ANTIMICROBIAL COATINGS

PRIOR APPLICATION INFORMATION

The present application claims the benefit of US Provisional Application 60/824,479, filed September 5, 2006, US Provisional Patent Application 60/939,698, filed May 23, 2007 and US Provisional Application 60/940,428, filed May 28, 2007.

BACKGROUND OF THE INVENTION

Raw poultry products can serve as a source of human pathogens such as Salmonella and Campylobacter that may cross-contaminate other foods. When appropriate rearing and shipping practices are followed, most poultry contamination by these organisms occurs during or after slaughter and processing (Slader et al., 2002; Zhao et al., 2001). Carcass washing with approved antimicrobials (AMs) has had limited success because many microorganisms are physically hidden in the feather follicles and skin folds which protect them from the action of AMs (Mehyar et al., 2005; Schneider et al., 2002; Wang et al., 1997; Xiong et al., 1998). Furthermore, increased line speed reduces the antimicrobial contact time with target microorganisms, and the moisture on chicken skin surface can act as a diluent, reducing antimicrobial effectiveness (Oyarzabal et al., 2004). An alternative approach to extending the contact time would be increasing the effectiveness of AMs. To obtain improved effectiveness without changing process speeds in the plant, edible gels containing AMs could be sprayed on chicken surfaces. In theory, the agents would gradually diffuse from the gels or coating material into skin irregularities and if applied early (after defeathering), provide increased contact time with target microorganisms and yield improved effectiveness. Most food-related antimicrobial coatings have been tested only for their quantitative antimicrobial effectiveness (Janes et al., 2002; Natrajan and Sheldon, 2000a, b; Siragusa and Dickson, 1992). No report has been found which relates the antimicrobial activity of the coatings to their surface properties or absorption into contaminated foods. Studying these physio-chemical properties will help in determining the minimum quantities of AMs required to eliminate pathogens from foods using methods which have beneficial economic and environmental consequences.

Chicken skin consists of two layers, the upper layer called the epidermis and the lower layer called the dermis (Lucas and Stettenheim, 1972). The epidermis is divided into the Stratum corneum (cuticle) and Stratum germinativum. The cuticle of the epidermis consists of waxy material which covers the skin surface, whereas the lower region is composed of cell layers that can be differentiated to become a part of the cuticle in

response to damage. Scalding at high temperature removes the cuticle layer from the skin which will affect skin adhesiveness characteristics (Lucas and Stettenheim, 1972). Indeed, a thinner cuticle layer increases skin hydrophilicity and makes microbial contamination more likely whereby organisms may be deposited within the skin and its folds (Suderman and Cunningham, 1980). The contact angle of a liquid drop on a smooth surface has been used to characterize the surface energies of solids (Choi and Han, 2002; Han and Krochta, 1999). In this study, this surface chemistry has been used to measure the adhesion force of coatings to the skin. In addition, it is also known that the contact angle of a liquid drop is affected by the extent of roughness of the target surface, and such effects could be substantial on a rough surface like chicken skin. The determination of contact angles can be used to explain solid surface properties in terms of both surface energy and roughness (Han and Krochta, 2001). The dermal layer of chicken skin contains collagen which readily absorbs water from the skin surface and swells, causing changes in skin microtopography (Thomas and McMeekin, 1982). Liquid absorption rate and maximum absorptiveness can be measured to reflect how fast and how much of an applied liquid penetrates and is absorbed by the skin.

Consumer interest in unprocessed foods preserved with natural ingredients has significantly increased recently (Cagri et al., 2004; Debeaufort et al., 1998). Development of edible films and coatings which have comparable properties with synthetic preservative ingredients is an approach taken to satisfy this interest (Mehyar and Han, 2004). Both starch and alginate have been shown to be structurally compatible with alkaline and acidic agents (Siragusa and Dickson, 1992; Ratnayake et al., 2002). The goal of the present work was to model the effectiveness of trisodium phosphate (TSP) and acidified sodium chlorite (ASC) in pea starch (PS) and alginate coatings, when applied to broiler carcasses during processing for their ability to reduce surface contamination by Salmonella. Since current standards require that carcasses should be free of any residual additives before shipping from the processing plant, the effect of these chemical applications on skin pH and persistence of coatings on the chicken skin were also determined, targeting 60 min for completion of carcass chilling and neutralization of the additives.

Hydrogel is a network of hydrophilic polymer chains which are able to hold up water but are kept from dissolution by either physical or chemical cross-links. There has been an increasing interest in physically cross-linked hydrogel, in lieu of chemically cross- linked hydrogel, which may involve the use of toxic agents. Several physical interactions have been exploited in the design of hydrogel, such as electrostatic attraction (Bodmeier and Wang, 1993, J Pharmaceut Sci 82: 191-194; Bodmeier et al., 1989, Pharmaceut Res 6: 413-417; Doria-Serrano et al., 2001 Biomacromolecules 2: 568-574; Grant et al, 1973,

FEBS Lett 32: 195-198; Seely and Hart, 1974, Macromolecules 7: 706-701 ; Ortega and Perez-Mateos, 1998, J Chem Technol Biotechnol 73: 7-12), hydrogen bonding (Durrani and Donald, 1995, Polym Gels Networks 3: 1-27; Goodfellow and Wilson, 1990, Biopolymers 30: 1183-1189; Ring et al., 1987, Carbohydr Res 162: 277-293; Liu and Han, 2005, J Food Sci 70: E31-E36), and antigen-antibody binding (Miyata et al., 1999, Macromolecules 32: 2082-2084). Basically, it is required that polymers possess an abundance of functional groups (e.g. -OH, -COO ^" , -NH, -SH) to achieve inter- and intramolecular interactions in the formation of hydrogel.

As a major storage polysaccharide in plants, starch is a compound of amylose and amylopectin, with its composition depending on the plant origin. Amylose is a nearly linear polymer of D-1 ,4 anhydroglucose units, with molecular weight of 10 ⁵ -10 ⁶ (Durrani and Donald, 1995; Galliard and Bowler, 1987 in Starch: Properties and Potential (Galliard, ed; John Wiley and Sons: New York, p 57-78)). In contrast, amylopectin is a highly branched polymer consisting of short α-1 ,4 chains linked by α-1 ,6 glucosidic branching points occurring every 25-30 glucose units, with molecular weight of 10 ⁷ -10 ⁹ (Durrani and Donald, 1995; Galliard and Bowler, 1987). When heated in water at 60 °C or above, starch granules gelatinize, characterized by granular swelling, amylose exudation and disruption of long-order crystalline structure (Liu, 2005 in Innovations in Food Packaging (J. H. Han ed., Academic Press: New York, p318-337)). Suspension of gelatinized starch starts gelling upon cooling as a result of inter- and intra-moiecular hydrogen bonding of amylose and linear branches on amylopectin (Goodfellow and Wilson, 1990; Liu and Han, 2005). Macroscopically, starch gel is a three-dimensional network constructed mainly by springlike strands of polymeric chains (Ring et al., 1987).

Alginate in a form of free acid or sodium salt is a collective term for a family of polysaccharide prepared mostly from brown algae (Smidsrod and Grasdalen, 1984, Hydrobiologia 116-117: 19-28). Chemically, alginate is a mixture of poly(β-D- mannuronate), poly(α-L-guluronate), and poly(β-D-mannuronate α-L-guluronate), with its exact composition depending on algal source. Similar to starch gel, alginate gel features a 3-D network structure (Ahearne et al., 2005, J R Soc Interface 2: 455-463; Doria-Serrano et al., 2001 ; Decho, 1999, Carbohydr Res 315: 330-333; Walkenstrom et al., 2003, Food Hydrocol 17: 593-603). However, alginate forms hydrogel by polymeric chains interacting with Ca ²⁺ and other divalent and trivalent metal ions (Donati et al., 2005, Biomacromolecules 6: 1031-1040; Rees and Samuel, 1967, J Chem Soc C Organic 22: 2295-2298), according to the so-called "egg-box" model (Grant et al., 1973). As a result of ionic interaction, the presence of di- or multivalent cations enable the formation of junction

zones between helical chains of guluronic blocks, those of mannuronic blocks, and those of mannuronic-guluronic blocks (Donati et al., 2005).

In addition to many other biomedical applications such as enzyme immobilization (Ortega and Perez-Mateos, 1998) and tissue engineering (Ahearne et al., 2005; Li et al., 2005, Biomaterials 26: 3919-3928), hydrogel is useful for drug release (Rajaonarivony et al., 1993, J Pharmaceut Sci 82: 912-917; Bodmeier and Wang, 1993). Drug release from hydrogel occurs mainly due to gel swelling, which can be controlled by the formulation chemistry of polymeric network (e.g., functional groups, degree of cross-linking) and by the environmental conditions (e.g., pH, temperature, ionic strength, etc.) (Peppas et al., 2000, Annu Rev Biomed Eng 2: 9-29). The swelling of hydrogel in water permits the entrapped drug to diffuse throughout the entire network and release from the gel. The release rate is primarily determined the degree of swelling (Prokop et al., 2002, Adv Polym Sci 160: 119-173).

Due to its ability to sustain the release of antimicrobials, hydrogel has become a potent carrier of antimicrobials in the meat and poultry industries (Natrajan and Sheldon, 2000, J Food Prot 63: 1189-1196; Natrajan and Sheldon, 2000, J Food Prot 63: 1268- 1272). Herein, the swelling and rheological properties of starch and alginate hydrogels in physiological saline and the release of antimicrobials from the hydrogels to the saline solution, which simulates the fluidic condition on the surfaces of chicken skin, pork and beef.

