1255

Journal of Food Protection, Vol. 64, No. 8, 2001, Pages 1255–1260

Copyright , International Association for Food Protection

Research Note

Antibacterial Activity in Extracts of Camellia japonica L. Petals

and Its Application to a Model Food System
KEUN Y. KIM,1 P. MICHAEL DAVIDSON, 2 AND HEE J. CHUNG 1*

1 Department of Food Science and Technology, College of Agriculture, Chonnam National University, Kwangju 500-757, Korea; and 2Department of
Food Technology and Science, 2509 River Drive, University of Tennessee, Knoxville, Tennessee 37996-4500, USA

MS 00-267: 3 August 2000/Accepted 27 February 2001

ABSTRACT

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The potential presence of naturally occurring antimicrobials in petals of Camellia japonica L., a member of the tea family,
was investigated against foodborne pathogens in microbiological media and food. Petals of the camellia  ower (C. japonica
L.) were extracted with methanol and fractionated into basic, acidic, and neutral fractions. The acidic fraction (equivalent to
1.0 g of raw sample per disk) produced an inhibitory zone of 14 to 19 mm (diameter) in a disk assay against the pathogens
Salmonella Typhimurium DT104, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on agar
plates. Silica gel adsorption column chromatography, Sephadex LH-20 column chromatography, and preparative puriŽ cation
by high-pressure liquid chromatography were used to purify compounds in the fraction. The mass spectrum of the antibacterial
compound isolated had a molecular ion (M1 ) of m/z 116 and showed good conformity with the spectrum of fumaric acid
(HOOC-CH5CH-COOH). An aqueous extract from the petals of C. japonica L. had an inhibitory effect on growth of all
pathogens at 378C in microbiological media by increasing the lag phase. None of the microorganisms was inhibited completely.
Milk was used as a model food system. Aqueous extract at a concentration of 100 mg/ml was bacteriostatic against all the
foodborne pathogens in the milk stored at 258C for up to 4 days.

