Food Hydrocolloids 89 (2019) 674–681

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Smart gelatin films prepared using red cabbage (Brassica oleracea L.) extracts T
as solvent
Yanina S. Musso, Pablo R. Salgado, Adriana N. Mauri*
Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA, CONICET-CCT La Plata and Facultad de Ciencias Exactas- Universidad Nacional de La
Plata), 47 y 116 S/N, B1900JJ, La Plata, Argentina

ARTICLE INFO ABSTRACT

Keywords: This work studied the preparation of smart edible films based on gelatin and anthocyanins. Films were prepared
Smart packaging by casting using aqueous or alcoholic extracts of red cabbage (Brassica oleracea L.) as solvent. The addition of
Anthocyanins these extracts to formulation provided films with important antioxidant properties (evaluated by ABTS.+and
Edible films FRAP assays) but not antimicrobial activity and the capacity to sense environmental pH changes through the
pH indicators
modification of its coloring. The response of these materials against pH changes was evaluated with buffers of
Gelatin
Food spoilage sensor
different pH (1–14) and simulating their contact with liquid and semisolid foods, and with a container headspace
Red cabbage at acid and alkaline pH. Alcoholic extracts were more effective than aqueous ones due to the higher antho-
cyanins concentration and variety, which exerted a plasticizing effect on the protein matrix. This effect was
observed in an increase in film's elongation and solubility and a decrease in its Young's modulus. On the other
hand, aqueous extracts seemed to favor the protein cross-linking, as they improved films' mechanical behavior.
These materials could be used as active and intelligent packaging of food products, especially those susceptible
to oxidation or whose deterioration is accompanied by pH changes.

1. Introduction effectiveness as smart packaging and its potential application area.

Nowadays there is a special interest in the development of smart
food packaging capable to extend products shelf-life improving safety
or sensory properties, whereas able to monitor by itself the condition of
packaged foods. In order to fulfill these requirements different additives
are incorporated into the materials formulation such as antioxidants,
antimicrobial, probiotics, colorants, flavors, and also others that at-
tempt to sense environmental changes or specific compounds generated
during food packaging or storage in order to inform the freshness or
microbiological quality of food (Biji, Ravishankar, Mohan, & Srinivasa
Gopal, 2015).
pH can be considered as a potential indicator of food spoilage as it
changes during food storage as a response to microorganisms' grow and
metabolism through the development of organic acids and/or volatile
amines. In previous works gelatin films containing synthetic acid-base
indicators such as methyl orange, neutral red and bromocresol green or
natural ones such as curcumin proved to be able to change their color
when being in contact with gaseous, liquid and semisolid media of
different pH (Musso, Salgado, & Mauri, 2016, 2017). The pH range in
which the indicator changes its color, as well as the chemical affinity
with the polymer matrix in which is incorporated would determine its
Food grade additives should be used if packaging would be in
contact with food (Janjarasskul & Krochta, 2010). Anthocyanins are
among food grade dyes sensible to pH changes. They represent a large
group of natural, water-soluble and nontoxic pigments (Janik,
Cozzolino, Dambergs, Cynkar, & Gishen, 2007) responsible for the red,
blue and purple colors of many fruits, vegetables and grains (Wrolstad,
Giusti, & Kalt, 2016). Recently, anthocyanins have attracted a great
deal of interest in the food and health industry due to their various
potential benefits attributed to their antioxidant activity (Awika,
Rooney, & Waniska, 2005; Wu, Gu, Prior, & McKay, 2004), such as
visual improvement, coronary heart disease, cancer, diabetes, cardio-
protective, antimutagenic, anti-inflammatory; and also antimicrobial
effects (De Pascual-Teresa & Sánchez-Ballesta, 2008). These properties
as well as their functional role as colorants placed anthocyanins as
potential agents in the production of products with high added value for
human consumption.
The use of natural extracts containing anthocyanins as solvent in the
preparation of food packaging films can be an interesting and eco-
nomical way to achieve new materials properties.
Red cabbage (Brassica Oleracea L.) extracts are rich sources of mono-
or diacylated cyanidin anthocyanins possessing strong antioxidant

*
Corresponding author.
E-mail addresses: anmauri@quimica.unlp.edu.ar, anmauri@hotmail.com (A.N. Mauri).

