Chitosan-Oregano Essential Oil Blends Use as Antimicrobial Packaging Material

Chitosan-Oregano Essential Oil Blends Use as Antimicrobial Packaging Material

Chapter 44 Chitosan-Oregano Essential Oil Blends Use as Antimicrobial Packaging Material M.Z. Elsabee⁎, R.E. Morsi† and M. Fathy† Cairo University, C...

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Chapter 44

Chitosan-Oregano Essential Oil Blends Use as Antimicrobial Packaging Material M.Z. Elsabee⁎, R.E. Morsi† and M. Fathy† Cairo University, Cairo, Egypt, †Egyptian Petroleum Research Institute, Cairo, Egypt

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44.1 INTRODUCTION In our quest for a perfect package with an eye on the environmental point of view, edible films come to the forefront. In the long term, edible films tend to have the potential to replace conventional synthetic oxygen and gas barriers, which are currently in use. Why do we need edible films? Most food consumed comes directly from nature, where many of them can be eaten immediately as we take them from the tree, vine, or ground. However, with increased transportation distribution systems, storage needs, and the advent of ever larger supermarkets and warehouse stores, foods are not only consumed close to processing facilities. It takes considerable time for a food product to reach the table of the consumer. During the time-consuming steps involved in handling, storage, and transportation, products start to dehydrate, deteriorate, and lose appearance, flavor, and nutritional value. If no special protection is provided, damage can occur within hours or days, even if this damage is not immediately visible (Embuscado and Huber, 2009). Natural, nontoxic, biodegradable polymers have been used in a variety of applications in the food industry as a new edible packaging material to control food quality; it can form transparent films, which may find application. During the past several decades, chitosan has received increased attention for its commercial applications in the biomedical, food, and chemical industries (Muzzarelli, 1977; Knorr, 1985). However, despite the extensive use of chitosan as edible films for coating, it still suffers from high water vapor permeation (WVP), which lowers its protective action. Therefore, the addition of oils may increase its hydrophobicity and improve its WVP. An excellent candidate is oregano essential oil (OEO), which has increasingly gained the interest of researchers as a potential “natural preservative” used in food processing. OEOs showed a high level of antimicrobial activity against different strains of Gram-positive and Gram-negative bacteria (Sivropoulou et al., 1996). In addition, OEOs have medical properties that help to improve human health. It has been reported to have several pharmaceutical uses such as an antibacterial, fungicidal, antiviral, biocidal, antioxidant (Bernath, 1996), antimicrobial (Şahin et al., 2004), antihyperglycemic (Lemhadri et al., 2004), and antithrombic activities, and possess activity against cancer (Goun et al., 2002). It also has nematicidal (Bernath, 1996) and insecticidal activities (Traboulsi et al., 2002). In some countries such as Jordan, Origanum syriacum leaves are used to cure eye ailments, burns, and stomach troubles, and the seeds are used as sedative (Oran and Al-Eisawi, 1998). Multiple research in recent years focused on chitosan and OEO antimicrobial activity and their use in food packaging as edible films.

44.2  EDIBLE FILMS Edible film as such is usually transparent film made from biobased material. It has the potential to coat food surfaces, separate different components, or serve the purpose of a casing, pouch, or wrap. It can effectively safeguard product quality by the formation of oxygen, oil, or moisture barriers; carry functional ingredients such as antioxidants or antimicrobials; and improve appearance, structure, and handling. Edible films or coatings have been investigated for their abilities to retard moisture, oxygen, aromas, and solute transports (Gennadios and Weller, 1990). Edible and biodegradable films are not always meant to totally replace the synthetic packaging films (Krochta and de Mulder-Johnston, 1997), though it is one of the most effective methods of maintaining food quality. Usually film-forming substances are based on proteins, polysaccharides, lipids, and resins or a combination of these (Donhowe and Fennema, 1994). Edible packaging must have some functional and specific properties. They have to be selective toward mass transfers. Selective properties of edible films and coatings are responsible for retarding organic vapors (aromas, solvents), water Antimicrobial Food Packaging. http://dx.doi.org/10.1016/B978-0-12-800723-5.00044-9 © 2016 Elsevier Inc. All rights reserved.