The microbiological safety of food continues to be a
major concern to consumers, regulatory agencies, and food
industries throughout the world. Microorganisms such as
Escherichia coli O157:H7, Listeria monocytogenes, Sal-
monella Typhimurium, and Staphylococcusaureus continue
to be involved in foodborne disease and economic losses
due to regulatory recalls. The inactivation or inhibition of
these microorganisms using physical and chemical food
preservation methods is important to the maintenance of
safe food. In some cases, physical preservation methods
such as heat, cold, or irradiation are not always desirable.
Chemical antimicrobials or preservatives are one alternative
to physical methods. Chemical food antimicrobials are of-
ten divided into synthetic (traditional, regulatory approved)
and naturally occurring groups. Traditional antimicrobials
include the organic acids (acetic, benzoic, lactic, propionic,
and sorbic acids) and nitrite and sulŽ tes (2, 3, 9). These
compounds have been used for many years to control
growth of microorganisms in foods. In recent years, there
has been a growing interest in natural antimicrobials. There
are two primary driving forces behind this increased inter-
est. The Ž rst force is a desire in the food industry to expand
the selection and spectrum of available regulatory-approved
food antimicrobials. Currently, there is a very limited
choice of antimicrobials approved by international regula-
tory agencies for use in foods and those available are often
* Author for correspondence. Tel: 82-62-530-2144; Fax: 82-62-530-2149;
E-mail: chunghj@chonnam.chonanm.ac.kr.
useful only in foods with low pH. The second force is to
meet the demand of consumers for more ‘‘natural’’ foods
and food components, which results in a more ‘‘green’’
food label. For these reasons, alternative sources of safe,
effective, and acceptable natural preservatives need to be
discovered.
Many plant food products contain naturally occurring
compounds that have antimicrobial activity (2, 4, 9). In the
natural state, these compounds may play a role in extending
the shelf life of a food product. In addition, a number of
these naturally occurring compounds have been studied for
their potential as direct food antimicrobials. For example,
it is known that enzymes, proteins, organic acids, fatty ac-
ids, essential oils, spices, pigments, hop extracts, oleuro-
pein, caffeine, theophylline, theobromine, and phytoalexins
possess preservative effects (4, 9).
The purpose of this study was to investigate the pos-
sible presence of naturally occurring antimicrobial com-
pounds in the petals of Camellia japonica L. (common
name, camellia), a  owering tree common in Korea and
other parts of the world and a member of the tea family.
The objectives were as follows: (i) to assay the antimicro-
bial activity of extracts of the  ower against several food-
borne pathogenic bacteria, (ii) to attempt to purify and iden-
tify potential antibacterial substances contained in the  ow-
er petals, and (iii) to apply a crude extract of the petals to
microbiological media and a model milk system to deter-
mine potential antimicrobial activity against several food-
borne pathogens.
1256 KIM ET AL.
FIGURE 1. Extraction and fractionation procedures of petals of
C. japonica L.
MATERIALS AND METHODS
Sample preparation. Fresh petals of C. japonica L. were
harvested from Yeosoo, Korea, in March 1997. The harvested
samples were kept on ice, transported to the laboratory, and frozen
at 2408C until used.
Extraction and solvent fractionation. The extraction meth-
od was based on the procedures described by MacMillan (6). Ten
kilograms of sample was homogenized for 15 s on full speed using
a homogenizer (NISSEI BM-2, Nihonseiki Kaisha Ltd., Tokyo,
Japan) and then extracted for 24 h with 30 liters of methanol
(MeOH) at room temperature. The extract was Ž ltered (Whatman
no. 2 Ž lter paper) under vacuum and the Ž ltrate concentrated in a
rotary vacuum evaporator at 458C. The MeOH extract was sys-
tematically fractionated with ethyl acetate (EtOAc) and various
buffer solutions (Fig. 1). Antibacterial activity of each fraction
was assayed at each puriŽ cation step as described below.
Strains and culture media. Strains of Salmonella Typhi-
murium DT104, E. coli O157:H7, L. monocytogenes, and S. au-
reus were used in all tests. All strains were obtained from the
Department of Food Science and Toxicology culture collection,
University of Idaho, donated by Dr. Dale Hancock or Dr. Tom
Besser, College of Veterinary Medicine, Washington State Uni-
versity, or purchased from the American Type Culture Collection
