Development of antioxidant food packaging materials containing eugenol for extending display life of fresh beef

Accepted Manuscript Development of antioxidant food packaging materials containing eugenol for extending display life of fresh beef

Vesta Navikaite-Snipaitiene, Liudas Ivanauskas, Valdas Jakstas, Nadine Rüegg, Ramune Rutkaite, Evelyn Wolfram, Selçuk Yildirim PII: DOI: Reference:

S0309-1740(17)30985-3 doi:10.1016/j.meatsci.2018.05.015 MESC 7565

To appear in:

Meat Science

Received date: Revised date: Accepted date:

5 July 2017 8 March 2018 22 May 2018

Please cite this article as: Vesta Navikaite-Snipaitiene, Liudas Ivanauskas, Valdas Jakstas, Nadine Rüegg, Ramune Rutkaite, Evelyn Wolfram, Selçuk Yildirim , Development of antioxidant food packaging materials containing eugenol for extending display life of fresh beef. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mesc(2017), doi:10.1016/j.meatsci.2018.05.015

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ACCEPTED MANUSCRIPT Development of antioxidant food packaging materials containing eugenol for extending display life of fresh beef Vesta Navikaite-Snipaitienea, Liudas Ivanauskasb, Valdas Jakstasc, Nadine Rüeggd, Ramune Rutkaitea, Evelyn Wolframe, Selçuk Yildirimd a

Department of Polymer Chemistry and Technology, Kaunas University of Technology, Radvilenu Rd. 19, LT-50254 Kaunas,

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Lithuania Department of Analytical and Toxicological Chemistry, Lithuanian University of Health Sciences, Eiveniu Str. 4, LT-50161

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Kaunas, Lithuania

Department of Pharmaceutical Chemistry and Pharmacognosy, Lithuanian University of Health Sciences, Eiveniu Str. 4, LT-

Institute of Food and Beverage Innovation, Zurich University of Applied Sciences, Einsiedlerstrasse 29-34, 8820 Wädenswil,

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50161 Kaunas, Lithuania

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Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Einsiedlerstrasse 29-34, 8820 Wädenswil,

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Switzerland

ACCEPTED MANUSCRIPT Abstract In this study, clove essential oil (CL) or eugenol (EU) containing cellulose acetate (CA) or acrylic component/hydrophobically modified starch (AC/S) coatings on corona treated oriented polypropylene film (OPP) were designed and investigated for their possible applications as antioxidant packaging materials for fresh meat. The antioxidant properties of the coatings were investigated by Vapour Phase-

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DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. The CA coatings containing CL or EU showed 43-92 % and 43-94 % inhibition against DPPH free radicals through the vapour phase, respectively, whereas

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AC/S/CL and AC/S/EU coatings resulted in DPPH inhibition of 21-65 % and 25-84 %, respectively.

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AC/S/EU and CA/EU coatings on OPP containing from 0.32 ± 0.03 to 6.40 ± 0.14 g/m 2 of EU were used to prepare packaging for fresh beef (Longissimus thoracis). After 14 days, the lipid oxidation in beef

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steaks kept in control and antioxidant packages was 3.33 and 1.00-1.22 mg of malondialdehyde per

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kilogram of meat, respectively. Moreover, red color of beef in antioxidant packages was retained.

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Keywords: Vapour Phase DPPH assay, eugenol, clove essential oil, antioxidant packaging, beef.

ACCEPTED MANUSCRIPT 1. Introduction In the last few years, numerous research efforts have aimed to develop new packaging technologies to better protect the quality of perishable foods. Active packaging is one of the innovative packaging concepts that has been introduced in response to the continuous changes in consumer demands and market trends (Vermeiren, Devlieghere, Beest, Kruijf, & Debevere, 1999). This technology is based on

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the concept of incorporation of certain components into packaging systems that release or absorb substances from or into the packaged food or the environment surrounding to extend the shelf-life or to

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maintain or improve the condition of the packaged food (Regulation (CE) No. 450/2009 (29/05/2009)).

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One of the main active packaging technologies is the antioxidant packaging which releases synthetic or natural antioxidants to prevent food oxidation (Lee, 2014). Compared to synthetic

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antioxidants, natural antioxidants such as herbs and spices are of great interest because of their safety and

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health characteristics (Lee, 2014; Fernandes et al., 2016; Lorenzo, González-Rodríguez, Sánchez, Amado, & Franco, 2013). Among the natural essential oils clove (Syzygium aromaticum) essential oil has been

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studied as one of the strongest natural antioxidants, with activity even higher than some synthetic

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antioxidants like BHT or butylated hydroxyanisole (Jirovetz, Buchbauer, Stoilova, Stoyanova, Krastanov, & Schmidt, 2006; Wei, & Shibamoto, 2010). Gas chromatographic analysis of commercial clove essential oil indicates the presence of three major compounds, namely eugenol (67.6 %), aceteugenol (16.8 %) and

