International Journal of Biological Macromolecules 155 (2020) 1307–1316

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International Journal of Biological Macromolecules

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

Active edible furcellaran/whey protein films with yerba mate and white
tea extracts: Preparation, characterization and its application to fresh soft
rennet-curd cheese
Agnieszka Pluta-Kubica a,⁎, Ewelina Jamróz b, Agnieszka Kawecka c, Lesław Juszczak d, Paweł Krzyściak e
a
Department of Animal Products Processing, University of Agriculture in Krakow, Balicka 122, 30-149 Krakow, Poland
b
Institute of Chemistry, University of Agriculture in Krakow, Balicka 122, 30-149 Krakow, Poland
c
Department of Product Packaging, Cracow University of Economics, Rakowicka 27, 31-510 Krakow, Poland
d
Department of Food Analysis and Evaluation of Food Quality, University of Agriculture in Krakow, Balicka 122, 30-149 Krakow, Poland
e
Department of Mycology, Collegium Medicum, Jagiellonian University, Czysta 18, 31-121 Krakow, Poland

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

Article history: Biopolymer films based on furcellaran-whey protein isolate (FUR/WPI) and furcellaran-whey protein isolate in-
Received 16 October 2019 corporated with yerba mate extract (FUR/WPI + YM) and with white tea extract (FUR/WPI + WT) were success-
Received in revised form 6 November 2019 fully developed and investigated as active packaging materials for fresh soft rennet-curd cheese. YM improved
Accepted 11 November 2019
water vapour permeability, water content, solubility, modulus elasticity, puncture strength and thermal stability
Available online 12 November 2019
of film. Water content and water activity decreased (P ≤ .05) during storage in cheese packed in each kind of the
Keywords:
biopolymer films. These parameters did not change (P N .05) in control wrapped in linear low-density polyeth-
Biopolymer film ylene (LLDPE). Likewise, pH decreased with the exception of the application of LLDPE and FUR/WPI + YM. In
Active packaging those cheese samples pH did not differ (P N .05) during storage. Total bacteria count decreased (P ≤ .05) in the
Soft rennet-curd cheese cheese samples wrapped in edible films during storage. However, their levels were not significantly lower
Shelf life than control samples when compared within the storage week. The yeast and mould counts did not differ (P N
.05) during storage for the cheese packed in FUR/WPI + WT, whereas it increased in other cheese samples.
The coliform bacteria count decreased (P ≤ .05) during storage in all examined cheese samples. Overall organo-
leptic quality was more often rated as desirable and very desirable in cheese packed in edible films than in LLDPE.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction
Natural films based on proteins and polysaccharides are an interest-
ing alternative for plastic packaging. Their properties positively affect
both the packaged product and the environment as they are biocompat-
ible and biodegradable. Mono-component films (polysaccharides or
protein) may have poor barrier and mechanical properties, therefore
the researchers mixed biopolymers together to improve the functional
properties of natural packaging coatings [1–3]. Whey protein can be
used to create transparent, odourless and flexible films. The use of
WPC or WPI for the production of edible packaging materials increases
whey management. However, the hydrophilic properties of whey pro-
tein cause some limitations in relation to moisture [4]. Therefore, an at-
tempt was made to connect the whey protein with the polysaccharide.
Furcellaran is a negatively charged polysaccharide obtained from red
⁎ Corresponding author.
E-mail addresses: agnieszka.pluta-kubica@urk.edu.pl (A. Pluta-Kubica),
ewelina.jamroz@urk.edu.pl (E. Jamróz), kaweckaa@uek.krakow.pl (A. Kawecka),
rrjuszcz@cyf-kr.edu.pl (L. Juszczak), pawel.krzysciak@uj.edu.pl (P. Krzyściak).
algae Furcellaria lubricalis. Due to its film-forming properties, our re-
search group extensively used furcellaran as one of the components
for the film-forming matrix [1,5–7].
Active packaging has gained increased attention, as it not only pro-
vides passive protection for products, but also, due to its antioxidant
and antibacterial properties, it can influence the quality and extend
the shelf life of products. Plant extracts can act as an active additive
for biopolymer films. Paraguayan holly plant (Ilex paraguariensis A. St.
Hil.) from which yerba mate is produced, grows naturally in South
America [8]. Due to its antioxidant properties, the yerba mate extract
is an ideal addition to biopolymer films [9–12]. White tea contains poly-
phenols such as epigallocatechingallate (EGCG) which have very strong
antioxidant activity [13]. The main steps in obtaining white tea extract
apply to a prolonged drying process without enzyme inactivation and
fermentation processes [14].
Numerous studies have shown that active films obtained from bio-
polymers with plant extracts improve shelf life of the following: cashew
nuts [15], meat and sausages [16–20], bread slices [21], fish and shrimp
[22–27] and vegetables [28]. However, little information is available on
this topic in relation to cheese.

