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Antimicrobial Agents of Animal Origin

Animals produce several antimicrobial compounds, as first line of defense, which are also found in products of animal origin such as, milk and eggs (Straus and Hancock 2006). The potential of several antimicrobials derived from animal sources as food preservatives is increasingly being reported. Some of the well-characterized antimicrobials of animal origin are described in this section.

Chitosan

Chitosan, a complex polysaccharide naturally present in the exoskeletons of crustaceans and arthropods, alternatively, can be produced from some fungi (A. niger, Mucorrouxii, P notatum) (Tayel et al., 2010). It has gained considerable attention for commercial applications in food (Leleu et al., 2011). Moreover, it has been found to be nontoxic, biodegradable, biofunctional, and biocompatible; moreover, several researchers reported that the chitosan has strong antimicrobial and antifungal activities (Hague et al., 2005; Kim and Rajapakse, 2005). The biological activity of chitosan depends on its molecular weight, deacetylation degree, chitosan derivatization, degree of substitution, length and position of a substituent in glucosamine units of chitosan, pH of chitosan solution, and, of course, the target organism. It is generally found that yeasts and molds are the most sensitive group to chitosan, followed by Gram-positive and Gram-negative bacteria (Aider, 2010). A study on the synergy of MAP and chitosan was conducted successfully on stracciatella cheese (Gammariello et al., 2011). In this work, the addition of different amounts of chitosan (0.010, 0.015, 0.020%) during cheese making, combined with MAP prolonged stracciatella cheese shelf life (about 7 days), if compared with the control sample (more or less 3 days). The results showed that the Pseudomonas spp. growth was inhibited by combination of chitosan with MAP.

Del Nobile et al. (2009a) studied an integrated approach to prolong the shelf life of Fior di latte cheese. The investigated strategy was based on the combination of chitosan in the manufacture, either coating or active coating (lysozyme and ethylenediamine tetraacetic acid disodium salt), combined with MAP. The authors reported that the integrated approach developed allowed us to obtain a significant shelf life prolongation to 5 days in comparison with the traditional packaging the latter showed a very short shelf life limited to more or less 1 day. The use of chitosan and extracts of lemon and sage was assessed during Fior di latte cheese making by Gammariello et al. (2010). The active compounds were added to working milk at different concentrations. The results highlighted that the presence of lemon extract and chitosan improved Fior di latte cheese shelf life, whereas, the addition of sage extract negatively affected the sensory properties. In particular, the chitosan at the concentration of 240 mg/Kg was effective against coliforms and Pseudomonadaceae.

Coma et al. (2002) investigated the effect of edible film based on chitosan matrix and tested her antimicrobial activity against L. monocytogenes. The chitosan film showed 100% of L. monocytogenes inhibition for at least 8 days. The latter results were validated on Emmenthal cheese samples using L. innocua as model strain. The results showed that the chitosan-free samples exhibited 10 times higher colony forming units of bacteria compared to chitosan-coated ones. Besides, after 132 hours of storage, no colonies were detected from chitosan-treated samples. The chitosan was used also as antifungal agent on Kariesch cheese, in this work the cheese was treated with 0.5% and 1.0% chitosan solutions. The authors found that the chitosan-treated cheese showed an improvement of shelf life extended up to the 18th day of storage with respect to the control. The results indicated that the application of chitosan on the Kareish cheeses improves the mycological quality, in fact, a reduction of two logaritmic cycles in the molds and yeasts counts was observed in cheese samples treated with chitosan 1% compared with the control at the end of storage period (El-Diasty et al., 2012). Di Pierro et al. (2011) conducted a study extending the shelf life of ricotta cheese by using a chitosan/whey protein coating. The data proved that the viable numbers of mesophilic and psychrotrophic microorganisms were significantly lower in coated ricotta cheese than in control samples; besides, the coating delayed the development of undesirable acidity, better maintained the texture, and did not seem to modify sensory characteristics. As reported by Duan et al. (2007), the antimicrobial activities of chitosan-lysozyme (CL) composite films and coatings were tested against microorganisms inoculated (L. monocytogenes, E. coli, P fluorescens) onto the surface of mozzarella cheese. Three different package applications (CL film, CL lamination on film, and CL coating) were assessed. The authors reported that the CL composite films and coatings showed the greatest antimicrobial— in fact, they significantly reduced the growth of the microorganisms inoculated and molds in mozzarella cheese, although they had a lesser antimicrobial effect on yeast.

