Dual Nature of Bacteriophages: Friends or Foes in Minimally Processed Food Products—A Comprehensive Review

Abstract

The increasing consumer demand for minimally processed foods (MPFs) has highlighted the need for innovative preservation methods that ensure both safety and quality. Among promising biocontrol tools, bacteriophages—viruses that selectively destroy bacteria—have gained significant attention. This review explores the dual role of bacteriophages in the food industry. On one hand, they offer a natural, highly specific, and environmentally friendly means of controlling both pathogenic and spoilage bacteria in MPFs, contributing to improved food safety, extended shelf life, and reduced reliance on antibiotics and chemical preservatives. Their use spans primary production, bio-sanitization, and biopreservation. On the other hand, phages pose significant risks in fermentation-based industries such as dairy, where they can disrupt starter cultures and impair production. This review also examines the regulatory, technological, and safety challenges involved in phage application, including concerns about antibiotic resistance gene transfer, the presence of endotoxins, and scale-up limitations. Ultimately, this paper argues that with proper strain selection and regulation, bacteriophages can become valuable allies in sustainable food systems, despite their potential drawbacks. The application of strictly virulent bacteriophages as part of “green biotechnology” could enhance food quality and improve consumer health safety. By implementing the “farm to fork” strategy, bacteriophages may contribute to the production of health-promoting and sustainable food.

1. Introduction

Malnutrition arises from deficiencies or poor absorption of essential nutrients, and it encompasses both undernutrition and overnutrition, including obesity—a major global cause of disease and mortality [1,2]. Diet is a key modifiable risk factor for various conditions, influencing blood pressure, glucose, cholesterol, and body weight [3,4]. Alongside poor dietary habits, smoking and inactivity significantly contribute to disease development [5,6]. Food processing also affects health outcomes [2,7]. The NOVA classification, introduced in 2009, categorizes foods based on processing level and purpose into four groups: (a) unprocessed and minimally processed foods; (b) processed culinary ingredients; (c) processed foods, and (d) ultra-processed foods [3,6,8,9]. Though endorsed by the World Health Organization (WHO), Food and Agriculture Organization (FAO), and Pan American Health Organization (PAHO), NOVA has faced criticism in the U.S. and lacks Food and Drug Administration (FDA) and United States Department of Agriculture (USDA) recognition [10,11].

Growing awareness of the link between diet and health has shifted consumer preferences toward fresh, minimally processed foods (MPFs) due to their role in preventing degenerative diseases such as heart disease and cancer [12,13,14]. However, maintaining both safety and nutritional quality in MPFs remains a challenge. Traditional methods often compromise one for the other, prompting interest in biological solutions like bacteriophages—viruses that specifically target bacteria. This review explores their dual role as tools for enhancing safety and serving as potential disruptors in MPF systems.

2. Minimally Processed Food

The concept of “minimal processing” generally refers to using the least amount of processing needed to achieve the desired outcome [14]. It often emphasizes preserving nutritional and sensory qualities while reducing reliance on heat [12]. Modern definitions highlight techniques that ensure shelf life and meet consumer expectations for freshness and convenience [15]. The NOVA classification offers a distinct view, defining MPFs as natural items treated with basic preservation methods (e.g., drying, cooking, freezing) without added salt, sugar, fats, or other substances. These processes aim to retain the food’s natural state, aid storage, and ensure safety. Many foods classified in the first NOVA group reflect home-cooked meals and support a healthy diet when eaten in balanced combinations [8,11,16]. Despite variations in how “minimally processed food” is interpreted, several common characteristics can be identified: (a) they retain the freshness of raw ingredients—such as texture, color, taste, and aroma—due to gentle heat treatment and preservation methods; (b) they maintain nutrients that are sensitive to processing, particularly vitamins, provitamins, phytonutrients, and minerals; (c) they often involve combined preservation methods that incorporate mild processing techniques alongside physicochemical or biological approaches; (d) packaging involves modified conditions and materials specifically tailored to the product; and (e) they require refrigeration throughout the production and distribution process to maintain quality and safety [17,18,19].

2.1. Microbiological Hazards in Minimally Processed Food Products

The interest in and demand for fresh, natural foods have been steadily increasing for many years, with consumers seeking low-processed products that offer high nutritional value [19,20]. This growing preference for minimally processed and functional foods presents a significant challenge for food technologists. Producing safe, MPFs with an adequate shelf life is complex and requires the development of innovative, non-thermal processing methods within the food industry [21,22,23].

MPFs can be broadly categorized into two main groups: (a) plant-based products, such as fruits and vegetables, and (b) animal-based products, including meat, fish, and seafood. In addition to these primary categories, the expanding market for low-processed foods also encompasses other product types, such as whole-grain foods, ready-to-eat meals, and items preserved using advanced techniques like cook–chill, cook–freeze, and sous-vide methods [19,24].

Minimally processed technologies often involve mechanical operations such as cutting or peeling, which can cause tissue damage and the release of cell contents at injury sites [25,26]. The leakage of cell juices during processing increases water activity in the product, creating favorable conditions for microbial growth [25,27]. Specifically, latex secreted from cut lettuce surfaces can stimulate the proliferation of enterohemorrhagic Escherichia coli O157:H7 (EHEC), a verotoxin-producing bacterium [28]. The elevated microbial load in MPF products often stems from the high level of microorganisms present in the raw materials. In addition to monitoring pathogenic bacteria that pose a risk to consumer health, it is equally important to address the presence of saprophytic bacteria. Their rapid growth can lead to a decline in product quality and a shortened shelf life [29,30].

2.2. Methods of Preserving Minimally Processed Foods

To guarantee the safety and high quality of minimally processed foods, the food industry is advancing various preservation techniques. Common methods include thermal and non-thermal processing, refrigerated storage, innovative packaging solutions (modified atmosphere packaging, controlled atmosphere packaging, edible coatings), the application of natural antimicrobials or chemicals, and the use of hurdle technology [12,17,19].

In recent years, there has been a growing shift away from physical and chemical food preservation methods in favor of biological approaches. Among the most promising biocontrol agents for extending the shelf life of fresh-cut fruits and vegetables are lactic acid bacteria (LAB) [20,31], which have demonstrated antibacterial activity against pathogens such as Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium, and Shigella sonnei [32]. Both Gram-positive and Gram-negative bacteria produce bacteriocins—protein-based compounds with bactericidal or bacteriostatic properties [33,34]. Unlike antibiotics, which are produced during the dying phase of microbial growth (secondary metabolites), bacteriocins are synthesized during the stationary phase as primary metabolites [35]. These compounds target bacterial cytoplasmic membranes, disrupting proton movement, which leads to ion leakage, a loss of ATP, the release of genetic material (RNA or DNA), and ultimately cell death [36].

Another promising biological preservation method involves the use of bacterio-phages—highly specific, strictly lytic viruses that target bacteria. The application of phage-based biopreparations for food biopreservation is discussed in Section 4.4.

Figure 1 illustrates the primary physical, chemical, and biological methods employed in preserving MPF products.

Figure 1. Methods of preserving minimally processed foods. The figure was prepared in Canva.

