J Food Sci Technol (January 2016) 53(1):461–470

DOI 10.1007/s13197-015-2002-1

ORIGINAL ARTICLE

Thermal and pH degradation kinetics of anthocyanins in natural

food colorant prepared from black rice bran
Patiwit Loypimai 1 & Anuchita Moongngarm 1 & Pheeraya Chottanom 1

Revised: 28 June 2015 / Accepted: 18 August 2015 / Published online: 25 August 2015
# Association of Food Scientists & Technologists (India) 2015

Abstract The study of the stability of anthocyanins in food increasing temperature and pH value. The degradation of
colorant powder is important to predict the quality changes cyanidin-3-O-glucoside and total anthocyanins showed a strong
occurring as the food products are processed, to prevent and positive correlation with C*. The changes in visual color may be
control the degradation of the anthocyanins. The objectives of used as an on-line quality control indicator during thermal pro-
this study were to identify anthocyanin components in natural cessing of food products containing rice bran colorants which
food colorants obtained from black rice bran, and investigate have high anthocyanin content.
their thermal stability at 60, 80, and 100 °C, pH stability from
2.0 to 5.0 and also their correlation with visual color, L*, C*, and Keywords Black rice bran . Anthocyanins . Degradation .
h°. Results showed that only six types of anthocyanins, Stability . Food colorants
cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, delphinidin,
cyanidin, pelargonidin and malvidin were present in raw black
rice bran (BRB) and black rice bran colorant powder (BCP). Introduction
The thermal degradation of both the visual color and the antho-
cyanin content in the BCP followed a first-order kinetic reaction The use of natural anthocyanin pigments as coloring agents in
model. The temperature-dependent degradation was adequately food products is receiving increasing attention as they are attrac-
fitted to the Arrhenius equation. In terms of the pH stability, tive to consumers and have positive health benefits (Chou et al.
increasing pH values resulted in lower activation energies (Ea) 2007). Anthocyanin pigments are permitted as natural food col-
and higher half-life (t1/2) values for both color parameters and orants in the USA under the category of fruits (21 CFR 73.250)
individual anthocyanins when heating from 60 to 100 °C. and vegetables (21 CFR 73.260), and the EU classification num-
Moreover, the degradation rate constant (k) increased with ber is E163 (Lipman 1996; Wrolstad 2000). They are water-
soluble glycosides and acylglycosides of the anthocyanidins of
Highlights many fruits, vegetables and cereal grains. They are found in the
• Natural food colorant powder was prepared from black rice bran.
• Thermal and pH stability of the colorant powder were investigated. form of polyhydroxylated and or methoxylated heterosides,
• Correlations between anthocyanin degradation and visual color were which are derived from the flavylium ion or 2-
investigated. phenylbenzopyrilium (Wu et al. 2004; Castañeda-Ovando
• Six types of anthocyanins were found in black rice bran and colorant et al. 2009). Currently, some 250 anthocyanins have been iden-
powder.
• The degradation of total anthocyanins showed a strong positive tified, however, only six of these, pelargonidin, cyanidin,
correlation with C*. peonidin, delphinidin, petunidin and malvidin, are commonly
found in fruits and cereal grains (Escribano-Bailon et al.
* Anuchita Moongngarm 2004). These compounds have been recognized as health-
[email protected] enhancing substances, owing to their antioxidant activity, anti-
inflammatory properties and hypoglycaemic effects (Nam et al.
1
Department of Food Technology and Nutrition, Faculty of
2006; Philpott et al. 2004). They also show other biological
Technology, Mahasarakham University, Maha Sarakham 44150, effects, including antimutagenic and anticarcinogenic activities
Thailand (Hyun and Chung 2004).
462 J Food Sci Technol (January 2016) 53(1):461–470

