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International Journal of Food Microbiology 121 (2008) 123 – 138

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Review
Application of bacteriocins in vegetable food biopreservation
Luca Settanni 1 , Aldo Corsetti ⁎
Dipartimento di Scienze degli Alimenti, Sezione di Microbiologia Agro-Alimentare ed Ambientale,
Università degli Studi di Teramo, V. C.R. Lerici 1, 64023 Mosciano Sant'Angelo (TE), Italy

Received 15 May 2007; received in revised form 15 August 2007; accepted 3 September 2007

Abstract

Bacteriocins are generally recognized as “natural” compounds able to influence the safety and quality of foods. In the past years, a lot of works
have been aimed to the detection, purification and characterisation of bacteriocins, as well as to their use in food preservation strategies. A list of
review articles dealing with the application of bacteriocins to the protection of foods of animal origin is also available in literature, but it lacks for a
summary on the utilization of bacteriocins in vegetable foods. These biopreservatives can be used in a number of ways in food systems and this
paper mainly focuses on the state-of-the-art application of bacteriocins from lactic acid bacteria (LAB) to promote the microbial stability of both
fermented and non-fermented vegetable food products using bacteriocinogenic strains as starter cultures, protective cultures or co-cultures and the
employment of pure bacteriocins as food additives. In addition, applications of bacteriocins from non-LAB are also reviewed. The scopes of future
directions of research are summarised.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Bacteriocins; Biopreservation; Food additives; Vegetable foods

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
2. Classification, determination of activity, mode of action and vegetable matrices of isolation of bacteriocins . . . . . . . . . . . . 125
3. Application of bacteriocinogenic LAB strains as co-culture, protective or starter cultures . . . . . . . . . . . . . . . . . . . . . . 126
3.1. Fermented vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
3.1.1. Table olives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
3.1.2. Sourdough . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3.1.3. Miso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3.1.4. Sauerkrauts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3.2. Non-fermented vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
3.2.1. Refrigerated pickles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
3.2.2. Mungbean sprouts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
4. Interactions between bacteriocins and undesired bacteria in model systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
5. Application of bacteriocins from LAB as food additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.1. Fermented vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.1.1. Kimchi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.1.2. Cider . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

⁎ Corresponding author. Tel.: +39 0 861 266896; fax: +39 0 861 266915.
E-mail address: acorsetti@unite.it (A. Corsetti).
1
Present address: Istituto Agrario di San Michele all'Adige (IASMA), Centro Sperimentale-Dipartimento Qualità Agro-Alimentare, Unità Microbiologia e
Tecnologie Alimentari, V. E. Mach 1, 38010 San Michele a/A (TN), Italy.

0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2007.09.001
124 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138

5.2. Non-fermented vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

5.2.1. Mashed potatoes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.2.2. Soy milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.2.3. Fruit and vegetable juice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
5.2.4. Canned vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
5.2.5. Fresh-cut products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
5.2.6. Zucchini purée . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
6. Combined effect of pure bacteriocins and bacteriocin-producing strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
7. Application of non-LAB bacteriocinogenic strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
8. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

1. Introduction Biopreservation refers to the extension of the shelf-life and

Nowadays, consumers are particularly aware of the health
concerns regarding food additives; the health benefits of
“natural” and “traditional” foods, processed with no added
chemical preservatives, are becoming more and more attractive.
Thus, in the last decades, due to consumer demand for higher
quality and naturalness of foods, as well as strict government
requirements for guarantees of food safety, food producers have
faced conflicting challenges. Chemical additives have generally
been used to combat specific microorganisms. Historically, the
birth of modern microbiology is dated back to 1929 when
Alexander Fleming discovered the antibiotic properties of
penicillin, but the need of controlling pathogenic and spoilage
microorganisms is dated back to the previous century since the
studies of Louis Pasteur.
In the case of fermented foods, lactic acid bacteria (LAB)
have been essential for millennia. Thanks to some of their
metabolic properties, LAB are generally employed because they
significantly contribute to the flavour, texture, and, in many
cases, to the nutritional value of the food products (McKay and
Baldwin, 1990). LAB play a defining role in the preservation
and microbial safety of fermented foods (Caplice and Fitz-
gerald, 1999), thus promoting the microbial stability of the final
products of fermentation (Mensah et al., 1991). Protection of
foods is due to the production of organic acids, carbon dioxide,
ethanol, hydrogen peroxide and diacetyl (Atrih et al., 2001; De
Vuyst and Vandamme, 1994), antifungal compounds such as
fatty acids (Corsetti et al., 1998) or phenyllactic acid
(Lavermicocca et al., 2000), bacteriocins (De Vuyst and
Vandamme, 1994) and antibiotics such as reutericyclin (Höltzel
et al., 2000).
However, growth of LAB has been also found to show
positive effects in non-fermented foods, e.g. vacuum-packaged
meat products (Castellano et al., 2004; Vold et al., 2000).
Actually, in that specific case, LAB such as Carnobacterium
spp., Lactobacillus spp. and Leuconostoc spp. are the main
spoilage organisms (Borch et al., 1996; Korkeala and Björkroth,
1997; Nychas and Drosinos, 2000), but a selective growth
promotion of LAB capitalising on their ability to control meat-
borne pathogens with a preferential growth of benign strains
would minimise their spoilage effects.
improvement of the safety of foods using microorganisms and/
or their metabolites (Ross et al., 2002). The term “bacteriocin”
was coined in 1953 to define colicin produced by Escherichia
coli. Like LAB, also bacteriocins have been consumed for
millennia by mankind as products of LAB and, for this reason,
they may be considered as natural food ingredients. As reported
by Cotter et al. (2005) “bacteriocins can be used to confer a
rudimentary form of innate immunity to foodstuffs”. Bacter-
iocins are ribosomally synthesized, extracellularly released low-
molecular-mass peptides or proteins (usually 30–60 amino
acids) which have a bactericidal or bacteriostatic effect on other
bacteria (Klaenhammer, 1988; Tagg et al., 1976), either in the
same species (narrow spectrum) or across genera (broad
spectrum) (Cotter et al., 2005). Thanks to their typical
characteristics, bacteriocins, although showing antibiotic prop-
erties, are not termed antibiotics in order to avoid confusion and
concern with therapeutic antibiotics that can determine allergic
reactions in humans (Cleveland et al., 2001). Since the
discovery of the first bacteriocin by Gratia in 1925 (Garneau
et al., 2002), bacteriocin production has been found in nume-
rous species of bacteria, among which, due to their “generally
recognised as safe” (GRAS) status, LAB have attracted great
interest in terms of food safety. In fact, LAB bacteriocins enjoy
a food-grade and this offers food scientists the possibility of
allowing the development of desirable flora in fermented foods
or preventing the development of specific unwanted (spoilage
and pathogenic) bacteria in both fermented and non-fermented
foods by using broad- and narrow-host-range bacteriocins,
respectively. De Vuyst and Vandamme reported in 1994 that the
majority of bacteriocins described at the time of writing were
those produced by Lactobacillus spp., followed by Enterococ-
cus, Pediococcus and Leuconostoc spp.; nowadays, other
bacteriocins from the group of LAB, such as those produced
by Lactococcus, Streptococcus and Carnobacterium have
been described. However, the only bacteriocin approved for
utilization as a preservative in many foods by the US Food
and Drug Administration is nisin (Federal Register, 1988),
commercially available as Nisaplin™ (Danisco, Copenhagen,
Denmark). Nisin is the first antibacterial polypeptide found in
LAB (Rogers, 1928); at the time of discovery, the producer
strains were identified as Streptococcus lactis [later classified as
 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138
Lactococcus lactis (Schleifer et al., 1985)]. Another commer-
cially produced bacteriocin is pediocin PA-1 produced by Pe-
diococcus acidilactici and marketed as ALTA™ 2431 (Kerry
Bioscience, Carrigaline, Co. Cork, Ireland).
