Plasticized Poly(lactic acid)âPoly(hydroxybutyrate) (PLAâPHB) Blends

Article pubs.acs.org/JAFC

Plasticized Poly(lactic acid)−Poly(hydroxybutyrate) (PLA−PHB) Blends Incorporated with Catechin Intended for Active FoodPackaging Applications Marina Patricia Arrieta,*,†,# María del Mar Castro-López,§ Emilio Rayón,‡ Luis Fernando Barral-Losada,§ José Manuel López-Vilariño,§ Juan López,† and María Victoria González-Rodríguez§ †

Instituto de Tecnologı ́a de Materiales, Universitat Politècnica de Valencia, E-03801 Alcoy-Alicante, Spain Grupo de Polı ́meros-Centro de Investigacións Tecnológicas (CIT), Departamento de Fı ́sica, Escuela Universitaria Politécnica, Universidade de A Coruña (UDC), Campus de Ferrol, 15471 Ferrol, Spain ‡ Instituto de Tecnologı ́a de Materiales, Universitat Politècnica de Valencia, E-46022 Valencia, Spain # Catholic University of Cordoba, Camino a Alta Gracia Km 71/2, 5017 Córdoba, Argentina §

ABSTRACT: Active biobased packaging materials based on poly(lactic acid)-poly(hydroxybutyrate) (PLA−PHB) blends were prepared by melt blending and fully characterized. Catechin incorporation, as antioxidant compound, enhanced the thermal stability, whereas its release was improved by the addition of acetyl(tributyl citrate) (ATBC) as plasticizer. Whereas the incorporation of ATBC resulted in a reduction of elastic modulus and hardness, catechin addition produced more rigid materials due to hydrogen-bonding interactions between catechin hydroxyl groups and carbonyl groups of PLA and PHB. The quantification of catechin released into a fatty food simulant and the antioxidant effectiveness after the release process were demonstrated. The effect of the materials’ exposure to a food simulant was also investigated. PHB-added materials maintained their structural and mechanical properties after 10 days in a test medium that represents the worst foreseeable conditions of the intended use. Thus, plasticized PLA−PHB blends with catechin show their potential as biobased active packaging for fatty food. KEYWORDS: poly(lactic acid) (PLA), poly(hydroxybutyrate) (PHB), catechin, active antioxidant packaging, biodegradable

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With regard to the field of application, plasticizers are frequently incorporated into PLA-based materials intended for food packaging to overcome somewhat ductility.11,12 Particularly, PLA could be melt processed with PHB due to their similar melting temperatures,3 and their processability could be improved by the addition of plasticizers at the same time as resulting in more flexible materials.7,8,13 Nevertheless, only nontoxic substances approved for food contact can be considered as modifying agents for PLA. Moreover, the modifiers should be miscible with PLA and not be too volatile to avoid evaporation at the elevated temperatures used for PLA industrial processing.14 In this sense, acetyl(tributyl citrate) (ATBC) has been suggested as one of the most efficient plasticizers for PLA,13 which does not raise a safety concern for food contact materials.15 Moreover, it has been shown that PLA−PHB plasticized with ATBC offers good perspective for the biodegradable food packaging industry by improving polymer performance in film manufacturing and use, showing significantly higher values of elongation at break (∼180%) than other plasticizers such as PEG (∼10%)8 and D-limonene (∼8%),7 whereas it also accelerates disintegration under composting conditions10 in relation to their environmentally friendly post-use.

INTRODUCTION An increasing attitude toward the reduction of the environmental impact produced by plastic waste, in particular from the food packaging sector, has increased research and industry attention on the development of new biodegradable materials. In this sense, the most promising raw material for the production of biodegradable food packaging is poly(lactic acid) (PLA) due to its high mechanical strength and superior transparency. However, PLA is highly brittle1 and, thus, its range of processability is reduced,2 which limits PLA use in the food-packaging industry. Thus, many research studies have been focused on PLA modification for extending its industrial applications in the food-packaging sector, such as the addition of modifiers, copolymerization, or blending. The modification of PLA by blending with another more crystalline biobased and/or biodegradable polymer has many advantages because it offers the opportunity to tune the physical properties over a wide range through a relatively simple approach.3,4 It has been reported that PLA melt blended with 25 wt % of PHB showed optimal miscibility between both polymers and improved mechanical properties compared with those of pure PLA5 by enhancing the PLA crystallinity6,7 because PHB acts as nucleating agent for PLA.5 In previous works, it was shown that PLA−PHB (75:25) blends showed improved properties of the final formulation,3 particularly significant in the packaging field, such as oxygen barrier and surface wettability,7−9 whereas their disintegrability in composting conditions suggested a valuable end-life option for PLA−PHB packaging materials.7,9,10 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 10170

June 24, 2014 September 23, 2014 September 25, 2014 September 25, 2014 dx.doi.org/10.1021/jf5029812 | J. Agric. Food Chem. 2014, 62, 10170−10180