Quality of fresh poultry offered at retail depends greatly on the microbiological quality of fresh eviscerated chicken (Mehyar and others 2005). Most research has been concerned with the contamination of chicken carcasses and poultry products by Salmonella or Campylobacter which are predominant pathogens, and Pseudomonas which are the major psychotropic spoilage bacteria of refrigerated poultry products (Smith and others 2005a; Mehyar and others 2005; Uyttendaele and others 2006). A Belgian survey in 2001 , as an example, showed that 18% of chicken fillets and 35% of chicken carcasses were contaminated by Campylobacter, and this number has remained at a high level (Uyttendaele and others 2006). Campylobacter numbers on poultry are much higher than that of Salmonella, which are estimated to be 102 - 107 and 1 - 102 cfu/bird, respectively (Jorgensen and others 2002; Zhao and others 2001). Poultry processing lines operate at high-speed, often processing over 150 bird/min. At this high speed poultry meat is very vulnerable to cross-contamination. Consequently, much effort is spent to maintain good sanitation during processing, and these efforts involve optimization of specific unit operating procedures, and adoption of good manufacturing practice (GMP) and HACCP- based quality systems.

Various processing methods are used to reduce levels of undesired microorganisms on broiler carcasses in poultry processing lines. Among them, one of the important unit processes is washing using an inside-outside bird washer before immersion or air chilling (Smith and others 2005a; 2005b). Recently, immersion at 75 - 80 ⁰ C before cold water immersion chilling (Corry and others 2007), extended immersion time (24 h) in cold chlorine water (Cason and others 2006), and the use of large amounts of cold water during immersion chilling (Northcutt and others 2006) have been tried to reduce poultry carcass contamination. However, hot water immersion and day-long cold immersion, or the use of a large quantity of water are not commercially feasible processes although these approaches reduced the numbers of some pathogens. It appears that after washing followed by chilling, there is no unit process in use which can satisfactorily remove pathogens or spoilage microorganisms from poultry carcasses.

An attractive antimicrobial procedure would be one where a nonthermal treatment was used to reduce the number of microorganisms just prior to or during the packaging process. Such nonthermal treatments may include combinations of modified atmosphere packaging (MAP) with antagonistic cultures, electron beam irradiation, high pressure processing, or antimicrobial packaging/coating (Han 2007). MAP of pre-cooked chicken meats inhibited spoilage microorganisms (i.e., Pseudomonas, yeast and molds) compared to air packaging (Patsias and others 2006). Electron beam treatment also reduced the number of Escherichia coli O157:H7 in chicken meat products and has the potential to control other pathogens (Black and Jaczynski 2006).

Edible coatings are produced from edible biopolymers and food-grade additives. Film-forming biopolymers can be selected from proteins, polysaccharides (carbohydrates and gums), or lipids (Gennadios and others 1997). Various antimicrobial agents may be incorporated into edible coating materials to produce antimicrobial coating systems, as they allow a slow migration of the antimicrobial agents from the coating materials and extend the shelf-life of coated foods. Common edible antimicrobial agents include organic acids (e.g., acetic acid, and fatty acids), phenolics (e.g., benzoic acids and cinnamaldehyde), bacteriocins (e.g., nisin, lacticin and others), enzymes (e.g., lysozyme and glucose oxidase), monoglycerides (e.g., monolaurin and monocaprin), and various plant extracts from herbs and spices (Han 2003; 2005).

A variety of antimicrobial coating systems have been applied to chicken carcasses and poultry meat products. Starch and calcium alginate gels incorporating trisodium phosphate and acidified sodium chlorite, respectively, effectively inhibited an inoculated Salmonella cocktail on chicken wings (Mehyar and others 2007). Nisin was mixed with protein and carbohydrate coating materials and reduced the number of Salmonella and

Listeria on chicken meats (Janes and others 2002; Natrajan and Sheldon 2000a, b). Edible polymers in coating materials carrying active agents increased the viscosity of coating materials. The agents extended the contact time of incorporated agents when placed against chicken surfaces, and consequently improved the antimicrobial efficiency of the coating systems against pathogenic and spoilage microorganisms.

Among available antimicrobial agents, oils of plant or spice extracts are attractive since they are natural ingredients (which require no or a reduced label declaration), are accepted by consumers (Cagri and others 2004; Debeaufort and others 1998; Han 2003, 2005) and they can be extracted easily from herbs, spices and aromatic plants by solvents or steam distillation. Many of these essential oils contain antimicrobial as well as antioxidant activity. Examples include rosemary, clove, thyme, oregano and basil oils, plus horseradish and mustard extracts. They are mostly phenolics or terpenes while the latter two contain isothiocyanates (Burt 2004; Holley and Patel 2005).

Thyme oil mainly contains thymol, p-cymene and carvacrol, which demonstrate antimicrobial and antioxidant activities (Kaloustian and others 2005; Sasso and others 2006; Youdim and others 2002). Thyme oil has been reported to inhibit the growth of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, Penicillium spp. and many other bacteria (Friedman and others 2006; Smith and others 2001 ; Sasso and others 2006; Singh and others 2003; Suhr and Nielsen 2003). The antimicrobial activity of thyme oil was adversely affected by food composition, especially lipid content (Singh and others 2003; Smith and others 2001).

SUMMARY QF THE INVENTION

According to a first aspect of the invention, there is provided an antibacterial coating comprising a hydrophilic polymer and a hydrophilic water soluble antimicrobial.

According to a second aspect of the invention, there is provided a method of protecting a perishable food surface from microbial contamination comprising: providing an antibacterial coating comprising a hydrophilic polymer and a hydrophilic water soluble antimicrobial; and applying the antimicrobial coating to the perishable food surface wherein the concentration of the hydrophilic polymer is such that it forms a solution that is viscous during coating and forms a gel during drying.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Application of coatings to chicken drumettes: A) 3.5 % (w/v) pea starch (PS) containing 10 % (w/v) trisodium phosphate (TSP): B) 1 % (w/v) alginate containing

1200 ppm acidified sodium chlorite (ASC). Solution (a) contained 1 (w/v) % CaCI ₂ plus ASC; solution (b) contained 1 % (w/v) sodium alginate.

Figure 2. Effect of inclusion of commercial AMs in polymeric coatings on survival of inoculated Salmonella on chicken skin during storage at 4 ⁰ C for 5 d. TSP = trisodium phosphate, ASC = acidified sodium chlorite, PS = pea starch. Columns with different letters at the same sampling time are significantly (P ≤ 0.05) different.

Figure 3. Surface pH of chicken drumettes dipped in 10 % (w/v) trisodium phosphate (TSP) and 1200 ppm acidified sodium chlorite (ASC) with and without inclusion in 3.5 % (w/v) pea starch (PS) or 1.0 % (w/v) calcium alginate (Algn), respectively during storage at 4 ⁰ C.

Figure 4. Effect of antimicrobial pea starch (PS+TSP) coating viscosity (prepared with different concentrations of PS) on the initial contact angle of coating drops applied to the chicken skin surface.

Figure 5. Effect of pea starch (PS) concentration change in the antimicrobial pea starch (PS+TSP) coatings on the initial contact angle of the coating drops on the chicken skin surface.

Figure 6 Schematic assembly used for preparing calcium alginate gel

Figure 7 Dimensionless mass of solids (M _s /M _s0 ) in starch gels (a) and alginate gels (b) as a function of time (f) of immersion in saline solution, with fitted curves based on Fikian diffusion

Figure 8 Dimensionless mass of water (MJM^) in starch gels (a) and alginate gels (b) as a function of time (f) of immersion in saline solution, with fitted curves based on Fikian diffusion

Figure 9 Concentration of antimicrobials (C) released from PS+TSP and ALG+ASC gels into the saline solution, as a function of immersion time (t), with fitted curves based on Fikian diffusion

Figure 10 Dimensionless storage moduli (G' IGO) for starch gels (a) and alginate gels (b) as a function of time (t) of immersion in saline solution

Figure 11 Dimensionless solids content (SCISCo) of starch gels (a) and alginate gels (b) as a function of time (t) of immersion in saline solution

Figure 12 Consistency profile of pea starch gels with and without thyme oil at 25 ⁰ C.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the

invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

Described herein is an antibacterial coating comprising a hydrophilic polymer and a hydrophilic water soluble antimicrobial.

In some embodiments, as discussed below, the concentration of the hydrophilic polymer is such that it forms a solution that is viscous during coating and forms a gel during drying.

In a preferred embodiment, the antimicrobial coating comprises 0.1-10% or 0.1-5% hydrophilic polymer and 0.1-25% or 0.5-25% or 1-25% antimicrobial.

The hydrophilic polymer may be selected from the group consisting of microcrystalline cellulose, (pre-)gelatinized starch, modified starch, dextrin, maltodextrin, pectin, iota-carrageenan, lambda-carrageenan, gum arabic, gum acacia, gum ghatti, guar gum, xanthan gum, gellan gum, pullulan and combinations thereof. As discussed below, in a preferred embodiment, the hydrophilic polymer is pea starch.

As will be apparent to one of skill in the art, in the instant invention, the polymer allows for the slow release of antimicrobials (or sanitizers) which in turn extends the effective antimicrobial period. Thus, the polymer provides sustained delivery of antimicrobial agents using gel type coating materials consisting of edible polymers. In a preferred embodiment, the antimicrobial coating is used for covering perishable food surfaces, thereby protecting the foods from contamination by environmental microbial hazards, and also eliminating microorganisms which may have previously existed on the food surfaces.

It is of note that animal carcasses are one example of a perishable food surface. However the antimicrobial coating may be used for any perishable solid foods or any foods which are susceptible to surface contamination during processing through cross- contamination. In addition to meat products, these include any solid foods which are subject to reprocessing or post-processing such as shredding, slicing, cutting, grinding and the like. These include for example but by no means limited to cheeses, fruits, vegetables, and any frozen/refrigerated foods.

In some embodiments, as discussed below, the concentration of the hydrophilic polymer is such that it forms a solution that is viscous during coating and forms a gel during drying.

In a preferred embodiment, the antimicrobial coating comprises 0.1-10% or 0.1-5% hydrophilic polymer and 0.1-25% antimicrobial.

