Science of the Total Environment 825 (2022) 154058

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Science of the Total Environment

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

Combined antimicrobial effect of bacteriocins with other hurdles of

physicochemic and microbiome to prolong shelf life of food: A review
⁎
Guorong Liu , Rong Nie, Yangshuo Liu, Arshad Mehmood
Beijing Advance Innovation Center for Food Nutrition and Human Health, Beijing Laboratory of Food Quality and Safety, Beijing Engineering and Technology Research Center of
Food Additives, Beijing Technology and Business University (BTBU), Beijing 100048, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Combination of bacteriocins with other

hurdles markedly improve food safety.
• Synergistic effects are observed between
bacteriocin and other hurdles.
• Combination of bacteriocins with other
hurdles could expand the antibacterial
spectrum.
• Combination of bacteriocins with other
hurdles could reduce usage.
• Combination of bacteriocins with other
hurdles could extend shelf life.

A R T I C L E I N F O A B S T R A C T

Article history: Bacteriocins are ribosomally synthesized peptides to inhibit food spoilage bacteria, which are widely used as a kind of
Received 18 October 2021 food biopreservation. The role of bacteriocins in therapeutics and food industries has received increasing attention
Received in revised form 24 January 2022 across a number of disciplines in recent years. Despite their advantages as alternative therapeutics over existing strat-
Accepted 17 February 2022
egies, the application of bacteriocins suffers from shortcomings such as the high isolation and purification cost, narrow
Available online 22 February 2022
spectrum of activity, low stability and solubility and easy enzymatic degradation. Previous studies have studied the
Editor: Vijai Kumar Kumar synergistic or additive effects of bacteriocins when used in combination with other hurdles including physics,
chemicals, and microbes. These combined treatments reduce the adverse effects of chemical additives, extending
Keywords: the shelf life of food products while guaranteeing food quality. This review highlights the advantages and disadvan-
Bacteriocins tages of bacteriocins in food preservation. It then reviews the combined effect and mechanism of different hurdles
Combination and bacteriocins in enhancing food preservation in detail. The combination of bacterioncins and other hurdles provide
Hurdle technologies potential approaches for maintaining food quality and food safety.
Food preservation

1. Introduction
The ever increasing globalization of food trade and a strive towards lon-
ger shelf-lives imply that novel food preservation technologies are needed
due to trends of convenience and durability to support long-distance trans-
port (Elsser-Gravesen and Elsser-Gravesen, 2013). Meanwhile, foodborne
pathogens threat the safety of international public health and safety
(IPHS) (Manea et al., 2017). A report published by the Centers for Disease
Control and Prevention (CDC) documented that annual foodborne illnesses
in the United States result in around 76 million infections, 325,000 hospi-
talizations, and 5000 deaths. Over 90% of deaths are caused by Bacillus ce-
reus, Staphylococcus aureus, Listeria monocytogenes, Campylobacter spp.,
Salmonella spp., and Escherichia coli O157:H7 (Scallan et al., 2011; Van
Cauteren et al., 2017). Consumer demands are varying for low salt, less

⁎ Corresponding author.
E-mail address: liuguorong@th.btbu.edu.cn (G. Liu).

http://dx.doi.org/10.1016/j.scitotenv.2022.154058
0048-9697/© 2022 Elsevier B.V. All rights reserved.
G. Liu et al. Science of the Total Environment 825 (2022) 154058

sugar and fat in food in pursuit of healthy diet while higher pH for milder
taste, minimal processing for fresher appearance of the food. These de-
mands result in a friendlier and less adverse environment for unwanted mi-
croorganisms (Elsser-Gravesen and Elsser-Gravesen, 2013). This
significantly affects the industry when maintaining food quality. To guaran-
tee food safety while preserving the nutritions and sensory properties of
food, various preservation techniques are developed including heat treat-
ment (heating, boiling, drying, etc.), cold storage (chilling, frozen, etc.),
modern preservation methods (pasteurization, canning, etc.), chemical pre-
servatives and biopreservation to retard the growth of spoilage microorgan-
isms. However, heat treatment and modern preservation methods may
change the sensory properties and destroy the nutritients in food (Smelt
and Brul, 2014); chilling to an unsuitable low temperature is detrimental
to some foods of plant origin causing chilling injury (Leistner, 2000); chem-
ical preservatives were proven to cause allergies and catalyzing the forma-
tion of carcinogenic end-products (Pisoschi et al., 2018) (Table 1). These
contradictory trends and demands put food biopreservation under the spot-
light. A “Hurdle Concept”, two or more suitable preservation techniques are
combined to have an additive or synergistic effect, could be useful to meet
current consumer demand.
Bacteriocins are natural antibacterial peptides capable of inhibiting the
growth of foodborne pathogens or spoilage bacteria and some of them
could be potential biopreservatives for food industries, as i) bacteriocins
markedly retard the growth of C. perfringens, E. coli, Salmonella enteritidis,
B. cereus, and L. monocytogenes (Yang et al., 2012); ii) most bacteriocins
are safe for human microbiome, because they only inhibit sensitive bacteria
but leave beneficial bacteria intact since most bacteriocins can be degraded
by enzymes in human body; iii) most of foodborne pathogens and spoilage
microorganisms are not bacteriocin-resistant, and some bacteriocins can in-
hibit methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-
resistant E. faecalis strains (Kruszewska et al., 2004; Millette et al., 2008);
iv) bacteriocins have minimal effects on the nutrients and sensory proper-
ties of food. However, only a few bacteriocins have been commercialized:
i) nisin has been approved by regulatory authorities including the World
Health Organisation (WHO)/Food Development Authority (FDA) in the
USA and the European Food Safety Authority (ESFA) in Europe as a pure

Table 1
Overview on the advantages and disadvantages of current technologies for food preservation.
Type Hurdles Advantages Disadvantages

⟡ Heat resistance of spores

⟡ Cost-effective ⟡ Affect the taste, texture, smell and color of food
Heat
⟡ Widely available ⟡ Do not satisfy demand for fresh-like products
⟡ Energy consumption
⟡ Slow down the chemical and biological processes in
foods ⟡ Rely on mechanical refrigeration systems or ice
Chilling
⟡ Minimal effects on the nutritional values and sensory ⟡ Chilling injury
properties of food products
Physical Pulsed electric ⟡ Energy efficient
⟡ High cost
hurdles field ⟡ Minimal losses of flavor and food quality
⟡ Widely available
High pressure ⟡ Pressure resistance strains
⟡ Do not affect the taste, texture, smell and color of food
processing ⟡ High cost
⟡ Reduce allergic substances
⟡ Stability in physiological solution
⟡ High surface area-to volume ratio ⟡ Complexified combination processing
Nanotechnology ⟡ Easy to synthesize with low production cost ⟡ Unavailability of proper in vivo experimental information
⟡ Non-toxic nature at low concentrations ⟡ Limited research on the toxic nature of the combinations
⟡ Do not cause rapid resistance of bacteria
⟡ Broad antimicrobial spectrum ⟡ Unacceptable organoleptic properties
Essential oils ⟡ Biodegradable ⟡ Complicated content
⟡ GRAS ⟡ Pose hazards to pregnant women
⟡ Disruption of the outer membrane of some G− bacteria ⟡ Toxicity at relevant concentrations
EDTA
⟡ Make G− bacteria sensitive to bacteriocins ⟡ Environment unfriendly
Organic acid ⟡ Wide antimicrobial spectrum ⟡ Affect taste of food
Chemical ⟡ Wide and effective antimicrobial spectrum
Salts of organic ⟡ Side effects to human
hurdles ⟡ Low cost
acid ⟡ Cause concerns of costumers
⟡ High stability
⟡ Antimicrobial system naturally present in raw milk
Lactoperoxidase
⟡ Wide inhibition spectrum ⟡ Less stable under acidic conditions
system
⟡ Resistant to proteolytic enzymes
⟡ Antimicrobial effect of G+ bacteria
Lysozyme ⟡ High resistance of G− bacteria
⟡ Non-toxic and harmless, high safety
⟡ Wide inhibition spectrum
⟡ Water soluble substance ⟡ Poor inhibition of L. monocytogenes
Reuterin
⟡ Active at a wide range of pH values ⟡ High prurition cost
⟡ Resistant to proteolytic and lipolytic enzymes
⟡ Effective inhabitation of G− bacteria ⟡ Toxicity at relevant concentrations
Polymyxin B
Microbial ⟡ High solubility ⟡ Fail to inhabit G+ bacteria
hurdles Surface layer ⟡ Fail to affect G+ bacteria
⟡ Endopeptidase activity against cell wall of G− bacteria
protein ⟡ Hardly extract
⟡ Highly specific
⟡ Narrow host range
⟡ Low cost
Bacteriophages ⟡ Potential for negative consumer perception regarding the use of the terminology
⟡ Do not affect taste, texture, smell and color of food
“viruses” or “virulent phages” in foods
⟡ Widespread in the natural environment
⟡ Effective antimicrobial activity
⟡ GRAS ⟡ Relatively narrow spectrum of antibacterial activity
⟡ Highly specific ⟡ Rapid but not long bactericidal effect
Bacteriocins ⟡ Minimal effects on the nutritional values and sensory ⟡ Low yield and high production and purification cost
properties of food products ⟡ Low solubility and diffusivity in food matrix
⟡ Widespread in the natural environment ⟡ Adverse interactions of bacteriocins and food matrix
⟡ Low toxicity towards eukaryotic cells