Journal of Agricultural and Food Chemistry

Article

Table 1. Film Formulations formulation

PLA (wt %)

PLA (control) PLA−ATBC PLA−CAT PLA−ATBC−CAT PLA−PHB PLA−PHB−ATBC PLA−PHB−CAT PLA−PHB−ATBC−CAT

99.8 84.8 94.8 79.8 74.85 63.6 71.1 59.85

PHB (wt %)

ATBC (wt %) 15 15

24.95 21.2 23.7 19.95

On the other hand, current worldwide trends to reduce the use of food additives have given way to investigations of incorporating additives, such as antimicrobial or antioxidant compounds, into food packaging. These systems are known as active food packaging and offer the opportunity to provide continuous release of active compound from packaging material to the foodstuffs, having an important effect on the shelf life extension without the need of direct incorporation of additives into the final food product. However, active agents could show higher affinity for the polymer matrix than the foodstuffs. Consequently, the design of active packaging systems is aimed to obtain an effective release capacity of active agent from the polymer matrix. It is known that the plasticizer presence increases the polymer chain mobility in PLA−PHB blends,7,8 and for this reason it is expected that the mass transport of the active agent should be modified,16 resulting in an effective way to improve the release of the active substance from the polymer matrix.17 One of the main drawbacks of polymer modifiers is the negative effect of the additives on the thermal stability. Thermal degradation is responsible for serious damage to many polymeric materials during processing.11 In this sense, antioxidants are chosen as active substances not only to protect food deterioration and extend its shelf life17,18 but also to protect the polymer matrices from thermo-oxidative degradation during processing18,19 because antioxidants are able to act with the free radicals formed during thermo-oxidation, blocking further polymer degradation.20 Currently, synthetic additives are being replaced by natural preservatives due to safety16 and environmental concern. Most of the common natural antioxidants are phenolic compounds derived from flavonoids,18,20 which are sourced primarily from renewable resources and have been in use for centuries. For instance, catechins are flavonoids with multiple biological effects18 that have gained considerable attention due to their potential benefits on human health21 related to antimutagenic, antidiabetic, anti-inflammatory, and prevention activities against several kinds of cancer.22 From a technological aspect, catechins have been suggested as effective natural antioxidants in many polyolefins for active food-packaging applications.16,18,21 Catechin, one of the major active constituents of green tea, represents a natural food-grade additive that inhibits lipid oxidation, increasing the shelf life of foods,23 and has a positive acceptance among consumers owing its well-known healthy properties. With regard to the polymers’ transformation, the extrusion process is one of the most widely used processing technologies in the packaging industry.24 Because PLA−PHB blends should be processed in the region from at least 160 °C to below 200 °C,3 catechin, which is a nonvolatile compound, is optimal for melt processing to avoid or reduce its loss during polymer processing.18

15 15

I168 (wt %) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

CAT (wt %)

5 5

5 5

The main objective of this work was to develop a biodegradable active packaging based on PLA−PHB blends with added catechin, as antioxidant agent, intended for fatty food-packaging application. These systems were plasticized with ATBC with the dual objective of overcoming ductility required for film manufacturing and uses as well as improving the kinetics of release of catechin from the polymer matrix to the foodstuff. The developed materials were characterized with regard to their structure and nanomechanical and thermal properties. The release capacity of catechin into a fatty food simulant after the release process was tested, as well as its antioxidant effectiveness, and the morphological, structural, and nanomechanical properties were also followed during the material exposure to the selected food simulant.

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MATERIALS AND METHODS

Materials and Chemicals. Poly(lactic acid) (PLA 2003D, Mn = 98000 g mol−1, 4 wt % D-isomer) was supplied by NatureWorks (USA), and poly(hydroxybutyrate) (PHB, under the trade name PHI) was acquired from NaturePlast (France). Catechin dehydrate (CAT), (−)-epicatechin (EC), Irgafos 168 [tris(2,4-ditert-butylphenyl)phosphate] (I168), acetyl tri-n-butyl citrate (ATBC) (M = 402 g mol−1, Tm = −80 °C, 98% purity), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) 95% free radical were purchased from Sigma-Aldrich (Madrid, Spain). Methanol (MeOH) and ethanol (EtOH) of HPLC grade were supplied by Merck (Darmstadt, Germany). Water was purified by means of a Milli-Q ultrapure water purification system (Millipore, Bedford, MA, USA). Sample Preparation. Samples were prepared by melt-blending the materials in a microextruder equipped with twin conical corotating screws (MiniLab Haake Rheomex CTW5, Thermo Scientific) with a capacity of 7 cm3. A screw rotation rate of 40 rpm, temperature of 190 °C, and residence time of 1 min were used. Irgafos 168 was incorporated in all cases at 0.2% to protect the polymer during processing steps. The concentration ratios of the obtained films are summarized in Table 1. Sample Characterization. Field Emission Scanning Electron Microscopy (FESEM). The film cross sections performed in liquid nitrogen were coated with a thin palladium−gold layer to be observed by FESEM (Supra 25-Zeiss). Thermogravimetric Analysis (TGA). TGA was performed with a thermogravimetric analyzer TGA/SDTA-851e Mettler Toledo (Schwarzenbach, Switzerland). Tests were run under dynamic mode from 30 to 600 °C at 10 °C min−1 in air (50 mL min−1). The initial degradation temperatures (T0) were determined at 5% mass loss, whereas temperatures at the maximum degradation rate (Tmax) were calculated from the first derivative of the TGA curves (DTG). Differential Scanning Calorimetry (DSC). Dynamic DSC experiments were performed in a Mettler Toledo calorimeter by using two different atmospheres of air (250 mL/min) and nitrogen (50 mL/min) with sample weight about 4 mg in hermetic aluminum pans. Samples analyzed under air conditions were heated from 30 to 350 °C at a heating rate of 10 °C min−1 to determine the maximum temperature at which the polymers degrade (onset oxidation temperature, OOT). OOT values were calculated as the intersection 10171

