A novel biodegradable film based on κ‐carrageenan activated with olive leaves extract

Abstract This research focused on the development of carrageenan based biodegradable films incorporated with olive leaves extract (OLE). OLE microbial inhibition and its total phenolics (TP) were evaluated. Carrageenan films were produced by casting technique and were characterized by thickness, water vapor permeability (WVP), color, mechanical properties, and infrared spectroscopy. In order to apply as lamb meat packaging, the antimicrobial effect of the films was investigated. Results showed that OLE inhibited E. coli growth and presented excellent TP (41.40 mgGAE/g). Thicknesses of the film with OLE (CAR‐OLE) were approximately 28% higher than film without OLE. Addition of OLE reduced the WVP by approximately 54%. CAR‐OLE was less resistant to breakage and more flexible showing darker color. FTIR showed interaction of carrageenan with OLE. Results revealed that CAR‐OLE promoted 167‐fold reduction in initial count of aerobic mesophiles indicating shelf‐life extension of lamb meat and promising use as antimicrobial food packaging.

antimicrobial packaging based on biopolymers, which may be in the form of biodegradable films, is presented as an important and emerged alternative (Robertson, 2013). Antimicrobial packaging is a type of active packaging, which reduces, inhibits, or slows the growth of microorganisms, unlike conventional packaging, which there are basic functions, interacts with the same for its conservation, and extends shelf-life while maintaining its quality. The biodegradable films become an antimicrobial package when antimicrobial agents are added in their formulation (Appendini & Hotchkiss, 2002;Labuza & Breene, 1989).
Carrageenans are biopolymers of the polysaccharide family, which have the ability to form good biodegradable films (Abdou & Sorour, 2014;Park, 1996). From the biotechnological point of view, sub-Antarctic macroalgae are present in a variety and very large availability (Mansilla, Ávila, & Yokoya, 2012). In this context, several products of algal origin are explored. In particular, Gigartina skottsbergii is a red seaweed from Chile that is used in the extraction of carrageenan for food products (Mansilla et al., 2012). They belong to sulfated galactan hydrocolloids extracted from red algae (Rhodophyta) being generally distributed in three categories: kappa (κ)-carrageenan, lambda (λ)-carrageenan, and iota (ι)-carrageenan (Williams, 2015). Carrageenans have properties that give it commercial application as additives used by the food industry, being gelling agents, emulsifiers, stabilizers, and thickeners (Williams & Phillips, 2009), but they have been gradually gaining recognition as a source of valuable materials, so it is necessary to intensify fundamental research, in order to discover novel functionality, taking full advantage of its potential, such as the production of biodegradable films.
The incorporation of bio compounds, such as phenolic compounds, in biodegradable films represents an interesting alternative. Aimed at the reduction of the use of chemical additives in the food industry, there has been a growing interest in the use of natural additives obtained from plant extracts from material considered as waste (Leuschner & Zamparini, 2002;Schmidt, Taylor, & Davidson, 2013). The olive leaf extract has been shown potential for application in biodegradable films, since olive leaves contain significant amounts of phenolics compounds, such as oleuropein, important factors for their antimicrobial capacity. (Benavente-García, Castillo, Lorente, Ortuño, & Del Rio, 2000). Furthermore, another advantage of using olive leaves is that these leaves are considered as a by-product of the olive oil industry, these can represent between 5% and 10% by weight, of the olive that enters for processing (Boudhrioua, Bahloul, Slimen, & Kechaou, 2008;El & Karakaya, 2009).
In the presented context, the development of biodegradable films from carrageenans and olive leaf extract can present a great potential in the diversification of the formulation of the films. Therefore, the aims of this study are develop and characterize biodegradable carrageenan films incorporated with olive leaves extract and evaluate their antimicrobial activity when used as lamb meat packaging.

