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Utilization of thymol as an antimicrobial agent for biodegradable poly(butylene succinate)
Materials Chemistry and Physics xxx (2015) 1e7
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Utilization of thymol as an antimicrobial agent for biodegradable poly(butylene succinate) Nawadon Petchwattana a, *, Phisut Naknaen b a Division of Polymer Materials Technology, Faculty of Agricultural Product Innovation and Technology, Srinakharinwirot University, Sukhumvit 23, Wattana, Bangkok 10110, Thailand b Division of Food Science and Nutrition, Faculty of Agricultural Product Innovation and Technology, Srinakharinwirot University, Sukhumvit 23, Wattana, Bangkok 10110, Thailand
h i g h l i g h t s PBS was softer and tougher due to the plasticization effect derived from thymol. OTR increased with increasing thymol due to the increased amorphous region. Thymol was found to effectively inhibit foodborne pathogens growth. Release kinetics showed that thymol was effective over 15 days studied.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2015 Received in revised form 14 July 2015 Accepted 18 July 2015 Available online xxx
The poly(butylene succinate) (PBS)/thymol film was successfully prepared by using a blown film extruder at five different thymol concentrations ranging from 2 to 10 wt%. Experimental results indicated that PBS was softer and tougher due to the plasticization effect derived from thymol. The oxygen transmission rate (OTR) increased slightly with increasing thymol content due to the increased amorphous region in PBS structure. Under heating process, the blends exhibited lower crystallization temperature (Tc), enthalpy of crystallization (DHc), enthalpy of melting (DHm) and degree of crystallinity (Xc) than that observed in neat PBS. Thymol was found to effectively inhibit foodborne pathogens growth. Its antimicrobial activity against Staphylococcus aureus was evidence at 6 wt% while Escherichia coli did at 10 wt% thymol. Over 15 days studied, release of thymol showed some differences depend on food simulant. Maximum migration was obtained when the film was immersed in isooctane at all test duration. Release kinetics indicated that the incorporation of 10 wt% thymol to PBS films were effective over 15 days. © 2015 Elsevier B.V. All rights reserved.
Keywords: Polymers Differential scanning calorimetry (DSC) Mechanical testing
1. Introduction Nowadays, food packaging is one of the major plastic wastes accumulating in the environment due to its non-biodegradabillity [1,2]. Major part of this waste comes from short-serviced life food such as dairy, meat and vegetable products. In recent years, the utilization of biodegradable polymers has drawn much attention from both industries and research institutions especially for non-
* Corresponding author. E-mail address: [email protected] (N. Petchwattana).
durable applications [2e4]. Poly(butylene succinate) (PBS) is one of the biodegradable polymer derived from glucose fermentation and butanediol. Literature reviews indicated that PBS is one of a tough polymer with mechanical and thermal properties comparable to some petroleum based polymers [4]. Thus, PBS is possibly to be one of the commercial biopolymer in the near future. During storage, some properties of food usually change due to the microbiological activity and others [2,5]. These changes allow further undesired deteriorations and consumer rejection [5e8]. To solve these problems, the preservatives have been applied to retard the food spoilage [9e11]. However, most of them affected the taste, the appearance or the odor of foods.
http://dx.doi.org/10.1016/j.matchemphys.2015.07.052 0254-0584/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: N. Petchwattana, P. Naknaen, Utilization of thymol as an antimicrobial agent for biodegradable poly(butylene succinate), Materials Chemistry and Physics (2015), http://dx.doi.org/10.1016/j.matchemphys.2015.07.052
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In the past, the interaction between food and package was avoided due to its possibility of changing the food quality [5e8]. To date, it has been proved that some interactions do not affect the quality of food but capable to retard the food deterioration [6,7]. Antimicrobial packaging technology is one of the novel concepts which provide the interaction between food and packaging material while maintaining nutritional and sensorial qualities as well as safety [5e8]. This technology has drawn much attention from many researchers to produce polymer based antimicrobial packaging especially the use of bio-based essential oil and biopolymer. Essential oil was initially used in Egypt, India and Persia more than 2000 years ago. Its antimicrobial inhibitory was discovered around a century ago [12]. Nowadays, many types of essential oils were used as antimicrobial agent in food packaging materials. The essential oil extracted from cinnamon was used as an antimicrobial agent in poly(ethylene terephthalate) (PET) film. It was found that the active PET showed the inhibition of Aspergillus flavus growth [13]. After incorporated to poly(lactic acid) (PLA)/poly(trimethylene carbonate) (PTMC) film, thymol was found to inhibit the growth of Escherichia coli, Staphylococcus aureus, Listeria, Bacillus subtilis, and Salmonella [14]. Thymol (2-isopropyl-5-methylphenol) is an essential oil extracted from thyme (Thymus vulgaris), onions (Allium cepa), garlic (Allium sativum) or other plants. It has been utilized as antimicrobial agent in polymer film due to its efficiency and physical properties suitable for blown film process. Numerous reports have found the potential of thymol when it was incorporated in polymer films. Rota et al. [15] concluded that thymol was an effective antimicrobial agent for preserving the food spoilage and increasing the shelf-life. Under microencapsulated condition, thymol showed significant inhibited the Saccharomyces cerevisiae, Listeria innocua, E. coli and S. aureus growths [16]. Although many literatures have reported the antimicrobial efficiency of thymol but no report exists in the PBS/thymol blends. This study focuses on the development of PBS films with thymol. A blown film process was employed to produce the active PBS/ thymol films. Evaluation of the E. coli and S. aureus inhibitory action was compared with various thymol concentrations ranging from 2 to 10 wt%. Other characterizations were carried out by determination tensile, thermal and oxygen barrier properties and release kinetics of the films. 2. Materials and methods 2.1. Materials and processing A blown film grade PBS (FZ91PD) was used as a polymer matrix. Its melting temperature and melt flow rate were 110 C and 6 g/ 10 min respectively. Thymol was selected as an antimicrobial agent to reduce the growth rate of E. coli and S. aureus. Fig. 1 illustrates the chemical structure of (a) PBS and (b) thymol. Formulations of PBS were prepared with various thymol concentrations of 2, 4, 6, 8 and 10 wt%. Thymol was firstly dry-blended with the PBS by using a high speed mixer (Thermo Prism Pilot 3) at 500 rpm for 30 s to disperse thymol powder in PBS matrix. The dryblended compositions were then melt-blended by using a twin screw extruder (Labtech Engineering, LTE20-40). The barrel temperature was set at 100e150 C and at the screw speed of 100 rpm. Each formulation was then pelletized and blown to obtain film of 100 mm in thickness for testing and characterizations. 2.2. Microorganisms Microorganisms obtained from the culture collection of the
Fig. 1. Chemical structure of (a) PBS and (b) thymol.
