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PROCESSING, PRODUCTS, AND FOOD SAFETY |
Department of Food Science, Faculty of Agricultural and Foods Sciences, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada
1 Corresponding author: hanjh{at}ms.umanitoba.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: antimicrobial coating • broiler • Salmonella • calcium alginate • pea starch
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Present understanding of poultry skin properties suggests that scalding at 
60°C removes the surface cuticle, which affects skin adhesiveness (Lucas and Stettenheim, 1972), making the surface more hydrophilic and facilitating microbial contamination (Suderman and Cunningham, 1980). During washing, collagen in the underlying dermal layer swells and provides further opportunity to shield surface contaminants (Thomas and McMeekin, 1982). Given these parameters, work in the present study was designed to characterize interactions of candidate coatings (containing AM) with poultry skin. For evaluation of coating suitability, Choi and Han (2002) and Han and Krochta (1999) measured the cohesive force between coatings and the skin surface. This was done by calculating the angle formed by the tangent of the surface of a coating droplet at the edge of its contact with the skin being tested. Such measurements take into account the surface energy of the coated material, which is roughly analogous to the surface tension of a liquid. When the contact of an applied liquid at a surface equals zero, the interfacial surface energy between the surface and the liquid is equal to the surface tension of the liquid applied (Michalski et al., 1997). The liquid absorption rate and maximum absorptiveness can be used to characterize the relationship of the coating to its substrate.
Consumer interest in unprocessed foods preserved with natural ingredients has significantly increased recently (Debeaufort et al., 1998; Cagri et al., 2004). Development of edible films and coatings having more desirable properties than synthetic preservatives is an approach taken to satisfy this interest (Mehyar and Han, 2004). Starch and alginate, respectively, 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) in pea starch (PS) and acidified sodium chlorite (ASC) in alginate as coatings, when applied to broiler carcasses during processing for their ability to reduce surface contamination by Salmonella. Because 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 24 h for completion of carcass processing and neutralization of the additives.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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A 100-mL dispersion of 3.5% (wt/vol) PS (Nutri-Pea Ltd., Portage-la-Prairie, Manitoba, Canada) 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 TSP (Sigma Chemical Co., St. Louis, MO) was added (10% wt/vol), mixed, and homogenized by a Powergen-700 (Model GLH 115, PG700, Fisher Scientific International Inc., Hampton, NH) for 5 s at 20,000 rpm. This yielded PS + TSP coating solution.
Calcium alginate coating (alginate + ASC) consisted of 2 solutions of 100 mL each. Solution (a) was 1% (wt/vol) calcium chloride (Sigma Chemical Co.) in ASC (1,200 ppm) prepared by mixing equal portions of the acid and salt parts of Sanova provided by Alcide Corp. (Redmond, WA). This solution was used within 30 min as recommended by the manufacturer. Solution (b) contained 1% (wt/vol) sodium alginate (Sigma Chemical Co.) dissolved in water and mixed. Coatings free of AM (water controls) were prepared following the same procedures but without TSP addition to PS and without ASC addition to alginate. The PS + TSP solutions containing 0.5, 1.5, 2.0, 3.5, 4.0, or 4.8 % (wt/vol) PS, and alginate + ASC with 0.5, 1.0, or 1.5% (wt/vol) alginate were prepared as outlined above. These solutions were used for absorptiveness, contact angle, and viscosity measurements.
Chicken Treatment
Scalded, unchilled chicken thighs and drumettes (Mehyar et al., 2005) were obtained from a local processing plant where they were portioned from the carcass immediately after the inside carcass washer before chilling. The warm thighs were used within 30 min after arrival at the laboratory 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) provided by R. Ahmed (Canadian Science Centre for Human and Animal Health, Winnipeg, Manitoba, Canada). The 3 strains were grown separately in tryptic soy broth (TSB; Difco division of Becton Dickinson, Sparks, MD) for 24 h at 37°C. Cultures were standardized to an optical density at 600 nm of 0.80 using sterile TSB to yield about 9 log cfu/mL (confirmed by plating on TSB agar) 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 0.2 to 0.25 min. 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% wt/vol); (2) ASC (1,200 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% (wt/vol) PS without AM; or (6) 1% (wt/vol) calcium alginate without AM. Drumettes were weighed before and directly after dipping using a digital balance (Model TR-203, Denver Instrument Co., Denver, CO; ±0.00005 g). The drumettes were hung inside a covered glass chamber with 85% relative humidity and incubated at 4°C for 120 h. Triplicate drumettes were withdrawn for testing after 1, 24, 72, and 120 h incubation, and experiments were repeated twice (n = 6).
