Antimicrobial Peptides: The New Generation of Food Additives

Laila Ben said, Ismaı̈l Fliss, Clément Offret, and Lucie Beaulieu, Institut sur la nutrition et les aliments fonctionnels (INAF),
Département des sciences des aliments, Université laval, Québec, QC, Canada
© 2018 Elsevier Inc. All rights reserved.

Introduction 1
Origins of Natural Antimicrobial Peptides (AMPs) and Their Different Functions 2
Plant 2
Bacteria 2
Fish and Marine Products 2
Milk and Milk Products 2
Algae and Mushrooms 3
Mechanisms of Action 3
Extracellular-Targeting AMPs 3
Outer Surface Lipids 3
Outer Membrane Proteins 4
Intracellular-Targeting AMPs 4
Inner Membrane 4
Nucleic Acids 4
Intracellular Proteins 4
Applications of Natural AMPs as Food Additives 4
Lactic Acid Bacteria Producing Strains 4
Active Packaging Material Encapsulating AMPs 5
Food Matrix Interaction and Bioavailability of AMPs 5
Food Additive Regulation and Approval of AMPs 5
Conclusion 5
References 6
Relevant Websites 7

Introduction

Increasing human population and the demand for safe and nutritious food impose food security challenges worldwide (Bajagai
et al., 2016). It has been reported that an estimated 600 million, which means almost 1 in 10 people in the world, fall ill after eating
contaminated food that can cause notably severe diarrhea and 420000 die every year (WHO, 2017). To face this threat it is critical to
ensure food safety throughout the entire food chain from the production to the consumption of the end-products (WHO, 2014).
Foodborne illnesses are usually caused by bacteria, viruses, and parasites entering the body through microorganisms-
contaminated foodstuffs (Pisoschi et al., 2018). Bacteria can account for both food spoilage and toxicity (Gram et al., 2002). Indeed,
major foodborne illnesses are caused by Salmonella sp, Campylobacter sp, Escherichia coli, Listeria sp and Vibrio cholera (WHO, 2017).
Administration of antibiotics is still the main practical strategy to treat these infections (Hassan et al., 2012). However, their overuse
and misuse in veterinary and human medicine has been linked to the emergence of resistant foodborne pathogens, rendering the
treatment of infectious diseases ineffective in animals and humans (Dong et al., 2007).
Microbial resistance development is one of the main fears to modern medicine and it has driven the search for new antimicrobial
agents to replace conventional antibiotics (Hassan et al., 2012). Hence, both pharmaceutical and food industries are looking for safe
new types of antimicrobials from natural sources (Boisvert et al., 2015). Besides, there is a growing consumer demand for natural
alternatives to replace synthetic food preservatives such as nitrates, benzoates, sulfites, sorbates, and formaldehyde, which are
known for their life-threatening side effects (Sultana et al., 2014). The search for an alternative to chemical preservations has led
to defined novel methods like biopreservation that ensure food safety (Pisoschi et al., 2018). Plant and animal-derived antimicro-
bials, natural or controlled microflora, such as lactic acid bacteria (LAB) and their antibacterial metabolites such as lactic acid, bacte-
riocins demonstrated ability to prevent lipid oxidation, inhibited colour loss, prolonged shelf life and ensured food security
(Pisoschi et al., 2018). Antimicrobial peptides (AMPs) such as bacteriocins have attracted particular attention as their producer
organisms could obtain GRAS (generally recognized as safe) status and are naturally present in many food products (Tahiri
et al., 2009). Either low toxicity, thermostability or high specificity make these molecules of clear interest to the food industries
(de Castro and Sato, 2015).
Moreover, there is an urgent need for innocuous antimicrobials in food preservation, which has led numerous research activities
towards AMPs (Boisvert et al., 2015; Beaulieu et al., 2015; Ennaas et al., 2015a, 2015b, 2016). AMPs are components of the innate
immune defenses and exhibit high structural diversity (Reddy et al., 2018; Shabir et al., 2018). They generally have amphipathic