As discussed below, in some embodiments, the antibacterial is preferably thymol, carvacrol, linalool, geraniol, thujanol, terpineol or a combination thereof. As will be appreciated by one of skill in the art, many natural oils are high in thymol and pinene, for example but by no means limited to thyme oil, rosemary oil, clove oil, basil oil, mint oil, Eucalyptus oil, tea tree oil and oregano oil. In the examples discussed below, thyme oil is used but it is to be understood that any suitable source of thymol, carvacrol, linalool, geraniol, thujanol-4, and/or terpineol may be used within the invention. In other embodiments, the antimicrobial is trisodium phosphate (TSP) ₁ acidified sodium chlorite or another such suitable antimicrobial known in the art as discussed herein.

As discussed below, it is believed that the thymol, carvacrol, linalool, geraniol, thujanol, and terpineol in thyme oil enhances the intermolecular interaction of the polymer, for example, high-amylose pea starch, resulting in a film solution which has much higher yield stress.

In the present study, thyme oil was incorporated into a polymer, for example, high- amylose pea starch gel and applied on chicken breast meats pre-inoculated with spoilage or pathogenic microoranisms. The objective was to characterize: (1) the Theological characteristics of the starch-based coating material with and without thyme oil; and (2) the antimicrobial effectiveness of thyme oil in a starch-based coating material against food borne pathogens and spoilage bacteria on chicken meat. The goal of this project was to determine whether the formation of an antimicrobial coating containing thyme oil applied to chicken carcasses would be suitable to reduce the effects of contamination by a highspeed poultry line, enhance the safety of poultry products and extend their shelf-life.

Fresh chickens are processed at plants using high-speed processing lines which are vulnerable to rapid cross-contamination of large amounts of product. Antimicrobial coating on chicken carcasses may reduce the effects of this contamination during processing and improve product shelf-life and safety. Thyme oil, a natural antimicrobial flavor, was mixed at 0.5% (v/v) with a pre-gelatinized pea starch coating solution. The coating solution was spread on chicken breast meat after inoculation with Salmonella Typhimurium plus S. Heidelberg, and also Campylobacter jejuni, Listeria monocytogenes, or Pseudominas aeruginosa. After inoculation at 6 log cfu/g, the chicken meats were packaged in plastic bags and stored at 4 ⁰ C. During 12 d storage, total aerobic bacteria, lactic acid bacteria and inoculated organisms were counted at 4 d intervals. Thyme oil treatments reduced the viability of Salmonella as well as the growth of Listeria and Pseudomonas by 2 log cfu/g, and appeared to eliminate inoculated Campylobacter during storage. The addition of thyme oil increased the viscosity of the pre-gelatinized pea starch solution, but these effects may be minimized by the use of a suitable washer pressure at

application. The results suggested that thyme oil inclusion in an edible starch coating may be a satisfactory delivery system to enhance the safety of processed fresh meat.

Thyme oil reduced C. jejuni viability below detectable levels, significantly inhibited the growth of S. enterica serovars as well as L. monocytogenes, and delayed the growth of P. aeruginosa on chicken breast meats. Pea starch coating was used as a delivery vehicle for thyme oil and also served as a viscosity enhancer to extend the contact of thyme oil with the chicken meat surface. This study has shown that thyme oil either alone or in a gelatinized pea starch coating was effective in delaying growth of spoilage and pathogenic bacteria on chicken meat surfaces during refrigerated storage. These treatments were effective in essentially eliminating large numbers of C. jejuni from the chicken meat and significantly reduced the viability of S. Typhimurium. The pea starch coating may be a useful vehicle for application of natural antimicrobials to control undesirable organisms on chicken carcasses.

Antimicrobial Effectiveness, Drumette Weight and Surface pH Changes

PS+TSP and alginate+ASC coatings on chicken appeared clear, continuous and homogenous (Figure 1). Alginate+ASC coating imparted a pale yellowish color to the drumettes while the PS+TSP coating did not induce any noticeable visual changes. Figure 2 shows the reduction in Salmonella on drumettes over 120 h at 4 ⁰ C. PS not only maintained the antimicrobial activity of TSP longer but also increased its antimicrobial activity compared to the TSP treatment without PS. Because of the viscosity of PS, the TSP + PS solution has longer contact time to chicken surface compared to the TSP solution without PS. This extended contact time increased the effectiveness of TSP. Enhanced antimicrobial activity was also exhibited in the alginate+ASC coating. Coatings with TSP and ASC had significantly (P < 0.05) greater antimicrobial activity than the corresponding solutions without polymers after 24 h. AMs in aqueous solution and in antimicrobial-free coatings were unable to cause > 1.0 log cfu/g reductions.

Most (88 %) of the PS+TSP coating containing TSP appeared to drip from the skin within 1 h (Table 1 ), whereas the coating without TSP was better retained on the surface for 24h. This suggests that TSP may have reduced the viscosity of the PS coatings and accelerated its drip from the skin, which could have occurred as a result of starch degradation under alkaline conditions (BeMiller 1965). Calcium alginate coatings with and without ASC were more stable throughout incubation. Initial weight gains of 7.9 and 6.9 % during alginate treatments were also greater than that of untreated controls (water) at the end of the tests (Table 1). It was suggested that the acidic nature (pH 5.0) of ASC increased the viscosity of the alginate matrix by enhanced charging of calcium ions and

protonation of carboxyl groups (King 1982). Under these conditions calcium ions can more readily form bridges with the negatively-charged alginate matrix and the repulsion between protonated carboxyl groups of alginate is lowered, which promotes the formation of cross- linked networks (King 1982).

Figure 3 shows that TSP increased and ASC decreased the initial pH of the chicken skin. Although AMs in solution caused significant (P ≤ 0.05) initial changes in the skin pH, the effects were transient and did not last more than 24 h. TSP and ASC in coatings significantly changed the surface pH which was maintained up to 120 h and 72 h, respectively (Figure 3). Gelatinized starch is soluble in aqueous environments (Ratnayake et al., 2002). It slowly dissolves within the pores and follicles of the skin and ostensibly releases TSP into skin, which improves its antimicrobial action. The alginate matrix seemed to be more stable but chlorous acid (HCIO ₂ ) which is formed by sodium chlorite acidification during ASC formulation, may gradually diffuse inside the matrix. As it reaches the higher pH of the skin, chlorous acid is dissolved into the skin structure (King 1982; Oyarzabal et al., 2004; Schneider et al., 2002). From the results of this study, it is shown that the PS and alginate coatings can prolong the exposure of surface bacteria to the TSP and ASC at high and low pH, respectively, thereby interfering with cell metabolic activity (Siragusa and Dickson, 1992).

Coating Absorptiveness

Both the rate and amount in absorption of PS+TSP and alginate+ASC coatings to the skin depended on the polymer content of the coatings (Table 2 and 3). At concentrations > 3.5 % PS and > 0.5 % alginate, the absorptiveness was significantly (P ≤ 0.05) reduced during 60 min possibly because the polymers are hydrophilic. At the lowest PS concentration (0.5 %), the amount of coating absorbed by the skin was higher than that of water (Table 2) because the polymers are diluted and do not exist as a separate layer on the skin. At low concentration, the hydrophilic polymers adhered on the chicken skin and increased the water absorptiveness. In addition, these values are comparable to the amounts of absorbed water during commercial immersion chilling for 30 min (Thomas and McMeekin, 1984). Retention of residual polymers inside skin crevices, folds and follicles which would not be removed by surface wiping may have contribution to extra weight gain. PS+TSP coatings were absorbed quicker than alginate+ASC coating as indicated by the higher absorption rate values (i.e., the slope of the absorption curve) in Table 3. Both the rate and quantity of PS absorbed was higher compared to alginate at concentrations that exerted antimicrobial effectiveness (3.5 % and 1.0 %, respectively) (Table 2 and 3). This may explain the higher and more prolonged (120 h) antimicrobial

effectiveness of the PS+TSP coating compared to the alginate+ASC coating (Figure 2). This may also explain the greater antimicrobial activity of TSP in aqueous media against Salmonella on chicken skin (Mehyar et al., 2005). In addition, gelatinized PS at low viscosity may more easily fill skin follicles and pores, bringing TSP directly in contact with more surface bacteria that may have been protected by irregularities in skin surface topography. Alginate+ASC exhibited higher antimicrobial activity than ASC alone only at ≤ 72 h of treatment (Figure 2B). This could have been due to the method of its application when the skin was first dipped in calcium chloride solution with ASC followed by dipping in an aqueous solution of sodium alginate. The formation of an ASC gradient in the alginate coating may have occurred which altered the amount of ASC exposed to targeted bacteria.

Coating Adhesion and Skin Wetting Properties

Although the contact angle technique was successfully used to determine the critical surface energy of solids such as coated paper surfaces using probe liquids (Han and Krochta 2001), the method was less successful on chicken skin. None of the probe solutions formed drops on the skin regardless of their surface tension values which indicates that other factors beside surface energy, such as surface roughness, affected the initial contact angle. Nonetheless, measurements of initial contact angle were successfully used to determine adhesion of liquid materials to food surfaces (Michalski et al., 1997). In the present tests, the formation of discrete drops by the PS+TSP coating solution allowed contact angle measurement. However, stable drops with measurable angles were unobtainable from alginate+ASC coatings. Due to low viscosity calcium chloride and sodium alginate solutions diffused over the skin and yielded a thin film. This means that the contact angle method was not available to measure the surface energy of chicken skins. However, this indicates that any hydrophilic coating layer can adhere on the surface of chicken and form a film structure with surface covering.

