**1.2 Antimicrobial agents as additives to active packaging materials**

Natural antimicrobial agents/compounds refer to a class of substances extracted from plants and animals or produced by microorganisms. These active agents may perform antagonistic actions against bacteria, viruses, yeast, or molds [18]. They also show anti-insect and antioxidant activity [19]. According to their biological origin, they can be divided into three categories: plant-derived antimicrobial agents, animal-derived antimicrobial agents, and microbial-derived natural antimicrobial agents. The food industry has used typically mainly chemical preservatives, such as hydrogen peroxide, sorbate, sorbic acid, benzoate, benzoic acid, and nitrite, to inhibit the growth of microorganisms responsible for food spoilage. Commercial preservatives may extend the shelf-life of food products; however, they may have unfavorable effects on the sensory properties of food [18, 20]. In order to extend the

shelf-life of food and reduce health hazards, natural antimicrobial compounds, such as essential oils, propolis, lactoferrin, glucose oxidase enzyme bacteriocins, and probiotics extracted from animals, plants or produced by microorganisms could replace typical chemical preservatives. They could be used by the food packaging industry due to their nontoxic character [18]. Essential oils (EOs) play an important role in the design of antimicrobial packaging materials, as they exhibit a high and specific antimicrobial efficacy against a broad range of foodborne pathogens [3]. EOs are extracted from leaves, bark, flowers, and seeds of aromatic plants and are "generally recognized as safe," (GRAS) [19]. Eos, such as thyme, clove, cinnamon and tea tree, peppermint, oregano, lemongrass, and citronella, are plant-derived compounds that exhibit promising inhibitory effects [3, 18, 21]. EOs from the *Myrtaceae* family such as cloves, eucalyptus, galbanum, thyme, and tea tree contain eugenol and terpinen-4-ol as major bioactive components. It has been reported that these active agents offer antifungal effects. The results indicate that they inhibit glycolysis, which in turn influences cell energy metabolism, therefore disturbing the normal physiological activity of fungal pathogens [21]. According to several studies, EOs from *Lauraceae* and *Lamiaceae* (e.g., thyme, rosemary, oregano, cinnamon, etc.), exhibit essential antibacterial activities against foodborne pathogens and are generally rich in phenolic compounds, such as carvacrol, thymol, cinnamaldehyde, or eugenol [20]. Typically, the antibacterial mechanism is based on chemical interaction with the bacterial membrane and mitochondria leading to altering of their permeability, destroying their structural order, resulting in a massive loss of cell contents, important ions and molecules, and eventually leading to the death of the cell [18, 20, 21]. Moreover, many studies have demonstrated that the minor components of EOs play an outstanding role in the antimicrobial activity of essential oil, probably by a synergistic effect with the other EO components. One known example is the synergistic interaction between p-cymene and carvacrol, while p-cymene barely inhibits bacterial cell growth. On the other hand, carvacrol itself has a proven antibacterial effect against a wide range of microorganisms. It has been shown that the growth of microorganisms was significantly inhibited by a mixture of p-cymene and carvacrol. Interestingly, this activity was significantly lower when each terpene acts separately on bacteria growth [20, 22]. To summarize, at present, according to their high antimicrobial activity, they are mainly essential plant oils that act as antimicrobial and antioxidant compounds and they are widely used in smart or bioactive packaging materials to prevent the surface growth of microorganisms in foods [18, 23]. Natural antimicrobial agents can be extracted from plants, such as black currant, apple, pomegranate, grape, and quince, as well as chokeberry, bilberry, raspberry, mulberry, blueberry, yerba mate, green tea, sour cherry, walnut, rosemary, thyme, cinnamon, oregano, cumin, and many others. Plant extracts contain a wide range of bioactive components that include, polyphenols, iridoids, amides, saponins, alkaloids, and glycosides, as well as tannins, terpenoids, and quinones, which all have been reported to have a broad spectrum of antimicrobial properties. [18, 24–27]. The generic and quantitative contents of active compounds in plants vary widely. There are many factors that influence the composition and concentration of the active substances in plants and plant extracts, such as the organ, cultivar, and many various growth conditions, including weather. These factors all have a significant influence on the antimicrobial effect of the plant extracts [26, 27]. There are three main antimicrobial action mechanisms by plant extracts: (a) the inhibition of cytoplasmic membrane function (the destruction of the cell membrane and membrane proteins and causing damage to the cell wall of the microorganisms);

