Food Control 22 (2011) 457e461

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Combined effect of reuterin and lactic acid bacteria bacteriocins on the

inactivation of food-borne pathogens in milk
Juan L. Arqus*, Eva Rodrguez, Manuel Nuez, Margarita Medina
Dpto. de Tecnologa de Alimentos, INIA, Carretera de La Corua km 7, 28040 Madrid, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 28 April 2010
Received in revised form
15 September 2010
Accepted 21 September 2010

Antimicrobial activity of reuterin in combination with different bacteriocins from lactic acid bacteria
against food-borne pathogens in milk was investigated. A strong synergistic effect of reuterin in
combination with nisin, lacticin 481 or enterocin AS-48 on Listeria monocytogenes was observed. Only
nisin increased the antimicrobial activity of reuterin against Staphylococcus aureus. Bactericidal activity of
reuterin towards Escherichia coli O157:H7, Salmonella enterica, Yersinia enterocolitica, Aeromonas hydrophila and Campylobacter jejuni was not enhanced signicantly by the addition of any of the bacteriocins
investigated. The synergism of reuterin and nisin against L. monocytogenes and S. aureus was also found
at refrigeration temperatures, where the pathogens were completely inactivated. Refrigerated milk
treated with both natural antimicrobials would mean a feasible system to control pathogenic
contaminants.
2010 Elsevier Ltd. All rights reserved.

Keywords:
Reuterin
LAB-bacteriocins
Food-borne pathogens
Combined treatments

1. Introduction
Reuterin (b-hydroxypropionaldehyde) is a molecule with antimicrobial activity towards a broad spectrum of food-borne pathogens and spoilage organisms. Reuterin is soluble in water, resistant
to heat, proteolytic and lipolytic enzymes, and stable over a wide
range of pH values (Axelsson, Chung, Dobrogosz, & Lindgren, 1989;
Vollenweider, Grassi, Knig, & Puhan, 2003). The use of reuterin to
control Gram-positive and Gram-negative pathogens has been
investigated in milk and dairy products (Arqus, Rodrguez, Nuez, &
Medina, 2008; El-Ziney & Debevere, 1998) and in meat products (ElZiney, van den Tempel, Debevere, & Jakobsen, 1999). In all studies,
reuterin has been shown to have a higher antimicrobial activity on
Gram-negative than on Gram-positive pathogenic bacteria.
Bacteriocins of lactic acid bacteria (LAB) are small peptides that
show a narrow or broad antimicrobial activity spectrum against
Gram-positive bacteria. The use of bacteriocins to control foodborne pathogens and spoilage bacteria has been reported in a variety
of foods including milk and dairy products (Rodrguez, Arqus, Gaya
Nuez, & Medina, 2001; Zottola, Yezzi, Ajao, & Roberts, 1994). A
number of bacteriocins with industrial potential have been puried
and characterized. LAB-bacteriocins have a common mechanism of
action on sensitive cells by the formation of transitory poration
complexes or ionic channels in the cytoplasmic membrane that

* Corresponding author. Fax: 34 91 3572293.

E-mail address: arques@inia.es (J.L. Arqus).
0956-7135/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2010.09.027

causes total or major dissipation of the proton motive force (Bruno &
Montville, 1993). Their action is generally inactive against Gramnegative bacteria because of the presence of the outer membrane.
Nisin, the only bacteriocin authorized as a food preservative in over
50 countries wordwide, is produced by some Lactoccocus lactis and it
is active against undesirable Gram-positive bacteria associated with
food. The combination of bacteriocins with other antimicrobials in
order to reduce the selection for resistance to bacteriocins in target
strains or to extend its inhibitory activity to Gram-negative bacteria
has been reported (Helander, von Wright, & Mattila-Sandholm,
1997; Stevens, Sheldom, Klapes, & Klaenhammer, 1991).
The use of natural antimicrobials in the food industry can help to
reduce the addition of chemical preservatives, offering an alternative to satisfy the increasing consumer demand for safe, freshtasting, ready-to-eat, minimally-processed foods and also to
develop novel food products. Nevertheless, the chemical and
physical properties of a food such as pH, enzymes, fat and additives
can limit the antimicrobial activity of natural compounds. Research
into synergistic effects of the combined action of natural preservatives to increase microbial lethality could achieve an improved
level of product safety according to the hurdle concept of food
preservation that would benet both consumers and producers.
Here we sought combinations of reuterin with LAB-bacteriocins
in order to enhance individual antimicrobial activity against foodborne pathogens in milk. Subsequently, the inhibitory potential of
combining reuterin and nisin on Listeria monocytogenes and
Staphylococcus aureus in milk stored at two refrigeration temperatures has also been investigated.

