Development of Active Films From Pectin and

Fruit Extracts: Light Protection, Antioxidant

Capacity, and Compounds Stability
Kaliana S. Eça, Mariana T. C. Machado, Miriam D. Hubinger, and Florencia C. Menegalli

C: Food Chemistry
Abstract: Pectin films containing fruit extracts were developed and tested in relation to ultraviolet light transmission,
phytochemical contents, and antioxidant capacity during 90 d shelf life storage. Aqueous and alcoholic extracts from 5
different fruits (acerola, cashew apple, papaya, pequi, and strawberry) were obtained. Because the alcoholic extracts from
acerola, cashew apple, and strawberry presented the highest phytochemical content and antioxidant capacity, they were
incorporated into pectin films individually or as a mixture. Incorporation of these extracts into pectin films provided
antioxidant capacity while retaining the physical properties. The pectin films containing fruit extract acted as adequate
light barrier and prevented photooxidation. Among the prepared films, the pectin film containing acerola extract afforded
the highest antioxidant capacity, with a half-life of 99 d. Overall, the results revealed that incorporation of fruit extracts
into pectin films potentially produces antioxidant films and coatings for different food applications.

Keywords: antioxidant capacity, extraction, films, phytochemicals

Practical Application: The production of pectin films incorporated with fruit extract is based on combination of the
antioxidant activity, natural color, and optical barrier properties from fruit phytochemical components to the active film.
This film could be potentially used as active packing on food products in order to protect their nutrients against free
radicals action and photooxidation and, hence, preserve the quality, integrity, and safety of food during the storage period.

Introduction
Pectin films containing natural extracts
The development of films containing biodegradable polymers
combined with natural additives is a promising technology
in the food industry. Cellulose, starch, chitosan, carrageenan,
alginate, arabic gum, and pectin are the most often used
biodegradable components in film matrixes. Pectin-based films
are inexpensive, and their renewability and sustainability render
them “green-packaging” materials. In addition, the FDA regards
pectin as generally safe (GRAS), so this polymer can afford
low-calorie edible films and coatings (FDA 2013). Pectin films
and coatings display crystalline and/or amorphous regions, which
are suitable for incorporation of phytochemical compounds and
for immobilization of water in the film structure, to enable
hydrophilic compound retention ( De’Nobili and others 2013).
In fact, incorporating natural extracts, vitamins, colorings, flavors,
and spices into pectin films and coatings could provide them with
antimicrobial, nutritive, and antioxidant properties (Oms-Oliu
and other 2008; Bierhalz and others 2012; Pérez and others 2012;
De’Nobili and others 2013). These so-called active films preserve
the quality, integrity, and safety of food products during storage.
Along time, the oxidation of organic molecules causes several
undesirable changes in food properties, such as decreased nutri-
tional quality and altered taste. In turn, antioxidants can scav-
enge free radicals, decompose peroxides, quench singlet oxygen,
and inhibit enzymes, thereby preventing the deleterious reactions
MS 20150583 Submitted 8/4/2015, Accepted 8/16/2015. Authors are with Dept.
of Food Engineering, School of Food Engineering, Univ. of Campinas, Campinas, SP,
Brazil. Direct inquiries to author Eça (E-mail: kaliana.se@gmail.com).
C 2015 Institute of Food Technologists
 R
of nutrients and extending product shelf life (Huang and others
2005).
The most important natural sources of antioxidants are fruits and
vegetables. They contain high levels of phytochemical compounds
such as vitamins and secondary metabolites (phenolic compounds,
carotenoids, sterols, glucosinolates, and saponins), known to act as
free radical scavengers (Bravo 1998; Skibsted 2012). Fruits offer
the advantage of synergism, that is, the combined action of its
constituents, which enhances their antioxidant capacity (Righetto
and others 2005). Indeed, studies have indicated that ascorbic acid,
phenolic compounds, and carotenoids can act together against ox-
idative stress in food. Moreover, these compounds can regenerate
each other via different reaction mechanisms (Dai and others 2008;
Skibsted 2012).
Another strategy to prevent oxidative processes in films is to use
light absorbers. Antioxidant compounds in fruits could enhance
the film light barrier properties (Akhtar and others 2010) because
these compounds can absorb the most harmful light wavelength
(ultraviolet radiation) and thus reduce photooxidation (Li and oth-
ers 2014). In this context, evaluating fruit extracts as film-forming
ingredients is an interesting topic. This strategy would allow for
combination of the antioxidant activity, natural color, and optical
barrier properties of the fruit phytochemical components. Study-
ing the stability of fruit extracts bioactive compounds in the film
is also important to evaluate the filmogenic matrix ability to pro-
mote the sustained release of these compounds and to maintain
their antioxidant capacity.
In this scenario, this work had 2 major objectives: (1) to obtain
aqueous (WE) and alcoholic (AE) extracts from different fruits,
characterize the antioxidant capacity of each sample, and quan-
tify the major bioactive compounds in the extracts; and (2) to
investigate how the addition of fruit extracts with higher an-
tioxidant capacity to pectin-based films affects film properties.

