Application of active films with natural extract for beef hamburger preservation

Edible chitosan-based films, in pure form (CH100), or blended with gelatin (GEL50:CH50), with and without boldo extract (B) were prepared. The oxygen permeability of the films, as well as color changes and antioxidant and antimicrobial activities on beef hamburgers samples were evaluated. CH100+B film exhibited optimal oxygen barrier properties (6.40±0.170cc-mL/[m2-day]), and this value was significantly increased in blended films (GEL50:CH50+B = 12.99±1.50cc-mL/[m2-day]) (P<0.05). All films provoked color changes in hamburger samples during storage, and the samples covered with CH100+B film showed a lower brightness and a higher redness. All films effectively protected beef hamburger from lipid oxidation, and boldo extract incorporation in pure and blended films enabled significant protection, when compared to the control sample (155.78±0.85meq O2/kg) (P<0.05). Finally, all films proved to be effective in microbial growth control, suggesting that these films are an effective alternative in improving food safety and quality of fresh meat.


INTRODUCTION
Meat is recognized as one of the most perishable foods (DOULGERAKI et al., 2012). Fresh beef meat has an extremely short shelf life, ranging from three to five days when stored at 4ºC (SIROCCHI et al., 2017), due to its high water content and chemical composition, which favor microbial growth to unacceptable levels, contributing significantly to meat spoilage (HAUTE et al., 2016;CASABURI et al., 2015;BONILLA et al., 2014). In addition, oxygen has a negative effect on meat quality (MOURE et al., 2001), where lipid oxidation, after microbial growth, is characterized as the primary cause of food decomposition (YUAN et al., 2016).
Application of active-edible films containing antioxidants and antimicrobials agents for food preservation represents a novel approach to improving the shelf life of food products (SIROCCHI et al., 2017). In this sense, diverse biopolymers have been employed for film production. Gelatin has been used for obtaining biodegradable films due to its edible and biodegradable nature, low cost and wide availability of sources (CLARKE et al., 2016). It retains optimal compatibility with other polymers, such as poly (vinyl alcohol) (ALVES et al., 2011), polysaccharides such as chitosan (BONILLA & SOBRAL 2016), and others proteins such as sodium caseinate (BONILLA & SOBRAL, 2017a). Conversely, chitosan has received significant consideration due to its biodegradability, biocompatibility, low cytotoxicity (DUTTA et al., 2009), and intrinsic antimicrobial activity, demonstrated in meat products (BONILLA et al., 2014;LEKJING, 2016;UPADHYAY et al., 2015). Chitosan has been approved by the United States Food and Drug Administration (FDA) as a Generally Recognized as Safe (GRAS) food additive (FOOD AND DRUG ADMINISTRATION, 2013).
According to HAUTE et al., (2016) andSIROCCHI et al., (2017), the demand for natural agents (e.g. essential oils or natural extracts) to replace synthetic additives (e.g. sorbate and benzoate) has increased in foodstuffs, due to their safety, biocompatibility, biodegradability and low toxicity. ATARÉS et al., (2010) informed that bioactive agents, with antioxidant activity, showed two different mechanisms of action: 1) oxygen barrier effect (reducing oxygen availability in the product) and 2) the particular antioxidant activity. Conversely, natural bioactive ingredients can present antibacterial properties against foodborne pathogens (such as Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella typhimurium) (BURT, 2004).
Peumus boldus Molina (Chilean boldo) is a native tree from the central region of Chile. Phenolic constituents (primarily proanthocyanidins and flavonol glycosides), alkaloids, and essential oils are the main bioactive compounds of boldo leaf extracts, of which boldine is the main constituent. It retains GRAS status, conferred by the FDA, and is easily decomposed, making it environmentally friendly (GIRARDI et al., 2016).
BONILLA & SOBRAL (2017b) studied the antioxidant and antimicrobial activity of boldo extract, among other extracts. Boldo extract at 1% was picking out for future studies due to the results obtained. Then, BONILLA & SOBRAL (2016) developed active films based on gelatin-chitosan blends using boldo extract. The physical-chemical properties (such as optical, mechanical, microstructural and water vapor permeability, and antioxidant and antimicrobial activity) were determined, and the optimal antioxidant and antimicrobial properties of these films were reported. Previous results obtained in these different studies were the basis for choosing the formulations (gelatin50:chitosan50, boldo extract at 1% and glycerol at 0.2%) used in the present study of application in food product.
Thus, the objective of the present research was to apply films based on pure chitosan (CH100) or blended with gelatin (GEL50:CH50), with and without boldo extract (B) on beef hamburger samples stored at 4ºC up to 10 days, to study their preservation (food safety and the quality of fresh meat) in this conditions. Films were first evaluated regarding their oxygen permeability, and then color changes, lipid oxidation and microbial growth on beef hamburger samples were determined.

