ARTICLE IN PRESS

Food Microbiology 25 (2008) 762 770

Contents lists available at ScienceDirect

Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Inhibition of Bacillus cereus and Bacillus weihenstephanensis in raw vegetables

by application of washing solutions containing enterocin AS-48 alone and in
combination with other antimicrobials
Antonio Cobo Molinos a, Hikmate Abriouel a, Rosario Lucas Lopez a, Nabil Ben Omar a, Eva Valdivia b,c,
Antonio Galvez a,
rea de Microbiologa, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jaen, 23071 Jaen, Spain
A
Departamento de Microbiologa, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain
c
Instituto de Biotecnologa, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain
a

a r t i c l e in f o

a b s t r a c t

Article history:
Received 11 February 2008
Received in revised form
29 April 2008
Accepted 4 May 2008
Available online 13 May 2008

Enterocin AS-48 is a broad-spectrum cyclic antimicrobial peptide produced by Enterococcus faecalis. In

the present study, the bacteriocin was tested alone and in combination with other antimicrobials for
decontamination of Bacillus inoculated on alfalfa, soybean sprouts and green asparagus. Washing with
enterocin AS-48 solutions reduced viable cell counts of Bacillus cereus and Bacillus weihenstephanensis by
1.01.5 and by 1.52.38 log units right after application of treatment, respectively. In both cases, the
bacteriocin was effective in reducing the remaining viable population below detection levels during
further storage of the samples at 6 1C, but failed to prevent regrowth in samples stored at 15 or 22 1C.
Application of washing treatments containing enterocin AS-48 in combination with several other
antimicrobials and sanitizers (cinnamic and hydrocinnamic acids, carvacrol, polyphosphoric acid,
peracetic acid, hexadecylpyridinium chloride and sodium hypochlorite) greatly enhanced the
bactericidal effects. The combinations of AS-48 and sodium hypochlorite, peracetic acid or
hexadecylpyridinium chloride provided the best results. After application of the combined treatments
on alfalfa sprouts contaminated with B. cereus or with B. weihenstephanensis, viable bacilli were not
detected or remained at very low concentrations in the treated samples during a 1-week storage period
at 15 1C. Inhibition of B. cereus by in situ produced bacteriocin was tested by cocultivation with the AS-48
producer strain E. faecalis A-48-32 inoculated on soybean sprouts. Strain A-48-32 was able to grow and
produce bacteriocin on sprouts both at 15 and 22 1C. At 15 1C, growth of B. cereus was completely
inhibited in the cocultures, while a much more limited effect was observed at 22 1C. The results
obtained for washing treatments are very encouraging for the application of enterocin AS-48 in the
decontamination of sprouts. Application of washing treatments containing AS-48 alone can serve to
reduce viable cell counts of bacilli in samples stored under refrigeration, while application of combined
treatments should be recommended to avoid proliferation of the surviving bacilli under temperatureabuse conditions.
& 2008 Elsevier Ltd. All rights reserved.

Keywords:
Bacillus cereus
Bacillus weihenstephanensis
Bacteriocins
Biopreservation
Vegetable foods
Sprouts

1. Introduction
Endospore-forming bacteria of the Bacillus cereus group are
often considered hazardous to food safety because of their capacity
to produce enterotoxins, together with their wide distribution in
nature and frequent contamination of raw materials used in food
production. B. cereus is a common soil inhabitant that is often
present in a variety of foods, such as rice, spices, milk and dairy

 Corresponding author. Tel.: +34 953 212160; fax: +34 953 212943.

E-mail address: agalvez@ujaen.es (A. Galvez).

