Monitoring of a starter culture for fermented sausages by molecular methods

Abstract

The ability of a commercial starter culture to perform a sausage fermentation was evaluated by culture –dependent and –independent methods. The starter culture, as well as the sausage during fermentation, was sampled and strains of lactic acid bacteria (LAB) and coagulase negative cocci (CNC) were isolated. After identification it was determined that Lactobacillus plantarum, contained in the inoculated starter, was the main LAB representative that conducted the transformation, while Staphylococcus xylosus, not declared in the label of the starter culture, was able to colonize the sausages studied. Molecular characterization of isolated Lb. plantarum and S. xylosus highlighted that the commercial mix contained several strains of the same species, and their behavior during the fermentation was different. Analysis of the nucleic acids extracted directly from the sausages confirmed the performance of Lb. plantarum, which was present and active throughout the fermentation, and highlighted the contribution of Lactobacillus curvatus. Introduction For fermented sausage production, the starter cultures are represented by mixtures of lactic acid bacteria (LAB) and coagulase-negative cocci (CNC). LAB are the main population responsible for the pH drop, followed by a second step in which CNC are neutralizing the organic acids produced by LAB, through production of peptides and aminoacids from their proteolytic activity. In addition, CNC are able to induce the release of various aromatic substances due to their capability to produce lipases (Montel et al., 1996). Recently, direct amplification of DNA and RNA by polymerase chain reaction (PCR) followed by denaturing gradient gel electrophoresis (DGGE) (Muyzer et al., 1993) showed very good applicability in monitoring the microbial ecology of Italian fermented sausages (Cocolin et al., 2001; Rantsiou et al., 2005). In this paper we wanted to validate the use of a commercial starter for the production of fermented sausages by molecular methods. DGGE was used to analyze PCR and RT-PCR products obtained from DNA and RNA extracted directly from the sample to monitor the bacterial dynamics during transformation. Moreover, LAB and CNC were isolated both from the starter culture and during fermentation and subsequently subjected to RAPD analysis to understand if the strains inoculated were able to conduct the fermentation process considered in the study. Materials and Methods Fermented sausages were prepared in a local meat factory using traditional techniques as previously described (Comi et al., 2005) and inoculated with 20 g starter culture containing S. carnosus and Lb. plantarum (Biostart SL1-200, Wiesby GMBH Co., Niebull, Germany). The ripening was conducted for 28 days. The fermented sausages were analyzed in triplicates at 0, 3, 5, 7, 14 and 28 days. Potentiometric measurements of pH were made using a pin electrode of a pH-meter (Radiometer Copenhagen pH M82, Cecchinato, Italy). The starter culture (20 g), of the same batch that was used in the production followed, was resuspended in 200 ml sterile water and left at room temperature for 30 min and serial dilutions were prepared in saline/peptone water (8 g/l NaCl, 1 g/l bacteriological peptone, Oxoid, Milan, Italy) and analyzed on MRS agar (Oxoid) incubated with a double layer at 30°C for 48 h and on Mannitol Salt Agar (MSA, Oxoid) incubated at 30°C for 48 h. Fifteen colonies of LAB were isolated from MRS agar and the same number of CNC were selected from MSA agar. The fermented sausages were analyzed to monitor the dynamic changes in the populations responsible for the ripening of fermented sausages, as well as their hygienic quality. In particular, 25 g of each sample were transferred into a sterile stomacher bag and 225 ml of saline/peptone water were added and mixed for 1 min and 30 s in a Stomacher machine (PBI, Italy). Further decimal dilutions were made and the following analyses were carried out on duplicate agar plates: a) total bacterial count (TBC) on Gelisate Agar (Oxoid) incubated for 48–72 h at 30°C; b) LAB on MRS agar; c) CNC on MSA (Oxoid);