Black rice (Oryza sativa L.) is becoming increasingly pop- Et Province, Thailand. The raw bran from the milling process
ular and is widely consumed in China, Japan, Korea and other was immediately passed through a 20-mesh sieve to remove
East Asian countries such as Thailand (Pereira-Caro et al. broken pieces of rice and husks. The moisture content was
2013; Hou et al. 2013). Black rice bran (BRB), a waste prod- determined according to the method of AOAC (2000)
uct from the rice milling process, has gained recent attention (10.21 %).
for its potential use as a functional food because it contains Standards of cyanidin-3-O-glucoside chloride, dephinidin,
high levels of polyphenols, especially anthocyanins, which pelargonidin, cyanidin, malvidin, cyanidin-3-O-rutinoside
are mainly found in the pericarp and aleurone layers of the chloride, and maltodextrin (DE 4-7) were purchased from
bran fraction removed from the rice during the milling process Sigma-Aldrich Chemical Co., (St. Louis, USA). High-
(Jang and Xu 2009; Yawadio et al. 2007). Moreover, the bran performance liquid chromatography (HPLC) grade methanol,
is a rich source of other bioactive substances such as tocoph- acetonitrile, hexane, acetic acid, ethyl acetate and ethanol
erols, tocotrienols and γ-oryzanol (Loypimai et al. 2009; were purchased from BDH Chemicals (Poole, UK). All
Ryynänen et al. 2004), which are well known as beneficial chemicals and reagents were of analytical grade.
compounds for human health. However, the major current
problem for the use of anthocyanins as food colorants has Preparation of black rice bran colorant powder (BCP)
been limited by their relative instability under varying light,
oxygen, temperature, enzymes, thermal treatment, co-pigment The bran sample was added to deionised water to adjust the
and pH conditions as well as other factors (Hou et al. 2013; moisture content (MC) to 40 % (%wet basis) following
Zhang et al. 2013). Preventing and controlling the degradation optimal conditions and then placed in a chamber using
of anthocyanins in natural colorants is critical. In order to ohmic heating, as reported by our previous study
predict the quality changes occurring during food processing (Loypimai et al. 2015). Immediately after heating, the bran
such as thermal processing and altering pH value, this study sample was removed from the chamber, cooled to room
investigated the stability of the anthocyanins in food colorant temperature, placed in a polyethylene bag and kept at
powder prepared from black rice bran under several food pro- 4 °C. The treated bran was extracted following the method
cessing conditions. Thermal degradation has been previously reported by Duangmal et al. (2008) with some modifica-
reported for BRB anthocyanins (Hou et al. 2013), blackberry tions. The 20 g sample from the ohmic treatment was ex-
juice and concentrate (Wang and Xu 2007), purple-fleshed tracted with 100 ml of acidified hydroalcoholic solution
sweet potato anthocyanins (Jie et al. 2013), and freeze-dried (water: 95 %, ethanol: 1:1, acidified with 0.1 M HCl to
Roselle colorants (Duangmal et al. 2008). Visual color is an obtain a pH value of 2.5). The bran and solution were
important physical and sensory property for product quality mixed using a mixer (Velp scientific, Europe) for 1 min
evaluation. The correlation between visual color and anthocy- before shaking in an orbital shaker (Gerhardt LS500, UK)
anin content during thermal processing in Urmu mulberry at 100 rpm for 3 h. The slurry was filtered through a V-700
concentrate (Kara and Ercelebi 2013), blood orange juice vacuum pump (Buchi, Switzerland) using Whatman No. 4
(Shao-qian et al. 2011), and purple corn (Zea mays L.) filter paper. The extract was added to maltodextrin (2 g/
(Yang et al. 2008) has been reported. This study was carried 100 ml) and frozen at −50 °C before freeze-drying in a
out to investigate the stability of the anthocyanins by focusing freeze dryer (FTS system Dura-DryTm, USA) under a 27–
on the effect of temperature, time and pH on the degradation 33 Pa vacuum at a condenser temperature of −50 °C for
of individual the anthocyanins obtained from the BRB. The 20 h. The dried sample was weighed, ground into a pow-
relationship between changes in visual color (L*, C*, and h°) der, and passed through a 50 mesh sieve. The colorant
and anthocyanin degradation during the thermal treatment of powder was kept in a brown glass bottle (45 ml) and placed
BRB colorant was examined. The findings will be useful in in a desiccator for storage at 4 °C until required for
preparing appropriate food colorants and assist in establishing analysis.
thermal and pH processing guidelines for food products.
Visual color is an on-line quality parameter which can be used Qualitative and quantitative analysis using
to predict anthocyanin degradation during thermal processing. an HPLC-PDA (photodiode array detector)

The HPLC system consisted of a Shimadzu (Kyoto, Japan)

Materials and methods LC-20AC series pumping system, a SPD-M20A diode array
detector, and a SIL-10AD series auto-injector. The column
Materials and chemicals was an Apollo C18 (Alltech Associates, Deerfield, IL, USA)
(ø4.6 × 250 mm, 5 μm) protected with a Inertsil ODS-3 guard
A BRB (Oryza sativa L.; waxy type) sample was obtained column (ø 4.0 × 10 mm, 5 μm; GL Science Inc., Tokyo,
from a rice-milling factory (10 % degree of milling) in Roi- Japan). A 20 μl aliquot of each sample was used. The elution
J Food Sci Technol (January 2016) 53(1):461–470 463

