Biopreservation strategies using bacteriocins to control meat spoilage and foodborne outbreaks

Abstract Fresh meat is highly perishable, presenting challenges in spoilage mitigation and waste reduction globally. Despite the efforts, foodborne outbreaks from meat consumption persist. Biopreservation offers a natural solution to extend shelf life by managing microbial communities. However, challenges include the effective diffusion of bacteriocins through the meat matrix and the potential inhibition of starter cultures by bacteriocins targeting closely related lactic acid bacteria (LAB). LAB, predominant in meat, produce bacteriocins – small, stable peptides with broad antimicrobial properties effective across varying pH and temperature conditions. This review highlights the recent advances in the optimization of bacteriocin use, considering its structure and mode of action. Moreover, the strengths and weaknesses of different techniques for bacteriocin screening, including novel bioengineering methods, are described. Finally, we discuss the advantages and limitations of the modes of application of bacteriocins toward the preservation of fresh, cured, and novel meat products. Introduction Meat is a source of high-quality proteins, minerals, and vitamins (Geiker et al., 2021). However, it is especially prone to spoilage, as it undergoes microbial (e.g., microbiological spoilage), chemical (e.g., autolytic enzymatic reactions), and physical (e.g., slime and liquid formation) deterioration; in fact, estimates show that as much as 23% of the annual production in the meat sector is lost and wasted (Luong et al., 2020; Odeyemi et al., 2020; Karwowska et al., 2021). Regarding the safety of meats, recent cases (2016-2021) of foodborne outbreaks in the European Union have been linked to the consumption of contaminated meats, for instance, with Salmonella spp. (n=289), Clostridium perfringens (n=102), Staphylococcus aureus toxins (n=34), and Listeria monocytogenes (n=17), resulting in a total of 1363 hospitalizations and economic losses of up to US$90 billion annually (Scharff, 2020; EFSA, 2023). Research on meat preservation considers not only the extension of the product’s organoleptic features but also its microbiological safety (European Commission, 2005 - EC No. 2073/2005). In the domain of biopreservation, natural or controlled microbial communities, and their antibacterial products are used as an approach for controlling microbial growth. An integral component of the initial microbial community of meat is lactic acid bacteria (LAB), which rapidly develops under chill-stored, post-processed, and vacuum-packed/modified atmosphere conditions (Nauman et al., 2022). LAB are classified as Gram-positive, catalase-negative, anaerobic, with a varied morphology (rods or cocci), and play a crucial role in fermentation. Moreover, LAB can be used as biocontrol agents by the generation of metabolites with antimicrobial properties against pathogens, including organic acids (e.g., lactic acid, acetic acid), short-chain fatty acids, proteases/peptidases, and bacteriocins (Ibrahim et al., 2021). This review highlights the recent advances in the optimization of bacteriocin use, considering the bacteriocin’s structure and mode of action. Moreover, the strengths and weaknesses of different techniques for bacteriocin screening, including novel bioengineering methods, are described. Italian Journal of Food Safety 2024; volume 13:12558 [Italian Journal of Food Safety 2024; 13:12558] [page 361] Biopreservation strategies using bacteriocins to control meat spoilage and foodborne outbreaks Nathália Fernandes,1,2 Fouad Achemchem,3Ursula Gonzales-Barron,1,2 Vasco Cadavez1,2 1Mountain Research Center, Campus of Santa Apolónia, Polytechnic Institute of Bragança, Portugal; 2Laboratory for Sustainability and Technology in Mountain Regions, Campus of Santa Apolónia, Polytechnic Institute of Bragança, Portugal; 3LASIME Laboratory, Agadir Superior School of Technology, Ibn Zohr University, Agadir, Morocco Correspondence: Vasco Cadavez, Mountain Research Center, Campus of Santa Apolónia, Polytechnic Institute of Bragança, 5300253 Bragança, Portugal. Tel.: +351 273 303 325. E-mail: [email protected] Key words: antimicrobial peptides, biopreservation, meat, lactic acid bacteria. Contributions: NF, VC, UGB, conceptualization; VC, UGB, resources, project administration, funding acquisition; NF, data curation; NF, writing – original draft preparation; NF, FA, VC, UGB, writing – review and editing; FA, VC, UBG, supervision. All authors have read and agreed to the published version of the manuscript. Conflict of interest: the authors declare no conflict of interest. Ethics approval and consent to participate: not applicable. Funding: the research was funded by the Foundation for Science and Technology (FCT, Portugal) financial support through national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2021) and through funding of the PAS-AGRO-PAS (PRIMA/0014/2022). Availability of data and materials: data and materials are available from the corresponding author upon request. Received: 11 April 2024. Accepted: 9 July 2024. Early access: 17 October 2024. This work is licensed under a Creative Commons AttributionNonCommercial 4.0 International License (CC BY-NC 4.0). ©Copyright: the Author(s), 2024 Licensee PAGEPress, Italy Italian Journal of Food Safety 2024; 13:12558 doi:10.4081/ijfs.2024.12558 Publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Non-commercial use only Finally, we discuss the advantages and limitations of different bacteriocins’ modes of application toward the preservation of fresh, cured, and novel meat products. General overview Bacteriocins are small peptides synthesized in the ribosome and can be categorized into four groups, according to their size, structure, and function: class I or lantibiotics (<5 kDa), class II (<10 kDa), class III or bacteriolysins/non-lytic (>30 kDa), and class IV (reclassified as bacteriolysins) (Simons et al., 2020; Barcenilla et al., 2023). These