International Journal of Biological Macromolecules 114 (2018) 1094–1101

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Investigation of the physical properties, antioxidant and antimicrobial

activity of ternary potato starch-furcellaran-gelatin films incorporated
with lavender essential oil
Ewelina Jamróz a,⁎, Lesław Juszczak b, Mateusz Kucharek a
a
Institute of Chemistry, University of Agriculture, Balicka Street 122, 30-149 Cracow, Poland
b
Department of Food Analysis and Evaluation of Food Quality, Faculty of Food Technology, University of Agriculture in Cracow, Balicka Street 122, 30-149 Cracow, Poland

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

Article history: Lavender essential oil (OEL) was added to starch, furcellaran and gelatin (S/F/G) films in concentrations of 2%, 4%
Received 28 December 2017 and 6%. The films were examined in terms of physical properties (thickness, density, water solubility, water ab-
Received in revised form 23 March 2018 sorption and degree of swelling) and mechanical properties (tensile strength, elongation at break). The test re-
Accepted 4 April 2018
sults show that the film thickness increased upon addition of OEL. The solubility, water absorption and degree
Available online 5 April 2018
of swelling of the film decreased with increasing concentration of oils. Tensile strength (TS) decreased consider-
Keywords:
ably with increasing concentration of oil, which resulted in lower mechanical strength. Parameters of elongation
Furcellaran at break (EAB) were not changed. The thermal behaviour of the film was affected by OEL. Possible interaction be-
Biofilms tween film matrix and lavender oil was confirmed using FTIR. Antioxidant properties proved to be significantly
Biomaterials enhanced with increasing OEL concentration. The microbiological analysis confirmed antimicrobial properties
of S/F/G with OEL. S/F/G films with OEL can provide new formulation of active packaging with potential food-
technology applications.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction
Films with antimicrobial/antioxidant properties may comprise three
groups of substances: biopolymers (polysaccharides, proteins, fats), ad-
ditives (plasticizers, emulsifiers), and natural or synthetic Antimicro-
bial/Antioxidant (A/A) ingredients. Increasing interest is observed
with respect to biodegradable films made of natural, renewable raw
materials, such as proteins, polysaccharides and essential oils, which de-
stroy or inhibit the growth of microorganisms [1]. The use of biopoly-
mers for the production of packaging offers new opportunities, e.g.
films can be edible, or may be carriers of biologically active substances
[2–4]. In addition, the introduction of essential oils in packaging films
creates strong bactericidal and fungicidal properties, positively affecting
the storage of food products [5–7].
To our knowledge, no data is accessible about three-component
films with the addition of essential oil. The furcellaran/gelatin complex
was added to the starch film-forming base, and then antioxidant and
antimicrobial properties were improved by the addition of lavender es-
sential oil. In this work, the starch is used as a base for the production of
biodegradable films, because in terms of macro-structure it is similar to
⁎ Corresponding author.
E-mail addresses: ewelina.jamroz@urk.edu.pl, (E. Jamróz), rrjuszcz@cyf-kr.edu.pl.
(L. Juszczak).
that of synthetic polymers. It can be used for plasticizing, mixing with
other materials, undergoing genetic and chemical modifications, or a
combination of these processes. Thanks to good mechanical properties,
adequate stability, cohesive strength and gas permeability, starch pro-
vides a good base for films and membranes [8]. Protein–polysaccharide
complexes are important self-assembling structures occurring naturally
in various biological systems. The compatibility of furcellaran and fish
gelatin is due to the establishment of electrostatic interaction when
the two macromolecules are oppositely charged at appropriate pH con-
ditions. Furcellaran could interact with oppositely charged macroions,
including bovine serum albumin [2,9] and gelatin [10]. Furcellaran is a
polysaccharide extracted from red algae Furcellaria lumbricalis, nega-
tively charged. It is a linear polymer composed of units consisting of a
fragment of (1 → 3) β-D-galactopyranose with a sulphate group at C-4
and (1 → 4)-3.6-anhydro-α-D-galactopyranose [11]. Furcellaran is clas-
sified together with Carrageenan (E407) and complies with EU directive
2009/10/EC, Food Chemical Codex and JECFA standards. Carrageenan is
a potential film-forming material [12], but preparation of films using
furcellaran is not abundant in literature. Furcellaran is potentially suit-
able as the one of components for a core of nanocapsules [2]. Gelatin
is composed of an amino-acid sequence, is rich in glycine, proline and
hydroxyproline, which is important due to its gelling effect [13]. The
main reasons for the use of gelatin as a component of biopolymer
films include its high biocompatibility, plasticity, biodegradability and

https://doi.org/10.1016/j.ijbiomac.2018.04.014
0141-8130/© 2018 Elsevier B.V. All rights reserved.