Thailand Institute of Scientific and Technological Research (TISTR) included E. coli (TISTR 780) and S. aureus (TISTR 1466). 2.3. Testing and characterizations Tensile test was conducted by using a Universal testing machine (Instron 5567) equipped with a 1 kN load cell performed on rectangular films of 10 100 mm2. Tensile modulus, tensile strength and tensile elongation at break were determined from the stressestrain curves according to ASTM D882. Test results were the average of five replicated specimens. A dart drop film impact test was conducted in accord with ASTM D 1709. An oxygen permeation tester (Mocon OX-TRAN, 2/21) was employed to measure the oxygen transmission rate (OTR) according to ASTM D 3985 with an oxygen flow rate of 40 cm3/min at 23 C and 0% relative humidity. To estimate the antimicrobial efficiency of thymol entrapped in PBS films, E. coli (gram-negative, TISTR 780) and S. aureus (gram-positive, TISTR 1466) were selected and measure the viability by using the agar diffusion method. The inhibition zone was determined after the PBS/thymol films were placed on the agar surface at 37 C after 24 h incubation. A differential scanning calorimeter (DSC) (PerkinElmer, DSC6000) was employed to evaluate the transition temperatures of PBS and PBS/thymol blends under nitrogen atmosphere. At the first heating step, the sample was heated from 30 to 150 C at a heating rate of 10 C/min to remove the thermal history and followed by the isothermal holding at 150 C for 10 min. The sample was then cooled from 150 to 30 C at the identical heating rate. A second heating was then performed at the same conditions as the first heating. Finally, samples were cooled to room temperature. The degree of crystallinity (Xc) was calculated by using Equation (1) [17].
Xc ¼
DHc 100 DHf XPBS
(1)
where DHc and XPBS are the crystallization enthalpy and mass
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Table 1 Effect of thymol on the mechanical properties of PBS. Thymol content (wt%)
Film impact strength (J/m)
Neat PBS 2 4 6 8 10
22.49 24.76 27.32 29.54 30.41 32.11
± ± ± ± ± ±
Tensile modulus (GPa)
0.53 0.60 0.39 0.51 0.20 0.98
0.72 0.73 0.64 0.58 0.53 0.48
± ± ± ± ± ±
fraction of PBS respectively. DHf is the heat of fusion, defined as the melting enthalpy of 100% crystalline PBS, which was 110.3 J/g [18]. 2.4. Migration tests 2.4.1. Release tests The release of thymol from PBS films was conducted in five food simulants namely distilled water, acetic acid 3%, ethanol 10%, ethanol 95% and fatty acid isooctane. The test procedures and conditions were performed in accord with EN 13130-2005 standard. Quantitative evaluation of the release was extracted from the samples immersed in the food simulants at 2, 6, 12, 24, 48 h and 5, 10 and 15 days [9] by using a High Performance Liquid Chromatography (HPLC, Agilent 1100 series). The mobile phase composed of 0.05 M ortho-phosphoric acid:acetronitrile, 40:60 (vol/vol). The injection volume and the flow rate were 5 ml and 1 ml/min respectively. 2.4.2. Release kinetics To estimate the release kinetics of thymol, the mass transport parameters were calculated by means of Fick's law of diffusion. The assumptions for this equation are i) the release is controlled by means of Fickian diffusion, ii) the thymol in PBS film is homogeneously dispersed, iii) the initial concentration of thymol at all five food simulants is zero, iv) no interactions between food simulant and PBS/thymil film and v) no degradation of thymol and PBS during the test duration. The diffusion coefficient (D, m2/s) of thymol was determined by using Equations (2)e(4) [19,20].
"
∞ X MF;t 2að1 þ aÞ ¼1 exp MF;∞ 1 þ a þ a2 q2n n¼1
# Dq2n t
39.98 35.64 31.05 26.95 24.77 23.03
± ± ± ± ± ±
1.25 0.90 0.87 0.95 0.67 0.49
Elongation at break (%) 17.46 17.10 19.30 21.85 21.05 22.58
± ± ± ± ± ±
1.46 1.56 1.35 1.60 1.67 2.90
partition coefficient of the active compound between the food simulant and the PBS which can be calculated by using Equation (4). CF,∞ and CP,∞ are the concentrations of thymol in the food simulant and in the PBS at equilibrium respectively. VF and VP are the volumes of the food simulant and the PBS respectively. qn is the positive roots of tan qn which can be determined by using Equation (5). The determination was done by plotting f(qn) ¼ tan qn þ aqn as a function of qn and then observing the points where f(qn) become zero [3]. Equation (2) was simplified and proposed by Chung et al. [9,10]. Thus the diffusion coefficient, D, value can be estimated from Equation (6) [20].
1 1 MF;t $ p a MP;0
0:5
D0:5 0:5 1 ¼ $t þ 0:5 a$LP p
(6)
where MP,0 is the initial amount of migrant in the PBS. For complete migration, partitioning and resistance to mass transfer are negligible. MF,∞ is equals to MP,0 [19]. In case of the ratio MF,t/MP,0 not higher than 0.6, Equation (6) can be simplified to Equations (7) and (8), applied for short and long contact times respectively.