PS+TSP coating at low viscosity (below 0.37 N s m ^"2 ) linearly affected the contact angle. At higher viscosity PS+TSP formed a gel at room temperature and the contact angle was no longer dependent on the viscosity (Figure 4). When the concentration of polymers is very high in the coating solution, the coating solution turns into what is effectively a gel. It is hard to use this gelled coating solution for the coating process. To obtain better coating, the coating solution should be a viscous solution when it is coated on the surface, and form a gel as it dries. Therefore, the coating solution should contain polymers at the concentration lower than the gelation concentration for coating process. The effect of PS concentrations on the contact angle as an indicator of coating

adhesiveness to the skin is shown in Figure 5. In general, increasing the PS concentration increased coating adhesion to the skin. At a low concentration of PS (< 0.5 %) the measurement of the contact angle was not possible, but between 0.5 and 1.5 % PS, the contact angle increased with concentration. At PS levels ranging from 1.5 % through 3.5 %, the contact angle was not affected (P > 0.05). At 4.0 %, the contact angle increased to 70 °, whereas at higher concentrations the solutions began to gelatinize to form a soft solid, which invalidated estimation of adhesion by contact angle measurement. Several factors could influence the changes in the initial contact angles shown in Figures 4 and 5. Skin roughness was believed to be responsible for generating unstable liquid drops of the PS+TSP coating solution at low PS concentrations (< 0.5 %). Under these conditions the drops were quickly absorbed and disappeared in the skin. Increasing the PS concentration from 0.5 % to 1.5 % increased the coating viscosity from 0.004 to 0.37 N s m ^"2 , which resulted in proportional increases in the initial contact angle.

The increase in viscosity gave the coating drops the strength to overcome the effects of skin roughness and become stabilized on the surface. At 1.5 % to 3.5 % PS the initial contact angle was not affected by the increases in viscosity (from 0.37 to 1.0 N s m ^{" 2} ) and the resulting contact angle could account for the difference in the surface energies between the skin and the coating solution. In order for the probe solutions to accurately measure critical surface energy of the skin, they should have a viscosity in the range of 0.37 to 1.0 N s m ^"2 . At high levels of PS (> 4.0 %) the solutions started to gelatinize and the initial contact angle measured was independent of the surface energy difference. Overall, the adhesion of the coating to the skin depended on PS concentration and solution viscosity. As will be appreciated by one of skill in the art, just a reduction of polymer concentration can decrease viscosity. The polymer concentration is the main factor to control the viscosity of the antimicrobial coating layer and effectiveness of the antimicrobial activity.

Stabilizing TSP and ASC in PS and alginate coatings, respectively, enhanced their antimicrobial activity against Salmonella on chicken skin. PS+TSP caused significant reductions of the bacterial numbers for longer periods than alginate+ASC. This could have been caused by several factors including: distribution of the AMs within the coatings; prolonged effects of the treatments on skin pH; coating absorptiveness; and coating adhesion to the skin. Although PS+TSP was more effective, it was less stable on the skin. The coating tended to drip from the skin but also absorbed quicker than the alginate+ASC coating. Since they had transient (< 60 min) stability on the skin surface, but had good skin adhesion, with low absorption and significant antimicrobial activity, 5 - 15 % TSP in

coatings of 1 - 5 % (w/v) PS may be of industrial value in applications to reduce numbers of Salmonella on poultry skin.

Solids loss and water uptake

Hydrogels in contact with solution lose solids and take up water (Figs.7 and 8). After immersion in the saline solution for 3 hr, hydrogels lost 40% or more of the initial solids while absorbing more water at the same time. The solids loss and water uptake were largely a process of Fickian diffusion, as shown by good fittings. However, the noticeable scattering of data points about the Fickian curves may imply the concurrent gel erosion and swelling in an oscillatory manner (Makino et al., 1996, Colloids Surf B Biointerfaces 8: 93-100). The presence of TSP in starch gel aggravated the loss of solids (Fig.7a), whereas ASC made little difference in the solids loss of alginate gels (Fig.7b). In contrast, the presence of TSP or ASC in the gel substantially affected the degree of water uptake (Fig.8). For example, gels with antimicrobials absorbed about 45% more water than those without antimicrobials after 3-hr immersion in the saline solution. Due to their high charge density, phosphate anions also tend to structure water by hydrogen bonding (Jane, 1993, Starch/Starke 45: 161-166), and facilitate the water uptake of starch gel. It is likely that those electrolytes by electrostatic interactions open up the cross-linked gel structures, which become more accessible to water molecules. Meanwhile, more solids would be lost in a more open gel structure, since it imposes less hindrance for small molecules (e.g. antimicrobials) and/or dangling clusters to leach out.

Antimicrobial release

As shown in Fig. 9, the release of antimicrobials from hydrogels into the saline solution followed Fikian diffusion. All R-squared values for the non-linear fitting were greater than 0.95. The apparent diffusivity for TSP in starch gel was 2.72x10 ⁹ m ² /s, much lower than the apparent diffusivity of the solids (10.3x10 ⁹ m ² /s) but close to the water diffusivity (2.88 ^χ 10 ^~9 m ² /s). Similarly, the apparent diffusivity for ASC (6.58* 10 ^'9 m ² /s) in alginate gel was lower than the apparent diffusivity of the solids (9.22x10 ^'9 m ² /s) but fairly close to the water diffusivity (5.21 ^χ 10 ^"9 m ² /s). On this basis, the antimicrobials were most likely unattached to polymer chains in the gel, but rather liberated in the water phase. Therefore, the release of antimicrobials TSP and ASC resulted from the osmotic pressure, rather than dissolution of solids. Due to higher solids content of the starch gel compared to the alginate gel, the denser gel structure imposes a greater block for the antimicrobial to get out (or water to get in), resulting in a slower release rate of TSP. Therefore, the

PS+TSP gel would be of particular interest to applications where sustained release of the antimicrobial agent is needed.

Storage modulus of hydrogel

The dimensionless storage modulus (GYGO) of hydrogel in the saline solution decreased with immersion time in a trend of exponential decay (Fig.10). Substantially decreased solids content (Fig.11) due to both solids loss (Fig.7) and water uptake (Fig.8) was largely responsible for the softening of gels as the immersion prolonged. Since the solids content of PS+TSP gel decreased faster than that of the PS-TSP gel (Fig.11a), it is not unexpected that storage modulus of the PS+TSP gel decreased faster than that of the PS-TSP gel (Fig.10a). However, the ALG+ASC gel showed significantly slower modulus reduction than the ALG-ASC gel (Fig.10b), even though both gels had little difference in the change in dimensionless solids content with time (Fig.11 b). The stabilization effect of ASC on the alginate gel presumably results from the immobilization of Ca ²⁺ in the gel by citrate from ASC. Otherwise Ca ²⁺ would be prone to ion exchange with Na ⁺ in the saline solution, as in the ALG-ASC gel.

The presence of antimicrobials substantially influenced the rheological properties of hydrogels by accelerating solids loss and water gain. Since the release of antimicrobials was slower than the loss of total solids in the gel, and antimicrobials and water had the same level of diffusivity, it is suggested that the release of antimicrobial TSP in starch gel or ASC in alginate gel is largely controlled by osmotic-pressure-induced gel swelling (water in and ions out), rather than dissolution of polymer chains in the gel structure. This work implies that water diffusivity in hydrogel could be used as a monitor of drug release when the drug is known not to strongly interact with polymer chains in the hydrogel. There are two main mechanisms of release (1) diffusion and (2) erosion. Most gels may have either one or a combination of these two mechanisms. Diffusion is the release of active agent from the matrix gels through diffusion, and erosion means that the release is caused by the degradation of the matrix gels. Since all biodegradable polymers will be eroded eventually, the mechanism of early stage release is important to control the release rate so as to maximize effectiveness.

Antimicrobial Coatings

A 100 ml dispersion of 3.5 % (w/v) pea starch was prepared in cold water. The mixture was heated to boiling with mixing and held for 5 min to complete starch gelatinization. The solution was then cooled to room temperature and trisodium phosphate

(TSP) was added (10 % w/v), mixed and homogenized by a Powergen-700 for 5 s at 20000 rpm. This yielded PS+TSP coating solution.

Calcium alginate coating (alginate+ASC) consisted of two solutions of 100 ml each. Solution (a) was 1 % (w/v) calcium chloride in acidified sodium chlorite (ASC, 1200 ppm) prepared by mixing equal portions of the acid and salt parts of Sanova provided by Alcide Corp. This solution was used within 30 min as recommended by Alcide Corp. Solution (b) contained 1 % (w/v) sodium alginate dissolved in water and mixed. Coatings free of AMs were prepared following the same procedures but without TSP addition to PS and without ASC addition to alginate. PS+TSP solutions containing 0.5, 1.5, 2.0, 3.5, 4.0 or 4.8 % (w/v) PS, and alginate+ASC with 0.5, 1.0 or 1.5 % (w/v) alginate were prepared as outlined above. These solutions were used for absorptiveness, initial contact angle and viscosity measurements.

Chicken Treatment

Unchilled chicken thighs and drumettes (Mehyar et al., 2005) were obtained from a local processing plant immediately after slaughtering and used within 30 min after their arrival. The warm thighs were used for contact angle tests. The drumettes were inoculated with an ampicillin-resistant Salmonella cocktail. Bacterial cultures used to inoculate drumettes were: Salmonella entericia serovars Typhimurium (# 02-8425 and # 02-8421) and Heidelberg (# 271 ). The three strains were grown separately in tryptic soy broth (TSB) for 24 h at 37°C. Cultures were standardized to an OD ₆₀₀ of 0.80 using sterile TSB to yield about 9 log cfu/ml and were combined in equal portions. Inoculations were performed by dipping drumettes in triplicate into 300 ml bacterial suspension containing 7 log cfu/ml for < 15 sec. The drumettes were hung for 10 min to allow bacterial attachment before being dipped for 0.25 min in one of the following solutions: (1) TSP (10% w/v); (2) ASC (1200 ppm); (3) PS+TSP coating; (4) calcium chloride in ASC (solution a) then dipped in sodium alginate solution (solution b) to form the alginate+ASC coating; (5) coatings of 3.5 % (w/v) PS without AMs; or (6) 1 % (w/v) calcium alginate without AMs. Drumettes were weighed before and directly after dipping using a digital balance (± 0.00005 g). The drumettes were hung inside a covered glass chamber with 85 % relative humidity and incubated at 4 ⁰ C for 120 h. Samples were withdrawn in triplicate for testing after 1 , 24, 72 and 12O h incubation.