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v­ apor, solute (lipids, salts, additives, pigments), and gases (oxygen, carbon dioxide, nitrogen). The water barrier is desirable to retard the surface dehydration of fresh (meat, fruits, and vegetables) or frozen products. The control of gas exchanges, particularly of oxygen, allows better control of the ripening of fruits or to significantly reduce the oxidation of oxygensensitive foods and the rancidity of polyunsaturated fats. Organic vapor transfers have to be minimized to retain aroma compounds in the product during storage or to prevent solvent penetration in foods, which induces toxicity or off flavoring. Further, the effect of ultraviolet light that involves radical air reactions in foods could also be significantly reduced. In the latter case, the efficiency of the film to prevent light effect can be improved by the addition of pigments or light absorbers. Edible packaging can improve mechanical properties of food to facilitate handling and carriage. Functional efficiency strongly depends on the nature of components and film composition and structure. The choice of film-forming substance and/or active additive is made based on the objective, the nature of the food product and/or the application method. A number of polymer films are already in use for food packaging, such as polyethylene (PE), polyvinyl alcohol (PVA), polyvinyl chloride (PVC), polylactic acid (PLA), and others. To acquire antimicrobial properties, active agents have to be added either by simply spreading antimicrobial solutions onto the polymer surface or by more sophisticated means such as combining the antimicrobials with binders such as cellulose or an acrylic copolymer. These agents can also be covalently attached, with natural and synthetic cross-linkers like genipin, glutaraldehyde, and formaldehyde. For some films, plasticizers were considered necessary, and for this purpose glycerol, propylene glycol (PG), or polyethylene glycol (PEG) are added. As compared with other biobased food packaging materials, chitosan has the advantage of being able to incorporate functional substances such as minerals or vitamins and possesses antibacterial activity (Möller et al., 2004; Jeon et al., 2002; Chen et al., 2002). In view of these qualities, chitosan films have been used as a packaging material for the quality preservation of a variety of food (Tsai and Su, 1999; Suyatma et al., 2005; Park and Zhao, 2004).

44.3 CHITOSAN According to the “Google scholar” database, 483,000 references reporting chitosan and its properties and applications have been published as of April 2015. Chitosan is a modified natural carbohydrate polymer derived by deacetylation of chitin [poly-β-(1 → 4)-N-acetyl-d-glucosamine], a major component of the shells of crustaceans such as crabs, shrimp, and crawfish and the second-most abundant natural biopolymer after cellulose (Abdou et al., 2008; No et al., 2007). Figure 1 shows the structure of chitosan and appearance of its film.

FIGURE 1  Structure of chitosan and a picture of its film, cast from a 4% solution and thicknesses of 20 mils (0.02 in. or 508 mm) and 40 mils (0.04 in. or 1016 mm) (Embuscado and Huber, 2009).

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During the past several decades, chitosan has received increased attention for its commercial applications in the biomedical, food, and chemical industries (Muzzarelli, 1977; Knorr, 1985). Chitosan is now widely produced commercially from crab and shrimp shell wastes with different deacetylation grades and molecular weights (thus, viscosities of chitosan solutions) and, hence, different functional properties (No et al., 2007; Cho et al., 1998).

44.3.1  Chitosan Antimicrobial and Film-Forming Properties The antimicrobial activity of chitosan has been considered in a wide variety of fungi and bacteria (Hernández-Lauzardo et al., 2008; İkinci et al., 2002; No et al., 2002). Taking into account these data, the ability of chitosan to extend the storage life of fruits and vegetables has been demonstrated (Campaniello et al., 2008; Fernandez-Saiz et al., 2009; Sebti et al., 2005). It has been postulated that the antimicrobial action of chitosan occurs as a result of several mechanisms. The interaction between positively charged chitosan molecules and negatively charged microbial cell membranes could lead to their disruption and leakage of essential intracellular constituents (İkinci et al., 2002; Helander et al., 2001; Kong et al., 2008). Chitosan has been extensively studied for coating applications because of its film-forming properties. Chitosan films do not dissolve in water, and they exhibit good wet tensile strength. The methods of preparation of chitosan gels can be broadly divided into four groups: solvent evaporation, neutralization, cross-linking, and ionotropic gelation. The first of the methods is mainly used for the preparation of films. A solution of chitosan in organic acid is cast onto a plate and allowed to dry, if possible, at elevated temperature. Upon drying, the film is usually neutralized with a dilute NaOH solution and crosslinked to avoid disintegration in acidic solutions (Krajewska, 2004). Film formation also may be followed by precipitation after increasing the pH of a chitosan solution with an alkali. Another possibility is the use of a cross-linking agent such as glutaraldehyde to facilitate gel formation. Several studies have reported that chitosan edible films have greater antimicrobial effect for bacteria than for molds (No et al., 2007; Ponce et al., 2008; Zivanovic et al., 2005). Chitosan films are able to disperse homogeneously different compounds, so diffusion is relatively fast (Rhim et al., 2006). Park et al. (2005) reported inhibition of molds by chitosan films applied to strawberries, due to changes of the internal atmosphere inside the fruit. Sebti et al. (2005) reported inhibition of Aspergillus niger with chitosan films placed on plates of agar along with the mold; because no drying film was required, chitosan acted freely inhibiting the microorganism. Nevertheless, the use of plasticizers like Tween 20, which act as emulsifiers, favors the solubility in water and reduces the interaction with chitosan molecules (Suppakul et al., 2003). Recently, a chitosan-starch film has been prepared using microwave treatment that may find potential application in food packaging (Tripathi et al., 2008a). Other varieties of chitosan-based antimicrobial films have recently been well documented (Tripathi et al., 2008b).

