Effectiveness of Selected Antimicrobial Agents and Their Possible Commercial Implementation

Transcription

1 Effectiveness of Selected Antimicrobial Agents and Their Possible Commercial Implementation Kay Cooksey, Clemson University Abstract Active packaging is defined as a package that senses and responds to stimuli and thus alters the package correspondingly. Many active packaging systems, such as antimicrobial packaging continue to be examined with hopes of future implementation. An overview of the challenges and factors for commercial implementation as well as some in depth results of antimicrobial packaging research on benzoic acid, nisin and chlorine dioxide will be presented. Overview of Antimicrobials used in Food Packaging Research A variety of antimicrobial packaging systems have been reviewed (1,2,3,4,5). Table 1 shows a representation of some of the antimicrobial food packaging agents that have been studied over the years. In general, antimicrobial packaging systems may be classified in two categories, surface active and non-surface active. Surface active antimicrobial materials, usually require direct surface contact to be most effective and include a carrier or support system that allows it to be incorporated into the package. For example, some films have incorporated the antimicrobial agent directly into the polymer (6), while others have used biopolymer films as effective carriers of antimicrobial agents (7,8). Many of these biopolymer films are cellulose-based and because of their water-soluble nature, 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 that has inherent antimicrobial properties and doesn t require a carrier. It can be used as a coating or can also be cast into stand alone films with good strength and barrier properties (9). Non-surface active antimicrobial packaging systems may also require a carrier or support system to deliver the antimicrobial but do not require direct surface contact for effectiveness. An example would be a sachet which would release a vapor active compound upon absorbing moisture from the product in the package. Vapor active compounds such as allyl isothiocyanate (AIT) can also be incorporated into the adhesive of labels applied to the inside surface of package and continually release into the package through a porous surface. (10) Table 1 List of types of antimicrobials studied Type Enzymes Chitosan Bacteriocins Antibiotics Organic acids Spices Citrus extracts Isothiocyanates Metals Fungicides Oxidizers Specific examples or derivatives Lyzoyme, Peroxidase Derived from Shellfish Nisin, Pediocin Imazalil benzoic, sorbic acid, ascorbic, propionic Rosemary, Garlic, Thymol Grapefruit seed extract, limonene Allyl isothiocyanate derived from derived from mustard, wasabi and/or horseradish Silver ions Benomyl, Ethanol Ozone, Chlorine dioxide Summary of Research on Nisin, Benzoic Acid and Chlorine dioxide Nisin The majority of research on antimicrobial food packaging materials has involved the incorporation of nisin into a cellulose-based coating for inhibition of Listeria monocytogenes. Initially, work was done to determine the optimum level of nisin necessary to inhibit Listeria monocytogenes in a solution prior to incorporation into the packaging coating. This was done with a spot on lawn assay which involved placing 10μL of solution containing a buffer solution (as the control) and antimicrobial agent. The droplet is placed upon the agar seeded with Listeria monocytogenes. The plates were incubated at 37oC for 48h and zones of inhibition around the droplet were