3. Food as a Carrier of Antibiotic-Resistant Bacteria

The antibiotic era began in the 20th century with Alexander Fleming’s discovery of penicillin in 1928 [37,38]. The period from 1950 to 1970 is often referred to as the “golden age” of antibiotic discovery, during which half of the antibiotics we use today were identified. However, since then, no new classes of antibiotics have been discovered [39,40]. Unfortunately, poor antibiotic stewardship (AMS) has contributed to the onset of what is now known as the post-antibiotic era [41,42,43]. Antibiotics are becoming less effective in treating bacterial infections, and the rise of antibiotic resistance in microorganisms is closely linked to the presence of antibiotic resistance genes (ARGs) in the environment [44,45]. These resistance genes can be transferred to pathogenic bacteria through horizontal gene transfer (HGT) [46,47]. Conjugation, one of the primary mechanisms for spreading ARGs, bypasses the usual genetic barriers to gene transfer [44,47]. Some conjugative modules even enable resistance plasmids to be exchanged between unrelated bacterial species [44]. Numerous studies highlight that food is a significant reservoir of ARGs (Figure 2).

Figure 2. A schematic representation of the problem of antibiotic-resistant bacteria within the food chain. The figure was prepared in Canva.

The rapid spread of ARGs is mainly driven by excessive antibiotic use in agriculture and HGT [44,45]. For instance, streptomycin use in the 1950s to control Erwinia amylovora in fruit trees led to resistance development; it remains approved in some countries outside the EU, including Israel, Canada, Mexico, and New Zealand [44]. Agricultural antibiotic use far exceeds medical use—about 80% of U.S. antibiotic sales are for farming [48]. Resistant bacteria from treated animals are excreted into the environment via feces, contaminating manure-fertilized fields and aquatic ecosystems [49]. ARGs have since been found in meat and minimally processed plant-based foods. The dairy industry also contributes to environmental ARG spread [44,47].

Inappropriate human antibiotic use—such as prescribing for viral infections or improper dosage—further worsens resistance [50,51,52]. This has led to increased prevalence of MDR strains and reduced antibiotic effectiveness [53,54]. Notably, methicillin-resistant Staphylococcus aureus (MRSA) causes more annual deaths in the U.S. than emphysema, Parkinson’s, or AIDS [55,56].

In response, the EU banned antibiotics as growth promoters in 2006 [57,58], and resistance now ranks among the top public health priorities in EU programs [59]. Member states must develop national action plans. In Poland, the National Antibiotic Protection Programme, now partly integrated into the National Health Programme (2021–2025), addresses this issue [60].

Eliminating all drug resistance genes is not feasible, as some have other functions unrelated to antimicrobial resistance (AMR). Therefore, efforts should focus on controlling the emergence, selection, and spread of these genes, particularly in bacteria that interact with humans, animals, and plants. Researchers are intensively working on methods to prevent HGT. Another promising approach involves combining principles from developmental biology, evolution, and ecology to combat AMR spread [44]. Increasing attention is also being given to the use of bacteriophages as a potential solution to fighting MDR strains (discussed in Section 4) [61,62,63].

3.1. Antibiotics and Mechanisms of Their Action

Antibacterial drugs are a broad group of compounds. The original definition of “antibiotics” referred to natural, secondary metabolites produced by microorganisms with antibacterial properties, while chemotherapeutics were considered synthetic chemical compounds that did not have a natural equivalent. Today, the term “antibiotics” encompasses all antibacterial drugs, including both naturally derived and synthetic chemotherapeutic agents [64,65]. Antibiotics with similar molecular structures tend to have similar efficacy, toxicity, and potential side effects [65,66]. Figure 3 illustrates the classification of antibiotics based on how they act on or within bacterial cells.

Figure 3. The mechanism of action of selected drug groups. The figure was created in BioRender. Wójcicki, M. (2025) https://BioRender.com/r74m836, accessed on 27 May 2025 (license no.: KO27XLU1BJ). Symbols in the figure: PABA—p-aminobenzoic acid; DHF—dihydrofolate; THF—tetrahydrofolate. The asterisk (*) indicates antibiotics that are active only against Gram-positive bacteria.

3.2. Antibiotic Resistance and Mechanisms of Bacterial Resistance

The development of new antibiotics has driven the emergence of drug-resistant bacterial strains. Acquired resistance arises from genomic changes, either through spontaneous mutations (e.g., DNA polymerase errors) or via transposable elements. HGT also facilitates resistance spread through conjugation, transformation, and transduction. Key contributors include mobile genetic elements (e.g., plasmids, phages, integrons), recombination processes, and selective pressure [64]. Definitions of acquired antibiotic resistance were established by experts from the European Centre for Disease Prevention and Control (ECDC) in Stockholm and the US Centers for Disease Control and Prevention (CDC):

i.

MDR (multidrug resistance): the strain shows resistance to at least one antibiotic from three or more different classes of antibacterial drugs;

ii.

XDR (extensively drug resistance): the strain is resistant to at least one antibiotic from all but a maximum of two classes of antibiotics used to treat infections caused by the microorganism;

iii.

PDR (pandrug resistance): the strain is resistant to all available, approved antibiotics from all categories used to treat infections caused by the specific microorganism species [67,68,69,70].

Over time, bacteria have evolved various mechanisms to resist antibiotics, including (a) target site alteration; (b) reduced membrane permeability; (c) drug-inactivating enzymes; (d) efflux pumps; (e) alternative pathways or enzymes; and (f) protective proteins shielding drug targets [70,71].

3.3. Natural Resistance of Bacteria to Antibiotics

In addition to acquired resistance, bacteria also exhibit natural resistance, which is commonly observed within a particular family, genus, or species. This type of resistance is not linked to prior antibiotic exposure and does not involve HGT. Natural resistance arises from (a) the absence of a target site for the drug within the bacterial cell, (b) the presence of structures that block the drug from reaching its target, such as lipopolysaccharides in the outer membrane of Gram-negative bacteria, or (c) the activity of specific resistance mechanisms, where effectors like enzymes or membrane transporters are encoded by chromosomal genes characteristic of the bacterial species [70,72,73].

4. Bacteriophages as a Natural Method of Biocontrol of Bacterial Microbiota in Food

The growing prevalence of MDR strains and the increasing frequency of infections caused by these bacteria have prompted the search for alternative antibacterial agents to traditional antibiotics [74,75]. One potential solution is the use of bacteriophages—natural enemies of bacteria. Phage therapy is not a new concept, as it was practiced even before the discovery of antibiotics [76,77,78]. For many years, experimental, targeted phage therapy has been carried out in Poland at the Phage Therapy Unit of the Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences (HIIET PAS) in Wroclaw [79,80,81].

The rising demand for minimally processed food, where maintaining structure and nutritional value is essential, has led the food industry to explore new, non-invasive preservation methods [82,83]. The use of bacteriophages throughout the food production chain—such as directly eliminating bacteria from food surfaces and processing equipment in contact with the product—could help improve the microbiological safety of minimally processed food [84,85,86,87]. Since bacteriophages are obligate bacterial parasites, they naturally coexist with the food microbiota and could serve as natural biological agents targeting their bacterial hosts [88,89,90].

