Physical and Antibacterial Properties of Ac¸a´ı Edible Films Formulated with Thyme Essential Oil and Apple Skin Polyphenols Paula J. P. Espitia, Roberto J. Avena-Bustillos, Wen-Xian Du, Bor-Sen Chiou, Tina G. Williams, Delilah Wood, Tara H. McHugh, and Nilda F. F. Soares

Thyme essential oil (TEO) and apple skin polyphenols (ASP) are natural compounds considered as generally recognized as safe by FDA, with biological effects against bacteria and fungi. This work aimed to evaluate physical and antimicrobial properties of ac¸a´ı edible films formulated with TEO and ASP at 3% and 6% (w/w) individually or combined at 3% (w/w) each. Physical properties studied include mechanical resistance, water vapor permeability (WVP), color, and thermal resistance. Antimicrobial activity against Listeria monocytogenes was determined using the overlay diffusion test. Addition of ASP resulted in improved mechanical properties. TEO at 6% (w/w) resulted in increased elongation. ASP films had significant higher WVP than control film. ASP films were lighter and had more red color than other films. Incorporation of ASP resulted in improved film thermal stability, whereas TEO caused rapid thermal decomposition. Presence of clusters was observed on the surface of films. Addition of ASP resulted in a smother surface, whereas addition of TEO led to the formation of crater-like pits on the film surface. Ac¸a´ı edible film incorporated with 6% (w/w) TEO presented the highest antimicrobial activity. However, both antimicrobials are necessary in the ac¸a´ı films in order to obtain edible films with suitable physical-mechanical properties. The results of the present study showed that TEO and ASP can be used to prepare ac¸a´ı edible films with adequate physical-mechanical properties and antimicrobial activity for food applications by direct contact. Keywords: ac¸a´ı, antimicrobial activity, edible film, mechanical properties, pectin, polyphenols, thermal stability, thyme

essential oil Practical Application: Developed ac¸a´ı edible films presented antimicrobial activity against L. monocytogenes and good physical-mechanical properties, showing the potential use of ac¸a´ı edible films in food preservation.

Introduction Consumer concerns for a healthy, nutritive, and safe diet have resulted in fast development of edible films research. Moreover, environmental issues created by plastic accumulation have contributed to development in this area of research. In this way, although recycling has been considered as an option to diminish contamination by plastic accumulation, less than 5% of plastics are recycled while the production of plastic resins is constantly growing (Sutherland and others 2010). As a result of these concerns, biopolymers have been studied as substitute materials for plastics due to their film-forming properties, which are desirable for the elaboration of edible films intended as food packaging (Azeredo and others 2009). Edible films can be prepared from polysaccharides, such as pectin. Pectin is a polysaccharide that is able to form cohesive MS 20131183 Submitted 8/20/2013, Accepted 2/4/2014. Authors Espitia and Soares are with Food Packaging Laboratory, Food Technology Dept, Federal Univ. of Vic¸osa, Av. P. H. Rolfs s/n, Campus Universit´ario, 36570-000. Vic¸osa, Minas Gerais, Brazil. Authors Avena-Bustillos, Du and McHugh are with Processed Foods Research Unit, Western Regional Research Center, U.S. Dept. of Agriculture, Agricultural Research Service, 800 Buchanan St, Albany, CA, 94710, USA. Authors Chiou, Williams and Wood are with Bioproduct Chemistry and Engineering Research, Western Regional Research Center, U.S. Dept. of Agriculture, Agricultural Research Service, 800 Buchanan St., Albany, CA, 94710, USA. Direct inquiries to author Espitia (E-mail: [email protected]).