doi: 10.1111/1750-3841.13074 Vol. 80, Nr. 11, 2015 r Journal of Food Science C2389
Further reproduction without permission is prohibited
 Development of active films from pectin and fruit extracts . . .
Characterization included determination of the mechanical, phys-
ical, and chemical properties and of the antioxidant stability of the
films.
Material and Methods
Extract: preparation and characterization
C: Food Chemistry
Fruit extraction. Acerola (Malpighia punicifolia L.), cashew
apple (Anacardium occidentale L.), papaya (Carica Papaya L.), pe-
qui (Caryocar brasiliense Camb.), and strawberry (Fragaria vesca)
were purchased from CEASA (Central Supply S.A., Campinas,
SP, Brazil). The fruits were selected on the basis of size uniformity,
maturity stage (based on skin color and firmness), and absence
of physical damage. Extraction was conducted according to the
method described by Roesler and others (2007), with some mod-
ifications. Briefly, with the aid of a mixer, the fruits were ground
and homogenized with the solvent (distilled water or absolute
ethanol:water 95:5 v:v) at 20000 rpm for 2 min; a sample:solvent
1:2 ratio (w:w) was used. The resulting mixture was filtered, and a
strainer was used to remove rough particles. Then, the residue was
reextracted in the same conditions, to give maximum compounds
recovery from the fruit material. The combined extracts (from the
first and second extraction, 400 mL) were concentrated to a final
volume of 150 mL in a rotary evaporator (40 °C and 700 mm Hg).
The entire procedure was performed in the absence of light. The
concentrate was considered the final product and was subjected to
chemical analysis.
Total carotenoids determination. The total carotenoids
content of the fruit extracts was determined according to the
method described by Rodriguez-Amaya (1999). The carotenoids
were extracted with acetone, separated in petroleum ether, diluted
in a volumetric flask, and analyzed on a spectrophotometer (model
SQ-2800 UV/VIS, UNICO, United Products & Instruments Inc,
N.J., U.S.A.) at a wavelength of 450 nm. Equation (1) was used
for quantification:
Abs × V × 106
Total − carotenoids = , (1)
A1%
1cm × m × 100
where Total_carotenoids is expressed as µg of β-carotene/g of dry
extract, Abs refers to the maximum absorbance at 450 nm, V
corresponds to the dilution volume (mL), A1% 1cm is the absorption
coefficient for β-carotene in petroleum ether (2592 according to
Rodriguez-Amaya 1999), and m is the mass of dry extract (g).
Total anthocyanins determination. The total anthocyanins
content of the fruit extracts was determined via the pH differential
method described by Giusti and Wrolstad (2001). Two sample
dilutions were prepared: one in hydrochloric acid:potassium
chloride buffer pH = 1.0; the other in sodium acetate:acetic acid
buffer pH = 4.5. After 15 min, the absorbance of each equilibrated
solution was measured in a spectrophotometer (model SQ-2800
UV/VIS, UNICO, United Products & Instruments Inc) at the
maximum absorption wavelength (Amax ) and at 700 nm (A700 ) for
haze correction; glass cells with 1 cm path length (l) were used.
The dilution factor (DF) was determined. The difference in the
absorbance values obtained at pH 1.0 and 4.5 was directly propor-
tional to the total anthocyanins concentration. The anthocyanins
content was calculated as cyanidin-3-glycoside (MW = 449.2
g/mol and ɛ = 26.900 L/mol/cm). The results are expressed as
mg of cyanidin-3-glycoside/g of dry extract. The absorbance of
the diluted sample (A) and the total anthocyanins content (TA)
were calculated by using Eq. (2) and (3), respectively:
A = (Am a x − A700 ) p H1,0 − (Am a x − A700 ) p H4,5 (2) and absorbance at 734 nm was recorded at 6 min after mixing.
C2390 Journal of Food Science r Vol. 80, Nr. 11, 2015
(A.MW.DF.1000)
TA = . (3)
ε.l
Ascorbic acid determination–HPLC methodology.
Ascorbic acid was analyzed by high performance liquid chro-
matography coupled to a diode-array detector (HPLC-DAD).
For this purpose, an EC-C18 Poroshell 120 column (2.7 µm,
100 × 4.6 mm) was used at 22 °C. The mobile phase con-
sisted of MeOH:KH2 PO4 50 mM (30:70, v:v); the flow rate was
0.3 mL/min; and the total analysis time was 5 min. The spectra
were obtained between 200 and 600 nm; the chromatograms were
processed at 254 nm. Before analysis, the samples were appropri-
ately diluted with methanol and filtered (0.45 µm). Ascorbic acid
was quantified by comparison with an external standard; 5-point
analytical curves (in duplicate) with concentrations ranging from
5 to 130 ppm (R2 = 0.99) were used.
Total phenolic compounds determination. The total phe-
nolic compounds content of all the extracts was quantified by 2
different methods: the Folin-Ciocalteau colorimetric method and
the chromatographic method as described by Waterhouse (2001)
and Chistéand others (2012), respectively.
For the colorimetric method, 40 µL of the diluted extract
(1:10 extract:solvent) was mixed with 3.16 mL of distilled water
and 200 µL of the Folin-Ciocalteau reagent. After 3 min, 600 µL
of sodium carbonate solution (20%) was added. After 2 h at room
temperature in the dark, the absorbance at 765 nm was read on
a spectrophotometer. The total phenolic compounds content is
expressed as mg of gallic acid equivalents (GAE)/g of dry extract.
For the chromatographic method, the phenolic compounds
were analyzed by HPLC-DAD. The phenolic compounds were
separated on an EC-C18 Poroshell column (2.7 µm, 100 ×
4.6 mm) at 29 °C. The mobile phase consisted of water/formic
acid (99.5:0.5, v:v) (solvent A) and acetonitrile:formic acid
(99.5:0.5, v:v) (solvent B), used as linear gradient from A:B 99:1
to A:B 50:50 in the first 50 min, followed by linear gradient from
A:B 50:50 to A:B 1:99 in the following 5 min, at a flow rate
of 0.9 mL/min. The latter A:B ratio (1:99) was maintained for
additional 5 min. The spectra were obtained between 200 and
600 nm, and the chromatograms were processed at 271, 320, and
367 nm. Phenolic compounds were quantified by comparison
with external standards, with the aid of 5-point analytical curves
(in duplicate). Concentrations ranged from 25 to 200 ppm for
gallic acid, caffeic acid, transcinnamic acid, chlorogenic acid,
ellagic acid, ferulic acid, protocatechuic acid, p-coumaric acid,
4-hydroxybenzoic acid, sinapic acid, syringic acid, apigenin,
catechin, chrysin, flavone, kaempferol, luteolin, rutin, and
quercetin. For all of these compounds, R2 was equal to 0.99.
The phenolic compounds content determined by HPLC-DAD is
expressed as µg/g of dry extracts.
Antioxidant capacity determination. The antioxidant ca-
pacities of the extracts were determined by 3 assays: ABTS, DPPH,
and FRAP (RUFINO and others 2010). For all the methods, the
samples were previously diluted in the extraction solvent at an
extract:solvent 1:10 ratio.
ABTSࢫ+ radical cations were produced by reacting 5 mL of
ABTS 7 mM stock solution with 88 µL of potassium persulfate
140 mM and allowing the mixture to stand in the dark and at
room temperature for 16 h before use. The ABTSࢫ+ solution was
diluted with ethanol to an absorbance of 0.70 ± 0.05 at 734 nm.
After addition of 30 µL of the sample, pure solvent (control) or
trolox standard was added to 3 mL of diluted ABTSࢫ+ solution,
Development of active films from pectin and fruit extracts . . .

