Journal Pre-proof

Whey protein isolate edible films incorporated with essential oils: Antimicrobial activity
and barrier properties

Hülya Çakmak, Yeşim Özselek, Osman Yağız Turan, Ebru Firatligil, Funda
Karbancioğlu Güler

PII: S0141-3910(20)30217-2
DOI: https://doi.org/10.1016/j.polymdegradstab.2020.109285
Reference: PDST 109285

To appear in: Polymer Degradation and Stability

Received Date: 31 December 2019

Revised Date: 15 June 2020
Accepted Date: 27 June 2020

Please cite this article as: Çakmak Hü, Özselek Yeş, Turan OsmanYağı, Firatligil E, Güler
FundaKarbancioğ, Whey protein isolate edible films incorporated with essential oils: Antimicrobial
activity and barrier properties, Polymer Degradation and Stability (2020), doi: https://doi.org/10.1016/
j.polymdegradstab.2020.109285.

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 Credit author statement

Hülya ÇAKMAK: Investigation, Resources, Writing - Original Draft

Yeşim ÖZSELEK: Investigation, Resources, Writing - Original Draft
Osman Yağız TURAN: Investigation, Writing - Original Draft, Writing - Review & Editing,
Visualization, Formal analysis
Ebru FIRATLIGİL: Project administration, Conceptualization, Methodology
Funda KARBANCIOĞLU GÜLER: Project administration, Conceptualization, Methodology
1 WHEY PROTEIN ISOLATE EDIBLE FILMS INCORPORATED WITH
2 ESSENTIAL OILS:
3 ANTIMICROBIAL ACTIVITY AND BARRIER PROPERTIES

4 Hülya ÇAKMAK, Yeşim ÖZSELEK, Osman Yağız TURAN, Ebru FIRATLIGİL, Funda
5 KARBANCIOĞLU GÜLER

6 Istanbul Technical University, Food Engineering Department, 34469, Istanbul, Turkey.

7 E-mail: turanos@itu.edu.tr
8 Abstract
9 Improvement of innovative biopolymers obtained from agricultural sources or food-waste
10 products is one of the leading technologies to preserve quality, freshness and safety of food
11 materials. The use of biopolymers in the packaging applications and their film forming ability
12 reveal the idea of edible films which are also environment friendly. Starch, cellulose derivatives,
13 chitosan/chitin, gums, and animal or plant-based proteins can be accepted as an option to obtain
14 edible films for extending the shelf life of the foods. Film forming proteins provide mechanical
15 stability and can be obtained from animals (casein, whey, gelatin, egg albumin) or plant sources
16 (corn, wheat, rice). Flexibility of the protein network can be enhanced with plasticizers,
17 additionally water permeability can be altered by the addition of oils or beeswax which are
18 hydrophobic materials. Essential oils, in the concentration range of 0.05-0.1%, have
19 demonstrated activity against pathogens. Whey protein isolate of 96% protein ratio (at 8%
20 (w/v)), glycerol, lemon and bergamot essential oils were used for film preparation. Optimum
21 concentrations of constituents of edible film were determined with response surface
22 methodology as 39.2% for glycerol and 4.5% for essential oil. Essential oil concentration is
23 higher than the value which was determined after MIC determination so this amount is effective
24 against S. aureus and E. coli.
25 Keywords: Edible films; Antimicrobial; Response surface methodology; whey protein; Essential
26 oils
27 Introduction
28 Food quality and safety have always been crucial concerns for producers and researchers while
29 preserving the natural state of the material is a simultaneous challenge. New technologies are
30 continually being studied to overcome these challenges. One important technology to provide
31 fresh, high quality and safe food product is the use edible biopolymers obtained from agricultural
32 commodities or food wastes. Starch, proteins, gums or cellulose derivatives can be used to form
33 edible films and utilized in food packaging operations [1]. Edible films are used as packaging
34 materials to improve the fresh quality of food products, form a gas barrier against the
35 surrounding atmosphere, lower the moisture loss of food material and reduce the use of
36 petroleum based non-degradable packaging materials [2].