and transparent films (Alves and others 2011). This polysaccharide is a structural component of cell walls, which consists primarily of partially methyl esterified poly α-d-1,4-linked galacturonic acid (homogalacturonan, “smooth” ordered regions). Pectin also has kinks of (1→2)-linked α-l-rhamnose residues as “hairy” regions due to side chains of arabinogalactan I, constituting the disordered regions (Pérez and others 2009). Moreover, pectin is an ingredient used in the food industry and is considered as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA) (FDA 2012). Pectin has been used in combination with fruits to produce edible films. In those studies, apple (Du and others 2008a; Mild and others 2011), tomato (Du and others 2008b), carrot and hibiscus (Ravishankar and others 2012) were used as the primary ingredients for the preparation of edible films. However, to the best of our knowledge, there are no studies using ac¸a´ı as polymeric matrix for the development of edible films in food packaging. Recently, ac¸a´ı (Euterpe oleracea), a tropical fruit from Brazil, has received great attention due to the presence of bioactive compounds. Ac¸a´ı is a palm berry, round and dark purple when mature, with an average diameter of 2 cm. Ac¸a´ı berries are characterized by a nutty flavor with lingering metallic undertones and a creamy, as well as oily texture (Schreckinger and others 2010). According to Azeredo and others (2009), edible films produced with fruit can have sufficient mechanical and barrier properties along with the color and flavor provided by the pigments and

Published 2014. This article is a U.S. Government work and is in the public domain in the USA. doi: 10.1111/1750-3841.12432 Further reproduction without permission is prohibited

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Abstract:

Properties of ac¸a´ı edible films . . .

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volatile compounds of the fruit. Thus, ac¸a´ı has potential to be used in conjunction with pectin as the polymeric matrix for the production of edible films with desired color and flavor in antimicrobial food packaging. Antimicrobial packaging is a type of packaging that interacts with the product to reduce, inhibit or retard the growth of microorganisms that may be present on food surfaces (Soares and others 2009). To obtain this type of active packaging, several antimicrobials have been incorporated in polymeric matrixes. These include apple skin polyphenols (ASP), which are flavoring components listed as GRAS by the FDA. The antimicrobial activity observed on apple components has been reported against foodborne pathogens, such as Escherichia coli and Staphylococcus aureus. This is due to a variety of phytochemicals in apples, such as quercetin, catechin, phloridzin, and chlorogenic acid, with strong antioxidant and antimicrobial activities (Boyer and Liu 2004; Alberto and others 2006). Moreover, essential oils, such as thyme essential oil (TEO), are natural substances also considered as GRAS by the FDA (L´opez and others 2007). Their biological effects, including antibacterial and antifungal activity, as well as pharmaceutical and therapeutic potentials, have been previously reported (Edris 2007; Bakkali and others 2008; Espitia and others 2012). In this study, ac¸a´ı edible films incorporated with ASP and/or TEO were developed. Physical-mechanical properties of ac¸a´ı edible films, including mechanical resistance, water vapor permeability (WVP), color properties, thermal stability, and microstructure, were evaluated. Also, the antimicrobial activity of these edible films was tested against L. monocytogenes.

Materials and Methods Ac¸a´ı edible film preparation Ac¸a´ı puree (Amafruits, Orland Park, Ill., U.S.A.) and pectin were the primary ingredient in all ac¸a´ı-based film forming solutions. Glycerol, also known as vegetable glycerin (Starwest Botanical, Rancho Cordova, Calif., U.S.A.), was added as a plasticizing agent. R , Lindon, Utah, U.S.A.) and citric acid Ascorbic acid (Bronson (Archer Daniels Midland Co., Decatur, Ill., U.S.A.) were used as browning inhibitors. Pectin solution (3% w/w) was prepared with high methoxyl (1400) pectin (Tic Gum, White Marsh, Md., U.S.A.) and added to ac¸a´ı puree. The ac¸a´ı film-forming solution was prepared using the Kitchen Aid mixer by adding ac¸a´ı puree (26% w/w), citric acid (0.25% w/w), ascorbic acid (0.25% w/w), and vegetable glycerin (3% w/w) to the pectin solution (70.5% w/w) and mixing at low speed for 15 min. The film-forming solution was homogenized in the Kinematica Polytron (Beckman Instruments Inc., Westbury, N.Y., U.S.A.) for 3 min at 20000 to 24000 rpm. The solution was then degassed under vacuum for 30 min before film casting. The ac¸a´ı edible films were prepared by placing a polyethylene terephthalate film (PET) on a glass plate (30.5 × 30.5 cm), followed by pouring the film-forming solution (60 ± 1 g) on the PET film. The film was then cast using a draw down bar (45 mil = 1.143 mm). The edible films were dried for approximately 12 ± 1 h at room temperature.