1
2 Antimicrobial Peptides: The New Generation of Food Additives

structure facilitating their attachment and the formation of pores in the cytoplasmic membrane of microorganisms (Mor, 2000).
This characteristic, unlikely to lead to the development of the microorganism’s resistance, has stimulated their studies for alternative
antimicrobial agents (Mor, 2000). Furthermore, the newly identified AMPs should be specifically active against foodborne patho-
gens, and they are expected not to influence negatively the consumer’s own microbiome (Pisoschi et al., 2018). In addition, AMP-
containing formulations have to exhibit the expected antimicrobial effects, with least chemical or biological degradation of peptides
(Nordström and Malmsten, 2017).
This chapter will focus on the potential of AMPs as one of the key novel food preservatives for the future.

Origins of Natural Antimicrobial Peptides (AMPs) and Their Different Functions

Plant
Plant AMPs are small cysteine-rich peptides that have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of
species (Terras et al., 1995). They are grouped into several families and many share general features, such as an overall positive
charge, the presence of disulfide bonds, which stabilize the structure and a mechanism of action targeting outer membrane struc-
tures, such as ion channels. Based on amino acid sequence homology, AMPs were classified mostly as a-defensins, thionins, lipid
transfer proteins, cyclotides, snakins and hevein-like (Broekaert et al., 1997). In addition to their role in host defense and their
appeal as simple models for studying the molecular mechanism of antimicrobial peptide action, AMPs have the potential to inhibit
pathogens, including those showing increased resistance to conventional antimicrobial compounds. These peptides usually have
a broad-spectrum of antimicrobial activity and therefore, representing promising candidates for plants applications (Nawrot
et al., 2014). PhytAMP is a database that provides a very useful interactive platform that enables easy access to systematized infor-
mation about all families of plant antimicrobial peptides. PhytAMP also provides useful tools that facilitate analysis of these
peptides and search for new members of the plant AMP classes (http://phytamp.hammamilab.org/).

Bacteria
Most families of bacteria are able to produce AMPs namely bacteriocins. Bacteriocins of gram-positive bacteria are more abundant
and more diverse than those produced by gram-negative bacteria. Archeae are also able to produce their own bacteriocin-like-anti-
microbials known as archaeocins (Riley and Wertz, 2002). Bacteriocins are ribosomally synthesized molecules and have bacterio-
static or bactericidal effects. Bacteriocins have narrow or broad spectra of activity that inhibit the growth of bacteria of the same
species or other genera including pathogenic and antibiotic-resistant strains (Cotter et al., 2012). Bacteriocins represent a heteroge-
neous family of small, heat-stable peptides in terms of size, microbial spectrum of activity, modes of action and release and mech-
anism of bacterial immunity (Gordon et al., 2007). All this information has been assembled in a database named Bactibase, which
is a web-accessible database that contains calculated or predicted physicochemical properties of several bacteriocins produced by
both Gram-positive and Gram-negative bacteria. The information in this database is very easy to extract and allows rapid prediction
of structure/function relationships and target organisms of these peptides and therefore, better exploitation of their biological
activity in both the medical and food sectors https://www.ncbi.nlm.nih.gov/pubmed/17941971).

Fish and Marine Products

Many marine organisms such as crustaceans, crab, mollusks and fish are potential sources of bioactive peptides such as antimicro-
bial peptides (Kang et al., 2015; Cheung et al., 2015). Marine AMPs belong to a particular group of peptides. They have high cysteine
content and are positively charged in their active forms. They play a crucial role as a first line of defence protecting the hosts against
a broad range of pathogenic infections (da Costa et al., 2015). Marine AMPs differ in length, isoelectric point, secondary structure,
hydrophobicity, and amphipathicity, and in their antimicrobial activities. Marine AMPs may be classified into four main categories,
namely: (a) linear-helical peptides; (b) linear or helical peptides with abundance of one amino acid (proline, tryptophan, histidine
or glycine rich peptides); (c) peptides forming hairpin-like-sheet or -helical/-sheet mixed structures stabilized by intramolecular
disulfide bonding; and (d) cyclic peptides (Falanga et al., 2016). Most of the marine AMPs are described in APD (http://aps.
unmc.edu/AP/), a database that contains information for 116 antimicrobial peptides identified from fish.