 E. Jamróz et al. / International Journal of Biological Macromolecules 114 (2018) 1094–1101
low production cost [14]. For the production of biodegradable packag-
ing film, fish skin gelatin can be used – these are non-edible parts of
fish, however, gelatin film production allows management of waste
and thus reduces environmental issues. An additional advantage of
using fish gelatin as a raw material for the production of film is health
security, because it does not create threats attributed to bovine gelatin.
Recently, Jamróz et al. [10] have reported the functional properties of
starch-furcellaran-gelatin films. The selection of the composition of
the film under study proceeded with extensive studies of the biofilms
composed of potato starch, fish skin gelatin and furcellaran combined
in a wide range of proportions. Among the prepared films, the S/F/G
films had the best functional properties (solubility, mechanical proper-
ties), so the study focused on the films with three components.
Due to health and current environmental issues, research focuses on
sustainable packaging development based on renewable natural bio-
polymers [14]. In order to reduce the use of chemical additives, in the
food industry there is a growing interest in combinations of biopolymer
films with essential oils having antibacterial and antioxidant properties,
which have no adverse effect on human health [15]. Lavender essential
oil is produced from Lavendula angustifolia. Lavender essential oil has
been extensively investigated in pharmaceutical preparations and in
the food industry (as a natural flavoring agent for sweets, ice creams,
chewing gum, beverages etc.) [16]. One of the most important proper-
ties of these essential oils is antioxidant activity [14]. Lavender oil is
not only applied in food processing as an antioxidant to protect food
products from oxidative rancidity [17]. Beside their antioxidant activity,
essential oils can also improve the functional properties of films. This is
the first report on the TAC (Total Antioxidant Capacity) measurement of
films using the QUENCHER-CUPRAC method (QUick, Easy, New, CHEap
and Reproducible treatment involving forced solubilization of bound
phenolics by oxidizing TAC reagent) [18]. As a result, a novel method
is proposed to give a reliable estimate of the total antioxidant capacity
of S/F/G films with OEL.
The scope of work included:
• obtaining a film of native potato starch, gelatin acquired from fish
skin, and furcellaran
• modifying the resulting films with lavender essential oil at various
concentrations
• examining the performance of the film: thickness, density, water sol-
ubility, water content, swelling degree, tensile strength, elongation at
break
• investigating antimicrobial and antioxidant activity
To the best of our knowledge, no information regarding the effect of
lavender essential oil on the properties of S/F/G films has been reported.
2. Materials and methods
2.1. Materials
The chemicals and the solvents were supplied by Sigma-Aldrich (St.
Louis, MO, USA) in ASC purity, used without further purification.
Furcellaran (Mw = 255 kDa, 9,3% moisture content; pH 7.79), was pur-
chased from Est-Agar AS (Karla village, Estonia); local potato starch
(~15–30% amylose content) was purchased from Superior Standard
(WPPZ S.A, Luboń, Poland); gelatin (Mw = 29.6 kDa; pH 4.50) was iso-
lated from Canadian fish skin (Sigma-Adrich, Poznań, Poland, catalogue
no. G7765); lavender essential oil (Lavendula angustifolia oil) – product
of Etja (Elbląg, Poland).
The distilled water used in all experiments was obtained with the
three-stage Millipore Direct-Q 3UVpurification system (EMD Millipore,
MO, USA). The experiments were performed at room temperature
295 K.
1095
2.2. Synthesis and modification of the film
Potato starch (0.6% w/w) was dissolved in 50 ml of distilled water
at 90οC for 20 min. A blend of furcellaran (0.2% w/w) and gelatin (0.2%
w/w) in 50 ml H2O was 30 min heated at 60 °C followed by an admix-
ture of glycerol (0.05% w/w). The ingredients were mixed near to the
isoelectric point of the furcellaran/gelatin complex (in ratio 1:1) pH-
4.0. When starch formed a homogeneous solution, the mixture of gel-
atin and furcellaran was added. The amount of starch/furcellaran/gela-
tin was chosen based on previous work [10]. After 15 min. of mixing
the film-forming solution, lavender essential oil at different concentra-
tions were added (0–6% m / m - 0%, 2%, 4%, 6%). Tween 80 was added
as an emulsifier in quantities proportional to the essential oil (0.1%,
0.2% and 0.3% w/w) to help distribute and completely incorporate
the oil.