Preparation of films
Chitosan (CH) and pigskin gelatin (GEL) solutions were prepared separately, according to BONILLA & SOBRAL (2016). Chitosan (1% w/w) was dispersed in an aqueous solution of glacial acetic acid (1.0% v/w). In parallel, gelatin (4g GEL/100 mL of distilled water) was hydrated for 30 min, and solubilized afterward in a thermostatic bath (Marconi ® MA-184, Piracicaba, SP, Brazil) at 55ºC (±0.5ºC). Next, glycerol (0.2%) was added in GEL solution. Boldo extract (B), at 1%, was prepared according to BONILLA & SOBRAL (2016). Film-forming solutions (FFS) were prepared mixing both solutions for obtained the following formulations: films based on pure (CH100) and blended (GEL50:CH50) biopolymers without extracts, and films based on pure (CH100+B) and blended (GEL50:CH50+B) biopolymers with B extract. The pure gelatin (GEL100) film was only prepared for oxygen permeability determination. All films were prepared using a casting technique and dried at 30ºC -60% RH for about 24h, in a climate oven with controlled atmosphere (Marconi ® MA 835/UR, Piracicaba, SP, Brazil). Next films were peeled off and conditioned for seven days at 25ºC-53% RH in chambers containing saturated solutions of Mg(NO 3 ) 2 . Thickness of all films was around 80µm, measured with a micrometer (Mitutoyo Mexicana S.A. de C.V., State of Mexico, Mexico) at six random positions.

Oxygen permeability of the films
The oxygen permeability (OP) test was carried out following the ASTM D3985-95 (1995), at 25ºC-50% RH, and using a MOCON OX-TRAN ® 2/21 (Mocon ® , Minneapolis, USA) device. Films (50cm 2 ) were clamped in the diffusion chamber. Pure oxygen (99.9% purity) was introduced into the upper half of the sample chamber, while pure nitrogen (99.9% purity) was injected into the lower half, where an oxygen sensor was placed. Three replicates were carried out per formulation.

Beef hamburger preparation
The bovine meat (Longissimus dorsi) was obtained from the slaughterhouse at Universidade de São Paulo (USP), Pirassununga campus, and it was processed after four hours of slaughter deboning, to produce beef hamburger samples of six cm in diameter and 0.5mm of thickness. Both surfaces (top and bottom) of the hamburger samples were coated with the different film formulation. No-coated (control) and coated samples were placed in foam trays (length: 10.5cm, width 8.5cm, height 1cm), wrapped with a commercial stretch plastic film (PVC) and subsequently stored in refrigerated conditions (4°C), with light. Different trays with samples were prepared for analysis at 0, 3, 7 and 10 days. Four analytical replicates were carried out per formulation.

Color determination of beef hamburger samples.
Color measurements were analyzed using a MiniScan MSEZ 1049 (HunterlLab ® , Reston, VA, USA) colorimeter, with a spectral range from 400 to 700nm, D65 (day light) lamp, angle of 10º and a measuring cell with an opening of 30 cm. The CIEL ab system measurements (L * for luminosity, and a * and b * for the color-opponent dimensions) were obtained from sample reflection spectra. For beef hamburger samples analysis, the PVC and prepared films on both surfaces (top and bottom) were removed, and two minutes later, the color changes were measured on hamburger surface. Three replicates were carried out per formulation.

