Lucilla Iacumin

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.

Description of the Microflora of Sourdoughs by Culture-Dependent and Culture-Independent Methods

Four types of sourdoughs (L, C, B, Q) from artisanal bakeries in Northern Italy were studied using culture-dependent and culture-independent methods. In all samples, the yeast numbers ranged from 160 to 10(7)cfu/g, and the numbers of lactic acid bacteria (LAB) ranged from 10(3) to 10(9)cfu/g. The isolated LAB were sequenced, and a similarity was noted between two samples (C, Q), both in terms of the species that were present and in terms of the percentage of isolates. In these two samples, Lactobacillus plantarum accounted for 73% and 89% of the bacteria, and Lactobacillus brevis represented 27% and 11%. In the third sample (B), however, the dominant LAB isolate was Lb. brevis (73%), while Lb. plantarum accounted for only 27%. The fourth sourdough (L) was completely different from the others. In this sample, the most prominent isolate was Weisella cibaria (56%), followed by Lb. plantarum (36%) and Pediococcus pentosaceus (8%). In three out of four samples (L, C and Q), all of the yeasts isolated were identified as Saccharomyces cerevisiae, yet only Candida humilis (90%) and Candida milleri (10%) were isolated in the fourth sample (B). The microbial ecology of the sourdoughs was also examined with direct methods. The results obtained by culture-independent methods and DGGE analysis underline a partial correspondence between the DNA and RNA analysis. These results demonstrate the importance of using a combined analytical approach to explore the microbial communities of sourdoughs.

Direct Identification in Food Samples of Listeria spp. and Listeria monocytogenes by Molecular Methods

ABSTRACT A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.

Moulds and ochratoxin A on surfaces of artisanal and industrial dry sausages.

The use of moulds as a seasoning for sausage can have both desirable and undesirable consequences. The desirable consequences are the creation of a successful product that appeals to consumers. The undesirable consequences are due to the growth of undesirable moulds that produce highly toxic secondary metabolites referred to as mycotoxins. The aim of the paper was to investigate the presence of moulds producing ochratoxin A (OTA) on the surface of sausages from northern Italy. A total of 757 mould strains were isolated from sausage casings. The most frequently identified species were Penicillium nalgiovense, Penicillium oxalicum, Eurotium amstelodami, Penicillium olsonii, Penicillium chrysogenum, Penicillium verrucosum, Penicillium viridicatum, and Eupenicillium crustaceum. Aspergillus ochraceus was detected in only one production lot. Approximately 45% of these samples were positive for the presence of OTA. On the casings of the investigated sausages, the lowest and highest OTA values were 3 and 18 microg/kg, respectively. The OTA concentration was reduced to below the limit of detection (LOD) by brushing and washing the sausages prior to sale. From these data it appears that the presence of OTA on the surface of sausage (on the casings) is not indicative of any health risk for human consumption of sausage, since OTA was not identified inside the dry meat.

Molecular Detection and Identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in Spoiled Wines

ABSTRACT In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B. anomalus. Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from other Brettanomyces spp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B. bruxellensis and B. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection of B. bruxellensis and B. anomalus in wines suspected to be spoiled by Brettanomyces spp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.

Molecular and technological characterization of Staphylococcus xylosus isolated from naturally fermented Italian sausages by RAPD, Rep-PCR and Sau-PCR analysis.

Coagulase-negative cocci (CNC) are important microorganisms in fermented sausages because they release lipases and proteases that are able to free short-chain fatty acids and peptides and aminoacids, respectively, that are responsible for the aroma of fermented sausage. The purpose of this study was to characterize Staphylococcus xylosus strains isolated from naturally fermented sausages, produced in three different processing plants in the Friuli Venezia Giulia region in the Northeast of Italy. Two hundred and forty-nine strains of S. xylosus were identified by species-specific PCR and subjected to molecular and technological characterization. RAPD-PCR with primer M13, Rep-PCR using primer (GTG)(5) and Sau-PCR with primer SAG(1) were used for the molecular analysis, while the capability of the strains to grow at different temperatures and in the presence of NaCl and their lipolytic and proteolytic activity were tested in order to define the technological characteristics. The results obtained allowed us to differentiate strains coming from different plants, thereby admitting the presence of strains that are plant-specific.

Assessment of aerobic and respiratory growth in the Lactobacillus casei group.

One hundred eighty four strains belonging to the species Lactobacillus casei, L. paracasei and L. rhamnosus were screened for their ability to grow under aerobic conditions, in media containing heme and menaquinone and/or compounds generating reactive oxygen species (ROS), in order to identify respiratory and oxygen-tolerant phenotypes. Most strains were able to cope with aerobic conditions and for many strains aerobic growth and heme or heme/menaquinone supplementation increased biomass production compared to anaerobic cultivation. Only four L. casei strains showed a catalase-like activity under anaerobic, aerobic and respiratory conditions and were able to survive in presence of H2O2 (1 mM). Almost all L. casei and L. paracasei strains tolerated menadione (0.2 mM) and most tolerated pyrogallol (50 mM), while L. rhamnosus was usually resistant only to the latter compound. This is the first study in which an extensive screening of oxygen and oxidative stress tolerance of members of the L. casei group has been carried out. Results allowed the selection of strains showing the typical traits of aerobic and respiratory metabolism (increased pH and biomass under aerobic or respiratory conditions) and unique oxidative stress response properties. Aerobic growth and respiration may confer technological and physiological advantages in the L. casei group and oxygen-tolerant phenotypes could be exploited in several food industry applications.

Effect of TiO2 photocatalytic activity in a HDPE-based food packaging on the structural and microbiological stability of a short-ripened cheese.

A high density polyethylene (HDPE)/calcium carbonate (CaCO(3)) film containing TiO(2) was prepared via blown film extrusion process. The photocatalytic properties of this film were evaluated by voltammetric, UV-Vis spectrophotometric and gas chromatographic measurements following the decomposition rate of suitably selected molecular probes, such as 4-hydroxybenzoic acid and methylene blue. The film containing 1% w/w of TiO(2) displayed a profitable and reproducible photoinduced degradation activity towards target organic compounds. The effect of packaging photocatalytic activity on the structural and microbiological stability of a short-ripened cheese was studied. Cheese structure was assessed by dynamic, small deformation rheological tests. A container consisting of a multilayer material, where the layer brought in contact with the food, made from the HDPE+CaCO(3)+TiO(2) composite matrix, was able to provide a greater maintenance of the original cheese structure than a rigid container currently used, mainly due to the inhibition of lactic acid bacteria and coliforms.