Development and Characterization of an Antimicrobial Packaging Film Coating Containing Nisin for Inhibition of Listeria monocytogenes

Transcription

1 475 Journal of Food Protection, Vol. 67, No. 3, 2004, Pages Copyright q, International Association for Food Protection Development and Characterization of an Antimicrobial Packaging Film Coating Containing Nisin for Inhibition of Listeria monocytogenes JENNIFER L. GROWER, 1 * KAY COOKSEY, 1 AND KELLY J. K. GETTY 2 1 Department of Packaging Science, B212 P & A Building, Clemson University, Clemson, South Carolina 29634; and 2 Food Science Institute, 148 Waters Hall, Kansas State University, Manhattan, Kansas 66506, USA MS : Received 30 May 2003/Accepted 18 September 2003 ABSTRACT The purpose of this study was to develop and characterize a packaging lm coating containing nisin. A spot-on-lawn assay was used to determine the effect of acid type (ascorbic, acetic, hydrochloric, lactic) and nisin level (equal increments from 10,000 IU to 9 IU) to be used in the formulation of the lm coating. Zones of inhibition were measured after incubation on tryptic soy agar (378C, 48 h). Low-density polyethylene lms coated with differing levels of nisin were characterized by eld emission scanning electron microscopy, tensile strength, elongation, and water vapor transmission rate. The MIC of nisin in solution was 157 mg/ml. All acids were equally inhibitory (P. 0.05), but acetic acid produced the largest zone of inhibition (21 mm). Field emission scanning electron microscopy con rmed that the cloudy appearance of the lms was due to sodium chloride found in the commercially prepared nisin. Tensile strength increased as nisin concentration increased, which also corresponded to increasing lm thickness. The nisin coatings (10,000 and 2,500 IU/ml) did not have a signi cant effect (P. 0.05) on the water vapor transmission rate of the low-density polyethylene lm. Listeria monocytogenes is a gram-positive pathogen responsible for many foodborne illness outbreaks. Common in nature, this pathogen has been found in milk, cheeses, raw vegetables, and raw and fermented meat and sh. Ready-to-eat foods such as hot dogs and deli-style meats also have been linked to numerous recalls and identi ed as the source in a number of outbreaks (6 8, 15). L. monocytogenes has a particular ability to circumvent common inhibitory conditions such as low ph, low temperature, salty conditions, and anaerobic environments; therefore, new hurdles are needed to inhibit its growth in food. One method to control L. monocytogenes in foods is the use of nisin, a bacteriocin effective against gram-positive bacteria. Nisin has GRAS (generally recognized as safe) status in the United States for use in processed cheese spreads. The antimicrobial activity of nisin depends on many factors, including the type and number of microorganisms present, the amount of nisin, ph level, conditions of application, and composition of food components (18). The antimicrobial activity of nisin is dependent on ph, with low ph systems being more effective than neutral or alkaline conditions (4). The solubility and stability of nisin are dependent on the ph of the solution (13). At ph 2.5, Hurst (12) reported a solubility of 12%, decreasing to 4% at ph 5.0 and even lower solubility at neutral ph (9). As the ph decreases, the solubility of nisin increases. This increased solubility allows nisin to be more effective against bacteria. Because nisin is dissolved in an acidic environment, * Author for correspondence. Tel: ; Fax: ; jgrower@clemson.edu. the type of acid used to dissolve the nisin might have a synergistic effect against bacteria. Undissociated organic acids have a greater antimicrobial activity against a broad range of microorganisms than their dissociated forms (11). Undissociated organic acids can pass through the cell membrane, dissociate inside the cytoplasm, and interfere with metabolic processes of the cell (5). Studies have shown that various organic acids have different levels of antimicrobial effectiveness against L. monocytogenes depending on temperature, ph, and salt content (17, 19). For example, Ahamad and Marth (1) found that, depending on the temperature, acetic acid was more inhibitory than lactic and citric acid against L. monocytogenes. In addition, other studies have shown synergistic effects among various organic acids and antimicrobial agents. Oh and Marshall (17) found a greater inhibitory effect against L. monocytogenes when combining monolaurin, a broad-spectrum antimicrobial additive, with organic acids such as lactic or citric acid. Therefore, combining nisin with different organic acids might enhance inhibition of L. monocytogenes when used in a coating solution. The overall objective of this study was to develop and characterize a packaging lm coating solution containing nisin. The rst step was to determine the type of acid and level of nisin for optimal inhibition of L. monocytogenes in a solution with the use of a spot-on-lawn assay. These nisin ef cacy results were used to produce a cellulose-based solution carrying nisin that was coated onto low-density polyethylene (LDPE) lm. The next objective was to study the surface topography and chemical composition with eld emission scanning electron microscopy (SEM). Finally, the

2 476 GROWER ET AL. mechanical properties of the lm coating solutions were measured to determine whether the coatings had any effect on the properties of LDPE lm. MATERIALS AND METHODS Bacterial culture. The L. monocytogenes (ATCC avirulent strain, rabbit isolate) used in this study was obtained from the Food Microbiology Laboratory at Clemson University. The frozen stock culture was stored in brain heart infusion broth with 20% glycerol at 2708C. The working stock cultures were maintained in tryptic soy broth at 48C and grown at 378C for 24 h. All microbiological media was purchased from Sigma Chemical Co. (St. Louis, Mo.). Media preparation. All three replicates used tryptic soy agar (TSA) pour plates modi ed to ph 5.0 with 2.0 N HCl (Fisher Scienti c, Fair Lawn, N.J.). A 24-h culture of L. monocytogenes was diluted to 6 log CFU/ml with 0.1% buffered peptone water, mixed with TSA, and poured into petri plates. Nisin concentration spot-on-lawn assay. The effect of different nisin concentrations and the MIC of nisin was determined with a stock solution and serial dilutions ranging from 10,000 to 8 IU/ml. A stock solution of 10,000 IU/ml was obtained by adding 1 mg of nisin (2.5% nisin concentration balanced with sodium chloride and denatured milk solids, Sigma Chemical Co.) to 1 ml of HCl (ph 2.0). From the stock solution, dilutions of 5,000, 2,500, 1,250, 625, 313, 156, 78, 39, 19, and 9 IU/ml were made. A 10-ml drop of each nisin-containing solution was placed onto the inoculated TSA pour plates. All plates were incubated at 378C for 48 h and zones of inhibition were measured in millimeters with a ruler in both the horizontal and vertical directions. Results from the two directions were averaged. The experiment was replicated three times and reported as zones of inhibition (mm). Different acids spot-on-lawn assay. To measure the effect of different acids on nisin activity, solutions containing four different types of acids were prepared. Each acid ascorbic, acetic, lactic, and hydrochloric (Fisher Scienti c) was adjusted to 0.02 N (all acids averaged ph 2.5 to 3.0). One milligram of nisin (10,000 IU/ml) was added to 1 ml of each of the four acids. One milligram of nisin (10,000 IU/ml) was added to 1 ml sterile water as a control. A 10-ml drop of each solution was placed onto the inoculated TSA pour plates. All plates were incubated at 378C for 48 h, and zones of inhibition were measured in millimeters. The experiment was replicated three times, and statistical differences were analyzed with a one-way analysis of variance (P, 0.05) (StatView5121, Calabasas, Calif.). Film coating solution preparation and coating method. Preliminary testing was done to determine the optimal conditions for nisin ef cacy prior to incorporation into the lm coating solution. On the basis of preliminary results, nisin was most effective when dissolved in acetic acid at ph 2.0 to 2.5. These results were similar to previous studies that identi ed the increased effectiveness of nisin at lower ph levels to increased solubility and increased environmental stress on the bacteria, allowing the nisin to be more effective (4). Nisin is less soluble at ph 7.0, which impedes its effectiveness (4), but has greater solubility at lower ph values (20). Coating solutions were prepared with the formulation shown in Figure 1, adapted from Kamper and Fennema (14). The coating solution was mixed at 7,000 rpm for 2 min by a Vertishear homogenizer with a 20-mm shaft (Vertis Co., Gardner, N.Y.). LDPE lm was taped to a glass plate (20 