2 measured with a caliper. The range of nisin used was determined based on the maximum level of nisin allowed for use in cheese products which is 10,000 IU/mL. The study indicated that 156 IU/mL was the minimum inhibitory concentration of nisin and established the lowest effective range of nisin to use in future studies (11). The next step was to incorporate nisin into a coating that could be applied to low density polyethylene. Based on earlier studies by (11), a cellulose-based coating was selected. The coating was applied to low density polyethylene using a thin layer chromatography (TLC) coater set at a thickness of 500μm. Two methods were used to determine the effectiveness of the film coating containg nisin for inhibition of Listeria monocytogenes. The first method involved submersion of the film (containing 10,000, 7,500, 5,000, 2,500 or 0 IU/cm 2 nisin) in a buffer solution from which samples (10 μl) were removed at time intervals ranging from 1, 30, 60 minutes, 8, 24 hours, 4, and 8 days and placed on trypic soy agar (TSA) plates seeded with Listeria monocytogenes. Inhibition was determined by measuring zones of inhibition around each sample droplet placed upon the agar after incubation at 37 o C for 48h. Films containing 5,000, 7,500 and 10,000 IU/cm 2 nisin) inhibited Listeria monocytogenes after 30 min., films with 7,500 and 10,000 IU/cm 2 nisin inhibited Listeria monocytogenes after 60 min. and 8 h. No zones of inhibition were observed after 24h and 4 days for all films. After 8 days, zones of inhibition were observed for films with all levels of nisin except 2,500 IU/cm 2. Films with 0 and 2,500 IU/ cm 2 nisin did not produce zones of inhibition throughout the study. Overall, the coated film samples were effective for inhibition of Listeria monocytogenes, particularly when 7,500 and 10,000 IU/cm 2 nisin were used but the release of nisin was not controlled and appeared to be dependent upon dissolution of the coating into the buffer solution (12). The second method involved a technique referred to as film on lawn whereupon 10mm x 10mm squares of film were placed on agar plates spiral plated with a lawn of Listeria monocytogenes. Effectiveness of film coatings was determined by measuring zones of inhibition. Film coatings containing no nisin and IU/mL of nisin had no inhibitory effect on Listeria monocytogenes grown on TSA or modified oxford agar (MOX) at 37 C for 48 hours and 4 C for 17 days. Films coatings containing 2,500, 7,500, and 10,000 IU/mL of nisin were effective for inhibiting Listeria monocytogenes on both agars at both incubation conditions (13) The final study in this series involved determining the effectiveness of packaging films coated with a methylcellulose/hydroxypropyl methylcellulose (MC/HPMC) based solution containing 10,000, 7,500, 2,500 or IU/ml of nisin for controlling Listeria monocytogenes on the surface of vacuum-packaged hot dogs. 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 nisin-containing pouches, and inoculated with a five-strain Listeria monocytogenes cocktail (approximately 5 log CFU/package), vacuum-sealed, and stored for intervals of 2 hours, 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 modified oxford (MOX) agar and tryptic soy agar (TSA) and incubated to obtain counts reported as CFU/package. Listeria monocytogenes counts on hot dogs packaged with IU/mL nisin level films decreased slightly (approximately 0.5 log reduction) through day 15 of refrigerated storage but was statistically the same (p>0.05) as hot dogs packaged in films without nisin after 60 days of storage. Packaging films coated with a cellulose-based solution containing 10,000 and 7,500 IU/ml of nisin significantly decreased (p<0.05) Listeria monocytogenes populations on the surface of hot dogs by greater than two logs CFU/package throughout the 60 day study. Similar results were observed for hot dogs packaged with 2,500 IU/ml nisin level films; however, Listeria monocytogenes populations were observed to be approximately 4 log CFU/package after 60 days of refrigerated storage from plate counts on TSA and MOX (14). Current studies involving nisin are using similar techniques but utilize a nisin-rosemary blend rather than nisin alone (Matthews, 2006). Spot on lawn assays indicated that levels as low as 2.75mg/ml were effective for reducing Listeria monocytogenes on TSA agar. However, when the blend was incorporated into a cellulose coating and applied to a packaging film, results showed that a higher level of 5.5mg/ml were needed for inhibition and showed the same level of inhibition as levels as high as 22 mg/ml. The rosemary-nisin blend was further tested in a food challenge study whereupon the fresh beef cubes were inoculated with a 5 log CFU/ml population of Listeria monocytogenes and packaged in vacuum bags containing cellulose-based coating with no nisin-rosemary blend, or 5.5 mg/ml or 22mg/ml on the inside surface. Beef cubes were stored are refrigeration temperature and analyzed for microbial population on days 0,6,12,18, 24, 30 and 36. The results indicated that the beef contained in the nisinrosemary treated bags had significantly lower (1-1.5 log CFU/ml) populations of Listeria monocytogenes when measured on modified oxford agar. Overall, nisin-rosemary blend was less effective in the food challenge study compared to the film on lawn and spot on lawn methods which is consistent with the findings of the previous work