4.1. Phage Particle Structure

Bacteriophages (phages), like eukaryotic viruses, lack the ability to amplify independently and do not have a functional metabolism or cellular structure [91,92]. Despite this, phages undergo evolutionary processes and exhibit variability. Their genome is composed of nucleic acids, which may include configurations not commonly found in cellular genomes, such as double-stranded RNA (dsRNA) [91,93]. The unique characteristics of phages place them at the intersection of living and non-living matter [92]. Virus classification typically relies on analyzing virion morphology through transmission electron microscopy (TEM) and genome sequencing methods [94,95]. The International Committee on Taxonomy of Viruses (ICTV) is responsible for the taxonomic classification of viruses, including bacteriophages [94,96]. However, there are no universal criteria for defining the genus or species of individual viruses. The ICTV adopts a polythetic species concept, where a species is identified by a set of common features, some of which may be absent in certain members of the species [97]. According to ICTV guidelines, subfamilies are only created when they provide additional hierarchical information [98]. A genus is defined as a group of viruses with over 70% nucleotide sequence similarity, distinct from other viral types, while a species is a monophyletic group of viruses distinguishable by multiple criteria. A species is the lowest taxonomic level recognized by the ICTV [99].

The primary demarcation criterium for defining bacteriophage species is a genome sequence similarity of at least 95%, meaning that two viruses within the same species can differ by no more than 5% in nucleotide sequence alignment [98]. In 2020, the ICTV adopted a binomial naming system for virus species, where the genus name and species epithet together create a unique species name [100,101]. The Bacterial Viruses Subcommittee (BVS) implemented a fully open binomial format, allowing research groups and authors of taxonomic proposals to determine the final species name format [102]. Classifying bacteriophages based on experimental data is both time-consuming and labor-intensive, which has led to the exploration of machine learning as an appealing alternative [103,104,105]. New tools for supporting bacteriophage classification include applications like INPHARED, GRAViTy, vConTact2, virClust, PhaGCN2, and PhageAI [106,107,108,109,110].

Phage particles are composed of genetic material in the form of either single-stranded or double-stranded DNA or RNA, encased in structural proteins that form a capsid. The capsid can have a helical (filamentous or rod-like; such as phage M13), isometric (icosahedral or spherical, such as phage ΦX174), or icosahedral shape, often connected to a tail, forming a complex (tailed) structure [111,112,113]. The phage genome contains all the necessary information for replication within the bacterial host cell, the synthesis of phage proteins, and the assembly of progeny phages capable of infecting other bacterial cells [114]. The protein capsid is highly resistant to most external environmental factors, safeguarding the genetic material inside [111,115]. Many phage nucleocapsids are surrounded by an additional lipid membrane, which is a fragment of the host cytoplasmic membrane, forming an envelope connected to the capsid by matrix proteins. In contrast, non-enveloped (naked) viruses lack this lipid membrane [116,117].

Typical examples of phages with complex structures are virions belonging to the Caudoviricetes class, which includes both bacterial and archaeal viruses containing a double-stranded DNA (dsDNA) genome [118,119,120]. In 2022, the BVS recognized the Caudoviricetes class, removing the Caudovirales order, including previous morphological Myoviridae, Podoviridae, and Siphoviridae families, and introducing a binomial system for species naming [102]. Research on the classification of newly discovered bacteriophage sequences is ongoing, and as of 30 April 2025, the Caudoviricetes class includes 11 orders, 105 families, 132 subfamilies, 1680 genera, and 5799 species of bacterial and archaeal viruses [121].

4.2. Bacteriophage Replication Cycles

The first step in phage infection is the adsorption of virions to the surface of the bacterial host cell [122,123]. This process varies depending on the type of bacteria. In Gram-negative bacteria, phage attachment occurs in two stages. Initially, the phage binds reversibly to lipopolysaccharides (LPSs) that contain the polymannose O antigen, and then it attaches permanently to the ferrichrome transporter [124,125,126]. Ferrichromes are siderophores, compounds that bacteria secrete to capture iron ions from the environment [127]. For Gram-positive bacteria, phage attachment involves the binding of the phage to specific sugar receptors in the bacterial cell wall. These receptors may include glucose, galactose, rhamnose, glucosamine, or galactosamine [124,128]. In some cases, the interaction between the phage and bacterial host may require the involvement of bacterial phage infection proteins (PIPs) or membrane proteins [129,130].

Environmental factors, as well as the physiological and genetic traits of the bacterial host, can lead to variations in phage replication pathways [129]. Phage replication strategies exist on a spectrum, from highly productive infections that result in the release of new virions to persistent nonproductive infections that do not generate new phages but instead spread prophage genomes within the bacterial population (transferred from parent to daughter cells) [131,132]. The two most well-known and studied phage replication pathways are the lytic and lysogenic cycles [95,133]. A variation of the lysogenic cycle is the carrier state, also known as the pseudolysogenic cycle [124]. Another form of phage–host interaction is chronic infection [124,131]. Figure 4 illustrates the differences between these various phage infection strategies.

Figure 4. Possible phage–bacterial host interactions. The figure was created in BioRender. Wójcicki, M. (2025) https://BioRender.com/r74m836, accessed on 27 May 2025 (license number: FR27XLSFWZ).

4.3. Benefits and Risks of Using Bacteriophages

Phage therapy refers to the use of phages and/or their enzymes to treat bacterial infections. This approach offers several advantages over antibiotic therapy [124,134]. However, despite its benefits, phage application also carries certain risks that must be considered when selecting phages for the development of effective biocontrol preparations.