Table 1–Phenolic compounds, vitamin C, anthocyanin, and carotenoids contents and antioxidant activity (DPPH, FRAP, and
ABTS) of the fruit extracts.

Anthocyanin
Phenolic (mg of Carotenoids DPPH FRAP ABTS
compounds Vitamin C cyanidin-3- (µg of β (mg of (mg of (mg of
(mg of GAE/g (mg of AA/g of glycoside/g of carotene/g Trolox/g of dry Trolox/g of dry Trolox/g of dry
of dry extract) dry extract) dry extract) dry extract) extract) extract) extract)

C: Food Chemistry
AAE 236 ± 1a 8.8 ± 0.4a 3.09 ± 0.03b 332 ± 4a 92.4 ± 0.3b 969.0 ± 0.7ª 114.24 ± 0.01ª
AWE 179 ± 1b 6.14 ± 0.02b 2.5 ± 0.1c 252 ± 9b 98.3 ± 0.2ª 300.01 ± 0.01b 65 ± 2b
CAE 27.1 ± 0.7c 1.41 ± 0.04de – 23 ± 5d 41.14 ± 0.04c 163.6 ± 0.2c 29.3 ± 0.2c
CWE 12.30 ± 0.06e 0.56 ± 0.01f – – 17.00 ± 0.06f 89.1 ± 0.4e 16.7 ± 0.1e
PAE 5.51 ± 0.03fg 0.45 ± 0.01f – 326 ± 25a 1.56 ± 0.03j 25.8 ± 0.5g 1.3 ± 0.1g
PWE 7.4 ± 0.1f 0.36 ± 0.01f – 278 ± 9b 2.14 ± 0.01i 22.5 ± 0.1h 2.44 ± 0.03g
PPAE 3.9 ± 0.2g 1.38 ± 0.01de – 336 ± 9a 9.1 ± 0.1h 31.0 ± 0.2f 1.3 ± 0.1g
PPWE 4.19 ± 0.07g 1.14 ± 0.01e – 108 ± 9c 10.0 ± 0.1g 23.2 ± 0.8h 10.0 ± 0.3f
SAE 18.15 ± 0.09d 1.63 ± 0.01cd 4.97 ± 0.05a – 32.4 ± 0.4d 138.1 ± 0.2d 30.7 ± 0.1c
SWE 19.03 ± 0.09d 1.93 ± 0.02c 1.8 ± 0.6d – 24.4 ± 0.2e 138.3 ± 0.5d 21 ± 3d
(–), content below the detection limit. Different letter superscripts in the same column indicate a statistically significant difference (P < 0.05).
A, acerola; C, cashew apple; P, pequi; PP, papaya; S, strawberry; AE, alcoholic extract; WE, water extract.

Table 2–Identification and quantification of total phenolic compounds in the investigated fruit extracts by HPLC.

Phenolic compounds concentrations (µg/g of dry extract)

Acerola Cashew apple Strawberry Pequi
Phenolic compounds WE AE WE AE WE AE WE AE
Transcinnamic acid – – 20.2 ± 0.1 177 ± 16 39 ± 2.71 – – –
Ellagic acid – – – – – 1696 ± 139 – 2.51 ± 0.29
Gallic acid – – 55.9 ± 0.5 221 ± 15 – – – 113 ± 9
p-coumaric acid – – – 72 ± 8 – – – 20.55 ± 0.08
Syringic acid – – – – – – – –
Catechin – 337 ± 34 – – 173 ± 11 925 ± 47 25 ± 1.51 29 ± 4
Rutin 152 ± 13 413 ± 36 24 ± 2 356 ± 26 – 2228 ± 187 – –
TOTAL 152 750 101 829 212 4850.55 25.84 165.19
(–), no identified compound.

