See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/11936873 Antimicrobial Effects of Corn Zein Films Impregnated with Nisin, Lauric Acid, and EDTA Article in Journal of food protection · July 2001 DOI: 10.4315/0362-028X-64.6.885 · Source: PubMed CITATIONS READS 121 311 3 authors, including: Inyee Y Han Paul L Dawson Clemson University Clemson University 37 PUBLICATIONS 839 CITATIONS 105 PUBLICATIONS 1,882 CITATIONS SEE PROFILE SEE PROFILE All content following this page was uploaded by Paul L Dawson on 04 December 2016 The user has requested enhancement of the downloaded file All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately 885 Journal of Food Protection, Vol 64, No 6, 2001, Pages 885–889 Copyright q, International Association for Food Protection Research Note Antimicrobial Effects of Corn Zein Films Impregnated with Nisin, Lauric Acid, and EDTA K L HOFFMAN, I Y HAN, AND P L DAWSON* Department of Food Science and Human Nutrition, Clemson University, Clemson, South Carolina 29634–0371, USA MS 00-353: Received October 2000/Accepted 21 January 2001 ABSTRACT Bacterial growth during food transport and storage is a problem that may be addressed with packaging materials that release antimicrobials during food contact In a series of ve experiments, EDTA, lauric acid (LA), nisin, and combinations of the three antimicrobial agents were incorporated into a corn zein lm and exposed to broth cultures of Listeria monocytogenes and Salmonella Enteritidis for 48 h (sampled at 2, 4, 8, 12, 24, and 48 h) Four experiments used starting cultures of 108 CFU/ml in separate experiments tested against each bacterium; the fth experiment examined the inhibitory effect of selected antimicrobial agents on Salmonella Enteritidis with an initial inoculum of 104 CFU/ml L monocytogenes cell numbers decreased by greater than logs after 48 h of exposure to lms containing LA and nisin alone No cells were detected for L monocytogenes (8-log reduction) after 24-h exposure to any lm combination that included LA Of all lm agent combinations tested, none had greater than a 1-log reduction of Salmonella Enteritidis when a 108-CFU/ml broth culture was used When a 104 CFU/ml of Salmonella Enteritidis initial inoculum was used, the lms with EDTA and LA and EDTA, LA, and nisin were bacteriostatic However, there was a 5-log increase in cells exposed to control within 24 h The results demonstrate bacteriocidal and bacteriostatic activity of lms containing antimicrobial agents Direct addition of antimicrobials results in some loss of activity at the surface because of leaching into the food core, enzymatic activity, and reaction with other food components such as lipids and proteins (5, 9, 17) Listeria monocytogenes is a concern in food with extended shelf life because of the ability to tolerate salt, pH changes, inadequate pasteurization, and refrigerated temperatures (2, 3) If an antimicrobial can be released from the package during an extended period, the activity can be extended into the transport and storage phase of food distribution Non–food grade antimicrobial polymers are available, several of which use Triclosan (18), silver (1), and quaternary ammonium salts (18) In the United States, the number of approved antimicrobial agents for food contact are limited at present and much of the work has focused on using organic acids and bacteriocins Ming et al (11) coated cellulose casings with pediocin to inhibit the growth of L monocytogenes on cooked meats Salmonella Typhimurium–inoculated poultry skin was reduced by 0.4 to 2.1 logs after exposure to nisin-coated polymer lms (12) Siragusa et al (20) incorporated nisin into polyethylene-based lms and observed 1.4-log reduced growth of Brochothrix thermosphacta on vacuum-packaged beef surfaces compared with lms not impregnated with nisin Nisin activity is restricted to gram-positive bacteria (17, 20) but can be active against gram-negative bacteria when combined with chelators and surfactants (21, 22) Larsen (10) found that 15 * Author for correspondence Tel: 864-656-1138; Fax: 864-656-0331; E-mail: pdawson@clemson.edu mM EDTA was effective against Escherichia coli