dough were reduced to 4.59 and 3.72, respectively
(Table 4). The increased temperature and baking time slightly
decreased the pH values of both control and acid-modified cookies
in relation to their dough.
Amic, Baranac, and Vukadinovic (1990) reported that the red-
colored flavylium cation is the predominant anthocyanin species
in strongly acidic media (pH 1). Between pH values of 2 and 4, the
uncharged blue quinonoidal unstable species prevails, while at pH
5 and 6, unacylated anthocyanins are unstable and decolorize
quickly by hydration at the position 2 of the anthocyanidin skeleton
forming the carbinol pseudobase and chalcone structures. Ac-
cording to our results, blue popping corn and blue-standard corn
cookies with citric acid had pH values of 4.41 and 3.55 and
appeared to be pink and pale pink (Fig. 1), respectively. In addition,
lightness (CIE L*) and redness (CIE a*) of blue popping corn and
blue-standard corn cookies with citric acid were increased as
compared to color of their dough (Table 4). Due to decolourisation
of anthocyanins at pH above 7.8, color of control doughs of red
popping corn, blue popping corn and blue-standard corn was black
and gray, while the brown surface of the control cookies was a
result of thermal anthocyanins degradation, as well as the
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S. Zili

Table 4
pH values and color of pigmented corn flours, their dough and cookies.

Corn samples Recipe Baking conditions Color pH

L* a* b*

Dark-red popping corn Flour 6.70e

1 Dough 11.64 ± 1.04h 2.23 ± 0.10f 1.65 ± 0.01g 7.86ab
1 200
C/7 min 32.75 ± 0.56d 0.64 ± 0.43e 15.25 ± 0.80d 7.54cd
1 200
C/10 min 25.35 ± 1.78fg 4.73 ± 0.28d 18.91 ± 1.20c 7.38d
Blue popping corn Flour 5.60f
1 Dough 25.60 ± 1.20fg 4.96 ± 0.24g 1.12 ± 0.18g 7.89ab
1 200
C/7 min 30.93 ± 1.55de 0.74 ± 0.49e 16.23 ± 1.53cd 7.73bc
1 200
C/10 min 26.62 ± 3.08ef 5.82 ± 0.24d 22.83 ± 1.80b 7.39d
2 Dough 40.73 ± 0.99c 7.98 ± 0.37c 5.52 ± 0.34f 4.59g
2 150
C/12 min 42.03 ± 1.25c 18.75 ± 0.32a 3.49 ± 0.16fg 4.41g
Blue-standard corn Flour 5.44f
1 Dough 21.33 ± 2.02g 4.16 ± 0.16 6.19 ± 0.19f 8.05ab
1 200
C/7 min 48.38 ± 1.63b 0.83 ± 0.10e 32.63 ± 1.69a 7.54cd
1 200
C/10 min 34.58 ± 2.38 4.53 ± 0.62d 30.66 ± 1.85a 7.40d
3 Dough 48.28 ± 2.81b 5.30 ± 0.42d 16.88 ± 0.30cd 3.72h
3 150
C/12 min 58.85 ± 1.56a 10.44 ± 0.31b 15.13 ± 0.32d 3.55h
4 Dough 21.69 ± 0.32g 5.63 ± 0.02g 9.01 ± 0.14e 8.11a
4 150
C/12 min 56.25 ± 1.23a 4.39 ± 0.78g 18.85 ± 1.58c 7.63cd

Superscript letter in each column indicate statistically significant difference (p < 0.05). The measurements were done in duplicate.

formation of brown polymers of the Maillard reaction called mel-
anoidins. The usual trend for CIE L* is to decrease, and for CIE a* and
CIE b* to increase during browning proceeds (Romani, Rocculi,
Mendoza, & Dalla Rossa, 2009), which is in agreement with our
results. The decrease in pH along with the lower temperatures
restricted Maillard reaction and thus non-enzymatic browning was
avoided during baking of corn cookies. In low pH media amino
groups found in amino acids and proteins present in protonated
form and their reactivity towards carbonyl compounds become
limited. Therefore the rate of Maillard reaction becomes lower. On
the other hand higher pH enhances the Maillard reaction providing
remarkable browning.
In order to determine the effect of the high pH value of dough
and ammonium bicarbonate on the phenolic compounds stability
and the color formation in blue-standard corn cookies, the con-
centration of ammonium bicarbonate, as often used leavening
agent, was increased from 0.5 (Recipe 1) to 1.25 g/100 g on flour
basis (Recipe 4). No significant increase in the pH of the dough was
observed however increased concentration of ammonium bicar-
bonate had a negative effect on both stability and content of total
flavonoids and anthocyanins. Expressed per kg of flour, the content
of the total flavonoids and anthocyanins in the blue-standard corn
cookies with 1.25 g/100 g of ammonium bicarbonate baked at
150
C for 12 min was decreased by 35 and 7%, respectively, in
relation to that in control cookies with 0.5 g/100 g of ammonium
bicarbonate baked at 200
C for 7 min. Moreover, decrease in total
flavonoids and anthocyanins were found to be 65 and 51%,
values and pale green color on the surface (Fig. 1) Light color of
cookies was the result of a low baking temperature, as well as the
absence of high fructose corn syrup and sodium chloride, which
enhance Maillard reaction and caramelization.
The results in Table 5 show the effect of applied baking condi-
tions, as well as various pH values on total antioxidant capacity
of corn cookies. It was evident that the phenolic compounds in
corn flours contributed to its antioxidant capacity. Among used
raw materials the dark-red popping corn flour had the highest
antioxidant capacity (44.04 mmol Trolox/kg). Baking treatments
increased antioxidant capacity of control cookies, on flour basis.
The increase in total antioxidant capacity of control cookies pre-
pared from dark-red popping corn, blue popping corn and blue-
standard corn dough with slightly alkaline pH values could be
partially explained by an increased release of bound phenolics. The