For the DPPH method, 0.3 mL of the sample was mixed
with 0.3 mL of ethanol solution containing 1,1-diphenyl-2-
picrylhidrazyl (DPPH) 0.5 mM and 2.4 mL of ethanol (99.5%).
For the control, 0.3 mL of the pure solvent was used instead
of the diluted extract. The absorbance was read at 517 nm after
60 min of incubation at room temperature.
For the FRAP method, 100 µL of the sample or pure solvent
(control) was mixed with 3 mL of freshly prepared FRAP reagent
(2,4,6-Tris(2-pyridyl)-s-triazine, FeCl3 , acetate buffer). The ab-
sorbance was read at 593 nm after 30 min of incubation at 37 °C.
All the analyses were carried out in the dark. The antioxidant
capacities are expressed as mM trolox/g of dry extract.
Pectin films: preparation, characterization, and stability
After extract characterization, the extracts with the best antiox-
idant capacities were selected for incorporation into pectin films.
Five formulations were proposed for the assays: pectin film with-
out additive (control), pectin film with acerola alcoholic extract,
pectin film with cashew apple alcoholic extract, pectin film with
strawberry alcoholic extract, and pectin film with a mixture of the
3 latter extracts, in a concentration of 0.17 g of total solids/g of
pectin (dry basis) of each selected extracts.
Preparation of pectin films containing fruit extracts.
Pectin-based biodegradable films were prepared by casting in 2-
stage contact with calcium ion as described by Norajitand others
(2010). CP KelcoBrasil S/A (Limeira, São Paulo, Brazil) supplied
food grade pectin with low degree of methylation (GENU Pectin
Type LM-102 AS-BNB). Pectin (2 g) was dissolved in 100 mL
of distilled water, and glycerol (1.5 g/g1 of pectin) and calcium
chloride (0.005 g/g of pectin) were added to the suspension. The
suspension was then heated to 70 °C under stirring, until all the
solids were dissolved and a homogeneous suspension was achieved.
Next, the suspension was cooled to 40 °C, and the fruit extracts
were incorporated at a concentration of 0.5 g of total solids/g
of pectin (dry basis). A control treatment without additives was
prepared for comparison purposes.
The film-forming solution was casted on an acrylic-coated plate
and dried at 40 °C and 50% relative humidity (RH) in a B.O.D.
Incubator (Model MA-415UR, Marconi, Brazil) equipped with
a control system for drying temperature and RH. After drying,
65 mL of 2% CaCl2 solution was poured onto the dry pectin film
for 30 s. Then, the film was redried at 40 °C until films that could
be easily removed from the plate were formed. Before analysis, the
films were conditioned in desiccators under 58% RH, at 25 °C,
for 48 h.
Film thickness. Film thickness was measured with a digital
micrometer (Tesa Technology, Renens, Switzerland). The thick-
ness was measured in 10 randomly selected points on each film;
an average value was calculated.
Moisture content. Film samples (0.2 g) were weighed and
dried at 105 °C, in an oven, for 24 h. The sample weight loss
was determined, and the moisture content was calculated as the
percentage of water that was removed from the system.
Solubility and swelling degree. Film solubility and swelling
degree were determined according to the methods described
by Gontard and others (1992) and Bierhalz and others (2012),

Vol. 80, Nr. 11, 2015 r Journal of Food Science C2391

 Development of active films from pectin and fruit extracts . . .

Table 3–Thickness, moisture content, solubility, swelling degree, water vapor permeability (WVP), tensile strength (TS), elongation
at break, and elastic modulus (EM) of the prepared pectin films.

Thickness Moisture Solubility Swelling WVP Elongation

Pectin films (mm) (g/100 g of film) (%) (g water/g film) (g mm/h m2 kPa) TS (MPa) (%) EM (MPa)

Acerola 0.064 ± 0.005ª 32 ± 2a 65.3 ± 1.5a 1.23 ± 0.07a 0.123 ± 0.009bc 10.7 ± 0.6a 10.5 ± 0.8a 391 ± 26ab
Cashew apple 0.065 ± 0.004ª 33 ± 1ª 73.1 ± 4.3a 1.34 ± 0.03a 0.148 ± 0.004ab 8.3 ± 0.4bc 10.8 ± 1.7a 244 ± 5bc
C: Food Chemistry

Strawberry 0.066 ± 0.006ª 31 ± 3ª 70.5 ± 3.7a 1.34 ± 0.11a 0.104 ± 0.006c 10.0 ± 0.7ab 6.8 ± 0.1b 384 ± 52ab
Mixture 0.06 ± 0.01ª 32 ± 2ª 70.2 ± 3.8a 1.02 ± 0.18a 0.135 ± 0.017abc 7.0 ± 0.4c 11.3 ± 0.2a 235 ± 48c
Control 0.078 ± 0.004ª 27 ± 4ª 65.4 ± 2.1a 1.25 ± 0.02a 0.160 ± 0.005a 12.1 ± 0.4a 8.9 ± 0.2ab 480 ± 35a
Different letter superscripts in the same column indicate a statistically significant difference (P < 0.05).