Milk and Milk Products

Milk and milk products are shown to be good sources of a variety of bioactive compounds such as AMPs (Mohanty et al., 2016).
AMPs have already been isolated and characterized from major milk proteins such as caseins, lactoferrin, a-lactalbumin and b-lacto-
globulin (Meisel, 1998). They were shown to have a broad spectrum of activity against pathogenic organisms like bacteria, yeast,
fungi, and some parasites (Mohanty et al., 2016). Milk-derived AMPs may be found in inactive form within the sequence of the
precursor protein but may also be produced by enzymatic digestion especially during the gastrointestinal digestion (Gobbetti
et al., 2007). Recently, a database, MilkAMP (http://milkampdb.org/), has been created to assembly all information about natural
and artificial (synthetic or modified) antimicrobial peptides derived from amino acid sequences of dairy proteins of different origins
and provides. This database contains most complete information on peptide structure/function relationships, inhibitory activity,
 Antimicrobial Peptides: The New Generation of Food Additives 3

spectrum of action and minimal inhibitory concentration (MIC) determined for each tested microbial strain. It also allows rapid
prediction of structure/function relationships and target organisms and hence should lead to better use of the biological activities
of peptides in both the pharmaceutical and food sectors.

Algae and Mushrooms

Only a few studies concerning the inhibitory activities of macroalgal AMPs have been carried out. The lectin is an example of anti-
bacterial algal protein reported in the literature (Liao et al., 2003; Holanda et al., 2005). In addition, a cyclic depsipeptide obtained
from green seaweed Bryopsis sp. has demonstrated activity against Mycobacterium tuberculosis (Hamann and Scheuer, 1993; Hamann
et al., 1996), while a dipeptide derivative originated from red delesseriacean macroalgae has displayed antibacterial effect against
Serratia marcescens and Salmonella typhi XLD (N’Diaye et al., 1996). More recently, AMPs have been extracted from the brown macro-
alga Saccharina longicruris in order to recover antibacterial peptides by enzymatic hydrolysis with trypsin. The protein hydrolysate
fraction exhibited activity against the bacterium Staphylococcus aureus (Beaulieu et al., 2015).
Mushrooms reported as producers of a large number of proteins and peptides with interesting biological activities, such as lec-
tins, fungal immunomodulatory proteins (FIP), ribosome-inactivating proteins (RIP), antimicrobial proteins, ribonucleases, and
laccases. These bioactive components have become popular sources of natural antimicrobial, antiviral, antioxidative, antitumor
and immunomodulatory agents (Xu et al., 2013; Jose Alves et al., 2013; Lavanya and Subhashini, 2013). Some mushrooms-
derived AMPs are described in APD (http://aps.unmc.edu/AP/), a database that contains information for 2 antimicrobial peptides
identified from mushrooms.

Mechanisms of Action

AMPs have diverse structures and functions and interact with cell membranes of invader cells by disturbing the membrane integrity.
As shown in Fig. 1, adapted from Brogden (2005), AMPs may be divided into different groups based on their mechanisms of action.
Some of them have the cell membrane as target and may act by inhibition of cell membrane synthesis, alteration of the cytoplasmic
membrane and inhibition of cell septum formation or activation of the cell autolysis. Other peptides seem to act on intracytoplas-
mic targets. For example, arginine-rich peptides have been shown to act either by inhibiting the synthesis of the cell membrane,
DNA, RNA or proteins. Other peptides such as histatins act rather by inhibiting enzymatic activity.
AMPs mechanisms of action could be classified in two main groups, AMPs targeting external processes or internal processes of
microorganisms.

