Antimicrobial Agents of Microbial Origin

Biopreservation can be defined as the extension of shelf life and food safety by the use of natural or controlled microbiota and/or their antimicrobial compounds (Angiolillo et al., 2014). Lactic acid bacteria (LAB) have antagonistic properties that make them particularly useful as biopreservatives.

When LAB compete for nutrients, their metabolites often include active antimicrobials such as lactic and acetic acid, hydrogen peroxide, and peptide bacteriocins. LAB bacteriocins can be used for biopreservation and their combination with other preservative techniques could be a way to control spoilage bacteria and other pathogens, and inhibit the activities of a wide spectrum of organisms, including inherently resistant Gram-negative bacteria. Bacteriocins can be classified into five major classes:

  • • Class I: Lantibiotics, which are small (2-5 kDa), contain lanthionine (e.g., nisin)
  • • Class II: unmodified, which are small (<5 kDa), also called Listeria active peptide (e.g., Pediocin PA1, and Leucocin A)
  • • Class III: Large (>30 kDa), heat labile and are least characterized group (e.g., Helvetin J and Enterolysin)
  • • Class IV: complex, composed of protein plus one or more chemical compound (lipid or carbohydrate) (e.g., Leuconosin S, and Lactococcin 27)
  • • Class V: cyclic, head-to-tail cyclic backbone (e.g., Gassericin A, and AS-48) (Cleveland et al., 2001).

Most of the LAB bacteriocins identified so far are thermostable cationic molecules that have up to 60 amino acid residues and hydrophobic patches. Electrostatic interactions with negatively charged phosphate groups on target cell membranes are thought to contribute to the initial binding, forming pores and killing the cells after causing lethal damage and autolysin activation to digest the cellular wall. LAB bacteriocins, have many attractive characteristics that make them suitable candidates for use as food preservatives:

  • • Protein nature, inactivation by proteolytic enzymes of gastrointestinal tract
  • • Nontoxic to laboratory animals tested and generally nonimmunogenic
  • • Inactive against eukaryotic cells
  • • Generally thermo-resistant (with antimicrobial activity after pasteurization and sterilization)
  • • Broad bactericidal activity affecting most of the Gram-positive bacteria and some, damaged, Gram-negative
  • • Genetic determinants generally located in plasmid, which facilitates genetic manipulation to increase the variety of natural peptide analogues with desirable characteristics

The generally recognized as safe (GRAS) bacteriocin nisin produced by Lactococcus lactis subsp. Lactis isolated from milk was the first antibacterial peptide described in LAB active against Gram-negative and Gram-positive bacteria. Nisin is a peptide composed of 34 amino acid residues, classified as a class-Ia bacteriocin or lantibiotic (Cleveland et al., 2001). To date, it is the only bacteriocin that has been approved by the World Health Organization for use as a food preservative. Nisin has been shown to be effective in the microbial control of a number of dairy products, and its use has been widely assessed in cheese manufacturing at low pH. The use of nisin producing and nisin resistant starter cultures appears to be a viable means of incorporating and maintaining this bacteriocin, through the cheese-making process, to control foodborne pathogenic and spoilage bacteria.

Pinto et al. (2011) used nisin at different concentration (0, 100 and 500 IU mL) against S. aureus in Minas traditional serro cheese, that is a traditional semi-hard Brazilian cheese manufactured with raw milk. Nisin effectively reduced S. aureus count with a 1.2 and 2.0 log reduction cycles.

Nisin was added also to a chilled high-fat milk pudding dessert, one of the most popular desserts in Japan, previously inoculated with spores of Bacillus thuringiensis, by Oshima et al. (2014). They showed that nisin A inhibited spiked bacteria.

Nisin and other bacteriocines can be applied directly by incorporation in liquid systems (Gallo and Jagus, 2006) or in the case of food solid surfaces, using different techniques like spraying, dipping, or brushing. According to Ture et al. (2011), direct application of additives can have limited benefits because a loss of activity should be present due to the interaction or reaction with other additives or components present in the food matrix. Incorporation of antimicrobials in food interfaces by means of the use of edible films where they are entrapped helps to decrease the rate of diffusion from the surface to the bulk of the product (Kristo et al., 2008). For these reasons, Martins et al. (2010) used an edible coatings made of galactomannans from Gleditsia triacanthos with nisin against L. monocytogenes in ricotta cheese. Results showed that the cheese coated with nisin-added galactomannan film presented the best results in terms of microbial growth delay. Sakacin C2 is a novel bacteriocin with a broad inhibitory spectrum secreted by Lactobacillus sakei with an antimicrobial activity against some food- borne spoilage and pathogenic bacteria, including not only Gram-positive but also Gram-negative bacteria such as E. coli and S. typhimurium. Yurong et al. (2012) studied the effects of chemical composition including milk fat, the addition of emulsifiers and preservatives, and homogenization on activity of sakacin C2 against E. coli in milk, laying the groundwork for the use of sakacin as a biopreservative in dairy products. They found that milk fat in pasteurized and homogenized milk products (low-fat milk and whole milk) decreased the activity of sakacin C2 against E. coli ATCC 25922. Although underutilized in the majority of cases, some enterocins (class II batteriocin) produced by enterococci are among the most active bacteriocins in combating L. monocytogenes. However, direct application of enterocins may result in decrease or the complete loss of antimicrobial activity due to problems related to interaction with food components (Chollet et al., 2008). Alternatively, the incorporation of live bacteriocin-producing strain(s), either through direct addition to the food or in an immobilized form on packaging, may present a potential benefit in controlling L. monocytogenes in dairy products.