0% to 10% (w/w) (Du and others 2011). However, 6% ASP edible films had the highest antimicrobial activity against Listeria monocytogenes even after 48 h of exposure to this microorganism. Thus, to compare the antimicrobial effect of ASP and TEO, both antimicrobial compounds were incorporated individually in the edible films at concentration of 0%, 3%, and 6% (w/w), respectively. Moreover, to test the possible combined antimicrobial effect of these 2 compounds both antimicrobials were incorporated at 3% (w/w) into the ac¸a´ı edible film. The control film was prepared according to the methodology previously described for the elaboration of ac¸a´ı edible films, without the incorporation of any antimicrobial.

Film thickness Film thickness was measured with a digital micrometer (Mitutoyo Manufacturing, Tokyo, Japan) at 5 random positions on the film samples for further tests. Mechanical properties of film Mechanical properties of films were studied by characterizing the tensile properties: maximum load, tensile strength at break, elongation at break, and Young’s modulus (elastic modulus). Tensile properties were measured according to standard method D88209 (ASTM 2009) using an Instron Model 55R4502 Universal Testing Machine (Instron, Canton, Mass., U.S.A.) with a 100 N load cell. The test speed was 10 mm/min and the distance between the grips was 10 cm. Ten specimens of edible films from each treatment were used for measuring tensile properties. Water vapor permeability (WVP) The WVP of edible films was determined using the gravimetric modified cup method according to McHugh and others (1993) based on the standard method ASTM (1980). Eight specimens of ac¸a´ı edible films from each treatment were used. Cabinets used for measuring WVP were preequilibrated to 0% relative humidity R (RH) using calcium sulfate desiccant (Drierite W.A. Hammond Drierite, Xenia, Ohio, U.S.A.). Test cups made of polymethylmethacrylate (PlexiglasTM Evonik Industries AG, Darmstadt, Germany) were filled with 6 mL of deionized water to expose the film to a high water activity inside the test cups. Ac¸a´ı edible films from each treatment were placed between the cup and its lid (a ring of poly(methyl methacrylate) with an opening of 19.6 cm2 ). Next, each cup was sealed with its lid using 4 screws symmetrically located around the cup circumference (Figure 1). Then, samples

Antimicrobial compounds ASP powder was an apple skin extract produced by Apple Poly LLC (Morrill, Nebr., U.S.A.). TEO was obtained from Lhasa Karnak Herb Co. (Berkeley, Calif., U.S.A.). Previous work has Figure 1–Scheme of the gravimetric modified cup method according to reported the incorporation of ASP at concentrations ranging from McHugh and others (1993) for WVP measurement of ac¸a´ı edible films. M2 Journal of Food Science r Vol. 00, Nr. 0, 2014

Properties of ac¸a´ı edible films . . . Table 1–Mechanical properties of ac¸a´ı edible films incorporated with ASP and TEO∗ . Treatment Control 3%ASP 3%TEO 3%ASP + 3%TEO 6%ASP 6%TEO

Maximum load (N) 1.42 1.22 1.65 1.23 5.50 1.21

± ± ± ± ± ±

0.32b 0.01b 0.47b 0.03b 0.31a 0.00b

Tensile Strength (MPa) 1.43 1.00 1.03 0.80 2.74 0.59

± ± ± ± ± ±

0.35b 0.07bc 0.42bc 0.11c 0.50a 0.11c

Elongation (%) 99.13 27.58 114.31 33.92 67.34 89.18

± ± ± ± ± ±

42.54a 20.84b 57.03a 6.86b 15.80ab 9.23a

Elastic modulus (MPa) 6.72 13.36 3.98 5.22 9.38 3.17

± ± ± ± ± ±

1.36n.s. 13.36n.s. 1.68n.s. 1.39n.s. 6.39n.s. 0.59n.s.