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J.L. Arqus et al. / Food Control 22 (2011) 457e461

2. Materials and methods

2.4. Inhibitory activity of reuterin combined with nisin against

Gram-positive pathogens in milk at refrigeration temperatures

2.1. Microorganisms and culture conditions

Escherichia coli O157:H7 ATCC 43894, Salmonella enterica subsp.
enterica CECT 409, Campylobacter jejuni LMG 6629, Yersinia enterocolitica CECT 559, Aeromonas hydrophila subsp. hydrophila CECT
839, L. monocytogenes Ohio serotype 4b (from R.G. Crawford, Food
and Drug Administration, Cincinnati, OH 45226, USA) and S. aureus
CECT 4013 were used as test microorganisms. The strains were
propagated in Tryptic Soy Broth (TSB, Biolife, Milano, Italy) at 37  C
(at 30  C in the case of A. hydrophila) for 18 h. C. jejuni was grown in
basal medium with the addition of 5% (v/v) lysed horse blood and
incubated in a microaerophilic atmosphere of 5% oxygen and 10%
carbon dioxide generated by Gas Generating Kit (Oxoid, Unipath
Ltd., Basingstoke, UK). Test bacteria were subcultured in sterile
reconstituted skim milk supplemented with 0.3% yeast extract
before use in milk assays. Lacticin 481-producing L. lactis subsp.
cremoris TAB 24, enterocin I-producing Enterococcus faecalis TAB 52
and enterocin AS-48-producing E. faecalis TAB 70 from the INIA
(Instituto Nacional de Investigacin y Tecnologa Agraria y Alimentaria, Madrid, Spain) culture collection were propagated in
MRS broth (Biolife) at 30  C for 18 h. All the strains were maintained
in a frozen stock at 80  C with 15% glycerol and propagated twice
before used in experiments.
2.2. Biopreservatives
The reuterin-producing L. reuteri INIA PRO 137 was used for
reuterin production in a process carried out in two steps, biomass
generation and reuterin production as previously described (Arqus
et al., 2004). Nisin (Nisaplin, Danisco, Copenhagen, Denmark) was
resuspended in 0.02 N HCl to 10000 IU/ml and stored at 40  C. The
nisin stock was diluted prior to use in experiments. LAB-bacteriocins
were obtained from bacteriocinogenic cultures. After cultivation the
supernatants obtained by centrifugation (10,000 g, 15 min, 4  C)
were ltered (0.22 mm, Millipore Corporation, Bedford, MA, USA)
and adjusted to pH 6 with 1 N NaOH. Inhibitory activity of reuterin
and bacteriocins (lacticin 481, enterocin I and enterocin AS-48) was
determined with the modied assay of Chung, Axelsson, Lindgren,
and Dobrogosz (1989) using E. coli K12 CECT 433 as indicator
strain for reuterin and L. monocytogenes Ohio as indicator strain for
LAB-bacteriocins. Arbitrary units (AU) were dened as the reciprocal
of the highest serial two-fold dilution which did not show inhibition
of the indicator strain.
2.3. Inhibitory activity of reuterin in combination with LABbacteriocins against pathogens in milk
Bacterial strains were separately inoculated at approximately
104 cfu/ml into asks containing 15 ml of UHT skim milk with 0.04%
fat (Pascual, Aranda del Duero, Spain). Biopreservatives were added
individually or in combination immediately after inoculation. Reuterin and nisin were added at an estimated nal activity of 8 AU/ml
and 100 IU/ml, respectively, and lacticin 481, enterocin I and enterocin AS-48 at 170 AU/ml. Milk inoculated with each pathogen with
no addition of reuterin or LAB-bacteriocins served as control. Flasks
were incubated at 37  C for 24 h except for A. hydrophila which was
incubated at 30  C. Microbiological counts were determined after 4
and 24 h on Tryptic Soy Agar (TSA, Biolife) plates. C. jejuni was
determined on Blood Agar Base No 2 plates (Oxoid) supplemented
with 5% laked horse blood and Campylobacter growth supplement
(Oxoid). Two separate experiments were carried out. Microbiological
analyses were performed in duplicate.