Changes in Drumette pH, Weight, and Viable Salmonella after Coating
At each sampling day, the surface pH of the coated drumettes was measured at 3 different locations using a pH meter equipped with an Isfet surface probe (Type Titan, Sentron Europe B. V., Roden, the Netherlands; sanitized in chlorine between use), and their average values were recorded. Drumettes were then weighed aseptically, their skins were excised and placed in stomacher bags with buffered peptone water (10 g of peptone, 5 g of NaCl, 3.5 g of Na2HPO4, 1.5 g of KH2PO4 per L) and homogenized for 3 min to prepare 10–1 homogenates. The homogenates were then serially diluted and plated on prepoured XLD agar (Oxoid, Ltd., Nepean, Ontario, Canada) containing 100 ppm of ampicillin (Oxoid, Ltd.). Salmonella were counted after 24 h at 35°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 4 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 (Wo) and 5 mL of the PS + TSP coating solution, or 2.5 mL of 1% (wt/vol) 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 and experiments were repeated twice (n = 6). Absorption was terminated by wiping away the excess coating solutions that remained on the skin surface with a tissue at each sampling time. The weights of the apparatus holding the skin were recorded before (Wwet) and after drying (Wdry). The absorptiveness (% At) was defined as:
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where We is the weight of an empty apparatus without skin.
Contact Angle and Skin Wetting Properties
The contact angles of probe liquids [HPLC grade water (Fisher Scientific International Inc.), glycerol (Sigma Chemical Co.), ethylene glycol (Fisher Scientific International Inc.), and dimethyl sulfoxide (Sigma Chemical Co.)] and the coating solutions on the skin were used to try to determine the surface energy of the skin (equal to the surface tension of an applied liquid when the contact angle is zero) and adhesiveness characteristics of the PS and alginate coating solutions. The surfaces of fresh, un-chilled chicken thighs were wiped with a dry tissue to remove residual water. The thighs were cut lengthwise to the bone with a razor blade and about one-third of the skin and flesh was removed. Cut thighs including the bone were placed on a rack with adjustable height and attached using plastic putty (Play Dough, Hasbro Canada, Longueuil, Quebec, Canada), which hardened upon exposure to air. A digital microscope (Intel play QX3 computer microscope, Santa Clara, CA; 10x magnification) was aimed horizontally to observe the cut chicken surface at a 90° angle. Drops of 10 µL of the probe liquids or coating solutions were placed separately at the edge of the uncut skin surface using a microsyringe, and side images of the liquid drops were recorded by a computer after confirming the horizontal level position of samples. The angle formed by the tangent of the drop circumference (surface) and the surface of the skin was measured and described as the contact angle. To account for any asymmetry of the image caused by irregularity in leveling, the contact angles of both sides of each liquid drop were measured and the average values were recorded. Surface energy, which can be equated to surface tension of an applied liquid, was only measurable when the contact angle was zero. All measurements were completed within 5 min and 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 (Sigma Chemical Co.). 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 (AR 1000, TA Instruments Inc., New Castle, DE). The instrument was operated with parallel plate geometry (plate diameter = 20 mm, gap = 1 mm). Coating solutions were placed in the apparatus and allowed to equilibrate at 25°C prior to analysis. Measurements were conducted at 3 Pa shear stress and 1 Hz frequency. The relationships between changes in contact angle and the viscosity of PS + TSP coating solutions with different concentrations of PS (0.5 to 6.0%; wt/vol) were determined.
Statistical Analysis
Data obtained were the average values from 3 replicates in each of 2 separate experiments. The statistical analytical system (Version 8.2, SAS Institute Inc., Cary, NC) was used to generate an ANOVA and a comparison of the treatment means for each set of samples. 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 spreadsheet (Microsoft Excel 2002, Redmond, WA) for the absorption curves (weight vs. time).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The PS + TSP and alginate + ASC coatings on chicken appeared clear, continuous, and homogenous. The alginate + ASC coating imparted a pale yellowish color to the drumettes, whereas the PS + TSP coating did not cause any noticeable visual changes. Figure 1
shows the reduction in Salmonella on drumettes over 120 h at 4°C. The PS not only maintained the AM activity of TSP longer but also increased its AM activity compared with the TSP treatment without PS. Enhanced AM activity was also exhibited by the alginate + ASC coating. Coatings with TSP and ASC had significantly (P 0.05) greater AM activity than the corresponding solutions without polymers after 24 h. The AM in aqueous solution and AM-free coatings were unable to cause >1.0 log cfu/g reductions. Previously, Mehyar et al. (2005) reported greater reductions of Salmonella using similar experimental conditions, however, a longer dipping time (1.0 min) was used than in the present study. Results obtained in this study were similar to those reported by Wang et al. (1997), Schneider et al. (2002), and Oyarzabal et al. (2004).
