Meat Science 120 (2016) 118–132

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Meat Science

journal homepage: www.elsevier.com/locate/meatsci

Bacteriocins from lactic acid bacteria and their applications in meat and
meat products
Weerapong Woraprayote a, Yuwares Malila a, Supaluk Sorapukdee b, Adisorn Swetwiwathana c,
Soottawat Benjakul d, Wonnop Visessanguan a,⁎
a
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phahonyothin Road, Pathum Thani 12120, Thailand
b
Faculty of Agricultural Technology, King Mongkut's Institiute of Technology Ladkrabang (KMITL), Chalong-krung Road, Ladkrabang, Bangkok 10520, Thailand
c
Faculty of Agro-industry, King Mongkut's Institiute of Technology Ladkrabang (KMITL), Chalong-krung Road, Ladkrabang, Bangkok 10520, Thailand
d
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Meat and meat products have always been an important part of human diet, and contain valuable nutrients for
Received 1 February 2016 growth and health. Nevertheless, they are perishable and susceptible to microbial contamination, leading to an
Received in revised form 8 March 2016 increased health risk for consumers as well as to the economic loss in meat industry. The utilization of bacterio-
Accepted 6 April 2016
cins produced by lactic acid bacteria (LAB) as a natural preservative has received a considerable attention. Inoc-
Available online 13 April 2016
ulation of bacteriocin-producing LAB cell as starter or protective cultures is suitable for fermented meats, whilst
Keywords:
the direct addition of bacteriocin as food additive is more preferable when live cells of LAB could not produce bac-
Lactic acid bacteria teriocin in the real meat system. The incorporation of bacteriocins in packaging is another way to improve meat
Bacteriocins safety to avoid direct addition of bacteriocin to meat. Utilization of bacteriocins can effectively contribute to food
Natural antimicrobials safety, especially when integrated into hurdle concepts. In this review, LAB bacteriocins and their applications in
Meats meat and meat products are revisited. The molecular structure and characteristics of bacteriocins recently discov-
Meat products ered, as well as exemplary properties are also discussed.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction: the need for natural antimicrobials in meat
application
Microbial contamination causes serious safety and quality problems
in meat industry. Meat and meat products, particularly fresh meat, con-
tain adequate amount of water and abundance of proteins and essential
nutrients with favorable pH for supporting microbial growth. The mi-
croorganisms present on meat and its products are in broad spectrum,
ranging from bacteria to yeasts, molds and viruses, depending on type
of the products. By far, microbial issues in meat industry have arisen
mostly due to bacteria (Hui, 2012). As reviewed by Jayasena and Jo
(2013), the main spoilage bacteria in meat include Pseudomonas,
Acinetobacter, Brochothrix thermosphacta, Moraxella, Enterobacter, Lacto-
bacillus, Leuconostoc, and Proteus. Upon a substantial growth of those
spoilage organisms, proteins and lipids of meat and meat products un-
dergo degradation, adversely changing appearance, texture and flavor
of the products (Borch, Kant-Muermans, & Blixt, 1996). Normally, spoil-
age microbes do not harmfully affect health but they can stimulate gas-
trointestinal disturbances when consumed in high concentrations
(Jayasena & Jo, 2013).
⁎ Corresponding author.
E-mail address: wonnop@biotec.or.th (W. Visessanguan).
In addition to microbial spoilage, meat and its products are also
prone to contamination by pathogenic microorganisms. Nine major
pathogenic bacteria associated with meat and meat products include
Salmonella spp., thermophilic Campylobacter jejuni, enterohemorrhagic
Escherichia coli O157:H7, Clostridium perfringens, anaerobic Clostridium
botulinum, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus,
and Yesinia enterocolitica, causing illness or even death in humans (Hui,
2012). The outbreaks caused by contamination of those pathogens in
meat are steadily occurred. An example of the recent foodborne out-
break results from the contamination of Salmonella in pork reported
during April to September 2015 from five states along the West Coast
of the US (Center of Disease Control and Prevention, 2015), leading to
a major recall of more than 520,000 pounds of pork from the responsi-
ble company (Johnston, 2015). This multistate outbreak resulted in a
total of 192 ill people, 30 were hospitalized. Luckily, no death was re-
ported in this case. The incidence, however, instantly became headlines
in global-wide media, raising consumer suspicion in safety of meat and
its products.
Application of physical and chemical technologies has been
employed to inactivate microbes in meat and meat products. Physical
processes, such as freezing, refrigerating or thermal processing, howev-
er, may not completely assure safety of meat and its products and meet
consumer satisfaction. Antimicrobial agents have been widely used.
Incorporation of antimicrobial compounds with non-thermal

http://dx.doi.org/10.1016/j.meatsci.2016.04.004
0309-1740/© 2016 Elsevier Ltd. All rights reserved.
 W. Woraprayote et al. / Meat Science 120 (2016) 118–132
processing is of interest as this alternative hurdle technique can en-
hance microbial inactivation and allow preservation of desirable
characteristics of most foods (Ross, Griffiths, Mittal, & Deeth, 2003).
The active compounds provide preventive effects during the pro-
cessing as well as against post-process microbial contamination,
extending shelf life of the products. According to an increased nega-
tive perception towards chemical agents, natural antimicrobial
agents have been extensively screened and tested for their effective-
ness in foods. The approved antimicrobial agents in meat, their us-
ages and challenges are discussed in the next section.
2. Antimicrobial agents used in meat and meat products
Antimicrobial agents have long been directly applied as food addi-
tives or used as processing aids with primary intention of prolonging
shelf life and preserving quality of meat and meat products. In general,
food additives refer to substances not consumed as foods but intention-
ally added to a food, and it or its by-products become components of the
food. Processing aids are also substances that are not normally con-
sumed as food by itself but intentionally used in the processing of raw
materials, foods or their ingredients, for a certain technological purpose
during treatment or processing. Application of processing aids may re-
sult in the unintentional but technically unavoidable presence of resi-
dues or its derivatives provided in the final product. Nevertheless, the
resulting residues or derivatives do not have any technological effect
on the final product. The use of antimicrobial agents in meat and meat
products has been approved in 21 Code of Federal Regulations for use
in meat, poultry, and egg products as food additives (USFDA, 2015).
For European Union (EU), the use of food additives in meat preparations
is stated in the Commission Regulation (EU) No. 601/2014 (The
European Commission, 2014).
Addition of nitrites, organic acids (i.e. lactic, ascorbic, benzoic and
sorbic acids) and its salts as food additives in meat and meat products
are approved by Codex Committee on Food Additives as stated in Gen-
eral Standard for Food Additives (GSFA). Blends of propionic acid,
caprylic acid and acetic acid are generally used in marinated meat
(Smith, 2012). Although sorbic acid/sorbates are not allowed in meat
and meat products as specified by GSFA, the solution of 10% potassium
sorbate may be applied to unrefrigerated dry sausages in the US
(Stopforth, Sofos, & Busta, 2005). Application of organic acids is limited
due to their potential negative impact on flavor and color of the prod-
ucts (Smith, 2012).
Organic acids, inorganic phosphates and oxidizers, are also applied
as processing aids for quality control. Those chemical antimicrobial
agents can be used in combination with hot water and steam treat-
ments for carcass and fresh meat decontamination (Simpson & Sofos,
2009). In the US, the use of antimicrobials for such applications is
approved by the Food Safety and Inspection Service (FSIS), if the
chemicals (i) are generally recognized as safe (GRAS), (ii) do not
lead to adulteration, (iii) do not create labeling issues, (iv) are scien-
tifically proven to be efficacious, and (v) do not pose human health
issues to worker or consumer (Koutsoumanis, Geornaras, & Sofos,
2006; Simpson & Sofos, 2009). According to the EU regulation (EU)
No 101/2013, beef carcass can be treated with lactic acid to reduce
microbiological surface contamination (The European Commission,
2013).
Today, the application of chemical preservatives has been
questioned because of the potential toxic and carcinogenic effects (Sax
& Lewis, 1989; Schaubschläger, Becker, Schade, Zabel, & Schlaak, 1991;
Tompkin, 2005). Not only pressure of the consumer health concerns,
but also a trend towards natural food additives so called “clean-label-
ing” has driven exploring of natural antimicrobial compounds as an al-
ternative to synthetic food additives (Castellano, Belfiore, Fadda, &
Vignolo, 2008; Deegan, Cotter, Hill, & Ross, 2006).
Among natural antimicrobial compounds, numbers of plant-derived
extracts have been widely studied (Cowan, 1999; Lucera, Costa, Conte, &
119
Del Nobile, 2012).The phytochemicals are plant secondary metabolites
playing roles in defense mechanisms against microbes and predators
as well as contributing to flavor and aroma (Cowan, 1999). The plant-
based antimicrobial substances can be divided into groups of phenolics,
terpenoids (essential oils), alkaloids, lectins and polyacetylenes
(Cowan, 1999). Extracts of spices and herbs commonly used in foods re-
ceive tremendous attention. In meat and meat products, the plant ex-
tract can be used alone or combined with the other extracts or with a
minimal process for synergistic output. Recent studies, for example, in-
dicated inhibitory effects of clove and cinnamon oils in ground chicken
against L. monocytogenes (Hoque, Bari, Juneja, & Kawamoto, 2008),
thyme and balm oils in fresh chicken breast (Fratianni et al., 2010)
and hop extracts in marinated pork (Kramer et al., 2015) against
broad-spectrum of meat spoilage bacteria. Phytochemicals are certified
as GRAS (Lucera et al., 2012), and pleasantly accepted by majority of
consumers in comparison with synthetic preservatives. It is worth not-
ing that properties of meat and process condition can significantly inter-
fere with the antimicrobial efficacy of the plant antimicrobials.
Compared to in vitro assays, a greater concentration of phytochemicals
is required to achieve the same effect in food (Jayasena & Jo, 2013;
Kramer et al., 2015; Sultanbawa, 2011). In addition to their strong fla-
vor, usage of the phytochemicals at high concentration is subjected to
critical scrutiny on the safety due to limited toxicological information
(Sultanbawa, 2011).
Advancement in protein and peptide research has led to discoveries
of natural antimicrobial proteins and peptides. To date, lysozyme,
lactoferrin, and nisin are the only three proteins and peptides approved
for application in meat and meat products (Davidson & Branen, 2005).
Lysozymes, a natural lytic enzyme present in egg white, possess capabil-
ity to hydrolyze the glycosidic bonds linking peptidoglycans, causing
the leakage of bacterial cell wall (Losso, Nakai, & Charter, 2000). The al-
lowable usage of egg-white lysozyme is 2.5 mg/lb of sausage casings or
2.0 mg/lb of ready-to-eat meat or poultry products (USFDA, 2000a). As
for lactoferrin, the milk-derived iron-binding glycoprotein competes
with the iron acquisition of bacteria, inhibiting bacterial growth.
Lactoferrin can block microbial adhesion on biosurfaces by disrupting
outer-membrane proteins of Gram-negative pathogens, preventing mi-
crobial colonization on the meat surfaces (Atef Yekta et al., 2010; Naidu,
2002). Low concentration of lactoferrin was shown to detach viable or
dead tissue-bound bacterial cells (Naidu, 2002). Although milk-
derived lactoferrin is considered as GRAS, the ingredient must be la-
beled when beef carcasses and cuts are treated with water-based
spray containing up to 2% milk-derived lactoferrin. Thereby, milk-
allergic individuals will be aware of the presence of milk-based ingredi-
ent. No labeling is required when the lactoferrin treatment is followed
by sufficient rinse to leave a residual concentration lower than
800 ppb (USFDA, 2003). While lysozyme effectively inhibits gram-
positive bacteria, lactoferrin exhibits broad-range antibacterial and anti-
viral activities (Naidu, 2002). Similar challenges of lysozyme and
lactoferrin are the reduced efficacy when directly added into food ma-
trices. Denaturation induced under harsh processing conditions also de-
creases the antimicrobial effect.
In contrast to plant extracts and the other protein-based antimi-
crobial preservatives, bacteriocins tolerate high thermal stress, are
active over a wide pH range and remain effective at fairly low con-
centration (Cleveland, Montville, Nes, & Chikindas, 2001). Applica-
tion of the bactericidal peptides does not alter sensory quality of
food products, as the peptides present colorless, odorless, and taste-
less characteristics. Based upon their advantageous characteristics,
bacteriocins have been attracting considerable interest as an alterna-
tive natural food preservative to extend shelf life and safety of meat
and meat products. The main objective of this review is to update
current status of bacteriocins and their application in meat and
meat products. Novel bacteriocins discovered in our laboratory,
together with their structures, characteristics, as well as exemplary
properties, will also be addressed herein.
120 W. Woraprayote et al. / Meat Science 120 (2016) 118–132