Lysozyme

Lysozyme is an enzyme that is naturally present in avian eggs and mammalian milk and is generally recognized as safe (GRAS). It has been used both in the pharmaceutical and food industries. The white lysozyme of hen eggs is commonly used as a preservative for meat, meat products, fish, fish products, milk and dairy products, and fruits and vegetables (Cegielska-Radziejewska et al., 2009). It is well known that lysozyme is bactericidal against Gram-positive microorganisms (Boland et al., 2003), whereas it is essentially ineffective against Gram-negative bacteria, owing to the presence of a lipopolysaccha- ride layer in outer membrane. However, its effectiveness could be increased through the use of some chelating agents (EDTA) as membrane disrupting agents.

Different concentrations of lysozyme (0.25, 0.50, and 1.00 mg mL1) +50 mM of Ethylene-Diamine Tetraacetic Acid (EDTA), incorporated in a sodium alginic acid- based coating, were evaluated on Fior di latte cheese shelf life (Del Nobile et al., 2009b). The same concentrations of active compound were used in brine to package the controls. As reported by the authors, an increase in the shelf life (104%) was recorded for the coated samples, respect to controls packaged in brine without active compounds. This shelf life increase was slightly lower than that recorded with samples packaged in the active brine (151%), as a result of a more pronounced microbial proliferation. Conte et al. (2011) studied the effects of lysozyme/EDTA disodium salt (Na2-EDTA) combined with MAP on shelf life of burrata cheese. Three concentrations of enzyme (150, 250, and 500 mg/kg) were added to the burrata samples. The results confirmed that the combination of lysozyme/Na2-EDTA and MAP prolonged cheese shelf life; in particular, the enzyme at the highest concentration was effective against Pseudomonas spp.

Doosh and Abdul-Rahman (2014) examined the effect of hen egg white lysozyme (250 and 300 mg/kg) as a natural antimicrobial to prolong the shelf life of soft cheese made from buffalo milk. The results revealed that the enzyme gave better result at 300 mg/Kg concentration, contributing clearly to reduce the development of total count of bacteria also the count of psychrophilic bacteria and yeast and mould. The antimicrobial activity of lysozyme combined with EDTA against spoilage microorganisms (coli- forms and Pseudomonas spp.) of dairy products was confirmed by Sinigaglia et al. (2008). Mozzarella cheeses containing lysozyme (0.25 mg mL-1) and different amounts of Na2-EDTA (10, 20 and 50 mmol L-1) were studied. The authors found that the lysozyme and Na2-EDTA significantly inhibited the growth of coliforms and Pseudomonadaceae during the first 7 days of storage, whereas the lactic acid bacteria were not affected. An alginate/lysozyme nanomultilayer coating in Coalho cheese was evaluated (Medeiros et al., 2014). The authors found that an shelf life extension of cheese was obtained—in particular, mesophilic and psychrotropic microbial counts and the visual valuation of fungal contamination were also found to be lower on coated cheese than on uncoated cheese. The lysozyme, incorporated in zein and zein-wax composite films, was tested only or in combination with catechin and gallic acid against L. monocytogenes inoculated on fresh Kashar cheese. The results confirmed the antimicrobial effect of the enzyme; all lysozyme containing films prevented the increase of L. monocytogenes counts in Kashar cheese, but it was only the zein-wax composite films with sustained lysozyme-release rates which caused a significant reduction (-0.4 decimals) in initial microbial load. The mixture of catechin and gallic acid showed no considerable antimicrobial effect in cheese (Unalan et al., 2013). Lysozyme is also used to prevent the late gas blowing of hard cheeses that is caused by the growth of Clostridium tyrobutyricum in Gauda, Edam, Provolone and Emmentaler in United States (Currel and Dam-Mieras, 2014).

