Food Chemistry 196 (2016) 220–227

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Pigment and color stability of beetroot betalains in cow milk during

thermal treatment
Onur Günesßer
Usak University, Engineering Faculty, Department of Food Engineering, 1 Eylul Campus, 64200 Usak, Turkey

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

Article history: Thermal stability of beetroot betalains in cow milk was determined during heating at 70–90 °C. Changes
Received 3 May 2015 in color values of colored milk were also investigated. Degradation of betalains followed first order kinet-
Received in revised form 6 September 2015 ics while changes of L⁄, Hue angle and Chroma values fitted zero order and first order kinetic, respectively.
Accepted 10 September 2015
Reaction rate for degradation of betalains, L⁄, Hue angle and Chroma values ranged between 1.588–
Available online 11 September 2015
30.975
103 min1, 90.50–379.75 L⁄ min1, 0.581–5.008 Hue angle min1 and 3.250–19.750
103
min1, respectively. Between 70 and 90 °C, activation energy for the degradation of betalains was
Keywords:
42.449 kJ mol1. L⁄ values was more stable than Hue angle and Chroma color values in colored milk dur-
Beetroot extract
Thermal stability
ing heating. 74.150 kJ mol1, 111.174 kJ mol1 and 93.311 kJ mol1 of activation energy values were
Reaction kinetics found for L⁄, Hue angle and Chroma values of milk, respectively. Significant positive and negative linear
Color values correlations were determined between betalains and color values. Multiple regression models were also
established to predict the content of betalains in milk during thermal process by using color values.
Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction
The color of foods results from its raw material characteristics
which contain natural color pigments such as anthocyanins/
chlorophylls etc. or caused by colorants as food additives which
are added to food during food processing (Downham & Collins,
2000). Nowadays, many foods have already been colored by syn-
thetic food colorants. Consumer’s preferences of natural com-
pounds regarding health concerns and unwillingness of
consumers against synthetic food additives have been provided a
great impact on natural food colorants obtained from fruits, veg-
etable, plants extract or insect extract (e.g. Cochineal) (Delgado-
Vergas, Jimenez, & Paredes-Lopez, 2000; Downham & Collins,
2000; Francis, 1992).
Betalains is one of the natural color pigments as well as antho-
cyanins. They are commonly found in beetroot (Beta vulgaris), dju-
lis (Chenopodium formosanum), amaranth (Amaranthaceae), cactus
pear (Opuntia spp.), pitayas (Stenocereu ssp.) and pitahayas (Hylo-
cereus undatus) (Saenz, Cancino, & Robert, 2012). Betalains are
water soluble color pigments that are immonium derivatives of
betalamic acid. Betalains was synthesized from shikimic acid or
from tyrosine amino acid within two groups that are betacyanins
and betaxanthins. While betacyanins give from red to purple col-
ors, color of betaxanthins changes from yellow to orange
E-mail address: onur.guneser@usak.edu.tr
(Delgado-Vergas et al., 2000; Kumar & Sinha, 2004; Saenz et al.,
2012; Wissgott & Bortlik, 1996). Betalains are commercially
obtained from beetroot. Major betalains of beetroot are betanin
and isobetanin. They both give red color. Beetroot extract is used
as a red colorant and has E number E162 as a food additive in many
types of foods such as dairy products, candies, jelly beans, non-
alcoholic beverages and some emulsified types of meat products
(Azeredo, 2009; Herbach, Stintzing, & Carle, 2006a).
The major factors affecting the stability of natural colorants in
foods are the concentration of pigments, pH and water activity of
food, oxygen, light, metallic ions, enzymes, temperature and time
of processing and storage conditions (Delgado-Vergas et al.,
2000; Henry, 1992). At the point of studies on natural food col-
orants, there are several scientific researches carried out on identi-
fication of color pigments in natural plant and fruit and/or
vegetables extracts (Attia-Gamilia, Moussa, & Sheashea, 2013;
Cai, Sun, & Corke, 2005; Fernandez-Lopez, Gimenez, Angosto, &
Moreno, 2012; Kujula, Loponen, & Pihlaja, 2001; Tsai, Sheu, Wu,
& Sun, 2010). Many studies were also conducted on the effects of
some factors such as light, pH, oxygen, heat on the stability of nat-
ural color pigments in juices, plant extracts or in their aqueous
solutions (Herbach, Rohe, Stintzing, & Carle, 2006; Kırca, Özkan,
& Cemeroğlu, 2007, 2006). Nevertheless, limited studies have dis-
cussed the stability of natural color pigments in real food systems
(Altamirano et al., 1993; Castellar, Obon, & Fernandez-Lopez, 2006;
Herbach, Stintzing, & Carle, 2006b). Evaluation of the stability of