MF;t 2 Dt ð0:5Þ ¼ MP;0 LP p
(7)
! MF;t 8 p2 Dt ¼ 1 2 exp MP;0 p 4L2P
(8)
(2)
L2p
3. Results and discussions
K V a ¼ FP F VP KFP ¼
Tensile strength (MPa)
0.07 0.11 0.78 0.50 0.41 0.60
(3)
CF;∞ CP;∞
(4)
tanqn ¼ aqn
(5)
where MF,t and MF,∞ are the mass of the migrant in the food at a particular time t and at equilibrium respectively. MF,∞ can be assumed to be equal to MP,0. LP is a film thickness. KFP is the
3.1. Mechanical properties of PBS/thymol films The tensile and impact properties of PBS/thymol blends, as well as of pure component, are listed in Table 1. The tensile modulus and the tensile strength of neat PBS were 0.72 and 39.98 MPa respectively. Adding thymol to PBS reduced both the tensile modulus and tensile strength by around 10e40% depending on thymol content. Compared to neat PBS, the blends were less stiff due to the plasticization effect derived from thymol. This allowed PBS to be stretched or deformed easier than the un-modified one. With the presence of 10 wt% thymol, the tensile elongation at break
Table 2 Effect of thymol on the OTR of PBS. Thymol content (wt%)
Thickness (mm)
Neat PBS 2 4 6 8 10
101 100 102 101 102 101
± ± ± ± ± ±
1.02 0.92 1.12 1.30 0.81 0.65
Oxygen transmission rate (ml mil/(m2 day atm)) 63.19 65.52 66.79 69.50 70.30 70.35
± ± ± ± ± ±
0.33 0.12 0.19 0.20 0.50 0.35
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elongated at higher rate and longer distance. Numerous literatures have found the plasticization effects of the essential oils on the food packaging films. In polypropylene (PP) film, the plasticization effect was found to decrease the tensile strength and increase the elongation at break [20]. Kavoosi et al. [23] incorporated 8 wt% thymol to gelatin film and found the reduction of the tensile strength from 2.9 to 1.2 N/m2. Furthermore, the tensile elongation at break was raised from 57 to 170%. Low density polyethylene (LDPE) was also less stiff when carvacrol was added as an antimicrobial agent [24]. Due to the plasticization effect, PLA was also softer and tougher when thymol [25] and limonene [26] were incorporated. 3.2. Barrier and thermal properties PBS/thymol films
Fig. 2. DSC thermograms of neat PBS and PBS/thymol blends (a) crystallization exotherm and (b) melting endotherm.
increased from 17.46 for neat PBS to 22.58%. Thymol was believed to behave like plasticizer when it was blended to PBS. It penetrated between the PBS chains and reduced the intermolecular forces making them disentangle easier [21,22]. This allowed PBS could be
Barrier property is one of the most important factors for food packaging which protect or reduce food contamination from the external contaminants [27]. As exhibited in Table 2, Neat PBS showed the OTR value of 63.19 g mil/(m2 day atm). Of the thymol concentration studied, the OTR tended to increase slightly and obtain the maximum value at 10 wt% thymol. Due to the plasticization effect, the amorphous segment in the PBS was increased thereby allowing the oxygen molecules to transport easier through the free volumes. M. Ramos et al. [9] produced antimicrobial PP film with essential oil (thymol and carvacrol) for food packaging application. They found that the OTR was increased with increasing thymol and carvacrol concentrations. Both essential oils were believed to have modified PP chain structure and consequently reduced the oxygen permeation resistance. In the starch/glycerol blends, the oxygen permeability was linearly increased with increasing glycerol content [28]. In general, the oxygen permeation in a semi-crystalline polymer like PBS is primarily a function of the amorphous phase, while the crystalline section is regularly assumed to be impermeable [29,30]. To confirm this structure-property relation, it is necessary to observe the change in crystallization of the PBS/thymol blends. Fig. 2 illustrates the heating and cooling thermograms of neat PBS and PBS/thymol blends. The data of the crystallization temperature (Tc), crystallization enthalpy (DHc), melting temperature (Tm), enthalpy of melting (DHm) and degree of crystallinity (Xc) for the neat PBS and PBS/thymol blends were indicated in Table 3. As shown in Fig. 2(a), blending thymol to PBS decreased both Tc and DHc compared to that of neat PBS. Tc reached the minimum value of 82.06 C when thymol was added at 10 wt%. This confirmed that the plasticizing effect lubricated the PBS chain and facilitated higher mobility [30]. As illustrated in Fig. 2(b), neat PBS showed double melting peaks, due to two different crystal structures, at 100.78 (Tm1) and 112.93 (Tm2). Vega-Baudrit et al. [17,31] explained that lower endotherm (Tm1) is relates to the melting of the original crystallites and higher endotherm (Tm2) corresponds to the melting of the recrystallized one. With increasing the thymol content, both Tm1 and Tm2 tended to shifted but only minutely. The Tm1 was found to shift to higher position around 4 C while Tm2 did at only 1 C. With increasing thymol content, total enthalpy of melting was decreased gradually from 57.44 to 47.04 J/g. This related to the
Table 3 Crystallization temperature (Tc), crystallization enthalpy (DHc), melting temperature (Tm) and melting enthalpy (DHm) for the neat PBS and PBS/thymol blends. Thymol content (wt%)
Tc ( C)
DHc (J/g)
Tm1 ( C)
Tm2 ( C)
DHm (J/g)
Xc (%)
Neat PBS 2 4 6 8 10
84.10 84.05 83.52 82.97 82.56 82.06
61.33 60.30 59.24 58.77 57.70 57.09
100.78 100.71 100.97 102.62 104.76 104.60
112.93 112.65 112.61 112.58 112.41 112.05
57.44 53.67 51.89 51.31 49.08 47.04
55.60 54.67 53.71 53.28 52.31 51.75
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Fig. 3. Antimicrobial activities of PBS and PBS/thymol blends against E. coli and S. aureus.
increased amorphous region of the blends which could be confirmed by the reduced Xc. This is in agreement with the work on PBS plasticization reported by Zhao and co-workers [32]. They concluded that blending the epoxidized soybean oil to PBS tended to increase the amorphous region of the blends enhancing the PBS chain mobility.
