13th ASEAN Food Conference, 9-11 September 2013, Singapore

Meeting Future Food Demands: Security & Sustainability

Effect of Temperature on Color Transition of Anthocyanin

and Possible-Application of Time-Temperature Indicator

Surachai Khankaew1, Waraporn Boonsupthip2, Chanassa Nandhivajrin1,

Chiravoot Pechyen1, Panuwat Suppakul1,*
1
Department of Packaging and Materials Technology, Faculty of Agro-Industry,
Kasetsart University, Bangkok 10900, Thailand
2
Department of Food Science and Technology, Faculty of Agro-Industry,
Kasetsart University, Bangkok 10900, Thailand
* Corresponding author: panuwat.s@ku.ac.th

Abstract Time–temperature indicators (TTIs) have been designed to provide information about
time–temperature history in food products during distribution and storage. Recently, some literatures found
in the effect of temperature and light on some natural dyes from plant extracts. The use of their natural dyes
as colorimetric bio-indicator is of increasing interest due to the perceived lower risk of this bio-indicator to
the consumer. Studies aimed at estimating the temperature effect on color transition of bio-colorants during
time interval. The experiment tested on 3 different concentration of bio-colorants solution which were asian
pigeonwings (Clitoria ternatea L.) (AP) and red cabbage (Brassica oleracea var. rubra) (RC). The
bio-colorants solution was mixed with 10% (w/v) of cellulose ether solution. These solutions were separated
for coated on card papers and casted as a thin film. The bio-colorant as a coated and thin film were changed
irreversibly the color tone; dark blue to bright blue for AP and dark purple to bright purple for RC. The total
color difference (TCD) parameter was increased upon time, temperature (25-45 ˚C) and UV-light exposure
time (0-360 min.). This study envisages the possible application in intelligent packaging technology as a
novel bio- time-temperature indicator.

Keywords Intelligent packaging, Time-temperature indicator, Natural dye, Bio-colorant, color transition

1. Introduction
Food safety is a constant concern for humans. Besides adulteration and contamination, another
major threat comes from the spontaneous spoilage of perishable products, which is basically
inevitable and highly dependent on the temperature history during the custody chain.

Besides the microbial growth and possible foodborne illness, the high temperature (above room
temperature, 21 ˚C) accelerates physical changes and chemical reactions promoting deterioration on
foods. Some nutritional changes in different environmental factors such as high temperatures,
moisture, light, water activity and others, causing loss of vitamins, protein, amino acid, change in
sensorial, color and off-flavor etc.

Temperature greatly affects the quality and safety of food and pharmaceutical products during
distribution and storage [1]. In general, appropriate packaging provides effective barrier to gases,
moisture and light, but temperature control depends strongly on the transport and storage conditions.
For advanced quality control and assessment, time–temperature indicators (TTIs) can be monitoring
and recording the temperature conditions during distribution and storage [2].

Many new types of time-temperature indicators have been developed recently. By et al. [3] reported
new enzyme reaction-diffusion TTI for milk; it has good stability and reliability at dynamic storage
conditions. Chahattuche et al. (2010) developed a prototype of TTI based on the diffusion of lactic
acid, they could be applied to show the time-temperature history of some foods, especially fruits
and vegetables [4]. Vinicius et al. [5] developed a colorimetric temperature indicator based on a
natural heat-sensitive pigment (Anthocyanin, ATH) [6].

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 13th ASEAN Food Conference, 9-11 September 2013, Singapore
Meeting Future Food Demands: Security & Sustainability

Natural colorants used as colorimetric bio-indicator is interesting due to the perceived lower risk to
the consumer. However, there are few published data in the scientific literature, related to
bio-indicator for time-temperature indicator application.

Many kinds of natural colorants can be used as natural indicator of temperature in intelligent
packaging systems due to its characteristics of color change when exposed with different
temperatures and light [5]. The natural colorants such as curcumin from turmeric (curcuminoid),
lycopene from tomatoes (carotenoid) and anthocyanin from blue flowers [7], vegetables and fruits
(flavonoid), etc. [8].