44.3.2  Factors Affecting the Antimicrobial Activity of Chitosan There are various factors, such as intrinsic and extrinsic, that affect the antimicrobial activity of chitosan. It has been demonstrated that lower-molecular-weight chitosan (of less than 10 kDa) have greater antimicrobial activity than native chitosan (Uchida, 1989). Furthermore, a degree of polymerization of at least seven is required; lower-molecular-weight fractions have little or no activity (Uchida, 1989; Ralston et al., 1964). Highly deacetylated chitosan are more antimicrobial than those with a higher proportion of acetylated amino groups, due to increased solubility and higher charge density (Sekiguchi et al., 1993). Surrounding matrix is the greatest single influence on antimicrobial activity. Being cationic, chitosan has the potential to bind to many food components such as alginates, pectins, proteins, and inorganic polyelectrolytes such as polyphosphate (Kubota and Kikuchi, 2004). Solubility can be decreased by using high concentrations of low-molecular-weight electrolytes such as sodium halides, sodium phosphate, and organic anions (Roberts, 1992). A greater emphasis on safety features associated with the addition of antimicrobial agents is gaining ground as one of the emerging areas for development in packaging technology, and it is likely to play a major role in the next generation of “active” packaging systems (Brody, 2001). Active packaging is a packaging system possessing attributes beyond basic barrier properties that are achieved by adding active ingredients in the packaging system and/or using functionally active polymers. For that, chitosan/essential oil films rise as an amazing option.

44.4  CHITOSAN AND ESSENTIAL OILS Antimicrobial activity of essential oils (EOs) was recognized long ago, but their application as natural antimicrobials has recently received increased attention in the food industry (Draughon, 2004). The specific advantage of EOs appears to be in the synergistic effects of their compounds as evidenced in greater activity when applied as a natural EO compared with

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summary of the effects of the individual substances (Beckstrom-Sternberg and Duke, 1994). EOs are classified as generally recognized as safe by the U.S. Food and Drug Administration. In the EU EOs are considered as safe food additives at concentrations <2 mg/kg b.w./day (ESFA, 2010). The incorporation of EO in chitosan films may not only enhance the film's antimicrobial properties but also reduce water vapor permeability and slow lipid oxidation of the product on which the film is applied (Yanishlieva et al., 1999; Botsoglou et al., 2002). Some of the main chemical compounds of EOs include alcohols, aldehydes, esters, ethers, ketones, phenols, and terpenes. Although each type of EO consists of more than 100 compounds, usually just a few of them dominate. Thus, anethole comprises more than 90% of anise EO (Soliman and Badeaa, 2002); methyl chavicol and linalool are the principal constituents of basil EO; α-pinene, borneol, linalool, and cineole dominate in coriander EO (Delaquis et al., 2002); and carvacrol and thymol are the principal constituents of oregano EO (Lambert et al., 2001). Generally, phenolics and terpenes are major contributors to antimicrobial effects of EOs. A number of reports established a good correlation between strong antibacterial activity and the presence of monoterpenes, eugenol, cinnamaldehyde, carvacrol, and thymol in EOs (Suresh et al., 1992; Kim et al., 1995a; Lis-Balchin and Deans, 1997; Ouattara et al., 1997). Vargas et al. (2006) reported data on the effect of unsaturated oils, such as olive oil, on the properties of chitosan-based films and the interactions between chitosan and olive oil or chitosan and olive oil components, and on the improved physicochemical quality of strawberries when coated with the edible coatings. Muzzarelli et al. (2000) reported that the capacity of chitosan to alter the composition of olive oil is due to the percolation of the oil through a bed of chitosan powder. Google scholar cites 3400 works related to chitosan EOs used in packaging and food applications due to antimicrobial properties. Georgantelis et al. (2007) studied the effect of rosemary extract, α-tocopherol and chitosan (10 g/kg) on microbial parameters and lipid oxidation of fresh pork sausages. Shelf life of samples containing chitosan was almost doubled compared to the remaining samples, and the best antimicrobial and antioxidant effects were obtained from the combination of chitosan with rosemary extract. Kanatt et al. (2008) studied the preservative effect of mint and chitosan (0.1%) mixture on pork cocktail salami. Results showed that addition of chitosan to mint extract did not interfere with the antioxidant activity of mint and that the shelf life of pork cocktail salami, as determined by bacterial count and oxidative rancidity, was enhanced in chitosan-mint treated samples stored at 0-3 °C.

