Development of PLA-PBSA based biodegradable active film and its application to salmon slices

Food Packaging and Shelf Life 22 (2019) 100393

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Development of PLA-PBSA based biodegradable active film and its application to salmon slices Chunxiang Yanga, Haibing Tanga, Yifen Wangb, Yuan Liuc, Jing Wanga, Wenzheng Shia, Li Lia,

T ⁎

a

Engineering Research Center of Food Thermal-Processing Technology, College of Food Science and Technology, Shanghai Ocean University, Shanghai, 201306, China Biosystems Engineering Department, Auburn University, Auburn, AL, 36849-5417, USA c School of Agriculture and Biology, Shanghai Jiao Tong University (SJTU), Shanghai, 200240, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Biodegradable films Active packaging Release kinetics Carvacrol Thymol Salmon slice

Due to negative impacts of non-biodegradable plastic films to the environment, biodegradable packaging materials have become a research hotspot. Novel biodegradable active films based on polylactic acid (PLA) blended with poly (butylene succinate adipate) (PBSA), carvacrol, and thymol were developed. Mechanical properties of films including tensile strength (TS), elongation at break (EAB), optical properties including transmittance (T) and haze (H), barrier properties including water vapor permeability (WVP), oxygen transmission rate (OTR), and releasing rates of active compound from films and antioxidant efficiency of films were investigated to evaluate the films. A shelf life test of salmon slices packed with PLA-PBSA bags (with and without active compound) were carried out. The results showed that PLA-PBSA based films had better mechanical properties than those of similar biodegradable films such as PLA-PHB, and even better than EVOH in terms of two major mechanical properties, TS and EAB. As to active properties, the results also showed a high release of active compound and high antioxidant efficiency of PLA-PBSA films with either carvacrol or thymol. The shelf life test of salmon slices showed the antibacterial and antioxidant properties of the PLA–PBSA films were enhanced when the active compound were released into salmon slices at equilibrium. As a result, spoilage and deterioration of the salmon slices were reduced, which extended the shelf life of salmon slices by 3–4 days during cold storage. Thus, the biodegradable PLA–PBSA films with active compound could prolong shelf life of fisheries products in particular, and food in general.

1. Introduction Active food packaging systems are designed to protect food from the environment and maintain food quality and safety. These systems are divided into two main categories, namely, nonmigratory and migratory (Mastromatteo, Mastromatteo, Conte, & Del Nobile, 2010). Nonmigratory active packaging is prepared by grafting active compound onto the surface of a film. On the other hand, in migratory active packaging, active compound are added to the film and can be released from the film onto the surface of the food. There are numerous studies focusing on simulating the release of active compound (antibacterial agent or antioxidant) into food simulants (Chang et al., 2016; De Dicastillo et al., 2011; Lopez, Dicastillo, Vilariño, & Rodríguez, 2013; Wu et al., 2017). However, the active compound released from packaging films into real food have been rarely investigated (García Ibarra, Sendón, & Rodríguez-Bernaldo de Quirós, 2016). Therefore, the releasing model for active compound corresponding to the effect on

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food preservation should be investigated. In the modern food industry, essential oil extracted from plants are frequently incorporated into polymer materials as active compound for active food packaging systems to protect food from environmental impact and to control the growth of microorganisms in food (Alboofetileh, Rezaei, Hosseini, & Abdollahi, 2014; Boumail et al., 2013; Chen, Li, Ma, McDonald, & Wang, 2019; Ma, Li, & Wang, 2017). Carvacrol and thymol, the major components of oregano essential oils, are legally registered as flavoring substances with reported antibacterial properties (Krepker et al., 2017). These components offer high natural potential for preservation and antioxidation of perishable food (Hosseini, Rezaei, Zandi, & Farahmandghavi, 2015). With the current increasing level of global plastic pollution in landscapes and bodies of water, biodegradable packaging materials have become a research hotspot (Geyer, Jambeck, & Law, 2017; Hillmyer, 2017). The most widely accepted environmentally friendly polymer materials are synthetic biodegradable polymers, such as

Corresponding author. E-mail address: [email protected] (L. Li).

https://doi.org/10.1016/j.fpsl.2019.100393 Received 28 September 2018; Received in revised form 24 August 2019; Accepted 26 August 2019 2214-2894/ © 2019 Elsevier Ltd. All rights reserved.

