REVIEW

published: 08 April 2016

doi: 10.3389/fmicb.2016.00461

Bacteriocins: Novel Solutions to Age

Old Spore-Related Problems?
Kevin Egan 1 , Des Field 1 , Mary C. Rea 2, 3 , R. Paul Ross 3, 4 , Colin Hill 1, 3 and Paul D. Cotter 2, 3*
1
School of Microbiology, University College Cork, Cork, Ireland, 2 Teagasc Food Research Centre, Moorepark, Fermoy,
Ireland, 3 APC Microbiome Institute, University College Cork, Ireland, 4 College of Science, Engineering and Food Science,
University College Cork, Cork, Ireland

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria,

which have the ability to kill or inhibit other bacteria. Many bacteriocins are produced
by food grade lactic acid bacteria (LAB). Indeed, the prototypic bacteriocin, nisin, is
produced by Lactococcus lactis, and is licensed in over 50 countries. With consumers
becoming more concerned about the levels of chemical preservatives present in food,
bacteriocins offer an alternative, more natural approach, while ensuring both food
safety and product shelf life. Bacteriocins also show additive/synergistic effects when
Edited by:
used in combination with other treatments, such as heating, high pressure, organic
Amit Kumar Tyagi, compounds, and as part of food packaging. These features are particularly attractive
The University of Texas MD Anderson
from the perspective of controlling sporeforming bacteria. Bacterial spores are common
Cancer Center, USA
contaminants of food products, and their outgrowth may cause food spoilage or
Reviewed by:
Bradley D. Jones, food-borne illness. They are of particular concern to the food industry due to their
The University of Iowa, USA thermal and chemical resistance in their dormant state. However, when spores germinate
Stella Maris Reginensi Rivera,
Universidad de la República, Uruguay
they lose the majority of their resistance traits, making them susceptible to a variety
Riadh Hammami, of food processing treatments. Bacteriocins represent one potential treatment as they
Laval University, Canada
may inhibit spores in the post-germination/outgrowth phase of the spore cycle. Spore
Beatriz Martínez,
Consejo Superior de Investigaciones eradication and control in food is critical, as they are able to spoil and in certain cases
Científicas, Spain compromise the safety of food by producing dangerous toxins. Thus, understanding
*Correspondence: the mechanisms by which bacteriocins exert their sporostatic/sporicidal activity against
Paul D. Cotter
paul.cotter@teagasc.ie
bacterial spores will ultimately facilitate their optimal use in food. This review will focus
on the use of bacteriocins alone, or in combination with other innovative processing
Specialty section: methods to control spores in food, the current knowledge and gaps therein with regard to
This article was submitted to
Food Microbiology,
bacteriocin-spore interactions and discuss future research approaches to enable spores
a section of the journal to be more effectively targeted by bacteriocins in food settings.
Frontiers in Microbiology
Keywords: antimicrobial peptide, bacteriocin, spore, Bacillus, Clostridium, food processing, LAB
Received: 28 January 2016
Accepted: 21 March 2016
Published: 08 April 2016
INTRODUCTION
Citation:
Egan K, Field D, Rea MC, Ross RP,
Control and eradication of Bacillus and Clostridium spores is one of the most challenging aspects
Hill C and Cotter PD (2016)
Bacteriocins: Novel Solutions to Age
of microbial control faced by the modern food industry. Traditionally, spores have been controlled
Old Spore-Related Problems? using extreme treatments such as high heat alone or in combination with chemical additives.
Front. Microbiol. 7:461. However, modern consumers are more conscious than previous generations of the negative health
doi: 10.3389/fmicb.2016.00461 effects associated with the consumption of certain chemical preservatives and of the significant

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Egan et al. Inhibiting Spores with Bacteriocins

effects of heat on the nutritional value and flavor of many foods. spore composition of an ingredient. Spores are also highly
With ready-to-eat and minimally processed foods becoming adhesive and may remain on the surfaces of equipment and
a staple of the modern diet, the food industry is faced with contribute to problems long after their initial contamination
an unprecedented challenge to provide food that is: (i) low of the facility. Reducing these initial spore loads is critical in
in synthetic chemical additives, (ii) low in salt/sugar, (iii) avoiding problems downstream. However, it is important to note
nutritionally beneficial, and (iv) stable and safe, from a microbial that spores are often selected for in food processing as their
perspective, over an extended period of time. As a result, the thermal resistance allows them to endure any heating steps (see
food industry is under pressure to employ innovative processing review by: Carlin, 2011).
methods to meet consumer and regulatory demands. One As early as 1956 (Stuy, 1956), the induction of spore
potential innovation that has been intensively researched over germination was identified as a strategy that could facilitate
the last number of decades, and is well positioned to provide a spore eradication. When threshold levels of nutrients (such as
safe and effective alternative to existing processing technologies, amino acids, sugars, and nucleosides) are present, they bind to
involves the use of bacteriocins. This review will examine the Ger complexes, located on the inner membrane of the spore.
efficacy of bacteriocins alone, and in combination with other This strategy takes advantage of the loss of the resistance
processing technologies, to control spores in food. properties that a dormant spore possesses. It has been shown
that once spores have germinated, they become more sensitive
than dormant spores to: heat (Durban et al., 1970), X-Ray
THE BACTERIAL SPORE and UV radiation (Stuy, 1956; Munakata, 1974), and copper
(Wheeldon et al., 2008). Interestingly the process of spore
Metabolically dormant spores of Gram-positive Clostridium and germination is not 100% efficient, due to the heterogeneity in
Bacillus species are formed during sporulation. This sporulation germination rates among members of the spore population in
process is typically a response to cellular nutrient starvation response to a particular nutrient germinant. Previous studies
and involves a complex cascade of enzyme reactions. This have highlighted the specificity of germinant receptors (GRs):
process of sporulation has been extensively described over showing that GerA will respond to L-alanine and L-valine, while
the last number of decades in the model spore former B. GerB and GerK will respond to a mixture of L-asparagine,
subtilis (see review by: Tan and Ramamurthi (2014). Spores D-glucose, D-fructose, and potassium ions (Moir et al., 1994;
consist of a core surrounded by a coat and/or endosporium. Atluri et al., 2006). The binding of the nutrients to their
The spore core consists of DNA, enzymes, and dipicolinic appropriate GRs results in the irreversible commitment of the
acid (DPA). DPA plays a role in maintaining spore dormancy, spore to germination.
providing resistance to DNA damaging substances and is usually Commonly spores termed superdormant have been isolated
bound to divalent cations such as Ca2+ at a 1:1 ratio in the from populations of B. subtilis following saturation with nutrient
core (Setlow, 2014b). The composition and structure of the germinant. This super dormancy is attributed to the lag
metabolically inactive, dehydrated, spore confers resistance to in initiation of the rapid loss of Ca2+ -DPA stage in spore
changes in pH (Blocher and Busta, 1983), wet and dry heat, germination. Following the initiation of rapid loss of Ca2+ -
UV radiation, desiccation (Nicholson et al., 2000), and various DPA from its core, the spore is no longer superdormant
toxic chemicals (Russell, 1990; Cortezzo and Setlow, 2005). and its germination will proceed in a similar manner as
A spore may be viable after extended periods of dormancy dormant spores (Figure 1). This superdormancy may be an issue
(Cano and Borucki, 1995), monitoring its environment for for antimicrobials (e.g. nisin) whose effect is only exhibited
favorable growth conditions and when suitable, germination on those spores that have reached the end of stage II
and outgrowth occur, ultimately resulting in a vegetative of germination (Figure 1; Chen et al., 2014). Superdormant
cell (Figure 1). Endospore-forming bacteria vary considerably spores may, however germinate, in response to an alternative
with respect to genotype and phenotype and, with respect germinant that utilizes an alternative GR. A different strategy,
to phenotype, consist of aerobic, facultative anaerobic, and which can be used to increase germination of super dormant
obligate anaerobic, psychrophilic, mesophilic, thermophilic, spores, is by using higher heat activation temperatures than
psychotropic and thermotolerant strains (see review by: Doyle is required for those non-superdormant spores (Ghosh et al.,
et al., 2015). This phenotypic heterogeneity of spore-forming 2009). Treatment of spores with sublethal heat (also called
bacteria means that virtually all types of food are potential heat activation) has been shown to increase the rate of
targets for spore contamination and spore outgrowth, with germination of a number of spore species. Luu et al. (2015)
potentially severe consequences with respect to food quality and suggested that although the main target of heat activation
safety. is the spore’s GRs, this may only be indirect and that the
There are many pathways via which spores can gain access sublethal heat is having a more direct effect on the inner
to the food chain. Food products are composed of multiple membrane of the spore in which the GRs are situated, ultimately
ingredients, potentially from different international origins, resulting in increased spore germination. Therefore decisive
each contributing their own specific quantity and diversity of triggering of the spore germination process, will allow food
spores into the final formulation. Factors such as microbial processors to render spores sensitive to a variety of inactivation
ecology, farming practices, the local climate, hygiene of the methods that are ineffective against highly resistant dormant
processing facility and animal feeding practices determine the spores.

