Effectiveness of antimicrobial food packaging materials

Transcription

1 Food Additives and Contaminants, October 2005; 22(10): Effectiveness of antimicrobial food packaging materials K. COOKSEY Department of Packaging Science, Clemson University, 229 Poole Ag Center, Box , Clemson, SC , USA Abstract Antimicrobial additives have been used successfully for many years as direct food additives. The literature provides evidence that some of these additives may be effective as indirect food additives incorporated into food packaging materials. Antimicrobial food packaging is directed toward the reduction of surface contamination of processed, prepared foods such as sliced meats and Frankfurter sausages (hot dogs). The use of such packaging materials is not meant to be a substitute for good sanitation practices, but it should enhance the safety of food as an additional hurdle for the growth of pathogenic and/or spoilage microorganisms. Studies have focused on establishing methods for coating low-density polyethylene film or barrier films with methyl cellulose as a carrier for nisin. These films have significantly reduced the presence of Listeria monocytogenes in solutions and in vacuum packaged hot dogs. Other research has focused on the use of chitosan to inhibit L. monocytogenes and chlorine dioxide sachets for the reduction of Salmonella on modified atmosphere-packaged fresh chicken breasts. Overall, antimicrobial packaging shows promise as an effective method for the inhibition of certain bacteria in foods, but barriers to their commercial implementation continue to exist. Keywords: Antimicrobial packaging, nisin films, chitosan, chlorine dioxide, modified atmosphere Introduction Antimicrobial packaging has been an area of interest for many years. As research in the area continues, more systems are showing promise for commercial application. The concept behind antimicrobial packaging is to enhance the safety and quality measures already used by the food industry. It is not meant as a substitute for good manufacturing and handling practices, but is meant to serve as an additional hurdle for bacteria to overcome. Several conditions should be considered when designing an antimicrobial packaging system. First, the regulatory status of the antimicrobial agent is important. Some antimicrobial systems rely on diffusion or release of the antimicrobial agent in order for it to be effective. This would place the packaging material under the classification of an indirect food additive and the material would require review by the US Food and Drug Administration (FDA) along with similar scrutiny in other countries. Generally, approval may not be difficult if the additive has already been effectively used as a direct food additive as long as migratory concentrations and conditions of use are addressed. Another issue is the cost-to-benefit ratio. Some antimicrobial systems can be effective, but if produced on a large scale, they might require expenses beyond the benefits obtained by an extended shelf life or improvement in quality. Lastly, there are numerous technical challenges related to coating methods, the rate of curing, the ease of heat sealing, the effects on physical and mechanical properties of film, the effects on colour, the texture or flavour of the food, and the ability of the antimicrobial agent to provide effectiveness throughout the package/ product life cycle. The challenges can be daunting, but as research in the field progresses, there is promise for many systems to meet these challenges. A variety of antimicrobial packaging systems have been reviewed (Kesler and Fennema 1986; Krochta and De Mulder-Johnston 1997; Han 2000; Cooksey 2001; Brody 2002). While some films have incorporated the antimicrobial agent into the polymer (Siragusa et al. 1999), others have used Correspondence: K. Cooksey. kcookse@clemson.edu ISSN X print/issn online ß 2005 Taylor & Francis DOI: /

