Marisa Manzano

Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages

ABSTRACT In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from the sausages and subjected to PCR and reverse transcription-PCR, and the amplicons obtained were analyzed by DGGE. Lactic acid bacteria (LAB) were present together with other organisms, mainly members of the family Micrococcaceae and meat contaminants, such as Brochothrix thermosphacta andEnterococcus sp., during the first 3 days of fermentation. After 3 days, LAB represented the main population, which was responsible for the acidification and proteolysis that determined the characteristic organoleptic profile of the Friuli Venezia Giulia fermented sausages. The PCR-DGGE protocol for studying sausage fermentation proved to be a good tool for monitoring the process in real time, and it makes technological adjustments possible when they are required.

An application of PCR-DGGE analysis to profile the yeast populations in raw milk

Four different zones from the Friuli Venezia Giulia region, North East of Italy, were sampled for the study of the yeast bio-diversity in raw milk. Samples were analysed by traditional methods to isolate different yeast strains that were subjected to identification by sequencing the D1–D2 domains of the 26S rRNA gene. Twelve different species of yeast were identified, six of them belonging to the genera Candida and two to the genera Kluyveromyces. The identified strains were then used for the optimization of a method based on polymerase chain reaction and denaturing gradient gel electrophoresis that was used for a direct monitoring of the populations in the samples. Applying the method to the DNA extracted directly from the raw milk samples, new bands appeared in the gel underlining a different bio-diversity in respect to the traditional method. The approach described is a powerful and reliable tool to monitor directly yeast ecology in milk and milk products without the need of traditional isolation and it could be used to follow specific populations to prevent spoilage or to control contamination.

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.

A novel polymerase chain reaction (PCR) - denaturing gradient gel electrophoresis (DGGE) for the identification of Micrococcaceae strains involved in meat fermentations. Its application to naturally fermented Italian sausages.

A new molecular method consisting of polymerase chain reaction (PCR) amplification and denaturing gradient gel electrophoresis (DGGE) of a small fragment from the 16S rRNA gene identified the Micrococcaceae strains isolated from natural fermented Italian sausages. Lactic acid bacteria, total aerobic mesophilic flora, Enterobacteriaceae and faecal enterococci were also monitored. Micrococcaceaea control strains from international collections were used to optimise the method and 90 strains, isolated from fermented sausages, were identified by biochemical tests and PCR-DGGE. No differences were observed between the methods used. The results reported in this paper prove that Staphylococcus xylosus is the main bacterium involved in fermented sausage production, representing, from the tenth day of ripening, the only Micrococcaceaea species isolated.

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.

Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides differentiation using a PCR-RE technique

A method was developed to differentiate between Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis using the polymerase chain reaction combined with a restriction endonuclease (PCR-RE) technique. This fast and simple protocol, applied to pure culture strains, was developed using the gyrB DNA sequence, as previously proposed by other authors. Strains from international collections were used to optimize the method which was then applied to the identification of strains isolated from food samples. Amplifications were specific for the B. cereus group. Only Staphylococcus aureus gave the same size PCR product, but it was easily differentiated from strains in the B. cereus group by using restriction analysis, based on digestion with the RsaI, Sau3AI and EcoRI endonucleases. Specific amplifications and good differentiations were obtained using pure strains, suggesting the possibility of using the method described to identify the B. cereus group directly in food samples.

Development of a rapid method for the identification of Lactobacillus spp. isolated from naturally fermented Italian sausages using a polymerase chain reaction–temperature gradient gel electrophoresis

L. COCOLIN, M. MANZANO, C. CANTONI and G. COMI.2000. A rapid method for the identification of Lactobacillus spp. isolated from naturally fermented Italian sausages was developed. It is based on the amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis (TGGE). Lactobacillus sakei, L. curvatus, L. alimentarius, L. casei, L. plantarum and L. brevis, obtained from International Collections, were used to optimize the method. Thiry‐nine strains of Lactobacillus spp. were isolated from naturally fermented sausages and, after traditional identification, were tested by the PCR–TGGE protocol developed. No differences were observed comparing the results obtained, apart from five strains identified as L. curvatus that showed a PCR–TGGE profile identical to L. sakei.

Persistence of Listeria monocytogenes strains isolated from products in a Polish fish-processing plant over a 1-year period

Abstract Seventy-one presumptive Listeria monocytogenes strains were isolated over a year from 152 samples comprising raw fish (salmon, seatrout) and their products (mainly, vacuum-packed cold-smoked sliced salmon) in a selected Polish fish-processing plant. Contamination of raw materials was at the level of 4.3–15.4%, whereas final products revealed significantly higher contamination (up to 77.8%) than regarded by other studies as typical (up to 40%). Strains were identified using conventional microbiological methods (including API®LISTERIA tests) and the PCR technique (aimed at iap gene fragment detection). A random amplification polymorphic DNA (RAPD) technique was applied to analyse their intraspecies diversity. RAPD typing revealed an incidence of eight RAPD types. Three of them were isolated over 8–10 months during the plant monitoring. It suggested that they were a persistent element of ‘in-house’ microflora and the applied typing technique produced evidence that fish products could be probably contaminated at the last stages of fish processing (e.g. smoking, slicing, and/or packaging). Their occurrence was probably supported by clone selection caused by ineffective application of cleaning and sanitizing procedures. The possibility of colonization of the production environment by fish-originated L. monocytogenes was also proven. Strains that belonged to a dominant RAPD type were additionally subjected to restriction fragment length polymorphism-pulsed field gel electrophoresis (RFLP-PFGE). RFLP-PFGE confirmed intraspecies similarity of strains belonging to a dominant RAPD type. A subset of strains from salmon samples was also characterized by serotyping. Contrary to earlier reports, they belonged mainly (91.7%) to the serotype 4.

Effects of copper and cadmium on growth, superoxide dismutase and catalase activities in different yeast strains.

1. Three strains of Saccharomyces cerevisiae have been adapted in vitro upon treatment with copper or cadmium. Growth rate, cellular size, metal uptake, superoxide dismutase and catalase activities were measured. 2. Growth rate and metal uptake are quite different among the yeast strains and also for copper and cadmium treatment. At the employed concentrations, only cadmium chiefly affects the cellular volume. 3. Cu, ZnSOD activity is stimulated in the presence of copper, while it is lightly inhibited in the presence of cadmium. Catalase level remains almost unchanged in the conditions tested. This lack of correlation is then discussed.

Use of polymerase chain reaction and restriction enzyme analysis to directly detect and identify Salmonella typhimurium in food

A primer set of oligonucleotides (Salm 3 and Salm 4) from the invA gene of Salmonellae has been evaluated for the specific detection of Salmonella spp. by the polymerase chain reaction (PCR). This primer set amplified 33 Salmonella serovars but did not amplify 16 non‐Salmonella bacteria. Moreover, after PCR amplification, it was possible to identify Salm. typhimurium by restriction enzyme analysis. The PCR‐RE method developed could represent a helpful tool for detecting Salmonella spp., and for directly and rapidly identifying Salm. typhimurium in food.