Bacteriophages, as viruses that infect prokaryotic cells, can replicate only within bacterial hosts, making them harmless to human, animal, and other eukaryotic cells [124,135]. They are a natural part of the human gut microbiome [92,136,137] and have also been found in blood, the genitourinary tract, the respiratory system, the skin, and cerebrospinal fluid [138,139]. The gut microbiota is a complex ecosystem containing trillions of microbial cells, including bacteria, archaea, eukaryotes, and viruses. It plays a crucial role in human health, contributing to the biosynthesis of vitamins, amino acids, and short-chain fatty acids (SCFAs), supporting immune system development, and providing protection against pathogens [140,141]. Disruptions in the gut microbiome have been linked to various diseases, such as inflammatory bowel disease, obesity, diabetes, and neurological disorders [142,143]. Bacteriophages are the most abundant component of the gut microbiota, with an estimated 10¹⁵ phage particles in the human gastrointestinal tract. They significantly influence the composition, function, and modulation of the gut microbiome [144,145]. Each individual has a unique “phageome”, with only about 5% of phages forming a core common to all humans [146]. The structure and function of the gut phage community are shaped by multiple factors, including environmental influences, gut microbiota composition, and host immune responses [92,146,147,148]. Phages and bacteria exhibit a distinct distribution within the intestines. Temperate phages and numerous bacteria are found closer to the intestinal lumen, while deeper layers of the mucosa contain lytic phages and fewer bacteria resistant to phage infection [92,147,148]. Despite their high morphological, structural, and genetic diversity, intestinal phages share certain characteristics. Most are non-enveloped and possess DNA genomes, though some RNA phages are present, likely originating from ingested plant-based food [136,149]. A promising emerging approach involves using lytic phages as probiotics or dietary supplements to target and eliminate harmful bacteria. The key distinction between bacterial probiotics and lytic phage-based probiotics, known as phagobiotics, lies in their mechanisms of action. While bacterial probiotics utilize nonpathogenic bacteria to inhibit the colonization and pathogenicity of harmful bacteria, phagobiotics employ lytic phages to selectively eliminate specific pathogens while preserving the beneficial microbial community. Due to their high specificity, phagobiotics are expected to have minimal impact on the overall microbiota and may even act synergistically with traditional probiotics [150]. Numerous studies have explored potential interactions between bacteriophages and eukaryotic cells, demonstrating their influence on biochemical and physiological processes [139,151]. Although phages do not trigger acute immune responses, they can affect tissue and organ function. Research has shown that phages can enter breast epithelial cells via endocytosis and reach the nucleus, suggesting a role in transcytosis across epithelial barriers [136,139]. Additionally, phages have been found to modulate gene expression in eukaryotic cells [139,152]. The therapeutic effects of phage preparations may extend beyond their bactericidal activity, as they can also stimulate immune system cells and enhance proliferation in response to mitogens [153]. Phage lysates, which contain bacterial remnants following lysis—such as endotoxins—can also influence immune responses [152,154]. Studies indicate that phages may exhibit immunomodulatory properties by affecting phagocyte functions, including phagocytosis, reactive oxygen species (ROS) production, and T lymphocyte proliferation [139,151,155]. Additionally, they possess immunogenic properties, stimulating the production of specific antibodies against phage antigens. Bacteriophages interact with innate immune cells, inducing phagocytosis and influencing antimicrobial protein levels, as well as modulating cytokine production by other immune cells. Due to their nucleoprotein structure, phages are recognized by immune system cells, leading to their neutralization and clearance from the body [156]. Furthermore, they can influence the adaptive immune response by stimulating the production of antiphage antibodies [136,139].

One of the key advantages of bacteriophages is their ability to increase their dose automatically. As self-amplifying entities, phages replicate in the presence of their bacterial hosts, accumulating precisely where they are needed [157,158]. A significant benefit of phage therapy is its effectiveness against multidrug-resistant bacteria, helping to control infections caused by resistant strains [158,159]. Additionally, bacteriophages through enzymes encoded in their genomes can penetrate and disrupt bacterial biofilms, improving access for other antibacterial agents [160,161,162]. Phages are highly specific to their bacterial targets, which can be both an advantage and a limitation. Their precise targeting makes them valuable for therapeutic applications and biocontrol in the agri-food industry. However, to ensure broad efficacy, phage-based treatments should consist of cocktails containing multiple phages with a polyvalent specificity [163,164]. On the other hand, this strain specificity prevents phages from harming beneficial bacteria used in biotechnological and food industries, such as dairy starter cultures [165,166]. Using phage cocktails or polyvalent phages (capable of infecting multiple bacterial strains) may introduce complex pharmacological interactions. In some cases, these interactions can lead to antagonistic effects, reducing phage efficacy and resulting in bacteriostatic rather than bactericidal activity [163].

For both therapeutic applications and the development of biopreparations for the agri-food industry and bioremediation, only virulent phages should be identified and selected [167,168]. This is because temperate phages, which integrate into the bacterial genome during the lysogenic cycle, can facilitate HGT, including the spread of antibiotic resistance genes [155,169,170,171]. Prophages often encode virulence factors within pathogenicity islands (PAIs), a subset of genome islands (GEIs), and lysogenic conversion can contribute to the pathogenicity of certain bacteria [172,173]. A notable example is Vibrio cholerae, where virulence is driven by transducing phages CTXΦ and VPIΦ, known as choleraphages, which carry virulence-related genetic elements in their genomes [174,175]. The virulence cassette within these phages contains four key genes that contribute to bacterial pathogenicity: (a) ctxAB—encodes cholera toxin (CT), a cytotoxic AB-type enterotoxin. Its A subunit induces intracellular cAMP (cyclic adenosine monophosphate) production, disrupting ion transport and causing severe fluid loss; (b) zot—encodes the zonula occludens toxin (ZOT), which disrupts intercellular junctions, increasing intestinal permeability; (c) ace—encodes the accessory cholera enterotoxin (ACE), which contributes to fluid accumulation in the intestine; and (d) cep—encodes fimbriae, aiding bacterial colonization of the small intestine [174,176,177].

Over the course of evolution, bacteria have developed various defense mechanisms against bacteriophage infections, acting at different stages of the infection process [178,179]. One such mechanism is adsorption inhibition, which prevents phage attachment by physically masking receptors, altering their structure, or eliminating them entirely. The masking process often involves the synthesis of extracellular polysaccharides, which form an additional protective layer that blocks phage recognition sites. Additionally, spontaneous mutations in bacterial genes responsible for receptor synthesis or transport can lead to the loss or malfunction of these receptors, making bacterial cells resistant to phage adsorption [179,180]. Other bacterial defense strategies include blocking phage DNA injection through the superinfection exclusion (Sie) system, which prevents foreign phage genomes from entering the cytoplasm [180]. Another important mechanism is abortive infection (Abi), which operates within the cytoplasm to disrupt various stages of phage replication, ultimately leading to the programmed cell death (apoptosis) of infected bacteria. This self-sacrificial strategy prevents the spread of phages within the bacterial population, ensuring the survival of uninfected cells [180,181,182,183,184]. Additionally, phages themselves can encode membrane-bound Sie proteins which protect against the entry of genetic material from competing lytic phages [182]. The restriction–modification (R-M) system is another bacterial defense mechanism that involves the recognition and destruction of foreign DNA through endonucleolytic digestion. Bacterial DNA is protected from degradation by specific methylation modifications introduced by enzymes with methyltransferase activity [183,185,186]. A more advanced bacterial immune defense mechanism is the CRISPR-Cas system (clustered regularly interspaced short palindromic repeats), which enables bacteria to recognize and degrade foreign genetic material. This system consists of short DNA fragments (spacers) homologous to phage genomes, separated by palindromic sequences within the bacterial genome. The associated Cas proteins degrade the invading phage DNA through a three-stage process: (1) adaptation—the bacterium incorporates a new spacer sequence from the phage genome into its CRISPR array; (2) expression—the CRISPR array is transcribed into CRISPR RNA (crRNA), which contains a single spacer sequence; and (3) interference—the crRNA associates with effector proteins that scan invading DNA for complementarity. If a match is found, the phage genome is degraded by Cas nucleases, preventing bacterial lysis [179,187,188,189,190]. Other bacterial defense systems include the DISARM (defense island system associated with restriction–modification), which consists of five genes encoding a DNA methyltransferase, DrmA helicase, DrmB (a protein of unknown function), phospholipase D domain nuclease (DrmC), and an accessory protein. This system functions by methylating bacterial DNA while restricting and modifying invading phage genomes [181,191,192]. Similarly, the BREX (bacteriophage exclusion) system prevents phage replication and integration through bacterial DNA methylation [193,194]. Recent discoveries have revealed additional bacterial antiphage defense systems, though their exact molecular mechanisms remain unknown and require further research [179,195,196].