Hence, this article presents the effect of temperature and UV-light on natural colorants for using as a
colorimetric bio-indicator for monitoring product quality during distribution and storage and
possible application in intelligent packaging technology as a time-temperature indicator has also
been demonstrated.

2. Materials and Methods

2.1. Materials

The bio-colorants were used as colorimetric bio-indicator are asian pigeonwings (Clitoria ternatea
L.) from Nakhonpathom and red cabbage extract (Brassica oleracea var. rubra) was purchased from
Qingdao Bio-tech Resource (China). Ethanol (Sigma–Aldrich, USA). Methylcellulose (MC)
(Methocel®, Dow Chemical, USA) was used as a bio-based polymer. Uncoated card paper 100 g
m-2 was obtained from Thai paper Co., Ltd. (Thailand) and distilled water were used as a solvent.

2.2. Preparation of bio-colorimetric solution and coating binder

Bio-colorimetric solutions was mixed by used a natural colorants powder are contain many of
anthocyanin (ATH) and can be purchased with commercial grade, which were asian pigeonwings
(AP), and red cabbage (RC) at 10.0% (w/v) concentrations with distilled water, warm at 80-90 ˚C
and stirrer with magnetic stirrer for 15 min. After finished, filtration the solution by used
filter-paper in a Büchner funnels for two times and cooling at room temperature (25 + 1 ˚C), for
turmeric, diluted the solution with ethanol to 20% (v/v) before testing.

A coating binder was prepared by mixed 10% (w/v) methylcellulose with distilled water, warm at
40-60 ˚C and stirrer with hot-plate magnetic stirrer for 20 min. Later on, cool down and degas at
room temperature for 24 hr.

2.3. Preparation of natural anthocyanin (ATH) colorimetric coating and thin film

2.3.1. Coating Natural ATH-colorimetric solution was mixed in a concentration of 1.25%, 2.50%,
and 5.00% (w/w) with coating binder, the mixture was stirred by magnetic stirrer until complete
dissolution have been achieved. The coating was coated on uncoated card paper surface using a 60
µm by bar coater (4-sides applicator, BYK Gardner, USA) forming the bio-coated paper. The coated
paper sheets were dried in room temperature for 60 min.
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 13th ASEAN Food Conference, 9-11 September 2013, Singapore
Meeting Future Food Demands: Security & Sustainability

2.3.2. The Natural ATH-colorimetric films was prepared by mixed coatings with distilled water in a
ratio of 1:1, degased and immediately poured onto the Petri dish, Later on, dried at room
temperature for 24 hr. After finished, cut to small circle (1-2 cm diameter).

2.2. Determination of the effect of temperature and light

Bio-coated paper and bio-colorimetric film were cut to small circle (1-2 cm diameter) and placed in
to A4 uncoated paper. Different conditions were tested by varied of temperature and UV-light
fallowing the Table 1.

Table 1. Conditions of temperature and UV-light.

Tests* UV-Light exposure time (min) Temperature (˚C) Storage time (day)
#1 0-360 (step 60 min) - -
#2 - 25, 35, 45 7
#3 360 (before storage) 25, 35, 45 7
*of asian pigeonwings (AP) and red cabbage (RC).

Experiments were run in triplicate and was recorded after each time interval by a chroma meter
spectrodensitometer (SpectroDens, Techkon®, Königstein, Germany).

The L*, a*, b* chroma system, which uses the corresponding value of total color difference (∆E) as
dynamic parameters, was used to analyzed the dynamic change in the bio-colorimetric solution. The
total color difference (TCD) is expressed as follows:

∆E = [(∆L*)2 + (∆a*)2 + (∆b*)2]0.5 (1)

where ∆L* is the brightness difference between initiation and each time interval, ∆a* is the
redness-greenness difference between initiation and each time interval, and ∆b* is the
yellowness–blueness difference between initiation and each time interval [9]

3. Results and Discussions

3.1. Effect of UV-light exposure on color transition

As exposed with UV light, the TCD value gradually increased. The TCD value of RC was higher
than AP when tested with the ATH-coated paper. Especially for RC, it tended to increase with
increasing exposure time. In contrast, the TCD of AP when determined with the ATH-incorporated
film yielded a higher value than that of RC. At the AP concentrations of both 1.25 and 2.5 % w/w, it
revealed a constant line of tendency after exposure to 180 min, whereas the TCD of AP slightly
changed (Figure 1).

