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microorganisms as well as an efficient antioxidant agent that
prevents browning and spoilage of some foods (Facio and
Warner, 1990). However, due to possible health risks for
Antimicrobial activity of nisin against Oenococcus oeni
and other wine bacteria
Beatriz Rojo-Bezares, Yolanda Sáenz, Myriam Zarazaga, Carmen Torres, Fernanda Ruiz-Larrea⁎
Department of Food and Agriculture, Faculty of Science, University of La Rioja, Av. Madre de Dios 51, 26006 Logroño, Spain
Received 1 March 2006; received in revised form 7 September 2006; accepted 10 December 2006
Nisin is a bacteriocin used against food spoilage bacteria. Sulphur dioxide is a potent antioxidant as well as an antimicrobial agent widely used
in the wine industry. In this study we describe the effect of these important antibacterial agents on the growth of a collection of 64 lactic acid
bacteria (23 Oenococcus, 29 Lactobacillus, 3 Leuconostoc and 9 Pediococcus), 23 acetic acid bacteria and 20 yeast isolates, most of them
recovered from wine. Minimal inhibitory concentrations (MIC) and minimal bactericide concentrations of nisin, potassium metabisulphite and
ethanol were determined. Nisin MIC50values for the tested isolates were as follows: 0.024, 12.5, 200 and ≥400 μg/ml for oenococci, lactobacilli–
pediococci–leuconostoc, acetic acid bacteria and yeasts, respectively. Synergistic effects on bacterial growth inhibition were observed, and
potassium metabisulphite MIC50values decreased from one to three orders of dilution when it was combined with subinhibitory concentrations of
nisin in the growth media. This effect was observed in all lactic acid bacteria species of our study. Significant differences in nisin sensitivity were
observed between Gram-positive and Gram-negative bacteria, and between Oenococcus oeni and other species of lactic acid bacteria. It is
concluded that appropriate combinations of nisin and metabisulphite could control the growth of spoilage bacteria in wine and therefore allow a
decrease in the levels of sulphur dioxide currently used by the wine industry.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Nisin; Bacteriocin; Oenococcus oeni; Lactic acid bacteria; Acetic acid bacteria; Wine; Preservatives; Metabisulphite
Nisin is a commercially available bacteriocin of the
lantibiotic group, which contains 35-amino acids and has
been approved in more than 40 countries for use in products
such as cheese, beer and canned foods (Carl et al., 2004). Nisin
has been reported to primarily act upon the cytoplasmic
membrane of Gram-positive bacteria, including Staphylococ-
cus aureus and Listeria monocytogenes and has become the
most thoroughly studied and characterised lactic acid bacteria
(LAB) produced antimicrobial. Sulphur dioxide is used as a
chemical preservative to control the growth of unwanted
sulphite-sensitive individuals, there is an ever-increasing
consumer demand to reduce sulphur dioxide levels in foods
and beverages. During the process of wine making, sulphur
dioxide is added to the prefermented grapes and to the end
product, to prevent unwanted microbial growth. It also acts as a
reducing agent and maintains the benefits of antioxidant
properties of the polyphenols of wine (Oliveira et al., 2002).
The objective of this study was to investigate the possibility
of controlling bacterial growth during wine making and
preservation by the combined use of sulphur dioxide and
subinhibitory concentrations of nisin, so that sulphur dioxide
levels could be reduced to minimal levels. In the present study
we determined minimal inhibitory concentration (MIC) and
minimal bactericide concentration (MBC) of ethanol, metabi-
sulphite and food-grade nisin for a selected number of LAB,
acetic acid bacteria (AAB) and yeast isolates of enological
importance. We tested these three antimicrobial agents alone
and in combination.
International Journal of Food Microbiology 116 (2007) 32–36
⁎Corresponding author. Tel.: +34 941 299749; fax: +34 941 299721.
E-mail address: email@example.com (F. Ruiz-Larrea).
