Production , antioxidant characterization and application of active starch-based films containing essential oils for beef packaging

The objective of this work was to study the antioxidant activity of biodegradable and active packaging containing essential oils. Seven types of active packaging were produced. In beef, lipid oxidation was measured using the thiobarbituric acid reactive substance test (TBARS); a microbiological analysis in beef was performed for Salmonella spp., coagulate positive Staphylococcus spp., Clostridium sulphite reducer and E. coli; the measurements were carried out on days 0, 3, 6, 9, 12 and 15 in triplicate. All packaging had exhibited antioxidant capacities and acted as an antioxidant in meat. The packages containing clove exhibited the highest total phenolic and antioxidant capacity (P < 0.05), and when used in beef were most effective against lipid oxidation. The use of oregano, clove and rosemary incorporated in active biodegradable starch based reduced microbial development and acted effectively against rancidity by maintaining the quality of meat stored under refrigerated conditions for 15 days.


Introduction
Microbial growth and lipid oxidation are the main causes of deterioration in meat quality (Radha et al., 2014). In order to minimise this effect, the use of oils and extracts from plants has been studied in animal diets Rivaroli et al., 2016) or for direct application in meat and meat products, as these treatments can generate changes, mainly in the odor and flavor sensory characteristics Vital et al., 2016). In this context, new alternatives such as the application of antimicrobial and antioxidant products in packaging have been tested (Appendini & Hotchkiss, 2002;Bolumar et al., 2011;Conde et al., 2011). Active and intelligent packaging introduces new perspectives for food packaging, such as the concept of intentional substance migration; for instance, the migration of preservatives and antioxidants from the package into the food (Gómez-Estaca et al., 2014;Realini & Marcos, 2014).
Allied to this, there is the growing demand by consumers for natural antioxidant and antimicrobial products due the potential toxicological effects of synthetic antioxidants. Essential oils (EOs) are rich in phenolic compounds. Recent studies have revealed that oregano, clove, rosemary, and other essential oils are active against many microorganisms and delay lipid oxidative degradation (Anthony et al., 2012;Burt, 2004;Javier Camo et al., 2011;Kempinski et al., 2017;Shah et al., 2014). Many essential oils exhibit antioxidant and antimicrobial activities due to their high content of phenolic compounds. Anthony et al. (2012) ranked 423 essential oils; the most effective antioxidants were present in eight of the tested botanical families, including oregano (thymol and carvacrol), clove (eugenol) and rosemary (α-pinene). These compounds act to delay the onset or to slow down the rate of oxidation, and are linked to damage to the membrane of bacterial cells, resulting in an increase in disintegration and permeability (Appendini & Hotchkiss, 2002;Burt, 2004).
Thus, the objective of this work was to study the antioxidant activity in biodegradable and active packaging containing essential oils, and the application of packaging in beef, stored under refrigerated conditions for 15 days.
Longissimus lumborum (LL) (from the 10 th to 12 th ribs) was obtained from eight young bulls ( 1 /2 Simmental vs. 1 /2 Nellore) finished in a feedlot for 168 days and fed with a high concentrate diet according to Eiras et al. (2016). Animal slaughter was performed in a slaughterhouse near of experimental farm. Animals had an average weight of 480 kg, carcass dressing of 54% and pH of 5.74, measured 24 hours after slaughter Eiras et al. (2016).

Film production and experimental design
The extrusion process was applied to develop the packaging material and was divided in two stages: the extrusion process and reprocessing the pellets were extruded again for film formation. The films were produced at the Laboratory of Food Science and Technology Department at the State University of Londrina. Starch (Indemil, Brazil) and glycerol (Synth P.A, Brazil) were used to obtain a thermoplastic starch (TPS); added poly (butylene adipate-co-terephthalate) (PBAT; BASF, Germany) by Ecoflex ® S BX 7025. Oregano, clove and rosemary oils were homogenised during the first stage. Were films extruded directly from the mixture of TPS (13% glycerol and 45% starch), Ecoflex (40%) and 2% essential oils, these mixtures were then extruded in order to obtain pellets by using twin screw extruder (model D-20 series 9002.001, BGM, São Paulo, Brazil). In the second stage, the extruder was fed manually with the pellets (blends) and the film formed through the blowing technique (film balloons) by injecting compressed air inside and outside the balloon, both polyurethane rollers speed and coil were held constant as described by Cestari et al. (2015).
Seven different types of packaging were prepared according to the experimental design mix by partially replacing starch by oregano, clove, rosemary oils and their mixtures (Table 1). After obtaining the films, they were manually cut and heat-sealing machine to make pouches (15 cm x 20 cm). The Longissimus lumborum samples was cut into six steaks at a thickness of 3.5 cm (300 g each) with a knife and placed in active packaging randomly, sealed and stored chilled at 4° C.