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if the active compound was equal to or more than 10%, film could not be casted due to the liquidish condition of the raw material. If the active compound was equal to or less than 6%, the release of active compound from films into food simulants was not efficient. Therefore, the following ingredients of films A–C were selected with film A as control: A: 90 wt% PLA +10 wt% PBSA B: 82.8 wt% PLA +9.2 wt% PBSA +8 wt% Carvacrol C: 82.8 wt% PLA +9.2 wt% PBSA +8 wt% Thymol

polylactic acid (PLA), poly (hydroxybutyrate) (PHB) and poly (butylene succinate adipate) (PBSA). Besides biodegradability and compostability, PLA pocesses relative good mechanical properties such as tensile strength (TS) and elongation at break (EAB) but it is brittle if it is used alone (Ma, Li, & Wang, 2018). PLA modification by blending with either PHB or PBSA brings some advantages because the latter ones can improve the physical and mechanical properties of pure PLA (Ma et al., 2018; Pradeep et al., 2017). To the best of our knowledge, there is study only on thermal stabilities of PLA/ PBSA blends, not on physical and mechanical properties for food packaging purpose so far. Therefore, the main objective of this work was to develop biodegradable active films based on PLA-PBSA containing active compound for aquatic products preservation in particular, and food in general. In order to fulfill the main objective, the following sub-objectives were carried out: a) preparation of PLA-PBSA based films containing 8 wt% of active compound (either carvacrol or thymol); (b) investigation of scanning electron microscopy (SEM) image and fourier transform infrared spectroscopy (FTIR) spectra, their physical and mechanical properties such as TS, EAB, barrier properties including water vapor permeability (WVP), oxygen transmission rate (OTR), optical properties such as haze (H) and transmittance (T); (c) determination of the release of active compound from films into 4 different food simulants and its antioxidant efficiency; and (d) the correlation between the release process into salmon slices and the preservation of salmon slices.

2.4. Film characterization 2.4.1. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) The cross-sections of the films were coated with a gold layer of 5 nm (50 s at the rate of 0.1 nm/s) and images were observed by SEM under high-vacuum mode at an operating voltage of 5 kV (SEM, Hitachi S3400, Tokyo, Japan). The image scales were set at 1:1000 and 1:5000, respectively. The FTIR spectras of the PLA–PBSA films were obtained between wavenumbers 500 and 4000 cm−1 using a Nicolet Nexus Avater 370 FTIR spectrophotometer (Thermo Nicolet Corporation, Madison, WI, U.S.A.) to assess the chemical structure of the films. The absorption bands of the main and additional functional groups were identified and observed for any optical changes (Samsudin, Soto-Valdez, & Auras, 2014).

2. Materials and methods 2.4.2. Mechanical and optical properties The tensile strength (TS) and elongation at break (EAB) of the films (10 cm × 2.5 cm) were measured at 25 °C and 100% RH by a XLW (EC)1502 auto tensile tester (Labthink Instrument Co., Ltd., Jinan, China) in accordance with ASTM D882-12(2012) (ASTM, 2012). In accordance with ASTM D1003-00(2000) (ASTM, 2000), the optical properties, including the transmittance (T) and haze (H) of the films, were measured with a WGT/S haze and transmittance testing machine (Shanghai Precision Instrument Co., Shanghai, China).

2.1. Chemicals and reagents PLA (PLA 2003D, Mn = 98,000 g/mol, 4 wt% D-isomer) was supplied by NatureWorks (Minnesota, MN, U.S.A.), and PBSA was purchased from Showa Denko (Tokyo, Japan). Carvacrol (≥ 98%) and thymol (≥ 98%) were supplied by Aladdin (Shanghai, China). Highperformance liquid chromatography (HPLC) grade acetonitrile, acetic acid, ethanol, boric acid, methyl red, bromocresol green, light magnesium oxide, thiobarbituric acid, sodium chloride and 2,2-diphenyl-1picrylhydrazyl (DPPH) 95% free radical were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Salmon was purchased from RT-Mart supermarket in Pudong New District (Shanghai, China). Water was purified by a Milli-Q Plus purification system (Millipore, Shanghai, China).

2.4.3. Barrier properties The water vapor permeability (WVP) of the films was determined at 37 °C and 100% RH with PERMATRAN-W1/50 G (Mocon, Minn., Germany) in accordance with ASTM E398-13 (2013) (ASTM, 2013). Oxygen transmission rate (OTR) was determined at 23 °C and 50% RH with a G2/132 oxygen permeation analyzer (Labthink Instruments Co., Ltd., Jinan, China).

2.2. HPLC system and operating conditions

2.4.4. Thermal analysis Differential Scanning Calorimetry (DSC) of the samples were determined using a Q2000 DSC from TA Instruments (Delaware, U.S.A.) under nitrogen atmosphere (50 mL min−1), with the samples (8 mg) placed in hermetic aluminum pans. Thermal analysis was conducted to investigate the influence of the addition of active compound on the thermal stability of the PLA-PBSA matrix. The first stage of heating was from 25 °C to 250 °C with 10 °C min−1 heating rate, followed by cooling to 25 °C at 30 °C min−1 and a second heating stage to 250 °C. Glass transition temperature (Tg), crystallization temperature (Tc), and melting temperature (Tm) were obtained in the second heating stage (De Dicastillo, Pernas, Lopez, Vilarino, & Rodriguez, 2013).