| Materials
The carrageenan was extracted from the red algae Gigartina skottsbergii collected in Fuerte Bulnes, the sub-Antarctic region of Magallanes, Chile (53º37'55.6"S, 70º55'17.9"W). Macroalgae specimens were collected manually during low tide at depths between 30 cm and 1.20 m from the intertidal and subtidal zone. The samples were transported in coolers filled with ice to laboratory refrigerators. In the laboratory, the samples were washed with Milli-Q deionized water to remove epiphytes, salt, and foreign matter. The samples were dried at room temperature (20°C) for 5 days.
The extract of olive leaves was obtained from leaves collected from the type Arbequina (31º30'04.0"S, 53º30'42.0"W), located in Pinheiro Machado, Rio Grande do Sul, Brazil.
The chemicals used in this study were glycerol (Mistura da Terra, Brazil), used as plasticizer, and Folin Ciocalteu's phenol reagent (Sigma Aldrich). For microbiological, analyzes were used: nutrient broth, Müller-Hinton broth, brilliant green bile broth 2%, agar for counting plaque microorganisms-PCA agar and peptone (HiMedia), as well as distilled water, all sterilized in a vertical autoclave (Prismatec-CS). These analyses were conducted in a biological safety cabinet (Filterflux-Class II A2). All other reagents used were of analytical grade.
The bacterial strain Escherichia coli ATCC 11229 were supplied by Fiocruz-Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. Stock cultures of the studied bacteria were grown on nutrient broth at 35°C for 24 hr before the tests.

| Carrageenan extraction
The carrageenan extraction was performed according to (Pereira, Amado, Critchley, Velde, & Ribeiro-Claro, 2009), (Sokolova et al., 2013), (Webber, Carvalho, Ogliari, Hayashi, & Barreto, 2012) and (Yermak et al., 2012). Red algae of the species Gigartina skottsbergii, previously dried were washed in tap water. 10 g of the sample was weighed and soaked in distilled water (800 ml) for 1 hr. The samples were then crushed with water in a blender. The solution was placed in a water bath at 74°C for 4 hr. Filtration for separation of the carrageenans from the residue was carried out in vacuum system. Nylon was used as filter medium. The filtered solution was oven dried at 60°C for 24 hr, thereby obtaining the solid carrageenan.

| Preparation, characterization, and application of olive leaf extract (OLE)
The olive leaves were sanitized in running water, commercial solution of 2% to 2.5% sodium hypochlorite and sterilized distilled water.
Subsequently, they were dried in an oven with forced air circulation at 40°C and comminuted in an analytical mill. The fraction that passed through in the 60 mesh sieve was used, obtaining a powder from the leaves. Thereafter, 10 g of the powder was macerated in 100 ml water and allowed to extract for 24 hr with shaking of 240 rpm and temperature of 25°C. The extract obtained was filtered by simple filtration with nylon (Nytex) with 100 µm of pore size and 44% open area.
For the determination of the total phenolic compounds, the spectrometric method adapted from (Singleton & Rossi, 1965) was used. An aliquot (0.5 ml) of OLE were mixed with 10 ml of distilled water and 1 ml of Folin Ciocalteu's. After 5 min, 8 ml of 7.5% (w/v) aqueous solution of sodium carbonate was added. The mixture was stored in the dark for two hours. Then the absorbance of the mixture was measured at wavelength of 765 nm with a spectrophotometer (Ultraspec1000, Amersham Pharmacia Biotech). The results of total phenolic compounds were expressed in milligrams of gallic acid equivalent per gram of dry matter. The analysis was performed in triplicate.
The bacterial inhibition assays were performed using the broth micro-dilution method, using 96-well microtitration plate, adapted to that described in the standard M07-A10 of Clinical and Laboratory Standards Institute (CLSI) (Balouiri, Sadiki, & Ibnsouda, 2016;CLSI, 2015;Liu, McKeever, & Malik, 2017). Briefly, 135 μl of OLE were placed in a 96-well plate along with 145 μl of sterile Muller-Hinton broth and 20 μl of the E. coli culture. The plate was incubated for a period of 16 hr at 35°C. Two absorbance readings at the wavelength of 630 nm (OD630) were recorded, one prior to incubation (0 hr) and one after the incubation period (16 hr), using a microplate reader (Celer-Polaris, Brazil). Wells without OLE and sterilized water were used as controls. Percent growth inhibition was calculated by Equation 1.
where I is the inhibition (%),OD extract2 is the OD630 for the sample after the incubation period, OD extract1 is the OD630 for the sample before the incubation period, OD control2 is the OD630 for control after the incubation period, and OD control1 is the OD630 for the control before the incubation period. Each experiment was repeated three times. More than that, E. coli is recognized as an important causative agent of food-borne disease. Especially for the scope of this work, meats are generally conducive to the development of bacteria. In particular, lamb meat due to its higher pH as compared to beef and pork, comprises an excellent substrate for the growth of pathogens including Staphylococcus aureus, Listeria monocytogenes, and E. coli (Karabagias, Badeka, & Kontominas, 2011