peptides play a crucial role in the competition for colonization sites and are able to influence the dynamics of the microbiome (Umu et al., 2017). For instance, bacteriocinogenic LAB inhibit target bacteria by interacting with the negatively charged cell membrane, a process mediated by their cationic and amphiphilic motifs (Lei et al., 2019; Heilbronner et al., 2021). Mode of action The bioactivity of bacteriocins can be either of a narrow spectrum (if the inhibition is exclusive to species that are closely related) or a broad spectrum, which is the case for lantibiotics (Woraprayote et al., 2016); in addition to presenting either bacteriostatic (inhibition) or bactericidal (killing) effects (Negash and Tsehai, 2020). The general mode of action of these antimicrobial peptides is given by their specialized functional domains: substrate binding site, translocation, and catalytic site (Gillor et al., 2008). The binding domain attaches to specific receptors on bacterial membranes, the translocation domain interacts with specific proteins integral to the cell membrane, and the effector domain executes lethal action: DNA degradation and/or induction of pores in the membrane (Davidson et al., 2008). Nisin Lantibiotics act by blocking the lipid II cycle (Figure 1) (Hsu et al., 2004), preventing correct cell wall synthesis, and inducing pore formation by interacting with the outer membrane (Diep et al., 2009). Nisin (grey) is composed of 34 amino acids and has a positive charge (+4), which allows it to interact with the anionic lipid II that constitutes the peptidoglycan layer in the bacterial cell membrane. The peptidoglycan is formed by a chain composed of N-acetylglucosamine (green), N-acetyl muramic acid (MurNAc; red), a pentapeptide (not shown), two pyrophosphate molecules (blue), and a prenyl chain (black). The lipid II mediated pathway of nisin starts with the recognition of lipid II MurNAc and isoprene units. The dehydrobutyrine and α-aminobutyric acid of nisin then establish a hydrogen bond with the pyrophosphate molecules of lipid II. The assembly of nisin occurs without a canonical secondary structure, where two lanthionine rings fold to form a cage-like structure with the nisin backbone amides and the lipid II pyrophosphate, with a molar ratio of 8:4 for nisin and lipid II, respectively. Finally, the cage-like structure induces pore formation, resulting in disruption of the cell membrane. Pediocin On the other hand, class II or non-lantibiotic bacteriocins (Figure 2), divided into four subclasses, utilize the mannose phosphotransferase system (man-PTS) to permeabilize the membrane, disrupt proton motive force, and deplete adenosine triphosphate pools (Diep et al., 2007). Class IIb bacteriocins activity depends on two-component, α and β subunits, which fold into α-helical structures and insert themselves into target bacterial membranes to alter their permeability, resulting in ion leakage and cell death (NissenMeyer et al., 2011; Proutière et al., 2023). Pediocin PA-1 is composed (Figure 2) (Zhu et al., 2022) of a hydrophilic and cationic N-terminal (brown) which consists of a three-stranded-β-sheet (containing the pediocin-box) linked by disulfide bridges (black). The region between Nand C-terminal is a flexible region composed of Asp/Asn in residue 17. The C-terminal is hydrophobic and is organized in an α-helix (blue), coupled with a hairpin-like tail (light pink). The N-terminal β-sheet of pediocin PA-1 links with the extracellular man-PTS core domain (red). Normally, this transporter protein composed of a v-motif (green) and a core domain (red) switches from position depending on the transport of mannose. The change in conformation occurs by an elevator movement. The binding occurs with the effector domain of pediocin PA-1 (pediocin-box and positively charged Lysn II and His 12) and the core domain of the man-PTS (Val 7, Cys 9, Cys 14, Tyr 3) establishing a linkage that blocks the elevator movement of man-PTS, with a molar ratio of pediocin PA-1 and man-PTS at 3:3. The hairpin-like tail of pediocin makes pi-stacking interactions with Trp 18 (white) of man-PTS and stabilizes the pediocin-manPTS structure, leading to disruption of the cell membrane (Zhu et al., 2022). Screening of bacteriocins The screening of bacteriocins can be divided into three stages: search for the presence of bacteriocin-encoding genes, evaluation of bacteriocin expression, and assessment of the bacteriocin Review [page 362] [Italian Journal of Food Safety 2024; 13:12558] Figure 1. Nisin mode of action. Figure 2. Pediocin-PA-1 mode of action. Non-commercial use only antimicrobial activity. For cultivable bacteria, the search for a bacteriocinogenic LAB strain begins with the isolation of autochthonous strains from the meat matrix. Then, the bacteriocinencoding genes can be amplified by polymerase chain reaction (PCR), and their expression is evaluated in real-time using realtime quantitative PCR (RT-qPCR). After purification, the bacteriocin’s antimicrobial activity can be assessed by antagonism tests (Figure 3) against indicator microorganisms. For instance, the in vitro assessment of bacteriocins or bacteriocin-like substances entails methodologies such as agar well diffusion (Bungenstock et al., 2020), agar spot (Moraes et al., 2010; Selman et al., 2021), turbidimetric (Yang et al., 2018; Piazentin et al., 2022), enzyme-linked immunosorbent assay (Martínez et al., 2000; Surati, 2020) and RT-qPCR (Dortu et al., 2009; Balutis, 2014; Wan et al., 2015). Moreover, the bacteriocin does not have an antimicrobial effect on the producer strain. These mechanisms are constituted of self-immunity proteins that competitively antagonize the putative bacteriocin receptors by anchoring the membrane surface (antagonism), being embedded in the membrane (repulsion), or producing metalloproteases that degrade the bacteriocin (Deegan et al., 2006; Bastos et al., 2015). Culture-based methods for assessing antimicrobial activity mostly relying on outdated protocols persist in being the most