Bacteriocins Offer Enormous Promise in Food Packaging Safety Advances

By Dr. Haiqiang Chen and John Williams Jr.

Historically, chemical preservatives and other traditional barriers have been used in food products to inhibit microbial growth. However, burgeoning consumer demands for faster, healthier, and ready-to-eat products have largely relegated the use of more traditional preservatives and additives to the sidelines, stimulating heightened research interest in finding natural, but effective preservatives. Bacteriocins, produced by lactic acid bacteria, may be considered natural preservatives that fulfill these requirements.

Bacteriocins

Bacteriocins are ribosomally-synthesized polypeptides possessing bacteriocidal activity that are rapidly digested by proteases in the human digestive tract. They generally possess narrow specificity of action against strains of the same or closely related species (1). Numerous bacteriocins have been characterized. Although some bacteriocins, such as pediocin PA-1 and lacticin 3147, have been developed for possible approval and use, nisin remains the most commercially important bacteriocin because of its relatively long history of safe use and documented effectiveness against important gram-positive foodborne pathogens and spoilage agents (2).

International acceptance of nisin was given in 1969 by the Joint Food and Agriculture Organization / World Health Organization (FAO / WHO) Expert Committee on Food Additives (3). In 1988, the Food and Drug Administration granted approval for the use of nisin in pasteurized processed cheese spreads and established the legal precedent for application of bacteriocins as food additives in the U.S. (4).

Purified nisin has been evaluated for toxicological effect and found harmless or at least with very low toxicity using rat and guinea pig models (5). The FAO / WHO Committee recommended a maximum daily intake of nisin for a 70-kg person to be 60 mg of pure nisin or 33,000 units; however, nisin is permitted in processed cheeses in Australia, France and Great Britain with no maximum limit. In the U.S., the maximum limit is 10,000 IU/g and use of nisin-producing starter cultures has never been regulated since lactococci are considered GRAS or generally regarded as safe (6).

Nisin usually has no effect on gram-negative bacteria, yeasts and molds, although gram-negative bacteria can be sensitized to nisin by sublethal heating, freezing and chelating agents. Normally only gram-positive bacteria are affected, and these types include lactic acid bacteria, vegetative pathogens such as Listeria, Staphylococcus and Mycobacterium, and the sporeforming bacteria, Bacillus and Clostridium. The spores of bacilli and clostridia are actually more sensitive to nisin than their vegetative cells although the antagonism is sporostatic, not sporicidal, thus requiring the continued presence of nisin to inhibit outgrowth of the spores (1).

Packaging Advances

Recent research studies evaluating the effectiveness of antimicrobial active packaging have garnered much attention from food scientists, processors, and manufacturers. In this technology, antimicrobial agents are incorporated into packaging material to actively control microbial growth and extend product shelf-life.

Antimicrobial packaging film prevents microbial growth by direct contact of the package with the surface of foods, such as meats and cheese. The gradual release of bacteriocins from a packaging film to the food surface may have an advantage over dipping and spraying foods with nisin. In the latter processes, antimicrobial activity may be lost or reduced due to inactivation of bacteriocins by food components or dilution below active concentration due to migration into the foods (7).

Two methods have been commonly used to prepare packaging films with bacteriocins (7). One is to incorporate bacteriocins directly into polymers. For example, Siragusa et al. (8) incorporated nisin into a polyethylene-based plastic film that was used to vacuum-pack beef carcasses. Nisin retained activity against Lactobacillus helveticus and Brochothrix thermosphacta inoculated in carcass surface tissue sections. An initial reduction of 2-log10 cycles of Brochothrix thermosphacta was observed with nisin-impregnated packaged beef within the first 2 days of storage at 4 °C. After 20 days of refrigerated storage at 4 or 12 °C (to simulate temperature abuse), Brochothrix thermosphacta populations from nisin-impregnated plastic-wrapped samples were significantly less than control (without nisin). Coma and others (9) incorporated nisin into edible cellulosic films made with hydroxypropylmethylcellulose by adding nisin to the film-forming solution. Inhibitory effect could be demonstrated against Listeria innocua and Staphylococcus aureus.