1
 1 Protein based edible films are favored for their exceptional properties such as solubility in water,
2 action as emulsifiers and excellent nutritional value. Proteins provide films with good
3 mechanical properties, however poor moisture barrier properties limit their use thus additional
4 applications or additions may be required depending on the aspect of use. Different types of oils
5 can be added to improve barrier and optical properties [3]. Whey proteins, acquired from cheese
6 production as a side product and used in sports foods and infant formulas, exhibit beneficial
7 properties when used to develop edible films. They feature good film forming abilities and
8 mechanical properties, and provide good gas barrier properties at low relative humidity.
9 However the hydrophilic nature of whey limits the moisture barrier characteristics so a necessity
10 to improve whey protein based films arises [4].
11 Essential oils (EOs) are naturally occurring antimicrobial compounds which are employed to
12 obtain minimally processed food products with longer shelf life. Essential oils such as cinnamon,
13 lemon, bergamot, tea tree or anise were incorporated into edible films to provide antimicrobial
14 effectiveness against pathogenic and spoilage microorganisms and improve barrier properties of
15 the films against moisture transfer regarding the hydrophilic nature of EOs [5]. The role of
16 essential oils in nature are to protect the plants by functioning as antibacterial, antifungal or
17 antiviral agents. These activities are promising for food industry to benefit EOs against
18 pathogens and foodborne contaminants [6].
19 In this study, whey protein isolates (WPI) based edible films incorporated with essential oils
20 (bergamot and lemon oils) were developed. Study consists of two major parts which are
21 microbiological analyzes of essential oils and obtaining edible films using response surface
22 methodology (RSM). The aim was to obtain an edible food packaging film that completely
23 consists of natural ingredients, WPI, glycerol and essential oils with the function of protecting
24 foods against any type of pathogen bacteria and fungi, and extending shelf life without using
25 chemical additives.
26 Materials and Methods
27 Materials
28 Whey protein isolate (WPI) HiPro Iso Whey was supplied from HardLine Nutrition which has
29 96.0 (2% lipid, 2% moisture) grams of whey protein per 100 grams of powder. Glycerol was
30 supplied from Emboy Kimya. Lemon and bergamot essential oils were provided by Aromsa
31 A.Ş.. Malt Extract Agar (Merck), Mueller Hinton Agar (Merck), Tween-80, peptone (Merck) and
32 all the required chemicals were provided from Istanbul Technical University food microbiology
33 department. Yıldız Technical University donated cultures of Escherichia coli and
34 Staphylococcus aureus, in addition, cultures of Aspergillus niger were from food microbiology
35 laboratory of Istanbul Technical University.
36 Antimicrobial activity of essential oils

2
 1 Lemon and bergamot essential oils are efficient inhibitors against E. coli, S. aureus and A. niger,
2 and the experiments were done for both oils to determine the antimicrobial activity.
3 Antimicrobial activities of essential oils were determined by using disc diffusion method
4 described in previous researches. Suspensions of tested microorganisms are prepared first and
5 concentrations were altered for bacteria as 0.5 McFarland units (Biosan DEN-1 Densitometer) by
6 using peptone water and 104 cells/ml for molds by using Tween-80. 100 µl of suspension of
7 bacteria were spreaded onto solid media plates containing Mueller Hinton Agar (MHA),
8 separately for E. coli and S. aureus. Same procedure was applied for A. niger, with petri dishes
9 containing Malt Extract Agar (MEA). Filter paper discs (6 mm in diameter), which are cut and
10 sterilized previously, impregnated with 10 µl essential oil (both lemon and bergamot, separately)
11 are placed on inoculated petri dishes. Bacteria containing plates are incubated (Nüve Incubator
12 EN055) at 37°C for 24 h, mold containing plates are incubated at 25°C for 48 h. Diameters of
13 inhibition zones were measured in millimeters. Procedures were conducted in three replicates [7,
14 8].
15 Antimicrobial activity of edible films with essential oils were investigated by disc diffusion
16 method as described above. Edible film discs with 1 cm of diameter were cut and placed on
17 mediums and inhibition zones were measured after respective incubation periods.
18 Determination of minimum inhibitory concentration (MIC)
19 The minimum inhibitory concentration (MIC) is defined as the lowest essential oil concentration
20 resulting in the lack of visible microorganism growth. MIC is determined to specify the
21 concentration of essential oils in edible film formulations. Alcoholic solutions of essential oils
22 were prepared in the concentrations between 5 – 640 µl/1000 µl and 10 µl of solutions were
23 added onto disks as previously described for each inoculated petri dish. Petri dishes were
24 incubated in respective temperatures.