(ATCC; Rockville, Md.). Strains of Salmonella Typhimurium and
E. coli O157:H7 were grown in Trypticase soy broth (Difco Lab-
oratories, Detroit, Mich.), whereas strains of L. monocytogenes or
S. aureus were grown in tryptose phosphate broth (Difco) or brain
heart infusion broth (Difco), respectively. Agar media were pre-
J. Food Prot., Vol. 64, No. 8
pared by adding 1.8% agar (Difco) to broth media and were used
for assay of antibacterial activity. Cultures were maintained on
slants at 48C and transferred monthly to maintain viability. A
working culture was prepared by inoculating a loopful of culture
into 5 ml of appropriate microbiological medium and incubating
at 378C for 18 h.
Antibacterial assay. The disk diffusion method (1) was used
to screen the antibacterial activity of extracts of petals of C. ja-
ponica L. Bacterial cells were grown to the beginning of station-
ary phase and were serially diluted in 0.1% (wt/vol) peptone to
obtain approximately 4 log CFU/ml for use as an inoculum. One
milliliter of diluted culture and 20 ml of molten agar media main-
tained at 508C or less were mixed in a petri dish. Known concen-
trations of test fractions were then added to sterile Ž lter paper
disks (6-mm diameter, Difco), which were then air dried and
placed on the surface of the previously inoculated plate. The plate
was incubated under optimum conditions for the test bacteria at
378C for 24 h. Butylated hydroxyanisole (Sigma Chemical Co.,
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St. Louis, Mo.) in 95% ethanol was used as a positive control
inhibitor in this and subsequent experiments, since it was thought
to possibly resemble naturally occurring phenolic antimicrobials
that might be isolated from the plant (3). The appropriate solvent
without plant extract was used as a negative control.
PuriŽ cation and identiŽ cation of active fraction. Chro-
matographic puriŽ cation of the active fraction was performed ac-
cording to the method of Ma et al. (5). A chromatographiccolumn
(4.1 by 50 cm diameter) was packed with 250 g of activated silica
gel 60 (70 to 230 mesh, Merck, Darmstadt, Germany). The extract
was dissolved in MeOH, loaded on the column, and then eluted
with an increasing concentration of MeOH in CHCl 3 by a stepwise
elution method at a  ow rate of 2.0 ml/min. Each eluting fraction
was assayed for the antibacterial activity. A second column (2.0
by 50 cm) was packed with 50 g of Silica gel 60 (70 to 230 mesh,
Merck) and the procedure repeated. The extract was dissolved in
MeOH-CHCl 3 (4:1, vol/vol) and components separated on Se-
phadex LH-20 (25 to 100 mesh, Pharmacia, Stockholm, Sweden)
using MeOH-CHCl3 (4:1, vol/vol) as the eluting solvent. As be-
fore, each fraction was assayed for the antimicrobial activity. Fur-
ther puriŽ cation of the active fraction was performed by high-
pressure liquid chromatography (HPLC; Jasco Pu-980, Jasco Co.,
Tokyo, Japan) with a Bonda-PAK C18 column (3.9 by 300 mm).
The active fraction was dissolved in 5 ml of MeOH and Ž ltered
through syringe Ž lter (Gelman Acrodisc CR PTFE, 0.45 m). An
aliquot (20 l) of this solution was injected into a HPLC and
eluted with the solvent MeOH-H2O (70:30, vol/vol) as a mobile
phase. Each fraction was evaporated to dryness with a rotary
evaporator at 408C. Finally, the sample was analyzed by electron
impact (70-eV) ionization mass spectrometry (Model Thermo-
beam TR, Waters, Milford, Mass.) equipped with a thermospray
interface.
Application of aqueous extract to a microbiological media
and a model food system. One kilogram of petals of C. japonica
L. was homogenized (NISSEI) in deionized water for 15 s and
then extracted with 5 liters of water. The extract was Ž ltered
(Whatman no. 2 Ž lter paper) under vacuum and centrifuged at
2,500 3 g for 10 min. Supernatant was frozen at 2508C for 3 h
and dried at 0.13 torr vacuum for 2 days in a freeze drier (Il-shin
Engineering, Seoul, Korea). A freeze-dried sample was stored in
tightly capped plastic vials at 248C until used. Growth inhibition
by the aqueous extract over time against the bacterial foodborne
pathogens in culture media was performed with selected strains
of Salmonella Typhimurium, E. coli O157:H7, L. monocytogenes,
J. Food Prot., Vol. 64, No. 8
TABLE 1. Antimicrobial activity of extract fractions from petals of C. japonica L. against selected foodborne pathogenic bacteria
Microorganism Methanol
Salmonella Typhimurium DT104 WSU 2380 19
Salmonella Typhimurium DT104 WSU 2576 18
Salmonella Typhimurium DT104 WSU 2582 18
Escherichia coli O157:H7 WSDH WSU 54 17
E. coli O157:H7 WSDH WSU 55 18