The mixture was poured to polyester Petri dishes (ϕ = 80 mm) then
dried for two days in the oven at 50 °C. The dry films were peeled from
dishes and conditioned at 20 °C and 50% relative humidity in desiccators
before evaluating.
2.3. Film thickness
Thickness of the films was taken using manual instrument Mitotuyo,
No. 7327 (Kawasaki, Japan). The measurements were performed with
the 1 μm precision in 5 points equally distributed around the circle
10 mm from its edge. Average value of these estimations was accepted
as the film thickness.
2.4. Film density
To determine film density, samples of 1 cm × 1 cm were maintained
in a desiccator with calcium sulphate desiccant (0% RH) for 20 days and
weighed, following the method of Shen & Kamdem [19]. Dry-matter
densities were calculated using Eq. (1):


density g=cm3 ¼ m=A δ ð1Þ
where A is the film area (1 cm2), δ is the film thickness (cm), m is the
film dry mass (g). The film density was expressed as the average of
three determinations.
2.5. Water content, solubility and swelling degree
The water content, solubility and swelling degree of film samples
in water was determined according to the methods of Kavoosi et al.
[20] and Souza et al. [21]. Three randomly selected specimens of
each type of film (3 cm × 3 cm) weighted (precision 0,0001 g) −initial
weight (W1); the samples were then dried in oven at 70 °C for 24 h to
determine the initial dry matter (W2). Each film was immersed into
30 ml of Milli-Q water, covered and stored for 24 h at room tempera-
ture (25 °C ± 2 °C). The film samples were removed after 24 h and
dried with filter paper and weighted (W3). After that, the film samples
were dried in an oven at 70 °C for 24 h to determine undissolved final
dry weight (W4). Three measurements were taken for each film sam-
ple to calculate the average value of the parameters. Water content,
film solubility and swelling degree were calculated by the Eqs. (2)–
(4), respectively:
water content ¼ ðW1 −W2 =W1 Þ  100% ð2Þ
solubility ¼ ðW2 −W4 =W2 Þ  100% ð3Þ
swelling degree ¼ ðW3 −W2 =W2 Þ  100% ð4Þ
1096 E. Jamróz et al. / International Journal of Biological Macromolecules 114 (2018) 1094–1101

2.6. Surface color measurement
Color parameters in the system of CIE L * a * b * were determined by
reflection method, using the spectrometer Color i5 (X-Rite, USA) with
the following settings: measuring geometry d/8, illuminant D65, ob-
server 10o, measuring slot 25 mm. The measurements were run in 5
replications. In addition, the total color difference value (ΔE) was calcu-
lated (Eq. (5)).
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ΔE ¼ ðΔLÞ2 þ ðΔaÞ2 þ ðΔbÞ
where ΔL*, Δa*, and Δb* are the differences between the color value pa-
rameters of S/F/G film without OEL and those of the S/F/G films contain-
ing OEL with different concentration.
2.7. Mechanical properties
Mechanical properties were performed with TAxT2i Stable Micro
System (Surrey, UK) texturometer. The mechanical properties of the
biopolymer films was characterized with a Tensile strength (TS) and
Elongation at break (EAB) parameters (Eq. (6)–(7)) available from the
diagram for the monoaxial stretching:
TS ½MPa ¼ Fmax =S
EAB ½% ¼ ðL f =L0 Þ  100
where TS is the mechanical endurance [MPa], Fmax is a breaking load [N]
and S is an area of the foil cross-section [mm]; EAB is elongation at
break; Lf is final length of the film sample at the point of break, L0 is ini-
tial length of the film sample.
Thus, 25 × 100 mm film samples were conditioned for 7 days at 20 °C
and 50% relative humidity RH. Conditioned films were fixed in the instru-
ment clamps mounted on the distance of 30 mm. The rate of deformation
was 0.2 mm/s. The measurements were run in 5 replications.
2.8. DSC
The thermodynamic characteristic of films was determined by using
a differential scanning calorimeter DSC 204F1 (Phoenix Netsch, Ger-
many). The calorimeter was calibrated by indium standard (Tm =
156.6 °C i ΔHm = 28.45 J/g). Samples of film (approx 2.5 mg) were
closed hermetically in aluminum pans and heated in the calorimeter
from 30 to 220 °C at a rate of 10 °C/min. Empty aluminum pan was
used as reference. Temperatures and enthalpy of thermal transitions
were determined with the use of instrument's software Proteus Analy-
sis (Netzsch-Gerätebau, Selb, Germany). DSC analysis was conducted in
triplicate.