used (Balouiri et al., 2016), despite recent advancements in genomics, namely the whole genome sequencing (WGS), RNA sequencing (RNA-seq), and PCR-based techniques. While agar-based tests offer cost-effectiveness and simplicity, particularly at an initial sample screening, they lack the depth necessary to fully explore a strain’s bacteriocinogenic potential. Moreover, utilizing different agar-based tests within a single study may introduce intra-study variability and errors due to differing experimental conditions and subjective interpretation of results (Hossain, 2024). In the case of unculturable microbes, the use of metagenomics tools is utilized since it is estimated that 99% of microorganisms are not possible to culture in isolation (Ayrapetyan and Oliver, 2016). The DNA of the food sample is extracted, followed by library preparation and then sequencing of all of the DNA present in the sample. This sequencing data enables the visualization of the microbial community composition within the food matrix, providing insights into the relationship between meat preservation and 16S rRNA diversity analysis as the microbiota continually changes during storage (Dorn-In et al., 2024). Shotgun metagenomics enables the discovery of the microbial profile of samples, allowing for the detection of spoilage and pathogenic organisms, as well as differentiation at the strain level (Srinivas et al., 2022). This method can be used to monitor the supply chain for agents of concern, providing valuable insights that incentivize food manufacturers to invest in preventative control measures (Imanian et al., 2022). The development of new in silico techniques has made it possible to analyze bacteriocins in a high-throughput manner (Nedyalkova et al., 2024). In contrast with classical assays for antimicrobial activity determination, the current methods for predicting bacteriocin gene clusters are high-speed and can be automated. BAGEL is a web mining tool that uses whole-genome sequence data to analyze the technological potential of bacterial strains (van Heel et al., 2018). Mining genomes with automated software (Wosinska et al., 2022; Sowers et al., 2023) for the identification of bacteriocins reveal gene loci that can be functional or not. The data generated from this type of analysis has the potential to be exploited by bioengineering, including de novo design of novel bacteriocins (Deo et al., 2022; Kordi et al., 2024). The recent increase in RNA-seq data, which describes the presence and quantity of RNA in a biological sample, demonstrates that RNA-seq can be used to follow the survival of target bacteria in the presence of the bacteriocin, by measurement of the expression of genes in food systems. Moreover, it is useful for analyzing the influence displayed by various environmental conditions on gene expression and fine-tuning them to achieve optimal conditions (Yang et al., 2024). Representative genomes on the NCBI platform have been used in comparative genomics to predict peptide expression and secretion by the bacteria (Marques et al., 2023). These tools in combination with molecular dynamics analysis allow automated assessment of the binding mechanism of action performed by bacteriocins (Walsh, 2017; Leslie et al., 2021; Rodrigues Blanco et al., 2022;). The computational tools include docking software and three-dimensional structure modeling of the putative peptides (Frederix et al., 2018; Nain et al., 2020; Xin et al., 2020; Das et al., 2021; Palmer et al., 2021; Krishnamoorthi et al., 2022; Wang et al., 2023). The development of these novel methodologies enables the fast discovery of structural and functional characteristics of specifReview Figure 3. Diagram for different in vitro methods for bacteriocin activity screening from foodstuffs. [Italian Journal of Food Safety 2024; 13:12558] [page 363] Non-commercial use only ic amino acid residues, which can be associated with binding sites in the bacterial membrane (Bindu and Lakshmidevi, 2021; Chen et al., 2022; Marques et al., 2023; Oftedal, 2023). By understanding the mode of action of bacteriocins, given the varied nature of their structures, strategies to enhance and potentiate the bacteriocin’s antimicrobial activity can be further developed (Amarh et al., 2023). Lantibiotics, for instance, are subjected to post-translational processing (Figure 4) (Hsu et al., 2004), featuring compact structures with modified amino acids (e.g., lanthionine bridges, βmethyllanthionine, and dehydrated amino acids) and thioether ring structures. In the case of nisin, its structure comprises a globular chain consisting of lanthionine and dehydrated serine residues. These elements undergo post-translational modifications and proteolytic cleavage during the peptide processing phase, contributing to the unique structure of nisin. Lactocin S, however, is prone to oxidation due to the sulfide bonds in its α and βrings, which results in its inactivation; therefore, the synthesis of this bacteriocin occurs under anaerobic conditions, which could be improved by replacement of the sulfide bonds with hydrocarbon chains in analogs of lactocin S leading to more oxidative stability (Ross et al., 2012; Tsukano et al., 2024). Lantibiotics active sites include the amino acid residues such as the catalytic site and the substrate binding site, with the F(ND)L(DEN)(LVI) motif (Figure 5) being conserved across different bacteriocins from this class. There is a gap in the literature on the correlation between the conserved motifs of bacteriocins and their mode of action considering their 3D structure and its influence on the bacteriocin’s activity. Figure 5 depicts predicted 3D structures produced by the α Fold algorithm and visualized with Chimera X (Jumper et al., 2021; Varadi et al., 2022; Meng et al., 2023;), Nand C-terminus indicated in the figure, region highlighted in red indicates the conserved region FNDLV, the conserved region of the three peptides appear differently across the space on the 3D structure format. Class IIa bacteriocins are