effectiveness of barrier film with a cellulose coating that carries nisin blends for the inhibition...

Copyright © 2010 John Wiley & Sons, Ltd.

Effectiveness of Barrier Film with a Cellulose Coating that Carries Nisin Blends for the Inhibition of Listeria Monocytogenes

By Brian Matthews,1 Sunil Mangalasary,2 Duncan Darby2 and Kay Cooksey2*

1Sealed Air Corporation, Cryovac Division, Duncan, SC 29334, USA2Department of Packaging Science, Clemson University, Clemson, SC 29634, USA

SUMMARY

The objective of this study was to determine the effectiveness of two nisin blend antimicrobial agents (Guardian NR 250, Novagard CBI) incorporated into a cellulose coating that was applied onto a barrier fi lm against Listeria monocytogenes. The minimum inhibitory concentration (MIC) of the agents in solution against L. monocytogenes was found to be 2.74 mg/ml for the two nisin blends. The concentrations tested for both nisin blend treatments were 5.49, 10.9, 16.4 and 21.9 mg/ml. Guardian NR 250 resulted in wider zones of inhibition compared to the Novagard CBI at all levels tested. The MIC for Guardian NR 250 in the fi lm was 5.49 mg/ml. Films containing Novagard CBI did not show any antimicrobial activity. A food challenge study was conducted using the fi lm containing Guardian NR 250 at levels of 5.49 and 21.9 mg/ml. Inoculated fresh beef cubes were individually packaged with pre-made barrier fi lm pouches that had an interior cellulose coating containing the antimicrobial agent and stored at 4°C for 36 days. Bacterial colonies were enumerated every 6 days on modifi ed Oxford agar. There was no signifi cant difference in the L. monocytogenes population between two levels of Guardian NR 250 throughout the study. There was statistically signifi cant inhibition of L. monocytogenes for both levels of Guardian NR 250 during 18–30 days of storage compared to a control fi lm without the antimicrobial agent. Copyright © 2010 John Wiley & Sons, Ltd.

Received 15 October 2008; Revised 2 February 2010; Accepted 15 February 2010

KEY WORDS: nisin blends; cellulose coating; Listeria monocytogenes

INTRODUCTION

Listeria monocytogenes is a foodborne pathogen of public health signifi cance. It produces listeriosis, a disease with higher mortality rate compared to other common foodborne diseases such as salmonel-losis and campylobacteriosis.1 The population groups most commonly affected by listeriosis are pregnant women, neonates, the elderly and people with suppression of immune system, such as AIDS and cancer or transplant patients.2 In 2002, a major outbreak of listeriosis in the northeastern USA associated with consumption of sliceable turkey deli meat resulted in 46 cases including seven deaths.3 Ready-to-eat meat and poultry products are primarily indicated in most of the Listeria outbreaks.3,4 The occurrence of L. monocytogenes in fresh beef was also established by several studies.5–7 L. mono-cytogenes is a ubiquitous organism, able to survive and multiply at refrigeration temperatures, in up to 12–13% salt, while certain strains may grow in water activity down to 0.9 and a pH value down

PACKAGING TECHNOLOGY AND SCIENCEPackag. Technol. Sci. 2010; 23: 267–273

Published online 4 May 2010 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/pts.894

* Correspondence to: K. Cooksey, Department of Packaging Science, B 212, P & AS, Clemson University, Clemson, SC 29634, USA.

E-mail: [email protected]

268 B. MATTHEWS ET AL.

Copyright © 2010 John Wiley & Sons, Ltd. Packag. Technol. Sci. 2010; 23: 267–273 DOI: 10.1002/pts

to 4.4.8 Therefore, effective measures are constantly sought for the control of contamination of foods by this organism. Various preservation methods such as use of chemical preservatives including natural antimicrobial compounds, irradiation, high-pressure processing, pulsed electric fi elds and in-package thermal pasteurization have been studied and found effective in reducing the contamination of food by L. monocytogenes.9

The use of packaging fi lms as a carrier of antimicrobial compounds to control spoilage and patho-genic bacteria on food products has been found effective. Various methods have been tested for the use of edible and polymer fi lms to deliver antimicrobial compounds to a variety of food surfaces including meat and meat products.10 Different types of antimicrobial agents including organic acids and their salts, nitrites, antibiotics, alcohols, enzymes, bacteriocins, especially nisin have been incorporated into packaging fi lms.11 Franklin et al.12 found that packaging fi lms coated with a cellulose-based solution containing 10 000 and 7500 IU/ml nisin signifi cantly decreased L. monocy-togenes populations on the surface of hot dogs by greater than 2 log CFU/package throughout the 60 day study.