3. Bacteriocins from lactic acid bacteria Table 1

LAB bacteriocins and their producing species reported (up to the Year 2015).

Bacteriocins are commonly referred to as ribosomally synthesized Numbers of bacteriocin reported in each class
antimicrobial peptides that usually display a high degree of target spec- Producer species
I IIa IIb IIc IId Un-classified Total
ificity against strains of bacteria closely related and/or broad range anti-
Carnobacterium divergens – – – – – 2 2
microbial activity. Generally, various Gram-positive bacteria are capable
Carnobacterium maltaromaticum – – – 1 – 2 3
of producing bacteriocins. Yet, lactic acid bacteria (LAB) bacteriocins are Carnobacterium piscicola – 5 – – – 1 6
the most studied due to the fact that most of bacteriocin-producing LAB Enterococcus avium – 1 – – – – 1
are isolated from food origins and are considered as GRAS bacteria. LAB Enterococcus durans – – – – 1 – 1
bacteriocins are also expected to be safe (Deegan et al., 2006; Zendo, Enterococcus faecalis 3 2 2 4 – – 11
Enterococcus faecium – 8 3 4 3 4 22
2013). The LAB bacteriocins present suitable characteristics to be used Enterococcus mundtii – 2 – – – 2 4
as food preservatives. The peptides exhibit antimicrobial activity at con- Enterococcus sp. – – – – – 1 1
centration as low as picomolar to nanomolar. In addition to being non- Lactobacillus acidophilus – – – – – 6 6
toxic, the LAB bacteriocins can be digested by proteases, thus having no Lactobacillus amylovorus – – – – – 1 1
Lactobacillus casei – – – – – 2 2
or little influence on the gut microbiota. The peptides are primary me-
Lactobacillus crispatus – – – – – 1 1
tabolites with simple biosynthetic mechanisms; thus, their antimicrobi- Lactobacillus curvatus – – – – – 3 3
al activity and specificity can easily be improved through genetic Lactobacillus gasseri – – – 1 – – 1
engineering (Cleveland et al., 2001; Deegan et al., 2006; Gálvez, Lactobacillus helveticus – – – – – 2 2
Abriouel, López, & Omar, 2007; Perez, Zendo, & Sonomoto, 2014; Lactobacillus paracasei – – – – – 4 4
Lactobacillus pentosus – – – – - 1 1
Zendo, 2013). Lactobacillus plantarum – – 4 – – 13 17
LAB bacteriocins are diverse in terms of molecular mass, post- Lactobacillus sake – – – – – 3 3
translational modification, presence of modified amino acids, chemical Lactobacillus sakei – 2 – – – 7 9
structure as well as mode of action. To our knowledge, up to the year Lactococcus garvieae – – – 1 1 1 3
Lactococcus lactis 6 1 4 – 5 6 22
of 2015, 185 LAB bacteriocins have been isolated and reported. Howev-
Lactococcus sp. – – – 1 – – 1
er, only 53% of them were well-characterized and sequenced at the pro- Leuconostic carnosum - – – – – 2 2
tein or DNA levels. Several approaches have been proposed to classify Leuconostoc gelidum – - – – – 2 2
bacteriocins. The latest classification schemes have been extended to Leuconostoc mesenteroides – – – 1 – 4 5
cover several bacteriocin groups that are produced from both gram- Leuconostoc pseudomesenteroides – 1 – – 2 – 3
Pediococcus acidilactici – 3 – – – 1 4
positive and gram-negative bacteria (Cotter, Ross, & Hill, 2013) or Pediococcus pentosaceus – 2 – – – 2 4
even detected in silico in publicly available LAB genomes which no rep- Streptococcus bovis 1 – – – – 1 2
resentative from a LAB has yet been reported (Alvarez-Sieiro, Streptococcus cremoris – – – – – 1 1
Montalbán-López, Mu, & Kuipers, 2016). Although these new classifica- Streptococcus macedonicus 1 – – – – – 1
Streptococcus mutans 6 – 2 – – 1 9
tions seem good, it needs more time to be accepted. Therefore, the
Streptococcus pyogenes 2 – – – – – 2
scheme by Cotter, Hill, and Ross (2005) seems better for LAB bacterio- Streptococcus rattus 2 – – – – 1 3
cins at this moment. Accordingly, LAB bacteriocins can be classified Streptococcus salivarius 3 – – – – 1 4
into two major classes: class I lantibiotics (lanthionine-containing anti- Streptococcus thermophilus – – 2 – – 2 4
biotics) and class II which can further be grouped into four subclasses: Streptococcus uberis 1 1 – 1 – – 3
Weissella cibaria – – – – – 2 2
IIa, IIb, IIc, and IId, respectively (Table 1). Weissella hellenica – – – – 3 3 6
Weissella paramescenteroides – – – – – 1 1
3.1. Class I bacteriocins Total 25 28 17 14 15 86 185