Regarding the application of bacteriocin-producing starter
strains in food fermentation, the major problem is related to the
in situ antimicrobial efficacy which can be negatively
influenced by various factors, such as binding of the
bacteriocins to food components (fat or protein particles) and
food additives (e.g. triglyceride oils), inactivation by proteases
or other inhibitors, changes in solubility and charge, changes in
the cell envelope of the target bacteria (Aesen et al., 2003;
Degnan and Luchansky, 1992; Gänzle et al., 1999; Jung et al.,
1992; Leroy and De Vuyst, 1999). Furthermore, unlike their
general behaviour represented by being active against closely
related species, some LAB bacteriocins have also been found to
be active against Gram-negative bacteria (Abriouel et al., 2001;
Caridi, 2002; Ko and Ahn, 2000; Messi et al., 2001; Todorov
and Dicks, 2005a), hence the interest in their application is also
forwarded to the gastrointestinal tract. Regarding food safety,
bacteriocin-producing LAB cultures that are not effective
against Gram-negative bacteria, protected by an outer mem-
brane, should not be used as the primary processing step or
barrier to prevent the growth or survival of pathogens, but rather
they can be used in hurdle technology strategies to reduce the
likelihood of food-borne disease (Deegan et al., 2006).
The most recent food application of bacteriocins encompass
their binding to polymeric packaging, a technology referred to
as active packaging. This kind of packaging, defined as an
intelligent or smart system involving interactions between
package or package components and food or internal gas
atmosphere, fully encounters consumer demand for prolonged
shelf-life of foods (Labuza and Breene, 1989). Due to this
proposal, some patents have been issued (Daeschel and
McGuire, 1995; Ming et al., 1997; Siragusa et al., 1999;
Wilhoit, 1996, 1997).
Up to date, numerous recent review articles focused on the
classification (basically based on three classes), biochemical
and genetic characterisation and the mode of action of LAB
bacteriocins, as well as on some of their (mainly of animal
origin) food applications (Breukink, 2006; Chen and Hoover,
2003; Cleveland et al., 2001; Cotter et al., 2005; Deegan et al.,
2006; Drider et al., 2006; Ennahar et al., 1999; Garneau et al.,
2002; Guinane et al., 2005; Messens and De Vuyst, 2002;
Rodgers, 2001, 2004; Ross et al., 2002; Saito, 2004; Työppönen
et al., 2003; Zhu et al., 2005), but literature lacks of updated
reviews dealing with the use of bacteriocins or bacteriocin-
producing strains for the protection of vegetable food products.
Bacteriocin potential for enhancing food safety and prolonging
the shelf-life of final products is routinely investigated and is
still under study. In general, the focus of studies on food safety
using bacteriocins as preservatives follows this scheme:
isolation of bacteria from raw materials or final products;
screening for bacteriocin production; characterisation of
bacteriocins; production of bacteriocins in food model systems;
in situ application. In this review, the application of bacteriocins
as agents of food biopreservation is analysed in the context of
125
foods (including drinks and beverages) of vegetable origin, thus
providing a complete and updated overview of this topic.
2. Classification, determination of activity, mode of action
and vegetable matrices of isolation of bacteriocins
Bacteriocins most frequently studied are those from LAB.
They have been classified in three classes on the basis of
common, mainly structural, characteristics (Nes et al., 1996).
Many review articles have dealt with the classification of LAB
bacteriocins as well as their main characteristics (please see
Introduction section for references cited). In general, bacter-
iocins target the cell envelope, and with the exception of the
larger proteins (N 20 kDa) that degrade the murein layer (e.g.
lysins and muramidases), use non-enzymatic mechanisms to
cause the depolarization of the target cell membrane and/or
inhibit cell wall synthesis (Abee et al., 1995). When the
presumptive bacteriocins are under study they should be
referred to as bacteriocin-like inhibitory substances (BLIS)
until their amino acid sequence determination.
The traditional determination of the antagonism of a
bacteriocin-producing strain against a sensitive strain, generally
indicated as “producer” and “indicator”, respectively, can be
performed in different ways (see Parente and Ricciardi, 1999 for a
review). In general, the methods most frequently used are the
agar-spot deferred test and the well diffusion assay (Schillinger
and Lücke, 1989). In the first method, colonies of the strains to be
tested for bacteriocin production are allowed to grow, generally
overnight, on the surface of the optimal agar medium. The
indicator strain is inoculated into the optimal soft agar medium
and poured on the plate where growth of the producers occurred.
After incubation, inhibition is scored positive in the presence of a
detectable clearing zone around the colony of the producer strain.
For the well diffusion assay, the agar base medium is overlaid with
soft agar medium containing the indicator strain, as above.
Thereafter, wells are cut into the agar and the cell-free supernatant
of the potential producer strains is placed into each well. In the
latter test, the inhibitory effect of lactic acid and/or H2O2, is
eliminated by the adjustment of supernatants to neutral pH and
treatment with catalase, respectively. In general, prior to
incubation, plates are refrigerated for a while to allow the radial
diffusion of the compounds contained in the supernatant.
Bacteriocin activity of the supernatants is commonly evaluated
by the critical dilution assay of Barefoot and Klaenhammer
(1983) defined as the reciprocal of the highest dilution showing
clear inhibition of the indicator strains and is expressed as activity
units per millilitre (AU/ml). Agar based antagonistic assays may
be replaced by quicker tools for bacteriocin detection: Rose et al.
(1999) developed a rapid method to examine culture supernatants
for the presence of some bacteriocins, such as brochocins A and
B, enterocins A and B, nisin and pediocin, by means of the matrix-
assisted laser desorption/ionization time-of-flight mass spectrom-
etry (MALDI-TOF MS). Furthermore, PCR methods have also
been used to detect genes coding for bacteriocins in pure cultures
(Bennik et al., 1997; Foulquié Moreno et al., 2003; Garde et al.,
2001; Moreno et al., 2002) and fermentation broth (Mugochi
et al., 2001).
126 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138

Fig. 1. Bactericidal effect of BLIS WGJ28.1 (•) and bacteriostatic effect of BLIS WGJ36.1 (▵), WGJ6.1 (□), WGW33.2 (○), WGJ5.1 (▴) and WGJ1.2 (◼) produced
by E. faecium WGJ28.1, P. pentosaceus WGJ36.1 and WGJ6.1, E. faecium WGW33.2 and P. pentosaceus WGJ5.1 and WGJ1.2, respectively, isolated from wheat
(Triticum durum) kernels and non-conventional flours against L. monocytogenes ATCC 19114T as determined in BHI for 72 h by means of Bioscreen C MBR (Growth
Curves AB Ltd., Piscataway, NJ, USA) (from Corsetti et al., in press). Control (broken line) is represented by growth of L. monocytogenes ATCC 19114T in BHI added
with concentrated supernatant of strain Enterococcus casseliflavus WGW22.1 (which does not produce any antimicrobial compound).