Another method to incorporate bacteriocins into packaging films is to coat or adsorb bacteriocins to polymer surfaces. Examples include nisin / methylcellulose coatings for polyethylene films and nisin coatings for poultry, adsorption of nisin on polyethylene, ethylene vinyl acetate, polypropylene, polyamide, polyester, acrylics and polyvinyl chloride (7). Bower and others (10) demonstrated that nisin adsorbed onto silanized silica surfaces inhibited the growth of Listeria monocytogenes. Nisin films were exposed to a medium containing Listeria monocytogenes and the contacting surfaces were evaluated at 4-h intervals for 12 h. Cells on surfaces that had been in contact with a high concentration of nisin (40,000 IU/ml) exhibited no signs of growth and many displayed evidence of cellular deterioration. Surfaces contacted with a lower concentration of nisin (4,000 IU/ml) had a smaller degree of inhibition. In contrast, surfaces contacted with films of heat-inactivated nisin allowed Listeria monocytogenes to grow. The efficacy of bacteriocin coatings on the inhibition of pathogens has also been demonstrated in other studies. For example, coating of pediocin onto cellulose casings and plastic bags has been found to completely inhibit growth of inoculated Listeria monocytogenes in meats and poultry through 12-week storage at 4°C (11).

Although shelf-life is extended in food products as populations of food spoilage organisms are reduced or inhibited when antimicrobial packaging films are used, the primary thrust is towards control of specific anticipated pathogens in the product. In this regard, antimicrobial casings containing nisin have been developed and used in the hot dog industry. Given the large product recalls experienced by major meat brands as the result of product contaminated with Listeria monocytogenes, the added cost of these casings is considered an economically sound investment.

The ubiquitous nature of Listeria monocytogenes, its hardiness, and ability to grow at refrigeration temperatures and under anaerobic conditions renders it a threat to food safety. Despite ongoing efforts to eradicate the organism from RTE foods, Listeria monocytogenes continues to be a leading public health concern (Figure 1).

The U.S. Department of Agriculture has established a rigid policy regarding Listeria monocytogenes and set a zero tolerance level for this organism in RTE foods (12). Many studies have been carried out to control Listeria monocytogenes in meat products since it is common within slaughterhouse and meat packing environments.

Validation Studies

To determine the efficacy of antimicrobial packaging films in inhibiting the growth of foodborne pathogens, a microbiological challenge study can be conducted in which samples with films and samples with normal packaging films are inoculated with antimicrobial packaging appropriate for pathogens. The samples are then analyzed for the challenged organism at different time intervals. The efficacy of the antimicrobial packaging films can be determined by comparing counts in these two treatments.

Sensory qualities are the primary criteria used by consumers to judge a food's acceptability. These qualities begin to change as microorganisms in the food grow and metabolize available nutrients. To determine the efficacy of antimicrobial packaging films in shelf-life extension, performing a shelf-life is highly recommended. In a shelf-life study, samples with antimicrobial packaging films and samples with normal packaging films are stored for certain period of time and the counts of microorganisms, such as aerobic bacteria, yeast, mold and lactic acid bacteria, are determined at different time intervals. The shelf-life extension can be determined by comparing counts in these two treatments.

If you are interested in investigating the efficacy of antimicrobial packaging, our research scientists can design and customize shelf-life and challenge studies for your product. Our research professionals have years of practical and technical experience with a diverse range of products, including RTE meats and dairy products. To learn more about our validation studies, contact us.

1. Joerger R.D., Hoover D.G., Barefoot S.F., Harmon K.M., Grinstead D.A., and Nettles-Cutter, C,G. 2000. Bacteriocins. In: Lederberg, editor. Encyclopedia of microbiology, vol. 1, 2nd edition. San Diego: Academic Press, Inc. P 383-397.

2. Chen, H. and Hoover, D.G. 2003. Bacteriocins and their food applications. Comprehensive Rev in Food Sci. and Food Safety. In press.