The milk assays at refrigeration temperatures were performed

as described above using L. monocytogenes and S. aureus as test
microorganisms. Flasks were stored at 4  C and 8  C for 12 d.
Microbiological counts were determined after 8 h and at 1, 3, 5, 7
and 12 d on duplicate plates of TSA. Two separate experiments
were carried out.
2.5. Statistical analysis
Data were subjected to ANOVA with treatment and time of
refrigeration as main effects on pathogen counts at each temperature using the SPSS program Win version 12.0 (SPSS Inc., Chicago,
IL, USA). Signicant differences in pathogen counts in milk between
different treatments for a given pathogen and time of refrigeration
were further analyzed using Tukeys test with a signicance level of
a 0.01, using the same program.
3. Results
3.1. Inhibitory activity of reuterin combined with LAB-bacteriocins
against pathogens in milk
Inhibitory activities of reuterin, LAB-bacteriocins and their
combination on Gram-positive and Gram-negative pathogens in
milk are shown in Tables 1 and 2, respectively. The counts of the
different pathogens in milk were signicantly inuenced by the
treatment (P < 0.001) and the incubation time (P < 0.001),
according to ANOVA. After 4 h, antimicrobial activity of reuterin
was bacteriostatic, since the initial bacterial levels did not decrease,
or slightly bactericidal on all the pathogens, except on C. jejuni
where a strong bactericidal activity was observed. Nisin, lacticin

Table 1
Log counts (cfu/ml) of L. monocytogenes and S. aureus in milk without biopreservatives (C) or with reuterin (R), nisin (N), lacticin 481 (L481), enterocin I (EI),
enterocin AS-48 (AS48) or their combination (R N, R L481; R EI; R AS48)
after incubation at 37  C for 4 and 24 h.
Time (h)

L. monocytogenes

S. aureus

3.91

4.10

4.66f
3.77e
0.15a
3.22d
4.40f
2.00b
nda
0.15a
2.70c
0.24a

5.89d
4.19c
1.21a
5.88d
5.88d
5.97d
3.54b
4.15c
4.22c
4.18c

8.95e
4.81b
7.44d
5.99c
8.61e
7.28d
nda
nda
4.68b
nda

8.89d
3.46b
8.52c
9.07d
9.11d
9.09d
2.59a
3.40b
3.59b
3.41b

0
C
4
C
R
N
L481
EI
AS48
RN
R L481
R EI
R AS48
24
C
R
N
L481
EI
AS48
RN
R L481
R EI
R AS48

Values with different superscripts indicate statistically signicant (P < 0.01)

differences between treatments for a given organism and time. nd, below detection
limit (1 cfu/ml).

J.L. Arqus et al. / Food Control 22 (2011) 457e461

Table 2
Log counts (cfu/ml) of Gram-negative pathogens in milk without biopreservatives
(C) or with reuterin (R), nisin (N), lacticin 481 (L481), enterocin I (EI), enterocin AS48 (AS48) or their combination (R N, R L481; R EI; R AS48) after incubation
at 37  C for 4 and 24 h.
Time (h)