44.5  OREGANO ESSENTIAL OIL Among the EOs from various aromatic plants, OEO has increasingly gained the interest of researchers as a potential “natural” antimicrobial to be used in food processing. The disinfectant and antimicrobial properties of OEO were first recognized in ancient Greece, where they were often used for treating bacterial infections on the skin or in wounds, and it was also employed to protect food from bacteria. It is a plant native to higher altitudes and normally grows in the mountains, which is how it got the name “Oregano,” which means “Delight of the Mountains.” It is extracted through steam distillation of fresh oregano leaves, which bear the scientific name Origanum vulgare. Most reports about oregano are related to Origanum vulgare, which is found on the Mediterranean coast. The spice known as Mexican oregano belongs to the Verbenaceae family and is known as Lippia berlandieri Schauer or Lippia graveolens HBK. It has higher EO content than European oregano; thymol content is higher and, therefore, has a stronger odor and slightly different antimicrobial activities (Arcila-Lozano et al., 2004).

44.5.1  Antimicrobial Activity of OEO Different hypotheses were suggested about the antimicrobial mechanism of the phenolic compounds or the EO components on microorganisms. Ultee et al. (1998) found that both carvacrol and cymene caused destabilization of the membrane of bacteria cells (Bacillus cereus) and decreased membrane potential. So, the most probable mechanism for the antimicrobial activity of carvacrol could be through the decrease in pH gradient across the cytoplasmic membrane due to hydroxyl groups and delocalized electrons. So if the metabolism is aerobic, this will reduce a proton motive force and effect on ATP synthesis. The absence of ATP inside the microorganism will lead to a change in the cell process and may cause cell death. In addition, researchers found that increases in the membrane fluidity and leakage of protons and ions were observed when bacteria were exposed to antimicrobial compounds (Sikkema et al., 1995; Heipieper et al., 1996). However, certain concentrations of these compounds are needed to cause membrane leakage (Mendoza-YEPES et al., 1997). The mode of action of these phenolic compounds still needs more investigation to understand their antimicrobial effects. Recently, the food industry has been showing a great interest in using plant-derived extract as antimicrobial compounds in the food to suppress the growth of foodborne pathogens. Carvacrol and thymol are the main components of the OEO responsible for their antimicrobial activity (Kim et al., 1995b; Skandamis and Nychas, 2000). The inhibition may be related to cell membrane damage, pH homeostasis, and inorganic ions equilibrium. Carvacrol is a major component of EOs that were

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extracted from plant sources such as thyme (Thymus vulgaris) and oregano (Origanum vulgare) (Adam et al., 1998; Burt, 2004). Its percentage in the OEO is highly dependent on the Origanum species, the extraction method, storage conditions, and laboratory method analysis. Carvacrol (2-methyl-5-(1-methlethyl) phenol) is a hydrophobic phenolic compound synthesized from p-cymene and α-terpinene (Poulose and Croteau, 1978). Their antimicrobial activity against several types of pathogens such as B. cereus (Ultee et al., 1998), Escherichia coli O157:H7 (Friedman et al., 2004; Pérez-Conesa et al., 2011), Staphylococcus aureus (Lambert et al., 2001; Knowles et al., 2005), and Salmonella enterica (Friedman et al., 2002; Nazer et al., 2005) are well documented. Fractions of OEO have been demonstrated to have activity against several species of bacteria, such as Salmonella (Helander et al., 1998; Paster et al., 1990) and E. coli O157: H7 (Burt and Reinders, 2003). Seydim and Sarikus (2006) reported that edible whey protein-based films combined with OEO are more effective against E. coli O157:H7, Staphylococcus aureus, Salmonella enteritidis, Listeria monocytogenes, and Lactobacillus plantarum than rosemary and garlic oils. In addition, EOs from different strains of oregano showed different antimicrobial activities. Horošová et al. (2006) found that oregano oils showed strong antibiotic effects on Lactobacilli and E. coli when combined with antibiotics such as apramycin, streptomycin, and neomycin. Chouliara et al. (2007) reported that OEO showed a shelf life extension effect on fresh chicken breast meat when combined with modified atmosphere packaging. With regard to the use of EOs to control Listeria monocytogenes (Elgayyar et al., 2001), an in vitro study showed that among several EOs used, OEO strongly inhibited the growth of Listeria monocytogenes. Tsigarida et al. (2000) added 0.8 ml/100 g of OEO to raw beef inoculated with 3.5 log cfu/g of Listeria monocytogenes and reported an initial reduction in Listeria monocytogenes by ca.1 log cfu/g. After 8 days of storage, treated samples had Listeria monocytogenes counts lower by ca. 1.5 log cfu/g compared to controls. On the other hand, Firouzi et al. (2007), in an in vitro study, investigated the effect of plant EOs (oregano and nutmeg at 1e3 ml/g), on the survival of Listeria monocytogenes and Yersinia enterocolitica (inoculum, 6-7 log cfu/g) in ready-to-eat barbecued chicken. Results showed that the population of both bacteria did not differ significantly (for various oregano and nutmeg EO concentrations and storage temperatures) when compared to control samples after 3 days.