Bacteriophages and bacteria are engaged in a continuous evolutionary arms race, each adapting to outcompete the other for survival. Phages evolve to overcome bacterial defense mechanisms by recognizing and developing alternatives to altered bacterial receptors, allowing them to effectively adhere to host cells [182]. When bacteria modify their receptors, phages counteract by producing polysaccharide-degrading enzymes, such as lyases and hydrolases, to break through bacterial defenses [197]. Phages can also bypass bacterial modification-dependent systems and deploy their own anti-CRISPR mechanisms to evade acquired bacterial resistance [182]. Additionally, they can escape CRISPR-Cas defenses through mutations in protospacer-adjacent motif (PAM) sequences, preventing recognition and cleavage by bacterial nucleases [198]. Some phages have evolved protective genomic modifications, such as glucosylated hydroxymethylcytosine (HMC) or methylated DNA, which shield them from bacterial restriction enzymes. For example, Staphylococcus phage K can eliminate Sau3A restriction sites on both DNA strands to evade degradation [199,200]. Similarly, bacteriophage T4 protects itself by glucosylating HMC residues, making its genome resistant to bacterial restriction–modification systems [201]. Phages can also produce proteins that inhibit bacterial defenses. For instance, some phages synthesize a 76-amino-acid restriction nuclease inhibitor (IPI*), which binds to bacterial GmrS and GmrD proteins, preventing phage DNA digestion [202]. Mutations in nucleotide metabolism genes allow phages to escape the Abi system, particularly the AbiQ mechanism [182,203]. To counteract bacterial toxin–antitoxin (TA) systems, bacteriophage ΦTE produces pseudoantitoxin RNA (pseudo-ToxI), which mimics bacterial antitoxins and neutralizes apoptosis-associated defense responses [204].

4.4. Invisible Guardians: The Role of Bacteriophages in Food Safety

Phages can be applied across three key sectors of the agri-food industry: (a) primary production, including animal farming and crop cultivation; (b) bio-sanitization, primarily in manufacturing facilities to prevent biofilm formation on equipment surfaces; and (c) biopreservation, which focuses on extending the microbiological shelf life of food by inhibiting the growth of both pathogenic and spoilage bacteria [83,205].

Given the potential risks associated with bacteriophage applications, phage preparations should comply with strict safety criteria: (a) they should be classified as organisms generally recognized as safe (GRAS); (b) they must be strictly lytic (virulent); (c) for pathogenic bacterial biocontrol, they should be capable of replicating in nonpathogenic host cells; and (d) their genomes must be free of toxin- and antibiotic resistance-related genes [206,207]. Additionally, bacteriophages used in biopreparations should exhibit a broad bacterial host range, high stability under various storage conditions (resistance to pH fluctuations, temperature changes, and gastrointestinal enzymes), and the ability to replicate efficiently, producing high phage titers [208,209,210,211].

4.4.1. Primary Production

Bacterial phytopathogens significantly reduce crop yields and thus pose a threat to food supply [212]. The use of bacteriophages is considered a potential biocontrol measure in sustainable primary production. Phage preparations specifically targeting major bacterial plant pathogens (including Xanthomonas, Pseudomonas, Erwinia, or Pectobacterium) in field cultivation could be applied in the form of sprays or surface treatments [213,214]. Unlike pesticides, phages as biodegradable particles do not leave harmful chemical residues in the environment [215,216]. Compared to antibiotics or copper-based sprays, they also exert lower selective pressure for resistance [213]. Phages have already been tested or applied to combat diseases such as bacterial fruit blotch, goss wilt, soft rot and blackleg, fire blight, bacterial canker, bacterial leaf blight, or pepper bacterial spot [212]. However, regulatory hurdles and the need for cost-effective large-scale production still limit their widespread adoption. Despite these challenges, some commercial phage products, such as AgriPhage in the USA, have been registered as biopesticides by the U.S. Environmental Protection Agency and are being marketed. AgriPhage phage cocktails combat bacterial spot and speck disease in tomatoes and peppers (“AgriPhage”), tomato bacterial canker (“AgriPhage-CMM”), fire blight in apple and pear (“AgriPhage-Fire blight”), and citrus canker (“Agriphage-Citrus canker”) [212,217]. Another preparation available in the US is “Xylphi-PD”, which targets Pierce’s disease of grape (related to Xylella fastidiosa infection) [217]. In Europe, only a very limited number of commercial phage bioproducts are available. The Scottish company APS Biocontrol sells the post-harvest food processing aid “BioLyse-PB”, which prevents Pectobacterium infections in potato tubers, while the Hungarian company Enviroinvest has received authorization for local sales of a phage cocktail targeting E. amylovora (“Erwiphage Plus”) [212,217]. However, to date, the EFSA has not registered any phage-based product as a plant protection agent or biopesticide.

Phages may also find application in targeted approaches to controlling bacterial infections such as Salmonella, E. coli, and Clostridium in livestock. By reducing the bacterial load and preventing disease outbreaks, phages can help improve survival rates, growth performance, and feed efficiency, which are key in breeding programs [218,219,220,221]. In addition, phages can selectively modulate the gut microbiome, promoting beneficial bacteria that enhance nutrient absorption and immune function, thereby indirectly supporting better reproductive outcomes. An important advantage of using phages is the reduction in antibiotic use, which helps lower the risk of developing antibiotic-resistant bacteria—a major global issue in both human and veterinary medicine. Phages may also play a role in improving reproductive health by treating infections in the reproductive system, potentially increasing fertility rates in breeding animals [222,223]. Current research focuses on developing phage-based feed additives, sprays, and modified phages with a broader host range or enhanced activity, as well as integrating phages into comprehensive herd health management strategies [224,225]. The Polish biotechnology company Proteon Pharmaceuticals specializes in developing commercial bacteriophage preparations aimed at improving animal health, reducing antibiotic use, and supporting sustainable livestock farming and aquaculture. It offers feed additives for poultry targeted at combating Salmonella (BAFASAL^® and BAFASAL^® + G) and avian pathogenic E. coli (BAFACOL^®). Additionally, it produces products for aquaculture. The BAFADOR^® preparation, targeting Aeromonas and Pseudomonas, aims to maintain gut microbiota balance, support fish welfare, and increase the economic profitability of farming. Proteon Pharmaceuticals’ products are commercially available in selected markets in Southeast Asia, South America, and Africa [226,227,228,229,230].