conditions, described previously by Durst and Wrolstad anthocyanins, or half-life (t1/2 in h) was calculated using the
(2001), were used with modifications. The mobile phase following equations:
consisted of solvent A (acetonitrile, CH3CN) and solvent B
C t ¼ C 0 expðk:t Þ ð1Þ
(4 % phosphoric acid, H3PO4) with the following gradient: 94
to 75 % B from 0 to 65 min, 75 to 94 % B from 65 to 70 min, t 1 =2 ¼ ln0:5=k ð2Þ
and isocratic at 94 % B from 70 to 75 min to equilibrate the
column for the next injection. Spectral data were recorded Dependence of the degradation rate constant on tempera-
from 200 to 600 nm and the anthocyanin chromatograms were ture was determined by applying the Arrhenius equation:
monitored at 520 nm. The operating conditions were column E a

temperature 40 °C, injection volume 20 μl, detection at K T ¼ K 0 e RT ð3Þ

520 nm, and flow rate of 1.0 ml/min. Quantification was per-
Where C0 is the initial color parameter or anthocyanin
formed by comparing the retention times and the spectra as
content and Ct is the color value and anthocyanin content
well as by the addition of standards. The chromatograms were
after t minutes of heating at a desired temperature. Ea is the
recorded and the peak areas were used to calculate the con-
activation energy (kJ.mol−1), ln (kT) is the kinetic constant
centration of the anthocyanins against the calibration curve of
at specified temperature, k 0 is a pre-exponential factor
the external standards.
(min−1), R is the universal gas constant (8.314 J/mol.K),
and T is the absolute temperature (K). Activation energies
Stability study
were calculated by plotting ln (kT) against the reciprocal of
the absolute temperature (1/T), where the slope of the linear
The kinetics of colorless and individual anthocyanins in the
graph is equivalent to E
RT .
a
BCP were studied at 60, 80, 100 °C and at four different pH
values (2.0, 3.0, 4.0, and 5.0). The BCP solution was pre-
pared as previously described by Hou et al. (2013), with Statistic analysis
slight modifications. Three grams of BCP were dissolved
in 1000 ml of 2.0 M acetate buffer. The pH values were The stability of the color parameters and the anthocyanins of
adjusted to 2.0, 3.0, 4.0, and 5.0, respectively. Aliquots of the BCP were analyzed using a linear regression model to
10 ml of each colorant solution were transferred to 15 ml obtain the degradation rate constants (k) and activation energy
brown glass vials and covered with a plastic cap to avoid (Ea). Analysis of variance (ANOVA) was performed using an
evaporation of the thermally sensitive compounds. They SPSS program (trial version). Mean values were compared
were then placed in a thermoelectric water bath using Duncan’s multiple range tests, and significant difference
(PolyScience®, USA) and preheated to the desired temper- was defined at P < 0.05.
ature (60, 80, and 100 °C). For each temperature, 28 vials
(seven vials for each pH) were randomly taken at 20 min
intervals (0, 20, 40, 60, 80, 100, and 120 min) and cooled in Results and discussion
an ice bath to stop thermal degradation. An aliquot of each
sample was passed through a 0.45 μm nylon syringe filter Identification of anthocyanins
(Whatman, USA) and injected into the HPLC system for
analysis of the anthocyanin degradation. The anthocyanins are the major flavonoids in BRB. These
The visual color of the solution sample was measured in compounds are responsible for the dark colors of black or
terms of the CIELAB L*, a*, and b*values using a Hunter Lab purple, and are located mainly in the aleurone and pericarp
instrument (MiniScan, USA). L* represents the lightness layers of the rice bran. Anthocyanins and the visual color of
(L* = 0 yields black and L* = 100 indicates diffuse white). the rice bran and BCP are listed in Table 1. The results indicate
The Chroma (C*) represents the color intensity, which is the that only six types of anthocyanins, namely cyanidin-3-O-glu-
distance of a color from the origin (a* = b* = 0) in the a* and b* coside (Cy-3-glu), cyanidin-3-O-rutinoside (Cy-3-rut),
plane. Hue angle (h°) is expressed in degrees from 0 to 360°, delphinidin (De), cyanidin (Cy), pelargonidin (Pe), and
where 0° (red) is located on the +a* axis, and then rotates malvidin (Mv), were detected. The major anthocyanin in the
anticlockwise to 90° (yellow) for the +b* axis, 180° (green) BRB and BCP was Cy-3-glu (62.5 % in BRB and 57.7 % in
for –a*, and 270° (blue) for –b*. BCP), followed by De and Pe, respectively. In this study, the
Previous studies (Jie et al. 2013; Hou et al. 2013; Yang et al. BCP showed a sevenfold increase in Cy-3-glu and a fourfold
2008), indicated the first-order reaction model for the degrada- increase in the total content, compared with that of the BRB.
tion of most natural anthocyanin pigments from various Mv was only found in a small amount (51.56 μg/g in BRB and
sources. The first-order reaction rate constants (k in min−1), 101.8 μg/g in the BCP). Results also showed that the total
the time needed for 50 % degradation in the visual color and anthocyanins (12,540.8 μg/g) in the BCP had a slightly higher
464 J Food Sci Technol (January 2016) 53(1):461–470