3 on nisin alone. Additionally, the nisin-rosemary blend was less effective than the nisin used alone for hot dogs performed by (15), probably due to the additional preservatives found in the hot dogs and lower levels of nisin used in the fresh beef cube study. Benzoic acid Organic acids have been proven effective as direct additives for food preservation and have also been studied in antimicrobial food packaging applications. A spot on lawn assay showed inhibition against Listeria monocytogenes at concentrations of 0.75, 1.5 and 3.0% when tested on TSA agar. The same levels were incorporated into a novel absorbent film and were also found to be effective for inhibition of Listeria monocytogenes on both TSA and MOX agars. In the final stage of the study the films were soaked in a solution of 10% benzoic acid, hot dogs were inoculated with a 5 log population and placed inside the film and sealed. The bags were stored at refrigeration temperature for 28 days and sampled for populations of Listeria monocytogenes. Listeria monocytogenes was completely inhibited (5 log reduction) compared to the control samples which maintained a 5 log population of Listeria monocytogenes throughout the study. Other studies support the effectiveness of organic acids and have also attributed their effectiveness to a ph effect (16). Chlorine dioxide Chlorine dioxide (ClO 2 ) is a gas that is rapidly gaining attention for use as an antimicrobial agent in active packaging systems. It has received self-affirmed GRAS status for certain applications with food and studies involving its use to reduce or eliminate microbial loads in a wide variety of food products such as fruits and vegetables is generating increased interest in the food industry. The objective of the research was to observe the effect of ClO 2 and modified atmosphere packaging on the quality of fresh chicken breasts under refrigerated storage for 15 days. Each chicken breast was inoculated with 4 log CFU/ml 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 o C. Microbial analysis, CIE L.a.b. color and sensory (appearance and aroma) was performed every 3 days for 15 days. Total plate counts for chicken 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 to 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 one log) compared to chicken which did not contain ClO 2 sachets. The ClO 2 adversely affected the color of the chicken, in areas close to the sachet. No off odor was detected by the sensory panelists (17). Challenges and Factors for Commercial Implementation Production challenges are one of the problems for commercial implementation of antimicrobials in food packaging materials. Those that can be incorporated into the polymer during extrusion are the most likely to be successful but questions still exist regarding release from such polymers and possible modifications to the antimicrobial that render it ineffective. Technologies that require soaking, coating, sachets or labels are also possible but require additional steps in the process which add cost. Alteration of polymer characteristics such heat seal strength or clarity may also occur when antimicrobial agents are added to the packaging materials. Regulatory approval is another serious issue. Some of the antimicrobials used in research studies are approved for direct food application but that doesn t necessarily mean that they can used in food packaging. The main question is whether the additive migrates intentionally or unintentionally and for what conditions is it to be approved. While the food contact notification process for new materials expedites commercialization of materials compared to the old process, it still requires proof that the material will be safe for use. Even if a company is willing to go through the process of applying for a non-objection status for use of their material, many are not willing to take on the liability of a material that was proven reliable under lab conditions but could be unreliable when used in the field. Ability to release or control release of the antimicrobial additives from the packaging system is another problem. Studies are finding better solutions to this problem and adapting technologies used by the pharmaceutical industry. Whether the additive requires release depends upon the type of package and level of contact the food has with the packaging. If the food is in intimate contact with the packaging and only requires reduction of bacteria on those surfaces, then release isn t as necessary as with a product contained within a package with headspace and not able to contact the antimicrobial agent.

4 More work needs to be done regarding the sensory and acceptability effects of the antimicrobial agents with foods. For example, antimicrobials released as volatiles have shown to have effects on either the color, flavor or aroma depending upon the level used. Other antimicrobials such as organic acids may change the ph of the food and therefore alter flavor, color or texture. Chitosan which is derived from shellfish could cause allergic reactions in people who are sensitive. Furthermore, bacteria may also be altered by repeated exposure to antimicrobial additives, leading to resistance which could be a serious problem in the future. Cost to benefit ratio is probably the biggest issue regarding commercial implementation. It s clear that antimicrobial packaging is a possibility but whether the level of protection against possible food borne illness is enough to justify the cost is debatable. However each time an incident occurs where death is involved, it brings up the question of how we can provide better, safer foods. In conclusion, it is important to remember that antimicrobial food packaging should never be considered a replacement for good manufacturing practices but, it can be considered an additional hurdle for bacteria and may find a niche market in value added products. References 1. Krochta J. M. and De Mulder-Johnston C., Edible and Biodegradable Polymer Films: Challenges and Opportunities, Food Technology 51(2), 61-74(1997). 2. Kesler J.J. and Fennema O., Edible Films and Coatings: A Review, Food Technology 48:47-59,(1986). 3. Han J.H., 2000, Antimicrobial Food Packaging, Food Technology, 54(3): 56-65(2000). 4. Cooksey K., Antimicrobial Food Packaging, Food, Cosmetics and Drug Packaging, 24: (2001). 5. Brody, A., Active Packaging: Beyond Barriers Proceedings of 2003 Packaging Strategies Conference on Active Packaging. Ponte Vedra Beach, FL. 6. Siragusa, G. R., Cutter, C. N., and Willett, J. L., Incorporation of Bacteriocin in Plastic Retains Activity and Inhibits Surface Growth of Bacteria on Meat, Food Microbiology 16: (1999). 7. Padgett, T., Han, I. Y., and Dawson, P. L., Incorporation of Food-Grade Antimicrobial Compounds into Biodegradable Packaging Films. Journal of Food Protection, 61: (1998). 8. Coma V, Sebti I, Pardon P, Deschamps A, Pichavant H., Anti-microbial Edible Packaging Based on Cellulosic Ethers, Fatty Acids and Nisin Incorporation to Inhibit Listeria innocua and Staphylococcus aureus Journal of Food Protection, 59: (2001). 9. Wiles, J.L., The Effect of Relative Humidity on the Steady State Water Vapor Permeability of Chitosan Films Ph.D. Dissertation. Department of Food Technology. Clemson University. Clemson, SC (2000). 10. Brody, A.L., Strupinsky, E.R., and Kline, L.R., Active Packaging Food Applications. CRC Press, Boca Raton, 2001, Chap. 10, pp Grower, J., Cooksey, K. and Getty, K., Development and Characterization of an Antimicrobial Packaging Film Coating Containing Nisin for Inhibition of Listeria monocytogenes Journal of Food Protection. 67: (2004). 12. Vojdani, F. and Torres, J. A., Potassium Sorbate Permeability of Methylcellulose and Hydroxypropyl Cellulose Coatings: Effect of Fatty Acids. Journal of Food Science 55: (1990). 13. Grower, J., Cooksey, K. and Getty, K., Release of nisin from methylcellulose-hydroxygpropyl methylcellulose film formed on low density polyethylene film, Journal of Food Science. 69: (2004).