4.4.2. Bio-Sanitization

Phages are becoming innovative tools in the bio-sanitation of food production lines. Unlike traditional chemical disinfectants, phages are highly specific, targeting only host bacteria without disrupting beneficial microbial communities or leaving chemical residues on equipment or surfaces [231,232]. This makes them particularly valuable when used against persistent bacterial pathogens such as L. monocytogenes, Salmonella spp., and E. coli, which can form biofilms on production line surfaces and resist conventional cleaning methods [232,233,234,235]. Phage-based bio-sanitation involves directly applying phage preparations onto food contact surfaces and production equipment [219,236,237]. These preparations can penetrate biofilms and effectively reduce bacterial loads, enhancing food safety while complying with regulatory and environmental standards. Moreover, phages are generally considered safe, making them a viable addition to integrated sanitation protocols in the food industry [232,237]. This approach enhances food safety, supports clean-label requirements, and may help combat antimicrobial resistance. Ongoing research aims to optimize phage application methods, formulations, and combinations with other sanitation strategies to improve efficacy and minimize the risk of bacterial resistance. As demand grows for safer, more sustainable food production, phage bio-sanitation stands out as an innovative tool for maintaining hygiene and ensuring the microbiological safety of food products [156,238,239].

4.4.3. Biopreservation

In the food industry, commercial phage preparations have emerged as promising biocontrol tools to enhance food safety by targeting foodborne bacterial pathogens such as L. monocytogenes, Salmonella spp., E. coli O157:H7, and Campylobacter spp. [225,240,241]. Information on commercially available phage preparations for food biocontrol and legal aspects is presented in Section 4.7.

Research institutions worldwide are exploring the potential of phage biopreparations as a means of preserving MPF products. Recent selected studies on the preservation of plant-based MPF products conducted over the past five years are summarized in Table 1.

Table 1. Biocontrol of saprophytic and pathogenic bacteria in minimally processed plant-based food products based on single phages and/or their cocktails.

Saprophytic Bacteria
Food Matrices (Storage Conditions)	Bacteria	Phage/Phage Cocktail (Application Method)	Main Results	Reference
alfalfa sprouts kale sprouts lentil sprouts sunflower sprouts radish sprouts (20 °C/350 mbar)	natural bacterial microbiota of the product	cocktail of 18 phages (spraying and an absorption pad)	The spraying method was significantly more effective, achieving a maximum reduction of 1.5 log CFU g−1 after 48 h, while phage-soaked pads reduced bacterial counts by only 0.27–0.79 log CFU g−1.	[77]
rucola (20 °C/protective atmosphere)	natural bacterial microbiota of the product	cocktail of 43 phages (spraying, and an absorption pad)	The spray and absorbent pad applications reduced TNB* growth similarly after 6 h. During storage, control samples saw a 3-log increase, while the phage cocktail applications reduced TNB by 99% (2-log reduction).	[242]
mixed-leaf salad with carrot (20 °C/protective atmosphere)			Spray and absorbent pad applications showed no significant bacterial reduction at 6 h but significantly reduced TNB at 48 h. The absorbent pad was more effective on the food product. During storage, microbial counts in phage spray tests increased by 1 log, while both methods reduced TNB by 99.9% compared to controls.
mixed-leaf salad with beetroot (20 °C/protective atmosphere)			The spray and absorbent pad had little effect on TNB at 6 h but significantly reduced growth at 48 h. Spraying was more effective on the product. Both methods reduced TNB by 99% (2-log reduction) compared to controls.
washed spinach (20 °C/protective atmosphere)			The spray and absorbent pad significantly reduced bacterial growth at 6 h, but no inhibitory effect was observed at 24 h and 48 h.
unwashed spinach (20 °C/protective atmosphere)			Applying the spray and absorbent pad to unwashed spinach significantly reduced TNB growth at 6 h and 24 h, but not at 48 h.
broccoli sprouts (20 °C/protective atmosphere)	natural bacterial microbiota of the product	cocktail of 29 phages (spraying and an absorption pad)	The application of the spray and absorbent pad notably inhibited microbial growth in the product environment after 24 h. During storage, TNB in the control sample increased by less than half a logarithmic unit, whereas in the phage-treated samples, it decreased by a comparable amount.	[243]
spinach leaves (20 °C/protective atmosphere)			Both spraying and absorbent pad application effectively suppressed microbial growth in the product environment within 6 h compared to the control. The application of the phage cocktail on spinach leaves led to a reduction in TNB by half to nearly one log unit, depending on the method used.
freshly squeezed carrot–celery juice (20 °C/protective atmosphere)		cocktail of 29 phages (phage suspension (5% of the volume of juice))	Throughout the 48 h storage period, TNB in the control sample remained largely unchanged. However, applying the phage suspension to the juice significantly reduced TNB within 6 h. During storage, TNB in the product treated with the phage mixture continued to decline steadily.
fresh-cut mixed vegetables (iceberg lettuce, carrot, and purple cabbage) (20 °C/nitrogen gas)	E. coli strain K12	cocktail of coliphages (Escherichia phage SUT_E420, Escherichia phage SUT_E520, Escherichia phage SUT_E1520, and Escherichia phage SUT_E1620) (phage suspension)	When the coliphage cocktail was applied, no E. coli were detected in treated samples on day 0, showing a reduction of approximately 3.8 log CFU mL−1. However, E. coli levels gradually increased to 0.9–1.4 log CFU mL−1. Despite this, bacterial counts in treated samples remained significantly lower than in untreated samples throughout the experiment, with a 2.4 log CFU mL−1 reduction still observed by the end of day 3.	[244]
lettuce leaves (Lactuca sativa var iceberg) (no data)	Enterobacter kobei strain AG07E	bacteriophage FENT2 (single-phage treatment by immersion)	Immersing lettuce leaves in the phage suspension effectively reduced the presence of the E. kobei strain AG07E on their surface, resulting in a significant reduction of 1 ± 0.06 log.	[245]
Bacterial Pathogens
Food Matrices (Storage Conditions)	Bacteria	Phage/Phage Cocktail (Application Method)	Main Results	Reference
radish sprouts (20 °C/normal atmosphere)	S. Virchow strain KKP 997	cocktail of four salmophages (KKP 3265, KKP 3266, KKP 3267, and KKP 3332) (spraying, and an absorption pad)	In all cases, the absorbent pad more effectively inhibited Salmonella growth in the food matrix, reducing it by up to 2 log units, compared to the spray method.	[246]
	S. Itami strain KKP 1001
	S. Enteritidis strain KKP 3078
	S. Typhimurium strain KKP 3079
HHP-preserved carrot–mango–apple juice(4 °C)	S. enterica subsp. enterica serovar 6,8:l,-:1,7 strain KKP 1762	single or phage cocktail: Salmonella phage KKP 3822, Salmonella phage KKP 3829, Salmonella phage KKP 3830, and Salmonella phage KKP 3831 (phage suspension)	Applying either individual phages or a phage cocktail to juice significantly reduced Salmonella growth, except when Salmonella phage KKP 3830 was added to juice infected with S. enterica strain KKP 1762, where no significant reduction occurred. Pathogen growth was most effectively restricted by the phage targeting the specific bacterial strain. The phage cocktail reduced the growth of both bacterial pathogen strains by approximately one log unit (90%) by the end of refrigerated storage, compared to untreated control samples.	[247]
	S. Typhimurium strain KKP 3080
raw carrot–apple juice (4 °C or 20 °C)	S. enterica subsp. enterica serovar 6,8:l,-:1,7 strain KKP 1762	phage cocktail: Salmonella phage KKP 3822, Salmonella phage KKP 3829, Salmonella phage KKP 3830, and Salmonella phage KKP 3831 (phage suspension)	Addition of a phage cocktail to juice infected with S. enterica strain KKP 1762 and storage at 4 °C for 24 h significantly reduced the pathogen level. For S. Typhimurium strain KKP 3080, a significant reduction was observed after 48 h. At 20 °C, control samples showed a one-fold increase in Salmonella counts after 24 h, while phage-treated samples showed a significant reduction. After two days at 20 °C or seven days at 4 °C, no Salmonella were detected in either control or phage-treated samples (strong pH changes during storage).
	S. Typhimurium strain KKP 3080
alfalfa sprout seeds (22 °C/in the dark)	S. Enteritidis strain S5-483, S. Newport strain S5-639, S. Muenchen strain S5-504, and S. Typhimurium strain S5-5336	phage cocktail (SE14, SE20, SF6) (single-phage treatment or repeated-phage treatment via washing)	The results indicate that S. Enteritidis was the most susceptible to both bacteriophage cocktails, showing a reduction of approximately 2.5 log CFU mL−1 on day 0 with cocktails SE14, SF5, and SF6. While S. enterica populations across all strains continued to grow despite daily bacteriophage applications, their growth rate was significantly lower compared to a single bacteriophage application. The extent of reduction varied depending on the S. enterica strain, but the findings suggest that repeated-phage treatment during sprout germination are more effective at reducing S. enterica populations than single-phage application.	[248]
		phage cocktail (SE14, SF5, SF6) (single-phage treatment or repeated-phage treatment via washing)
green juice (4 °C and 25 °C)	Shiga toxin-producing E. coli O157:H7 (STEC) strain ATCC 43895	bacteriophage vB_ESM-pEJ01 (phage suspension)	The phage’s effectiveness was evaluated by measuring viable host cell counts and Shiga toxin genes (stx1, stx2) abundance. After 24 h at 4 °C, phage treatment reduced host cells by 1.5 log CFU mL−1. At 25 °C, a 2.7 log CFU mL−1 reduction occurred after 12 h, but bacterial regrowth was observed at 24 h, suggesting better efficacy at 4 °C. Phage-treated samples also showed a significant decrease in stx gene abundance at 4 °C after 24 h and at 25 °C after 12 h.	[249]
Romaine lettuce (10 °C)	E. coli O157 (STEC)	single coliphages (VE04, VE05, and VE07)	All tested phages successfully inhibited E. coli O157 on Romaine lettuce. After three days of storage at 10 °C, the log reduction compared to control groups ranged from 2.6 log CFU cm−2 to approximately 6 log CFU cm−2.	[250]
celery (4 °C)	L. monocytogenes	cocktail of three lytic phages: LMPC01, LMPC02, and LMPC03 (spraying)	During 7 days of refrigerated storage, the phage cocktail with MOI = 10 reduced L. monocytogenes by 2.2 log CFU g−1.	[251]
enoki mushroom (4 °C)			During 7 days of refrigerated storage, the phage cocktail with MOI = 10 reduced L. monocytogenes by 1.8 log CFU g−1.
baby spinach (8 °C, 12 °C, and 25 °C)	L. monocytogenes strain 3053	phage vB_LmoH_P61 (spraying)	After 6 days of storage at 8, 12, and 25 °C, the concentration of L. monocytogenes decreased by 1.93, 2.06, and 3.3 log CFU g−1, respectively.	[252]
orange juice (4 °C)	L. monocytogenes strain LM008	phage vB-LmoM-SH3-3 (phage suspension)	Phage treatment resulted in an approximately 3.9-log decrease in L. monocytogenes.	[253]

