Release kinetics of cinnamaldehyde, eugenol, and thymol from sustainable and biodegradable active packaging films

Food Packaging and Shelf Life 24 (2020) 100484

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Release kinetics of cinnamaldehyde, eugenol, and thymol from sustainable and biodegradable active packaging films

T

Bade Tonyalia, Austin McDaniela, Jayendra Amamcharlaa,b, Valentina Trinettaa,b, Umut Yucela,b,* a b

Food Science Institute, Kansas State University, Manhattan, KS, 66506, United States Animal Sciences and Industry, Kansas State University, Manhattan, KS, 66506, United States

A R T I C LE I N FO

A B S T R A C T

Keywords: Antimicrobial packaging Active packaging Controlled release Terpenoids Pullulan GC-MS Emulsion Solid lipid nanoparticles

Active packaging films can be formulated with biodegradable polymer loaded with natural antimicrobial compounds (NAC) as biodegradable alternatives to traditional plastic packaging for food. Essential oil compounds, naturally found in the plants, have antimicrobial activity against microorganisms. In this work, they are encapsulated in liquid lipid nanoparticles (LLN) or solid lipid nanoparticles (SLN) to control and enhance their solubility and stability and control kinetic release. In this study, the release kinetics of thymol, cinnamaldehyde, and eugenol, from pullulan-based films were studied as a function of lipid structure (liquid vs solid) and carrier particle concertation. The crystallization temperature of SLN (10.2 °C) decreased with incorporation of NAC (6.8 °C in SLN loaded with 1 % cinnamaldehyde). Increasing NAC significantly decreased (p < 0.05) the crystallization temperature of SLN. The SLN structure was similar in films to emulsions as their melting and crystallization temperatures were similar (p > 0.05). The NAC release rate from SLN films was twice than that from LLN films due to higher aqueous partitioning. The release rate in SLN films increased 100 %, 50 %, and 5 % for thymol, eugenol, cinnamaldehyde, respectively, when NAC concertation in the particles increased from 1 % to 2 %. Thymol had highest release rate (0.93 ppm/min) from SLN loaded with 2 % NAC films than that of eugenol and cinnamaldehyde (0.40 and 0.15 ppm/min, respectively). This study shows that the NAC-loaded pullulan films can be used as active antimicrobial packaging film for food applications with controlled release of active compounds.

1. Introduction The main purpose of food packaging is to protect the food enclosed beside its other functions such as handling, and communication with the customers (Guarda, Rubilar, Miltz, & Galotto, 2011; Ribeiro-Santos, Andrade, Melo, de, & Sanches-Silva, 2017). There is a large need to design active packaging systems with antimicrobial activity from Generally Recognized as Safe (GRAS) ingredients to further improve their protective function, preferentially using sustainable and economical sources. Recently, biodegradable packaging films increasingly gaining attention in the food industry. They are often based on biopolymers that are sustainable, environmentally-friendly and cost-effective alternatives to traditional plastic packaging materials (Del Nobile, Conte, Incoronato, & Panza, 2008; Hassannia-Kolaee, Khodaiyan, Pourahmad, & Shahabi-Ghahfarrokhi, 2016; López, Sánchez, Batlle, & Nerín, 2007; Ma, Hu, Wang, & Wang, 2016; Noronha, De Carvalho, Lino, & Barreto,

2014). Biodegradable films were successfully formulated by using polysaccharides, such as pullulan (Hassannia-Kolaee et al., 2016; Trinetta, Cutter, & Floros, 2011), carrageenan (Shahbazi, Rajabzadeh, Ettelaie, & Rafe, 2016), starch (Basiak, Galus, & Lenart, 2015; Jaramillo, Gutiérrez, Goyanes, Bernal, & Famá, 2016; Qiu et al., 2015; Saberi et al., 2016), and chitosan (Bonilla, Poloni, Lourenço, & Sobral, 2018; Noshirvani et al., 2017; Rao, Kanatt, Chawla, & Sharma, 2010; Robledo et al., 2018). In particular, pullulan is a water soluble polysaccharide produced by fungus Aureobasidium pullulans, and known for its good film-forming abilities (e.g., oral film strips) (Kristo & Biliaderis, 2007). It was also used in the food industry for thickening and encapsulation purposes (Hassannia-Kolaee et al., 2016). Pullulan-based films are transparent, tasteless, odorless, and have good oxygen barrier properties (Hassannia-Kolaee et al., 2016; Trinetta et al., 2011). The functional properties of these films may be enhanced by blending pullulan with other polymers (i.e., gums) (Trinetta et al., 2011). The biopolymer-based films can further be functionalized to have

⁎ Corresponding author at: Food Science Institute & Department of Animal Sciences and Industry, Kansas State University, 1530 Mid-Campus Dr. North, Manhattan, KS, 66056, United States. E-mail address: [email protected] (U. Yucel).

https://doi.org/10.1016/j.fpsl.2020.100484 Received 18 September 2019; Received in revised form 23 December 2019; Accepted 8 February 2020 2214-2894/ © 2020 Elsevier Ltd. All rights reserved.