2.9. FTIR-ATR spectrophotometry
The FTIR-ATR spectrum of biocomposite films was recorded at the
range of 4000–500 cm−1 with complete 32 scans at resolution of
4 cm−1 using a MATTSON 3000 FT-IR (Madison, Wisconsin, USA)
spectrophotometer.
Application of the QUENCHER technique allows to omit complicated
and time-consuming procedures related to extraction and hydrolysis
[18].
To the test tube containing sample 1 mol pH 7.0 ammonia-acetate
buffer in water-ethanol (1:1) solution, 7.5 mmol neocuproine and
0.01 mol Cu2+ were added sequential in 1 ml aliquot. After 2 h of shak-
ing at 25 °C samples were centrifuged (10 min at 4000 ×g) and trans-
ferred to 96-well plates. TAC was measured spectrophotometrically at
a wavelength of λ = 450 nm and was expressed as in nmol TE/cm2.
ð5Þ
2.10.2. Antimicrobial test
Antibacterial activity on films was assessed using the agar diffusion
method. Films were aseptically cut into a 9 mm diameter disc using a
circular knife and then placed on agar plates. MH agar (Mueller Hinton
agar) surface on Petri dish was covered with a mixture of 60 μl of 24 h
bacterial cultures (E. coli ATCC 25922 and S. aureus ATCC 25923) in
the exponential phase of growth, and 3 ml of MH medium (Mueller
Hinton medium). The plates were then incubated at 37 °C for 24 h.
After 24 h of incubation, the inhibition zones were measured and
photographed in each Petri dish. Each assay was performed by triplicate
on two separate experimental runs.
2.11. Statistical analysis
ð6Þ
All data were expressed as mean ± standard deviation (n ≥ 3). Dif-
ð7Þ ferences among data mean values were tested for statistical significance
at the p b 0.05 level using analysis of variance and the Fisher's test. Sta-
tistical analysis was carried out using Statistica v12.0 software (StatSoft,
Tulsa, USA).
3. Results and discussion
3.1. Physical properties of S/F/G-OEL composite films
In the present study, starch-furcellaran-gelatin (S/F/G) based
biocomposite films with lavender essential oil (OEL) were developed
by a solution casting method. By combining these ingredients, changes
in the structure and physical properties of the film could be observed.
The addition of essential oils causes changes in the polymer film matrix,
depending on the applied concentration. This produced a series of films
with concentrations of 0%, 2%, 4% and 6% for OEL. The physical proper-
ties of S/F/G films, with or without the addition of OEL, including thick-
ness, density, solubility, water content, degree of swelling, tensile
strength and elongation at break are shown in Table 1. The results
showed that the addition of OEL to biopolymer film increased its thick-
ness. The thickness of the film is affected by its composition, the pres-
ence of plasticizers or other additives. The S/F/G films with OEL varied
between the range of 0.17 mm–0.21 mm. This result indicated that
the agglomerations of small particles of essential oil in the film had little
effect on the thickness variation of the prepared film. Moreover, addi-
tion of OEL into the S/F/G matrix, might contribute to the formation of
Table 1
The physical properties of S/F/G films, with or without the addition of OEL.
Essential oils conc. (% w/w)*

2.10. Bioactivities assay 0% 2% 4% 6%

Thickness (mm) 0.17 ± 0.01a 0.20 ± 0.01b 0.21 ± 0.02c 0.21 ± 0.01c
2.10.1. Film total antioxidant capacity Density (g/cm3) 1.04 ± 0.02a 1.12 ± 0.01b 1.20 ± 0.02c 1.21 ± 0.03c
After freezing at −80 °C film samples were pulverized (MM400, Solubility (%) 45.25 ± 1.05a 40.88 ± 1.25b 39.85 ± 2.19b 36.99 ± 2.22c
Swelling 73.24 ± 2.98a 32.54 ± 3.43b 30.45 ± 1.90b 24.89 ± 2.56c
Retch, Germany) and 5 mg samples were assayed by means of
degree (%)
QUENCHER-CUPRAC method [18]. The CUPRAC (CUPric Reducing Anti- Water 74.48 ± 5.04a 72.28 ± 9.75a,b 70.16 ± 8.07b 68.81 ± 6.44c
oxidant Capacity) method of antioxidant activity measurement is based content (%)
on the absorbance measurement of Cu(I)-neocuproine complex formed TS (MPa) 70.2 ± 2.8a 62.8 ± 1.9b 57.3 ± 2.5b,c 50.8 ± 2.8c
as a result of the redox reaction of chain-breaking antioxidants present EAB (%) 20.1 ± 3.0a 19.5 ± 2.6a 21.4 ± 3.8a 20.3 ± 2.0a

in the sample with the CUPRAC reagent (Cu(II)-neocuproine complex). *Values in the columns marked with various letters are significantly different at p b 0.05.