of ac¸a´ı edible films in individual cups were placed in preequilibrate 5 min. Following this, one edible film disk (12 mm diameter) was placed on the center of each previously inoculated TSA plate cabinets and 8 weights were taken for each cup at 2 h intervals. with the film’s shiny side down. The plates were incubated at 37 °C for 24 h. The inhibition radius around the film disk (colonyColorimetric analysis Color of edible films was measured using a Minolta Chroma free perimeter) was measured with a digital caliper (Neiko Tools, Meter (Model CR-400, Minolta, Inc., Tokyo, Japan). The color Ontario, Calif., U.S.A.) in triplicate after 24 h of incubation. The was measured using the CIE L∗ , a∗ , and b∗ coordinates and illumi- inhibition area was then calculated. nant D65 and 10° observer angle. The instrument was calibrated using a Minolta standard white reflector plate. A total of 10 films Statistical analysis were evaluated for each treatment and 5 readings were made for Data from antimicrobial activity and physical-mechanical propeach replicate. erties of ac¸a´ı edible films were evaluated by analysis of variance (ANOVA) and Tukey’s multiple comparison tests at 95% confiThermogravimetric analysis dence level using the Statistical Analysis System (SAS) version 9.1 A thermogravimetric analyzer from TA Instruments TGA 2950 (SAS Inc., Cary, N.C., U.S.A.). (New Castle, Del., U.S.A.) was used to characterize the thermal stability of edible films. Sample from each treatment (10 ± 1 mg) Results and Discussion was heated to 800 °C at a rate of 10 °C/min. The sample chamber was purged with nitrogen gas at a flow rate of 40 cm3 /min. Mechanical properties Weight loss of samples was measured as a function of temperature. Mechanical properties of packaging materials incorporated with The derivative of thermogravimetric analysis (TGA) curves was antimicrobials are essential for practical application. The mechaniobtained using TA analysis software. cal performance was characterized by determining their maximum load, tensile strength, elongation, and elastic modulus of ac¸a´ı edField emission scanning electron microscopy (FESEM) ible films incorporated with 6% (w/w). The results showed that Morphological analyses of edible films were done using a Hi- these properties were significantly different between treatments, tachi S-4700 Field Emission Scanning Electron Microscope (FE- except for the elastic modulus (Table 1). SEM, Hitachi, Tokyo, Japan). Samples were prepared by dropping Addition of 6% (w/w) ASP resulted in the highest maximum a piece of film (1 cm2 ) into liquid nitrogen and allowing the piece load. The tensile strength was also improved with the addition of to equilibrate in the liquid nitrogen. The film piece was then ASP. These results are probably due to the presence of fiber in the fractured into several smaller pieces. Selected smaller pieces were ASP powder. Several researchers have reported that the dietary mounted edge-up on a small aluminum cube, which was then fiber content is higher in apple peel when compared to other edmounted on a specimen stub using double adhesive coated carbon ible parts of the fruit (Gorinstein and others 2002; Leontowicz tabs (Ted Pella, Inc., Redding, Calif., U.S.A.). The samples were and others 2003). Dietary fiber consists mainly of cellulose, hemicoated with gold-palladium in a Denton Desk II sputter coating celluloses, lignins, pectins, and gums (Sudha and others 2007). unit (Denton Vacuum, LLC, Moorestown, N.J., U.S.A.). Finally, Henr´ıquez and others (2010) found that total dietary fiber in apac¸a´ı edible film samples were viewed in the FESEM. Images were ple peel represented about 47.8% of the dry weight of Granny captured at 2650 × 1920 pixel resolution. Smith apple peel. Studies have shown that incorporation of fibers in biodegradAntimicrobial activity able polymers can improve their mechanical properties. Luo and L. monocytogenes was obtained from Univ. of California, and was Netravali (1999) reported that pineapple fibers improved the isolated from cheese associated with an outbreak. Frozen cultures tensile strength of poly(hydroxybutyrate-co-valerate) (PHBV), a of L. monocytogenes were streaked on trypticase soy agar (TSA) biodegradable polymer produced from a wide range of microorand then incubated at 37 °C for 24 h. One isolated colony was ganisms. Also, they indicated that compared to virgin PHBV resin, restreaked on TSA and then incubated at 37 °C for 24 h. This was composites incorporated with 30% pineapple fibers showed an infollowed by inoculating one isolated colony into a tube with 5 mL crease in Young’s modulus. Moreover, the incorporation of cotton trypticase soy broth (TSB) and incubating at 37 °C for 24 h with fiber or coconut husk fiber (whisker) into edible fruit films imagitation. The microbial broth was then serially diluted (10×) in proved their overall tensile properties (Azeredo and others 2012). In contrast, the incorporation of 6% (w/w) TEO resulted in 0.1% peptone water. For overlay diffusion tests, 0.1 mL of 105 CFU/mL of bacterial highest value of elongation. Similar effect was observed by Espitia culture was plated onto each of the TSA plates. The inoculum and others (2011), after the incorporation of oregano, cinnamon, was spread evenly throughout each plate and then let to dry for and lemongrass essential oil in cellulose acetate films. They found Vol. 00, Nr. 0, 2014 r Journal of Food Science M3