E. coli

Sal. enterica

C. jejuni

A. hydrophila

Y. enterocolitica

4.16

3.61

3.92

3.90

3.60

7.01b
4.09a
7.05b
7.03b
7.04b
7.15b
3.99a
3.95a
4.01a
4.03a

5.41b
3.09a
5.28b
5.40b
5.45b
5.50b
3.11a
3.22a
3.13a
3.14a

3.97b
nda
4.11b
4.00b
4.08b
4.07b
nda
nda
nda
nda

5.91b
3.41a
5.92b
5.98b
5.99b
6.06b
3.32a
3.39a
3.51a
3.51a

5.12b
3.48a
5.14b
5.12b
5.13b
5.09b
3.45a
3.48a
3.53a
3.47a

8.95c
3.17a
8.96c
8.95c
8.92c
8.92c
3.01ab
2.89a
3.00ab
3.00ab

8.98b
2.07a
8.99b
8.95b
9.11b
9.02b
2.16a
1.97a
2.12a
1.92a

6.72c
nda
6.63c
6.61c
6.67c
6.17b
nda
nda
nda
nda

8.97b
nda
9.05b
9.02b
9.05b
9.06b
nda
nda
nda
nda

8.63b
1.79ab
8.59b
8.49b
8.51b
8.54b
1.64ab
1.38a
2.00b
1.63ab

0
C
4
C
R
N
L481
EI
AS48
RN
R L481
R EI
R AS48
24
C
R
N
L481
EI
AS48
RN
R L481
R EI
R AS48

Values with different superscripts indicate statistically signicant (P < 0.01)

differences between treatments for a given organism and time. nd, below detection
limit (1 cfu/ml).

481 and enterocin AS-48 individually added to milk resulted in

a reduction of L. monocytogenes, with counts 4.51, 1.44 and 2.66 log
units lower with respect to levels in control milk, respectively. In
milk with nisin, counts 4.68 log units lower for S. aureus than in
control milk were detected, but no signicant differences were
observed in milk with other LAB-bacteriocins tested with respect to
control milk. L. monocytogenes was completely inactivated after 4 h
in milk with the combination of reuterin and nisin. The combined
effect of reuterin and lacticin 481 or enterocin AS-48 lowered the
counts of this pathogen by 4.51 and 4.42 log units, respectively,
compared with control milk. Reuterin in combination with nisin
achieved a reduction in the counts of S. aureus, with a level 2.35 log
units lower than in control milk, while in combination with lacticin
481, enterocin I or enterocin AS-48 levels were similar to those
found in milk with addition of reuterin only. As expected, no
inhibitory activity of any bacteriocin against the Gram-negative
pathogens investigated was recorded. Signicantly similar counts
of the Gram-negative pathogens were detected in milk with reuterin and in milk with the combination of reuterin and any LABbacteriocin.
After 24 h, a reduction in the counts of L. monocytogenes and S.
aureus was observed in milk with reuterin, with the counts being
4.14 and 5.43 log units lower than in control milk. L. monocytogenes
restarted the growth after the early bactericidal activity of nisin,
lacticin 481 and enterocin AS-48, which could be attributed to the
presence of resistant cells, with levels 1.51, 2.96 and 1.67 log units
lower in milk treated with that bacteriocins with respect to levels in
control milk, respectively. Similar results were obtained for S.
aureus in milk with nisin added individually, with levels only
0.37 log units lower than those in control milk. Sensitivity to reuterin of Gram-negative pathogens tested was higher than that of
Gram-positive, showing a bactericidal activity with counts 5.78,
6.91 and 6.84 log units lower in milk with reuterin than in control
milk for E. coli O157:H7, Sal. enterica and Y. enterocolitica, respectively. A. hydrophila and C. jejuni were not detected in milk with