Nisin is a bacteriocin which was approved for use in food in 1969 and was awarded generally recognized as safe status in the USA in 1988.13 Nisin is effective in a number of food systems, inhibit-ing the growth of a wide range of gram-positive bacteria, including many important foodborne patho-gens such as L. monocytogenes.14 Combined antimicrobial effect of nisin and other natural antimicrobial compounds like enzymes, plant extracts, animal-derived compounds, fatty acids and their derivatives has been extensively studied.15,16 Rosemary (Rosemary offi cinalis) extract functions as an antimicro-bial and antioxidant agent. Lysozyme is a natural enzyme found in egg white and other body fl uids which exhibit signifi cant antibacterial activity against gram-positive bacteria.

Different variations of cellulose like methyl cellulose, hydroxypropyl methylcellulose and carboxy-methyl cellulose are Food and Drug Administration-approved food additives.17 Because of the water-soluble nature of those compounds, they can effectively release antimicrobial compounds on contact with foods containing high moisture levels.18

The objective of this study was to evaluate the anti-listerial activity of two nisin blends, Novagard CB1 (combination of nisin and lysozyme) (N–L) and Guardian NR 250 (combination of nisin and rosemary extract) (N–RM), in solution and on fresh beef when incorporated into a cellulose coating that was applied onto a barrier fi lm. Even though nisin had been studied, the antimicrobial effect of the combinations of nisin with the above chemicals is yet to be tested in antimicrobial food packaging applications.

MATERIALS AND METHODS

Preparation of fi lm coating solutions

Initially, seven levels (0.345, 1.37, 2.74, 5.49, 10.9, 16.4, 21.9 mg/ml) of Novagard CB1 and Guardian NR 250 (Danisco USA Inc., Madison, WI, USA) along with 3.5 g methylcellulose and 1.5 g hydroxy-propyl methylcellulose (Sigma, St. Louis, MO, USA) were added to a solvent consisting of 100 ml each of ethanol and sterile water with 0.5 ml of acetic acid (Sigma). Three milliliters of polyethylene glycol (Sigma) was added as the plasticizer. The contents were mixed thoroughly using a Vertis vertishear (The Virtis Company Inc., Gardiner, NY, USA) for 2 min. Control solutions containing only coating ingredients (without antimicrobials) were also mixed for 2 min.

Film coating

The prepared coating solutions were then applied to Cryovac (Cryovac Division, Sealed Air Corpora-tion, Simpsonville, SC, USA) barrier fi lm (B140T standard Gauge Product SE 194: T Plain) taped to a glass plate, using a thin layer chromatography (TLC) plate coater (Camag, Muttenz, Switzerland) with a 500 μm gate. The coated fi lm was then allowed to dry for 24 h at room temperature. The Cryovac barrier fi lm had an oxygen transmission rate of 6 cc/m2/day at 40°C according to manufac-ture’s data.

FILM CARRYING NISIN BLENDS 269


Culture preparation

Frozen stock cultures of L. monocytogenes American type culture collection (ATCC 15313) were obtained from the Food Microbiology Laboratory at Clemson University. The frozen culture was thawed at room temperature and 0.1 ml was transferred to 9.9 ml of brain–heart infusion (BHI) broth (Becton Dickinson, Sparks, MD, USA) and incubated aerobically for 16–18 h at 37°C with agitation. The inoculum was prepared by harvesting the cells by centrifugation and resuspending them in 0.1% peptone water. The inoculum size was established from a bacterial growth curve experiment carried out separately. The inoculated (0.1 ml) tubes containing 9.9 ml of BHI broth were incubated at 37°C in an agitated water bath. The cell density of the inoculum was verifi ed by measuring the optical density before each experimental run with the help of an established growth curve.

Spot on lawn assay

The fi lm coating solution containing antimicrobials and a control solution without the antimicrobials were used for spot on lawn assay. An aliquot of 0.05 ml of each solution was placed onto Tryptic Soy Agar (TSA) plates inoculated using a Spiral Autoplate 4000 (Spiral Biotech, Norwood, MA, USA) with a Listeria suspension containing approximately 108 CFU/ml. The zones of inhibition were measured after incubating the plates at 37°C for 48 h. The minimum inhibitory concentration (MIC) for each antimicrobial compound was determined as the lowest concentration of the agent resulting in a clear zone of inhibition and this was used as the starting point of various concentrations of anti-microbial agents used in coating the fi lms used in the fi lm on lawn assay.