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Egan et al. Inhibiting Spores with Bacteriocins

FIGURE 1 | Germination dependent inhibition of spore outgrowth by bacteriocins. Dormant spores may germinate after being activated by a variety of means;
most commonly sub-lethal heat being used. Heat is believed to activate the dormant spores by making the germinant receptors (GR) more accessible to nutrient
germinants. Once the GR-nutrient binding occurs, the spore is now committed to germination even if the germinant is removed. Stage 1 of germination consists of
H+ , K+ , and Na+ ion release followed by Ca2+ -DPA release. This release of Ca2+ -DPA triggers stage II of germination where the cortex is degraded, allowing the
germ cell wall to expand and take up water. At the end of stage II the spore core is hydrated and has expanded along with the cortex. This rise in water content
signals the end of stage II of germination and the beginning of the outgrowth phase. At this point bacteriocins that are not active against dormant spores become
active, inhibit outgrowth and reduce viable counts from the germinated spore population. This figure is adapted from Setlow (2014a).

BACTERIOCINS throughput, culture-based screens can also be valuable (Rea et al.,

2010).
Bacteriocins are a class of ribosomally synthesized antimicrobial
peptides (AMPs) produced by bacteria. These small and naturally
produced peptides can kill other bacteria, which are closely Bacteriocins from the LAB Are Suitable for
(narrow spectrum) or distantly (broad spectrum) related to Food Preservation
the producing bacteria (Cotter et al., 2005). It is hypothesized Although there are many Gram-negative and Gram-positive
that the production of bacteriocins is a strategy to control microorganisms which produce bacteriocins, those produced by
competing bacteria in the hunt for nutrients and space in the lactic acid bacteria (LAB) are of particular interest to the food
an environmental niche. Therefore, it is not surprising that it industry. Many of these bacteria already play a crucial role in
has been estimated that many bacteria produce at least one a variety of food fermentations by converting lactose to lactic
bacteriocin (Riley and Wertz, 2002), which may help them acid, as well as producing a variety of additional antimicrobial
to influence the surrounding population dynamics. Although molecules such as other organic acids, diacetyl, acetoin, hydrogen
many bacteriocin-producing bacteria in the biosphere have been peroxide, antifungal peptides, and bacteriocins. The best
investigated, it is still the case that there remain many are that are known LAB genera are Lactococcus, Streptococcus, Lactobacillus,
still to be discovered (Yang et al., 2014). Indeed, bioinformatic Pediococcus, and Enterococcus, though a number of other,
mining of publically available genomes, along with other rapid generally regarded as more peripheral and less frequently applied
techniques, are beginning to bridge this gap in initial discovery, from an industrial perspective, genera also exist. LAB offer several
by overcoming the previous dependence on the expensive, time key properties which make their bacteriocins highly desirable for
consuming, culture-dependent nature of bacteriocin discovery use in food: (i) the LAB are Generally Regarded As Safe (GRAS)
and purification (Sandiford, 2015). BAGEL3 (BActeriocin and there are perceived by the public as having health promoting
Genome mining tooL) (van Heel et al., 2013) and antiSMASH features, (ii) their bacteriocins are sensitive to digestive proteases
3.0 (antibiotics and Secondary Metabolite Analysis Shell) (Weber such as pancreatin complex, trypsin and chymotrypsin, and thus
et al., 2015) are examples of web based genome mining tools don’t impact negatively on the gut microbiota, (iii) they are
that detect putative bacteriocin biosynthetic gene clusters. Liquid non-toxic to eukaryotic cells (iv) they are often active across
chromatography/mass spectrometry has also been used to rapidly a range of pH values and are, in many cases, not temperature
detect bacteriocins in as little as 25 µl of culture supernatant and sensitive (Table 1), (v) they are gene encoded and therefore
is sensitive enough to distinguish between variants of the same highly amenable to genetic manipulation where desired (Field
bacteriocin e.g., nisins A, Z, and Q (Zendo et al., 2008). High et al., 2015), (vi) not all of the bacteriocins produced by the LAB

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Egan et al. Inhibiting Spores with Bacteriocins

TABLE 1 | Bacteriocins that are active against vegetative cells of Gram-positive spore-forming bacteria.

Bacteriocin Class Producer Size (Da) Spectrum Heat stability Active Sensitive References
pH Spore-formers

Acidocin LCHV IId Lactobacillus 1158.2c Broad Heat stable 3–8 B. cereus Mkrtchyan et al., 2010
acidophilus n.v. Er B. subtilis
317/402 strain narine

Acidocin IIb Lactobacillus gasseri 3500-5000a Broad Heat stable 2–9 B. cereus Bogovic-Matijasić
LF221A LF221 C. sporogenes et al., 1998
Acidocin C. tyrobutyricum
LF221B

Bac217 IId Lactobacillus paracasei 7000a Broad Heat stable 3–12 B. cereus Lozo et al., 2004
subsp. paracasei B. fragilis
BGBUK2-16 B. subtilis

BacC1 ND Enterococcus faecium 10,000a Broad Heat stable 2–6 B. cereus Goh and Philip, 2015
C1

Bacteriocin IIa Lactobacillus salivarius 3454 Broad ND ND C. perfringens Svetoch et al., 2011
L-1077 1077

Bifidocin B IIb Bifidobacterium bifidum 4432.9c Narrow Heat stable 2–10 B. cereus Yildirim and Johnson,
NFBC 1454 1998b; Yildirim et al.,
1999

Bificin C6165 ND Bifidobacterium 3395.1c Narrow Moderate 3.5–6.5 A. acidoterrestris Pei et al., 2013
animalis subsp.
animalis CICC 6165

Brevicin 925A IId Lactobacillus brevis ND Narrow Heat resistant ND B. coagulans Wada et al., 2009
925A

Divergicin 750 IId Carnobacterium 3447.7 Broad ND ND C. perfringens Holck et al., 1996
divergens 750

Duranicin IId Enterococcus durans Q 5227.8c Narrow Moderate 2–10 B. coagulans Hu et al., 2008
TW-49M 49 B. circulans
B. subtilis
G. stearothermophilus

Enterocin IId Enterococcus faecalis 7A 5200.8c Broad ND ND C. butyricum Liu et al., 2011
7A/7B 710C 7B 5206.65c C. botulinum
C. perfringens
C. sporogenes

Enterocin A IIa Enterococcus faecium 3829c Broad Heat stable 2–10 B. coagulans Aymerich et al., 1996;
CTC492, Enterococcus B. subtilis Casaus et al., 1997; Hu
faecium T136 C. sporogenes et al., 2010, 2014
C. tyrobutyricum

Enterocin IIc Enterococcus faecalis 7140c Broad Heat stable ND Alicyclobacillus spp. Lucas et al., 2006b;
AS-48 A-48-32 B. cereus Burgos et al., 2014
B. coagulans
B. licheniformis
B. subtilis
C. perfringens
C. sporogenes
C. tetani
G. stearothermophilus
Paenibacillus spp.

Enterocin B IId Enterococcus faecium 5463c Broad Heat stable ND B. coagulans Casaus et al., 1997; Hu
T136 Ent B. subtilis et al., 2010
C. sporogenes
C. tyrobutyricum

(Continued)

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Egan et al. Inhibiting Spores with Bacteriocins

TABLE 1 | Continued

Bacteriocin Class Producer Size (Da) Spectrum Heat stability Active Sensitive References
pH Spore-formers

Enterocin EJ97 IId Enterococcus faecalis 5340c Broad Heat stable 2–9.5 B. circulans Gálvez et al., 1998;
EJ97 B. coagulans Garcia et al., 2004
B. macrolides
B. megaterium
B. moroccanus
B. subtilis
G. stearothermophilus
Paenibacillus macerans

Enterocin L50 IIb Enterococcus faecium A: 5190c Broad Heat stable 2–11 B. cereus Cintas et al., 1995;
L50 B: 5178c B. subtilis Basanta et al., 2010

Enterocin IT IId Enterococcus faecium 6390c Narrow ND ND B. subtilis Izquierdo et al., 2008
IT62

Enterocin MR10 IIb Enterococcus faecalis A: 5201.6b Broad Heat stable 4.6–9 B. cereus Martín-Platero et al.,
MRR10-3 B: 5207.5b B. licheniformis 2006

Enterocin IIc Enterococcus faecium 6316.42c Broad Heat stable 2–10 B. circulans Himeno et al., 2015
NKR-5-3B NKR-5-3 B. coagulans
B. subtilis

Enterocin RM6 IId Enterococcus faecalis 7145c Broad ND ND B. cereus Huang et al., 2013
OSY-RM6

Enterocin P IId Enterococcus faecium 4493b Broad Heat stable 2–11 B. cereus Cintas et al., 1997
P13 C. botulinum
C. perfringens
C. sporogenes
C. tyrobutyricum

Enterocin IIa Enterococcus faecalis 5356.2c Narrow Heat stable 3–11 B. subtilis Eguchi et al., 2001
SE-K4 K-4 C. beijerinckii

Gassericin A IIc Lactobacillus gasseri 3800a Broad Heat stable 2–12 B. cereus Nakamura et al., 2013
LA 39

Gassericin KT7 ND Lactobacillus gasseri ND Broad Heat stable 2.5–9 B. cereus Zhu et al., 2000
KT7 B. subtilis
C. botulinum
C. perfringens