dx.doi.org/10.1021/jf5029812 | J. Agric. Food Chem. 2014, 62, 10170−10180


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coupled in series with a fluorescence detector (FL, model 2475, Waters) with an excitation wavelength of 280 nm and an emission wavelength of 310 nm.21 Output signals were monitored and processed with a personal computer operated under Empower software (Waters). Catechin and epicatechin were identified by means of retention time and UV spectrum. Release data were expressed as the percentage of catechin released into the food simulant after the contact period: amount of catechin released per kilogram of film with reference to the initial amount of catechin loaded per kilogram of film formulation. The stability of catechin was also evaluated in the simulant under selected exposure conditions by storing a solution of the additive in the simulant in parallel with the release tests and quantified using the same procedure as for the samples.17 The release process is normally described by the kinetics of the diffusion of the antioxidant in the film and is expressed by the diffusion coefficient (D). D is usually estimated using the Fickian diffusion law.16,17 When the release of antioxidant reached equilibrium, a rigorous model for describing the migration controlled by Fickian diffusion in a packaging film is used as explained in eq 2:

point between the baseline and the tangent to the degradation peak. Meanwhile, samples analyzed under nitrogen conditions were subjected to a cycle program consisting of a first heating stage from 30 to 180 °C at a heating rate of 10 °C min−1, followed by a cooling process to 30 °C at 30 °C min−1 and subsequent heating to 250 °C to erase the materials’ thermal history. The glass transition temperature (Tg) was taken at the midpoint of heat capacity changes. The melting temperature (Tm) and cold crystallization temperature (Tcc) were obtained from the second heating, and the degree of crystallinity (χc) was calculated through eq 1

⎡ ΔHm − ΔHc ⎤ 1 χc = 100% × ⎢ ⎥× ΔHmc ⎣ ⎦ WPLA

(1)

where ΔHm is the melting enthalpy, ΔHcc is the cold crystallization enthalpy, and ΔHmc is the melting heat associated with pure crystalline PLA (93 J g−1) and WPLA is the proportion of PLA in the blend.25 X-ray Diffraction (XRD). The crystalline phases of the nanocomposite films were studied by using XRD equipment (Bruker AXS D5005, SCSIE Universitat de València). Scanning was performed on square film surfaces (15 mm × 15 mm) mounted in an appropriate sample holder. The patterns for profile fitting were obtained from a diffractometer using Cu Kα radiation with a scanning step of 0.02° between 2.5° and 40° in 2θ with a collection time of 10 s per step, and the voltage was held at 40 kV. Wettability. The surface wettability of films was evaluated by measuring the water contact angle with an Easy-Drop Standard goniometer FM140 (Krüss GmbH, Hamburg, Germany) equipped with a camera and analysis software (Drop Shape Analysis SW21; DSA1). Samples for wettability measurements were prepared as disk films with an average thickness between 180 and 230 μm by a compression molding process at 180 °C in a hot press (Mini C 3850, Caver, Inc., Wabash, IN, USA). The contact angle was calculated by randomly adding 5 drops of distilled water (2 μL) with a syringe onto the film surface at room temperature, and the average values of 10 measurements for each drop were used. Release Studies. Release studies of the antioxidant flavonoid catechin were conducted by the determination of the specific migration into a solution of 50% ethanol as a fatty food simulant (simulant D1)26 at 40 °C. Release tests were performed by total immersion of preweighed rectangular strip film pieces (80 ± 0.099 mm × 3.40 ± 0.26 mm × 1.20 ± 0.14 mm) in 10 mL of food simulant contained in glassstoppered tubes with PTFE closures at 40 °C. For fatty food products that will be stored at ambient conditions (temperatures between 20 and 40 °C) during not more than 30 days, legislation orders that food packaging materials should be assayed at contact temperature in worst foreseeable use of 40 °C for 10 days. In this work, the release test was conducted during 20 days. Thus, samples were taken at 1, 5, 10, and 20 days. Working standard catechin solution in the food simulant and pure simulant were run simultaneously with the release test to check for interferences, and all samples were performed in triplicate. Measurement of Released Antioxidant. The released catechin was measured after the contact period; an aliquot of each simulant was filtered through an Acrodisc PTFE CR 13 mm, 0.2 μm filter (Waters, Milford, MA, USA) and quantified by HPLC-PDA-FL. A Waters 2695 HPLC system with a gradient pump and automatic injector was used for high-performance liquid chromatography (HPLC) analysis equipped with a stainless steel column packed with SunFire C18 (150 mm × 3.0 mm, 3.5 μm) (Waters) kept at 35 °C following the methodology described by Castro López et al.16 The method consisted of a two-solvent gradient elution at a flow rate of 0.5 mL min−1. Mobile phase was composed by water (A) and methanol (B). The following gradient elution profile was used: Mobile phase composition started at 25% B and was maintained for 0.5 min. Then, it was linearly increased to 40% B in 4.5 min, to 60% B in 1 min, and to 100% B in 2 min. Finally, it was maintained for 3 min and brought back to recover the initial chromatographic conditions. Detection was performed on a photodiode array detector (PDA, model 996 UV) set in the range of 200−400 nm (277 nm output signal as a quantification wavelength)