459

reuterin. No inhibitory activity on Gram-negative pathogens was

detected in milk with the addition of any of the LAB-bacteriocins
investigated. The combination of reuterin with nisin, lacticin 481
and enterocin AS-48 acted synergistically and resulted in a reduction of L. monocytogenes counts below the detection limit (1 cfu/ml)
after 24 h. A synergistic reduction was also observed for S. aureus, as
the combined effect of reuterin and nisin (6.30 log units) was
higher than the sum of reductions achieved by reuterin and nisin
individually. The combination of reuterin and any LAB-bacteriocin
did not result in a signicant reduction in the counts of the different
Gram-negative pathogens tested with respect to individual addition of reuterin to the milk.
3.2. Inhibitory activity of reuterin combined with nisin against
Gram-positive pathogens in milk at 4  C
Psychrotrophic L. monocytogenes grew up to 6.63 log cfu/ml after
12 days in milk at 4  C, whereas S. aureus was not able to grow in
control milk during storage. Levels of both pathogens were significantly (P < 0.001) inuenced by the biopreservatives added and
the 12 d of storage at 4  C (Table 3), according to ANOVA results.
Reuterin was slightly bactericidal against L. monocytogenes, with
differences of 2.96 log units compared with the control milk.
Bactericidal activity of nisin was observed during the rst three-ve
days of refrigeration, but afterwards, the growth was resumed and
this pathogen reached counts 3.45 log units lower than those of
control milk at the end of the refrigeration period. The weak
bactericidal effect of reuterin against S. aureus observed at 37  C
turned into greater inhibitory effect after 12 days at 4  C, with levels
3.42 log units lower than in control milk. Nisin led to S. aureus
counts 1.06 log units lower than in control milk after 12 days. The
highest inactivation of L. monocytogenes and S. aureus was achieved
in milk with reuterin and nisin, which resulted in levels of both
pathogens below the detection limit after 12 days.
3.3. Inhibitory activity of reuterin combined with nisin against
Gram-positive pathogens in milk at 8  C
The result of ANOVA revealed signicant effects (P < 0.001) of
the biopreservatives employed and the refrigeration time at 8  C on
the levels of L. monocytogenes and S. aureus in milk (Table 4).
L. monocytogenes reached counts of 8.42 log cfu/ml, while
S. aureus did not increase in count after 12 days at 8  C. In milk with
reuterin L. monocytogenes counts were 4.14 log units lower than in
control milk. Nisin was less effective in reducing the counts, which
were 1.56 units lower than in control milk. As recorded for milk
refrigerated at 4  C, the antimicrobial activity of reuterin against

Table 3
Log counts (cfu/ml) of Listeria monocytogenes and Staphylococcus aureus in milk
without biopreservatives (C) or with reuterin (R), nisin (N) or their combination
(R N) during refrigeration at 4  C.
0h
L. monocytogenes
C
4.21
R
N
RN
S. aureus
C
4.01
R
N
RN

8h

1d

3d

5d

7d

12 d

4.15b
4.13b
2.95a
2.76a

4.25c
4.17c
2.31a
2.82b

4.26c
4.22c
1.66a
1.81a

4.47d
4.29c
1.70b
1.00a

5.07d
4.17c
2.20b
0.70a

6.63d
3.67c
3.18b
nda

3.82a
3.89a
3.93a
3.91a

3.83a
3.95a
3.89a
3.90a

4.00b
3.71a
3.84ab
3.64a

4.01d
2.99b
3.70c
2.73a

3.87d
2.05b
3.49c
1.57a

3.85d
0.43b
2.79c
nda

Values with different superscripts indicate statistically signicant (P < 0.01)

differences between treatments for a given organism and time. nd, below detection
limit (1 cfu/ml).

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J.L. Arqus et al. / Food Control 22 (2011) 457e461

Table 4
Log counts (cfu/ml) of Listeria monocytogenes and Staphylococcus aureus in milk
without biopreservatives (C) or with reuterin (R), nisin (N) or their combination
(R N) during refrigeration at 8  C.
0h
L.monocytogenes
C
4.25
R
N
RN
S. aureus
C
4.08
R
N
RN

8h

1d

3d

5d

7d

12 d

4.19c
4.19c
2.58b
2.30a

4.22c
4.19c
1.35b
0.51a

5.36d
3.55c
2.23b
0.71a

6.59d
4.55c
3.54b
0.50a

7.99d
4.46b
4.98c
0.30a

8.42d
4.28b
6.86c
nda

4.03a
3.97a
3.98a
4.04a

4.08b
3.99ab
3.88a
3.91a

4.10c
3.78b
3.83b
3.54a

4.10c
3.35b
3.72c
2.79a

4.07d
2.68b
3.54c
1.99a

4.13d
0.24b
3.46c
nda

Values with different superscripts indicate statistically signicant (P < 0.01)

differences between treatments for a given organism and time. nd, below detection
limit (1 cfu/ml).