Table 4–Color parameters (L∗ , a∗ and b∗ ) and transparency of carried out in quadruplicate, and film solubility in water (%) was
the prepared pectin films. calculated according to Eq. (4):
Transparency
Pectin films log (T600/x) L∗ a∗ b∗ W f − Wi
Solubility = . (4)
Acerola 3.2 ± 0.1ª 93.4 ± 0.7bc –0.27 ± 0.02b 7.1 ± 0.4b Wi
Cashew apple 3.13 ± 0.06ª 92.8 ± 0.5c –0.06 ± 0.01b 4.9 ± 0.2c
Strawberry 3.18 ± 0.04ª 88.2 ± 0.2e 11.3 ± 0.4ª 5.7 ± To analyze swelling, film discs (diameter = 20 mm) were
0.3c
Mixture 3.22 ± 0.09ª 90.6 ± 0.5d –0.80 ± 0.02c 14.4 ± weighted to obtain the initial dry mass (Wi ). Then, the films
1.2a
Control 3.12 ± 0.02ª 94.6 ± 0.3a –0.04 ± 0.01b 3.0 ± 0.3d
were immersed into 100 mL of distilled water, for 30 min. Next,
Different letter superscripts in the same column indicate a statistically significant the films were removed from water, dried superficially with fil-
difference (P < 0.05). ter paper, and weighed (Wf ). The swelling degree was calcu-
lated as the ratio of the adsorbed water mass and the initial
mass.
Table 5–Antioxidant capacity of pectin films. Water vapor permeability. Water vapor permeability (WVP)
DPPH (µgTrolox/g of dry material) was determined gravimetrically by following the standard method
E96-00 (ASTM, 2000) with modifications. For this purpose, the
Films Theoretical Measured in film Recovery (%)
film (diameter = 0.06 m) was placed on the circular opening (area
Acerola 50 ± 1aA 36 ± 2ªB 72.55 = 0.00196 m2 ) of a permeation cell and sealed with sealant ring, to
Cashew apple 5.83 ± 0.07dA 1.67 ± 0.05dB 28.69 ensure that humidity migrated through the film only. The interior
Strawberry 11.25 ± 0.06cA 5.7 ± 0.2cB 50.99
bA bB of the cell was filled with saturated MgCl2 solution (33% RH),
Mixture 15.98 ± 0.05 11.4 ± 0.6 71.42
and the system was stored in a desiccator containing saturated
Different lowercase letters in the same column indicate a statistically significant NaNO2 solution (65% RH) at 25 °C. The weight gain of the cells
difference among films containing different extracts (P < 0.05). Different capital letters
in the same line indicate a statistically significant difference among the theoretical and was monitored every 30 min, for 8 h. Analyses were conducted
measured antioxidant capacity in the same film (P < 0.05). in triplicate, and WVP was calculated on the basis of Eq. (5) and
expressed in g/mm/m2 /h/kPa:

respectively, with some modifications. To analyze solubility, 3
discs (diameter = 20 mm) of each film were weighed, to obtain
the initial weight (Wi ), and then immersed into 50 mL water at
25 °C for 24 h, under sporadic agitation. After this period, the
solution containing the film discs was filtered, and the insoluble
matter was dried at 105 °C for 24 h. The resulting material
was weighed to determine the final weight (Wf ). Analyses were
Figure 1–Light transmission of pectin films.
w δ
WV P = . , (5)
t A.P
where w/t is the angular coefficient determined graphically by
plotting the weight gain (w) as a function of time (t) (g/s), δ is
the mean sample thickness (m), A is the sample permeation area
(m2 ), and P is the difference in water vapor pressure through the
sample at 25 °C (Pa).
Mechanical properties. The tensile properties were inves-
tigated with a texture analyzer (Stable Micro Systems, model
TA.TXplus, Surrey, England) according to the standard method
D882-02 (ASTM, 2002); the average of 10 determinations was
taken for each film. The samples were cut into 25 mm wide and
115 mm long strips with a scalpel and mounted between the tensile
grips of the instrument. The initial grip spacing and the crosshead
speed were set at 80 mm and 1.0 mm/s, respectively. The ten-
sile strength (force/initial cross-sectional area) and the elongation
at break were computed directly from the strength x elongation
curves by using the Texture Exponent 32 software; Young’s mod-
ulus was calculated as the slope of the initial linear portion of this
curve.
Color. Film color was assessed on a Hunter Lab colorimeter
(model Color Quest II) equipped with the CIELab scale (L∗ ,
a∗ , b∗ , and Haze) and working in the transmittance mode; D65

C2392 Journal of Food Science r Vol. 80, Nr. 11, 2015

Development of active films from pectin and fruit extracts . . .

Table 6–Phytochemical compounds content in the films. Statistical analysis

Acerola Cashew apple Strawberry Mixture The measurements were conducted at least in triplicate. All the
results were calculated as mean and standard deviations. Statistical
Vitamin C  R
analyses were performed by ANOVA; the statistical software SAS
(mg asc. acid/g 16.9 ± 0.2a 0.11 ± 0.01c 0.33 ± 0.02c 3.55 ± 0.06b
of dry films) (Statistical Analysis System) was used. The significance level was
Phenolic set at P < 0.05.
compounds

C: Food Chemistry
(mg GAE/g of 32.7 ± 0.5a 16 ± 1d 26.1 ± 1c 30 ± 1b
dry films)
Different letter in the same line indicates a statistically significant difference (P < 0.05).

illuminant and a 10° observer angle were used as reference system.