Mt =1− MF, ∞

∞

∑ n=0

⎡ ⎤ − D(2n + 1)2 π 2t ⎥ 8 exp⎢ 2 2 2 ⎥⎦ (2n + 1) π Lp ⎣⎢

(2)

Mt is the mass of the migrant in the food simulant at a particular time t (s), MF,∞ is the mass of the migrant in the food at equilibrium, Lp is the film thickness (cm), D is the diffusion coefficient (cm2 s−1), and t (s) is time. Nevertheless, when release is slow and equilibrium is not reached at the end of the experiment, with the boundary condition of Mt/Mp < 0.6, eq 3 can be used: 1/2 Mt 4 ⎛⎜ Dt ⎞⎟ = Mp Lp ⎝ π ⎠

(3)

Mp is the initial loading of antioxidants in the film, D is estimated from the slope of the plot of Mt/Mp versus t1/2, and the intersection with the x-axis represents the time lag. Antioxidant Activity. The antioxidant effectiveness of development materials was measured according to the DPPH method by determining the absorbance of release medium at different times by means of a UV−vis spectrophotometer at 517 nm. The antioxidant activity was obtained according to eq 4

⎛ Abscontrol − Abssample ⎞ I (%) = ⎜ ⎟ × 100% Abscontrol ⎝ ⎠

(4)

where I (%) is the percentage of inhibition. The results were calculated in percent of inhibition as the mean of three replicates, and they were expressed as the equivalent of gallic acid (GA) concentration (ppm) by using a calibrated curve of gallic acid concentration versus I (%). Nanomechanical Analysis. The mechanical hardness (H) and elastic modulus (E) properties of films before release studies and after 10 days of contact with food simulant D1 were determined by a nanoindenter machine G-200 (Agilent Technologies, Santa Clara, CA, USA). All samples were indented in the same experiment. All samples before release studies and those after 10 days in food simulant were indented during the same test. Indentations were performed at maximum 2000 nm constant depth using a Berkovich diamond tip. An array of 16 indentations was performed for each sample. The area function that is used to estimate the contact area at low depths was previously calibrated in fused silica. The stiffness required to calculate the contact area beyond indenter and elastic modulus was obtained by means of the continuous stiffness measurement (CSM) technique.27,28 The CSM technique provides in-depth profiles of the stiffness response and, subsequently, in-depth profiles of H and E. Statistical Analysis. Experimental data were statistically analyzed with OriginPro 8 software. One-way analysis of variance (ANOVA) was carried out, and significant differences among formulations were 10172



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Figure 1. Microstructure of fracture surface of unplasticized (left side) and plasticized films (right side).

and PLA−PHB films incorporated with CAT appear to be more porous without a plain smoothness (Figure 1c,g). Thermal Characterization. Thermogravimetric Analysis. The thermal decomposition was studied by TGA and DTG (Figure 2). Stabilized with catechin and unstabilized PLA and plasticized PLA materials decomposed in a one-step process (Figure 2a), whereas PLA−PHB blend counterparts decomposition took place in two well-separated steps. In the first degradation process the total weight loss is close to 25% and corresponds to the degradation of PHB, and the second stage is related with the degradation of PLA. Figure 2b shows that PHB decreases the thermal stability of the final formulation in accordance with previous results.7,8 Table 2 also summarizes the main thermal parameters obtained by TGA and DTG. Whereas ATBC shifted the T0 to

recorded at the 95% confidence level according to Tukey’s post hoc test.