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ASP, apple skin polyphenol; TEO, thyme essential oil; n.s., no significant differences between films. ∗ Data reported are mean values ± standard deviation, mean values in the same column followed by different letters are significantly different at P < 0.05, according to the Tukey test.

Properties of ac¸a´ı edible films . . . Table 2–Water vapor permeability of ac¸a´ı edible films incorporated with ASP and TEO∗ .

Treatment

Relative humidity Water vapor Treatment L∗ inside the permeability 25.86 ± 0.17c 4.45 cups (% RH) (g•mm/kPa•h•m2) Control 3%ASP 27.24 ± 0.17ab 6.95 0.006d 80.3 ± 2.4bc 2.56 ± 0.44b 3%TEO 25.16 ± 0.24d 4.28 cd c ab 0.008 78.3 ± 1.9 3.22 ± 0.53 3%ASP+3%TEO 27.16 ± 0.33b 7.38 0.001b 84.5 ± 0.3a 3.03 ± 0.06ab 6%ASP 27.67 ± 0.15a 7.99 0.016cd 79.3 ± 1.4c 3.29 ± 0.37ab 6%TEO 25.40 ± 0.38d 3.74 a a a 0.039 84.9 ± 0.4 3.55 ± 0.63 ASP, apple skin polyphenol; TEO, thyme essential oil. 0.013bc 82.3 ± 2.1ab 3.13 ± 0.53ab ∗

Thickness (mm)

Control 0.104 3%ASP 0.116 3%TEO 0.165 3%ASP+3%TEO 0.127 6%ASP 0.201 6%TEO 0.144

± ± ± ± ± ±

Table 3–Effect of ASP and TEO on color parameters of ac¸a´ı edible films∗ .

ASP, apple skin polyphenol; TEO, thyme essential oil. ∗ Data reported are mean values ± standard deviation. Mean values in the same column followed by different letters are significantly different at P < 0.05, according to the Tukey test.

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that the elongation at break of the films increased with the addition of oregano or lemongrass essential oil individually. This result was attributed to the plasticizing effect of these essential oils in the polymeric matrix. Moreover, Bonilla and others (2012) have pointed out that the incorporation of essential oil in chitosanbased films produces discontinuities in the film structure, resulting in decreased values of tensile strength and elastic modulus. These diminished values derive from lack of cohesion among polymeric chains. However, similar to results of Bonilla and others (2012) we observed a notable stretchability in ac¸a´ı edible films since the elongation of the films with TEO was comparable to control film and higher than ac¸a´ı film with ASP (Table 1). Thus, our results are considered favorable due to the plasticizing effect of incorporated essential oil, which as explained by Bonilla and others (2012) results in a smoother displacement of polymeric chains while stretching the films, allowing the deformation of the packaging material without damage.

Film thickness and WVP The film thickness results showed significant differences among treatments (Table 2). Ac¸a´ı edible films incorporated with 6% (w/w) ASP had the highest thickness, while the incorporation of TEO alone or in combination with ASP resulted in intermediate thickness. These results indicated that the addition of antimicrobials altered the thickness and microstructure of the films. This was later confirmed by microscopic analysis. Ac¸a´ı edible films incorporated with 6% (w/w) ASP showed significant higher WVP than other developed films (Table 2). This film also had the highest RH at the film underside. Colorimetric analysis Color is one of the main characteristics of food packaging design due to its function of drawing attention of consumers and influencing their food purchase (Ares and Deliza 2010). Moreover, Piqueras-Fiszman and Spence (2011) have indicated that packaging color is a major feature in the food industry because consumers associate a food product with some of its probable qualities based solely on packaging color. In addition, around 60% to 90% of consumer buying selection is motivated by packaging color (Piqueras-Fiszman and others 2012). Thus, color of packaging constitutes one of the main factors on consumer’s sensory expectations. For ac¸a´ı films, the ideal color of developed films should match or be closely related to the colorimetric parameters of ac¸a´ı berry or ac¸a´ı pulp. In this way, according to Cipriano (2011), the colorimetric parameters for fresh ac¸a´ı pulp are L∗ = 25.46; a∗ = 1.02; M4 Journal of Food Science r Vol. 00, Nr. 0, 2014