Extracellular-Targeting AMPs
Outer Surface Lipids
AMPs must first be attracted to bacterial surfaces, and one obvious mechanism is electrostatic bonding between anionic or cationic
peptides and structures on the bacterial surface such as lipids that constitute the outer membrane (de Leeuw et al., 2010). Defensins,
nisin and other lantibiotics were able to interact with lipids constituting bacterial cells, and notably the lipid II that is a central
component of the bacterial cell wall synthesis (de Leeuw et al., 2010; Sass et al., 2010; Hsu et al., 2004). The interaction of cationic

Figure 1 Modes of action for cellular antimicrobial peptide activity. adapted from Brogden (2005).
4 Antimicrobial Peptides: The New Generation of Food Additives

AMPs is well understood and be explained by their initial electrostatic attraction with the outer leaflet of Gram-negative bacteria
initiating the passage of AMPs across the outer membrane through a self-promoted uptake mechanism (Hale and Hancock,
2007). Such as the Cecropin P1, this attachment to the bacterial membrane could induce in many cases it disruption and membrane
permeabilization, leading to cell-killing (Strauss et al., 2010).

Outer Membrane Proteins

Inside the outer membrane of bacteria, it is known that several proteins play an important role as a receptor for AMPs. As describe by
Lin et al. (2010), outer membrane protein I (OprI) plays a critical role in maintaining the integrity of the outer membrane of Pseu-
domonas aeruginosa and this protein serves as the receptor for the cationic-helical AMPs, SMAP-29 and CAP-18. In consequence, the
bacterial membrane became permeable to metabolites. Other AMPs showed a non-membrane-lytic mechanism of action such as
analogs of protegrin I that are able to target the b-barrel protein LptD, a protein implicated in the outer-membrane biogenesis
(Srinivas et al., 2010).

Intracellular-Targeting AMPs
Inner Membrane
Membrane interactions remain important even for intracellular-targeting peptides because they must have a means of translocation
(Nguyen et al., 2011). AMPs could create pores in the cell membrane, through different transmembrane pore-forming mechanisms
such as toroidal pores, carpet or barrel stave (Hale and Hancock, 2007; Nguyen et al., 2011). All these mechanisms of action were
directly correlated to the peptide structure and were the most common and studied among all the mechanisms of action known and
identified from AMPs. Once in the cytoplasm, translocated peptides can alter the cytoplasmic membrane septum formation, and
inhibit synthesis of the cell wall, nucleic acids, proteins or enzymatic activity.

Nucleic Acids
While many AMPs kill bacteria through membrane permeabilization, a smaller subset of AMPs is believed to inhibit bacterial
growth by interfering with essential intracellular functions (Hale and Hancock, 2007). For example, Buforin II and DesHDAP1
(histone-derived AMPs) are both able to target DNA of bacterial cells (Uyterhoeven et al., 2008; Sim et al., 2017) by electrostatic
attraction to negatively charged phosphodiester bonds in the nucleic acid backbone (Sim et al., 2017). The AMP indolicidin inter-
acted with DNA by covalent links with a basic site of single or double DNAs of the HIV-1 viral cDNA (Marchand et al., 2006). As
they are able to bind DNA, AMPs such as the fish peptide pleurocidin, interact with RNA and thus inhibit macromolecular synthesis
and all protein synthesis (Patrzykat et al., 2002).

Intracellular Proteins
All the interactions with nucleic acids of bacterial cells lead indirectly to disruption of macromolecular process mechanisms, but
AMPs also directly target macromolecules and proteins. The most detailed information on inhibition of protein synthesis derived
from the studies of proline-rich AMPs, a group of AMPs with high content of proline and arginine (Scocchi et al., 2016). These AMPs
exhibited in some cases, a polyproline helix II structure, which permits it to bind ribosome subunits and inhibit protein synthesis
(Scocchi et al., 2011). Daptomicin is known as altering bacterial envelope architecture before killing but this lysis occurs as
a secondary event, so it may be the late consequence of a general cell dysfunction and not the main action of this lipopeptide
(Pogliano et al., 2012). However, in most other cases, the intracellular targets of AMPs are either unknown or need to be confirmed.
All the mechanisms of action underline the complexity of interactions occurring at the most molecular level between AMPs and
microbial structures.