0740-0020/$ - see front matter & 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2008.05.001

products, vegetables, meat, cakes and other desserts (Granum,

2001). B. cereus produces several toxins (Schoeni and Wong, 2005),
including emetic toxin (Agata et al., 1995) and at least four other
enterotoxins: hemolysin BL or Hbl (Beecher and Wong, 1997), nonhemolytic enterotoxin or Nhe (Lindback et al., 2004) and cytotoxin
K or CytK (Lund et al., 2000). Bacillus weihenstephanensis is a
psychrotolerant bacterium belonging to the B. cereus group
(Lechner et al., 1998). Some strains may be highly cytotoxic, and
have been shown to carry at least some of the genetic determinants
of the enterotoxins as well as the emetic toxin described in B. cereus
(Pru et al., 1999; Stenfors et al., 2002; Thorsen et al., 2006; Baron
et al., 2007). B. cereus has been isolated from sprouts and sprouting

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A. Cobo Molinos et al. / Food Microbiology 25 (2008) 762770

et al., 2004, 2007; Ananou et al., 2005a, b; Grande et al., 2005,

2006, 2007a, b; Lucas et al., 2006). Enterocin AS-48 may be an
interesting bacteriocin for decontamination of raw vegetables. In
lettuce juice, added enterocin AS-48 caused a strong inhibition of
Staphylococcus aureus and completely inactivated L. monocytogenes and B. cereus (Grande et al., 2005). Enterocin AS-48 was
tested previously in washing treatments for decontamination of
seed sprouts against L. monocytogenes (Cobo Molinos et al., 2005).
The antilisterial activity of AS-48 was enhanced greatly by several
antimicrobial compounds, reducing the population of viable
listeria below detection limits in the treated sprouts after
treatment and preventing regrowth of the listeria during storage
of the treated samples (Cobo Molinos et al., 2005). Results from
our previous studies indicate that the effectiveness of AS-48
treatments greatly depends on the target bacteria and the food
system. While added bacteriocin completely inactivated B. cereus
in rice-based foods (Grande et al., 2006), higher bacteriocin
concentrations were required to control mixed populations of
bacilli in purees (Grande et al., 2007b). In washing treatments, the
bacteriocin concentration in samples will decrease after treatment, limiting the protection of samples during storage. The
purpose of the present study was to determine the effectiveness of
AS-48 applied in washing treatments against two toxicogenic
species of the B. cereus group alone and in combination with other
antimicrobials in search for synergistic effects against the bacilli.

seeds (Kim et al., 2004; Pao et al., 2005). In soybean sprouts,

endospore-forming bacteria were found in the order of 2 logunits/g,
and 53 out of 55 B. cereus isolates were found to produce diarrheic
enterotoxins (Kim et al., 2004). B. cereus has also been isolated from
raw vegetables used to prepare refrigerated minimally processed
foods (Valero et al., 2002). The use of sanitizers to reduce the
B. cereus levels in fruits and vegetables has been proposed as a
safety measure (Beuchat et al., 2004).
Bacteriocins of lactic acid bacteria (LAB) have seldom been
used for decontamination of fresh produce (reviewed by Galvez
et al., 2008). Nisin and pediocin individually or in combination
with antimicrobial agents (sodium lactate, potassium sorbate,
phytic acid and citric acid) were tested as possible sanitizer
treatments for reducing the population of Listeria monocytogenes
on cabbage, broccoli and mung bean sprouts (Bari et al., 2005).
Washing solutions consisting of LAB bacteriocins produced on a
lettuce extract (nisin Z, coagulin and a nisin:coagulin cocktail)
reduced viable cell counts of L. monocytogenes on fresh-cut
iceberg lettuce stored in microperforated plastic bags, but did
not prevent further growth of survivors during refrigeration
storage (Allende et al., 2007). Enterocin AS-48 is a cyclic peptide
with a broad antibacterial spectrum (Galvez et al., 1986, 1989;
Maqueda et al., 2004). This bacteriocin has been puried on a
semi-preparative scale (Abriouel et al., 2003), and is now being
oz
tested as a natural preservative in different food systems (Mun

8
15C

22C

0
7

3
5
Time (days)

3
5
Time (days)
22C

Log CFU/ml

Log CFU/ml
7

0
Time (days)

15C

6
6C

0
0

6
Log CFU/ml

Log CFU/ml

15C

3
5
Time (days)

Time (days)

6
6C

Time (days)

0
0

Time (days)

2
0

Log CFU/ml

22C
Log CFU/ml

Log CFU/ml

Log CFU/ml

6C

Log CFU/ml

763

6
4
2
0

Time (days)

