MS : Received 12 August 2009/Accepted 26 January 2010 ABSTRACT

Transcription

1 812 Journal of Food Protection, Vol. 73, No. 5, 2010, Pages Incorporation of Preservatives in Polylactic Acid Films for Inactivating Escherichia coli O157:H7 and Extending Microbiological Shelf Life of Strawberry Puree 3 TONY JIN,* HOWARD ZHANG, AND GLENN BOYD Food Safety Intervention Technologies Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania MS : Received 12 August 2009/Accepted 26 January 2010 ABSTRACT Antimicrobial films of polylactic acid polymer incorporated with nisin, EDTA, sodium benzoate (SB), potassium sorbate (PS), and their combinations were developed, and their antimicrobial effects on the inactivation of Escherichia coli O157:H7 and natural background microflora (total aerobic bacteria, molds, and yeasts) in strawberry puree at 10 and 22uC were determined. Direct addition of SBzPS to strawberry puree was also used as a comparison with SBzPS film treatment. The combination treatment reduced the cell populations of E. coli O157:H7 from 3.5 log CFU/ml to undetectable levels (,1 CFU/ml) after 14 days and 1 day at 10 and 22uC, respectively, while the cells of E. coli O157:H7 in control samples survived up to 48 days at 10uC and more than 14 days at 22uC. The SBzPS film treatment produced a greater reduction of population of E. coli O157:H7 cells than did the SBzPS direct addition treatment. Similar results were observed for inactivation of natural microflora. In general, the antimicrobial effect was in the following order: film combination. SBzPS film. SBzPS direct addition. EDTA film. nisin film. The data obtained in this study suggest two approaches toward the development of control interventions against E. coli O157:H7 and extension of the microbiological shelf life of strawberry puree: (i) using antimicrobial packaging and (ii) using combinations of preservatives. The film formulas developed here can be used to make bottles or as coatings on the surface of bottles for use in liquid food packaging. Enteric foodborne pathogens including Escherichia coli O157:H7 have been reported to be capable of survival in raw fruit and vegetable juices (2, 28, 30, 35). Microorganisms associated with fruit juice outbreaks in the United States include E. coli O157:H7 and Salmonella (3 5, 8, 9). Although thermal processing (heat pasteurization) has been long recognized as an effective method to eliminate pathogenic vegetative cells in fluid foods such as juices, it may extensively damage the organoleptic, nutritional, and physicochemical properties of food (16, 18, 24). To avoid excess thermal processing, preservatives are generally introduced in a food to prevent or delay spoilage. Potassium sorbate (PS) and sodium benzoate (SB) have been used effectively against yeasts in fruit products in the past (6, 39). In addition, sorbate and benzoate were found to be effective against E. coli O157:H7 in apple cider (29, 40). Zhao et al. (50) reported that SB (0.1%) caused a 5-log-unit reduction in E. coli O157:H7 populations during 2 to 10 days of storage at 8uC. The use of nisin in combination with food grade chelating agents such as EDTA to inactivate gram-negative bacteria such as Salmonella and E. coli O157:H7 did meet with some success (11, 31, 32, 41 43, 47). Ukuku and Fett * Author for correspondence. Tel: ; Fax: ; tony.jin@ars.usda.gov. { Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. (44) reported that treatment of whole and fresh-cut cantaloupe and honeydew melon with a combination of nisin and EDTA significantly reduced the natural microflora and extended its shelf life. Zhao et al. (50) found SB to be more effective in E. coli O157:H7 inactivation than PS. They also indicated that combinations of the two preservatives exhibited enhanced antimicrobial activity in cider held at 8uC when compared with SB added alone. However, few published data are available regarding the use of the combination of nisin or EDTA with SB and PS to reduce E. coli O157:H7 populations in strawberry puree. Strawberries are a major dietary source of ellagic acid containing compounds (7), which demonstrate anticancer activity, anti-inflammatory effect, and prevention of heart disease by decreased oxidation of low-density lipoprotein and platelet aggregation (20). Strawberry purees are produced commercially; some are consumed directly, while others are used as intermediate products and ingredients for juices, smoothies, fruit fillings, yogurt, jams or jellies, etc. In a bar or restaurant setting, strawberry purees are often used directly without any further thermal treatment (e.g., added to smoothies and used as ice cream toppings). Therefore, it is important to ensure that the purees contain no pathogens. A traditional approach to controlling the growth of undesirable microorganisms in foods is adding preservatives such as nisin to foods by formulation. Direct addition of nisin into foods results in an immediate reduction of bacterial populations but may not prevent the recovery of