http://dx.doi.org/10.1016/j.foodchem.2015.09.033
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
 O. Günesßer / Food Chemistry 196 (2016) 220–227
natural color pigments in real food matrix during processing and/
or storage of foods in terms of kinetics approaches is essential to
determine the shelf life of food product regarding visual quality
and the usage feasibility of the natural pigments in foods.
The purpose of this study were to investigate the thermal
stability of beetroot betalains in cow milk during heating by
considering a chemical kinetics approaches and to reveal the
relationship between betalains content and color values of milk
added beetroot extract during heating by multiple linear
regression and correlation.
2. Material and methods
2.1. Materials
Raw cow milk was obtained from a local milk producer (Decem-
ber 2014). Milk sample was carried in a thermo bag with an ice
pack at 10 °C in our laboratory. Then, milk was filtered through
cheesecloth to remove solid impurities prior to heat treatments
and analyses.
2.2. Compositional analysis
The titratable acidity (lactic acid, %), pH, dry matter (%), total
protein (%), and ash (%) contents of the milk samples were deter-
mined according to procedures described by Bradley et al.
(1992). The fat content of the milk samples was determined using
a method by Gerber-Van Gulik 3059 (Bradley et al., 1992).
Fig. 1. Milk samples added beetroot extract without heating (A), milk samples heated at 90 °C for 35 min (B), milk samples heated at 80 °C for 70 min (C), milk samples
heated at 70 °C for 140 min (D).
221
2.3. Addition of beetroot betalains into milk
5 L of cow milk was colored with beetroot extract (NANTE
Chemical Company, Istanbul, Turkey) at a rate of 0.3% (w/v)
(Fig. 1A). The addition rate of beetroot extract was determined
according to the pre-coloring experiments of milk and NANTE
Chem. Company recommends. The colored milk was mixed well
at 5000 rpm by Ultraturrax (IKA-WERKE GmbH, Germany).
2.4. Degradation studies and heat treatment
Heat treatment was widely applied to milk temperature ranges
from 65 to 135 °C depends on time in dairy industry. Moreover,
natural color pigments stabilities during the heat treatment were
mostly studied between 50 and 90 °C in previous studies
(Ahmed, Shivhare, & Raghavan, 2004; Chandran, Nisha, Singhal, &
Pandit, 2014; Tsai et al., 2010). Therefore, thermal degradation of
betalains (betanin + isobetanin) and color changes of milk were
studied at 70, 80, 90 °C to realize and see the effect of high temper-
ature on color changes and betalains degradation in milk system.
For heating treatment, 30 mL of milk was filled into Pyrex tubes
of 24 m diameter, 200 mm length and 1 mm thickness. The tubes
were well capped to avoid evaporation and placed in a thermo-
static oil bath pre heated to given temperature. When the temper-
ature of milk reached the target temperature (within in
approximately 2–3 min), zero time (t = 0) sample was taken. The
milk samples were taken at regular time intervals (20 min inter-
vals for 140 min at 70 °C; 10 min intervals for 70 min at 80 °C,
and 5 min intervals for 35 min at 90 °C). After samples were
removed from oil bath, they were rapidly cooled in an ice water
bath. The heat treatment was conducted in duplicate (Fig. 1).
222 O. Günesßer / Food Chemistry 196 (2016) 220–227

2.5. Determination of betalains in milk by high performance liquid Table 1

chromatography (HPLC) Composition of cow milk.

Properties Cow milk (Mean ± S.D.)

The extraction of betalains was achieved using trichloroacetic pH 6.69 ± 0.07
acid (4% TCA in water) solution. For the sample preparation, Acidity (lactic acid %) 0.14 ± 0.01
4.0 mL of milk was poured into a 15 mL glass tube; 4 mL of TCA Dry matter (%) 11.13 ± 0.02
solution were added, and the mixture was vortexed for 3 min Fat (%) 3.55 ± 0.07
Total protein (%) 3.31 ± 0.05
and centrifuged (Hettich-EBA 20, Germany) for 10 min at Ash (%) 0.65 ± 0.03
6000 rpm at 25 °C. The clear liquid phase was filtered through a
0.45-lm PTFE filter, and 15 lL of filtrate was directly injected into S.D.: standard deviation.