https://doi.org/10.1016/j.foodhyd.2018.11.036
Received 8 October 2018; Received in revised form 16 November 2018; Accepted 17 November 2018
Available online 22 November 2018
0268-005X/ © 2018 Elsevier Ltd. All rights reserved.
Y.S. Musso et al.
activity. Arapitsas, Sjöberg, and Turner (2008) and Pliszka, Huszcza-
Ciołkowska, Mieleszkoa, and Czaplicki (2009) reported the possibility
of finding between 9 and 24 different anthocyanins from red cabbage.
Due to this broad composition, its extracts can exhibit a wide spectrum
of color, ranging from orange through red to purple and blue based
upon the pH of the environment. These extracts were used to develop
pH indicator films prepared mainly by polysaccharides (Mendes Bento,
Pereira, Chaves, & Stefani, 2015; Pourjavaher, Almasi, Meshkini, Pirsa,
& Parandi, 2017; Silva-Pereira, Teixeira, Pereira-Junior, & Stefani,
2015). As far as we know, these extracts were not used to activate
protein films. Recently, Uranga, Etxabide, Guerrero, and de la Caba
(2018) used anthocyanins, extracted from red cabbage and lyophilized,
to prepare antioxidant fish gelatin films by compression molding.
The aim of this work was to develop gelatin films capable of mod-
ifying their color against medium pH changes using different red cab-
bage (Brassica Oleracea L.) extracts as solvent, and to evaluate the ac-
tivation of the resulting materials with antioxidant and antimicrobial
properties.
2. Materials and methods
2.1. Materials
Bovine gelatin with 240 Bloom (Kraft Foods, Argentina) was used as
protein source. Its protein content, as measured by the Kjeldahl method
(AOAC, 1995), was 87.8 ± 0.6% (w/w, dry weight; N × 5.5). Glycerol
(Anedra, Argentina) and red cabbage (Brassica Oleracea L.) obtained
from a local shop were used as films plasticizer and anthocyanins'
source respectively. All the other reagents used in this study were of
analytical grade.
2.2. Preparation of anthocyanin extracts
Two extracts were prepared from red cabbage using water and
ethanol as solvents. 50 g of red cabbage were cut and mixed in a
chopper (Moulinex, Argentina) with 50 ml of water or ethanol (96%).
Then, 50 ml of additional solvent were added to each extract and they
were stirred for half an hour at 300 rpm. Finally, they were first filtered
with a strainer to remove coarse particles and then with filter paper
(Whatman n° 1). These extracts were named as Aw and Ae depending
on whether the solvent used to extract anthocyanins was water (w) or
ethanol (e).
2.3. Red cabbage extracts characterization
2.3.1. Total anthocyanins concentration
The concentration of total anthocyanins in both extracts Aw and Ae
was determined using the pH difference method (Fuleki & Francis,
1968). Briefly, a 200 μL aliquot of anthocyanin extract was mixed with
7 ml of buffer at pH 1.0 (0.025 M potassium chloride adjusted with
hydrochloric acid) and another with 7 ml of a buffer at pH 4.5 (acetate
of sodium 0.4 M, adjusted with acetic acid). The absorbance difference
at 530 nm (maximum absorption wavelength) of both buffers is pro-
portional to the anthocyanin content (Wrolstad et al., 2016). Mea-
surements were performed on a UV-VIS plate reader (SYNERGY
HT–SIAFRT, Biotek Instruments, USA) using distilled water as blank.
Corrections due to the presence of degraded compounds or interfering
substances were done measuring the absorbance at 700 nm. Taking into
account that anthocyanins of red cabbage are derived from cyanidin
glucoside, concentrations were expressed as mg cyanidin-3-glucoside
per 100 mL extract and calculated according to equations (1) and (2)
(Hrazdina, Iredale, & Mattick, 1977):
A. MW average . FD . 1000
Anthocyanin concentration =
.L (1)
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Food Hydrocolloids 89 (2019) 674–681
A = (A530 A700 ) pH : 1,0 (A530 A700 )pH: 4,5 (2)
Where ΔA is the difference among absorbance change at 530 and
700 nm in buffers at pH 1 and 4.5; MW average is the molecular mass for
cyanidin-3-glucoside (449.2 g/mol); FD is the dilution factor; Ɛ is the
molar extinction coefficient for cyanidin-3-glucoside
(26900 cm−1.M−1); L is the optical path (cm); and 1000 is the con-
version factor of grams to milligrams. Measurements were performed in
triplicate.
2.3.2. Anthocyanins profile
Anthocyanins extracts (Aw and Ae) were filtered through a syringe
with a 0.45 μm filter (Millipore Corporation, Bedford, MA, USA) and
analyzed by high performance liquid chromatography (HPLC) using a
Waters Model 6000A LC system (Milford, MA, USA) equipped with a
diode array detector (Waters 2998). A 250 × 4.6 mm i.d. C18 column
(Phenomenex, CA, USA) was used for separation. Elution was per-
formed at 40 °C using mobile phase A (5% formic acid aqueous solu-
tion) and mobile phase B (methanol). The elution gradient used was:
0–2 min: 5% B; 2–10min: 5–20% B; 10–15 min: 20% B; 15–40 min:
20–30% B; 40–45 min: 5% B (Wu et al., 2004; Zaro, Keunchkarian,
Chaves, Vicente, & Concellón, 2014). The flow rate was 0.5 mL/min
and detection was performed at 520 nm. Measurements were per-
formed in duplicate.
2.3.3. UV–visible absorption spectra