2 J. Food Prot., Vol. 73, No. 5 ANTIMICROBIAL FILMS INACTIVATE E. COLI O157:H7 AND NATURAL MICROFLORA 813 TABLE 1. Formulas of antimicrobial films Amt (g) Amt (mg) Amt (ml) Antimicrobial films PLA a Nisin b EDTA c PS d SB d Methylene chloride e PLA only (PLA film) 1 15 PLA z nisin (nisin film) PLAzEDTA (EDTA film) PLAzSBzPS (SBzPS film) PLAzEDTA z nisin z SBzPS (combined film) a From Natureworks, Minnetonka, MN. b 2.5% purity; Sigma Chemical Co., St. Louis, MO. c Na 2 H 2 EDTA; Sigma Chemical Co. d Supplied by American Beverage Marketers, New Albany, IN. e From Fisher Scientific, Fairlawn, NJ. injured cells or the growth of cells that were not destroyed by direct addition if residues of the antimicrobial are rapidly depleted (49). Our previous studies demonstrated that nisin incorporated in polylactic acid (PLA) or pectin-pla films retained more activity against foodborne pathogens over 48 h than did a direct addition of a similar total dose and suggested that the incorporation of preservatives into the PLA polymer could provide a possible delivery system for improving the efficacy of preservatives in food applications (25, 26). The objective of this work was to test the hypotheses that incorporation of preservatives into PLA films would retain more antimicrobial activity than direct addition and that the combination of the preservatives would generate synergistic effects. In this study, films of PLA polymer incorporated with nisin, EDTA, PS, SB, and their combinations were developed. The effects of these antimicrobials on the inactivation of E. coli O157:H7, total aerobic bacteria, molds, and yeasts in strawberry puree were determined. MATERIALS AND METHODS Food sample. Single-strength strawberry puree without preservatives was supplied by American Beverage Marketers (New Albany, IN). Puree samples were shipped frozen and thawed 1 day prior to use. The ph of the strawberry puree was 3.7. Film preparation. One gram of PLA resin and a predetermined amount of antimicrobials (Table 1) were accurately weighed and dispersed in 15 ml of methylene chloride. This mixture was stirred by a magnetic bar until the polymer was totally dissolved and then distributed to Teflon petri dishes 10 cm in diameter and 1.5 cm deep (Performance Plastics, Wayne, NJ). The methylene chloride was allowed to evaporate at room temperature under a chemical hood. Each dish was stored over a desiccant for 24 h, and then films were peeled from the dishes and placed in a sealed container until time of use (within 1 week). Bacterial inoculum. E. coli O157:H7 Oklahoma was obtained from the culture collection of the U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center. Stock cultures were maintained at 280uC. The inoculating cultures were propagated on tryptic soy agar (Difco, Becton Dickinson, Sparks, MD) at 37uC and maintained at 0 to 2uC until use. Prior to inoculation, E. coli O157:H7 cells were grown in tryptic soy broth (Remel, Inc., Lenexa, KS) aerobically at 37uC for 16 to18 h. Antimicrobial study. One-half (surface area of ca. 24 cm 2 )of each resultant film was placed in a glass bottle with 50 ml of strawberry puree, creating a ratio of 2.08 ml of liquid per 1 cm 2 of exposed polymer surface. Film samples in each bottle had 500 mg of PLA and contained 125 mg of nisin (2.5% purity), 125 mg of EDTA, or 22.5 mg of PS plus 37.5 mg of SB for nisin film, EDTA film, or SBzPS film, respectively. The combined film contained 125 mg of nisin, 125 mg of EDTA, 22.5 mg of PS, and 37.5 mg of SB. For the direct-addition test (SBzPS direct treatment), 22.5 mg of PS plus 37.5 mg of SB was added to 50 ml of strawberry puree sample. The medium in the bottle was inoculated with 1 ml of an overnight culture of E. coli O157:H7 and shaken at 10 or 22uC at 150 rpm. The initial cell populations inoculated in samples were approximately CFU/ml. Samples without any film or preservative treatment served as controls. The strawberry puree samples without inoculation of E. coli O157:H7 were used for total plate count and mold