44.5.2  Chitosan Films with OEO A simple method for preparation of chitosan films enriched with oregano is by using chitosan stock solution with 1.5% (w/w) chitosan in 1.5% (v/v) acetic acid. The solution is stirred overnight at room temperature, filtered, and sterilized at 121 °C for 15 min. The EO is mixed with Tween 20 and then added to the chitosan stock solution. The final film-forming solutions are homogenized under aseptic conditions at 21,600 rpm for 1 min and poured into sterile petri dishes. The films were dried under 5 psi vacuum at 30 °C (Chi, 2006). Heng Yin et al. studied the effects of chitosan on the content of secondary polyphenols in Greek oregano. Four chitosan treatments (50, 200, 500, and 1000 ppm) were used in a field experiment. The 50 and 200 ppm treatments of chitosan upregulated the content of polyphenols significantly (38% and 29%, respectively). The chitosan treatments induced H2O2 generation in Greek oregano leaves (Yin et al., 2012). Petrou et al. investigated the antimicrobial activity of chitosan, oregano, and their combination on the shelf life of modified atmosphere packaged chicken breast meat stored at 4 °C. Treatments were M (control samples stored under modified atmosphere packaging MAP), MO (samples treated with oregano oil 0.25% (v/w), stored under MAP), MCH (samples treated with chitosan 1.5% (w/v), stored under MAP), and MCHO (treated with chitosan 1.5% (w/v) and oregano oil 0.25% (v/w), stored under MAP). As shown in Figure 2 treatment of MCHO significantly affected mesophilic total plate counts (TPC), lactic acid bacteria (LAB), Brochothrix thermosphacta, Enterobacteriaceae, Pseudomonas spp., and yeasts-molds during the storage period. Lipid oxidation (as determined by MDA values) of control and treated chicken samples was in general low and below 0.5 mg MDA/kg, showing no oxidative rancidity during the storage period. The addition of chitosan to the chicken samples produced higher (p < 0.05) lightness (L*) values as compared to the control samples. The results of this study indicate that the shelf life of chicken fillets can be extended using either oregano oil singly and/or chitosan by approximately 6 (MO) and >15 (MCH and MCHO) days. Interestingly, chitosan (MCH) or chitosan-oregano (MCHO) treated chicken samples were sensor ally acceptable during the entire refrigerated storage period of 21 days. It is noteworthy that the presence of chitosan in MCH and MCHO samples did not negatively influence the taste of chicken samples, with MCH samples receiving a higher score (compared to MCHO), probably as a result of a distinct and “spicy” lemon taste of chitosan that was well received by the panelists. Based primarily on sensory data (taste attribute), MCH and MO treatments extended the shelf life of chicken fillets by 6 days, whereas MCHO treatment resulted in a product with a shelf life of 14 days (Petrou et al., 2012).

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FIGURE 2  Changes in mesophilic TPC (a) LAB, (b) B. thermosphacta, (c) Enterobacteriaceae, (d) Pseudomonas spp., (e) and yeasts/molds, (f) of chicken breast meat during refrigerated storage under MAP (M, ■), under MAP with oregano oil (0.25%, v/w; MO treatment, ▲), under MAP with chitosan (1.5%, w/v; MCH treatment, ♦) and under MAP with oregano oil (0.25%, v/w) and chitosan (1.5%, w/v; MCHO treatment, ●). Each point is the mean of three samples taken from two replicate experiments (n = 3 × 2 = 6). Error bars show SD (Petrou et al., 2012).

Nevena Krkić studied the effect of a chitosan coating with oregano (Origanum vulgare) on lipid oxidation of dry fermented sausage (Petrovská klobása). Fatty acid profile, aldehyde contents, and sensory analysis of odor and flavor were determined after drying and during 7 months of storage. Between coated and control sausage, a difference was observed after 2 months storage in fatty acid profiles (myristic, oleic, and linoleic acids), but after 7 months of storage there was no difference. A decrease in polyunsaturated acid content was observed (from 17.25% to 15.70%), as well as an increase in total aldehydes (from 4.54 to 31.80 μg/g), due to lipid oxidation during storage. After 7 months of storage, the content of most aldehydes was significantly lower in coated sausage than in the control. Sensory characteristics of odor and flavor were better for coated sausage after 7 months of storage. Results suggest that chitosan-oregano coating can be successfully applied to protect dry fermented sausages from lipid oxidation (Krkić et al., 2013). Pelissari et al. studied the physicochemical and antimicrobial properties of starch-chitosan films incorporated with OEO against B. cereus, E. coli, Salmonella enteritidis, and Staphylococcus aureus that were determined by the disk inhibition