https://doi.org/10.1016/j.ijbiomac.2019.11.102
0141-8130/© 2019 Elsevier B.V. All rights reserved.
1308 A. Pluta-Kubica et al. / International Journal of Biological Macromolecules 155 (2020) 1307–1316
Fresh soft rennet-curd cheese is manufactured using mesophilic lac-
tic acid bacteria and rennet. Unlike mould- or smear-ripened soft
rennet-curd cheese it is intended for direct consumption without matu-
ration. There are also other kinds of fresh cheese, e.g. acid curd fresh
cheese [29], low-fat fresh cheese manufactured from reconstituted
skim milk powder [30] and fresh acid/rennet curd cheese with uniform
texture [31]. However, fresh soft rennet-curd cheese was chosen for this
study as it is popular in our region and commonly wrapped in packaging
made of plastic.
The objective of this study was to use furcellaran, whey protein iso-
late and yerba mate or white tea extracts to obtain active packaging ma-
terials. The physicochemical characterization of the films was
determined using FTIR and DSC. Rheological and mechanical properties
were also evaluated, as well as water behaviour and antioxidant activity.
Biopolymer films were applied as packaging for fresh soft rennet-curd
cheese stored for 3 weeks in refrigerated conditions to determine
their influence on cheese quality. The results were compared to the ef-
fect of linear low-density polyethylene (LLDPE) film.
2. Materials and methods
2.1. Materials
Furcellaran (FUR) was purchased from Est-Agar AS (Estonia) (carbo-
hydrates 79.61%; protein 1.18%; fat 0.24%). Whey protein isolate (WPI)
was obtained from MYPROTEIN (United Kingdom) (protein 82.0%; car-
bohydrates 4.0%; fat 7.5%; salt 0.5%). White tea China Pai Mu Tan (Pro-
gressive Agata Szurlej, Poland) and Yerba Mate Ilex paraguariensis
Taragui Sin Palo (Las Marias, Argentina) were purchased from a local
market (Krakow, Poland). Mesophilic culture FD-DVS CHN-19 (Chr.
Hansen, Denmark) and Fromase 2200TL (DSM, Netherlands) were
used. The rest of chemicals were of analytical grade obtained from
Sigma-Aldrich (Poznań, Poland).
2.2. Preparation of active films
White tea (WT) and yerba mate (YM) extract solutions were pre-
pared by mixing tea powder in distilled water (6 g/300 ml H2O) con-
trolled at 90 °C for 20 min. The water extract of tea was filtered
through Whatman No.1 filter paper. The yield (%) of WT and YM was
9.29 ± 0.33 and 13.79 ± 0.58, respectively.
The films were prepared in three independent series using the cast-
ing method. Furcellaran (FUR 0.66 g/50 ml H2O) and whey protein iso-
late (WPI 1.34 g/50 ml H2O) were prepared separately, then stirred at
250 rpm on a magnetic stirrer set at 45 °C and then mixed to obtain con-
trol films. The active film forming solution was obtained by adding WT
and YM extracts to the FUR/WPI solutions to reach a final concentration
of 20% w/w. The final concentration of WT and YM was carried out ac-
cording to preliminary experiments (data not shown). In every type of
film forming solution, glycerol (1% w/w) was added as plasticiser.
The prepared solutions were poured onto Petri dishes (15 cm diam-
eter) and then allowed to dry under a fume hood for 48 h. The dried
films were peeled with care from the casting surface and used for future
experiments.
WPI-based films were also prepared in preliminary studies. How-
ever, the obtained films were fragile and hardly separated from the cast-
ing surface. Therefore, the FUR/WPI films were prepared and used in the
presented study.
2.3. Physicochemical properties of the active films
2.3.1. Fourier transform infrared spectroscopy (FTIR)
FTIR analysis was performed in triplicate in the range of
4000–700 cm−1 using a MATTSON 3000 FT-IR (Madison, Wisconsin,
USA) spectrophotometer. The films were analysed under transmission
mode, with 32 complete scans at a resolution of 4 cm−1.
2.3.2. Surface colour measurement
The film colour was determined using a Color i5 (X-Rite, USA) spec-
trometer with measuring geometry d/8, illuminant D65, observer 10o
and measuring slot 25 mm. The measurements were run in 5 replica-
tions. In addition, the total colour difference value (ΔE) was calculated
(Eq. (1)).
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ΔE ¼ ðΔLÞ2 þ ðΔaÞ2 þ ðΔbÞ ð1Þ
where ΔL, Δa, and Δb are the differences between the colour value pa-
rameters of FUR/WPI films without extracts and those of the FUR/WPI
films containing WT and YM.
2.3.3. Thermal properties by differential scanning calorimetry (DSC)
The thermodynamic characteristic of films was determined by using
a differential scanning calorimeter DSC 204F1 (Phoenix Netsch,
Germany). The calorimeter was calibrated by indium standard (Tm =
156.6 °C, ΔHm = 28.45 J/g). Approximately 2.0 mg of film was measured
into an aluminium pan and heated in the calorimeter from 25 to 300 °C
at a rate of 10 °C/min, and then cooled at 40 °C/min. An empty alumin-
ium pan was used as the reference. Temperatures and enthalpy of ther-
mal transitions were determined with the use of the instrument's
Proteus Analysis software (Netzsch-Gerätebau, Selb, Germany). DSC
analysis was conducted in triplicate.
2.3.4. Mechanical and rheological properties
2.3.4.1. Modulus elasticity and elongation at break. The mechanical prop-
erties were determined in triplicate using two parameters: modulus of
elasticity (expressed in MPa) and elongation at break (expressed in %)
using standards for plastics [32]. The tests were carried out using the
Shimadzu EZ Test (Kyoto, Japan). Rectangular samples (width-
15 mm) were prepared, which had been previously conditioned for
24 h (humidity 50% and temperature 23 °C).
2.3.4.2. Puncture strength. The puncture strength test was carried out in
triplicate using Shimadzu EZ Test (Kyoto, Japan) with the correct
adapter for testing the strength according to PN-EN 14477:2005 stan-
dard [33]. During the measurement, the sample in the shape of a circle
was clamped in the bottom ring-shaped holder (22 mm diameter).
From the top, the material was pierced with an exchangeable penetrator
with a rounded tip (1 mm diameter) and the maximum piercing force of
the films was measured.
2.3.4.3. Rheological properties. The rheological characterization of films
was performed in triplicate using a Mars II rheometer (Thermo-Haake,
Germany) with serrated plate/plate measuring geometry (diameter
35 mm, gap size 0.05 mm). Using a measuring element with a serrated
surface avoided the slippage effect. A sweep frequency test was per-
formed in the linear viscoelastic range at a constant strain of 0.5% in
the angular frequency range of 0.1–100 rad/s. The creep curves were de-
termined in the range where strain was proportional to stress. The con-
stant shear stress in the creep phase amounted to 500 Pa during 180 s.
The measurements were made at 25.0 ± 0.3 °C.
2.3.5. Water behaviour
2.3.5.1. Water content, solubility and swelling degree. The water behaviour
of films was determined in triplicate according to methodology de-
scribed by Kavoosi et al. [34] and Souza et al. [35]. The samples of each
type of film were weighed (initial weight-W1), then dried for 24 h at
70 °C then weighed again (initial dry weight-W2). Each sample was
placed in 30 ml of water and stored. After 24 h, the films were dried
using tissue paper and weighed (W3). Subsequently, the film samples
were dried for 24 h at 20 °C and weighed (undissolved final weight-
 A. Pluta-Kubica et al. / International Journal of Biological Macromolecules 155 (2020) 1307–1316
W4). The water behaviour of films was calculated using Eqs. (2)–(4), re-
spectively:
water content ½% ¼ ðW1−W2=W1Þ  100
solubility ½% ¼ ðW2−W4=W2Þ  100
swelling degree ½% ¼ ðW3−W2=W2Þ  100
2.3.5.2. Water vapour transmission rate (WVTR). The study of water va-
pour transmission rate was made in triplicate according to ISO
2528:2017 standard [36]. A special glass vessel was filled with a drying
substance (silica gel), then covered with the analysed material and
placed in a chamber with an adjustable microclimate. Standard condi-
tions were used for this specific test in accordance with standard re-
quirements C (temperature 25 °C, humidity 75%). Weight gain was
used to estimate the WVTR. Samples for testing were prepared and sub-
jected to air conditioning in a normative manner. The test was carried
out in accordance with these requirements. The calculations are
shown in Eq. (5):
 