and yeast analyses. Microbiological analysis. At different storage times after film or preservative treatments, a 1-ml portion of strawberry puree was sampled and the specimens were serially diluted by sterile Butterfield s phosphate buffer (ph 7.2; Hardy Diagnostics, Santa Maria, CA) and then pour plated onto different agars. MacConkey sorbitol agar (Becton Dickinson) with cefixime tellurite supplement (Invitrogen Dynal, Oslo, Norway) was used to enumerate E. coli O157:H7 counts in each sample. The plates were incubated at 37uC for 24 h to determine plate counts. The plate count agar (Becton Dickinson) plates were incubated at 37uC for up to 48 h to determine total aerobic mesophilic bacterial populations. Yeasts and molds were enumerated on dichloran rose bengal chloramphenicol agar (Becton Dickinson) prepared according to the manufacturer s recommendations. The plates were incubated at 25uC for 5 days. Statistical analysis. Antimicrobial experiments were conducted in triplicate. Data points were expressed as the mean log CFU per milliliter standard deviation. Data were analyzed by analysis of variance from SAS version 9.1 software (SAS Institute, Cary, NC). Duncan s multiple range tests were used to determine the significant difference of mean values. Unless stated otherwise, significance was expressed at the 5% level. RESULTS Inactivation of E. coli O157:H7 in strawberry puree. Figure 1 shows the survival of E. coli O157:H7 in strawberry puree stored at 10uC. When inoculated with 3.5 log CFU of E. coli O157:H7 per ml and stored at 10uC, the

3 814 JIN ET AL. J. Food Prot., Vol. 73, No. 5 FIGURE 1. Survival of E. coli O157:H7 in strawberry puree at 10uC. Fifty-milliliter samples of strawberry puree were treated with nisin film (125 mg of nisin), EDTA film (125 mg of EDTA), SBzPS film (22.5 mg of PS plus 37.5 mg of SB), combined film (125 mg of nisin plus 125 mg of EDTA plus 22.5 mg of PS plus 37.5 mg of SB), and PLA film (no preservatives). SBzPS direct treatment contained 22.5 mg of PS plus 37.5 mg of SB. Error bars represent the standard deviations of the means. cell numbers in the control gradually decreased to undetectable levels (,1 CFU/ml) during the 48-day storage period. Preservative treatments reduced the cells at a greater rate: the organism was reduced to undetectable levels after 28 days by nisin film; after 21 days by EDTA film, SBzPS film, and SBzPS direct addition treatments; and after 14 days by the combination film treatments. In general, the combination film treatment had the greatest effect and the nisin film treatment had the least effect on the reduction of E. coli O157:H7 populations in strawberry puree. Significant differences (P, 0.05) of cell reduction between SBzPS film and SBzPS direct addition treatments were observed, as the SBzPS film treatment reduced more cells of E. coli O157:H7 at 7 and 14 days. There was no significant difference between the nisin treatment and the control treatments on the first 14 days. However, from day 21 to day 28, nisin significantly (P, 0.05) reduced the pathogen compared to the controls. Similarly, the combination film treatment proved to be the most effective and the nisin film treatment the least effective against E. coli O157:H7 when the samples were stored at 22uC (Fig. 2). All samples were initially inoculated with approximately CFU of E. coli O157:H7 per ml. Two hours after the addition of films into strawberry puree samples, the treatments of nisin, EDTA, and SBzPS films reduced the cell counts of E. coli O157:H7 approximately 1 to 2.5 log CFU/ml, with the latter being further reduced to undetectable levels at 7, 3, and 2 days of storage, respectively. The combination film treatment reduced the pathogen s population by 2 log CFU/ml initially, and to an undetectable level at day 1. Storage at a lower temperature (10uC) has been shown to enhance the survival of E. coli O157:H7 in strawberry purees. During the 7-day storage, cells of E. coli O157:H7 in control samples decreased by 1 log CFU/ml at 22uC (Fig. 2) and by only 0.5 log CFU/ml at 10uC (Fig. 1). It took 14, 21, or 28 days at 10uC for preservative treatments FIGURE 2. Survival of E. coli O157:H7 in strawberry puree at 22uC. Fifty-milliliter samples of strawberry puree were treated with nisin film (125 mg of nisin), EDTA film (125 mg of EDTA), SBzPS film (22.5 mg of PS plus 37.5 mg of SB), and combined film (125 mg of nisin plus 125 mg of EDTA plus 22.5 mg of PS plus 37.5 mg of SB). SBzPS direct treatment contained 22.5 mg of PS plus 37.5 mg