Garvieacin Q IId Lactococcus garvieae 5339c Broad Heat stable 2–8 B. coagulans Tosukhowong et al.,
BCC 43578 2012

Lacticin 3147 I Lactococcus lactis ltnA1: 3305c Broad Heat stable 5–9 B. cereus McAuliffe et al., 1998;
subsp. lactis DPC3147 ltnA2: 2847c B. subtilis Martinez-Cuesta et al.,
C. sporogenes 2010; Iancu et al., 2012
C. tyrobutyricum

Lacticin 481 I Lactococcus lactis 2901c Narrow Heat stable ND C. tyrobutyricum Piard et al., 1990, 1993
subsp. lactis CNRZ
481

Lacticin LC14 ND Lactococcus lactis 3333.7c Broad Heat stable 2–10 B. cereus Lasta et al., 2012
BMG6. 14 B. thuringiensis

Lacticin Q IId Lactococcus lactis 5926.5c Broad Heat stable 2–10 B. cereus Fujita et al., 2007
QU 5 B. circulans
B. coagulans

(Continued)

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Egan et al. Inhibiting Spores with Bacteriocins

TABLE 1 | Continued

Bacteriocin Class Producer Size (Da) Spectrum Heat stability Active Sensitive References
pH Spore-formers

Lacticin Z IId Lactococcus lactis QU 5968.9c Broad Heat stable 2–10 B. subtilis Iwatani et al., 2007
14 B. circulans
B. coagulans

Lactococcin BZ ND Lactococcus lactis 5500a Broad Heat stable 2–7 B. cereus Sahingil et al., 2011
subsp. lactis B. subtilis

Lactococcin R ND Lactococcus cremoris 2500a Broad Heat stable 2–9 B. cereus Yildirim and Johnson,
subsp. cremoris R B. subtilis 1998a
C. perfringens
C. sporogenes

Leucocin H IIb Leuconostoc MF215B ND Broad ND ND B. cereus Blom et al., 1999

C. perfringens

Leucocyclicin Q IIc Leuconostoc 6115.59c Broad ND ND B. cereus Masuda et al., 2011

mesenteroides B. coagulans
TK41401 B. subtilis

Lactocyclin Q IIc Lactococcus sp. strain 6062c Broad Heat stable 3–9 B. cereus Sawa et al., 2009;
QU 12 B. coagulans Masuda et al., 2011
B. subtilis

Mesentericin ND Leuconostoc ND Broad Heat stable 2–12 B. subtilis Todorov and Dicks,
ST99 mesenteroides ST99 2004

Macedocin I Streptococcus 2795c Broad Heat stable 4–9 B. cereus Georgalaki et al., 2002
macedonicus B. subtilis
C. sporogenes
C. tyrobutyricum

Macedovicin I Streptococcus 3428.8c Broad ND ND B. licheniformis Georgalaki et al., 2013

macedonicus ACA-DC C. sporogenes
198 C. tyrobutiricum

Nisin I Lactococcus lactis 3353.53c Broad Heat stable 2–6 A. acidoterrerstris Meghrous et al., 1999;
subsp. lactis B. anthracis Pirttijärvi et al., 2001;
B. amyloliquefaciens Wijnker et al., 2011;
B. cereus Hofstetter et al., 2013;
B. coagulans Oshima et al., 2014;
B. fliexus Aouadhi et al., 2015
B. licheniformis
B. pumilus
B. sporothemodurans
C. beigerinckii
C. butyricum
C. perfringens
C. sporogenes
C. tyrobutyricum
Paenbacillus jamilae

Nisin Z I Lactococcus lactis 3330.93 Broad Heat stable 2–6 B. cereus Rollema et al., 1995;
NIZO 22186 B. pumilus Meghrous et al., 1999;
B. subtilis Noonpakdee et al.,
C. butyricum 2003; Park et al., 2003;
C. perfringens Rilla et al., 2003;
C. sporogenes Rumjuankiat et al.,
C. tyrobutyricum 2015

Nisin Q I Lactococcus lactis 3327.5 Broad Heat stable ND B. circulans Zendo et al., 2003
61-14 B. coagulans
B. subtilis

(Continued)

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TABLE 1 | Continued

Bacteriocin Class Producer Size (Da) Spectrum Heat stability Active Sensitive References
pH Spore-formers

Pediocin A IIa Pediococcus 80,000a Broad Heat sensitive ND B. cereus Piva and Headon, 1994
pentosaceus FBB61 C. sporogenes
C. tyrobutyricum

Pediocin IIa Pediococcus 4624c Broad Heat stable 2–10 B. cereus Marugg et al., 1992;
AcH/PA-1 acidilactici PAC 1.0 C. butyricum Meghrous et al., 1999;
C. perfringens Rodríguez et al., 2002;
C. sporogenes Nieto-Lozano et al.,
C. tyrobutyricum 2010

Pediocin AcM IIa Pediococcus 4618c Broad Heat stable 1–12 B. cereus Elegado et al., 1997
acidilactici M B. coagulans
C. perfringens

Pediocin L50 IId Pediococcus 5250c Broad Heat stable 2–11 B. cereus Cintas et al., 1995
acidilactici L50 C. botulinum
C. perfringens
C. sporogenes
C. tyrobutyricum

Pentocin TV35b ND Lactobacillus pentosus 3930 Broad Heat stable 1–10 C. sporogenes Okkers et al., 1999
TV35b C. tyrobutyricum

Plantaricin 163 IId Lactobacillus 3553.2 Broad Heat stable 2–10 B. cereus Hu et al., 2013
plantarum 163

Plantaricin 423 IIa Lactobacillus 3932c Narrow Heat stable 1–10 B. cereus van Reenen et al.,
plantarum 423 C. sporogenes 1998; Mills et al., 2011
Lactobacillus
plantarum LMG
P-26358

Plantaricin C Ì Lactobacillus 2880.3c Broad Heat stable <8 B. subtilis Gonzalez et al., 1994
plantarum LL441 C. sporogenes
C. tyrobutyricum

Plantaricin IId Lactobacillus 3497.97c Broad Heat stable 2–12 B. cereus Rumjuankiat et al.,
KL-1Y plantarum KL-1 B. coagulans 2015
B. subtilis

Plantaricin ND Lactobacillus 1000 - 5000a Broad Heat stable ND B. cereus Suma et al., 1998
LP84 plantarum NCIM 2084 B. licheniformis
B. subtilis

Plantaricin IId Lactobacillus 2572.9c Broad Heat stable 2–6 B. subtilis Song et al., 2014
PZJ5 plantarum ZJ5

Plantaricin S IIb Lactobacillus α 2904c Broad Heat stable 3–7 C. tyrobutyricum Soliman et al., 2011
plantarum LPC010 β 2873c

Plantaricin ND Lactobacillus 2755c Broad Heat stable 3–8 B. subtilis Todorov et al., 1999
ST31 plantarum ST31

Plantaricin ND Lactobacillus 2500a Broad Heat stable 1–9 B. cereus Hernández et al., 2005;
TF711 plantarum TF711 C. sporogenes González and Zárate,
2015

Plantaracin ND Lactobacillus 3000- Narrow Heat stable 3.5–8 B. cereus Enan et al., 1996
UG1 plantarum UG1 10,000a C. perfringens
C. sporogenes

(Continued)

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Egan et al. Inhibiting Spores with Bacteriocins

TABLE 1 | Continued

Bacteriocin Class Producer Size (Da) Spectrum Heat stability Active Sensitive References
pH Spore-formers

Plantaricin ND Lactobacillus 1334.77 Broad Heat stable 2–8 B. subtilis Zhu et al., 2014
ZJ008 plantarum ZJ008

Salivaricin D I Streptococcus 3467.55 Broad Heat stable ND B. subtils Birri et al., 2012
salivarius 5M6c C. butyricum
C. bifermentans

Thermophilin I Streptococcus 3700a Broad Heat stable 3–10 B. cereus Kabuki et al., 2007
1277 thermophilus SBTI1277 C. butyricum
C. sporogenes
C. tyrobutyricum

Themophilin 13 IIb Streptococcus 5776c Broad ND ND B. cereus Marciset et al., 1997

thermophilus SFi13 B. subtilis
C. botulinum
C. tyrobutyricum

Thermophilin T ND Streptococcus 2500a Narrow Heat stable 1–12 C. sporogenes Aktypis et al., 1998
thermophilus ACA-DC C. tyrobutyricum
0040

VJ13B IIa Pediococcus 4000a Broad Moderate 2–8 B. cereus Vidhyasagar and
pentosaceus VJ13 B. subtilis Jeevaratnam, 2013
C. perfringens
C. sporogenes

Weissellicin Y IId Weisella hellenica Q13 4925c Broad Heat stable 3–11 B. cereus Masuda et al., 2012
B. circulans
B. subtilis
B. coagulans

Weissellicin M IId Weisella hellenica Q13 4968c Broad Moderate 3–11 B. cereus Masuda et al., 2012
B. circulans
B. coagulans
B. subtilis

a Mass estimated using SDS-PAGE.

b Mass calculated based on amino acid sequence.
c Mass obtained using mass spectrometry.