The concentrations of active compound released into simulants and salmon slice were quantified using a Waters 2695 HPLC equipped with a 2998 photodiode array detector (Model 2998, SpectraLab Scientific Inc., Toronto, Canada) at 274 nm for carvacrol and thymol on the basis of standard curves made from known concentrations (0–250 mg/kg). Standards and extracts were analyzed using an HPLC on a Sunfire C18 column (4.6 mm × 150 mm, 5 μm particle) (Waters, Massachusetts, U.S.A.) with 10 μL of mobile phase consisting of acetonitrile and Milli-Q water (3:2, v/v; 0.1% acetic acid–water solution (v/ v)) at a flow rate of 1.0 mL/min by gradient elution. The column temperature was 30 °C ± 1 °C, and the analysis time was 10 min. Quantitative analysis of carvacrol and thymol were based on an external standard method. 2.3. Film preparation

2.5. Release of active compound into food simulants and antioxidant efficiency of films

Films A–C with average thickness of 60 μm, 58 μm and 60 μm were prepared by extrusion-casting method. Screw rotation rate was set at 45 rpm, and the temperature levels in the seven heating zones of the corotating twin-screw extruder were set at 90 °C, 165 °C, 170 °C, 170 °C, 170 °C, 170 °C, and 165 °C. All film samples were stored in aluminum foil bags for short time before analysis. Based on our preliminary tests,

2.5.1. Release tests into food simulants For assessing the release of carvacrol and thymol from the active PLA–PBSA films into the food simulants, pure water, 3% acetic acid (v/ v), 10% ethanol (v/v), and 95% ethanol (v/v) were selected as simulants of aqueous food, acidic food, alcoholic food, and fatty food, respectively. The film samples (100 mm × 150 mm) were placed in 2

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Fig. 1. HPLC chromatogram of 10 mg/kg carvacrol and thymol standard.

500 mL conical flasks in contact with 250 mL of each simulants for maceration in the dark (Hu et al., 2017). Exactly 1 mL of film sample extract was removed and filtered through a 0.22 μm nylon membrane filter at selected time intervals and analyzed by HPLC (De Dicastillo et al., 2013). An HPLC chromatogram of 10 mg/kg carvacrol and thymol standard was shown in Fig. 1. The experimental data of release were fitted to Eq. (1) (Siepmann & Peppas, 2011) to investigate the dynamics involved in the release process of active compound through the contact of PLA–PBSA films with different simulants.

2.5.2. Antioxidant properties The antioxidant effectiveness of the films was measured according to 1,1-diphenyl-2-picrylhydrazyl (DPPH•) method. DPPH radicalscavenging activity in the presence of the film extract solution (Sakanaka, Tachibana, Ishihara, & Juneja, 2004) was monitored at 517 nm by a UV–vis spectrophotometer (Hitachi Ltd, Tokyo, Japan). The film was extracted in 95% ethanol at 65 °C for 3 h. The antioxidant activities of the films were determined by DPPH• method, and scavenging activity was obtained using Eq. (3):

SA (%) =

MF , t = kt α MF , ∞

(1)

∞

∑n=0

8 ⎡ (2n + 1)2π ⎤ exp ⎢− Dt⎥ 2 2 (2n + 1) π Lp2 ⎣ ⎦

Ao

× 100%

(3)

where Ai – absorbance of the DPPH solution mixed with the film extract solution Aj – absorbance of the film extract solution mixed with 95% ethanol Ao – absorbance of the DPPH solution mixed with 95% ethanol

where MF,t is the concentration of the active compound in the release medium over time, MF,∞ is the maximum concentration of the active compound in the release medium at equilibrium, k is the rate of mass transfer per unit area per unit concentration difference incorporating the characteristics of the matrix during the diffusion process, and α is the diffusional exponent providing information about the dynamics involved in release process. If α is 0.5, then the release occurs through Fick’s diffusion, whereas if α is lower than 0.5, then quasi-Fick’s diffusion can be considered for the active release (Requena, Vargas, & Chiralt, 2017). Similarly, the diffusion of carvacrol and thymol from films into simulants were determined using Eq. (2), which is based on Fick’s second law (Lopez et al., 2013). This equation describes the release rate of active compound from films into simulants in contact with the polymer:

MF , t =1− MF , ∞

1 − (Ai − Aj )

2.6. Preservation and characterization of salmon slices 2.6.1. Salmon slices Skin, bones, and guts of fresh salmon were removed and the meat was sliced into uniform weight (50 ± 1 g) each for 81 slices. These 81 slices of salmon meat were packed into 81 PLA-PBSA bags (3 types of films × 3 parallel experiments × 9 days of storage) and stored at 4 °C ± 1 °C in a refrigerator for the following tests. 2.6.2. Release tests on salmon slices The release of carvacrol and thymol from the active PLA − PBSA films to salmon slices was investigated through a preservation experiment at 4 °C ± 1 °C. All salmon slices samples and film samples were accurately weighed. After 1, 2, 3, 4, 5, 6, 7, 8, and 9 days of contact between the active PLA–PBSA films and the salmon slices samples, the samples were retrieved and homogenized with FJ200-SH homogenizer

(2)

Where D (m2 s−1) is the diffusion coefficient, t is time (s), and Lp is film thickness (m). 3