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), whereas the coating without TSP was better retained on the surface for 24 h. 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 7.86 and 6.88% 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).
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shows that TSP increased and ASC decreased the initial pH of the chicken skin. Although AM in solution caused significant (P 0.05) initial changes in the skin pH, the effects were transient and substantial changes did not last more than 24 h. The TSP and ASC in coatings significantly changed the surface pH, which was maintained up to 120 and 72 h, respectively (Figure 2). Gelatinized starch is soluble in aqueous environments (Ratnayake et al., 2002). It is capable of slowly dissolving within the pores and follicles of the skin and ostensibly releasing TSP into the skin, which improves its AM action. The alginate matrix seemed to be more stable, but chlorous acid (HClO2), 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; Schneider et al., 2002; Oyarzabal et al., 2004). From this part of the present study, it was shown that PS and alginate coatings have the potential to prolong the exposure of surface bacteria to TSP and ASC at high and low pH, respectively, and interfere with cell metabolic activity (Siragusa and Dickson, 1992). Industry adoption of coating technology will be dependent upon predictable control over gel stability to ensure gel drip from carcasses before shipment.
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The rate and amount of PS + TSP and alginate + ASC coatings absorbed by the skin depended on the polymer content of the coatings (Tables 2 and 3). At concentrations >3.5% PS and >0.5% alginate, the absorptiveness was significantly (P 0.05) reduced during 60 min. At the lowest PS concentration (0.5%), the amount of coating absorbed by the skin was higher than that of water (Table 2). In addition, these values were 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 that would not be removed by surface wiping may have contributed to additional weight gain. The 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. The rate and quantity of PS absorbed were higher compared with alginate at concentrations that exerted AM effectiveness (3.5 and 1.0%, respectively; Tables 2 and 3). This may explain the higher and more prolonged (120 h) antimicrobial effectiveness of the PS + TSP coating compared with the alginate + ASC coating (Figure 1
). Clearly, TSP in aqueous media had greater AM activity than ASC against Salmonella on chicken skin (Mehyar et al., 2005). In addition, gelatinized PS at low viscosity may more easily fill skin follicles and pores than the alginate gel, bringing TSP directly in contact with surface bacteria that may have been protected by irregularities in skin surface topography. Alginate + ASC exhibited higher AM activity than ASC alone only at 72 h of treatment (Figure 1B). This could have been due to the method of its application where the skin was first dipped in a calcium chloride solution containing 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.
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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 contact angle. Nonetheless, measurements of contact angles have been 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 enabled contact angle measurement. However, stable drops with measurable angles were unobtainable with alginate + ASC coatings. Because of low viscosity and surface irregularity, mixed calcium chloride and sodium alginate solutions diffused over the skin and yielded a thin film.
The PS + TSP coatings at low viscosity (below 0.37 N s·m–2) showed a linear response in terms of 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 3). The effect of PS concentrations on the contact angle as an indicator of coating adhesiveness to the skin is shown in Figure 4. In general, increasing the PS concentration decreased coating adhesion to the skin. At a low concentration of PS (<0.5%) 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 contact angles shown in Figures 3 and 4. Skin roughness was believed 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 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 contact angle was not affected by 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 the interfacial surface energy at the skin, they should have a viscosity in the range of 0.37 to 1.0 N s·m–2. Unfortunately, at high levels of PS (>4.0%) the solutions started to gelatinize with the result that the contact angles measured became independent of the surface energy difference and could not be used to measure surface energy differences. Overall, the adhesion of the coating to the skin depended on PS concentration and solution viscosity.
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24 h) stability on the skin surface but had good skin adhesion with low absorption and significant AM activity, 10% TSP in coatings of 1.5 to 2.5% (wt/vol) PS may be of industrial value in applications to reduce numbers of Salmonella on poultry skin. Work to design AM PS coatings with limited integrity is underway with the goal to maximize AM effectiveness and minimize detectable residues at carcass shipment.
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ACKNOWLEDGMENTS |
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Received for publication January 26, 2006. Accepted for publication September 18, 2006.
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REFERENCES |
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