Anthocyanins extracts (Aw and Ae) were diluted (1:2) using the
corresponding solvent, water or ethanol respectively. The UV–Vis ab-
sorption spectra of these dilutions were recorded from 200 to 700 nm
(SYNERGY HT–SIAFRT, Biotek Instruments, USA). Triplicates were
made for each extract.
2.4. Preparation of films
5 g of gelatin were dispersed in 50 mL of distilled water at 100 °C
under magnetic stirring, and then mixed with 1.25 g of glycerol (Musso,
Salgado, & Mauri, 2017) and 50 ml of each extract, aqueous (Aw) or
alcoholic (Ae) at room temperature. pH of each dispersion was adjusted
to 6 with 2 mol/L NaOH and stirred during additional 30 m. Finally,
10 mL of aqueous and hydroalcoholic film-forming dispersions, GAw y
GAe respectively, were cast onto polystyrene Petri dishes (64 cm2) and
dried in an oven with air flow circulation (Yamato, DKN600, USA) at
60 °C for 3 h. The resulting films were preconditioned during 48 h at
20 °C and 58% relative humidity (in desiccators with saturated solu-
tions of NaBr) just before being peeled from the casting surface and
characterized. Furthermore, control gelatin films (Gw and Ge) were
prepared as described previously adding water or ethanol instead of red
cabbage extracts respectively. Two independent batches for each type
of gelatin film were performed.
2.5. Physicochemical properties of films
2.5.1. Thickness
Film thickness was measured with a digital coating thickness gauge
(Check Line DCN-900, USA). Measurements were done at five positions
along the rectangular strips for the tensile test, and at the center and at
eight positions round the perimeter for the water vapor permeability
(WVP) determinations. The mechanical properties and WVP were cal-
culated using the average thickness for each film replicate.
2.5.2. Moisture content (MC)
Small specimens of films were collected after conditioning, cut and
weighed before and after oven drying at 105 °C for 24 h according to
ASTM D644-99 (ASTM, 2004). MC values were determined in triplicate
for each film, and calculated as the percentage of weight loss relative to
the original weight.
Y.S. Musso et al.
2.5.3. Water solubility (WS)
WS was determined as was described by Gontard, Duchez, Cuq, and
Guilbert (1994) with slight modifications. Three pieces of films (dia-
meter = 2 cm; ≈ 0.03–0.05 g) were weighed and immersed in 50 mL of
distilled water. The system was sealed, shaken at 100 rpm for 24 h at
20 °C (Ferca, TT400 model, Argentina), and then filtered through
Whatman n°1 filter paper (previously dried and weighed) to recover the
remaining undissolved film, which was desiccated at 105 °C for 24 h.
WS was calculated as follows:
WS = [(P0. (100 – MC)) – Pf]. 100 / [P0. (100 – MC)] (3)
Where P0 = initial film weight (g), Pf = final dry film weight (g),
MC = moisture content (%). All tests were carried out in triplicate.
2.5.4. Water vapor permeability (WVP)
Water vapor permeability tests were conducted according to ASTM
method E96-00 (ASTM, 2004) with some modifications. Each film
sample was sealed over a circular opening of 0.00185 m2 in a per-
meation cell that was stored at 20 °C in desiccators. To maintain a 75%
relative humidity (RH) gradient across the film, anhydrous silica (0%
RHc) was placed inside the cell and a saturated NaCl solution (75%
RHd) was used in the desiccators. The RH inside the cell was always
lower than outside, and water vapor transport was determined from the
weight gain of the permeation cell. When steady-state conditions were
reached (about 1 h), eight weight measurements were made over 5 h.
Changes in the weight of the cell were recorded and plotted as a
function of time. The slope of each curve (Δm/Δt, g H2O s−1) was ob-
tained by linear regression and the water vapor transmission rate
(WVTR) was calculated from the slope divided by the permeation cell
area (A, in m2). WVP (g H2O Pa−1 s−1 m−1) was calculated as:
WVP = [WVTR/(PVH 2O (RHd RHc ))] d (4)
Where: WVTR = water vapor transmission rate (g H2O s −1
m ), −2
PVH2O = saturation water vapor pressure at test temperature
(2339.27 Pa at 20 °C), RHd - RHc = relative humidity gradient across
the film -expressed as a fraction- (0.75), A = permeation area (m2), and
d = film thickness (m). Each WVP value represents the mean value of
three samples taken from different films.
2.5.5. Mechanical properties
Tensile tests were carried out using a texture analyzer TA.XT2i
(Stable Micro Systems, Surrey, England) equipped with a tension grip
system A/TG following the procedures outlined in the ASTM method
D882-02 (ASTM, 2004). Films probes of 90 mm length and 6 mm width
were used. The initial grip separation was set at 50 mm and the
crosshead speed at 0.4 mm s−1. Measurements were performed at 20 °C
in a temperature-controlled room. The curves of force (N) as a function
of distance (mm) were recorded by the Texture Expert V.1.15 software
(Stable Micro Systems, Surrey, England). Tensile properties were cal-
culated from the plot of stress (tensile force/initial cross-sectional area)
versus strain (ε, elongation as a percentile of the original length).