2 Effectiveness of antimicrobial food packaging materials 981 biopolymer films as effective carriers of antimicrobial agents (Padgett et al. 1998; Coma et al. 2001). Many of these biopolymer films are cellulose-based, and because of their water-soluble nature, they effectively release additives when combined with foods of high water content. For example, upon contact, a cellulose-based matrix degrades and releases the antimicrobial agent from the matrix to the surface of the food product resulting in bacterial inhibition. However, chitosan is a biopolymer with inherent antimicrobial properties and it does not require a carrier. It can be used as a coating or cast into films with good strength and barrier properties (Wiles 2000). Unlike the previous systems mentioned, vapour-active antimicrobial packaging systems do not require direct surface contact. Vapour-active antimicrobial agents include allyl isothiocyanate, ethanol and chlorine dioxide. Nisin has Generally Recognized as Safe (GRAS) status in the USA for use in processed cheese spreads. It is an lantibiotic produced by Lactobacillus lactis and destroys target cells by incorporating itself into their cytoplasmic membranes, which leads to a loss of intracellular ions and disruption of the ph gradient and proton motive force (Klaenhammer 1993; Breukink and De Kruijff 1999; Jydegaard et al. 2000). The initial step of the antibacterial action is the binding of nisin molecules to the cell membrane. Studies have shown that nisin preferentially binds to membranes containing anionic lipids. Gram-positive bacteria generally have higher concentrations of anionic lipids in their plasma membrane than Gram-negative bacteria, which may explain the increased antimicrobial activity towards Gram-positive bacteria (Breukink and De Kruijff 1999). Nisin is also more effective in an acidic environment and some studies have shown that there may be synergistic effects depending upon the type of acid used with nisin in solution. Chitosan is derived from crustacean shells, insect exoskeletons and cells walls of fungi. It possesses antimicrobial activity due to its polycationic nature which allows it to react readily with negatively charged molecules and surfaces such as microbial cell walls. Chitosan is a deacetylated version of chitin and has an abundance of highly reactive amine groups (Wang 1992; No et al. 2002). The more amino groups present, the more deacetylated the chitosan. It is not possible to produce fully deacetylated chitosan. Commercially available chitosan is typically available in the range 70 95% deacetylation (DA). Chlorine dioxide (ClO 2 ) is a gas that is rapidly gaining attention for use as an antimicrobial agent in active packaging systems. Unlike the previous methods, it does not rely on direct surface contact to be effective. It has FDA approval and its use to reduce or eliminate microbial loads in a wide variety of food products such as fruits and vegetables is becoming more intriguing (Rulis 2001). The following is an overview of several studies performed in our laboratories over the past 4 years. The overall objective of the research has been to develop antimicrobial films and test their effectiveness for food-packaging applications. Materials and methods Coating of nisin onto films The first phase of the study was to develop the coating that was to act as a carrier for the nisin. Based on a review of the literature, a cellulose-based solution was chosen. The packaging film coating was prepared by blending methyl cellulose (0.875 g) and hydroxypropyl methyl cellulose (0.375 g) with ethanol (25 ml) and PEG 400 (0.75 ml). Total biopolymer concentration in final solution was 70/30% MC/HPMC on a dry weight basis. When nisin was added to the coating, it was first dissolved in 0.02 N acetic acid (ph 2) and added to distilled water for initial activation of nisin and then blended with the coating solution. Initially, the maximum level of nisin allowed for use in cheese products was chosen as the highest level to be tested in the coating solution. A minimum concentration of effectiveness of nisin in solution was determined by Grower et al. (2004a). Nisin film-coating solutions were prepared containing a maximum level of IU ml 1 (2.5 g) as a stock solution and serial dilutions ranging from to 8IUml 1 were used. Once an optimal coating solution was developed, it was applied to the surface of a packaging film (Franklin et al. 2004; Grower et al. 2004a, b). Low-density polyethylene (LDPE) was used as the base film since it is one of the most commonly used packaging films. Film samples were taped to a cm glass plate and nisin-based (50 ml) or control solutions (no nisin) were cast onto the film with a thin-layer chromatography (TLC) plate coater (CAMAG, Muttenz, Switzerland) to obtain a 500-mm coating thickness. The coated LDPE was dried at ambient conditions (22 C, 28% relative humidity). Before testing, nisin-treated film and control samples were cut into mm squares and treated under ultraviolet light with a Zeta TM 7400 (Loctite Corp., Newington, CT, USA) for 5 min to sterilize any contaminates on the film introduced during production. The degree to which nisin could diffuse from the coating into a solution was studied by Grower et al. (2004b). LDPE film was coated