by 20 cm), and the nisin-based FIGURE 1. Film coating formulation on the basis of 50-ml liquid portion, which coated 32 plates measuring 20 by 20 cm (1,400 cm 2 ). solutions (50 ml) or control solutions were cast onto the lm with a 500-mm coating thickness with the use of a manually operated thin layer chromatography plate coater (CAMAG, Muttenz, Switzerland). SEM preparation. The coatings were separated from the LDPE substrate with tweezers, mounted onto aluminum stubs with double-sided carbon tape, and sputter coated with platinum for 3 min. A sample of LDPE lm and a sample of nisin powder also were prepared in the same manner. The coated samples were examined with the Hitachi-S-4700 eld emission scanning electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 3 kv, and the images were captured with PCI software (Ontario, Canada). Properties of coated lm. Tensile strength and percent elongation of lms with nisin-containing coatings and control coating (no nisin) were compared with a Satec model T10000 (Grove City, Pa.) according to ASTM D (2). The grips of the SATEC were 2 in. apart and separated at a rate of 10 in./min. The water vapor transmission rate of the lms were compared with the use of a Mocon Permatran W600 (Minneapolis, Minn.) at 37.88C and 100% relative humidity according to ASTM F (2). Analysis of variance, least signi cant difference test, and Tukey s test were conducted by the Statistical Analysis System (SAS) program (Cary, N.C.). RESULTS AND DISCUSSION Effect of nisin concentration on antimicrobial activity. The antimicrobial effect of nisin against L. monocytogenes was tested with different nisin concentrations (Fig. 2). These results show that in a range of nisin concentrations (9 to 10,000 IU/mL), antimicrobial activity decreases as the concentration of nisin decreases. This was observed by measuring the zones of inhibition, which became smaller in size as the concentration of nisin lessened. At 10,000 IU/ ml, zones of inhibition were 30 mm in diameter and decreased to 0 mm at 78 IU/mL. The range of nisin tested in this study was used to determine a high and low concentration for use in the packaging coating. The high level was determined on the basis of the maximum allowable concentration (10,000 mg/kg, 2.5% nisin preparation) for pasteurized processed cheese spread in the United States (3). The MIC was identi ed as the lowest concentration of nisin resulting in complete inhibition of visible growth beneath the droplet. The lowest concentration that inhibited the growth of L. monocytogenes in 0.02 N HCl was IU/ml (Fig. 2), with a mean

3 ANTIMICROBIAL FILM COATING FOR INHIBITION OF L. MONOCYTOGENES 477 FIGURE 2. Inhibition of Listeria monocytogenes ATCC by different concentrations of nisin (IU/ml). zone of inhibition measuring 4 mm. At 78 IU/ml, bacterial growth was observed under the droplet, indicating that this nisin concentration was not inhibitory. Therefore, the MIC of nisin against L. monocytogenes was identi ed as IU/ml. Other studies using different strains and conditions report different MICs; therefore, these results only apply to the strain and testing conditions stated. Meghrous et al. (16) determined the MICs of three bacteriocins nisin A, nisin Z, and pediocin in 30 different bacterial species and strains. There was considerable variation among species and even among strains of the species, as seen in the nine strains of L. monocytogenes. Sensitivity to bacteriocins varied with the bacteriocin, the strain tested, and the growth medium. Benkerroum and Sandine (4) found that the concentration of nisin required for complete inhibition of L. monocytogenes in deman, Rogosa, and Sharpe (MRS) and tryptic soy broth media varied from 105 to 740 IU/ml depending on the strain and culture medium used (4, 16). Effect of different acids on nisin activity. When dissolving 10,000 IU/ml nisin, the ascorbic, acetic, lactic, and hydrochloric acids were found to be equally antagonistic against L. monocytogenes and produced average zones of FIGURE 3. LDPE lm coated with cellulose-based coating only (shown on left) and LDPE lm with cellulose-based and 10,000 IU/g nisin (shown on right). FIGURE 4. Surface of lm coating containing 10,000 IU/cm2 nisin. inhibition of 17, 21, 20, and 19 mm, respectively. All four acids produced similar antimicrobial activities against the pathogen, and no signi cant differences in zones of inhibition (P. 0.05) were