International Journal of Biological Macromolecules 174 (2021) 370–376

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Colorimetric indicator based on purple tomato anthocyanins and

chitosan for application in intelligent packaging
Yana Li ⁎, Kaixuan Wu, Beihai Wang, Xuezhong Li
School of Mechanical Engineering, Wuhan Polytechnic University, Wuhan 430023, China

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

Article history: Intelligent colorimetric indicator films were prepared to monitor freshness/spoilage of milk and fish by
Received 9 January 2021 incorporating purple tomato anthocyanin (PTA) into chitosan (CS) matrix via solution casting method
Received in revised form 27 January 2021 with PTA concentration (w/w, based on CS) of 10%, 30%, and 50%, respectively. The pH-response, UV absorp-
Accepted 28 January 2021
tion, Swelling Index, and the mechanical properties of CS/PTA films were determined. It was found that the
Available online 2 February 2021
color of the original CS/PTA films became darker with an improvement of PTA content and expressed well
Keywords:
pH-sensitivity. With increasing of pH, the color of the CS/PTA films exposed to pH = 3–11 solutions became
Purple tomato anthocyanin darker and the change in color of the CS/10% PTA film was the most discernable. The tensile strength and
Colorimetric indicator Young's modulus of the CS/PTA film was much lower than that of CS film, however, the elongation at break-
Intelligent packaging ing and Swelling Index were both improved by adding PTA. The intelligent films with 10% PTA changed their
color during progressive spoilage of milk or fish, revealing their potential application for monitoring food
freshness/spoilage.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction
There is currently a growing interest in the development of colori-
metric pH indicator films for application in intelligent packaging to
monitor the freshness of food. This indicator changes color in response
to a change in the pH of the food. Consequently, without opening the
package, consumers can quickly distinguish between fresh and spoiled
food, which may lead to improvements in food quality and reductions
in the amount of food wasted [1]. These films are usually prepared by
combining biodegradable polymers with pH-sensitive dyes. The poly-
mers used include polysaccharides, gums, protein, gelatin and polyvinyl
alcohol [1–6] because they are recyclable, decompose in a short period
of time, are non-toxic, and are environment friendly.
However, the use of synthetic dyes as pH indicators is limited due to
potential toxicity and mutagenicity [7]. Hence, current research focuses
on natural pigments derived from plant products and food wastes be-
cause they satisfy the safety requirements of the packaging materials
and their potential bioactive function [2,8]. These natural pigments in-
clude anthocyanins [9], curcumin [10], betalains [11], chlorophyll [12]
and carotenoids [13]. Of these, the anthocyanins represent a large
group of naturally occurring plant-based pigments found abundantly
in different parts of plants [14,15].
Studies mentioning colorimetric indicators based on anthocyanins
from red cabbage [16], purple sweet potato [17], blueberry [18], black
⁎ Corresponding author.
E-mail address: myllyn@126.com (Y. Li).
carrot [3] and other dark purple and black vegetables [19] have been re-
ported. The results showed that the anthocyanin source greatly influ-
ences its functional and physical properties [7]. Chitosan is the second
most abundant natural biopolymer after cellulose, which is edible and
has the advantages of biodegradability, biocompatibility and excellent
film-forming ability, so it is applied widely in food packaging [20]. In
this study, the anthocyanins extracted from a new source - purple toma-
toes (Solanum lycopersicum) was used to develop colorimetric pH indi-
cator films for intelligent packaging applications by combining chitosan
(CS). The film properties such as pH-response, swelling activity, me-
chanical properties as a function of purple tomato anthocyanins (PTA)
content were evaluated. The indicator was also utilized to monitor the
freshness/spoilage of milk and fish.
2. Materials and methods
2.1. Preparation of the CS/PTA composite films
The PTA was donated by Prof. Ding Xinhua of Shandong Agricultural
University. Those were extracted from purple tomato fruits (Solanum
lycopersicum BS003) using an efficient purification method [21] with
54% (w/w) of the purity. CS/PTA composite films were prepared using
a solution casting method [22].
CS solution was prepared by dissolving 1 g of CS (Sinopharm
Chemical Reagents Ltd., DDA = 92.5%, Mw = 50 kDa) in 100 mL of
acetic acid solution (0.1% v/v) with vigorous stirring at room temper-
ature for 3 h. A certain content of PTA (0.1 g, 0.3 g, or 0.5 g) was then