containing 10 wt% thymol was the most effective in the E. coli (gram-negative) growth inhibition. At this thymol content, the clear zone was estimate by only 2 cm. Lower than this concentration, no inhibition zone was observed. Another antimicrobial activity was performed against S. aureus (gram-positive). The clear zone was observed by around 1.8 cm when thymol was blended at 6 wt%. Beyond this concentration, the growth inhibition zone
3.3. Antimicrobial activity of PBS/thymol films Fig. 3 illustrates the antimicrobial activity performed against foodborne pathogens namely S. aureus and E. coli. The antimicrobial evaluation was performed by placing the PBS/thymol film in direct contact with pathogenic bacteria. Further investigation was made by measuring clear zone produced by a film containing thymol and clearly shown in Table 4. Under the test duration, thymol was expected to release from PBS into the agar and then produced the inhibition zone around the active films. The minimum inhibition concentration (MIC) value of thymol against E. coli and S. aureus was 10 and 6 wt% respectively. As illustrated in Fig. 3(a)e(f), PBS
Table 4 Inhibition zone diameter of the PBS/thymol films. Thymol concentration (wt%)
Inhibition zone diameter (cm) E. coli
S. aureus
Neat PBS 2 4 6 8 10
nd nd nd nd nd 2.09 ± 0.11
nd nd nd 1.83 ± 0.27 4.43 ± 0.10 5.52 ± 0.06
nd: No inhibition zone detected.
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Fig. 4. Release of thymol from PBS into different food simulants over 15 days (a) distilled water, (b) acetic acid, (c) isooctane, (d) ethanol 10% and (e) ethanol 95%. (C) Experimental data and (d) Simulation results obtained from Equation (2).
gradually increased by 4.4 and 5.5 cm for 8 and 10 wt% thymol respectively. In comparison, the film was found to have lower antimicrobial performance against E. coli. This was expected because of the outer membrane of the gram-negative bacteria helped them stronger. To overcome this feature, larger amounts of antimicrobial agent is required. In PP, thymol was also found to inhibit the S. aureus growth. The inhibition clear zone diameter of 3.7 cm was observed when 8 wt% thymol was added [9]. Thymol was also found to inhibit the E. coli, S. aureus, Listeria, B. subtilis and Salmonella. However, the inhibition was directly depended on thymol concentration [14]. 3.4. Release kinetics of thymol from PBS films The fractional release (MF,t/MP,0) versus time curves in Fig. 4 was obtained by fitting Equation (2) to the experimental data. It shows the migration of thymol from PBS films with various incubation times and food simulants i.e. distilled water, acetic acid, isooctane, ethanol 10% and ethanol 95%. Good agreements were obtained between experimental data and proposed model prediction. This suggested that the release kinetic could be described by the Fick's law of diffusion. With increasing the incubation time, thymol was rapidly migrated from PBS into the food simulants. It required 50e60 h to reach equilibrium at all food simulants studied. The type of food simulants were expected to be the key factor which influence the migration [20]. At thermodynamic equilibrium,
isooctane allowed thymol to migrate with the largest fractional release at around 0.8. This was expected because of their identical polarity. Several approaches have been reported to explain the phenomenon occurred in the release of an active compound from a polymer matrix. Suppakul et al. [33] proposed the swellingcontrolled model of the polyethylene (PE)/antimicrobial agent blends when they were immersed in the food simulant. This model indicated that the food simulant was firstly diffused into PE matrix and then dissolved the antimicrobial agent thereby enabling its migration. In zein film/thymol system, the thymol release kinetics took place over three steps namely; the diffusion of food simulant, macromolecular matrix relaxation kinetic and diffusion of the active compound through the swollen polymeric network [34]. In this case, the release of thymol from PBS matrix took place over three steps like other systems discussed above. The first one was the penetration of the food simulant molecules through the Table 5 Estimated diffusivities for PBS films containing thymol. Food simulant
Diffusivity (m2/s)
Distilled water Acetic acid Isooctane Ethanol 10% Ethanol 95%
1.31 2.23 8.43 9.70 5.86
1015 1015 1014 1015 1014
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PBS. Secondly, the thymol molecules were dissolved in the food simulants thereby transferring mass to the PBS surface. Finally, the dissolved thymol was completely migrated to the food simulants until the thermodynamic equilibrium was reached. This release was facilitated by the plasticization effect derived from thymol which could be confirmed by the decreased Xc. The diffusivity (D), in Table 5, shows that the diffusion process in different food simulants was ranging from 1.31 1015 to 8.43 1014 m2/s. Maximum D was found in isooctane due to its non-polarity like thymol. The identical polarity facilitated thymol to dissolve easier and faster mass transport. Compared to other antimicrobial polymer systems, PBS/thymol blends met the needs of short cycle food packaging application such as meat, vegetable and fruit products. Antimicrobial activity of thymol was found to active over 15 days studied and the MIC against E. coli and S. aureus were 10 and 6 wt% respectively. Mastromatteo et al. [35] applied thymol-coated polystyrene as shrimp package. They found that the antimicrobial activity was effective at around 14 days. In polypropylene/thymol blends, the MIC against S. aureus was observed at 8 wt% [9]. In zein film, thymol was released in water over 4 days studied and the release rate depended on the thymol concentration [34]. In PLA, the inhibition clear zone was found when thymol was added at 8 wt% [25]. 4. Conclusions In the current paper, thymol was melt-blended with PBS to produce the antimicrobial food packaging. The film was softer and tougher due to the plasticization effect derived from thymol. The OTR tended to increase slightly with increasing thymol content due to the increased amorphous region. Thymol was found to effectively inhibit foodborne pathogens growth. Its antimicrobial activity against S. aureus was evidence when 6 wt% while E. coli did at 10 wt% thymol. Over 15 days studied, release of thymol showed some differences depend on food simulant. Maximum migration was obtained when the film was immersed in isooctane at all test duration. Release kinetics indicated that the incorporation of 10 wt % thymol to PBS films were effective over 15 days. Acknowledgments The authors would like to acknowledge the research grant supported by Srinakharinwirot University (Contract no. 143/2556144/2556). Thanks are extended to Mr. Jirawat Mala and Miss Sasinee Wiburanawong for the antimicrobial and the OTR test.