Tensile strength (TS) and elongation at break (εb) were determined
directly from the stress-strain curves, and the Young's modulus (YM)
was determined as the slope of the initial linear portion of this curve. At
least twelve replications taken from different films for each formulation
were performed.
2.6. Antioxidant and antimicrobial properties of films
2.6.1. Antioxidant capacity of films
The antioxidant activity of the supernatants obtained in a solubility
test (2.5.3) of the resulting films were characterized by its radical
scavenging ability (ABTS assay) and its ferric ion reducing capacity
(FRAP assay). The ABTS.+ radical (2,2′-Azino-bis(3-ethylbenzothiazo-
line-6-sulfonic acid)) scavenging capacity was determined according to
676
Food Hydrocolloids 89 (2019) 674–681
a modified version of the method of Re et al. (1999). The stock solution
of ABTS radical, consisted of 7 mM ABTS (Sigma-Aldrich) in potassium
persulfate 2.45 mM (Sigma-Aldrich), was kept in the dark at room
temperature for 12–16 h. An aliquot of the stock solution was diluted
with distilled water in order to prepare the working solution of ABTS.+
radical with absorbance at 734 nm of 0.70 ± 0.02 (SYNERGY HT-
SIAFRT, Biotek Instruments, USA). A 50 μL aliquot of the samples was
mixed with 950 μL of ABTS reagent. The mixture was then left to stand
at 30 °C for 10 min and the absorbance values were read at 734 nm.
Results were expressed as milligrams of ascorbic acid per gram of
protein film based on a standard curve of vitamin C (Sigma-Aldrich),
which relates the concentration of vitamin C to the amount of absor-
bance reduction caused by vitamin C. All determinations were per-
formed at least in triplicate.
FRAP (ferric-reducing ability of plasma) is a measure of the redu-
cing ability of samples and was performed according to the method
described by Pulido, Bravo, and Saura-Calixto (2000). Samples (30 μL)
were incubated at 37 °C with 90 μL of distilled water and 900 μL of
FRAP reagent (containing 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ, Sigma-
Aldrich) and FeCl3) in sodium acetate buffer pH 3.6. Absorbance values
were read at 595 nm after 30 min (SYNERGY HT-SIAFRT, Biotek In-
struments, USA). Results were expressed as mmol FeSO4.7H2O
equivalents/g of protein film based on a standard curve which relates
its concentration to the absorbance at 595 nm. All determinations were
carried out in duplicate.
2.6.2. Antimicrobial properties of films
The antimicrobial activity of films was determined by the agar
diffusion method against Salmonella enteritidis, Escherichia coli, Bacillus
cereus, and Staphylococcus aureus, selected by its importance for being
responsible for food spoilage. Briefly, gelatin films were aseptically cut
into 10 mm diameter disks and laid onto the surface of nutrient agar
(Biokar diagnostics) previously inoculated with 100 μL of each micro-
organism (containing 108 CFU/mL). Plates were incubated at 37 °C for
48 h. After incubation, the inhibition (clear) area —considered the
antimicrobial activity— was measured with a gauge. Results were ex-
pressed as percentage of growth inhibition respecting to the total plate
surface. Tests were done in duplicate.
2.7. Films’ response to pH changes
The response of the resulting films to pH changes were evaluated by
two assays. First, droplets of buffers at different pH 1–14 were placed
on GAw and GAe films. Phosphate buffers were used to cover pH from 1
to 7 and borate buffers to cover pH from 8 to 14; all of them with
0.15 M ionic strength (Ross, 1991). The responses of the materials to
the pH changes were recorded by means of photographs taken with a
digital camera (Kodak M853, USA).
Next, each film was faced with liquid, semisolid and gaseous media
of different pH: i) adding a drop of 2 mol/L HCl or 2 mol/L NaOH
directly on films; ii) placing the films in contact with gels prepared from
gelatin solutions at 7.5% w/v at pH = 2.5, and 11; and iii) exposing the
films to gaseous atmospheres generated by acetic acid glacial (C2H4O2,
pKã4.8, Anedra, Argentina) and ammonia (NH3, pKã9.3, Anedra,
Argentina) (Musso et al., 2016). Photographs of films before and after
(30 min) contacting it with those media of different pH were taken with
a digital camera (Kodak M853, USA).
2.8. Statistical analysis
Results were expressed as mean ± standard deviation and were
analyzed by analysis of variance (ANOVA). Means were tested with the
Tukey's HSD (Honestly Significant Difference) test for paired compar-
ison, with a significance level α = 0.05, using the Statgraphics Plus
version 5.1 software (Statgraphics, USA).
Y.S. Musso et al.
3. Results and discussion
3.1. Characterization of aqueous and alcoholic extracts of red cabbage
(Brassica Oleracea L.)
Aqueous and alcoholic extracts of red cabbage (Aw and Ae) showed
a final pH of 6,0 ± 0,1 and 5,7 ± 0,2 respectively, and were trans-
lucent and colored. Their appearance is shown in Fig. 1. The aqueous
extract had a pink tone while the alcoholic one had a more violaceous
coloration. The UV–Visible spectrum of both extracts is also shown in
Fig. 1. Peaks at 205, 230, 245 and 325 nm are observed in both sam-