Changes in Drumette pH, Weight and Viable Salmonella after Coating

At each sampling day, the surface pH of the coated drumettes was measured at three different locations using a pH meter equipped with an lsfet surface probe and their

average values were recorded. Drumettes were then weighed and their skins were excised and placed in stomacher bags with buffered peptone water (10 g peptone, 5 g NaCI, 3.5 g Na ₂ HPO ₄ , 1.5 g KH ₂ PO ₄ per liter) and homogenized for 3 min to prepare 10 ^'1 homogenates. The homogenates were then serially diluted and plated on pre-poured XLD agar containing 100 ppm ampicillin. Salmonella were counted after 24 h at 35 ⁰ C. Logarithmic reductions were determined by calculating the differences in Salmonella numbers between the control and the treated samples.

Coating Absorptiveness

The method of Han and Krochta (1999) was modified to measure the coating absorption into chicken skin. A plastic ring specimen holder with four screws, similar to that used by Han and Krochta (1999), was used to fix skin samples. Skins of unchilled chicken thighs were excised and used within 10 min. The outer surface of the skin was placed between the base and the ring (diameter 5.8 cm) facing upward in the holder and the ring was secured with screws. The holder with the skin was then weighed (W ₀ ) and 5 ml of the PS+TSP coating solution, or 2.5 ml of 1 % (w/v) calcium chloride in ASC (solution a) and 2.5 ml of solution b were applied on the top of the skin. Nine samples were prepared for each coating and the holding units were placed on a flat plate at room temperature to allow the skin samples to absorb the coating solutions. Samples were withdrawn in triplicate at 10, 30 and 60 min after application. Absorption was terminated by wiping away the excess coating solutions which remained on the skin surface with a tissue at each sampling time. The weights of the apparatus holding the skin were recorded before (W _wet ) and after drying (W _dry ). The absorptiveness (% A ₁ ) was defined as:

% A, = (Ww ₆ , - W _dry )/(W ₀ - W _e ) x 100 where W _e is the weight of an empty apparatus without skin.

Contact Angle and Skin Wetting Properties

The initial contact angles for the various probe liquids and the coating solutions on the skin were used to determine critical surface energy of skin and absorption profile of coating solutions, respectively. Fresh, unchilled chicken thighs were used and their surfaces were wiped by a dry tissue to remove any residual water. The thighs were cut on one side lengthwise to the bone with a razor blade and a portion of the skin and flesh was removed from the thigh. For testing, the specimens were placed on a rack with adjustable height, and attached to the rack using plastic putty. A digital microscope (10 X magnification) was aimed horizontally to observe the cut chicken surface. Drops of 10 μL of the probe liquids or coating solutions were placed on the skin surface using a

microsyringe and the side images of the liquid drops were recorded by a computer after confirming the horizontal level position of samples. In order to account for any asymmetry of the image caused from improper leveling, the contact angles of both sides of each liquid drop were measured and the average values were recorded. All measurements were done inside a closed chamber equipped with an electric fan to circulate the internal air which was equilibrated to 85 % relative humidity with a saturated solution of zinc sulfate. The probe liquids used were HPLC grade water, glycerol, ethylene glycol and dimethyl sulfoxide. In order to study the effect of PS viscosity on the contact angle, the dynamic viscosity of PS+TSP solutions with different PS concentrations was determined using a rheometer. The instrument was operated with parallel plate geometry (plate diameter = 20 mm and gap = 1 mm). Samples were placed in the apparatus and allowed to equilibrate at 25 ⁰ C prior to analysis. Measurements were conducted at 3 Pa shear stress and 1 Hz frequency. The relationships between the initial contact angle and PS concentration of PS+TSP coating solution, and between the initial contact angle and the PS+TSP coating solution viscosity were determined.

Flow properties of starch-based coating solution

Figure 12 shows the shear stress-strain curve of the pea starch coating solution with and without thyme oil. From this figure the consistency index and power law flow behavior index were calculated, and these results are summarized in Table 4. The consistency of the gelatinized pea starch coating solution was affected significantly by the presence of thyme oil, which caused increased viscosity at low shear rate range. The addition of thyme oil decreased the power law flow behavior index and made the starch gel more viscous and pseudoplastic.

Figure 12 shows that both starch coating solutions, regardless of thyme oil addition, exhibited shear-thinning pseudoplastic behavior below 100 s-1 of shear rate. However, above 100 s-1 , the pseudoplastic characteristics were converted to Newtonian behavior, specifically Bingham flow. Starch solutions possess intermolecular interactions and form elastic starch gels when the deformation is not significant, such as occurred below 100 s-1 of shear. However, above this critical shear, the intermolecular interaction of starch gels could not be maintained and were converted from an elastic gel to a viscous solution. The corresponding critical shear stresses of 100 s-1 shear rate were approximately 20 Pa and 5 Pa for pea starch with and without thyme oil, respectively. Yield stresses (the Y-intercept of Bingham) were 22.4903 Pa and 5.3486 Pa for pea starch solutions with and without thyme oil, respectively, which reflects the dramatic increase in the yield stress of the starch solutions caused by thyme oil addition. This result implies

that thyme oil enhanced the intermolecular interaction of starch, perhaps by the formation of starch (amylose)-lipid complexes. Han and others (2006) found that the addition of beeswax to gelatinized pea starch did not change the starch structure and related characteristics until 30% (w/w) of beeswax had been added to the starch gel. Therefore, the changes in visco-elastic properties of pea starch gels by 5% thyme oil are remarkable. Thyme oil contains mostly phenolic compounds that have very small molecular weight compared to those of beeswax. It is hypothesized that the small hydrophobic molecules can be incorporated within the amylose helix much easier than macromolecular lipids, and consequently form a high-degree amylose-lipid complex. For the practical application of a thyme oil-starch coating for poultry processing, it is suggested that an inside-outside bird washer be used. The washer would spray the starch coating solution at both high pressure and high speed feeding rate. Therefore, within the practical operating range of feeding, which will be definitely over 100 s-1 shear rate, the thyme oil-starch solution will behave as a Bingham fluid. A minimum 22.49 Pa of pressure is required for the bird washer to initiate the flow of the starch coating containing thyme oil. The higher yield stress produces a thicker coating weight. Since the yield stress of the coating solution increased 5 times after thyme oil addition, theoretically on a smooth surface hanging vertically (e.g., chicken carcass on an overhead conveyor), the thickness of the coating containing thyme oil will be 5 times greater than that of a starch coating without thyme oil. Therefore, understanding the effects of yield stress upon coating viscosity is critical to optimize coating application and uniformity. After washing, chicken carcasses are warm and the antimicrobial coating solution can be sprayed at ambient processing room temperature.

Microbial viability on Salmonella-inoculated chicken

Application of the starch coating to chicken cubes had little effect on the numbers of total organisms, the lactic acid bacteria present, and the viability of inoculated (ampicillin resistant) Salmonella during 12 d storage at 4 ⁰ C (Table 5). Numbers of total organisms (psychrotrophs) and lactic acid bacteria increased similarly in the presence or absence of the starch coating. MRS agar is a non-selective enriched medium and Salmonella were able to form colonies on this agar. Salmonella numbers decreased by about 1 log cfu/g during refrigerated storage in treatments with and without the starch coating. Inclusion of thyme oil in the coating delayed the growth of psychrotrophs until day 4 and the lactic acid bacteria until after day 8. Thyme oil inclusion in the coating had a significant negative effect on Salmonella viability with recoveries being 2 log cfu/g lower at day 4 and this reduction was increased to 3 log cfu/g at days 8 and 12.

Microbial viability on Campylobacter-inoculated chicken

As with the previously reported trial (Table 5), the starch coating had essentially no effect on the growth of psychrotrophs and lactic acid bacteria during storage of the chicken meat at 4 ⁰ C for 12 d (Table 6). However, addition of starch coating containing thyme oil significantly reduced the extent of both psychrotrophic and lactic acid bacterial growth by 2 and 3 log cfu/g at days 8 and 12, respectively. Direct addition of thyme oil as a water emulsion without the coating caused a similar delay in psychrotrophic bacterial growth, but had a greater initial inhibitory effect on the lactic acid bacteria. These latter recovered by day 8 to reach about the same numbers as were present on chicken coated with starch containing thyme oil. These latter levels were 2 to 3 log cfu/g less than in treatments where thyme oil was not used. Campylobacter were absent from the chicken meat used in this study, and following inoculation their numbers were relatively stable during storage at 4 ⁰ C. A very slight reduction in Campylobacter viability was noted in response to starch coating at day 12, but use of thyme oil alone or use of thyme oil following its incorporation into the starch coating caused an immediate reduction in Campylobacter viability to below detectable levels, and this inhibitory or lethal effect was maintained for the remainder of the study (Table 6).

Microbial viability on Listeria-inoculated chicken

As noted in Tables 5 and 6, psychrotrophic and lactic acid bacteria naturally present on uninoculated chicken grew rapidly and reached 7 to 8 log cfu/g by 12 d of storage at 4 °C (Tables 7 and 8). There was little difference in bacterial recoveries (psychrotrophs, lactic acid bacteria or inoculated L. monocytogenes) among the media used when starch-coated chicken (with or without L. monocytogenes inoculation) was stored at 4 ⁰ C for 12 d. L. monocytogenes was able to grow on the MRS medium used for lactic acid bacteria recovery, and contributed to the number of colonies recovered as lactic acid bacteria.

The extent of bacterial growth on BHI and MRS agars was reduced in treatments containing thyme oil, and inhibition caused by direct addition of thyme oil was only slightly greater than that caused by the thyme oil-starch coating (Table 7). The inhibitory effects were not as great as noted with Campylobacter (Table 6).

L. monocytogenes was not recovered on Listeria selective agar from uninoculated chicken during storage, but following its inoculation the organism increased one log cfu/g during storage. In addition, growth of L. monocytogenes was unaffected by the presence of the starch coating as noted with Salmonella and Campylobacter. Thyme oil alone or

when incorporated into the starch coating was inhibitory to L. monocytogenes (on Listeria agar) to about the same extent (> 1 log cfu/g reduction) by 12 d storage.