Time (days)

Fig. 1. Effect of immersion treatments with solutions containing enterocin AS-48 on survival and proliferation of B. cereus LWL1 inoculated on soybean sprouts (AC), alfalfa
sprouts (DF) and green asparagus (GI). After immersion for 5 min at working temperatures in solutions containing nal bacteriocin concentrations of 0 (O) and 25 mg/ml
(m), samples were incubated at 6 1C (A, D, G), 15 1C (B, E, H) or 22 1C (C, F, I). Data represent the average of two independent experiments plus standard deviation (error bars).

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A. Cobo Molinos et al. / Food Microbiology 25 (2008) 762770

2. Materials and methods

2.1. Bacterial strains and cultivation conditions
Enterococcus faecalis A-48-32 (Martinez Bueno et al., 1990) was
used for the production of enterocin AS-48, and E. faecalis S-47
from our collection was used for standard determination of
bacteriocin activity. B. cereus LWL1 is a psychrotrophic and
enterotoxigenic strain (Dufrenne et al., 1995), kindly supplied by
Dr. F.M. van Leusden (Microbiological Laboratory for Health
Protection, Natl. Inst. Publ. Health and Environ., The Netherlands).
B. weihenstephanensis CECT 5894 was obtained from the Spanish
type culture collection (CECT). All strains were cultivated
routinely on brainheart infusion (BHI, Scharlab, Barcelona, Spain)
broth at 37 1C, and stored at 4 1C on BHI-agar slants.

dipped for 5 min. at room temperature in 5 ml of sterile distilled

water (controls) or distilled water containing enterocin AS-48 (at
nal concentrations of 5, 12.5 or 25 mg/ml). Immersion solutions
were held at room temperature for at least 1 h before use.

6C

Log CFU/ml

764

2.2. Preparation of bacteriocin and chemical preservatives

0
0

3
Time (days)

15C

6
Log CFU/ml

Enterocin AS-48 was recovered from cultured broths of the

producer strain E. faecalis A-4832 in CMG-medium by cation
exchange chromatography as described elsewhere (Abriouel et al.,
2003). Bacteriocin concentrates were ltered through 0.22 mm
pore size low protein-binding lters (Millex GV; Millipore
Corporation, Belford, MA, USA) under sterile conditions and tested
for bacteriocin activity against the indicator strain E. faecalis S-47
by the agar well diffusion method (Abriouel et al., 2003).
Immersion solutions were prepared by diluting bacteriocin
concentrates (500 mg/ml) in sterile distilled water or in aqueous
solutions of the chemical preservatives to be tested in the case of
combined treatments.
Lactic acid, sodium lactate, tri-sodium trimetaphosphate,
polyphosphoric acid, hexadecylpyridinium chloride, n-propyl
p-hydroxybenzoate, and p-hydoxybenzoic acid methyl esther
were purchased from Sigma (Madrid, Spain). Cinnamic acid,
hydrocinnamic acid, carvacrol and peracetic acid were from Fluka
(Madrid). Sodium hypochlorite was a commercial concentrated
bleach (ConejoTM, Henkel Iberica, Barcelona, Spain). The commercial solutions or concentrated stock solutions prepared by
dissolving the solid compounds in sterile distilled water or in
ethanol were diluted at least 20-fold in sterile distilled water in
order to prepare the immersion solutions to be used for the
washing treatments. All solutions were prepared fresh before use.

0
0

Time (days)