 E. Jamróz et al. / International Journal of Biological Macromolecules 114 (2018) 1094–1101
Table 2
The color of S/F/G films with the addition of OEL.
Parameter OEL concentration (v/v) in biocomposite film*
0%
Luminosity (L*) 40.14 ± 0.12a
Chromaticity parameter a* −0.42 ± 0.03a
Chromaticity parameter b* 1.82 ± 0.12a
Color differences ΔE) – 0.63
*Values are expressed as mean ± standard deviation. Different letters in the same rows indicate significant differences (p b 0.05).
an uncompacted texture with pores, indicating an increase in film thick-
ness. Table 1 shows the results of the solubility of the film at concentra-
tions of 0%, 2%, 4% and 6% OEL. Compared to the control sample, addition
of 2% OEL decreased the solubility of 9.66%, at 4% OEL a decrease of
11.9%, and with 6% OEL – a decreased of 18.25%. The water resistance
of films was determined in terms of degree swelling exhibited in Table
1. The degree of swelling of S/F/G with OEL decreased by 66.02% (in
6% ODH), compared with control films. The S/F/G films are a hydrophilic
material and so showed a higher swelling capacity. Incorporation of OEL
into S/F/G films could reduce the degree of swelling, which might be re-
lated to the hydrophobic nature of OEL. Similar results were observed by
Peng and Li [22] in chitosan films with different essential oils (lemon,
thyme, cinnamon). The degree of swelling of chitosan films with cinna-
mon and lemon essential oils was 11.30% (a decrease of about 78.61%
compared to the control film). Hosseini et al. [23] studied fish gelatin
and chitosan films, in combination with oregano essential oil. These
films exhibit low solubility (38%), similar to that described by Jeya
Shakila et al. [24]. Ghasemlou et al. [25] studied the effect of essential
oils on the solubility of starch film, which confirmed that hydrophobic
compounds, such as essential oils, decrease the solubility of the film,
while hydrophilic ones increase their solubility. This trend was ob-
served in a study conducted by Sanchez-Gonzalez et al. [26] in cellulose
films, after the addition of tea tree oil, also by Pires et al. [27] in hake
protein films, after the addition of various essential oils, and by Atarés
et al. [28], who included cinnamon and ginger oils to films with sodium
caseinate. Essential oils, as complex mixtures of various chemical com-
pounds, show that the hydrophobic character is a variable characteris-
tic, which affects their performance and barrier properties of the film
[15]. Equally important is the choice of the film components, which af-
fects the nature of the interaction between its components. Table 1
shows the results of the water content of the film S/F/G at concentra-
tions of 0%, 2%, 4% and 6% OEL. Compared to the control sample, there
was a decrease in water absorption in the film with the addition of
OEL. The water content of the film with the addition of OEL was in the
range of 68.81–72.28%. The films with the highest concentration of oil
(6%) had a water absorption order of 68.81%. Water content of the con-
trol film was 74.48%. The difference between the decrease in water ab-
sorption films with a concentration of 2%, 4% and 6% OEL and the control
sample is respectively 2.95%, 5.8% and 7.61%. This may be due to interac-
tions between the functional groups of OEL, potato starch, furcellaran
and gelatin which limit polysaccharide-water-protein interactions,
thus resulting in a decrease in water content. The solubility and degree
of swelling of the film in water is a very important parameter during the
selection of the application. When water resistance for packaged foods
Table 3
Thermal characteristic of S/F/G films incorporated with OEL.
Film type Essential 1st transition 2nd transition
oils conc.