The color measurements are expressed in terms of lightness L∗
and the chromaticity parameters a∗ and b∗ . Measurements were
conducted at least 5 times.
Light transmission and transparency. The barrier prop-
erties of pectin films against ultraviolet (UV) and visible light
were measured at selected wavelengths between 200 and 800 nm,
on a UV–visible spectrophotometer (model SQ-2800 UV/VIS,
UNICO, United Products & Instruments Inc), according to the
procedure described by Fang and others (2002). Film transparency
was calculated by the equation: transparency = A600/x or log
(T600/x), where A600 is the absorbance at 600 nm, T600 is the
% transmittance at 600 nm, and x is the film thickness in millime-
ters (HAN and FLOROS 1997).
Biocompounds content. Film samples (1 g) were mixed with
10 mL of methanol and stirred vigorously in a shaker (model
TE420, Tecnal, Sao Paulo, Brazil) with controlled temperature
(25 °C) and agitation (100 rpm) for 1 h, in a dark room. Then,
the methanol solution was analyzed. The ascorbic acid content, the
total phenolic compounds content, and the antioxidant capacity
(DPPH) of the film solution were measured as described in sec-
tions “Ascorbic acid determination–HPLC methodology,” “Total
phenolic compounds determination,” and “Antioxidant capacity
determination,” respectively.

Biocompounds stability in the pectin films

The films were characterized with respect to their ability to (1)
incorporate the phytochemical compounds, (2) promote sustained
release of these compounds, and (3) maintain their antioxidant
capacity.
Pectin films were conditioned in closed packings and stored
under controlled temperature (25 °C), in the dark. The biocom-
pounds stability was monitored for 90 d and analyzed at storage
days 0, 7, 20, 40, 70, and 90. The film samples were prepared ac-
cording to section “Pectin films: preparation, characterization, and
stability.” The results are expressed as percentage of biocompounds
loss. For the antioxidant capacity loss, the first-order reaction rate
constants (k) and half-lives (t1/2 ) were calculated according to the
following equations:

Ct = C0 exp(−kt ), (6)

ln 2
t1/2 = , (7)
k

where C0 is the initial antioxidant capacity, and Ct is the antioxi-

dant capacity at the reaction time t. Figure 2–Percentage of (A) phenolic compounds, (B) vitamin C, and (C)
antioxidant activity loss (DPPH) during 90-d storage for the pectin films
containing: acerola, cashew, strawberry, and a mixture of fruit extracts.