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Utilization of thymol as an antimicrobial agent for biodegradable poly(butylene succinate) Nawadon Petchwattana a, *, Phisut Naknaen b a Division of Polymer Materials Technology, Faculty of Agricultural Product Innovation and Technology, Srinakharinwirot University, Sukhumvit 23, Wattana, Bangkok 10110, Thailand b Division of Food Science and Nutrition, Faculty of Agricultural Product Innovation and Technology, Srinakharinwirot University, Sukhumvit 23, Wattana, Bangkok 10110, Thailand
h i g h l i g h t s PBS was softer and tougher due to the plasticization effect derived from thymol. OTR increased with increasing thymol due to the increased amorphous region. Thymol was found to effectively inhibit foodborne pathogens growth. Release kinetics showed that thymol was effective over 15 days studied.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2015 Received in revised form 14 July 2015 Accepted 18 July 2015 Available online xxx
The poly(butylene succinate) (PBS)/thymol film was successfully prepared by using a blown film extruder at five different thymol concentrations ranging from 2 to 10 wt%. Experimental results indicated that PBS was softer and tougher due to the plasticization effect derived from thymol. The oxygen transmission rate (OTR) increased slightly with increasing thymol content due to the increased amorphous region in PBS structure. Under heating process, the blends exhibited lower crystallization temperature (Tc), enthalpy of crystallization (DHc), enthalpy of melting (DHm) and degree of crystallinity (Xc) than that observed in neat PBS. Thymol was found to effectively inhibit foodborne pathogens growth. Its antimicrobial activity against Staphylococcus aureus was evidence at 6 wt% while Escherichia coli did at 10 wt% thymol. Over 15 days studied, release of thymol showed some differences depend on food simulant. Maximum migration was obtained when the film was immersed in isooctane at all test duration. Release kinetics indicated that the incorporation of 10 wt% thymol to PBS films were effective over 15 days. © 2015 Elsevier B.V. All rights reserved.
Keywords: Polymers Differential scanning calorimetry (DSC) Mechanical testing
1. Introduction Nowadays, food packaging is one of the major plastic wastes accumulating in the environment due to its non-biodegradabillity [1,2]. Major part of this waste comes from short-serviced life food such as dairy, meat and vegetable products. In recent years, the utilization of biodegradable polymers has drawn much attention from both industries and research institutions especially for non-
* Corresponding author. E-mail address: [email protected] (N. Petchwattana).
durable applications [2e4]. Poly(butylene succinate) (PBS) is one of the biodegradable polymer derived from glucose fermentation and butanediol. Literature reviews indicated that PBS is one of a tough polymer with mechanical and thermal properties comparable to some petroleum based polymers [4]. Thus, PBS is possibly to be one of the commercial biopolymer in the near future. During storage, some properties of food usually change due to the microbiological activity and others [2,5]. These changes allow further undesired deteriorations and consumer rejection [5e8]. To solve these problems, the preservatives have been applied to retard the food spoilage [9e11]. However, most of them affected the taste, the appearance or the odor of foods.
http://dx.doi.org/10.1016/j.matchemphys.2015.07.052 0254-0584/© 2015 Elsevier B.V. All rights reserved.
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In the past, the interaction between food and package was avoided due to its possibility of changing the food quality [5e8]. To date, it has been proved that some interactions do not affect the quality of food but capable to retard the food deterioration [6,7]. Antimicrobial packaging technology is one of the novel concepts which provide the interaction between food and packaging material while maintaining nutritional and sensorial qualities as well as safety [5e8]. This technology has drawn much attention from many researchers to produce polymer based antimicrobial packaging especially the use of bio-based essential oil and biopolymer. Essential oil was initially used in Egypt, India and Persia more than 2000 years ago. Its antimicrobial inhibitory was discovered around a century ago [12]. Nowadays, many types of essential oils were used as antimicrobial agent in food packaging materials. The essential oil extracted from cinnamon was used as an antimicrobial agent in poly(ethylene terephthalate) (PET) film. It was found that the active PET showed the inhibition of Aspergillus flavus growth [13]. After incorporated to poly(lactic acid) (PLA)/poly(trimethylene carbonate) (PTMC) film, thymol was found to inhibit the growth of Escherichia coli, Staphylococcus aureus, Listeria, Bacillus subtilis, and Salmonella [14]. Thymol (2-isopropyl-5-methylphenol) is an essential oil extracted from thyme (Thymus vulgaris), onions (Allium cepa), garlic (Allium sativum) or other plants. It has been utilized as antimicrobial agent in polymer film due to its efficiency and physical properties suitable for blown film process. Numerous reports have found the potential of thymol when it was incorporated in polymer films. Rota et al. [15] concluded that thymol was an effective antimicrobial agent for preserving the food spoilage and increasing the shelf-life. Under microencapsulated condition, thymol showed significant inhibited the Saccharomyces cerevisiae, Listeria innocua, E. coli and S. aureus growths [16]. Although many literatures have reported the antimicrobial efficiency of thymol but no report exists in the PBS/thymol blends. This study focuses on the development of PBS films with thymol. A blown film process was employed to produce the active PBS/ thymol films. Evaluation of the E. coli and S. aureus inhibitory action was compared with various thymol concentrations ranging from 2 to 10 wt%. Other characterizations were carried out by determination tensile, thermal and oxygen barrier properties and release kinetics of the films. 2. Materials and methods 2.1. Materials and processing A blown film grade PBS (FZ91PD) was used as a polymer matrix. Its melting temperature and melt flow rate were 110 C and 6 g/ 10 min respectively. Thymol was selected as an antimicrobial agent to reduce the growth rate of E. coli and S. aureus. Fig. 1 illustrates the chemical structure of (a) PBS and (b) thymol. Formulations of PBS were prepared with various thymol concentrations of 2, 4, 6, 8 and 10 wt%. Thymol was firstly dry-blended with the PBS by using a high speed mixer (Thermo Prism Pilot 3) at 500 rpm for 30 s to disperse thymol powder in PBS matrix. The dryblended compositions were then melt-blended by using a twin screw extruder (Labtech Engineering, LTE20-40). The barrel temperature was set at 100e150 C and at the screw speed of 100 rpm. Each formulation was then pelletized and blown to obtain film of 100 mm in thickness for testing and characterizations. 2.2. Microorganisms Microorganisms obtained from the culture collection of the
Fig. 1. Chemical structure of (a) PBS and (b) thymol.