ples. But while Aw showed a higher absorbance near the red zone,
between 550 and 700 nm; Ae showed a higher absorbance in the ul-
traviolet zone, between 300 and 400 nm. In Aw spectra, a peak at
265 nm and another at 600 nm are also observed while in that corre-
sponding to Ae two other peaks at 275 and 540 nm became more no-
ticeable and the absorbance of that at 325 nm increased significantly.
These differences should be attributed to different anthocyanins con-
centration and composition. Colors are related with anthocyanins
structure (Ikan, 1991) and with the number and orientation of the
hydroxyl and methoxy groups of the molecule, as well as the glycosidic
and acylation substitution (Stintzing, Carle, Frei, & Wrolstad, 2002).
Ikan (1991) reported that the violet coloration of anthocyanins at
neutral pH is influenced by the number of hydroxyl groups, that the
orange-red color is attributed mainly to pelargonidin, the intense red to
cyanidin and the bluish to the derivatives of delphinidin.
Total anthocyanins concentration in the alcoholic extract
(4,0 ± 0,4 mg/L) was almost seven times higher than that corre-
sponding to the aqueous one (0,6 ± 0,1 mg/L), demonstrating a higher
extractive efficiency of ethanol compared with water, which was cor-
responded with an increase in the tone of the solution. Similar results
were reported by Chandrasekhar, Madhusudhan, and Raghavarao
(2012) when extracting anthocyanins from red cabbage in different
solvents –ethanol, methanol, water and their mixtures– at different pH.
Fig. 2 shows the chromatograms obtained by HPLC from both red
cabbage extracts. The one obtained using water as solvent (Aw) pre-
sented a single peak at a retention time of 29.3 min while the alcoholic
one (Ae) showed two peaks, the first one at 26.9 min and the second at a
similar retention time (29.6 min) than that of Aw. Taking into account
the separation characteristics of the column used, both peaks could be
attributed to different forms of anthocyanins likely derived from cya-
nidins (Wu & Prior, 2005). The areas ratio of the chromatograms for
both extracts (3:1) confirmed the greater effectiveness of ethanol ex-
traction, which apparently, in addition to extracting the anthocyanins
present in the aqueous extract, would be extracting other forms present
in the red cabbage. Ahmadiani, Robbins, Collins, and Giusti (2014)
analyzed extracts of seven varieties of red cabbage extracted with an
acetone/chloroform mixture by HPLC assays with a gradient of formic
acid (4.5% w/v) and acetonitrile, and identified derivatives of cya-
niding-3-O-diglucoside-5-O-glucoside, which can be non-acylated,
mono-acylated or di-acylated with p-coumaric, caffeic, ferulic and
Fig. 1. Visual appearance and UV–Visible spectrum of aqueous and alcoholic
anthocyanins extracts (Aw and Ae, respectively) of red cabbage: ─ Aw, ─ Ae.
677
Food Hydrocolloids 89 (2019) 674–681
sinapic acids, according with works previously published by Wu et al.
(2004). Arapitsas et al. (2008) also found mono- or di-glycosylated
cyanidins.
3.2. Films'appearance
Fig. 3 shows films' photographs. Films prepared or not with red
cabbage extracts as solvent were transparent, and easily removed from
the plates and manipulated. Although the filmogenic dispersions con-
taining anthocyanins showed an intense coloration, this color was no
longer observed in the films obtained after the drying process at 60 °C.
This characteristic of anthocyanins was reported by other authors.
Some pigments present in anthocianins extracts lose their coloration
when being exposed to light or temperatures higher than 30 °C
(Alighourchi & Barzegar, 2009; Cevallos-Casals & Cisneros-Zevallos,
2004; Shaked-Sachray, Weiss, Reuveni, Nissim-Levi, & Oren-Shamir,
2002). Wiczkowski, Szawara-Nowak, and Topolska (2015) showed a
decrease in the content of all the anthocyanins analyzed in red cabbage
extracts after being submitted to fermentation and heating processes,
and stated that this effect was dependent on the chemical structure of
each compounds. Silva-Pereira et al. (2015) took advantage of this
behavior and developed chitosan and polyvinyl alcohol films added
with red cabbage anthocyanins capable of responding to temperature
changes.
3.3. Films response to pH changes
Fig. 4 shows response of gelatin films prepared using red cabbage
extracts as solvent (pH 6) after being in contact with buffers of different
pH: 1–14. Films became colored with buffers at extreme pH: pink with
those at pH < 4 or yellow for those at pH > 11. These color changes
could be clearly seen with films prepared with the alcoholic extract Ae
(GAe) due to their higher concentration of anthocyanins and/or to a
different response as the result of a different anthocyanins composition.
Fig. 5 shows the response of gelatin films added with anthocyanins
from both extracts, GAw and GAe, after being in contact with liquids,
semisolids and gases of different pH, used as simulants of liquid or
semisolid food, or the headspace of a food container. Films' coloration
changed from transparent to pink after being in contact with acidic