WVTR g=m2  d ¼ 240  ðweight of water=surface penetrationÞ
 24
2.3.6. Antioxidant properties
Antioxidant properties of edible films were evaluated in three series
in six replicates for DPPH radical scavenging activity, according to
Jamróz et al. [6], with slight modifications. The films were cut into
small squares with a surface area of about 4–5 mm2. Then 50 mg of
film was dissolved in 10 ml of distilled water, shaken for 20 min using
a laboratory shaker and centrifuged at 10,000g for 10 min. Afterwards,
0.2 ml of the acquired supernatant was mixed with 2.6 ml of distilled
water and 0.2 ml of 0.1 mM ethanolic solution of DPPH. The blank was
prepared by addition of distilled water instead of film extract. The mix-
tures were incubated in the dark for 30 min and their absorbance was
then measured at 517 nm (Helios Gamma, USA). The obtained results
were expressed as the percentage of radical scavenging effect.
2.4. Preparation of cheese
Cheese was produced in laboratory conditions in three independent
series. The raw bovine milk was obtained in one season (winter). The
production process was as follows: normalisation to fat content
(3.0%), pasteurisation (72 °C, 15 s), cooling to 32 °C, addition of calcium
chloride and mesophilic mixed strain starter culture, fermentation (32
°C, 1 h), addition of rennet, coagulation (32 °C, 1.5 h), cutting, stirring
(32 °C, 0.5 h), moulding, draining (28 °C, 1 h; 20 °C, 21 h) brining
(16% NaCl, 16–18 °C, pH 5.1–5.2, 0.5 h), dripping (4–6 °C, 22 h) and stor-
ing for 3 weeks (4–6 °C). After dripping the cheese was divided into four
groups: control (wrapped in LLDPE film), FUR/WPI (wrapped in FUR/
WPI film), FUR/WPI + YM (wrapped in FUR/WPI + YM film) and
FUR/WPI + WT (wrapped in FUR/WPI + WT film).
2.5. Analysis of cheese
2.5.1. Chemical composition and physicochemical properties of cheese
The chemical composition and physiochemical properties were de-
termined in triplicate. The content of water, total protein, fat, fat in dry
matter, NaCl and ash were analysed according to AOAC [37] and ISO
3433:2008 [38]. The pH was assessed electrometrically using a pH-
meter (CP-411, Elmetron, Poland). Water activity was measured using
LabMaster-aw (Novasina AG, Switzerland). All parameters of the groups
of cheese were evaluated the next day after brining. The content of
1309
water, water activity and pH were also determined after 1, 2 and
3 weeks of storage.
ð2Þ 2.5.2. Microbiological quality of cheese
Test samples, initial suspension and decimal dilutions of cheese
ð3Þ were prepared according to PN-EN ISO 6887-5:2010 [39]. Buffered pep-
tone water (BioCorp, Warsaw, Poland) was used for suspension and di-
lutions. Total counts of bacteria, yeast, moulds, and coliforms were
ð4Þ
determined the following day after brining, and after 1, 2, and 3 weeks
of storage. The methods used were according to PN-EN ISO 4833-
1:2013-12 [40], PN-ISO 21527-1:2009 [41] and PN-ISO 4832:2007
[42], respectively. PCA, DRBC and VRBL medium were purchased from
BioCorp (Poland). The plates were incubated at 30 °C for 72 h, 25 °C
for 120 h and 30 °C for 24 h, respectively.
2.5.3. Organoleptic quality of cheese
The organoleptic evaluation of cheese samples was performed on
the fifth day after wrapping using a 9-point hedonic scale (1 – extremely
undesirable, 2 – very undesirable, 3 – undesirable, 4 – slightly undesir-
able, 5 – neither undesirable nor desirable, 6 – slightly desirable, 7 – de-
sirable, 8 – very desirable, 9 – extremely desirable). The following
quality properties were evaluated: appearance in the packaging mate-
rial, appearance after removing the packaging material, smell, consis-
tency, taste and overall quality. Every group of cheese was evaluated
ð5Þ by 8 trained panellists.
2.6. Statistical analysis
The obtained results were statistically analysed using Statistica ver-
sion 12 (StatSoft Inc., USA). Means and standard deviations were calcu-
lated. A one-way ANOVA was employed and the significance of
differences between the means was established using the Tukey's test.
3. Results
3.1. Structural analysis (FTIR) of films
FTIR spectra of FUR, WPI and FUR/WPI films are shown in Fig. 1A. The
peaks at 1622 cm−1, 1502 cm−1 and 1214 cm−1 were due to amides I, II
and III of WPI, respectively.
The peaks at 1022 cm−1 were assigned to C\\O stretching and the
peaks observed in 911 cm−1 and 832 cm−1 were due to the stretching
mode of the SO groups, which are typical for the structure of furcellaran
[6]. The bands at the range of 700 to 1000 cm−1 represent the finger-
printing region, which is characteristic of each polysaccharide [43]. Gen-
erally, these peaks are weak in spectra of protein [44].
In the spectral range of FUR/WPI films studied at 4000 to 700 cm−1
were relevant peaks, which corresponded to the structure of WPI and
FUR. The IR spectrum of FUR/WPI films differed from the spectrum of in-
dividual components, which may indicate their good compatibility,
which can lead to electrostatic interactions between positively charged
amide groups of WPI and negatively charged sulfate groups of FUR.
These characteristic peaks demonstrated the successful preparation of
FUR/WPI films. The influence of addition of YM and WT on structural
characterization of FUR/WPI films are shown in Fig. 1B. The FTIR spectra
of FUR/WPI + YM and FUR/WPI + WT films look similar to spectra of
control film, which may indicate that the addition of both WT and YM
did not affect the FUR/WPI film structure. Similar results were observed
in addition of grape seed extract and carvacrol into chitosan films [45].
3.2. Thermal properties of films
The characterization of FUR/WPI films and FUR/WPI films with YM
or WT are presented in Table 1. The thermal properties of packaging ma-
terials in the food and pharmaceutical industries are one of the most im-
portant parameters of the films, as it is required that the materials
1310 A. Pluta-Kubica et al. / International Journal of Biological Macromolecules 155 (2020) 1307–1316