when used in corn zein lms with different nisin concentrations Lauric acid (LA) at concentrations of 4% (400 mg/ml) and 8% (800 mg/ml) in corn zein lms effectively inhibited Lactobacillus plantarum (16) Nisin, at various concentrations alone and with other antimicrobial agents incorporated into lms, was effective against various organisms, including L plantarum, L monocytogenes, E coli, and Salmonella sp (4, 10, 15) The study objective was to determine the effect of corn zein lms impregnated with nisin, LA, and EDTA on L monocytogenes and Salmonella Enteritidis MATERIALS AND METHODS Five experiments were conducted to ful ll the objective Experiments and compared EDTA, LA, and nisin (singly) in corn zein lms for inhibiting L monocytogenes and Salmonella Enteritidis, respectively Experiments and compared the combinations of EDTA and LA; EDTA and nisin; and EDTA, LA, and nisin in corn zein lms for inhibiting the same two bacteria, respectively Experiment tested lower inoculum levels (104 CFU/ml) of Salmonella Enteritidis suspended in a growth medium exposed to lms containing LA; EDTA and LA; and EDTA, LA, and nisin Film formation The corn zein lms were formed using the casting method described by Gennadios and Testin (6) Corn zein (6.75 g) (F-4000 regular grade, Freeman Industries, Tuckahoe, N.Y.) was dissolved in 40.6 ml of 95% ethanol with stirring Glycerin (1.9 ml) (Fisher Scienti c, Fairlawn, N.J.) was added as a plasticizer and the mixture was heated slowly to a boil The mix- 886 HOFFMAN ET AL J Food Prot., Vol 64, No ture was boiled to help reduce gas bubbles in the lms during casting For antimicrobial-incorporated lms, antimicrobial agents were mixed with 10 ml of the lm solution in glass tubes just before casting A mortal and pestle were used to reduce the particle size of the EDTA and nisin, then the ne particles were dispersed in the lm solution using a sterile glass rod LA was dissolved into the lm solution Five milliliters of the lm mixtures was pipetted into level petri dishes (100-mm diameter by 15-mm depth) and allowed to dry at room temperature (23 to 258C) overnight After casting, ve measurements were made on each sample using an electronic micrometer (Model 49–60, Testing Machines, Inc., Amittyville, N.Y.), and the mean thickness was calculated to the nearest 0.005 mm Antimicrobial agents EDTA (J.T Baker Inc., Phillipsburg, N.J.), the acid form of LA (J.T Baker), and nisin (Sigma Chemical Co., St Louis, Mo.) were added separately to the three glass tubes containing 10 ml of lm mixture at concentrations of 5.58, 40, and 0.0375 mg/ml The nal concentration of each antimicrobial compound per lm was 27.9, 200, and 0.188 mg for EDTA, LA, and nisin, respectively Films with combinations of the antimicrobial agents were also formed using the concentrations stated: EDTA and LA, EDTA and nisin, LA and nisin, and all three together With each test, a control lm was also formed with no antimicrobial agents added Nisin activity The nisin was reported to be 2.5% pure, with the remaining components being listed as sodium chloride and milk solids The activity of the nisin was measured using an arbitrary unit (AU) dilution method A total of 1.5 mg of the stock nisin was mixed in ml of distilled, deionized water A 10-ml aliquot was taken from this 1.5-mg/ml solution and diluted at 1: 1, 1:2, 1:4, and 1:8; then 10 ml of these were spotted on a lawn of L plantarum The lowest dilution showing a clear zone was 1: 4; therefore, 10 ml of the 1.5-mg/ml solution was equated to AU This converts to 400 AU/ml of the 1.5-mg/ml solution or 400 AU/1.5 mg of the stock nisin Calculation of activity is therefore 266.6 AU/mg of stock nisin or 2.67 105 AU/g Enumeration L monocytogenes ATCC 15313 and Salmonella Enteritidis phage type 13 resistant to nalidixic acid (Southeastern Poultry Research Laboratory, Athens, Ga.) were used L monocytogenes was grown in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.) and incubated aerobically for 16 h at 378C Two 10-ml tubes of L monocytogenes were then centrifuged for 20 at 1,500 g, decanted, washed with 0.1% peptone (Difco), centrifuged 20 min, and decanted The