https://doi.org/10.1016/j.ijbiomac.2021.01.182
0141-8130/© 2021 Elsevier B.V. All rights reserved.
Y. Li, K. Wu, B. Wang et al. International Journal of Biological Macromolecules 174 (2021) 370–376

added into the CS solution with continuous stirring for 30 min to ob- 2.2.2. UV–Vis spectra
tain CS/PTA mixtures with PTA concentrations of 10%, 30%, and 50% The UV–Vis absorption of samples was measured from 200 nm to
(w/w, based on CS), respectively. Finally, 70 mL of the mixtures or 800 nm with an ultraviolet spectrophotometer (UV1800/UV1800 PC,
CS solution were cast on a petri dish (15 cm diameter) and dried at Shanghai Jinghua Instrument Co., China).
35 °C for 48 h, consequently the films with thickness of 40 ± 2 μm
were peeled off and conditioned into a desiccator (RH = 50%) at 2.2.3. Swelling index
23 °C for at least seven days in the dark before testing. CS film was Swelling Index (SI) of films was determined by the method de-
used as the control. scribed by Pereira et al. [22]. The sample films (2 cm × 2 cm) were
placed in a desiccator for seven days with silica gel to remove the
2.2. Characteristics of the CS/PTA composite films water from them and the dry weight of each sample was determined.
Then the samples were soaked in 250 mL of distilled water for 0.5, 1,
2.2.1. Color response 3, 5, 7, 10, 15, and 20 min, respectively, at 23 °C. Finally, the sample
The finished CS/PTA films (2 cm × 2 cm) were exposed to buffer so- was removed from the solutions, any excess water removed and their
lutions with a pH of 3, 4, 5, 6, 7, 8, 9, 10 and 11. The L* (brightness), a* weight was determined. These experiment steps were repeated three
(red/green), and b* (yellow/blue) of each sample were measured times. The SI was calculated by Eq. (2):
using a colorimeter (CR-10, Konica Minolta, Japan) [3]. The value of
the total color difference (ΔE) was calculated using Eq. (1) [5]: Finally weight−Initial weight
SI ¼  100% ð2Þ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Initial weight

2  2 

2
ΔE
¼ L standard −L
sample þ a
standard −a
sample þ b standard −b sample
ð1Þ
2.2.4. Mechanical properties
where, L* standard, a* standard and b* standard are color values of the The mechanical properties of the CS/PTA films were measured with
standard CS/PTA film (pH 7). a universal electronic tester (SANS JTM4104, China) at a crosshead

(a)
(b)

(c)

10% PTA 30% PTA 50% PTA

(d) 2.5
3.0 1.8
2.0 1.6
2.5
1.4
1.5 2.0 1.2
Abs.

Abs.

1.0
Abs.

1.5
1.0 0.8
1.0 0.6
0.5 0.4
0.5
0.2
0.0 0.0 0.0
300 400 500 600 700 800 300 400 500 600 700 800 300 400 500 600 700 800
Wavelength (nm) Wavelength (nm) Wavelength (nm)

Fig. 1. (a) The anthocyanins were extracted from purple tomatoes. (b) The PTA powder was used in the present study. (c) CS/PTA films and (d) those UV–Vis spectra.

371
Y. Li, K. Wu, B. Wang et al. International Journal of Biological Macromolecules 174 (2021) 370–376

speed of 20 mm/min at 23 °C [23]. The samples were 100 × 15 mm rect- 3. Results and discussion
angular pieces of the film. Six samples were measured and the average
value was used as the final test result. 3.1. Original films