0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
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YPD broth was used for yeasts. MIC was defined as the smallest
amount of antimicrobial agent needed to totally inhibit the
growth of the bacteria strain after incubation for 48 h. MBC was
determined as the minimal concentration of the antimicrobial
agent that killed more than 99.9% of the initial inoculum after
incubation from 48 to 72 h. MIC50and MIC90were defined as
the MIC that inhibited 50% and 90%, respectively, of the tested
microorganisms. Analogous definitions were applied to MBC50
2. Materials and methods
2.1. Bacteria and yeast strains
64 LAB, 23 AAB and 20 yeast isolates were included in this
study. Most LAB (62 out of 64) and AAB isolates (20 out of 23)
were recovered from wine, nine yeast isolates were also obtained
from wine and musts, and the remaining isolates were obtained
from the Spanish Culture Type Collection (CECT), the collection
food (two isolates) as shown in Table 1. Bacterial species
identification was performed according to previously recom-
(Quere et al., 1997; Zapparoli et al., 1998), and other LAB and
AAB species were confirmed by sequencing PCR amplicons of
yeasts were identified by sequencing their corresponding
amplicons of the 26s rDNA variable region (Kurtzman and
Robnett, 1998). Sequence analysis and comparison were
performed using the BLAST program.
2.2. Culture and growth conditions
LAB, except O. oeni, were cultivated for 48 h onto MRS agar
atmosphere containing 5% CO2. O. oeni was cultivated for 3–
4 days onto MLO–agar plates (35 g/l MLO, 15 g/l agar, 1 ml/
l polisorbate 80, 100 ml/l tomato serum) (Scharlau Chemie S. A.)
at 30 °C under strict anaerobic conditions (anaerobic system
BR038B, Oxoid Ltd., Basingstoke, England) (7–10% final CO2
concentration). AAB were cultivated for 48 h onto mannitol agar
plates [25 g/l n-mannitol (Panreac Química S.A., Barcelona,
Spain), 5 g/l yeast extract (Scharlau Chemie S. A.), 3 g/l peptone
and 15 g/l agar (Difco, Becton, Dickinson and Co., Le Pont de
Claix, France)]. Yeasts were cultivated for 24 h on YPD agar
[10 g/l yeast extract (Scharlau Chemie S. A.), 20 g/l peptone
(Difco, Becton, Dickinson and Co.), 20 g/l glucose (Panreac
Química S.A.), 20 g/l agar (Difco, Becton, Dickinson and Co.)].
2.3. Determination of minimal inhibitory concentrations and
minimal bactericide concentrations
MICs were determined by microtiter dilution method, using
serial double dilutions of the antimicrobial agents and initial
inocula of 5×105CFU/ml for all the studied microorganisms.
Bacterial growth was determined by absorbance at 600 nm.
MRS broth was used for LAB, except for O. oeni that was
assayed in MLO broth. Mannitol broth was used for AAB, and
and MBC90. Statistical analysis was performed by the non-
parametric U Mann–Whitney test.
Nisin stock solution (800 μg/ml) was prepared dissolving
Bacteria and yeasts included in the study
(number of strains)
LAB (n=64) Lactobacillus
2 Yeast (n=20)
aLAB: lactic acid bacteria; AAB: acetic acid bacteria.
bCECT: Spanish Culture Type Collection; IFI: Spanish Institute of Industrial
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3. Results and discussion
prior to use in experiments. Nisin concentrations were tested in
the concentration range from 0.012 to 400 μg/ml (double
dilutions). Potassium metabisulphite (Scharlau Chemie S. A.)
was tested in the concentration range from 12.5 to 12,800 μg/ml
(double dilutions) at pH 3.5, except for O. oeni isolates, which
Química S.A.) was diluted in sterile water to obtain final
assay. In experiments combining two antimicrobials, the
subinhibitory concentrations that were utilised were as follows:
ethanol subinhibitory concentration of 6% for LAB and yeasts,
3.1. Ethanol antimicrobial activity
The LAB isolates (including O. oeni) showed resistance to
ethanol under the experimental growth conditions described
above, as expected from bacteria isolated from wine. Ethanol
MIC90values were ≥10% (vol/vol). The range of MICs for
ethanol was between 6 and N20% (Table 2). It is worth noting
that two L. plantarum and one L. hilgardii isolates showed MIC
values of 18%, and five isolates (one L. hilgardii, one
L. paracasei, one Pediococcus parvulus, and two L. plantarum
isolates) showed ethanol MIC values higher than 20%. AAB
behaviour in the presence of ethanol was rather uniform and the
majority of the isolates (15 out of 23) showed ethanol MIC
values between 4 and 8%. Six Acetobacter isolates and
remarkably two Gluconobacter oxydans isolates showed MIC
values for ethanol of 10%, which is the highest reported
threshold for AAB ethanol resistance (Ribereau-Gayon et al.,
1998). Ethanol resistance of the yeast collection (20 isolates)
was in the range from 4 to N20% as shown in Table 2. Only
three isolates showed the highest ethanol resistance, with MIC
values of N20%. They were the species of Rhodotorula rubra,
Saccharomyces cerevisiae and Brettanomyces sp.