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trans-caryophyllene (10.8 %) (Teixeira, Marques, Ramos, Neng, Nogueira, Saraiva, et al., 2013). The

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antioxidant activity of clove essential oil is mainly attributed to eugenol. The antioxidant efficiency of eugenol has been evaluated in lipid model systems, by studying the formation of primary (hydroperoxydienes) and secondary (malonaldehyde) components of the oxidative process (Ruberto, & Baratta, 2000; Wei, & Shibamoto, 2010). Antioxidants can be applied in the packaging systems in different forms such as sachets, labels, coatings or could be immobilized on packaging material (Realini, & Marcos, 2014). Furthermore, biopolymers can be used as safe and biodegradable carriers for the incorporation and release of active compounds to the food (Cha, & Chinnan, 2004). Research has shown that incorporation of extracts and essential oils such as rosemary, clove, eugenol into biopolymers like

ACCEPTED MANUSCRIPT chitosan or modified cellulose can secure their high antioxidant properties, and such materials potentially could be applied as an active material for food packaging (Abdollahi, Rezaei, & Farzi, 2012; Phoopuritham, Thongngam, Yoksan, & Suppakul, 2012; Woranuch, & Yoksan, 2013). Meat packaging is an interesting application for antioxidant packaging, since it can prevent lipid oxidation, color changes and discoloration of meat and meat products. Up to now only a few studies have

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been published, where essential oil containing antioxidant packaging was used to protect the quality of fresh lamb, beef or foal meat (Camo, Antonio Beltrán, & Roncalés, 2008; Camo, Lorés, Djenane, Beltrán,

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& Roncalés, 2011; Lorenzo, Batlle, & Gómez, 2014; Nerin, Tovar, Djenane, Camo, Salafranca, Beltran,

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et al. 2006).

In this work new active packaging materials containing biopolymers (modified starch, cellulose

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acetate) and clove essential oil or eugenol were designed, and their antioxidant properties through a newly

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developed tailored “Vapour Phase - DPPH method” were investigated. Additionally, the influence of use of such antioxidant packaging materials on the quality and display life of fresh beef during a storage of 14

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days was evaluated.

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2. Materials and Methods 2.1. Materials

Clove essential oil, eugenol, 2-2-diphenyl-1-picrylhydrazyl (DPPH), methanol, cellulose acetate

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were purchased from Sigma-Aldrich and used without further purification. Hydrophobically modified

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waxy maize starch was purchased from Ingredion GmbH (Germany). Premo® Coat BR20 (food contact approved acrylic component) was supplied by Flint Group (Finland). Acetone was purchased from Acros Organics. Corona treated oriented-polypropylene (OPP) film (30 µm) and PET/PE/EVOH/PE peel film were supplied by SÜDPACK® (Switzerland). 2.2. Preparation of acrylic component/starch coatings containing clove essential oil or eugenol The aqueous emulsion consisting of water, 25 % (w/v) hydrophobically modified waxy maize starch (S) and 12.5 % (w/v) of clove essential oil (CL) was prepared by using rotor-stator homogenizer (Polytron® PT 2500 E, disperse element PT-DA 12/2EC-E157, Switzerland) at 15,000 rpm for 2 min.

ACCEPTED MANUSCRIPT Alike the aqueous emulsion consisting of water, 25 % (w/v) S and 12.5 % (w/v) or 25 % (w/v) of eugenol (EU) was prepared. The concentration of starch and essential oil were chosen based on results of our previous experiments (unpublished results). The emulsions were mixed with acrylic component (AC) in certain quantities to obtain different amounts of CL or EU in the coatings (see Table 1). The preparations were cast on corona treated oriented-polypropylene films (OPP) by using a coating machine (Zehntner,

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ZAA 2300, Switzerland) and profile rods (20.14 μm or 100 μm thickness of wet film). The coated OPP films were dried at room temperature for 1 h. The final content of CL or EU in the AC/S coatings was

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ranging from 2.5 to 20 wt%. Thus, the average amount of active components on OPP varied from 0.11 ±

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0.02 to 6.40 ± 0.14 g/m2.

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Table 1. Preparation of AC/S/CL and AC/S/EU coatings Preparation of coating Composition of dry coating composition (g) (wt %) AC Emulsion AC S CL or EU 92.0 8.0 92.5 5 2.5 84.2 15.8 85 10 5 68.6 31.4 70 20 10 65.2 34.8 60 20 20

Average amount of active component on OPP (g/m2) CL EU 0.11±0.02 0.14±0.01 0.23±0.02 0.26±0.03 0.50±0.05 0.54±0.01 6.40±0.14

2.3. Preparation of cellulose acetate coatings containing clove essential oil or eugenol

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Firstly, 12 g of cellulose acetate (CA) was dissolved in respective amount of acetone for each

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composition and three different quantities of CL or EU were added under stirring to prepare coating compositions (see Table 2). The obtained coating preparations were cast on corona treated OPP films by