ND, Not determined.

have similar/the same mode of action, and (vii) they are active over 50 countries to improve food safety and extend shelf life.
against a range of food pathogenic and spoilage bacteria. Other important members of this class include: the two peptide
This review will focus solely on bacteriocins produced by the lantibiotic lacticin 3147 produced by L. lactis DPC 3147 (Suda
LAB because these bacteriocins possess the greatest promise with et al., 2012), subtilin produced by Bacillus subtilis ATCC 6633
respect to use in the food industry. (Lee and Kim, 2011), and lacticin 481 produced by L. lactis
CNRZ 481 (Piard et al., 1993). Lantibiotics undergo extensive
Classification of Bacteriocins Produced by post-translational modifications, resulting in the presence of
the LAB unusual amino acids such as lanthionine, β-methyllanthionine,
LAB bacteriocins may be classified into two separate classes based dehydrobutyrine, and dehydroalanine. Covalent bonds are
on their modification status: Modified (class I), and minimally formed between these non-standard residues, resulting in
modified or cyclic (class II; Rea et al., 2011; Cotter et al., 2013). internal rings which are important for its potent activity (Rink
Class I are comprised of all peptides that undergo post- et al., 2007).
translational modification during biosynthesis and include the Class II bacteriocins are <10 kDa, heat stable and non-
subclass of lantibiotics among others. While several other modified that can be further subdivided into four subgroups: IIa
subclasses within class I have been described (Arnison et al., pediocin like, IIb two peptide bacteriocins, IIc cyclic bacteriocins,
2013; Cotter et al., 2013), this review will focus mainly on those and IId single linear non-pediocin bacteriocins. Members of class
with relevance to the food industry. The commercially important IIa are Listeria-active peptides which contain a conserved amino
bacteriocin nisin is produced by L. lactis and is the prototypical acid consensus sequence across all members of this group: Y-
member of the class I lantibiotics. Nisin is currently used in G-N-G-V-X1 -C-X2 -K/N-X3 -X4 -C (where X is any amino acid)

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Egan et al. Inhibiting Spores with Bacteriocins

(Cui et al., 2012). This consensus sequence is often referred to to vancomycin (Gut et al., 2011). Other bacteriocins that exert
as the “pediocin box” and is present at the N-terminal region of their bactericidal mechanism of action by inhibition of cell wall
the class IIa bacteriocins. Class IIb bacteriocins are unmodified biosynthesis are mersacidin, which inhibits transglycosylation
two peptide bacteriocins, which interact to give full activity; (Brötz et al., 1995), and lactococcin 972 which targets septum
having little or no activity in isolation. Class IIc bacteriocins biosynthesis via lipid II (Martínez et al., 2008). While lipid
are covalently linked from their N to C termini during post- II is an important receptor for certain bacteriocins, there are
translational modification resulting in a circular backbone. Class however other receptors to which bacteriocins bind on the Gram-
IId are a heterogeneous group, made up of bacteriocins which are positive cell such as: the mannose PTS system, the maltose ABC
linear, do not contain a pediocin box and do not require another transporter, Zn-dependent metallopeptidase, and undecaprenyl
peptide for full activity. pyrophosphatase phosphatase (see review by: Cotter, 2014).
Indeed these bacteriocin-receptor complexes play an important
Using Bacteriocins Produced by role in specifying a bacteriocins spectrum of activity. The
outer cell membrane of Gram-negative bacteria provides an
Enterococcus in Food
effective barrier to bacteriocins from binding their lipid II targets.
The bacteriocins produced by Enterococcus species are diverse,
However, Gram-negative bacteria can be sensitized toward
both in terms of their classification and inhibitory spectrum
bacteriocins if treated with agents or chemicals that destabilize
(Table 1). While most LAB are GRAS, and thus their associated
the outer cell membrane (such as sodium phosphate buffer or
bacteriocins can be considered for food applications, the status
EDTA).
of enterococci is less clear. Indeed, many strains are clearly
Bacteriocins may also kill or damage cells by pore formation in
not food grade. Although Enterococcus species have been used
the cell membrane. This pore formation is achieved by insertion
as artisanal cultures in a variety of foods, their suitability
of the bacteriocin into the cell membrane, forming a membrane
for use in food is questionable as they have been sometimes
pore. This pore results in depolarization of the membrane
associated with pathogenicity. Indeed, cases of urinary tract
potential and diffusion of low molecular cytosolic compounds
infections, bacteremia and endocarditis have been associated
out of the cell; ultimately rendering the bacterial cell non-
with Enterococcus species (Franz et al., 1999; Kayser, 2003). De
viable. Enterocin AS-48 is predicted to form aggregates which
Vuyst et al. (2003) suggested that Enterococcus species could
insert into the bacterial membrane, resulting in accumulation of
be safely used in food if virulence genes are absent (cytolysin,
positive charge along the cell surface, destabilizing the membrane
vancomycin resistance, etc.). However, in a review by Franz
potential, leading to pore formation and cellular leakage. Other
et al. (2011), the ability of Enterococcus to acquire virulence
bacteriocins that form pores include: streptococcin SA-FF22,
and antibiotic resistance genes on mobile genetic elements was
lacticin F, and lactococcin A (Héchard and Sahl, 2002).
identified as a significant barrier to their use in food. Recently,
There are a number of members of the bacteriocins that
Jaouani et al. (2015) examined the safety of previously identified
exhibit dual modes of antimicrobial action by both: forming
bacteriocinogenic enterococci, by examining the presence of
pores and inhibiting cell wall biosynthesis. The ability of such
virulence and antibiotic resistance genes. Using these criteria, it
bacteriocins to act through two mechanisms of action can
was concluded that 22/55 of the strains tested were safe for use
prevent the development of bacteriocin resistance. Moreover, it is
in food. Ultimately, Enterococcus are an important reservoir for
worth noting that microorganisms that are resistant to antibiotics
bacteriocin discovery and therefore developing a comprehensive
generally do not display cross-resistance to bacteriocins (Jordan
set of guidelines/considerations for their safe use would be highly
et al., 2014). Nisin (Wiedemann et al., 2001), pediocin PA-
valuable when considering their suitability for use in food.
1 (Diep et al., 2007), lacticin 3147 (Wiedemann et al., 2006),
epidermin (Götz et al., 2014), and gallidermin (Götz et al., 2014)
Bacteriocin Mode of Action against are examples of bacteriocins that display a dual mode of action,
Vegetative Cells making their activity particularly potent against their targets.
Mechanistically, bacteriocin molecules produced by the LAB act
by one, or both, of two different mechanisms: (i) inhibition of cell
wall biosynthesis, and (ii) pore formation. BACTERIOCIN-SPORE INTERACTIONS
At the cell envelope, lipid II plays a key role in the
synthesis of peptidoglycan as it transports cell wall subunits In comparison to the vast knowledge available with respect to
across the bacterial cytoplasmic membrane. Lipid II delivers its bacteriocin interactions with vegetative cells, it is safe to say
peptidoglycan subunit cargo from the cytosol to an exterior that there is considerably less known about bacteriocin/spore
multi-enzyme complex which is responsible for polymerization interactions. However, a small number of bacteriocins (Table 2)
of that subunit into the peptidoglycan cell wall. The halting for which activity against a variety of bacterial spores has
of cell wall biosynthesis by sequestering lipid II is a strategy been demonstrated. Phase contrast microscopy can be utilized
employed by a number of antimicrobial compounds which to determine at what stage in the spore cycle (Figure 1) the
results in cell death (see review by: Oppedijk et al., 2015). The bacteriocin exhibits its anti-spore activity by combining the
important clinical antibiotic vancomycin also targets lipid II, bacteriocins with dormant (phase bright) and germinated (phase
though its lipid II binding site is distinctly different to the dark) spores. Spore viability can then be examined following the
lantibiotic nisin. The alternative binding site for nisin results treatment with bacteriocin to determine the bacteriocins effect
in the ability of nisin to kill bacterial cells which are resistant on the spore. Two outcomes may ensue: the bacteriocin (i) does

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Egan et al. Inhibiting Spores with Bacteriocins

TABLE 2 | Bacteriocins that display inhibitory action against bacterial spores.

Bacteriocin Sensitive spores References

Nisin A. acidoterrestris, B. amyloliquefaciens B. anthracis, B. licheniformis, Komitopoulou et al., 1999; Mansour et al., 1999; Wandling et al., 1999;
B. sporothermodurans, B. subtilis, B. cereus, G. stearothermophilus, Pol et al., 2000; Black et al., 2008; Gut et al., 2008; Udompijitkul et al.,
C. perfringens, C. sporogenes, C. botulinum, C. difficile, C. beijerinckii 2012; Hofstetter et al., 2013; Nerandzic and Donskey, 2013; Aouadhi
et al., 2015
Enterocin AS-48 A. acidoterrestris, B. cereus, B. licheniformis, G. stearothermophilus Abriouel et al., 2002; Lucas et al., 2006
Bificin C6165 A. acidoterrestris Pei et al., 2014
Lacticin 3147 C. tyrobutyricum Martinez-Cuesta et al., 2010
Plantaricin TF711 C. sporogenes González and Zárate, 2015
Thurincin H B. cereus Wang et al., 2014

TABLE 3 | Bacteriocin mode of action against bacterial spores is heterogeneous.