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Hayashibara (Okayama, Japan); glycerin from VWR (Batavia, IL, USA); xanthan gum from TCI America (Portland OR, USA); locust bean gum from CP Kelco (Lille Skensved, Denmark), and sodium caseinate from Alfa Aesar (Ward Hill, MA, USA). Medium Chain Triglycerides (MCT) refined coconut oil and hydrogenated palm oil were obtained from Better Body Foods (Lindon, UT, USA) and Cargill (Minneapolis, MN, USA), respectively. Tryptic Soy Agar (TSA) was obtained from Becton, Dickinson and Company (Sparks, MD); 2-Cyclohexylcyclohexanone from Sigma-Aldrich (Milwaukee, WI, USA), and screw cap vials (10 mL Thread Vial and silicone, 1.3 mm caps) from La-Pha-Pack (Part of Thermo-Fisher Scientific, Germany). All chemicals were reagent grade and used without further modification.

antimicrobial activity using, natural antimicrobial compounds (NAC) such as essential oils (Noshirvani et al., 2017; Robledo et al., 2018). There is a plethora of articles on the antimicrobial effectiveness of essential oils as they are sustainable and GRAS compounds (Kuorwel, Cran, Sonneveld, Miltz, & Bigger, 2011). This form of active packaging shown promising results on controlling the growth of microorganism (Kuorwel et al., 2011). The essential oil compounds, thymol, eugenol, and cinnamaldehyde, which are naturally found in thyme, clove, and cinnamon essential oils, respectively, were shown to have strong antimicrobial effectiveness due to their phenolic structures (Del Nobile et al., 2008). The antimicrobial mode of action is related to their ability to disturb the cell membrane of microorganisms (Del Nobile et al., 2008; Marchese et al., 2017; Noshirvani et al., 2017; Robledo et al., 2018). These compounds are hydrophobic in nature, and therefore, need a form of delivery strategy to homogenously incorporate them into largely aqueous matrixes. At the same time, they are labile to thermal and chemical stresses (Trinetta, Morgan, Coupland, & Yucel, 2017). We previously showed that encapsulating these active compounds in liquid lipid nanoparticles (LLN) can improve their stability and control their release (Trinetta et al., 2017; Yucel, Elias, & Coupland, 2012). The encapsulated systems enhanced antimicrobial activity by controlling the distribution of the active ingredients throughout the system (Trinetta et al., 2017; Yucel et al., 2012). This enhancement is related to increased surface area-to-volume ratio and homogenous distribution (Donsì & Ferrari, 2016; Trinetta et al., 2017). Furthermore, solid lipid nanoparticles (SLN) can be formed by crystallization of lipid droplets in fine emulsions to further control the distribution and release of encapsulated ingredients (Yucel, Elias, & Coupland, 2013). The crystallization even can lead to expulsion of the ingredients from the droplet core, a phenomenon known as the burst release. Although it is often regarded as an instability problem, this instantaneous release of active compounds can enhance their antimicrobial effectiveness (e.g., limonene) by increasing their availability via interfacial interactions (Trinetta et al., 2017). Therefore, the antimicrobial activity of these compounds are largely determined by the release kinetics of entrapped active agents from film matrices. Other researcher investigated release of thymol from zein films (Del Nobile et al., 2008), citronella essential oil from soy protein lignin blend (Arancibia, Giménez, López-Caballero, Gómez-Guillén, & Montero, 2014; Arancibia, López-Caballero, Gómez-Guillén, & Montero, 2014), and cinnamon essential oil from polysaccharide bilayer films (Arancibia, Giménez et al., 2014; Arancibia, López-Caballero et al., 2014). Overall, conflicting results have been reported in the literature for the antimicrobial effectiveness of these compounds even in a liquid model environment (i.e., emulsions). Some of this discrepancy is related to the lack of knowledge for the release of these compounds as a function of physicochemical properties of the carrier systems; yet none in active packaging films formulated with liquid or solid lipid nanoparticles to encapsulate NAC and compound concentrations. Recently, our group studied the application of pullulan-based films to control postharvest disease in small berries, where the enhanced antimicrobial activity of NAC was greatly enhanced by controlling the internal structure of the carrier particles (McDaniel, Tonyali, Yucel, & Trinetta, 2019). Therefore, the objective of this work was to investigate the release kinetics of selected natural antimicrobial compounds (thymol, cinnamaldehyde, and eugenol) from the pullulan-based biodegradable films as a function of lipid nano-particles structure and crystallinity, and compound concentration.

