There has been an increased interest in the development of food colorants from
natural sources as alternatives to synthetic dyes. Anthocyanins are the naturally
occurring, water-soluble compounds that impart many of the orange, red, magenta,
violet, purple and blue colors to a variety of fruits, vegetables and plants.
Plants rich in anthocyanins are blackberry, blackcurrant, chokeberry, cherry,
eggplant, blue grape, vaccinium and red cabbage. The interest in anthocyanins
derives not only from their coloring effect but also from their beneficial properties,
including antioxidising activity, improvement in the tightness of capillary
blood vessels and prevention of thrombocyte aggregation, all of which reduce
the risk of circulatory diseases (Walkowiak-Tomczak and
Czapski, 2007). Despite the great potential of application that anthocyanins
represent for food, pharmaceutical and cosmetic industries, their use has been
limited because of their relative instability. Color stability of anthocyanin
influenced by many factors include structure and concentration, pH, temperature,
light, presence of co-pigments, self association, metallic ions, enzymes, oxygen,
ascorbic acid, sugar and their degradation products, proteins and sulphur dioxide
(Ersus and Yurdagel, 2007).
The role of anthocyanins as food coloring agents becomes very important since
they form the reds and the blues of many fruits and vegetables and provide the
attractive color of many fruit juices, wines, jams and preserves (Fossen
et al., 1998). Their color is easily affected by a number of reactions
occurring in food products and the major problem associated with the storage
of anthocyanins is their instability caused by pH, storage temperature, chemical
structure, concentration, light, oxygen, solvents the presence of enzymes, flavonoids,
proteins and metallic ions. The main focus of this study is to determine the
color behavior of anthocyanin towards various pH levels. Generally, increasing
of pH during storage increased the degradation rates of anthocyanins.
Anthocyanins can be found in different chemical forms which depend on the pH
of the solution as shown in Fig. 1 (Castaneda-Ovando
et al., 2009). At pH 3 or lower, the flavylium cation is the predominant
species and contributes to purple, orange and red colors. As the pH increase,
kinetic and thermodynamic competition occurs between the hydration reaction
of the flavylium cation and the proton transfer reactions related to its acidic
While the first reaction gives colorless carbinol pseudo-bases (pH 5) which
can undergo ring opening to yellow retro-chalcones (pH 6), the latter reactions
give rise to more violet quinonoidal bases (pH 4). Further deprotonation of
the quinonoidal bases can take place at pH between 6 and 7 with the formation
of more bluish resonance-stabilised quinonoid anions. At pH values higher than
7, the anthocyanins are degraded depending on their substituent groups. It is
well known that anthocyanin properties, including color expression, are highly
influenced by anthocyanin structure and pH. According to Torskangerpoll
and Andersen (2005), the parameters employed for describing color variation
of anthocyanin solutions have mainly been shifts of the visible λmax
as a measure for hue variations and absorptivity changes for variations of color
Color is an important factor influencing consumers acceptability of food
products. This is due to the fact that consumers always associate food color
with other qualities such as freshness, ripeness and food safety. However, natural
food colorants are not stable in food products. They can be decolorized and
degraded due to many factors as mentioned above. There are many techniques in
order to improve the color stability of anthocyanins. Co-pigmentation, compartmenting,
encapsulation and association are considered to cause stabilization of the anthocyanins
(Heins et al., 2001).
Color is an important feature of agricultural and food products due to its
close association with the perceived quality of the product. It is a widely
used parameter in the assessment of their maturity, freshness, nutritional condition
and growth factors. At the present time, the color judgment of agricultural
and food products still depends mainly on human eyes and brain. These are superbly
sensitive organs but the judgment is subjective and inevitably affected by human
physical condition and environmental elements. Modern instruments are necessary
to provide consistent measurement and quantitative expression of color (Zhang
et al., 1998). Methods available for color measurement can be categorized
into three broad types; visual systems, tristimulus colorimetry and spectrophotometry.
Visual systems involve comparison with colored references under controlled illumination,
tristimulus instruments use three filters to provide the red, green and blue
components of a color and the spectrophotometric method measures color by determining
a reflection or transmission spectrum (Zhang et al.,
CIE L*a*b* (CIELAB) is the most complete color model used conventionally to describe all the colors visible to the human eye. It was developed for this specific purpose by the International Commission on Illumination (Commission Internationale d'Eclairage). The asterisk (*) after L, a and b are part of the full name, since they represent L*, a* and b*, derived from L, a and b. CIELAB is an Adams Chromatic Value Space. The three parameters in the model represent the lightness of the color (L* = 0 yields black and L* = 100 indicates white), its position between magenta and green (a*, negative values indicate green while positive values indicate magenta) and its position between yellow and blue (b*, negative values indicate blue and positive values indicate yellow).
MATERIALS AND METHODS
Experimental procedures: The materials used were powdered anthocyanins from red cabbage (Brassica oleracea) and juice obtained by extracting crude red cabbage employing solvent extraction technique. In this study, it can be divided into three parts which were extraction of anthocyanin from crude red cabbage, preparation of buffer solutions for various pH and color measurement for both powdered and juice anthocyanin for acidic, neutral and basic conditions.
Anthocyanin extraction: Red cabbage leaves were shredded and soaked in 20% ethanol/water for 24 h. Then it was boiled for 5-10 min until purple color appeared.