Bacteriocin Spore Effect on Effect on Requires Other remarks References

germination dormant germination
rate spores to be active

Nisin B. anthracis None None Yes Lipid II becomes available for Gut et al., 2008, 2011
nisin to bind following
germination, followed by pore
formation in the outgrowing
spore.
B. sporothermodurans Decreased None Yes Aouadhi et al., 2015
rate
B. licheniformis None None Yes Mansour et al., 1999
C. butyricum ND None Yes Ramseier, 1960
C. botulinum Increases None Yes Mazzotta and Montville, 1999
rate
C. difficile None None Yes Nerandzic and Donskey, 2013
C. perfringens None None Yes Udompijitkul et al., 2012

Enterocin AS-48 A. acidoterrestris ND Sporicidal No Grande et al., 2005

B. cereus None None Yes Abriouel et al., 2002
B. coagulans ND None Yes Lucas et al., 2006
B. licheniformis ND None Yes Grande et al., 2006a
G. stearothermophilus ND None Yes Viedma et al., 2009

Thurincin H B. cereus None None Yes Wang et al., 2014

ND, Not determined.

not require germination and will be sporicidal against dormant germination, as examined on the basis of spore refractility, with
spores, or (ii) will be sporostatic to dormant and germinated or without nisin. Conversely, the presence of 25 µg/ml of nisin
spores but requires germination to inhibit spore outgrowth. has been shown to have a progerminant activity for C. botulinum
Bacteriocins can also affect the germination rate of the spore, spores, as when it was present in the germination medium, the
which can be examined by measuring the drop in absorbance germination rate was doubled. However, the presence of nisin
(OD600 nm ) of a dormant spore suspension as it transitions to a (125 µg/ml) has been shown to decrease the germination rate of
germinated spore suspension over a time period. These outcomes B. sporothermodurans spores (Aouadhi et al., 2015).
are however heterogeneous (Table 3), with differences occurring With respect to anti-B. anthracis activity, it has been reported
at species level where the same bacteriocin was used, and will be that nisin exerts its inhibitory effect after germination initiation,
further discussed below. where nisin binds lipid II in the germinating spore and
prevents it from becoming metabolically active by interfering
Nisin with the establishment of a membrane potential and oxidative
Previous studies have shown that for B. anthracis (Gut metabolism. Germination initiation is required for this lipid II
et al., 2008, 2011), B. licheniformis (Mansour et al., 1999), binding to occur, as nisin is unable to associate with the dormant
C. difficile (Nerandzic and Donskey, 2013), and C. perfringens spore due to the absence of lipid II on the exterior of the spore
(Udompijitkul et al., 2012), nisin had no impact on the process of (Gut et al., 2011). When investigating the effects of nisin on C.
germination, as it neither initiated, inhibited, or altered the rate of perfringens spores, it was observed that, as for studies involving

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Egan et al. Inhibiting Spores with Bacteriocins

B. anthracis and C. butyricum, nisin exhibited its inhibitory activated spores below the level of detection in rice gruel after 24
action during the stage of spore outgrowth (Udompijitkul et al., h at three different temperatures (6, 15, and 37◦ C; Grande et al.,
2012). Using a truncated nisin derivative consisting of rings A, 2006b).
B and C (which could bind lipid II but not form pores) and Outgrowth inhibition of the important thermophilic spore-
fluorescently labeled unmodified nisin, it was shown that lipid former Geobacillus stearothermophilus has also been shown using
II binding alone was insufficient to inhibit spore outgrowth. This enterocin AS-48. G. stearothermophilus is regularly identified as
was further investigated using the double mutants N20P/M21P a spoilage agent in low acid canned food, being highly heat
and M21P/K22P, which were unable to form pores, but could resistant with a D121◦ C value of 1 min, so its removal from canned
bind lipid II. These nisin mutants were again shown to be products require an extensive heat treatment (Durand et al.,
unable to inhibit spore outgrowth. Through the use of the double 2015). Viedma et al. (2009) tested the efficacy of enterocin AS-
mutant and truncated nisin, it is clear that pore formation is 48 in inhibiting spore outgrowth of G. stearothermophilus using
the essential mechanism by which nisin inhibits spore outgrowth three food models, canned corn, canned peas and coconut milk,
while lipid II is the target for nisin (acting as a receptor using a cocktail of two G. stearothermophilus strains. Here it was
for nisin) to inhibit outgrowth in the germinating spore (Gut shown that AS-48, used at 1.75 µg/ml, reduced the viable counts
et al., 2008, 2011). While it has been shown that truncated of heat treated spores below the level of detection after 24 h. B.
nisin consisting of rings A, B, and C does not inhibit spore licheniformis was controlled in a commercial cider by AS-48 at a
outgrowth in B. anthracis, it has been reported elsewhere that this level of 5 µg/ml at 30◦ C. A significant reduction was observed
peptide does inhibit outgrowth of B. subtilis (Rink et al., 2007). in a population of germinated spores following treatment with
While the mechanisms underlying these differing results have AS-48 (Grande et al., 2006a).
not yet been completely elucidated, some possible explanations The genus Alicyclobacillus has in recent years become a
given were (i) differences in outgrowth measurement methods problem in the food industry. Members of this genus have an
and (ii) potential spore structure variations (Gut et al., 2011). ability to grow at high temperatures (50–70◦ C), and at low pH
Nisin however displays sporicidal activity against dormant values (3.0–3.5), which makes their eradication from certain
B. sporothermodurans (Aouadhi et al., 2013), in contrast foods problematic. A. acidoterrestris is a particular problem in
with the sporostatic activity against other targets described acidic juice products such as apple, tomato and orange, amongst
above. others (Steyn et al., 2011). Inhibition of A. acidoterrestris spores
The ability of microorganisms to develop resistance by enterocin AS-48 has been observed at concentrations as low as
mechanisms to bacteriocins is a concern that could impede 2.5 µg/ml. At this concentration a reduction of 6 Log10 spores/ml
their widespread use in food (see review by: Draper et al., was achieved. Using electron microscopy it was observed that
2015). Nisin resistance has been reported for toxigenic spores the enterocin AS-48 treated spore structures sustained substantial
of C. botulinum which had the ability to germinate and grow in damage supporting the hypothesis that the bacteriocin adsorbs
levels of nisin that reduced levels of sensitive germinating spores to the spores negatively charged surface groups. This interaction
by 7–8 logs10 /ml (Mazzotta and Montville, 1999). The exact with A. acidoterrestris would suggest a sporicidal rather than the
mechanism by which these spores exhibited nisin resistance is sporostatic mechanism of action that is suggested for B. cereus
unknown but, interestingly it has been noted that nisin resistant (Grande et al., 2005).
strains have an altered fatty acid composition, which is consistent
with a more rigid membrane. It has also been observed that nisin Lacticin 3147
resistant strains of C. botulinum display cross-resistance to class Lacticin 3147, produced by L. lactis subsp. lactis DPC3147, has
II bacteriocins (Mazzotta et al., 1997). been shown to inhibit spores of C. tyrobutyricum in milk. This
Clostridium species is responsible for late blowing in hard cheese,
Enterocin AS-48 as their spores can survive heat treatments and germinate in the
Enterocin AS-48 produced by Enterococcus faecalis A-48-32 is ripening cheese. Previously nitrate was used to combat clostridia
a class IIc cyclic bacteriocin that is active against a number of but has been banned by the European Food Safety authority
Bacillus and Clostridium sp. (Table 1). Unlike nisin, the exact (EFSA) in an effort to reduce nitrosamines in food (Bassi et al.,
molecular mechanism by which enterocin AS-48 interacts with 2015). When used at a concentration of 45 µg/ml, lacticin
bacterial spores is unknown. It was observed that spores of B. 3147 was also able to completely inactivate 4–5 Log10 spores/ml
cereus became more sensitive to enterocin AS-48 gradually after over a 24 h period. Additionally, when lacticin 3147 was added
germination and were sensitive to 25–50 µg/ml 10 min after following a 24 h incubation of the spores, total inactivation
germination initiation. The greatest effect of enterocin AS-48 was 6 days post addition of the bacteriocin was observed. In situ
observed 90–120 min after germination initiation, when cellular production of lacticin 3147, in a model curd system, has also
growth occurred (Abriouel et al., 2002). Enterocin AS-48 has been shown to significantly reduce (3 Log10 spores/g) the number
also been shown to be effective in inhibiting spore outgrowth of Clostridium spores after 13 days, when compared to a non-
using heat activated spores of B. cereus. In a boiled rice substrate, bacteriocin producing control. After 60 days of ripening, lacticin
25 µg/ml of enterocin AS-48 reduced heat activated spores 3147 produced in situ was shown to be effective in reducing the
incubated at 37 and 15◦ C, below the level of detections after levels of artificially contaminated clostridia (introduced prior to
3 days, whereas at 6◦ C, this reduction took 14 days. A higher ripening) from 8 to 2 Log10 spores/g (Carmen Martínez-Cuesta
concentration of 35 µg/ml of enterocin AS-48, reduced the heat et al., 2010).