Table 1 Anthocyanin content and visual color of raw black rice bran a 0.00
(BRB) and black rice bran colorant powder (BCP)

Characteristics BRB BCP -0.20

Anthocyaninsa (μg/g)
-0.40

Anthocyanin content
Cyanidin-3-O-glucoside 1042.4 ± 10.5 7235.5 ± 18.3
Cyanidin-3-O-rutinoside 27.3 ± 3.19 93.25 ± 10.2

ln (C/C0)
-0.60
Delphinidin 451.3 ± 7.33 723.8 ± 19.7
Cyanidin 184.3 ± 8.35 638.4 ± 23.3
-0.80
Pelargonidin 334.9 ± 10.2 1654.2 ± 54.2 Temp. 60
Malvidin 51.6 ± 3.98 101.8 ± 11.2 Temp. 80
-1.00 Temp. 100
Total contents 2947.3 ± 22.5 12,540.8 ± 85.9
Predicted (Eq.1)
Color value
* -1.20
L 37.2 ± 0.84 39.4 ± 0.95 0 20 40 60 80 100 120 140
C* 15.2 ± 0.38 17.2 ± 0.48 Heating time (min)

h° 310.9 ± 6.61 348.8 ± 6.74

b 12.0
a
Values are means ±SD of triplicate samples (n = 3) (on a wet weight
basis) 10.0

content (9480 μg/g) than those reported by Nontasan et al. 8.0

(2012). According to the study of Jang and Xu (2009), Cy-
3-glu is the predominate anthocyanin in the black rice. In 6.0
-ln k

contrast, lower concentrations, and some different anthocya-

4.0
nin profiles, were found in Japanese black–purple rice
(Pereira-Caro et al. 2013), Chinese and Korean black rice 2.0
powder (Hou et al. 2013; Frank et al. 2012), and American
black rice (Zhang et al. 2013). The variation of anthocyanins 0.0
may be attributed to different cultivars of pigmented rice, var- 0.0026 0.0027 0.0028 0.0029 0.003 0.0031
1/T (1/K)
iable growing conditions, and the degree of rice milling.
The visual color measurements in terms of the L*, *C and Fig. 1 Degradation of cyanidin-3-O-glucoside in black bran colorant
during heating at 60, 80, and 100 °C, and at pH = 2.0 (a), and its
h° values of the BCP were 39.41, 17.19, and 348.8, respec- Arrhenius pots for degradation at pH = 2.0 (b)
tively. The BCP obtained was dark purple in color, and a
darker shade than that of the BRB (Table 1). This finding is
the total contents clearly followed the first-order reaction ki-
in agreement with the results documented by Mozetic et al.
netic model with a high regression coefficient
(2004), who reported that changes in C* values strongly cor-
(0.8647 < R2 < 0.9887). This result was in agreement with
related to changes in anthocyanin content, and can be consid-
previous studies that reported the first-order reaction model
ered a good indicator of anthocyanin concentration. This may
for the degradation of the anthocyanin content in BRB (Hou
be attributed to the higher number of anthocyanins in the
et al. 2013), purple-fleshed sweet potato anthocyanins (Jie
colorant form.
et al. 2013), blackberry juice and concentrate (Wang and Xu
2007) as well as freeze-dried Roselle colorants (Duangmal
Anthocyanin stability et al. 2008).
The kinetic rate constant (k) is an indicator that enables the
Thermal degradation of anthocyanins in the BCP was inves- prediction of the thermal degradation of anthocyanins. The
tigated at 60, 80, and 100 °C and under different acidity con- lower the k value, the better the anthocyanin stability. The k
ditions (pH 2.0, 3.0, 4.0, and 5.0). Relative changes in indi- values of individual anthocyanins and the total content signif-
vidual anthocyanin content, with reference to the uncondi- icantly increased (p < 0.05) as the temperature increased from
tioned sample (control) (Ct/C0) whilst heating, were plotted 60 to 100 °C. The highest k value was observed at 100 °C with
against a regular time interval of 20 min (Fig. 1a). The kinetic a pH value of 5.0 (p < 0.05). The increase in the stability of
parameters of degradation of the anthocyanins, including Cy- anthocyanin pigment with a decline in the half-life value (t1/2)
3-glu, Cy-3-rut, De, Pe, Cy, and Mv, and the total anthocyanin was also less significant (p < 0.05). The greatest t1/2 value of
content while heating are displayed in Table 2. It was observed the colorant was observed in Cy-3-glu (26.9 h), followed by
that the thermal degradation of individual anthocyanins and total anthocyanins (19.6 h) and Pe (15.9 h), respectively. This
Table 2 Influence of temperature and pH level on the kinetic rate constant (k) and half-life time (t1/2) values of individual anthocyanins and total content degradation in black rice bran colorant powder
(BCP)