| Preparation and characterization of carrageenan biodegradable films
The carrageenan films were prepared according to the casting method, involving the formation of a film-forming solution, which is subsequently dehydrated. Biodegradable films were prepared with carrageenan and plasticizer, as follows: 1% (w/v) of carrageenan, 37.5% (w/w) glycerol (based on the carrageenan mass), and 75% (v/v) of OLE (based on the final volume of the film solution in relation to the amount of water added). These proportions result in 0.5 g of carrageenan, 0.3 g of glycerol, 37.5 ml of extract, and 12.5 ml of water.
The formula was dissolved under constant agitation (110 rpm) on hot plate and magnetic stirrer (Quimis-Q261M23) at temperature of 70ºC for 15 min.
These conditions were determined in preliminary tests (Martiny, 2017), that were carried out to determine the most suitable plasticizer and OLE concentrations. The results demonstrated that films without the addition of plasticizer were brittle while those with high proportions of glycerol were sticky and difficult to remove from the plates. It was tested that films obtained with the OLE at concentrations below 75% (v/v) showed low or no antimicrobial activity.
Carrageenan has this feature in small amounts to provide the desired effect, since it goes through the gelation process. In other studies, this proportion is usually employed. For example, Shojaee-aliabadi, Hosseini, and Amin, (2013) used 1% carrageenan and 50% plasticizer. Glycerol was chosen as a plasticizer because it is one of the most widely used plasticizing agents in the production of biodegradable films, due to its compatibility and stability with the biopolymer chains. In addition, results in thinner films show very similar functional films attributes regarding their application as food wrappings and decrease film fragility (Fonseca et al., 2018;Saberi et al., 2016).
Finally, the film-forming solution was poured into polystyrene Petri dishes (150 mm diameter) and biodegradable films were obtained by solvent evaporation in an oven with air circulation at 40ºC for 24 hr. After drying, film samples were peeled from Petri dishes and conditioned at room temperature for 48 hr in a desiccator containing sulfuric acid solution with a relative humidity of 50% before testing. The biodegradable carrageenan films produced with the olive leaf extract were designated [CAR-OLE]. The biodegradable films of carrageenan without the addition of olive leaf extract were produced as controls [CAR-control].
The biodegradable films thickness was measured by a digital micrometer (Insize-IP65). Mean thickness was calculated from ten measurements taken at different locations on the biodegradable films samples.
Water vapor permeability (WVP) of biodegradable films was determined gravimetrically using the ASTM Standard E96/E96M (2012). Samples in the disks form were fixed on permeation cells containing granular anhydrous calcium chloride (CaCl 2 )-0% RH. (1) These cells were placed in desiccators with 50% relative humidity at room temperature. Anhydrous calcium chloride mass gain was determined at 7 days during the experiment, and thus, it was possible to determine the water vapor transferred through the biodegradable film according to Equation 2. where the WVP is the water vapor permeability (g/mPa -1 s -1 ), and w ab is the amount of absorbed moisture (g) which was measured by the difference of the initial weighing and final weighing of the experimental apparatus used in the analysis, t the time (s), e is the film thickness (m), A is the area of the exposed film surface (m 2 ) and ΔP the partial pressure difference across the film ( where L * , a * , and b * are the color parameter values of the standard and L * s , a * s , and b * s are the color parameter values of the sample. L* parameter ranges from 0 (black) to 100 (white). The a* parameter measures the degree of red (+a) or green (-a) color and the b* parameter measures the degree of yellow (+b) or blue (-b) color.
The Attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR) was used for the chemical characterization of carrageenan and to observe the structural interaction of carrageenan-based films with added OLE. A Perkin-Elmer spectrometer (UATR Two), in the range of 400 cm -1 to 4,000 cm -1 , was used with 32 scans per spectrum and with a resolution of 4 cm -1 . The samples were cut into small squares and then inserted into the sample portal of the FTIR-ATR apparatus to obtain the spectra to be analyzed.