2.2. Preparation of emulsions Coconut oil and palm fat emulsions were prepared using a hot homogenization technique as described by Yucel et al. (2013). In brief, the antimicrobial compounds (cinnamaldehyde, thymol, or eugenol) were individually mixed with one of the two carrier lipids (coconut oil or palm fat). Afterwards, 2 % w/w sodium caseinate solution was added to obtain final ratio of 1:9:90 or 2:8:90 of antimicrobial compound: carrier lipid: sodium caseinate solution. The coarse emulsion was obtained by mixing the solution using a high-shear mixer Brinkmann Polytron, Brinkmann Instruments Inc., Westbury, NY, USA at 11,000 rpm for 30 s. The final emulsions were prepared by homogenizing the premix in a two-stage valve homogenizer (Panda Plus 2000, GEA Niro Soavi, Parma, Italy) at 500 bars for 3 passes. The samples were kept in a water bath at 80 °C for 20 min for sterilization prior to film preparation. The particle size of emulsions was measured by using the DelsaMax Pro particle size analyzer equipped with a DelsaMax Assist unit (Beckman Coulter, Indianapolis, IN, USA). 2.3. Preparation of pullulan-based films Pullulan-based films were prepared following the procedure described by Trinetta et al. (2011). Two sets of emulsions were used to formulate films with the same final NAC concertation in the films: 1 % and 2 % NAC loaded SLN and LLN as follows: The first film set were prepared by mixing pullulan (50 g), glycerol (5 g), xanthan gum (5 g), and locust bean gum (5 g) with 500 mL of water at 70−80 °C for 20 min. The mixture was autoclaved at 121 °C for 5 min, and then mixed with emulsions containing 2 % NAC (500 mL) when it was still hot. The second film set was obtained by mixing the same amount of dry ingredients directly with the emulsion containing 1 % NAC (1 L) and stirred for 5 min at 90 °C. Both mixtures contain the same amount of NAC at the end but different number of carrier particles (i.e., 1 % NAC films contain double the number of particles in 2 % NAC films). The film solution is then were poured onto ultraviolet light-sterilized aluminum sheet pans (43 × 28 cm). Films were kept at 21 °C and 40 % relative humidity for 24 h to dry. After drying, the coconut-oil films were transferred to airtight containers and stored at room temperature, while the palm-oil films were kept at -20 °C until analysis. The experimental design was shown in Table 1. 2.4. Differential scanning calorimeter (DSC) analysis The crystallization and melting characteristics of palm-oil based films and emulsions, and coconut-oil films were studied by using a differential scanning calorimeter (DSC 2000, TA Instruments, DE, USA). 20 mg of the emulsion samples were put in hermetically sealed pans and were heated to 60 °C, cooled to -5 °C, and then heated to 60 °C again at a rate of 5 °C/minutes. 10 mg of the film samples were put in hermetically sealed pans and were heated to 150 °C, cooled to -5 °C, and then heated to 150 °C again at a rate of 5 °C/minutes.

2. Materials and methods 2.1. Materials Cinnamaldehyde, eugenol, and thymol were purchased from SigmaAldrich (Milwaukee, WI, USA). Pullulan was obtained from 2

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Table 1 Experimental variables with concentrations of ingredients used to develop the emulsions (on the left) and the films (on the right). NAC indicates natural antimicrobial compounds; THY, EUG, and CIN indicates thymol, eugenol, and cinnamaldehyde, respectively. Emulsions

Films

INGREDIENTS

Set 1 (wt%)

Set 2 (wt%)

INGREDIENTS

Set 1

Set 2

NAC (THY, EUG, or CIN)

2

1

Carrier Lipid (Palm or Coconut)

8

9

50 5 5 5

50 5 5 5

Sodium Caseinate

90

90

Pullulan (g) Glycerol (g) Xanthan Gum (g) Locust Bean Gum (g) Set 1 Emulsion (mL) Set 2 Emulsion (mL) Water (mL)