Buffer solution preparation: Buffer solutions for 14 different pH values were prepared. The accurate pH for each buffer solution was measured with a portable pH Meter. The spectral behavior of anthocyanins is dependent on pH and substances present in the solutions may influence the color.
Color analysis: The effect of pH on both anthocyanin juice and powdered was studied at pH 1 (acidic), pH 7 (neutral) and pH 13 (basic). Samples were kept in screw-capped bottles and shaken continuously in controlled temperature. The absorbance of the solution at 380 nm was monitored and degradation of cabbage red color was determined. A HACH DR/4000 U spectrophotometer (model HACH PROGRAM 1666) was used to measure the color using CIELAB color scale method. CIELAB parameters (L*, a* and b*) at same wavelength, λ (380nm) was recorded from the spectrophotometer. The color behavior was determined using CIELAB system L*, C*and H0. L* designates the color lightness, C* is the color intensity and H0 is the color tone. Color parameters were recorded every two days at a period for 10 days. Samples were kept in a refrigerator (4°C) between the measurements.
RESULTS AND DISCUSSION
Color of anthocyanin: By boiling the red cabbage leaves, a class of
pigment molecules called anthocyanins will dissolve into the liquid. Anthocyanin
molecules will change their color depending upon the pH of their environment
thus it may serve as a pH indicator. The anthocyanin turns red-pink in acids
(pH 1-6), reddish-purple in neutral solutions (pH 7) and green in alkaline or
basic solutions (pH 8-14) (Fossen et al., 1998).
A change of red cabbage color solutions was observed when it was exposed to
various pH conditions. The effect of pH on the sample can be determined by the
CIELAB parameter values through the color of the solution.
Color characteristics of the samples: Quantitative color measurements
have been carried out by spectrophotometry in solutions of anthocyanins previously
isolated from red cabbage. From the spectrophotometry readings, chromatic analyses
were employed to calculate the intensity and color lightness of the sample following
the CIE (Commission International de lEclairage) system of 1976 (Goncalves
et al., 2007). Values of L*, a* and b* were measured to describe
a three dimensional color space and interpreted as follows: L* indicates lightness
read from 0 (completely opaque or black) to 100 (completely
transparent or white). A positive a* value indicates redness
(-a* is greenness) and a positive b* value yellowness (-b* is blueness).
Hue (H0) is color tone whether it is blue, red, green, yellow and
etc. Chroma (C*) is color intensity whether it bright or dull and Lightness
(L*) is the amount of color whether it light or dark. Equation
1 and 2 represent the equation for H0 and C*,
respectively which were calculated by the transformation of a*and b* values:
Based on the hue circle, H0 values are stepped from 0 to 360°
(red). The other reference values are 90° (yellow), 180° (bluish green)
and 270° (blue) (Ersus and Yurdagel, 2007). Differences
in hue angle (H0) could be attributed to both differences in anthocyanin
and phenols composition (Reyes and Cisneros-Zevallos, 2007).
Changes in the H0 parameter describe the direction of changes of
the color tone. Generally, during storage, H0 value increased with
an increase in the level of independent variables (storage time). This indicates
the change of the color towards yellow (high pH) which is associated with the
development of products of anthocyanin degradation. Color intensity (color purity)
indicates if color is pure or contaminated by the presence of another pigment
(Gonnet, 1999). The loss of lightness was reflected
by a reduction of L* and was directly related to the humidity during storage
(Heredia et al., 1998).
According to the CIELAB parameters storage, their color stabilities depend highly on pH and anthocyanin structure. Juice anthocyanin was found to be significantly more brighter color than powdered anthocyanin at most pH values, showing very high L* and C* values after storage for 10 days (Fig. 2 and 3). The maximum color intensity (C*) for both juice and powdered anthocyanin was observed in the alkaline region at pH 13. From the result, it indicates that powdered anthocyanin color intensity was consistently stable compared to juice anthocyanin at a wide pH range but the color lightness (L*) was slightly decreased with increasing storage time. This is due to degradation of anthocyanins.
From the analyses, H° values of both powdered and juice anthocyanin samples
have reddish color at the pH 1. Increasing the pH values from 1 to 7, the color
gradually changed toward more purplish tones. At higher pH values, the hue angles
decreased dramatically indicated by the changes of color to green. As revealed
in Fig. 4, there were no changes in hue values for powdered
anthocyanin and small changes for juice anthocyanin at various pH after storage
for 10 days.
||Color lightness of powdered and juice anthocyanin at various
||Color intensity of powdered and juice anthocyanin at various
||Color tone of powdered and juice anthocyanin at various pH
This suggests that powdered anthocyanin have relative stable hue compared with
Generally, powdered anthocyanin showed greater color stability, expressed by H°, C* and L* parameters than juice anthocyanin at all pH values.
In conclusion, anthocyanin occurred in the most intense reddish color in acidic aqueous solutions. Neutral solutions will result in a purplish color and basic solutions will appear in the green-yellow region of color. Powdered anthocyanin is more stable than juice anthocyanin at most pH values, showing no changes in C* and H0 and little change in L* values at a period of 10 days storage.
Powdered anthocyanins have better color stability at specific pH which allows it to be used as pH color indicator in food packaging. Because of its wide range of color tones in relation to pH, as well as high color stability, anthocyanin extracted from red cabbage is also highly recommended for food colorant. The color variations exhibit from the results obtained emphasize the importance of future work to develop anthocyanin as pH color indicator in food packaging system.
Thanks are due to all who are involved in this work for their guidance and support.