Shelf-life extension of meat products by cellulose acetate antimicrobial film incorporated with oregano’s essential oil

This study aimed to apply cellulose acetate (CA) films incorporated with oregano essential oil (OEO) to inhibit bacteria growth associated with spoilage of meat products (Weissella viridescens (microaerophilic) and Pseudomonas fluorescens (aerobic)) and evaluate its effect on the shelf life of vacuum-packed sliced ham (VPSH). CA films were produced using acetone solvent, adding 25, 35, 50, or 75 mg of OEO per film. Antimicrobial activity and mechanical properties of films were determined. CA films in Petri dishes showed a better antimicrobial effect against W. viridescens than P. fluorescens. As VPSH, presents a microaerophilic environment, product shelf life was determined fitting Baranyi and Roberts’ model to W. viridescens’ growth experimental data, at 8 °C. OEO did not modify films’ mechanical properties. Application of the CA film with 75 mg of OEO decreased value of W. viridescens, increased its value, resulting in a ham` shelf-life increased by eight days, demonstrating excellent application potential.


Introduction
The control of microbial growth in meat products is crucial to extend products´ shelf life and prevent foodborne diseases Jacob, Mathiasen, & Powell, 2010). Lactic acid bacteria (LAB) is one of the main spoilage microorganisms present in meat products stored under vacuum or modified atmosphere. Weissella viridescens is a Grampositive LAB that can grow in microaerophilic conditions, been strongly stimulated by temperature fluctuation throughout the cold chain . Pseudomonas spp. is Gram-negative, aerophilic, and psychrotrophic bacteria that can grow under refrigeration conditions. These characteristics make them the main spoilage microorganisms in meat products, especially at low temperatures, under aerobic conditions (Pseudomonas spp.) and vacuum packaging (W. viridescens) (Oh et al., 2014;Sousa et al., 2012).
The antimicrobial packaging is an alternative to delay the growth or inactivate bacterial cells, increasing food safety, and shelf life in a broad range of applications (Jafarzadeh et al., 2020;Kapetanakou & Skandamis, 2016). Active films containing antibacterial essential oils interact with the product surface by direct contact or gradual release of the active molecules previously added to the film (Espitia et al., 2013;Woranuchet al., 2015).
Several essential oils, and some isolated chemical molecules naturally present in them, had their antimicrobial activity tested against a wide range of foodborne microorganisms (Boskovic et al., 2020;Boskovic et al., 2015;Busatta et al., 2007).
Essential oils from oregano, thyme, lemon, and lavender are very active against Gram-positive and Gram-negative foodborne bacteria (Correa et al., 2017). Its antimicrobial and antioxidant activity widely recognizes the oregano essential oil (OEO), attributed to phenolic compounds, such as thymol, carvacrol, and eugenol (Burt, 2004;Jafarzadeh et al., 2020). The antimicrobial activity of films added with OEO has been demonstrated in previous studies (Caetano et al., 2017;Munhuweyi et al., 2018), including extending the shelf life of fish fillets using OEO/polybutylene adipate-co-terephthalate films (Cardoso et al., 2017).
One of the biggest challenges in active packaging is to reach a suitable material that meets sustainable and affordable characteristics. Cellulose acetate (CA) is a useful eco-friendly polymer once it is biodegradable, amorphous, non-toxic, and odorless (Rudaz & Budtova, 2013). Films based on CA have shown applications in food packaging, including active films (Pola et al., 2016;Rodríguez et al., 2014).
The use of mathematical models describing microbial growth has become an essential tool in predicting food shelf life, risk assessments, and quality (Koutsoumanis et al., 1999;Mataragas et al., 2006). Mathematical modeling has been widely applied to describe bacteria growth associated with the spoilage of meat products Silva et al., 2017).
Previous studies report the use of mathematical modeling to assess the shelf life of vacuum-packed ham, considering the growth of LAB (Menezes et al., 2018;Slongo et al., 2009).
Thus, the aims of this study were: i) to apply CA films incorporated with OEO to inhibit the growth of the bacteria W.viridescens (microaerophilic) and Pseudomonas fluorescens (aerobic) in a culture medium, ii) to characterize the mechanical properties of the resulting films, iii) to model the effect of the active films on the increase of the shelf life of vacuum-packed cooked sliced ham under isothermal conditions (8°C), based on the growth of the W. viridescens.