Applications of Natural AMPs as Food Additives

AMPs are gaining much attention as potential active components of food products. However, some limited application could be
related to their selective intestinal uptake and physiological instability when consumed orally (Udenigwe and Fogliano, 2017).
AMPs are subject to structural changes and to interact with the food matrix during product development and as a result possibly
reducing their bioaccessibility, bioavailability as well as biological activities (Udenigwe and Fogliano, 2017). The selection of
friendly processing methods and less reactive matrices is imperative to preserve AMPs integrity and stability.
Many approaches exist for preparation of AMP-containing formulations such as inoculating food products with LAB producing
strains and using their antimicrobial metabolites to inhibit the growth of undesirable microorganisms (Tahiri et al., 2009), delivery
systems by controlling AMP release rate (Nordström and Malmsten, 2017) or active packaging material incorporating AMPs to
extend storage life (Meira et al., 2017).

Lactic Acid Bacteria Producing Strains

The use of LAB with known antimicrobial activity as biopreservatives has been largely studied and reviewed in O’Bryan et al. (2015).
Nisin was the first bacteriocin approved for use in food (FDA, 1998) and a commercial crude fermentation product of LAB
 Antimicrobial Peptides: The New Generation of Food Additives 5

containing pediocin has also been manufactured by Quest International (Alta 2341). LAB may be present as natural flora of the
food, or be intentionally incorporated as starter cultures in an industrial fermentation process. Several applications of LAB in foods
such as poultry and meat (Woraprayote et al., 2016), seafood (Calo-Mata et al., 2008), fruits and vegetables (Hanning et al., 2009)
have been studied to increase safety or maintain quality in foods. For instance, the potential of bacteriocin-producing LAB of tradi-
tional Thai fermented meat has been studied in order to decrease risks due to Salmonella spp., S. aureus, and Listeria monocytogenes
(Swetwiwathana and Visessanguan, 2015). In addition, it has been clearly demonstrated that Carnobacterium divergens M35 culture
as well as divergicin M35 bio-ingredient could be applied to the inactivation of L. monocytogenes in ready-to-eat seafood (Tahiri et al.,
2009), which have been enabled by Health Canada’s Food Directorate as food additives. Recently, the effects of a starter culture for
controlling kimchi fermentation, a traditional Korean lactic acid-fermented vegetable, have been determined to prolong shelf life
(Moon et al., 2018).

Active Packaging Material Encapsulating AMPs

Another strategy to control the microbial contamination in foods that have received much attention in recent years is the develop-
ment of innovative films enriched with bacteriocins (Woraprayote et al., 2018). The important role of packaging system is main-
taining the concentration of bacteriocins above the active concentration and constantly releasing bacteriocins to kill target
contaminant microorganisms (Appendini and Hotchkiss, 2002). For instance, an antimicrobial biodegradable film containing
bacteriocin to control the growth of Aeromonas, Escherichia, Pseudomonas, Staphylococcus, Listeria and Salmonella in pangasius fish
fillets have been successfully developed (Woraprayote et al., 2018). Additionally, the inhibition of L. monocytogenes by liposomal
nanovesicles containing bacteriocins produced by Lactobacillus sakei has been shown to efficiently retard bacterial growth in UHT
goat milk (Malheiros et al., 2016).