Fig. 1. FTIR spectrum of (A) FUR, WPI and FUR/WPI films, (B) FUR/WPI films incorporated with YM and WT.

withstand the heating process when using the products [46]. The DSC
parameters of the peak melting temperature (Tm) and the enthalpy
change (ΔH) were measured as shown in Table 1. Two major transitions
were found in FUR/WPI films with or without YM or WT.
The 1st transition is associated with the phenomenon of melting.
The addition of YM or WT did not cause changes in Tm but caused
changes in ΔH. The increase in the ΔH value may have been due to
the interaction between the FUR/WPI matrix and the phenolic com-
pounds of YM and WT. Our previous study also indicated that green
tea and pu-erh extracts had influence on the thermal stability of
furcellaran-gelatine films [6]. Interactions between the -OH groups of
biopolymers and -OH, -COOH and =CO groups of tea extracts via hydro-
gen bonding can cause stiffness of the film, therefore more energy is
needed to destroy the reinforced film structure [47,48].
The 2nd transition, during cooling, could have been associated with
crystallization. The changes in the Tm and ΔH with the incorporation of
YM or WT might have been related to the films' increased crystallinity.
The increase in ΔH of the films after the addition of extracts may have
 A. Pluta-Kubica et al. / International Journal of Biological Macromolecules 155 (2020) 1307–1316 1311

Table 1
The influence of addition of YM and WT on the thermal and physical parameters of FUR/WPI films.