of SB. Error bars represent the standard deviations of the means. to reduce the E. coli cells to undetectable levels (Fig. 1), while only 1, 2, 3, or 7 days were needed at 22uC (Fig. 2). Similar results were obtained by Zhao et al. (50), who reported that E. coli O157: H7 in apple cider without any treatments survived from 10 to 31 days at 8uC or 2 to 3 days at 25uC. For the film-treated samples, the results may indicate that higher temperature likely increases the release of preservatives from films and enhances their antimicrobial activities against E. coli O157:H7, as a shorter time was required to reduce the populations of E. coli O157:H7 to undetectable levels for each preservative treatment at 22uC than at 10uC. Similar temperature dependence of nisin released from film was also observed by Dawson et al. (13). PLA film without any preservatives (PLA only) did not contribute to any antimicrobial activity against E. coli O157:H7 (Fig. 1), which was also observed in our previous study (26); therefore, the data from this treatment were not shown in Fig. 2. Inactivation of aerobic bacteria and molds and yeasts in strawberry puree. The first experiment was conducted at 22uC to determine the effects of nisin, EDTA, and PSzPB in film formula on the inactivation of natural microflora in strawberry purees. The survival of aerobic bacteria and molds and yeasts in strawberry purees after film treatments is shown in Figures 3 and 4, respectively. Total aerobic bacterial counts (TPC) in control samples and in samples treated with EDTA film and nisin film slightly increased after 3 days and then decreased during 28 days of storage. TPC in SBzPS film treated samples decreased to 1.1 log CFU/mg at 28 days, whereas control, nisin film, and EDTA film samples had 2.8, 2.5, and 2.2 log CFU/ml, respectively (Fig. 3). As shown in Figure 4, molds and yeasts in the control sample grew rapidly to 6.5 log CFU/ml after 14 days and then gradually decreased to 5 log CFU/ml at the end of the 28-

4 J. Food Prot., Vol. 73, No. 5 ANTIMICROBIAL FILMS INACTIVATE E. COLI O157:H7 AND NATURAL MICROFLORA 815 FIGURE 3. Survival of total aerobic bacteria in strawberry puree at 22uC. Fifty-milliliter samples of strawberry puree were treated with nisin film (125 mg of nisin), EDTA film (125 mg of EDTA, SBzPS film (22.5 mg of PS plus 37.5 mg of SB), and PLA film (no preservatives). Error bars represent the standard deviations of the means. day storage time. Similar growth patterns were observed for all the treatments; that is, the cells increased and then decreased during the storage period. However, the maximum cell populations of molds and yeasts were obtained at 17, 7, and 3 days for nisin film, EDTA film, and SBzPS film treatments and were 5.9, 4.8, and 4.1 log CFU/ml, respectively. By the end of storage, SBzPS film treatment reduced the cell populations to 0.8 log CFU/ml, which was significantly (P, 0.05) lower than those of the EDTA film treatment (3.0 log CFU/ml) and the nisin film treatment (4.8 log CFU/ml). The results indicated that the film treatment of PSzPB had more effect on aerobic bacteria and molds and yeasts than the treatments of EDTA and nisin films. The second experiment was conducted at 10uC with the same treatments as those for E. coli O157:H7; that is, nisin film, EDTA film, SBzPS film, direct addition of SBzPS, and the combination film treatments. FIGURE 5. Survival of total aerobic bacteria in strawberry puree at 10uC. Fifty-milliliter samples of strawberry puree were treated with nisin film (125 mg of nisin), EDTA film (125 mg of EDTA), SBzPS film(22.5mgofpsplus37.5mgofsb),andcombinedfilm(125mg of nisin plus 125 mg of EDTA plus 22.5 mg of PS plus 37.5 mg of SB). SBzPS direct treatment contained 22.5 mg of PS plus 37.5 mg of SB. Error bars represent the standard deviations of the means. Figure 5 shows the survival of total aerobic bacteria in strawberry puree stored at 10uC. TPC decreased during the 48-day storage period in all treated and control samples. After 48 days of storage, the TPCs were 1.6, 1.1, 0.8, and 0.3 log CFU/ml for control, nisin film, SBzPS direct addition, and EDTA film treatments, respectively. There were no TPCs observed in the samples treated by the combination and SBzPS films. The combination film treatment exhibited the most effect on the reduction of aerobic bacteria in strawberry puree at 10uC, followed by SBzPS film treatment. The effects of preservative treatments on survival of molds and yeasts in strawberry puree stored at 10uC are shown in Figure 