Microbial viability on Pseudomonas-inoculated chicken

The microbial growth profile on chicken inoculated with P. aeruginosa as monitored on BHI and MRS agars (Table 8) did not differ from results obtained with the other inoculated organisms when thyme oil was not used (Tables 5, 6 and 7). In addition, the pea starch coating did not further alter bacterial recoveries on these media or Pseudomonas agar during storage at 4 ⁰ C. Thyme oil along or when incorporated in the pea starch coating significantly delayed the growth of bacteria on chicken monitored with all three media. These differences were from one to 2 log cfu/g and were noted at 12 d of storage (Table 8), however, there was no significant difference in effectiveness of thyme oil action before or after incorporation in the starch coating.

Antimicrobial effectiveness of thyme oil

Thyme oil has been shown to one of several potently antimicrobial essential oils during tests against a range of spoilage and pathogenic bacteria. Its major component, thymol, was as effective as eugenol and carvacrol against most of the pathogens tested in the present study (Burt 2004). Generally, essential oils are more effective against Gram positive bacteria, but Gram negative bacteria can be vulnerable (Burt 2004; Holley and Patel 2005). In the present work delayed growth of aerobic psychrotrophs and lactic acid bacteria was not unexpected. Inhibition of L. monocytogenes growth and reduction in Salmonella viability in the presence of thyme oil reported here are consistent with the results from other studies where different substrates and temperatures of incubation were used (Burt 2004). The delayed growth of P. aeruginosa reported here is a positive finding since Pseudomonas frequently show resistance to essential oil treatment (Holley and Patel 2005), however, it is likely that during longer storage P. aeruginosa would recover from the inhibitory effects of thyme oil exposure. One of the more important observations made here was the drastic reductions in numbers of C. jejuni which occurred immediately upon exposure to thyme oil alone or to the starch-thyme oil coating, which was sustained during 12 d storage. Surprisingly little work is reported in the literature concerning C. jejuni inhibition by thyme oil. In a study by Friedman and others (2002) thyme oil was found to be as effective as cinnamaldehyde, eugenol, carvacrol, citral, geranol, and benzaldehyde against C. jejuni in a microplate assay.

In the C. jejuni and L. monocytogenes tests reported here where thyme oil was directly added to the chicken meat surface, a more immediate inhibitory effect was found

against the lactic acid bacteria, however, this difference was not evident at 12 d storage. In P. aeruginosa tests the starch-thyme oil coating initially showed a greater inhibitory effect but this difference was resolved by day 8 of storage. In a separate test it was found that Salmonella, L. monocytogenes, and P. aeruginosa were able to form small colonies on MRS agar. Thus, lactic acid bacterial recoveries may have been over-estimated to some extent. However, this observation does not affect the overall conclusions from the study.

Statistical Analysis

Data obtained were the average values of three replicates for treatments. Each treatment was conducted twice in separate experiments. The statistical analytical system was used to compare means of the replicates at each sampling time. A significance level of 5 % was used for all analyses. Linear regression analysis for absorption rate was conducted using the data analysis option of a spread sheet for the absorption curves (weight vs. time).

Preparation of starch and alginate hydrogels

3 grams of pea starch (PS, 37% amylose, Nutri-Pea Ltd., Portage-la-Prairie, MB) was dispersed in 100 ml cold water. The dispersion was heated to boiling with mixing and held for 5 min when starch granules were almost fully gelatinized. The solution was then cooled to room temperature (23°C), and 10 grams of trisodium phosphate (TSP, Sigma Chemical Co., St. Louis, MO) was added in, followed by homogenization with a Powergen- 700 (Fisher Scientific International Inc., Whitby, ON) for 5 s at 20,000 rpm. The solution was then poured into two 200 ml beakers, with 50 ml solution in each beaker, and left overnight at room temperature to allow the stabilization of gel structure. PS hydrogel without TSP was also prepared and used as control.

Two solutions were used to prepare calcium alginate (ALG) hydrogel. Solution (a) was an acidified sodium chlorite (ASC) solution containing 1 % w/v of calcium chloride (CaCI ₂ , Sigma Chemical Co., St. Louis, MO). The ASC solution was prepared by mixing equal portions of citric acid solution (900 ppm) and sodium chlorite solution (1100 ppm) (Sanova, Alcide Corp., Redmond, WA), and was used within 30 min after preparation. Solution (b) contained 0.5% w/v sodium alginate (Product No.180947, CAS 9005-38-3, Sigma Chemical Co., St. Louis, MO) dissolved in water at room temperature.

Calcium alginate gel was prepared using a plastic assembly consisting of a mold and two fixative rings (Fig.6). A piece of CaCI ₂ permeable membrane (Dialysis Tubing, Fisher Brand regenerated cellulose, Fisher Scientific, Nepean, ON) was first attached onto

the mold by one fixative ring. Solution (b) of 50ml was poured into the mold, and the mold was covered by another piece of the membrane, which was fixed onto the mold using the other ring. Two assemblies containing the sodium alginate solution were then immersed in solution (a) of 500 ml and taken out after 24h. Self-standing calcium alginate gels containing ASC were obtained after the removal of the rings and membranes. Calcium alginate gels without ASC were also prepared by the same procedure except that solution (a) used was a pure CaCI ₂ solution with the same concentration of 1% w/v.

Rheological properties of hydrogels in air and in saline

Freshly prepared hydrogel was cut into a cylinder using a plastic borer with a height of 10 mm and internal diameter of 20 mm. The gel cylinder was then sliced into specimens with a thickness of 5 mm by a sharp blade. Rheological analysis was carried out using a controlled stress rheometer (AR-1000, TA Instruments Inc., New Castle, DE) with 20-mm parallel plate geometry. After a specimen was centered on the base platen, the upper platen was programmed to move down at a decelerating speed until it came in contact with the specimen in order to avoid any pre-loading deformation. Oscillatory stress sweeps from 0.1 Pa to 10 Pa at a frequency of 1 Hz were done at a temperature of 25°C to determine the linear viscoelastic range for hydrogels in air. Since both gels exhibited linear elastic regions at stress below 2 Pa, a stress of 1 Pa was chosen for the following time-sweep experiments.

A physiological saline was prepared by dissolving 0.8 g of sodium chloride (Sigma Chemical Co., St. Louis, MO) in 100 ml tap water, followed by adjusting pH to 6.8. After a gel specimen was centered in a bath (height 30 mm, inner diameter 34 mm, outer diameter 64 mm) glued to the base platen, a saline solution double the specimen's volume was filled inside the bath while the upper plate came in contact with the specimen. Oscillatory time sweeps (1 Pa at 1 Hz) were run for all specimens in saline. A series of dynamic storage moduli (G') as a function of immersion time (t) were obtained from the control software.

Determination of TSP and ASC concentrations in saline

After the specimen was loaded, saline solution of 0.5 ml was withdrawn periodically and collected in a vial. The samples, diluted 1000 fold, were analyzed by an ion chromatography system (Dionex Corporation, Sunnyvale, CA). The injection volume and flow rate were maintained at 50 μl and 1 ml/min, respectively, throughout the analysis. External standards (0, 5, 25, 50, 75 and 100 μg/ml) for both phosphate and chlorite anions were used for calibration. NaOH solution of 30 mM was used as eluent for all samples.

The concentration of antimicrobial (C) in the saline was determined by the peak area for the elution which was calculated by Chromeleon Chromatography Management Systems (Dionex Corporation, Sunnyvale, CA). The concentration of ASC was determined by subtracting the peak area for chloride anions in the pre-load saline solution from the peak area for both chloride and chlorite anions in the samples containing ASC, since both chlorite anions from ASC and chloride anions from NaCI eluted at the same time (3 mt ^' n). The concentration of TSP was determined directly by the peak area for phosphate anions in the samples. The concentration of antimicrobial after 12-hr immersion in the saline solution was taken as the equilibrium concentration (C ₀₀ ).

Solids loss and water uptake during gel swelling

A freshly prepared gel sample (diameter 20 mm and thickness 5 mm) was weighed before {M) and after (M _ύ ) drying at 105 ⁰ C to constant weight (about 5 hr). The initial solids content (SC ₀ ) of the fresh gel was determined as MdM ₁ . A gel sample, after weighing (M ₀ ), was immersed in a saline bath (the sample size, the inner diameter of the bath, the concentration and amount of the saline solution same as those used in the rheological determination) for a period of time up to 8 hr. The swollen gel was then weighed (λ/f _sw ), followed by drying at 105 ⁰ C to constant weight (M _s ). The amount of solids (M _s0 ) and water (Mw ₀ ) in the pre-swelling gel were M ₀ SC ₀ and /W ₀ (1-SC ₀ ), respectively. The amount of solids (M _s ) and water (M _w ) in the post-swelling gel were M _s and M _aw -M _s , respectively. The solids content (SC) of the swollen gel was M _S IM _SVI . All samples are duplicated.

Apparent diffusivities of solids, water and antimicrobials

Solids loss, water uptake and antimicrobial release were all assumed to follow Fikian diffusion. A simple form of the solution (Schwartzberg and Chao, 1982, Food Technol 36: 73-86) is: = c _{l E} xp(-!^) (1)

X, - X. ' ^ R ² ' where X can be the amount of solids (M _s ) or water (M _w ), or the concentration of antimicrobial released (C), and the subscripts 0 and ∞ stand for at zero and infinite time, respectively. The constants C ₁ and Qi are correlated. For infinite cylinder q _l = , where the stripping factor D = 2 (the volume of saline solution divided by the volume of gel). R is the radius of gel sample (10mm), and t the immersion time. The apparent diffusivity (D) of solids, water and antimicrobial were

obtained by 3-parameter non-linear fitting of /W _s /M _s0 ~ f, MJM^ ₀ ~ t, and CIC _∞ ~ t, based on Equation 1.