Characterization of films Type of film

FUR/WPI FUR/WPI + YM FUR/WPI + WT

The thermal properties

Peak temperature (Tm)[°C] 218.2a ± 1.1 219.5a ± 1.5 219.4a ± 2.3
1st transition – endothermic
Enthalpy (ΔH) [J/g] −96.53a ± 2.02 −118.00b ± 0.72 −122.20c ± 1.82
Peak temperature (Tm)[°C] 160.7a ± 5.3 176.9b ± 1.4 176.2b ± 0.9
2nd transition - exothermic
Enthalpy (ΔH) [J/g] 16.15a ± 0.51 29.79b ± 1.57 30.75b ± 0.20

The colour properties

L 40.44a ± 0.10 38.65b ± 0.16 39.76a ± 0.07
a −0.37a ± 0.03 −1.50b ± 0.04 −0.19c ± 0.02
Surface colour measurement
b 2.49a ± 0.09 7.16b ± 0.10 3.67c ± 0.20
ΔE – 5.13 1.38

The appearance of film

The water properties

Solubility [%] 47.40a ± 1.83 46.91a ± 1.17 51.86b ± 0.38
Water content [%] 14.29a ± 0.91 12.04b ± 0.17 14.15a ± 0.35
Swelling degree [%] 285.62a ± 45.43 336.06b ± 8.16 265.94a ± 15.78
WVTR [g/m2·d] 708.07a ± 23.60 679.44b ± 25.09 693.61ab ± 24.25

The mechanical properties

Modulus elasticity [MPa] 198.03a ± 31.13 238.13b ± 34.30 214.09a ± 31.68
Elongation at break [%] 25.99a ± 3.32 22.17b ± 2.86 25.13a ± 2.79
Puncture strength [N] 1.36a ± 0.32 1.69b ± 0.29 1.31a ± 0.20

Mean values ± SD within a row with different superscript letter are significantly different (P ≤ .05).