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using a coating machine (Zehntner, ZAA 2300, Switzerland) and profile rod (40.15 μm thickness of wet film). The coated OPP films were dried at room temperature for 0.5 h. The final content of CL and EU in

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the CA coatings was ranging from 7 to 23 wt%. Thus, the average amount of active components on OPP varied from 0.18 ± 0.03 to 0.82 ± 0.13 g/m2. Table 2. Preparation of CA/CL and CA/EU coatings Preparation of coating Composition of dry coating composition (g) (wt %) CA Acetone CL or EU CA CL or EU 12.00 84.40 3.60 93 7 12.00 86.20 1.80 87 13 12.00 87.10 0.90 77 23

Average amount of active component on OPP (g/m2) CL EU 0.18±0.02 0.19±0.05 0.31±0.05 0.44±0.09 0.77±0.10 0.82±0.13

2.4. Determination of antioxidant properties by using Vapour Phase - DPPH assay The antioxidant properties of AC/S/CL, AC/S/EU, CA/CL and CA/EU coatings were investigated through the vapour phase by Vapour Phase - DPPH method (VP-DPPH) in tightly closed petri dishes

ACCEPTED MANUSCRIPT (Eppendorf cell culture dishes, 60 mm). Coating sample (55 mm of diameter size) was stuck by double sticky tape to the inside of the lid of the petri dish. 2.5 mL of 0.1 mM DPPH methanol solution was placed onto the bottom of plate (non-direct contact coating). The coatings without CL or EU were used as negative controls. Petri dishes were tightly closed by placing a rubber ring between the lid and the bottom plate, sealed with ParafilmM® and kept in linear shaker (80 rpm) at room temperature in the dark. After a

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certain incubation time 240 µL of sample were pipetted into sample well and the absorbance of DPPH solution at 517 nm was measured by using a plate reader (Synergy HT, BioTek Instruments, Switzerland).

(Eq. 1)

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Inhibition (%) = ((A0 – AS) / A0)) · 100

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The inhibition of the stable DPPH radical was calculated by using the following equation:

where A0 is the absorbance of the initial DPPH solution at 517 nm, and AS is the absorbance of DPPH

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solution exposed to the sample.

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2.5. Determination of concentration and release of eugenol

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AC/S/CL (0.30 ± 0.04 g/m2), AC/S/EU (0.31 ± 0.04 g/m2), CA/CL (0.76 ± 0.11 g/m2) and CA/EU

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(0.79 ± 0.06 g/m2) coatings on OPP were used for determination of eugenol release. The amount of EU in the coatings and release of EU into the vapour phase were analyzed using ordinary and headspace methodologies on SHIMADZU GC-MS-QP2010 Ultra chromatography system equipped with AOC-5000

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injection system and fused silica capillary column (Rxi-5ms, Restek Corporation; 30m x 0.25 mm, 0.25

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µm film coating). The GC oven temperature was programmed from 80 ºC for 1 min, then 8 ºC /min to 170 ºC for 1 min, then 30 ºC/min to 270 ºC and held constant for 2 min. The injector temperature was 260 ºC, helium used as carrier gas at 1.43 mL/min, injection mode was split less (1.5 min), and headspace injection volume was 500 µL, liquid injection volume was 1 µL. Mass spectra scan range was of 35-500 amu. Identification of the EU was done on the basis of retention and mass spectrum of reference, m/z 164 was used for calibration (nmol vs peak area) and quantitation. The amount of EU in the coated films were established after dissolution of samples in the acetone (10 mL). The release of EU from the coated films into a headspace was monitored using coated film samples putted into a headspace vials (20 mL) and

ACCEPTED MANUSCRIPT sealed. The vials were maintained at room temperature for defined periods. The vials were stored at 30 ºC for 1 min before the headspace injection. Three headspace injections from separate vials were made per sample. The partition coefficient (K) of the amount ratio of the EU between two phases was calculated by using the following equation: (Eq. 2)

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K=Cg/Cf

where Cg represents amount (nmol) of the EU in the gas phase and Cf - established amount (nmol) of the

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EU in the coating

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2.6. Preparation of antioxidant food packaging for beef samples

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The steaks (Longissimus thoracis) of fresh beef of about 90 g of weight (1.5 cm thick) were sliced using cutting machine. Each steak was placed into high barrier tray (PS/EVOH/PE) of 204×147×250 mm size. The fresh beef samples were packed under modified atmosphere (MAP) with a gas mixture

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consisting of 80 % O2 and 20 % CO2 (supplied by PanGas, Switzerland) and sealed with PET/PE/EVOH/PE 57µm peel film (Südpack, Germany) by using tray sealer (Multivac T200, Switzerland). Before the tray sealing, the OPP film with active coating (187 cm2) containing different

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amounts of eugenol (AC/S/EU1 - 0.32 ± 0.03 g/m2; AC/S/EU2 - 6.40 ± 0.14 g/m2; CA/EU - 0.65 ± 0.08

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g/m2) was attached to peel film. As control, meat was packaged without active coating. The samples were displayed in a refrigerated cabinet under illumination (HE 21 W/827, Osram Lumilux Interna, Italy) at 2 ± 1 °C for 24 h, thereafter in the dark for 14 days. During the storage change in meat colour were monitored and lipid oxidation in meat was quantified. The experiments for each sample kept in packaging with different coating were performed in triplicates for each sample point.