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Egan et al. Inhibiting Spores with Bacteriocins

Bificin C6165 bacteriocins which suggests that these peptides may in fact be
Bificin C6165 produced by Bifidobacterium animalis subsp. bacteriolysins (Yin et al., 2003).
animalis CICC 6165 was shown to inhibit species such as
Lactobacillus, Bifidobacterium, Enterococcus, Staphylococcus, and Comparing the Sensitivity of Spores and
Alicyclobacillus acidoterrestris. Indeed, from an anti-sporeformer
perspective, it is notable that bificin C6165 inhibited 20/20 strains
Vegetative Cells to Bacteriocins
To date there has been conflicting reports as to whether
of A. acidoterrestris tested. Bificin C6165 could also reduce a
germinated spores are more or less resistant to bacteriocins than
population of A. acidoterrestris spores and was more effective as
vegetative cells. Heat activated spores of B. sporothermodurans
the concentration of the bacteriocin increased (Pei et al., 2013).
are less sensitive to nisin (1.25 µg/ml), than vegetative cells
Another important characteristic of bificin C6165 which makes it
of B. sporothermodurans (Aouadhi et al., 2015). The Minimum
an ideal candidate for inhibition of A. acidoterrestris is its activity
Inhibitory Concentration (MIC) of nisin for vegetative cells of
at acidic pH 3.5–6.5 (Pei et al., 2014).
C. butyricum, C. perfringens, C. sporogenes, and C. tyrobutyricum
was found to be 0.17, 0.75, 38.4, and 4.8 µg/ml, respectively.
Plantaricin TF711
However, 23 µg/ml of nisin prevented outgrowth of heat
Plantaricin TF711, produced by Lactobacillus plantarum TF711,
activated Clostridium spores for up to 10 days. Unfortunately in
is active over a broad pH range and is active against vegetative
this study it is unclear whether the vegetative cells were more or
cells of B. cereus and C. sporogenes (Hernández et al., 2005).
less resistant than their spores to the nisin treatment as no MIC
C. sporogenes acts as a research surrogate for proteolytic C.
for spores was carried out (Meghrous et al., 1999). Another study
botulinum as these two species are closely related. This species
found that vegetative cells of C. sporogenes were less resistant
has also been associated with late blowing of hard cheese (Bassi
to nisin than heat activated spores, yielding MICs of 0.23 and
et al., 2015). Plantaricin TF711 was shown to reduce C. sporogenes
1.11 µg/ml, respectively. In contrast, it was revealed that heat
spore counts significantly from 7 days onwards when introduced
activated C. beijerinckii spores were less resistant with an MIC
in the form of an adjunct culture producing the bacteriocin in
of 1.09 µg/ml while their vegetative cells exhibited an MIC of
situ. The bacteriocin was shown to be present at highest levels at
1.3 µg/ml (Hofstetter et al., 2013). At odds with these findings,
day 21, after which its activity declined. This decline in activity
however, were the results obtained by Ávila et al. (2014), which
could be due to loss of stability, depletion of the bacteriocin in
compared the sensitivity of spores and vegetative cells of four
the cheese, or reduced production of the bacteriocin (González
clostridia: C. tyrobutyricum, C. butyricum, C. beijerinckii, and
and Zárate, 2015).
C. sporogenes. Using four representatives of each species, they
Thurincin H showed that spores had a higher MIC, and thus were more
Thurincin H produced by B. thuringiensis SF361 has been resistant to nisin, than their vegetative counterparts in 15 of
shown to be sporostatic against dormant B. cereus spores the 16 strains tested. The only exception was displayed by C.
and sporicidal against germinated B. cereus spores. Similarly tyrobutyricum CET 4011 strain where the vegetative and spore
to other bacteriocins, thurincin H displays sporicidal activity MIC values were equal at 0.39 µg/ml. It is also important to note
after germination, while it was sporostatic to dormant spores. that in this case all the MIC values were below the maximum
Although not an LAB bacteriocin, it has been suggested that permissible limit for nisin, which is 12.5 µg/ml in Europe.
Thurincin H may have potential for use in food (Wang et al., Spores of A. acidoterrestris were found to be more sensitive
2014). to nisin than their vegetative cells. The MIC values for both
spores and vegetative cells were carried out in mYPGA at two
Other Bacteriocins Active against Bacterial different pH values (pH 3.4 and pH 4.2). Interestingly, at pH 3.4,
all spores were more sensitive (7/7) than their vegetative cells.
Spores However, at pH 4.2 (3/7) spores had equal MIC-values to their
There are a number of other bacteriocins that have shown
vegetative cells (Yamazaki et al., 2000). Whether this is due to the
potential. Some of these are described here. Soria and Audisio
(i) enhanced activity of nisin at lower pH, (ii) negative effects of
(2014) revealed that heat activated B. cereus spores could be
pH on the spore or (iii) a combined activity of both, has yet to be
inhibited by the cell free supernatant of E. faecium SM21
determined. These findings were further confirmed by Ruiz et al.
containing an enterocin which produced a bacteriostatic effect
(2013), who found the MIC of spores and vegetative cells of A.
at both pH 5 and pH 6. Bacteriocin production by Streptococcus
acidoterrestris to be 7.81 and 31.25 µg/ml, respectively.
thermophilus 580 was capable of inhibiting C. tyrobutyricum
gas production in a ripening curd model for up to 14 days,
when compared to controls which produced gas after 14 days Inhibition of Spore Outgrowth Prevents
(Mathot et al., 2003). Pentocin L and pentocin S, are produced Toxin Formation
by Pediococcus pentosaceus L and S, respectively. Both of Toxin formation is an important feature of a number Clostridium
these bacteriocins are inhibitory against a variety of vegetative and Bacillus species. There are two types of toxin with which
Bacillus and Clostridium strains (Table 1). Furthermore, these B. cereus strains are frequently associated: (i) heat labile
bacteriocins were shown to be sporostatic by inhibiting the diarrheal enterotoxin and/or (ii) heat-stable emetic enterotoxin.
germination of three different strains of non-heat activated Beuchat et al. (1997) showed that the production of diarrheal
B. cereus spores. These active proteins are larger than typical enterotoxin produced in beef gravy inoculated with B. cereus

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Egan et al. Inhibiting Spores with Bacteriocins

spores could be inhibited by addition of nisin. Enterotoxin 4◦ C in chocolate milk for 15 and 24 h, resulted in significantly
production normally occurred after 3 and 9 days for heat reduced D130◦ C values of 20.5 and 25.1%, respectively, compared
activated B. cereus spores incubated at 15 and 8◦ C, respectively. to those spores not pretreated with nisin. When the nisin
Addition of 1 µg/ml of nisin inhibited enterotoxin production pretreatment was raised to 100 µg/ml this did not cause a
completely at 8◦ C, whereas a higher concentration of 5 µg/ml significant reduction over the lower concentration of 50 µg/ml
was needed to inhibit enterotoxin production at 15◦ C over (Beard et al., 1999). B. amyloliquefaciens spores were rapidly
a 14 day period. The levels of nisin required to prevent inactivated when treated with 90◦ C and 16 µg/ml of nisin, in
enterotoxin production from a spore inoculum also ensured contrast to the results when a 90◦ C treatment was used, alone,
that the final cell numbers did not exceed 4.03 and 6.23 where there was no inactivation of spores (Hofstetter et al.,
Log10 CFU/g at 8 and 15◦ C, respectively. Without nisin, 2013).
enterotoxin was produced when cell numbers exceeded 6.78 A reduction of 2 Log10 spores/ml was observed when spores
and 7.1 Log10 CFU/ml at 15 and 8◦ C, respectively. This is in of C. sporogenes spores were subjected to a heat treatment of
agreement with the strategy of keeping the B. cereus population 90◦ C for 2 h in the presence of 16 µg/ml nisin vs. a 90◦ C
below ∼7 Log10 CFU/g to prevent enterotoxin production heat treatment without nisin. Additionally there was 30% greater
(Christiansson et al., 1989). It would be interesting to see if DPA release when spores of C. sporogenes were heat treated at
the cell numbers in the presence of nisin were allowed to 90◦ C with nisin than those spores which were not treated in any
exceed these numbers would enterotoxin be still be produced or way. However, when C. beijerinckii was subjected to the same
would the enterotoxin production cease due to the presence of conditions (90◦ C for 2 h and 16 µg/ml nisin), no increased
nisin. inactivation was observed. The ability of nisin to increase the
Enterocin AS-48 was also shown to have an effect on permeability of resting spores of C. sporogenes and C. beijerinckii
enterotoxin production by psychrotrophic vegetative cells of was observed using DAPI staining. Fluorescence was observed
B. cereus. Enterocin AS-48 completely inhibited enterotoxin after a treatment at 90◦ C with nisin, whereas a heat treated
production and bacterial growth for at least 72 h when used spore without nisin that did not fluoresce (Hofstetter et al.,
at 7.5 µg/ml. When enterocin AS-48 was used at subinhibitory 2013). These findings are consistent with the hypothesis that
concentrations (2.5 or 5 µg/ml) the growth of the cells were nisin lowers the heat resistance of spores by permeabilizing their
severely subdued and enterotoxin titres were 10-fold lower than exterior.
non-bacteriocin treated controls (Abriouel et al., 2002). Response surface technology (RSM) is an empirical modeling
technique that can be used to examine and predict the
COMBINING BACTERIOCINS WITH OTHER relationship between the response variable and the test variable.
RSM can be used to predict optimum processing conditions to
HURDLES
achieve a pre-determined reduction in spores (Table 5).
Bacteriocins in Combination with Heating Dormant B. coagulans spores were shown to be resistant to
The thermal resistance of bacterial spores makes their eradication enterocin AS-48 in that use of 6 µg/ml bacteriocin resulted in
from food by heat a major problem during food processing. an approximately one log reduction in the number of viable
Nisin at various concentrations has been shown to reduce cells when dormant spores were treated with the bacteriocin
the decimal reduction times (D-values) and thus the thermal in three food models: (i) tomato paste, (ii) syrup from canned
resistance of bacterial spores. Therefore, nisin has been described peach, and (iii) juice from canned pineapple. However, using
as a compound with a “two-fold beneficial effect”: (i) it enterocin AS-48 at 3 and 6 µg/ml in combination with
enhances the heat sensitivity of the bacterial spore (Table 4) heat treatments (5 min at a minimum of 80◦ C) showed a
and (ii) it prevents the outgrowth of spores which survive the significant reduction in the number of viable cells in both
heat treatment (Komitopoulou et al., 1999). Pre-exposing heat food models. When spores were incubated at 22◦ C for 48 h
activated G. stearothermophilus spores to nisin (50 µg/ml) at with bacteriocin, then heat treated at both 80 and 95◦ C,