Lipid oxidation on beef hamburger samples.
Beef lipid was extracted following the BLIGH & DYER (1959) method. Beef hamburger samples (30g) were homogenized in 210 mL of a chloroform: methanol:distilled water (60:120:30) mixture, using a rotorstator homogenizer (T25 digital ULTRA-TURRAX ® ) at a speed of 10,000rpm for 1 min, at 4°C. The homogenate was diluted with 60mL of chloroform and homogenized at 10,000rpm for 30 seconds. Then, 60mL of distilled water was added, and the mixture was homogenized again for 30 seconds. The homogenate was centrifuged at 4,500g for 10 min, at 4°C, and the supernatant was transferred into a separating flask. Chloroform phase was drained off into a 250mL Erlenmeyer flask containing from 2-5g of anhydrous sodium sulfate, shaken well and decanted into a round-bottom flask through Whatman No. 4 filter paper. Lastly, the solvent was evaporated by a drying and sterilization oven, at 100°C. The extracted lipid was then subjected to peroxide value (PV) analysis. PV determination was performed according to the BRAZILIAN ANIMAL FEED COMPENDIUM (2013).Beef hamburger lipid (3 g) was dissolved in a 2:3v/v mixture of chloroform and acetic acid. Subsequently, an oversaturated KI solution (0.5mL) was added. The mixture was stirred for 1 min, followed by the addition of 30mL of distilled water. Next, titration with sodium thiosulfate solution (0.001N) was carried out, using starch as an indicator. Peroxide value was expressed as milliequivalents of peroxide index per kilogram of lipid. All analyses were performed in triplicate.

Microbiological analyses on beef hamburger samples.
Beef hamburger samples (10g) were aseptically obtained and homogenized in a stomacher with 90mL of sterile buffered peptone water, for two min. Aliquots were serially diluted in buffered peptone water. Then 1 mL of each dilution was placed onto Petrifilm ™ Plates, according to the following official methods: AOAC 2003.07 (2012) (S. aureus), AOAC 991.14 (2012) (total coliforms), and AOAC 990.12 (2012) (aerobic plate count). The plates were incubated at 37°C and the colonies formed were analyzed after 24 hours for S. aureus and total coliforms, and 48 hours for aerobic microorganisms. All analyses were performed in triplicate.

Statistical analyses
Analysis of variance (ANOVA) was applied using the Statgraphics ® Plus 5.1 software (Manugistics Corp., Rockville, MD, USA). Effects of storage time and different packaging treatments in beef hamburger samples were studied. Multiple means comparisons were performed using the Fisher's Least Significant Difference (LSD) test, at 95% level of confidence level. Additionally, standard error of the mean (SEM) calculation was based on Mean Square Error of ANOVA analysis.

Oxygen permeability of the films
In figure 1, it can be observed that pure CH100 and GEL100 films displayed similar and rather low oxygen permeability (OP) values (9.73±2.10cc -mil/ [m² -day]) and 9.74±2.10cc -mil/[m² -day] respectively), in comparison to those reported by other authors (DI PIERRO et al. 2011, CASARIEGO et al. 2009) (P>0.05). Unfortunately, the film based on blended biopolymers without extract (GEL50:CH50) exhibited a significant increment in these values (16.7±2.10cc -mL/[m² -day]), when compared to pure films (P<0.05). These results can be due to glycerol plasticizer incorporation in the blend films, not present in the pure chitosan films. Addition of glycerol made the films more susceptible to moisture, contributing to promote mass transfer processes due to the promotion of molecular mobility, and then showing lower barrier properties compared to pure films.
Conversely, the presence of B extract in the pure chitosan (CH100+B) and blended (GEL50:CH50+B) films resulted in significant reduction of OP values (6.40±2.10cc -mil/[m² -day] and 13.0±2.10cc -mil/[m² -day] respectively), when compared to the same formulations without extracts (P<0.05). These results can be due to the polar chemical structure of the polyphenolic compounds in the extract, reducing the ability of the nonpolar oxygen molecules to condense, by increasing the tortuosity and making it more difficult for oxygen to diffuse through the films.
Similar results were reported by WANG ET AL. (2016), who studied the OP of flexible soy protein isolate films by incorporating chestnut bur extracts, and concluded that when the extract content in the film was increased, the OP values of the film decreased (P<0.05). TALÓN ET AL. (2017) studied the OP of films based on chitosan and starch containing tannic acid and thyme extracts and observed that extract addition improved the oxygen barrier property of the films, which is consistent with the development of a tighter more closed structure.