4.5. The Dark Side of Phages: Challenges in Food Production and Fermentation

Bacteriophages may find applications in many branches of the agri-food industry. However, in the dairy industry, bacteriophages pose a serious problem because they can cause lysis of the starter culture, leading to slowed fermentation or complete inhibition of microbial growth [205,254,255]. This, in turn, leads to a decrease in the quality of the final product or its spoilage [165,256]. It is estimated that about 70% of technological process disturbances in the dairy industry (in the production of curd and rennet cheeses) are related to phage infections. The biotechnological processes carried out in the plant are susceptible to infections because the raw material is not sterile. The use of the same starter culture (bacterial inoculum) further supplies host cells for phage development [257]. The primary sources of bacteriophages in the dairy industry are raw milk and starter cultures, while secondary contamination can occur via inadequate hygiene practices by employees (e.g., contaminated clothing) or production and ventilation equipment. Many phages retain their activity during pasteurization and spray drying processes. Starter cultures, particularly in the case of Lactococcus spp. strains, may in turn contain prophages capable of induction. Especially sensitive is Lactococcus lactis subsp. lactis var. diacetylactis, which is responsible for shaping the sensory characteristics of many dairy products [256]. Raw milk delivered to dairies may contain up to 10⁴ PFU mL⁻¹ of bacteriophages. The phage titer in whey can reach up to 10⁹ PFU mL⁻¹, making it the main secondary source of bacteriophages in the dairy plant. Aerosols generated from whey can lead to air contamination at levels up to 10⁸ PFU m⁻³ [258].

Phage detection in dairy production relies on monitoring acidification and performing lysogeny tests. In addition, qPCR or PCR methods (with a detection limit of 10³ PFU mL⁻¹) are employed, as well as flow cytometry, which requires prior removal of lipid droplets from the samples [205,254]. Compliance with hygiene standards in production, the use of starter culture rotation, and the acquisition of phage-resistant strains reduce the risk of phage infections. Modern phage infection control methods include the use of phage-resistant starter culture strains free of prophages, the use of lyophilized or frozen concentrated starter cultures (DVI/DVS), and early addition of rennet, which, in combination with elevated temperature, limits the absorption of certain phages. The possibility of controlled selection of phage-resistant strains may be provided by the use of the CRISPR/Cas system [259]. Media that inhibit phage replication and aseptic inoculation systems are also used, along with separation of the CIP (clean-in-place) systems between starter culture preparation and production areas, and the use of HEPA filters. Destruction of phages with chemical agents can involve treatment with 0.05% sodium hypochlorite or potassium permanganate solutions for 5 min at room temperature [165].

4.6. Risks and Implications of Contaminants in Phage-Based Preparations

The application of bacteriophages in the food industry has gained increasing interest due to their specificity and effectiveness in controlling foodborne pathogens. However, the production and use of phage-based preparations are associated with several safety concerns, primarily related to the presence of contaminants originating from the bacterial host or the culture medium [260,261,262,263,264]. One of the most critical risks involves endotoxins, particularly LPS, which are components of the outer membrane of Gram-negative bacteria frequently used as host strains in phage amplification. These endotoxins are known for their pyrogenic and immunostimulatory properties and can pose significant health risks if not properly removed, even when phages are applied externally to food products [261,262].