two pellets of organisms were placed into 99 ml of peptone water to obtain an inoculum of approximately 108 CFU/ml The 108-CFU/ ml count was veri ed by serial dilutions of the inoculum suspended in peptone water and plating in BHI agar Fifteen milliliters of the inoculum were added to each of the petri plates containing the lms The lm with no added antimicrobials was used as a control The plates were put on an orbital shaker and rotated at room temperature (238C) at 50 rpm After 0, 2, 4, 8, 12, 24, and 48 h, 0.1-ml samples were taken from the petri dishes, diluted, and plated in duplicate on BHI agar The plates were incubated at 378C in an aerobic chamber for 48 h The number of colonies on each plate was counted and reported as CFU/ml The same tests were performed using Salmonella Enteritidis resistant to nalidixic acid grown in Trypticase soy broth (Difco) at 378C for 16 h Shaking was necessary and only one tube (pellet) of Salmonella Enteritidis in 99 ml of peptone water was needed to obtain 108 CFU/ml Also, 3.0 ml of nalidixic acid (Sigma) per 200 ml of media was added to the Trypticase soy agar when plat- FIGURE Effects of EDTA, LA, and nisin in corn zein lms on a 108-CFU/ml L monocytogenes starting population Asterisk indicates P value; n 6; SEM 0.54 ing the Salmonella Enteritidis samples to ensure that only nalidixic acid–resistant cells would be included in enumeration Another experiment was conducted using reduced concentrations of Salmonella Enteritidis (104 CFU/ml) A 104-CFU/ml inoculum was suspended in 50% peptone water and 50% growth medium (Trypticase soy broth) The Trypticase soy broth was added to determine if there was an increase in cell numbers during the duration of the experiment Fifteen milliliters of solution was placed on each lm and tested as stated above Statistical analysis Each experiment was replicated three times on different days, with two observations per lm treatment for each replication (n 6) A general linear model was used and an analysis of variance was conducted to determine the signi cance of the main effects of lm treatment, exposure time (2, 4, 8, 12, 24, and 48 h), and their interaction Replication, lm treatment, exposure time, and the treatment-by-time interaction were included in the model with the residual interaction effects comprising the error term Where the main effects were signi cant, the means were separated using the pdiff (SAS term to generate P values) and std err (SAS term used to generate standard error) of SAS (19) Where the treatment-by-time interaction was significant (P # 0.05), the lsmeans (SAS term to generate least significant means) for the interaction was sliced by treatment and time to determine which individual treatments or times were contributing to the interaction RESULTS AND DISCUSSION Film thickness ranged from 0.61 to 0.81 mm, with an average of 0.68 0.06 mm The addition of antimicrobial compounds had no effect on the lm thickness or the variation of lm thickness compared with lms with no added compounds Effects of EDTA, LA, and nisin-impregnated lm effects on L monocytogenes The control lm without antimicrobial agents and the lms containing EDTA caused no change (P 0.05) in L monocytogenes populations after 48 h of exposure (Fig 1) This was not surprising since EDTA is a chelating agent that enhances the effect of other antimicrobials but has little antagonistic effect itself (21, 22) LA and nisin had signi cant antibacterial effects J Food Prot., Vol 64, No 887 ANTIMICROBIAL EFFECTS OF CORN ZEIN FILMS TABLE Effect of different antimicrobial lms exposed to Salmonella Enteritidis suspended in 0.1% peptone watera Antimicrobial treatment Control EDTA Lauric acid Nisin EDTA and lauric acid EDTA and nisin Lauric acid and nisin EDTA, lauric acid and nisin FIGURE Effects of EDTA and LA (EL); EDTA and nisin (EN); LA and nisin (LN); and EDTA, LA, and nisin (All) in corn zein lms on a 108-CFU/ml L monocytogenes starting population Asterisk indicates P value; n 6; SEM 0.08 (P , 0.0001) LA reduced the population of L monocytogenes by 0.1 log (CFU/ml) at h, 0.8 log at 12 h, 2.9 logs at 24 h, and to 5.0 logs after 48 h Padgett