Production of CA antimicrobial film incorporated with OEO
Films were produced by the casting method, which consists of pouring and drying a colloidal film-forming solution (FS) on a flat surface (e.g., Petri plate). For the preparation of the FS, CA was solubilized in acetone. Our research group used the acetone solution as solvent-based in previous studies. The films from the acetone solution were less dense, mechanically rigid, and more hydrophobic than the other tested solvent. Besides, it evaporates more quickly, resulting in less time to obtain the films.
The FS was prepared with a CA concentration of 5 g per 100 mL of solvent. The CA was added to the solvent and kept under mechanical stirring at room temperature until the polymer was completely solubilized. Then, 25, 35, 50, or 75 mg of OEO were added to the FS (10 mL per Petri dish) and kept under stirring for 10 min. The OEO quantity was chosen based on previous studies with OEO carried out in our group.
A predetermined volume of 10 mL of the FS was added in the glass Petri dishes (9 cm in diameter) capped, forming a film with an area of approximately 63,6 cm 2 . The films were dried in a glass chamber (21 °C and relative humidity around 65 -70%) containing blue silica with a lower opening to avoid the solvent saturation internally for 48 h. The films were named F25, F35, F50, and F75, to represent the quantities of OEO per film (25, 35, 50, and 75 mg of OEO).

Bacterial strain and inoculum preparation
Freeze-dried pure cultures of W. viridescens (CCT 5843 ATCC 12706, Lot 22.07) and P. fluoresces (CCT 7393 ATCC 13525, Lot 25.06) were used. W. viridescens inoculum was prepared in Man, Rogosa, and Sharpe broth (MRS) at 30 °C for 18 h, resulting in a cell concentration around 10 8 CFU/mL. P. fluorescens inoculum was prepared in Brain Heart Infusion broth (BHI) at 30 °C for 24 h, resulting in a cell concentration close to 10 7 CFU/mL.

Antimicrobial activity of the CA antimicrobial film incorporated with OEO in the culture medium
The antimicrobial activity of the films F25, F35, F50, and F75 was tested against W. viridescens, while films F50, F75 were tested against P. fluorescens (OEO concentration was chosen based on previous studies with OEO carried out in our group). Serial dilutions were prepared in 0.1% peptone water and adjusted to 10 4 CFU/mL. Active films were then placed on the lids of Petri dishes previously inoculated with 100 µL of each suspension in five points dropped on agar surface (Petri dish + agar + suspension / Petri dish lid + antimicrobial film). Next, the plates were incubated at 30 °C for 48 h. A control analysis was carried out. The results were compared with the control experiment's growth (without the antimicrobial film) and assessed visually. Research, Society and Development, v. 10, n. 16, e271101623335, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.23335

Characterization of ham
Physicochemical composition analysis of ham samples was carried out by measuring pH, water activity (aw), and sodium chloride concentration (NaCl). The pH values were measured using a portable digital pH-meter model 205 (TESTO, Sparta, USA). The aw was performed with a dew-point hygrometer (Aqualab, SERIES 3TE, Pullman, USA). The concentration of NaCl was determined by the analysis of chlorides and conversion in sodium chloride, according to the methodology proposed by Aliño et al. 2011. Samples of ham (2 g) were previously ground with distilled water in Ultra Turrax homogenizer (IKA, model T25 Digital) at 20,000 rpm for 1 min. The solution was made up to 100 mL and then centrifuged (Centrifuge Sigma, model 4k15) for 10 min at 9000 rpm. A previously calibrated automatic chlorine analyzer analyzed an aliquot of 0.5 mL of the supernatant (Cole Parmer, model 926). The results were expressed in milligrams of Chlorine per liter of solution.