Air-chilled fresh chicken breast meats were obtained from a local poultry processing plant (Dunn-Rite, Winnipeg, Manitoba, Canada) about 4 h before the experiment. The meats were cut into 2 cm x 2 cm cubes (10 g ± 1 g) with a knife disinfected in 70% ethanol. Starch extracted from Canadian yellow field peas (Pisum sativum L. Miranda) by a conventional wet milling process was supplied by Nutri-Pea Ltd. (Portage-La-Prairie, Manitoba, Canada). Pea starch is a C-type starch containing 37 - 40% amylose. One gram of phosphatidyl choline (Fisher Scientific, Nepean, Ontario, Canada) was dissolved in 15 mL of thyme oil (Sigma Chemicals Co., St. Louis, MO) and stored at 4 ⁰ C until used.

Ampicillin resistant Salmonella entericia serovars (i.e., Typhimurium and Heidelberg) and Campylobacter jejuni were obtained from R. Ahmed, Canadian Centre for Human and Animal Health (Winnipeg, Manitoba, Canada). Listeria monocytogenes and Pseudomonas aeruginosa were obtained from the culture collections of the Department of Food Science and the Department of Microbiology, respectively, at the University of Manitoba (Winnipeg, Manitoba, Canada).

Consistency profile of starch coating solution

Fully gelatinized 2.5 % (w/v) aqueous starch solution (prepared by boiling 20 min) containing 1.25% (w/v) glycerol was mixed with 5% (v/v) thyme oil at room temperature, and its consistency was determined using a rheometer (AR 1000, TA Instruments, New Castle, DE). The volume of samples was 0.99 mL. Operating conditions of the rheometer were 25 ⁰ C using a 60 mm diameter 1° angle steel cone. Initial shear rate was 1.275 s-1 and was ramped to 1000 s-1. Shear rate was increased by steady state flow mode with a logarithmic ramp pattern. Consistency index and fluid behavior index were calculated using the power law equation by parameter estimate of regression analysis. Each treatment was tested in triplicate.

Bacterial inoculum preparation

All bacterial cultures were maintained in BHI (brain heart infusion) broth and enumerated on BHI agar (Difco Division, Becton Dickinson Co., Sparks, MD) after incubation at 35 °C for 24 to 48 h. For Campylobacter culture BHI agar and broth media were used with 0.5% (w/v) yeast extract and 10% (w/v) laked horse blood (Oxoid Ltd., Nepean, Ontario, Canada), and were incubated at 35 ⁰ C under microaerophilic conditions

created by the CampyPak Plus system (Becton Dickinson Co., Cockeysville, MD) for 48 h. Bacterial culture broth was centrifuged at 300Og for 15 min at 10 ⁰ C (Sorvall RC2-B Refrigerated Centrifuge, Du Pont, Newtown, CT). The sedimented culture pellet was suspended in 0.85% sterile saline solution to wash and was recentrifuged. The pellet was diluted to yield an optical density of 0.80 at 600 nm and the live bacterial population was determined using a spiral plating unit (Autoplate 4000, Spiral Biotech, Bethesda, MD). The equivalent number of bacteria for 0.8 optical density units was 109 cfu/mL The two Salmonella cultures were mixed at equal numbers of cells to obtain a cocktail of S. Typhimurium and S. Heidelberg.

Antimicrobial pea starch coating

Pea starch suspension was prepared by mixing 25 g pea starch and 12.5 g glycerol (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada) in 1 L sterile cold distilled water. This suspension was boiled for 20 min with agitation to gelatinize pea starch, and cooled in a water bath at 50 ⁰ C. The thyme oil and phosphatidyl choline mixture was blended into the pea starch coating solution to give a 5% (v/v) concentration and stirred for 5 min.

Inoculation of chicken meat

Chicken meat cubes (approximately 2 kg) were placed in a sterile aluminum tray and 2 L of inoculum containing 106 cfu/mL of each of the test organisms and the Salmonella cocktail were separately poured on the chicken cubes. The tray was shaken 2 to 3 times during 15 min exposure to allow the meat to adsorb bacteria, then the excess liquid was drained. The inoculated meats were dried for 5 min in the tray. One quarter of the inoculated cubes (approximately 0.5 kg) were enclosed in a high-barrier plastic bag (Deli ^* 1 , WinPak, Winnipeg, Manitoba, Canada) composed of nylon/ethylene vinyl alcohol/polyethylene, and heat-sealed. The film was 75 μm thick with an oxygen transmission rate of 2.3 cm ³ m ^"2 d ^'1 at 23°C, and water vapor transmission rate of 7.8 g m ^"2 d ^"1 at 37.8°C and 98% relative humidity. The second quarter of the inoculated cubes was transferred onto a sterile tray and 1 L of pea starch coating solution was poured onto the cubes. After shaking for 1 to 2 min, the excess starch solution was drained. The coated cubes were dried for 1 h in the tray, and each cube was packaged in the high-barrier plastic bag. The third quarter of inoculated cubes was placed in a sterile tray, and 1 L of pea starch coating solution containing 5% thyme oil was poured on the chicken cubes. The last quarter of inoculated chicken cubes was mixed with 1 L sterile water containing

5% thyme oil. Both thyme oil treatments were mixed, dried and packaged as described earlier. Chicken meats without inoculation and coating were packaged as control samples (i.e., no treatment). All samples were stored at 4 ⁰ C.

Viable numbers of bacteria

At 0, 4, 8 and 12 d of storage after inoculation, three bags per treatment were opened aseptically and 90 mL of 0.1 % peptone water was added. This bag was placed in a stomacher and pummeled for 1 min. After appropriate serial dilutions, the samples were plated on agar media using the spiral plating unit, and incubated. All plates were counted in duplicate from each sample (total 6 analyses per treatment). Types of agar media used and incubation conditions used for inoculated bacteria were:

Total aerobes: BHI agar at 35 ⁰ C for 24 h

Lactic acid bacteria: MRS agar (Difco) at 32 ⁰ C for 48 h

Salmonella: XLD agar (Difco) containing 100 ppm ampicillin (Sigma-Aldrich) at 35 ⁰ C for

24 h

Campylobacter: Karmali agar (Oxoid Ltd.) containing a growth supplement (Oxoid SR 139) at 35 ⁰ C for 48 h under microaerophilic conditions

Listeria: Listeria selective agar (Oxford selective fomulation, Oxoid Ltd.) at 35 ⁰ C for 24 h

Pseudomonas: Pseudomonas agar (Oxoid Ltd.) with a supplement (Oxoid SR 103) at 35

⁰ C for 24 h

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

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Table 1. Weight changes ¹ of chicken drumettes dipped in 10 % (w/v) trisodium phosphate (TSP) with or without 3 % (w/v) pea starch (PS), or in 1200 ppm acidified sodium chlorite (ASC) with or without 1 % (w/v) calcium alginate during storage at 4 ⁰ C Treatment % weight change during storage (means ± SD)

Oh Th 24h 72h ϊ20h

PS+TSP 5.12 ±0.48 0.62 ± 0.2 0.12 ±0.19 0.35 ±0.14 -0.68 ±0.13

PS 4.84 ± 0.49 3.86 ± 0.37 3.64 ± 0.36 0.91 ± 0.31 0.89 ± 0.31

TSP 1.49 ±0.18 0.52 ±0.21 -0.89 ±0.19 ND ² ND

Alginate+ASC 7.86 ± 0.84 5.32 ±1.0 5.25 ±1.07 3.98 ± 0.84 4.05 ±1.2

Calcium alginate 6.88 ± 0.47 4.98 ± 0.29 4.1 ±0.30 2.5 ±0.19 2.6 ±0.11

ASC 1.43 ±0.15 1.25 ±0.16 -0.55 ±0.13 ND ND

Water (control) 1.47 ± 0.04 1.66 ± 1.1 -1.93 ± 0.60 ND ND

¹ Weight gained or lost/ initial weight x 100.

² Not determined.

Table 2. Percentage absorptiveness (% A _t ) ¹ of antimicrobial pea starch (PS+TSP) and calcium alginate (alginate+ASC) coatings containing different polymer concentrations applied to chicken skin and held at room temperature for ≤ 60 min

Treatment Polymer concentration %A _t (means ± SD) after holding ) (min.)

(% w/v) 10 30 60

PS+TSP 0.5 2.40 ± 0.61 ^a 4.51± 0.36 ^a 5.81 ± 0.66 ^a

3.5 1.98 ± 0.23 ^a 3.81± 0.55 ^ab 4.73 ± 0.49 ^a

4.8 0.93 ± 0.20 ^b 1.23 ± 0.26 ° 1.61 ± 0.30 ^b

Alginate+ASC 0.5 0.98 ± 0.26 ^b 1.15 ± 0.15 ^c 1.21 ± 0.65 ^b

1.0 0.62 ± 0.15 ^c 0.75 ± 0.15 ^c 0.92 ± 0.30 ^b

1.5 0.45 ± 0.21 ^c 0.32 ± 0.21 ^d 0.51 ± 0.36 ^b

Water (control) 0.0 1.8 ± 0.20 ^a 2.7 ± 0.36 ^b 4.8 i : 0.96 ^{a a c} Means within the same column with common letters are not significantly (P > 0.05) different.

¹ %A _t = (W _wer W _dr y)/(W ₀ -W _e ) x 100; W _wet and W _dr y are weights of absorptiveness apparatus holding the skin before and after drying, respectively; W ₀ initial weight of the skin; W _e weight of the empty apparatus.

Table 3. Linear regression analysis ¹ of changes in weight of chicken skin coated with antimicrobial pea starch (PS+TSP) and calcium alginate (alginate+ASC) coatings containing different polymer concentrations with time

Treatment Polymer concentration Absorption rate Y-intercept R ²

(% w/v) (g/min) (initial weight g)

PS+TSP 0.5 0.066 2.00 0.94

3.5 0.053 1.74 0.91

4.8 0.013 0.81 0.99

Alginate+ASC 0.5 0.005 0.96 0.86

1.0 0.006 0.56 0.79

1.5 0.001 0.46 0.51

Water (control) 0.0 0.060 1.10 0.98 equation: Y = ax + b; Y is weight of sample; x is time in min.; a is absorption rate; b is initial absorption.