the Agilent 1260 HPLC system (Agilent Technologies Inc., Folsom,

CA). The recovery of betalains for this extraction was 95.94%. All
had 11.36% of dry matter, 2.82% of protein, 3.42% of fat, 0.65% of
samples were prepared in a dark room to eliminate the effects of
ash and 4.47% of lactose.
light. Degradation of betalains in milk during heating was followed
by determining the content of betalains (betanin + isobetanin) by
3.2. Degradation kinetics of betalains in milk and the changes of color
HPLC method of Naderi, Ghazali, Meor Hussin, Amid, and Manap
values during heating
(2010). Chromatographic separation of betalains was performed
by Agilent Zorbax SB-C18 column (4.6
250 mm, particle size of
Betalain contents of milk samples were plotted in a semi loga-
5 lm) with isocratic flow. The mobile phase contained 0.5% triflu-
rithmic graph as a function of heating time at various tempera-
oroacetic acid (5 mL of TFA in 1 L of water) and acetonitrile (90:10).
tures. The linear relationship indicated that thermal degradation
The flow rate was 1 mL/min. Ultraviolet detection was at 540 nm,
of betalains (betanin and isobetanin) in milk followed first order
and the column temperature was 20 °C. Betalains were identified
reaction kinetics at 70–90 °C (Fig. 2) with higher determination
by comparing the retention time of betalain standard (Sigma–
coefficients (R2 > 0.95). The findings of the present study are in
Aldrich) and quantified using external standard methods. The
good agreement with previous studies reporting the first order
repeatability of the method was 0.66 (RSD %). The limit of detec-
reaction model for the thermal degradation of betalains (Cai, Sun,
tion (LOD) and limit of quantification (LOQ) were 5.23 mg L1
& Corke, 1998; Herbach et al., 2006a,b). Herbach et al. (2006b)
and 17.44 mg L1, respectively.
determined that degradation of betanin in its water solution
adjusted to pH 4 followed first order kinetics at 85 °C. Hence, Cai
2.6. Color measurement et al. (1998) indicated that the degradation of betacyanins pig-
ments from Amaranthus betacyanins fitted first-order at 40, 60,
Color measurement was also conducted to evaluate the kinetics 80 and 100 °C. In contrast to the findings of study by Chandran
of color changes in milk added beetroot betalains. Minolta Cr-400 et al. (2014); Moreno, Betancourt, Pitre, Garcia, Bethlehem, and
(Minolta Co. Ltd., Tokyo, Japan) was used to measure the color Medina (2007) indicated that degradation of tuna (Opuntia elatior)
parameters of milk samples. CIELab color space was used for the pulp betalains and beet root (B. vulgaris) betalains in citrus bever-
color parameters. Colorimeter was calibrated by a white standard ages at 7 ± 0.1 °C during 21 days followed zero order kinetics.
plate. L⁄, a⁄ and b⁄ values were measured from the milk samples Rate constant (k) and half-life (t1/2) for degradation of betalains
with light projection holder. To better describe the color, Hue angle and changes of color values were calculated by using following
and Chroma value were calculated from a⁄ and b⁄ values and used equations:
for kinetic calculations. Illuminant condition was standard illumi- Zero order reaction;
nant C and standard observer angle of 2° was used. Measurements C ¼ Co  k
t ð1Þ
were taken two times for each sample by ISO-CIE Standard (2008).
Co
t1=2 ¼ ð2Þ
2.7. Calculation of kinetic parameters and statistical analysis 2ko
First order reaction;
Evaluation of degradation of betalains and changes of color val-
ues were considered in terms of a chemical kinetics approach by
calculating kinetics parameters of reaction order, reaction rate con-
stants (k) and half-life (t1/2). Temperature dependence of degrada-
tion of betalains was evaluated by calculating activation (Ea)
energy and temperature quotient (Q10) values using Arrhenius
equation (van Boekel, 2008). Relations of color values and betalains
content of milk were also determined by multiple regression and
Pearson’s correlation analysis. SPPS for Windows (version 15.0)
was used for all statistical analyses.

3. Results and discussion

3.1. Compositional analysis

Composition of the milk sample used in the present study was

presented in Table 1. The chemical composition of the cow milk is
in normal range for cow milk composition and the results are in
agreement with previous findings by Ceballos et al. (2009).
Ceballos et al. (2009) reported that Holstein Friesian cow’s milk Fig. 2. Degradation of betalains in milk at 70, 80 and 90 °C.
 O. Günesßer / Food Chemistry 196 (2016) 220–227 223