6. Molds and yeasts grew in the control FIGURE 4. Survival of molds and yeasts in strawberry puree at 22uC. Fifty-milliliter samples of strawberry puree were treated with nisin film (125 mg nisin), EDTA film (125 mg of EDTA), SBzPS film (22.5 mg of PS plus 37.5 mg of SB), and PLA film (no preservatives). Error bars represent the standard deviations of the means. FIGURE 6. Survival of molds and yeasts in strawberry puree at 10uC. Fifty-milliliter samples of strawberry puree were treated with nisin film (125 mg of nisin), EDTA film (125 mg of EDTA), SBzPS film (22.5 mg of PS plus 37.5 mg of SB), and combined film (125 mg of nisin plus 125 mg of EDTA plus 22.5 mg of PS plus 37.5 mg of SB). SBzPS direct treatment contained 22.5 mg of PS plus 37.5 mg of SB. Error bars represent the standard deviations of the means.

5 816 JIN ET AL. J. Food Prot., Vol. 73, No. 5 sample from 4.1 to 6.6 log CFU/ml after 14 days at 10uC and then remained constant through the 48-day storage period. Nisin film treatment reduced the mold and yeast counts by 0.5 log CFU/ml after 2 h of treatment, but they then had a growth rate similar to that of the control and reached 5.8 log CFU/ml at 48 days. All other treatments significantly (P, 0.05) reduced the growth rates of molds and yeasts. No cells of mold or yeast were observed at 48, 28, and 14 days for treatments of SBzPS direct addition, PSzPB film, and combination film, respectively. The EDTA film treatment reduced the mold and yeast counts by 2 log CFU/ml, but there were cell levels of 1.1 log CFU/ml observed at the end of storage. Similar to what was observed with E. coli O157:H7, among the treatments, the combination film treatment was the most effective treatment, and SBzPS film treatment was the second most effective treatment with regard to antimicrobial activities against natural microflora. Similar to what was observed for E. coli O157:H7, the PLA-only treatment did not affect the survival of the natural background microflora in strawberry puree (Figs. 3 and 4); hence, the data were not reported in Figures 5 and 6. DISCUSSION SB and PS are generally recognized as safe preservatives for miscellaneous and general-purpose usage. A maximum level of 0.30% SB is allowed for use in foods (17). Effective antimicrobial concentrations of sorbates in most foods are in the range of 0.05 to 0.30% (38). Zhao et al. (50) reported that 0.1% SB was an effective antimicrobial agent against E. coli O157:H7 at 8uC, reducing the population of the organism to undetectable levels (reduction of.4 log CFU/ml) within 7 to 15 days. Enhanced antimicrobial activity at 8uC was observed when 0.1% PS and 0.1% SB were used in combination, with survival times being reduced by approximately 50%. However, concentrations of benzoate higher than % reportedly impart noticeable tastes (37). Hence, using the lowest effective concentration of preservatives would be beneficial to consumers and the beverage industry. In this study, the antimicrobial effectiveness of SB and PS released to a liquid medium was affected by the mode of addition. The preservatives released from films reduced more microbial cells than those instantly added at each corresponding storage time. Our previous study showed that nisin in pectin-pla film also retained more activity against Listeria monocytogenes over 48 h than by direct addition of nisin in brain heart infusion broth (25). Similarly, Salmaso et al. (36) observed that nisin-loaded PLA particles prolonged nisin activity in a liquid medium up to 40 days while instantly added nisin samples displayed antibacterial activity only for 7 days. Antimicrobials can be added to food formulations directly or through slow release from packaging materials. When added instantly to a food formulation, an antimicrobial is eventually exhausted from the foods due to interactions with the food matrix and bacterial cells (48). Consequently, adding antimicrobials by the formulation approach alone often requires excessive amounts of antimicrobials for effective inhibition of pathogens. Although very limited information is available about the decrease of SB and/or PS activity in the direct addition model, several studies involving nisin have been published. Zhang et al. (49) studied the effectiveness of nisin against Listeria in the simulated liquid food models, examining (i) the formation of nisin in liquid foods and (ii) the slow release of nisin from packaging materials to liquid foods. They discovered that cells treated with instantaneously added nisin developed