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The FTIR spectra of the control PLA–PBSA film (Fig. 3(1)) showed bending at 1748 cm−1 due to C]O carbonyl stretching. CHe bending was also observed at 1451, 1080, and 1180 cm−1, which corresponds to CeOCe symmetric and asymmetric stretching vibrations (Tawakkal, Cran, & Bigger, 2016). Similar bending characteristics were observed in the FTIR spectra of the active PLA–PBSA films. In addition, bending appeared close to 808 cm−1, which corresponds to CHe bending of the rings in the structures of carvacrol and thymol because they are isomers. This bending indicated that carvacrol and thymol were successfully incorporated into the PLA–PBSA films without chemical modification and these substances showed good miscibility (Fig. 2) (Alvarado et al., 2017; Chieng, Ibrahim, Then, & Loo, 2014).

(Shanghai Specimen and Model Factory, Shanghai, China). Salmon fish paste (5 g) was placed into a 50 mL centrifuge tube with 20 mL of acetonitrile and centrifuged for 6 min at 8000 rpm by using a highspeed refrigerated centrifuge (Xiangyi Centrifuge Instrument Co., Ltd. Xiangyi, China) (Armorini et al., 2016). Subsequently, acetonitrile extract was removed, and the centrifugation process was repeated. Next, two batches of acetonitrile extracts were combined and maintained at a constant volume in a 50 mL volumetric flask. After the acetonitrile extract solution was evaporated to almost dry by a rotary evaporator (Shensheng Technology Co., Ltd., Shanghai, China), the dried residue was dissolved by 5 mL of acetonitrile, and filtered through a 0.22 μm nylon membrane filter and subjected to HPLC for analysis (Otero-Pazos et al., 2014). All assays were conducted in triplicate. Acetonitrile was selected as solvent owing to its features as a powerful extractor of active compound.

3.1.2. Mechanical and optical properties The mechanical and optical properties of the PLA–PBSA films are given in Table 1. The TS and EAB of PLA-PBSA film (Film A) were 48.61 ± 1.22 MPa and 55.70 ± 3.56%, respectively, which were greater than TS (30.8 ± 2.0 MPa) and EAB (13.6 ± 1.22) of PLA-PHB film reported by Ma et al. (2018). And the EAB value of control PLA–PBSA film (Film A) is higher than the values (2%−6%) for pure PLA films reported by Javidi, Hosseini, and Rezaei (2016). The addition of carvacrol and thymol caused a considerable increase (p < 0.05) in EAB (352.22 ± 12.3% for Film B and 353.80 ± 24.80% for Film C), meanwhile no significant differences were observed between the active PLA–PBSA films. These EAB was even much better than that of EVOH (86.9 ± 2.0%) (Ma et al., 2018), which is currently widely used packaging film due to its great mechanical properties. This difference may be attributed to the similar effect caused by the addition of the isomers carvacrol and thymol into the polymer matrix. The presence of active compound improved the mobility and reduced the intermolecular forces of the PLA–PBSA polymer chains resulting in an increase in the flexibility and extensibility of the PLA–PBSA films (Chieng et al., 2014). Table 1 also shows that the TS values of films B and C decreases of approximately 9.69%, and 11.93%, respectively compared with the control film A. This phenomenon may be due to the plasticizing toughening and elastomeric effects of the active compound on PLA–PBSA films (Erdohan, Çam, & Turhan, 2013). The optical properties of the PLA–PBSA films are also presented in Table 1. Compared with the control, the active PLA–PBSA films B and C demonstrated a slight increase (p < 0.05) in T values. No significant changes in the H values were observed between the control and active PLA–PBSA films. Thus, the active compound added into the PLA–PBSA matrix improved the arrangement of polymer molecules and light refraction resulting in smooth and uniform surfaces of the PLA–PBSA films. This result is in agreement with the SEM images of the films mentioned above.

2.6.3. Microbial analysis The total viable counts (TVC) of the salmon slices were investigated following the methodology described by Hu et al. (2017). Each salmon slice sample (10 g) was mixed and diluted at 1:10 with 0.85% NaCl saline and homogenized in a stomacher for 2 min. After serial dilutions prepared from the homogenized mixture, samples (1 mL) of each dilution were inoculated into plate count agar (PCA) and incubated at 37 °C for 48 h after 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 days of storage. All counts were expressed as log CFU/g and performed in triplicate. 2.6.4. Thiobarbituric acid-reactive substances (TBARS) TBARS was determined spectrophotometrically according to the method described by Barbosa-Pereira et al. (2013) with slight modification. The salmon samples (5 g) were homogenized in 25 mL of 7.5% trichloroacetic acid (containing 0.1% EDTA) for 30 min and filtered. Aliquots (10 mL) of the filtrate were removed and mixed with 10 mL of 0.02 mol/L thiobarbituric acid in a test tube with a screw cap, then heated in a water bath at 90 °C for 40 min. Afterward, the extract solution was cooled to room temperature and centrifuged for 5 min at 1600 rpm. Chloroform (5 mL) was added to the extract solution. After stratifying, the clear supernatant extract was used for analysis, and absorbance was measured with a UV–vis spectrophotometer (Hitachi Ltd, Tokyo, Japan) at 532 nm and 600 nm. The TBARS value was obtained using Eq. (4):

TBARS (mg MDA/100g ) =

A532 − A600 × 726 155

(4)

2.7. Statistical analysis Analysis of the experimental data was performed with Origin 8 software. The results were subjected to ANOVA, and statistically significant difference was defined at p < 0.05.