a∗ ± ± ± ± ± ±

b∗ 0.08d 0.09c 0.11d 0.38b 0.34a 0.14e

2.57 2.84 2.53 2.86 2.20 2.37

± ± ± ± ± ±

0.11abc 0.12ab 0.11bc 0.34a 0.16d 0.17cd

Data reported are mean values ± standard deviation, mean values in the same column followed by different letters are significantly different at P < 0.05, according to the Tukey test.

b∗ = –0.29; and Alexandre and others (2004) have indicated the colorimetric parameters (L∗ = 21.79; a∗ = 4.01; b∗ = 0.88) for acidified and heat-treated ac¸a´ı pulp. The colorimetric parameters analysis showed significant differences among ac¸a´ı edible films (Table 3). The color parameter L∗ is a measurement of the lightness or darkness of the film. Its value ranges from 0 to 100 as indication of dark to light. The incorporation of ASP resulted in lighter films, whereas the addition of TEO resulted in darker films when compared to the control. Although these results evidence statistical difference among films, the L∗ value of developed films is notably close to the L∗ values for fresh and heat-treated ac¸a´ı pulp, thus meeting consumer expectations. Positive values of the colorimetric parameter a∗ indicate the redness of the material, whereas negative values indicate greenness. The addition of ASP resulted in the highest value of a∗ . This result was expected since the natural color of ASP is red. The control film had an intermediate value, whereas the presence of 6% (w/w) TEO showed a decrease in this color parameter. The natural color of ac¸a´ı berries when ripe is dark purple (Pompeu and others 2009) due to the presence of 2 predominant anthocyanins (cyanidin-3-rutino-side and cyanidin-3-glucoside). These anthocyanins are responsible for most blue, red, and related colors of ac¸a´ı and other fruits, and are a major source of color in ac¸a´ı-containing juices and beverages (Pacheco-Palencia and Talcott 2010). Thus, the natural red color of ac¸a´ı edible films resulted from anthocyanins present naturally in ac¸a´ı pulp. The red color of ASP also contributed to the increase in this parameter. In addition, positive values of b∗ indicate yellowness, whereas negative values indicate blueness. The incorporation of both antimicrobial compounds at 3% (w/w) resulted in increased value of b∗ when compared to control, indicating the tendency of films incorporated with TEO and ASP to yellowness. However, when antimicrobials were incorporated individually resulted in low values of b∗ , with a tendency to blueness.

Thermogravimetric analysis Thermal degradation and stability of packaging materials are of special concern to the food industry, and these features have been studied using several techniques including the TGA. TGA provides insights of the thermal resistance of packaging materials, for example, temperature range of polymer decomposition, by measuring polymer weight variation depending on temperature and/or time, meanwhile the sample is exposed to progressive increase of temperature (Hernandez-Izquierdo and Krochta 2008; Espitia and others 2014). TGA has been previously used in the characterization of biopolymer and edible films including extruded starch–nanoclay nanocomposites, composites based on a blend of starch/EVOH/glycerol incorporated with coconut fibers

Properties of ac¸a´ı edible films . . . sition (Mangiacapra and others 2006). The high decomposition temperature observed for ac¸a´ı is related to ac¸a´ı fiber. According to Martins and others (2008), cellulose and lignin have decomposition peaks around 340 °C. Ac¸a´ı and ASP were the most thermally stable components. Although ac¸a´ı had the highest thermal decomposition temperature, ASP and pectin powder suffered lower total weight losses. Pure TEO had the lowest thermal decomposition temperature due to its volatile nature. Moreover, ac¸a´ı edible films prepared for each treatment followed similar trend as observed in the thermograms of individual components (Figure 2B). Below 150 °C, all developed ac¸a´ı films presented an initial weight decrease related to water loss. However, ac¸a´ı edible film incorporated with ASP presented notably less

Figure 2–Thermograms of main components used for the production of ac¸a´ı edible films (A) and thermograms of different treatments of ac¸a´ı edible films (B): edible film incorporated with 6% (w/w) ASP; and 6% (w/w) TEO and control film.