2
G. Liu et al.
bacteriocin preparation; ii) MicroGARDTM range and ALTA 2431 which
both contain pediocin PA-1 have been approved by FDA as bacteriocin con-
taining fermentates; iii) bacteriocins can be used as bacteriocin cultures, in-
cluding the BactofermTM range, HOLDBAC® protective cultures and
Micocin® (O’Connor et al., 2020).
Only a very few of bacteriocins have been commercialized out of those
discovered and preserved in laboratory. Bacteriocins facing an application
difficulty because i) some bacteriocins are known to exhibit a relatively nar-
row spectrum of antibacterial activity; ii) enterococcal cytolysin has a wide
spread cytotoxic activity (Cox et al., 2005) and gram-negative bacteriocins
when producing strains may also produce some endotoxins resulting in del-
eterious effect, thus requiring rigorous purification procedure; iii) the per-
meability of the bacteriocins in food matrix, the pH of the food matrix,
enzymes and other adverse interactions of bacteriocins and food matrix in-
fluence the efficacy of pure bacteriocins; iv) it is reported that strains of
L. monocytogenes and S. aureus are becoming resistant to nisin, plantaricin
C19 and sakacin A (Gálvez et al., 2007). Therefore, bacteriocins alone is
not enough to control food safety, let along its high monetary cost (Fig. 1).
To expand the antibacterial spectrum, reduce usage, enhance antibacte-
rial effect and avoid the resistance, the hurdle technology that involves
many physicochemical treatments with the bacteriocin has attracted con-
siderable attention. As suggested, hurdle technology could help adjust the
level of bacteriocin to maximize the viability loss of the target bacteria,
thus minimizing the occurance of resistances in food preservation. The hur-
dles combined with bacteriocins include (i) physical hurdles: temperature
(Aras et al., 2020), pulsed electric fields (Pol et al., 2001), high-pressure
processing (Ramaroson et al., 2018), nanotechnology (Sulthana and
Archer, 2021); (ii) chemical hurdles: essential oils (Alves et al., 2016; Du
et al., 2019; Shi et al., 2017), EDTA (Ay and Tuncer, 2016; Castellano
et al., 2011; Liang et al., 2020), organic acids (Zhao et al., 2017), animal-
derived enzymes (Arqués et al., 2008a, 2008b); (iii) microbial hurdles:
other bacteriocins, reuterin, bacteriophages (Duc et al., 2020; Heo et al.,
2018). Previous research has observed a synergistic effect when bacterio-
cins are combined with above hurdles, which gets a maximum lethality
against micro-organisms with the minimum damage to the sensory param-
eters of the food.
This paper sets out to describe the combined antimicrobial effect of bac-
teriocins with other hurdles as well as strategic usage in food system. In ad-
dition, this study also preliminary summary their combination mechaism. It
is necessary to enhance the study of bacteriocins and other hurdles to facil-
itate bacteriocins application in food industries.
2. Source and classification of bacteriocins
Microbes of different groups demonstrate a widely ranging capabilities
bacteriocins production, which can be divided into Gram-positive, Gram-
Fig. 1. Characteristics of bacteriocins from different group of microbes.
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Science of the Total Environment 825 (2022) 154058
negative bacteria and archaea (Johnson et al., 2018) (Fig. 1). Among the
gram-positive bacteria, lactic acid bacteria (LAB) can produce bacteriocins
with most prepositive antimicrobial peptides to preserve food in a natural
way. Threfore, LABs are recognized to be “Generally Regarded As Safe
(GRAS)” by the FDA (Chen and Hoover, 2003). Moreover, some LAB bacte-
riocins like enterocin AS-48, enterocin P, paracin 1.7, plantaricin MG and
lactococcin BZ can inhibit the growth of Salmonella which have resistance
to most bacteriocins (Chalón et al., 2012). Since LAB is generally present
in devious fermented food, LAB for bacteriocins production could be used
as a star culture without goting through rigorous purification procedure
(Mills et al., 2017). Bacteriocins from Gram-negative bacteria like
microcins are less studied as food preservatives, because they are produced
by E. coli which is potentially threatening food safety and has a narrow
spectrum of antibacterial activity (Chalón et al., 2012). Only a few bacterio-
cins produced by archaea were reported, including sulfolobicins from
Sulfolobus and halocin from Halogeometricum sp., and most studies are re-
lated to clinical application (O’Connor and Shand, 2002).
Bacteriocins are classified based on the chemical structures, types of
producing organism, physical properties, mode of action, stability and mo-
lecular size. Class I bacteriocins are mainly 19–50 amino acids in size,
consisting of highly post-translationally modified emanate of the non-
standard amino acids (Parada et al., 2007). Class II bacteriocins comprise
small non-modified peptides that are heat-stable. The Cass II is further di-
vided into pediocin-like bacteriocins (Class IIa), two-peptide unmodified
bacteriocins (Class IIb), circular bacteriocins (Class IIc), and unmodified,
linear, non-pediocin-like bacteriocins (Class IId) (Drider et al., 2006;
Oppegård et al., 2007). Class III bacteriocins include heat-labile proteins
with large-molecular-weight (>30 kDa) (Parada et al., 2007). Class IV bac-
teriocins which originally refer to antimicrobial peptides containing large
peptides combined with carbohydrates or lipids are cancelled as the com-
plex structures are not thoroughly purified (Güllüce and Karaday, 2013).
The representative member of Class I, II, III are nisin, pediocin and colicin,
respectively.
3. Mode of action of bacteriocins
The main mode of action of bacteriocins is disturbing the cytoplasmic
membrane with pores, or causing cell wall degradation (Fig. 2). Apart
from this, bacteriocins may affect living cells in some other ways, such as
microcin B17 inhibits DNA synthesis, colicins D and E5 can increase ex-
haustion of tRNA which slows down protein synthesis (Sulthana and
Archer, 2021), microcin J25 inhibit RNA polymerase inhibitor (Lopez
et al., 2007). Nevertheless, bacteriocins' modes of action has not been
fully discovered, especially those that are active against Gram-negative bac-
teria (Pérez-Ramos et al., 2021). Although nisin and pediocin PA-1 have
been commercialized as food additives, other bacteriocins like enterocin
G. Liu et al.
Fig. 2. Antimicrobial mechanism of bacteriocins alone and combined with other technologies.
AS-48 also have the potential to be used as biopreservatives in food based
on existing studies. The specific mechanism of above bacteriocins are
thereby introduced to compare the different modes between independently
used bacteriocins and the combination of bacteriocins with other hurdles.
3.1. Nisin and its mode of action
Nisin is a Class I bacteriocin produced by Lactococcus lactis used in
cheese manufacture. It has effective antimicrobial activity against a wide
range of Gram-positive foodborne pathogens, while its antimicrobial effect
on Gram-negative bacteria is small. Nisin A was first discovered in 1928
and has been applied to commercial food preservation since 1953 till
today. Besides, FDA approved to use nisin in pasteurized processed cheese
spreads established the legal precedent in the U.S. for application of bacte-
riocins as food additives (Chen and Hoover, 2003). Since then several nisin
variants (nisin Z, F, P, Q, H, U, U2) have been identified. The difference of
structure between nisin A and Nisin Z is just a single amino acid residue at
position 27, which inducing Nisin Z with a higher rate of diffusion and sol-
ubility under neutral pH conditions (Ellis, 2018). However, the antimicro-
bial effect does not show significant difference. Nisin act by forming a
pore in membranes of target cells, leading to leakage of essential small mol-
ecules at end. The C-terminal region of nisin is responsible for the initial in-
teraction of nisin with the target membrane (Breukink et al., 1998). Firstly,
nisin is inserted and partially penetrated into the cytoplasmic membrane of
target bacteria (Breukink and de Kruijff, 2006). Then, nisin binds to
phosphatidylglycerol (universal receptor) by the C-terminal region
(Breukink and de Kruijff, 2006; Giffard et al., 1997). Followed by the mem-
brane insertion, subsequent pore formation results in abrupt cell death
(Breukink and de Kruijff, 2006; Cotter et al., 2005).
3.2. Pediocin and its mode of action
Pediocin is Class IIa bacteriocin produced by Pediococcus pentosaceus,
showing extremely strong inhibition activity against L. monocytogenes
which could grow at 4 °C and cause various foodborne illness. Although
pediocin PA-1 has been commercialized in food preservatives, it has not
been legally approved yet to be applied in food preservation worldwide
(Settanni and Corsetti, 2008). Like nisin, pediocin also causes target cell
death when forming pores or channels, but in a different pore formation
method. The antimicrobial effect of pediocin is caused by its ability of
permeabilizing lipid vesicles, but the author think it only occurres in high
pediocin concentrations (Chen et al., 1997a, 1997b; Chikindas et al., 1993).
4
Science of the Total Environment 825 (2022) 154058
3.3. Enterocin and its mode of action
The enterocins produced by different strains of Enterococcus faecium
have different structures. To be specific, enterocin A, P are Class IIa bacte-
riocins, enterocin 1071 is Class IIb bacteriocin, and enterocin AS-48 is
Class IIc bacteriocin. The E. faecium has the potential to be used as start cul-
tures to provide enterocins and organic acid, enhancing the control of
L. monocytogenes in fermented products. Among enterocins, the action of
enterocin AS-48 has been studied in detail. Enterocin AS-48 takes
cytoplasmatic membrane as the target, destabilizing the membrane to dissi-
pate the proton motive force and cause cell death. The simulation results
suggested that the protein inserted could organize itself to generate stable
pores (Sidhu and Nehra, 2019, 2021), while the supportive experiments
are absent. The action of enterocins in Class IIa bacteriocin is similar to
pediocin. Enterocins of Class IIb bacteriocin act on the membranes of
their targets by rendering them permeable to certain cations such as Na+,
K+, Li+ or H+.
4. Combination of bacteriocin with other hurdles for food preserva-
tion
Compared with preservation methods that use single bacteriocin, hur-
dle technology is a novel and promising technique that controls food safety
with minimal impact. It improves the antimicrobial effect, reduces the
amounts of preservations used, saves energy, and expands the shelf life
(Fig. 3). Therefore, we reviewed studies published over the past two de-
cades to know more about the combined effect of bacteriocins with other
hurdles against various microorganisms. According to the origin of the hur-
dles, they could be divided into physical (Table 2), chemical (Table 3) and
microbial hurdles (Table 4).
4.1. Combination of bacteriocins and physical hurdles against foodborne patho-
gens and spoilage microorganisms
4.1.1. Bacteriocins and heat
Traditional heat processes are the most commonly used and effective
way to control foodborne microorganisms. Traditional heat processes inac-
tivate microorganisms via protein unfolding and denaturation (Smelt and
Brul, 2014). In traditional canning methods, extensive heat process was
used to destruct all spores or inhibit the grow of spores below 40 °C during
the storage. To meet the consumer demands for fresh-like products, milder
heat treatment is adopted as low or middle temperature would not destruct
G. Liu et al.
Fig. 3. Combined effect of bacteriocins with other hurdles for food preservation.
the heat resistance of spores and vegetative cells. The combination of bac-
teriocins and heat treatment could increase the heat sensitivity of spore, ex-
tend the time of antimicrobial effect and broaden the spectrum of
antibacterial activity.
The combination of bacteriocins and heat treatment could alter the ther-
mal resistance of bacterial spores. Clostridium sporogenes ATCC 7955 spores
were rapidly inactivated when being treated in 16 μg/mL nisin at 90 °C. In
contrast, when it is only treated at 90 °C, no spores were inactivated. Ac-
cordingly, percentage of dipicolinic acid (DPA) which contributes to ther-
mal resistance as a component of spores release was higher when spores
were treated in nisin at 90 °C, relative to a treatment of 90 °C alone
(Hofstetter et al., 2013a, 2013b). Moreover, the divergent effects of nisin
on spore membrane fluidity provide a rationale for their divergent effects
on heat-induced spore inactivation and DPA release (Hofstetter et al.,
2013a, 2013b). Similarly, adding nisin (2000 IU/mL) to skim milk before
heat processing (103 °C) enhances the control of spores of the mesophile
B. cereus T and the thermophile Bacillus stearothermophilus ATCC 12980.
Nisin enhances the spore sensitivity to heat and the presence of residual
nisin inhibits the spore survival and growth (Wandling et al., 1999).
The combination of bacteriocins and heat processing could stop the re-
growth of foodborne pathogens and spoilage bacteriocins, thereby extend-
ing the shelf-life of food product. Ananou et al. observed that enterocin AS-
48 (40,60μg/g) alone significantly reduces the viable counts of
L. monocytogenes CECT 4032, but L. monocytogenes CECT 4032 regrows dur-
ing the 60 days storage period of cooked ham at 5/15 °C. Meanwhile,
S. aureus CECT 976 in cooked ham is slightly sensitive to enterocin AS-48
and might be out of control at 15 °C with 40μg/g enterocin AS-48. While
sublethal heat (60 °C, 2 min) remarkably increased enterocin AS-48 activity
against S. aureus CECT 976 and L. monocytogenes CECT 4032 in cooked ham
(Ananou et al., 2010a).
In addition, proper heat treatment combined bacteriocins would solve
the problem of narrow spectrum of antibacterial activity. P. aeruginosa
and the two salmonellae are not sensitive to nisin, while heating at 55 °C
produce transient sensitivity to nisin. Particularly, these strains are only
sensitive if nisin is present during heating. These strains would normally
survive heating in an injured state, while being inactivated by nisin during
heating. Furthermore, the increase in hydrophobicity, lipopolysaccharides
release and permeability can be detected in the heating treatment, indicat-
ing that heating can produce transient injury to the outer membrane perme-
ability barrier, so nisin gains a temporary access to the cytoplasmic
5
Science of the Total Environment 825 (2022) 154058
membrane. However, the outer membrane recovers rapidly, so does the
nisin resistance (Boziaris and Adams, 2001).
Therefore, the combination of bacteriocins and heat treatment makes
spore, which used to be heat-resistant, become sensitive which saves the en-
ergy. Solving the instant antimicrobial effect of bacteriocins can extend the
shelf life of food and broaden the spectrum of bacteriocins. The combina-
tion enhances action effect because heat could injure the outer membrane
allowing bacteriocins enter the cell membrane.
4.1.2. Bacteriocins and chilling
Chilling treatment generally refers to temperatures above 0 °C and slow
down the growth rate of microorganisms as well as the chemical and bio-
logical processes in foods. Current studies only focus on the antibacterial ef-
fects of the cooling rates alone which indicated that very low (<5 °C/min)
and intermediate cooling rates (180–5000 °C /min) induce obviouse cell in-
juries or death, while low (5–180 °C/min) or very high cooling rates
(>5000 °C/min) may help cell survival (Cao-Hoang et al., 2010). Further-
more, sensitizing Gram-negative bacteria to bacteriocins will enhance the
antibacterial effect of the bacteriocins-and-chilling combination, and the
mechanism of chilling makes nisin-resistant L. monocytogenes sensitive to
nisin is described as followed.
The chilling rate is an important factor influencing the sensitivity of
Gram-negative bacteria to nisin, but the critical rate of chilling is yet to be
found. Rapid chilling and slow chilling from room temperature to 0.5 °C
in the nisin (50, 500, 2500 IU/mL) showed opposite effects on injury and
inactivation of Gram-negative bacteria (S. enteritidis PT4 and PT7, E. coli
ATCC 25922, P. aeruginosa USCC 2186 and Pseudomonas fragi ATCC
4973), but no effect is observed in the combination of slow chilling. How-
ever, rapid chilling shows a dose-dependent increase in lethality with
nisin. P. aeruginosa USCC 2186 is the most sensitive in combinations, show-
ing a 1.5–2.5 log reduction. Though reductions resulting from the chilling-
nisin combination is less than the reduction in broth experiments, adding
EDTA to the combination reduces P. aeruginosa USCC 2186 populations
by 1.3 log CFU (Boziaris and Adams, 2000). As for E. coli TG, it is reported
that slow chilling (2 °C/ min, reached 0 °C) does not induce transient sus-
ceptibility to nisin. However, E. coli TG in both exponential and stationary
growth phases are sensitive to nisin if there is nisin during the rapid chilling
(2000 °C/ min, reached 0 °C) or nisin is added a few minutes after the treat-
ment (Cao-Hoang et al., 2008). Subsequently, under an obvious synergistic
effect, exponentially growing E. coli TG in 100 IU/mL nisin under rapid
chilling shows a reduction of more than 6 logs, while the stationary grow-
ing E. coli TG in 1000 IU/mL nisin with rapid chilling shows a 2 logs reduc-
tion (Cao-Hoang et al., 2008). Similarly, the survived P. aeruginosa, S.
Enteritidis USCC2186, S. Enteritidis PT4 and PT7 are not sensitive to
nisin that is added a few minutes after the rapid chilling (7.3 °C/ min,
reached 0.5 °C) (Boziaris and Adams, 2001).
Apart from changing Gram-negative bacteria’ sensitivity to nisin, the
combination of chilling and bacteriocins make nisin-resistant Gram-
positive bacteria sensitive to nisin. After long-time low temperature (10
°C) treatment, L. monocytogenes Scott A, the nisin-resistant strain, becomes
sensitive to nisin due to the modified membrane fluidity. Cell membranes
of L. monocytogenes Scott A do adopt a lower gel-fluid phase transition tem-
perature to maintain sufficient membrane fluidity at 10 °C. Although the
lower temperature (10 °C) significantly increased the membrane rigidity
of the lipid vesicles, L. monocytogenes Scott A is still sensitive to nisin after
a long-term low temperature treatment (Li et al., 2002). In another study,
when wild-type cells are switched to a lower temperature (4 °C), the
order of the fatty acids makes the membrane more rigid, reducing nisin
Z's action. Meanwhile, the K+ efflux rates also decreased with droping tem-
peratures (Abee et al., 1994). In conclusion, low temperature could make
the membrane more rigid, negatively affecting the nisin's action, while
the sensitivity of target cells to nisin remains unchanged because the anti-
microbial activity is subject to multiple factors. However, the study mainly
focuses on nisin and chilling instead of researching the antimicrobial effect
of other bacteriocins with chilling treatment.
G. Liu et al. Science of the Total Environment 825 (2022) 154058