Sample preparation
Cooked ham pieces (about 3 kg) (Seara®, São Paulo, Brazil), purchased from a local supermarket and stored at 4 °C, were cut aseptically in a slicer (Metvisa,model CFIE 250,Brusque,Brazil), with thickset to 1.5 mm, resulting in 5.32 g ± 0.73 g per slice. Aliquots with 100 μL bacterial suspension of W. viridescens (around 10 5 CFU/mL) were inoculated and spread on the surface of the ham slices. After inoculation, the film was placed on the surface of the sample and folded. The control samples were inoculated and folded. The samples were put into a sterile mixer bag, packaged in a plastic vacuum bag, and stored in a temperature-controlled incubator (Dist, Florianopolis, Brazil) at 8 °C. The temperature of the samples was measured by data-loggers (Testo 174, Lenzkirch, Germany) every 10 min. For simplicity, samples of cooked, sliced, and vacuum-packed ham will sometimes be called ham samples.

Microbial analyses
Samples were taken at selected times to measure the W. viridescens cells concentration. Each vacuum-packaged sample was aseptically homogenized with peptone water (1% w/v) in the ratio 9:1 [volume peptone water (mL): ham mass (g)] for 60 s in a stomacher (ITR model 1204) to carry out the first dilution. Then, series dilutions were prepared, and 1 mL of each dilution was transferred to sterile Petri dishes with a double layer of agar MRS (Difco Laboratories, Detroit, USA). All the procedures were carried out in a laminar flow chamber. Finally, the plates were incubated at 30 °C for 48 h. The W. viridescens concentration was expressed in CFU/g of ham.

Primary model
The primary model proposed by Baranyi and Roberts (1994) was fitted to the experimental data of W. viridescens growth on the samples of vacuum packaged ham, stored under isothermal condition (8 °C). The curves describe the logarithm of the microbial concentration = log (N) during the time ( ). The parameters of equation 1 are i) that is the logarithm of the initial microbial concentration, ii) that is the specific growth rate (h -1 ), that is the maximum population (CFU/g), and that is the lag phase duration (h). The whole mathematical modeling is described by Equations 1, 2 and, 3, which is related to the physiological state of the cells (Equation 2), and is the parameter concerning the initial physiological state of the cell that is related to the duration of the lag phase ( ) (Equation 3). The fitting procedure was performed in the MATLAB R2013a (The MathWorks Inc®, Natick, USA) using the algorithm developed by Longhi et al. (2013) (1) (3)

Statistical analysis
The statistical indices used to evaluate primary model ability of fitting to experimental data were the Coefficient of Determination ( (Equation 4)), the Root-mean-square Error ( (Equation 5)), and the Bias ( (Equation 6)) and Accuracy factors ( (Equation 7)) (Ross, 1996). The are the values predicted by the model, are the experimental growth data, and n is the number of experimental data, y is the value of microbial cell concentration, is the arithmetic mean of all values of y. (4)

Mechanical properties
The film samples that showed better efficiency and a control film (CA film without the addition of OEO) were analyzed immediately after their production. Film thicknesses were determined with a digital micrometer (Digimatic MDC-Lite, Japão). Tensile strength (TS), Elongation at break (EB), and Young's modulus (YM) were measured using a texturometer (TA.HD.plus Texture Analyse) assisted by the Stable Micro Systems program. Film samples were cut into rectangular strips (25x60 mm) and tested with an initial claw distance of 40 mm and a test velocity of 0.8 mm/s. The tests were performed in triplicate.

Oregano essential oil
The results of CG-MS and CG-FID analysis in the OEO are shown in Table 1. The presence of 21 major compounds in OEO was observed. Both analyses showed a high concentration of phenolic compounds, especially carvacrol (76.1%/GC-MS and 53.76%/GC-FID), considered an active compound against microorganisms (Burt, 2004). The profile of OEO obtained in the present study was very similar to the previous results reported by Boskovic et al. (2020), who also found carvacrol as a major compound.

