Use of a Bacteriocin-producer Lactobacillus Sakei for Fermented Sausages Production

The aim of this paper was the technological characterization of a Lactobacillus sakei strain able to produce the bacteriocin sakacin P. Experiments were conducted in vitro, using MRS-based medium, and in vivo, when the strain was inoculated as starter culture in real sausage fermentation. The results obtained underlined that the strain was able to grow in conditions that are commonly used in the production line, and only lactose and high concentrations of NaCl (5% w/v) affected the capability for bacteriocin production. When inoculated in sausages, it showed a good performance, being able to colonize rapidly the ecosystem. A high number of isolates, producing sakacin P, where found already from the third day of fermentation, and this number remained stable throughout the fermentation. The strain inoculated affected also the microbial trends, in fact total bacterial count and fecal enterococci showed a rapid decrease at the end of the fermentation. Introduction In the last decade, a new approach to food stabilization, based on the antagonism displayed by one microorganism towards another, was established linking the lactic acid bacteria (LAB) and protective cultures with biopreservation. According to Stiles (1996), biopreservation refers to extended storage life and enhanced safety of food using their natural or controlled microflora and (or) their antibacterial products. The microbial interference caused by LAB is due to the production of organic acids and the pH decrease, the competition for nutrients but it also correlates to the production of bacteriocins. Adding a pure culture of the viable bacteriocin-producing LAB to a food comodity represents an example of biopreservation. This practice offers an indirect way to incorporate bacteriocins in meat products and the success of the operation depends on the capability of the added strain to grow and produce the bacteriocin under the fermentation conditions. The production of a certain bacteriocin in laboratory media does not imply its effectiveness in a food system. When evaluating a bacteriocin-producing culture for sausage fermentation or biopreservation, it is important to consider that meat and meat products are complex systems with a number of factors influencing the microbial growth and the metabolite production. Therefore, the influencing of formula and fermentation technology on the performance of bacteriocin-producing strains needs to be tested (Hugas, 1998). Within the frame of the European project “Safety of traditional fermented sausages: research on protective cultures and bacteriocins”, contract n. ICA4-CT-2002-10037, we isolated a strain of Lb. sakei that possessed antimicrobial activity towards Listeria monocytogenes. It was determined that the bacteriocin produced was sakacin P (Urso et al., 2004). In this paper, the technological characterization of the strain, in connection with its bacteriocin production, was carried out, as well as the evaluation of its ability to conduct sausage fermentations. Materials and Methods The growth of Lb. sakei, and its capability to produce bacteriocin, was tested in different conditions resembling the fermented sausage production line. Temperatures of 10, 14, 18 and 25°C, pH values of 6.0, 5.7 and 5.4, NaCl concentrations of 2, 3.5 and 5% (w/v), glucose concentrations of 0.5, 1.0, 1.5% (w/v), lactose concentrations of 0.25, 0.5 and 1% (w/v) and sucrose concentrations of 0.5, 1.0 and 1.5% (w/v) were selected. Growth was followed by measuring the optical density (OD) of the cultures at 600 nm with the SmartSpec 3000 spectrophotometer (Biorad, Milan, Italy), while quantification of the bacteriocin was performed by the critical dilution method, as suggested by Barefoot and Klaenhammer (1983), using as indicator organism Listeria monocytognes, strain NCTC 10527. The experiments were performed twice and samples were collected in duplicates. Fermented sausages were prepared in a local meat factory using traditional techniques. The 200 kg batch was inoculated with about 7.5 x 10 cells/g. The ingredients were: pork meat (60 kg), lard (40 kg), a mix of sodium chloride (2.5 kg) and black pepper (70 g), sugars (1.5 kg), and nitrite/nitrate (200 ppm). After amalgamation, stuffing of natural casings produced fresh sausages 25 cm long and 5 cm in diameter. The ripening was performed as follows: the first stage consisted of 2 days drying with the relative humidity (RH) of 85% and a temperature of 22°C that was