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FIGURE 3  Inhibition zones for (a) cassava starch film, (b) cassava starch-chitosan film, and (c–f) cassava starch-chitosan films incorporated with 1% oregano against the microorganisms (c) S. enteritidis, (d) E. coli, (e) B. cereus, and (f) S. aureus (because in (a) and (b) no inhibition zones were observed, only one representative plate for each case is shown) (Pelissari et al., 2009).

zone method. Films added with oregano effectively inhibited the four microorganisms tested and demonstrated improved barrier properties with the formation of more flexible films as seen in Figure 3. The tensile strength of the biofilms was affected by the addition of oregano. The presence of oregano caused the reduction of the tensile strength in the films, which was most likely due to plasticizing capacity. The addition of oregano also significantly affected elongation at break. Concentrations of 0.1% and 0.5% OEO increased elongation at break from 27.18% to 40.73%, respectively, reaching a maximum of 48.40% for 1% oregano. The addition of chitosan and oregano led to a significant reduction of Young's modulus (p < 0.05) and, therefore, the formation of less-rigid films. The film produced with only starch and glycerol presented a higher Young's modulus, 140.36 MPa, which is related to the effect of extrusion conditions (high temperature, pressure, and shearing) in the starch, allowing for the approximation and interaction of the chains and favoring the formation of a denser and more rigid matrix. The WVP ratio environment of the films decreased significantly (p < 0.05) with the addition of chitosan. The cassava starch film (control) had the highest WVP value, 1.39 × 10−10 g/Pa m s, which may be attributed to the larger number of free hydroxyl groups and, consequently, the increased interaction with water, favoring permeability. However, the addition of chitosan resulted in an increased interaction with starch, due to the formation of hydrogen bonds between the NH2 present in chitosan and the OH− of cassava starch, reducing the availability of the hydrophilic groups and decreasing the WVP to 1.00 × 10−10 g/Pa m s. WVP also decreased significantly (p < 0.05) with an increase in the concentration of the WVP for the maximum concentration of oregano (1.0%) was 0.62 × 10−10 g/Pa m s (Pelissari et al., 2009). Raúl Avila-Sosa et al. studied the inhibition by vapor contact of A. niger and Penicillium digitatum by selected concentrations of Mexican oregano (Lippia berlandieri Schauer), cinnamon (Cinnamomum verum) or lemongrass (Cymbopogon citratus) EOs added to amaranth, chitosan, or starch edible films. Amaranth, chitosan, and starch edible films were formulated with EO concentrations of 0.00%, 0.25%, 0.50%, 0.75%, 1.00%, 2.00%, or 4.00%. Antifungal activity was evaluated by determining the mold radial growth on agar media as seen in Figure 4 inoculated with A. niger and P. digitatum after exposure to vapors arising from EOs added to amaranth, chitosan, or starch films using the inverted lid technique. Chitosan films exhibited better antifungal effectiveness (inhibition of A. niger with 0.25% of Mexican oregano and cinnamon EO; inhibition of P. digitatum with 0.50% EOs) than amaranth films (2.00% and 4.00% of cinnamon and Mexican oregano EO were needed to inhibit the studied molds, respectively) (Avila-Sosa et al., 2012). Avila-Sosa et al. studied the inhibition of A. niger and Penicillium spp. by selected concentrations of Mexican oregano (Lippia berlandieri Schauer) EO added to amaranth, chitosan, or starch edible films. He formulated amaranth, chitosan, and starch edible films with EO concentrations of 0.00%, 0.25%, 0.50%, 0.75%, 1%, 2%, and 4%. Antifungal effectiveness of edible films was starch > chitosan > amaranth. In starch edible films, both studied molds were inhibited with 0.50% of EO (Avila-Sosa et al., 2010). Bao et al. (2013) investigated the antimicrobial activity and scavenging of O2−, ·OH, DPPH radical and reducing power of chitosan-PE bilayer films incorporated with (100 μl/100 ml OEO). The results showed that the scavenging effect of film solution on O2−, ·OH, and DPPH was 80.87%, 85.56%, and 87.24%, respectively. The reducing power of film increased with the improvement of OEO. The antibacterial activity against E. coli and B. subtilis increased from 80.77% to 96.15%