3.2. Metabisulphite antimicrobial activity
Our results showed that metabisulphite susceptibility of
O. oeni was higher than that of the other wine LAB species
(pb0.001), as it had been previously reported (Romano and
Values of minimal inhibitory concentrations and minimal bactericide concentrations of the tested antimicrobial agents in the series of lactic acid bacteria, acetic acid
bacteria and yeasts included in the study
MIC (μg/ml)MBC (μg/ml)
aLAB⁎: lactic acid bacteria except O. oeni; AAB: acetic acid bacteria.
bEthanol concentration is expressed as % (vol/vol).
cND: not determined.
dEthanol subinhibitory concentrations were as follows: 6% for LAB⁎, O. oeni and yeasts, and 3% for AAB.
eMetabisulphite subinhibitory concentration was 50 μg/ml for all tested microorganisms.
fNisin subinhibitory concentrations were as follows: 0.39 μg/ml for LAB⁎, 0.01 μg/ml for O. oeni and 1.5 μg/ml for AAB.
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MIC50value for AAB was 200 μg/ml either in the absence or in
the presence of subinhibitory concentrations of ethanol in the
growth medium (Table 2). All of our AAB wine isolates,
belonging to both genera Acetobacter and Gluconobacter,
showedthe same susceptibility to nisin and no major differences
were observed among their MIC values (data not shown).
Regarding yeasts, nisin had no effect on the tested strains and
nisin MIC values were out of range (≥400 μg/ml) both in the
Suzzi, 1994). Metabisulphite MICs were in the range ≤12.5–
100 μg/ml for O. oeni, and 50–N12,800 μg/ml for the other
LAB species (Table 2), with Leuconostoc mesenteroides being
the most resistant species of our collection (MICN12,800 μg/
ml). When a subinhibitory concentration of ethanol (6%) was
added to the growth medium, O. oeni strains became more
resistant to metabisulphite, and thus MIC50value increased
from 50 μg/ml in the absence of ethanol, to 100 μg/ml in the
presence of 6% ethanol (pb0.001) as shown in Table 2. In
contrast, the other LAB isolates of our study demonstrated a
decreased resistance to metabisulphite in the presence of 6%
ethanol (pb0.001), as the results for MIC50and MIC90indicate
(Table 2). A previous report by our group had indicated a clear
activation of O. oeni growth in the presence of up to 7% ethanol
in the growth medium (G-Alegría et al., 2004), and this
activation could account for the increased resistance to
metabisulphite shown by our O. oeni isolates when grown in
the presence of low concentrations of ethanol.
The AAB strains tested showed lower resistance to
metabisulphite than LAB (pb0.001). Thus MIC50values were
one to three orders of dilution lower than those shown by LAB
(Table 2). A former study on AAB resistance to sulphur dioxide
(Du-Toit and Lambrechts, 2002) focused mainly on grape juice
AAB. A later study (Du-Toit et al., 2005) reported the inhibitory
effect of sulphur dioxide on the viability of one Acetobacter
pasteurianus strain. A threshold minimal level to prevent
growth of this strain was 0.8 μg/ml of free molecular sulphur
The metabisulphite MIC50 value for yeast isolates was
100 μg/ml and the MIC ranged from 50 to 1600 μg/ml
(Table 2). These values are quite high indicating that yeasts
have a higher tolerance to metabisulphite than O. oeni. When a
subinhibitory concentration of ethanol was added to the
medium (6%) no effect was observed on yeast growth as the
metabisulphite MIC50value was not changed (Table 2). This
could be expected as yeast strains are able to grow in wine and
adapt to some concentration of ethanol in their growth medium.
3.3. Nisin antimicrobial activity
Our results revealed nisin as an efficient antimicrobial agent
against wine LAB, with a MIC50value of 0.024 μg/ml for
O. oeni, and 12.5 μg/ml for the other wine LAB species
(Table 2). O. oeni was clearly the most susceptible species to
nisin (pb0.001). Nisin inhibitory effect was slightly enhanced
in the presence of low concentrations of ethanol (6%) and thus,
MIC50values decreased one order of dilution for O. oeni in the
presence of ethanol.