observed when nisin was dissolved in these different acid types. Acetic acid was used to prepare the lm-coating solutions because it produced the largest zones of inhibition of all the acids, although not statistically greater. SEM. After the formulation of the nisin-containing solution was completed, the results were used to produce a coating solution that was applied to the surface of LDPE lm (Fig. 3). Films containing no nisin (control) and containing 2,500, 5,000, 7,500, and 10,000 IU/cm2 nisin were produced. Visual observation of the lm showed a cloudy appearance, which was analyzed using eld emission SEM. Scanning electron micrographs (Figs. 4 and 5) show crystalline structures on the surface of the lms coated with 10,000 and 2,500 IU/cm2 nisin. Films containing 5,000 and 7,500 IU/cm 2 are not shown but had similar presence of crystalline structure. In addition to the branching crystals, both nisin-containing lms have random deposits of smaller, highly de ned crystals. The surface topography of the FIGURE 5. Surface of lm coating containing 2,500 IU/cm2 nisin.

4 478 GROWER ET AL. FIGURE 6. Surface of control lm coating containing no nisin. control lm (without nisin) appears to be relatively free of surface structures (Fig. 6). With the use of energy-dispersive spectroscopy, the crystals of both nisin-containing lms were identi ed as sodium chloride. Energy-dispersive spectroscopy dot mapping con rmed both the branching crystals and the smaller crystalline deposits as sodium chloride. The sodium chloride comes from the nisin preparation itself. During the manufacturing process, nisin is balanced with NaCl and whey protein, hence accounting for the crystalline structures found in the coating. The presence of nisin was not identi able with energy-dispersive spectroscopy. The initial purpose of using SEM was to identify nisin within the coating matrix, to con rm its presence, and to determine whether it was evenly dispersed. However, this technique was not successful for identifying nisin, but it identi ed the sodium chloride in the coating solution responsible for the cloudy appearance of the lm. The importance of the SEM data was strictly related to analyzing the visual properties of the lm coating. Performance properties of lm samples. It was important to examine the mechanical properties of the coated lm to establish whether the coating would have an effect on the properties of the lm. When choosing a packaging lm for any application, it is important to select the lm on the basis of the needs of the product. Some effect on barrier properties was expected; however, mechanical properties were not expected to be affected. Films containing only 2,500 and 10,000 IU/cm2 were analyzed and compared to uncoated and coated LDPE lm. The thickness of the lms varied according to the coating (Fig. 7). Coating material increased the thickness of all lms compared to uncoated LDPE; however, 10,000 IU/ cm2 nisin-coated lms had the greatest thickness because of the presence of sodium chloride crystals. The 2,500 IU/ cm2 nisin-coated lm also had sodium chloride crystals but contained only one quarter the amount of nisin compared with 10,000 IU/cm2 nisin-coated lm. This allowed fewer salt crystals to form, resulting in decreased thickness of the 2,500 IU/cm2 nisin-coated lm. These results are not un- FIGURE 7. Mean and standard deviations of thickness (mils) of various coating treatments: LDPE without coating and LDPE lm coated with a cellulose-based coating (control), with 2,500 IU/ cm2, and with 10,000 IU/cm2 nisin in the machine and cross directions. Cross direction refers to material oriented 908 from the machine direction. expected but relate to the differences observed in tensile strength. The tensile strength of all lm samples was signi cantly different (P, 0.05) between each lm treatment (LDPE, control, 2,500 IU/cm2, 10,000 IU/cm2 ) in the machine direction and the same treatment in the cross direction (Fig. 8). The tensile strength of machine direction for all treatments was signi cantly greater than the tensile strength of the cross direction for the corresponding sample. For example, the tensile strength of the 10,000 IU/cm2 lm in FIGURE 8. Mean tensile strength (psi) among various lm coating treatments: LDPE lm without coating and coated with a cellulose-based coating (control), with 2,500 IU/cm2, or with 10,000 IU/cm2 nisin in the cross and machine directions.