C perature and time, types of matrix in which pigment found and

ln ¼ k
t
Co
ln 2
t 1=2 ¼
k
Co and C are values of determined properties (betalains content,
color values) at the beginning of the reaction and after t minutes
of heating at given temperature, respectively.
Calculated first order rate constant (k) and half-life (t1/2) for
degradation of betalains at 70, 80 and 90 °C were shown in Table 2.
As expected, degradation rate of betalains increased with
increasing temperature and time. k values for the degradation of
betalains in milk system ranged between 13.588 and
30.975
103 min1, while t1/2 values were between 22 and
51 min at 70–90 °C. Fernandez-Lopez, Angosto, Gimenez, and
Leon (2013) reported that k values for the degradation of beta-
cyanins in aqueous solution of reed beet extract (pH 5.5) were,
3.16 and 5.33
103 min1 at 70 and 90 °C, respectively. First
order rate constants (k values) for the degradation of betanin in
aqueous solution in atmospheric pressure were also found as
0.95, 2.1 and 7.83
103 min1 at 45, 55 and 65 °C, respectively.
Researchers also indicated that a high pressure with CO2 treatment
above 30 MPa with heat treatment led to increase k values of
degradation of both betalains and they become less stable (Liu,
Gao, Xu, Wang, & Yang, 2008). In other study by Serris and
Biliaderis (2001), the degradation kinetics of encapsulated beetroot
pigment in three different natural polymeric materials (pullulan
and two maltodextrin types) under various water activity (0.23–
0.84) and storage temperature (30–50 °C). The highest k values
for betalain degradation were observed at 0.64 aw for all type poly-
meric materials during storage at 30, 40 and 50 °C. It was deter-
mined that k values varied between 179.28–367.47
103 day1.
Compared to the previous studies, we found higher k values for
the degradation of betalains in milk system than in fruit puree,
juices (Coskuner, Turker, Ekiz, Aksay, & Karababa, 2000; Reynoso,
Garcia, Morales, & Gonzalez de Meija, 1997; Tsai et al., 2010) or
pigments in aqueous solution (Fernandez-Lopez et al., 2013). This
could be attributed to many factors such as heating or storage tem-
ð3Þ
reaction conditions. It was thought that range of pH of fruit juice
and purees (6–6 pH) and some antioxidant compounds especially
ascorbic acids and isoascorbic acids, and matrix structure of fruit
ð4Þ juice and purees (e.g. pectin) led to a higher stability of betalains
during heating than in milk system (Herbach et al., 2006a). As
mentioned before, degradation or retention of natural pigments
in food system depends on the physicochemical properties of food
matrices. Many researchers pointed out that stability of betalains
in food and model systems depends on pH, temperature, light,
water activity, metal ions and oxygen in particular. This factors
have been discussed extensively by Azeredo (2009) and Delgado-
Vergas et al. (2000). It was emphasized that the degradation of
betalains occurred in many types of reaction. These reactions
include isomerization, deglycosylation, hydrolysis, decarboxyla-
tion, and dehydrogenation reactions. Each reaction causes different
chromatic and structural changes in betalains (Herbach et al.,
2006a). For examples, heating at 85 °C during 8 h of thermal treat-
ment of beetroot juice resulted isobetanin and neobetanin forma-
tion from betanin (isomerization reaction) (Herbach, Stintzing, &
Carle, 2004). Moreover, it was proposed that prolonged thermal
treatment of betalains causes especially decarboxylation, dehydro-
genation and hydrolysis. Hydrolysis of the betanin gives bright yel-
low betalamic acid and the colorless cyclo-dopa5-O-b-glucoside,
while decarboxylation of betanin can give 17-decarboxybetanin
associated orange-red color (Azeredo, 2009; Herbach, Stintzing, &
Carle, 2005, 2006a).
L⁄, Hue angle and Chroma values were also plotted as a function
of heating time at 70, 80 and 90 °C (Fig. 3). It was determined that a
significant increase in L⁄ and Hue angle values in milk was
observed while Chroma values decreased during heating Hence,
the linear relationship indicates changes of L⁄ and Chroma values
followed zero-order reaction while the changes of Hue angle fol-
lowed first order reaction. Calculated rate constants were shown
Table 2.
k values for the changes of L⁄, Hue angle and Chroma values
during heating ranged between 90.50–379.75
103 L⁄ min1,
0.58–5.00 Hue angle min1 and 3.25–19.750
103 min1, respec-
tively. Half lives (t1/2) of Chroma value in milk were calculated

Table 2
The kinetics parameters for betalains degradation and changes of color values in milk during heating.

Properties Temperature (°C) First-order t1/2 (min) Activation energy (Ea) Q10
k
103 (min1) ± S.D. (kJ mol1) ± S.D. 70–80 °C 80–90 °C
Betalains 70 13.588 ± 0.001 (0.975) 51.06 42.449 ± 3.142 (0.976) 1.364 1.67
80 18.539 ± 0.001 (0.979) 37.39
90 30.975 ± 0.003 (0.953) 22.5
Temperature (°C) Zero-order t1/2 (min) Activation energy (Ea) Q10
k
103 (L min1) ± S.D. (kJ mol1) ± S.D. 70–80 °C 80–90 °C
L⁄ value 70 90.500 ± 0.002 (0.989) – 74.150 ± 1.683 (0.984) 1.788 2.346
80 161.850 ± 0.004 (0.967) –
90 379.750 ± 0.001 (0.860) –
Temperature (°C) Zero-order t1/2 (min) Activation energy (Ea) Q10
k (Chroma min1) ± S.D. (kJ mol1) ± S.D. 70–80 °C 80–90 °C
Hue angle 70 0.581 ± 0.033 (0.908)a – 111.174 ± 3.174 (0.955) 2.024 4.258
80 1.176 ± 0.002 (0.941) –
90 5.008 ± 0.020 (0.986) –
Temperature (°C) First order t1/2 (min) Activation Energy (Ea) Q10
k
103 (min1) ± S.D. (kJ mol1) ± S.D. 70–80 °C 80–90 °C
Chroma value 70 3.250 ± 0.212 (0.953) 213.23 93.311 ± 4.438 (0.980) 2.049 2.965
80 6.660 ± 0.707 (0.884) 104.054
90 19.750 ± 0.354 (0.860) 35.088

S.D.: standard deviation.

a
Numbers in parentheses are determination coefficient (R2), –: not calculated.
224 O. Günesßer / Food Chemistry 196 (2016) 220–227

Fig. 3. The changes of L⁄ (A), Hue angle (B) and Chroma (C) values in milk during heating.