Table 2
Strategic usage of the combination of bacteriocins and physical hurdles.
Type Combination Food Positive effect Target microorganisms Reference
system
bacteriocins other hurdles

C. sporogenes ATCC 7955, Hofstetter

Nisin Heat – Inactivate endospores C. beijerinckii ATCC 8260, et al., 2013a,
C. difficile 3195 2013b
B. cereus T, Wandling
Nisin Heat Skim milk Reduce D values of spores
Heat + B. stearothermophilus ATCC 12980 et al., 1999
bacteriocins Cooked Avoid regrowth of food S. aureus CECT 976, Ananou et al.,
Enterocin AS-48 Sublethal heat
ham pathogens during 60 days L. monocytogenes CECT 4032 2010a,2010b
Show synergistic inhibitory L. innocua ATCC 51742,
White Al-Holy et al.,
Nisin Sublethal heat activity and avoid regrowth of L. innocua ATCC 33090,
chesses 2012
food pathogens L. innocua ATCC 33091
S. enteritidis PT4 and PT7,
Show enhancement of E. coli ATCC 25922, Boziaris and
Nisin Rapid chilling –
antimicrobial effect P. aeruginosa USCC 2186, Adams, 2001
Chilling + P. fragi ATCC 4973
bacteriocins Show synergistic inhibitory
Nisin Rapid chilling – E. coli TG Bi et al., 2018
effect
Long-term treatment with Sensitize nisin-resistant cell to
Nisin – L. monocytogenes Scott A Li et al., 2002
low temperature nisin
Show synergistic inhibitory Pol et al.,
Nisin PEF Skim milk B. cereus IFR-NL94–25
effect 2001
Plused electric Show obviously synergistic Sobrino-Lopez
Enterocin AS-48 + nisin PEF Milk S. aureus CECT 240
fields (PEF) + inhibitory effect et al., 2009
bacteriocins Fresh
Shear stress-moderate Show synergistic inhibitory E. coli K12, Mok et al.,
Nisin apple-kale
electric field effect L. innocua 2020
blend juice
Expand the antibacterial Rodriguez
Nisin HPP Cheese E. coli O157:H7
spectrum of nisin et al., 2005
Expand the antibacterial
High pressure carbon
Nisin – spectrum of nisin and show E. coli O157:H7 NCTC 12900 Bi et al., 2018
dioxide
high synergistic effect
E. coli parent strain MG1655 and
Reduce pressure-resistant E. García-Graells
Nisin HPP Milk pressure-resistant mutants LMM1010,
Coli et al., 1999
LMM1020 and LMM1030
Orange Eliminate the HHP-resistance Alpas and
High-pressure Nisin HPP S. aureus 485
juice of S. Aureus 485 Bozoglu, 2000
processing
Grade A
(HPP) + Nisin Reduce the HHP-resistance of Alpas and
HPP pasteurized S. aureus 485
bacteriocins Pediocin PA-1 S. Aureus 485 Bozoglu, 2000
milk
Low-acid Show synergistic inhibitory S. aureus,
Ananou et al.,
Enterocin AS-48 HPP fermented activity and don't accept L. monocytogenes,
2010a, 2010b
sausage growth of LAB S. Typhimurium
L. monocytogenes CTC1010,
Cooked Avoid regrowth of food Jofré et al.,
Nisin HPP L. monocytogenes CTC1011,
ham pathogens during 3 months 2008
L. monocytogenes CTC1034
Avoid regrowth of food Dallagnol
Lactocin AL705 HPP Pork loin L. innocua 7
pathogens during 40 days et al., 2017
Soluble soybean Fresh L. monocytogenes,
Luo et al.,
Nisin polysaccharide-based tomato Extend shelf life B. subtilis,
2019
nanoparticles juice S. aureus
Large
Zohri et al.,
Nisin Chitosan nanoparticles yellow Extend shelf life S. aureus
2010
croaker
E. coli, Vukomanović
Nisin Gold nanoparticles – Reduce MIC of nisin
P. aeruginosa et al., 2017
Nanotechnology
Bacteriocins produced by
+ bacteriocins S. aureus, Sidhu and
Lactobacillus spp. Silver nanoparticles – Enhance antimicrobial effect
S. flexneri Nehra, 2020
Bac4463 and Bac22
Show higher antimicrobial
García-Toledo
Pediocin Liposome – activity, stability and L. innocua AST-062
et al., 2019
controlled release
Dual coated liposome Enhance the stability and
Gomaa et al.,
Microcin MCCJ25 (polymeric network of – protect the bacteriocin from S. Enteritidis
2017
pectin and whey proteins) enzymatic degradation

4.1.3. Bacteriocins and pulsed electric fields
Pulsed electric field (PEF) is a commonly used non-thermal method of
liquid food preservation. According to existing studies, it has the potential
to be an alternative to heat processing. PEF treatment has been proven to
inactivate microorganisms while better maintaining the original flavor
and quality. Besides, as the method is performed at low processing temper-
atures which turns out to be energy efficient. Bacterial cells experience a
transmembrane potential across their membrane when being applied an
electrical field is applied (Yogesh, 2016). PEF has been used with bacterio-
cins to increase antimicrobial effect against foodborne pathogens
(Calderón-Miranda et al., 1999; Sobrino-Lopez et al., 2009; Terebiznik
et al., 2002).
The combination of PEF and bacteriocins leads different results. On the
one hand, the combination of nisin and PEF showed slight synergy effect.