In addition to endotoxins, there is a risk of contamination with exotoxins, which are toxic proteins secreted by certain bacterial hosts during growth. These substances, if retained in crude lysates, may contribute to toxicity or allergenicity. Furthermore, bacterial cell debris, host DNA, enzymes, and residual proteins may persist through insufficient purification processes and negatively impact the safety or regulatory compliance of the final product [262,263,264]. Residues from nutrient-rich culture media, such as peptones or yeast extract, may also remain in the preparation, potentially affecting product stability or triggering unwanted reactions. Another concern is the inadvertent inclusion of non-target or temperate bacteriophages, which could facilitate HGT or unpredictably disrupt microbial communities [260,263].

Scaling up bacteriophage production for food industry use also presents significant technological challenges [265,266]. In upstream processes, the selection of suitable host strains is crucial; these strains must be nonpathogenic, genetically stable, and incapable of producing harmful byproducts. Controlled bacterial lysis is essential to ensure efficient phage release while minimizing the liberation of cellular contaminants. Downstream processing, which includes purification, concentration, and formulation, is often complex and costly. Techniques such as ultrafiltration, chromatography, and ultracentrifugation are employed to reduce endotoxin levels and remove debris, but these methods can reduce phage viability and overall yield [260,267,268].

Concentration and stabilization of phage preparations to reach high titers (typically 10⁹–10¹¹ PFU mL⁻¹) without compromising their infectivity is another critical hurdle [260]. The formulation must also ensure long-term stability under various environmental conditions, particularly for industrial food applications where shelf life and robustness are key. In addition, the absence of standardized production protocols leads to variability in product quality, making regulatory approval and batch-to-batch consistency more difficult to achieve [269].

These limitations have important implications for the food industry. Contaminants, even in trace amounts, may affect regulatory acceptance by bodies such as the FDA or EFSA and could result in delays, recalls, or loss of consumer trust. Moreover, the high costs of advanced purification technologies and the need for rigorous quality control present economic barriers to large-scale implementation.

In summary, while bacteriophages offer a promising and natural alternative for pathogen control in food systems, their safe and effective use requires overcoming substantial technological and regulatory challenges. Addressing contamination risks and optimizing large-scale production processes will be essential steps toward integrating phage-based solutions into mainstream food safety practices.

4.7. Legal Aspects Related to the Use of Bacteriophages in the Agri-Food Industry

Currently, only a few centers in the EU use bacteriophages for therapeutic purposes, the leading ones being the Phage Therapy Unit of the Medical Centre of the HIIET PAS in Wroclaw [73,270,271] and the Queen Astrid Military Hospital in Brussels [272,273]. Despite extensive research on bacteriophage biology and their effects on the human immune system, phage therapy in Poland remains an experimental approach, with no government funding to support its clinical use [270,274]. In the agri-food sector, the use of phages as antibacterial agents is also highly restricted. While there have been successful applications of bacteriophages in food biocontrol, their incorporation into food products is only allowed in certain countries, with regulations applying exclusively to specific commercial phage-based products [275]. Within the EU, phage preparations have not yet been approved for direct contact with food [205,276,277]. However, several non-EU countries, including the United States, Brazil, Israel, Canada, Switzerland, Australia, and New Zealand, permit the use of phages in the food industry. Commercial biotechnology companies offer phage-based biopreparations targeted at food safety, with many of these products receiving FDA approval. Notable FDA-approved phage preparations include Listex^TM P100, Secure Shield E1, EcoShield^TM, ListShield^TM, ShigaShield^TM, and SalmoFresh^TM, and USDA-approved products include Ecolicide^®, SalmoFresh^TM, and Finalyse^® [278,279,280]. Ten commercial phage preparations have earned GRAS status from the FDA, ensuring that their endotoxin content is below 250 endotoxin units per milliliter [281]. For phage therapy solutions administered intravenously, the permissible endotoxin level is set at 5 EU per kilogram of body weight per hour [282,283]. Given the empirical nature of phage dosing, further research is necessary to fully understand the pharmacokinetics of phage biopreparations [282]. Some commercial phage biopreparations are Kosher- and Halal-certified and are approved for use in organic food production [278]. The Polish company Proteon Pharmaceuticals S.A. (Łódź, Poland) also develops and markets phage biopreparations for use as feed additives in animal husbandry [79]. One of their products, Bafasal^®, containing four strictly lytic bacteriophages specific to S. enterica serovar Gallinarum strain B/00111, has received a positive opinion from the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), at the request of the European Commission. Bafasal^® is designed to be used as a zootechnical additive in drinking water and complementary liquid feed for all bird species [284,285].

In 2016, the European Food Safety Authority (EFSA) published a report [286] confirming the toxicological safety and efficacy of Listex™ P100 by Micreos Food Safety, while recommending further studies to assess the preparation’s effectiveness. In the United States, when Listex™ P100 (targeting L. monocytogenes) is used on ready-to-eat products, meat, and poultry, the manufacturer is not required to include information about the preparation on the food label, provided permissible doses are followed. The approved limit is up to 10⁹ PFU g⁻¹, with a maximum of 50 ppm of potassium lactate (as Listex™ P100 contains potassium lactate) [287,288]. While Listex™ P100 is a single-phage preparation (utilizing phage P100), another commercially available biopreparation for eliminating L. monocytogenes is ListShield™ produced by Intralytix, Inc. This product contains six bacteriophages: LIST–36, LMSP–25, LMTA–34, LMTA–57, LMTA–94, and LMTA–148. ListShield™ can be used as a food biopreservative and for food processing in products like fish and shellfish, fresh and processed fruits, vegetables, and dairy products at a concentration of 10⁶ PFU g⁻¹ [275,289].

In October 2023, the European Medicines Agency (EMA) published the “Guideline on quality, safety and efficacy of veterinary medicinal products specifically designed for phage therapy” [290], while in August 2024, the EFSA issued the “EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain” [291], which opens up the possibility of using phage biopreparations in the agri-food industry in the future.

5. Conclusions

Bacteriophages play a dual role in the food industry—acting as both beneficial agents and potential threats. On the positive side, their usefulness is most evident in ensuring food safety, especially for minimally processed plant- and animal-based products. Phages applied in biocontrol, bio-sanitation, and biopreservation effectively combat pathogenic and spoilage bacteria, helping to extend shelf life and reduce the need for chemical preservatives. Moreover, thanks to their high specificity, they do not harm beneficial microorganisms and can target antibiotic-resistant strains, making them a valuable tool in the fight against antimicrobial resistance.

However, phages also have a dark side. In fermentation industries, particularly in dairy production, their presence poses a serious threat. By infecting starter cultures, phages can slow down or completely halt fermentation processes, resulting in reduced product quality or spoilage. Many dairy plants must invest in costly measures to limit the risk of phage contamination in production lines. Additionally, not all phages are suitable for industrial applications—temperate phages carry the risk of transferring antibiotic resistance or virulence genes, so only strictly lytic phages, which lead to complete bacterial cell lysis, should be used for biocontrol purposes.

In summary, bacteriophages can be both friends and foes of the food industry. With proper selection, quality control, and advanced purification technologies, their potential as natural, effective biocontrol agents clearly outweighs the risks. As a result, they may become an important part of strategies to enhance food safety, reduce chemical use, and address the global challenge of antibiotic resistance.