Film on lawn assay

Films coated with or without antimicrobial solutions (control) were used in this assay. Samples mea-suring 10 mm × 10 mm were cut from the fi lms and sterilized with a ultraviolet (UV) light (Loctite Corporation, Newington, CT, USA) for 5 min. The cut fi lm strips were placed onto TSA plates inocu-lated with a Listeria suspension containing approximately 108 CFU/ml. The zones of inhibition were measured after incubating the plates at 37°C for 48 h.

Food challenge study

Fresh lean muscle tissue from a beef chuck primal cut was obtained from the Clemson University Meat lab and divided into three separate test groups of fresh beef chunks. Each beef chunk was cut into 1 inch cubes which were used as the experimental units. The meat samples were sterilized with a UV light for 4 min per side under a laminar fl ow hood (Labconco Corporation, Kansas City, MO, USA). After sterilization, the samples were dipped into a Listeria suspension containing 105 CFU/ml for 5 min. Samples were allowed to sit for 15 min for the attachment of cells. Samples were then individually placed into a barrier fi lm pouch made using the coated fi lm. Pouches were made in such a way that the coated side formed the interior of the pouch. The treatments used were control (coated with a solution containing no antimicrobials) and fi lms coated with solutions containing two levels of N–RM (5.49 and 21.9 mg/ml). The pouches were vacuum-sealed (KOCH Supplies Inc., Kansas City, MO, USA) and stored at 4°C. The samples were analysed for Listeria population on days 0, 6, 12, 18, 24, 30 and 36. On each experimental day, three treatment samples were homogenized, serially diluted in 0.1% peptone water and plated onto modifi ed Oxford agar. Colonies were counted after incubating the plates at 37°C for 48 h and reported as CFU/ml.

Statistical analysis

The experiments were replicated three times. The treatment means were compared against each other in spot and fi lm on lawn assays and against days of storage and each other in food challenge study. The data were analysed using the general linear model procedure of SAS (SAS Institute, 2003, Cary, NC, USA) and results were compared at a signifi cance level of 95%.



RESULTS AND DISCUSSION

Spot on lawn assay

For the initial screening of antimicrobial effect of the agents, a spot on lawn assay using coating solutions was carried out. The results are shown in Figure 1. The inhibitory effect of the antimicrobial agents was measured as the difference in the diameter of the inhibitory zone obtained for the control (without any antimicrobial agents) and the solutions containing antimicrobial agents since control solution also produced clear zones of inhibition on the agar plates. The zones produced in the control group may be due to the inhibitory effect of ethyl alcohol on L. monocytogenes which is an ingredient of the coating solution. As seen in Figure 1, there was an increase in the zone of inhibition along with the increase in concentration of both the antimicrobial agents. Both N–L and Guardian N–RM inhibited the organism at concentrations as low as 2.74 mg/ml (1250 IU/ml).

At the same concentration levels, N–RM produced wider zones of inhibition compared to N–L. Since nisin, rosemary and lysozyme individually have antimicrobial activity against Gram positive organisms5,19,20 and both rosemary and lysozyme have shown synergistic antibacterial effect when used along with nisin against gram-positive organisms,16,21 the difference in the activity of two com-pounds could be due to an enhanced synergistic action of the combination of nisin and rosemary over nisin and lysozyme combination. Thomas and Isac21 found that the combination of rosemary and nisin showed synergistic effect against gram-positive bacteria. Rosemary extracts enhanced both the bactericidal and bacteriostatic activities of nisin against L. monocytogenes. Based on the MIC deter-mined, further testing with N–L and N–RM in a fi lm application was limited to concentrations of 2.74 mg/ml as the lowest test level.

Figure 1. The inhibitory effect of Novagard (N-L) and Guardian (N-RM) against Listeria monocytogenes measured as the diameter of the inhibition zone (after subtracting the diameter of

inhibition zone obtained for the control sample) in a spot on lawn assay.



Film on lawn assay

Barrier fi lms coated with solutions containing the antimicrobial agents as well as with solutions without antimicrobials (control) were tested for their anti-listerial activity by a fi lm on lawn assay. The diameter of the zone of inhibition obtained for different levels of N–RM are given in Table 1. Different concentrations of N–L used in this assay failed to produce any inhibitory zone. Since N–L produced greater inhibitory zones compared to the control sample in spot on lawn assay, the lack of inhibitory zone in fi lm on lawn assay could be attributed by the failure of this agent to be released from the coating to the agar surface. A similar situation of lack of antibacterial activity may probably occur when applying this fi lm on a food product.