been due to an increase of the inter- and intra-molecular interaction
[49]. The increased thermal stability of the films may have been due to
the interaction between phenolic compounds and the whey protein,
which led to increased energy activation and better thermal stability.
Specific interactions may have involved hydrogen bonds that arose be-
tween negatively charged nitrogen and oxygen atoms of amino and hy-
droxyl groups and positively charged hydrogen atoms from an adjacent
hydroxyl or amino group of polyphenol or protein [50].
3.3. The colour parameters of films
The colour parameters of films are shown in Table 1. The higher b
values, which indicate a yellowish colour, were found when YM was in-
corporated into FUR/WPI films.
The green intensity is highlighted by negative values of a parameter.
The clearest shade of green was seen in the films with YM, which can be
explained by the greenish colour of the extract due to the presence of
natural pigments found in the leaves of Ilex paraguariensis. The addition
of YM to the film caused an increase in darkness of the FUR/WPI film (L
value). The phenolic components of this extract may have caused diffu-
sion and refraction of light, which resulted in darker films.
3.4. The water parameters of films
Solubility and water content affects the capacity of water-resistant
properties, as well as their subsequent use. Highly soluble films can be
used as edible coatings, whereas insoluble films can provide a protective
function for products with high humidity [51]. The water content, solu-
bility and swelling degree are shown in Table 1. There was no difference
between the solubility of control and films with YM, which can be at-
tributed to the presence of interactions between polymers and polyphe-
nols. However, higher solubility of FUR/WPI film was observed after the
addition of WT. This can be attributed to the hydrophilic character of
polyphenols, which can easily interact with water [51,52]. The strong
interaction between the phenolic compounds of YM and group of
furcellaran and whey protein decreased the availability of water
interaction with the polymer chain. The same trend was observed in
chitosan films with mango leaf extract [15].
The water vapour transition rate (WVTR) depends on the crystallin-
ity of the film, the tortuosity of the path and the presence of surface and
structural defects, as well as the balance between the hydrophobic and
hydrophilic ratio of the film components [53,54]. The WVTR value de-
creased when the YM extract was added to the FUR/WPI films. The ad-
dition of WT did not significantly affect the WVTR of FUR/WPI films. The
reduction of solubility, water content and WVTR after the addition of
yerba mate to the film was caused by the hydrophobic nature of the ex-
tract and the strong interactions of hydrogen bonds between groups of
furcellaran-whey protein and the polar components of yerba mate. The
same results were observed in cassava starch/yerba mate films [11,55].
The swelling degree results suggest that YM tends to increase the values
of this parameter. However, there were no significant differences be-
tween FUR/WPI films and FUR/WPI films with WT.
3.5. The mechanical parameters of films
The mechanical properties were measured using modulus elasticity,
puncture strength and elongation at break (Table 1). The puncture
strength was interesting when the films were used as packaging mate-
rial, e.g. for food products or for container parts with sharp edges.
Modulus elasticity and puncture strength of YM reinforced films in-
creased compared to the control film. The decreased elongation during
the break after the addition of YM may have been caused by the reduced
mobility of the chain by stronger interactions of the polyphenolic com-
pounds with the polymer chain. Non-significant differences were de-
tected in elongation at break, modulus elasticity and puncture
strength of FUR/WPI films with WT and control films. Apparently the
addition of WT did not cause stronger interactions with the polymer
matrix.
The results of modulus of elasticity and puncture strength demon-
strated that addition of YM had a positive effect on mechanical proper-
ties of FUR/WPI films. This could be attributed to the strong interaction
between the phenolic compounds of YM with the functional group of
FUR and WPI, which resulted in the rigid structure of the films.
1312 A. Pluta-Kubica et al. / International Journal of Biological Macromolecules 155 (2020) 1307–1316
Moreover, these interactions may have resulted in stronger interfacial
adhesion, which led to more effective resistance to mechanical stresses
[56].
3.6. The rheological properties of films
The obtained films were also characterised in terms of rheological
properties by plotting the sweep frequency curves (Fig. 2A). The univer-
sal values which characterised the viscoelastic properties of the material
are modules: storage or elastic (G′) modulus and loss or viscous (G″)
modulus. The storage modulus represents that part of the energy sup-
plied to the material that is stored, while the loss modulus corresponds
to that part of the energy that is dissipated in the viscous flow.
All obtained films were characterised by larger values of the G′ con-
serving module than the loss modulus G′, the values of which were also
slightly dependent on the frequency of oscillations. In all cases the tan-
gent values of the phase shift angle (tan δ = G″/G′) were lower than 0.1
(Fig. 2B) which indicated the properties characteristic of strong gels. The
addition of tea extracts modified the rheological properties of the ob-
tained films and significantly increased the values of the conserving
module G′ (Fig. 2A).
However, in the case of the loss modulus, the impact on its value of
tea extracts was not as clear. The increased value of the G′ modulus re-
sulted in a significant decrease in the tangent value of the phase shift
(Fig. 2B), which indicated that the addition of tea extracts influenced
strengthening of the film structure which may have resulted from the
interaction of the polyphenolic components of the extract with the
polysaccharide-protein matrix. These results also confirmed the in-
creased melting enthalpy and modulus of elasticity (Table 1). However,
lower tan δ values were found for the film with the addition of white tea
extract.
Fig. 2. (A) The influence of addition of YM and WT extracts on the sweep frequency curves of FUR/WPI films, (B) The influence of addition of YM and WT extracts on the tangent of phase-
shift angle of FUR/WPI films; (C) The creep curves of FUR/WPI films and samples with addition of YM and WT extracts.
The creep test was also used in the assessment of rheological prop-
erties of the investigated films, and its results as dependence of suscep-
tibility (J) from the time the constant tangential stress was applied
(Fig. 2C). The results obtained confirmed the observations obtained in
the sweep frequency test. The control film was the most susceptible to
the applied stress. The addition of tea extracts significantly reduced
the susceptibility of the films obtained to the applied stress. This indi-
cated the possibility of the structure of the film having been strength-
ened, presumably due to the effects of tea polyphenols with the
polysaccharide-protein matrix.
3.7. Antioxidant properties of films
FUR/WPI showed almost no antioxidant activity – 1.47 ± 1.27%.
Whey proteins showed some antioxidant properties [57]. However,
their share in the obtained extract was relatively small. The average rad-
ical scavenging effects of FUR/WPI + YM and FUR/WPI + WT were sig-
nificantly higher (P ≤ .01) and equalled 36.96 ± 5.96% and 38.68 ±
4.38%, respectively. This can be attributed to the antioxidant activity of
the YM and WT extracts, which are both known to contain polyphenols
[12,13].
3.8. Chemical composition and physicochemical properties of cheese
samples
The content of water, total protein, fat, fat in dry matter, NaCl, ash,
water activity and pH evaluated in the cheese samples the following
day after brining were as follows: 64.8 ± 0.5%, 15.1 ± 1.2%, 15.9 ±
0.3%, 45.3 ± 0.5%, 1.1 ± 0.2%, 2.5 ± 0.3%, 0.974 ± 0.001 and 4.84 ±
0.02, respectively.
 A. Pluta-Kubica et al. / International Journal of Biological Macromolecules 155 (2020) 1307–1316 1313

