Rosalinda Urso

Culture-Dependent and -Independent Methods To Investigate the Microbial Ecology of Italian Fermented Sausages

ABSTRACT In this study, the microbial ecology of three naturally fermented sausages produced in northeast Italy was studied by culture-dependent and -independent methods. By plating analysis, the predominance of lactic acid bacteria populations was pointed out, as well as the importance of coagulase-negative cocci. Also in the case of one fermentation, the fecal enterocci reached significant counts, highlighting their contribution to the particular transformation process. Yeast counts were higher than the detection limit (>100 CFU/g) in only one fermented sausage. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns and sequencing of the bands allowed profiling of the microbial populations present in the sausages during fermentation. The bacterial ecology was mainly characterized by the stable presence of Lactobacillus curvatus and Lactobacillus sakei, but Lactobacillus paracasei was also repeatedly detected. An important piece of evidence was the presence of Lactococcus garvieae, which clearly contributed in two fermentations. Several species of Staphylococcus were also detected. Regarding other bacterial groups, Bacillus sp., Ruminococcus sp., and Macrococcus caseolyticus were also identified at the beginning of the transformations. In addition, yeast species belonging to Debaryomyces hansenii, several Candida species, and Willopsis saturnus were observed in the DGGE gels. Finally, cluster analysis of the bacterial and yeast DGGE profiles highlighted the uniqueness of the fermentation processes studied.

Characterisation of naturally fermented sausages produced in the North East of Italy.

In the Friuli Venezia Giulia region, in the North East of Italy, a traditional fermented sausage is produced without the use of microbial starters. It is characterized at the end of the ripening period by accentuated acidity, slight sourness and elastic, semi-hard consistency. In this study, three fermentations, carried out in different seasons (winter, spring and summer) were followed analyzing the microbiological, physicochemical and sensory aspects of this product. The sausages were characterized by an important microbial activity of lactic acid bacteria and micro/staphylococci that resulted in a product with a final pH of about 5.6-5.7. An interesting aspect was the high number of fecal enterococci that can play an important role in the definition of the organoleptic profile of the final product. No Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus were ever isolated from the raw materials or the fermented sausages during the maturation, underlining the safety of this product. The final water activity of the product was 0.91-0.92. One hundred and fifty lactic acid bacteria were isolated and identified by molecular methods to understand which species were more predominant in the product. Lactobacillus curvatus and Lactobacillus sakei were the most numerous (54 and 64 strains isolated, respectively) and they were the only species common to all three fermentations. A cluster analysis of the profiles obtained from these strains after RAPD-PCR highlighted a population distribution that was fermentation-specific.

Study of the Ecology of Fresh Sausages and Characterization of Populations of Lactic Acid Bacteria by Molecular Methods

ABSTRACT In this study, a polyphasic approach was used to study the ecology of fresh sausages and to characterize populations of lactic acid bacteria (LAB). The microbial profile of fresh sausages was monitored from the production day to the 10th day of storage at 4°C. Samples were collected on days 0, 3, 6, and 10, and culture-dependent and -independent methods of detection and identification were applied. Traditional plating and isolation of LAB strains, which were subsequently identified by molecular methods, and the application of PCR-denaturing gradient gel electrophoresis (DGGE) to DNA and RNA extracted directly from the fresh sausage samples allowed the study in detail of the changes in the bacterial and yeast populations during storage. Brochothrix thermosphacta and Lactobacillus sakei were the main populations present. In particular, B. thermosphacta was present throughout the process, as determined by both DNA and RNA analysis. Other bacterial species, mainly Staphylococcus xylosus, Leuconostoc mesenteroides, and L. curvatus, were detected by DGGE. Moreover, an uncultured bacterium and an uncultured Staphylococcus sp. were present, too. LAB strains isolated at day 0 were identified as Lactococcus lactis subsp. lactis, L. casei, and Enterococcus casseliflavus, and on day 3 a strain of Leuconostoc mesenteroides was identified. The remaining strains isolated belonged to L. sakei. Concerning the yeast ecology, only Debaryomyces hansenii was established in the fresh sausages. Capronia mansonii was initially present, but it was not detected after the first 3 days. At last, L. sakei isolates were characterized by randomly amplified polymorphic DNA PCR and repetitive DNA element PCR. The results obtained underlined how different populations took over at different steps of the process. This is believed to be the result of the selection of the particular population, possibly due to the low storage temperature employed.

Microflora of Feta cheese from four Greek manufacturers

The components of the microflora of four Feta cheeses, produced by different Greek manufacturers, were determined by culture dependent and independent techniques. Isolates from microbiological media were first grouped by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and then representatives of each DGGE group were sequenced for identification purposes. DNA and RNA, extracted directly from the cheese, were subjected to PCR-DGGE. Moreover, Feta cheeses were subjected to FISH analysis in order to identify viable bacterial populations. The microbial ecology, as represented by the Lactic Acid Bacteria (LAB) and yeast populations, was different for the four cheeses. The main LAB species isolated were Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus coryniformis and Lactobacillus fermentum. However, some inconsistencies were observed between the results obtained with the culture dependent and the culture independent approach. In the case of the yeasts, the results obtained by PCR-DGGE compared very well with those obtained by the conventional microbiological analysis and the main species found were Kluyveromyces lactis, Pichia fermentans and C. zeylanoides. FISH analysis highlighted viable but not culturable populations of Streptococcus thermophilus and Lactococcus spp. RAPD-PCR performed on the L. plantarum isolates revealed a cheese specific distribution and a temperature dependent clustering.