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RESULTS AND DISCUSSION

Morphological Characterization. Figure 1 shows FESEM images of cryofractured surfaces of unplasticized (left side) and plasticized films (right side). Whereas unplasticized films showed a smooth fracture, plasticized samples showed a clear plastic behavior with no apparent phase separation. These results show that ATBC is well incorporated into the PLA and PLA−PHB matrices in agreement with previous observations.8 The characteristic plastic behavior was more evident in PLA− ATBC (Figure 1b) and PLA−ATBC−CAT (Figure 1d) than in plasticized PLA−PHB counterparts (Figure 1f ,h). The PLA 10173



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Figure 2. TGA and DTG of PLA-based (a, c) and PLA−PHB-based (b, d) films.

Table 2. TGA and DSC Thermal Parameters of PLA and PLA−PHB Samples T0 (°C) PLA PLA−ATBC PLA−CAT PLA−ATBC−CAT PLA−PHB PLA−PHB−ATBC PLA−PHB−CAT PLA−PHB−ATBC−CAT

315.7 276.0 329.3 308.8 275.0 268.4 279.3 275.1

Tmax PHB (°C)

Tmax PLA (°C)

OOT (°C)

Tg (°C)

Tcc (°C)

Tm (°C)

ΔHcc (J/g)

ΔHm (J/g)

χc (%)

279.3 279.4 289.5 289.5

345.5 346.5 349.6 350.1 339.7 323.8 337.8 309.6

265.7 232.0 320.2 299.3 267.7 271.8 280.0 281.2

63.8 45.81 64.7 54.4 63.2 60.6 62.1 57.7

118.1 95.9 129.6 115.8 130.0 106.3 150.0 126.0

153.4 153.8 154.1 145.0 158.6 157.0 155.4 152.5

24.5 22.3 12.6 24.5 8.5 19.0 0.1 5.3

27.9 25.0 15.3 27.1 11.9 22.4 7.2 8.1

3.6 3.5 3.1 3.4 4.8 5.7 4.9 5.1

lower degradation temperatures, catechin led to an increment in T0, delaying the beginning of the thermal decomposition process in all cases indicative of an effective stabilizing effect. As well, catechin presence leads to a displacement of the Tmax of PLA toward slightly higher values in PLA and plasticized PLA samples (PLA−CAT and PLA−ATBC−CAT). However, in PLA−PHB blends at the same time as the Tmax corresponding to the degradation of PHB showed a considerable increment of about 10 °C with catechin addition, the Tmax of PLA was shifted to lower values, and this behavior was particularly noticeable in PLA−PHB−ATBC−CAT, where the reduction was about 14 °C. The antioxidant capacity of catechin protects the polymer thermal degradation at the first stage leading the Tmax of the blends to higher values, and then the degradation at higher temperatures is accelerated. Differential Scanning Calorimetry. The OOT was used to evaluate the effectiveness of catechin to increase the polymer stability under oxidative atmosphere, and results are summarized in Table 2. OOT values clearly increased with the addition of catechin in all cases, showing its effectiveness to protect PLA, PLA−PHB, plasticized PLA, and plasticized PLA−PHB against

oxidation, as was already commented for TGA results above. However, PLA-based materials stabilized with catechin showed higher values of OOT than PLA−PHB counterparts, in good accordance with T0 values. It must be noted that whereas ATBC decreases the thermal stability of PLA, it slightly increases the thermal stability of PLA−PHB blend due to the fact ATBC improves the interaction between both polymers.8 Consequently, catechin could be considered as a natural stabilizer for PLA, PLA−PHB, and their plasticized systems. Panels a and b of Figure 3 show the thermograms for the DSC second heating where it is possible to observe the glass transition temperature (Tg), the cold crystallization exotherm (Tcc), and the melting endotherm (Tm) of the PLA-based (Figure 3a) and PLA−PHB-based (Figure 3b) films, showing that the cooling conditions applied did not produce the crystallization of PLA. DSC thermal properties are also summarized in Table 2. ATBC induced the depression in Tg of all samples, confirming its plasticizing effect also reported and discussed in a previous study.8 Moreover, the lowest Tg values were presented by PLA−ATBC followed by PLA− ATBC−CAT due to their higher plastic behavior, in good 10174



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Figure 3. DSC second heating scan of (a) PLA-based films and (b) PLA−PHB-based films. (c) X-ray diffraction patterns of (I) PLA, (II) PLA− ATBC, (III) PLA−CAT, (VI) PLA−ATBC−CAT, ( V) PLA−PHB, (VI) PLA−PHB−CAT, and (VII) PLA−PHB−ATBC−CAT. (d) Contact angle measurements of PLA-based and PLA−PHB-based films. Different letters on the bars indicate significant differences between formulations (p < 0.05).