Class I bacteriocin is a group of small peptides (b 5 kDa) that possess

unusual post-translational modified residues such as lanthionine or 3-
methyllanthionine which result in characteristic inner-molecular-ring
structures. One of the oldest and the most extensively characterized
bacteriocins is nisin A, which was discovered by Rogers (1928). Nisin
A is produced by many strains of Lactococcus lactis, a species widely
used for cheese manufacture. It has a broad antimicrobial spectrum
against a wide range of Gram-positive genera, including staphylococci,
streptococci, Listeria spp., bacilli, and enterococci. Nisin A has been
used in the food industry as a biopreservative for more than 50 years
without inducing microbial resistance (Cotter et al., 2005). The antimi-
crobial effect of nisin is caused by its interaction with the precursor of
peptidoglycan, lipid II on bacterial cell wall. Upon binding to the target
molecule, nisin–lipid II complex inserts itself into bacterial cell mem-
brane and then forms pores, creating leakage of essential cellular mate-
rials and, ultimately, cell death (Delves-Broughton & Weber, 2011).
To date, eight natural nisin variants have been discovered (O'Connor
et al., 2015). These include nisins A, Z, F, and Q, which have been isolated
from lactococci, nisin H from Streptococcus hyointestinalis, nisins U and
U2, from Streptococcus uberis, and nisin P, which is encoded on nisin op-
erons present in both Streptococcus gallolyticus subsp. pasteurianus and
Streptococcus suis. Alignment of the amino acid sequences of nisins A,
Z, F, Q, H, U, U2, and P shows an increasing number of amino acid chang-
es from the prototypical lactococcal nisin A, ranging from 1 amino acid
position for nisin Z up to 13 positions for streptococcal nisin P. While
these nisin variants have comparable biochemical features and antimi-
crobial activity spectra, the difference in amino acid sequence contrib-
utes to greater structural stability and superior stability. For example,
the inherent higher stability of nisin Q against oxidation than nisins A
and Z is brought about by the absence of a methionine at the hinge re-
gion (Perez et al., 2014). The increased oxidative resistance of nisin Q
compared to the other variants suggests its usefulness in certain appli-
cations where oxidation is common.
3.2. Class II bacteriocins
3.2.1. Class IIa bacteriocins
Unlike class I, class II bacteriocin includes a very large group of small
(b 10 kDa) heat-stable peptides, which do not undergo extensive post-
translational modification (Perez et al., 2014). Class IIa or pediocin-like
bacteriocins can be considered as the major subgroup among all charac-
terized LAB bacteriocins. These bacteriocins are cationic and display
anti-Listeria activity. However, recent reports have indicated that
many species within the genus Enterococcus are capable of producing
pediocin-like bacteriocins that are heterogeneous in structure and differ
in their inhibition spectra.
Pediocin PA-1, produced by Lactobacillus plantarum, is a well-known
class IIa bacteriocin. All pediocin-like bacteriocins contain disulfide brid-
ges and a common YGNG(V/L) motif (pediocin box) which forms an S-
 W. Woraprayote et al. / Meat Science 120 (2016) 118–132
shaped, sheet-like structure followed by a hinge and a somewhat more
hydrophobic and diverse helix-containing C-terminal half (Johnsen,
Fimland, & Nissen-Meyer, 2005). The C-terminal domains are less con-
served and are thought to determine the non-listerial antimicrobial
spectrum. Class-IIa bacteriocins may be divided into three subgroups
based on differences in the C-terminal; subgroup I (pediocin PA-1/
AcH, sakacin P and enterocin A), subgroup II (leucocin A and
mesentericin Y105) and subgroup III (curvacin A and carnobacteriocin
B2) (Fimland, Johnsen, Dalhus, & Nissen-Meyer, 2005).
Pediocin PA-1 performs its function by permeabilizing the cytoplas-
mic membrane of the receptor bacteria, resulting in a leakage of ions
and small molecules. Previous studies have demonstrated that pediocin
PA-1 interacts with its receptor by binding an extracellular loop of the
mannose-phosphotransferase system (MPTs) which located in the
membrane (Sun, Song, & Zheng, 2015). In this process, the conserved
N-terminal domain of pediocin PA-1 mediates the initial binding with
MPTs by electrostatic interaction and then the hydrophobic C-terminal
domain penetrates into the membrane of target cell, playing a crucial
role in receptor membrane leakage.
3.2.2. Class IIb bacteriocins
Class IIb or two-peptide bacteriocins, as the name suggests, consist
of two different individual peptide molecules that require equal peptide
ratio of each peptide to exert its optimal antimicrobial activity
(Garneau, Martin, & Vederas, 2002), whilst each peptide displays very
low activity when tested individually. Lactococcin G and lactacin F pro-
duced by L. lactis and L. johnsonii are the first reported two-peptide
bacteriocins with a narrow spectrum of inhibition against Clostridium,
E. faecalis and lactobacilli. The best characterized two-peptide systems
are plantaricins EF/JK and plantaricin S produced by L. plantarum C11
and LPCO10, respectively (Anderssen, Diep, Nes, Eijsink, &
Nissen-Meyer, 1998; Jiménez-Díaz et al., 1995). On the other hand,
lactocin 705 secreted by L. curvatus CRL705 (formerly L. casei), isolated
from Argentine fermented sausages, is the first two-peptide bacteriocin
reported from a meat-associated strain. Lactocin 705 showed to be an-
tagonistic toward other LAB and B. thermosphacta when assayed in
meat systems (Castellano, Holzapfel, & Vignolo, 2004; Castellano &
Vignolo, 2006). Three novel bacteriocins belonging to this class:
lactococcin Q isolated from L. lactis QU 4, enterocin X from E. faecium
KU-B5, and enterocin NKR-5-3AZ from E. faecium NKR-5-3 have been
described by Perez et al. (2014).
3.2.3. Class IIc bacteriocins
Class IIc bacteriocins are grouped based on their unique structural
feature of a head-to-tail cyclization of their backbones (Gabrielsen,
Brede, Nes, & Diep, 2014; van Belkum, Martin-Visscher, & Vederas,
2011). A number of LAB circular bacteriocins have been identified to
date (Acedo et al., 2015), including enterocin AS-48, gassericin A,
uberolysin, carnocyclin A, lactocyclicin Q, garvicin ML, leucocyclicin Q,
and acidocin B. Based on the calculated isoelectric point (pI) of the ma-
ture peptide, circular bacteriocins are classified into two subgroups.
Members of subgroup I, for examples carnocyclin A and enterocin AS-
48, have high pI values (~10), Among the circular bacteriocins identi-
fied, carnocyclin A and enterocin AS-48 are classified in subgroup I in
which a common saposin-like fold was observed. The saposin-like pro-
teins are known to interact with lipids and are composed of 4 or 5 adja-
cent α-helices that are folded into two leaves (Bruhn, 2005). While
those in subgroup II have low pI values (~7 or lower), gassericin A and
butyrivibriocin AR10, are composed of four α-helices that are also
folded to resemble the structure of the saposins (Martin-Visscher,
Gong, Duszyk, & Vederas, 2009). The circular nature of their structure
provides greater structural stability, higher thermal stress resistance,
and superior stability against proteolytic digestion, compared to their
linear peptides (Perez et al., 2014). Their biosynthetic mechanisms
and mode of action of these circular bacteriocins were recently pub-
lished (Gabrielsen et al., 2014).
121
3.2.4. Class IId bacteriocins
Class IId bacteriocins include the remaining well-characterized bac-
teriocins, combined as miscellaneous, which are now including non-
pediocin like single linear peptides (Cotter et al., 2005), sec-dependent
bacteriocins that are synthesized and translocated with a typical N-
terminal signal sequence of a sec-dependent pathway (Cintas et al.,
2000) and leaderless bacteriocins that are atypically synthesized with-
out an N-terminal leader sequences and are active immediately after
translation (Klaenhammer, 1993). A number of novel leaderless bacte-
riocins have been recently reported (Perez et al., 2014), for examples,
lacticin Q produced by L. lactis QU 5 and its homologue lacticin Z pro-
duced by L. lactis QU 14, and weissellicins Y and M produced by
Weissella hellenica QU 13.
4. Novel bacteriocins: Garvieacin Q and Bacteriocin 7293
In our laboratory, three novel class-IId bacteriocins produced by LAB
were isolated from Thai fermented pork sausages known as nham.
Garvieacin Q or GarQ, produced by Lactococcus garvieae BCC 42578, con-
tains 70 amino acids in length of which 20 amino acids is N-terminal
leader sequence which is cleaved at the Gly-Gly site to generate the ma-
ture GarQ with molecular mass of 5339 Da, pI of 9.73 and net 2 positive
charges (Tosukhowong et al., 2012). GarQ was classified as a class-IId
bacteriocin since it did not contain any modified amino acid, no con-
served YGNGVXC motif in molecule and exhibited its antimicrobial ac-
tivity without the need of other complementary peptides. GarQ, in the
concentration range of micromolar, exhibited antibacterial spectrum
against various strains of the closely related bacteria L. garvieae, as
well as the potentially pathogenic Enterococcus faecium and Listeria
monocytogenes. However, GarQ was not active against Staphylococcus
aureus and other gram-negative meat-borne pathogens, including
Escherichia coli and Salmonella Typhimurium. GarQ retained its antimi-
crobial activity over a wide pH range of 2 to 8, even following heat treat-
ment at 100 °C for 15 min. A complete loss of its antimicrobial activity
was observed when heated at 121 °C for 15 min. Purified GarQ, up to
1 mg/mL, was not cytotoxic to Vero (African green monkey kidney),
HepG2 (human liver hepatocarcinoma), and Caco-2 (human colon ade-
nocarcinoma) cell lines as detected by MTT [3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay.
Apart from GarQ, the other two novel bacteriocins, Bacteriocins
7293A (Bac7293A) and B (Bac7293B), from Weissella hellenica BCC
7293 have been recently discovered (Woraprayote et al., 2015). Unlike
GarQ, these two bacteriocins inhibit broad spectrum of bacteria, includ-
ing foodborne pathogenic Listeria monocytogenes, Staphylococcus aureus,
Salmonella Typhimurium, Escherichia coli, as well as food-spoilage Pseu-
domonas aeruginosa, and Aeromonas hydrophila. Both exhibited activity
through bactericidal mode of action against both gram-positive and
gram-negative bacteria without cell-lysis. The Bac7293A showed great-
er antimicrobial activity but lower pH and thermal stability compared
with those properties of Bac7293B. The antimicrobial activity of both
bacteriocins were completely inactivated by proteolytic enzymes, i.e.
trypsin, α-chymotrypsin, pepsin and protease K, but the peptides
remained active after treated with lipase and amylase, suggesting
their proteinaceous nature. Organic solvents (ethanol, isopropanol, ace-
tone, acetonitrile) and surfactants (Tween 20, Tween 80 and Triton X-
100) did not affect the antimicrobial activity of these bacteriocins.
Based on electrospray ionization mass spectrometry, molecular masses
of Bac7293A and B were determined to be 6,249.302 and 6,489.716 Da,
respectively. The structures and biosynthesis of Bac7293A and B will re-
quire further investigation for better understanding about their molec-
ular characteristics. Since the traditional method for amino acid
sequencing by Edman degradation could not provide significant amino
acid sequences of both bacteriocins, proteogenomic approach has
been used to access their primary structure. To date, amino acid se-
quence of Bac7293A has been currently revealed (Tosukhowong et al.,
submitted for publication).
122 W. Woraprayote et al. / Meat Science 120 (2016) 118–132

Fig. 1. Homology-based 3D structures predicted by the SWISS-MODEL Homology Modeling and helical wheel projections analyzed by HeliQuest of the mature peptides of GarQ and
Bac7293A. The one letter code for amino acids is used. Hydrophobic residues are shown in yellow, serine and threonine in purple, basic residues in dark blue, acidic residues in red,
asparagine and glutamine in pink, alanine and glycine in grey and histidine in light blue circles. The arrow in the helical wheel corresponds to the hydrophobic moment. aa is the
amino acid residues. H and μH are the mean hydrophobicity and the hydrophobic moment calculated by HeliQuest (Gautier, Douguet, Antonny, & Drin, 2008), respectively. The net
charge (z) was calculated at pH = 7.4, under the assumption that histidine is neutral and that the N-terminal amino group and the C-terminal carboxy group of the sequence are
uncharged. The isoelectric point (pI) was calculated using online analysis tools on the ExPASy server.