3.1.3. Barrier properties The values of OTR and WVP of all the films are presented in Table 1 as well. The OTR values of the active PLA–PBSA films significantly increased with the addition of carvacrol and thymol, reaching 347.63 cm3/m2.24 h.0.1 MPa and 300.92 cm3/m2.24 h.0.1 MPa, respectively. This phenomenon can be explained by the increases in chain mobility and free volume in the PLA–PBSA matrix caused by the plasticization effect of the active compound (Yang et al., 2016), and the chemical reaction between the active compound and the polymer (Sothornvit & Krochta, 2000). These findings are in agreement with previously reported results on the oxygen barrier property of PLA-based films (Jamshidian et al., 2011; Ramos, Jiménez, Peltzer, & Garrigós, 2014). These results are also consistent with the result of the DSC analysis, in which the crystallinity of the polymers decreased with the addition of carvacrol and thymol. In addition, the WVP values of the active PLA–PBSA films increased from 5.26 10−15 (g/m.Pa.s) to 6.92 and 6.64 × 10−15 (g/m.Pa.s). The increase in the free volume of the PLA–PBSA films due to the hydrophobic property reduction caused by the presence of hydroxy groups in active compound, which resulted in the decrease in the resistance of films to water vapor transmission

3. Results and discussion 3.1. Film characterization 3.1.1. SEM and FTIR analysis SEM images of the cross-sectional surfaces of the active PLA–PBSA films are presented in Fig. 2. The surfaces of the active PLA–PBSA films containing carvacrol and thymol demonstrated mainly smooth, compact, and homogeneous (Fig. 2(2)–(3) and (5)–(6)). By contrast, the image of the control (Fig. 2(1)) showed a relatively flat film with burrs on both sides of the cross-section. In agreement with previous observations (Yang et al., 2016), these results showed that active compound was well incorporated into the PLA–PBSA matrix and improved the mechanical and physical properties of polymer. Furthermore, no notable modifications in the FTIR spectra of the active PLA–PBSA packaging films were detected compared with the control (Fig. 3(1)). 4

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Fig. 2. Cross-sectional SEM images of developed films: (1) control film (image scales: 1:1000); (2) PLA - PBSA - 8% Carvacrol film (image scales: 1:1000); (3) PLA - PBSA - 8% Thymol film (image scales: 1:1000); (4) control film (image scales: 1:5000); (5) PLA - PBSA 8% Carvacrol film (image scales: 1:5000); (6) PLA - PBSA - 8% Thymol film (image scales: 1:5000).

(Burgos et al., 2017). This outcome indicates the effects of carvacrol and thymol on the barrier to water vapor are similar to those on oxygen barrier property. Meanwhile, the OTR values and WVP values of the active PLA–PBSA films with carvacrol additive were significantly higher than those of films with thymol additive. 3.1.4. Thermal characterization The thermal property of the PLA–PBSA films was analyzed by DSC, which measures the amount of heat energy absorbed or released when a polymer matrix is heated. Three parameters were determined: glass transition temperature (Tg), crystallization temperature (Tc), and melting temperature (Tm). In Fig. 3(2), the control PLA–PBSA films (film A) showed an endothermic peak of melting at 149 °C, and a decrease in melting temperature (Tm) of 6 °C–9 °C was observed. These observations indicated that the melting temperature (Tm) of the PLA–PBSA films was also affected by the addition of carvacrol and thymol. The glass transition temperature (Tg) and crystallization temperature (Tc) values of the control films were higher than those of the active PLA–PBSA films (films B and C). The decreases of Tg and Tc for active PLA-PBSA based films may be due to the plasticizing effect of carvacrol and thymol, which enhanced mobility of PLA–PBSA chains and consequently improved the flexibility and ductility of active PLAPBSA based films (Javidi et al., 2016; Ramos et al., 2014). Moreover, crystallization of the material decreased significantly with the addition of carvacrol and thymol indicating the changes in the mechanical and barrier properties of the PLA–PBSA films. Thus, the interactions between the active compound and the PLA–PBSA polymer caused a decrease in the crystallinity of active films. Similar results were previously reported with the addition of active compound (Ramos, Jimenez, Peltzer, & Garrigos, 2012). 3.2. Release of active compound into food simulants and antioxidant efficiency of films 3.2.1. Release in food simulants Fig. 4 shows the amounts of carvacrol and thymol released from the active PLA–PBSA films (Films B and C) into four food simulants at 4 °C for 288 h of contact. The extent of release depended on the compatibility of carvacrol and thymol with the PLA–PBSA polymer matrix and food simulants. High solubility of the active compound in the simulant indicated high values of the release (Lopez et al., 2013). As shown in Fig. 4, the amounts of released carvacrol and thymol differed between the active PLA–PBSA films and increased with contacting time. Moreover, the release amounts of carvacrol and thymol presented the highest

Fig. 3. The physical, thermal performance properties of PLA–PBSA films. (1) FTIR spectra of PLA–PBSA films; (2) DSC thermograms of PLA–PBSA films.

