Status, Antimicrobial Mechanism, and Regulation of Natural Preservatives in Livestock Food Systems

Plant polyphenol extracts have been used as natural meat preservatives, including extracts from oregano, cranberry, sage, rosemary, grape seed, and others. Polyphenols can act as reducing agents and metal ion chelators in the presence of various hydroxyl radicals.

Oregano and cranberry extracts were evaluated for antimicrobial activity against L. monocytogenes in laboratory media, beef, and fish (Lin et al., 2004). These phenolicbased plant extracts are widely used in food preparation and are classified as Generally Regarded as Safe (GRAS). The effects of neem oil on the meat pathogens Carnobacterium maltaromaticum, Brochothrix thermosphacta, E. coli, and Pseudomonas fluorescens, were investigated as a preservative for fresh retail meat (Del Serrone et al., 2015a, Del Serrone et al., 2015b). Citrus species extracts were investigated as antifungal agents against spoilage fungi including Mucor sp. and Rhizophus sp. (Mohanka and Priyanka, 2014). Ethanol extract of Citrus species showed a higher antifungal effect than water extract did, and the minimum inhibitory concentration of the extract ranged from 6.25 to 25 mg/mL. Inula britannica ethanol extract showed an antimicrobial effect against five B. cereus strains in low fat milk, and the antimicrobial effect depended on terpene and polyphenol compounds (Lee et al., 2012). Brassica juncea extract showed an antiviral effect against influenza virus A/H1N1 in nonfat milk (Lee et al., 2014). Chestnut inner shell extract containing gallic acid and quercetin was shown an antimicrobial effect against C. jejuni in chicken meat at 1 and 2 mg/mL (Lee et al., 2016). Eight different flavonoids [quercetin, kaempferol, apigenin, luteolin, 5,4-dihydroxy-7-methnozyflavone (genkwanin), narigenin, hesperetin and hesperidin] were tested for antimicrobial effects against B. cereus strains (P14 and KCCM 40935) (Lee et al., 2011). Among these flavonoids, only kaempferol and apigenin were inhibitory, and kaempferol showed the greatest antimicrobial effect at 100 μM.

Essential oil or terpenes are secondary metabolites that provide flavor and aroma. The addition of adding essential oils from marjoram and rosemary was investigated in beef patties (Mohamed and Mansour, 2012). These essential oils were beneficial for antioxidant activity and sensory evaluation.

Plant antimicrobial peptides (pAMPs) were discovered in 1942 as natural defense compounds against pathogens (Hintz et al., 2015). The pAMPs were named as thionins, plant defensins, lipid transfer proteins (LTPs), myrosinase-binding proteins (MBPs), hevein- and knottin-like peptides, snakins, cyclotides, and peptides from hydrolysates. These substances have been isolated from Triticum aestivum (wheat), Impatients balsamina, Hordeum vulgare (barley), Arabidopsis thaliana, Hevea brasiliensis, Solanum tuverosum (potato), Oldenlandia offinis, etc.

The antimicrobial mechanisms of phenol compounds depend on their concentration. Phenols affect enzyme activity related to energy production at low concentrations; however, they cause protein denaturation at high concentrations (Fig. 1). These abilities affect microbial cell permeability, thereby interfering with membrane function (material transport, nucleic acid synthesis, and enzyme activity) (Bajpai et al., 2008; Fung et al., 1977; Rico-Munoz et al., 1987). The high antibacterial activity of phenolic compounds can be due to alkyl substitution into the phenol nucleus, forming phenoxy radicals, which does not occur in more stable molecules such as the ethers myristicin or anethole (Dorman and Deans, 2000; Gutierrez et al., 2008). Catechol and pyrogallol possess phenolic toxicity to microorganisms through enzyme inhibition by oxidized compounds, possibly by reacting with sulfhydryl groups or through more nonspecific interactions with proteins (Mason and Wasserman, 1987). The antimicrobial targets of quinones may include surface-exposed adhesins, cell wall polypeptide, and membrane-bound enzymes (Cowan, 1999). The antimicrobial activities of isothiocynates derived from Brassicaceae vegetables, such as cauliflower, broccoli, mustard, and cabbage are related to 1) loss of cell membranes integrity, 2) inhibiting enzyme or regulatory activity by quorum sensing (in Helicobacter pylori, Pseudomonas aeruginosa, Chromobacterium violaceum, etc.), 3) inhibition of respiratory enzymes, 4) induction of heat-shock and oxidative stress, and 5) induction of a stringent response (Dufort et al., 2015). Carvacrol, (þ)-carvone, thymol, and trans-cinnamaldehyde decrease the intracellular ATP (adenosine triphosphate) content of E. coli O157:H7 cells (Helander et al., 1998).