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FIGURE 4  Effect of chitosan edible films added with EOs at selected concentrations (0.00% (▲), cinnamon 0.25% (■), Mexican oregano 0.25% (●), or lemongrass 4.00% (□)) on A. niger (a) and P. digitatum (b) growth. Dt is the average colony diameter at time t, and Do is the average colony diameter at initial time (Avila-Sosa et al., 2012).

and from 93.02% to 98.10%, respectively. The preservation experiments showed that chitosan-PE film incorporated with OEO could extend the shelf life of pork to 8-10 days at 4 °C. In another study, Zivanovic et al. (2005) incorporated EOs (oregano, anise, basil, and coriander) in chitosan films to enhance the antimicrobial properties of the film against two pathogens (Listeria monocytogenes and E. coli O157:H7) inoculated on bologna slices. Pure chitosan films reduced Listeria monocytogenes by 2 logs. There are two possible reasons for these results. First, the inoculum in this experiment was 106 CFU per petri dish, whereas others have used much lower inoculum (<102 CFU/petri dish) for similar experiments (Coma et al., 2002). Thus, the high number of bacteria may exceed the inhibition activity of chitosan. Another explanation may be in the fact that chitosan must be dissolved to act as an antimicrobial. It is possible that chitosan molecules were tightly bound within the film, which prevented expression of the antimicrobial action. Inhibition of Listeria monocytogenes and E. coli O157:H7 with chitosan films enriched with OEO is seen in Figure 5. Films with 1% and 2% oregano EO decreased the numbers of Listeria monocytogenes by 3.6-4 logs and E. coli by 3 logs. The inhibition effects of oregano EO incorporated into the chitosan films were lower than those of pure EO. Similar to the results with the pure EO, oregano EO incorporated into the chitosan films, regardless of the concentration applied, exhibited the strongest inhibition of tested pathogens compared with other EO. Oil of anise, basil, and coriander had weak inhibition

Width of inhibition zone (mm)

14 12

L. monocytogenes E. coli 0157:H7

10 8 6 4 2 0 0

1 2 3 Concentration of oregano essential oil (% in FFS)

4

FIGURE 5  Inhibition of Listeria monocytogenes and E. coli O157:H7 with chitosan films enriched with OEO. Plates were inoculated with 106 ­colony-forming units (CFU)/plate and incubated for 48 h at 35 °C. The initial disk diameter was 6.6 mm, and the inside diameter of petri dish was 84 mm (Zivanovic et al., 2005).