2.7. Colour analysis

ACCEPTED MANUSCRIPT The colour analysis of beef steaks surface was performed by using a chroma meter (Konica Minolta CR-400, illuminant D65) 30 min after pack opening in order to allow colour stabilization on air exposure. CIE L* (lightness), a* (redness) and b* (yellowness) parameters were recorded. The average value for each parameter was the mean of 10 determinations for each sample. Hue angle (hab) and chroma (C*)

hab=tan−1(b*/a*)

(Eq. 3)

C*=(a*2 + b*2)1/2

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parameters were calculated using the following equations:

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(Eq. 4)

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2.8. Lipid oxidation (TBARS) assay

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Evaluation of lipid oxidation was carried out using Food TBARS Assay Kit (Oxford Biomedical

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Research, Inc.) according to the protocol with some modifications. 5 g of meat and 5 mL of deionized water were added into a 50 mL centrifuge tube and homogenized by using homogenizer (Polytron® PT

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2500 E, disperse element PT-DA 12/2EC-E157, Switzerland) for 1 min at 12,000 rpm/min and 1 min at

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15,000 rpm/min in order to obtain a smooth suspension. Afterwards, sample volume was adjusted to 10 mL by adding distilled water.

Lipid oxidation analyses. 1 mL of indicator solution (5 % 2-thiobarbituric acid (TBA) solution in

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DMSO containing acid catalyst) was added into a tube with 1 g of the suspension. Solution without TBA

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reagent was used as a blank. All samples were agitated vigorously for one minute on a vortex mixer. Afterwards, tubes of samples were kept to react for 60 minutes at room temperature and then centrifuged at 16,000 x g for 5 min at 22-25 ºC. After centrifugation, aqueous layer was taken out, recentrifuged and then transferred to a cuvette. The absorbance at 532 nm was measured by using UV-vis spectrophotometer (Genesys™ 10S UV-Vis, USA). Thiobarbituric acid reactive substances (TBARS) values were calculated from a standard calibration curve of malondialdehyde (MDA) and expressed as milligrams of MDA per kilogram of meat. For each sample triplicates were performed. The lipid oxidation was measured at 0, 3, 7, 10, 14 days.

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2.9. Statistical analyses

Data were statistically handled by the one-way analysis of variance (ANOVA for Excel, vers. 2.2). All experiments were carried out in triplicate, and the results were expressed as Mean ± SD. Duncan’s

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multiple-range test was applied for the calculation of the significant differences among the values of

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characteristic parameters at probability level P<0.05.

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3. Results and discussion

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3.1. Antioxidant properties of active packaging materials

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Antioxidant properties of materials, especially radical scavenging activities, are very important due to the deleterious role of free radicals in foods and biological systems (Sanhez-Moreno, 2002). Free

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radical scavenging is one of the known mechanisms by which antioxidants inhibit lipid oxidation

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(Hatano, Edamatsu, Mori, Fujita, Yasuhara, Yoshida, T., et al., 1989). Therefore, DPPH methods have been widely used to test the ability of compounds to act as free radical scavengers and thus to evaluate the antioxidant activity in vitro. Generally, when using those methods the tested substances are in direct

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contact with DPPH solution (Alam, Bristi, & Rafiquzzaman, 2013). However, most food products such as

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meat, fish or bakery products are packaged without direct contact with upper packaging material (sealed closure film) in plastic trays. For this reason, the development and assessment of the films or their coatings acting through the vapor phase are of high importance. In this study, acrylic component/hydrophobically modified starch (AC/S) or cellulose acetate (CA) coatings containing from 0.11 ± 0.02 to 0.77 ± 0.10 g/m2 of CL and from 0.14 ± 0.01 to 0.82 ± 0.13 g/m2 of EU were prepared on corona treated OPP. Afterwards their applications as antioxidant packaging materials were investigated. The antioxidant activity results, obtained by using VP-DPPH method, are presented in Figs. 1 and 2. As depicted in Figs. 1a and 1b the antioxidant activity of AC/S/CL and

ACCEPTED MANUSCRIPT AC/S/EU coatings increased gradually (P<0.05) during the incubation time. When of CL in AC/S coating was used 21 % of DPPH inhibition was achieved after 1h and the antioxidant activity increased gradually up to 41 % after 5 hours (Fig. 1a). Increase in the concentration of CL to 0.23 ± 0.02 g/m2 resulted in a slight increase in the inhibition of DPPH (25%) after one hour. However, final antioxidant activity reached about 62 % after 5h. The highest activities were achieved using 0.50 ± 0.05 g/m2 amount of CL.