Vol. 80, Nr. 11, 2015 r Journal of Food Science C2393

 Development of active films from pectin and fruit extracts . . .
Results and Discussion
Extracts characterization
Table 1 shows the results concerning the phenolic compounds,
vitamin C, anthocyanin, and carotenoids contents as well as the
antioxidant activity (DPPH, FRAP, and ABTS) of the several
extracts.
C: Food Chemistry
Acerola contains a large amount of micronutrients; indeed, it
contains the highest values of phenolic compounds, vitamin C,
and carotenoids among the fruits investigated herein. The acerola
composition shown in Table 1 agrees with data from other au-
thors, who have reported that acerola is one of the Brazilian fruits
with the highest content of phytochemical compounds (Righetto
and others 2005; MEZADRI and others 2008). In other words,
the acerola aqueous and alcoholic extracts present the highest an-
tioxidant capacity, which is at least twice the activity verified for
the other investigated fruit extracts.
The cashew apple and strawberry extracts display higher antiox-
idant capacity than the pequi and papaya extracts. As far as nutri-
tional value is concerned, the former extracts constitute excellent
sources of phenolic compounds and vitamin C. The strawberry
alcoholic extract presents higher amount of anthocyanins than the
parent aqueous extract. Puértolasand others (2013) reported better
extraction of anthocyanins from purple-fleshed potato when they
used 96% ethanol solution.
Pequi, papaya, and acerola contain a large amount of
carotenoids. The alcoholic extracts of these fruits exhibit higher
carotenoids content than the aqueous counterparts because
carotenoids are soluble in less polar solvents. Although the cel-
lular structure protects carotenoids such as phenolic compounds,
alcohol destroys this barrier more effectively than water, thereby
releasing more fruit components into the alcoholic solution
(Lapornik and others 2005).
Although the papaya and pequi extracts have virtually the same
carotenoids content as the acerola extracts, they contain lower vita-
min C and phenolic compounds content and less pronounced an-
tioxidant activity. In fact, the antioxidant capacity of fruit extracts
seems to depend on the phenolic compounds content rather than
on the carotenoids content. Chistéand others (2012) have stud-
ied carotenoids and phenolic compounds extraction from piquiá
(Caryocar villosum); these authors reported that extracts with the
highest carotenoids content do not present any antioxidant ac-
tivity. Mezadriand others (2008) and Righetto and others (2005)
have stated that the antioxidant activity of a certain extract de-
pends on the synergistic action of the constituents of its dif-
ferent fractions. They also reported that phenolic compounds
and vitamin C underlie the antioxidant activity of an extract.
Other authors have also verified that vitamin C and phenols are
the major contributors to the antioxidant activity displayed by
fruit extracts. These compounds can function as indicators of
the antioxidant capacity of an extract, as attested by the high
correlation between these components and the antioxidant activ-
ity obtained in all antioxidant assays (Viuda-Martos and others
2008).
Table 2 summarizes the identification and quantification of the
phenolic compounds extracted from the investigated fruits, on the
basis of a chromatographic method.
Most of the assayed extracts contain rutin and catechin; straw-
berry AE presents the highest concentrations of these compounds.
Among the extracts, strawberry AE displays higher concentration
of phenolic compounds—besides rutin and catechin, this extract
contains ellagic acid at 1696 µg/g of dry extract.
C2394 Journal of Food Science r Vol. 80, Nr. 11, 2015
A larger number of identifiable compounds occur in the cashew
apple and pequi extracts. As for the papaya extracts, they do not
contain any of the standard compounds used in the chromato-
graphic method.
As previously mentioned, acerola and cashew apple extracts have
higher phenolic compounds (by colorimetric method) and vitamin
C contents. However, the chromatographic method revealed lower
concentration of phenolic compounds, probably due to lack of
peak identification in the chromatogram.
Mezadriand others (2008) have detected chlorogenic acid,
epigallocatechingallate, epicatechin, and rutin in acerola pulp,
whereas Righettoand others (2005) have identified gallic acid, cat-
echin, caffeic acid, p-coumaric acid, syringic acid, and ferulic acid
in the pulp of this same fruit. In the present work, we have found
rutin and catechin in the acerola extracts, but not the other com-
pounds. Broiniziand others (2007) have identified 9 phenolic acids
in cashew apple pulp, including gallic, p-coumaric, and cinnamic
acids, as identified in this work. Liu and Lin (2013) have reported
that strawberry alcoholic extract contains quercetin, p-coumaric
acid, and rutin, whereas Holzwarthand others (2012) have identi-
fied catechin, ellagic acid, and cinnamic acid in strawberry pulp,
the same compounds we have detected herein. Chistéand oth-
ers (2012) have identified ellagic acid and gallic acid in pequi
pulp. Although we have not detected phenolic compounds in the
papaya extracts, Gayosso-Garcı́a Sancho and others (2011) have
verified that caffeic acid, p-coumaric acid, and ferulic acid occur
in this fruit. These distinct findings may stem from different fruit
ripening, pre- and postharvest conditions, climate, soil, extrac-
tion methods, and analytical methods ( Righetto and others 2005;
Queiroz and others 2011). Besides that, in our case we evapo-
rated the extract at 40 °C, which may have modified the phenolic
compounds composition due to hydrolysis of the bioactive com-
pounds.
The decision to incorporate strawberry, cashew apple, and
acerola into the pectin films was based on the fact that these fruit
extracts contain the highest amount of vitamin C and phenolic
compounds and display the greatest antioxidant capacity.
Biodegradable films characterization
Film properties. Table 3 presents the film properties (thick-
ness, moisture content, solubility, swelling degree, WVP, and me-
chanical properties). Samples containing distinct types of extract
do not differ significantly from the control film in terms of film
moisture content, thickness, solubility, or swelling degree (P <
0.05), as a result of the similar hydrophilic nature of the extracts
and film composition (pectin and glycerol).
Regarding WVP, extract incorporation into the pectin films
reduces the free volume of the polymeric structure due to disper-
sion of the hydrophilic and hydrophobic compounds (phenolic
compounds, anthocyanins, vitamin C, and carotenoids) present in
the extracts. This enhances matrix organization and obstructs the
passage of water vapor through the film. Reis and others (2015),
Peng and others (2013), and Wu and others (2013) have verified
the same behavior. Park and Zhao (2006) developed pectin films
incorporated with cranberry pomace extracts and obtained WVP
of 3.1 g/mm/m2 /h/kPa, which is around 10 times higher than
the WVP value observed in the present work. Considering that
WVP is one of the most important film properties for food preser-
vation, our results show that extract incoporation into pectin films
improves the film barrier properties, mainly in the case of the
strawberry and acerola films.
Development of active films from pectin and fruit extracts . . .
Table 3 also brings the mechanical properties of pectin films.
Compared with the control film, incorporation of fruit extracts
into pectin films mantained or slightly decreased the tensile
strength and elastic modulus without affecting elongation, as a
result of the different molecular interactions between the polymer
chains and the extract components in the film matrix. Bierhalz