The content of water, water activity and pH determined after 1, 2
and 3 weeks of storage are presented in Table 2. These parameters did
not differ significantly during the entire storage period in control,
whereas they decreased (P ≤ .05) in all cheese groups packed in edible
films (except pH in FUR/WPI + YM).
The comparison of the water content within the storage week re-
vealed that cheese wrapped in edible films had lower moisture than
the control, however they did not differ significantly according to the
type of active packaging. The same dependencies were observed in
water activity after 2 and 3 weeks of storage. After 1 week, only FUR/
WPI + WT was characterised by water activity lower than the control.
Notwithstanding, pH of the cheese samples did not differ (P N .05)
within the storage week.
The significant decrease in water content was caused by much
higher WVTR of edible films than LLDPE [58]. Lowering of water activity
was a consequence of decreased moisture.
3.9. Microbiological quality of cheese samples
The results of total bacterial (TBC), yeast, moulds and coliforms
count are shown in Table 2. TBC during storage did not differ from con-
trol and decreased in the cheese samples wrapped in edible films. How-
ever, the comparison of TBC within the storage week revealed that all
examined cheese samples did not differ (P N .05), even after 3 weeks.
We obtained satisfactory results because mainly lactic acid bacteria
are included in TBC in this kind of cheese, which are not spoilage
microorganisms.
The yeast and mould count increased (P ≤ .05) during storage in al-
most all cheese samples, except FUR/WPI + WT, in which it remained
constant. The cheese samples packed in edible films did not differ signif-
icantly within the storage week neither in water content, water activity
nor pH, therefore these results suggested that the yeast and mould
counts did not increase due to the application of FUR/WPI + WT. Similar
results were previously reported when pomegranate peel extract incor-
porated with zein film was applied on fresh Himalayan cheese [59]. The
obtained results also demonstrated that the yeast and mould counts in-
creased more rapidly in the control than in FUR/WPI and FUR/WPI
+ YM. Significantly higher yeast and mould counts were found in the
control after 2 and 3 weeks of storage, respectively. These groups of mi-
croorganisms are responsible for spoilage of fresh soft rennet-curd
cheese. Thus, the obtained results definitely showed, that the applica-
tion of edible films resulted in extension of shelf life of examined cheese.
The coliform count decreased during storage in all examined cheese
samples. No coliform bacteria were found in control and FUR/WPI after
3 and 2 weeks of storage, respectively, while these microorganisms
were not detected after 1 week in FUR/WPI + YM and FUR/WPI
+ WT. These results denoted that the packaging material that exhibited
antioxidant properties inhibited the growth of coliforms better than
FUR/WPI. This is beneficial because the presence of these microorgan-
isms is undesirable in food.
3.10. Organoleptic quality of cheese samples
The results of the evaluation of the organoleptic quality of cheese
sample on the fifth day after wrapping are presented in Fig. 3.
All quality characteristics were more often rated positively (6–9) than
negatively (1–4), regardless of the type of packaging material. However,
the appearance of the film, the appearance after removing the film and
the smell were assessed negatively more often in the cheese samples
wrapped in edible films, especially FUR/WPI + YM, than in the control.
This could have been caused by the fact that FUR/WPI + YM film had
the most intensive green colour, was the darkest and stained the cheese
surface green. Cheese samples wrapped in FUR/WPI and FUR/WPI
+ WT resembled smoked cheeses, thanks to which their appearance
was perceived as better. On the other hand, the consistency of the cheeses
packed in edible films was rated only positively, whereas the consistency

Table 2
The water content, physicochemical properties and microbiological quality of cheese wrapped in LLDPE (control) and edible films during storage.

Parameters Type of film Storage weeks

0 1 2 3

The water content and the physicochemical properties

Water content [%] Control 64.8a ± 0.5 64.7a1 ± 1.7 64.5a1 ± 0.6 64.3a1 ± 1.6
FUR/WPI 54.7b2 ± 1.1 48.2c2 ± 2.3 37.6d2 ± 2.9
FUR/WPI + YM 55.3b2 ± 4.0 48.5bc2 ± 2.8 40.4c2 ± 4.9
FUR/WPI + WT 54.7b2 ± 1.1 46.3c2 ± 2.2 34.6d2 ± 4.2
Water activity Control 0.974a ± 0.001 0.973a1 ± 0.001 0.974a1 ± 0.001 0.973a1 ± 0.001
FUR/WPI 0.958b12 ± 0.006 0.952b2 ± 0.003 0.927c2 ± 0.007
FUR/WPI + YM 0.957ab12 ± 0.011 0.943bc2 ± 0.003 0.928c2 ± 0.013
FUR/WPI + WT 0.951b2 ± 0.009 0.938b2 ± 0.004 0.912c2 ± 0.010
pH Control 4.84a ± 0.02 4.83a1 ± 0.06 4.77a1 ± 0.04 4.80a1 ± 0.03
FUR/WPI 4.79ab1 ± 0.05 4.76b1 ± 0.01 4.73b1 ± 0.01
FUR/WPI + YM 4.79a1 ± 0.02 4.79a1 ± 0.01 4.74a1 ± 0.07
FUR/WPI + WT 4.81ab1 ± 0.06 4.77ab1 ± 0.04 4.72b1 ± 0.03