S. aureus was enhanced at 8  C with respect to 37  C, with counts

close to the detection limit. A slight inhibitory activity of nisin was
detected throughout storage on S. aureus, with counts 0.67 log units
lower after 12 d in milk with the bacteriocin than in control milk. A
strong bactericidal effect on L. monocytogenes was detected in milk
with reuterin and nisin in combination (more than 8.42 log cfu/ml
reduction), which acted synergistically. When this combination
was added to milk, L. monocytogenes and S. aureus were absent at
the end of the refrigeration period.
4. Discussion
The effect of reuterin in combination with well-known LABbacteriocins on different Gram-positive and Gram-negative foodborne pathogens in milk as a model system was investigated. L.
monocytogenes not only survives, but grows at temperature at or
below 7  C in food (Donnelly & Briggs, 1986). Antimicrobial activity
of reuterin on L. monocytogenes was mainly bacteriostatic at all
temperatures studied, with differences lower than 0.70 log units
with respect to the initial inoculums throughout the incubation/
refrigeration periods. Similar bacteriostatic effect on L. monocytogenes by reuterin added at 8 AU/ml has been reported in milk
held at 37  C (Arqus et al., 2004). However, previous results
showed that antimicrobial activity of reuterin is concentration
dependent (Rasch, Metris, Baranyi, & Bjorn, 2007). The pathogen
was completely inactivated in milk refrigerated at 7  C with reuterin at 150 AU/ml (El-Ziney & Debevere, 1998). S. aureus did not
grow in control milk at 4 or 8  C in the present work. Addition of
reuterin to milk at these temperatures enhanced considerably the
bactericidal effect observed at 37  C. On the contrary, higher efciency of reuterin against E. coli at increasing temperatures
(10e30  C) was previously reported (Rasch, 2002).
L. monocytogenes was signicantly affected by nisin, lacticin 481
and enterocin AS-48, but not by enterocin I. However, nisin was the
only bacteriocin able to reduce the counts of S. aureus. Nisin and
lacticin 481 belong to class I bacteriocins (Dufour, Hindr, Haras, &
Pennec, 2007), while enterocin I (Floriano, Ruiz-Barba, & JimnezDaz, 1998) also described as enterocin L50, belongs to class II
(Criado et al., 2006; Dezwaan et al., 2007). Class I, characterized by
their unusual and posttranslationally modied aminoacids, and
class II bacteriocins, are small heat-stable peptides that, because of
their abundance and potential application in industrial processes,
are the most comprehensively studied. In addition, enterocin AS-48
is the most extensively studied cyclic bacteriocin produced by LAB
due to the fact that it shows great potential as a food preservative
(Ananou et al., 2005; Viedma et al., 2009). We did not observe in
our experiments an effect of the bacteriocins on Gram-negative