6
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Table 3
Strategic usage of the combination of bacteriocins and chemical hurdles.
Classification Hurdle technology Food system Positive outcomes Target Reference
microorganisms
Bacteriocins Other hurdles

L.
Cinnamaldehyde, monocytogenes
Alves et al.,
Nisin carvacrol, eugenol, and Cow milk Show synergistic inhibitory effect ATCC 15313,
2016
thymol S. aureus ATCC
25923
E. coli O157:
H7, Campion et al.,
Nisin variants Carvacrol Apple juice Expand the antibacterial spectrum
C. sakazakii 2017
NCTC 8155
Show synergistic antimicrobial effect, expand Ghrairi and
Enteriocin A Thyme – E. coli O157:H7
the antibacterial spectrum, decrease MIC of EOs Hani, 2015
Show synergistic antimicrobial effect, decrease 14 strains of S.
Nisin Cinnamaldehyde Milk Shi et al., 2017
MIC of EOs aureus
Grapefruit seed extract, Show antibacterial effect and reduce dosage of L.
Nisin Pork loin Yu et al., 2019
cinnamaldehyde nisin and EOs monocytogenes
E. coli O157:
H7,
L.
Solomakos
monocytogenes
Essential oils + Nisin Thyme Minced beef Increase the shelf life of minced beef et al., 2008a,
Scott A,
bacteriocins 2008b
L.
monocytogenes
Lmk
Minced sheep Reduce unacceptable organoleptic properties of Govaris et al.,
Nisin Oregano essential oil S. Enteritidis
meat EOs 2010
Cooked Show enhanced antimicrobial effect and protect L. Zhao et al.,
Nisin Grape seed extract
shrimps color during prolonged shelf life monocytogenes 2020a,2020b
L.
Bologna Churklam
Nisin Carvacrol Show synergistic inhibitory effect monocytogenes
sausages et al., 2020
10403S
L.
monocytogenes
ATCC 15313,
L.
Grape fruit seed extract Show antibacterial effect and reduce MIC of
Nisin Lettuce monocytogenes Yu et al., 2019
and cinnamaldehyde nisin and EOs
H7962,
L.
monocytogenes
NADC 2045
Frozen
Bacteriocins form L. curvatus Castellano
EDTA ground-beef Expand the antibacterial spectrum E. coli O157:H7
EDTA + CRL705 and L. lactis CRL1109 et al., 2011
patties
bacteriocins
EDTA and Field et al.,
Nisin A – Expand the antibacterial spectrum E. coli
cinnamaldehyde 2017
S. aureus ATCC
29213,
Zhao et al.,
Nisin Citric acid Milk Enhance antimicrobial effect L.
2017
monocytogenes
Organic acid +
ATCC 19115
bacteriocins
Infant Campion et al.,
Nisaplin® Citric acid Enhance antimicrobial effect C. sakazakii
formula 2017
Enhance antimicrobial effect and no color Ajingi et al.,
Nisin Formic acid Potato B. subtilis
affected 2020
Sodium lactate, sodium L. Lungu and
Nisin Turkey breast Enhance antimicrobial effect
diacetate monocytogenes Johnson, 2005
Cold-smoked Enhance antimicrobial effect and reduce MIC of L. Neetoo et al.,
Nisin Sodium lactate
salmon pate nisin and sodium lactate monocytogenes 2008
L.
monocytogenes
CTC1010,
Salts of organic acid
L.
+ bacteriocins Jofré et al.,
Nisin Sodium lactate Cooked ham Show synergistic inhibitory effect monocytogenes
2007
CTC1011,
L.
monocytogenes
CTC1034
Cold-smoked Enhance antimicrobial effect and extend shelf L. Nykänen et al.,
Nisin Sodium lactate
rainbow trout life monocytogenes 2000
L.
Arqués et al.,
Animial-drived Nisin Lactoperoxidase system Skim milk Avoid regrowth of food pathogens monocytogenes
200
enzymes + Scott A
bacteriocins Infant Oshima et al.,
Nisin or lacticin 3147 Lactoperoxidase system Inhibit outgrowth of C. Sakazakii C. sakazakii
formula 2012

(continued on next page)

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G. Liu et al. Science of the Total Environment 825 (2022) 154058

Table 3 (continued)

Classification Hurdle technology Food system Positive outcomes Target Reference

microorganisms
Bacteriocins Other hurdles

lactic acid Chung and

Nisin Lysozyme Pork loin Prolong the shelf life to 6 weeks
bacteria Hancock, 2000

The addition of nisin (0.04 mg/mL) individually reduces 1.0 log unit of
B. cereus IFR-NL94–25 and the use of PEF treatment (16.7 kV/cm, 20 pulses
of 2 ms duration, 10 min) alone cause about 0.7 log unit, while the combi-
nation of nisin and PEF leads to a reduction of 1.9 log unit (Pol et al., 2001).
Under individual shear stress-moderate electric field treatment for 10 min,
maximum inactivating levels of E. coli K12 and Listeria innocua are 5.4 and
5.2 log CFU/mL respectively. Nisin (100 IU/mL) treatment individually in-
activates E. coli K12 and L. innocua by 1.0 and 2.6 log CFU/mL, respectively.
The combination of shear stress-moderate electric field and nisin reduces
E. coli K12 and L. innocua by 5 log units in 5 min, which is less effective
than the method that uses shear stress-moderate electric field before adding
nisin as a post-treatment in 0– 10 min (Mok et al., 2020). On the other hand,

Table 4
Strategic usage of the combination of bacteriocins and microbial hurdles.
Type Hurdle technology Food system Positive outcomes Target Reference
microorganisms
Bacteriocins Other hurdles

Nisin Reuterin Raw milk Enhance antimicrobial effect E. coli K12,

L. monocytogenes
ATCC 13932,
E. faecalis NCDC
135,
Serratia marcescens
ATCC 13880,
S. Typhi NCDC Kumar et al.,
Pediocin Reuterin Raw milk Enhance antimicrobial effect 113, 2020
Yersinia
enterocolitica ATCC
130715,
Klebsiella
pneumoniae NCDC
138,
P. acidilactici LB42
Microbial secondary
S. aureus LMGT
metabolites +
3242,
bacteriocins Chi and Holo,
Nisin+garvicin KS Farnesol – Show synergistic antimicrobial effect A. baumannii
2018
B1162,
E. coli LMGT 3704
A. baumannii
B1162,
Show synergistic antimicrobial effect and Chi and Holo,
Garvicin KS Polymyxin B – A. iwoffii B1163,
expend the antibacterial spectrum 2018
A. calcoaceticus
B1165
Show synergistic antimicrobial effect and L. innocua HPB13, Naghmouchi
Nisin Polymyxin B –
expend the antibacterial spectrum E. coli RR1 et al., 2010
S. Newport,
Surface layer S. aureus ATCC Prado-Acosta
Nisin – Show synergistic antimicrobial effect
protein 6538, et al., 2010
B. cereus 6A1
Surface layer Show synergistic antimicrobial effect and
Nisin Chicken meat S. saprophyticus P2 Sun et al., 2017
protein extended the shelf life of chicken meat
S. aureus
ATCC25923, Sheoran and
Enterocin LD3 Plantaricin LD4 – Show synergistic antimicrobial effect
S. Typhimurium Tiwari, 2021
Bacteriocins + ATCC13311
bacteriocins S. aureus LMGT Chi and Holo,
Garvicin KS Nisin – Show synergistic antimicrobial effect
3242 2018
Producer of nisn A Show synergistic antimicrobial effect and L. innocua
Plantaricin Cheese Mills et al., 2017
and lacticin 3147 more resistant to bacteriophage attack DPC6578
Bacteriocin from S.
Phage P4 and A3 – Show synergistic antimicrobial effect C. perfringens Heo et al., 2018
Hyointestinalis B19
Smoked salmon, hake L. monocytogenes Baños et al.,
Enterocin AS-48 Phage P100 Avoid regrowth of food pathogens
and salmon fillets CECT 4032 2016
Show synergistic antimicrobial effect and L. monocytogenes Rodríguez-Rubio
Coagulin C23 Phage FWLLm1 Milk
Bacteriophages + lower rate of resistance development 2000/47 et al., 2015
bacteriocins L. monocytogenes
1/2a,
Phage LM-103 and Fresh-cut melons and L. monocytogenes Leverentz et al.,
Nisin Enhance antimicrobial activity
LMP-102 apples 1/2b, 2003
L. monocytogenes
4b