The microbiological quality

TBC [log cfu/g] Control 8.8a ± 0.2 8.6a1 ± 0.3 8.1a1 ± 0.5 7.9a1 ± 0.3
FUR/WPI 8.8a1 ± 0.2 8.2ab1 ± 0.4 7.3b1 ± 0.6
FUR/WPI + YM 8.6a1 ± 0.5 8.0ab1 ± 0.6 7.0b1 ± 0.6
FUR/WPI + WT 8.5a1 ± 0.4 8.1ab1 ± 0.7 7.1b1 ± 0.5
Yeasts count [log cfu/g] Control 0.8a ± 0.8 2.7b1 ± 0.9 4.4bc1 ± 0.5 5.6c1 ± 0.5
FUR/WPI 1.3a1 ± 0.6 1.6ab2 ± 0.2 2.9b2 ± 0.2
FUR/WPI + YM 1.5ab1 ± 0.1 1.6ab2 ± 0.2 2.1b2 ± 0.1
FUR/WPI + WT 1.8a1 ± 0.1 1.9a2 ± 1.0 2.1a2 ± 1.3
Moulds count [log cfu/g] Control 0.4a ± 0.3 1.1a1 ± 0.1 1.8ab1 ± 1.0 3.0b1 ± 0.7
FUR/WPI 1.3ab1 ± 0.5 1.4b1 ± 0.3 1.4b2 ± 0.1
FUR/WPI + YM 0.5ab1 ± 0.2 1.2ab1 ± 0.5 1.3b2 ± 0.3
FUR/WPI + WT 0.9a1 ± 0.2 0.9a1 ± 0.3 1.4a2 ± 0.6
Coliforms count [log cfu/g] Control 2.3a ± 0.2 1.7ab1 ± 0.4 1.1b1 ± 0.4 0.0c1 ± 0.0
FUR/WPI 1.3b1 ± 0.1 0.0c2 ± 0.0 0.0c1 ± 0.0
FUR/WPI + YM 0.0b2 ± 0.0 0.0b2 ± 0.0 0.0b1 ± 0.0
FUR/WPI + WT 0.0b2 ± 0.0 0.0b2 ± 0.0 0.0b1 ± 0.0

Mean values ± SD within a row with different superscript letter and mean values ± SD within a column with different superscript numerals are significantly different (P ≤ .05).
1314 A. Pluta-Kubica et al. / International Journal of Biological Macromolecules 155 (2020) 1307–1316

Appearance in the packaging material Appearance after removing the packaging

12 material

Number of observations
14

Number of observations
10
12
8 10
6 8
6
4
4
2 2
0 0
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

Smell Consistency
12 12

Number of observations
Number of observations

10 10
8 8
6 6
4 4
2 2
0 0
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

Taste Overall quality

14 12
Number of observations
Number of observations

12 10
10 8
8
6
6
4
4
2 2
0 0
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

Fig. 3. Organoleptic quality of cheeses determined in the 9-point hedonic scale.

of the control was considered slightly undesirable four times. All packag-
ing materials had similar influence on the taste of cheese samples. This
characteristic was most often rated as desirable in the control and FUR/
WPI + YM and very desirable in FUR/WPI and FUR/WPI + WT. Notwith-
standing the above, the results clearly demonstrated that the cheese sam-
ples wrapped in the films which contained YM and WT extract were
characterised by better overall quality and received no negative evalua-
tions. Moreover, this characteristic was more often rated as desirable
and very desirable in the cheese samples packed in edible films than in
the LLDPE. Mushtaq et al. [59] who studied the effect of zein film with
pomegranate peel extract on the sensory evaluation of stored fresh Hima-
layan cheese came to similar conclusions.
The application of edible films as packaging had a beneficial effect on
consistency and overall quality of cheese. However, the films were not
colourless and odourless. A few panellists found that undesirable. These
results indicate that if the developed films were tested on other kinds of
cheese in the future it would be interesting to perform additional descrip-
tive sensory methods, e.g. the check-all-that-apply (CATA) questionnaire
[60,61] and projective techniques [62,63].
4. Conclusions
Active FUR/WPI films with YM or WT extracts were successfully de-
veloped. FT-IR analysis showed compatibility between FUR, WPI and
YM or WT. The addition of YM or WT impacted the thermal stability of
FUR/WPI films. The addition of YM caused reduction in water solubility,
water content and water vapour transition rate. It was observed that
WT did not significantly improve the physical and mechanical proper-
ties of films.
The application of FUR/WPI, FUR/WPI + YM and FUR/WPI + WT
films as a packaging material of soft cheese prolonged its shelf life. The
cheese samples wrapped in edible films were characterised by a lower
yeast and mould count than the cheese packed in LLDPE. Active packag-
ing also inhibited the growth of coliform bacteria better than the one
made of plastic. These microorganisms were not detected after 1 week
in the cheese samples wrapped in FUR/WPI + YM and FUR/WPI
+ WT, and after 2 weeks of storage in the samples packed in FUR/WPI
film. The edible films had a small negative impact on the appearance
of the film, the appearance after removing the film and the smell of
the cheese samples. However, they positively influenced the consis-
tency of the cheese samples. Nevertheless, the overall organoleptic
quality was more often rated as desirable and very desirable in the
cheese samples packed in edible films than in LLDPE.
The developed FUR/WPI, FUR/WPI + YM and FUR/WPI + WT films
are innovative active edible packaging materials which were success-
fully applied as an aid to prolong the shelf life of soft rennet-curd cheese.
The use of such packaging does not have a negative impact on the envi-
ronment and increases the use of whey. However, its use in the dairy
 A. Pluta-Kubica et al. / International Journal of Biological Macromolecules 155 (2020) 1307–1316
industry will most likely require more consumer research and some ex-
planation to consumers, as the films are not colourless and odourless.
Funding
This work was supported by the Ministry of Science and Higher Ed-
ucation of the Republic of Poland (BM-2704/KTGiK/18).
Declaration of competing interest
None.
Acknowledgement
The authors would like to thank Ewa Słotwińska for her assistance in
the microbial analysis of cheese.
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