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and fish gelatin films (Chiou and others 2007; Chiou and others 2009; Rosa and others 2009). The thermal stability of ac¸a´ı edible films and individual components used in the preparation of the edible films was investigated by TGA. The thermograms (Figure 2A) indicate that each component had an initial weight loss at temperatures ranging from 50 to 100 °C, which corresponds to water loss. After this, a maximum decomposition step was observed at a temperature around 226 °C for pectin powder, 347 °C for ac¸a´ı, 274 °C for pure ASP and 125 °C for pure TEO from the derivative thermogravimetric curves (data not shown). These results are in agreement with Gohil (2011), who reported that maximum weight loss of pectin occurred at 220 °C. Pectin decomposition in the range from 200 to 400 °C was related to degradation from pyrolytic decompo-

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Properties of ac¸a´ı edible films . . . weight loss when compared to control ac¸a´ı film and ac¸a´ı film with TEO. At the end of the analysis, the ac¸a´ı edible film incorporated with 6% (w/w) ASP had the highest remaining weight (22.9%), indicating the highest thermal stability. This is probably due to the presence of fibers from apple peel. Previous studies have shown the relationship between increased thermal stability and fiber content in films. Lu and others (2008) observed that the incorporation of microfibrillated cellulose resulted in slightly increased thermal stability of polyvinyl alcohol composite films. Moreover, Visakh and others (2012) reported that the thermal stability of natural rubber

(latex) nanocomposites improved with increasing content of fiber from waste bamboo cellulose pulp. The control film showed intermediate thermal stability when compared to ASP and TEO films. The slightly higher thermal stability of the control compared to the TEO film resulted from the absence of the highly volatile oil in the polymeric matrix and the presence of ac¸a´ı fibers. The fiber content in ac¸a´ı have been previously reported by Cipriano (2011) as 34 g/100 g of ac¸a´ı, and Martins and others (2008) have reported the presence of rough fiber layers in ac¸a´ı berries microstructure. The lowest

M: Food Microbiology & Safety Figure 3–FESEM photomicrograph of ac¸a´ı edible film (a and b) and neat pectin film (c to e).

Figure 4–FESEM photomicrograph of ac¸a´ı edible film incorporated with 6% (w/w) of ASP (a and b) and 6% (w/w) of TEO (c and d).

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Properties of ac¸a´ı edible films . . . thermal stability of ac¸a´ı film with TEO is probably due to the volatile nature of the essential oil. Similar result was observed by Tongnuanchan and others (2012), who showed that fish skin gelatin film incorporated with citrus essential oil had lower thermal degradation temperature and higher weight loss when compared to the control film.

Table 4–Antimicrobial activity of ac¸a´ı edible films incorporated with ASP and TEO.

ing structures with the presence of clusters, probably as a result of the fleshy ac¸a´ı skin and fiber (Figure 3A). The protrusions were more evident in the cross section of the film (Figure 3B). The images showed that the protrusions were large, thickened portions of the film. The formation of the thickened areas was due to ac¸a´ı rather than pectin. This was further verified by the elaboration of an extra film constituted by neat pectin, which was more homogenous and lacked the thickened areas found in the ac¸a´ı films (Figure 3C and D). The photomicrograph of the ac¸a´ı edible film containing 6% (w/w) ASP had a smoother surface than the control film (Figure 4A). However, there are pits in the inner structure of the edible film (Figure 4B). The surface of the ac¸a´ı edible film containing 6% (w/w) TEO showed crater-like pits on its surface (Figure 4C) and linear structures in the cross-section (Figure 4D). The presence of crater-like pits is probably resulted from agglomeration of ASP or due to remaining droplets of TEO in the microstructure of ac¸a´ı films. Similar results were observed by Bonilla and others (2012), who reported that chitosan-based films incorporated with basil and TEO presented void in the cross-section of films probably due to oil droplets remaining in film microstructure.

ASP, apple skin polyphenol; TEO, thyme essential oil. ∗ Data reported are mean values ± standard deviation, mean values followed by the different letters are significantly different at P < 0.05.

