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
Trang 1Antimicrobial 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
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Trang 2Journal 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 9 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
monocy-togenes 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 104CFU/ml L monocytogenes cell numbers decreased by greater than 4 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 104CFU/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
com-ponents such as lipids and proteins (5, 9, 17) Listeria
mon-ocytogenes is a concern in food with extended shelf life
because of the ability to tolerate salt, pH changes,
inade-quate 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
quater-nary ammonium salts (18) In the United States, the number
of approved antimicrobial agents for food contact are
lim-ited at present and much of the work has focused on using
organic acids and bacteriocins Ming et al (11) coated
cel-lulose casings with pediocin to inhibit the growth of L.
monocytogenes on cooked meats Salmonella
Typhimu-rium–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
ther-mosphacta on vacuum-packaged beef surfaces compared
with lms not impregnated with nisin Nisin activity is
re-stricted to gram-positive bacteria (17, 20) but can be active
against gram-negative bacteria when combined with
che-lators 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
concentra-tions alone and with other antimicrobial agents incorporated into lms, was effective against various organisms,
includ-ing L plantarum, L monocytogenes, E coli, and
Salmo-nella 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
Enteriti-dis
MATERIALS AND METHODS
Five experiments were conducted to ful ll the objective Ex-periments 1 and 2 compared EDTA, LA, and nisin (singly) in
corn zein lms for inhibiting L monocytogenes and Salmonella
Enteritidis, respectively Experiments 3 and 4 compared the com-binations of EDTA and LA; EDTA and nisin; and EDTA, LA, and nisin in corn zein lms for inhibiting the same two bacteria, respectively Experiment 5 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 Glyc-erin (1.9 ml) (Fisher Scienti c, Fairlawn, N.J.) was added as a plasticizer and the mixture was heated slowly to a boil The
Trang 3mix-J Food Prot., Vol 64, No 6
886 HOFFMAN ET AL.
FIGURE 1 Effects of EDTA, LA, and nisin in corn zein lms on
a 108-CFU/ml L monocytogenes starting population Asterisk in-dicates P value; n 5 6; SEM 5 0.54.
ture was boiled 5 min 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,
Test-ing 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
antimicro-bial 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
ar-bitrary unit (AU) dilution method A total of 1.5 mg of the stock
nisin was mixed in 1 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 4
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 3 105AU/g
Enumeration L monocytogenes ATCC 15313 and
Salmo-nella Enteritidis phage type 13 resistant to nalidixic acid
(South-eastern 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 min at 1,500 3 g, decanted, washed with 0.1%
peptone (Difco), centrifuged 20 min, and decanted The two
pel-lets of organisms were placed into 99 ml of peptone water to
obtain an inoculum of approximately 108CFU/ml The 108-CFU/
ml count was veri ed by serial dilutions of the inoculum
sus-pended in peptone water and plating in BHI agar Fifteen
milli-liters of the inoculum were added to each of the petri plates
con-taining 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 108CFU/ml Also, 3.0 ml of nalidixic acid (Sigma) per
200 ml of media was added to the Trypticase soy agar when
plat-ing the Salmonella Enteritidis samples to ensure that only
nali-dixic acid–resistant cells would be included in enumeration Another experiment was conducted using reduced
concentra-tions of Salmonella Enteritidis (104CFU/ml) A 104-CFU/ml in-oculum was suspended in 50% peptone water and 50% growth medium (Trypticase soy broth) The Trypticase soy broth was
add-ed 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 5 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 treat-ment, exposure time, and the treatment-by-time interaction were included in the model with the residual interaction effects com-prising 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 icant (P # 0.05), the lsmeans (SAS term to generate least
signif-icant means) for the interaction was sliced by treatment and time
to determine which individual treatments or times were contrib-uting to the interaction
RESULTS AND DISCUSSION
Film thickness ranged from 0.61 to 0.81 mm, with an average of 0.68 6 0.06 mm The addition of antimicrobial compounds had no effect on the lm thickness or the vari-ation 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
an-timicrobial 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
Trang 4FIGURE 2 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
As-terisk indicates P value; n 5 6; SEM 5 0.08.