Determination of thickness films
Film thickness was determined using a manual micrometer (Mitutoyo, Japan). The final thickness was determined by the arithmetic mean of 10 measurements random conditioned for 48 h at 64% RH.

Water vapor permeability (WVP)
To determine the WVP of the films, the gravimetric method adapted from the standard E 96-95 of ASTM was used. The gradient relative humidity was used 2% -53%. The vapor permeation ratio (WVPR) was obtained with Equation 1.
Where m/t is the angular coefficient of the mass gain line (g) versus time (day), and A (m 2 ) is the sample permeation area. The WVP (g Pa day −1 m −1 ) was calculated (Equation 2).
Where st is the mean sample thickness (m), sp is the water vapor saturation pressure at the assay temperature (Pa), RH1 is the relative humidity of the desiccator and RH2 is the relative humidity in the interior of the permeation cell. The tests were conducted in duplicate.

Active packaging extract preparation
The packaging was cut manually with scissors in small pieces, then immersed in solution 50% ethanol/H2O (v/v) in a proportion of 1:10 (w/v) under magnetic stirring for 1 h in the dark room according to (Michiels et al., 2012), with modifications.
After filtration, the ethanolic extracts were used for antioxidant analysis.

Total soluble phenolic compound content
The content of soluble phenols of samples were determined in triplicate according to the Folin-Ciocalteu method (Singleton & Rossi, 1965), with modifications described by (Wu et al., 2005). gram of sample (µmol GA g -1 ).

Oxygen radical absorbance capacity (ORAC)
The ORAC methodology was performed as described by (Zulueta et al., 2009)  The wavelengths of excitation and emission were 485 and 515 nm, respectively.
The final ORAC values were calculated using linear regression: y = ax + b between the Trolox concentration (μmol L −1 ) and the net area under the fluorescein decay curve according to (Ou et al., 2001). The results are expressed as µmol Trolox equivalents (TE) g -1 of essential oil (µmol TE g -1 ).

Microbiological analysis
On days 0, 3, 6, 9, 12 and 15, triplicate packages from each treatment were aseptically opened and crushed; a 25 g portion was homogenised with 225 mL of peptone water (Merck) for 1 min to carry out the initial dilution (10 -1 ), then proper serial dilutions (until 10 -5 ) were performed. For the enumeration of Clostridium sulphite reducers at 46º C, at the time of use 1 mL of sterile 4% D-cycloserine was added to 100 mL of tryptose-sulphite-cycloserine agar (TSC, Merck). Aliquots of 1 mL were placed in sterile plates and 10 mL of TSC agar was added. After the agar solidified on a flat surface, a second layer of the same medium was added. The plates were incubated inverted in anaerobic jars (BBL GasPak) at 46º C for 48 h. All microbial counts were converted to logarithms of colony-forming units per gram (log CFU/g) (FDA, 2015).

Statistical analysis
Data were assessed through variance analysis (ANOVA). Least squares differences were used to compare the mean values for treatments and Tukey's HSD test was used to identify significant differences between treatments. Regression analysis was performed for the treatment and storage time interaction, both with a significance level of 5%. SAS 9.1 was used to perform the analysis (SAS, 2004). Table 2 show the results of tthickness and water vapor permeability (WVP). Regardless of the type of oil and its amount, the addition of essential oils did not affect the thickness and permeability to water vapor (P > 0.05). The presence of oil, due to its hydrophobic nature, may decrease the permeability to water vapor of biodegradable films, but in large quantity the presence of oil can cause discontinuity in the polymer chain, consequently increasing the permeability to water vapor (Shahbazi, 2017).