5 ANTIMICROBIAL FILM COATING FOR INHIBITION OF L. MONOCYTOGENES the machine direction was 1,836 psi, whereas the tensile strength of the cross direction was 1,691 psi. This difference was attributed to the properties of the overall tensile strength of LDPE, which served as the base material for all coating treatments. In general, polymer lms have greater tensile strength in the machine direction because of a stronger orientation of the bonds in this direction. There were signi cant differences (P, 0.05) among the lm treatments, except between the tensile strength of the control coated lm and LDPE. The signi cant difference in tensile strength among the coated treatments re ects the increased thickness of the coatings. The water vapor transmission rate of the LDPE lm coatings and control lm coatings were signi cantly different (P, 0.05); however, there was no signi cant difference among any of the other treatments (12.20 and g/m2/day for lms containing nisin concentrations of 2,500 and 10,000 IU/cm2, respectively). The control lm had a water vapor transmission rate of g/m2/day, whereas the LDPE lm without any coating had a water vapor transmission rate of g/m2 /day. Although signi cantly different, this difference was relatively small and may be attributed to the poor barrier properties of methylcellulose and hydroxypropyl methylcellulose. LDPE was a good moisture barrier, but hydroxyl groups on methylcellulose and hydroxypropyl methylcellulose might have attracted water, which decreased the moisture barrier properties. Overall, the differences between the coated lms were minimal, indicating that nisin concentrations had no effect on water vapor permeability. Water vapor transmission rates for all coated lms were similar to the ndings of researchers who examined methylcellulose lms (10, 14). A packaging lm coating solution containing nisin was developed and tested with a spot-on-lawn assay. Low-density polyethylene lms made with 10,000 IU/cm2 of nisin exhibited a snowy appearance. On examination with eld emission SEM, salt was found to be the major contributor of the crystal structures found on the surface of the lms. Differences in the tensile strength and percent elongation between lms were linked to coating thickness. The coating did not have a signi cant effect on the water vapor transmission rate of the LDPE lm. Overall, a nisin-containing coating was developed that did not adversely affect the properties of the LDPE lm and could be used effectively for food packaging applications. Levels of nisin made no difference in the performance characteristics of lm. This study developed a viable antimicrobial lm coating that could be used as an enhanced food safety measure delivered through an active packaging system. Further research is needed to determine the ef cacy of such antimi- 479 crobial packaging with various food products. Additionally, the mechanism of release and development of a controlledrelease system should be investigated. ACKNOWLEDGMENTS Special thanks to Glen Quattlebaum and Linda McCaskill for their technical assistance. REFERENCES Ahamad, N., and E. H. Marth Behavior of Listeria monocytogenes at 7, 13, 21, and 358C in tryptose broth with acetic, citric, or lactic acid. J. Food Prot. 52: American Society of Testing Materials Annual book of ASTM standards. ASTM, Philadelphia. Anonymous Nisiplin technical information. Integrated Ingredients, Rosemont, Ill. Benkerroum, N., and W. E. Sandine Inhibitory action of nisin against Listeria monocytogenes. J. Dairy Sci. 71(12): Brul, S., and P. 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