Bacteriocins have generally a cationic character and easily
interact with Gram-positive bacteria that have a high content of
anionic lipids in the membrane determining the formation of
pores (see Cleveland et al., 2001 and Chen and Hoover, 2003
for reviews). Pores in the cytoplasmic membrane clearly affect
the energetic status of the cell, i.e. dissipation of proton motive
force causes an arrest of ΔpH and Δψ (transmembrane
electrical potential) dependent (e.g. transport) processes while
certain bacteriocins cause ATP efflux (Moll et al., 1999). A
bacteriocin producer protects itself against its own antimicrobial
compound by means of a system referred to as immunity, which
is expressed concomitantly with the antimicrobial peptide (Nes
et al., 1996). The mode of action of bacteriocins can be
bactericidal or bacteriostatic determining death or extension of
lag phase, respectively (Fig. 1).
Bacteriocins or BLIS from LAB associated to or found in
vegetable food matrices are reported in Table 1.
3. Application of bacteriocinogenic LAB strains as
co-culture, protective or starter cultures
3.1. Fermented vegetables
In general, starter cultures for fermented foods are mainly
selected on the basis of their technological potential, such as acid
and exopolysaccharides production and contribution to flavour
development. Moreover, other features are being taken into
account, e.g. the ability to improve nutritional quality and to
reduce anti-nutritional and/or toxic compounds of raw materials
and probiotic attitude. The production of antimicrobial activities
may constitute a secondary effect of starter cultures while it
represents the unique characteristic requested for protective
cultures or co-cultures which must not alter or negatively interfere
with the wanted fermentation process and should not affect the
final product's sensorial properties. Applications of protective
cultures and co-cultures are considered as additional safety factors
warranting the microbiological stability of the resulting foods
reducing risks of growth and survival of food-borne pathogens
and food spoilage organisms (Holzapfel et al., 1995).
3.1.1. Table olives
In the process of selection of starter cultures for olive
fermentation, antimicrobial production is a desired character.
Jiménez-Díaz et al. (1993) and Franz et al. (1996) approached the
selection of potential starter cultures focusing on the screening of
table olive-associated LAB for bacteriocin production: the most
interesting bacteriocin-producing strains were Enterococcus
faecium BFE 900 (Franz et al., 1996) and Lactobacillus
plantarum LPCO10 (Jiménez-Díaz et al., 1993).
L. plantarum LPCO10 was then included in a project aimed
at optimizing the fermentation of green olives (Leal et al., 1998;
Leal-Sánchez et al., 2003; Ruiz-Barba et al., 1994). Firstly, the
authors evaluated the ability of strain LPCO10 to dominate the
epiphytic microflora of fermented olives and they assumed that
such a dominance was due to the production of bacteriocin
(Ruiz-Barba et al., 1994). L. plantarum LPCO10 and its
non-bacteriocin-producing, bacteriocin-immune derivative,
L. plantarum 55-1 were evaluated separately for growth and
persistence in natural green olive fermentations. Spontaneously
occurring lactobacilli and lactic acid bacterial cocci of brines
were sensitive to L. plantarum LPCO10 bacteriocin. The final
concentration of lactic acid in brines inoculated with
L. plantarum LPCO10 was higher than that of brines inoculated
with L. plantarum 55-1 or non-inoculated, indicating that
L. plantarum LPCO10 could be a good starter culture for
the lactic acid fermentation of Spanish-style green olives.
 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138
Subsequently, a medium resembling the natural environment of
L. plantarum LPCO10, named “olive juice broth” (OJB), was
obtained from green olives and it was optimal for bacteriocin
production by the above strain (Leal et al., 1998). In that work,
unlike L. plantarum 55-1, L. plantarum LPCO10 was able to
dominate over a L. plantarum strain (128/2) of olive origin
used as indicator. On the basis of those results, the authors
suggested that the OJB could be a valuable substitute for the
olive fermentation brine for successful Spanish-style olive
fermentations with strain LPCO10. A third piece of work was
aimed to better investigate on the fermentation profile of green
olives obtained with L. plantarum LPCO10 by means of a
factorial study (Leal-Sánchez et al., 2003). In all different
conditions, including different salt concentration, inoculum
size (CFU/ml), inoculum carrier, inoculation time, homogeni-
zation, type of acid used to decrease the basic pH of brines, and
initial pH, L. plantarum LPCO10 was able to dominate over the
natural population of LAB and led to a faster decrease of pH
and a faster acidification than the spontaneous process during
the first 25 days after brining. The authors determined that, in
order to have the best final product, L. plantarum LPCO10
should be suspended at 107 CFU/ml in brine and the brine
should contain 4% w/v of NaCl and be adjusted for its initial pH
with acetic acid.
3.1.2. Sourdough
Corsetti and Settanni (2007) have reviewed the contribution
of bacteriocins from LAB to the stability of sourdoughs over
consecutive refreshments, reporting on the in situ activity of
BLIS 2MF8 from L. pentosus 2MF8 (Corsetti et al., 2004),
lacticin 3147-like bacteriocin from L. lactis M30 (Settanni
et al., 2005) and amylovorin L471 from Lactobacillus
amylovorus DCE 471 (De Vuyst et al., 2004). In addition,
Menteş et al. (2007) recently published a paper dealing with the
effect of sourdoughs produced with L. plantarum LMO25 and
Lactobacillus alimentarius LMO7, previously characterised
for their inhibitory activity against rope-forming Bacillus
strains, in wheat bread. The addition of 15% or 20% low pH
(pH 3.5–4.0) sourdough to bread dough prevented the
generation of visual rope caused by Bacillus subtilis and Ba-
cillus licheniformis while sourdoughs with a higher pH (N4)
were not effective in preventing rope caused by both B. subtilis
and B. licheniformis at concentrations lower than 20%.
According to the authors, production of bacteriocins by the
starter strains may explain the rope inhibition in breads with
final pH values above 4.8 and they discussed their results as
consistent with prior data showing that the bacteriocins
produced by Lactobacillus strains have optimal activities at
pH 3.0–4.0 (Menteş et al., 2005).
3.1.3. Miso
Miso, a fermented soybean paste, is a very important
seasoning for Japanese and Asian cooking (Fukushima, 1981).
Rice-koji miso is prepared using a certain percentage of rice-
koji fermented by Aspergillus oryzae (Yamabe et al., 2007).
Rice miso has presented a big concern regarding the growth of
Bacillus spp. during koji fermentation which prevents the
127
development of the fermenting aspergilli and produces off-
flavours, thus, causing quality defects of the resulting rice miso
(Ebine, 1984) and the persistence of alive Bacillus spores. The
spores cause spoilage of processed foods seasoned with miso.