3 982 K. Cooksey with a solution containing a high or low viscosity methylcellulose and hydroxypropyl methylcellulose. Films contained , 7500, 5000, 2500 or 0IUcm 2 nisin (control). Film samples were placed into peptone water and 10-ml samples were removed and placed onto spiral plated lawns of L. monocytogenes. A modification of the spot on lawn assay called a film on lawn assay was used to determine the effectiveness of the nisin-containing LDPE coated film for inhibition of L. monocytogenes on tryptic soy agar (TSA) plate and modified oxford plates (Franklin 2002). LDPE film was coated with a cellulose-based solution containing no nisin or , 7500, 2500 or IU ml 1 nisin. Modified oxford (MOX) and TSA plates were spiral plated with L. monocytogenes populations ranging from approximately 7 10 log CFU cm 2. Film samples (10 10 mm) were placed onto the inoculated plates. The effectiveness of film coatings was determined by measuring the zones of inhibition. The main objective of the next study was to determine the effectiveness of packaging films coated with a methyl cellulose/hydroxypropyl methyl cellulose (MC/HPMC) based solution containing , 7500, 2500 or IU ml 1 nisin for controlling L. monocytogenes on the surface of vacuum-packaged hot dogs (Franklin et al. 2004). Barrier film coated with MC/HPMC-based solution containing nisin or no nisin (control) was heat sealed to form individual pouches. Hot dogs were placed in control and nisincontaining pouches and inoculated with a five-strain L. monocytogenes cocktail (approximately 5 log CFU/ package), vacuum sealed and stored for intervals of 2 h, 7, 15, 21, 28 and 60 days at 4 C. After storage, hot dogs and packages were rinsed with 0.1% peptone water. Diluent was spiral plated on MOX agar and TSA and incubated to obtain counts reported as CFU/package. Chlorine dioxide in modified atmosphere packaging Research was carried out to observe the effect of chlorine dioxide and modified atmosphere packaging on the quality of fresh chicken breasts under refrigerated storage for 15 days (Ellis 2003). Each chicken breast was inoculated with 4 logs cfu ml 1 culture of Salmonella typhimurium NAR (nalidixic acid-resistant strain) and placed into a barrier foam tray. Fast or slow release chlorine dioxide sachets were placed next to the chicken in each package. A control set of packages that did not contain a chlorine dioxide sachet was also included in the study. Packages were flushed with either 100% N 2 or 75% N 2 /25% CO 2 and stored at 3 C. Microbial analysis and sensory (appearance and aroma) analysis were performed every 3 days for 15 days. Results and discussion Nisin films The range of effectiveness of nisin against L. monocytogenes is shown in Figure 1. The results indicate that a nisin level of 156 IU ml 1 was the minimum inhibitory concentration necessary to inhibit L. monocytogenes. Therefore, 156 IU ml 1 became the lowest amount of nisin to be added to the coating for further studies. In addition to the nisin level, the effect of different organic acids were tested (Figure 2) to determine if they enhanced the inhibitory effect of nisin, but all four acids produced similar antimicrobial activity against the pathogen and no significant differences in zones of inhibition ( p > 0.05) were observed (Grower et al. 2004a). Grower et al. also examined the physical properties of the film. For example, film coatings containing increasing levels of nisin appeared cloudier than those with lower levels of nisin or no nisin. Note that the composition of commercially prepared nisin Chitosan films The specific objective was to study the antimicrobial effects of chitosan solutions with different