T peak (°C) ΔH (J/g) T peak, (°C)
(% w/w)*
Control 0% 127.0 ± 2.8a 16.7 ± 1.4a 181.6 ± 1.6a
2% 136.0 ± 1.8b 10.1 ± 0.8b 178.7 ± 2.3b
OEL 4% 139.4 ± 1.9b 10.0 ± 0.8b 182.9 ± 3.0a
6% 150.8 ± 3.4c 11.3 ± 0.8b 191.5 ± 1.6c
*Values in the columns marked with various letters are significantly different at p b 0.05.
1097
2% 4% 6%
40.67 ± 0.10a 40.25 ± 0.36a 40.51 ± 0.25a
−0.41 ± 0.01a −0.47 ± 0.04a −0.41 ± 0.03a
1.48 ± 0.05a 1.50 ± 0.16a 1.35 ± 0.08a
0.62 0.60
with high moisture content is required, the solubility of the film is a dis-
advantage. Also, mechanical properties (tensile strength - TS and elon-
gation at break - EAB) of S/F/G film were examined, with or without
the addition of OEL (Table 1). The control film reached TS of approx.
70.2 MPa. With respect to films containing OEL, the values of TS ranged
from 50.8 MPa to 62.8 MPa. The addition of OEL to the film significantly
affects parameters of TS. Adding the highest oil concentration (6%) re-
sulted in a decrease in the mechanical strength parameter. The study
also examined the effect of concentration of essential oil on the param-
eter of EAB of the film (Table 1). The addition of OEL to S/F/G film did not
cause significant changes in this parameter. Control films and films with
OEL at varying concentrations reported value of the parameter EAB
within a range of 19.5% to 21.4%. Numerous studies have been published
on the effect that essential oils have on the mechanical properties of
films [5,7,25,28–32]. The addition of an essential oil or a lipid to protein
and polysaccharide films may hinder the interaction between polymer
chains, and create flexible areas (domains) in the film [33]. The addition
of essential oil reduces the interaction between the molecules of a bio-
polymer, and probably hinders chain-chain interaction of polymers,
which consequently leads to a decrease in TS [19]. The microstructure
of the film, which depends on the interaction between the film compo-
nents and drying conditions, seriously affects the physical properties of
the film, such as mechanical properties.
The color parameters of S/F/G films incorporated with OEL at various
concentrations are shown in Table 2. No marked effect on the color pa-
rameters of those films was obtained when OEL was added. The same
trend was observed in many works [28,30,34]. The control samples (S/
F/G without OEL) appeared slightly yellow, deriving from gelatin and
furcellaran. The addition of OEL did not significantly affect the color pa-
rameters (L*, a*, b*) of the film. The calculated values of the total color
difference (ΔE) also indicate a lack of visual effect (ΔE b 1) of the OEL
on the S/F/G film coloration. These results are compatible with visual
observation. However, there are studies that show the opposite effect
[22,29,32]. These results suggested that the incorporation of essential
oils influenced film color, but the changes depended on the type of es-
sential oil. Martucci et al. [14] showed that the incorporation of OEL to
gelatin films caused an increase in yellowish coloration.
ΔH (J/g)
243.7 ± 21.8a
165.1 ± 2.5b
125.7 ± 4.4c
112.6 ± 1.0c
Fig. 1. DSC thermograms of S/F/G film with OEL.
1098 E. Jamróz et al. / International Journal of Biological Macromolecules 114 (2018) 1094–1101

3.2. Structural and thermal characteristic of S/F/G-OEL composite films
DSC studies of S/F/G films with OEL were examined to understand
the structure and interaction between polymers and essential oil. The
occurrence of two endotherms as observed on DSC thermograms sug-
gested that the macromolecules in the system are not thermodynami-
cally compatible, and thus there is a separation of phases [35]. The
first transition occurs in the temperature range 108 °C–150 °C with
maximum at 127.0 ± 2.8 °C (Table 3). It could be associated with
helix-coil transition of some fraction of the gelatin. The presence of
this change for pigskin gelatin has been observed previously [36]. The
addition of OEL significantly influenced the change in the characteristic
temperatures of this transformation, as shown by the peak temperature
(Table 3), whose value increased with the increasing of OEL in the film
matrix. Ma et al. [37] observed the same trend. These authors stated that
the inclusion of olive oil in film matrix resulted in the increase the helix–
coil transition temperature of gelatin. They concluded, that olive oil con-
stituted non-miscible emulsified phases in the films. Thus, the protein
chains may segregate to form a protein-rich phase, which may favor
protein-protein interactions to a great extent. As a consequence, the
films possessed higher helix-coil transition temperature in comparison
with those of the films without oil. Also, the presence of the oil in the
film significantly reduced the value of the enthalpy of transition (ΔH)
compared to the film control, although the amount of added oil did
not have a significant impact here. Lower values of ΔH for the film
with the addition of OEL may indicate that for the transformation of

Fig. 2. FTIR spectra for S/F/G films with and without OEL.