Homology-based three-dimensional structures of mature GarQ and
Bac7293A predicted by the SWISS-MODEL are shown in Fig. 1. GarQ
and Bac7293A was predicted to contain one and two helices, respective-
ly. GarQ is composed of a C-terminal α-helix that extends from amino
acid residues 28 to 44 (Fig. 1a, top), whereas Bac7293A has both N-
terminal helix, from amino acid residues 1 to 7, and C-terminal α-
helix, from amino acid residues 34 to 51 (Fig. 1b, top). The helical
wheel projection of the C-terminal helices of GarQ and Bac7293A sug-
gest their inherent ability to form an amphipathic α-helix that allows
the peptides to partition into the bacterial membrane lipid bilayer
(Hancock & Sahl, 2006) (Fig. 1a &1b, bottom). The hydrophobic mo-
ment (μH), quantification of a helix amphipathicity, of GarQ and
Bac7293A are similar to that of the C-terminal amphipathic α-helical
segment of Carnobacteriocin B2 which proposed to be the region deter-
mining target specificity through binding of the putative receptor pro-
tein (Wang et al., 1999). With a higher mean hydrophobicity,
hydrophobic moment, and net charge, the C-terminal helices of GarQ
and Bac7293A are likely to play a major role in antimicrobial action of
the bacteriocin. Several studies revealed that the reduction in antimi-
crobial activity was observed when the hydrophobic moment of peptide
was decreased (Dathe & Wieprecht, 1999). Together with high pI value,
the increase in peptide charge of Bac7293A may enhance the antimicro-
bial activity against gram-positive as well as gram-negative bacteria
(Dathe & Wieprecht, 1999). This could also explain why Bac7293A ex-
hibited around 7-fold stronger antimicrobial activity against
L. monocytogenes ATCC 19115 than GarQ. Like other cationic peptides,
the positive charge, amphipathic α-helix and hydrophobic residues
can play a critical role in membrane binding, most likely by partitioning
into the hydrophobic core of the lipid bilayer. They may also help coun-
teract alternative targeting effects when the basic residues confer affin-
ity for other intracellular destinations. The antimicrobial action may
follow either one of the two mechanisms: (i) transmembrane pore for-
mation via a “barrel-stave” mechanism, and (ii) membrane destruction/
solubilization via a “carpet-like” mechanism, both leading to bacterial
membrane permeation and cell death as described by Oren and Shai
(1998).
5. Current applications of LAB bacteriocins in meats and meat
products
Bacteriocins from class I and IIa are among the best biochemically
and genetically characterized antimicrobial peptides and the most likely
to be used in food applications due to their target specificity (Table 1).
Among hundreds of bacteriocins, nisin is the only commercial bacterio-
cin approved for application in meat, poultry, ready-to-eat meat prod-
ucts and sausage casing (USFDA, 2000b). Despite no official approved
use, pediocin has been widely studied and applied in meat and meat
products. In meat applications, nisin (Franklin, Cooksey, & Getty, 2004;
Luchansky & Call, 2004; Mohamed, Elnawawi, & Yousef, 2011;
Nguyen, Gidley, & Dykes, 2008) and pediocin PA-1/AcH (Hartmann,
Wilke, & Erdmann, 2011; Kingcha et al., 2012; Ming, Weber, Ayres, &
Sandine, 1997; Woraprayote et al., 2013) are usually used to decontam-
inate or to control the growth of L. monocytogenes, one of the most path-
ogen of concern, especially in RTE meat products. Although anti-Listeria
efficiency of nisin and pediocin significantly differed depending on the
producing or indicator strains, the sample preparation method, and the
 W. Woraprayote et al. / Meat Science 120 (2016) 118–132 123

Table 2
Applications of bacteriocin-producing LAB in meat and meat products (2000–present).

Application Bacteriocin-producing Product Features Reference

approach strain

Direct inoculation Lactobacillus curvatus DF126
(starter culture) (Curvacin DF126)
Lactobacillus plantarum 423
(Plantaricin 423)
Lactobacillus curvatus DF38
(Curvacin DF38)
Pediococcus pentosaceus BCC 3772
(Pediocin PA-1/AcH)
Lactococcus lactis supsp. lactis 69
(Nisin Z)
Lactobacillus sakei C2
(Sakacin C2)
Lactobacillus curvatus 54M16
(Sakacins X, T and P)
Direct inoculation Leuconostoc carnosum 4010
(Protective culture) (Leucocins 4010)
Leuconostoc carnosum 4010
(Leucocins 4010)
Lactobacillus curvatus CWBI-B28
(Sakacin P)
Lactobacillus sakei CWBI-B1365
(Sakacin G)
Lactobacillus curvatus CRL705
(Lactocin 705 and lactocin AL705)
Lactobacillus curvatus ACU-1
(Sakacin Q)
Lactobacillus curvatus MBSa2
(Sakacins P and X)
Ostrich meat salami
Salami from ostrich, beef, mutton,
Blesbok (Damaliscus dorcas
phillipsi) and Springbok
(Antidorcas marsupialis)
Salami from beef, horse, mutton,
Blesbok (Damaliscus dorcas
phillipsi) and Springbok
(Antidorcas marsupialis)
Nham, Thai traditional fermented
pork sausage
Charqui, Brazilian traditional
salted and dried meat
Fermented pork sausage
Fermented sausage
Vacuum-packed sliced cooked
meats/Surface inoculation
Gas-packed sliced cooked
saveloys/ two methods were
applied (i) mixed with saveloys
batter before casing, incubation
and heat treatment, and (ii)
sprayed onto the surface of sliced
saveloys
Raw beef/Surface inoculation
Raw beef/Surface inoculation
Vacuum-packed fresh
beef/Spraying
with protective culture
Cooked meat/Immersion in cell
suspension
Salami/Mixing with salami batter
before casing and incubation
Anti-Listeria activity Dicks et al. (2004)
Anti-Listeria activity Dicks et al. (2004);
Todorov et al. (2007)
Anti-Listeria activity Todorov et al. (2007)
Anti-Listeria activity without significant Kingcha et al. (2012)
changes in sensory characteristics and
consumer acceptability of the product
Reduced halotolerant and spoilage Biscola et al. (2014)
bacteria during Charqui fermentation
Anti-Listeria and Anti-Enterobacteriacae Gao et al. (2014)
activity, reduced malondialdehyde and
nitrite content in the product
Reduced the number of Staphylococci Casaburi et al. (2016)
and Enterobacteriaceae
Anti-Listeria activity Budde et al. (2003)
Both application approaches exhibited Jacobsen et al. (2003)
anti-Listeria activity. Spraying the
protective culture onto the product
surface was more effective than
incorporation in sausage batter.
Anti-Listeria activity Dortu, Huch, Holzapfel,
Franz, & Thonart (2008)
Anti-Listeria activity Dortu et al. (2008)
Inhibited the growth of Listeria innouca Castellano, González,
and Brochothrix thermosphacta Carduza, & Vignolo (2010);
Castellano & Vignolo
(2006)
Anti-Listeria activity Rivas et al. (2014)
Anti-Listeria activity De Souza Barbosa et al.
(2015a)

bacteriocin assay conditions, pediocin is likely to have higher activity and
acts more specifically against L. monocytogenes than nisin (Cintas, Casaus,
Fernández, & Hernández, 1998). In addition, pediocin PA-1/AcH, in con-
trast to both nisins A and Z, has potential to inhibit Listeria without
disturbing other bacteria including beneficial ones (Blay, Lacroix, Zihler,
& Fliss, 2007). Pediocin PA-1/AcH is, therefore, a good candidate for the
control of L. monocytogenes in meat and meat products.
The emergence of bacteria naturally resistant to pediocin and other
class II bacteriocins seems to occur more frequently than those of nisin
(Rodríguez, Martínez, & Kok, 2002). Other than nisin-resistant strains
obtained in laboratory settings, no reports of emerging resistance to-
wards nisin have yet appeared despite its prolonged used as a preserva-
tive in food industry (Kramer et al., 2004). This might become a
potential obstacle to the application of pediocin and other class II bacte-
riocins as food biopreservatives. To overcome this problem, it has been
suggested that the level of bacteriocin used should be adjusted to obtain
maximum viability loss of the target bacteria which consequently min-
imize the emergence of resistances when used as a food preservative
(Rodríguez et al., 2002).
Bacteriocins can be applied in meats and meat products in three
major approaches including (i) direct inoculation of bacteriocin-
producing LAB cells into meat and meat products as either starter or
protective cultures (Table 2), (ii) direct application of cell free superna-
tant (CFS), purified or partially purified bacteriocins as a food additive
(Table 3) and (iii) use purified/partially purified bacteriocins in the
form of packaging (Table 4).
5.1. Direct inoculation of bacteriocin-producing LAB
Direct utilization of bacteriocin-producing cells is one of the most
practical strategies that seem to be more feasible from an economic
point of view and lesser legal restrictions compared to the direct addi-
tion of purified bacteriocins. As a starter culture, inoculated cells mainly
contribute to produce acids and bacteriocins and hydrolyze proteins in
meat thus dramatically changes the properties and organoleptic charac-
teristics of meat products. As a protective culture, the objective use of
bacteriocin-producing LAB cell is to inhibit the growth of unwanted bac-
teria without the cause of sensorial changes of meat or meat products.
Concerns associated with the use of bacteriocin-producing LAB as inoc-
ulum are (i) a lack of compatibility between bacteriocin-producer and
other starter cultures in multi-strain starters system, (ii) undesirable
quality of meat products due to incomplete fermentation provided by
using bacteriocin-producing LAB as single-strain starter, and (iii) no
protective effect of bacteriocin-producing LAB due to the interference
of food matrix and the un-optimized conditions for its growth and
bacteriocin(s) production.
L. plantarum 423 and Lactobacillus curvatus DF126, plantaricin 423
and curvacin DF126 producer, respectively, were used as starter culture
together with Micrococcus sp. MC50 in multi-strain starters system for
ostrich meat salami production (Dicks, Mellett, & Hoffman, 2004).
Both bacteriocin-producers were reported to be compatible with Micro-
coccus sp. MC50 for ostrich meat salami fermentation since their pro-
duced bacteriocins did not inhibit Micrococus sp. MC50. Both starter
124 W. Woraprayote et al. / Meat Science 120 (2016) 118–132

Table 3
Applications of LAB bacteriocin preparations in meat and meat products (2000–present).