per cent DA values (90, 85, 80%) and determine whether viscosity had an effect on the antimicrobial properties of chitosan solutions with three different per cent DA values (Campbell 2003). A spot on lawn assay was used to determine the effectiveness of the chitosan with 90, 85 and 80% DA (high and low viscosity of each) against L. monocytogenes. Films were made using 1 and 2% solutions of each type of chitosan and statistical comparisons ( p < 0.05) were made using a one-way analysis of variance. Mean zones of inhibition (mm) Nisin concentration (IUml 1 ) Figure 1. Inhibition of Listeria monocytogenes using different concentrations of nisin (IU ml 1 ). Zones of inhibition for nisin concentrations 78, 39, 19 and 9 IU ml 1 are zero

4 Effectiveness of antimicrobial food packaging materials 983 consists of 2.5% nisin, 77.5% sodium chloride (NaCl), 12% protein (in the form of milk solids), 6% carbohydrate and 2% moisture. Field-emission scanning electron microscopy was unsuccessful for determining whether nisin was homogenously dispersed within the cellulose matrix. However, it was successful in confirming that the cloudiness Mean zones of inhibition (mm) Ascorbic Acetic Lactic HCl Acids Figure 2. Means and standard deviations of zones of inhibition (mm) measured against Listeria monocytogenes to determine the effect of nisin dissolved in different types of acids. No significant differences ( p > 0.05) were observed among acids. of the film was attributed to the salt and milk solids contained within the nisin preparation and not nisin itself. To determine whether nisin was released from the cellulose-based coating, coated LDPE film samples containing differing levels of nisin were placed in peptone solution. As shown in Table I, nisin diffused from the coating solution from 1 min to 8 h, did not diffuse after 24 h and 4 days but exhibited zones of inhibition after 8 days once the coating was completely dissolved. The viscosity of the cellulosebased carrier had no effect (Grower et al. 2004b). These results indicated that nisin can be released from the coating into solution, but the rate of release is not controlled. Franklin (2002) determined the degree to which L. monocytogenes could be inhibited on solid media. Film coatings containing no nisin and IU ml 1 nisin had no inhibitory effect on L. monocytogenes grown on TSA or MOX at 37 C for 48 h and 4 C for 17 days (Tables II and III). Films coatings containing 2500, 7500, and IU ml 1 nisin were effective for inhibiting L. monocytogenes on both agars at both storage conditions. Zones of inhibition were greater for Table I. Mean zones of inhibition (mm) measuring the rate of release of nisin in solution over time from film coatings made from high (H) and low (L) viscosities of methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC) containing various concentrations of nisin. 1 min 30 min 60 min 8 h 24 h 4 days 8 days Control L L L 2.75 b 2.25 c b,c 7500 L 6.75 a 3.50 a c 5.00 a 3.50 b a,b L 7.38 a 7.00 a 6.33 a 6.50 a a Control H H H 2.25 b 2.50 b,c b,c 7500 H 5.00 a,b 3.50 a,b,c 4.50 a 3.25 b a H 7.38 a 6.75 a,b 5.50 a 5.75 b a a,b,c Superscripts indicate significant differences ( p < 0.05) among film coating treatments during each period. Treatments that did not produce zones of inhibition were not included in statistical analyses. Table II. Mean zones of inhibition (mm) from LDPE coated with IU ml 1 nisin solution. No zones of inhibition were observed from LDPE coated with solutions containing IU ml 1. Population (log CFU cm 2 ) TSA (37 C, 48 h) Zone of inhibition (mm) IU ml 1 TSA (4 C, 17 days) MOX (37 C, 48 h) MOX (4 C, 17 days) A,x B,y * ** A,a,x B,y 25.4 b,z ** A,a,x B,y b,z ** a,x y b,z ** A,B Superscripts indicate significant differences ( p < 0.05) when comparing between the same agar type and different incubation temperatures (37 C vs. 4 C). a,b Superscripts indicate significant differences ( p < 0.05) when comparing between the different agar types (TSA vs. MOX) and the same incubation temperature. x,y,z Superscripts indicate significant differences ( p < 0.05) when comparing the same agar types (TSA or MOX) and different populations (log 7 10 CFU cm 2 ). * LM growth not sufficient to measure zones of inhibition. ** LM eliminated from nisin-containing film side of agar.