Total phenolic and antioxidant activity of the packaging
In all seven packaging the composition of total phenolic compounds were detected and expressed as content of soluble phenols (Table 3). The packaging containing clove oil (CL2) had the highest amount (P < 0.05) of total phenolic compounds, followed by the packaging containing ORCL1, CLRO1 and ORCLRO1/3. The packaging containing rosemary oil (RO2) had the lowest means, i.e., the least amount of phenolic compounds (P < 0.05). The packaging antioxidant capacities (Table 3) were evaluated by DPPH and ORAC. The results show that packaging containing CL2, ORCL1 and CLRO1 had the highest (P < 0.05) antioxidant activity (μmol TE g -1 ) by both methodologies, while lower activity was determined for OR2 (4.06 μmol TE g -1 ) by DPPH and RO2 (76.41 μmol TE g -1 ) by ORAC.
There was a positive correlation between antioxidant activity and total phenolic content, as samples with high antioxidant activity also had a high phenolic content. Comparing the essential oils used and the interaction between them, we observed the best results for clove (content of soluble phenols and antioxidant capacity). This can be explained by previous publications showing that clove has a high antioxidant activity (J Camo et al., 2008;Wojdyło et al., 2007).
Due to the variable compositions of EOs, some authors have postulated a potential synergistic effect between the components (Di Pasqua et al., 2005;Hernández-Ochoa et al., 2014). This effect may be due sequential inhibition of a common biochemical pathway, inhibition of protective enzymes, combinations of cell wall active agents, and the use of cell wall active agents to enhance uptake, as described by (Macwan et al., 2016). In this work, a synergistic effect between different oils was observed. The same packaging containing OR2, CL2 and RO2 were composed of 2% isolated oils, while the treatments ORCL1, ORRO1, CLRO1 and ORCLRO1/3 contained a mixture of EOs. The mixtures, especially in the packaging containing ORCL1 (1% oregano and 1% clove oil) and CLRO1 (1% clove plus 1% rosemary oil) resulted in lower values of mg MDA kg -1 ; for both ORCL1 and CLRO1, the results are lower than 0.056 after 15 days of storage. This result is in agreement with the results obtained for the total phenolic and antioxidant activity of active packaging biodegradable in this study. It was noted that the films that presented high total phenolic and antioxidant activity inhibited lipid oxidation when applied to beef.
Others authors have succeeded in extending the shelf life of meat and meat products using active packaging with natural antioxidants (Barbosa- Pereira et al., 2014;J Camo et al., 2008;Contini et al., 2011). (Park et al., 2012) Park et al. (2012 developed vacuum active packaging containing antioxidant agents (thymol, carvacrol and eugenol) and applied it to beef patties, stored under refrigeration (4° C). The packaging effectively inhibited lipid oxidation and positively affected colour stability.

Microbiological analysis
Salmonella spp. was absent in 25 g of all samples on days 0, 3 and 15. Coagulate positive Staphylococcus spp. was negative, Clostridium sulphite reducers were less than 10 CFU/g and Escherichia coli were not detected on all evaluation days.
Total coliforms were found at <1 log CFU/g on days 0 and 3 and <0.5 log CFU/g on days 6, 9 12 and 15 for all treatments, with a reduction of more than 50% after 6 days.
This can be explained because EOs containing phenolic compounds that act mainly on the cell cytoplasmic membrane, i.e. compounds as carvacrol, thymol, p-cymene and ɤ-terpinene, are effective against Gram-positive and Gram-negative bacteria. This is, in part, due to the hydrophobicity of these EOs, making the structures of the cell membrane more permeable (Burt, 2004;Burt et al., 2005). Additionally, Burt et al. (2005) revealed that oregano and thyme EOs are active against E. coli. There is some evidence from studies with EOs in concentrations from 500 mg/L and 750 mg/L to be effective in meat against Escherichia coli, Salmonella, Clostridium perfringens and Staphylococcus aureus (Hernández-Ochoa et al., 2014).
In other studies, oregano and potassium sorbate in an active film reduced the total viable counts and total coliforms and delayed E. coli growth on low sodium restructured chicken steaks during 150 days of freezing storage (Cestari et al., 2015;Emiroğlu et al., 2010) incorporated (0, 1, 2, 3, 4 and 5%) of oregano (OR) and thyme (TH) EOs in active packaging and evaluated their antimicrobial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Lactobacillus plantarum during 12 days of refrigerated storage in ground beef and found that more than 3% of oregano and thyme EOs was effective in inhibiting these microorganisms.

Conclusion
All active packaging prolonged shelf life and sustained the oxidative and microbiological quality, and thus have possible uses as meat preservatives. The most effective packaging included clove oil (CL2, ORCL1 and CLRO1). These treatments demonstrated the highest total phenolic content and provided the greatest antioxidant activity compared to oregano and rosemary oils, and consequently was the most effective in retarding lipid oxidation in beef compared to other treatments.