2.3. Assay of enterocin AS-48 in vegetables articially contaminated

with bacilli

22C

6
Log CFU/ml

The effect of immersion in solutions containing enterocin AS48 (25 mg/ml, nal concentration) on survival and growth of
B. cereus LWL1 inoculated onto fresh alfalfa, soybean sprouts and
green asparagus as well as B. weihenstephanensis CECT 5894 on
soybean sprouts was investigated at storage temperatures of 6, 15
and 22 1C. Cultures of bacilli grown overnight in BHI broth at 37 1C
were diluted 1:100 in sterile saline solution to a nal cell density
of approx. 5.5 log CFU/ml. This Bacillus cell suspension was used to
articially contaminate the vegetables being tested. Fresh green
asparagus (Mary Washington variety, 510 mm diameter), soybean sprouts (Alleuras Industries, Madrid) and alfalfa sprouts
(Productos Fanya, Madrid) were purchased from local supermarkets. Asparagus were cut onto 2 cm pieces before treatment
application. Samples of the vegetable being tested (2.5 g each)
were deposited inside sterile capped 50 ml polypropylene tubes
(Sterilin, Stone, UK) and dipped for 5 min in 5 ml sterile distilled
water (negative controls) or in 5 ml of Bacillus cell suspension at 6,
15 and 22 1C. Then, they were deposited on a sterile lter paper to
drain excess water. The articially contaminated samples were

0
0

Time (days)
Fig. 2. Effect of immersion treatments with solutions containing enterocin AS-48
on survival and proliferation of B. weihenstephanensis CECT 5894 inoculated on
soybean sprouts. After immersion for 5 min at working temperatures in solutions
containing nal bacteriocin concentrations of 0 (O) and 25 mg/ml (m), samples
were incubated at 6 1C (A), 15 1C (B) or 22 1C (C). Data represent the average of two
independent experiments plus standard deviation (error bars).

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765

Combined treatments of enterocin AS-48 and chemical preservatives were carried out on Bacillus articially contaminated
food samples at room temperature essentially as described above,
by using immersion solutions containing enterocin AS-48 (25 mg/ml
nal concentration) and/or the corresponding chemical compounds
(at the nal concentrations indicated in previous heading). Viable
cell counts of bacilli were determined as described above following
immersion treatment (time zero) and after 24 h incubation at 22 1C.
Conrmation of B. cereus was done by PCR amplication of the sspE
gene with specic primers sspE1-F (50 -GAGAAAGATGAGTAAAAAA
CAACAA-30 ) y sspE1-R (50 -CATTTGTGCTTTGAATGCTAG) as described by Kim et al. (2004), followed by detection of the 71-bp
amplicon by agarose gel electrophoresis.

Following immersion treatments, excess immersion solution was

removed as above, and samples were stored in sterile capped
50 ml polypropylene test tubes placed in refrigerated storage or
incubation chambers (Memmert, Schwabach, Germany) at desired
incubation temperatures (6, 15, 22 1C) for different periods of time.
At each step, samples (2.5 g) were mixed with 5 ml of sterile saline
solution (0.85% NaCl) and pummelled for 3 min in a Stomacher 80
(Biomaster) before they were serially diluted in sterile saline
solution and spread in triplicate on plates of B. cereus agar
(Scharlab, Barcelona) supplemented with egg yolk and polymixin
B (Panreac, Barcelona). Plates were incubated at 37 1C for 48 h, and
the number of colonies showing features typical of B. cereus were
determined in order to calculate viable cell counts.

Control
AS-48
Lactic acid (0.5%)

Lactic acid + AS-48

Sodium lactate (0.5%)

Sodiumlactate + AS-48

Sodium hypochlorite (100 ppm)

Sodium hypochlorite + AS-48

+ *

+ *

Trisodium trimetaphosphate (0.5%)

Trisodium trimetaphosphate + AS-48

Hexadecylpyridinium + AS-48
Peracetic acid (40 ppm)
Treatment

+ *

+
+

Hexadecylpyridinium (0.5%)

+
+ *
+

Peracetic acid + AS-48

+ *
+ *
+

Polyphosphoric acid (0.1%)

Polyphosphoric acid + AS-48

+ *

+ *

Polyphosphoric acid(0.5%)

+ *
+ *

Polyphosphoric acid + AS-48

Carvacrol (0.3%)

+
+ *
+ *

Carvacrol + AS-48
Cinnamic acid (0.3%)

+
+ *
+ *
+
+

Cinnamic acid + AS-48

Hydrocinnamic acid (0.5%)
+ *
+ *

Hydrocinnamic acid + AS-48

n-Propyl-p-hydroxybenzoate (0.5%)

+
+

n-Propyl-p-hydroxybenzoate + AS-48

p-Hydroxybenzoic acid methyl esther (0.5%)

+
+

p-Hydroxibenzoic acid methyl esther + AS-48

0

+
3

Log CFU/g
Fig. 3. Effect of enterocin AS-48 (25 mg/ml) in combination with chemical antimicrobial compounds on the viability of B. cereus LWL1 in alfalfa sprouts. Viable cell counts
were determined following application of treatment (open bars) and also after 24 h (closed bars) of storage at 15 1C. Standard deviation is shown by error bars. +,
statistically signicant reduction (Po0.05) compared with the untreated control. *, statistically signicant reduction (Po0.05) of the combined treatment compared with
treatment with the chemical agent alone. Data represent the average of two independent experiments plus standard deviation (error bars).

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A. Cobo Molinos et al. / Food Microbiology 25 (2008) 762770

Cells from an overnight culture (grown at 37 1C in BHI broth) of

the enterocin AS-48 producer strain E. faecalis A-48-32 were
recovered by centrifugation (5000g for 10 min at room temperature) and resuspended in sterile saline solution to the initial
culture volume. This suspension was added to the bacilli
contamination suspensions to obtain a co-culture suspension
with an approximate enterococci-to-bacilli ratio of 100:1. Vegetable samples (2.5 g each) were immersed in the co-culture
suspension for 5 min at room temperature as above and excess
suspension was removed as above. At desired intervals of
incubation at the test temperature (15 or 22 1C), samples were
plated for viable cell counts of bacilli and enterococci. For
the bacilli, B. cereus agar was supplemented with ampicillin
(80 mg/ml, nal concentration) in order to inhibit growth of
enterococci. Bacilli were conrmed by PCR amplication as
described above. Viable cell counts of enterococci were also
carried out, by spreading samples in triplicate on kanamicin
esculin azide agar (KAA, Scharlab), followed by 48 h incubation at
37 1C. Conrmation of the AS-48 producer strains from KAA plates
was done by testing colonies isolated at random for bacteriocin
production by the spot on a lawn method with E. faecalis S-47 as
the sensitive indicator strain and A-48-32 strain as the AS-48resistant indicator strain. Colonies that showed inhibition against
strain S-47 but failed to inhibit strain A-48-32 were considered to
be AS-48 producers.
Bacteriocin activity in samples was determined by testing
100 ml of the pummelled vegetable suspension by the agar-well
diffusion method.
2.5. Statistical analyses
The average data from duplicate trials7standard deviations
were determined with Excel programme (Microsoft Corp., USA). In
order to determine the statistical signicance of the data, a t-test
was performed at the 95% condence interval with Statgraphics
Plus version 5.1 (Statistical Graphics Corp., USA). The signicance
of combined treatments was determined by comparison of data
from the same incubation time.

counts by 1.52.38 log units at all temperatures tested. In the

bacteriocin-treated samples, no viable bacilli were detected after
day 1 of storage at 6 1C (Fig. 2A). However, the surviving

Log CFU/ml

2.4. Co-cultivation experiments

0
0

3
Time (days)

3
Time (days)

3
Time (days)

Log CFU/ml

766

3. Results

The effect of washing treatments with solutions containing

25 mg/ml AS-48 was studied on B. cereus LWL1 in soybean, alfalfa
sprouts and green asparagus (Fig. 1) stored at 6, 15 and 22 1C. Best
results were obtained for samples refrigerated at 6 1C. In all cases,
statistically signicant (Po0.05) reductions of viable cell counts of
1.06, 1.3 and 1.59 log units were obtained after washing treatments, and the remaining viable population was reduced below
detection levels after days 13 of storage (Fig. 1A, D and G). By
contrast, reductions of viable cell counts obtained after treatments at 15 or 22 1C were not statistically signicant for the three
types of food tested, and the remaining viable cells multiplied as
the untreated controls at least for the rst day of storage. During
prolonged storage, viable cell counts of treated samples were
signicantly lower than the untreated controls only for alfalfa
sprouts and asparagus at day 3 and for soybean sprouts after day 3
of storage.
Washing treatments with AS-48 (25 mg/ml) were also applied
to B. weihenstephanensis CECT 5894 inoculated on soybean sprouts
(Fig. 2). The bacteriocin treatment signicantly reduced viable cell

Log CFU/ml

3.1. Effect of washing treatments containing enterocin AS-48 alone

Fig. 4. Effect of enterocin AS-48 (25 mg/ml) in combination with the phenolic
compounds carvacrol (A), cinnamic acid (B) and hydrocinnamic acid (C) on the
viability of B. cereus LWL1 in alfalfa sprouts stored at 15 1C for 1 week. Controls (J),
samples treated with AS-48 (m), phenolic compound (), or AS-48+phenolic
compound (K). Data represent the average of two independent experiments plus
standard deviation (error bars).

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population multiplied during storage at higher temperatures,

especially at 15 1C and to a less extent also at 22 1C (Fig. 2B and C).
3.2. Effect of washing treatments containing enterocin AS-48 in
combination with other antimicrobial compounds
Enterocin AS-48 (25 mg/ml) was tested on B. cereus in
combination with several other antimicrobial compounds in
alfalfa sprouts stored at 15 1C. At this concentration, the
bacteriocin alone did not reduce signicantly the number of
viable B. cereus after treatment or during the following 24 h of
storage (Fig. 3).
Many of the antimicrobial compounds tested individually
reduced the numbers of viable cells signicantly (Po0.05)
compared with the untreated controls, either at time 0 or after
24 h storage or both (Fig. 3). Comparison of viable cell counts
obtained for each chemical preservative and the combination
bacteriocinchemical preservative indicated that the bactericidal
effect of treatments was enhanced signicantly by addition of AS48 (Po0.05) for cinnamic and hydrocinnamic acids, carvacrol,
polyphosphoric acid (at 0.1% and 0.5%), peracetic acid, hexadecylpyridinium chloride and sodium hypochlorite, both at 0 and
24 h of storage, and also for trisodium trimetaphosphate at 24 h.
Interestingly, some of the combined treatments (with sodium
hypochlorite, peracetic acid, polyphosphoric acid and hydrocinnamic acid) reduced the viable cell counts of B. cereus below
detection levels.

767

alfalfa sprout samples stored at 15 1C for 1 week. In samples

treated with carvacrol, cinnamic and hydrocinnamic acids in
combination with AS-48, viable cell counts of B. cereus LWL1
remained signicantly lower compared with each individual
treatment (Po0.05) during most part or the whole storage period
(Fig. 4). Best results were obtained for the combined treatment
with hydrocinnamic acid, for which no viable cells were detected
except at day 2 (Fig. 4C).
The chemical compounds sodium hypochlorite, hexadecylpyridinium chloride, peracetic acid and polyphosphoric acid reduced
the population of B. cereus below detection levels for the whole or
at least most of the storage period when tested in combination
with AS-48 (Fig. 5). However, none of the compounds was able to
completely eliminate B. cereus when tested without bacteriocin.
Antimicrobial compounds were also tested against B. weihenstephanensis CECT 5894 inoculated on alfalfa sprouts stored a
15 1C. Treatment with bacteriocin alone reduced viable cell counts
below detection levels from the beginning, but did not prevent
further proliferation of the surviving bacteria (Fig. 6). By contrast,
the combinations of 25 mg/ml AS-48 and hydrocinnamic acid,
peracetic acid, sodium hypochlorite as well as hexadecylpyridinium chloride reduced the population of B. weihenstephanensis
below detection levels for the whole or at least most of the storage
period (Fig. 6). In the case of polyphosphoric acid, a higher
concentration of 0.5% was required to achieve such a degree of
inactivation (Fig. 6E and F). Addition of the antimicrobial
compounds alone had much more limited effects in most cases.

3.3. Preservation of sprouts after treatment

3.4. Inhibition of B. cereus in sprouts by cocultivation with a

bacteriocin-producing strain

The efcacy of combinations of selected preservatives and

enterocin AS-48 for prolonged inhibition of bacilli were tested in

B. cereus LWL1 was inoculated on soybean sprouts in combination with the bacteriocin-producing strain E. faecalis A-48-32 as a

Log CFU/ml

Log CFU/ml

0
0

Time (days)

Log CFU/ml

Log CFU/ml

Time (days)

0
0

Time (days)

3
5
Time (days)

Fig. 5. Effect of enterocin AS-48 (25 mg/ml) in combination with the chemical compounds sodium hypochlorite (A), hexadecylpyridinium chloride (B), peracetic acid (C) and
polyphosphoric acid (D) on the viability of B. cereus LWL1 in alfalfa sprouts stored at 15 1C for 1 week. Controls (J), samples treated with AS-48 (m), chemical compound
(), or AS-48+chemical compound (K). Data represent the average of two independent experiments plus standard deviation (error bars).

ARTICLE IN PRESS
768

A. Cobo Molinos et al. / Food Microbiology 25 (2008) 762770

Log CFU/ml

Log CFU/ml

0
0

3
5
Time (days)

Log CFU/ml

0
0

Time (days)

Time (days)

Log CFU/ml

Log CFU/ml

3
5
Time (days)

Log CFU/ml

0
0

3
5
Time (days)

3
5
Time (days)

Fig. 6. Effect of enterocin AS-48 (25 mg/ml) in combination with hydrocinnamic acid (A), peracetic acid (B), sodium hypochlorite (C), hexadecylpyridinium chloride (D), 0.1%
polyphosphoric acid (E) and 0.5% polyphosphoric acid (F) on the viability of B. weihenstephanensis CECT 5894 in alfalfa sprouts stored at 15 1C for 1 week. Controls (J),
samples treated with AS-48 (m), chemical compound (), or AS-48+chemical compound (K). Data represent the average of two independent experiments plus standard
deviation (error bars).

protective culture at 15 and 22 1C (Fig. 7). Enterococci multiplied

rapidly on sprouts both at 15 and 22 1C. In both cases, bacteriocin
activity could be recovered from the sprouts during days 13, but
not at other points of storage. In coculture samples stored at 15 1C,
growth of B. cereus was completely inhibited for the whole storage
period, with viable cell counts being signicantly lower than
monocultures for the rst 5 days of storage (Fig. 7A). By contrast,
cocultivation with the producing strain at 22 1C only produced
some growth inhibition of B. cereus, during the rst 3 days of
storage (Fig. 7B).

4. Discussion
B. cereus is the main aerobic mesophilic endospore former of
concern in the food industry (Schoeni and Wong, 2005). The
spores of B. cereus survive pasteurization processes of 100 1C
during 2.25.4 min and, in addition, vegetative cells from some

strains can grow at low temperatures, of 45 1C (Dufrenne et al.,

1995; Choma et al., 2000). Seed sprouts can contain spores of B.
cereus coming from diverse sources (vegetal substrate, earth,
fertilizers), that can germinate and reach high population
densities greater than risk levels. The closely related psychrotolerant species B. weihenstephanensis is now being investigated as it
may also produce food-poisoning toxins. The need to include
additional hurdles for the control of B. cereus in the food chain
could be solved by using natural antibacterial substances like
bacteriocins.
Previous studies have demonstrated that the addition of
enterocin AS-48 presents a good inhibitory activity against this
bacteria in foods of milk origin, boiled rice and vegetable purees
oz et al., 2004; Grande et al., 2006, 2007b). Nevertheless, the
(Mun
potential of this bacteriocin in the control of B. cereus as well as B.
weihenstephanensis in sprouts had still not been investigated. The
results obtained in the present study are clearly promising for the
decontamination of these two species, mainly for samples stored

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15

12

8
6

10

AS-48 (mm)

Log CFU/ml

10

2
0

5
0

3
5
Time (days)

12

15

8
6

10

AS-48 (mm)

Log CFU/ml

10

2
0

5
0

3
5
Time (days)

Fig. 7. Cocultivation of B. cereus LWL1 and the enterocin AS-48 producer strain E.
faecalis A-48-32 on soybean sprouts stored at 15 1C (A) and 22 1C (B). Viable cell
counts of B. cereus LWL in control monocultures (J) and in cocultures (K) are
shown. Growth of strain E. faecalis A-48-32 in cocultures (m) and bacteriocin
production (bars). Data represent the average of two independent experiments
plus standard deviation (error bars).

under refrigeration. However, the treatment with bacteriocin did

not provide any protection under abuse temperature conditions of
15 or 22 1C. For this reason, in order to increase the temperature
safety margins it would be necessary to apply combined
treatments with other antimicrobial agents. Results from the
preliminary screening carried out in the present study were highly
satisfactory for the combined treatments of AS-48 with the
cinnamic and hydrocinnamic acids, carvacrol, polyphosphoric
acid, peracetic acid, hexadecylpyridinium chloride and sodium
hypochlorite. For most of them, viable cell counts of B. cereus were
reduced below the detection limits. In other cases (lactic acid,
lactate sodium, n-propyl p-hydroxybenzoate and p-hydoxybenzoic acid methyl ester), viable cell counts in the bacteriocintreated samples were signicantly lower compared to the nontreated controls, but not with the chemical preservatives alone,
indicating that there was no synergism with the bacteriocin.
While previous reports have studied the synergistic effects of
antimicrobial compounds with bacteriocins (reviewed by Galvez
et al., 2007), this is the rst example of application of washing
treatments against endospore-forming bacteria and also for
application of a bacteriocin against B. weihenstephanensis.
One of the main problems of decontamination treatments is
that the reductions of viable cell counts obtained may not be

769

sufcient to avoid growth of survivors and sublethally injured

cells during storage. In the present study, those combinations of
AS-48 that were more effective in reducing viable cell counts
during treatment also provided a very good protection against
B. cereus and B. weihenstephanensis during 1 week storage of
sprouts at 15 1C, reducing the concentration of viable cells below
the detection limits for most of the samples. These results suggest
that such combinations could be used as highly effective
decontamination treatments against Bacillus in germinated seed
sprouts.
Another possible alternative to control undesired microorganisms in foods is based on the application of competitive exclusion
techniques, in which the bacteriocin-producing strains are used to
inhibit the growth of pathogenic or toxicogenic bacteria. In this
sense, the use of producing strains of enterocin AS-48 has shown
good results in the control of B. cereus and S. aureus in cheese as
oz
well as of S. aureus and L. monocytogenes in a meat system (Mun
et al., 2004, 2007; Ananou et al., 2005a, b). Enterococci are part of
the normal microbiota of the human gastrointestinal tract, and are
also found in many different types of foods (revised by Foulquie
Moreno et al., 2006). Since there is now a concern that enterococci
may be involved in the spread of antibiotic resistance and
virulence traits in foods, the strains used for food applications
should be devoid of such traits. In this respect, previous results
have shown that the genetic traits coding for enterocin AS-48 can
be transferred articially into suitable strains devoid of virulence
traits and with suitable technological performance (Fernandez et
al., 2007). The results obtained in the present study against B.
cereus in soybean sprouts also indicate that the AS-48 producing
strains could serve to control the growth of B. cereus under
suitable temperature conditions that allow production of sufcient amounts of bacteriocin without favouring an excessive
growth of B. cereus. It is noteworthy the good implantation shown
by the bacteriocinogenic strain A-48-32 in soybean sprouts,
reaching elevated cell densities within the three rst days of
storage of the samples at 15 and 22 1C. However, the application of
this strain as a protective culture would be limited by the low
bacteriocin production under refrigeration and the lack of
effectiveness shown at 22 1C. Therefore, addition of ex-situ
produced bacteriocin (either alone or in combination with other
antimicrobials) remains the best choice to control B. cereus and B.
weihenstephanensis on sprouts.

Acknowledgments
This work was supported by the Spanish Ministry of Education
(Research Project AGL2005-07665-C02-02/ALI). We also acknowledge the Research Programme of the University of Jaen, and the
Research Plan of the Junta de Andaluca (Research Group AGR230).
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