Color determination
The film formulation was the factor that retained the highest influence on color changes (Table 1). The color of hamburger samples showed initial values (day 0) of 37.5±0.150, 18.6±0.200, and 15.8±0.160 regarding luminosity L * , a * , and b * measurements, respectively.
Regarding the L * parameter, up to 10 days of storage, all film formulations affected the beef hamburger color, enabling a slightly significant decrease in the luminosity of the hamburger samples covered with pure chitosan films with (CH100+B=29.8±0.150) and without (CH100=32.0±0.150) B extract added (P<0.05), with respect to non-coated samples (control) and other films. No change was observed regarding storage time.
Regarding to the a * parameter, up to 10 storage days and considering the redness of the hamburger samples, a significant increment on this value (more vivid color) was observed in hamburger samples covered with CH100+B film (  the conversion of myoglobin into metmyoglobin, due to the high oxygen pressure induced by the films, in which, in the case of CH100+B films, the process was slower. Considering the b * parameter (yellow-green color scale), neither the film formulations nor the storage days resulted in any significant differences in the color of all hamburger samples, varying between 14.1±0.160 and 17.3±0.160, for non-coated and coated samples with GEL50:CH50 film, respectively, up to 10 days of storage (P>0.05). SIROCCHI et al. 2017, BONILLA et al. 2014, LEKJING, 2016and GARRIDO et al. 2011 have reported color changes in beef or pork meat, coated with coatings or films for shelf life extension of these products.
VARGAS et al. (2016) added rosemary and pitanga extracts directly onto beef patties to evaluate color stability at 4ºC during 4 days, and observed that pitanga extracts had a negative influence on meat color (redness and metmyoglobin formation), probably due to high chlorophyll content in extract, suggesting that this pigment affects myoglobin oxidation. LEKJING (2016) examined chitosan-based coatings, with or without clove oil, on cooked pork sausages, in refrigerated storage (4±2ºC) during 20 days. He reported that, during storage, the L * , a * , and b * values of the control samples were not significantly altered by the treatment at any single storage time, probably due to chitosan and clove oil acting as preventive antioxidants on the meat product.

Lipid oxidation
Active films enriched with natural extracts are promising food preservation tools, due to the presence of bioactive compounds that have free radical scavenging actions, which can reduce the oxidation process, and, as a consequence, an extension of shelf life can be achieved (SIROCCHI et al. 2017). Meat has low oxidative stability and is very susceptible to rancidity during production and storage. Numerous studies have indicated that lipid oxidation in meat products may be controlled or minimized by using antioxidants (KANATT et al. 2008).
In the present research, it is remarkable that, at any control time (3, 7, and 10 days), all-edible films had a protective effect against lipid oxidation, when compared to the uncoated samples. This effect was more appreciable at the end of the storage period, when a greater advance of the process has occurred (Figure 2).
The initial peroxide value was 53.3±0.850 meq/1000g of fat. At the end of the 10-day storage period, the uncovered samples exhibited a 3-fold increase in the initial PV value (156±0.850meq/1000g of fat fat and GEL50:CH50+B = 17.1±0.850meq/1000g of fat) when compared to the covered samples without extract, revealing the antioxidant chemical effect of the active compounds of the extract, and the lower oxygen permeability values of these films containing the extract. YUAN ET AL. (2016) reported that the antioxidant capacity of chitosan might be due to the ability of residual free amino groups of the polysaccharide to react with free radicals forming stable macromolecular radicals and ammonium groups. Moreover, O'BRIEN et al. (2006) described that boldo leaves contain tannins, essential oils (mainly ascaridole and cineole), and flavonoids, of which catechin was recently reported to be the most abundant of the metabolites. Therefore, the mentioned compounds appear to contribute in great extent to the total antioxidant capacity of B extracts and infusions. LEKJING (2016) studied peroxide value in the lipid fraction in cooked pork sausages during 25 days of storage at 4°C, and concluded that the best antioxidant effect was obtained with the combination of chitosan and clove oil, possibly since chitosan acts as a chelator of transition metal ions that initiate lipid peroxidation and start chain reactions.