500 – 500

– 1000 –

2.5. Release of NAC from pullulan-based films The static NAC headspace concentration as a function of aqueous NAC concentration in emulsions was generated from a series of NAC loaded emulsions diluted in water (0.1–10 %). The internal standard, 2Cyclohexylcyclohexanone, (10 ppm) was used for quantification. The headspace analysis was performed following the method described in Trinetta et al. (2017) with modifications. The headspace concentration was analyzed using a Varian 431 gas chromatography (Agilent Technologies, CA, USA) equipped with a Varian 220 MS mass spectrometry unit (Agilent Technologies, CA, USA) and an HP-5MS capillary column (30 m x0.25 mm x0.25 μm, Agilent Technologies, CA, USA). The vials were incubated at 35 °C in a heater block (Multi-Blok Heater, Baxter Scientific Lab-Line Instruments, Thermo Scientific, USA) for one hour to allow equilibrium. The headspace from the vials (100 u L) was sampled with a gas tight syringe. The temperature program for the gas chromatography was: injection temperature, initial temperature 60 °C for 1 min, followed by gradients of 20 °C/min to 110 °C, 2 °C/min to 124 °C, and then 20 °C/min to reach 190 °C. The injector temperature was 280 °C and flow rate of helium as the carrier gas was 1 mL/min. The experimental conditions were determined from preliminary experiments. The one-dimensional release kinetics of NAC from the films were analyzed using similar headspace measurements at discrete time intervals. The film samples were prepared as circles (diameter: ca. 1 cm) for the release study. TSA (5 mL) containing internal standard of 2Cyclohexylcyclohexanone (1 ppm) was poured into the screw cap vials. Once the agar was cooled, the film circles were put onto top of it. The vials were screwed tight and left at room temperature during their incubation time (10, 20, 30 min and 1, 2, 4, 8 h). After the incubation period, the films were removed, and the caps were immediately screwed back. The vials were left overnight at room temperature to reach equilibrium. The release rates were determined from headspace NAC concentrations as described above.

Fig. 1. Particle size distribution of A) LLN (liquid lipid nanoparticles) and B) SLN (solid lipid nanoparticles) emulsions containing 0 and 2 % NAC (natural antimicrobial compounds), thymol (THY, ), cinnamaldehyde (CIN, ee), eugenol (EUG, • e), and control ().

3. Results and discussion 3.1. Emulsion and film characterization The particle size of LLN and SLN emulsions were measured using dynamic light scattering. All emulsions showed a monodispersed particle size distribution (Fig. 1). The Sauter mean diameter (d32) of control emulsions of LLN and SLN were slightly but significantly different (p < 0.05) as 172 ± 2 and 190 ± 10 nm, respectively. This is related to narrow particle size distribution (i.e., small polydispersity index) and particle shape change in nanoparticles when crystallized (Trinetta et al., 2017). The addition of NAC did not significantly (p < 0.05) affect the particle size of LLN and SLN emulsions without NAC. This is irrespective of the NAC type used in this study. Similarly, our previous work showed that addition of limonene to nano-emulsions does not affect the particle size distribution (Trinetta et al., 2017). However, the particle size of cinnamaldehyde loaded SLN (1 and 2 % NAC) were found to be significantly higher (ca. 200 nm) than other SLN (ca. 168 nm same for thymol and eugenol). In Acevedo-Fani, Salvia-Trujillo, Rojas-Graü, and Martín-Belloso (2015)’s study, thyme oil loaded emulsions had larger droplet size than sage oil loaded emulsions. It was attributed to their different affinity for water and oil phases as well as their chemical compositions. Hence, the chemical composition and water solubility of cinnamaldehyde (1.85 g/L at 25 °C) in the emulsion system might affect the particle size. In our previous work (Trinetta et al., 2017), we showed that incorporation of LLN or SLN did not alter the original pullulan film properties. In the current work, we investigated the effect of incorporation of selected NAC on the mechanical and structural properties of biodegradable pullulan films. The control films (i.e., 0 % NAC) had similar moisture content (p > 0.05) to NAC loaded films (ca. 3.5 wt%). Increasing NAC concentration or NAC type did not affect (p < 0.05) the moisture content of the films. The incorporation of NAC

2.6. Statistical analysis All experiments were performed in triplicate, unless otherwise was stated. The results were presented as mean ± standard deviation and analyzed by analysis of variance (ANOVA) using MINITAB software (Minitab Version16, State College, Pa., USA). Tukey`s multiple comparison test was applied to evaluate the differences between treatments. The differences were considered significant when p < 0.05.