Bacteriocin, producer Product Features Reference

Enterocins A and B, Enterococcus faecium
CTC492
Leucocins 4010, Leuconostoc carnosum 4010
Lactocin 705, Lactobacillus curvatus CRL705
Lactocin AL705, Lactobacillus curvatus CRL705 Vacuum-packed fresh beef/Spraying with
Bacteriocin, Pediococcus acidilactici
Pentocin 31-1, Lactobacillus pentosus 31-1
Enterocin AS-48, Enterococcus faecalis
A-48-32
Nisin, Sigma
Bacteriocins MT 104 and MT 162,
Enterococcus faecium
Sakacin Q, Lactobacillus curvatus ACU-1
Nisin, commercial nisin
Nisin, NiprosinTM
Cooked ham (applied onto meat surface), minced All exhibited anti-Listeria activity Aymerich et al. (2000)
pork (mixed with meat batter), pâté (mixed with
meat batter), and espetec (mixed with meat
batter).
Gas-packed sliced cooked saveloy/Two methods Both exhibited anti-Listeria activity. Application Jacobsen et al. (2003)
were applied (i) mixed partially purified of bacteriocin onto meat surface was more
bacteriocin with saveloys batter before casing and effective approach to control Listeria during
heat treatment and, (ii) applied partially purified storage at 5 °C.
bacteriocin solution onto both sides of sliced
saveloys.
Vacuum-packed fresh beef/ Spraying with Inhibited the growth of Brochothrix Castellano & Vignolo
bacteriocin solution thermosphacta (2006)
Anti-Listeria activity Castellano & Vignolo
bacteriocin solution (2006)
Raw pork/Immersion in the concentrated culture Anti-Listeria activity, reduced the growth of Nieto-Lozano et al.
supernatant Clostridium perfringen (2006)
Tray-packed chilled pork/Immersion in bacteriocin Suppress the growth of Listeria monocytogenes Zhang et al. (2010)
solution and Pseudomonas fluorescens
Low acid fermented sausage called fuets/Mixing Reduced the growth of Listeria monocytogenes Ananou et al. (2010)
with bacteriocin and Salmonella
Raw meat/Immersion in nisin solution Anti-Listeria activity. Gamma-radiation Mohamed et al. (2011)
enhanced its antimicrobial effectiveness.
Meat sausage/Mixing with bacteriocin Anti-Listeria activity. Addition of nisin and Turgis et al. (2012)
gamma-radiation improved the antimicrobial
activity of bacteriocins MT 104 and MT 162.
Cooked meat/Immersion in CFS, or freeze-dried CFS Both exhibited anti-Listeria activity. The Rivas et al. (2014)
reconstituted in distilled water freeze-dried reconstituted CFS was more
effective one.
Vacuum-packed sliced cooked ham/Brine injection Reduced the total count of LAB Kalschne et al. (2014)
Ham/Coating with free and microencapsulated All exhibited anti-Listeria activity. Huq et al. (2015)
antimicrobials, nisin and combination of nisin and Microencapsulated nisin + essential oil
essential oil exhibited the highest activity. Gamma-radiation
enhanced antimicrobial effectiveness of the
antimicrobials.

cultures effectively reduced the numbers of L. monocytogenes artificially
contaminated in salami from approx, 107 to approx. 104 CFU/g within
the first 9 h of fermentation at 16–18 °C. L. plantarum 423 was sug-
gested, by the authors, to be a potentially good starter culture since
the specific activity of its bacteriocin, plantaricin 423, increased as the
culture pH decreased during fermentation.
The above statement was confirmed by the study of Todorov et al.
(2007). L. plantarum 423 was used as a good starter culture in the pro-
duction of salami from several types of meat, including beef, mutton,
Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas
marsupialis). All salami produced with this bacterium yielded a good
meat aroma, color, texture, venison like flavor, sour meat flavor and
oily mouthfeel. The numbers of L. innocua artificially contaminated in
salami was reduced in the presence of L. plantarum 423. The effective-
ness of L. curvatus DF38 for salami production was also evaluated in
this study. It was reported to exhibit more anti-Listeria activity in all
meat salami when compared with L. plantarum 423. However, based
on organoleptic characteristics, salami produced with L. curvatas DF38
was considered inferior to that produced with L. plantarum 423.
In our laboratory, the use of indigenous bacteriocin-producing LAB
for control of pathogenic microorganisms during meat fermentation
was assessed (Kingcha et al., 2012). Pediococcus pentosaceus BCC 3772,
an indigenous pediocin PA-1/AcH producer, was applied as a starter cul-
ture for Nham, Thai traditional fermented pork sausage. Inoculation of
P. pentosaceus BCC 3772 in Nham caused a significant decrease of 3.2
log in population of the spiked L. monocytogenes within 18–24 h of fer-
mentation, in comparison with the initial count, with no significant
changes in sensory characteristics and consumer acceptability of
the final fermented Nham products. The authors suggested that not
only carbon dioxide, short-chain organic acids, hydrogen peroxide
and diacetyl, but also bacteriocin produced by P. pentosaceus BCC
3772 was the factor which play significant role in the reduction of
L. monocytogenes during Nham fermentation. The results indicated
that the indigenous P. pentosaceus BCC 3772 should be useful as a
functional starter culture for control of L. monocytogenes without
compromising the unique quality of Nham.
The spoilage potential of charqui, Brazilian traditional salted and
dried meat product, was reduced by the addition of the L. lactis subsp.
lactis 69, the indigenous bacteriocinogenic bacteria, as starter culture
during charqui fermentation (Biscola et al., 2014). L. lactis 69 significant-
ly reduced the populations of halotolerant and spoilage bacteria, thus
reduced the spoilage potential, without any effect on the counts and di-
versity of LAB during charqui manufacturing and storage up to 45 days.
Moreover, the tested virulence genes of L. lactis 69, including genes
encoding vancomycin resistance (vanB), endocarditis antigen (efaA),
aggregation substance (asa1), gelatinase (gelE), cytolysin (cylA), hyal-
uronidase (hyl), histidine decarboxylase (hdc), tyrosine decarboxylase
(tdc) and ornithine decarboxylase (odc), were not detected suggesting
its safety for food applications.
In some cases, non-indigenous bacteriocin-producing LAB could also
be used as good starter culture with high antimicrobial effectiveness.
Lactobacillus sakei C2, sakacin C2 producer, isolated from Chinese tradi-
tional fermented cabbage, was used as a starter culture for the
fermented pork sausage. During the sausage fermentation, L. sakei C2
could quickly establish and effectively control the growth of harmful
microorganisms, L. monocytogenes and Enterobacteriacae present in
the products. In addition to the product safety improvement, this bacte-
rium could reduce malondialdehyde and nitrite content and improve
sensory characteristics, color, flavor and overall acceptability (Gao, Li,
& Liu, 2014). This study points out that the appropriate selection of
 W. Woraprayote et al. / Meat Science 120 (2016) 118–132 125

Table 4
Applications of LAB bacteriocins in meat packaging through direct incorporation into film matrix/coating/adsorption (2000–present).

Application Bacteriocin/ packaging material Product Features References

technique

Enterocins A and B/alginate film
Nisin, commercial/palmitoylated
alginate-based film
Pediocin ALTA® 2351/cellulose
acetate film
Lactocin 705 and lactocin AL705
mixture/synthetic polymer-based
and wheat gluten-based films
Nisin Z/pullulan film
Coating Nisin, commercial/barrier film
Nisin, commercial/cellulose casing
Enterocin 416K1/low-density
polyethylene film
Nisin, commercial with
EDTA/plastic bags
Adsorption Nisin, Nisaplin(R)/cellulose-based
packaging paper
Nisin, Nisaplin®/cellophane film
Nisin, Sigma/bacterial cellulose film
Pediocin PA-1/AcH/poly(lactic
acid)/sawdust particle
biocomposite film
Chilled air-packed and vacuum-packed Anti-Listeria activity. Vacuum-packed treatment Marcos et al. (2007)
cooked sliced ham was more effective than air-packed treatment.
Chilled sliced beef Reduced the growth of Staphylococcus aureus Millette et al. (2007)
Chilled vacuum-packed sliced ham Anti-Listeria and anti-Salmonella activity Santiago-Silva et al.
(2009)
Chilled vacuum-packed Wieners Anti-Listeria activity with slightly inhibition of Massani et al. (2014)
Lactobacillus plantarum. Active synthetic
polymer-based film was more effective than active
wheat gluten-based film
Chilled vacuum-packed raw beef and deli Antimicrobial activity against Salmonella spp., Pattanayaiying et al.
ham L. monocytogenes, S. aureus, and E. coli was (2015)
observed in both meat samples. The addition of
lauric arginate improved the antimicrobial
effectiveness of nisin film.
Chilled vacuum-packed hot dogs Anti-Listeria activity Franklin et al. (2004)
Chilled vacuum-packed frankfurters Anti-Listeria activity Luchansky & Call
(2004)
Chilled frankfurters Anti-Listeria activity. Iseppi et al. (2008)
Chilled vacuum-packaged beef chops Inhibited the growth of Brochothrix thermosphacta Ferrocino et al. (2013)
and Carnobacterium spp.
Chilled MAP-packed cooked ham Antimicrobial activity against Staphylococcus Scannell et al. (2000)
aureus, Listeria innocua and LAB
Chilled fresh veal meat Inhibited total endogenic aerobic bacteria in fresh Guerra et al. (2005)
veal meat
Chilled vacuum-packaged frankfurters Anti-Listeria activity. Nguyen et al. (2008)
Chilled raw sliced pork Anti-Listeria activity. Woraprayote et al.
(2013)