2.3. Freshness/spoilage monitoring of milk or fish
Pasteurized milk (20 mL) was poured in glass petri-dish under an
aseptic condition and maintained at 23 °C until the milk got spoiled.
At different time points (0, 24 and 48 h), a CS/10%PTA film
(2 cm × 2 cm) was immersed in milk for 30s. At similar intervals,
the color of the CS/10%PTA samples was pictured and the corre-
sponding pH value of the milk were measured using digital pH
meter [2].
A sample of fish (10 g) was placed inside a pasteurized Petri dish
and sealed using a Petri dish cover which contained a sample
(2 cm × 2 cm) of CS/10%PTA film attached to its inside top side,
and stored at 23 °C for 48 h to monitor fish spoilage during storage
[24]. The change in the color of the CS/10%PTA film was evaluated
using color photographs taken at 0 h, 24 h and 48 h. Simultaneously,
the total volatile essential nitrogen (TVB-N) of the fish sample was
determined according to Cao et al. [25] with a semi-micro Kjeldahl
distillation instrument.
Fig. 1 illustrates the source of the PTA (Fig. 1(a) and (b)) and the
original CS/PTA films (Fig. 1(c)) containing 10%, 30%, and 50% PTA re-
spectively. An increase in the concentration of PTA resulted in the film
having a darker color (Fig. 1(c)). The CS/10%PTA film was light green,
the CS/30%PTA film was translucent to blackish green, and the CS/50%
PTA film was an opaque dark green. And they all had the similar UV–
Vis spectra with the maximum adsorption at around 300 nm and
600 nm (Fig. 1(d)).
3.2. Color response
Fig. 2 illustrates the CS/PTA films with PTA concentrations of 10%,
30% and 50%, respectively, in solutions with different pH values of 3 to
11 at increments of pH = 1. It was found that both the UV–Vis spectros-
copy and colorimetric data revealed that the color of CS/PTA composites
was strongly pH-dependent. Specifically, the peaks from Fig. 2(a) in the
UV–Vis spectra depended on pH that the maximum absorbance peak in
acidic conditions was observed around 300 nm, while the maximum

(b) 10% PTA

˄a˅
3.0 3

2.5 5

2.0
Abs.

1.5 4
6
1.0 9
7 10
11
0.5 8

0.0
300 400 500 600 700 800
Wavelength (nm)
(c) 30% PTA (d) 50% PTA

Fig. 2. UV–Vis spectra of CS/10%PTA (a) and color variations of CS/PTA films containing 10% (b), 30% (c) and 50% (d) PTA respectively, at pH values of 3, 4, 5, 6, 7, 8, 9, 10, 11.

372
Y. Li, K. Wu, B. Wang et al.
absorption peak reduced in height and shifted to higher wavelengths
with increasing pH.
Moreover, the change in color of the CS/10%PTA film was from
pink (pH = 3) → light purple (pH = 5) → bright green (pH =
7) → light green (pH = 9) → light yellow (pH = 11). The change in
color of the CS/30%PTA film was from fuchsia (pH = 3) → deep purple
(pH = 5) → dark blue (pH = 7) → green (pH = 9) → yellow-green
(pH = 11). The change in color of the CS/50%PTA film was from fuchsia
(pH = 3) → blue → green (pH = 11). The change in color of the CS/50%
PTA film was not easy to discern and this was probably because of
the darker color of the original film (Fig. 1(c)) due to its high PTA
content.
Research has demonstrated that anthocyanins decompose rapidly
and that its chemical properties are unstable. The stability of anthocya-
nins varies with differences in pH, storage temperature, enzymes, and
the light, and oxygen conditions of their environment. When the struc-
ture and concentration of anthocyanins differed, the differences in sta-
bility were found to be enormous. In response to this, Brouillard [26]
determined that in dilute acid solutions anthocyanins exist in the form
of flavonoid cations, and that double bonds are prone to extended con-
jugation, forming a colorless pseudoalkaline and a balanced reaction of
red flavonoid cations. The Handerson-Hassel Balch equation [27]
(Fig. 3) can be used to analyze and demonstrate the reaction equilib-
rium of anthocyanins.
Anthocyanins are more stable in acidic than in alkaline solutions.
Under acidic conditions anthocyanins form four substances: colorless
chalcones, colorless carbinol pseudo-base, red flavonoid cations, and
blue qinonoidal alkali. The four substances form a dynamic equilibrium.
When the solution's alkalinity increases, the nucleophilic reaction
between the red flavonoid cations and water forms colorless carbinol.
This is because the total amount and concentration of the red flavonoid
Fig. 3. Structural changes of anthocyanins at different pH values.
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International Journal of Biological Macromolecules 174 (2021) 370–376
cations are reduced and the color intensity is correspondingly reduced.
Consequently, when anthocyanins are in an acidic medium, the
flavylium cations play a major role and the films appear red. With an in-
crease in pH, the anthocyanins become quinoid alkali caused by a loss of
cations from the carbon and oxygen ring, and the composite films ap-
pear blue [28].
When evaluating an intelligent pH indicator system, an important
point is the change in the ΔE with pH. It is necessary that an indicator
of ΔE vary enough to be discernable, so that consumers will easily be
able to see this color change and obtain information on the condition
of the product.
The change in the ΔE of the CS/PTA composite films is illustrated
in Fig. 4. As illustrated in Fig. 3, the ΔE strongly dependent on PTA
amount. The ΔE of the CS/30%PTA and CS/50%PTA did not vary signif-
icantly with a change in pH from 3 to 5 and 9–11, but changed inten-
sively at pH = 5–7 and pH = 8–10. However, the CS/10%PTA
composite films changed dramatically in ΔE with a change in
pH 3–11. Therefore, of the three films tested, the CS/10%PTA was
the best pH-response in color. That indicates the CS/PTA composite
film is potential in intelligent packaging application due to its easy
visualization of the color change.
3.3. UV–Vis spectra
The UV–Vis adsorption of CS, PTA, and CS/PTA is shown in Fig. 5. It
is found that the CS film has no apparent maximum absorption peak.
The maximum absorption for PTA in the UV–Vis region was at
300 nm and 540 nm respectively and those were characteristic of
anthocyanins in the neutral range. In each molecule of anthocyanins,
there are two benzene rings forming a conjugated system with max-
imum absorption around 280 nm and 500–550 nm in the UV and Vis
Y. Li, K. Wu, B. Wang et al. International Journal of Biological Macromolecules 174 (2021) 370–376