E. coli O157:H7 WSDH WSU 251 17
Listeria monocytogenes ATCC 19114 15
L. monocytogenes ATCC 19115 15
L. monocytogenes ATCC 19116 16
Staphylococcus aureus ATCC 12600 14
S. aureus ATCC 13566 15
S. aureus ATCC 25923 14
a Butylated hydroxyanisole (added as a positive control).
and S. aureus. The microorganisms were inoculated at an initial
level of approximately 3 log CFU/ml and incubated at 378C for
24 h. Viable cells were enumerated on appropriate nonselective
media using the pour plate method every 3 h. The assay for
growth inhibition by the aqueous extract in a food system was
described by Ulate-Rodriguez et al. (10). Milk was selected as the
model food system for evaluation of the antibacterial effect be-
cause it is easy to handle and contains potential antimicrobial
interference compounds, including protein, fat, and minerals. Five
grams of freeze-dried C. japonica L. powder was dissolved in 10
ml of distilled water and the mixture sterilized using a 0.45- m
membrane Ž lter. The sterilized extract was added to sterilized
strawberry milk (Namyang Co., Seoul, Korea; fat content, 3.4%;
pH 6.5) to obtain a Ž nal concentration of approximately 100 mg/
ml. Selected strains of Salmonella Typhimurium, E. coli O157:
H7, L. monocytogenes, and S. aureus were inoculated into the
milk system at an initial level of approximately 3 to 4 log CFU/
ml and stored at 7 or 258C for 7 days. Viable cells were enumer-
ated on appropriate nonselective media using the pour plate meth-
od every 24 h.
RESULTS AND DISCUSSION
Antibacterial activity of petal fractions. All test bac-
teria were inhibited (14- to 19-mm-diameter clear zones)
by the MeOH extract of the C. japonica L. petals at a con-
centration of 1.0 g eq (equivalent to 1 g of petals) per disk
(Table 1). Salmonella Typhimurium and E. coli O157:H7
demonstrated slightly greater inhibition than the gram-pos-
itive L. monocytogenes and S. aureus. The MeOH extract
was then separated into basic, acid, and neutral fractions
and assayed for antimicrobial activity (Table 1). The basic
fraction did not inhibit the growth of any of the bacteria.
The acid fraction inhibited the growth of all of the bacteria
tested at a concentration of 1.0 g eq/disk and was the most
effective antibacterial fraction of the three based on zone
size. Since the pH of the media is approximately 7.3, there
would be little effect of low pH on antimicrobial activity
away from the disk. These results agree with Ma et al. (5),
who reported the acidic fraction of Aralia elata had strong
antimicrobial activity against various foodborne pathogens.
PuriŽ cation and identiŽ cation of acidic fraction. At-
tempts were made to characterize the identity of the anti-
ANTIBACTERIAL ACTIVITY OF C. JAPONICA L. PETALS 1257
Inhibitory zone (mm)
Basic Acid Neutral BHAa
0 19 10 11
0 16 8 12
0 17 8 11
0 17 8 11
0 17 10 11
0 16 8 11
0 14 8 11
0 14 8 12
0 15 8 12
0 14 8 11
0 14 8 12
0 13 8 11
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microbial(s) in the acidic fraction using the silica gel ad-
sorption column chromatography, Sephadex LH-20 column
chromatography, and preparative HPLC. Salmonella Ty-
phimurium was used to assay the antimicrobial activity at
each step. The acidic fraction (10.90 g) was subjected to
silica gel adsorption column chromatography. Eluting step-
wise with an increasing concentration of MeOH in CHCl3
showed the 30 to 50% fraction had potential antibacterial
activity (Fig. 2A). The active fraction (4.64 g) was further
puriŽ ed by silica gel column chromatography using the
same solvent system, and the active fraction was found in
15 to 30% CHCl3 /MeOH eluate (Fig. 2B). The active frac-
tion (3.48 g) was puriŽ ed by Sephadex LH-20 column chro-
matography, using a MeOH-CHCl3 (4:1, vol/vol) solvent
system as the mobile phase. The eluate with a elution vol-
ume/total volume of 1.26 to 1.32 showed antibacterial ac-
tivity against Salmonella Typhimurium (Fig. 2C). This re-
sult indicates that the active compound(s) could be expected
to have a molecular weight of less than 300. The active
fraction (57.8 mg) was further puriŽ ed by HPLC in a re-
verse-phase octadecylsilane column with a MeOH-H2 O
(70:30, vol/vol) as the eluent termed compound I (retention
time 5 2.8, 1.0 mg) was obtained from the acidic fraction
as the major natural antibacterial substance. The elution
pattern indicated the compound had very high polarity. The
mass spectrum of the antibacterial compound isolated from
the petals of C. japonica L. was molecular ion (M1 ) of m/
z 116. The base fragment ion was at m/z 98, leaving H2O
of molecular ion. The mass spectrum gave (M-OH-H 2O)1