characterized by a conserved sequence at the N-terminal (YGNGV), also known as pediocinbox (Figure 6) (Waterhouse et al., 2009), a sequence related to strong anti-listerial activity first described in pediocin PA-1 (Cui et al., 2012). Class IIb is composed of two short chains. Class IIc are circular bacteriocins that lack the leader peptide sequence and are dependent on the general secretion pathway (sec) for transportation across the cytoplasmic membrane (Perez et al., 2014; Choi et al., 2023). Class IId bacteriocins are typically constituted of NGY residues at the Nterminus and central YxVTK motifs (Yoo et al., 2023); this class covers the remaining single-peptide and nonpediocin-like bacteriocins (Iwatani et al., 2011). Class II (pediocin-like) suffers cleavage of a leader peptide (Figure 6) (Waterhouse et al., 2009) in the Nterminus in order to turn into a mature bacteriocin, which can be seen from the representation of class IIa bacteriocins isolated from meat aligned by the CLUSTAL W algorithm (Thompson et al., 1994; Drider et al., 2006; Lee and Kim, 2011). Sequences were fetched from UniProt. The pediocin-box is highlighted in a black square, hydrophobic (blue), positive charge (red), polar (green) glycine (orange), prolines (yellow), and aromatic (cyan) residues. 3D primary structures of class IIa bacteriocins (Figure 7), generated using the α fold algorithm and visualized with Chimera X, show that the conserved region YGNGV exhibits distinct spatial arrangements within the 3D structures of the different class IIa bacteriocins. This variation suggests that although the sequence is conserved, the spatial positioning and orientation of this region can differ significantly from one peptide to another (Figure 7). Such differences in the three-dimensional conformation could influence how these bacteriocins interact with their target receptors, potentially affecting their antimicrobial activity and specificity (Jumper et al., 2021; Varadi et al., 2022; Meng et al., 2023). Bacteriocins exhibit robust resistance to high temperatures and low pH due to their specific amino acid composition, a high number of disulfide bridges, and ion pairs (Szilágyi and Závodszky, 2000). The solubility of these peptides increases at low pH due to a net charge change that facilitates greater diffusion through bacterial membranes (Yu et al., 2023). Lantibiotics, in particular, Review Figure 4. The amino acid composition of nisin. Figure 5. Lantibiotics three-dimensional primary structures. A) Nisin A (P13068); B) nisin Z (P29559); C) lactocin S (P23826). [page 364] [Italian Journal of Food Safety 2024; 13:12558] Non-commercial use only demonstrate strong resistance under extreme conditions. Additionally, owing to their proteinaceous nature, bacteriocins are susceptible to proteolytic enzymes—such as pancreatin complex, trypsin, and chymotrypsin—found in the gastrointestinal tract (Aljohani et al., 2023). The characteristic nature of bacteriocins can be determined by testing their sensitivity to an array of proteolytic enzymes, producing a pattern of protease sensitivity (Bromberg et al., 2004). WGS has made it possible to identify conserved open reading frames and understand the organization of gene loci encoding the bacteriocin and its immunity genes, in addition, it allowed to predict the promoter and terminator sequence of the peptide from the DNA data by predicting the RNA-polymerase binding motif, which can be useful for improving expression of bacteriocin encoding genes (Ruiz Puentes et al., 2022). Promotion of bacteriocin synthesis can be obtained by constitutive expression of genes or by regulating gene expression as a response to the metabolite production from competing strains (González and Keshavan, 2006; Ng and Bassler, 2009). The bacteriocin synthesis gene clusters have been found to be located in the chromosome and in mobile elements such as plasmids and/or transposons (Achemchem et al., 2005; Lahiri et al., 2022). These clusters encode genes for the expression of the bacteriocin itself, enzymes, and self-immunity regulators of bacteriocin production and are organized in operons and/or regulons that undergo rapid evolution and are susceptible to high rates of horizontal transfer and spontaneous loss (Mørtvedt and Nes, 1990; Noda et al., 2018; Almeida-Santos et al., 2021). A series of databases have been developed specifically for information on bacteriocins, for instance, the open-access database BACTIBASE (http://bactibase.hammamilab.org) with information on bacteriocins based on published literature extracted from PubMed (Hammami et al., 2010). LABiocin (https://labiocin.univlille.fr/) is a database of LAB bacteriocins containing 517 entries extracted from literature searches on Scopus, PubMed/Medline, and ScienceDirect with articles published up until 2017 (Kassaa et Review [Italian Journal of Food Safety 2024; 13:12558] [page 365] Figure 6. Representation of class IIa bacteriocins isolated from meat aligned by the CLUSTAL W algorithm and visualized in Jalview. Figure 7. 3D primary structures of class IIa bacteriocins. A) Pediocin PA-1 (P29430); B) divergicin 750 (Q46597); C) carnobacteriocin A (P38578). Non-commercial use only al., 2019). These databases provide valuable information on structure, amino acid sequence, gene sequence, purification, and physicochemical characteristics of bacteriocins; homology search is an additional feature comprised in these databases which allows for sequence alignment using algorithms such as BLAST (McGinnis and Madden, 2004). Meat application Gram-positive bacteria, particularly LAB, are the most studied source of bacteriocins from the meat environment (da Costa et al., 2019). The bacteriocins isolated from LAB in meat and meat products belong to different species, for instance, Lactobacillus sakei from vacuum-packed lamb meat (Holck et al., 1994), Carnobacterium piscicola from spoiled meat (Jack et al., 1996), Leuconostoc gelidum from processed packaged meat (Hastings et al., 1991), Leuconostoc carnosum from packaged