Thailand Institute of Scientific and Technological Research (TISTR) included E. coli (TISTR 780) and S. aureus (TISTR 1466). 2.3. Testing and characterizations Tensile test was conducted by using a Universal testing machine (Instron 5567) equipped with a 1 kN load cell performed on rectangular films of 10 100 mm2. Tensile modulus, tensile strength and tensile elongation at break were determined from the stressestrain curves according to ASTM D882. Test results were the average of five replicated specimens. A dart drop film impact test was conducted in accord with ASTM D 1709. An oxygen permeation tester (Mocon OX-TRAN, 2/21) was employed to measure the oxygen transmission rate (OTR) according to ASTM D 3985 with an oxygen flow rate of 40 cm3/min at 23 C and 0% relative humidity. To estimate the antimicrobial efficiency of thymol entrapped in PBS films, E. coli (gram-negative, TISTR 780) and S. aureus (gram-positive, TISTR 1466) were selected and measure the viability by using the agar diffusion method. The inhibition zone was determined after the PBS/thymol films were placed on the agar surface at 37 C after 24 h incubation. A differential scanning calorimeter (DSC) (PerkinElmer, DSC6000) was employed to evaluate the transition temperatures of PBS and PBS/thymol blends under nitrogen atmosphere. At the first heating step, the sample was heated from 30 to 150 C at a heating rate of 10 C/min to remove the thermal history and followed by the isothermal holding at 150 C for 10 min. The sample was then cooled from 150 to 30 C at the identical heating rate. A second heating was then performed at the same conditions as the first heating. Finally, samples were cooled to room temperature. The degree of crystallinity (Xc) was calculated by using Equation (1) [17].
Xc ¼
DHc 100 DHf XPBS
(1)
where DHc and XPBS are the crystallization enthalpy and mass
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Table 1 Effect of thymol on the mechanical properties of PBS. Thymol content (wt%)
Film impact strength (J/m)
Neat PBS 2 4 6 8 10
22.49 24.76 27.32 29.54 30.41 32.11
± ± ± ± ± ±
Tensile modulus (GPa)
0.53 0.60 0.39 0.51 0.20 0.98
0.72 0.73 0.64 0.58 0.53 0.48
± ± ± ± ± ±
fraction of PBS respectively. DHf is the heat of fusion, defined as the melting enthalpy of 100% crystalline PBS, which was 110.3 J/g [18]. 2.4. Migration tests 2.4.1. Release tests The release of thymol from PBS films was conducted in five food simulants namely distilled water, acetic acid 3%, ethanol 10%, ethanol 95% and fatty acid isooctane. The test procedures and conditions were performed in accord with EN 13130-2005 standard. Quantitative evaluation of the release was extracted from the samples immersed in the food simulants at 2, 6, 12, 24, 48 h and 5, 10 and 15 days [9] by using a High Performance Liquid Chromatography (HPLC, Agilent 1100 series). The mobile phase composed of 0.05 M ortho-phosphoric acid:acetronitrile, 40:60 (vol/vol). The injection volume and the flow rate were 5 ml and 1 ml/min respectively. 2.4.2. Release kinetics To estimate the release kinetics of thymol, the mass transport parameters were calculated by means of Fick's law of diffusion. The assumptions for this equation are i) the release is controlled by means of Fickian diffusion, ii) the thymol in PBS film is homogeneously dispersed, iii) the initial concentration of thymol at all five food simulants is zero, iv) no interactions between food simulant and PBS/thymil film and v) no degradation of thymol and PBS during the test duration. The diffusion coefficient (D, m2/s) of thymol was determined by using Equations (2)e(4) [19,20].
"
∞ X MF;t 2að1 þ aÞ ¼1 exp MF;∞ 1 þ a þ a2 q2n n¼1
# Dq2n t
39.98 35.64 31.05 26.95 24.77 23.03
± ± ± ± ± ±
1.25 0.90 0.87 0.95 0.67 0.49
Elongation at break (%) 17.46 17.10 19.30 21.85 21.05 22.58
± ± ± ± ± ±
1.46 1.56 1.35 1.60 1.67 2.90
partition coefficient of the active compound between the food simulant and the PBS which can be calculated by using Equation (4). CF,∞ and CP,∞ are the concentrations of thymol in the food simulant and in the PBS at equilibrium respectively. VF and VP are the volumes of the food simulant and the PBS respectively. qn is the positive roots of tan qn which can be determined by using Equation (5). The determination was done by plotting f(qn) ¼ tan qn þ aqn as a function of qn and then observing the points where f(qn) become zero [3]. Equation (2) was simplified and proposed by Chung et al. [9,10]. Thus the diffusion coefficient, D, value can be estimated from Equation (6) [20].
1 1 MF;t $ p a MP;0
0:5
D0:5 0:5 1 ¼ $t þ 0:5 a$LP p
(6)
where MP,0 is the initial amount of migrant in the PBS. For complete migration, partitioning and resistance to mass transfer are negligible. MF,∞ is equals to MP,0 [19]. In case of the ratio MF,t/MP,0 not higher than 0.6, Equation (6) can be simplified to Equations (7) and (8), applied for short and long contact times respectively.