between 35.08 min and 213.23 min. During heating, it was ers reported that color of beetroot puree changed from deep violet
observed that the color of milk changed from red purple to yellow- red color to yellowish brown during heating at 50–120 °C. More-
ish and pinkish orange depending on heating temperature (Fig. 1). over, it was found that the redness color degradation (Hunter
Similar results were observed by Chandran et al. (2014), research- a/b) for beetroot puree followed first-order reaction at 50–120 °C
 O. Günesßer / Food Chemistry 196 (2016) 220–227
and half life of color degradation varied between 365 and 21 min
depending on heating temperature. Similarly, Fernandez-Lopez
et al. (2013) indicated that the DE value degradation of red beet
extract in aqueous solution (pH 5.5) followed first order reaction
kinetics at 50–90 °C and t1/2 values ranged between 196.8 and
110.4 min. In a study by Narkprasom, Wang, Hsiao, and Tsai
(2012), rate of color loss (redness value) Djulis extract in various
alcohol model systems (0–60%) during storage at 20–50 °C was
observed to follow first-order reaction kinetics. Researcher also
indicated that higher ethanol concentration and temperature led
to the higher rate of color loss.
3.3. Temperature dependence of degradation of betalains and changes
of color values in milk
The temperature dependence of degradation of betalains and
changes of color values of milk were evaluated by calculating acti-
vation energies (Ea) and temperature quotient (Q10) values from
the following equations:
k ¼ ko eEa =RT ðArrhenius equationÞ
k2
Q 10 ¼
k1
Ea: activation energy (kJ mol1, R: ideal gas constant
(8.314 J mol1 K1), T: absolute temperature (K), ko: total number
of collisions (1 s1), k: reaction rate.
The temperature sensitivity of the reaction was expressed by
activation energy value (Ea). A higher activation energy showed
that a small temperature elevation brings faster degradation or for-
mation for compounds (van Boekel, 2008). At 70–90 °C, the calcu-
lated Ea value for the degradation of betalains in milk was
42.449 kJ mol1 while, Ea values of 74.150, 111.174 and
93.311 kJ mol1 were found for the changes of L⁄, Hue angle and
Chroma values, respectively. Q10 values indicated that changes
color values of milk were more affected by temperature increasing
in higher heating temperature (Table 2). Moreover, it was observed
that temperature elevation (10 °C) induce approximately 1.5-fold
higher reaction rate for degradation of betalains in milk In case
of color values, it can be said that reaction rate for the decreasing
of Chroma value of milk was higher approximately two fold. Unlike
L⁄ and Chroma values, it was found that Hue angle was more
effected by temperature elevation from 80 °C to 90 °C and reaction
rate for decreasing of Hue angle increased about 4-fold (Table 2).
Several Ea values were reported for the degradation of beetroot
betalains in different heating and storage conditions in previous
studies. Similarly Ea value for betalains observed in the present
study, Merin, Gagel, Popel, Berstein, and Rosenthal (1987) reported
that degradation of betacyanin pigment obtained from prickly-
pear fruit followed pseudo-first order reaction at 50–90 °C and Ea
Table 3
Relationship between betalains content and color values of milk during heating.
Temperature (°C) Multiple regression model
⁄
70 Betalains (ppm) = 21,000–279
L value + 21.3
Hue angle + 10.9
Chroma
80 Betalains (ppm) = 11,337–150
L⁄ value + 9.04
Hue angle + 60.8
Chroma
90 Betalains (ppm) = 8102–84.4
L⁄ value  0.56
Hue angle  36.2
Chroma
Correlations coefficients
L⁄ value
Hue angle
Chroma
a
Numbers in parentheses are P values for correlations analysis.
225
values for diluted and concentrated pigment were calculated by
as 32.26 kJ mol1 and 44.85 kJ mol1, respectively by researchers.
Serris and Biliaderis (2001) found that average Ea values for the
degradation of beetroot betalains encapsulated with maltodextrin
(GlucoDry 200) was 41.8 kJ mol1 between 0.23 and 0.84 aw at
30–50 °C of storage temperature. Fernandez-Lopez et al. (2013)
reported a slightly lower Ea value (35.37 kJ mol1) for beetroot
betalains in aqueous solutions (pH 5.5). In early study by
Liaotrakoon et al. (2013), a lower Ea was calculated as
38.50 kJ mol1 for red dragon fruit puree at 50–90 °C. However,
our Ea value for betalains is lower than those reported for
yellow-orange pigments from Opuntia ficus-indica (65.1 kJ mol1)
at 50–90 °C at pH 5 (Coskuner et al., 2000) and betanin pigment
in aqueous solution (144.8 kJ mol1) at pH 5 at 60–86 °C (Drdak
& Vallova, 1990). Reynoso et al. (1997) also reported a higher Ea
value (87.09 J mol1) for pigments (betaxanthins, betanin and
isobetanin) of Garambullo (Myrtillocactus geometrizans) fruits for