3

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Fig. 2. Pictures of 10 % A) SLN (solid lipid nanoparticles) formulated films with 1 % NAC (natural antimicrobial compounds) emulsion, B) SLN formulated films with 2 % NAC emulsion, C) LLN (liquid lipid nanoparticles) formulated films with 1 % NAC emulsion, D) LLN formulated films with 2 % NAC emulsion. From left to right: Control, Thymol, Eugenol, and Cinnamaldehyde.

to the films resulted in certain color changes and loss of translucency with NAC addition in increasing by order of thymol, eugenol, cinnamaldehyde (Fig. 2). The appearance of both films formulated with 1 % and 2 % NAC emulsions looked similar, and the number of droplets containing the NAC didn’t affect the color (i.e., the final NAC concentrations are the same in films). The color change, however, was affected by the crystallinity of the particles, and was more pronounced in SLN films than LLN films. This difference is related to the partitioning of the NAC and expulsion form the core of SLN to the surface with crystallization.

when it was homogenized to small droplets (Fig. 3B). This is due to small particle size of emulsions, which need more cooling for the crystallization due to homogenous nucleation mechanisms and excess the interfacial free energy further modulating the crystallization enthalpy (Coupland, 2002; McClements, Decker, & Weiss, 2007). When NAC was incorporated into the emulsions, the crystallization temperature further decreased as a function of NAC amount. 1 % NAC addition decreased the crystallization temperature 46 %, while 2 % NAC addition caused 83 % decrease in the crystallization temperature of SLN compared to control SLN in emulsions (Table 2). Similar observations were seen in the films. The crystallization temperature of SLN in control film was at 10 °C and it shifted to around 5 °C with incorporation of 1 % NAC (Table 2). Muriel-Galet, Cran, Bigger, Hernández-Muñoz, and Gavara (2015) suggested that incorporation of additives, i.e., essential oils and natural antimicrobial compounds, into the film matrix could interfere with the crystallization process and

3.2. Thermal properties of emulsions and films Melting and crystallization behavior of LLN and SLN in the emulsions and films were studied using DSC (Fig. 3). The crystallization temperature of bulk palm fat decreased from 30 °C to around 12 °C 4

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Fig. 3. A) Heating and B) Cooling thermograms of 10 % SLN (solid lipid nanoparticles) formulated with 1 % Thymol (THY, ), Cinnamaldehyde (CIN, ), Eugenol (EUG, ) in the emulsions as compared to control SLN (no NAC,); and C) heating and D) Cooling thermograms of 10 % LLN (liquid lipid nanoparticles) formulated with 1 % Thymol, Cinnamaldehyde, Eugenol in the emulsions as compared to control LLN (no NAC,).

therefore shift the peaks. Ahmed, Mulla, and Arfat (2016) observed a significant decrease in crystallization temperature of polylactide films with increasing cinnamon oil content. They attributed this shift in

temperature to increased chain mobility. Nostro et al. (2013) suggested that the essential oils behave like plasticizers since they decrease the intermolecular forces of polymer chains. The crystallization

5

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Table 2 Melting and crystallization temperature of bulk palm fat, and SLN (solid lipid nanoparticles) in emulsions and films. CIN, EUG, and THY indicates cinnamaldehyde, eugenol, and thymol, respectively. Melting Temperature (°C) 54.0 ± 0.6w

Bulk Fat SLN Emulsions 1% 2% SLN films 1% 2%

Crystallization Temperature (°C)