5 984 K. Cooksey Nisin concentration (IU ml 1 ) Table III. Mean zones of inhibition (mm) from LDPE coated with 7500 and 2500 IU ml 1 nisin solution. Population (log CFU cm 2 ) Zone of inhibition (mm) TSA (37 C, 48 h) TSA (4 C, 17 days) MOX (37 C, 48 h) MOX (4 C, 17 days) A,a,x B,x b ** A,a,x B,x b ** A,a,x B,x b ** A,a,x * b ** A,a,x B,y b ** A,a,x B,y b ** A,a,x B,y b ** A,B Superscripts indicate significant differences ( p < 0.05) when comparing between the same agar type and different incubation temperatures (37 C vs. 4 C). a,b Superscripts indicate significant differences ( p < 0.05) when comparing between the same LM populations and different agar types (TSA vs. MOX) at the same incubation temperature for both 7500 and 2500 IU ml 1 films. x,y Superscripts indicate significant differences ( p < 0.05) when comparing between the same agar type and population at different nisin concentrations (2500 and 7500 IU ml 1 ). * LM growth not sufficient to measure zones of inhibition. ** LM eliminated from nisin-containing film side of agar. Table IV. Listeria monocytogenes (five-strain cocktail) populations on the surface of hot dogs packaged in film coated with methyl cellulose/ hydroxypropyl methyl cellulose solutions containing , 7500 and 2500 IU ml 1 nisin or no nisin (control) when enumerated on tryptic soy agar (TSA). Storage (days) Nisin (IU ml 1 ) þ 0.26 a,x 5.51 þ 1.16 a,x 6.13 þ 0.50 a,x 6.33 þ 0.24 a,y 8.01 þ 1.83 a,y 9.11 þ 0.93 a,y þ 0.45 a,x 4.9 þ 0.21 b,x 4.90 þ 0.16 b,x 5.37 þ 0.45 b,y 7.50 þ 2.29 b,y 9.52 þ 0.43 a,y 2500 n.d. b n.d. c n.d. c n.d. c n.d. c n.d. b 7500 n.d. b n.d. c n.d. c n.d. c n.d. c n.d. b n.d. b n.d. c n.d. c n.d. c n.d. c n.d. b n.d., Populations were below the detectable limit (<2.9 log CFU/package) throughout the study. a c Significant differences ( p < 0.05) between nisin treatments (no nisin, 2500 and 7500 IU ml 1 ) for each day of storage. x,y Significant differences ( p < 0.05) between the storage day for each treatment. Table V. Listeria monocytogenes (five-strain cocktail) populations on the surface of hot dogs packaged in film coated with methyl cellulose/ hydroxypropyl methyl cellulose solutions containing , 7500, 2500 IU ml 1 nisin or no nisin (control) when enumerated on modified oxford agar (MOX). Storage (days) Nisin (IU ml 1 ) þ 0.25 a,w 5.45 þ 1.21 a,w 5.82 þ 0.64 a,w 6.07 þ 0.37 a,w 7.95 þ 1.84 a,x 9.06 þ 0.90 a,x þ 0.50 b,w 4.49 þ 0.33 b,w 4.42 þ 0.13 b,w 5.34 þ 0.64 b,x 7.50 þ 2.50 b,y 9.40 þ 0.43 a,z 2500 n.d. c n.d. c n.d. c n.d. c n.d. c n.d. b 7500 n.d. c n.d. c n.d. c n.d. c n.d. c n.d. b n.d. c n.d. c n.d. c n.d. c n.d. c n.d. b n.d., Populations were below the detectable limit (<2.9 log CFU/package) throughout the study. a c Significant differences ( p < 0.05) between nisin treatments (no nisin, 2500 and 7500 IU ml 1 ) for each day of storage. w z Significant differences ( p < 0.05) between storage day for each treatment. film coatings containing 2500, 7500 and IU ml 1 nisin incubated for 17 days at 4 C (MOX and TSA) compared with those incubated for 48 h at 37 C (MOX and TSA). Franklin et al. (2004) also studied the effectiveness of nisin-coated barrier bags for inhibition of L. monocytogenes in vacuum-packaged hot dogs. L. monocytogenes counts on hot dogs packaged with IU ml 1 nisin level films decreased slightly (approximately 0.5 log reduction) through day 15 of refrigerated storage, but it was statistically the same ( p > 0.05) as hot dogs packaged in films without nisin after 60 days of storage (Tables IV and V). Packaging films coated with a cellulose-based solution containing and 7500 IU ml 1 nisin significantly decreased ( p < 0.05) L. monocytogenes populations on the surface of hot dogs by more than 2 logs CFU/package throughout the 60-day study. Similar results were observed for hot dogs packaged with 2500 IU ml 1 nisin level films;