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Inhibition of DPPH of 43 % was achieved after 1h which increased to 65 % after 5 h. Use of EU resulted in higher antioxidant activities compared to those of CL coating with similar concentrations (Fig. 1b).

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Inhibition of DPPH of 65 – 84% was achieved after 5 h.

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The cellulose acetate coatings containing CL or EU showed high antioxidant activities (Fig. 2). DPPH inhibition increased during the incubation time and reached values of 87 % to 92 % after 3 h with

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the CL concentration of 0.18 ± 0.03 to 0.77 ± 0.10 g/m2, respectively (Fig. 2a). On the other hand, EU

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concentration from 0.19 ± 0.05 to 0.82 ± 0.13 g/m2 resulted in similar antioxidant activities reaching an inhibition of DPPH after 3h ranging from 90 to 94 %, respectively (Fig. 2b).

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Comparing DPPH radical-scavenging activity of AC/S and CA coatings when similar

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concentrations of CL or EU were used CA coatings showed higher antioxidant activities. This is probably due to the faster release of active compounds from CA than AC/S. After 3 h incubation, CA and AC/S coatings containing 0.19 ± 0.05 g/m2 and 0.14 ± 0.01 g/m2 of EU showed an inhibition of DPPH of 90 %

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and 45 %, respectively. Moreover, the final antioxidant activities of CA/CL and CA/EU coatings were

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higher than those of AC/S/CL or AC/S/EU. It can emphasised that developed novel VP-DPPH method was successfully applied to characterise the antioxidant activities of both types of coatings and can be regarded as beneficial and rapid technique for antioxidant activity screening of packaging materials when testing them though the vapour phase. Further studies are still needed to optimise VP-DPPH method for MAP conditions in relation to the real packaging applications.

3.2. Eugenol release into headspace

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In order to understand the release rates of active substance from different coatings, active coatings were stored and the release of EU into the headspace of sealed containers was measured using GC-MS (see Fig. 3). As depicted in Fig. 3a, EU was released very rapidly within 1 day and the saturation of headspace was reached already after 3 days in all samples. CA/CL and CA/EU coatings containing

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highest amount of EU in solid phase (Fig. 3b) had also highest saturation concentration of active substance in the headspace.

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The partition coefficient (K) was used to compare the ratio of EU amount in the vapor phase and

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solid phase. Average K value for AC/S/CL and AC/S/EU coatings was found to be 9.9 ± 0.1×10 -3 and 10.9 ± 0.7×10-3, respectively. Whereas, K value of CA/CL and CA/EU coatings was 3.5 ± 0.1×10 -3 and

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4.2 ± 0.2×10-3, respectively. High values of K are related to rapid release of volatile compounds into the

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surrounding aerial medium. Conversely, low values of K are linked to prolongation of release. However, it wouldn’t be correct to directly compare K values of AC/S/CL, AC/S/EU, CA/CL and CA/EU due to

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different amount of EU in solid phase. Prolonged release of active volatiles from container system is

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expected more attractive for food and nonfood products during their shelf life period. CA/EU samples are characterized by low K and high concentration of saturation and therefore may be linked to rapid and prolonged effects by this type of coating.

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3.3. Antioxidant food packaging for fresh beef

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After showing the antioxidant activity of the coated OPP films and characterizing the release of EU from AC/S/CL, AC/S/EU, CA/CL and CA/EU coatings, EU was chosen as the most effective substance for the antioxidant food packaging applications. Therefore, AC/S/EU(0.32 ± 0.03 g/m2 (EU1) and 6.40 ± 0.14 g/m2 (EU2)) and CA/EU(0.65 ± 0.08 g/m2) coatings were used for packaging of fresh beef steaks. OPP films without active coating were used as control. The colour changes and lipid oxidation (TBARS method) of beef were monitored during the storage period of 14 days.

3.3.1. Fresh beef colour analysis

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Changes in colour of fresh beef samples stored in active or control packaging during the period of 14 days are presented in Table 3. Table 3. Effects of active packaging on colour changes of fresh beef packed under modified atmosphere during the storage at 2 ± 1 ºC a 3 41.24 ± 2.82aAB 40.64 ± 1.68aAB L* 41.27 ± 1.50aAB 41.52 ± 1.74aB 30.59 ± 1.68dB 32.17 ± 1.55cD a* 29.20 ± 1.86cA 31.25 ± 1.41bBCD Control 14.87 ± 0.58ab 18.36 ± 1.35dBC AC/S/EU1 14.87 ± 0.58a 18.60 ± 1.07cC b* a AC/S/EU2 14.87 ± 0.58 17.63 ± 1.42dA a CA/EU 14.87 ± 0.58 18.35 ± 0.97cBC a Control 22.80 ± 0.75 30.96 ± AC/S/EU1 22.80 ± 0.75a 1.32bBCD hab AC/S/EU2 22.80 ± 0.75a 30.02 ± 0.86eA a CA/EU 22.80 ± 0.75 31.10 ± 1.10bD 30.42 ± 0.58bAB e Control 38.36 ± 0.62 35.69 ± 1.99dB d AC/S/EU1 38.36 ± 0.62 37.17 ± 1.80cD d C* AC/S/EU2 38.36 ± 0.62 34.12 ± 2.25cA d CA/EU 38.36 ± 0.62 36.25 ± 1.68cBCD a Content of EU in the coatings: EU1 - 0.32 ± 0.03 g/m2; EU2