media (liquid, gaseous and semisolid), whereas it changed to yellow in
contact with alkaline liquids and to green with alkaline gaseous and
semisolid media. These differences in the color acquired in contact with
alkaline media could be attributed to the pH value reached. In this
sense, Yoshida, Maciel, Mendonça, and Franco (2014) reported that at
pH above 11 anthocyanin coloration turns yellow while at lower pH
values the color turns greenish. Furthermore it should be noted that all
the observed changes were reversible, according to previous studies
(Musso et al., 2016 y 2017).
Although both films showed the ability to indicate pH changes
through their color change, these changes were very tenuous for films
prepared with aqueous extracts (GAw) and most significant for those
prepared with anthocyanins ethanolic extracts (GAe).
3.4. Films' physicochemical properties
Table 1 shows films' thickness, moisture content, water solubility
and water vapor permeability.
Thickness was unaffected by the presence of anthocyanins in the
formulation, since no significant differences (p > 0.05) were observed
by replacing each solvent with its respective red cabbage extract. But
films prepared from aqueous dispersions were thinner than those from
hydroalcoholic ones (p < 0.05). Similar results were observed pre-
viously (Musso et al., 2017) working with gelatin films activated with
curcumin prepared from hidroalcoholic and aqueous film-forming dis-
persions. Ethanol can denature proteins by disrupting the side-chain
intramolecular hydrogen bonding, and allowing the formation of new
Y.S. Musso et al.
Absorbance
(520 nm)
15 20
Fig. 2. HPLC chromatograms of aqueous and alcoholic anthocyanins extracts (Aw and Ae, respectively) of red cabbage: ─ Aw, ─ Ae.
Fig. 3. Appearance of gelatin-based films (G) and those with anthocyanin ex-
tract added (GA) obtained with different solvents –water (w) and an ethanol-
water mixture (e)– at pH = 6.
Fig. 4. Response of gelatin films prepared using aqueous or alcoholic red
cabbage extracts as solvent (GAw and GAe respectively) after being in contact
with buffers of different pH: 1–14. The arrows indicate the buffer pH at which
films coloration change.
hydrogen bonds between alcohol molecules and protein side chains.
Protein conformation in film-forming dispersions affects the physico-
chemical properties of films (Salgado et al., 2017). It is evident that
gelatin dispersed in ethanol-water mixtures produced films with a lower
degree of compaction, suggesting different chain molecular unfolding
or cross-linking within the protein network of the film (Denavi et al.,
2009).
Moisture content of gelatin films decreased to a similar value
(≈18%) when using ethanol as solvent (p < 0.05) or by incorporating
the compounds present in the aqueous extract of red cabbage to the
formulation (p < 0.05); while their water solubility was only modified
in films prepared with alcoholic extract (GAe), increasing it by at least
58% (p < 0.05). Menawhile, WVP was unaffected by the presence of
anthocyanins in the formulation (p > 0.05), but were higher for films
prepared from hydroalcoholic dispersions that showed a higher thick-
ness (p < 0.05). The increase of WVP with thickness is a characteristic
of hydrophilic films (McHugh, Avena-Bustillos, & Krochta, 1993). It
should be noted that WVP values of the studied films are in the range of
those reported for good protein films, although this property is the
greatest weakness of natural biopolymer based materials respect to
synthetic polymers widely used in packaging, such as LDPE and HDPE
(WVP ≈ 10−13 gH2O s−1m−1Pa−1) (Garavand, Rouhi, Razavi,
Food Hydrocolloids 89 (2019) 674–681
25 30 35 40
Time (min)
Fig. 5. Response of gelatin films prepared using aqueous or alcoholic red
cabbage extracts as solvent (GAw and GAe respectively) after being in contact
with liquid, gaseous and semisolid media of different pH.
Cacciotti, & Mohammadi, 2017; Suderman, Isa, & Sarbon, 2018;
Wihodo & Moraru, 2013). So regarding these properties related to film's
susceptibility to water, the addition of anthocyanins extracted in water
only caused a slight decrease in the water content of the films, while
those extracted in ethanol only caused an increase in the solubility,
suggesting a lower cross-linking or an interference in protein cross-
linking.
Fig. 6 shows films' stress-strain curves obtained by mechanical
tensile tests. Gelatin films prepared from aqueous dispersions (Gw)
showed interesting mechanical properties considering that they were
protein materials (TS ≈ 3 MPa, εb ≈ 160% and YM ≈ 0, 15 MPa)
(Gennadios, 2002). Those prepared from hydroalcoholic dispersions
(Ge) showed lower values of Young's modulus (≈57%) (p < 0.05), but
similar tensile strength and elongation at break than those prepared
using water as solvent (Gw) (p > 0.05). This decrease in YM suggested
a lower cross-linking, probably due to a different initial protein con-
formation, that would also be responsible of the lower degree of
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Y.S. Musso et al. Food Hydrocolloids 89 (2019) 674–681