meat (Raimondi et al., 2021), Enterococcus faecium from dry fermented sausages (Casaus et al., 1997), Carnobacterium divergens from vacuumpacked meat (Zhang et al., 2019) and Carnobacterium maltaromicus from vacuum-packed chilled meat (Quadri et al., 1994). In the last 30 years, bacteriocins have been screened and applied in meat to control microbial decay and spoilage, acting as natural inhibitors and extending the shelf life of meat products (da Costa et al., 2019). However, there are still challenges associated with this approach, including the variability in peptide function depending on the nature of the meat matrix – especially in more fibrous matrices, effective inhibition of target microorganisms, resistance development, and compatibility with surrounding LAB (Sionek et al., 2024). Regarding the nature of the meat matrix, challenges arise when applying bacteriocins in meat products primarily due to the hydrophobic nature of the meat and its instability at neutral pH, its interaction with phospholipids derived from meat products and other emulsifiers that make it difficult to distribute and solubilize the bacteriocin at pH values higher than 6.0. For instance, in fresh meat, three glutathione molecules are able to conjugate with one nisin molecule resulting in activity loss. However, it is possible to regulate the amount of free sulfhydryl groups present in the matrix, such as with the process of cooking the meat which reduces the free sulfhydryl groups and prevents the formation of the nisin-glutathione complex (Rose et al., 2002). Strains resistant to a specific class of bacteriocins express immunity genes that confer protection against antimicrobial peptides from different classes, for instance, nisin resistant strain of L. monocytogenes has been reported to show cross-resistance to pediocin PA-1 and leuconocin S (Crandall and Montville, 1998; Darbandi et al., 2021). To overcome the resistance development related to bacteriocins in food systems, it is advisable to apply a combined strategy of peptides in multi-hurdle strategies, for instance, in combination with additives, pH and atmosphere control, recipe modifications with spices and condiments, natural extracts and essential oils as ingredients (Kaur et al., 2013; Soltani et al., 2021). When classifying bacteriocins isolated from fresh and processed meat products, two main classes based on biochemical structure emerge: lantibiotics and pediocin-like peptides (Woraprayote et al., 2016). There are different methods regarding their practical application as biocontrol agents (Figure 8) and each comes with its set of advantages and disadvantages; for instance, crude preparations are tasteless, colorless, and odorless, however, their activity may be limited by a narrow spectrum reach, limited diffusion in solid matrices, and cross-resistance generation (Morata, 2015; Urso et al., 2006). In the next section, we describe the types of bacteriocin applications regarding meat preservation. Mixed starters The use of LAB as inoculum is the most commonly used mode of application for preservation of foodstuffs. For meat preservation, bacteriocins can be applied as an inoculum of pure or mixed cultures (Baillo et al., 2023), as a crude bacteriocin preparation (Xin et al., 2023), and as a purified or semi-purified formulation (da Costa et al., 2019). A selection of two autochthonous LAB strains (6.3 log CFU/g) isolated from spontaneously fermented Spanish sausages Review Figure 8. Diagram of lactic acid bacteria biofilm and bacteriocin for different kinds of applications in the processing and conservation of meat and meat products. [page 366] [Italian Journal of Food Safety 2024; 13:12558] Non-commercial use only (salchichón) – Lactiplantibacillus paraplantarum BPF2 (producer of leucocin K) and P. acidilactici ST6 (producer of pediocin PA-1) were used as starter and reduced levels of rancidity in aroma and taste and improved the intensity and the persistence of the sausage’s characteristic flavor (García-López et al., 2023). Commercial bacteriocin products such as Nisaplin©(Aplin and Barrett Ltd.) and Bactoferm©(Chr. Hansen AS) often consist of a mixture of crude preparations and organic acids in bioactive powder form. These products offer a mix of lyophilized starters for use in meat processing industries, such as for the production of salami, pepperoni, dry and cured meats, which are frequently used combined in multi-hurdle strategies with curing, drying and smoking preservative methods (Soltani et al., 2021). Traditional Iberian cold-smoked fermented sausages underwent testing to assess the antilisterial effect of Bactoferm F-LC©. While Bactoferm F-LC©exhibited bacteriostatic activity at 10ºC, L. sakei CTC494 showed a more pronounced and rapid inhibition of Listeria. This resulted in a significant reduction of the pathogen by 2 log counts (Ortiz et al., 2014). The application of mixed starters is beneficial because it combines strains that shorten fermentation time with those that enhance the meat’s organoleptic properties. Additionally, strains that produce antimicrobial metabolites can be included to combat hazardous and pathogenic bacteria, thereby increasing the food’s microbial shelf life. However, compatibility between strains remains a significant issue, as the antimicrobial metabolites produced by biocontrol strains can be detrimental to the survival of strains promoting fermentation. Therefore, the choice of strains must consider their competitiveness with surrounding bacteria, the expression levels of bacteriocin synthesis, and their compatibility with the fermenting culture (Zacharof and Lovitt, 2012). Pure culture A novel multi-hurdle strategy was developed to extend the shelf life of the Portuguese fermented sausage alheira, combining mild high-pressure processing (300 MPa, 5 min at 10ºC), 0.1% (v/w) Pediococcus acidilactici (producer of pediocin PA-1), and 0.1% (v/w) phage Listex. This approach displayed no significant differences in color, texture, or lipid peroxidation between unprocessed