TABLE 4 | Nisin addition to food reduces spore D-values.

Spore Nisin Conc µg/ml Food model Dx◦ C % decrease in D-value compared References
to non-nisin treated control

B. cereus 25 Milk D80◦ C 40 Vessoni and Moraes, 2002

D90◦ C 16
D97.8◦ C 46
B. cereus 50 Milk D97◦ C 32 Wandling et al., 1999
D100◦ C 20
D103◦ C 42
G. stearothermophilus 100 Milk D130◦ C 20 Wandling et al., 1999
A. acidoterrestris 1.25 Apple juice D80◦ C 42 Komitopoulou et al., 1999

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Egan et al. Inhibiting Spores with Bacteriocins

TABLE 5 | RSM models can be used to predict a treatment to achieve a specific spore reduction in food.

Spore Predicted reduction Treatment Food model employed References

Pressure Temperature Time Nisin

(MPa) (◦ C) (min) (µg/ml)

B. sporothermodurans 5 Log10 spore/ml 95 12 3.125 Water Aouadhi et al., 2014

B. sporothermodurans 5 Log10 spore/ml 100 13 3.35 Milk
B. sporothermodurans 5 Log10 spore/ml 100 15 3.375 Chocolate milk
B. sporothermodurans 5 Log10 spore/ml 472 53 5 5.025 Water Aouadhi et al., 2013
C. perfringens 6 Log10 spore/ml 654 74 13.6 8.2 UHT milk Gao et al., 2011

there was a significant difference in the number of viable but without inactivation, was observed in milk when a higher
cells obtained following both treatments relative to the non- treatment of 500 MPa was used. When spores were treated
heat treated controls or those that were heat treated without with a combination of HP (600 MPa) and nisin (12.5 µg/ml),
bacteriocin (Lucas et al., 2006). A relationship between heat spore germination and inactivation increased to 6 and 3 Log10
temperature and survivors was observed, showing that viable spores/ml, respectively. When cycled twice with nisin there was
counts in samples supplemented with bacteriocin decreased as a further increase in spore germination and inactivation of
the temperature was increased. This relationship was further 8 and 6 Log10 spores/ml, respectively. High pressure-induced
evidenced by the significant reduction in viable counts obtained germination is known not to require the presence of nutrient
from bacteriocin treated spores heat treated at 95◦ C over those receptors and is characterized by a rapid release in DPA-Ca2+
heat treated at 80◦ C. This relationship was observed in all three from the core. Nisin can be characterized as a potent pro-
food models previously discussed (Lucas et al., 2006). Ultimately, germinant in the presence of germinants (naturally present
this study nicely highlights the efficacy of bacteriocins to (i) in milk) such as L-alanine and L-cysteine. Interestingly, nisin
reduce the severity of heat treatments and (ii) increase the doubled the rate of germination in C. botulinum spores, while it
effectiveness of heat treatments, when used to inactivate spores had no effect on nisin resistant spores (Mazzotta and Montville,
in food. 1999). It was hypothesized that inactivation of spores by HHP
Another bacteriocin discussed previously, bificin C6165, has and nisin could be due to (i) nisin and HP acting synergistically
been shown to reduce the D90◦ C value of A. acidoterrestris as to inactivate spores or (ii) HP inducing germination after which
the bacteriocin concentration increased from 0 to 160 µg/ml. nisin exerts its lethal effect on the germinated spore (Black et al.,
Addition of 80 and 160 µg/ml of bificin C6165 was shown to 2008). C. sporogenes spores were also shown to be inhibited
reduce the D90◦ C A. acidoterrestris CFD1 by 32.7 and 42.7%, rapidly by a treatment of nisin and 600 MPa at 90◦ C, relative to a
respectively (Pei et al., 2014). treatment of 90◦ C alone (Hofstetter et al., 2013).
More recently, several studies have used response surface
Bacteriocins in Combination with High methodology (RSM) to test the effectiveness of high pressure,
Pressure heat and nisin. Aouadhi et al. (2013) used RSM to investigate
High-pressure processing (HPP) is a “non-thermal” food the effects of high pressure, in combination with moderate
preservation technique that inactivates harmful pathogens and heat and nisin treatment, on B. sporothermodurans spores.
vegetative spoilage microorganisms by using pressure rather than The authors showed that spore inactivation was concentration
heat to effect pasteurization. HPP utilizes intense pressure (about dependent and that 1.25 and 125 µg/ml caused an inactivation
400–600 MPa or 58,000–87,000 psi) at chilled or mild process of 0.4 and 4 Log10 spores/ml, respectively. Aouadhi et al.
temperatures (<45◦ C), allowing most foods to be preserved (2014) and Gao et al. (2011) showed that RSM (Table 5) could
with minimal effects on taste, texture, appearance, or nutritional be effectively implemented to design an optimum treatment,
value. Microorganisms do however display a variability in their involving multiple parameters to reduce spores loads by a
sensitivity to HHP in the order: Gram-negative bacteria > Gram- predetermined amount.
positive bacteria > bacterial spores. While HPP is an effective Interestingly, superdormant spores of B. cereus and B. subtilis
method used for the destruction of microorganisms in food, it have been shown to germinate similarly to dormant spores when
is not sufficient alone to inactivate spores and therefore must be treated with pressure of 150MPa regardless of whether they were
combined with other hurdles, such as bacteriocins, to increase heat-activated or non-heat-activated. There have, however, been
its efficacy. Indeed, treating food with bacteriocins may be an conflicting reports regarding the ability of pressure treatments to
excellent combination as HHP can induce germination, which cause germination of an entire spore population. This uncertainty
can facilitate the germination-dependent sporicidal activity of has impeded the widespread use of high pressure. It has been
bacteriocins. Black et al. (2008) showed that treatment of 8 Log10 hypothesized that spores which remain superdormant after high
spores/ml of B. subtilis with low pressure (100 MPa i.e., not HHP) pressure may do so via a distinct mechanism from that involved
at 40◦ C in milk resulted in germination and inactivation of 4 and in making some spores superdormant to nutrient germinants
1 Log10 spores/ml, respectively. A similar level of germination, (Wei et al., 2010).

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Egan et al. Inhibiting Spores with Bacteriocins