3. WHO. 1969. Specifications for identity and purity of some antibiotics. World Health Organization / Food Add. 69.34:53-67.

4. FDA. Federal Register. 1988. Nisin preparation: Affirmation of GRAS status as a direct human food ingredient. 21 CFR Part 184, Fed. Reg. 53:11247-11251.

5. Frazer, A.C., Sharratt, M., and Hickman J.R. 1962. The biological effects of food additives. I. Nisin. J. Sci. Food Agric. 13:32-42. Shtenberg, A.J. and Ignatev, A.D. 1970. Toxicological evaluation of some combinations of food preservatives. Food Cosmet. Toxicol. 8:369-380.

6. Hurst, A. and Hoover, D.G. 1993. Nisin. In: Davidson P.M. and Branen, A.L., editors. Antimicrobials in foods, New York: Marcel Dekker, Inc. p. 369-407. Chikindas M.L., Montville, T.J. 2002. Perspectives for application of bacteriocins as food preservatives. In: Juneja, V.K. and Sofos, J.N., editors. Control of foodborne microorganisms. New York: Marcel Dekker, Inc. p. 303-321.

7. Appendini, P., Hotchkiss J.H. 2002. Review of antimicrobial food packaging. Innov. Food Sci. Emerg. Technol. 3:113-126.

8. Siragusa, G.R., Cutter, C.N., and Willett J.L. 1999. Incorporation of bacteriocin in plastic retains activity and inhibits surface growth of bacteria on meat. Food Microbiol. 16:229-235.

9. Coma, V., Sebti, I., Pardon, P., Deschamps, A. and Pichavant, F.H. 2001. Antimicrobial edible packaging based on cellulosic ethers, fatty acids, and nisin incorporation to inhibit Listeria innocua and Staphylococcus aureus. J. Food Prot. 64:470-475.

10. Bower, C., McGuire, J. and Daeschel, M. 1995. Suppression of Listeria monocytogenes colonization following adsorption of nisin onto silica surfaces. Appl Environ Microbiol 61:992-997.

11. Ming, X., Weber, G., Ayres, J. and Sandine, W. 1997. Bacteriocins applied to food packaging materials to inhibit Listeria monocytogenes on meats. J Food. Sci. 62:413-415.

12. Jay, J.M. 1996. Modern food microbiology. 5th ed. New York: Chapman and Hall. p. 635.

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The 24-Hour Petrifilm Staph. Express Count plate method, developed by 3M™ Microbiology, for the enumeration of Staphylococcus aureus in meat, poultry, and seafood was recently granted official method status by AOAC INTERNATIONAL. Industry-wide, the enumeration of S. aureus is often used as a food safety and quality indicator.

To obtain AOAC approval for the new method (2003.11), the Silliker, Inc. Corporate Research Center was one of 12 labs contracted by 3M™ to compare the method to an existing 72-hour method (975.55) for the enumeration of S. aureus in selected foods.

In the collaborative study, the laboratories analyzed four foods -- cooked, diced chicken, cured ham, smoked salmon, and pepperoni -- for the existence of S. aureus. The Silliker study, headed by researchers Victoria Aleo, Ann Schultz, and Wendy McMcMahon, concluded that the method was equivalent to the 72-hour standard method in terms of repeatability and reproducibility.

Findings from the study, "3M™ Petrifilm™ Staph Express Count Plate Method for the Enumeration of Staphylococcus aureus in Selected Types of Meat, Seafood, and Poultry: Collaborative Study," were presented during poster sessions at the recent AOAC INTERNATIONAL annual meeting in Atlanta, GA. The study will also be featured in an upcoming issue of the Journal of AOAC International.

The Silliker, Inc. Corporate Research Center can help companies compare the accuracy and reliability of new testing methods against existing standards through pre-collaborative and collaborative studies. Over the past three decades, our research scientists have earned an international reputation for their significant contributions to the development and validation of rapid methods. To learn more about our studies, click here.

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