and minimally processed samples (Komora et al., 2023). In this study, homogenization was manually performed by gently massaging the sample for approximately 3 minutes, which did not impact the texture. Although the acidifying capacity of LAB can increase the firmness of sausages, the selected LAB in this case was not a strong acidifier, which may explain why the texture remained unchanged. In contrast, The E. lactis Q1, producer of enterocin P, added as a pure culture (107CFU/g) on raw beef reduced L. monocytogenes counts by 6 log units after 1 week of cold storage and improved the sensorial characteristics such as color, odor, and appearance, especially since Enterococci are good acidifiers, particularly in meat products (Ben Braïek et al., 2020). When comparing mixed and pure cultures, mixed cultures offer greater advantages due to their diverse LAB, which can be fine-tuned for better product quality. Moreover, while pure LAB cultures are effective in reducing pathogen growth in meats, their efficacy is enhanced when used alongside other hurdle strategies. Crude preparation The application of the peptide (1280 AU/g) onto the surface of ham resulted in a 1.74 log reduction of L. monocytogenes counts (p<0.05); therefore, potentially increasing the ham shelf life to 1 month in refrigerated storage (4ºC). Incorporating the peptide in the meat paste produced an inhibition of background spoilage bacteria resulting in significantly lower counts (p<0.01). However, L. monocytogenes strains were reported to develop resistance to plantaricin UG1 in subsequent generations (Enan, 2006). A combination of lactocin and high hydrostatic pressure treatments increased L. monocytogenes cell death in chilled vacuumpacked pork loin slices (Dallagnol et al., 2017) by a 6 log CFU/g reduction. By applying 200 AU/mL of the pure bacteriocinogenic culture in the salami batter, L. monocytogenes was reduced by 2 log CFU/g (de Souza Barbosa et al., 2015). Pediocin PA-1 reduced the counts of L. monocytogenes inoculated in raw chicken meat from 5 log CFU/g to 3.8 log CFU/g when stored at 4 ºC for 1 month, however, re-growth was observed after this period, which could be due to the actions of proteases derived from the meat (Kiran and Osmanagaoglu, 2014). Choosing between a pure culture of bacteriocin-producing bacteria and a crude bacteriocin preparation involves a nuanced assessment of their respective advantages and disadvantages. A pure culture offers the benefit of continuous bacteriocin production, potentially increasing antimicrobial activity over time and reducing initial preparation costs. However, using live bacteria can lead to compatibility issues, where different strains may inhibit each other’s growth and compete for nutrients, resulting in inconsistent efficacy. Additionally, regulatory concerns arise with live cultures, particularly in food applications, due to potential health risks and spoilage. In contrast, a crude bacteriocin preparation provides predictable and controlled antimicrobial effects, as the activity of the bacteriocins is well-characterized and targeted against specific bacteria. This method bypasses compatibility issues and is generally more acceptable from a regulatory perspective. However, the process of purifying bacteriocins is labor-intensive and costly, with challenges in achieving effective concentrations across different food matrices and target microorganisms. Moreover, maintaining the stability of crude bacteriocin preparations requires careful handling and storage. Ultimately, the choice depends on the specific application, balancing cost, regulatory considerations, and the need for precise antimicrobial activity. Encapsulation Challenges with the bacteriocin adsorption in the matrix can be addressed by using immobilized preparations, such as encapsulation on gel coatings, films, silica particles, or liposomes (Gálvez et al., 2007). Encapsulation of bacteriocins involves incorporating these antimicrobial peptides into a protective matrix or carrier to enhance their stability, control their release, and improve their effectiveness in various applications. The application of encapsulated bacteriocins combined with citrus extract, and thyme essential oil led to a synergistic antimicrobial activity for meatballs protection against L. monocytogenes and all tested LAB; however, there was no effect observed on the inhibition of S. Typhimurium (Sarmast et al., 2023). Moreover, the anti-listerial activity of cell-adsorbed bacteriocins combined with oregano essential oil had a synergistic effect on the reduction of L. monocytogenes counts and delayed the growth rebound by 2 weeks in pork meat during storage at 4 ºC (Ghalfi et al., 2007). Smart packaging represents an advanced approach to packaging design, integrating technology to augment the functionality, safety, and user experience of packaged products. A recent advancement in this domain is the incorporation of bacteriocins Review [Italian Journal of Food Safety 2024; 13:12558] [page 367] Non-commercial use only into packaging materials, utilizing natural antimicrobial agents to enhance food safety and preservation. Typically, this method employs crude bacteriocin preparations rather than inoculum. Nevertheless, significant gaps remain in the current field, particularly concerning the varying characteristics of different matrices for the establishment of effective smart packaging solutions. Edible films For instance, whey protein-based edible films, enriched with the cell-free supernatant of Lactobacillus sakei strains were applied in beef, resulting in a decrease from 3.5 log + 0.2 CFU/g to 0.3 + 0.1 log CFU/g of Escherichia coli counts after 36 h of refrigerated storage. Moreover, sensory evaluation of grilled beef wrapped with the antimicrobial films demonstrated no significant differences in flavor and color as assessed by the panelists, the overall acceptability was high (Beristain-Bauza et al., 2017). Polythylene-based films A plantaricin solution applied in an active package made with