at m/z 81 and (M-CO-OH-H 2 O)1 at 53. These masses of
compound I showed good accordance with the spectrum of
fumaric acid (HOOC-CH5CH-COOH) as a reference sub-
stance.
Antibacterial activities of compound I and fumaric acid
standard (pH not determined) were compared by the paper
disk method (Table 2). At 0.2 mg/disk, compound I and
fumaric acid showed similar effectiveness against Salmo-
nella Typhimurium and E. coli O157:H7. At the concen-
trations evaluated, L. monocytogenes and S. aureus were
not inhibited in the disk assay. Inhibitory zones associated
1258 KIM ET AL.
FIGURE 2. Distribution of antibacterial activities in (A) silica gel
adsorption, (B) additional silica gel adsorption, and (C) Sephadex
LH-20 column chromatography of the acidic fraction from petals
of C. japonica L.
with fumaric acid increased for all microorganisms, with
increasing concentration to 1.0 mg/disk for all bacteria test-
ed. Since the pHs of the various culture media are approx-
imately 7.3, it would be expected that there would be little
effect of low pH on activity of the antimicrobials away
from the disk. The high pH would also indicate that fumaric
J. Food Prot., Vol. 64, No. 8
acid (pKa1 5 3.03, pKa2 5 4.54) was mostly dissociated.
For the extract, this suggests that sufŽ cient undissociated
acid remained to cause inhibition, that fumaric acid acted
as an antimicrobial in the dissociated form, or that another
compound, perhaps in concert with fumaric acid, was an
inhibitor. Fumaric acid has been shown to have antimicro-
bial activity in high-pH meat systems. Podolak et al. (8)
demonstrated that fumaric acid (5%) in ground beef signif-
icantly inhibited growth of coliforms and psychrotrophic
bacteria during storage for up to 14 days at 48C and that
the compound was more effective than lactic acid. In ad-
dition, Podolak et al. (7) showed that 1.0 or 1.5% fumaric
acid reduced viable L. monocytogenes, Salmonella Typhi-
murium, and E. coli O157:H7 by an average 1.02, 1.31,
and 1.85 log CFU/g, respectively, on the surface of lean
beef dipped in the acid for 15 s at 558C. Fumaric acid was
more effective as a sanitizer dip than acetic or lactic acid
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against the three pathogens on lean beef. During a 14-day
storage period, both the 1.0 and 1.5% fumaric acid treat-
ments caused a continued reduction of L. monocytogenes
by approximately 1.5 logs. In contrast, the gram-negative
bacteria increased slightly by 0.6 to 0.8 log (7). In the pres-
ent study, the gram-negative Salmonella Typhimurium and
E. coli O157:H7 also appear to be slightly more sensitive
to fumaric acid. Differences between the two studies could
be due to strains used and the presence of the meat com-
ponents.
Activity of the aqueous extract of petals. Although
the petals were methanol extracted and fractionated to iden-
tify potential active components, the most useful commer-
cial natural antimicrobial would likely be a simple crude
water extract of a natural antimicrobial source (9). There-
fore, the camellia petals were extracted using only water to
determine if any of the antimicrobial activity remained and
if it was useful in a food product. The inhibitory effective-
ness of the aqueous extract on the selected pathogens in
microbiological media was determined over time at 378C
with concentrations found in previous studies to demon-
strate some inhibition of each culture (data not shown). The
microorganism and concentration used were as follows:
Salmonella Typhimurium DT104, 100 mg/ml; E. coli
O157:H7, 100 mg/ml; L. monocytogenes, 125 mg/ml; and
S. aureus, 225 mg/ml. In the control media, the growth of
bacteria showed conventional growth patterns with lag
phases of approximately 3 h (Fig. 3A through 3D). In con-
trast, the lag phases of pathogens in the presence of the C.
japonica L. aqueous extracts were approximately 9 h for
the gram-negative bacteria and 6 h for the gram-positive
bacteria. It is presumed that part, or all, of the lag phase
inhibition is due to the antimicrobial factors, potentially in-
cluding fumaric acid, present in the aqueous extract.
The inhibitory effect in model milk system of 100 mg/
ml of aqueous extract against Salmonella Typhimurium, E.
coli O157:H7, L. monocytogenes, and S. aureus was mon-
itored at 7 and 258C for 7 days (Fig. 4A through 4D). There
was no signiŽ cant difference in inhibitory effect between
control and aqueous extract at 78C due to the lack of growth
of the microorganisms at this temperature. In addition, the
J. Food Prot., Vol. 64, No. 8 ANTIBACTERIAL ACTIVITY OF C. JAPONICA L. PETALS 1259