7 42.52 ± 2.20bB 40.87 ± 1.93aA 41.08 ± 2.00aA 41.28 ± 1.20aA 25.15 ± 1.06cA 29.99 ± 1.30bC 26.06 ± 1.11aB 25.94 ± 1.02aB

10 43.81 ± 1.40bcdAB 43.39 ± 2.34cAB 43.83 ± 2.28cB 43.27 ± 1.91cAB 23.28 ± 1.92bA 26.80 ± 1.29aBC 27.40 ± 1.72bC 26.51 ± 1.95aBC

14 44.33 ± 3.38dB 44.06 ± 2.12cAB 44.06 ± 1.88cAB 43.86 ± 1.91cAB 18.44 ± 3.36aA 26.53 ± 1.08aBC 26.10 ± 1.51aBC 26.03 ± 1.07aC

15.59 ± 1.32cA 16.89 ± 0.92bB 15.75 ± 0.92bA 15.28 ± 0.80aA 31.76 ± 1.79bD 29.38 ± 1.02dA 31.15 ± 1.37bBCD 30.49 ± 0.63bB

15.51 ± 2.06bcA 17.00 ± 0.86bBC 17.11 ± 1.48dC 16.59 ± 1.40bBC 33.60 ± 2.74cB 32.38 ± 0.56cA 31.96 ± 1.62dA 32.04 ± 1.82dA

14.21 ± 1.19aA 16.60 ± 0.92bC 16.26 ± 0.94bBC 16.38 ± 1.02bBC 38.11 ± 4.96dB 32.03 ± 1.29cA 31.93 ± 1.03dA 31.70 ± 1.19dA

29.60 ± 1.42cA 34.43 ± 1.47bC 30.46 ± 1.24aB 30.10 ± 1.25aAB

28.01 ± 2.41bA 31.74 ± 1.52aBC 32.31 ± 2.08bC 31.29 ± 2.20bBC

23.36 ± 2.99aA 31.30 ± 1.23aC 30.75 ± 1.69aBC 30.55 ± 1.32abBC

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Control AC/S/EU1 AC/S/EU2 CA/EU Control AC/S/EU1 AC/S/EU2 CA/EU

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Days of storage 0 41.08 ± 0.91a 41.08 ± 0.91a 41.08 ± 0.91a 41.08 ± 0.91a 35.36 ± 0.54e 35.36 ± 0.54d 35.36 ± 0.54d 35.36 ± 0.54c

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Packaging

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Colour parameter

- 6.40 ± 0.14 g/m2; EU - 0.65 ± 0.08 g/m2.

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: the different

lowercase letters within the same rows for each sample show that the results are significantly different (P<0.05; Duncan test); The different uppercase letters within the same column for each sample show that the results are significantly different

(P<0.05; Duncan test).

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A-D:

L*, a* and b* values of beef were 41.08 ± 0.91, 35.36 ± 0.54 and 14.87 ± 0.58 at the start of the

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experiment, respectively. Significant changes did not occur in L* (lightness) values of different samples within the storage time that were around 44 at the day 14th. Meanwhile, the values of a* (redness) decreased for all samples, whereas the control beef sample showed a steady decrease (P<0.05), and reached a* value of 18.44 ± 3.36 at day 14th which indicated loss of red colour (see Fig. 4). Meanwhile, beef samples kept in active packaging containing eugenol showed red colour stability until the end of storage and a* values were around 26. This is agreement with the results reported by Camo et al. (2011) and Nerin et al. (2006) who studied beef meat in antioxidant packaging containing oregano and rosemary extract, respectively. In addition, no significant changes in a* values were found independently of the

ACCEPTED MANUSCRIPT active packaging system used (see Table 3). Meanwhile, marginal protective effect against meat discoloration in relation to the extract amount on the packaging was observed in other study (Camo et al., 2011). As could be seen, b* (yellowness) values of beef samples slightly changed at the end of storage period. The final values of b* were around 14 and 16.5 when storing beef samples in control and active packaging, respectively.

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Colour characteristics of the samples may also be evaluated from the values of the hue angle (hab) and chroma (C*) calculated from a* and b* values. Hue values show the colour changes from red to

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purple. Meanwhile, the chroma value is used to indicate the vividness of colour. By using both parameters

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colour differences can be calculated and compared objectively (Tapp, Yancey, & Apple, 2011). As could be seen, the calculated values of hab and C* were 22.80 ± 0.75 and 38.36 ± 0.62 at the day 0, respectively.