polyvinylidene chloride film in pork fresh meat inhibited L. monocytogenes growth by 1.4 log CFU/g after 7 days of cold storage (Xie et al., 2018). Plantaricin BM-1 solution was used to soak polyethylene-based films applied in meat artificially inoculated with L. monocytogenes and exerted antimicrobial activity that inhibited the pathogen growth during storage for 120 days at 25ºC (Zhang et al., 2017). Curvaticin 32Y produced by L. curvatus 32Y isolated from dry sausages has been shown to reduce viable counts of L. monocytogenes by 1 log when applied in a polythene film by soaking, spraycoating as a preservative for artificially inoculated pork steaks (Mauriello et al., 2004). Moreover, in bioactive packaging made of sawdust particles and poly lactic acid biocomposite film, the adsorption of pediocin PA1-AcH enhanced raw sliced pork meat protection against L. monocytogenes, with counts reduced by ~2 log units after storage at 4ºC for 14 days (Woraprayote et al., 2013). Cellophane coating Nisin has been shown to have greater antimicrobial potential in meat preparation when used in combination with organic acids and salts; for instance, nisin reduced the total aerobic bacteria counts in 0.1 log CFU/g from veal meats when applied in a cellophane coating packaging and extended the shelf life of chopped meat under refrigerated storage (Guerra et al., 2005). Alginate matrix As a component in antimicrobial packaging, nisin had an inhibitory effect against microbial decay and extended the shelf life of refrigerated chicken meat up to 15 days more than the control when applied and incorporated in an alginate matrix (Carrión et al., 2023). A packaging composed of alginate films and containing immobilized viable enterocin-producing E. faecium Smr18 reduced S. Typhimurium counts by 3 log CFU/g in chicken meat after 34 days at cold storage (Rashid et al., 2023). Cultivated meat Meat grown from animal cells in a laboratory setting is called ‘cultivated meat’ (CM). CM appears as a new solution to several safety issues with livestock farming such as the zoonotic transfer of viruses and infection through human consumption (Ramani et al., 2021). The development of CM is dependent on the retrieval of animal cells by biopsy, creation of a bank of cells, growth, and differentiation by reprogramming stem cells into skeletal muscle cells, harvesting the cells, and processing them into tissues. CM meat can become contaminated by bacteria, fungi, and viruses, which are managed by the addition of antibiotics during cell growth, as well as by the addition of cryoprotectants during cell storage. Moreover, the risk for contamination during further downstream processing is expected to be similar to the case of traditional meat products (Broucke et al., 2023). CM production systems are considered to be more sustainable and safer in comparison to conventional meat production systems, but they may have a completely different risk profile; such a risk coming from antibiotic application in vitro to promote the growth of cells still consists of a gap in the field and much attention would require to be paid to the safety of added substrates and other compounds of the culture medium to the human health. As it will be easier to keep control of pathogenic contamination in cultured meat production, CM is associated with fewer risks with respect to microbial contamination. The application of LAB as a starter for CM production is expected to influence the final product similarly than it already affects the meat that incorporates starter cultures, having a positive effect on organoleptic features and on microbial safety (Kolodkin-Gal et al., 2024). Conclusions and future perspectives Fresh meat is particularly susceptible to spoilage, posing a significant challenge in reducing losses and waste associated with meat and its products on a global scale. Furthermore, despite concerted efforts, foodborne outbreaks linked to the consumption of meat products persist as an unresolved issue. In this context, biopreservation emerges as a natural alternative for extending the shelf life of meat products by managing their inherent microbial communities; however, proper diffusion of the bacteriocin through the meat matrix remains a challenge, in addition to the inhibition by the bacteriocins against closely related LAB, which could cause loss of starter culture effectiveness. LAB are the dominant group present in meat and produce a variety of metabolites with antimicrobial effects. Bacteriocins are secondary metabolites produced by LAB with antimicrobial properties, these small peptides are stable in extreme temperatures and pH. While culture-based methods remain prevalent in assessing antimicrobial activity due to their cost-effectiveness and simplicity, they often rely on outdated protocols that may not fully explore the bacteriocinogenic potential of strains. Recent advancements in genomics, such as WGS, RNA-seq, and PCR-based techniques, offer more comprehensive insights into antimicrobial mechanisms. Moreover, computational tools, including docking software and three-dimensional structure modeling, now enable automated assessment of bacteriocin binding mechanisms. This advanced understanding of bacteriocin structures and their modes of action holds promise for developing strategies to enhance and optimize their antimicrobial efficacy in practical applications. The development for higher efficiency of bacteriocin diffusion from packaging surfaces or application sites into meat, considers multiple factors, including meat texture and the thoroughness of mixing between the bacteriocin and the meat, which significantly influence homogeneous distribution within the meat matrix. The choice of application method is influenced by factors such as the Review [page 368] [Italian Journal of Food Safety 2024; 13:12558] Non-commercial use only complexity of the food matrix, scalability, and cost-effectiveness of production. Looking forward, advancements in 3D printing and CM could profit from integrating LAB and