bacteria. Most of the studies reporting the effective use of LABbacteriocins against Gram-negative bacteria are those in which
bacteriocins were used in combination with treatments that
permeate the outer membrane (Stevens et al., 1991; Vaara, 1992) or
cause sub-lethal damage (Boziaris & Adams, 2001; Kalchayanand,
Hanlin, & Ray, 1992). Combination of reuterin with any of the
LAB-bacteriocins studied did not enhance the antimicrobial effect
of reuterin on Gram-negative pathogens in milk in the present
work. These data conrmed and extended previous observations
that showed no additional inhibition of Gram-negative pathogens
when reuterin is combined with nisin, compared to reuterin added
individually (Arqus et al., 2004).
A synergistic effect was evident when reuterin was combined
with nisin, lacticin 481 and enterocin AS-48 against L. monocytogenes
at optimum growth temperature of 37  C, while nisin was the only
LAB-bacteriocin tested that showed a synergistic antimicrobial
activity with reuterin against S. aureus. The synergistic antimicrobial
effect between reuterin and nisin on L. monocytogenes persisted at
refrigeration temperatures, where the pathogen was completely
inactivated after 12 d. Similar results were obtained for S. aureus at
refrigeration temperatures, where the highest rate of inactivation
was achieved in milk with reuterin and nisin, resulting in no
detectable levels of the pathogen.
Antimicrobial effect of LAB-bacteriocins in combination with
other antimicrobials such us nitrite, organic acids and their salts,
chelating agents, ethanol, essential oils, lysozyme, lactoferrin or
lactoperoxidase system on food-borne pathogens have been reported by different authors (Glvez, Abriouel, Lucas & Ben Omar, 2007).
Reuterin has a great potential application in the food industry as
biopreservative, nevertheless, very few studies have addressed the
combination of reuterin with other antimicrobials. Lactic acid (5%,
vol/vol) enhanced the inhibitory activity of reuterin against E. coli
O157:H7 and L. monocytogenes during meat decontamination, while
addition of ethanol (40% vol/vol) to the mixture of reuterin and lactic
acid had no additional effect (El-Ziney et al., 1999). In milk at
refrigeration temperatures, a strong synergistic bactericidal activity
on Gram-negative pathogens of reuterin in combination with the
lactoperoxidase-thiocyanate-hydrogen peroxide system, an inhibitory system that occurs naturally in milk, has been described
(Arqus et al., 2008).
Combinations of biopreservatives with respect to optimization of
practical applications require a detailed study, where it is important
to take into account the inuence of main environmental factors
such as temperature, pH and salt, on their antimicrobial effect. ElZiney and Debevere (1998) reported that increasing the NaCl
content from 1% to 3% increased inhibitory effect of reuterin against
L. monocytogenes. However, variations in pH (4.5e6.5) and salt
content (0.5e3%) did not inuence the inhibitory activity of reuterin
on E. coli K12 (Rasch, 2002). It has also been reported that presence
of NaCl could enhance the antimicrobial effect of bacteriocins
(Ananou, Valdivia, Martnez Bueno, Glvez, & Maqueda, 2004;
Parente, Giglio, Riccardi, & Clementi, 1998), or oppositely be antagonistic with them (Bell & de Lacy, 1985; Bouttefroy, Mansour, Linder,
& Milliere, 2000). Moreover, the increase in net charge of bacteriocins at low pH could facilitate translocation of the bacteriocins
through the cell wall and enhance their antibacterial activity.
Therefore, the combination of hurdles to be applied would depend
on the food systems and potential pathogens and/or spoilage
microorganism associated.
Our study showed clear synergistic effects of reuterin with some
LAB-bacteriocins against Gram-positive pathogens, but the mode of
action of the combinations is not known. Reuterin, which might
inhibit the activity of ribonucleotide reductase and thioredoxin
(Vollenweider & Lacroix, 2004), and LAB-bacteriocins, causing
dissipation of the proton motive force (Bruno & Montville, 1993), do

J.L. Arqus et al. / Food Control 22 (2011) 457e461

not appear to act on the same cellular target. Nevertheless, the

multiple cell damages caused by the combination of the two antimicrobials would prevent the development of resistance, and
would require much higher energy cost for the repair, that may
nally lead to the cell death. On the contrary, LAB-bacteriocins did
not increase the rate of reductions observed for reuterin on the
Gram-negative pathogens tested. These results suggest that reuterin does not permeate the outer membrane of Gram-negative
microorganisms and bacteriocins are not able to reach the target
cytoplasmic membrane.
The combination of reuterin with nisin in milk at refrigeration
temperatures led to the complete elimination of L. monocytogenes
and S. aureus, two Gram-positive pathogens of major concern for
the dairy industry. Thus, the addition of both biopreservatives
could be exploited in the control of undesirable pathogens potentially present before or during the manufacture and storage of dairy
products or other refrigerated foods.
Acknowledgements
This work was supported by projects RM2004-00013-00-00 and
AGL2007-65235-C02-01.
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