55 10%PTA
160 30%PTA
50

45 140

40 120

Swelling Index /%
35 100
ΔE

30 80

25 60

20 10% 40
30%
15 20
50%
10 0
2 3 4 5 6 7 8 9 10 11 12 0.5 1 3 5 7 10 15 20

pH Time/min

Fig. 4. Total color difference (ΔE) of CS/PTA films containing 10%, 30% and 50% PTA Fig. 6. Swelling Index of CS/PTA films.
respectively, as a function of pH.

3.5. Mechanical properties

regions respectively [29]. Those that the CS/PTA had absorption
peaks at 310 nm and 600 nm were close to the absorption peaks of
anthocyanins with a slight shift towards the Vis range. These results
confirmed that the PTA had been combined with the CS to form the
CS/PTA composite.
3.4. Swelling index
Fig. 6 shows the results of the SI, however, the SI could not be mea-
sured for CS/50%PTA film because it completely dissolved in the aque-
ous solution at 0.5 min. From Fig. 6, it is observed the SI increased as a
function of PTA concentration in CS films and it initially increased and
then decreased indicating that a hydration equilibrium period occurred
from 1 to 7 min for both CS/10%PTA and CS/30%PTA films. The final
values of the SI of CS/10%PTA and CS/30%PTA films was 41.0% and
93.0%, respectively, which were less than the highest recorded hydra-
tion value of 63.5% for CS/10%PTA and 160.8% for CS/30%PTA. This phe-
nomenon can be explained by the fact that after a period of hydration
the chain loses its fluidity which hinders the entry of solvents into the
film [22].
The mechanical properties of the CS/PTA composite films are shown
in Fig. 7. It is found compared with CS film, the addition of PTA signifi-
cantly reduced the tensile strength and Young's modulus of the CS/
PTA film, however, it had a higher elongation at break. That suggests
film's strength and stiffness decreased significantly, and the flexibility
increased. This may be attributed to the increased interfacial force be-
tween the PTA and the CS polymer matrix through H-bonds formation
[30,31].
The higher elongation at break indicated that these CS/PTA films are
more ductile than CS. This may be related to there being more available
sites that improve the interaction of water molecules with the film-
forming matrix [32]. In addition, the inclusion of PTA in the CS matrix
may act as a plasticizer based on the favourable hydrophilicity of PTA
and reduce the interaction between macromolecules [33], which in
turn leads to a decrease in tensile strength and Young's modulus. This
effect was enhanced by increasing the concentration of PTA embedded
in the CS matrix.
3.6. Freshness/spoilage monitoring of milk or fish
Table 1 shows the results of CS/10%PTA films to monitor fresh-
ness/spoilage of milk or fish as colorimetric indicators. It was found

Tensile strength/MPa
Tensile strength /MPa Young's Modulus /MPa
Elongation atatbreak
Elongation break/%/%
60
2000
2.5 CS/PTA
50
2.0 1500
40
1.5
Abs.