results of Zili c et al. (2014) showed that wheat grain phenolic
compounds in the bound form, as dominant, exert lower antioxi-
dant capacity in comparison with its hydrolysed and isolated free
forms. Despite this, our results show that the Maillard reaction,
which takes place in a higher rate under alkaline conditions, had
crucial influence on the increased antioxidant capacity of control
Table 5
Antioxidant capacity of pigmented corn flour and their cookies.
Corn samples Recipe Baking Total antioxidant capacity
conditions (mmol Trolox Eq/kg)

respectively, compared to that in raw flour (Table 2). Our results are Per kg Per kg of flour
in agreement with study of Corte s, Salinas, San Martín-Martinez, of cookies
and Martínez-Bustos (2006), who analyzed the effect of pH value Dark-red Flour 44.04 ± 1.31c
by different concentrations of calcium hydroxide on anthocyanins popping corn 1 200
C/7 min 22.02 ± 2.11bc 48.88 ± 4.58c
stability after nixtamalization. However, combined with the tem- 1 200
C/10 min 28.22 ± 2.21a 62.57 ± 4.91a
Blue popping corn Flour 28.46 ± 2.43ef
perature, the leavening agent increased solubility of ferulic and p- 1 200
C/7 min 22.30 ± 0.11bc 50.27 ± 0.25bc
coumaric acids (Table 3). The study of Friedman and Jürgens (2000) 1 200
C/10 min 25.05 ± 2.41ab 56.75 ± 5.45ab
demonstrates that the susceptibility of different plant phenolic 2 150
C/12 min 10.98 ± 0.52f 24.12 ± 1.14
compounds to pH strongly depends on the phenol structure. Ac- Blue-standard corn Flour 23.40 ± 0.07f
1 200
C/7 min 15.98 ± 0.37d 35.98 ± 0.83d
cording to results of these authors, ferulic acid with a single OH
1 200
C/10 min 20.04 ± 0.07c 45.08 ± 0.16c
group appears to be stable at high pH, presumably because it 3 150
C/12 min 12.22 ± 0.59ef 26.08 ± 1.31ef
cannot form quinone oxidation products. The color of cookie dough 4 150
C/12 min 14.13 ± 1.50de 31.05 ± 3.29de
with 1.25 g/100 g of ammonium bicarbonate was almost green with Superscript letter in each column indicate statistically significant difference
the CIE L*, a* and b* values of 21.69, 5.63 and 9.01, respectively. (p < 0.05). The measurements were done in duplicate per each replications of baking
After baking, cookies had more than twice higher the L* and b* experiment.

c et al. / LWT - Food Science and Technology 65 (2016) 597e603
S. Zili
cookies. The formed melanoidins are probably the predominant
contributors to the control cookies antioxidant capacity. As a result
of Maillard reaction inhibition at low pH and temperature, anti-
oxidant capacity of anthocyanins-rich blue popping corn and blue
standard corn cookies with 0.5 and 1 g/100 g citric acid was lower
by 56 and 39%, respectively, compared to antioxidant capacity of
their control cookies baked at 200
C for 10 min.
4. Conclusion
The present study showed that applied baking conditions has
great potential to release phenolic compounds associated to the
dietary fibers. However, in control corn cookies the content of total
flavonoids and anthocyanins, as free water-soluble phenolic com-
pounds, was reduced by applied temperature and baking times. As
result of thermal anthocyanins degradation and the formation of
the Maillard reaction products, surface of the control cookies was
brown. Adding of citric acid to pigmented corn dough increased the
phenolic compounds in corn cookies by anthocyanins stabilization.
Our results showed that citric acid affected total anthocyanins in
corn cookies dough and final cookies by lowering pH and by acyl-
ation of their sugar residues or flavylium cation. In addition to the
stabilizing effect, citric acid improved accessibility of the anthocy-
anins. The blue popping corn and blue-standard corn cookies with
0.5 and 1 g/100 g citric acid and their pH values of 4.41 and 3.55
appeared to be pink and pale pink, respectively. In addition, the
present study showed that the Maillard reaction had crucial
influence on the increment of antioxidant capacity in cookies.
Considering that replacement of synthetic colorants is the main
requirement of the food industry and that it can be done by acyl-
ated anthocyanins, more research is needed to evaluate the effects
of different organic acids and their concentrations on corn antho-
cyanins stabilization. In addition, although corn cookies with citric
acid had a pleasant smell of popcorn and good crispness, detailed
laboratory measurements and evaluation of textural and sensory
characteristics will be the subject of future research.
Acknowledgments
This work was financially supported by the Ministry of Educa-
tion and Science of the Republic of Serbia (Grants no. TR-31069).
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