Bacteriocin in Combination with Pulsed Bacteriocins in Food Packaging

Electric Field The preservation of sausage casings of preserved intestines
Pulsed electric field (PEF) is an innovative food preservation of animals has been practiced for centuries. However, this
method, which may be suitable for reducing spore loads in preservation method has been modernized to suit modern
liquid food. One of the distinct advantages of PEF is that the consumer desires. Such a modernization is the binding of
thermal impacts on food are minimized as this treatment is nisin to sausage casing in order to control Clostridium spore
relatively non-thermal. Any heat produced is directly influenced outgrowth. Wijnker et al. (2011) showed that nisin, at 100
by the energy input of the treatment. While it is known that µg/ml, when bound to casings and placed on agar plates seeded
vegetative cells of B. cereus are sensitive to PEF and nisin with Clostridium spores, produced zones whereas those casings
(Pol et al., 2000) and that this combination is sporostatic with only 50 µg/ml did not. They also observed that addition
but this treatment did not initiate germination nor did they of nisin at 50 µg/ml to the casings delayed C. sporogenes
affect the viability of the dormant spores. After germination, spore outgrowth between 1 and 8 days. Furthermore, at this
B. cereus immediately became sensitive to nisin (1.25 µg/ml) concentration of 50 µg/ml, this sporostatic activity was observed
but it was longer (50 min) before they became sensitive to for 30 days. In contrast, Meghrous et al. (1999) showed that a
PEF (27 kV/cm, 302-µs pulses; flow rate, 10 ml/min). Unlike lower concentration of nisin, 23 µg/ml, delayed clostridial spore
the synergistic activity of nisin and PEF against vegetative cells outgrowth by 10 days. It should also be noted that Wijnker et al.
(Pol et al., 2000), when spores when treated with both PEF (2011) used 106 spores/ml whereas Meghrous et al. (1999) used
and nisin this synergistic activity was not observed as the 103 spores/ml. The reason that nisin at 50 µg/ml could inhibit
reduction was comparable to nisin alone (Pol et al., 2001). While outgrowth in vitro but not on the casings could be due to the
this combination is not synergistic against spores, food rarely irreversible binding of nisin to the collagen matrix of the casing
contains spores alone but rather a mixed population of spores wall. This would suggest that if outgrowth is to be prevented the
and vegetative cells. Therefore, this combination may still be casings need to contain a higher concentration of nisin in order
an effective way of maintaining dormant spore numbers yet to overcome the deleterious effect of irreversible binding to the
reducing the population of vegetative cells for increased food casing.
safety and shelf life.
Bacteriocins in Combination with Plant
Extracts
Plants contain innumerable constituents and are valuable sources
Bacteriocins in Combination with Osmotic of new and biologically active molecules possessing antimicrobial
Activation properties. The plant family Piperaceae are found in tropical
Stimulation of dormant bacterial spore germination followed and subtropical regions and are commonly used as to generate
by subsequent inactivation, as previously discussed, is a medicinal herbs. Ruiz et al. (2013) showed that a combination of
promising method used for spore inactivation. Small, non-polar, nisin and Piper aduncum exhibited a strong antibacterial activity
hydrophobic solutes that permeate the plasma membrane have against spores of A. acidoterrestris and also exhibited a synergism
been shown to stimulate B. cereus germination (Preston and (FIC = 0.24) against A. acidoterrestris vegetative cells. Prenylated
Douthit, 1984). Inhibition of non-heat activated C. difficile spores chromone was identified as the active compound in this plant
was significantly increased when treated with nisin and single extract. Piperaceae extract is a natural food preservation method
osmotic activators (ammonium, glycerol, and Tris) compared that may be combined with nisin to lower (if any) heat treatment
to heat activated spores treated with nisin and solutes in a needed to reduce and inhibit spores outgrowth.
germination medium. For example, nisin in combination with
heating resulted in a 1–2.5 log10 spores/ml decrease in viable Nisin in Combination with Fatty Acid Esters
spores but when nisin was combined with osmotic activators Sucrose fatty esters are approved internationally for use as
this increased to >3.5 log10 spores/ml (Nerandzic and Donskey, emulsifiers and these non-toxic molecules have also been
2013). Using flow cytometry, it was observed that the membrane reported to inhibit Gram-positive bacteria. A combination of
permeability of spores was significantly increased when treated nisin and the fatty acid ester, sucrose palmitate (P-1570),
with osmotic activators. Spores treated with both nisin and displayed synergism against spores of B. cereus whereas sucrose
solute transitioned to phase dark (as spores germinate they fatty acid esters alone caused no decrease in growth (Thomas
appear phase dark using phase contrast microscopy), whereas et al., 1998). Total inhibition of B. licheniformis spore outgrowth
those incubated with nisin and osmotic activators separately was achieved when nisin (0.75 µg/ml) was combined with the
did not transition to phase dark (Nerandzic and Donskey, fatty acid ester monolaurin (100 µg/ml) whereas when these
2013). The proposed synergistic ability of nisin and osmotic treatments were used separately at higher concentrations they
activators to inhibit outgrowth was attributed to the osmotically only partially inhibited outgrowth (Mansour et al., 1999).
induced loss of membrane integrity. Although C. difficile is
of clinical importance, this use of osmotic activation could be Nisin in Combination with Potassium
used to overcome limitations of the germination dependent Sorbate
activity of bacteriocins with other food related strains of Sorbates are extensively used in the food industry, as they
clostridia. are able to inhibit, or delay growth of, spores and vegetative

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Egan et al. Inhibiting Spores with Bacteriocins

populations of bacteria. Although their mechanism of action bacteriocins has yet to be elucidated and a better understanding
is not full defined for bacterial spores, it is has been shown of the methods by which bacteriocins kill bacteria will facilitate
that potassium sorbate inhibits the growth of spores of Bacillus a solid basis for engineering new and more potent derivatives
species (Oloyede and Scholefield, 1994). A combination of with optimized potency and stability. Given that spores must
nisin (1.25 µg/ml) and potassium sorbate (2% w/v) has been germinate to exert their adverse effects, future research should
shown to cause a synergistic reduction in the number of heat focus on stimulating spore germination to enable spores to
activated B. sporothermodurans spores. After 8 h there was be more effectively targeted by bacteriocins in food settings.
∼3 Log10 spores/ml reduction in spores. This reduction in Indeed, recent research provides stimulating evidence for using
spores continued albeit at a slower rate until 5 days where total a germination step prior to spore destruction for promoting
inhibition of B. sporothermodurans spores occurred (Aouadhi inactivation of Bacillus and Clostridial spores (Gut et al., 2008).
et al., 2015). When tested separately at these levels, both nisin Furthermore, although numerous components of the spore
and potassium sorbate inhibited spore outgrowth. Nisin was germination machinery are conserved between spore forming
not sporicidal but rather sporostatic, inhibiting spore outgrowth. members of bacilli and clostridia, significant differences between
While potassium sorbate was not sporicidal, it did significantly the germination of spores of Clostridium perfringens and that of
perturb germination of B. sporothermodurans and inhibited the spores of a number of Bacillus species, both in the proteins and
outgrowth of spores (Aouadhi et al., 2015). This ability of in the signal transduction pathways involved have been revealed
potassium sorbate to inhibit spore germination has previously (Abhyankar et al., 2014; Setlow, 2014a; Olguín-Araneda et al.,
been reported for spores of B. cereus and C. botulinum (Smoot 2015). Indeed, as the number of microbial genome sequences
and Pierson, 1981). has increased dramatically, bioinformatics data contained in
the large number of spore-forming Bacillales and Clostridiales
DISCUSSION genomes that have been sequenced and the information gained
from their analysis, can be used to guide researchers to
While spores are a widely recognized problem in the food develop novel strategies to achieve a complete and permanent
industry the majority of bacteriocin-related studies have focused loss of the spore’s ability to germinate and grow in food
on the elimination of vegetative cells from food. The removal of products.
spores and inhibition of their outgrowth in food is important Regardless of the specific bacteriocin of choice, it is clear
for (i) increasing shelf life and (ii) protecting the consumer that there is considerable evidence of the potential value of
from harmful pathogenic spore-formers. Although, there are bacteriocins with respect to controlling sporeforming bacteria in
numerous bacteriocins which have been characterized as safe and food. In the case of spores, while this activity more frequently
effective molecules for use in food, to date, nisin is the only tends to be sporostatic, there are also examples of sporicidal
bacteriocin which is authorized for use as a food preservative. effects. As is the case for vegetative cells, the mechanisms
While this bacteriocin provides an effective and safe method to via which bacteriocins inhibit spores may be heterogeneous
reduce spore outgrowth in food, it is important to recognize that but ultimately it is apparent that in general bacterial spores
this molecule has its limitations. Bacteriocins in food may be can be controlled using bacteriocins, and their application in
limited by: molecule specific solubility, the active pH range of the combination with other novel non-thermal treatments makes
bacteriocin, inactivation by proteases in food, and the possible their efficacy even greater. The use of the bacteriocins with other
negative interactions that occur between certain bacteriocins and food processing hurdles, such as those previously described, thus
certain food components. One such limitation of nisin is its loss has the potential to satisfy consumer demands for “clean label”
of activity as the pH of the food increases. There are a variety of products, enabling processors to produce foods of optimal quality
bacteriocins which are more active than nisin at higher pH, such and shelf life.
as gassericin A, pediocin AcM, and thermophilin T (Table 1),
however they still need to be further characterized before their AUTHOR CONTRIBUTIONS
use in food may be authorized.
In the majority of cases nisin is only sporicidal against those KE drafted the manuscript. DF, MR, RR, CH, and PC revised and
spores in the outgrowth phase and therefore has no effect on approved the final manuscript.
those spores in the dormant phase. Although this model of nisin
(and other bacteriocins) use in food suggests that germination is FUNDING
a prerequisite for its activity, it is important to note that there
are relatively few studies which investigate bacteriocin/spore KE, DF, CH, PC, MR, RR are supported by the Irish Government
interactions. Furthermore, it should be recognized that the under the National Development Plan, through the Food
only detailed mechanism for bacteriocins/spore interaction is Institutional Research Measure, administered by the Department
that of B. anthracis (Figure 2). Indeed, the limited number of of Agriculture, Fisheries and Food, Ireland (DAFM 13/F/462) to
existing studies highlights the need for further research in this PC and MR, a Science Foundation Ireland (SFI) Technology and
area. Understanding these interactions and mechanisms will Innovation Development Award (TIDA 14/TIDA/2286) to DF,
ultimately lead to a more precise and optimal use of bacteriocins SFI-PI funding (11/PI/1137) to PDC and the APC Microbiome
in food. Undeniably, the mode of action for a great many Insitute under Grant Number SFI/12/RC/2273.

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Egan et al. Inhibiting Spores with Bacteriocins

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