1000
30

1.0
PTA 20 500

0.5
10
CS 0
0.0
0
300 400 500 600 700 800 0 10 30 50
Wavelength (nm) PTA concentration/ %

Fig. 5. UV–Vis spectra of CS film, PTA solution, and CS/PTA film. Fig. 7. Mechanical properties of the CS/PTA composite films.

374
Y. Li, K. Wu, B. Wang et al.
Table 1
Monitoring milk or fish freshness/spoilage using CS/10%PTA indicator films during storage time.
Storage time/h Milk
pH Color changes Freshness/spoilage
0 6.8 ± 0.1 Fresh
24 6.1 ± 0.1 Medium fresh
48 5.0 ± 0.1 Spoiled
All values were expressed as mean ± standard error (n = 3).
that the pH of the fresh milk was weakly acidic. When milk is stored
at a temperature higher than the cooling temperature (7 °C), it de-
composes due to insufficient stability. The subsequent accumulation
of lactic acid due to microbial contamination reduces the pH of the
milk. In the present study, the pH of the milk decreased from 6.8
(0 h) to 6.1 (24 h), and 5.0 (48 h). Correspondingly, the decreasing
pH of the milk was indicated by the change in color of the samples
of CS/PTA film. For fresh milk with pH 6.8 the CS/PTA film was
green. As the pH decreased from 6.8 to 6.1, the color of the CS/PTA
film changed to yellow. After 48 h at a pH = 5.0 the milk had a dis-
tinct smell and the CS/PTA film was pink, which clearly indicated
that the milk was spoiled.
While for fish, it was fresh with the TVB-N content of 7.1 mg/100 g at
0 h according to Chinese standard for fish (GB 2733-2015), which was
specified that the TVB-N content of fresh fish should be less than
15 mg/100 g. At that time, the CS/PTA film showed dark green. As the
TVB-N content reached 16.7 mg/100 g at storage time of 24 h it indi-
cated that the quality of fish had declined rapidly, and the CS/PTA film
was green. The TVB-N content of the fish reached 38.8 mg/100 g at
48 h and the color of CS/PTA film turned to light green. A clear exudate
appeared on the surface of the fish and it was no longer suitable for
human consumption. During the storage of fish, microorganisms, and
temperature will cause biogenic amines, which will increase the pH
value [34], thereby changing the color of the CS/PTA film from dark
green to green and light green, respectively. Therefore, our results
showed that the anthocyanins from purple tomatoes can be used in chi-
tosan films to monitor food safety and quality.
4. Conclusions
In the present study, the anthocyanins extracted from purple toma-
toes were used to fabricate the CS/PTA films containing 10%, 30%, and
50% PTA. The color of the CS/PTA film gradually darkened with an in-
crease in the concentration of PTA. The change in color of the CS/10%
PTA film was from pink (pH = 3) → light purple (pH = 5) → bright
green (pH = 7) → light green (pH = 9) → light yellow (pH = 11).
The change in color of the CS/30%PTA film was from fuchsia (pH =
3) → deep purple (pH = 5) → dark blue (pH = 7) → green (pH =
9) → yellow-green (pH = 11). The change in color of the CS/50%PTA
film was from fuchsia (pH = 3) → blue → green (pH = 11). Among
them, the CS/10%PTA film had the most discernable color change
under different pH values. UV–Vis spectroscopy confirmed that the
PTA had been incorporated into the CS film. As the concentration of
PTA increased, the elongation at break of the film improved, while the
tensile strength, Young's modulus and SI% decreased sharply. The CS/
10%PTA film in milk or on fish changed in color depending on the fresh-
ness or spoilage of the milk or fish. That indicated that the CS/PTA film
has potential as colorimetric indicators in intelligent packaging.
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International Journal of Biological Macromolecules 174 (2021) 370–376
Fish
TVB-N content (mg/100 g) Color changes Freshness/spoilage
7.1 ± 0.3 Fresh
16.7 ± 0.2 Medium fresh
38.8 ± 0.5 Spoiled
CRediT authorship contribution statement
Li Yana: Conceptualization, Methodology, Software, Roles/Writing –
original draft, Writing – review & editing.
Wu Kaixuan: Data curation, Investigation, Formal analysis.
Wang Beihai: Visualization, Investigation, Resources.
Li Xuezhong: Supervision, Validation.
Acknowledgements
This research was financially supported by Research and Innovation
Initiatives of WHPU (2020Y08).
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