Antimicrobial effect of with CA antimicrobial film incorporated with OEO against W. viridescens and P.fluorescens
The visual analysis of applications of the films F25, F35, F50, and F75 and control on W. viridescens and P. fluorescens growth are presented in Table 2. The films incorporated with OEO resulted in a growth reduction of W. viridescens and P. fluorescens, which decreased with the increase of OEO quantity. Different responses were obtained for each bacterium. The active films F50 and F75 led to complete inhibition for W. viridescens. The films F25 and F35 led to results that were not different from the control experiment. The film F50 also resulted in the same pattern observed for the control experiment for P. fluorescens, while the film F75 showed a growth reduction when compared to the control.

Control (P.fluorescens) F25
⁎⁎ ⁎⁎ -- The results showed that the Gram-negative bacteria, P. fluorescens, was more resistant to the OEO CA-film than the Gram-positive bacteria, W. viridescens. Marino et al. (2001) reported that EO from sage, mint, hyssop, camomile, and oregano were more effective against Gram-positive bacteria. Escherichia coli O157:H7 was the most sensitive species of Gramnegative bacteria, while Listeria innocua and the Bacillaceae were the most sensitive among the Gram-positive species.
However, the bactericidal activity was more pronounced against the Gram-negative bacteria. Ouattara et al. (1997) observed that several EOs, including OEO, caused similar effects in Gram-negative and Gram-positive bacteria after a 24-hour treatment. Although, after 48 hours of treatment, the inhibition effect appeared to be higher against Gram-negative bacteria.
The antibacterial effect of OEO against bacteria can be attributed to its hydrophobicity that damages the membrane cell, increasing their permeability and nutrient losses (Burt, 2004). Most studies agree that EOs are slightly more active against Gram-positive than Gram-negative bacteria. Gram-negative microorganisms have an outer membrane surrounding the cell wall that restricts the diffusion of hydrophobic compounds through their lipopolysaccharide cover. This fact can make these organisms less susceptible to the action of Eos (Burt, 2004). Dorman & Deans (2000) reported that individual components of The results obtained in the present study showed that the OEO had good antimicrobial activity. Incorporating EOs in packaging films is of great interest to increase shelf life and food safety (Laird & Phillips, 2012;Pola et al., 2016). A case study with vacuum-packaged ham is presented in the following, considering W. viridescens as the target microorganism.

Characterization of ham
The samples of cooked ham used in this work present water activity 0.975 (sd=±0.004, cv=0.4%), pH 6.267 (sd=±0.049, cv=0.7%), and sodium chloride 2.126 (sd=±0.048, cv=2.25%) (% in mass). The results showed low standard deviations (sd) and coefficients of variation (cv), indicating homogeneous samples. Those found agree with Bressan et al. (2007) that reported sliced meat products to have salt contents of 2 to 4%. Kalschne et al. (2014) reported a ham aw of 0.970, while Garrido et al. (2010) reported that the pH range appropriate to cooked ham is 6.00 to 6.73. These results agree with the previous find by Menezes et al. (2018) that indicates pH 6.22 (sd=± 0.04). Therefore, the samples of ham used in the present study had the expected characteristics for this product and are adequate to study the action of antimicrobial films on its shelf life increase.