pH Temperature (°C) Cy-3-glu Cy-3-rut De Cy Pe Total contents

k × 103 (min−1) t1/2 (h) k × 103 (min−1) t1/2 (h) k × 103 (min−1) t1/2 (h) k × 103 (min−1) t1/2 (h) k × 103 (min−1) t1/2 (h) k × 103 (min−1) t1/2 (h)

2.0 60 0.43a (0.9314)X 26.9a 0.77a (0.9356) 15.0a 0.94a (0.9578) 12.3a 0.85a (0.9639) 13.6a 0.73a (0.9729) 15.9a 0.59a (0.9284) 19.6a
80 0.52a (0.8752) 22.1b 0.99b (0.9308) 11.6bc 1.79b (0.9692) 6.45b 1.03a (0.9225) 11.2b 1.68b (0.9707) 6.88b 1.04b (0.8795) 11.1c
J Food Sci Technol (January 2016) 53(1):461–470

100 1.16c (0.9014) 9.96cd 2.42e (0.9524) 4.77de 4.36d (0.9355) 2.65d 3.78f (0.8954) 3.06f 3.96e (0.9568) 2.92d 3.81e (0.9552) 3.03f
3.0 60 0.98b (0.9653) 11.9c 0.83ab (0.9708) 13.9ab 1.73b (0.9015) 6.68b 1.84b (0.9005) 6.28c 2.87c (0.9028) 4.03c 0.71a (0.9629) 16.3b
80 1.19c (0.9306) 9.71cd 1.71d (0.9528) 6.76d 2.44c (0.9245) 4.73c 2.17c (0.9125) 5.32cd 3.46d (0.9285) 3.34cd 1.26c (0.9066) 9.17d
100 1.85e (0.9313) 6.24def 3.61g (0.8946) 3.20ef 6.32g (0.9039) 1.83def 5.93h (0.9228) 1.95g 7.18g (0.9271) 1.61e 4.12f (0.8863) 2.80g
4.0 60 1.31cd (0.9425) 8.82cd 1.21c (0.9528) 9.55c 2.46c (0.9244) 4.70c 2.67d (0.9139) 4.33de 3.46d (0.8647) 3.34cd 2.16d (0.9213) 5.35e
80 2.47f (0.8944) 4.68efg 2.82f (0.9254) 4.10def 4.75e (0.9412) 2.43de 3.12e (0.9293) 3.70ef 7.76h (0.9002) 1.49f 3.67e (0.9509) 3.15f
100 3.16g (0.9484) 3.66fg 4.84h (0.9246) 2.39ef 9.71i (0.9021) 1.19fg 7.26i (0.8994) 1.59gh 9.45i (0.9334) 1.22g 4.87g (0.8759) 2.37h
5.0 60 1.41d (0.9224) 8.19cde 2.44e (0.9301) 4.73de 5.98f (0.9215) 1.93def 4.37g (0.9025) 2.64fg 6.73f (0.9035) 1.72e 5.34h (0.9248) 2.16i
80 3.85h (0.8848) 3.00fg 5.18i (0.9208) 2.23ef 7.51h (0.9435) 1.54ef 5.87h (0.8941) 1.97g 10.4j (0.9110) 1.11g 8.59i (0.9075) 1.34j
100 4.73i (0.8966) 2.44g 7.33j (0.8842) 1.58f 18.8j (0.9184) 0.61g 15.2j (0.9212) 0.76h 16.1k (0.8868) 0.72h 12.3j (0.9887) 0.94k
% CV 6.85 7.67 8.97 4.52 9.11 7.31 4.68 4.59 6.71 8.10 8.23 3.68

Values are means of triplicate samples (n = 3)