### *Overview of Food Antimicrobial Packaging DOI: http://dx.doi.org/10.5772/intechopen.108666*

(b) inhibition of nucleic acid synthesis (extracts may suppress DNA synthesis by inhibiting DNA gyrase activity); (c) inhibition of energy metabolism (extracts can inhibit ATP synthesis). Finally, plant extracts may influence biofilm formation by influencing the quorum sensing mechanism, pigment production, and bacteria swarming motility, as well as altering the structure of the biofilm itself. Although active compounds from extracts can have an inhibitory effect at higher concentrations, in the case of lower concentrations they may have a stimulatory effect, indicating a bacterial defense mechanism [18, 26, 27]. Due to strong antimicrobial and antioxidant properties, plant extracts rich in active compounds, such as polyphenols, can be used within the food industry as natural preservatives and limit the nowadays use of chemical preservatives. They can also be used within the food packaging industry to extend the shelf life of food products by inhibiting microorganism growth and spoilage processes [18, 26, 27]. Antimicrobial peptides (AMPs) can be synthesized artificially in the laboratory or produced by bacteria [28]. Antimicrobial peptides are mostly composed of 12 ~ 60 amino acids that offer antimicrobial activity and participate in the host defense system [18, 28]. AMPs may be synthesized via three main methods: enzymatic synthesis, chemical synthesis, and biosynthesis, using a DNA recombinant technique. Among these methods, chemical synthesis is very common and has attracted increasing attention in the food packaging industry. AMPs obtained from bacteria are known as bacteriocins [28]. These AMPs can offer broad activity to directly inhibit the growth of yeasts, molds, bacteria, viruses, or even cancer cells [18, 28]. Typically, so-called antibacterial peptides (ABPs) have been mainly found to be active against bacteria. They have a clear influence on the bacterial cell membrane and create pores on their surface, resulting in the leakage of the intracellular matrix. They may also penetrate the cell membrane and interact with intracellular structures and disturb many activities including DNA/RNA or protein synthesis, resulting in bacterial cell death. ABPs are mainly positively charged molecules and they exhibit a high ratio of hydrophobic amino acids, allowing them to selectively bind to negatively charged bacterial membranes. The action mechanism of ABPs leads to the perforation of the cell membranes and their death [18, 29]. This is quite different from the bactericidal mechanism of antibiotics and does not lead to the microorganism becoming resistant. Furthermore, AMPs do not easily bind to mammalian cell membranes, which finally could be very harmful. It should be emphasized that antimicrobial peptides generally do not have any toxic side effects, though some have antioxidant functions and have been seen to scavenge free radicals. Lysostaphin and nisin are good examples of bactericidal peptides that suppress the growth of gram-positive bacteria. Antifungal peptides (AFPs) also function as antimicrobial proteins. They interact with the cell membrane, resulting in the disruption and finally death of the cell. Good examples of AFPs are echinocandin, defensin, and heliomycin [28]. As emphasized in the work of Ramos [30], innovative solutions have been proposed to improve the structural and functional properties of biopolymer-based packaging materials, including the incorporation of low amounts of specific nanoparticles (NPs) devoid of any relevant alteration of their migration to ensure their suitability for their application in food packaging. The authors stressed that the use of metallic-based nanoparticles (NPs), rather