The MIC for N–RM in fi lm on lawn assay was 5.49 mg/ml of coating solution. This higher value obtained compared to the MIC obtained for N–RM in spot on lawn method could be due to the slow diffusion of antimicrobial compound from the fi lm. A previous study on the antimicrobial effect of nisin-containing cellulose fi lm has theorized that a hydroxypropyl methyl cellulose/methyl cellulose (HPMC/MC) fi lm and the plasticizer polyethylene glycol (PEG) will form a bond that limits antimi-crobial release.22 The diffusion or release of antimicrobial agent from the fi lm to the agar surface seeded with the organism is a critical factor in producing inhibition zones in fi lm on lawn assays.

Food challenge study

This part of the study used the lowest effective quantity tested for N–RM (5.49 mg/ml) and the highest effective level (21.9 mg/ml) of N–RM (for use according to the manufacturer’s data) on fresh beef inoculated with L. monocytogenes. Since the meat was sterilized prior to inoculation, there were no competitive bacteria which could have increased or decreased the effectiveness of the antimicrobials.

The growth pattern of the organism for different treatments during 36 days of refrigerated storage is shown in Figure 2. There was an increase in Listeria population during storage for all the treatments in the study including a control fi lm where no antimicrobial agent was added to the fi lm. In the control sample, after a steady increase, the population remained without much change through days 24–36. There was no signifi cant difference (p > 0.05) in bacterial growth in beef samples packaged with fi lms containing different levels of the antimicrobial agent throughout the storage. But compared to the control sample, the bacterial population was signifi cantly lower (p < 0.05) for beef samples packaged with two levels of N–RM through days 18–30. This reduction in bacterial population starting from day 18 could be a result of delayed release of the antimicrobial agent to the food surface from the fi lm coating. During extended storage times, the exposure of the coating to surface water from the meat product may allow the coating to dissolve and thereby release the antimicrobial agent. The primary objective of the antimicrobial packaging is to have an extended antimicrobial activity on the food product which could be attained by the slow and continuous diffusion of the antimicrobial com-pound from the packaging fi lm on to the food product. In this experiment, the bacterial reduction resulting from two levels of N–RM occurred later than expected. Controlled release of antimicrobial compounds from the fi lm would be an ideal way to achieve a continuous antibacterial activity on the food product for an extended period. A few studies22–25 had been conducted to study the release kinet-ics of the antimicrobials from the packaging fi lms. Results from those studies indicate that release of

Table 1. Antimicrobial activity of different levels of N-RM against Listeria monocytogenes in fi lm on lawn assay.

N-RM (mg/ml) Inhibition zone diameter (mm)

21.9 11.24 ± 0.1216.4 11.05 ± 0.4310.9 10.88 ± 0.57 5.49 10.23 ± 0.19 2.74 0.00



antimicrobials can be controlled through modifi cation of the fi lm structure, changes in temperature and other fi lm-forming ingredients. United States Department of Agriculture/Food Safety Inspection Service (USDA/FSIS) has accepted N–RM for use as an Alternative 1 anti-listerial treatment for frankfurters and similar meat/poultry products at dosage between 277 and 550 mg/g.26 The increase of L. monocytogenes over time in both levels of N–RM could be due to the low level of the agent used to have an optimal antibacterial effect. Therefore, incorporation of this compound at a higher concentration level to a suitable fi lm structure resulting in a steady diffusion on to the food surface would be an effective intervention against L. monocytogenes contamination of meat and meat products.

REFERENCES

1. Wing EJ, Gregory SH. Listeria monocytogenes: clinical and experimental update. The Journal of Infectious Diseases 2002; 185(suppl. 1): S18–S24.

2. Gerba CP, Rose JB, Hass CN. Sensitive populations: who is at the greatest risk? International Journal of Food Microbiol-ogy 1996; 30: 113–123.

3. CDC (Centers for Disease Control and Prevention). Public health dispatch: outbreak of listeriosis – northeastern United States. Morbidity and Mortality Weekly Report 2002; 51: 950–951.

4. CDC (Centers for Disease Control and Prevention). Multistate outbreak of listeriosis – United States. Morbidity and Mor-tality Weekly Report 2000; 49: 1129–1130.