bacteriocins to improve their safety and functionality. Continued research into bacteriocins is crucial for advancing meat safety and expanding functional meat product offerings in the future. References Achemchem F, Martínez-Bueno M, Abrini J, Valdivia E, Maqueda M, 2005. Enterococcus faecium F58, a bacteriocinogenic strain naturally occurring in Jben, a soft, farmhouse goat’s cheese made in Morocco. J Appl Microbiol 99:141-50. Aljohani AB, Al-Hejin AM, Shori AB, 2023. Bacteriocins as promising antimicrobial peptides, definition, classification, and their potential applications in cheeses. Food Sci Technol 43:e118021. Almeida-Santos AC, Novais C, Peixe L, Freitas AR, 2021. Enterococcus spp. as a producer and target of bacteriocins: a double-edged sword in the antimicrobial resistance crisis context. Antibiotics 10:1215. Amarh MA, Laryea MK, Borquaye LS, 2023. De novo peptides as potential antimicrobial agents. Heliyon 9:e19641. Ayrapetyan M, Oliver JD, 2016. The viable but non-culturable state and its relevance in food safety. Curr Opin Food Sci 8:127-33. Baillo AA, Cisneros L, Villena J, Vignolo G, Fadda S, 2023. Bioprotective lactic acid bacteria and lactic acid as a sustainable strategy to combat Escherichia coli O157:H7 in meat. Foods 12:231. Balouiri M, Sadiki M, Ibnsouda SK, 2016. Methods for in vitro evaluating antimicrobial activity: a review. J Pharm Anal 6:719. Balutis AM, 2014. Quantification of bacteriocin gene expression in Carnobacterium maltaromaticum ATCC PTA-5313. Available from: https://era.library.ualberta.ca/items/294af8ad-8bbe4ae5-95dd-680c22caf4df. Barcenilla C, Puente A, Cobo-Díaz JF, Alexa EA, Garcia-Gutierrez E, O’Connor PM, Cotter PD, González-Raurich M, López M, Prieto M, Álvarez-Ordóñez A, 2023. Selection of lactic acid bacteria as biopreservation agents and optimization of their mode of application for the control of Listeria monocytogenes in ready-to-eat cooked meat products. Int J Food Microbiol 403:110341. Bastos MCF, Coelho MLV, Santos OCS, 2015. Resistance to bacteriocins produced by Gram-positive bacteria. Microbiology 161:683-700. Ben Braïek O, Smaoui S, Ennouri K, Ben Ayed R, Hani K, Mastouri M, Ghrairi T, 2020. In situ Listeria monocytogenes biocontrol and sensory attributes enhancement in raw beef meat by Enterococcus lactis. J Food Process Preserv 44:e14633. Beristain-Bauza SC, Mani-López E, Palou E, López-Malo A, 2017. Antimicrobial activity of whey protein films supplemented with Lactobacillus sakei cell-free supernatant on fresh beef. Food Microbiol 62:207-11. Bindu A, Lakshmidevi N, 2021. In vitro and in silico approach for characterization of antimicrobial peptides from potential probiotic cultures against Staphylococcus aureus and Escherichia coli. World J Microbiol Biotechnol 37:172. Bromberg R, Moreno I, Zaganini CL, Delboni RR, de Oliveira, J, 2004. Isolation of bacteriocin-producing lactic acid bacteria from meat and meat products and its spectrum of inhibitory activity. Braz J Microbiol 35:137-44. Broucke K, Van Pamel E, Van Coillie E, Herman L, Van Royen G, 2023. Cultured meat and challenges ahead: a review on nutritional, technofunctional and sensorial properties, safety and legislation. Meat Sci 195:109006. Bungenstock L, Abdulmawjood A, Reich F, 2020. Evaluation of antibacterial properties of lactic acid bacteria from traditionally and industrially produced fermented sausages from Germany. PLoS One 15:e0230345. Casaus P, Nilsen T, Cintas LM, Nes IF, Hernández PE, Holo H, 1997. Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. Microbiology 143:2287-94. Carrión MG, Corripio MAR, Contreras JVH, Marrón MR, Olán GM, Cázares ASH, 2023. Optimization and characterization of taro starch, nisin, and sodium alginate-based biodegradable films: antimicrobial effect in chicken meat. Poul Sci 102:103100. Chen H, Ma L, Dai H, Fu Y, Wang H, Zhang Y, 2022. Advances in rational protein engineering toward functional architectures and their applications in food science. J Agric Food Chem 70:4522-33. Choi GH, Holzapfel WH, Todorov SD, 2023. Diversity of the bacteriocins, their classification and potential applications in combat of antibiotic resistant and clinically relevant pathogens. Crit Rev Microbiol 49:578-97. Crandall AD, Montville TJ, 1998. Nisin resistance in Listeria monocytogenes ATCC 700302 is a complex phenotype. Appl Environ Microbiol 64:231-7. Cui Y, Zhang C, Wang Y, Shi J, Zhang L, Ding Z, Qu X, Cui H, 2012. Class IIa bacteriocins: diversity and new developments. Int J Mol Sci 13:16668-707. da Costa RJ, Voloski FLS, Mondadori RG, Duval EH, Fiorentini ÂM, 2019. Preservation of meat products with bacteriocins produced by lactic acid bacteria isolated from meat. J Food Qual 2019:e4726510. Dallagnol AM, Barrio Y, Cap M, Szerman N, Castellano P, Vaudagna SR, Vignolo G, 2017. Listeria inactivation by the combination of high hydrostatic pressure and lactocin AL705 on cured-cooked pork loin slices. Food Bioprocess Technol 10:1824-33. Darbandi A, Asadi A, Mahdizade Ari M, Ohadi E, Talebi M, Halaj Zadeh M, Darb Emamie A, Ghanavati R, Kakanj M, 2021. Bacteriocins: properties and potential use as antimicrobials. J Clin Lab Anal 36:e24093. Das P, Sercu T, Wadhawan K, Padhi I, Gehrmann S, Cipcigan F, Chenthamarakshan V, Strobelt H, Dos Santos C, Chen PY, Yang YY, Tan JPK, Hedrick J, Crain J, Mojsilovic A, 2021. Accelerated antimicrobial discovery via deep generative models and molecular dynamics simulations. Nat Biomed Eng 5:613-23. Davidson AL, Dassa E, Orelle C, Chen J, 2008. Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol Mol Biol Rev 72:317-64. de Souza Barbosa M, Todorov SD, Ivanova I, Chobert JM, Haertlé T, de Melo Franco BDG, 2015. Improving safety of salami by application of bacteriocins produced by an autochthonous Lactobacillus curvatus isolate. Food Microbiol 46:254-62. Deegan LH, Cotter PD, Hill C, Ross P, 2006. Bacteriocins: biological tools for bio-preservation and shelf-life extension. Int Dairy J 16:1058-71. Review [Italian Journal of Food Safety 2024; 13:12558] [page 369] Non-commercial use only