25 Determination of optimum composition of edible films using Response Surface

26 Methodology (RSM)
27 Response surface methodology is used to determine the optimum concentrations of glycerol as
28 plasticizer and essential oils as antimicrobial agents. Concentrations of film forming agents were
29 independent variables in RSM as shown in Table 1, where X1 and X2 are coded variables, x1 and
30 x2 are concentration variables: glycerol and essential oil proportions (%, w/w), respectively. Four
31 responses of the study were determined as antimicrobial and barrier properties, which were water
32 vapor and oxygen permeability, and antimicrobial activity of films against E. coli and S. aureus
33 [9].
34 Table 1. Experimental design of process variables

3
 Coded Variables Encoded Variables
Run X1 X2 x1 x2
1 -1 -1 30 2
2 1 -1 50 2
3 -1 1 30 5
4 1 1 50 5
5 -1.414 0 26 3.5
6 1.414 0 54 3.5
7 0 -1.414 40 1.38
8 0 -1.414 40 5.62
9 0 0 40 3.5
10 0 0 40 3.5
11 0 0 40 3.5
12 0 0 40 3.5
13 0 0 40 3.5

1 Film preparation
2 Whey protein isolate aqueous solutions with a concentration of 8% (w/w) were prepared by
3 stirring dry whey powder with distilled water until WPI is dissolved completely. Solutions were
4 heated to 95°C and kept for 20 minutes to denature the protein. Solutions then cooled to room
5 temperature, various amounts of glycerol (26-54 % w/w) and essential oils added. Films are
6 casted by weighing 17±0.05 g of solution on glass petri plates with inner diameter of 11 cm and
7 allowed to dry for 16 h at 50°C in oven. After 16 h, dried films are peeled intact from the casting
8 plates. All films are conditioned in conditioning cabinet (Memmert, HPP 110) at 50% RH and
9 25°C for 24 h prior to tests.
10 Water vapor permeability (WVP)
11 Water permeability tests were conducted by the instructions given in ASTM E96 standard [10].
12 Desiccant (silica gel) containing beakers with a volume of 175 ml were covered with edible films
13 and placed in a conditioning cabinet at 50% relative humidity. Weight gain of beakers were
14 recorded for 54 hours and vapor permeability is calculated with the following equation:

15 = Eq.1
( )

16 Where S refers to the saturation vapor pressure of water (Pa) at the temperature of analysis
17 (25℃). R1 is the relative humidity of conditioning cabinet, R2 is the relative humidity of beaker
18 atmosphere and d is the thickness of films (m). Water Vapor Transmission Rate (WVTR) was

4
1 calculated by the linear regression of the slope of weight gain versus time graph and then by
2 dividing the slope by exposed area of the films (g/h.m2).
3 Oxygen permeability
4 A variable-pressure constant-volume method was employed to determine the gas permeation
5 properties of edible films with a gas permeability tester (GDP-C Brügger, Müchen). Samples
6 were placed in a gas transmission cell with a diameter 10 cm, in order to form a sealed
7 semibarrier between two chambers. One chamber contains the test gas at a specific high pressure,
8 and the other chamber, at a lower pressure, receives the permeating gas. O2 permeability of films
9 were expressed in cm3/m2dbar at standard temperature (20℃).

10 Results and Discussion

11 The study was conducted in two parts. Firstly, microbiological analyses were done. It was
12 decided to use lemon and bergamot essential oils, and disc diffusion method was applied in order
13 to determine their antimicrobial activity. Inhibition zones showed that bergamot is more effective
14 than lemon essential oil. So, study was continued with bergamot oil and minimum inhibitory
15 concentration of bergamot was also evaluated to determine the range in film formulation. In the
16 second part, edible films were prepared depending on concentrations which were obtained with
17 RSM. Optimum combination of antimicrobial edible film constituents were determined by using
18 responses; water vapor permeability, antimicrobial activity, and oxygen permeability.

19 Antimicrobial activity of essential oils

20 Effects of essential oils on different microorganisms were evaluated by comparing their
21 inhibition zones around filter paper discs. In this part essential oils were not diluted, 10 µl of oil
22 was dropped onto discs which are located on inoculated petri dishes. Inhibition zones of bacteria
23 containing petri dishes with respect to bergamot oil and lemon oil are shown in Figure 1 and 2,
24 respectively. While 6 mm white spots show filter paper discs, other whitish colored areas
25 demonstrate the growth of bacteria.

5
1
2 Figure 1. Inhibition zones of bergamot essential oil against bacteria: (A) E. coli, (B) S. aureus

3
4 Figure 2. Inhibition zones of lemon essential oil against bacteria: (A) E. coli, (B) S. aureus

5 It can be seen in figures that different essential oils may exhibit different influences on the same
6 microorganisms. Although bergamot had a strong antimicrobial activity on both Escherichia coli
7 and Staphylococcus aureus, lemon oil was not a powerful antimicrobial against S. aureus but

6
1 inhibited growth of E. coli. Inhibition zones of essential oils against Aspergillus niger is also
2 showed in Figure 3.