bacteriocin-producing LAB, even not the indigenous bacteria, for use as
starter culture could be a tool to improve food safety and improve/pre-
serve the typical characteristics of the fermented meat products.
L. curvatus 54M16, sakacins X, T and P producer, was applied as start-
er culture for fermented sausage. Its use was reported to improve the
quality and safety of the traditional fermented sausages prepared with-
out antimicrobial additives. The antimicrobial efficiency of this bacteri-
um during sausage fermentation against naturally contaminated
pathogens indicated the reduction of Staphylococci and Enterobacteria-
ceae when compared with sausage without L. curvatus 54M16
(Casaburi, Di Martino, Ferranti, Picariello, & Villani, 2016).
As a protective culture, the addition of Leuc. carnosum 4010, leucocins
A, B and C producer, to the vacuum-packed sliced cooked meats immedi-
ately reduced the population of the spiked L. monocytogenes to level
below the detection limit and no increase in L. monocytogenes was ob-
served during storage at 5 °C for 21 days. Moreover, this protective culture
did not produce any undesirable flavors in the products. These results
demonstrated that Leuc. carnosum 4010 is suitable as a protective culture
for vacuum-packed sliced cooked meat products during chilled storage
(Budde, Hornbæk, Jacobsen, Barkholt, & Koch, 2003).
L. curvatus CRL705 was used as a protective culture in fresh beef. This
bacterium effectively inhibited L. monocytogenes and B. thermosphacta
as well as the indigenous contaminant LAB, retained its inhibitory effect
at low temperature and had a negligible effect on meat pH (Castellano
et al., 2008; Castellano & Vignolo, 2006).
To alleviate the effect of some food components specially proteolytic
enzymes that may interfere or inhibit bacteriocin activity, bacteriocin
production and growth of protective culture. Kouakou et al. (2008) ob-
served L. monocytogenes growth inhibition in raw and cooked pork meat
after treated with L. curvantus CWBI-B28wt. However, the growth re-
bound of L. monocytogenes occurred after two weeks, coinciding with
loss of 70% of bacteriocin activity. The major factor that caused the re-
duction of bacteriocin activity was reported to be the action of proteo-
lytic enzymes isolated from both L. curvatus CWBI-B28wt and the
meat matrix. To reduce the effect of meat proteases, chelating agents
such as EDTA was supposed to retard protease activity thus maintain
the intact bacteriocin. Flour and/or an appropriate protein for which
meat proteases have an affinity might be alternatives to prevent the
loss of bacteriocin applied in fresh meat.
The entrapment of protective culture was also expected to protect
them from the adverse conditions in the meat matrix. L. curvatus
MBSa2, bacteriocin-producing LAB isolated from salami, was entrapped
in calcium alginate and tested for functionality in salami artificially con-
taminated with L. monocytogenes AL602/08. During 30 days of simulated
salami processing conditions, encapsulation of bacteriocin-producing LAB
in liposomes was suggested by the authors as a promising technological
alternative for the control of L. monocytogenes in food products (De
Souza Barbosa, Todorov, Jurkiewicz, & de Melo Franco, 2015a).
5.2. Direct application of bacteriocin as food additive
The incorporation of bacteriocins as an ingredient is the commonly
used approach that has also been shown to be effective in the control
of pathogenic and spoilage microorganisms in meat and meat products
(Table 3). This application technique is more preferable when live cells
of LAB could not produce bacteriocin in the real meat systems. Bacteri-
ocin preparations used can be any forms ranging from cell-free superna-
tant, partially-purified, and purified ones.
E. faecium CTC492 could not be used effectively as starter or protec-
tive culture for fermented meat products due to the growth interference
from product ingredients. Whilst, its bacteriocins, enterocins A and B,
could be applied in different types of meat products, including cooked
ham, minced pork, pâté and slightly fermented sausages called espetec,
to significantly reduce the population of Listeria innocua during chilled
storage (Aymerich et al., 2000).
Live cells of Leuc. carnosum 4010 were more effective than its partial-
ly purified leucocins 4010 for the growth inhibition of L. monocytogenes
on sliced gas packed pork saveloy (Jacobsen, Budde, & Koch, 2003).
Purified lactocin AL705 as well as its producer, L. curvatus CRL705,
used as a bioprotective culture were reported to be similarly effective
126 W. Woraprayote et al. / Meat Science 120 (2016) 118–132
in preventing the growth of L. innocua in meat throughout the storage
period (Castellano & Vignolo, 2006).
Bacteriocin produced by Pediococcus acidilactici was partially puri-
fied and applied on raw meat surface artificially contaminated with
L. monocytogenes and C. perfringens. This treatment reduced
L. monocytogenes and exhibited bacteriostatic effect against
C. perfringens during storage at 15 °C (Nieto-Lozano, Reguera-Useros,
Peláez-Martínez, & de la Torre, 2006).
The application of pentocin 31-1, bacteriocin produced by Lactoba-
cillus pentosus 31-1 in tray-packaged chilled pork could suppress the
growth of microflora, especially L. monocytogenes and Pseudomonas
fluorescens, could improve physicochemical parameters and sensory
characteristics of products and could extend the shelf life of the treated
product up to 15 days (Zhang, Liu, Li, & Qu, 2010).
The partially purified enterocin AS-48, bacteriocin produced by En-
terococcus faecalis A-48-32, was applied in a low acid fermented sausage
called fuet to control the growth of pathogenic microorganism during
fuet ripening. Enterocin AS-48 caused a drastic 5.5 log CFU/g decrease
in L. monocytogenes and a significant inhibition for Salmonella
(1.79 log CFU/g) at the end of ripening (10 days). The significant im-
provement of Salmonella-inhibition was observed when enterocin AS-
48 was used in combined with high hydrostatic pressure (Ananou
et al., 2010) suggesting the promising way to enhance antimicrobial ef-
fectiveness of bacteriocin by the combination with other hurdle
technology.
Bacteriocins MT 104 and MT 162 produced by E. faecium significantly
reduce the growth of L. monocytogenes on meat sausage after 5 days of
storage at 4 °C. Together with other hurdle technology, γ-irradiation,
synergistic anti-listeria effect was observed for bacteriocin MT 104,
whilst only additive anti-listeria effect was observed for bacteriocin
MT 162 (Turgis et al., 2012).
Sakacin Q produced by L. curvatus ACU-1 was used to control the
growth of L. innocua artificially inoculated on cooked meat surface dur-
ing chilled storage. Four forms of bacteriocin applications, including
protective culture, cell-free supernatant (CFS), a mixture of both and
freeze-dried reconstituted CFS, were investigated. The use of freeze-
dried reconstituted CFS was the most effective one to control Listeria
growth within the studied systems (Rivas, Castro, Vallejo, Marguet, &
Campos, 2014).
Direct addition of nisin in vacuum-packed sliced cooked ham could
significantly reduce the total LAB count (2 to 3 log cycles) in meat prod-
ucts when compared with control (without nisin) during chilled storage
for 60 days (Kalschne, Geitenes, Veit, Sarmento, & Colla, 2014).
Partially purified sakacins P and X produced by L. curvatus MBSa2
was applied to the batter for salami production. By the means of chal-
lenge test, the addition of bacteriocins caused 2 log CFU/g reduction of
L. monocytogenes in the final product when compared to salami without
addition of bacteriocins. The findings highlighted that application of
these bacteriocins can be an additional method for improving the safety
of salami and other RTE meat products with regards to L. monocytogenes
(De Souza Barbosa et al., 2015b).
E. coli O157:H7, a food borne pathogen reported to cause the cross-
contamination during grinding of ground beef, was inhibited by the cul-
ture supernatant of Leuconostoc isolate DGB-1040. The application of
DGB-1040 culture supernatant significantly reduced the number of
E. coli O157:H7 artificially inoculated in ground beef during storage at
5 and 10 °C (Koo, Kim, & Kang, 2015).
Recently, multiple technologies including irradiation and microen-
capsulation of bacteriocin together with other food preservatives were
used as an advanced process to improve the food safety for RTE meat
product. Huq, Vu, Riedl, Bouchard, and Lacroix (2015) developed the
anti-Listeria formulations from the combination of nisin and essential
oil(s). To preserve the antimicrobial efficiency during storage, the for-
mulations were microencapsulated with alginate based polymer. The
γ-irradiation, applied to RTE meat products previously treated with
the microencapsulated antimicrobial formulations, was found to exhibit
the synergistic antimicrobial effect against L. monocytogenes on RTE
meat products during chilled storage. This treatment could reduce the
number of L. monocytogenes artificially inoculated into meat sample
(~ 5–6 log CFU/g) to below the detection limit (≤ 50 CFU/g) since the
first day of storage. This strong inhibitory effect still remained until
28 days of refrigeration. This study suggested the feasibility of the use
of bacteriocin with other hurdles, microencapsulation technology and
irradiation, for meat safety improvement.
5.3. Application of bacteriocins in antimicrobial packaging
The direct addition of bacteriocins into meat matrix and/or onto
meat surfaces may lead to some loss of its activity because of the dilu-
tion effect which can result in a depletion of bacteriocin effectiveness
(Quintavalla & Vicini, 2002). Bacteriocins may be inactivated by some
components of the meat they are presented in (Kouakou et al., 2008;
Rose, Sporns, Stiles, & McMullen, 1999). Since microbial contamination
of meat and meat products occurs primarily at the surface, the use of
packaging system containing bacteriocins could be more efficient, by
continuous release of bacteriocins from the packaging to the surface of
the products, thus helping maintain its effective concentrations
(Quintavalla & Vicini, 2002). Packaging system has been reported to
protect bacteriocins from the inactivation by interaction with food com-
ponents such as lipids and enzymes. Moreover, the application of bacte-
riocins in food-contact materials required lower amounts of
bacteriocins compared to the direct addition into the whole meat vol-
ume, thus reducing the use of the preservative. The incorporation of
bacteriocins into packaging could avoid the direct addition of bacterio-
cins to meat. This technique can meet demand of consumers who search
for the foods that are free of additives. Two major methods of incorpo-
ration of bacteriocins into meat packaging usually used are (i) direct in-
corporation of bacteriocins into film matrix, and (ii) indirect
incorporation by coating bacteriocins onto the film surface (Table 4).