PHB slightly increased Tm temperature with respect to the neat PLA,7 suggesting miscibility between both polymers ascribed to a transesterification reaction between PLA and PHB chains during melt blending.4 Although PLA can crystallize showing different polymorphisms, namely, the alpha (α), beta (β), and gamma (γ) forms, from the melt PLA crystallizes in the α form.29 Neat PLA showed the Tm corresponding to the melt of stable α homocrystals developed during the heating process. Meanwhile, plasticized PLA systems (PLA−ATBC and PLA−ATBC−CAT) showed a multimelting behavior ascribed to the presence of different PLA crystals. Disordered α′ crystals are formed when the Tcc is below 110 °C.30 PLA is known for its slow crystallization rate.31,32 However, the crystallization rate of PLA is higher at temperatures between 100 and 118 °C,33 at which disorderto-order phase transition (α′ to α) takes place.30 Similar findings were observed in PLA plasticized with oligomeric lactic acid (OLA).34 On the other hand, the presence of multimelting temperatures in PLA−PHB blends, after the PLA melting peak, at around 175 and 185 °C corresponds to the as-formed PHB crystals during blending process and recrystallized PHB crystals formed during DSC heating, respectively.3,5 Different behavior was observed in plasticized PLA−PHB blends (PLA−PHB− ATBC and PLA−PHB−ATBC−CAT). PLA−PHB−ATBC showed the Tcc below 110 °C and as a consequence the multimelting PLA peak as well as the as-formed and recrystallized PHB crystals, whereas the multimelting behavior was not detected for PLA−PHB−ATBC−CAT. The CAT presence considerably shifted the Tcc from 106.3 °C in PLA− PHB−ATBC to 126.0 °C in PLA−PHB−ATBC−CAT, avoiding the formation of disordered PLA α′ crystals. The nonappearance of as-formed and recrystallized PHB crystals

Figure 4. Proposed molecular interaction between PLA, PHB, ATBC, and catechin.

accordance with FESEM micrographs. ATBC also shifted the Tcc to lower values, whereas catechin shifted the Tcc to higher temperatures probably as a result of increased crystal nuclei density, which requires more thermal energy to crystallize PLA. Catechin presence also promoted faster crystallization of PLA and PLA−PHB and the plasticized systems, most likely due to hydrogen-bonding interactions between catechin hydroxyl groups and carbonyl groups of PLA and PHB. Figure 4 shows the proposed molecular interaction. Similar findings were previously reported by López de Dicastillo et al., who found an induction effect of catechin on the crystal nucleation of maleic anhydride modified poly(propylene) (MAPP) and also the formation of intermolecular hydrogen bonds between catechin and poly(propylene) (PP).18 10175



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of PLA and PLA−PHB remained practically unchanged (p > 0.05) with the addition of 5 wt % of catechin in PLA−CAT and PLA−PHB−CAT. PLA−PHB−ATBC−CAT film resulted in material with a surface hydrophobicity increased (p < 0.05) with respect of those of PLA−PHB−ATBC and PLA−PHB−CAT. One possible explanation for this behavior could be that there is a strong interaction of catechin hydroxyl groups with PLA and PHB chains and the interaction is also improved with the presence of ATBC. As a consequence, catechin hydroxyl groups did not show surface orientation affecting the surface chemical properties, leading to an increase in the surface hydrophobicity. Catechin Release. The release of the catechin from PLAbased and PLA−PHB-based developed materials expressed as the percentage of catechin and epicatechin released into food simulant D1 at 1, 5, 10, and 20 days and their diffusion kinetics obtained by HPLC-PDA are shown in Figure 5, panels a and b, respectively. The incorporation of ATBC showed a significant increase of the release capacity from PLA and PLA−PHB matrices. After 10 contact days, levels of released catechin increased about 3 times in both PLA and PLA−PHB with respect to the corresponding films without plasticizer. CastroLopez et al. also showed an improvement in the release of antioxidants from a polypropylene matrix due to the use of plasticizers in the formulations with increased diffusion coefficients between 1 and 2 orders of magnitude.16 Diffusion coefficients were estimated from Figure 5b and from the slope of the curve obtained by eq 3, and they also showed the positive effect of the presence of plasticizer on the release of catechin from PLA-based and PLA−PHB-based materials. Increased values from 1.9 × 10−12 cm2 s−1 in PLA−CAT to 8.3 × 10−11 cm2 s−1 in PLA−CAT−ATBC and from 2.6 × 10−11 cm2 s−1 in PLA−PHB−CAT to 3.5 × 10−10 cm2 s−1 in PLA−PHB− CAT−ATBC were observed due to the ability of plasticizer to increase the polymer chain mobility and, thus, the release of catechin. It is expected that the higher amount of crystalline regions of PLA−PHB blends slowed the diffusion of catechin. However, the addition of PHB showed somewhat an increment in the release of active compound in comparison with those formulations without PHB, particularly noticeable in PLA− PHB−ATBC−CAT film (Figure 5a). The incorporation of PHB produced only an induction period in the diffusion process, due to the limited polymer chain mobility in PLA− PHB-based blends, leading to an increase in time lag from 30 s in PLA−CAT to 209 s and to 252 s in PLA−PHB−CAT and PLA−PHB−ATBC−CAT, respectively. This increase in the time lag can be justified by the increase of interactions between catechin and the PLA−PHB blend matrix. However, once the diffusion process started, the release of catechin became faster in PHB-added samples as shown by the increment in 1 order of magnitude of the diffusion coefficient with respect to PLAbased material counterparts. This behavior could be ascribed to the higher affinity of the simulant D1 with less polar catechinadded PLA−PHB blends in comparison with catechin-added PLA equivalents, as was already commented for the wettability test. Thus, the higher penetration capacity of simulant D1 in the PLA−PHB matrix than in PLA matrix, after the required time lag, accelerated the catechin release process as a function of time. Additionally, after the 10 contact days, enhanced crystallinity of all tested materials was observed by XRD (Figure 6). The diffraction patterns clearly show an increased intensity of the