Table 1
Thickness, moisture content (MC), water solubility (WS) and water vapor permeability (WVP) of gelatin-based films (G) and those with anthocyanin extract added
(GA) obtained with different solvents –water (w) and an ethanol-water mixture (e)– at pH = 6.
Film Gw GAw Ge GAe

Thickness (μm) 49.7 ± 4.4a 51.3 ± 4.6a 60.1 ± 6.2b 55.5 ± 5.3ab
MC (%) 21.8 ± 0.5b 18.7 ± 1.1a 17.2 ± 0.6a 19.1 ± 1.0a
WS (%) 39.2 ± 4.3a 37.2 ± 2.6a 35.6 ± 0.6a 62.0 ± 3.7b
WVP. 1011 (gH2O s−1m−1Pa−1) 6.8 ± 0.4a 6.5 ± 0.6a 11.1 ± 3.4b 12.0 ± 1.0b

Reported values for each gelatin film are means ± standard deviation (n = 9 for thickness, n = 3 for MC, WS and WVP). Different letters in the same row indicate
significant differences between samples (p < 0.05), according to Tukey's test.

compaction reflected in films thickness. Similar results were obtained
previously when replacing water by ethanol-water mixtures in film
forming dispersions of the same gelatin (Musso et al., 2017).
Mechanical properties were affected differently when replacing part
of the solvent by both red cabbage anthocyanins extracts. Films added
with aqueous extracts (GAw) showed higher tensile strength and
Young's modulus (152% and 80% higher) (p < 0.05) and similar
elongation at break (p > 0.05) than Gw; while those added with the
alcoholic extract (GAe) showed lower Young modulus (88%), higher
elongation (16%) and similar tensile strength than Ge. It is evident that
the different composition of anthocyanins in both extracts (observed by
HPLC analysis), led to an inverse effect on the mechanical properties.
Anthocyanins in the aqueous extracts would favor protein cross-linking,
as their presence improved the mechanical strength of films. This effect
was reported by other authors when adding anthocyanins and other
polyphenols present in green tea, Centella asiatica (L.) urban, pome-
granate and pine bark extracts containing polyphenolic compounds
such as oligomeric procyanidins to film-forming dispersions of gelatin,
zein and soybean proteins (Han, Yu, & Wang, 2018; Mushtaq, Gani,
Gani, Punoo, & Masoodi, 2018; Rasid, Nazmi, Isa, & Sarbon, 2018; Wu
et al., 2013) respectively, mainly as the result of hydrogen bonds among
these active compounds and protein matrixes. Meanwhile, alcoholic
extracts of red cabbage would be plasticizing the protein matrix, an
effect that would also explain the increase in water solubility of these
materials. Taking into account that this extract showed two peaks in the
HPLC chromatogram, one of them similar to the only one in the chro-
matogram of aqueous extract, the species present in the other peak,
which eluted at shorter times would be responsible for the net behavior
observed. Probably these less polar species were less affine with gelatin,
and instead of improving the cross-linking among polypeptide chains,
they interfered with it, leading to less resistant, more elongated and
more water soluble films. Bodini, Sobral, Favaro-Trindade, and
Carvalho (2013) reported a similar effect working with alcoholic pro-
polis extract in gelatin films; and Musso et al. (2017) observed that
gelatin films activated with curcumin and prepared from hydroalco-
holic film forming dispersions showed lower tensile strength and higher
water solubility than similar films without curcumin addition.
8 for 3 h carried out to their preparation, that made films became almost
6 serve to indicate some change in the quality of the foods associated with
4 foods whose deterioration occurs through oxidation reactions.
2 red cabbage anthocyanins (extracted with different solvents) against
0 S. Typhymurium and M. Luteus. S. Aureus, P. Aeruginosa, and K.
0 50 100 150 200
Fig. 6. Stress (σ)–strain (ε) curves of gelatin-based films (G) and those with
anthocyanin extract added (GA) obtained with water (w) or an ethanol-water
mixture (e) as solvents at pH = 6 (— Gw, - - GAw, —Ge, and - - GAe).
3.5. Films' antioxidant and antimicrobial properties
Fig. 7 shows the antioxidant properties of studied gelatin films as
assessed by different methods: ABTS•+ (A and B panels) and FRAP (C
and D panels). Gelatin films prepared from aqueous film forming dis-
persions (Gw) exhibited a low antioxidant capacity in both methods
tested, that could be attributed in part to some gelatin amino acids that
can act as electron donors, reacting with free radicals to give rise to
more stable products in ABTS assay or reducing ferric ion in FRAP assay
(Salgado, López-Caballero, Gómez-Guillén, Mauri, & Montero, 2012).
Gelatin films prepared from hydroalcoholic dispersions (Ge) showed a
significant higher antioxidant activity than Gw These differences could
be attribute, at least in part, to differences in protein conformation and
cross-linking, discussed above (Section3.4) according to Musso et al.
(2017).
The addition of red cabbage extracts to the formulation increased
the antioxidant properties of resulting films significantly (p < 0.05).
But although these increments were significantly higher for GAw than
for GAe when compared with the corresponding films without antho-
cyanins (Gw and Ge respectively) by both methods evaluated, the an-
tioxidant properties of GAe were significantly higher than that of GAw.
This should be attributed to the higher content and varieties of an-
thocyanins in Ae (Section 3.1), and to the lower cross-linking of their
corresponding films, since it is worth pointing out that GAe films were
the ones that showed the greatest solubility and elongation along with
the lower Young's module. These differences in protein cross-linking,
and the changes in the hydrophilic-hydrophobic nature of the protein
matrix, should modify the retention or release of active principles af-
fecting the antioxidant properties of the resulting films.
Antioxidant activity of anthocyanins varies enormously in the lit-
erature (Singh et al., 2006). Results obtained in this work can be
compared with those analyzed by Jacob, Mahal, Mukherjee, and
Kapoor (2011) who evaluated the antioxidant properties of green and
red cabbage extracts obtained by different procedures. These authors
reported that antioxidant properties of Brassica plants were dependent
on several factors such as the variety, the maturity at harvest, and on
the state and conservation conditions of soil after harvest. But it should
be noted that films prepared with both anthocyanins extracts showed
an important antioxidant capacity despite the heating process at 60 °C
colorless. Therefore, beyond the fact that the developed films could
the pH changes, they could also function as an active container for
Finally, although it has been reported the antimicrobial activity of
different bacterial strains such as E. Coli, B. Subtilis, V. Parahaemolyticus,
Pneumoniae; and also antifungal activity against T. Rubrum and A.
Terreucomo (Boo et al., 2012; Hafidh et al., 2011); films studied in this
work did not show activity against S. Enteritidis, E. coli, B. cereus, and S.
Aureus under the tested conditions. This could be probably attributed,
at least partly, to the low concentration of anthocyanins in Aw and Ae
extracts, as antifungal and antimicrobial properties reported by Hafidh