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After 14 days storage period the final values of hab were around 31-32 and 38.11 ± 4.96 when storing beef

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samples in active and control packaging, respectively. Larger hue angles indicated a red colour decrease. In addition, vividness of red colour after 14 days was higher (P<0.05) for beef samples kept in active

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packaging (around 30-31) then in control packaging (23.36 ± 2.99). The visual changes of fresh beef

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steaks at the end of storage period are shown in Fig. 4. As can be seen from the pictures, beef samples stored in active packaging (Fig. 4 b, c and d) secured bright red colour. Although MAP packaging conditions have been developed to maintain color stability of red meat,

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high-oxygen environment also induces lipid and protein oxidation (Bao, Puolanne, & Ertbjerg, 2016).

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Changes in meat color are due to oxidation of red oxymyoglobin to brownish metmyoglobin. Lipid oxidation is a major contributor to metmyoglobin formation. These both processes often appear to be linked and the oxidation of one of these leads to the formation of chemical species that can exacerbate oxidation of the other and lead to fresh meat discoloration (Faustman et al., 2010). The coincidence of meat discoloration and lipid oxidation has been confirmed in beef meat, lamb, and fish (Insausti, Beriain, Lizaso, Carr, & Purroy, 2008; Schafer, Liu, Faustman, & Yin, 1995; Luciano, Monahan, Vasta, Pennisi, Bella, & Priolo, 2009; Maqsood, & Benjakul, 2011). The incorporation of antioxidants into packaging protects from lipid oxidation and may stabilize oxymyoglobin from oxidation (O’Grady, Monahan, &

ACCEPTED MANUSCRIPT Brunton, 2001). Consequently, the release of the antioxidant eugenol molecules from the coatings and scavenging of free radicals in the food or package headspace may be suggested as hypothesis for a possible mechanism of action when preserving red colour of MAP packed fresh beef steaks in our study.

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3.3.2. Lipid oxidation assay

Fats, oils, and other lipids react with oxygen to form peroxides, which then further decompose to

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give aldehydes, including malonaldehyde (MDA) (Hammond, & White, 2011). The 2-thiobarbituric acid

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reactive substances (TBARS) assay was used for detecting the content of malonaldehyde in fresh beef steaks. TBARS values were expressed as milligrams of malondialdehyde per kilogram of beef (mg

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MDA/kg). The effects of different types of packaging systems on the lipid oxidation stability of beef steaks during the storage period of 14 days under modified atmosphere conditions at 2 ± 1 °C are

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presented in Fig. 5.

The initial TBARS value of fresh beef steaks was found to be 0.32 ± 0.03 mg MDA/kg. During

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storage TBARS value of beef kept in control packaging increased significantly (P<0.05) and after 7 days was above 1.5 mg of MDA/kg. It has been reported that value of 1.5 is closely related to perceptible and unacceptable off-odour of meat (Martínez, Djenane, Cilla, Beltrán, & Roncalés, 2006). TBARS value in

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control packaging reached 3.33 ± 0.22 mg of MDA/kg after 14 days. Whereas meat kept in antioxidant

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packaging the TBARS value increased to 0.852 ± 0.152 – 0.966 ± 0.256 mg of MDA/kg within the first 3 days of storage and no significant increase in TBARS value was observed afterwards. In addition, no significant differences in final TBARS values were determined for the samples when concentration of eugenol on packaging film was varied from 0.32 ± 0.03 to 6.40 ± 0.14 g/m2. The inhibition of lipid oxidation in fresh meat kept in active packaging containing oregano or rosemary extracts have been reported in other studies (Camo et al., 2008; Camo et al., 2011; Nerin et al., 2006). Essential oils as volatile antioxidants are thought to work through inhibition of gas-phase oxidation reactions with headspace free radicals and subsequent autooxidation in the food matrix with indirect migration (Camo et al., 2008; Camo et al., 2011). Therefore, it might be suggested that release of

ACCEPTED MANUSCRIPT eugenol from packaging film with active coating to the package headspace and scavenging of vaporphase free radicals in the package and in the food is responsible for the inhibition of lipid oxidation in fresh beef samples in our study. The studies of Pezo, Salafranca, and Nerín (2008) demonstrated that active films reacted with headspace free radicals and reinforce the latter hypothesis.

this

work

we

have

demonstrated

that

cellulose

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In

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4. Conclusions

acetate

(CA)

or

acrylic

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component/hydrophobically modified starch (AC/S) can be successfully used to incorporate natural antioxidants such as clove essential oil and eugenol. The resulting films showed rapid release of active

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substances into the headspace. This resulted in high antioxidant activities which could be quantified with

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new VP-DPPH method we have developed in this work. Use of eugenol containing CA and AC/S films for packaging of beef reduced the lipid oxidation in beef as well as protected its color. The display life of