5.3.1. Direct incorporation of bacteriocins into film matrix
Researches and developments in incorporation technique for bacte-
riocins have been usually focused on casting or solvent compounding
process to avoid any severe heat treatment. Although some bacteriocins
are relatively heat-resistant, solvent compounding may be a more suit-
able method for their incorporation into packaging (Appendini &
Hotchkiss, 2001). The effect of film-forming method (casting and
heat-pressing) on the retention of biologically active nisin (Nisaplin®)
was investigated by Dawson, Hirt, Rieck, Acton, and Sotthibandhu
(2003). It was found that activity of cast film (solvent compounding)
retained three times greater than that of heat-pressed films. Even in sol-
vent compounding processing, severe heat treatment of bacteriocins
and/or film forming solution containing bacteriocins should be avoided.
As observed by Beristain-Bauza, Mani-López, Palou, and López-Malo
(2016), heat treatment of cell-free supernatant of Lactobacillus
rhamnosus NRRL B-442 at 92 °C for 30 min before adding into film
forming solution significantly decreased the antimicrobial activity of
the film when compared with that produced without heat treatment.
In solvent compounding, bacteriocins are needed to be compatible
with film polymers and/or needed to be soluble in the same solvent of
the film. Biopolymers, such as protein-based and carbohydrate-based
polymers, are good candidates for this type of film forming process
since they are soluble in water, ethanol and other solvents which are
compatible with bacteriocins (Dawson et al., 2003). A number of studies
have shown that biopolymers as film forming materials in solvent
compounding process to produce the antimicrobial films impregnated
with bacteriocins for meat packaging such as the incorporation of
enterocin produced by E. faecium CTC492 into alginate, zein and polyvi-
nyl alcohol film (Marcos, Aymerich, Monfort, & Garriga, 2007), incorpo-
ration of nisin into modified alginate film (Millette, Le Tien,
Smoragiewicz, & Lacroix, 2007), and incorporation of commercial
 W. Woraprayote et al. / Meat Science 120 (2016) 118–132
pediocin, ALTA® 2351, into cellulose acetate film (Santiago-Silva et al.,
2009) is a feasible technique.
The antimicrobial efficiency of cellulose acetate film incorporated
with pediocin ALTA®2351 on preservation of sliced ham was investigat-
ed. 25% and 50% of pediocin ALTA®2351 was added directly into cellu-
losic base film by solvent compounding method. The antimicrobial
efficiency of the films against L. innocua ATCC 33090 and Salmonella
Typhi ATCC 6539 on sliced ham was evaluated by means of a challenge
test. Under the vacuum-packed condition, the antimicrobial films were
more effective against L. innocua. The 50% pediocin-film caused a reduc-
tion of 2 log cycles in relation to control treatment after 15 days of
storage. Whilst, the 25% and 50% pediocin-films had similar perfor-
mance on Salmonella sp. inhibition, both presenting 0.5 log cycle reduc-
tion in relation to control, after 12 days of storage. The authors
suggested that the films incorporated with pediocin exhibited potential
use as one hurdle technology added in the storage period among others
good manufacturing practices for preservation of sliced ham (Santiago-
Silva et al., 2009).
This study is a case that shows the potential of direct incorporation
technique to apply bacteriocins directly into meat product packaging
to produce the effective antimicrobial meat packaging. The morpholog-
ical characteristic of the pediocin-films produced in this study, however,
was poorer when compared with the non-pediocin containing film. The
direct incorporation of pediocin into film matrix showed the adverse ef-
fect on transparency, thickness and some mechanical properties of the
produced films such as force in rupture and deformation in rupture
(Santiago-Silva et al., 2009).
To improve film properties, especially film transparency, film thick-
ness, and antimicrobial properties of the film incorporated with bacteri-
ocin, glycerol as a hydrophilic plasticizer was added into the cellulosic
matrix of the film (Imran, El-Fahmy, Revol-Junelles, & Desobry, 2010).
In this study, the antimicrobial film was produced using casting process
with direct incorporation of commercial nisin, Nisaplin®, into film
forming solution. The incorporation of Nisaplin® into cellulose deriva-
tive, i.e. HPMC-based film, dramatically increased the film thickness
due to salt crystallization while glycerol could normalize its effect by ho-
mogenous dispersibility. The tensile strength of nisin-films decreased,
whilst, ultimate elongation was increased significantly. The transparen-
cy of antimicrobial film with glycerol (10–30% glycerol) was not signif-
icantly different from control film (without nisin and glycerol), whilst,
the film incorporated with nisin without glycerol reduced the film
transparency. These results demonstrated that the addition of glycerol
as a plasticizer can improve stretch-ability, transparency and film ho-
mogeneity. In addition, the antimicrobial efficiency of active film was
still high. The antimicrobial film significantly inhibited the growth of
16 strains of indicators (Listeria, Staphylococcus, Bacillus and Enterococ-
cus). Glycerol was found to have no adverse effect on the release of
nisin from the film (Imran et al., 2010). However, plasticizer generally
causes the increased water permeability of the film so it must be
added at a certain amount to obtain the films with improved flexibility,
thickness and transparency without significant decrease of mechanical
strength and barrier property to mass transfer (Brindle & Krochta,
2008).
Synthetic polymer-based and gluten-based films were used as a car-
rier for lactocin 705 and lactocin AL705, bacteriocins produced by
L. curvatus CRL705, for control the growth of spoilage LAB and Listeria
(Massani et al., 2014). Bacteriocins directly incorporated into both
types of film effectively inhibited Listeria in the artificially contaminated
Wieners (fat content of 20–30%) during 45 days of chilled storage,
whilst LAB was only slightly inhibited by the active films. The authors
suggested that the low inhibitory effectiveness against LAB is in correla-
tion with the low activity observed for lactocin 705 in the presence of
fat. The results supported the feasibility of the application of bacterio-
cins through meat packaging to control pathogens in meat products
and highlighted the importance of evaluating antimicrobial packaging
systems for each particular meat product application.
127
In attempt to increase the antimicrobial efficacy or broaden the anti-
microbial spectrum of meat packaging, other food grade antimicrobials
were applied to the film to act synergistically with bacteriocins. The par-
tially purified nisin Z, produced by L. lactis subsp. lactis I8-7-3, together
with lauric arginate (LAE) were directly incorporated into pullulan film
for use as fresh and processed meat packaging during chilled storage.
The result showed that the antimicrobial activity, against Salmonella
spp., L. monocytogenes, S. aureus, and E. coli, of the film containing the
combination of nisin Z and LAE was significantly improved when com-
pared to the film containing nisin Z or LAE alone. While control film
(without nisin and/or LAE) could not inhibit all tested pathogens
(Pattanayaiying, H-Kittikun, & Cutter, 2015).
5.4. 3.2. Indirect incorporation of bacteriocins onto film surface
Incompatibility between bacteriocin preparations and polymers is
the major problem for direct incorporation or mix bacteriocins directly
into petroleum-based films. To overcome this problem, the indirect in-
corporation by coating bacteriocins onto plastic film surface was used
(Iseppi et al., 2008). Cast edible films, for example, have been used as
carriers for bacteriocins and applied as coatings onto packaging mate-
rials. Examples include pediocin-containing milk-based powder
adsorbed onto cellulose casings and barrier bags (Ming et al., 1997),
nisin coated onto the surface of polyethylene, ethylene vinyl acetate,
polypropylene, polyamide, polyester, acrylics and polyvinyl chloride
films (Coma, 2008), and enterocin 416K1 doped hybrid coatings coated
onto polymeric film (Iseppi et al., 2008).
Polymeric film coated with hybrid coatings doped with enterocin
416K1 for use as anti-listeria food packaging was developed (Iseppi
et al., 2008). Enterocin 416K1, produced by Enterococcus casseliflavus
IM416K1, was mixed with an organic-inorganic hybrid coating and
then was coated onto a low-density polyethylene (LDPE) film surface.
The antimicrobial efficiency of the produced film against
L. monocytogenes was evaluated by means of challenge test using frank-
furters as a model. The result showed that enterocin-coated film signif-
icantly reduced L. monocytogenes viable counts in frankfurters in the
first 24 h compared to the control uncoated film during chilled storage.
The listeria count of frankfurters packed with enterocin-coated film was
significantly lower than that of one packed with uncoated film through-
out the storage period of 28 days at 4 and 22 °C. This suggested the po-
tential of its use as a good meat packaging for control L. monocytogenes.
A further advantage of this bacteriocin application method was that the
coated films showed the same transparency of the uncoated one, and
that the bioactive coating has good adhesion to the plastic films even
without any preliminary treatment of the plastic film surface. All these
results, along with the relatively low cost of reactants and the easy ap-
plication to plastic substrates, confirmed the possible industrial use of
this technology in the meat packaging field.
The combination of nisin with EDTA was applied to the internal sur-
face of plastic film for use as beef chops packaging at 1 °C under vacuum
condition. When compared with the un-activated film, film coated with
nisin-EDTA significantly reduced the loads of spoilage bacteria,
B. thermosphacta and Carnobacterium spp., and reduced the release of
metabolites in the headspace of beef with a probable positive impact
on meat quality (Ferrocino et al., 2013).
To avoid the use of carrier, diffusion coating or adsorption of bacte-
riocins to the packaging surface has been attempted. The key factor
affecting bacteriocin adsorption is the compatibility of bacteriocin and
film surface characteristics, the purity of bacteriocin preparations,
adsorption temperature and adsorption time. Massani, Vignolo,
Eisenberg, and Morando (2013) reported that the impurities in bacteri-
ocin preparations, generated during growth of bacteriocin producer,
strongly affected the adsorption of lactocin 705 on the multilayer-
linear low density polyethylene (LLDPE) films. The adsorption of
Nisaplin®, commercial nisin, to the cellophane surface was investigated
to be affected by adsorption temperature. The increase in adsorption
128 W. Woraprayote et al. / Meat Science 120 (2016) 118–132