| Antimicrobial activity of biodegradable films in the packaging of lamb meat
The evaluation of the inhibitory effect of CAR-OLE films was performed by analyzing the growth of total coliforms and aerobic mesophiles during the storage of lamb meat. The sample of chilled lamb meat was purchased locally. The lamb meat was used fresh and raw, without any previous preparation. First, the microbiological analyzes (total coliforms and aerobic mesophiles) of the lamb meat sample at the initial time were performed. Three other samples were packaged in duplicate with different films-the first with the CAR-control film, the second with the CAR-OLE film and the last with commercial film. The commercial film used was polyvinyl chloride (PVC), which was purchased from Royal Pack, Brazil. The lamb meat samples were packaged in a way that attempted to reproduce the conditions under which the fresh meat is marketed, that is, exposed in styrofoam trays and wrapped superficially with PVC film. This form of packaging allows the antimicrobial agents to be released gradually on the food surface by diffusion. The samples were kept packed for two days, and this period is generally given to chilled meat cuts.
Subsequently, the packed samples were stored at 7°C for 48 hr.
After this 48 hr period, the microbiological (total coliforms and aerobic mesophilic) analyses of the lamb meat sample were performed.

| Statistical analysis
Experimental data were analyzed by Statistica software (Stat Soft Inc., 10). The Student t test was applied for determining differences at 95% significance level.

| Phenolics and antimicrobial activity of olive leaves extract
The total phenolic content was 41.40 mg GAE /g (d.b.) greater than ob-

| Characteristics of biodegradable films
The biodegradable films CAR-control and CAR-OLE appeared homogenous, uniform, nonbrittle, flexible and were easily removed from the support (plate) (Figure 1). Table 1 shows the effect of OLE incorporation on carrageenan based films. The addition of the olive leaf extract in the biodegradable films of carrageenan caused an increase in the thickness of the films. This result was already expected due to the increase in mass of the filmogenic solution due to the incorporation of the extract (Sobral, 2000). These data were corroborated by Ma, Zhang, and Zhong (2016) (2017) reported that produced gelatin films with incorporated olive leaves extract showed WVP in the range of 1.83 × 10 -10 g/ms -1 Pa -1 -4.67 × 10 -10 g/ms -1 Pa -1 . These values are higher than found in this study, which may indicate that the use of the polymeric matrix of carrageenan is most advantageous.