Lactoferrin

Lactoferrin is a multifunctional iron-binding protein belonging to the transferrin family and it is found on mucosal surfaces and in biological fluids, including milk, saliva and seminal fluid, indicating that it may play a protective role in the innate immune response. Lactoferrin and its peptides are a promising class of antimicrobial compounds in the fight against pathogenic microorganisms, including S. aureus, E. coli, and L. monocytogenes (Dionysius and Milne, 1997), Streptococcus mutans and Vibrio cholerae (Farnaud and Evans, 2003). The bactericidal function of bovine lactoferrin is partially the result of the direct interaction between the positive charged regions with anionic molecules present on the surface of some microorganisms, which causes an increase in the membrane's permeability, inflicting damage to the bacteria (Haversen, 2010).

The antibacterial activity of lactoferrin added at doses of 2% and 4% to Minas fres- cal cheese inoculated with S. aureus was estimated. Data revealed that the lactoferrin prevented the increase of the S. aureus population in the cheeses at the two concentrations tested, and the antimicrobial effect showed to be dose-dependent. Furthermore, initially the enzyme presented an action bacteriostatic, then a bactericidal action was observed after the 15th day of storage when lactoferrin was added at a concentration of 2%, and after the 8th day at the concentration of 4% (Santana da Silva et al., 2012). Another study on Minas frescal cheese-added lactoferrin (2 and 4 mg/g) was conducted by Inay et al. (2012). Psychotropic population in the cheeses added by lactoferrin did not differ from control cheese, demonstrating that no antimicrobial activity occurred in the products. Quintieri et al. (2012) studied antimicrobial efficacy of pepsin-digested bovine lactoferrin (LFH) on spoilage bacteria contaminating traditional high-moisture mozzarella cheese. These natural substances were effective when tested in vitro against five potential spoilage bacteria contaminating cold stored mozzarella cheese. Only the fraction containing lactoferricins, were effective against E. coli as the whole pepsin-digested hydrolysate. The LFH tested on cold-stored commercial mozzarella cheese delayed significantly the growth of pseudomonads and coliforms in comparison with the untreated samples. The pepsin- digested bovine lactoferrin was used also to prevent blue discoloration of mozzarella cheese caused by P. fluorescens (Caputo et al., 2015). The authors discovered that the mozzarella cheese samples treated with LFH and inoculated with a selected P fluorescens strain showed no pigmentation and changes in casein profiles up to 14 days of cold storage. Furthermore from day 5, the count of P. fluorescens spoiling strain was steadily ca. one log cycle lower than that of LFH-free samples. A plasma coating functionalized by bovine lactoferrin (LB) and lactoferricin (LfcinB) was studied to make an active packaging useful to control cheese spoilage by Pseudomonas. The LfcinB immobilized on plasma confirmed its higher antimicrobial activity with respect to BLF, against strains belonging to three Pseudomonas species (Quintieri et al., 2013). Shashikumar and Puranik (2011) used lattoferrin at different levels (10, 15 and 20 ppm) in Paneer, a traditional Indian milk. It was observed that, as the level of lactofer- rin in the product increased, there was a significant decrease in the bacterial growth when compared to the control.

Lactoperoxidase

Lactoperoxidase is a native enzyme in animal secretions such as saliva, tear-fluid, and milk. Lactoperoxidase catalyzes the oxidation of thiocyanate ion by peroxide into shortlived reactive oxidation products, such as hypothiocyanite anion (Garcia-Graells et al., 2000). This compound, in turn, oxidizes enzymes and other proteins in the bacterial cell membrane that have exposed sulfhydryl groups, which inhibit the growth of microorganisms (Kussendrager and Van Hooijdonk 2000). Lactoperoxidase system has been proposed as a biopreservative in milk and other products. Arques et al. (2008) studied the effect of lactoperoxidase combined with nisin and reuterin against L. monocytogenes and S. aureus in enhancing shelf and safety of cuajada, a semi-solid cheese manufactured in Spain. The most pronounced decrease in pathogen counts was achieved by the triple combination, which acted synergistically on the inactivation of L. monocytogenes and S. aureus in cuajada over 12 days at 10°C.

 
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