0–100 °C at pH 5.5 than our calculated Ea value for betalains from
beetroot. Vaillant, Perez, Davila, Dornier, and Reynes (2005) calcu-
lated a higher Ea values for the degradation of betacyanin in red–
ð5Þ purple pitahaya juices at natural (pH 5) and acidified pHs (pH 4
and 3.5) as 71.06 kJ mol1, 81.02 kJ mol1 and 112.02 kJ mol1,
respectively. Furthermore, when our findings Ea for color values
ð6Þ
were examined, higher Ea value (70.16–81.04 kJ mol1) was found
by Narkprasom et al. (2012) for red color loss (a/ao) in Djulis
extracts at 20–50 °C and in 0–60% of ethanol concentrations.
Chandran et al. (2014) calculated a lower Ea as 37.54 kJ/mol for
red color degradation in beetroot puree at 50–120 °C.
3.4. Relationships between color values and betalains content of milk
Revelation of relations between betalains content and color val-
ues is essential to estimate retention betalains in colored milk pro-
cess by using simply and fast color measurement rather than HPLC
methods. Therefore, multiple regression and Pearson’s correlation
coefficients were calculated in the present study. Table 3 shows
the results of multiple regression analysis and correlations coeffi-
cients between betalains content and color values at different heat-
ing temperatures. It was found that multiple regression model was
significant for all heating temperatures (P < 0.05) and determina-
tion coefficient (R2) was higher than 90% for 70 °C and 80 °C. How-
ever, determination coefficient (R2 = 72.7) for 90 °C was lower than
other heating temperatures. These results showed that a good esti-
mation for betalains content in milk during heating process can be
done with these models by determining color values. Revealing the
relationship of betalains content and color values of milk added
beetroot extract were provided for first time with this study. It
was thought that the results of the study may provide valuable
contributions for the determination of betalains content of milk
during heating processes.
R2 P value
96.9 0.001
94.01 0.002
72.7 0.043
Betalains L⁄ value Hue angle
0.846
(0.001)a
0.209 0.06
(0.326) (0.779)
0.857 0.75 0.505
(0.001) (0.001) (0.012)
226 O. Günesßer / Food Chemistry 196 (2016) 220–227
Significant positive and negative good linear correlations were
also determined between betalains content and L⁄ and Chroma val-
ues (P < 0.05). A negative correlation was observed between beta-
lains content and L⁄ values while Chroma value has a positive
correlation with betalains content. No significant correlation was
also observed between Hue angle and betalains contents in colored
milk (P = 0326). According to correlation results, it can be said that
an increase in the Chroma values of colored milk indicates a higher
betalains content and a lower betalains content in colored milk also
indicates a higher L⁄ and Hue angle values during heating (Table 3).
There are many studies on relationships between color values
and natural pigments and similar observations with our findings
were reported in previous studies (Ahmed et al., 2004;
Narkprasom et al., 2012). Narkprasom et al. (2012) recommended
that L⁄ and b⁄ values of the dragon (Hylocereus spp.) fruit purees
can be used as online quality control indicators and total color
changes can be used to predict the betacyanins content in dragon
fruit purees. In a study by Ahmed et al. (2004), the linear relation-
ship between the hunter color values and the anthocyanin content
in plum was revealed during heating. Researchers expressed that
decreasing anthocyanin content of plum puree during heating lead
to decrease in hunter L, a⁄ and b⁄ values of plum puree. Meleandez-
Martianez, Isabelm, and Heredia (2003) explored the correlations
between the CIE color parameters (L⁄, a⁄, b⁄ L⁄, Chroma⁄ and Hue)
and the carotenoids content in Valencia late orange juices. They
found that a significant were found between b⁄, L⁄, Chroma⁄ and
Hue and the content of b-cryptoxanthin, lutein + zeaxanthin and
b-carotene in orange juice. They also established multiple regres-
sion model for the calculation of some carotenoid levels including
such as neoxanthin, violaxanthin, antheraxanthin as a function of
color parameters. Moreover, Arias, Lee, Logendra, and Janes
(2000) established exponential regression for the determination
of lycopene content in Laura tomatoes by using a⁄/b⁄ color value.
4. Conclusion
The degradation of betalains in milk increased with increasing
temperature and time. Degradation of betalains followed first
order kinetics while, changes of L⁄, Hue angle and Chroma values
fitted zero order kinetics. Kinetic parameters (k and t1/2) showed
that betalains from beetroot had moderate stability in milk at pro-
cessing temperatures. Hence, it was found that L⁄ values was more
stable than Hue angle and Chroma color values in colored milk