CIN 49.9 47.2 CIN 52.1 49.3

EUG B, xy

± 0.1 ± 1.1B,y

± 0.4AB,wx ± 0.6B,y

49.3 46.5 EUG 51.9 49.9

30.9 ± 0.1w THY

B,y

± 0.1 ± 0.1B,z

± 0.2B,x ± 0.4B,y

49.6 45.9 THY 51.1 48.5

Control B,x

± 1.1 ± 0.6B,y ± 0.5B,x ± 0.5B,xy

52.2 ± 52.2 ± Control 53.6 ± 53.6 ±

CIN A,x

0.1 0.1A,x 0.2A,wx 0.2A,wx

6.8 ± 2.3 ± CIN 6.8 ± 4.9 ±

EUG B,x

0.9 1.3B,y 2.7A,x 2.2B,xy

7.0 ± 3.1 ± EUG 7.1 ± 5.1 ±

THY B,x

0.3 0.7B,y 1.8A,x 0.5B,xy

5.3 ± 1.1 ± THY 1.2 ± 1.2 ±

Control B,x

1.4 0.1B,y 0.1B,y 0.2B,y

11.7 ± 11.7 ± Control 10.2 ± 10.2 ±

0.3A,x 0.3A,x 1.1A,x 1.1A,x

Data is presented as mean ± standard deviation. Different capitalized letters (A, B, C) in the same row indicate significant difference for melting and crystallization temperatures, separately (p < 0.05). Different lower-case letters (w, x, y, z) in the same column indicate significant difference for melting and crystallization temperatures as compared to bulk fat (p < 0.05).

3.3. Release characteristics of NAC from active packaging films

temperatures for 1 % and SLN loaded with 2 % NAC in films were 51 and 63 % less than control SLN in films. All DSC thermograms of SLN in emulsions and films showed one endothermic peak in the range of 45−55 °C which was attributed to the melting peak. The melting temperature of bulk palm fat was around 54.0 °C and it decreased to 52.2 °C after homogenization (Fig. 3A). The melting temperature of bulk palm fat was in agreement with Xiao et al. (2016)’s finding. The addition of 1 and 2 % NAC to SLN significantly decreased (p < 0.05) the melting point of the droplets in the emulsions (5.1 and 10.8 % lower than the control SLN in emulsions for 1 and 2 % NAC addition, respectively). Moreover, increasing the NAC concentration to 2 % showed a significant further decrease (p < 0.05) in the melting temperatures of SLN in emulsions. There was no significant difference in the melting points of SLN in the emulsions among different NAC for both concentrations (Table 2). The film samples showed a similar decreasing trend as emulsions. The melting temperature of SLN in control film significantly (p < 0.05) shifted to lower temperatures (53.6 °C to 51.7 °C) with incorporation of 1 % NAC, having a decrease of 3.5 %. Salarbashi et al. (2014) and Nisar et al. (2018) observed a decrease in melting point after integration of essential oils in soluble soybean polysaccharide films and citrus pectin films, respectively. This decrease is attributed to the effect of molecular structure of the essential oils on the chain mobility (Salarbashi et al., 2014). The films formed with 2 % NAC SLN emulsions had significantly lower (p < 0.05) melting temperature (ca. 4.7 %) than the films formed with 1 % NAC SLN. Šuput et al. (2016) and Nisar et al. (2018) previously observed a decrease in the melting temperature with increasing the essential oil concentrations. Hosseini, Rezaei, Zandi, and Farahmandghavi (2015) mentioned the decrease in the melting point to lower temperatures might be due to the plasticizing effect of essential oils. Essential oil creates more free volume within the biodegradable polymer network as well as more mobility in the polymer chains. In Tongnuanchan, Benjakul, Prodpran, and Nilsuwan (2015)’s study, the melting temperature of palm oil droplets increased with increasing palm oil content in the droplets. The authors hypothesized that the oil phase underwent stronger order-phase fraction with gelatin in the presence of higher palm oil content. Similarly, in our study, SLN loaded with 1 % NAC in emulsions and films had higher melting points than SLN loaded with 2 % NAC in emulsions and films. In SLN emulsion matrices, the SLN loaded with 1 % NAC have higher palm oil content (9 %) than SLN loaded with 2 % NAC (8 %). Additionally, in SLN film matrices, the films formed from 2 % NAC emulsions have fewer particles due to water dilution during the film preparation step. The melting temperature of the bulk coconut oil was around -3.2 °C. As expected, we did not observe a phase change, i.e., melting and crystallization peaks, when the coconut oil was homogenized in emulsions, and the LLN were incorporated in the film matrix at the experimental conditions (Fig. 3C and D).