et al (16) reported lms containing LA alone did not have a signi cant effect on L plantarum when using a zone of inhibition method, but LA reduced the population of L plantarum by logs in h when the lm was in contact with a liquid broth The lm containing nisin reduced L monocytogenes population by 1.0 log at h and 5.5 logs after 48 h Nisin concentrations in heat-pressed corn zein lms as low as 0.1 mg/g inhibited L monocytogenes growth (7) Orr et al (14) reported refrigerated milk samples inoculated with L monocytogenes had a log reduction of approximately logs when exposed for 48 h to cast corn zein lms containing nisin In a separate experiment combining the antimicrobials, the control lm reduced L monocytogenes by 1.5 logs (Fig 2) The EDTA and LA lm reduced L monocytogenes populations by logs after 12 h The log reduction values for the EDTA and nisin lm were 1.7 CFU/ml at h and 5.7 CFU/ml at 48 h The LA and nisin lm gave log reduction values of 0.8 CFU/ml at h and reduced cells to ‘‘too few to count’’ (,102 CFU/ml) by 12 h As with the EDTA and LA lm, no cells were detected at 12 or 48 h LA or nisin incorporated into corn zein lms were effective inhibitors of L plantarum; however, as the concentration of LA increased, the effectiveness of nisin decreased (16) In the present study, the lm with all of the antimicrobial agents (EDTA, LA, and nisin) had an effect similar as the LA and nisin lm and the EDTA and LA lm The log reduction was 1.39 CFU/ml at h, and cell population decreased to less than 102 by 12 and 48 h of lm exposure Thus, any of the lms containing combinations that included LA reduced cell numbers to below 102 after 12 h of exposure The LA alone caused a 5-log reduction in L monocytogenes, indicating that some synergy occurred when LA was combined with nisin or EDTA Effect of lm impregnated with EDTA, LA, and nisin on Salmonella Enteritidis No statistically signi cant (P 0.05) antimicrobial treatment and time interactions a b Mean log counts (CFU/ml) 8.14 8.11 8.02 8.16 7.89 8.02 8.02 7.82 AB b B C A D C C E n 36; SE 0.02 Means having the same superscript are not signi cantly different (P 0.05) occurred; therefore, only the main effects for treatment and the main effects for time were compared Salmonella Enteritidis displayed little growth during exposure to the control lm up to 48 h The effect of the control lm was not different (P 0.05) from the EDTA- or nisin-containing lms (Table 1) The results of the present study were similar to those found by Stevens et al (21), in which EDTA and nisin (not incorporated into lms) were tested against Salmonella Typhimurium with no inhibition The corn zein lm containing LA displayed no increase in culture population, and although different (P # 0.05) from the other lm treatments, LA should be considered ineffective in reducing Salmonella Enteritidis under these conditions The effect of the control lm was not different (P 0.05) from that of the lms containing EDTA and nisin or LA and nisin Stevens et al (21) reported that after h of direct exposure to 50 mg/ml of nisin and 20 mM EDTA, Salmonella Enteritidis Puerto Rico no was reduced 3.6 logs The results of the present study may differ from those reported by Stevens et al (21) because a different Salmonella sp culture was used, the nisin treatment was applied directly in the previous study rather than in a lm, and different concentrations of antimicrobials were used (lower concentration of nisin, higher concentration of EDTA) Broth cultures exposed to corn zein lm containing EDTA and LA and EDTA, LA, and nisin had lower (P # 0.05) populations than cultures exposed to other lms after 48 h; however, these differences were not signi cant from a practical standpoint At 2, 4, and h, Salmonella Enteritidis log counts were 7.93, 7.99, and 8.13 CFU/ml, respectively The mean log reduction values were not different (P 0.05) from 8.18 CFU/ml at 12 h to 8.19 CFU/ml at 48 h The results suggest no bacteriocidal effect on Salmonella Enteritidis by the lms containing single antimicrobial agents For some antimicrobial agents, the outer membrane lipopolysaccharide portion of gram-negative bacteria is dif