Figure 5. (a) Catechin (%) released from PLA-based and PLA−PHBbased materials calculated by means of HPLC-PDA. (b) Diffusion kinetics of catechin from PLA-based and PLA−PHB-based materials. (c) Antioxidant activity of catechin expressed as gallic acid concentration (ppm) measured by DPPH radical scavengers. Different letters on the bars within the same day indicate significant differences between formulations (p < 0.05).

could be related with an improvement in the interfacial adhesion between PLA, PHB, and ATBC due to CAT presence. X-ray Diffraction. Figure 3c shows a comparative plot of the crystalline profiles of films. PLA had one broad diffraction peak centered at 2θ = 15° and a smaller one centered at 2θ = 31°. The X-ray patterns of PLA−ATBC, PLA−CAT, and PLA− ATBC−CAT are very similar to that of neat PLA. The presence of crystalline PHB showed an increase in the crystallinity of PLA in the PLA−PHB blend, in good accordance with the increase of the degree of crystallinity calculated by DSC (Table 2). The characteristic reflection peaks of PHB at 2θ = 13.5° and 2θ = 16.9°, as well as the weak peaks at higher 2θ, are observed because the crystal growth rate of PHB is higher and faster than that of PLA.35 Wettability. The surface hydrophobicity is an important issue in materials intended for food-packaging applications because they are required to protect foodstuffs from humidity during transport, handling, and storage.9 Thus, water contact angle measurements were conducted to study the water absorption of these material surfaces (Figure 3d). The line at 65 θ° represents the limit value between a hydrophilic surface (θ° < 65°) and a hydrophobic surface (θ° > 65°). All studied formulations showed values higher than 65°, showing a poor affinity of the water to the material surfaces and highlighting their potential use for packaging applications. The addition of 15 wt % ATBC and 25 wt % of PHB significantly increased the water resistance of PLA (p < 0.05). Meanwhile, the wettability 10176



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Figure 6. X-ray diffraction patterns of PLA-based and PLA−PHB-based films before and after 10 days of contact with simulant D1.

peak at 2θ = 16.5° for all formulations, attributed to the typical α crystal form of the crystalline phase of PLA,34 showing the highest intensity in plasticized samples. Thus, the higher penetration capacity of simulant D1 in PLA−PHB blends at the same time as the increase of crystallinity of amorphous PLA based materials led to comparable catechin release profiles for PLA−ATBC−CAT and PLA−PHB−CAT at 5, 10, and 20 contact days in simulant D1. The highest diffusion process was observed for PLA−PHB− ATBC−CAT samples between 0 and 10 days. However, at the highest period of contact time studied here of 20 days, a reduction of catechin concentration occurred, showing the not possible lower value than those obtained at 5 and 10 days. This unexpected result could be explained by the feasible interaction between the free catechin molecules in the food simulant, possible forming dimers or oligomers, which cannot be

quantified by the experimental methodology used here. In fact, in a food simulant the released compound is dispersed in the food simulant where no oxidation processes are happening, as can occur in a real packaged food environment. The molecules of catechin released from the packaging are expected to interact with food components, jamming oxidation processes. It should be taken into account that the inherent biodegradability of PLA-based materials could be a drawback in food packaging, where the material structure needs to be guaranteed at least during the food’s shelf life.36 For this reason, the morphology of cross sections of the films after 10 and 20 days exposed to simulant D1 was also studied by FESEM (Figure 7). PLA-based and PLA−PHB-based materials preserved their structure at 10 days of the migration test. Meanwhile, PLA, PLA−ATBC (Figure 7a), and PLA−PHB and 10177



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Figure 7. FESEM images of (a) PLA-based materials and (b) PLA−PHB-based materials after 10 and 20 days of exposure to simulant D1.