TABLE 1 Effect of different antimicrobial lms exposed to Sal-monella Enteritidis suspended in 0.1% peptone water a
Antimicrobial treatment
Mean log counts (CFU/ml) Control
EDTA Lauric acid Nisin EDTA and lauric acid EDTA and nisin Lauric acid and nisin EDTA, lauric acid and nisin
8.14ABb
8.11B
8.02C
8.16A
7.89D
8.02C
8.02C
7.82E
a n 5 36; SE 5 0.02.
bMeans having the same superscript are not signi cantly different
(P 0.05).
(P , 0.0001) LA reduced the population of L
monocy-togenes by 0.1 log (CFU/ml) at 2 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
5 logs in 6 h when the lm was in contact with a liquid
broth The lm containing nisin reduced L monocytogenes
population by 1.0 log at 2 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
mon-ocytogenes had a log reduction of approximately 2 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
pop-ulations by 8 logs after 12 h The log reduction values for
the EDTA and nisin lm were 1.7 CFU/ml at 2 h and 5.7
CFU/ml at 48 h The LA and nisin lm gave log reduction
values of 0.8 CFU/ml at 2 h and reduced cells to ‘‘too few
to count’’ (,102CFU/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
in-creased, 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 2 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
re-duced cell numbers to below 102 after 12 h of exposure
The LA alone caused a 5-log reduction in L
monocyto-genes, indicating that some synergy occurred when LA was
combined with nisin or EDTA
Effect of lm impregnated with EDTA, LA, and
nis-in on Salmonella Enteritidis No statistically signi cant
(P 0.05) antimicrobial treatment and time interactions
occurred; therefore, only the main effects for treatment and
the main effects for time were compared Salmonella
En-teritidis displayed little growth during exposure to the con-trol lm up to 48 h The effect of the concon-trol 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
Sal-monella Typhimurium with no inhibition The corn zein
lm containing LA displayed no increase in culture
popu-lation, and although different (P # 0.05) from the other
lm treatments, LA should be considered ineffective in
re-ducing 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 1 h of direct exposure to 50 mg/ml of nisin and 20 mM EDTA,
Salmo-nella Enteritidis Puerto Rico no 1 was reduced 3.6 logs.
The results of the present study may differ from those
re-ported by Stevens et al (21) because a different Salmonella
sp culture was used, the nisin treatment was applied di-rectly in the previous study rather than in a lm, and dif-ferent 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 prac-tical standpoint
At 2, 4, and 8 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 an-timicrobial 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
dis-rupt 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
Trang 5J Food Prot., Vol 64, No 6
888 HOFFMAN ET AL.
FIGURE 3 Effects of LA; EDTA and LA (EL); EDTA and nisin
(EN); and EDTA, LA, and nisin (All) in corn zein lms on a 104
-CFU/ml Salmonella Enteritidis starting population Asterisk
in-dicates P value; n 5 6; SEM 5 0.01.
have a killing effect A statistically signi cant increase in
the Salmonella Enteritidis population was observed at and
after 8 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 5 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 0 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 104CFU/ml to 1010CFU/ml
With the EDTA and nisin lm, Salmonella Enteritidis cell
numbers increased 4 logs after 48 h The EDTA and LA
and the EDTA, LA, and nisin lms held the increase in cell
numbers to 1 log after 48 h, which was 5 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
re-searchers 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
prop-erties 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
How-ever, the nisin incorporated into the lms was very effective
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 mi-gration 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 pop-ulations but direct addition may not address the recovery
of injured cells or the growth of cells that were not de-stroyed by direct addition Thus, antimicrobial lms may have applications for both uid and semisolid foods by in-hibiting bacterial growth hours and days after packaging The growth and death rates of bacteria will vary for each growth medium; therefore, conclusions on how antimicro-bial lms will perform with a food product must be deter-mined for each food application
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