Antimicrobial activity of the CA antimicrobial film incorporated with OEO in vacuum-packed slices of ham
Based on previous results, the antimicrobial effect of CA active film F50 was assessed in samples of sliced ham, inoculated with W. viridescens, vacuum-packed, and stored at 8 °C. According to the literature (Kreyenschmidt et al., 2010;Slongo et al., 2009), a LAB concentration of 10 7 CFU/g (7 (log CFU/g)) can be the criterion to determine the end of the ham shelf life. Growth evaluation of W. viridescens in ham using F50 showed no significant difference in time to reach the concentration of 10 7 CFU/g (7 (log CFU/g)) when compared to the control (results not shown). Then, the growth of W. viridescens in vacuum-packed cooked sliced ham using F75, stored at 8 °C, was investigated. In Figure 1, it is possible to Research, Society andDevelopment, v. 10, n. 16, e271101623335, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.23335 8 observe that the film F75 inhibited the growth of W. viridescens.   Source: Authors.
The literature reported the effect of oregano's essential oil on the shelf life of vacuum-packed cooked sliced ham, based on the growth of LAB natural microbiota under isothermal conditions. The use of this essential oil led to an increase of , a decrease of , and a consequent increase in the ham shelf life compared to the control samples (Menezes et al., 2018).
The precise parameter estimation is essential to develop kinetic models, characterize the antimicrobial effects of commercial and new natural compounds, predict the microorganism-count-based shelf life, and even in fermentation technology Silva et al., 2017). Previous studies have stated that the lag and exponential phases during microbial growth are of most significant interest in food preservation because the spoilage occurs before the stationary phase (Augustin & Carlier, 2000;Baranyi & Roberts, 1994).
In the present study, the bacterial concentration in ham, using F75, stayed below 7 log CFU/g (limit count to the end of the shelf life for ham) until the 17th day of storage, while the control reached 7 log CFU/g after 9 days of storage (Table 3).
The lag values were extended from 2.2 days to 5.2 days by using F75. Correa et al. (2017) reported that polyhydroxybutyrate/polycaprolactone biodegradable films activated with nisin present a bacteriostatic inhibition effect on Lactobacillus plantarum inoculated on ham. The results showed 3.4 log cycles reduction on the 21st day of storage at 5 ºC, and 2.6 log cycles reduction at the end of the experiment (28th day). The authors reported an extended lag phase from 2.17 to 5.18 days due to active film with OEO.
Our results showed that the active films could be an excellent alternative to increase meat product shelf life. Ouattara et al. (2000) reported that antimicrobial films might be more efficient than the direct use of antimicrobial compounds in foods.
The slow and gradual migration of the active compound from the packaging to the surface of the food could maintain the necessary concentration to inhibit the development of microorganisms (Jafarzadeh et al., 2020). In most fresh or processed foods, microbial contamination occurs at higher levels on the surface, requiring effective control of microbial growth at this local.

Mechanical properties
The mechanical properties of the films without (control) and containing 50 and 75 mg of OEO were determined. Table 4 shows the thickness, tensile strength (TS), elongation at break (EB), and Young modulus (YM) of these three films.
From a visual analysis, both films formed with OEO were transparent, uniform, and continuous, without ruptures. The films presented constant thickness, which is essential to their functionality. Pola et al. (2016) reported that the film thickness influences its mechanical properties and that a constant thickness indicates an appropriate production process, which can form uniform films. Reported values are the means (± standard deviation). Different letters (a, b) in the same column indicate significant differences (p<0.05). Source: Authors.
The TS, EB, and YM results did not show significant differences between the control and the two formulated films (p < 0.05). However, it was possible to observe that the EB increased with increasing OEO concentration in the films. Llana- -Cabello et al. (2018) reported that the EB was affected by the incorporation of OEO. Lee et al. (2019) also noted that incorporating OEO nanoemulsions (5% v/v) in hydroxypropyl methylcellulose-based films increased EB compared to the control films.

Ruiz
Our results agree with those reported by Aguirre et al. (2013) and Jouki et al. (2014). The increase in the films' EB showed that the OEO had a typical plasticizing effect. The plasticizing effect of OEO in CA-based films is due to the low molecular weight of essential oils when compared to the macromolecules of AC. The OEO molecules entered between the polymer chains, weakening the intermolecular interactions (Pola et al., 2016).

Conclusion
The CA films incorporated with OEO showed antimicrobial effects against the microorganisms. The F75 films partially reduced the growth of P. fluorescens. On the other hand, F50 and F75 films completely inhibit W. viridescens growth in the culture medium.
Baranyi and Roberts' model can describe the growth of W. viridescens in ham with the application of the OEO active CA film. The CA films incorporated with OEO present mechanical properties close to the control film, which is essential for its practical application. In ham, F75 films decrease the value of , increases the value of , which increases the ham's shelf life from 9 to 17 days. Thus, OEO active CA films demonstrate excellent potential and could be used to prolong the shelf life of vacuum-packed ham. The use of oregano essential oil microencapsulation in active packaging is suggested for potential future research.