Chitosan-Oregano Essential Oil Blends Chapter | 44   547

of both bacteria study (0.33 and 0.81 mm, 2.19 and 4.39 mm, 1.26 and 0.77 mm for chitosan films with 4% of anise, basil, and coriander EO against Listeria monocytogenes and E. coli, respectively). The possible reason for the decrease in activity of the EO incorporated in the chitosan films compared with activity of pure EO may be due to potential partial loss of highly volatile compounds of the EO during film preparation. The other reason may be due to slower/controlled release of active compounds from the chitosan film than from cellulose filter paper. Similar to the effects of the pure oil, Listeria monocytogenes appeared to be more sensitive to oregano films than E. coli O157:H7. The intensity of antilisterial activity was in the following order: oregano > coriander > basil > anise. Even though experimental conditions in the aforementioned study are drastically different than those used in the present one, it is clear that in both cases chitosan in combination with OEO exhibits an additive/synergistic antilisterial effect. Chitosan films made from film-forming solution with no EO were transparent and colorless. Incorporation of the emulsifier and EO resulted in thicker and opaque films. Pure chitosan films with 10 mg chitosan/cm2 were 89 m thick, whereas the addition of 2% oregano EO in film-forming solution resulted in more than a threefold increase in film thickness. The films easily absorbed water, and after application on bologna during 5 days at 10 μC, thickness further increased. However, the enlargement was the highest in the films with the addition of emulsifier only and the lowest with addition of EO. These results were expected because EOs, although being complex mixtures, are highly hydrophobic, and the increase in hydrophobicity of the film matrix should reduce water absorption. Similarly, water vapor permeability decreased with increased fraction of the hydrophobic compound. This activity offers the possibility not only to control the antimicrobial efficiency of the films but also to improve the barrier properties of chitosan films by EOs. N,O-carboxymethyl chitosan (NOCC)/OEO coats to chicken fillet samples resulted to the complete inhibition of Listeria monocytogenes after 2 days in the low inoculum experiment and 4 days in the high inoculum experiment. The combination of both antimicrobial agents resulted to a 6-day shelf life extension of chicken fillets. The combination prepared was of 1 g/100 ml N,O-carboxymethyl chitosan and 1% OEO dip. Results showed that total viable count exceeded 7 log cfu/g after day 6 and 10 for control samples and samples treated with OEO, respectively. Samples treated with either NOCC or OEO plus NOCC never reached 7 log cfu/g throughout storage. NOCC had a substantially stronger antimicrobial effect as compared to OEO. A 1.2 and 2.8 log cfu/g reduction in Listeria monocytogenes (low inoculum) in comparison to control samples was recorded for the OEO- and NOCC-treated samples, respectively, after 14 days of storage. Respective values for the high Listeria inoculum were 1.5 and 3.3 log cfu/g (Khanjari et al., 2013). Hosseini et al. (2013) encapsulated OEO in chitosan nanoparticles by a two-step method, that is, oil-in-water emulsion and ionic gelation of chitosan with sodium tripolyphosphate. In the case of antimicrobials, the nano-level encapsulation can increase the concentration of the bioactive compounds in food areas where microorganisms are preferably located, for example, water-rich phases or liquid-solid interfaces (Weiss et al., 2009). OEO-loaded chitosan particles and OEO-loaded chitosan nanoparticles were prepared according to a method modified from the ones described by Calvo et al. (1997) and Yoksan et al. (2010). Briefly, aqueous and oil phase solutions were produced. Chitosan solution (1% (w/v) was prepared by agitating chitosan in an aqueous acetic acid solution (1% (v/v)) at ambient temperature (23-25 °C) overnight. The mixture was then centrifuged for 30 min at 9000 rpm; the supernatant was removed then and filtered through 1 μm pore size filters. Tween 80 (HLB 15.9, 0.45 g) was then added as a surfactant to the solution (40 ml) and stirred at 45 °C for 2 h to obtain a homogeneous mixture. The obtained nanoparticles exhibited a regular distribution and spherical shape with size range of 40-80 nm as observed by scanning electron microscopy (SEM) in Figure 6 and atomic force microscopy. As determined by thermogravimetric analysis technique, the encapsulation efficiency and loading capacity of OEOloaded chitosan nanoparticles were about 21-47% and 3-8%, respectively, when the initial OEO content was 0.1-0.8 g/g chitosan. In vitro release studies showed an initial burst effect, followed by a slow drug release. The in vitro release profiles of OEO from the nanoparticles, prepared using a different weight ratio of chitosan to OEO, are shown in Figure 7. The amount of OEO released at different times was measured at 275 nm. Drug or oil release from nanoparticles and microparticles takes place by several mechanisms, including surface erosion, disintegration, diffusion, and desorption (Hariharan et al., 2006). The in vitro release profile of OEO from chitosan nanoparticles can be described as a two-step biphasic process, that is, an initial burst release followed by a subsequent slower release. The initial burst release was attributed to the OEO molecules adsorbed on the surface of the particles and oil entrapped near the surface, as the dissolution rate of the polymer near the surface is high, the amount of drug released will be also high (Anitha et al., 2011). Figure 6 shows the release profile as a function of OEO concentration, which was found to be concentration dependent. At low concentration of OEO (0.1 g/g chitosan), burst effect occurred within 3 h, and about 82% encapsulated OEO was released from the nanoparticles. This could be mainly attributed to the particle size of this formulation. Chitosan nanoparticles with smaller particle size would have greater surface-to-volume ratio and thus may result in fast release of OEO adsorbed on the surface. Similar results with an initial release of 85% encapsulated α-tocopherol were reported with chitosan nanoparticles coated with zein and later followed slow release at a constant but different rate (Luo et al., 2011). As OEO

548   Antimicrobial Food Packaging

FIGURE 6  SEM images of (a) chitosan nanoparticles and (b) OEO-loaded chitosan nanoparticles prepared using an initial weight ratio of chitosan to OEO of 1:0.4 (Hosseini et al., 2013).

Cumulative release of OEO (%)

100

80

60

40

20

0

0

24

48

72 Time (h)

96

120

144

FIGURE 7  In vitro release profiles of OEO from chitosan nanoparticles prepared using different weight ratios of chitosan to OEO: (♦) 1:0.1, (■) 1: 0.2, (▲) 1:0.4, and (×) 1:0.8. Values were expressed as mean ± standard deviation (n = 3) (Hosseini et al., 2013).

concentration increased, the burst effect was dramatically alleviated, and the accumulative release after 3 h was reduced from 82% to 12%, as OEO concentration reached 0.8 g/g chitosan. In the second stage, the release rate was relatively slow, or we could say that the release of OEO reached plateau at this stage (Figure 6). This might be due to the diffusion of the OEO dispersed into the polymer matrix as the dominant mechanism. This stage has a slower rate and thus results in nearly no additional release of OEO at this stage. Further release of OEO required the swelling and degradation of the compact chitosan-TPP nanoparticles. Hence, the results indicate that the chitosan-TPP nanosystem is suitable for controlling the release of OEO.

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