MF;t 2 Dt ð0:5Þ ¼ MP;0 LP p
(7)
! MF;t 8 p2 Dt ¼ 1 2 exp MP;0 p 4L2P
(8)
(2)
L2p
3. Results and discussions
K V a ¼ FP F VP KFP ¼
Tensile strength (MPa)
0.07 0.11 0.78 0.50 0.41 0.60
(3)
CF;∞ CP;∞
(4)
tanqn ¼ aqn
(5)
where MF,t and MF,∞ are the mass of the migrant in the food at a particular time t and at equilibrium respectively. MF,∞ can be assumed to be equal to MP,0. LP is a film thickness. KFP is the
3.1. Mechanical properties of PBS/thymol films The tensile and impact properties of PBS/thymol blends, as well as of pure component, are listed in Table 1. The tensile modulus and the tensile strength of neat PBS were 0.72 and 39.98 MPa respectively. Adding thymol to PBS reduced both the tensile modulus and tensile strength by around 10e40% depending on thymol content. Compared to neat PBS, the blends were less stiff due to the plasticization effect derived from thymol. This allowed PBS to be stretched or deformed easier than the un-modified one. With the presence of 10 wt% thymol, the tensile elongation at break
Table 2 Effect of thymol on the OTR of PBS. Thymol content (wt%)
Thickness (mm)
Neat PBS 2 4 6 8 10
101 100 102 101 102 101
± ± ± ± ± ±
1.02 0.92 1.12 1.30 0.81 0.65
Oxygen transmission rate (ml mil/(m2 day atm)) 63.19 65.52 66.79 69.50 70.30 70.35
± ± ± ± ± ±
0.33 0.12 0.19 0.20 0.50 0.35
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elongated at higher rate and longer distance. Numerous literatures have found the plasticization effects of the essential oils on the food packaging films. In polypropylene (PP) film, the plasticization effect was found to decrease the tensile strength and increase the elongation at break [20]. Kavoosi et al. [23] incorporated 8 wt% thymol to gelatin film and found the reduction of the tensile strength from 2.9 to 1.2 N/m2. Furthermore, the tensile elongation at break was raised from 57 to 170%. Low density polyethylene (LDPE) was also less stiff when carvacrol was added as an antimicrobial agent [24]. Due to the plasticization effect, PLA was also softer and tougher when thymol [25] and limonene [26] were incorporated. 3.2. Barrier and thermal properties PBS/thymol films
Fig. 2. DSC thermograms of neat PBS and PBS/thymol blends (a) crystallization exotherm and (b) melting endotherm.
increased from 17.46 for neat PBS to 22.58%. Thymol was believed to behave like plasticizer when it was blended to PBS. It penetrated between the PBS chains and reduced the intermolecular forces making them disentangle easier [21,22]. This allowed PBS could be
Barrier property is one of the most important factors for food packaging which protect or reduce food contamination from the external contaminants [27]. As exhibited in Table 2, Neat PBS showed the OTR value of 63.19 g mil/(m2 day atm). Of the thymol concentration studied, the OTR tended to increase slightly and obtain the maximum value at 10 wt% thymol. Due to the plasticization effect, the amorphous segment in the PBS was increased thereby allowing the oxygen molecules to transport easier through the free volumes. M. Ramos et al. [9] produced antimicrobial PP film with essential oil (thymol and carvacrol) for food packaging application. They found that the OTR was increased with increasing thymol and carvacrol concentrations. Both essential oils were believed to have modified PP chain structure and consequently reduced the oxygen permeation resistance. In the starch/glycerol blends, the oxygen permeability was linearly increased with increasing glycerol content [28]. In general, the oxygen permeation in a semi-crystalline polymer like PBS is primarily a function of the amorphous phase, while the crystalline section is regularly assumed to be impermeable [29,30]. To confirm this structure-property relation, it is necessary to observe the change in crystallization of the PBS/thymol blends. Fig. 2 illustrates the heating and cooling thermograms of neat PBS and PBS/thymol blends. The data of the crystallization temperature (Tc), crystallization enthalpy (DHc), melting temperature (Tm), enthalpy of melting (DHm) and degree of crystallinity (Xc) for the neat PBS and PBS/thymol blends were indicated in Table 3. As shown in Fig. 2(a), blending thymol to PBS decreased both Tc and DHc compared to that of neat PBS. Tc reached the minimum value of 82.06 C when thymol was added at 10 wt%. This confirmed that the plasticizing effect lubricated the PBS chain and facilitated higher mobility [30]. As illustrated in Fig. 2(b), neat PBS showed double melting peaks, due to two different crystal structures, at 100.78 (Tm1) and 112.93 (Tm2). Vega-Baudrit et al. [17,31] explained that lower endotherm (Tm1) is relates to the melting of the original crystallites and higher endotherm (Tm2) corresponds to the melting of the recrystallized one. With increasing the thymol content, both Tm1 and Tm2 tended to shifted but only minutely. The Tm1 was found to shift to higher position around 4 C while Tm2 did at only 1 C. With increasing thymol content, total enthalpy of melting was decreased gradually from 57.44 to 47.04 J/g. This related to the
Table 3 Crystallization temperature (Tc), crystallization enthalpy (DHc), melting temperature (Tm) and melting enthalpy (DHm) for the neat PBS and PBS/thymol blends. Thymol content (wt%)
Tc ( C)
DHc (J/g)
Tm1 ( C)
Tm2 ( C)
DHm (J/g)
Xc (%)
Neat PBS 2 4 6 8 10
84.10 84.05 83.52 82.97 82.56 82.06
61.33 60.30 59.24 58.77 57.70 57.09
100.78 100.71 100.97 102.62 104.76 104.60
112.93 112.65 112.61 112.58 112.41 112.05
57.44 53.67 51.89 51.31 49.08 47.04
55.60 54.67 53.71 53.28 52.31 51.75
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Fig. 3. Antimicrobial activities of PBS and PBS/thymol blends against E. coli and S. aureus.
increased amorphous region of the blends which could be confirmed by the reduced Xc. This is in agreement with the work on PBS plasticization reported by Zhao and co-workers [32]. They concluded that blending the epoxidized soybean oil to PBS tended to increase the amorphous region of the blends enhancing the PBS chain mobility.
containing 10 wt% thymol was the most effective in the E. coli (gram-negative) growth inhibition. At this thymol content, the clear zone was estimate by only 2 cm. Lower than this concentration, no inhibition zone was observed. Another antimicrobial activity was performed against S. aureus (gram-positive). The clear zone was observed by around 1.8 cm when thymol was blended at 6 wt%. Beyond this concentration, the growth inhibition zone
3.3. Antimicrobial activity of PBS/thymol films Fig. 3 illustrates the antimicrobial activity performed against foodborne pathogens namely S. aureus and E. coli. The antimicrobial evaluation was performed by placing the PBS/thymol film in direct contact with pathogenic bacteria. Further investigation was made by measuring clear zone produced by a film containing thymol and clearly shown in Table 4. Under the test duration, thymol was expected to release from PBS into the agar and then produced the inhibition zone around the active films. The minimum inhibition concentration (MIC) value of thymol against E. coli and S. aureus was 10 and 6 wt% respectively. As illustrated in Fig. 3(a)e(f), PBS
Table 4 Inhibition zone diameter of the PBS/thymol films. Thymol concentration (wt%)
Inhibition zone diameter (cm) E. coli
S. aureus
Neat PBS 2 4 6 8 10
nd nd nd nd nd 2.09 ± 0.11
nd nd nd 1.83 ± 0.27 4.43 ± 0.10 5.52 ± 0.06
nd: No inhibition zone detected.