than antimicrobial organic agents, offered some advantages, such as high antimicrobial activity, lack of negative influence on food sensory properties, and compatibility with harsh polymer processing conditions, making NPs highly suitable for food spoilage control. Many nanomaterials have been used for food packaging, such as silver NPs, copper NPs, zinc oxide NPs, and titanium dioxide NPs, as well as silicon dioxide NPs or mixtures of antimicrobial agents containing NPs. Several studies have confirmed that Ag-NPs, ZnO-NPs, and TiO2-NPs are often used in the packaging industry as antimicrobial agents [4, 30–34]. Ag-NPs were found to exhibit antioxidant activity and offer antibacterial effects against grampositive bacteria, such as *S. aureus,* including methicillin-resistant *S. aureus* (MRSA), gram-negative *E. coli,* and *Pseudomonas aeruginosa* [35]. It has been shown that nano-biocomposite films based on PLA with modified cellulose nanocrystals (s-CNC) and Ag-NPs demonstrated high antimicrobial activity against *E. coli* and *S. aureus.* These materials showing homogeneous Ag dispersion in the polymer matrix, while also not affecting the PLA transparency, showed a significant improvement in barrier properties and antimicrobial activity [36, 37]. One should stress that consideration regarding human safety, as well as the environmental effects of packaging materials containing Ag-NPs in direct contact with food, have led several studies to report that these types of substances could be used for food preservation, due to their quality and safety [30, 36, 37]. The authors of several studies demonstrated that Ag nanoparticles migration levels were significantly below the legislative migration limits in Europe set by EU Regulation No. 10/2011 (for plastic materials intended to come into contact with food), such as in PLA nanocomposites and in poly(vinyl chloride) (PVC) [30, 38–40]. One of the most attractive nanoparticles that could be used in the packaging industry is nanosized ZnO. ZnO-NPs with unique morphologies, such as nanohelix and nanorings, may be easily synthesized and are cost-effective [32]. These nanoparticles have been explored as antimicrobial substances, used in antimicrobial food packaging as one of five various zinc compounds, which are regarded as being safe (GRAS) by the United States food and drug administration (USFDA, 21CFR182.8991) [41–43]. It was reported [32] that ZnO-NPs were highly reactive and induced reactive oxygen species (ROS), which causes single-stranded DNA breaks at a relatively low concentration of 10 mg/ml. Due to their high activity, ZnO nanoparticles offered a broad bactericidal effect on grampositive and gram-negative bacteria and bacterial spores that typically are resistant to high pressure and high temperature [44–46] as well as yeasts and molds (Noshirvani et al. 2017). An additional advantage of ZnO-NPs is their UV-blocking properties [32]. The application of nanoparticles can improve the UV-shielding of all respective packaging film materials [47–49]. TiO2 nanoparticles have been reported by several authors [33, 50] to exhibit antimicrobial activity when exposed to UV light by generating reactive oxygen and hydroxyl radical (OH•) species (ROS) on its surface, resulting in the oxidation of the polyunsaturated phospholipids of microorganism cell membranes. As a consequence, the microorganism was inactivated. TiO2 nanoparticles have been used to inactivate a wide spectrum of microorganisms, such as *E. coli, P. aeruginosa, Enterococcus faecalis, Cyanobacteria, Lactobacillus helveticus, Legionella pneumophila, Clostridium perfringens, Salmonella enterica Choleraesuis, Vibrio parahaemolyticus* and *L. monocytogenes* [50]. The process of microorganism species inhibition by CuO-NPs was influenced by the concentration and size of nanoparticles. The CuO-NPs were confirmed to be active against gram-negative and gram-positive bacteria by transiting the microorganism cell membrane and then destroying their enzymes. The antifungal action of CoO-NPs was also observed. It should be mentioned that copper is very important in the case of active packaging because of its high activity against a wide spectrum of microorganisms. When copper ions are attached to a microbial cell they immediately donate and accept electrons, as a result, they show increased redox ability and the capability to inactivate the cell components and kill them [51].