 E. Jamróz et al. / International Journal of Biological Macromolecules 114 (2018) 1094–1101 1099

the helix–coil of some protein fractions, less energy is needed, because

the addition of the oil causes dilution of the environment, which may
limit the interactions between the molecules. On the thermogram, the
second transition in the temperature range 178 °C–188 °C was observed
(Fig. 1, Table 3) and was related to the melting of the film. The values of
Tm peak (Table 3) were slightly affected by the addition of 2 and 4% OEL.
Only the 6% addition of OEL caused a significant increase in the temper-
ature of the maximum transition. The control films showed the highest
ΔH, compared with others (Table 3). Lower enthalpy values (ΔH) are
correlated with the amorphous phase in film and its thermal stability
[38]. The thermal stability corresponded to the crystalline structure,
meaning that the higher crystal degree relates to higher thermal stabil-
ity [39]. Essential lavender oil incorporated to the film might increase
the non-order structure of films. Lower thermal stability was observed
in weaker structure of films, resulting in lower values of transformation
enthalpy. This was associated with lower values of TS. Similar results
were observed in works [23,40].
The FTIR pattern of S/F/G films with and without addition of OEL in
different concentration are shown in Fig. 2. The amide I band
(~1628 cm−1, stretching carbonyl vibrations) and amide II band
(~1531 cm−1, stretching C\\N vibrations) were characteristic for gela-
tin. The band at ~1232 cm−1 observed in spectra, indicating ester sul-
phate groups [41], characteristic for furcellaran are present in the
spectrum of all the film (with or without OEL). The band at
~1073 cm−1, attributed to C\\O stretching vibrations is characteristics
to polysaccharide compounds (furcellaran and starch) and glycerol. A
strong peak was observed at ~3267 cm−1, representing the hydrogen-
bonded O\\H stretching.
The amplitude of the peaks near 2921 cm−1 (C\\H stretching) in-
creased when OEL was added to the S/F/G films, indicating the presence
of essential oil containing hydrocarbons in the film matrix. Aldehyde,
ketone or ester carbonyl groups are the one of main functional groups
in essential oils and C_O stretching vibration of this components oc-
curred at wavenumbers of ~1732 cm−1 [38,42]. The FT-IR spectrum of
the lavender oil reveals that, the peak such as 1729 cm−1, represents
the presence of two major constituents: linalool and linalyl acetate
[43]. For films with OEL the C_O groups were observed at wavenumber
of 1732 cm−1. Moreover, the spectra of lavender oil showed major
peaks at 2950 cm−1 and 1463 cm−1 (alkane group to the C\\H
stretching and bending vibrations) [43]. In S/F/G films with OEL a new
bands at ~2930 cm−1 and ~1446 cm−1 were observed, respectively.
The results suggested that the addition of essential oils influenced the
molecular interaction of polymers chain in the film.
3.3. Antioxidant activity of S/F/G-OEL composite films
Lipid oxidation is the main problem in food spoilage. The application
of S/F/G films with OEL can solve this problem. Antioxidative activity
expressed as the QUENCHER-CUPRAC method of S/F/G based films in
the presence 6% OEL is shown in Fig. 3. The QUENCHER-CUPRAC method
(QUick, Easy, New, CHEap and Reproducible treatment involving forced
solubilization of bound phenolics by oxidizing TAC reagent) is a modifi-
cation of CUPRAC, which allows the examination of the TAC of food sam-
ples without prior extraction, thus making it possible to take into
account interactions on the boundary between the solid phase (a matrix
with bound antioxidants) and the liquid phase (dissolved oxidants such
as ROS or indicator substances) [44]. This method has many advantages,
for example: it provides better selectivity compared to the FRAP
method, has comparatively low interference, the CUPRAC reagent has
relative insensitivity to air, sunlight and solvent type (as opposed to
DPPH), simplicity of performance and ease of use in a routine analysis
[45]. To the best of our knowledge, a study of the antioxidant properties
of biopolymer films using this method has not been reported yet. The S/
F/G films without OEL has antioxidant capacity, probably because of
furcellaran had naturally occurring polyphenols. Similar results was
found for carrageenan films [12]. As excepted, when essential oil
Fig. 3. Antioxidant capacity of S/F/G films with and without OEL.
where present in the films, the total antioxidant activity was signifi-
cantly improved by 88.97%, as compared with the control film (Fig.