Kato et al. (1999) investigated on the inhibition of growth of
B. subtilis by nisin-producing lactococci in fermented soybeans.
From this proposal, nisin-producing L. lactis ssp. lactis
IFO12007, a strain that does not tolerate the presence of
NaCl, was used. Strain IFO12007 rapidly proliferated to more
than 109 cells/g in cooked soybeans, but it did not determine an
excessive pH decrease. However, the growth of B. subtilis,
inoculated at a concentration of 106 cells/g, was inhibited till
72 h. Thus, L. lactis ssp. lactis IFO12007, whose nisin activity
was determined to be 1.28 × 105 AU/g in cooked soybean, was
used as a starter culture to produce soybean miso. The problem
regarding over-acidification of miso by L. lactis was solved by
adding salt. Later, Kato et al. (2001) focused on the growth of
nisin-producing lactococci in cooked rice supplemented with
soybean extract to inhibit B. subtilis in rice miso. LAB growth
in cooked rice supplemented with soybean extract was not
negatively affected and no off-flavour or undesirable coloration
were generated. L. lactis ssp. lactis IFO12007 proliferated till
109 cells/g showing in situ nisin activity inhibiting bacilli,
including B. subtilis and it did not inhibit the growth of A.
oryzae during koji fermentation. The resulting rice miso,
obtained after 12 weeks of ageing, was characterised by
favourable acidity, taste and colour.
3.1.4. Sauerkrauts
The most common among the fermented Brassica (e.g.
cabbage) products is sauerkraut, which is manufactured either
by natural or controlled fermentation (Fleming et al., 1995) in
the presence of NaCl. The correct sequence of organisms is
essential in achieving a stable product with flavour and aroma
typical of sauerkraut (Pederson and Albury, 1969).
Harris et al. (1992) developed a paired starter culture system
for sauerkraut, consisting of the nisin-resistant Leuconostoc
mesenteroides NCK293 and the nisin-producing L. lactis
NCK401. The authors used a filter-sterilized cabbage juice
broth (CJB), previously demonstrated (Stamer et al., 1971) to be
a satisfactory experimental substitute for shredded cabbage. The
two strains were first evaluated separately and in combination
for growth and nisin production in a model sauerkraut
fermentation. The growth and survival of L. mesenteroides
were similar in the presence and absence of L. lactis. The growth
of L. lactis was reduced and the population decline was more
pronounced in the presence of L. mesenteroides. Nisin level was
almost constant over the 12-day test period. However, the
maximum cell populations and nisin levels achieved could be
altered by changing the initial cell ratios of L. mesenteroides and
L. lactis. Nisin level produced in mixed culture was sufficient to
delay the growth of the L. plantarum strain used as an indicator.
Although in a model system, the above work represents the
first application of a bacteriocin-producing strain as a co-
culture/protective culture to a vegetable food fermentation.
Furthermore, as highlighted by the authors themselves, that
research, from a microbiological point of view, offered an
128 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138

Table 1
Bacteriocin-producing bacteria isolated from vegetable raw materials or foods
 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138
Table 1 (continued )
N.R., not reported.
excellent model system for the study of mixed culture ecology.
They concluded that, at the time of writing, the lack of literature
available on the application of starter cultures for sauerkraut
production was, in part, due to the difficulties involved in
studying a natural mixed culture fermentation.
3.2. Non-fermented vegetables
When no fermentation is required, preservation with live
(protective) cultures provides the unique temperature-respon-
sive protection.
3.2.1. Refrigerated pickles
Refrigerated pickles are made from cucumbers packed into
brine with appropriate spices and salt. The product is not heat
processed. The shelf-life depends on the refrigeration temper-
ature and the presence of vinegar and preservatives such as
sodium benzoate: in general, about 3 weeks without any
addition to three months with vinegar and preservatives added
to the brine (Reina et al., 2005). Reina et al. (2005), in order to
characterise spontaneous LAB for their biopreservation poten-
tial, subjected refrigerated pickles to various thermal abuse (16,
25 and 30 °C) after storage for three weeks at 5 °C. A total of 10
isolates were found to be bacteriocin-producers, among which
three showed anti-Listeria activities. At the time of writing, the
authors showed the willingness to perform future trials of
minimally processed pickle products co-inoculated with Lis-
teria monocytogenes and LAB cultures producing bacteriocins.
3.2.2. Mungbean sprouts
Mungbean sprouts are commonly sold in modified atmosphere
packaging (MAP). As minimally processed food, the shelf-life of
this product mainly counts on the refrigeration at which it is
stored. However, the growth of L. monocytogenes may occur.
Bacteriocinogenic strains of LAB (Enterococcus mundtii ATO6
and Pediococcus parvulus ATO34 and ATO77) were investigated
for their ability to inhibit L. monocytogenes MAP mungbean
sprouts (Bennik et al., 1999). Firstly, E. mundtii ATO6 and
P. parvulus ATO34 and ATO77 were evaluated for their growth
and acid and bacteriocin production in APT broth and vegetable
agar medium at different temperatures and CO2 concentrations.
129
E. mundtii ATO6 was selected, since it showed a significantly
higher maximum specific growth rate than P. parvulus strains at
concentrations of CO2 relevant for storage of vegetables under
MAP. The latter strain was then evaluated for its potential to in
situ control the growth of L. monocytogenes LDCD861 and
LDCD1087. The interactions between the mundticin producer
E. mundtii ATO6 and L. monocytogenes strains, after spraying
10 ml of approximately 106–108 CFU/ml cell suspension per kg
of produce, was studied in fresh mungbean sprouts under 1.5%
O2/20% CO2/78.5% N2 at 8 °C. The treatment did not show the
same defining effect of the selected lactic acid bacterium on the
pathogen as observed on vegetable agar. However, the application
of pure mundticin (200 AU/ml) to the vegetables during a
washing step or a coating procedure with an alginate film was
successful against L. monocytogenes.
4. Interactions between bacteriocins and undesired bacteria
in model systems
Food model systems are artificial media that mimic the com-
position of natural food complex matrices or sterile food matrices
useful to study the basic interactions between microorganisms.
Foods are not sterile, thus the results that are retrieved by antag-
onistic assays can be influenced by a mixed microbial population.
Furthermore, it has to be noted that the heat-treatments necessary
to kill any microbial form in foods might modify the chemical
composition of the matrices used for bacterial inhibition test and
that the results can also be influenced by a different substrate
availability and/or a different interaction between antimicrobials
and food chemicals. However, since model systems have the
advantage of being sterile, it is easy to evidence any reciprocal
effect of the different species inoculated or the effect of anti-
microbials on the undesired microorganisms.
Jamuna et al. (2005) evaluated the antibacterial efficacy of
bacteriocin LABB and LABP from appam batter and vegetable
pickles Lactobacillus isolates, respectively, and nisin individ-
ually and in combination against several Gram-positive and
Gram-negative food spoilage and pathogenic organisms in a
liquid medium. The authors found that when employed
individually, nisin was a potent inhibitor of Clostridium
sporogenes while bacteriocins LABB and LABP were effective
130 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138
against L. monocytogenes and Staphylococcus aureus. Bacter-
iocins LABB and LABP in combination with nisin were more
potent than alone and these dual bacteriocin combinations were
also able to inhibit Gram-negative E. coli and Pseudomonas
spp. On the basis of these results, the authors addressed their
attention towards the inhibition of L. monocytogenes and
S. aureus in a vegetarian food model system represented by a
partially cooked vegetable pulav, prepared using rice, vegetable
and spices, packed in paper aluminium foil and polypropylene
pouches and autoclaved. The bacteriocin LABB was found to
be more effective as there was considerable inhibition of both
L. monocytogenes and S. aureus as compared to either nisin or
LABP, whereas the combination of LABB with nisin proved to
be better as there was maximum inhibition of both organisms
and no bloating of the sealed pouches was observed up to
14 days. The authors concluded that combinations of bacter-
iocins may improve microbial quality, safety and shelf-life of
vegetarian foods.