during heating. A higher correlation was observed between beta-
lains content and L⁄ and Chroma values in colored milk during
heating. The multiple regression analysis showed that a good pre-
diction for the determination of betalains content might be done
by using color values during heating processing of milk colored
with beetroot extract. Further studies are needed on degradation
kinetics of natural color pigment in several real food systems to
conceive their relationship between color changes and shelf life
of food products during processing and/or storage.
Conflict of interest
The author certifies that there is no conflict of interest with any
organization regarding the material discussed in the manuscript.
Acknowledgments
This study was funded by The Scientific Research Fund of Usak
University (Usßak, Turkey, Project No: 2014/MF020). The author
thanks the NANTE Chemical Company for providing beetroot
extract. The author also would like to thank Dr. Yonca Karagul
Yuceer and Dr. Aysegul Kirca Toklucu for editing of the manuscript.
References
Ahmed, J., Shivhare, U. S., & Raghavan, G. S. V. (2004). Thermal degradation kinetics
of anthocyanin and visual colour of plum puree. European Food Research and
Technology, 218, 525–528.
Altamirano, R. C., Drdak, M., Simon, P., Rajniakova, A., Karovicova, J., & Preclik, L.
(1993). Thermal degradation of betanine in various water alcohol model
systems. Food Chemistry, 46, 73–75.
Attia-Gamilia, Y., Moussa, M. E. M., & Sheashea, E. R. (2013). Characterization of red
pigments extracted from red beet (Beta vulgaris) and its potential uses as
antioxidant and natural food colorants. Egyptian Journal of Agricultural Research,
91, 1095–1110.
Arias, R., Lee, T. C., Logendra, L., & Janes, H. (2000). Correlation of lycopene measured
by HPLC with the L⁄, a⁄, b⁄ color readings of a hydroponic tomato and the
relationship of maturity with color and lycopene content. Journal of Agricultural
and Food Chemistry, 48, 1697–1702.
Azeredo, M. H. C. (2009). Betalains: Properties, sources, applications, and stability –
A review. International Journal of Food Science & Technology, 44, 2365–2376.
Bradley, J. R. L., Arnold, J. E., Barbano, D. M., Semerad, R. G., Smith, D. E., & Vines, B. K.
(1992). Chemical and physical methods. In R. T. Marshall (Ed.), Standard
methods for the examination of dairy products (pp. 433–531). Washington, DC:
American Public Health Association.
Cai, Y., Sun, M., & Corke, H. (1998). Colorant properties and stability of Amaranthus
betacyanin pigments. Journal of Agricultural and Food Chemistry, 46, 4491–4495.
Cai, Y., Sun, M., & Corke, H. (2005). HPLC characterization of betalains from plants in
the Amaranthaceae. Journal of Chromatographic Science, 43, 454–460.
Castellar, M. R., Obon, J. M., & Fernandez-Lopez, J. A. (2006). The isolation and
properties of a concentrated red-purple betacyanin food colourant from Opuntia
stricta fruits. Journal of the Science of Food and Agriculture, 86, 122–128.
Ceballos, S., Morales, E. R., Torre Adarve, G., Castro, J. D., Martinez, L. P., & Sanz
Sampelayo, M. R. (2009). Composition of goat and cow milk produced under
similar conditions and analyzed by identical methodology. Journal of Food
Composition and Analysis, 4, 322–329.
Chandran, J., Nisha, P., Singhal, R. S., & Pandit, A. B. (2014). Degradation of colour in
beetroot (Beta vulgaris L.): A kinetics study. Journal of Food Science and
Technology, 51, 2678–2684.
Coskuner, Y., Turker, N., Ekiz, H. I., Aksay, S., & Karababa, E. (2000). Effect of pH and
temperature on the stability of prickly pear (Opuntia ficus-indica) yellow-orange
pigments. Nahrung, 44, 261–263.
Delgado-Vergas, F., Jimenez, A. R., & Paredes-Lopez, O. (2000). Natural pigments:
Carotenoids, anthocyanins, and betalains – Characteristics, biosynthesis,
processing, and stability. Critical Reviews in Food Science and Nutrition, 40,
173–289.
Downham, A., & Collins, P. (2000). Colouring our foods in the last and next
millennium. International Journal of Food Science & Technology, 35, 5–22.
Fernandez-Lopez, J. A., Angosto, J. M., Gimenez, P. J., & Leon, G. (2013). Thermal
stability of selected natural red extracts used as food colorants. Plant Foods for
Human Nutrition, 68, 11–17.
Fernandez-Lopez, J. A., Gimenez, P. J., Angosto, J. M., & Moreno, J. I. (2012). A Process
of recovery of a natural yellow colourant from Opuntia fruits. Food Technology
and Biotechnology, 50, 246–251.
Francis, F. J. (1992). A new group food colorants. Trends in Food Science & Technology,
3, 27–30.
Drdak, M., & Vallova, M. (1990). Kinetics of the thermal degradation of betanine.
Nahrung, 34, 307–310.
Henry, B. S. (1992). Natural food colours. In G. A. F. Hendry & J. D. Houghton (Eds.),
Natural food colorants (pp. 39–78). New York: Blackie.