8
G. Liu et al.
the combination of enterocin AS-48 (28 AU/mL) and PEF (35 kV/cm, 1200
μs) did not show any additive or synergistic effect. Despite the addition of
enterocin AS-48 (28 AU/mL), nisin (20 IU/mL) individually caused no var-
iation in S. aureus CECT 240's survival in milk at its natural pH and pH 5.0 of
storage at 4 or 22 °C. Moreover PEF (35 kV/cm) treatment in milk at its nat-
ural pH and pH 5.0 for 1200 μs resulted in 3.5 log reductions of S. aureus
CECT 240. However, nisin (20 IU/mL) with enterocin AS-48 (28 AU/mL)
and PEF (35 kV/cm) treatment at pH 6.8 in milk reduced S. aureus CECT
24 by 6.3 log units (Sobrino-Lopez et al., 2009).
4.1.4. Bacteriocins and high-pressure processing
High-pressure processing (HPP) is a non-thermal processing technique
that typically uses water as a pressure transmission medium while preserv-
ing the food quality (such as functions, sensory, and nutrition) to the max-
imum. HPP is commonly used in the food processing, including meats, fruit
juices, and guacamole. HPP utilizes isostatic pressure (about 100 to 1000
MPa) which is equally transmitted into food matrices, leading to microbial
membrane damage (Barba et al., 2017; Gayán et al., 2012; Guerrero-
Beltrán et al., 2005; Huang et al., 2017). Microorganisms have varing sen-
sitivities to HPP in an order of Gram-negative bacteria > Gram-positive bac-
teria > bacterial spores. The application of HPP method requires a longer
processing time or higher pressure to kill vegetative bacteria that adversely
affects food quality and increases cost (Gayán et al., 2012). To improve the
HPP method, we need to develop new strategies to kill microorganisms.
Previous studies show that a combination of HPP and other hurdles, such
as bacteriocins, can effectively retard the growth of microorganisms while
preserving high food quality (Komora et al., 2020; Lee et al., 2003; Pérez-
Baltar et al., 2019; Rodriguez et al., 2005). In addition, the combination
can also reduce the population of pressure-resistant microorganism.
The combination of HPP treatment and bacteriocins could broaden the
antibacterial spectrum of bacteriocins. The Gram-negative bacteria, E. coli
and Pseudomonas fluorescens, are both nisin-resistant, but HPP sensitizes
E. coli and P. fluorescens to the action of nisin (500 IU/mL). Further studies
found that adding nisin (500 IU/mL) before HPP treatment (500 MPa for 5
min) has a synergy effect, while only a small number of P. fluorescens cells
became sensitive to nisin and most of cells were still nisin-resistant. In
this report, the L. innocua population decreased due to the combination,
but it shows no traits of synergy effect (Black et al., 2008). As for another
Gram-negative bacteria E. coli O157:H7, it was also resistant to nisin. HPP
treatment (300Mpa, 10 °C,10 min) alone results in a reduction of E. coli
O157:H7 population (1.3 log units). However, the combined effect of
bacteriocin-producing LAB and HPP treatments is much higher (3.4 log
unit reduction) than HPP treatment individually in cheese. The synergistic
effects are widely observed in bacteriocin-producing LAB, including lacticin
481 producing strain Lactococcus lactis TAB 24, nisin Z producing strain
L. lactis TAB 26, nisin Z producing strain L. lactis TAB 26, TAB 57 producing
strain L. lactis TAB 57, TAB 7 producing strain E. faecium TAB 7, Enterocin I
producing strain E. faecalis TAB 52 and enterocin AS-48 producing strain
E. faecalis INIA 4 (Rodriguez et al., 2005). These results indicates that
bacteriocin-producing LAB could be added as additives to the starter to con-
trol foodborne pathogens. When E. coli O157:H7 NCTC 12900 is treated
under high pressure carbon dioxide (HPCD) at 5 MPa for 40– 60 min before
adding 200 μL nisin (20,000 IU/mL) for 5 min (HPCD→nisin), the counts of
E. coli O157:H7 NCTC 12900 are significantly decreases by about 0.25–1.0
log10 cycles than using HPCD individually (Bi et al., 2018). Similar results
are found in the report that the treatment of HPP (276 MPa, 15 min) in-
duces E. coli sensitive to nisin (Kalchayanand et al., 1998). To gain a
much stronger synergistic effect, the combination of HPCD and nisin
could cause an extra 0.5–3 log10 cycles reduction on E. coli than using
HPCD individually. The difference is reflected by the sequence of HPCD
and nisin treatments. These results suggested that it is important to add bac-
teriocins and other hurdles in a designed order.
The combination of HPP treatment and bacteriocins could reduce
pressure-resistant strains. Garcia-Graells et al. observed that pressure-
resistant E. coli LMM1010, LMM1020 and LMM1030 reduced in skim
milk when nisin or lysozyme (400 IU/mL) were added prior to HPP (550
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Science of the Total Environment 825 (2022) 154058
MPa) (García-Graells et al., 1999). However, whole milk (pH 6.7) showed
protection of both E. coli MG1655 and its pressure-resistant mutants in
the HPP treatment (300,400,500,600,700 Mpa for 15 min). Based on
that, in the test of antibacterial effect on the above strains when using
nisin/HPP used individually and in combination in milk with different con-
centration of fat (3.6%, 1.55%, 0.05% fat), it was suggested that the combi-
nation of nisin (400 IU/mL) and HPP (600Mpa) destructed more target
microorganism which was positive correlated to fat content of the milk.
Further studies showed that cyclic treatment (3×10 min) always results
in a much higher inhibition effect (>1000 folds) than continuous treatment
(1×30 min) (García-Graells et al., 1999). Similarly, S. aureus 485 could not
be detected in orange juice (pH 3.76), but it survives after HPP treatment
(345 MPa, 50 °C, 5 min) in Grade A pasteurized milk (pH 6.65). To elimi-
nate the HPP-resistance of S. aureus 485, nisin in combination with a
pediocin PA-1 (total 5000 AU/mL) and HPP treatment (345 MPa, 50 °C, 5
min) significantly reduced the S. aureus 485 population in Grade A pasteur-
ized milk when being storaged at 25 °C (Alpas and Bozoglu, 2000). In an-
other study, the activity of enterocin AS-48 and HPP when being used
individually and in combination against S. aureus, L. monocytogenes and S.
Typhimurium was investigated in low-acid fermented sausage (fuet) during
ripening and storage. HPP has a modest effect on L. monocytogenes when
being used individually, but the S. Typhimurium and L. monocytogenes pop-
ulation dropped dramatically when HPP was unsed in combination with
enterocin AS-48 (Ananou et al., 2010b).
The combination of HPP treatment and bacteriocins could extend shelf
life of food because bacteriocin cause an immediate bactericidal effect, and
when it was used individually, the effect was less remarkable in the long-
term storage. The vacuum packed cooked ham slice with nisin (800 AU/
g) at 6 °C is the least effective against L. monocytogenes (CTC1010,
CTC1011 and CTC1034) and after 3-months the population is not signifi-
cantly different from the control group. However, the level of
L. monocytogenes sustains under 10 CFU/g in refrigerated storage (6 °C)
after the combined treatment of HPP (600 MPa) and nisin (800 AU/g).
Moreover, these treatments only reduce the population of pathogens but
did not affect the LAB (Jofré et al., 2008). In another study, HPP treatment
(600 MPa) alone was not enough to prevent the regrowth of L. innocua 7 in
cured-cooked pork loin at the 4 °C storaging condition. While a complete in-
hibition of L. innocua 7 in pork loin after the combined treatment with
lactocin AL705(105 AU/mL) and HPP (600 MPa), no cells regrew up to
40-day storage. Furthermore, cell membrane/wall damage are observed
in all treatments, while complete cell lysis is only found in binary combina-
tion treatments (Dallagnol et al., 2017).
On the one hand, the combination of HHP treatment and bacteriocins
could broaden the antibacterial spectrum of bacteriocins, inactivate the re-
growth of target cells in long storage period. On the other hand, the
pressure-resistance strains starts to reduce after the combination treatment.
Moreover, the combination did not inhibit the growth of LAB.
4.1.5. Bacteriocins and nanotechnology
Nanotechnology is the manipulation of functional materials and struc-
tures on the nanoscale size (with diameters ranging from 1 to <1000 nm).
The basic element of nanotechnology is nanoparticles which have unique
physical, chemical and biological properties on nanoscale. Most nanoparti-
cles do not have antimicrobial effect, while some nanoparticles such as sil-
ver nanoparticles show a wide range of antimicrobial action (Sulthana and
Archer, 2021). Various studies have documented the enhanced antimicro-
bial activity of bacteriocins against food spoilage bacteria when combined
with conjugated nanoparticles (Sidhu and Nehra, 2019; Sulthana and
Archer, 2021; Vukomanović et al., 2017). Additionally, bacteriocins com-
bined with nanoparticles could encounter drawbacks of bacteriocins,
which include broading the antimicrobial spectrum of bacteriocins, reduc-
ing the dosage of bacteriocins, protecting bacteriocins from degradation
with proteolytic enzymes and increasing their survival during long-term
storage (Sulthana and Archer, 2021).
The combination of nanoparticles and bacteriocins extends the shelf life
by enhancing antimicrobial effect and prevents regrowth of the survival.
G. Liu et al.
Nisin packed in soluble soybean polysaccharide-based nanoparticles could
inhibit the growth of L. monocytogenes, B. subtilis and S. aureu in tomato
juice, thereby extend the shelf life (Luo et al., 2019). The combination of
chitosan and nisin preserve large yellow croaker better than chitosan indi-
vidually. Compared with nisin that is used individually, the chitosan nano-
particles with nisin showes double antimicrobial effect against S. aureus
(Zohri et al., 2010). Besides, the MIC of nisin packed in nanoparticles de-
creased from 2 mg/mL to 0.5 mg/mL. The combination of nisin and gold
nanoparticles are capable of disintegrating the wall of Gram-negative bacte-
ria, thereby inhibiting the growth of E. coli and P. aeruginosa (Vukomanović
et al., 2017). Similarly, the combination of bacteriocins (produced by Lacto-
bacillus spp. Bac4463 and Bac22) and silver nanoparticles enhances the ac-
tivity against S. aureus and Shigella flexneri (Sidhu and Nehra, 2020).
The combination of nanoparticles and bacteriocins enhance the stability
of nisin which can be further improved by encapsulating nisin in
nanoliposomes at 25– 70 °C and in extreme pH conditions (Taylor et al.,
2007). Meanwhile, nanoliposomes keep nisin to be antimicrobial active
for a longer time, Teixeira et al. found that bacteriocin-like substance
retained its original antibacterial effect for more than 16 days compared
with free nisin in the same condition (Teixeira et al., 2008). Similar results
showed that pediocin encapsulated in liposome has higher capability of
inhibiting L. innocua AST-062, remaining stability and controlling release
of pediocin (García-Toledo et al., 2019). Microcin MCCJ25 encapsulated
in dual coated liposome (polymeric network of pectin and whey proteins)
enhanced the stability, realized the gradual release and protectd the bacte-
riocin from gastrointestinal enzymes in vitro (Gomaa et al., 2017).
4.2. Combination of bacteriocins and chemical hurdles against foodborne patho-
gens and spoilage microorganisms
4.2.1. Bacteriocins and essential oils
Essential oils (EOs) are volatile secondary metabolites produced by
plants, mainly comprising the volatile and aromatic compounds naturally
present all parts of the plants including bark, stem, flowers, seeds, peel
