Application of Bacteriocins in Biopreservation of Salami

LAB are considered to be normal microflora of fermented meat products and bac- teriocins from LAB have been tested for application in biopreservation of salami in only a limited number of studies.

One of the first reports in Brazil on bacteriocinogenic LAB, was by Maciel et al. (2003). In this work, bacteriocinogenic LAB isolated during the processing of Italian salami, obtained from two different processing plants, in the State of Parana were reported. A total of 484 isolates were tested for their antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, Salmonella enteritidis and Escherichia coli. From these samples, 115 isolates inhibited at least two of the pathogens. However, the authors of this study have not gone for a deeper and systematic study of the produced antimicrobial agents, and therefore it is only possible to speculate on a potential production of bacteriocin/s or other antimicrobial compounds.

Barbosa et al. (2015) isolated Lactobacillus curvatus from salami produced in Brazil and tested its potential biopresevation properties with the control of Listeria monocytogenes contaminants of salami. Sudirman et al. (1993) isolated Lactobacillus spp. strains obtained from semi-dry sausages; Cintas et al. (1995) reported LAB isolates from Spanish dry-fermented sausages. Aymerich et al. (2000) failed in the isolation of LAB from fuet, chorizo and salchichon. Belgacem et al. (2008) reported on LAB isolated from gueddid, a Tunisian fermented meat. In the work of Carvalho et al. (2006), anti-listerial activity of LAB isolated from salami and sausages and the study of the development of anti-Listerial resistance was evaluated. Vermeiren et al. (2004) obtained LAB from meat products. Todorov et al. (2013) reported on Lactobacillus sakei isolated from Portuguese fermented meat products. Todorov et al. (2007) reported the application of Lactobacillus plantarum in control batch of Listeria monocytogenes in salami prepared from game meat in South Africa. However, even if LAB are natural inhabitants of salami and have an important role in the fermentation processes and development of the organoleptical properties of salami, the role of the expressed bacteriocin/s during the maturation stage is difficult based on the negative effect of the limited conditions for production and expression of the bacteriocin/s. Some of these factors (nutritional, temperature, pH, presence of lipids, etc.) have been reviewed in detail by Favaro et al. (2015).

Despite the potential difficulties in the expression of bacteriocin/s during the fermentation and maturation of salami, some works have been showing the potential application of bacteriocinogenic LAB in control of food-borne pathogens in the final fermented products. Barbosa et al. (2015a) evaluated potential of bacteriocin produced Lactobacillus curvatus strain isolated from Italian-type salami on control of Listeria monocytogenes during manufacturing of salami in a pilot scale. In this study two isolates (differentiated by RAPD-PCR) showed activity against high numbers of Listeria monocytogenes in addition to several other Gram-positive bacteria. In addition, on the basic features of the expressed bacteriocins, Barbosa et al. (2015) performed a three-step purification procedure and indicated that both strains produced the same two active peptides (4457.9 Da and 4360.1 Da), homlogous to saka- cins P and X, respectively. Addition of the semi-purified bacteriocins produced by Lactobacillus curvatus MBSa2 to the batter for the production of salami, experimentally contaminated with Listeria monocytogenes (104-105 CFU/g), caused 2 log and 1.5 log reductions in the counts of the pathogen in the product after 10 and 20 days respectively, highlighting the interest in application of these bacteriocins to improve the safety of salami during its manufacture (Barbosa et al. 2015a).

As has been shown previously (Favaro et al. 2015), environmental conditions can interfere with the stability and survival of the bacteriocins producers when applied in the real food production systems. In order to protect the LAB and to have a better effect of the expressed bacteriocin/s, Barbosa et al. (2015b) encapsulated the bacteriocin producer Lactobacillus curvatus, previously isolated from salami. Lactobacillus curvatus was entrapped in calcium alginate and tested for functionality in MRS broth and in salami experimentally contaminated with Listeria monocytogenes AL602/08 (a meat isolate), during 30 days of simulating manufacture process conditions, including fermentation and maturation steps. The entrapment process did not affect bacteriocin production by Lactobcillus curvatus MBSa2 in MRS broth and in salami. Both, free and entrapped Lactobacillus curvatus MBSa2 caused reduction in a similar manner in the counts of Listeria monocytogenes AL602/08 in salami during the manufacture process (Barbosa et al. 2015b). The entrapment of Lactobacillus curvatus in calcium alginate did not effect bacteriocin production when strain was applied in salami. Consequently, no improvement in inhibition of Listeria monocytogenes in this meat product could be achieved, when compared to a free and encapsulated bacteriocin producer, however, entrapped cells showed better survival (Barbosa et al. 2015b).

In a different study Barbosa et al. (2014) evaluated bacteriocin potential of Lactobacillus sakei MBSa1, isolated from that produced in Brazil salami, including the genetic features of the producer strain. Expressed bacteriocin by Lactobacillus sakei MBSal exhibited heat and pH stability with remarkable activity against Listeria monocytogenes. However, the expressed bacteriocin MBSa1 did not inhibit the tested probiotic strains (e.g. Lactobacillus acidophilus La 5) nor starter cultures (e.g. Lactobacillus acidophilus La-14). This suggests an interesting potential for technological applications in fermented foods for control of Listeria, without affecting starter or probiotic cultures. Expressed bacteriocin was purified by cation-exchange reversed-phase HPLC, molecular mass (4303.3 Da) and amino acid sequence (SIIGGMISGWAASGLAG), similar to that recorded to sakacin A, and which determined maximal production of bacteriocin MBSa1 (1600 AU/ml) in MRS broth and occurred after 20 hours at 25°C. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins Ta, Tp, X, P, G and Q).

According to Castro et al. (2011), bacteria belonging to Lactobacillus species are common in fermented meat products. Particularly, Lactobacillus sakei is specially adapted to the meat environment and has already been used as a starter culture for the production of different meat products (Carr et al. 2002). Chaillou et al. (2005) reported on the determination of the complete genome sequence of the French sausage isolate of Lactobacillus sakei 23K, demonstrating that this particular strain has a specialized metabolic repertoire that may contribute to its competitive ability in these foods.

Based on the fact that Lactobacillus sakei can be a producer of different antimicrobial compounds, including lactic and acetic acids, diacetyl, hydrogen peroxide and bacteriocins, some Lactobacillus sakei strains possess interesting biotechnological potential application for food biopreservation (Carr et al. 2002). Numerous bacterio- cins produced by Lactobacillus sakei strains have been identified, such as sakacin A (Schillinger and Lucke 1989; Holck et al. 1992); sakacin M (Sobrino et al. 1992), bavaricin A (Larsen et al. 1993; Messens and de Vuyst 2002); sakacin P (Holck et al. 1994; Tichaczek et al. 1994; Vaughan et al. 2001; Urso et al. 2006; Carvalho et al. 2010), sakacin K (Hugas et al. 1995), bavaricin MN (Kaiser and Montville 1996), sakacins 5T and 5X (Vaughan et al. 2001), sakacin G (Simon et al. 2002), sakacin Q (Mathiesen et al. 2005), sakacin C2 (Gao et al. 2010) and sakacin LSJ618 (Jiang et al. 2012).

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