3.). There are studies which reported that the main components of lav-
ender essential oil (1,8-cineol, linalool and camphor) do not exhibit an-
tioxidant activity [46]. This fact suggests that the antioxidant activity
exhibited may result from synergistic compounds with other com-
pounds present in lavender essential oil that could improve its bioactive
properties [47].
3.4. Antimicrobial properties of S/F/G-OEL composite films
The antimicrobial properties of the prepared films at different con-
centration of OEL were evaluated against Gram-positive (Staphylococcus
aureus) and Gram-negative bacteria (Escherichia coli) via the agar disc
diffusion assay. The inhibition zones of S/F/G films with or without
OEL are shown in Table 4 and Fig. 4. The S/F/G films with OEL in different
concentration are effective against both E. coli and S. aureus, while they
are more effective Gram positive bacteria rather than to Gram negative
bacteria. The lipoteichoic acids in cell membrane of gram (+) bacteria
may help the penetration of hydrophobic components of essential oils,
while the inherence of an extrinsic membrane of gram (−) bacteria
limits the diffusion rate of hydrophobic compounds through the lipo-
polysaccharide layer. This is the reason why gram negative bacteria
are more resistant to the effects of essential oils than gram positive
ones [6]. The proposed mechanism of the antimicrobial activity of EOs
is their attack on the phospholipid cell membrane, which causes in-
creased permeability and leakage of cytoplasm, or in their interaction
with enzymes located on the cell wall [49]. Martucci and cooworkers
[14] reported that linalool have significant antimicrobial activity of the
lavender oil against E. coli and S. aureus.
Table 4
Antimicrobial activity of S/F/G films incorporated with OEL in different concentration.
Essential oils conc. (% w/w) Inhibitor zone [mm]*
E. coli S. aureus
0% ND1 ND
2% 2.1 ± 0.1a 8.9 ± 0.9a
4% 10.5 ± 0.5b 10.4 ± 0.87a
6% 15.1 ± 0.65c 24.5 ± 0.5b
*Values are measurements of diameter of inhibitory zone and expressed in mm. Means (n
= 3) in same column with different letters are significantly different (p ≤ 0.005). Data re-
ported are mean values ± SD.
1
ND: Not detected.
1100 E. Jamróz et al. / International Journal of Biological Macromolecules 114 (2018) 1094–1101
Fig. 4. Qualitative antimicrobial activity of S/F/G films with OEL in different concentration over S. aureus. Inhibition zone for S/F/G films with OEL and examples of: a) absence (0%
concentration) and b) presence of these zone (2% concentration).
4. Conclusion
Starch, fish skin gelatin and furcelleran are suitable components to
form biopolymer films (S/F/G film). The addition of lavender essential
oil (OEL) changes the physical (thickness, density, solubility, water con-
tent, degree of swelling and light transmission), mechanical (tensile
strength and elongation at break), antioxidant and antimicrobial prop-
erties of the film. The OEL was distributed throughout the S/F/G matrix
as observed by FTIR spectra. DSC analysis clearly showed that the addi-
tion of OEL at various concentrations lead to a different thermal stability
compared to the control films. S/F/G films with OEL showed antioxidant
and antimicrobial ability which is an advantage to retard the oxidation
reaction in food, and can be used for prolonging the shelf life of packed
foods. The use of films containing S/F/G with OEL in food preservation
could be limited because of their physical properties such as high values
of solubility and low values of TS. The higher values of solubility of S/F/G
films with OEL is disadvantageous for their application in foods with a
high water content. Information about polysaccharide-protein films,
conjugate with essential oils, requires further research, since the effects
of adding essential oils to the polymer matrix are variable, and depend
on specific interactions between the components of oil and the compo-
nents of the film. Further studies are necessary to examine the potential
toxicity of lavender essential oil liberated from the S/F/G matrix and
their migration to the food system. Additionally, to have detailed char-
acterization of S/F/G films with OEL, other properties should be tested,
such as barrier properties, antifungal activity and antimicrobial proper-
ties against various microorganisms. However, research is focused on
controlled release and application in food systems.
Acknowledgment
Financial support from University of Agriculture in Cracow (BM
4710) is gratefully acknowledged.
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