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 13th ASEAN Food Conference, 9-11 September 2013, Singapore
Meeting Future Food Demands: Security & Sustainability

Figure 1. Total color difference (TCD) of (a) natural ATH-coated paper

and (b) natural ATH-incorporated film.

3.2. Effect of temperature and UV-light activate with temperature on color transition

For the AP-naturally-derived ATH, it was found that the TCD value rose when increasing the time
of exposure, especially in the case of coated paper. The TCD immediately increased after exposure
to UV light for 360 min. It could distinct for the coated paper rather than the film with the
concentrations of 2.5-5.0 % w/w. The ATH-incorporated film showed a minor change in color with
the TCD less than 5 in the 7th day of investigation. It happened in a similar manner in every tested
temperature conditions. This is in accordance with the study of Vinicius et al. [5].

The TCDs of RC-ATH-coated paper and RC-ATH-incorporated film did not significantly change
with tested temperatures in a range of 25-35 ˚C. But the TCD of UV-pre-exposed RC-ATH-coated paper
rapidly increased at 45 ˚C, especially with the concentrations of 2.5-5.0 % w/w. On the contrary to the paper,
the RC-ATH incorporated film yielded a significant increase in the TCD value. It is interesting to note that
UV pre-exposure treatments did not pay attribute to the TCD value of the RC-ATH incorporated film, as
shown in Figure 2.

(25 ˚C) (35 ˚C) (45 ˚C)

(a) AP-ATH coated paper

(25 ˚C) (35 ˚C) (45 ˚C)

(b) AP-ATH incorporated film

(25 ˚C) (35 ˚C) (45 ˚C)

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 13th ASEAN Food Conference, 9-11 September 2013, Singapore
Meeting Future Food Demands: Security & Sustainability

(c) RC-ATH coated paper

(25 ˚C) (35 ˚C) (45 ˚C)

(d) RC-ATH incorporated film

Figure 2. Total color difference (TCD) of natural ATH-coated paper influenced from
temperature and UV-light activate conditions.

3.3. Color transition of natural ATH

Homogeneous ATH-colorimetric solutions were characterized by both a deep blue color system for
AP and a deep purple color system for RC, respectively, forming either a coated card paper or an
incorporated film without a bubbles or defects. Color shades of these materials are dependent upon
the concentrations of their colorants.

As shown in Table 2, it was found that both AP and RC naturally derived ATH immediately changed
in color after coating onto the paper. It happened in a similar fashion according to various
concentrations of ATH. The color led to the blue shade as figured out in the negative b value. This is
due to the immediate reaction between the ATH and alkaline residual in paper. It resulted in a basic
form of ATH as revealed in the blue color.

In addition, UV light affected the degradation of ATH found in AP and RC, as observed by unaided
eyes. Their color tended to fade out after 360 min UV-light exposure. Later on, there was no change
in color shade when treated with different temperatures. At the end of investigation (the 7th day), it
was found that the treatments without UV-light exposure gave a higher negative b* value than those
of the UV pre-exposed treatments, tested at 25-45 ˚C, in turn, a blue tone as observed by unaided
eyes.

For both AP-ATH and RC-ATH-incorporated films, it could be observed as changing in color after
7th days of investigation, especially in the case of higher concentrations of 2.5-5.0 % w/w.
RC-ATH-incorporated film yielded in an easier observation in the color change, compared to the
AP-ATH-incorporated film. However, it was found that both ATH-incorporated films revealed a
lesser color change in comparison with the ATH-coated papers, as demonstrated in Table 2.

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 13th ASEAN Food Conference, 9-11 September 2013, Singapore
Meeting Future Food Demands: Security & Sustainability

3.3. Color transition of natural ATH

Table 2. L*a*b* color transition of natural ATH color on finished (7th days).