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Table 1 Physico-chemical, barrier and mechanical properties of films.

A B C

EAB %

TS MPa

T %

H %

OTR cm3/m2.24 h.0.1 MPa

WVP 10−15 (g/m Pa s)

55.70 ± 3.56a 352.22 ± 12.36b 353.80 ± 24.80b

48.61 ± 1.22a 38.92 ± 1.41b 36.68 ± 1.74b

86.90 ± 1.12a 88.56 ± 0.42b 90.25 ± 0.51c

0.50 ± 0.25a 0.29 ± 0.22a 0.25 ± 0.13a

266.39 ± 3.44a 347.63 ± 3.69b 300.92 ± 4.01c

5.26 ± 0.25a 6.92 ± 0.01b 6.64 ± 0.01c

n = 6, mean ± SD. Significant difference (p < 0.05) among the values within the same column indicated by different superscripts (a–c).

levels (229.03 ± 0.54 mg/kg and 218.81 ± 0.31 mg/kg) in the 95% ethanol simulant when the release reached equilibrium after a certain period of time which was about four times as much as active compound (CIN) releasing from PLA-PHB film (59.0 μg/mL) (Ma et al., 2018) and from non-biodegradable EVOH film (54.45 ± 60.94 μg/mL) (Ma et al., 2017). According to the data, the increased solubility of carvacrol and thymol accounted for the high release into the 95% ethanol simulant. In general, the release values (V) of active compound obtained at a similar temperature and time follows the order: V 95% ethanol > V 3% acetic acid > V 10% ethanol > V water. These results may be attributed to carvacrol and thymol being nonpolar compound. Thus, carvacrol and thymol both showed low solubility in water, which is a polar substance. In addition, the release of carvacrol and thymol may be related to the extent of swelling of the PLA–PBSA matrix in the different food simulants. Therefore, changes in the food matrix markedly affect the release of active compound from films (Silva, Freire, Sendon, Franz, & Losada, 2009). However, a certain amount of carvacrol and thymol can be lost during film preparation according to the data of release into 95% ethanol. Table 2 shows that the α values of active compound released

Table 2 Diffusivity and diffusional exponent of carvacrol and thymol in films contact to different simulants. Active

Simulant

α

k

R2

D (m2 s−1)

Carvacrol

water 3 % acetic acid 10 % ethanol 95 % ethanol

0.35726 0.15068 0.24653 0.08371

−1.8269 −0.85873 −1.53685 −0.12886

0.97836 0.9738 0.9771 0.95068

3.71 × 10−15 2.48 × 10−14 3.96 × 10−15 6.45 × 10−13

Thymol

water 3 % acetic acid 10 % ethanol 95 % ethanol

0.32416 0.26034 0.48471 0.09761

−1.87333 −1.43529 −2.74607 −0.12995

0.97895 0.98795 0.99833 0.92386

2.84 × 10−15 5.08 × 10−15 7.32 × 10−16 6.04 × 10−13

were not higher than 0.5 in all simulants, therefore, Fick’s diffusion equation can be used to predict amount of carvacrol and thymol in the active PLA–PBSA films. The diffusivity (D) values of carvacrol and thymol in films in contact with simulants were calculated using Eq. (2), and the results are shown in Table 2. The highest D values were

Fig. 4. Release of carvacrol and thymol from active PLA–PBSA films (group B and C) into food simulant: water (a); 3% acetic acid (b); 10% ethanol (c); 95% ethanol (d). 6

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Fig. 6. Release of carvacrol and thymol from active PLA–PBSA films (group B and C) into salmon samples.

Fig. 5. DPPH radical scavenging activity of PLA–PBSA films in 95% ethanol at 65 °C for 3 h.

released in real food is less than the maximum release value in the simulant. Although the selection of food simulants is a common practice to determine migration of bioactive elements (Chen et al., 2019; Hu et al., 2017; Ma et al., 2017, 2018) into food, the simulants still can not reflect the real release rate precisely. Water and 95% ethanol can be used as simulants here but Tenax may be more appropriate to mimic solid food (Guazzotti et al., 2015; Triantafyllou, Akrida-Demertzi, & Demertzis, 2007). And also, the thickness of the salmon slices is important for release of active compound.