5 14. Franklin, N., Nisin containing packaging film to control Listeria monocytogenes on modified oxford and tryptic soy agar in individually packaged hot dogs. M.S. Thesis. Clemson University, Clemson, SC., Franklin, N., Cooksey, K., and Getty, K., Inhibition of Listeria monocytogenes on the Surface of Individually Packaged Hot Dogs with a Packaging Film Coating Containing Nisin, Journal of Food Protection. 67: (2004). 16. Allen, R.. Incorporation of antimicrobial agents in non-food and food systems using novel absorbent films. M.S. Thesis. Clemson University, Clemson, SC, Ellis, M.F., Improvement of quality of fresh strawberries and fresh chicken breasts using chlorine dioxide sachets. M.S. Thesis. Clemson University, Clemson, SC, 2003.

6 Effectiveness of Selected Antimicrobial Agents and their possible Commercial Implementation Presented by: Kay Cooksey, Professor Clemson University 2006 PLACE Conference September Cincinnati, Ohio Type Enzymes Chitosan Bacteriocins Antibiotics Organic acids Spices Citrus extracts Isothiocyanates Metals Fungicides Oxidizers Types of Antimicrobials Studied Specific examples or derivatives Lyzoyme, Peroxidase Derived from Shellfish Nisin, Pediocin Imazalil Benzoic, Sorbic acid, Ascorbic, Propionic Rosemary, Garlic, Thymol Grapefruit seed extract, limonene Allyl isothiocyanate Silver ions Benomyl,, Ethanol Ozone, Chlorine dioxide Antimicrobial Packaging Direct contact Biopolymer films as carriers of antimicrobial agents. Ex.) cellulose based Biopolymer films as antimicrobial agents themselves. Ex.) chitosan 1

7 Antimicrobial Packaging Indirect contact Delivery system for use in existing packaging systems. Ex.) Sachet or label with vapor active agent Nisin Methylcellulose and Hydroxypropyl methylcellulose coating as a carrier LDPE or barrier bags used as substrate Tested antimicrobial potential Drop assay Diffusion Film on Lawn Inoculated hot dogs, individually wrapped in coated film Spot on Lawn Assay for Nisin 10μL drop taken from tubes containing film samples in buffer solution buffer solution only 2

8 MIC of nisin in solution Mean Zones of Inhibition (mm) ,000 5,000 2,500 1, Nisin Concentration (IU/mL) Nisin LDPE film coated with cellulose-based coating No nisin 10,000 IU/g nisin Release of Nisin from Film Coating into Peptone H L 7500 H 7500 L 5000 H 5000 L 5000 L 5000 H 7500 L 7500 H L H hour 24 hour 4 day 8 day minute minute minute Time period Mean zones of inhibition (mm) 3