and whole plants (Sánchez-González et al., 2011). EOs are widely used in
various countries as food preservatives and medicine, cosmetics and per-
fumes. EOs and their phytoconstituents have been reported to exert a
broad spectrum of biological activities (such as antifungal, insecticidal, an-
tibacterial, and antiviral) (Fitzgerald, 2003; Schnitzler et al., 2007). Re-
search delving into the application of EOs as antimicrobial agents has
increased due to their wide spectrum of activities and the property of
being GRAS (generally recognized as safe) with natural origins
(Nedorostova et al., 2009). However, high EO level in food showed unac-
ceptable organoleptic properties. If the level of EO decreases to preserve
the color of food, antibacterial activity effect will be undermined. It has
been documented that EOs combined with other antimicrobial agents,
such as bacteriocins, have an enhanced or synergistic effect while both
the EOs and bacteriocins can be used in lower volume (Alves et al., 2016;
Churklam et al., 2020; Turgis et al., 2012). Moreover, the combination of
bacteriocins and EOs could inhibit the regrowth of survival cells and even
expand the antibacterial spectrum. This section will discuss the combined
effect of EOs and bacteriocins against food spoilage organisms.
It is widely known that EOs and bacteriocins act in an additive or syner-
gistic manner to inhibit the growth of foodborne pathogens and spoilage
bacteria. The antimicrobial potential of pediocin, nisin, and two bacterio-
cins produced from E. faecium (MT 104 and MT 162) and six essential oils
(Cymbopogon nardus, Cinnamomum cassia, Satureja montagna, Origanum
vulgare, Thymus vulgaris, and Brassica hirta) against two spoilage bacteria
(Pseudomonas putida and L. sakei) and five pathogenic bacteria
(L. monocytogenes, S. aureus, S. Typhimurium, B. cereus, and E. coli) is re-
ported in a previous study. The results indicated that O. vulgare EO and
nisin have a synergistic effect on the inhibition of L. monocytogenes, whereas
nisin combined with T. vulgaris EO had a synergistic effect on the inhibition
of S. Typhimurium, while S. montagna EO combined with pediocin had a
synergistic effect on the inhibition of E. coli (Turgis et al., 2012). The
enterocin KT2W2G and cinnamon mix at the ratio of 6:4 displayed a
10
Science of the Total Environment 825 (2022) 154058
synergistic and broad-spectrum action against the tested spoilage microor-
ganisms. However, enterocin KT2W2G alone did not show antimicrobial
activity against 18 spoilage microorganisms from spoiled banana peel
(Klebsiella pneumoniae, Serratia marcescens, E. faecalis, L. lactis subsp. lactis,
K. variicola, Kodamaea ohmeri, Hanseniaspora opuntiae, Pichia kudriavzevii,
P. fermentans, and Candida metapsilosis) (Issouffou et al., 2018). The poly-
phenolic compounds (cinnamaldehyde, carvacrol, eugenol, and thymol)
and nisin have synergy effect against pathogens L. monocytogenes ATCC
15313 and S. aureus ATCC 25923 in milk (Alves et al., 2016). Besides, the
synergistic interaction of EOs and bacteriocins against L. monocytogenes
can be found in various food, such as sliced bologna sausages (Churklam
et al., 2020), whole, low and skim milk (Bajpai et al., 2014), lettuce (Yu
et al., 2019), raw pork loin (Yu et al., 2019), shrimp (Zhao et al., 2020b).
However, the exact action mechanism of EO and bacteriocins against
L. monocytogenes has yet been studied in depth. The underlying mechanism
of grape seed extract and nisin against L. monocytogenes has been elucidated
through a metabolomics approach based on nuclear magnetic resonance
spectroscopy (NMR) (Zhao et al., 2020a). The combination of nisin and
grape seed extract potently reduces the L. monocytogenes population (4.49
log CFU/mL). Confocal laser scanning microscopy results reveals that cell
membrane permeability varies dramatically and the leakage of protein
and nucleic acid increases. Furthermore, the results of NMR-based metabo-
lomics coupled with multivariate analysis showed lower threonine, ade-
nine, cysteine, adenosine triphosphate (ATP), and nicotinamide adenine
dinucleotide phosphate (NADP), while γ-aminobutyric acid and lactic
acid increases are observed in a nisin and grape seed extract treatment.
Pathway analysis indicates that the nisin and grape seed extract combina-
tion inhibites L. monocytogenes by blocking amino acid biosynthesis, the
TCA cycle (citric acid cycle) and the energy-producing pathway. The com-
bination of nisin and grape seed extract was applied in shrimp and it mark-
edly inhibits the L. monocytogenes population (1.79 log CFU/g) (Zhao et al.,
2020b).
Specifically, the combination of bacteriocins and EOs could broaden the
antibacterial spectrum. Three EOs (derived from basil, sage, and ajowan)
showed greater antibacterial activity against S. aureus ATCC 29213 but
very marginal effects on E. coli ATCC 25922, while the antimicrobial effect
of the three EOs used in combination with nisin shows synergic activities
with FIC of 0.06,0.03,0.06 against E. coli ATCC 25922, respectively. How-
ever, all these combinations showed no interactive effect on S. aureus
ATCC 29213 (Mehdizadeh et al., 2016). Furthermore, nisin variants (60
μM) used in the combination with carvacrol EOs (0.03%) reduces by 3
log units E. coli O157:H7 and 4 log units C. sakazakii NCTC 8155 compared
to nisin A carvacrol treatment. Furthermore, the nisin variants (30 μM) and
carvacrol EOs (75 μg/mL) causes complete inactivation of E. coli O157:H7
in apple juice within 3 h at 25 °C compared to that of the equivalent nisin
A combination (Campion et al., 2017). As for enteriocin A, when it was
used individually, the E. coli O157:H7 is not inhibited from growth. How-
ever, the addition of thyme EOs (0.71 μg/mL) and enterocin A (3.4
μg/mL) showed a synergistic antimicrobial effect against E. coli O157:H7,
and MIC of thyme EOs decreases from 2.2 to 0.71 μg/mL (Ghrairi and
Hani, 2015).
In addition, mixed natural preservative can be more cost-effective in
terms of food preparation due to smaller dose of natural preservative.
Cinnamaldehyde (CA), an essential oil isolated from cinnamon bark, and
nisin both have antimicrobial activity against 14 strains of S. aureus,
while the combination of nisin (8 μg/mL) and CA (0.25 mg/mL) not only
exhibits synergistic antimicrobial activity against S. aureus in milk, but
also reduced the does of nisin used by about 8– 16 folds and the use of
CA by about 5 folds. Furthermore, the combination of nisin and CA resulted
in about 54.5% cell membrane-damage, which is nearly 2 folds of the out-
come of using the nisin or CA treatment individually. Similar results are
found in the damage of cell wall. Severe demages of cell membrane and
cell wall have caused the synergistic effect of the combination against
S. aureus (Shi et al., 2017). Enterocin A combined with thyme EOs showed
bactericidal effect of L. monocytogenes EGDe after 18 h treatment. More-
over, the combination could reduce the MIC of enterocin A (4.57 to 0.9
G. Liu et al.
μg/mL) and thyme EOs (3.6 to 1.2 μg/mL) (Ghrairi and Hani, 2015). The
combination of nisin (5 to 6 ppm), grapefruit seed extract (6 to 8 ppm)
and cinnamaldehyde (15 to 20 ppm) increases the antibacterial effect
against L. monocytogenes on pork loin. The level of nisin decreases from
250 ppm to 15 ppm, the level of grapefruit seed extract reduces from
31.25 ppm to 6 ppm, and the level of cinnamaldehyde fell from 500 ppm
to 15 ppm (Zhao et al., 2020b).
The combination of EOs and bacteriocins weakens the unacceptable or-
ganoleptic properties of EOs. Most of the time, high level EOs showed unac-
ceptable organoleptic properties in food, while low level EOs possesses a
weak antibacterial activity, and the thyme EO (0.9%) showes unacceptable
organoleptic properties in minced meat. To reduce the side effect and con-
trol foodborne pathogens, studies have investigated the effect of thyme EO
and nisin used individually or in combination against E. coli O157:H7 and
L. monocytogenes Scott A and Lmk. The results show that thyme EO
(0.6%) with nisin (1000 IU/g) exerts an additive effect against the patho-
gens and extends the shelf life of minced beef storaged at 4 °C (Solomakos
et al., 2008a, 2008b). Sensory evaluation showed that the adding 0.6 or
0.9% of oregano essential oil (OEO) in minced sheep meat is organolepti-
cally acceptable, and OEO is easier to be accepted at 0.6% than at 0.9%.
OEO enriched with carvacrol and thymol at 0.6%-0.9% and nisin (500 or
1000 IU/g) used individually and in combination are studied for activity
against S. Enteritidis in minced sheep meat stored at 4–10 °C for 12 days.
The results reveals that OEO or nisin when used alone fails to kill S.
Enteritidis, but when used in combination, OEO and nisin exhibits more po-
tent antimicrobial activity against S. Enteritidis. The author suggested that
OEO has strong a potentail to inactivate S. Enteritidis when being used in
combination with nisin (Govaris et al., 2010). In another study, the combi-
nation of nisin and grape seed extract (1:1) reduces L. monocytogenes in the
cooked shrimp from 6.1 to 4.3 log CFU/g in 15 min. Though the food color
is slightly affected at the beginning of storage after the treatment with grape
seed extract, it provids an additional protection to color from the degrada-
tion during prolonged shelf life. Furthermore, atomic force microscopy
analysis reveals greater morphological changes when the components of
the treatment are used in combination (Zhao et al., 2020a).
The activity of bioengineered nisin variants is usually superior over the
wild type nisin A equivalent when used in combination with a variety of es-
sential oils. In a study investigated the effect of semi-purified nisin A and
nisin V along with EOs (trans-cinnamaldehyde, carvacrol, and thymol)
against L. monocytogenes, it is noticed that the growth curves for combined
treatment with nisin V shows a delayed lag phase comparing with nisin A
used in combination with EOs (Field et al., 2015). The antimicrobial activ-
ity of more nisin variants (nisin V and nisin S29A) combined with carvacrol
EOs is higher than the wild type nisin A-EOs treatments (Campion et al.,
2017).
In summary, the combination of EOs and bacteriocins not only shows a
wide range of antimicrobial effect and inactivates the regrowth of
foodborne pathogens, but also reduces the dosage of bacteriocins and
weakens unacceptable organoleptic properties of EOs. The potential mech-
anism of the combination is changing cell membrane permeability and
causes leakage of protein and nucleic acid in target cells.
4.2.2. Bacteriocins and EDTA/organic acids/salts of organic acids
Studies on chelating agents are growing as a topic in food processing re-
search because the agents are able to bind metal ions and stabilize food.
Chelating agents help bacteriocins to kill Gram-negative pathogens. The cy-
toplasmic membrane of Gram-negative bacteria is protected by an outer
membrane (OM) composed of a phospholipid bilayer. The lipopolysaccha-
ride of Gram-negative bacteria layer forms a tight shield barricading bacte-
riocins while the divalent cations, particularly Mg2+ and Ca2+ stabilize the
anionic lipopolysaccharide layer. If these cations are removed, lipopolysac-
charide molecules will be released from the outer membrane, and the
Gram-negative cells will be sensitive to bacteriocins (Martin-Visscher
et al., 2011). Sequestrants form chelate complexes with polyvalent metal
ions, which can serve a variety of advantages in assuring food safety and
quality (Bohrer, 2019). Ethylenediaminetetraacetic acid (EDTA),
11