The one-dimensional release kinetics of each NAC from films to a model matrix (i.e., TSA) were evaluated at 37 °C over a period of 8 h using GC–MS headspace analysis. The amount of NAC in the model (Fig. 5) was quantified from the NAC peak areas per IS peak area of standard curve (Fig. 4). The NAC incorporated LLN in the emulsions increased linearly for peak area and increasing oil concentration in the range of 0.01 to 0.5 % (Fig. 4). The film release kinetics targeted in this study are within this linear range. In general, the headspace NAC concentrations followed the differences in their boiling points (b.p.). Thymol (b.p. 233 °C) had highest headspace concentrations among LLN loaded emulsions at each oil concentration associated to its lower boiling point than cinnamaldehyde (b.p. 253 °C) and eugenol (b.p. 252−253 °C). This observation was also in agreement with the results of headspace analysis of SLN and LLN incorporated films. The release rate of the NAC from the films were determined by the amount of NAC diffused to TBA with time and was function of physicochemical properties of NAC and the structure of the carrier emulsion particles. The release rate of thymol was highest in all films then cinnamaldehyde and thymol (Figs. 5), although the logP value of thymol (logP 3.3) is higher than the later (logP 2 for both eugenol and cinnamaldehyde). Thymol loaded SLN incorporated films had highest release rate (∼10 fold and ∼3 fold than cinnamaldehyde and eugenol in NAC loaded SLN incorporated films, respectively), although the logP value of thymol (logP 3.3) is higher than the later (logP 2 for both eugenol and cinnamaldehyde). Indeed, the rate constant of cinnamaldehyde and thymol are not different from each other due to similar physiochemical properties. This observation suggests that thymol was carried in nanoemulsion particles together with the carrier lipid rather than molecular diffusion mechanisms. The particle size of thymol loaded SLN was smaller than cinnamaldehyde loaded SLN in emulsions, which might also contribute to this argument and enhance the release kinetics (Fig. 1). Similarly, Sakulku et al. (2009) and Yuan, Gao, Zhao, and Mao (2008) observed the same trend. Therefore, the reason of higher release of thymol than cinnamaldehyde and eugenol might be the particle size. On the other hand, Mastromatteo, Barbuzzi, Conte, and Del Nobile (2009) stated the film thickness can also affect the release rate of thymol from zein films. In the current study, we kept the film-forming polymer composition and film thickness constant and is not a significant variable to affect the headspace NAC concentrations or release rates. In addition, there was no significant difference in moisture content of the films that it was not expected to impact the release characteristics. Similarly, Kashiri et al. (2017) observed higher release of thymol than carvacrol from zein films into anaqueous medium (i.e., containing 10 % ethanol or 3 % acetic acid) since the aqueous solubility of thymol is higher than carvacrol. However, the large amount of zein protein can strongly bind to small molecules at varying extents modulating their transport phenomena. Nostro et al. (2012, 2013) observed 6

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Fig. 4. Headspace peak areas of 10 % LLN (liquid lipid nanoparticles) films formulated with A) 1 % or B) 2 % Thymol (THY, •••), Cinnamaldehyde (CIN, • e), Eugenol (EUG,) from the emulsions.

LLN loaded with 1 % NAC (Fig. 5), although both films contain the same NAC amount. The number of SLN and LLN loaded with 1 % NAC in the films was double the number of SLN and LLN loaded with 2 % NAC films, and the final NAC concentration in the films were ca. 5 %. This difference in films formulated with LLN were marginal that the difference was not significant for any of the NAC (p > 0.05). This is as expected since in LLN systems the majority of the hydrophobic compounds partition to the droplet core, and therefore the volume effect overcomes the surface effects. When droplets crystallized into SLN, there was a significant increase in the release rates for all the films. SLN: This increase was more pronounced in the films made from SLN loaded with 2 % NAC: the release rate constant was doubled for cinnamaldehyde SLN films (p < 0.05), 50 % increased for eugenol SLN films (p < 0.05), and 5 % increased (not significant, p > 0.05) for thymol SLN films as compared to films made from SLN loaded with 1 % NAC. The expulsion of the hydrophobic small molecules from the core to resulted in loss of significance of the volume effects, and therefore the release behavior is determined by the number of particles present in the system, or the total interfacial area. In other words, in films made from SLN loaded with 2 % NAC there is half the number of particles than films made from SLN loaded with 1 % NAC. Therefore, more NAC is associated per surface area of carrier particles. Indeed, this surface association was determined by the physicochemical characteristics of the NAC used, and highest for the

higher amount of release of cinnamaldehyde and eugenol than carvacrol and citronellol, respectively, due to hydrophobic structures. The NAC concentrations in headspace showed a noticeable increase at 240 min for all the films. This is attributed to the change in their brittle structure at that time based on the visual observation of the films. The film loses its brittle structure due to the dsiffusion of water to the film matrix (Fernández-Pan, Maté, Gardrat, & Coma, 2015). The diffusion of water increases the mobility of the polymeric chains and promotes the diffusion (Benbettaïeb et al., 2016). Crystallization of SLN in the films exposed the NAC to the interfacial region, which resulted higher NAC concentration in headspace than that of LLN in the films. Similar results were reported in essential oil and aroma compounds added to eicosane emulsions (Ghosh, Peterson, & Coupland, 2006; Trinetta et al., 2017) and octadecane emulsions (Longyuan et al., 2010). In Yucel et al. (2012) showed that the hydrophobic compound PTMIO expulsed from the core upon crystallization of eicosane droplets but localized at SLN surfaces. This behavior increases the availability of NAC for bacterial attachment with higher contact area and concentration of NAC in aqueous medium. This hypothesis is confirmed with our previous study (McDaniel et al., 2019), where films from SLN showed better antifungal activity compared to films from LLN. In general, the films formulated with SLN and LLN loaded with 2 % NAC showed higher release rates than films formulated with SLN and 7