temperature resulted in a decrease in the amount of adsorbed nisin. For

this system, maximum load of nisin was obtained at 8 °C. This nisin
adsorbed cellophane film effectively reduced the total endogenic bacte-
ria in fresh veal meat during chilled storage for 12 days (Guerra, Macías,
Agrasar, & Castro, 2005).
Recently, a novel packaging system of poly(lactic acid) (PLA)/saw-
dust particle (SP) biocomposite film to facilitate the incorporation of
bacteriocin into packaging materials without the need of bacteriocin
carrier or coating polymer has been developed in our laboratory
(Woraprayote et al., 2013). PLA, a renewable polymer, was selected as
the packaging material according to the trend toward green, environ-
mental friendly and biodegradable packaging. PLA film was elaborated
by blow film extrusion followed by the film pre-conditioning and diffu-
sion coating of bacteriocin (Fig. 2). Antimicrobial evaluation of the PLA
film diffusion coated with pediocin PA-1/AcH (PLA + Ped) indicated
that the film did not exhibit the antimicrobial activity against target mi-
croorganism (Fig. 3). In accordance with protein adsorption determina- Fig. 3. Antimicrobial activity of poly(lactic acid) film (PLA) and poly(lactic acid)/sawdust
tion, PLA film could not adsorb any protein, including pediocin PA-1/ particle biocomposite film (PLA/SP) diffusion coated with partially purified pediocin
(Ped), commercial nisin from Sigma (Nisin) and partially purified bacteriocin 7293
AcH (data not shown). This may be explained by the incompatibility be-
(Bac7293). Antimicrobial activity of the films was tested against Listeria monocytogenes
tween hydrophilic bacteriocin solution and more hydrophobic PLA film. by means of the agar diffusion method. Antimicrobial activity was expressed as the clear
To overcome this limitation, modified sawdust particles (SP) with high zone of inhibition.
hydrophilic property were incorporated into PLA film to play a role in
embedding partially purified pediocin PA-1/AcH into the hydrophobic
PLA film during diffusion coating process. The maximum pediocin ad- Not only for pediocin, this developed packaging system was also ef-
sorption (11.63 ± 3.07 μg protein/cm2) was observed after the pre- fective to adsorb other bacteriocins including nisin as well as bacteriocin
treated PLA/SP film was soaked in pediocin solution for 30 min. The pro- 7293, a novel bacteriocin produced by Weissella hellenica BCC 7293, to
duced PLA/SP film coated with pediocin PA-1/AcH (PLA/SP + Ped) ef- provide a novel type of antimicrobial biodegradable films with high an-
fectively inhibited the growth of L. monocytogenes both in vitro (Fig. 3) timicrobial effectiveness and broader antimicrobial spectra against var-
and in model food system. The use of PLA/SP + Ped as a food-contact ious meat spoilage and meat borne pathogens. Not surprisingly, PLA
antimicrobial packaging for raw sliced pork suggested a potential inhi- film without SP could not adsorb nisin and bacteriocin 7293 (data not
bition of L. monocytogenes (99% of total listerial population) on raw shown). Bacteriocin 7293, as previously mentioned, was reported to
sliced pork during the chilled storage, supporting the feasibility of be active against several unwanted bacteria both gram positive and
using pediocin-coated PLA/SP film to reduce the initial load of gram negative including L. monocytogenes, S. aureus, S. Typhimurium,
L. monocytogenes on the surface of raw pork. E. coli, Pseudomonas aeruginosa, and A. hydrophila (Woraprayote et al.,

Fig. 2. Scheme of poly(lactic acid) (PLA) and poly(lactic acid)/sawdust particle (PLA/SP) film elaboration and antimicrobial testing. PLA polymer was initially compounded with modified
sawdust particles (SP) and then was blown to form the film. PLA/SP films were then pre-conditioned by dry-heat treatment at 90 °C for 2 h and diffusion coated with bacteriocin solution
before antimicrobial tested by agar diffusion method. PLA film without SP was used as a control.
 W. Woraprayote et al. / Meat Science 120 (2016) 118–132
2015). According to the JIS Z 2801:2000 standard method for antimicro-
bial testing of the film (Japanese Standard Association, 2000), PLA/SP
film coated with partially purified bacteriocin 7293 (PLA/
SP + Bac7293) was evaluated to be effective antimicrobial packaging
since it significantly reduced the number of tested pathogens
(L. monocytogenes, S. aureus, S. Typhimurium, E. coli, P. aeruginosa, and
A. hydrophila) for more than 2 log CFU/cm2 when compared with con-
trol uncoated PLA/SP film (data not shown). The antimicrobial activity
of bacteriocin 7293 adsorbed onto PLA/SP film remained unchanged
even after storage at 25 °C for 12 months, whilst free form of bacteriocin
7293 completely lost its activity in the same storage condition,
highlighting the additional role of PLA/SP film in preserving antimicro-
bial effectiveness of bacteriocin. The antimicrobial effectiveness of
PLA/SP + Bac7293 in meat and its effect on meat and meat products
qualities are being evaluated.
Until now, no regulation specifically on the amount of bacteriocins
released from packaging to food has been established. However, the fol-
lowing guidance may be considered when meat packaging incorporated
with bacteriocins is going to be developed and commercialized. In the
United States, antimicrobial packaging compounds that do not migrate
into the food, and hence are not considered food additive, can be candi-
dates for approval through the food-contact substance notification pro-
cess. Whilst, compounds that migrate into food are considered food
additives and must meet the food additive standards or must be subject-
ed to the food additive petition process (Zhang et al., 2011).
In Europe, all the compounds used as food-contact materials (FCMs),
including food packaging, need to be approved and included in ap-
proved lists of FCMs. Nisin is also the only bacteriocin approved by the
European Community as a safe preservative for food contact, coded as
E 234 (Perez-Espitia et al., 2012). The amount of bacteriocins released
from packaging to food may be considered to meet the overall migration
limit (OML), typically 60 mg/kg food, according to the commission di-
rective 20002/72/EC (The Commission of the European Communities,
2002). However, Valdés, Mellinas, Ramos, Garrigós, and Jiménez
(2014), suggested that the amount of active compounds released from
packaging materials should not be included in the calculation of the
OML and could exceed the overall migration requirements indicated
in the EU or national legislations. The active compounds should be sub-
jected to safety assessment by the EFSA (European Food Safety Author-
ity) before they are authorized for their use (Valdés et al., 2014).
Covalent bonding of bacteriocins to packaging materials seems to
alter the conformation and limit the mobility of bacteriocins, conse-
quently reduces the antimicrobial effectiveness of the peptide (Espitia
et al., 2012). Moreover, at or before actual time of use, it may be neces-
sary to treat the surface of packaging so as to break the covalent bonds
linking the bacteriocin molecules to the surface, thereby rendering the
molecules capable of detaching from the packaging and of attacking tar-
get bacteria (Daeschel & McGuire, 1995). These make the packaging co-
valently bonded with bacteriocins complicate to be used in food
application. To the best of our knowledge, there have no report on the
incorporation of bacteriocins in meat and other food packaging via co-
valent bonding.
6. Conclusion
The advancement of science and technology has led to an increase in
bacteriocin research. As a consequence, a number of novel LAB bacterio-
cins with unique and promising properties have been discovered.
Nevertheless, further comprehensive research is still required to gain
insight into the molecular mechanisms involved in bacteriocin produc-
tion, immunity and mode of action, which is necessary for safe and
effective exploitation of the discovered bacteriocins. Moreover, toxico-
logical data and the fate of the molecule after ingestion are also required
to establish the GRAS status and legal approval of bacteriocins for food
application.
129
To guarantee safety and quality of meat and meat products, not only
L. monocytogenes, but also other meat spoilage and meat-borne patho-
gens must be taken into account. Nisin and pediocin are not active or ex-
hibit low efficacy to inhibit gram-negative bacteria. In this regards, the
use of newly discovered bacteriocins can be promising alternatives to
assure the safety of meat and meat products as they demonstrated a
great intervention over broad spectrum of bacteria. Other GRAS micro-
organisms of Bifidobacterium and Bacillus genus, in addition to LAB, have
been known to produce bacteriocins with antimicrobial activity against
broad spectrum of both gram-positive and gram-negative food borne
pathogens, and in some cases against yeast and fungi. The important
and industrial value of Bacillus-bacteriocins has been largely
underestimated. To our knowledge, the application of bacteriocins
from Bacillus species in meat and meat products, as well as other
foods, has just received attention and lately been investigated
(Khochamit, Siripornadulsil, Sukon, & Siripornadulsil, 2015; Mouloud,
Daoud, Bassem, Atef, & Hani, 2013; An et al., 2015; Guo et al., 2016).
The use of bacteriocins in combination with other preservation
methods can create a series of hurdles during the manufacturing to re-
duce food spoilage caused by microorganisms. It has been proven that
the application of chemical preservatives, physical treatments (heat),
or new mild non-thermal physical methods (pulsed electric field, high
hydrostatic pressure (HHP), vacuum, or modified atmosphere packag-
ing) positively enhances the activity of many bacteriocins. Using an ad-
equate mix of hurdles is not only economically attractive but also
improves safety, sensory and nutritional qualities of meat and meat
products. Practically, the combined use of several preservation factors
can produce a synergistic effect.
Last but not least, the innovative techniques or technology to incor-
porate bacteriocins by means of antimicrobial/active packaging are still
considered challenging. Increasing demand for safe, minimally proc-
essed, fresh food products brings about the major challenges to the
food packaging industry to develop antimicrobial packaging technology,
especially with the aid of bacteriocin, for maintaining the safety and
quality of packaged foods. Recent relevant studies have demonstrated
a success in an incorporation of LAB bacteriocins into packaging
materials. Development of such active packaging may be a promising
technique. Still, the technology requires further investigation for large-
scale production.
Acknowledgements
The authors would like to thank the Thailand Research Fund (TRF)
for financial support under The TRF Distinguished Research Professor
Grant (Grant No. DPG5880002) to Soottawat Benjakul, the National
Center for Genetic Engineering and Biotechnology (BIOTEC) for provid-
ing research funding, laboratory equipment and facilities, Meat Tech-
nology Research Network Center (MTRNC) under the supported by
King Mongkut's Institute of Technology Ladkrabang (KMITL) and The
Animal Husbandry Association of Thailand.
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