Control 3% ASP 3% TEO 3% ASP+3%TEO 6% ASP Field emission scanning electron microscopy (FESEM) The control film showed a heterogeneous surface and protrud- 6% TEO

Antimicrobial activity Ac¸a´ı edible films incorporated with ASP and TEO showed antimicrobial activity while control film showed no activity against L. monocytogenes. Moreover, ac¸a´ı edible film incorporated with 6% (w/w) TEO presented the highest inhibition zone (Figure 5). Statistical analysis indicated that antimicrobial activity was signif-

Inhibition zone (mm2 )∗ 44.2 135.1 115.4 108.5 844.4

0.0 ± 4.7b ± 21.2b ± 8.8b ± 21.6b ± 120.0a

icantly different (P < 0.05) among ac¸a´ı edible films incorporated with the antimicrobial compounds, with 6% TEO being more effective against L. monocytogenes (Table 4). The high antimicrobial activity of plant extracts has been recognized for centuries, resulting in their use as natural medicine. Recently, the antimicrobial activity of TEO has been widely studied, with thymol and carvacrol being the 2 major flavor components in the oil responsible for its antimicrobial activity (Burt 2004; Tajkarimi and others 2010). Additionally, polyphenols are natural compounds with recognized antimicrobial, antioxidative, and antiinflammatory properties. These molecules are abundant in apples and are especially concentrated in the peel (Pastene and others 2009). ASP constitute ࣙ80% polyphenols, 5% to 8% phloridzin, 15% to 18% chorogenic acid, and ࣙ4% proanthocyanidins B2 (data provided by supplier). Main polyphenols include procyanidin, catechin, epicatechin, chlorogenic acid, and phlorizin; however, their fractions differ with variety, ripening degree, storage, processing, and the degree to which the polyphenols are standardized (Boyer and Liu 2004; Du and others 2011). Although ac¸a´ı edible film incorporated with 6% (w/w) TEO presented the highest antimicrobial activity, both antimicrobials are necessary in the films in order to obtain ac¸a´ı edible films with suitable physical-mechanical properties.

Figure 5–Antimicrobial activity against L. monocytogenes of edible films incorporated with 3% (w/w) ASP (A), 3% (w/w) TEO (B), 3% (w/w) ASP + 3% (w/w) TEO (C), 6% (w/w) ASP (D), 6% (w/w) TEO (E), and control film (F). Vol. 00, Nr. 0, 2014 r Journal of Food Science M7

M: Food Microbiology & Safety

Treatment

Properties of ac¸a´ı edible films . . .

Conclusions A new edible film based on ac¸a´ı berries and pectin was successfully developed. The study of physical-mechanical properties of edible films is essential for industrial application when working with new polymer matrix, such as ac¸a´ı. However, few works have combined studies of antimicrobial activity with the effects of antimicrobials on packaging physical-mechanical properties. This work showed main features of ac¸a´ı edible films alone and when incorporated with TEO and ASP by means of mechanical properties, WVP, color analysis, TGA assessment, and FESEM observation. Finally, the study of physical-mechanical properties together with antimicrobial activity of developed ac¸a´ı edible films indicated that both compounds are necessary in the films in order to obtain ac¸a´ı edible films with suitable physical properties and antimicrobial activity for the control of L. monocytogenes.

Acknowledgments The authors thank Mr. Carl Olsen (PFR Unit, USDA/ARS) for providing technical support. Author Espitia gratefully acknowledges financial support from Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES – Brazil) through PEC-PG agreement.

Author Contributions

M: Food Microbiology & Safety

P. J. P. Espitia, R. J. Avena-Bustillos, and W-X Du designed the study and drafted the manuscript. P. J. P. Espitia, R. J. Avena-Bustillos, W-X Du, T. G. Williams, D. Wood, and B-S Chiou collected test data and interpreted the results. T. H. McHugh and N. F. F. Soares supervised the study and provided technical/financial support.

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Physical and antibacterial properties of açaí edible films formulated with thyme essential oil and apple skin polyphenols.

Thyme essential oil (TEO) and apple skin polyphenols (ASP) are natural compounds considered as generally recognized as safe by FDA, with biological ef...
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