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Fig. 4. Release of thymol from PBS into different food simulants over 15 days (a) distilled water, (b) acetic acid, (c) isooctane, (d) ethanol 10% and (e) ethanol 95%. (C) Experimental data and (d) Simulation results obtained from Equation (2).
gradually increased by 4.4 and 5.5 cm for 8 and 10 wt% thymol respectively. In comparison, the film was found to have lower antimicrobial performance against E. coli. This was expected because of the outer membrane of the gram-negative bacteria helped them stronger. To overcome this feature, larger amounts of antimicrobial agent is required. In PP, thymol was also found to inhibit the S. aureus growth. The inhibition clear zone diameter of 3.7 cm was observed when 8 wt% thymol was added [9]. Thymol was also found to inhibit the E. coli, S. aureus, Listeria, B. subtilis and Salmonella. However, the inhibition was directly depended on thymol concentration [14]. 3.4. Release kinetics of thymol from PBS films The fractional release (MF,t/MP,0) versus time curves in Fig. 4 was obtained by fitting Equation (2) to the experimental data. It shows the migration of thymol from PBS films with various incubation times and food simulants i.e. distilled water, acetic acid, isooctane, ethanol 10% and ethanol 95%. Good agreements were obtained between experimental data and proposed model prediction. This suggested that the release kinetic could be described by the Fick's law of diffusion. With increasing the incubation time, thymol was rapidly migrated from PBS into the food simulants. It required 50e60 h to reach equilibrium at all food simulants studied. The type of food simulants were expected to be the key factor which influence the migration [20]. At thermodynamic equilibrium,
isooctane allowed thymol to migrate with the largest fractional release at around 0.8. This was expected because of their identical polarity. Several approaches have been reported to explain the phenomenon occurred in the release of an active compound from a polymer matrix. Suppakul et al. [33] proposed the swellingcontrolled model of the polyethylene (PE)/antimicrobial agent blends when they were immersed in the food simulant. This model indicated that the food simulant was firstly diffused into PE matrix and then dissolved the antimicrobial agent thereby enabling its migration. In zein film/thymol system, the thymol release kinetics took place over three steps namely; the diffusion of food simulant, macromolecular matrix relaxation kinetic and diffusion of the active compound through the swollen polymeric network [34]. In this case, the release of thymol from PBS matrix took place over three steps like other systems discussed above. The first one was the penetration of the food simulant molecules through the Table 5 Estimated diffusivities for PBS films containing thymol. Food simulant
Diffusivity (m2/s)
Distilled water Acetic acid Isooctane Ethanol 10% Ethanol 95%
1.31 2.23 8.43 9.70 5.86
1015 1015 1014 1015 1014
Please cite this article in press as: N. Petchwattana, P. Naknaen, Utilization of thymol as an antimicrobial agent for biodegradable poly(butylene succinate), Materials Chemistry and Physics (2015), http://dx.doi.org/10.1016/j.matchemphys.2015.07.052
N. Petchwattana, P. Naknaen / Materials Chemistry and Physics xxx (2015) 1e7
PBS. Secondly, the thymol molecules were dissolved in the food simulants thereby transferring mass to the PBS surface. Finally, the dissolved thymol was completely migrated to the food simulants until the thermodynamic equilibrium was reached. This release was facilitated by the plasticization effect derived from thymol which could be confirmed by the decreased Xc. The diffusivity (D), in Table 5, shows that the diffusion process in different food simulants was ranging from 1.31 1015 to 8.43 1014 m2/s. Maximum D was found in isooctane due to its non-polarity like thymol. The identical polarity facilitated thymol to dissolve easier and faster mass transport. Compared to other antimicrobial polymer systems, PBS/thymol blends met the needs of short cycle food packaging application such as meat, vegetable and fruit products. Antimicrobial activity of thymol was found to active over 15 days studied and the MIC against E. coli and S. aureus were 10 and 6 wt% respectively. Mastromatteo et al. [35] applied thymol-coated polystyrene as shrimp package. They found that the antimicrobial activity was effective at around 14 days. In polypropylene/thymol blends, the MIC against S. aureus was observed at 8 wt% [9]. In zein film, thymol was released in water over 4 days studied and the release rate depended on the thymol concentration [34]. In PLA, the inhibition clear zone was found when thymol was added at 8 wt% [25]. 4. Conclusions In the current paper, thymol was melt-blended with PBS to produce the antimicrobial food packaging. The film was softer and tougher due to the plasticization effect derived from thymol. The OTR tended to increase slightly with increasing thymol content due to the increased amorphous region. Thymol was found to effectively inhibit foodborne pathogens growth. Its antimicrobial activity against S. aureus was evidence when 6 wt% while E. coli did at 10 wt% thymol. Over 15 days studied, release of thymol showed some differences depend on food simulant. Maximum migration was obtained when the film was immersed in isooctane at all test duration. Release kinetics indicated that the incorporation of 10 wt % thymol to PBS films were effective over 15 days. Acknowledgments The authors would like to acknowledge the research grant supported by Srinakharinwirot University (Contract no. 143/2556144/2556). Thanks are extended to Mr. Jirawat Mala and Miss Sasinee Wiburanawong for the antimicrobial and the OTR test.
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Please cite this article in press as: N. Petchwattana, P. Naknaen, Utilization of thymol as an antimicrobial agent for biodegradable poly(butylene succinate), Materials Chemistry and Physics (2015), http://dx.doi.org/10.1016/j.matchemphys.2015.07.052