6 Effectiveness of antimicrobial food packaging materials 985 however, L. monocytogenes populations were observed to be approximately 4 log CFU/package after 60 days of refrigerated storage from plate counts on TSA and MOX (Franklin et al. 2004). Chitosan solutions As shown in Table VI, all six types of chitosan solutions equally inhibited growth of L. monocytogenes on TSA and MOX agar for both 1 and 2% solutions (Campbell 2003). Films were also produced using the same chitosan solutions, and although films with good tensile, elongation and permeation properties were produced (data not shown), it was difficult to test their antimicrobial properties using the film on lawn assay due to excess curling of the film. A solution to this problem is to use the film overlay method. Table VI. Inhibition of Listeria monocytogenes by 1 and 2% chitosan solutions on TSA and MOX agar. 1 Log CFU ml 1 Deacetylation (%) Viscosity High þ þ þ 90 Low þ þ þ 85 High þ þ þ 85 Low þ þ þ 80 High þ þ þ 80 Low þ þ þ þ, Inhibition only at the film/medium interface within the 7 8 mm diameter of the applied chitosan droplet. 1 Data represent results that were identical for both types of agar. Chlorine dioxide modified atmosphere packaging Total plate counts for chicken (Figure 3) increased steadily after 6 9 days of storage regardless of package atmosphere or ClO 2 treatment. However, those treated with ClO 2 sachets had log cfu/chicken breast lower total plate counts compared with those without ClO 2 sachets. After 15 days, samples treated with ClO 2 (fast and slow release sachets) had significantly lower S. typhimurium NAR populations (approximately 1 log) compared with chicken which did not contain ClO 2 sachets (Figure 4). The ClO 2 adversely affected the colour Figure 3. Total plate count of chicken breasts (log cfu/chicken breast) stored at 3 C. Day 0 means were 5.51 log cfu/chicken breast for all treatments.

7 986 K. Cooksey Figure 4. Salmonella typhimurium (NAR strain) counts (mean þ pooled standard error) of chicken breasts (log cfu/chicken breast) stored at 3 C. Day 0 means were 3.95 log cfu/chicken breast for all treatments. ClO 2 release Table VII. Sensory panel aroma values (mean þ pooled standard error) of chicken breasts stored at 3 C % N 2 25% CO 2 100% N 2 25% CO 2 100% N 2 25% CO 2 100% N 2 25% CO 2 75% N 2 / 75% N 2 / 75% N 2 / 75% N 2 / None a,y f,y a,x f,x b,y f,y b,y g,y Fast a,x f,x a,w f,w a,x f,x a,x f,x Slow a,z f,z a,y f,z a,z f,z a,z f,g,z Day 0 means 0.52 for all treatments. 0 ¼ fresh (a fresh reference sample was provided); 15 ¼ off odour. a,b Significant differences within columns (100% N 2 atmosphere) for each day of storage. f,g Significant differences within columns (75% N 2 / 25% CO 2 atmosphere) for each day of storage. w,x,y Significant differences within rows (ClO 2 release rates) for each day of storage. of the chicken, as areas close to the sachet were brown or green. No off odour (Table VII) was detected by the sensory panellists (Ellis 2003). Conclusions Based on the above studies, nisin was found to be an effective antimicrobial agent and the package coating solution successfully served as a carrier for the nisin. The effectiveness of the nisin was reduced when incorporated into the coating matrix as compared with a solution, but levels of 2500 and 7500 IU ml 1 significantly reduced the population of L. monocytogenes on hot dogs for 60 days of refrigerated storage. This also demonstrates the importance of testing an antimicrobial packaging system using solutions, agar and food systems for best overall results. Chitosan was also an effective antimicrobial material, but there was no advantage in using differing levels of the percent DA or viscosities with regard to inhibition of L. monocytogenes. Chlorine dioxide combined with modified atmosphere packaging reduced the population of S. typhimurium NAR in fresh chicken, but it was less effective against the natural microflora and had an adverse effect on the colour of the chicken placed close to the sachets. All antimicrobial packaging systems showed promise for reducing the population of selective bacteria in food and each also had some conditions that require further study to make them practical for industrial applications.

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