Values with the same letters along the same columns are not significantly different (p < 0.05)
CV Coefficient of variation
Cy-3-glu cyanindin-3-O-glucoside
Cy-3-rut cyanindin-3-O-rutinoside
De delphinidin
Cy cyanidin
Pe pelargonidin
X
correlation coefficient (R2 )
465
466 J Food Sci Technol (January 2016) 53(1):461–470

observation showed that Cy-3-glu was more stable than the Zevallos (2004) reported that anthocyanins from the extracts
other derivative anthocyanins for the black rice bran colorant. of red sweet potato, purple corn, and commercial purple carrot
However, the t1/2 values of Cy-3-glu and Cy-3-rut were higher colorants were more stable at pH 3.0 than at pH 1.0, at a
than those reported by Hou et al. (2013). This may be attrib- temperature of 98 °C. In addition, the stability of the anthocy-
uted to the fact that the colorant powder prepared in this study anin pigment is affected by several factors other than the pH
contained a stabilizer (maltodextrin), which was used to en- value and heat treatment, such as storage temperature, chem-
capsulate the pigment, resulting in enhanced anthocyanin sta- ical structure, concentration, light, oxygen, solvents, the pres-
bility. The combination of the flavylium cation form of the ence of enzymes, flavonoids, proteins, and metallic ions (Rein
anthocyanins and dextrin retarded their transformation to oth- 2005).
er less-stable forms (Chandra et al. 1993). Moreover, the col-
orant powder with the maltodextrin addition may prevent a Degradation of visual color
change in state from powder to a sorption gel. In this study, pH
ranges could be another influencing factor on the anthocyanin The color change of BCP following the first-order reaction
degradation. Results are similar to those of Fleschhut et al. kinetics with the determined coefficient (R2) greater than
(2006), and Kennedy and Waterhouse (2000), showing that 0.8943 (Table 3) clearly showed that either pH range or heat
the anthocyanin pigment was found in different chemical treatment had a significant influence on the stability of the
forms, depending on the pH value of the solution. At a pH color values (L*, C*, and h0) (P < 0.05). During heating, a
value of 1.0, the flavylium cation (red color) is the predomi- change in C* value with high k and t1/2values was observed,
nant form and contributes to the purple and red colors. At pH whereas the L*and h0 values showed only small changes, as
values between 2.0 and 4.0, the quinoidal blue species are shown in Fig. 2. In addition, the k and t1/2 values increased
predominant. At pH values between 5.0 and 6.0, only two with an increase in temperature and pH value. The increasing
colorless species can be detected, carbinol pseudobase and temperature and time resulted in anthocyanin pigments of all
chalcone. Torskangerpoll and Andersen (2005) indicated that samples becoming darker in all pH values, corresponding to
the pigment degradation of anthocyanin depended highly on significant decreases (P < 0.05) in L* and C* values as indi-
the pH and anthocyanin structure. Hou et al. (2013) reported cators for browning. This conclusion was supported by
that the t1/2 values of black rice anthocyanins diluted with Lozano and Ibarz (1997) who reported that the change in L*
citrate–phosphate buffer at pH 1.0 were greater than at could be used to measure the browning of a heat-treated con-
pH 2.0–6.0. On the contrary, Cevallos-Casals and Cisneros- centrated fruit pulp.

Table 3 Influence of temperature and pH level on the kinetic rate constant (k) and half-life time (t1/2) values of visual color degradation in black rice
bran colorant (BCP)

pH Temperature (°C) L* C* ho

k × 103 (min−1) t1/2 (h) k × 103 (min−1) t1/2 (h) k × 103 (min−1) t1/2 (h)

2.0 60 1.53a (0.9669)X 7.55a 2.93a (0.9749) 3.94a 1.01a (0.9359) 11.4a
80 3.27b (0.9804) 3.53b 5.55b (0.9596) 2.08c 1.51bc (0.9729) 7.65bc
100 4.30c (0.9497) 2.69c 7.12c (0.9888) 1.62d 1.79c (0.9795) 6.45cd
3.0 60 3.01b (0.9435) 3.84b 3.57a (0.9133) 3.24b 1.39b (0.9679) 8.31b
80 5.54d (0.9189) 2.09d 9.82e (0.9864) 1.18de 2.37de (0.9766) 4.87ef
100 6.64e (0.9201) 1.74e 16.1h (0.9675) 0.72gh 3.74g (0.9779) 3.09gh
4.0 60 4.73c (0.9088) 2.44c 7.61c (0.9250) 1.52d 1.85c (0.9729) 6.24cde
80 7.73f (0.9817) 1.49f 11.3f (0.9125) 1.02efg 2.20d (0.9842) 5.25def
100 8.65g (0.9629) 1.34g 17.1i (0.9218) 0.68gh 2.38de (0.9566) 4.85ef
5.0 60 8.75gh (0.9025) 1.32g 8.34d (0.9296) 1.39de 2.60ef (0.9396) 4.44fg
80 9.15gh (0.9258) 1.26h 15.2g (0.9547) 0.76fgh 2.81f (0.9723) 4.11fg
100 9.28h (0.8943) 1.24h 24.8j (0.9084) 0.47h 4.43h (0.9128) 2.61h
% CV 9.56 10.5 7.56 7.65 8.52 5.10