O

O r-- o

O Table 4 Flow characteristics of gelatinized pea starch coating material with and without thyme oil at 25 ⁰ C

<

U

H U

Values in parentheses are coefficients of variance (CV). * Viscosity and yield stress of Bingham were obtained from data over 100 s ^'

C5 of shear rate.

90 r--

SO

O

90 O O

O

Table 5 Effects of thyme oil treatments on the numbers (log cfu/g) of Salmonella Typhimurium and S. Heidelberg on chicken breast meat at 4 ⁰ C.

Treatments DayO Day 4 Day 8 Day 12 ^•

Total aerobes

No treatment 3.1+0.2 4.6 ±0.1 6.1 ±0.2 6.6 ± 0.3

Salmonella inoculation 4.7 ± 0.0 5.0 ±0.1 6.6 ±0.5 7.1 ±0.1

Salmonella + Pea starch coating 4.8 ± 0.05 5.2 ±0.3 7.0 + 0.4 7.5 ±0.1

Salmonella + Pea starch coating + 4.0 ± 0.05 ^a 3.4 + 0.5 b 5.8 +0.3 7.2 ± 0.4

Thyme oil

Lactic acid bacteria

No treatment 2.6 + 0.3 3.6 ±0.1 5.2 ±0.6 5.4 ±0.7

Salmonella inoculation 4.7 ± 0.0 4.7 ± 0.2 5.3 ±0.3 6.1 ±0.6

Salmonella + Pea starch coating 4.9 ± 0.2 4.8 ±0.1 5.4 ± 0.2 6.9 ±0.1

Salmonella + Pea starch coating + 3.9 ± 0.1 ^b 3.0 ±0.0 C 2.8 + 0.7° 5.8 ± 0.4 a

Thyme oil

Salmonella

No treatment 0.0 + 0.0 0.0 ±0.0 0.0 ± 0.0 0.0 ± 0.0

Salmonella inoculation 5.2 ± 0.0 ^a 4.5 ±0.1 b 4.3 ± 0.2 ^b 4.2 ±0.1 b

Salmonella + Pea starch coating 5.1 ±0.1' 4.2 ± 0.2 b 4.4 ± 0.2 ^b 3.9 ±0.1 C

Salmonella + Pea starch coating + 4.3 +0.4 ^a 2.9 ±0.2 b 2.0±1.7 ^b 2.2 ± 0.4 b

Thyme oil

Experiments with Salmonella + H ₂ O + thyme oil treatment were not conducted. Different superscripts indicate a significant difference of values in rows (t-test, n = 6, p < 0.05).

Table 6 Effects of thyme oil treatments on the survival (log cfu/g) of Campylobacter jejuni on chicken breast meat at 4 °C.

Treatments Day O Day 4 Day 8 Day 12 Total aerobes

No treatment 3.2 ± 0.0 5.0 ± 0.1 7.7 + 0.1 7.5 + 0.1

Campylobacter inoculation 3.4 ± 0.2 4.9 ± 0.1 6.9 ± 0.1 7.2 ± 0.3

Campylobacter + Pea starch coating 3.5 ± 0.3 4.9 ± 0.1 7.7 ± 0.0 7.8 ± 0.1

Campylobacter + H ₂ O + Thyme oil ND 3.4 + 0.4 5.3 + 0.5 5.3 ± 0.6

Campylobacter + Pea starch coating 2.0 ± 0.0 2.4 ± 0.1 5.0 ± 0.05 5.7 ± 1.0 + Thyme oil

Lactic acid bacteria

No treatment 2.4 ± 0.1 4.3 ± 0.0 6.3 ± 0.2 6.9 ± 0.0

Campylobacter inoculation 3.2 ± 0.0 4.4 + 0.0 6.1 ± 0.2 7.0 ± 0.1

Campylobacter + Pea starch coating 3.4 ± 0.2 4.2 ± 0.1 6.3 + 0.1 7.5 + 0.2

Campylobacter + H ₂ O + Thyme oil ND 2.0 + 0.0 2.3 ± 0.0 4.1 ± 0.5

Campylobacter + Pea starch coating ND ND 2.3 ± 0.0 4.7 + 0.2 + Thyme oil Campylobacter jejuni

No treatment 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

Campylobacter inoculation 4.6 ± 0.1 ^a 4.2 ± 0.1 b 3.8 ± 0.2° 3.7 ± 0.1 ^c

Campylobacter + Pea starch coating 4.2 + 0.5 ^a 3.4 ± 0.1 b 4.6 ± 0.3 ^a 2.5 + 0.1 ^c

Campylobacter + H ₂ O + Thyme oil 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

Campylobacter + Pea starch coating 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 + Thyme oil

Different superscripts indicate a significant difference of values in rows (t-test, n = 6, p < 0.05). ND stands for not detectable (< 100 cfu/g).

Table 7 Effects of thyme oil treatments on the growth (log cfu/g) of Listeria monocytogenes on chicken breast meat at 4 ⁰ C.

Treatments Day O Day 4 Day 8 Day 12

Total aerobes

No treatment 3.0 ±0.6 4.6 ±0.1 6.8 ±0.1 7.7 ±0.0

Listeria inoculation 5.6 ± 0.0 5.2 ± 0.4 6.9 ± 0.2 8.1+0.9

Listeria + Pea starch coating 4.710.1 6.1 ±0.1 7.2 ±0.1 8.3 ±0.0

Listeria + H ₂ O + Thyme oil 4.0 + 0.4 ^a 3.5 + 0.1 ^b 5.3 ±0.6 5.1 ±0.6

Listeria + Pea starch coating + 4.5 ± 0.3 5.1 ±0.1 5.9 ±0.8 6.8 ±0.5

Thyme oil

Lactic acid bacteria

No treatment 2.5 ±0.5 4.6 ±0.1 6.7 ±0.2 7.6 ±0.1

Listeria inoculation 5.5 ±0.1 5.6 ±0.0 6.9 ± 0.2 7.7 ± 0.2

Listeria + Pea starch coating 4.8 ±0.1 5.8 ±0.2 7.0 + 0.4 7.8 ±0.1

Listeria + H ₂ O + Thyme oil 3.9 ± 0.4 ^a 3.3 ± 0.1 ^b 3.9 ± 0.6 ^a 5.0 ± 0.1 ^b

Listeria + Pea starch coating + 4.5 ± 0.3 4.5 ±0.5 5.3 ±0.3 5.5 ±1.0

Thyme oil

Listeria monocytogenes

No treatment 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

Listeria inoculation 5.5 ±0.1 5.5 ±0.0 5.9 ±0.1 6.4 + 0.2

Listeria + Pea starch coating 4.7 ± 0.0 5.9 ±0.3 6.6 ± 0.3 7.2 ± 0.2

Listeria + H ₂ O + Thyme oil 6.0 + 0.4 ^a 3.1 ±0.0 ^d 3.6 ± 0.3 ^c 5.1 ±0.0 ^b

Listeria + Pea starch coating + 4.3+0.3 4.6 + 0.5 4.8 + 0.1 5.1+0.2

Thyme oil

Different superscripts indicate a significant difference of values in rows (t-test, n = 6, p < 0.05).

Table 8 Effects of thyme oil treatments on the growth (log cfu/g) of Ps eudomonas aeruginosa on chicken breast meat at 4 ⁰ C.

Treatments DayO Day 4 Day 8 Day 12

Total aerobes

No treatment 3.2 ±0.1 5.5 ±0.0 6.9 ± 0.2 7.9 ± 0.2 Pseudomonas inoculation 5.1 ±0.1 5.6 ±0.6 7.0 ± 0.3 7.5 ± 0.6 Pseudomonas + Pea starch coating 4.8 ±0.1 5.5 ± 0.6 6.9 ±0.1 8.2 ± 0.1 Pseudomonas + H ₂ O + Thyme oil 4.2 ± 0.0 4.6 ± 0.5 4.9 ±0.6 6.8 ± 0.4 Pseudomonas + Pea starch coating 44..00 ±± 00..33 b 3.1 ±0.7 a 5.1 ±0.8° 5.6 ± l.l ^c + Thyme oil

Lactic acid bacteria

No treatment 2.4 ± 0.4 4.9 ± 0.3 6.0 ± 0.2 7.3 ± 0.3 Pseudomonas inoculation 5.0 ± 0 i.l 4.9 + 0.1 6.3 ± 0.2 6.9 ± 0.3 Pseudomonas + Pea starch coating 4.2 ±1. ,2 4.8 ± 0.5 5.9 ± 0.5 7.4 ± 0.0 Pseudomonas + H ₂ O + Thyme oil 4.1 ±0. ,1 4.3 ± 0.5 4.6 ± 0.5 5.9 ± 0.5 Pseudomonas + Pea starch coating 3.9 + 0. ,3 ^b 2.7 ± 0.8 a 4.5 ±0.7 b 5.1 ± 0.9 ^b ' ^c + Thyme oil

Pseudomonas aeruginosa

No treatment 3.2 ±0.1 5.0 ±0.2 7.6 ±0.1 7.9 ±0.5

Pseudomonas inoculation 5.1 ± 0.2 5.3 ± 0.3 7.8 ± 0.0 7.7 ± 0.2

Pseudomonas + Pea starch coating 4.8 + 0.1 5.2 ±0.3 7.6 ±0.1 8.4 ±0.1

Pseudomonas + H ₂ O + Thyme oil 4.1 ±0.0 4.5 ±0.5 6.0 ±0.6 6.8 ±0.1

Pseudomonas + Pea starch coating 4.0 ± 0.2 ^b 2.8 ± 0.9 ^a 5.6 + 1.9 ^b ' ^c 6.0 ± 1.0 ° + Thyme oil Different superscripts indicate a significant difference of values in rows (t-test, n = 6, p < 0.05).