AAB isolates showed higher nisin MICs than LAB. Nisin
absence or presence of ethanol in the growth medium (Table 2).
These results were as expected from Gram-negative bacteria
and yeasts, and the fact that nisin has been described to act upon
Gram-positive bacteria (De Vuyst and Vandamme, 1994).
To our knowledge this is the first systematic study of the
lantibiotic nisin as inhibitor of wine LAB growth, and
specifically of O. oeni strains. Bauer et al. (2002) described
nisin inhibitory effect on the formation of O. oeni biofilms onto
stainless steel surfaces and reported the successful use of nisin,
as well as pediocin PD-1 and plantaricin 423, to remove
biofilms from stainless steel surfaces. Pediocin PD-1 was as
well reported as a method to control O. oeni growth in wine,
although no combinations with metabisulphite were evaluated
(Bauer et al., 2003). Nisin effect on a wide range of other LAB
species has been extensively studied, and Carl et al. (2004)
reported considerable variations in nisin sensitivity among
distinct clones of the same Lactobacillus species. The combined
effect of nisin together with lysozyme against LAB was studied
by Chung and Hancock (2000), but no O. oeni, nor other wine
LAB bacteria were included in their study. They reported a
synergistic effect of both nisin and lysozyme and suggested the
benefits of using mixtures of both to prevent food spoilage.
Yurdugül and Bozoglu (2002) reported growth inhibition of
wine LAB strains by some bacteriocin-like inhibitory sub-
stance, nevertheless, they did not study O. oeni and their report
restricted to growth inhibition of two Lactobacillus spp. strains.
3.4. Combined effect of metabisulphite and nisin
Our results show a synergistic effect on LAB growth
inhibition by nisin and metabisulphite: in the presence of
subinhibitory concentrations of nisin (0.01 μg/ml for O. oeni
and 0.39 μg/ml for the other LAB), metabisulphite MIC50value
decreased from 50 to 25 μg/ml in the case of O. oeni, and from
200 to 25 μg/ml in the case of the other wine LAB (pb0.001). It
should be pointed out that nine O. oeni isolates did not survive
in the presence of subinhibitory concentration of nisin (0.01 μg/
ml nisin), and therefore, MIC50value was obtained only from
14 O. oeni isolates.
In summary, we determined the MIC of ethanol, metabisul-
phite and nisin, as well as different binary combinations of these
antimicrobials, for 64 LAB isolates, 23 AAB and 20 yeast
isolates from our collection. Our results showed a distinct
behaviour of O. oeni species when compared to the other wine
LAB species, in that it demonstrated a much higher sensitivity
to nisin than the other LAB species, as well as higher sensitivity
to metabisulphite. In contrast, O. oeni species was activated by
the presence of low concentrations of ethanol (6%), which
increased its resistance to metabisulphite. Our results showed a
synergistic effect of nisin and metabisulphite on growth
inhibition of wine LAB strains, and suggest that appropriate
mixtures of lower concentrations of metabisulphite than what
are currently utilised, together with subinhibitory concentra-
tions of nisin (≤0.39 μg/ml) could effectively prevent LAB
growth in wines and constitute a new method for wine
preservation. It should be noted that at present the addition of
nisin to wine is not approved. However, nisin could provide a
35B. Rojo-Bezares et al. / International Journal of Food Microbiology 116 (2007) 32–36
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safe partial replacement for sulphur dioxide which is currently
added to must and wine and thus contribute to significantly
lower levels of sulphur dioxide in wine.
This work was supported by grant AGL2002-02293 of the
Spanish Ministry of Education and Science and FEDER of the
European Community, and by the I.N.I.A. grant VIN03-043-
C3-2. We acknowledge the valuable help of Prof. Fernando
Antoñanzas and Reyes Lorente of the Department of Economy
and Management of the University of La Rioja, for carrying out
the statistical analysis of data. B. Rojo-Bezares was supported
by a doctorate grant of the Spanish Ministry of Education and
Science (ref. BI-UR-03/16586.840), and Y.Sáenz had a contract
of technician supported by grant PTA-2003-02-00002 of the
Spanish Ministry of Education and Science.
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