Science of the Total Environment 825 (2022) 154058
carboxylic acid and tartaric acid are the most commonly used chelating
agents in the food industry. Many studies have reported that using bacterio-
cins with chelating agents could extend the shelf life of food products
(Castellano et al., 2011; Liang et al., 2020; Morsy et al., 2018).
To study the effect of two bacteriocin-producing strains (L. curvatus
CRL705 and L. lactis CRL1109) combining with disodium ethylenediamine-
tetraacetic acid (Na2EDTA) against E. coli O157:H7, the contaminated fro-
zen ground-beef patties at 5 °C over 9 days are used. Both bioprotective
cultures (ca. 107 CFU/g) combined with EDTA (18 g/kg of beef) greatly
contain the growth of E. coli. However, the treatments alone failed to inhibit
the growth of E. coli. Furthermore, no significant effect on ground-beef pat-
ties color was produced after the treatment of bioprotective cultures, while
a darker color observed on patties in the presence of EDTA (Castellano
et al., 2011). However, the combination of chrisin (a commercial nisin
preparation) with EDTA was effective against all Gram-positive bacteria
tested at a range of concentrations, but the combination was not effective
against all of the Gram-negative bacteria at the concentrations tested. Re-
cent studies reported that nisin with three commercial chelating resins
(Dowex MAC-3, Chelex 100, and Lewatit TP260) did not show significant
inhabitation on the growth of Alicyclobacillus acidoterrestris ATCC 49025
in apple juice (Herskovitz et al., 2019). Such oppsite results indicate the im-
portance of applications testing in real food matrices.
Organic acids (acetic acid, lactic acid, and citric acid etc.) and sodium
salts of the low molecular weight organic acids (sodium lactate, sodium
diacetate etc.) have been used as chemical additives to control microbial
growth, improve sensory attributes and extend the shelf life of various
food systems including meat, poultry and fish (Figueiredo and Almeida,
2017). The combined use of nisin M21A (78 μg/mL), a bioengineered de-
rivative of nisin A, and citric acid (1170 μg/mL) against L. monocytogenes
F6854 has a much higher effect than the combination of nisin A (78
μg/mL) and citric acid (1170 μg/mL) (Zhao et al., 2017). In addition, the
combined use of nisin M21A (0.1 μg/mL) with citric acid (175 μg/mL),
nisin A (0.1 μg/mL) with citric acid (175 μg/mL) and Nisaplin® (30
μg/mL) with citric acid (15 μg/mL) eradicated the biofilms of
L. monocytogenes F6854 (Smith et al., 2016). Similar results were observed
in the synergistic interaction of nisin (0.25 mg/mL) and citric acid (16
mg/mL) against S. aureus ATCC 29213 and L. monocytogenes ATCC 19115
in milk (Zhao et al., 2017), and the main action targets of the combination
were cell wall and membrane. Another study shows that the combined use
of Nisaplin® (10 mg/mL) and citric acid (30 mM) significantly reduced the
C. sakazakii count in infant formula (Campion et al., 2017). Except citric
acid, other organic acids (formic, lactic, cirtic, malic, fumaric and tartaric)
combined with nisin have synergistic interaction to pathogens
(P. aeruginosa, E. coli, S. Typhimurium, S. aureus, and S. faecalis) (Ajingi
et al., 2020). Meanwhile, in the evaluation of the antimicrobial potential
of formic acid in combination with nisin towards B. subtilis on potato, the
results showed that the combination of nisin (0.016 mg/mL) and formic
acid (0.25%) inhibited B. subtilis counts while the color changed slightly
(Ajingi et al., 2020).
The combined antimicrobial activity of sodium lactate and bacteriocins
have recorded different results. For example, Figueiredo and Almeida re-
ported that the it was no difference of the combination of nisin and sodium
lactate (nisin (0.0012 μg/g) and sodium lactate (0.5 μg/g) and individually
used in inhibiting L. monocytogenes on ready-to-eat sliced pork ham
(Figueiredo and Almeida, 2017). Similar results were observed by Lung
and Johnson, who applied nisin (640 AU/mL), sodium lactate (0.06
g/mL) and sodium diacetate (0.06 g/mL) in combination against
L. monocytogenes in turkey breast (Lungu and Johnson, 2005). On the
contray, some studies reported that the combination of nisin and sodium
lactate significantly decreased the L. monocytogenes count in cooked ham,
cold-smoked salmon pate and cold-smoked rainbow trout (Jofré et al.,
2007; Neetoo et al., 2008; Nykänen et al., 2000). Since the nisin and potas-
sium sorbate combination exerted a bacteriostatic effect on
B. sporothermodurans in milk (Aouadhi et al., 2015), the combination of
nisin, potassium sorbate and lactocin AL705 coult perform potent anti-
L. innocua activity (Verdi et al., 2020).
G. Liu et al.
To sum up, the combination of chelating agents and bacteriocins re-
moved lipopolysaccharide of Gram-negative cell membrane to make
Gram-negative bacteria sensitize to bacteriocins. The combination of or-
ganic acid and bacteriocins shows synergistic antimicrobial effect, extend-
ing the shelf life, however, bacteriocins combined with salt of organic
acid showed opposite effects on antibacterial.
4.2.3. Bacteriocins and animal-derived enzymes
Lactoperoxidase (EC 1.11.1.7) (LP) is a member of the peroxidase fam-
ily, a group of natural enzymes in bovine milk. LP can catalyse the oxidation
of certain molecules using H2O2 to generate reactive product with a wide
antimicrobial activity. LP-system (LPS), consisting of LP, SCN− and H2O2,
is used as natural biopreservatives in food. LPS can kill Gram-negative bac-
teria such as pseudomonads, coliforms, salmonellae and shigellae, but only
inhibit Gram-positive bacteria like streptococci and lactobacilli
(Kussendrager and van Hooijdonk, 2000). Studies have reported that bacte-
riocins induce an rapid but not lasting bactericidal effect, while the com-
bined LPS with nisin will contain the regrowth of L. monocytogenes in
skim milk at 25 or 30 °C (Boussouel et al., 2000; Zapico et al., 1998). In
comparison, LPS combined with reuterin (2 AU/mL) is less effective than
that combined with nisin (100 IU/mL) (Arqués et al., 2008). However,
when strains produced with nisin (L. lactis ATCC 11454, L. lactis ESI 515)
and strains produced with enterocin 4 (E. faecalis INIA 4) were used in com-
bination with LPS in raw milk at 4 °C or 8 °C, the counts of L. monocytogenes
was reduced significantly (Rodríguez et al., 1997). In addition, the anti-
Listeria effect of nisin and LSP was compared between being added simulta-
neously and in two steps. The results showed that the effect of nisin and LPS
added in sequence was greater than the effect of being added simulta-
neously, while the antimicrobial activity depended on the treating time of
first inhibitor and target microorganism. When nisin (200 IU/mL) and
L. monocytogenes has been interacted for 4 h than LPS was added, it showed
a bactericidal effect within 48 h and which lasted for 15 days. However, the
longer treatment of nisin and L. monocytogenes firstly (12h) would lead to
re-grow of L. monocytogenes after 200 h of incubation. When LPS added
firstly then nisin was added 24 h later, no viable L. monocytogenes cells
would be detectable within 10 days. When nisin was added 72 h after
LPS, counts of L. monocytogenes reduced by 8 log units in milk compared
with the control (Boussouel et al., 2000). In another study, when LPS was
added after 3 h and nisin after 5 h, counts of L. monocytogenes decreased
by 4.5 log in 24 h (Zapico et al., 1998). However, the adding nisin and
LPS simultaneously or in two steps do not affect the inhibitory effect of
E. coli, Acinetobacter, Flavobacterium indologenes and Psychrobacter immobilis
strains in fish (Elotmani and Assobhei, 2004). As for the combined effect
of bacteriocins (nisin or lacticin 3147) and LPS, C. sakazakii in rehydrated
infant formula was inhibited outgrowth at 37 °C for 8 h (Oshima et al.,
2012).
Hen egg-white lysozyme has been the only lysozyme allowed in the
food industry. It can cleave the β-(1,4)-glycosidic bond between N-
acetylmuramic acid and N-acetylglucosamine of bacterial cell wall peptido-
glycans, leading to the antimicrobial efficiency of Gram-positive bacteria.
However, when being used individually, lysozyme exhibits weak inhibitory
effects against Gram-negative bacteria (Wu et al., 2019). A combination of
lysozyme and nisin (3:1,w/w) whose concentration is approximately 260
μg/cm2 at the surface of vacuum packaged pork loins is effective in
inhibiting the growth of lactic acid bacteria at 2 °C for up to 6 weeks
(Nattress, 2003). Chung and Hancock found that the combination of lyso-
zyme and nisin is active against LAB (Chung and Hancock, 2000), while
no such interactions were observed in study of Gill and Holley (Gill and
Holley, 2003), so the action is probably because the two studies used differ-
ent growth media (MRS and ATP). Moreover, the antimicrobial effect of ly-
sozyme and nisin was enhanced when MRS was diluted to 1/8 (a nutrient-
deficient environment), while there was no enhanced bactericidal effect in
a pork juice medium under the same conditions (Chung and Hancock,
2000). Therefore, the nutrient condition of evaluating model is important
influencing factors on control food spoilage microorganism. The results of
scanning electron microscopy and the increase of optical density and cell
12
Science of the Total Environment 825 (2022) 154058
depolarization showed that surface disruption of cells could cause superior
effect of the combination.
Above all, the combination of animal-derived enzymes and bacteriocins
would change the spectrum of antimicrobial activities. In addition, the an-
timicrobial activities were affected by nutrient condition because proteins
or fats protect foodborne pathogens.
4.3. Combination of bacteriocins and microbial hurdles against foodborne path-
ogens and spoilage microorganisms
4.3.1. Bacteriocins and microbial secondary metabolites/other bacteriocins
Reuterin (β-hydroxypropionaldehyde) is a small organic compound bio-
synthetically produced by L. reuteri during anaerobic fermentation of glyc-
erol. It possesses a broad spectrum of antimicrobial activities against
foodborne pathogens and spoilage microorganisms. The combination of
reuterin (8 AU/mL) and nisin (100 IU/mL) showed a significant synergistic
effect on L. monocytogenes and a slight additive effect on S. aureus after 24 h
in milk at 37 °C (Arqués et al., 2004). In another similar study, the combi-
nation of reuterin (150 AU/mL) and nisin (100 IU/mL) and the combina-
tion of reuterin (100 AU/mL) and pediocin (2185 AU/mL) reduced
coliform counts by 2.2 log CFU/mL and 1.5 log CFU/mL in raw milk within
3 h at 37 °C, respectively (Kumar et al., 2020). It was reported that the com-
bination of reuterin (8 AU/mL) with nisin (100 IU/mL) did not enhance
the antimicrobial effect of reuterin during the storage of UHT skimmed
milk at 4 or 8 °C, although the combination effectively killed Campylobacter
jejuni LMG 6629 and Aeromonas hydrophila subsp. hydrophila CECT 839 in
milk.
Farnesol, a cheap and harmless compound, has been considered a prom-
ising adjuvant for antibiotics. When being used in combination with nisin
and garvicin KS, it showed a synergistic effect against S. aureus LMGT
3242, A. baumannii B1162 and E. coli LMGT 3704 (Chi and Holo, 2018).
Polymyxin B is a drug with strong inhibitory effect on Gram-negative bac-
teria, but it could be toxic at certain concentrations. Synergistic effect was
observed in the combination of polymyxin B and garvicin KS, which inhibit
A. baumannii B1162, A. iwoffii B1163 and A. calcoaceticus B1165. Moreover,