Food Matrix Interaction and Bioavailability of AMPs

For evidencing the technological applicability of AMPs, tests on toxicity, functionality and consequences to sensorial properties
when added to different food matrices are necessary. Compared to other antimicrobial compounds such as oil and phenolics,
AMPs tend to introduce less negative influence on the sensory qualities of food (Gould, 1996). As peptides, AMPs could be easily
digested in the stomach, avoiding residue build-up and some of them may even contribute benefits to health (Wang et al., 2015). In
the food matrix, the stability of AMPs has to be recognized through evaluation of different parameters such as insensitivity to salt,
adaptation of a wide range of pH, thermal resistance and low cytotoxicity or hemolytic activity (Wang et al., 2015).

Food Additive Regulation and Approval of AMPs

In the food sector, each AMP must be subjected to a very rigorous evaluation by regulatory agencies, such as the Food and drug
administration (http://www.fda.gov/FoodGuidances) or Health Canada (https://www.canada.ca/fr/sante-canada/services/
aliments-nutrition/legislation-lignes-directrices/documents-reference.html#add) before being recognized as safe (GRAS status)
and approved as a food additive. The applicant must provide the following information during the evaluation and the approval
process:
1. Scientific name
2. Proposed name
3. Composition and distinctive characteristics
4. Method of manufacture
5. Quantity used, proposed uses and purposes thereof, detailed directions for use, and recommendations
6. Data on the effectiveness of the antimicrobial bio-preservative
7. Detailed report on product safety under recommended conditions of use including gastrointestinal stability, allergenic
potential, effect of a long-term exposure
8. Data on the quantity of residue that might remain on or in the food product
9. Proposed limit for residue on or in the food product method of detecting and monitoring the bio-preservative in the food
product
10. Example of a proposed label for the additive

Conclusion

Identification of AMPs has attracted a lot of attention in recent years. Their biological activity as well as their GRAS status makes
them very attractive and offers a very promising alternative for use in food, animal and human sectors. As described in this
book chapter, AMPs are widely distributed in nature and are relatively well documented in the literature. However, very few
AMP-based products are currently authorized by regulatory agencies for food, medical or veterinary use despite the increasingly
urgent demand for this type of compound. Research on both fundamental and applied aspects to characterize AMPs, to understand
6 Antimicrobial Peptides: The New Generation of Food Additives

their mechanisms of action, to study their behavior and biological activity in complex matrices, is still necessary to reach their true
potential.
Future work should focus on the following priority targets:
- Evaluation of the extent of the spectrum of action of AMPs not only against bacteria but also fungi and viruses;
- Characterization of the nature of antimicrobial compounds and their complete physicochemical property. At present, only few
AMPs have been relatively well studied;
- Study of the behavior and the biological activity of AMPs under the real conditions of use. Moreover, the interactions of these
compounds with the endogenous microbial ecosystem (animal or food) must be well studied;
- Study of their safety and their potential undesirable effects for the animal and the human;
- The development of technological processes enabling large scale, efficient and cost-effective production and stabilizing of AMPs.
These studies will generate essential knowledge for a more efficient and better targeted use of AMPs in different sectors. This knowl-
edge is crucial in the present context where the use of chemical additives is increasingly challenged in foods and that the problem of
resistance to antibiotics as well as the emergence of new pathogens are increasing continuously in the veterinary and medical sectors.

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Relevant Websites
Database
- APD (http://aps.unmc.edu/AP/).
- Bactibase (https://www.ncbi.nlm.nih.gov/pubmed/17941971)
- CAMPR3 (Collection of Anti-Microbial Peptides) (http://www.camp3.bicnirrh.res.in/).
- MilkAMP (http://milkampdb.org/).
- PhytAMP database (http://phytamp.hammamilab.org/).

Organizations
- FAO (Food and Agriculture Organization of the United Nations) (http://www.fao.org/documents/card/en/c/I8240EN).
- Food and Drug Administration (http://www.fda.gov/FoodGuidances).
- Health Canada (https://www.canada.ca/fr/sante-canada/services/aliments-nutrition/legislation-lignes-directrices/documents-reference.html#add).
- WHO (World Health Organization), 2017. Food Safety. Fact Sheet. http://www.who.int/mediacentre/factsheets/fs399/en/.