679
Y.S. Musso et al. Food Hydrocolloids 89 (2019) 674–681

A) B)
0.16 18

15
0.12

(mg/ml/g film)
(mg/ml/g film)

Ascorbic acid
Ascorbic acid
12

0.08 9

6
0.04
3
0 0
Gw GAw Ge GAe
C) D)
150 1500

1250

FeSO4 (mM/ g film)

120
FeSO4 (mM/ g film)

1000
90
750
60
500
30 250

0 0
Gw GAw Ge GAe

Fig. 7. Antioxidant properties of gelatin-based films (G) and those with anthocyanin extract added (GA) obtained with water (w, A and C panels) or an ethanol-water
mixture (e, B and D panels) as solvents at pH = 6, measured by ABTS.+ (A and B panels) and FRAP (C and D panels) assays.

et al. (2011) were found with extracts containing 700 mg/ml of an-
thocyanins, a significant higher concentration than the one of film
forming dispersions used in this work. Beyond that strain character-
istics, or specific interactions among anthocyanins and gelatin in the
film matrix could be also the cause of the lack of antimicrobial activity.
4. Conclusions
Aqueous and alcoholic extracts of red cabbage, rich in anthocyanins,
were used as a solvent for the preparation of edible films based on
gelatin. The resulting materials showed important antioxidant proper-
ties and the ability to modify their coloration against changes in the pH
of the surrounding medium. Both behaviors were more significant with
films prepared with the alcoholic extract, which presented a greater
concentration and variety of anthocyanins that ended exerting a plas-
ticizing effect in the protein matrix, unlike those present in the aqueous
extract that promoted protein cross-linking and improved their me-
chanical properties.
These materials could be used as active and intelligent packaging of
food products, especially those susceptible to oxidation or whose de-
terioration is accompanied by pH changes.
Acknowledgements
The authors are thankful to the National Agency of Scientific and
Technological Support of Argentina (PICT-2010-1837, PICT-2013-
2124) and the National University of La Plata (11/X618 and 11/X750)
for their financial support.
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