Fig. 1. Antimicrobial mechanisms of natural preservatives. AMPs, antimicrobial peptides; pAMPs, plant antimicrobial peptides.

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Essential oils have multiple cellular targets. Their hydrophobicity results in reactions with lipids on bacterial and fungal cell membranes, increasing membrane permeability and disturbing the original cell structure (Hintz et al., 2015; Pinto et al., 2009). In addition, antiviral effects are achieved by inhibiting viral protein synthesis at multiple stages of viral infection and replication (Wu et al., 2010).

The antimicrobial mechanism of most pAMPs involves cell membranes of targeted organisms and is driven by net positive charge, flexibility, and hydrophobicity to enable interaction with bacterial membranes (Jessen et al., 2006). Their antifungal mechanisms are involved in cell lysis, interference with fungal cell wall synthesis, permeabilization, binding to ergosterol/cholesterol in the membrane, depolymerization of the actin cytoskeleton, and targeting intracellular organelles such as mitochondria. Antiviral activity is also related to viral adsorption and entry processes.

Lysozyme is obtained from chicken egg whites, and is known as a bacteriolytic enzyme. Lysozyme has been used commercially under the name Inovapure, and can be used against a wide range of food spoilage organisms for extending the shelf life of various food products including raw and processed meats, cheese, and other dairy products (Tiwari et al., 2009).

The lactoperxoidase is a naturally active enzyme in milk with strong antimicrobial effects against both Gramnegative and -positive bacteria (de Wit and van Hooydonk, 1996; Russell, 1991) and fungi. Lactoperoxidase catalyzes the hydrogen peroxide (H₂O₂) oxidation of several acceptors; it has been utilized in industries such as dairy, oral care, cosmetics, cancer, and viral infection.

Lactoferrin and ovotranferrin are glycoproteins derived from bovine milk and hen egg respectively, that can bind iron, thereby restricting or preventing bacterial growth. Lactoferrin shows strong antimicrobial effects against various Gram-negative and -positive bacteria, fungi, and parasites in neutral pH and refrigeration temperature (Al-Nabulsi and Holley, 2005). Ovotranferrin peptide fragment OTAP-92 has strong bactericidal activity against both S. aureus and E. coli strains through membrane damage (Ibrahim et al., 2000). However, these transferrin peptides cannot be utilized in food systems because of their high cost.

AMPs are widely distributed in nature and are essential components of nonspecific host defense systems (Park et al., 1997; Tossi et al., 2000). The AMPs produced by animal cells include magainin (Zasloff et al., 1988), MSI-78 (Ge and Yan, 2002), PR-39 (Shi et al., 1996), pleurocidin (Cole et al., 1997), and dermaseptin S4 (Mor and Nicolas, 1994). AMPs are considered a promising solution for antibiotic resistance because of their non-specific molecular targets and fast membrane destruction. Pleurocidin is isolated from the winter flounder (Pleuronectes americanus) is active against Gram-negative and -positive bacteria (Cole et al., 2000). It is stable in heat and salt; however, it is unstable in supraphysiological concentrations to magnesium and calcium. An antimicrobial effect of pleurocidin was reported in foodborne organisms including Vibrio parahemolyticus, L. monocytogenes, E. coli O157: H7, Saccharomyces cerevisiae, and Penicillium expansum (Burrowes et al., 2004). Defensins are widely found in mammalian epithelial cells from chicken, turkey, and others (Brockus et al., 1998). Their antimicrobial spectrum included Gram-negative and -positive bacteria, fungi, and enveloped viruses (Ganz, 2003; Murdock et al., 2007).

Chitosan is a natural biopolymer obtained from the exoskeletons of crustaceans and arthropods, and has been used as an antifungal and antimicrobial agent (Ben-Shalom et al., 2003; Je and Kim, 2006; Liu et al., 2006). Chitooligosaccharides have a bacteriostatic effect on Gramnegative bacteria, E. coli, Vibrio cholera, Shigella dysenteriae, and Bacteriodes fragilis (Benhabiles et al., 2012). Chitosan (0.25, 0.5, and 1%) was studied as an antimicrobial ingredient in fresh pork sausage (Bostan and ’Isin Mahan, 2011).