Microbial counts
Besides lipid oxidation, spoilage of meat can be considered a result of the microbial activity of a wide variety of microorganisms, since meat nutrient composition, pH (5.5-6.5), and high moisture content enable the growth and survival of microorganisms (CASABURI et al. 2015). Numerous methods are available to control spoilage and thus extend the shelf life of meat, among which the use of natural antimicrobial agents, since it was reported that bacterial counts are suppressed in meat stored aerobically in the presence of these compounds (DOULGERAKI et al. 2012). Figure 3 shows the effect of different films, containing or not boldo extract, on the growth and survival of total aerobic, total coliform, and S. aureus microorganisms.
The initial microbial load of total aerobic microorganisms was 3.30±0.900Log CFU/g. At the end of the storage period (10 days), the chitosan-based films, with (CH100+B) or without (CH100) boldo extract added, maintained the same microbial load (3.20±0.900Log CFU/g and 4.00±0.900 Log CFU/g respectively) as the initial day (P>0.05), exhibiting significantly lower values than non-coated samples (8.80±0.900Log CFU/g). Also, the blended films (GEL50:CH50 = 5.30±0.900Log CFU/g and GEL50:CH50+B = 6.40±0.900Log CFU/g) exhibited a reduction in growth microbial, with respect to the control samples, but with significantly higher values than all CH100 films (P<0.05) (Figure 3). Nevertheless, the initial microbial load of coliform microorganisms was 2.90±0.200Log CFU/g. At 10 days of storage, all the films were effective in inhibiting the growth of coliforms on beef hamburgers samples when compared with the control sample (8.10±0.200Log CFU/g) (P<0.05) ( Figure 3). As described previously, the chitosan-based films, with or without addition of boldo extract (CH100+B = 3.20±0.200Log CFU/g and CH100 = 2.30±0.200Log CFU/g) exhibited lower values of microbial loads than blended films (GEL50:CH50 = 3.50±0.200Log CFU/g and GEL50:CH50+B 4.80±0.200Log CFU/g) at the end of the storage period (P>0.05). These results suggested that the antimicrobial effect of the films was directly associated with the chitosan concentration, of which antibacterial properties have been proven in previous studies with pork meat products (BONILLA et al. 2014).
Lastly, the initial microbial load of S. aureus microorganisms was 2.20±0.600Log CFU/g. At 10 days of storage, differences in the antimicrobial effect of all films were described. The chitosan-based films (CH100 = 2.30±0.600Log CFU/g and CH100+B = 2.30±0.600Log CFU/g) and blended films without extract (GEL50:CH50 = 2.30±0.600Log CFU/g) maintained the initial microbiological load of S. aureus. Significant differences regarding GEL50:CH50+B films (3.20±0.600Log CFU/g) and non-coated beef hamburger samples (4.00±0.600Log CFU/g) were observed (P<0.05) ( Figure  3). These results suggested that reducing the ratio of chitosan by 50%, the antimicrobial activity of the blended films showed the same effectiveness, which was not improved by the addition of the B extract.
In general, all of the obtained results reflect that chitosan (CH), alone or in the presence of B extract, retains remarkable antimicrobial activity on beef hamburgers, and the presence of gelatin (GEL) only contributed to improving preservation quality due to its antioxidant activity, with the incorporation of B extract. JRIDI et al. (2018) studied the effect of active gelatin coated with henna (L. inermis) extract on beef meat quality during chilled storage (4 ºC), and concluded that the combined coating (gelatin + extract) increased the shelf life of meat by decreasing the total and psychrophilic vial bacteria counts at the end of storage (8 days). And, XU et al. (2018) developed tapioca starch active nanocomposite films loaded with cellulose nanocrystal and with two grape pomace extracts (Cabernet Franc [red variety] and Viognier [white variety]) using a solvent casting method. They applied these films on ready-to-eat chicken meat and observed that films with grape pomace extracts exhibited a stronger inhibitory effect against S. aureus (ATCC 29213) compared to L. monocytogenes (ATCC 7644). Further application of the films on readyto-eat chicken meats indicated that starch/cellulose nanocrystal/Viognier films were the most effective against L. monocytogenes inoculated on the meat samples during the 10 days at 4 °C.
Bonilla et al.

CONCLUSION
Pure chitosan (CH100) and pure gelatin (GEL100) films exhibited lower values of OP than blended films (GEL50:CH50), and B extract addition reduced these values significantly. All the films affected the surface color of beef hamburger samples, showing a slight decrease in luminosity and a significant increment of redness of the meat covered with CH100+B film at the end of the storage period. All edible films retained a protective effect against lipid oxidation and growth inhibition of microorganisms on beef hamburger samples at 4ºC. At any storage time, the PV values of meat samples coated with films containing B extract (CH100+B and GEL50:CH50+B) showed lower values when compared to the other samples (CH100, GEL50:CH50, and the control). Regarding microbiological analyses, chitosan films with (CH100+B) or without (CH100) boldo extract added were the most efficient in growth inhibition of total aerobic microorganisms. Also, GEL50:CH50+B blended films enabled a slight reduction in microbial load for coliform microorganisms, when compared to the other films. Regarding S. aureus microorganisms, differences in the antimicrobial effect were observed between pure CH and blended films (CH100; CH100+B, GEL50:CH50 and GEL50:CH50+B) when compared to the control. Considering the obtained results, active-edible films can be used as an effective alternative packaging technology for improving food safety and fresh meat quality.