resistance to nisin, in contrast to cells treated with slowly added nisin at the same total amount of the antimicrobial; hence, the antimicrobial effectiveness of instantaneously added nisin decreased. Others reported that direct addition of nisin could result in more loss of its activity because of instant reaction with other food components such as lipids or proteins (12, 19, 22, 25, 46). The application of antimicrobial films or coatings allows for the migration of the antimicrobial to the film surface and then to liquid media, providing a continuous antimicrobial effect on the food during extended exposure. Therefore, the use of packaging films or coatings containing antimicrobial agents could be made more efficient by a controlled migration of the agents into the food; this would allow not only initial inhibition of undesirable microorganisms but also residual activity over time during the transport and storage of food during distribution (10, 33). These data further confirmed that antimicrobial packaging provides an approach to improve the effectiveness for control interventions developed to reduce pathogenic and spoilage microorganisms. The results of this study showed that nisin, EDTA, and SBzPS, when used individually, also exhibited an antimicrobial effect on E. coli O157:H7 and native microflora, and among them, the nisin treatment showed the least antimicrobial effect. Other researchers have indicated that nisin possesses antimicrobial activity against a wide range of gram-positive bacteria but shows little or no activity against gram-negative bacteria, yeasts, or molds (23, 34, 38). However, our data show that the effect of combined treatments was significantly greater than that of each preservative used alone. Our results agreed with those of others using the combination of nisin with EDTA (1, 41 43). Larsen (27) found that 15 mm EDTA was effective against E. coli when used in corn zein films with different nisin concentrations. Zhang and Mustapha (47) reported that nisin combined with EDTA resulted in a 0.8-log reduction of E. coli O157:H7 compared with a control over a 30-day period. It is well recognized that the antimicrobial effect of nisin is caused by its interaction with phospholipid components of the cytoplasmic membrane followed by an interference with the membrane function (21), and EDTA, a food grade chelator, binds ions on the outer membrane of bacterial cells and destroys the cell wall structure (45). Addition of SBzPS to nisin and EDTA would help to further destroy microbial cells because the undissociated benzoic acid molecule interferes with the permeability of the microbial cell membrane (15). Therefore, a synergistic effect clearly played a role in preventing growth of

6 J. Food Prot., Vol. 73, No. 5 ANTIMICROBIAL FILMS INACTIVATE E. COLI O157:H7 AND NATURAL MICROFLORA 817 microorganisms. Because of this synergism, it may be possible that the concentration of each preservative in the film formula could be reduced while a similar reduction of spoilage and pathogenic microorganisms could be achieved. Further study is warranted to optimize the film formulation and to achieve maximum or certain microbial reduction with a minimum addition of preservatives. Yeasts, molds, and lactic acid bacteria have been implicated in spoilage of fruit juices (14). Molds and yeasts are able to grow over wider ranges of ph and temperature than bacteria; lower storage temperature does not prevent their growths in high-acid foods. The data in this study suggested that the incorporation of preservatives into polymer films and the use of a combination of preservatives provided effective ways to reduce the natural aerobic background microflora and presumably extend the shelf life of strawberry puree. In conclusion, this study demonstrated that SBzPS retained more antimicrobial activity when they were incorporated into PLA films, while nisin, EDTA, SB, and PS in combination achieved significant reductions of native microflora and inoculated populations of E. coli O157:H7. The developed film formulas can be used in liquid food packaging, either directly in bottle manufacturing or as a coating material on the bottle inner surface. Antimicrobial packaging for liquid foods is an alternative approach to ensure food safety and extend food shelf life. ACKNOWLEDGMENTS The authors thank Drs. Joshua Gurtler and Dike Ukuku for their thoughtful reviews of the manuscript and Anita Parameswaran for technical support. 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