observed for carvacrol and thymol released from the films into 95% ethanol (6.45 × 10−13 m2 s−1 and 6.04 × 10−13 m2 s−1, respectively). These results illustrated that 95% ethanol is more effective than other food simulants on readily penetrating into the PLA–PBSA films and leading to swelling of the PLA–PBSA matrix and increased release rate of the active compound in the films. Moreover, the D values were consistent with the release rate of each active compound in the four food simulants. 3.2.2. Antioxidant activity of films The antioxidant characteristics of the PLA–PBSA based films with and without active compound were revealed by the scavenging activity of their ethanol extracts against the DPPH radical (Fig. 5). Ma et al. (2018) reported a radical-scavenging activity of 15.7% for a film made of 5% CIN and 95% PLA-PHB, of 39.01% for a non biodigredable film made of 5% CIN and 95% EVOH (w/w) (Ma et al., 2017). Compared with those of the control, the extracts of the active PLA–PBSA films in this study exhibited notably stronger radical-scavenging activity (p < 0.05) of 57.9 ± 2.8%, and 58.85 ± 0.95%. The reason is that the antioxidant activity of active compound was induced by the presence of the phenolic group in carvacrol and thymol molecules. Furthermore, the capability of hydroxyl groups in the active compound reduced the DPPH radical by donating hydrogen atoms (López-Mata et al., 2013). Therefore, carvacrol and thymol can act as effective antioxidant compounds in the PLA–PBSA films.

3.3.2. Microbial growth Fig. 7(1) shows that the initial TVC value of fresh salmon samples was lower than 2 log CFU/g at the time of packaging, which agrees with previous reports (Pettersen, Bardet, Nilsen, & Fredriksen, 2011). Subsequently, the TVC values increased gradually with the extension of storage period. No significant difference in TVC values was observed between the control and treated groups within the first 2 days of storage. However, on 3–4 days of storage, the increase in the TVC value of the control was faster than those of the treated groups (p < 0.05). Afterward, the TVC value of the control PLA–PBSA film group reached a relatively high level (5.4 log CFU/g) on day 9, whereas those of treated groups B and C increased from 1.8 log CFU/g to 2.85 and 2.98 log CFU/ g, respectively. This improvement indicates that the preservation effect on salmon slices in the early storage period was insignificant due to the minimal release of active compound. However, when the active compound in the PLA–PBSA films was released onto the surface of the salmon samples at equilibrium, the antibacterial effects of the active compound on the cellular membranes were significantly enhanced which effectively inhibited bacterial growth (Cerisuelo et al., 2013; Rubilar, Cruz, Khmelinskii, & Vieira, 2013; Ruiz-Navajas, ViudaMartos, Sendra, Perez-Alvarez, & Fernandez-López, 2013) and extended the shelf life for 3–4 days compared with the control. In addition, the release of carvacrol and thymol into salmon slices during cold storage inhibited the activity of enzymes and hindered the decomposition of proteins and volatile basic components (such as trimethylamine and biogenic amines) (Souza et al., 2010; Vatavali, Karakosta, Nathanailides, Georgantelis, & Kontominas, 2013). This phenomenon agreed with the TVB-N (Fig. S1), pH (Fig. S2), and texture (Fig. S4) results for the salmon slices samples. However, the color (Fig. S3) of the samples was approximately constant (Vatavali et al., 2013), and it could also be seen from the actual preservation of salmon silces (Fig. S5).

3.3. Preservation and characterization of salmon slices 3.3.1. Release of active compound into Salmon slices The release values of carvacrol and thymol obtained from active PLA–PBSA films into salmon slices within a certain storage period at 4 °C ± 1 °C are shown in Fig. 6. It showed that the delivery of carvacrol and thymol into the salmon slices decelerated over time, and reached sustainable concentration values after 48 h; this outcome is in agreement with the observation that 44 h is needed for active compound to be released into fatty foods and consequently achieve antimicrobial purpose (Burt, 2004). Nevertheless, the active compound are difficult to release completely from the packaging films into salmon slices (solid food), and the equilibrium concentration of carvacrol and thymol were 3.66 and 3.70 mg/kg, respectively. Furthermore, the high moisture of salmon possibly impeded the release of carvacrol and thymol from films and caused the low concentrations at equilibrium. That is, the amount 7

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Fig. 7. Changes in physicochemical properties for the salmon slice samples during storage. (1) Changes in TVC values in salmon slice stored at 4 °C ± 1 °C during 9 days; (2) Changes in TBARS values in salmon slice stored at 4 °C ± 1 °C during 9 days.

valuable information provided in this study might help accelerate process of replacing nonbiodegradable packaging material with biodegradable ones.