5. Application of bacteriocins from LAB as food additives
Bacteriocins can be incorporated into foods as a concen-
trated, though not purified, preparation made with food-grade
techniques.
5.1. Fermented vegetables
5.1.1. Kimchi
Kimchi is a generic term indicating a group of traditional
lactic acid-fermented vegetable foods in Korea (Lee et al.,
2005). The major raw material (oriental cabbage or radish) is
salted after pre-brining, blended with various spices (red pepper,
garlic, green onion, ginger, etc.) and other minor ingredients
(seasonings, salted seafoods, fruits and vegetables, cereals, fish
and meats, etc.), and then fermented. Its fermentation is
extremely difficult to control because of the unique character-
istics of the fermenting microflora, among which are mainly
species of LAB.
Choi and Park (2000) reported on the use of nisin to control
the lactobacilli responsible for the over-ripening of kimchi. L.
mesenteroides represents the main species during kimchi
fermentation at temperatures under 10 °C, producing kimchi
with a better organoleptic quality than that made at higher
temperatures at which L. plantarum is dominant and kimchi
becomes too acidic for consumption ([Lee et al., 1992]). With
this in mind, Choi and Park (2000) tested pure nisin against 40
lactobacilli, including L. plantarum (n = 28), L. brevis (n = 7),
Lactobacillus malefermentans (n = 4) and an unidentified Lac-
tobacillus spp. Most of lactobacilli were sensitive to nisin at a
concentration of 100 AU/ml, while two strains appeared to be
resistant. Thus, the concentration of 100 AU/ml of nisin was
chosen for evaluation in kimchi. In those experimental
conditions, the growth of Lactobacillus spp. was inhibited
more than the growth of Leuconostoc spp., as evaluated by plate
counts and confirmed by scanning electron micrograph
observations. The predominance of cocci in kimchi containing
nisin suggested that, at recommended levels, nisin can be used
to preserve kimchi by inhibiting lactobacilli more effectively
than LAB positively involved in kimchi fermentation.
5.1.2. Cider
Cider can be spoiled by the growth of exopolysaccharide-
producing bacteria, e.g. B. licheniformis, which are responsible
for the formation of slime, giving the resulting drink a ropy
appearance (Larpin et al., 2002). In order to preserve their
characteristic organoleptic properties, raw materials that cannot
be heat-treated need alternative methods to inactivate undesir-
able bacteria. Grande et al. (2006) proposed the use of enterocin
AS-48, whose stability in vegetable juice was prior determined
(Grande et al., 2005a), to inhibit the endospore-forming
B. licheniformis LMG 19409. The bacteriocin, produced by
Enterococcus faecalis A-48-32 (formerly Streptococcus fae-
calis subsp. liquefaciens S-48) isolated from a human wound
exudate (Gálvez et al., 1989) was tested in glucose–MRS broth,
fresh-made apple juice and two commercial apple ciders. The
authors found that a concentration of 0.5 μg/ml of enterocin
AS-48 was enough to inactivate B. licheniformis LMG 19409
in glucose–MRS broth, while 3 μg/ml was necessary to obtain
the inhibition in fresh apple juice. Furthermore, enterocin AS-
48 was able to increase the heat sensitivity of spores and the
combination of heat-treatment and enterocin AS-48 was de-
monstrated to determine a reduction of the complete inactiva-
tion time of intact spores in cider.
5.2. Non-fermented vegetables
5.2.1. Mashed potatoes
Potatoes represent the main ingredient of many convenience
foods. The cooking process does not inactivate heat-resistant
bacterial spores and, consequently, endospore-forming Bacillus
spp. and Clostridum spp. can be found in the final products,
even during storage at low temperature (≤ 5 °C) (Gould, 1995).
Clostridium botulinum and Bacillus cereus belong to this
genera that are reported as the cause of several food-borne
outbreaks (Angulo et al., 1998; Doan and Davidson, 2000),
thus, their inhibition by bacteriocins has a relevant significance.
Nisin has been tested for its useful contribution to control Ba-
cillus and Clostridium growth in potato-based products by
Thomas et al. (2002). Addition of 6.25 μg of nisin per g of
cooked mashed potatoes retarded the growth of B. cereus and
B. subtilis, previously inoculated in the product not vacuum
packaged, for at least 27 days at 8 °C and the growth of
C. sporogenes and Clostridium tyrobutiricum, added as spores
in the product and then vacuum packaged, for at least 58 days at
25 °C. Nisin remained at active levels after pasteurization, but
the authors highlighted that, in order to be effective against
temperature abuse and in extending shelf-life of final products,
nisin must be well mixed to the various ingredients.
5.2.2. Soy milk
Soy powder milk is one of the product of soybean that is
becoming very popular among consumers, not only among
those intolerant to animal milk. Similarly to milk products, in
general, the etiological agents of concern in soy milk are mainly
 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138
represented by L. monocytogenes and S. aureus. In order to
prevent the possible contamination and/or to determine their
elimination, Lauková and Czikková (1999) tested enterocin
CCM 4231 for controlling the growth of the above two species
in soy milk. The addition of enterocin CCM 4231 at 3200 AU/
ml inhibited the L. monocytogenes indicator strain for 24 h,
while concentration of S. aureus was reduced of 3.55 log
cycles. The mode of action of the bacteriocin was presumptively
bactericidal against L. monocytogenes, whereas it was proven to
be bacteriostatic against S. aureus. Since enterocin CCM 4231
did not affect pH of soy milk, it was suggested as a valid
candidate to preserve its stability. Furthermore, the authors also
verified the ability of E. faecium CCM 4231 to grow and to
produce bacteriocin in soy milk. It was found to be an in situ
bacteriocin-producing strain: after 2 h from inoculation,
bacteriocin was detected at 100 AU/ml.
5.2.3. Fruit and vegetable juice
Growth of unwanted bacteria in fruit and vegetable juices
during storage may cause spoilage due, in particular, to the
formation of ropiness and off-flavours. In order to face this
problem, Grande et al. (2005a) evaluated the stability of enterocin
AS-48 in fruit and vegetable juices as a preliminary step for its
food applications. Enterocin AS-48 was first concentrated and
then applied 10-fold diluted in juices. It showed different
interactions with the juices tested, from complete to negligible
activity losses. Under refrigeration storage, the bacteriocin was
stable within the first 24–48 h in vegetable juices (avocado,
cabbage, cauliflower, celery, green beans and lettuce), while its
stability was longer (at least 15 days) in fresh fruit juices (apple,
grapefruit, kiwi, orange, pear and pineapple) and mixed juices. In
the latter, bacteriocin activity was detectable after 30 days.
Stability of enterocin AS-48 did not change for up to 120 days in
case of refrigerated commercial fruit juices (apple, orange, peach
and pineapple) whereas its inactivation increased with tempera-
ture. However, at 15 °C, in case of lettuce juice, enterocin AS-48
reduced viable counts of L. monocytogenes, B. cereus and
S. aureus. Moreover, loss of activity of enterocin AS-48 was
reduced by increasing its concentration, diluting the juices or heat-
pretreatment of juices. The results of the study also indicated
compatibility of the bacteriocin with food-grade dyes and
thickening agents, making it suitable for food bioprotection.