F I G U R E 1 Biodegradable carrageenan films produced. (a) CAR-control. (b) CAR-OLE (a) (b)
It is observed from Table 1  high tensile strength of the formulated film is required; however, the flexibility of the film, indicated by the elongation, is a very important parameter. That is, the longer the film elongation, the better its applicability as packaging will be (Teck Kim, Min, & Won Kim, 2013). As shown in Table 2, the addition of extract significantly decreases the L* parameter; that means, its transparency decreases.
And the parameters a* and b* increased significantly with the ad-

F I G U R E 2 FTIR-ATR extracted carrageenan
Variations in the components of the carrageenan structure influence the hydration, strength, texture, and melting of the gel (dos Pereira, 2004).
The analyzed spectrum had strong absorption bands in the region of 1000-1100 cm -1 , characteristic of polysaccharides, the region between 1,010 and 1,080 cm -1 is attributed to the glycosidic bonds present in carrageenans. The spectrum shows bands of strong absorption in the region 927 cm -1 (C-O of 3,6-anhydrogalactose) and in the region 843 cm -1 (C-O-SO 4 of galactose-4-sulfate), typical of κ-carrageenan. It also shows reduced absorbance in the 805 cm -1 region, which is associated with the sulfate group of the 3,6-anhydrogalactose unit, which means the presence of small amounts of  The proximal analysis of a variety of olive leaves performed by Boudhrioua et al. (2008) revealed that on average there are 40.49% carbohydrates. The broad band around 2,800 and 3,000 cm -1 was assigned to C-H stretching, it is also more pronounced in the CAR-OLE film spectrum, due to additional groups extract. The region 1800-1500 cm -1 corresponding to stretching vibration of C = O and C = C (ester, acid, carboxylate, aromatic ring). In particular, for the CAR-OLE the range 1500-1200 cm -1 is very complex, in which CH and OH deformation vibrations and CO stretching vibrations (phenols) can be found. Intense bands between 1,150 and 950 cm -1 correspond mainly to C-O stretching vibration endocyclic and exocyclic carbohydrate (Aouidi et al., 2012). The spectra of the films show several bands, dos Pereira (2004)

| ANTIMI CROB IAL AC TIVIT Y OF B I ODEG R ADAB LE FILMS IN THE PACK AG ING OF L AMB ME AT
The lamb meat was checked for the microorganisms at the initial time, and the result was 100 MPN of total coliforms/g. After two days of storage, the results of 9.3-46 MPN of total coliforms/g of lamb meat for the sample packed with CAR-OLE and > 110 MPN of total coliforms/g of lamb meat packed with CAR-control. From the analysis of total coliforms, it was obtained that the lamb meat sample packed with CAR-OLE had the microbial population decreased in relation to the initial population; the packaging showed inhibitory activity in the packed meat product. The sample packed with the CAR-control did not stop the proliferation of the microorganisms, presenting a MPN of total coliforms/g of lamb meat higher than the one obtained initially. In this regard, Husna et al., (2019)  Contrary to some cited references that used a chemical preservative to confer the additional antimicrobial activity, or synthetic polymer in the formulation of the films, the present study used the extract of olive leaves (natural product), that in the same way promoted an inhibitory effect when applied in biodegradable films based on carrageenan. Thus, the extract of olive leaves becomes a promising in the formulation of biodegradable films.

| CON CLUS IONS
The development of this research allowed the elaboration and characterization of active biodegradable films, as well as their application in the conditioning of lamb meat. The extract produced in this study from olive leaf constitutes a promising alternative for valorizing olive by-products.
The results indicate that the addition of OLE in biodegradable films based on carrageenan promote changes in film properties and confer antimicrobial activity to them against aerobic mesophiles and total coliforms. OLE was highly effective against E. coli. The impact of the addition of OLE on the mechanical properties and WVP of the biodegradable carrageenan films was positive, as it increased the elongation at break, decreased the tensile strength and decreased the WVP. For the color analysis, the films produced with OLE presented a tendency to red and yellow coloration and differentiated from the control in approximately 80%. CAR-OLE reduced the growth of aerobic mesophiles in lamb meat during storage. The results of this study suggest that OLE as a natural antimicrobial has potential for use in biodegradable carrageenan films, which have been shown to be suitable for packaging according to the characterization results. The films developed in this study have a great prospect of application in the sector of active food packaging.

ACK N OWLED G M ENTS
The