3
4 Figure 3. Inhibition zones of essential oils against A. niger: (A) bergamot, (B) lemon

5 It was seen that lemon oil did not show crucial inhibition in the experiments related with A.
6 niger, however it can be concluded that bergamot oil has an antimicrobial effect on A. niger.
7 Additionally, diameters of inhibition zones including filter papers were measured and evaluated
8 by taking an average value of two inhibition zones in each petri dish, and results are given in
9 Table 2.

10 Table 2. Inhibition zones of essential oils against microorganisms

Inhibition zones (cm)
(Average of replications)
Bergamot oil Lemon oil
Escherichia coli 2.10 1.60
Staphylococcus aureus 2.05 0.95
Aspergillus niger 3.50 0.00

11 As a result, it was concluded that antimicrobial activity of bergamot essential oil was higher than
12 lemon essential oil against all the specified microorganisms. So, bergamot was chosen as the
13 antimicrobial to use in film formulation, and the following steps of the study was developed on
14 bergamot essential oil for both microbiological analysis and edible film formation processes.

15 Minimum Inhibitory Concentration (MIC) Determination

16 Minimum inhibitory concentration was previously defined as the lowest concentration of

17 essential oil that any microbial growth was not observed. Because of powerful antimicrobial
18 activity of bergamot, it was diluted to specific concentrations by using ethanol. Agar disc
19 diffusion method was applied for MIC determination and filter paper discs were absorbed with

7
1 specified concentrations of essential oils, pure alcohol and pure bergamot oil. Inhibition
2 diameters of various bergamot oil concentrations are shown in Table 3 for bacteria.

3 Table 3. Inhibition zones of various essential oil concentrations against bacteria (cm)

% Staphylococcus
Escherichia coli
Essential aureus
oil (v/v) 1st zone 2nd zone 1st zone 2nd zone
0.0 1.00 1.15 0.90 0.95
0.5 1.50 1.35 0.80 0.70
1.0 1.45 1.60 0.80 0.85
2.0 1.25 1.50 0.80 0.90
4.0 1.45 1.50 0.95 1.00
8.0 1.55 1.45 1.05 1.00
16.0 1.50 1.50 1.10 1.15
32.0 1.50 1.45 1.10 1.15
64.0 1.40 1.60 1.10 1.10
100.0 2.4 1.55 2.60 2.90

4 Ethanol has an antimicrobial activity on microorganisms, and in order to disregard its effect in
5 diluted solutions, concentration of 0.0 % (v/v) which is pure alcohol is tested first. Inhibition
6 zone diameter of ethanol varies from 0.8 to 1.0 cm. The concentration which exceeds inhibition
7 zone of ethanol was accepted as the lowest concentration. For Escherichia coli, change from 0.0
8 to 1.5 cm showed a sudden increase, so it showed that minimum inhibitory concentration was
9 between 0.0 and 0.5 % (v/v). Concentrations between 0.0 and 0.5 % were tested and 0.125% was
10 found to be the minimum inhibitory concentration for E. coli. For Staphylococcus aureus,
11 antimicrobial activity of ethanol was observed from 0.0 to 4.0 % (v/v), while effect of bergamot
12 oil was not seen. But at the concentration of 4.0% (v/v), an increase was experienced which
13 arises from the impact of bergamot oil, thus minimum inhibitory concentration was determined
14 as 4.0% (v/v) for S. aureus. Inhibition zones for MICs were given in Figure 4.

8
1
2 Figure 4. Inhibition zones of bergamot oil (MIC) against E. coli (A) and S. aureus (B).

3 For Aspergillus niger, rapid growth of microorganisms were observed and no MIC was
4 determined in specified range. Due to the fact that minimum inhibitory concentration had to
5 cover all the microorganisms, MIC was determined as 4.0 % (v/v) for all the microorganisms
6 which specified in this study.
7 Determination of optimum composition of edible films using Response Surface
8 Methodology (RSM)
9 Films were prepared at different concentrations of glycerol and essential oil as previously
10 described and responses were analyzed. Responses of the analysis were water vapor
11 permeability, oxygen permeability and antimicrobial activity of films.
12 Water vapor and oxygen permeability
13 Water vapor permeability is one of the most important attributes of a food packaging film. Water
14 vapor transmission rates and water vapor permeability values of the films studied are shown in
15 Table 4. It is expected that the plasticizer and essential oil composition both affect the rate of
16 water vapor permeability. Regarding the tested values of EO rates, using a 3.5% EO in the film
17 composition would result a better barrier property against water vapor. The change in the vapor
18 permeability is due to the decrease of hydrophilic portion of the films by the incorporation of
19 essential oil [11]. Studies suggest that increasing glycerol ratio would lead to reorganization of
20 polymeric network and generate a less dense film causing more space for water vapor
21 transmission [12]. Thus, using relatively high amounts of plasticizer (~50%) would have adverse
22 effects on expected contribution of EOs on low water vapor permeability.
23 Table 4. Water vapor permeability rates of films
Glycerol Essential Thickness WVTR Permeability
% oil % (mm) (g/h·m2) (g·mm/h·m2·kPa)
26 3.5 0.9 18.75 5.77
30 2.0 0.9 17.90 5.50
30 5.0 0.45 18.76 2.88