Alicyclobacillus acidoterrestris is a spore-forming bacterium
known to cause problems in fruit juices and fruit juice-based
drinks either not heat-treated or pasteurized (Pettipher et al.,
1997). Komitopoulou et al. (1999) studied the growth of
A. acidoterrestris in fruit juice and its sensitivity to heat-
treatment and nisin. The spores were confirmed to be heat-
resistant after 10 min at 80 °C, 2 min at 90 °C and l min at 95 °C
in orange, grapefruit and apple juice. The resistance was
reduced with decrement of pH of juices, although the effect was
less marked at higher temperatures. Nisin addition (100 AU/ml)
completely prevented A. acidoterrestris under all temperature
and time of storage conditions. In particular, the presence of
nisin during heating decreased the decimal reduction time up
to 40% and its minimal inhibition concentration against
A. acidoterrestris spores was only 5 AU/ml at 25 °C.
131
Control of A. acidoterrestris in fruit juice was also ap-
proached with enterocin AS-48 (Grande et al., 2005b). Vege-
tative cells of A. acidoterrestris DSMZ 2498 were inactivated
by 2.5 μg/ml of this bacteriocin in natural orange and apple
juices incubated at 37 °C. No growth was detected in both juices
until the 15th day of observation. Commercial orange, apple,
pineapple, peach and grapefruit juices were then added to the
same concentration of enterocin AS-48 and inoculated with
vegetative cells or endospores of strain DSMZ 2498 and main-
tained at different incubation temperatures (4, 15 and 37 °C) for
three months. In this cases, no viable cells were observed during
the whole incubation, except for apple, peach and grapefruit
juices at 37 °C containing vegetative cells which, however, were
stable for up to 60 days. Treatment with enterocin AS-48, as
revealed by electron microscopy, determined cell damage and
bacterial lysis and disorganization of endospore structure in all
fruit juices object of the study. These findings showed that
enterocin AS-48 can be a valid substitute of the intense heat-
treatments necessary for inactivation of A. acidoterrestris endo-
spores without altering chemical composition of fruit juices.
5.2.4. Canned vegetables
Canned foods represent a continuous supply of seasonal
vegetables which are preserved by acidification and thermal
treatments. Bacteria that tolerate these conditions can proliferate
during storage and may cause spoilage; for this reason,
endospore-forming bacteria (e.g. Bacillus spp.) are a major
problem because the endospores require more intense treat-
ments for inactivation than vegetative cells. Bacillus coagulans
is the microorganism most frequently associated to spoilage of
canned vegetables with pH between 4 and 4.5 (Mallidis et al.,
1990). A big concern with the presence of B. coagulans in low
pH foods is represented by the increment of food pH to values
allowing germination of the food pathogen C. botulinum spores
(Anderson, 1984).
The purpose of the work published by Lucas et al. (2006) was
to test the efficacy of enterocin AS-48 in canned vegetable foods
at different final pH [tomato paste (pH 4.64), syrup from canned
peaches (pH 3.97) and juice from canned pineapple (pH 3.65)]
against B. coagulans. Enterocin As-48 was added at concentra-
tions of 3 and 6 μg/ml. At the highest bacteriocin concentration,
no viable cells were recovered from any sample after 15 days.
Actually, a strain of B. coagulans (CECT 561) showed a poor
survival in tomato paste, but AS-48 inhibit the growth of
surviving cells. The bacteriocin was effective against vegetative
cells of another B. coagulans strain (CECT 12), even though it
did not show significant inhibition of its spores. Furthermore,
addition of lactic acid (1,5%), glucose (10%) and sucrose (20%)
increased bacteriocin activity against vegetative cells and the
combination of bacteriocin and heat-treatment (80–95 °C for
5 min) enhanced the effect of thermal treatments on spore in-
activation. The results of that work showed that enterocin AS-48
can reduce the concentration of vegetative cells of B. coagulans
until levels at which they do not represent any risk of spoilage.
Since the efficacy of bacteriocin AS-48 is enhanced by other
additives, it has been proposed as an additional hurdle in order to
protect canned vegetables by B. coagulans damages.
132 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138
5.2.5. Fresh-cut products
Vegetables, during growth in field, are frequently in contact
with soil (Beuchat, 1995) and their surfaces can be contami-
nated. In order to eliminate or reduce the presence of pathogenic
and spoilage microorganism, physical and chemical treatments
are used in food processing (Ukuku and Fett, 2002a; Ukuku and
Sapers, 2001; Ukuku et al., 2004). Bacteriocins, in combination
with other food additives, are being commonly employed as
protective agents in this kind of products. In particular, Ukuku
and Fett (2002a) first studied the effectiveness of chlorine and
nisin–EDTA treatments on whole melons and fresh-cut pieces
in reducing native microflora and then the combined effect of
nisin, EDTA, sodium lactate and potassium sorbate in reducing
Salmonella on fresh-cut cantaloupe (Ukuku and Fett, 2004),
while Bari et al. (2005) focused on the combination of nisin,
pediocin, sodium lactate, citric acid, phytic acid, potassium
sorbate and EDTA to L. monocytogenes on fresh-cut cabbage,
broccoli and mungbean sprouts. Furthermore, the use of H2O2
in combination with nisin, sodium lactate and citric acid to
reduce the transfer of bacterial pathogens from whole melon
surfaces to fresh-cut pieces was also reported (Ukuku et al.,
2005).
In the first study (Ukuku and Fett, 2002b), the attachment and
survival of L. monocytogenes on cantaloupe, their resistance to
chlorine and H2O2 treatments, were investigated and their
transfer from unsanitised and sanitised rinds to fresh-cut tissues
during cutting. The authors concluded that sanitising with
chlorine and H2O2 has the potential to reduce or eliminate the
transfer of L. monocytogenes on melon surfaces to fresh-cut
pieces. In a second work of the same authors (Ukuku and Fett,
2004), whole melons and fresh-cut pieces were inoculated with
five Salmonella strains (Salmonella Poona RM2350, Salmo-
nella Stanley H0558, Salmonella Newport H1275, Salmonella
Anatum F4317 and Salmonella Infantis F4319) and stored at
5 °C for 7 days. All combinations of nisin and chemicals in-
cluded in the experiment resulted in the reduction of approxi-
mately 3 log cycles for whole melon, whereas when tested alone,
all compounds, along with water washes, were ineffective. None
of the five combinations completely avoided the transfer of
pathogen survivors to fresh-cut pieces. However, washing with
the combination nisin–sodium lactate–potassium sorbate was
significantly more effective in inhibiting salmonellas than the
other mixed solutions, but it was less acceptable in terms of
appearance, odour and overall acceptability. A similar ap-
proach was used, a year later (Bari et al., 2005), to inhibit L.
monocytogenes. Also in this case study, a five-strain cocktail of
the pathogen object of study (L. monocytogenes ATCC 43256,
ATCC 49594, JCM 7676, JCM 7672 and JCM 7671) was
inoculated in fresh-cut vegetables and left at 25 °C for 4 h before
antimicrobial (individual or combined) treatments were applied.