Values are means of triplicate samples (n = 3)

Values with the same letters along the same columns are not significantly different (p < 0.05)
CV Coefficient of variation
X
correlation coefficient (R2 )
J Food Sci Technol (January 2016) 53(1):461–470 467

a 0.00 levels (range 2.0 to 5.0), and the effect on several parameters,
were determined. The activation energy (Ea) was calculated by
-0.20 plotting ln(kT) against the reciprocal of the absolute temperature
(1/T), where the slope of the linear graph is equivalent to E
RT to
a

-0.40 fit the Arrhenius equation, (Eq. 3, Fig. 1b). The Ea of black rice
Color value

bran colorant anthocyanins during heating was highest at

ln (L* /L*0)

-0.60 pH 2.0; the values were 31.60 (Cy-3-glu), 37.87 (Cy-3-rut),

39.45 (De), 37.94 (Cy), 43.60 (Pe), and 46.75 (total contents)
-0.80 kJ mol−1, respectively (Table 4.). The Ea of Pe and total con-
Temp. 60
Temp. 80
tents were highest, which indicated that they were the most
-1.00
Temp. 100 stable at low pH values. In addition, a high pH level was more
Predicted (Eq.1) sensitive to temperature changes, with high anthocyanin degra-
-1.20
0 20 40 60 80 100 120 140 dation. These results agreed with the findings of Hou et al.
Heating time (min) (2013). The anthocyanins in BRB, in a reaction with higher
activation energy, were less stable, even though there were only
b 3.50 small changes in temperature. In contrast, a higher stability of
anthocyanins was obtained in aqueous solutions with pH 3.0
3.00
and 4.0 for apple and pear juices (Jie et al. 2013).
2.50 Regarding color change analysis, the Eavalue for color, that
is, for changes in L*, C*, and h° at pH 2.0, was better than
2.00
those at other pH values (Fig. 2b and Table 5). The C* change
-ln k

1.50
had the highest value ofEa. At higher pH values (4.0–5.0), the
Ea values of color changes in L*, C*, and h° ranged from 15.75
1.00 to 1.53 kJmol−1, 23.09 to 20.87 kJmol−1, and 13.56 to 6.55
0.50
kJmol−1, respectively. From the Ea parameter studied, it can be
determined that color changes and anthocyanin degradation
0.00 are strongly correlated to the Ea value. They were more stable
0.00265 0.0027 0.00275 0.0028 0.00285 0.0029 0.00295 0.003 0.00305
1/T (1/K)
at lower temperatures with higher Ea values.
Fig. 2 Degradation of L* value in black rice bran colorant solution during
heating at 60, 80, and 100 °C, and at pH = 3.0 (a), and the Arrhenius plot Relationship between visual color and anthocyanin
for degradation at pH = 3.0 (b) content

The dependence of color and anthocyanin degradation of During heating of black rice bran colorant solution at different
BCP at temperatures of 60, 80, and 100 °C and various pH pH values, the correlation between changes in the visual color

Table 4 Influence of pH level on the activation energy (Ea(kJ.mol-1)) of anthocyanin degradation in black rice bran colorant (BCP)

pH Cy-3-glu Cy-3-rut De Cy Pe Total content

2.0 31.60a (0.8942)X 37.87a (0.9982) 39.45a (0.9844) 37.94a (0.9203) 43.60a (0.9983) 47.75a (0.9362)
3.0 25.36b (0.8712) 35.90b (0.9222) 35.38b (0.9968) 31.83b (0.8963) 26.21b (0.9099) 45.05b (0.9477)
4.0 22.90c (0.9539) 29.19c (0.8862) 33.12c (0.9142) 29.77c (0.9295) 23.38c (0.8745) 21.09c (0.9802)
5.0 16.40d (0.9407) 28.57c (0.9691) 29.20d (0.8731) 25.46d (0.9403) 22.49c (0.9987) 17.54d (0.9977)
% CV 5.91 9.68 5.12 7.50 8.65 4.41

Values are means of triplicate samples (n = 3)

Values with the same letters along the same columns are not significantly different (p < 0.05)
CV Coefficient of variation
Cy-3-glu cyanindin-3-O-glucoside
Cy-3-rut cyanindin-3-O -rutinoside
De delphinidin
Cy cyanidin
Pe pelargonidin
X
correlation coefficient (R2 )
468 J Food Sci Technol (January 2016) 53(1):461–470

Table 5 Influence pH level on the activation energy (Ea (kJ.mol−1)) of 0.90

color degradation in black rice bran colorant (BCP) Temp. 60
0.80
Temp. 80
pH L* C* h° 0.70 Temp. 100
Predicted
0.60
2.0 26.89a (0.9470)X 39.10a (0.9736) 25.56a (0.9918)