9 Inhibition of LM using a packaging film coating containing nisin Inhibition of LM using a packaging film coating containing nisin Population (log CFU/cm 2 ) Zone of Inhibition (mm) 10,000 IU/mL TSA (37 C, 48h) A,,x A, a,x A, a,x a,x TSA (4 C, 17d) B,y B,y B,y y MOX (37 C, 48h) * 25.4 b,z b,z b,z MOX (4 C, 17d) ** ** ** ** Inhibition of LM using a packaging film coating containing nisin Zone of Inhibition (mm) Nisin Population (log CFU/cm2) TSA (37C, 48h) TSA (4C, 17d) MOX (37C, 48h) MOX (4C, 17d) ± 1.28 A, a, x ± 2.28 B, x ± 1.34 b ** 7,500 IU/mL ± 1.51 A, a, x ± 1.79 B, x ± 1.52 b ** ± 1.51 A, a, x Similar results observed for 2,500 IU/mL ± 1.79 B, x ± 1.52 b ** 4

10 Nisin Inhibition of LM in Hot Dogs using Nisin Coated Packaging Film Nisin levels tested 10000, 7500, 2500 and 156 Coated onto barrier bags, hot dogs individually vacuum packaged 5 strain LM cocktail, 5 log inoculum Measured LM populations at days 0, 7, 15, and 60 days. Listeria monocytogenes (5 strain cocktail) populations on the surface of hot dogs enumerated on TSA Nisin (IU/mL ml) 0 7 Days of Storage a,x 5.51 a,x 6.l3 a,x 6.33 a,y 8.01 a,y 9.11 a,y a,x 4.9 b,x 4.90 b,x 5.37 b,y 7.50 b,y 9.52 a,y 2500 ND b ND b 7500 ND b ND b 10,000 ND b ND b Nisin Nisin-Rosemary Blend Spot on Lawn Assay 2.75mg/ml MIC Film on Lawn Assay 5.5mg/ml needed for inhibition Food Challenge Study Fresh Beef Cubes 5.5 mg/ml or 22mg/ml reduced Listeria by log for 36 days of storage. 5

11 Nisin Overall summary Effective at 2500 IU/mL or above Diffuses from cellulose carrier over time Affects visual and heat sealing properties Blend with rosemary didn t t improve effectiveness of nisin used alone. Spot on Lawn Benzoic Acid Inhibition of Listeria monocytogenes using Benzoic Acid on TSA Agar Zone of Inhibition (mm) Benzoic Acid % Benzoic Acid Film on Lawn Inhibition of Listeria monocytogenes using film 7139 and benzoic acid on TSA agar, 37C soak, 37C incubation Zone of Inhibition (mm) Benzoic Acid % 6

12 Benzoic Acid Hot Dog Challenge Study Inhibition of Listeria monocytogenes using benzoic acid with film # E E E+05 CFU / ml 1.50E E E E E E+05 control benzoic acid 0.00E+00 Days Quality of Chicken using ClO 2 and MAP Packaging Fresh chicken breasts inoculated with 4 log population S. typhimurium NAR Sachets: Fast release (6.6mg, 26 hours) Slow release (2.25mg, 22 days) Package atmosphere: 100% N 2 or 75% N 2 /25% CO 2 TSA, TSA w/na, L.a.b. color and sensory (odor and color) on days 0, 3, 6, 9, 12 and 15 of refrigerated storage. Quality of Chicken using ClO 2 and MAP Packaging Total Plate Count 10 Log CFU/chicken breast None N2 Fast N2 Slow N2 N2 None N2/CO2 Fast Fast N2/CO2 N2/CO2 Slow Slow N2/CO2 N2/CO Days of Storage (3 o C) 7

13 Quality of Chicken using ClO 2 and MAP Packaging S. typhimurium NAR populations Log CFU/chicken breast Days of Storage (3 o C) None N2 Fast N2 Slow N2 None N2/CO2 Fast N2/CO2 Slow N2/CO2 Research - ClO 2 Chicken breasts after 15 days at 2.8 C Quality of Chicken using ClO 2 and MAP Packaging Color affected in areas close to sachet Odor was significantly reduced by ClO 2, a concern with regard to indication of spoilage. 8

14 Challenges and Factors for Commercial Implementation Production Regulatory Release/control Affects on Food Properties Cost Benefit Ratio Conclusion Find an antimicrobial based on needs of food. Consider combination systems. Additional hurdle concept. Niche market for value added products. Thank You PRESENTED BY Kay Cooksey Professor Duncan Darby Associate Professor Clemson University Please remember to turn in your evaluation sheet... 9