9
 40 1.4 0.98 18.06 6.05
40 3.5 0.54 16.83 3.11
40 5.6 0.5 19.51 3.33
50 2.0 0.53 19.70 3.57
50 5.0 0.9 14.20 4.37
54 3.5 1.05 13.33 4.78

1 A variable-pressure constant-volume method was employed to determine the gas permeation

2 properties of edible films with different concentrations. Before oxygen permeability
3 measurement, films which are placed in equipment were vacuumed in order to remove air
4 bubbles in films. Oxygen permeability of the films are given in Table 5.
5 Table 5. Oxygen permeability of films
Oxygen
Glycerol Essential Thickness
Permeability
% oil % (mm)
(mol/m·s· Pa)
26 3.5 0.9 1.15
30 2.0 0.9 1.40
30 5.0 0.45 0.16
40 1.4 0.98 2.10
40 3.5 0.54 0.37
40 5.6 0.5 1.20
50 2.0 0.53 1.20
50 5.0 0.9 1.16
54 3.5 1.05 1.46

6 Protein isolate films have lower oxygen permeability than non-ionic polysaccharide films due to
7 the lower free space between polymer chains. Incorporation of oil based substances in the
8 composition of such films shape a heterogeneous matrix consisting of protein and oil, thus
9 leading to a higher gas permeability [12, 13].

10 Antimicrobial activity of the films

11 Edible films with essential oil had antimicrobial activity against bacterium and the activity
12 against E.coli was greater than S. aureus as bigger zones around discs were observed. Inhibition
13 zones of edible films against Aspergillus niger could not be measured because the molds grown
14 excessively on plates. According to inhibition rate of microorganism, zones were measured and
15 inhibition area were calculated. Area of inhibition zones were given in Table 6.
16 Table 6. Average inhibition zone areas of edible films against bacteria

10
 Glycerol Essential
E. coli S. aureus
% oil %
26 3.5 15.48 0.33
30 2.0 13.10 0.01
30 5.0 13.07 0.31
40 1.4 13.07 0.35
40 3.5 18.20 0.40
40 5.6 21.74 0.40
50 2.0 24.76 0.43
50 5.0 15.84 0.55
54 3.5 26.62 0.01

1 The antimicrobial effects of essential oils were being studied for years due to the increasing
2 demand for natural alternatives to synthetic food additives. The EOs interact with the lipid
3 constituents of the cell membranes, changing the membrane permeability and causing the
4 leakage of microbial cell [14, 15]. Regarding the microbial inhibition zone and permeability
5 responses of the study, optimum concentrations were calculated as 39.2% for glycerol and 4.5%
6 for bergamot oil using RSM.

7 Conclusions
8 Response surface methodology was effective in understanding and determining an optimum
9 concentration of glycerol and oil regarding the barrier and antimicrobial responses of whey
10 protein isolate film. Protein concentration of edible films kept constant at 8% while variable
11 essential oil and glycerol concentrations were studied during experiments. Optimum
12 concentrations after analysis were determined as 39.2% for glycerol and 4.5% for essential oil.
13 Essential oil concentration was higher than the value which detected via MIC determination, thus
14 this amount is noted as effective against S. aureus and E. coli. Besides, edible films with given
15 concentrations show remarkable responses against water vapor and oxygen permeability. Data on
16 the influence of process variables over the barrier and antimicrobial properties of whey protein
17 isolate-based edible films are significant in applicability of edible films having antimicrobial
18 features in food industry.
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3

13
Highlights

• Whey protein isolate (at 8% (w/v)), glycerol, lemon and bergamot essential oils were
used.
• Essential oils, in the range of 0.05-0.1%, have demonstrated activity against pathogens.

• Optimum concentrations were determined with RSM as 39.2% for glycerol and 4.5% for
essential oil.
• Films showed remarkable responses against water vapor and oxygen permeability.
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: