Food Microbiology 26 (2009) 289293

Contents lists available at ScienceDirect

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

Inactivation of Geobacillus stearothermophilus in canned food and coconut milk

samples by addition of enterocin AS-48
Pilar Martnez Viedma a, Hikmate Abriouel a, Nabil Ben Omar a, Rosario Lucas Lopez a, Eva Valdivia b,
Antonio Galvez a, *
a
b

rea de Microbiologa, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jaen, 23071 Jaen, Spain
A
Department of Microbiology, University of de Granada, 18071 Granada, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 22 October 2008
Received in revised form
18 December 2008
Accepted 21 December 2008
Available online 6 January 2009

The cyclic bacteriocin enterocin AS-48 was tested on a cocktail of two Geobacillus stearothermophilus
strains in canned food samples (corn and peas), and in coconut milk. AS-48 (7 mg/g) reduced viable cell
counts below detection levels in samples from canned corn and peas stored at 45  C for 30 days. In
coconut milk, bacterial inactivation by AS-48 (1.75 mg/ml) was even faster. In all canned food and drink
samples inoculated with intact G. stearothermophilus endospores, bacteriocin addition (1.75 mg per g or
ml of food sample) rapidly reduced viable cell counts below detection levels and avoided regrowth
during storage. After a short-time bacteriocin treatment of endospores, trypsin addition markedly
increased G. stearothermophilus survival, supporting the effect of residual bacteriocin on the observed
loss of viability for endospores. Results from this study support the potential of enterocin AS-48 as
a biopreservative against G. stearothermophilus.
2008 Elsevier Ltd. All rights reserved.

Keywords:
Bacteriocin
Preservation
Canned foods
Flat sour
Geobacillus

1. Introduction
Spoilage of canned vegetables is usually caused by thermophilic
spore-forming bacteria (Brackett, 2001). Flat sour spoilage will
likely occur if canned products contaminated with thermophilic
spores are stored at temperatures above 43  C. Thermophilic
bacteria were initially reported to be the prime cause of spoilage of
canned corn (Denny, 1981). Bacillus stearothermophilus was identied as responsible for spoilage of canned asparagus (Lin et al.,
1968). Geobacillus stearothermophilus (formerly B. stearothermophilus) is a Gram-positive, non-pathogenic, spore-forming
bacterium that thrives in high temperature environments. It typically survives canning and sterilization procedures of food products. Spores of G. stearothermophilus are extremely heat resistant,
up to 20 times more resistant than Clostridium botulinum spores
(Lin et al., 1968; Feeherry et al., 1987; Lopez et al., 1997; Watanabe
et al., 2003; Iciek et al., 2008). G. stearothermophilus spores can and
do survive thermal processing in a commercially sterile product
(Cameron and Esty, 1926; Denny, 1981; Brackett, 2001), and may

* Corresponding author. Present address: Area de Microbiologa, Departamento

de Ciencias de la Salud, Facultad de Ciencias Experimentales, Edif. B3, Universidad
de Jaen, Campus Las Lagunillas s/n, 23071 Jaen, Spain. 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.2008.12.007

cause spoilage problems especially where foods must be stored at

elevated temperatures for a long time (e.g., canned food vending
machines or military operations in tropical climates). Growth of
G. stearothermophilus spores results in at sour spoilage because
acid is produced but with little or no gas generated (Brackett, 2001).
Low-acid foods such as meat and marine products, milk, vegetables,
meat and vegetable mixtures (such as soups) can be spoiled by
G. stearothermophilus under improper storage conditions (Ayres
et al., 1980). Furthermore, the capacity of this bacterium to adhere
to stainless steel and grow in biolms appears to be a likely cause of
contamination of manufactured dairy products (Flint et al., 2001).
Feeherry et al. (1987) noted that many food products cannot
withstand the heat treatment needed to inactivate thermophilic
spores (to achieve at least a 6D reduction). Therefore, other
measures such as the control of contamination of ingredients by
thermophilic organisms, rapid cooling below 43  C after thermal
processing, and controlled storage are required to prevent spoilage.
Since bacterial endospores are also resistant to a variety of other
treatments such as irradiation, high hydrostatic pressure, pulsed
electric elds, and chemicals, alternative methods for inactivation
of endospore-formers should be of great value to the food processing industry. Among the proposed methods to avoid the
problems of bacterial food spoilage is the application of bacteriocins, as part of hurdle technology (Cleveland et al., 2001; Devlieghere et al., 2004; Galvez et al., 2007). So far, nisin is the only

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P.M. Viedma et al. / Food Microbiology 26 (2009) 289293

bacteriocin reported to inhibit thermophilic bacteria in canned

vegetable foods (Thomas et al., 2000). Enterocin AS-48 is a broadspectrum cyclic antimicrobial peptide (reviewed by Maqueda et al.,
2004) which is active against several food spoilage and pathogenic
oz et al., 2004; Ananou et al., 2005; Cobo Molinos
bacteria (Mun
et al., 2005; Grande et al., 2005, 2006a,b, 2007a,b; Lucas et al.,
oz et al., 2007). This bacteriocin was very active against
2006; Mun
B. coagulans in canned foods (Lucas et al., 2006), B. licheniformis in
apple cider (Grande et al., 2006a), B. cereus in rice-based foods
(Grande et al., 2006b), and Alicyclobacillus acidoterrestris in fruit
juices (Grande et al., 2005). Treatment with enterocin AS-48
decreased the heat resistance of bacterial endospores for B. coagulans, B. licheniformis and B. cereus (Grande et al., 2006a,b; Lucas
et al., 2006), while endospores of A. acidoterrestris became inactivated by bacteriocin treatment at room temperature (Grande et al.,
2005). These results would suggest that the bacteriocin may also be
effective against other endospore-formers in food systems. With
these premises, the purpose of the present study was to determine
the efcacy of enterocin AS-48 against G. stearothermophilus
vegetative cells and endospores in different types of canned fruit
juice and vegetable foods during storage under temperature abuse
conditions.
2. Materials and methods
2.1. Bacterial strains and growth conditions
G. stearothermophilus CECT 43 (type strain) from deteriorated
canned food, and G. stearothermophilus CECT 49 from canned food
ola de
with at sour spoilage were supplied by the Coleccion Espan
Cultivos Tipo (CECT, Burjasot, Valencia, Spain). Enterococcus faecalis
A-48-32 (Martinez-Bueno et al., 1990) was used to produce enterocin AS-48. E. faecalis S-47 was used as an indicator strain for
determination of bacteriocin activity. Geobacilli were cultivated
routinely on tryptic soy broth (TSB, Scharlab, Barcelona) or tryptic
soy agar (TSA, Scharlab) at 45  C. Enterococci were grown on Brain
Heart Infusion broth (BHI, Scharlab) at 37  C.

examination under light microscopy. Once sporulation had

reached at least 95%, the plates were ooded with sterile
distilled water and the growth was scraped off with a sterile
swab in order to collect the spores. Spores were washed three
times in distilled water and separated by centrifugation (13,000
g, for 5 min), and then stored in distilled water at 20  C until
use. The spore concentration was determined by serial dilution
and viable cell count on TSA.
2.4. Food and drink samples
Canned corn (containing corn grains, water and salt; pH 6.37;
Bonduelle Iberica, Alcobendas, Spain), canned peas (containing
peas, water, and salt; pH 6.19; Bonduelle Iberica) and coconut milk
(containing 33% coconut extract and 67% water; pH 6.37; TIGER
TIGER Marketing, Nottingham, UK) were bought from local supermarkets. Cans were opened under aseptic conditions and the
content of cans was used for bacteriocin tests.
2.5. Bacteriocin treatments
Food and beverage samples (5 g, or 5 ml) in duplicate were
inoculated with a cocktail of vegetative cells or endospore
suspensions (3.03.5  106 CFU/ml) from strains CECT 43 and 49
prepared as described above, and then supplemented with enterocin AS-48 at desired nal concentrations (1.757 mg per g or ml of
sample). Untreated controls and bacteriocin-treated samples were
placed in an incubation chamber with a controlled temperature of
45  C (Memmert, Schwabach, Germany). At desired intervals, peas
and corn samples were homogenised with a Stomacher 80 (Biomaster, Seward, UK) at maximum speed for 30 s. Drink samples
were homogenised by vortexing. Samples were serially diluted
with sterile saline solution and then plated in triplicate on TSA agar
plates and incubated at 45  C for 5 days. The average number of
colonies was used to calculate the viable cell concentrations. These
were expressed in log10 colony-forming units (CFU) per gram or ml
of solid or liquid food sample, respectively. The detection limit was
10 CFU.

2.2. Bacteriocin preparation

2.6. Trypsin rescue of bacteriocin-treated endospores
Enterocin AS-48 was obtained from cultured broths of the
producer strain E. faecalis A-48-32 after concentration by cation
exchange chromatography as described by Abriouel et al. (2003).
Bacteriocin concentrates were desalted through 2000 mw cut-off
dialysis tubing (Sigma) and ltered through 0.22 mm pore size low
protein binding lters (Millex GV; Millipore Corp., Belford, MA,
USA) under sterile conditions. Samples were serially diluted and
tested (100 ml) for bacteriocin activity against the indicator strain
E. faecalis S-47 by the agar well diffusion method using stainless
steel cylinders of 8 mm (outer) diameter (Abriouel et al., 2003). One
arbitrary unit (AU) was dened as the highest dilution producing
a visible (9 mm diameter) zone of inhibition. The bacteriocin
concentration of samples was determined from the previously
reported specic activity value of 3.5 AU/mg protein (Abriouel et al.,
2003).

Since intact endospores treated with the bacteriocin did not

produce viable counts, a treatment with trypsin (which inactivates
AS-48) was applied in order to determine whether the bacteriocin
adsorbed on the spores would be responsible for inactivation
during the germination and outgrowth phases, or if intact spores
could be inactivated directly by the bacteriocin before germination.
Intact endospores of G. stearothermophilus CECT 43 were inoculated
in TSB broth with or without enterocin AS-48 (0.75 mg/ml) and
incubated at 30  C. After 5 min incubation, the samples were supplemented with trypsin (Sigma, Madrid) (1 mg/ml in 0.1 M phosphate buffer) to a nal concentration of 100 mg/ml, or with
phosphate buffer alone. After 20 min of further incubation at 30  C
(with or without trypsin), spore suspensions were serially diluted
in sterile saline solution and plated for viable counts.

2.3. Preparation of inocula and endospore suspensions

2.7. Statistical analysis

G. stearothermophilus exponential phase vegetative cells were

grown in TSB at 45  C for 48 h before they were used for inoculation of food and drink samples. In order to obtain endospore
preparations, G. stearothermophilus cultures were grown in TSA
for approximately 1015 days at 45  C. The level of sporulation
was monitored by staining the spores with a solution of 5%
malachite green and then with 0.5% safranin followed by

The average data from duplicate trials  standard 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.