Coelho et al. (2014) discovered that bacteriocin activity was only detected in the whey of fresh cheese inoculated with two Enterococcus strains, but all cheeses made with bacteriocin-producing strains inhibited L. monocytogenes growth in the agar diffusion bioassay. To test the effect of in situ bacteriocin production against L. monocytogenes, fresh cheese was made from pasteurized cows' milk inoculated with bacteriocin-producing LAB and artificially contaminated with approximately 106 CFU/mL of L. monocytogenes. All strains controlled the growth of L. monocytogenes, although some Enterococcus were more effective in reducing the pathogen counts. After 7 days, this reduction was of approximately 4 log units compared to the positive control. In addition to bacteriocins, LAB exert their antimicrobial activity by their fermentation activity and with the production of other substances with antimicrobial action.

Angiolillo et al. (2013) stated that the addition of Lactobacillus rhamnosus in an edible sodium alginate coating applied on the surface of Fior di latte cheese exerted an antimicrobial activity against Pseudomonas and Enterobacteriaceae. In another study, Angiolillo et al. (2014) elaborated a sodium alginate coating containing Lactobacillus reuteri in combination with glycerol applied on the surface of Fior di latte cheese in order to extend its shelf life by means of in situ production of reuterin. They demonstrated that the active coating with L. reuteri was effective in prolonging Fior di latte microbial quality improving also its final taste.

Some strains of L. reuteri have been recognized for their ability to produce reuterin (b-hydroxypropionaldehyde; b-HPA) during anaerobic metabolism of glycerol (Rodriguez et al., 2003). Reuterin is an antimicrobial compound soluble in water, resistant to heat and stable over a wide range of pH values, that inactivates Gram-negative and Gram-positive bacteria (Vollenweider et al., 2003). Direct addition of reuterin to control foodborne pathogens such Salmonella spp., Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus has been investigated in milk and dairy products (Arques et al., 2008). Natamycin (or pimaricin) is a polyene antifungal antibiotic produced by Streptomyces natalensis. It is used to control fungus growth in the surface of most cheese and is not effective against bacteria or viruses. Natamycin has been used for many years in a large number of countries throughout the world as an authorized preservation treatment for cheeses. It is commonly employed in dairy-based food products to prevent yeast and mold contamination (El-Diasty et al., 2008).

Moreira et al. (2007) incorporated natamycin in a cellulose-based film and evaluated the antimicrobial efficiency of natamycin-incorporated film in the production process of Gorgonzola cheese. They found that films with 2% and 4% natamycin presented satisfactory results for fungus inhibition. Ture et al. (2011) studied the effect of wheat gluten (WG) and methyl cellulose (MC) biopolymers containing natamycin on the growth of A. niger and P. roquefortii on the surface of fresh kashar cheese during storage at 10°C for 30 days. Wrapping of A. niger-inoculated cheese with MC films containing 5-20 mg NA per 10 g resulted in approximately 2-log reductions in spore count. Two mg NA per 10 g included into WG films was sufficient to eliminate A. niger on the surface of cheese.

Fajardo et al. (2010) studied chitosan coatings containing natamycin on the physicochemical and microbial properties of semi-hard Saloio cheese. They proved that nata- mycin-coated samples presented a decrease on molds/yeasts of 1.1 log10 CFU g-1 compared to control after 27 days of storage. Dairy propionic bacteria are commercially important in the production of the “eye” and typical flavors in Swiss-type cheeses. They also produce organic acids, leading to an effective “natural” antifungal ingredient that could be used in dairy industry. Tawfok et al. (2004) added lyophilized P thoenii P-127 metabolites by 1.5% to Domiati cheese milk with a consequent prolongation of soft cheese shelf life.

Natural antimicrobials are rarely used as single compounds; they are usually used in combination with others to provide hurdles for the growth of microorganisms without affecting sensorial and nutritional characteristics. Pires et al. (2008) used a combination of nisin and natamycin incorporated into a cellulose-based film and applied on mozzarella cheese against molds and yeasts, Staphylococcus sp. and psychrotrophic bacteria.

By the ninth day of storage at 12° ± 2°C, the count of yeasts and molds on cheese covered with the antimicrobial film decreased 2 log10 units compared with the count on cheese with control film. In a study, Resa et al. (2014) evaluated the effectiveness of natamycin and nisin supported in tapioca starch films against Saccharomyces cerevisiae and L. innocua in a mixed culture present on the surface of a model system and of Port Salut cheese was evaluated. It was observed that the preservatives incorporated in starch films controlled growth of both microorganisms present together on the surface of the cheese during storage.

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