When tested alone, all compounds determined a reduction of
about 2–4 log cycles. The combinations nisin–phytic acid and
nisin–pediocin–phytic acid caused significant reductions of
L. monocytogenes on cabbage and broccoli, but not on mung-
bean sprouts. Furthermore, pediocin treatment alone or in com-
bination with organic acids showed a stronger effect than nisin
treatment alone. Although none of the treatments tested in the
two above works completely eliminated the pathogens arbitrari-
ly added to the vegetables, the authors suggested that some
treatments could be useful to improve the microbiological safety
of fresh-cut vegetables.
Regarding the use of mixed chemicals and nisin for reducing
transfer of bacterial pathogens from whole melon surfaces to
fresh-cut pieces, Ukuku et al. (2005) inoculated whole
cantaloupes and honeydew melons with E. coli O157:H7 and
L. monocytogenes at concentrations of approximately 3–5 log
CFU/cm2 and incubated them at 5 °C for 7 days. Whole melons
at day 0 and 7 were subjected to antimicrobial [H2O2 (2.5%) alone
or H2O2 (1%) in combination with nisin (25 μg/ml), sodium
lactate (1%), and citric acid (0.5%)] washing treatments and
surviving bacterial populations and the numbers transferred to
fresh-cut pieces were determined: the combination of inhibitors
significantly reduced the numbers of both pathogens by 3 to 4 log
CFU/cm2 on both types of whole melon and it was also found to
be significantly more effective than treatment with 2.5% H2O2.
The authors proposed the above mixture as a washing treatment to
decontaminate whole melon surfaces and, thus, improve the
microbial safety and quality of fresh-cut melons.
Recently, Molinos et al. (2005) tested the effectiveness of
immersion solutions containing enterocin AS-48 for decontam-
ination of raw vegetables. In particular, the authors focused on the
application of immersion solutions alone or in combination with
chemical preservatives to inhibit L. monocytogenes CECT 4032
growth on fresh green asparagus and alfalfa and soybean sprouts.
L. monocytogenes CECT 4032 was inoculated onto fresh vege-
tables at a concentration ranging from 4.69 to 4.72 log CFU/g.
Each produce was immersed for 5 min at room temperature in
solutions containing 25 μg/ml of enterocin AS-48. In case of
alfalfa and soybean sprouts, L. monocytogenes counts were
reduced of 2.0–2.4 log CFU/g compared to a control immersion
treatment in distilled water, while the treatment showed a limited
effect when applied on green asparagus. However, during storage,
L. monocytogenes viable counts were reduced below the detection
limits at days 1 to 7 not only for alfalfa and soybean sprouts at 6
and 15 °C, but also for green asparagus at 15 °C. At 22 °C the
inhibition of L. monocytogenes CECT 4032 on alfalfa sprouts and
green asparagus was not significant. Treatment with solutions
containing enterocin AS-48 and chemicals such as lactic acid,
sodium lactate, sodium nitrite, sodium nitrate, trisodium phos-
phate, trisodium trimetaphosphate, sodium thiosulphate, n-propyl
p-hydroxybenzoate, p-hydoxybenzoic acid methyl ester, hexade-
cylpyridinium chloride, peracetic acid, or sodium hypochlorite
reduced L. monocytogenes viable counts below the detection
limits and significant increases of antimicrobial activity were
found for AS-48 in some combination with potassium perman-
ganate, acetic acid, citric acid, sodium propionate, and potassium
sorbate.
5.2.6. Zucchini purée
Endospore-forming bacteria, especially Bacillus spp., repre-
sent the main bacterial population of vegetable purées (Carlin
et al., 2000). On the basis of a study reporting on Bacillus
macroides/Bacillus maroccanus as being favoured in zucchini
purée stored at 4 °C (Guinebretiere et al., 2001), García et al.
 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138
(2004) studied the effectiveness of enterocin EJ97 to inhibit
strains (INRA P51-5 and INRA P53-2) of zucchini purée origin
belonging to the above species. The bacteriocin had a bacteri-
cidal effect on strain INRA P53-2 (chosen as test organism on the
basis of its higher sensitivity to enterocin EJ97 with respect to
strain INRA P51-5) after several incubation conditions (4 h at
37 °C, 24 h at 15 °C and 48 h at 4 °C); its activity was reduced at
pH 5.0 and 9.0 and enhanced by sodium nitrite, sodium
benzoate, sodium lactate and sodium tripolyphosphate. The in
situ efficacy of pure enterocin EJ97 was obtained with a 10-fold
higher concentration, whereas no inhibition of strain INRA P53-
2 was detected with the application of E. faecalis EJ97 as a
developing bacterium in purée, although it was able to produce
the bacteriocin in situ. Thus, the enterocin EJ97 has a potential to
preserve food spoiled by B. macroides/B. maroccanus if used in
concentrated pure form.
6. Combined effect of pure bacteriocins and
bacteriocin-producing strains
Schillinger et al. (2001) studied the potential of nisin in
combination with bacteriocinogenic LAB (E. faecium BFE 900-
6a, Lactobacillus sakei Lb 706-1a and L. lactis BFE 902) in the
preservation of highly perishable food products, in order to
confirm their previous observation of the enhancement of the
anti-listerial efficacy of nisin by combination with a bacter-
iocinogenic protective culture in a laboratory medium at 30 °C
(Schillinger et al., 1998). Tofu, a non-fermented soybean
product consumed in Far-Eastern countries which may allow
the growth of L. monocytogenes at refrigeration temperatures,
was chosen as the test food system. The efficacy of nisin against
Listeria spp. may be compromised by the emergence of spon-
taneous nisin-resistant mutants. Tests performed in vitro demon-
strated that E. faecium BFE 900-6a, L. sakei Lb 706-1a and
L. lactis BFE 902 used as protective cultures in combination with
nisin were able to suppress the proliferation of L. monocytogenes
cells not killed by nisin at 10 °C. Furthermore, when used in
combination with live strains, lower amounts of nisin were
required for an effective inhibition of L. monocytogenes Scott A.
In particular, the combination nisin/E. faecium BFE 900-6a or
nisin/L. lactis BFE 902 resulted in a complete suppression of
L. monocytogenes growth in homemade tofu stored at 10 °C for
1 week, whereas the combination nisin/L. sakei Lb 706-1a was
found to be less effective and it did not prevent a slight increase of
L. monocytogenes Scott A numbers during storage. In conclusion,
the author found that when the protective culture is used in
combination with nisin, L. monocytogenes is kept at a low level
by nisin for several days, during which the protective culture can
proliferate until reaching concentrations sufficient to suppress
growth of survivors of nisin.
7. Application of non-LAB bacteriocinogenic strains
In the last few years, the potential of starter cultures for
controlling and safeguarding the fermentation of traditional
fermented foods has been emphasised also with regard to non-
LAB species (Beaumont, 2002). In particular, the benefits
133
through the use of non-LAB bacteriocinogenic starter cultures or
co-cultures for the production of traditional fermented products,
such as condiments, are mainly represented by improved safety,
as compared to spontaneous and uncontrolled traditional
fermentations; this assumes a basic significance in case of
countries with poor hygienic conditions (Beaumont, 2002).