PLA−PHB−ATBC (Figure 7b) showed increased plastic behavior at 20 days caused by the plasticization effect produced by the simulant dispersion into the polymer matrix. In most cases, the materials become rougher with increasing time in contact with food simulant. As well, at 20 days some cavities were formed in PLA catechin-incorporated samples (Figure 7a), whereas many cavities were observed in PLA−PHB catechin incorporated samples (Figure 7b) as a result of the higher loss of catechin in those samples. This observation supported our conclusion that higher levels of catechin than those obtained here should be released from PLA−PHB− ATBC−CAT sample after 20 days in contact with food simulant. All catechin-incorporated materials showed an effective release of catechin in worst foreseeable use conditions between 0 and 10 days as indicated by current legislation for fatty food intended to be transported, handled, and stored at ambient conditions. Antioxidant Activity. The effectiveness of the developed materials as antioxidant packaging systems was demonstrated by the reduction of stable free radical DPPH caused by catechin presence in the food simulant, and the obtained results are expressed as gallic acid (GA) concentration (Figure 5c). As expected, the antioxidant activity shows comparative tendency to the catechin release. It is known that the solubility characteristics of the antioxidant can determine its effectiveness.37 Catechin shows high solubility in ethanol (50 g L−1),18 and consequently catechin-incorporated materials usually show high activity in ethanol-containing simulants.16,18,22,38,39 Thus, the high affinity of catechin for fatty food simulants and its high antioxidant activity give good reasons for its addition in plasticized PLA and PLA−PHB materials for fatty foodstuff. Mechanical Features of the Films. The mechanical behavior of all samples before and after 10 days in contact with food simulant D1 was assayed by means of a nanoindenter test. The mean of elastic modulus (E) and hardness (H) values for each

Figure 8. Nanomechanical results of materials essayed before and after 10 days in contact with simulant D1: (a) elastic modulus (E); (b) nanohardness (H).

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in the food at equilibrium; Lp, film thickness; I (%), percentage of inhibition; GA, gallic acid; H, hardness; E, elastic modulus; CSM, continuous stiffness measurement; OOT, oxidation onset temperature; MAPP, maleic anhydride grafted into polypropylene; PP, polypropylene; OLA, oligomeric lactic acid

sample was calculated in the range from 500 to 1000 nm. The obtained values are shown in Figure 8. As expected, plasticized samples before release studies showed lower H and E values than unplasticized ones, showing the effectiveness of ATBC to plasticize PLA and PLA−PHB systems.8,10 On the other hand, catechin-added samples showed increased H and E values with respect to their counterparts without catechin. This result confirms that catechin produced a somewhat reinforcing effect blocking the elastic and plastic deformation mechanisms owing to the enhancement of the materials’ interfacial adhesion in the blend, in accordance with DSC experiments (see Differential Scanning Calorimetry). An increase in PLA and PLA−PHB modulus was also observed in PLA−PHB blends reinforced with cellulose nanocrystals9 ascribed to an enhancement in the interface interaction between PLA and PHB due to nanocellulose presence.3 The nanomechanical results of assayed samples after release studies showed scattered values, probably due to residues of simulant D1 and/or released components from the polymer matrices on their surfaces. This activated surface is able to affect the short distance forces between the surface and nanoindenter tip. After 10 days in contact with simulant D1, catechin-added materials revealed in general a drastic diminution of E and H values as a result of the release of catechin to the food simulant, with the exception of PLA−PHB−ATBC−CAT, which mainly maintained the mechanical properties without significant changes. This result highlights an additional positive effect of catechin incorporation to plasticized PLA−PHB systems.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*(M.P.A.) E-mail: [email protected]. Funding

This work has been supported by the Spanish Ministry of Economy and Competitiveness (MAT2011-28648-C02-01 and MAT2011-28468-C02-02). M.P.A. is a recipient of a Santiago Grisolı ́a Fellowship (Generalitat Valenciana) (GRISOLIA/ 2011/007). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Prof. M. Dolores Salvador (Polytechnic University of Valencia) for her assistance with nanomechanical analysis.

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ABBREVIATIONS USED PLA, poly(lactic acid); PHB, poly(hydroxybutyrate); ATBC, acetyl(tributyl citrate); CAT, catechin dehydrate; EC, (−)-epicatechin; I168, Irgafos 168 [tris(2,4-ditert-butylphenyl)phosphate]; DPPH, 2,2-diphenyl-1-picrylhydrazyl; MeOH, methanol; EtOH, ethanol; FESEM, field emission scanning electron microscopy; TGA, thermogravimetric analysis; DTG, first derivative of the TGA curve; DSC, differential scanning calorimetry; Tg, glass transition temperature; Tm, melting temperature; Tcc, crystallization temperature; χc, degree of crystallinity; ΔHm, melting enthalpy; ΔHcc, cold crystallization enthalpy; ΔHmc, melting heat of pure crystalline PLA; XRD, Xray diffraction; HPLC, high-performance liquid chromatography; PDA, photodiode array detector; FL, fluorescence detector; D, diffusion coefficient; Mt, mass of the migrant in the food simulant at time t; t, time; MF,∞, mass of the migrant 10179



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