the combination reduced the resisitance of A. iwoffii B1163 to polymyxin B.
Similar synergistic antibacterial effects were observed when nisin
combinated with polymyxin B (Chi and Holo, 2018). In addition, the com-
bination significantly reduced the MIC of polymyxin B and removed the
toxicity to human. Besides, the usage of nisin considerably decreased in
the combination of polymyxin B on the effect of anti-Listeria
(Naghmouchi et al., 2010).
The surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356 has an
endopeptidase activity against cell wall of Gram-negative bacteria, but it
did not affect Gram-positive bacteria. When S-layer is used with nisin, it
showed synergistically inhibitory effect on the growth of S. enterica serovar
Newport, S. aureus ATCC 6538 and B. cereus 6A1. The S-layer causes pepti-
doglycan of cell wall hydrolysis, thus enhancing the antimicrobial activity
of nisin by enabling it to cross the cell wall then acting on the cell mem-
brane (Prado-Acosta et al., 2010). Another surface layer protein, SlpB, iso-
lated from Lactobacillus crispatus, also showed enhancement to the
antimicrobial activity of nisin. The combination of nisin and SlpB show syn-
ergistic antibacterial effect against S. saprophyticus and extends the shelf life
of chicken meat. However, the mechanism of SlpB/nisin is different from
the combination of S-layer and nisin. Cell lysis has not been observed
after any treatment of SlpB/nisin, while membrane damage and structural
disorganization are observed, enhancing nisin access to cell membrane,
which could cause the dramatic release of micro-molecules from the cell
(Sun et al., 2017).
Additionally, the combination of different bacteriocins is a cost-efficient
method for food industry by reducing MIC of bacteriocins. The MIC of
enterocin LD3 in combination was decreased by 8 folds against S. aureus
ATCC25923 and S. Typhimurium ATCC13311 and the MIC of plantaricin
LD4 in combination reduced by 3 folds against the above target strains.
The results of transmission electron microscopy and FTIR spectra showed
that the higher disruption of cell membrane in the combined bacteriocin-
G. Liu et al.
treated cells as compared with alone effects, and the two bacteriocins have
different mode of action which caused synergistic activity against target
cells (Sheoran and Tiwari, 2021). It was reported that garvicin KS and
nisin acted synergistically against S. aureus, reducing the strains counts
(Chi and Holo, 2018). Mills et al. constructed a double-bacteriocin-
producing starters of L. lactis which produced nisin A and lacticin 3147 sta-
bly, although the antimicrobial activity was as effective as its single-
producing strains, while double-bacteriocin-producing strain was more re-
sistant to bacteriophage attack. Interestingly, the double producer com-
bined with the plantaricin producer as start culture in cheese exhibited a
considerable reduction in L. innocua DPC6578 (Mills et al., 2017).
Therefore, the combination of microbial secondary metabolites
(reuterin, farnesol, polymyxin B and surface layer protein) and bacteriocins
enhanced the antimicrobial effect and reduced dosage in use, thus lowering
the cost of food preservation. In addition, the bacteriocins of different ac-
tion mode will easily produce synergistic effects.
4.3.2. Bacteriocins and bacteriophages
Bacteriophages or phages are viruses that infect and replicate within the
bacteria, causing host lysis and cell death. These natural killers of bacteria
are widely distributed around us from sewage plants, silage to food process-
ing environments. Assessment has proved that these viruses can be utilized
as new tools to reduce levels of foodborne pathogens and spoilage bacteria.
For example, the first phage-based product (ListShield™) was approved to
control L. monocytogenes in meat and poultry products (Bren, 2007). Until
now, there is no evidence that phages may harm humans or animals
(Endersen et al., 2014). However, bacteriophages or bacteriocins individu-
ally did not contain the regrow of foodborne pathogens. To inhibit the re-
growth of pathogens, the combined bacteriocins with bacteriophages
were used in food.
Although treatment with bacteriocin or phage alone results in rapid an-
timicrobial effects against C. perfringens, the survivals of C. perfringens re-
grow during the storge. However, when bacteriocin from S. hyointestinalis
B19 combined with P4 and A3 phages, it resulted in synergistic effect and
eradicate C. perfringens completely (Heo et al., 2018). Similar results show
that the phage P100 (2.3 × 107 PFU/cm2) and enterocin AS-48 (0.37
μg/cm2) against eliminate L. monocytogenes CECT 4032 from hake and
salmon fillets from 2 and 1 days, respectively. The treatment of P100/AS-
48 reduce L. monocytogenes CECT 4032 below detection levels from 1 to
15 days in smoked salmon (Baños et al., 2016). In another study, the Listeria
phage FWLLm1 (MOI of 100) in combination with bacteriocin coagulin
C23 (584 AU/mL) was more effective than treatment alone for the
inactivating or reducing L. monocytogenes counts in milk at 4 °C
(Rodríguez-Rubio et al., 2015). Moreover, study has shown that the combi-
nation of phages and the bacteriocin nisin enhanced antimicrobial activity
by against L. monocytogenes in artificially contaminated fresh-cut melons
and apples (Leverentz et al., 2003) and against S. aureus in pasteurized
milk (Martínez et al., 2008), proving that this approach is feasible in food
biopreservation (Rodríguez-Rubio et al., 2015).
As the results show, the combination of bacteriophages and bacteriocins
has enhanced the antimicrobial effect against various food spoilage micro-
organisms. Due to practical constraints, this paper cannot provide a com-
prehensive review of mechanism behind the possible synergy of
bacteriocins with bacteriophages. Future studies on this topic is are there-
fore recommended.
5. Conclusion
Though bacteriocins as antimicrobial peptides have planty of advan-
tages, there are several drawbacks when bacteriocins is used as
biopreservatives individually for the food industries. The combination of
bacterioncins and other hurdles provide an attractive option for maintain-
ing food quality and food safety. Briefly, bacteriocins and other hurdles
showed synergistic or additive antimicrobial effect with a lower dosage, re-
ducing economic cost of food preservation, while mitigating the advese ef-
fect such as toxicity or unacceptal organoleptic properties caused by high
13
Science of the Total Environment 825 (2022) 154058
concentration. Moreover, synergistic effect usually occurs when one tech-
nology addresses the barriers of another technology while the latter one
has different mode of action. The combination of bacteriocins and hurdles
extends shelf life of food as the combination increases the stability of bacte-
riocins and inhibit the regrowth of food pathogens and spolige bacteria.
More importantly, the two catagories of combination of bacteriocins
and other hurdles could broaden the spectrum of antimicrobial activity.
The first is the combination of bacteriocins and food preservaties with a
wide range antimicrobial activities such as EOs, the other is the combina-
tion of bacteriocins and treatment which destroy cell membrane of Gram-
negative bacteria such as EDTA. In addition, different adding sequence of
hurdles and evaluation system will lead to opposite antimicrobial effect.
The determination of appropriate adding sequence is lack of adequate
research, so future investigations will need to be undertaken. There is a con-
sensus that the evaluation system is an important factor in food safety re-
search. Furthur work is required to provide greater insight into the effect
of the adverse interactions between bacteriocins and food ingredients.
6. Future perspectives
The early results show that the combination of bacteriocins and other
food preservation techniques have the potential to be applied in food indus-
try. However, the governing principles of bacteriocins and other hurdles are
absent and most strategic usages of hurdle technologies have been merely
blind attempts. The mechanism of the combination is pending deeper inves-
tigation. It shows that the combination of different preservation methods
may lead to synergistic antimicrobial effect as one technique may mititage
the adverse impact caused by another method, thus enhancing the action.
Moreover, the complicated real food matrix protects foodborne pathogens
and spoilage bacteria by reducing the efficiency of bacteriocins. Under-
standing the interactions and mechanisms will be helpful to find more pre-
cise and optimal utilization of bacteriocins in food, while proteomic,
genomic and transcriptomic tools could facilitate the discovery of underly-
ing mechanisms. In addition, it is necessary to have experiments in real
food production, since the antimicrobial activity in broth and media gener-
ally need less dose for preservation. As for target microorganism, there have
been sufficient studies on foodborne pathogens like L. monocytogenes, while
food spoilage bacteria, a common and main reason for food spoilage, need
further research. In addition to food safety, future research with more focus
on nutritions and sensory properties of food is therefore suggested. More-
over, more research is needed to better understand the effect on the taste
and texture of food products. Indeed, when applying hurdle technology, se-
quence of addition should be considered because a minor change in the
adding sequence of bacteriocins and other hurdles would lead to a hugely
different antimicrobial activity. Additionally, current research suffers
from insufficient studies on the combination of bacteriocin-producing
strain and hurdle. However, adding bacteriocin-producing strain directly
can make the isolation and purification process more cost-efficient. There-
fore, it is of crucial importance to study the fundamental mechanism behind
the hurdle technology which involves bacteriocins. Based on that, it will be
possible to design an effective, environment friendly and practical food
safety control hurdle concept based strategies in the future.
CRediT authorship contribution statement
No conflict of interest exits in the submission of this manuscript, and
manuscript is approved by all authors for publication. I would like to de-
clare on behalf of my co-authors that the work described was original re-
search that has not been published previously, and not under
consideration for publication elsewhere, in whole or in part. All the authors
listed have approved the manuscript that is enclosed.
Guorong Liu: Writing- Original draft preparation, Writing- Reviewing
and Editing. Rong Nie: Conceptualization, Data curation. Yangshuo Liu:
Data curation, Formal analysis. Arshad Mehmood: Conceptualization.
G. Liu et al.
Funding
This project was funded by National Natural Science Foundation of
China (No. 31871772; No. 31671832); National Key R & D program of
China (2019YFC1606200); Beijing Natural Science Foundation (Grant
No. 6192003); School Level Cultivation Fund of Beijing Technology and
Business University for Distinguished and Excellent Young Scholars
(BTBUYP2022); 2021 Postgraduate Scientific Research Ability Improve-
ment Project of Beijing Technology and Business University, and Graduate
Innovation Project of School of Food and Health in 2021.
Declaration of competing interest
The authors declare that they have no known competing financial inter-
ests or personal relationships that could have appeared to influence the
work reported in this paper.
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