3.3.3. TBARS TBARS is a measure of malondialdehyde (MDA), which is the end product of lipid oxidation (Fernandez, Perez-Alvarez, & FernandezLopez, 1997). The TBARS values for all salmon slices samples are shown in Fig. 7(2). No significant difference was observed in TBARS values between the control and treated groups in the early storage period. Subsequently, after day 4, the TBARS values of the control were significantly (p < 0.05) higher than the corresponding values of the treated samples, but the increase rate in TBARS values of the treated groups were lower. This result agrees with the release behavior of carvacrol and thymol into salmon slices during storage. The initial TBARS value of salmon was 0.021 mg MDA/100 g; and this value increased to 0.205, 0.098 and 0.107 mg MDA/100 g after 9 days. This phenomenon can be attributed to the increased oxidation of unsaturated fatty acids and partial dehydration of the salmon slice (Kilincceker, Dogan, & Kucukoner, 2009). With the release of carvacrol and thymol into salmon slices, the antioxidant properties of the active PLA–PBSA films and the antioxidant capacities of carvacrol and thymol in the active PLA–PBSA films were gradually enhanced. These improvements are attributed to the films’ phenolic compounds with antioxidant activity that hampered lipid oxidation in the treated samples. Furthermore, the TBARS values of the salmon slices are also dependent on the contact of films with the salmon slices. In general, the release of carvacrol and thymol from the active PLA–PBSA films alleviated the oxidation of unsaturated fatty acids in the salmon slices, indirectly maintained the nutrition of the salmon slice, and extended the preservation of salmon slices by 3–4 days during cold storage.

Declaration of Competing Interest The Authors declare that there are no conflicts of interest. Acknowledgements This work was funded by National Natural Science Foundation of China (Grant No. 31622042, 31371790, 31271900, 31471685) and The National Key R&D Program of China (2016YFD0400803, 2016YFD0401501). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fpsl.2019.100393. References ASTM (2000). Standard test method for haze and luminous transmittance of transparent plastics. West Conshohoken, Pa., U.S.A. p. D1003–D00. ASTM (2012). Standard test method for tensile properties of thin plastic sheeting. West Conshohocken, Pa., U.S.A. p. D882–D12. ASTM (2013). Standard test method for water vapor transmission rate of sheet materials using dynamic relative humidity measurement. West Conshohocken, Pa. U.S.A., p. E398–E313. Alboofetileh, M., Rezaei, M., Hosseini, H., & Abdollahi, M. (2014). Antimicrobial activity of alginate/clay nanocomposite films enriched with essential oils against three common foodborne pathogens. Food Control, 36(1), 1–7. https://doi.org/10.1016/j. foodcont.2013.07.037. Armorini, S., Yeatts, J. E., Mullen, K. A. E., Mason, S. E., Mehmeti, E., Anderson, K. L., et al. (2016). Development of a HS-SPME-GC-MS/MS method for the quantitation of thymol and carvacrol in bovine matrices and to determine residue depletion in milk and tissues. Journal of Agricultural and Food Chemistry, 64, 7856–7865. https://doi. org/10.1021/acs.jafc.6b02899. Alvarado, N., Romero, J., Torres, A., de Dicastillo, C. L., Rojas, A., Galotto, M. J., et al. (2017). Supercritical impregnation of thymol in poly(lactic acid) filled with electrospun poly(vinyl alcohol)-cellulose nanocrystals nanofibers: Development an active food packaging material. Journal of Food Engineering, 217, 1–10. https://doi.org/10. 1016/j.jfoodeng.2017.08.008. Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods—A review. International Journal of Food Microbiology, 94(3), 223–253. https:// doi.org/10.1016/j.ijfoodmicro.2004.03.022. Boumail, A., Salmieri, S., Klimas, E., Tawema, P. O., Bouchard, J., & Lacroix, M. (2013). Characterization of trilayer antimicrobial diffusion films (ADFs) based on methylcellulose–polycaprolactone composites. Journal of Agricultural and Food Chemistry, 61(4), 811–821. https://doi.org/10.1021/jf304439s. Barbosa-Pereira, L., Cruz, J. M., Sendon, R., de Quiros, A. R. B., Ares, A., Castro-Lopez, M., et al. (2013). Development of antioxidant active films containing tocopherols to extend the shelf life of fish. Food Control, 31, 236–243. https://doi.org/10.1016/j. foodcont.2012.09.036. Burgos, N., Armentano, I., Fortunati, E., Dominici, F., Luzi, F., Fiori, S., et al. (2017).

4. Conclusions This study was to develop biodegradable active films based on PLAPBSA with active compound for food products. Mechanical and thermal properties of films including TS, EAB, T and H, WVP, OTP and DSC were investigated as well as structural analysis by SEM and FTIR. No effect of active compounds (carvacrol and thymol) on the microstructure and chemical structure of PLA-PBSA films were found. This illustrated that active compound had a good compatibility with PLAPBSA material. However, the addition of active compound enhanced mobility of PLA–PBSA chains, and improved the flexibility and ductility of active PLA-PBSA based films, consequently improved TS and EAB—two major mechanical properties of packaging films. In active property analysis, it was observed that the higher releasing rate of active compound into food simulant was, the greater D value was. Finally, the results of shelf life test of salmon slices packed with PLA-PBSA films showed a strong correlation between the release of active compound into salmon slices and the preservation of the salmon slices: the more active compound released, the longer shelf life was extended. The 8

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