and others (2012) and Rubilar and other (2013) obtained similar
results.
Table 4 depicts the color characteristics (L∗ , a∗ , and b∗ ) and
the transparency of the 5 film samples. All the pectin films have
transparency around 3, with no statistical differences (P ˂ 0.05).
Compared with the control film, incorporation of the extracts
into the pectin film decreases lightness slightly (lower L∗ values).
The film containing strawberry extract is slightly red, and it has
higher a∗ value; the other films exhibit a∗ values close to zero and
are yellowish. The film containing the mixture of extracts has the
highest b∗ value.
Figure 1 shows the light transmission properties of pectin films at
selected wavelengths lying between 200 and 800 nm. Although the
films display the characteristic color of the incorporated extract,
all the 5 films present high light transmission in the visible spectral
region (380 to 780 nm). However, the films containing fruit extract
transmit less light than the control film. The strawberry film affords
the greatest light transmission levels—transmission between 550
and 800 nm is 100%.
The control film does not absorb in the UV region. Neverthe-
less, enrichment of pectin films with fruit extracts increases UV
light absorption (from 200 to 380 nm). The presence of double
bonds and the cyclic structures of phenolic compounds account
for the ability of fruit extracts to act as UV light absorbers. Li
and others (2014), who incorporated phenolic compounds into
gelatin films, have reported similar results.
Commonly used UV light absorbers are potential migrants and
therefore have limited use in polymeric packaging that is in direct
contact with food (ALLEN and others 2004). Hence, natural ex-
tracts could be an interesting replacement for these absorbers, with
the advantage that they can protect both the packaging material
and its content against photooxidation.
Norajitand others (2010) have verified similar results regarding
light properties. These authors incorporated ginseng extract into
alginate films and observed that the resulting film transmits less
light and absorbs more UV light as compared with the control
film.
Biocompounds in pectin films. Table 5 shows the theo-
retical and measured antioxidant capacity and the retention of
this capacity in the prepared films. The theoretical value refers to
the antioxidant capacity of the extract incorporated into the film;
the measured value corresponds to the antioxidant capacity deter-
mined by spectrophotometric analyses after methanol extraction
from the film.
Losses occur during film preparation. These losses may have
stemmed from 3 steps of film formation: during the shaking and
drying process, oxidation may have taken place; in the second
Ca+2 cross-linking process, compounds may have leached.
The phytochemical composition of the films aligns well with
the corresponding extract contents. Among the prepared films,
pectin films containing acerola extract and a mixture of extracts
present the greatest amount of vitamin C and phenolic compounds
(Table 6). The amount of phytochemical compounds measured in
these films is also consistent with the experimental antioxidant
capacity. Films bearing fruit extract have high antioxidant capacity
Table 7–Kinetic parameters for the antioxidant activity degra-
dation in pectin films.
Films k (d−1 ) t1/2 (d) R2
Acerola 0.007 99 0.966
Cashew apple 0.010 67 0.828
Strawberry 0.009 82 0.843
Mixture 0.012 60 0.950
C: Food Chemistry
as a result of synergistic, antagonistic, and additive effects of the
phytochemicals present in the fruits (Skibsted and others 2012).
Stability of biocompounds in pectin films
Figure 2 presents the percentage of phenolic compounds, vita-
min C, and antioxidant activity loss in films stored in the dark, at
room temperature (25 °C), for 90 d.
The pectin film containing acerola extract displays the highest
capacity to retain phenolic compounds on the 90th storage day;
the other films have 20% higher phenolic compounds loss.
On the 90th storage day, the pectin film containing the acerola
extract, the cashew apple extract, a mixture of the extracts, and the
strawberry extract retain 70%, 60%, 45%, and 35% of vitamin C,
respectively. Bastosand others (2009) have achieved similar results:
65% of pure ascorbic acid retention in calcium alginate-Capsul
film stored at room temperature, in the dark, for 90 d.
According to Queirozand others (2011) and Jang and Moon
(2011), ascorbic acid potentially inhibits enzymes. This could ex-
plain the higher retention of phenolic compounds in acerola films
(the extract that contains the highest amount of vitamin C) during
storage.
For all the prepared films, the phenolic compounds are less sta-
ble than vitamin C during storage. Comparison of the molecular
weight (MW) and affinity for water of the extracted phytochem-
ical compounds reveals that ascorbic acid has lower MW or MW
similar to (176.12 g/mol) the MW of most phenolic compounds
(from 100 to 30000 g/mol) (Bravo 1998; López-de-Dicastillo and
others 2012). Ascorbic acid is also more hydrophilic than pheno-
lic compounds. The combination of pectin and calcium chloride
forms a reticulate polymer that retains water and entraps the extract
compounds in the film matrix ( León and Rojas 2007; Bastos and
others 2009; De’Nobili and others 2013). Hence, ascorbic acid
retention in the polymeric matrix might be higher as compared
with the retention of phenolic compounds, and the latter com-
pounds may have been more susceptible to oxidation. As expected,
the films that lose a larger amount of phytochemical compounds
present lower antioxidant activity.
The k and half-life (t1/2 ) values for antioxidant activity were
calculated for each film on the basis of the first-order kinetics
degradation. Half-life corresponds to the time at which the an-
tioxidant capacity drops by 50% with respect to time zero (Desobry
and others 1997). See Table 7 for the results.
Among the prepared films, the pectin films containing the
acerola and the strawberry extract display longer half-life. How-
ever, the film containing the acerola extract presents the highest
proportion of variance (R2 ). The extracts have the characteristic
heterogeneity of food systems: besides phytochemical compounds,
they contain lipids, protein, and enzymes. Therefore, chemical re-
actions may take place in the films during storage, which affords
degradation products that can accelerate the degradation of phy-
tochemical compounds.
Vol. 80, Nr. 11, 2015 r Journal of Food Science C2395
 Development of active films from pectin and fruit extracts . . .
Conclusions
Fruit extracts can potentially replace synthetic antioxidants used
in the chemical, food, and pharmaceutical industries. Among the
fruit extracts tested herein, the acerola, cashew apple, and straw-
berry extracts have the highest content of phytochemicals with
proven antioxidant activity.
Compared with the control pectin film, the films containing
C: Food Chemistry
incorporated fruit extracts do not present significantly different
moisture content, thickness, transparency, or light transmission
between 550 and 800 nm. On the basis of UV light absorption
results, these films are potentially applicable as packaging because
they provide both the packaging material and its content with
enhanced protection against photooxidation.
In addition, the incorporation of extracts into the pectin films
provides these materials with higher antioxidant capacity. Among
the studied films, the pectin film with incorporated acerola extract
exhibits the highest antioxidant capacity retention and is a potential
ingredient to produce antioxidant films or coatings for various food
applications.
Acknowledgments
The authors acknowledge the financial support from CAPES,
CNPq (143885/2011-1 and 304475/2013-0), and FAPESP.
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