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beef is of significant importance in the retail marketplace. High oxygen (80% O2/20% CO2) case-ready

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packs of fresh beef have demonstrated display life under normal commercial conditions of 2-4 days only (Belcher, 2006). Consequently, results of this work allow to envisaging antioxidant packaging with eugenol as a promising technology for increasing the display life of beef or other meats. However,

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additional studies such as microbiological and sensory analysis including acceptability of eugenol odour

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when used as antioxidant packaging for meat application should be carried out to fully evaluate industrial feasibility of proposed technologies. Acknowledgements

The financial support of the Rector’s Conference of Swiss the Universities in the form of SciexNMSch Fellowship to V. Navikaite-Snipaitiene is highly acknowledged. The authors are grateful to the Research Council of Lithuania for the financial support of the project MIP-055/2015.

ACCEPTED MANUSCRIPT References Abdollahi, M., Rezaei M., & Farzi, G. (2012). Improvement of active chitosan film properties with rosemary essential oil for food packaging. International Journal of Food Science & Technology, 47, 847853. Alam, M. N., Bristi, N. J., & Rafiquzzaman, M. (2013). Review on in vivo and in vitro methods

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Faustman, C., Sun, Q., Mancini, R., & Suman, S. P. (2010). Myoglobin and lipid oxidation interactions: Mechanistic bases and control. Meat Science, 86, 86-94. Fernandes, R. P. P., Trindade, M. A., Tonin, F. G., Lima, C. G., Pugine, S. M. P., Munekata, P. E. S., et al. (2016). Evaluation of antioxidant capacity of 13 plant extracts by three different methods: cluster analyses applied for selection of the natural extracts with higher antioxidant capacity to replace synthetic antioxidant in lamb burgers. Journal of Food Science and Technology, 53, 451-460. Hammond, E. G., & White, P. J. (2011). A Brief History of Lipid Oxidation. Journal of the American Oil Chemists' Society, 88, 891-897.

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ACCEPTED MANUSCRIPT Nerin, C., Tovar, L., Djenane, D., Camo, J., Salafranca, J., Beltran, J. A., et al. (2006). Stabilization of beef meat by new active packaging containing natural antioxidants. Journal of Agricultural and Food Chemistry, 54, 7840-7846. O’Grady, M. N., Monahan, F. J., & Brunton, N. P. (2001). Oxymyoglobin oxidation and lipid oxidation in bovine muscle. Mechanistic studies. Journal of Food Science, 66, 386-392.

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fluorescence detection. Journal of Chromatography A, 1178, 126-133.

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Foods and Biological Systems. Food Science and Technology International, 8(3), 121-137. Schafer, D. M., Liu, Q. P., Faustman, C., & Yin, M. C. (1995). Supranutritional administration of vitamins E and C improves oxidative stability of beef. The Journal of Nutrition, 125, 1792S-1798S.

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Meat Science, 89, 1-5.

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ACCEPTED MANUSCRIPT Woranuch, S., & Yoksan, R. (2013). Eugenol-loaded chitosan nanoparticles: II. Application in bio-based

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plastics for active packaging. Carbohydrate Polymers, 96, 586-592.

ACCEPTED MANUSCRIPT Fig. 1. Antioxidant properties of AC/S/CL (a) and AC/S/EU (b) coatings through the vapour phase by VP-DPPH method using different concentrations (g/m2) of CL or EU (indicated in Figures);

a-e:

the different letters for the same sample within the

incubation time show that the results are significantly different (P<0.05; Duncan test). Fig. 2. Antioxidant properties of CA/CL (a) and CA/EU (b) coatings trough the vapour phase by VP-DPPH method using different concentrations (g/m2) of CL or EU (indicated in Figures);

a-d:

the different letters for the same sample within the

incubation time show that the results are significantly different (P<0.05; Duncan test).

a-d:

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Fig. 3. Amount of eugenol (nmol) in headspace (a) and solid phase (b) of different types coatings during the storage time; the different letters for the same sample within the storage time show that the results are significantly different (P<0.05;

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Duncan test).

Fig. 4. Images for fresh beef stored in control (a) and AC/S/EU1 (0.32 ± 0.03 g/m2) (b) AC/S/EU2 (6.40 ± 0.14 g/m2) (c),

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CA/EU (0.65 ± 0.08 g/m2) (d) packaging under modified atmosphere for 14 days at 2 ± 1 ºC. Fig. 5. TBARS values of fresh beef steaks kept in different packaging under modified atmosphere during the storage time at 2

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± 1 ºC. The lipid oxidation was measured at 0, 3, 7, 10, 14 days.

ACCEPTED MANUSCRIPT Highlights Coatings containing clove essential oil and eugenol on polypropylene film were designed.

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The antioxidant properties of coatings were investigated through the vapour phase.

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Eugenol inhibited lipid oxidation in fresh beef and increased its display life.

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Figure 1

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Figure 5