Unlike majority of fermented foods in which LAB dominates,
okpehe, a traditional Nigerian soup condiment produced from
Prosopis africana seeds (Guill, Perr) Taub. (Sanni and Onilude,
1999), is mainly fermented by Bacillus spp., with B. subtilis
being commonly isolated (Oguntoyinbo et al., 2007). Some
strains of the species B. subtilis are, indeed, known as spoilage
agents, e.g. wheat bread due to rope formation (Kirschner and
Von Holy, 1989), or as cause of food-borne illness if present at
levels over 105 CFU/g (Kramer and Gilbert, 1989). However,
B. subtilis has been recently proposed as a starter culture for this
kind of product (Oguntoyinbo et al., 2007). In that study, the
selection of starter cultures for fermentation of P. africana seeds
started from Bacillus and Enterococcus strains isolated from
traditional okpehe. The choice of starter strains was then
narrowed to two B. subtilis strains (BFE 5301 and BFE 5372),
determined to be the best starter combination because of rapid
growth, high amylolytic and proteolytic activities, high levels of
polyglutamic acid production by strain BFE 5372, bacteriocin
production by strain BFE 5301, as well as non-virulent
characteristics. The employment of B. subtilis BFE 5372 alone
produced okpehe with very good sensory characteristics, but it
did not prevent the growth of B. cereus, hence the subtilisin-
producing B. subtilis BFE 5301, which was able to delay
B. cereus growth and showing poor proteinase activity and
polyglutamic acid production, was used as a co-culture.
Another example of the use of a non-LAB bacteriocin in
food biopreservation is represented by variacin from Kocuria
varians to control the growth of B. cereus in chilled dairy
products (O'Mahony et al., 2001).
8. Future prospects
With the growing consumer refusal of chemical additives to
combat undesired bacterial growth in foods and beverages, there
is a growing demand for alternative antimicrobial treatments and
bacteriocins are well accepted natural means of selective
microbial inhibition. Moreover, the increasing demand of mini-
mal food processing is providing an opportunity for their wide-
spread application. However, although many bacteriocins, such as
lacticin 3147, pediocin PA-1, and enterocin AS-48, have been
shown to exhibit a large spectrum of food applications, due to
legal restrictions, nisin is still the sole bacteriocin permitted as
biopreservative in the food industries of about 50 countries. The
incidence of nisin-resistant mutants is determining novel
strategies for the inhibition of nisin non-sensitive strains, basically
represented by the combination of more bacteriocins or bacte-
riocins and bacteriocinogenic strains. In the latter strategy, in case
of non-fermented foods, bacteriocinogenic strains have to be used
as protective cultures.
In the past, there had been a great attraction of food scientists
towards the creation of bacterial strains exhibiting over expression
134 L. Settanni, A. Corsetti / International Journal of Food Microbiology 121 (2008) 123–138
of bacteriocins by genetic manipulation (Horn et al., 1999) or the
introduction of this character into eukaryotic organisms (Van
Reenen et al., 2003). This branch of research is still on the
increase. However, although particularly interesting, this strategy
cannot represent an applicative future direction of research
because of the restrictive legal regulations and the skeptical
behaviour of consumers with regards to genetically modified
organisms.
In addition to the three approaches identified by Schillinger
et al. (1996) as commonly used in the application of
bacteriocins for biopreservation of foods (inoculation with
bacteriocinogenic bacteria, adjunct in purified form and use
of a product previously fermented with bacteriocinogenic
bacteria as ingredient in food processing), a fourth recent food
application of bacteriocins is represented by their binding to
polymeric packaging. Up to date, this kind of bacteriocin
application has been reported only for preserving foods of
animal origin such as meat and poultry (Ming et al., 1997),
frankfurters (Ercolini et al., 2006) and raw and pasteurized
milk (Mauriello et al., 2005); from our perspective, it has a
great potential as a biopreservation technology for vegetable
origin foods as well.
Lysozyme and nisin are both antimicrobial proteins effective
against Gram-positive bacteria and their use in combination
with chelating agents (e.g. EDTA) displays increased effective-
ness against Gram-negative bacteria (Padgett et al., 1998).
Furthermore, bacteriocins often have synergies with other
treatments (Cleveland et al., 2001). Thus, bacteriocin-activated
packaging films alone or in a hurdle technology program
represent one of the most attractive directions of future research
on vegetable food protection.
9. Conclusions
The application of bacteriocins as biopreservatives for
vegetable food matrices started approximately 20 years ago. In
these years, a lot of studies have focused on the inhibition of
spoilage and/or human pathogen bacteria vehiculated with
vegetable foods and beverages by bacteriocins and their appli-
cation appeared as a good alternative to chemical compounds
and antibiotics. Whether deliberately added or produced in situ,
bacteriocins have been found to play a defining role in the
control of undesirable flora, as well as in the establishment of
beneficial bacterial populations. However, the effect of bacter-
iocins, bacteriocinogenic strains or their combinations would not
alleviate the practical food safety issues associated with a large
variety of vegetable foods, e.g. they may be efficient only in a
narrow pH range, which excludes their utilization in many food
products. Thus, a single bacteriocin-based technique could fit
with a single food matrix and its application should be tested on a
“product by product” basis. Furthermore, it can be concluded
that in addition to the traditional hurdle technology represented
by low temperature and vacuum packaging or MAP, the exploi-
tation of bacteriocinogenic cultures, as well as their pure
bacteriocins holds a great potential for extension of shelf-life and
improvement of microbiological safety of vegetable raw
materials and final products.
Acknowledgement
The authors wish to thank Dr. Elena Critaro (Library of the
Agricultural Faculty-University of Teramo, Italy) for her
valuable help in retrieving the bibliographic material.
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strains from ben saalga, a traditional fermented gruel from Burkina Faso.
International Journal of Food Microbiology 112, 44–50.
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producing Lactococcus lactis subsp. lactis from bean-sprouts. Journal of
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Choi, H.-J., Lee, H.-S., Her, S., Oh, D.-H., Yoon, S.-S., 1999. Partial
characterisation and cloning of leuconocin J, a bacteriocin produced by
Leuconostoc sp. J2 isolated from the Korean fermented vegetable kimchi.
Journal of Applied Microbiology 86, 175–181.
Choi, H.-J., Cheigh, C.-I., Kim, S.-B., Pyun, Y.-R., 2000. Production of a nisin-
like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from
kimchi. Journal of Applied Microbiology 88, 563–571.
Corsetti, A., Gobbetti, M., Smacchi, E., 1996. Antimicrobial activity of sourdough
lactic acid bacteria: isolation of a bacteriocin-like inhibitory substance from
Lactobacillus sanfrancisco C57. Food Microbiology 13, 447–456.
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activity of Lactobacillus amylovorus DCE 471 and large scale isolation of its
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Hartnett, D.J., Vaughan, A., Van Sinderen, D., 2002. Antimicrobial producing
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characterisation of two bacteriocin-producing bacteria isolated from garlic
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Bacteriocin production by lactic acid bacteria isolated from Rioja red wines.
Journal of Applied Microbiology 88, 44–51.
Onda, T., Yanagida, F., Tsuji, M., Shinohara, T., Yokotsuka, K., 2003.
Production and purification of a bacteriocin peptide produced by Lacto-
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Todorov, S.D., Dicks, L.M.T., 2004a. Partial characterisation of bacteriocins
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Todorov, S.D., Dicks, L.M.T., 2004b. Characterisation of mesentericin ST99, a
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