Herbach, K. M., Stintzing, F. C., & Carle, R. (2004). Impact of thermal treatment on
color and pigment pattern of red beet (Beta vulgaris L.) preparations. Journal of
Food Science, 69, C491–C498.
Herbach, K. M., Stintzing, F. C., & Carle, R. (2005). Identification of heat-induced
degradation products from purified betanin, phyllocactin and hylocerenin by
high-performance liquid chromatography/electrospray ionization mass
spectrometry. Rapid Communications in Mass Spectrometry, 19, 2603–2616.
Herbach, K. M., Rohe, M., Stintzing, F. C., & Carle, R. (2006). Structural and chromatic
stability of purple pitaya (Hylocereus polyrhizus [Weber] Britton & Rose)
betacyanins as affected by the juice matrix and selected additives. Food
Research International, 39, 667–677.
Herbach, K. M., Stintzing, F. C., & Carle, R. (2006a). Betalain stability and degradation
– Structural and chromatic aspects. Journal of Food Science, 71, R41–R50.
Herbach, K. M., Stintzing, F. C., & Carle, R. (2006b). Stability and color changes of
thermally treated betanin, phyllocactin, and hylocerenin solutions. Journal of
Agricultural and Food Chemistry, 54, 390–398.
ISO-CIE Standard (2008). Colorimetry, part 4: CIE 1976 L⁄a⁄b⁄ colour space, 11664–4.
Kırca, A., Özkan, M., & Cemeroğlu, B. (2007). Effect of temperature, solid content and
pH on the stability of black carrot anthocyanins. Food Chemistry, 101, 212–218.
Kırca, A., Özkan, M., & Cemeroğlu, B. (2006). Stability of black carrot anthocyanins in
various fruit juices and nectars. Food Chemistry, 97, 598–605.
Kujula, T., Loponen, J., & Pihlaja, K. (2001). Betalains and phenolics in red beetroot
(Beta vulgaris) peel extracts: Extractions and characterisation. Zeitschrift für
Naturforschung C, 56c, 343–348.
Kumar, J. K., & Sinha, A. K. (2004). Resurgence of natural colourants: A holistic view.
Formerly Natural Product Letters, 18, 59–84.
Liaotrakoon, W., De Clercq, N., Van Hoed, V., Van de Walle, D., Lewille, B., &
Dewettinck, K. (2013). Impact of thermal treatment on physicochemical,
 O. Günesßer / Food Chemistry 196 (2016) 220–227
antioxidative and rheological properties of white-flesh and red-flesh dragon
fruit (Hylocereus spp.) purees. Food Bioprocess and Technology, 6, 416–430.
Liu, X., Gao, Y., Xu, H., Wang, Q., & yang, B. (2008). Impat of high-pressure carbon
dioxide combined with thermal treatment on degredation of red beet (Beta
vulgaris L) pigments. Journal of Agricultural and Food Chemistry, 56, 6480–6487.
Meleandez-Martianez, A. J., Isabelm, V. M., & Heredia, F. J. (2003). Application of
tristimulus colorimetry to estimate the carotenoids content in ultrafrozen
orange Juices. Journal of Agricultural and Food Chemistry, 51, 7266–7270.
Merin, U., Gagel, S., Popel, G., Berstein, S., & Rosenthal, I. (1987). Thermal
degradation kinetics of prickly pear fruit red pigment. Journal of Food Science,
52, 485–486.
Moreno, M., Betancourt, M., Pitre, A., Garcia, D., Bethlehem, D., & Medina (2007).
Stability of Citric beverages pigmented with natural sources of betalains:
tunapulp and beetroot. Biagro(online), 19, 149–159.
Naderi, N., Ghazali, H. M., Meor-Husssin, A. S. M., Amid, M., & Manap, Y. M. (2010).
Characterization and quantification of dragon fruit (Hylocereus polyrhizus)
betacyanin Pigments extracted by two procedures. Pertanika Journal of Tropical
Agricultural Science, 35, 33–40.
Narkprasom, K., Wang, S. P., Hsiao, S. M., & Tsai, P. J. (2012). Kinetics of color loss of
Djulis (Chenopodium formosanum Koidz.) extracts during storage in different
concentrations of alcohol and temperature. APCBEE Procedia, 2, 32–36.
227
Reynoso, R., Garcia, F. A., Morales & Gonzalez de Mejia, E. (1997). Stability of
betalains pigments from a Cactaceae fruit. Journal of Agricultural and Food
Chemistry, 45, 2884–2889.
Saenz, C., Cancino, B., & Robert, P. (2012). Red betalains from Opuntia spp.: Natural
colorants with potential applications in foods. Israel Journal of Plant Sciences, 60,
291–299.
Serris, G. S., & Biliaderis, C. G. (2001). Degradation kinetics of beetroot pigment
encapsulated in polymeric matrices. Journal of the Science of Food and
Agriculture, 81, 691–700.
Tsai, P. J., Sheu, C. H., Wu, P. H., & Sun, Y. F. (2010). Thermal and pH stability of
betacyanin pigment of Djulis (Chenopodium formosanum) in Taiwan and their
relation to antioxidant activity. Journal of Agricultural and Food Chemistry, 58,
1020–1025.
van Boekel, M. A. J. S. (2008). Kinetic modeling of reactions in foods (1st ed.).
Newyork: CRC Press (Chapter 4).
Vaillant, F., Perez, A., Davila, I., Dornier, M., & Reynes, M. (2005). Colorant and
antioxidant properties of red-purple pitahaya (Hylocereus sp.). Fruits, 60, 3–12.
Wissgott, U., & Bortlik, K. (1996). Prospects for new natural colorants. Trends in Food
Science & Technology, 7, 298–302.