cult to penetrate When EDTA disrupts the lipopolysaccharide layer, there is an increase in cell permeability (22), allowing nisin to disrupt cell activity and eventually resulting in cell lysis It was concluded that EDTA and nisin in this study needed to be in simultaneous contact with the bacterial culture to 888 HOFFMAN ET AL FIGURE Effects of LA; EDTA and LA (EL); EDTA and nisin (EN); and EDTA, LA, and nisin (All) in corn zein lms on a 104CFU/ml Salmonella Enteritidis starting population Asterisk indicates P value; n 6; SEM 0.01 have a killing effect A statistically signi cant increase in the Salmonella Enteritidis population was observed at and after h; however, these are again fractions of log values and are not noteworthy from a practical standpoint Effect of lm impregnated with LA; EDTA and LA; EDTA and nisin; and EDTA, LA, and nisin on a 104 CFU/ml Salmonella Enteritidis population The starting 108 -CFU/ml population would not be expected to increase in cell density much beyond the 108 level; therefore, the bacteriostatic properties of these treatments at lower initial inoculation levels were evaluated in experiment The most inhibitory antimicrobial treatments from the 108 -CFU/ ml experiment were used with a 104 -CFU/ml Salmonella Enteritidis inoculum For all of the lm samples, Salmonella Enteritidis grew from to 48 h (Fig 3) The control and LA lms did not differ (P 0.05) in cell growth, with Salmonella Enteritidis cell numbers increasing from 104 CFU/ml to 1010 CFU/ml With the EDTA and nisin lm, Salmonella Enteritidis cell numbers increased logs after 48 h The EDTA and LA and the EDTA, LA, and nisin lms held the increase in cell numbers to log after 48 h, which was logs less than the control lm at 48 h Natrajan and Sheldon (12) reported a 0.4- to 2.1-log reduction of Salmonella Typhimurium for inoculated broiler skin exposed to polymer lms coated with nisin formulations In a separate experiment, these researchers obtained up to a 4.6-log reduction by broiler drumsticks coating with agar or alginate gels containing a nisin mixture (13) The presence of moisture at the meat surface was determined to be bene cial for the ef cacy of nisin formulations (12, 13) Temperature, pH, time, growth medium, and lm properties may have affected the antimicrobial activity of the agents tested For example, nisin may be less effective in corn zein lms because of binding with the corn proteins Also, nisin is less stable and soluble at higher pH ranges (8) The lm solution pH was approximately 4.6, whereas the cell solution on the lms had a pH of 6.0 to 7.0 However, the nisin incorporated into the lms was very effective J Food Prot., Vol 64, No against L monocytogenes, which suggests that it was not binding (or had very little binding) with the corn proteins of the lms and was stable and active at the pH ranges used in this study The application of package-based biocides to reduce postprocess growth of food pathogens has promise The application of antimicrobial lms might allow for migration of the antimicrobial to the lm surface and therefore a continued antimicrobial effect at the food surface during extended exposure Direct addition of antimicrobials to food will result in an immediate reduction of bacterial populations but direct addition may not address the recovery of injured cells or the growth of cells that were not destroyed by direct addition Thus, antimicrobial lms may have applications for both uid and semisolid foods by inhibiting bacterial growth hours and days after packaging The growth and death rates of bacteria will vary for each growth medium; 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EDTA and nisin (EN); LA and nisin (LN); and EDTA, LA, and nisin (All) in corn zein lms on a 108-CFU/ml... and after h; however, these are again fractions of log values and are not noteworthy from a practical standpoint Effect of lm impregnated with LA; EDTA and LA; EDTA and nisin; and EDTA, LA, and. .. lm, and different concentrations of antimicrobials were used (lower concentration of nisin, higher concentration of EDTA) Broth cultures exposed to corn zein lm containing EDTA and LA and EDTA,