P.M. Viedma et al. / Food Microbiology 26 (2009) 289293

3. Results

3.1. Effect of enterocin AS-48 in samples from canned foods

and coconut milk inoculated with vegetative cells of
G. stearothermophilus

3.3. Trypsin rescue of bacteriocin-treated endospores

When endospores of G. stearothermophilus CECT 43 were challenged with enterocin AS-48 without addition of trypsin, the short
bacteriocin treatment was sufcient to achieve a 3.0 log reduction
of viable counts (Table 1). By contrast, viable counts from samples
challenged with AS-48 and then treated with trypsin were 2.8 log
units higher (P < 0.05) compared to samples treated with AS-48
alone. These results indicate that trypsin caused inactivation of AS48 before the bacteriocin could kill the spores, and suggest that
residual bacteriocin or bacteriocin adsorbed on the spores was
responsible for killing after the short-time treatment in the samples
treated only with bacteriocin.
4. Discussion
Consumers demands increasingly aim at high-quality, minimally processed, nutritious and fresh-like products. Traditional
thermal processing methods cause loss of desirable properties

Log CFU/g

0
0

10

15

20

25

30

25

30

25

30

Time (days)

10

3.2. Effect of enterocin AS-48 in foods and coconut milk inoculated

with endospores of G. stearothermophilus

0
0

10

15

20

Time (days)

Log CFU/ml

In food and coconut milk samples inoculated with a cocktail of

G. stearothermophilus strains CECT 43 and CECT 49 endospores,
viable cell counts in the untreated controls increased rapidly during
the rst day of incubation for the samples from canned corn, canned peas and also coconut milk (Fig. 2A, B, C). The highest cell
concentrations reached in canned peas inoculated with endospores
(day 3 of storage) were lower compared to the corresponding
samples inoculated with vegetative cells. In all the food and drink
samples tested, a bacteriocin concentration as low as 1.75 mg per g
or ml of food was sufcient to reduce viable cell counts below
detection levels from day 1 to the end of the storage period.

10

Log CFU/g

G. stearothermophilus vegetative cells multiplied rapidly both in

the samples of canned corn and canned peas, reaching high cell
concentrations (ca. 8 log CFU/g) within the rst day of incubation.
This was followed by a progressive decay of the viable population
during the subsequent 30-days incubation (Fig. 1A, B). In the canned corn samples, a bacteriocin concentration of 1.75 mg/g caused
some growth inhibition during the rst day of incubation and
reduced the viable cell concentration below detection levels by day
3 (Fig. 1A). A higher bacteriocin concentration (3.5 mg/g) signicantly reduced (P < 0.05) the viable cell concentrations by 1.4 log
units at day 1, while 7.0 mg/g completely inactivated all detectable
cells. Remarkably, no viable cells were detected in the bacteriocintreated samples during further storage for up to 30 days. Results
obtained in samples from canned peas were very similar, with
reductions of viable cell counts from 1.5 to 2.1 log units or
a complete inactivation of the bacilli within the rst day for
bacteriocin concentrations of 1.75, 3.0, and 7.0 mg/g respectively
(Fig. 1B).
In coconut milk, G. stearothermophilus showed a much lower
capacity to grow, reaching 5.4 log CFU/ml at day 1 (Fig. 1C).
However, some viable cells (1.3 log CFU/ml) were still detected at
day 15 of storage. At the lowest bacteriocin concentration tested
(1.75 mg/ml), enterocin AS-48 reduced viable cell counts below
detection levels within the rst 24 h of storage (Fig. 1C).

291

10

15

20

Time (days)
Fig. 1. Effect of enterocin AS-48 in food samples of canned corn (A), canned peas (B)
and coconut milk (C) inoculated with a cocktail of vegetative cells from G. stearothermophilus strains CECT 43 and CECT 49, incubated at 45  C. Open symbols:
untreated controls. Closed symbols: samples supplemented with enterocin AS-48 at
nal concentrations of 1.75 (:), 3.5 (-) and 7.0 mg/g or ml (C).

related to texture, avour, colour, and nutrient value. However, the

most serious commercial problems with product sterility are
caused by thermally resistant spores. In an attempt to provide
alternatives to solve these problems, we have tested enterocin AS48 on a thermophilic spoilage bacterium known for the high
thermal resistance of its endospores. The results obtained indicated