Lipids of animal origin have antimicrobial activity against a wide range of microorganisms. Free fatty acids at mucosal surfaces have been shown to inactivate S. aureus (Bibel et al., 1989). Milk lipids are active against Gram-positive bacteria including S. aureus, C. botulinum, B. subtilis, B. cereus, and L. monocytogenes, and Gramnegative bacteria such as P. aeruginosa, E. coli, and Salmonella enteritidis (Isaacs et al., 1990; Lampe et al., 1998; Wang and Johnson, 1997).

AMPs, transferrins, and lipids can influence cell membranes and peptide synthesis (Fig. 1) (Brogden, 2005; Zasloff, 2002). AMPs can interact directly with the microbial cell membrane and result in the leaching out of vital cell components (Cole et al., 2000; Hancock, 1997). Lipids mainly inhibit bacterial cell walls or membranes, intracellular replication, or intracellular targets. Lysozymes inhibit bacterial cell membranes by hydrolyzing β-1,4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine in bacterial peptidoglycan.

Lactic acid bacteria produce antimicrobial compounds like organic acids, diacetyl, hydrogen peroxide, and proteinaceous bacteriocins (Lee et al., 2013). Bacteriocins are antimicrobial peptides or proteins produced by mainly lactic acid bacteria; these compounds are small and ribosomally synthesized. Most bacteriocins have potential as food preservatives because of their antimicrobial effect against food pathogens. The representative bacteriocins are nisin, diplococcin, acidophilin, bulgaricin, helveticin, lactacin, pediocin, and plantarin. Of these bacteriocins, nisin and pediocin have been used as commercial natural preservatives.

Nisin is a representative bacteriocin produced by various Lactococcus lactis strains, and has activity against food pathogens including Alicyclobacillus spp., L. monocytogenes, Bacillus spp., Micrococcus spp., Clostridium spp., Pediococcus spp., Desulfotomaculus spp., S. aureus, Enterococcus spp., Streptococcus haemolyticus, Lactobacillus spp., Sporolactobacillus spp., and Leuconostoc spp. Nisin is proteinaceous polypeptide that is most stable in acidic conditions. Nisin is soluble in aqueous conditions and is unstable in alkali solutions and heat. It has been used in various food products alone or in combination with other compounds. Nisin is the most widely used bacteriocin approved by the FDA as a food preservative. Dairy and meat products are applied with doses of 50-200 mg/kg. In the USA, nisin is used to inhibit outgrowth of C. botulinum spores and toxin formation in pasteurized processed cheese spread with fruits, vegetables or meats with a limited dose of about 250 ppm in finished products.

Pediocin is GRAS bacteriocin produced by strains of Pediococcus acidilactici (AcH, PA-1, JD, and 5) and P. pentosaceus (A, N5p, ST18, and PD1) (Anastasiadou et al., 2008). Most pediocins are stable in heat and a wide range of pH vlues. Pediocin AcH is effective against both spoilage and pathogenic organisms, including L. monocytogenes, Enterococcus faecalis, S. aureus, and Clostridium perfringens (Bhunia et al., 1988).

Natamycin is an antifungal substance produced by Streptomyces natalensis that is effective against almost all molds and yeasts; however, it has little or no effect on bacteria (EFSA, 2009). Natamycin has been used in dairy, meats, and other foods for antifungal effects, and its use as a surface preservative is regulated (E 235).

Reuterin (β-hydroxypropionaldehyde), an antimicrobial compound produced by Lacotobacillus reuteri, is a watersoluble nonproteinaceous metabolite of glycerol (Axelsson et al., 1989). Its broad antimicrobial spectrum includes Gram-negative and -positive bacteria, yeasts, and filamentous fungi (Nom and Rombouts, 1992).

The antimicrobial mechanism of bacteriocin involves pore formation in the cytoplasmic membrane of target microorganisms (Fig. 1). This leads to cell death by loss of intracellular molecules and a collapse of the proton motive force (Driessen et al., 1995). Bacteriocin originating from Gram-positive bacteria is only effective for Grampositive bacteria, and is less effective on Gram-negative bacteria due to their selective membrane permeability (Lee et al., 2003). These disadvantages could be compensated for by using other preservatives and preservative methods.

Natamycin has an antimicrobial effect by binding to ergosterol, a cell membrane sterol, in fungal membranes (EFSA, 2009). The structure of natamycin contains a large lactone ring with a rigid lipophilc chain containing conjugated double bonds, and a flexible hydrophilic portion bearing several hydroxyl groups. The hydrophobic groups form a polar pore with ergosterol in the membrane, and this complex affects material passage (K⁺, H⁺, amino acids, and other metabolites) (Deacon, 1997).