Yeast biodiversity and dynamics during sweet wine production as determined by molecular methods

In this study we investigated yeast biodiversity and dynamics during the production of a sweet wine obtained from dried grapes. Two wineries were selected in the Collio region and grapes, grape juices and wines during fermentations were analyzed by culture-dependent methods (plating on WLN medium) and culture-independent methods (PCR-DGGE). Moreover, the capability of the Saccharomyces cerevisiae starter cultures to take over the fermentation was assessed by RAPD-PCR. On WLN agar several species of non-Saccharomyces yeasts (Hanseniaspora, Metschnikowia, Pichia, Candida, Torulaspora and Debaryomyces), but also strains of S. cerevisiae, were isolated. After inoculation of the starter cultures, only colonies typical of S. cerevisiae were observed. Using PCR-DGGE, the great biodiversity of moulds on the grapes was underlined, both at the DNA and RNA level, while the yeast contribution started to become important only in the musts. Here, bands belonging to species of Candida zemplinina and Hanseniaspora uvarum were visible. Lastly, when the S. cerevisiae isolates were compared by RAPD-PCR, it was determined that only in one of the fermentations followed, the inoculated strain conducted the alcoholic fermentation. In the second fermentation, the starter culture was not able to promptly implant and other populations of S. cerevisiae could be isolated, most likely contributing to the final characteristics of the sweet wine produced.

Lactic acid bacteria ecology of three traditional fermented sausages produced in the North of Italy as determined by molecular methods.

In this study the bacterial biodiversity during the maturation process of three traditional sausages produced in the North of Italy (Salame bergamasco, Salame cremonese and Salame mantovano) was investigated by using culture-dependent and -independent methods. Eleven plants, in the three provinces considered here, were selected because starter cultures were never used in the production. The bacterial ecology, as determined by plate counts, was dominated by lactic acid bacteria (LAB), with minor contribution of coagulase negative cocci and yeasts. After molecular identification of 486 LAB strains, the species more frequently isolated were Lactobacillus sakei and Lactobacillus curvatus. This evidence was also confirmed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE). All the samples analyzed were characterized by the constant presence of L. sakei and L. curvatus bands. A richer biodiversity was only detected at the beginning of maturation. The results obtained by the molecular characterization of the L. sakei and L. curvatus and by the cluster analysis of the DGGE profiles highlighted a plant-specific population, rather than a geographic characterization of the products, underlining how the environmental and processing conditions are able to select specific microbiota responsible for the main transformations during the fermentation and ripening of the sausages.

Spoilage of blood sausages morcilla de Burgos treated with high hydrostatic pressure

In this study, the microbial ecology of the blood sausage morcilla de Burgos, subjected to high hydrostatic pressure treatment (HPP), was studied by culture-dependent and -independent methods. Morcilla de Burgos is the most traditional and famous blood sausage in Spain. The producers are interested in extending its shelf-life in order to expand their market and to reduce losses attributed to spoilage. Sausage batter prior to stuffing and blood sausages HPP treated or not (control) were analyzed at 0, 9, 14, 21, 28 and 35 days of storage at 4 degrees C. Lactic acid bacteria, Pseudomonas spp. and aerobic mesophilic bacteria were investigated by traditional plating. PCR-denaturing gradient gel electrophoresis (DGGE) was used to analyze the DNA and the RNA extracted directly from the blood sausages, as well as bulk cells of LAB and Pseudomonas spp. The results showed that HPP improved the shelf life of morcilla de Burgos to 28 days in comparison with control samples. The populations responsible for spoilage, namely LAB, remained lower in HPP treated samples when compared with the control samples. Only at 35 days of storage they reached values of 10(8) cfu/g, leading to the spoilage of the product. Although, HPP affected the LAB population, they were able to recover the injury provoked by the treatment. Lastly, HPP seemed to affect differently LAB species detected. While Leuconostoc mesenteroides was completely inactivated by HPP, Weissella viridescens was able to recover and carry out the typical spoilage of the product. Pseudomonas spp. remained under detection level (<10(2) CFU/g) after the HPP treatment.

Multiphasic approach to study the bacterial ecology of fermented sausages inoculated with a commercial starter culture.

ABSTRACT In this paper, the ability of a commercial starter culture to perform a sausage fermentation is evaluated. Molecular analysis revealed the presence of several strains of the same species contained in the starter culture with different behavior during the fermentation, and the contribution of Lactobacillus curvatus, which was only marginally isolated during the transformation.

Sequencing and expression analysis of the sakacin P bacteriocin produced by a Lactobacillus sakei strain isolated from naturally fermented sausages.

A Lactobacillus sakei strain, designated as I151 and isolated from naturally fermented sausages, was found to produce the sakacin P bacteriocin which is active against Listeria monocytogenes. In this study, we performed the sequencing of the gene cluster involved in the production of the sakacin P, and we followed the expression of the sppA gene, encoding for the bacteriocin, in vitro, using Rogosa–Sharpe medium, and in situ, inoculating the strain in fermented sausages as starter culture. The results obtained underlined the high similarity (>99%) of the entire sakacin P gene cluster from the L. sakei studied here with others present in strains of L. sakei already described. Moreover, from the expression experiments, it was shown that the gene is expressed during the exponential phase and that production procedures typical of fermented sausages are not turning off the expression of the gene encoding the bacteriocin. The capability of the strain studied to produce sakacin P during production is considered an advantage for its use as starter culture to improve the safety aspect of traditional fermented sausages produced in Italy.