TABLE 2. Comparison of the antibacterial effect of compound I isolated from petals of C. japonica L. to fumaric acid against selected
foodborne pathogenic bacteria
Inhibitory zone (mm)

Compound I Fumaric acid BHAa

Microorganism 0.2 mg/disk 0.2 mg/disk 1.0 mg/disk 0.2 mg/disk

Salmonella Typhimurium DT104 WSU 2380 9 8 12 11

Escherichia coli O157:H7 WSDH WSU 54 8 8 12 10
Listeria monocytogenes ATCC 19116 0 0 9 9
Staphylococcus aureus ATCC 12600 0 0 9 10
a Butylated hydroxyanisole (added as a positive control).

results indicated that the extract has no bactericidal effect that the extracts may be useful against both gram-negative
(i.e., no reduction in numbers) at low temperatures. In con- and gram-positive bacterial pathogens. This is a great ben-
trast, growth inhibition was observed in the model milk eŽ t since most approved food antimicrobials are more ef-
system at 258C. The lag phases of Salmonella Typhimurium fective against gram-positive bacteria (3). The active anti-

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and E. coli O157:H7 were increased by 2 to 3 days and
those of L. monocytogenes and S. aureus by 1 to 2 days.
In addition, the growth rate was slower and the Ž nal growth
level reached after 7 days was reduced for all pathogens in
the presence of the extracts.
In conclusion, high concentrations of aqueous extracts
of C. japonica L. petals appear to have potential to inhibit
foodborne pathogens not only in microbiological media but
also in the presence of food components. It also appears
microbial component(s) likely include fumaric acid but are
probably composed of other compounds yet to be identiŽ ed.
Obviously, this is only the Ž rst step in developing a natural
antimicrobial since information on the toxicology and sen-
sory effects of the extracts is lacking.
ACKNOWLEDGMENT
The authors wish to acknowledge the Ž nancial support of the Korea
Research Foundation made in the program year of 1997.

FIGURE 3. Growth inhibition by water extract from petals of C. japonica L. in broth medium at 378C against (A) Salmonella Typhi-
murium DT104 WSU 2380, (B) E. coli O157:H7 WSDH 54, (C) L. monocytogenes ATCC 19116, and (D) S. aureus ATCC 12600.
1260 KIM ET AL.
FIGURE 4. Growth inhibition by water extract from petals of C. japonica L. in milk at 7 and 258C against (A) Salmonella Typhimurium
DT104 WSU 2380, (B) E. coli O157:H7 WSDH 54, (C) L. monocytogenes ATCC 19116, and (D) S. aureus ATCC 12600.
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