C* /C* 0
3.0 20.61b (0.9285) 28.16b (0.9931) 14.87b (0.9616) 0.50

4.0 15.75c (0.9041) 23.09c (0.9543) 13.56b (0.8833) 0.40

5.0 1.53d (0.9344) 20.87d (0.9979) 6.55c (0.9676)
0.30
% CV 6.58 5.52 8.57
0.20
Values are means of triplicate samples (n = 3) 0.10
Values with the same letters along the same columns are not significantly
different (p < 0.05) 0.00
0.00 0.20 0.40 0.60 0.80 1.00
CV Coefficient of variation C/C0
X
correlation coefficient (R2 )
0.60
and the anthocyanin degradation was described using a linear Temp. 60
Temp. 80
relationship, as shown in Fig. 3.The graph shows the relation- 0.50
Temp.100
ship between color values and Cy-3-glu concentration which Predicted
is the major anthocyanin in black rice bran colorant. 0.40

Degradation of each anthocyanin and total content was posi-

tively correlated with L*, C*, and h° values. Interestingly, the L* /L* 0 0.30

C* value showed high correlation with Cy-3-glu (R2 > 0.9342)

0.20
and total anthocyanins (R2 > 0.9211). This excellent linear
correlation inferred that the visual color parameters (L*, C*,
0.10
and h°) may also be used instead of anthocyanins for black
rice bran colorant. This might be due to the decrease in visual 0.00
color especially C*predominantly caused by the destruction of 0.00 0.20 0.40 0.60 0.80 1.00
anthocyanin pigments upon heating, resulting in transforma- C/C0
tion of BCP solution from dark to other colorless forms. A
similar result was recorded by Mozetic et al. (2004), who 0.30
Temp. 60
reported that changes in C* strongly related to changes in Temp. 80
0.25
anthocyanin content. Duangmal et al. (2008) found that the Temp. 100
degradation of a drink with Roselle anthocyanin powder Predicted
0.20
added was highly correlated to changes in L* and C*. A similar
hº /hº0

result of the changes in a*, b*, and L* values was also reported 0.15
by Kara and Ercelebi (2013) in Urmumulberry concentrate.
This study was carried out on a model solution. However, the 0.10

colorant powder with high anthocyanin content is possible to

0.05
use in acidic foods and beverages. This has been confirmed by
our study on the application of the colorant powder to prepare 0.00
functional yogurt high in anthocyanin. Moreover, visual color 0.00 0.20 0.40 0.60 0.80 1.00
in terms of C* relating to the pigment concentration may be C/C0
considered as an indicator for an on-line quality control to Fig. 3 Relationship between visual color (C*, L*, and h° values) and
predict the anthocyanin degradation during thermal process- anthocyanin content (Cy-3-glu) of black rice bran colorant solution
ing. Further studies are still needed to prove these suggestions. during thermal treatment at pH 2.0

in Cy-3-glu and fourfold increase in total anthocyanin content

compared to BRB.
Conclusions This study indicated that thermal degradation of color and
anthocyanins in BCP followed first-order reaction kinetics.
Only six types of anthocyanin, Cy-3-glu, Cy-3-rut, De, Cy, Pe, The degradation of visual color and individual anthocyanin
and Mv were present in the BRB and BCP. The major antho- content also depended on temperature and pH level. A higher
cyanin identified was Cy-3-glu. BCP had a sevenfold increase stability of color and anthocyanin pigments of BCP was
J Food Sci Technol (January 2016) 53(1):461–470 469

achieved at low temperature (60 °C) and a pH value of 2.0. Kara S, Ercelebi AE (2013) Thermal degradation kinetics of anthocyanins
and visual colour of Urmu mulberry (Morus nigra L.). J Food Eng
The degradation of anthocyanins showed a strong positive
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lishing appropriate thermal processing guidelines and molecular mass grape phenolics using ion-pair, normal-phase
predicting anthocyanin degradation using visual color as an high-performance liquid chromatography. J Chromatogr A 866(1):
25–34
on-line quality control indicator.
Lipman AL (1996) Current regulations for certification exempt color
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Acknowledgments The authors gratefully acknowledge the Faculty of symposium on natural colorants INF/COL II. Acapulco, Mexico:
Technology, Mahasarakham University and Bansomdejchaopraya The Hereld Oraganization, S.I.C. Publishing.
Rajabhat University, Thailand, for financial support. Loypimai P, Moonggarm A, Chottanom P (2009) Effects of ohmic
heating on lipase activity, bioactive compounds and antioxi-
dant activity of rice bran. Aust J Basic Appl Sci 3(4):3642–
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