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Fig. 5. NAC release from 10 % LLN (liquid lipid nanoparticles) films formulated with A) 1 % or B) 2 % Thymol (THY, ◼), Cinnamaldehyde (CIN, ▲), Eugenol (EUG, ●); and from 10 % SLN (solid lipid nanoparticles) formulated with C) 1 % or D) 2 % Thymol (◼), Cinnamaldehyde (▲), Eugenol (●).

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cinnamaldehyde. A similar behavior was observed in our parallel study where we investigated the antimicrobial effectiveness of these compounds through volatile transport (McDaniel et al., 2019). In parallel to the findings of the current study, he antimicrobial effectiveness of cinnamaldehyde was significantly affected by the number of particles in SLN films, but not in LLN films. In a similar way, there was no difference for the antimicrobial activity of the films made from SLN loaded either 1 % or 2 % thymol. Earlier, Ghosh et al. (2006) showed the effect of hydrophobic nature of model flavor molecules on their surface binding to emulsion droplets to simialr conclusions. 4. Conclusion Thymol, eugenol, and cinnamaldehyde active compounds loaded LLN and SLN showed potential to be used in the food industry for active packaging applications. The NAC release was faster in films made from particles loaded with 2 % NAC than the films from particles loaded with 1 % NAC. Higher number of particles increased the surface area in 1 % NAC films to limit diffusion due to surface associations. This increase was more pronounced in SLN films. Moreover, NAC loaded SLN in films showed higher NAC concentration than NAC loaded LLN in films, to allow temperature to be used as a trigger in developing active antimicrobial packaging films for food applications. This result is complimented with DSC results where the crystallization temperatures of SLN in films were higher than the crystallization temperature of LLN in films. These results are useful in providing guidance for selecting volatile antimicrobial compounds, their concentrations, and carrier lipid particles with different physicochemical properties in temperaturecontrolled active packaging applications and manufacturing in large scale. CRediT authorship contribution statement Bade Tonyali: Writing - original draft, Data curation, Formal analysis, Investigation. Austin McDaniel: Writing - review & editing, Investigation. Jayendra Amamcharla: Writing - review & editing, Resources. Valentina Trinetta: Funding acquisition, Supervision, Writing - review & editing. Umut Yucel: Funding acquisition, Supervision, Writing - original draft, Conceptualization. Acknowledgements The authors wish to thank the Kansas Department of Agriculture for the funding support and the USDA National Institute of Food and Agriculture Hatch/Multi-state projects 1014385 and 1014344. Contribution no. 19-331-J from the Kansas Agricultural Experiment Station. 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.2020.100484. References Acevedo-Fani, A., Salvia-Trujillo, L., Rojas-Graü, M. A., & Martín-Belloso, O. (2015). Edible films from essential-oil-loaded nanoemulsions: Physicochemical characterization and antimicrobial properties. Food Hydrocolloids, 47, 168–177. https://doi. org/10.1016/j.foodhyd.2015.01.032. Ahmed, J., Mulla, M. Z., & Arfat, Y. A. (2016). Thermo-mechanical, structural characterization and antibacterial performance of solvent casted polylactide/cinnamon oil composite films. Food Control, 69, 196–204. https://doi.org/10.1016/j.foodcont. 2016.05.013. Arancibia, M., Giménez, B., López-Caballero, M. E., Gómez-Guillén, M. C., & Montero, P. (2014). Release of cinnamon essential oil from polysaccharide bilayer films and its use for microbial growth inhibition in chilled shrimps. LWT – Food Science and Technology, 59(2P1), 989–995. https://doi.org/10.1016/j.lwt.2014.06.031. Arancibia, M. Y., López-Caballero, M. E., Gómez-Guillén, M. C., & Montero, P. (2014). Release of volatile compounds and biodegradability of active soy protein lignin blend

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