The simulate color at the last of testing time

L*a*b*
Colorant UV-light UV-light with Temp. Temper
(Initial color)
(360 min.) 25 ˚C 35 ˚C 45 ˚C 25 ˚C
Natural ATH-coated paper
AP1.25% 92.70/1.59/-5.79 93.52/1.59/-2.15 93.52/1.59/-2.15 94.13/1.71/-2.72 94.27/1.52/-1.84 93.45/1.57/-7.02 93.02/
AP2.50% 91.64/1.14/-6.81 92.95/1.57/-1.96 92.95/1.57/-1.96 92.88/1.56/-2.15 93.43/1.46/-1.67 89.34/0.65/-9.21 91.83/
AP5.00% 59.85/0.97/-8.55 92.33/1.38/-1.91 92.33/1.38/-1.91 92.60/1.74/-2.61 92.39/1.46/-1.81 86.98/0.61/-10.24 90.48/
RC1.25% 92.26/1.80/-6.11 92.82/1.81/-1.69 92.82/1.81/-1.69 92.89/2.02/-2.11 93.23/1.87/-1.34 92.28/1.77/-6.67 91.44/
RC2.50% 89.28/1.65/-7.55 90.63/1.87/-1.10 90.63/1.87/-1.1.0 91.64/2.50/-1.69 91.49/2.27/-0.87 90.53/1.47/-7.90 89.23/
RC5.00% 81.04/1.91/-11.35 88.62/1.93/-1.12 88.62/1.93/-1.12 90.02/3.28/-1.50 89.74/3.01/-0.75 78.43/-0.57/-12.85 78.79/
Natural ATH-incorporated film
AP1.25% 81.70/-3.06/-9.30 82.47/-3-32/-5.02 82.47/-3.32/-5.02 82.56/-2.50/-6.13 82.50/-3.32/-4.85 84.05/-2.83/-9.25 82.63/
AP2.50% 73.07/-6.56/-10.30 80.99/-3.34/-4.57 80.99/-3.34/-4.57 78.52/-3.76/-4.61 74.59/-5.49/-4.96 74.40/-5.97/-11.75 73.62/
AP5.00% 62.82/-5.92/-23.85 73.53/-4.35/-9.26 73.53/-4.35/-9.26 67.73/-5.72/-14.10 66.50/-5.28/-14.60 64.17/-7.65/-16.73 60.56/
RC1.25% 79.69/10.99/-6.55 78.10/11.64/-2.94 78.10/11.64/-2.94 80.52/9.53/-3.41 78.52/10.97/-2.93 80.99/9.32/-6.72 76.53/
RC2.50% 68.89/22.16/-8.36 72.64/17.48/-4.67 72.64/17.48/-4.67 62.61/26.03/-6.07 67.21/24.36/-5.96 71.24/19.69/-7.21 62.81/
RC5.00% 53.22/42.74/-9.83 51.74/54.05/-6.17 51.74/43.05/-6.17 54.09/40.91/-7.61 52.58/43.73/-7.18 54.49/39.13/-8.91 50.06/

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 13th ASEAN Food Conference, 9-11 September 2013, Singapore
Meeting Future Food Demands: Security & Sustainability

4. Conclusions

This present study revealed that both UV-light and temperature affected on the color transition of
the naturally-derived anthocyanin (ATH) colorants including Asian pigeonwing (AP) and Red
cabbage (RC). It is interesting to note that the color of ATH-coated paper could be dramatically
faded out when exposed to UV-light. However, the temperature could be impacted on both of
ATH-coated paper and ATH-incorporated film, with a lesser degree of degradation in comparison
with the UV-light effect. In addition, these safe ATH colorants could be adapted to intelligent
packaging application in terms of time-temperature indicator.

Acknowledgements

This work was financial support from the Thailand research fund through the Royal Golden Jubilee
(RGJ.) Ph.D. Program (grant no. PHD/2554/0054) is acknowledged.

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13th ASEAN Food Conference, 9-11 September 2013, Singapore
Meeting Future Food Demands: Security & Sustainability