292

P.M. Viedma et al. / Food Microbiology 26 (2009) 289293

10

Log CFU/g

0
0

10

15

20

25

30

20

25

30

Time (days)

10

Log CFU/g

0
0

10

15

Time (days)

Log CFU/ml

that vegetative cells from a cocktail of two strains isolated from

spoiled canned foods were very sensitive to AS-48, requiring very
low bacteriocin concentrations for inactivation in the four different
food systems tested. In canned vegetable food samples, the
minimum bactericidal concentration (MBC) capable of reducing
viable cell concentrations below detection levels within 24 h of
incubation was 7 mg/g. This MBC is much lower compared to the
values obtained for B. cereus in rice foods (25 mg/g), and close to the
values obtained for B. licheniformis in apple cider (10 mg/g; Grande
et al., 2006a) or B. coagulans in low-acid canned foods (6 mg/g;
Lucas et al., 2006). In addition, the bacteriocin was even more active
in coconut drinks, with a MBC value of 1.75 mg/g. This value is
similar to the MBC of 2.5 mg/ml reported for A. acidoterrestris in fruit
juices (Grande et al., 2005).
One of the most interesting aspects of the present study is the
rapid inactivation observed in samples inoculated with G. stearothermophilus endospores after bacteriocin treatment. Previous
studies have shown that intact endospores of B. cereus, B. licheniformis and B. coagulans (Abriouel et al., 2002; Grande et al.,
2006a,b; Lucas et al., 2006) were completely resistant to AS-48,
although B. cereus endospores become gradually sensitive within
the course of germination (Abriouel et al., 2002). Endospore resistance may be related to the mechanism of action of enterocin
AS-48, which disrupts the bacterial cytoplasmic membrane
permeability (Galvez et al., 1991). In combined treatments, enterocin AS-48 also increased the heat sensitivity of endospores
(Grande et al., 2006a,b; Lucas et al., 2006), similarly to results
reported for nisin (Beard et al., 1999; Wandling et al., 1999). In the
present study, inactivation of enterocin AS-48 by trypsin markedly
counteracted the effect of the bacteriocin on G. stearothermophilus
endospores during a short-time treatment. These results suggest
that the bacteriocin has no effect on intact endospores, and also
that adsorbed bacteriocin molecules are responsible for loss of
viability during the course of germination and outgrowth, similarly
to nisin (Thomas et al., 2000). According to a previous study,
endospores of A. acidoterrestris (another thermophilic endosporeformer) were highly sensitive to enterocin AS-48 (Grande et al.,
2005). Bacteriocin-treated A. acidoterrestris endospores seemed to
be arrested at early-stage germination and exhibited substantial
structural damages under electron microscopy examination.
In conclusion, the high activity of enterocin AS-48 against
G. stearothermophilus (in foods inoculated with either vegetative
cells or endospores) opens new possibilities to explore the usefulness of this bacteriocin in the food industry to protect against this
spoilage-causing thermophilic endospore-former.

Acknowledgements
0
0

10

15

20

25

30

Time (days)
Fig. 2. Effect of enterocin AS-48 in food samples of canned corn (A), canned peas (B)
and coconut milk (C) inoculated with a cocktail of intact endospores from G. stearothermophilus strains CECT 43 and CECT 49, incubated at 45  C. Open symbols:
untreated controls. Closed symbols: samples supplemented with enterocin AS-48 at
a nal concentration of 1.75 mg/g or ml (:).

Table 1
Effect of trypsin addition (100 mg/ml) on survival of G. stearothermophilus CECT 43
endospores in TSB upon treatment with enterocin AS-48 (0.75 mg/ml).
Treatment

Control

Trypsin

AS-48

AS-48 Trypsin

Viable counts (log CFU/ml)

4.04  0.02

4.06  0.03

1.0  0.11

3.80  0.09

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, from which
Pilar Martnez Viedma received a fellowship, and the Research Plan
of the Junta de Andaluca (research group AGR230).

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