5. Zhang S, Musthapha A. Reduction of Listeria monocytogenes and Escherichia coli O157:H7 numbers on vacuum-packaged fresh beef treated with nisin or nisin combined with EDTA. Journal of Food Protection 1999; 62: 1123–1127.

6. Barros MAF, Nero LA, Silva LC et al. Listeria monocytogenes: Occurrence in beef and identifi cation of the main con-tamination points in processing plants. Meat Science 2007; 76: 591–596.

7. Takahashi T, Ochiai Y, Matsudate H et al. Isolation of Listeria monocytogenes from the skin of slaughtered beef cattle. The Journal of Veterinary Medical Science 2007; 69: 1077–1079.

Figure 2. Average Listeria monocytogenes populations on fresh beef packaged with barrier fi lm coated with three levels of N-RM (0, 5.49, 21.9 mg/ml) during storage at 4°C.



8. Farber JM, Peterkin PI. Listeria monocytogenes, a food pathogen. Microbiological Reviews 1991; 55: 476–551. 9. Zhu M, Du M, Cordray J, Ahn DU. Control of Listeria monocytogenes contamination in ready to eat meat products.

Comprehensive Reviews in Food Science and Food Safety 2005; 4: 34–42.10. Cutter CN, Willet JL, Siragusa GR. Improved activity of nisin incorporated polymer fi lms by formulation change and

addition of food grade chelator. Letters in Applied Microbiology 2001; 33: 325–328.11. Guerra NP, Macías CL, Agrasar AT, Castro LP. Development of bioactive packaging cellophane using nisaplin as bio-

preservative agent. Letters in Applied Microbiology 2005; 40: 106–110.12. Franklin NB, Cooksey KD, Getty KJK. Inhibition of Listeria monocytogenes on the surface of individually packed hot

dogs with a packaging fi lm coating containing nisin. Journal of Food Protection 2004; 67: 480–485.13. FDA (Food and Drug Administration). Nisin preparation: affi rmation of GRAS status as a direct human food ingredient.

Federal Register 1988; 53: 11247–11251.14. Tagg JR, Dajani AS, Wannamaker LW. Bacteriocins of Gram positive bacteria. Bacteriological Reviews 1976; 40: 722–

756.15. Dykes GA, Amarowicz R, Pegg RB. Enhancement of nisin antibacterial activity by bearberry leaf extract. Food Microbi-

ology 2003; 20: 211–216.16. Chung W, Hancock REW. Action of nisin and lysozyme mixtures against lactic acid bacteria. International Journal of

Food Microbiology 2000; 60: 25–32.17. FDA (Food and Drug Administration). Food additive status list. http://www.cfsan.fda.gov/~dms/opa-appa.html [accessed

24 July 2006].18. Grower JL, Cooksey K, Getty K. Release of nisin from methylcellulose – hydroxypropyl methylcellulose fi lm formed on

low-density polyethylene fi lm. Journal of Food Science 2004; 69: 107–111.19. Campo JD, Amiot MJ, Nguyen, MJ. Antimicrobial effects of rosemary extracts. Journal of Food Protection 2000 63:

1359–1368.20. Proctor VA, Cunningham FE. The chemistry of lysozyme and its use as a food preservative. Critical Reviews in Food

Science and Nutrition 1988; 26: 359–395.21. Thomas LV, Isak T. Nisin synergy with natural antioxidant extracts of the herb rosemary. Acta Horticulturae 2006; 709:

109–114.22. Cha DS, Choi JH, Chinnan MS, Park HJ. Release of nisin from various heat-pressed and cast fi lms. Lebensmittel-

Wissenschaft und-Technologie 2003; 36: 209–213.23. Ouattara B, Simard RE, Piette G, Begin A, Holley RA. Diffusion of acetic and propionic acids from chitosan based anti-

microbial fi lms. Journal of Food Science 2006; 65: 768–773.24. Buonocore GG, DelNobile MA, Panizza A, Corbo MR, Nicolais L. A general approach to describe the antimicrobial agent

release from highly swellable fi lms intended for food packaging applications. Journal of Controlled Release 2003; 90: 97–107.

25. Joerger RD. Antimicrobial fi lms for food applications: a quantitative analysis of their effectiveness. Packaging Technology and Science 2007; 20: 231–273.

26. Danisco. Food protection ‘It is everyone’s business.’; http://www.infost.org/reports-resources/technical_reports/Technical_ Report/Shanghai/presentation.pdf [accessed 18 August 2008].

effectiveness of barrier film with a cellulose coating that carries nisin blends for the inhibition...

Documents